CCAAT/enhancer binding protein β mediates the activation of the murine α₁(I) collagen promoter by acetaldehyde

Acetaldehyde was previously shown to activate the α₁(I) and α₂(I) collagen promoters and to increase collagen production in activated stellate cells. Also, CCAAT/enhancer binding protein β (C/EBPβ) binds and activates the mouse α(I) collagen promoter. This study investigates the role of C/EBPβ in mediating the activation of the α(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 α₁(I) collagen promoter. The four complexes were identified to represent C/EBPβ binding to the oligonucleotide by supershift with C/EBPβ antibody. The principal C/EBP isoform found in the nuclear extracts from stellate cells was C/EBP/β, with very low amounts of C/EBPα detected. Acetaldehyde (200 μM) increased C/EBPβ protein in stellate nuclear extracts, increased its binding to the promoter, and activated the α₁(I) collagen promoter in transfected stellate cells. Mutation of the C/EBPβ 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/EBPβ expression vector. This study shows that C/EBPβ is the predominant C/EBP isoform found in activated stellate cells and that increased C/EBPβ protein and C/EBPβ binding to a proximal C/EBP binding site in the promoter mediates the activating effect of acetaldehyde. (C) 2000 Academic Press.