The objectives are to obtain a better understanding of the molecular effects of alcohol upon hepatic function as well as the pathogenesis of hepatic fibrosis in alcoholic liver disease. Two general areas of inquiry will be investigated: (1) What levels of gene regulation and specifically collagen gene regulation are affected in alcoholic liver disease? This will involve an analysis of mRNA steady state levels by cell free protein synthesis and RNA-DNA hybridization. Quantitative determinations of changes in several specific mRNA species including types I, III, and IV procollagen, albumin, fibronectin and -actin will be undertaken. Nuclear transcriptional run-off analysis will be performed in order to examine the basis for observed changes in steady state mRNA contents. Collagen gene promoter structure and function will be determined by constructing partial genomic libraries from cirrhotics and non-cirrhotic alcoholics, isolating and purifying the collagen promoter, and analyzing it by means of S1 nuclease sensitivity methylation, DNA sequencing, and use of a transient expression vector (pSV-0-CAT). These latter studies may explain variations in an individual's fibrotic response to alcohol. (2) What factors predispose an individual to develop alcoholic cirrhosis? This will involve a genetic analysis of the collagen gene using DNA polymorphisms and analysis of the role of sex hormones and corticosteroids in hepatic collagen synthesis using cell culture techniques, molecular hybridization studies and in vivo transient expression vector analysis. The results of such experiments will help us expand our knowledge of the molecular mechanisms responsible for hepatic fibrosis in man. This knowledge will help in developing diagnostic methods for evaluating fibrogenesis predicting susceptibility to develop cirrhosis, and provide the basis for better therapy. These studies will also facilitate my long term objective to become an independent investigator in academic hepatology with expertise in current areas of molecular biology.
