Biosci. Biotechnol. Biochem.

, 70 (9), 21452153, 2006

Endogenous Prostaglandins E2 and F2 Serve as an Anti-Apoptotic

Factor against Apoptosis Induced by Tumor Necrosis Factor-
in Mouse 3T3-L1 Preadipocytes
Kohji N ISHIMURA,1; y Tsutomu SETOYAMA,2 Hirofumi T SUMAGARI,2 Nana M IYATA,2
Yoko H ATANO,2 Li X U,2 Mitsuo J ISAKA,2 Tsutomu N AGAYA,2 and Kazushige Y OKOTA2; y
1

Department of Molecular and Functional Genomics, Center for Integrated Research in Science,
Shimane University, Nishikawatsu-cho, Matsue, Shimane 690-8504, Japan
2
Department of Life Science and Biotechnology, Shimane University, Nishikawatsu-cho,
Matsue, Shimane 690-8504, Japan
Received February 28, 2006; Accepted April 26, 2006; Online Publication, September 7, 2006
[doi:10.1271/bbb.60106]

Adipocytes can function as endocrine cells secreting

a variety of adipocytokines including tumor necrosis
factor (TNF)-. Treatment of cultured mouse 3T3-L1
preadipocytes with TNF- induced apoptosis, as was
evident from increases in nuclear condensation and
caspase-3 activity, but dierentiated adipocytes during
the maturation phase showed resistance to apoptosis by
TNF-. Antioxidants eectively reduced TNF--induced
apoptosis in preadipocytes, indicating the involvement
of reactive oxygen species. Exposure of preadipocytes
to calcium ionophore A23187 reduced TNF--induced
apoptosis, which was accompanied by increased production of prostaglandins (PGs) E2 and PGF2 . TNF-
preferentially promoted gene expression of cyclooxygenase (COX)-2 without aecting that of COX-1. Consistently, NS-398, a COX-2 inhibitor, stimulated TNF-induced apoptosis, which was reversed by exogenous
PGE2 and PGF2 . These results indicate that endogenous PGE2 and PGF2 synthesized by preadipocytes
through the induction of COX-2 can serve as antiapoptotic factors against apoptosis by TNF-.
Key words:

apoptosis; cyclooxygenase; prostanoid;

3T3-L1; tumor necrosis factor (TNF)-

Obesity is a risk factor for severe diseases such as

noninsulin-dependent diabetes, arteriosclerosis, high
blood pressure, coronary heart disease, and certain
y

cancers.1,2) The biological events leading to obesity are

characterized by changes in the cell properties of
adipocytes as represented by an increase in cell
numbers, size, or both.3) Adipocytes have a life cycle
including cell proliferation, induced dierentiation,
maturation, cell death, and changes in insulin sensitivity.4,5) Adipose tissues are known to play a critical role
in the storage and mobilization of lipids. Moreover,
recent advances have provided evidence that adipocytes
can function as endocrine cells by secreting leptin,
adiponectin, free fatty acids, and other cytokines. These
factors secreted from adipose tissues target adipocytes
and other tissues in autocrine and paracrine ways
respectively. Some of them can control blood sugar
levels by aecting directly the insulin signaling pathway.6)
TNF-
is a well-known cytokine produced from
various types of cells, and this secreted factor plays a
critical role in inammation, apoptosis, and various
biological events.7) In adipose tissues, TNF-
is increasingly released from adipocytes with the progression of
dierentiation from preadipocytes into mature adipocytes.8) Recent reports have shown that TNF-
suppressed adipogenesis in a feedback manner. TNF-
also
attenuates the uptake of blood glucose by adipocytes and
muscle cells through blockage of the insulin signaling
pathway, leading to the development of insulin-resistance.9) In addition, TNF-
has been reported to induce

To whom correspondence should be addressed. Kohji NISHIMURA, Tel: +81-852-32-6288; Fax: +81-852-32-6109; E-mail: knishimu@
shimane-u.ac.jp; Kazushige YOKOTA, Tel: +81-852-32-6576; Fax: +81-852-32-6576; E-mail: yokotaka@life.shimane-u.ac.jp
Abbreviations: AscA, ascorbic acid; ASK1, apoptosis signal-regulating kinase 1; COX, cyclooxygenase; DMEM-HEPES, Dulbeccos modied
Eagles medium with 25 mM HEPES; DEVD-pNa, acetyl-Asp-Glu-Val-Asp-p-nitroaniline; DMSO, dimethyl sulfoxide; 15d-PGJ2 , 15-deoxy-
12;14 prostanglandin J2 ; dNTP, deoxyribonucleoside 50 -triphosphates mixed solution; ELISA, enzyme-linked immunosorbent assay; FCS, fetal calf serum;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HETE, hydroxyeicosatetraenoic acid; LOX, lipoxygenase; MDCK, Madin-Darby canine
kidney; 2-ME, 2-mercaptoethanol; PBS (
), phosphate-buered saline without Ca2 or Mg2 ions; PG, prostaglandin; PLA2 , phospholipase A2 ;
PPAR, peroxisome proliferator-activated receptor; ROS, reactive oxygen species; RT-PCR, reverse transcriptase-polymerase chain reaction; TNF,
tumor necrosis factor

2146

K. NISHIMURA et al.
10)

apoptosis in human adipocytes, eventually resulting in

the regulation of cell numbers of adipose tissues in an
apoptotic manner.
Arachidonic acid can be converted into a series of
lipid mediators to control diverse biological events such
as cell proliferation and dierentiation, the immune
system, and inammation through a biosynthetic pathway called the arachidonate cascade, which includes
cyclooxygenase (COX) and lipoxygenase (LOX) pathways.1116) In addition, recent reports have shown that
COX and LOX metabolites play conicting roles in
regulating cell survival and apoptosis depending on
certain types of cells and signaling molecules.1724)
Some sorts of prostanoids are known to be regulators
of adipocyte dierentiation in cultured cells. For
example, exogenous 15-deoxy-
12;14 -PGJ2 (15d-PGJ2 )
has been shown to have a potent activity to induce
adipocyte dierentiation in NIH-3T3 cells forced to
undergo ectopic expression of peroxisome proliferatoractivated receptor (PPAR)-.25) 15d-PGJ2 can bind
directly to PPAR-.26) In addition, a PPAR-response
element has been identied in the regulatory region of
the COX-2 gene.27) Alternatively, a recent study
provided evidence that COX-2 might be involved in
body-fat regulation.28) Heterolozygous COX-2 decient
mice showed the increased body weight with fat pads as
compared with those from wild-type and COX-1decient mice, suggesting a specic role for COX-2 in
the generation of endogenous prostanoids controlling
adipogenesis in body fats. Nevertheless, the roles of
endogenous PGs produced by COX isoforms in the life
cycle of adipocytes remain unclear.
We have been employing cultured mouse 3T3-L1
cells, a preadipogenic cell line, as a useful model system
to study the eects of dietary lipids and metabolic
factors on the control of the life cycle of adipocytes.
3T3-L1 preadipocytes, which were established from
Swiss mouse embryo 3T3 broblasts, can be dierentiated into adipocytes in culture after exposure to a
standard mixture of dexamethasone, 1-methyl-3-isobutylxanthine, and insulin.29) Mouse preadipogenic 3T3-L1
cells produced several types of PGs during adipogenesis
as well as cell proliferation.30) Exogenous 15d-PGJ2 has
been shown to induce adipocyte dierentiation in other
cultured preadipogenic cells.25) On the other hand,
PGF2
has been reported to inhibit adipogenesis of
3T3-L1 cells.31,32) Our previous study evaluated the role
of the arachidonate cascade in the life cycle of 3T3-L1
adipocytes by employing specic COX inhibitors.33) The
results suggested that the COX pathway was involved in
promoting adipogenesis during the maturation phase and
also in attenuating apoptosis of preadipocytes induced
by TNF-
.
Here we provide novel evidence that TNF-
promotes
apoptotic cell death of preadipocytes through generation
of ROS, whereas mature adipocytes are almost entirely
insensitive to TNF-
. In addition, we indicate that
endogenous PGE2 and PGF2
produced through de novo

synthesis of COX-2 and activation of phospholipases

have a protective role in attenuating TNF-
-induced
apoptosis in 3T3-L1 preadipocytes.

Materials and Methods

Chemicals. Dulbeccos modied Eagles medium
with 25 mM N-2-hydroxyethylpiperazine-N0 -2-ethanesulfonic acid (DMEM-HEPES), glutaraldehyde, Hoechst
33342, A23187, and TNF-
were purchased from Sigma
(St. Louis, MO). Ascorbic acid and 2-mercaptoethanol
were purchased from Wako (Osaka, Japan). Fetal calf
serum (FCS) was purchased from Biological Industries
(Kibbutz Beth Haemek, Israel). Acetyl-Asp-Glu-ValAsp-p-nitroaniline (DEVD-pNa) was purchased from
CosmoBio (Tokyo). TrueScript II reverse transcriptase
and ribonuclease inhibitor were purchased from Sawady
Technology (Tokyo). KOD-Plus
DNA polymerase was
purchased from Toyobo (Osaka, Japan), and authentic
PGs, NS-398, and SC-560 from Cayman Chemicals
(Ann Arbor, MI). All other chemicals were of reagent or
tissue culture grade.
Cell culture and apoptosis induction. A mouse
preadipogenic 3T3-L1 cell line (JCRB9014) was obtained from the Japanese Cancer Research Resources
Bank (JCRB) (Tokyo). 3T3-L1 cells were cultured in
growth medium containing DMEM-HEPES supplemented with 10% FCS and 200 mM ascorbic acid at 37  C
under 7% CO2 . For induction of apoptosis, 3T3-L1 cells
were grown until near 80% conuence. Then the culture
medium was replaced with fresh growth medium to
which the reagents to be tested were added. Ethanol used
as a vehicle was included in the culture medium at nal
concentrations of 0.10.5% (v/v). Mature adipocytes
were prepared after inducing dierentiation, as described previously.33) After the cells were cultured in
maturation medium for 5 d, the medium was replaced
with fresh growth medium containing 100 ng/ml of
TNF-
. To determine the eects of COX inhibitors,
prostanoids, or antioxidants on TNF-
-induced apoptosis, 3T3-L1 cells were grown until 80% conuence in
a 60-mm dish containing 4 ml of the growth medium.
3T3-L1 preadipocytes were pretreated for 1 h with COX
inhibitors, A23187, or antioxidants and then stimulated
for 24 h with 40 ng/ml TNF-
along with each chemical,
whereas various prostanoids to be tested were administrated 1 h after the addition of TNF-
. After 3T3-L1
cells were treated with various reagents for the indicated
times, the apoptotic characteristics, such as nuclear
condensation and activated caspase-3 activity, were
determined as described previously.33,34) For quantitative determination of apoptosis, the number of cells
showing nuclear condensation was counted using uorescent photomicrographs after the monolayer cells were
stained with Hoechst 33342. The index of apoptotic cell
death was represented as the percentage of apoptotic
cells among total cells.

Endogenous Prostanoids as Anti-Apoptotic Factors

2147

Caspase-3 activity
(% of maximum)

100

50

100 ng/ml TNF-

( )

( )

( )

Preadipocytes

( )

Mature adipocytes

Fig. 1. Activation of Caspase-3 Activity during Apoptosis by TNF-
in Preadipocytes but Not in Mature Adipocytes.
3T3-L1 cells were grown to 80% conuence in the growth medium. The resulting cells were used as preadipocytes. According to the standard
procedures, 3T3-L1 cells were grown to conuence in the growth medium, treated with the dierentiation medium, and then cultured in the
maturation medium for 4 d. The resulting cells on d 4 of the maturation medium were used as mature adipocytes. The cultured preadipocytes and
the mature adipocytes were exposed to 100 ng/ml TNF-
for 24 h in fresh growth medium. The homogenates of the cells were subjected to assay
of caspase-3 activity, as described in Materials and Methods. Data represent the mean  SEM (n 4).  P < 0:05 between the two groups
indicated.

Enzyme-linked immunosorbent assay (ELISA) for

PGE2 and PGF2
. To determine the stimulated synthesis
of PGE2 and PGF2
by cultured 3T3-L1 preadipocytes in
response to TNF-
and A23187, ELISA for PGE2 and
PGF2
was carried out as follows: At the indicated times,
the cells were treated for 24 h with a mixture of 20 ng/
ml TNF-
and 0.1 mM A23187 at 37  C in a CO2
incubator. The culture medium of 3T3-L1 cells was
harvested and centrifuged to collect the supernatant,
which was subjected to ELISA for PGE2 and PGF2
, as
described previously.35,36)

for 1 min. The resulting 5-ml aliquot was subjected to

agarose gel electrophoresis on 1.5% gel. The primers for
COXs and GAPDH used in this study were desrcribed
previously.38)
Statistical analysis. Each experiment was performed
three or four times under conditions in which the test
agents showed no detectable cytotoxicity. Statistical
signicance of dierences between two groups was
determined by Students t test.

Results
Reverse transcriptase-polymerase chain reaction (RTPCR). Total RNA was extracted using an acid guanidinium-thiocyanate-phenol-chloroform mixture and
used for RT-PCR.37) A 10-ml RT reaction mixture
contained 0.5 mg of total RNA, 1  buer (Sawady
technology), a 1 mM deoxyribonucleoside 50 -triphosphates mixed solution (dNTP), 0.5 mM oligo (dT)12{18 ,
0.5 mM random hexamer, 1 unit/ml ribonuclease inhibitor, and 5 units/ml TrueScript II reverse transcriptase.
After a 1-h incubation at 43  C, the reaction mixture
was heated for 5 min at 98  C and diluted to a nal 30 ml
with sterile water. The resulting 1-ml aliquot was
subjected to PCR amplication in a total volume of
20 ml containing 1  PCR buer for KOD-Plus
DNA
polymerase, 200 mM dNTP, 0.3 mM sense primer, 0.3 mM
antisense primer, 1 mM MgSO4 , and 0.04 units/ml of
KOD-Plus
DNA polymerase. After heating at 94  C
for 2 min, amplication was performed for 35 cycles
involving denaturation at 94  C for 15 s, primer annealing at 55  C for 30 s, and polymerase reaction at 68  C

Stimulation of apoptosis in 3T3-L1 preadipocytes by

TNF-
through the generation of reactive oxygen
species (ROS)
Preadipocytes in the growth phase or adipocytes after
the dierentiation and maturation phases were exposed
to 100 ng/ml TNF-
for 24 h. Then the resulting
caspase-3 activity was determined using DEVD-pNa
as a tetrapeptide substrate (Fig. 1). Treatment of
preadipocytes with TNF-
caused a marked activation
of caspase-3, whereas adipocytes showed resistance to
the addition of TNF-
. These results were supported by
Hoechst staining analysis. Apoptotic cells were found
at a proportion of only 1.2% in 3T3-L1 preadipocytes
without TNF-
, whereas treatment with 40 ng/ml TNF
resulted in an increase in the percentage of apoptotic
cells to 14.8%. We also found resistance to the induction
of apoptosis in the serum-depleted medium (data not
shown).
Antioxidants such as ascorbic acid (AscA) and 2-

2148

K. NISHIMURA et al.

Caspase-3 activity
(% of maximum)

100

50

0
( )

50 M AscA 50 M 2-ME
( )

( )

50 M AscA 50 M 2-ME
40 ng/ml TNF-

Fig. 2. Eects of Various Antioxidants on Caspase-3 Activity Induced by TNF-
.

Cultured 3T3-L1 preadipocytes, as in Fig. 1, were pretreated for 1 h with various antioxidants, followed by incubation for 24 h with or without
of 40 ng/ml TNF-
along with each antioxidant in the growth medium. The antioxidants used in this study were ascorbic acid (AscA) and 2mercaptoethanol (2-ME) at a concentration of 50 mM. Caspase-3 activity was determined as described in Materials and Methods. Data
represent the mean  SEM (n 4).  P < 0:05 between the two groups indicated.

mercaptoethanol (2-ME) have been useful for study of

the involvement of ROS in cell culture systems.38) To
investigate the mediation of ROS in apoptosis induced
by TNF-
, cultured 3T3-L1 preadipocytes were pretreated for 1 h with various antioxidants, including 50 mM
AscA and 50 mM 2-ME, and then the cells were
incubated for 24 h with 40 ng/ml TNF-
along with
each antioxidant (Fig. 2). Both AscA and 2-ME eectively reduced the caspase-3 activity induced by TNF-
,
suggesting that the generation of ROS is responsible for
the activation of caspase-3.
Attenuation of apoptosis and stimulated synthesis of
endogenous prostanoids in the presence of TNF-
and
A23187
Calcium ionophore A23187 is known to stimulate an
increase in the concentration of intracellular calcium
ions that activate phospholipase A2 to release free
arachidonic acid. In addition, prostanoids have been
found to play dierent roles in adipogenesis.25,26,39) To
bring about delayed synthesis of prostanoids, 3T3-L1
preadipocytes were stimulated for 24 h in the presence
of 40 ng/ml TNF-
and varying concentrations of
A23187 (Fig. 3). A23187 reduced caspase-3 activity in
the preadipocytes in a dose-dependent manner, suggesting a contribution of elevated endogenous prostanoids to
protection of the cells from the apoptotic cell death
induced by TNF-
.
To determine the delayed synthesis of endogenous
prostanoids by ELISA for PGE2 and PGF2
, the
preadipocytes were treated with 20 ng/ml TNF-
in
the presence or absence of 0.1 mM A23187 (Fig. 4).
TNF-
markedly stimulated the formation of PGE2

(Fig. 4A) and PGF2
(Fig. 4B). The eect of TNF-
in

stimulating the delayed synthesis of both prostanoids
was more potently enhanced along with A23187. TNF-

at concentrations from 10 ng/ml to 40 ng/ml was
similarly eective in stimulating the production of both
prostanoids in 3T3-L1 preadipocytes. The eect of
TNF-
at those concentrations was also synergistically
enhanced along with 0.1 mM A23187 (data not shown).
Next we examined whether TNF-
induces gene
expression of COX isoforms during exposure of the cells
to TNF-
for 3 h (Fig. 4C). TNF-
eciently induced
gene expression of COX-2 while the mRNA level of
COX-1 remained nearly constitutive. This nding
indicates that stimulated synthesis of endogenous PGE2
and PGF2
serving as anti-apoptotic factors required the
de novo synthesis of COX-2 by TNF-
.
Eect of exogenous prostanoids on apoptosis induced
by TNF-
and NS-398, a COX-2 inhibitor
To conrm the contribution of endogenous prostanoids synthesized through the mediation of COX-2 to
the attenuation of TNF-
-induced apoptosis, cultured
3T3-L1 preadipocytes were exposed to 40 ng/ml TNF-

with or without 10 mM NS-398, a specic COX-2
inhibitor. Then nuclear condensation was analyzed
using a uorescence microscope by visualizing preadipocytes stained with Hoechst 33342. This analysis
revealed that TNF-
-induced apoptosis was stimulated
more highly with NS-398 (Fig. 5A), indicating synthesis
of endogenous prostanoids serving as anti-apoptotic
factors by the action of COX-2. Moreover, to determine
the eect of exogenous prostanoids on apoptotic cell
death induced by a mixture of TNF-
and NS-398, 3T3-

Endogenous Prostanoids as Anti-Apoptotic Factors

2149

Caspase-3 activity
(% of maximum)

100

50

0
A23187 (M)

0.1

0.1

40 ng/ml TNF-

( )

Fig. 3. Eect of Calcium Ionophore A23187 on Caspase-3 Activity Induced by TNF-
.

3T3-L1 preadipocytes, as in Fig. 1, were pretreated for 1 h with varying concentrations of A23187 and then the cells were stimulated for 24 h
with 40 ng/ml TNF-
together with A23187. Caspase-3 activity was determined as described in Materials and Methods. Data represent the
mean  SEM (n 4).  P < 0:05 between the two groups indicated.

15
PGE2 (ng/mg protein)

C
COX-1

10
COX-2
5
GAPDH
40 ng/ml
TNF-

( ) ( )

0.1 M A23187

( )

( )

( )

20 ng/ml TNF-

( )

( )

( )

0.1 M A23187

( )

( )

( )

20 ng/ml TNF-

( )

( )

( )

B
PGF2 (ng/mg protein)

20

10

Fig. 4. Synthesis of PGE2 and PGF2
by Preadipocytes in Response to TNF-
and A23187.
3T3-L1 preadipocytes, as in Fig. 1, were treated for 24 h with 20 ng/ml TNF-
and 0.1 mM A23187. The culture medium was collected and
subjected to ELISA of PGE2 (A) and PGF2
(B), as described in Materials and Methods. Data represent the mean  SEM (n 3).

P < 0:05 between the two groups indicated. C, Gene expression of COX isoforms. 3T3-L1 preadipocytes, as in Fig. 1, were treated for 3 h
with 40 ng/ml TNF-
. Total RNA was extracted and subjected to RT-PCR analysis using primers specic for COX-1 and COX-2, as
described in Materials and Methods. The amplied cDNA fragments for COX-1, COX-2, and GAPDH were 1017 bp, 912 bp, and 667 bp
respectively.

2150

K. NISHIMURA et al.

( )

40 ng/ml TNF-

( )

10 M
PGE2

10 M
PGF2

10 M
PGD 2

10 M
15d-PGJ 2

40 ng/ml TNF + 10 M NS-398

Caspase-3 activity (%)

200

150

100

50

0
10 M PGE2

( )

( )

( )

( )

( )

( )

10 M PGF2

( )

( )

( )

( )

( )

( )

10 M NS-398

( )

( )

( )

( )

( )

( )

40 ng/ml TNF-

( )

( )

( )

( )

( )

( )

Fig. 5. Eect of Exogenous Prostanoids on Apoptosis Induced by a Mixture of TNF-
and NS-398.
3T3-L1 preadipocytes were pretreated for 1 h with 10 mM NS-398 and then exposed to 40 ng/ml TNF-
together with NS-398 for 24 h in the
presence of one or the other type of prostanoids at 10 mM. Prostaglandins were added to the culture medium after 1 h of stimulation with TNF-
.
A, Fluorescence microscopic images of nuclear condensation (100 magnication). The cells were stained with Hoechst 33342. Typical
apoptotic cells are indicated by arrows. B, Analysis of caspase-3 activity. Enzymatic activity was determined as described in Materials and
Methods. Data represent the mean  SEM (n 4).  P < 0:05 between the two groups indicated.

L1 preadipocytes were exposed to various prostanoids at

10 mM for 24 h in the presence of 40 ng/ml TNF-
and
10 mM NS-398. PGE2 and PGF2
were eective in
suppressing induced apoptotic cell death. By contrast,
adipogenic prostanoids, including PGD2 and 15d-PGJ2 ,
a dehydration product of the former compound, exhibited almost no eects (Fig. 5A). These results were

also conrmed by determination of caspase-3 activity

(Fig. 5B). TNF-
stimulated caspase-3 activity in
preadipocytes by about 15-fold as compared with the
control without TNF-
. The TNF-
-induced enzyme
activity was furthermore stimulated by approximately
1.8-fold with NS-398. But when cultured preadipocytes
were exposed to either PGE2 or PGF2
together with

Endogenous Prostanoids as Anti-Apoptotic Factors

TNF-
and NS-398, the increased caspase-3 activity fell

to the level of cells treated with TNF-
alone. These
ndings were further supported by uorescent microscopic analysis using Hoechst staining. We found that
23.9% of preadipocytes were apoptotic cells when the
cells were exposed to 40 ng/ml TNF-
and 10 mM NS398. In contrast, the addition of PGE2 and PGF2
at 10
mM markedly reduced the percentage of apoptotic cells,
to levels of 5.6% and 8.5% respectively. But it remains
unclear whether the apoptosis cell death induced by a
mixture of 40 ng/ml TNF-
and 10 mM NS-398 can be
blocked completely by these prostanoids at more than
10 mM. Taken together, our results provide evidence that
TNF-
induces increases in the formation of PGE2 and
PGF2
through the induction of COX-2, and that these
endogenous prostanoids serve as anti-apoptotic factors
for 3T3-L1 preadipocytes in an autocrine manner.

Discussion
The present study indicates that TNF-
induced
apoptosis in 3T3-L1 preadipocytes, as was evident from
increased caspase-3 activity (Fig. 1) and nuclear condensation (Fig. 5A). On the other hand, 3T3-L1 adipocytes exhibited resistance to this type of apoptosis
(Fig. 1). Accordingly, mature adipocytes showed more
potent resistance to apoptotic cell death than undierentiated preadipocytes did. In agreement with our
results, mature adipocytes have been reported to be
more resistant to apoptotic cell death induced by serumdepletion through increased levels of an anti-apoptotic
protein, Bcl-2, as well as lower deoxyribonuclease
activity.40) Therefore, activation of Bcl-2 might have
been involved in the protection of 3T3-L1 adipocytes
from apoptosis induced by TNF-
in our experimental
system. The detailed mechanism of the resistance to
apoptosis in dierentiated adipocytes remains obscure.
As shown in Fig. 2, we found that antioxidants,
including AscA and 2-ME, suppressed the induction of
apoptosis by TNF-
in 3T3-L1 preadipocytes. Supporting our nding, a recent study reported the generation of
ROS in TNF-
signaling.41) Moreover, apoptosis signalregulating kinase 1 (ASK1), a member of the MAP
kinase cascade, has been reported to play a crucial role
through the activation by ROS in the signal transduction
pathway responsible for apoptosis induced by oxidative
stress or TNF-
in various types of cells.42) Although we
did not determine the activation of ASK1, this kinase
appears to be a candidate molecule for signal transduction responsible for TNF-
-induced apoptosis in
3T3-L1 preadipocytes.
Adipocytes can produce various endogenous prostanoids depending on the stage of the life cycle, implying
a critical role of such prostanoids in the regulation of
adipocyte functions, including proliferation, dierentiation, maturation, and apoptosis. Recently we found that
PGE2 was produced during cell proliferation, whereas
PGD2 can be synthesized during the progression of

39)

2151

adipogenesis. In addition, PGF2
decreased the accumulation of lipid droplets, whereas PGD2 stimulated the
storage of lipids. Thus endogenous prostanoids generated from adipocytes have the potential to regulate the
life cycle of adipocytes in a dierent manner. Previously
we reported that COX inhibitors stimulated TNF-
induced apoptosis in 3T3-L1 preadipocyte, suggesting a
protective role of some endogenous prostanoids in
apoptosis.33) The present study clearly provided evidence that exposure of 3T3-L1 preadipocytes to calcium
ionophore A23187 caused attenuation of TNF-
-induced apoptosis (Fig. 3), accompanied by stimulated
production of endogenous PGE2 (Fig. 4A) and PGF2

(Fig. 4B). A recent report provided evidence that several
types of phospholipase A2 (PLA2 ) are expressed in the
life cycle of 3T3-L1 cells.43) The
type of cytosolic
PLA2 (cPLA2
) was expressed in the proliferating
phase, and the mRNA level decreased during adipogenesis. By contrast, the  and  types of calciumindependent PLA2 (iPLA2  and iPLA2 ), which were
less expressed in the growth phase, were increasingly
transcribed with the progression of adipocyte dierentiation. Therefore, stimulated production of PGE2 and
PGF2
in 3T3-L1 preadipocytes by A23187 probably
involves predominant activation of cPLA2
by the
calcium ionophore. We found increased gene expression
of COX-2 induced by TNF-
(Fig. 4C). Thus activation
of cPLA2
as well as the induction of COX-2
contributed mainly to elevated production of endogenous PGE2 and PGF2
, functioning to attenuate TNF-
induced apoptosis. To support these results, we conrmed that exogenous PGE2 and PGF2
attenuated
apoptosis induced by TNF-
and NS-398, a specic
COX-2 inhibitor (Fig. 5). Recently, we also reported
that PGE2 and PGF2
attenuated apoptosis induced in
the presence of 12-O-tetradecanoyl phorbol--acetate
and nordihydroguaiaretic acid in Madin-Darby canine
kidney cells, indicating common protective roles of
PGE2 and PGF2
in some types of apoptosis,38) but the
previous study revealed that PGF2
was much more
eective than PGE2 . On the other hand, the present
study indicates the equal potency of PGE2 and PGF2
in
protecting 3T3-L1 preadipocytes from apoptosis, suggesting dierences in their action. Taken together, these
ndings suggest that endogenous PGE2 and PGF2
serve
as anti-apoptotic factors in 3T3-L1 preadipocytes in an
autocrine manner.
The nuclear hormone receptor PPAR- is known to
play a pivotal role in the dierentiation and adipogenesis of adipocytes,44) and to be closely linked with
hypertrophy and insulin resistance in vitro and in vivo.45)
For activation of PPAR-, ligands serving agonists must
be provided to the adipocytes. Eective exogenous
ligands include not only antidiabetic drugs but also
naturally occurring compounds. Of these, the PGD2
dehydration product, 15d-PGJ2 , has been shown to be a
potent activator of PPAR-.25,26) 15d-PGJ2 has been
shown to cause apoptotic cell death in certain mamma-

2152

K. NISHIMURA et al.
20)

lian cells such as human cancer cell lines. In contrast,

we did not detect any stimulatory eect of 15d-PGJ2 on
apoptosis with a mixture of TNF-
and NS-398 in 3T3L1 preadipocytes, although PGE2 and PGF2
, which
acted through cell-surface receptors, were eective
(Fig. 5). In terms of adipogenesis, 15d-PGJ2 and PGF2

have been shown to exhibit opposite eects, since 15dPGJ2 is pro-adipogenic while PGF2
is anti-adipogenic.
Thus the role of PPAR- in apoptosis is complicated and
remains elusive.
In summary, we found that TNF-
stimulated apoptosis of 3T3-L1 preadipocytes by activation of caspase-3
through the generation of ROS. TNF-
induced gene
expression of COX-2, resulting in the stimulated synthesis of endogenous PGE2 and PGF2
in the presence of
A23187. These endogenous prostanoids serve as antiapoptotic factors against TNF-
-induced apoptosis in
preadipocytes.

Acknowledgments
We are grateful to Professor T. Kawada in Kyoto
University, Dr. N. Takahashi of the National Institute for
Physiological Sciences of Japan, and Dr. J. Yamauchi of
the National Research Institute for Child Health and
Development of Japan for helpful discussion. In this
study, K.Y. was supported by Grants-in-Aid for Scientic Research (C) 14560099 from the Japan Society for
the Promotion of Science. This work was also supported
by the Program for Promotion of Basic Research
Activities for Innovative Biosciences (PROBRAIN) of
Japan.

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