ISOLATION AND ACTION OF ADULT HEPATOCYTE MITOGENS

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The long term objective of this research is to understand how blood-borne substances control liver regeneration. To accomplish this goal, hepatic mitogens will be isolated and purified, characterized structurally, and analyzed for their mechanisms of action. Two hypotheses will be tested: (1) Rat plasma Factor X, a coagulation zymogen with a putative homology to the primary sequence of epidermal growth factor ("EGF"), stimulates hepatocyte proliferation by binding to specific hepatic cell surface receptors; and (2) Factor X and/or EGF receptors mediate sodium ion-dependent prereplicative events (proto-oncogene c-fos activation) required for proliferative transitions. Factor X will be purified from rat plasma (by ion exchange and/or immunoaffinity chromatography (using an anti-X mouse monoclonal antibody ("MCA") selected for its ability to block the Factor Xo Xa conversion)), characterized structurally (subunit M/r (SDS-PAGE); peptide maps (cellulose TLC); primary sequence (deduced from rat liver cDNA sequences cloned and detected with anti-X-MCA in the expression vector wavelength gtll)), and bioassayed for its mitogenic properties (using validated primary cultures of adult rat hepatocytes in the presence or absence of other known mitogens). Factor X receptors will be identified kinetically by monitoring the binding of 125 I-labelled Factor X to plasma membranes (or membrane fractions) from liver tissue and hepatocytes isolated or cultured under varying conditions. Kinetic binding constants (k1, k2, k/d, b/max) will be determined from non-equilibrium rate studies and Scatchard plots. The ability of Factor X to stimulate fos expression (increases in fos mRNA levels (detected by hybridization of 32P-v-fos probes on Northern blots), fos protein synthesis (detected by immunoprecipitation of 35S-met-p55 fos using a new c-fos-specific MCA), and %fos-producing hepatocytes (dual immunocytochemical detection with anti-fos MCA and anti- albumin antibody) will be tested in cultured hepatocyte bioassays. The prereplicative kinetics of fos expression--and its dependence on extracellular sodium ions--will be determined precisely and compared to fos activation responses obtained with EGF (in the presence or absence of other growth factors). Anti-fos MCA or anti-sense fos mRNA will be inserted into hepatocytes by rbc ghost/needle microinjection or by retroviral gene transduction, respectively, in attempts to block mitogen-activated fos expression (or function) and subsequent hepatocyte growth. Results from these studies will provide fundamental insights into the mechanisms that control and link hepatic growth and function.