Activation of CCAAT/Enhancer-binding Protein (C/EBP) α Expression by C/EBPβ during Adipogenesis Requires a Peroxisome Proliferator-activated Receptor-γ-associated Repression of HDAC1 at the C/ebpα Gene Promoter
2006; Elsevier BV; Volume: 281; Issue: 12 Linguagem: Inglês
10.1074/jbc.m510682200
ISSN1083-351X
AutoresYing Zuo, Li Qiang, Stephen R. Farmer,
Tópico(s)Adipokines, Inflammation, and Metabolic Diseases
ResumoStudies have shown that CCAAT/enhancer-binding protein β (C/EBPβ) can stimulate adipogenesis in noncommitted fibroblasts by activating expression of peroxisome proliferator-activated receptor-γ (PPARγ). Other investigations have established a role for C/EBPα as well as PPARγ in orchestrating the complex program of adipogenic gene expression during terminal preadipocyte differentiation. Consequently, it is important to identify factors regulating transcription of the C/ebpα gene. In this study, we demonstrated that inhibition of PPARγ activity by exposure of 3T3-L1 preadipocytes to a potent and selective PPARγ antagonist inhibits adipogenesis but also blocks the activation of C/EBPα expression at the onset of differentiation. Ectopic expression of C/EBPβ in Swiss 3T3 mouse fibroblasts (Swiss-LAP cells) induces PPARγ expression without any significant enhancement of C/EBPα expression. Treatment of Swiss-LAP cells with a PPARγ agonist induces adipogenesis, which includes activation of C/EBPα expression. To further establish a role for PPARγ in regulating C/EBPα expression, we expressed C/EBPβ in PPARγ-deficient mouse embryo fibroblasts (MEFs). The data show that C/EBPβ is capable of inducing PPARγ in Pparγ+/- MEFs, which leads to activation of adipogenesis, including C/EBPα expression following exposure to a PPARγ ligand. In contrast, C/EBPβ is not able to induce C/EBPα expression or adipogenesis in Pparγ-/- MEFs. Chromatin immunoprecipitation analysis reveals that C/EBPβ is bound to the minimal promoter of the C/ebpα gene in association with HDAC1 in unstimulated Swiss-LAP cells. Exposure of the cells to a PPARγ ligand dislodges HDAC1 from the proximal promoter of the C/ebpα gene, which involves degradation of HDAC1 in the 26 S proteasome. These data suggest that C/EBPβ activates a single unified pathway of adipogenesis involving its stimulation of PPARγ expression, which then activates C/EBPα expression by dislodging HDAC1 from the promoter for degradation in the proteasome. Studies have shown that CCAAT/enhancer-binding protein β (C/EBPβ) can stimulate adipogenesis in noncommitted fibroblasts by activating expression of peroxisome proliferator-activated receptor-γ (PPARγ). Other investigations have established a role for C/EBPα as well as PPARγ in orchestrating the complex program of adipogenic gene expression during terminal preadipocyte differentiation. Consequently, it is important to identify factors regulating transcription of the C/ebpα gene. In this study, we demonstrated that inhibition of PPARγ activity by exposure of 3T3-L1 preadipocytes to a potent and selective PPARγ antagonist inhibits adipogenesis but also blocks the activation of C/EBPα expression at the onset of differentiation. Ectopic expression of C/EBPβ in Swiss 3T3 mouse fibroblasts (Swiss-LAP cells) induces PPARγ expression without any significant enhancement of C/EBPα expression. Treatment of Swiss-LAP cells with a PPARγ agonist induces adipogenesis, which includes activation of C/EBPα expression. To further establish a role for PPARγ in regulating C/EBPα expression, we expressed C/EBPβ in PPARγ-deficient mouse embryo fibroblasts (MEFs). The data show that C/EBPβ is capable of inducing PPARγ in Pparγ+/- MEFs, which leads to activation of adipogenesis, including C/EBPα expression following exposure to a PPARγ ligand. In contrast, C/EBPβ is not able to induce C/EBPα expression or adipogenesis in Pparγ-/- MEFs. Chromatin immunoprecipitation analysis reveals that C/EBPβ is bound to the minimal promoter of the C/ebpα gene in association with HDAC1 in unstimulated Swiss-LAP cells. Exposure of the cells to a PPARγ ligand dislodges HDAC1 from the proximal promoter of the C/ebpα gene, which involves degradation of HDAC1 in the 26 S proteasome. These data suggest that C/EBPβ activates a single unified pathway of adipogenesis involving its stimulation of PPARγ expression, which then activates C/EBPα expression by dislodging HDAC1 from the promoter for degradation in the proteasome. The differentiation of preadipocytes into adipocytes is regulated by an elaborate network of transcription factors that control expression of many hundreds of proteins responsible for establishing the mature fat cell phenotype (1Rosen E.D. Walkey C.J. Puigserver P. Spiegelman B.M. Genes Dev. 2000; 14: 1293-1307Crossref PubMed Google Scholar). The most notable among these factors are members of the C/EBP 2The abbreviations used are: C/EBP, CCAAT/enhancer-binding protein; PPAR, peroxisome proliferator-activated receptor; LAP, liver-enriched transcriptional activator protein; MIX, isobutylmethylxanthine; DEX, dexamethasone; PBS, phosphate-buffered saline; HDAC, histone deacetylase; MEF, mouse embryo fibroblast; ChIP, chromatin immunoprecipitation; TET, tetracycline; FBS, fetal bovine serum; pol, polymerase. and PPAR families of transcription factors. In fact, it is now well accepted that both PPARγ and C/EBPα function as critical regulators of adipogenesis because deficiency of either of these proteins prevents the development of white adipose tissue in the mouse (2Linhart H.G. Ishimura-Oka K. DeMayo F. Kibe T. Repka D. Poindexter B. Bick R.J. Darlington G.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12532-12537Crossref PubMed Scopus (252) Google Scholar, 3Rosen E.D. Sarraf P. Troy A.E. Bradwin G. Moore K. Milstone D.S. Spiegelman B.M. Mortensen R.M. Mol. Cell. 1999; 4: 611-617Abstract Full Text Full Text PDF PubMed Scopus (1669) Google Scholar, 4Barak Y. Nelson M.C. Ong E.S. Jones Y.Z. Ruiz-Lozano P. Chien K.R. Koder A. Evans R.M. Mol. Cell. 1999; 4: 585-595Abstract Full Text Full Text PDF PubMed Scopus (1659) Google Scholar). Studies performed in cell culture have positioned PPARγ and C/EBPα in the center of this network of factors where they orchestrate the many functions associated with the mature adipocyte (1Rosen E.D. Walkey C.J. Puigserver P. Spiegelman B.M. Genes Dev. 2000; 14: 1293-1307Crossref PubMed Google Scholar, 5Morrison R.F. Farmer S.R. J. Nutr. 2000; 130: S3116-S3121Crossref PubMed Google Scholar). Some functions appear to be governed exclusively by PPARγ such as lipogenesis, whereas others such as insulin-dependent glucose transport and adiponectin expression are dependent on simultaneous expression of both proteins (6El-Jack A.K. Hamm J.K. Pilch P.F. Farmer S.R. J. Biol. Chem. 1999; 274: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 7Wu Z. Rosen E.D. Brun R. Hauser S. Adelmont G. Troy A.E. McKeon C. Darlington G.J. Spiegelman B.M. Mol. Cell. 1999; 3: 151-158Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar, 8Park B.-H. Qiang L. Farmer S.R. Mol. Cell. Biol. 2004; 24: 8671-8680Crossref PubMed Scopus (165) Google Scholar). Consequently, in our efforts to gain a complete understanding of the processes regulating the function of adipocytes, it is important to identify the mechanisms regulating transcription of both PPARγ and C/EBPα. Several investigators have demonstrated a direct role for C/EBPβ along with C/EBPδ in inducing expression of PPARγ2 through association with C/EBP regulatory elements in the Pparγ2 gene promoter (9Wu Z. Bucher N.L.R. Farmer S.R. Mol. Cell. Biol. 1996; 16: 4128-4136Crossref PubMed Google Scholar, 10Yeh W.C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (813) Google Scholar, 11Clarke S.L. Robinson C.E. Gimble J.M. Biochem. Biophys. Res. Commun. 1997; 240: 99-103Crossref PubMed Scopus (193) Google Scholar, 12Elberg G. Gimble J.M. Tsai S.Y. J. Biol. Chem. 2000; 275: 27815-27822Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). A similar role for these C/EBPs in inducing transcription of the C/ebpα gene has not been established. It is generally assumed, however, that C/EBPβ does induce C/EBPα based on data from in vitro assays showing transactivation of a C/ebpα minimal promoter reporter gene in different cell types by C/EBPβ (13Rana B. Xie Y. Mischoulon D. Bucher N.L.R. Farmer S.R. J. Biol. Chem. 1995; 270: 18123-18132Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 14Tang Q.Q. Jiang M.S. Lane M.D. Mol. Cell. Biol. 1999; 19: 4855-4865Crossref PubMed Scopus (128) Google Scholar, 15Wiper-Bergeron N. Wu D. Pope L. Schild-Poulter C. Hache R.J. EMBO J. 2003; 22: 2135-2145Crossref PubMed Scopus (119) Google Scholar). Earlier studies aimed at characterizing the importance of C/EBPβ in inducing adipogenesis failed to demonstrate induction of C/EBPα expression because the NIH-3T3 fibroblasts used in the experiments do not express C/EBPα (10Yeh W.C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (813) Google Scholar, 16Wu Z. Xie Y. Bucher N.L.R. Farmer S.R. Genes Dev. 1995; 9: 2350-2363Crossref PubMed Scopus (481) Google Scholar). In those studies, however, ectopic expression of C/EBPβ was capable of inducing PPARγ2 expression, and following exposure to appropriate ligands the NIH-3T3 cells underwent conversion into lipid-laden adipocytes that were unresponsive to insulin because they lacked C/EBPα (6El-Jack A.K. Hamm J.K. Pilch P.F. Farmer S.R. J. Biol. Chem. 1999; 274: 7946-7951Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Other studies performed in cells capable of expressing C/EBPα have demonstrated that PPARγ can activate transcription of the C/ebpα gene in the absence of ongoing protein synthesis (17Hamm J.K. Park B.H. Farmer S.R. J. Biol. Chem. 2001; 276: 18464-18471Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), suggesting that the transcriptional cascade responsible for initiating terminal adipogenesis involves induction of PPARγ2 by C/EBPβ, and PPARγ2 then is responsible for inducing C/ebpα along with other adipogenic genes. Activation of gene expression involves a complex multistep process that includes docking of select transcription factors on regulatory elements within the promoter and/or enhancers of the target genes, which initiates recruitment of a variety of nuclear factors involved in reorganization of surrounding chromatin as well as assembly of the transcriptional machinery at the promoter. It also appears that in some cases transcription factors can dock on various genes without initiating transcription. These factors appear to recruit corepressors and associated proteins to the gene that suppress transcription until an effector(s) dislodges the repressors to facilitate recruitment of appropriate coactivators. In the case of C/EBPβ, there is evidence to suggest that it can dock on the promoters of C/ebpα and Pparγ genes prior to their activation during the early phase of adipogenesis. Adipogenic effectors then facilitate association of the chromatin remodeling complex SWI/SNF with C/EBPβ on the Pparγ gene (18Salma N. Xiao H. Mueller E. Imbalzano A.N. Mol. Cell. Biol. 2004; 24: 4651-4663Crossref PubMed Scopus (151) Google Scholar), whereas glucocorticoids are responsible for dislodging an mSin3a-HDAC1 complex from the C/EBPβ site on the C/ebpα gene (15Wiper-Bergeron N. Wu D. Pope L. Schild-Poulter C. Hache R.J. EMBO J. 2003; 22: 2135-2145Crossref PubMed Scopus (119) Google Scholar). In an attempt to define the role of PPARγ along with C/EBPβ in regulating C/EBPα expression during adipogenesis, we ectopically expressed each of the proteins in nonadipogenic fibroblasts and analyzed adipogenic gene expression. The data demonstrate that C/EBPβ is capable of docking on the C/ebpα gene promoter but is incapable of inducing C/EBPα transcription in the absence of PPARγ. Furthermore, treatment of cells expressing C/EBPβ with glucocorticoids is not capable of inhibiting HDAC1 activity at the C/ebpα gene, whereas activation of PPARγ facilitates targeting of the HDAC1 that is associated with the C/ebpα gene to the proteasome and thereby inducing transcription. Expression Vectors and Cell Lines—The p34C/EBPβ(LAP)-pBI-G plasmid and pBabe-Puro-LAP and pRevTRE-C/EBPα-Myc-His retroviral vectors were produced as described previously (8Park B.-H. Qiang L. Farmer S.R. Mol. Cell. Biol. 2004; 24: 8671-8680Crossref PubMed Scopus (165) Google Scholar, 17Hamm J.K. Park B.H. Farmer S.R. J. Biol. Chem. 2001; 276: 18464-18471Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar, 19Moldes M. Zuo Y. Morrison R.F. Silva D. Park B.H. Liu J. Farmer S.R. Biochem. J. 2003; 376: 607-613Crossref PubMed Scopus (250) Google Scholar). The LAP-pBI-G vector was transfected into Swiss mouse 3T3 fibroblasts constitutively expressing the Tet-Off activator protein (Swiss Tet-Off cells; Clontech) along with a puromycin selection plasmid (pBabe-puro). Colonies of cells resistant to 3 μg/ml puromycin were selected and analyzed for expression of the pBI-G vector on the basis of tetracycline-responsive β-galactosidase production. The initial selection gave rise to several nonhomogeneous colonies because only 10–20% of the cells expressed β-galactosidase activity. Therefore, one of these colonies was subjected to serial dilution single cell cloning. A colony (LAP-A cells) was selected in which almost the entire population of cells expressed β-galactosidase activity in a tetracycline-responsive manner. To establish retrovirus-producing cell lines, human embryonic kidney 293T cells were seeded at 80% confluence in a 60-mm diameter dish on the day of transfection. Individual cultures of cells were transfected with FuGENE 6 (Roche Applied Science) and 2 μg of either the pRevTRE-C/EBPα-Myc-His or pBabe-Puro-LAP vector along with 2 μg each of vesicular stomatitis virus G and GP expression plasmids (pVpack; Stratagene). Two days after transfection, culture medium containing high titer virus was harvested and filtered through a 0.45-μm pore size filter. The viral filtrate supernatant was used to infect Pparγ+/- or Pparγ-/- mouse embryo fibroblasts (20Rosen E.D. Hsu C.H. Wang X. Sakai S. Freeman M.W. Gonzalez F.J. Spiegelman B.M. Genes Dev. 2002; 16: 22-26Crossref PubMed Scopus (1118) Google Scholar). Cells were seeded in a 60-mm dish at 25% confluence on the day of infection and incubated with retrovirus overnight in the presence of 4 μg of Polybrene/ml (Sigma), and the infection was allowed to proceed for an additional 2 days in fresh growth medium. At this stage, 100 μg of hygromycin and/or 2 μg/ml puromycin was added to facilitate selection of stable cell lines, which required culture for 2–3 weeks in the presence of the antibiotic. Cell Culture—For experiments, cells were grown in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal bovine serum (FBS). Induction of differentiation was achieved by treatment of post-confluent cells with dexamethasone (DEX, 1 μm), 3-isobutyl-1-methylxanthine (MIX, 0.5 mm), and insulin (1.67 μm). The Swiss-LAP A cells and the other cell lines were differentiated by the same method as for 3T3-L1 preadipocytes (8Park B.-H. Qiang L. Farmer S.R. Mol. Cell. Biol. 2004; 24: 8671-8680Crossref PubMed Scopus (165) Google Scholar) and maintained in the presence or absence of 5 μm troglitazone (Parke-Davis) or 1 μm GW7845 (a highly selective PPARγ agonist) obtained from GlaxoSmithKline. To block PPARγ activity, cells were exposed to T0070907 (a selective PPARγ antagonist) as described previously (21Lee G. Elwood F. McNally J. Weiszmann J. Lindstrom M. Amaral K. Nakamura M. Miao S. Cao P. Learned R.M. Chen J.-L. Li Y. J. Biol. Chem. 2002; 277: 19649-19657Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar) and obtained from AdipoGenix (Boston). Cells were refed every 2 days. Cell Extracts and Western Blot Analysis of Proteins—Isolation and Western blot analysis of whole-cell proteins was performed as described previously (8Park B.-H. Qiang L. Farmer S.R. Mol. Cell. Biol. 2004; 24: 8671-8680Crossref PubMed Scopus (165) Google Scholar). Antibodies employed in the analysis were as follows: anti-C/EBPα, anti-C/EBPβ, anti-PPARγ (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-HDAC1–3 (Upstate Biotechnology, Inc., Lake Placid, NY); anti-β-catenin (BD Transduction Laboratories); anti-Acrp30/adiponectin (Affinity BioReagents, Golden, CO); and anti-perilipin (Dr. Andy Greenberg, New England Medical Center, Tufts University, Boston). Immunoprecipitation—Cell extracts prepared as described previously (8Park B.-H. Qiang L. Farmer S.R. Mol. Cell. Biol. 2004; 24: 8671-8680Crossref PubMed Scopus (165) Google Scholar) were incubated overnight at 4 °C with 3–5 μg of the antibody or the same amount of a nonspecific mouse or rabbit IgG. The following day, 30-μl protein-G beads were added, and extracts were incubated with rotation for 3 h at 4°C. The beads were then washed three times with lysis buffer, and antigens were eluted by incubation with 30 μl of Laemmli sample buffer with or without 50 mm dithiothreitol. RNA Analysis—Total RNA was extracted with TRIzol® (Invitrogen) according to the manufacturer's instructions. After quantification, 20–25 μg from each RNA sample was subjected to Northern blot analysis as described previously (17Hamm J.K. Park B.H. Farmer S.R. J. Biol. Chem. 2001; 276: 18464-18471Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Chromatin Immunoprecipitation Assays—Cells were fixed by addition of 37% formaldehyde to a final concentration of 1% formaldehyde and incubation at room temperature for 10 min. Cross-linking was stopped by addition of glycine to a final concentration of 0.125 m. Cells were then trypsinized, scraped, washed with phosphate-buffered saline (PBS), and swelled in hypotonic buffer (10 mm Hepes, pH 7.6, 1.5 mm MgCl2, 10 mm KCl, 0.2 mm phenylmethylsulfonyl fluoride). Nuclei were pelleted by microcentrifugation and lysed by incubation in nuclear lysis buffer (50 mm Tris-HCl, pH 8.1, 10 mm EDTA, 1% SDS) on ice for 10 min. The resulting chromatin solution was sonicated with three 30-s pulses at maximum power. After microcentrifugation, the supernatant was pre-cleared with blocked protein A-agarose beads. The chromatin fractions were then immunoprecipitated with 1–2μg of the following antibodies: anti-C/EBPβ, anti-C/EBPα, anti-acetylated histone H3, anti-acetylated histone H4, anti-HDAC1, anti-RNA polymerase II, or IgG (anti-acetyl-histone H3, anti-acetyl-histone H4, anti-RNA polymerase II, and normal rabbit IgG were purchased from Upstate Biotechnology, Inc.). After incubation at 4 °C overnight, the DNA-protein complexes were immunoprecipitated with protein A-agarose. After washing the DNA-protein complexes, DNA was extracted with phenol/chloroform, precipitated, redissolved, and used as templates for PCR. Different PCR cycles (ranging from 24 to 32) were used to evaluate each assay, and the lowest possible cycle was chosen for presentation. Input and antibody controls were performed at the same number of PCR cycles as the immunoprecipitated complexes. The primers used for the PCR correspond to regions flanking the C/EBP-binding site within the C/ebpα gene promoter and are as follows: sense, 5′-CTG GAA GTG GGT GAC TTA GAG G-3′; antisense, 5′-GAG TGG GGA GCA TAG TGC TAG-3′. Oil Red O Staining—The cells were seeded in 35-mm plates, and at the specified stage of differentiation they were rinsed with PBS and fixed with 10% formalin in PBS for 15 min. After two washes in PBS, cells were stained for at least 1 h in freshly diluted Oil Red O solution (6 parts Oil Red O stock solution and 4 parts H2O; Oil Red O stock solution is 0.5% Oil Red O in isopropyl alcohol). The stain was then removed, and cells were washed twice with water, with or without counterstain (0.25% Giemsa for 15 min), and then photographed. To determine whether activation of C/EBPα expression during the early phase of adipogenesis requires PPARγ activity, we treated 3T3-L1 preadipocytes with T0070907 (a selective and potent antagonist of PPARγ activity) at day 1 following exposure of the confluent cells to the adipogenic inducers DEX, MIX, insulin, and 10% FBS. Fig. 1 shows a Western blot analysis of total proteins extracted from the cells at the indicated days following exposure to the antagonist. It is quite apparent that the PPARγ antagonist completely blocks expression of perilipin (Fig. 1, compare lane 9 with lane 10), a known downstream target of PPARγ (22Arimura N. Horiba T. Imagawa M. Shimizu M. Sato R. J. Biol. Chem. 2004; 279: 10070-10076Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 23Nagai S. Shimizu C. Umetsu M. Taniguchi S. Endo M. Miyoshi H. Yoshioka N. Kubo M. Koike T. Endocrinology. 2004; 145: 2346-2356Crossref PubMed Scopus (57) Google Scholar, 24Dalen K.T. Schoonjans K. Ulven S.M. Weedon-Fekjaer M.S. Bentzen T.G. Koutnikova H. Auwerx J. Nebb H.I. Diabetes. 2004; 53: 1243-1252Crossref PubMed Scopus (172) Google Scholar), and also inhibits differentiation into mature adipocytes (data not shown). More important is the observation that blocking PPARγ activity also leads to a significant attenuation of C/EBPα expression. In fact, the antagonist appears to prevent the activation of C/EBPα during the initial 3 days of adipogenesis (Fig. 1, compare lane 5 with lane 6) prior to the expression of the terminal genes such as perilipin. It is interesting that the antagonist also attenuates expression of PPARγ2. This is likely because of the fact that C/EBPα normally feeds back on the Pparγ2 gene (7Wu Z. Rosen E.D. Brun R. Hauser S. Adelmont G. Troy A.E. McKeon C. Darlington G.J. Spiegelman B.M. Mol. Cell. 1999; 3: 151-158Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar). There appears to be minimal effect of the PPARγ antagonist on C/EBPβ expression. The fact that T0070907 is a specific inhibitor of PPARγ activity suggests that PPARγ contributes to the initial activation of C/EBPα during the differentiation of 3T3-L1 preadipocytes. Because our previous studies had shown that inhibition of C/EBPβ activity by ectopic expression of a dominant negative C/EBPβ protein (LIP) blocks both C/EBPα and PPARγ expression (17Hamm J.K. Park B.H. Farmer S.R. J. Biol. Chem. 2001; 276: 18464-18471Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar), we next addressed the role of C/EBPβ in regulating C/EBPα expression. Earlier studies have demonstrated that C/EBPβ is capable of inducing expression of PPARγ2 in NIH-3T3 fibroblasts (9Wu Z. Bucher N.L.R. Farmer S.R. Mol. Cell. Biol. 1996; 16: 4128-4136Crossref PubMed Google Scholar, 16Wu Z. Xie Y. Bucher N.L.R. Farmer S.R. Genes Dev. 1995; 9: 2350-2363Crossref PubMed Scopus (481) Google Scholar). Unfortunately, it was not possible to assess the role of C/EBPβ in activating C/EBPα expression because the NIH-3T3 cells do not transcribe the C/ebpα gene (10Yeh W.C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (813) Google Scholar, 16Wu Z. Xie Y. Bucher N.L.R. Farmer S.R. Genes Dev. 1995; 9: 2350-2363Crossref PubMed Scopus (481) Google Scholar). To assess the contribution of both C/EBPβ and PPARγ to the induction of C/EBPα during adipogenesis, we ectopically expressed C/EBPβ in Swiss mouse fibroblasts using the Tet-Off conditional expression system (Swiss-LAP A cells). The Swiss-LAP A cells were cultured for several days in the absence of tetracycline in order to stimulate expression of the ectopic C/EBPβ protein. At confluence, the cells were exposed to medium containing 10% FBS and various combinations of the adipogenic inducers, DEX, MIX, or insulin, in the presence or absence of a potent PPARγ agonist (GW7845) for 2 days at which time the cells were maintained in medium containing 10% FBS, insulin, and the corresponding quantity of the PPARγ agonist for an additional 3 days. Fig. 2 shows abundant expression of the ectopic C/EBPβ protein, which results in activation of PPARγ2 expression under all conditions except when cells were cultured in tetracycline to suppress the C/ebpβ gene (Fig. 2, lanes 17 and 18). The ectopic C/EBPβ, in addition to activating PPARγ2, was also capable of inducing perilipin expression (marker of adipogenesis) in response to exposure of these Swiss fibroblasts to DEX (Fig. 2, lanes 4, 10, 12, and 16). Exposure to insulin and/or MIX failed to activate the perilipin gene to any significant extent (Fig. 2, lanes 6, 8, and 14). Of interest is the observation that C/EBPβ did not stimulate expression of C/EBPα in the presence of any of these inducers (Fig. 2, lanes 4, 6, 8, 10, 12, 14, and 16). Importantly, activation of PPARγ2 activity by treatment with the PPARγ agonist (GW7468) induces expression of C/EBPα as well as perilipin to levels normally expressed in mature adipocytes (data not shown) regardless of the presence of the other effectors (DEX, MIX, or insulin). Fig. 2 also demonstrates the reciprocal relationship between β-catenin and adipogenesis in these fibroblasts as observed previously (19Moldes M. Zuo Y. Morrison R.F. Silva D. Park B.H. Liu J. Farmer S.R. Biochem. J. 2003; 376: 607-613Crossref PubMed Scopus (250) Google Scholar, 25Ross S.E. Hemati N. Longo K.A. Bennett C.N. Lucas P.C. Erickson R.L. MacDougald O.A. Science. 2000; 289: 950-953Crossref PubMed Scopus (1539) Google Scholar, 26Liu J. Farmer S.R. J. Biol. Chem. 2004; 279: 45020-45027Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Taken together, these data suggest that activation of PPARγ2 expression only requires C/EBPβ, whereas expression of C/EBPα requires ligand-dependent activation of PPARγ along with C/EBPβ. To substantiate further the importance of PPARγ in regulating C/EBPα expression, we stably expressed the p34 LAP isoform of C/EBPβ in mouse embryo fibroblasts lacking a functional Pparγ gene (Pγ-/- MEFs) and in a corresponding population of heterozygous (Pγ+/-) MEFs. The cell lines (Pγ+/- and Pγ-/-) were exposed to DEX, MIX, and insulin in the presence or absence of troglitazone for 2 days and were then cultured for an additional 3 days in the presence or absence of troglitazone. The cells were either stained with Oil Red O for morphological analysis or harvested for Western blot analysis of total cellular proteins. Fig. 3, A and B, demonstrates that ectopic expression of C/EBPβ induces some lipid accumulation in the MEFs that contain one functional allele of PPARγ (Pγ+/- cells) following exposure to DEX, MIX, and insulin, and this effect is enhanced manyfold in the presence of the troglitazone. As expected, C/EBPβ failed to induce any lipid accumulation in the PPARγ-deficient cells (Pγ-/-) with or without troglitazone. The absence of adipogenesis in the Pγ-/- cells did not necessarily mean that C/EBPβ was not capable of inducing expression of C/EBPα in these cells because other studies have shown that C/EBPα is incapable of inducing lipid accumulation in the absence of PPARγ (20Rosen E.D. Hsu C.H. Wang X. Sakai S. Freeman M.W. Gonzalez F.J. Spiegelman B.M. Genes Dev. 2002; 16: 22-26Crossref PubMed Scopus (1118) Google Scholar). Consequently, we analyzed expression of C/EBPα in response to C/EBPβ in both Pγ+/- and Pγ-/- cells. Fig. 4A shows abundant expression of the ectopic C/EBPβ in both cell lines. Treatment of the Pγ+/- cells with DEX, MIX, and insulin induces a low but detectable level of PPARγ, C/EBPα, perilipin, and adiponectin expression (Fig. 4A, lane 3), and exposure to the PPARγ agonist enhances expression of all four proteins to the abundant levels (lane 4) normally expressed in mature adipocytes. Most importantly, exposure of the PPARγ-deficient cells to DEX, MIX, and insulin with or without PPARγ ligand was incapable of stimulating C/EBPα, perilipin, or adiponectin even though the cells produced abundant quantities of the ectopic C/EBPβ protein (Fig. 4A, lanes 1 and 2). The Northern blot analysis of these cells demonstrate that the absence of C/EBPα expression in Pparγ-/- MEFs is because of a lack of expression of the corresponding mRNA (Fig. 4B, compare lane 2 with lane 4). These data support the notion that C/EBPβ activates a single unified pathway of adipogenesis (20Rosen E.D. Hsu C.H. Wang X. Sakai S. Freeman M.W. Gonzalez F.J. Spiegelman B.M. Genes Dev. 2002; 16: 22-26Crossref PubMed Scopus (1118) Google Scholar) involving its induction of PPARγ, which then activates C/EBPα expression along with other terminal adipogenic programs. To confirm that the only defect in the Pparγ-/- MEFs was the lack of PPARγ, we retrovirally expressed PPARγ2 in these cells that already expressed the ectopic C/EBPβ and showed that its expression facilitated induction of C/EBPα along with the other markers of the adipogenic program (Fig. 4C).FIGURE 4C/EBPβ induces C/ebpα gene expression in Pγ+/- MEFs, but not in Pγ-/- MEFs. Pγ+/- and Pγ-/- MEFs expressing an ectopic C/EBPβ were induced to differentiate for 6 days as described in Fig. 3. At this time, cells were harvested for either total proteins or RNA as described under “Experimental Procedures.” A, equal amounts of total cellular protein were subjected to Western blot analysis of C/EBPβ, PPARγ, C/EBPα, perilipin, and adiponectin. B, equal amounts of total RNA were analyzed by Northern blot hybridization using cDNA probes corresponding to PPARγ and C/EBPα. The membrane was also stained with methylene blue to visualize the rRNA species and verify the presence of equal amounts of RNA. C, ectopic PPARγ2 rescues expression of C/EBPα in Pγ-/- cells expressing C/EBPβ. Pparγ-/- MEFs expressing an ectopic C/EBPβ (LAP) were infected with retrovirus vector containing PPARγ2 cDNA (pBabe-Pγ) or the empty vector (pBabe). The cell lines were cultured in growth medium for 3 days until confluent and were then induced to differentiate as described under “Experimental Procedures.” At day 5, cells were harvested for Western blot analysis of C/EBPβ, PPARγ, C/EBPα, perilipin, aP2, and adiponectin.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Other studies have
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