| Hepatitis C virus (HCV) has been shown to cause a myriad of liver diseases starting with chronic inflammation, steatosis (fatty liver), fibrosis, cirrhosis, and ultimately progressing to hepatocellular carcinoma (HCC). Cytokine array analysis showed that HCV infection induced a significant increase in the secretion of transforming growth factor-beta 1 (TGF-beta1) among other fibrogenic, angiogenic, and inflammatory cytokines and chemokines (Presser et al., 2011). During de novo infection of hepatocytes, HCV induced the multi-functional protein TGF-beta1, which is processed and secreted in its bioactive form where it can bind hepatocytes in an autocrine manner, or hepatic stellate cells (HSC) in a paracrine manner, triggering HSC activation, proliferation and production of extracellular matrix (ECM) molecules leading to liver fibrosis.;In this study, we demonstrated the molecular mechanisms of TGF-beta1 induction, proteolytic activation in HCV (JFH-1)-infected cells, and activation and proliferation of HSCs. Our studies showed that the expression of HCV nonstructural (NS) proteins NS3/4A, and NS5A can induce TGF-beta1 using cell-based luciferase assay. Furthermore, mutational analysis revealed that the functionally active protease domain of NS3 and N terminus domain of NS5A are required for TGF-beta1 activity. We demonstrated that the synthesis and secretion of TGF-beta1 in HCV-infected cells was reduced in the presence of Ca2+ chelators, an inhibitor of mitochondrial Ca2+ uptake, and antioxidants.;We also demonstrate the induction of TGF-beta1 promoter-luciferase reporter in HCV-infected human hepatoma cells. Using various deletion mutants of TGF-beta1 promoter-luciferase reporters, we demonstrated the role of transcription factors AP-1, Sp1, NF-kappa, and STAT-3 in TGF-beta1 promoter activation. Furthermore, we observed that the activation of TGF-beta1 promoter was abrogated in the presence of pharmacological inhibitors of AP-1, Sp1, and NF-kappaB Using a series of inhibitors and dominant negative mutants, we show that HCV-induced activation of TGF-beta1 promoter is mediated by the activation of cellular kinases, including p38 MAPK, Src, JNK, and MEK1/2.;Using siRNA approach we demonstrated that HCV-induced furin and thrombospondin-1 (TSP-1) are involved in the proteolytic activation of TGF-beta1 Next, we demonstrated the fibrogenic potential of HCV-induced TGF-beta1 on HSCs. We observed that HCV-induced secreted bioactive TGF-beta1 is responsible for activation of HSCs. Using siRNA approach, we demonstrated that HCV-induced furin, TSP-1 are critical for bioactive TGF-beta1 secretion and HSCs activation. Evidence is also presented for the potential role of HCV-induced TGF-beta1 in HSC invasion in Boyden chamber. Our results also suggest the role of HCV-induced TGF-beta1 in HCV replication and egress.;Collectively, these observations provide insight into the mechanism of TGF-beta1 promoter activation, as well as HSC activation/proliferation, which likely manifests in liver fibrosis associated with HCV infection.;Overall, my studies highlight the importance of TGF-beta1 in HCV infected cells and liver fibrosis. These studies elucidate the molecular mechanisms of HCV-induced TGF-beta1 and identify pathways induced by HCV infection that trigger an increase in TGF-beta1 induction and secretion. These studies also highlight a potential role for TGF-beta1 in the HCV life cycle. We also demonstrate a role for secreted bioactive TGF-beta1 from HCV infected hepatocytes in HSC activation which elucidates the role of HCV in liver fibrosis from infection of hepatocytes to activation of HSCs. The outcome of these studies offer insight into HCV biology which may lead to identification of potential novel therapeutic targets to control HCV infection and associated liver fibrogenesis. |
