Mechanism of Canalicular Bile Formation and Cholestasis

描述（由申请人提供）：本提案的长期目标是了解小管胆汁形成和胆汁淤积的机制。我们目前对胆汁淤积症发病机制的理解是基于研究，以确定参与胆汁形成的转运蛋白的生理调节及其在胆汁淤积症中的失调。在最后一个授权期间，我们已经确定了aPKCz、Rab4和磷酸化在Ntcp易位中的作用，并开始确定nPKCd、nPKCe和p38 MAPK在Mrp2易位中的作用。目前的建议扩展这些研究，以进一步确定蛋白激酶C，p38 MAPK和Rab蛋白在Ntcp和Mrp2易位的作用。关键目标之一是确定这些激酶在肝细胞中的对抗作用所涉及的机制。提出了以下假设：1）nPKCd-pThr505通过cAMP介导Ntcp/NTCP的易位以及通过cAMP和TUDC介导Mrp2/MRP 2的易位，2）nPKCe通过磷酸化MARCKS和/或Mrp2/MRP 2介导TLC诱导的Mrp2/MRP 2从质膜的回收，并且cAMP和TUDC通过抑制TLC诱导的nPKCe活化来逆转该效应，3）cAMP和TUDC通过激活p38a MAPK刺激MRP 2易位，TLC通过激活p382 MAPK诱导MRP 2恢复，和4）Rab4通过将驱动蛋白募集到含有NTCP的囊泡中促进cAMP诱导的NTCP易位，cAMP和TUDC通过激活Rab4和/或Rab11刺激MRP 2易位，TLC通过激活Rab5诱导MRP 2恢复。拟定研究将在大鼠肝细胞和肝细胞系中进行。各种激酶和Rab蛋白的作用将通过使用化学抑制剂、野生型质粒、组成型活性质粒和显性阴性质粒以及Si和/或shRNA操纵其活性和表达来评估。将在从特定激酶敲除小鼠中分离的灌注肝脏和肝细胞中进行有限的研究，以进一步证实激酶的作用。免疫荧光和免疫共沉淀研究将用于确定所需蛋白质在亚细胞器中的共定位以及与其他蛋白质的共定位。总的来说，拟议的研究应进一步确定PKC和p38 MAPK亚型和Rab蛋白在Ntcp和Mrp2易位中的作用。由于激酶可能以同种型特异性方式产生有益或毒性作用，因此更好地理解同种型特异性作用应允许更好的药物设计，从而避免潜在的肝毒性。公共卫生相关性：该提案的长期目标是了解小管胆汁形成和胆汁淤积的机制。本提案的目的是进一步确定在生理和病理（胆汁淤积）条件下溶质从血液到胆汁的载体转运中涉及的细胞内信号传导机制的作用。更具体地说，该建议将试图确定蛋白激酶C和p38 MAPK的亚型在胆汁形成和胆汁淤积中的作用。通过定义参与胆汁淤积作用的亚型，我们应该能够设计选择性逆转胆汁淤积作用而不影响有益作用的治疗剂。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.