Abstract
Osteopontin (OPN) is a secreted phosphoprotein, originally characterized in malignant-transformed epithelial cells. OPN is associated with tumor metastasis of several tumors and is overexpressed in hepatocellular carcinoma (HCC) tissue involving HCC invasion and metastasis. Importantly, OPN is significantly up-regulated in liver injury, inflammation, and hepatitis C virus (HCV)-associated HCC. However, the underlying mechanisms of OPN activation and its role in HCV-mediated liver disease pathogenesis are not known. In this study, we investigated the mechanism of OPN activation in HCV-infected cells. We demonstrate that HCV-mediated Ca(2+) signaling, elevation of reactive oxygen species, and activation of cellular kinases such as p38 MAPK, JNK, PI3K, and MEK1/2 are involved in OPN activation. Incubation of HCV-infected cells with the inhibitors of AP-1 and Sp1 and site-directed mutagenesis of AP-1- and Sp1-binding sites on the OPN promoter suggest the critical role of AP-1 and Sp1 in OPN promoter activation. In addition, we show the in vivo interactions of AP-1 and Sp1 with the OPN promoter using chromatin immunoprecipitation assay. We also show the calpain-mediated processing of precursor OPN (∼75 kDa) into ∼55-, ∼42-, and ∼36-kDa forms of OPN in HCV-infected cells. Furthermore, we demonstrate the critical role of HCV-induced OPN in increased phosphorylation of Akt and GSK-3β followed by the activation of β-catenin, which can lead to EMT of hepatocytes. Taken together, these studies provide an insight into the mechanisms of OPN activation that is relevant to the metastasis of HCV-associated HCC.
Highlights
The mechanism underlying Hepatitis C virus (HCV)-induced OPN remains elusive
The results show increased phosphorylation of Akt, GSK-3, and -catenin in HCV-infected cells compared with mock cells, which was reduced in HCV-infected cells transfected with siOPN, siCD44, and si3 but not with siGFP
HBV and HCV chronic infections account for 75% of hepatocellular carcinoma (HCC), whereas nonviral etiologies such as alcohol and genetic or metabolic disorders represent less than 25% of cases
Summary
The mechanism underlying HCV-induced OPN remains elusive. Results: HCV-induced Ca2ϩ signaling, oxidative stress, and activation of AP-1 and Sp1 play a critical role in OPN activation. Conclusion: OPN plays important roles in EMT and cell migration induced by HCV through the activation of Akt, GSK-3, and -catenin. HrCs.V-related HCC has been reported to be associated with tocytes Taken together, these studies provide an insight into the an increased recurrence after liver resection, signifying that mechanisms of OPN activation that is relevant to the metastasis HCV may promote tumor growth and metastasis [8]. Recent studies have shown the correlation of serum OPN levels with hepatic inflammation and fibrosis in association with alcohol intake [17] Several viruses such as murine polyoma virus middle T antigen, HBV X protein, and HIV have been shown to induce OPN, which can lead to cell invasion and metastasis (18 –20). These observations provide a novel role of HCV-induced OPN in EMT and migration of hepatocytes
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