Decreased activity and enhanced nuclear export of CCAAT-enhancer-binding protein β during inhibition of adipogenesis by ceramide

Abstract

To identify novel molecular mechanisms by which ceramide regulates cell differentiation, we examined its effect on adipogenesis of 3T3-L1 preadipocytes. Hormonal stimulation of 3T3-L1 preadipocytes induced formation of triacylglycerol-laden adipocytes over 7days; in part, via the co-ordinated action of CCAAT-enhancer-binding proteins α, β and δ (C/EBP-α, -β and -δ) and peroxisome-proliferator-activated receptor γ (PPARγ). The addition of exogenous N-acetylsphingosine (C2-ceramide) or increasing endogenous ceramide levels inhibited the expression of C/EBPα and PPARγ, and blocked adipocyte development. C2-ceramide did not decrease the cellular expression of C/EBPβ, which is required for expression of C/EBPα and PPARγ, but significantly blocked its transcriptional activity from a promoter construct after 24h. The ceramide-induced decrease in the transcriptional activity of C/EBPβ correlated with a strong decrease in its phosphorylation, DNA-binding ability and nuclear localization at 24h. However, ceramide did not change the nuclear level of C/EBPβ after a period of 4 or 16h, suggesting that it was not affecting nuclear import. CRM1 (more recently named ‘exportin-1') is a nuclear membrane protein that regulates protein export from the nucleus by binding to a specific nuclear export sequence. Leptomycin B is an inhibitor of CRM1/exportin-1, and reversed the ceramide-induced decrease in nuclear C/EBPβ at 24h. Taken together, these data support the hypothesis that ceramide may inhibit adipogenesis, at least in part, by enhancing dephosphorylation and premature nuclear export of C/EBPβ at a time when its maximal transcriptional activity is required to drive adipogenesis.