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

Abstract

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 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.

Combinatorial Transcription Factor Regulation of the Cyclic AMP-response Element on the Pgc-1α Promoter in White 3T3-L1 and Brown HIB-1B Preadipocytes

“…Expression of Ucp1 was significantly (p Ͻ 0.01) increased by treatment with forskolin, compared with basal DMSO-treated cells at confluence ( Fig. 2A), as we have previously demonstrated (25). This response disappeared 12 and 24 h after inducing differentiation but returned by 36 h and increased to 7 days after induction, when lipid droplets can be observed in HIB-1B cells.…”

“…Plasmids-The firefly luciferase reporter gene construct containing 264 bp (264PGC1␣-pGL3) from the region upstream of the rat Pgc-1␣ transcription start site ligated to the pGL3-Basic vector (Promega) has been described (25). The pMSVC/EBP␤ (mouse) expression plasmid that contains the respective cDNAs under the control of the mouse sarcoma virus long terminal repeat was kindly provided by S. McKnight (University of Texas Southwestern Medical Center, Dallas, TX).…”

“…Western Blotting-Western blotting on whole cell lysates was performed as reported previously (25). Immunological detection was performed using antibodies directed against CREB (1:1000 dilution), ATF-2 (1:500 dilution), CHOP10 (1:350 dilution), C/EBP␤ (1:500 dilution), and actin (1:250 dilution; Sigma).…”

“…Protein kinase A activation of PGC-1␣ expression has been proposed to be mediated by interaction of ATF-2 and CREB, in brown adipose tissue and liver, respectively. Our previous study has demonstrated that C/EBP␤ binding to the CRE on the Pgc-1␣ promoter is able to induce cAMPresponsive expression of Pgc-1␣ and Ucp1 in the white preadipocyte cell line 3T3-L1 (25).…”

Brown and beige fat: development, function and therapeutic potential

“…3). Loss of C/ebpβ is associated with defective thermogenesis, whereas increasing the amounts of C/ebpβ in white fat cells triggers a brown fat transcriptional profile 42,[112][113][114] .…”