TRUSTEES OF TUFTS COLLEGE INC
The long-term goal of this proposal is to understand the mechanism of canalicular bile formation and cholestasis. Our present understanding of the pathogenesis of cholestasis is based on studies to define the physiological regulation of transporters involved in bile formation and their deregulation in cholestasis. During the last granting period we have defined the role of aPKC?, Rab4 and phosphorylation in Ntcp translocation and started defining the role of nPKC?, nPKC? and p38 MAPK in Mrp2 translocation. The present proposal extends these studies to further define the role of p38 MAPK and Rab proteins in Ntcp and Mrp2 translocation. One of the key goals is to define the mechanism involved in opposing effects of these kinases in hepatocytes. The following hypotheses are proposed: 1) cAMP and TUDC stimulate MRP2 translocation by activating p38? MAPK, and TLC induces MRP2 retrieval by activating p38? MAPK, and 2) Rab4 facilitates cAMP-induced NTCP translocation by recruiting kinesin to NTCP containing vesicles, cAMP and TUDC stimulate MRP2 translocation by activating Rab4 and/or Rab11, and TLC induces MRP2 retrieval by activating Rab5. Proposed studies will be conducted in rat hepatocytes and hepatic cell lines. Role of various kinases and Rab proteins will be evaluated by manipulating their activity and expression using chemical inhibitors, wild type-, constitutively active- and dominant negative-plasmids and Si- and/or ShRNA. Limited studies will be conducted in perfused livers and hepatocytes isolated from specific kinase knockout mice to confirm physiological relevance. Immunofluorescence and co-immunoprecipitation studies will be used to determine colocalization of desired proteins in subcellular organelles and with other proteins. Collectively, proposed studies should further define the role of p38 MAPK isoforms and Rab proteins in Ntcp and Mrp2 translocation. Since a kinase may produce beneficial or toxic effect in an isoform specific manner, a better understanding of isoform specific effects should allow for a better drug design that could avoid potential liver toxicity.