The Orphan Nuclear Receptor Rev-Erbα Is a Peroxisome Proliferator-activated Receptor (PPAR) γ Target Gene and Promotes PPARγ-induced Adipocyte Differentiation
2003; Elsevier BV; Volume: 278; Issue: 39 Linguagem: Inglês
10.1074/jbc.m304664200
ISSN1083-351X
AutoresCoralie Fontaine, Guillaume Dubois, Yannick Duguay, Torben Helledie, Ngoc Vu‐Dac, Philippe Gervois, Fabrice Soncin, Susanne Mandrup, Jean‐Charles Fruchart, Jamila Fruchart‐Najib, Bart Staels,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoRev-Erbα (NR1D1) is an orphan nuclear receptor encoded on the opposite strand of the thyroid receptor α gene. Rev-Erbα mRNA is induced during adipocyte differentiation of 3T3-L1 cells, and its expression is abundant in rat adipose tissue. Peroxisome proliferator-activated receptor γ (PPARγ) (NR1C3) is a nuclear receptor controlling adipocyte differentiation and insulin sensitivity. Here we show that Rev-Erbα expression is induced by PPARγ activation with rosiglitazone in rat epididymal and perirenal adipose tissues in vivo as well as in 3T3-L1 adipocytes in vitro. Furthermore, activated PPARγ induces Rev-Erbα promoter activity by binding to the direct repeat (DR)-2 response element Rev-DR2. Mutations of the 5′ or 3′ half-sites of the response element totally abrogated PPARγ binding and transcriptional activation, identifying this site as a novel type of functional PPARγ response element. Finally, ectopic expression of Rev-Erbα in 3T3-L1 preadipocytes potentiated adipocyte differentiation induced by the PPARγ ligand rosiglitazone. These results identify Rev-Erbα as a target gene of PPARγ in adipose tissue and demonstrate a role for this nuclear receptor as a promoter of adipocyte differentiation. Rev-Erbα (NR1D1) is an orphan nuclear receptor encoded on the opposite strand of the thyroid receptor α gene. Rev-Erbα mRNA is induced during adipocyte differentiation of 3T3-L1 cells, and its expression is abundant in rat adipose tissue. Peroxisome proliferator-activated receptor γ (PPARγ) (NR1C3) is a nuclear receptor controlling adipocyte differentiation and insulin sensitivity. Here we show that Rev-Erbα expression is induced by PPARγ activation with rosiglitazone in rat epididymal and perirenal adipose tissues in vivo as well as in 3T3-L1 adipocytes in vitro. Furthermore, activated PPARγ induces Rev-Erbα promoter activity by binding to the direct repeat (DR)-2 response element Rev-DR2. Mutations of the 5′ or 3′ half-sites of the response element totally abrogated PPARγ binding and transcriptional activation, identifying this site as a novel type of functional PPARγ response element. Finally, ectopic expression of Rev-Erbα in 3T3-L1 preadipocytes potentiated adipocyte differentiation induced by the PPARγ ligand rosiglitazone. These results identify Rev-Erbα as a target gene of PPARγ in adipose tissue and demonstrate a role for this nuclear receptor as a promoter of adipocyte differentiation. Adipocyte differentiation is a complex biological process, which is reflected at the molecular level by the transcriptional activation of a number of adipocyte-specific genes and by the acquisition of the ability to accumulate cytoplasmic lipid droplets (1Cornelius P. MacDougald O.A. Lane M.D. Annu. Rev. Nutr. 1994; 14: 99-129Crossref PubMed Scopus (575) Google Scholar, 2Spiegelman B.M. Flier J.S. Cell. 1996; 87: 377-389Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar, 3Rosen E.D. Walkey C.J. Puigserver P. Spiegelman B.M. Genes Dev. 2000; 14: 1293-1307Crossref PubMed Google Scholar). The nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ, 1The abbreviations used are: PPAR, peroxisome proliferator-activated receptor; DMEM, Dulbecco's modified Eagle's medium; EMSA, electrophoretic mobility shift assay; RSG, rosiglitazone; DR, direct repeat; IBMX, 3-methyl-1-isobutylxanthine. NR1C3) (4Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3133) Google Scholar, 5Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (2005) Google Scholar) and members of the CCAAT enhancer-binding protein (C/EBP) family (6Christy R.J. Yang V.W. Ntambi J.M. Geiman D.E. Landschulz W.H. Friedman A.D. Nakabeppu Y. Kelly T.J. Lane M.D. Genes Dev. 1989; 3: 1323-1335Crossref PubMed Scopus (467) Google Scholar, 7Freytag S.O. Geddes T.J. Science. 1992; 256: 379-382Crossref PubMed Scopus (251) Google Scholar, 8Freytag S.O. Paielli D.L. Gilbert J.D. Genes Dev. 1994; 8: 1654-1663Crossref PubMed Scopus (393) Google Scholar, 9Wu Z. Xie Y. Bucher N.L. Farmer S.R. Genes Dev. 1995; 9: 2350-2363Crossref PubMed Scopus (481) Google Scholar, 10Wu Z. Bucher N.L. Farmer S.R. Mol. Cell. Biol. 1996; 16: 4128-4136Crossref PubMed Google Scholar, 11Yeh W.C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (813) Google Scholar, 12Holst D. Grimaldi P.A. Curr. Opin. Lipidol. 2002; 13: 241-245Crossref PubMed Scopus (79) Google Scholar) play key roles in this adipogenic process. In addition, the adipocyte differentiation and determination factor-1 (SREBP-1/ADD1) appears to promote adipocyte differentiation by activating the expression of PPARγ and increasing the synthesis of endogenous PPARγ ligands (13Kim J.B. Spiegelman B.M. Genes Dev. 1996; 10: 1096-1107Crossref PubMed Scopus (850) Google Scholar, 14Tontonoz P. Kim J.B. Graves R.A. Spiegelman B.M. Mol. Cell. Biol. 1993; 13: 4753-4759Crossref PubMed Scopus (536) Google Scholar, 15Kim J.B. Wright H.M. Wright M. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4333-4337Crossref PubMed Scopus (562) Google Scholar). Members of the PPAR family bind as heterodimers with the retinoid X receptors (RXR) to specific response elements termed peroxisome proliferator response elements (PPRE) (for review see Ref. 16Barbier O. Torra I.P. Duguay Y. Blanquart C. Fruchart J.C. Glineur C. Staels B. Arterioscler. Thromb. Vasc. Biol. 2002; 22: 717-726Crossref PubMed Scopus (383) Google Scholar). These PPREs usually consist of a direct repeat of the PuGGTCA motif spaced by one nucleotide (DR1). The transcriptional activity of the PPARs is activated by a number of different fatty acid metabolites, most notably products of the cycloxygenase and lipoxygenase pathways. In addition, a large number of synthetic compounds are known to be potent and subtype specific PPAR ligands. For example, thiazolidinedione compounds used as insulin sensitizers in the treatment of type II diabetes are high affinity PPARγ ligands (17Willson T.M. Lambert M.H. Kliewer S.A. Annu. Rev. Biochem. 2001; 70: 341-367Crossref PubMed Scopus (542) Google Scholar). Rev-Erbα (NR1D1) is another nuclear receptor, the expression of which is induced during adipocyte differentiation (18Chawla A. Lazar M.A. J. Biol. Chem. 1993; 268: 16265-16269Abstract Full Text PDF PubMed Google Scholar). Rev-Erbα is highly expressed in adipose tissue but also in skeletal muscle, liver and brain (18Chawla A. Lazar M.A. J. Biol. Chem. 1993; 268: 16265-16269Abstract Full Text PDF PubMed Google Scholar, 19Forman B.M. Chen J. Blumberg B. Kliewer S.A. Henshaw R. Ong E.S. Evans R.M. Mol. Endocrinol. 1994; 8: 1253-1261Crossref PubMed Scopus (184) Google Scholar, 20Lazar M.A. Hodin R.A. Darling D.S. Chin W.W. Mol. Cell. Biol. 1989; 9: 1128-1136Crossref PubMed Scopus (264) Google Scholar, 21Miyajima N. Horiuchi R. Shibuya Y. Fukushige S. Matsubara K. Toyoshima K. Yamamoto T. Cell. 1989; 57: 31-39Abstract Full Text PDF PubMed Scopus (200) Google Scholar). Since no ligand has been identified so far, Rev-Erbα is considered as an orphan member of the nuclear receptor superfamily. Rev-Erbα has been shown to act as a negative regulator of transcription (22Laudet V. Adelmant G. Curr. Biol. 1995; 5: 124-127Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) binding either as monomer on nuclear receptor half-site motifs flanked 5′ by an A/T rich sequence (A/T PuGGTCA), or as a homodimer to a direct repeat of the PuGGTCA motif spaced by two nucleotides (DR2). We have previously shown that PPARα activates the expression of Rev-Erbα through an atypical PPRE, a DR-2 element, in the Rev-Erbα promoter (23Gervois P. Chopin-Delannoy S. Fadel A. Dubois G. Kosykh V. Fruchart J.C. Najib J. Laudet V. Staels B. Mol. Endocrinol. 1999; 13: 400-409PubMed Google Scholar). Transcriptional activation by PPARγ through a DR-2 element has so far not been reported. However, since Rev-Erbα is induced during the course of adipocyte differentiation, we decided to investigate whether PPARγ could be involved in transcriptional induction of Rev-Erbα expression in adipocytes. Furthermore, we wanted to investigate whether Rev-Erbα plays a modulatory role in the process of adipogenesis. Our results from both in vivo and in vitro studies demonstrate that treatment with the PPARγ agonist rosiglitazone increases Rev-Erbα gene expression and that PPARγ activates Rev-Erbα transcription via the Rev-DR2 response element present in the human Rev-Erbα promoter. Finally, we show that ectopic expression of Rev-Erbα in 3T3-L1 preadipocytes significantly augments the adipogenic activity of the PPARγ selective ligand rosiglitazone. Animals—Male Sprague-Dawley rats (10 weeks old) were treated for 14 days by gavage with rosiglitazone (10 mg/kg/d) suspended in 1% carboxymethylcellulose solution. Control animals received an equal volume (5 ml/kg/d) of carboxymethylcellulose solution. At the end of the experiments, animals were sacrified by exsanguination under ether anesthesia. Adipose tissues were removed immediately and frozen in liquid nitrogen. RNA Analysis—RNA extraction of mice adipose tissues and 3T3-L1 cells and Northern blot analysis were performed as previously described (24Staels B. van Tol A. Andreu T. Auwerx J. Arterioscler. Thromb. 1992; 12: 286-294Crossref PubMed Google Scholar) using rat Rev-Erbα (20Lazar M.A. Hodin R.A. Darling D.S. Chin W.W. Mol. Cell. Biol. 1989; 9: 1128-1136Crossref PubMed Scopus (264) Google Scholar), mouse PPARγ (25Barak 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), rat aP2 (26Ross S.R. Graves R.A. Greenstein A. Platt K.A. Shyu H.L. Mellovitz B. Spiegelman B.M. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9590-9594Crossref PubMed Scopus (184) Google Scholar), chicken β-actin (27Cleveland D.W. Lopata M.A. MacDonald R.J. Cowan N.J. Rutter W.J. Kirschner M.W. Cell. 1980; 20: 95-105Abstract Full Text PDF PubMed Scopus (1312) Google Scholar), or rat 36B4 (28Masiakowski P. Breathnach R. Bloch J. Gannon F. Krust A. Chambon P. Nucleic Acids Res. 1982; 10: 7895-7903Crossref PubMed Scopus (702) Google Scholar) cDNA probes. For extraction of RNA, Trizol reagent (Invitrogen) was used following the manufacturer's instructions. Northern analysis was performed using Ultrahyb hybridization solution (Ambion, Austin, TX). Hybridization and washes were done according to the manufacturer's directions. Reverse transcription was performed with MMLV reverse transcriptase starting with 1 μg of total RNA following the manufacturer's instructions (Invitrogen). For quantitative PCR, reverse transcribed were quantified by real time PCR on a MX4000 apparatus (Stratagene, La Jolla, CA), using specific oligonucleotide primers indicated in Table I. PCR amplification was performed in a volume of 25 μl containing 100 nm of each primers, 4 mm MgCl2, the Brilliant Quantitative PCR Core reagent Kit mix as recommended by the manufacturer (Stratagene, La Jolla, CA). The conditions were 95 °C for 10 min, followed by 40 cycles of 30 s at 95 °C, 30 s at 55 °C, and 30 s at 72 °C. mRNA levels were normalized to 28S rRNA. The values presented are means ± S.D. of triplicates.Table IOligonucleotide primersNameOrientationSequenceh/m Rev-ErbαForward5′-GACATGACGACCCTGGACTC-3′h/m Rev-ErbαReverse5′-GCTGCCATTGGAGTTGTCAC-3′m Rev-ErbαForward5′-TGGCCTCAGGCTTCCACTATG-3′m Rev-ErbαReverse5′-CCGTTGCTTCTCTCTCTTGGG-3′m aP2Forward5′-GAATTCGATGAAATCACCGCA-3′m aP2Reverse5′-CTCTTTATTGTGGTCGACTTTCCA-3′m PPARγForward5′-TGCTGTTATGGGTGAAACTCTGGG-3′m PPARγReverse5′-CGCTTGATGTCAAAGGAATGCG-3′m CEBPαForward5′-CTGCGAGCACGAGACGTCTATAG-3′m CEBPαReverse5′-TCCCGGGTAGTCAAAGTCACC-3′m CEBPβForward5′-CCAAGGCCAAGGCCAAGAAG-3′m CEBPβReverse5′-AAGTTCCGCAGGGTGCTGAG-3′m SREBP-1Forward5′-GTTACTCGAGCCTGCCTTCAGG-3′m SREBP-1Reverse5′-GCTGCCATTGGAGTTGTCAC-3′h/m 28SForward5′-GCACATCGGGGTTGAAGAGG-3′h/m 28SReverse5′-AAACTCTGGTGGAGGTCCGT-3′ Open table in a new tab Transfection Experiments—The human Rev-Erbα promoter constructs were described previously (29Adelmant G. Begue A. Stehelin D. Laudet V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3553-3558Crossref PubMed Scopus (100) Google Scholar), 3T3-L1 and COS cells were obtained from ATCC (Manassas, VA). Cells were grown in DMEM, supplemented with 2 mm glutamine and 10% (v/v) fetal calf serum in a 5% CO2 humidified atmosphere at 37 °C. All transfections were carried out as described previously (23Gervois P. Chopin-Delannoy S. Fadel A. Dubois G. Kosykh V. Fruchart J.C. Najib J. Laudet V. Staels B. Mol. Endocrinol. 1999; 13: 400-409PubMed Google Scholar). 3T3-L1 preadipocytes were transfected using the cationic lipid RPR 120535B as described previously (30Raspe E. Madsen L. Lefebvre A.M. Leitersdorf I. Gelman L. Peinado-Onsurbe J. Dallongeville J. Fruchart J.C. Berge R. Staels B. J. Lipid Res. 1999; 40: 2099-2110Abstract Full Text Full Text PDF PubMed Google Scholar). Luciferase activities were determined on total cell extracts using a luciferase assay system (Promega, Madison, WI). Transfection experiments were performed in triplicate and repeated at least three times. In Vitro Translation and EMSAs—pSG5hPPARγ, pSG5mRXRα, and pSG5hRev-Erbα were in vitro transcribed with T7 polymerase and translated using the rabbit reticulocyte lysate system (Promega, Madison, WI). Electrophoretic mobility shift assays (EMSAs) with Rev-Erbα, PPARγ, and/or RXRα were performed exactly as described previously (23Gervois P. Chopin-Delannoy S. Fadel A. Dubois G. Kosykh V. Fruchart J.C. Najib J. Laudet V. Staels B. Mol. Endocrinol. 1999; 13: 400-409PubMed Google Scholar). For competition experiments, increasing amounts of the indicated cold probe were added just before the labeled oligonucleotide. The complexes were resolved on 5% polyacrylamide gels in 0.25× TBE buffer (90 mm Tris borate, 2.5 mm EDTA, pH 8.3) at room temperature. Gels were dried and exposed overnight at –70 °C to x-ray film (XO-MAT-AR, Eastman Kodak, Rochester, NY). Viral Production and Infection—GP+E86 virus-encapsidating cells (31Markowitz D. Goff S. Bank A. J. Virol. 1988; 62: 1120-1124Crossref PubMed Google Scholar) were cultured in DMEM (4.5 g/liter glucose) containing 10% heat-inactivated calf serum (Hyclone), 8 μg/ml gentamycin, 50 units/ml penicillin, 50 μg/ml streptomycin at 37 °C in a 5% CO2/95% air humidified atmosphere. In order to generate cell lines that constitutively overexpress Rev-Erbα, the coding sequence of human Rev-Erbα was inserted in front of the internal ribosome entry site and the neomycin resistance gene (pCITE, Novagen) of the MFG retrovirus plasmid (32Dranoff G. Jaffee E. Lazenby A. Golumbek P. Levitsky H. Brose K. Jackson V. Hamada H. Pardoll D. Mulligan R.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 3539-3543Crossref PubMed Scopus (2670) Google Scholar) using the NcoI-BamHI sites to generate pMFG-Rev-Erbα. A similar construction lacking the Rev-Erbα sequence was made and used throughout the study as a negative control (pMFG-Neo). The bicistronic construct was designed to allow separate expression of the Rev-Erbα protein and the neomycin resistance by the infected cells. GP+E86 cells (15,000/cm2) were transfected with the MFG plasmid constructs (2 μg) using LipofectAMINE (Invitrogen) and selected for resistance using the geneticin analog G418 (0.8 mg/ml, Invitrogen). 3T3-L1 cells were infected with the GP+E86-produced MFG-Neo or MFG-Rev viruses essentially as previously described (33Mattot V. Vercamer C. Soncin F. Calmels T. Huguet C. Fafeur V. Vandenbunder B. Oncogene. 2000; 19: 762-772Crossref PubMed Scopus (36) Google Scholar). Geneticin-resistant infected cell pools were used for the studies within three passages after infection. Cell Culture and Differentiation—3T3-L1 cells were cultured in growth medium containing DMEM and 10% calf serum. The cells were differentiated by the method of Bernlohr et al. (34Bernlohr D.A. Angus C.W. Lane M.D. Bolanowski M.A. Kelly Jr., T.J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 5468-5472Crossref PubMed Scopus (244) Google Scholar). 2 days post-confluent cells (designated day 0) were switched to differentiation medium (DMEM, 10% fetal calf serum, 1 μm dexamethasone, 10 μg/ml insulin, and 0.5 mm 3-methyl-1-isobutylxanthine (IBMX) (Sigma)) for 2 days. Thereafter, the cells were cultured in post-differentiation medium (DMEM, 10% fetal calf serum, insulin) with or without rosiglitazone (1 μm). The medium was changed every day. Retroviral infected 3T3-L1 preadipocytes were cultured under the same conditions, but differentiated either with (standard differentiation conditions) or without (rosiglitazone-dependent differentiation conditions) dexamethasone. After treatment the cells were fixed with 10% formaldehyde in phosphate-buffered saline and stained with Oil Red O (Sigma) or total RNA was extracted as described above (35Tontonoz P. Hu E. Devine J. Beale E.G. Spiegelman B.M. Mol. Cell. Biol. 1995; 15: 351-357Crossref PubMed Google Scholar). Activation of PPARγ Increases Rev-Erbα mRNA Levels in Adipose Tissue—In order to determine whether PPARγ activation affects Rev-Erbα expression in vivo, rats were treated for 14 days with rosiglitazone, a highly specific and potent PPARγ ligand (36Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3469) Google Scholar). The expression of Rev-Erbα was analyzed in epididymal and perirenal adipose tissue by Northern blot analysis. Compared with control treated rats, rosiglitazone treatment strongly increased Rev-Erbα mRNA levels both in epididymal and perirenal adipose tissue (Fig. 1), whereas no change in control β-actin mRNA levels was observed. These experiments demonstrate that PPARγ activators increase Rev-Erbα mRNA levels in adipose tissue in vivo. PPARγ Activation Induces Rev-Erbα mRNA in 3T3-L1 Preadipocytes—To study the molecular and cellular mechanisms of this induction, we next investigated the regulation of Rev-Erbα mRNA expression by rosiglitazone in the 3T3-L1 preadipocyte cell line. Cells were grown until confluency in medium containing calf serum. 2 days post-confluent cells (designated day 0), cells were transferred to medium containing fetal calf serum and differentiated with the classic differentiation mixture containing dexamethasone, IBMX, and insulin. From day 2, either 1 μm rosiglitazone or vehicle was added. RNA was harvested at day 0, 2, 4, 6, and 8 and used for Northern analysis. As previously reported (18Chawla A. Lazar M.A. J. Biol. Chem. 1993; 268: 16265-16269Abstract Full Text PDF PubMed Google Scholar), Rev-Erbα mRNA levels increased upon differentiation of preadipocytes into adipocytes (Fig. 2, A and B). However, compared with the standard differentiation treatment, Rev-Erbα mRNA levels were induced earlier in the presence of rosiglitazone. Moreover, Rev-Erbα mRNA levels were higher after 8 days in fully differentiated 3T3-L1 adipocytes treated with rosiglitazone compared with controls (Fig. 2, A and B). Rosiglitazone is known to be a potent inducer of differentiation, thus in order to distinguish between direct effects of rosiglitazone on Rev-Erbα gene expression and indirect effects mediated via increased differentiation, we investigated whether rosiglitazone was able to induce Rev-Erbα in fully differentiated day 10 adipocytes. As shown in Fig. 2, C and D, rosiglitazone activated the expression of Rev-Erbα as well as the adipocyte lipid-binding protein (ALBP/aP2), a well-characterized PPARγ target gene (5Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (2005) Google Scholar), in mature adipocytes. Inhibition of protein synthesis by cycloheximide caused a superinduction of Rev-Erbα and could therefore not be used to investigate whether the induction of Rev-Erbα was mediated directly by PPARγ (data not shown). Nevertheless, these experiments demonstrate that activation of PPARγ increases Rev-Erbα mRNA during in vitro differentiation of 3T3-L1 preadipocytes as well as in mature adipocytes. PPARγ Induces Rev-Erbα Expression at the Transcriptional Level—PPARα has previously been shown to activate the human Rev-Erbα promoter in hepatocytes via a DR-2 element (23Gervois P. Chopin-Delannoy S. Fadel A. Dubois G. Kosykh V. Fruchart J.C. Najib J. Laudet V. Staels B. Mol. Endocrinol. 1999; 13: 400-409PubMed Google Scholar), through which Rev-Erbα represses also its own transcription (29Adelmant G. Begue A. Stehelin D. Laudet V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3553-3558Crossref PubMed Scopus (100) Google Scholar). To determine whether this site also mediates the activation of the Rev-Erbα promoter by PPARγ in adipocytes, transfection assays were carried out using luciferase reporter constructs driven by the Rev-Erbα promoter. 3T3-L1 cells were cotransfected with the PPARγ expression vector (pSG5-hP-PARγ) or empty vector (pSG5) and treated with rosiglitazone or vehicle. Rev-Erbα promoter activity was induced by PPARγ cotransfection, an effect that was enhanced in the presence of rosiglitazone (Fig. 3). By contrast, Rev-Erbα promoter activity was unaffected by rosiglitazone in the absence of overexpressed PPARγ, probably due to the insufficient levels of endogenous PPARγ in non-confluent preadipocytes. These data indicate that Rev-Erbα gene transcription is induced by rosiglitazone via PPARγ. To further investigate the importance of the DR-2 element in the induction by PPARγ, Rev-Erbα promoter constructs in which the Rev-DR2 site was mutated, were tested next. Mutations affecting the 5′-AGGTCA motif (pGL2hRev-Erbα Δ as described (29Adelmant G. Begue A. Stehelin D. Laudet V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3553-3558Crossref PubMed Scopus (100) Google Scholar) (Fig. 4A)) of the Rev-DR2 site resulted in the loss of Rev-Erbα promoter inducibility by rosiglitazone and PPARγ (Fig. 4B). These results indicate that the Rev-DR2 site mediates the transcriptional induction of Rev-Erbα by PPARγ. To assess whether the Rev-DR2 site could also function as a PPARγ-responsive element in front of a heterologous promoter, transient transfection experiments were performed using constructs containing the wild-type or mutated versions of the Rev-DR2 site (Fig. 4B) cloned in front of the heterologous SV40 promoter (Rev-DR2, Rev-DR2M5′ and Rev-DR2M3′). In COS cells, cotransfection of pSG5-hPPARγ on the Rev-DR2 driven SV40 reporter vector led to a 2.5-fold induction of transcription activity compared with empty pSG5 vector cotransfection (Fig. 4C). This effect was enhanced in the presence of rosiglitazone. By contrast, PPARγ did not activate the Rev-DR2 site mutated in its 5′-AGGTCA half-site (Rev-DR2M5′). Furthermore, mutation of the 3′-half-site (Rev-DR2M3′) of the DR2 site also abolished transactivation by PPARγ. These results clearly demonstrate that the Rev-Erbα human promoter is regulated by PPARγ and that this induction is mediated via the Rev-DR2 site. PPARγ Binds as a Heterodimer with RXRα to the Rev-DR2 Site—To investigate whether PPARγ binds directly to the Rev-DR2 site, electrophoretic mobility shift assays (EMSAs) were performed using in vitro synthesized PPARγ and RXRα proteins. Since the Rev-DR2 site was previously described as a Rev-Erbα response element, binding of Rev-Erbα was assayed as a control (Fig. 5A). As expected (29Adelmant G. Begue A. Stehelin D. Laudet V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3553-3558Crossref PubMed Scopus (100) Google Scholar), Rev-Erbα bound both as homo- and as monomer to the Rev-DR2 site. Furthermore, no binding was observed on the Rev-DR2 oligonucleotide carrying a mutation in the 5′-half-site (M5′), whereas Rev-Erbα bound only as a monomer to the Rev-DR2 carrying a mutation in the 3′-half-site (M3′) in accordance with previous observations (29Adelmant G. Begue A. Stehelin D. Laudet V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3553-3558Crossref PubMed Scopus (100) Google Scholar). RXRα or PPARγ alone did not bind to either wild-type or mutated Rev-DR2 sites confirming that PPARγ cannot bind as a monomer. By contrast, binding to the Rev-DR2 site was observed when PPARγ was incubated in the presence of RXRα. The binding was specific since it was competed out by excess of unlabeled oligonucleotide (Fig. 5A). The specificity of the binding complex was verified by the addition of a specific anti-PPARγ antibody (38Fajas 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. J. Biol. Chem. 1997; 272: 18779-18789Abstract Full Text Full Text PDF PubMed Scopus (1087) Google Scholar), which inhibited formation of the PPARγ/RXRα complex. The binding was prevented by mutation of both the 5′-(M5′) or 3′-(M3′) half-sites of the Rev-DR2 element. To determine the relative binding affinity of the PPARγ/RXRα heterodimer for the Rev-DR2 site, cross-competition EMSA experiments were performed comparing binding of PPARγ/RXRα to the natural DR-1 site in the aP2 promoter (5Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (2005) Google Scholar) and the Rev-DR2 site. As shown in Fig. 5B, competition with the cold Rev-DR2 site decreased PPARγ/RXRα binding to both the radiolabeled Rev-DR2 and aP2 DR-1 sites. These experiments demonstrate that PPARγ binds as a heterodimer with RXRα to the Rev-DR2 site of the Rev-Erbα promoter, albeit with significantly lower affinity compared with the aP2 PPRE site (Fig. 5B). Rev-Erbα Increases the Adipogenic Activity of PPARγ Agonists—To assess a potential role of Rev-Erbα in adipogenesis, full-length human Rev-Erbα was cloned into a retroviral vector, and 3T3-L1 preadipocytes were infected with the resulting virus (MFG-Rev) or the control MFG-Neo virus. Pools of cells stably transduced, but not clonal selected 3T3-L1 cells were subsequently cultured with an incomplete differentiation mixture, which requires PPARγ activation with rosiglitazone for optimal differentiation, and either rosiglitazone (1 μm) or vehicle was added from day 2 to day 8. The presence of Rev-Erbα in MFG-Neo or MFG-Rev infected cells was analyzed by Western blot (Fig. 6A) and Northern blot analysis (Fig. 6B) of 3T3-L1 cells treated for 6 days with the differentiation mixture. Western blot analysis demonstrated the presence of ectopic expressed human Rev-Erbα protein in MFG-Rev infected, but not in the control cells (Fig. 6A). Since the antibody used was raised against a peptide, its affinity was too low to detect endogenous Rev-Erbα protein. However, both ectopic and endogenous Rev-Erbα expression was detected by Northern blot analysis in MFG-Rev cells, whereas MFG-Neo cells only expressed endogenous Rev-Erbα (Fig. 6B). The effect of ectopic Rev-Erbα on adipocyte differentiation was investigated using Oil Red O staining to assess triglyceride accumulation in 3T3-L1 cells induced to differentiate in incomplete medium (Insulin and IBMX, but no dexamethasone). Under these conditions, full differentiation is dependent on the presence of the PPARγ agonist rosiglitazone. In the absence of rosiglitazone, ectopic expression of Rev-Erbα induced only a slight increase in triglyceride accumulation (Fig. 6, C and D). However, when rosiglitazone was added, a major increase in triglyceride accumulation was observed in the cells expressing Rev-Erbα compared with control cells. These morphological changes were accompanied by a pronounced induction of mRNA levels of the adipocyte-specific marker, and PPARγ target gene, aP2, whose expression was strongly induced by rosiglitazone and Rev-Erbα (Fig. 7). Moreover, a strong increase in PPARγ mRNA levels was observed in MFG-Rev compared with MFG-Neo cells, which may be a reflection of the differentiation status of the cells. In addition, rosiglitazone treatment induced PPARγ expression due to PPARγ auto-induction (39Mueller E. Drori S. Aiyer A. Yie J. Sarraf P. Chen H. Hauser S. Rosen E.D. Ge K. Roeder R.G. Spiegelman B.M. J. Biol. Chem. 2002; 277: 41925-41930Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar). Similarly, mRNA levels of C/EBPα, another differentiation marker, were also induced by Rev-Erbα over-expression. Moreover, as expected, C/EBPα mRNA level was induced by rosiglitazone in MFG-Neo cells, likely due to the cross regulation of PPARγ and C/EBPα (40Wu Z. Rosen E.D. Brun R. Hauser S. Adelmant 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). However, in Rev-Erbα expressing cells, rosiglitazone treatment did not further enhance C/EBPα expression. Although the expression levels of C/EBPβ, an early inducer of adipocyte differentiation, and SREBP-1/ADD1 were higher upon rosiglitazone treatment, likely due to the optimal differentiation status of the cells under these conditions, the mRNA levels of these transcription factors were not influenced by Rev-erbα expression (Fig. 7). Next, the influence of Rev-Erbα on the expression of aP2, PPARγ and C/EBPα was determined when 3T3-L1 cells were differentiated under classical conditions (insulin, IBMX, and dexamethasone). As expected, induction levels of aP2, PPARγ, C/EBPα, and endogenous Rev-Erbα were more pronounced in the complete compared with the incomplete mixture without dexamethasone (compare Fig. 7 to Fig. 8). Induction levels of these adipogenic markers were identical between non-infected cells and MFG-Neo cells indicating that the retrovirus-infected cells differentiate normally (data not shown). Interestingly, Rev-Erbα infected cells expressed Rev-Erbα mRNA levels similar to th
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