Selective cyclo-oxygenase-2 inhibitors impair adipocyte differentiation through inhibition of the clonal expansion phase

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 γ 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. 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 γ 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. 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 (1Coleman R.A. Smith W.L. Narumiya S. International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes.Pharmacol. Rev. 1994; 46: 205-229PubMed Google Scholar). 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. (2Marnett L.J. Kalgutkar A.S. Cyclooxygenase 2 inhibitors: discovery, selectivity and the future.Trends Pharmacol. Sci. 1999; 20: 465-469Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar)]. 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. (3Dubois R.N. Abramson S.B. Crofford L. Gupta R.A. Simon L.S. Van De Putte L.B. Lipsky P.E. Cyclooxygenase in biology and disease.FASEB J. 1998; 12: 1063-1073Crossref PubMed Scopus (2213) Google Scholar)]. Adipocyte differentiation is a complex and staged process (4Fajas L. Fruchart J.C. Auwerx J. Transcriptional control of adipogenesis.Curr. Opin. Cell Biol. 1998; 10: 165-173Crossref PubMed Scopus (251) Google Scholar). 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/EBPβ and δ (5Reusch J.E. Colton L.A. Klemm D.J. CREB activation induces adipogenesis in 3T3–L1 cells.Mol. Cell. Biol. 2000; 20: 1008-1020Crossref PubMed Scopus (259) Google Scholar, 6Aubert J. Saint-Marc P. Belmonte N. Dani C. Negrel R. Ailhaud G. Prostacyclin IP receptor up-regulates the early expression of C/EBPbeta and C/EBPdelta in preadipose cells.Mol. Cell. Endocrinol. 2000; 160: 149-156Crossref PubMed Scopus (52) Google Scholar), which then induce the expression of peroxisome proliferator-activated receptor γ (PPARγ). 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 PPARγ (7Fajas L. Landsberg R.L. Huss-Garcia Y. Sardet C. Lees J.A. Auwerx J. E2Fs regulate adipogenesis.Dev. Cell. 2002; 3: 39-49Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Both C/EBPs and PPARγ 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 (8Reichert M. Eick D. Analysis of cell cycle arrest in adipocyte differentiation.Oncogene. 1999; 18: 459-466Crossref PubMed Scopus (101) Google Scholar). 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 (8Reichert M. Eick D. Analysis of cell cycle arrest in adipocyte differentiation.Oncogene. 1999; 18: 459-466Crossref PubMed Scopus (101) Google Scholar). 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/EBPβ and δ in preadipose cells (6Aubert J. Saint-Marc P. Belmonte N. Dani C. Negrel R. Ailhaud G. Prostacyclin IP receptor up-regulates the early expression of C/EBPbeta and C/EBPdelta in preadipose cells.Mol. Cell. Endocrinol. 2000; 160: 149-156Crossref PubMed Scopus (52) Google Scholar). More controversial is the participation of prostaglandin E2 (PGE2). Whereas its antilypolytic effect has been established in differentiated adipocytes (9Richelsen B. Prostaglandins in adipose tissue–with special reference to triglyceride metabolism.Dan. Med. Bull. 1991; 38: 228-244PubMed Google Scholar), PGE2 has been reported to either activate (10Shillabeer G. Kumar V. Tibbo E. Lau D.C. Arachidonic acid metabolites of the lipoxygenase as well as the cyclooxygenase pathway may be involved in regulating preadipocyte differentiation.Metabolism. 1998; 47: 461-466Abstract Full Text PDF PubMed Scopus (35) Google Scholar) or inhibit (11Lepak N.M. Serrero G. Inhibition of adipose differentiation by 9 alpha, 11 beta-prostaglandin F2 alpha.Prostaglandins. 1993; 46: 511-517Crossref PubMed Scopus (25) Google Scholar) early adipogenesis. Finally, prostaglandin J2 (PGJ2) has a positive role in terminal adipocyte differentiation currently thought to be mediated by increasing the activity of PPARγ, a nuclear receptor that also can be activated by a number of synthetic compounds, such as the thiazolidinediones [reviewed in ref. (12Debril M.B. Renaud J.P. Fajas L. Auwerx J. The pleiotropic functions of peroxisome proliferator-activated receptor gamma.J. Mol. Med. 2001; 79: 30-47Crossref PubMed Scopus (211) Google Scholar)]. 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. 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. 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 (7Fajas L. Landsberg R.L. Huss-Garcia Y. Sardet C. Lees J.A. Auwerx J. E2Fs regulate adipogenesis.Dev. Cell. 2002; 3: 39-49Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). 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. SDS-PAGE and electrotransfer were performed as described (13Fajas L. Auboeuf D. Raspe E. Schoonjans K. Lefebvre A.M. Saladin R. Najib J. Laville M. Fruchart J.C. Deeb S. Vidal-Puig A. Flier J. Briggs M.R. Staels B. Vidal H. Auwerx J. Organization, promoter analysis and expression of the human PPARγ gene.J. Biol. Chem. 1997; 272: 18779-18789Abstract Full Text Full Text PDF PubMed Scopus (1077) Google Scholar). 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 (14Rocchi S. Picard F. Vamecq J. Gelman L. Potier N. Zeyer D. Dubuquoy L. Bac P. Champy M.F. Plunket K.D. Leesnitzer L.M. Blanchard S.G. Desreumaux P. Moras D. Renaud J.P. Auwerx J. A unique PPARgamma ligand with potent insulin-sensitizing yet weak adipogenic activity.Mol. Cell. 2001; 8: 737-747Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). PGE2 and 15-deoxy Δ PGJ2 levels were determined with the Enzyme Immunoassay Kit exactly as described by the manufacturer (Assay Designs, Inc., Ann Arbor, MI). 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 PPARγ (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 PGF1α, 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 PPARγ agonist 15-deoxy Δ 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 PGF1α 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 PPARγ 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 PPARγ 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 PPARγ activation, but was rather affecting any process prior to PPARγ expression. It has been previously reported that COX-2 actively participates in cell cycle progression and proliferation (15Sheng G.G. Shao J. Sheng H. Hooton E.B. Isakson P.C. Morrow J.D. Coffey Jr., R.J. DuBois R.N. Beauchamp R.D. A selective cyclooxygenase 2 inhibitor suppresses the growth of H-ras-transformed rat intestinal epithelial cells.Gastroenterology. 1997; Full Text PDF PubMed Scopus Google Scholar, H. Shao J. Isakson P. Coffey R.J. Morrow J. Beauchamp R.D. DuBois R.N. Inhibition of human cell growth by selective inhibition of 1997; PubMed Scopus Google Scholar). inhibition of COX-2 in cell cycle arrest (15Sheng G.G. Shao J. Sheng H. Hooton E.B. Isakson P.C. Morrow J.D. Coffey Jr., R.J. DuBois R.N. Beauchamp R.D. A selective cyclooxygenase 2 inhibitor suppresses the growth of H-ras-transformed rat intestinal epithelial cells.Gastroenterology. 1997; Full Text PDF PubMed Scopus Google Scholar, H. Shao J. Isakson P. Coffey R.J. Morrow J. Beauchamp R.D. DuBois R.N. Inhibition of human cell growth by selective inhibition of 1997; PubMed Scopus Google Scholar). Because inhibition of cell cycle reentry during clonal expansion adipocyte differentiation (8Reichert M. Eick D. Analysis of cell cycle arrest in adipocyte differentiation.Oncogene. 1999; 18: 459-466Crossref PubMed Scopus (101) Google Scholar), we that inhibition of COX-2 inhibit cell cycle progression during clonal expansion of adipocyte differentiation. this we first analyzed cell cycle progression in proliferating 3T3-L1 The number of cyclin (Fig. BrdU (Fig. and cells was decreased when the COX-2 inhibitor was as by immunofluorescence analysis, with cells incubated in the absence of COX-2 inhibitors (Fig. to proliferating induced 3T3-L1 preadipocytes incubated in the presence of sc-58236 were from the cell cycle, as indicated by decreased cyclin expression and BrdU (Fig. In all no in cell were as determined by cell (data not shown). This is inhibition of proliferation result in decreased cell we have observed increased cell differentiation in the absence of COX which for the increased cell number as a result of clonal suggested that COX-2 inhibition adipocyte differentiation by the clonal expansion phase. In to the involved in the of adipogenesis by we added the COX-2 inhibitors either before or 2 after hormonal Consistent with 3T3-L1 which were induced to in the presence of COX-2 were not to (Fig. and not the marker aP2 (Fig. In adipocyte differentiation and aP2 expression were not when COX-2 inhibitors were added after the clonal expansion phase (Fig. Furthermore, COX-1 inhibitors a combination of COX-1 and COX-2 inhibitors had any effect in the differentiation process when added after the clonal expansion phase (Fig. The number of and cells was with the observed adipocyte (Fig. that the role of COX-2 in adipogenesis is to the control of the cell cycle in the early clonal expansion phase. Inhibition of COX-2 activity in the of prostaglandins in the of specific prostaglandins the by inhibition of COX-2 this 3T3-L1 cells were induced to with the hormonal mix in the absence or in the presence of the specific COX-2 inhibitor, and specific prostaglandins. Cells were then to for days. It is important to that cells were in the presence of prostaglandins only for 2 the required for the clonal expansion phase. In this of prostaglandins not related to clonal expansion were immunofluorescence analysis was performed to BrdU at the day of differentiation, cells incubated sc-58236 a increase of which is with the reentry of the cells into cell cycle (Fig. In a of cells was observed when COX-2 was (Fig. of PGE2 and 6-keto PGF1α in an increase of cells similar to that observed when cells were in the absence of COX-2 inhibitors (Fig. and PGJ2, were to the in cells COX-2 inhibition (Fig. only cells that cell cycle, and clonal expansion, were to PPARγ, and into adipocytes at day (Fig. that proliferative prostaglandins, such as PGE2 and the PGI2 6-keto PGF1α, to the clonal expansion phase of adipogenesis. PGF1α is a PGI2 which is a that the observed of this prostaglandin could be mediated at in by Because expression of was not in preadipocytes (data not we the of activation in these cells. Interestingly, of the synthetic agonist L165041 cell cycle reentry and was also to the by the COX-2 inhibitor in adipocyte differentiation (Fig. Arachidonic acid, the of is converted into prostaglandins, which are of cell differentiation, and [reviewed in ref. (3Dubois R.N. Abramson S.B. Crofford L. Gupta R.A. Simon L.S. Van De Putte L.B. Lipsky P.E. Cyclooxygenase in biology and disease.FASEB J. 1998; 12: 1063-1073Crossref PubMed Scopus (2213) Google Scholar)]. In this we demonstrate that inhibition of COX-2 adipocyte differentiation, which is with the previously reported adipogenic potential of some prostaglandins R. D. Ailhaud G. Prostacyclin as a potent of J. PubMed Scopus Google Scholar, D. Negrel R. M. Ailhaud G. and role of arachidonic acid in the differentiation of J. PubMed Scopus Google Scholar). first participation of COX-2 in adipogenesis was that it the synthesis of prostaglandins, of PGJ2 has been demonstrated to adipocyte differentiation activation of PPARγ L.B. is a ligand for peroxisome proliferator-activated receptor γ Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, P. J. prostaglandin J2 is a ligand for the adipocyte Full Text PDF PubMed Scopus Google Scholar). Therefore, of COX-2 could result in the of PGJ2 in the PPARγ and cells to a analysis of the of COX-2 inhibition demonstrated that adipocyte differentiation was before PPARγ was This suggested that were the in adipocyte differentiation by selective COX-2 inhibition. this was that inhibition of prostaglandin synthesis during terminal adipocyte differentiation, when the clonal expansion phase was not the adipogenic PGJ2, as a PPARγ has adipogenic the that inhibition of COX-2 activity at stages of adipogenesis had no effect on terminal differentiation the also of the importance of the participation of this prostaglandin in the adipocyte differentiation process. this is the that the PPARγ ligand 15-deoxy Δ PGJ2 in 3T3-L1 cells independent of the differentiation stage (data not shown). The that the PGJ2 is only at levels in is with a role of this as well as its in adipose biology M. Y. N. H. K. and of its in of PubMed Scopus Google Scholar). an role of COX-2 as a of PPARγ in terminal differentiation, we to the of COX-2 and prostaglandins in the early phases of adipocyte differentiation. of preadipocytes into cell cycle in to hormonal is a for adipocyte differentiation, the inhibition of synthesis at this stage in the of differentiation (8Reichert M. Eick D. Analysis of cell cycle arrest in adipocyte differentiation.Oncogene. 1999; 18: 459-466Crossref PubMed Scopus (101) Google Scholar). that COX-2 inhibition clonal expansion, of the cells not the cell cycle, as by BrdU and cyclin expression. Consistent with only prostaglandins, which a proliferative were to overcome the in adipogenesis by the COX-2 inhibitor. particular physiological is the that both PGE2 and the of PGI2, 6-keto PGF1α (Fig. which are the prostaglandins by adipose and adipose cells (9Richelsen B. Prostaglandins in adipose tissue–with special reference to triglyceride metabolism.Dan. Med. Bull. 1991; 38: 228-244PubMed Google Scholar), could trigger reentry into cell cycle and therefore adipogenesis. PGI2 is a positive of adipocyte differentiation, its effect cAMP levels in the pathway N. F. P. B. Saint-Marc P. J. J. K. A. Narumiya S. Ailhaud G. Dani C. of and the expression of binding and in Endocrinol. 2001; Google Scholar). This the proliferative of the cell and therefore promotes clonal expansion N. F. P. B. Saint-Marc P. J. J. K. A. Narumiya S. Ailhaud G. Dani C. of and the expression of binding and in Endocrinol. 2001; Google Scholar). Interestingly, PGI2 could be a positive of adipogenesis regulating it has been previously shown that PGI2 this nuclear receptor R.A. J. DuBois R.N. activation of peroxisome proliferator-activated receptor in Sci. 2000; PubMed Scopus Google Scholar). This is with the reported positive role of during the clonal expansion phase of adipogenesis H. 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DuBois R.N. activation of peroxisome proliferator-activated receptor in Sci. 2000; PubMed Scopus Google Scholar), it was that can the of prostaglandins in the whereas were suggested to inhibit inhibit activity B. is an of nonsteroidal anti-inflammatory 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). The of of human cells in a role of B. of the of human Sci. 2001; PubMed Scopus Google Scholar). PGE2 plays also a role in differentiated adipocytes inhibiting whereas its role in preadipocytes is We here that PGE2 proliferation of 3T3-L1 cells induced to that PGE2 positively adipocyte differentiation the clonal expansion phase (Fig. of PGE2 in proliferative processes, such as after has been previously L.J. Prostaglandins are required for CREB activation and proliferation during Sci. 2001; PubMed Scopus Google Scholar). It has also been demonstrated that this effect is mediated the PGE2 receptor which the cAMP pathway M. S. M. receptor synthesis and proliferation in of rat the of 2001; PubMed Scopus Google Scholar). Interestingly, preadipocytes a of receptor with adipocytes J.D. S.B. Ailhaud G. Negrel R. B. expression of prostaglandin receptor during adipose cell 1999; PubMed Scopus Google Scholar). We also the effect of prostaglandins in adipogenesis. Consistent with the adipocyte differentiation process (11Lepak N.M. Serrero G. Inhibition of adipose differentiation by 9 alpha, 11 beta-prostaglandin F2 alpha.Prostaglandins. 1993; 46: 511-517Crossref PubMed Scopus (25) Google Scholar, Prostaglandins and adipogenesis on peroxisome proliferator-activated receptor gamma.J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus (259) Google Scholar), whereas no effect was observed for PGJ2, or (Fig. 4 and not shown). In contrast to a study a role of COX-1 and COX-2 in adipocyte differentiation H. A. M. Lau D.C. of COX-1 and COX-2 in adipogenesis in 3T3–L1 Full Text Full Text PDF PubMed Scopus Google Scholar). In this inhibition of COX activity in a slight increase in adipogenesis. it is not in this study COX inhibitors were added before or after clonal Furthermore, COX-1 and activity was not in this study at particular of the differentiation process. It is therefore not to clonal expansion was is a problem in developed and is characterized by an increase in of the as a result of and an increase in the number of as a result of adipocyte differentiation. inhibitors are currently used for the treatment of inflammatory diseases and are for the treatment of K. A.M. a for the and treatment of 2001; 8: PubMed Scopus Google Scholar). Interestingly, obesity has been suggested to be a inflammatory obesity an inflammatory 2001; PubMed Scopus Google Scholar). Therefore, inhibition of the inflammatory may also the of We here to the that of the activity of the COX-2 enzyme might open up new perspectives in the control of adipogenesis and for the treatment of obesity and related metabolic diseases. This was by from the Union the the and the of