| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
-mediated apoptosis by activation of phosphoinositol 3-kinase and 
-catenin
1 Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA
2 Jefferson Medical School, Thomas Jefferson University, Philadelphia, PA, USA
3 Department of Microbiology and Immunology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA, USA
Correspondence
Mark A. Feitelson
mfeitelson1{at}yahoo.com
or
feitelso{at}temple.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) is also an important mediator of chronic hepatitis, and partially shares signalling with Fas, experiments were designed to test whether URG7 blocks TNF killing of HepG2X cells. HepG2X cells expressing URG7 and HepG2 cells overexpressing URG7 in the absence of HBxAg were resistant to TNF killing compared with HepG2CAT cells. URG7 small interfering RNA restored the sensitivity of HepG2X cells to TNF killing. Killing was associated with the activation of caspases 3 and 8, suggesting that URG7 blocked these caspases. This resistance was also associated with activation of phosphoinositol 3-kinase/Akt. Given that Akt and HBxAg also activate 
-catenin, experiments were designed to determine whether URG7 blocked apoptosis via activation of -catenin. Both HBxAg and URG7 activated fragments of the -catenin promoter, and also promoted expression of -catenin target genes. Hence, URG7 inhibits TNF-mediated killing by blocking one or more caspases in the apoptotic pathway and by activating phosphoinositol 3-kinase and -catenin, thereby overriding the apoptotic signalling of TNF. This suggests that URG7 helps to protect virus-infected hepatocytes during chronic hepatitis B virus infection.
Present address: Suite 409 BioLife Science Building, Department of Biology, College of Science and Technology, Temple University, 1900 North 12th Street, Philadelphia, PA 19122, USA. 
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). These people are at high risk for the development of hepatitis, cirrhosis and hepatocellular carcinoma (HCC) (Beasley & Hwang, 1984). The pathogenesis of chronic infection is immune-mediated (Chisari & Ferrari, 1995), yet HBV is rarely cleared despite the persistence of cell-mediated and humoral immune responses against virus-infected cells. The findings that acute, resolving hepatitis is associated with strong, polyspecific and persistent immune responses, while chronic hepatitis is associated with weak, monospecific and transient immune responses (Chisari & Ferrari, 1995), suggest that the carrier state and chronic liver disease (CLD) depend upon the quality, timing and amplitude of immune responses that develop following acute infection.
HBV encodes a small trans-activating protein, referred to as hepatitis B x antigen (HBxAg), that contributes to the establishment of the carrier state and to the pathogenesis of CLD. For example, woodchucks are naturally infected with an HBV-like woodchuck hepatitis virus, and when an infectious molecular clone of the virus was used for experimental infection of neonatal animals, most became carriers, developed CLD, and then HCC (Popper et al., 1987). However, when the X gene was mutated in this clone, so that no corresponding protein was made, experimental infection consistently failed to give rise to carriers and no liver disease developed (Chen et al., 1993; Zoulim et al., 1994). This work suggested that X antigen trans-activation of virus gene expression and replication was important for the development of the carrier state. Independent work showed a direct correlation between X antigen staining in woodchuck and human infections, and the intensity of CLD (Wang et al., 1991a, b; Feitelson et al., 1993; Jin et al., 2001), suggesting that X antigen may also protect infected hepatocytes from immunologically mediated killing. If so, X antigen would help preserve replication space for the virus despite ongoing immune responses aimed at the elimination of virus-infected hepatocytes.
Given that HBxAg is a trans-activating protein, it is possible that the upregulated expression of one or more host proteins contributes to the apparent resistance of infected cells to immune-mediated killing (Feitelson & Duan, 1997). To test this, cultures of HepG2 cells have been stably transduced with recombinant retrovirus encoding HBxAg or the bacterial chloramphenicol acetyltransferase (CAT) gene as a control (Lian et al., 1999). When the differential expression of cellular mRNAs was studied by PCR select cDNA subtraction, an uncharacterized gene, provisionally designated upregulated gene, clone 7 (URG7), was found to partially protect cells against anti-Fas-mediated killing (Lian et al., 2001). Given that Fas killing contributes to the pathogenesis of CLD (Mochizuki et al., 1996; Luo et al., 1997; Roskams et al., 2000), it is likely that the upregulated expression of URG7 in HBxAg-positive cells provides partial protection against immune clearance. Tumour necrosis factor alpha (TNF) is also an important mediator of inflammation in CLD (Lau et al., 1991; Hussain et al., 1994; Marinos et al., 1995; Fang et al., 1996), and shares part of its signalling cascade with Fas [at the level of the Fas-associated death domain (FADD) and caspase 8], raising the possibility that URG7 may protect against TNF killing as well. Hence, experiments were designed to test the hypothesis that HBxAg, through URG7, blocked TNF killing, and to elucidate some of the mechanistic steps associated with resistance to these killing signals.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) (ATCC), was cultured on tissue culture dishes or plates coated with type-1 rat tail collagen (Becton Dickinson). Cells were grown in Earle's modified Eagle's medium supplemented with 10 % heat inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 µM non-essential amino acids, 1 mM sodium pyruvate, and standard concentrations of penicillin plus streptomycin (all from Mediatech). HepG2X and HepG2CAT cells were prepared and characterized as described previously (Lian et al., 1999), and cultured at 37 °C in 5 % CO2. The human hepatocellular carcinoma cell line, Huh7 (Nakabayashi et al., 1982), was also used to create Huh7X and Huh7CAT cultures in parallel experiments.
Plasmids and transient transfections.
The retroviral plasmid, pSLXCMVneo, was used to clone URG7 cDNA (Lian et al., 1999). This was done by PCR, amplifying the 652 bp fragment encoding full-length URG7, flanked by MluI and BglII restriction endonuclease sites, which facilitated cloning to the corresponding sites within the pSLXCMV polylinker. Correct cloning was verified by DNA sequencing in the appropriate facility at the Kimmel Cancer Institute of Thomas Jefferson University.
To measure NF-
B activity, the reporter plasmid pGL2-HIV-LTR-luciferase was used as described previously (Lian et al., 1999). To measure AP-1 activity, the plasmid p-AP-1-luciferase was purchased from Stratagene. To test for -catenin promoter activity, two fragments of the -catenin promoter cloned into pSEAP-basic (a gift from Dr Frans van Roy, University of Ghent) (Nollet et al., 1996) were used. The fragments were FRAG 2 (–298 to +139) and FRAG 3 (the 6 kb fragment adjacent to the 5' end of the gene). Promoter activity was evaluated by measuring secreted human placental alkaline phosphatase (SEAP) at 24 and 48 h after transfection using a Phospha-Light chemiluminescent reporter gene assay (Soriano et al., 1991). To measure -catenin activity on cognate promoters, cells were transiently transfected with the T-cell factor reporter plasmids p-TOPFLASH (which is -catenin responsive) or p-FOPFLASH (a mutant that is -catenin unresponsive) (both from Upstate Technology).
For transient transfections, cells were seeded in six-well plates (4x105 cells per well) and incubated overnight at 37 °C in 5 % CO2. Reporter plasmids (0.5 µg per transfection) were transiently transfected by standard calcium phosphate precipitation. After overnight incubation with the DNA precipitates, cells were washed with PBS and incubated in fresh complete medium for another 24 h. Luciferase activity in 10 µg total protein lysate from each sample was measured by using a luciferase assay kit (Promega) according to the manufacturer's instructions.
Preparation of HepG2URG7 cells.
Recombinant retrovirus encoding URG7 was prepared as described previously (Lian et al., 1999), and then used to stably transduce HepG2 cells. Cells were selected in G418 (1 mg ml–1) for 3 weeks, and all drug-resistant cells were passaged without selection of individual colonies. Lysates prepared from 5x106 HepG2URG7 cells were assayed for URG7 by Western blot analysis with a mixture of peptide antibodies, as described previously (Lian et al., 2001). Some experiments were performed using peptide antibodies with the same specificities that were kindly provided by Dr Ling-Xun Duan (Aviva Biosystems).
Western blot analysis.
Cell lysates were prepared with lysis buffer containing 50 mM Tris/HCl (pH 7.4), 250 mM NaCl, 5 mM EDTA, phosphatase inhibitors (50 mM NaF, 0.1 mM Na3VO4), protease inhibitors (1 mM PMSF, 10 µg leupeptin ml–1 and 10 µg pepstatin ml–1) and 1 % Triton X-100. Each sample was analysed on a 4–20 % Tris/HCl Read gel (Biorad Laboratories), and the proteins then transferred to PVDF membranes (Millipore). After blocking, Western blot analysis was performed with rabbit anti-URG7 or anti-URG11 peptide antibodies (Lian et al., 2001, 2006), with rabbit anti-phospho-AKT (ser473; Cell Signalling Technology), with a mouse monoclonal -catenin antibody (E-5; Santa Cruz Biotechnology, which recognized wild-type and truncated -catenin in HepG2 cells), with mouse anti-GSK3 (for total GSK3 levels; Santa Cruz), with mouse anti-phospho-GSK3 (at serine 6; Santa Cruz), or with a mouse monoclonal antibody recognizing only activated wild-type -catenin (clone 8E7; Upstate Cell Signalling). All antibodies (except anti-URG7; 1 : 5000) were used at a 1 : 1000 dilution. The secondary antibodies were horseradish peroxidase-(HRP) conjugated goat anti-rabbit Ig (diluted 1 : 4000; Accurate), or HRP-conjugated goat anti-mouse Ig (diluted 1 : 3000; Accurate), and the results were visualized using enhanced chemiluminescence (ECL; Amersham). Mouse anti-human -actin monoclonal antibody (Clone AC-15; Sigma) was used at 1 : 5000 dilution as an internal control.
Inhibition of phosphoinositol 3-kinase (PI3K).
To evaluate the affects of the PI3K inhibition on p-AKT levels, cells were pretreated with 50 µM Ly294002 (Cell Signalling Technology) for 24 h, lysed, and then analysed for p-AKT by Western blotting, as described above.
RNA isolation and Northern blot analysis.
Total cellular RNA was isolated using the RNA Mini kit (Qiagen). A 10 µg aliquot of RNA from each sample was analysed on 1 % denaturing agarose gels using formaldehyde, and the integrity was assessed with rRNA. Samples were then blotted onto nytran nylon membranes (Schleicher & Schuell). Northern blot analysis was carried out using a URG7 probe obtained from a pSLXCMV URG7 fragment insert that was radiolabelled with [-32P]-labelled dCTP using the Prime-a-Gene labelling system (Promega). Following autoradiographic exposure, membranes were stripped and rehybridized with a radiolabelled glyceraldehyde-3-phosphate dehydrogenase (G3PDH) probe (Clontech). The G3PDH signal served to normalize the URG7 mRNA levels following gel scanning.
TNF treatment, signalling inhibitors and cell viability.
Approximately 18 000 cells, in 90 µl of complete culture medium, were seeded into each well of a 96-well plate and incubated overnight at 37 °C in 5 % CO2. To each well, 10 µl of 10x solutions of TNF and cycloheximide (CHX) were added to achieve final concentrations of 1 ng ml–1 and 10 µg ml–1, respectively. After 24 h, cells were analysed using the MTT assay [20 µl per well, CellTiter 96 AQueous One Solution cell proliferation assay (Promega)], and absorption was read 2–4 h later with an ELISA plate reader. Cells treated with only CHX were used as controls. The percentage cell survival was calculated as follows: (average OD490 of wells treated with TNF plus CHX/average OD490 of wells treated with CHX only)x100.
In some experiments, cells were pretreated with the caspase inhibitors Z-VAD-FMK or Z-IETD-FMK, or with the PI3K inhibitor Ly294002 (all from Cell Signalling Technology), for 1 h prior to the addition of TNF and CHX. A 1 µl aliquot from 100x stocks of each inhibitor was added to each well to give a final inhibitor concentration of 50 µM.
Caspase-3 assay.
Cells were cultured in six-well plates (8x105 per well) in complete medium overnight. Cultures were divided into five groups and treated with complete medium, TNF, CHX, TNF plus CHX, or TNF and CHX plus Z-VAD-FMK. After 6 h incubation, cells were lysed and 30 µg of total protein from each sample was evaluated for caspase-3 activity using a commercially available colorimetric assay (Promega CaspACE assay system). The specific activity of caspase-3 [pmol p-nitroaniline (pNA) liberated h–1 per µg protein] was calculated as per the manufacturer's instructions.
URG7 and URG11 small interfering RNAs (siRNAs).
To verify the contribution of upregulated URG7 to cell survival, cells were pretreated with URG7-specific or control siRNA. Accordingly, cells were seeded in 96-well plates (1x104 per /well) in antibiotic-free complete medium and cultured overnight. URG7 siRNA (sense sequence: CAAAGCCAAGAUGGUAGCUdTdT) was transfected into the cells with DharmaFECT1 (DF1, Dharmacon) according to the manufacturer's instructions. In some experiments, URG11 siRNAs (residues 420–438: CAGACGGAUUGCUGUACUU and residues 1385–1403: ACACAGACUUUACCUACAA) were used. For transfection, 100 nM siRNA and 0.2 µl DF1 were added to each well. Parallel wells were transfected with siControl Non-Targeting #1 siRNA (Dharmacon), or with transfection reagent only. After 48 h incubation, the medium was replaced with CHX with or without TNF. Cell viability was measured by using the MTT assay 24 h later.
To verify that URG7 siRNA suppressed URG7 or p-AKT, Western blots analyses were performed after the transfection of cells with URG7 siRNA. Briefly, 3.5x105 cells per well were cultured overnight in six-well plates, and then transfected with 100 nM siRNA and 6 µl DF1. Cell lysates were prepared 48 h later and 40 µg total protein was analysed by Western blotting. Transfection with siControl #1 or with transfection reagent alone provided additional controls.
Statistical analysis.
Comparisons of HepG2X, HepG2CAT and HepG2URG7 cells with regard to sensitivity to TNF killing, relative caspase 3 or 8 activities, relative phospho-Akt levels, relative levels of -catenin promoter activity, and of -catenin effector genes, were made using the Student's t-test. A significant difference was scored when P<0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
-mediated apoptosis by blocking caspases 3 and 8). By comparison, HepG2X cells had 4.2±0.44-fold more URG7 mRNA than HepG2CAT cells (Fig. 1a), indicating that the levels of URG7 in HepG2URG7 and HepG2X were similar, and that both were significantly elevated compared with HepG2CAT cells (P<0.001). Similar observations were obtained by Western blot analysis, which showed that URG7 protein levels in HepG2URG7 and HepG2X cells were 4.8±0.3-fold and 4.6±0.4-fold higher, respectively, than in HepG2CAT cells (P<0.001) (Fig. 1b).
|
, these cultures were treated with TNF plus CHX for 24 h. Live cells were measured by MTT assay in TNF plus CHX-treated cultures and compared to the same cultures treated with CHX alone. The results showed that the survival of HepG2URG7 (72 %) and HepG2X cells (82 %) was significantly greater than that of HepG2CAT cells (49 %, P<0.005, Fig. 1c
), suggesting that HBxAg, perhaps mostly through URG7, blocks the ability of TNF to trigger killing of HepG2 cells. Pretreatment of these cells with the broad spectrum caspase inhibitor, Z-VAD-FMK, completely protected these cells from TNF killing. Pretreatment of these cells with the caspase 8 inhibitor, Z-IETC-FMK, also offered protection against TNF killing, suggesting that URG7 blocked TNF signalling by inhibiting caspase 8 (Fig. 1c). Given that HBxAg blocks caspase 3 (Gottlob et al., 1998), and that this effector caspase is downstream of caspase 8 in TNF signalling, it was expected that this would also be the case for URG7. The results showed that caspase 3 was not activated by medium, by TNF alone, or by CHX alone, but required the combination of TNF plus CHX, and that caspase 3 activity was suppressed 2.6±0.19-fold in HepG2X and 2.1±0.23-fold in HepG2URG7 compared with HepG2CAT cells (P<0.005; Fig. 2). Hence, it appears that HBxAg, through URG7, provides protection against TNF killing by blocking the action of one or more caspases in the TNF signalling pathway that triggers apoptosis.
|
-mediated apoptosis by activation of PI3K/Akt signalling; Chen et al., 1998; Gibbs & Grabbe, 1999). Since PI3K is also activated by HBxAg (Lee et al., 2001), experiments were designed to ask whether URG7 overexpression activated PI3K. Accordingly, HepG2X, HepG2URG7 and HepG2CAT cells were analysed for the phosphorylated (activated) form of Akt, a substrate for PI3K. HepG2X and HepG2URG7 cells had 4.6±0.52- and 4.3±0.48-fold more phosphorylated Akt than HepG2CAT cells (P<0.001, Fig. 3a). When these cells were treated with the PI3K inhibitor, Ly294002, no phosphorylated Akt was detected (Fig. 3a). When these cells were incubated with Ly294002 for 1 h, before the addition of TNF plus CHX, significant reductions in viability were observed for HepG2X (P<0.005) and HepG2URG7 (P<0.01), while the decrease in viability among HepG2CAT cells did not reach statistical significance (P>0.1, Fig. 3b), suggesting that Akt activation in HepG2URG7 and HepG2X cells was associated with increased resistance to TNF killing.
|
killing-mediated killing, HepG2X and HepG2URG7 cells were transiently transfected with URG7-specific or control siRNA. The results showed that the introduction of URG7-specific siRNA suppressed URG7 expression several fold in the cultures tested (Fig. 4a, lane 2), compared with mock-transfected cells (lane 1) or cells transfected with an irrelevant siRNA (lane 3), demonstrating that it was active in downregulating expression of URG7 (P<0.001). In parallel experiments, treatment with URG7-specific siRNA significantly depressed the levels of phosphorylated (activated) Akt roughly sixfold in HepG2URG7 (P<0.001) and roughly fivefold in HepG2X (P<0.001) compared with the same cells treated with an irrelevant siRNA (Fig. 4b). When the experiments were repeated and cell viability was monitored after transient transfection, only cells transfected with URG7-specific siRNA were highly sensitive to TNF killing (Fig. 4c). Given that this work was performed in HepG2 cells, parallel experiments were conducted with the human hepatoma cell line, Huh7, stably transduced with the X or CAT genes. Transient transfection of URG7-specific but not control siRNA into Huh7X and Huh7CAT cells blocked endogenous URG7 polypeptide expression and Akt phosphorylation (data not shown but similar to Fig. 4a and b, respectively). Treatment of Huh7X and Huh7CAT with TNF plus CHX along with URG7-specific siRNA blocked the ability of HBxAg to protect cells from TNF killing, while control siRNA-transfected cells did not block the ability of HBxAg to protect cells from TNF (Fig. 4c), suggesting that this activity was not restricted to HepG2 cells. In addition, the fact the cultures tested were not of clonal origin, underscores the generality of these findings. These results confirm that elevated URG7 is associated with activation of Akt, and that the latter is also associated with resistance to TNF.
|
-catenin signalling (GSK3) is inactivated after phosphorylation by Akt, resulting in the stabilization of -catenin (Morin, 1999). Given that HBxAg activates -catenin by inactivation of GSK3 and by trans-activating the -catenin promoter (Lian et al., 2006), experiments were designed to test whether the URG7-activation of Akt did the same. The results showed that GSK3 was strongly phosphorylated in HepG2URG7 and HepG2X cells compared with HepG2CAT cells (P<0.001, Fig. 5a), suggesting that HBxAg inactivated GSK3 through URG7. To determine whether HBxAg trans-activated the -catenin promoter through URG7, cells were transiently transfected with reporter plasmids expressing luciferase under the control of two overlapping fragments of the -catenin promoter (Nollet et al., 1996). The results showed that both HBxAg and URG7 stimulated the -catenin promoter by 3–4-fold (P<0.005, Fig. 5b). Co-transfection of URG7-specific or control siRNAs into HepG2URG7 cells showed that upregulated -catenin promoter activity was a function of URG7 expression (Fig. 5b). Given that both HBxAg and URG7 stimulate PI3K/Akt (Figs 3a and 4b), that HBxAg activates NF-B (Sliva, 2004; Amiri & Richmond, 2005), and that there are NF-B-binding sites in the -catenin promoter (Nollet et al., 1996; Li et al., 2004), these results suggest that -catenin may be transcriptionally targeted by these mechanisms. This was supported by Western blot analysis using an antibody that specifically bound activated -catenin, which showed a 4–5-fold increase in activated -catenin in HepG2URG7 and HepG2X cells relative to HepG2CAT (Fig. 5c). To see whether the accumulation of -catenin resulted in the activation of its downstream target genes, the -catenin-responsive TOPFLASH reporter and -catenin-non-responsive FOPFLASH reporter constructs were transiently transfected into HepG2URG7, HepG2X and HepG2CAT cells. The results showed very strong activation of -catenin-responsive genes in HepG2URG7 (more than 50-fold) and HepG2X cells (more than 80-fold) compared to these same cultures transfected with the control FOPFLASH vector (P<0.001) (Fig. 5d). In contrast, HepG2CAT cells showed only baseline activation of TOPFLASH (Fig. 5d). Co-transfection of URG7-specific or control siRNAs into HepG2URG7 cells showed that pTOPFLASH activity was a function of URG7 expression (Fig. 5d). This suggested that URG7 significantly stimulated -catenin signalling and turned on -catenin-responsive genes.
|
-catenin (Carruba et al., 1999), that both HBxAg and URG7 inactivate GSK3 (Fig. 5a) and that both stimulate wild-type -catenin (Fig. 5c), raises the question as to the contribution of mutant -catenin under these circumstances. To address this, GSK3 was targeted by transfection of specific siRNA into HepG2CAT, HepG2X and HepG2URG7 cells. The results showed that GSK3-specific siRNA was effective in blocking the expression of GSK3 in all the cultures tested (Fig. 6a, lanes 2, 4 and 6), while control siRNA was ineffective (Fig. 6a, lanes 1, 3 and 5). The levels of phosphorylated GSK3 decreased in parallel (Fig. 6b). Western blot analysis for total -catenin showed a modest decrease in wild-type -catenin, but no change in mutant -catenin, upon treatment of cells with GSK3-specific siRNA (Fig. 6d). This profile was similar to that obtained when Western blot analysis was conducted with another antibody that only detected active -catenin (Fig. 6e). Given that the truncated mutant of -catenin lacks the GSK3-binding site (Carruba et al., 1999), it is not surprising that there is no change in mutant -catenin levels in Fig. 6(d). Hence, it is likely that the functional changes observed in Fig. 5(d) are due to changes in the levels and activities of wild-type -catenin.
|
killing-catenin activity (Fig. 5; Lian et al., 2006), suggests that URG11 may contribute to the TNF resistance observed in HBxAg-expressing or URG7-overexpressing cells. Accordingly, when the levels of URG11 were assessed in HepG2X, HepG2URG7 and HepG2CAT cells, they were observed to be elevated 4.9±0.4-fold in HepG2X cells and 2.1±0.25-fold in HepG2CAT cells compared with HepG2URG7 cells (Fig. 7a). This confirms that HBxAg promotes the stability of -catenin, which is associated with the upregulated expression of URG11 (Lian et al., 2006). In contrast, there seems to be an inverse relationship between intracellular levels of URG7 and URG11 in HepG2URG7 compared with HepG2CAT cells. When each of these cultures were treated with TNF along with URG11-specific siRNA, URG11 expression was blocked (data not shown), and the viability of HepG2URG7 cells was significantly less than that of HepG2CAT or HepG2X cultures, suggesting that URG11 also contributes to the resistance of liver cells to apoptosis (P<0.001, Fig. 7b).
|
B and AP-1 signalling signalling, through RIP and TRAF-2, may stimulate the anti-apoptotic AP-1 and NF-B pathways. HBxAg has also been observed to stimulate AP-1 and NF-B signalling (Kekule et al., 1993; Natoli et al., 1994). To see if either or both of these pathways were activated by URG7; HepG2X, HepG2URG7 and HepG2CAT cells were transiently transfected with pGL2-HIV-LTR-luciferase to measure NF-B activity, or with p-AP-1-luciferase to measure AP-1 activity. The results show that only HepG2X cells stimulate these reporter plasmids (Fig. 8), indicating that URG7 does not stimulate these signalling pathways.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
), and that both TNF- and Fas-mediated apoptotic pathways play central roles. For example, strong Fas/FasL and TNF/TNF receptor 1 (TNFR) expression are often observed in hepatocytes around areas of intense inflammation (Mochizuki et al., 1996; Luo et al., 1997) adjacent to HCC nodules (Roskams et al., 2000), and generally correlate with the severity of CLD (Lau et al., 1991; Hussain et al., 1994; Marinos et al., 1995; Fang et al., 1996). Importantly, the distribution of HBxAg is similar (Wang et al., 1991a, b; Feitelson et al., 1993; Jin et al., 2001), suggesting that HBxAg may modulate Fas and TNF expression and/or signalling. Interestingly, HBxAg trans-activates TNF expression in cell culture (Lara-Pezzi et al., 1998). Although this suggests that HBxAg may promote apoptosis, the accumulation of HBxAg-positive hepatocytes during CLD (Wang et al., 1991a; Jin et al., 2001) suggests that HBxAg is protecting infected cells from TNF killing at the expense of uninfected hepatocytes. HBxAg also upregulates the expression of FasL in liver and in hepatoma cell lines (Shin et al., 1999), which may kill cytotoxic T cells, and permit the escape of HBxAg-positive cells from immune-mediated killing. In addition, HBxAg activates NF-B (Pan et al., 2001), SAPK/JNK (Diao et al., 2001) and PI3K (Suzuki et al., 2000; Lee et al., 2001), which protect HBxAg-positive cells from Fas-mediated killing. The finding in this report, that HBxAg protects cells from TNF killing through the upregulated expression of URG7 and URG11, identifies key cellular genes that are likely to contribute to the survival of virus-infected cells during chronic infection.
In addition to direct killing through the Fas/FasL pathway, which is often mediated by activated T cells, hepatocytes, Kupffer cells, other inflammatory cells, and peripheral blood mononuclear cells produce TNF (and other cytokines) at elevated levels in the serum of HBV carriers (Sheron et al., 1991; Gonzalez-Amaro et al., 1994). A general characteristic of these inflammatory cytokines is that they trigger the generation of reactive oxygen intermediates (ROI) during CLD. ROI stimulate selected signalling pathways, such as AP-1 and NF-B. The findings that HBxAg also stimulates these pathways (Kekule et al., 1993; Natoli et al., 1994), and that URG7 is a target gene for NF-B (Lian et al., 2001), imply that in the presence of HBxAg, the levels of NF-B activation pass a threshold whereby the URG7 gene becomes upregulated. URG7 would then stimulate PI3K/Akt signalling (Figs 3 and 4), resulting in the inactivation of GSK3, and the stabilization of wild-type -catenin (Figs 5 and 6). While these, and perhaps other pathways, may override the apoptotic signals triggered by TNF, the finding that URG7 blocks caspase 8 and downstream caspase 3 activities (Figs 1 and 2) provides a mechanism whereby apoptotic pathways are shut off at the same time that survival-related pathways are turned on. The blockage of caspase 8 may also contribute importantly to the mechanism of how HBxAg-positive cells become resistant to anti-Fas-mediated killing (Fig. 1), since TNF, through the TNFR-associated death domain, and anti-Fas, through FADD, both converge and share caspase 8 as an important link whereby these mechanisms trigger apoptosis.
It is intriguing that overexpression of URG7 does not promote the growth of HepG2 (or Hep3B or Huh7) cells in soft agar or accelerate tumourigenesis in nude mice (Lian et al., 2001) even though high levels of URG7 expression are associated with the activation of -catenin (Figs 5 and 6). In this context, there is an increasing role for upregulated -catenin in blocking apoptosis, independent of its role in promoting tumourigenesis (Mikami et al., 2005; Ormestad et al., 2006; Yang et al., 2006), and it is proposed here that one of the ways this could happen is by the stimulated expression of URG7. Importantly, HBxAg upregulates another novel cellular gene, URG11, that also stabilizes/upregulates -catenin, but in the latter case, the upregulated expression of -catenin was shown to strongly stimulate growth in soft agar and tumour formation in nude mice (Lian et al., 2003, 2006). Evidence presented here suggests that URG11 also contributes to the resistance of URG7 overexpressing cells to apoptosis (Fig. 7), suggesting it may have a dual role in chronic HBV infection. Hence, HBxAg upregulates the expression of two cellular genes that help to protect cells from immune-mediated killing, which may promote virus persistence in the context of CLD.
The model whereby URG7 acts as outlined above may be only part of the picture. For example, caspase 8 catalyses the cleavage of the anti-apoptotic Bid to the proapoptotic tBid (Li et al., 1998), which translocates to the mitochondria, where it triggers the release of cytochrome c, leading to the activation of caspase 9, and then caspase 3 (Roy & Nicholson, 2000). If caspase 8 activity is inhibited by URG7, it would not only block extrinsic (receptor-mediated) apoptosis, but also endogenous (mitochondrial-based) apoptosis. In fact, activation of -catenin is known to block mitochondria-mediated apoptosis (Yang et al., 2006). In addition, the expression of Bid is lower in HCC than in the surrounding non-tumour liver (Chen et al., 2001a), and a significant decrease in Bid has also been observed in hepatoma cells transfected with HBxAg (Chen et al., 2001b). PI3K/Akt is also known to phosphorylate the proapoptotic molecule BAD converting it to the anti-apoptotic molecule pBAD, so again, the question becomes whether URG7 could mediate the phosphorylation of BAD. Furthermore, the finding that the combined activation of PI3K and NF-B in several tumour types is associated with the upregulation of BCL-2 and cell survival (Catz & Johnson, 2003) raises the question as to whether the combined actions of HBxAg and URG7 upregulate BCL-XL in the liver during chronic infection. In other systems, activation of NF-B by PI3K is associated with highly invasive cancers (Sliva, 2004; Amiri & Richmond, 2005). The fact that URG7 does not activate NF-B (or AP-1; Fig. 8) may indicate that activation of PI3K/Akt and -catenin under these circumstances promotes cell survival by blocking apoptosis, while the additional activation of NF-B by the combination of ROI, HBxAg and PI3K promotes tumourigenesis as well.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Amiri, K. I. & Richmond, A. (2005). Role of nuclear factor- B in melanoma. Cancer Metastasis Rev 24, 301–313.[CrossRef][Medline]
Beasley, R. P. & Hwang, L. Y. (1984). Epidemiology of hepatocellular carcinoma. In Viral Hepatitis and Liver Disease (Proceedings of the 1984 International Symposium on Viral Hepatitis), pp. 209–224. Edited by G. N. Vyas, J. L. Dienstag & J. H. Hoofnagle. Orlando: Grune and Stratton.
Berra, E., Diaz-Meco, M. T. & Moscat, J. (1998). The activation of p38 and apoptosis by the inhibition of Erk is antagonized by the phosphoinositide 3-kinase/Akt pathway. J Biol Chem 273, 10792–10797.
Carruba, G., Cervello, M., Miceli, M. D., Farruggio, R., Notarbartolo, M., Virruso, L., Giannitrapani, L., Gambino, R., Montalto, G. & Castagnetta, L. (1999). Truncated form of -catenin and reduced expression of wild-type catenins feature HepG2 human liver cancer cells. Ann N Y Acad Sci 886, 212–216.[CrossRef][Medline]
Catz, S. D. & Johnson, J. L. (2003). BCL-2 in prostate cancer: a minireview. Apoptosis 8, 29–37.[CrossRef][Medline]
Chen, H. S., Kaneko, S., Girones, R., Anderson, R. W., Hornbuckle, W. E., Tennant, B. C., Cote, P. J., Gerin, J. L., Purcell, R. H. & Miller, R. H. (1993). The woodchuck hepatitis virus X gene is important for establishment of virus infection in woodchucks. J Virol 67, 1218–1226.
Chen, R. H., Su, Y. H., Chuang, R. L. & Chang, T. Y. (1998). Suppression of transforming growth factor--induced apoptosis through a phosphatidylinositol 3-kinase/Akt-dependent pathway. Oncogene 17, 1959–1968.[CrossRef][Medline]
Chen, G. G., Lai, P. B. S., Chak, E. C. W., Xu, H., Lee, K. M. & Lau, W. Y. (2001a). Immunohistochemical analysis of proapoptotic Bid levels in chronic hepatitis, hepatocellular carcinoma and liver metastases. Cancer Lett 172, 75–82.[CrossRef][Medline]
Chen, G. G., Lai, P. B. S., Chan, P. K. S., Chak, E. C. W., Yip, J. H. Y., Ho, R. L. K., Leung, B. C. S. & Lau, W. Y. (2001b). Decreased expression of Bid in human hepatocellular carcinoma is related to hepatitis B virus X protein. Eur J Cancer 37, 1695–1702.[CrossRef][Medline]
Chisari, F. V. & Ferrari, C. (1995). Hepatitis B virus immunopathology. Springer Semin Immunopathol 17, 261–281.[Medline]
Diao, J., Khine, A. A., Sarangi, F., Hsu, E., Lorio, C., Tibbles, L. A., Woodgett, J. R., Penninger, J. & Richardson, C. D. (2001). X protein of hepatitis B virus inhibits Fas-mediated apoptosis and is associated with up-regulation of the SAPK/JNK pathway. J Biol Chem 276, 8328–8340.
Fang, J. W., Shen, W. W., Meager, A. & Lau, J. Y. (1996). Activation of the tumor necrosis factor- system in the liver in chronic hepatitis B virus infection. Am J Gastroenterol 91, 748–753.[Medline]
Feitelson, M. A. & Duan, L. X. (1997). Hepatitis B virus x antigen in the pathogenesis of chronic infections and the development of hepatocellular carcinoma. Am J Pathol 150, 1141–1157.[Abstract]
Feitelson, M. A., Lega, L., Duan, L. X. & Clayton, M. (1993). Characteristics of woodchuck hepatitis X antigen in the livers and sera from chronically infected animals. J Hepatol 17 (Suppl. 3), S24–S34.
Gibbs, B. F. & Grabbe, J. (1999). Inhibitors of PI 3-kinase and MEK kinase differentially affect mediator secretion from immunologically activated human basophils. J Leukoc Biol 65, 883–889.[Abstract]
Gonzalez-Amaro, R., Garcia-Monzon, C., Garcia-Buey, L., Moreno-Otero, R., Alonso, J. L., Yague, E., Pivel, J. P., Lopez-Cabrera, M., Fernandez-Ruiz, E. & Sanchez-Madrid, F. (1994). Induction of tumor necrosis factor production by human hepatocytes in chronic viral hepatitis. J Exp Med 179, 841–848.
Gottlob, K., Fulco, M., Levrero, M. & Greessmann, A. (1998). The hepatitis B virus HBx protein inhibits caspase 3 activity. J Biol Chem 273, 33347–33353.
Hussain, M. J., Lau, J. Y., Williams, R. & Vergani, D. (1994). Hepatic expression of tumour necrosis factor- in chronic hepatitis B virus infection. J Clin Pathol 47, 1112–1115.
Jin, Y. M., Yun, C., Park, C., Wang, H. J. & Cho, H. (2001). Expression of hepatitis B virus X protein is closely correlated with the high periportal inflammatory activity of liver diseases. J Viral Hepat 8, 322–330.[CrossRef][Medline]
Kekule, A. S., Lauer, U., Weiss, L., Luber, B. & Hofschneider, P. H. (1993). HBV transactivator HBx uses a tumour promoter signalling pathway. Nature 361, 742–745.[CrossRef][Medline]
Lara-Pezzi, E., Majano, P. L., Gomez-Gonzalo, M., Garcia-Monzon, C., Moreno-Otero, R., Levrero, M. & Lopez-Cabrera, M. (1998). The hepatitis B virus X protein up-regulates tumor necrosis factor gene expression in hepatocytes. Hepatology 28, 1013–1021.[CrossRef][Medline]
Lau, J. Y. N., Sheron, N., Nouri-Aria, K. T., Alexander, G. J. M. & Williams, R. (1991). Increased tumor necrosis factor- receptor number in chronic hepatitis B virus infection. Hepatology 14, 44–50.[Medline]
Lee, Y. I., Kang-Park, S., Do, S. I. & Lee, Y. I. (2001). The hepatitis B virus-X protein activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade. J Biol Chem 276, 16969–16977.
Li, H., Zhu, H., Xu, C. J. & Yuan, J. (1998). Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94, 491–501.[CrossRef][Medline]
Li, Q., Dashwood, W.-M., Zhong, X., Al-Fageeh, M. & Dashwood, R. H. (2004). Cloning of the rat -catenin gene (Ctnnb1) promoter and its functional analysis compared with the Catnb and CTNNB1 promoters. Genomics 83, 231–242.[CrossRef][Medline]
Lian, Z., Pan, J., Liu, J., Zhu, M., Arbuthnot, P., Kew, M. C. & Feitelson, M. A. (1999). The translation initiation factor, SUI1, may be a target of HBxAg in hepatocarcinogenesis. Oncogene 18, 1677–1687.[CrossRef][Medline]
Lian, Z., Liu, J., Pan, J., Tufan, N. L. S., Zhu, M., Arbuthnot, P., Kew, M., Clayton, M. M. & Feitelson, M. A. (2001). A cellular gene up-regulated by hepatitis B virus encoded X antigen promotes hepatocellular growth and survival. Hepatology 34, 146–157.[CrossRef][Medline]
Lian, Z., Liu, J., Li, L., Li, X., Tufan, N. L., Clayton, M., Wu, M. C., Wang, H. Y., Arbuthnot, P. & other authors (2003). Up-regulated expression of a unique gene by hepatitis B x antigen promotes hepatocellular growth and tumorigenesis. Neoplasia 5, 229–244.[Medline]
Lian, Z., Liu, J., Li, L., Li, X., Clayton, M., Wu, M. C., Wang, H. Y., Arbuthnot, P., Kew, M. & other authors (2006). Enhanced cell survival of Hep3B cells by the hepatitis B x antigen effector, URG11, is associated with up-regulation of -catenin. Hepatology 43, 415–424.[CrossRef][Medline]
Luo, K. X., He, H. T., Zhu, Y. F. & Zhang, L. (1997). Hepatocyte Fas expression and DNA damage in chronic hepatitis B. Chin J Intern Med 35, 750–752.
Marinos, G., Naoumov, N. V., Rossol, S., Torre, F., Wong, P. Y. N., Gallati, H., Portmann, B. & Williams, R. (1995). Tumor necrosis factor receptors in patients with chronic hepatitis B virus infection. Gastroenterology 108, 1453–1463.[CrossRef][Medline]
Mikami, I., You, L., He, B., Xu, Z., Batra, S., Lee, A. Y., Mazieres, J., Reguart, N., Uematsu, K. & other authors (2005). Efficacy of Wnt-1 monoclonal antibody in sarcoma cells. BMC Cancer 5, 53[CrossRef][Medline]
Mochizuki, K., Hayashi, N., Hiramatsu, N., Katayama, K., Kawanishi, Y., Kasahara, A., Fusamoto, H. & Kamada, T. (1996). Fas antigen expression in liver tissues of patients with chronic hepatitis B. J Hepatol 24, 1–7.[CrossRef][Medline]
Morin, P. J. (1999). -catenin signaling and cancer. Bioessays 21, 1021–1030.[CrossRef][Medline]
Nakabayashi, H., Taketa, K., Miyano, K., Yamane, T. & Sato, J. (1982). Growth of human hepatoma cell lines with differentiated functions in chemically defined medium. Cancer Res 42, 3858–3863.
Natoli, G., Avantaggiati, M. L., Chirillo, P., Puri, P. L., Ianni, A., Balsano, C. & Levrero, M. (1994). Ras- and raf-dependent activation of c-Jun transcriptional activity by the hepatitis B virus transactivator pX. Oncogene 9, 2837–2843.[Medline]
Nollet, F., Berx, G., Molemans, F. & van Roy, F. (1996). Genomic organization of the human -catenin gene (CTNNB1). Genomics 32, 413–424.[CrossRef][Medline]
Ormestad, M., Astorga, J., Landgren, H., Wang, T., Johansson, B. R., Miura, N. & Carlsson, P. (2006). Foxf1 and Foxf2 control murine gut development by limiting mesenchymal Wnt signaling and promoting extracellular matrix production. Development 133, 833–843.
Pan, J., Duan, L. X., Sun, B. S. & Feitelson, M. A. (2001). HBV X protein decreases the anti-Fas induced apoptosis in human liver cells by inducing NF-B. J Gen Virol 82, 171–182.
Popper, H., Roth, L., Purcell, R. H., Tennant, B. C. & Gerin, J. L. (1987). Hepatocarcinogenicity of the woodchuck hepatitis virus. Proc Natl Acad Sci U S A 84, 866–870.
Roskams, T., Libbrecht, L., van Dame, B. & Desmet, V. (2000). Fas and Fas ligand: strong co-expression in human hepatocytes surrounding hepatocellular carcinoma; can cancer induce suicide in peritumoral cells?. J Pathol 191, 150–153.[CrossRef][Medline]
Roy, S. & Nicholson, D. W. (2000). Cross-talk in cell death signaling. J Exp Med 192, F21–F25.
Sheron, N., Lau, J., Daniels, H., Goka, J., Eddleston, A., Alexander, G. J. & Williams, R. (1991). Increased production of tumour necrosis factor alpha in chronic hepatitis B virus infection. J Hepatol 12, 241–245.[CrossRef][Medline]
Shin, E. C., Shin, J. S., Park, J. H. & Kim, S. J. (1999). Expression of Fas ligand in human hepatoma cell lines: role of hepatitis B virus X (HBX) in induction of Fas ligand. Int J Cancer 82, 587–591.[CrossRef][Medline]
Sliva, D. (2004). Signaling pathways responsible for cancer cell invasion as targets for cancer therapy. Curr Cancer Drug Targets 4, 327–336.[CrossRef][Medline]
Soriano, P., Montgomery, C., Geske, R. & Bradley, A. (1991). Targeted disruption of the c-src proto-oncogene leads to osteoperosis in mice. Cell 64, 693–702.[CrossRef][Medline]
Suzuki, A., Hayashida, M., Kawano, H., Sugimoto, K., Nakano, T. & Shiraki, K. (2000). Hepatocyte growth factor promotes cell survival from Fas-mediated cell death in hepatocellular carcinoma cells via Akt activation and Fas-death-inducing signaling complex suppression. Hepatology 32, 796–802.[CrossRef][Medline]
Tiollais, P., Pourcel, C. & Dejean, A. (1985). The hepatitis B virus. Nature 317, 489–495.[CrossRef][Medline]
Wang, W. L., London, W. T., Lega, L. & Feitelson, M. A. (1991a). HBxAg in liver from carrier patients with chronic hepatitis and cirrhosis. Hepatology 14, 29–37.[CrossRef][Medline]
Wang, W. L., London, W. T. & Feitelson, M. A. (1991b). HBxAg in HBV carrier patients with liver cancer. Cancer Res 51, 4971–4977.
Yang, F., Zeng, Q., Yu, G., Li, S. & Wang, C.-Yu. (2006). Wnt/-catenin signaling inhibits death receptor-mediated apoptosis and promotes invasive growth of HNSCC. Cell Signal 18, 679–687.[CrossRef][Medline]
Zoulim, F., Saputelli, J. & Seeger, C. (1994). Woodchuck hepatitis virus X protein is required for viral infection in vivo. J Virol 68, 2026–2030.
Received 4 June 2007;
accepted 20 August 2007.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
