Role of cyclooxygenases COX-1 and COX-2 in modulating adipogenesis in 3T3-L1 cells

Cyclooxygenase (COX) catalyses the rate-limiting step of prostanoid biosynthesis. Two COX isoforms have been identified, COX-1, the constitutive form, and COX-2, the inducible form. While COX-2 has been implicated in body fat regulation, the underlying cellular mechanism remains to be elucidated. The present study was undertaken to examine the potential role of COX in modulating adipogenesis and to dissect the relative contribution of the two isoenzymes in this process. COX-2 was found to be expressed in undifferentiated 3T3-L1 cells and down-regulated during differentiation, whereas the cellular level of COX-1 remained relatively constant. Abrogating the activity of either of these two isoenzymes by selective COX inhibitors accelerated cellular differentiation, suggesting that both COX isoenzymes negatively influenced differentiation. Tumor necrosis factor-α (TNFα) significantly up-regulated COX-2 expression (∼2-fold) in differentiating 3T3-L1 cells, whereas similar effect was not observed with COX-1 expression. Abrogating the induced COX-2 activity reversed the TNFα-induced inhibition of differentiation by ∼70%, implying a role for COX-2 in mediating TNFα signaling.Hence, both COX isoforms were involved in the negative modulation of adipocyte differentiation. COX-2 appeared to be the main isoform mediating at least part of the negative effects of TNFα. Cyclooxygenase (COX) catalyses the rate-limiting step of prostanoid biosynthesis. Two COX isoforms have been identified, COX-1, the constitutive form, and COX-2, the inducible form. While COX-2 has been implicated in body fat regulation, the underlying cellular mechanism remains to be elucidated. The present study was undertaken to examine the potential role of COX in modulating adipogenesis and to dissect the relative contribution of the two isoenzymes in this process. COX-2 was found to be expressed in undifferentiated 3T3-L1 cells and down-regulated during differentiation, whereas the cellular level of COX-1 remained relatively constant. Abrogating the activity of either of these two isoenzymes by selective COX inhibitors accelerated cellular differentiation, suggesting that both COX isoenzymes negatively influenced differentiation. Tumor necrosis factor-α (TNFα) significantly up-regulated COX-2 expression (∼2-fold) in differentiating 3T3-L1 cells, whereas similar effect was not observed with COX-1 expression. Abrogating the induced COX-2 activity reversed the TNFα-induced inhibition of differentiation by ∼70%, implying a role for COX-2 in mediating TNFα signaling. Hence, both COX isoforms were involved in the negative modulation of adipocyte differentiation. COX-2 appeared to be the main isoform mediating at least part of the negative effects of TNFα. Cyclooxygenase (COX) catalyzes the rate-limiting step of prostanoid biosynthesis and controls the first committed step of prostanoid formation. The availability of this enzyme directly relates to the abundance of prostaglandin (PG) production. Two COX isoforms have been identified, COX-1, the constitutive form, and COX-2, the inducible form. While the former is believed to be responsible for homeostasis, the latter is thought to exert actions mainly in pathological states (1Smith W.L. Dewitt D.L. Prostaglandin endoperoxide H synthases-1 and -2.Adv. Immunol. 1996; 62: 167-215Google Scholar, 2Smith W.L. DeWitt D.L. Garavito R.M. Cyclooxygenases: structural, cellular, and molecular biology.Annu. Rev. Biochem. 2000; 69: 145-182Google Scholar). The divergent function of these two COX isoforms may be attributable to the availability of the enzyme under particular settings or the functional coupling with different downstream terminal PG synthases (3Smith W.L. Langenbach R. Why there are two cyclooxygenase isozymes.J. Clin. Invest. 2001; 107: 1491-1495Google Scholar, 4Ueno N. Murakami M. Tanioka T. Fujimori K. Urade Y. Kudo I. Coupling between cyclooxygenases, terminal prostanoid synthases and phospholipase A{sub2}s.J. Biol. Chem. 2001; 276: 34918-34927Google Scholar). A recent study suggested that COX-2 might be involved in body fat regulation (5Fain J.N. Ballou L.R. Bahouth S.W. Obesity is induced in mice heterozygous for cyclooxygenase-2.Prostaglandins Other Lipid Mediat. 2001; 65: 199-209Google Scholar). Mice heterozygous for the COX-2 gene showed increased body weight by about 30%, with fat pads enlarged 2–3-fold when compared with those from the wild-type animals. In comparison, mice lacking COX-1 gene appeared normal in phenotype and body fat content. These findings suggested that COX-2 might be involved in body fat regulation in vivo, whereas the role of COX-1 remains obscure. The underlying cellular mechanism for the COX-2 effect remains to be elucidated. Adipogenesis is a crucial aspect in controlling body fat mass. The acquisition of the mature adipocyte phenotype is a highly regulated process in which preadipocytes undergo differentiation resulting in both increased size and number of mature adipocytes in the adipose tissue. Our previous study showed that COX pathway might be involved in regulating this process (6Shillabeer 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: 461-466Google Scholar). The present study was undertaken to further elucidate the underlying cellular mechanisms. 3T3-L1 cells were used as the model system and a pharmacological inhibition approach using highly selective COX inhibitors was employed to dissect the relative contributions of the two COX isoenzymes. The role of COXs under stimulation of tumor necrosis factor-α (TNFα), an adipokine abundantly produced by adipocytes and a potent negative regulator of adipogenesis, was also explored. Dulbecco’s modified Eagle’s medium (DMEM), calf serum, and fetal bovine serum were purchased from Gibco BRL (Burlington, ON). Insulin, 1-methyl-3-isobutylxanthine (MIX), and dexamethasone (Dex) were obtained from Sigma Chemical Co. (St. Louis, MO). Rosiglitazone was purchased from SmithKline Beecham Pharma (Oakville, Canada). The selective COX-2 inhibitor celecoxib was a kind gift from Dr. Ching-Shih Chen (University of Kentucky). The other specific COX-2 inhibitor, NS-398, and COX-1 inhibitor, SC-560, were purchased from Cayman Chemicals (Ann Arbor, MI). Rabbit adipocyte fatty acid-binding protein (aP2) antiserum was obtained from Alpha Diagnostic International, Inc. (San Antonio, TX). Goat polyclonal antibodies against COX-1, COX-2, glucose transporter-4 (GLUT4), rabbit polyclonal antibodies against peroxisome-proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (c/EPBα), the blocking peptide for COX-1, and the horseradish peroxidase (HRP) conjugated secondary antibodies (anti-goat IgG and anti-rabbit IgG) were products of Santa Cruz Biotechnology (Santa Cruz, CA). 3T3-L1 cells were obtained from American Type Culture Collection (Rockville, MD). Cells were maintained in DMEM containing 10% calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. Differentiation induction was performed at 2 days post-confluence [day 0 of differentiation (DD0)] in DMEM supplemented with 10% fetal bovine serum. For standard induction of differentiation, cells were exposed to 1.7 μM insulin, 0.5 mM MIX, and 1 μM dexamethasone for 2 days, followed by 0.4 μM insulin and 1 μM dexamethasone for another 2 days. To avoid the potential interference of dexamethasone (Dex) on COX expression, a modified differentiation cocktail was used in most experiments, in which Dex was replaced by 2 μM of rosiglitazone in the hormonal cocktail described earlier. By using this protocol, about 60–70% of cells underwent differentiation by day 4 of differentiation. In order to accentuate the COX effect on differentiation, cells were differentiated in one set of experiments under conditions that would induce partial maturation (40% vs. ∼95%): 0.17 μM insulin, 0.5 mM MIX, and 0.1 μM Dex for 2 days, followed by 0.04 μM insulin and 0.1 μM Dex for another 2 days. Following different treatments as indicated, cells were washed thrice with phosphate-buffered saline (PBS), harvested in 10 mM Tris-EDTA buffer (pH 7.4), and sonicated. Following centrifugation at 100,000 g for 10 min at 4°C, the supernatant was collected. Protein content was determined by the Bradford method (Bio-Rad Laboratories, Inc., Mississauga, ON), and glycerol-3-phosphate dehydrogenase (GPDH) activity was quantified according to the method of Kozak and Jensen (7Kozak L.P. Jensen J.T. Genetic and developmental control of multiple forms of L-glycerol 3-phosphate dehydrogenase.J. Biol. Chem. 1974; 249: 7775-7781Google Scholar). One unit of specific enzyme activity corresponded to the oxidation of 1 nmol of NADH/min/mg protein. Cells were rinsed thrice with PBS, and scraped into lysis buffer [125 mM NaCl, 2 mM EDTA, 50 mM HEPES (pH 7.4), 1% Triton X-100, 1 mM DTT] supplemented with pepstatin (5 μg/ml), leupeptin (5 μg/ml), and phenylmethylsulfonyl fluoride (1 mM). After centrifugation at 12,000 g for 15 min at 4°C, the soluble fraction was collected, and protein content was determined. Twenty micrograms of total protein was separated by SDS-PAGE (10–12% gel) and electroblotted onto polyvinylidene difluoride (PVDF) membranes (Bio-Rad Laboratories, Inc.) Pausau S staining was performed after transfer to confirm sample loading and transfer efficiency. After blocking with 5% skimmed milk [in TBS containing 0.05% Tween 20 (TBST)] for 30–60 min at room temperature (RT), the membrane was incubated with the primary antibody at the appropriate dilution (goat anti-COX-2, 1:2,000; goat anti-COX-1 and anti-GLUT4, 1:1,000; rabbit anti-PPARγ and anti-c/EBPα, 1:1,000; rabbit antiserum against aP2, 1:2,000) at 4°C overnight or at RT for 1 h. After washing thrice with TBST, the membrane was probed with HRP conjugated secondary antibody (anti-goat or anti-rabbit IgG at 1:2,000 for COX-2 and aP2, and 1:1,000 for COX-1, PPARγ, c/EBPα, and GLUT4) at RT for 1 h. The membrane was then washed thrice with TBST, and signal was visualized by enhanced chemiluminescence (Amersham, Buckinghamshire). After exposure to Kodak X-OMAT AR film, the immunoblot exposures were scanned, and bands were quantified using NIH Image 1.55. Quantitative data were expressed as means ± SD from at least three independent experiments, and analyzed by the two-tailed Student’s t-test. The expression of COX-1 and COX-2 during differentiation was determined by immunoblotting. COX-2 was found to be expressed in undifferentiated 3T3-L1 cells, and the expression was down-regulated during differentiation. COX-1 expression, in comparison, remained relatively stable over the 7-day period (Fig. 1A). As Dex, a key ingredient in the standard differentiation cocktail, has been shown to down-regulate COX-2 expression by affecting its mRNA stability (8Lasa M. Brook M. Saklatvala J. Clark A.R. Dexamethasone destabilizes cyclooxygenase 2 mRNA by inhibiting mitogen-activated protein kinase p38.Mol. Cell. Biol. 2001; 21: 771-780Google Scholar), we examined the direct effect of Dex on COX-2 expression in 3T3-L1 cells. COX-2 protein expression was unaltered up to 5 days following Dex treatment (Fig. 1B). To further ascertain the notion that decreased COX-2 was a differentiation-dependent event, a modified Dex-free hormonal cocktail containing rosiglitazone was used to induce differentiation. Similar expression patterns of COXs were observed under this condition (Fig. 1C). Both COX-1 and COX-2 inhibitors were found to enhance differentiation, as suggested by the augmented GPDH-specific activity (Fig. 2A). A more profound effect was observed when cells were induced to undergo partial differentiation, as indicated by the enhanced expression of PPARγ2, c/EBPα, aP2, and GLUT4 (Fig. 2B), as well as increased GPDH-specific activity (data not shown). Adipogenesis is subject to both positive and negative regulation in vivo (9MacDougald O.A. Lane M.D. Transcriptional regulation of gene expression during adipocyte differentiation.Annu. Rev. Biochem. 1995; 64: 345-373Google Scholar, 10Smas C.M. Sul H.S. Control of adipocyte differentiation.Biochem. J. 1995; 309: 697-710Google Scholar, 11Gregoire F.M. Smas C.M. Sul H.S. Understanding adipocyte differentiation.Physiol. Rev. 1998; 78: 783-809Scopus (1843) Google Scholar). While COX-1 and COX-2 both exerted an inhibitory effect on differentiation in the presence of positive adipogenic stimuli, the next question was whether they functioned in concert in response to negative signals. To this end, we examined the role of these two COX isoenzymes in the presence of TNFα, a potent negative regulator of adipogenesis. TNFα was found to up-regulate COX-2 expression in a dose- and time-dependent fashion, with the earliest induction seen at 6 h and a maximum induction of up to 2-fold (Fig. 3). No induction was observed with COX-1 expression (Fig. 3A). To further examine whether the induced COX-2 activity mediated the TNFα signaling, specific COX-2 inhibitors were employed. Abrogating the induced COX-2 activity by specific COX-2 inhibitors reversed the TNFα-induced differentiation inhibition by ∼70%, as assessed by GPDH-specific activity (Fig. 4A)as well as aP2 and GLUT4 expression (Fig. 4B).Fig. 4COX-2 inhibitors partly reversed TNFα-induced differentiation inhibition. 3T3-L1 cells were induced to undergo differentiation by the modified hormonal cocktail (Dex free). TNFα (125 pM) was concomitantly added to the differentiation medium in the presence or absence of the specific COX-2 inhibitors (0.125 μM celecoxib or 0.1 μM NS-398). Cells were harvested at differentiation day 8. GPDH-specific activity was quantified (A) and protein expression of aP2 and GLUT4 was determined by immunoblotting (B). Quantitative data represent the mean ± SD of three experiments. Double asterisks denote P < 0.01 versus TNFα treated cells.View Large Image Figure ViewerDownload (PPT) Prostaglandins are a class of lipid mediators comprised of PGE2, PGI2, PGD2, PGF2α, and their metabolites. COX catalyzes the first committed step of PG biosynthesis and plays a central role in PG production (12Smith W.L. Marnett L.J. Prostaglandin endoperoxide synthase: structure and catalysis.Biochim. Biophys. Acta. 1991; 1083: 1-17Google Scholar). Two COX isoforms have been identified, COX-1, the constitutive form, and COX-2, the inducible form (1Smith W.L. Dewitt D.L. Prostaglandin endoperoxide H synthases-1 and -2.Adv. Immunol. 1996; 62: 167-215Google Scholar, 2Smith W.L. DeWitt D.L. Garavito R.M. Cyclooxygenases: structural, cellular, and molecular biology.Annu. Rev. Biochem. 2000; 69: 145-182Google Scholar). The two isoforms are encoded by different genes located at different chromosomes and appear to exert different actions in the organism (13Langenbach R. Loftin C. Lee C. Tiano H. Cyclooxygenase knockout mice: models for elucidating isoform-specific functions.Biochem. Pharmacol. 1999; 58: 1237-1246Google Scholar). Our earlier work and that of others (6Shillabeer 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: 461-466Google Scholar, 14Mater M.K. Pan D. Bergen W.G. Jump D.B. Arachidonic acid inhibits lipogenic gene expression in 3T3–L1 adipocytes through a prostanoid pathway.J. Lipid Res. 1998; 39: 1327-1334Google Scholar, 15Casimir D.A. Miller C.W. Ntambi J.M. Preadipocyte differentiation blocked by prostaglandin stimulation of prostanoid FP2 receptor in murine 3T3–L1 cells.Differentiation. 1996; 60: 203-210Google Scholar) have demonstrated that the COX pathway is involved in the regulation of adipogenesis. However, the relative contributions of the two COX isoenzymes to the differentiation program as well as the underlying mechanisms remain to be elucidated. In the present study we mapped the COX protein expression profile during differentiation in 3T3-L1 cells. Both COX-1 and COX-2 proteins were detectable in undifferentiated 3T3-L1 cells. COX-1 protein level remained unchanged, whereas COX-2 expression was down-regulated during differentiation. The decrement in COX-2 expression appeared to be a differentiation-dependent event based on two separate observations. First, cells differentiated by different induction cocktails, in the absence or presence of Dex, showed similar expression patterns. Second, Dex did not exert direct influence on COX-2 protein expression in 3T3-L1 cells. Thus, COX-2 appeared to the COX isoform regulated during differentiation. It is known that COX-2 expression is highly regulated at transcription level, mainly via the NF-κB pathway (2Smith W.L. DeWitt D.L. Garavito R.M. Cyclooxygenases: structural, cellular, and molecular biology.Annu. Rev. Biochem. 2000; 69: 145-182Google Scholar). Recent studies showed that the NF-κB pathway is suppressed by PPARγ (16Setoguchi K. Misaki Y. Terauchi Y. Yamauchi T. Kawahata K. Kadowaki T. Yamamoto K. Peroxisome proliferator-activated receptor-gamma haploinsufficiency enhances B cell proliferative responses and exacerbates experimentally induced arthritis.J. Clin. Invest. 2001; 108: 1667-1675Google Scholar), the key regulator of adipocyte differentiation. It is thus plausible that the constitutive activation of PPARγ during differentiation, possibly via suppression of the NF-κB pathway, down-regulates COX-2 expression. This cellular mechanism may explain how COX-2 expression is down-regulated during differentiation and warrants further investigation. To our knowledge, this is the first report that has examined COX protein expression, coupled with functional studies, during differentiation. A previous study has reported COX transcription profile in OB177A cells, but functional studies were not carried out (17Borglum J.D. Richelsen B. Darimont C. Pedersen S.B. Negrel R. Expression of the two isoforms of prostaglandin endoperoxide synthase (PGHS-1 and PGHS-2) during adipose cell differentiation.Mol. Cell. Endocrinol. 1997; 131: 67-77Google Scholar). The detection of COX-2 in 3T3-L1 cells in the basal state was somewhat unexpected, as COX-2 is generally considered to be the inducible form of COX, present mainly in the stimulated state. However, recent studies suggest that this isoform may indeed be present under unstimulated conditions in such tissues as brain (18Yamagata K. Andreasson K.I. Kaufmann W.E. Barnes C.A. Worley P.F. Expression of a mitogen-inducible cyclooxygenase in brain regulation by activity and Scholar), Y. M.D. is with the of and with Clin. Invest. Scholar), and M. E. R. C. expression of prostaglandin endoperoxide but not in an cells in 1996; Scholar). As COX-1 and COX-2 showed expression patterns during differentiation (Fig. one would that they might exert different effects on adipose cell differentiation. However, blocking the activity of either of the two COX isoforms with specific inhibitors similar Differentiation was augmented in both suggesting that both COX-1 and COX-2 were involved in the negative modulation of differentiation. the on differentiation was under both conditions (Fig. suggesting that both COX-1 and COX-2 to a similar preadipocyte differentiation. As both isoforms negatively influenced the for differentiation, would appear that downstream products by the COX pathway would exert a positive effect on adipose cell differentiation. Hence, our findings the question of the of a downstream prostanoid of the COX pathway to be an of PPARγ and a potent of differentiation J.M. I. D.C. J.M. A prostaglandin proliferator-activated receptor and adipocyte 1995; Scholar). The that of has been in vivo further for the potential role of COX-1 and COX-2 as negative of adipose cell differentiation. The different expression patterns of COX-1 and COX-2 during differentiation suggested that these isoforms might different in adipose cell While both COX isoenzymes appeared to function in the presence of positive adipogenic stimuli, the that they might in response to negative signals. used TNFα, a negative regulator of adipogenesis, in our next of experiments to this TNFα is a produced by adipocytes and is present in abundance in the states expression of tumor necrosis direct role in insulin Scholar). It is known to adipocyte The role of in adipocyte Biol. 1999; Scholar) and has also been shown to be a potent for COX-2 but not for the constitutive COX-1 Chen expression in of the phospholipase protein kinase and B kinase pathway.J. Immunol. 2000; Scholar). It is thus that COX-2, but not COX-1, might function as a in the presence of negative such as TNFα. our experiments demonstrated that COX-2 was up-regulated by TNFα in differentiating 3T3-L1 cells, whereas a similar effect was not observed with COX-1 expression. Abrogating the induced COX-2 activity by specific COX-2 inhibitors reversed the TNFα-induced inhibition of differentiation by about suggesting that COX-2 was the mediating TNFα A similar modulating effect of COX-2 has also been reported in T. H. M. M. K. J. T. Y. Y. regulation of fat cell in via and Clin. Invest. Scholar). Our present findings are in with the reported in knockout mice in the COX Mice heterozygous for the COX-2 gene were found to be (5Fain J.N. Ballou L.R. Bahouth S.W. Obesity is induced in mice heterozygous for cyclooxygenase-2.Prostaglandins Other Lipid Mediat. 2001; 65: 199-209Google Scholar), with more body fat the wild-type animals. The phenotype for COX-1 knockout mice was to the with in body fat content. Hence, the responses of the two COX isoenzymes to negative may explain the different in between the COX-2 and COX-1 knockout animals. In both COX-1 and COX-2 negatively influenced adipose cell differentiation. COX-2 was the isoform involved in mediating of the TNFα negative effects on adipogenesis. It be that COX-2 may a more role in body fat regulation in of the of this and the underlying mechanisms regulating COX-2 may into the control of and total body fat and potential for the treatment of This study was by a from the and of was by the of and by a from the The Dr. J. for and on this The of and is adipocyte fatty acid-binding protein CCAAT/enhancer-binding protein α cyclooxygenase day of differentiation dexamethasone modified medium glucose transporter-4 glycerol-3-phosphate dehydrogenase horseradish peroxidase 1-methyl-3-isobutylxanthine prostaglandin peroxisome-proliferator-activated receptor γ 2 tumor necrosis factor-α

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