Intracellular signaling pathways and virus induced biliary atresia

描述（由申请人提供）：胆道闭锁（BA）是儿童终末期肝病最常见的原因，也是儿童肝移植的第一适应症。由于在患病儿童的肝脏中发现了致病病毒，因此提出的胆道闭锁的病因是围产期病毒感染引发胆道上皮的炎症破坏。小鼠胆道闭锁模型支持病毒发病机制，因为感染了恒河轮状病毒（RRV）的新生小鼠在门道内发生炎症和肝外胆管阻塞。我们的首要假设是胆道闭锁是由一种病毒感染胆管细胞引起的，这种病毒重新编程其细胞内环境，引发免疫介导的胆道阻塞。我们的重点是确定RRV感染胆管细胞的机制，RRV如何在胆管细胞内进行复制，以及被感染的胆管细胞如何通过分泌生物活性分子来改变微环境。确定这些相互关联事件的机制基础对于了解病毒诱发BA的发病机制至关重要。本提案的目的是发现RRV如何利用胆管细胞内的细胞内信号通路来最大化复制。针对这一应用，我们提出了细胞外调节激酶1和2 （ERK1/2）信号级联控制RRV复制的假设。ERK 1/2级联是丝裂原活化蛋白激酶（MAPK）细胞内信号通路之一，我们实验室产生的新数据支持了这一假设，其中抑制MAPK通路降低了RRV在胆管细胞内复制的能力。我们将通过研究RRV感染胆管细胞如何激活MAPK通路，以及MAPK通路的激活如何反过来控制RRV的复制，来确定这一途径如何参与病毒诱导BA的发病机制。我们将使用病毒结合试验、western blot分析、siRNA和MAPK通路的特异性抑制剂来实现这些目标。

DESCRIPTION (provided by applicant): Biliary atresia (BA) is the most common cause of pediatric end stage liver disease and the number one indication for pediatric liver transplantation. Because pathogenic viruses have been found in the liver of afflicted children, a proposed etiology for biliary atresia is a perinatal viral infection triggering inflammatory destruction of the biliary epithelium. The murine model of biliary atresia supports a viral pathogenesis as newborn mice infected with rhesus rotavirus (RRV) develop inflammation within the portal tract and extra-hepatic bile duct obstruction. Our overarching hypothesis is that biliary atresia results from the infection of cholangiocytes by a virus which re-programs its intracellular environment triggering immune-mediated biliary obstruction. Our focus is to determine the mechanisms used by RRV to infect cholangiocytes, how RRV undergoes replication within the cholangiocyte and how infected cholangiocytes modify the microenvironment by secretion of bioactive molecules. Determination of the mechanistic basis of these inter-related events is essential to understanding the pathogenesis of virus induced BA. The objective of this proposal is to discover how RRV utilizes the intracellular signaling pathways within the cholangiocyte to maximize replication. Specific for this application, we propose the hypothesis that the extracellular regulated kinase 1 and 2 (ERK1/2) signal cascade governs RRV replication. The ERK 1/2 cascade is one of the mitogen activated protein kinase (MAPK) intracellular signaling pathways, and this hypothesis is supported by novel data generated in our laboratory in which inhibition of the MAPK pathways reduced the ability of RRV to replicate within the cholangiocyte. We will determine how this pathway contributes to the pathogenesis of virus induced BA by the examining how cholangiocyte infection by RRV activates the MAPK pathways and how activation of MAPK pathways in turn govern RRV replication. We will use viral binding assays, western blot analysis, siRNA and specific inhibitors of the MAPK pathways to accomplish these goals. PUBLIC HEALTH RELEVANCE: Biliary atresia (BA) is the most common cause of pediatric end stage liver disease and the number one indication for pediatric liver transplantation. Because pathogenic viruses have been found in the liver of afflicted children, a proposed etiology for biliary atresia is a perinatal viral infection triggering inflammatory destruction of the biliary epithelium. Our goal is to determine the mechanistic basis for this process and in so doing develop new treatment strategies to alter the course of this challenging disease. This project is in complete accord with the NIH mission to reduce illness and disability.