Intracellular signaling pathways and virus induced biliary atresia

描述(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，因此提出的胆道闭锁的病因是围产期病毒感染，引发胆管上皮的炎性破坏。胆道闭锁的小鼠模型支持一种病毒致病机制，即感染恒河猴轮状病毒(RRV)的新生小鼠发展为门静脉内炎症和肝外胆管梗阻。我们的主要假设是，胆道闭锁是由病毒感染胆管细胞所致，病毒对细胞内环境进行重新编程，引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。确定这些相互关联的事件的机制基础对于理解病毒诱导的BA的发病机制是至关重要的。这项建议的目的是发现RRV如何利用胆管细胞内的细胞内信号通路来最大限度地复制。针对这一应用，我们提出了一种假设，即细胞外调节的激酶1和2(ERK1/2)信号级联调控RRV的复制。ERK1/2级联是细胞内丝裂原活化蛋白激酶(MAPK)信号通路之一，本实验室的新数据支持这一假说，抑制MAPK通路降低了RRV在胆管细胞内复制的能力。我们将通过检测RRV感染的胆管细胞如何激活MAPK通路，以及MAPK通路的激活如何反过来调控RRV的复制，来确定该通路在病毒诱导的BA的发病机制中的作用。我们将使用病毒结合分析、免疫印迹分析、siRNA和MAPK途径的特定抑制剂来实现这些目标。 公共卫生相关性：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，因此提出的胆道闭锁的病因是围产期病毒感染，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，并通过这样做来开发新的治疗策略来改变这种具有挑战性的疾病的进程。该项目完全符合美国国立卫生研究院减少疾病和残疾的使命。

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.