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

蛋白激酶 C 异构体​​在胆汁形成和胆汁淤积中的作用

• 批准号: 8019397
• 负责人: MOHAMMED SAWKAT ANWER
• 金额: $ 58.3万
• 依托单位: TUFTS UNIVERSITY BOSTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-30 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8019397
• 关键词: Abbreviations; Affect; Anions; Bile Acids; Bile fluid; Blood; Cell Line; Cell membrane; Cells; Chemicals; Cholestasis; Co-Immunoprecipitations; Cyclic AMP; Dominant-Negative Mutation; Down-Regulation; Goals; Grant; Hepatocyte; Hepatotoxicity; Human; Immunofluorescence Immunologic; Impairment; Liver; MAP Kinase Gene; MAPK14 gene; MARCKS gene; Mediating; Membrane; Mus; Organelles; Pathogenesis; Phosphorylation; Phosphotransferases; Physiological; Plasmids; Protein Dephosphorylation; Protein Isoforms; Protein Kinase C; Proteins; Rattus; Regulation; Retrieval; Role; Signal Transduction; Signaling Molecule; Testing; Therapeutic Agents; Therapeutic Intervention; Toxic effect; base; bile acid transporter; bile formation; choleretic; design; inhibitor/antagonist; novel; solute

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. 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 protein kinase C isoforms 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其机制。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。在最后一个授予期间，我们定义了APKC，RAB4和磷酸化在NTCP易位中的作用，并开始定义NPKC，NPKC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NPKC和NPKC在NTCP/NTCP和MRP2/MRP2易位/检索中的作用。关键目标之一是定义这些激酶在肝细胞中涉及的机制。 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, cAMP和TUDC通过抑制TLC诱导的NPKC激活而逆转了这种效果。拟议的研究将在大鼠肝脏，原发性人肝细胞和肝细胞系的灌注肝脏和肝细胞中进行。此外，将在从NPKC - / - 和NPKC - / - 小鼠中分离出的灌注肝和肝细胞中进行研究，以进一步确认这些激酶的作用。这些激酶的作用将通过使用化学抑制剂，野生型，组成性活性和显性负质粒以及si-和/或shRNA来评估它们的活性和表达。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。总的来说，拟议的研究应进一步定义NPKC在NTCP/NTCP和MRP2/MRP2易位，NPKC在MRP2/MRP2检索中的作用以及TLC诱导的CAMP和TUDC造成的胆汁疾病的作用。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治疗剂，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同工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。