Although enhanced hepatic lipid peroxidation and overproduction of collagen type I by hepatic lipocytes are critical steps in the development of liver cirrhosis, the regulation of collagen alpha-1 (I) gene expression remains unclear. We have shown that products of lipid peroxidation, such as reactive aldehydes, stimulate collagen gene transcription, and that lipocyte collagen alpha-1 (I) gene expression is enhanced by lipid peroxidation in the liver, in coculture with hepatocytes, and when cultured alone. Moreover, we demonstrated that lipid peroxidation induces activation of lipocytes, an indispensable phenotypic change for these cells to express collagen-type I. We have identified in transgenic mice and in primary lipocytes and fibroblasts bearing regulatory elements of the collagen alpha-1 (I) gene, specific cis-regulatory elements critical for the differential transcription of the collagen alpha-1 (I) gene in activated lipocytes and fibroblasts. Also we found that induction of c-myb is both sufficient and necessary for lipocyte activation, and that oxidative stress plays a critical role in this biological process, which is the basis of fibrogenesis. To gain insight into the mechanisms underlying the activation of lipocytes we will assess the cascade induced by lipid peroxidation (protein Kinase C activation, NFkB nuclear translocation, and c-myb expression) leading to lipocyte proliferation. In addition we will determine the role of cell cycle inhibitors (as well as trans-dominant negative modulators) from the bZip and helix-loop-helix families of differentiation factors. The pathophysiological relevance of these findings will be ascertained by blocking lipocyte activation and collagen gene expression in animal models of hepatic lipid peroxidation and fibrogenesis. We will use retroviral vectors expressing c-myb antisense or collagen alpha-1 (I) antisense under the direction of the specific enhancer of the alpha- smooth muscle actin (which is induced in activated lipocytes).
The specific aims of this proposal are: a. The characterization of the cis-regulatory region of the collagen alpha-1 (I) in activated lipocytes. b. The identification of the gene(s) encoding the factor(s) that transactivates the collagen alpha-1 (I) gene in activated lipocytes. c. The characterization of the molecular mechanisms responsible for lipocyte activation. d. The identification of the gene(s) encoding the factor(s) that confers lipocyte differentiation. e. The inhibition of collagen gene expression in animal models of hepatic fibrosis.
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