CCAAT/enhancer binding protein beta mediates the activation of the murine alpha1(I) collagen promoter by acetaldehyde.Arch Biochem Biophys. 2000 Jun 01; 378(1):57-64.AB
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.