Testis Receptor 4 - GR Resistance Modulator - J Clin Endo Metabol 2016

ORIGINAL ARTICLE
Testicular Receptor-4: Novel Regulator of

Glucocorticoid Resistance
Dongyun Zhang1, Li Du1,2, and Anthony P. Heaney 1,3
Departments of 1 Medicine and 3 Neurosurgery, David Geffen School of Medicine, University of

California, Los Angeles, CA; 2Beckman Research Institute City of Hope, Duarte, CA
Context: Glucocorticoids are powerful steroid hormones that regulate development, metabolism,
and immune response. However, glucocorticoid unresponsiveness or resistance is observed in the
treatment of inflammatory, autoimmune and lymphoproliferative diseases, and significantly limits
their efficacy.
Objective: In Cushing’s disease (CD), although some glucocorticoid-mediated suppression of pi-

tuitary-derived adrenocorticotrophic hormone (ACTH) is seen, corticotroph tumors exhibit relative
resistance to glucocorticoid action. We previously demonstrated that testicular orphan receptor 4
(TR4) binds to the pro-opiomelanocortin (POMC)-promoter to induce corticotroph tumor POMC
expression and ACTH secretion, and hypothesized that TR4 may interact with glucocorticoid sig-
naling to modulate POMC expression and action.
Results: Here we demonstrate that TR4 abrogates glucocorticoid receptor (GR)- or dexamethasone
(Dex)-mediated POMC and AP-1 trans-repression in both murine and human pituitary corticotroph
tumor cells. Co-immunoprecipitation studies indicate that TR4 and GR interact directly with each
other resulting in TR4-mediated disruption of GR binding to the POMC promoter.
Conclusion: These results demonstrate that TR4 binds GR to play an important role in glucocorti-
coid-directed corticotroph tumor POMC regulation in addition to modulating glucocorticoid ac-
tions on other GR targets. Characterization of this pathway may offer important insights into
glucocorticoid resistance and may identify a novel approach for the treatment of Cushing’s disease
and the glucocorticoid resistant states.
G lucocorticoids (GCs), a group of lipophilic steroid

hormones produced by the adrenal cortex, play a
central role in development, metabolism, immunosup-
(2). Under normal physiological conditions, GCs inhibit
their own synthesis by transrepression of hypothalamic
corticotrophic releasing hormone (CRH) and pituitary ad-
pression, and the stress response (1). The powerful actions renocorticotrophic hormone (ACTH) via the GR (2). Pro-
of GCs are mainly mediated by glucocorticoid receptor opiomelanocortin (POMC), the precursor peptide to
(GR), which is ubiquitously expressed (2). Upon ligand ACTH, is a well-defined GR responsive gene and is tran-
binding, the GR disassociates from its cytoplasmic oligo- srepressed by liganded GR via a negative GRE (nGRE,
meric complex, and translocates to the nucleus where the – 69!-61) located proximally in the POMC promoter (3).
homodimers bind to palindromic promoter glucocorti- Cushing’s disease (CD) is characterized by excess pitu-
coid response elements (GREs) to modulate target gene itary tumor-derived ACTH secretion (4). The consequent
expression (3). Maintenance of GC homeostasis depends adrenal-derived excess cortisol secretion leads to disabling
on complex feedback interactions within the hypo- symptoms, including diabetes, hypertension, osteoporosis
thalamo-pituitary-adrenal (HPA) axis to coordinate reg- and obesity, resulting in a !4-fold increased mortality (5).
ulation of immune, endocrine and neurological responses Although the recent findings of ubiquitin-specific protease
ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations: ACTH, adrenocorticotrophic hormone; AP-1, activator protein-1; CD, Cush-
Printed in USA ing’s disease; CRH, corticotrophic releasing hormone; Dex, dexamethasone; GCs, gluco-
Copyright © 2016 by the Endocrine Society corticoids; GR, glucocorticoid receptor; GREs, glucocorticoid response elements; HPA,
Received February 13, 2016. Accepted May 26, 2016. hypothalamo-pituitary-adrenal; POMC, Pro-opiomelanocortin; TR4, testicular receptor 4.
doi: 10.1210/jc.2016-1379 J Clin Endocrinol Metab press.endocrine.org/journal/jcem 1
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 07 June 2016. at 01:55 For personal use only. No other uses without permission. . All rights reserved.
2 TR4 binds GR to regulate target gene expression J Clin Endocrinol Metab
8 mutations (6, 7) and increased expression of heat shock Real-time PCR. Total RNA was extracted with RNeasy (Qiagen,
protein 90 (8) in pituitary corticotroph tumors provide Germantown, MD). RNA quantification and integrity were as-
sessed by measurement of absorbance at 260 and 280 nm. Total
new insight into this disorder, the molecular mechanisms
RNA was reverse transcribed into first-strand cDNA using
underlying CD in many patients remain largely unsolved. cDNA synthesis kit (Life Technologies, Carlsbad, CA). Quanti-
Furthermore, although novel therapies, such as the next tative PCR reactions were carried out using CFX Real-time PCR
generation somatostatin receptor ligand pasireotide and Detection System (Bio-Rad Laboratories Inc. Irvine, CA). Primer
the glucocorticoid receptor blocker mifepristone are now sequences (Life Technologies, Carlsbad, CA) were as follows:
approved treatment (9, 10), the management of many pa- mouse actin forward primer, 5#- GGC TGT ATT CCC CTC CAT
CG –3#; mouse actin reverse primer, 5#- CCA GTT GGT AAC
tients with CD remains to be challenging.
AAT GCC ATG T –3#; mouse pomc forward primer, 5#- CCA
Our recent findings show that testicular receptor 4 TAG ATG TGT GGA GCT GGT G –3#; mouse pomc reverse
(TR4, also known as NR2C2), an orphan nuclear recep- primer,5#- CAT CTC CGT TGC CAG GAA ACA C –3#.
tor, is strongly expressed in the nucleus of corticotroph
pituitary tumors and that TR4 potently induces POMC ACTH ELISA Assay. Murine and human ACTH ELISA kits were
transcription as well as murine corticotroph tumor cell purchased from Calbiotech (Spring Valley, AC). The ELISA as-
says were performed in triplicates.
proliferation and invasion (11). Given the essential roles of
the GR in transrepression of POMC and the importance of Plasmid constructs. The plasmid containing 1kb human POMC
GC resistance in CD, leukemia, inflammatory and auto- promoter fused with luciferase reporter was a gift from Dr.
immune disorders, we sought to better understand the po- Jacques Drouin (Institute de Recherches Cliniques de Montreal,
tential interactions between TR4 and GR in POMC Montréal, Québec, Canada). The AP-1 luciferase reporter was
regulation. from Stratagene (La Jolla, CA). The plasmids of pCMX-TR4
encoding human TR4 and the empty vector pCMX were gifts
from Dr. Chawnshang Chang (University of Rochester Medical
Center, Rochester, NY). The plasmids of V5-TR4 and V5-GR
Materials and Methods containing mouse TR4 or GR! fused to the 5# of V5 tag respec-
tively were generously provided by Dr. Michael R. Downes and
Cell culture and reagents. AtT20/D16V-F2 murine corticotroph Dr. Ronald M. Evans (The Salk Institute for Biological Studies,
tumor cells secreting ACTH were obtained from ATCC (Ma- San Diego, CA). The plasmids encoding the different regions of
nassas, VA) and cultured as monolayer in Dulbecco’s Modified GR including 1–278aa, 1– 432aa, 427–792aa, 501–792aa, and
Eagles Medium (DMEM, Life Technologies, Inc., Carlsbad, CA) 518 –792aa were generated by the QuikChange II XL site-di-
containing 10% fetal bovine serum (FBS, Sigma-Aldrich, St. rected mutagenesis kit (Stratagene, La Jolla, CA) using the above
Louis, MO), and penicillin/streptomycin (Life Technologies, mentioned V5-GR plasmid as template. The pairs of primers for
Inc.) at 37°C, 5% CO2 (11). Dexamethasone (Dex), bovine se- construction of these GR truncation variants were listed as fol-
rum albumin (BSA), collagenase, and hyaluronidase were pur- lows: V5-GR 1–278aa forward primer 5#-CCT AAA ATT CAG
chased from Sigma-Aldrich (St. Louis, MO). GAT ACT GGA GAA AAG GGC AAT TCT GCA GAT-3# and
reverse primer 5#-ATC TGC AGA ATT GCC CTT TTC TCC
Primary Cultures of Human Corticotroph Tumor. Surgically re- AGT ATC CTG AAT TTT AGG-3#; V5-GR 1– 432aa forward
sected human ACTH producing pituitary tumor tissues (n " 5) primer 5#- CAC AGC AAC GGG ACC AGA AAA GGG CAA
were washed, minced, and digested with DMEM containing TTC TG-3# and reverse primer 5#-CAG AAT TGC CCT TTT
0.5% BSA, 0.35% collagenase, and 0.1% hyaluronidase at 37°C CTG GTC CCG TTG CTG TG-3#; V5-GR 427–792aa forward
for 20 minutes. After centrifugation, cell pellets were resus- primer 5#-CCT TCC ACC ATG GAC TCC ACA GCA ACG
pended in culture medium and cultured in incubator for 24 GGA-3# and reverse primer 5#-TCC CGT TGC TGT GGA GTC
hours. Then cells were transfected with the pCMX-TR4 plasmid CAT GGT GGA AGG-3#; V5-GR 501–792aa forward primer
encoding human TR4 (11) or the empty vector pCMX in com- 5#-CCC TTC CAC CAT GGA CGG AAT GAA CCT GGA
bination with V5-GR plasmid and/or AP-1-Luciferase reporter AG-3# and reverse primer 5#-CTT CCA GGT TCA TTC CGT
(Stratagene, La Jolla, CA). Twenty-four to forty-eight hours CCA TGG TGG AAG GG-3#; V5-GR 518 –792aa forward
later, cells were harvested to quantitate POMC mRNA expres- primer 5#-CCT TCC ACC ATG GAC CAA GCC ACT GCA
sion and AP-1 luciferase activity. Endogenous TR4-GR associ- GGA-3# and reverse primer 5#-TCC TGC AGT GGC TTG GTC
ation was evaluated by immunoprecipitation using an anti-GR CAT GGT GGA AGG-3#. The plasmids containing various re-
antibody (Cell Signaling Technology, Danvers, MA) in primary gions of TR4 were generated by insertion of the respective PCR
cultures of human pituitary corticotroph tumors. products into the KpnI/XhoI site of pcDNA 3.1/V5-HisB vector.
The pairs of primers for construction of these TR4 truncation
Transfection and reporter assays. Murine corticotroph tumor variants were listed as follows: V5-TR4 1–106aa forward primer
AtT20 cells were transfected with POMC-, or AP-1-Luciferase 5#-CGG GGT ACC GAG CTC GGA TCC ACT A-3# and reverse
reporters, V5-TR4, V5-GR and control vector pcDNA3.1/V5- primer 5#-CCG CTC GAG TTT GCC TTC CCC AGC AAA
HisB plasmids using Lipofectamine 2000 (Life Technologies, CGC TCCA-3#; V5-TR4 1–329aa forward primer 5#-CGG GGT
Inc., Carlsbad, CA). As TR4 has been reported to alter viral ACC GAG CTC GGA TCC ACT A-3# and reverse primer 5#-
promoter activity (10), protein concentration was used as inter- CCG CTC GAG TAT GGA GGT GAA GCA CTA TCT GTG
nal control to normalize firefly luciferase results. GTA-3#; V5-TR4 205–596aa forward primer 5#-CGG GGT
doi: 10.1210/jc.2016-1379 press.endocrine.org/journal/jcem 3
ACC GCT GCT TCC ACT GAG AAG ATC-3# and reverse Differences were assessed using student t test. P values less than
primer 5#-CCG CTC GAG TAT AGA CTG GCT CCA GTG 0.05 were considered significant.
ATC TG-3#. All sequences were confirmed by automatic DNA
sequencing.
Results
Co-Immunoprecipitation and Western blotting. Following
cotransfection of V5-tagged mouse TR4 and/or GR plasmids TR4 attenuates dexamethasone- and
into AtT20 cells for 48 hours, cells were harvested and lysed in
cell lysis buffer (Cell Signaling Technology, Danvers, MA) con-
glucocorticoid receptor-mediated POMC
taining complete protease inhibitor (Roche, Basel, Switzerland) suppression and AP-1 transrepression
and 100 mM PMSF (Cell Signaling Technology, Danvers, MA). Our previous studies demonstrated that TR4 induces
The majority (85%) of the cell lysates were then incubated with POMC expression by binding to the POMC promoter
Protein A/G agarose beads (Santa Cruz Biotechnology, Inc. Dal- (11). To investigate potential interactions of TR4 on GC-
las, Texas) and anti-V5 antibody (Life Technologies, Carlsbad, mediated POMC repression, murine corticotroph tumor
CA) or mouse IgG at 4°C overnight with agitation. The resin was
then washed 3 times with PBS. The pulled-down protein com-
AtT20 cells were cotransfected with POMC-promoter lu-
plexes were run on SDS-PAGE gels and immunoblotted using ciferase reporter and TR4-expressing (pCMX-TR4) or
anti-V5 antibody and anti-TR4 antibodies. The remaining cell control (pCMX) plasmids. Eighteen hours later, AtT20
lysates (Input, 15%) were also analyzed on SDS-PAGE gels and transfectants were treated with Dex (10 nM and 100 nM)
immunoblotted in similar fashion. Anti-TR4 antibody or vehicle for an additional 6 hours. As previously re-
(ab109301, Abcam, Cambridge, MA) and normal rabbit IgG
ported by us (11) and depicted in Figure 1A, TR4 over-
(Santa Cruz Biotechnology, Inc., Dallas, Texas) were used for
endogenous immunoprecipitation, and individual membranes
expression resulted in a ! 2 fold increase in POMC pro-
were immunoblotted with anti-GR (M-20), antimineralocorti- moter reporter activity compared to control vector
coid receptor (MCR, H-300) and antiprogesterone receptor (PR, transfected cells (Ctrl). As expected, addition of Dex sup-
C-19) antibodies (Santa Cruz Biotechnology, Inc. Dallas, Texas). pressed POMC promoter activity by ! 30% in control
The densitometric analyses of the protein bands vs. the individ- vector transfected cells (Figure 1A). In contrast, Dex-treat-
ual loading controls were performed using the ImageQuant 5.2
ment of corticotroph tumor cells transfected with TR4 did
software (GE Healthcare, Pittsburgh, PA). The results shown
were representative of three independent experiments. not result in POMC promoter suppression (Figure 1A). To
further validate the observed actions of TR4 on POMC
Chromatin Immunoprecipitation Assay. To characterize the transcription, we compared actions of a specific shRNA to
binding region of TR4 on the POMC promoter, AtT20 cells were TR4 on POMC transcription (11) to a nonsense control in
transiently transfected with V5-TR4 and empty vector pcDNA the murine corticotroph tumor cells. Consistently, Dex
3.1-His B. Twenty-four hours later, ChIP assay was carried out treatment inhibited POMC promoter reporter activity by
using EZ-ChIP™ Chromatin Immunoprecipitation Kit (EMD
!20% in control cells. In contrast, TR4 knockdown in-
Millipore, Billerica, MA) and anti-V5 antibody or mouse IgG
control (Life Technologies, Carlsbad, CA). Coimmunoprecipi- duced an !80% reduction in POMC promoter reporter
tated DNA was analyzed by real time PCR using paired POMC- activity, which was more pronounced than Dex-treatment
promoter–specific primers, including primer pair 1 (forward 5#- (Figure 1B), suggesting that abrogation of corticotroph
GCC AGG TGA CAC ACT CC-3# and reverse 5#-GTT TTG tumor TR4 expression effectively represses POMC ex-
TTT GGG GGA TTC C-3#), primer pair 2 (forward 5#-AGA pression. We also tested actions of TR4 on glucocorticoid
TTA GGC AGG CAC CCC GAC TG-3# and reverse 5#- GAA
receptor (GR)-mediated POMC transcription by measur-
TGG TCT GGG TGG GGA TTG TCT G-3#), primer pair 3
(forward 5#-TCT TGC AGA TCG GAG TGG AAT TAC C-3# ing POMC-promoter luciferase reporter activities after
and reverse 5#-TGG CGC TGG TGG TTA GGA AGA ACT overexpression of the glucocorticoid receptor (GR) either
T-3#), primer pair 4 (forward 5#-TTT CCA GGC AGA TGT alone or in combination with TR4. As shown in Figure 1C,
GCC TTG CGC T-3# and reverse 5#-CAG GGT TGG GTG GGT POMC-promoter luciferase activity was suppressed by ei-
GAG CCT TGG A-3#). To examine interactions of TR4 on GR ther GR (Figure 1C, lane 3) alone or by combination of
binding to the POMC promoter, AtT20 cells were transiently
transfected with V5-TR4 and empty vector pcDNA 3.1-His B.
Dex and GR (Figure 1C, lane 4). In contrast, TR4 over-
Twenty-four hours later, ChIP assay was carried out using EZ- expression in murine corticotroph tumor cells blocked GR
ChIP™ Chromatin Immunoprecipitation Kit (EMD Millipore, (Figure 1C, lane 7) and combination of Dex and GR (Fig-
Billerica, MA) and anti-GR antibody or rabbit IgG control (Cell ure 1C, lane 8) induced POMC luciferase suppression.
Signaling Technology, Danvers, MA). Coimmunoprecipitated RT-PCR quantification of POMC mRNA in TR4 over-
DNA was analyzed by real time PCR using primer pair 4. The expressing or control vector transfected cells confirmed
results shown were representative of three independent
experiments.
the reporter assay experiments, whereby Dex treatment
inhibited POMC mRNA level !50% of control cells, and
Statistics. At least three independent experiments were per- TR4 overexpression induced a 2.5 fold increase in POMC
formed for all studies. Results were expressed as mean $ SEM. mRNA levels compared to control (Figure 1D). In con-
Figure 1. TR4 attenuates the Dex- or GR-mediated POMC suppression. (A), Murine pituitary corticotroph tumor AtT20 cells were cotransfected
with POMC-promoter luciferase reporter and TR4-expressing or control plasmids for 18 hours followed by treatment with Dex (10 or 100 nM) or
vehicle for additional 6 hours. The results are expressed as POMC promoter induction relative to vector control. B, shRNA TR4 stable transfectants
or nonsense control transfected AtT20 cells were cotransfected with POMC-promoter luciferase reporter. Twenty-four hours later, cells were
treated with vehicle or Dex (10 or 100 nM) for another 4 hours, after which POMC luciferase activities were measured and expressed as POMC
promoter induction relative to vector control. C, POMC luciferase activities were measured in AtT20 cells transfected with either TR4 or GR alone,
or TR4 in combination with GR in the presence or absence of Dex-treatment for 4 hours. (D and E), POMC mRNA expression was measured by RT-
PCR in AtT20 cells overexpressing TR4 (D) or knockdown TR4 (E) following treatment with vehicle or Dex for 6 hours. F, AtT20 cells were
cotransfected with TR4 or GR plasmids alone, or in combination. Twenty four hours later, cells were treated with vehicle or Dex for 6 hours, and
trast, Dex-treatment did not suppress POMC mRNA ex- in a time-dependent manner with maximal 90% inhibition
pression in TR4 overexpressing corticotroph tumor cells by 72 hours (Figure 2B). Dex-treatment alone had weak
(Figure 1D). In support of these findings, TR4 knockdown effect on the AP-1 luciferase reporter activity (Figure 2C,
not only inhibited basal POMC mRNA level, but also led lane 2), whereas GR overexpression alone (Figure 2C, lane
to an enhancement of Dex-induced POMC mRNA sup- 3) or in combination with Dex-treatment (Figure 2C, lane
pression (Figure 1E). We further tested murine cortico- 4) suppressed the AP-1 activity by 45% and 55% respec-
troph tumor POMC mRNA changes following Dex-treat- tively. In contrast, TR4 overexpression not only induced
ment with or without co-overexpression of GR either in AP-1 transactivation, but abrogated both the GR- and
native cells or in TR4 overexpressing murine corticotroph Dex-mediated suppressive effects on AP-1 luciferase (Fig-
tumor transfectants. As shown in Figure 1F, POMC ure 2C, lanes 6 – 8). Similar effects were observed in pri-
mRNA level was suppressed by either Dex (Figure 1F, lane mary cultures of human pituitary corticotroph tumor
2) or GR overexpression (Figure 1F, lane 3) alone or by the cells, where TR4 increased AP-1 activity at the 24 and 48
combination of Dex and GR overexpression (Figure 1F, hours time points (Figure 2D). AP-1 transrepression was
lane 4). However, both Dex- and GR-mediated POMC observed in human corticotroph primary cultures follow-
suppression was blocked by coexpression of TR4 (Figure ing either Dex-treatment alone, GR overexpression alone
1F, lanes 6 – 8). In full support of our findings of TR4 or the combination of Dex and GR (Figure 2E, lanes 2– 4
actions to abrogate both Dex- and GC-mediated actions vs. 1), but this AP-1 repression was largely blocked by
on POMC promoter reporter and POMC mRNA, we also concomitant TR4 overexpression (Figure 2E, lanes 6 – 8
demonstrated that corticotroph TR4 overexpression ab- vs. 5), supporting our findings in the murine AtT20 cells.
rogated Dex- and GR-mediated actions on murine corti- In summary, these results indicate that TR4 acts to mod-
cotroph tumor ACTH secretion. As shown in Figure 1G, ulate glucocorticoid and GR-directed actions on a variety
ACTH secretion was suppressed by Dex-treatment and of GR transcriptional targets, including POMC and AP-1.
GR overexpression alone or in combination (Figure 1G,
lanes 2– 4 vs. 1), but Dex- and GR-mediated suppression TR4 interacts with GR
of ACTH secretion was blocked by coexpression of TR4 To further analyze the mechanism of TR4 effects on GR
(Figure 1G, lanes 6 – 8 vs. lane 5). In primary cultures of function, we tested if TR4 physically interacted with GR.
freshly resected human pituitary corticotroph tumor cells AtT20 cell lysates were immunoprecipitated using either
(from 5 individual patients), we observed a similar action an anti-TR4 antibody or normal rabbit IgG. As illustrated
of TR4 to abrogate either GR- (Figure 1H), or Dex- (Fig- in Figure 3A, immunoblot of the TR4 precipitation com-
ure 1I) mediated actions on POMC mRNA levels or hu- plex demonstrated that GR was present in the protein
man corticotroph tumor ACTH secretion (Figure 1J). complex. In contrast, TR4 did not immunoprecipitate the
These results indicated that TR4 acted to blunt both GR- progesterone receptor (PR) and the mineralocorticoid re-
and Dex-mediated POMC transrepression in both murine ceptor (MCR), indicating specificity of the TR4-GR in-
and human corticotroph tumors in vitro. teraction. In support of the findings in the murine corti-
We next sought to determine if the TR4-mediated ac- cotroph tumors, immunoblot of a TR4 precipitation
tions to block Dex-and GR-suppression of POMC we had complex from primary cultures of human corticotroph
demonstrated were specific to the POMC gene or whether tumor cells confirmed the TR4-GR interaction (Figure
TR4 might have a broader role in modifying glucocorti- 3B). Cotransfection of AtT20 cells with a pCMX-TR4
coid actions. Activator protein-1 (AP-1) interacts with the plasmid and a V5-tagged GR plasmid followed by immu-
GR and is negatively regulated by GCs to modulate both noprecipitation with an anti-V5 antibody or mouse IgG
inflammatory responses and cell proliferation (12). As control demonstrated that TR4 was present in the V5-GR
demonstrated in Figure 2A, TR4 overexpression markedly precipitated complex (Figure 3C). Reciprocal immuno-
increased murine corticotroph tumor cells AP-1 transac- precipitation studies using an anti-TR4 antibody demon-
tivation in a dose-dependent manner. Consistent with the strated that GR was present in the TR4 precipitated com-
latter finding, TR4 knockdown inhibited AP-1 activation plex, confirming that TR4 and GR were present together
Legend to Figure 1 Continued. . .

mRNA was harvested for real-time PCR analysis of POMC mRNA expression. G,. Cell supernatants from latter cells were collected for measurement
of ACTH secretion by ELISA assay. H–J, Primary cultures of freshly resected human corticotroph tumor cells (representative of 5 individual tumors
studied) were transfected with either TR4 or GR alone, or TR4 in combination with GR for 18 hours with or without Dex-treatment. POMC mRNA
expression was measured by RT-PCR (H and I). ACTH secretion was detected using ACTH ELISA kit (J). Data shown were representative of at least
three independently repeated experiments. Bars indicate the mean $ standard error of the mean of triplicate tests. * P % .05; ** P % .01; ***P %
.005; ns: not significant.
(Figure 3D). Collectively, these studies provide evidence We next tested if TR4 alters GR translocation in response
for a physical interaction between the TR4 and GR to glucocorticoid binding and if the TR4-GR interaction
proteins. could be modulated by glucocorticoid binding to the GR.
To examine this, we extracted cytosolic and nuclear pro-
Dex treatment regulates TR4 and GR interaction tein lysates following Dex-treatment for 2 and 16 hours in
dynamically both TR4 overexpressing and vector control AtT20 cell
Our previous immunocytochemical studies in cortico- transfectants. A swift translocation of GR from the cytosol
troph pituitary tumors demonstrated that TR4 is predom- to the nucleus was observed following Dex-treatment be-
inantly located in the nucleus (10). In contrast, prior stud- ginning at 2 hours and reducing by 16 hours (Figures 4A,
ies demonstrate that the GR protein dynamically shuttles control: lanes 1– 6 and quantified in 4B), the latter was
between the cytosol and nucleus upon ligand binding (13). consistent with Dex-directed regulation of GR protein
Figure 2. TR4 blocks the Dex- or GR-regulated AP-1 transrepression. (A), V5-TR4 expression plasmid (250 ng and 500 ng) was transiently
transfected into murine corticotroph tumor AtT20 cells stably expressing AP-1 luciferase reporter, and AP-1 luciferase activities determined 72
hours after transfection. B, An AP-1 luciferase reporter and shRNA TR4 or nonsense control plasmids were transiently transfected into AtT20 cells
and harvested at 24 hours, 48 hours and 72 hours post transfection to quantitate AP-1 luciferase activity. C, Mouse corticotroph tumor AtT20 cells
were cotransfected with AP-1 luciferase reporter and/or GR- and TR4-expressing or control plasmids for 48 hours and then treated with Dex (100
nM) or vehicle for an additional 6 hours. Results are expressed as AP-1 luciferase induction relative to vector control. D, An AP-1 luciferase reporter
and TR4 expression plasmids were transiently transfected into primary cultures of freshly resected human pituitary corticotroph tumor cells (n " 5)
and harvested at 24 hours and 48 hours post transfection to quantitate AP-1 luciferase activity. E, primary cultures of human corticotroph tumor
cells (n " 5) were cotransfected with AP-1 luciferase reporter and/or GR- and TR4-expressing or control plasmids and then treated with Dex (100
nM) or vehicle for an additional 48 hours to detect AP-1 luciferase activities. Data shown are representative of at least three independently
conducted experiments. Bars indicate the mean $ standard error of the mean of triplicate tests. * P % .05; ns: not significant.
degradation (13). TR4 overexpression (V5-TR4) did not Interaction of TR4 with GR requires GR N-terminal
alter the observed Dex-directed GR translocation or its domain and TR4 DNA binding domain
protein stability (Figures 4A, V5-TR4: lanes 7–12 and GR and TR4 are both highly conserved nuclear recep-
quantified in 4B). Next, we examined if Dex-treatment tors, and contain several functional protein regions, in-
would alter GR content in the TR4 immunoprecipitated cluding an N-terminal domain, a DNA binding domain, a
complex. As depicted in Figures 4C and quantified in 4D, hinge region, a ligand binding domain and a C-terminal
in concert with the timing of the Dex-directed GR nucleus domain (illustrated schematically in Figure 5A for GR;
translocation, the TR4-GR interaction was also dynami- Figure 5C for TR4). To begin to characterize which re-
cally regulated by Dex-treatment with increased GR pres- gions of the GR protein were involved in its interaction
ent in the TR4 immuoprecipitated complex after 2 hours with TR4, protein lysates were harvested from AtT20 cells
Dex-treatment followed by decreased GR in the TR4 im- transfected with V5-tagged murine GR plasmids encoding
muoprecipitated complex by 16 hours (Figures 4C and the GR regions of 427–792aa, 501–791aa, and 518 –
4D). Altogether these studies demonstrated that in un- 792aa as well as the full length GR protein. Proteins were
stimulated conditions, the low level nuclear located GR then immunoprecipitated with anti-V5 antibody as before
readily interacts with TR4; and following glucocorticoid and probed with anti-TR4 antibody (Figure 5B). In con-
treatment, enhanced availability of nuclear GR results in trast to the full length GR protein which showed a strong
increased TR4-GR interaction. This provides a mechanis- interaction with TR4 (Figure 5B, lane 2), no interaction
tic basis for the regulation of POMC by TR4 with or with- with TR4 was seen with the other GR fragments (Figure
out glucocorticoid treatment. 5B, lanes 3–5). These results demonstrated that TR4 in-
Figure 3. TR4 binds GR (A and B), Murine (AtT20, A) or human pituitary corticotroph tumor cells (B) were lysed and subjected to
immunoprecipitation using specific anti-TR4 or anti-GR antibodies, after which immunocomplexes were analyzed by immunoblot using anti-GR,
PR, MCR and TR4 specific antibodies. (C and D), AtT20 cells transfected with plasmids encoding TR4 and V5-tagged GR were harvested and
immunoprecipitated with mouse IgG, anti-V5 (GR) antibody (C) or anti-TR4 antibody (D). The immunocomplexes were then analyzed by
immunoblotting using specific antibodies to TR4 and V5 (GR). Data shown were representative of at least three independently repeated
experiments.
teracts with the GR via its N-terminal region. In parallel, POMC promoter, or alternatively a quenching mecha-
reciprocal immunoprecipitation studies were carried out nism. To this end, we performed CHIP assays using an
using various truncation mutants of TR4, including the anti-TR4 antibody to characterize TR4 binding sites on
N-terminal region (1–106aa), DNA binding domain (1– the POMC promoter. Four pairs of primers covering
329aa), and its ligand binding domain (205–596aa) as !1000 bp proximal mouse POMC promoter region (P1-
illustrated in Figure 5C. In V5-TR4 immunoprecipitate P4) were tested (illustrated in Figure 5E). As show in Fig-
complexes, we observed that both full length TR4 (Figure ure 5F, anti-TR4 –immunoprecipitated DNA with the en-
5C, lane 2) and a 1–329 aa TR4 region (Figure 5C, lane 4) riched locus was strongly amplified by primer pair 2 (P2),
bound to the GR protein. In contrast, the TR4 N-terminal indicating specific TR4 binding to the POMC promoter
region (1–106aa) and its ligand binding domain (205– around this region, which is distinct from the reported GR
596aa) did not bind the GR (Figure 5D, lanes 3 and 5), binding consensus, nGRE presented in the primer pair 4
indicating that the 106 –205aa protein sequence (predom- region (P4, 23). We also used an anti-GR antibody in the
inantly the DNA binding domain) of TR4 is crucial for the CHIP assay to examine TR4 actions on GR binding to the
TR4-GR interaction. POMC promoter. As shown in Figure 5G, when TR4 was
Next we sought to examine how our demonstrated overexpressed, the binding of GR to the nGRE on POMC
TR4-GR interaction modifies POMC transcription, and promoter was greatly inhibited. Therefore, we concluded
address whether it involves direct TR4 and GR competi- that by direct protein-protein interaction, TR4 overrides
tion for overlapping DNA binding sequences on the the inhibitory activity of GR on POMC transcription,
Figure 4. Dex treatment dynamically regulates TR4 and GR interaction. (A and B), AtT20 cells were transiently transfected with V5-TR4 or control
plasmid, and treated with Dex (100 nM) for 2 and 16 hours. Individual cytosolic and nuclear protein lysates were prepared from the above cells
and probed with specific antibodies. Lamin B and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as nuclear and cytosol markers
(A). Protein bands were quantitated by scanning densitometry and compared to controls using the ImageQuant 5.2 software (B). (C and D),
Immunoprecipitation was then carried out using an anti-V5 antibody, and presence of GR in the immunocomplex detected by immunoblot using
specific anti-GR antibody (C). Protein bands were quantitated by scanning densitometry and compared to controls using the ImageQuant 5.2
software (D). The results shown were representative of three independent experiments.
leading to up-regulation of POMC expression. Given that activators, such as Nur77 to the POMC promoter thereby
TR4 and GR bind different regions of the POMC pro- replacing GR (Figure 5H-b).
moter, we hypothesize that TR4 inhibits the GR binding
to POMC by a direct quenching type action (as depicted in
Figure 5H-a) or by facilitating the access of other trans-
Figure 5. Interaction of TR4 with GR requires the N-terminal GR domain and TR4 DNA binding domain. (A and C), Schematic illustration of the
GR protein (A) and TR4 protein (C) functional regions. B, Protein lysates were harvested from AtT20 cells transfected with V5-tagged mutant GR
plasmids encoding the C-terminal regions 427–792aa, 501–792aa, and 518 –792aa as well as full length GR protein. Protein lysates were then
immunoprecipitated with anti-V5 antibody and immunocomplexes were analyzed by immunoblot using a specific anti-TR4 antibody. D, AtT20 cells
were transfected with either V5-tagged full length TR4, or 1–106aa, 1–329aa, or 205–596aa deletion mutants, and transfectants harvested and
subjected to immunoprecipitation with anti-V5 antibody. The immunocomplexes were then analyzed by immunoblot using a specific anti-GR
antibody. E, Four pairs of primers covering !1000 bp proximal mouse POMC promoter region (P1–P4) were designed to identify the TR4-binding
POMC promoter region. F, AtT20 cells were transiently transfected with V5-TR4 plasmid and empty vector pcDNA 3.1-His B. Twenty-four hours
later, ChIP assay was carried out using primers to individual POMC promoter region P1 (-1043!-911), P2 (-854!-637), P3 (-276!-146) and P4
(-103!&23) (F). G, AtT20 cells were transiently transfected with V5-TR4 and empty vector pcDNA 3.1-His B. Twenty-four hours later, ChIP assay
was carried out using an anti-GR antibody or rabbit IgG control to quantify GR binding compared to control. *** P % .005; **** P % .01. Data
shown are representative of at least three independently repeated experiments. H, Schematic showing the proposed molecular mechanisms
underlying TR4 inhibition of negative regulatory GR action on POMC transcription.
Discussion POMC transcription (21). In our studies, unlike Nur77,

TR4-induced activation of POMC transcription cannot be
In the normal physiological state, pituitary derived ACTH
antagonized by GR expression or Dex-treatment in cor-
is under negative regulation by feed-back from adrenal
ticotroph tumor cells. Based on our Co-IP studies which
derived cortisol resulting in a carefully balanced availabil-
demonstrate that TR4 binds the GR via its N-terminal
ity of glucocorticoids. This rapid and specific negative reg-
domain, we hypothesize that TR4 binding to GR prevents
ulation of ACTH mediated by glucocorticoid action in the
the GR accessing the nGRE to mediate POMC suppres-
anterior pituitary is largely attributable to the binding of
sion (Figure 5H, Schematic a). However, we cannot en-
the liganded-glucocorticoid receptor (GR) to the 5#-flank-
tirely exclude other mechanisms. For example, it is pos-
ing region of the POMC gene (14). In Cushing’s disease,
sible that upon TR4 disrupts GR binding to POMC
although some glucocorticoid-mediated suppression of pi-
tuitary-derived ACTH is seen, corticotroph tumors ex- promoter, other transactivators bind to the same GR bind-
hibit relative resistance to this glucocorticoid action (15). ing sequences and abolish GR-mediated POMC suppres-
We have recently shown that TR4 is strongly expressed in sion (Figure 5H, Schematic b). Nonetheless, our demon-
human corticotroph tumors and that it induces murine stration that both V5-tagged TR4 and endogenous TR4
and human corticotroph tumor POMC transcription and immunoprecipitated the GR protein is more supportive of
ACTH secretion (11). In the current studies, we demon- a direct action of TR4 to disrupt GR action. Our studies
strate that TR4 acts to attenuate the effects of both glu- also demonstrate a novel feature of the TR4-GR interac-
cocorticoids and the glucocorticoid receptor to inhibit tion, namely the requirement of N-terminal region of GR
POMC gene transcription via a direct protein-protein in- for protein-protein interaction. When GR forms het-
teraction. We also demonstrated that TR4 potentially in- erodimers with other nuclear receptors, such as COUP-
duces AP-1 transactivation, providing further mechanistic TFII (22), mineralocorticoid receptor (23) and androgen
insight into the potential mode of action by which TR4 receptor (24), the hinge domain or DNA domain of GR
induces proliferation (11). Additionally, we show here has been implicated for maintaining the protein-protein
that TR4 also acts to block GR-mediated AP-1 transre- interaction. Given the GR N-terminal region is involved
pression pointing to a broader significance of the TR4-GR with its interactions with cofactors to modulate gene ex-
interaction we describe. pression (25), we hypothesize that TR4 binding to the
Orphan nuclear receptors represent a diverse group of N-terminal GR impairs its ability to bind other cofactors.
the nuclear receptor superfamily, which are highly con- In tandem, given TR4 itself is a strong coactivator of
served throughout evolution. They function as ligand-me- POMC (18), this combined action may totally abrogate
diated transcription factors to modulate numerous phys- the gene repressive effect of GR, providing a dual molec-
iological and pathophysiological processes (16). TR4 ular mechanism for the negative effect of TR4 on GR
belongs to the subfamily 2 of nuclear receptors, and to- actions.
gether with its homology gene TR2 play key roles in sper- Based on our results, we believe that TR4 modulates
matogenesis, lipoprotein regulation and central nervous glucocorticoid and glucocorticoid receptor actions on a
system (CNS) development (17). In addition to direct
variety of GR targets and play an important role in glu-
DNA binding, TR4 can serve as a coactivator or corepres-
cocorticoid resistance. Further characterization of this
sor by interacting with other nuclear receptors (18). Al-
pathway may offer important insights into glucocorticoid
though the mechanism(s) for increased TR4 expression in
resistance in Cushing’s diseases and other diseases such as
corticotroph tumors are not fully understood, it is possible
steroid resistant asthma, inflammatory bowel disease and
that increased phosphorylated active TR4 could result
glucocorticoid-resistant leukemia.
from enhanced activity of growth factor signaling as has
been recently highlighted due to USP8 mutation in human
corticotroph tumors (6, 7).
GR-mediated gene repression occurs via several mech- Acknowledgments
anisms, which may be mediated by either direct DNA- We thank Dr. Jacques Drouin (Institute de Recherches Cliniques
binding or independent of DNA binding (19). GR medi- de Montreal) for his generous gift of plasmid human POMC
ated-POMC repression involves antagonism of NurRE luciferase reporter; Dr. Michael R. Downes and Dr. Ronald M.
activity by the GR physically interacting with Nur77 pro- Evans (The Salk Institute for Biological Studies, San Diego, CA)
tein to disrupt its binding to the POMC promoter (20). for their gifts of plasmids of V5-TR4 and V5-GR; Dr. Chawn-
Additionally, the GR can also bind the nGRE located up- shang Chang (University of Rochester Medical Center, Roches-
stream of the TATA-box in the POMC promoter to inhibit ter, NY) for the gift of pCMX-TR4 plasmid.
Address all correspondence and requests for reprints to: An- ticoid Receptors In Vitro: Chromatin Recycling and Nuclear Ex-
thony P. Heaney, Phone: 310 –267-4980, Fax: 310 –267-1899, port. The Journal of Cell Biology. 1997;137:523–538.
E-mail: aheaney@mednet.ucla.edu. 14. Wallace AD, Cidlowski JA. Proteasome-mediated Glucocorticoid
Disclosure Summary: The authors have nothing to disclosure. Receptor Degradation Restricts Transcriptional Signaling by Glu-
This work was supported by . cocorticoids. Journal of Biological Chemistry. 2001;276:42714 –
42721.
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Testis Receptor 4 - GR Resistance Modulator - J Clin Endo Metabol 2016

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Testis Receptor 4 - GR Resistance Modulator - J Clin Endo Metabol 2016

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ORIGINAL ARTICLE

Testicular Receptor-4: Novel Regulator of

Dongyun Zhang1, Li Du1,2, and Anthony P. Heaney 1,3

Departments of 1 Medicine and 3 Neurosurgery, David Geffen School of Medicine, University of

Objective: In Cushing’s disease (CD), although some glucocorticoid-mediated suppression of pi-

G lucocorticoids (GCs), a group of lipophilic steroid

doi: 10.1210/jc.2016-1379 J Clin Endocrinol Metab press.endocrine.org/journal/jcem 1

Legend to Figure 1 Continued. . .

Discussion POMC transcription (21). In our studies, unlike Nur77,

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