C/EBPβ Reprograms White 3T3-L1 Preadipocytes to a Brown Adipocyte Pattern of Gene Expression

Georgios Karamanlidis, Angeliki Karamitri, Kevin Docherty, David G. Hazlerigg, Michael A. Lomax
2007 Journal of Biological Chemistry  
cAMP-dependent protein kinase induction of PPAR␥ coactivator-1␣ (PGC-1␣) and uncoupling protein 1 (UCP1) expression is an essential step in the commitment of preadipocytes to the brown adipose tissue (BAT) lineage. We studied the molecular mechanisms responsible for differential expression of PGC-1␣ in HIB1B (BAT) and 3T3-L1 white adipose tissue (WAT) precursor cell lines. In HIB1B cells PGC-1␣ and UCP1 expression is cAMP-inducible, but in 3T3-L1 cells, expression is reduced and is
more » ... and is cAMP-insensitive. A proximal 264-bp PGC-1␣ reporter construct was cAMP-inducible only in HIB1B cells and was suppressed by site-directed mutagenesis of the proximal cAMP response element (CRE). In electrophoretic mobility shift assays, the transcription factors CREB and C/EBP␤, but not C/EBP␣ and C/EBP␦, bound to the CRE on the PGC-1␣ promoter region in HIB1B and 3T3-L1 cells. Chromatin immunoprecipitation studies demonstrated that C/EBP␤ and CREB bound to the CRE region in HIB1B and 3T3-L1 cell lysates. C/EBP␤ expression was induced by cAMP only in HIB1B cells, and overexpression of C/EBP␤ rescued cAMP-inducible PGC-1␣ and UCP1 expression in 3T3-L1 cells. These data demonstrate that differentiation of preadipocytes toward the BAT rather than the WAT phenotype is controlled in part by the action of C/EBP␤ on the CRE in PGC-1␣ proximal promoter. Downloaded from FIGURE 5. Inducibility of C/EBP isomer mRNA during differentiation or by cAMP in HIB1B and 3T3-L1 cells. A, C/EBP␣/␤/␦ mRNA expression at 0 (confluence) and 7 days after induction to differentiate. B, confluent cells were treated with forskolin for 3 h. Values assayed by real-time PCR and normalized against ␤-actin expression. Error bars represent S.E. of triplicate observations of one of three independent experiments. C/EBP␤ Reprograms 3T3-L1 AUGUST 24, 2007 • VOLUME 282 • NUMBER 34 JOURNAL OF BIOLOGICAL CHEMISTRY 24665 by guest on July 25, 2018 http://www.jbc.org/ Downloaded from FIGURE 6. Overexpression of C/EBP␤ rescues PGC-1␣ proximal promoter transcriptional activity responses to cAMP in 3T3-L1 cells. HIB1B or 3T3-L1 cells were transiently co-transfected with an empty control vector p-cDNA3 or 264PGC1␣-p-GL3, p-MSV-C/EBP␣, p-MSV-C/EBP␤, p-MSV-C/EBP␦, and 6CRE-pGL3 as indicated, and at confluence were treated with forskolin for 12 h. A, Western blot showing overexpression of C/EBP isomers. B, 264PGC1␣-pGL3 promoter-reporter activity. C, 6CRE-pGL3 luciferase constructs driven by a 6-CRE in tandem. Error bars represent S.E. of triplicate observations of one of three independent experiments. C/EBP␤ Reprograms 3T3-L1
doi:10.1074/jbc.m703101200 pmid:17584738 fatcat:at2p2uk2xzdg7bedkeg6rgyjty