Role of CREB in transcriptional regulation of CCAAT/enhancer-binding protein beta gene during adipogenesis

The proximal promoter of the C/EBPbeta gene possesses dual cis regulatory elements (TGA1 and TGA2), both of which contain core CREB binding sites. Comparison of the activities of C/EBPbeta promoter-reporter genes with 5'-truncations or site-directed mutations in the TGA elements showed that both are required for maximal promoter function. Electrophoretic mobility shift and chromatin immunoprecipitation (ChIP) analyses with antibodies specific to CREB and ATF1 showed that these CREB family members associate with the proximal promoter both in vitro and ex vivo. Immunoblotting and ChIP analysis revealed that other CREB family members, CREM and ATF1, are up-regulated and associate with the proximal C/EBPbeta promoter in mouse embryonic fibroblasts (MEFs) from CREB(-/-) mice. ChIP analysis of wild-type MEFs and 3T3-L1 preadipocytes revealed that interaction of phospho-CREB, the active form of CREB, with the C/EBPbeta gene promoter occurs only after induction of differentiation of 3T3-L1 preadipocytes and MEFs. Consistent with the interaction of CREB and ATF1 at the TGA regulatory elements, expression of constitutively active CREB strongly activated C/EBPbeta promoter-reporter genes, induced expression of endogenous C/EBPbeta, and caused adipogenesis in the absence of the hormonal inducers normally required. Conversely, expression of a dominant-negative CREB blocked promoter-reporter activity, expression of C/EBPbeta, and adipogenesis. When subjected to the standard adipocyte differentiation protocol, wild-type MEFs differentiate into adipocytes at high frequency, whereas CREB(-/-) MEFs exhibit greatly reduced expression of C/EBPbeta and differentiation. The low level of expression of C/EBPbeta and differentiation in CREB(-/-) MEFs appears to be due to up-regulation of other CREB protein family members, i.e. ATF1 and CREM.     

Post-transcriptional control of CCAAT/enhancer-binding protein beta (C/EBPbeta) expression: formation of a nuclear HuR-C/EBPbeta mRNA complex determines the amount of message reaching the cytosol

In 3T3-L1 cells, HuR is constitutively expressed and prior to induction of differentiation localized predominantly to the nucleus. Within minutes of induction of differentiation, nuclear HuR binds to its target ligand mRNAs, and the complexes appear to move to the cytosol. One ligand mRNA is the CCAAT/enhancer-binding protein beta (C/EBPbeta) message. To examine the function and importance of the HuR-C/EBPbeta interaction, retroviral expression constructs were created in which the HuR binding site was altered by deletion (betadel) or deletion and substitution (betad/s). Expression of these constructs in murine embryonic fibroblasts resulted in significant adipose conversion relative to those cells expressing wild type C/EBPbeta. C/EBPbeta protein content was increased markedly in both betadel and betad/s, which correlated with the acquisition of the adipocyte phenotype. Analysis of the betad/s cell line demonstrated a robust expression of C/EBPalpha coincident with peroxisome proliferator-activated receptor gamma expression. Total C/EBPbeta mRNA accumulation indicated no difference between cells harboring either the wild type C/EBPbeta cDNA or betad/s construct. However, cytosolic C/EBPbeta mRNA in the cells expressing the betad/s construct was maintained at levels between 2- and 7-fold greater than in the cells expressing the wild type construct. Alteration in mRNA half-life was not responsible for the increased accumulation. Mechanistically, these data suggest that HuR binding results in nuclear retention of the C/EBPbeta mRNA and is consistent with HuR control, at least in part, of mRNA processing.     

CCAAT/enhancer-binding protein beta isoforms and the regulation of alpha-smooth muscle actin gene expression by IL-1 beta

The role of IL-1beta in inflammation is amply documented, but its ability to inhibit myofibroblast differentiation and, in particular, the suppression of alpha-smooth muscle actin (alpha-SMA) gene expression is less well understood. Because IL-1beta can induce C/EBPbeta expression, the role of C/EBPbeta isoforms in IL-1beta regulation of alpha-SMA gene expression was investigated in rat lung myofibroblasts. The results showed that IL-1beta inhibited alpha-SMA expression in a dose-dependent manner, which was associated with stimulation of the expression of both C/EBPbeta isoforms, liver-enriched activating protein (LAP) and liver-enriched inhibitory protein (LIP). However, a greater increase in LIP relative to LAP expression resulted in a reduced LAP/LIP ratio after IL-1beta treatment. Transfection with an LAP-expressing plasmid stimulated, whereas an LIP-expressing plasmid inhibited, alpha-SMA expression. Cells from C/EBPbeta-deficient mice had reduced levels of alpha-SMA expression and promoter activity, which failed to respond to IL-1beta treatment. Sequence analysis identified the presence of a C/EBPbeta consensus binding sequence in the alpha-SMA promoter, which, when mutated, resulted in diminished promoter activity and abolished its responsiveness to IL-1beta treatment. EMSA revealed binding of C/EBPbeta to this C/EBPbeta consensus binding sequence from the alpha-SMA promoter. Finally, IL-1beta enhanced the expression of eukaryotic initiation factor 4E, a stimulator of LIP expression, which may account for a mechanism by which IL-1beta could alter the LAP/LIP ratio. These data taken together suggest that C/EBPbeta isoforms regulate alpha-SMA gene expression, and that its inhibition by IL-1beta was due to preferential stimulation of LIP expression.