Inhibition Of Hepatitis B Virus Core Protein(HBc) On Fas-mediated Apoptosis Of Hepatoma Cells

TNF-α/TNF-R, Fas L/Fas and TRAIL/DR5 are three major death ligand/receptor pathways involved in hepatocyte apoptosis, among which Fas L/Fas apoptotic pathway plays a vital role in regulation of hepatocyte apoptosis. Fas L/Fas system includes Fas ligand and Fas receptor. Furthermore, Fas receptor consists of two forms including m Fas(membrane-bound Fas) and s Fas(soluble Fas), in which m Fas induces apoptosis, while the s Fas inhibits apoptosis. HBc(Hepatitis B virus core protein) was reported to be able to regulate the hepatocytes apoptosis. As a gene regulatory protein, HBc may down-regulate DR5 receptor to inhibit TRAIL(TNF-related apoptosis-inducing ligand) mediated apoptosis of hepatocytes. HBc can also sensitize hepatocytes to TNF-α-induced apoptosis by suppression of the phosphorylation of MKK7. However, the effect of HBc on Fas-mediated apoptosis of hepatocytes is unknown. The aim of this study is to investigate the effects of HBc on Fas–mediated apoptosis of hepatocytes and elucidate the mechanism involved.The first part of this study is to investigate the effects of HBc on Fas-mediated apoptosis of hepatocytes. To address this issue, we generated stable Hep G2(wildtype p53) cell lines, i.e., Hep G2-pc DNA3.1 and Hep G2-HBc cells, which harboring either the empty vector pc DNA3.1/Hygro(+) or pc DNA3.1-HBc. The expression of HBc in thses two cell lines was confirmed by Western blot analysis using an antibody raised against the core antigen of HBV. The results showed that HBc could significantly protected Hep G2 cells from anti-Fas CH11-evoked cytotoxicity, as determined by methods of CCK-8 and TUNEL, which suggesting that HBc was able to decrease the sensitivity of Hep G2 cells to anti-Fas CH11-induced apoptosis and likely to act as an inhibitor of Fas/Fas L induced apoptosis of hepatocytes.The second chapter of this section is to elucidate the effect of p53 on HBc-mediated Fas/Fas L signaling pathway. Huh7(p53 mutant) and Hep3B(p53 null) cells were stably transfected with pc DNA3.1-HBc or control plasmid pc DNA3.1/Hygro(+) and Western blotting analysis demonstrated that Huh7-p HBc and Hep3B-p HBc could steadily express HBc. Interestingly, in the absence of p53, CH11 treatment did not produce effective and differential cytotoxicity to either Huh7-p HBc or Hep3B-p HBc cells as determined by a CCK-8 assay or TUNEL assay. To further ascertain the central role of p53 in Fas-mediated apoptosis, p53 wild-type Hep G2-pc DNA3.1 and Hep G2-p HBc cells were treatedwith the p53 inhibitor PFT-α or the p53 inducer Bleomycin followed by exposure to anti-Fas CH11 and then assayed by TUNEL staining. As expected, pretreatment with PFT-α fully abrogated CH11-induced apoptotic cell death regardless of the presence of HBc, whereas Bleomycin substantially augmented the frequency of apoptotic cells with the effect being greater in the Hep G2-pc DNA3.1 than the Hep G2-p HBc cells. Taken together, these results clearly indicated that reduction of Fas-mediated apoptosis by HBc was p53 dependent.The second part of this study is to elucidate the effect of HBc on the expression of p53, total Fas, m Fas and Fas L. The relative expression of proapoptotic proteins in Hep G2-pc DNA3.1 and Hep G2-HBc cells on Bleomycin treatment was quantified by q RT-PCR and Western blot analysis. The results demonstrated tha exposure of Hep G2-pc DNA3.1 and Hep G2-HBc cells to Bleomycin resulted in enhanced expression of p53 and m Fas at both the m RNA and protein levels, indicating the effectiveness of the treatment. However, the augmentation was less pronounced in Hep G2-p HBc cells compared with that in control cells. Similar results were obtained with Fas L and total Fas m RNA levels.The third part of this study is to elucidate the effect of HBc on s Fas expression. The results showed that in the absence of Bleomycin, the level of s Fas in the culture medium of Hep G2-p HBc cells as measured by ELISA was higher than that in control cells. Treatment of the cells with Bleomycin further increased the s Fas levels. This was confirmed by semiquantitative PCR analysis. The expression of HBc in Hep G2-p HBc cells did increase s Fas m RNA levels. Fas splicing assay results also showed that relative m RNA levels of Fas exon 5/7(representative of s Fas) to Fas exon 5–7 in Hep G2-HBc were significantly elevated compared with those in control cells, and treatment of Bleomycin reduced the magnitude of this effect. These data support the notion that HBc enhances the expression of s Fas via increasing transcripts of an alternatively spliced variant that specifically encodes the soluble form of the receptors.The second chapter of this section is to elucidate mechanisms of Fas alternative m RNA splicing regulation by HBc. In the absence of Bleomycin, Hep G2-p HBc cells expressed higher levels of PTBP1 protein compared with control with no significant difference in the expression of PTBP2 or PTBP3 in the absence or presence of Bleomycin. Interestingly, m RNA expression levels of all the PTB isoforms remained unchanged between Hep G2-p HBc and control cells either treated or untreated with Bleomycin. The steady state levels of active caspase-3 were significantly reduced in Hep G2-p HBc cells compared with the control cells. Less activation of caspase-3 was also seen in Hep G2-p HBc cells following exposure to Bleomycin compared with control, although Bleomycin elevated the caspase-3 level in both cells.Consistently, the changes in the protein levels of active caspase-3 were accompanied by a proportional alterations in the enzyme activity of caspase-3. HBc inhibited FASTK expression with the levels of TIA-1 and TIAR unaffected, whereas treatment of Bleomycin had no effect on the expression. A reduction in the FASTK m RNA level was also observed in Hep G2-p HBc cells compared with the control, indicating that HBc may regulate FASTK gene expression at the transcriptional level. Cells were transfected with a FASTK promoter-driven luciferase reporter construct p GL4.10-FASTK to evaluate changes in possible HBc-regulated promoter activation. Hep G2-p HBc cells demonstrated a significant decrease in reporter gene activity relative to control cells. Taken together, these results implied that HBc might up-regulate PTBP1 dependent of p53 to increase Fas alternative m RNA splicing as well as selectively suppress FASTK transcripts independent of p53 to block the synergistic effects of FASTK with TIA-1/TIAR in Fas splicing.