Mechanism of Canalicular Bile Formation and Cholestasis

胆小管形成和胆汁淤积的机制

基本信息

批准号：
7916586
负责人：
MOHAMMED SAWKAT ANWER
金额：
$ 46.35万
依托单位：
TUFTS UNIVERSITY BOSTON
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-01 至 2011-06-30
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：该提案的长期目标是了解胆汁形成和胆汁淤积的机制。我们目前对胆汁淤积发病机理的理解是基于研究的研究，以定义涉及胆汁形成的转运蛋白的生理调节及其在胆汁淤积中的消耗管制。在最后的授予期间，我们定义了APKCZ，RAB4和磷酸化在NTCP易位中的作用，并开始定义NPKCD，NPKCE和P38 MAPK在MRP2易位中的作用。本提案扩展了这些研究，以进一步定义PKC，p38 MAPK和RAB蛋白在NTCP和MRP2易位中的作用。关键目标之一是定义这些激酶在肝细胞中涉及的机制。提出了以下假设：1）NPKCD-PTHR505通过CAMP和TUDC通过营地介导NTCP/NTCP的易位，MRP2/MRP2的易位，2）NPKCE介导了TLC诱导的MRP2/MRP2从plaSmaMmbrane和MRCKS/MRCS/MRC中介导MRP2/MRP2的检索。 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和/或RAB11，TLC通过激活RAB5诱导MRP2检索。建议的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，组成型活性和显性负质粒以及SI-和/或SHRNA来处理其活性和表达来评估。有限的研究将在从特定的激酶基因敲除小鼠中分离出的灌注肝脏和肝细胞中进行，以进一步证实激酶的作用。免疫荧光和共免疫沉淀研究将用于确定亚细胞器和其他蛋白质中所需蛋白的共定位。总体而言，提出的研究应进一步定义PKC和P38 MAPK同工型和RAB蛋白在NTCP和MRP2易位中的作用。由于激酶可能以同工型特定方式产生有益或毒性的作用，因此对同工型特异性效应的更好理解应允许更好的药物设计，以避免潜在的肝毒性。公共卫生相关性：该提议的长期目标是了解大肠胆汁形成和胆汁淤积的机制。当前建议的目的是进一步定义细胞内信号传导机制在生理和病理（胆汁淤积）条件下从血液到胆汁的静脉传输中涉及的作用。更具体地说，该建议将尝试定义蛋白激酶C和p38 MAPK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同工型，我们应该能够设计出有选择地逆转胆汁淤积作用而不会影响有益作用的治疗剂。