Decreased activity and enhanced nuclear export of CCAAT-enhancer-binding protein beta during inhibition of adipogenesis by ceramide.
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 7 days; in part, via the co-ordinated action of CCAAT-enhancer-binding proteins alpha, beta and delta (C/EBP-alpha, -beta and -delta) and peroxisome-proliferator-activated receptor gamma (PPARgamma). The addition of exogenous N-acetylsphingosine (C2-ceramide) or increasing endogenous ceramide levels inhibited the expression of C/EBPalpha and PPARgamma, and blocked adipocyte development. C2-ceramide did not decrease the cellular expression of C/EBPbeta, which is required for expression of C/EBPalpha and PPARgamma, but significantly blocked its transcriptional activity from a promoter construct after 24 h. The ceramide-induced decrease in the transcriptional activity of C/EBPbeta correlated with a strong decrease in its phosphorylation, DNA-binding ability and nuclear localization at 24 h. However, ceramide did not change the nuclear level of C/EBPbeta after a period of 4 or 16 h, 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/EBPbeta at 24 h. 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/EBPbeta at a time when its maximal transcriptional activity is required to drive adipogenesis.
Sprott
Kam M
KM
Department of Pharmacology and Toxicology, University of Kansas, Lawrence, 5064 Malott Hall, KS 66045, USA.
Chumley
Michael J
MJ
Hanson
Janean M
JM
Dobrowsky
Rick T
RT
D016475:3T3 Cells; D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D000818:Animals; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000378:metabolism; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D004247:DNA / Q000378:metabolism; D006801:Humans; D051379:Mice; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology*; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation / Q000187:drug effects
D022763:CCAAT-Enhancer-Binding Protein-alpha; D022782:CCAAT-Enhancer-Binding Protein-beta; C064769:N-acetylsphingosine; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D004247:DNA; D013110:Sphingosine