COMMUNICATION
Prostaglandins Promote and Block Adipogenesis through Opposing Effects on Peroxisome Proliferator-activated Receptor gamma *

Mauricio J. Reginato, Samuel L. Krakow, Shannon T. Bailey, and Mitchell A. LazarDagger

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

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 gamma  (PPARgamma ), a nuclear hormone receptor that is central to adipogenic determination. We report here that PGF2alpha blocks adipogenesis through activation of mitogen-activated protein kinase, resulting in inhibitory phosphorylation of PPARgamma . Both mitogen-activated protein kinase activation and PPARgamma phosphorylation are required for the anti-adipogenic effects of PGF2alpha . 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 PGF2alpha and PGJ2 signaling may thus be central to the development of obesity and diabetes.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-Delta 12,14-PGJ2 (15d-PGJ2) bind and activate members of the nuclear hormone receptor superfamily (8) called peroxisome proliferator-activated receptors (PPARs) alpha  and gamma  (9-12).

Obesity is due to increased size and number of adipocytes. PPARgamma , the nuclear receptor for PGJ2 derivatives, plays a central role in adipogenesis (12-14). PPARgamma is the target of thiazolidinediones, an exciting new class of antidiabetic drugs that function as direct ligands for PPARgamma and have also been shown to be adipogenic (10, 15-17). An endogenous PPARgamma 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 PGF2alpha (7). PGF2alpha is known to be synthesized by preadipocytes but does not activate PPARgamma (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 PPARgamma , PGF2alpha 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 PPARgamma and inhibits adipogenesis (25-27). Although insulin can induce phosphorylation of ectopic PPARgamma 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 PPARgamma phosphorylation is unknown. Thus we considered whether PGF2alpha might work through this pathway. Here we show that PGF2alpha induces phosphorylation of PPARgamma via activation of MAP kinase and that this is required for inhibition of 3T3-L1 adipogenesis by PGF2alpha .

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 PPARgamma 2 or PPARgamma 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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

PGF2alpha Activates MAP Kinase-- We first determined whether PGF2alpha activates MAP kinase. Fig. 1A shows that indeed, treatment of 3T3-L1 cells with PGF2alpha leads to a dose-dependent increase in activated MAP kinase. Similar MAP kinase activation was observed with fluprostenol, a specific ligand for the PGF2alpha 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 PGF2alpha and fluprostenol.


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Fig. 1.   MAP kinase activation is required for inhibition of adipogenesis by PGF2alpha . A, PGF2alpha 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 PGF2alpha effects and not those of RA. Morphology of 3T3-L1 cells following differentiation in the presence of vehicle control, PGF2alpha (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 PGF2alpha 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.

MAP Kinase Activation Is Required for Inhibition of Adipogenesis by PGF2alpha -- We next tested whether MAP kinase activation was required for inhibition of adipogenesis by PGF2alpha . Fig. 1B shows the morphology of 3T3-L1 cells 7 days after exposure to differentiation medium. As expected, PGF2alpha , 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 PGF2alpha 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 PGF2alpha 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 PGF2alpha and by RA. Consistent with the cellular morphology, MAP kinase inhibition prevented inhibition of the adipocyte-specific genes by PGF2alpha 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).

PGF2alpha Induces Phosphorylation of PPARgamma -- Given the ability of PGF2alpha to activate MAP kinase and the inhibitory effects of MAP kinase phosphorylation on the activity of PPARgamma , we next tested the effects of PGF2alpha on PPARgamma phosphorylation. Phosphorylation of PPARgamma 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 PPARgamma 1 and PPARgamma 2 protein, with about equal amounts of the hypophosphorylated and phosphorylated forms of each. Treatment with PGF2alpha increased the phosphorylation state of the PPARgamma 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 PPARgamma 1 or PPARgamma 2, and PD98059 had no independent effect. Prostacyclin (PGI2) also had no effect on PPARgamma phosphorylation (data not shown). Thus PGF2alpha activation of MAP kinase resulted in phosphorylation of endogenous PPARgamma in 3T3-L1 cells. PGF2alpha -induced phosphorylation of PPARgamma inhibited its transcriptional activity as previously shown for phosphorylation of PPARgamma by other agents (data not shown).


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Fig. 2.   PGF2alpha -induced phosphorylation of PPARgamma correlates with inhibition of adipogenesis. A, agonists of FP receptor (PGF2alpha , fluprostenol) phosphorylate PPARgamma 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 PPARgamma . B, phosphorylation of PPARgamma during adipocyte differentiation, and the effects of treatment with PGF2alpha . PGF2alpha (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 PPARgamma (P). C, addition of PGF2alpha 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 PGF2alpha -induced phosphorylation of PPARgamma correlated with inhibition of adipogenesis. Fig. 2B shows that PPARgamma protein was not reproducibly detectable on day 0, and the differentiation process involves amplification of PPARgamma expression by a feed-forward mechanism. The ability of PPARgamma ligands to induce adipogenesis (14, 34) strongly suggests that functional PPARgamma is present in the preadipocyte, and thus hyperphosphorylation by PGF2alpha would inhibit their function. However, PGF2alpha inhibition of adipogenesis also blocked the feed-forward induction of PPARgamma , preventing analysis of PPARgamma phosphorylation due to technical limitations (data not shown). Nevertheless, we were able to test the correlation between inhibition of adipocyte differentiation and PPARgamma phosphorylation by addition of PGF2alpha at times when PPARgamma protein was expressed. Fig. 2B shows that PPARgamma protein was induced between days 1 and 2 of adipogenesis and reached maximum levels by days 3 and 4. Both PPARgamma and PPARgamma 2 were present in nonphosphorylated and phosphorylated states, with similar ratios of nonphosphorylated to phosphorylated PPARgamma throughout differentiation. Addition of PGF2alpha 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). PPARgamma induction is itself a marker of adipocyte differentiation, and indeed PPARgamma levels were lower in the PGF2alpha -treated cells (Fig. 2B). Thus the ability of PGF2alpha to inhibit adipogenesis correlated with the phosphorylation of endogenous PPARgamma .

PGF2alpha Selectively Inhibits Adipogenesis Due to Phosphorylated Form of PPARgamma -- Thus far we have shown that the ability of PGF2alpha to activate MAP kinase is essential for its ability to inhibit adipogenesis, that MAP kinase activation by PGF2alpha is sufficient to phosphorylate PPARgamma in 3T3-L1 cells, and that this phosphorylation correlates with inhibition of differentiation. We next tested whether the ability to phosphorylate PPARgamma was necessary for PGF2alpha to inhibit adipogenesis. We previously showed that ectopic expression of PPARgamma 2 results in adipocytic differentiation of 3T3-L1 cells (35). We hypothesized that PGF2alpha -induced phosphorylation of the ectopic PPARgamma 2 would inhibit its ability to induce adipogenesis, whereas adipocytic differentiation induced by PPARgamma 2 (S112A) would be unaffected by activation of MAP kinase by PGF2alpha . Indeed, Fig. 3A shows that PGF2alpha completely prevented adipogenesis due to PPARgamma 2 expression but had little or no effect upon adipogenesis caused by PPARgamma 2 (S112A). In contrast, 15d-PGJ2 potentiated the adipogenicity of both wild type and mutant PPARgamma . Fig. 3B shows that the wild type PPARgamma was nearly completely phosphorylated by PGF2alpha , whereas the PPARgamma 2 (S112A) was not phosphorylated at all. Fig. 3C shows that this difference in susceptibility to the effects of PGF2alpha was reflected in the expression of the adipocyte marker adipsin. PGF2alpha completely prevented the ability of PPARgamma 2 to induce adipsin but had no effect on adipsin induction by the nonphosphorylatable form of PPARgamma 2. Again, 15d-PGJ2 functioned as an activating ligand of both forms of PPARgamma .


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Fig. 3.   PPARgamma phosphorylation is required for anti-adipogenic effects of PGF2alpha . A, PGF2alpha inhibits differentiation of 3T3-L1 cells induced by PPARgamma 2 but not PPARgamma 2 (S112A). Morphology of 3T3-L1 cells infected with retrovirus causing ectopic expression of PPARgamma 2 or PPARgamma 2 (S112A). 2 days post-confluency (day 0) cells were grown in the presence of vehicle control, PGF2alpha (100 nM), or 15-deoxy-Delta 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, PGF2alpha treatment leads to phosphorylation of ectopic PPARgamma 2 but not PPARgamma 2 (S112A). Cells were harvested 1 h after treatment with PGF2alpha (100 nM) on day 0. C, PGF2alpha inhibits adipsin induction due to PPARgamma 2 but not PPARgamma 2 (S112A). Actin and 28 S rRNA are shown as loading controls.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 PPARalpha , and these signaling pathways are cooperative (9-12, 36-38). In contrast, PGF2alpha 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 PPARgamma as in the model shown in Fig. 4. 3T3-L1 cells are known to produce PGF2alpha , and in accordance with recent suggestions our model proposes that a PPARgamma 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 PPARgamma 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 (PGF2alpha ) or to nuclear hormone receptors (PGJ2 and derivatives). Despite the different types of receptors bound by PGF2alpha and PGJ2 derivatives, both the inhibition of PPARgamma activity by PGF2alpha -driven phosphorylation as well as activation of PPARgamma by noncovalent PG ligand binding could be physiologically relevant not only in adipocytes but also in other cell types that express PPARgamma , 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 PGF2alpha and PGJ2 derivatives in adipocyte differentiation. PGF2alpha interacts with the FP receptor, which activates MAP kinase resulting in phosphorylation of PPARgamma 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 PPARgamma ligands. In any case, these ligands interact directly with PPARgamma to stimulate its activity. The net effects on PPARgamma activity result in repression or stimulation of the adipogenic gene program. Similar antagonistic effects on PPARgamma function could also alter PPARgamma activity in the mature adipocyte.

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 PGF2alpha 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 PPARgamma . Because thiazolidinediones that activate PPARgamma are potent antidiabetic compounds (47), the ability of PGF2alpha to inhibit the activity of PPARgamma suggests that the FP receptor may be a novel therapeutic target for diabetes.

    ACKNOWLEDGEMENT

We thank Dalei Shao for helpful discussions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK49780.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: University of Pennsylvania, 611 CRB, 415 Curie Blvd., Philadelphia, PA 19104-6149. Tel.: 215-898-0198; Fax: 215-898-5408; E-mail: lazar{at}mail.med.upenn.edu.

1 The abbreviations used are: AA, arachidonic acid; PG, prostaglandin; GPCR, G-protein-coupled receptor; PPAR, peroxisome proliferator-activated receptor; COX, cyclooxygenase; MAP, mitogen-activated protein; RA, retinoic acid; PAGE, polyacrylamide gel electrophoresis; 15d-PGJ2, 15-deoxy-Delta 12,14-PGJ2.

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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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