Hepatitis B Virus Surface Antigen (HBsAg) Enhances Fas- Mediated Hepatocyte Apoptosis And Liver Injury Via Suppression Of AKT Phosphorylation

Fas receptor/ligand?Fas/FasL?system plays a prominent role in hepatocyte apoptosis.When FasL binds to Fas,it transmits apoptosis signals through the Fas-associated death domain?FADD?,and recruits procaspase-8 to form a death-inducing signaling complex?DISC?and then activates downstream caspases to induce hepatocyte apoptosis.Surface antigen?HBsAg?is the most abundant HBV-encoded protein in liver and peripheral blood of patients with chronic hepatitis B virus?HBV?infection.However,the role of HBsAg in Fas-regulated hepatocyte apoptosis has not been elucidated.Therefore,this dissertation research aimed to study the effect and mechanism of HBsAg on Fas-mediated hepatocyte apoptosis.The first part of this study was to investigate the effect of HBsAg on Fas-mediated apoptosis of hepatocytes.Firstly,we generated stable HepG2 cell lines,HepG2-pHBsAg and HepG2-pcDNA3.1,and confirmed the expression of HBsAg.Through CCK-8,TUNEL and AnnexinV binding assay,it was found that HBsAg increased the sensitivity of HepG2 cells to apoptosis induced by human Fas monoclonal antibody anti-Fas CH11.In addition,HBsAg promoted apoptosis of human primary hepatocytes induced by anti-Fas CH11 and FasL.These results suggest that HBsAg can promote Fas-mediated apoptosis of hepatocytes.The second part of the study was to investigate the mechanism underlying HBsAg-enhanced Fas-mediated apoptosis of hepatocyte.In the first chapter of this part,we examined the effects of HBsAg on the expression of p53,mFas,sFas,FasL and palmitoylation of Fas.To this end,real-time RT-PCR,semi-quantitative RT-PCR and western blot analysis were employed to assess the different expression of p53,mFas,sFas and FasL mRNA and protein levels between HepG2-pHBsAg and HepG2-pcDNA3.1cells.The effect of HBsAg on modification of Fas palmitoylation was detected by acyl-biotin exchange?ABE?technique.The results showed that HBsAg had no effect on the expression of p53,mFas,sFas,FasL as well as Fas palmitoylation.The second chapter of this part was focused on the effect of HBsAg on DISC.In this regard,we used western blot to evaluate the influence of HBsAg on Fas aggregation and the expression of FADD,FLIP_L/S and procaspase-8.Immunoprecipitation?IP?was used to detect the effects of HBsAg on the recruitment of DISC components and the activities of caspase 8,9,3/7 were measured by the caspase enzymatic activity assay.The results showed that under anti-Fas CH11 treatment,HepG2-pHBsAg cells has higher aggregation of Fas,more activation of procaspase-8 but no difference in the expression of FADD as compared to the control cells.Further immunoprecipitation by Fas antibody demonstrated that in HepG2-pHBsAg cells the aggregation of Fas and the recruitment of both FADD and procaspase-8 cells was higher but the recruitment of FLIP_L/S was lower as compared with the control cells.Moreover,the activities of caspase 8,9,3/7 in HepG2-pHBsAg cells was higher than that in control cells.These results indicate that HBsAg promotes the activation of procaspase-8 by enhancing the formation of Fas aggregation and reducing the recruitment of FLIPL/S at the DISC.The third chapter of this part was to explore the role of AKT phosphorylation in Fas-mediated hepatocyte apoptosis promoted by HBsAg.To this end,AKT-specific activator SC79 and the overexpression of AKT were introduced.Annexin V was used to examine the effect of HBsAg on the reduction of Fas-mediated apoptosis induced by AKT activation.Western blot was used to examine the effect of HBsAg on the components of DISC,negatively regulated by AKT activation.The results showed that the apoptosis rate of cells was decreased after SC79 treatment or AKT overexpression in the presence of anti-Fas CH11 stimulation.However,the apoptotic rate of HBsAg-expressing HepG2-pHBsAg cells was still higher than that of HepG2-pcDNA3.1 cells.After AKT activation,the level of Fas receptor aggregation and procaspase-8 cleavage were decreased whereas the level of FLIP_L/S/S was increased.However,the level of Fas receptor aggregation and procaspase-8 cleavage in HBsAg-expressing HepG2-pHBsAg cells were still higher than that in HepG2-pcDNA3.1 cells,and the amount of FLIPL/S was lower than HepG2-pcDNA3.1 cells.These results indicate that HBsAg can reverse the reduction of Fas-mediated apoptosis induced by AKT activation via restoring Fas receptor aggregation and procaspase-8 cleavage,as well as suppression of FLIP_L/S expression.There results clearly indicated that HBsAg-promoted Fas-mediated apoptosis of hepatocytes is AKT dependent.The fourth chapter of this part was to elucidate the mechanism by which HBsAg down-regulates AKT phosphorylation.To this end,western blot was used to examine the differences in the protein levels of AKT upstream effectors PDPK1,pPDPK1?Ser241?,mTOR,pmTOR?Ser2481?and PTEN between HepG2-pHBsAg and HepG2-pcDNA3.1cells.The levels of pAKT?Thr308?,pAKT?Ser473?and their upstream regulatory factors pPDPK1?Ser241?and pmTOR?Ser2481?in HepG2-pHBsAg cells were found to be lower than those in the control cells.The endoplasmic reticulum inhibitor 4-PBA can increase the expression of pAKT?Thr308?,pAKT?Ser473?,pPDPK1?Ser241?and pmTOR?Ser2481?in HepG2-pHBsAg cells.Annexin V results showed that anti-Fas CH11-mediated hepatocyte apoptosis promoted by HBsAg could be partially alleviated by 4-PBA.Immunoprecipitation results showed that HBsAg did not interact with AKT,PDPK1,mTOR and PTEN.These results indicate that HBsAg could attenuate AKT phosphorylation through deactivation of PDPK1 and mTORC2 by inducing ER stress.In the fifth chapter of this part,we study the effect of HBsAg mutation on Fas-mediated apoptosis of hepatocytes.To this end,we selected 14 commonly occurring HBsAg mutants,generated stable HepG2 cell lines and confirmed the expression.Five HBsAg mutant cell lines?L49T,M133I,G145R,S204R and M1213I?with reduced extracellular secretion and significant intracellular retention were established.The protein levels of GRP94,p-eIF2?,eIF2?AKT,pAKT?Thr308?and pAKT?Ser473?were detected by western blot analysis.The spliced XBP1?S?mRNA levels were detected by semi-quantitative RT-PCR.The Fas-mediated apoptosis and the protein levels of active caspase-8,tBID,Cytochrome C?cytoplasmic/membrane?and active caspase-3 were assessed by Annexin V and western blot.The results showed that the protein levels of GRP94 and p-eIF2?and the mRNA level of XBP1?S?in HepG2-pHBsAg mutants were higher than those of HepG2-pHBsAg whereas the levels of pAKT?Thr308?and pAKT?Ser473?were lower than those of HepG2-pHBsAg.The apoptotic rate of HepG2-pHBsAg mutants and the protein levels of active caspase-8,tBID,Cytochrome C?cytoplasmic?and active caspase-3 were higher than those of HepG2-pHBsAg,and the levels of BID and Cytochrome C?membrane?were lower than those of HepG2-pHBsAg.These results indicate that mutations of L49T,M133I,G145R,S204R and M213I lead to increased intracellular retention of HBsAg and enhance endoplasmic reticulum stress thus further promoting Fas-mediated apoptosis of hepatocytes.The third part of this study was to investigate the effect of HBsAg on Fas-mediated liver function in mice.To this end,expression of HBsAg including G145R and S204R mutants in the liver of mice was established by tail intravenous injection of AAV8,and an acute liver failure model of mice was established by intraperitoneal injection of anti-Fas Jo2.The serum HBsAg levels of AAV8-G145R and AAV8-S204R mice were significantly lower than those of AAV8-HBsAg mice while the intrahepatic retention was more obvious.The phosphorylation level of AKT in the liver tissues of AAV8-HBsAg mice was lower than that of AAV8-control,and the phosphorylation levels of AKT in AAV8-G145R and AAV8-S204R mice were lower.The levels of GRP94 and p-eIF2?in the liver tissues of AAV8-HBsAg mice were higher than those of AAV8-control,and the levels of GRP94 and p-eIF2?in AAV8-G145R and AAV8-S204R mice were higher.Under the treatment of anti-Fas Jo2,the ALF survival rate and survival time of AAV8-HBsAg mice were lower than AAV8-control while the survival rate and survival time of AAV8-G145R and AAV8-S204R mice were even lower.SC79 pretreatment could alleviate mouse ALF induced by anti-Fas Jo2.In addition,the results of this study also showed that under the treatment of anti-Fas Jo2,the serum ALT/AST,hepatocyte apoptosis rate and caspase 8,9,3/7 activity of AAV8-HBsAg mice were higher than those of AAV8-control mice with even higher those parameters observed in AAV8-G145R and AAV8-S204R mice.SC79 pretreatment can attenuate hepatocyte apoptosis and liver injury.These results indicate that HBsAg can promote endoplasmic reticulum stress in mouse liver tissue,inhibit the phosphorylation of AKT,trigger severe hepatocyte apoptosis and liver injury,shorten the survival timeand increase the mortality of ALF in mice.HBsAg G145R and S204R mutations can further enhance this effect.The study also suggested that pretreatment with AKT activator SC79 can alleviate anti-Fas Jo2-induced ALF in mice,which implicates that AKT activator can protect Fas-mediated ALF and strongly suggests that the use of this drug may improve the survival rate of ALF patients clinically.