CCAAT/Enhancer-binding Protein α Is Required for Transcription of the β3-Adrenergic Receptor Gene during Adipogenesis
2001; Elsevier BV; Volume: 276; Issue: 1 Linguagem: Inglês
10.1074/jbc.m008440200
ISSN1083-351X
AutoresTonya M. Dixon, Kiefer W. Daniel, Stephen R. Farmer, Sheila Collins,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoThe β3-adrenergic receptor (β3AR) is expressed predominantly in adipocytes, and it plays a major role in regulating lipolysis and adaptive thermogenesis. Its expression in a variety of adipocyte cell models is preceded by the appearance of CCAAT/enhancer-binding protein α (C/EBPα), which has been shown to regulate a number of other adipocyte-specific genes. Importantly, it has been demonstrated that several adipocyte cell lines that fail to express C/EBPα exhibit reduced insulin sensitivity, despite an apparent adipogenic phenotype. Here we show that transcription and function of the β3AR correlates with C/EBPα expression in these adipocyte models. A 5.13-kilobase pair fragment of the mouse β3AR promoter was isolated and sequenced. This fragment conferred a 50-fold increase in luciferase reporter gene expression in adipocytes. Two putative C/EBP binding sites exist at −3306 to −3298 and at −1462 to −1454, but only the more distal site is functional. Oligonucleotides corresponding to both the wild-type and mutated −3306 element were inserted upstream of a thymidine kinase luciferase construct. When cotransfected in fibroblasts with a C/EBPα expression vector, reporter gene expression increased 3-fold only in the wild-type constructs. The same mutation, when placed into the intact 5.13-kilobase pair promoter, reduced promoter activity in adipocytes from 50-fold to <10-fold. Electrophoretic mobility shift analysis demonstrated that the site at −3306 generated a specific protein-oligonucleotide complex that was supershifted by C/EBPα antibody, while a probe corresponding to a putative site at −1462 did not. These results define C/EBPα as a key transcriptional regulator of the mouse β3AR gene during adipogenesis. The β3-adrenergic receptor (β3AR) is expressed predominantly in adipocytes, and it plays a major role in regulating lipolysis and adaptive thermogenesis. Its expression in a variety of adipocyte cell models is preceded by the appearance of CCAAT/enhancer-binding protein α (C/EBPα), which has been shown to regulate a number of other adipocyte-specific genes. Importantly, it has been demonstrated that several adipocyte cell lines that fail to express C/EBPα exhibit reduced insulin sensitivity, despite an apparent adipogenic phenotype. Here we show that transcription and function of the β3AR correlates with C/EBPα expression in these adipocyte models. A 5.13-kilobase pair fragment of the mouse β3AR promoter was isolated and sequenced. This fragment conferred a 50-fold increase in luciferase reporter gene expression in adipocytes. Two putative C/EBP binding sites exist at −3306 to −3298 and at −1462 to −1454, but only the more distal site is functional. Oligonucleotides corresponding to both the wild-type and mutated −3306 element were inserted upstream of a thymidine kinase luciferase construct. When cotransfected in fibroblasts with a C/EBPα expression vector, reporter gene expression increased 3-fold only in the wild-type constructs. The same mutation, when placed into the intact 5.13-kilobase pair promoter, reduced promoter activity in adipocytes from 50-fold to <10-fold. Electrophoretic mobility shift analysis demonstrated that the site at −3306 generated a specific protein-oligonucleotide complex that was supershifted by C/EBPα antibody, while a probe corresponding to a putative site at −1462 did not. These results define C/EBPα as a key transcriptional regulator of the mouse β3AR gene during adipogenesis. adrenergic receptor CCAAT/enhancer-binding protein peroxisome proliferator-activated receptor adipocyte fatty acid-binding protein base pair(s) kilobase pair(s) Dulbecco's modified Eagle's medium serum-free medium peroxisome proliferator-activated receptor γ response element The β3-adrenergic receptor (β3AR)1 is a unique member of the βAR family because, unlike the β1AR and β2AR, it is expressed predominantly in adipocytes and regulates both lipolysis and nonshivering thermogenesis (reviewed in Ref. 1Collins S. Cao W. Soeder K.J. Snedden S.K. Ntambi J.M. Adipocyte Biology and Hormone Signaling. 37. IOS Press, Washington, D. C.2000: 51-62Google Scholar). In genetic and dietary models of obesity, progressive accumulation of adipose tissue is associated with defects in the ability of catecholamines to mobilize lipid stores (2Laudat M.H. Pairault J. Eur. J. Biochem. 1975; 56: 583-589Crossref PubMed Scopus (24) Google Scholar, 3Shepherd R.E. Malbon C.C. Smith C.J. Fain J.N. J. Biol. Chem. 1977; 252: 7243-7248Abstract Full Text PDF PubMed Google Scholar, 4Collins S. Daniel K.W. Petro A.E. Surwit R.S. Endocrinology. 1997; 138: 405-413Crossref PubMed Scopus (187) Google Scholar). We have previously shown that the expression and function of the adipocyte βARs are blunted in most models of obesity (5Collins S. Daniel K.W. Rohlfs E.M. Ramkumar V. Taylor I.L. Gettys T.W. Mol. Endocrinol. 1994; 8: 518-527PubMed Google Scholar, 6Collins S. Daniel K.W. Rohlfs E.M. Int. J. Obesity. 1999; 23: 669-677Crossref PubMed Scopus (67) Google Scholar). Nevertheless, a curious aspect of β3AR biology is that, despite defects in β3AR expression and function, selective agonists for this receptor have been shown to prevent or reverse obesity (4Collins S. Daniel K.W. Petro A.E. Surwit R.S. Endocrinology. 1997; 138: 405-413Crossref PubMed Scopus (187) Google Scholar, 7Arch J.R.S. Ainsworth A.T. Cawthorne M.A. Piercy V. Sennitt M.V. Thody V.E. Wilson C. Wilson S. Nature. 1984; 309: 163-165Crossref PubMed Scopus (645) Google Scholar, 8Himms-Hagen J. Cui J. Danforth Jr., E. Taatjes D.J. Lang S.S. Waters B.L. Claus T.H. Am. J. Physiol. 1994; 266: R1371-R1382Crossref PubMed Google Scholar, 9Largis E.E. Burns M.G. Muenkel H.A. Dolan J.A. Claus T.H. Drug Dev. Res. 1994; 32: 69-76Crossref Scopus (50) Google Scholar, 10Sasaki N. Uchida E. Niiyama M. Yoshida T. Saito M. J. Vet. Med. Sci. 1998; 60: 465-469Crossref PubMed Scopus (27) Google Scholar). The efficacy of these drugs is related to increased brown adipose tissue thermogenesis and a restoration of expression of the β3AR and β1AR in white adipose tissue depots (4Collins S. Daniel K.W. Petro A.E. Surwit R.S. Endocrinology. 1997; 138: 405-413Crossref PubMed Scopus (187) Google Scholar). For these reasons it is important to understand the tissue-specific and hormonal factors that regulate the expression of this receptor.Two groups of transcription factors are known to be responsible for initiating and maintaining adipocyte differentiation: the CCAAT/enhancer-binding proteins (C/EBP) (11Freytag S.O. Paielli D.L. Gilbert J.D. Genes Dev. 1994; 8: 1654-1663Crossref PubMed Scopus (391) Google Scholar, 12Darlington G.J. Ross S.E. MacDougald O.A. J. Biol. Chem. 1998; 273: 30057-30060Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 13Wu Z. Xie Y. Morrison R. Bucher N. Farmer S. J. Clin. Invest. 1998; 101: 22-32Crossref PubMed Scopus (297) Google Scholar, 14Wu Z. Xie Y. Bucher N.L.R. Farmer S.R. Genes Dev. 1995; 9: 2350-2363Crossref PubMed Scopus (475) Google Scholar, 15Yeh W.C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (806) Google Scholar, 16Schwarz E.J. Reginato M.J. Shao D. Krakow S.L. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1552-1561Crossref PubMed Google Scholar, 17Lin F. Lane M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8757-8761Crossref PubMed Scopus (381) Google Scholar) and PPARγ (18Brun R.P. Tontonoz P. Forman B.M. Ellis R. Chen J. Evans R.M. Spiegelman B.M. Genes Dev. 1996; 10: 974-984Crossref PubMed Scopus (407) Google Scholar, 19Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (1981) Google Scholar, 20Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3091) Google Scholar). From a large body of work in model adipocyte cell lines, such as 3T3-L1, it has been shown that the C/EBPs are expressed in a cascade-like fashion during the early stages of adipocyte differentiation, with C/EBPβ and C/EBPδ preceding the appearance of C/EBPα (15Yeh W.C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (806) Google Scholar, 21Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1332) Google Scholar). More recent studies indicate that the expression of the adipogenic transcription factor PPARγ is partially under the control of the C/EBP family of transcription factors and vice versa (22Wu Z. Rosen E.D. Brun R. Hauser S. Adelmant G. Troy A.E. MeKeon C. Darlington G.J. Spiegelman B.M. Mol. Cell. 1999; 3: 151-158Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar, 23Wu Z. Puigserver P. Andersson U. Zhang C. Adelmant G. Mootha V. Troy A. Cinti S. Lowell B. Scarpulla R.C. Spiegelman B.M. Cell. 1999; 98: 115-124Abstract Full Text Full Text PDF PubMed Scopus (3115) Google Scholar). Additionally, insulin-sensitive glucose uptake has been shown to be impaired in adipogenic cells that lack C/EBPα, due to deficits in insulin receptor and insulin receptor substrate-1 (22Wu Z. Rosen E.D. Brun R. Hauser S. Adelmant G. Troy A.E. MeKeon C. Darlington G.J. Spiegelman B.M. Mol. Cell. 1999; 3: 151-158Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar, 24El-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 (167) Google Scholar). Like other adipocyte-specific genes, the β3AR is not expressed in preadipocytes, but appears late in the adipogenic program of both white and brown adipocytes (Refs. 25Feve B. Emorine L. Lasnier F. Strosberg D. Pairault J. J. Biol. Chem. 1991; 266: 20329-20336Abstract Full Text PDF PubMed Google Scholar and 26Cannon B. Nedergaard J. Biochem. Soc. Trans. 1996; 24: 40-45Crossref Scopus (19) Google Scholar; this report). Because of the pivotal role of C/EBPα in activating many adipocyte-specific genes, the focus of our studies was to determine the role of C/EBPα in initiating β3AR gene transcription during adipogenesis. Our results show that C/EBPα is required for the adipocyte-dependent expression of the mouse β3AR gene, and we define the C/EBP binding site in the β3AR promoter that is responsible for this regulation.DISCUSSIONIn this study we have shown that the mouse β3AR gene is specifically activated by C/EBPα and that this activation is correlated with the binding of C/EBPα to an element residing between −3306 and −3298 bp upstream of the β3AR gene transcription start site. By utilizing several cell lines that contain various combinations of the C/EBPs and PPARγ, we showed that only adipocytes expressing C/EBPα possess β3AR transcripts and concomitant functional activity. A 5.13-kb promoter fragment of the mouse β3AR gene containing two putative C/EBP binding sites, at −3306 to −3298 and at −1462 to −1454, confers robust expression of a luciferase reporter preferentially in adipocytes. This transcriptional activity is significantly decreased upon deletion of the more distal C/EBP element, while removal of the more proximal element had no further effect. We also showed that this C/EBP element at −3306 conveys transcriptional activity to C/EBPα in vitro. Finally, electrophoretic mobility shift assays provided evidence that C/EBPα interacts specifically with the element at −3306 and not the element at −1462 in the mouse β3AR promoter. In every case, mutation of the −3306 C/EBP site eliminates these responses.Our observation that expression of the β3AR gene is positively regulated by C/EBPα is consistent with numerous reports showing the role of this transcription factor in adipogenesis. For example, ectopic expression of C/EBPα is sufficient to induce adipocyte differentiation in a number of cell lines, while the expression of an antisense C/EBPα construct in 3T3-L1 adipocytes blocks differentiation (43Lin F.-T. Lane M.D. Genes Dev. 1992; 6: 533-544Crossref PubMed Scopus (274) Google Scholar). Consistent with these in vitrostudies, C/EBPα null mice fail to develop white adipose tissue (44Wang N. Finegold M. Bradley A. Ou C. Abdelsayed S. Wilde M. Taylor L. Wilson D. Darlington G. Science. 1995; 269: 1108-1112Crossref PubMed Scopus (827) Google Scholar).In addition to C/EBPα, C/EBPβ and C/EBPδ have also been shown to be critical regulators of adipocyte differentiation. C/EBPβ and C/EBPδ are transiently expressed and precede the appearance of C/EBPα (12Darlington G.J. Ross S.E. MacDougald O.A. J. Biol. Chem. 1998; 273: 30057-30060Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). Interestingly, overexpression of C/EBPβ, but not C/EBPδ, in preadipocytes converts them to adipocytes, suggesting that C/EBPβ can substitute for C/EBPα (15Yeh W.C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (806) Google Scholar). However, the β/δ39 cells, which constitutively express C/EBPβ and C/EBPδ, acquire an adipocyte phenotype, as evidenced by the presence of PPARγ and aP2, but fail to express either C/EBPα or β3AR. As shown in our gel shift assays, it appears that C/EBPβ is capable of binding the C/EBP element at −3306 in the mouse β3AR promoter. Perhaps this is not surprising because C/EBP isoforms have been shown to bind to a common DNA consensus sequence (21Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1332) Google Scholar, 41Elberg G. Gimble J.M. Tsai S.Y. J. Biol. Chem. 2000; 275: 27815-27822Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Despite this interaction, it is clear that C/EBPβ or C/EBPδ alone is insufficient to induce β3AR.Although we show a pivotal role for C/EBPα in transactivating the β3AR gene during adipogenesis, tissues containing high levels of C/EBPα, such as liver and lung, do not contain appreciable amounts of β3AR. In addition, we show that the NIH-α cells do not express β3AR in the preadipocyte state, despite the constitutive expression of C/EBPα. Therefore, it is clear that some other transcription factor(s) are important in directing the adipocyte-specific expression of the β3AR gene. Another key regulator of adipogenesis is PPARγ. Constitutive expression of PPARγ in fibroblasts can induce the conversion to the adipocyte phenotype (20Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3091) Google Scholar). PPARγ ligands, such as the thiazolidinediones, which are a class of insulin-sensitizing agents, convert fibroblasts and multipotential stem cells to adipocytes (13Wu Z. Xie Y. Morrison R. Bucher N. Farmer S. J. Clin. Invest. 1998; 101: 22-32Crossref PubMed Scopus (297) Google Scholar, 29Kliewer S.A. Lenhard J.M. Wilson T.M. Patel I. Morris D.C. Lehmann J.M. Cell. 1995; 83: 813-819Abstract Full Text PDF PubMed Scopus (1859) Google Scholar). As well, PPARγ (−/−) animals completely lack adipose tissue (45Barak 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 (1629) Google Scholar, 46Rosen 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 (1626) Google Scholar, 47Kubota N. Terauchi Y. Miki H. Tamemoto H. Yamauchi T. Komeda K. Satoh S. Nakano R. Ishii C. Sugiyama T. Eto K. Tsubamoto Y. Okuno A. Murakami K. Sekihara H. Hasegawa G. Naito M. Toyoshima Y. Tanaka S. Shiota K. Kitamura T. Fujita T. Ezaki O. Aizawa S. Nagai R. Tobe K. Kimura S. Kadowaki T. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1212) Google Scholar). Previous studies showed that, despite expression of PPARγ and development of an adipocyte morphology, a lack of C/EBPα results in decreased insulin-stimulated glucose transport (22Wu Z. Rosen E.D. Brun R. Hauser S. Adelmant G. Troy A.E. MeKeon C. Darlington G.J. Spiegelman B.M. Mol. Cell. 1999; 3: 151-158Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar, 24El-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 (167) Google Scholar). Despite this apparent cooperation between C/EBPα and PPARγ in adipocyte differentiation, our initial attempts to locate a DR-1 PPARγ response element (PPRE) within our mouse β3AR promoter fragment or to demonstrate transactivation of the β3AR by PPARγ have been unsuccessful. It is plausible that a PPRE lies outside of the promoter region that we have isolated and studied. However, it is known that PPREs in several PPARγ target genes deviate significantly from the consensus DR-1 site (18Brun R.P. Tontonoz P. Forman B.M. Ellis R. Chen J. Evans R.M. Spiegelman B.M. Genes Dev. 1996; 10: 974-984Crossref PubMed Scopus (407) Google Scholar). For this reason we are currently investigating some regions that weakly resemble a PPRE half-site. To fully understand the role of the β3AR in obesity, it will be important for us to determine not only what other critical transcription factor(s) is/are required for the regulation of the β3AR gene, but how their effects on β3AR expression may contribute to obesity. The β3-adrenergic receptor (β3AR)1 is a unique member of the βAR family because, unlike the β1AR and β2AR, it is expressed predominantly in adipocytes and regulates both lipolysis and nonshivering thermogenesis (reviewed in Ref. 1Collins S. Cao W. Soeder K.J. Snedden S.K. Ntambi J.M. Adipocyte Biology and Hormone Signaling. 37. IOS Press, Washington, D. C.2000: 51-62Google Scholar). In genetic and dietary models of obesity, progressive accumulation of adipose tissue is associated with defects in the ability of catecholamines to mobilize lipid stores (2Laudat M.H. Pairault J. Eur. J. Biochem. 1975; 56: 583-589Crossref PubMed Scopus (24) Google Scholar, 3Shepherd R.E. Malbon C.C. Smith C.J. Fain J.N. J. Biol. Chem. 1977; 252: 7243-7248Abstract Full Text PDF PubMed Google Scholar, 4Collins S. Daniel K.W. Petro A.E. Surwit R.S. Endocrinology. 1997; 138: 405-413Crossref PubMed Scopus (187) Google Scholar). We have previously shown that the expression and function of the adipocyte βARs are blunted in most models of obesity (5Collins S. Daniel K.W. Rohlfs E.M. Ramkumar V. Taylor I.L. Gettys T.W. Mol. Endocrinol. 1994; 8: 518-527PubMed Google Scholar, 6Collins S. Daniel K.W. Rohlfs E.M. Int. J. Obesity. 1999; 23: 669-677Crossref PubMed Scopus (67) Google Scholar). Nevertheless, a curious aspect of β3AR biology is that, despite defects in β3AR expression and function, selective agonists for this receptor have been shown to prevent or reverse obesity (4Collins S. Daniel K.W. Petro A.E. Surwit R.S. Endocrinology. 1997; 138: 405-413Crossref PubMed Scopus (187) Google Scholar, 7Arch J.R.S. Ainsworth A.T. Cawthorne M.A. Piercy V. Sennitt M.V. Thody V.E. Wilson C. Wilson S. Nature. 1984; 309: 163-165Crossref PubMed Scopus (645) Google Scholar, 8Himms-Hagen J. Cui J. Danforth Jr., E. Taatjes D.J. Lang S.S. Waters B.L. Claus T.H. Am. J. Physiol. 1994; 266: R1371-R1382Crossref PubMed Google Scholar, 9Largis E.E. Burns M.G. Muenkel H.A. Dolan J.A. Claus T.H. Drug Dev. Res. 1994; 32: 69-76Crossref Scopus (50) Google Scholar, 10Sasaki N. Uchida E. Niiyama M. Yoshida T. Saito M. J. Vet. Med. Sci. 1998; 60: 465-469Crossref PubMed Scopus (27) Google Scholar). The efficacy of these drugs is related to increased brown adipose tissue thermogenesis and a restoration of expression of the β3AR and β1AR in white adipose tissue depots (4Collins S. Daniel K.W. Petro A.E. Surwit R.S. Endocrinology. 1997; 138: 405-413Crossref PubMed Scopus (187) Google Scholar). For these reasons it is important to understand the tissue-specific and hormonal factors that regulate the expression of this receptor. Two groups of transcription factors are known to be responsible for initiating and maintaining adipocyte differentiation: the CCAAT/enhancer-binding proteins (C/EBP) (11Freytag S.O. Paielli D.L. Gilbert J.D. Genes Dev. 1994; 8: 1654-1663Crossref PubMed Scopus (391) Google Scholar, 12Darlington G.J. Ross S.E. MacDougald O.A. J. Biol. Chem. 1998; 273: 30057-30060Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar, 13Wu Z. Xie Y. Morrison R. Bucher N. Farmer S. J. Clin. Invest. 1998; 101: 22-32Crossref PubMed Scopus (297) Google Scholar, 14Wu Z. Xie Y. Bucher N.L.R. Farmer S.R. Genes Dev. 1995; 9: 2350-2363Crossref PubMed Scopus (475) Google Scholar, 15Yeh W.C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (806) Google Scholar, 16Schwarz E.J. Reginato M.J. Shao D. Krakow S.L. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1552-1561Crossref PubMed Google Scholar, 17Lin F. Lane M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8757-8761Crossref PubMed Scopus (381) Google Scholar) and PPARγ (18Brun R.P. Tontonoz P. Forman B.M. Ellis R. Chen J. Evans R.M. Spiegelman B.M. Genes Dev. 1996; 10: 974-984Crossref PubMed Scopus (407) Google Scholar, 19Tontonoz P. Hu E. Graves R.A. Budavari A.I. Spiegelman B.M. Genes Dev. 1994; 8: 1224-1234Crossref PubMed Scopus (1981) Google Scholar, 20Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3091) Google Scholar). From a large body of work in model adipocyte cell lines, such as 3T3-L1, it has been shown that the C/EBPs are expressed in a cascade-like fashion during the early stages of adipocyte differentiation, with C/EBPβ and C/EBPδ preceding the appearance of C/EBPα (15Yeh W.C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (806) Google Scholar, 21Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1332) Google Scholar). More recent studies indicate that the expression of the adipogenic transcription factor PPARγ is partially under the control of the C/EBP family of transcription factors and vice versa (22Wu Z. Rosen E.D. Brun R. Hauser S. Adelmant G. Troy A.E. MeKeon C. Darlington G.J. Spiegelman B.M. Mol. Cell. 1999; 3: 151-158Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar, 23Wu Z. Puigserver P. Andersson U. Zhang C. Adelmant G. Mootha V. Troy A. Cinti S. Lowell B. Scarpulla R.C. Spiegelman B.M. Cell. 1999; 98: 115-124Abstract Full Text Full Text PDF PubMed Scopus (3115) Google Scholar). Additionally, insulin-sensitive glucose uptake has been shown to be impaired in adipogenic cells that lack C/EBPα, due to deficits in insulin receptor and insulin receptor substrate-1 (22Wu Z. Rosen E.D. Brun R. Hauser S. Adelmant G. Troy A.E. MeKeon C. Darlington G.J. Spiegelman B.M. Mol. Cell. 1999; 3: 151-158Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar, 24El-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 (167) Google Scholar). Like other adipocyte-specific genes, the β3AR is not expressed in preadipocytes, but appears late in the adipogenic program of both white and brown adipocytes (Refs. 25Feve B. Emorine L. Lasnier F. Strosberg D. Pairault J. J. Biol. Chem. 1991; 266: 20329-20336Abstract Full Text PDF PubMed Google Scholar and 26Cannon B. Nedergaard J. Biochem. Soc. Trans. 1996; 24: 40-45Crossref Scopus (19) Google Scholar; this report). Because of the pivotal role of C/EBPα in activating many adipocyte-specific genes, the focus of our studies was to determine the role of C/EBPα in initiating β3AR gene transcription during adipogenesis. Our results show that C/EBPα is required for the adipocyte-dependent expression of the mouse β3AR gene, and we define the C/EBP binding site in the β3AR promoter that is responsible for this regulation. DISCUSSIONIn this study we have shown that the mouse β3AR gene is specifically activated by C/EBPα and that this activation is correlated with the binding of C/EBPα to an element residing between −3306 and −3298 bp upstream of the β3AR gene transcription start site. By utilizing several cell lines that contain various combinations of the C/EBPs and PPARγ, we showed that only adipocytes expressing C/EBPα possess β3AR transcripts and concomitant functional activity. A 5.13-kb promoter fragment of the mouse β3AR gene containing two putative C/EBP binding sites, at −3306 to −3298 and at −1462 to −1454, confers robust expression of a luciferase reporter preferentially in adipocytes. This transcriptional activity is significantly decreased upon deletion of the more distal C/EBP element, while removal of the more proximal element had no further effect. We also showed that this C/EBP element at −3306 conveys transcriptional activity to C/EBPα in vitro. Finally, electrophoretic mobility shift assays provided evidence that C/EBPα interacts specifically with the element at −3306 and not the element at −1462 in the mouse β3AR promoter. In every case, mutation of the −3306 C/EBP site eliminates these responses.Our observation that expression of the β3AR gene is positively regulated by C/EBPα is consistent with numerous reports showing the role of this transcription factor in adipogenesis. For example, ectopic expression of C/EBPα is sufficient to induce adipocyte differentiation in a number of cell lines, while the expression of an antisense C/EBPα construct in 3T3-L1 adipocytes blocks differentiation (43Lin F.-T. Lane M.D. Genes Dev. 1992; 6: 533-544Crossref PubMed Scopus (274) Google Scholar). Consistent with these in vitrostudies, C/EBPα null mice fail to develop white adipose tissue (44Wang N. Finegold M. Bradley A. Ou C. Abdelsayed S. Wilde M. Taylor L. Wilson D. Darlington G. Science. 1995; 269: 1108-1112Crossref PubMed Scopus (827) Google Scholar).In addition to C/EBPα, C/EBPβ and C/EBPδ have also been shown to be critical regulators of adipocyte differentiation. C/EBPβ and C/EBPδ are transiently expressed and precede the appearance of C/EBPα (12Darlington G.J. Ross S.E. MacDougald O.A. J. Biol. Chem. 1998; 273: 30057-30060Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). Interestingly, overexpression of C/EBPβ, but not C/EBPδ, in preadipocytes converts them to adipocytes, suggesting that C/EBPβ can substitute for C/EBPα (15Yeh W.C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (806) Google Scholar). However, the β/δ39 cells, which constitutively express C/EBPβ and C/EBPδ, acquire an adipocyte phenotype, as evidenced by the presence of PPARγ and aP2, but fail to express either C/EBPα or β3AR. As shown in our gel shift assays, it appears that C/EBPβ is capable of binding the C/EBP element at −3306 in the mouse β3AR promoter. Perhaps this is not surprising because C/EBP isoforms have been shown to bind to a common DNA consensus sequence (21Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1332) Google Scholar, 41Elberg G. Gimble J.M. Tsai S.Y. J. Biol. Chem. 2000; 275: 27815-27822Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Despite this interaction, it is clear that C/EBPβ or C/EBPδ alone is insufficient to induce β3AR.Although we show a pivotal role for C/EBPα in transactivating the β3AR gene during adipogenesis, tissues containing high levels of C/EBPα, such as liver and lung, do not contain appreciable amounts of β3AR. In addition, we show that the NIH-α cells do not express β3AR in the preadipocyte state, despite the constitutive expression of C/EBPα. Therefore, it is clear that some other transcription factor(s) are important in directing the adipocyte-specific expression of the β3AR gene. Another key regulator of adipogenesis is PPARγ. Constitutive expression of PPARγ in fibroblasts can induce the conversion to the adipocyte phenotype (20Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3091) Google Scholar). PPARγ ligands, such as the thiazolidinediones, which are a class of insulin-sensitizing agents, convert fibroblasts and multipotential stem cells to adipocytes (13Wu Z. Xie Y. Morrison R. Bucher N. Farmer S. J. Clin. Invest. 1998; 101: 22-32Crossref PubMed Scopus (297) Google Scholar, 29Kliewer S.A. Lenhard J.M. Wilson T.M. Patel I. Morris D.C. Lehmann J.M. Cell. 1995; 83: 813-819Abstract Full Text PDF PubMed Scopus (1859) Google Scholar). As well, PPARγ (−/−) animals completely lack adipose tissue (45Barak 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 (1629) Google Scholar, 46Rosen 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 (1626) Google Scholar, 47Kubota N. Terauchi Y. Miki H. Tamemoto H. Yamauchi T. Komeda K. Satoh S. Nakano R. Ishii C. Sugiyama T. Eto K. Tsubamoto Y. Okuno A. Murakami K. Sekihara H. Hasegawa G. Naito M. Toyoshima Y. Tanaka S. Shiota K. Kitamura T. Fujita T. Ezaki O. Aizawa S. Nagai R. Tobe K. Kimura S. Kadowaki T. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1212) Google Scholar). Previous studies showed that, despite expression of PPARγ and development of an adipocyte morphology, a lack of C/EBPα results in decreased insulin-stimulated glucose transport (22Wu Z. Rosen E.D. Brun R. Hauser S. Adelmant G. Troy A.E. MeKeon C. Darlington G.J. Spiegelman B.M. Mol. Cell. 1999; 3: 151-158Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar, 24El-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 (167) Google Scholar). Despite this apparent cooperation between C/EBPα and PPARγ in adipocyte differentiation, our initial attempts to locate a DR-1 PPARγ response element (PPRE) within our mouse β3AR promoter fragment or to demonstrate transactivation of the β3AR by PPARγ have been unsuccessful. It is plausible that a PPRE lies outside of the promoter region that we have isolated and studied. However, it is known that PPREs in several PPARγ target genes deviate significantly from the consensus DR-1 site (18Brun R.P. Tontonoz P. Forman B.M. Ellis R. Chen J. Evans R.M. Spiegelman B.M. Genes Dev. 1996; 10: 974-984Crossref PubMed Scopus (407) Google Scholar). For this reason we are currently investigating some regions that weakly resemble a PPRE half-site. To fully understand the role of the β3AR in obesity, it will be important for us to determine not only what other critical transcription factor(s) is/are required for the regulation of the β3AR gene, but how their effects on β3AR expression may contribute to obesity. In this study we have shown that the mouse β3AR gene is specifically activated by C/EBPα and that this activation is correlated with the binding of C/EBPα to an element residing between −3306 and −3298 bp upstream of the β3AR gene transcription start site. By utilizing several cell lines that contain various combinations of the C/EBPs and PPARγ, we showed that only adipocytes expressing C/EBPα possess β3AR transcripts and concomitant functional activity. A 5.13-kb promoter fragment of the mouse β3AR gene containing two putative C/EBP binding sites, at −3306 to −3298 and at −1462 to −1454, confers robust expression of a luciferase reporter preferentially in adipocytes. This transcriptional activity is significantly decreased upon deletion of the more distal C/EBP element, while removal of the more proximal element had no further effect. We also showed that this C/EBP element at −3306 conveys transcriptional activity to C/EBPα in vitro. Finally, electrophoretic mobility shift assays provided evidence that C/EBPα interacts specifically with the element at −3306 and not the element at −1462 in the mouse β3AR promoter. In every case, mutation of the −3306 C/EBP site eliminates these responses. Our observation that expression of the β3AR gene is positively regulated by C/EBPα is consistent with numerous reports showing the role of this transcription factor in adipogenesis. For example, ectopic expression of C/EBPα is sufficient to induce adipocyte differentiation in a number of cell lines, while the expression of an antisense C/EBPα construct in 3T3-L1 adipocytes blocks differentiation (43Lin F.-T. Lane M.D. Genes Dev. 1992; 6: 533-544Crossref PubMed Scopus (274) Google Scholar). Consistent with these in vitrostudies, C/EBPα null mice fail to develop white adipose tissue (44Wang N. Finegold M. Bradley A. Ou C. Abdelsayed S. Wilde M. Taylor L. Wilson D. Darlington G. Science. 1995; 269: 1108-1112Crossref PubMed Scopus (827) Google Scholar). In addition to C/EBPα, C/EBPβ and C/EBPδ have also been shown to be critical regulators of adipocyte differentiation. C/EBPβ and C/EBPδ are transiently expressed and precede the appearance of C/EBPα (12Darlington G.J. Ross S.E. MacDougald O.A. J. Biol. Chem. 1998; 273: 30057-30060Abstract Full Text Full Text PDF PubMed Scopus (604) Google Scholar). Interestingly, overexpression of C/EBPβ, but not C/EBPδ, in preadipocytes converts them to adipocytes, suggesting that C/EBPβ can substitute for C/EBPα (15Yeh W.C. Cao Z. Classon M. McKnight S.L. Genes Dev. 1995; 9: 168-181Crossref PubMed Scopus (806) Google Scholar). However, the β/δ39 cells, which constitutively express C/EBPβ and C/EBPδ, acquire an adipocyte phenotype, as evidenced by the presence of PPARγ and aP2, but fail to express either C/EBPα or β3AR. As shown in our gel shift assays, it appears that C/EBPβ is capable of binding the C/EBP element at −3306 in the mouse β3AR promoter. Perhaps this is not surprising because C/EBP isoforms have been shown to bind to a common DNA consensus sequence (21Cao Z. Umek R.M. McKnight S.L. Genes Dev. 1991; 5: 1538-1552Crossref PubMed Scopus (1332) Google Scholar, 41Elberg G. Gimble J.M. Tsai S.Y. J. Biol. Chem. 2000; 275: 27815-27822Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Despite this interaction, it is clear that C/EBPβ or C/EBPδ alone is insufficient to induce β3AR. Although we show a pivotal role for C/EBPα in transactivating the β3AR gene during adipogenesis, tissues containing high levels of C/EBPα, such as liver and lung, do not contain appreciable amounts of β3AR. In addition, we show that the NIH-α cells do not express β3AR in the preadipocyte state, despite the constitutive expression of C/EBPα. Therefore, it is clear that some other transcription factor(s) are important in directing the adipocyte-specific expression of the β3AR gene. Another key regulator of adipogenesis is PPARγ. Constitutive expression of PPARγ in fibroblasts can induce the conversion to the adipocyte phenotype (20Tontonoz P. Hu E. Spiegelman B.M. Cell. 1994; 79: 1147-1156Abstract Full Text PDF PubMed Scopus (3091) Google Scholar). PPARγ ligands, such as the thiazolidinediones, which are a class of insulin-sensitizing agents, convert fibroblasts and multipotential stem cells to adipocytes (13Wu Z. Xie Y. Morrison R. Bucher N. Farmer S. J. Clin. Invest. 1998; 101: 22-32Crossref PubMed Scopus (297) Google Scholar, 29Kliewer S.A. Lenhard J.M. Wilson T.M. Patel I. Morris D.C. Lehmann J.M. Cell. 1995; 83: 813-819Abstract Full Text PDF PubMed Scopus (1859) Google Scholar). As well, PPARγ (−/−) animals completely lack adipose tissue (45Barak 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 (1629) Google Scholar, 46Rosen 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 (1626) Google Scholar, 47Kubota N. Terauchi Y. Miki H. Tamemoto H. Yamauchi T. Komeda K. Satoh S. Nakano R. Ishii C. Sugiyama T. Eto K. Tsubamoto Y. Okuno A. Murakami K. Sekihara H. Hasegawa G. Naito M. Toyoshima Y. Tanaka S. Shiota K. Kitamura T. Fujita T. Ezaki O. Aizawa S. Nagai R. Tobe K. Kimura S. Kadowaki T. Mol. Cell. 1999; 4: 597-609Abstract Full Text Full Text PDF PubMed Scopus (1212) Google Scholar). Previous studies showed that, despite expression of PPARγ and development of an adipocyte morphology, a lack of C/EBPα results in decreased insulin-stimulated glucose transport (22Wu Z. Rosen E.D. Brun R. Hauser S. Adelmant G. Troy A.E. MeKeon C. Darlington G.J. Spiegelman B.M. Mol. Cell. 1999; 3: 151-158Abstract Full Text Full Text PDF PubMed Scopus (812) Google Scholar, 24El-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 (167) Google Scholar). Despite this apparent cooperation between C/EBPα and PPARγ in adipocyte differentiation, our initial attempts to locate a DR-1 PPARγ response element (PPRE) within our mouse β3AR promoter fragment or to demonstrate transactivation of the β3AR by PPARγ have been unsuccessful. It is plausible that a PPRE lies outside of the promoter region that we have isolated and studied. However, it is known that PPREs in several PPARγ target genes deviate significantly from the consensus DR-1 site (18Brun R.P. Tontonoz P. Forman B.M. Ellis R. Chen J. Evans R.M. Spiegelman B.M. Genes Dev. 1996; 10: 974-984Crossref PubMed Scopus (407) Google Scholar). For this reason we are currently investigating some regions that weakly resemble a PPRE half-site. To fully understand the role of the β3AR in obesity, it will be important for us to determine not only what other critical transcription factor(s) is/are required for the regulation of the β3AR gene, but how their effects on β3AR expression may contribute to obesity. We thank the following people for gifts of plasmids and other reagents: Steve Kliewer, Jurgen Lehmann, Steve McKnight, Pamela Mellon, Bruce Spiegelman, Ormand MacDougald, Donald McDonnell, and Tom Gettys. We also thank Steven Kingsman and Michael Seldin for the mouse genomic DNA library in the λ-DASH vector, Putting Xu and Sheridan Snedden for help with library screening, and other members of the Collins laboratory for advice and helpful discussions.
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