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-catenin depending on the status of cellular p53
Division of Biological Sciences, College of Natural Sciences, Pusan National University, Busan 609-735, Korea
Correspondence
Kyung Lib Jang
kljang{at}pusan.ac.kr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-catenin is considered to be a strong driving force in hepatocellular carcinogenesis; however, the mechanism of -catenin accumulation in tumours is unclear. Here, it was demonstrated that hepatitis B virus X protein (HBx) differentially regulates the level of -catenin through two ubiquitin-dependent proteasome pathways depending on p53 status. In the presence of p53, HBx downregulated -catenin through the activation of a p53–Siah-1 proteasome pathway. For this purpose, HBx upregulated Siah-1 expression at the transcriptional level via activation of p53. In the absence of p53, however, HBx stabilized -catenin through the inhibition of a glycogen synthase kinase-3-dependent pathway. Interestingly, HBx variants with a Pro-101 to Ser substitution were unable to activate p53 and thus could stabilize -catenin irrespective of p53 status. Based on these findings, a model of -catenin regulation by HBx is proposed whereby the balance between the two opposite activities of HBx determines the overall expression level of -catenin. Differential regulation of -catenin by HBx depending on host (p53 status) and viral factors (HBx sequence variation) helps not only to explain the observation that cancers accumulating -catenin also exhibit a high frequency of p53 mutations but also to understand the contradictory reports on the roles of HBx during hepatocellular carcinogenesis.
These authors contributed equally to this work. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-Catenin has critical functions in several normal and malignant intracellular processes. In cell-adhesion signalling, -catenin binds the cytoplasmic domain of cadherin adhesion receptors along with 
-catenin to transmit signals from cadherins to the underlying actin cytoskeleton (Gottardi & Gumbiner, 2001
). In addition, -catenin binds to T-cell factor/lymphoid-enhancing factor (Tcf/Lef) in the nucleus and acts as its co-activator to stimulate the transcription of target genes such as c-myc and cyclin D1 (He et al., 1998; Tetsu & McCormick, 1999). The intracellular -catenin level is mainly regulated by the ubiquitin–proteasome system (Nelson & Nusse, 2004; Peifer & Polakis, 2000). According to the Wnt pathway, Axin and adenomatous polyposis coli (APC) serve as scaffolds to facilitate the phosphorylation of -catenin by glycogen synthase kinase-3 (GSK-3) (Nelson & Nusse, 2004; Peifer & Polakis, 2000) and the phosphorylated -catenin is targeted for degradation by the ubiquitin–proteasome system (Aberle et al., 1997). Activation of classic Wnt signalling leads to inhibition of GSK-3-dependent phosphorylation and degradation of -catenin (Nelson & Nusse, 2004). The alternative pathway for -catenin degradation involves mammalian homologues of the Drosophila seven in absentia protein (Siah), which bind ubiquitin-conjugating enzymes, and Ebi (‘shrimp’ in Japanese), an F-box protein, which binds -catenin independently of GSK-3-mediated phosphorylation (Liu et al., 2001; Matsuzawa & Reed, 2001).
The activated Wnt/-catenin pathway is now considered to be one of the main driving forces of hepatocarcinogenesis. Up to 62 % of all hepatocellular carcinomas (HCCs) examined so far show abnormal -catenin protein accumulation in the cytoplasm and nucleus (Wong et al., 2001; Taniguchi et al., 2002). In addition, tumours with -catenin accumulation are associated with a dismal prognosis due to a poorly differentiated morphology (Devereux et al., 2001; Wong et al., 2001), large tumour size (Laurent-Puig et al., 2001; Wong et al., 2001) and vascular invasion (Endo et al., 2000). Mutations in Axin, APC or the GSK-3 phosphorylation site of -catenin result in accumulation of -catenin in various tumours (Polakis, 2007). However, the mechanism responsible for -catenin accumulation in HCC is poorly understood. Axin mutations in HCC range from 5 to 10 % (Satoh et al., 2000; Taniguchi et al., 2002). An APC mutation has been described recently in a case report (Katoh et al., 2006), but it is unusual in HCC (Colnot et al., 2004). -Catenin exon 3 mutations have been associated only with nuclear -catenin accumulation ranging from 12 to 44 % (Cui et al., 2003; Wong et al., 2001). Thus, genetic alteration alone seems not to be sufficient to account for the accumulation of -catenin in HCC.
Hepatitis B virus (HBV) is strongly associated with the development of HCC (Block et al., 2003). HBx is encoded by the smallest open reading frame of the HBV genome, termed X, and is the most frequently integrated viral sequence found in HCCs (Paterlini et al., 1995). HBx is a multifunctional regulatory protein that can activate several transcription factors including AP-1, NF-
B, CREB and TBP (Benn et al., 1996; Maguire et al., 1991; Qadri et al., 1995). In addition, HBx has been implicated in the activation of several signal transduction pathways that lead to the transcriptional upregulation of a number of cellular genes, including those of growth factors and oncogenes (Benn et al., 1996; Lee & Yun, 1998; Shih et al., 2000). Moreover, HBx is able to induce HCC in transgenic mice (Kim et al., 1991). However, some lineages of HBx transgenic mice fail to develop liver tumours unless exposed to additional hepatocarcinogenic influences (Slagle et al., 1996; Terradillos et al., 1997). Although HBx is considered to play an important role in HBV-mediated hepatocellular carcinogenesis, the mechanism by which HBx mediates its role is still controversial.
Recently, it has been demonstrated that HBx can stabilize -catenin by suppressing GSK-3 activity (Cha et al., 2004; Ding et al., 2005). However, according to our previous reports (Ahn et al., 2002; Kwun & Jang, 2004), HBx, like other oncogenic proteins, upregulates levels of p53, which is known to induce proteasomal degradation of -catenin (Cagatay & Ozturk, 2002; Matsuzawa & Reed, 2001; Sadot et al., 2001). Thus, in the present study, we explored the possibility that HBx promotes -catenin degradation via p53 and investigated the mechanism involved in this process. We also examined whether HBx could stabilize -catenin in the absence of p53. In addition, we investigated whether HBx variants differentially regulate the level of -catenin. Based on the opposing effects of HBx on -catenin depending on p53 status, we propose a working model for -catenin regulation by HBx.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The Tcf reporter plasmids TOPFlash (optimal Tcf-binding site) and FOPFlash (mutated Tcf-binding site) were obtained from Upstate Biotechnology. For construction of -catenin-expressing plasmids, cDNA encoding either the wild-type or mutant form (S37A) of -catenin was inserted into BamHI and XbaI sites in pCMV-3xHA1 (Lee et al., 1998). Plasmid pHA-ubiquitin encoding HA-tagged ubiquitin was a gift from Y. Xiong (University of North Carolina at Chapel Hill, USA). Plasmid pSiah
1–75, which expresses a Siah-1 dominant-negative mutant (Siah-1DN), has been described previously (Matsuzawa & Reed, 2001). Both wild-type and mutant (R248Q) forms of p53-expressing plasmids were kindly provided by C.-W. Lee (Sungkyunkwan University, Korea).
Cell culture and transient transfection.
HepG2 (KCLB 58065) and Hep3B (KCLB 58064) were obtained from the Korean Cell Line Bank. Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10 % fetal calf serum. For transient expression, 2x105 cells per 60 mm diameter plate were transfected with 1 µg of appropriate plasmid(s) with the use of a Fugene 6 transfection kit (Roche) or WelFect-EX PLUS (WelGENE), following the manufacturer's instructions.
Luciferase assay.
Cells were transiently transfected with Tcf reporter plasmids along with effectors. To control the variation in transfection efficiency, 0.1 µg of pCH110 (Pharmacia) containing the Escherichia coli lacZ gene under the control of the simian virus 40 promoter was co-transfected as an internal control. After 48 h, cells were lysed in passive lysis buffer and luciferase activity was monitored in the cell lysate with the use of luciferase assay reagents (Promega) as described by the manufacturer. The value obtained was normalized to the -galactosidase activity measured in the corresponding cell extract.
Western blot analysis.
Cells were lysed in buffer [50 mM Tris/HCl (pH 8.0), 150 mM NaCl, 0.1 % SDS, 1 % NP-40] supplemented with protease inhibitors. Twenty micrograms of cell extract was separated by SDS-PAGE and transferred to a nitrocellulose membrane (Hybond PVDF; Amersham). Membranes were incubated with anti--catenin antibody (#610153; BD Transduction Laboratories); anti-Siah-1, anti-c-myc and anti-p53 antibodies (sc-5505, sc-40 and sc-126, respectively; Santa Cruz Biotechnology); anti-HA antibody (#11 583 816 001; Roche) and anti-tubulin antibody (T-6557; Sigma) and subsequently with the appropriate horseradish peroxidase-conjugated secondary antibodies: goat anti-mouse IgG(H+L)–HRP (#170-6516; Bio-Rad) and rabbit anti-goat IgG–HRP (sc-2768; Santa Cruz Biotechnology). An ECL kit (Amersham) was used to visualize protein bands.
Immunoprecipitation.
HepG2 cells were co-transfected with HA-tagged ubiquitin-encoding plasmid and increasing amounts of HBx-expressing plasmid as described above. Whole-cell lysates were pre-cleared with A/G PLUS-Agarose beads (Santa Cruz) for 30 min at 4 °C and incubated with anti-HA tag antibody (Santa Cruz) for an additional hour and then with beads overnight at 4 °C. After intensive washing and centrifugation, immune complexes were separated by SDS-PAGE and probed with anti--catenin antibody by Western blotting.
Semi-quantitative RT-PCR.
For RT-PCR, total cellular RNA was extracted from HepG2 cells with TRIzol reagent (Gibco). DNase I-digested RNA (3 µg) was reverse transcribed with the corresponding antisense primer. One-quarter of the reverse-transcribed RNA was amplified with Taq polymerase (95 °C for 5 min; 30 cycles at 95 °C for 1 min, 56 °C for 1 min and 72 °C for 30 s; 72 °C for 5 min) using the sense primers, 5'-ATGGCTACTCAAGCTGATTTG-3', 5'-GACTGGCACAACTGCATCCA-3', 5'-ACCGAATTCCCATGGCTGCT-3' and 5'-ATGGGGAAGGTGAAGGTCGG-3' and antisense primers 5'-TTACAGGTCAGTATCAAACCA-3', 5'-AGCCAAGTTGCGAATGGATC-3', 5'-AACTCTAGATGATTAGGCAGAGGT-3' and 5'-TGGAGGGATCTCGCTTG-3' for -catenin, Siah-1, HBx and glyceraldehyde 3-phosphate dehydrogenase, respectively.
RNA interference.
Based on the target sequence of siRNA for Siah-1 (5'-AACTCCTGCCTCCTTATGTATTT-3') and the non-silencing siRNA (control siRNA) sequence (5'-AAGAGCCGTCAGACTGCTACA-3'), siRNA duplexes were synthesized and purified by Qiagen. A plasmid-based RNA interference system (SilenCircle RNAi system; Allele Biotech.) was employed to knock down p53 gene expression. Based on the target sequence of siRNA for p53 (5'-GACTCCAGTGGTAATCTAC-3'), siRNA inserts composed of both sense and antisense sequences separated by a central common sequence were designed. The siRNA inserts were cloned into the pre-cut pSilenCircle vector.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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-catenin depending on the status of cellular p53-catenin, we transiently transfected an HBx expression plasmid (HBX3) (Kwun & Jang, 2004) into a p53-positive hepatoma cell line, HepG2 (Ahn et al., 2002). Both full-length and truncated forms of -catenin in HepG2 cells (de La Coste et al., 1998) were downregulated by HBx in a dose-dependent fashion (Fig. 1a, left). Interestingly, however, the opposite effect was observed in Hep3B cells, which are p53 negative (Park et al., 2000), showing that HBx upregulated the level of -catenin (Fig. 1a, right).
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-catenin by HBx resulted in corresponding changes in the transcriptional activity of -catenin, we transfected each cell line with reporter plasmids containing wild-type (TOPFlash) and mutant (FOPFlash) binding sites for Tcf (van de Wetering et al., 1991). As expected, HBx differentially regulated Tcf reporter activity in HepG2 and Hep3B cells (Fig. 1b
). In addition, HBx decreased the level of c-myc (a target gene of Tcf) in HepG2 cells but increased it in Hep3B cells in a dose-dependent manner (Fig. 1a). Thus, the alteration in the level of -catenin in the presence of HBx resulted in physiological changes in both cell lines.
To exclude the possibility that the opposing effects of HBx resulted from other genetic differences between the two cell lines, we examined whether the effect of HBx on -catenin could be altered in the same cells depending on the status of p53. We first attempted to knock down p53 in HepG2 cells by introducing a specific siRNA against p53. Under this condition, HBx could not decrease -catenin but slightly increased it (Fig. 1c, lane 3). Expression of p53 alone was sufficient to downregulate -catenin in Hep3B cells (Fig. 1c, lane 5). HBx could downregulate the levels of -catenin (Fig. 1c, lanes 7 and 8) and Tcf reporter activity (Fig. 1d) in Hep3B cells in the presence of p53. In contrast, a naturally occurring p53 mutant, R248Q, which has a fatal substitution in the DNA-binding domain at codon 248 (Arg
Gln) (Morgan et al., 2000) could not reverse the effect of HBx on -catenin in Hep3B cells (Fig. 1c, lanes 7 and 9), suggesting that a functional p53 is required for the downregulation of -catenin by HBx. Based on these observations, we concluded that HBx downregulates -catenin in a p53-dependent manner, whereas it upregulates -catenin in the absence of p53.
Differential regulation of -catenin by HBx variants depends on the status of p53
According to our previous report (Kwun & Jang, 2004), HBx natural variants have opposing effects on the expression of p21Waf1, a downstream target of p53. Thus, we compared the effects of two natural variants, HBX3 and hbx2, on the level of -catenin in HepG2 cells. According to Fig. 2 (a, b), both Tcf reporter activity and -catenin levels in HepG2 cells were differentially affected by the two HBx variants. As HBX3 but not hbx2 downregulated -catenin, we hypothesized that the unique amino acid residues in HBX3 are critical for the observed effect. Among the six amino acid residues that are different between HBXX3 and hbx2 (Fig. 2a), the two substitutions, S101P and K130M, are known to be critical for the differential regulation of p21Waf1 expression (Kwun & Jang, 2004). Therefore, we investigated whether these two amino acid residues were also responsible for the opposing effects on the level of -catenin. For this purpose, we employed three artificial hbx2 variants, each of which contained a substituted amino acid residue at position 101 and/or 130 in hbx2 (Fig. 2a). We found that only the hbx2 derivatives containing Ser-101 instead of Pro-101 (hbx2P101S and hbx2P101S/M130K) downregulated the -catenin level, to the level obtained with HBX3 (Fig. 2a, b). In addition, HBx variants with Ser-101 could upregulate p53 (Fig. 2b) because they can protect p53 from MDM2-mediated degradation (Kwun & Jang, 2004). Thus, the Ser-101 residue in HBx is critical for the downregulation of -catenin in HepG2 cells. In the absence of p53, however, all HBx variants examined upregulated -catenin (Fig. 2c). Taken together, we concluded that HBx variants differentially regulate -catenin depending on their potential to activate p53, as well on as the status of cellular p53.
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-catenin-catenin in HepG2 cells. As levels of -catenin transcripts were barely affected by HBx in either HepG2 or Hep3B cells (Fig. 3a), it is unlikely that HBx regulates -catenin expression at the transcription level. Instead, HBx may downregulate -catenin by stimulating its proteasomal degradation, as the effect of HBx on -catenin was almost completely abolished in the presence of a proteasome inhibitor, MG132 (Fig. 3b). As ubiquitination is usually a necessary step preceding proteasomal degradation (Ciechanover & Ben-Saadon, 2004), we examined whether HBx enhanced ubiquitination of -catenin. In our immunoprecipitation assay, HBx increased the amount of ubiquitin-complexed -catenin in a dose-dependent manner (Fig. 3c), indicating that HBx induces -catenin degradation at the level of ubiquitination or upstream of it.
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-independent degradation of -catenin-catenin through either the GSK-3-dependent or GSK-3-independent ubiquitin–proteasome system (Matsuzawa & Reed, 2001; Sadot et al., 2001). As part of the ligase complex, -TrCP1 recognizes -catenin as a substrate for ubiquitination only when it is phosphorylated by GSK-3 at both Ser-33 and Ser-37 residues (Calender et al., 1987). Indeed, the use of non-phosphorylable mutants of -catenin and the pharmacological inhibitor of GSK-3, LiCl, has demonstrated activation of -catenin through inhibition of its ubiquitin-dependent degradation (Everly et al., 2004; Jang et al., 2005). When a non-phosphorylable form of -catenin, S37A, which has Ala substituted for Ser at position 37, was introduced into HepG2 cells, it was more stable and transcriptionally more active than wild-type -catenin (Fig. 4a, b). Similar results were obtained in the presence of LiCl; inhibition of GSK-3 activity increased the wild-type -catenin protein levels (Fig. 4c), as well as Tcf reporter activity (Fig. 4d). Under this condition, the truncated form was barely affected, as the large deletion (aa 25–140) in the truncated -catenin removes the potential regulatory GSK-3 sites (de La Coste et al., 1998). Taken together, these data led to the conclusion that GSK-3-dependent degradation machinery for -catenin is active in HepG2 cells.
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-catenin (Fig. 4a, lane 4), as well as the transcriptional activity of the -catenin–Tcf complex (Fig. 4b, column 4). Moreover, HBx abolished protein accumulation and transcriptional activity of -catenin induced by inhibition of GSK-3 with LiCl (Fig. 4c, lane 4, and 4d, column 4). These results imply that, in addition to the GSK-3-dependent pathway, some other destruction machinery for -catenin is responsible for the observed effect.
HBx downregulates -catenin via the p53–Siah-1 pathway
Another pathway for -catenin degradation involves Siah ubiquitin ligases (Matsuzawa & Reed, 2001). In contrast to the GSK-3-dependent pathway, ubiquitination by Siah-1 does not require -catenin phosphorylation (Liu et al., 2001; Matsuzawa & Reed, 2001). In addition, it is known to mediate -catenin degradation initiated by p53 activation (Liu et al., 2001; Iwai et al., 2004; Matsuzawa & Reed, 2001). Therefore, we explored whether Siah-1 was involved in HBx-mediated -catenin downregulation. As a result, we found that, with increasing amounts of HBx, the protein levels of Siah-1 increased, which directly correlated with the levels of p53 (Fig. 5a, lanes 1–3). The addition of wild-type p53 resulted in a further increase in the level of Siah-1 (Fig. 5a, lane 4). Whilst the dominant-negative form of p53, R248Q, abolished Siah-1 activation mediated by HBx (Fig. 5a, lane 6), indicating that HBx activates Siah-1 expression via p53.
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). Therefore, we investigated whether HBx activated Siah-1 at the transcriptional level. According to a semi-quantitative RT-PCR assay, HBx increased the levels of Siah-1 mRNA in a dose-dependent manner (Fig. 5b). This effect was almost completely abolished by the addition of the dominant-negative form of p53, R248Q, indicating that activated p53 in the presence of HBx stimulated Siah-1 transcription.
To examine whether the ubiquitin ligase activity of Siah-1 was important for -catenin degradation mediated by HBx, we introduced a Siah-1-inactive mutant, Siah-1DN (Matsuzawa & Reed, 2001), into HepG2 cells. The functionally inert Siah-1 mutant almost completely regenerated the transcriptional activity of -catenin (Fig. 5d, compare columns 2 and 4), as well as protein levels (Fig. 5c, compare lanes 2 and 4) in the presence of HBx, indicating that Siah-1 ubiquitinating activity plays a critical role in downregulation of -catenin by HBx in these cells. In addition, we employed Siah-1-specific siRNA to knock down the expression of endogenous Siah-1. As a result, the inhibitory effect of HBx on -catenin was abolished when Siah-1 expression was silenced by the siRNA (Fig. 5e), consistent with the results of the Siah-1 dominant-negative experiment. Taken together, our results demonstrate that HBx participates in the destabilization of -catenin in human hepatocytes through the activation of Siah-1-mediated destruction.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, resulting in stabilization of -catenin (Desbois-Mouthon et al., 2001; Holnthoner et al., 2002). As HBx activates several growth signalling pathways (Benn et al., 1996; Lee & Yun, 1998; Shih et al., 2000), it may stabilize -catenin via one of these signalling pathways. Indeed, recently, it has been demonstrated that HBx can stabilize -catenin by suppressing GSK-3 activity via activation of Src (Cha et al., 2004). Activation of ERK signalling also primes GSK-3 inactivation, resulting in upregulation of -catenin (Ding et al., 2005). These studies suggest that HBx participates at least in part in the stabilization of -catenin in HCC. The present study, however, argues that the regulation of -catenin by HBx is not a simple pathway but rather a complex mechanism.
Responding to genotoxic stresses, p53 exhibits anti-proliferative effects through a variety of mechanisms (Levine, 1997). Downregulation of -catenin by stimulating its proteasomal degradation should be one such mechanism (Matsuzawa & Reed, 2001; Sadot et al., 2001). Several oncogenic proteins such as Myc, Ras, adenovirus E1A and -catenin are known to induce p53 (Albrechtsen et al., 1999; Janus et al., 1999; Levine, 1997). HBx also stabilizes p53 by protecting it from MDM2-mediated degradation (Kwun & Jang, 2004). The present study showed that HBx destabilizes -catenin through the Siah-1-mediated ubiquitin–proteasome pathway. HBx has been shown to upregulate both RNA and protein levels of Siah-1 (Fig. 5a, b). These effects were further augmented by the supplementation of wild-type p53. Based on these observations, we suggest that HBx upregulates Siah-1 expression via upregulation of p53 transcriptional activity. As Siah-1 is itself a target for ubiquitination and proteasomal degradation (Hu & Fearon, 1999), HBx might enhance the stability of Siah-1 protein. However, this possibility could be excluded because the upregulation of Siah-1 expression by HBx was almost completely abolished in the presence of a dominant-negative form of p53 (Fig. 5a, b).
Recently, Ding et al. (2005) reported the opposing observation that HBx upregulates levels of -catenin in HepG2 cells. We also observed that some HBx variants with Pro-101 instead of Ser-101, which cannot stabilize p53 (Kwun & Jang, 2004; Fig. 2b), increased the levels of -catenin in HepG2 cells (Fig. 2b). Thus, it is possible to speculate that HBx, depending on its sequence variation, can regulate -catenin in an opposing manner in the same type of cells. Moreover, all HBx variants examined in this study could increase -catenin in p53-negative cells. These results suggest that HBx can upregulate -catenin expression in a p53-independent pathway. This activation could be due to the inhibition of GSK-3 by HBx, as also demonstrated by other groups (Cha et al., 2004; Ding et al., 2005).
Based on the ubiquitin-dependent -catenin degradation pathways (Matsuzawa & Reed, 2001), we suggest a working model for -catenin regulation by HBx (Fig. 6). At least two ubiquitin–proteasome pathways for -catenin degradation are active in hepatoma cells. The GSK-3-independent pathway can induce degradation of both non-phosphorylable mutant and wild-type -catenin. HBx destabilizes -catenin by activating this pathway; the increase in p53 by HBx induces expression of Siah-1, which subsequently stimulates ubiquitin–proteasomal degradation of -catenin. In contrast, the alternative pathway requires GSK-3-mediated phosphorylation of -catenin and is responsible for the degradation of wild-type -catenin. HBx may stabilize -catenin by inhibiting GSK-3 activity through activation of growth signal transducers such as Src and Erk (Cha et al., 2004; Ding et al., 2005). Taken together, the balance between the opposite activities may determine the overall effect of HBx on -catenin expression.
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-catenin downregulation by HBx in vivo is unknown. Excessive wild-type p53 activity leads to a variety of cellular outcomes, most notably cell-cycle arrest and apoptosis, which can reduce the incidence of cancer through elimination of cancer-prone cells from the replicative pool (Levine, 1997). Thus, modulation of p53 and -catenin by HBx may simply reflect the host defence strategy to overcome oncogenic stress during an early stage of viral infection. In addition, the observation that the expression of p53 is increased in cases of chronic severe viral hepatitis (Elmore et al., 1997) suggests its possible role in the development of hepatitis, although it has not yet been verified in vivo. In any case, the downregulation of -catenin by HBx may serve as a selective pressure for loss of p53 activity during hepatocellular carcinogenesis.
During a long period of HBV replication in the liver of hepatitis patients, several mutations can accumulate in the HBx-coding region. Some HBx variants may lose their ability to stabilize p53, as demonstrated with some HBx variants in the present study. In addition, expression of HBx may enhance liver-cell susceptibility to carcinogen-induced mutagenesis, potentially through downregulation of DNA excision repair (Jia et al., 1999; Prost et al., 1998). Moreover, mutational inactivation of the p53 gene is very common (30–55 %) in HCCs (Sohn et al., 2000). Thus, mutation in either HBx or p53 can abolish the potential of HBx to activate Siah-1 expression. Indeed, Siah-1 has been shown to be significantly downregulated in advanced-stage tumours (Matsuo et al., 2003). Under this condition, HBx could induce stabilization and excessive accumulation of -catenin, which may induce rapid and uncontrolled cell proliferation that may further facilitate accumulation of more mutations in other oncogenes or tumour-suppressor genes, ultimately leading to HCC.
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ACKNOWLEDGEMENTS |
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Received 8 January 2007;
accepted 12 March 2007.
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