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Originally published In Press as doi:10.1194/jlr.M300248-JLR200 on July 1, 2003
Journal of Lipid Research, Vol. 44, 1652-1659, September 2003
Copyright © 2003 by American Society for Biochemistry and Molecular Biology
Selective cyclo-oxygenase-2 inhibitors impair adipocyte differentiation through inhibition of the clonal expansion phase
Lluis Fajas1,*,
Stéphanie Miard*,
Michael R. Briggs and
Johan Auwerx2,*
* Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, B. P. 163, F-67404 Illkirch cedex, C.U. de Strasbourg, France
Pharmacia Discovery Research, St. Louis, MO 63198
Published, JLR Papers in Press, July 1, 2003. DOI 10.1194/jlr.M300248-JLR200
1 Present address for L. Fajas: Equipe Avenir, INSERM U540, Endocrinologie Moléculaire et Cellulaire des Cancers, 60 rue de Navacelles, 34090 Montpellier, France. 
2 To whom correspondence should be addressed. e-mail: auwerx{at}igbmc.u-strasbg.fr
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ABSTRACT
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Selective cyclo-oxygenase-2 (COX-2) inhibitors are nonsteroidal antiinflammatory drugs used in the management of inflammatory diseases. We demonstrate here that inhibition of the COX-2 enzyme impairs adipocyte differentiation. The inhibition of adipogenesis occurs in the early clonal expansion phase. In particular, COX-2 inhibition limits cell cycle reentry required before terminal adipocyte differentiation. This inhibition of adipogenesis is independent of the production of the peroxisome proliferator activated receptor ligand prostaglandin J2, but dependent on the production of proliferative prostaglandins, such as prostaglandin E2.
Modulation of the activity of the COX-2 enzyme via COX-2 selective inhibitors might open up new perspectives in the control of obesity and related metabolic diseases.
Supplementary key words peroxisome proliferator activated receptor cell cycle insulin resistance obesity
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INTRODUCTION
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Cyclo-oxygenase (COX) is the key enzyme required for the conversion of arachidonic acid to prostaglandins. COX catalyses the enzymatic conversion of 20-carbon polyunsaturated fatty acids, such as arachidonic acid, into prostaglandin G2 and H2 (PGH2), which are subsequently converted into a variety of eicosanoids by specific prostaglandin synthases (1). Two COX isoforms have been described. COX-1 is constitutively expressed in almost all tissues, and its activity depends only on the availability of the substrate. In contrast to COX-1, COX-2 behaves as an immediate early gene subjected to transcriptional regulation. Most of the currently used nonsteroidal anti-inflammatory drugs (NSAIDs) are not specific for any of these COX isoforms and have a number of side effects, mainly linked to their activity on COX-1. Selective COX-2 inhibitors were therefore developed for the management of inflammatory diseases [reviewed in ref. (2)]. Targeting the COX-2 isoform seems to overcome some of the observed side effects, such as the irritation of the gastric mucosa, a problem often encountered with the classical NSAIDs.
Prostaglandins participate in a large number of physiological processes, such as inflammation, immune response, kidney function, bone metabolism, ovulation, and adipocyte differentiation [reviewed in ref. (3)]. Adipocyte differentiation is a complex and staged process (4). Upon reaching confluence, proliferating preadipocytes become growth arrested by contact inhibition. After hormonal induction, those contact-inhibited preadipocytes undergo clonal expansion, characterized by reentry into cell cycle. At this stage, increased cAMP levels elicit the expression of the CCAAT enhancer binding protein transcription factors, C/EBPß and (5, 6), which then induce the expression of peroxisome proliferator-activated receptor (PPAR ). In addition, the activity of the E2F family of transcription factors, observed during early phases of adipogenesis, participates in the induction of the expression of PPAR (7). Both C/EBPs and PPAR will then trigger the terminal differentiation process. The importance of the early clonal expansion stage is underscored by the observation that either decrease of cAMP levels or inhibition of cell cycle impairs terminal adipocyte differentiation (8).
The role of particular prostaglandins in adipocyte differentiation is suggested to be dependent on the specific stages of this differentiation process. Prostacyclin (PGI2) has been shown to positively influence adipocyte differentiation (8). The stable analog of PGI2, carbacyclin, promotes terminal adipocyte differentiation by eliciting the required increase in cAMP levels as well as the release of intracellular Ca2+. Moreover, PGI2 increased the expression of both C/EBPß and in preadipose cells (6). More controversial is the participation of prostaglandin E2 (PGE2). Whereas its antilypolytic effect has been established in differentiated adipocytes (9), PGE2 has been reported to either activate (10) or inhibit (11) early adipogenesis. Finally, prostaglandin J2 (PGJ2) has a positive role in terminal adipocyte differentiation currently thought to be mediated by increasing the activity of PPAR , a nuclear receptor that also can be activated by a number of synthetic compounds, such as the thiazolidinediones [reviewed in ref. (12)].
By inhibiting prostaglandin synthesis using specific COX-2 inhibitors at different stages of adipogenesis, we demonstrate here that COX-2 plays an important role in early adipocyte differentiation, regulating the entry into cell cycle, whereas its role in terminal differentiation is dispensable.
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MATERIALS AND METHODS
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Materials
All chemicals, except if stated otherwise, were purchased from Sigma (St. Louis, MO). Antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), except for the anti-bromodeoxyuridine (BrdU) antibody, which was from Sigma. COX-2 inhibitor sc-58236 and COX-1 inhibitor sc-58560 were synthetised by Pharmacia. L165041 was a gift from Merck.
Cell culture and protein extracts
3T3-L1 cells were grown in Dulbecco's modified Eagle medium (DMEM) and 10% fetal calf serum. Cells were differentiated with DMEM, 10% serum, 0.5 mM 3-isobutyl-1methylxanthine, 10 µg/ml insulin, and 1 µM dexamethasone for 2 days. From day 3 on, cells were incubated with DMEM, 10% serum, and 10 µg/ml insulin. Nuclear and whole-cell extracts preparation, Oil red O staining, and quantification are described elsewhere (7).
Immunofluorescence
For all immunofluorescence experiments, cells were grown on coverslips. For BrdU incorporation, cells were incubated for 8 h in the presence of BrdU, and an additional treatment of the cells with 1.5 N HCl for 10 min at 21°C was performed. After fixation and permeabilization with methanol, cells were incubated with the indicated antibodies. Preparations were then incubated with a combination of Texas Red-conjugated anti-mouse IgG and fluorescein isothiocyanate-conjugated anti-rabbit IgG.
Western and Northern blot analyses
SDS-PAGE and electrotransfer were performed as described (13). The membranes were blocked overnight in blocking buffer (20 mM Tris, 100 mM NaCl, 1% Tween-20, and 10% skim milk). Filters were first incubated for 4 h at 21°C with the indicated antibody, and then for 1 h at 21°C with a peroxidase conjugate secondary antibody. The complex was visualized with 4-chloro-1-naphthol. RNA extraction and analysis, as well as aP2 and 36B4 probes, were described previously (14).
Prostaglandins measurement
PGE2 and 15-deoxy PGJ2 levels were determined with the Enzyme Immunoassay Kit exactly as described by the manufacturer (Assay Designs, Inc., Ann Arbor, MI).
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RESULTS
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COX-2 inhibitors block adipogenesis in 3T3-L1 cells
Western blot analysis of nuclear extracts from differentiating 3T3-L1 preadipocytes demonstrated that COX-2 is absent in quiescent preadipocytes and is robustly induced during the clonal expansion phase of adipogenesis preceding the expression of PPAR (Fig. 1A)
. E2F1 and cyclin A protein expression were used as markers of the clonal expansion stage (Fig. 1A). The timing of COX-2 expression suggested that it could play a role in this phase of adipocyte differentiation. Therefore, inhibition of COX-2 activity might result in differences in the adipogenic potential of 3T3-L1 cells. 3T3-L1 cells were hence incubated with the specific COX-2 inhibitor sc-58236 for 24 h prior to the induction of adipocyte differentiation with hormonal mix. Consistent with a positive role in adipogenesis, inhibition of COX-2 impaired differentiation in a dose-dependent manner, as evidenced by a decrease in Oil red O staining (Fig. 1B, C) and a decrease in the expression of the adipocyte marker aP2 (Fig. 1D). This inhibitory effect of sc-58236 on adipocyte differentiation correlated with a decrease in the production of PGE2 and 6-keto PGF1 , indicating that the COX-2 enzyme was indeed inactivated (Fig. 1E). Interestingly, when we attempted to measure the effect of the COX-2 inhibitor on the production of the PPAR agonist 15-deoxy PGJ2, we found barely detectable levels of PGJ2 in any stage of 3T3-L1 differentiation (data not shown). No effect on the production of PGE2 or only a slight effect on the production of 6-keto PGF1 was observed when the specific COX-1 inhibitor, sc-58560, was used in a similar study (Fig. 1E). Furthermore, sc-58560 had no effect on the differentiation of the 3T3-L1 preadipocytes (data not shown).
Because PPAR is a key player in adipocyte differentiation, we analyzed its expression 48 h after induction of differentiation in the absence or in the presence of 10-6 M of the COX-2 inhibitor. Expression of PPAR was found to be decreased about 3-fold when sc-58236 was added to the differentiation mix (Fig. 1F, G). This result suggested that participation of COX-2 in the differentiation process was not related to PPAR activation, but was rather affecting any process prior to PPAR expression.
COX-2 inhibitors repress the clonal expansion phase of adipogenesis
It has been previously reported that COX-2 actively participates in cell cycle progression and proliferation (15, 16). Consequently, inhibition of COX-2 results in cell cycle arrest (15, 16). Because inhibition of cell cycle reentry during clonal expansion inhibits adipocyte differentiation (8), we postulated that inhibition of COX-2 would inhibit cell cycle progression during clonal expansion of adipocyte differentiation. To test this hypothesis, we first analyzed cell cycle progression in proliferating 3T3-L1 preadipocytes. The number of cyclin E (Fig. 2A)
and BrdU (Fig. 2B) -positive, and thus -proliferating, cells was decreased when the COX-2 inhibitor was used, as measured by immunofluorescence analysis, compared with cells incubated in the absence of COX-2 inhibitors (Fig. 2A, B). Similarly to proliferating cells, hormonally induced 3T3-L1 preadipocytes incubated in the presence of sc-58236 were prevented from reentering the cell cycle, as indicated by decreased cyclin E expression and BrdU incorporation (Fig. 2C, D). In all cases, no changes in cell density were observed, as determined by cell counting under light microscopy (data not shown). This is somehow surprising, because inhibition of proliferation should result in decreased cell number. However, we have consistently observed increased cell death upon triggering differentiation in the absence of COX inhibitors, which would compensate for the increased cell number as a result of clonal expansion. These results suggested that COX-2 inhibition restrains adipocyte differentiation by repressing the clonal expansion phase.

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Fig. 2. Inhibition of clonal expansion by COX-2 inhibitors. Analysis of cyclin E expression (A and C) or quantification of bromodeoxyuridine (BrdU) incorporation (B and D) by immunofluorescence in either proliferating 3T3-L1 cells (A and B) or cells induced to differentiate with the hormonal mix (C and D) in the absence or presence of 10-6 M of the COX-2 inhibitor. Cyclin E-expressing cells are labeled in green. Nuclei were visualized with Hoechst staining. At least 500 cells were quantified for BrdU incorporation studies. An asterisk indicates statistically significant differences (P < 0.05).
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COX-2 is not required during terminal adipocyte differentiation
In order to more precisely determine the mechanisms involved in the regulation of adipogenesis by COX-2, we added the COX-2 inhibitors either immediately before or 2 days after hormonal induction. Consistent with our previous results, 3T3-L1 cells, which were induced to differentiate in the presence of COX-2 inhibitors, were not able to differentiate (Fig. 3A)
, and consequently did not express the adipocyte-specific marker aP2 (Fig. 3B). In contrast, adipocyte differentiation and aP2 expression were not affected when COX-2 inhibitors were added after the clonal expansion phase (Fig. 3A, B). Furthermore, neither COX-1 inhibitors nor a combination of COX-1 and COX-2 inhibitors had any effect in the differentiation process when added after the clonal expansion phase (Fig. 3A, B). The number of PPAR - and BrdU-positive cells was consistent with the observed adipocyte phenotype (Fig. 3C). These results suggest that the role of COX-2 in adipogenesis is restricted to the control of the cell cycle in the early clonal expansion phase.

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Fig. 3. No effect of COX-2 inhibition in terminal adipocyte differentiation. A: Oil red O staining comparing lipid accumulation in 3T3-L1 cells induced to differentiate into adipocytes in the absence (mix) or presence of 10-6 M of sc-58236 (COX2i), added 24 h prior to addition of the differentiation mix (before mix), or in the presence of either sc-58236, sc-58560 (COX1i), or both together, added 48 h after the differentiation mix (after mix). B: Northern blot showing the expression of aP2 mRNA of 3T3-L1 cells used in A. Lane numbers correspond to the conditions used in A. C: Quantification of the number of BrdU-positive, PPAR -positive, and Oil red O-positive cells analyzed by immunofluorescence or light microscopy under the same conditions tested in A. At least 500 cells were counted in each of three independent experiments. Statistically significant differences (P < 0.05) are indicated by an asterisk.
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Mitogenic prostaglandins and PPARß/ agonists bypass the block imposed by COX-2 inhibitors on adipogenesis
Inhibition of COX-2 activity results in the depletion of prostaglandins in the cell. Consequently, addition of specific prostaglandins should bypass the block imposed by inhibition of COX-2 activity. To this end, 3T3-L1 cells were induced to differentiate with the regular hormonal mix in the absence or in the presence of the specific COX-2 inhibitor, sc-58236, and specific prostaglandins. Cells were then allowed to differentiate for 7 days. It is important to note that cells were cultured in the presence of prostaglandins only for 2 days, the time required for the clonal expansion phase. In this way, further effects of prostaglandins not related to clonal expansion were minimized. When immunofluorescence analysis was performed to quantify BrdU incorporation at the second day of differentiation, cells incubated without sc-58236 showed a substantial increase of BrdU-positive cells, which is consistent with the reentry of the cells into cell cycle (Fig. 4A, B)
. In contrast, a clear reduction of BrdU-positive cells was observed when COX-2 was inhibited (Fig. 4A, B). Addition of PGE2 and 6-keto PGF1 resulted in an increase of BrdU-positive cells similar to that observed when cells were cultured in the absence of COX-2 inhibitors (Fig. 4A, B). PGA2 and PGJ2, however, were unable to reverse the reduction in BrdU-positive cells upon COX-2 inhibition (Fig. 4A, B). Strikingly, only cells that reentered cell cycle, and thus underwent clonal expansion, were able to express PPAR , incorporate lipids, and consequently differentiate into adipocytes at day 7 (Fig. 4A, B). These results suggest that proliferative prostaglandins, such as PGE2 and the PGI2 derivative, 6-keto PGF1 , contribute to the clonal expansion phase of adipogenesis. 6-Keto PGF1 is a PGI2 metabolite, which is a PPARß/ activator, suggesting that the observed effects of this prostaglandin could be mediated at least in part by PPARß/ . Because expression of PPARß/ was not affected in COX-2-inhibited preadipocytes (data not shown), we evaluated the effects of PPARß/ activation in these cells. Interestingly, addition of the synthetic PPARß/ agonist L165041 promoted cell cycle reentry and was also able to bypass the block imposed by the COX-2 inhibitor in adipocyte differentiation (Fig. 4A, B).
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DISCUSSION
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Arachidonic acid, through the action of COX, is converted into prostaglandins, which are major regulators of cell growth, differentiation, and homeostasis [reviewed in ref. (3)]. In this study, we demonstrate that inhibition of COX-2 attenuates adipocyte differentiation, which is consistent with the previously reported adipogenic potential of some prostaglandins (17, 18). Our first hypothesis regarding participation of COX-2 in adipogenesis was that it facilitates the synthesis of prostaglandins, ligands of PPAR . Notably, PGJ2 has been demonstrated to promote adipocyte differentiation through activation of PPAR (19, 20). Therefore, inactivation of COX-2 could result in the depletion of PGJ2 in the cell. Consequently, PPAR would remain inactive, and cells would fail to differentiate. However, a more detailed analysis of the effects of COX-2 inhibition demonstrated that adipocyte differentiation was attenuated before PPAR was expressed. This suggested that other PPAR -independent mechanisms were causing the block in adipocyte differentiation imposed by selective COX-2 inhibition. An argument supporting this hypothesis was that inhibition of prostaglandin synthesis during terminal adipocyte differentiation, when the clonal expansion phase was completed, did not affect the adipogenic potential. Although PGJ2, as a PPAR ligand, has adipogenic potential, the fact that inhibition of COX-2 activity at later stages of adipogenesis had no major effect on terminal differentiation raises the question also of the importance of the participation of this prostaglandin in the adipocyte differentiation process. Supporting this idea is the fact that the PPAR ligand 15-deoxy PGJ2 remains undetectable in 3T3-L1 cells independent of the differentiation stage (data not shown). The previous report that the PGJ2 precursor, PGD2, is only present at very low levels in epididymal fat is consistent with a minor role of this prostaglandin, as well as its downstream products in adipose tissue biology (21).
Having excluded an eventual role of COX-2 as a supplier of PPAR ligands in terminal differentiation, we turned our attention to the eventual effects of COX-2 and prostaglandins in the early phases of adipocyte differentiation. Reentry of growth-arrested preadipocytes into cell cycle in response to hormonal stimulation is a prerequisite for adipocyte differentiation, because the inhibition of DNA synthesis at this stage results in the attenuation of differentiation (8). Our data clearly suggest that COX-2 inhibition abrogates clonal expansion, because most of the cells did not reenter the cell cycle, as measured by BrdU incorporation and cyclin E expression. Consistent with this, only prostaglandins, which exhibited a proliferative potential, were able to overcome the block in adipogenesis imposed by the COX-2 inhibitor. Of particular physiological relevance is the fact that both PGE2 and the metabolite of PGI2, 6-keto PGF1 (Fig. 4), which are the two major prostaglandins produced by adipose tissue and adipose precursor cells (9), could trigger reentry into cell cycle and therefore adipogenesis. PGI2 is a positive effector of adipocyte differentiation, exerting its effect through elevating cAMP levels in preadipocytes, activating the CREB/ATF pathway (22). This facilitates the proliferative response of the cell and therefore promotes clonal expansion (22).
Interestingly, PGI2 could be a positive effector of adipogenesis through regulating PPARß/ activity. Indeed, it has been previously shown that PGI2 activates this nuclear receptor (23). This is furthermore consistent with the reported positive role of PPARß/ during the clonal expansion phase of adipogenesis (24, 25) and the fact that in the present study the synthetic PPARß/ agonist L165041 overcame the effect of COX-2 inhibition on cell proliferation. Furthermore, significant amounts of 6-keto PGF1 are produced during adipocyte differentiation (Fig. 1E). These effects of PPARß/ activation on clonal expansion in adipocyte differentiation are furthermore in line with the proliferative effects of PPARß/ activation in other systems, such as colon (26). In the colon, PPARß/ was shown to be a target gene for the ß-catenin/Tcf-4 transcription complex, which is formed when the adenomatous polyposis coli tumor suppressor protein is mutated. In view of the coexpression of PPARß/ and COX-2 (23), it was proposed that PPARß/ can mediate the protumorigenic effects of prostaglandins in the colon, whereas NSAIDs were suggested to inhibit tumorigenesis because they inhibit PPARß/ activity (26). The lack of tumorigenicity of PPARß/ -/- human colorectal cells in nude mice supported a protumorigenic role of PPARß/ (27).
PGE2 plays also a clear role in differentiated adipocytes inhibiting lipolysis, whereas its role in preadipocytes is less studied. We show here that PGE2 stimulates proliferation of 3T3-L1 cells induced to differentiate, suggesting that PGE2 positively influences adipocyte differentiation through triggering the clonal expansion phase (Fig. 4). Participation of PGE2 in proliferative processes, such as liver regeneration after partial hepatectomy, has been previously documented (28). It has also been demonstrated that this effect is mediated through the PGE2 receptor EP1, which activates the cAMP pathway (29). Interestingly, preadipocytes express a higher level of EP1 receptor compared with mature adipocytes (30). We also tested the effect of other prostaglandins in adipogenesis. Consistent with previous studies, PGF2 inhibited the whole adipocyte differentiation process (11, 31), whereas no effect was observed for PGA2, PGD2, PGJ2, or TXB2 (Fig. 4 and data not shown).
In contrast to our results, a recent study documented a negative role of COX-1 and COX-2 in adipocyte differentiation (32). In this study, inhibition of COX activity resulted in a slight increase in adipogenesis. However, it is not clear in this study whether COX inhibitors were added before or after clonal expansion. Furthermore, COX-1 and -2 activity was not measured in this study at each particular time of the differentiation process. It is therefore not possible to know whether clonal expansion was abrogated.
Obesity is a major health problem in developed countries and is characterized by an increase in size of the adipocytes, as a result of lipid accumulation, and an increase in the number of adipocytes, as a result of accelerated adipocyte differentiation. COX-2-selective inhibitors are currently being used for the treatment of inflammatory diseases and are under clinical trials for the treatment of cancer (33). Interestingly, obesity has recently been suggested to be a low-grade systemic inflammatory disease (34). Therefore, inhibition of the inflammatory response may also attenuate the formation of fat. We present here evidence to support the idea that modulation of the activity of the COX-2 enzyme might open up new perspectives in the control of adipogenesis and consequently for the treatment of obesity and related metabolic diseases.
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ACKNOWLEDGMENTS
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This work was supported by grants from Centre Nationale de la Recherche Scientifique, Institut Nationale pour la Santé et la Recherche Médicale, Hopitaux Universitaires de Strasbourg, Pharmacia, the European Union RTD program, the Association de Recherche contre le Cancer, the Fondation pour la Recherche Médicale, and the National Institutes of Health (NIH).
Manuscript received June 10, 2003
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J. Biol. Chem.,
September 12, 2008;
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I. Tzameli, H. Fang, M. Ollero, H. Shi, J. K. Hamm, P. Kievit, A. N. Hollenberg, and J. S. Flier
Regulated Production of a Peroxisome Proliferator-activated Receptor-{gamma} Ligand during an Early Phase of Adipocyte Differentiation in 3T3-L1 Adipocytes
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