Intracellular signaling pathways and virus induced biliary atresia

描述（由申请人提供）：胆道闭锁（BA）是儿科终末期肝病的最常见原因，也是儿科肝移植的头号适应症。由于在患病儿童的肝脏中发现了致病病毒，因此胆道闭锁的病因学被认为是围产期病毒感染引发胆管上皮的炎性破坏。胆道闭锁的小鼠模型支持病毒发病机制，因为感染恒河猴轮状病毒（RRV）的新生小鼠在门脉道内发生炎症和肝外胆管阻塞。我们的总体假设是胆道闭锁是由病毒感染胆管细胞引起的，病毒重新编程其细胞内环境，触发免疫介导的胆道梗阻。我们的重点是确定RRV感染胆管细胞的机制，RRV如何在胆管细胞内复制，以及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。这些相互关联的事件的机制基础的确定是必不可少的，以了解病毒引起的BA的发病机制。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，以最大限度地复制。具体针对这一应用，我们提出了细胞外调节激酶1和2（ERK 1/2）信号级联调控RRV复制的假设。ERK 1/2级联是促分裂原活化蛋白激酶（MAPK）细胞内信号传导途径之一，并且该假设得到了我们实验室中产生的新数据的支持，其中MAPK途径的抑制降低了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复制来确定该通路如何有助于病毒诱导的BA的发病机制。我们将使用病毒结合试验、蛋白质印迹分析、siRNA和MAPK通路的特异性抑制剂来实现这些目标。 公共卫生相关性：胆道闭锁（BA）是小儿终末期肝病的最常见原因，也是小儿肝移植的头号适应症。由于在患病儿童的肝脏中发现了致病病毒，因此胆道闭锁的病因学被认为是围产期病毒感染引发胆管上皮的炎性破坏。我们的目标是确定这一过程的机制基础，并在此过程中开发新的治疗策略，以改变这种具有挑战性的疾病的进程。该项目与NIH减少疾病和残疾的使命完全雅阁。

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.