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 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.

Ceramide Negatively Regulates GlutathioneS-transferase Gene Transactivation via Repression of Hepatic Nuclear Factor-1 That Is Degraded by the Ubiquitin Proteasome System

“…Oltipraz induces GSTA2 through enhanced nuclear translocation of HNF1 and HNF1 binding to the HRE (M. K. Cho and S. G. Kim, unpublished data) and through activating C/EBP␤ binding to the C/EBP response element (Kang et al, 2003). Ceramide decreased the transcriptional activity of C/EBP␤ with a strong decrease in its phosphorylation (Sprott et al, 2002;Kostova and Wolf, 2003). We also found that ceramide stimulates degradation of C/EBP␤ in H4IIE cells (I. N. Park, I. J. Cho, and S. G. Kim, unpublished data).…”

Molecular Symbiosis of CHOP and C/EBPβ Isoform LIP Contributes to Endoplasmic Reticulum Stress-Induced Apoptosis

“…Nuclear localization and nuclear export sequences were previously described for C/EBP␤, and it was experimentally proven that the LAP isoforms are able to shuttle to the cytoplasm in some conditions, such as in tumor necrosis factor alpha-treated primary hepatocytes (2) or 3T3-L1 preadipocytes treated with ceramide (40). Here, we sought to determine the cellular compartment in which LIP is being degraded during the ER stress, since this might prove to be relevant for its function.…”

Inverse Relationship between Adipocyte Differentiation and Ceramide Level in 3T3-L1 Cells

“…Addition of exogenous C 2 ceramide or D-e-MAPP, an alkaline ceramidase inhibitor, to preadipocytes inhibited the expression of CCAAT-enhancer binding protein alpha (C/EBPa) and peroxisome proliferator activated receptor gamma (PPARg) and then blocked adipocyte development. 28) EGCG, a polyphenol compound, inhibited adipocyte differentiation [29][30] and elevated endogenous ceramide content in 3T3-L1 cells (Fig. 3).…”