Role of Protein Kinase C in Isoforms in Bile formation and Cholestasis

说明(申请人提供)：本提案的长期目标是了解。我们目前对胆汁淤积发病机制的理解是基于对胆汁形成过程中转运体的生理调节以及它们在胆汁淤积中的失控的研究。在最后的授予期间，我们已经确定了aPKC、Rab4和磷酸化在Ntcp易位中的作用，并开始定义nPKC、nPKC和p38MAPK在MRP2易位中的作用。本提案扩展了这些研究，以进一步确定nPKC和nPKC在Ntcp/Ntcp和MRP2/MRP2易位/检索中的作用。研究的主要目标之一是确定在肝细胞中参与这些激酶相反作用的机制。提出以下假设：1)nPKC-pThr505通过cAMP介导Ntcp/NTCP的转位，并通过cAMP和TUDC将MRP2/MRP2转位到质膜；2)nPKC通过磷酸化Marcks和/或MRP2/MRP2，介导TLC诱导的MRP2/MRP2从质膜上恢复，cAMP和TUDC通过抑制TLC诱导的nPKC激活而逆转这一作用。拟议的研究将在灌流的肝脏和来自大鼠肝脏的肝细胞、原代人类肝细胞和肝细胞系中进行。此外，将在nPKC-/-和nPKC-/-小鼠的灌流肝脏和分离的肝细胞中进行研究，以进一步证实这些激酶的作用。这些激酶的作用将通过使用化学抑制剂、野生型、结构性活性和显性负质粒以及Si和/或shRNA来调节它们的活性和表达来评估。免疫荧光和免疫共沉淀研究将被用来确定所需蛋白质在亚细胞细胞器中的共存以及与其他蛋白质的共存。总之，建议的研究应进一步明确nPKC在Ntcp/Ntcp和MRp2/MRP2易位中的作用，nPKC在MRp2/MRP2恢复中的作用，以及cAMP和TUDC逆转TLC诱导的胆汁淤积的机制。由于一种激酶可能以一种异构体特异性的方式产生有益的或毒性的效应，对异构体特异性效应的更好的理解应该允许更好的治疗药物，从而避免潜在的肝脏毒性。

DESCRIPTION (provided by applicant): The long-term goal of this proposal is to understand the mechanism of. 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 nPKC and nPKC in Ntcp/NTCP and Mrp2/MRP2 translocation/retrieval. 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) nPKC -pThr505 mediates translocation of Ntcp/NTCP by cAMP and translocation of Mrp2/MRP2 by cAMP and TUDC to the plasma membrane, 2) nPKC 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 nPKC activation. Proposed studies will be conducted in perfused livers and hepatocytes from rat livers, primary human hepatocytes and hepatic cell lines. In addition, studies will be conducted in perfused livers and hepatocytes isolated from nPKC -/- and nPKC -/- mice to further confirm the role of these kinases. Role of these kinases 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. 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 nPKC in Ntcp/NTCP and Mrp2/MRP2 translocation, the role of nPKC in Mrp2/MRP2 retrieval, and mechanism of reversal of TLC-induced cholestasis by cAMP and TUDC. Since a kinase may produce beneficial or toxic effect in an isoform specific manner, a better understanding of isoform specific effects should allow for better therapeutic agents that could avoid potential liver toxicity.