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Highly related insulin response sequences (IRSs) mediate effects of insulin on the expression of multiple genes in the liver, including insulin-like growth factor binding protein-1 (IGFBP-1) and phosphoenolpyruvate carboxykinase (PEPCK). Gel shift studies reveal that oligonucleotide probes containing an IRS from the IGFBP-1 or PEPCK gene form a similar complex with hepatic nuclear proteins. Unlabeled competitors containing the IGFBP-1 or PEPCK IRS or a binding site for C/EBP proteins inhibit the formation of this complex. Antibody against C/EBPbeta (but not other C/EBP proteins) supershifts this complex, and Western blotting of affinity purified proteins confirms that C/EBPbeta is present in this complex. Studies with affinity purified and recombinant protein indicate that C/EBPbeta does not interact directly with the IRS, but that other factors are required. Gel shift assays and reporter gene studies with constructs containing point mutations within the IRS reveal that the ability to interact with factors required for the formation of this complex correlates well with the ability of insulin to regulate promoter activity via this IRS (r = 0.849, p < 0.01). Replacing the IRS in reporter gene constructs with a C/EBP-binding site (but not an HNF-3/forkhead site or cAMP response element) maintains the effect of insulin on promoter activity. Together, these findings indicate that a nucleoprotein complex containing C/EBPbeta interacts with IRSs from the IGFBP-1 and PEPCK genes in a sequence-specific fashion and may contribute to the ability of insulin to regulate gene expression.  相似文献   

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Acetaldehyde was previously shown to activate the alpha1(I) and alpha2(I) collagen promoters and to increase collagen production in activated stellate cells. Also, CCAAT/enhancer binding protein beta (C/EBPbeta) binds and activates the mouse alpha1(I) collagen promoter. This study investigates the role of C/EBPbeta in mediating the activation of the alpha1(I) collagen promoter by acetaldehyde. Nuclear extracts isolated from cultured activated rat hepatic stellate cells formed four protein-DNA complexes on electrophoretic mobility shift assay with an oligonucleotide including the C/EBP binding site between -365 and -335 in the alpha1(I) collagen promoter. The four complexes were identified to represent C/EBPbeta binding to the oligonucleotide by supershift with C/EBPbeta antibody. The principal C/EBP isoform found in the nuclear extracts from stellate cells was C/EBPbeta, with very low amounts of C/EBPalpha detected. Acetaldehyde (200 microM) increased C/EBPbeta protein in stellate nuclear extracts, increased its binding to the promoter, and activated the alpha1(I) collagen promoter in transfected stellate cells. Mutation of the C/EBPbeta binding site markedly decreased nuclear protein binding. A transfected promoter, mutated at the C/EBP binding site, had decreased basal activity, was not activated by acetaldehyde, and was not activated when cotransfected with a C/EBPbeta expression vector. This study shows that C/EBPbeta is the predominant C/EBP isoform found in activated stellate cells and that increased C/EBPbeta protein and C/EBPbeta binding to a proximal C/EBP binding site in the promoter mediates the activating effect of acetaldehyde.  相似文献   

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CCAAT/enhancer binding protein (C/EBP) family members are known to transactivate the gene encoding cytosolic phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) in hepatocytes via promoter proximal C/EBP response elements. PEPCK is also expressed in adipocytes; however, fibroblasts that are homozygous null for C/EBPbeta cannot express PEPCK when induced to differentiate into adipocytes (Tanaka et al., EMBO J. 16, 7432-7443, 1997). This along with our previous observation that an upstream adipocyte-specific enhancer contains multiple putative C/EBP binding elements suggested the possibility that C/EBPbeta transactivates the PEPCK gene in adipocytes via distal elements. We report here that C/EBPbeta transactivates a PEPCK-luciferase chimera in transient transfection assays. C/EBPbeta acted independently of peroxisome proliferator-activated receptor gamma (PPARgamma) which is required for function of the enhancer. C/EBPbeta in nuclear extracts and recombinant C/EBPbeta bound three of the putative C/EBP-binding elements within the enhancer. C/EBPbeta binding to these three elements was strongly cooperative. However, mutation of all three elements did not affect reporter transactivation by C/EBPbeta suggesting that additional elements participate in PEPCK regulation or that the effects of C/EBPbeta are indirect.  相似文献   

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The contribution of C/EBP proteins to Epstein-Barr virus (EBV) lytic gene expression and replication in epithelial cells was examined. Nasopharyngeal carcinoma cell lines constitutively expressed C/EBPbeta and had limited C/EBPalpha expression, while the AGS gastric cancer cell line expressed significant levels of both C/EBPalpha and C/EBPbeta. Induction of the lytic cycle in EBV-positive AGS/BX1 cells with phorbol ester and sodium butyrate treatment led to a transient stimulation of C/EBPbeta expression and a prolonged increase in C/EBPalpha expression. In AGS/BX1 cells, endogenous C/EBPalpha and C/EBPbeta proteins were detected associated with the ZTA and oriLyt promoters but not the RTA promoter. Electrophoretic mobility shift assays confirmed binding of C/EBP proteins to multiple sites in the ZTA and oriLyt promoters. The response of these promoters in reporter assays to transfected C/EBPalpha and C/EBPbeta proteins was consistent with the promoter binding assays and emphasized the relative importance of C/EBPs for activation of the ZTA promoter. Mutation of the oriLyt promoter proximal C/EBP site had little effect on ZTA activation of the promoter in a reporter assay. However, this mutation impaired oriLyt DNA replication, suggesting a separate replication-specific contribution for C/EBP proteins. Finally, the overall importance of C/EBP proteins for lytic gene expression was demonstrated using CHOP10 to antagonize C/EBP DNA binding activity. Introduction of CHOP10 significantly impaired induction of the ZTA, RTA, and BMRF1 proteins in chemically treated AGS/BX1 cells. Thus, C/EBPbeta and C/EBPalpha expression are associated with lytic induction in AGS cells, and expression of C/EBP proteins in epithelial cells may contribute to the tendency of these cells to exhibit constitutive low-level ZTA promoter activity.  相似文献   

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