Mechanism of Canalicular Bile Formation and Cholestasis

描述(申请人提供)：本提案的长期目标是了解胆小管胆汁形成和胆汁淤积的机制。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aPKCz、Rab4和磷酸化在Ntcp易位中的作用，并开始定义nPKCd、nPKCe和p38MAPK在mrp2易位中的作用。本研究扩展了这些研究以进一步确定PKCs、p38MAPK和Rab蛋白在Ntcp和MRp2易位中的作用。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。提出如下假设：1)nPKCd-pThr505通过cAMP介导Ntcp/Ntcp的易位，通过cAMP和TUDC介导mrp2/MRP2的易位；2)nPKCe通过磷酸化Marcks和/或mrp2/MRP2介导TLC诱导的mrp2/MRP2的移位，cAMP和TUDC通过抑制TLC诱导的nPKCe的激活而逆转这一作用；3)cAMP和TUDC通过激活p38A MAPK来刺激MRP2的易位，TLC通过激活p382 MAPK来诱导MRP2的恢复；4)Rab4通过招募激动素到含有NTCP的囊泡中促进cAMP诱导的Ntcp的易位，而TUDC通过激活Rab4和/或Rab4来刺激MRP2的易位而TLC通过激活Rab5来诱导MRP2的恢复。拟议的研究将在大鼠肝细胞和肝细胞系中进行。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来调节各种激酶和Rab蛋白的活性和表达来评估它们的作用。有限的研究将在灌流的肝脏和从特定的激酶基因敲除小鼠分离的肝细胞中进行，以进一步证实激酶的作用。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，拟议的研究应进一步确定PKC和p38MAPK亚型以及Rab蛋白在Ntcp和MRp2易位中的作用。由于一种激酶可能以一种异构体特异性的方式产生有益或有毒的效应，对异构体特异性效应的更好的理解应该允许更好的药物设计，从而避免潜在的肝脏毒性。公共卫生相关性：这项建议的长期目标是了解胆小管胆汁形成和胆汁淤积的机制。本研究的目的是进一步明确在生理和病理(胆汁淤积)条件下，细胞内信号机制在溶质从血液到胆汁的载体运输中的作用。更具体地说，这项提议将试图确定蛋白激酶C和p38MAPK的异构体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异构体，我们应该能够设计出选择性逆转胆汁淤积作用而不影响有益效果的治疗药物。

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.