CCAAT/enhancer-binding protein alpha is required for transcription of the beta 3-adrenergic receptor gene during adipogenesis.
The beta(3)-adrenergic receptor (beta(3)AR) 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 alpha (C/EBP alpha), 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/EBP alpha exhibit reduced insulin sensitivity, despite an apparent adipogenic phenotype. Here we show that transcription and function of the beta(3)AR correlates with C/EBP alpha expression in these adipocyte models. A 5.13-kilobase pair fragment of the mouse beta(3)AR 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/EBP alpha 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/EBP alpha antibody, while a probe corresponding to a putative site at -1462 did not. These results define C/EBP alpha as a key transcriptional regulator of the mouse beta(3)AR gene during adipogenesis.
Dixon
T M
TM
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Daniel
K W
KW
Farmer
S R
SR
Collins
S
S
D016475:3T3 Cells; D000262:Adenylyl Cyclases / Q000378:metabolism; D000273:Adipose Tissue / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D058667:Adrenergic beta-3 Receptor Agonists; D000818:Animals; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D019556:COS Cells; D002454:Cell Differentiation; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004149:Dioxoles / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D018507:Gene Expression Regulation, Developmental; D017930:Genes, Reporter; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009928:Organ Specificity; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D022702:Receptors, Adrenergic, beta-3 / Q000235:genetics*; D020218:Response Elements / Q000235:genetics; D017422:Sequence Analysis, DNA; D014162:Transfection
D058667:Adrenergic beta-3 Receptor Agonists; D022762:CCAAT-Enhancer-Binding Proteins; D004149:Dioxoles; D012333:RNA, Messenger; D022702:Receptors, Adrenergic, beta-3; C076126:disodium (R,R)-5-(2-((2-(3-chlorophenyl)-2-hydroxyethyl)-amino)propyl)-1,3-benzodioxole-2,3-dicarboxylate; D004247:DNA; D000262:Adenylyl Cyclases