Hepatitis C virus core protein modulates TRAIL-mediated apoptosis by enhancing Bid cleavage and activation of mitochondria apoptosis signaling pathway

Hepatitis C virus (HCV) is a major human pathogen causing chronic liver disease, which leads to cirrhosis of liver and hepatocellular carcinoma. The HCV core protein, a viral nucleocapsid, has been shown to affect various intracellular events, including cell proliferation and apoptosis. However, the precise mechanisms of the effects are not fully understood. In this study, we show that HCV core protein sensitizes human hepatocellular carcinoma cell line, Huh7, conferred sensitivity to TRAIL-, but not Fas ligand-mediated apoptosis. Huh7 cells are resistant to TRAIL, despite the induction of caspase-8 after TRAIL engagement. However, HCV core protein induces TRAIL apoptosis signaling via sequential induction of caspase-8, Bid cleavage, activation of mitochondrial pathway, and effector caspase-3. HCV core protein also induces activation of caspase-9 after TRAIL engagement, and the induction of TRAIL sensitivity by HCV core protein could be reversed by caspase-9 inhibitor. Therefore, the HCV core protein-induced TRAIL-mediated apoptosis is dependent upon activation of caspase-8 downstream pathway to convey the death signal to mitochondria, leading to activation of mitochondrial signaling pathway and breaking the apoptosis resistance. These results combined indicate that the HCV core protein enhances TRAIL-, but not Fas ligand-mediated apoptotic cell death in Huh7 cells via a mechanism dependent on the activation of mitochondria apoptosis signaling pathway. These results suggest that HCV core protein may have a role in immune-mediated liver cell injury by modulation of TRAIL-induced apoptosis.     

Inhibition of cytochrome c release in Fas-mediated signaling pathway in transgenic mice induced to express hepatitis C viral proteins

Persistent hepatitis C virus (HCV) infection often progresses to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Numerous viruses have been reported to escape from apoptotic mechanism to maintain persistent infection. In the present study, we characterized the effect of HCV proteins on the Fas signal using HCV transgenic mice, which expressed core, E1, E2, and NS2 proteins, regulated by the Cre/loxP switching system. The transgene expression of HCV transgenic mice caused resistance to Fas antibody stimulated lethality. Apoptotic cell death in the liver of HCV protein expressing mice was significantly reduced compared with nonexpressing mice. Histopathological analysis and DNA fragmentation analysis revealed that the HCV proteins suppressed Fas-mediated apoptotic cell death. To identify the target pathway of HCV proteins, we characterized caspase activity. The activation of caspase-9 and -3/7 but not caspase-8 was inhibited by HCV proteins. Cytochrome c release from mitochondria was inhibited in HCV protein expressing mice. These results indicated that the expression of HCV proteins may directly or indirectly inhibit Fas-mediated apoptosis and death in mice by repressing the release of cytochrome c from mitochondria, thereby suppressing caspase-9 and -3/7 activation. These results suggest that HCV may cause persistent infection, as a result of suppression of Fas-mediated cell death.     

Hepatitis B virus alters the antioxidant system in transgenic mice and sensitizes hepatocytes to Fas signaling

Hepatitis B virus (HBV) is a major etiological factor of hepatocellular carcinoma (HCC). However, the precise pathogenetic mechanisms linking HBV infection and HCC remain uncertain. It has been reported that decreased antioxidant enzyme activities are associated with severe liver injury and hepatocarcinogenesis in mouse models. It is unclear if HBV can interfere with the activities of antioxidant enzymes. We established a HBV transgenic mouse line, which spontaneously developed HCC at 2 years of age. We studied the activities of the antioxidant enzymes in the liver of the HBV transgenic mice. Our results showed that the antioxidant enzymes glutathione peroxidase and superoxide dismutase 2 were down-regulated in HBV transgenic mice and correlated with JNK activation. HBV enhanced the Fas-mediated activation of caspase 6, caspase 8 and JNK without enhancing the activation of caspase 3 and hepatocellular apoptosis. As a proper redox balance is important for maintaining cellular homeostasis, these effects of HBV on the host antioxidant system and Fas-signaling may play an important role in HBV-induced hepatocarcinogenesis.     

Hepatitis C virus infection causes cell cycle arrest at the level of initiation of mitosis

Chronic infection with the hepatitis C virus (HCV) is associated with increased risk for hepatocellular carcinoma (HCC). Chronic immune-mediated inflammation is likely to be an important factor in the development of HCV-associated HCC, but direct effects of HCV infection on the host cell cycle may also play a role. Although overexpression studies have revealed multiple interactions between HCV-encoded proteins and host cell cycle regulators and tumor suppressor proteins, the relevance of these observations to HCV-associated liver disease is not clear. We determined the net effect of these interactions on regulation of the cell cycle in the context of virus infection. Flow cytometry of HCV-infected carboxyfluorescein succinimidyl ester-labeled hepatoma cells indicated a slowdown in proliferation that correlated with abundance of viral antigen. A decrease in the proportions of infected cells in G(1) and S phases with an accumulation of cells in G(2)/M phase was observed, compared to mock-infected controls. Dramatic decreases in markers of mitosis, such as phospho-histone H3, in infected cells suggested a block to mitotic entry. In common with findings described in the published literature, we observed caspase 3 activation, suggesting that cell cycle arrest is associated with apoptosis. Differences were observed in patterns of cell cycle disturbance and levels of apoptosis with different strains of HCV. However, the data suggest that cell cycle arrest at the interface of G(2) and mitosis is a common feature of HCV infection.     

Mechanisms of liver injury. III. Oxidative stress in the pathogenesis of hepatitis C virus

Hepatitis C virus (HCV) is a major cause of viral hepatitis that can progress to hepatic fibrosis, steatosis, hepatocellular carcinoma, and liver failure. HCV infection is characterized by a systemic oxidative stress that is most likely caused by a combination of chronic inflammation, iron overload, liver damage, and proteins encoded by HCV. The increased generation of reactive oxygen and nitrogen species, together with the decreased antioxidant defense, promotes the development and progression of hepatic and extrahepatic complications of HCV infection. This review discusses the possible mechanisms of HCV-induced oxidative stress and its role in HCV pathogenesis.     

Immunological and molecular aspects of liver fibrosis in chronic hepatitis C virus infection

Chronic C hepatitis represents a major health problem worldwide, mainly because progression of the tissue damage leads to the development of cirrhosis and hepatocellular carcinoma. In this review we discuss the molecular mechanisms underlying the development of liver fibrosis. In particular we consider some immunologic aspects that regulate the interaction between HCV and the host immune defense. Reflections are made about the roles played by the host capacity to respond to the viral infection during therapy and the consequences of the deposition of extracellular matrix (ECM) proteins leading to the development of fibrosis. The involvement of inflammatory cytokines in regulating the proteolytic remodeling of the liver and the ECM turn-over is essential for the activation of hepatic stellate cells (HSCs), that have an important role in the progression of liver fibrosis. Finally, we analyze one of the aspects involved in the activation of the HSCs, namely the proteolytic remodeling of the surrounding environment.     

E2 of hepatitis C virus inhibits apoptosis

Hepatitis C virus (HCV) is the major causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, and can be involved in very long chronic infections up to 30 years or more. Therefore, it has been speculated that HCV possesses mechanisms capable of modulating host defense systems such as innate and adaptive immunity. To investigate this virus-host interaction, we generated HCV replicons containing various HCV structural proteins and then analyzed the sensitivity of replicon-containing cells to the apoptosis-inducing agent, TRAIL. TRAIL-induced apoptosis was monitored by cleavage of procaspase-3 and procaspase-9 as well as that of their substrate poly(ADP-ribose) polymerase. TRAIL-induced apoptosis was inhibited in cells expressing HCV E2. Moreover, expression of HCV E2 enhanced the colony forming efficiency of replicon-containing cells by 25-fold. Blockage of apoptosis by E2 seems to be related to inhibition of TRAIL-induced cytochrome c release from the mitochondria. Based on these results, we propose that E2 augments persistent HCV infection by blocking host-induced apoptosis of infected cells.     

Iron Regulator Hepcidin Exhibits Antiviral Activity against Hepatitis C Virus

Hepatitis C viral infection affects 170 million people worldwide. It causes serious chronic liver diseases. HCV infection has been implicated in iron accumulation in the liver and iron overload has been shown to be a potential cofactor for HCV associated hepatocellular carcinoma progression. The underlying mechanisms are not understood. Human hepcidin, a 25 amino acid peptide mainly produced by hepatocytes, is a key regulator of iron metabolism. Alteration of hepcidin expression levels has been reported in the setting of chronic HCV infection and hepatocellular carcinoma. In this study, we aim to examine the interactions between HCV infection and hepcidin expression in liver cells. We found that hepcidin expression was suppressed in HCV infected cells. The suppressive effect appears to be regulated by histone acetylation but not DNA methylation. Moreover, we found that hepcidin had a direct antiviral activity against HCV replication in cell culture. The antiviral effect is associated with STAT3 activation. In conclusion, hepcidin can induce intracellular antiviral state while HCV has a strategy to suppress hepcidin expression. This may be a novel mechanism by which HCV circumvents hepatic innate antiviral defense.     

Hepatitis C virus core protein: intriguing properties and functional relevance

Hepatitis C virus (HCV) often causes a prolonged and persistent infection, and an association between hepatocellular carcinoma (HCC) and HCV infection has been noted. The pathogenesis of liver damage is at least in part related to virus-mediated factors. Understanding the molecular basis of pathogenesis is a major challenge in gaining insight into HCV-associated disease progression. Recent experimental evidence using HCV cloned genomic regions suggests that the core protein has numerous functional activities. These include its likely role in encapsidation of viral RNA, a regulatory effect on cellular and unrelated viral promoters, interactions with a number of cellular proteins, an modulatory role in programmed cell death or apoptosis under certain conditions, involvement in cell growth promotion and immortalization, induction of HCC in transgenic mice, and a possible immunoregulatory role. These intriguing properties suggest that the core protein, in concert with cellular factors, may contribute to pathogenesis during persistent HCV infection.     

Activation of the CKI-CDK-Rb-E2F pathway in full genome hepatitis C virus-expressing cells

Hepatitis C virus (HCV) causes persistent infection in hepatocytes, and this infection is, in turn, strongly associated with the development of hepatocellular carcinoma. To clarify the mechanisms underlying these effects, we established a Cre/loxP conditional expression system for the precisely self-trimmed HCV genome in human liver cells. Passage of hepatocytes expressing replicable full-length HCV (HCR6-Rz) RNA caused up-regulation of anchorage-independent growth after 44 days. In contrast, hepatocytes expressing HCV structural, nonstructural, or all viral proteins showed no significant changes after passage for 44 days. Only cells expressing HCR6-Rz passaged for 44 days displayed acceleration of CDK activity, hyperphosphorylation of Rb, and E2F activation. These results demonstrate that full genome HCV expression up-regulates the CDK-Rb-E2F pathway much more effectively than HCV proteins during passage.     

Extracellular hepatitis C virus core protein activates STAT3 in human monocytes/macrophages/dendritic cells via an IL-6 autocrine pathway

Hepatitis C virus (HCV) infection is highly efficient in the establishment of persistent infection, which leads to the development of chronic liver disease and hepatocellular carcinoma. Impaired T cell responses with reduced IFN-γ production have been reported to be associated with persistent HCV infection. Extracellular HCV core is a viral factor known to cause HCV-induced T cell impairment via its suppressive effect on the activation and induction of pro-inflammatory responses by antigen-presenting cells (APCs). The activation of STAT proteins has been reported to regulate the inflammatory responses and differentiation of APCs. To further characterize the molecular basis for the regulation of APC function by extracellular HCV core, we examined the ability of extracellular HCV core to activate STAT family members (STAT1, -2, -3, -5, and -6). In this study, we report the activation of STAT3 on human monocytes, macrophages, and dendritic cells following treatment with extracellular HCV core as well as treatment with a gC1qR agonistic monoclonal antibody. Importantly, HCV core-induced STAT3 activation is dependent on the activation of the PI3K/Akt pathway. In addition, the production of multifunctional cytokine IL-6 is essential for HCV core-induced STAT3 activation. These results suggest that HCV core-induced STAT3 activation plays a critical role in the alteration of inflammatory responses by APCs, leading to impaired anti-viral T cell responses during HCV infection.     

ATP controls neuronal apoptosis triggered by microtubule breakdown or potassium deprivation.

BACKGROUND: Early loss of neurites followed by delayed damage of neuronal somata is a feature of several neurodegenerative diseases. Death by apoptosis would ensure the rapid removal of injured neurons, whereas conditions that prevent apoptosis may facilitate the persistence of damaged cells and favor inflammation and disease progression. MATERIALS AND METHODS: Cultures of cerebellar granule cells (CGC) were treated with microtubule disrupting agents. These compounds induced an early degeneration of neurites followed by apoptotic destruction of neuronal somata. The fate of injured neurons was followed after co-exposure to caspase inhibitors or agents that decrease intracellular ATP (deoxyglucose, S-nitrosoglutathione, 1-methyl-4-phenylpyridinium). We examined the implications of energy loss for caspase activation, exposure of phagocytosis markers, and long-term persistence of damaged cells. RESULTS: In CGC exposed to colchicine or nocodazole, axodendritic degeneration preceded caspase activation and apoptosis. ATP-depleting agents or protein synthesis inhibition prevented caspase activation, translocation of the phagocytosis marker, phosphatidylserine, and apoptotic death. However, they did not affect the primary neurite loss. Repletion of ATP by enhanced glycolysis restored all apoptotic features. Peptide inhibitors of caspases also prevented the apoptotic changes in the cell bodies, although the axodendritic net was lost. Under this condition cell demise still occurred 48 hr later in a caspase-independent manner and involved plasma membrane lysis at the latest stage. CONCLUSIONS: Inhibition of the apoptotic machinery by drugs, energy deprivation, or endogenous mediators may result in the persistence and subsequent lysis of injured neurons. In vivo, this may favor the onset of inflammatory processes and perpetuate neurodegeneration.     

Mechanisms of viral hepatitis induced liver injury

Among seven human hepatitis viruses (A to E, G and TT virus), hepatitis B (HBV) and C (HCV) viruses are able to persist in the host for years and principally contribute to the establishment of chronic hepatitis. During the course of persistent infection, continuous intrahepatic inflammation maintains a cycle of liver cell destruction and regeneration that often terminates in hepatocellular carcinoma (HCC). While the expression and retention of viral proteins in hepatocytes may influence the severity and progression of liver disease, the mechanisms of liver injury in viral hepatistis are defined to be due not to the direct cytopathic effects of viruses, but to the host immune response to viral proteins expressed by infected hepatocytes. In the process of liver injury, hepatocellular death (apoptosis) induced by the proapoptotic molecules of T cells activated following antigen recognition triggers a cascade of antigen nonspecific effector systems and causes necroinflammatory disease. Accordingly, the regulation of the immune response, e.g., via the cell death pathways, in chronically infected patients should prevent the development of HCC.     

Unique sequence of a high molecular weight myosin light chain kinase is involved in interaction with actin cytoskeleton

Resolution of neutrophil mediated inflammation is achieved, in part, through induction of neutrophil apoptosis. This constitutively expressed programme can be delayed by inflammatory mediators and induced by ligation of the Fas receptor. However, functional activation of the neutrophil results in resistance to Fas signalled death. We evaluated the effects of Fas antibody engagement on caspase activation and mitochondrial permeability, and the impact of co-stimulation by lipopolysaccharide (LPS) or granulocyte macrophage-colony stimulating factor (GM-CSF) on these events. Fas engagement by an agonistic anti-Fas antibody resulted in enhanced caspase 3 and 8 activity and increased mitochondrial permeability. Studies with pharmacological inhibitors of caspase activity showed that activation of caspase 8 occurred before, and activation of caspase 3 occurred after mitochondrial disruption. The mitochondrial stabilising agent bongkrekic acid also inhibited caspase activation and apoptosis. LPS, GM-CSF and increased glutathione stabilised the mitochondria and inhibited caspase 3. Caspase 8 activity was also inhibited by co-stimulation through a mechanism independent of mitochondrial stabilisation. Glutathione directly inhibited caspase 3 and 8 activity. We conclude inhibition of Fas antibody induced apoptosis by inflammatory proteins is associated with augmented mitochondrial stability and reduced caspase 3 activity that may be glutathione mediated.     

The hepatitis C virus persistence: how to evade the immune system?

Hepatitis C virus (HCV) is an emerging virus of medical importance. A majority of HCV infections become chronic and lead to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV usually induces robust immune responses, but it frequently escapes the immune defense to establish persistent infection. The fact that HCV exists as an evolving quasispecies plays an important role in the selection of escape mutants. Furthermore, several viral proteins interfere with cellular functions, in particular, those involved in the immune response of the host. Several HCV proteins also modulate cell signalling through interaction with different effectors involved in cell proliferation and apoptosis, or in the interferon-signalling pathway. In addition, HCV infects immune cells such as B and T cells, and thus affects their normal functions. These various strategies used by HCV to counter the immune response of the host are reviewed here. A better understanding of these mechanisms would help design new therapeutic targets.     

Redox regulation of hepatitis C in nonalcoholic and alcoholic liver

Hepatitis C virus (HCV) is an RNA virus of the Flaviviridae family that is estimated to have infected 170 million people worldwide. HCV can cause serious liver disease in humans, such as cirrhosis, steatosis, and hepatocellular carcinoma. HCV induces a state of oxidative/nitrosative stress in patients through multiple mechanisms, and this redox perturbation has been recognized as a key player in HCV-induced pathogenesis. Studies have shown that alcohol synergizes with HCV in the pathogenesis of liver disease, and part of these effects may be mediated by reactive species that are generated during hepatic metabolism of alcohol. Furthermore, reactive species and alcohol may influence HCV replication and the outcome of interferon therapy. Alcohol consumption has also been associated with increased sequence heterogeneity of the HCV RNA sequences, suggesting multiple modes of interaction between alcohol and HCV. This review summarizes the current understanding of oxidative and nitrosative stress during HCV infection and possible combined effects of HCV, alcohol, and reactive species in the pathogenesis of liver disease.     

Hepatitis C virus core protein interacts with 14-3-3 protein and activates the kinase Raf-1

Persistent hepatitis C virus (HCV) infection is a major cause of chronic liver dysfunction in humans and is epidemiologically closely associated with the development of human hepatocellular carcinoma. Among HCV components, core protein has been reported to be implicated in cell growth regulation both in vitro and in vivo, although mechanisms explaining those effects are still unclear. In the present study, we identified that members of the 14-3-3 protein family associate with HCV core protein. 14-3-3 protein bound to HCV core protein in a phosphoserine-dependent manner. Introduction of HCV core protein caused a substantial increase in Raf-1 kinase activity in HepG2 cells and in a yeast genetic assay. Furthermore, the HCV core-14-3-3 interaction was essential for Raf-1 kinase activation by HCV core protein. These results suggest that HCV core protein may represent a novel type of Raf-1 kinase-activating protein through its interaction with 14-3-3 protein and may contribute to hepatocyte growth regulation.