Hepatitis C virus and liver fibrogenesis

描述（由申请人提供）：本提案的主要目标是了解丙型肝炎病毒（HCV）感染导致肝纤维化的分子机制。丙型肝炎病毒（HCV）是世界范围内慢性肝病的主要原因。HCV感染通常导致高达60-80%的受感染成人发生慢性肝炎，并进展为肝纤维化、肝硬化和肝细胞癌。慢性丙型肝炎肝损伤和纤维化的机制尚不清楚。HCV基因组是正义单链RNA分子，其编码约3000个氨基酸的多蛋白前体，其在感染后被宿主和病毒编码的蛋白酶切割成结构蛋白（核心、E1和E2）和非结构蛋白（NS 2、NS 3、NS 4A、NS 4 B、NS 5A、NS 5 B）。最近，我们已经显示了在表达HCV非结构蛋白的培养细胞中ROS水平升高。该提案的主要假设是HCV诱导的氧化应激和Ca 2+信号传导诱导转化生长因子-21（TGF-21）的合成和活化及其在HCV复制和肝纤维化中的潜在作用。为了验证这一假设，我们的目标是使用最近描述的HCV细胞培养感染系统来描绘诱导TGF-21的信号通路。具体目的是：1）确定不同HCV基因型（1b型与2a型）激活TGF-21的动力学。2)分析HCV诱导的Ca 2+信号和氧化应激在刺激TGF-21合成中的作用。3)明确细胞激酶和转录因子在TGF-21基因表达反式调节中的作用。4)探讨HCV诱导的内源性TGF-21对HCV复制的影响。HCV诱导的氧化应激和Ca 2+信号传导对TGF- 21表达的刺激将通过分析来自HCV感染的细胞和用抗氧化剂和Ca 2+螯合剂处理的细胞的TGF-21的RNA和蛋白质水平来监测。接下来，将通过使用定量RT-PCR测量HCV RNA水平来监测TGF- 21介导的HCV复制调节.将通过使用来自用TGF-21 siRNA沉默的HCV表达细胞的RNP复合物的两步亲和纯化来检查响应于TGF-21的HCV核糖核蛋白复合物（RNP）的组装。拟议的研究将对导致肝纤维化、肝硬化的促纤维化因子的激活机制产生新的见解，并为描绘肝癌发生前的通路铺平道路。公共卫生相关性：HCV感染通常导致慢性肝炎，进而发展为肝纤维化、肝硬化和肝细胞癌。HCV感染的肝细胞显示氧化应激，这可能提供HCV感染与导致纤维化进展的TGF-21活化之间的联系。从这些研究中得到的信息有可能为HCV诱导的氧化应激和钙信号传导如何诱导促纤维化因子TGF-21、肝纤维化和最终肝癌提供线索。

DESCRIPTION (provided by applicant): The major goal of this proposal is to understand the molecular mechanism(s) of liver fibrosis by hepatitis C virus (HCV) infection. Hepatitis C virus (HCV) is the leading cause of chronic liver disease worldwide. HCV infection often leads to chronic hepatitis in up to 60-80% of infected adults and progresses to liver fibrosis, cirrhosis and hepatocellular carcinoma. The mechanisms underlying the liver injury and fibrosis in chronic hepatitis C are unclear. The HCV genome is a positive-sense single stranded RNA molecule that encodes a polyprotein precursor of ~3000 amino acids which is post- translationally cleaved by host and virus-encoded proteases into structural proteins (core, E1, and E2) and nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, NS5B). Recently, we have shown the elevated levels of ROS in cultured cells expressing HCV nonstructural proteins. This proposal's main hypothesis is that HCV-induced oxidative stress and Ca2+ signaling induce the synthesis and activation of transforming growth factor-21 (TGF-21) and its potential role in HCV replication and liver fibrosis. To test this hypothesis, the goal is to delineate the signaling pathways in induction of TGF-21 using recently described HCV cell culture infection system. The specific aims are to: 1) To define the kinetics of TGF-21 activation by different HCV genotypes (type 1b vs. 2a). 2) To analyze the role of HCV-induced Ca2+ signaling and oxidative stress in stimulating TGF-21 synthesis. 3) To define the role of cellular kinases and transcription factors on trans-regulation of TGF-21 gene expression. 4) To determine the effect of HCV-induced endogenous bioactive TGF-21 on HCV replication. The stimulation of TGF- 21 expression by HCV-induced oxidative stress and Ca2+ signaling, will be monitored by analyzing the RNA and protein levels of TGF- 21 from HCV infected cells and those treated with antioxidant and Ca2+ chelators. Next, TGF- 21 mediated regulation of HCV replication will be monitored by measuring the HCV RNA levels using quantitative RT- PCR. The assembly of HCV ribonucleoprotein complex (RNP) in response to TGF-21 will be examined by using two-step affinity purification of RNP complexes from HCV expressing cells silenced with TGF-21 siRNA. The proposed studies will yield novel insights into mechanisms of activation of profibrogenic factors that leads to hepatic fibrosis, cirrhosis and pave the way for delineating the pathways preceding liver cancer. PUBLIC HEALTH RELEVANCE: HCV infection often leads to chronic hepatitis which progresses to liver fibrosis, cirrhosis and hepatocellular carcinoma. HCV infected hepatocytes display oxidative stress which may provide a link between HCV infection and activation of TGF-21 that leads to progression of fibrosis. The information resulting from these investigations has a potential to provide clues to how HCV-induced oxidative stress and calcium signaling may induce profibrogenic factor, TGF-21, liver fibrosis and ultimately liver cancer.