Ethanol induction of class I alcohol dehydrogenase expression in the rat occurs through alterations in CCAAT/enhancer binding proteins beta and gamma.
Alcohol dehydrogenase (ADH) is the principal ethanol-metabolizing enzyme. Ethanol induces rat Class I ADH mRNA and activity by an as yet unknown mechanism. In the current study, adult male rats were fed an ethanol-containing diet by continuous intragastric infusion for 42 days. Hepatic Class I ADH mRNA, protein, and activity levels in the ethanol-infused rats increased 3.9-, 3.3-, and 1.7-fold, respectively (p <0.05). Cis-acting elements within the proximal promoter region of the ADH gene were studied by electrophoretic mobility shift assay (EMSA). Hepatic nuclear extract (HNE) binding to either the consensus or ADH-specific CCAAT/enhancer binding protein (C/EBP) sites was >2.4-fold greater in ethanol-fed rats (p <0.05) than controls. Antibody-specific EMSA assays demonstrated binding of the transcription factor C/EBPbeta to the C/EBP site. Western blot immunoblot analysis of HNEs demonstrated 3.5- and 2.3-fold increases in C/EBPbeta (LAP) and C/EBPdelta (p <0.05), respectively, in ethanol-fed rats compared with controls, whereas levels of the truncated C/EBPbeta (LIP) and C/EBPgamma were lower in ethanol-fed rats (p <0.05). HNE from ethanol-fed rats increased (3-fold) the in vitro transcription of rat Class I ADH (p <0.05), and mutation of the C/EBP element in the proximal promoter region blocked this effect. Antisera against LIP or C/EBPgamma enhanced transcription efficiency (p <0.05). These data provide the first evidence for the mechanism by which ethanol regulates rat hepatic Class I ADH gene expression in vivo. This mechanism involves the C/EBP site and the enhancer binding proteins beta and gamma.
He
Ling
L
Arkansas Children's Nutrition Center, Little Rock 72202, USA.
Ronis
Martin J J
MJ
Badger
Thomas M
TM
D000426:Alcohol Dehydrogenase / Q000096:biosynthesis*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D015152:Blotting, Northern; D015153:Blotting, Western; D022782:CCAAT-Enhancer-Binding Protein-beta / Q000378:metabolism*; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002492:Central Nervous System Depressants / Q000494:pharmacology; D000431:Ethanol / Q000494:pharmacology*; D008297:Male; D009154:Mutation; D009841:Oligonucleotides / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D014158:Transcription, Genetic
D022782:CCAAT-Enhancer-Binding Protein-beta; D022762:CCAAT-Enhancer-Binding Proteins; C405462:CCAAT-enhancer-binding protein-gamma; D002492:Central Nervous System Depressants; D009841:Oligonucleotides; D012333:RNA, Messenger; D000431:Ethanol; D000426:Alcohol Dehydrogenase