Hepatitis C virus (HCV) constitutively activates STAT-3 via oxidative stress: role of STAT-3 in HCV replication

The hepatitis C virus (HCV) causes chronic hepatitis, which often results in liver cirrhosis and hepatocellular carcinoma. We have previously shown that HCV nonstructural proteins induce activation of STAT-3 via oxidative stress and Ca2+ signaling (G. Gong, G. Waris, R. Tanveer, and A. Siddiqui, Proc. Natl. Acad. Sci. USA 98:9599-9604, 2001). In this study, we focus on the signaling pathway leading to STAT-3 activation in response to oxidative stress induced by HCV translation and replication activities. Here, we demonstrate the constitutive activation of STAT-3 in HCV replicon-expressing cells. The HCV-induced STAT-3 activation was inhibited in the presence of antioxidant (pyrrolidine dithiocarbamate) and Ca2+ chelators (BAPTA-AM and TMB-8). Previous studies have shown that maximum STAT-3 transactivation requires Ser727 phosphorylation in addition to tyrosine phosphorylation. Using a series of inhibitors and dominant negative mutants, we show that HCV-induced activation of STAT-3 is mediated by oxidative stress and influenced by the activation of cellular kinases, including p38 mitogen-activated protein kinase, JNK, JAK-2, and Src. Our results also suggest a potential role of STAT-3 in HCV RNA replication. We also observed the constitutive activation of STAT-3 in the liver biopsy of an HCV-infected patient. These studies provide an insight into the mechanisms by which HCV induces intracellular events relevant to liver pathogenesis associated with the viral infection.     

Hepatitis B virus X protein stimulates the mitochondrial translocation of Raf-1 via oxidative stress

The human hepatitis B virus (HBV) X protein (HBx) plays a crucial role(s) in the viral life cycle and contributes to the onset of hepatocellular carcinoma (HCC). HBx caused the mitochondrial translocation of Raf-1 kinase either alone or in the context of whole-viral-genome transfections. Mitochondrial translocation of Raf-1 is mediated by HBx-induced oxidative stress and was dependent upon the phosphorylation of Raf-1 at the serine338/339 and Y340/341 residues by p21-activated protein kinase 1 and Src kinase, respectively. These studies provide an insight into the mechanisms by which HBV induces intracellular events relevant to liver disease pathogenesis, including HCC.     

The X protein of hepatitis B virus activates hepatoma cell proliferation through repressing melanoma inhibitory activity 2 gene

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer deaths globally. Chronic hepatitis B virus (HBV) infection accounts for over 75% of all HCC cases; however, the molecular pathogenesis of HCC is not well understood. In this study, we found that the expression of the newly identified gene melanoma inhibitory activity 2 (MIA2) was reduced by HBV infection in vitro and in vivo, and that HBV X protein (HBx) plays a major role in this regulation. Recent studies have revealed that MIA2 is a potential tumor suppressor, and that, in most HCCs, MIA2 expression is down-regulated or lost. We found that the knock-down of MIA2 in HepG2 cells activated cell growth and proliferation, suggesting that MIA2 inhibits HCC cell growth and proliferation. In addition, the over-expression of HBx alone induced cell proliferation, whereas MIA2 over-expression impaired the HBx-mediated induction of proliferation. Taken together, our results suggest that HBx activates hepatoma cell growth and proliferation through repression of the potential tumor suppressor MIA2.     

Hepatitis B virus X protein activates a survival signaling by linking SRC to phosphatidylinositol 3-kinase

We have previously shown that transactivation-proficient hepatitis virus B X protein (HBx) protects Hep 3B cells from transforming growth factor-beta (TGF-beta)-induced apoptosis via activation of the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt signaling pathway. This work further investigated how HBx activates PI 3-kinase. Src activity was elevated in Hep 3B cells following expression of transactivation-proficient HBx or HBx-GFP fusion proteins. The Src family kinase inhibitor PP2 and C-terminal Src kinase (Csk) both alleviated HBx-mediated PI 3-kinase activation and protection from TGF-beta-induced apoptosis. Therefore, HBx activated a survival signal by linking Src to PI 3-kinase. Systemic subcellular fractionation and membrane flotation assays indicated that approximately 1.5% of ectopically expressed HBxGFP was associated with periplasmic membrane where Src was located. However, neither nucleus-targeted nor periplasmic membrane-targeted HBxGFP was able to upregulate Src activity or to augment PI 3-kinase survival signaling pathway.     

Ubiquitin-dependent and -independent proteasomal degradation of hepatitis B virus X protein

The hepatitis B virus X protein (HBX) plays key regulatory roles in viral replication and the development of hepatocellular carcinoma. HBX is an unstable protein; its instability is attributed to rapid degradation through the ubiquitin-proteasome pathway. Here, we show that the middle and carboxyl-terminal domains of HBX, independently fused to GFP, render the recombinant proteins susceptible to proteasomal degradation, while the amino-terminal domain has little effect on the ubiquitination or stability of HBX. Mutation of any single or combination of up to five of six lysine residues, all located in the middle and carboxyl-terminal domain, did not prevent HBX from being ubiquitinated, ruling out any specific lysine as the sole site of ubiquitination. Surprisingly, HBX in which all six lysines were mutated and showed no evidence of ubiquitination, was still susceptible to proteasomal degradation. These results suggest that both ubiquitin-dependent and -independent proteasomal degradation processes are operative in HBX turnover.     

乙型肝炎病毒X蛋白对细胞凋亡的影响存在剂量依赖性

乙型肝炎病毒(HBV)X蛋白(HBx)与HBV相关肝细胞肝癌(HCC)的发生和发展密切相关.深入研究HBx在HCC形成中的作用将为探索HBV致癌机制提供重要依据.HBx是多功能蛋白,其对细胞凋亡的影响至今仍存在分歧.许多研究表明,HBx既有促进细胞凋亡又有抑制细胞凋亡的功能,但原因不清楚.本研究中,将表达HBx的质粒短...     

Hepatitis B virus X protein activates the p38 mitogen-activated protein kinase pathway in dedifferentiated hepatocytes

Hepatitis B virus X protein (pX) is implicated in hepatocarcinogenesis by an unknown mechanism. Employing a cellular model linked to pX-mediated transformation, we investigated the role of the previously reported Stat3 activation by pX in hepatocyte transformation. Our model is composed of a differentiated hepatocyte (AML12) 3pX-1 cell line that undergoes pX-dependent transformation and a dedifferentiated hepatocyte (AML12) 4pX-1 cell line that does not exhibit transformation by pX. We report that pX-dependent Stat3 activation occurs only in non-pX-transforming 4pX-1 cells and conclude that Stat3 activation is not linked to pX-mediated transformation. Maximum Stat3 transactivation requires Ser727 phosphorylation, mediated by mitogenic pathway activation. Employing dominant negative mutants and inhibitors of mitogenic pathways, we demonstrate that maximum, pX-dependent Stat3 transactivation is inhibited by the p38 mitogen-activated protein kinase (MAPK)-specific inhibitor SB 203580. Using transient-transreporter and in vitro kinase assays, we demonstrate for the first time that pX activates the p38 MAPK pathway only in 4pX-1 cells. pX-mediated Stat3 and p38 MAPK activation is Ca(2+) and c-Src dependent, in agreement with the established cellular action of pX. Importantly, pX-dependent activation of p38 MAPK inactivates Cdc25C by phosphorylation of Ser216, thus initiating activation of the G(2)/M checkpoint, resulting in 4pX-1 cell growth retardation. Interestingly, pX expression in the less differentiated hepatocyte 4pX-1 cells activates signaling pathways known to be active in regenerating hepatocytes. These results suggest that pX expression in the infected liver effects distinct mitogenic pathway activation in less differentiated versus differentiated hepatocytes.