Abstract
Cyclin D1 expression is elevated and Wnt10b is repressed by cAMP during the first few hours of adipogenesis. cAMP-responsive element-binding protein (CREB) is a primary target for cAMP signaling, and we have shown that activation of CREB promotes adipogenesis and adipocyte survival. Here we tested the impact of CREB on expression of cyclin D1 and wingless-related mouse mammary tumor virus integration site 10b (Wnt10b) in 3T3-L1 cells. Forced depletion of CREB blocked Bt(2)cAMP-stimulated cyclin D1 expression and basal Wnt10b gene expression. Two CREB-binding sites were identified in the Wnt10b promoter region. Ablation of either site partially blocked promoter activity, while mutation of both sites completely suppressed promoter activity. These results suggest that CREB activates transcription from both the cyclin D1 and Wnt10b gene promoters. What accounts for the differential regulation of cyclin D1 and Wnt10b genes by cAMP? Chromatin immunoprecipitation revealed CREB bound to the Wnt10b promoter in untreated preadipocytes but not following treatment with Bt(2)cAMP. CREB binding to the cyclin D1 promoter was detected in untreated cells and post-Bt(2)cAMP. Differences between CREB binding to the two genes correlated with increasing methylation of the Wnt10b promoter following Bt(2)cAMP treatment, whereas no methylation of the cyclin D1 promoter was observed. Treatment of cells with the methylase inhibitor 5-azacytidine restored CREB binding to the Wnt10b gene promoter and prevented the inhibition of Wnt10b RNA expression by Bt(2)cAMP. We conclude that cAMP stimulates phosphorylation and binding of CREB to the cyclin D1 gene promoter. Simultaneously, hypermethylation of the Wnt10b gene promoter suppresses binding of CREB, allowing adipogenesis to proceed.
Highlights
Adipogenesis is the process by which mature, insulin-responsive adipocytes are generated from undifferentiated preadipocytes and mesenchymal progenitor cells [1]
Cyclin D1 expression is elevated and Wnt10b is repressed by cAMP during the first few hours of adipogenesis. cAMP-responsive element-binding protein (CREB) is a primary target for cAMP signaling, and we have shown that activation of CREB promotes adipogenesis and adipocyte survival
We previously reported that the activity of the transcription factor CREB was stimulated by cAMP mimetics and insulin in both preadipocytes [8], suggesting that CREB might play a role in adipogenic conversion
Summary
C/EBP, CCAAT/enhancer binding protein; CRE, cAMP response element; CREB, CRE-binding protein; PPAR, peroxisomeproliferator-activated receptor; siRNA, small interfering RNA; RT, reverse transcription; DMEM, Dulbecco’s modified Eagle’s medium. Cyclic AMP-induced cyclin D1 expression and MDI-stimulated preadipocyte proliferation were suppressed in CREB-deficient cells This indicated that CREB was required for cyclin D1 expression in response to cAMP. The effect of CREB on Wnt10b gene expression appears to be mediated by two putative cAMP response elements (CREs) in the Wnt10b gene promoter This suggested that CREB was required to maintain basal Wnt10b gene transcription rather than repress Wnt10b expression in response to cAMP mimetics. The differential binding of CREB to these gene promoters appears to be modulated in part by DNA methylation
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