title: CCAAT/enhancer binding protein beta mediates the activation of the murine alpha1(I) collagen promoter by acetaldehyde.
abstract: 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.
authors.affiliation: Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
authors.forename: F A
authors.initials: FA
authors.lastname: Attard
authors.forename: L
authors.initials: L
authors.lastname: Wang
authors.forename: J J
authors.initials: JJ
authors.lastname: Potter
authors.forename: L
authors.initials: L
authors.lastname: Rennie-Tankersley
authors.forename: E
authors.initials: E
authors.lastname: Mezey
mesh_terms: D000079:Acetaldehyde / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D022762:CCAAT-Enhancer-Binding Proteins; D003094:Collagen / Q000235:genetics*; D017931:DNA Primers / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects*; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008297:Male; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014162:Transfection
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D003094:Collagen; D000079:Acetaldehyde