Abstract
Osteopontin (OPN) is a secreted phosphoprotein which has been linked to tumor progression and metastasis in a variety of cancers including hepatocellular carcinoma (HCC). Previous studies have shown that OPN is upregulated during liver injury and inflammation. However, the role of OPN in hepatitis C virus (HCV)-induced liver disease pathogenesis is not known. In this study, we determined the induction of OPN, and then investigated the effect of secreted forms of OPN in epithelial to mesenchymal transition (EMT), migration and invasion of hepatocytes. We show the induction of OPN mRNA and protein expression by HCV-infection. Our results also demonstrate the processing of precursor OPN (75 kDa) into 55 kDa, 42 kDa and 36 kDa forms of OPN in HCV-infected cells. Furthermore, we show the binding of secreted OPN to integrin αVβ3 and CD44 at the cell surface, leading to the activation of downstream cellular kinases such as focal adhesion kinase (FAK), Src, and Akt. Importantly, our results show the reduced expression of epithelial marker (E-cadherin) and induction of mesenchymal marker (N-cadherin) in HCV-infected cells. We also show the migration and invasion of HCV-infected cells using wound healing assay and matrigel coated Boyden chamber. In addition, we demonstrate the activation of above EMT markers, and the critical players involved in OPN-mediated cell signaling cascade using primary human hepatocytes infected with Japanese fulminant hepatitis (JFH)-1 HCV. Taken together, these studies suggest a potential role of OPN in inducing chronic liver disease and HCC associated with chronic HCV infection.
Highlights
hepatitis C virus (HCV) infection is the leading cause of chronic hepatitis which often results in liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC) [1]
HCV Induces OPN Expression and Secretion To determine the kinetics of OPN activation in HCV-infected cells, cellular lysates from mock (Huh7.5) and HCV-infected cells at various time points were subjected to western blot analysis
We observed significantly increased expression of precursor form of OPN (,75 kDa) at days 3 to 8 (Fig. 1A, lane 4–9) followed by its cleavage into various forms of OPN (,55 kDa,42 kDa,36 kDa) in HCV-infected cells compared to mock-infected cells
Summary
HCV infection is the leading cause of chronic hepatitis which often results in liver fibrosis, cirrhosis and HCC [1]. The development of a productive JFH-1-HCV (genotype 2a) infection system provided a major breakthrough that allows the production of infectious virions in cell culture [3,4]. HCC is a highly aggressive carcinoma of the liver, and is the third most common cause of cancer related death worldwide. Cirrhosis of any etiology is the most common risk factor for HCC development. Over 90% of HCCs develop in a cirrhotic liver resulting from chronic hepatitis B virus (HBV) or HCV infections, alcoholic cirrhosis, or nonalcoholic steatohepatitis [5]. The rate of HCC development among HCV-infected persons ranges from 1% to 4%. Identifying the primary contributors to the metastatic cascade, especially at the early stages of cellular invasion, may present opportunities for reducing the severity of HCC through new therapeutic intervention
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