Portal de Periódicos da CAPES

The Peroxisome Proliferator-activated Receptor-γ Regulates Murine Pyruvate Carboxylase Gene Expression in Vivo and in Vitro

2005; Elsevier BV; Volume: 280; Issue: 29 Linguagem: Inglês

10.1074/jbc.m503836200

1083-351X

Sarawut Jitrapakdee, Marc Slawik, Gema Medina‐Gómez, Mark Campbell, John C. Wallace, Jaswinder K. Sethi, Stephen O’Rahilly, Antonio Vidal‐Puig,

Metabolism, Diabetes, and Cancer

Pyruvate carboxylase (PC) plays a crucial role in various metabolic pathways, including gluconeogenesis, lipogenesis, and glucose-induced insulin secretion. Here we showed for the first time that the PC gene is transcriptionally regulated by peroxisome proliferator-activated receptor-γ (PPARγ) in vitro and in vivo in white and brown adipose tissue. PC mRNA and protein are markedly increased during differentiation of 3T3-L1 cells and HIB-1B, in parallel with the expression of the adipogenic transcription factors, CCAAT-enhancer binding protein α, PPARγ1, and PPARγ2. Tumor necrosis factor-α, a cytokine that blocks differentiation of 3T3-L1 cells, suppressed PC expression. Co-transfection studies in 3T3-L1 preadipocytes or HEK293T cells with a 2.3-kb promoter fragment of mouse PC gene linked to a luciferase reporter construct and with plasmids overexpressing retinoid X receptor α/PPARγ1 or retinoid X receptor α/PPARγ2 showed a 6-8-fold increase above the basal promoter activity. Furthermore, treatment of these transfected cells with the PPARγ agonist doubled the promoter activity. Mutation of the putative PPAR-response element-(-386/-374) of this 2.3-kb PC promoter fragment abolished the PPARγ response. Gel shift and chromatin immunoprecipitation assays demonstrated that endogenous PPARγ binds to this functional PPAR-response element of the PC promoter. Mice with targeted disruption of the PPARγ2 gene displayed ∼50-60% reduction of PC mRNA and protein in white adipose tissue. Similarly, in brown adipose tissue of PPARγ2-deficient mice subjected to cold exposure, PC mRNA was 40% lower than that of wild type mice. Impaired in vitro differentiation of white adipocytes of PPARγ2 knock-out mice was also associated with a marked reduction of PC mRNA. Our findings identified PC as a PPARγ-regulated gene and suggested a role for PPARγ regulating intermediary metabolism. Pyruvate carboxylase (PC) plays a crucial role in various metabolic pathways, including gluconeogenesis, lipogenesis, and glucose-induced insulin secretion. Here we showed for the first time that the PC gene is transcriptionally regulated by peroxisome proliferator-activated receptor-γ (PPARγ) in vitro and in vivo in white and brown adipose tissue. PC mRNA and protein are markedly increased during differentiation of 3T3-L1 cells and HIB-1B, in parallel with the expression of the adipogenic transcription factors, CCAAT-enhancer binding protein α, PPARγ1, and PPARγ2. Tumor necrosis factor-α, a cytokine that blocks differentiation of 3T3-L1 cells, suppressed PC expression. Co-transfection studies in 3T3-L1 preadipocytes or HEK293T cells with a 2.3-kb promoter fragment of mouse PC gene linked to a luciferase reporter construct and with plasmids overexpressing retinoid X receptor α/PPARγ1 or retinoid X receptor α/PPARγ2 showed a 6-8-fold increase above the basal promoter activity. Furthermore, treatment of these transfected cells with the PPARγ agonist doubled the promoter activity. Mutation of the putative PPAR-response element-(-386/-374) of this 2.3-kb PC promoter fragment abolished the PPARγ response. Gel shift and chromatin immunoprecipitation assays demonstrated that endogenous PPARγ binds to this functional PPAR-response element of the PC promoter. Mice with targeted disruption of the PPARγ2 gene displayed ∼50-60% reduction of PC mRNA and protein in white adipose tissue. Similarly, in brown adipose tissue of PPARγ2-deficient mice subjected to cold exposure, PC mRNA was 40% lower than that of wild type mice. Impaired in vitro differentiation of white adipocytes of PPARγ2 knock-out mice was also associated with a marked reduction of PC mRNA. Our findings identified PC as a PPARγ-regulated gene and suggested a role for PPARγ regulating intermediary metabolism. Adipose tissue not only plays a crucial role in lipid metabolism by storing circulating free fatty acids as triglycerides, but it is also a site where de novo fatty acid synthesis occurs (1Mackall J.C. Student A.K. Polakis S.E. Lane M.D. J. Biol. Chem. 1976; 251: 6462-6464Abstract Full Text PDF PubMed Google Scholar). The differentiation of preadipocytes to mature adipocytes is a complex process involving biochemical and morphological changes. These changes are associated with the sequential activation of pro-adipogenic transcription factors, including the CCAAT-enhancer binding protein family, C/EBPβ/δ, 1The abbreviations used are: C/EBPα, CCAAT-enhancer binding protein α; PC, pyruvate carboxylase; PPARγ, peroxisome proliferator activated receptor-γ; TNFα, tumor necrosis factor-α; RXRα, retinoid X receptor-α; PPRE, PPAR-response element; EMSA, electrophoretic mobility shift assay; ChIP, chromatin immunoprecipitation; mPC, murine PC; E2F, elongation factor 2F. and ADD1/SREBP1 followed by C/EBPα and peroxisome proliferator-activated receptor-γ (PPARγ) expression. This transcriptional activation cascade in turn switches on several lipogenic genes, resulting in preadipocyte growth arrest and development of mature adipocytes (2Cowherd R.M. Lyle R.E. McGehee Jr., R.E. Semin. Cell. Dev. Biol. 1999; 10: 3-10Crossref PubMed Scopus (240) Google Scholar, 3Rosen E.D. Walkey C.J. Puigserver P. Spiegelman B.M. Genes Dev. 2000; 14: 1293-1307Crossref PubMed Google Scholar, 4Rosen E.D. Spiegelman B.M. Annu. Rev. Cell Dev. Biol. 2000; 16: 145-171Crossref PubMed Scopus (1061) Google Scholar). PPARγ, a member of the PPAR subfamily of nuclear hormone receptors, heterodimerizes with the retinoid X receptor α (RXRα) and regulates the expression of pro-adipogenic genes (3Rosen E.D. Walkey C.J. Puigserver P. Spiegelman B.M. Genes Dev. 2000; 14: 1293-1307Crossref PubMed Google Scholar). Binding of PPARγ-RXRα to the PPAR-response element (PPRE) of the promoter recruits co-activators and releases corepressors that in turn results in transcriptional activation (5Rosen E.D. Spiegelman B.M. J. Biol. Chem. 2001; 276: 37731-37734Abstract Full Text Full Text PDF PubMed Scopus (1087) Google Scholar). PPARγ consists of two isoforms, PPARγ1 and PPARγ2, the latter of which contains 30 additional amino acids at its N terminus as the result of alternative splicing at the 5′-end of the gene (6Fajas L. Auboef D. Raspe E. Schoonjans K. Lefebvre A.M. Salaadin R. Najlb J. Laville M. Fruchart J.C. Deeb S. Vidal-Puig A. Flier J. Briggs M.R. Staels B. Vidal H. Auwerx J. J. Biol. Chem. 1997; 272: 18779-18789Abstract Full Text Full Text PDF PubMed Scopus (1089) Google Scholar, 7Zhu Y. Qi C. Korenberg J.R. Chen X.N. Noya D. Rao M.S. Reddy J.K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7921-7925Crossref PubMed Scopus (606) Google Scholar). Although many tissues express PPARγ1 at low levels, PPARγ2 is restricted to white and brown adipose tissue (8Braissant O. Foufelle F. Scotto C. Dauca M. Wahli W. Endocrinology. 1996; 137: 354-366Crossref PubMed Scopus (1737) Google Scholar), where each isoform represents 50% of the total PPARγ. Pyruvate carboxylase (PC), a member of the biotin-containing enzyme family, catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate (9Jitrapakdee S. Wallace J.C. Biochem. J. 1999; 340: 1-16Crossref PubMed Scopus (191) Google Scholar). The level of PC is highest in gluconeogenic tissues, i.e. kidney cortex and liver (10Jitrapakdee S. Walker M.E. Wallace J.C. Biochem. Biophys. Res. Commun. 1996; 223: 695-700Crossref PubMed Scopus (27) Google Scholar, 11Wexler I.D. Du Y. Lisgaris M.V. Manda L.S.K. Freytag S.O. Yang B.S. Liu T.C. Kwon M. Patel M.S. Kerr D.S. Biochim. Biophys. Acta. 1994; 1227: 46-52Crossref PubMed Scopus (46) Google Scholar), but PC activity, protein, and mRNA are also highly expressed in adipose tissue as well as in differentiated adipocytes (12Mackall J.C. Lane M.D. Biochem. Biophys. Res. Commun. 1977; 79: 720-725Crossref PubMed Scopus (59) Google Scholar, 13Freytag S.O. Utter M.F. J. Biol. Chem. 1983; 258: 6307-6312Abstract Full Text PDF PubMed Google Scholar, 14Lynch C.J. McCall K.M. Billingsley M.L. Bohlen L.M. Hreniuk S.P. Martin L.F. Witters L.A. Vannucci S.J. Am. J. Physiol. 1992; 262: E608-E618PubMed Google Scholar, 15Zhang J. Xia W.-L. Ahmad F. Biochem. J. 1995; 306: 205-210Crossref PubMed Scopus (19) Google Scholar, 16Jitrapakdee S. Gong Q. MacDonald M.J. Wallace J.C. J. Biol. Chem. 1998; 273: 34422-34428Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The role of PC in lipogenesis is thought to provide substantial amounts of an acetyl group and NADPH required for de novo fatty acid synthesis (17Ballard F.J. Hanson R.W. J. Lipid Res. 1967; 8: 73-79Abstract Full Text PDF PubMed Google Scholar). Acetyl-CoA is generated in the mitochondria by the oxidative decarboxylation of pyruvate, and after condensation with oxaloacetate, acetyl groups are transported to the cytoplasm as citrate, which undergoes ATP-dependent cleavage to yield acetyl-CoA and oxaloacetate. This pathway requires a continuous supply of oxaloacetate, which is provided by the activity of PC. Acetyl-CoA, a building block for long chain fatty acids, is then converted into malonyl-CoA by acetyl-CoA carboxylase. The cytoplasmic oxaloacetate generated from citrate is reduced by NADH to malate, which is decarboxylated to yield pyruvate and NADPH, with the latter being necessary for de novo fatty acid synthesis. Pyruvate carboxylation was shown to be necessary in hamster brown adipose tissue for maximal oxygen consumption in norepinephrine-stimulated respiration, even when drainage of the citric acid cycle for amino acid synthesis is eliminated, suggesting that the provision of oxaloacetate promotes the oxidation of acetyl-CoA from fatty acid degradation (18Cannon B. Nedergaard J. Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4701) Google Scholar, 19Cannon B. Nedergaard J. Eur. J. Biochem. 1979; 94: 419-426Crossref PubMed Scopus (42) Google Scholar). Here we show that the levels of PC protein and mRNA are highly correlated with the expression of PPARγ during adipocyte differentiation. Promoter analysis and transient transfection experiments with reporter constructs show that the 2.3-kb promoter fragment of the mouse PC gene contains a functional PPRE that directly mediates a PPARγ response. Mutational analysis, EMSA, and ChIP assay also support our in vitro data. PPARγ2 null mice (20Medina-Gomez G. Virtue S. Lelliott C. Boiani R. Campbell M. Christodoulides C. Perrin C. Jimenez-Linan M. Blount M. Dixon J. Zahn D. Thresher R.R. Aparicia S. Carlton M. Colledge W.H. Kettunen M.I. Seppanen-Laakso T. Sethi J.K. O'Rahilly S. Brindle K. Cinti S. Burcelin R. Vidal-Puig A. Diabetes. 2005; 54: 1706-1716Crossref PubMed Scopus (137) Google Scholar) also show a marked reduction of PC protein and PC mRNA both in white and brown adipose tissues. Animals—PPARγ2-/- male mice with a mixed background 129SV/C57Bl6 and their wild type littermates (18Cannon B. Nedergaard J. Physiol. Rev. 2004; 84: 277-359Crossref PubMed Scopus (4701) Google Scholar) were used in this study. After weaning (age of 3 weeks), the knock-out and the wild type mice were fed with chow or a high fat diet for 4 months before being sacrificed. Fasting and refeeding experiments were carried out as described previously (20Medina-Gomez G. Virtue S. Lelliott C. Boiani R. Campbell M. Christodoulides C. Perrin C. Jimenez-Linan M. Blount M. Dixon J. Zahn D. Thresher R.R. Aparicia S. Carlton M. Colledge W.H. Kettunen M.I. Seppanen-Laakso T. Sethi J.K. O'Rahilly S. Brindle K. Cinti S. Burcelin R. Vidal-Puig A. Diabetes. 2005; 54: 1706-1716Crossref PubMed Scopus (137) Google Scholar). Epididymal fat pads were removed and snap-frozen in liquid nitrogen. For cold exposure, 4-month-old knock-out mice and their wild type littermates fed with chow diet were maintained at 4 °C or at room temperature (25 °C) for 12 days before they were sacrificed. Interscapular brown adipose tissues were removed and snap-frozen in liquid nitrogen before the RNA and protein were extracted. Cell Culture—Murine 3T3-L1 preadipocytes were routinely grown in complete media (Dulbecco's modified Eagle's medium (Sigma) supplemented with 10% (v/v) fetal bovine serum (Invitrogen) and 100 units/ml penicillin, 100 μg/ml streptomycin). For differentiation of preadipocytes to mature adipocytes, 2 × 105 cells were plated in 6-well plates and maintained in the above medium until they reached confluence. At day 2 post-confluence (designated as day 0), the cells were induced to differentiate with complete medium supplemented with 0.5 mm 3-isobutylmethylxanthine, 1 μm dexamethasone, and 10 μg/ml insulin. After 2 days in induction medium, 3T3-L1 cells were maintained in complete medium supplemented with 10 μg/ml insulin. The medium was changed every 2 days. Human embryonic kidney (HEK293T) cells and brown adipocyte cell line (HIB-1B) (21Ross S.R. Choy L. Graves R.A. Fox N. Solevjeva V. Klaus S. Ricquier D. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7561-7565Crossref PubMed Scopus (113) Google Scholar) were also routinely grown in the complete medium above. Preadipocyte Isolation and Culture—Preadipocyte isolation, culture, and differentiation were performed as described previously (20Medina-Gomez G. Virtue S. Lelliott C. Boiani R. Campbell M. Christodoulides C. Perrin C. Jimenez-Linan M. Blount M. Dixon J. Zahn D. Thresher R.R. Aparicia S. Carlton M. Colledge W.H. Kettunen M.I. Seppanen-Laakso T. Sethi J.K. O'Rahilly S. Brindle K. Cinti S. Burcelin R. Vidal-Puig A. Diabetes. 2005; 54: 1706-1716Crossref PubMed Scopus (137) Google Scholar). At 0, 2, 4, and 8 days after differentiation in the absence or presence of 0.1 μm rosiglitazone, cells were harvested, and RNAs were extracted. RNA Isolation, cDNA Synthesis, and Real Time PCR—Total RNA was extracted from 3T3-L1 cells or primary cultures using an RNeasy kit (Qiagen) or from frozen adipose tissues with STAT-60 (AMS Biotechnology) and quantified by GeneQuant (Amersham Biosciences). The quality of extracted RNA was assessed by formaldehyde gel electrophoresis. cDNA synthesis was carried out at 37 °C for 1 h in a 20-μl reaction volume containing 0.5 μg of total RNA, 100 ng of random primers (Promega), 1× reverse transcriptase buffer (50 mm Tris, pH 8.3, 75 mm KCl, 3 mm MgCl2, 10 mm dithiothreitol), 1 mm each of dNTP, and 200 units of Moloney murine leukemia virus-reverse transcriptase (Promega). PCR was carried out with real time PCR with a TaqmanR probe (Applied Biosystems). Real time PCR was carried out in a 96-well optical plate (Applied Biosystems). Each well contained 12 μl of reaction mixture consisting of 1× ABI master mix (Applied Biosystems), 0.25 μm each of forward and reverse primers, 0.125 μm fluorogenic probe in which its 5′- and 3′-ends were modified with 6-carboxy-fluorescein and 6-carboxy-tetramethyl-rhodamine, respectively, 2 μl of 1/10 dilution of cDNA. 18 S rRNA was also amplified as the internal control using 18 S ribosomal RNA control reagent (Applied Biosystems) with 1/200 dilution of cDNA samples. The primer and probe sets used to detect various mRNAs included mPC, mPPARγ1, mPPARγ2, and mC/EBPα. They were synthesized by Sigma Genosys and are shown in Table I. The amplification profile consisted of an initial incubation at 50 °C for 2 min and 95 °C for 10 min followed by 40 cycles of denaturation at 95 °C for 10 s, annealing and extension at 60 °C for 1 min. The relative quantities of amplified cDNAs were analyzed by the SDS software (Applied Biosystems). The abundance of mRNA was normalized with 18 S rRNA, and the value was expressed as "relative gene expression."Table IPrimers and fluorogenic probes used for real time PCRGene nameForward primerReverse primerFluorogenic probemPC5′-GATGACCTCACAGCCAAGCA-3′5′-GGGTACCTCTGTGTCCAAAGGA-3′5′-CCCTGGTGGCCTGTACCAAAGGG-3′mPPARγ15′-TTTAAAAACAAGACTACCCTTTACTGAAATT-3′5′-AGAGGTCCACAGAGCTGATTCC-3′5′-AGAGATGCCATTCTGGCCCACCAACTT-3′mPPARγ25′-GATGCACTGCCTATGAGCACTT-3′5′-AGAGGTCCACAGAGCTGATTCC-3′5′-AGAGATGCCATTCTGGCCCACCAACTT-3′mC/EBPα5′-GGAACAGCTGAGCCGTGAAC-3′5′-GCGACCCGAAACCATCCT-3′5′-AAGGCCATGACTGCGCGTGAGGCG-3′ Open table in a new tab Reporter Construct and Mutagenesis—The 2.3-kb fragment of the lipogenic promoter of the mouse PC gene (22Jitrapakdee S. Petchamphai N. Sunyakumthorn P. Wallace J.C. Boonsaeng V. Biochem. Biophys. Res. Commun. 2001; 287: 411-417Crossref PubMed Scopus (22) Google Scholar) was isolated from mouse genomic DNA by PCR. The PCR was carried out in a 50-μl reaction mixture containing 1× High Fidelity PCR buffer (Roche Applied Science), 0.25 mm each of dNTP, 100 ng of mouse genomic DNA, 0.25 μm of forward primer (5′-GGGCACGCGTGGGATGAATCCCCAGGTGGGACAG-3′) and reverse primer (5′-GTGCTGCACACAGAGGACGTGATAGAG-3′), and 2.5 units of Expanded High Fidelity Polymerase (Roche Applied Science). The reaction was subjected to 35 cycles of amplification in which each cycle consists of denaturation at 94 °C for 15 s, annealing at 55 °C for 30 s, and extension at 72 °C for 3 min. The PCR product was gel-purified using a gel extraction kit (Qiagen) and ligated to pGEM-T Easy vector (Promega). The nucleotide sequence of the insert was verified by automated sequencing using Big Dye (Applied Biosystems) before being restricted with MluI and BamHI and subsequently ligated to MluI- and BglII-digested pGL-3 basic (Promega). This resulting construct was designated pGL-wt mPC. The mutation of putative PPRE on a wild type promoter fragment was carried out with XL-Quick change site-directed mutagenesis kit (Stratagene) with PPREmF1 (5′-CAGCCTTCAGGCTAATGCAGCTCTCACCCTCCATCATGCCTGG-3′) and PPREmR1 (5′-GTGCCAGGCATGATGGAAAGCAAAAATCACATTAGCCTGAAGG-3′) as the mutagenic primers. The mutagenic reaction was performed following the manufacturer's instructions, and the correct mutagenic sequence was verified by DNA sequencing. Transient Transfection and Transactivation Study—Briefly, 2 × 105 cells of 3T3-L1 cells or 1 × 105 cells of HEK293T were plated in 24-well plates in antibiotic-free Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum 1 day before transfection. Transfection was carried out the next day using Lipofectamine 2000 reagent (Invitrogen), with 0.5 pmol of firefly luciferase reporter construct alone or with equivalent amounts of pcDNA-PPARγ1, pcDNA-PPARγ2, or pRSV-RXRα plasmids overexpressing PPARγ1, PPARγ2, and RXRα, respectively (23Meirhaeghe A. Crowley V. Lenaghan C. Lelliott C. Green K. Stewart A. Hart K. Schinner S. Sethi J.K. Yeo G. Brand M.D. Cortright R.N. O'Rahilly S. Montague C. Vidal-Puig A.J. Biochem. J. 2003; 373: 155-165Crossref PubMed Scopus (173) Google Scholar). Renilla luciferase reporter plasmids (1 ng) driven by the thymidine kinase promoter (pRL-TK) (Promega) were also included in all transfections as an internal control. The luciferase assay was performed with 20 μg of cell lysate using the dual luciferase assay system (Promega) in a Berthold luminometer. The firefly luciferase activity was normalized with Renilla luciferase and expressed as "relative luciferase activity." EMSA—Six day differentiated 3T3-L1 or HEK293T cells transiently transfected with plasmids overexpressing RXRα/PPARγ1 or RXRα/PPARγ2 were used as the source of nuclear extract. Briefly, 2 × 106 cells were washed with phosphate-buffered saline containing 1 mm phenylmethylsulfonyl fluoride before being scraped and centrifuged at 1,200 rpm at 4 °C. The cell pellet was subjected to crude nuclear protein preparation using a cytosolic and nuclear protein extraction kit (Pierce). EMSA was performed using LightShift chemiluminescent EMSA kit (Pierce). The probes were prepared by annealing oligonucleotides with their 3′-end labeled with biotin. The oligonucleotides include mouse aP2 PPRE-For (5′-GATCTGTGACCTTTGACCTAGTAAG-3′; underline indicates putative PPRE sequence), aP2 PPRE-Rev (5′-CTTACTAGGTCAAAGGTCACAGATC-3′) (24Tontonoz P. Graves R.A. Budavari A.I. Erdjument-Bromage H. Lui M. Hu E. Tempst P. Spiegelman B.M. Nucleic Acids Res. 1994; 22: 5628-5634Crossref PubMed Scopus (331) Google Scholar), mPC PPRE-For (5′-CTAATGTGACCCTTGCCCTCCATCA-3′), and mPC PPRE-Rev (5′-TGATGGAGGGCAAGGGTCACATTAG-3′). The DNA-protein binding assay was performed at room temperature for 20 min in a final volume of 20 μl containing 1× binding buffer (10 mm Tris, pH 7.5, 50 mm KCl, 1 mm dithiothreitol), 2.5% (v/v) glycerol, 5 mm MgCl2, 1 μg of poly(dI-dC), 0.05% (v/v) Nonidet P-40, 8 pmol of double-stranded biotinylated probe, and 10 μg of nuclear extract. The DNA-protein complexes were separated by 5% PAGE in 0.5× TBE at 200 V at 4 °C for 2 h. DNA-protein complexes in gel were transferred to Hybond N+ nylon membrane (Amersham Biosciences) by electroblotting with 0.5× TBE at 350 mA for 1.5 h. DNA-protein complexes were fixed to the membrane by UV cross-linker and detected by a nonradioactive nucleic acid detection kit (Pierce). For the competition assay, 5×, 10×, or 20× more concentrated double-stranded DNAs were included in the binding reaction. For supershift EMSA, 2 μl of PPARγ monoclonal antibody (E8) or 2 μl of RXRα polyclonal antibody (D20) (Santa Cruz Biotechnology) were preincubated in the binding reaction for 10 min before the probe was added. ChIP Assay—Briefly, 2 × 105 cells of 3T3-L1 plated in a 35-mm well were used in the assay. The DNA and protein were cross-linked in situ with 0.5% (v/v) formaldehyde at 37 °C for 5 min. Soluble chromatin was prepared using a chromatin immunoprecipitation assay kit (Upstate Biotechnology, Inc.). The lysate was sonicated four times for 10 s at 4 °C. The lysates were precipitated with either 50 μl of anti-E2F or 10 or 50 μl of anti-PPARγ (E8) mouse monoclonal antibodies (Santa Cruz Biotechnology) overnight before protein A-agarose beads were added. The proteins were removed from DNA by digesting with 10 μg/ml proteinase K at 45 °C for 30 min. The DNA was further purified by a QIAquick PCR purification kit (Qiagen). The DNA was eluted in 50 μl of sterile water. One microliter of eluted DNA was used to amplify a 250-bp amplicon with F1 (5′-CCTTGTCTTGTGTCTGGCAGTGC-3′) and R1 (5′-GCTAGAAAGCAGCTGCAGACTT-3′) primers that flanked the PPRE of the mPC or to amplify a 150-bp amplicon with the "negative control primers" F2 (5′-CCTGAACCTGAAGGAGCTGGAG-3′) and R2 (5′-GGGTCCTTGAAGAAAGAGACGAG-3′) that are located >3 kb downstream of the PPRE for the mPC. PCR was performed for 35 cycles for which linear amplification was obtained. Ten microliters of PCR products were analyzed by 2% agarose gel electrophoresis. Protein Extraction and Western Analysis—Total protein lysates of 3T3-L1 from each well were extracted in 100 μl of RIPA buffer (150 mm NaCl, 50 mm Tris, pH 7.4, 1% (v/v) Nonidet P-40, 0.25% (w/v) sodium deoxycholate, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride and protease inhibitor mixture (Roche Applied Science)) for 5 min on ice. The protein lysates were recovered by centrifugation at 13,000 rpm for 5 min at 4 °C. Aliquots (50 μg) of protein lysates were subjected to reducing SDS-PAGE (25Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207856) Google Scholar) (10% SDS-PAGE for PPARγ1, PPARγ2, and C/EBPα or 7.5% SDS-PAGE for PC). Proteins were transferred to polyvinylidene difluoride membranes by electroblotting. The blots were blocked in 4% (w/v) skim milk in PBS-T overnight. PC, PPARγ, and C/EBPα bands were detected by incubating the blots with PC polyclonal antibody (26Rohde M. Lim F. Wallace J.C. Arch. Biochem. Biophys. 1991; 290: 197-201Crossref PubMed Scopus (37) Google Scholar), PPARγ monoclonal antibody (E-8), or C/EBPα polyclonal antibody (14AA) from Santa Cruz Biotechnology, respectively. The polyclonal rabbit anti-p85α subunit of phosphoinositol-3-phosphate kinase was a gift from Professor K. Siddle, Department of Clinical Biochemistry, University of Cambridge. Immunoreactive bands were visualized upon adding goat anti-rabbit or goat anti-mouse polyclonal antibodies (Dako Cytomation) conjugated with horseradish peroxidase followed by ECL Western blot detection system (Amersham Biosciences). PC Is Expressed Concomitant with Adipogenic Transcription Factors during 3T3-L1 and HIB-1B Differentiation—It has been reported previously that PC activity and mRNA are induced during the conversion of 3T3-L1 preadipocyte to mature adipocyte by hormonal induction (1Mackall J.C. Student A.K. Polakis S.E. Lane M.D. J. Biol. Chem. 1976; 251: 6462-6464Abstract Full Text PDF PubMed Google Scholar, 13Freytag S.O. Utter M.F. J. Biol. Chem. 1983; 258: 6307-6312Abstract Full Text PDF PubMed Google Scholar, 15Zhang J. Xia W.-L. Ahmad F. Biochem. J. 1995; 306: 205-210Crossref PubMed Scopus (19) Google Scholar), suggesting PC may play a lipogenic role during terminal differentiation. However, the molecular mechanism controlling PC expression during adipocyte differentiation has yet to be elucidated. To address this, we correlated the window of PC expression with that of PPARγ and C/EBPα, two major pro-adipogenic transcription factors, during differentiation of 3T3-L1. As shown in Fig. 1A, Western analysis clearly showed that PC expression is barely detectable in confluent preadipocytes but is rapidly increased at day 2 after cells were induced to differentiate. The level of PC reached a maximum by day 6 and remained unchanged thereafter. This expression pattern is also similar to those of PPARγ1, PPARγ2, and C/EBPα. To determine whether PC induction is dependent on adipocyte differentiation, we used TNFα, a cytokine known to block adipocyte differentiation through a PPARγ-dependent mechanism. As shown in Fig. 1A, TNFα not only markedly inhibited expression of C/EBPα, PPARγ1, and PPARγ2 by >90% but also dramatically suppressed PC expression. However, the suppression of PC expression was more directly correlated with the expression profile of PPARγ, i.e. PC and PPARγ levels were completely inhibited within the first 2 days of differentiation, and their expression was partially restored after day 4 and beyond. Real time RT-PCR analysis demonstrated that the induction of PC expression during differentiation occurred at the transcriptional level, i.e. the PC mRNA was increased ∼20-fold 2 days after differentiation and reached maximum (35-fold) at day 6 (Fig. 2). The rise of PC mRNA was also closely correlated with that of PPARγ1, PPARγ2, and C/EBPα (Fig. 2). The induction of PC expression during adipocyte differentiation was not restricted to this white adipocyte cell line as this effect was also seen in the brown adipocyte cell line HIB-1B. During confluence-induced spontaneous differentiation of HIB-1B, PC level was also markedly increased, reaching a maximal level at day 6 and remaining unchanged thereafter (Fig. 1B). This pattern was also closely related to those of PPARγ1, PPARγ2, and C/EBPα as seen during differentiation of 3T3-L1 cells. PPARγ but Not C/EBPα Transactivated the mPC Promoter—Because PC was expressed concomitant with C/EBPα and PPARγ1 and PPARγ2, we investigated whether PC was transcriptionally regulated by these transcription factors during adipogenesis. Therefore, we cloned a 2.3-kb DNA fragment upstream of the transcription start site of the mPC gene. This DNA fragment was extended ∼1.5 kb further from the 5′-end of the 0.7-kb fragment than we have characterized previously (22Jitrapakdee S. Petchamphai N. Sunyakumthorn P. Wallace J.C. Boonsaeng V. Biochem. Biophys. Res. Commun. 2001; 287: 411-417Crossref PubMed Scopus (22) Google Scholar). The 2.3-kb promoter fragment was cloned into the firefly luciferase reporter plasmid (PC-Luc) and was transiently cotransfected with the plasmids overexpressing C/EBPα, SREBP1c, PPARγ1 or PPARγ2, respectively, in HEK293T cells. As shown in Fig. 3A, the relative luciferase activity was unchanged when the PC-Luc construct was co-transfected with plasmids overexpressing C/EBPα or SREBP1c. However, the luciferase activity was increased 3-fold relative to the basal condition (cells transfected with PC-Luc alone), when PC-Luc construct was co-transfected with either PPARγ1 or PPARγ2 constructs. The luciferase activities were further increased to ∼6-7-fold when the plasmid overexpressing RXRα, a heterodimer partner of PPARγ, was co-transfected with either PPARγ1 or PPARγ2 constructs. Treating transfected cells with 0.1 μm rosiglitazone (PPARγ agonist) for 24 h doubled the luciferase activities of cells co-transfected with RXRα constructs together with PPARγ1 or PPARγ2 constructs to 12- or 14-fold, respectively. Similar results were obtained when the above assays were performed with 3T3-L1 preadipocytes (Fig. 3B). Taken together these data suggest the 2.3-kb mPC promoter fragment may contain a PPARγ-response element (PPRE). The mPC Promoter Contains Functional PPRE Element at Positions -386/-374—The 2.3-kb nucleotide sequence of the mPC promoter was searched for the putative transcription factor binding sites by the TESS data base (www.cbil.upenn.edu/tess/). As shown in Fig. 4A, we identified a putative PPRE (AGGGCAAGGGTCA, underline indicates two direct repeats (DR1 and DR2 respectively) separated by one nucleotide) on the opposite strand, at positions -386/-374 relative to transcription start site. This site was located upstream of the initiator site, HIP1, and two downstream GC boxes, potential binding sites for the general transcription factor(s), Sp1/Sp3. The nucleotide sequence of this putative PPRE of PC appears to be conserved between mouse and rat (22Jitrapakdee S. Petchamphai N. Sunyakumthorn P. Wallace J.C. Boonsaeng V. Biochem. Biophys. Res. Commun. 2001; 287: 411-417Crossref PubMed Scopus (22) Google Scholar, 27Jitrapakdee S. Booker G.W. Cassady A.I. Wallace J.C. J. Biol. Chem. 1997; 272: 20520-20528Abstract Full Text Full Text PDF Scopus (54) Google Scholar) genes (Fig. 4B). The PPRE of the mPC contains one nucleotide of each DR1 and DR2 that did not match the consensus PPRE site, i.e. AGGTCANAGGTCA (underline indicates DR1 and DR2) (5Rosen E.D. Spiegelman B.M. J.