The main goal of the proposal is to understand the role of Hepatocyte Growth Factor (HGF), its receptor (cMet) and the hepatic biomatrix as determinants of hepatic growth, regeneration, tissue differentiation and neoplasia. Converging studies from our lab and others have identified HGF and matrix as key parameters in all these processes. Based on previous work, we have identified different roles and sources for HGF for the first 3 hours after partial hepatectomy (PHx), primarily from pre-existing matrix stores, and following 3 hours after PHx, primarily from newly synthesized HGF. Animal (rat and mouse) models, including conditional homozygous deletion of hepatic HGF gene, are to be employed to study the functions of HGF in liver regeneration. The same models will also be employed to understand the role of hepatic HGF during development of hepatic neoplasia. New HGF at 3 hours post PHx is synthesized not only in liver but also in remote sites. Since we found that norepinephrine stimulates synthesis of new HGF and also rises in the plasma after PHx, will pursue studies to explore the role of norepinephrine as the messenger for HGF synthesis throughout the body after PHx. In studies from the previous period we found that beta-catenin is subjected to tyrosine phosphorylation by cMet. We also now have evidence that beta-catenin is a key regulator of the early stages after PHx and it is subjected to tyrosine phosphorylation by cMet. Studies are proposed to understand the interaction between cMet and beta-catenin, including transgenic mice in which beta-catenin is expressed under the albumin promoter. Since beta-catenin mutations are important in liver cancer in humans, the proposed studies will provide information on the function of beta-catenin and its interaction with cMet as regulators of hepatic neoplasia. We have designed organoid cultures in which HGF, EGF and corticosteroids interact in a complex medium (HGM) to allow growth of hepatocytes and non-parenchymal cells, so that they form reproducible and recognizable structures of hepatic histology in a completely defined environment. We have found that corticosteroids and HGF are essential for hepatocyte maturation. HGF and EGF are essential for formation of the complex connective tissue and the fully mature biliary epithelium seen in these cultures. This system allows development biology studies impossible in mouse genetic systems. Proposed studies aim to use this """"""""in vitro embryology"""""""" system and provide answers on the role of HGF and matrix in hepatic tissue development and differentiation. Finally, we have succeeded in isolating relatively pure material on a key transcription factor (HNFx, for the purposes of the grant) that appears to regulate matrix- and HGF-induced hepatocyte differentiation in culture. Preliminary studies suggest that HNFx is related or identical to Ear2 transcription factor. Studies are described to determine the nature of HNFx and its regulation by growth factors and matrix during hepatocyte differentiation.
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