Molecular Mechanisms of Intrahepatic Cholestasis

描述（由申请人提供）：进行性家族性肝内胆汁淤积症1型（PFIC 1; Byler病，OMIM 211600）是一种全身性疾病，其主要表现为肝脏。PFIC 1中胆汁盐的胆汁排泄减少，导致进行性胆汁淤积性肝病。PFIC 1的小鼠模型的特征在于不适当地增强了胆盐的肠摄取。ATP 8B 1的缺陷是PFIC 1的基础，其机制尚未确定。FIC 1是一种P型ATP酶膜蛋白，存在于肝、肠和胰腺中。在肝膜和CHOK 1细胞中的功能研究将氨基磷脂翻转酶活性归因于FIC 1。我们实验室的初步研究表明，法尼醇X受体（FXR）的活性在FIC 1疾病儿童和FIC 1反义治疗的Caco-2细胞中降低。FIC 1活性的缺失与FXR核定位的破坏有关。FXR活性降低可能通过影响这些转运蛋白的启动子，导致肝小管胆汁酸排泄减少和回肠胆汁酸转运蛋白增强。FIC 1是一种整合膜蛋白，可以改变脂质双层中氨基磷脂的不对称性。我们假设FIC 1表达的变化改变了膜的不对称性，并改变了一种未知的细胞内信号通路，改变了FXR的翻译后修饰。将利用siRNA和FIC 1表达构建体在细胞系（例如Caco-2、CHO）中研究对FIC 1表达变化的调节反应。通过微阵列分析鉴定下游靶标，并通过北方印迹或RT/PCR进行确认。将在存在或不存在FIC 1的情况下，利用充分表征的抑制剂、抗体和蛋白质组学方法鉴定相关信号转导途径。FXR的特异性翻译后修饰将通过生物化学和突变分析来确定。这些创新的研究将描绘一组新的机制，PFIC 1的发病机制的基础，并将开发一个新的领域的细胞调节脂质膜不对称性。

DESCRIPTION (provided by applicant): Progressive familial intrahepatic cholestasis type 1 (PFIC1; Byler's disease, OMIM 211600) is a systemic disease, whose principal manifestation is hepatic. Biliary excretion of bile salts is diminished in PFIC1 resulting in progressive cholestatic liver disease. A mouse model of PFIC1 is characterized by inappropriately enhanced intestinal uptake of bile salts. Defects in ATP8B1 underlie PFIC1 by as yet to be determined mechanisms. FIC1, a P-type ATPase membrane protein, is found in liver, intestine and pancreas. Functional studies in liver membranes and CHOK1 cells ascribe an aminophospholipid flippase activity to FIC1. Preliminary studies in our laboratory have shown that activity of the Farnesoid X-Receptor (FXR) is diminished in children with FIC1 disease and in FIC1 anti-sense treated Caco-2 cells. Absence of FIC1 activity is associated with a disruption in the nuclear localization of FXR. Diminished FXR activity may lead to reduced hepatic canalicular bile acid excretion and enhanced ileal bile acid transporter via effects on the promoters for these transporters. FIC1 is an intergral membrane protein that may alter the asymmetry of aminophospholipids in the lipid bilayer. We hypothesize that changes in FIC1 expression alter membrane asymmetry and transduce an unknown intracellular signaling pathway that alters the post-translational modification of FXR. Regulatory response to alterations in FIC1 expression will be studied in cell lines (e.g. Caco-2, CHO) utilizing siRNA and FIC1 expression constructs. Downstream targets will be identified by microarray analysis with confirmation by northern blot or RT/PCR. Relevant signal transduction pathways will be identified in the presence or absence of FIC1 utilizing well characterized inhibitors, antibodies and proteomic approaches. The specific post-translation modifications of FXR will be determined by biochemical and mutational analysis. These innovative studies will delineate a novel set of mechanisms that underlie the pathogenesis of PFIC1 and will develop a new area of cellular regulation of lipid membrane asymmetry.