From the Division of Endocrinology, Diabetes, and Metabolism,
Department of Medicine and Departments of Genetics and Pharmacology,
University of Pennsylvania Medical Center,
Philadelphia, Pennsylvania 19104
Fat cell differentiation is a critical aspect of
obesity and diabetes. Dietary fatty acids are converted to arachidonic
acid, which serves as precursor of prostaglandins (PGs). PGJ2
derivatives function as activating ligands for peroxisome
proliferator-activated receptor
(PPAR
), a nuclear hormone
receptor that is central to adipogenic determination. We report here
that PGF2
blocks adipogenesis through activation of
mitogen-activated protein kinase, resulting in inhibitory
phosphorylation of PPAR
. Both mitogen-activated protein kinase
activation and PPAR
phosphorylation are required for the
anti-adipogenic effects of PGF2
. Thus, PG signals generated at a
cell surface receptor regulate the program of gene expression required
for adipogenesis by modulating the activity of a nuclear hormone
receptor that is directly activated by other PG signals. The balance
between PGF2
and PGJ2 signaling may thus be central to the
development of obesity and diabetes.
 |
INTRODUCTION |
Altered levels of free fatty acids or their metabolites commonly
occur in obesity and diabetes (1, 2), and fatty acid uptake is
increased in these disorders (3). Levels of arachidonic acid
(AA),1 which is derived from
dietary essential fatty acids, are high relative to other fatty acids
in obesity and diabetic states (4), and high levels of AA may
exacerbate diabetes by negatively regulating glucose uptake (5). AA
serves as precursor for eicosanoid signaling molecules including
leukotrienes, hydroxyeicosatetraenoic acids, and prostaglandins (PGs)
(6). Many eicosanoids signal via cell surface G-protein-coupled
receptors (GPCRs) (7). Others including 8 S hydroxyeicosatetraenoic
acids, leukotriene B4, and a number of PGs including PGJ2 and
derivatives such as 15-deoxy-
12,14-PGJ2 (15d-PGJ2) bind and activate
members of the nuclear hormone receptor superfamily (8) called
peroxisome proliferator-activated receptors (PPARs)
and
(9-12).
Obesity is due to increased size and number of adipocytes. PPAR
, the
nuclear receptor for PGJ2 derivatives, plays a central role in
adipogenesis (12-14). PPAR
is the target of thiazolidinediones, an
exciting new class of antidiabetic drugs that function as direct ligands for PPAR
and have also been shown to be adipogenic (10, 15-17). An endogenous PPAR
ligand is therefore likely to be an important metabolic regulator.
The rate-limiting step in PGJ2 biosynthesis is catalyzed by
cyclooxygenase (COX) (18). The actions of different enzymes upon the
COX product PGH2 lead to numerous PGs that have different effects on
growth, differentiation, and function of many tissues, including
PGF2
(7). PGF2
is known to be synthesized by preadipocytes but
does not activate PPAR
(9) and by contrast has a potent inhibitory
effect upon adipocyte differentiation (19, 20). Thus, products of AA
metabolism downstream of COX have opposing effects upon adipogenesis.
Although the adipogenic effects of PGJ2 derivatives involve direct
activation of nuclear PPAR
, PGF2
utilizes a specific GPCR on the
cell surface to initiate intracellular signal transduction (21-24). We
and others recently showed that activation of the MAP kinase pathway
phosphorylates PPAR
and inhibits adipogenesis (25-27). Although
insulin can induce phosphorylation of ectopic PPAR
in cells
expressing ectopic insulin receptor (27), insulin is adipogenic, and
its ability to activate MAP kinase does not play an important role
during adipogenesis (28). Indeed, the physiological inducer of PPAR
phosphorylation is unknown. Thus we considered whether PGF2
might
work through this pathway. Here we show that PGF2
induces
phosphorylation of PPAR
via activation of MAP kinase and that this
is required for inhibition of 3T3-L1 adipogenesis by PGF2
.
 |
MATERIALS AND METHODS |
Cell Culture and Differentiation--
3T3-L1 cells were obtained
from American Type Culture Collection (Rockville, MD). Cells were
cultured in Dulbecco's modified Eagle's medium containing 10% bovine
calf serum (HyClone). Cell were differentiated into mature adipocytes
as described previously (29). In some experiments PGs (Cayman
Chemical), PD98059 (Calbiochem), or retinoic acid (RA) (Sigma) were
added simultaneously with the differentiation medium at day 0 or as
indicated and maintained throughout. Compounds were dissolved in either
ethanol or Me2SO according to manufacturer instructions.
For acute treatment with PGs, cells were treated for 30 min and then
harvested for protein. Retroviral gene transduction of 3T3-L1 cells
with PPAR
2 or PPAR
2 (S112A) were performed as described
previously (26). Briefly, infected 3T3-L1 cells were selected in G418
and grown to confluence in growth medium (Dulbecco's modified Eagle's
medium containing 10% iron-enriched fetal bovine serum). 2 days
post-confluency (day 0) vehicle control or PGs were added to medium and
maintained throughout. Morphology and RNA were analyzed at day 10.
Northern Analysis--
Isolation of total RNA and Northern blots
were performed as described previously (29). The cDNA probes for
aP2, adipsin, and actin were labeled with 32P using random
hexamers.
Western Analysis--
Whole cell extracts were prepared from
3T3-L1 cells using RIPA lysis buffer supplemented with pepstatin (5 µg/ml), leupeptin (5 µg/ml), and phenylmethylsulfonyl fluoride (2 mM). Extracts were incubated at 4 °C for 30 min and then
centrifuged 30 min at 4 °C. 100 µg of soluble protein was
separated by SDS-PAGE (10% gel, acrylamide:bis-acrylamide ratio of 100 with 4 M urea), and immunoblots were performed as described
previously (29). Phospho-specific MAP kinase antibody (New England
BioLabs) was used at a 1:400 dilution.
 |
RESULTS |
PGF2
Activates MAP Kinase--
We first determined whether
PGF2
activates MAP kinase. Fig.
1A shows that indeed,
treatment of 3T3-L1 cells with PGF2
leads to a
dose-dependent increase in activated MAP kinase. Similar MAP kinase activation was observed with fluprostenol, a specific ligand
for the PGF2
receptor (FP receptor) (23), but not with 15d-PGJ2.
Note that PD98059, the specific inhibitor of MAP kinase kinase (30),
also prevented the activation of MAP kinase by PGF2
and
fluprostenol.

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Fig. 1.
MAP kinase activation is required for
inhibition of adipogenesis by PGF2 . A, PGF2 activates
MAP kinase in 3T3-L1 cells. Cells were treated with PGs for 30 min
prior to harvesting for protein. As indicated, cells were pretreated
with the inhibitor of MAP kinase PD98059 (50 µM) for 30 min prior to addition of PGs. Cell lysates were separated by SDS-PAGE
and immunoblotted with a phospho-specific MAP kinase antibody.
Arrow indicates phosphorylated MAP kinase
(P-MAPK). B, inhibitor of MAP kinase activity
blocks PGF2 effects and not those of RA. Morphology of 3T3-L1 cells following differentiation in the presence of vehicle control, PGF2
(100 nM), fluprostenol (100 nM), 15d-PGJ2 (10 µM), or RA (10 µM) in the absence or the
presence of PD98059 (25 µM). All compounds were added on
day 0 and maintained during differentiation protocol. C,
PD98059 blocks inhibitory effects of PGF2 on induction of
adipocyte-markers aP2 and adipsin. Northern analysis of aP2, adipsin,
and actin at day 7 after exposure to adipogenic stimulus in the
presence or the absence of indicated compounds are shown.
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MAP Kinase Activation Is Required for Inhibition of Adipogenesis by
PGF2
--
We next tested whether MAP kinase activation was required
for inhibition of adipogenesis by PGF2
. Fig. 1B shows the
morphology of 3T3-L1 cells 7 days after exposure to differentiation
medium. As expected, PGF2
, fluprostenol, and RA blocked acquisition
of the adipocyte phenotype, whereas 15d-PGJ2 did not. The MAP kinase inhibitor PD98059 had little effect on its own. However, it completely prevented inhibition of adipogenesis by PGF2
and by fluprostenol, indicating that MAP kinase activation was required for the
anti-adipogenic effects of these compounds. This effect of PD98059 was
specific for inhibition by PGF2
and fluprostenol, because PD98059
had no effect on the ability of RA to inhibit adipogenesis, consistent with evidence that RA acts by antagonizing the effects of C/EBP (29), a
mechanism that would be predicted to be independent of MAP kinase. We
also assessed the effects of MAP kinase inhibition on the induction of
fatty acid binding protein aP2 and adipsin, two adipocyte-specific
genes that serve as markers of the differentiation process (31-33).
Fig. 1C shows that induction of aP2 and adipsin was blocked
by PGF2
and by RA. Consistent with the cellular morphology, MAP
kinase inhibition prevented inhibition of the adipocyte-specific genes
by PGF2
but not by RA. Indeed, MAP kinase inhibition caused a
reproducible increase in aP2 expression, consistent with a recent report that MAP kinase activation is anti-adipogenic (28).
PGF2
Induces Phosphorylation of PPAR
--
Given the ability
of PGF2
to activate MAP kinase and the inhibitory effects of MAP
kinase phosphorylation on the activity of PPAR
, we next tested the
effects of PGF2
on PPAR
phosphorylation. Phosphorylation of
PPAR
2 on serine 112 can be detected by differences in
electrophoretic mobility of the phosphorylated and nonphosphorylated forms. Fig. 2A shows that
3T3-L1 cells on day 4 of a standard differentiation protocol express
abundant PPAR
1 and PPAR
2 protein, with about equal amounts of the
hypophosphorylated and phosphorylated forms of each. Treatment with
PGF2
increased the phosphorylation state of the PPAR
isoforms in
a concentration-dependent manner, and the phosphorylation
was blocked by the MAP kinase inhibitor. Similar results were obtained
with fluprostenol. By contrast, 15d-PGJ2 had no effect upon the
phosphorylation state of PPAR
1 or PPAR
2, and PD98059 had no
independent effect. Prostacyclin (PGI2) also had no effect on PPAR
phosphorylation (data not shown). Thus PGF2
activation of MAP kinase
resulted in phosphorylation of endogenous PPAR
in 3T3-L1 cells.
PGF2
-induced phosphorylation of PPAR
inhibited its
transcriptional activity as previously shown for phosphorylation of
PPAR
by other agents (data not shown).

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Fig. 2.
PGF2 -induced phosphorylation of PPAR
correlates with inhibition of adipogenesis. A, agonists of
FP receptor (PGF2 , fluprostenol) phosphorylate PPAR in 3T3-L1
cells. Mature adipocytes (day 7) were treated with PGs as in Fig.
1B. Cell lysates were separated by SDS-PAGE and analyzed by
Western mobility shift assay using antibody to PPAR . B,
phosphorylation of PPAR during adipocyte differentiation, and the
effects of treatment with PGF2 . PGF2 (100 nM) was
added to differentiating 3T3-L1 cells at day 3 and maintained
throughout. Protein extracts were harvested at day indicated and
analyzed by Western blot. Arrows indicates phosphorylated PPAR (P). C, addition of PGF2 on day 3 inhibits induction of adipocyte-marker aP2. Northern analysis of aP2
mRNA at day 7 after exposure to indicated treatments. Ethidium
bromide staining of 28 S is shown to confirm loading of equal RNA
amounts.
|
|
We next tested whether PGF2
-induced phosphorylation of PPAR
correlated with inhibition of adipogenesis. Fig. 2B shows
that PPAR
protein was not reproducibly detectable on day 0, and the differentiation process involves amplification of PPAR
expression by
a feed-forward mechanism. The ability of PPAR
ligands to induce adipogenesis (14, 34) strongly suggests that functional PPAR
is
present in the preadipocyte, and thus hyperphosphorylation by PGF2
would inhibit their function. However, PGF2
inhibition of
adipogenesis also blocked the feed-forward induction of PPAR
, preventing analysis of PPAR
phosphorylation due to technical limitations (data not shown). Nevertheless, we were able to test the
correlation between inhibition of adipocyte differentiation and PPAR
phosphorylation by addition of PGF2
at times when PPAR
protein
was expressed. Fig. 2B shows that PPAR
protein was
induced between days 1 and 2 of adipogenesis and reached maximum levels by days 3 and 4. Both PPAR
and PPAR
2 were present in
nonphosphorylated and phosphorylated states, with similar ratios of
nonphosphorylated to phosphorylated PPAR
throughout differentiation.
Addition of PGF2
at day 3 caused a marked reduction in adipocyte
differentiation, as indicated by a dramatic (70%) reduction in aP2
gene expression on day 7 (Fig. 2C) as well as by a similar
reduction in adipogenesis as assessed by cell morphology (data not
shown). PPAR
induction is itself a marker of adipocyte
differentiation, and indeed PPAR
levels were lower in the
PGF2
-treated cells (Fig. 2B). Thus the ability of PGF2
to inhibit adipogenesis correlated with the phosphorylation of
endogenous PPAR
.
PGF2
Selectively Inhibits Adipogenesis Due to Phosphorylated
Form of PPAR
--
Thus far we have shown that the ability of
PGF2
to activate MAP kinase is essential for its ability to inhibit
adipogenesis, that MAP kinase activation by PGF2
is sufficient to
phosphorylate PPAR
in 3T3-L1 cells, and that this phosphorylation
correlates with inhibition of differentiation. We next tested whether
the ability to phosphorylate PPAR
was necessary for PGF2
to
inhibit adipogenesis. We previously showed that ectopic expression of PPAR
2 results in adipocytic differentiation of 3T3-L1 cells (35). We
hypothesized that PGF2
-induced phosphorylation of the ectopic PPAR
2 would inhibit its ability to induce adipogenesis, whereas adipocytic differentiation induced by PPAR
2 (S112A) would be unaffected by activation of MAP kinase by PGF2
. Indeed, Fig. 3A shows that PGF2
completely prevented adipogenesis due to PPAR
2 expression but had
little or no effect upon adipogenesis caused by PPAR
2 (S112A). In
contrast, 15d-PGJ2 potentiated the adipogenicity of both wild type and
mutant PPAR
. Fig. 3B shows that the wild type PPAR
was
nearly completely phosphorylated by PGF2
, whereas the PPAR
2
(S112A) was not phosphorylated at all. Fig. 3C shows that
this difference in susceptibility to the effects of PGF2
was
reflected in the expression of the adipocyte marker adipsin. PGF2
completely prevented the ability of PPAR
2 to induce adipsin but had
no effect on adipsin induction by the nonphosphorylatable form of
PPAR
2. Again, 15d-PGJ2 functioned as an activating ligand of both
forms of PPAR
.

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Fig. 3.
PPAR phosphorylation is required for
anti-adipogenic effects of PGF2 . A, PGF2 inhibits
differentiation of 3T3-L1 cells induced by PPAR 2 but not PPAR 2
(S112A). Morphology of 3T3-L1 cells infected with retrovirus causing
ectopic expression of PPAR 2 or PPAR 2 (S112A). 2 days
post-confluency (day 0) cells were grown in the presence of vehicle
control, PGF2 (100 nM), or 15-deoxy- 12,14-PGJ2 (10 µM) in growth medium. Cells were analyzed on day 10. The
photographs show representative phase contrast views of the cells. This
experiment was repeated three times with similar results. B,
PGF2 treatment leads to phosphorylation of ectopic PPAR 2 but not
PPAR 2 (S112A). Cells were harvested 1 h after treatment with
PGF2 (100 nM) on day 0. C, PGF2 inhibits adipsin induction due to PPAR 2 but not PPAR 2 (S112A). Actin and
28 S rRNA are shown as loading controls.
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 |
DISCUSSION |
Our results shed important new light on the role of cell surface
and nuclear receptors for eicosanoids. Some eicosanoids, such as
leukotriene B4, interact with both cell surface receptors and nuclear
PPAR
, and these signaling pathways are cooperative (9-12, 36-38).
In contrast, PGF2
and PGJ2 derivatives are different products of AA
metabolism with opposing biological effects mediated by cell surface
and nuclear receptors, respectively. These converge on the PG nuclear
receptor PPAR
as in the model shown in Fig. 4. 3T3-L1 cells are known to produce
PGF2
, and in accordance with recent suggestions our model proposes
that a PPAR
ligand related to PGJ2 is endogenously produced during
adipogenesis. However, the expression of PGD synthase in adipocytes has
not been established, and it is likely that prostaglandins or other PPAR
ligands derived from paracrine, endocrine, as well as
pharmacological sources outside the fat cell could also influence this
process. In any case, in a single cell type, PGs can function both by
binding to GPCRs (PGF2
) or to nuclear hormone receptors (PGJ2 and
derivatives). Despite the different types of receptors bound by PGF2
and PGJ2 derivatives, both the inhibition of PPAR
activity by
PGF2
-driven phosphorylation as well as activation of PPAR
by
noncovalent PG ligand binding could be physiologically relevant not
only in adipocytes but also in other cell types that express PPAR
,
such as lung (39), colon (40, 41), and hematopoietic cells (42). Moreover, because other eicosanoids function via cell surface and
nuclear receptors, similar opportunities exist for convergent regulation of biological processes by eicosanoids that either bind
directly to or modulate the phosphorylation state of nuclear hormone
receptors.

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Fig. 4.
Model of opposing actions of PGF2 and PGJ2
derivatives in adipocyte differentiation. PGF2 interacts with
the FP receptor, which activates MAP kinase resulting in
phosphorylation of PPAR and inhibition of its activity. PGJ2
derivatives are depicted as being endogenously produced but could also
be derived from extracellular paracrine, endocrine, or pharmacological
sources. Thiazolidinediones could also serve as PPAR ligands. In any
case, these ligands interact directly with PPAR to stimulate its
activity. The net effects on PPAR activity result in repression or
stimulation of the adipogenic gene program. Similar antagonistic
effects on PPAR function could also alter PPAR activity in the
mature adipocyte.
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Chronic exposure of adipocytes to AA causes a state of insulin
resistance associated with down-regulation of the insulin-responsive glucose transporter (43). Interestingly, AA-derived PGF2
levels are
increased in diabetic humans (44, 45). The link between free fatty acid
levels and the pathogenesis of noninsulin-dependent diabetes has been recognized for many years (46). Our results provide a
potential molecular mechanism relating fatty acids to both increased
and decreased activity of the adipogenic nuclear receptor PPAR
.
Because thiazolidinediones that activate PPAR
are potent
antidiabetic compounds (47), the ability of PGF2
to inhibit the
activity of PPAR
suggests that the FP receptor may be a novel
therapeutic target for diabetes.