HBx combined with AFB1 triggers hepatic steatosis via COX‐2‐mediated necrosome formation and mitochondrial dynamics disorder

Hepatitis B virus (HBV) infection and aflatoxin B1 (AFB1) exposure have been recognized as independent risk factors for the occurrence and exacerbation of hepatic steatosis but their combined impacts and the potential mechanisms remain to be further elucidated. Here, we showed that exposure to AFB1 impaired mitochondrial dynamics and increased intracellular lipid droplets (LDs) in the liver of HBV‐transgenic mice in vivo and the hepatitis B virus X protein (HBx)‐expressing human hepatocytes both ex vivo and in vitro. HBx combined with AFB1 exposure also up‐regulated receptor interaction protein 1 (RIP1), receptor interaction protein 3 (RIP3) and activated mixed lineage kinase domain like protein (MLKL), providing evidence of necrosome formation in the hepatocytes. The shift of the mitochondrial dynamics towards imbalance of fission and fusion was rescued when MLKL was inhibited in the HBx and AFB1 co‐treated hepatocytes. Most importantly, based on siRNA or CRISPR/Cas9 system, we found that the combination of HBx and AFB1 exposure increased cyclooxygenase‐2 (COX‐2) to mediate up‐regulation of RIP3 and dynamin‐related protein 1 (Drp1), which in turn promoted location of RIP3‐MLKL necrosome on mitochondria, subsequently exacerbated steatosis in hepatocytes. Taken together, these findings advance the understanding of mechanism associated with HBx and AFB1‐induced hepatic necrosome formation, mitochondrial dysfunction and steatosis and make COX‐2 a good candidate for treatment.     

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.     

Hepatitis B virus X protein induces TNF-alpha expression via down-regulation of selenoprotein P in human hepatoma cell line,HepG2

Human hepatitis B virus X protein (HBx) is associated with the induction of oxidative stress, which is considered significant in the development of liver damage. In this study, we investigated the molecular mechanisms by which HBx induced lipid peroxidation and tumor necrosis factor-alpha (TNF-alpha) expression through regulation of selenoprotein P (SeP) expression in the human hepatoma cell line, HepG2. Forced expression of HBx significantly down-regulated the expression of SeP mRNA and protein in both the cell lysates and the culture medium. Lipid peroxidation increased 2.5-fold when expression of the SeP protein was blocked with a SeP antisense vector. Also, HBx transfection increased lipid peroxidation by 3.0-fold, whereas the hepatitis B virus core protein (HBc) had no significant effects. The induction of lipid peroxidation due to the block in SeP protein expression or treatment with ferric chloride (FeCl(3)) up-regulated the expression levels of TNF-alpha mRNA and protein. The pattern of HBx-induced lipid peroxidation and TNF-alpha up-regulation was reversed by SeP introduction. These results suggest that HBx induces lipid peroxidation via down-regulation of SeP expression, resulting in increased expression of TNF-alpha in the human hepatoma cell line, HepG2.     

C-C Chemokine Receptor 2 Inhibitor Ameliorates Hepatic Steatosis by Improving ER Stress and Inflammation in a Type 2 Diabetic Mouse Model

Hepatic steatosis is the accumulation of excess fat in the liver. Recently, hepatic steatosis has become more important because it occurs in the patients with obesity, type 2 diabetes, and hyperlipidemia and is associated with endoplasmic reticulum (ER) stress and insulin resistance. C-C chemokine receptor 2 (CCR2) inhibitor has been reported to improve inflammation and glucose intolerance in diabetes, but its mechanisms remained unknown in hepatic steatosis. We examined whether CCR2 inhibitor improves ER stress-induced hepatic steatosis in type 2 diabetic mice. In this study, db/db and db/m (n = 9) mice were fed CCR2 inhibitor (2 mg/kg/day) for 9 weeks. In diabetic mice, CCR2 inhibitor decreased plasma and hepatic triglycerides levels and improved insulin sensitivity. Moreover, CCR2 inhibitor treatment decreased ER stress markers (e.g., BiP, ATF4, CHOP, and XBP-1) and inflammatory cytokines (e.g., TNFα, IL-6, and MCP-1) while increasing markers of mitochondrial biogenesis (e.g., PGC-1α, Tfam, and COX1) in the liver. We suggest that CCR2 inhibitor may ameliorate hepatic steatosis by reducing ER stress and inflammation in type 2 diabetes mellitus.     

Hepatitis B virus replication is associated with an HBx-dependent mitochondrion-regulated increase in cytosolic calcium levels

The nonstructural hepatitis B virus (HBV) protein HBx has an important role in HBV replication and in HBV-associated liver disease. Many activities have been linked to HBx expression; however, the molecular mechanisms underlying many of these activities are unknown. One proposed HBx function is the regulation of cytosolic calcium. We analyzed calcium levels in HepG2 cells that expressed HBx or replicating HBV, and we demonstrated that HBx, expressed in the absence of other HBV proteins or in the context of HBV replication, elevates cytosolic calcium. We linked this elevation of cytosolic calcium to the association of HBx with the mitochondrial permeability transition pore.     

Hepatitis B virus regulatory HBx protein binds to adaptor protein IPS-1 and inhibits the activation of beta interferon

Hepatitis B virus (HBV) encodes the regulatory HBx protein, which is required for virus replication, although its specific role(s) in the replication cycle remains under investigation. An immunoprecipitation/mass spectrometry approach was used to identify four novel HBx binding proteins from the cytoplasmic fraction of HBx transgenic mouse livers. One of these HBx binding partners is beta interferon promoter stimulator 1 (IPS-1), an adaptor protein that plays a critical role in mediating retinoic acid-inducible gene I (RIG-I) signaling, which leads to the activation of beta interferon (IFN-β). The HBx-IPS-1 protein interaction was confirmed in plasmid-transfected HepG2 cells by reciprocal coimmunoprecipitation and Western blotting. We hypothesized that HBx might alter IPS-1 function since proteins of hepatitis C virus and hepatitis A virus similarly bind IPS-1 and target it for inactivation. The effect of HBx on IPS-1-mediated IFN-β signaling was tested in transfected 293T and HepG2 cells, and we show that HBx inhibits double-stranded DNA (dsDNA)-mediated IFN-β activation in a dose-dependent manner when expressed either alone or within the context of HBV replication. However, HBx does not inhibit poly(I:C)-activated IFN-β signaling. These results demonstrate that HBx interferes with the RIG-I pathway of innate immunity. Hepatitis B virus now joins hepatitis C virus and hepatitis A virus in targeting the same innate immune response pathway, presumably as a shared strategy to benefit replication of these viruses in the liver.     

Lipid peroxidation product 4-hydroxy-trans-2-nonenal causes endothelial activation by inducing endoplasmic reticulum stress

Lipid peroxidation products, such as 4-hydroxy-trans-2-nonenal (HNE), cause endothelial activation, and they increase the adhesion of the endothelium to circulating leukocytes. Nevertheless, the mechanisms underlying these effects remain unclear. We observed that in HNE-treated human umbilical vein endothelial cells, some of the protein-HNE adducts colocalize with the endoplasmic reticulum (ER) and that HNE forms covalent adducts with several ER chaperones that assist in protein folding. We also found that at concentrations that did not induce apoptosis or necrosis, HNE activated the unfolded protein response, leading to an increase in XBP-1 splicing, phosphorylation of protein kinase-like ER kinase and eukaryotic translation initiation factor 2α, and the induction of ATF3 and ATF4. This increase in eukaryotic translation initiation factor 2α phosphorylation was prevented by transfection with protein kinase-like ER kinase siRNA. Treatment with HNE increased the expression of the ER chaperones, GRP78 and HERP. Exposure to HNE led to a depletion of reduced glutathione and an increase in the production of reactive oxygen species (ROS); however, glutathione depletion and ROS production by tert-butyl-hydroperoxide did not trigger the unfolded protein response. Pretreatment with a chemical chaperone, phenylbutyric acid, or adenoviral transfection with ATF6 attenuated HNE-induced monocyte adhesion and IL-8 induction. Moreover, phenylbutyric acid and taurine-conjugated ursodeoxycholic acid attenuated HNE-induced leukocyte rolling and their firm adhesion to the endothelium in rat cremaster muscle. These data suggest that endothelial activation by HNE is mediated in part by ER stress, induced by mechanisms independent of ROS production or glutathione depletion. The induction of ER stress may be a significant cause of vascular inflammation induced by products of oxidized lipids.     

Protective Role of Sirtuin3 (SIRT3) in Oxidative Stress Mediated by Hepatitis B Virus X Protein Expression

Background/Aim

The hepatitis B virus (HBV) infection is accompanied by the induction of oxidative stress, especially mediated by HBV X protein (HBx). Oxidative stress has been implicated in a series of pathological states, such as DNA damage, cell survival and apoptosis. However, the host factor by which cells protect themselves under this oxidative stress is poorly understood.

Methodology/Principal Findings

In this study, we first confirmed that HBV infection significantly induced oxidative stress. Moreover, viral protein HBx plays a major role in the oxidative stress induced by HBV. Importantly, we found that mitochondrial protein SIRT3 overexpression could decrease reactive oxygen species (ROS) induced by HBx while SIRT3 knockdown increased HBx-induced ROS. Importantly, SIRT3 overexpression abolished oxidative damage of HBx-expressing cells as evidenced by γH₂AX and AP sites measurements. In contrast, SIRT3 knockdown promoted HBx-induced oxidative damage. In addition, we also observed that oxidant H₂O₂ markedly promoted HBV replication while the antioxidant N-acetyl-L-cysteine (NAC) inhibited HBV replication. Significantly, SIRT3 overexpression inhibited HBV replication by reducing cellular ROS level.

Conclusions/Significance

Collectively, these data suggest HBx expression induces oxidative stress, which promotes cellular oxidative damage and viral replication during HBV pathogenesis. Mitochondrial protein SIRT3 protected HBx expressing-cells from oxidative damage and inhibited HBV replication possibly by decreased cellular ROS level. These studies shed new light on the physiological significance of SIRT3 on HBx-induced oxidative stress, which can contribute to the liver pathogenesis.