HORMONAL REGULATION OF ETHANOL METABOLISM

Johns Hopkins University, Baltimore MD

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Abstract

The objectives of the proposal are twofold: 1) To investigate the mechanism of the effect of hormones on the regulation of transcription of the rat class I alcohol dehydrogenase gene and 2) To study the mechanism whereby acetaldehyde stimulates hepatic fibrogenesis. Alcohol dehydrogenase is increased by growth hormone and suppressed by dihydrotestosterone. Glucagon and epinephrine enhance alcohol dehydrogenase and this effect is most likely mediated by cAMP. The effect of growth hormone in activating alcohol dehydrogenase is mediated by binding of C/EBPbeta to the promoter. It will now be determined whether growth hormone also affects transcription of alcohol dehydrogenase by decreasing binding of rat liver negative regulatory elements, namely analogues to the Drosophila adult enhancer factor-1 (AEF-1) which inhibits C/EBPalpha action, and elements binding between -400 and -230 on the promoter, since growth hormone activates the 230 bp, but not the 400 bp alcohol dehydrogenase promoter. Studies of the inhibitory effect of dihydrotestosterone on alcohol dehydrogenase will determine whether it occurs at the level of transcription or is the result of enhanced enzyme degradation. The effects of cAMP on alcohol dehydrogenase will be determined by measurements of enzyme activity, message, and rate of transcription and if the rate of transcription is found altered, mediating cis-acting and trans-acting elements will be studied. Investigation of the mechanism whereby acetaldehyde enhances collagen formation will be carried out in rat myofibroblast-like cells and in NIH 3T3 fibroblasts in culture. Initial studies will determine whether the effects of acetaldehyde are mediated by transforming growth factor-beta1. The mediating role of cis- acting elements on the alpha2(I) collagen promoter will be assessed by transfection of the wild-type promoter and promoters which contain deletions and/or point mutations in regions where transcription factors are known to bind. The role of nuclear factor I (NF-I) in mediating the effect of acetaldehyde in enhancing the alpha2(I) collagen promoter will be determined in cotransfection experiments with a NF-I expression vector and the above promoters. The effects of exposure of cultured cells to acetaldehyde will be determined on the binding of nuclear proteins to the alpha2(I) collagen promoter and to oligonucleotides containing binding sequences to specific trans-acting factors such as NF-I. Finally, it will be determined whether extracellular added acetaldehyde forms adducts with specific nuclear proteins since such adduct formation may result in alterations of the regulation of the expression of alcohol dehydrogenase and other genes.