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 α1(I) collagen promoter. This study investigates the role of C/EBPβ in mediating the activation of the α1(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/EBPβ, 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.