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: 8152120
• 负责人: MOHAMMED SAWKAT ANWER
• 金额: $ 47.9万
• 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/8152120
• 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; public health relevance; 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制的理解是基于对参与胆汁形成的转运蛋白的生理调节及其在胆汁淤积中的失调的研究的基础上的。在上一个授权期间，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。目前的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。关键目标之一是确定肝细胞中这些激酶的相反作用所涉及的机制。提出以下假设：1) nPKC -pThr505 介导 cAMP 引起的 Ntcp/NTCP 易位以及 cAMP 和 TUDC 引起的 Mrp2/MRP2 易位至质膜，2) nPKC 通过磷酸化 MARCKS 和/或 Mrp2/MRP2 介导 TLC 诱导的 Mrp2/MRP2 从质膜的修复， cAMP 和 TUDC 通过抑制 TLC 诱导的 nPKC 激活来逆转这种效应。拟议的研究将在大鼠肝脏、原代人肝细胞和肝细胞系的灌注肝脏和肝细胞中进行。此外，还将在从 nPKC -/- 和 nPKC -/- 小鼠分离的灌注肝脏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、组成型活性和显性失活质粒以及Si-和/或ShRNA操纵它们的活性和表达来评估。免疫荧光和免疫共沉淀研究将用于确定亚细胞细胞器中所需蛋白质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，因此更好地了解同种型特异性效应应该允许更好的治疗剂，从而避免潜在的肝毒性。 公众健康相关性：该提案的长期目标是了解胆小管形成和胆汁淤积的机制。当前提案的目标是进一步定义在生理和病理（胆汁淤积）条件下参与溶质从血液到胆汁矢量运输的细胞内信号传导机制的作用。更具体地说，该提案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效应的异构体，我们应该能够设计出选择性逆转胆汁淤积效应而不影响有益效果的治疗药物。