Hepatic fibrogenesis and cirrhosis are associated with the activation of the hepatic Ito cell into a proliferating, collagen matrix producing cell. This cell is the major effector during the collagen deposition phases of hepatic fibrosis. Efforts to limit the collagen deposition associated with cirrhosis have been hampered in part by an incomplete understanding of the regulation of Ito cell activation. The long range objective of this laboratory is to identify the intracellular mechanisms that regulate Ito cell activation. Towards that end, the current proposal will specifically focus on the factors which regulate Ito cell proliferation in response to platelet-derived growth factor (PDGF). The activated Ito cell expresses the PDGF surface receptor and thereby becomes responsive to this prototypic mitogen which is present during liver injury and fibrogenesis in vivo. The PDGF signal transduction pathway will be examined with emphasis on the nuclear steps which follow raf and MAPK activation. These two essential PDGF-induced cytoplasmic serine/threonine kinases lead to nuclear fos and jun activation which then dimerize and function as transcription factors. The major fos/jun DNA binding site is a DNA sequence termed the activator protein-1 (AP-1) site. Stimulation of the AP-1 site (present in the regulatory region of numerous genes) is closely linked to cellular proliferation. We have characterized several novel agents capable of potentiating (e.g.- 1.25 dihydroxyvitamin D3 ) or inhibiting (e.g.retinoic acid, prostaglandin E, and lipoxygenase inhibition) the mitogenic response to PDGF by different mechanisms. A detailed examination of their mechanisms of action should provide considerable insight into the central regulatory features of the activation which occurs during hepatic fibrogenesis. Preliminary studies demonstrate that these novel modulators all function at a nuclear level distal to raf and MAPK. The focus of the current proposal will be to dissect the PDGF cascade at the proximal nuclear step which follows raf and MAPK activation. These modulators will be systematically examined for their effects on the critical Ito cell nuclear signalling which is induced by PDGF in vitro as well as following carbon tetrachloride administration and bile duct ligation in vivo (rat models of Ito cell proliferation). The main hypothesis of the current proposal is that: Specific modulators of rat Ito cell proliferation alter AP-1 mediated gene transcription. The agents will be used as tools to specifically examine Ito cell fos/jun activation, AP-1 transcriptional activity and AP-1 binding. These studies should provide insight into the factors which regulate Ito cell activation as it relates to the induction of cell cycle progression and mitogenesis. The findings should ultimately impact on the rational design of anti- fibrogenic therapeutics as well as significantly further our knowledge regarding the fibrotic process in general which is operative in a wide variety of disease states.
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