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 aPKCz, Rab4 and phosphorylation in Ntcp translocation and started defining the role of nPKCd, nPKCe and p38 MAPK in Mrp2 translocation. The present proposal extends these studies to further define the role of PKCs, 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) nPKCd-pThr505 mediates translocation of Ntcp/NTCP by cAMP and translocation of Mrp2/MRP2 by cAMP and TUDC, 2) nPKCe mediates TLC-induced retrieval of Mrp2/MRP2 from the plasma membrane by phosphorylating MARCKS and/or Mrp2/MRP2, and cAMP and TUDC reverse this effect by inhibiting TLC-induced nPKCe activation, 3) cAMP and TUDC stimulate MRP2 translocation by activating p38a MAPK, and TLC induces MRP2 retrieval by activating p382 MAPK, and 4) 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 further confirm the role of kinases. 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 PKC and 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.
The long-term goal of this proposal is to understand the mechanism of canalicular bile formation and cholestasis. The goal of the current proposal is to further define the role of intracellular signaling mechanisms involved in vectorial transport of solutes from blood to bile under physiological and pathological (cholestasis) conditions. More specifically, this proposal will attempt to define the role of isoforms of protein kinase C and p38 MAPK in bile formation and cholestasis. By defining the isoforms that are involved in the cholestatic effect, we should be able to design therapeutic agents that selectively reverse cholestatic effect without affecting beneficial effects.
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