The overall aim of this proposal is to obtain a better understanding of the molecular mechanisms of hepatic fibrosis by examining the effects of certain cytokines on the process of collagen synthesis in the liver. Three general areas will be investigated: (1) What effects do tumor necrosis factor (TNF) and transforming growth factor-beta (TGF-beta) have on hepatic collagen synthesis? Hepatocytes, hepatic fibroblasts, and hepatic adipocytes treated with these two cytokines will be examined for total collagen synthesis, steady-state mRNA levels, and transcriptional rates for types I, III, and IV procollagen mRNA. Ito cells (hepatic adipocytes) treated with TNF and TGF-beta will also be specifically studied for the ability of these two substances to affect adipocyte phenotype (and hence collagen synthesis) by examining the expression of adipocyte-specific genes. In vivo experiments will also be done by injecting TNF and TGF-beta into normal animals and studying them for the existence of hepatic fibrosis, as well as by examining animal models of fibrosis for increased gene expression of these two cytokines. (2) How do gamma-interferon (gamma-IFN) and corticosteroids inhibit collagen synthesis and what are their effects in vivo? The three hepatic cell types will be treated with gamma-IFN alone or with gamma-IFN together with either TNF or TGF-beta to determine the inhibitory effects of gamma-IFN on collagen production and TNF and TGF-beta. Gamma-IFN's effects in vivo will be studied in the fibrogenic animal models. Corticosteroids will be examined by means of the CAT assay for the presence of a glucocorticoid- repressor element. Steroids' inhibitory effects on TNF and TGF- beta on cells in culture will also be delineated. (3) What cell type produces collagen in vivo? The technique of in situ hybridization will be used to determine the cell type(s) responsible for collagen synthesis in normal and diseased livers. The sum of these individual experiments will enable us to expand our knowledge of the process of hepatic fibrosis. Such information is essential to preventing or developing a therapy for this disease state.
