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Hepatitis B Virus X Protein Inhibits Transforming Growth Factor-β-induced Apoptosis through the Activation of Phosphatidylinositol 3-Kinase Pathway*

Open AccessPublished:August 18, 2000DOI:https://doi.org/10.1074/jbc.M003578200
      Transforming growth factor-β (TGF-β) is a potent inducer of apoptosis in Hep 3B cells. This work investigated how hepatitis B virus X protein (HBx) affects TGF-β-induced apoptosis. Trypan blue exclusion and colony formation assays revealed that HBx increased the ID50 toward TGF-β. In the presence of HBx, TGF-β-induced DNA laddering was decreased, indicating that HBx had the ability to block TGF-β-induced apoptosis. Furthermore, HBx did not alter the expression levels of type I and type II TGF-β receptors. HBx did not affect TGF-β-induced activation of promoter activities of the plasminogen activator inhibitor-1 (PAI-1) gene. These results indicate that HBx interferes with only a subset of TGF-β activity. In the presence of phosphatidylinositol (PI) 3-kinase inhibitors, wortmannin or LY294002, the HBx-mediated inhibitory effect on TGF-β-induced apoptosis was alleviated. In addition, the tyrosine phosphorylation levels of the regulatory subunit p85 of phosphatidylinositol 3-kinase (PI 3-kinase) and PI 3-kinase activity were elevated in stable clones with HBx expression. Transactivation-deficient mutants of HBx lost their ability to inhibit TGF-β-induced apoptosis. Phosphorylation of the p85 subunit of PI 3-kinase and Akt, a downstream target of PI 3-kinase, was not observed in stable clones with transactivation-deficient HBx mutant's expression. Thus, the anti-apoptotic effect of HBx against TGF-β can be mediated through the activation of the PI 3-kinase signaling pathway, and the transactivation function of HBx is required for its anti-apoptosis activity.
      HBx
      hepatitis B virus X
      ERK
      extracellular signal-regulated kinase
      MEKK
      mitogen-activated protein kinase (MEK)/ERK kinase
      JNK
      Jun N-terminal kinase
      Jak
      Janus kinase
      STAT
      signal transducers and activators of transcription
      TGF-β
      transforming growth factor-β
      PI
      phosphatidylinositol
      GFP
      green fluorescence protein
      PAI-1
      plasminogen activator inhibitor-1
      PMT
      polyoma middle T antigen
      Hepatitis B virus X protein (HBx)1 has been demonstrated to function as a transcriptional transactivator of a variety of viral and cellular promoter/enhancer elements (
      • Yen T.S.B.
      ,
      • Murakami S.
      ). Although not binding directly to DNA, HBx can transactivate transcription through multiple cis-acting elements including AP-1, AP-2, ATF/CREB, NF-κB, C/EBP, and Egr-1 binding sites. However, the exact mechanism of transactivation still remains unsolved. Previous investigations have demonstrated that HBx interacts in the nucleus with components of the basal transcription machinery, including RPB5, a subunit of all three mammalian RNA polymerases, and several transcription factors (
      • Cheong J.
      • Yi M.
      • Lin Y.
      • Murakami S.
      ,
      • Qadri I.
      • Maguire H.F.
      • Siddiqui A.
      ,
      • Haviv I.
      • Shamay M.
      • Doitsh G.
      • Shaul Y.
      ,
      • Lin Y.
      • Nomura T.
      • Cheong J.
      • Dorjsuren D.
      • Iida K.
      • Murakami S.
      ). Thus, HBx may exert its effect by mimicking the cellular coactivator function. Another proposed mechanism for HBx activity involves the activation of signal transduction pathways such as the Ras/Raf/ERK, and MEKK-1/JNK cascades, leading to the induction of AP-1, NF-κB, and probably other transcription factors (
      • Benn J.
      • Schneider R.J.
      ,
      • Natoli G.
      • Avantaggiati M.L.
      • Chirillo P.
      • Puri P.
      • Ianni A.
      • Balsano C.
      • Levrero M.
      ,
      • Doria M.
      • Klein N.
      • Lucito R.
      • Schneider R.J.
      ,
      • Chirillo P.
      • Falco M.
      • Puri P.L.
      • Artini M.
      • Balsano C.
      • Levrero M.
      • Natoli G.
      ,
      • Su F.
      • Schneider R.J.
      ,
      • Klein N.P.
      • Schneider R.J.
      ). HBx has been discovered to be distributed not only in the cytoplasm but also to some extent in the nucleus of transfected cells (
      • Doria M.
      • Klein N.
      • Lucito R.
      • Schneider R.J.
      ). Thus, HBx may have a dual function: one, related to its cytoplasmic localization, which can mediate the activation of signal transduction pathways, and another, a nuclear function, that may account for the interaction with transcription factors and components of the transcription apparatus to enhance the binding or activity of these proteins (
      • Doria M.
      • Klein N.
      • Lucito R.
      • Schneider R.J.
      ). In addition to its well known transcriptional transactivation ability through interaction with different cellular targets, HBx has been reported to affect DNA repair (
      • Lee T.H.
      • Elledge S.J.
      • Butel J.S.
      ,
      • Sitterlin D.
      • Lee T.H.
      • Prigent S.
      • Tiollais P.
      • Butel J.S.
      • Transy C.
      ,
      • Becker S.A.
      • Lee T.H.
      • Butel J.S.
      • Slagle B.L.
      ,
      • Koike K.
      • Moriya K.
      • Yotsuyanangi H.
      • Iino S.
      • Kurokawa K.
      ,
      • Benn J.
      • Su F.
      • Schneider R.J.
      ), cell cycle control (
      • Wang X.W.
      • Gibson M.K.
      • Vermenlen W.
      • Forrester K.
      • Sturzbecher H.W.
      • Hoeijmaker J.H.J.
      • Harris C.C.
      ,
      • Chirillo P.
      • Pagano S.
      • Natoli G.
      • Puri P.L.
      • Burgio U.L.
      • Balsano C.
      • Levrero M.
      ), and apoptosis (
      • Su F.
      • Schneider R.J.
      ,
      • Kim H.
      • Lee H.
      • Yun Y.
      ,
      • Gottlob K.
      • Fulco M.
      • Levrero M.
      • Graessmann A.
      ,
      • Bergametti F.
      • Prigent S.
      • Luber B.
      • Benoit A.
      • Tiollais P.
      • Sarasin A.
      • Transy C.
      ). Therefore, the pleiotropic activities of HBx are potentially relevant to the development of hepatocellular carcinoma.
      Transforming growth factor-β (TGF-β) is a potent inducer of apoptosis in hepatocytes and several hepatoma cell lines (
      • Lin J.-K.
      • Chou C.-K.
      ,
      • Oberhammer F.A.
      • Pavelka M.
      • Sharma S.
      • Tiefenbacher R.
      • Purchio A.F.
      • Bursch W.
      • Flermann R.S.
      ,
      • Fan G.
      • Ma X.
      • Kren B.T.
      • Steer C.J.
      ). TGF-β exerts its action through transmembrane serine/threonine kinase receptors. These receptors propagate the signal by phosphorylating the intracellular targets, Smads. Phosphorylated Smad2 or Smad3 can form a stable complex with Smad4, which then translocates to the nucleus to regulate the transcriptional response to TGF-β (
      • Heldin C.H.
      • Miyazono K.
      • ten Dijke P.
      ,
      • Zhang Y.
      • Derynck R.
      ). However, the mechanism(s) whereby TGF-β induces apoptosis is not fully characterized. Nevertheless, induction of oxidative stress (
      • Sanchez A.
      • Alvarez A.M.
      • Benito M.
      • Fabregat I.
      ), activation of caspase 3 (
      • Chen R.-H.
      • Chang T.-Y.
      ), and inhibition of Rb expression (
      • Fan G.
      • Ma X.
      • Kren B.T.
      • Steer C.J.
      ) have been implicated in mediating TGF-β-induced apoptosis. In liver cells, insulin and insulin-like growth factor-1 (
      • Tanaka S.
      • Wands J.R.
      ), as well as interleukin-6 (
      • Chen R.-H.
      • Chang M.-C.
      • Su Y.-H.
      • Tsai Y.-T.
      • Kuo M.-L.
      ), all block TGF-β-induced apoptosis. Recent studies have revealed that phosphatidylinositol 3-kinase (PI 3-kinase) and its downstream target, Akt, are responsible for the anti-apoptotic activity of these factors against TGF-β (
      • Chen R.-H.
      • Chang M.-C.
      • Su Y.-H.
      • Tsai Y.-T.
      • Kuo M.-L.
      ,
      • Chen R.-H.
      • Su Y.-H.
      • Chuang R.L.C.
      • Chang T.-Y.
      ).
      To elucidate the correlation between the HBx gene and its response to apoptotic stimuli, the effect of HBx gene expression on TGF-β-induced apoptosis in the Hep 3B cell line was examined. Cells with constitutive or inducible expression of wild or mutant HBx were generated and tested. Transactivation-proficient HBx inhibited TGF-β-induced apoptosis. The PI 3-kinase/Akt signaling pathway was involved in the HBx-mediated anti-apoptotic effect.

      DISCUSSION

      This study has demonstrated that HBx effectively suppresses TGF-β-induced apoptotic death of hepatoma cells. The HBx-mediated anti-apoptotic effect was not mediated through decreased expression of TGF-β receptors. Two specific inhibitors of PI 3-kinase, wortmannin and LY294002, blocked the anti-apoptotic effect of HBx, implying that HBx might affect the PI 3-kinase signaling pathway in mediating the effect. In cells expressing HBx, the PI 3-kinase activity and not its protein level was elevated. An increased phosphorylation of Akt at Ser-473 resulted. The anti-apoptotic mechanism of HBx was attributed, at least in part, to the activation of PI 3-kinase signaling cascades.
      The effects of HBx on cell death or apoptosis have been studied by several groups. p53-dependent apoptosis was prevented by microinjection of HBx into primary fibroblasts (
      • Wang X.W.
      • Gibson M.K.
      • Vermenlen W.
      • Forrester K.
      • Sturzbecher H.W.
      • Hoeijmaker J.H.J.
      • Harris C.C.
      ). Chirillo et al. (
      • Chirillo P.
      • Pagano S.
      • Natoli G.
      • Puri P.L.
      • Burgio U.L.
      • Balsano C.
      • Levrero M.
      ) demonstrated that after DNA damage, HBx induced p53-dependent apoptosis in NIH3T3 cells transiently expressing HBx. In Chang liver cells, HBx failed to induce apoptosis; however, it did sensitize cells to apoptosis triggered by TNF-α (
      • Su F.
      • Schneider R.J.
      ). Upon the induction of HBx expression mediated by theCre/loxP recombination system, liver cell apoptosis was observed independently of the p53 pathway (
      • Shintani Y.
      • Yotsuyanangi H.
      • Moriya K.
      • Fujie H.
      • Tsutsumi I.
      • Kanegae Y.
      • Kimura S.
      • Saito I.
      • Koike K.
      ). The liver cells derived from a transgenic mouse were more susceptible to diverse apoptosis insults, and this phenomenon was not dependent upon p53 (
      • Terradillos O.
      • Pollicino T.
      • Lecoeur H.
      • Tripodi M.
      • Gougeon M.L.
      • Tiollais P.
      • Buendia M.A.
      ). These seemingly contradictory results of HBx on apoptotic events might be attributable to the utilization of different cells and expression systems. However, these results suggest that HBx affects the apoptotic processes by multiple mechanisms, including the inactivation of the p53 functions, interference of DNA repair ability, or modulation of cellular signaling cascades. Our findings on the modulation of PI 3-kinase signaling by HBx in mediating its anti-apoptotic effect offered a new mechanism.
      The molecular mechanism by which HBx activates PI 3-kinase is addressed hereafter. Because of its well known transactivation function through the increased expression of cytokines or cognate receptors, HBx might establish an autocrine or paracrine loop. HBx was reported to stimulate the expression of cytokines (e.g. interleukin-6 (
      • Lee Y.
      • Park U.S.
      • Choi I.
      • Yoon S.K.
      • Park Y.M.
      • Lee Y.I.
      ) and insulin-like growth factor-II (
      • Lee Y.I.
      • Lee S.
      • Lee Y.
      • Bong Y.S.
      • Hyun S.W.
      • Yoo Y.D.
      • Kim S.J.
      • Kim Y.W.
      • Poo H.R.
      )) as well as cytokine receptors (e.g. insulin-like growth factor-I receptor (
      • Kim S.O.
      • Park J.G.
      • Lee Y.I.
      ) and epidermal growth factor receptor (
      • Menzo S.
      • Clementi M.
      • Alfani E.
      • Bagnarelli P.
      • Lacovacci S.
      • Manzin A.
      • Dandri M.
      • Natoli G.
      • Levrero M.
      • Carloni G.
      )). Interleukin-6 was demonstrated to inhibit apoptosis through the PI 3-kinase signaling pathway in hepatoma cells (
      • Chen R.-H.
      • Chang M.-C.
      • Su Y.-H.
      • Tsai Y.-T.
      • Kuo M.-L.
      ). The phosphorylated tyrosine residues, generated on receptors (e.g. epidermal growth factor receptor) or their associated substrate molecules (such as IRS-1/2 in signaling by insulin and insulin-like growth factor), form the docking sites for the Src homology-2 domains of p85. The interaction mediates the translocation of PI 3-kinase to the receptor tyrosine kinases and their substrate and assists in positioning p110, the catalytic subunit of PI 3-kinase, close to the membranes that contain the lipid substrates. Using Northern blot analysis or a ribonuclease protection assay, HBx did not alter the mRNA level of interleukin-6, TNF-α, or interferon-β (data not shown). The addition of media collected from the overnight culture of HBx-expressing cells did not protect Hep 3B cells from TGF-β-induced apoptosis (data not shown). Although not excluded, the hypothesis that the observed activation of PI 3-kinase by HBx might be due to the secondary results of the primary activation of cytokines or growth factors was not favored.
      Rather, HBx might work through the modulation of signaling cascades to activate PI 3-kinase. Related investigations reported that HBx modulates several signaling cascades (
      • Benn J.
      • Schneider R.J.
      ,
      • Natoli G.
      • Avantaggiati M.L.
      • Chirillo P.
      • Puri P.
      • Ianni A.
      • Balsano C.
      • Levrero M.
      ,
      • Doria M.
      • Klein N.
      • Lucito R.
      • Schneider R.J.
      ,
      • Chirillo P.
      • Falco M.
      • Puri P.L.
      • Artini M.
      • Balsano C.
      • Levrero M.
      • Natoli G.
      ,
      • Su F.
      • Schneider R.J.
      ,
      • Klein N.P.
      • Schneider R.J.
      ). Benn and Schneider (
      • Benn J.
      • Schneider R.J.
      ) indicated that HBx activated Ras-GTP complex formation. Activation of Src family kinases was demonstrated to be indispensable for HBx-mediated activation of Ras (
      • Klein N.P.
      • Schneider R.J.
      ). The activated Ras-GTP complex binds to p110, the catalytic subunit of PI 3-kinase, resulting in the activation of PI 3-kinase (
      • Rodriguez-Viciana P.
      • Warne P.H.
      • Dhand R.
      • Vanhaesebroeck B.
      • Gout I.
      • Fry M.J.
      • Waterfield M.D.
      • Downward J.
      ). The binding of the Src homology-3 domain of Src family kinases to a proline-rich region within the p85 of PI 3-kinase resulted in the activation of PI 3-kinase (
      • Pleiman C.M.
      • Marc Hertz W.
      • Cambier J.C.
      ). Lately, HBx has been shown to interact with Jak1 and activate Jak-STAT signaling (
      • Lee Y.H.
      • Yun Y.
      ). Both Jak (
      • Sharfe N.
      • Dadi H.K.
      • Roifman C.M.
      ,
      • Oh H.
      • Fujio Y.
      • Kunisada K.
      • Hirota H.
      • Matsui H.
      • Kishimoto T.
      • Yamauchi-Takihara K.
      ,
      • Al-Shami A.
      • Naccache P.H.
      ) and STAT (
      • Pfeffer L.M.
      • Mullersman J.E.
      • Pfeffer S.R.
      • Murti A.
      • Shi W.
      • Yang C.H.
      ) interact with p85 and activate PI 3-kinase signaling pathway. Therefore, through the activation of Ras, Src family kinases, or JAK-STAT, HBx might be able to achieve its effect on PI 3-kinase. In addition, HBx might directly activate PI 3-kinase. In a glutathione S-transferase pull-down assay, HBx was found to be associated with p110 (data not shown). The contribution of this interaction in the observed elevation of PI 3-kinase activity requires further investigation.
      It is noteworthy that wortmannin and LY294002 partially blocked the anti-apoptotic activity of HBx (Fig. 4). Although activation of the PI 3-kinase/Akt signaling pathway mediated the observed phenomenon, the interference of other molecules by HBx cannot be excluded. A recent investigation confirmed that HBx can inhibit caspase 3 activity (
      • Gottlob K.
      • Fulco M.
      • Levrero M.
      • Graessmann A.
      ). Chen and Chang (
      • Chen R.-H.
      • Chang T.-Y.
      ) reported that caspase 3 was involved in TGF-β-induced apoptosis. However, the contribution of the inhibitory effect of HBx on caspase 3 in our system remains to be elucidated.
      Polyoma middle T antigen (PMT) is identified as the tumorigenic component of the polyoma virus. PMT forms a complex with pp60c-src and PI 3-kinase, subsequently activating PI 3-kinase (
      • Courtneidge S.A.
      • Heber A.
      ). Several studies have inferred that a PI 3-kinase signaling pathway is required for PMT-mediated tumorigenesis (
      • Whitman M.
      • Kaplan D.R.
      • Schaffhausen B.
      • Cantley L.
      • Roberts T.M.
      ,
      • Dahl J.
      • Jurczak A.
      • Cheng L.A.
      • Baker D.C.
      • Benjamin T.C.
      ). Our study clearly demonstrated the activation of PI 3-kinase signaling by HBx, subsequently triggering anti-apoptotic signaling. As with PMT, inhibition of apoptosis by HBx could disrupt the normal cellular surveillance mechanism for removing damaged cells, thereby providing a clonal selective advantage for hepatocytes expressing this integrated viral gene during the early stages of human liver carcinogenesis. Mutations that affected the transactivation activity of HBx inhibited its ability to activate PI 3-kinase/Akt signaling pathway and failed to block apoptosis. These observations indicate that transactivation and anti-apoptotic activity of HBx are linked. However, Gottlob et al. (
      • Gottlob K.
      • Pagano S.
      • Levrero M.
      • Graessmann A.
      ) have reported that transactivation activity is not required for the transforming activity of HBx in REV2 cells. Therefore, additional studies are required to further define whether PI 3-kinase activation and subsequently anti-apoptotic activity are a prerequisite for HBx-mediated transformation.

      Acknowledgments

      We thank Dr. Rey-Hwa Chen for valuable comments and Dr. H.-F. Yang-Yen for providing helpful instructions on the PI 3-kinase assay. We also acknowledge Mr. Ted Knoy for revision of the manuscript.

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