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H e l e n L . H e n ry postdoctoral work in Paul D.

Boyer’s group at UCLA,


in 1964 he joined the Department of Biochemistry at
University of California, Riverside
the University of California, Riverside, as an assistant
professor. From 1976 to 1981, he served as chair of
HELEN L. HENRY received her Ph.D. in 1970 from
the department and currently holds a Presidential
Washington University, St. Louis, and did postdoc-
Chair and is a Distinguished Professor of Biochem-
toral work in animal reproduction at Ohio State
istry and Biomedical Sciences. Dr. Norman has also
University. Following further postdoctoral work at
been active for some 25 years in medical education on
the University of California, Riverside, she joined the
the UC Riverside campus and at UCLA through
faculty and is currently a professor of biochemistry.
participation in the UC Riverside/UCLA Program in
From 1990 to 1996, she served as Associate Dean of
Biomedical Sciences, of which he was Dean and
Biological Sciences in the College of Natural and
Director from 1986 to 1991.
Agricultural Sciences.
Dr. Norman’s biomedical research career has
Dr. Henry’s laboratory has made major contri-
focused on the mechanism of action of the vitamin
butions to the understanding of vitamin D metab-
D family of steroids. His chief contributions to these
olism, particularly regulation of the production of the
areas of cellular and molecular endocrinology have
active vitamin D hormone by the kidney. She
played a pivotal role in defining the boundaries of this
pioneered the use of cell culture systems to study
research domain via discoveries that have opened
renal vitamin D metabolism. A related area of
new areas of investigation. The first of these was the
research focus is the regulation of gene expression
discovery in 1968, and chemical characterization in
in the kidney by 1,25-dihydroxyvitamin D3. In
1971, of the hormonally active form of vitamin D,
addition to peer-reviewed research articles, Dr.
1a,25(OH)2-vitamin D3. Subsequent achievements
Henry has authored chapters for the books Vitamin
include the discovery and characterization of the
D and Handbook of Physiology, as well as the article
nuclear receptor for 1a,25(OH)2D3, the clinical
“Vitamin D Metabolism” for this encyclopedia.
evaluation of 1a,25(OH)2D3 in renal osteodystrophy,
Dr. Henry was awarded a National Institutes of
articulation of the concept of the vitamin D
Health (NIH) Research Career Development Award in
endocrine system, the importance of 1a,25(OH)2D3
1977. She received the Fuller Albright Award from the
to insulin secretion, and the discovery of a new
American Society for Bone and Mineral Research in
rapid, nongenomic, signal transduction process for
1984 and has served this society as a member of the
1a,25(OH)2D3.
council and on several scientific program committees.
Dr. Norman has been the recipient of awards that
Dr. Henry has been a member of the NIH General
include a Fulbright Fellowship, 1970; Public Health
Medicine B Study Section (1989 – 1993) and the
Service Career Development Award, 1970; Mead
National Science Foundation Panel on Integrative
Johnson Award, American Institute of Nutrition,
Biology (1994 – 1996). She has served on the editorial
1977; Ernst Oppenheimer Award, Endocrine Society,
boards of several scientific journals, including Endo-
1977; Visiting Lecturer, Australian Society of Endo-
crinology, American Journal of Physiology, and
crinology, 1978; Visiting Faculty Member, Mayo
Journal of Bone and Mineral Research. She is currently
Clinic, 1981; Prix Andre Lichtwitz (INSERM, Paris,
a member of the Endocrine Society, the American
France), 1981; Faculty Research Lecturer, UC River-
Society of Biochemistry and Molecular Biology, the
side, 1982; MERIT Award from National Institutes of
American Society for Bone and Mineral Research, and
Health, 1986; David Curnow Plenary Lecturer,
the American Institute of Nutritional Sciences.
Australian Society for Clinical Biochemistry, 1989;
Osborne and Mendel Award, American Institute of
Nutrition, 1990; Visiting Professor, Catholic Univer-
A n t h o n y W. N o r m a n sity of Leuven, Belgium, 1992; William F. Neuman
University of California, Riverside Award, American Society for Bone and Mineral
Research, 1995; Fellow of American Association for
ANTHONY W. NORMAN received an A.B. from Oberlin the Advancement of Science, 1995; and Visiting
College in 1959, and an M.S. and Ph.D. in Professor, Department of Biochemistry, UC San
biochemistry in 1961 and 1963, respectively, from Francisco/Presidential Chair in Biochemistry, UC
the University of Wisconsin, Madison. Following Riverside, 1999.
A n to n y W. Bu r g e s s P. M i c h a e l C o n n
Ludwig Institute for Cancer Research, Melbourne, Oregon National Primate Research Center,
Australia Beaverton, Oregon

ANTONY W. BURGESS has had an interest in protein P. MICHAEL CONN is Associate Director and Senior
chemistry since his early studies on the conformation- Scientist of the Oregon National Primate Research
al determinants of peptides and proteins. He received Center and Special Assistant to the President and
a Ph.D. at the University of Melbourne; after Professor of Physiology and Pharmacology at Oregon
postdoctoral studies at Cornell University and the Health and Science University. After receiving a B.S.
Weizmann Institute between 1972 and 1974, and teaching certification from the University of
Dr. Burgess returned to Australia, to the Walter and Michigan in 1971, an M.S. from North Carolina
Eliza Hall Institute, to study the growth factors that State University in 1973, and a Ph.D. from Baylor
stimulate blood cell formation. He and his colleagues College of Medicine in 1976, Dr. Conn did a
purified the first colony-stimulating factor (CSF) and fellowship at the NIH/National Institute of Child
discovered growth and differentiation factors con- Health and Human Development. He then joined the
trolling the development of blood cells. This work led faculty in the Department of Pharmacology at Duke
to the molecular cloning of a CSF and the initial University Medical Center, where he was promoted to
clinical studies with recombinant forms of these Associate Professor in 1982. In 1984, he became
factors. Professor and Head of Pharmacology at University of
Dr. Burgess was involved in the detection and Iowa College of Medicine, a position he held for 11
biology of the cell surface receptors for the CSFs years.
before taking up studies on the epidermal growth Dr. Conn is presently Editor-in-Chief of Endo-
factor (EGF)/receptor system. These studies have crine, Methods, Contemporary Endocrinology, and
contributed to the understanding of the three- Contemporary Drug Therapy; prior Editor-in-Chief
dimensional structure of EGF and its receptor, as of Endocrinology, Molecular and Cellular Neuro-
well as the mechanisms associated with signal sciences, Methods in Neuroscience, and Recent
transduction from the different EGF receptor com- Progress in Hormone Research; and prior Editor of
plexes. At present, Dr. Burgess is attempting to Journal of Clinical Endocrinology and Metabolism.
develop improved approaches to cancer therapy He has edited texts in the fields of pharmacology
through the use of EGF receptor inhibitors. He has (Essentials of Pharmacology), neuroscience (Neuro-
an active research program on the molecular basis of science in Medicine), neuroendocrinology (Neuro-
colon cancer, in particular the biology and molecular endocrinology in Physiology and Medicine),
biology of the apc protein. endocrinology (Endocrinology: Basic and Clinical
Presently on staff at the Ludwig Institute for Principles), and molecular endocrinology (Principles
Cancer Research in Melbourne, Dr. Burgess is a of Molecular Regulation), as well as more than 100
committee member of the ARC Selection Committee volumes in endocrinology and neuroscience.
for Centres of Excellence, a past president of the Best known for his research in the area of
Australian Society for Biochemistry and Molecular neuroendocrinology, Dr. Conn has focused on the
Biology, a former chairperson of the Board of the cellular basis of action of gonadotropin-releasing
Biomolecular Research Institute, a former board hormone action in the pituitary and central nervous
member of the International Society of Differen- system. He has authored or coauthored nearly 300
tiation, and a former World Committee member publications in this area. The work of his laboratory
of the Society for Research into Comparative has been recognized with the MERIT Award from the
Leukemic and Associated Diseases. He has been NIH, the J.J. Abel Award of the American Society for
awarded honors that include the Australian Acad- Pharmacology and Experimental Therapeutics, the
emy of Science Gottschalk Medal, 1981; Australian Weitzman and Oppenheimer awards of the Endocrine
Academy of Science Fellow, 1993; the Amgen Prize, Society, the National Science Medal of Mexico
1996; Companion of the Order of Australia, 1998; (the Miguel Aleman Prize), and the Stevenson Award
and the MOG/AMRAD Cancer Achievement of Canada. Dr. Conn has served on the National
Award, 1999. Board of Medical Examiners, including two years as
Chairman of the Reproduction and Endocrinology the Max Planck Institute for Biophysical Chemistry
Committee, and is a previous member of council (1976 –1979). In 1980, Dr. Kelly joined the Depart-
for the American Society for Cell Biology and a ment of Physiology at the University of Pittsburgh
past president of the Endocrine Society. Conn is a School of Medicine and moved to OHSU in 1982. His
member of the Mexican Institute of Medicine and an area of research is the cellular neurophysiology and
honorary investigator of the Mexican Institute of neuropharmacology of hypothalamic neurons that
Social Security. control neuroendocrine functions, motivation, and
reward in the female. Dr. Kelly has been the recipient
of two research scientist development awards from
G e o r g e H . G r e e l e y, J r . the NIH, the Research Career Development Award
University of Texas Medical Branch, Galveston, from the National Institute of Child Health and
Human Development (1987 – 1992), and the
Texas
Research Scientist Development Award from the
National Institute on Drug Abuse (1994 –1999).
GEORGE H. GREELEY, Jr. earned his Ph.D. in endocrin-
Dr. Kelly was the first to demonstrate rapid
ology at the Medical College of Georgia in 1974 and
signaling of estrogen in the central nervous system
was recently recognized by the school as alumni of the
and to show how it alters reproductive function in the
year. Dr. Greeley did postdoctoral training in neuro-
female. In particular, he found that 17b-estradiol can
endocrinology at the University of North Carolina in
directly alter the excitability of gonadotropin hor-
Chapel Hill. Presently, he directs an internationally
mone-releasing hormone (GnRH) neurons, which are
recognized research program in physiology of gastro-
critical for the control of female reproduction. He also
intestinal hormones in the Department of Surgery at
found that the rapid activation by estrogens of protein
the University of Texas Medical Branch in Galveston.
kinase activity in hypothalamic opioid and dopamine
Dr. Greeley has authored or coauthored more than
neurons alters the coupling of neurotransmitter
190 peer-reviewed journal articles and serves on the
receptors to their effector systems (e.g., channels) in
editorial boards of Endocrinology, American Journal
the female. These results have significant ramifications
of Physiology, and Regulatory Peptides. His primary
in terms of stress responses, appetite control, fluid
areas of research include feedback mechanisms
balance, and motivated behaviors, and may explain
underlying regulation of gut hormone secretion, a
many of the gender differences in these functions.
new stomach hormone called ghrelin, and luminal
Dr. Kelly and colleagues are also interested in
regulation of cholecystokinin (CCK) secretion.
characterizing the membrane properties of proopio-
Honors awarded Dr. Greeley include the NATO
melanocortin (POMC), dopamine, and GnRH neur-
Collaborative Research Grant, 1989 to present;
ons in males and females, and the effects of
Invited Participant, Second Galveston International
neurotransmitters on these neurons. Using guinea
Symposium, Galveston, Texas, 1989; Invited Partici-
pigs and transgenic mice as models, they are trying to
pant, International Conference of Gut Hormones,
identify the phosphorylated target proteins that are
Shizuoka, Japan, 1993; Mentor in the APS – NIDDK
altered by neurotransmitters and steroids in hypo-
Travel Fellowship Program for minority students,
thalamic neurons that are critical for the control of
Experimental Biology Meetings, 1995, 1996; and
neuroendocrine functions, motivation, and reward in
Mentor, American Gastroenterological Association,
the female. Dr. Kelly’s recent manuscripts include
Endocrine Society, Summer Student Research Fellow-
“Rapid Actions of Plasma Membrane Estrogen
ship Recipients, 1995 – 1998.
Receptors” with E. R. Levin in Trends in Endocrin-
ology and Metabolism and “Rapid Membrane Effects
of Estrogen in the CNS” with O. K. Rønnekleiv in the
M a rt i n J. K e l ly Academic Press book Hormones, Brain and Behavior.
Oregon Health and Science University, Portland,
Oregon
Pau l B . L a rs e n
MARTIN J. KELLY is a professor of physiology and University of California, Riverside
pharmacology at Oregon Health and Sciences Uni-
versity (OHSU). He obtained his Ph.D. in 1976 PAUL B. LARSEN received his Ph.D. from Purdue
from the University of Texas Southwestern Medical University in 1994, did postdoctoral work at Cornell
School in Dallas and did his postdoctoral training at University (1994 – 1997) and at the University of
Maryland (1997 – 2000), where he was also a U.S. defective cytokine signaling and that mutations in the
Department of Agriculture Fellow. Dr. Larsen is Janus family tyrosine kinase, Jak3, which associates
presently an assistant professor of biochemistry in with gc, cause a similar clinical phenotype. Most of
the College of Natural and Agricultural Sciences at his research focuses on signal transduction and gene
the University of California, Riverside. regulation related to gc-dependent cytokines.
Dr. Larsen’s laboratory focuses on two topics of Dr. Leonard is currently Vice President and
plant biology. The first deals with the elucidation of President-elect of the International Cytokine Society,
the mechanism responsible for the signal transduction a member of the American Association of Immunol-
of ethylene, a plant hormone that regulates many ogists, American Society for Clinical Investigation,
physiological processes throughout plant growth and and the American Association of Physicians, and a
developement. Using Arabidopsis thaliana as a model fellow of the American Association for the Advance-
genetic system, he has been responsible for identifi- ment of Science. He is a current or past member of
cation of novel components of the ethylene signaling major editorial boards, including Immunity, Journal
pathway, an approach that may ultimately give the of Immunology, Journal of Biological Chemistry, and
means to better control such ethylene-regulated Cytokine. He has helped organize major international
processes as fruit ripening, tissue senescence, and meetings in the cytokine field, including a Keystone
induction of pathogen defenses. Additionally, Dr. Symposium and an annual meeting of the Inter-
Larsen’s group is exploring the mechanisms that national Cytokine Society. Dr. Leonard has received
plants utilize to cope with abiotic stress, particularly a number of major awards, including the Outstanding
that of aluminum toxicity in acid soil. Aluminum Investigator Award of the American Federa-
toxicity in acid soils is a global problem limiting crop tion for Clinical Research Foundation. (Note that
productivity for more than 30% of agriculturally Dr. Leonard’s contributions to this book were per-
available land. As for ethylene signaling, Dr. Larsen is formed in his private capacity, and the contents of this
also using Arabidopsis as a model system for book do not necessarily reflect the views of NIH.)
identification of genes that are required for plant
growth in aluminum toxic environments.
Dr. Larsen has presented papers in his areas of G e r a l d L i t wac k
expertise at prestigious seminars at Cambridge Thomas Jefferson University College of Medicine,
University and at the American Society of Plant Philadelphia, Pennsylvania
Physiologists and has authored or coauthored a
number of articles published in scientific journals GERALD LITWACK obtained his Ph.D. in biochemistry
such as Plant Physiology, Plant Molecular Biology, from the University of Wisconsin in 1953. After a
and the Proceedings of the National Academy of postdoctoral year at the Biochemical Laboratories of
Science. He also authored the article, “Ethylene” in the Sorbonne in Paris, he spent the early part of his
this Encyclopedia of Hormones. academic career at Rutgers University and the
University of Pennsylvania, where he was the
recipient of a National Institutes of Health Career
Wa r r e n J. L e o n a r d Development award. In 1964, Dr. Litwack became
National Institutes of Health, Bethesda, Maryland Professor of Biochemistry at the Fels Institute for
Cancer Research and Molecular Biology at Temple
WARREN J. LEONARD is Chief, Laboratory of Molecu- University School of Medicine. In the 1980s, he
lar Immunology, at the National Heart, Lung, and became Deputy Director of the Institute and Laura H.
Blood Institute of the National Institutes of Health. Carnell Professor of Biochemistry and received the
Dr. Leonard was a pioneer in the interleukin-2 field, Faculty Research Award of Temple University.
having cloned the IL-2 receptor a chain in 1983. Dr. In 1991, Dr. Litwack moved to the Jefferson
Leonard has published more than 200 articles and Medical College as Chairman of the Department of
reviews, most of which are related to the IL-2 family Pharmacology and Deputy Director of the Jefferson
of cytokines. His main research focus relates to IL-2 Cancer Institute (now the Kimmel Cancer Institute),
and other cytokines with receptors that contain the an appointment he continued when, in 1996, he
common cytokine receptor g chain, gc, which Dr. became Chair of the newly fused Department of
Leonard’s group demonstrated is the protein that is Biochemistry and Molecular Pharmacology and
mutated in X-linked severe combined immunodefi- Associate Dean for Scientific Affairs. In 2000, he
ciency. He demonstrated that this is a disease of was appointed Vice Dean for Research.
Dr. Litwack’s research has centered on regulation, Dr. Raikhel is the author of more than 100
particularly by hormones, and, during the past 15 research papers and reviews on molecular aspects of
years, the mechanism and regulation of apoptosis. insect reproduction. He has served as a member of the
The Litwack laboratory group characterized the World Health Organization Advisory Committee and
mammalian glucocorticoid receptor and co-discov- has organized several symposia on the topic of insect
ered “ligandin,” subsequently found to be the reproduction and endocrinology, including several
glutathione S-transferase family of enzymes. Dr. Keystone Symposia in Molecular Insect Science. He is
Litwack has published more than 300 papers in the recipient of numerous awards for his research
these areas and is a co-discoverer on several patents achievements. Dr. Raikhel is Editor-in-Chief of
covering many of the caspases in the apoptotic Insect Biochemistry and Molecular Biology and is a
cascade. His service includes participation on scien- member of the editorial board of Annual Reviews of
tific advisory boards (the Diabetes Center of the Entomology.
University of Pennsylvania) and on grant review
panels of the National Science Foundation, National
Institutes of Health, U.S. Army, Israel Cancer
Research Fund, and American Cancer Society. His
R . Pau l Ro b e rt s o n
editorial board service includes Endocrinology, Can- University of Washington, Seattle, Washington
cer Research, Anticancer Research, Oncology R. PAUL ROBERTSON, M.D. is President, Scientific
Research, Proceedings of Experimental Biology and Director, and CEO of the Pacific Northwest Research
Medicine, Journal of Nutrition, Growth and Cancer, Institute. He became Professor of Medicine and
ISI Atlas of Science, Cancer Communications, Pharmacology at the University of Washington in
Chemtracts, Oncology Reports, Critical Reviews in 1980, where he received most of his postgraduate
Eukaryotic Gene Expression, and Apoptosis. training. He has been elected into membership of the
The books and publications Dr. Litwack has American Society for Clinical Investigation and the
authored, coauthored, or edited include Experimen- Association of American Physicians.
tal Biochemistry (1960, John Wiley & Sons), Dr. Robertson has been Editor-in-Chief of Dia-
Biochemical Actions of Hormones (1970 – 1987, betes, the research journal of the American Diabetes
Academic Press, a serial in 14 volumes), Actions Association, and is currently on the editorial board of
of Hormones on Molecular Processes (1964, Aca- the Journal of Biological Chemistry. Honors received
demic Press), Receptor Purification (1989 – 1990, by Dr. Robertson include the endowed Pennock Chair
Humana Press), Receptor (renamed Receptors & for Diabetes Research at the University of Minnesota,
Signal Transduction; 1990 – 1998, a journal founded Banting and Best Lecturer at the Joslin Clinic, the
by G. Litwack and Editor-in-Chief, Humana Press), Moses Barron Award of the Minnesota Affiliate of the
Hormones (1987, 1997, Academic Press), and Vita- American Diabetes Association, and the Albert
mins and Hormones (Academic Press, the publisher’s Renold Award of the American Diabetes Association.
longest running serial). He has published more than 260 manuscripts with
primary emphasis on beta cell function in humans,
animals, and clonal cell lines. His most recent research
activities are centered on studies of glucose toxicity of
Alexander S. Raikhel the beta cell as seen in type 2 diabetic patients. He is
University of California, Riverside also very active in studies of the metabolic conse-
quences of successful pancreas and islet transplan-
ALEXANDER S. RAIKHEL is Professor at the University tation in patients with type 1 diabetes mellitus.
of California, Riverside. He received his Ph.D. in
1975 from the Zoological Institute of the Academy of
Science in St. Petersburg, Russia. Dr. Raikhel is the
leading authority in molecular endocrinology of C h a r l e s E u g e n e Ro s e l l i
insects. His research focuses on endocrine control of Oregon Health and Science University, Portland,
insect reproduction. In particular, he and his collab- Oregon
orators have elucidated a complex network of nuclear
receptors that mediate the action of a steroid CHARLES EUGENE ROSELLI is a professor in the
hormone ecdysone in gene activation and repression Department of Physiology and Pharmacology at the
during egg maturation in mosquitoes. Oregon Health and Science University, where he has
been on the faculty since 1985. He received his Ph.D. This has provided insights into the role of estrogens in
in 1981 from Hahnemann University in Philadelphia, the physiology and pathophysiology of both males
Pennsylvania, and did his postdoctoral training at the and females, revealing many unexpected and non-
Oregon Regional Primate Research Center (1981 – sexually dimorphic roles for estrogens unrelated to
1984). Dr. Roselli’s research focuses on the neuro- sexual differentiation or reproduction.
biological activity of androgens. His work has Dr. Simpson has authored more than 300 peer-
contributed to an integrated understanding of the reviewed articles and some 70 book chapters and
subcellular signaling pathways, the steroid-sensitive nonreviewed publications. He has been a featured
brain circuitry, and the neurochemical mechanisms speaker at events ranging from closed workshops
that are responsible for the behavioral and neuro- such as the Wyeth Ayerst sponsored symposium on
endocrine actions of androgens. Frontiers in Estrogen Action to international and
A major emphasis of Dr. Roselli’s research has national society meetings. Currently, he is Chairman
been directed at the characterization of the aromatase of the International Organizing Committee of the
(CYP19, or estrogen synthetase) signaling pathway in International Congress of Hormonal Steroids and
neural tissue, characterizing the distribution and Hormones and Cancer, and until last year was
regulation of CYP19, and defining its role in neural Chairman of the International Aromatase Confer-
development, adult reproductive behavior, and gon- ence. In 1998, he was Chairman of the Program
adotropin secretion. Dr. Roselli and his colleague Dr. Organizing Committee of the U.S. Endocrine Society
Resko were the first to demonstrate that androgens Annual Meeting. He is a member of the council of the
regulate their own efficacy in the mammalian brain Endocrine Society of Australia, Editor-in-Chief of the
through androgen receptor-dependent positive feed- Journal of Molecular Endocrinology, and Associate
back of the aromatization pathway. His research also Editor of Endocrine Reviews.
demonstrated for the first time that gender differences Dr. Simpson’s honors and awards include the
in neural responsiveness to androgens are expressed Transatlantic Medal Lecturer for the U.K. Society for
at the subcellular level through the differential Endocrinology in 1990, and in 2003 he is the society’s
expression of androgen receptors and aromatase. Asia and Oceania Medal Lecturer; he received the
Dr. Roselli and collaborators recently identified a President’s Scientific Achievement Award from the
sexually dimorphic preoptic nucleus in the ovine Society for Gynecological Investigation (United
brain and found that the volume of this nucleus States). He has been a guest lecturer at several
correlates with sexual partner preference in rams. institutions, including the University of Western
They are currently studying the neuroendocrine and Ontario, the University of Kansas, and Johns
neuroanatomical basis of naturally occurring vari- Hopkins University, and at Princess Takematsu’s
ations in sexual partner preference. Annual Symposium on Breast Cancer in Tokyo.

E va n R . S i m p s o n G u i d o Ve r h o e v e n
Prince Henry’s Institute of Medical Research, Catholic University of Leuven, Leuven, Belgium
Clayton, Australia
GUIDO VERHOEVEN was born in Antwerpen, Belgium,
EVAN R. SIMPSON began his career in 1964 and is on March 26, 1945. In 1970, he completed medical
presently Director of Prince Henry’s Institute of studies at the Catholic University of Leuven, Belgium,
Medical Research of Monash University, Australia. and in 1974 obtained a Ph.D. from the same
Historically, Dr. Simpson’s research has been in three institution. He is a Registered Specialist in Clinical
major fields: the regulation of steroid hormone Chemistry with authorization for nuclear medicine
biosynthesis in the adrenal cortex and in the ovary, in vitro. In 1974, he received a Biomedical Fellowship
the role of lipoprotein cholesterol as precursor for of the Population Council in New York and worked
steroid hormone biosynthesis, and the study of for one year in the laboratory of Professor Jean
estrogen biosynthesis, in particular the regulation of D. Wilson at the University of Texas Southwestern
biosynthesis of the enzyme aromatase, responsible for Medical School in Dallas on the problem of androgen
the biosynthesis of estrogens. Most recently, Dr. insensitivity syndromes. Professor Verhoeven
Simpson’s research group developed the aromatase received several postdoctoral fellowships from the
knockout mouse as a model of estrogen insufficiency. National Fund for Scientific Research from Belgium
and became Full Professor at the Catholic University Methods in Enzymology (1982, Academic Press),
of Leuven in 1986, where he teaches pathophysiology Steroid Hormone Receptors, Structure and Function
and general medicine both at the Medical School and (1983, Elsevier), Laboratory Methods Manual for
at the School for Pharmacy. Hormone Action and Molecular Endocrinology,
Professor Verhoeven has been Chairman of the (1989, Houston Biological Associates), Receptor
Department of Developmental Biology for 14 years. Purification (1990, Humana Press), Endocrinology,
He is Full Member of the Belgian Royal Academy of 4th Edition (2000, Saunders Company), and
Medicine and has served in a variety of capacities Encyclopedia of Molecular Medicine (2002, Wiley
within the scientific community. These include and Sons).
Secretary of the Belgian Contact Group on Steroid Dr. Weigel has served on a number of review
Hormones and Secretary and Vice President of the panels and editorial boards and has participated in
Belgian Society for Endocrinology. He is a member of many invited lectures around the United States and
the Endocrine Society, an academician of the Euro- Europe, most recently for the fifth consecutive year at
pean Academy of Andrology, and a member of the Frontiers in Reproduction in Woods Hole, Massa-
Permanent Scientific Committee of the European chusetts. She has chaired the American Cancer
Testis Workshops. In 1984, he organized the 8th Society Tumor Biochemistry and Endocrinology
European Workshop on the molecular and cellular Study Section, is an editorial board member for
endocrinology of the testis in De Panne, Belgium. Steroids, Endocrinology, and Journal of Biological
Professor Verhoeven’s main research interests are Chemistry, and is a member of the Endocrine
cell –cell interactions and androgen action in the Society, the American Association for Cancer
testis, and androgens and the control of proliferation Research, Women in Cancer Research, the American
and differentiation in the normal prostate and in Society for Bone and Mineral Research, the
prostate tumor cells. In recent years, his research has American Society for Biochemistry and Molecular
increasingly focused on prostate cancer. Professor Biology, the American Association for the Advance-
Verhoeven has published nearly 200 papers in inter- ment of Science, and Women in Endocrino-
national peer-reviewed journals. logy, serving as Secretary – Treasurer from 1998
until 2001.

Na n cy L . We i g e l
Baylor College of Medicine, Houston, Texas Roy E . We i s s
University of Chicago, Chicago, Illinois
NANCY L. WEIGEL earned undergraduate degrees at
Cornell University and a Ph.D. from Johns Hopkins ROY E. WEISS is Professor of Medicine at the
University. Dr. Weigel was named a National University of Chicago and is Associate Director of
Institutes of Health Postdoctoral Fellow (1979 – the Clinical Research Center. Since the mid-1980s,
1981) and a Searle Scholar (1983 –1986). Following Dr. Weiss has been involved in efforts to understand
postdoctoral work in steroid receptors, she joined the the molecular basis of thyroid hormone action. As an
faculty of Baylor College of Medicine, where she is active clinician, educator, and molecular biologist, he
presently a professor in the Department of Molecular has studied the clinical and physiological abnormal-
and Cellular Biology. ities in patients with the syndrome of resistance to
Dr. Weigel is engaged in a variety of research thyroid hormone, to understand mutant thyroid
projects, including the regulation of human steroid hormone receptor isoform and cofactor interaction.
receptors by phosphorylation, the role of androgen Dr. Weiss was the first to demonstrate that this
receptors in prostate cancer, and the effects of vitamin syndrome could be diagnosed at birth and the effect
D on both bone loss and prostate cancer. She has of treatment on outcome. Dr. Weiss has also demon-
authored or coauthored more than 100 scientific strated the importance of nuclear coactivators in
journal publications. Her manuscripts are also thyroid hormone action in vivo. Currently, he is
included in a number of books: Gene Regulation by working on understanding the basis for resistance to
Steroid Hormones (1982, Springer-Verlag), Mechan- thyroid hormone in patients with normal thyroid
isms of Steroid Action (1981, Macmillan Press Ltd.), hormone receptors.
FOREWORD

The discipline of endocrinology was born with the follicle-stimulating hormone, to thrytropin-releasing
discovery of hormones, but the concept of endocrin- hormone, to growth hormone, to name a few. For
ology has been substantially expanded by the more over a decade, little attention was given to the more
recent discovery of paracrine and autocrine regula- difficult task of understanding the functions of their
tors. The field of hormone action was formed to receptors and intracellular signaling pathways.
understand the molecular mechanisms by which Nevertheless, the advent of this assay methodology,
hormones act in cells, and continues to expand including the ability to synthesize radiolabeled pep-
explosively. In the late 1960s, the prevailing view of tide hormones, eventually allowed the identification
hormone action ranged from effects on membrane and quantification of cell surface receptors for
transport of nutrients and precursors for RNA and peptide and amine-containing hormones. The time
protein synthesis to effects on the translation of of this application was about 1970. Researchers
mRNA at the level of ribosomes. Nevertheless, a demonstrated that cAMP levels were induced in
cadre of voices predicted a possible nuclear action on concert with ligand occupation of certain membrane
mRNA synthesis. These voices were correct in that receptors, and the second messenger cAMP was
steroid hormones, acting via their receptors, indeed postulated to initiate intracellular phosphorylation
were proved to regulate gene transcription. To the of unknown targets. At this point, the field of peptide
best of my knowledge, the first paper to be presented hormone action also was born.
at the national endocrine meetings in a new field of The two distinct but related fields, steroid
hormone action was in 1967, and it dealt with hormone action and peptide hormone action, devel-
hormonal stimulation of oviductal protein synthesis. oped together for much of the next decade. Hormone
It was about this time that a small group of scientists action conferences invariably contained talks on both
interested in hormone effects in cells attended a types of receptors and progress was rapid and in
Gordon Conference in New Hampshire; this was one concert with the development of molecular biology.
of the first conferences to focus an entire program on In the steroid field, progress was more rapid initially,
hormone action and mechanisms. The attendees were but by the mid 1980s, the peptide field attained equal
primarily involved in aspects of steroid hormone and mechanistic status.
thyroid hormone actions; peptide hormone action Investigators of steroid hormone action concen-
was yet to experience its own birth and a similar trated on first understanding the “pathway of action”
expansive growth. Only a short time previously, for their hormones. Scientists looked for model
Elwood Jensen had discovered the estrogen-binding systems showing large responses to steroids. One
protein that eventually became the “estrogen recep- approach was to assess changes in enzyme levels in
tor,” thus it was logical that the conference dealt cultured cells. The intact chicken oviduct was another
mainly with steroid receptors; there were also a few of the more notable systems, in that regard because of
papers presenting data that steroid hormones could the ability of sex steroids (estrogen, progesterone) to
induce specific enzyme/protein synthesis in target induce large increases both in certain egg-white
cells. The mechanisms of these effects were the subject proteins and in their respective mRNAs. Viral proteins
of great debate at this first conference on steroid also were shown to be induced by glucocorticoids. The
hormone action, a meeting that persists to this day in finding that purified steroid receptors could bind to
New England each summer. DNA directly led to a new understanding of the
Following the monumental discovery of peptide pathway of steroid, to intracellular receptor, to DNA,
immunoassays, workers in the peptide field were to mRNA, to protein, and to function. Still, many
immersed in the work of measuring hormones, complexities remained to be sorted out when the
ranging from insulin, to luteinizing hormone, to receptor cDNAs were cloned in the 1980s.

xlvii
xlviii FOREWORD

Our concept of hormones expanded considerably transcription factors, numbering 48 in humans. The
with the advent of growth factors and cytokines. availability of cloned cDNAs and other reagents
Arguably, the myriads of growth factors only allowed mutational analyses of receptors, followed by
represent an additional list of peptidelike hormones their reintroduction into cells to monitor effects on
that often act within the tissue of their origin; they synthetic reporter genes; structure –function relation-
have a strong predilection for growth and cell cycle ships proved the existence of receptor domains for
control. Cytokines have both local and distal actions transcriptional activation, nuclear translocation, and
and are particularly oriented to processes such as DNA binding. Now at a frenetic pace, information on
smooth muscle function and inflammation and dimeric DNA binding and heterodimeric partners
apoptosis. (retinoic acid X receptor), receptor crosstalk with
For a decade, it seems as if the peptide action peptide pathways, ligand-independent receptor acti-
researchers were unduly fixated on cAMP induction vation, and receptor phosphorylation was accumu-
and protein kinase A activation. The complexity of lated. More definitive appreciation of the biology of
signaling pathways emanating from membrane recep- classical and orphan receptors was accomplished by
tors increased logarithmically with the discovery of the emerging transgenic technologies and gene knock-
the numerous protein kinases that phosphorylated out strategies. The ability to screen for new ligands for
serine and tyrosine, the kinase-kinases, the phospha- orphan receptors extended the range of hormones to
tases, the calcium and diacylgycerol regulators, and lipids (peroxisome proliferator-activated receptors)
the regulators of all of the phosphorylation inter- and other previously unsuspected metabolic regula-
mediates. The types of receptors that proliferated tors. The yearly stream of publications on the
ranged from seven membrane (protein kinase A), physiology of orphan receptors and their novel
growth factors (tyrosine kinase, protein kinase C), ligands continued to bring excitement and more
cytokine, and eventually even chemokine in nature. expansion to the field. Pharmaceutical companies
The appreciation of G-proteins as upstream targets of salivated at the possibilities of new drugs acting at the
the cAMP pathway was key to eventual solutions of nuclear level. The tamoxifen paradox (it acts in one
the signaling cascade. The discoveries of the ras/raf tissue as an agonist and in another as an antagonist)
pathway for mediating the effects of mitogens, and provided encouragement for the generation of a
the JAK/Stat pathway as mediator for cytokines and successful search for selective receptor modulators
certain peptide hormone effects, were also important that contain tissue- and function-specific profiles.
milestones in unraveling signal transduction in Still, the field clamored for a greater molecular
eukaryotic cells. The realization that CREB and Stat understanding of how the nuclear receptors worked
proteins were regulatable transcription factors that at the level of DNA. The discovery of receptor-
eventually acted on DNA united the steroid –peptide associated regulatory proteins provided this missing
fields, in part, at the level of the nucleus. That order link and changed the field of hormone action
was made out of apparent chaos is a striking tribute further. We moved from a situation wherein many
to the intellectual prowess and perseverance of the believed that intracellular receptors carried out the
workers in this field. Most importantly, the signaling transcriptional regulation inherent to the actions of
pathways emanating from the membrane have steroid/thyroid hormones and vitamins, to an under-
brought new insights to pathologies such as cancer, standing that the receptor-associated “coregulators”
and have led to an explosive development of new are the primary mediators of this genetic response.
pharmaceutical stimulators and inhibitors, with good We now know that the receptor co-activators act
promise for therapies of neuropsychiatric disorders, as powerful transducers of hormone action, either
cancer, and other human disorders. through inherent enzyme activities or by serving as a
After a consolidation period in the 1970s, the scaffold for recruitment of additional co-activator
steroid action field heated up again with the cloning of proteins. The coregulators can be divided loosely into
the steroid, thyroid, vitamin D, and retinoic receptors. two camps: co-activators and co-repressors. Taken
Certain unpredictable events occurred. Investigators together, these molecules mediate the two main
began to clone (by cross-screening) numerous mol- tasks of receptors: stimulation and repression of
ecules that were similar to steroid receptors, but that gene expression.
were not known to be activated by an existing ligand. It is perhaps fitting that a burst of recent attention
The term “orphan receptors” was born and the has focused again on the membrane, where both
deduction was made that the steroid receptors were steroids and their receptors have been postulated to
part of a giant superfamily of nuclear receptor have biologically important effects. Actions of nuclear
FOREWORD xlix

receptors, and also of free hormones binding to in many species. This effort allows the reader the
traditional membrane receptors or ion channels, are opportunity to survey the state of both membrane
likely to be of increasing future interest to workers receptor-initiated signaling and nuclear receptor-
investigating hormonal function. In this respect, initiated signaling from the viewpoints of a wide
we have traveled full circle. I have no doubt that variety of leading investigators. The history and
the future will see many additional examples of breadth of this field are evident within the articles of
membrane reinforcement of nuclear gene regulation, these volumes. The ambitious project will stand
of important ligand-independent activities of recep- as a major reference source for the field, and
tors initiated at the membrane, and of pathway I predict that readers will have no problem
crosstalk among the varieties of hormones and their savoring the current rapid progress and heightened
intracellular pathways. excitement that exists in this vast field of molecular
These volumes of the Encyclopedia of Hormones endocrinology.
represent one of the most ambitious projects com-
pleted to date in the field of hormones and their BERT W. O’MALLEY
actions. The Editors have assembled articles that Thompson Professor and Chair
address a full spectrum of the biology and cellular Department of Molecular and Cellular Biology,
physiology of numerous hormones and their actions Baylor College of Medicine Houston, Texas
P R E FA C E

The publication of the Encyclopedia of Hormones is The Encyclopedia of Hormones is intended to


intended to provide a comprehensive reference work serve as a useful and comprehensive source of
on all known hormones in vertebrate animals, insects, information spanning all aspects of the general
and plants. The list of classical hormones that had been subject of hormones. It consists of nearly 300 articles
discovered and characterized over the interval from that collectively describe hormones from several key
1914 to 1985 numbered approximately 55; however, perspectives: (1) the cellular and subcellular sites of
as a consequence of the application of modern functioning of the hormone, (2) the major physio-
chemical characterization techniques and molecular logical system(s) in which it is operative (e.g.,
biology methodology, this list now exceeds 150 and is reproductive, immune, neuroendocrine, digestive,
still expanding. In fact, during the production interval and developmental), (3) the nature of the receptor
for the Encyclopedia, several new hormones were and signal transduction pathway(s) used by the
discovered and new activities for existing hormones hormone (e.g., nuclear or membrane signal transduc-
were clearly defined. In addition, enormous strides tion), and (4) for the vertebrate hormones, the
have been made in our understanding of the detailed important diseases of deficiency or excess or other
actions of hormones at the molecular and cellular instances for which there is unusual molecular insight
levels. There have been dramatic applications of this available. We expect that the Encyclopedia of
new knowledge in the medical arena with respect to Hormones will be as useful to the scientific expert
both diagnosis and treatment of diseases; for the plant concerned with cutting-edge questions as it will be to
and insect hormones, new applications have arisen in students and interested nonscientists.
the realms of agricultural biotechnology and biologi- Given the broad scope of such a major reference
cal control. work, it was essential to assemble a team of Associate
Some comment is appropriate concerning the Editors. Each of these 14 individuals has dedicated his
definition of a hormone that has been utilized in or her professional career to researching scholarly
compiling entries in the Encyclopedia. Of course, the endeavors in a specific domain of hormones and, as a
classical definition of a hormone is that it is a chemical consequence of the breadth and depth of achievement
messenger in the body: it is secreted by an endocrine in this area, is an acknowledged leader in their field.
gland and is delivered through the circulatory system These interests include the hormone domains of
to target cells that possess receptors specific for the adrenal cortex, calcium-regulating hormones, cyto-
hormone. Occupancy of the receptor by its cognate kines, female reproduction, male reproduction, gas-
hormone leads to the initiation of signal transduction trointestinal hormones, growth factors, thyroid,
processes that result in generation of specific biologi- membrane signal transduction, neuroendocrinology,
cal responses. But in this post-human-genome era, and nuclear signal transduction, pancreas, plant hor-
with the rich array of technologies used to study and mones, and insect hormones.
define at the cellular and molecular levels the The Encyclopedia was launched at a two-day
enormous array of signal transduction pathways meeting of the Editors, Associate Editors, and
employed by cells, the Editors have adopted a broader Elsevier–Academic Press representatives in La Jolla,
definition of hormone. Hormones can now be California, in April, 2001. Here the preliminary list of
considered to include not only chemical messengers article titles prepared by the Editors was refined and a
in the classical sense, but also local paracrine and list of potential authors created. Each Associate
autocrine signals. Thus, the Encyclopedia includes Editor was then responsible for the crucial process
articles on many growth factors, interleukins, and of recruiting authors for the individual entries. As
intracellular mediators of signal transduction. the manuscripts were received by Academic Press,

li
lii PREFACE

they were critically reviewed by the Associate Editors If the Encyclopedia has merit, it is due largely to
and Editors, as well as by the editorial staff at the contributions of the authors of all the articles, and
Academic Press. The final total of 296 articles entered as well to the dedication of the Associate Editors.
production in only 16 months. Shortcomings are, of course, the responsibility of the
All of the articles are formatted according to the Editors and we would appreciate having them
same blueprint and each is intended to be a self- brought to our attention. The completion of this
contained presentation. Each article begins with a large project in the relatively short time, from launch
brief topical content outline that provides the reader meeting to the actual printing of the Encyclopedia
with a listing of the major topics presented in the (only 23 months), is the result of much hard work and
article. The article body begins with an introductory dedication. Certainly the primary credit must go to
paragraph that defines the topic under discussion and the some 500 authors who prepared their contri-
summarizes the content of the article. Following the butions in a timely fashion. The board of Associate
article are reference citations to provide the reader Editors also provided exceptional leadership and
with access to further in-depth consideration of the service. The Editors thank them all.
topic at hand and a cross-reference to related entries Finally, thanks are due to the staff of Elsevier–
in the Encyclopedia. A glossary list defines key terms Academic Press, including Tari Paschall, Judy Meyer,
that may be unfamiliar to the reader and are Chris Morris, and Carolan Gladden, who each
important to an understanding of the article. A provided skillful and friendly ongoing management
compilation of all glossary terms appearing in the of the project.
complete multivolume Encyclopedia is presented in
the final volume as a dictionary of subject matter HELEN L. HENRY
relevant to hormones. ANTHONY W. NORMAN
A
biosynthesis, degradation, inactivation, transport,
Abscisic Acid and subcellular compartmentation of the hormone.
SÉBASTIEN MONGRAND , PETER D. HARE , AND
NAM -HAI CHUA A. Structure
Rockefeller University The 15 carbon atoms of the sesquiterpene ABA
configure an aliphatic ring with one double bond,
I. INTRODUCTION three methyl groups, and an unsaturated chain
II. STRUCTURE AND OCCURRENCE containing the carboxyl group (Fig. 1). The cis and
III. SYNTHESIS trans isomers differ in the orientation of the carboxyl
IV. DEGRADATION group, and the asymmetric carbon at the 10 position
V. TRANSPORT of the ring distinguishes between the S(þ ) and R(2 )
VI. ROLES OF ABA enantiomers. The different forms of ABA occur in
VII. THE ABA SIGNAL TRANSDUCTION PATHWAY
different proportions in plants and can have different
VIII. BIOTECHNOLOGICAL FEATURES
IX. CONCLUSIONS
activities. The S-cis form is the most abundant
naturally occurring form and is the active form in
fast responses such as stomatal closure. Both enan-
The development of strategies that enable tiomers are active in long-term responses such as
growth without excessive consumption of changes in gene expression and protein synthesis. In
water was vital to the evolution of terrestrial contrast to the cis-trans isomers, the S and R forms
plants. It is now well established that phyto- cannot be interconverted in planta. The exact ABA
hormones, in particular, abscisic acid, regulate
chemical structure is essential for its physiological
plant water status.
activity, and the loss of a carboxyl group, a tertiary
hydroxyl group, a 2-cis 4-trans-pentadienoic side
chain, a 40 -ketone, or a double bond in the
I. INTRODUCTION cyclohexane ring greatly reduces activity.
During the 1960s, two independent groups identified
a compound active in the initiation of bud dormancy B. Occurrence
in sycamore and cotton boll abscission, naming it
ABA is widespread in vascular plants, occurring in
dormin and abscisin II, respectively. Following its
mosses, ferns, liverworts (where a similar compound,
purification from cotton fruits, the chemical structure
lunaric acid, plays a similar role), and all algal classes,
of this compound was determined in 1965 and it was
including photosynthetic prokaryotes such as cyano-
renamed abscisic acid (ABA). Shortly after this, it was
bacteria. Some pathogenic fungi make ABA, but the
discovered that ABA levels increase considerably
biosynthetic pathway appears to be quite different
when plants wilt and that ABA causes stomatal
from that of higher plants. ABA is also reported to
closure. These two discoveries highlighted the import-
occur in the mammalian brain, although its role there
ance of ABA in mediating responses of vegetative
is not known.
tissues to environmental stresses such as drought, high
salinity, and low temperature. ABA is also required for
the accumulation of seed nutrient reserves, the III. SYNTHESIS
acquisition of desiccation tolerance, and the arrest of
embryonic development during seed maturation. The endogenous ABA concentration can rise and fall
Despite its name, ABA is not a major regulator of dramatically in response to environmental or devel-
absicission, which is primarily controlled by ethylene. opmental cues. It appears that ABA is synthesized in
almost all cells containing chloroplasts or amylo-
plasts (i.e., plastids), but the regulatory controls
appear to differ between tissues. Not only do absolute
II. STRUCTURE AND OCCURRENCE
ABA concentrations increase dramatically during
Like all hormones, ABA responses depend not only embryogenesis, but the ABA content of leaves and
on the sensitivity of the tissue to ABA, but also on roots increases 10- to 50-fold when water potentials
local ABA concentration. This is regulated by the fall below 2 1.0 MPa (approximately 2 10.0 bar).

1
2 ABSCISIC ACID

FIGURE 1 Chemical structures of the S and R forms of cis-ABA and the (S)-2-trans form of ABA.

The concentration of ABA in the xylem sap of well- at the level of plastidic xanthoxin production, which
watered plants is 1.0 – 15.0 nM and can increase to appears to be rate-limiting in ABA biosynthesis.
3.0 mM after water stress. The main rise in ABA Transgenic experiments indicated that ZEP is primar-
caused by water loss occurs some 2 – 3 h after the ily involved in the regulation of ABA synthesis during
onset of wilting. The ability of cycloheximide to seed development.
block this process indicates a requirement for de novo The final steps are not yet completely defined.
protein synthesis and thus implicates an up-regu- Xanthoxin is converted to ABA in the cytosol via
lation of ABA biosynthesis in stressed tissues. either AB-aldehyde or xanthoxic acid. Genetic
The plant ABA biosynthetic pathway represents a evidence suggests that xanthoxin is first oxidized to
minor branch of the carotenoid pathway and begins AB-aldehyde, although the involvement of xanthoxic
in plastids. In contrast with isoprene biosynthesis in acid as a precursor has not been completely elimi-
animal cells, the main precursor of ABA, isopentenyl nated. Arabidopsis thaliana aba3 and tomato flacca
diphosphate (IPP), is generated by the methyl and sitiens mutants are defective in the last oxidation
erythritol phosphate pathway and not from mevalo- step and are thus unable to oxidize AB-aldehyde to
nic acid. Eight IPP residues are combined to form
geranylgeranyl diphosphate, the precursor for the
biosynthesis of the C40 compound b-carotene. Both
rings of b-carotene are hydroxylated to form the
xanthophyll zeaxanthin, which can be regarded as the
first intermediate in ABA synthesis (Fig. 2). The role
of xanthophylls as intermediates in ABA biosynthesis
is supported by the reduced ABA content of maize vp
(viviparous) mutants that are blocked in early steps of
carotenoid synthesis. Zeaxanthin is then oxidized to
antheraxanthine and to all-trans-violaxanthin by
zeaxanthin epoxidase (ZEP), which is absent in the
Nicotiana plumbaginifolia aba2 mutant. ZEP con-
tains a putative N-terminal transit sequence for
targeting to chloroplasts. ABA2/ZEP expression is
detected in stems, leaves, roots, and seeds and it is
strongly induced by drought stress in roots but not
leaves.
The oxidative cleavage of the 9-cis-epoxycarote-
noid precursor generates the 15C skeleton of ABA.
The maize vp14 mutant is deficient in the chloroplas-
tic 9-cis-epoxycarotenoid dioxygenase (NCED)
responsible for the cleavage of the 9-cis-isomers, but
not the all-trans-isomers, to xanthoxin. Therefore,
the precise order of isomerization-type reactions
remains uncertain. The gene is expressed constitu-
tively in embryos and roots and in contrast with
ABA2/ZEP transcripts, NCED transcripts accumu- FIGURE 2 The ABA biosynthetic pathway. The metabolic
late to high levels in water-stressed leaves. Thus, ABA blocks in various ABA-deficient mutants are indicated in italics.
accumulation in wilted leaves is primarily regulated Adapted from Taylor et al. (2000).
ABSCISIC ACID 3

ABA. This last step involves an enzyme that requires enzymes involved in ABA catabolism nor the genes
a molybdenum cofactor. Arabidopsis aba3 and that encode them have been isolated.
N. plumbaginifolia aba1 mutants cannot produce
the functional molybdate cofactor required by AB-
aldehyde oxidase (AO). This last enzyme of ABA V. TRANSPORT
synthesis is not highly substrate-specific and interest-
ingly, AO can also catalyze synthesis of another plant ABA is secreted by cells into the apoplast (i.e.,
growth regulator, the auxin indole-3-acetic acid. In intercellular space) and is easily transported in both
Arabidopsis, a multigene family comprising at least xylem and phloem sap to most plant parts, especially
four members encodes AO, only one of which stems, leaves, roots, and ripening fruits. Since roots
appears to act specifically in ABA synthesis. are the primary sites of perception of water deficit,
ABA synthesized in roots can be transported to shoot
tissues via the transpiration stream, where it triggers
A. Alternative Pathways stomatal closure to reduce water loss from leaves.
Within leaves, ABA is redistributed as a function of
Although ABA2 is a single-copy gene, the ABA content pH. In a well-watered plant, the xylem sap is more
of the N. plumbaginifolia aba2 null mutant is acidic (approximately pH 6.3) and ABA occurs in
23 – 48% that of the wildtype (WT). Moreover, ABA its protonated form (ABAH). During drought stress,
is present in tomato flacca and sitiens mutants that the sap becomes slightly alkaline (approximately
lack an effective AO. These results suggest either that pH 7.2), favoring the deprotonation of ABAH to
there is more than one biosynthetic pathway or that ABA. As a result, less ABA is taken up by mesophyll
there is some redundancy in part of the primary cells and more is diverted to guard cells. Therefore,
biosynthetic pathway. The 2-trans-ABA-alcohol accu- even though absolute ABA levels may not change, the
mulated during water stress in flacca and sitiens pH-dependent redistribution of root-derived ABA to
mutants could be formed via a P450 mono-oxygenase guard cells can induce stomatal closure.
able to add a second oxygen atom to the C1 position A similar redistribution may exist within cells.
and can be further slowly converted to ABA. This When photosynthesis is active, the pH of the
reaction may also occur to a small extent in WT plants. chloroplast stroma increases as protons are pumped
Unlike plants, fungi are able to synthesize ABA directly into the thylakoid lumen. A prevalence of deproto-
from the 15C compound farnesyl pyrophosphate. nated ABA limits its ability to cross the chloroplast
membrane, causing the accumulation of ABA in the
stroma. During drought stress, photosynthetic rates
IV. DEGRADATION decrease. The resulting drop in stromal pH increases
After wilted leaves regain turgor, ABA can be levels of ABAH, which can traverse membranes and
inactivated by oxidation to phaseic acid and dihy- be released for transport to guard cells.
drophaseic acid or by conjugation to glucose to form
a glucose ester. In the first case, catabolic inactivation
proceeds via hydroxylation at the 80 position to form VI. ROLES OF ABA
an unstable intermediate that subsequently forms ABA is unquestionably involved in a plant’s response
phaseic acid. The ABA-80 -hydroxylase considered as to stress and in the initiation and maintenance of seed
the pivotal enzyme in ABA degradation is a mem- dormancy. However, it also influences many other
brane-associated cytochrome P450 mono-oxygenase. aspects of plant physiology, often by interacting
It is expressed at high levels in plant tissues recovering synergistically/antagonistically with hormones such
from hyperosmotic stresses. Although phaseic acid is as ethylene, gibberellins, cytokinins, auxin, jasmonic
still able to trigger stomatal closure in some species, acid, and brassinosteroids or by modulating meta-
its activity is much weaker than that of ABA. In bolic sensing pathways such as those monitoring
contrast, dihydrophaseic acid, which is the reduced cellular sugar status.
form of phaseic acid, has no detectable activity.
Conjugation of ABA to glucose not only renders ABA A. ABA Triggers Stomatal Closure During
inactive but also changes its distribution in the cell.
Water Stress
Whereas free ABA is mainly cytosolic, ABA-b-D -
glycosyl ester accumulates in vacuoles and could be a Stomata are pores, found on the aerial surfaces of
storage form of the hormone. Until now, neither the plants, that allow CO2 uptake for photosynthesis and
4 ABSCISIC ACID

at the same time the loss of water, which drives the occurs in the last stages of seed maturation. Seeds
transpiration stream. Stomatal pore diameter is prepare for desiccation by accumulating nutritive
regulated by turgor changes of the two surrounding reserves and proteins that allow the cell to survive the
guard cells. Unlike most other cells in higher-plant ensuing loss of up to 90% of its water content. As a
tissues, the absence of plasmodesmata in mature consequence of dehydration, seeds become dormant.
guard cells renders them independent of surrounding The ABA content of seeds increases during the first
cells and enables them to respond autonomously half of seed development and decreases during the
to stimuli such as CO2, water status, temperature, second phase involving seed maturation.
red/blue light, and plant pathogens. Applied ABA ABA strongly induces genes that encode abun-
inhibits the opening and promotes the closure of dantly expressed seed storage proteins (e.g., zein,
stomata. The increased transpiration rates observed conglycinin, and lectin proteins) as well as proteins
in ABA-biosynthetic mutants and the accumulation involved in desiccation tolerance. The highly con-
of ABA in stressed leaves with reduced transpiration served water-soluble and basic late-embryogenesis
are consistent with the view that endogenous ABA abundant (LEA) proteins are rich in glycine/lysine
normally plays an important role in the reduction of and low in hydrophobic residues and are thought to
water loss by transpiration. Expression of an anti- stabilize other proteins when the cell is dehydrated.
ABA antibody in transgenic tobacco plants retained They are related to members of the DHN (dehydrin)
ABA in the endoplasmic reticulum and caused leaves and RAB (Responsive to ABA) protein families.
to wilt by impairing stomatal closure.
The molecular mechanism by which ABA induces
C. ABA Inhibits Germination and Seedling
stomatal closure has been studied using genetic,
biochemical, single-cell, and electrophysiological
Growth
approaches. Opening and closing of stomata is Seed germination can be defined as the resumption of
thought to provoke turgor and volume changes in growth of the embryo following dormancy. As
guard cells. During water stress, the increase in cellular possibly the most critical developmental transition
ABA or in apoplastic ABA at guard cell surfaces in the plant life cycle, germination is contingent on
mediates guard cell closure by triggering a net efflux of suitable environmental conditions. However, dor-
Kþ and Cl2 from the vacuole to the cytoplasm and mant seeds will not germinate even under normally
from the cytoplasm to the apoplast. Additionally, permissive conditions of temperature or water, light,
sucrose and malate are metabolized to osmotically and oxygen availability. Seed dormancy introduces a
inactive starch, all of which function to reduce the delay in germination that provides additional time for
osmolarity in the guard cells. During stomatal open- geographical dispersal and also maximizes seedling
ing, guard cells swell following the accumulation of survival by preventing germination under adverse
Kþ, anions, and sucrose. The resulting out-bowing of conditions.
the guard cell pair increases pore aperture and allows ABA appears to be the most important mediator
reestablishment of transpiration. of seed dormancy. The ability of exogenous ABA to
prevent seed germination in many species has been
B. ABA Promotes Seed Maturation and used to isolate several abi (ABA-insensitive) Arabi-
dopsis mutants (Table 1). Exogenous ABA can also
Dormancy
inhibit the precocious germination of immature
One of the clearest effects of ABA is to prepare embryos in culture. ABA-deficient Arabidopsis (aba)
the seed for desiccation and to impose embryo mutants are nondormant at maturity, and embryos of
dormancy to prevent premature germination. Seeds maize vp mutants germinate directly on the cob while
of ABA-deficient mutants or transgenic plants still attached to the mother plant. This precocious
depleted of endogenous ABA by expression of an germination, named vivipary, suggests that ABA
ABA-specific antibody fail to mature fully and normally constrains developing embryos in an early
acquire dormancy. developmental stage. In contrast with maize, ABA
Seed development can be divided into two phases deficiency in Arabidopsis does not cause vivipary
of equal duration. The first includes growth and because the rigid seed coat prevents embryo growth
development of the embryo and the endosperm. The while the seed is in the seed pod. Nevertheless,
second phase begins with the arrest of cell division vivipary occurs when Arabidopsis abi3 mutant
and the accumulation of storage reserves and is embryos are dissected out of the seed coat before
followed by preparation for desiccation, which complete desiccation.
ABSCISIC ACID 5

TABLE 1 Mutations Affecting ABA Biosynthesis and Signal Transduction Pathway in Plants
a
Plant species Mutations Gene product/function

ABA-deficient mutants
Zea mays vp2,5,7– 9 Carotenoid biosynthesis
Z. mays vp14 9-cis-Epoxycarotenoid dioxygenase
Chlamydomnas reinhardtii M526 ABA xanthophyll biosynthesis
Arabidopsis thaliana aba1 Zeaxanthin epoxidase
A. thaliana aba2 Xanthoxin oxidase
A. thaliana aba3 Molybdenum cofactor biosynthesis
A. thaliana aao3 Aldehyde oxidase
Nicotiana plumbaginifolia aba2 Zeaxanthin epoxidase
N. plumbaginifolia aba1/ckr1 Molybdenum cofactor biosynthesis
N. plumbaginifolia aba2 ABA xanthophyll biosynthesis
N. plumbaginifolia cnxA Molybdenum cofactor biosynthesis
Lycopersicum esculentum notabilis 9-cis-Epoxycarotenoid dioxygenase
L. esculentum flacca Molybdenum cofactor biosynthesis
L. esculentum sitiens Aldehyde oxidase
Solanum phureja droopy Aldehyde oxidase
Hordeum vulgare nar2a Molybdenum cofactor biosynthesis
Pisum sativum wilty ND
ABA-insensitive mutants
A. thaliana abi1 (SD) Type 2C protein phosphatase
A. thaliana abi2 (SD) Type 2C protein phosphatase
A. thaliana abi3 Seed-specific putative transcription factor
A. thaliana abi4 Transcription factor
A. thaliana abi5 bZIP transcription factor
A. thaliana axr2 (D) Auxin response factor (allelic to the auxin mutant iaa7)
A. thaliana gca1– 8 ND
A. thaliana gpa1 Heterotrimeric G-protein a-subunit
Z. mays vp1 Seed-specific bZIP transcription factor
Z. mays rea ND
Hordeum vulgare cool (ND) ND
Craterostigma plantagineum cdt-1 (D) Regulatory RNA or short peptide
ABA-hypersensitive mutants
A. thaliana abh1 Subunit of a nuclear RNA cap-binding complex
A. thaliana bri1 Steroid receptor kinase
A. thaliana era1 Farnesyltransferase b-subunit
A. thaliana era2 ND
A. thaliana era3 Novel transmembrane protein (allelic to the ethylene mutant
ein2)
A. thaliana fiery1 Inositol polyphosphate 1-phosphate
A. thaliana jar1 ND
A. thaliana jin4 ND
A. thaliana hyl1 Double-stranded RNA-binding protein
A. thaliana sax ND

a
Unless indicated, all mutations are recessive (SD, semidominant; D, dominant; ND, not determined).

Germination is regulated by an antagonism D. ABA Controls Root and Shoot Growth


between ABA, which promotes dormancy, and
gibberellic acid (GA), which counteracts the effects ABA shows different effects on root and shoot
of ABA by promoting growth and the mobilization of growth depending on plant water status. Under
storage reserves. An elegant demonstration of this water stress, ABA depresses both shoot and root
antagonism is the recovery of mutants defective in growth, but the overall effect is a dramatic increase in
ABA synthesis in a screen for revertants of GA- the root:shoot ratio, which facilitates water conser-
deficient mutants. vation.
6 ABSCISIC ACID

E. ABA Mediates Wound Responses activates two types of plasma membrane anion-efflux
channels. One of these shows voltage-dependent slow
After mechanical wounding, a specific set of defense- activation (S-type), whereas the other shows rapid
related proteins, such as protease inhibitors I and II, transient activation (R-type). The two types may
cathepsin D inhibitor, and threonine deaminase, reflect different states of a single channel. (3) The
accumulate both at the site of injury and systemically conjugate actions of these channels lead to a transient
throughout the plant. ABA, together with jasmonic or sustained depolarization and the alkalinization of
acid, appears to play a role in the induction of these the guard cell cytoplasm, which (4) deactivates ðKþ in Þ
genes. channels and also contributes to the opening of
voltage-gated Kþ out channels. The ensuing long-term
VII. THE ABA SIGNAL TRANSDUCTION efflux of both anions and Kþ from guard cells
PATHWAY contributes to loss of turgor and to stomatal closure.
Clues as to how the ABA signal is transduced to 2. Ca21 Channels
mediate its physiological and developmental pro- Considerable evidence indicates that ABA produces
cesses are now beginning to emerge. It should be repetitive, transient increases or oscillations in
emphasized that although many individual com- intracellular Ca2þ levels. These encode information
ponents have been identified mainly by molecular required for stomatal closure. Ca2þ-induced [Ca2þ]cyt
genetic approaches, the complete network has not yet oscillations include a repetitive Ca2þ influx across the
been elucidated. plasma membrane coupled to Ca2þ release from an
intracellular compartment. Stomatal closure is abol-
A. Receptor(s) ished in guard cells when a nonoscillating Ca2þ
ABA is thought to initiate its effects by binding to a plateau is imposed experimentally. Many other
receptor(s) that triggers the signal transduction stimuli responsible for stomatal closure (cold shock,
cascade. Currently, the identity of the receptor(s) is oxidative stress, and increases in CO2) also cause
unknown. Cells may possess at least two sites of ABA [Ca2þ]cyt elevations. Anion channel regulation and
perception, one of which is located at the plasma stomatal movement phenotypes of Arabidopsis
membrane and is triggered by extracellular ABA. abi1-1 or abi2-1 mutants are suppressed by exper-
Biophysical studies indicate that ABA effects in imentally elevating [Ca2þ]cyt.
stomatal guard cells also involve intracellular recep- The mechanisms by which ABA activates guard
tors accessible to the protonated form, ABAH, which cell plasma membrane Ca 2þ channels remain
readily permeates membranes. unknown. In Arabidopsis guard cells, ABA causes a
rapid increase of reactive oxygen species (ROS) that
B. Downstream Signaling Events activate hyperpolarization-activated Ca2þ-permeable
channels. ROS-induced stomatal closure and Ca2þ
Recently, considerable insights have been gained into
activation are abolished in the ABA-insensitive
the identities of molecular components of the com-
mutant gca2. The origin of the Ca2þ required to
plex signaling network that mediates the actions of
elevate [Ca2þ]cyt in response to ABA is unclear, but is
ABA. In particular, ion channels and fairly ubiquitous
probably mediated by inositol 1,4,5-trisphosphate
small second messengers have been implicated in
(InsP3) and/or cyclic ADP-ribose (cADPR). RAB18
ABA action.
expression in Arabidopsis suspension culture cells
1. Ion Channels Regulated by ABA Control requires rapid ABA-induced Ca2þ influx and S-type
Stomatal Aperture anion channel activation. ABA-induced membrane
Electrophysiological studies, either by whole cell depolarization in radish seedlings and tobacco
impalement or by patch clamping of the plasma epidermal and mesophyll cells indicates that these
membrane of guard cell protoplasts or isolated mechanisms are of general importance for ABA
vacuoles, have identified a number of membrane ion signaling in different cell types. A Ca2þ-independent
channels. The sequence of events in ABA-induced pathway also appears to exist.
stomatal closure is thought to be the following: (1)
ABA induces release of Ca2þ into the cytosol from an 3. H1 Channels
internal store, e.g., the vacuole. (2) The resultant Inhibition of the plasma membrane Hþ-ATPase
increase in cytosolic Ca2þ inhibits plasma membrane mediated by both cytosolic alkalinization and the
Hþ pumps and inward Kþ ðKþ in Þ channels, but increase in cytosolic Ca2þ may also contribute to
ABSCISIC ACID 7

membrane depolarization. The origin of ABA- InsP3. Whether InsP6 causes [Ca2þ]cyt elevations and
induced cytosolic alkalinization is unknown. whether both messengers function in the same or
separate signaling branches is unknown.
4. Cyclic Nucleotides (cAMP, CGMP, CADPR) Phosphatidic acid (PtdOH) generated from phos-
Cyclic ADP-ribose (cADPR) plays a central role in pholipase D (PLD) increases transiently following
ABA responses. Microinjection of hypocotyl cells of ABA treatment of Vicia faba guard cells. PtdOH
the tomato aurea mutant with both potential promotes stomatal closure and inactivates Kþ in chan-
intermediates in the ABA signaling cascade and nel currents but does not elicit a [Ca2þ]cyt increase,
fusions of the Arabidopsis RD29A and KIN2 suggesting that PLD acts either in a parallel Ca2þ-
promoters to a reporter gene suggested that ABA independent pathway or downstream of Ca2þ release.
triggers a transient accumulation of cADPR, which Both U-73122 (a PI-PLC inhibitor) and 1-butanol (a
induces a release of Ca2þ from internal stores such as PLD inhibitor) only partially inhibit ABA-dependent
vacuoles and the endoplasmic reticulum. Microinjec- stomatal closure, and simultaneous application of
tion of mutant abi1-1 protein inhibited ABA-, both inhibitors does not have additive effects. Thus,
cADPR-, and Ca2þ-induced gene expression, and PLC and PLD appear to act in the same pathway that
these effects were reversed by an excess of WT ABI1 requires the cooperation of an additional pathway(s)
protein. to attain the complete effects of ABA. This may be
Other cyclic nucleotides may also act in a Ca2þ- mediated by cADPR, since simultaneous application
dependent stomatal opening pathway. For example, of 1-butanol with the cADPR antagonist nicotina-
cAMP or the membrane-permeable cyclic GMP mide increases the extent to which ABA-induced
analog 8-Br-cGMP stimulates stomatal opening. stomatal closure is reduced.
cGMP-induced stomatal opening is inhibited by Sphingosine-1-phosphate (S1P) is another lipid-
chelation of external Ca2þ or by inhibitors of derived Ca2þ-mobilizing agent capable of inducing
intracellular Ca2þ release. stomatal closure. An increase in S1P levels occurs in
leaves following drought stress, but disruption of S1P
5. Lipid-Derived Second Messengers production causes only partial inhibition of ABA-
Various lines of evidence suggest that ABA stimulates induced stomatal closure.
phosphoinositide metabolism. ABA-treated guard cell
protoplasts showed a slight increase in InsP3. The 6. Protein Kinases and Protein Phosphatases
release of caged InsP3 into the cytosol of guard cells Phosphorylation/dephosphorylation events are cen-
caused [Ca2þ]cyt increases, inhibition of Kþ
in channels, tral mediators in ABA signaling. In guard cells, the
and stomatal closure. The Arabidopsis fry1 ( fiery1) Ca2þ signal is possibly relayed by specific protein
mutant, which is defective in an inositol polyphos- kinases and phosphatases. Protein kinase inhibitors
phate 1-phosphatase, accumulates more InsP3 than abolish the activation of S-type anion channels and
WT plants after ABA treatment and is hypersensitive thus block ABA-induced stomatal closure. Recipro-
to ABA in germination and gene expression assays. cally, the protein phosphatase inhibitor okadaic acid
Similarly, overexpression of a different InsP3 phos- (OKA) maintains guard cell S-type channels in the
phatase blocked the inhibition of germination and active state. In-gel phosphorylation assays demon-
seedling growth in Arabidopsis. Overexpression of a strated that ABA rapidly activates a Ca2þ-indepen-
stress- and ABA-inducible phosphatidylinositol- dent 48 kDa Ser/Thr protein kinase in V. faba guard
specific phospholipase C (PI-PLC) in Arabidopsis cells. ABA fails to activate anion channels or induce
suggests that although increased InsP3 levels are stomatal closure in guard cells that express a
necessary for maximal ABA-induced gene expression dominant loss-of-function allele of this kinase.
in vegetative tissues, the AtPLC1 isoform is normally Several stress- and ABA-inducible protein kinases
latent and probably participates in secondary ABA have been identified. In epidermal peels of Pisum
responses. A reduction in InsP3 levels in transgenic sativum, the ABA-induced accumulation of a DHN
lines expressing antisense AtPLC1 correlated with transcript was reduced by K-252a (an inhibitor of
their insensitivity to ABA in germination and seedling Ser/Thr protein kinases) and also by OKA or
growth assays. cyclosporin A (an inhibitor of Ser/Thr protein
ABA also stimulates production of myo-inositol- phosphatases type 2B). In barley aleurone proto-
hexakisphosphate (InsP6) in guard cells to a greater plasts, the stimulation of a MAP kinase activity
extent than InsP3. InsP6 inhibits Kþ in channels in a appeared to be correlated with the induction of
Ca2þ-dependent manner with greater efficiency than RAB16 transcript. OKA inhibited the induction
8 ABSCISIC ACID

of HVA1 and RAB16 transcripts by ABA, and soluble signaling proteins to membranes. In addition
phenylarsine oxide (an inhibitor of Tyr protein to enhancing ABA signaling, loss of ERA1 function
phosphatases) blocked RAB16 induction. affects several other signaling pathways and develop-
The analysis of abi1 and abi2 has shed new light mental programs, including meristem development.
on the involvement of phosphorylation events in ABA Thus, although ERA1 targets are not restricted to
signaling. These two dominant ABA-insensitive ABA action, a factor that normally suppresses ABA
mutants, originally isolated in a screen for mutants responses requires farnesylation. The exact relation-
able to germinate and grow in nonpermissive ABA ship between ERA1 and the ABI loci remains
concentrations, have phenotypes reminiscent of ABA unknown.
deficiency viz. reduced seed dormancy, improper
regulation of stomatal aperture, and decreased 8. RNA Binding and ABA
expression of various ABA-inducible genes. ABI1 Arabidopsis abh1 mutants are hypersensitive to ABA-
and ABI2 encode Ser/Thr protein phosphatases type mediated inhibition of germination as well as
2C (PP2C). The dominant mutant alleles abi1-1 and induction of stomatal closure and increases in
abi2-1 have point mutations that substitute a [Ca2þ]cyt. ABH1 is expressed in stomata and encodes
conserved Gly with Asp, probably disrupting the a nuclear transcript cap-binding protein that appar-
conformation of a site required for Mg2þ-binding or ently functions in a heterodimeric complex. ABH1,
phosphatase activity. Several downstream responses by analogy to yeast and mammalian RNA cap-
to ABA are impaired in abi1-1 and abi2-1, including binding proteins, is proposed to regulate the strength
Kþ þ
out and Kin channel regulation, anion channel of ABA signaling by transcript modification of early
activation, and increases in [Ca2þ]cyt. Because the signaling components.
mutations are dominant, it remains unclear whether
the ABI1 and ABI2 are positive or negative regulators
of ABA signaling or, indeed, whether they affect ABA 9. Heterotrimeric G-Protein Action
signaling at all in WT plants. However, because Heterotrimeric G-proteins are central to many
intragenic revertants of abi1-1 and abi2-1 have signaling processes. Arabidopsis contains only a
reduced or no phosphatase activity in vitro and a single gene (GPA1) encoding a prototypic Ga
double mutant of both revertants is hypersensitive to subunit. ABA-mediated inhibition of stomatal open-
ABA, ABI1 and ABI2 are probably negative regula- ing, but not ABA-controlled promotion of stomatal
tors of ABA signaling. Accordingly, overexpression of closure, is impaired in gpa1 null mutants. GPA1 is
WT ABI1 in maize mesophyll protoplasts blocks ABA required for negative regulation of Kþ
in channels and
regulation of gene expression. The precise roles of the pH-independent activation of anion channels.
kinases and phosphatases in ABA signaling and the
identities of their protein substrates have not been 10. Role of the Actin Cytoskeleton in Stomatal
clearly established. Movements
A reorganization of the actin cytoskeleton of guard
7. Farnesylation cells has been observed after ABA treatment. Cyto-
Although researchers have focused mainly on posi- chalasin D (an actin filament-depolymerizing agent)
tively acting components of the ABA signaling activates Kþin channels, while phalloidin (an actin
pathway, inactivation of negative regulators of ABA filament stabilizer) inhibits Kþ channel currents. ABA
signaling should result in an enhanced response to treatments reorganize actin cytoskeleton architecture
ABA. An Arabidopsis mutant era1 (enhanced from a radial arrangement to a randomly oriented
response to A BA) was isolated based on its inability and fragmented pattern. A small Arabidopsis GTP-
to germinate in the presence of low concentrations of binding protein, AtRac1, is a negative regulator in
ABA (0.3 mM) that do not inhibit germination of WT ABA-induced actin reorganization. The inactivation
seeds. The era1 mutation markedly increases seed of AtRac1 by ABA is impaired in abi1-1.
dormancy and ABA hypersensitive activation of
S-type anion currents in this mutant increases
C. Regulation of Gene Expression by ABA
stomatal closure, reducing water loss during drought.
The ERA1 gene encodes the b-subunit of a hetero- ABA regulates the expression of numerous genes
dimeric farnesyltransferase. Farnesyltransferases cat- during embryogenesis and seed maturation as well as
alyze the attachment of a 15-carbon farnesyl lipid to under stress conditions such as heat shock, low
C-terminal target sequences, which localizes specific temperature, drought, and high salinity.
ABSCISIC ACID 9

1. ABA-Inducible Genes activators, the loss of which affects several aspects of


The use of ABA-deficient and ABA-insensitive seed maturation, including the expression of storage
mutants has demonstrated that ABA contributes to proteins and LEA genes. VP1/ABI3-like proteins
the regulation of numerous genes involved in seed appear to activate transcription by distinct mechan-
maturation and/or the response of vegetative tissues to isms depending on the target cis elements. VP1
hyperosmotic stress. Characterization of the promo- interacts directly with the sph element of the C1
ters of ABA-responsive genes has enabled identifi- promoter and acts on ABREs via association with a
cation of the cis- and trans-acting elements that act at distinct trans-acting factor(s).
the termini of branches in the ABA signaling cascade.
Considerable evidence indicates the existence of
ABA-independent dehydration and cold-induced sig- D. Novel Genetic Screens
naling pathways.
Quantitative and mechanistic characterization of new
signaling mutants is necessary for a complete
2. Cis-Acting Elements
molecular understanding of the ABA signaling
Gene activation is mediated by the binding of
cascade. Several mutants have been isolated in screens
transcription factors to ABA-responsive elements
for deregulated ABA control of ABREs fused to
(ABREs) located in the promoters of ABA-induced
reporter genes. Eight gca (growth control by ABA)
genes. To date, more than 20 functional ABREs have
mutants are characterized by reduced sensitivity to
been found in ABA-inducible genes that are abun-
dantly expressed in desiccating seeds and/or are the inhibition of seedling growth by exogenous ABA
responsive to drought stress and ABA in vegetative and aberrant stomatal regulation. An elegant screen
tissues. that uses small differences in leaf temperature to
The first type of ABRE defined was a sequence of distinguish transpiration rates in mutants and WT
8 – 10 bp that shares a conserved ACGT core motif, plants is likely to identify new elements which
named the G-box. The sequence flanking the ACGT mediate ABA action in guard cells. Screens for
core is important for in vivo and in vitro function. enhancer or suppressor mutations offer one approach
Some ACGT elements confer developmental and to identify genes which interact genetically with
tissue-specific expression on a minimal promoter. In known participants in ABA signaling.
a natural promoter context, an ABRE functions with
a coupling element (CE). ABA-responsive complexes
comprising an ABRE and a CE can confer ABA- VIII. BIOTECHNOLOGICAL FEATURES
inducible transcription upon a minimal promoter.
The sph element, first identified in the promoter of the Fresh water scarcity is currently one of the principal
C1 gene involved in anthocyanin synthesis in maize threats to global food security. Plants account for
endosperm, is a second category of cis-acting element approximately 65% of global fresh water use. Losses
distinct from the G-box. in agricultural yields resulting from the desiccation of
crops and horticultural plants during periods of
3. Trans-Acting Factors drought have severe social and economic repercus-
Yeast one-hybrid assays to identify ABRE-binding sions. Unfortunately, because of the high cost of
proteins (AREBs) have enabled cloning of several synthesis and its instability in UV light, there are no
homologous transcription factors of the basic leucine practical uses of ABA. However, synthetic ABA
zipper (bZIP) family. ABA-regulated transcription analogs such as the acetyleneacetal-type compounds
factors of the homeodomain leucine zipper, basic LAB 173 711 and LAB 144143 reduce crop water use
helix-loop-helix leucine zipper, and MYB classes have and increase cold-hardiness. Engineering the ABA
also been identified. AREBS are capable of activating signal transduction network in guard cells to control
reporter genes fused to ABREs and their induction by CO2 intake and water loss could contribute substan-
ABA at the transcript level frequently precedes the tially to more sustainable water use under adverse
induction of other ABA-responsive genes. Arabidopsis environmental conditions. The manipulation of seed
ABI5, the only bZIP AREB recovered in a genetic maturation and dormancy in certain species by
screen, is also subject to posttranscriptional modifi- modification of ABA-regulated developmental pro-
cation by ABA. Maize VP1 and Arabidopsis ABI3 grams may also be of considerable agricultural
appear to be orthologous seed-specific transcriptional significance.
10 ACTIVATING AND INACTIVATING RECEPTOR MUTATIONS

IX. CONCLUSIONS Further Reading


Recent advances have filled in many gaps concerning Assmann, S., and Wang, X.-Q. (2001). From milliseconds to
the biochemistry and subcellular localization of ABA millions of years: Guard cells and environmental responses.
Curr. Opin. Plant Biol. 4, 421–428.
synthesis as well as demonstrating the unquestionable
Busk, P., and Pagès, M. (1998). Regulation of abscisic acid-induced
involvement of ion channels, cytosolic pH, protein transcription. Plant Mol. Biol. 37, 425–435.
(de)phosphorylation, and cADPR- and phosphoinosi- Gazzarrini, S., and McCourt, P. (2001). Genetic interactions
tide-mediated increases in [Ca2þ]cyt in transducing between ABA, ethylene and sugar signaling pathways. Curr.
the ABA signal. Substantial progress has been made in Opin. Plant Biol. 4, 387–391.
characterization of the terminal signaling elements Giraudat, J., Parcy, F., Bertauche, N., Gosti, F., Leung, J., Morris,
P. C., Bouvier-Durand, M., and Vartanian, N. (1994). Current
involved in ABA-mediated transcriptional regulation. advances in abscisic acid action and signaling. Plant Mol. Biol.
Nonetheless, the mechanism(s) of ABA perception 26, 1557– 1577.
and early signaling events that result in cADPR Leung, J., and Giraudat, J. (1998). Abscisic acid signal transduc-
synthesis or InsP3 release remain to be resolved. tion. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 199 –222.
Considering the multitude of physiological responses Milborrow, B. V. (2001). The pathway of biosynthesis of abscisic
acid in vascular plants: A review of the present state of
modulated by ABA, it will be interesting to assess the
knowledge of ABA biosynthesis. J. Exp. Bot. 52, 1145–1164.
extent of overlap in the signaling events involved in Schroeder, J. I., Allen, G. J., Hugouvieux, V., Kwak, J. M., and
well-characterized ABA effects such as the regulation Waner, D. (2001). Guard cell signaling transduction. Annu.
of stomatal closure, the inhibition of seed germina- Rev. Plant Physiol. Plant Mol. Biol. 52, 627 –658.
tion, and the induction of stress-responsive gene Taylor, I. B., Burbidge, A., and Thompson, A. J. (2000). Control of
abscisic acid synthesis. J. Exp. Bot. 51, 1563–1574.
expression. The striking degree of phenotypic pleio-
tropy observed in many mutants recovered in screens Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
for altered sensitivity to ABA indicates extensive
overlap between ABA action and other signaling
pathways. Better insight into the regulation of ABA
concentrations and the cellular capacity for response ACTH
to ABA will provide a more complete picture of its
importance to growth and development throughout
the plant life cycle. See Adrenocorticotropic Hormone

Glossary
Activating and Inactivating
abscission The rejection of plant organs at an abscission
zone where hydrolytic enzymes reduce cell adhesion. Receptor Mutations
dormancy A resting condition with reduced metabolic
rate found in ungerminated seeds and nongrowing ILPO T. HUHTANIEMI
buds. University of Turku, Finland and Imperial
stomata Small openings located in the epidermal layers of College London
plants allowing uptake of CO2 and loss of water.
Stomata are surrounded by two guard cells that control
I. INTRODUCTION
the pore size.
II. ACTIVATING MUTATIONS
stress The consequence of suboptimal environmental con-
III. INACTIVATING MUTATIONS
ditions that significantly decrease plant growth and/or
IV. DOMINANT NEGATIVE MUTATIONS
reproductive capacity below potential.
V. SUMMARY
vivipary The ability of a plant embryo to bypass dormancy
and proceed directly from embryogenesis to germina-
tion if rescued from the normal dehydration that occurs Human hereditary diseases can be divided
during seed maturation. according to the Mendelian inheritance pattern
into three categories: autosomal dominant,
autosomal recessive, and X-linked recessive.
See Also the Following Articles The same division applies to the mutations that
are known today in hormone receptor genes.
Auxin . Brassinosteroids . Cytokinins . Ethylene The autosomal dominant mutations of recep-
. Gibberellins . Jasmonates . Salicylic Acid tor genes cause activating or gain-of-function
10 ACTIVATING AND INACTIVATING RECEPTOR MUTATIONS

IX. CONCLUSIONS Further Reading


Recent advances have filled in many gaps concerning Assmann, S., and Wang, X.-Q. (2001). From milliseconds to
the biochemistry and subcellular localization of ABA millions of years: Guard cells and environmental responses.
Curr. Opin. Plant Biol. 4, 421–428.
synthesis as well as demonstrating the unquestionable
Busk, P., and Pagès, M. (1998). Regulation of abscisic acid-induced
involvement of ion channels, cytosolic pH, protein transcription. Plant Mol. Biol. 37, 425–435.
(de)phosphorylation, and cADPR- and phosphoinosi- Gazzarrini, S., and McCourt, P. (2001). Genetic interactions
tide-mediated increases in [Ca2þ]cyt in transducing between ABA, ethylene and sugar signaling pathways. Curr.
the ABA signal. Substantial progress has been made in Opin. Plant Biol. 4, 387–391.
characterization of the terminal signaling elements Giraudat, J., Parcy, F., Bertauche, N., Gosti, F., Leung, J., Morris,
P. C., Bouvier-Durand, M., and Vartanian, N. (1994). Current
involved in ABA-mediated transcriptional regulation. advances in abscisic acid action and signaling. Plant Mol. Biol.
Nonetheless, the mechanism(s) of ABA perception 26, 1557– 1577.
and early signaling events that result in cADPR Leung, J., and Giraudat, J. (1998). Abscisic acid signal transduc-
synthesis or InsP3 release remain to be resolved. tion. Annu. Rev. Plant Physiol. Plant Mol. Biol. 49, 199 –222.
Considering the multitude of physiological responses Milborrow, B. V. (2001). The pathway of biosynthesis of abscisic
acid in vascular plants: A review of the present state of
modulated by ABA, it will be interesting to assess the
knowledge of ABA biosynthesis. J. Exp. Bot. 52, 1145–1164.
extent of overlap in the signaling events involved in Schroeder, J. I., Allen, G. J., Hugouvieux, V., Kwak, J. M., and
well-characterized ABA effects such as the regulation Waner, D. (2001). Guard cell signaling transduction. Annu.
of stomatal closure, the inhibition of seed germina- Rev. Plant Physiol. Plant Mol. Biol. 52, 627 –658.
tion, and the induction of stress-responsive gene Taylor, I. B., Burbidge, A., and Thompson, A. J. (2000). Control of
abscisic acid synthesis. J. Exp. Bot. 51, 1563–1574.
expression. The striking degree of phenotypic pleio-
tropy observed in many mutants recovered in screens Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
for altered sensitivity to ABA indicates extensive
overlap between ABA action and other signaling
pathways. Better insight into the regulation of ABA
concentrations and the cellular capacity for response ACTH
to ABA will provide a more complete picture of its
importance to growth and development throughout
the plant life cycle. See Adrenocorticotropic Hormone

Glossary
Activating and Inactivating
abscission The rejection of plant organs at an abscission
zone where hydrolytic enzymes reduce cell adhesion. Receptor Mutations
dormancy A resting condition with reduced metabolic
rate found in ungerminated seeds and nongrowing ILPO T. HUHTANIEMI
buds. University of Turku, Finland and Imperial
stomata Small openings located in the epidermal layers of College London
plants allowing uptake of CO2 and loss of water.
Stomata are surrounded by two guard cells that control
I. INTRODUCTION
the pore size.
II. ACTIVATING MUTATIONS
stress The consequence of suboptimal environmental con-
III. INACTIVATING MUTATIONS
ditions that significantly decrease plant growth and/or
IV. DOMINANT NEGATIVE MUTATIONS
reproductive capacity below potential.
V. SUMMARY
vivipary The ability of a plant embryo to bypass dormancy
and proceed directly from embryogenesis to germina-
tion if rescued from the normal dehydration that occurs Human hereditary diseases can be divided
during seed maturation. according to the Mendelian inheritance pattern
into three categories: autosomal dominant,
autosomal recessive, and X-linked recessive.
See Also the Following Articles The same division applies to the mutations that
are known today in hormone receptor genes.
Auxin . Brassinosteroids . Cytokinins . Ethylene The autosomal dominant mutations of recep-
. Gibberellins . Jasmonates . Salicylic Acid tor genes cause activating or gain-of-function
ACTIVATING AND INACTIVATING RECEPTOR MUTATIONS 11

mutations. In these cases, the structure of the sensitivity of the receptor to its normal ligand, (3)
receptor protein is usually altered in such a fashion relaxed specificity of the receptor to ligands, and
that its signal transduction is constitutively (4) acquired novel functions of the mutated receptor.
activated in the absence of ligand hormone, and Category 1 is most common among the activating
one altered allele is sufficient to cause the receptor mutations. Some of these mutations are
phenotype. The autosomal recessive mutations
inherited via the germ line, but some, in particular
are usually of the inactivating or loss-of-function
those encountered in tumors, are somatic (Table 1).
type; i.e., the mutant allele encodes a functionally
inactive receptor protein. In most cases, the normal
allele is sufficient to maintain normal hormonal A. Nuclear Receptors
function, but in some situations the heterozygous Only a few cases of gain-of-function mutations of
carrier has a phenotype, usually in the form of mild
nuclear receptors are currently known, although it is
hormone resistance. X-linked recessive mutations
cause a phenotype in all males and in homozygous possible to produce constitutively activated nuclear
females, and in some cases heterozygous females receptors through site-directed mutagenesis in vitro.
have a milder phenotype. A special case of The reason for their paucity may be the fundamental
inactivating receptor mutations is the dominant role that many of the nuclear receptors play in cellular
negative mutations, where the mutated receptor functions and because their inappropriate activation
inhibits the function of that encoded by the wild- may be embryo-lethal. In fact, almost the only germ-
type allele. This is the only situation where an line mutations known in nuclear receptors are those
inactivating mutation causes the phenotype in of the androgen receptor (AR), not necessary for life.
dominant fashion. Besides the above hereditary The extensions of the polyglutamine (CAG) repeat in
(germ line) mutations, somatic mutations are also the N-terminal part of AR (see below) in Kennedy’s
known in hormone receptors. They are invariably
disease apparently bring about novel neurotoxic
found in tumors, are of the activating type, and
play a role in tumor formation or growth. In functions for the mutated receptor protein. Con-
addition, numerous polymorphisms are known in versely, if this repeat is shortened, as is found in
receptor genes; they either cause no phenotype or somatic mutations in advanced forms of prostatic
may alter susceptibility to certain diseases. cancer, the mutated AR either is constitutively
activated or has relaxed ligand specificity. Activating
mutations of retinoic acid receptors may play a role in
I. INTRODUCTION the pathogenesis of certain leukemias and hepatomas,
due to chromosomal rearrangements within the
A large number of hormone receptor mutations are retinoic acid receptor genes, resulting in fusion
known today, and this article presents appropriate proteins with novel functions. The only other piece
examples of the different mechanisms by which the of information on constitutively activated nuclear
mutated receptor brings about either receptor inac- receptors concerns estrogen receptors that may
tivation or constitutive activation. Hormone recep- contribute to the formation of estrogen-independent
tors can be subdivided into two functional categories cell clones in breast cancer.
on the basis of their cellular location, i.e., those
present on the plasma membrane and those present B. Cell Membrane Receptors
inside the cell, in the nucleus and/or cytoplasm.
Because of differences in the function of these two The receptors for the glycoprotein hormones,
receptor types, they are presented separately, under i.e., luteinizing hormone (LH), follicle-stimulating
the headings of activating and inactivating mutations. hormone (FSH), and thyroid-stimulating hormone
Table 1 summarizes the phenotypes, types of func- (TSH), constitute a good example of gain-of-function
tional alteration, and modes of inheritance of most of mutations of cell membrane receptors. They all
the currently known hormone receptor mutations. belong to the family of G-protein-associated seven-
transmembrane domain receptors. Normal receptor
activation entails binding of the ligand to the
extracellular receptor domain, whereby a conforma-
II. ACTIVATING MUTATIONS
tional change occurs in the transmembrane domain,
Activating or gain-of-function mutations can be allowing activation of the intracellular second-
classified, according to the functional alteration, into messenger generation and ultimately the functional
four categories: (1) constitutive receptor activation response of the target cell to hormonal stimulation.
in the absence of ligand hormone, (2) increased Accordingly, most of the activating mutations in these
12 ACTIVATING AND INACTIVATING RECEPTOR MUTATIONS

TABLE 1 Currently Known Mutations in Hormone Receptor Genes


Receptor Type of mutation Type of inheritance Phenotype

Nuclear receptors
Thyroid hormone Dominant negative D Thyroid hormone resistance
Inactivating R Thyroid hormone resistance
Estrogen-a Inactivating R Male: unfused epiphyses, poor sperm, osteoporosis,
insulin resistance
Activating S Receptor positive, hormone-resistant breast cancer
Androgen Inactivating XR Lack of male sexual differentiation (testicular feminization)
Activating XR Kennedy’s disease
S Advanced prostate cancer
Glucocorticoid Inactivating R Variable symptoms of glucocorticoid resistance
S Nelson’s syndrome (pituitary adenoma)
Mineralocorticoid Inactivating D Autosomal dominant pseudohypoaldosteronism I
Vitamin D Inactivating R Hypocalcemic vitamin D-resistant rickets
Retinoic acid Activating S Various malignant tumors
Dominant negative D
Plasma membrane
receptors
TSH Activating S Toxic thyroid adenomas
D Toxic thyroid hyperplasia (hereditary)
Inactivating R Euthyroid, elevated TSH (mild)
R Hypothyroidism (severe)
LH Activating D Males: gonadotropin-independent precocious puberty
Females: no phenotype
S Leydig cell tumors
Inactivating R Males: lack of male sexual differentiation
Females: anovulatory infertility
FSH Activating D Normal spermatogenesis in the absence of gonadotropins
Inactivating R Females: infertility with arrest of follicular maturation
Males: suppressed spermatogenesis
ACTH Inactivating R Familial glucocorticoid deficiency
Activating D Normal cortisol, low ACTH
MSH Inactivating R Red hair and light skin
Vasopressin Inactivating XR Nephrogenic diabetes insipidus
GnRH Inactivating R Hypogonadotropic hypogonadism
Ca2þ-sensing Inactivating R Familial hypocalciuric hypercalcemia, neonatal severe
hyperparathyroidism
Activating D Hypoparathyroidism, hypocalcemia
PTH/PTHrP Activating D Jansen’s metaphyseal chondrodysplasia
GH Inactivating R Dwarfism
Dominant negative D Dwarfism
GHRH Inactivating R Dwarfism
Insulin Inactivating R Leprechaunism, Rabson –Mendenhall syndrome
Dominant negative D Type A insulin resistance
Erythropoietin Activating D Primary polycythemia
Leptin Inactivating R Obesity, pituitary dysfunction

D, dominant; R, recessive; S, somatic; X, X-linked; TSH, thyroid-stimulating hormone; LH, luteinizing hormone; FSH, follicle-stimulating
hormone; ACTH, adrenocorticotropic hormone; MSH, melanocyte-stimulating hormone; GnRH, gonadotropin-releasing hormone; PTH,
parathyroid hormone; PTHrP, parathyroid hormone-related peptide; GH, growth hormone; GHRH, growth hormone-releasing hormone.

receptors have been discovered in the third intracellu- tor domain, allowing activation of signal transduction
lar loop or the sixth transmembrane region of the without preceding ligand binding. The functional
receptor, assumed to be the hot spot for activating alteration can be demonstrated by transfecting
mutations. The mutation apparently brings about a cDNA that encodes the mutated receptor into a
conformational change in the transmembrane recep- cell line, which then displays the cellular response,
ACTIVATING AND INACTIVATING RECEPTOR MUTATIONS 13

for instance, cAMP production, in the absence of mutation is known. The latter may be due to the fact
ligand hormone (Fig. 1). that activating FSHR mutations do not cause a clear
Both activating germ-line and somatic mutations phenotype or to the fact that the phenotype is
have been observed in the TSH receptor (R), and the unexpected and the right types of patients have not
latter mutations explain the molecular pathogenesis of been investigated. Gain-of-function mutations of the
a large proportion of toxic thyroid adenomas; they LHR cause in males gonadotropin-independent early
have also been implicated in some follicular thyroid onset precocious puberty (testotoxicosis), but in
carcinomas. Tens of this type of mutations are women, for reasons not clearly understood, there is
currently known in the TSHR, but why this receptor no phenotype. Most of the activating LHR mutations
in particular is prone to somatic mutations remains bring about a clear, approximately 10-fold increase in
unclear. Activating germ-line mutations of the TSHR basal cAMP production in cell lines expressing the
are found in hereditary and sporadic toxic thyroid mutated receptor. This explains why testosterone
hyperplasia. The hereditary form is characterized production in affected boys is activated well before
by autosomal dominant transmission, hyperthyroid- the normal age of puberty. An interesting special case is
ism with variable age of onset, hyperplastic steadily an activating somatic LHR mutation that was recently
growing goiter, and absence of stigmata of autoimmu- discovered in Leydig cell tumors. This mutation
nity. The sporadic cases present with congenital activated, in addition to cAMP, the inositol phosphate
hyperthyroidism, due to neomutations in the TSHR signaling pathway, providing an example of an
gene. The mutations found in toxic adenomas and activating mutation with acquired novel function.
neomutations overlap, but another set of mutations
are found in hereditary hyperthyroidism. The latter
present with milder phenotypes and, hence, the auton- III. INACTIVATING MUTATIONS
omous receptor activation is milder than in the other
An inactivating mutation can cause the following
two cases with more severe phenotypes. Natural selec- functional aberrations in the receptor protein: (1)
tion may explain this difference. The sporadic cases decreased synthesis, (2) aberrant intracellular process-
display such strong TSHR activation that carriers of ing, (3) impaired or missing ligand-binding activity,
the same mutations in the germ line would apparently (4) impairment or lack of signal transduction, (5)
not have survived until fertile age in the past. inability to anchor to the plasma membrane, (6)
The other examples of activating mutations in inability to dimerize, if needed for signal transduc-
cell membrane receptors are those of the LHR. tion, or (7) increased degradation.
Approximately 15 activating mutations of the LHR
are currently known, but interestingly, not a single
unequivocally documented case of activating FSHR A. Nuclear Receptors
A good example of a nuclear receptor with inactivat-
ing mutations is the AR, a member of the steroid
hormone/thyroid hormone/retinoic acid receptor
family. It is extremely polymorphic and more than
250 AR mutations are known today. Because the AR
gene is located on the X chromosome, mutational
AR defects exist only in men (X-linked dominant
inheritance). A homozygous female is impossible due
to obligatory infertility of men carrying a mutated
AR gene. The AR protein is a single polypeptide
chain, containing, like all nuclear receptors, three
functional domains, i.e., the transactivation domain,
DNA-binding domain, and ligand-binding domain.
FIGURE 1 Schematic presentation of hormone-stimulated The phenotypes with androgen insensitivity vary
response, for instance. cAMP production, in cells transfected
from mild defects of virilization (Reifenstein’s syn-
with cDNA encoding the wild-type receptor and one carrying
an activating or inactivating mutation. The elevated response in
drome) to complete female phenotype (testicular
the absence of hormonal stimulation is typical for activating feminization), and the extent and location of the AR
mutation. In inactivating mutation the response is totally or mutation often predict the phenotype. Splicing
nearly completely missing. defects, frameshifts, immature termination codons,
14 ACTIVATING AND INACTIVATING RECEPTOR MUTATIONS

and partial or complete gene deletions invariably asymptomatic to isolated chronic fatigue and hyper-
result in total androgen insensitivity. Single amino tension with or without hypokalemic alkalosis and/or
acid substitutions, in contrast, result in a wide variety hyperandrogenism. All GR mutations detected cause
of phenotypes, with severity depending on location of partial hormone resistance, since complete inacti-
the mutation. Substitutions in the DNA-binding vation of glucocorticoid function would probably be
domain are a relatively homogenous group in which lethal. Differences in impact of the various mutations
ligand binding is not altered, but the capability of on glucocorticoid sensitivity of the different target
the AR to modulate androgen-responsive genes is organs probably explain the variability of pheno-
impaired. Mutations in the hormone-binding domain types encountered in this syndrome. A mild form of
have more varying effects. The mutated receptor is autosomal dominant hypoaldosteronism (type I) has
seldom totally unable to bind hormone, whereas the been found to be due to inactivating mutations in the
ligand binding may be qualitatively altered or has low mineralocorticoid receptor. Inactivating mutations
affinity. The phenotype of these mutations is often of thyroid hormone receptors cause, as expected,
difficult to predict. Mutations in the transactivation thyroid hormone resistance. The vitamin D receptor
domain are relatively rare. This part of the AR is the mutations cause hypocalcemic vitamin D-resistant
least conserved and it possibly allows greater vari- rickets. In this case, the mechanism of receptor inacti-
ability in structure without hampering receptor func- vation falls into three categories: (1) suppressed DNA
tion. In addition, no abnormalities in ligand binding binding/nuclear localization, (2) suppressed hormone
can be detected in a large proportion of the patients binding, and (3) inhibition of heterodimerization
despite their clear phenotype of androgen insensi- with retinoic acid X receptor (RXR). The latter effect
tivity. Some other genes involved in androgen action, is incompatible with the mechanism of vitamin D
e.g., those of AR co-activators, may be mutated in action, where the vitamin D-receptor complex must
these cases. heterodimerize with RXR to evoke a biological re-
AR has been found to have an intriguing type of sponse.
structural polymorphism, a polyglutamine repeat
(CAG at the DNA level), in exon 1 of its N-terminus.
B. Cell Membrane Receptors
Short repeats (20 residues) are detected in advanced
forms of prostatic cancer, apparently as somatic An inactivating mutation of a cell membrane-associ-
mutations, in which case the ligand specificity of the ated receptor causes hormone resistance usually in
AR is relaxed and various steroidal and nonsteroidal the recessive mode of inheritance. Large deletions of
ligands can activate it. The normal length of the the gene often predict the inactivation mechanism
repeat is 23 – 27 residues. Slight androgen insensi- of the receptor, but especially if a point mutation is
tivity is apparent when the repeat length is 28, as can discovered, it is difficult to decipher from the small
occur in men with oligo-azoospermia. Finally, clearly structural alteration the type of functional impact
extended polyglutamine repeats (40 repeats) exist in of the mutation. Functional analysis of synthesis,
spinal and bulbar muscular atrophy due to degener- intracellular processing, and function of the mutated
ation of motor neurons (Kennedy’s disease) where receptor protein in cell culture transfections is there-
the receptor function is more drastically altered, and fore needed to prove its functional significance and to
it may have acquired novel toxic effects on motor solve the molecular basis of the inactivation mecha-
neurons. nism. A typical finding with an inactivating receptor
Inactivating mutations are also known in other in functional analysis is schematically presented in
steroid receptors. Only one case of estrogen receptor-a Fig. 1. Similar functional tests are available for nuclear
mutations has been described. This was detected in receptors.
an adult male with tall stature, incomplete closure Mutations of the growth hormone receptor (GHR)
of epiphyses, osteoporosis, and insulin resistance. provide good examples of the various mechanisms of
The receptor inactivation was due to a point muta- receptor inactivation. More than 30 GHR mutations
tion that induced a premature stop codon and hence are currently known, mostly in the area encoding
formation of nonfunctional receptor protein. No the extracellular receptor domain, including exonic
progesterone receptor mutations have been described, deletions and nonsense, frameshift, splice-site, and
possibly since they would be effectively eliminated missense mutations. As is well known, the phenotype
from the genetic pool due to their adverse effect of this condition is GH resistance (Laron dwarfism),
on reproduction. Glucocorticoid receptor (GR) muta- which is usually inherited in an autosomal recessive
tions present a variable range of phenotypes from manner. The various categories of GHR inactivation
ACTIVATING AND INACTIVATING RECEPTOR MUTATIONS 15

in this syndrome can be grouped according to of receptor inactivation, whether a defect in synthesis,
their effects on circulating GH-binding protein intracellular processing, transport to the plasma
(GHBP), which is a proteolytic cleavage product of membrane, ligand binding, or signal transduction,
the plasma membrane GHR. In some of the cases, the has not been studied.
GHBP level is suppressed, as a sign of failure of the As with the LHR, inactivating mutations of
mutated GHR protein to bind GH or to be expressed at the FSHR show correlation between the severity of
the cell membrane. In other types of this syndrome, the phenotype and the completeness of receptor acti-
GHBP levels are either normal or elevated. One of vation. Complete receptor inactivation in women
them, with normal GHBP levels and a point mutation causes primary hypergonadotropic amenorrhea with
in the extracellular domain, was found to be caused impaired pubertal development, lack of follicular
by inactivated GHR due to impairment of expression maturation, and infertility, whereas the phenotype of
and signaling. The third type of GHR mutation incomplete receptor activation is secondary amenor-
has increased GHBP levels, and in this case a point rhea with arrest of follicular maturation. In men, a
mutation resulted in alternative splicing and a surprisingly mild phenotype with variable derange-
premature stop codon, with the resulting receptor ment of spermatogenesis, but no obligatory infer-
protein lacking the transmembrane domain and most tility, is observed with complete FSHR inactivation.
of the intracellular domain. The truncated receptor The impairment of function of the mutated FSHR
was unable to anchor to the cell membrane, but is primarily due to sequestration of the synthesized
was instead secreted in large amounts into the receptor protein inside the cell. The few mutated
circulation, thus causing the elevated GHBP levels receptors expressed at the plasma membrane of trans-
and lack of GH response. In addition, there are cases fected cells seem to display a normal cAMP response to
of GH insensitivity with normal GHR function, hormonal stimulation.
explained by abnormalities in postreceptor signaling
mechanisms. A fourth mechanism of GHR inacti-
vation, through a dominant negative mutation, will be
IV. DOMINANT NEGATIVE MUTATIONS
discussed below.
Another example of inactivating receptor The concept of dominant negative mutation means
mutations is that of the TSHR. A number of mutations that a heterozygously inherited mutated receptor has
are known, and depending on the extent of recep- the capability of blocking the action of functional
tor inactivation, their phenotypes vary greatly. The receptors encoded by the wild-type allele. Abolition of
mildest cases represent euthyroidism with elevated hormone action occurs thus even though only one
TSH levels; the mutation lowers only the affinity of the allele is mutated, and therefore, the mode of inheri-
TSHR-ligand complex, and higher TSH levels can tance is dominant despite a loss-of-function mutation.
maintain euthyroidism, since otherwise the receptor This type of inactivation mechanism is encountered
function is normal. In more severe cases, the affected with receptors that need to be homo- or heterodimer-
individual is hypothyroid, and the mutation causes ized in order to achieve their biological effects. The
sequestration of the mutated receptor inside the cells different modes in which a dominant negative
and near total abolition of its function. mutation brings about inactivation of the wild-type
A third example of inactivating receptor receptor are presented in Fig. 2.
mutations concerns those of the gonadotropins LH In thyroid hormone resistance, typical loss-of-
and FSH. The inactivating LHR mutation in the male function mutations with recessive inheritance are
causes, depending on the extent of receptor inacti- rare. Usually, the mutation causes receptor inacti-
vation, an array of phenotypes ranging from micro- vation in a dominant negative fashion. In most cases,
penis and hypospadias to total lack of male sexual the patients have an inactivating mutation in one of
differentiation (XY, pseudohermaphroditism). The their thyroid hormone receptor (TR) b alleles. The
female phenotype is much milder, entailing only mutated receptor interferes negatively with func-
anovulatory infertility. In functional analyses of the tional receptor proteins encoded by the two normal
LHR mutations there is a good correlation between TRa alleles and one TRb allele. Unliganded TR is
the severity of phenotype and the completeness of bound as a homodimer, or as a heterodimer with
receptor inactivation, as demonstrated by the vari- RXR, to the thyroid hormone response element of a
ably suppressed functional responses to gonadotropin target gene and is associated with a complex of co-
stimulation in cells expressing the various forms of repressor proteins, being transcriptionally inactive.
mutated receptors. In most cases, the real mechanism Mutations usually abolish ligand-binding activity
16 ACTIVATING AND INACTIVATING RECEPTOR MUTATIONS

Dominant negative mutations can explain hor-


mone resistance in the molecular pathogenesis of a rare
syndrome with insulin resistance, acanthosis nigricans
and polycystic ovary syndrome (type A insulin resis-
tance). Another example of a dominant negative
mutation in cell membrane receptors concerns GHR.
In this case, a particular single base substitution can
cause skipping of exon 9 of the GHR upon transcrip-
tion with consequent formation of a receptor without
the intracellular domain, which is an essential part for
signal transduction. The mutated receptor was unable
to transmit signal, but it effectively heterodimerized
with a wild-type receptor to form dimers devoid of
signal transduction capability (Fig. 2D).

V. SUMMARY
The hormone receptor mutations can be divided in
two categories: (1) the constitutively activating muta-
tions that cause inappropriate activation of a hormon-
al effect in the absence of ligand hormone and (2) the
inactivating mutations that are the most common
cause of hormone resistance syndromes. Mutations
have already been detected in a number of hormone
receptor genes, and new mutations are expected to be
found. However, it should be kept in mind that the
receptor mutations may represent only the tip of the
iceberg. All genes encoding the various components
of a hormonal regulatory cascade, from synthesis of
ligand to specific cellular responses downstream of
receptor activation, are prone to mutations. Con-
ditions with a clear phenotype of activating or inacti-
FIGURE 2 Models for mechanisms of receptor inactivation vating receptor mutation, but intact receptor function,
by dominant negative mutations of nuclear (A –C) and cell are candidates for mutations of the other genes
membrane (D) receptors. In each case, this type of receptor
involved in hormone action. Due to the large number
inactivation requires that the wild-type receptor forms homo-
or heterodimers in order to exert its biological action. of such candidate genes it will be a long time before
all aspects of the molecular pathogenesis of inap-
propriate hormone action and hormone resistance are
of the TR. Normally, ligand binding promotes re- fully understood.
placement of co-repressor with co-activators, which
results in receptor activation. Since the mutated Glossary
receptor dimerizes with wild-type TR, it keeps the
complexes transcriptionally inactive, explaining the activating (gain-of-function) mutation Structural altera-
dominant negative effect. Mutated TR may also make tion of the receptor makes it constitutively activated,
stable dimers with RXR at the TR recognition site, i.e., in the absence of ligand hormone. Activating
thus blocking the binding of wild-type TR, needed for mutation can also increase the receptor’s affinity for
the ligand hormone, alter its binding specificity, or
activation of the heterodimer.
permit new functions.
In retinoic acid receptors, chromosomal rearrange- cell membrane receptor A hormone-binding trans-
ments and other types of mutations have been membrane protein. Binding of the ligand hormone
described in connection with various malignancies; triggers a cascade of responses: conformational change
some of the mutated genes function in dominant of the receptor ! activation of the intracellular second-
negative fashion and some in gain-of-function fashion. messenger response(s) ! secondary intracellular
ACTIVIN RECEPTOR SIGNALING 17

response(s) ! functional target cell response(s) to McPhaul, M. J. (1999). Molecular defects of the androgen receptor.
hormone stimulation. J. Steroid Biochem. Mol. Biol. 69, 315 –322.
dominant negative mutation A mutation in a receptor Taylor, J. A., Lewis, K. J., and Lubahn, D. B. (1998). Estrogen
that must be dimerized to evoke the functional receptor mutations. Mol. Cell. Endocrinol. 145, 61– 66.
response. When the mutated receptor dimerizes with Themmen, A. P. N., and Huhtaniemi, I. T. (2000). Mutations of
gonadotropins and gonadotropin receptors: Elucidating the
wild-type receptor, the heterodimer remains function-
physiology and pathophysiology of pituitary–gonadal func-
ally inactive.
tion. Endocr. Rev. 21, 551–583.
hormone resistance Disease condition in which a specific Tritos, N. A., and Mantzoros, C. S. (1998). Clinical review 97:
hormone is unable to exert its biological actions; it is Syndromes of severe insulin resistance. J. Clin. Endocrinol.
usually caused by an inactivating receptor mutation. Metab. 83, 3025–3030.
inactivating (loss-of-function) mutation A mutation that Vassart, G. (1998). Hypo- and hyperthyroidism caused by
inactivates the function of a receptor by one of the mutations of the TSH receptor. In “Contemporary Endocrin-
following mechanisms: decreased synthesis, aberrant ology: G Proteins, Receptors and Disease” (A. L. Spiegel, ed.),
intracellular processing, impaired or missing ligand pp. 119–138. Humana Press, Totowa, NJ.
binding, impaired or missing signal transduction, Yen, P. M., and Chin, W. W. (1994). Molecular mechanisms of
inability to anchor to plasma membrane (cell membrane dominant negative activity by nuclear hormone receptors. Mol.
receptors), inability to dimerize (if needed for action), or Endocrinol. 8, 1450– 1454.
increased degradation.
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
nuclear receptor A hormone receptor present inside the
cell, in either the cytoplasm or the nucleus, binding
hormones that are able to pass through the cell
membrane (e.g., steroid and thyroid hormones). The
hormone-receptor complex binds in dimerized form to Activin Receptor Signaling
specific promoter sequences of hormone-responsive
genes, thus functioning as a transcription factor. PETER C. K. LEUNG * AND CHUN PENG †
University of British Columbia, Vancouver . †York
p

See Also the Following Articles University, Toronto

Glucocorticoid Receptor, Natural Mutations of I. ACTIVIN RECEPTORS


. Glucocorticoid Resistance . Luteinizing Hormone II. SMADS AS INTRACELLULAR MEDIATORS
Receptor Signaling . Membrane Receptor Signaling in III. MODULATION OF ACTIVIN SIGNALING
Health and Disease . Receptor– Receptor Interactions IV. SUMMARY
. Signaling Pathways, Interaction of . Thyroid Hormone
Receptor, TSH and TSH Receptor Mutations
Activins are dimeric proteins consisting of two
inhibin b-subunits. Homo- and heterodimeri-
Further Reading zation of two isoforms of b subunits, bA and
Aittomäki, K., Dieguez Lucena, J. L., Pakarinen, P., Sistonen, P., bB, result in three forms of activins, activin-A,
Tapanainen, J., Gromoll, J., Kaskikari, R., Sankila, E.-M., -B, and -AB. It is now recognized that activins
Lehväslaiho, H., Reyes Engel, A., Nieschlag, E., Huhtaniemi, function mainly as autocrine/paracrine factors
I., and de la Chapelle, A. (1998). Mutation in the follicle- to regulate the proliferation, differentiation,
stimulating hormone receptor gene causes hereditary hyper- and apoptosis of many types of cells and are
gonadotropic ovarian failure. Cell 82, 959–968. involved in a variety of physiological pro-
Allolio, B., and Reincke, M. (1997). Adrenocorticotropin receptor
cesses. Activins also play important roles during
and adrenal disorders. Horm. Res. 47, 273–278.
Clayton, P. E., Freeth, J. S., and Norman, M. R. (1999). Congenital embryonic development. Activins elicit their
growth hormone insensitivity syndromes and their relevance to biological effects through interaction with a
idiopathic short stature. Clin. Endocr. 50, 275–283. receptor complex that contains two types of
Geller, D. S., Rodriguez-Soriano, J., Vallo Boado, A., Schifter, S., structurally related transmembrane serine/
Bayer, M., Chang, S. S., and Lifton, R. P. (1998). Mutations in threonine kinases referred to as type I and type
the mineralocorticoid receptor gene cause autosomal dominant II receptors, respectively. The signal is then
pseudohypoaldosteronism type I. Nat. Genet. 19, 279–281. propagated from the receptors at the cell surface
Huhtaniemi, I. (2000). Activating and inactivating hormone
to target genes in the nucleus by intracellular
receptor mutations. Horm. Res. 56 (Suppl. 3), 9– 16.
Lazar, M. A. (1993). Thyroid hormone receptors: Multiple forms,
mediators, the Smad family of proteins. This sig-
multiple possibilities. Endocr. Rev. 14, 184– 193. naling cascade can be modulated at the cell sur-
Malloy, P. J., Pike, J. W., and Feldman, D. (1999). The vitamin D face by interfering with the interaction between
receptor and the syndrome of hereditary 1,25-dihydroxyvita- activins and their receptors and in the cyto-
min D-resistant rickets. Endocr. Rev. 20, 156 –188. plasm by facilitating or blocking the activation
ACTIVIN RECEPTOR SIGNALING 17

response(s) ! functional target cell response(s) to McPhaul, M. J. (1999). Molecular defects of the androgen receptor.
hormone stimulation. J. Steroid Biochem. Mol. Biol. 69, 315 –322.
dominant negative mutation A mutation in a receptor Taylor, J. A., Lewis, K. J., and Lubahn, D. B. (1998). Estrogen
that must be dimerized to evoke the functional receptor mutations. Mol. Cell. Endocrinol. 145, 61– 66.
response. When the mutated receptor dimerizes with Themmen, A. P. N., and Huhtaniemi, I. T. (2000). Mutations of
gonadotropins and gonadotropin receptors: Elucidating the
wild-type receptor, the heterodimer remains function-
physiology and pathophysiology of pituitary–gonadal func-
ally inactive.
tion. Endocr. Rev. 21, 551–583.
hormone resistance Disease condition in which a specific Tritos, N. A., and Mantzoros, C. S. (1998). Clinical review 97:
hormone is unable to exert its biological actions; it is Syndromes of severe insulin resistance. J. Clin. Endocrinol.
usually caused by an inactivating receptor mutation. Metab. 83, 3025–3030.
inactivating (loss-of-function) mutation A mutation that Vassart, G. (1998). Hypo- and hyperthyroidism caused by
inactivates the function of a receptor by one of the mutations of the TSH receptor. In “Contemporary Endocrin-
following mechanisms: decreased synthesis, aberrant ology: G Proteins, Receptors and Disease” (A. L. Spiegel, ed.),
intracellular processing, impaired or missing ligand pp. 119–138. Humana Press, Totowa, NJ.
binding, impaired or missing signal transduction, Yen, P. M., and Chin, W. W. (1994). Molecular mechanisms of
inability to anchor to plasma membrane (cell membrane dominant negative activity by nuclear hormone receptors. Mol.
receptors), inability to dimerize (if needed for action), or Endocrinol. 8, 1450– 1454.
increased degradation.
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
nuclear receptor A hormone receptor present inside the
cell, in either the cytoplasm or the nucleus, binding
hormones that are able to pass through the cell
membrane (e.g., steroid and thyroid hormones). The
hormone-receptor complex binds in dimerized form to Activin Receptor Signaling
specific promoter sequences of hormone-responsive
genes, thus functioning as a transcription factor. PETER C. K. LEUNG * AND CHUN PENG †
University of British Columbia, Vancouver . †York
p

See Also the Following Articles University, Toronto

Glucocorticoid Receptor, Natural Mutations of I. ACTIVIN RECEPTORS


. Glucocorticoid Resistance . Luteinizing Hormone II. SMADS AS INTRACELLULAR MEDIATORS
Receptor Signaling . Membrane Receptor Signaling in III. MODULATION OF ACTIVIN SIGNALING
Health and Disease . Receptor– Receptor Interactions IV. SUMMARY
. Signaling Pathways, Interaction of . Thyroid Hormone
Receptor, TSH and TSH Receptor Mutations
Activins are dimeric proteins consisting of two
inhibin b-subunits. Homo- and heterodimeri-
Further Reading zation of two isoforms of b subunits, bA and
Aittomäki, K., Dieguez Lucena, J. L., Pakarinen, P., Sistonen, P., bB, result in three forms of activins, activin-A,
Tapanainen, J., Gromoll, J., Kaskikari, R., Sankila, E.-M., -B, and -AB. It is now recognized that activins
Lehväslaiho, H., Reyes Engel, A., Nieschlag, E., Huhtaniemi, function mainly as autocrine/paracrine factors
I., and de la Chapelle, A. (1998). Mutation in the follicle- to regulate the proliferation, differentiation,
stimulating hormone receptor gene causes hereditary hyper- and apoptosis of many types of cells and are
gonadotropic ovarian failure. Cell 82, 959–968. involved in a variety of physiological pro-
Allolio, B., and Reincke, M. (1997). Adrenocorticotropin receptor
cesses. Activins also play important roles during
and adrenal disorders. Horm. Res. 47, 273–278.
Clayton, P. E., Freeth, J. S., and Norman, M. R. (1999). Congenital embryonic development. Activins elicit their
growth hormone insensitivity syndromes and their relevance to biological effects through interaction with a
idiopathic short stature. Clin. Endocr. 50, 275–283. receptor complex that contains two types of
Geller, D. S., Rodriguez-Soriano, J., Vallo Boado, A., Schifter, S., structurally related transmembrane serine/
Bayer, M., Chang, S. S., and Lifton, R. P. (1998). Mutations in threonine kinases referred to as type I and type
the mineralocorticoid receptor gene cause autosomal dominant II receptors, respectively. The signal is then
pseudohypoaldosteronism type I. Nat. Genet. 19, 279–281. propagated from the receptors at the cell surface
Huhtaniemi, I. (2000). Activating and inactivating hormone
to target genes in the nucleus by intracellular
receptor mutations. Horm. Res. 56 (Suppl. 3), 9– 16.
Lazar, M. A. (1993). Thyroid hormone receptors: Multiple forms,
mediators, the Smad family of proteins. This sig-
multiple possibilities. Endocr. Rev. 14, 184– 193. naling cascade can be modulated at the cell sur-
Malloy, P. J., Pike, J. W., and Feldman, D. (1999). The vitamin D face by interfering with the interaction between
receptor and the syndrome of hereditary 1,25-dihydroxyvita- activins and their receptors and in the cyto-
min D-resistant rickets. Endocr. Rev. 20, 156 –188. plasm by facilitating or blocking the activation
18 ACTIVIN RECEPTOR SIGNALING

of Smads. Finally, activin signaling can be termi-


nated through ubiquitin-mediated degradation
of Smads.

Activins share structural similarities with transform-


ing growth factor-b (TGF-b) and are recognized as
members of the TGF-b superfamily. In addition to
activins and TGF-bs, bone morphogenetic proteins
FIGURE 1 Schematic representation of activin receptors.
(BMPs), growth and differentiation factors, inhibins, Both type I and type II receptors have an extracellular region
Müllerian inhibiting hormone, glial cell line-derived where the ligand-binding domain (LBD) is located, a trans-
neurotrophic factors, and Nodal are also members of membrane (TM) region, and an intracellular region containing
this superfamily. a juxtamembrane region, a serine/threonine kinase domain
Originally, activin-A was isolated from por- (KD), and a C-terminal tail. The LBD has 10 cysteine residues
cine follicular fluids and identified as a stimulator (indicated by vertical lines) and one or two glycosylation sites
( ). Type I receptors have a GS domain in the juxtamembrane
of pituitary follicle-stimulating hormone (FSH). This
region and a shorter C-terminal tail.
molecule was found to be identical to a protein that
exhibited potent differentiating effects on erythroleu-
kemia cells and was designated erythroid differen- cloned and designated ActR-IIB. The human ActRII-
tiation factor. A gene is mapped to 2q22.2– q23.3 while the ActR-
IIB gene is located in 3p22. When type II receptors
are expressed alone in mammalian cells, they are
I. ACTIVIN RECEPTORS able to bind to activins with high affinity. The
Initial affinity labeling and cross-linking studies using Kd value of activin type II receptors ranged from 0.1
activin-A as a ligand revealed the presence of two to 0.7 nM.
activin receptor complexes with molecular weights of The extracellular domain of activin type II
approximately 65 and 85 kDa, designated type I and receptors has two N-glycosylation sites and 10
type II activin receptor complexes, respectively. cysteine residues (Fig. 1). The spacing between each
Subsequently, cDNAs and genes for type I and type cysteine is identical between ActR-IIA and ActR-IIB
II activin receptors were cloned. Receptors that can and is conserved in different groups of vertebrates.
bind to activin in transfection assays include activin Studies on the ActR-IIA have shown that deglycosyla-
type I (ActR-I, also known as ActR-IA or activin tion of the receptor resulted in only a moderate
receptor-like kinase 2, ALK-2), ActR-IB (or ALK-4), decrease in receptor affinity, suggesting that it is
ActR-II (also named ActR-IIA), and ActR-IIB. How- not critical for receptor binding. On the other hand,
ever, as discussed below, these receptors can also bind disulfide bonds formed by cysteine residues are
to and mediate the actions of other ligands of the important in maintaining the proper confirmation
TGF-b superfamily. required for activin binding. In ActR-IIA, five disulfide
bonds are formed from the cysteines in the following
arrangement: C1 – C3, C2 –C4, C5 – C8, C6 –C7, and
A. Type II Receptors C9 – C10. The crystal structure of the extracellular
Using an expression cloning strategy, in 1991 ligand-binding domain has been solved. This region
Mathwes and Vale cloned the first activin receptor exhibits a three-finger toxin-like fold formed by seven
from AtT20 mouse corticotropic cells. The cDNA disulfide-cross-linked b sheets. Further analyses have
encodes a protein of 513 amino acids. The mature revealed that three conserved hydrophobic residues
peptide has 494 amino acids and contains an (Phe42, Trp60, and Phe83) interact with one another
extracellular ligand-binding domain, a single trans- to form a binding site on ActR-IIA for activin-A.
membrane domain, and an intracellular region The kinase domain of ActR-IIA and ActR-IIB
containing a juxtamembrane region, a kinase domain contains sequence similar to that of serine/threonine
predicted to be a serine/threonine kinase, and a protein kinases. Studies have shown that they indeed
C-terminal tail (Fig. 1). Based on its ability to bind have kinase activities. Substrates of type II receptor
activin and its size, the receptor was identified as kinases include the receptors themselves and type I
the type II activin receptor and is now referred to activin receptors. Type II receptors are constitutively
as ActR-IIA. Another activin receptor that showed a phosphorylated, even in the absence of ligands.
structure similar to that of ActR-IIA was subsequently The phosphorylation of type II receptors is, at least
ACTIVIN RECEPTOR SIGNALING 19

in part, the result of autophosphorylation. The The GS domain is present in all type I receptors of the
phosphorylation occurs predominantly on serine TGF-b superfamily. The C-terminal tail in the type I
residues and to some extent on threonine. receptors is shorter than that in the type II receptors.
Gene knock-out studies in mice have shown that The human ActR-IA is mapped to 2q23 –q24 and the
deletion of the ActR-IIA gene resulted in severe ActR-IB gene is located at 12q13.
suppression of FSH secretion and impaired reproduc- Unlike type II receptors, type I receptors do not
tive functions. Female mice showed defects in folli- bind to activins on their own. They can bind to ligands
culogenesis and were infertile. Male mice showed only in the presence of activin type II receptors. On the
decreased sperm production and delayed fertility. other hand, type I receptors are essential for receptor
These findings demonstrate that ActR-IIA is a signaling to downstream mediators. It is well estab-
functional receptor that mediates the action of activin lished now that activins first bind to type II receptors,
on FSH secretion. On the other hand, the ActR- which then recruit type I receptors to form a complex
IIA-deficient mice had several developmental defects that contains two or more of each of the type I and
that are quite distinct from those found in activin- type II receptors. Type II receptors subsequently
deficient mice, suggesting that other ligands, rather phosphorylate type I receptors, primarily, but not
than activins, signal through ActR-IIA to regulate exclusively, at the GS domain, which then in turn
embryonic development. Targeted disruption of the propagate the signals to downstream targets, namely,
mouse ActR-IIB gene caused abnormal left – right Smads.
axis formation and lateral asymmetry. Mice deficient Although initial studies demonstrated that ActR-
in the ActR-IIB gene died shortly after birth due to IA, when co-expressed with type II activin receptors,
complicated cardiac defects. The developmental can bind to activins, increasing evidence has indicated
abnormality of axial formation observed in ActR- that the physiological ligand for this receptor is not an
IIB knockout mice is a characteristic nodal activity, activin. In Xenopus laevis embryos, ActR-IA mimics
suggesting that ActR-IIB mediates Nodal signaling the role of BMP-4, but not activins. Many other
during embryonic development. reports have documented that ActR-IA binds to and/
Type II activin receptors have a broad specificity. or mediates the signaling of other members of the
In addition to binding to activins, they have been TGF-b superfamily, including Müllerian inhibiting
shown to bind to BMPs, such as BMP2 and BMP7, substance, BMP-2, BMP-4, and BMP-7. Also, ActR-
and Nodal when co-expressed with appropriate type I IA activates Smad1, which is known to mediate BMP,
receptors. Studies using truncated ActR-IIA and IIB but not activin, signaling. On the other hand, the role
have shown that these receptors mediate both activin of ActR-IB in activin signaling has been confirmed.
and BMP signaling. As discussed above, gene knock- ActR-IB mediates the growth-inhibitory effects of
out experiments also support the notion that other activin in several cell lines. Also, the regulatory
ligands signal through type II activin receptors to effects of activins on target gene expression, such as
regulate vertebrate embryonic development. plasminogen activator inhibitor-1, are conferred by
ActR-IB.
B. Type I Receptors
II. SMADS AS INTRACELLULAR MEDIATORS
Following the cloning of type II receptors, the sequen-
ces of type I activin receptors were also obtained. Once type I receptors are phosphorylated by type II
Similar to type II receptors, the type I receptors receptors, they become active kinases and can
also have an extracellular ligand-binding domain, a phosphorylate downstream signaling molecules.
transmembrane domain, and an intracellular kinase Smad proteins are intracellular mediators of the
domain. The extracellular domains of type I receptors TGF-b superfamily. They function as effector mol-
also have 10 cysteine residues; however, the spacing ecules to transduce the signals of the TGF-b super-
among these residues is different between ActR-IA family from the cell surface to the nucleus. The first
and ActR-IB and also differs from that in the type II member of the Smad family was discovered in
activin receptors. A unique feature of the type I Drosophila melanogaster through genetic screenings
receptors is that they contain a segment of about 30 and was named Mad (mother against dpp). A related
residues that is rich in serine, threonine, and glycine, protein was also identified in Caenorhabditis elegans
located between the transmembrane and kinase and is referred to as Sma. Subsequently, homologues
domains. This segment has a characteristic sequence of Mad and Sma were found in various vertebrates
of SGSGSG and therefore was named the GS domain. and are now called Smads.
20 ACTIVIN RECEPTOR SIGNALING

A. Structural Features of Smads that are found to associate with this region include
Smad themselves to form oligomers and many tran-
To date, eight Smads have been identified in ver- scription factors. In the case of Smad2 and Smad3,
tebrates and they are classified into three subfamilies this domain also contains phosphorylation sites for
based on their structural and functional character- ActR-IB and a binding site for a Smad anchor for
istics: receptor-regulated Smads (R-Smads), common receptor activation (SARA), which recruits Smad2
Smads (Co-Smads), and inhibitory Smads (I-Smads). and 3 to activin and TGF-b receptors. The linker
The R-Smads, Smad1, 2, 3, 5, and 8, are activated by region, although poorly conserved, also has impor-
specific type I receptors of the TGF-b superfamily tant functions. This region is known to contain the
through phosphorylation. Smad4, also referred to as phosphorylation sites for mitogen-activated protein
“deleted in pancreatic carcinoma” (DPC4), is the only kinases. The linker region of Smad4 has a Smad acti-
Co-Smad in vertebrates and can form a complex with vation domain that is important for transcriptional
phosphorylated R-Smads. The I-Smads (Smad6 and 7) activation.
function as inhibitors of the receptor-regulated
Smads. Figure 2 shows the major structural features
of Smads. In R-Smads and Co-Smads, there are two B. Signal Transduction via Smads
highly conserved regions, MH1 and MH2. These two
Upon activin binding, Smad2 and (or) Smad3 are
domains, located at the N-and C-terminals, respec-
transiently associated with activin receptor com-
tively, are linked by a less conserved linker region. The plexes. Both Smad2 and Smad3 can be phosphory-
MH1 domain is much shorter in the inhibitory Smads lated by ActR-IB. Subsequently, the activated
than in the R-Smads and Smad4. R-Smads also have Smad2 and (or) Smad3 form a complex with Smad4
a characteristic phosphorylation site, SSXS, at their and are translocated into the nucleus. This Smad
C-termini. All Smads lack intrinsic enzyme activity. complex then interacts with DNA to regulate target
They function primarily through regulation of pro- gene expression. Although the Smad complex can
tein – protein and protein –DNA interactions. bind to DNA directly, the affinity and specificity of
Smads that have been identified as being involved such binding are low and therefore DNA-binding
in activin receptor signaling include Smad2, 3, 4, partners are required for regulation of specific target
and 7. This set of Smads also mediates TGF-b signal gene expression. The best-characterized transcription
transduction. In human, genes for Smad2, Smad4, factor that interacts with Smads to regulate activin-
and Smad7 are all mapped to the region 18q21.1, and induced transcription is the forkhead activin signal
the Smad3 gene is found at 15q21 –q22. The MH1 transducer (FAST), a winged helix forkhead tran-
domain of Smad3 can bind to DNA directly, but the scription factor. Activin induces the transcription of
MH1 domain of Smad2 lacks DNA-binding activity. homeobox genes Mix. 2 in Xenopus and goodecoid in
This is apparently due to an insertion of 30 residues mouse during embryonic development. Activation of
in the MH1 domain of the Smad2. This insertion these genes by activin involves the formation of
is thought to alter the conformation of the MH1 a transcription complex comprising Smad2, Smad4,
domain, thereby preventing the direct interaction of and FAST. The protein complex binds to specific ele-
Smad2 with DNA. The MH1 domain of Smad4 also ments in the promoter regions of these genes through
has DNA-binding activity. The MH2 domain of both FAST- and Smad-binding sites. Following the
Smad2, Smad3, Smad4, and Smad7 is mainly binding of Smad and DNA-binding partners to the
responsible for protein– protein interactions. Proteins DNA, Smads can further recruit transcription co-
activators or co-repressors to positively or negatively
regulate gene expression.

III. MODULATION OF ACTIVIN SIGNALING


A. Inhibition of Signaling at the Cell Surface
FIGURE 2 Structural features of Smads. R-Smads and Co- Activin signaling can be blocked at the receptor level
Smad (Smad4) have two conserved domains, MH1 (gray) and by two types of molecules, follistatins and inhibins.
MH2 (vertical rules), connected by a linker region (black). Follistatins are binding proteins of activins that
I-Smads have a much shorter MH1 domain. neutralize activin actions in many biological systems
ACTIVIN RECEPTOR SIGNALING 21

by controlling the accessibility of activins to their therefore, antagonize activin actions. The binding
receptors. There are two major forms of follistatins, affinity of inhibins to ActR-IIA is 10-fold lower than
FS-288 and FS-315, generated from alternative that between activins and ActR-IIA; however, it has
splicing of the same gene. Each b-subunit can bind been recently shown that betaglycan, a proposed
to one follistatin and therefore, the activin –follistatin TGF-b type III receptor, can bind to inhibins with
complex consists of one activin molecule and two high affinity and that the binding of inhibins to
follistatin molecules. The affinity of follistatin binding betaglycan results in an increased affinity of inhibin
to activins is similar to that between activins and their binding to ActR-IIA. It was therefore suggested that
receptors. Follistatins neutralize activin bioactivity by inhibins bind to betaglycan and ActR-IIA and block
inhibiting the binding of activins to their type II activin signaling by competing with activins for their
receptors. In addition, FS-288 inhibits activin action type II receptors.
by increasing the endocytotic degradation of activin
through the association with cell surface heparan
sulfate.
B. Modulation in Cytoplasm
Inhibins are structurally related to activins by The interaction between activin receptors and Smads
sharing the same b-subunit. It is well documented in the cytoplasm is facilitated by some proteins with a
that inhibins block many actions of activins and FYVE (Fab1p, YOTB, VAC1p, EEAI) domain, such
function as activin antagonists. Several studies have as SARA and Hgs. It has been shown that SARA and
shown that inhibins can bind to ActR-IIA and, Hgs (Hrs) cooperate to recruit Samd2 and Smad3 to

FIGURE 3 Activin signaling via the Smad pathway. Activin first binds to the type II receptors, which in turn recruit
and phosphorylate type I receptors. The type I receptors then phosphorylate Smad2 and/or Smad3. The phosphorylated
R-Smad forms a complex with Smad4 and is then translocated to the nucleus. The R-Smad– Smad4 complex interacts
with other DNA-binding co-factors to regulate gene expression. Follistatin inhibits activin signaling by binding to
activin and making it unavailable to its receptor. Inhibin blocks activin signaling by competing with activin for type II
receptors. SARA and Hgs enhance activin signaling by recruiting R-Smads to the activin receptors while Smad7 inhibits
activin signaling by preventing phosphorylation of R-Smads by the activin receptor. Finally, Smad signaling may be
turned off via ubiquitin-mediated degradation.
22 ACTIVIN RECEPTOR SIGNALING

the activin receptor complex. Overexpression of between R-Smads and receptors. Finally, Smad
SARA and Hgs enhances activin signaling by increas- signaling can be terminated through the proteolytic
ing the phosphorylation and activation of Smad2 and degradation of Smads via the ubiquitin-dependent
Smad3 by ActR-IB. On the other hand, overexpres- pathway.
sion of a nonfunctional mutant of SARA or Hag
decreases activin signaling.
Glossary
Smad7 inhibits activin signaling by binding stably
to the activated receptor complex and thereby block- activins Protein hormones belonging to the transforming
ing the association of Smad2 and/or Smad3 with the growth factor-b superfamily that regulate many devel-
receptors and preventing the phosphorylation and opmental and physiological processes, particularly
activation of R-Smads. Smad7 may also facilitate the reproduction. They are either homo- or heterodimers
of two related inhibin b-subunits, bA and bB.
degradation of the active receptor complex. Interest-
receptor serine/threonine kinase A membrane receptor that
ingly, the Smad7 gene is induced by activin. Therefore,
phosphorylates serines and/or threonines on target
Smad7 likely plays a role in the negative feedback proteins. The receptor has an extracellular domain, a
control of activin signaling. Smad7 has been shown to transmembrane region, and an intracellular kinase
attenuate the actions of activin on cell growth, domain.
apoptosis, and Xenopus embryonic development. Smad proteins A group of intracellular proteins that
mediate signaling by members of the TGF-b super-
C. Termination of Signaling in the Nucleus family. They are vertebrate homologues of Drosophila
Mad (mother against dpp) and Caenorhabditis elegans
The activated Smad2 has been shown to be degraded Sma. They include receptor-mediated Smads that are
through the ubiquitin-dependent 26S proteasome phosphorylated and activated by type I receptors,
pathway. Although the precise involvement of this common Smads that form complexes with receptor-
pathway in activin signaling requires further studies, mediated Smads, and inhibitory Smads that antagonize
degradation of activated Smads in the nucleus could signaling by members of the TGF-b superfamily.
type I activin receptor A component of the activin receptor
provide a mechanism to switch off activin signaling.
complex. It is a serine/threonine kinase that phosphory-
lates intracellular mediators after being phosphorylated
by a type II activin receptor.
IV. SUMMARY
type II activin receptor A component of the activin receptor
Although activin receptors were discovered almost a complex. It binds to activins and in turn phosphorylates
decade ago, the signaling mechanisms underlying type I receptors via its intracellular serine/threonine
activin actions have been uncovered only in recent kinase domain.
years. As shown in Fig. 3, activins bind to cell surface
serine/threonine kinase receptors via two activation See Also the Following Articles
steps. First, activins bind to constitutively active type
Activins . Angiotensin II Receptor Signaling . Follicle
II receptors and this complex then recruits type I
Stimulating Hormone (FSH) . Inhibin Receptor Signaling
receptors. Second, type II receptors phosphorylate
. Inhibins, Activins, and Follistatins . Luteinizing Hormone
type I receptors at the GS domain. Following recep- Receptor Signaling . Protein Kinases
tor activation, the activin signal is transmitted into
the nucleus via intracellular mediators, Smads. The
type I receptor, ActR-IB, phosphorylates Smad 2 and/ Further Reading
or Smad 3. The phosphorylated Smad2 and (or) Attisano, L., Silvestri, C., Izzi, L., and Labbe, E. (2001). The
Smad3 then form(s) a complex with Smad4, and the transcriptional role of Smads and FAST (FoxH1) in TGFb and
complex enters the nucleus. The Smad complex then activin signalling. Mol. Cell. Endocrinol. 180, 3–11.
interacts with other transcription factors, such as Chang, H., Lau, A. L., and Matzuk, M. M. (2001). Studying TGF-b
superfamily signaling by knockouts and knockins. Mol. Cell.
FAST-1, and recruits transcription co-activators or Endocrinol. 180, 38–46.
co-repressors to regulate the expression of target Gaddy-Kurten, D., Tsuchida, K., and Vale, W. (1995). Activins and
genes. Activin signaling can be blocked at the the receptor serine kinase superfamily. Rec. Prog. Horm. Res.
receptor level by their binding proteins, follistatins, 50, 109–129.
and by their antagonists, inhibins. In the cytoplasm, Lebrun, J.-J., Chen, Y., and Vale, W. (1997). Receptor serine
kinases and signaling by activins and inhibins. In “Inhibin,
SARA and Hgs facilitate activin signaling by recruit- Activin and Follistatin Regulatory Functions in System and
ing R-Smads to activin receptors while Smad7 Cell Biology” (T. Aono, H. Sugina, and W. W. Vale, eds.),
inhibits activin signaling by blocking the interaction pp. 1–20. Springer-Verlag, Berlin/New York.
ACTIVINS 23

Massagué, J. (1998). TGF-b signal transduction. Annu. Rev. (TGF-b) superfamily of growth and differentiation
Biochem. 67, 753–791. factors. Since these proteins were first described as
Massagué, J., and Chen, Y.-G. (2000). Controlling TGF-b
signaling. Genes Dev. 6, 627 –644.
regulators of the release of follicle-stimulating hor-
Mather, J. P., Moore, A., and Li, R. (1997). Activins, inhibins and mone (FSH) from the pituitary, multiple actions have
follistatins: Further thoughts on a growing family of regula- been assigned to them in a variety of tissues. An
tors. Proc. Soc. Exp. Biol. Med. 215, 209–222. additional three activin b-subunit proteins have been
Miura, S., Takeshita, T., Asan, H., Kimura, Y., Murata, K., described, i.e., bC, bD, and bE, and there is a growing
Yoshiteru, S., Hanai, J.-I., Beppu, H., Tsukazaki, T., Wrana,
J. L., Miyaono, K., and Sugamura, K. (2000). Hgs (Hrs), a
family of recognized activin- and inhibin-binding
FYVE domain protein, is involved in Smad signaling through proteins, receptors, and signaling molecules (Table 1).
cooperation with SARA. Mol. Cell. Biol. 20, 9346–9355.
Oh, S. P., and Li, E. (1997). The signaling pathway mediated by the
type IIB activin receptor controls axial patterning and lateral
asymmetry in the mouse. Genes Dev. 11, 1812–1826. II. EXPRESSION AND BIOSYNTHESIS
Peng, C., and Mukai, S. (2000). Activins and their receptors in OF ACTIVINS
female reproduction. Biochem. Cell Biol. 78, 261– 279.
Wrana, J. L. (2000). Regulation of Smad activity. Cell 100, The TGF-b superfamily of growth factors includes
189–192. bone morphogenetic proteins (BMPs) and Müllerian
inhibitory substance (MIS), and currently over 45
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
members of this family have been identified. Struc-
tural similarities between activins and other members
of the TGF-b superfamily are based on the conserva-
Activins tion of the number and spacing of the cysteines within
each subunit and the disulfide linkages between the
two subunits that form the characteristic cysteine
GAIL P. RISBRIDGER
knots. Other similarities relate to dimer formation,
Monash University, Melbourne the location of the bioactive peptide in the carboxy-
terminal region of the precursor molecule, and the
I. INTRODUCTION similarities in intracellular signaling mechanisms.
II. EXPRESSION AND BIOSYNTHESIS OF ACTIVINS The biosynthesis of activins requires the formation
III. SIGNALING OF ACTIVIN LIGANDS
of homo- or heterodimers of disulfide-linked bA- or
IV. ACTIVINS IN PHYSIOLOGY
V. ACTIVINS IN PATHOLOGY AND DISEASE
bB-subunit proteins that share 63% amino acid
VI. CLINICAL APPLICATIONS identity. The assembly of different combinations of
VII. SUMMARY activin subunits bA and bB is reflected in their
nomenclature; thus, activin A is a dimer composed
of bAbA, activin B is composed of bBbB, and activin
Activins, acting in opposition to inhibins, are AB is composed of bAbB. There is an independent
involved in diverse physiological activities, pattern of synthesis of the two subunit genes, which
including stimulating pituitary, hypothalamic,
are located on different chromosomes; the activin
and gonadal hormones, insulin secretion, germ
cell development and differentiation, erythroid bA-subunit gene is located on 7p15 – 7p14 and the
differentiation, nerve cell survival, and embryo- activin bB-subunit gene on 2qcen– 2q13.
nic development. Activin b-subunit types A –E Another subset of activin b-subunits (bC, bD, and
form homodimers and heterodimers that play bE) was identified more recently, based on their
different roles in health and disease in different homology to subunits bA and bB; this subset of
tissues throughout the body. Assays that allow subunits shares 62% amino acid identity with each
comparisons of specific subunit activities are other. The activin bC-subunit shows close similarity
necessary to understand the complex roles of to the activin bE-subunit in terms of genomic
each of the activin subunits. organization and chromosomal localization on
12q13.1, and subunits bC, bD, and bE are considered
to be a separate subset of activins. The bC-subunit
I. INTRODUCTION
dimerizes with itself in vitro and with subunits bA and
Activin subunits are present in numerous human bB to form the putative activins, activin C, AC, and
tissues of both endocrine and nonendocrine organs. BC. Activin subunits bD and bE have been isolated
Homo- or heterodimers of activin bA or bB subunits from Xenopus and mouse cDNA libraries, respect-
are members of the transforming growth factor-b ively, but their capacity to form activin heterodimers
ACTIVINS 23

Massagué, J. (1998). TGF-b signal transduction. Annu. Rev. (TGF-b) superfamily of growth and differentiation
Biochem. 67, 753–791. factors. Since these proteins were first described as
Massagué, J., and Chen, Y.-G. (2000). Controlling TGF-b
signaling. Genes Dev. 6, 627 –644.
regulators of the release of follicle-stimulating hor-
Mather, J. P., Moore, A., and Li, R. (1997). Activins, inhibins and mone (FSH) from the pituitary, multiple actions have
follistatins: Further thoughts on a growing family of regula- been assigned to them in a variety of tissues. An
tors. Proc. Soc. Exp. Biol. Med. 215, 209–222. additional three activin b-subunit proteins have been
Miura, S., Takeshita, T., Asan, H., Kimura, Y., Murata, K., described, i.e., bC, bD, and bE, and there is a growing
Yoshiteru, S., Hanai, J.-I., Beppu, H., Tsukazaki, T., Wrana,
J. L., Miyaono, K., and Sugamura, K. (2000). Hgs (Hrs), a
family of recognized activin- and inhibin-binding
FYVE domain protein, is involved in Smad signaling through proteins, receptors, and signaling molecules (Table 1).
cooperation with SARA. Mol. Cell. Biol. 20, 9346–9355.
Oh, S. P., and Li, E. (1997). The signaling pathway mediated by the
type IIB activin receptor controls axial patterning and lateral
asymmetry in the mouse. Genes Dev. 11, 1812–1826. II. EXPRESSION AND BIOSYNTHESIS
Peng, C., and Mukai, S. (2000). Activins and their receptors in OF ACTIVINS
female reproduction. Biochem. Cell Biol. 78, 261– 279.
Wrana, J. L. (2000). Regulation of Smad activity. Cell 100, The TGF-b superfamily of growth factors includes
189–192. bone morphogenetic proteins (BMPs) and Müllerian
inhibitory substance (MIS), and currently over 45
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
members of this family have been identified. Struc-
tural similarities between activins and other members
of the TGF-b superfamily are based on the conserva-
Activins tion of the number and spacing of the cysteines within
each subunit and the disulfide linkages between the
two subunits that form the characteristic cysteine
GAIL P. RISBRIDGER
knots. Other similarities relate to dimer formation,
Monash University, Melbourne the location of the bioactive peptide in the carboxy-
terminal region of the precursor molecule, and the
I. INTRODUCTION similarities in intracellular signaling mechanisms.
II. EXPRESSION AND BIOSYNTHESIS OF ACTIVINS The biosynthesis of activins requires the formation
III. SIGNALING OF ACTIVIN LIGANDS
of homo- or heterodimers of disulfide-linked bA- or
IV. ACTIVINS IN PHYSIOLOGY
V. ACTIVINS IN PATHOLOGY AND DISEASE
bB-subunit proteins that share 63% amino acid
VI. CLINICAL APPLICATIONS identity. The assembly of different combinations of
VII. SUMMARY activin subunits bA and bB is reflected in their
nomenclature; thus, activin A is a dimer composed
of bAbA, activin B is composed of bBbB, and activin
Activins, acting in opposition to inhibins, are AB is composed of bAbB. There is an independent
involved in diverse physiological activities, pattern of synthesis of the two subunit genes, which
including stimulating pituitary, hypothalamic,
are located on different chromosomes; the activin
and gonadal hormones, insulin secretion, germ
cell development and differentiation, erythroid bA-subunit gene is located on 7p15 – 7p14 and the
differentiation, nerve cell survival, and embryo- activin bB-subunit gene on 2qcen– 2q13.
nic development. Activin b-subunit types A –E Another subset of activin b-subunits (bC, bD, and
form homodimers and heterodimers that play bE) was identified more recently, based on their
different roles in health and disease in different homology to subunits bA and bB; this subset of
tissues throughout the body. Assays that allow subunits shares 62% amino acid identity with each
comparisons of specific subunit activities are other. The activin bC-subunit shows close similarity
necessary to understand the complex roles of to the activin bE-subunit in terms of genomic
each of the activin subunits. organization and chromosomal localization on
12q13.1, and subunits bC, bD, and bE are considered
to be a separate subset of activins. The bC-subunit
I. INTRODUCTION
dimerizes with itself in vitro and with subunits bA and
Activin subunits are present in numerous human bB to form the putative activins, activin C, AC, and
tissues of both endocrine and nonendocrine organs. BC. Activin subunits bD and bE have been isolated
Homo- or heterodimers of activin bA or bB subunits from Xenopus and mouse cDNA libraries, respect-
are members of the transforming growth factor-b ively, but their capacity to form activin heterodimers
24 ACTIVINS

TABLE 1 Activin Ligands: Purified Homo- or Heterodimers of Activin b-Subunit Proteins


Subgroups of activin b-subunits Purified proteins Evidence of putative dimeric proteins
a
bA Activin A —
a a
bB Activin B , activin AB —
b c c
bC Activin C Activin AC , activin BC
bD ? —
bE Monomeric activin bE —

a
Bioactive and detected in biological fluids.
b
Noknown bioactivity.
c
Heterodimeric proteins formed in vitro; currently there is no evidence for their existence in vivo.

with subunits bA and bB or between themselves is The interaction between the R-Smads and the receptor
unknown. complex involves a membrane-bound protein named
Homo- and heterodimers of activin subunits bA Smad anchor for receptor activation (SARA). After
and bB were originally isolated from follicular fluid phosphorylation, the R-Smads are released and
as inactive full-length precursor proteins. Similar to form heteromeric complexes with Smad4, a common
other growth and differentiation factors, the pre- mediator (Co-Smad). The R-Smad and Co-Smad com-
cursor activins require intracellular processing for full plexes then translocate to the nucleus to regulate
bioactivity. Precursor activins are 110-kDa dimers and gene expression. A third class of Smads, the inhibitory
consist of a signal peptide, a glycosylated prodomain Smads (I-Smads; Smad6 and Smad7), antagonize
(Pro-b), and a mature C-terminal domain (b) that is the signaling events just described and prevent
bioactive. Proteolytic cleavage releases the pro and access and phosphorylation of the R-Smads or
signal peptides, resulting in mature, unglycosylated interfere with the formation of the R-Smad/ Co-Smad
activin subunits. complexes.
Dimers of the activin b-subunits form activin In the nucleus, Smads target specific gene promo-
ligands, but heterodimers of activin b-subunits with ters with low binding affinity for DNA, and Smad
the inhibin a-subunit result in the formation of inhibin binding alone is insufficient for gene activation.
proteins (inhibin A or B). The inhibin a-subunit gene Smads use members of the forkhead activin signal
is located on 2q33 – q36 and the pattern of expression transducer (FAST; also called FoxH1) family as DNA-
of the inhibin a-subunit is independent of the activin binding partners to regulate gene transcription. Other
b-subunits. As is the case for activins, the inhibins are transcription factors likely to be involved in the Smad
synthesized as precursor proteins and proteolytic pathway include fos, jun, and the vitamin D receptor.
cleavage occurs intracellularly as well as in serum. However, in contrast to FAST/FoxH1, many of the
other DNA binding partners can function indepen-
dently of Smads, whereas FAST/FoxH1 target genes
require Smad function for transcriptional activation.
III. SIGNALING OF ACTIVIN LIGANDS Thus, the binding of activin ligands to the membrane-
The high degree of homology between the ligands bound receptor initiates a cascade of protein– protein
extends to the elements in their signaling pathways. interactions that controls gene expression and specific
At the cell surface, activin ligands interact with a dual biological responses.
receptor system involving a family of transmembrane
serine/threonine kinase receptors classified as type I
or type II receptors. Activin binding to the type II IV. ACTIVINS IN PHYSIOLOGY
receptor (ActRII) leads to the recruitment of the type I
A. Biological Actions of Activins
receptor (ActRI) and formation of a heteromeric
complex. Formation of this complex induces phos- Activins were originally isolated based on the
phorylation of the type I receptor, leading to stimulation of FSH production and secretion by rat
activation of the receptor-regulated Smad (R-Smad). pituitary cells in vitro. It is now recognized that
R-Smads are ligand specific, with Smad2 and activins have a range of biological activities that
Smad3 mediating activin and TGF-b signaling and include, but are not limited to, mesoderm induction
Smad1, Smad5, and Smad8 mediating BMP signaling. in Xenopus laevis embryos, reproduction through
ACTIVINS 25

the regulation of pituitary FSH production, bone demonstrated in vivo actions of dimeric ligands.
growth, nerve cell survival, wound healing, and tissue However, significant progress in understanding the
differentiation in pancreas, kidney, and heart. roles for activins has been made using other
In some instances, the actions of activins are approaches. Knockout and knock-in mouse models
antagonized by inhibins. The most well-known have been critical in the evaluation of the functional
opposing actions of activin and inhibin relate to effects effects of activins, and many of these were developed
on the reproductive system, including the regulation in the Matzuk laboratory. As well, the development
of pituitary FSH and gonadal steroidogenesis. Both of specific immunoassays for the different forms of
ligands also oppose each other during chondrogenesis activins, follistatins, and inhibins has been essential
in chick limb bud mesoderm and in T-cell prolifer- in evaluating the expression and roles for specific
ation. However, there are other instances when the ligands or binding proteins in physiology and in
effects of activins are not opposed by inhibin, such as disease.
during mesoderm induction, in neuronal cell survival,
and in some developmental processes. In these B. Knockout and Knock-in Mouse Models
circumstances, other factors, such as activin-binding
A number of activin b-subunit knockout mice that
proteins (e.g., follistatins) are powerful antagonists of
exhibit unique phenotypes have been generated
activins. In general, inhibins are regarded as endocrine
(Table 3). These studies confirm an essential role
factors that function primarily in the regulation of
for activins in normal development of reproductive
FSH, whereas activins are local paracrine and/or
organs as well as other tissues. Activins are mesoderm-
autocrine growth factors. inducing factors, but mice deficient in subunits bA or
As predicted by their wide pattern of expression bB develop to term. Activin bA-subunit-deficient mice
throughout the body and during all stages of life, the lack whiskers and lower incisors, have defects in their
bioactivities of activins have been recorded in many secondary palates, and die within 24 h of birth. Mice
cells and tissues (Table 2). In vitro model systems of deficient in activin bB are viable and fertile but have
cell growth (using cell lines and organ cultures) are defects in eyelid development. The defects are additive
common means to demonstrate significant effects in mice deficient in both subunits bA and bB and
that involve proliferative or antiproliferative actions, suggest that the subunits do not have overlapping
as well as those that alter cell differentiation. Most functions up to this age of development. To determine
studies have been performed with activin A, less with if the unique phenotypes of these mice are related to
activin B, and even fewer have reported the actions of differences in the temporal and spatial expression of
activin AB; there are no reports of bioactivity of other the two subunits or to specific ligand – receptor
putative activin dimers or heterodimers. Many of the signaling interactions, mice were generated in which
effects of activins in vivo are inferred by expression of activin bB was expressed in the spatial and temporal
subunit mRNA or protein; due to the limited supply pattern of activin bA (i.e., activin bB knock-in to the
of purified ligands, very few studies have directly activin bA locus). The craniofacial malformations

TABLE 2 Examples of Biological Fluids and Tissues in Which Activins A or AB or Follistatins Have Been Detected
or Measured
Protein Fluid or tissue

Activin A Sera, e.g., from pubertal boys and girls, women with pre-eclampsia, or patients with chronic hepatitis
Extracts, e.g., from gonadal, placental, choriodecidual, and allantoic tissues or fluids
Media from cell lines, e.g., human prostate tumor cells, gonadotropin-releasing hormone-secreting neurons, and
pituitary-derived folliculo-stellate cells
Fluids, e.g., follicular fluid, seminal plasma, and amniotic fluid in normal and Down syndrome pregnancies
Activin AB Follicular fluid from human, bovine, ovine, and porcine tissues
Media from granulosa cells
Follistatin Sera from pubertal girls, pre-eclamptic pregnancies, hypertensive pregnant women, postmenopausal women with
epithelial ovarian cancer, and patients with chronic hepatitis
Extracts from amnion, choriodecidual, and placental tissues
Supernatants from prostate tumor cell line cells
Amniotic fluid in normal and Down syndrome pregnancies
26 ACTIVINS

TABLE 3 Summary of Some of the Transgenic Mouse Model Phenotypes Bearing Modifications to Activin Subunits
or Intermediates in Activin Signaling
Genetic modification Phenotype

bA knockout Craniofacial defects, e.g., lack whiskers and lower incisors; cleft
palate; die , 24 h after birth
Overexpression of bA Sterility in male and females; testicular degeneration, but no
testicular tumors
bB knockout Viable and fertile but defects in eyelid development
bA and bB combined knockout Additive defects; mice develop to term with defects in whiskers,
incisors, and secondary palate formation
bB knock-in to bA locus Rescues craniofacial malformations and neonatal lethality of bA
deficiency, but defects in hair, gonads, and external genitalia
Activin type II receptor knockout Gonadal defects and altered fertility; some mice die due to
mandible defects
Follistatin knockout Craniofacial defects, growth retardation, and skin defects;
neonatal lethal
Follistatin overexpression Defects in testes, ovary, and hair
Inhibin a knockout Gonadal and adrenal tumors develop, followed by cachexia-like
wasting syndrome
Inhibin a þ activin type II receptor knockout Tumors develop, but without cachexia
Inhibin a þ overexpression of follistatin Reduced rate of tumor development and reduced symptoms of
cachexia

and neonatal lethality were rescued and the results Groome laboratory. The activin A assay measures
implied that activin subunits signaled through the total activin A, i.e., bound and unbound forms of the
same receptors in these tissues; further analyses ligand, and is used to measure activin A in sera and
revealed a greater level of complexity. In older mice, other biological fluids (e.g., follicular fluid) in a
during testicular growth, B-subunit does not replace number of species, including humans. A specific assay
the A-subunit function and the knock-in mice show for activin B is unavailable, but an enzyme-linked
delayed testicular maturation and onset of fertili- immunosorbent assay (ELISA) for activin AB detects
ty. Similarly, there are other defects in hair, ovarian the homodimer in some biological fluids. A specific
growth, development of external genitalia, and antibody for subunit bC has been developed by
somatic growth. Thus, although some actions of the Mellor et al., who have also shown that bC dimerizes
A subunit are replaced by B, others are not and the with subunit bA or bB. The field awaits the develop-
importance of the spatial and temporal patterns of ment of new activin assays to expand our under-
expression of the subunits has been confirmed. The standing of the role and expression of the different
success of the approach of generating null mice for activin ligands in diverse tissues and systems. By
functional analyses of the activin subunits is less infor- analogy, the use of specific assays for inhibin A and B
mative for the other activin subunits, C and E. Mice has been fundamental to understanding the roles of
lacking these subunits alone or in combination are the inhibins in men and women, particularly in the
viable and have no obvious abnormalities. Unequiv- field of reproductive physiology.
ocal evidence for a functional role for activin homo-
or heterodimers of C or E or combinations with the A or
B subunits has yet to be revealed. D. Overlapping Functions of Activins and Other
Ligands in the TGF-b Superfamily
The relative redundancy between the ligands relates
C. Assays for Activins
to the demonstration that bB can replace bA in some
The development of specific assays to measure the circumstances. The degree of overlap in functions of
activin ligands in biological fluids and tissues, subunits that are less similar to subunits bA and bB is
including serum, has significantly increased our unknown, although activin subunit bC can dimerize
understanding of the role of these ligands. Many, with bA and bB in vitro. The degree of overlap in
but not all, of the assays were developed in the the functions of activins and other members of the
ACTIVINS 27

TGF-b family is also unclear. TGF-b and activins V. ACTIVINS IN PATHOLOGY AND DISEASE
signal through type I and type II receptors and utilize
Smad2 and Smad3, but it is uncertain if the biological The role and regulation of activin ligands in disease
responses to the ligands are the same or different. have received little attention in comparison to almost
Apparently, not all of the actions of activins are a decade of research into the use of inhibins for
mimicked exactly by TGF-b or vice versa. For the detection of ovarian cancer or Down syndrome.
example, separate studies have shown that TGF-b Almost exclusively, the investigations center on
inhibits cell proliferation in human prostate tumor activin A.
cell lines PC3 and DU145, whereas the same cell lines
fail to respond to activin A. Conversely, the LNCaP A. Pregnancy
androgen-dependent human prostate tumor cells are
In pregnancy, activin A (as well as inhibin A) is a
growth inhibited by activin A, but are unresponsive to
product of the healthy placenta, but changes that have
TGF-b. Relatively few studies have directly compared
been reported to occur in pathological conditions
the ligands in the same biological systems, and the
might lead to diagnostic applications. Follistatins are
question of the redundancy between activins and
also consistently higher at all stages of pregnancy,
TGF-b remains unexplored.
suggesting that activin A is present in bound form
during a healthy pregnancy. Higher levels of activin A
E. Follistatin-Binding Proteins have been reported in women with pre-eclampsia,
gestational hypertension, and chronic hypertension.
Prior to any signaling event, activins can be bound to In established cases of pre-eclampsia, several groups
binding proteins such as follistatins (FSs). In many have shown that activin A (and inhibin A) levels are
tissues, the interplay between activins and FSs , 10 times higher, but it is not clear that changes in
provides a potent mechanism to regulate the access activin A are useful to predict the onset of pre-
of the ligands to their receptors. Follistatins are eclampsia in asymptomatic women. Research con-
encoded from a single gene located on chromosome tinues to assess if activin A, in combination with other
5q11.2 and alternative splicing of the mRNA markers, may be useful. The normal elevation in
generates different molecular weight isoforms. The activin A in late pregnancy is related to the onset of
FS proteins include an “FS domain” of 10-cysteine parturition, but might also be used for the detection
structures and an extracellular calcium-binding of preterm labor. There is no evidence of the usefulness
domain that makes them structurally homologous of any activins as markers for Down syndrome, similar
with epidermal growth factor (EGF), a group of to that reported for inhibin A.
enzyme inhibitors of the Kazal family, and other pro-
teins such as SPARC, agrin, testican, and follistatin-
related protein (FSRP). The function of the FS domains B. Inflammation
is not known but binding to activins differentiates
follistatins from other members. Activin A and FS have been implicated in inflamma-
The biological activity of FS is mediated via its tory processes. In synovial fluid from patients with the
high-affinity binding to activins. The binding is nearly inflammatory condition of rheumatoid arthritis, levels
irreversible and prevents ligand access to receptors on of activin A are elevated compared to those patients
the cell surface, making FS binding a critical regulator with degenerative osteoarthritis. An increased expres-
of activin bioactivity in cells in which FS is expressed. sion of activins related to change in bA-subunit
FS also contains a heparin-binding domain that expression is implied in patients with inflammatory
permits high-affinity association with cell surface bowel diseases such as Crohn’s disease and in ulcera-
heparin-sulfated proteoglycans (HSPGs). When acti- tive colitis. Indirect evidence for a role of activins has
vins are bound to FS –HSPG complexes at the cell emerged from the detection of elevated levels of FS in
surface or in the extracellular matrix, access to the patients with sepsis and meningitis, although activin
receptors is restricted and hence the paracrine actions levels in these conditions are unknown.
of locally or systemically derived ligands are inhib-
ited. As well as activins, FS binds and neutralizes the
C. Kidney/Renal Ischemia
mesoderm-inducing actions of other members of the
TGF-b superfamily, such as BMP-2, -4, and -7, Evidence for a role of activin A in renal regeneration
although the affinity of FS for the BMPs is signifi- after ischemic injury has emerged from animal studies.
cantly lower than that for the activins. Activin A, produced in the tubules, delays tubular
28 ACTIVINS

regeneration by inducing apoptosis, and FS antag- are implicated in the promotion of cachexia. Mutant
onizes this effect. However, the interplay between mice bearing deletions of both the inhibin a-subunit
activins and FS in the pathophysiology of renal and the ActRII receptor develop gonadal tumors, but
diseases such as chronic glomerulonephritis remains cachexia is reduced relative to animals with a single
unknown. gene deletion.

D. Bone
VI. CLINICAL APPLICATIONS
The osteogenic actions of activin A in bone have been
demonstrated in vivo and in vitro. In animal models, In contrast to the assays for inhibins or the a-subunit,
activin A promotes fracture healing, and the detection assays for activins remain to be validated for use as
of significant levels of activin A in bone matrix indi- new diagnostic tools in clinical medicine.
cates an involvement in bone resorption and for-
mation. Thus, activins (as well as FS) have potential
roles to play in osteoporosis. VII. SUMMARY
There is substantial evidence for a role for activins in
E. Tissue and Wound Repair normal physiological systems and in disease. How-
Mechanisms involved in tissue repair often closely ever, much of the information about these ligands
resemble those involved in embryonic development. relates to activin A (not activin B or activin AB), and
A number of studies have implicated activins in repair even less is known about the biological roles of the
or regeneration of kidney, skin, and hair. During other subset of activins (C and E) or the putative
cutaneous wound repair in mice, activin A regulates ligands formed between the groups of subunits. The
formation of granulation tissue and extracellu- overlap or redundancy between activins and other
lar matrix deposition. In animal models of acute members of the superfamily is largely unexplored and
brain injury, basic fibroblast growth factor (bFGF) is few studies have made direct comparisons of the
neuroprotective and neurotropic and induction of different members of the superfamily. For example, the
activin A appears to be a part of the signaling cascade bioactivities of TGF-b and activin A or B are seldom
for bFGF in this process. A potential role for activins in compared in the same models, and the regulatory
the prevention of neuronal loss during ischemic and actions of binding proteins such as follistatins, which
traumatic brain injury in humans remains unknown. bind both activins and BMPs, are unknown. Develop-
ment of new assays and purified ligands to study these
ligands and direct comparisons of activin and other
F. Cancer TGF-b family members using new mouse models and
Activin, like TGF-b, can inhibit or stimulate tumor cell new systems will facilitate and promote our under-
growth. For example, activins have growth inhibitory standing of the wide and diverse roles for activins in
effects on breast, liver, and prostate cancer cells and biology and disease.
pituitary adenomas, whereas growth stimulatory
actions are described in ovary and testis. The role of
activins in cancers is unclear and may be different in Glossary
different organs and tissues or may vary even within bone morphogenetic protein (BMP) Member of the trans-
the same tissue type. In tissues in which the effects are forming growth factor-b superfamily.
growth inhibitory, it is relevant to ask if the tumors follistatins (FS) Activin-binding proteins.
become resistant to activins due to mutations in the Müllerian inhibitory substance (MIS) Member of the
signaling pathway, but very little is known about transforming growth factor-b superfamily.
mutations in activin receptors or signaling factors transforming growth factor-b (TGFb) Superfamily of
specific for activin in cancer, compared to TGF-b. In growth and differentiation factors.
tissues in which growth-promoting effects of activins
have been documented, the sustained synthesis of the
ligands in malignancy may contribute to tumorigen- See Also the Following Articles
esis due to other effects of activins, e.g., on immune Activin Receptor Signaling . Anti-Müllerian Hormone
suppression, tissue remodeling, and angiogenesis. . Bone Morphogenetic Proteins . Inhibins . Inhibins,
Apart from local effects on tumor tissues, activins Activins, and Follistatins
ADIPOKINETIC HORMONES AND CARBOHYDRATE METABOLISM 29

Further Reading Insect adipokinetic hormones (AKHs) belong to


a large AKH/red pigment-concentrating hor-
Attisano, L., and Wrana, J. L. (2000). Smads as transcriptional co- mone family, whose members are involved in
modulators. Curr. Opin. Cell Biol. 12(2), 235 –243. the mobilization of energy substrates for flight
Chang, H., Brown, C. W., and Matzuk, M. M. (2003). Genetic activity. This article provides a survey of the
analysis of the mammalian transforming growth factor-b way in which adipokinetic and other related
superfamily. Endo. Rev. 23(6), 787–823. hormones play a key role in the mobilization of
Chang, H., Lau, A. L., et al. (2001). Studying TGF-beta super-
carbohydrate reserves as flight fuels. Special
family signaling by knockouts and knockins. Mol. Cell.
emphasis is put on the hormonal control of
Endocrinol. 180(1–2), 39–46.
Groome, N. P., Tsigou, A., et al. (2001). Enzyme immunoassays for
the enzymes involved and on the mechanism
inhibins, activins and follistatins. Mol. Cell. Endocrinol. of intracellular transduction of the hormonal
180(1–2), 73–77. signals.
Itoh, S., Itoh, F., et al. (2000). Signaling of transforming growth
factor-beta family members through Smad proteins. Eur.
J. Biochem. 267(24), 6954–6967.
Matzuk, M. M. (2000). Revelations of ovarian follicle biology I. CARBOHYDRATE MOBILIZATION
from gene knockout mice. Mol. Cell. Endocrinol. 163(1–2),
Carbohydrate is the major fuel in insects that fly only
61–66.
McPherson, S. J., Thomas, T. Z., et al. (1997). Growth inhibitory short distances, such as flies and bees, and it also
response to activin A and B by human prostate tumour cell provides most of the energy for the initial period of
lines, LNCaP and DU145. J. Endocrinol. 154(3), 535–545. flight in long-term flying insects, such as locusts.
Mellor, S. L., Cranfield, M., et al. (2000). Localization of activin Reserves of carbohydrate are stored in the fat body in
betaA, betaA and betaC subunits in human prostate and the form of glycogen, which upon flight is degraded by
evidence for formation of new activin heterodimers of betaC
the action of the enzyme glycogen phosphorylase.
subunit. J. Clin. Endocrinol. Metab. 85(12), 4851–4858.
Pangas, S. A., and Woodruff, T. K. (2000). Activin signal Unlike glucose in vertebrates and most invertebrates,
transduction pathways. Trends Endocrinol. Metab. 11(8), the carbohydrate transport form in insects is trehalose,
309–314. a nonreducing disaccharide that contains two D -glu-
Piek, E., Heldin, C. H., et al. (1999). Specificity, diversity, and cose residues. The mobilization of fat body glyco-
regulation in TGF-beta superfamily signaling. FASEB J. gen and the subsequent release of trehalose into the
13(15), 2105–2124.
hemolymph is controlled by adipokinetic hor-
Phillips, D. J. (2000). Special issue: Proceedings of the International
Workshop on Inhibins, Activins and Follistatins, Melbourne, mone/red pigment-concentrating hormone (AKH/
Australia, 2000. Mol. Cell. Endocrinol. 180, 1 –202. RPCH) peptides, which are released by the corpus
Risbridger, G. P., Schmitt, J. F., et al. (2001). Activins and cardiacum upon the onset of flight. The involvement of
inhibins in endocrine and other tumors. Endocr. Rev. 22(6), these hormones in carbohydrate metabolism was
836–858. demonstrated in vivo by the activation of fat body
ten Dijke, P., Miyazono, K., et al. (2000). Signaling inputs converge
glycogen phosphorylase or by increases of trehalose in
on nuclear effectors in TGF-beta signaling. Trends Biochem.
Sci. 25(2), 64–70. hemolymph after hormone injection and in fat body
Wrana, J. L., and Attisano, L. (2000). The Smad pathway. Cytokine in vitro by activation of the enzyme or by increased
Growth Factor Rev. 11(1–2), 5–13. trehalose release into the medium when isolated fat
body tissue was incubated in the presence of hormone.
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
The hormonal stimulation of carbohydrate mobili-
zation in an insect does not necessarily lead to increased
trehalose concentrations in the hemolymph under the
appropriate physiological conditions. In the locust, for
Adipokinetic Hormones and instance, in which the trehalose utilization rate is
Carbohydrate Metabolism increased during prolonged flight, the enhanced rate of
trehalose release does not lead to hypertrehalosemia.
WIL J. A. VAN MARREWIJK
Utrecht University, The Netherlands
II. HORMONAL ACTIVATION OF GLYCOGEN
I. CARBOHYDRATE MOBILIZATION PHOSPHORYLASE
II. HORMONAL ACTIVATION OF GLYCOGEN
The rate of glycogenolysis in the fat body depends on
PHOSPHORYLASE
the activity of glycogen phosphorylase. Hormonal
III. HORMONAL SIGNAL TRANSDUCTION
stimulation of glycogenolysis leading to an increased
ADIPOKINETIC HORMONES AND CARBOHYDRATE METABOLISM 29

Further Reading Insect adipokinetic hormones (AKHs) belong to


a large AKH/red pigment-concentrating hor-
Attisano, L., and Wrana, J. L. (2000). Smads as transcriptional co- mone family, whose members are involved in
modulators. Curr. Opin. Cell Biol. 12(2), 235 –243. the mobilization of energy substrates for flight
Chang, H., Brown, C. W., and Matzuk, M. M. (2003). Genetic activity. This article provides a survey of the
analysis of the mammalian transforming growth factor-b way in which adipokinetic and other related
superfamily. Endo. Rev. 23(6), 787–823. hormones play a key role in the mobilization of
Chang, H., Lau, A. L., et al. (2001). Studying TGF-beta super-
carbohydrate reserves as flight fuels. Special
family signaling by knockouts and knockins. Mol. Cell.
emphasis is put on the hormonal control of
Endocrinol. 180(1–2), 39–46.
Groome, N. P., Tsigou, A., et al. (2001). Enzyme immunoassays for
the enzymes involved and on the mechanism
inhibins, activins and follistatins. Mol. Cell. Endocrinol. of intracellular transduction of the hormonal
180(1–2), 73–77. signals.
Itoh, S., Itoh, F., et al. (2000). Signaling of transforming growth
factor-beta family members through Smad proteins. Eur.
J. Biochem. 267(24), 6954–6967.
Matzuk, M. M. (2000). Revelations of ovarian follicle biology I. CARBOHYDRATE MOBILIZATION
from gene knockout mice. Mol. Cell. Endocrinol. 163(1–2),
Carbohydrate is the major fuel in insects that fly only
61–66.
McPherson, S. J., Thomas, T. Z., et al. (1997). Growth inhibitory short distances, such as flies and bees, and it also
response to activin A and B by human prostate tumour cell provides most of the energy for the initial period of
lines, LNCaP and DU145. J. Endocrinol. 154(3), 535–545. flight in long-term flying insects, such as locusts.
Mellor, S. L., Cranfield, M., et al. (2000). Localization of activin Reserves of carbohydrate are stored in the fat body in
betaA, betaA and betaC subunits in human prostate and the form of glycogen, which upon flight is degraded by
evidence for formation of new activin heterodimers of betaC
the action of the enzyme glycogen phosphorylase.
subunit. J. Clin. Endocrinol. Metab. 85(12), 4851–4858.
Pangas, S. A., and Woodruff, T. K. (2000). Activin signal Unlike glucose in vertebrates and most invertebrates,
transduction pathways. Trends Endocrinol. Metab. 11(8), the carbohydrate transport form in insects is trehalose,
309–314. a nonreducing disaccharide that contains two D -glu-
Piek, E., Heldin, C. H., et al. (1999). Specificity, diversity, and cose residues. The mobilization of fat body glyco-
regulation in TGF-beta superfamily signaling. FASEB J. gen and the subsequent release of trehalose into the
13(15), 2105–2124.
hemolymph is controlled by adipokinetic hor-
Phillips, D. J. (2000). Special issue: Proceedings of the International
Workshop on Inhibins, Activins and Follistatins, Melbourne, mone/red pigment-concentrating hormone (AKH/
Australia, 2000. Mol. Cell. Endocrinol. 180, 1 –202. RPCH) peptides, which are released by the corpus
Risbridger, G. P., Schmitt, J. F., et al. (2001). Activins and cardiacum upon the onset of flight. The involvement of
inhibins in endocrine and other tumors. Endocr. Rev. 22(6), these hormones in carbohydrate metabolism was
836–858. demonstrated in vivo by the activation of fat body
ten Dijke, P., Miyazono, K., et al. (2000). Signaling inputs converge
glycogen phosphorylase or by increases of trehalose in
on nuclear effectors in TGF-beta signaling. Trends Biochem.
Sci. 25(2), 64–70. hemolymph after hormone injection and in fat body
Wrana, J. L., and Attisano, L. (2000). The Smad pathway. Cytokine in vitro by activation of the enzyme or by increased
Growth Factor Rev. 11(1–2), 5–13. trehalose release into the medium when isolated fat
body tissue was incubated in the presence of hormone.
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
The hormonal stimulation of carbohydrate mobili-
zation in an insect does not necessarily lead to increased
trehalose concentrations in the hemolymph under the
appropriate physiological conditions. In the locust, for
Adipokinetic Hormones and instance, in which the trehalose utilization rate is
Carbohydrate Metabolism increased during prolonged flight, the enhanced rate of
trehalose release does not lead to hypertrehalosemia.
WIL J. A. VAN MARREWIJK
Utrecht University, The Netherlands
II. HORMONAL ACTIVATION OF GLYCOGEN
I. CARBOHYDRATE MOBILIZATION PHOSPHORYLASE
II. HORMONAL ACTIVATION OF GLYCOGEN
The rate of glycogenolysis in the fat body depends on
PHOSPHORYLASE
the activity of glycogen phosphorylase. Hormonal
III. HORMONAL SIGNAL TRANSDUCTION
stimulation of glycogenolysis leading to an increased
30 ADIPOKINETIC HORMONES AND CARBOHYDRATE METABOLISM

biosynthesis and release of trehalose has been inves- AKH peptides has been demonstrated in locusts,
tigated extensively in cockroaches, which rely mainly the moth Manduca sexta, and the beetle Pachnoda
on carbohydrate as a fuel for flight activity. Hyper- sinuata. In the locust Locusta migratoria, all three
trehalosemic hormones (HTHs), representatives of known AKHs (AKH-I, -II, and -III) are able to activate
the AKH/RPCH family, were shown to activate fat glycogen phosphorylase in a bioassay, although with
body glycogen phosphorylase and to stimulate treha- different potencies. In vertebrates, phosphorylase is
lose synthesis in Periplaneta americana and Blaberus regulated by reversible phosphorylation/dephos-
discoidalis. Activation of fat body phosphorylase by phorylation between nonphosphorylated inactive

FIGURE 1 Fractionation of locust fat body phosphorylase by DEAE –Sephacel chromatography. Elution profiles were
obtained with partially purified enzyme from the pooled fat bodies of 80 locusts at rest (Rested) and of 120 flown locusts
(Flown).
ADIPOKINETIC HORMONES AND CARBOHYDRATE METABOLISM 31

phosphorylase b and phosphorylated active phos- cAMP is directed more toward carbohydrate mobili-
phorylase a. Also in locusts, flight activity as well zation than lipid mobilization. The involvement of
as AKH injection induces the conversion of fat body cAMP in the stimulation of glycogen mobilization is
phosphorylase b into the a form. This activating con- not a general feature of insects, since the HTHs of
version indicates phosphorylation of each of the two several cockroach species studied do not utilize this
subunits of the enzyme, which takes place in two second messenger to activate trehalose synthesis in
steps, thereby giving rise to an intermediate hybrid the fat body.
ab form (Fig. 1). Activation of fat body phosphorylase Experiments on locust fat body using cholera
by flight activity or hormone injection, combined with toxin (CTX) and pertussis toxin (PTX) suggest that
the occurrence of three forms of the enzyme including the AKH receptor(s) is coupled to the Gs-protein (and
the partially phosphorylated phosphorylase ab, has not to Gi). The demonstration that AKH-I-, AKH-II-,
also been demonstrated in M. sexta and P. americana. and AKH-III-stimulated phosphorylase activation is
It has been suggested that the presence of this hybrid ablated by the universal G-protein inhibitor guano-
phosphorylase would provide for a sophisticated way sine-50 -O-(2-thiodiphosphate) strongly substantiates
of regulating carbohydrate metabolism in insects. this suggestion.

B. Calcium
III. HORMONAL SIGNAL TRANSDUCTION The relative importance of Ca2þ in signal transduc-
Binding of the AKH/RPCH peptides to their plasma tion in the fat body is not equal in several closely
membrane receptor(s) in the insect fat body results related insect species. For example, the influx of
in the induction of a variety of signal transduction extracellular Ca2þ into the fat body of L. migratoria
events that ultimately lead to the activation of target has a much stronger stimulating effect on glycogen
enzymes. As discussed above, the target enzyme in phosphorylase activity than the release of calcium
the mobilization of carbohydrate reserves is glyco- ions from intracellular stores, but in B. discoidalis the
gen phosphorylase, which initiates the conversion of opposite result holds. The presence of extracellular
glycogen into trehalose. Signal transduction mechan- Ca2þ ions has been shown to be indispensable for the
isms have been investigated mainly in locusts and induction of fat body phosphorylase by AKH in
cockroaches, with focus on GTP-binding (G) proteins, L. migratoria and by HTH in P. americana. In the
cyclic AMP (cAMP), Ca2þ, inositol phosphates, absence of Ca2þ in the medium, none of the three
signaling cross-talk, and hormone receptors. Some locust AKHs is capable of enhancing cAMP pro-
major results are discussed in this article. duction and inducing glycogen phosphorylase acti-
vation in the fat body, but 1.5 mM Ca2þ (which is the
Ca2þ concentration in hemolymph) is sufficient for
A. Cyclic AMP and G-Proteins complete activation (Fig. 2). Since the induction of
In the fat body of L. migratoria, an accumulation
of the second messenger cAMP, brought about by
AKH-I has been demonstrated both in vivo and
in vitro. Moreover, this accumulation of cAMP leads
to the activation of glycogen phosphorylase, which is
evidence in favor of a role of cAMP in AKH signal
transduction. Each of the AKHs stimulates cAMP
production in a dose-dependent manner in the fat
body within 1 min. At a physiological dose of 4 nM,
AKH-III is the most potent and AKH-I the least
potent peptide hormone in stimulating cAMP pro-
duction, and the same order of potency holds for the
activation of glycogen phosphorylase by this hormon-
al dose. The observation that AKH-II is somewhat
stronger than AKH-I in activating glycogen phos- FIGURE 2 Dependence of AKH-induced activation of locust
phorylase is in line with suggestions that the second fat body phosphorylase on extracellular Ca2þ. Fat body was
AKH would be the major trigger for carbohydrate incubated for 15 min in the presence or absence of 40 nM
mobilization from the fat body and that the action of AKH-I. Similar results were obtained with AKH-II and -III.
32 ADIPOKINETIC HORMONES AND CARBOHYDRATE METABOLISM

phosphorylase by cAMP in fat body was shown to


be independent of extracellular Ca2þ, data suggest
that at least part of the action of extracellular Ca2þ
is at a site proximal to cAMP, e.g., the binding of
the hormones to their receptor(s) or a Ca2þ-sensitive
adenylyl cyclase.
All three locust AKHs are capable of stimulating
the Ca2þ inflow into the fat body cells within 30 s
with equal potency. This hormone-induced Ca2þ
influx appeared to be mediated through depletion of
intracellular Ca2þ stores, suggesting the functioning
of a store-operated or capacitative Ca2þ entry
mechanism. Simultaneously, the AKHs also enhance
the efflux of Ca2þ from the fat body. At a physiologi-
cal dose, AKH-III caused the strongest efflux and
AKH-I the weakest; at a massive dose, their efficacy
was equal. As the influx of Ca2þ exceeded the efflux, it
is feasible that the intracellular Ca2þ concentration
rises as a result of incubation of fat body with AKH.

FIGURE 3 Effect of AKH on the formation of inositol phos-


C. Inositol Phosphates phate isomers from locust fat body. Fat body tissue prelabeled
with myo-[2-3H]inositol was incubated for 1 min in the
For a maximal effect of AKH on glycogen phos- presence or absence (controls) of 40 nM AKH-I; then InsPn
phorylase activity in locust fat body, the release of were isolated and separated by high-performance liquid
chromatography and their radioactivity was measured. Results
Ca2þ from intracellular stores is required in addition
are expressed as disintegrations per minute per milligram of
to the availability of extracellular calcium. The same protein.
holds for the activation of glycogen phosphorylase
and the stimulation of trehalose synthesis by HTH in
B. discoidalis fat body. In the regulation of Ca2þ the effects of this hormone may be stronger than
mobilization from intracellular stores, inositol phos- estimated from its relative amount in the circulation.
phates (InsPn) play a pivotal role, and the formation Ins(1,4,5)P3 levels are greatly increased by HTH
of these putative second messengers is induced in the fat body of B. discoidalis in a time- and dose-
by the locust AKHs. Each of the Locusta AKHs dependent manner, which, along with the strong
stimulates the synthesis of total InsPn within 1 min evidence for Ca2þ as component in the HTH second-
with different potencies: AKH-II barely induces any messenger cascade, argues strongly for InsP3 as a
InsPn and AKH-III is more potent than AKH-I. The primary second messenger in response to HTH
observation that the activation of glycogen phos- followed by the mobilization of intracellular Ca2þ.
phorylase by each of the AKHs is dampened by the
phospholipase C (PLC) inhibitor U73122 suggests the
involvement of InsPn (and/or diacylglycerol) in AKH D. Signaling Crosstalk
signaling in the locust fat body. Crosstalk between signal transduction cascades pro-
All individual forms of InsPn are elevated by the vides the cell with a complex intracellular system
AKHs, with InsP3 and InsP4 being the most interesting for fine-tuning of hormone-induced signals. In locust
because of their presumed Ca2þ mobilizing actions fat body an elevation of cAMP levels does not
(Fig. 3). With respect to InsP3, AKH-III is again more influence the intracellular InsPn content, indicating
potent than AKH-I, and the AKH-II-enhanced InsP3 that the basal PLC activity is not regulated by this
formation is quite small. The most prolonged effect on cyclic nucleotide. Moreover, none of the signal
InsP3 is caused by AKH-III. The high potency and transducing elements between the AKH receptor and
prolonged effects of AKH-III with respect to the PLC is affected by cAMP, since preincubation of fat
induction of various second-messenger systems appar- body tissue with forskolin or dibutyryl-cAMP does
ently compensate (in part) for its low abundance not have an impact on AKH-induced InsPn pro-
relative to that of the other AKHs, and therefore, duction. Proof of a direct linkage between the AKH
ADIPOKINETIC HORMONES AND CARBOHYDRATE METABOLISM 33

FIGURE 4 Tentative model for the coupling of AKH signaling pathways mediating the mobilization of energy
substrates in the locust fat body cell. R, receptor; ER, endoplasmic reticulum; AC, adenylyl cyclase; PLC, phospholipase
C; Phos, phosphorylase; G1P, glucose 1-phosphate; TAG, triacylglycerol; DAG, diacylglycerol.

receptor(s) and PLC (instead of a route via cAMP) molecule, using orthophosphate as a co-substrate and
came from the substantiation that the G-protein releasing glucose-1-phosphate.
activator aluminum fluoride increases InsPn levels. hemolymph Blood of insects, which circulates in the
Experiments using CTX, PTX, and GP antagonist- body cavity between the various organs, bathing them
directly. It consists of a fluid plasma in which the blood
2A, a specific inhibitor of Gq, preclude the involve-
cells or hemocytes are suspended.
ment of Gi and a Gs-sensitive isoform of PLC and
hypertrehalosemia Condition characterized by an increase
evidence the involvement of Gq in the transduction in the level of trehalose in the hemolymph (insect blood)
of AKH signals toward fat body PLC (Fig. 4). to values that significantly exceed the normal resting
level.
hypertrehalosemic hormone Peptide hormone from the
Glossary corpus cardiacum involved in the mobilization of fat
body glycogen to fuel flight activity. For transport to the
adipokinetic hormone Peptide hormone in insects involved
flight muscles, glycogen is converted into the disac-
in the mobilization of substrates (predominantly lipids
charide trehalose, the carbohydrate transport form in
and carbohydrates) for energy generation needed by
insects, whose level in hemolymph is raised during
contracting flight muscles.
flight.
corpus cardiacum Neuroendocrine organ situated caudal
red pigment concentrating hormone Peptide hormone from
to the brain to which it is connected by paired nerves.
the eyestalk of crustaceans; member of the adipokinetic
The organ combines the functions of neurohemal
hormone/red pigment-concentrating hormone family
storage and adipokinetic/hypertrehalosemic hormone
that comprises structurally related but functionally
production.
diverse peptides.
fat body Organ whose functional role in insects is
signaling cross-talk Occurrence of communication/interac-
analogous to the combined functions of liver and
tion between different signaling pathways, resulting
adipose tissue in mammals. Fat body is the chief site
in an integrated response to a hormone-induced signal.
of intermediary metabolism, detoxification, storage of
trehalase Enzyme that catalyzes the hydrolytic reaction by
nutrient reserves, and, in some species, deposition of
which trehalose is brought into the glycolytic main-
nitrogenous waste products.
stream: Trehalose þ H2O ! 2D -glucose.
glycogenolysis Breakdown of glycogen; in insect fat
body, the resulting glucose monomers are used for the
biosynthesis of the disaccharide trehalose, the carbo- See Also the Following Articles
hydrate transport form in insects.
glycogen phosphorylase Enzyme that catalyzes the sequen- Adipokinetic Hormones and Lipid Mobilization
tial removal of glycosyl residues from the nonreducing . Adipokinetic Hormones: Structure and Biosynthesis
end (i.e., with a free 40 -OH group) of the glycogen . Insect Endocrine System
34 ADIPOKINETIC HORMONES AND LIPID MOBILIZATION

Further Reading In the migratory locust, Locusta migratoria, the


flight activity-induced release of adipokinetic
Becker, A., Liewald, J. F., and Wegener, G. (1998). Signal hormones (AKHs; AKH-I, -II, and -III) from the
transduction in isolated fat body from the cockroach Blaptica neurosecretory cells of the corpus cardiacum, a
dubia exposed to hypertrehalosaemic neuropeptide. J. Comp. neuroendocrine gland located caudal to the insect
Physiol. 168B, 159–167. brain, has been studied extensively. The direct
Keeley, L. L., and Hesson, A. S. (1995). Calcium-dependent
actions of these hormones on their fat body target
signal transduction by the hypertrehalosemic hormone in
cells trigger signal transduction processes that lead
the cockroach fat body. Gen. Comp. Endocrinol. 99,
373–381.
to the mobilization of lipid (diacylglycerol). This
Orr, G. L., Gole, J. W. D., Jahagirdar, A. P., Downer, R. G. H., and substrate fulfills a major role in energy metabo-
Steele, J. E. (1985). Cyclic AMP does not mediate the action of lism of the contracting flight muscles during
synthetic hypertrehalosemic peptides from the corpus cardia- sustained flight activity. The molecular mechanism
cum of Periplaneta americana. Insect Biochem. 15, 703–709. of diacylglycerol transport in insect blood
Park, J. E., and Keeley, L. L. (1996). Calcium-dependent action of involves a reversible conversion of the insect
hypertrehalosemic hormone on activation of glycogen phos- lipoprotein lipophorin, which has revealed a novel
phorylase in cockroach fat body. Mol. Cell. Endocrinol. 116, concept for lipid transport in the circulatory
199–205. system.
Steele, J. E. (1999). Activation of fat body in Periplaneta americana
(Blattoptera: Blattidae) by hypertrehalosemic hormones
(HTH): New insights into the mechanism of cell signalling.
Eur. J. Entomol. 96, 317–322. I. INTRODUCTION
Van der Horst, D. J., Van Marrewijk, W. J. A., and Diederen, J. H.
B. (2001). Adipokinetic hormones of insect: Release, The flight activity of insects provides an exception-
signal transduction, and responses. Int. Rev. Cytol. 211, ally suitable yet relatively simple model system for
179–240. studying the regulatory phenomena involved in
Van Marrewijk, W. J. A., and Van der Horst, D. J. (1998). Signal energy expenditure during extreme physical activity.
transduction of adipokinetic hormone. In “Recent Advances Similar to the processes used by vertebrates to
in Arthropod Endocrinology” (G. M. Coast and S. G.
generate energy for sustained locomotion, long-
Webster, eds.), pp. 172– 188. Cambridge University Press,
Cambridge, UK. distance flying insects mobilize endogenous lipid
Vroemen, S. F., De Jonge, H., Van Marrewijk, W. J. A., and Van der (triacylglycerol) reserves to fuel oxidative metabolism
Horst, D. J. (1998). The phospholipase C signaling pathway in during migratory flight. The fat body, which combines
locust fat body is activated via Gq and not affected by cAMP. many of the properties and functions of vertebrate
Insect Biochem. Mol. Biol. 28, 483–490. liver and adipose tissue, plays a fundamental role in
Vroemen, S. F., Van Marrewijk, W. J. A., Schepers, C. C. J., and Van
der Horst, D. J. (1995). Signal transduction of adipokinetic
lipid storage, as well as in the process of lipolysis
hormones involves Ca2þ fluxes and depends on extracellular controlled by adipokinetic hormones (AKHs). This
Ca2þ to potentiate cAMP-induced activation of glycogen article discusses the action of AKHs on lipid mobili-
phosphorylase. Cell Calcium 17, 459– 467. zation in fat body cells and the eventual AKH-induced
transformation of the transport system of lipids in the
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
insect blood, which will be exemplified using mainly
the migratory locust (Locusta migratoria) as an insect
model.
Adipokinetic Hormones and
Lipid Mobilization II. ADIPOKINETIC HORMONE SIGNAL
TRANSDUCTION IN INSECT FAT BODY
DICK J. VAN DER HORST AND ROB C. H. M. OUDEJANS Binding of the peptide adipokinetic hormones to their
Utrecht University, The Netherlands plasma membrane receptor(s) at the fat body cells
results in the induction of a variety of signal transduc-
I. INTRODUCTION tion events that ultimately lead to the activation of
II. ADIPOKINETIC HORMONE SIGNAL TRANSDUCTION target key enzymes. The first insect AKH receptors
IN INSECT FAT BODY have recently been identified at the molecular level.
III. EFFECT OF ADIPOKINETIC HORMONE ON LIPID The signal transduction mechanism of the three locust
MOBILIZATION
AKHs has been studied extensively and involves
IV. ADIPOKINETIC HORMONE-INDUCED LIPOPHORIN
stimulation of cAMP production, which is dependent
CONVERSIONS
on the presence of extracellular Ca2þ. Additionally,
34 ADIPOKINETIC HORMONES AND LIPID MOBILIZATION

Further Reading In the migratory locust, Locusta migratoria, the


flight activity-induced release of adipokinetic
Becker, A., Liewald, J. F., and Wegener, G. (1998). Signal hormones (AKHs; AKH-I, -II, and -III) from the
transduction in isolated fat body from the cockroach Blaptica neurosecretory cells of the corpus cardiacum, a
dubia exposed to hypertrehalosaemic neuropeptide. J. Comp. neuroendocrine gland located caudal to the insect
Physiol. 168B, 159–167. brain, has been studied extensively. The direct
Keeley, L. L., and Hesson, A. S. (1995). Calcium-dependent
actions of these hormones on their fat body target
signal transduction by the hypertrehalosemic hormone in
cells trigger signal transduction processes that lead
the cockroach fat body. Gen. Comp. Endocrinol. 99,
373–381.
to the mobilization of lipid (diacylglycerol). This
Orr, G. L., Gole, J. W. D., Jahagirdar, A. P., Downer, R. G. H., and substrate fulfills a major role in energy metabo-
Steele, J. E. (1985). Cyclic AMP does not mediate the action of lism of the contracting flight muscles during
synthetic hypertrehalosemic peptides from the corpus cardia- sustained flight activity. The molecular mechanism
cum of Periplaneta americana. Insect Biochem. 15, 703–709. of diacylglycerol transport in insect blood
Park, J. E., and Keeley, L. L. (1996). Calcium-dependent action of involves a reversible conversion of the insect
hypertrehalosemic hormone on activation of glycogen phos- lipoprotein lipophorin, which has revealed a novel
phorylase in cockroach fat body. Mol. Cell. Endocrinol. 116, concept for lipid transport in the circulatory
199–205. system.
Steele, J. E. (1999). Activation of fat body in Periplaneta americana
(Blattoptera: Blattidae) by hypertrehalosemic hormones
(HTH): New insights into the mechanism of cell signalling.
Eur. J. Entomol. 96, 317–322. I. INTRODUCTION
Van der Horst, D. J., Van Marrewijk, W. J. A., and Diederen, J. H.
B. (2001). Adipokinetic hormones of insect: Release, The flight activity of insects provides an exception-
signal transduction, and responses. Int. Rev. Cytol. 211, ally suitable yet relatively simple model system for
179–240. studying the regulatory phenomena involved in
Van Marrewijk, W. J. A., and Van der Horst, D. J. (1998). Signal energy expenditure during extreme physical activity.
transduction of adipokinetic hormone. In “Recent Advances Similar to the processes used by vertebrates to
in Arthropod Endocrinology” (G. M. Coast and S. G.
generate energy for sustained locomotion, long-
Webster, eds.), pp. 172– 188. Cambridge University Press,
Cambridge, UK. distance flying insects mobilize endogenous lipid
Vroemen, S. F., De Jonge, H., Van Marrewijk, W. J. A., and Van der (triacylglycerol) reserves to fuel oxidative metabolism
Horst, D. J. (1998). The phospholipase C signaling pathway in during migratory flight. The fat body, which combines
locust fat body is activated via Gq and not affected by cAMP. many of the properties and functions of vertebrate
Insect Biochem. Mol. Biol. 28, 483–490. liver and adipose tissue, plays a fundamental role in
Vroemen, S. F., Van Marrewijk, W. J. A., Schepers, C. C. J., and Van
der Horst, D. J. (1995). Signal transduction of adipokinetic
lipid storage, as well as in the process of lipolysis
hormones involves Ca2þ fluxes and depends on extracellular controlled by adipokinetic hormones (AKHs). This
Ca2þ to potentiate cAMP-induced activation of glycogen article discusses the action of AKHs on lipid mobili-
phosphorylase. Cell Calcium 17, 459– 467. zation in fat body cells and the eventual AKH-induced
transformation of the transport system of lipids in the
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
insect blood, which will be exemplified using mainly
the migratory locust (Locusta migratoria) as an insect
model.
Adipokinetic Hormones and
Lipid Mobilization II. ADIPOKINETIC HORMONE SIGNAL
TRANSDUCTION IN INSECT FAT BODY
DICK J. VAN DER HORST AND ROB C. H. M. OUDEJANS Binding of the peptide adipokinetic hormones to their
Utrecht University, The Netherlands plasma membrane receptor(s) at the fat body cells
results in the induction of a variety of signal transduc-
I. INTRODUCTION tion events that ultimately lead to the activation of
II. ADIPOKINETIC HORMONE SIGNAL TRANSDUCTION target key enzymes. The first insect AKH receptors
IN INSECT FAT BODY have recently been identified at the molecular level.
III. EFFECT OF ADIPOKINETIC HORMONE ON LIPID The signal transduction mechanism of the three locust
MOBILIZATION
AKHs has been studied extensively and involves
IV. ADIPOKINETIC HORMONE-INDUCED LIPOPHORIN
stimulation of cAMP production, which is dependent
CONVERSIONS
on the presence of extracellular Ca2þ. Additionally,
ADIPOKINETIC HORMONES AND LIPID MOBILIZATION 35

the AKHs enhance the production of inositol


phosphates including inositol 1,4,5-trisphosphate,
which may mediate the mobilization of Ca2þ from
intracellular stores. This depletion of Ca2þ from
intracellular stores stimulates the influx of extracellu-
lar Ca2þ, indicative of the operation of a capacitative
(store-operated) calcium entry mechanism. The inter-
actions between the AKH signaling pathways ulti-
mately result in the mobilization of stored reserves as
fuels for flight. Although the carbohydrate (mainly
trehalose) in the insect blood (hemolymph) provides
the energy for the initial period of flight, additional
trehalose is mobilized from fat body glycogen reserves
by the AKH-induced activation of glycogen phos-
phorylase. At the same time, the concentration of lipid
(diacylglycerol, DAG) in the hemolymph is increased
progressively at the expense of stored triacylglycerol FIGURE 1 Dose– response curves for the lipid-mobilizing
(TAG) reserves in the fat body and gradually effects of AKH-I, -II, and -III in L. migratoria. Adult male
constitutes the major substrate during prolonged locusts were injected with different doses of AKH or, in the
flight. controls, with saline, and after 120 min, the lipid content in
hemolymph was determined. Responses represent increases in
hemolymph lipid in AKH-injected locusts, expressed as grams
per liter. For clarity, data points and standard error bars were
III. EFFECT OF ADIPOKINETIC HORMONE omitted. ED50, effective dose eliciting 50% of the maximum
ON LIPID MOBILIZATION response.

The namesake action of the AKHs on insect fat body


cells results in the mobilization of lipid (TAG) stores. sensitive lipase (HSL) controls mobilization of TAG
The increased production of DAG reflected by the stores in adipose tissue, and although, in contrast to
increased concentration of DAG in the hemolymph
the situation in insects, free fatty acids (FFA) are
indicates hormonal activation of the key enzyme, fat
released into the blood for uptake and oxidation in
body TAG lipase. In a bioassay, all three L. migratoria
muscle, there is a clear functional similarity between
AKHs (-I, -II, and -III) are able to stimulate lipid
vertebrate adipose tissue HSL and insect fat body
mobilization, although their relative potencies are
TAG lipase. In vertebrate HSL, specific regulatory
different, as inferred from the dose – response curves
sites are phosphorylated in vitro by cAMP-dependent
shown in Fig. 1. This recalls the concept of a hor-
protein kinase (PKA), resulting in activation of HSL.
monally redundant system involving multiple regu-
latory molecules with overlapping actions. Results The mechanism behind this activation of HSL upon
obtained with combinations of two or three AKHs, phosphorylation by PKA is not well understood, but
which are likely to occur together in locust hemo- seems to involve translocation of HSL from the
lymph under physiological conditions in vivo, cytosol to the lipid droplet as well as conformational
revealed that the maximal responses for the lipid- changes in the HSL molecule. Different phosphory-
mobilizing effects were much lower than the lation sites in HSL may play different roles in the
theoretically calculated responses based on the process of translocation and the increase in specific
dose – response curves for the individual hormones. activity of the enzyme. Additionally, perilipins, a
In the lower (probably physiological) range, however, family of unique proteins intimately associated with
combinations of the AKHs were more effective than the limiting surface of neutral lipid storage droplets,
the theoretical values calculated from the responses are acutely polyphosphorylated by PKA on stimu-
elicited by the individual hormones. lation of lipolysis, hinting at a role in this lipolytic
The mechanism by which TAG lipase catalyzes process. Conformational changes of phosphorylated
AKH-controlled production of the DAG on which perilipins may expose the neutral lipid cores of the
long-distance flight depends is only poorly under- lipid droplets, facilitating the ensuing hydrolysis.
stood, mainly due to technical problems in isolating Even though a translocation of the insect fat body
or activating the lipase. In vertebrates, hormone- TAG lipase has not been established, the involvement
36 ADIPOKINETIC HORMONES AND LIPID MOBILIZATION

of AKHs in the process of lipolysis is beyond dispute. mechanism for lipid transfer is a premier issue.
The effect of AKHs was demonstrated not only in vivo The action of AKH on lipid release has revealed a
from enhanced levels of DAG in hemolymph of insects novel concept for lipid transport in the circulation of
injected with the hormones as shown above, but also animal organisms. Insect hemolymph generally con-
in vitro by the accumulation of DAG in isolated tains abundant amounts of a single multifunctional
L. migratoria fat body tissue that was incubated in the lipoprotein particle, high-density lipophorin (HDLp),
presence of AKH. In later in vitro experiments, both that performs the tasks of transporting dietary and
cAMP and Ca2þ were shown to play an important role endogenously produced lipids to peripheral tissues
in the effect of AKH on lipolysis. The involvement of during all developmental stages. A characteristic
cAMP suggests a role for PKA in the phosphorylation feature of HDLp is its ability to function as a reusable
and activation of fat body TAG lipase. In addition, vehicle for a variety of lipids by the selective loading
an increase of fat body TAG lipase activity was and unloading of lipid components at target tissues.
measured in gregarious locusts in vivo when injected In the flying insect, however, the increased transport
with AKH-I, whereas in solitary locusts, which do of the released DAG in the hemolymph appears to
not store sufficient amounts of TAG in the fat require an AKH-stimulated transformation of the
body, AKH-I administration had no significant effect lipophorin particle, which is capable of alternating
on lipase activity. The reason that the measured between the relatively lipid-poor HDLp form and a
factorial increases of lipase activity after stimulation lipid-enriched (low-density lipophorin, LDLp) form.
with lipolytic agents are relatively modest in these and In this reversible conversion the exchangeable
other similar studies may reside in the applied apolipoprotein apoLp-III, which exists in a lipid-
experimental procedure, in which the enzyme is free form and a lipid-bound form, plays an essential
assayed in extracts of fat body or adipose tissue in role. A schematic overview of the process is depicted
the presence of optimally accessible TAG substrate. in Fig. 2. Recent advances on the structural properties
In this way, only the effects of (de)phosphorylation or of HDLp and apoLp-III demonstrate a remarkable
other conformational changes of the enzyme on its similarity to their counterparts in the mammalian
lipolytic activity will be measured, and other potential system (the two nonexchangeable apolipoproteins of
effects such as enzyme translocation and the involve- HDLp are similar to apoB; apoLp-III is similar to
ment of lipid droplet-associated proteins, factors apoE). However, the functioning of the insect
that may be highly important for lipase activity as lipoprotein as a shuttle mechanism operating in
discussed above for vertebrate HSL, remain con- energy transport during flight activity is intriguingly
founded. This may also explain why phosphorylation different, since in mammals lipoproteins do not
by PKA did not change the activity of TAG lipase that play a role as carriers of mobilized lipids during
had been purified from fat body of the adult exercise.
hawkmoth, Manduca sexta. Both HDLp and apoLp-III are abundantly
In two insect species that rely on lipid mobili- present in the hemolymph of resting insects. The
zation during sustained flight activity, L. migratoria loading of DAG onto HDLp particles at the fat body
and M. sexta, it has been shown that DAG, which is cell plasma membrane induces the association of
released from the fat body by the action of AKH, is multiple copies of apoLp-III with the expanding
stereospecific and has the sn-1,2-configuration. lipoprotein surface. The resulting LDLp, which is
Although conclusive evidence on the pathway for both larger and lipid-enriched, travels in the circula-
the stereospecific synthesis of this sn-1,2-DAG is still tion to the flight muscles, where a lipophorin lipase
lacking, the most probable route involves stereo- specifically hydrolyzes the LDLp-carried DAG. The
specific hydrolysis of TAG into sn-1,2-DAG by a liberated FFA are imported into the muscle cells
stereospecific lipase acting at the sn-3 position of and oxidized to yield energy. Once the lipid content
the TAG. of the LDLp has diminished, apoLp-III dissociates
from the particle, and finally, its HDLp constituent is
recovered. Both HDLp and apoLp-III are recycled to
IV. ADIPOKINETIC HORMONE-INDUCED the fat body and reutilized for DAG uptake (see
Fig. 2). This concept of a reusable lipoprotein
LIPOPHORIN CONVERSIONS
shuttle for lipid transport during flight activity was
For insect species that recruit fat body TAG depots to initially reported for the locust, but thereafter found
power their flight muscles during the vast distances to be present in many other long-distance flying
covered nonstop by migratory flight, an efficient insects.
ADIPOKINETIC HORMONES AND LIPID MOBILIZATION 37

FIGURE 2 Schematic overview of AKH-controlled substrate mobilization from insect fat body during flight activity.
AKH, adipokinetic hormones; HDLp, high-density lipophorin; LDLp, low-density lipophorin; apoLp-III, apolipophorin
III; FFA, free fatty acids.

This shuttle mechanism is specific to the adult stage Glossary


of the insect. During all stages of locust development,
high circulating levels of HDLp are present to adipokinetic hormone Peptide hormone in insects involved
in the mobilization of substrates (predominantly lipids
transport lipid components between tissues. However,
and carbohydrates) for energy generation needed by
the apoLp-III gene is expressed only after the first week
contracting flight muscles.
of adult life and is apparently related to the adult- apolipophorin III (apoLp-III) Exchangeable apolipoprotein
specific capacity of the insect to fly. The molecular (mol wt 17 – 20 kDa) in the hemolymph of insects
basis of the interaction of apoLp-III with the lipopro- engaging in long-distance flights for which lipids are
tein surface is of great interest and value since the used as an energy source. In the resting blood,
locust apoLp-III represents the only full-length apoli- apolipophorin III forms a stable particle consisting of
poprotein for which a three-dimensional structure has a bundle of five amphipathic a-helices; during lipid
been disclosed. In the absence of lipid, apoLp-III loading of lipophorin the conformation of apoLp-III
changes and allows the peptide to unfold and reversibly
reveals a globular bundle of five amphipathic
bind to the expanding particle.
a-helices. These helices are oriented such that the
fat body Organ whose functional role in insects is analogous
hydrophobic amino acid side chains are buried in the to the combined functions of liver and adipose tissue in
bundle interior and the hydrophilic residues face mammals. Fat body is the chief site of intermediary
outward. A plausible model postulates that lipid metabolism, detoxification, storage of nutrient reserves,
binding induces a significant reorganization of the and, in some species, deposition of nitrogenous waste
helical segments, allowing interaction of the hydro- products.
phobic face of the helices with the lipoprotein lipid HDLp High-density lipophorin (density approximately
surface. A critical event in lipid surface recognition 1.12 g/ml), the abundant and generally single lipopro-
tein particle in insect hemolymph, transporting several
seems to be the disturbance of the phospholipid
classes of lipids between organs and tissues in the
monolayer by the appearance of patches of DAG
resting situation.
molecules in the lipoprotein surface. Mechanistic LDLp Low-density lipophorin (density approximately
details underlying the opening of the amphipathic 1.04 g/ml), the adipokinetic hormone-induced form of
helix bundle and the lipid-binding activity of lipoprotein in the hemolymph of insects engaging in
apoLp-III, however, remain to be defined. long-distance flights for which lipids are used as an
38 ADIPOKINETIC HORMONES: STRUCTURE AND BIOSYNTHESIS

energy source. The particle consists of lipid (diacylgly-


cerol)-loaded and apoLp-III-associated HDLp.
lipophorin The abundant and generally single lipoprotein Adipokinetic Hormones:
particle in insect hemolymph. The density of lipophorin
(lipid-bearing protein) is similar to that of human high- Structure and Biosynthesis
density lipoprotein (HDL); for the insect lipophorin,
HDLp is used to distinguish it from HDL. ROB C. H. M. OUDEJANS AND
DICK J. VAN DER HORST
See Also the Following Articles Utrecht University, The Netherlands

Adipokinetic Hormones and Carbohydrate Metabolism I. INTRODUCTION


. Adipokinetic Hormones: Structure and Biosynthesis II. PRIMARY STRUCTURE OF ADIPOKINETIC HORMONES
. Insect Endocrine System III. BIOSYNTHESIS OF ADIPOKINETIC HORMONES
IV. STORAGE, RELEASE, AND INACTIVATION OF
ADIPOKINETIC HORMONES
Further Reading
Goldsworthy, G. J., Lee, M. J., Luswata, R., Drake, A. F., and
Hyde, D. (1997). Structures, assays and receptors for locust The structure and biosynthesis of the adipokin-
adipokinetic hormones. Comp. Biochem. Physiol. 117B, etic hormones (AKHs) are reviewed and their
483–496. storage, release, and inactivation in insect blood
Ogoyi, D. O., Osir, E. O., and Olembo, N. K. (1998). Fat body (hemolymph) are discussed. Since most of our
triacylglycerol lipase in solitary and gregarious phases of knowledge on the biosynthesis of the AKHs has
Schistocerca gregaria (Forskal) (Orthoptera: Acrididae). been obtained from locust species, these insects
Comp. Biochem. Physiol. 119B, 163 –167. will serve as a general model system.
Oudejans, R. C. H. M., Dijkhuizen, R. M., Kooiman, F. P., and
Beenakkers, A. M. T. (1992). Dose–response relationships of
adipokinetic hormones (Lom-AKH-I, II and III) from the
migratory locust, Locusta migratoria. Proc. Sect. Exp. Appl. I. INTRODUCTION
Entomol. Neth. Entomol. Soc. 3, 165–166.
Pines, M., Tietz, A., Weintraub, H., Applebaum, S. W., and Insect long-distance flight activity involves extremely
Josefsson, L. (1981). Hormonal activation of protein kinase high metabolic rates, and insect flight muscles are
and lipid mobilization in the locust fat body in vitro. Gen.
among the most energy-demanding tissues known.
Comp. Endocrinol. 43, 427–431.
Ryan, R. O., and Van der Horst, D. J. (2000). Lipid transport The energy substrates needed to fuel the flight muscles
biochemistry and its role in energy production. Annu. Rev. are stored in the fat body and their mobilization is
Entomol. 45, 233–260. under the control of adipokinetic hormones (AKHs).
Staubli, F., Jørgensen, T. J. D., Cazzamali, G., Williamson, M., Additional functions of the AKHs are revealed during a
Lenz, C., Søndergaard, L., Roepstorff, P., and Grimmelikhuij-
variety of other metabolic “stress” situations (energy-
zen, C. J. P. (2002). Molecular identification of the insect
adipokinetic receptors. Proc. Natl. Acad. Sci. USA 99, consuming processes), such as starvation, diapause,
3446–3451. and molting. The AKHs are peptide hormones that
Van der Horst, D. J., Van Marrewijk, W. J. A., and Diederen, J. H. B. (with a few exceptions) are synthesized and stored in
(2001). Adipokinetic hormones of insect: Release, signal the so-called adipokinetic cells of the glandular lobes
transduction, and responses. Int. Rev. Cytol. 211, 179–240.
of the corpus cardiacum, a neuroendocrine organ
Van Marrewijk, W. J. A., and Van der Horst, D. J. (1998).
Signal transduction of adipokinetic hormone. In “Recent situated in the insect head and connected to the central
Advances in Arthropod Endocrinology” (G. M. Coast and nervous system.
S. G. Webster, eds.), pp. 172– 188. Cambridge University Press,
Cambridge, UK.
Vroemen, S. F., Van der Horst, D. J., and Van Marrewijk, W. J. A.
(1998). New insights into adipokinetic hormone signaling. II. PRIMARY STRUCTURE OF ADIPOKINETIC
Mol. Cell. Endocrinol. 141, 7–12. HORMONES
Wang, Z., Hayakawa, Y., and Downer, R. G. H. (1990). Factors
influencing cyclic AMP and diacylglycerol levels in fat body of Since the elucidation of the structure of the first AKH
Locusta migratoria. Insect Biochem. 20, 325–330. in 1976 from 3000 locust corpora cardiaca, analytical
Ziegler, R., Eckart, K., and Law, J. H. (1990). Adipokinetic techniques have been revolutionized and today it is
hormone controls lipid metabolism in adults and carbohydrate possible to determine the structure of a new AKH from
metabolism in larvae of Manduca sexta. Peptides 11,
1037–1040.
just a few picomoles of the hormone. To date, the
structures of some 37 different AKHs are known
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved. from representatives of most insect orders (Table I).
38 ADIPOKINETIC HORMONES: STRUCTURE AND BIOSYNTHESIS

energy source. The particle consists of lipid (diacylgly-


cerol)-loaded and apoLp-III-associated HDLp.
lipophorin The abundant and generally single lipoprotein Adipokinetic Hormones:
particle in insect hemolymph. The density of lipophorin
(lipid-bearing protein) is similar to that of human high- Structure and Biosynthesis
density lipoprotein (HDL); for the insect lipophorin,
HDLp is used to distinguish it from HDL. ROB C. H. M. OUDEJANS AND
DICK J. VAN DER HORST
See Also the Following Articles Utrecht University, The Netherlands

Adipokinetic Hormones and Carbohydrate Metabolism I. INTRODUCTION


. Adipokinetic Hormones: Structure and Biosynthesis II. PRIMARY STRUCTURE OF ADIPOKINETIC HORMONES
. Insect Endocrine System III. BIOSYNTHESIS OF ADIPOKINETIC HORMONES
IV. STORAGE, RELEASE, AND INACTIVATION OF
ADIPOKINETIC HORMONES
Further Reading
Goldsworthy, G. J., Lee, M. J., Luswata, R., Drake, A. F., and
Hyde, D. (1997). Structures, assays and receptors for locust The structure and biosynthesis of the adipokin-
adipokinetic hormones. Comp. Biochem. Physiol. 117B, etic hormones (AKHs) are reviewed and their
483–496. storage, release, and inactivation in insect blood
Ogoyi, D. O., Osir, E. O., and Olembo, N. K. (1998). Fat body (hemolymph) are discussed. Since most of our
triacylglycerol lipase in solitary and gregarious phases of knowledge on the biosynthesis of the AKHs has
Schistocerca gregaria (Forskal) (Orthoptera: Acrididae). been obtained from locust species, these insects
Comp. Biochem. Physiol. 119B, 163 –167. will serve as a general model system.
Oudejans, R. C. H. M., Dijkhuizen, R. M., Kooiman, F. P., and
Beenakkers, A. M. T. (1992). Dose–response relationships of
adipokinetic hormones (Lom-AKH-I, II and III) from the
migratory locust, Locusta migratoria. Proc. Sect. Exp. Appl. I. INTRODUCTION
Entomol. Neth. Entomol. Soc. 3, 165–166.
Pines, M., Tietz, A., Weintraub, H., Applebaum, S. W., and Insect long-distance flight activity involves extremely
Josefsson, L. (1981). Hormonal activation of protein kinase high metabolic rates, and insect flight muscles are
and lipid mobilization in the locust fat body in vitro. Gen.
among the most energy-demanding tissues known.
Comp. Endocrinol. 43, 427–431.
Ryan, R. O., and Van der Horst, D. J. (2000). Lipid transport The energy substrates needed to fuel the flight muscles
biochemistry and its role in energy production. Annu. Rev. are stored in the fat body and their mobilization is
Entomol. 45, 233–260. under the control of adipokinetic hormones (AKHs).
Staubli, F., Jørgensen, T. J. D., Cazzamali, G., Williamson, M., Additional functions of the AKHs are revealed during a
Lenz, C., Søndergaard, L., Roepstorff, P., and Grimmelikhuij-
variety of other metabolic “stress” situations (energy-
zen, C. J. P. (2002). Molecular identification of the insect
adipokinetic receptors. Proc. Natl. Acad. Sci. USA 99, consuming processes), such as starvation, diapause,
3446–3451. and molting. The AKHs are peptide hormones that
Van der Horst, D. J., Van Marrewijk, W. J. A., and Diederen, J. H. B. (with a few exceptions) are synthesized and stored in
(2001). Adipokinetic hormones of insect: Release, signal the so-called adipokinetic cells of the glandular lobes
transduction, and responses. Int. Rev. Cytol. 211, 179–240.
of the corpus cardiacum, a neuroendocrine organ
Van Marrewijk, W. J. A., and Van der Horst, D. J. (1998).
Signal transduction of adipokinetic hormone. In “Recent situated in the insect head and connected to the central
Advances in Arthropod Endocrinology” (G. M. Coast and nervous system.
S. G. Webster, eds.), pp. 172– 188. Cambridge University Press,
Cambridge, UK.
Vroemen, S. F., Van der Horst, D. J., and Van Marrewijk, W. J. A.
(1998). New insights into adipokinetic hormone signaling. II. PRIMARY STRUCTURE OF ADIPOKINETIC
Mol. Cell. Endocrinol. 141, 7–12. HORMONES
Wang, Z., Hayakawa, Y., and Downer, R. G. H. (1990). Factors
influencing cyclic AMP and diacylglycerol levels in fat body of Since the elucidation of the structure of the first AKH
Locusta migratoria. Insect Biochem. 20, 325–330. in 1976 from 3000 locust corpora cardiaca, analytical
Ziegler, R., Eckart, K., and Law, J. H. (1990). Adipokinetic techniques have been revolutionized and today it is
hormone controls lipid metabolism in adults and carbohydrate possible to determine the structure of a new AKH from
metabolism in larvae of Manduca sexta. Peptides 11,
1037–1040.
just a few picomoles of the hormone. To date, the
structures of some 37 different AKHs are known
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved. from representatives of most insect orders (Table I).
ADIPOKINETIC HORMONES: STRUCTURE AND BIOSYNTHESIS 39

TABLE I Amino Acid Sequences of Peptide Hormones of the AKH/RPCH Family


Hormone
(by acronym) Amino acid sequence

Miv-CC pGlu-Ile-Asn-Phe-Thr-Pro-Asn-Trp-NH2
Phm-AKH-III pGlu-Ile-Asn-Phe-Thr-Pro-Trp-Trp-NH2
Poa-HrTH pGlu-Ile-Thr-Phe-Thr-Pro-Asn-Trp-NH2
Lom-AKH-II pGlu-Leu-Asn-Phe-Ser-Ala-Gly-Trp-NH2
Tem-HrTH pGlu-Leu-Asn-Phe-Ser-Pro-Asn-Trp-NH2
Pab-RPCH pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2
Scg-AKH-II pGlu-Leu-Asn-Phe-Ser-Thr-Gly-Trp-NH2
Pya-AKH pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-NH2
Ers-AKH pGlu-Leu-Asn-Phe-Thr-Pro-Ser-Trp-NH2
Lom-AKH-III pGlu-Leu-Asn-Phe-Thr-Pro-Trp-Trp-NH2
Mem-CC pGlu-Leu-Asn-Tyr-Ser-Pro-Asp-Trp-NH2
Pht-HrTH pGlu-Leu-Thr-Phe-Ser-Pro-Asp-Trp-NH2
Pea-CAH-II pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH2
Taa-AKH PGlu-Leu-Thr-Phe-Thr-Pro-Gly-Trp-NH2
Ona-CC-II PGlu-Phe-Asn-Tyr-Ser-Phe-Asp-Trp-NH2
Ona-CC-I PGlu-Tyr-Asn-Phe-Ser-Thr-Gly-Trp-NH2
Pea-CAH-I pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2
Ani-AKH pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-NH2
Grb-AKH pGlu-Val-Asn-Phe-Ser-Thr-Gly-Trp-NH2
Emp-AKH pGlu-Val-Asn-Phe-Thr-Pro-Asn-Trp-NH2
Psi-AKH pGlu-Val-Asn-Phe-Thr-Pro-Gly-Trp-NH2
Lia-AKH pGlu-Val-Asn-Phe-Thr-Pro-Ser-Trp-NH2

Mas-AKH pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp-Gly-NH2

Del-CC pGlu-Leu-Asn-Phe-Ser-Pro-Asn-Trp-Gly-Asn-NH2
Tea-HrTH pGlu-Leu-Asn-Phe-Ser-Thr-Gly-Trp-Gly-Gly-NH2
Phm-AKH-I pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Ser-NH2
Lom-AKH-I pGlu-Leu-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2
Hez-HrTH pGlu-Leu-Thr-Phe-Ser-Ser-Gly-Trp-Gly-Asn-NH2
Phl-CC pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-Ser-NH2
Cam-HrTH-II pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2
Cam-HrTH-I pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2
l
Hexose
Taa-HoTH pGlu-Leu-Thr-Phe-Thr-Pro-Gly-Trp-Gly-Tyr-NH2
Bld-HrTH pGlu-Val-Asn-Phe-Ser-Pro-Gly-Trp-Gly-Thr-NH2
Plc-HrTH-II pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-Gly-Asn-NH2
Rom-CC-I pGlu-Val-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2
Lom-HrTH pGlu-Val-Thr-Phe-Ser-Arg-Asp-Trp-Ser-Pro-NH2

Vac-AKH pGlu-Leu-Thr-Phe-Thr-Ser-Ser-Trp-Gly-Gly-Lys

Variation per residue position

Position: 1 2 3 4 5 6 7 8 9 10 11

pGlu Ile Asn Phe Ser Ala Asn Trp Gly Asn Lys
Leu Thr Tyr Thr Arg Asp Ser Gly
Phe Phe Gly Pro
Tyr Pro Ser Ser
Val Ser Trp Thr
Thr Tyr

They are short peptides consisting of 8 – 11 amino acid based on the use of the first two characters of the genus
residues and constitute the so-called AKH/RPCH (red name and the first character of the species name of the
pigment-concentrating hormone) family. The nomen- animal from which the hormone was first isolated,
clature of the members of this family (by acronym) is supplemented by an abbreviation of its function.
40 ADIPOKINETIC HORMONES: STRUCTURE AND BIOSYNTHESIS

The name is followed by a roman numeral in case the cockroach). In many insect species a partic-
there is more than one hormone per function. So ular physiological function seems to be supported by
Lom-AKH-I, -II, and -III are the three AKHs isolated two, or in some locust and grasshopper species even
from Locusta migratoria, and Lom-AKH-I and three, hormones, suggesting sophisticated hormonal
Scg-AKH-II are the two AKHs from Schistocerca control. Whether there are quantitative or qualitative
gregaria. differences that result in the fine-tuning of apparently
In addition to the AKHs, the family contains identical or just pleiotropic functions is still under
hypertrehalosemic (HrTH) and hypotrehalosemic investigation.
hormones (HoTH), corpus cardiacum factors (CC),
etc. All members are found in insects, except
for RPCH, which is restricted to the X-organ-sinus
gland of crustaceans. Curiously, the variation in the III. BIOSYNTHESIS OF ADIPOKINETIC
structure of AKHs of insects is extremely large, HORMONES
whereas in all crustaceans investigated to date, just All AKHs studied thus far are translated from
one RPCH structure has been found. All members
separate mRNAs. They code for preprohormones
(except Vac-AKH) are fully blocked peptides: They
with a simple structure: a signal sequence, a mono-
are N-terminally blocked by a pyroglutamate (pGlu)
copy AKH sequence (starting with a Gln to form
residue and C-terminally blocked by an amide group.
pGlu), a Gly residue (to form the terminal AKH amide
Although the variation in structure has increased by
group), a dibasic processing site, and an AKH-asso-
an increasing number of new hormones, characteristic
ciated peptide sequence. In particular, in L. migratoria
features are still present. At the bottom of Table I the
the signal sequences of the three preprohormones are
variation per amino acid residue position is indicated.
cotranslationally cleaved, and the resulting prohor-
At position 1 a pGlu is always found, at position 8 a
mones of AKH-I and -II dimerize at random by
Trp is always found, and at position 4 an aromatic
oxidation of their Cys residues and give rise to two
amino acid residue is always found. At position 2
homodimeric precursor molecules (AKH-I/I and
most members contain a branched-chain amino
acid residue, and at position 5 either a Ser or a Thr AKH-II/II) and one heterodimeric precursor mole-
is present. Except for Lom-HrTH, all longer mem- cule (AKH-I/II). This dimerization is a rather unique
bers possess a Gly at position 9. Most variation phenomenon, first established for another locust
occurs at positions 6, 7, and 10. In only one member species (S. gregaria). Further proteolytic processing
(Cam-HrTH-I), a sugar moiety with an as yet un- of the dimeric products in the secretory granules of
known structure has been found as a posttranslational the AKH cells of the corpus cardiacum results in
modification. Except for a few members (Lom-HrTH, the bioactive hormones and one heterodimeric and
Mem-CC, Ona-CC-II, and Pht-HrTH), all peptides two homodimeric AKH-precursor-related peptides
are uncharged. (APRPs) with as yet unknown functions (see Fig. 1).
Adipokinetic activity of a substance can be The biosynthesis of AKH-III is not yet fully under-
shown in the locust (L. migratoria) by using a simple stood. Recently, dimerization of the AKH-III prohor-
bioassay of injecting the substance into resting mone with itself (it contains two Cys residues itself)
animals and measuring the increase in lipid content has been established. Dimerization with the prohor-
in the hemolymph. Potential hypertrehalosemic mones of AKH-I and -II has not been observed,
activity can be measured using a bioassay in the and whether a cyclic prohormone of AKH-III can
cockroach (Periplaneta americana). Interestingly, arise by formation of an internal disulfide bond is
all members of the AKH/RPCH family (including unknown.
RPCH itself) exert an adipokinetic effect in the In vitro studies on the biosynthesis of AKHs in
locust assay, suggesting that the AKH receptor(s) of locusts have revealed that the total time required for
L. migratoria is (are) less specialized or that all AKHs their biosynthesis from the starting point when
share structural similarities. In contrast to locusts and radiolabeled amino acids are made available to the
cockroaches, some insect species have a low response moment that radiolabeled bioactive hormones appear
to their own AKH and a higher response to the is 30 –60 min. In L. migratoria the time for biosyn-
hormone from other species. There are even members thesis of AKH-III is shorter than that for AKH-I and
of the family with an as yet unknown function (for -II, suggesting that there are two different pathways
instance, Lom-HrTH, which stimulates in a heterol- or different processing procedures for AKH-I/II, on
ogous assay the mobilization of carbohydrates only in the one hand, and for AKH-III, on the other.
ADIPOKINETIC HORMONES: STRUCTURE AND BIOSYNTHESIS 41

The situation in L. migratoria is even more


complex, since secretory granules of only a particular
age can be released. Newly formed granules con-
taining the AKHs must mature before they can release
their contents (or before they can fuse with the
plasma membrane). Granules more than 8 h old are
believed to enter a nonreleasable pool. Determination
of the total hormone content of a neuroendocrine
structure has therefore a limited physiological value,
because only the releasable amount of hormone is
relevant.
Finally, the balance between released hormones (a
mixture of many compounds present in the secretory
granule) and the rate of their inactivation prior to
reaching the target tissue will be of eminent impor-
tance for their ultimate effect. The three AKHs of
L. migratoria appear to be inactivated differentially
after their release, with half-lives during flight of 35,
37, and 3 min obtained for AKH-I, -II, and -III,
respectively.
FIGURE 1 The biosynthesis of the locust adipokinetic
hormones AKH-I and -II and the adipokinetic precursor-
related peptides APRP-1, -2, and -3. After cleavage of the signal
peptide from the preprohormones, the resulting monomeric Glossary
prohormones, which consist of the AKH sequence and the
AKH-associated peptide (AAP), form three dimeric prohor- adipokinetic hormone Peptide hormone in insects involved
mones, which are then processed to the AKHs and the APRPs. in the mobilization of substrates (predominantly lipids
and carbohydrates) for energy generation needed by
contracting flight muscles.
IV. STORAGE, RELEASE, AND INACTIVATION corpus cardiacum Neuroendocrine organ situated caudal
to the brain to which it is connected by paired nerves.
OF ADIPOKINETIC HORMONES
The organ combines the functions of neurohemal
AKHs are stored in the secretory granules of the storage and adipokinetic/hypertrehalosemic hormone
adipokinetic cells of the corpus cardiacum. In production.
L. migratoria the total content of the AKHs increases fat body Organ whose functional role in insects is analogous
to the combined functions of liver and adipose tissue in
continuously during the larval stages and throughout
mammals. Fat body is the chief site of intermediary
adult life. In aging locusts an increasing number of metabolism, detoxification, storage of nutrient reserves,
intracisternal granules that contain stores of AKH and, in some species, deposition of nitrogenous waste
prohormones/precursors are also found. The three products.
AKHs, colocalized and stored in the same secretory hemolymph Blood of insects, which circulates in the body
granules, are released during flight. Since the mem- cavity between the various organs, bathing them
brane of the pertinent secretory granule fuses with the directly. It consists of a fluid plasma in which the
plasma membrane, the total contents of the granule blood cells or hemocytes are suspended.
are released into the hemolymph: the bioactive AKHs, hypertrehalosemic hormone Peptide hormone from the
the APRPs, and possibly other end products. corpus cardiacum involved in the mobilization of fat
The release of the AKHs in L. migratoria is body glycogen to fuel flight activity. For transport to
the flight muscles, glycogen is converted into the di-
subjected to many regulatory substances, which are
saccharide trehalose, the carbohydrate transport form
of either a stimulatory or an inhibitory nature and
in insects, whose level in hemolymph is raised during
can be of both neural and humoral origin. A detailed flight.
description falls beyond the scope of this article. The red pigment concentrating hormone Peptide hormone from
only natural stimulus of release of the AKHs is flight the eyestalk of crustaceans, a member of the adipokin-
and the relative contributions of all known substances etic hormone/red pigment-concentrating hormone
effective in the release process remain to be established family that comprises structurally related but function-
in vivo. ally diverse peptides.
42 ADRENAL CORTEX ROLE IN MEDULLARY SYNTHESIS OF PNMT

See Also the Following Articles


Adipokinetic Hormones and Carbohydrate Metabolism
. Adipokinetic Hormones and Lipid Mobilization . Insect
Adrenal Cortex Role in
Endocrine System Medullary Synthesis of PNMT
I. C. MC MILLEN AND M. B. ADAMS
Further Reading University of Adelaide, Australia
Bogerd, J., Kooiman, F. P., Pijnenburg, M. A. P., Hekking, L. H. P.,
Oudejans, R. C. H. M., and Van der Horst, D. J. (1995). I. INTRODUCTION
Molecular cloning of three distinct cDNAs, each encoding a II. THE ADRENAL MEDULLA AND ADRENAL CORTEX—
different adipokinetic hormone precursor, of the migratory ANATOMY AND DEVELOPMENT
locust, Locusta migratoria. J. Biol. Chem. 270, 23038–23043. III. SYNTHESIS OF ADRENOMEDULLARY CATECHOLAMINES
Clynen, E., Baggerman, G., Veelaert, D., Cerstiaens, A., Van der
IV. GLUCOCORTICOIDS AND PNMT SYNTHESIS
Horst, D., Harthoorn, L., Derua, R., Waelkens, E., De Loof,
A., and Schoofs, L. (2001). Peptidomics of the pars intercer-
V. GLUCOCORTICOIDS AND PNMT SYNTHESIS DURING
ebralis–corpus cardiacum complex of the migratory locust, DEVELOPMENT
Locusta migratoria. Eur. J. Biochem. 268, 1929–1939. VI. SUMMARY
Diederen, J. H. B., Oudejans, R. C. H. M., Harthoorn, L. F., and
Van der Horst, D. J. (2002). Cell biology of the adipokinetic
hormone-producing neurosecretory cells in the locust corpus During development, two anatomically and
cardiacum. Microsc. Res. Techn. 56, 227–236. functionally distinct structures merge to form
Flanigan, J. E., and Gäde, G. (1999). On the release of the three the adrenal gland. These structures comprise the
locust (Locusta migratoria) adipokinetic hormones: Effect of central adrenal medulla, which synthesizes and
crustacean cardioactive peptide and inhibition by sugars. secretes the catecholamines noradrenaline and
Z. Naturforsch. 54C, 110 –118. adrenaline, and the peripheral adrenal cortex,
Gäde, G. (1996). The revolution in insect neuropeptides illustrated which synthesizes and secretes steroid hor-
by the adipokinetic hormone/red pigment concentrating
mones, most notably the mineralocorticoids
hormone family of peptides. Z. Naturforsch. 51C, 607 –617.
Goldsworthy, G. J., Lee, M. J., Luswata, R., Drake, A. F., and
(e.g., aldosterone) and the glucocorticoids (e.g.,
Hyde, D. (1997). Structures, assays and receptors for locust corticosterone or cortisol).
adipokinetic hormones. Comp. Biochem. Physiol. 117B,
483–496.
Hagbrechts, J., Clynen, E., Baggerman, G., De Loof, A., and
Schoofs, L. (2002). Isolation and indentification of the AKH III I. INTRODUCTION
precursor-related peptide from Locusta migratoria. Biochem.
Biophys. Res. Commun. 296, 1112– 1117. Although the adrenal medulla and cortex have
Oudejans, R. C. H. M., Harthoorn, L. F., Diederen, J. H. B., and different embryological origins and are regulated
Van der Horst, D. J. (1999). Adipokinetic hormones. Coupling
predominantly by separate reflex neurogenic and
between biosynthesis and release. Ann. N. Y. Acad. Sci. 897,
291–299. endocrine mechanisms, respectively, there is substan-
Oudejans, R. C. H. M., Vroemen, S. F., Jansen, R. F. R., and Van der tial functional cross talk between these two separate
Horst, D. J. (1996). Locust adipokinetic hormones: Carrier- components of the adrenal gland, which is important
independent transport and differential inactivation at physio- in the maintenance of an adequate physiological stress
logical concentrations during rest and flight. Proc. Natl. Acad.
response to a range of metabolic and other physio-
Sci., USA 93, 8654–8659.
Raina, A. K., and Gäde, G. (1988). Insect peptide nomenclature. logical stressors. One important aspect of the func-
Insect Biochem. 8, 785–787. tional interaction between the adrenal cortex and the
Rayne, R. C., and O’Shea, M. (1993). Structural requirements for medulla is the role of the adrenal cortex in the control
processing of pro-adipokinetic hormone I. Eur. J. Biochem. of the synthesis of the adrenaline-synthesizing hor-
217, 905–911.
mone, phenylethanolamine N-methyltransferase
Rayne, R. C., and O’Shea, M. (1994). Reconstitution of
adipokinetic hormone biosynthesis in vitro indicates steps in (PNMT). This article reviews the separate develop-
prohormone processing. Eur. J. Biochem. 219, 781–789. mental origins of the adrenal medulla and adrenal
Vullings, H. G. B., Diederen, J. H. B., Veelaert, D., and Van der cortex, the anatomical relationship between these
Horst, D. J. (1999). Multifactorial control of the release of two structures, and the role of the glucocorticoid
hormones from the locust retrocerebral complex. Microsc.
Res. Techn. 45, 142–143.
hormones in the induction and maintenance of
PNMT synthesis during different physiological
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved. conditions.
42 ADRENAL CORTEX ROLE IN MEDULLARY SYNTHESIS OF PNMT

See Also the Following Articles


Adipokinetic Hormones and Carbohydrate Metabolism
. Adipokinetic Hormones and Lipid Mobilization . Insect
Adrenal Cortex Role in
Endocrine System Medullary Synthesis of PNMT
I. C. MC MILLEN AND M. B. ADAMS
Further Reading University of Adelaide, Australia
Bogerd, J., Kooiman, F. P., Pijnenburg, M. A. P., Hekking, L. H. P.,
Oudejans, R. C. H. M., and Van der Horst, D. J. (1995). I. INTRODUCTION
Molecular cloning of three distinct cDNAs, each encoding a II. THE ADRENAL MEDULLA AND ADRENAL CORTEX—
different adipokinetic hormone precursor, of the migratory ANATOMY AND DEVELOPMENT
locust, Locusta migratoria. J. Biol. Chem. 270, 23038–23043. III. SYNTHESIS OF ADRENOMEDULLARY CATECHOLAMINES
Clynen, E., Baggerman, G., Veelaert, D., Cerstiaens, A., Van der
IV. GLUCOCORTICOIDS AND PNMT SYNTHESIS
Horst, D., Harthoorn, L., Derua, R., Waelkens, E., De Loof,
A., and Schoofs, L. (2001). Peptidomics of the pars intercer-
V. GLUCOCORTICOIDS AND PNMT SYNTHESIS DURING
ebralis–corpus cardiacum complex of the migratory locust, DEVELOPMENT
Locusta migratoria. Eur. J. Biochem. 268, 1929–1939. VI. SUMMARY
Diederen, J. H. B., Oudejans, R. C. H. M., Harthoorn, L. F., and
Van der Horst, D. J. (2002). Cell biology of the adipokinetic
hormone-producing neurosecretory cells in the locust corpus During development, two anatomically and
cardiacum. Microsc. Res. Techn. 56, 227–236. functionally distinct structures merge to form
Flanigan, J. E., and Gäde, G. (1999). On the release of the three the adrenal gland. These structures comprise the
locust (Locusta migratoria) adipokinetic hormones: Effect of central adrenal medulla, which synthesizes and
crustacean cardioactive peptide and inhibition by sugars. secretes the catecholamines noradrenaline and
Z. Naturforsch. 54C, 110 –118. adrenaline, and the peripheral adrenal cortex,
Gäde, G. (1996). The revolution in insect neuropeptides illustrated which synthesizes and secretes steroid hor-
by the adipokinetic hormone/red pigment concentrating
mones, most notably the mineralocorticoids
hormone family of peptides. Z. Naturforsch. 51C, 607 –617.
Goldsworthy, G. J., Lee, M. J., Luswata, R., Drake, A. F., and
(e.g., aldosterone) and the glucocorticoids (e.g.,
Hyde, D. (1997). Structures, assays and receptors for locust corticosterone or cortisol).
adipokinetic hormones. Comp. Biochem. Physiol. 117B,
483–496.
Hagbrechts, J., Clynen, E., Baggerman, G., De Loof, A., and
Schoofs, L. (2002). Isolation and indentification of the AKH III I. INTRODUCTION
precursor-related peptide from Locusta migratoria. Biochem.
Biophys. Res. Commun. 296, 1112– 1117. Although the adrenal medulla and cortex have
Oudejans, R. C. H. M., Harthoorn, L. F., Diederen, J. H. B., and different embryological origins and are regulated
Van der Horst, D. J. (1999). Adipokinetic hormones. Coupling
predominantly by separate reflex neurogenic and
between biosynthesis and release. Ann. N. Y. Acad. Sci. 897,
291–299. endocrine mechanisms, respectively, there is substan-
Oudejans, R. C. H. M., Vroemen, S. F., Jansen, R. F. R., and Van der tial functional cross talk between these two separate
Horst, D. J. (1996). Locust adipokinetic hormones: Carrier- components of the adrenal gland, which is important
independent transport and differential inactivation at physio- in the maintenance of an adequate physiological stress
logical concentrations during rest and flight. Proc. Natl. Acad.
response to a range of metabolic and other physio-
Sci., USA 93, 8654–8659.
Raina, A. K., and Gäde, G. (1988). Insect peptide nomenclature. logical stressors. One important aspect of the func-
Insect Biochem. 8, 785–787. tional interaction between the adrenal cortex and the
Rayne, R. C., and O’Shea, M. (1993). Structural requirements for medulla is the role of the adrenal cortex in the control
processing of pro-adipokinetic hormone I. Eur. J. Biochem. of the synthesis of the adrenaline-synthesizing hor-
217, 905–911.
mone, phenylethanolamine N-methyltransferase
Rayne, R. C., and O’Shea, M. (1994). Reconstitution of
adipokinetic hormone biosynthesis in vitro indicates steps in (PNMT). This article reviews the separate develop-
prohormone processing. Eur. J. Biochem. 219, 781–789. mental origins of the adrenal medulla and adrenal
Vullings, H. G. B., Diederen, J. H. B., Veelaert, D., and Van der cortex, the anatomical relationship between these
Horst, D. J. (1999). Multifactorial control of the release of two structures, and the role of the glucocorticoid
hormones from the locust retrocerebral complex. Microsc.
Res. Techn. 45, 142–143.
hormones in the induction and maintenance of
PNMT synthesis during different physiological
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved. conditions.
ADRENAL CORTEX ROLE IN MEDULLARY SYNTHESIS OF PNMT 43

II. THE ADRENAL MEDULLA AND ADRENAL blood directly from arteries known as arteriae
CORTEX—ANATOMY AND DEVELOPMENT medullae. The presence of arteriae medullae within
the adrenal gland is species specific, with relatively
In early development, ectodermal neural crest cells few being observed in the rat adrenal gland,
migrate ventrally from the apex of the neural tube to whereas substantially greater numbers are found in
the dorsal aorta, where they aggregate and differen- cat and bovine adrenals. The arteriae medullae
tiate to form sympathetic neurons, or to the adrenal derive from branches of the large capsular arteries
gland primordia, where they differentiate to form that pass directly through the adrenal cortex before
chromaffin cells. The migratory primitive chromaffin entering the medulla and branching into arterioles and
cells invade the medial side of the developing adrenal capillaries. These vessels then empty into the medul-
cortical anlage and pass between the cortical cells lary veins, which in turn empty into the central adrenal
and as the medulla occupy the center of the gland. vein.
In the developing mammalian adrenal medulla, From a range of studies in different species, it
three types of cells can be observed depending on the appears that the adrenal medullary cells are exposed
stage of development. The first type includes the to high concentrations of glucocorticoids delivered
primitive sympathetic migratory neurons, which are either through the vasculature or through paracrine
known as sympathogonia and are totipotent, being interactions within the gland. In the human adrenal, it
capable of differentiating into either sympathetic has been shown, for instance, that there are variously
neurons or chromaffin cells depending on migratory protrusions, clusters, islets, and single cortical cells
route and the levels of environmental factors, such as that occur diffusely within the adrenal medulla,
the presence of glucocorticoids, nerve growth factor providing the opportunity for paracrine interactions.
(NGF), and fibroblast growth factors. These cells are Similarly, specific immunostaining for the neuroendo-
characterized by a small rounded nucleus with only a crine protein chromogranin-A has identified the
very thin peripheral rim of cytoplasm. The second type occurrence of chromaffin cells within all three zones
of cell is the phaeochromoblast, which has large of the human adrenal cortex, again supporting a close
elongated nuclei and a cytoplasm that is devoid of functional relationship between these cell types. In
catecholamine secretory granules. The third and final species such as the sheep or cow, adrenomedullary
cell type is the mature “chromaffin” cell or phaeo- cells directly adjacent to the zona reticularis and zona
chromocyte, which possesses a smaller ovoid nucleus fasiculata of the adrenal cortex characteristically
and contains catecholamine-storing granules, with the synthesize large amounts of adrenaline and the area
cytoplasm giving a positive staining reaction to of the adrenaline-synthesizing zone varies directly
chromic acid due to the oxidation of the catechol- with the activity of the adrenal steroidogenic cells.
amines to melanin. The mammalian adrenal cortex Before discussing the range of mechanisms by which
has its embryological origins in the mesoderm arising adrenal glucocorticoids may regulate the synthesis of
from mesenchymal tissue adjacent to the coelomic adrenaline, it is necessary to review the synthetic
epithelium lying close to the urogenital ridge. pathway for both of the major adrenal catechol-
Mesothelial cells between the root of the mesentery amines, noradrenaline and adrenaline.
and the developing gonad undergo proliferation and Cholinesterase staining and nerve degeneration
subsequently penetrate the underlying mesenchyme. studies confirm that the majority of the nerve fibers
These cells then differentiate to form the adrenocor- that project to the chromaffin cells of the mammalian
tical masses, which are invaded during development adrenal medulla are preganglionic cholinergic sym-
by migrating sympathochromaffin cells. An important pathetic fibers arising from the splanchnic nerve.
feature of the anatomical juxtaposition of the adrenal Retrograde tracer studies reveal that the preganglionic
medulla and cortex is the fact that the continuous sympathetic fibers that innervate the adrenal gland
networks of blood vessels that supply the adrenal arise ipsilaterally from the intermediolateral horn
cortex converge and empty into the larger sinusoids of the spinal cord between thoracic level 3 (T3) and
present within the corticomedullary and medullary lumbar level 2, with the majority arising from
regions, so that the medulla is directly exposed to T8 –T11. There is a body of evidence to suggest that
blood that has passed through the adrenal cortex. The splanchnic nerve stimulation can enhance ACTH-
medullary sinusoids then drain into medullary veins. induced glucocorticoid output from the adrenal
Aside from the cortical effluent, which drains into cortex. Postganglionic sympathetic fibers containing
the medullary sinusoids, the medulla also receives catecholamines have been found using fluorescence
44 ADRENAL CORTEX ROLE IN MEDULLARY SYNTHESIS OF PNMT

histochemistry to run in close association with


vascular supply of the gland. The adrenal gland also
appears to have an intrinsic innervation with
ganglion cells having been identified within the
gland with their numbers being species specific. In
the rat, two populations of ganglion cells have been
identified. One population of cells, termed the type I
ganglion cells, are relatively large and exhibit proper-
ties consistent with a postganglionic, noradrenergic
phenotype. These cells are probably derived from
neural crest cells that invaded the cortical anlage but
unlike the chromaffin cells were exposed to a different
set of environmental factors that led to their
differentiation into neurons. Type II ganglion cells
are smaller, have a nonclassical peptidergic transmit-
ter phenotype, and express nitric oxide synthase.
Intra-adrenal ganglion cells have fibers that project to
cortical, medullary, and capsular regions of the gland,
with a number appearing in close proximity to blood
vessels and chromaffin cells. Little is known about the
physiological function of these intrinsic ganglion cells
but they may act to regulate adrenal blood flow and
FIGURE 1 Biosynthetic pathway of adrenomedullary
also secretory output from the adrenal medullar and catecholamines. Schematic diagram of the synthesis of cat-
adrenal cortex. echolamines within the chromaffin cells of the adrenal medulla,
with substrates and products, enzymes, and co-factors indi-
cated. Shaded area represents chromaffin granule; the unshaded
area depicts the cytosol of the chromaffin cell. DOPA,
III. SYNTHESIS OF ADRENOMEDULLARY dihydroxyphenylalanine; TH, tyrosine hydroxylase; AADC,
CATECHOLAMINES aromatic amino acid decarboxylase; DBH, dopamine
b-hydroxylase; PNMT, phenylethanolamine N-methyltransfer-
Catecholamines are synthesized in the chromaffin ase; BPt, tetrahydrobiopterine; SAM, S-adenosylmethionine.
cells of the adrenal gland from the dietary amino acid
tyrosine, as illustrated in Fig. 1. The initial step in the
synthesis of adrenomedullary catecholamines is the sence in the cytoplasm of the chromaffin cells. DOPA
oxidation of tyrosine to dihydroxyphenylalanine is subsequently decarboxylated by DOPA decarboxy-
(DOPA) by the enzyme tyrosine hydroxylase [TH; lase, also known as aromatic L -amino acid decarboxy-
tyrosine 3-monooxygenase; tyrosine, tetrahydropteri- lase (AADC; EC 4.1.1.28), to form dopamine.
dine:oxygen oxidoreductase (3-hydroxylating), EC AADC is not specific for catecholamine synthesis as
1.14.16.2]. TH is a mixed-function oxidase, which it is involved in the decarboxylation of a number of
requires molecular oxygen and utilizes a tetrahydro- aromatic L -amino acids and appears to have a wide
biopterine as a co-substrate. The tetrahydropteridine distribution in a number of tissues. Like TH, AADC
co-substrate is oxidized to dihydrobiopteridine in the activity in the chromaffin cell is found in the
conversion of tyrosine to DOPA; dihydropteredine water-soluble fraction of adrenal homogenates, indi-
reductase (EC 1.6.99.7) and nicotinamide adenine cating that it is located in the cytosol of chromaffin
dinucleotide phosphate are required to regenerate the cells. Dopamine is actively transported into the
pool of the reduced form of the biopterine. Tyrosine chromaffin granules and converted to noradrenaline
hydroxylase is the rate-limiting enzyme in the by dopamine b-hydroxylase [DBH; 3,4-dihydroxy-
catecholamine synthetic pathway, as TH has a phenylethylamine, ascorbate:oxygen oxidoreductase
substantially lower specific activity than any of the (b-hydroxylating), EC 1.14.17.1]. DBH is the only
other catecholamine synthetic enzymes and the pool enzyme in the catecholamine biosynthetic pathway
of tyrosine is greater than that of any of the other located within the chromaffin granules; all of the
catecholamine synthetic enzyme substrates. Tyrosine other enzymes are situated in the cytoplasm of the
hydroxylase is located in the soluble fractions of chromaffin cells. DBH is present in the chromaffin
adrenal medullary homogenates, indicating its pre- granules in one of two similar forms, either bound to
ADRENAL CORTEX ROLE IN MEDULLARY SYNTHESIS OF PNMT 45

the internal membrane or free in the internal matrix glucocorticoid-response elements on the PNMT gene
of the chromaffin granule. The relative proportions of to stimulate transcription. Furthermore, substantially
the enzyme that are free versus membrane-bound higher levels of GR immunoreactivity have been
vary between species, with the free form probably identified in the adrenaline-containing cells than in
being derived from a membrane-bound precursor the noradrenaline-containing cells of the adrenal
that undergoes proteolysis. Like TH, DBH is a mixed- medulla of the rat. There is evidence from a range
function oxidase and it catalyzes the oxidation of of studies that glucocorticoids appear to play a crucial
dopamine to noradrenaline, requires molecular oxy- role in the induction of adrenal PNMT activity and
gen, and utilizes ascorbate as a co-factor. The final mRNA expression that occurs in response to a range
step in the catecholamine biosynthetic pathway is the of stressors, thus demonstrating the importance of the
N-methylation of noradrenaline, which passively anatomical juxtaposition of the adrenal cortex and
permeates into the cytosol from chromaffin adrenal medulla in the generation of the adaptive
granules, to adrenaline. This is achieved by transfer stress response.
of the S-methyl group from S-adenosylmethionine to
the primary nitrogen group of noradrenaline by the
enzyme PNMT.
V. GLUCOCORTICOIDS AND PNMT
SYNTHESIS DURING DEVELOPMENT
IV. GLUCOCORTICOIDS AND PNMT Previous studies have shown that when dissociated
SYNTHESIS chromaffin cells from immature adrenals are cultured
in the absence of glucocorticoids they exhibit poor
Early endocrine ablation and replacement exper-
long-term survival in culture. The addition of NGF to
iments clearly demonstrated that glucocorticoids
these cultures is able to rescue many of the chromaffin
play a role in the maintenance of the activity of TH
cells; however, they subsequently undergo a pheno-
and PNMT. Hypophysectomy of the adult rat, which
typical change exhibiting extensive neurite out-
abolishes pituitary ACTH secretion and reduces
growth. Glucocorticoid administration is able to
adrenal corticosterone output, dramatically decreases
both adrenaline content and PNMT activity within block or delay NGF-induced neurite outgrowth.
the adrenal gland. Administration of corticosterone Long-term culture of rat adrenal chromaffin cells in
or the potent synthetic glucocorticoid dexamethasone the presence of NGF and the absence of glucocorti-
reverses the fall in PNMT activity after hypophysect- coids results in a complete transition to a sympathetic
omy. Glucocorticoids are also found to inhibit the neuron phenotype as assessed by a number of
enhanced degradation of the PNMT enzyme that morphological and biochemical criteria. Hence,
occurs as a consequence of hypophysectomy, by glucocorticoids appear to act as a survival factor for
stabilization of the enzyme co-substrate, S-adenosyl- chromaffin cells and repress the neuronal transdiffer-
methionine. A number of studies have found that entiation of these cells. The levels of corticosterone
glucocorticoid administration to hypophysectomized present in the fetal rat adrenal gland at the time of
rats also increases PNMT mRNA expression due to invasion by the SA progenitor cells would be more
increased transcription of the PNMT gene. Analysis than sufficient to suppress neural transdifferentiation
of the PNMT gene has revealed the presence of by these cells. It has therefore been considered that
consensus sequences for the glucocorticoid-response the sympathetic progenitor cells that migrate from the
element in the 50 upstream regulatory/promoter sympathetic ganglion primordium through the devel-
sequence. In primary cultures of isolated bovine oping adrenal anlage develop into chromaffin cells in
chromaffin cells that have been removed from any the adrenal medulla under the influence of the
cortical influence, dexamethasone is a powerful glucocorticoid-rich environment. Recent studies,
inducer of PNMT activity with an EC50 for PNMT however, have challenged this view as an analysis of
mRNA and activity induction of 1 –10 nM. Bovine mice carrying targeted mutations of the GR gene
chromaffin cells have been found to possess gluco- found that the mice lacking a GR gene product had
corticoid-binding receptors with a Kd of approxi- normal numbers of adrenal chromaffin cells. The GR
mately 1 nM, and binding of glucocorticoids to the mutant mice did, however, lack adrenomedullary
type II glucocorticoid receptors (GRs) present in PNMT. It therefore appears that the role of gluco-
chromaffin cells results in dimerization and transport corticoids in development is to modulate directly
into the nucleus, where the complex interacts with or indirectly the expression of PNMT, but that
46 ADRENAL CORTEX ROLE IN MEDULLARY SYNTHESIS OF PNMT

the expression of PNMT itself does not determine the Glossary


chromaffin phenotype.
There are also important interactions between the chromaffin cell The functional cell of the adrenal medulla;
it contains catecholamine-storing granules, with the
adrenal cortex and the adrenal medulla in those
cytoplasm giving a positive staining reaction with
species, such as sheep and human, that have a chromic acid due to the oxidation of the catechol-
prolonged gestation period and in which there are amines.
intact neuroendocrine responses to intrauterine dopamine b-hydroxylase The only enzyme in the catechol-
stressors present before birth. Classical studies in the amine biosynthetic pathway located within the
sheep fetus have shown that adrenaline content in the catecholamine-containing granules of the chromaffin
adrenal gland increased markedly during the 10 – 15 cell, where it catalyzes the conversion of dopamine to
days preceding parturition coincident with the pre- noradrenaline.
partum surge in adrenal cortisol output. Intrafetal glucocorticoid receptors Proteins that are present in
administration of adrenaline to the sheep halts lung chromaffin cells and which, after binding with the
liquid secretion and stimulates lung liquid absorption glucocorticoids, dimerize and are transported to the
nucleus of the cell, where the complex interacts with
by a b-adrenergic-dependent mechanism. Stimulation
the glucocorticoid-response elements on the phenyl-
of b2-adrenoreceptors in the fetal sheep and fetal ethanolamine N-methyltransferase gene to stimulate
rabbit lung with adrenaline and specific b2-adreno- transcription.
receptor agonists also elicits an increase in pulmonary phenylethanolamine N-methyltransferase An enzyme that
phospholipid synthesis and also the levels of phos- catalyzes the final step in the catecholamine biosyn-
pholipids in lung lavage fluid. Catecholamines, in thetic pathway, i.e., the N-methylation of noradrenaline
particular adrenaline, via b2-adrenoreceptor stimu- to result in the synthesis of adrenaline.
lation are able to stimulate an increase in pulmonary tyrosine hydroxylase The enzyme that catalyzes the oxi-
surfactant secretion and synthesis. The well-estab- dation of tyrosine to dihydroxyphenylalanine; it is the
lished actions of glucocorticoids on surfactant syn- rate-limiting enzyme in the catecholamine synthetic
pathway in the adrenal medulla.
thesis in the lungs are therefore enhanced by the
concomitant actions of the catecholamines. New-
borns that are delivered by caesarean section do not See Also the Following Articles
experience the substantial catecholamine surge that Adrenocorticosteroids and Cancer . Adrenocorticotropic
occurs in babies that are vaginally delivered and have Hormone (ACTH) and Other Proopiomelanocortin
a lower lung compliance after delivery. Peptides . Glucocorticoid Effects on Physiology and Gene
Expression . Mineralocorticoid Biosynthesis
. Mineralocorticoid Effects on Physiology and Gene
Expression
VI. SUMMARY
The functional interactions between the neighboring Further Reading
cells of the adrenal medulla and adrenal cortex are Anderson, D. J. (1993). Molecular control of cell fate in the neural
initiated early in development and are maintained by crest: The sympathoadrenal lineage. Annu. Rev. Neurosci. 16,
the paracrine and endocrine actions of the adreno- 129 –158.
cortical glucocorticoids on PNMT and hence adrena- Finotto, S., Krieglstein, K., Schober, A., Delming, F., Lindner, K.,
line synthesis in the adrenomedullary chromaffin Bruhl, B., Beier, K., Metz, J., Garcia-Arraras, J. E., Roig-Lopez,
J. L., Monaghan, P., Schmid, W., Cole, T. J., Kellendonk, C.,
cells. The array of strategies that exist across a Tronche, F., Schitz, G., and Unsicker, K. (1999). Analysis of
range of species to ensure that adrenomedullary cells mice carrying target mutations of the glucocorticoid receptor
that synthesize adrenaline are in close contact with gene argues against an essential role for glucocorticoid
the adrenocortical cells highlight the importance of a signalling for generating adrenal chromaffin cells. Develop-
coordinated adrenocortical – medullary response to a ment 126, 2935–2944.
Hodel, A. (2001). Effects of glucocorticoids on adrenal chromaffin
range of acute and chronic stressors from before birth cells. J. Neuroendocrinol. 13, 217–221.
and into adult life. The chromaffin cells are therefore Schober, A., Krieglstein, K., and Unsicker, K. (2000). Molecular
well placed to integrate both neurogenic and hormo- cues for the development of adrenal chromaffin cells and their
nal stimuli to ensure that adrenaline and the preganglionic innervation. Eur. J. Clin. Invest. 30, 87–90.
glucocorticoids act in a tightly regulated partnership Ungar, A., and Phillips, J. H. (1983). Regulation of the adrenal
medulla. Physiol. Rev. 63, 787–843.
to enhance the metabolic, cardiovascular, and respi- Unsicker, K. (1993). The chromaffin cell: Paradigm in cell,
ratory responses to physiological challenges that developmental and growth factor biology. J. Anat. 183,
threaten homeostasis. 207 –221.
ADRENOCORTICOSTEROIDS AND CANCER 47

Wong, D. L., Bildstein, C. L., Siddall, B., Lesage, A., and Yoo, Y. S. corticotropic hormone (ACTH, or corticotropin) or
(1993). Neural regulation of phenylethanolamine N-methyl- sex hormones. Children less than 6 years of age
transferase in vivo: Transcriptional and translational changes.
Mol. Brain Res. 18, 107– 114.
can present with evidence of virilization, precocious
Wong, D. L., Lesage, A., Siddall, B., and Funder, J. W. (1992). puberty, or Cushing’s syndrome. Adults (typically
Glucocorticoid regulation of phenylethanolamine N-methyl- between the ages of 40 and 50) can also present with
transferase in vivo. FASEB J. 6, 3310–3315. these tumors, as evidenced by hormonal syndromes
Wong, D. L., Siddall, B., and Wang, W. (1995). Hormonal control (feminization, virilization, hypercortisolism, or hyper-
of rat adrenal phenylethanolamine N-methyltransferase
enzyme activity, the final critical pathway. Neuropsychophar-
aldosteronism). Surgery is curative if total resection is
macology 13, 223 –234. possible, though 70% of patients present with positive
lymph nodes or distant metastasis and therefore
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved. require chemotherapy.

II. PARANEOPLASTIC SYNDROMES


Adrenocorticosteroids It should be noted that tumors outside the adrenal
and Cancer axis sometimes secrete adrenal corticosteroids. The
classic tumors with ectopic endocrine production
are some small-cell cancers of the lung and pituitary
THOMAS J. DILLING AND W. GILLIES MC KENNA
adenomas, though other less frequent tumors appear
University of Pennsylvania on this list as well. Pituitary ACTH overproduction
(also known as Cushing’s disease) is the most common
I. ADRENOCORTICAL TUMORS syndrome, occurring in 55 to 82% of patients, and
II. PARANEOPLASTIC SYNDROMES
adrenal dysfunction occurs in 5 to 32% of patients,
III. MANAGEMENT OF TUMOR SEQUELAE
ectopic ACTH production in 11 to 25% of patients,
IV. TREATMENT OF CANCER
V. MANAGEMENT OF SEQUELAE FROM CANCER and CRH overproduction in approximately 1% of
TREATMENT patients. The resultant paraneoplastic syndromes are
numerous. Cushing’s initial description of the periph-
eral effects of hyperfunctioning pituitary adenoma
Adrenocorticosteroids play several distinct roles included truncal obesity, purple striae, hypertension,
for the clinical oncologist. Rarely, patients fatigue, moon facies, buffalo hump, weakness,
may have a primary tumor of the adrenal gland
depression, amenorrhea, hirsutism, and edema. Com-
itself, leading to a clinical excess (or dearth)
of adrenocortical hormones. Sometimes, too, plete treatment of this vast topic is beyond the scope of
tumors outside the adrenal axis can produce this article, though the reader is encouraged to consult
these hormones, causing well-described clini- the citations under Further Reading for additional
cal syndromes. Expansile tumors in the brain information.
or spinal column are an oncologic emergency,
managed in part with adrenocorticosteroids.
Sometimes steroids are also used as a primary III. MANAGEMENT OF TUMOR SEQUELAE
chemotherapeutic agent in the treatment of the Two dreaded complications of malignancy include
cancer itself. Finally, despite the oncologist’s spinal cord compression and life-threatening brain
best efforts, patients sometimes experience side edema. In the former condition, a metastatic clone of
effects from chemotherapy and/or radiation,
malignant cells grows and expands within the spinal
particularly in the lung; these side effects can
effectively be managed by adrenocorticosteroids column, eventually compressing the spinal cord to the
in many cases. point that permanent paralysis can quickly ensue if
not treated urgently. Likewise, primary brain tumors
(or lesions metastatic to the brain) can expand within
the fixed volume of the skull and cause life-threaten-
I. ADRENOCORTICAL TUMORS
ing edema, leading to gross alteration of personality,
Adrenal cortical carcinoma is a very rare tumor, seizures, or death.
comprising 0.05 to 0.20% of all cancers. It may be Adrenocorticosteroids have a potent anti-inflam-
either functional or nonfunctional in terms of hor- matory activity that plays an important role in the
mone production. When functional, the carcinomas management of these conditions. Dexamethasone is
may secrete excessive amounts of either adreno- commonly prescribed in doses ranging from 16 to
ADRENOCORTICOSTEROIDS AND CANCER 47

Wong, D. L., Bildstein, C. L., Siddall, B., Lesage, A., and Yoo, Y. S. corticotropic hormone (ACTH, or corticotropin) or
(1993). Neural regulation of phenylethanolamine N-methyl- sex hormones. Children less than 6 years of age
transferase in vivo: Transcriptional and translational changes.
Mol. Brain Res. 18, 107– 114.
can present with evidence of virilization, precocious
Wong, D. L., Lesage, A., Siddall, B., and Funder, J. W. (1992). puberty, or Cushing’s syndrome. Adults (typically
Glucocorticoid regulation of phenylethanolamine N-methyl- between the ages of 40 and 50) can also present with
transferase in vivo. FASEB J. 6, 3310–3315. these tumors, as evidenced by hormonal syndromes
Wong, D. L., Siddall, B., and Wang, W. (1995). Hormonal control (feminization, virilization, hypercortisolism, or hyper-
of rat adrenal phenylethanolamine N-methyltransferase
enzyme activity, the final critical pathway. Neuropsychophar-
aldosteronism). Surgery is curative if total resection is
macology 13, 223 –234. possible, though 70% of patients present with positive
lymph nodes or distant metastasis and therefore
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved. require chemotherapy.

II. PARANEOPLASTIC SYNDROMES


Adrenocorticosteroids It should be noted that tumors outside the adrenal
and Cancer axis sometimes secrete adrenal corticosteroids. The
classic tumors with ectopic endocrine production
are some small-cell cancers of the lung and pituitary
THOMAS J. DILLING AND W. GILLIES MC KENNA
adenomas, though other less frequent tumors appear
University of Pennsylvania on this list as well. Pituitary ACTH overproduction
(also known as Cushing’s disease) is the most common
I. ADRENOCORTICAL TUMORS syndrome, occurring in 55 to 82% of patients, and
II. PARANEOPLASTIC SYNDROMES
adrenal dysfunction occurs in 5 to 32% of patients,
III. MANAGEMENT OF TUMOR SEQUELAE
ectopic ACTH production in 11 to 25% of patients,
IV. TREATMENT OF CANCER
V. MANAGEMENT OF SEQUELAE FROM CANCER and CRH overproduction in approximately 1% of
TREATMENT patients. The resultant paraneoplastic syndromes are
numerous. Cushing’s initial description of the periph-
eral effects of hyperfunctioning pituitary adenoma
Adrenocorticosteroids play several distinct roles included truncal obesity, purple striae, hypertension,
for the clinical oncologist. Rarely, patients fatigue, moon facies, buffalo hump, weakness,
may have a primary tumor of the adrenal gland
depression, amenorrhea, hirsutism, and edema. Com-
itself, leading to a clinical excess (or dearth)
of adrenocortical hormones. Sometimes, too, plete treatment of this vast topic is beyond the scope of
tumors outside the adrenal axis can produce this article, though the reader is encouraged to consult
these hormones, causing well-described clini- the citations under Further Reading for additional
cal syndromes. Expansile tumors in the brain information.
or spinal column are an oncologic emergency,
managed in part with adrenocorticosteroids.
Sometimes steroids are also used as a primary III. MANAGEMENT OF TUMOR SEQUELAE
chemotherapeutic agent in the treatment of the Two dreaded complications of malignancy include
cancer itself. Finally, despite the oncologist’s spinal cord compression and life-threatening brain
best efforts, patients sometimes experience side edema. In the former condition, a metastatic clone of
effects from chemotherapy and/or radiation,
malignant cells grows and expands within the spinal
particularly in the lung; these side effects can
effectively be managed by adrenocorticosteroids column, eventually compressing the spinal cord to the
in many cases. point that permanent paralysis can quickly ensue if
not treated urgently. Likewise, primary brain tumors
(or lesions metastatic to the brain) can expand within
the fixed volume of the skull and cause life-threaten-
I. ADRENOCORTICAL TUMORS
ing edema, leading to gross alteration of personality,
Adrenal cortical carcinoma is a very rare tumor, seizures, or death.
comprising 0.05 to 0.20% of all cancers. It may be Adrenocorticosteroids have a potent anti-inflam-
either functional or nonfunctional in terms of hor- matory activity that plays an important role in the
mone production. When functional, the carcinomas management of these conditions. Dexamethasone is
may secrete excessive amounts of either adreno- commonly prescribed in doses ranging from 16 to
48 ADRENOCORTICOSTEROIDS AND CANCER

100 mg/day, tapering over time. Studies on spinal have improved the efficacy of these treatments while
cord compression have failed to demonstrate a dis- simultaneously diminishing their side effects on the
tinct advantage for the higher doses, though patients patient. Occasionally, however, despite physicians’
do appear to experience diminished pain after 24 h on great determination, patients do experience problems
the higher doses. Lower doses are usually used to related to the cancer therapy itself. While the specifics
treat brain edema. In any case, radiation therapy or of all these potential problems are far beyond the
surgical decompression is then performed urgently to scope of this article, one requires mention here. The
debulk the tumor. lung is a relatively sensitive organ, and the tumors
that occupy it can be quite resistant to treatment,
requiring high doses of radiation or chemotherapy in
IV. TREATMENT OF CANCER an attempt to achieve patient cure. Clinicians, there-
fore, must balance these opposing problems by
In 1943, a corticosteroid (the so-called Compound E)
examining the therapeutic ratio—the ability to
was reported to cause atrophy of lymphatic tissue in
sterilize the tumor while sparing normal tissues.
mice. Cortisone acetate and adrenocorticotropic
Chemotherapy and radiation sometimes cause a
hormone became clinically available in 1948. In
hypersensitivity-type reaction called radiation pneu-
November 1949, a group of researchers published a
monitis. Chemotherapy has been shown to be a
report in which they noted cancer regression in a pair
radiation sensitizer in the lung, which is important to
of patients with different types of lymphoma. They
remember in this era of combination chemotherapy
stated that enlargement of lymph nodes occurred
and radiation. Approximately 43% of patients
approximately 6 weeks after ACTH was discontinued
demonstrate radiologic changes within the lung
in the patient with Hodgkin’s disease, but found that
from chemoradiotherapy treatment. Interestingly,
there was no recurrence in the patient with lympho-
symptomatic radiation pneumonitis occurs in only
sarcoma 10 weeks after discontinuation of therapy. A
approximately 7% of patients. Patient variability
number of studies followed in the 1950s and 1960s,
seems to play a role. The volume of lung actually
confirming the earlier results regarding the impor-
being irradiated, which can vary greatly depending
tance of prednisone in treating lymphoma.
upon the exact disease process, also determines
Although individual compounds were shown to
whether a patient will experience this problem. It
be helpful, they did not always completely cure
has also been shown that prior irradiation or
patients of these lymphomas or produce lasting
chemotherapy can predispose a patient to radiation
remission. In 1968, Vincent DeVita and co-workers
pneumonitis. Preexisting lung disease has also been
reported their landmark study, first published in
implicated. Likewise, it has been shown that sub-
1970, in which they combined four different chemo-
clinical radiation pneumonitis can become sympto-
therapeutics that had previously demonstrated indi-
matic after steroid withdrawal.
vidual activity against Hodgkin’s disease. They
Clinically, the syndrome generally manifests itself
combined cyclophosphamide, vincristine, procarba-
within about 2 to 3 months after the completion of
zine, and prednisone to create the MOPP chemo-
therapy. Occasionally it presents as quickly as 1
therapy regime. When tested on 43 patients, it
month or as late as 6 months after the completion of
induced complete remission in 35, 17 of whom had
treatment. Patients present with shortness of breath,
lasting remission—a vast improvement over any
which can vary from mild to severe. Cough is also
previously published results. In the years since,
typically prominent in about half of these patients.
prednisone has also been shown to have activity
Fever is also sometimes seen. The symptoms can be
against chronic leukemia and non-Hodgkin’s lym-
relatively minor if the area of lung treated is small. On
phoma. In addition, different combinations (most
the other hand, the clinical course can be fulminant,
notably CHOP, which also contains prednisone) have
leading to respiratory insufficiency and cyanosis,
been tested and have become standards in the
progressing to cor pulmonale (right heart failure) in
treatment of some lymphatic/leukemic cancers.
a matter of days.
The histopathology and cellular biology of this
V. MANAGEMENT OF SEQUELAE FROM disease process are fairly well elucidated. At 0 to 2
months after radiation, injury to small vessels and
CANCER TREATMENT
capillaries can be seen. This leads to vascular con-
In the years since chemotherapy and radiation gestion and increased capillary permeability. As a
therapy were introduced, physicians and scientists result, proteinaceous material is deposited within
ADRENOCORTICOTROPIC HORMONE AND OTHER PROOPIOMELANOCORTIN PEPTIDES 49

the alveoli of the lungs. In the range of 2 to 9 months DeVita, V. T., Jr, Serpick, A. A., and Carbone, P. P. (1970).
postradiation, one can note microscopic evidence of Combination chemotherapy in the treatment of advanced
Hodgkin’s disease. Ann. Int. Med. 73, 881–895.
obstruction of pulmonary capillaries. In addition, one
Fuller, B. G., Heiss, J. D., and Oldfield, E. H. (2001). Spinal cord
sees hyperplasia of the vascular endothelium, and the compression. In “Cancer: Principles and Practice of Oncol-
walls of the alveoli become infiltrated with fibroblasts. ogy” (V. T. DeVita, Jr., S. Hellman, and S. A. Rosenberg, eds.),
If the radiation injury is mild, these changes may pp. 2617–2633. Lippincott Williams & Wilkins, Philadelphia.
subside. However, if the injury is severe enough, it can Kofman, S., Perlia, C. P., Boeson, Eistenstein, R., and Taylor, S. G.
(1961). The role of corticosteroids in the treatment of
progress to a chronic stage.
malignant lymphomas. Cancer 15, 338– 345.
Corticosteroid administration dramatically Levin, V. A., Leibel, S. A., and Gutin, P. H. (2001). Neoplasms of
improves the physiologic changes in mice and the central nervous system. In “Cancer: Principles and Practice
decreases their mortality. When given prophylacti- of Oncology” (V. T. DeVita, Jr., S. Hellman, and S. A.
cally, the steroids failed to prevent radiation pneumo- Rosenberg, eds.), pp. 2100– 2160. Lippincott Williams &
nitis, but when given at the first sign of clinical Wilkins, Philadelphia.
Lowenthal, R. M., and Jestrimski, K. W. (1986). Corticosteroid
symptoms, they produced a clinical response. Other drugs: Their role in oncological practice. Med. J. Austr. 144,
reports, however, failed to show amelioration of 81–85.
severe radiation pneumonitis with steroid adminis- Movsas, B., Raffin, T. A., Epstein, A. H., and Link, C. J., Jr (1997).
tration. No controlled clinical trials have been Pulmonary radiation injury. Chest 111, 1061– 1076.
conducted, however, to prove their efficacy in humans. Norton, J. A., and Le, H. N. (2001). Adrenal tumors. In “Cancer:
Principles and Practice of Oncology” (V. T. DeVita, Jr., S.
Despite this fact, they are commonly given when the
Hellman, and S. A. Rosenberg, eds.), pp. 1770 – 1788.
diagnosis is reasonably ascertained, at a dose of Lippincott Williams & Wilkins, Philadelphia.
1 mg/kg. This dose is maintained for several weeks Rubin, P., and Casarett, G. W. (1968). “Clinical Radiation
and then slowly weaned. Pathology,” Vol. 1, pp. 423 – 470. W. B. Saunders Co.,
Philadelphia.

Glossary Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.

paraneoplastic syndromes Clinical syndromes experienced


by some cancer patients due to the production of hor-
mones or other substances by a tumor. The symptoms of
the syndrome depend upon the exact substance(s)
Adrenocorticotropic Hormone
produced. (ACTH) and Other
radiation pneumonitis Pathologically demonstrable series
of changes seen in the lung tissue of some patients after Proopiomelanocortin (POMC)
irradiation. The syndrome can range from subclinical to
highly toxic in severity and may be either self-limited or Peptides
chronic.
spinal cord compression Oncologic emergency in which a RICHARD G. ALLEN
clone of tumor cells expands within the spinal column, Oregon Health & Science University, Portland
leading to permanent paralysis if not treated urgently.
I. ACTH AND POMC PEPTIDES
See Also the Following Articles II. PHYSIOLOGY OF ACTH AND POMC-DERIVED PEPTIDES
III. REGULATION OF ACTH AND POMC-DERIVED PEPTIDES
Adrenal Cortex Role in Medulla Synthesis of PNMT IV. SUMMARY
. Adrenocorticotropic Hormone (ACTH) and Other
Proopiomelanocortin Peptides . Androgen Receptors and
Prostate Cancer . Apoptosis, Glucocorticoid-Induced Adrenocorticotropin (ACTH) is a 39-amino-acid
. Cancer Cells and Progrowth/Prosurvival Signaling peptide derived from the prohormone proopio-
. Estrogen and Progesterone Receptors in Breast Cancer melanocortin (POMC). ACTH stimulates adre-
. Glucocorticoid Effects on Physiology and Gene nocortical steroid synthesis in response to
Expression stress and circadian fluctuations. ACTH is
synthesized in corticotropes in the anterior
lobe of the pituitary gland and, to a much lesser
Further Reading extent, in melantropes of the intermediate
Abid, S. H., Malhotra, V., and Perry, M. C. (2001). Radiation- pituitary of many species with the exception of
induced and chemotherapy-induced pulmonary injury. Curr. humans. It was first thought that ACTH was
Opin. Oncol. 13, 242–248. exclusively an endocrine hormone; however,
ADRENOCORTICOTROPIC HORMONE AND OTHER PROOPIOMELANOCORTIN PEPTIDES 49

the alveoli of the lungs. In the range of 2 to 9 months DeVita, V. T., Jr, Serpick, A. A., and Carbone, P. P. (1970).
postradiation, one can note microscopic evidence of Combination chemotherapy in the treatment of advanced
Hodgkin’s disease. Ann. Int. Med. 73, 881–895.
obstruction of pulmonary capillaries. In addition, one
Fuller, B. G., Heiss, J. D., and Oldfield, E. H. (2001). Spinal cord
sees hyperplasia of the vascular endothelium, and the compression. In “Cancer: Principles and Practice of Oncol-
walls of the alveoli become infiltrated with fibroblasts. ogy” (V. T. DeVita, Jr., S. Hellman, and S. A. Rosenberg, eds.),
If the radiation injury is mild, these changes may pp. 2617–2633. Lippincott Williams & Wilkins, Philadelphia.
subside. However, if the injury is severe enough, it can Kofman, S., Perlia, C. P., Boeson, Eistenstein, R., and Taylor, S. G.
(1961). The role of corticosteroids in the treatment of
progress to a chronic stage.
malignant lymphomas. Cancer 15, 338– 345.
Corticosteroid administration dramatically Levin, V. A., Leibel, S. A., and Gutin, P. H. (2001). Neoplasms of
improves the physiologic changes in mice and the central nervous system. In “Cancer: Principles and Practice
decreases their mortality. When given prophylacti- of Oncology” (V. T. DeVita, Jr., S. Hellman, and S. A.
cally, the steroids failed to prevent radiation pneumo- Rosenberg, eds.), pp. 2100– 2160. Lippincott Williams &
nitis, but when given at the first sign of clinical Wilkins, Philadelphia.
Lowenthal, R. M., and Jestrimski, K. W. (1986). Corticosteroid
symptoms, they produced a clinical response. Other drugs: Their role in oncological practice. Med. J. Austr. 144,
reports, however, failed to show amelioration of 81–85.
severe radiation pneumonitis with steroid adminis- Movsas, B., Raffin, T. A., Epstein, A. H., and Link, C. J., Jr (1997).
tration. No controlled clinical trials have been Pulmonary radiation injury. Chest 111, 1061– 1076.
conducted, however, to prove their efficacy in humans. Norton, J. A., and Le, H. N. (2001). Adrenal tumors. In “Cancer:
Principles and Practice of Oncology” (V. T. DeVita, Jr., S.
Despite this fact, they are commonly given when the
Hellman, and S. A. Rosenberg, eds.), pp. 1770 – 1788.
diagnosis is reasonably ascertained, at a dose of Lippincott Williams & Wilkins, Philadelphia.
1 mg/kg. This dose is maintained for several weeks Rubin, P., and Casarett, G. W. (1968). “Clinical Radiation
and then slowly weaned. Pathology,” Vol. 1, pp. 423 – 470. W. B. Saunders Co.,
Philadelphia.

Glossary Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.

paraneoplastic syndromes Clinical syndromes experienced


by some cancer patients due to the production of hor-
mones or other substances by a tumor. The symptoms of
the syndrome depend upon the exact substance(s)
Adrenocorticotropic Hormone
produced. (ACTH) and Other
radiation pneumonitis Pathologically demonstrable series
of changes seen in the lung tissue of some patients after Proopiomelanocortin (POMC)
irradiation. The syndrome can range from subclinical to
highly toxic in severity and may be either self-limited or Peptides
chronic.
spinal cord compression Oncologic emergency in which a RICHARD G. ALLEN
clone of tumor cells expands within the spinal column, Oregon Health & Science University, Portland
leading to permanent paralysis if not treated urgently.
I. ACTH AND POMC PEPTIDES
See Also the Following Articles II. PHYSIOLOGY OF ACTH AND POMC-DERIVED PEPTIDES
III. REGULATION OF ACTH AND POMC-DERIVED PEPTIDES
Adrenal Cortex Role in Medulla Synthesis of PNMT IV. SUMMARY
. Adrenocorticotropic Hormone (ACTH) and Other
Proopiomelanocortin Peptides . Androgen Receptors and
Prostate Cancer . Apoptosis, Glucocorticoid-Induced Adrenocorticotropin (ACTH) is a 39-amino-acid
. Cancer Cells and Progrowth/Prosurvival Signaling peptide derived from the prohormone proopio-
. Estrogen and Progesterone Receptors in Breast Cancer melanocortin (POMC). ACTH stimulates adre-
. Glucocorticoid Effects on Physiology and Gene nocortical steroid synthesis in response to
Expression stress and circadian fluctuations. ACTH is
synthesized in corticotropes in the anterior
lobe of the pituitary gland and, to a much lesser
Further Reading extent, in melantropes of the intermediate
Abid, S. H., Malhotra, V., and Perry, M. C. (2001). Radiation- pituitary of many species with the exception of
induced and chemotherapy-induced pulmonary injury. Curr. humans. It was first thought that ACTH was
Opin. Oncol. 13, 242–248. exclusively an endocrine hormone; however,
50 ADRENOCORTICOTROPIC HORMONE AND OTHER PROOPIOMELANOCORTIN PEPTIDES

ACTH and its POMC relatives are expressed


abundantly in the nervous system. POMC is a
complex prohormone cleaved by specific pro-
hormone convertases. In addition to ACTH,
POMC contains the amino acid sequences of
b-lipotropic hormone (b-LPH), b-endorphin,
the melanocortins, corticotropin intermediate
lobe peptide, and joining peptide. POMC is also
highly expressed in neurons of the arcuate
nucleus of the hypothalamus and the nuclear
trachis solitacius. Even though the structure of
POMC is the same wherever it is expressed,
posttranslational cleavage of POMC can pro-
duce a different variety of biologically active
peptides in each cell.

FIGURE 1 Processing of human proopiomelanocortin


(POMC). The processing proceeds in stages, yielding a variety
I. ACTH AND POMC PEPTIDES of forms of secreted peptides; N-terminal peptide, adrenocorti-
The biologically active peptides derived from POMC cotropic hormone (ACTH), and b-lipotropin are the principal
circulating forms produced by the anterior pituitary cortico-
are the result of proteolytic processing by the
troph. In the rodent, approximately 40% of ACTH has a
prohormone convertases PC1/3 and PC2. Several posttranslational addition of a phosphate moiety. g-MSH,
such enzymes have been characterized and include the g-melanocyte-stimulating hormone; a-MSH, a-melanocyte-
PCs, as well as furan and PACE 4. These enzymes are stimulating hormone; b-LPH, b-lipotropin, CHO, carbo-
members of the family of kexin/subtilisin-like serine hydrate. In the CNS, ACTH is converted to a-MSH and CLIP.
proteases. PCs cleave at single or multiple basic
amino acid residues; however, as is the case with PC2,
an accessory protein may be required for activity. The exhibits the same lobe-specific POMC processing
basic amino acid sequence lysine, arginine (KR) is the found in rodents and not humans.
most often observed cleavage motif, but KK, RR, and In humans, each of the three principal products
RK will subserve this function as well. The PCs have of POMC contains a common tetrapeptide core
different affinities for these basic cleavage motifs and (His-Phe-Arg-Trp), which in turn is contained in the
thus generate different bioactivities from a common three melanocortin peptides, a-MSH, b-MSH, and
prohormone in a given cell type. g-MSH. In addition, ACTH itself contains the tetra-
Peptide processing studies established that the peptide core and has melanotropic (pigment-produc-
principal peptide derivatives of POMC in the anterior ing) activity. This tetrapeptide core is the recognition
lobe cells of humans are the N-terminal peptide, site for the ACTH and melanocortin receptors.
ACTH, and b-lipotropin (Fig. 1). The intermediate Previously, it was suggested that the hyperpigmenta-
lobe POMC cells, called melanotropes, are present in tion found in disorders of excess ACTH may be related
other mammals and in the human fetus, but largely to ACTH itself or to the other melanotropic deriv-
are vestigial in the human adult. The melanotropes atives of the POMC molecule. The melanotropic
produce additional products such as a-melanocyte- activities of these peptides, particularly g-MSH, have
stimulating hormone (a-MSH) and corticotropin- not been fully elucidated, and the circulating con-
like intermediate lobe peptide (CLIP) from ACTH, centrations of these melanotropin peptides have not
and b-endorphin from g-lipotropin. The smaller yet been determined simultaneously. Nonetheless,
POMC peptides are also produced in the human the absence of significant amounts of g-MSH, and
nervous system, with ACTH serving as a biosynthetic of b-MSH in humans, support ACTH as the likely
intermediate for the production of a-MSH. In candidate.
rodents, the biological activity of a-MSH is enhanced The cloning of five different melanocortin recep-
by a-N-acetylation; however, the acetylation state of tors (MC1 –5) began a new era in studying POMC
human a-MSHs is unclear at present. Human b-MSH peptides. All of the receptors for POMC peptides that
probably is not secreted as a distinct hormone but is a have been cloned belong to the seven-transmembrane
by-product of postsecretory proteolysis. It is also G-protein-coupled receptor superfamily. ACTH
interesting to note that the nonhuman primate binds to the adrenal ACTH receptor (MC2) with
ADRENOCORTICOTROPIC HORMONE AND OTHER PROOPIOMELANOCORTIN PEPTIDES 51

nanomolar affinity. It then activates adenylyl cyclase of circulation glucocorticoids. This occurs because
via Gas, increasing intracellular cAMP, activating stress increases neural activity in the CNS, stimulat-
protein kinase A, and thus resulting in the phos- ing the hypothalamic parvicellular neurons in the
phorylation of proteins. Activation of this pathway paraventricular nucleus to secrete corticotropin-
induces cortisol synthesis and secretion. releasing hormone (CRH) at a greater rate. If stress
The melanocortins (the MSHs) bind to MC1 and persists, the hypothalamic – pituitary – adrenal axis
MC3 –5 with nanomolar affinity to mediate a variety will function at a higher setpoint, maintaining higher
of physiological actions described below. All of the concentrations of ACTH and glucocorticoids, which
MC receptors stimulate cAMP production when may eventually have detrimental effects on the
activated by their respective ligands. In contrast, individual. The role of the intermediate lobe melano-
b-endorphin binds the m class of opioid receptors and trope in stress response is not clear. For instance, in all
inhibits cAMP production through the Gai/o class of nonhuman species studied, the majority of the
G-protein-coupled receptors. The receptor for b-LPH endorphins produced by the intermediate lobe are
has not been cloned. a-N-acetylated and thus do not bind the opioid
receptors. In lower organisms, circulating melano-
cortins may be involved in pigmentation responses to
stress.
II. PHYSIOLOGY OF ACTH AND POMC-
Whereas the first two decades of POMC research
DERIVED PEPTIDES
were heavily focused on endorphins, their expression
ACTH, released from the anterior pituitary cortico- in the pituitary, and the expression of these peptides
troph, is the major physiologic regulator of the in the CNS, recently there has been intense interest in
synthesis and secretion of glucocorticoids by the POMC peptides and feeding behavior. The cloning of
adrenal glands. Glucocorticoids act on many pro- the melanocortin and ACTH receptors contributed
cesses mainly by altering gene transcription and greatly to this interest as a-MSH had been implicated
protein synthesis in the target cells. In, addition, in food intake regulation for many years. The
glucocorticoids permit metabolic adaptation during melanocortins also have wide variety of physiological
fasting to prevent low blood sugar. They also play an effects including alterations in motor activity and
important role in the body response to physical and sexual behavior, analgesia, memory, nerve regener-
emotional stress. Other actions include their inhibi- ation, antipyretic actions, pigmentation, and groom-
tory effect on inflammation and regulation of ing behavior.
vascular responsiveness to norepinephrine. Though many studies have been performed in
ACTH rapidly stimulates steroidogenesis, which rodent models examining POMC peptides and
can result in a great rise in blood glucocorticoids obesity, only in the past 5 years has firm evidence
within seconds or minutes. To support this function, for POMC’s involvement in obesity emerged. Much
ACTH stimulates adrenal blood flow and the rate- of this information comes from genetic studies.
limiting step of glucocorticoid synthesis: the conver- A current hypothesis is that mutations in regulatory
sion of cholesterol to pregnenolone. It also exerts elements of the POMC gene decrease the levels of
several long-term tropic effects on adrenal cells that POMC expression in the brain, thus causing altera-
maintain the cellular machinery necessary to carry tions in energy metabolism.
out steroidogenesis. There have been a small number of children with
In addition to ACTH, b-lipotropin is cleaved from null mutations in the POMC gene resulting in
POMC in anterior pituitary corticotrophs. b-Lipo- undetectable levels of ACTH. The common pheno-
tropin has effects on lipid metabolism, but its type presented was red hair, adrenal insufficiency, and
physiologic function in humans has not yet been severe, early onset obesity. Furthermore, recessive
established. When stimulated, corticotrophs secrete and dominant mutations in the MC4 receptor
ACTH and b-lipotropin in a 1:1 ratio into the (MC4-R) gene have been seen in humans and these
bloodstream. mutations are proposed to cause 5% of childhood
The circulating levels of ACTH and glucocorti- obesity. These and other data suggest that obesity
coids are greatly influenced by stress. When an may be a genetic disease of the hypothalamus and
individual is exposed to a stressful situation, levels that POMC may be a major player.
of ACTH and glucocorticoids rise rapidly in the The POMC and neuropeptide Y systems are
blood. Stress stimulates the hypothalamic – pituitary– the most extensively studied regarding energy
adrenal axis regardless of the existing concentrations intake. The POMC system receives input detailing
52 ADRENOCORTICOTROPIC HORMONE AND OTHER PROOPIOMELANOCORTIN PEPTIDES

the nutritional status and energy storage via insulin


and leptin signaling mediated by receptors on POMC
neurons in the arcuate nucleus of the hypothalamus.
There is a complex interaction of food intake
inhibition by a-MSH and agouti-related protein at
the level MC4-R. CRH is the mediator of food
intake and environmental conditions. It inhibits food
intake and weight gain, activates the hypothalamic –
pituitary axis, and modulates energy storage in
adipose tissue. The link between the CRH system
and POMC neurons in the hypothalamus and other
brain areas remains to be completely defined.

III. REGULATION OF ACTH AND POMC-


DERIVED PEPTIDES
Pituitary ACTH secretion is under two primary
regulatory influences. There is a positive peptidergic
influence from the hypothalamus, mediated by CRH,
which varies diurnally and is subject to extreme
perturbation by stress. There also is a negative
feedback influence of glucocorticoids, acting both at
the pituitary level to inhibit the corticotrope and at
the hypothalamic level to inhibit CRH neurons.
FIGURE 2 Scheme for control of ACTH secretion. Ach,
Cortisol is the major physiologic glucocorticoid
acetylcholine; NE, norepinephrine; GABA, g-aminobutyric
inhibitor of ACTH secretion; there is no other acid; CRH, corticotropin-releasing hormone; AVP, arginine
important endogenous ACTH inhibitor in humans. vasopressin.
Figure 2 shows a scheme that depicts a variety of
neurotransmitter inputs to the hypothalamus that
have been postulated to regulate CRH release and synthesis and secretion. Glucocorticoids inhibit the
ultimately, the secretion of ACTH. Further, vasopres- release of CRH from the hypothalamus, which also
sin (AVP) and melatonin have been implicated in the decreases ACTH release. In addition, glucocorticoids
regulation of ACTH secretion. In particular, AVP and inhibit ACTH secretion by acting directly on the
melatonin released from the suprachiasmatic nucleus corticotroph to inhibit the action of CRH. As a result,
appear to be intimately involved in the circadian CRH becomes less effective at stimulating ACTH
rhythm of glucocorticoids. In addition, AVP released release. In species other than humans, the synthesis
from the neural lobe of the pituitary has long been and secretion of POMC peptides are tonically
thought to act synergistically with CRH to enhance inhibited by hypothalamic neurons releasing dopa-
ACTH secretion from the anterior lobe. mine. Intermediate lobe melanotropes do not express
In humans, ACTH has a diurnal rhythm, exhibit- the glucocorticoid receptor and therefore are not
ing the highest plasma levels at approximately 6 – 8 regulated by glucocorticoids.
AM and a nadir at approximately midnight. In rats, Cortisol and its synthetic analogues (e.g., pre-
this pattern is reversed, and both lighting and activity dnisone and dexamethasone) suppress ACTH
may alter this cycle. In humans, there is also a free- secretion. After the administration of a dose of a
running cycle of plasma cortisol that varies between glucocorticoid, ACTH secretion diminishes within
25 and 33 h. All of the other peptides derived from minutes (“fast feedback”) and, in a second phase
pituitary POMC demonstrate these cycles; however, (“delayed feedback”), is suppressed for hours or days,
due to differences in the half-lives of the peptides, the depending on the glucocorticoid used. It is unclear
plasma concentrations are not superimposable. whether the fast and delayed feedback actions are
The negative feedback effect of glucocorticoids on associated with different anatomic sites. It is likely,
ACTH secretion results from actions on both the however, that delayed feedback is an intracellular
hypothalamus and the corticotroph. Glucocorticoids event operating through changes in nuclear mechan-
act directly on the corticotrophs to decrease POMC isms, whereas fast feedback may be dependent on
ADRENOCORTICOTROPIC HORMONE AND OTHER PROOPIOMELANOCORTIN PEPTIDES 53

effects on the cell membrane mediated through other a-MSH, b-MSH, g-MSH a-, b-, and g-Melanocyte-
classes of receptors. stimulating hormones, collectively called the melano-
Although pituitary POMC regulation by circulat- cortins. These peptides are thought to stimulate
ing glucocorticoids is fairly well understood, much pigmentation and may be involved in appetite and
feeding behavior.
less is known about POMC peptide regulation in the
prohormone convertase A class of proteolytic enzymes
brain. In the rodent, a large cell group of POMC
responsible for cleaving proopioimelanocortin into
neurons is bilaterally distributed in the arcuate biologically active peptides.
nucleus of the hypothalamus and projects extensively prohormone A protein that is converted to biologically
to other regions of the brain. These projections pass active molecules via posttranslational processing by
through limbic structures, the diencephalon, and specific proteolytic enzymes or other modifications.
amygdala, as well as the thalamus and periaqueductal proopioimelanocortin A prohormone expressed in the
gray, areas associated with nociception and sensory neuroendocrine system.
integration. In addition, there are projections to the
brainstem, which are thought to be involved in the See Also the Following Articles
regulation of cardiovascular and respiratory systems.
Appetite Regulation, Neuronal Control . Corticotropin-
In the nucleus tractus solitarius, POMC neurons send
Releasing Hormone, Stress, and the Immune System
projections to the spinal cord and probably interact
. Endocrine Rhythms: Generation, Regulation, and
with local POMC circuits in the spinal cord itself. It Integration . Glucocorticoid Effects on Physiology and
appears that human central nervous system POMC Gene Expression . Melatonin in Humans . Neuropeptides
expression resembles that found in the rodent, thus and Control of Anterior Pituitary . Stress . Vasopressin
serving as a good model for future POMC peptide (AVP)
research.
Further Reading
IV. SUMMARY Allen, R. G., Hatfield, J. M., and Stack, J. (1988). Post-translational
processing of pro-opiomelanocortin (POMC)-derived peptides
POMC is a prohormone that gives rise to several during fetal monkey pituitary development. I. Adrenocorti-
biologically active peptides that are expressed pri- cotropin (ACTH) and a-melanotropins (a-MSHs). Dev. Biol.
marily in the pituitary and brain. ACTH, the 126, 156–163.
Allen, R. G., Herbert, E., Hinman, M., Shibuya, H., and Pert, C. B.
melanotropins, and endorphins are liberated by
(1978). Co-ordinate control of corticotrophin, a-lipotropin,
specific proteolytic enzymes called PCs. By expressing and b-endorphin release in mouse pituitary cell cultures. Proc.
the PCs in a cell-type-specific fashion, a variety of Natl. Acad. Sci. USA 75, 4972–4976.
POMC-derived products can be differentially Allen, R. G., Orwoll, E., Kendall, J. W., Herbert, E., and Paxton, H.
expressed. When combined with the tissue-specific (1980). The distribution of forms ACTH and b-endorphin in
normal, tumor, and autopsy human anterior pituitary tissue:
expression of POMC peptides and their cognate
Virtual absence of 13K ACTH. J. Clin. Endocrinol. Metab. 51,
receptors, an incredibly diverse array of physiological 376– 379.
responses can be produced. Since all of the peptides Berg, A. L., and Nilsson-Ehle, P. (1994). Direct effects of
derived from POMC are co-released from large corticotropin on plasma lipoprotein metabolism in man—
secretory granules, a myriad of biological activities Studies in vivo and in vitro. Metabolism 43, 90–97.
Eipper, B. A., and Mains, R. E. (1981). Further analysis of post-
are modulated simultaneously. In the case of POMC,
translational processing of beta-endorphin in rat intermediate
these are glucocorticoid production, food intake, pituitary. J. Biol. Chem. 256, 5689–5695.
stress responses, and modulation of pain perception. Farooqi, I. S., Yeo, G. S., Keogh, J. M., Aminian, S., Jebb, S. A.,
There is much to learn about how the modulation of Butler, G., Cheetham, T., and O’Rahilly, S. (2000). Dominant
these specific POMC-derived bioactivities evolved to and recessive inheritance of morbid obesity associated with
melanocortin 4 receptor deficiency. J. Clin. Invest. 106,
be regulated in a coordinated manner.
271– 279.
Keller-Wood, M. E., and Dallman, M. F. (1984). Corticosteroid
inhibition of ACTH secretion. Endocr. Rev. 5, 1– 24.
Glossary Krieger, D. T., Liotta, A. S., Brownstein, M. J., and Zimmerman,
E. A. (1980). ACTH, beta-lipotropin, and related peptides in
adrenocorticotropic hormone A peptide that elicits gluco-
brain, pituitary, and blood. Rec. Prog. Horm. Res. 36,
corticoid secretion from the adrenal cortex. 277– 344.
b-endorphin A peptide possessing analgesic properties Krude, H., Biebermann, H., Luck, W., Horn, R., Brabant, G., and
mediated through the opioid receptors. Gruters, A. (1998). Severe early-onset obesity, adrenal
b-LPH A peptide derived from proopioimelanocortin insufficiency and red hair pigmentation caused by POMC
containing g-MSH and b-endorphin. mutations in humans. Nat. Genet. 19, 155–157.
54 AMINO ACID AND NITRIC OXIDE CONTROL OF THE ANTERIOR PITUITARY

Marks, D. L., and Cone, R. D. (2001). Central melanocortins and hormones, whereas Glu neurons increase their
the regulation of weight during acute and chronic disease. Rec. secretion. Second-order NO neurons mostly
Prog. Horm. Res. 56, 359 –375. increase, but in one case decrease, anterior pitui-
Pacak, K., and Palkovits, M. (2001). Stressor specificity of central
tary tropic hormone secretion.
neuroendocrine responses: Implications for stress-related
disorders. Endocr. Rev. 22, 502 –548.
Robinson, S. W., Dinulescu, D. M., and Cone, R. D. (2000).
Genetic models of obesity and energy balance in the mouse. I. HYPOTHALAMIC CONTROL OF PITUITARY
Annu. Rev. Genet. 34, 687–745. GLAND FUNCTION
Rouille, Y., Duguay, S. J., Lund, K., Furuta, M., Gong, Q., Lipkind,
G., Oliva, A. A., Jr, Chan, S. J., and Steiner, D. F. (1995). A. Hypothalamic Releasing and Inhibiting
Proteolytic processing mechanisms in the biosynthesis of
neuroendocrine peptides: The subtilisin-like proprotein con-
Hormones
vertases. Front. Neuroendocrinol. 16, 322 –361. Neurons situated in the hypothalamus control
Taylor, A. L., and Fishman, L. M. (1988). Corticotropin-releasing
the synthesis and secretion of the hormones of the
hormone. N. Engl. J. Med. 319, 213– 222.
Wikberg, J. E., Muceniece, R., Mandrika, I., Prusis, P., Lindblom, anterior and posterior pituitary gland (Fig. 1). The
J., Post, C., and Skottner, A. (2000). New aspects on the posterior pituitary hormones, oxytocin and vasopres-
melanocortins and their receptors. Pharmacol. Res. 42, sin, are synthesized in the cell bodies of neurons
393–420. located in the hypothalamic supraoptic and paraven-
tricular nuclei. Axons of these neurons project to
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
terminals in the posterior lobe (PL), from which
oxytocin and vasopressin are released on depolari-
zation. In contrast, the anterior pituitary hormones
Amino Acid and Nitric Oxide are controlled by hypothalamic neurons, whose cell
bodies are located in arcuate nuclei (AN) and other
Control of the Anterior Pituitary
MICHAEL SELMANOFF
University of Maryland School of Medicine

I. HYPOTHALAMIC CONTROL OF PITUITARY GLAND


FUNCTION
II. HYPOTHALAMIC GABA NEURONS AND THE
NEUROENDOCRINE REGULATION OF LH SECRETION
III. HYPOTHALAMIC GLUTAMATE NEURONS AND THE
NEUROENDOCRINE REGULATION OF LH SECRETION
IV. HYPOTHALAMIC NITRIC OXIDE NEURONS AND THE
NEUROENDOCRINE REGULATION OF LH SECRETION
V. THE ROLE OF GABA, GLUTAMATE, AND NITRIC OXIDE
NEURONS IN THE NEUROENDOCRINE REGULATION OF
OTHER ANTERIOR PITUITARY TROPIC HORMONES

Hypothalamic neurosecretory neurons regulate


the secretion of the six anterior pituitary tropic
hormones. In turn, these first-order, final
common pathway neurons of the neuroendo-
crine system are immediately controlled by
FIGURE 1 Secretion of hypothalamic hormones. The hor-
second-order afferent neurons. Most of these mones of the posterior lobe (PL) are released into the general
hypothalamic afferent neurons utilize either the circulation from the endings of supraoptic and paraventricular
inhibitory neurotransmitter g-aminobutyric neurons, while hypophysiotropic hormones are secreted into
acid (GABA) or the excitatory neurotransmitter the hypophysial portal circulation from the endings of arcuate
glutamate (Glu). Second-order neurons also and other hypothalamic neurons. The hormones circulate in
regulate the neurosecretory neurons by elabo- the portal vessels to the anterior lobe (AL), where they control
rating the neuroactive gas nitric oxide (NO). the synthesis and secretion of the anterior pituitary tropic
Second-order GABA neurons uniformly decrease hormones. MB, Mamillary bodies; OC, optic chiasm. Modified
the secretion of the anterior pituitary tropic from Ganong (2001), with permission.
54 AMINO ACID AND NITRIC OXIDE CONTROL OF THE ANTERIOR PITUITARY

Marks, D. L., and Cone, R. D. (2001). Central melanocortins and hormones, whereas Glu neurons increase their
the regulation of weight during acute and chronic disease. Rec. secretion. Second-order NO neurons mostly
Prog. Horm. Res. 56, 359 –375. increase, but in one case decrease, anterior pitui-
Pacak, K., and Palkovits, M. (2001). Stressor specificity of central
tary tropic hormone secretion.
neuroendocrine responses: Implications for stress-related
disorders. Endocr. Rev. 22, 502 –548.
Robinson, S. W., Dinulescu, D. M., and Cone, R. D. (2000).
Genetic models of obesity and energy balance in the mouse. I. HYPOTHALAMIC CONTROL OF PITUITARY
Annu. Rev. Genet. 34, 687–745. GLAND FUNCTION
Rouille, Y., Duguay, S. J., Lund, K., Furuta, M., Gong, Q., Lipkind,
G., Oliva, A. A., Jr, Chan, S. J., and Steiner, D. F. (1995). A. Hypothalamic Releasing and Inhibiting
Proteolytic processing mechanisms in the biosynthesis of
neuroendocrine peptides: The subtilisin-like proprotein con-
Hormones
vertases. Front. Neuroendocrinol. 16, 322 –361. Neurons situated in the hypothalamus control
Taylor, A. L., and Fishman, L. M. (1988). Corticotropin-releasing
the synthesis and secretion of the hormones of the
hormone. N. Engl. J. Med. 319, 213– 222.
Wikberg, J. E., Muceniece, R., Mandrika, I., Prusis, P., Lindblom, anterior and posterior pituitary gland (Fig. 1). The
J., Post, C., and Skottner, A. (2000). New aspects on the posterior pituitary hormones, oxytocin and vasopres-
melanocortins and their receptors. Pharmacol. Res. 42, sin, are synthesized in the cell bodies of neurons
393–420. located in the hypothalamic supraoptic and paraven-
tricular nuclei. Axons of these neurons project to
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
terminals in the posterior lobe (PL), from which
oxytocin and vasopressin are released on depolari-
zation. In contrast, the anterior pituitary hormones
Amino Acid and Nitric Oxide are controlled by hypothalamic neurons, whose cell
bodies are located in arcuate nuclei (AN) and other
Control of the Anterior Pituitary
MICHAEL SELMANOFF
University of Maryland School of Medicine

I. HYPOTHALAMIC CONTROL OF PITUITARY GLAND


FUNCTION
II. HYPOTHALAMIC GABA NEURONS AND THE
NEUROENDOCRINE REGULATION OF LH SECRETION
III. HYPOTHALAMIC GLUTAMATE NEURONS AND THE
NEUROENDOCRINE REGULATION OF LH SECRETION
IV. HYPOTHALAMIC NITRIC OXIDE NEURONS AND THE
NEUROENDOCRINE REGULATION OF LH SECRETION
V. THE ROLE OF GABA, GLUTAMATE, AND NITRIC OXIDE
NEURONS IN THE NEUROENDOCRINE REGULATION OF
OTHER ANTERIOR PITUITARY TROPIC HORMONES

Hypothalamic neurosecretory neurons regulate


the secretion of the six anterior pituitary tropic
hormones. In turn, these first-order, final
common pathway neurons of the neuroendo-
crine system are immediately controlled by
FIGURE 1 Secretion of hypothalamic hormones. The hor-
second-order afferent neurons. Most of these mones of the posterior lobe (PL) are released into the general
hypothalamic afferent neurons utilize either the circulation from the endings of supraoptic and paraventricular
inhibitory neurotransmitter g-aminobutyric neurons, while hypophysiotropic hormones are secreted into
acid (GABA) or the excitatory neurotransmitter the hypophysial portal circulation from the endings of arcuate
glutamate (Glu). Second-order neurons also and other hypothalamic neurons. The hormones circulate in
regulate the neurosecretory neurons by elabo- the portal vessels to the anterior lobe (AL), where they control
rating the neuroactive gas nitric oxide (NO). the synthesis and secretion of the anterior pituitary tropic
Second-order GABA neurons uniformly decrease hormones. MB, Mamillary bodies; OC, optic chiasm. Modified
the secretion of the anterior pituitary tropic from Ganong (2001), with permission.
AMINO ACID AND NITRIC OXIDE CONTROL OF THE ANTERIOR PITUITARY 55

hypothalamic nuclei, and whose axons project to tropic hormone-secreting neurons by second-order,
terminals situated in the median eminence (ME). On afferent neurons, which synapse on them and increase
depolarization, these neurons release their hypotha- or decrease their firing rate in response to physio-
lamic releasing and inhibiting hormones into the logical stimuli, is an intensely active area of neuro-
hypophysial portal circulation, which carries them to endocrine research. It is now clear that some of these
the anterior lobe (AL). There the hormones bind to afferents are neurons utilizing inhibitory (GABA,
specific high-affinity, low-capacity receptors on the glycine, taurine, b-alanine) and excitatory (Glu,
AL cells, which synthesize and secrete the anterior aspartate, cysteic acid, homocysteic acid) amino
pituitary tropic hormones. Although there is some acid neurotransmitters, and neurons that elaborate
evidence that GABA, Glu, and NO affect anterior neuroactive gases (NO, carbon monoxide). Most
pituitary tropic hormone secretion by direct actions workers in this area to date have studied the principal
at the level of the pituitary, at the present time most of inhibitory and excitatory amino acid and neuroactive
this evidence is not compelling; hence, further gas neurotransmitters: GABA, Glu, and NO. Elec-
discussion here is restricted to the hypothalamic trophysiological and whole-cell patch-clamp work by
actions of these neurotransmitters. van den Pol and co-workers indicates that almost all
fast synaptic activity in the hypothalamus is gener-
B. Tropic Hormones of the Anterior ated by the release of the inhibitory transmitter
GABA or the excitatory transmitter Glu. Hence, this
Pituitary Gland
article focuses on three systems of neuronal afferents,
Six anterior pituitary tropic hormones (Fig. 2) control GABA, Glu, and NO neurons, which likely affect the
the functioning of the adrenal glands [adrenocortico- activity of all of the hypophysiotropic hormone-
tropic hormone (ACTH)], the thyroid gland [thyroid- secreting neurons. To date, the majority of the
stimulating hormone (TSH)], the mammary glands research in this area has focused on the regulation
[prolactin (PRL)], and the gonads [luteinizing hor- of the GnRH neurons by afferent GABA, Glu, and
mone and follicle-stimulating hormone (LH and NO neurons. Evidence for the regulation of GnRH is
FSH)] and exert widespread effects on body growth presented here in some detail; the effect of these
[growth hormone (GH)]. These AL tropic hormones afferent neurons on the neuroendocrine control of the
are regulated by the hypothalamic releasing and other anterior pituitary hormones is limited to a
inhibiting hormones corticotropin-releasing hormone summary table.
(CRH), thyrotropin-releasing hormone (TRH), pro-
lactin-inhibiting hormone (PIH or dopamine), pro-
C. The Hypothalamic GnRH Pulse Generator
lactin-releasing factor (PRF), gonadotropin-releasing
hormone (GnRH), growth hormone-releasing hor- GnRH is released in episodic bursts that produce the
mone (GHRH), and somatostatin (SS or growth pulses of LH observed in male and female mammals,
hormone-inhibiting hormone). The cell bodies of and in other vertebrate species as well. In humans, the
these neurons reside in several hypothalamic struc- LH pulses in men occur at a frequency of one every
tures (Fig. 3). The regulation of these hypophysio- 90 min; the LH pulse frequency in women ranges

FIGURE 2 The hypothalamic hypophysiotropic hormones that control the secretion of the anterior pituitary tropic
hormones. CRH, Corticotropin-releasing hormone; ACTH, adrenocorticotropic hormone; TRH, thyrotropin-releasing
hormone; TSH, thyroid-stimulating hormone; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; FSH,
follicle-stimulating hormone; GHRH, growth hormone-releasing hormone; SS, somatostatin (growth hormone-
inhibiting hormone); GH, growth hormone; PIH, prolactin-inhibiting hormone (dopamine); PRF, prolactin-releasing
factor (unidentified); PRL, prolactin. Modified from Ganong (2001), with permission.
56 AMINO ACID AND NITRIC OXIDE CONTROL OF THE ANTERIOR PITUITARY

GnRH stimulation and for generating the other


hormonal events responsible for reproductive cycles
in females and normal testicular function in males.
The hypothalamic GnRH pulse generator may be
composed solely of the GnRH neurons, or may also
consist of additional GnRH afferent circuitry, which,
in conjunction with the GnRH neurons, generate the
oscillatory bursting patterns of the GnRH neurons.
Hence, the GABA, Glu, and NO neurons may indeed
be components of the GnRH pulse generator, or may
instead be significant modulators of GnRH pulse
generator activity by virtue of their being GnRH
neuronal afferents. Even relatively small changes in
GnRH pulse generator function are associated with
menstrual disorders in females and with infertility in
both sexes. Dramatic changes in GnRH pulse
generator function also are responsible for control-
ling the onset of puberty, the waning of reproductive
function associated with menopause in women, and,
to a lesser extent, andropause in men.
The activity of the GnRH pulse generator is
modulated by numerous physiological stimuli,
including circulating hormones. For example, the
sex steroid hormones—estradiol and progesterone in
females and testosterone in males—exert negative
and positive feedback effects on GnRH pulse gen-
erator function as part of homeostatic, physiological
regulatory mechanisms coordinating the integrated
functions of the hypothalamic – pituitary – gonadal
(i.e., the reproductive) axis. Another example is the
FIGURE 3 Location of the cell bodies of the hypophysio-
tropic hormone-secreting neurons projected on a ventral view
adipose cell hormone leptin, which exerts a positive
of the hypothalamus and pituitary of the rat. The names of the effect on the GnRH pulse generator in its role in
hormones are enclosed in the boxes. The magnocellular communicating the nutritional status of the individ-
oxytocin and vasopressin neurosecretory neurons that project ual to the neurons controlling reproductive function.
to the posterior lobe (PL) are indicated on the left side of the Hence, the presence of sex steroid or leptin receptors
figure. The parvicellular neurosecretory neurons that control on hypothalamic GABA, Glu, or NO neurons
anterior lobe (AL) tropic hormone secretion are indicated on
thought to be GnRH afferents, or documented effects
the right side of the figure. The dashed line from the median
eminence (ME) to the AL represents the hypophysial portal
of these hormones on these GnRH afferents, may
circulation. AN, Arcuate nucleus; BA, basilar artery; CRH, both be considered lines of evidence indicating that
corticotropin-releasing hormone; DA, dopamine; GnRH, specific populations of GABA, Glu, and NO neurons
gonadotropin-releasing hormone; GHRH, growth hormone- are physiologically significant GnRH afferents.
releasing hormone; IC, internal carotid artery; IL, intermediate
lobe; MC, middle cerebral artery; PC, posterior cerebral
artery; Peri, periventricular nucleus; PVN, paraventricular II. HYPOTHALAMIC GABA NEURONS AND
nucleus; SON, supraoptic nucleus; SS, somatostatin; TRH,
THE NEUROENDOCRINE REGULATION OF
thyrotropin-releasing hormone. Modified from Ganong
(2001), with permission. Courtesy of L.W. Swanson and LH SECRETION
E.T. Cunningham, Jr.
A. Hypothalamic GABA Neurons
from one every 90 min during the follicular phase of GABA, a four-carbon amino acid, was discovered in
the menstrual cycle to one every 3 h during the luteal brain tissue in 1950 by Eugene Roberts and co-
phase. The frequency and amplitude of the GnRH workers. GABA, produced by the enzymatic decar-
secretory episodes are essential for maintaining the boxylation of glutamic acid by the rate-limiting
sensitivity of anterior pituitary gonadotropes to enzyme, glutamic acid decarboxylase (GAD), is
AMINO ACID AND NITRIC OXIDE CONTROL OF THE ANTERIOR PITUITARY 57

catabolized primarily to succinic semialdehyde by the TIGA neurons are observed closely apposed to the
GABA-transaminase (GABA-T) (Fig. 4). Whereas GnRH terminals. Double-label immunohistochemis-
monoaminergic (dopamine, norepinephrine, epineph- try at the EM level indicates that rostral MPOA
rine, and serotonin) and cholinergic neurons consti- GABA neurons form axodendritic and axosomatic
tute relatively small populations of neurons, GABA is synapses with the GnRH neurons. Hence, GABA
thought to be the most ubiquitous transmitter in neurons may provide inhibitory input to the GnRH
brain. GABA neurons comprise 30 – 50% of all nerve neurons in either the MPOA or the ME, or both.
cell bodies, and perhaps 20% of the estimated 1015 Indeed, recent quantitative EM work in the primate
central nervous system (CNS) synapses utilize GABA AN indicates that inhibitory (not excitatory) synapses
as their transmitter. In the hypothalamus, morpho- comprise the dominant input to the GnRH neurons.
metric analysis at the electron microscopic (EM) level van den Pol and co-workers have provided compel-
indicates that about 50% of all synapses are ling electrophysiological evidence that almost all fast
GABAergic. Most nuclei of the hypothalamus have inhibition in the hypothalamus is caused by GABA
high concentrations of GAD and GABA and this release. Despite GABA being a dominant neurotrans-
brain region has heavy concentrations of GABA cell mitter in the hypothalamus, this very large popu-
bodies. Medial (compared with lateral) hypothalamic lation of hypothalamic neurons has been
structures in particular are among the most densely understudied by neuroendocrine investigators.
GABA innervated regions in brain. It is in these GABA was first convincingly shown to be an
medial structures that most hypothalamic releasing inhibitory transmitter in 1967, and subsequent
and inhibiting hormone cell bodies and their den- iontophoretic studies indicate that GABA inhibits
drites are located (Fig. 3). GABA cells make up virtually every neuron in the adult CNS. GABA is
50 – 90% of all cells in the rostral hypothalamus [the thought to be released by a depolarization-induced,
medial preoptic area (MPOA)], where the GnRH cell Ca2þ-dependent exocytotic process, although physio-
bodies are located, and 15 – 50% of all cells in the logically significant GABA release also may occur by
arcuate nucleus. A tuberoinfundibular system of reversal of the Naþ-dependent presynaptic GABA
GABA (TIGA) neurons exists with cell bodies in the transporter (GAT) (Fig. 4). At the level of the
AN and axons that project to GABA terminals in the membrane, activation of the postsynaptic GABAA
external layer of the ME. In the ME the terminals of receptor and its integrated Cl2 channel produces

FIGURE 4 Schematic representation of a GABA synapse. The a-ketoglutarate formed in the Krebs cycle is
transaminated to glutamate (Glu) by mitochondrial GABA-transaminase (GABA-T). The transmitter GABA is formed
from Glu by glutamic acid decarboxylase (GAD). Released GABA may bind to postsynaptic GABAA receptors or to
presynaptic GABAB autoreceptors, or may be taken up by high-affinity GABA transporters (GAT) present on neurons
and glia. GS, Glutamine synthetase; vGAT, vesicular GABA transporter. Modified from Zigmond et al. (1999), with
permission.
58 AMINO ACID AND NITRIC OXIDE CONTROL OF THE ANTERIOR PITUITARY

membrane hyperpolarization, whereas presynaptic slices indicates that MPOA GABA neurons may
GABAB receptors control synaptic transmission by mediate estrogen negative feedback by reducing the
inhibiting Ca 2þ influx and/or by affecting K þ autoinhibition of GABA neurons brought about by
channels, thus inhibiting transmitter release at the presynaptic, GABA B autoreceptor stimulation
nerve terminal (Fig. 4). Less is known about the (Fig. 4). A better understood oscillator in the
recently cloned G-protein-coupled GABAB receptor, thalamus and the hypothalamic rhythm generator in
which is structurally similar to the Glu metabotropic the suprachiasmatic nucleus both rely on GABA
receptors, and the putative GABAC receptor. The neurons for their function. A network oscillator
GABAA receptor, a heteropentameric protein, is more model for hypothalamic pulse generators consisting
correctly referred to as a receptor complex because it of Glu and GABA neurons would function more
has separate binding sites for GABA agonists and reliably than would a single-cell oscillator (i.e.,
antagonists, benzodiazepines, barbiturates, alcohol, composed solely of the GnRH neurons). Finally,
steroids, and certain convulsants. This variety of reflecting on the overall function and importance of
binding sites on the GABAA receptor makes it highly GABA neurons in brain, Eugene Roberts concludes
susceptible to modulation by physiological and that “disinhibition, coupled to variability gener-
pharmacological parameters. It is composed of ation… is the major organizing principle in nervous
combinations of at least 20 different subunits and system function.”
their functional splice variants: a1 – 6, b1 – 3, g1 and 3, More physiologically oriented work in intact
g2L and 2S, d, 1, u, p, and r1 – 3. animals indicates that GABA has an inhibitory action
on LH secretion. Administration of GABA or the
B. GABA Regulation of GnRH Pulse GABAA receptor agonist muscimol (MUS) into the
MPOA or intracerebroventricularly (icv) inhibits
Generator Function
pulsatile LH release, and the GABAA receptor
Several lines of evidence are consistent with the antagonist bicuculline (BIC) blocks this effect.
hypothesis that hypothalamic GABA neurons inhibit GABAA agonists and antagonists inhibit and stimu-
the GnRH pulse generator, and may indeed comprise late, respectively, GnRH pulse generator function as
pulse generator circuitry. First, GnRH neurons in situ assessed by multiunit activity recordings from the
express the GABAA a1, a5, b1, b3, and g2 receptor AN – ME region. Systemic administration of the
subunit mRNAs, demonstrating that GnRH neurons GABA-T (Fig. 4) inhibitor aminooxyacetic acid
are targets for the neurotransmitter GABA. Second, (AOAA) blocks GABA degradation, resulting in
evidence indicates that MPOA and AN GABA increased brain GABA levels. This GABA increase is
neurons express estrogen receptor (ER) and pro- associated with inhibition of pulsatile LH secretion.
gesterone receptor (PR) mRNA and protein. In the MUS and AOAA administration also block LH surge
MPOA, about 40% of all estrogen-receptive cells release and BIC prevents the GABAA agonist effect.
immunostain for GAD, indicating they are GABA Sex steroid removal down-regulates, and sex steroid
neurons. Third, in several tissues, including brain, administration up-regulates, several components of
one of the physiological actions of estrogen is to GABA transmission in MPOA and TIGA neurons:
induce the progesterone receptor. The TIGA neurons GAD mRNA levels and enzymatic activity, GAT-1
are reported to express such estrogen-inducible PR, as mRNA levels and transport activity, GABAA receptor
well as the leptin receptor. Indeed, it is reported that binding and the levels of receptor subunit mRNAs,
all ER immunoreactive cells in the hypothalamus, and the rate of GABA turnover. In females, MPOA
including the MPOA and AN, also express leptin GAD mRNA levels, the rate of GABA turnover, and
receptor protein. GnRH neurons studied in hypo- the extracellular GABA concentrations in microdia-
thalamic slices in vitro are directly inhibited by lysates all decrease before and during the LH surge.
applied GABA, and this effect is GABAA receptor Most recently, a group reported that grafting geneti-
mediated. This additional evidence demonstrates that cally engineered astrocytes that release GABA in the
the GABAA receptors expressed in the GnRH neurons ME near GnRH terminals disrupts estrous cyclicity in
are, indeed, physiologically functional. Similarly, rats; this finding demonstrates that GABA released in
GnRH neurons studied in hypothalamic slices are the vicinity of the ME can inhibit the GnRH pulse
generally silent, suggesting that they are tonically generator, raising the possibility that similar local
inhibited, but also exhibit episodes of burst firing, alterations in GABA neurotransmission may contrib-
which may underlie pulsatile GnRH release. Recent ute to the pathology of hypothalamic amenorrhea/
electrophysiological evidence from hypothalamic oligomenorrhea in humans.
AMINO ACID AND NITRIC OXIDE CONTROL OF THE ANTERIOR PITUITARY 59

III. HYPOTHALAMIC GLUTAMATE NEURONS types: kainate (KA), a-amino-3-hydroxy-5-methyl-4-


AND THE NEUROENDOCRINE REGULATION isoxazole propionate (AMPA), and N-methyl-D -
OF LH SECRETION aspartate (NMDA). KA and AMPA receptors permit
Naþ influx and Kþ efflux; NMDA receptors pass
A. Hypothalamic Glu Neurons relatively large amounts of Ca2þ. The ionotropic
Iontophoresis of Glu onto many different mammalian receptors are either pentamers or tetramers, and to
neurons rapidly depolarizes them. Perhaps 75% of date six NMDA (NR1, NR2A – D, and NR3A), five KA
excitatory transmission in the brain is attributable to (GluR5 – 7 and KA1 and 2), and four AMPA (GluR1 – 4)
Glu synapses, and van den Pol and co-workers have subunits have been identified and their genes have
published evidence confirming that Glu is the been cloned. The Glu concentration required for half-
principal excitatory neurotransmitter in the hypo- maximal (EC50) stimulation of AMPA receptors is
thalamus. Utilizing Glu-specific antibodies, immuno- 200 mM, whereas the EC50 for NMDA receptors is
histochemical work reveals relatively dense Glu nerve only 10 –15 mM. Hence, Glu is a much more potent
terminal immunostaining throughout most of the activator of NMDA receptors, compared to AMPA
hypothalamus. In neurons, Glu is thought to be receptors.
synthesized principally from glutamine by the mito- Eight metabotropic receptor subunits have been
chondrial enzyme phosphate-activated glutaminase described (mGluR1 – 8). These receptors are G-protein-
and also from aspartate by the cytoplasmic enzyme coupled serpentine receptors that increase intracellu-
aspartate aminotransferase (Fig. 5). Presynaptic Glu lar inositol trisphosphate and diacylglycerol levels or
is sequestered in secretory vesicles at concentrations decrease intracellular cAMP levels. In addition to
exceeding 20 mM. When Glu nerve terminals are binding to these pre- and postsynaptic receptors,
depolarized, Glu is released in a Ca2þ-dependent released Glu may be taken up by neurons or
manner. surrounding glia via the Glu transporters: excitatory
Two kinds of Glu receptors are recognized: amino acid carrier-1 (EAAC1), glutamate transpor-
ionotropic and metabotropic. The ionotropic recep- ter-1 (GLT-1), or glutamate –aspartate transporter
tors are ligand-gated cation channels and are of three (GLAST) (Fig. 5).

FIGURE 5 Schematic representation of a glutamate synapse. Glutamate (Glu) is synthesized from glutamine (Gln) by
the action of phosphate-activated glutaminase (PAG), and from aspartate (Asp) by the action of aspartate
aminotransferase (AAT). Glu is concentrated in secretory granules by a vesicular transporter. Released Glu can bind
to postsynaptic N-methyl-D -aspartate (NMDA), a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), or
kainate (KA) receptors or to presynaptic metabotropic autoreceptors (mGluR), or can be taken up by the high-affinity
Glu transporters, excitatory amino acid carrier-1 (EAAC1), glutamate transporter-1 (GLT-1), or glutamate-aspartate
transporter (GLAST), which are present on neurons and glia as indicated. Modified from Zigmond et al. (1999), with
permission.
60 AMINO ACID AND NITRIC OXIDE CONTROL OF THE ANTERIOR PITUITARY

Double-label immunohistochemistry at the EM administration of Glu and the Glu receptor agonists
level indicates that Glu-immunoreactive neurons NMDA, KA, and AMPA induce a rapid release in LH
synapse with GnRH dendrites and that ME Glu secretion, and Grattan and co-workers demonstrated
terminals are closely apposed to GnRH terminals, that intravenous NMDA directly stimulates GnRH
suggesting that asynaptic neurotransmission may release into anterior pituitary perfusates collected by
occur in this structure. Autoradiographic mapping push –pull cannulae. NMDA microinfusion specifi-
demonstrates a relatively dense distribution of Glu cally into the MPOA stimulates LH release. Pulsatile
receptors in the hypothalamus, particularly in the LH release is suppressed by NMDA and AMPA/KA
AN. More specifically, NMDA (NMDAR1 and receptor blockers. Microdialysis studies suggest that
NMDAR2A – D subunits), AMPA (GluR1 – 4 subunits), Glu release increases at the time of the LH surge in the
KA (GluR5 and 6 and KA2 subunits), and mGlu MPOA but not in the AN –ME region. Adminis-
(mGluR1,2,3 and 5 subunits) receptor mRNAs and/or tration of NMDA or AMPA receptor blockers
proteins are found widely expressed in hypothalamic inhibits LH surge release. Also, sex steroid adminis-
neurons. In most hypothalamic regions the NMDA tration to gonadectomized animals up-regulates
receptor predominates. Finally, mRNA and protein components of hypothalamic Glu transmission,
for the Glu transporters EAAC1, GLT-1, and GLAST including Glu release, the activity of Glu transporters,
are detected in the hypothalamus. and the expression of Glu receptors. Specifically, Glu
transport into hypothalamic Glu nerve terminals
varies across the rat estrous cycle, and hypothalamic
B. Glu Regulation of GnRH Pulse
GluR1, GluR2/3, and AMPA receptor subunit proteins
Generator Function increase with sex steroid administration.
Several lines of evidence are consistent with the
hypothesis that hypothalamic Glu neurons stimulate
the GnRH pulse generator and may indeed comprise IV. HYPOTHALAMIC NITRIC OXIDE NEURONS
pulse generator circuitry. First, in situ, about 50% of AND THE NEUROENDOCRINE REGULATION
GnRH neurons express the kainic acid 2 (KA2) OF LH SECRETION
receptor subunit mRNA, whereas about 80%
A. Hypothalamic NO Neurons
express the NMDAR1 receptor subunit mRNA,
demonstrating that GnRH neurons are targets for In the 1980s it was determined that the potent
the neurotransmitter Glu. Second, double-label vasodilatory factor released by endothelial cells was
immunofluorescence work in several hypothalamic the gas nitric oxide; it was proposed that in brain, Glu
structures indicates that an average of 80% of Glu acting at the NMDA receptor caused rapid NO
neurons express ER and as many as of 93% of Glu synthesis and subsequent NO-induced cGMP level
neurons express PR. Third, Glu evokes currents in increases in nearby cells, and that an isoform of the
GnRH neurons studied in hypothalamic slices; enzyme producing NO, neuronal nitric oxide
subsequent patch-clamp work indicates these cur- synthase (nNOS), was present in neurons (Fig. 6).
rents are mediated by NMDA and AMPA channels, These data suggested that NO might be a neuro-
indicating that the Glu receptors expressed by GnRH transmitter in brain. If this were so, however, it was
neurons are physiologically functional. In addition, soon appreciated that its neurotransmitter character-
antisense oligodeoxynucleotides against the istics would be quite unconventional, because NO is
NMDAR2A receptor subunit mRNA decrease the not stored in synaptic vesicles, nor indeed in any other
protein levels of this receptor subunit and suppress cellular compartment, is not released by Ca2þ-
pulsatile GnRH secretion from MPOA – ME hypo- dependent exocytosis, does not bind to membrane
thalamic fragments studied in vitro. Another finding receptors on target cells, sometimes functions as a
is that NMDA, AMPA, and KA agonists all stimulate retrograde signaling molecule, and is not inactivated
GnRH release from hypothalamic fragments incu- by reuptake into neurons or glia or by enzymatic
bated in vitro. Finally, mathematical modeling of degradation. Instead, NO is synthesized in a one-step
hypothalamic pulse generators indicates that neuro- reaction in which nNOS converts L -arginine to NO
nal oscillators consisting of Glu and GABA neurons and citrulline (Fig. 6). nNOS is present in NO
exhibit greater stability than would a single-cell neurons but requires calmodulin-bound Ca2þ for
oscillator composed solely of GnRH neurons. activation. Once nNOS is activated, NO is immedi-
In vivo work in intact animals indicates that Glu ately synthesized; NO increases cGMP levels by
stimulates GnRH and LH secretion. Systemic or icv binding to and stimulating the enzyme guanylyl
AMINO ACID AND NITRIC OXIDE CONTROL OF THE ANTERIOR PITUITARY 61

FIGURE 6 Schematic representation of a nitric oxide (NO)-releasing neuron. NO is formed from arginine by the
action of neuronal nitric oxide synthase (nNOS). NO freely diffuses across cell membranes and can thereby influence
both presynaptic neurons, such as the glutamatergic presynaptic neuron in the figure, and other cells that are not directly
apposed to the nNOS-containing neuron, such as the other neuron and glial cell depicted. In each case NO stimulates
guanylyl cyclase, increasing cGMP and activating cGMP-sensitive intracellular targets. As indicated in the figure, Glu
can trigger NO formation by activating the NMDA receptor, which results in Ca2þ influx and Ca2þ activation of nNOS.
The half-life of NO is less than 30 s; it decays spontaneously to nitrite. Modified from Zigmond et al. (1999), with
permission.

cyclase. In this way cGMP levels increase and cGMP- NOS (miNOS) is observed in microglia, and some
sensitive targets are activated in the NO neuron or in endothelial NOS (eNOS) is expressed by CNS
the surrounding cells, including NO afferent neurons. vascular endothelial cells, nNOS is found to be the
Message termination rapidly occurs by spontaneous major NOS isoform present in the hypothalamus.
degradation to nitrite.
Although nNOS is present in only about 1% of B. NO Regulation of GnRH Pulse
brain neurons, the processes of these cells ramify so Generator Function
extensively that it is estimated that every neuron in
Several lines of evidence are consistent with the
brain is likely exposed to NO. Because vesicular hypothesis that hypothalamic NO neurons stimulate
release of NO is not regulated in neurons, as is the the GnRH pulse generator, may comprise pulse
case for other transmitters, it seems reasonable that generator circuitry, and may mediate NMDA-
the biosynthetic enzyme be highly regulated. Indeed, induced GnRH secretion. nNOS immunoreactive
no other enzyme is known to be regulated by so many neurons are visualized in several hypothalamic
factors. nNOS regulators include five oxidative – structures, including the MPOA, and some immu-
reductive cofactors, four phosphorylating enzymes, nostaining is also present in terminals in the ME.
and three binding proteins, and its mRNA is Hence, NO neurons may affect the GnRH neurons in
regulated by hormones and other factors. Although either the MPOA cell body and dendrite region or the
stored NO is not released in an impulse-dependent ME preterminal axon and terminal location. A few
manner, neuronal stimulation indeed causes NO GnRH neurons appear nNOS-positive and many
release, by the Glu and NMDA receptor mechanism nNOS immunoreactive neurons surround GnRH cell
(Fig. 6) and/or by other as yet unknown mechanisms. bodies in the MPOA. Some hypothalamic NO
In the hypothalamus, many nNOS immuno- neurons express ER and PR, and estradiol increases
reactive neuronal cell bodies, processes, and terminals hypothalamic nNOS mRNA and protein levels.
are observed. Although some macrophage-inducible Similarly, MPOA nNOS mRNA, protein, and enzyme
62 AMINO ACID AND NITRIC OXIDE CONTROL OF THE ANTERIOR PITUITARY

activity vary across the rat estrous cycle. NO TABLE 1 Net In vivo Effect of GABA, Glu, and NO on
stimulates MPOA and AN– ME cGMP in a dose- the Secretion of the Anterior Pituitary Tropic
related manner and this stimulation is blocked by Hormonesa
hemoglobin, an NO scavenger. Effector LH/FSH ACTH TSH GH PRL
In vivo work in intact animals indicates that NO
stimulates GnRH and LH secretion in rats. First, icv GABA # # # # #
administration of an nNOS inhibitor suppresses Glu " " " " "
NO " # ND " "
pulsatile LH secretion. Second, nNOS inhibitors
block LH surge release and icv administration of a
LH, Luteinizing hormone; FSH, follicle-stimulating hormone;
the NO precursor L -arginine potentiates the LH ACTH, adrenocorticotropic hormone; TSH, thyroid-stimulating
surge. Third, antisense oligodeoxynucleotides against hormone; GH, growth hormone; PRL, prolactin; " , increases; # ,
nNOS mRNA, delivered icv, decrease hypothalamic decreases; ND, not determined.
nNOS protein and diminish LH surge release. Fourth,
microinfusion of the NO precursor L -arginine into of this evidence is presented in reviews cited in the
the AN– ME region increases the amount of cGMP bibliography of this article. Evidence substantial
and GnRH recovered in AN– ME push– pull perfu- enough to form a reasoned judgment is included in
sates. Fifth, NO and GnRH are secreted in a Table 1, which summarizes the neuroendocrine effect
copulsatile fashion from ME fragments incubated in of GABA, Glu, and NO on the secretion of ACTH,
vitro, and the amplitude of these secretions varies TSH, GH, and PRL. So that the table is complete, the
across the estrous cycle. effects on LH and FSH secretion are included.
The precise neuronal mechanism whereby NO Although a great deal is known about hypothalamic
stimulates GnRH release is uncertain. NO may GABA, Glu, and NO neurons and how they function
directly stimulate guanylyl cyclase in GnRH neurons, as physiologically significant afferents to the hypo-
resulting somehow in GnRH release. However, there physiotropic hormone-secreting neurons, consider-
is more evidence for other indirect actions. Evidence ably more than that remains to be learned about these
exists suggesting that NO induction is required for neuroendocrine regulatory mechanisms.
Glu-induced GnRH secretion, that NO releases
prostaglandin E2 (which activates adenylate cyclase
and protein kinase A, and then GnRH secretion), and Glossary
that NO-stimulated GnRH secretion is mediated by afferent neuron Neuron that conveys incoming nerve
neuropeptide Y neurons. Clearly, a great deal of impulses to another neuron or nerve center with
additional work is required before it will be possible which it synapses.
to distinguish between these and other neuroendo- g-aminobutyric acid Principal inhibitory neurotransmitter
crine regulatory mechanisms. in the mammalian central nervous system (CNS),
inhibiting virtually every neuron in the adult CNS at
physiological concentrations.
glutamate Principal excitatory neurotransmitter in the
V. THE ROLE OF GABA, GLUTAMATE, AND mammalian central nervous system, stimulating vir-
NITRIC OXIDE NEURONS IN THE tually every neuron in the adult CNS at physiological
NEUROENDOCRINE REGULATION OF OTHER concentrations.
ANTERIOR PITUITARY TROPIC HORMONES gonadotropin-releasing hormone Hypothalamic releasing
hormone that stimulates luteinizing hormone and
The evidence indicating significant physiological roles follicle-stimulating hormone secretion from the anterior
for GABA, Glu, and NO neurons in the regulation of pituitary gland.
GnRH and LH secretion is well documented. Related hypophysiotropic hormone-secreting neurons Hypotha-
evidence suggests that these neurons are significant lamic neurons that secrete into the hypophysial portal
afferents to the other hypothalamic neurons that circulation various releasing or inhibiting hormones,
which in turn control secretion of the six anterior
secrete releasing and inhibiting hormones [CRH,
pituitary tropic hormones.
TRH, GHRH, SS (somatostatin), PIH, and PRF], luteinizing hormone and follicle-stimulating hormone An-
which in turn regulate the secretion of the anterior terior pituitary tropic hormones controlling the repro-
pituitary hormones (ACTH, TSH, GH, and PRL). ductive functions of the testes and ovaries.
However, the data supporting the latter role are fewer median eminence That portion of the medial basal
and in some cases substantially less well developed, hypothalamus from which the hypophysial portal
compared to the evidence for GnRH regulation. Some vessels arise.
AMPHIREGULIN 63

nitric oxide Neuroactive gas, released from neurons, which van den Pol, A. N., Wuarin, J.-P., and Dudek, F. E. (1990).
functions as a neurotransmitter, probably affecting Glutamate, the dominant excitatory transmitter in neuroendo-
every neuron in brain. crine regulation. Science 250, 1276–1278.
van den Pol, A. N., Wuarin, J.-P., and Dudek, F. E. (1996).
Glutamate neurotransmission in the neuroendocrine hypo-
See Also the Following Articles thalamus. In “Excitatory Amino Acids: Their Role in
Neuroendocrine Function” (D. W. Brann and V. B. Mahesh,
Adrenocorticotropic Hormone (ACTH) and Other eds.), pp. 1–54. CRC Press, Boca Raton, FL.
Proopiomelanocortin (POMC) Peptides . Cytokines and
Anterior Pituitary Function . Follicle-Stimulating Hormone Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
(FSH) . Gonadotropin-Releasing Hormone (GnRH)
. Luteinizing Hormone (LH)

Further Reading Amphiregulin


Brann, D. W., and Mahesh, V. B. (1997). Excitatory amino acids:
Evidence for a role in the control of reproduction and anterior EUNKYUNG CHUNG *, PAUL W. COOK †, AND
pituitary hormone secretion. Endocr. Rev. 18, 678–700. ROBERT J. COFFEY *
Decavel, C., and van den Pol, A. N. (1990). GABA: A dominant p
Vanderbilt University and VA Medical Center . †Cascade
neurotransmitter in the hypothalamus. J. Comp. Neurol. 302, Biologics, Inc., Oregon
1019– 1037.
Deutch, A. Y., and Roth, R. H. (1999). Neurotransmitters. In
“Fundamental Neuroscience” (M. J. Zigmond, F. E. Bloom, I. INTRODUCTION
S. C. Landis, J. L. Roberts, and L. R. Squire, eds.), II. STRUCTURE AND PROCESSING OF AR
pp. 193– 234. Academic Press, San Diego, CA. III. SELECTED ASPECTS OF AR SIGNALING
Dhandapani, K. M., and Brann, D. W. (2000). The role of IV. REGULATION OF AR EXPRESSION
glutamate and nitric oxide in the reproductive neuroendocrine V. BIOLOGICAL ROLES FOR AR
system. Biochem. Cell Biol. 78, 165–179. VI. SUMMARY
Dingledine, R., and McBain, C. J. (1999). Glutamate and aspartate.
In “Basic Neurochemistry: Molecular, Cellular and Medical
Aspects,” 6th Ed. (B. W. Agranoff, R. W. Albers, S. K. Fisher, The epidermal growth factor (EGF) family of
and M. D. Uhler, eds.), pp. 315 – 333. Lippincott-Raven polypeptide growth factors includes EGF,
Publishers, Philadelphia.
transforming growth factor-a, amphiregulin,
Ganong, W. F. (2001). “Review of Medical Physiology,” 20th Ed.
McGraw-Hill, New York.
heparin-binding EGF-like growth factor, beta-
Grattan, D. R., Park, S.-K., and Selmanoff, M. (1995). Orchidec- cellulin, epiregulin, epigen, heregulin, neure-
tomy and NMDA increase GnRH secretion as measured by gulin, and cripto.
push–pull perfusion of rat anterior pituitary. Am. J. Physiol.
268, E685–692.
Kordon, C., Drouva, S. V., Martı́nez de la Escalera, G., and Weiner,
R. I. (1994). Role of classic and peptide neuromediators in the I. INTRODUCTION
neuroendocrine regulation of luteinizing hormone and prolac-
tin. In “The Physiology of Reproduction,” Vol. 1 (E. Knobil The epidermal growth factor (EGF) receptor ligands
and J. Neill, eds.), pp. 1621–1682. Raven Press, New York. can interact with four known EGF receptor (EGFR)
Martin, D. L., and Olsen, R. W. (eds.) (2000). “GABA in the subtypes, and, in some cases, distinct intracellular
Nervous System: The View at Fifty Years.” Lippincott- signaling pathways can be activated in a ligand/
Williams & Wilkins, Philadelphia.
receptor-dependent fashion. Like the other EGFR
Mugnaini, E., and Oertel, W. H. (1985). An atlas of the distribution
of GABAergic neurons and terminals in the rat CNS as ligands, amphiregulin (AR) is synthesized in a pro-
revealed by GAD immunocytochemistry. In “Handbook of form and is inserted into the plasma membrane,
Chemical Neuroanatomy, Vol. 4, GABA and Neuropeptides in where it is cleaved in its ectodomain to release soluble
the CNS” (A. Björkland and T. Hökfelt, eds.), pp. 436–608. growth factor. AR was originally identified as an
Elsevier, Amsterdam. EGFR ligand from a phorbol ester-treated human
Olsen, R. W., and DeLorey, T. M. (1999). GABA and glycine. In
“Basic Neurochemistry: Molecular, Cellular and Medical breast adenocarcinoma cell line, MCF-7. Although
Aspects,” 6th Ed. (B. W. Agranoff, R. W. Albers, S. K. Fisher, the growth inhibitory properties of AR have not been
and M. D. Uhler, eds.), pp. 335 – 346. Lippincott-Raven subsequently validated, Shoyab and co-workers
Publishers, Philadelphia. named it “amphi” because of its growth inhibitory
Roberts, E. (1991). Living systems are tonically inhibited, effects on A431 and several other cancer cell lines and
autonomous optimizers, and disinhibition coupled to varia-
bility generation is their major organizing principle: Inhibitory
growth stimulation of many other cell lines, normal
command-control at levels of membrane, genome, metab- and transformed. This article will attempt to provide
olism, brain, and society. Neurochem. Res. 16, 409–421. a biological perspective on AR by discussing its
AMPHIREGULIN 63

nitric oxide Neuroactive gas, released from neurons, which van den Pol, A. N., Wuarin, J.-P., and Dudek, F. E. (1990).
functions as a neurotransmitter, probably affecting Glutamate, the dominant excitatory transmitter in neuroendo-
every neuron in brain. crine regulation. Science 250, 1276–1278.
van den Pol, A. N., Wuarin, J.-P., and Dudek, F. E. (1996).
Glutamate neurotransmission in the neuroendocrine hypo-
See Also the Following Articles thalamus. In “Excitatory Amino Acids: Their Role in
Neuroendocrine Function” (D. W. Brann and V. B. Mahesh,
Adrenocorticotropic Hormone (ACTH) and Other eds.), pp. 1–54. CRC Press, Boca Raton, FL.
Proopiomelanocortin (POMC) Peptides . Cytokines and
Anterior Pituitary Function . Follicle-Stimulating Hormone Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
(FSH) . Gonadotropin-Releasing Hormone (GnRH)
. Luteinizing Hormone (LH)

Further Reading Amphiregulin


Brann, D. W., and Mahesh, V. B. (1997). Excitatory amino acids:
Evidence for a role in the control of reproduction and anterior EUNKYUNG CHUNG *, PAUL W. COOK †, AND
pituitary hormone secretion. Endocr. Rev. 18, 678–700. ROBERT J. COFFEY *
Decavel, C., and van den Pol, A. N. (1990). GABA: A dominant p
Vanderbilt University and VA Medical Center . †Cascade
neurotransmitter in the hypothalamus. J. Comp. Neurol. 302, Biologics, Inc., Oregon
1019– 1037.
Deutch, A. Y., and Roth, R. H. (1999). Neurotransmitters. In
“Fundamental Neuroscience” (M. J. Zigmond, F. E. Bloom, I. INTRODUCTION
S. C. Landis, J. L. Roberts, and L. R. Squire, eds.), II. STRUCTURE AND PROCESSING OF AR
pp. 193– 234. Academic Press, San Diego, CA. III. SELECTED ASPECTS OF AR SIGNALING
Dhandapani, K. M., and Brann, D. W. (2000). The role of IV. REGULATION OF AR EXPRESSION
glutamate and nitric oxide in the reproductive neuroendocrine V. BIOLOGICAL ROLES FOR AR
system. Biochem. Cell Biol. 78, 165–179. VI. SUMMARY
Dingledine, R., and McBain, C. J. (1999). Glutamate and aspartate.
In “Basic Neurochemistry: Molecular, Cellular and Medical
Aspects,” 6th Ed. (B. W. Agranoff, R. W. Albers, S. K. Fisher, The epidermal growth factor (EGF) family of
and M. D. Uhler, eds.), pp. 315 – 333. Lippincott-Raven polypeptide growth factors includes EGF,
Publishers, Philadelphia.
transforming growth factor-a, amphiregulin,
Ganong, W. F. (2001). “Review of Medical Physiology,” 20th Ed.
McGraw-Hill, New York.
heparin-binding EGF-like growth factor, beta-
Grattan, D. R., Park, S.-K., and Selmanoff, M. (1995). Orchidec- cellulin, epiregulin, epigen, heregulin, neure-
tomy and NMDA increase GnRH secretion as measured by gulin, and cripto.
push–pull perfusion of rat anterior pituitary. Am. J. Physiol.
268, E685–692.
Kordon, C., Drouva, S. V., Martı́nez de la Escalera, G., and Weiner,
R. I. (1994). Role of classic and peptide neuromediators in the I. INTRODUCTION
neuroendocrine regulation of luteinizing hormone and prolac-
tin. In “The Physiology of Reproduction,” Vol. 1 (E. Knobil The epidermal growth factor (EGF) receptor ligands
and J. Neill, eds.), pp. 1621–1682. Raven Press, New York. can interact with four known EGF receptor (EGFR)
Martin, D. L., and Olsen, R. W. (eds.) (2000). “GABA in the subtypes, and, in some cases, distinct intracellular
Nervous System: The View at Fifty Years.” Lippincott- signaling pathways can be activated in a ligand/
Williams & Wilkins, Philadelphia.
receptor-dependent fashion. Like the other EGFR
Mugnaini, E., and Oertel, W. H. (1985). An atlas of the distribution
of GABAergic neurons and terminals in the rat CNS as ligands, amphiregulin (AR) is synthesized in a pro-
revealed by GAD immunocytochemistry. In “Handbook of form and is inserted into the plasma membrane,
Chemical Neuroanatomy, Vol. 4, GABA and Neuropeptides in where it is cleaved in its ectodomain to release soluble
the CNS” (A. Björkland and T. Hökfelt, eds.), pp. 436–608. growth factor. AR was originally identified as an
Elsevier, Amsterdam. EGFR ligand from a phorbol ester-treated human
Olsen, R. W., and DeLorey, T. M. (1999). GABA and glycine. In
“Basic Neurochemistry: Molecular, Cellular and Medical breast adenocarcinoma cell line, MCF-7. Although
Aspects,” 6th Ed. (B. W. Agranoff, R. W. Albers, S. K. Fisher, the growth inhibitory properties of AR have not been
and M. D. Uhler, eds.), pp. 335 – 346. Lippincott-Raven subsequently validated, Shoyab and co-workers
Publishers, Philadelphia. named it “amphi” because of its growth inhibitory
Roberts, E. (1991). Living systems are tonically inhibited, effects on A431 and several other cancer cell lines and
autonomous optimizers, and disinhibition coupled to varia-
bility generation is their major organizing principle: Inhibitory
growth stimulation of many other cell lines, normal
command-control at levels of membrane, genome, metab- and transformed. This article will attempt to provide
olism, brain, and society. Neurochem. Res. 16, 409–421. a biological perspective on AR by discussing its
64 AMPHIREGULIN

structure and processing, mechanisms of action,


factors that regulate its expression, and conditions
associated with its overexpression.

II. STRUCTURE AND PROCESSING OF AR


At the genomic level, human AR maps to chromo-
some 4q13 –q21 and is partitioned into six exons that
span 10.2 kb of genomic DNA. As deduced from the
cDNA, the transcript is 1.4 kb and the translation
product is 252 amino acids with a predicted mass of
25.9 kDa without glycosylation. Fig. 1 depicts the
chromosomal location of AR in human and mouse.
AR is contiguous with epiregulin in human and with
betacellulin in mouse, respectively. The tight linkage
between these EGFR ligands in both human and
mouse suggests that they may have arisen through a
tandem gene duplication event. In addition, AR is in
close proximity to the chemotactic cytokines for
neutrophils, B cells, and melanocytes, such as Gro1,
Gro2, Gro3, and interleukin-8 (IL-8) in human and
Gro1 in mouse. Of interest, AR has been shown to be
pro-inflammatory in the skin of keratin 14 promoter
(K14)-AR transgenic mice, in which there is targeted
overexpression of human AR in the basal layer of the
epidermis. FIGURE 2 Structure of AR precursor. AR is synthesized as a
As illustrated in Fig. 2, mature AR is initially 252-amino-acid, single-pass transmembrane glycoprotein.
synthesized as a 252-amino-acid transmembrane After the signal peptide (1– 19), there is an N-terminal pro-
precursor that is proteolytically cleaved to its mature region (amino acids 20 – 100) containing N- and O-linked
78- to 84-amino-acid form. Following the signal glycosylation sites and potential tyrosine sulfation sites,
followed by mature AR. Mature AR consists of two distinct
parts, a heparin-binding domain (HBD) and an EGF repeat.
TM represents the transmembrane domain (amino acids 199 –
221) that is followed by the cytoplasmic domain (amino acids
222– 252). Arrow at a distal site indicates a TACE-mediated
cleavage site, and two arrowheads show alternative proteolytic
processing sites that give rise to different isoforms of mature
AR.

peptide, the pro-region consists of potential N- or


O-linked glycosylation sites and three potential
tyrosine sulfation sites. Subsequent work from the
authors’ laboratory has revealed that N-linked
FIGURE 1 Chromosomal location of AR and adjacent genes glycosylation of the pro-region predominates over
in human and mouse genomes. Human AR is in close proximity O-linked glycosylation and that tyrosine sulfation
to epiregulin on chromosome 4q13 – q21, and mouse AR is occurs in this region. Mature AR consists of a
adjacent to betacellulin on chromosome 5 at 50– 52 cM. (In heparin-binding domain (HBD) made up of positively
human) IL-8, interleukin-8; GRO1, 2, 3 oncogene, melanoma charged amino acids and followed by the EGF
growth stimulating activity a; EREG, epiregulin; AREG, domain, which is composed of the conserved spacing
amphiregulin; p115, vesicle-docking protein p115; NUP54,
nucleoporin p54; FGF5, fibroblast growth factor 5. (In mouse)
of six cysteine residues (CX7CX4CX1CX8C) shared
Gro1, the mouse homologue of human GRO1; Btc, betacellu- among EGFR ligands. Plowman and co-workers have
lin; Areg, amphiregulin; Rps12-rs1, ribosomal protein S12; shown that the HBD of AR prevents the secretion of
Art3, ADP-ribosyl transferase 3. mature AR when the pro-region is deleted. In this
AMPHIREGULIN 65

study, deletion of the pro-region resulted in the (see below). Fig. 3 depicts various isoforms of AR in
prevention of secretion of AR into the medium. the cell lysates and medium following its posttransla-
Moreover, fusion of the HBD of AR to the EGF tional modification. Although some of these isoforms
repeats of transforming growth factor-a (TGF-a) and have been shown to possess biological activity, it is
EGF, along with TGF-b’s signal peptide, prevented not known whether all of these soluble AR forms are
secretion of these constructs. From these studies, an biologically active and, if so, whether they may differ
electrostatic model has been proposed in which the in their biological activities.
negatively charged pro-region of AR interacts with its
positively charged HBD to facilitate proper folding
and secretion. However, replacement of AR’s pro- III. SELECTED ASPECTS OF AR SIGNALING
region with the pro-region of heparin-binding EGF- The sorting and processing of AR in the context
like growth factor (HB-EGF) (which is positively of polarized epithelial cells have been examined.
charged) allowed secretion of mature AR, whereas This approach assumes biological relevance when it is
TGF-a’s pro-region (which is negatively charged) did recalled that the EGFR is localized preferentially to
not. Therefore, it may be that only specific negatively the basolateral surface of all polarized epithelial cells.
charged amino acid sequences can confer proper Studies have examined the trafficking of wild-type
folding and secretion of AR. and mutant forms of AR in polarized MDCK II
In the C-terminus of AR’s EGF repeat, a leucine cells that had been stably transfected with these
that is conserved in all other EGFR ligands is cDNAs, as well as trafficking of endogenous AR in
converted to methionine at residue 186, a change polarized HCA-7 cells, a human colon cancer cell line
that reduces binding to EGFR. The transmembrane that was generated from an individual with a well-
domain and cytoplasmic domain of AR are not differentiated rectal cancer. These HCA-7 cells, like
required for secretion of mature AR, although MDCK II cells, form a uniform polarizing monolayer
proteolytic removal of the N-terminal pro-region is when cultured on polycarbonate-coated Transwell
less efficient in the absence of the membrane anchor. filters. Both of these cells contain 20,000 – 40,000
However, the loss of basolateral sorting was observed basolateral EGFRs. In both lines, AR is delivered
by deletion of these regions in polarized Madin- preferentially to the basolateral surface, where it
Darby canine kidney (MDCK) II epithelial cells is cleaved by tumor necrosis factor a-converting

FIGURE 3 Various forms of cellular and secreted AR. Different forms of cell-associated and secreted AR are depicted
and the extent of glycosylation and the ability to bind heparin are described quantitatively. Cell surface, membrane-
anchored forms of AR are proteolytically processed to multiple soluble isoforms. TACE-mediated cleavage of the
ectodomain of 50 kDa pro-AR at a distal site releases 43 kDa soluble AR into conditioned medium. Alternatively, in the
absence of primary proteolytic cleavage at the distal site, secondary cleavage at proximal sites removes the N-terminal
domain, resulting in 28 and 26 kDa cell surface pro-AR forms, which appear as a doublet. These forms are then released
by TACE-mediated cleavage at the distal site. This soluble doublet probably represents the originally described mature
form of AR. In the absence of cleavage at the distal site, further proteolytic processing at a proximal site results in a
16 kDa cell surface form of AR, which has a reduced HBD and is not glycosylated. The 16 kDa cell surface form can be
cleaved by TACE at a distal site to release a 9 kDa soluble AR.
66 AMPHIREGULIN

enzyme/a disintegrin and metalloprotease 17 activation of alternative signaling pathways. It should


(TACE/ADAM17) (see Fig. 4). The basolateral be emphasized, however, that activation of such
sorting information resides in the cytoplasmic tail as alternative pathways has not been proven. In the
AR cDNA constructs that lack the tail are missorted following discussion, possible effects due to the
in polarized MDCK II cells. However, cell surface ability of AR to bind heparin are considered in light
processing of AR appears to be unaffected, suggesting of what has been established for the other heparin-
that information in the tail is not required for cell binding EGFR ligand, HB-EGF.
surface proteolytic cleavage. AR exhibits punctate Immunohistochemical studies have identified sev-
immunoreactivity at both the basal and the lateral eral binding proteins that may be involved in AR
surfaces of these polarized epithelial cells, likely due signaling. AR has been shown to co-localize with
to its ability to bind to heparan sulfate proteoglycans CD44, a hyaluronan receptor, which is modified by
(HSPGs) at the cell surface and in the extracellular HSPGs in its v3 region. This interaction with CD44
matrix. Levels of AR in the basolateral medium are may facilitate the juxtacrine activity of AR as has
not significantly altered after administration of an been shown for HB-EGF. It should be noted, however,
antibody to the EGFR that blocks ligand binding that CD44 also has been reported to co-localize with
(MAb 528 or 225). This contrasts with TGF-a levels EGFR, erbB-4, and matrilysin/matrix metallopro-
that increase promptly in the basolateral medium tease-7. CD9, a tetra-spanning transmembrane pro-
after EGFR MAb blockade, a finding that has been tein, has also been found to co-localize with AR by
interpreted as evidence that TGF-a is a locally acting immunofluorescence studies. It has been reported that
growth factor. These differences in post-cleavage CD9 co-localizes with a3b1 integrins, which, in turn,
disposition of TGF-a and AR underscore the possi- increases the juxtacrine activity of HB-EGF and
bility of distinct biological activities. possibly TGF-a, respectively; however, to date, this
A speculative view of possible participants in interaction has not been shown to increase the
endogenous AR signaling is presented in Fig. 5. juxtacrine activity of pro-AR. In addition, cell surface
Signaling pathways initiated by endogenous AR are HSPGs, members of the syndecan and glypican
thought to be complex due to a number of factors. family, have been postulated as possible binding
These include glycosylation, multiply processed partners of AR. In polarized epithelial cells, glyco-
forms, ability to bind HSPGs, and variable-affinity sylphosphatidylinositol (GPI)-linked glypican, which
binding to EGFR. Although all seven EGFR ligands is generally targeted to the apical domain, would be
bind to the EGFR and thereby activate conserved less likely to participate in AR-induced signaling than
downstream effectors, such as Ras-mitogen-activated syndecan-1, which is sorted to the basolateral
protein kinase and phosphatidylinositol 3-kinase – compartment of polarized epithelial cells like AR
Akt/protein kinase B, the complexity of the pro- and EGFR. It is possible that HSPGs on the cell
duction and processing of endogenous AR allows for surface and extracellular matrix (perlecan, agrin) act

FIGURE 4 Sorting and processing of AR in polarized epithelial cells. This simplified schematic depicts cell surface
cleavage of one AR molecule and its binding to EGFR. It should be noted that TACE, AR, and EGFR are all found within
the basolateral compartment. N, ER, and TGN represent the nucleus, endoplasmic reticulum, and trans-Golgi network,
respectively.
AMPHIREGULIN 67

FIGURE 5 Participants in AR signaling. A model of protein interactions that appear to regulate AR signaling is
presented (see text for details). Both CD44v3 and CD9 directly interact with AR. Whether TACE interacts directly with
AR is not known.

as depots for AR in its pro-AR form or after it is of mitogenic activity, and cell density-dependent
cleaved and released from the cell surface. These responsiveness in Balb MK-2 cells.
depot forms of AR may increase local concentrations More convincing evidence for distinct biological
of AR or regulate its access to the EGFR. In summary, actions of AR comes from mouse studies in which
these accessory molecules presumably influence the there was a targeted overexpression or disruption of
nature of the interaction between AR and EGFR, as AR. In the study of triple knockouts of EGFR ligands
there is no evidence at the present time for EGFR- (EGF, TGF-a, and AR), a specific role for AR was
independent signaling by AR. identified in mammary gland development with signal
What is the evidence that endogenous AR has transducers and activators of transcription (Stat) 5a
effects distinct from those of the other EGFR ligands? involved in this AR-specific effect. Two separate
In vitro, AR differed from EGF in its recruitment of groups used the K14 promoter to overexpress AR and
downstream molecules when these ligands were TGF-a to the basal layer of the epidermis. Both
administered to cells expressing an EGFR with a transgenic lines exhibited hyperplasia of the epider-
C-terminal truncation (C0 1000). It is also possible mis; however, only AR mice exhibited marked
that distinct actions of AR may ensue from differences inflammation of the skin and synovium with features
that arise after ligand binding to the EGFR. For strikingly similar to psoriasis and psoriatic arthritis.
example, Derynck’s group previously reported that These features included epidermal T-lymphocytic and
TGF-a dissociated from the EGFR earlier in the neutrophilic infiltration and tortuous dilated blood
endocytic pathway than EGF based on differences in vessels within the dermis, indicating that AR may
their isoelectric points (the pI for TGF-a is 5.9 and exert unexpected and unique pro-inflammatory and
that for EGF is 4.6). Thus, TGF-a is less likely to be angiogenic activities. This study also demonstrated
degraded and more likely to be recycled. AR has a pI that epidermally targeted AR expression in transgenic
of 9.84 and therefore would be predicted to dissociate mice stimulated psoriatic pathological processes in
from EGFR even earlier than TGF-a during endocy- the skin that were different from those of other
tosis. AR also possesses biological properties that in transgenically targeted cytokines and growth factors
some cases are distinct from those of TGF-a or EGF. such as vascular endothelial growth factor, keratino-
These properties include affinity for heparin and cyte growth factor, IL-1a, tumor necrosis factor a,
heparin-like molecules, heparin-dependent inhibition interferon-g (IFN-g), and IL-6. Additionally, because
68 AMPHIREGULIN

AR is significantly induced by barrier disruption as AR, TGF-a, and HB-EGF. EGFR blockade partially
well as placing human skin into explant culture, it has reverts the transformed morphology of oncogenic
been proposed that cutaneous injury (wounding) Ras RIE-1 cells and prevents the morphological
induces AR expression and initiates the isomorphic, transformation of parental RIE-1 cells caused by
Koebner response (wound-dependent lesion for- constitutively activated Ras-conditioned medium.
mation) in psoriatic individuals. Collectively, these Moreover, the growth inhibitory effects of the
K14 – AR transgenic studies suggest that AR may act farnesyltransferase inhibitor L744, 832 for oncogenic
as a cutaneous injury signaling factor that initiates Ras-transformed RIE-1 cells is preceded by decreased
epidermal proliferation, angiogenesis, and innate expression of AR, HB-EGF, and TGF-a. Also, in
immune responses in the skin. mouse models of skin carcinogenesis and melanoma,
EGFR signaling (with AR likely being an active
participant) plays a pivotal role in mediating the
IV. REGULATION OF AR EXPRESSION Ras-transformed phenotype. Thus, the oncogenic
Table 1 lists factors that affect AR expression. Ras-transformed phenotype is mediated, at least in
Multiple lines of evidence support increased EGF- part, through increased production of EGFR ligands
related peptide signaling and activation of the EGFR (including AR) that presumably bind to and activate
as important components of oncogenic Ras trans- EGFR. It is thought that EGFR signaling will
formation of rat intestinal epithelial cells (RIE-1 further augment Ras signaling as well as activate
cells). For example, oncogenic Ras-transformed RIE- Ras-independent signals that contribute to the
1 cells exhibit increased production of EGFR ligands Ras-transformed phenotype.

TABLE 1 In Vitro and In Vivo Conditions with Altered AR Expression


Condition Target cells mRNA Protein

In vitro conditions with altered AR expression


Up-regulation
Mutated Ras RIE-1, IEC-6 þ þ
EGFR ligands RIE-1, HK, LIM1215 þ þ
Insulin/IGF-I HK þ þ
IL-1a and TNFa Cervical epithelial cells þ þ
IFN-g NHBECs þ þ
Gastric injury Rat gastric epithelial cells þ þ
Helicobacter pylori MKN 28 þ þ
1,25(OH)2D3 SCC25, MCF-7, MDA-231 þ ND
Androgen LNCaP þ þ
E2 MCF-7 þ þ
Phorbol esters MCF-7, T-47D, MDA-231 þ þ
Gastrin Rat stomach cells þ ND
TARP PC3 þ ND
WT1 U20S þ ND
Down-regulation
TGF-b A549 2 2
PPAR-g agonists Ha-Ras-transformed IEC 2 2
In vivo conditions with increased AR expression
Psoriasis Skin þ þ
Neoplasia Pancreatic cancer, þ þ
GI cancer (gastric, CRC), þ þ
mammary gland cancer þ þ

Note: þ, induction; 2, down-regulation; ND, not determined. RIE-1, IEC-6, rat intestinal epithelial cells; HK, human keratinocytes;
NHBECs, normal human bronchial epithelial cells; LIM1215, colon adenocarcinoma cell line; MKN 28, gastric adenocarcinoma cell line;
1,25(OH)2D3, 1,25-dihydroxy vitamin D3; SCC25, head and neck squamous cell carcinoma cell line; MCF-7, estrogen receptor (ER)-positive
breast carcinoma cell line; T-47D, ER-positive breast carcinoma cell line; MDA-231 (MDA-MB-231), ER-negative breast carcinoma cell line;
LNCaP, androgen-sensitive prostate cancer cell line; E2, 17b-estradiol; phorbol esters, TPA (12-O-tetradecanoylphorbol-13-acetate); TARP,
T-cell receptor g-chain alternate reading frame protein; PC3, androgen-independent prostate cancer cell line; U20S, osteosarcoma cell
line; A549, lung adenocarcinoma cell line; PPAR-g agonists, peroxisome proliferator-activated receptor-g agonists (troglitazone,
resiglitazone); GI, gastrointestinal tract; CRC, colorectal cancer.
AMPHIREGULIN 69

In addition, there is extensive autoinduction and regulator during kidney differentiation through direct
cross-induction of EGFR ligands in RIE-1 cells and binding to the AR promoter. Furthermore, recombi-
other EGF growth-responsive epithelial cells. Under nant AR stimulates epithelial branching in organ
EGFR ligand stimulation, there is often a prompt and cultures of embryonic mouse kidney. Taken together,
robust induction of AR. Various pro-inflammatory these studies support a role for AR as an important
cytokines, such as IFN-g and IL-1a, and growth modulator of branching morphogenesis in developing
hormones, such as androgen, estrogen, and gastrin, lung, mammary gland, and kidney. AR appears to
have been reported to induce AR expression in various act on both epithelium and mesenchyme with the
cell types. TGF-b and peroxisome proliferator- participation of HSPGs.
activated receptor-g agonists have been found to
down-regulate AR expression. Table 1 also lists
in vivo conditions in which AR expression has been
VI. SUMMARY
reported to be increased. Human AR is a heparin-binding, heparin-inhibited,
polypeptide growth factor of the EGF family, which
was initially isolated from the conditioned medium of
V. BIOLOGICAL ROLES FOR AR human MCF-7 breast tumor cells that had been
treated with phorbol ester. The human AR gene is
A. AR as an Autocrine Growth Factor located on chromosome 4q13 – q21 and is expressed
AR has been shown to act as an autocrine growth as a 252-amino-acid precursor that is processed into
factor for normal human keratinocytes, mammary several different cell surface and soluble isoforms. In
epithelial cells, and bronchial epithelial cells. More- polarized epithelial cells, AR is preferentially deliv-
over, evidence has been presented for AR acting as an ered to and processed at the basolateral surface. AR is
autocrine growth factor in certain human cancer cell thought to associate with extracellular and cell-
lines. These include ovarian (OVCAR3, OVCAR8), associated HSPGs (e.g., syndecans, CD44) as well
prostatic (DU145 and PC3), colonic (HCA-7 and as other accessory molecules (e.g., CD9). Interaction
Caco-2), bladder, cervical, and gastric cell lines. with these molecules may facilitate a depot form of
AR and promote productive interactions between AR
and cell surface EGFRs.
B. AR as a Morphogen
AR displays some biological properties that are
Previous studies have shown that AR influences organ distinct from those of either TGF-a or EGF. These
development in the lung, mammary gland, and properties include affinity for heparin, heparin-
kidney. In lung development, AR contributes to mediated inhibition of mitogenic activity, and cell
the growth of the mesenchyme and epithelium, density-dependent responsiveness. Distinct biological
thus participating in the process of branching actions of AR were identified from a comparison of
morphogenesis. These effects are mediated through mice with targeted overexpression of AR and TGF-a
interactions with HSPGs, as glycosaminoglycan- in the skin, where AR mice exhibit an inflammatory
degrading enzymes abolished AR-induced mito- psoriasis-like phenotype. Furthermore, experiments
genesis and branching morphogenesis. The role of with AR null mice have revealed a role for AR in
AR in mammary gland development has been mammary gland development through activation of
demonstrated by studies of mice with targeted Stat5a. AR also participates in branching morpho-
disruption of AR, TGF-a, and EGF. Mice lacking genesis in the lung and kidney. Additional studies will
AR singly and in combination with TGF-a showed be needed to elucidate mechanisms underlying these
defects in ductal outgrowth in glands. In triple-null biological properties of AR. It is anticipated that AR
glands, alveoli were poorly organized and poorly will be an important target of the protease-mediated
differentiated, and milk protein gene expression was cleavage of EGFR ligands that underlie G-protein-
decreased. A role for AR in kidney differentiation was coupled receptor transactivation of the EGFR.
found initially in studies using an embryonic epi- AR has been shown to be regulated by a variety of
thelial cell line derived from c-met mice. In addition, agents and conditions that include up-regulation by
AR was identified in a search for genes with altered EGFR ligands and mutant Ras. AR has also been
expression following induction of the Wilms tumor shown to be up-regulated in pathological states such
suppressor (WT1), a zinc-finger transcription factor as psoriasis and a number of neoplasms, where it may
implicated in kidney differentiation and tumori- act in an autocrine manner to stimulate growth
genesis. It appears that WT1 acts as a transcriptional and/or prevent apoptosis.
70 ANDROGEN EFFECTS IN MAMMALS

Glossary the expression of cyclin D1 and EGF-like growth factors in ras-


transformed rat intestinal epithelial cells. Int. J. Cancer 94,
epidermal growth factor receptor (EGFR) Type I trans- 335 –342.
membrane protein that has tyrosine kinase activity in its Liou, A., Elias, P. M., Grunfeld, C., Feingold, K. R., and Wood,
cytoplasmic domain. There are four mammalian L. C. (1997). Amphiregulin and nerve growth factor
members of this receptor family. Signaling from the expression are regulated by barrier status in murine epidermis.
J. Invest. Dermatol. 108, 73–77.
EGFR is initiated by binding of its ligands, which then
Luetteke, N. C., Qiu, T. H., Fenton, S. E., Troyer, K. L., Riedel,
induces receptor dimerization and autophosphorylation
R. F., Chang, A., and Lee, D. C. (1999). Targeted inactivation
to propagate downstream signaling cascades. of the EGF and amphiregulin genes reveals distinct roles for
epidermal growth factor receptor (EGFR) ligands There EGF receptor ligands in mouse mammary gland development.
are currently seven members of the mammalian Development 126, 2739–2750.
epidermal growth factor family that exhibit conserved Niemeyer, C. C., Spencer-Dene, B., Wu, J. X., and Adamson, E. D.
spacing of six cysteine residues in the mature domain of (1999). Preneoplastic mammary tumor markers: Cripto and
the growth factor. The ligands are synthesized in a amphiregulin are overexpressed in hyperplastic stages of tumor
proform, delivered to the cell surface as a transmem- progression in transgenic mice. Int. J. Cancer 81, 588–591.
brane protein, and then cleaved to release mature Nylander, N., Smith, L. T., Underwood, R. A., and Piepkorn, M.
soluble growth factor. (1998). Topography of amphiregulin expression in cultured
human keratinocytes: Colocalization with the epidermal
psoriasis Hyperproliferative skin disorder with infiltrating
growth factor receptor and CD44. In Vitro Cell Dev. Biol.
lymphocytes and neutrophils in the epidermis and
Anim. 34, 182–188.
dermis. Sawa, M., Tsukamoto, T., Kiyoi, T., Kurokawa, K., Nakajima, F.,
tumor necrosis factor a-converting enzyme/a disintegrin and Nakada, Y., Yokota, K., Inoue, Y., Kondo, H., and Yoshino, K.
metalloprotease 17 (TACE/ADAM17) Enzyme that is (2002). New strategy for antedrug application: Development
involved in the cleavage of pro-transforming growth of metalloproteinase inhibitors as antipsoriatic drugs. J. Med.
factor-a and pro-amphiregulin at the cell surface. Chem. 45, 930–936.
Shoyab, M., Plowman, G. D., McDonald, V. L., Bradley, J. G., and
See Also the Following Articles Todaro, G. J. (1989). Structure and function of human
amphiregulin: A member of the epidermal growth factor
Epidermal Growth Factor (EGF) Family . Heparin-Binding family. Science 243, 1074– 1076.
Epidermal Growth Factor-like Growth Factor (HB-EGF) Torring, N., Jorgensen, P. E., Sorensen, B. S., and Nexo, E. (2000).
. HGF (Hepatocyte Growth Factor)/MET System . Nerve Increased expression of heparin binding EGF (HB-EGF),
Growth Factor (NGF) . Platelet-Derived Growth Factor amphiregulin, TGF a and epiregulin in androgen-independent
(PDGF) . Vascular Endothelial Growth Factor prostate cancer cell lines. Anticancer Res. 20, 91–95.
Wolfgang, C. D., Essand, M., Lee, B., and Pastan, I. (2001). T-cell
receptor g chain alternate reading frame protein (TARP)
Further Reading expression in prostate cancer cells leads to an increased growth
Akutsu, N., Bastien, Y., Lin, R., Mader, S., and White, J. H. (2001). rate and induction of caveolins and amphiregulin. Cancer Res.
Amphiregulin is a vitamin D3 target gene in squamous cell and 61, 8122– 8126.
breast carcinoma. Biochem. Biophys. Res. Commun. 281, Wong, L., Deb, T. B., Thompson, S. A., Wells, A., and Johnson,
1051–1056. G. R. (1999). A differential requirement for the COOH-
Berquin, I. M., Dziubinski, M. L., Nolan, G. P., and Ethier, S. P. terminal region of the epidermal growth factor (EGF) receptor
(2001). A functional screen for genes inducing epidermal in amphiregulin and EGF mitogenic signaling. J. Biol. Chem.
growth factor autonomy of human mammary epithelial cells 274, 8900–8909.
confirms the role of amphiregulin. Oncogene 20, 4019–4028. Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
Brown, C. L., Coffey, R. J., and Dempsey, P. J. (2001). The
proamphiregulin cytoplasmic domain is required for basolat-
eral sorting, but is not essential for constitutive or stimulus-
induced processing in polarized Madin-Darby canine kidney
cells. J. Biol. Chem. 276, 29538–29549. Androgen Effects in Mammals
Cook, P. W., Pittelkow, M. R., and Piepkorn, M. (1999).
Overexpression of amphiregulin in the epidermis of transgenic
mice induces a psoriasis-like cutaneous phenotype. J. Invest.
SHALENDER BHASIN
Dermatol. 113, 860. Charles R. Drew University of Medicine and Science,
Damstrup, L., Kuwada, S. K., Dempsey, P. J., Brown, C. L., Los Angeles
Hawkey, C. J., Poulsen, H. S., Wiley, H. S., and Coffey, R. J., Jr
(1999). Amphiregulin acts as an autocrine growth factor in
I. TESTOSTERONE SECRETION, TRANSPORT, AND
two human polarizing colon cancer lines that exhibit domain
selective EGF receptor mitogenesis. Br. J. Cancer 80,
METABOLISM
1012–1019. II. TESTOSTERONE AS A PROHORMONE
Kitamura, S., Miyazaki, Y., Hiraoka, S., Nagasawa, Y., Toyota, M., III. MECHANISM OF ANDROGEN’S ACTION
Takakura, R., Kiyohara, T., Shinomura, Y., and Matsuzawa, Y. IV. TESTOSTERONE’S EFFECTS ON REPRODUCTIVE
(2001). PPARg agonists inhibit cell growth and suppress ORGANS
70 ANDROGEN EFFECTS IN MAMMALS

Glossary the expression of cyclin D1 and EGF-like growth factors in ras-


transformed rat intestinal epithelial cells. Int. J. Cancer 94,
epidermal growth factor receptor (EGFR) Type I trans- 335 –342.
membrane protein that has tyrosine kinase activity in its Liou, A., Elias, P. M., Grunfeld, C., Feingold, K. R., and Wood,
cytoplasmic domain. There are four mammalian L. C. (1997). Amphiregulin and nerve growth factor
members of this receptor family. Signaling from the expression are regulated by barrier status in murine epidermis.
J. Invest. Dermatol. 108, 73–77.
EGFR is initiated by binding of its ligands, which then
Luetteke, N. C., Qiu, T. H., Fenton, S. E., Troyer, K. L., Riedel,
induces receptor dimerization and autophosphorylation
R. F., Chang, A., and Lee, D. C. (1999). Targeted inactivation
to propagate downstream signaling cascades. of the EGF and amphiregulin genes reveals distinct roles for
epidermal growth factor receptor (EGFR) ligands There EGF receptor ligands in mouse mammary gland development.
are currently seven members of the mammalian Development 126, 2739–2750.
epidermal growth factor family that exhibit conserved Niemeyer, C. C., Spencer-Dene, B., Wu, J. X., and Adamson, E. D.
spacing of six cysteine residues in the mature domain of (1999). Preneoplastic mammary tumor markers: Cripto and
the growth factor. The ligands are synthesized in a amphiregulin are overexpressed in hyperplastic stages of tumor
proform, delivered to the cell surface as a transmem- progression in transgenic mice. Int. J. Cancer 81, 588–591.
brane protein, and then cleaved to release mature Nylander, N., Smith, L. T., Underwood, R. A., and Piepkorn, M.
soluble growth factor. (1998). Topography of amphiregulin expression in cultured
human keratinocytes: Colocalization with the epidermal
psoriasis Hyperproliferative skin disorder with infiltrating
growth factor receptor and CD44. In Vitro Cell Dev. Biol.
lymphocytes and neutrophils in the epidermis and
Anim. 34, 182–188.
dermis. Sawa, M., Tsukamoto, T., Kiyoi, T., Kurokawa, K., Nakajima, F.,
tumor necrosis factor a-converting enzyme/a disintegrin and Nakada, Y., Yokota, K., Inoue, Y., Kondo, H., and Yoshino, K.
metalloprotease 17 (TACE/ADAM17) Enzyme that is (2002). New strategy for antedrug application: Development
involved in the cleavage of pro-transforming growth of metalloproteinase inhibitors as antipsoriatic drugs. J. Med.
factor-a and pro-amphiregulin at the cell surface. Chem. 45, 930–936.
Shoyab, M., Plowman, G. D., McDonald, V. L., Bradley, J. G., and
See Also the Following Articles Todaro, G. J. (1989). Structure and function of human
amphiregulin: A member of the epidermal growth factor
Epidermal Growth Factor (EGF) Family . Heparin-Binding family. Science 243, 1074– 1076.
Epidermal Growth Factor-like Growth Factor (HB-EGF) Torring, N., Jorgensen, P. E., Sorensen, B. S., and Nexo, E. (2000).
. HGF (Hepatocyte Growth Factor)/MET System . Nerve Increased expression of heparin binding EGF (HB-EGF),
Growth Factor (NGF) . Platelet-Derived Growth Factor amphiregulin, TGF a and epiregulin in androgen-independent
(PDGF) . Vascular Endothelial Growth Factor prostate cancer cell lines. Anticancer Res. 20, 91–95.
Wolfgang, C. D., Essand, M., Lee, B., and Pastan, I. (2001). T-cell
receptor g chain alternate reading frame protein (TARP)
Further Reading expression in prostate cancer cells leads to an increased growth
Akutsu, N., Bastien, Y., Lin, R., Mader, S., and White, J. H. (2001). rate and induction of caveolins and amphiregulin. Cancer Res.
Amphiregulin is a vitamin D3 target gene in squamous cell and 61, 8122– 8126.
breast carcinoma. Biochem. Biophys. Res. Commun. 281, Wong, L., Deb, T. B., Thompson, S. A., Wells, A., and Johnson,
1051–1056. G. R. (1999). A differential requirement for the COOH-
Berquin, I. M., Dziubinski, M. L., Nolan, G. P., and Ethier, S. P. terminal region of the epidermal growth factor (EGF) receptor
(2001). A functional screen for genes inducing epidermal in amphiregulin and EGF mitogenic signaling. J. Biol. Chem.
growth factor autonomy of human mammary epithelial cells 274, 8900–8909.
confirms the role of amphiregulin. Oncogene 20, 4019–4028. Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
Brown, C. L., Coffey, R. J., and Dempsey, P. J. (2001). The
proamphiregulin cytoplasmic domain is required for basolat-
eral sorting, but is not essential for constitutive or stimulus-
induced processing in polarized Madin-Darby canine kidney
cells. J. Biol. Chem. 276, 29538–29549. Androgen Effects in Mammals
Cook, P. W., Pittelkow, M. R., and Piepkorn, M. (1999).
Overexpression of amphiregulin in the epidermis of transgenic
mice induces a psoriasis-like cutaneous phenotype. J. Invest.
SHALENDER BHASIN
Dermatol. 113, 860. Charles R. Drew University of Medicine and Science,
Damstrup, L., Kuwada, S. K., Dempsey, P. J., Brown, C. L., Los Angeles
Hawkey, C. J., Poulsen, H. S., Wiley, H. S., and Coffey, R. J., Jr
(1999). Amphiregulin acts as an autocrine growth factor in
I. TESTOSTERONE SECRETION, TRANSPORT, AND
two human polarizing colon cancer lines that exhibit domain
selective EGF receptor mitogenesis. Br. J. Cancer 80,
METABOLISM
1012–1019. II. TESTOSTERONE AS A PROHORMONE
Kitamura, S., Miyazaki, Y., Hiraoka, S., Nagasawa, Y., Toyota, M., III. MECHANISM OF ANDROGEN’S ACTION
Takakura, R., Kiyohara, T., Shinomura, Y., and Matsuzawa, Y. IV. TESTOSTERONE’S EFFECTS ON REPRODUCTIVE
(2001). PPARg agonists inhibit cell growth and suppress ORGANS
ANDROGEN EFFECTS IN MAMMALS 71

V. ANDROGEN’S EFFECTS ON NONREPRODUCTIVE BODY luteinizing hormone (LH) receptor have been studied;
SYSTEMS these patients have varying degrees of genital
VI. TESTOSTERONE’S EFFECTS ON BEHAVIOR ambiguity and Leydig cell agenesis, pointing to the
VII. TESTOSTERONE REPLACEMENT IN ANDROGEN- important role of LH in the regulation of Leydig cell
DEFICIENT MEN
development in humans.
VIII. ANABOLIC EFFECTS OF ANDROGENS IN PATIENTS WITH
CHRONIC ILLNESSES AND AGE-RELATED FRAILTY In man, 3 –10 mg of testosterone is secreted daily
by the testis; direct secretion of testosterone by the
adrenal and the peripheral conversion of androstene-
Testosterone, a 19-carbon steroid secreted by dione, which is secreted by the adrenal and converted
the testis, is the predominant androgen in most to testosterone, collectively account for another
mammalian species. Androgens directly or
500 mg of testosterone daily. Leydig cells arise from
indirectly affect almost all body systems during
fetal and pubertal development and in adult life. poorly characterized mesenchymal precursor cells
The sexual differentiation of the mammalian under the influence of luteinizing hormone, insulin-
fetus is a complex, multifactorial process that like growth factor-I, transforming growth factor-b,
requires not only the genetic information carried transforming growth factor-a, interleukin 1, and
on the sex chromosomes, but also the hormonal basic fibroblast growth factor. Leydig cells exist in
secretions of the fetal testis, namely, testoster- two distinct generations in higher mammals, fetal and
one and Müllerian-inhibiting hormone. Testos- adult, that are separated in higher mammals by a
terone masculinizes the Wolffian structures and prepubertal period during which the testis is devoid of
causes the external genitalia to form a scrotum
Leydig cells.
and penis. In addition, increasing testosterone
levels during puberty promote somatic growth Testosterone secretion by Leydig cells is under the
and virilization of boys. Testosterone plays a control of LH, a pituitary glycoprotein hormone. LH
critical role in mammalian reproduction; it is binds to specific G-protein-coupled receptors on the
essential for maintaining sexual function, germ Leydig cells and activates the cyclic AMP pathway.
cell development, and accessory sex organs. Although LH also activates the phopholipase C
In the adult animal, testosterone has additional pathway, it is unclear whether this pathway is
effects on the muscle, bone, hematopoeisis, essential for LH-mediated stimulation of testosterone
coagulation, plasma lipids, protein and carbo- production. The rate-limiting step in testosterone
hydrate metabolism, and psychosexual and
biosynthesis is the delivery of cholesterol to the inner
cognitive function.
mitochondrial membrane, which is the site of the
cholesterol side chain cleavage complex that converts
I. TESTOSTERONE SECRETION, TRANSPORT, cholesterol to pregnenolone. A steroidogenesis acute
AND METABOLISM regulatory protein makes cholesterol available to the
cholesterol side chain complex and regulates the rate
A. Testosterone Secretion of testosterone biosynthesis. Peripheral benzo-
In males of most mammalian species, 95% of diazepine receptor, a mitochondrial cholesterol-
circulating testosterone is derived from testicular binding protein known to be involved in mediating
secretion. Only a small amount of dihydrotestoster- cholesterol transport, is present in high concen-
one (DHT; approximately 70 mg daily) is secreted trations in the outer mitochondrial membrane and
directly by the human testis; most circulating DHT is has also been proposed as an acute regulator of
derived from the peripheral conversion of testoster- Leydig cell steroidogenesis. In addition, another
one. Testosterone is produced in the testis by a group of poorly characterized mitochondrial proteins
heterogeneous group of cells that includes the adult has been implicated in the control of steroidogenesis
Leydig cells, Leydig cell precursors, and immature in Leydig cells.
Leydig cells. Studies in hypogonadotropic (hpg) mice Leydig cell production of testosterone is modu-
suggest that fetal development of both Sertoli and lated by a number of paracrine factors within the
Leydig cells is independent of gonadotropins; how- seminiferous tubule and the interstitium of the testis,
ever, normal differentiation and proliferation of the including insulin-like growth factor-I, insulin-like
adult Leydig cell population require the presence of growth factor-binding proteins, inhibins, activins,
gonadotropins. The number of Sertoli cells after birth transforming growth factor-b, epidermal growth
is regulated by gonadotropins. A number of 46,XY factor, interleukin-1, basic fibroblast growth factor,
male humans with inactivating mutations of the gonadotropin-releasing hormone, and vasopressin.
72 ANDROGEN EFFECTS IN MAMMALS

B. Androgen Transport in the Body blood and through a series of chemical reactions that
involve 5a- and 5b-reductases, 3a- and 3b-hydroxy-
Ninety-eight percent of circulating testosterone is steroid dehydrogenases, and 17b-hydroxysteroid
bound to plasma proteins: the sex hormone-binding dehydrogenase converts it into androsterone and
globulin (SHBG) and albumin. The sex hormone- etiocholanolone, which are both inactive metabolites,
binding globulin binds testosterone with much dihydrotestosterone, and 3-a-androstanediol. These
greater affinity than albumin. Only 1 to 3% of compounds undergo glucoronidation or sulfation
testosterone is unbound; although the dogma has before their excretion by the kidneys. Free and
been that only the unbound fraction is biologically conjugated androsterone and etiocholanaolone are
active, it has been argued that albumin-bound the predominant urinary metabolites of testosterone.
hormone may dissociate readily in the capillaries
and thus become bioavailable. In fact, Pardrige et al.
have demonstrated that albumin- and SHBG-bound II. TESTOSTERONE AS A PROHORMONE
androgens represent the major circulating pool of
Testosterone can be converted in many peripheral
bioavailable hormone for testis or prostate. Further-
tissues into its active metabolites, 17b-estradiol and
more, these investigators have argued that the
5a-DHT (Fig. 1). Aromatization of the A ring
SHBG – sex steroid complex may be nearly comple-
converts it into 17b-estradiol. In addition, reduction
tely available for influx through the blood – testis
of the d4 double bond can convert testosterone into
barrier or prostate plasma membrane; this view is not
5a-DHT. Testosterone’s actions in many tissues are
universally shared.
mediated through these metabolites. For instance,
SHBG is a glycoprotein, synthesized in the liver,
testosterone’s effects on trabecular bone resorption
that displays high-affinity binding for testosterone
and the sexual differentiation of the brain require its
and estradiol. Hepatic production of SHBG is
aromatization to estradiol. Testosterone’s effects on
regulated by insulin, thyroid hormones, dietary
plasma lipids, atherosclerosis progression, and some
factors, and the balance between androgens and
types of behaviors are also mediated through its
estrogens. SHBG is involved in the transport of sex
conversion to estrogen. Considerable gains in insight
steroids in plasma and its concentration is a major
into the role of estrogen in mammalian physiology
factor regulating their distribution between the
have been made through the study of three mouse
protein-bound and free states, although its functional
knockout models: the estrogen receptor-a, estrogen
role in testosterone delivery to target organs is not
receptor-b, and aromatase knockouts. These models
clear. Plasma SHBG concentrations are decreased by
of estrogen deficiency exhibit significant disruption of
androgen administration, obesity, insulin, and
spermatogenesis and fertility, elevated testosterone
nephrotic syndrome. Conversely, estrogen adminis-
and LH levels, decreased bone mass, and increased
tration, hyperthyroidism, many types of chronic
adiposity, indicating the important role of estrogens
inflammatory illnesses, and aging are associated
in the regulation of bone mass, gonadotropin
with high SHBG concentrations. A locus that is
regulation, body composition, and spermatogenesis.
associated with SHBG concentrations in African
A very small number of humans with inactivating
Americans and Caucasians has been mapped to
mutations of the CYP19 aromatase gene have been
1q44. In addition, several other loci in African
Americans exhibit linkage with SHBG concen-
trations, suggesting that many genes likely regulate
SHBG levels. The binding of testosterone to SHBG or
albumin is not essential for androgen action or steroid
homeostasis; rats that are deficient in both sex
hormone-binding globulin and albumin are fertile
and have normal mating behavior.

C. Testosterone Metabolism
Testosterone is metabolized predominantly in the
liver (50 – 70%) although some degradation also FIGURE 1 Effects of testosterone and its metabolites in men.
occurs in peripheral tissues, particularly the prostate DHT, dihydrotestosterone; HDL, high-density lipoprotein.
and the skin. Liver takes up testosterone from the Modified from Bhasin and Fisher (2002), with permission.
ANDROGEN EFFECTS IN MAMMALS 73

reported. Females with CYP19 gene mutations are A large body of information about the role of
masculinized, fail to undergo female pubertal devel- DHT has emerged from the study of patients with the
opment, have elevated levels of androgens, LH, and autosomal recessive genetic disorder of steroid 5a-
follicle-stimulating hormone, have polycystic ovaries, reductase deficiency. 46,XY males with this syndrome
and are tall in stature. Males with CYP19 aromatase contain normal male internal structures including
mutations have been characterized by osteoporosis, testes but exhibit ambiguous or female external
increased bone turnover, delayed epiphyseal fusion, genitalia at birth. At puberty, these individuals
tall stature, and increased testosterone but markedly undergo partial virilization, which includes develop-
decreased estradiol levels. ment of a male gender identity in many but not all
At least two isoenzymes of steroid 5a-reductase 46,XY individuals, even if they have been brought up
have been cloned and characterized. Type 1 steroid as females. Their development suggests that testos-
5a-reductase isoenzyme is expressed in many non- terone itself is able to stimulate psychosexual
genital tissues, has been mapped to chromosome behavior, libido, development of the embryonic
region 5p15, and has a pH optimum of 8. Type 2 Wolffian duct, muscle development, voice deepening,
steroid 5a-reductase isoenzyme is expressed in the spermatogenesis, and axillary and pubic hair growth.
prostate and other genital tissues, has been mapped to In contrast, DHT is required for prostate develop-
2p23, and has a pH optimum of 5.0. The biological ment and growth, the development of the external
role of 5a-reductase type 1 has not yet been genitalia, and male patterns of facial and body hair
ascertained. Gene-targeting experiments suggest that growth or male-pattern baldness. All the 5a-
type 1 enzyme plays a role in progesterone metab- reductase-deficient kindreds that have been studied
olism at the end of pregnancy. The mice lacking 5a- to date have been shown to have mutations in steroid
reductase enzyme type 1 have failure of cervical 5a-reductase type 2, the predominant form in the
ripening and fail to deliver. prostate. Many patients with congenital deficiency of
Testosterone’s effects on the prostate and seba- the type 2 isoform of 5a-reductase have low or low
ceous glands of the skin require its 5a-reduction to normal DHT levels at puberty, either due to the
DHT. DHT has been implicated in the pathophysiol- activity of type 1 isoenzyme or due to the residual low
ogy of benign prostatic hyperplasia and androgenic activity of type 2 isoenzyme. Therefore, it is not clear
alopecia. Type 2 isoenzyme is the predominant form whether the normal muscle development and male
in the prostate and has been implicated in the psychosexual behavior observed in many of these
pathophysiology of benign prostatic hypertrophy, patients after puberty are due to the low normal DHT
hirsutism, and possibly male-pattern baldness. levels or testosterone itself.
During embryonic life, testosterone controls the
differentiation of the Wolffian ducts into epididymis,
III. MECHANISM OF ANDROGEN’S ACTION
vasa deferentia, and seminal vesicles. The develop-
ment of structures from the urogenital sinus and the Most of androgen’s actions are mediated through its
genital tubercle such as the scrotum, penis, and penile binding to an intracellular androgen receptor that
urethra require the action of DHT. The role of 5a- acts as a ligand-dependent transcription factor. The
reduction of testosterone in mediating its effects on androgen receptor has homology to other nuclear
muscle and sexual function remains unclear. receptor proteins including the receptors for gluco-
Although testosterone and DHT can both exercise corticoids, progesterone, and mineralocorticoids. The
anabolic effects on the muscle, steroid 5a-reductase predominant 919-amino-acid, 110 – 114 kDa, andro-
activity is very low or absent in the skeletal muscle, gen receptor protein has three conserved functional
and it is not known whether 5a-reduction of domains: the steroid-binding domain, the DNA-
testosterone to DHT is obligatory for mediating binding domain, and the transcriptional activation
androgen’s effects on the muscle. Similarly, previously domain; of these, the central, cysteine-rich, DNA-
published data are unclear on whether androgen’s binding domain is the most conserved. The single-
effects on sexual function in adult men are mediated copy androgen receptor gene spans a 90 kb region on
through testosterone or its 5a-reduced metabolite, chromosomal region Xq11 – q12. In the absence of its
DHT. Although the enzyme 5a-reductase is expressed ligand, the androgen receptor protein is distributed
in the muscle at low levels, it is generally believed both in the nucleus and in the cytoplasm. However,
that conversion of testosterone to DHT is not androgen binding to the receptor causes it to
obligatory for mediating its anabolic effects on the translocate within the nucleus; the sequence between
muscle. amino acids 617 and 633 of the androgen receptor is
74 ANDROGEN EFFECTS IN MAMMALS

important for its nuclear migration and transactiva- length of the polyglutamine tract in exon 1 of the
tion function. There is emerging evidence that some androgen receptor has been associated with spinal
of the effects of androgens may be mediated through and bulbar muscular atrophy, also known as
nongenomic receptors on the cell membrane. Kennedy’s disease. Although some studies have
Binding of androgens by the androgen receptor reported an association of androgen receptor poly-
results in conformational changes in this protein; morphisms in the lengths of polyglutamine and
there is evidence that binding of anti-androgens to the polyglycine tracts with male infertility and risk of
androgen receptor might induce a different set of prostate cancer, other studies have not confirmed these
conformational changes. The androgen receptor can findings.
use two transactivation domains, AF1 and AF2. The
transactivation domain AF1 (including the so-called 1
and 5 regions) is located in the amino-terminal IV. TESTOSTERONE’S EFFECTS
portion of the receptor, whereas AF2 is located in ON REPRODUCTIVE ORGANS
the carboxy-terminal, ligand-dependent domain. In
the intact receptor, both AF1 and AF2 are ligand-
A. Testosterone and the Prostate
dependent and influenced by nuclear receptor co- Androgens are required for normal prostate develop-
activators. In contrast, in a truncated androgen ment; however, it is not known whether androgens
receptor that is missing the ligand-binding domain, play a role in the initiation of prostate neoplasms.
AF1 becomes constitutively active. Hormone binding Conversion of testosterone to DHT is obligatory for
to the androgen receptor results in the assembly of mediating its effects on the prostate. The prostate
tissue-specific co-activators and co-repressors that increases in size during the peripubertal period in
determine the specificity of hormone action. parallel with the rise in serum testosterone levels.
In a majority of androgen target tissues, either Androgen imprinting prior to puberty is an important
testosterone or its 5a-reduced metabolite, DHT, binds determinant of the number of stem cells within the
to the androgen receptor and regulates gene prostate and hence adult prostate size in rats and
expression. Testosterone binds to the androgen dogs. Castration before puberty, 5a-reductase
receptor with half the affinity of DHT, although the deficiency, and inactivating mutations of the andro-
maximal binding capacity is similar for both andro- gen receptor prevent subsequent development of
gens. The DHT– androgen receptor complex has benign prostatic hypertrophy. Many of the effects of
greater thermostability and a slower dissociation androgen on the prostatic epithelium are mediated
rate than the testosterone – receptor complex. This through the stromal cells; thus, the interaction
may confer greater potency to DHT in mediating between the stromal cells and the epithelial cells is
androgen’s effects in some androgen-sensitive tissues, important for normal prostatic development. Andro-
such as the prostate. However, the reason that 5a- gen’s effects on prostatic growth are mediated in part
reduction is necessary for mediating androgen’s effects through the regulation of apoptotic genes. Androgen
in some tissues and not others is unclear. Why this deficiency in adult males is associated with increased
metabolic conversion step evolved for testosterone expression of many apoptotic genes, resulting in
and not for other steroid hormones is also unknown. involution of the prostate.
The mutations of the androgen receptor gene Only the human and the dog develop benign
have been associated with a wide spectrum of pheno- prostatic hypertrophy. Androgens are essential, but
typic abnormalities. Some patients with complete not sufficient, for the development of benign prostatic
androgen insensitivity present with male pseudo- hypertrophy. Abnormal stromal activation and dys-
hermaphroditism characterized by female external regulation of several growth factors play a role in the
genitalia, a blind vaginal pouch, and well-developed pathogenesis of this complex disorder. Although
breasts. Other patients with androgen receptor androstanediol and DHT can induce prostatic enlar-
mutations may have a male phenotype and milder gement in the dog, physiologic testosterone adminis-
abnormalities such as hypospadias, gynecomastia, tration to hypogonadal men has not been associated
and infertility. with pathologic prostatic enlargement beyond the
A number of polymorphisms in the androgen levels observed in healthy, age-matched controls.
receptor gene have been described; the lengths of the Estradiol accentuates prostatic hypertrophy induced
CAG and GCG repeats in exon 1 of the androgen by androgen administration in the dog. The critical
receptor gene have been linked to transcriptional role of androgens in prostatic growth is supported by
activity of androgen receptor protein. An abnormal clinical findings that lowering serum testosterone
ANDROGEN EFFECTS IN MAMMALS 75

levels by surgical orchidectomy or by pharmacologic as proenkephalin, are down-regulated after castration


inhibitors of testosterone production decreases but are not restored by androgen replacement,
prostate volume in men with benign prostatic whereas expression of other genes, such as
hyperplasia. Similarly, clinical trials have established E-cadherin, is decreased by castration in a region-
that finasteride, an inhibitor of 5a-reductase enzyme, specific manner and is restored to varying degrees by
produces a clinically significant reduction in prostate androgen administration.
size in men with benign prostatic hypertrophy.

B. Testosterone and Spermatogenesis V. ANDROGEN’S EFFECTS ON


NONREPRODUCTIVE BODY SYSTEMS
High intratesticular testosterone concentrations are
required for the initiation and maintenance of A. Androgen’s Effects on Bone
spermatogenesis. Gonadotropin deficiency resulting
Androgens are important regulators of bone matu-
from hypophysectomy or administration of a
ration and maintenance of bone mass. Androgens
gonadotropin-releasing hormone (GnRH) antagonist
promote linear bone growth and endochondral
is associated with a marked depletion of intratesti-
ossification in rats. In humans, the peri-pubertal
cular testosterone concentrations and azoospermia in
bone accretion is androgen dependent; consequently,
the rat, monkey, and human male. High doses of
men with androgen deficiency during the peri-
testosterone can reinitiate and maintain spermato-
pubertal period have diminished peak bone mass.
genesis in rats made gonadotropin-deficient by
Adult men with testosterone deficiency have lower
hypophysectomy or GnRH antagonist treatment. In
bone density than age-matched, healthy controls;
men with acquired gonadotropin deficiency, luteiniz-
testosterone replacement therapy of hypogonadal
ing hormone can reinitiate and maintain spermato-
men increases but does not normalize bone density.
genesis by stimulating testosterone production within
Suppression of serum testosterone levels by adminis-
the testis. The mechanism by which testosterone
tration of GnRH agonist analogues or surgical
maintains germ cell development is not well under-
orchidectomy leads to a progressive decrease in
stood. Testosterone receptors are present on the
bone density. Testosterone’s effects on bone are
Sertoli and peritubular cells, some Leydig cells, and
mediated in part through its conversion to estradiol.
endothelial cells of the small arterioles. However, it
The critical role of aromatization in mediating
has not been established whether androgen receptors
androgen’s effects on bone is supported by obser-
are also present on germ cells. It is generally believed
vations that 46,XY men with mutations of the genes
that androgen’s effects on spermatogenesis are
that code for the estrogen receptor or the aromatase
mediated indirectly through Sertoli cells, although it
enzyme have delayed epiphyseal fusion and decreased
is possible that testosterone might also directly affect
bone density. Also, in epidemiological studies, bio-
germ cell development. Testosterone affects protein
available estradiol levels correlate better with bone
secretion by both round spermatids and Sertoli cells.
mineral density than total testosterone levels. Testos-
The expression of androgen receptors is maximal at
terone has additional direct effects in stimulating
stages VI – VII of the seminiferous epithelium; the
cortical bone formation. Androgen receptors have
effects of testosterone might be exerted on the germ
been demonstrated on the osteoblasts. Testosterone
cells as they pass through these stages. More recent
might also indirectly affect bone mineral density
evidence suggests that testosterone regulates germ cell
through stimulation of insulin-like growth factor-I
apoptosis in a stage-specific manner.
and by increasing muscle mass and muscle strength.
C. Testosterone’s Effects on the Epididymis
B. Androgen and Body Composition—Effects on
Androgens are important regulators of epididymal
Muscle and Fat Metabolism
structure and function. A variety of epididymal
biochemical processes, such as the transport of Testosterone is a major determinant of body compo-
small molecules across the epididymal epithelium, sition in male mammals. The sexual dimorphism of
DNA and RNA synthesis, protein synthesis, activity several androgen-responsive muscles such as the
of a number of epididymal enzymes, and maturation masseter and levator ani in several mammalian
and storage of spermatozoa, are under androgen species has been attributed to differences in androgen
control. The regulation of epididymal function by levels in males and females. Testosterone adminis-
androgens is highly region-specific. Some genes, such tration increases nitrogen retention in castrated males
76 ANDROGEN EFFECTS IN MAMMALS

of several mammalian species and in hypogonadal growth hormone secretory status, the existence of
men, women, and boys before puberty. Recent studies co-morbid conditions, cytokines, and exercise status.
have established that testosterone replacement in
hypogonadal men increases fat-free mass, muscle size, C. Testosterone’s Effects on Intermediary
and maximal voluntary strength. Supraphysiologic Metabolism and Cardiovascular Risk
doses of androgens further increase muscle mass and
Testosterone is an anabolic hormone that increases
strength in eugonadal men, especially when given in
muscle mass by stimulating fractional muscle protein
association with resistance exercise. Controlled clini-
synthesis. The mechanism by which testosterone
cal trials have demonstrated that testosterone sup-
increases protein synthesis is not known. It has been
plementation in older men with low testosterone
proposed that testosterone increases muscle protein
concentrations and in men with human immunode-
synthesis by increasing the expression of the insulin-
ficiency virus (HIV) infection or chronic obstructive
like growth factor-I gene within the muscle. It is not
lung disease and low testosterone concentrations is
known whether testosterone affects muscle protein
associated with significant gains in fat-free mass and
degradation. Androgen receptors are present on
maximal voluntary muscle strength.
adipocytes and testosterone stimulates lipolysis in
Widespread abuse of androgenic steroids by
some experimental models. The effects of testosterone
athletes and recreational body builders is based on
on lipid uptake and metabolism are region-specific.
the premise that androgens promote muscle hyper-
Cross-sectional studies of middle-aged men demon-
trophy. Testosterone’s effects on muscle mass,
strate a direct correlation of serum testosterone levels
strength, and leg power are dependent on the
with insulin sensitivity and an inverse correlation
administered dose of testosterone and the circulating
with visceral fat. However, physiologic replacement
testosterone concentrations. The effects of androgen
or slightly supraphysiologic doses of testosterone
administration on muscle performance and physical
have not been shown to affect glucose tolerance or
function have not been well studied, although the
insulin sensitivity in lean, young men.
available evidence indicates that testosterone does not
improve aerobic performance. There is some evidence
D. Androgen’s Effects on the Skin
that testosterone might reduce reaction time by its
effects on neuromuscular transmission. The hair follicles, sebaceous glands, and apocrine
A testosterone-induced increase in muscle mass is sweat glands in the skin are androgen-sensitive
associated with hypertrophy of both type I and type II structures. Increased testosterone levels during
muscle fibers but no change in muscle fiber number. pubertal development stimulate the growth of term-
Testosterone administration in healthy young men is inal hair in the axillary and pubic areas in both
also associated with an increase in the number of men and women and on the face in men. Excessive
myonuclei and satellite cells. Emerging evidence androgen production or action has been implicated in
suggests that testosterone stimulates the differen- the pathophysiology of clinical disorders such as acne,
tiation of muscle pluripotent cells into the myogenic hirsutism, androgenic alopecia, and suppurative
lineage and inhibits their differentiation into the hidradenitis. Many investigators believe that local
adipogenic lineage. abnormalities in androgen metabolism within the
Androgen-deficient men have a greater amount of skin in hirsute women make their skin more
fat than eugonadal men. In middle-aged men, serum sensitive to the effects of circulating androgens.
testosterone levels correlate inversely with visceral fat Testosterone’s effects on the skin are mediated
and cardiovascular risk. In healthy, young men, through its conversion to DHT. Type 1 isoenzyme is
administration of graded doses of testosterone leads the major form of steroid 5a-reductase in the
to a dose-dependent decrease in whole body fat; skin. The highest level of activity of the steroid
the decrease in fat mass occurs in both the trunk and 5a-reductase enzyme has been located in the apocrine
the appendices and is evenly distributed in both the glands and the sebaceous glands. Inhibitors of type 1
superficial subcutaneous and the deep intermuscular steroid 5a-reductase enzyme in the skin, such as
and visceral fat compartments. Physiologic testoster- aliphatic unsaturated fatty acids, are being explored
one replacement in middle-aged men with midseg- as therapeutic agents for the treatment of androgen-
ment obesity decreases visceral fat, glucose, and responsive skin disorders such as hirsutism and acne.
insulin levels. Androgen’s effects on body compo- Finasteride, a weak inhibitor of type 2 isoenzyme,
sition in men are modulated by pretreatment body has been approved by the Food and Drug Adminis-
composition, genetic and nutritional factors, the tration for the treatment of androgenic alopecia.
ANDROGEN EFFECTS IN MAMMALS 77

E. Androgen’s Effects on Hematopoiesis, the ment of hypogonadal men modestly lowers plasma
Coagulation System, and the Vascular System HDL cholesterol levels. Other studies using physio-
logic replacement doses in older men or HIV-infected
Testosterone increases red cell mass; this effect is men have reported either no change or only minor
mediated primarily by increasing the production of changes in plasma lipid profiles. Cross-sectional
erythroid precursors from the pluripotent stem cells epidemiological studies demonstrate a direct relation-
in the bone marrow. Testosterone has also been ship between serum testosterone levels and HDL
reported to increase erythropoietin production in the cholesterol levels and an inverse relationship between
kidney, although other data show that testosterone testosterone levels and visceral fat. Therefore, these
can increase red cell production independent of its data suggest that testosterone levels in the mid- to
effects on erythropoietin. There are numerous anec- high normal range may be optimum for cardiovas-
dotal reports of sudden death, vascular thrombosis, cular health in men. Indeed, physiologic testosterone
myocardial infarctions, and cardiomyopathy in rela- replacement decreases visceral fat, glucose, and
tively young athletes abusing large quantities of insulin levels in middle-aged men with visceral
anabolic steroids, leading to speculation that testos- obesity. It is not known what testosterone levels are
terone increases the thromobogenicity of plasma. consistent with optimum cardiovascular risk in men.
However, the effects of testosterone on the coagu- The prevalent dogma that physiologic testoster-
lation system remain controversial. Fibrinogen and one replacement increases cardiovascular risk and
plasminogen activator inhibitor concentrations in accelerates atherosclerosis progression is not sup-
plasma correlate directly with endogenous testoster- ported by data. Of the 30 cross-sectional studies
one; these changes may be viewed as favoring plasma reviewed by Alexanderson et al., 18 reported lower
thrombogenicity. However, androgens also increase testosterone concentrations in men with coronary
tissue plasminogen activator, protein C, and anti- artery disease than in age-matched controls, 11
thrombin III levels; these factors protect the body reported similar levels, and only 1 study reported
from vascular thrombosis. Testosterone and DHT higher concentrations of dehydroepiandrosterone
increase platelet aggregation at the site of endothelial sulfate in men with coronary artery disease than
trauma. Danazol, a weak synthetic androgen, has age-matched controls. In mice with low-density
been reported to increase platelet count. Androgens lipoprotein receptor mutations, surgical orchidect-
may increase vascular reactivity to trauma and omy is associated with accelerated atherogenesis, and
decrease aortic smooth muscle prostaglandin I2. testosterone replacement retards atherosclerosis pro-
Therefore, some effects of androgens appear to gression. Testosterone infusion has been reported to
favor hypercoagulability and others appear to oppose improve coronary blood flow in humans and dogs.
it. The net effect in a person may vary with the type Testosterone’s effects on blood vessel walls require its
and dose of androgen used, genetic predisposition, conversion to estradiol because concomitant admin-
simultaneous use of other medications, and the istration of an aromatase inhibitor can attenuate the
presence of co-morbid conditions. beneficial effects of testosterone in retarding athero-
genic lesions. The effects of testosterone replacement
on atherosclerosis progression in older men with low
F. Androgens and Plasma Lipids
testosterone levels have not been studied.
and Cardiovascular Risk
The effects of androgens on plasma lipids depend on
the dose (physiologic or supraphysiologic), the route VI. TESTOSTERONE’S EFFECTS ON BEHAVIOR
of administration (oral or systemic), and the type of
A. Androgen’s Effects on Sexual Function
androgen used (aromatizable or not aromatizable).
Supraphysiologic doses of testosterone and other Sexual function in men is a complex multicomponent
androgenic steroids decrease plasma high-density process that includes central mechanisms for the
lipoprotein (HDL) cholesterol levels in men. Andro- regulation of sexual desire and arousability and local
genic steroids that can be aromatized produce a mechanisms for penile tumescence, orgasm, and
smaller decrease in high-density lipoprotein levels ejaculation. The primary effects of testosterone are
than those that cannot be aromatized. Lowering on sexual interest and motivation. Observations that
serum testosterone levels experimentally by GnRH eunuchs and hypogonadal men can have erections are
antagonist administration increases plasma HDL consistent with the proposal that testosterone regu-
cholesterol levels; conversely, testosterone replace- lates libido rather than erectile function. Davidson
78 ANDROGEN EFFECTS IN MAMMALS

et al. demonstrated that although overall sexual well-being, independent of the change in erectile
activity is reduced in hypogonadal men, these function; this hypothesis has not been examined.
patients have a normal erectile response to visual In rats, mating behavior can be maintained at
erotic stimuli. Testosterone replacement of young, serum testosterone levels that are at the lower end of
androgen-deficient men improves a wide range of the normal male range. Similarly, in men in whom
sexual behaviors including frequency of sexual endogenous testosterone secretion has been sup-
activity, sexual daydreams, sexual thoughts, feelings pressed by a GnRH agonist, low doses of testosterone
of sexual desire, spontaneous erections, episodes of that bring serum testosterone levels to the lower end
nocturnal penile tumescence, and the duration of of the normal range can maintain sexual desire and
penile erections in response to visual erotic stimuli. activity and nocturnal erections. However, supraphy-
Testosterone regulates nitric oxide synthase activity siologic doses of testosterone might further increase
in the cavernosal smooth muscle, and it is possible some aspects of sexual arousability.
that achievement of optimal penile rigidity might The role of 5a-reductase in mediating androgen’s
require physiologic testosterone concentrations. effects on sexual function remains unclear. Although
Orgasm and ejaculation are androgen-independent. DHT can restore sexual behavior in castrated
In male mammals, relatively low normal levels of monkeys, patients with 5a-reductase deficiency have
serum testosterone can maintain sexual function. normal libido and erectile function. Furthermore, the
There is considerable confusion among clinicians relatively low frequency of sexual dysfunction in men
with respect to the relationship between androgen treated with finasteride, an inhibitor of 5a-reductase
deficiency and erectile dysfunction. In middle-aged enzyme, suggests that testosterone conversion to
and older men, erectile dysfunction and androgen DHT is not required for androgen’s effects on sexual
function. Important insights into the role of 5a-
deficiency are two common but independently
reduction in regulating male sexual behavior have
distributed clinical disorders that sometimes co-exist
emerged from the studies of a synthetic steroid, 7a-
in the same patient. Eight to 10% of men presenting
methyl-19-nortestosterone (MENT). This androgen
with erectile dysfunction have low testosterone levels.
binds the androgen receptor and exerts biologic
The prevalence of low testosterone levels is not
effects at some peripheral tissues with a greater
significantly different between middle-aged and older
potency than testosterone. In vivo, MENT does not
men with impotence and those without impotence.
undergo 5a-reduction. MENT, when administered to
Testosterone administration does not improve sexual
castrated rats, maintains full copulatory behavior
function in impotent men with normal testosterone
similar to that observed in testosterone-treated rats.
levels. On the other hand, many, but not all, older These studies of MENT suggest that 5a-reduction of
men with low testosterone levels experience improve- testosterone is not necessary for maintaining normal
ments in their libido and overall sexual activity with mating behaviors in the male rat. However, penile
androgen replacement therapy. The response to nitric oxide synthase activity in the rat is stimulated
testosterone supplementation in this group of men is by testosterone, and it is possible that conversion of
variable because of the co-existence of other disorders testosterone to DHT may be required for achieving
such as diabetes mellitus, hypertension, cardiovascu- penile rigidity.
lar disease, and psychogenic factors. A meta-analysis
of the usefulness of androgen replacement therapy
concluded that testosterone administration is associ- B. Testosterone’s Effects on Nonreproductive
ated with greater improvements in sexual function Behaviors
than placebo treatment in men with sexual dysfunc- Testosterone is aromatized to estradiol in the brain
tion and low testosterone levels. However, previous and some effects of testosterone may be mediated
studies of testosterone replacement in older men have through its conversion to estradiol. However, andro-
been flawed because of failure to include a placebo- gen receptors are expressed in specific regions of the
control group, small sample sizes, selection of men brain and likely mediate some of testosterone’s
with erectile dysfunction associated with multiple organizational effects during brain development and
systemic illnesses, which may attenuate the response some activational effects postnatally. There are
to testosterone therapy, and inclusion of men with gender differences in the distribution of androgen
normal testosterone levels. Testosterone might also receptor in the human hypothalamus.
favorably affect marital interactions and intimacy due During fetal life, testosterone is essential for
to an overall increase in sexual desire and sense of sexual differentiation of the brain along male lines.
ANDROGEN EFFECTS IN MAMMALS 79

There are structural sex differences in the volume and determine the effects of testosterone supplementation
the synaptic organization of many brain nuclei or over a wider range of doses on a number of domains
regions. Testosterone promotes aggression among of cognitive function.
males, particularly at the time of mate selection. The
scent marking behavior, a marker for territoriality, is
testosterone-dependent in many mammalian males. VII. TESTOSTERONE REPLACEMENT
The effects of testosterone on human aggression IN ANDROGEN-DEFICIENT MEN
remain controversial. There is a significant, though Testosterone, when administered by mouth, is
not strong, correlation between serum testosterone absorbed readily from the gastrointestinal tract;
levels and some aspects of aggressive behavior in men. however, most of it is destroyed during its first pass
A significant proportion of anabolic steroid users through the liver. Therefore, an appropriate delivery
report mood disorders, manic and hypomanic syn- system is needed to achieve sustained levels of
dromes characterized by aggressiveness, and other testosterone in the blood. Because of the potential
psychiatric disorders. Administration of high doses of contraceptive and anabolic applications of andro-
testosterone to men is associated with increased gens, there has been considerable interest in develop-
aggressive responses compared to placebo. However, ing more physiologic and long-acting testosterone
other well-controlled studies have failed to find clear delivery systems (Table 1).
increases in aggressive behaviors in androgen-treated
men. Testosterone affects the development of sex-
typed nonreproductive behaviors such as maze A. Testosterone Esters
learning and juvenile play. Androgens are considered Esterification of the 17b-hydroxyl group makes the
to be responsible for the greater visuospatial ability compound more hydrophobic. Therefore, when these
and lower verbal ability in men than in women. esters are injected in an oil suspension in the muscle,
Exposure to testosterone through its conversion to they are absorbed very slowly from the muscle depot
estradiol during the neonatal period is responsible for into the bloodstream. It is the slow release rather the
the development of brain structures that subserve slow deesterification that accounts for the extended
spatial functions. Alterations of androgen levels in duration of testosterone esters. The longer the side
adult animals do not affect spatial processing but may chain, the more hydrophobic the compound and the
influence memory storage and affective properties. longer the duration of action. Testosterone enanthate
The data on the relationship of circulating and cypionate, the two commonly used testosterone
androgen concentrations with cognitive function are esters, have identical kinetics. After injection of
conflicting. Androgen’s effects on cognitive function 200 mg testosterone enanthate or cypionate, the
are domain-specific. Observations that men outper- usual replacement dose in hypogonadal men, serum
form women in a variety of visuospatial skills suggest testosterone levels rise into the supraphysiologic
that androgens enhance visuospatial skills. Clinical range within 24 to 48 h and then gradually decline
trials in older men suggest that testosterone admin- into the hypogonadal range 10 –17 days later. These
istration might enhance visuospatial skills and verbal fluctuations in serum testosterone levels are associ-
memory. Hypogonadal men perform worse on tests ated with changes in the patient’s mood and energy
of verbal fluency than eugonadal men and show level and are a major drawback of this formulation.
improvement after testosterone replacement. In Variations in serum testosterone levels during treat-
transsexual males, administration of anti-androgen ment with testosterone esters can be minimized by
and estrogen decreases anger and aggression, sexual giving 100 mg of testosterone enanthate or cypionate
arousability, and spatial skills and increases verbal every week. With administration of physiologic
fluency. Conversely, testosterone administration to replacement doses, serum levels of estradiol and
female transsexuals decreases verbal fluency and DHT are normal. Given an adequate dose, testoster-
increases spatial skills. one esters can produce virilization, restore sexual
The reported literature on testosterone and cogni- function, and increase muscle mass, bone mass, and
tion is equivocal but these inconsistencies should hematocrit in hypogonadal men.
not be interpreted to mean that there is no effect.
Furthermore, previous studies of androgen sup-
B. Testosterone Transdermal Systems
plementation have focused on a very limited number
of domains. Prospective, randomized, placebo- Three transdermal testosterone systems are commer-
controlled trials are needed to comprehensively cially available to treat hypogonadal men: a scrotal
TABLE 1 Clinical Pharmacology of the Testosterone Formulations

Formulation Regimen Pharmacokinetic profile DHT and estradiol Advantages Disadvantages


Testosterone 100 mg im weekly or After a single im injection, DHT and E2 levels rise in Corrects symptoms of Requires im injection;
enanthate 200 mg im every 2 serum T levels rise into proportion to the increase androgen deficiency; peaks and valleys in
or cypionate weeks the supraphysiological in T levels; T:DHT and relatively inexpensive, serum T levels
(127 –129) range and then decline T:E2 ratios do not change if self-administered;
gradually into the flexibility of dosing
hypogonadal range by
the end of the dosing
interval (127–129)
Scrotal One scrotal patch Normalizes serum T levels Serum E2 levels are in the Corrects symptoms of To promote optimum
testosterone designed to nominally in many but not all physiological male range, androgen deficiency adherence of the patch,
patch deliver 6 mg over androgen-deficient men but DHT levels rise into scrotal skin needs to be
(130 –131) 24 h, applied daily the surpraphysiological shaved; high DHT levels
range; T:DHT ratio is
significantly lower than
in healthy men (130– 132)
Nongenital One or two patches, Restores serum T, DHT, T:DHT and T:E2 levels are Ease of application; Serum testosterone levels in
transdermal designed to nominally and estradiol levels into in the physiological male corrects symptoms of some androgen-deficient
system deliver 5– 10 mg T over the physiological male range androgen deficiency; men may be in the low
(133 –134) 24 h, applied daily on range mimics the normal normal range; these men
nonpressure areas diurnal rhythm of may need application of
testosterone secretion; two patches daily; skin
lesser increase in irritation at the application
hemoglobin than site may be a problem for
injectable esters some patients
Testosterone Testosterone gel Restores serum T and Serum DHT levels and Corrects symptoms of Potential of transfer to a
gel containing 50 – 100 mg estradiol levels into T:DHT ratios are lower androgen deficiency; female partner or child
testosterone, should be the physiological in hypogonadal men provides flexibility of by direct skin-to-skin
applied daily male range treated with the dosing; ease of contact; moderately high
testosterone gel than in application; good DHT levels
healthy eugonadal men skin tolerability
17a-Methyl Orally active, Orally active Clinical responses variable;
testosterone 17a-alkylated potential for liver toxicity;
(135) compound that should not be used for
should not be used treatment of androgen
because of potential deficiency
for liver toxicity
Testerone Three or four 200 mg Maintains serum T and Normal DHT and E2 Long duration Requires insertion through
pellets pellets inserted free T in normal range concentrations a trocar
under the skin for 3 –4 months
Testosterone 80 –160 mg daily Serum T increases transiently High DHT but normal Oral route Requires insertion through a
undecanoate into normal range, but E2 concentrations trocar; spontaneous extrusion;
needs 3– 4 times a day suboptimal clinical responses;
administration variability in serum T levels

Note. DHT, dihydrotestosterone; E2, 17b-estradiol; im, intramuscular; T, testosterone.


ANDROGEN EFFECTS IN MAMMALS 81

testosterone patch (Testoderm, ALZA Corp., Palo D. Novel Androgen Formulations under
Alto, CA) and two nongenital patches (Androderm, Development
Watson Pharmaceuticals, Pomona, CA; Testoderm
TTS, ALZA Pharmaceuticals, Mountainview, CA). Interest in developing more physiologic, sustained-
Scrotal patches deliver either 4 or 6 mg/day of release testosterone formulations has increased due to
testosterone depending on their size. Serum testoster- the potential application of testosterone as an
anabolic agent and as a male contraceptive. With
one levels in hypogonadal men peak in the midnormal
long-acting injectable testosterone preparations (tes-
range 4 to 8 h after application of the patch and then
tosterone buciclate, 600 mg intramuscularly every 3
decline through the day in a pattern similar to the
to 4 months), serum levels peak at 6 weeks and can
circadian pattern in normal young men. Hypogona-
remain in the normal range for 12 weeks. Testoster-
dal men treated with the scrotal patch have normal
one pellets (three 200 mg pellets or six 100 mg
serum estradiol levels but much higher DHT levels
pellets) are implanted under the skin and can provide
than untreated eugonadal men. This is likely the normal testosterone levels as well as physiologic
result of the high activity level of 5a-reductase in the levels of estradiol and DHT for up to 6 months. A
scrotal skin. It is not known whether long-term single intramuscular injection of a biodegradable
exposure to high serum DHT levels will have testosterone microsphere formulation produces nor-
deleterious effects on the prostate or whether mal levels of testosterone in hypogonadal men for up
intraprostatic DHT levels are also elevated. to 11 weeks; serum estradiol and DHT levels are
Two nongenital transdermal systems, Androderm maintained in the normal range. A number of
and Testoderm, can maintain physiologic testoster- transdermal, buccal, sublingual, parenteral, and oral
one levels in hypogonadal men. Four to 12 h after testosterone formulations are currently under
application of the patch, serum testosterone and development.
estradiol levels are in the midnormal range. The
nongenital patches also produce physiologic levels of
serum DHT and estradiol and normal DHT:testoster- VIII. ANABOLIC EFFECTS OF ANDROGENS
one and estradiol:testosterone ratios. Some patients IN PATIENTS WITH CHRONIC ILLNESSES
treated with the patches may experience skin irri- AND AGE-RELATED FRAILTY
tation at the site of patch application. Many chronic illnesses such as cancer, HIV infection,
chronic obstructive lung disease, and end-stage renal
disease are associated with substantial loss of muscle
C. Oral Testosterone Formulations mass and function. Although these diseases are not
yet curable, disease stability can often be achieved.
17a-Alkylation of testosterone makes it less suscep-
Muscle wasting in these illnesses produces debility
tible to hepatic degradation. However, these 17a-
and is associated with poor quality of life, adverse
alkylated androgens should not be used because of
disease outcomes, and increased utilization of health
the potential for hepatotoxicity. Testosterone unde-
care resources. There is a high prevalence of low
canoate, an oral form of testosterone, when adminis- testosterone levels in these sarcopenic disorders.
tered in oleic acid, is absorbed preferentially through Furthermore, low testosterone levels correlate with
the intestinal lymphatics into the lymphatic duct and loss of muscle mass and exercise capacity. Because
general circulation and is therefore spared the first- testosterone replacement in hypogonadal men
pass degradation in the liver. Its short half-life increases lean body mass and muscle strength, it has
necessitates dosing two to three times per day for been speculated that testosterone supplementation in
clinical effects. Circulating levels of testosterone men with chronic illnesses such as HIV infection will
vary among subjects receiving the same dose of this have similar anabolic effects. Clinical trials have
formulation; overnight levels are low and clinical shown that androgen administration in HIV-infected
response is not as good as with other forms of testo- men is associated with modest increases in fat-free
sterone. A sublingual preparation of cyclodextrin- mass, hematocrit, and some aspects of health-related
complexed testosterone is rapidly absorbed from the quality of life. However, further studies are needed to
sublingual mucosa into the bloodstream, leading to determine whether testosterone replacement can
rapid peaks in serum testosterone levels followed by a produce clinically meaningful changes in muscle
decline within 2 h. Frequent dosing several times a function or disease outcomes in patients with chronic
day is necessary to maintain normal serum levels. illnesses.
82 ANDROGEN EFFECTS IN MAMMALS

There is agreement that total and free testosterone disability, cognitive function, progression to demen-
levels and testosterone production rates are lower in tia, and psychosexual function have not been
older men than in young healthy men. Some studies rigorously evaluated in adequately powered studies.
that exclusively recruited healthy, middle-income Thus, the long-term risks and benefits of testosterone
older men failed to detect significant differences in supplementation in older men remain largely
serum testosterone levels between old and young unknown.
men. Recent studies with more representative popu-
lation samples have reconfirmed the age-related Glossary
decrease in serum testosterone levels. Interpretation anabolic steroid Any substance related to the male sex
of serum testosterone levels in the older men in these hormone testosterone that promotes the growth of
studies has been complicated by several confounding skeletal muscle (anabolic effects) and the development
factors: the cross-sectional nature of these studies, the of male sexual characteristics (androgenic effects).
loss of diurnal variation in serum testosterone levels andropause A controversial term used to describe the
in the elderly so that studies that obtain samples in the clinical syndrome associated with the age-related
afternoon underestimate the age-related changes in decline in serum testosterone concentrations in older
testosterone levels, and higher sex hormone-binding men. There is considerable debate over whether the age-
globulin levels in older men so that total testosterone related decline in serum testosterone concentrations is
clinically important and whether this should be
levels underestimate the greater decline in free
corrected by testosterone replacement.
testosterone levels (both dialyzable and bioavailable). aromatase CYP19 An enzyme associated with cytochrome
A meta-analysis of 44 studies of testosterone levels in P450 that catalyzes the final step in the biosynthesis of
older men demonstrated an unequivocal decrease in C-18 estrogens from C-19 steroids and converts the A
morning testosterone levels. Recent longitudinal ring into an aromatic ring.
studies of normal men have verified the age-related dietary steroidal supplements Compounds such as dehy-
decline in serum testosterone levels. Lower testoster- droepiandrosterone (DHEA) and androstenedione that
one levels are the result of changes at multiple levels can be purchased in the United States without a
of the hypothalamic – pituitary– gonadal axis. Testi- prescription through many health food stores. They
cular response to gonadotropins is diminished in are often taken because the user believes they have
older men, gonadotrope responsiveness to androgen anabolic effects. Steroidal supplements such as DHEA
and androstenedione can be converted into testosterone
suppression is attenuated, and the pulsatility of the
and other sex steroids in the body.
hypothalamic GnRH pulse generator is altered. In steroid 5a-reductase An enzyme complex that converts
addition, there are abnormalities of feed-forward and testosterone to 5a-dihydrotestosterone. It has two
feedback systems. Co-existing diseases, malnutrition, isoforms that have different biochemical properties
and concomitant medications also affect serum and tissue distribution.
testosterone levels. A number of clinical problems testosterone esters The compounds that result from esterfi-
prevalent in older men may be related to androgen cation of testosterone at the 17b-hydroxy position. Two
deficiency, including sexual dysfunction, muscle common, clinically used testosterone esters include
weakness and wasting, changes in body composition, testosterone enanthate and testosterone cypionate.
osteopenia, increased prevalence of hip and vertebral
fractures, decreased body hair, decreased hematopoi- See Also the Following Articles
esis, and memory loss. It has been speculated but not Androgen Receptor Crosstalk with Cellular Signaling
proven that androgen replacement may help prevent Pathways . Androgen Receptor-Related Pathology
or reverse these disorders. Four long-term studies of 1 . Androgen Receptors and Prostate Cancer . Androgen
to 3 years in duration have demonstrated that Receptor Structure and Function . Androgens:
testosterone supplementation in older men with low Pharmacological Use and Abuse . Dihydrotestosterone,
testosterone levels is associated with modest increases Active Androgen Metabolites and Related Pathology
. Steroid Hormone Receptor Family: Mechanisms of
in fat-free mass, grip strength, and hematocrit and a
Action
decrease in fat mass. The frequency of adverse events
associated with testosterone administration has been Further Reading
low in these studies, although these studies were not
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relationship of natural androgens to coronary heart disease in
cardiovascular event rates. Furthermore, the effects males: A review. Atherosclerosis 125, 1–13.
of physiologic testosterone replacement on health- Archer, J. (1991). The influence of testosterone on human
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ANDROGEN RECEPTOR CROSSTALK WITH CELL SIGNALING PATHWAYS 83

Bhasin, S., Fisher, C. E. (2002). American College of Physicians/ I. STEROID RECEPTORS AND NONSTEROIDAL ACTIVATION
American Society of Internal Medicine Disease Management II. PEPTIDE GROWTH FACTORS AND ANDROGEN RECEPTOR
Module on Male Hypogonadism. FUNCTION
Bhasin, S., and Buckwalter, J. G. (2001). Testosterone supplemen- III. ANDROGEN RECEPTOR ACTIVATION AND PROTEIN
tation of older men: A rational idea whose time has not yet KINASE A SIGNALING PATHWAY
come. J. Androl. 22, 718– 731. IV. REGULATION OF ANDROGEN RECEPTOR ACTIVITY BY
Bhasin, S., Woodhouse, L., Casaburi, R., Singh, A. B., Bhasin, D.,
PLEIOTROPIC COMPOUNDS
Berman, N., Magliano, L., Dzekov, C., Dzekov, J., Bross, R.,
V. WIDE SPECTRUM OF ANDROGEN RECEPTOR
Phillips, J., Sinha-Hikim, I., Shen, R., and Storer, T. W. (2001).
Testosterone dose response relationships in healthy young
ACTIVATORS
men. Am. J. Physiol. 281, E1172–E1181.
Bhasin, S., Storer, T. W., Berman, N., Callegari, C., Clevenger, B. A.,
The androgen receptor (AR) is a nuclear
Phillips, J., Bunnell, T., Tricker, R., Shirazi, A., and Casaburi,
R. (1996). The effects of supraphysiologic doses of testosterone
transcription factor that regulates the expres-
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Harman, S. M., Metter, E. J., Tobin, J. D., Pearson, J., and carcinoma and in heterologous cells that
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(1994). Estrogen resistance caused by a mutation in the but it might also occur in other tissues. Although
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Strom, B. L. (1999). Effect of testosterone treatment on body ators of AR function. It is hypothesized that
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Verhoeven, G., Swinnen, K., Cailleau, J., Deboel, L., Rombauts, L., N-terminus.
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Zhou, Z.-X., Wong, C.-I., Sar, M., and Wilson, E. M. (1994). The I. STEROID RECEPTORS AND NONSTEROIDAL
androgen receptor: An overview. Rec. Prog. Horm. Res. 49, ACTIVATION
249–274.
In the 1990s, it became clear that steroid receptors are
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved. activated not only in the classical way by their
respective ligands but also by a variety of nonster-
oidal compounds, which in turn stimulate intermedi-
ary signal transducers activating protein kinases.
Androgen Receptor Crosstalk Early studies on steroid receptor activation by cellular
regulators were focused mainly on the human
with Cell Signaling Pathways estrogen receptor-a, which is a classic example of
activation by growth factors and analogues of cAMP
ZORAN CULIG , HELMUT KLOCKER , GEORG BARTSCH , in the absence of ligand. In contrast, human
AND ALFRED HOBISCH progesterone and glucocorticoid receptor display
University of Innsbruck, Austria ligand-dependent activation in the presence of growth
ANDROGEN RECEPTOR CROSSTALK WITH CELL SIGNALING PATHWAYS 83

Bhasin, S., Fisher, C. E. (2002). American College of Physicians/ I. STEROID RECEPTORS AND NONSTEROIDAL ACTIVATION
American Society of Internal Medicine Disease Management II. PEPTIDE GROWTH FACTORS AND ANDROGEN RECEPTOR
Module on Male Hypogonadism. FUNCTION
Bhasin, S., and Buckwalter, J. G. (2001). Testosterone supplemen- III. ANDROGEN RECEPTOR ACTIVATION AND PROTEIN
tation of older men: A rational idea whose time has not yet KINASE A SIGNALING PATHWAY
come. J. Androl. 22, 718– 731. IV. REGULATION OF ANDROGEN RECEPTOR ACTIVITY BY
Bhasin, S., Woodhouse, L., Casaburi, R., Singh, A. B., Bhasin, D.,
PLEIOTROPIC COMPOUNDS
Berman, N., Magliano, L., Dzekov, C., Dzekov, J., Bross, R.,
V. WIDE SPECTRUM OF ANDROGEN RECEPTOR
Phillips, J., Sinha-Hikim, I., Shen, R., and Storer, T. W. (2001).
Testosterone dose response relationships in healthy young
ACTIVATORS
men. Am. J. Physiol. 281, E1172–E1181.
Bhasin, S., Storer, T. W., Berman, N., Callegari, C., Clevenger, B. A.,
The androgen receptor (AR) is a nuclear
Phillips, J., Bunnell, T., Tricker, R., Shirazi, A., and Casaburi,
R. (1996). The effects of supraphysiologic doses of testosterone
transcription factor that regulates the expres-
on muscle size and strength in men. N. Engl. J. Med. 335, 1–7.
sion of specific genes in male reproductive and
Brinkmann, A. O. (2001). Molecular basis of androgen insensitiv- visceral organs. The function of the human AR
ity. Mol. Cell. Endocrinol. 179(1/2), 105 –109. has been investigated in models of prostate
Harman, S. M., Metter, E. J., Tobin, J. D., Pearson, J., and carcinoma and in heterologous cells that
Blackman, M. R. (2001). Longitudinal effects of aging on transiently express wild-type or mutated steroid
serum total and free testosterone levels in healthy men. receptors. The AR can be activated in a ligand-
Baltimore Longitudinal Study of Aging. J. Clin. Endocrinol. independent manner by a number of cellular
Metab. 86, 724–731. regulators. AR activation by peptide growth
Nieschlag E. and Behre H. M. (eds.) (1999). “Testosterone: Action, factors, agents that stimulate protein kinase A
Deficiency, Substitution.” Springer-Verlag, Heidelberg, activity, interleukin-6, and the differentiation
Germany. agent phenylbutyrate has been investigated in
Russell, D. W., and Wilson, J. D. (1994). Steroid 5-a reductase: detail. Ligand-independent activation of the AR
Two genes/two enzymes. Annu. Rev. Biochem. 63, 25–61. has implications regarding the regulation of both
Sakiyama, R., Pardridge, W. M., and Musto, N. A. (1988). Influx of
proliferation and differentiation. Cellular regu-
testosterone-binding globulin (TeBG) and TeBG-bound sex
lators potentiate AR activity induced by low
steroid hormones into rat testis and prostate. J. Clin. Endo-
crinol. Metab. 67(1), 98– 103.
concentrations of androgenic hormones. This
Smith, E. P., Boyd, J., Frank, G. R., Takahashi, H., Cohen, R. M., mechanism is, at least in part, relevant to the
Specker, B., Williams, T. C., Lubahn, D. B., and Korach, K. S. failure of endocrine therapy in prostate cancer
(1994). Estrogen resistance caused by a mutation in the but it might also occur in other tissues. Although
estrogen receptor gene in a man. N. Engl. J. Med. 331, in some cases AR antagonists inhibit AR
1056– 1061. activation by intracellular kinase pathways,
Snyder, P. J., Peachey, H., Hannoush, P., Berlin, J. A., Loh, L., there are also examples of their diminished
Lenrow, D. A., Holmes, J. H., Dlewati, A., Santanna, C. J., and efficiency in the presence of nonsteroidal regu-
Strom, B. L. (1999). Effect of testosterone treatment on body ators of AR function. It is hypothesized that
composition and muscle strength in men over 65. J. Clin. nonsteroidal AR activators modulate recep-
Endocrinol. Metab. 84, 2647–2653. tor phosphorylation, most probably in the
Verhoeven, G., Swinnen, K., Cailleau, J., Deboel, L., Rombauts, L., N-terminus.
and Heyns, W. (1992). The role of cell –cell interactions in
androgen action. J. Steroid Biochem. Mol. Biol. 41(3/8),
487–494.
Zhou, Z.-X., Wong, C.-I., Sar, M., and Wilson, E. M. (1994). The I. STEROID RECEPTORS AND NONSTEROIDAL
androgen receptor: An overview. Rec. Prog. Horm. Res. 49, ACTIVATION
249–274.
In the 1990s, it became clear that steroid receptors are
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved. activated not only in the classical way by their
respective ligands but also by a variety of nonster-
oidal compounds, which in turn stimulate intermedi-
ary signal transducers activating protein kinases.
Androgen Receptor Crosstalk Early studies on steroid receptor activation by cellular
regulators were focused mainly on the human
with Cell Signaling Pathways estrogen receptor-a, which is a classic example of
activation by growth factors and analogues of cAMP
ZORAN CULIG , HELMUT KLOCKER , GEORG BARTSCH , in the absence of ligand. In contrast, human
AND ALFRED HOBISCH progesterone and glucocorticoid receptor display
University of Innsbruck, Austria ligand-dependent activation in the presence of growth
84 ANDROGEN RECEPTOR CROSSTALK WITH CELL SIGNALING PATHWAYS

factors or peptide hormones. The function of the front of the TATA box. KGF is a stromal growth
human androgen receptor (AR) has been studied in factor that is, in co-culture models, induced by
prostate cancer models and in heterologous cells that androgenic hormones and whose receptor is
are transiently transfected with AR cDNA. The AR is expressed on the surface of epithelial cells. Exper-
a nuclear transcription factor that is expressed in iments performed on explant cultures of developing
benign prostate tissue and nearly all prostate cancers rat seminal vesicle and prostate tissues provided
and is involved in the regulation of proliferation and evidence that the crosstalk between KGF and the AR
differentiation. This article will summarize findings is important in vivo. KGF-stimulated development
on the crosstalk between signaling pathways of was blocked by anti-androgens, in concordance with
peptide hormones and cytokines and that of the AR. findings previously reported for transfected prostate
It is particularly interesting that nonsteroidal acti- cancer cells. The growth of prostate primary epi-
vation of the AR has been observed both with thelial cultures was stimulated by KGF and the effect
compounds that stimulate and with those that retard was abolished by the anti-androgen hydroxyfluta-
tumor cell proliferation. Data regarding the action of mide. Taken together, these findings demonstrate up-
AR antagonists in the presence of nonsteroidal regulation of AR function by KGF.
receptor activators and the involvement of protein In addition to ligand-independent activation of
kinases will also be presented. the AR, growth factors potentiate receptor activation
via low concentrations of androgen. Synergistic
ligand-dependent activation of the AR was also
observed with other cellular regulators and is
II. PEPTIDE GROWTH FACTORS AND
pathophysiologically significant. Low concentrations
ANDROGEN RECEPTOR FUNCTION
of androgenic hormones persist in patients who
The growth of the prostate gland is regulated receive endocrine therapy for advanced carcinoma
by, in addition to steroid hormones, several growth of the prostate. AR activation in conditions in which
factors in a paracrine or an autocrine manner. In the androgen levels are substantially reduced might be, at
late stages of prostate cancer, expression of growth least in part, responsible for failure of androgen
factors or their receptors is frequently disregulated ablation. In PC-3 prostate cancer cells stably trans-
and therefore their potential interactions with other fected with the AR, dihydrotestosterone and EGF
signaling pathways are of special interest. Interactions regulated the proliferative response in a synergistic
between these polypeptides and the AR were initially manner. This might be due to both AR activation by
studied in DU-145 prostate cancer cells, which lack EGF and EGF receptor up-regulation by androgen.
endogenous steroid receptors. In transient transfec- HER-2/neu tyrosine kinase is an EGF receptor-
tion experiments, AR ligand-independent activation related molecule that is expressed at high levels
was caused by insulin-like growth factor-I (IGF-I), in androgen-independent sublines of the LAPC-4
keratinocyte growth factor (KGF), and epidermal prostate cancer xenograft. Similar to previously
growth factor (EGF). In that experimental system, reported effects of EGF, ligand-independent and
IGF-I was a more potent AR activator than KGF or synergistic activation of the AR by HER-2/neu was
EGF and it was effective with three different andro- documented (Fig. 1). This activation resulted in
gen-responsive reporters. Thus, although there are induction of expression of the endogenous andro-
structural and functional similarities between the AR gen-regulated prostate-specific antigen (PSA) gene.
and human progesterone and glucocorticoid recep- The AR is involved in the regulation of molecules
tors, only the AR could be activated in the absence of that govern cell cycle progression, such as cyclin-
ligand. Proliferation of prostate cells is stimulated by dependent kinases. Enhanced AR activity induced by
IGF-I, and IGF receptors are detectable in the growth factors and HER-2/neu might therefore
majority of prostate cancers. IGF-II, which binds to facilitate tumor progression. Interestingly, the non-
IGF receptor type I with lower affinity than IGF-I, was steroidal anti-androgens hydroxyflutamide and bica-
not effective in co-transfection – transactivation lutamide were less efficient at blocking AR activation
experiments. The importance of IGF-I activation of by HER-2/neu than at blocking AR activation
the AR could reflect the fact that elevated serum IGF-I induced by androgenic hormones. A substantial
is associated with prostate cancer risk. reduction of the ligand-independent effect was
The effect of KGF on the AR was seen with a achieved with an inhibitor of the mitogen-activated
reporter gene that was driven by a promoter protein kinase (MAPK) signaling pathway and
consisting of two androgen-responsive elements in MAPK phosphatase. The effect of HER-2/neu on
ANDROGEN RECEPTOR CROSSTALK WITH CELL SIGNALING PATHWAYS 85

FIGURE 1 Effects of HER-2/neu on androgen receptor function. (A) TS-13 hamster kidney epithelial cells were
transfected with PSA P/E luc in conjunction with plasmids expressing androgen receptor (AR) and/or HER-2 in various
combinations and in the presence or absence of 1.0 nM R1881 (þ or 2). Expression of AR was measured by
immunoblot analysis of whole-cell lysates. Luciferase results are expressed as the fold activation relative to TS13 cells
transfected with PSA P/E luc in the absence of AR, HER-2/neu, and R1881, which was designated as onefold. (B)
Similarly, TS-13 cells were transfected with the PSA E E4-CAT reporter construct in the presence or absence of AR,
HER-2/neu, and/or 1.0 nM R1881. CAT results were analyzed by thin-layer chromatography and quantitated using a
phosphorimager. Data are expressed as the fold activation relative to TS13 cells transfected with PSA E E4-CAT in the
absence of AR, HER-2/neu, and R1881, which was designated as onefold, and represent the mean of three independent
experiments. (C) Dose response of the PSA E E4-CAT reporter to R1881, generated by transfection of TS13 cells with
AR [HER-2/neu with R1881 at concentrations of 0.1, 0.3, and 1.0 nM (wedges)]. CAT activity was measured by thin-
layer chromatography. At higher doses of R1881, the PSA E E4-CAT construct was maximally activated and no
additional effect of HER-2/neu was observed (data not shown). (D) Dose response of a reporter containing a single
androgen-response element (ARE E4-CAT) to R1881 with AR [HER-2/neu with R1881 at concentrations of 0.01, 0.1,
and 1.0 nM (wedges)]. CAT activity was measured by thin-layer chromatography. The two rightmost lanes contain
higher doses of HER-2/neu plasmid (þþ ). Reprinted from Craft et al., 1999, Nat. Med. 5, 280 –285, with permission.

AR function was also seen in LNCaP cells in which pathway. The AR is phosphorylated at serine residues
AR protein levels did not change following transfec- 213 and 791 by Akt, which is downstream of PI
tion. The HER-2/neu effect on the AR requires 3-kinase. Consistent with these observations, consti-
functionality of the phosphatidylinositol (PI) 3-kinase tutively active Akt stimulated an androgen-inducible
86 ANDROGEN RECEPTOR CROSSTALK WITH CELL SIGNALING PATHWAYS

reporter, and a dominant-negative mutant abolished


the effect of HER-2/neu. Another example of the
inability of AR antagonists to reduce nonsteroidal
regulation of AR-mediated gene expression is acti-
vation by the protein kinase C pathway inducer,
phorbol ester. The outcome of experiments to
investigate nonsteroidal activation is cell type-
dependent. In CV-1 and HeLa cells, mainly andro-
gen-dependent effects of growth factors on AR
transcriptional activity were noted.

III. ANDROGEN RECEPTOR ACTIVATION AND


FIGURE 2 Induction of the activities of androgen-responsive
PROTEIN KINASE A SIGNALING PATHWAY promoters by forskolin and PKA. LNCaP cells were transfected
In addition to being influenced by growth factors, with 1.0 mg of PSA (2 630 to þ 12) (A), PB (B), or ARR3-tk-
luciferase reporter constructs (C) and either with or without the
prostate cell growth is affected by peptide hormones
expression vector for the catalytic subunit of PKA (PKAc,
and neurotransmitters that increase intracellular 0.5 mg). Cells were incubated with forskolin (FSK, 50 mM),
levels of cAMP and activate protein kinase A (PKA). R1881 (10 nM), or vehicle (Me2SO, 0.5%) for 48 h under
AR activation by forskolin (which activates adeny- serum-free conditions and harvested, and luciferase activities
lylcyclase to synthesize cAMP and stimulates PKA were measured. The normalized luciferase activities were
activity) in monkey kidney and prostate cancer cell divided by the normalized activity of cells transfected with
lines that transiently expressed the AR was investi- reporter plasmid and with empty PKAc plasmid to give the fold
induction. Reprinted from Sadar, 1999, J. Biol. Chem. 274,
gated. It was confirmed that the AR could be activated
7777 –7783, with permission.
in a ligand-independent manner and that AR antag-
onists block the activity in these circumstances.
Forskolin was maximally efficient at a concentration similar findings were reported for other steroid
of 1 mM. In the same series of experiments, forskolin receptors.
showed a synergistic rather than a ligand-independent
effect with the human progesterone receptor, thus
pointing to specificity of action. In transfected cells, IV. REGULATION OF ANDROGEN RECEPTOR
there were no changes in AR expression following
ACTIVITY BY PLEIOTROPIC COMPOUNDS
forskolin treatment. AR mutants that lack the DNA-
binding domain could not be activated by forskolin, Luteinizing hormone-releasing hormone (LHRH)
and there is also evidence that residues of the analogues are used for prostate cancer therapy
N-terminal region of the AR are needed for AR because their systemic application leads to long-
activation through the PKA pathway. The extent of term suppression of serum androgen levels. Prostate
ligand-independent activation of the AR depends on cancer cells express LHRH receptors and their
cell type, promoter, presence of an endogenous or a growth is differentially affected by LHRH. Modest
transfected AR, and the use of a particular inducer of AR activation by LHRH analogues was observed in
PKA activity. Forskolin was more efficient with transfected cells, and these compounds reduced the
reporter constructs that consisted of natural probasin concentration of androgen needed for maximal
and PSA promoters coupled to the luciferase gene than activation of the AR.
with the reporter that was driven by a promoter There is an increasing interest in AR activation by
consisting of three androgen-response elements interleukin-6 (IL-6). IL-6 is a multifunctional cyto-
(Fig. 2). It is clear that the PKA pathway has an kine that is involved in the regulation of immune and
important role in the regulation of function of the AR inflammatory responses and causes direct effects in
since forskolin also up-regulated PSA secretion in target tissues. In prostate cells, IL-6 either induces G1
prostate explants. From experiments with the PKA growth arrest or acts as an anti-apoptotic factor. IL-6
inhibitor PKI, it became obvious that the PKA signal is of special interest in human prostate cancer because
transduction cascade is also implicated in receptor its serum levels increase in patients with metastatic
activation by ligand. Administration of the inhibitor disease. IL-6 ligand-independent and synergistic
partially reduced androgen-induced AR activity and activation of the AR was demonstrated both in cells
ANDROGEN RECEPTOR CROSSTALK WITH CELL SIGNALING PATHWAYS 87

that transiently express the AR and in LNCaP cells in


which expression of the PSA gene was up-regulated in
response to IL-6. LNCaP cells were growth-inhibited
by IL-6 and it was concluded that IL-6-induced
activation of the AR is important for the maintenance
of prostatic differentiation function. There is no
evidence of changes in AR expression by a nonster-
oidal compound in any cell line transiently trans-
fected with AR cDNA. In contrast, IL-6 increases the
activity of the endogenous AR gene promoter, AR
mRNA, and protein in LNCaP cells. Thus, in that cell
line, IL-6-induced AR activity is associated with
increased protein expression. In concordance with
these observations, nuclear localization of the AR
was enhanced by IL-6. The AR nonsteroidal antag-
onists hydroxyflutamide and bicalutamide nearly
completely blocked AR activation. The two major
signaling pathways of IL-6 in target cells are those of
Janus kinases, which in turn lead to nuclear translo-
cation and activation of signal transducers and
activators of transcription (STAT) and MAPK.
Inhibitors of these pathways as well as those of
PKA and PKC down-regulate the effect of IL-6 on the
AR. In contrast, the inhibitor of the PI 3-kinase
pathway, wortmannin, was ineffective (Fig. 3).
The presence of IL-6 and its receptor was
investigated in prostate cells by immunohistochem- FIGURE 3 PSA promoter induction by IL-6 was blocked by
istry. It was revealed that, in benign prostate tissue, specific inhibitors to signal transducers. (A) LNCaP cells were
IL-6 expression is confined to basal cells, whereas co-transfected with pRL-CMV (for normalization using the
tumor cells themselves acquire the ability to produce dual lux assay) and p1.5kPSA-Lux. The cells were then
IL-6. Prostate stromal cells secrete large amounts of incubated with vehicle, SB202190 (5 mM), PD 98059
IL-6 into their supernatants. The IL-6 receptor, which (15 mM), wortmannin (100 nM), or tyrphostin AG879
(10 mM) for 30 min prior to IL-6 administration (10 ng/ml)
consists of the ligand-binding subunit gp 80 and the
for 24 h. After incubation, lux levels were measured using a
signal-transducing subunit gp 130, is present in luminometer. Data are reported as the mean (^SE) lux activity
benign and malignant prostate tissues. IL-6 binding relative to the basal activity, which was set as 100%, of three
to the receptor activates intracellular signaling by independent experiments. (B) The immunoblot of AR and
protein kinase pathways that leads to the activation GAPDH from 40 mg of cell lysate. LNCaP cells were cultured
of the AR. Because of the presence of IL-6 and the AR in medium containing IL-6 (lane 1) or IL-6 plus pretreatment of
in prostate tissues, one can conclude that the cross the inhibitor reagents described above (lanes 2– 5, respect-
ively). Reprinted from Lin et al., 2001, Clin. Cancer Res. 7,
talk between IL-6 and the AR is significant for
1773– 1781, with permission.
numerous cellular events. The crosstalk between
androgen and the IL-6 signaling cascade is bidirec-
tional; androgenic hormones enhance activation of tyrate. The butyrate derivative increases PSA gene
STAT3 and expression of classic IL-6-target genes. expression by activation of the AR and induces
Endogenous protein inhibitors of activated STAT3 androgen-inducible promoters to various extents.
down-regulate AR activity induced by IL-6. The binding of the AR to DNA-response elements is
enhanced by differentiation-promoting compounds.
Butyrate does not change total AR levels but does
increase nuclear AR.
V. WIDE SPECTRUM OF ANDROGEN
The level of AR ligand-dependent activation is
RECEPTOR ACTIVATORS
also enhanced by caveolin, which is implicated in the
One of the experimental therapies for prostate cancer development of therapy-resistant prostate disease.
is application of the differentiation agent phenylbu- AR co-immunoprecipitates with caveolin through
88 ANDROGEN RECEPTOR CROSSTALK WITH CELL SIGNALING PATHWAYS

interactions between the ligand-binding domain of a signal transducer and activator of transcription 3-dependent
the AR and the NH2-terminal domain of caveolin. pathway in LNCaP prostate cancer cells. Cancer Res. 60,
2132–2135.
Examples of nonsteroidal activation of the AR also Craft, N., Shostak, Y., Carey, M., and Sawyers, C. L. (1999). A
include cadmium; the thyroid gland hormone T3, mechanism for hormone-independent prostate cancer through
which shows a co-stimulatory effect on PSA with modulation of androgen receptor signaling by the HER-2/neu
androgen; MAPK kinase kinase I, whose overexpres- tyrosine kinase. Nat. Med. 5, 280–285.
sion induces apoptosis; and b-catenin. Culig, Z., Hobisch, A., Cronauer, M. V., Radmayr, C., Trapman, J.,
Hittmair, A., Bartsch, G., and Klocker, H. (2000). Androgen
In conclusion, ligand-independent and/or syner-
receptor activation in prostatic tumor cell lines by insulin-like
gistic activation of the AR by cellular regulators is growth factor-I, keratinocyte growth factor, and epidermal
important in advanced prostate cancer and it offers an growth factor. Cancer Res. 54, 5474–5478.
explanation of why androgen-responsive genes are Darne, C., Veyssiere, G., and Jean, C. (1998). Phorbol ester causes
up-regulated in late stages of the disease and in ligand-independent activation of the androgen receptor. Eur.
respective xenograft models. A better understanding J. Biochem. 256, 541 –549.
Hobisch, A., Eder, I. E., Putz, T., Horninger, W., Bartsch, G.,
of the mechanisms underlying activation of the AR in Klocker, H., and Culig, Z. (1998). Interleukin-6 regulates
the absence of ligand or synergistic regulation of AR prostate-specific protein expression in prostate carcinoma cells
activity is crucial for the development of novel by activation of the androgen receptor. Cancer Res. 58,
endocrine therapies. 4640–4645.
Hobisch, A., Rogatsch, H., Hittmair, A., Fuchs, D., Bartsch, G., Jr,
Klocker, H., Bartsch, G., and Culig, Z. (2000). Immunohisto-
Glossary chemical localization of interleukin-6 and its receptor in
benign, premalignant and malignant prostate tissue. J. Pathol.
androgen receptor Nuclear transcription factor that regu- 191, 239– 244.
lates expression of androgen-inducible genes. Lin, D. L., Whitney, M. C., Yao, Z., and Keller, E. T. (2001).
caveolin The principal protein component of caveolar Interleukin-6 induces androgen responsiveness in prostate
membranes. cancer cells through up-regulation of androgen receptor
growth factors Polypeptide hormones that regulate growth expression. Clin. Cancer Res. 7, 1773–1781.
in an autocrine and paracrine manner. Lu, M. L., Schneider, M. C., Zheng, Y., Zhang, X., and Richie, J. P.
IGF-I, IGF-II Growth factors with structures similar to that (2001). Caveolin-1 interacts with androgen receptor: A
of insulin. positive modulator of androgen receptor mediated transactiva-
interleukin-6 Proinflammatory cytokine that either inhibits tion. J. Biol. Chem. 276, 13442–13451.
Nazareth, L. V., and Weigel, N. L. (1996). Activation of the human
proliferation or suppresses apoptosis in target tissues.
androgen receptor through a protein kinase A signaling
phenylbutyrate Butyrate derivative used in differentiation
pathway. J. Biol. Chem. 271, 19900–19907.
therapy. Sadar, M. D. (1999). Androgen-independent induction of prostate-
protein kinase Enzymes that catalyze protein phosphoryl- specific antigen gene expression via cross-talk between the
ation. androgen receptor and protein kinase A signal transduction
xenograft Tumor maintained in host animals. pathways. J. Biol. Chem. 274, 7777–7783.
Sadar, M. D., and Gleave, M. E. (2000). Ligand-independent
See Also the Following Articles activation of the androgen receptor by the differentiation agent
butyrate in human prostate cancer cells. Cancer Res. 60,
Androgen Effects in Mammals . Androgen Receptor- 5825–5831.
Related Pathology . Androgen Receptors and Prostate Thomson, A. A., Foster, B. A., and Cunha, G. R. (1997). Analysis of
Cancer . Androgen Receptor Structure and Function growth factor and receptor mRNA levels during development
. Androgens: Pharmacological Use and Abuse . Estrogen of the rat seminal vesicle and prostate. Development 124,
Receptor Crosstalk with Cellular Signaling Pathways 2431–2439.
Wen, Y., Hu, M. C., Makino, K., Spohn, B., Bartholomeusz, G.,
. Insulin-like Growth Factor (Igf) Signaling . Interleukin-6
Yan, D. H., and Hung, M. C. (2000). HER-2/neu promotes
. Progesterone Receptor Structure/Function and Crosstalk
androgen-independent survival and growth of prostate cancer
with Cellular Signaling Pathways . Steroid Receptor cells through the Akt pathway. Cancer Res. 60, 6841–6845.
Crosstalk with Cellular Signaling Pathways Yeh, S., Lin, H. K., Kang, H. Y., Thin, T. H., Lin, M. F., and Chang,
C. (1999). From HER2/Neu signal cascade to androgen
Further Reading receptor and its coactivators: A novel pathway by induction
of androgen target genes through MAP kinase in prostate
Brass, A. L., Barnard, J., Ptai, B. L., Salvi, D., and Rukstalis, D. B. cancer cells. Proc. Natl. Acad. Sci. USA 96, 5458–5463.
(1995). Androgen up-regulates epidermal growth factor Zhang, S., Hsieh, M. L., Zhu, W., Klee, G. G., Tindall, D. J., and
receptor expression and binding affinity in PC3 cell lines Young, C. Y. (1999). Interactive effects of triiodothyronine and
expressing the human androgen receptor. Cancer Res. 55, androgens on prostate cell growth and gene expression.
3197–3203. Endocrinology 140, 1665–1671.
Chen, T., Wang, L. H., and Farrar, W. L. (2000). Interleukin 6
activates androgen receptor-mediated gene expression through Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
ANDROGEN RECEPTOR STRUCTURE AND FUNCTION 103

cytochrome P450 enzymes, some of which are also


used in the biosynthetic pathways for steroid hor-
Androgen Receptor Structure mones. Testosterone is the major circulating andro-
gen, the majority of which is bound to serum-binding
and Function proteins. Only approximately 1% of the total
testosterone is free and thus available to enter cells
ELIZABETH M. WILSON by diffusion. Most cells are exposed to similar levels
University of North Carolina, Chapel Hill of circulating androgen, an exception being the testis,
where levels are high due to testosterone production
I. BIOLOGICALLY ACTIVE ANDROGENS by the Leydig cells. Whether a particular cell responds
II. MECHANISMS OF ANDROGEN REGULATION to testosterone or dihydrotestosterone (DHT)
OF GENE EXPRESSION depends on the androgen concentration in the cell
III. DOMAIN STRUCTURE AND INTERACTIONS
and on the presence of the androgen receptor (AR).
OF THE ANDROGEN RECEPTOR
IV. ANDROGEN RECEPTOR MUTATIONS ASSOCIATED WITH
In some tissues that are highly responsive to
DISEASE androgen stimulation, such as the prostate and
V. ANDROGEN RECEPTOR ANTAGONISTS AND AGONISTS epididymis, testosterone is bioactivated to 5a-DHT
IN THE ENVIRONMENT by the cytochrome P450 enzyme 5a-reductase type 2.
VI. SUMMARY DHT is a more active androgen than testosterone in
part because it dissociates more slowly from the AR.
That testosterone cannot replace all of the functions
The androgen receptor (AR) is a ligand-
of DHT is dramatically revealed in the 5a-reductase
activated transcription factor that binds with
high affinity to the biologically active andro- syndrome. In this genetic disorder, a mutation in the
gens, testosterone and dihydrotestosterone. 5a-reductase gene results in a deficiency in the
Androgen binding targets the AR to the nucleus, conversion of testosterone to DHT during embry-
where it binds to specific androgen-response ogenesis. 46,XY genetic males with the 5a-reductase
element DNA sequences associated with syndrome are born with incomplete development of
promoter or intron sequences of androgen- the male genitalia due to reduced circulating levels of
regulated genes. AR-mediated gene activation is DHT. Remarkably, at puberty, increases in testoster-
required for male sexual development during one synthesis by the testes and an increase in the
embryogenesis and sexual maturation at activity of the 5a-reductase type 1 overcome, to a
puberty. The domain structure of the AR certain extent, the DHT deficiency, resulting in
parallels that of other steroid receptors. An
virilization of the male.
important aspect of AR structure and function is
an androgen-specific interaction between the A secondary source of androgen in the male is the
NH2-terminal and carboxyl-terminal regions adrenal gland, but this is not considered a highly
that contributes to stabilization of the AR. significant contribution to overall androgen levels
Genetic defects in the AR gene in humans under normal physiological conditions. In the female,
prevent normal male sexual development or by contrast, adrenal androgens contribute a signifi-
cause adult onset muscular atrophy. Androgens cant portion of circulating androgen levels, which are
are required in prostate development and low compared to those of the male under normal
growth and may contribute to prostate cancer conditions, minimizing the induction of androgen-
proliferation. responsive genes in the female. Adrenal androgens,
such as androstenedione, are produced by the same
steroidogenic enzyme pathways that function in the
I. BIOLOGICALLY ACTIVE ANDROGENS
testis. Adrenal androgens serve as precursors for
In the male, testosterone is produced in Leydig cells of testosterone production.
the testis in response to placental chorionic gonado- Certain endocrine conditions associated with
tropin during male sexual development of the fetus. excess adrenal androgen production are problematic
The testosterone concentration in circulating blood in the female. Congenital adrenal hyperplasia results
increases again at the time of puberty under from a genetic defect in an enzyme in the pathway to
stimulation by pituitary gonadotropin (luteinizing hydrocortisone (cortisol) biosynthesis. The most
hormone or interstitial cell-stimulating hormone) and frequently mutated gene that causes congenital
remains at relatively high levels until a decline later in adrenal hyperplasia is the 21-hydroxylase gene. This
life. Testosterone is synthesized by a series of enzyme defect reduces the synthesis of cortisol, which
104 ANDROGEN RECEPTOR STRUCTURE AND FUNCTION

results in the loss of negative feedback to the a reporter plasmid. Reporter plasmids contain a
hypothalamus and anterior pituitary. The conse- responsive DNA element (DNA sequence that binds
quence is an increase in the secretion of adrenocorti- the nuclear receptor) linked to a gene such as
cotropic hormone by the anterior pituitary and the luciferase or chloramphenicol acetyltransferase,
subsequent overstimulation of the adrenal glands. whose activity can be quantitated in in vitro assays.
This leads to accumulation of 17-hydroxyprogester- Co-transfection of expression plasmids for the inter-
one, which results in increased conversion to andros- acting proteins is used to determine their effects on
tenedione and testosterone in the adrenal gland with ligand-induced receptor regulation of gene
increased circulating testosterone. During embryonic transcription.
development, congenital adrenal hyperplasia causes All but a few tissues in the body express the AR.
differing degrees of virilization (development of male However, the levels of AR expression differ among
external genitalia) in the female fetus and increased tissues. The highest levels are observed in androgen-
virilization in affected males. responsive tissues, such as prostate, seminal vesicle,
epididymis, testis, and the genitalia. High AR levels
are also reported in the ovary; however, the role of
androgen in female reproduction remains to be
II. MECHANISMS OF ANDROGEN
clearly defined. Genes that are activated by the AR
REGULATION OF GENE EXPRESSION contain an androgen-response element sequence in
Under normal physiological conditions, the AR is the promoter or intron region that allows for selective
bound to a biologically active androgen, testosterone binding of AR to the DNA. In addition, there have
or DHT, which induces the AR to translocate to the been reports of nongenomic effects of the AR;
nucleus. In the nucleus, the AR binds as a homodimer however, it remains to be determined by what
to specific DNA sequences known as androgen- mechanisms these effects are mediated.
response elements. Thus, the AR is a ligand-inducible
transcription factor. The androgen-bound AR com-
plex recruits associated co-regulatory proteins that III. DOMAIN STRUCTURE AND INTERACTIONS
act during the process of chromatin modification
OF THE ANDROGEN RECEPTOR
necessary for gene activation. Many associated
proteins have been reported to have a role in AR- The AR is encoded on the X chromosome. Thus,
mediated gene activation. These proteins include the 46,XY genetic males have only one allele for the AR
family of p160 co-activators, named for their gene. 46,XX genetic females have two X chromo-
approximate size of 160 kDa and for their capacity somes but also express only one allele due to the
to increase the transcriptional response to ligand- random inactivation of the X chromosome. There
activated steroid receptors as measured in transcrip- appears to be a single AR gene and only one form of
tion assays. The p160 group of transcriptional the AR. This contrasts with other steroid receptors
co-activators include steroid receptor co-activator 1 for which there are multiple genes and forms. For
(SRC1), transcriptional intermediary factor 2 (TIF2, example, estrogen receptor a and b are encoded by
SRC2, GRIP1), and the SRC3/TRAM1/AIB1/pCI- different but related genes. The A and B forms of the
P/ACTR/RAC3 group of co-activators. Many of these progesterone receptor are synthesized from alterna-
are associated with histone acetyl transferase activity tive transcripts and have different cellular effects.
and can recruit CBP, pCAF, and other co-regulatory Recent evidence supports the existence of only one
proteins required for chromatin modification and human AR gene and only the full-length form of the
gene activation. human AR in cells. In some fish, however, there is
The use of two-hybrid screening methods in yeast evidence for more than one form of the AR.
expression systems has revealed many proteins in The AR shares with other steroid and nuclear
addition to the p160 co-activators that associate with receptors a common overall structural arrangement
the AR and are reported to increase AR-mediated of its functional domains (Fig. 1). A striking aspect of
transcriptional activation. The extent of increased these domains is a relatively high degree of functional
gene activation is typically measured using transient independence. Each of the domains can be expressed
transfection DNA transfer methods in cultured cell and shows its respective activity to a significant
lines. Plasmid DNA transfected into cells includes degree. However, as described below, important
expression vectors containing the DNA coding interactions that have an impact on AR function
sequence for the AR or other steroid receptors and occur among the domains.
ANDROGEN RECEPTOR STRUCTURE AND FUNCTION 105

One aspect of the NH2-terminal region that may


allow these amino acid repeat expansions to occur
without severe disruption of AR function could be a
lack of strict structural constraints in the region.
Binding of transcription factors to the NH2-terminal
FIGURE 1 Diagram of the structural domains of the human region may impose a more structured arrangement.
androgen receptor. Shown are activation functions 1 (AF1) and The allowable flexibility in length and position of the
2 (AF2), the DNA-binding domain (DBD), and the ligand- glutamine repeat relative to AR function suggests that
binding domain (LBD). Also shown are the N/C interaction the NH2-terminal region of AR is tolerant of a
motifs FXX LF and WXX LF. Reprinted from He et al. (2001),
significant amount of alteration in protein structure.
with permission.
This contrasts with the very strict structural require-
ments for the DNA- and ligand-binding domains.
A. NH2-Terminal Region The NH2-terminal region is nevertheless critical
for AR function because it contains the major
The NH2-terminal region is the largest domain in the
transactivation region referred to as the activation
AR, making up about 60% of the receptor protein.
function 1 (AF1) region. Deletion of the NH2-
This contrasts with other nuclear and steroid
terminal region results in the loss of its major
receptors in which the NH2-terminal region may be
transcriptional activation domain. The NH2-terminal
short. But like the AR, the glucocorticoid, progester-
region likely acts to recruit specific transcription
one, and mineralocorticoid receptors have a similarly factors, some of which have been reported. The AF1
large NH2-terminal region. The AR NH2-terminal region is located between amino acid residues 142
region is unusual because it contains several repeated and 337 in the 1- to 558-residue NH2-terminal region
sequences, the most striking being the glutamine and (Fig. 1). Although it is not well defined, the NH2-
glycine repeats. The glutamine repeat is encoded by a terminal AF1 region requires androgen binding for
CAG trinucleotide repeat whose length is poly- transcriptional activity and appears to be critical for
morphic in the normal population, ranging from 13 AR-mediated gene activation.
to 35 glutamine residues. An increase beyond 36
glutamine residues is associated with adult onset
muscular atrophy or Kennedy’s disease (see Section B. DNA-Binding Domain and Hinge Region
IV.B). The glycine repeat is usually 23 residues in The NH2-terminal region is contiguous with the
length and is less polymorphic than the glutamine centrally positioned DNA-binding domain that
repeat. Significant expansion or contraction of the allows for specific binding to androgen-response
glycine repeat has not been associated with a disease. DNA elements present in androgen-regulated genes
A short 8-residue proline repeat, the function of (Fig. 1). The AR DNA-binding domain is composed
which is not known, also occurs in the NH2-terminal of a two zinc-finger structure where two zinc atoms
region of the AR. are held in position by tetrahedral coordination with
The NH2-terminal region is the least conserved highly conserved cysteine residues. The zinc-finger
domain of the ARs of different species and is the least structure of the DNA-binding domain is character-
conserved domain among different steroid receptors. istic of all steroid and nuclear receptors. The DNA-
Homology comparisons of the amino acid sequence binding domain and flanking sequence can be
of the NH2-terminal region in primates indicates expressed as an isolated domain that binds DNA.
92% similarity between human and the prosimian A region of genomic DNA associated with androgen-
lemur AR amino acid sequence. Lemurs arose during regulated genes that interacts with the AR
primate evolution from the lower mammals. The DNA-binding domain is referred to as an androgen-
glutamine repeat at this position in the NH2-terminal response element, or hormone-response element.
domain expanded throughout primate evolution, Androgen-response elements vary in sequence, but
increasing from 4 CAG repeats in lemur AR to its for many genes, they adhere to the DNA consensus
present average length of 22 CAG repeats coding for sequence 50 -GGTACAnnnTGTTCT-30 , where n rep-
the glutamine repeat region in human AR. In lower resents an unspecified spacer nucleotide.
mammals, such as rat and dog, a variable-length A short hinge region at amino acid residues 624 –
glutamine repeat also occurs, but remarkably, this 676 (Fig. 1) is positioned between the AR DNA-
glutamine repeat, also encoded by a CAG repeat, is in binding domain and the carboxyl-terminal region.
a different position within the NH2-terminal region. Within the hinge region is the AR nuclear-targeting
106 ANDROGEN RECEPTOR STRUCTURE AND FUNCTION

signal that triggers the subcellular transport of AR amino acid. The AF2 region of the AR appears to
from the cytoplasm to the nucleus. Androgen binding have a lower binding affinity for the LXX LL motifs
to the AR activates this bipartite targeting sequence, of the p160 co-activators than do the other nuclear
which is composed of two closely spaced clusters of receptors when expressed as a DNA- and ligand-
the basic amino acids lysine and arginine. Mutation binding domain fragment. It is therefore not clear to
of these basic residues inhibits androgen-induced what extent the AF2 region in the AR ligand-binding
nuclear translocation of the AR. domain contributes to AR transcriptional activity in
vivo. A mutation at lysine residue 720 in the AR AF2
region of the ligand-binding domain decreased the
C. Ligand-Binding Domain interaction with p160 co-activators but did not
The carboxyl-terminal ligand-binding domain at significantly alter AR transactivation when deter-
amino acid residues 676 –919 (Fig. 1) is approxi- mined in a transient transfection assay, where
mately one-fourth the size of the AR protein and recombinant AR was expressed in cultured cells in
constitutes the high-affinity hormone-binding site. the presence of a reporter vector.
The ligand-binding region of AR and that of several
other nuclear receptors have been crystallized in the D. Interaction Between NH2- and Carboxyl-
presence of bound hormone. The carboxyl-terminal
Terminal Regions
ligand-binding domain forms a series of alpha-helical
folds that create a hydrophobic cavity for hormone As indicated above, two regions of the AR appear to
binding. The size of the binding cavity varies among be involved in androgen-induced transactivation of
the steroid receptors, with the smaller binding cavities genes, AF1 in the NH2-terminal region and AF2 in the
showing a higher degree of hormone specificity. The ligand-binding domain. In comparison to other
ligand-binding domain of the AR can function as an steroid receptors, AF2 in AR is weak, apparently
isolated domain with apparent high-affinity androgen reflecting a reduced ability to recruit p160 co-
binding when the recombinant protein is expressed in activators through their LXX LL motifs. In addition,
the absence of the NH2-terminal region. However, as inhibition of p160 co-activator binding results from
described below, the NH2-terminal region influences a strong androgen-induced NH2- and carboxyl-
the kinetics of androgen binding. terminal (N/C) interaction between an FXX LF
Steroid hormone binding in the hydrophobic motif in the NH2-terminus at amino acid residues
pocket of the ligand-binding domain is relatively 23 – 27 (Fig. 1) and the carboxyl-terminal AF2
specific for the ligand. For example, the AR shows hydrophobic binding surface. A second AR NH2-
highest binding affinity and specificity for the terminal N/C interaction site, WXX LF at amino acid
biologically active androgens testosterone and DHT. residues 433 – 437, also interacts with AF2 in the
However, AR and other steroid receptors also bind ligand-binding domain (Fig. 1).
additional ligands, but with lower affinity. For Androgen dissociation studies provided the first
example, the mineralocorticoid and glucocorticoid evidence that the NH2-terminal region of the AR
receptors bind each other’s preferred hormone, interacts with the ligand-binding domain. Deletion of
aldosterone and hydrocortisone, with similar affinity. the NH2-terminal domain increases the rate at which
A variety of mechanisms including receptor concen- bound androgen dissociates, whereas the apparent
tration and selective steroid metabolism contribute to equilibrium binding affinity is not altered, indicating
facilitating a specific hormone response. In the case of that the association rate is increased. These studies
the mineralocorticoid receptor, there are enzymes in were followed with a two-hybrid interaction assay
certain tissues, such as the placenta and kidney, that that supported a direct interaction between these
facilitate the rapid metabolism of cortisol to its regions. More recently, FXX LF and WXX LF motifs
inactive form, cortisone, thereby allowing the miner- in the NH2-terminal region that interact in the
alocorticoid receptor to bind aldosterone, the active presence of testosterone or DHT with the AF2 region
mineralocorticoid. The AR can bind other hormones in the ligand-binding domain were identified. Recent
and ligands depending on their concentration. studies suggest that the N/C interaction stabilizes the
Within the carboxyl-terminal region is an acti- AR protein against degradation and interferes with
vation region referred to as activation function 2 the binding of p160 co-activators to the AF2 region.
(AF2; Fig. 1). AF2 forms a hydrophobic binding site The FXX LF motif appears to have a higher affinity
for the LXX LL motifs of the p160 transcriptional co- for the AF2 region than do the LXX LL motif
activators, where L refers to leucine and X is any sequences of the p160 co-activators. A major effect
ANDROGEN RECEPTOR STRUCTURE AND FUNCTION 107

of the androgen-dependent FXX LF/WXX LF inter- missense mutations where a new amino acid residue
action with the ligand-binding domain is to slow the is substituted or nonsense mutations where a pre-
dissociation rate of bound androgen and to stabilize mature termination codon is introduced. Many of the
the AR against degradation. premature mutant stop codons occur within the NH2-
terminal region. A compilation of the more than 200
different AR mutations that cause partial or complete
IV. ANDROGEN RECEPTOR MUTATIONS androgen insensitivity is available on the Internet at
ASSOCIATED WITH DISEASE http://www.mcgill.ca/androgendb/. The frequency of
AR gene mutations in the human population is about
A. Androgen Insensitivity Syndrome 1 in 20,000 individuals. Females can be carriers of an
Steroid receptor-regulated gene transcription is AR gene mutation without significant effects to
required for cell proliferation, differentiation, and themselves probably because of the presence of one
development and involves the formation of active normal allele for the AR gene and random inacti-
transcription initiation complexes composed of a vation of the X chromosome.
specific steroid receptor and a host of co-activators. Partial forms of the androgen insensitivity syn-
The essential role of the AR in male sex differen- drome occur in a spectrum of forms, ranging from the
tiation is well established. Naturally occurring AR mildest form, which causes male infertility in the
gene mutations in 46,XY genetic males result in a adult, to a more severe form that leads to ambiguous
phenotypic spectrum known as the androgen insensi- genitalia in the newborn. The presence of the 46,XY
tivity syndrome, characterized by incomplete male male sexual karyotype with partial disruption or
sexual development in utero. Because the AR gene is complete loss of the male genital phenotype is the
located on the X chromosome (Xq11 –q12), there is hallmark of the androgen insensitivity syndrome. In
only one allele in the male. Thus, mutations that some cases, phenotypic expression of the same AR
abolish AR function can have a dramatic effect on the gene mutation varies between individuals from
response to androgen. Androgen insensitivity is an different families and among family members, illus-
end-organ defect because the individual cannot trating that other factors contribute to phenotypic
respond to the high circulating androgen levels due expression of a given AR gene mutation. Mutations
to a genetic defect in the AR protein. that significantly decrease androgen- or DNA-binding
The severity of the defect in male sexual develop- affinity result in the complete form of the syndrome.
ment reflects the importance of specific amino acid
residues in AR function. Partial or total loss of AR
B. Kennedy’s Disease
function can affect any one of a number of functional
properties of the AR including high-affinity androgen Neurodegenerative diseases are sometimes caused by
binding, DNA binding, and AR stability. Depending trinucleotide repeat expansions. The first trinucleo-
on the position and type of the substituted amino tide repeat expansion discovered was Kennedy’s
acid, the degree of phenotypic expression varies. disease or adult onset spinal/bulbar muscular atro-
Mutations causing partial androgen insensitivity can phy. Others include Huntington’s disease and spino-
alter steroid-binding affinity and specificity. cerebellar ataxia type 1. The diseases are caused by a
Mutations that severely disrupt androgen- or DNA- CAG repeat expansion that codes for glutamine.
binding affinity result in the complete form of the Differences occur in repeat length, stability, and
syndrome. This is characterized by a female genital location of the expansion. Within the NH2-terminal
phenotype in a 46,XY genetic male. These individuals domain of the AR is a CAG repeat that codes for a
lack a uterus and have abdominal testes that produce glutamine repeat that is polymorphic in the normal
normal to elevated male levels of testosterone. The population, ranging from 13 to 35 glutamine residues
majority of AR gene mutations are single-base in length. Expansion of the AR CAG repeat in the first
missense mutations in the DNA-binding or car- exon of the coding region of the NH2-terminal
boxyl-terminal regions (exons 2 –8) with more than domain to greater than 40 Gln residues results in X-
200 unique mutations reported. In some cases, there linked adult onset spinal/bulbar muscular atrophy or
is partial or complete deletion of the AR gene. Kennedy’s disease. Clinical features of Kennedy’s
Most of the naturally occurring AR gene disease are progressive muscle weakness after the age
mutations are in the DNA- or ligand-binding domain, of 25 –30 years, muscle atrophy associated with loss
with a surprising absence of missense mutations in the of lower motor and primary sensory neurons, and
NH2-terminal region. The genetic errors include partial androgen insensitivity, evidenced by elevated
108 ANDROGEN RECEPTOR STRUCTURE AND FUNCTION

serum gonadotropin levels, gynecomastia (breast greater response than wild-type AR to other ligands,
enlargement in the male), testicular atrophy, impo- such as estradiol, and to AR antagonists, such as
tence, and oligo- or azoospermia (low or absent hydroxyflutamide. It is not clear to what extent this
sperm production). Early onset, severity of muscle broadened ligand specificity contributes to the
weakness, and degree of androgen resistance corre- proliferation of prostate cancer in these cases.
late with longer repeats. Instability of triplet repeats Activation of the AR by other circulating hormones
associated with human hereditary disease may result increases the possibility for stimulation of tumor
from errors in DNA mismatch repair through the growth in the absence of the usual stimulating
formation of single-stranded hairpin structures and androgens, testosterone and DHT. This may be
DNA polymerase slippage during replication. Poly- particularly critical in late stage prostate cancer
morphisms of the AR CAG repeat are stable in the where patients have undergone androgen deprivation
normal population and can be useful as genetic therapy by castration or chemical treatment. Under
markers in clonality analysis and forensic science. these conditions of low androgen levels, other
How repeat expansions cause adult-onset muscle circulating ligands, such as the adrenal androgen
wasting is being actively investigated; current evi- dehydroepiandrosterone, might gain the ability to act
dence suggests that the expanded glutamine repeat as a more active androgen and promote the growth of
contributes to protein aggregation in the nucleus. late stage cancer in the androgen-deprived patient.
More recently, it was discovered that certain of
the p160 co-activators are highly expressed in the
C. Prostate Cancer majority of prostate cancer samples analyzed. These
There are conflicting reports in the literature con- highly expressed p160 co-activators include SRC1
cerning the incidence of AR gene mutations associ- and TIF2, whereas AIB1 (amplified in breast cancer 1)
ated with prostate cancer. Earlier reports suggested was not detected. p160 co-activator expression was
frequencies of AR mutations of up to 50% or more in low to undetectable in benign prostatic hyperplasia,
prostate cancer specimens. However, many of these which is a common form of enlarged prostate in the
early reports were confounded by the presence of aging male. It is conceivable that high-level
artifactual polymerase chain reaction DNA incorpor- expression of co-activators offers a mechanism for
ation errors. The actual frequency of AR mutations in AR activation through other ligands including the
prostate cancer specimens is probably not more than adrenal androgens or by ligand-independent
5% of cases. Additional studies will help to resolve mechanisms.
the conflicting reports but the preponderance of
recent information supports the view that AR gene
mutations are uncommon, particularly in early stage V. ANDROGEN RECEPTOR ANTAGONISTS
disease, but may become more prevalent in later
AND AGONISTS IN THE ENVIRONMENT
forms of the disease when overall genetic instability is
increased. Over the past several years, certain environmental
Despite their apparent low incidence, it is never- chemicals used as pesticides or herbicides in crop
theless of interest that the AR gene mutations that protection have been identified as precursors to
have been reported in prostate cancer specimens are chemicals with endocrine disruptor activity involving
sometimes associated with a broadened ligand the AR. A number of environmental chemicals were
specificity. In this case, the AR responds by increasing previously established to have estrogenic activity, but
gene transcription in response to not only the until recently, these activities related only to the
biologically active androgens, testosterone and estrogen receptor. At least five or more chemicals
DHT, but also to other circulating steroids. For previously or currently in use in the United States
example, an AR T877A mutation in the ligand- were found to be AR antagonists. An antagonist is a
binding domain is present in the LNCaP human chemical or pharmaceutical ligand that competes for
prostate cancer cell line. An AR H874Y gene hormone binding and fails to induce receptor-
mutation is present in a human prostate cancer mediated activation of gene transcription. Antagonist
xenograft. The xenograft is a transplant of a human activities are detected using in vitro assays to
prostate cancer into an immune-deficient mouse, demonstrate inhibition of androgen-induced AR
where it can be propagated for further study. These transcription activity and inhibition of DNA binding.
mutant ARs show a normal transcriptional response Thus far, it has been the in vivo metabolites of the
to testosterone and DHT but, in addition, have a parent chemicals that interfere with androgen binding
ANDROGEN RECEPTOR STRUCTURE AND FUNCTION 109

to the AR, resulting in antagonist activity. The precursors by the bacteria Mycobacterium smegmatis
apparent equilibrium binding affinity of a pesticide or Escherichia coli, which have been shown to
or herbicide metabolite is in the micromolar range as produce androstenedione from steroid precursors.
opposed to nanomolar binding affinity of androgens Conversion of plant sterols by bacterial metabolism
for the AR. Like androgens and other antagonists, provides a likely source for androstenedione in the
these chemicals bind to the ligand-binding domain in contaminated Florida river.
the carboxyl-terminal region of the AR and block
binding of androgen. Binding of these chemicals does
not elicit conformational changes in the AR, such as VI. SUMMARY
those required for the N/C interaction, and thus these The androgen receptor is a ligand-activated transcrip-
chemicals are ineffective in promoting agonist-related tion factor that binds testosterone or dihydrotestos-
effects on activation of gene transcription. terone. The androgen-activated AR binds to DNA
It is not known whether there is sufficient sequences known as androgen-response elements to
exposure from pesticide or fungicide residues on regulate gene transcription. The AR has a domain
fruits and vegetables or in farming practices to elicit structure characteristic of the nuclear receptor family
AR antagonist activity. The micromolar binding of transcription factors, with an NH 2-terminal
affinity indicates that relatively high levels of pesticide transactivation domain, a DNA-binding domain,
exposure would be required to disrupt androgen- and a carboxyl-terminal ligand-binding domain. An
induced effects. Of particular potential concern, unusual property of the AR is an androgen-induced
however, is the developing male fetus, which depends interdomain NH2- and carboxyl-terminal interaction
on the action of androgens produced from the fetal that stabilizes the AR against degradation and
testes during embryonic development for sexual inhibits p160 co-activator activation of AF2 in the
differentiation and development of the external ligand-binding domain. The AR gene is on the X
genitalia. Exposure of the pregnant mother to chromosome. In 46,XY genetic males, a single-
sufficient levels of chemicals with AR antagonist nucleotide base change causing a missense amino
activity during the critical period of male sexual acid mutation can inhibit normal male sexual
development could possibly inhibit development, development, resulting in the androgen insensitivity
resulting in incomplete formation of the external syndrome. Other diseases related to the AR are
genitalia. In some countries, an increasing incidence Kennedy’s disease and prostate cancer. Finally,
of incomplete masculinization has been observed, metabolites of certain chemicals used as pesticides
characterized by the hypospadias deformity, in which and herbicides have anti-androgen activity that could
the urethra opens on the shaft or base rather than at inhibit sexual development of the male fetus. On the
the tip of the penis. It is not known to what extent this other hand, androgens in the environment have been
relatively common form of incomplete masculiniza- reported to masculinize entire populations of fish.
tion is attributable to endocrine disruptor activity. Thus, the AR is a critical gene regulatory protein.
This type of defect can be repaired by surgery, Loss or gain of AR function through genetic mutation
resulting in full male sexual function. However, it is or environmental inhibition or activation can have a
not known whether other anti-androgen effects could wide range of adverse effects on sexual development.
occur in the brain to interfere with androgen
imprinting or in other organs of the developing male.
Glossary
Recent evidence suggests, in addition, the
presence of environmental androgens. Female East- activation function regions Segments in a steroid receptor
ern mosquitofish, Gambusia holbrooki, present in a that mediate transcriptional activation through their
Florida river downstream of a paper mill effluent interaction with co-activators or proteins of the basic
discharge were uniformly masculinized as evidenced transcriptional machinery of the cell.
by the presence of male-like elongated anal fins. androgen insensitivity syndrome 46,XY genetic males with
an androgen receptor gene mutation that results in
Androstenedione, a known precursor of testosterone,
infertility or partial or complete lack of masculinization
was confirmed by liquid chromatography mass
of the genitalia. The androgen insensitivity syndrome
spectrometry to be a component of the river water. has also been referred to as testicular feminization.
Androstenedione was present in the river at a androgen receptor A transcription factor that is activated
concentration of 0.14 nM. Phytosterols derived by binding a biologically active androgen, testosterone
from paper pulp, such as b-sitosterol, campesterol, or dihydrotestosterone; it is required for induction of
and stigmastanol, can be modified to androgen gene transcription in response to androgen.
110 ANDROGENS: PHARMACOLOGICAL USE AND ABUSE

hormone-response element Segment of DNA sequence that of androstenedione in a river containing paper mill effluent.
binds to the DNA-binding domain of a steroid receptor Environ. Toxicol. Chem. 20, 1325–1331.
and thereby facilitates the activation of a gene. Kelce, W. R., Stone, C. R., Laws, S. C., Gray, L. E., Kemppainen,
Androgen-response elements are nucleotide sequences J. A., and Wilson, E. M. (1995). Persistent DDT metabolite p,
p0 -DDE is a potent androgen receptor antagonist. Nature 375,
that bind the androgen receptor.
581 –585.
N/C interaction Interaction between the NH2-terminal Langley, E., Zhou, Z. X., and Wilson, E. M. (1995). Evidence for
and carboxyl-terminal (N/C) regions of the androgen an antiparallel orientation of the ligand activated human
receptor that is selectively induced by the binding of androgen receptor dimer. J. Biol. Chem. 270, 29983–29990.
a biologically active androgen, testosterone or dihy- La Spada, A. R., Wilson, E. M., Lubahn, D. B., Harding, A. E., and
drotestosterone. This interaction is mediated by Fischbeck, K. H. (1991). Androgen receptor gene mutations in
FXX LF- and WXX LF-binding motifs in the NH2- X-linked spinal and bulbar muscular atrophy. Nature 352,
terminal region and activation function 2 in the ligand- 77–79.
binding domain. Quigley, C. A., De Bellis, A., Marschke, K. B., El-Awady, M. K.,
p160 co-activators A group of co-regulatory proteins of Wilson, E. M., and French, F. S. (1995). Androgen receptor
defects: Historical, clinical and molecular perspectives.
approximately 160 kDa in size that interact with
Endocr. Rev. 16, 271–321.
nuclear receptors to increase the transcriptional Tan, J. A., Sharief, Y., Hamil, K. G., Gregory, C. W., Zang, D. Y.,
response. Sar, M., Gumerlock, P. H., deVere White, R. W., Pretlow, T. G.,
Harris, S. E., Wilson, E. M., Mohler, J. L., and French, F. S.
See Also the Following Articles (1997). Dehydroepiandrosterone activates mutant androgen
receptors expressed in the androgen dependent human prostate
Androgen Effects in Mammals . Androgen Receptor cancer xenograft CWR22 and LNCaP cells. Mol. Endocrinol.
Crosstalk with Cellular Signaling Pathways . Androgen 11, 450–459.
Receptor-Related Pathology . Androgens: Pharmacological Zhou, Z. X., Wong, C. I., Sar, M., and Wilson, E. M. (1994). The
Use and Abuse . Estrogen Receptor-a Structure and androgen receptor: An overview. Rec. Prog. Horm. Res. 49,
Function . Estrogen Receptor-b Structure and Function 249 –274.
. Steroid Hormone Receptor Family: Mechanisms of
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
Action

Further Reading
Choong, C. S., Kemppainen, J. A., and Wilson, E. M. (1998). Androgens: Pharmacological
Evolution of the primate androgen receptor: A structural basis
for disease. J. Mol. Evol. 47, 334–342. Use and Abuse
Choong, C. S., and Wilson, E. M. (1998). Trinucleotide repeats in
the human AR: A molecular basis for disease. J. Mol.
Endocrinol. 21, 235–257.
MARIA M. BYRNE AND EBERHARD NIESCHLAG
Gregory, C. W., He, B., Johnson, R. T., Ford, O. H., Mohler, J. L., University of Münster
French, F. S., and Wilson, E. M. (2001). A mechanism for
androgen receptor mediated prostate cancer recurrence after I. INTRODUCTION
androgen deprivation therapy. Cancer Res. 61, 4315–4319.
II. PHARMACOLOGY
Gregory, C. W., He, B., and Wilson, E. M. (2001). The putative
androgen receptor-A form results from in vitro proteolysis.
III. ANDROGEN USE
J. Mol. Endocrinol. 27, 309–319. IV. ANDROGEN ABUSE
He, B., Kemppainen, J. A., Voegel, J. J., Gronemeyer, H., and
Wilson, E. M. (1999). Activation function 2 in the human
androgen receptor ligand binding domain mediates interdo- Androgen preparations are widely prescribed
main communication with the NH2-terminal domain. J. Biol. for a variety of medical conditions in both men
Chem. 274, 37219–37225. and women. Misuse of androgens, however,
He, B., Kemppainen, J. A., and Wilson, E. M. (2000). FXX LF and has become an extensive problem of increasing
WXX LF sequences mediate the NH2-terminal interaction with proportion. Therefore, it is important for stu-
the ligand binding domain of the androgen receptor. J. Biol. dents, scientists, and clinicians to be familiar
Chem. 275, 22986–22994. with the risks and benefits of androgen use.
He, B., Bowen, N. T., Minges, J. T., and Wilson, E. M. (2001).
Androgen-induced NH2- and carboxyl-terminal interaction
inhibits p160 coactivator recruitment by activation function 2.
J. Biol. Chem. 276, 42293–42301.
I. INTRODUCTION
Heery, D. M., Kalkhoven, E., Hoare, S., and Parker, M. G. (1997). The principal medical use of androgens is for the
A signature motif in transcriptional co-activators mediates
binding to nuclear receptors. Nature 387, 733 –736.
treatment of male hypogonadism and delayed
Jenkins, R., Angus, R. A., McNatt, H., Howell, W. M., Kemppainen, puberty. However, many new indications for andro-
J. A., Kirk, M., and Wilson, E. M. (2001). Identification gen treatment are emerging. This article focuses on
ANDROGEN RECEPTOR-RELATED PATHOLOGY 89

phallus and the fusion of the genital ridges to form the


scrotum. In this process, the formation of DHT is
Androgen Receptor-Related particularly important to the virilization of the
external genitalia. In addition to events that are
Pathology mediated by the action of the androgens, a polypep-
tide hormone produced by the Sertoli cells of the
MICHAEL J. MC PHAUL testes, Müllerian inhibiting substance (MIS), induces
University of Texas Southwestern Medical School the regression of structures derived from the Müller-
ian ducts, particularly the uterus and fallopian tubes.
I. INTRODUCTION
II. PHENOTYPIC SPECTRUM OF ANDROGEN INSENSITIVITY
III. AR MUTATION TYPES
IV. AR MUTATIONS, FUNCTION, AND CLINICAL PHENOTYPE II. PHENOTYPIC SPECTRUM OF ANDROGEN
V. SPINAL AND BULBAR MUSCULAR ATROPHY INSENSITIVITY
VI. SUMMARY
Mutations of the AR associated with androgen
resistance have been described in a large number of
In mammals, testosterone and 5a-dihydrotes- patients with a wide range of phenotypic abnormal-
tosterone are the principal circulating male ities of male development. At least three different
hormones (androgens). These hormones serve influences are responsible for the relatively large
to modulate a number of important processes, number of clinically apparent defects of AR. The first
from the development of the male phenotype
is the chromosomal location of the AR gene. The AR
during embryogenesis to the regulation of
spermatogenesis and the secretion of gonado- gene is located on the human X chromosome and is
tropins in the adult. Mutations of the human present in only a single copy in 46,XY genotypic
androgen receptor gene have been implicated in males. As such, no second AR allele is present that
pathogenesis of several different diseases. might serve to mask or compensate the effect of any
mutation that impairs the function of the AR gene. In
addition, it is apparent that the actions of androgens,
although important for normal male sexual develop-
I. INTRODUCTION
ment, are not required for life. Even patients with
Biochemical and genetic data have demonstrated that complete defects of AR function mature to adult-
the male hormones, testosterone and 5a-dihydrotes- hood. Finally, many defects of AR function result in
tosterone (DHT), are not equivalent and that they abnormalities of sexual development that are evident
regulate some processes selectively. Although the at birth; such defects have prompted endocrine and
mechanism of such selective regulation is an area of genetic studies designed to identify the etiology.
active investigation, it is clear that the events The phenotypic spectrum of androgen insensitiv-
controlled by each hormone are exerted via the ity syndrome (AIS) is most easily conceived as
single, X-linked androgen receptor (AR). In mam- reflecting the degree to which the androgen-regulated
mals, the hormones produced by the gonads during events of male sexual development have been
specific periods of embryogenesis govern sexual disturbed. In individuals with the clinical phenotype
development. The peptide and steroid hormones of complete androgen insensitivity (complete testicu-
that are produced are, in turn, dictated by whether lar feminization), the function of the AR is completely
the bipotential gonad differentiates into a testis or defective and there is no development of internal or
ovary, a process that is governed by the fetal sex external male structures that are regulated by
chromosome composition and distinct genes encoded androgen. Such individuals show no signs of viriliza-
on autosomes. At approximately 9 weeks of devel- tion. The inability to respond to androgens is not
opment, the testes begin to secrete testosterone. In sufficient to explain the entire phenotype of complete
combination with its 5a reduced metabolite, 5a- androgen insensitivity, however. Although circulating
dihydrotestosterone, testosterone acts to induce the androgens cannot act via the AR, testosterone is
virilization of the internal and external genitalia. The aromatized to estrogen, which mediates feminization
Wolffian ducts grow, forming the pelvic portion of the because of the presence of the normal estrogen
urogenital sinus and giving rise to the seminal vesicles receptors. As such, affected individuals appear to be
and the epididymis, while the external genitalia normally developed women with normal breast
respond with the formation and enlargement of the development. Careful evaluation of such subjects
90 ANDROGEN RECEPTOR-RELATED PATHOLOGY

will identify testes within the labia majora or the evident in a substantial proportion of patients
abdominal cavity. In addition, because the testes in analyzed, even after the application of qualitative
such individuals produce Müllerian inhibiting sub- and quantitative assessments of AR binding. Second,
stance, the Müllerian-derived structures (the uterus it is clear that no direct relationship exists between
and fallopian tubes) are absent and the vagina is blind the clinical phenotype and the type of ligand binding
ending. abnormality. More detailed studies of the nature of
Other intermediate phenotypes have been the defects causing androgen insensitivity awaited
described and reflect differing levels of impairment characterization of the AR at the molecular level.
of AR function. These have been referred to using a
variety of terms, including incomplete testicular B. Structure of the AR
feminization, Reifenstein syndrome, and partial
The AR is a member of a large gene family, the
androgen insensitivity. The phenotypes of such
nuclear receptor family. In addition to the classic
individuals are characterized by varying degrees of
steroid receptors, such as the AR, this family also
virilization. Subjects may display a phenotype that is
contains a number of related proteins, including the
predominantly male in character but with defects of
receptors for thyroid hormone, vitamin D, and
urogenital development, such as perineal hypospadias
retinoic acid. Most members of this family display a
(the Reifenstein phenotype). Others exhibit pheno-
similar organization. Each contains a central DNA-
types that are predominantly female with varying
binding domain (DBD) and a carboxyl-terminal
degrees of clitoromegaly or labial fusion (incomplete
ligand-binding domain (LBD) domain that mediates
testicular feminization). In recent years, Quigley and
the high-affinity binding of ligand. In addition, each
colleagues have published a more detailed system
protein possesses an amino-terminal segment that is
with which to categorize patients with partial forms
of variable length and is required for full transcrip-
of androgen insensitivity.
tional activity. In the case of the human AR, the
Finally, in a small number of patients, specific
amino terminus is large and comprises nearly half of
processes that are regulated by androgen are dis-
the molecule (Fig. 1).
turbed, even though male sexual development
is normal or near normal. In some, subtle signs of
undervirilization, such as gynecomastia, may be III. AR MUTATION TYPES
present. In other individuals, infertility or oligosper-
Initial efforts categorized the syndromes of androgen
mia appear to be the only manifestations of defective
insensitivity syndrome on the basis of the associated
AR function.
abnormalities of ligand binding. At present, the
number of mutations of the AR that have been identi-
A. Characterization of AR Defects: Biochemical fied in patients with different forms of AIS permits
Studies additional information to be used in the classification
of such patients.
The phenotypic abnormalities that accompany the
androgen-resistant states can be viewed as a con-
A. Interruption of the AR Open Reading Frame
tinuum that relates directly to the extent to which the
androgen-mediated processes of male sexual devel- A number of distinct mechanisms have been identified
opment have been disturbed. In parallel with the that cause an interruption of the primary amino acid
description of the clinical syndromes, characteriza- sequence of the human AR. Large-scale deletions,
tion of the type of AR defect became possible using small-scale deletions, insertions, and alterations in
binding assays to measure the level and character of AR structure caused by changes in mRNA splicing
AR present in patient samples. The application of have been reported. Although mechanistically dis-
such techniques has permitted recognition of a tinct, in each instance the final protein product differs
number of quantitative and qualitative abnormalities in primary amino acid sequence from that of the
of androgen binding in patients with different forms normal human AR protein. This difference may be
of androgen insensitivity. caused by a premature truncation or by the addition
The use of these methods has permitted diagnosis or removal of one or more amino acids from the
and classification of many types of patients with receptor sequence. Because the LBDs and DBDs of
androgen insensitivity. Despite the utility of these the AR are located toward the C-terminus of the
techniques, the information derived from such receptor, mutations that result in a shift of the
methods is limited. First, no binding abnormality is open reading frame and premature termination of
ANDROGEN RECEPTOR-RELATED PATHOLOGY 91

FIGURE 1 Alterations of androgen receptor structure associated with human disease. Top: Schematic of the human
AR showing the relative positions of the segments critical to the binding of DNA and ligand by the receptor protein. The
predicted amino acid sequence of the AR also encodes a large amino-terminal segment that comprises approximately
half of the molecule. This region contains motifs composed of repeated glutamine, glycine, and proline residues.
Alterations of the length of the glutamine repeat have been associated with the pathogenesis of spinal and bulbar
muscular atrophy, a propensity to develop aggressive forms of prostate cancer, and infertility. Middle: Representative
mutations of the AR associated with androgen insensitivity syndrome. Categorization by type of genetic mutation is
shown on the left; the observed effects on androgen binding in patients with different forms of androgen insensitivity
syndrome are shown on the right. Bottom: A schematic of AR mutations that result in acquisition of novel biological
properties. In addition to expansion of the glutamine repeat within the amino terminus of the AR that is associated with
spinal and bulbar muscular atrophy (Q21 ! QEXP), also shown are examples of mutations that have been identified in
prostate cancer cell lines and tumors that result in novel responsiveness of the AR. (Modified with permission from
McPhaul, M. J., Marcelli, M., Zoppi, S., Griffin, J. E., Wilson, J. D. (1993). The spectrum of mutations in the androgen
receptor gene that causes androgen resistance. J. Clin. Endocrinol. Metab. 76, 17 – 23. Copyright The Endocrine
Society.)
92 ANDROGEN RECEPTOR-RELATED PATHOLOGY

the receptor have a dramatic effect on receptor receptor cause androgen insensitivity by interfering
function when introduced at virtually any position with a capacity of the receptor to recognize specific
within the primary amino acid sequence. By contrast, target DNA sequences. The published data suggest
mutations that result in the insertion or removal of that the extent to which DNA binding by the mutant
single or multiple amino acids from the normal AR receptors is impaired correlates with the degree of
sequence behave more like amino acid substitution receptor function and the phenotype that is observed.
mutations (see later) and cause syndromes of andro- The data of Nguyen et al. are interesting in this
gen resistance only when occurring within the DNA- regard. These scientists identified a mutation in the
or ligand-binding domains. P-box of the AR DBD in a patient with partial
androgen insensitivity. This study demonstrated that
the mutant AR (G577R; in the one-letter amino acid
B. Mutations in the DBD of the AR code, G is glycine and R is arginine) resulted in the
In the characterization of some patients, endocrine synthesis of a receptor protein that displayed selective
studies or family histories have been suggestive of impairment of binding of the AR to specific types of
androgen insensitivity, but abnormalities of ligand androgen response elements. It was further suggested
binding could not be identified (, 20% in one series). that such differential impairment of DNA binding
This group includes individuals with diverse pheno- might contribute to the diversity of androgen-
types, ranging from the most severely affected resistant phenotypes.
individuals to those with much lesser affected
phenotypes. At the time, these patients were postu- C. Mutations in the AR LBD
lated to harbor either subtle defects of the AR or
defects in other genes that are required for the normal Mutations that change single amino acid residues in
AR function. the LBD are the most frequent type of AR mutation.
In pedigrees in which the family history suggests This category accounts for approximately 60% of all
the inheritance of an X-linked trait, mutations within AR gene mutations causing clinical androgen resist-
the conserved DBD of the receptor are frequently ance. Mutations in this segment of the receptor have
detected. In one study of four unrelated subjects been identified in patients with all of the phenotypes
with complete or near complete forms of androgen associated with androgen insensitivity. These
resistance, amino acid substitutions were localized mutations fall into two categories: amino acid
to the DNA-binding region of the receptor protein substitutions causing absent ligand binding and
in each instance. When these mutant ARs were qualitative abnormalities of ligand binding.
analyzed in detail, they were found to bind androgen
with normal or near normal kinetics, as predicted 1. Amino Acid Substitutions Causing Absent
on the basis of binding studies performed in Ligand Binding
fibroblasts established from the individual patients. Two broad groups of amino acid substitutions in the
When assayed in transfection assays using a model AR LBD have been identified in patient fibroblasts
androgen-responsive reporter gene, these mutant ARs that exhibit undetectable levels of ligand binding.
were found to be markedly impaired. Studies of Only a few examples of the first type have been
fusion proteins containing the DBDs of the normal characterized and represent the replacement of
and mutant ARs demonstrated that in each instance residues critical to the formation of a LBD that is
the mutant ARs displayed an impaired capacity to capable of binding ligand with high affinity. When
bind to target DNA sequences. expressed and studied using eukaryotic or prokar-
AR mutations other than the replacement of yotic expression systems, such mutant ARs are
individual amino acids can have similar effects on incapable of binding ligand. The mutant AR contain-
receptor function when they disrupt the structure of ing a single amino acid substitution at residue 739
the DNA-binding domain while maintaining the AR (W739R; W, tryptophan) is one such mutation.
open reading frame. Such inferences are based on the Inspection of the crystal structures of the AR LBD
identification of patients in whom in-frame deletions reveals that this mutation results in the substitution of
or insertions have removed or inserted one or more a hydrophobic residue (tryptophan) at the amino
amino acid residues. The characterization of mutant terminus of helix 4 with a charged amino acid
ARs of this type has revealed that regardless of the (arginine). These structures demonstrate that this
nature of the causative mutation, changes that alter residue is localized in an area that makes important
the structure of only the DNA-binding domain of the contacts with the C ring of testosterone and is located
ANDROGEN RECEPTOR-RELATED PATHOLOGY 93

deep within the hydrophobic core of the LBD. In the mutant AR to induce the activity of androgen-
addition to disrupting important receptor– ligand responsive genes increased as ligand concentrations
contacts directly, the insertion of a charged residue were increased.
into this region of the receptor is likely to have A large number of the reported different amino
dramatic effects on the overall tertiary structure of the acid substitutions are associated with qualitative
LBD. abnormalities of ligand binding. In virtually all
More commonly, when analyses are performed to instances, the causative mutations are localized to
analyze such mutant ARs by expression in bacteria or the LBD of the receptor. An inspection of the
in eukaryotes, they frequently reveal that the AR is locations of mutations in the AR that cause qualita-
capable of interacting with ligand with measurable tive abnormalities of ligand binding and absent ligand
affinity, even when parallel studies performed in binding shows a similar distribution. This suggests
patient fibroblasts indicate that the mutant AR lacks that the degree to which the structure of the LBD is
the capacity to bind ligand. The mutant receptor disrupted dictates the type of binding abnormality
containing an amino acid replacement at residue 774 that is identified. Alterations that cause more
(R774C; C, cysteine) is an illustrative example. When dramatic changes of structure are associated with
analyzed in cultured fibroblasts, the levels of ligand absent ligand binding and those causing less extensive
binding that are measured are below the detection alterations are associated with qualitative defects of
limits of ligand-binding assays. When this mutant the AR. Studies of mutant ARs in which a single
receptor (R774C) is expressed in heterologous cells, residue has been mutated to different amino acid
however, the mutant receptor can be shown to bind residues in different pedigrees have supported this
ligand. The marked instability of the ligand binding concept. For example, Prior et al. identified a
that is detected in these assays is likely the reason that mutation that resulted in the replacement of an
ligand binding is undetectable in the patient samples. arginine residue by a cysteine residue (R774C) in a
Many additional mutant ARs that exhibit the same patient with complete androgen insensitivity. In
type of behavior as that of the R774C mutant have fibroblasts, this alteration is associated with an
been reported, in which normal or near normal-levels absence of ligand binding. By contrast, in a different
of immunoreactive receptor are expressed. This pedigree, a mutation that causes a replacement of this
discordance appears simply to reflect differences in same arginine by a histidine residue has been shown
the sensitivity of the assays employed and the higher to be associated with normal levels of AR that exhibit
levels of receptor that can be expressed using pronounced thermal instability. Similar studies of
eukaryotic and prokaryotic expression systems. additional subjects with androgen insensitivity have
characterized other mutant ARs in which different
2. Qualitative Abnormalities of Ligand Binding amino acid substitutions at the same residue lead to
In some samples obtained from patients with andro- different effects on ligand binding and receptor
gen insensitivity, the number of ARs measured in function.
ligand-binding assays is normal. In many instances, The study of mutant ARs that exhibit different
when such samples are carefully assessed, the AR that types of qualitative abnormalities and contain amino
is present can be shown to exhibit qualitative acid substitutions in the LBD has provided additional
abnormalities of ligand binding (e.g., affinity, reduced insights. In most target cells, enzymes (such as
increased thermal instability, and accelerated ligand members of the 17-hydroxysteroid dehydrogenase
dissociation). family) actively convert testosterone and 5a-dihydro-
The first study to identify the genetic basis of testosterone to inactive metabolites. Several studies
androgen insensitivity associated with a qualitative have noted that when mutant ARs containing amino
abnormality of the AR (increased Kd) was reported by acid substitutions in the LBD are stimulated with such
Lubahn and colleagues. These workers identified a androgens, the dosing, concentration, and type of
single nucleotide substitution mutation that resulted androgen employed have a dramatic effect on the
in a single amino acid substitution (V866M; V, valine; levels of AR function that are measured in functional
M, methionine) in the LBD of the mutant AR that assays. In the studies of Marcelli and co-workers,
was associated with a phenotype of complete andro- testosterone was the least potent, and dihydrotestos-
gen insensitivity. Functional studies of this mutant AR terone and mibolerone exhibited greater activity. In
demonstrated a reduced capacity to stimulate model several instances, these investigators were able to
androgen-responsive genes in transfection experi- show that repeated dosing of physiologic concen-
ments. Of interest, in these analyses the ability of trations of androgen could restore the function of
94 ANDROGEN RECEPTOR-RELATED PATHOLOGY

the mutant AR. These experiments have demon- The mechanism implicated in the analysis of the
strated the importance of the stability of the patient characterized by Choong and co-workers
hormone – AR complex. Conditions that enhance differs considerably. In this pedigree, the propositus
the formation and stability of these complexes exert was an individual with partial AIS in which reduced
major effects on the function of the mutant receptors levels of apparently normal AR were synthesized.
in functional assays. In addition, these results suggest Analysis of samples from affected subjects revealed a
that mutant receptors capable of binding hormone single nucleotide mutation that causes an amino acid
can be manipulated pharmacologically to normalize replacement at amino acid residue 2 (a lysine residue
AR function, inferences that have been supported in place of the normal aspartate residue at this
clinically in a limited number of instances. Finally, position). On the basis of cell transfection and in vitro
such experiments demonstrate that care must be translation studies, the authors postulated a reduced
taken when considering the relationship between efficiency of translation initiation that led to dimi-
measurements of AR function and patient phenotype. nished levels of AR.
In this regard, it is quite likely that minor changes in
the methods used (e.g., the use of different ligands) E. Additional Perspectives on the Function
could result in considerable differences in the levels of of Mutant ARs (Interaction of Termini)
receptor function that are measured.
Work in a number of laboratories has identified
ligand-dependent interactions occurring between the
D. Mutations That Result in the Synthesis amino and carboxyl termini of the AR. Initially
of Decreased Levels of AR inferred from the influence of changes in the length of
Two different mechanisms have been identified in the amino-terminal glutamine repeat on the activity
relationship to mutations that result in reduced of a mutant AR containing an amino acid substitution
quantities of AR, underscoring the diversity that is in the LBD of the receptor, several groups have
likely to be encountered in this category of genetic defined the basis for this interaction and have
alteration. The first mutation associated with this implicated it as an important component of normal
type of abnormality was reported by Zoppi et al. AR function. Despite considerable attention to
Using monolayer binding assays, affected individuals this interaction, experiments conducted by Chang
were found to express reduced amounts of AR. These and McDonnell have failed to demonstrate that
results contrasted dramatically with the results alterations of gene activation can be observed to ac-
established by immunoblot assays measuring AR in company the disruption of the amino- and carboxyl-
extracts of fibroblasts in affected family members. In terminal interaction using small peptides.
these assays, no immunoreactive AR protein was In one instance, these results have been extended
detected using antibodies directed at the amino to examine the interactions between the amino and
terminus of the AR (residues 1 – 20). This basis for carboxyl termini in mutant ARs containing LBD
this paradox was identified as an AR open reading mutations. There is evidence that the disrupted
frame mutation that introduced a premature termin- amino- and carboxyl-terminal interactions may rep-
ation codon in place of amino acid residue 60. The resent a marker of the potential molecular defect in
low level of AR detected in the initial fibroblast patients with AIS, in whom normal or near-normal
monolayer binding assays was shown to be the result androgen binding is present. Given the limited
of downstream initiation at Met-189. Functional number of AR mutations that have been analyzed in
characterization using transfection into heterologous this fashion, it is not clear whether such studies
cells suggested that the phenotype associated with provide information, or if they simply represent
this mutation in the pedigree (complete androgen additional properties that reflect the formation of an
insensitivity) was caused by a combination of reduced inherently unstable hormone –receptor complex.
receptor expression and a reduced function of the
receptor protein that is synthesized. Additional IV. AR MUTATIONS, FUNCTION,
studies demonstrate that this receptor protein,
AND CLINICAL PHENOTYPE
which is analogous to the A-form of the progesterone
receptor, is present in normal cells, although more It has become evident that the nature of the AR defect
recent reports have raised the possibility that the does not have a simple relationship to the phenotype
shortened AR (AR-A) is the result of protein exhibited by affected individuals. Despite this, the
degradation. large number of AR mutations that have been
ANDROGEN RECEPTOR-RELATED PATHOLOGY 95

reported in patients with various forms of androgen 5 AIS) and the second with a predominantly male
resistance permits a number of generalizations to be phenotype (perineal hypospadias, micropenis, and
made. First, truncation or interruption of the AR bifid scrotum; classified as grade 3 AIS). Investi-
protein is associated with a phenotype of complete gations of the structures of the AR genes in these
androgen resistance. This association is caused by the individuals identified identical AR mutations
location of the critical DNA- and ligand-binding (R855H; H, histidine) within the receptor LBD.
domains in the carboxyl-terminal segments of the AR Analyses of fibroblasts established from both subjects
open reading frame. For this reason, interruptions of revealed similar levels of androgen binding in both
the coding sequence remove one or both of these strains that were within the normal range. In these
important functional domains and result in the analyses, the mutant AR exhibited only a minor
synthesis of ARs that are inactive in assays of receptor reduction in binding affinity, compared to normal
function. By contrast, amino acid substitutions in the controls. In contrast to these findings, measurements
AR can cause variable effects on AR function and of 5a-reductase levels revealed substantial differences
have been associated with the complete range of between the strains. In the subject with the greater
androgen-resistant phenotypes. degree of virilization, levels of 5a-reductase were
The second generalization pertains to the relation- similar to the levels present in normal male and
ship between the AR gene defect and the observed female fibroblasts. By contrast, the level of 5a-
clinical phenotype. In some instances, no AR is reductase detected in the subject with the predomi-
expressed or the genetic mutation completely nantly female phenotype was in a range consistent
abolishes AR function. In these instances, the clinical with that observed in patients with 5a-reductase
phenotype—complete androgen insensitivity—agrees deficiency. This finding could not be explained on the
with assessments of receptor function and is consis- basis of mutations of 5a-reductase 2 gene and was felt
tent between pedigrees. Such considerations do not possibly to reflect “secondary” 5a-reductase defi-
apply when the AR that is expressed is not completely ciency, as had been observed in other AIS pedigrees.
defective. In these instances, the quantitation of the In any event, the differences in local formation of
degree of deficiency is considerably more difficult, DHT were proposed to underlie the differences in the
because the results of functional assays may show degree of virilization observed in these two subjects.
marked differences, depending on the conditions The postulated effect in these patients would be
under which such assays are performed. Although a expected to mirror the effects of DHT versus
number of examples of this phenomenon can be testosterone in cell transfection experiments using
found in the literature, the experiments of Marcelli different ligands to stimulate mutant receptors
are representative, i.e., changes in the incubation containing amino acid substitution in the LBD.
conditions or the type of the ligand used can lead to In some instances, the mutation identified appears
dramatic differences in the level of receptor function inconsistent with the observed phenotype. (e.g., a
measured. mutation causing premature termination of the AR in
Although in many instances similar phenotypes a subject with substantial levels of virilization) This is
are observed in different pedigrees in which the same the circumstance in the pedigree examined by
mutation is identified, a significant proportion of Holterhus and co-workers, who identified a prema-
patients with identical mutations will exhibit various ture termination codon occurring at amino acid 172
phenotypes. In the analysis of Gottlieb et al., as many in the open reading frame of the AR. Surprisingly, this
as 10 –15% of cases contained in the AR Mutation mutation was identified in a patient exhibiting subtle
Database have been reported to have distinctive signs of virilization (clitoromegaly). Investigation of
phenotypes but have identical mutations. The issues the level and nature of AR expressed in this patient
raised by these inferences have been more carefully indicated that the reduced level of ligand binding that
directly assessed in the Netherlands in a nationwide was detected reflected the presence of reduced
survey of patients with AIS. In this study, fully one- amounts of full-length normal AR. The derivation
third of families with partial AIS showed considerable of this normal AR was shown to be the result of a
variation in phenotype. At least two distinct mech- somatic mosaicism in which a proportion of the cells
anisms have been identified that may contribute to in this patient expressed normal AR. This reduced
such differences. level of normal AR was proposed to account for the
Boehmer et al. reported the characterization of degree of virilization that was observed in this
two siblings with partial forms of AIS: one with a patient. The investigators have identified additional
predominantly female phenotype (classified as grade pedigrees in which both normal and mutant ARs are
96 ANDROGEN RECEPTOR-RELATED PATHOLOGY

present. The proportion of mutant AR that is present glutamine residues (homopolymeric segment) within
is believed to influence the clinical phenotype. the open reading frame of the AR, from its normal
size (11 –33) to a larger size not observed in normal
individuals (. 38). This expansion is believed to
influence at least two distinct properties of the AR
V. SPINAL AND BULBAR MUSCULAR
that contribute to the overall phenotype that is
ATROPHY observed in individuals affected by spinal and bulbar
In 1968, Kennedy, Alter, and Sung reported the muscular atrophy (SBMA). First is the contribution of
description of two families with an adult-onset form this genetic change to the mild androgen resistance
of spinal and bulbar muscular atrophy. Although that is observed in a proportion of affected individ-
similar to other “muscular atrophies” that had been uals. Males carrying this genetic change develop
described previously, affected individuals in these normally and exhibit normal sexual function early in
pedigrees were noted to exhibit several unusual life. Despite this, a proportion of affected subjects
features. First, the disease became clinically evident begin to exhibit phenotypic changes characteristic of
in the fourth and fifth decades of life and was slowly mild androgen resistance as they enter adulthood. In
progressive. Affected individuals described low back- several series, 25 –50% of subjects had gynecomastia
ache and muscle cramps. These initial symptoms were and several had been investigated for infertility. The
followed by the appearance of overt muscle weakness expansion of the size of the glutamine repeat has been
and fasciculations. The muscle weakness was most suggested to contribute to these endocrine effects in
notable in the proximal musculature; distal muscular two different ways. First, it appears that the
involvement occurred later and was less pronounced. glutamine repeat expansion impairs the activity of
Bulbar involvement was present in affected individ- the receptor protein in transfection assays of receptor
uals and resulted in both dysarthria and dysphagia. function. When examined carefully, these changes
Nerve conduction velocities were normal and elec- appear to reflect true decreases in the specific activity
tromyelogram (EMG) analyses identified patterns of the receptor protein in modulating the activity of
consistent with degeneration of motor neurons within model reporter genes. In addition to these changes,
the anterior horns and cranial motor nuclei. Although however, it appears that the expansion of the
sensation and cerebellar function were judged to be glutamine repeats may in fact impair the efficiency
normal in clinical assessments, subsequent studies of translation of the AR mRNA, resulting in lower
identified abnormal sensory action potentials, leading levels of AR expression. Both of these changes may
some to term this disorder “bulbospinal neurono- well contribute to the evolution of the mild androgen
pathy.” Pathologic findings have supported that resistance that is a component of the SBMA
changes in ganglia are likely the basis for these phenotype. Perhaps with the decline of testicular
changes. Although such sensory abnormalities are function that occurs with age even in normal subjects,
consistently demonstrable in a proportion of affected the impairment of function of ARs containing the
subjects, symptoms and signs referable to the glutamine repeat expansion becomes evident
degeneration of affected spinal and bulbar motor clinically.
nuclei predominate. Importantly, only males are La Spada and colleagues were the first to link the
affected in these families, suggesting inheritance as a expansion of a glutamine repeat within the open
sex-linked recessive trait. In addition to these reading frame of a protein to the pathogenesis of a
neurological features, 3 of the 11 affected individuals disease. In the case of SBMA, they correlated this
were noted to have findings consistent with a defect of disorder to the expansion of a homopolymeric
androgen action (gynecomastia). domain within the amino terminus of the AR gene
Given the involvement of the AR in mediating the (from , 20 in normal controls to . 38 in affected
effects of androgen in virilization and fertility, the AR, individuals). Subsequent investigations have impli-
known to be localized to the human X chromosome, cated similar genetic changes in the pathogenesis of
represents a potential candidate gene for this disorder. several progressive neurological diseases, including
Seminal work by La Spada and colleagues has Huntington’s disease, spinocerebellar ataxia-type 1,
demonstrated that an expansion of a CAG triplet Machado – Joseph disease, and dentatorubral palli-
repeat in the amino terminus of the AR is the genetic doluysian atrophy.
change responsible for the development of Kennedy’s The mechanisms by which such glutamine repeat
syndrome. The increased size of this repeat segment expansions cause neurological symptoms remain
results in the expansion of a segment of repeated the subject of active investigation. Several lines of
ANDROGEN RECEPTOR-RELATED PATHOLOGY 97

evidence have suggested that the proteins containing subtle undervirilization or infertility. Between these
expanded polyglutamine tracts, such as the AR, extremes are individuals with substantial defects of
acquire a toxic property that is not possessed by the AR function that are associated with phenotypes
normal protein. Several groups have proposed that between that of a normal male and a normal female.
the central mechanism may involve accumulation of Although diverse at the molecular level, the genetic
peptides that are produced as a result of the presence alterations causing androgen insensitivity can be
of the glutamine repeat expansion. Supporting such a explained as reflecting loss-of-function mutations.
mechanism are studies that have identified intracellu- The same is not true for spinal and bulbar
lar inclusions containing fragments of ARs that muscular atrophy (Kennedy’s disease). This disease
contain the glutamine repeat expansion. In such is characterized by the appearance in adulthood of
models, the toxicity of these fragments is believed to signs and symptoms of mild androgen insensitivity
result in the degeneration of neurons within selected and the progressive deterioration of function of
spinal and bulbar nuclei. The mechanism of this muscles innervated by spinal and bulbar motor
toxicity may derive from the sequestration of critical nuclei. The pathogenesis of this disorder has been
proteins, such as transcription factors by the gluta- linked to the expansion of a triplet repeat encoding a
mine repeat-containing peptides. The toxicity may series of repeated glutamine residues. As with other
also involve distinct mechanisms that contribute to diseases caused by the expansion of glutamine
the neural pathology. It is likely that several different repeats, the basis of this toxic gain-of-function
properties of proteins containing glutamine repeat mutation has been the subject of intense mechanistic
expansions may contribute to the toxicity that these studies.
proteins exhibit. Further investigations will be needed
to clarify which of these properties represent critical
pathologic properties and which are essentially
ancillary findings. Acknowledgment
Despite the insights that such experiments have The original work in this article was supported by NIH grant
provided, a number of important questions still DKD3892.
remain pertaining to the mechanisms by which this
toxicity emerges. A particularly intriguing and
unexplained aspect of this disease is the timing of its Glossary
onset. The onset of the disease occurs later in life (i.e.,
middle age), in a time frame that is difficult to explain 17-hydroxysteroid dehydrogenases Group of enzymes
responsible for the reversible conversion of testosterone
based on what is known regarding the expression of
and 5a-dihydrotestosterone to inactive metabolites
the gene product (the AR) that has been implicated. (androstenedione and 5a-androstenedione, respect-
Finally, it is remarkable that the toxicity observed in ively). In specific tissues, selected members of this
SBMA is restricted to cells of neural lineage, enzyme group are responsible for synthesis of active
suggesting that some aspect of the neuronal cell androgens (e.g., 17b-hydroxysteroid dehydrogenase
type might make it particularly susceptible to the type 3 in the testis).
effects of proteins containing the expanded glutamine P-box Segment of the DNA-binding domain of nuclear
repeats. receptors that is responsible for the specificity of DNA
binding by the receptor protein.
5a-reductase Enzyme responsible for the conversion of
VI. SUMMARY testosterone to 5a-dihydrotestosterone.
target cell Type of cell in which a specific hormone exerts
Mutations of the human androgen receptor gene have
its actions (e.g., the prostate is a classic androgen target
been implicated in pathogenesis of several different tissue).
diseases. In androgen insensitivity, AR function is
defective to varying extents, causing a range of
abnormalities of male sexual development. At one See Also the Following Articles
end of this spectrum, individuals with complete Androgen Effects in Mammals . Androgen Receptor
androgen insensitivity (complete testicular feminiza- Crosstalk with Cellular Signaling Pathways . Androgen
tion) exhibit a normal female phenotype, with normal Receptors and Prostate Cancer . Androgen Receptor
breast development and female external genitalia. On Structure and Function . Androgens: Pharmacological Use
the other end of the spectrum, individuals with AR and Abuse . Dihydrotestosterone, Active Androgen
defects have male external genitalia, but exhibit either Metabolites and Related Pathology
98 ANDROGEN RECEPTORS AND PROSTATE CANCER

Further Reading defects: Historical, clinical, and molecular perspectives.


Endocr. Rev. 16, 271–321.
Boehmer, A. L., Bruggenwirth, H., van Assendelft, C., Otten, B. J., Thompson, J., Saatcioglu, F., Janne, O. A., and Palvimo, J. J.
Verleun-Mooijman, M. C., Niermeijer, M. F., Brunner, H. G., (2001). Disrupted amino- and carboxyl-terminal interactions
Rouwe, C. W., Waelkens, J. J., Oostdijk, W., Kleijer, W. J., van of the androgen receptor are linked to androgen insensitivity.
der Kwast, T. H., de Vroede, M. A., and Drop, S. L. (2001). Mol. Endocrinol. 15, 923–935.
Genotype versus phenotype in families with androgen insensi- Zoppi, S., Marcelli, J. P., Deslypere, Griffin, J. E., Wilson, J. D., and
tivity syndrome. J. Clin. Endocrinol. Metab. 86, 4151–4160. McPhaul, M. J. (1992). Amino acid substitutions in the DNA-
Boehmer, A. L., Brinkmann, A. O., Nijman, R. M., Verleun- binding domain of the human androgen receptor are a frequent
Mooijman, M. C., de Ruiter, P., Niermeijer, M. F., and Drop, cause of receptor-binding positive androgen resistance. Mol.
S. L. (2001). Phenotypic variation in a family with partial Endocrinol. 6, 409–415.
androgen insensitivity syndrome explained by differences in 5- Zoppi, S., Young, M., and McPhaul, M. J. (2001). Regulation of
alpha dihydrotestosterone availability. J. Clin. Endocrinol. gene expression by the nuclear receptor family. In “The
Metab. 86, 1240– 1246. Genetics of Steroid Biosynthesis and Function” (J. I Mason,
Chang, C.-Y., and McDonnell, D. P. (2002). Evaluation of ligand- ed.), pp. 369–405. Taylor & Francis, London and New York.
dependent changes in AR structure using peptide probes. Mol.
Endocrinol. 16, 647–660. Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.

Choong, C. S., Quigley, C. A., French, F. S., and Wilson, E. M.


(1996). A novel missense mutation in the amino-terminal
domain of the human androgen receptor gene in a family with
partial androgen insensitivity syndrome causes reduced effi-
ciency of protein translation. J. Clin. Invest. 98, 1423–1431.
Androgen Receptors and
Gottlieb, B., Beitel, L. K., and Trifiro, M. A. (2001). Variable
expressivity and mutation databases: The androgen receptor
Prostate Cancer
gene mutations database. Hum. Mutat. 17, 382–388.
Holterhus, P. M., Wiebel, J., Sinnecker, G. H., Bruggenwirth, H. T., DOLORES J. LAMB
Sippell, W. G., Brinkmann, A. O., Kruse, K., and Hiort, O. Baylor College of Medicine
(1999). Clinical and molecular spectrum of somatic mosaicism
in androgen insensitivity syndrome. Pediatr. Res. 46, 684–690.
La Spada, A. R., Wilson, E. M., Lubahn, D. B., Harding, A. E., and I.INTRODUCTION
Fischbeck, K. H. (1991). Androgen receptor gene mutations in II.CHANGES IN THE ANDROGEN RECEPTOR GENE
X-linked spinal and bulbar muscular atrophy. Nature 352, III.CHANGES IN THE RECEPTOR PROTEIN
77–79. IV. CHANGES IN INTERACTING FACTORS THAT MODULATE
Lubahn, D. B., Brown, T. R., Simental, J. A., Higgs, H. N., Migeon, RECEPTOR FUNCTION
C. J., Wilson, E. M., and French, F. S. (1989). Sequence of the V. CHANGES IN STEROID BIOAVAILABILITY
intron/exon junctions of the coding region of the human VI. ALTERNATIVE ROUTES TO GROWTH STIMULATION
androgen receptor gene and identification of a point mutation VII. SUMMARY
in a family with complete androgen insensitivity. Proc. Natl.
Acad. Sci. U.S.A. 86, 9534–9538.
Marcelli, M., Tilley, W. D., Zoppi, S., Griffin, J. E., Wilson, J. D., Diseases of the prostate account for an enor-
and McPhaul, M. J. (1991). Androgen resistance associated mous portion of the morbidity, mortality, and
with a mutation of the androgen receptor at amino acid 772
economics in health care of males in the United
(Arg – ! Cys) results from a combination of decreased
messenger ribonucleic acid levels and impairment of receptor
States. Prostate cancer represents a major
function. J. Clin. Endocrinol. Metab. 73, 318–325. health problem for adult men and is the second
Marcelli, M., Zoppi, S., Wilson, C. M., Griffin, J. E., and McPhaul, leading cause of cancer deaths in the United
M. J. (1994). Amino acid substitutions in the hormone-binding States. If men are castrated before puberty, pros-
domain of the human androgen receptor alter the stability of tate cancer does not develop. Accordingly,
the hormone receptor complex. J. Clin. Invest. 94, androgens are thought to be required for the
1642–1650. initiation and initial progression of prostate
Nguyen, D., Steinberg, S. V., Rouault, E., Chagnon, S., Gottlieb, B., cancer.
Pinsky, L., Trifiro, M., and Mader, S. (2001). A G577R
mutation in the human AR P box results in selective decreases
in DNA binding and in partial androgen insensitivity
syndrome. Mol. Endocrinol. 15, 1790– 1802. I. INTRODUCTION
Prior, L., Bordet, S., Trifiro, M. A., Mhatre, A., Kaufman, M.,
Pinsky, L., Wrogeman, K., Belsham, D. D., Pereira, F., and Androgens are required for the normal development
Greenberg, C. (1992). Replacement of arginine 773 by cysteine and differentiated function of the prostate. It is not
or histidine in the human androgen receptor causes complete
surprising that tumors that form in the prostate
androgen insensitivity with different receptor phenotypes. Am.
J. Hum. Genet. 51, 143–155. respond to androgens during the early stages of
Quigley, C. A., De Bellis, A., Marschke, K. B., el-Awady, M. K., disease. For tumors that have not spread outside of
Wilson, E. M., and French, F. S. (1995). Androgen receptor the prostate, radical prostatectomy is a favored form
98 ANDROGEN RECEPTORS AND PROSTATE CANCER

Further Reading defects: Historical, clinical, and molecular perspectives.


Endocr. Rev. 16, 271–321.
Boehmer, A. L., Bruggenwirth, H., van Assendelft, C., Otten, B. J., Thompson, J., Saatcioglu, F., Janne, O. A., and Palvimo, J. J.
Verleun-Mooijman, M. C., Niermeijer, M. F., Brunner, H. G., (2001). Disrupted amino- and carboxyl-terminal interactions
Rouwe, C. W., Waelkens, J. J., Oostdijk, W., Kleijer, W. J., van of the androgen receptor are linked to androgen insensitivity.
der Kwast, T. H., de Vroede, M. A., and Drop, S. L. (2001). Mol. Endocrinol. 15, 923–935.
Genotype versus phenotype in families with androgen insensi- Zoppi, S., Marcelli, J. P., Deslypere, Griffin, J. E., Wilson, J. D., and
tivity syndrome. J. Clin. Endocrinol. Metab. 86, 4151–4160. McPhaul, M. J. (1992). Amino acid substitutions in the DNA-
Boehmer, A. L., Brinkmann, A. O., Nijman, R. M., Verleun- binding domain of the human androgen receptor are a frequent
Mooijman, M. C., de Ruiter, P., Niermeijer, M. F., and Drop, cause of receptor-binding positive androgen resistance. Mol.
S. L. (2001). Phenotypic variation in a family with partial Endocrinol. 6, 409–415.
androgen insensitivity syndrome explained by differences in 5- Zoppi, S., Young, M., and McPhaul, M. J. (2001). Regulation of
alpha dihydrotestosterone availability. J. Clin. Endocrinol. gene expression by the nuclear receptor family. In “The
Metab. 86, 1240– 1246. Genetics of Steroid Biosynthesis and Function” (J. I Mason,
Chang, C.-Y., and McDonnell, D. P. (2002). Evaluation of ligand- ed.), pp. 369–405. Taylor & Francis, London and New York.
dependent changes in AR structure using peptide probes. Mol.
Endocrinol. 16, 647–660. Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.

Choong, C. S., Quigley, C. A., French, F. S., and Wilson, E. M.


(1996). A novel missense mutation in the amino-terminal
domain of the human androgen receptor gene in a family with
partial androgen insensitivity syndrome causes reduced effi-
ciency of protein translation. J. Clin. Invest. 98, 1423–1431.
Androgen Receptors and
Gottlieb, B., Beitel, L. K., and Trifiro, M. A. (2001). Variable
expressivity and mutation databases: The androgen receptor
Prostate Cancer
gene mutations database. Hum. Mutat. 17, 382–388.
Holterhus, P. M., Wiebel, J., Sinnecker, G. H., Bruggenwirth, H. T., DOLORES J. LAMB
Sippell, W. G., Brinkmann, A. O., Kruse, K., and Hiort, O. Baylor College of Medicine
(1999). Clinical and molecular spectrum of somatic mosaicism
in androgen insensitivity syndrome. Pediatr. Res. 46, 684–690.
La Spada, A. R., Wilson, E. M., Lubahn, D. B., Harding, A. E., and I.INTRODUCTION
Fischbeck, K. H. (1991). Androgen receptor gene mutations in II.CHANGES IN THE ANDROGEN RECEPTOR GENE
X-linked spinal and bulbar muscular atrophy. Nature 352, III.CHANGES IN THE RECEPTOR PROTEIN
77–79. IV. CHANGES IN INTERACTING FACTORS THAT MODULATE
Lubahn, D. B., Brown, T. R., Simental, J. A., Higgs, H. N., Migeon, RECEPTOR FUNCTION
C. J., Wilson, E. M., and French, F. S. (1989). Sequence of the V. CHANGES IN STEROID BIOAVAILABILITY
intron/exon junctions of the coding region of the human VI. ALTERNATIVE ROUTES TO GROWTH STIMULATION
androgen receptor gene and identification of a point mutation VII. SUMMARY
in a family with complete androgen insensitivity. Proc. Natl.
Acad. Sci. U.S.A. 86, 9534–9538.
Marcelli, M., Tilley, W. D., Zoppi, S., Griffin, J. E., Wilson, J. D., Diseases of the prostate account for an enor-
and McPhaul, M. J. (1991). Androgen resistance associated mous portion of the morbidity, mortality, and
with a mutation of the androgen receptor at amino acid 772
economics in health care of males in the United
(Arg – ! Cys) results from a combination of decreased
messenger ribonucleic acid levels and impairment of receptor
States. Prostate cancer represents a major
function. J. Clin. Endocrinol. Metab. 73, 318–325. health problem for adult men and is the second
Marcelli, M., Zoppi, S., Wilson, C. M., Griffin, J. E., and McPhaul, leading cause of cancer deaths in the United
M. J. (1994). Amino acid substitutions in the hormone-binding States. If men are castrated before puberty, pros-
domain of the human androgen receptor alter the stability of tate cancer does not develop. Accordingly,
the hormone receptor complex. J. Clin. Invest. 94, androgens are thought to be required for the
1642–1650. initiation and initial progression of prostate
Nguyen, D., Steinberg, S. V., Rouault, E., Chagnon, S., Gottlieb, B., cancer.
Pinsky, L., Trifiro, M., and Mader, S. (2001). A G577R
mutation in the human AR P box results in selective decreases
in DNA binding and in partial androgen insensitivity
syndrome. Mol. Endocrinol. 15, 1790– 1802. I. INTRODUCTION
Prior, L., Bordet, S., Trifiro, M. A., Mhatre, A., Kaufman, M.,
Pinsky, L., Wrogeman, K., Belsham, D. D., Pereira, F., and Androgens are required for the normal development
Greenberg, C. (1992). Replacement of arginine 773 by cysteine and differentiated function of the prostate. It is not
or histidine in the human androgen receptor causes complete
surprising that tumors that form in the prostate
androgen insensitivity with different receptor phenotypes. Am.
J. Hum. Genet. 51, 143–155. respond to androgens during the early stages of
Quigley, C. A., De Bellis, A., Marschke, K. B., el-Awady, M. K., disease. For tumors that have not spread outside of
Wilson, E. M., and French, F. S. (1995). Androgen receptor the prostate, radical prostatectomy is a favored form
ANDROGEN RECEPTORS AND PROSTATE CANCER 99

of treatment. If the tumor has spread and metasta- these alterations in the androgen receptor in prostate
sized, androgen ablation therapy, first proposed by cancer progression is also discussed.
Charles Huggins (who received the Nobel Prize in
1966 for this hypothesis), is a treatment for this II. CHANGES IN THE ANDROGEN
disease. Like metastatic breast cancers, initially the RECEPTOR GENE
tumors respond to steroid deprivation, with
regression resulting from apoptosis of androgen- A. Amplification of the Androgen
dependent prostate cancer cells. Although nearly all Receptor Gene
tumors continue to express the androgen receptor,
An increased copy number or amplification of the
some tumors may contain a heterogeneous mixture of androgen receptor gene has been observed in some
androgen-dependent and androgen-independent prostate cancers. Investigators have proposed that
cells. The androgen-independent cells will escape this amplification may lead to overexpression of the
apoptosis induced by androgen ablation. Other androgen receptor and, ultimately, to tumor growth
androgen-dependent cells may adapt to a state of stimulation at low levels of circulating androgens.
androgen depletion and then gain an androgen- Some investigators have questioned whether the
independent phenotype. Ultimately, prostate cancer amplification has physiologic relevance and it is
progresses to a state of androgen independence with somewhat controversial in the literature. An increase
uncontrolled growth. For the patient, this represents in androgen receptor copy number does not correlate
the failure of their therapy and a probable poor with the response to initial androgen ablation
outcome. The molecular basis for this progression to therapy, although it does appear to correspond to a
androgen independence is largely unknown at this positive outcome of secondary treatment with com-
time. One possibility is that mutations in the plete androgen blockade. The increase in androgen
androgen receptor contribute to this progression of receptor copy number was correlated with an
the prostate cancer to an androgen-independent state. increased secretion of prostate-specific antigen that
The androgen receptor is a member of the steroid did not correlate with tumor burden, again suggesting
receptor superfamily. The gene is located on the X a functional consequence of this amplification.
chromosome and there are eight exons that encode a Although androgen receptor gene amplification and
protein of 919 amino acids. Because the gene is an increased level of the androgen receptor gene
located on the X chromosome, mutation of only one expression occur in a significant percentage of
allele is required for a phenotypic change. Impor- samples obtained from hormone-independent
tantly, androgen action is dependent on not only a tumors, the frequency of androgen receptor gene
functioning receptor with high affinity and steroid- amplification is too low to account for all prostate
binding specificity, but also on the interactions of cancer progression to androgen independence.
growth factor signaling pathways and the complex
interaction of co-activators, co-repressors, and other B. Point Mutations Producing Superactive
factors to regulate specific gene expression. Changes Androgen Receptors
in any of these factors could influence androgen Theoretically, an androgen receptor mutation could
action. enhance the transcriptional activation of the receptor,
Mutant receptors with an altered function were especially at low ligand concentrations. In a trans-
first described in breast cancer, leading investigators genic adenocarcinoma of the prostate mouse,
to propose that point mutations in the androgen mutation was detected at residues (668)QPIF(671)
receptor may account for progression from androgen- at the boundary of the hinge and ligand-binding
dependent to androgen-independent growth in pros- domain, resulting in a receptor that exhibits two- to
tate cancer. Molecular changes in receptor structure fourfold increased activity compared with wild-type
or concentration may result in part in hormone- AR. This enhanced activity was noted in response to
independent tumor growth. The types of androgen administration of dihydrotestosterone, estradiol, pro-
receptor mutations/alterations and their functional gesterone, and adrenal androgens, as well as the
consequences are described here. In addition to androgen receptor antagonist hydroxy flutamide
mutations, there are a number of other potential without an apparent effect on receptor levels,
changes in the androgen receptor signaling pathway ligand-binding kinetics, or DNA binding. The recep-
that can stimulate androgen receptor activity at tor could be activated by low circulating levels of
suboptimal levels of hormone. The potential role of androgen. Thus, if such a mutation were present in
100 ANDROGEN RECEPTORS AND PROSTATE CANCER

a human prostate cancer, the growth of the tumor receptor that could bind steroid and translocate to
might be stimulated by the low levels of androgen the nucleus but was localized in abnormal discrete
remaining following androgen ablation. Preliminary foci, resulting in cytoplasmic and nuclear aggregates,
studies suggest that these types of mutations may perhaps due to protein misfolding. The mutated
occur in some advanced human prostate cancers, but androgen receptor could not activate androgen-
further investigations are required to confirm that this regulated transcription. Since androgens are normally
occurs and, if so, to define the frequency. This would thought to enhance prostate growth, the presence of
be considered to be a “gain of function” mutation. an “inactivating” mutation in advanced prostate
cancer may be surprising. Recently, Tindall and
C. Point Mutations Generating a Promiscuous colleagues showed that disruption of the androgen
receptor protein in prostate cancer cell lines inhibited
Receptor Protein That Is Activated by Ligands
their proliferation, suggesting that these loss of
Other Than Androgen function mutations may not provide a growth
Mutations that change steroid-binding specificity are advantage to the tumor. Accordingly, the mechanism
thought to be important in the progression of some of androgen regulation of prostatic growth is unclear,
prostate cancers. In fact, the first mutation found in and loss of androgen receptor function in the tumor
the androgen receptor in a prostate cancer was may result in a progressive loss of differentiated
identified in the LNCaP cell line. This cell line is an function and loss of growth control or it may inhibit
androgen-responsive cell line derived from a human tumor growth.
prostate cancer that was metastatic to bone. The
functional consequence of this mutation in exon 8 of E. Androgen Receptor Polymorphisms That
the androgen receptor is to change the steroid-binding
Modulate Transcriptional Activation
specificity of the androgen receptor, resulting in
estradiol and the anti-androgens hydroxy-flutamide, The length of a polymorphic polyglutamine tract in
nilutamide, and cyproterone acetate becoming ago- exon 1 of the androgen receptor influences the
nists. Since the identification of this first prostate relative transcriptional activity of the androgen
cancer androgen receptor mutant, mutations have receptor in vitro. The length of the tract is inversely
been identified in the androgen receptor in other correlated with the transcriptional activation of the
specimens of prostate cancer and cell lines, and these receptor by androgen. The shorter the tract, the more
mutations broaden or change the binding specificity active the receptor; receptor transcriptional acti-
of the androgen receptor for glucocorticoids, some vation is diminished as the tract lengthens. There
adrenal and weaker androgens, anti-androgens, are racial differences in the average length of the tract,
estrogens, and progesterone. In human prostate with African American males having approximately
cancer, the consequence of this type of mutation is 18 repeats, Caucasian males having an average of 21
obvious. Treatment of the patient with an anti- repeats, and Asian males having the highest repeat
androgen or an estrogen would have a detrimental number within the normal polymorphic range.
effect. Rather than inhibiting tumor growth, the Expansion of these repeats to 40 or more results in
antagonist has become an agonist for the mutated Kennedy disease (spinobulbar muscular atrophy), a
receptor and might enhance the growth of the tumor. progressive degenerative neuromuscular illness.
These mutations would all be considered to be in the Some, but not all, investigators have reported that
category of gain of function. the CAG repeat length (within the normal range)
might be related to the age of the patient at diagnosis
D. Inactivating Mutations of the Androgen of prostate cancer and their response to endocrine
therapy. It has been reported that a shorter poly-
Receptor
glutamine tract is associated with the presence of
A mutation in the androgen receptor may result in metastatic disease at the time of diagnosis, a younger
either the total loss of receptor protein (such as in a age of onset, increased incidence, higher mortality,
total deletion) or, more commonly, a truncation of the and a more aggressive nature of prostate cancer in the
receptor or a “loss of function” in one of the African American population; however, not all
functional domains of the protein. An example of authors agree. In general, it is accepted that the
this type of mutation is C619Y, found in a metastatic shorter polyglutamine repeat correlates with an
lymph node of a patient with prostate cancer. earlier age of onset and a diminished response of
This androgen receptor mutation resulted in a the prostate cancer to endocrine therapy.
ANDROGEN RECEPTORS AND PROSTATE CANCER 101

III. CHANGES IN THE RECEPTOR PROTEIN B. Ligand-Independent Activation of Androgen


Receptor
A. Intracellular Changes That Influence
Androgen Receptor Sensitivity or Protein The direct or indirect transcriptional activation of the
androgen receptor by growth factors, neurotransmit-
Stability
ters, or other agents that increase intracellular kinase
Receptor function may be modulated indirectly by activity or decrease phosphatase activity has been
changes in the expression of specific proteins, such as shown for chicken progesterone receptor and the
caveolin, that enhance androgen receptor action by human estrogen receptor. Similarly, the androgen
influencing androgen receptor sensitivity. Similarly, receptor can be activated in the absence of androgen
enhanced expression of the receptor, stabilization of by several growth factors thought to be acting
the protein, and nuclear localization of the receptor at through the mitogen-activated protein kinase path-
very low ligand concentrations enhanced androgen- way, through the protein kinase A signaling pathway,
mediated growth in human prostate cell lines tested in or through Her-2-neu overexpression signaling
vitro. These types of changes in receptor character- androgen receptor phosphorylation by Akt. Support
istics, whether through a direct action on the receptor for a role of kinases in androgen receptor action
or an indirect action, serve to enhance androgen comes from the observation that the tumor suppres-
receptor action at low concentrations of androgen, sor gene PTEN appears to inhibit androgen receptor
and presumably this contributes to enhanced growth action. As a result of this complex cross-talk between
of prostate cancer after endocrine ablation treatment. different signal transduction pathways, the androgen
receptor has the potential for activation.

IV. CHANGES IN INTERACTING FACTORS V. CHANGES IN STEROID BIOAVAILABILITY


THAT MODULATE RECEPTOR FUNCTION
A. Altered Steroid Bioavailability
A. Co-activator or Co-repressor Modulation of or Circulating Levels
Androgen Action in Advanced Prostate Cancer Only a small portion of circulating androgen is
Steroid receptors require co-activators to mediate unbound in the circulation and it is the “free”
their transcriptional activation functions with pro- testosterone that is available to diffuse into the cells,
teins in the general transcription complex to stimulate bind to the receptor, and initiate androgen action.
the steroid-regulated transcription of target genes. Changes in the relative concentrations of free to
These factors interact with either the amino- or the bound androgen may influence prostate cancer
carboxy-terminal regions of the receptor to enhance growth. There may be local increased or decreased
transcription. Some co-activators modulate a broad intraprostatic bioavailability of dihydrotestosterone
range of transcription factors, others stimulate the to activate the androgen receptor. Similarly, racial
activities of nuclear receptors in general, and others differences in the average levels of circulating andro-
show specificity for one or a few nuclear receptors. gens may lead to conditions more permissive of
Many proteins have been identified that enhance prostate cancer development and/or progression.
androgen receptor transcriptional activity in vitro. Thus, conditions that modulate the relative concen-
Loss of co-activator interaction may diminish andro- tration of androgen available to the cell may influence
gen receptor transcriptional activation or may prostate cancer.
broaden the ligand specificity of the receptor. Con-
versely, overexpression of specific co-activators, such
as steroid receptor co-activator 1 and transcriptional VI. ALTERNATIVE ROUTES TO GROWTH
intermediary factor 2, enhance androgen receptor STIMULATION
transactivation at physiological concentrations of A. Activation of Growth-Stimulating Signaling
adrenal androgen, providing an alternative mechan-
Pathways with the Ability to Bypass Androgen
ism for enhanced prostate cancer growth despite
androgen ablation therapy. It is clear that these co-
Receptor-Regulated Growth and Differentiation
activators play a critical role in androgen action and Growth factors may act directly to enhance prostate
this is an area of ongoing investigation in several cancer growth. Conceptually, androgens are normally
laboratories. considered to be the major stimulator of prostate
102 ANDROGEN RECEPTORS AND PROSTATE CANCER

cancer growth; however, it is known that there are prostate-specific antigen A protein that serves as a tumor
many alternative pathways (growth factors, cAMP, marker for prostate cancer; elevated concentrations in
kinases, apoptosis, etc.) involved in growth regu- the bloodstream may indicate the presence of prostate
lation. Accordingly, all of these pathways may cancer.
contribute to the loss of growth regulation by transgenic adenocarcinoma of the prostate (TRAMP)
androgens that occurs in advanced prostate cancer. mouse A transgenic mouse model of prostate cancer.
Since there is a limit to enhancement of the rate of cell
proliferation by these growth stimulatory agents,
maximal stimulation of growth by an alternative See Also the Following Articles
pathway may not be impacted by the loss of
androgens upon ablation therapy. Furthermore, the Adrenocorticosteroids and Cancer . Androgen Effects in
cells may adapt to a state of androgen deprivation Mammals . Androgen Receptor Crosstalk with Cellular
and seek to maximize their growth rate through these Signaling Pathways . Androgen Receptor-Related
alternative pathways. Pathology . Androgen Receptor Structure and Function
. Androgens: Pharmacological Use and Abuse . Apoptosis
. Cancer Cells and Progrowth/Prosurvival Signaling
VII. SUMMARY . Estrogen and Progesterone Receptors in Breast Cancer

These studies demonstrate the important, but rela-


tively poorly understood, function of the androgen
receptor in the development and maintenance of Further Reading
differentiated function of the normal prostate, as well
Buchanan, G., Yang, M., Harris, J. M., Han, G., Moore, N., Bentel,
as the development of prostate cancer and the J. B., Matusik, M. J., Horsfall, D. J., Marshall, V. R.,
progression of this disease. It is clear that there are Greenberg, N. M., and Tilley, W. D. (2001). Mutations at
multiple routes to androgen-independent disease. the boundary of the hinge and ligand binding domain of the
Alterations of the androgen receptor structure and androgen receptor confer increased transactivation function.
modulation of its function provide an important Mol. Endocrinol. 15, 46–56.
route for the development of new therapies to treat Chamberlain, N. L., Driver, E. D., and Miesfeld, R. L. (1994). The
length and location of CAG trinucleotide repeats in the
advanced prostate cancer.
androgen receptor N-terminal domain affect transactivation
function. Nucleic Acids Res. 22, 3181–3186.
Glossary Nazareth, L., and Weigel, N. (1996). Activation of the human
androgen receptor through a protein kinase A signaling
androgen A steroid hormone, such as testosterone or pathway. J. Biol. Chem. 271, 19900–19907.
dihydrotestosterone, that promotes the development Nazareth, L. V., Stenoien, D. L., Bingman, W. E., III, James, A. J.,
and maintenance of male secondary sex characteristics Wu, C., Zhang, Y., Edwards, D. P., Mancini, M., Marcelli, M.,
and structures. Lamb, D. J., and Weigel, N. L. (1999). A C619Y mutation in
androgen receptor A member of the steroid receptor the human androgen receptor causes inactivation and mis-
superfamily of genes that mediates the action of localization of the receptor with concomitant sequestration of
androgens on the target cell; a ligand-activated tran- SRC-1. Mol. Endocrinol. 13, 2065–2075.
scription factor. Tan, J., Sharief, Y., Hamil, K. G., Gregory, C. W., Zang, D. Y., Sar,
apoptosis Programmed cell death that requires gene M., Gumerlock, P. H., de Vere White, R. W., Pretlow, T. G.,
transcription and specific protein synthesis (physiologi- Harris, S. E., Wilson, E. M., Mohler, J. L., and French, F. S.
cally regulated event). The cells shrink and convolute, (1997). Dehydroepiandrosterone activates mutant androgen
the nucleus condenses, the DNA fragments, and the cell receptors expressed in the androgen-dependent human pros-
membrane swells and blebs. Ultimately, proteases are tate cancer xenograft CWR22 and LNCaP cells. Mol.
Endocrinol. 11, 450 –459.
activated.
Visakorpi, T., Hyytinen, E., Koivisto, P., Tanner, M., Keinanen, R.,
casodex, cyproterone acetate, hydroxyflutamide, and nilu-
Palmberg, C., Palotie, A., Tammela, T., Isola, J., and
tamide Androgen receptor antagonists that block
Kallioniemi, O. P. (1995). In vivo amplification of the
androgen receptor function. androgen receptor gene and progression of human prostate
co-activators and co-repressors Factors that positively or cancer. Nat. Genet. 9, 401–406.
negatively influence androgen receptor transcriptional Zegarra-Moro, O. L., Schmidt, L. J., Huang, H., and Tindall, D. J.
activity. (2002). Disruption of androgen receptor function inhibits
Kennedy disease (spinal bulbar muscular atrophy) A proliferation of androgen-refractory prostate cancer cells.
progressive degenerative neuromuscular disease of Cancer Res. 62(4), 1008– 1013.
genetic origin resulting from expansion of a polygluta-
mine repeat in the androgen receptor gene. Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
110 ANDROGENS: PHARMACOLOGICAL USE AND ABUSE

hormone-response element Segment of DNA sequence that of androstenedione in a river containing paper mill effluent.
binds to the DNA-binding domain of a steroid receptor Environ. Toxicol. Chem. 20, 1325–1331.
and thereby facilitates the activation of a gene. Kelce, W. R., Stone, C. R., Laws, S. C., Gray, L. E., Kemppainen,
Androgen-response elements are nucleotide sequences J. A., and Wilson, E. M. (1995). Persistent DDT metabolite p,
p0 -DDE is a potent androgen receptor antagonist. Nature 375,
that bind the androgen receptor.
581 –585.
N/C interaction Interaction between the NH2-terminal Langley, E., Zhou, Z. X., and Wilson, E. M. (1995). Evidence for
and carboxyl-terminal (N/C) regions of the androgen an antiparallel orientation of the ligand activated human
receptor that is selectively induced by the binding of androgen receptor dimer. J. Biol. Chem. 270, 29983–29990.
a biologically active androgen, testosterone or dihy- La Spada, A. R., Wilson, E. M., Lubahn, D. B., Harding, A. E., and
drotestosterone. This interaction is mediated by Fischbeck, K. H. (1991). Androgen receptor gene mutations in
FXX LF- and WXX LF-binding motifs in the NH2- X-linked spinal and bulbar muscular atrophy. Nature 352,
terminal region and activation function 2 in the ligand- 77–79.
binding domain. Quigley, C. A., De Bellis, A., Marschke, K. B., El-Awady, M. K.,
p160 co-activators A group of co-regulatory proteins of Wilson, E. M., and French, F. S. (1995). Androgen receptor
defects: Historical, clinical and molecular perspectives.
approximately 160 kDa in size that interact with
Endocr. Rev. 16, 271–321.
nuclear receptors to increase the transcriptional Tan, J. A., Sharief, Y., Hamil, K. G., Gregory, C. W., Zang, D. Y.,
response. Sar, M., Gumerlock, P. H., deVere White, R. W., Pretlow, T. G.,
Harris, S. E., Wilson, E. M., Mohler, J. L., and French, F. S.
See Also the Following Articles (1997). Dehydroepiandrosterone activates mutant androgen
receptors expressed in the androgen dependent human prostate
Androgen Effects in Mammals . Androgen Receptor cancer xenograft CWR22 and LNCaP cells. Mol. Endocrinol.
Crosstalk with Cellular Signaling Pathways . Androgen 11, 450–459.
Receptor-Related Pathology . Androgens: Pharmacological Zhou, Z. X., Wong, C. I., Sar, M., and Wilson, E. M. (1994). The
Use and Abuse . Estrogen Receptor-a Structure and androgen receptor: An overview. Rec. Prog. Horm. Res. 49,
Function . Estrogen Receptor-b Structure and Function 249 –274.
. Steroid Hormone Receptor Family: Mechanisms of
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved.
Action

Further Reading
Choong, C. S., Kemppainen, J. A., and Wilson, E. M. (1998). Androgens: Pharmacological
Evolution of the primate androgen receptor: A structural basis
for disease. J. Mol. Evol. 47, 334–342. Use and Abuse
Choong, C. S., and Wilson, E. M. (1998). Trinucleotide repeats in
the human AR: A molecular basis for disease. J. Mol.
Endocrinol. 21, 235–257.
MARIA M. BYRNE AND EBERHARD NIESCHLAG
Gregory, C. W., He, B., Johnson, R. T., Ford, O. H., Mohler, J. L., University of Münster
French, F. S., and Wilson, E. M. (2001). A mechanism for
androgen receptor mediated prostate cancer recurrence after I. INTRODUCTION
androgen deprivation therapy. Cancer Res. 61, 4315–4319.
II. PHARMACOLOGY
Gregory, C. W., He, B., and Wilson, E. M. (2001). The putative
androgen receptor-A form results from in vitro proteolysis.
III. ANDROGEN USE
J. Mol. Endocrinol. 27, 309–319. IV. ANDROGEN ABUSE
He, B., Kemppainen, J. A., Voegel, J. J., Gronemeyer, H., and
Wilson, E. M. (1999). Activation function 2 in the human
androgen receptor ligand binding domain mediates interdo- Androgen preparations are widely prescribed
main communication with the NH2-terminal domain. J. Biol. for a variety of medical conditions in both men
Chem. 274, 37219–37225. and women. Misuse of androgens, however,
He, B., Kemppainen, J. A., and Wilson, E. M. (2000). FXX LF and has become an extensive problem of increasing
WXX LF sequences mediate the NH2-terminal interaction with proportion. Therefore, it is important for stu-
the ligand binding domain of the androgen receptor. J. Biol. dents, scientists, and clinicians to be familiar
Chem. 275, 22986–22994. with the risks and benefits of androgen use.
He, B., Bowen, N. T., Minges, J. T., and Wilson, E. M. (2001).
Androgen-induced NH2- and carboxyl-terminal interaction
inhibits p160 coactivator recruitment by activation function 2.
J. Biol. Chem. 276, 42293–42301.
I. INTRODUCTION
Heery, D. M., Kalkhoven, E., Hoare, S., and Parker, M. G. (1997). The principal medical use of androgens is for the
A signature motif in transcriptional co-activators mediates
binding to nuclear receptors. Nature 387, 733 –736.
treatment of male hypogonadism and delayed
Jenkins, R., Angus, R. A., McNatt, H., Howell, W. M., Kemppainen, puberty. However, many new indications for andro-
J. A., Kirk, M., and Wilson, E. M. (2001). Identification gen treatment are emerging. This article focuses on
ANDROGENS: PHARMACOLOGICAL USE AND ABUSE 111

the pharmacology of the various androgen prep- CAG repeats are considered to play a modifying role
arations currently available and includes a discussion in prostate cancer risk. Therefore, polymorphisms in
of the variety of clinical indications (accepted and the AR may be as important as absolute testosterone
experimental) for androgen use. In addition, the levels in mediating androgen effects.
escalating problem of androgen abuse is addressed. Because of the short duration of action of
testosterone, pharmacological strategies have been
developed to produce more sustained blood levels of
II. PHARMACOLOGY testosterone, resulting in prolongation of its andro-
In human males, gonadotropin-releasing hormone genic action. These strategies include (1) new modes
(GnRH) released from the hypothalamus stimulates of testosterone administration and (2) chemical
the pituitary to secrete luteinizing hormone (LH) and modification of the testosterone molecule, including
follicle-stimulating hormone (FSH). LH stimulates esterification of the 17b-hydroxyl group or alkylation
the Leydig cells of the testes to secrete approximately of the 17a position of the D ring of the molecule, with
7 mg of testosterone per day. This amount is or without other modifications of the ring structure
necessary to induce and maintain secondary sexual (Fig. 1).
characteristics, sexual behavior, muscle development,
bone density, and male phenotype. The biological
action of testosterone is due to its direct binding to the A. Oral Testosterone
androgen receptor (AR), to its conversion to dihy- Orally administered unmodified testosterone is
drotestosterone (DHT) by the 5a-reductase enzyme, rapidly absorbed from the gastrointestinal tract into
or to its aromatization to estrogen. DHT has a higher the portal blood and then degraded by the liver (first-
affinity, compared to testosterone, for the AR and is pass effect), resulting in a minimal amount of
therefore a more potent androgen. The principal testosterone reaching the systemic circulation. Large
biological effects of testosterone include (1) stimu- doses (200 –400 mg/day), taken several times a day,
lation of prenatal differentiation and pubertal devel- are necessary to achieve physiological levels. Oral
opment of the testes, penis, epididymis, seminal testosterone induces production of the liver enzymes
vesicles, and prostate, (2) increased retention of that are responsible for testosterone metabolism.
nitrogen and increase in lean body mass and body Although long-term hepatotoxicity of large doses is
weight, (3) larynx growth, sebum production, beard unknown, unmodified oral testosterone is not rec-
growth, and pubic hair growth via DHT, (4) increased ommended to achieve physiological testosterone
bone mineral density (BMD) via estrogen, and (5) levels in blood.
reduction in high-density lipoprotein (HDL) and
increase in low-density lipoprotein (LDL) cholesterol
levels, with increases in triglycerides. The 5a- 1. Testosterone Undecanoate
reductase enzymes are most abundant in the prostate, Testosterone is esterified in the 17b position with a
skin, and reproductive tissues. DHT is required for long aliphatic side chain to produce an oral hydro-
fetal development of external genitalia, prostate, and phobic 17b-hydroxyl testosterone ester. It is adminis-
seminal vesicles, and adult secondary hair growth. tered in doses of 80 – 240 mg/day for androgen
Aromatization of testosterone to estradiol is necess- replacement therapy, and 40 mg/day for the treat-
ary for sexual differentiation of the brain, for bone ment of constitutionally delayed puberty. Due to the
mass accretion, and for fusion of the epiphyses at the nonpolar nature of the ester, it is preferentially
end of puberty. Testosterone circulates bound to sex absorbed through the lymphatic system, thereby
hormone-binding globulin (SHBG), with only 1 – 2% avoiding first-pass metabolism in the liver. Although
remaining unbound and hence biologically active. testosterone has been widely used for the treatment of
Whereas androgen status is typically defined solely on male hypogonadism in Australia, Canada, and
the basis of serum testosterone levels, the AR is Europe since the 1970s, its clinical use is limited by
clearly another important determinant of androgen a short half-life, necessitating multiple daily dosing.
action. The majority of studies demonstrate no Maximum serum levels were obtained 4 to 5 h after
correlation between endogenous testosterone levels ingestion, with huge interindividual variability. The
and the subsequent development of prostate cancer. DHT/testosterone ratio is increased secondary to
However, an inverse correlation exists between the intestinal 5a-reductase activity, and uncommon
length of the AR polyglutamate tract and both an gastrointestinal side effects include flatus, oily stool,
increased risk and an earlier onset of prostate cancer. and diarrhea.
112 ANDROGENS: PHARMACOLOGICAL USE AND ABUSE

FIGURE 1 Molecular structures of testosterone and some of its modifications.

2. 17a-Methyltestosterone and fluoxymestrone (addition of a fluorine and a


Alkylation at the 17a position reduces hepatic hydroxyl group into the steroid skeleton of testoster-
metabolism, resulting in improved systemic concen- one) are rarely used due to lack of potency and
trations of testosterone. 17a-Methyltestosterone is potential serious hepatotoxicity (hepatitis, cholesta-
rapidly absorbed, with maximal testosterone levels sis, peliosis, and benign and malignant hepatic
achieved 90 – 120 min after ingestion; plasma half-life tumors). Use of these derivatives has been discon-
is 150 min. 17a-Alkylated derivatives of testosterone tinued in Europe. Other 17a-alkylated derivatives
ANDROGENS: PHARMACOLOGICAL USE AND ABUSE 113

include oxandrolone, oxymetholone, stanozolol, and testosterone, with 60% being absorbed within the
danazol, which are less androgenic and more first 12 h, maintaining serum testosterone levels in the
anabolic. physiological range and mimicking the normal
Mesterolone, a 1a-methyl derivative of 5a-DHT, circadian rhythm. In contrast to scrotal TTS, DHT
contains only modifications in the A ring of testos- levels also remain within the physiological range. The
terone, without modification of position 17. Mester- main drawback of this patch is local skin irritation,
olone and DHT cannot be metabolized to estrogen which occurs in , 30% of patients compared to
and therefore will exert questionable clinical effects , 5% of patients using scrotal TTS. The patch can
on bone and brain. Mesterolone is considered to be a also cause an allergic contact dermatitis (, 12% of
relatively weak androgen and therefore not suitable patients). Simultaneous treatment with 0.1% triam-
for the treatment of male hypogonadism. cinolone acetonide under the patch will improve local
tolerability without altering the pharmacokinetics.
Skin irritation limits the acceptability of this treat-
B. Transdermal Testosterone ment, resulting in 10 – 15% of patients having to
The development of transdermal testosterone prep- discontinue treatment.
arations in the late 1980s was an important advance The recently approved nonscrotal patch Testo-
in androgen replacement therapy. Transdermal sub- derm TTS, which has a larger surface area but does
stitution of estradiol is a well-established method of not contain a reservoir, causes less skin irritation
treating ovarian insufficiency. However, only micro- (itching and erythema, in , 12% and , 3% of
grams of estradiol are required for replacement patients, respectively). However, adherence of the
therapy. In contrast, up to 7 mg/day of testosterone patch to the skin poses a problem in some patients,
is required for treating male hypogonadism. Due to especially those engaged in strenuous exercise. When
the superficial vascularity of the scrotal skin, there is a applied nightly it provides a steady-state delivery of
5- to 40-fold increase in steroid absorption when testosterone to the circulation that mimics the normal
compared to other skin sites. The scrotal transdermal diurnal rhythm. The long-term use of these transder-
testosterone system (TTS) is a 40 or 60 cm2 large mal patches has been efficacious in maintaining
polymeric membrane loaded with 10 or 15 mg of sexual function, secondary sexual characteristics,
testosterone. The patches are applied daily in the and bone and muscle mass in hypogonadal young
morning to the shaved scrotum and result in and elderly men.
physiological levels of testosterone (mimicking the The latest development in androgen replacement
normal circadian rhythm), rising to a maximum 2 – therapy is an open testosterone delivery system using
4 h after application and remaining within the normal a hydroalcoholic gel (Androgel 1%), which is now
range until the patch is removed. DHT levels rise licensed in the United States and several other
above normal within a few hours of application and countries for the treatment of hypogonadal men.
remain constantly elevated during the treatment When applied to the skin, the gel dries rapidly and the
period. This is the result of the high levels of 5a- steroid is absorbed into the stratum corneum, which
reductase activity in the scrotal skin. The clinical serves as a reservoir. Pharmacokinetic studies of this
significance of supraphysiological levels of DHT gel (50 or 100 mg) applied to hypogonadal men
remains currently unknown, but use over several indicate that testosterone levels increase into the
years has not resulted in untoward side effects. normal range within 30 min, with steady-state levels
Careful monitoring is still recommended. achieved by 24 h. Hypogonadal men treated for 6
In recent years the availability of nongenital months with this gel had improved sexual function,
patches has led to a decline in the use of scrotal increased lean body mass and muscle strength, and
preparations. Permeation-enhanced transdermal decreased fat mass. There was a significant dose-
delivery systems with a reservoir containing testos- related increase in hematocrit levels reported. Serum
terone (12.2 mg) in an alcohol base (Androderm) DHT levels were also increased. Transdermal gel is
have been shown to maintain serum testosterone user friendly, providing flexibility in dosing with little
levels more consistently within the normal range, skin irritation and thereby good compliance. One
compared to an equivalent intramuscular (im) dose. concern may be the contact transfer of androgens and
The recommended dosage for adult hypogonadism is the induction of androgenic side effects in females or
two 2.5-mg patches or one 5-mg patch applied at children.
night to nonscrotal skin (e.g., abdomen, thighs, upper DHT is available as a 2% hydroalcoholic gel that
arm, or back). This regimen delivers 5 mg/day of is applied to a large area of skin and washed off
114 ANDROGENS: PHARMACOLOGICAL USE AND ABUSE

10 min after application. This results in stable DHT Longer acting preparations, including testoster-
plasma levels for 24 h, an elevated DHT/testosterone one buciclate and testosterone undecanoate, are being
ratio, lack of aromatization to estrogen, and developed. Single injections of testosterone buciclate
suppression of endogenous estrogen and testoster- (600 mg) have been shown to maintain serum
one. There is also a risk of contact transfer to female testosterone levels in the low normal range for 12
partners. DHT has been shown to maintain sexual weeks in hypogonadal men. This is the longest
function in hypogonadal men but is less efficient than duration of action of any injectable ester tested so
testosterone in preventing bone loss. Prostate volume far and this ester appears to have great potential for
and prostate-specific antigen (PSA) remain clinical use. In China, injection of testosterone
unchanged with long-term DHT gel treatment in undecanoate dissolved in teaseed oil (125 mg/ml of
older men. A 0.7% hydroalcoholic dermal gel is teaseed oil) in doses of 500 –1000 mg results in serum
being developed for the treatment of aging men. testosterone levels within the normal range for 6 –8
Daily application for 3 months to elderly partially weeks. However, this is limited by an injection
androgen-deficient men results in stable pharmaco- volume of 8 ml, which renders its use impractical.
kinetic features with consistent negative feedback on Treatment of hypogonadal men with 1000 mg of
the pituitary– testicular axis and reduced body fat, testosterone undecanoate dissolved in 4 ml of castor
but minimal effects on muscle mass, strength, or oil (250 mg/ml), every 6 weeks for 6 months,
function. Prostate volume and PSA levels remain produced serum testosterone levels above the lower
unchanged. limit of normal, with good tolerability. The slowly
increasing testosterone levels at the end of the
injection interval suggested that the interval could
C. Intramuscular Testosterone be extended. A recent study has shown that injections
The most widely used testosterone replacement every 12 weeks for 2 years resulted in stable
therapy is in the form of im injection of testosterone testosterone levels within the normal range. Maximal
esters. Esterification at the 17b-hydroxyl group serum testosterone levels were lower than with the
increases the solubility of testosterone in oil and Chinese preparation and did not exceed 25 nmol/liter.
thereby slows its release into the circulation, prolong- Slight increases in body weight, hemoglobin, hem-
ing its duration of action. However, only the unester- atocrit, prostate volume, and PSA were observed.
ified testosterone is biologically active. Testosterone
propionate must be injected every 2– 3 days, but D. Testosterone Subcutaneous Implants
testosterone enanthate (doses of 200 –250 mg) and
A single implantation procedure delivering three to
testosterone cypionate have longer durations of action
six 200-mg pellets of unmodified testosterone pro-
and can be injected every 2 – 3 weeks, for replacement
vides stable, effective, and well-tolerated testosterone
therapy of hypogonadism.
replacement for 4 to 6 months. Disadvantages include
The disadvantage of all these esters is that they
the need for a minor surgical procedure and the
produce initially supraphysiological testosterone
cumbersome delivery system, requiring the use of a
levels, which then decline slowly to the hypogonadal
large trocar, leading to a small risk of bleeding and
range prior to the next injection. Some patients
infection. The major side effect is the extrusion of
correlate these fluctuations in testosterone levels with
pellets, which occurs after , 8.5% of implant
changes in well being, mood, and sexual activity. For
procedures. 7a-Methyl-19-nortestosterone (MENT),
these reasons, preparations containing a combination
a long-acting sc implant, is currently under investi-
of a short- and a long-acting 17b-hydroxyl testoster-
gation. This is a modified androgen that can be
one ester have been developed and are available in
aromatized to estrogen but not 5a-reduced. The
Europe (e.g., Testoviron Depot 50 and 100, contain-
relative sparing effects of MENT on the prostate,
ing a combination of testosterone propionate and
compared with testosterone, suggest that it may be
enanthate, and Sustanon 100 and 250, containing
useful for androgen therapy and for male
combinations of testosterone propionate, phenylpro-
contraception.
pionate, isocaproate, and decanoate). These prep-
arations result in even higher initial testosterone
E. Sublingual Testosterone
peaks with no prolongation in the duration of action
and are therefore not recommended ahead of Testosterone cyclodextrin (2.5 – 5.0 mg given three
testosterone enanthate or cypionate for the treatment times daily) is also being developed. Testosterone has
of male hypogonadism. been complexed with hydroxypropyl-b-cyclodextrin
ANDROGENS: PHARMACOLOGICAL USE AND ABUSE 115

to enhance its solubility and sublingual absorption. behavior, muscle development, bone density, and
Testosterone rises into the supraphysiological range male habitus. Testosterone will inhibit endogenous
in , 20 min and falls to baseline within 4 – 6 h. This gonadotropin secretion and thereby suppress sperma-
may be useful for the treatment of delayed puberty, of togenesis, resulting in infertility. The preparations
postmenopausal women with reduced libido, and of commonly used in the treatment of hypogonadism in
elderly men with hypogonadism. adult males are shown in Table 2.
Traditionally, long-acting esters such as testoster-
III. ANDROGEN USE one enanthate and testosterone cypionate have been
classically used im every 2 – 3 weeks (in doses of 200 –
The principal uses of androgens are for the treatment 250 mg) for the treatment of adult hypogonadism.
of adult male hypogonadism and for the treatment of Their use has been safe and free of significant side
delayed puberty. effects, and they are currently the cheapest form of
testosterone replacement. Patients can usually be
A. Treatment of Adult Male Hypogonadism taught to self-administer im injections, or a family
Before treating hypogonadal men, it is important to member or friend may be trained to give them. The
distinguish between primary hypogonadism (low disadvantage of these esters is that they produce
testosterone with elevated LH and FSH concen- supraphysiological testosterone levels for 2 –3 days
trations) and secondary hypogonadism (low testos- after injection. The levels then decline slowly into the
terone with serum LH and FSH concentrations that hypogonadal range prior to the next injection. In
are low, or low normal), as shown in Table 1. In males some men, these large fluctuations in testosterone
with primary hypogonadism, androgen-dependent levels result in undesired changes in mood, libido, and
processes can be stimulated and maintained by energy levels. This form of replacement therapy is not
exogenous hormone replacement; however, fertility optimal because it does not mimic stable physiologi-
cannot be induced by hormonal therapy. For second- cal levels and is associated with only minor diurnal
ary hypogonadism in males who are potentially variations. If patients have symptoms of androgen
fertile, there are two treatment options, exogenous deficiency with documented low serum testosterone
androgens or exogenous gonadotropins; these hor- levels a few days before the next injection, the dosage
mones can stimulate spermatogenesis and endogen- interval can be reduced. In elderly hypogonadal men
ous androgen secretion until paternity is achieved. with symptoms of bladder outlet obstruction or
The principle of testosterone replacement in the enlarged prostate glands, prostate carcinoma must
treatment of male hypogonadism is to induce and be excluded, and then it is recommended to
maintain secondary sexual characteristics, sexual commence therapy with 50 – 100 mg im every 2– 4

TABLE 1 Causes of Hypogonadism


Primary (hypergonadotropic) Secondary (hypogonadotropic) Combined (primary and secondary)

Klinefelter’s syndrome Panhypopituitarism Aging


XX males Hyperprolactinemia Hepatic cirrhosis
XY/XO mixed gonadal dysgenesis Isolated gonadotropin deficiency Sickle cell disease
XYY syndrome Kallman’s syndrome and variants
Ullrich –Noonan syndrome Idiopathic hypothalamic hypogonadism
Myotonic dystrophy Isolated LH or FSH deficiency
Sertoli-cell-only syndrome Genetic disorders
Enzymatic defects in testosterone biosynthesis Prader– Willi syndrome
Male pseudohermaphroditism (with AR defects Laurence –Moon – Biedl syndrome
or LH receptor defects) Severe systemic illness
Testicular feminization Massive obesity
Viral orchitis (mumps most common) Nutritional deficiency or starvation
Cryptorchidism Constitutional delay of puberty
Polyglandular autoimmune disease
Testicular trauma
After chemotherapy or testicular irradiation
Congenital or acquired anorchia
116 ANDROGENS: PHARMACOLOGICAL USE AND ABUSE

TABLE 2 Preparations Commonly Used in the Treatment of Adult Male Hypogonadism


Preparation Route Dosage

Currently approved
Testosterone undecanoate
(Restandol, Andriol) Oral 80 – 240 mg/day
Testosterone enanthate Intramuscular 200 –250 mg every 2– 3 weeks
Testosterone cypionate Intramuscular 200 mg every 2 weeks
Transdermal testosterone patch
(Testoderm) Scrotal skin 1 patch/day
(Androderm, Testoderm TTS) Nonscrotal 1 –2 patches/day
Testosterone gel
(Androgel 1%) Skin 50 – 100 mg/day
Dihydrotestosterone (Andractim) Skin 125 –150 mg/day
Testosterone pellets Subcutaneous implants 3 –6 doses of 200 mg every 6 months
Investigational
Testosterone undecanoate Intramuscular 1000 mg every 8– 12 weeks
Testosterone buciclate Intramuscular 600 mg every 12 weeks
Testosterone cyclodextrin Sublingual 2.5 – 5.0 mg twice daily
Dihydrotestosterone 0.7% gel Skin 16 – 64 mg/day
Obsolete
17 a-Methyltestosterone Oral 10 – 40 mg/day
Fluoxymesterone Oral 10 – 20 mg/day

weeks. Prostate examination and a PSA level assay relatively limited flexibility in dosing and are 10
are recommended after 3, 6, and 12 months and then times more expensive than testosterone esters. In
at yearly intervals for men , 45 years old and at comparison with testosterone esters, the patches
6-month intervals for men . 45 years old during provide similar symptomatic relief of androgen
testosterone replacement therapy. All patients should deficiency; increase energy, libido, and sexual func-
be informed about the expected somatic and beha- tion; and improve BMD.
vioral changes that occur during treatment. Daily administration of 50, 75, or 100 mg of 1%
Transdermal testosterone applied to scrotal (Tes- Androgel to hypogonadal men for 180 days results in
toderm) and nonscrotal skin (Testoderm, Andro- constant testosterone levels in the middle to upper
derm) is now widely used for the treatment of male physiological range throughout the entire day.
hypogonadism. However, some men find the scrotal Androgel provides flexibility in dosing and little
application cumbersome, and in men with hypogo- skin irritation. Within days or weeks of starting
nadism of prepubertal onset, the surface area of the therapy, men will notice an increase in libido,
scrotum may not be large enough to accommodate improved energy levels, and sense of well being.
the patch. Androderm is applied once nightly to Changes in physical appearance, including breaking
nonscrotal skin (e.g., abdomen, thighs, upper arm, or of the voice, will evolve over 6 months. Almost all
back) and the recommended dosage for adult studies of androgen replacement therapy in hypogo-
hypogonadism is two 2.5-mg patches or one 5-mg nadal men have shown a significant increase in
patch. This regimen delivers 5 mg/day of testoster- hematocrit, due to stimulation of erythropoiesis,
one, with 60% being absorbed within the first 12 h, which can potentially cause symptoms of hypervisc-
maintaining serum testosterone levels in the physio- osity, and a modest increase in prostate volume
logical range and mimicking the normal circadian comparable to that seen in age-matched eugonadal
rhythm. The main side effect of Androderm is local men. Despite an increase in PSA levels within the
skin irritation in , 30% of patients; however, normal range, there are no data to suggest a link
pretreatment of the skin with triamcinolone cream between testosterone administration and the induc-
reduces adverse skin reactions and only 10% of tion of prostate cancer, although longer term follow-
subjects have to discontinue therapy. Nonscrotal up is necessary. However, once a carcinoma has
Testoderm TTS is associated with less skin irritation developed, testosterone will promote its growth. The
but does not always adhere well to the skin. impact of androgens on lipids varies depending on
Testosterone patches have the disadvantage of the dose, route of administration, and whether
ANDROGENS: PHARMACOLOGICAL USE AND ABUSE 117

aromatizable or nonaromatizable androgens are abnormalities can be treated with 50 –100 mg of


used. Most studies describe a decrease in total testosterone enanthate or cypionate (im, every 4
cholesterol with a decrease in high-density lipopro- weeks for 3 months), whereas boys . 13 years old
tein cholesterol. Hepatotoxicity is generally limited to may be treated with 250 mg (im, every 4 weeks for 3
oral androgens. Therefore, patients should be care- months). After a 3-month “wait and see” period,
fully monitored by physicians with expertise in this another course of treatment may be offered if
area for changes in hematocrit, liver function, lipid pubertal development does not continue. An increase
parameters, and PSA. The prostate should be in testes size is the most important indicator of
evaluated by digital examination and by ultrasound spontaneous pubertal development, (testes volume
to detect changes in volume or development of . 3 ml). Overtreatment with testosterone may result
nodules or carcinoma. Absolute contraindications to in premature closure of the epiphyses of long bones,
testosterone use include prostate carcinoma and resulting in reduced adult height. Therefore, treat-
elevations in PSA. ment of patients who have not yet reached full adult
height has to be undertaken carefully. At the
B. Treatment of Aging Males beginning of therapy it is often difficult to distinguish
between boys with delay of growth and puberty, who
Although elderly men with hypogonadism are treated
require only temporary androgen replacement, and
like younger men according to the principles just
boys with idiopathic hypogonadotropic hypogonad-
described, it is currently disputed whether aging
ism, who require lifelong androgen therapy to
males in general should receive hormone substitution.
stimulate puberty and to maintain adult sexual
Male aging is associated with a gradual, progressive
function. However, boys with permanent hypogona-
decline in circulating testosterone concentrations.
dotropic hypogonadism will not have testicular
After decades of controversy, epidemiological studies
growth with androgen therapy. Because pubertal
clearly show that, in the general male population,
growth is a product of the interaction of growth
total testosterone concentrations decrease by up to
hormone (GH) and insulin-like growth factor I(IGF-I)
1% per year. This decline in testosterone is due to a
and the hypothalamic –pituitary –gonadal axis, boys
combination of a primary steroidogenic defect in the
with concomitant GH deficiency will require the
aging Leydig cells and a decreased pituitary secretory
simultaneous administration of GH and androgens
capacity and responsiveness to GnRH. Normal aging
for the treatment of delayed puberty.
is associated with decreased lean body mass, muscle
mass, and strength; diminished libido and erectile
function; decreased bone mass; increased fracture D. Treatment of Osteoporosis
risk; and diminished sleep quality. Controlled clinical
Testosterone and its conversion to estradiol are
trials of testosterone replacement in healthy older
important for the attainment of peak bone mass and
males with testosterone deficiency have demonstrated
for the maintenance of bone density. Delayed puberty
some equivocal moderate improvement in muscle
is associated with a reduction in peak cortical and
mass and strength. However, the clinical and func-
trabecular bone mass, and testosterone therapy is
tional relevance of these changes and the risks of
associated with an increase in BMD. Bone mass in
testosterone replacement therapy in elderly men in
men . 20 years old declines linearly with age, and the
general remain to be determined.
decrease in trabecular mass is greater than that in
cortical mass. Androgen deficiency is associated with
C. Treatment of Delayed Puberty in Boys
osteopenia and osteoporosis and increased risk of
Androgen replacement therapy in male adolescents vertebral and hip fractures. Administration of testos-
with constitutional delay of growth and adolescence terone to hypogonadal men improves levels of bone
has been shown to be beneficial psychologically as turnover markers (causing a dampening of bone
well as physiologically, and should be initiated resorption) and causes increases in particular trabe-
promptly on diagnosis. Boys with delayed puberty cular bone and also cortical BMD. However, it still
are at risk for not obtaining adequate peak bone mass remains unknown whether testosterone replacement
and for having deficiencies in developing social skills, reduces the fracture risk in osteoporotic hypogonadal
an impaired body image, and low self-esteem. men. Administration of androgens to eugonadal men
Younger boys with short stature, delayed bone age with idiopathic osteoporosis increases lumbar spine
(at least 10.5 years), and delayed pubertal develop- BMD, with no change in the femoral neck, trochan-
ment in the absence of other endocrinological ter, or total hip. Further long-term studies are
118 ANDROGENS: PHARMACOLOGICAL USE AND ABUSE

necessary to establish if testosterone therapy can be H. Experimental Use of Androgens


recommended for eugonadal men with idiopathic
osteoporosis or glucocorticoid-induced bone loss. 1. Male Contraception
Hormonal male contraceptives include exogenously
E. Treatment of Hereditary administered testosterone alone or in combination
Angioneurotic Edema with progestogens or GnRH analogues. Gonado-
tropin secretion is inhibited, resulting in inhibition of
Hereditary angioneurotic edema is characterized by spermatogenesis. Androgens are essential to inhibit
markedly reduced levels of the serum C1 esterase gonadotropin secretion and to replace endogenously
inhibitor and by a syndrome of episodic angioedema suppressed testosterone. Clinical trials using intra-
of the skin and mucous membranes, commonly muscular testosterone injections or subcutaneous
affecting the extremities, face, pharynx, and gastro- testosterone implants in combination with oral,
intestinal tract. 17a-Alkylated androgens stimulate intramuscular, or subcutaneously implanted gesta-
the production of C1 esterase inhibitor and can gens are under way and result in high rates of
therefore be used prophylactically in the treatment of azoospermia, the prerequisite for effective contra-
this condition. To minimize virilizing effects, danazol, ceptive protection. Self-administered oral or trans-
a weak androgen, is used in women. dermal regimes have been less successful in inducing
azoospermia.
F. Treatment of Hematological Disorders
Androgens enhance erythropoiesis via indirect stimu- 2. Treatment of Wasting Syndromes
lation of erythropoietin secretion from renal and Recent studies have shown that replacement doses of
extrarenal sources. Androgens also act directly on the testosterone in hypogonadal men and supraphysiolo-
bone marrow to increase red blood cell production in gical doses in eugonadal men increase fat-free mass,
response to erythropoietin. They are therefore ben- muscle size, and strength. It has therefore been
eficial for the treatment of anemia due to chronic proposed that androgens may be useful in conditions
renal failure, raising hemoglobin levels by approxi- associated with muscle wasting (sarcopenia) and
mately 1 –2 g/dl in the presence of adequate iron and protein catabolism. Interventions that prevent or
folate stores. With the availability of recombinant reverse the sarcopenia associated with aging, human
erythropoietin, androgens are now used infrequently immune deficiency virus (HIV) infection, cancer,
for the treatment of anemia associated with chronic chronic illness, major surgery, or burns could result
renal failure, especially in women, due to virilizing in an improved quality of life, improved disease
side effects. However, erythropoietin is very expens- outcomes, and reduced health care costs.
ive and androgens can be used in combination with Hypogonadism occurs in approximately 30% of
lower doses of erythropoietin. Androgens can also be HIV-infected men. Therapy with supraphysiological
beneficial to treat patients with aplastic anemia, doses of testosterone, nandrolone decanoate, or
Fanconi’s anemia, hemolytic anemia, and sickle cell oxandrolone can benefit HIV-infected men and
anemia, and some anemia secondary to hematologi- women with wasting and/or low levels of circulating
cal malignancies. Danazol (600 –800 mg/day) has androgen. However, it remains unclear whether
been used to treat patients with autoimmune hemo- physiological testosterone replacement can produce
lytic anemia and idiopathic thrombocytopenia pur- clinically meaningful changes in quality of life,
pura by directly lowering autoantibody titers. independent living, body composition, and muscle
function in HIV-infected patients.
G. Micropenis and Microphallus
Enlargement of a micropenis or microphallus can be 3. Overtall Stature
achieved by treatment with 25 –50 mg of testosterone In some European countries supraphysiological doses
enanthate or cypionate (im, every 3 to 4 weeks for 3 of testosterone (500 mg of testosterone enanthate, im,
months) or with 1.25 – 5% testosterone cream, 5% every 2 weeks for 1 – 4 years until epiphyseal closure)
DHT cream, or 10% testosterone propionate cream have been used to treat constitutional tall stature in
(twice daily for 3 months). High-dose androgen boys (predicted adult height of 205 cm). Because tall
therapy may be necessary to achieve some adrogen- stature is often desirable, this treatment is very rarely
ization in male pseudohermaphroditism due to 5a- indicated. Follow-up studies have shown that this
reductase deficiency and certain androgen receptor treatment has no long-term negative effect on
defects. testicular development.
ANDROGENS: PHARMACOLOGICAL USE AND ABUSE 119

4. Androgen Therapy in Women IV. ANDROGEN ABUSE


Evidence is emerging that testosterone plays a
physiological role in female brain development, The observation that androgens promote nitrogen
sexual function, mood, cognitive function, and well retention and muscle mass led to their use to improve
being. Premenopausally, 50% of testosterone is physical performance as early as the 1940s. Andro-
secreted directly by the ovaries and an equal amount genic anabolic steroids (AASs) have been classically
is produced by the peripheral conversion of andros- used by weight lifters, bodybuilders, and hammer and
tenedione and dehydroepiandrosterone (DHEA, pro- discus throwers, but today their use is more prevalent,
duced in the ovary and the adrenals). Testosterone extending to football players, swimmers, and track-
levels decline with increasing age in women during and-field participants (both male and female). AAS
the reproductive years such that levels in women aged abuse is also prevalent in peripubertal boys to
40 years are only 50% of those in women aged 21. enhance athletic performance and to improve appear-
This decline in androgen levels in females appears to ance. In one survey in the United States, 7% of male
be largely associated with increased age, rather than high school seniors reported having used AAS, and
being linked to menopause. DHEA sulfate (DHEAS) the majority had started at or before the age of 16
levels fall linearly with age, and probably contribute years. AASs are most frequently distributed illegally
to the decline of their main metabolite, testosterone. to athletes via a “black market.” Androgen sales due
The clinical indications for testosterone replacement to increasing misuse and abuse in the United States
therapy in women include adrenal insufficiency are reported to be increasing by 20 –30% each year.
and premature ovarian failure, including Turner’s It still remains controversial as to whether
syndrome, premenopausal iatrogenic androgen androgens improve athletic performance. It is known
deficiency (surgical menopause, chemotherapy, that androgens have muscle-growth-promoting
irradiation), and symptomatic deficiency following effects in boys, women, and hypogonadal men. The
natural menopause. Estrogen replacement therapy assumption that exogenous androgens have less
further reduces free testosterone levels and thereby effect on muscle growth in normogonadal men is
may enhance the symptoms of androgen deficiency based on the fact that the androgen receptor in
(i.e., reduced libido and sexuality). These symptoms striated muscle is down-regulated after puberty and is
are improved with androgen replacement therapy. nearly completely saturated, within physiological
Testosterone is usually recommended in combi- concentrations of testosterone. Therefore, the bio-
nation with estrogen. Examples of preparations logical response may well have reached a plateau
used in studies include oral esterified estrogen – within the physiological range. Nandrolone, the 19-
methyltestosterone, combined estradiol (E2) and nor analogue of testosterone, was the first compound
subcutaneous testosterone implants, transdermal to show enough myotrophic – androgenic dissociation
matrix delivery of testosterone, and intramuscular in animal experiments to justify its introduction
nandrolone decanoate, tibolone, and stanazol. For in clinical therapy as an anabolic steroid. Supra-
the treatment of adrenal insufficiency, 50 mg/day of physiological doses of testosterone enanthate
DHEA for months increased both androstenedione (600 mg/week) for 10 weeks, with or without
and testosterone levels, improved the sense of well resistance exercise training, have been shown to
being, and increased the frequency of sexual increase fat-free mass, muscle size, and muscle
thoughts, interest, and satisfaction. With regard to strength in normal men. The effects of exercise were
osteoporosis, the combined administration of testo- shown to be additive. However, the long-term effects
sterone and estrogen increases trabecular and cortical on muscle strength and the side effects of such doses
BMD in postmenopausal women to a greater extent remain unknown. Some of the reported enhancement
compared to estrogen alone. However, because there in performance may have been due to the effects of
are no prospective data confirming a reduction in anabolic steroids in increasing aggression and com-
fracture risk with this therapy, it cannot currently be petitiveness, to differences in intensity of training or
recommended for protecting against bone fracture. dietary intake, and to important placebo effects. The
Side effects include masculinization, hirsutism, acne, problem with studying the effects of androgens in
temporal balding, deepening of the voice, and athletes is that the athletes often take multiple
hepatotoxicity (particularly if 17a-alkylated steroids anabolic agents simultaneously (“stacking”). Fre-
are used). The effects on body composition, lipids, quently, the regimen starts with lower doses, with
risk of coronary heart disease (CHD), and breast doses progressively increasing over a period of several
cancer remain unclear. weeks. The most frequently misused androgens in
120 ANDROGENS: PHARMACOLOGICAL USE AND ABUSE

controlled competition sports are testosterone, nan- available to identify unambiguously doping with
drolone, stanozolol, and metandienone. Doses used exogenous steroids. Administration of human chor-
may be 100 times the doses used for hormone ionic gonadotropin (hCG) will stimulate testicular
replacement therapy. steroidogenesis without altering the testosterone:epi-
Although no systematic studies have been per- testosterone ratio. However, hCG can be detected by
formed (the difficulty being that many preparations immunoassay in the serum and urine. Synthetic
are abused simultaneously), the androgenic adverse androgens are extensively metabolized and doping
effects of anabolic steroids are known to include tests are focused on urinary metabolites. Derivatiza-
virilization (e.g., acne, hirsutism, and alopecia, tion methods for steroid analysis improve the detec-
sometimes irreversible) in women and premature tion limits for anabolic steroids. The potential risks of
epiphyseal closure with stunted final height, gyneco- high-dose anabolic steroids clearly outweigh their
mastia, decreased testicular volume, impaired sper- potential benefits.
matogenesis resulting in infertility (reversible),
increased sexual interest, and decreased HDL and
Glossary
cholesterol in men. The toxic effects of anabolic
steroids established from single reports include (1) anabolic androgenic steroids Synthetic derivatives of tes-
cardiovascular effects, including cardiomyopathy, tosterone having pronounced anabolic properties and
acute myocardial infarct, cerebral vascular accident, relatively weak androgenic properties.
and pulmonary embolism, (2) hepatic effects, includ- dehydroepiandrosterone The most abundant human ster-
ing cholestatic jaundice, peliosis hepatitis, and oid hormone and its sulfated derivative are produced by
the adrenal gland (approximately 30 mg/day), and are
tumors, and (3) psychological effects, including
universal precursors for androgenic and estrogenic
increased aggression, dysphoria, psychosis, addic- steroids. Conversion and metabolism occur in the
tion, withdrawal-like symptoms, and depression; a peripheral tissues.
fourth negative impact relates to needle sharing, i.e. dihydrotestosterone Testosterone is metabolized via 5a-
hepatitis and HIV infection. reductase to the potent androgen dihydrotestosterone,
Because the misuse of androgens is clearly an which binds directly to the androgen receptor and
escalating problem, there has been a marked increase cannot be aromatized to estrogen.
in urine testing prior to many athletic events. Many testosterone The major androgenic steroid hormone pro-
athletes previously stopped the administrations of duced by the Leydig cells of the testes (,7 mg/day in an
AASs in the last few weeks before a competition and adult man).
changed to androgens that are difficult to detect in the
urine or those that are rapidly metabolized and See Also the Following Articles
excreted within a few days (e.g., stanozolol and
testosterone). These deceptions were termed “pre- Androgen Effects in Mammals . Androgen Receptor
Crosstalk with Cellular Signaling Pathways . Androgen
competition bridging programs” in former Eastern
Receptor-Related Pathology . Androgen Receptor
Block countries. Today, the introduction of out-of- Structure and Function . Dihydrotestosterone, Active
competition testing has improved the detection rate Androgen Metabolites and Related Pathology . Sex
of androgen misuse. Analytically, testosterone admin- Hormones and the Immune System . Steroid Hormone
istration is much more difficult to detect compared to Receptor Family: Mechanisms of Action
doping with synthetic steroids. The ratio of testoster-
one to epitestosterone (an inactive metabolite of
testosterone secreted largely from the gonads, with Further Reading
only small amounts produced from the peripheral American Academy of Pediatrics (1997). Committee on Sports
metabolism of testosterone) is used to detect exogen- Medicine and Fitness. Adolescents and anabolic steroids: A
ous androgen misuse. The normal ratio is approxi- subject review. Pediatrics, 99, 904.
Bagatell, C. J., and Bremmer, W. J. (1996). Androgens in men—
mately 1:1 in men and women. A ratio of 6:1 is Uses and abuses. N. Engl. J. Med. 334, 707–714.
considered to be indicative of steroid abuse. However, Behre, H. M., von Eckardstein, S., Kliesch, S., and Nieschlag, E.
competitors are known to titrate their dose to below (1999). Long-term substitution therapy of hypogonadal men
the testosterone:epitestosterone ratio of 6, or to with transscrotal testosterone over 7–10 years. Clin. Endocri-
coadminister epitestosterone in an attempt to normal- nol (Oxf.) 50(5), 629– 635.
Bhasin, S., Storer, T. W., Berman, N., Callegari, C., Clevenger, B.,
ize their testosterone:epitestosterone ratio. Direct Phillips, J. et al. (1996). The effects of supraphysiological doses
methods such as gas chromatography/combustion/ of testosterone on muscle size and strength in normal men.
carbon isotope ratio mass spectrometry are currently N. Engl. J. Med. 335, 1–7.
ANGIOGENESIS 121

Bhasin, S., Bagatell, C. J., Bremner, W. J., Plymate, S. R., Tenover,


J. L., Korenman, S. G., and Nieschlag, E. (1998). Therapeutic
perspective: Issues in testosterone replacement in older men.
J. Clin. Endocrinol. Metab. 83, 3435–3448.
Angiogenesis
Buckler, H. M., Robertson, W. R., and Wu, F. C. W (1998). Which
androgen replacement therapy for women? J. Clin. Endocri- LENA CLAESSON -WELSH
nol. Metab. 83, 3920–3924. Uppsala University, Sweden
Hardy, D. O., Scher, H. I., Bogenreider, T., Sabbatini, P., Zhang,
Z. F., Nanus, D. M., and Catterall J. F. (1996). Androgen I. INTRODUCTION
receptor CAG repeat lengths in prostate cancer: Correlation II. FORMATION OF BLOOD VESSELS
with age of onset. J. Clin. Endocrinol. Metab. 81, 4400–4405. III. BLOOD VESSEL COMPOSITION
Hofbauer, L. C., and Khosla, S. (1999) Androgen effects on bone IV. ANGIOGENIC GROWTH FACTORS
metabolism: Recent progress and controversies. Eur. V. MOLECULAR MECHANISMS IN BLOOD VESSEL
J. Endocrinol. 140, 271–286. FORMATION
Kulin, H. E. (1996). Delayed puberty. J. Clin. Endocrinol. Metab. VI. CLINICAL THERAPIES TO SUPPRESS OR STIMULATE
81, 3460–3464. ANGIOGENESIS
Ly, L. P., Jimenez, M., Zhuang, T. N., Celermajer, D. S., Conway, VII. PERSPECTIVES
A. J., and Handelsman, D. J. (2001). A double-blind, placebo-
controlled, randomized clinical trial of transdermal dihydro-
testosterone gel on muscular strength, mobility, and quality of Physiological processes such as embryonic
life in older men with partial androgen deficiency. J. Clin. development, wound healing, and menstruation
Endocrinol. Metab. 86, 4078–4088. are known to require the formation of new blood
Nieschlag, E., and Behre, H. M. (1998). Pharmacology and clinical vessels to meet increased needs for oxygen and
uses of testosterone. In “Testosterone, Action Deficiency nutrients. Pathological processes involving
Substitution” (E. Nieschlag and H. M. Behre, eds.), growth of tissues, such as tumors and chronic
pp. 209– 228. Springer-Verlag, Berlin and Heidelberg. inflammatory diseases, also depend on the
Nieschlag, E., Behre, H. M., Engelmann, U., and Schwarzer, U. blood vessel supply.
(2000). Male contribution to contraception. In “Andrology:
Male Reproductive Health and Dysfunction” (E. Nieschlag
and H. M. Behre, eds.), pp. 399– 416. Springer-Verlag, Berlin I. INTRODUCTION
and Heidelberg.
Swerdloff, R. S., Wang, C., Cunningham, G., Dobs, A., Iranmanesh, A number of different growth factors are implicated
A., Matsumoto, A. M., Snyder, P. J., Weber, T., Longstreth, J., in blood vessel formation and one of these, vascular
Berman, N., and Testosterone Gel Study Group, (2000). endothelial growth factor (VEGF), is a focus of
Long-term pharmacokinetics of transdermal testosterone gel attention due to its critical and specific role both in
in hypogonadal men. J. Clin. Endocrinol. Metab. 85,
vasculogenesis (establishment of the vascular tree
4500– 4515.
Vermeulen, A. (2001). Commentary: Androgen replacement
during embryogenesis) and in angiogenesis (for-
therapy in the aging male—A critical evaluation. J. Clin. mation of new vessels from preexisting vessels). A
Endocrinol. Metab. 86, 2380–2390. number of approaches have been taken to inhibit the
von Eckardstein, S., and Nieschlag, E. (2002). Treatment of male function of VEGF in order to inhibit angiogenesis and
hypogonadism with testosterone undecanoate injected at thereby halt the progress of diseases such as cancer.
extended intervals of 12 weeks: A phase II study. J. Androl.
23, 419– 425.
Wang, C., Swerdloff, R. S., Iranmanesh, A., Dobs, A., Snyder, P. J., II. FORMATION OF BLOOD VESSELS
Cunningham, G., Matsumoto, A. M., Weber, T., and Berman,
N. (2000). Transdermal testosterone gel improves sexual During growth and development of the embryo, there
function, mood, muscle strength, and body composition is a continuously increasing need for oxygen and
parameters in hypogonadal men. J. Clin. Endocrinol. Metab. nutrients. This metabolic need, together with genetic
85, 2839–2853. programming, results in the maturation of endothelial
Wu, F. C. W. (1997). Endocrine aspects of anabolic steroids. Clin. cells from stem cells, the angioblasts. The maturation
Chem. 43, 1289–1292. probably occurs in discrete steps, but much work
Yu, Z., Gupta, S. K., Hwang, S. S., Kipnes, M. S., Mooradian,
remains to be done until this process is understood
A. D., Snyder, P. J., and Atkinson, L. E. (1997). Testosterone
completely. Once formed, the endothelial cells assem-
pharmacokinetics after application of an investigational
transdermal system in hypogonadal men. J. Clin. Pharmacol.
ble into crude vascular plexa with coarse tubes, which
12, 1139–1145. are remodeled into finer vessels. The “de novo”
establishment of the vascular tree is denoted vasculo-
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved. genesis. Interruption of vasculogenesis results in
ANGIOGENESIS 121

Bhasin, S., Bagatell, C. J., Bremner, W. J., Plymate, S. R., Tenover,


J. L., Korenman, S. G., and Nieschlag, E. (1998). Therapeutic
perspective: Issues in testosterone replacement in older men.
J. Clin. Endocrinol. Metab. 83, 3435–3448.
Angiogenesis
Buckler, H. M., Robertson, W. R., and Wu, F. C. W (1998). Which
androgen replacement therapy for women? J. Clin. Endocri- LENA CLAESSON -WELSH
nol. Metab. 83, 3920–3924. Uppsala University, Sweden
Hardy, D. O., Scher, H. I., Bogenreider, T., Sabbatini, P., Zhang,
Z. F., Nanus, D. M., and Catterall J. F. (1996). Androgen I. INTRODUCTION
receptor CAG repeat lengths in prostate cancer: Correlation II. FORMATION OF BLOOD VESSELS
with age of onset. J. Clin. Endocrinol. Metab. 81, 4400–4405. III. BLOOD VESSEL COMPOSITION
Hofbauer, L. C., and Khosla, S. (1999) Androgen effects on bone IV. ANGIOGENIC GROWTH FACTORS
metabolism: Recent progress and controversies. Eur. V. MOLECULAR MECHANISMS IN BLOOD VESSEL
J. Endocrinol. 140, 271–286. FORMATION
Kulin, H. E. (1996). Delayed puberty. J. Clin. Endocrinol. Metab. VI. CLINICAL THERAPIES TO SUPPRESS OR STIMULATE
81, 3460–3464. ANGIOGENESIS
Ly, L. P., Jimenez, M., Zhuang, T. N., Celermajer, D. S., Conway, VII. PERSPECTIVES
A. J., and Handelsman, D. J. (2001). A double-blind, placebo-
controlled, randomized clinical trial of transdermal dihydro-
testosterone gel on muscular strength, mobility, and quality of Physiological processes such as embryonic
life in older men with partial androgen deficiency. J. Clin. development, wound healing, and menstruation
Endocrinol. Metab. 86, 4078–4088. are known to require the formation of new blood
Nieschlag, E., and Behre, H. M. (1998). Pharmacology and clinical vessels to meet increased needs for oxygen and
uses of testosterone. In “Testosterone, Action Deficiency nutrients. Pathological processes involving
Substitution” (E. Nieschlag and H. M. Behre, eds.), growth of tissues, such as tumors and chronic
pp. 209– 228. Springer-Verlag, Berlin and Heidelberg. inflammatory diseases, also depend on the
Nieschlag, E., Behre, H. M., Engelmann, U., and Schwarzer, U. blood vessel supply.
(2000). Male contribution to contraception. In “Andrology:
Male Reproductive Health and Dysfunction” (E. Nieschlag
and H. M. Behre, eds.), pp. 399– 416. Springer-Verlag, Berlin I. INTRODUCTION
and Heidelberg.
Swerdloff, R. S., Wang, C., Cunningham, G., Dobs, A., Iranmanesh, A number of different growth factors are implicated
A., Matsumoto, A. M., Snyder, P. J., Weber, T., Longstreth, J., in blood vessel formation and one of these, vascular
Berman, N., and Testosterone Gel Study Group, (2000). endothelial growth factor (VEGF), is a focus of
Long-term pharmacokinetics of transdermal testosterone gel attention due to its critical and specific role both in
in hypogonadal men. J. Clin. Endocrinol. Metab. 85,
vasculogenesis (establishment of the vascular tree
4500– 4515.
Vermeulen, A. (2001). Commentary: Androgen replacement
during embryogenesis) and in angiogenesis (for-
therapy in the aging male—A critical evaluation. J. Clin. mation of new vessels from preexisting vessels). A
Endocrinol. Metab. 86, 2380–2390. number of approaches have been taken to inhibit the
von Eckardstein, S., and Nieschlag, E. (2002). Treatment of male function of VEGF in order to inhibit angiogenesis and
hypogonadism with testosterone undecanoate injected at thereby halt the progress of diseases such as cancer.
extended intervals of 12 weeks: A phase II study. J. Androl.
23, 419– 425.
Wang, C., Swerdloff, R. S., Iranmanesh, A., Dobs, A., Snyder, P. J., II. FORMATION OF BLOOD VESSELS
Cunningham, G., Matsumoto, A. M., Weber, T., and Berman,
N. (2000). Transdermal testosterone gel improves sexual During growth and development of the embryo, there
function, mood, muscle strength, and body composition is a continuously increasing need for oxygen and
parameters in hypogonadal men. J. Clin. Endocrinol. Metab. nutrients. This metabolic need, together with genetic
85, 2839–2853. programming, results in the maturation of endothelial
Wu, F. C. W. (1997). Endocrine aspects of anabolic steroids. Clin. cells from stem cells, the angioblasts. The maturation
Chem. 43, 1289–1292. probably occurs in discrete steps, but much work
Yu, Z., Gupta, S. K., Hwang, S. S., Kipnes, M. S., Mooradian,
remains to be done until this process is understood
A. D., Snyder, P. J., and Atkinson, L. E. (1997). Testosterone
completely. Once formed, the endothelial cells assem-
pharmacokinetics after application of an investigational
transdermal system in hypogonadal men. J. Clin. Pharmacol.
ble into crude vascular plexa with coarse tubes, which
12, 1139–1145. are remodeled into finer vessels. The “de novo”
establishment of the vascular tree is denoted vasculo-
Encyclopedia of Hormones. Copyright 2003, Elsevier Science (USA). All rights reserved. genesis. Interruption of vasculogenesis results in
122 ANGIOGENESIS

the death of the embryo. In the later stages of Different types of capillaries can be distinguished
development and during adulthood, new blood vessels (Fig. 1). Continuous capillaries, which occur in the
are formed from already existing blood vessels, by skin, muscles, lungs, and central nervous system, have
migration of endothelial cells away from the mother a low permeability, due to the presence of tight
vessel, followed by cell division and organization junctions between the cells. The tight junctions are
into a new vessel. The formation of blood vessels specialized structures that bridge adjacent cells and
from existing blood vessels is known as angiogenesis. keep them in close contact. A specialized variant of
Recent findings also indicate that the formation of these junctions, the blood –brain barrier, occurs in the
new blood vessels depends on or at least involves circu- central nervous system. Fenestrated capillaries are
lating, bone marrow-derived precursor cells. found in endocrine glands, kidney, intestine, and
Angiogenesis is important in wound healing and other tissues where large amounts of fluid or
for the normal growth of tissues, such as during metabolites enter or leave capillaries. Discontinuous
the later stages of embryo development. Growth of capillaries or sinusoids are found in the liver, spleen,
the endometrium during the menstrual cycle and the and bone marrow. These contain gaps between
process of ovulation are accompanied by angiogen- endothelial cells that are wide enough to allow large
esis. In recent years, there has been an increased proteins to cross the capillary wall.
awareness of the role of angiogenesis in a number of The capillaries fuse into venules and then into
diseases. These include diseases that are characterized larger vessels, veins, which transport the blood back
by the presence of too many blood vessels, such as to the heart and lungs for renewed oxygenation.
cancer, chronic inflammatory diseases, retinopathy, Similar to arteries, veins have a muscular coat,
and psoriasis. In these conditions, the tissues secrete although it is somewhat thinner. The pressure is
an increased amount of angiogenic growth factors. lower in veins than in arteries, and therefore, the
There are also conditions characterized by a lumen of veins is often collapsed on sectioning and
deficiency in the blood vessel supply. Such conditions histological analysis of the tissue. The veins of the
include coronary and peripheral ischemia. limbs contain paired valves, which ensure that the
blood does not move backward.
It is generally thought, but has not yet been
III. BLOOD VESSEL COMPOSITION formally proven, that endothelial cells from different
Blood vessels occur in three principal variants,
arteries, veins, and capillaries. All vessels contain a
single layer of endothelial cells, which face the lumen
of the vessel. Arteries transport the oxygenated blood
from the heart throughout the body. The main artery,
the aorta, branches out into finer vessels (arteriolae).
In arteries, several layers of contractile smooth
muscle cells, which are important for maintaining
the blood pressure, surround the endothelial cell
layer. Arterioles branch into a plexa of thread-thin
vessels, called capillaries. The exchange of oxygen
and nutrients occurs through the capillary wall. In
addition, new blood vessels are formed from capil-
laries and not from larger vessels. Capillaries are
composed of a single layer of endothelial cells,
surrounded by a specialized coat containing
extracellular matrix proteins (laminin, fibronectin,
collagen, perlecan, etc.), called the basement mem-
brane. Pericytes, which are supporting cells, may be FIGURE 1 Schematic illustration of a capillary composed of
endothelial cells surrounded by the basement membrane
embedded in the basement membrane or rest on the
(shaded). Different types of capillaries are indicated, such as
membrane. The function of pericytes is not clear, but sinusoid and fenestrated capillaries, which have gaps of
it has been suggested that both pericytes and the different sizes allowing the flow of proteins (sinusoid) and
basement membrane are critical for the stability of fluid and metabolites (fenestrated). In the continuous capil-
the capillary. laries, individual cells meet in the intercellular clefts.
ANGIOGENESIS 123

types of vessels, such as arteries, veins, and capil- many cells in the body in a manner that is dependent
laries, have different properties. These distinct prop- on the oxygen tension in the tissue. Thus, the
erties cannot be distinguished morphologically when promoter element in the VEGF-A gene contains a
endothelial cells are explanted and cultured in vitro. binding site for the hypoxia-induced factor. Solid
During development, arterial and venous specifica- tumors often have a central hypoxic core, which leads
tion can be traced before the onset of circulation by to increased VEGF-A production by the tumor cells
their specific expression of the growth factor ephrin- and therefore increased angiogenesis. Genetically
B2 and its receptor ephB-4 on primitive arteries and unstable cells, such as tumor cells, may also start to
veins, respectively. produce VEGF-A or other angiogenic growth factors
as a consequence of chromosomal rearrangements
that may lead to altered regulation of growth factor
IV. ANGIOGENIC GROWTH FACTORS production. As a rule, tumors can grow only up to a
Many growth factors have been implicated in the certain size, a few mm3, unless they start producing
stimulation of angiogenesis. Some of these are angiogenic growth factors, i.e., VEGF-A. This restric-
considered to act directly and some indirectly, tion point, the angiogenic switch, must be overcome
depending on whether or not receptors for these to allow further tumor growth. This is the rationale
growth factors have been detected on endothelial for the interest in developing therapies for treatment
cells. Growth factors bind to receptors that belong to of cancer based on inhibition of VEGF function.
a class of proteins called the tyrosine kinases. Thus, The additional members of the family are VEGF-
binding of the growth factor to the extracellular B, -C, and -D and placenta growth factor. The sixth
domain of the receptor leads to the activation of an family member is VEGF-E, which thus far has been
enzymatic activity that is harbored in the intracellular detected only as a virus-encoded protein and not in
domain of the receptor. This enzymatic activity, the mammalian cells. None of the VEGF family mem-
tyrosine kinase activity, catalyzes the transfer of a bers, except for VEGF-A, appear to be regulated by
phosphate group to particular tyrosine residues, hypoxia. The VEGF family members bind in a
either within the receptor molecule itself or on particular pattern to three receptors, which belong
cytoplasmic proteins, the so-called signal transduc- to the receptor tyrosine kinase family. Two VEGF
tion molecules. In many cases, these signal transduc- receptors, VEGF receptor-1 and -2, are expressed in
tion molecules are enzymes themselves, and vascular endothelial cells, whereas VEGF receptor-3
phosphorylation on tyrosine residues leads to their is expressed in endothelial cells in lymph vessels. The
activation. In this manner, a signal is propagated in essential role of VEGF-A and the VEGF receptors in
the cell, which eventually results in a cellular response vascular development has been demonstrated geneti-
of some kind, such as migration or division. cally, in experiments in which the individual genes for
The directly acting endothelial growth factors VEGF-A or the three receptors have been destroyed.
include the vascular endothelial growth factor/vas- Mice lacking expression of VEGF-A and VEGFR-2
cular permeability factor (VEGF/VPF) family and the die at approximately embryonic day 8.5 due to the
angiopoietins, for which there are receptors on lack of blood vessels. Mice lacking VEGFR-1 die
endothelial cells. However, there may not be recep- slightly later. In these animals, the endothelial cell
tors for these factors on all kinds of endothelial cells pool is enlarged and the cells fill the lumen of the
in, e.g., arteries, veins, and capillaries, or on vessels, rendering them nonfunctional. Elimination of
endothelial cells in vessels with specialized functions VEGFR-3 leads to embryonal death due to abnormal
such as in capillaries forming the blood –brain barrier. vessel formation.
Furthermore, receptors may not be expressed on Tyrosine phosphorylation sites in the VEGF
endothelial cells at all different developmental stages. receptors have been identified and to some extent
The VEGF family currently encompasses six signal transduction molecules interacting with these
members, and for several of these members, there sites have also been identified. A theme appears to be
are a number of splice variants that may have distinct that the signaling molecules bind with low affinity to
functions. The first identified VEGF family member the phosphorylated tyrosine residues. Phospholipase
was originally denoted VPF, but is now most Cg1 has been shown to be an important signaling
commonly referred to as VEGF-A. However, the molecule downstream of VEGFR-2. These types of
original nomenclature, VPF, indicates what is perhaps studies are complicated by the fact that the properties
the most important function of this factor, namely, to of VEGFR-2-mediated signal transduction appear to
induce vascular leakage. VEGF-A is produced by be correctly revealed only when primary endothelial
124 ANGIOGENESIS

cells are studied, rather than the more convenient receptor family described above, other important
standard tissue culture models based on transformed proteins are required, for example, growth modula-
cells. In addition, VEGFR-2 signal transduction tory factors such as the transforming growth factor-b
appears to be channeled through or modified by a family of ligands and receptors, the cell – cell adhesion
number of co-receptors, such as the neuropilins, protein vascular endothelial cadherin, and different
heparan sulfated proteoglycans, integrins, and cell – second-messenger proteins. Nuclear factors such as
cell adhesion molecules. the Id1– Id3 complex, which exert their effects by
The Tie family of receptor tyrosine kinases and regulating the binding of helix-loop-helix factors to
their ligands, the angiopoietins, are indispensable for DNA, are essential for proper vascular development.
normal embryonic blood vessel development, by their Angiogenesis involves the stimulation of endo-
critical function in the regulation of angiogenic thelial cells in capillaries by growth factors produced
remodeling and vessel stabilization. All known by the adjacent tissue or released by, e.g., platelets.
angiopoietins bind to one of the two known Tie Another possible growth factor source is a nearby,
receptors, Tie2. Interestingly, the Tie2 ligands have growing tumor. Morphologically, angiogenic capil-
opposing actions on endothelial cells. Accordingly, laries are composed of a stalk, where the endothelial
angiopoietin-1 and -4 function as activating ligands cells proliferate. The cells in the tip of the stalk do not
for Tie2, whereas angiopoietin-2 and -3 behave as proliferate, but send out cytoplasmic extensions
competitive antagonists. Activation of Tie2 facilitates (sprouts) toward the source of the stimulus. Prolifer-
the association between endothelial cells and adjacent ation of endothelial cells in response to growth factor
supporting cells and leads to decreased permeability stimulation is probably regulated in a manner similar
of the vessel, even in the presence of VEGF-A. to that shown for other cell types and involves
Other growth factors are assumed to act indirectly progression through the cell cycle, which eventually
on endothelial cells, but in most cases, the mechan- leads to cell division. The first important step in this
isms involved are unresolved. The exception may be process is the binding of the growth factor to its
platelet-derived growth factor-BB (PDGF-BB), which receptor, leading to activation of the receptor tyrosine
is known to act on supporting smooth muscle cells kinase and recruitment of signaling molecules. To
such as pericytes. The endothelial cells produce start the cell cycle, specific sets of signaling molecules
PDGF-BB, and the PDGF b-receptor is expressed on interact in a chain reaction, involving the small,
the pericytes. There are also growth stimulatory loops guanine triphosphate-hydrolyzing (GTPase) molecule
in the other direction, as the smooth muscle cells Ras, which eventually results in changes in the
produce angiopoietins, for which there are receptors transcriptional machinery and induction of cell
on the endothelial cells. cycling. It has been questioned whether Ras is critical
How fibroblast growth factor (FGF), hepatocyte for the proliferation of VEGF-A-stimulated endo-
growth factor, epidermal growth factor, and other thelial cells; it is possible that the signaling chain
growth factors act on endothelial cells is unclear. It is leading to proliferation in this case is dependent on
possible that an important mechanism for the phospholipase Cg1 rather than Ras. Thus, it is
angiogenic effect of these growth factors is the possible that the design of signaling chains is specific
stimulation of VEGF-A production. It is also possible to endothelial cells.
that endothelial cells in certain circumstances, for The cells in the tip of the angiogenic stalk are in
example, during development or in pathological the process of differentiation to form a new lumen-
conditions such as in tumors, express receptors for containing vessel, which is dependent on changes in
these growth factors. how the cytoskeleton is arranged. There are abundant
data indicating that cytoskeletal regulation in cell
types other than endothelial cells involves Ras-related
V. MOLECULAR MECHANISMS IN BLOOD GTPases of the Rho family. It is likely that these
proteins are also very important in endothelial cells.
VESSEL FORMATION
Much work remains to be done to d