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1型（PFIC1; Byler's disease, OMIM 211600）是一种以肝脏为主要表现的全身性疾病。胆汁盐的胆汁排泄在PFIC1中减少，导致进行性胆汁淤积性肝病。PFIC1小鼠模型的特征是肠道对胆盐的摄取不适当增加。ATP8B1的缺陷是PFIC1的基础，其机制尚未确定。FIC1是一种p型atp酶膜蛋白，存在于肝脏、肠道和胰腺中。肝膜和CHOK1细胞的功能研究将氨基磷脂翻转酶活性归因于FIC1。我们实验室的初步研究表明，在患有FIC1疾病的儿童和FIC1反义处理的Caco-2细胞中，Farnesoid x受体（FXR）的活性降低。FIC1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回肠胆汁酸转运体启动子导致肝小管胆汁酸排泄减少和回肠胆汁酸转运体增强。FIC1是一种完整的膜蛋白，可以改变脂质双分子层中氨基磷脂的不对称性。我们假设FIC1表达的变化改变了膜的不对称性，并转导了一个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FIC1表达构建体在细胞系（如Caco-2、CHO）中研究FIC1表达改变的调控反应。下游目标将通过微阵列分析确定，并通过northern blot或RT/PCR确认。相关的信号转导途径将在FIC1存在或不存在的情况下通过充分表征的抑制剂、抗体和蛋白质组学方法进行鉴定。FXR的具体翻译后修饰将通过生化和突变分析来确定。这些创新的研究将揭示PFIC1发病机制的一套新机制，并将开辟脂膜不对称细胞调控的新领域。

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.