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The Hepatitis B Virus-X Protein Activates a Phosphatidylinositol 3-Kinase-dependent Survival Signaling Cascade*

  • Yoon Ik Lee
    Affiliations
    From the Liver Cell Signal Transduction Research Unit
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  • Sukmi Kang-Park
    Affiliations
    From the Liver Cell Signal Transduction Research Unit
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  • Su-Il Do
    Affiliations
    From the Liver Cell Signal Transduction Research Unit

    Animal Cell and Medical Glycobiology Research Unit, Bioscience Research Division, Korea Research Institute of Bioscience and Biotechnology, P. O. Box 115, Yusong, Taejon 305-600, Korea
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  • Young Ik Lee
    Correspondence
    To whom correspondence should be addressed:
    Affiliations
    From the Liver Cell Signal Transduction Research Unit
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  • Author Footnotes
    * This work was supported by grants from the Korean Science and Engineering Foundation (Science Research Center Fund) and Korea Ministry of Science and Technology (grant NBM0080031).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:May 18, 2001DOI:https://doi.org/10.1074/jbc.M011263200
      The hepatitis B virus-X (HBx) protein is known as a multifunctional protein that not only coactivates transcription of viral and cellular genes but coordinates the balance between proliferation and programmed cell death, by inducing or blocking apoptosis. In this study the role of the HBx protein in activation of phosphatidylinositol 3-kinase (PI3K) was investigated as a possible cause of anti-apoptosis in liver cells. HBx relieved serum deprivation-induced and pro-apoptic stimuli-induced apoptosis in Chang liver (CHL) cells. Treatment with 1-d-3-deoxy-3-fluoro-myo-inositol, an antagonist to PI3K, which blocks the formation of 3′-phosphorylated phosphatidyl inositol in CHL cells transformed by HBx(CHL-X) but not normal Chang liver (CHL) cells, showed a marked loss of viability with evidence of apoptosis. Similarly, treatment with wortmannin, an inhibitor of PI3K, stimulated apoptosis inHBx-transformed CHL cells but not in normal cells, confirming that HBx blocks apoptosis through the PI3K pathway. The serine 47 threonine kinase, Akt, one of the downstream effectors of PI3K-dependent survival signaling was 2-fold higher inHBx-transformed CHL (CHL-X) cells than CHL cells. Phosphorylation of Akt at serine 473 and Bad at serine 136 were induced by HBx, which were specifically blocked by wortmannin and dominant negative mutants of Akt and Bad, respectively. We also demonstrated that HBx inhibits caspase 3 activity and HBx down-regulation of caspase 3 activity was blocked by the PI3K inhibitor. Regions required for PI3K phosphorylation on the HBx protein overlap with the known transactivation domains. HBx blocks apoptosis induced by serum withdrawal in CHL cells in a p53-independent manner. The results indicate that, unlike other DNA tumor viruses that block apoptosis by inactivating p53, the hepatitis B virus achieves protection from apoptotic death through a HBx-PI3K-Akt-Bad pathway and by inactivating caspase 3 activity that is at least partially p53-independent in liver cells. Moreover, these data suggest that modulation of the PI3K activity may represent a potential therapeutic strategy to counteract the occurrence of apoptosis in human hepatocellular carcinoma.
      PI3K
      phosphatidylinositol 3-kinase
      HBV-X
      hepatitis B virus-X
      HCC
      hepatocellular carcinoma
      CAT
      chloramphenicol acetyl transferase
      CHL
      Chang liver cell
      TUNEL
      terminal deoxynucleotidyl transferase-mediated dUTP end labeling
      CHL-X
      Chang liver cell-X
      DMEM
      Dulbecco's modified Eagle's medium
      FBS
      fetal bovine serum
      DAPI
      4′,6′-diamidino-2-phenylindole
      PBS
      phosphate-buffered saline
      PIP2
      phosphatidylinositol 4,5-biphosphate
      PH
      pleckstrin homology
      SH2
      Src homology 2 domain
      CPP32
      caspase 3
      bp
      base pair(s)
      Apoptosis is an active process of self-destruction that is important in both the development and maintenance of multicellular organisms (
      • Clem R.J.
      • Fechheimer M.
      • Miller L.K.
      ). Several viral gene products affect apoptosis by interacting directly with components of the highly conserved biochemical pathways, which regulate cell death. Most viruses have evolved strategies to block or induce apoptosis depending upon the cellular environment (
      • Shen V.
      • Shenk T.E.
      ,
      • Teodoro J.G.
      • Branton P.E.
      ). The human immunodeficiency virus Tat (
      • Li C.J.
      • Freidman D.J.
      • Wang C.
      • Metelev V.
      • Pardee A.B.
      ) and the human T-cell leukemia virus Tax (
      • Yamada T.
      • Yamaoka S.
      • Goto T.
      • Nakai M.
      • Tsujimoto Y.
      • Hatanaka M.
      ) induce apoptosis. On the other hand, the IE1 and IE2 proteins of the cytomegalovirus (
      • Zhu H.
      • Shen Y.
      • Shenk T.
      ) have the ability to block apoptosis. EIA/EIB of the adenovirus (
      • Zhu H.
      • Shen Y.
      • Shenk T.
      ,
      • Lowe S.W.
      • Ruley H.E.
      ) or the T-antigen of SV40 (
      • Fromm L.
      • Shawlot W.
      • Gunning K.
      • Butel J.S.
      • Overbeek P.A.
      ,
      • McCarthy S.A.
      • Symonds H.S.
      • Dyke T.V.
      ) may either block or induce apoptosis. Phosphatidylinositol 3-kinase (PI3K)1 is recruited and activated during the intracellular signal transduction of many growth factor receptors and has been implicated in the signaling of survival factors (
      • Yao R.
      • Cooper G.M.
      ). Altered phosphorylation and turnover of phosphatidylinositol (PI) has also been demonstrated in cells transformed by DNA and RNA tumor viruses (
      • Jackowski S.
      • Voelker D.R.
      • Rock C.O.
      ). PI3K phosphorylates inositol lipids that act as second messengers for several targets, including the serine-threonine Akt kinase (
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Toker A.
      • Cantley L.C.
      ). Akt was initially described as an oncogene and is activated by serum and a variety of growth factors sharing the ability to activate the PI3K, such as platelet-derived growth factor, epidermal growth factor, bovine fibroblast growth factor, insulin (
      • Franke T.F.
      • Yang S.I.
      • Chan T.O.
      • Datta K.
      • Kazlauskas A.
      • Morrison D.K.
      • Kaplan D.R.
      • Tsichlis P.N.
      ,
      • Burgering B.M.
      • Coffer P.J.
      ,
      • Andjelkovic M.
      • Jakubowicz T.
      • Cron P.
      • Ming X.F.
      • Han J.W.
      • Hemmings B.A.
      ,
      • Kohn A.D.
      • Kovacina K.S.
      • Roth R.A.
      ), insulin-like growth factor (
      • Haslam R.J.
      • Koide H.B.
      • Hemmings B.A.
      ), and interleukin 2 (
      • Mayer B.J.
      • Ren R.
      • Clark K.L.
      • Baltimore D.
      ). Activation of Akt is known to deliver a survival signal that inhibits apoptosis induced by growth factor withdrawal (
      • Kauffmann-Zeh A.
      • Rodriguez-Viciana P.
      • Ulrich E.
      • Gilbert C.
      • Coffer P.
      • Downward J.
      • Evan G.
      ). Akt phosphorylates Bad, thereby blocking it from binding and inactivating Bcl-2 and Bcl-XL, two antiapoptotic Bcl-2 family members (
      • Ahmed N.N.
      • Grimes H.L.
      • Bellacosa A.
      • Chan T.O.
      • Tsichlis P.N.
      ), which eventually suppress apoptosis of transformed cells. Activation of Akt ultimately leads to inhibition of the caspase activity and protection from apoptotic cell death (
      • Ahmed N.N.
      • Grimes H.L.
      • Bellacosa A.
      • Chan T.O.
      • Tsichlis P.N.
      ). In cardiac muscle cells, the activation of PI3K has been identified to suppress caspase 3 activation and inhibit the occurrence of apoptosis (
      • Wu W.
      • Lee W.L.
      • Wu Y.Y.
      • Chen D.
      • Lin T.
      • Jang A.
      • Sharma P.H.
      • Wang P.H.
      ). HBx is a causative agent of hepatocellular carcinoma, and during tumorigenesis, the HBx gene product is known to play an important role in the alteration of gene expression, sensitizing cells to apoptotic killing and deregulating cell growth arrest (
      • Su F.
      • Schneider R.J.
      ,
      • Benn J.
      • Schneider R.J.
      ). HBx has been shown to complex with p53, inhibiting sequence-specific DNA binding in vitro and p53-mediated transcriptional activationin vivo (
      • Wang Y.
      • Okan I.
      • Pokrovskaja K.
      • Wiman K.G.
      ), which eventually inhibits p53-dependent apoptosis. HBx was also shown to block apoptosis (
      • Wang X.W.
      • Gibson M.K.
      • Vermeulen W.Y.H.
      • Forrester K.
      • Stuzbecher H.W.
      • Hoeijmakers J.H.J.
      • Harris C.C.
      ) and induce transformation in NIH3T3 cells (
      • Shirakata Y.
      • Kawada M.
      • Fujiki Y.
      • Sano H.
      • Oda M.
      • Yaginuma K.
      • Kobayashi M.
      • Koike K.
      ). Besides the anti-apoptotic effect, HBx has also been shown to induce apoptosis in hepatoma cells (
      • Kim H.
      • Lee H.
      • Yun Y.
      ). Therefore, the fate of infected cells expressing HBx is likely to be determined by the balance between apoptotic and anti-apoptotic signals of viral, cellular, and environmental origin. The ability of HBx to modulate cell survival is potentially relevant for both viral pathogenicity in acute and chronic HBV infection as well as for the late development of hepatocellular carcinoma. In the long term, it is the anti-apoptotic function of HBx that is likely to be the major determinant for manifestation of the transformed phenotype. From this perspective, it is of central importance to define the molecular mechanisms underlying HBx-induced dysregulation of the cell death program. In this study we show that HBx activates PI3K and activated PI3K can lead to phosphorylation of Akt and Bad in CHL cells in a p53-independent manner. We also demonstrate that HBx suppressed the activation of caspase 3, through PI3K signaling, and suppresses apoptosis in CHL cells. In conclusion, we demonstrate that HBx acts as an anti-apoptosis agent through the HBx-PI3K-Akt-Bad pathway and inactivates caspase 3 activity in a PI3K-dependent manner that is at least partially p53 independent.

      DISCUSSION

      Phosphatidylinositol 3-kinase has been shown to mediate signaling induced by numerous growth factors and tumor antigens (
      • Auger K.R.
      • Serunian L.A.
      • Saltoff S.P.
      • Libby P.
      • Cantley L.C.
      ,
      • Carpenter C.L.
      • Cantley L.C.
      ). PI3K is a heterodimeric complex consisting of an 85-kDa regulatory subunit, p85, and a 110-kDa catalytic subunit, p110 (
      • Carpenter C.L.
      • Cantley L.C.
      ,
      • Kapeller R.
      • Cantley L.C.
      ). The p85 subunit contains two Src homology 2 (SH2) domains, which bind to tyrosine-phosphorylated receptors after stimulation of cells with growth factors and, in this manner, recruit the p85·p110 complex to the cell membrane. PI3K appears to induce a variety of cellular responses. These responses include the regulation of gene expression (
      • Beretta L.
      • Gingras A.C.
      • Svitkin Y.V.
      • Hall M.N.
      • Sorenberg N.
      ,
      • Hu Q.
      • Klippel A.
      • Muslin W.
      • Fantl W.J.
      • Williams L.T.
      ) and the activation of signaling kinases, which function in different pathways (
      • Didichemko S.A.
      • Tilton B.
      • Hemmings B.A.
      • Ballmer K.
      • Thelen M.
      ,
      • Klippel A.
      • Reinhard C.
      • Kavanagh M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ) as well as in membrane ruffling (
      • Martin S.S.
      • Rose D.W.
      • Saltiel A.R.
      • Klippel A.
      • Williams L.T.
      • Olefsky J.M.
      ), endocytosis (
      • Li G.
      • D'Souza-Schorey C.
      • Barbieri M.A.
      • Roberts R.L.
      • Klippel A.
      • Williams L.T.
      • Stahl P.D.
      ), glucose transport, and DNA synthesis (
      • Frevert E.U.
      • Kahn B.B.
      ,
      • Martin S.S.
      • Haruta T.
      • Morris A.J.
      • Klippel A.
      • Williams L.T.
      • Olefsky J.M.
      ). Recently, it was demonstrated that PI3K regulates cell survival in response to various apoptotic stimuli (
      • Marte B.M.
      • Downward J.
      ). Much evidence suggests that activation of PI3K may lead to suppress caspase 3 activity and DNA fragmentation in a variety of cells. Most of the evidence derived from studies using pharmacological inhibitors of PI3K (LY294002 and wortmannin) shows that inhibition of PI3K with these compounds leads to cell apoptosis or loss of the anti-apoptotic effect of growth factors (
      • Crowder R.J.
      • Freeman R.S.
      ,
      • Kulik G.
      • Weber M.
      ,
      • Ryu B.R.
      • Ko H.W.
      • Jou I.
      • Noh J.S.
      • Gwag B.J.
      ). Several other studies using dominant negative constructs of PI3K to inhibit PI3K signaling confirmed that PI3K is essential in maintaining cell survival (
      • Ryu B.R.
      • Ko H.W.
      • Jou I.
      • Noh J.S.
      • Gwag B.J.
      ,
      • Buerke M.
      • Murohara T.
      • Skurk C.
      • Nuss C.
      • Tomascelli K.
      • Lefer A.H.
      ). Recently, Wu et al. (
      • Wu W.
      • Lee W.L.
      • Wu Y.Y.
      • Chen D.
      • Lin T.
      • Jang A.
      • Sharma P.H.
      • Wang P.H.
      ) demonstrated that activation of PI3K alone is sufficient to protect cells from apoptotic induction and inhibition of caspase activation in cardiac muscle cells. By using either in vivo or cell free systems, it was shown that the phospholipids produced by PI3K mediate PI3K signaling (
      • Klippel A.
      • Reinhard C.
      • Kavanagh M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ,
      • Klippel A.W.
      • Kavanaugh W.M.
      • Pot D.
      • Williams L.T.
      ). One of the generated products phosphatidylinositol 3,4-diphosphate (
      • Klippel A.
      • Reinhard C.
      • Kavanagh M.
      • Apell G.
      • Escobedo M.A.
      • Williams L.T.
      ,
      • Swart R.
      • Ruf I.K.
      • Sample J.
      • Longnecker R.
      ), was able to induce the kinase activity of the PI3K effector Akt (protein kinase B)in vitro ∼10-fold (
      • Franke T.F.
      • Kaplan D.R.
      • Cantley L.C.
      • Toker A.
      ,
      • Marte B.M.
      • Downward J.
      ). Signaling intermediates, which bind phospholipids via domains homologous to pleckstrin such as G-protein exchange factors and GTPase-activating proteins, are also candidates for regulation by the products of PI3K (
      • Han J.
      • Luby-Phelps K.
      • Das B.
      • Shu X.
      • Xia Y.
      • Mosteller U.M.
      ,
      • Nimnual A.S.
      • Yatsula B.A.
      • Bar-Sagi D.
      ). Among these, the downstream target of PI3K that mediates the anti-apoptotic action of PI3K is Akt. The Akt pathway modulates sequential steps of apoptotic signaling. Activation of Akt may lead to phosphorylation of Bad (
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ), induction of Bcl-2 family of proteins (
      • Matsuzaki H.
      • Tamatani M.
      • Mitsuda N.
      • Nanmikawa K.
      • Kiyama H.
      • Miyake S.
      • Tohyama M.
      ), inhibition of cytochrome c release from mitochondria (
      • Kennedy S.G.
      • Kandel E.S.
      • Cross T.K.
      • Hay N.
      ) and phosphorylation and inactivation of caspase 9 (
      • Fujita E.
      • Jinbo A.
      • Matuzaki H.
      • Konishi H.
      • Kikkawa U.
      • Momoi T.
      ). However, some of these observations are dependent on the experimental system used. It appears that survival signaling may employ different antagonistic mechanisms in different cells. In cardiomyocytes the survival signals induced by insulin-like growth factor-I did not induce Bad phosphorylation, although they stimulated PI3K and Akt. In this case insulin-like growth factor-I induces anti-apoptosis in cardiomyocytes through PI3K/Akt signaling, which is independent of Bad (
      • Wu W.
      • Lee W.L.
      • Wu Y.Y.
      • Chen D.
      • Lin T.
      • Jang A.
      • Sharma P.H.
      • Wang P.H.
      ). In this study 1-d-3-deoxy-3-fluoro-myo-inositol, a potent antagonist of cells exhibiting constitutively activated PI3K (
      • Freuned R.
      • Dawe C.J.
      • Carroll J.P.
      • Benjamin T.L.
      ), and the PI3K inhibitor wortmannin was used. Apoptosis in CHL cells transformed by HBx is shown to be enhanced when PI3K activity is inhibited by either growth of cells with 1-d-3-deoxy-3-fluoro-myo-inositol or after treatment with wortmannin, indicating involvement of PI3K in blocking apoptosis. In contrast, normal CHL cells are much less dependent on PI3K for survival, as indicated by their resistance to death induced by the inositol analogue and by wortmannin. These results suggest that signal transduction via HBx through PI3K is important for survival and protection from apoptosis. HBx also activates and phosphorylates Akt and Bad through PI3K, because PI3K inhibitors block HBx activation and phosphorylation of Akt and Bad. These results suggest that HBx utilizes the PI3K/Akt/Bad survival pathway in the CHL cell line. Caspase 3 plays a pivotal role in execution of apoptosis; ES cells deficient in caspase 3 were resistant to apoptotic induction (
      • Colussi P.A.
      • Kumar S.
      ). In human cardiomyopathy, apoptosis of cardiac muscle cells is associated with release of cytochrome c and activation of caspase 3 (
      • Narula J.
      • Pandey P.
      • Arbustini E.
      • Haider N.
      • Narula N.
      • Kolodgie F.D.
      • Dal Bello B.
      • Semigran M.J.
      • Bielsa-Masdeu A.
      • Dec G.W.
      • Israels S.
      • Ballester M.
      • Virmani R.
      • Saxena S.
      • Kharbanda S.
      ). Using inhibitors of caspases, Bialik et al.(
      • Bialik S.
      • Cryns V.L.
      • Drincic A.
      • Miyata S.
      • Wollowick A.L.
      • Srinivasan A.
      • Kitsis R.U.
      ) showed that cardiomyocyte apoptosis induced by serum and glucose withdrawal can be attenuated. Our data show that HBx blocks caspase 3 activation during serum deprivation and caspase 3 activity is activated by the PI3K inhibitors, wortmannin, and LY294002 as shown in Fig. 6. These results imply that activation of caspase 3 during serum-deprived apoptosis is attenuated by HBx through the PI3K signaling pathway. The ability of a virus to delay host cell death is essential for viral growth (
      • Razvi E.S.
      • Welsh R.M.
      ). Some DNA viruses, such as the adenovirus and SV40, inhibit programmed cell death by inactivating p53. On the other hand, other DNA tumor viruses like the polyoma virus that has no known direct interaction with p53 (
      • Doherty J.
      • Frenun R.
      ) acts through PI3K and Akt and blocks apoptosis through the middle T-antigen (
      • Doherty J.
      • Frenun R.
      ). Interestingly, viruses that handle p53 directly by inactivation or degradation (i.e. adenovirus, SV40, and papilloma virus) lack direct mechanisms for activating PI3K. It has been proposed that a pro-apoptotic effect facilitates viral spread and allows for evasion of host-cell-mediated immunity (
      • Lau J.Y.N.
      • Xie X
      • Lai M.M.C.
      • Wu P.C.
      ). On the contrary, the inhibition of apoptosis would have the effect of allowing for the accumulation of potentially transforming mutations (
      • Lau J.Y.N.
      • Xie X
      • Lai M.M.C.
      • Wu P.C.
      ). The apparent discrepancy between the effects of HBx on apoptosis could reflect opposing concentration-dependent effects at different stages of natural HBV infection. A possibility is that HBx inhibits apoptosis during early hepatocyte infection and later on activates apoptosis to facilitate HBV replication and spread. Either stimulation or inhibition of apoptosis could lead to malignant transformation of hepatocytes. In HBV, the interactions between the HBx protein and p53 have been examined extensively (
      • Wang X.W.
      • Gibson M.K.
      • Vermeulen W.Y.H.
      • Forrester K.
      • Stuzbecher H.W.
      • Hoeijmakers J.H.J.
      • Harris C.C.
      ), although the activation of PI3K by HBx protein has not been reported. In this study, we identified that the activation of PI3K and subsequently of Akt and Bad by the HBx protein eventually causes anti-apoptosis in CHL cells. The PI3K activation domains in the HBx protein are similar to the promoter transactivation domains as showed in our previous study (
      • Kim Y.H.
      • Kang S.K.
      • Lee Y.I.
      ). CHL cells showed marked serum dependence for survival. In CHL cells expressing HBx proteins, significant protection against apoptosis upon serum withdrawal was identified. After transfection with p53val135, this protection was temperature-independent, implying that HBx-induced anti-apoptosis is p53-independent. Consistent with our findings, p53 independence of anti-apoptosis induced by serum withdrawal has also been observed in other cell types (
      • Auger K.R.
      • Serunian L.A.
      • Saltoff S.P.
      • Libby P.
      • Cantley L.C.
      ). Although some experimental evidence showed HBx inactivation of p53 in hepatocytes, the p53 independence of anti-apoptosis by the HBx protein through the PI3K-Akt-Bad pathway and inactivation of caspase 3 implies that HBx functions through a p53-independent pathway in inducing an anti-apoptosis phenomena in CHL cells.

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