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The Journal of Immunology, 2007, 178: 503-510.
Copyright © 2007 by The American Association of Immunologists, Inc.

Hepatitis B Virus Sensitizes Hepatocytes to TRAIL-Induced Apoptosis through Bax1

Xiaohong Liang2,*, Yugang Liu2,*, Qiu Zhang{dagger}, Lifen Gao*, Lihui Han*, Chunhong Ma*, Lining Zhang*, Youhai H. Chen3,{ddagger} and Wensheng Sun3,*

* Institute of Immunology, School of Medicine, {dagger} Institute of Environmental Research, Shandong University, Jinan, People’s Republic of China; and {ddagger} Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Hepatitis B virus (HBV) infection afflicts >300 million people worldwide and is a leading cause of hepatocyte death, cirrhosis, and hepatocellular carcinoma. While the morphological characteristics of dying hepatocytes are well documented, the molecular mechanisms leading to the death of hepatocytes during HBV infection are not well understood. TRAIL, the TNF-related apoptosis-inducing ligand, has recently been implicated in the death of hepatocytes under certain inflammatory but not normal conditions. To determine the potential roles of TRAIL in HBV-induced hepatitis, we examined the effects of HBV and its X protein (HBx) on TRAIL-induced hepatocyte apoptosis both in vivo and in vitro. We found that hepatitis and hepatic cell death in HBV transgenic mice were significantly inhibited by a soluble TRAIL receptor that blocks TRAIL function. We also found that HBV or HBx transfection of a hepatoma cell line significantly increased its sensitivity to TRAIL-induced apoptosis. The increase in TRAIL sensitivity were associated with a dramatic up-regulation of Bax protein expression. Knocking down Bax expression using Bax-specific small interference RNA blocked HBV-induced hepatitis and hepatocyte apoptosis. The degradation of caspases 3 and 9, but not that of Bid or caspase-8, was preferentially affected by Bax knockdown. These results establish that HBV sensitizes hepatocytes to TRAIL-induced apoptosis through Bax and that Bax-specific small interference RNA can be used to inhibit HBV-induced hepatic cell death.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Hepatic cell death is a leading cause of fatality for patients with hepatitis B virus (HBV)4 infection, which affects hundreds of millions of people worldwide. Although the morphological characteristics of dying hepatocytes during HBV infection are well documented, the molecular pathways leading to the death of hepatocytes are not well understood. Recent studies suggest that death ligands of the TNF family, which include TNF and Fas ligand (FasL), may play an important role in hepatitis. Thus, both FasL and TNF appear to be involved in mediating hepatic cell death in experimental models of hepatitis, and their blockade ameliorates the disease to various degrees (1, 2, 3, 4). TRAIL induces apoptosis of tumor cells but not most normal cells (5, 6). Its role in hepatic cell death has been controversial until recently. Initial studies using soluble recombinant TRAIL suggest that, unlike TNF and FasL, soluble TRAIL may not induce hepatic cell death in mice and nonhuman primates, although it kills sensitive tumor cells in these animals (7, 8). However, when mice were examined under conditions that induce hepatic inflammation, it became clear that TRAIL was also able to induce hepatocyte death (9, 10). Whether this is true for hepatocytes during HBV infection remains to be established.

Because death ligands and their receptors are constitutively expressed by a variety of cell types, their activities must be strictly controlled to prevent unwanted cytotoxicity. TRAIL can interact with two death receptors (DR4 and DR5, also known as TRAIL-R1 and TRAIL-R2, respectively) and two decoy receptors ((DcR)1, TRAIL-R3, and TRID) and decoy receptor 2 (DcR2, TRAIL-R4, and TRUNDD)) (6, 11, 12, 13, 14, 15, 16). Unlike DRs, the decoy receptors do not contain functional death domains, but can block DR-induced apoptosis (6, 14). In addition to decoy receptors, a variety of factors can modulate the cell’s sensitivity to TRAIL. These include intracellular regulators of the apoptotic pathway and extracellular cytokines as well as microbial products. For example, respiratory syncytial virus, HIV, and reovirus could all sensitize host cells to TRAIL-induced apoptosis (17, 18, 19). In contrast, human papillomavirus and adenovirus could inhibit TRAIL-mediated apoptosis (20, 21, 22). Among proteins encoded by the HBV genome, the X protein (HBx) has been shown to induce cell death and sensitize cells to apoptosis in addition to transactivating cellular oncogenes and promoting oncogenesis (23, 24, 25). However, the mechanisms through which HBx modulates hepatic cell death are unknown. The goal of this study is to determine for the first time whether TRAIL is involved in models of HBV-induced hepatitis, and if so, how its apoptotic role in hepatocytes is modulated by HBV gene products. We report here that HBV sensitizes hepatocytes to TRAIL-induced apoptosis through a novel mechanism that involves Bax.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Normal BALB/c mice were obtained from the Laboratory Animal Center of Shandong University. HBV complete genome (ayw subtype) transgenic BALB/c mice were purchased from the Transgenic Animal Central Laboratory (458 Hospital, Guang Zhou, China). The transgenic mice express high levels of HBV Ags, and their blood is positive for HBV DNA (26). All mice were housed in the Institute of Immunology Shandong University animal facilities under pathogen-free conditions. All procedures were preapproved by the Institutional Animal Care and Use Committee.

Plasmids

pcDNA3-1.1HBV was generated by inserting a 3.907-kb fragment of the p3.6II/HBV (provided by Prof. Y. Wang, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai, China) containing 110% of the HBV genome into the EcoRV site of the pcDNA3 (which contains a neomycin resistance gene under the control of the CMV promoter). pcDNA3-HBV plasmid was constructed by inserting one full copy of the HBV genome into the EcoRI restriction site of the pcDNA3. pcDNA3-HBx was generated by inserting a copy of the HBx gene into the HindIII and XbaI sites of the pcDNA3. Qiagen Plasmid Mini/Maxi kits were used to extract and purify the recombinant plasmids.

The mouse model of human HBV-mediated hepatitis

Five- to 6-wk-old male BALB/c mice were immunized with pcDNA3-HBV by i.m. injection. Mice injected with pcDNA3 vector served as controls. The expression of the anti-HBsAg in the serum was determined by ELISA and the presence of HBV-specific CTL in the spleen was confirmed by an in vitro CTL assay. Splenocytes from immunized mice were collected 4 wk after the immunization and were transferred into HBV transgenic BALB/c mice via tail vein (2.5 x 107 cells/mouse). Forty-eight hours later, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum were determined on a SpectraMax Plus spectrophotometer using the 3V Infinity Reagents (3V Biotech) per the manufacturer’s instructions. Liver and kidney were sectioned, stained by H&E, and examined by light microscopy.

TRAIL blockade in vivo

Recombinant human soluble DR5 (sDR5) was produced in Pichia Pastoris and purified as described previously (27). To block TRAIL effect in vivo, HBV transgenic mice were injected i.p. with 400 µg of purified sDR5 in 0.4 ml PBS 24 and 0 h before the splenocyte transfer.

Liposome-mediated transfection

Human hepatoma cell line BEL7402 was transfected with pcDNA3-1.1HBV, pcDNA3-HBx or pcDNA3 using LipofectAMINE 2000 (Invitrogen Life Technologies) per manufacturer’s instructions. Briefly, BEL7402 cells were seeded into 24-well plates with 1.5 x 105 cells/well and cultured for 20 h. The medium was removed, and cells were exposed to the transfection complexes (1 µg of DNA and 2 µl of liposome mixed in 100 µl of serum-free Opti-MEM) for 6 h at 37°C. Cells were then cultured in complete medium for 24 h. The transfected cells were selected in medium containing 380 µg/ml G418 (Invitrogen Life Technologies) for ~4 wk. The G418-resistant cells were used for the experiments described here.

TUNEL assay

Cells were seeded into 24-well plates with 2 x 105 cells/well and treated with different concentrations of recombinant soluble TRAIL (PeproTech) for 24 h. Apoptotic cells were labeled using an in situ apoptosis detection kit (Immunotech) by TUNEL. The degree of apoptosis was determined by flow cytometry.

Detection of TRAIL receptor expression by flow cytometry

Cells in the exponential phase of growth were trypsinized and washed twice in PBS. They were incubated with mouse anti-human DR4, DR5, DcR1, and DcR2 mAbs (Fourth Military Medical University) at 4°C for 30 min. After washing, cells were treated with FITC-conjugated goat anti-mouse IgG (Immunotech) for 30 min. The levels of the TRAIL receptors were determined by flow cytometry.

Western blot analysis

Cells from cultures were washed three times in PBS and incubated in the lysis buffer (50 mM Tris.Cl (pH 8.0), 150 mM NaCl, 0.1% SDS, 1% Nonidet P-40, 0.02% sodium azide, 100 µg/ml PMSF, 1 µg/ml peptin, and 1 µg/ml aprotinin) for 30 min on ice. Mouse liver tissues were ground in liquid nitrogen and homogenized in the lysis buffer (8 M urea, 4% CHAPS, 10 mM Tris.Cl (pH 8.0), 0.3 mg/ml EDTA, 1% DTT, 100 µg/ml PMSF, 1 µg/ml peptin, and 1 µg/ml aprotinin). The homogenate was centrifuged at 16,000 x g for 45 min at 4°C. Equal amount of protein from each lysate was subjected to 12% SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking for 1.5 h in 5% nonfat dried milk containing 0.1% Tween 20, the membrane was incubated at 4°C overnight in the presence of Abs to human or mouse caspase-3, caspase-8, caspase-9, Bax, beta-actin (Santa Cruz Biotechnology), or Abs to human Flip or TRAIL receptors (Upstate Biotechnology). The membrane was washed and further incubated for 1 h at room temperature with HRP-conjugated secondary Abs. Following washing, immunoreactive bands were detected using the DAB kit (Maixin) or Western Lightning Chemiluminescence Reagent (Amersham Biosciences).

Bax small interference RNA (siRNA) administration in vivo

Fifty micrograms of pmU6-Bax451 or control pmU6 plasmids was purified and diluted in saline with a volume equivalent to 8% of the mouse body weight. Plasmid solutions were injected into HBV transgenic mice via tail vein within 8 s, according to the hydrodynamic tail vein injection method for naked DNA as described by Liu et al. (28). pmU6-Bax451 encodes a Bax-specific siRNA that blocks Bax expression as described in Results.

Statistical analysis

The apoptosis rates and protein ratios were analyzed by {chi}2 test. Enzyme and protein levels were analyzed by ANOVA.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
TRAIL plays an important role in HBV-induced hepatitis

Although TRAIL may not be cytotoxic to normal hepatocytes, it has been shown to induce hepatocyte apoptosis in experimental models of hepatitis (9, 10). To determine the roles of TRAIL in HBV-mediated hepatic cell death, we tested the effect of TRAIL blockade in an HBV transgenic mouse model of hepatitis. In this model, the human HBV genes are constitutively expressed in the mouse liver, causing hepatocellular carcinoma in aged animals (26). The mice do not develop spontaneous hepatitis because the immune system is tolerized against the viral Ags. However, severe acute hepatitis can be induced upon transferring activated HBV-specific CTL into these mice. The latter cells can be generated in normal syngeneic mice by immunizing them with an expression plasmid that carries the HBV genome (29). Thus, hepatitis was induced in HBV transgenic mice by injecting into them activated splenocytes from HBV immunized mice (Fig. 1a, group 4). Significant blood levels of both ALT and AST were detected in these mice but not in the controls. Histochemical analysis of liver sections 2 days after splenocyte transfer revealed acute hepatitis in this group, which was characterized by leukocyte infiltration of liver parenchyma, acidophilic, and degenerative changes as well as death of hepatocytes (Fig. 1d). By contrast, no significant hepatitis or aminotransferase release were detected in mice that were pretreated with sDR5 (Fig. 1, a, group 5, and e). The marked inhibitory effect of sDR5 in this model is reminiscent of what was observed in other models of hepatitis in which blocking one death ligand significantly prevents the onset of the disease (30). This may be due to the interdependency of the death ligand functions in this model as recently demonstrated for TNF-{alpha} (30).


Figure 1
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  		FIGURE 1. TRAIL blockade prevents hepatitis in HBV transgenic mice. Six groups of HBV transgenic mice, five mice per group, were injected with or without splenocytes and/or sDR5 as indicated. Forty-eight hours after the cell transfer, serum ALT and AST levels were determined. a and b, Serum ALT and AST levels. Group 1, HBV transgenic mice; group 2, HBV transgenic mice received transfer of normal splenocytes from pcDNA3-treated mice; group 3, HBV transgenic mice injected with sDR5 as described in Materials and Methods; group 4, HBV transgenic mice received transfer of HBV primed splenocytes from pcDNA3-HBV immunized mice; group 5, mice in group 4 were injected with sDR5; and group 6, mice in group 4 were injected with human albumin. The differences between group 4 or 6 and other groups are statistically significant (p < 0.01 as determined by ANOVA). c–f, Histological profiles of the liver. Liver sections from groups 1 (c), 4 (d), 5 (e), and 6 (f) were stained with H&E. Original magnifications, x200.

 
HBV or HBx transfection increases the sensitivity of hepatoma cells to TRAIL-induced apoptosis

Because normal hepatocytes are not sensitive to TRAIL-induced apoptosis, the observed effect of TRAIL on HBV-induced hepatitis may be due to the altered sensitivity of HBV-infected cells. To test this theory, we transfected a widely used human hepatoma cell line BEL7402 with pcDNA3 or pcDNA3 plasmids that carry the entire HBV genome or the HBV X gene (HBx). Transfected cells were selected in G418-containing medium and tested for HBV gene expression before being used in this study. As shown in Fig. 2, transfection of BEL7402 cells with HBV but not control pcDNA3 plasmid significantly increased their sensitivity to TRAIL-induced apoptosis. Similar results were obtained when pcDNA3-HBx was used. Specifically, following incubation with 10 ng/ml TRAIL for 24 h, 12.6 ± 2.7% control BEL7402 cells underwent apoptosis; this was increased to 43 ± 5.4% for BEL7402 cells transfected with HBx (p < 0.001). These results establish that HBV and HBx are able to increase the sensitivity of BEL7402 hepatocytes to TRAIL- induced apoptosis.


Figure 2
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  		FIGURE 2. HBV transfection enhances the sensitivity of BEL7402 hepatocytes to TRAIL-induced apoptosis. The hepatoma cell line BEL7402, BEL7402 transfected with pcDNA3 (BEL7402-pcDNA3), or pcDNA3-1.1HBV (BEL7402-HBV) were treated with either PBS (a), 10 ng/ml TRAIL (b), or 10 ng/ml TRAIL plus 2 µg/ml sDR5 in PBS (c). Twenty-four hours later, cells were labeled by TUNEL and examined by flow cytometry as described in Materials and Methods.

 
HBV increases Bax expression in hepatocytes

To explore the mechanisms through which HBV increases the sensitivity of hepatocytes to TRAIL-induced apoptosis, we examined a panel of apoptosis mediators/regulators in BEL7402 cells that do or do not express HBV or HBx. We found that HBV or HBx transfection did not significantly alter the levels of DR4, DR5, DcR1, DcR2, Bid, FLIP, procaspases 3, 8, and 9 (Fig. 3 and our unpublished data). Upon treatment with TRAIL, procaspases 8 and 9 are slightly degraded in BEL7402 cells leading to a reduction in the levels of these proteins as compared with control cells (Fig. 3b). Degradation of both procaspases 8 and 9 was significantly enhanced in cells transfected with HBV (Fig. 3b), which is consistent with the apoptotic results shown in Fig. 2.


Figure 3
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  		FIGURE 3. HBV transfection does not alter the expression of TRAIL receptors, caspase-8, and caspase-9. a, BEL7402, BEL7402 transfected with pcDNA3, pcDNA3-1.1HBV, or pcDNA3-HBx were examined for TRAIL receptor expression by flow cytometry. b, BEL7402, BEL7402 transfected with pcDNA3, or pcDNA3-1.1HBV were treated with (+) or without (–) 10 ng/ml TRAIL. Procaspase-8 and procaspase-9 expressions were examined by Western blot analysis 24 h later. beta-Actin was used as a loading control.

 
Unexpectedly, Bax protein levels in both HBV and HBx transfected cells were markedly increased as compared with untransfected or pcDNA3-transfected cells (Fig. 4). Because HBx alone up-regulated Bax expression to a degree similar to that caused by HBV, we conclude that HBx may be the main mediator of Bax up-regulation in HBV transfected cells. HBx may up-regulate Bax expression at transcriptional, posttranscriptional, translational and posttranslational levels. By RT-PCR and real time PCR, we detected no significant differences in Bax mRNA levels between control and HBx transfected cells (data not shown). Thus, HBx may selectively up-regulate Bax protein expression without significantly altering its gene transcription.


Figure 4
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  		FIGURE 4. HBV transfection increases Bax expression in BEL7402 hepatocytes. BEL7402, BEL7402 transfected with pcDNA3, pcDNA3-1.1HBV, or pcDNA3-HBx were examined for Bax expression by Western blot analysis (a and b). The Bax/beta-actin protein ratios in c were determined by densitometry.

 
To determine whether HBV also regulates Bax expression in our model of HBV-induced hepatitis, we examined the hepatic levels of Bax protein in HBV transgenic mice and nontransgenic mice. We found that Bax protein levels in the liver were significantly higher in nontreated HBV transgenic mice (without hepatitis) than nontransgenic mice and that hepatitis further increased the Bax level in HBV transgenic mice (Fig. 5). Because nontreated HBV transgenic mice did not develop any detectable hepatic inflammation (Fig. 1c), the increase in Bax levels may be attributed to the direct effect of the HBV gene products as shown in cultured hepatocytes in Fig. 4. In contrast, the difference in hepatic Bax expression between mice with hepatitis and mice without hepatitis may be due to the inflammatory milieu in the former mice. To test this possibility, we also examined the hepatic Bax expression in mice that had developed aseptic hepatitis following injection of mitogen Con A as we described previously (10). We found that hepatic Bax protein level was increased by 2.1-fold in BALB/c mice 8 h after the onset of Con A-induced hepatitis as compared with control mice not treated with Con A (p < 0.01). This is consistent with our previous report that TRAIL also mediates hepatic cell death during Con A-induced hepatitis (10). Thus, both HBV gene products and inflammatory milieu may up-regulate Bax expression during HBV-induced hepatitis.


Figure 5
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  		FIGURE 5. HBV transgenic mice express increased levels of Bax in the liver. Livers were collected from the following groups of 6- to 7-wk-old BALB/c mice (n = 5): 1) HBV transgenic (Tg) mice injected with HBV-primed splenocytes as described in Materials and Methods 48 h earlier (group 1, HBV Tg + T cell), 2) control nontransgenic mice (group 2, control), and 3) nontreated HBV transgenic mice (group 3, HBV Tg). Bax expression in the liver was determined by Western blot (a). beta-Actin was used as a loading control. The Bax/beta-actin protein ratios were calculated by densitometry (b). The differences among the three groups are statistically significant (p < 0.05).

 
Bax knockdown blocks the effect of HBx on TRAIL-induced apoptosis of BEL7402 hepatocytes

Because Bax is a potent initiator of mitochondrial pathway of apoptosis, which can be activated by DRs in hepatocytes through Bid (31), its up-regulation may enhance TRAIL-induced apoptosis of HBV-transfected cells. To test this theory, we designed Bax-specific siRNAs to selectively knockdown Bax expression in HBx transfected cells. As shown in Fig. 6, one of the siRNAs, Bax382-Si, specifically and effectively diminished Bax mRNA and protein levels in BEL7402 cells. The control siRNA or Bax172-Si had no effect. To determine the effect of Bax knockdown on TRAIL-induced apoptosis of BEL7402 hepatocytes, TUNEL was performed on cells that did or did not express high levels of Bax (Fig. 7). We found that Bax knockdown significantly reduced the sensitivity of HBx transfected BEL7402 cells to TRAIL-induced apoptosis. Specifically, the rate of apoptosis in HBx transfected cells following TRAIL treatment was 43 ± 5.4%; this was reduced to 12.6 ± 2.7% in Bax knockdown group, a value similar to that of control BEL7402 cells not transfected with HBx.


Figure 6
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  		FIGURE 6. Bax expression in BEL7402 hepatocytes can be knocked down by a Bax-specific siRNA. BEL7402-HBx cells (BEL7402 cells stably expressing the HBx protein) were transfected with pSilencer3.1 plasmids that carry either a random sequence (pControl-Si) or Bax-specific siRNA sequences (pBax382-Si and pBax172-Si). Seventy-two hours later, Bax and beta-actin expressions were determined by RT-PCR (a) and Western blot analysis (b and c). Nontransfected cells were used as controls. The Bax/beta-actin protein ratios in c were determined by densitometry. The siRNA sequences used are as follows: pControl, GACTTCATAAGGCGCATGC; pBax382-Si, GGTGCCGGAACTGATCAGA; and pBax172-Si, GCTGAGCGAGTGTC TCAAG.

 

Figure 7
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  		FIGURE 7. Bax knockdown blocks the effect of HBx on TRAIL-induced apoptosis. BEL7402-HBx cells transfected with or without pControl-Si or pBax382-Si were treated with PBS or 10 ng/ml TRAIL in PBS. Twenty-four hours later, cells were labeled by TUNEL and examined by flow cytometry as described in Materials and Methods (a). The difference in TRAIL-induced apoptosis between pControl-Si and pBax382-Si-treated groups is statistically significant (p < 0.001) (b).

 
Bax knockdown blocks TRAIL-induced degradation of caspases 3 and 9 but not Bid or caspase-8

Caspases and Bid degradations are biochemical hallmarks of apoptosis. To determine the effect of Bax knockdown on caspases and Bid degradations, we examined the levels of caspases and Bid in total cell extracts by Western blot analysis. We found that TRAIL induced significant degradation (or reduction in the level) of procaspases 3, 8, and 9, as well as Bid in control BEL7402-HBx cells (Fig. 8). Remarkably, Bax knockdown using pBax382-Si selectively blocked the degradation of caspases 3 and 9 but not Bid or caspase-8 (Fig. 8). This is consistent with the theory that Bax regulates the mitochondrial but not nonmitochondrial pathway of apoptosis. Because TRAIL can activate the mitochondrial pathway of apoptosis in hepatocytes through Bid, Bax up-regulation is able to enhance TRAIL-induced apoptosis in these cells.


Figure 8
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  		FIGURE 8. Bax knockdown blocks TRAIL-induced degradation of caspase-3 and caspase-9. BEL7402-HBx cells transfected with pControl-Si or pBax382-Si were treated with (+) or without (–) 10 ng/ml TRAIL for 24 h as in Fig. 7. The levels of procaspase-3, procaspase-8, procaspase-9, and Bid were determined by Western blot analysis. beta-Actin was used as a loading control.

 
Bax knockdown in vivo ameliorates HBV-induced hepatitis and hepatocyte apoptosis

Results described above indicate that Bax may play a crucial role in HBV-induced hepatitis and that Bax blockade may be effective in ameliorating the disease. To test this theory, we examined the consequence of Bax knockdown in our model of HBV-induced hepatitis. As shown in Fig. 9, hydrodynamic administration of a plasmid encoding Bax siRNA significantly and specifically reduced hepatic Bax levels in mice with HBV-induced hepatitis. This was accompanied by a significant reduction of ALT and AST in the blood (Fig. 10). Histochemical analysis of the liver sections revealed less inflammation and reduced number of apoptotic cells in Bax siRNA treated group as compared with control groups (Fig. 10 and our unpublished data). Western blot analysis of hepatic extracts revealed that Bax siRNA preferentially blocked the degradation of procaspases 3 and 9 but not that of procaspase-8, an effect similar to that of Bax siRNA in vitro as shown in Fig. 8 (our unpublished data). The blockade of the caspase degradation was not complete either in vivo or in vitro, which may explain why hepatocyte apoptosis, although significantly reduced, still occurred in the presence of Bax siRNA (Figs. 7a and 10c).


Figure 9
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  		FIGURE 9. Bax-specific siRNA effectively blocks hepatic Bax expression in vivo. Groups of HBV transgenic BALB/c mice (n = 5) were injected as described in Materials and Methods with 1) HBV-primed splenocytes (group 1, T cell), 2) HBV-primed splenocytes plus pmU6Bax451 (group 2, T cell + pmU6Bax), and 3) HBV-primed splenocytes plus the control pmU6 plasmid (group 3, T cell + pmU6). Forty-eight hours later, mice were sacrificed, and Bax expression in the liver was quantified by Western blot analysis (a). The differences between pmU6Bax-treated group and other groups are statistically significant (p < 0.001) (b).

 

Figure 10
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  		FIGURE 10. In vivo administration of Bax-specific siRNA blocks hepatitis and hepatocyte apoptosis. Groups of HBV transgenic BALB/c mice (n = 5) were injected as described in Materials and Methods with 1) PBS (control), 2) HBV-primed splenocytes (T cell), 3) HBV-primed splenocytes plus pmU6Bax451 (T cell + pmU6Bax), and 4) HBV-primed splenocytes plus the control pmU6 plasmid (T cell + pmU6). Forty-eight hours later, mice were sacrificed and their liver and blood collected. a and b, Serum ALT and AST levels. c, Percentage of apoptotic cells in the liver as determined by TUNEL immunohistochemistry (10 ). The differences between pmU6Bax-treated group and other T cell-treated groups are statistically significant (p < 0.01).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
TRAIL is a promising new drug for cancer therapy because of its selective cytotoxicity to tumor cells (7, 8). However, the roles of TRAIL in physiological and pathological conditions remain poorly characterized. While TRAIL does not appear to kill hepatocytes under physiological conditions, it may do so under inflammatory conditions. HBV infection is a common inflammatory condition of the liver, which is the leading cause of cirrhosis and hepatocellular carcinoma. In this study, we show that HBV sensitizes hepatocytes to TRAIL-mediated apoptosis through up-regulating Bax. We propose that this explains why normal hepatocytes are not sensitive to TRAIL but HBV-infected cells are. Recently, Janssen et al. (24) reported that HBV transfection of hepatoblastoma cell line Hep G2 up-regulates DR4 expression, which may help promote TRAIL-induced apoptosis of these cells. However, we did not detect any change in DR4 expression in our BEL7402 cells following HBV or HBx transfection. This discrepancy may relate to the different cell lines used in these studies and suggest that HBV may sensitize hepatocytes to TRAIL through more than one mechanism. We elected to use BEL7402 cells in the current study because the Hep G2 cells from our laboratory appear to be different from the Hep G2 cells used by Janssen et al. (24, 32) with regard to DR expression and sensitivity to TRAIL. Our Hep G2 cells express more but not less DR than Hep G2.2.15 cells that carry the HBV genes and are more sensitive to TRAIL-induced-apoptosis (32). Because both Hep G2 and Hep G2.2.15 are long-term cultured cell lines, differences between them may or may not be directly caused by the HBV transgenes. The effect of spontaneous gene mutation and selection in the culture cannot be excluded. To circumvent this problem, only short-term transfected cells were used in this study. Using these cells, we discovered that HBV and HBx are potent enhancers of Bax expression.

Apoptosis can be mediated through two distinct pathways: the DR (extrinsic) pathway and the stress (mitochondria-dependent, intrinsic) pathway. The DR pathway requires FADD, which activates caspase-8/caspase-3 cascade leading to apoptosis. In contrast, stress factors, such as reactive oxygen species, induce apoptosis through mitochondria, which release cytochrome c that in turn activates the caspase cascade through Apaf-1 and caspase-9. According to the current dogma, there is little cross-talk between the two pathways at the initiating stage of apoptosis with the exception of caspase-8-Bid-mediated activation of the stress pathway in hepatocytes (31). Thus, in hepatocyte, Bcl-2-related proteins can regulate both the stress pathway and the DR pathway of apoptosis. The Bcl-2 family includes three classes of proteins that can either promote or inhibit apoptosis. The first class is called Bcl-2-like anti-apoptotic factors. These factors all contain three to four Bcl-2 homology (BH) domains, which are required for their antiapoptotic function. This group includes Bcl-2, Bcl-xL, Bcl-w, A1/Bfl-1, Boo/Diva/Bcl-B, and Mcl-1. The second class is called Bax-like proapoptotic factors. These factors contain BH1-BH3 domains, which act exclusively in a proapoptotic fashion. This group consists of Bax, Bak, and Bok/Mtd. The third class is called BH3-only proapoptotic factors. These members share only the short BH3 domain with the other Bcl-2 family members. They include Bim, Bid, Bad, Bik/Nbk, Blk, Hrk/DP5, Noxa, Puma/Bbc3, Bnip3, Bmf, and Bcl-GS (33, 34, 35). Although it is well recognized that Bcl-2 family of proteins plays an essential role in the stress pathway of apoptosis, the roles of most of these proteins in hepatic cell death remain to be established. Bcl-2 is the best-characterized member of this family. It blocks apoptosis by directly or indirectly preserving the integrity of the outer mitochondrial membrane, thus preventing cytochrome c release. Bcl-2 may prevent hepatocyte apoptosis induced by DR Fas through Bid (36, 37, 38). The proapoptotic member Bax induces apoptosis by causing cytochrome c release. Mice deficient in Bax are viable, suggesting that Bax is not required for development (39). However, male Bax–/– mice are sterile due to a developmental arrest in spermatogenesis. Bax-deficient animals also have increased numbers of sympathetic and motor neurons. In vitro, sympathetic neurons from Bax-deficient mice are resistant to apoptosis induced by growth factor deprivation (39). The roles of Bax in hepatic cell death during hepatitis are not known. Our demonstration that Bax knockdown in HBx-infected hepatocytes significantly blocks TRAIL-induced apoptosis indicates that Bax is a critical regulator of hepatocyte death.

In addition to the DR-Bid connection, TRAIL may also activate the mitochondrial pathway of apoptosis through TNFR-associated factor-2 and/or receptor interacting protein, which in turn activates JNK (40, 41, 42). JNK is a member of the MAPK superfamily, which can directly activate the mitochondria-dependent apoptosis pathway through Bim (43, 44, 45). Results from several laboratories indicate that the JNK-dependent pathway of apoptosis is directly involved in DR5-induced cell death (46, 47, 48). To what degree these are true during HBV infection remains to be established.

In summary, we discovered that HBV renders hepatocytes susceptible to TRAIL-induced apoptosis through Bax. Because this may contribute to HBV-induced fulminant hepatic failure and/or progressive chronic hepatitis, inhibition of TRAIL function may be beneficial in patients. Conversely, when HBV-infected hepatocytes are not adequately removed by the immune system, bolstering TRAIL’s effect pharmacologically (e.g., via exogenous TRAIL administration) may be desirable.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by grants from the Natural Science Foundation of China (30128023, 30440040, and 30371342) and National Education Ministry of China (20030422056). Back

2 X.L. and Y.L. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Youhai H. Chen, 614 BRB-II/III, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 421 Curie Boulevard, Philadelphia, PA 19104; E-mail address: yhc{at}mail.med.upenn.edu or Dr. Wensheng Sun, Institute of Immunology, School of Medicine, Shandong University, Jinan, 250012 People’s Republic of China; E-mail address: wsw{at}sdu.edu.cn Back

4 Abbreviations used in this paper: HBV, hepatitis B virus; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BH, Bcl-2 homology; DcR, decoy receptor; DR, death receptor; FasL, Fas ligand; sDR5, soluble DR5; siRNA, small interference RNA. Back

Received for publication January 12, 2006. Accepted for publication October 16, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Ksontini, R., D. B. Colagiovanni, M. D. Josephs, C. K. Edwards, III, C. L. Tannahill, C. C. Solorzano, J. Norman, W. Denham, M. Clare-Salzler, S. L. MacKay, L. L. Moldawer. 1998. Disparate roles for TNF-{alpha} and Fas ligand in concanavalin A-induced hepatitis. J. Immunol. 160: 4082-4089. [Abstract/Free Full Text]
  2. Ando, K., K. Hiroishi, T. Kaneko, T. Moriyama, Y. Muto, N. Kayagaki, H. Yagita, K. Okumura, M. Imawari. 1997. Perforin, Fas/Fas ligand, and TNF-{alpha} pathways as specific and bystander killing mechanisms of hepatitis C virus-specific human CTL. J. Immunol. 158: 5283-5291. [Abstract]
  3. Kondo, T., T. Suda, H. Fukuyama, M. Adachi, S. Nagata. 1997. Essential roles of the Fas ligand in the development of hepatitis. Nat. Med. 3: 409-413. [Medline]
  4. Chirmule, N., A. D. Moscioni, Y. Qian, R. Qian, Y. Chen, J. M. Wilson. 1999. Fas-Fas ligand interactions play a major role in effector functions of cytotoxic T lymphocytes after adenovirus vector-mediated gene transfer. Hum. Gene Ther. 10: 259-269. [Medline]
  5. Wiley, S. R., K. Schooley, P. J. Smolak, W. S. Din, C. P. Huang, J. K. Nicholl, G. R. Sutherland, T. D. Smith, C. Rauch, C. A. Smith, R. G. Goodwin. 1995. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3: 673-682. [Medline]
  6. Pan, G., J. Ni, Y. F. Wei, G. Yu, R. Gentz, V. M. Dixit. 1997. An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 277: 815-818. [Abstract/Free Full Text]
  7. Walczak, H., R. E. Miller, K. Ariail, B. Gliniak, T. S. Griffith, M. Kubin, W. Chin, J. Jones, A. Woodward, T. Le, et al 1999. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat. Med. 5: 157-163. [Medline]
  8. Ashkenazi, A., R. C. Pai, S. Fong, S. Leung, D. A. Lawrence, S. A. Marsters, C. Blackie, L. Chang, A. E. McMurtrey, A. Hebert, et al 1999. Safety and antitumor activity of recombinant soluble Apo2 ligand. J. Clin. Invest. 104: 155-162. [Medline]
  9. Mundt, B., F. Kuhnel, L. Zender, Y. Paul, H. Tillmann, C. Trautwein, M. P. Manns, S. Kubicka. 2003. Involvement of TRAIL and its receptors in viral hepatitis. FASEB J. 17: 94-96. [Abstract/Free Full Text]
  10. Zheng, S., P. Wang, G. Tsabary, Y. H. Chen. 2004. Critical roles of TRAIL in hepatic cell death and hepatic inflammation. J. Clin. Invest. 113: 58-64. [Medline]
  11. Pan, G., J. Ni, G. Yu, Y. F. Wei, V. M. Dixit. 1998. TRUNDD, a new member of the TRAIL receptor family that antagonizes TRAIL signalling. FEBS Lett. 424: 41-45. [Medline]
  12. Schneider, P., J. L. Bodmer, M. Thome, K. Hofmann, N. Holler, J. Tschopp. 1997. Characterization of two receptors for TRAIL. FEBS Lett. 416: 329-334. [Medline]
  13. Sheikh, M. S., T. F. Burns, Y. Huang, G. S. Wu, S. Amundson, K. S. Brooks, A. J. Fornace, Jr, W. S. el-Deiry. 1998. p53-dependent and -independent regulation of the death receptor KILLER/DR5 gene expression in response to genotoxic stress and tumor necrosis factor {alpha}. Cancer Res. 58: 1593-1598. [Abstract/Free Full Text]
  14. Sheridan, J. P., S. A. Marsters, R. M. Pitti, A. Gurney, M. Skubatch, D. Baldwin, L. Ramakrishnan, C. L. Gray, K. Baker, W. I. Wood, et al 1997. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 277: 818-821. [Abstract/Free Full Text]
  15. Walczak, H., M. A. Degli-Esposti, R. S. Johnson, P. J. Smolak, J. Y. Waugh, N. Boiani, M. S. Timour, M. J. Gerhart, K. A. Schooley, C. A. Smith, et al 1997. TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J. 16: 5386-5397. [Medline]
  16. Screaton, G. R., J. Mongkolsapaya, X. N. Xu, A. E. Cowper, A. J. McMichael, J. I. Bell. 1997. TRICK2, a new alternatively spliced receptor that transduces the cytotoxic signal from TRAIL. Curr. Biol. 7: 693-696. [Medline]
  17. Kotelkin, A., E. A. Prikhod’ko, J. I. Cohen, P. L. Collins, A. Bukreyev. 2003. Respiratory syncytial virus infection sensitizes cells to apoptosis mediated by tumor necrosis factor-related apoptosis-inducing ligand. J. Virol. 77: 9156-9172. [Abstract/Free Full Text]
  18. Clarke, P., S. M. Meintzer, A. C. Spalding, G. L. Johnson, K. L. Tyler. 2001. Caspase 8-dependent sensitization of cancer cells to TRAIL-induced apoptosis following reovirus-infection. Oncogene 20: 6910-6919. [Medline]
  19. Jeremias, I., I. Herr, T. Boehler, K. M. Debatin. 1998. TRAIL/Apo-2-ligand-induced apoptosis in human T cells. Eur. J. Immunol. 28: 143-152. [Medline]
  20. Hu, B., H. Zhu, S. Qiu, Y. Su, W. Ling, W. Xiao, Y. Qi. 2004. Enhanced TRAIL sensitivity by E1A expression in human cancer and normal cell lines: inhibition by adenovirus E1B19K and E3 proteins. Biochem. Biophys. Res. Commun. 325: 1153-1162. [Medline]
  21. Tollefson, A. E., K. Toth, K. Doronin, M. Kuppuswamy, O. A. Doronina, D. L. Lichtenstein, T. W. Hermiston, C. A. Smith, W. S. Wold. 2001. Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins. J. Virol. 75: 8875-8887. [Abstract/Free Full Text]
  22. Kabsch, K., A. Alonso. 2002. The human papillomavirus type 16 E5 protein impairs TRAIL- and FasL-mediated apoptosis in HaCaT cells by different mechanisms. J. Virol. 76: 12162-12172. [Abstract/Free Full Text]
  23. Terradillos, O., A. de La Coste, T. Pollicino, C. Neuveut, D. Sitterlin, H. Lecoeur, M. L. Gougeon, A. Kahn, M. A. Buendia. 2002. The hepatitis B virus X protein abrogates Bcl-2-mediated protection against Fas apoptosis in the liver. Oncogene 21: 377-386. [Medline]
  24. Janssen, H. L., H. Higuchi, A. Abdulkarim, G. J. Gores. 2003. Hepatitis B virus enhances tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) cytotoxicity by increasing TRAIL-R1/death receptor 4 expression. J. Hepatol. 39: 414-420. [Medline]
  25. Lin, N., H. Y. Chen, D. Li, S. J. Zhang, Z. X. Cheng, X. Z. Wang. 2005. Apoptosis and its pathway in X gene-transfected HepG2 cells. World J. Gastroenterol. 11: 4326-4331. [Medline]
  26. Xiong, Y., Y. Jia, H. Wang. 2000. High-level hepatitis B virus expression in transgenic mice. Infect. Dis. Info. 13: 164-165.
  27. Song, K., Y. Chen, R. Goke, A. Wilmen, C. Seidel, A. Goke, B. Hilliard, Y. Chen. 2000. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is an inhibitor of autoimmune inflammation and cell cycle progression. J. Exp. Med. 191: 1095-1104. [Abstract/Free Full Text]
  28. Liu, F., Y. Song, D. Liu. 1999. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther. 6: 1258-1266. [Medline]
  29. Schirmbeck, R., N. Dikopoulos, M. Kwissa, F. Leithauser, K. Lamberth, S. Buus, K. Melber, J. Reimann. 2003. Breaking tolerance in hepatitis B surface antigen (HBsAg) transgenic mice by vaccination with cross-reactive, natural HBsAg variants. Eur. J. Immunol. 33: 3342-3352. [Medline]
  30. Costelli, P., P. Aoki, B. Zingaro, N. Carbo, P. Reffo, F. J. Lopez-Soriano, G. Bonelli, J. M. Argiles, F. M. Baccino. 2003. Mice lacking TNF{alpha} receptors 1 and 2 are resistant to death and fulminant liver injury induced by agonistic anti-Fas antibody. Cell Death Differ. 10: 997-1004. [Medline]
  31. Yin, X. M., K. Wang, A. Gross, Y. Zhao, S. Zinkel, B. Klocke, K. A. Roth, S. J. Korsmeyer. 1999. Bid-deficient mice are resistant to Fas-induced hepatocellular apoptosis. Nature 400: 886-891. [Medline]
  32. Han, L., W. Sun, C. Ma, L. Zhang, Y. Cao, J. Song, Y. Chen. 2002. The influence of HBV infection on TRAIL-induced apoptosis and its mechanism. Zhonghua Yi Xue Za Zhi 82: 597-600. [Medline]
  33. Puthalakath, H., A. Strasser. 2002. Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins. Cell Death Differ. 9: 505-512. [Medline]
  34. Marsden, V. S., A. Strasser. 2003. Control of apoptosis in the immune system: Bcl-2, BH3-only proteins and more. Annu. Rev. Immunol. 21: 71-105. [Medline]
  35. Coultas, L., A. Strasser. 2003. The role of the Bcl-2 protein family in cancer. Semin. Cancer Biol. 13: 115-123. [Medline]
  36. Lacronique, V., A. Mignon, M. Fabre, B. Viollet, N. Rouquet, T. Molina, A. Porteu, A. Henrion, D. Bouscary, P. Varlet, et al 1996. Bcl-2 protects from lethal hepatic apoptosis induced by an anti-Fas antibody in mice. Nat. Med. 2: 80-86. [Medline]
  37. de la Coste, A., M. Fabre, N. McDonell, A. Porteu, H. Gilgenkrantz, C. Perret, A. Kahn, A. Mignon. 1999. Differential protective effects of Bcl-xL and Bcl-2 on apoptotic liver injury in transgenic mice. Am. J. Physiol. 277: G702-G708. [Medline]
  38. Yin, X. M.. 2000. Bid, a critical mediator for apoptosis induced by the activation of Fas/TNF-R1 death receptors in hepatocytes. J. Mol. Med. 78: 203-211. [Medline]
  39. Knudson, C., K. S. Tung, W. G. Tourtellotte, G. A. Brown, S. J. Korsmeyer. 1995. Bax-deficient mice with lymphoid hyperplasia and male germ cell death. Science 270: 96-99. [Abstract/Free Full Text]
  40. Chaudhary, P. M., M. Eby, A. Jasmin, A. Bookwalter, J. Murray, L. Hood. 1997. Death receptor 5, a new member of the TNFR family, and DR4 induce FADD-dependent apoptosis and activate the NF-{kappa}B pathway. Immunity 7: 821-830. [Medline]
  41. Hu, W. H., H. Johnson, H. B. Shu. 1999. Tumor necrosis factor-related apoptosis-inducing ligand receptors signal NF-{kappa}B and JNK activation and apoptosis through distinct pathways. J. Biol. Chem. 274: 30603-30610. [Abstract/Free Full Text]
  42. Lin, Y., A. Devin, A. Cook, M. M. Keane, M. Kelliher, S. Lipkowitz, Z. G. Liu. 2000. The death domain kinase RIP is essential for TRAIL (Apo2L)-induced activation of I{kappa}B kinase and c-Jun N-terminal kinase. Mol. Cell. Biol. 20: 6638-6645. [Abstract/Free Full Text]
  43. Harris, C. A., E. M. Johnson, Jr. 2001. BH3-only Bcl-2 family members are coordinately regulated by the JNK pathway and require Bax to induce apoptosis in neurons. J. Biol. Chem. 276: 37754-37760. [Abstract/Free Full Text]
  44. Putcha, G. V., S. Le, S. Frank, C. G. Besirli, K. Clark, B. Chu, S. Alix, R. J. Youle, A. LaMarche, A. C. Maroney, E. M. Johnson, Jr. 2003. JNK-mediated BIM phosphorylation potentiates BAX-dependent apoptosis. Neuron 38: 899-914. [Medline]
  45. Whitfield, J., S. J. Neame, L. Paquet, O. Bernard, J. Ham. 2001. Dominant-negative c-Jun promotes neuronal survival by reducing BIM expression and inhibiting mitochondrial cytochrome c release. Neuron 29: 629-643. [Medline]
  46. Herr, I., D. Wilhelm, E. Meyer, I. Jeremias, P. Angel, K. M. Debatin. 1999. JNK/SAPK activity contributes to TRAIL-induced apoptosis. Cell Death Differ. 6: 130-135. [Medline]
  47. Muhlenbeck, F., P. Schneider, J. L. Bodmer, R. Schwenzer, A. Hauser, G. Schubert, P. Scheurich, D. Moosmayer, J. Tschopp, H. Wajant. 2000. The tumor necrosis factor-related apoptosis-inducing ligand receptors TRAIL-R1 and TRAIL-R2 have distinct cross-linking requirements for initiation of apoptosis and are non-redundant in JNK activation. J. Biol. Chem. 275: 32208-32213. [Abstract/Free Full Text]
  48. Ohtsuka, T., D. Buchsbaum, P. Oliver, S. Makhija, R. Kimberly, T. Zhou. 2003. Synergistic induction of tumor cell apoptosis by death receptor antibody and chemotherapy agent through JNK/p38 and mitochondrial death pathway. Oncogene 22: 2034-2044. [Medline]



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