FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2061369977> ?p ?o ?g. }

• W2061369977 endingPage "22803" @default.
• W2061369977 startingPage "22797" @default.
• W2061369977 abstract "The leucine-rich nuclear export signal (NES) is used to shuttle large cellular proteins from the nucleus to the cytoplasm. The nuclear export receptor Crm1 is essential in this process by recognizing the NES motif. Here, we show that the oncogenic hepatitis B virus (HBV) X protein (HBx) contains a functional NES motif. We found that the predominant cytoplasmic localization of HBx is sensitive to the drug leptomycin B (LMB), which specifically inactivates Crm1. Mutations at the two conserved leucine residues to alanine at the NES motif (L98A,L100A) resulted in a nuclear redistribution of HBx. A recombinant HBx protein binds to Crm1 in vitro. In addition, ectopic expression of HBx sequesters Crm1 in the cytoplasm. Furthermore, HBx activates NFκB by inducing its nuclear translocation in a NES-dependent manner. Abnormal cytoplasmic sequestration of Crm1, accompanied by a nuclear localization of NFκB, was also observed in hepatocytes from HBV-positive liver samples with chronic active hepatitis. We suggest that Crm1 may play a role in HBx-mediated liver carcinogenesis. The leucine-rich nuclear export signal (NES) is used to shuttle large cellular proteins from the nucleus to the cytoplasm. The nuclear export receptor Crm1 is essential in this process by recognizing the NES motif. Here, we show that the oncogenic hepatitis B virus (HBV) X protein (HBx) contains a functional NES motif. We found that the predominant cytoplasmic localization of HBx is sensitive to the drug leptomycin B (LMB), which specifically inactivates Crm1. Mutations at the two conserved leucine residues to alanine at the NES motif (L98A,L100A) resulted in a nuclear redistribution of HBx. A recombinant HBx protein binds to Crm1 in vitro. In addition, ectopic expression of HBx sequesters Crm1 in the cytoplasm. Furthermore, HBx activates NFκB by inducing its nuclear translocation in a NES-dependent manner. Abnormal cytoplasmic sequestration of Crm1, accompanied by a nuclear localization of NFκB, was also observed in hepatocytes from HBV-positive liver samples with chronic active hepatitis. We suggest that Crm1 may play a role in HBx-mediated liver carcinogenesis. hepatocellular carcinoma nuclear export signal nuclear localization signal hepatitis B viral X protein hepatitis B virus normal human fibroblasts leptomycin B mitogen-activated protein kinase tumor necrosis factor-α hemagglutinin polymerase chain reaction green fluorescent protein guanosine 5′-3-O-(thio)triphosphate Hepatocellular carcinoma (HCC)1 is one of the most prevalent malignant diseases worldwide and hepatitis B virus (HBV) is the major etiologic factor for HCC (1Di Bisceglie A.M. Rustgi V.K. Hoofnagle J.H. Dusheiko G.M. Lotze M.T. Ann. Intern. Med. 1988; 108: 390-401Crossref PubMed Google Scholar, 2Montesano R. Hainaut P. Wild C.P. J. Natl. Cancer Inst. 1997; 89: 1844-1851Crossref PubMed Scopus (227) Google Scholar, 3Brechot C. Gozuacik D. Murakami Y. Paterlini-Brechot P. Semin. Cancer Biol. 2000; 10: 211-231Crossref PubMed Scopus (252) Google Scholar). HBV is a DNA tumor virus, which encodes four open reading frames, S/preS, C/preC, P, and HBx (4Ganem D. Varmus H.E. Annu. Rev. Biochem. 1987; 56: 651-693Crossref PubMed Scopus (812) Google Scholar). The oncogenicity of HBV is largely the result of HBx, the smallest gene encoding a 17-kDa protein (3Brechot C. Gozuacik D. Murakami Y. Paterlini-Brechot P. Semin. Cancer Biol. 2000; 10: 211-231Crossref PubMed Scopus (252) Google Scholar, 5Unsal H. Yakicier C. Marcais C. Kew M. Volkmann M. Zentgraf H. Isselbacher K.J. Ozturk M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 822-826Crossref PubMed Scopus (148) Google Scholar, 6Paterlini P. Poussin K. Kew M. Franco D. Brechot C. Hepatology. 1995; 21: 313-321Crossref PubMed Google Scholar, 7Robinson W.S. Parsonnet J. Microbes and Malignancy: Infection as a Cause of Human Cancers. Oxford University Press, New York1999: 232-266Google Scholar, 8Kim Y.C. Song K.S. Yoon G. Nam M.J. Ryu W.S. Oncogene. 2001; 20: 16-23Crossref PubMed Scopus (78) Google Scholar). One major cellular function of HBx is its promiscuous transcriptional activation activity, a property that is believed to contribute to its oncogenicity (9Andrisani O.M. Barnabas S. Int. J. Oncol. 1999; 15: 373-379PubMed Google Scholar). A wide range of cellular genes can be up-regulated or down-regulated by HBx (9Andrisani O.M. Barnabas S. Int. J. Oncol. 1999; 15: 373-379PubMed Google Scholar). However, HBx is localized in the cytoplasm mostly and does not bind to double-stranded DNA. A “universal” effect of HBx, on otherwise totally different types of promoters with no obvious consensus sequence, has led to the hypothesis that HBx may regulate gene expression by interacting directly with host general transcription factors (10Cheong J.H. Yi M. Lin Y. Murakami S. EMBO J. 1995; 14: 143-150Crossref PubMed Scopus (239) Google Scholar, 11Qadri I. Maguire H.F. Siddiqui A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1003-1007Crossref PubMed Scopus (185) Google Scholar, 12Qadri I. Conaway J.W. Conaway R.C. Schaack J. Siddiqui A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10578-10583Crossref PubMed Scopus (129) Google Scholar), or indirectly via the activation of protein kinase C and RAS-RAF mitogen-activated protein kinase (MAPK) signaling pathways (13Kekulë A.S. Lauer U. Weiss L. Luber B. Hofschneider P.H. Nature. 1993; 361: 742-745Crossref PubMed Scopus (334) Google Scholar, 14Benn J. Schneider R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10350-10354Crossref PubMed Scopus (402) Google Scholar, 15Wang H.D. Trivedi A. Johnson D.L. Mol. Cell. Biol. 1997; 17: 6838-6846Crossref PubMed Scopus (69) Google Scholar). Although HBx can induce neoplastic transformation, presumably by preventing p53-mediated apoptosis (16Wang X.W. Forrester K. Yeh H. Feitelson M.A. Gu J.R. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2230-2234Crossref PubMed Scopus (629) Google Scholar, 17Wang X.W. Gibson M.K. Vermeulen W. Yeh H. Forrester K. Sturzbecher H.W. Hoeijmakers J.H.J. Harris C.C. Cancer Res. 1995; 55: 6012-6016PubMed Google Scholar, 18Elmore L.W. Hancock A.R. Chang S.F. Wang X.W. Chang S. Callahan C.P. Geller D.A. Will H. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14707-14712Crossref PubMed Scopus (302) Google Scholar), it also can induce apoptosis in a p53-dependent or -independent manner (19Chirillo P. Pagano S. Natoli G. Puri P.L. Burgio V.L. Balsano C. Levrero M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8162-8167Crossref PubMed Scopus (192) Google Scholar,20Kim H. Lee H. Yun Y. J. Biol. Chem. 1998; 273: 381-385Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), 2M. Forgues, A. J. Marrogi, E. A. Spillare, C-G. Wu, Q. Yang, M. Yoshida, and X. W. Wang, unpublished data.2M. Forgues, A. J. Marrogi, E. A. Spillare, C-G. Wu, Q. Yang, M. Yoshida, and X. W. Wang, unpublished data. or sensitize cells to tumor necrosis factor α (TNFα)-induced apoptosis (21Su F. Schneider R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8744-8749Crossref PubMed Scopus (286) Google Scholar). Therefore, the precise mechanism related to its effector remains unknown and none of these studies satisfactorily explain the pleiotropic effects associated with HBx. Close inspection of the HBx sequence revealed a short, hydrophobic, leucine-rich nuclear export signal motif (NES) (Fig. 1 A). An NES is located in the center region of HBx (residues 89–100). The center region of HBx is retained in HCC frequently and is essential for its transactivation (22Kumar V. Jayasuryan N. Kumar R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5647-5652Crossref PubMed Scopus (113) Google Scholar, 23Ritter S.E. Whitten T.M. Quets A.T. Schloemer R.H. Virology. 1991; 182: 841-845Crossref PubMed Scopus (36) Google Scholar, 24Takada S. Koike K. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5628-5632Crossref PubMed Scopus (126) Google Scholar). This region also is conserved among HBx from different subtypes (Fig. 1 A). Several viral proteins including HIV-1 Rev, HTLV-1 Rex, and adenovirus E4 34-kDa proteins contain functional NESs (Fig. 1 A). Similar to HBx, Rev and Rex also are potent viral and cellular transactivators with no apparent DNA binding property (25Fischer U. Huber J. Boelens W.C. Mattaj I.W. Luhrmann R. Cell. 1995; 82: 475-483Abstract Full Text PDF PubMed Scopus (976) Google Scholar, 26Dobbelstein M. Roth J. Kimberly W.T. Levine A.J. Shenk T. EMBO J. 1997; 16: 4276-4284Crossref PubMed Scopus (150) Google Scholar). In addition, NESs also have been identified in cellular proteins, many of which are involved in transcription, cell signaling cascade, oncogenic transformation, and cell cycle regulators. Examples include protein kinase inhibitor, MAP kinase kinase (MAPKK), TFIIIA, Mdm2, p53, IκBα, NF-AT, cyclin B1, c-Abl, and 14-3-3 (reviewed in Ref.27Ullman K.S. Powers M.A. Forbes D.J. Cell. 1997; 90: 967-970Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). The activities of these proteins are tightly regulated by their NESs. The nuclear export receptor Crm1 and its cofactor Ran GTPase are essential in this process by recognizing NESs and mediating nuclear protein export (27Ullman K.S. Powers M.A. Forbes D.J. Cell. 1997; 90: 967-970Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar, 28Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1724) Google Scholar). In addition, previous results indicate that Crm1 may be involved in maintaining chromosomal integrity (29Adachi Y. Yanagida M. J. Cell Biol. 1989; 108: 1195-1207Crossref PubMed Scopus (195) Google Scholar) and Ran may play a key role in regulating mitosis initiation by stimulating spindle formation (30Wilde A. Zheng Y. Science. 1999; 284: 1359-1362Crossref PubMed Scopus (319) Google Scholar, 31Ohba T. Nakamura M. Nishitani H. Nishimoto T. Science. 1999; 284: 1356-1358Crossref PubMed Scopus (247) Google Scholar). Mutation of the hydrophobic leucine residues to alanines have been shown to disrupt NES function in a number of proteins, including HIV-rev, E4 34-kDa, p53, Mdm2, and cyclin B1 (25Fischer U. Huber J. Boelens W.C. Mattaj I.W. Luhrmann R. Cell. 1995; 82: 475-483Abstract Full Text PDF PubMed Scopus (976) Google Scholar, 26Dobbelstein M. Roth J. Kimberly W.T. Levine A.J. Shenk T. EMBO J. 1997; 16: 4276-4284Crossref PubMed Scopus (150) Google Scholar, 32Roth J. Dobbelstein M. Freedman D.A. Shenk T. Levine A.J. EMBO J. 1998; 17: 554-564Crossref PubMed Scopus (531) Google Scholar, 33Toyoshima F. Moriguchi T. Wada A. Fukuda M. Nishida E. EMBO J. 1998; 17: 2728-2735Crossref PubMed Scopus (276) Google Scholar). In this study, we have investigated the hypothesis that the pleiotropic effects associated with HBx may be contributed by the presence of a NES motif, and HBx may activate cellular gene expression and induce oncogenicity through the modulation of Crm1-mediated functions. We have identified a functional NES motif in HBx. This motif is necessary for HBx-induced cytoplasmic sequestration of Crm1, and subsequently, the nuclear translocation and activation of NFκB. Cytoplasmic retention of Crm1 also is found in liver samples with chronic active hepatitis infected with HBV, a condition that is predisposing individuals to the development of HCC. We suggest that the inactivation of the Crm1-mediated pathway may be an early step during viral hepatitis-mediated liver carcinogenesis. The plasmid pcDNA3-HBxadr-Hatag was constructed by the insertion of a C-terminal hemagglutinin (HA) epitope-tagged full-length HBx into the BamHI andHindIII sites of a pcDNA3 vector (Invitrogen). GFP-HBx and GFP-HBx-NESM were constructed as follows: a 529-base pair fragment released from the digestion of pcDNA3-HBxadr-Hatag withHindIII/ApaI and inserted into pEGFP-C2 (CLONTECH) at theHindIII/ApaI sites. The resulting GFP-HBx is an N-terminal fusion of HBx with GFP and a C-terminal fusion with HA tag. GFP-HBx-NESM was made by a PCR-based site-directed mutagenesis protocol using GFP-HBx as a template. To generate the HBx-NES mutant (L98A,G99A,L100A), 2 HBx fragments were amplified by PCR using primer pairs (5′-GCTGCTGCATCAGCAATGTCAACAACCGAC-3′/5′-ATGCTCTAGAGGCAGAGGTGAAAAAGTTGCATGG-3′ and 5′-TGCAGCAGCAGTCCTCTTATGTAAGAGCTT-3′/5′-TATAAAGCTTGGTACCGAGCTCGGATCTGATGGC-3′). The 2 PCR DNA fragments were denatured and reannealed. Then the HBx-NES mutant was generated by PCR with primers (5′-TATAAAGCTTGGTACCGAGCTCGGATCTGATGGC-3′ and 5′-ATGCTCTAGAGGCAGAGGTGAAAAAGTTGCATGG-3′) and was subcloned into the pEGFP-C2 vector. Mutations were verified by DNA sequencing. The GFP-NLS-NES and GFP-NLS-NESM constructs were made by the strategy described previously (34Stade K. Ford C.S. Guthrie C. Weis K. Cell. 1997; 90: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar). Briefly, the SV40 NLS motif (residues 125–133) was fused to GFP by the cloning of oligos TNLS1 (5′-TCGAGATCCCCCCAAGAAGAAGCGCAAGGTGGAGCA-3′/TNLS2 5′-AGCTTGCTCCACCTTGCGCTTCTTCTTGGGGGGATC-3′) into plasmid pGFPF-C1 (CLONTECH) to generate GFP-NLS. GFP-NES and GFP-NESM were constructed by cloning oligos XNESW1 (5′-AATTCTCAGGTCTTGCCCAAGCTCTTACATAAGAGGACTCTTGGACTCTCAGCAATG-3′)/XNESW2 (5′-GATCCATTGCTGAGAGTCCAAGAGTCCTCTTATGTAAGAGCTTGGGCAAGACCTGAG-3′) or XNESM3 (5′-AATTCTCAGGTCTTGCCCAAGCTCTTACATAAGAGGACTGCTGGAGCCTCAGCAATG-3′)- /XNESM4 (5′-GATCCATTGCTGAGGCTCCAGCAGTCCTCTTATGTAAGAGCTTGGGCAAGACCTGAG-3′), respectively, into GFP-NLS, resulting in a GFP-NLS-NES or GFP-NLS-NESM fusion protein. The NES motif corresponds to the HBx residues 87–103, while NESM is the HBx-NES motif with mutations at the conserved leucine residues (L98A and L100A). pSVCM contains a GFP-CRM1 fusion gene under the control of a CMV promoter (28Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1724) Google Scholar). pNFκB-Luc, provided by John Brady (NCI), contains a luciferase gene under the promoter containing 4 NFκB responsive elements. Normal primary human fibroblasts were obtained from Coriell Institute for Medical Research (Camden, NJ). These cells were grown in Ham's F-10 medium supplemented with 15% fetal bovine serum and were used before passage 12. A telomerase-immortalized human fibroblast line (NHF-hTERT), a gift of Dr. Judy Campisi, was grown in Dulbecco's modified Eagle's media supplemented with 10% fetal bovine serum. Normal primary human hepatocytes were purchased from Clonetics (BioWittaker), grown in LCM (BioWittaker), and used only at passage 1. Hep3B cells were grown in Eagle's minimum essential medium containing 10% fetal bovine serum. Microinjection of NHF cells was done essentially as described previously (35Wang X.W. Vermeulen W. Coursen J.D. Gibson M. Lupold S.E. Forrester K. Xu G. Elmore L. Yeh H. Hoeijmakers J.H.J. Harris C.C. Genes Dev. 1996; 10: 1219-1232Crossref PubMed Scopus (309) Google Scholar). Transfection of NHF-hTERT and Hep3B cells was carried out by the LipofectAMINE Plus reagent based on the recommended protocol described by the manufacturer (Life Technologies, Inc., Gaithersburg, MD). For the luciferase reporter assay, cells were seeded into 6-well dishes (Costar) at 50% confluence, co-transfected with 0.2 μg of pNFkB-Luc or pNFAT-Luc, along with 2 μg of each of various HBx constructs, in the presence or absence of TNFα (10 ng/ml). A total of 0.5 μg of pRL-null or 0.003 μg of pRL-CMV was added into each transfection as an internal control. pEGFPC1 was used to keep the total amount of plasmid DNA constant. Cells were then incubated for 24 h prior to harvesting. The luciferase activity was carried out by the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions and were measured in a Monolight 2010 luminometer (Analytical Luminescence Laboratory). The luciferase activity was expressed as fold activation against the untreated reporter by itself, which was normalized by Renilla luciferase activity for the transfection efficiency. The reported results represent at least three independent transfections. For immunocytochemistry analysis, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline for 10 min, followed by methanol for 20 min at 24 h postmicroinjection, and washed in phosphate-buffered saline-plus (0.15% glycine, 0.5% bovine serum albumin in phosphate-buffered saline). The GFP fusion proteins can be visualized directly in a fluorescence microscope equipped with an fluorescein isothiocyanate filter with or without fixation. The HBx expression also was verified by an anti-HBx monoclonal antibody (data not shown). NFκB was detected using an anti-NFκB polyclonal antibody (1:100) (Santa Cruz) and Crm1 was detected using an anti-Crm1 polyclonal antibody (1:100). Both antibodies were incubated for 1 h at 37 °C. Anti-rabbit IgG conjugated to fluorescein or Texas Red (Vector Laboratories) was used (1:300) for 1 h at room temperature. Nuclei were stained with 4,6-diamidino-2-phenylindole. The following criteria were used to define the subcellular localization, preferentially cytoplasmic localization: protein signals mostly intense in the cytoplasm; preferentially nuclear localization: protein signals mostly intense in the nucleus. Some of the slides were analyzed with a blind fashion to avoid any bias. For GFP-NLS reporter expression, cells on the same coverslip were microinjected with GFP-NLS, GFP-NLS-XNES, or GFP-NLS-XNESM construct and incubated for 6 h. Cells were monitored under a Zeiss Axioskop fluorescence microscope and representative images were taken using a high-performance CCD imaging system (IP Lab Spectrum) with the same exposure time. For co-localization of Crm1 and HBx analysis, cells were co-stained with anti-HBx and anti-Crm1 antibodies with the corresponding secondary antibodies conjugated with Texas Red or fluorescein, and analyzed by a Bio-Rad MRC 1024 confocal system. Sequential excitation at 488 and 568 nm was provided by a krypton-argon gas laser. Emission filters of 598/40 and 522/32 were used for sequentially collecting red and green fluorescence in channels 1 and 2, respectively. Z-sections were collected at 0.5-μm intervals for each cell using LaserSharp software. Images were analyzed by Confocal Assistant software. In vitro binding between HBx and hCrm1 was determined with the in vitrotranslated HBx and hCrm1 (16Wang X.W. Forrester K. Yeh H. Feitelson M.A. Gu J.R. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2230-2234Crossref PubMed Scopus (629) Google Scholar). The HA-tagged HBx and the hCrm1 proteins were made by the one-step TnT in vitro transcription and translation system (Promega), as described previously (16Wang X.W. Forrester K. Yeh H. Feitelson M.A. Gu J.R. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2230-2234Crossref PubMed Scopus (629) Google Scholar), from pcDNA-HA-HBx and hCrm1Blue744 vectors (gift of Dr. Gerard Grosveld), respectively. Aliquots of HBx (25 μl) and hCrm1 (45 μl) were mixed together in CBBL buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 5 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, and 0.1% Nonidet P-40) in the presence of 0.2 mm GTPγS and 80 μl of anti-HA affinity matrix (Roche Molecular Biochemicals, Indianapolis, IN). The mixture was incubated at 37 °C for 60 min. After centrifugation, beads were washed four times with CBBL buffer. The bound proteins were separated on SDS-polyacrylamide gel electrophoresis and Crm1 was detected by Western blotting using anti-Crm1 antibody and followed by the Amersham ECL system. A 10% of in vitrotranslated hCrm1 was loaded in parallel as the input. A total of 12 samples were included in this part of the study. Histopathologically, they included 5 normal liver samples, 7 viral hepatitis cases showing grades 1 to 2 activity according to the criteria of Scheuer (36Scheuer P.J. J. Hepatol. 1991; 13: 372-374Abstract Full Text PDF PubMed Scopus (1356) Google Scholar). In addition to H & E light microscopy, tissue sections were cut from frozen samples with a thickness of 4 μm and mounted on electrically charged glass slides. The sections were fixed in absolute alcohol and then washed with 2 changes of phosphate-buffered saline for 5 min each. They were then quenched with 3% hydrogen peroxide to block the endogenous peroxidase activity for 30 min. Following incubation with 10% horse serum to block the nonspecific binding, the sections were incubated overnight at 4 °C with anti-CRM1 antibodies. The sections were prepared in duplicate and two anti-CRM1 antibodies were used, including a goat anti-Crm1 antibody from Santa Cruz at a 1:10 dilution (Santa Cruz) and a rabbit anti-Crm1 polyclonal antibody (Gift from Gerard Grosveld) at a 1:100 dilution. A rabbit anti-NFκB antibody (anti-p65) (Santa Cruz) was used at a 1:100 dilution for staining NFκB. Biotinylated secondary antibodies and streptavidin peroxidase complex (LSAB) were used. Chromogenic development was obtained by the immersion of sections in 3,3′-diaminobenzidine solution (0.25 mg/ml with 3% hydrogen peroxide). The slides were counterstained with Harris' hematoxylin and re-hydrated with alcohol and xylene. Slides were evaluated in a blind fashion. To test if HBx-NES functions in nuclear protein export, we first investigated whether the subcellular localization of HBx is sensitive to LMB, an antitumor agent that selectively binds to and blocks Crm1-mediated nuclear export (37Wolff B. Sanglier J.J. Wang Y. Chem. Biol. 1997; 4: 139-147Abstract Full Text PDF PubMed Scopus (570) Google Scholar, 38Kuerbitz S.J. Plunkett B.S. Walsh W.V. Kastan M.B. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7491-7495Crossref PubMed Scopus (1838) Google Scholar). Consistent with previous findings (18Elmore L.W. Hancock A.R. Chang S.F. Wang X.W. Chang S. Callahan C.P. Geller D.A. Will H. Harris C.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 14707-14712Crossref PubMed Scopus (302) Google Scholar), HBx was localized in the cytoplasm predominately with a punctate staining pattern in appearance when expressed transiently in normal human fibroblasts (NHF) (Fig. 1) and in a HCC-derived cell line (Hep3B) (data not shown). However, LMB treatment causes an increase in nuclear accumulation of HBx (Fig. 1,B and C). Next, we used a GFP protein export reporter assay described previously (34Stade K. Ford C.S. Guthrie C. Weis K. Cell. 1997; 90: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar) to further test if the HBx-NES oligopeptide functions in nuclear protein export (34Stade K. Ford C.S. Guthrie C. Weis K. Cell. 1997; 90: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (922) Google Scholar). This reporter uses a nuclear localization signal (NLS) fused to a GFP, thereby inducing nuclear localization of the fusion protein. Consistently, the GFP-NLS reporter was localized in the nucleus of NHF exclusively. However, when an HBx-NES was fused to this reporter, the resulting GFP-NLS-XNES fusion protein can be found in the cytoplasm with a diffused staining pattern in appearance, although no nuclear exclusion of this reporter protein could be observed (Fig. 1 D). In contrast, GFP-NLS-XNESM that contains a mutated HBx-NES oligopeptide (L98A,L100A) does not localize in the cytoplasm (Fig. 1 D). To further examine if cytoplasmic localization of a full-length HBx is dependent on the presence of an NES, a GFP-HBx fusion gene (HBx) or an NES-mutated GFP-HBx fusion gene (HBxNESM) was constructed. Again, GFP-HBx was localized in the cytoplasm predominantly with a punctate pattern in appearance, while HBxNESM was found mainly in the nucleus with a diffused pattern (Fig. 1 E). Taken together, these data demonstrate that HBx-NES is functional in Crm1-mediated nuclear protein export. Nuclear export receptor Crm1 is known to interact with proteins containing NESs and to mediate nuclear protein export. The majority of Crm1 protein is found in the nucleus of normal cells (39Kudo N. Khochbin S. Nishi K. Kitano K. Yanagida M. Yoshida M. Horinouchi S. J. Biol. Chem. 1997; 272: 29742-29751Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Consistently, every NHF cell displays an endogenous nuclear-diffused Crm1 distribution (Fig.2). When HBx was expressed transiently in NHF, Crm1 was often found in the cytoplasm with a punctate staining pattern. Approximately 39% ± 2 of the HBx-expressing NHF cells displayed co-localization of Crm1, and over 99% of cytoplasmic punctate Crm1 signals were co-localized with HBx (Fig. 2 A). The punctate staining patterns containing both Crm1 and HBx signals were not always the same in appearance (Fig. 2 A, panels c-l). These results suggest that the cytoplasmic HBx and Crm1 are not necessarily associated with a single subcellular component. In contrast, no cytoplasmic co-localization was found in NHF cells expressing HBxNESM. These data indicate that HBx was able to partially retain Crm1 in the cytoplasm and suggest that HBx binds to Crm1in vivo. Consistently, in vitro translated HBx can physically interact with in vitro translated Crm1 (Fig.2 B). HBx can activate NFκB and other cellular transcription factors including NF-AT and MAP kinase cascade, although the mechanism of activation is unknown (14Benn J. Schneider R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10350-10354Crossref PubMed Scopus (402) Google Scholar, 40Meyer M. Caselmann W.H. Schluter V. Schreck R. Hofschneider P.H. Baeuerle P.A. EMBO J. 1992; 11: 2991-3001Crossref PubMed Scopus (179) Google Scholar, 41Cross J.C. Wen P. Rutter W.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8078-8082Crossref PubMed Scopus (145) Google Scholar). Because NFκB is inactivated normally through its binding to IκBα which is sequestrated in the cytoplasm by Crm1-mediated nuclear export (42Tam W.F. Lee L.H. Davis L. Sen R. Mol. Cell. Biol. 2000; 20: 2269-2284Crossref PubMed Scopus (133) Google Scholar), we examined whether HBx-NES is responsible for the activation of NFκB. Over 97% of normal dividing NHF cells display a predominant localization of NFκB in the cytoplasm (Fig. 3 A). In contrast, ∼55 ± 7% of HBx-expressing NHF cells show a preferentially nuclear distribution of NFκB, while an NES mutated HBx (HBxNESM) has lost this activity (Fig. 3, A andB). Next, we used a luciferase reporter that contains NFκB responsive elements (pNFkB-Luc) to investigate if the HBx-induced nuclear localization of NFκB correlates with its activity. pNFκB-Luc was co-transfected with HBx or HBxNESM into an hTERT-immortalized NHF (NHF-hTERT) or a hepatocellular carcinoma cell line (Hep3B) in the absence or presence of TNFα, an agent known to sensitize HBx-mediated transactivation (9Andrisani O.M. Barnabas S. Int. J. Oncol. 1999; 15: 373-379PubMed Google Scholar). Consistently, HBx alone induced the luciferase activity weakly which could be further enhanced by the treatment of TNFα in both NHF-hTERT and Hep3B cells. In contrast, HBxNESM is devoid of its ability to increase luciferase activity even in the presence of TNFα (Fig. 3 C). To investigate if Crm1 is sequestrated cytoplasmically in liver samples from chronic active hepatitis patients with HBV infection, we performed immunohistochemistry analysis of Crm1 in frozen tissue sections or in paraffin-embedded sections by anti-Crm1 polyclonal antibody. A total of 12 liver samples with samples from 5 normal individuals were included in this study. The presence of HBV DNA including Core, preS, and HBx genes in these samples was verified by PCR (data not shown). Among 7 HBV positive cases, liver samples show chronic active hepatitis with grades 1 to 2 activity (TableI). While 5/5 normal samples revealed nuclear Crm1 staining exclusively, 6/7 HBV samples showed cytoplasmic Crm1 staining (Fig. 4 and Table I) in hepatocytes. An anti-NFκB antibody was also used to determine the status of NFκB in these liver samples. Again, normal liver samples revealed mainly a cytoplasmic localization of NFκB while HBV positive liver samples showed a nuclear distribution of NFκB. The above findings provide an in vivo physiological significance for the presence of a cytoplasmic sequestration of Crm1 accompanied by an activation of NFκB associated with HBV infection.Table ICytoplasmic sequestration of Crm1 and nuclear localization of NFκB associated with HBV-infected liver samplesCase numberHistologyGrade 1-aHistology was determined according to the criteria of Scheuer. CAH, chronic active hepatitis.Virus Status 1-bThe virus status was determined by serological tests and by determining the presence of preS, HBx, and core genes in these tissues by PCR. HBV, hepatitis B virus.Crm1NFκBLocalization 1-cFrozen sections were subjected to immunohistochemical analysis by the anti-Crm1 or anti-NFκB antibodies. Anti-Crm1 or anti-NFκB reactivity was evaluated when crisp brown nuclear or cytoplasmic staining was detected, and scored as the following: a score of 0, no staining; 1+, when <10% of cells were stained; 2+, >10 to <25% of cells were stained; 3+, >26 to <50%; and 4+, >50%. A score of (1+) staining or higher was considered reactive. The following criteria were used in determining protein distribution: nuclear, nuclear distribution only; cytoplasmic, cytoplasmic distribution only; nuclear/cytoplasmic, predominant nuclear with some cytoplasmic distribution; cytoplasmic/nuclear, predominant cytoplasmic with some nuclear distribution.ScoreLocalizationScore1Normal0NegativeNuclear4+Cytoplasmic3+2Normal0NegativeNuclear4+Cytoplasmic2+3Normal0NegativeNuclear4+Cytoplasmic1+4Normal0NegativeNuclear4+Cytoplasmic3+5Normal0NegativeNuclear4+Cytoplasmic2+6CAH2HBVCytoplasmic4+Cytoplasmic/nuclear2+7CAH2HBVNuclear/cytoplasmic4+Nuclear2+8CAH2HBVCytoplasmic4+Nuclear1+9CAH2HBVCytoplasmic4+Nuclear1+10CAH2HBVCytoplasmic4+Nuclear2+11CAH1HBVCytoplasmic4+Nuclear2+12CAH1HBVCytoplasmic4+Nuclear/cytoplasmic2+1-a Histology was determined according to the criteria of Scheuer. CAH, chronic active hepatitis.1-b The virus status was determined by serological tests and by determining the presence of preS, HBx, and core genes in these tissues by PCR. HBV, hepatitis B virus.1-c Frozen sections were subjected to immunohistochemical analysis by the anti-Crm1 or anti-NFκB antibodies. Anti-Crm1 or anti-NFκB reactivity was evaluated when crisp brown nuclear or cytoplasmic staining was detected, and scored as the following: a score of 0, no staining; 1+, when <10% of cells were stained; 2+, >10 to <25% of cells were stained; 3+, >26 to <50%; and 4+, >50%. A score of (1+) staining or higher was considered reactive. The following criteria were used in determining protein distribution: nuclear, nuclear distribution only; cytoplasmic, cytoplasmic distribution only; nuclear/cytoplasmic, predominant nuclear with some cytoplasmic distribution; cytoplasmic/nuclear, predominant cytoplasmic with some nuclear distribution. Open table in a new tab We have demonstrated that the oncogenic HBx contains a functional NES, and that this NES is required for the HBx-mediated activation of the NFκB signaling cascade. We also have shown that HBx is able to bind to and sequester Crm1 in the cytoplasm, which correlates with the nuclear localization and activation of NFκB. Furthermore, liver samples from HBV-infected patients with chronic active hepatitis, a condition predisposing individuals for the development of liver cancer, also display cytoplasmic sequestration of Crm1 and nuclear localization of NFκB. It is possible that HBx may use a Crm1-dependent mechanism to modulate cellular gene transcription. Our data provide a plausible model whereby HBx may induce oncogenicity through the modulation of the Crm1-mediated pathway. The studies described in this report may offer a common mechanism to explain the pleiotropic effects of HBx. First, Crm1 transports and controls many cellular proteins including NFκB, NF-AT, MAPKK, and p53, whose activities are known to be regulated by HBx (14Benn J. Schneider R.J. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10350-10354Crossref PubMed Scopus (402) Google Scholar, 17Wang X.W. Gibson M.K. Vermeulen W. Yeh H. Forrester K. Sturzbecher H.W. Hoeijmakers J.H.J. Harris C.C. Cancer Res. 1995; 55: 6012-6016PubMed Google Scholar, 40Meyer M. Caselmann W.H. Schluter V. Schreck R. Hofschneider P.H. Baeuerle P.A. EMBO J. 1992; 11: 2991-3001Crossref PubMed Scopus (179) Google Scholar, 41Cross J.C. Wen P. Rutter W.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8078-8082Crossref PubMed Scopus (145) Google Scholar,43Lara-Pezzi E. Armesilla A.L. Majano P.L. Redondo J.M. Lopez-Cabrera M. EMBO J. 1998; 17: 7066-7077Crossref PubMed Scopus (87) Google Scholar). Second, Crm1 also regulates cytoplasmic mRNA transport. Inactivation of Crm1 by HBx, in principle, would alter the stability of mRNA thereby influencing gene expression. Consistent with this hypothesis is our findings that HBx is able to sequester Crm1 in the cytoplasm. The HBx-NES is essential for sequestering Crm1 in the cytoplasm and for the activation of NFκB. Inactivation of Crm1-mediated nuclear protein export is known to activate this target (44Rodriguez M.S. Thompson J. Hay R.T. Dargemont C. J. Biol. Chem. 1999; 274: 9108-9115Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Therefore, the promiscuous transactivation-mediated by HBx may be a secondary effect resulting from alteration of Crm1 function. An alternative mechanism for HBx to activate NFκB is through the degradation of IκBα in the cytoplasm by inducing its phosphorylation and ubiquitination. However, we did not observe any detectable degradation of IκBα in cells expressing HBx by Western blot analysis with anti-IκBα antibody (data not shown). This also correlates with our results that HBx alone only displays a weak activation of NFκB that can be enhanced significantly by TNFα (Fig.3 C). It is possible that a small degree of dissociation of the NFκB-IκBα complex, followed by the degradation of IκBα induced by HBx in the cytoplasm, may contribute to the nuclear localization of NFκB. A recent study by Weil and colleagues (45Weil R. Sirma H. Giannini C. Kremsdorf D. Bessia C. Dargemont C. Brechot C. Israel A. Mol. Cell. Biol. 1999; 19: 6345-6354Crossref PubMed Google Scholar) indicate that HBx may induce the nuclear import of NFκB/IκBα by binding to IκBα. Interestingly, the region of IκBα that is critical for HBx-induced nuclear import of IκBα contains a functional NES (45Weil R. Sirma H. Giannini C. Kremsdorf D. Bessia C. Dargemont C. Brechot C. Israel A. Mol. Cell. Biol. 1999; 19: 6345-6354Crossref PubMed Google Scholar). These findings are consistent with our data that HBx-induced nuclear localization of NFκB/IκBα is dependent on the IκBα-NES. HBx is thought to be small enough to diffuse passively through the nuclear pore complex. The fact that this oncogenic viral protein has acquired NES activity, is preferentially localized in the cytoplasm, and modified nuclear export implies that nuclear export may be an important target for viral-mediated oncogenesis. Interestingly, cellular oncoproteins such as c-Abl and Mdm2 are known to be involved in a Crm1-dependent nuclear export. Analogous to viral hepatitis oncoprotein, the cellular oncoproteins, in principle, may induce neoplastic transformation also through the disruption of the Crm1-mediated mechanism. It is known that TPR and CAN are nuclear pore complex-associated proteins that act as the docking sites and are essential for Crm1-mediated nuclear export (27Ullman K.S. Powers M.A. Forbes D.J. Cell. 1997; 90: 967-970Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Earlier findings demonstrated that the TPR and CAN genes are mutated through translocation in thyroid carcinoma, gastric carcinoma, and acute myeloid leukemia (46von Lindern M. van Baal S. Wiegant J. Raap A. Hagemeijer A. Grosveld G. Mol. Cell. Biol. 1992; 12: 3346-3355Crossref PubMed Scopus (354) Google Scholar, 47von Lindern M. Fornerod M. van Baal S. Jaegle M. de Wit T. Buijs A. Grosveld G. Mol. Cell. Biol. 1992; 12: 1687-1697Crossref PubMed Scopus (326) Google Scholar, 48Greco A. Pierotti M.A. Bongarzone I. Pagliardini S. Lanzi C. Della Porta G. Oncogene. 1992; 7: 237-242PubMed Google Scholar, 49Park M. Dean M. Cooper C.S. Schmidt M. O'Brien S.J. Blair D.G. Vande Woude G.F. Cell. 1986; 45: 895-904Abstract Full Text PDF PubMed Scopus (428) Google Scholar, 50Soman N.R. Correa P. Ruiz B.A. Wogan G.N. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4892-4896Crossref PubMed Scopus (163) Google Scholar, 51Fornerod M. van Deursen J. van Baal S. Reynolds A. Davis D. Murti K.G. Fransen J. Grosveld G. EMBO J. 1997; 16: 807-816Crossref PubMed Scopus (396) Google Scholar, 52Bangs P. Burke B. Powers C. Craig R. Purohit A. Doxsey S. J. Cell Biol. 1998; 143: 1801-1812Crossref PubMed Scopus (85) Google Scholar). We also found that cytoplasmic sequestration of Crm1 is associated frequently with HCC (data not shown). In addition, Crm1 and Ran may play a role in mitosis initiation (29Adachi Y. Yanagida M. J. Cell Biol. 1989; 108: 1195-1207Crossref PubMed Scopus (195) Google Scholar, 30Wilde A. Zheng Y. Science. 1999; 284: 1359-1362Crossref PubMed Scopus (319) Google Scholar, 31Ohba T. Nakamura M. Nishitani H. Nishimoto T. Science. 1999; 284: 1356-1358Crossref PubMed Scopus (247) Google Scholar). It is possible that the inactivation of Crm1 and Ran may induce genomic instability, a predisposing factor for cancer development. Thus, we postulate that the inactivation of the Crm1-mediated nuclear export is a common event during viral and cellular oncogenesis. We thank Dr. Gerard Grosveld for generous gifts of anti-Crm1 antibody and the Crm1 cDNA, Dr. Barbara Wolff for the leptomycin B, Dr. John Brady for the pNFκB-Luc plasmid, Dr. Judy Campisi for the NHF-hTERT cell line, and Dr. Rodney Markin for the human liver samples. We are grateful to Dr. Curtis Harris and members of his group for scientific support, Susan Garfield for assistance on the confocal imaging analysis, and Dorothea Dudek for excellent editorial assistance." @default.
• W2061369977 created "2016-06-24" @default.
• W2061369977 creator A5004179280 @default.
• W2061369977 creator A5012099509 @default.
• W2061369977 creator A5040706368 @default.
• W2061369977 creator A5040761742 @default.
• W2061369977 creator A5063797441 @default.
• W2061369977 creator A5070838435 @default.
• W2061369977 creator A5081684786 @default.
• W2061369977 date "2001-06-01" @default.
• W2061369977 modified "2023-10-01" @default.
• W2061369977 title "Interaction of the Hepatitis B Virus X Protein with the Crm1-dependent Nuclear Export Pathway" @default.
• W2061369977 cites W109080479 @default.
• W2061369977 cites W1933138417 @default.
• W2061369977 cites W1945507556 @default.
• W2061369977 cites W1964576498 @default.
• W2061369977 cites W1966723276 @default.
• W2061369977 cites W1969730825 @default.
• W2061369977 cites W1977638924 @default.
• W2061369977 cites W1983496475 @default.
• W2061369977 cites W1985022589 @default.
• W2061369977 cites W1986676942 @default.
• W2061369977 cites W1987536971 @default.
• W2061369977 cites W2001600181 @default.
• W2061369977 cites W2003977276 @default.
• W2061369977 cites W2007755820 @default.
• W2061369977 cites W2008922773 @default.
• W2061369977 cites W2015702695 @default.
• W2061369977 cites W2026540294 @default.
• W2061369977 cites W2030283142 @default.
• W2061369977 cites W2032785100 @default.
• W2061369977 cites W2034296773 @default.
• W2061369977 cites W2037968180 @default.
• W2061369977 cites W2043455507 @default.
• W2061369977 cites W2044697187 @default.
• W2061369977 cites W2050274505 @default.
• W2061369977 cites W2052779181 @default.
• W2061369977 cites W2057708151 @default.
• W2061369977 cites W2058349670 @default.
• W2061369977 cites W2062612693 @default.
• W2061369977 cites W2064062498 @default.
• W2061369977 cites W2077311317 @default.
• W2061369977 cites W2078131820 @default.
• W2061369977 cites W2079715135 @default.
• W2061369977 cites W2083028663 @default.
• W2061369977 cites W2095777941 @default.
• W2061369977 cites W2098431648 @default.
• W2061369977 cites W2130744590 @default.
• W2061369977 cites W2139024239 @default.
• W2061369977 cites W2150001528 @default.
• W2061369977 cites W2153988632 @default.
• W2061369977 cites W2155085170 @default.
• W2061369977 cites W2155510029 @default.
• W2061369977 cites W2157448594 @default.
• W2061369977 cites W2171258458 @default.
• W2061369977 cites W2346898172 @default.
• W2061369977 cites W61009196 @default.
• W2061369977 doi "https://doi.org/10.1074/jbc.m101259200" @default.
• W2061369977 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11287420" @default.
• W2061369977 hasPublicationYear "2001" @default.
• W2061369977 type Work @default.
• W2061369977 sameAs 2061369977 @default.
• W2061369977 citedByCount "80" @default.
• W2061369977 countsByYear W20613699772012 @default.
• W2061369977 countsByYear W20613699772013 @default.
• W2061369977 countsByYear W20613699772014 @default.
• W2061369977 countsByYear W20613699772015 @default.
• W2061369977 countsByYear W20613699772016 @default.
• W2061369977 countsByYear W20613699772017 @default.
• W2061369977 countsByYear W20613699772018 @default.
• W2061369977 countsByYear W20613699772019 @default.
• W2061369977 countsByYear W20613699772020 @default.
• W2061369977 countsByYear W20613699772021 @default.
• W2061369977 countsByYear W20613699772022 @default.
• W2061369977 crossrefType "journal-article" @default.
• W2061369977 hasAuthorship W2061369977A5004179280 @default.
• W2061369977 hasAuthorship W2061369977A5012099509 @default.
• W2061369977 hasAuthorship W2061369977A5040706368 @default.
• W2061369977 hasAuthorship W2061369977A5040761742 @default.
• W2061369977 hasAuthorship W2061369977A5063797441 @default.
• W2061369977 hasAuthorship W2061369977A5070838435 @default.
• W2061369977 hasAuthorship W2061369977A5081684786 @default.
• W2061369977 hasBestOaLocation W20613699771 @default.
• W2061369977 hasConcept C159047783 @default.
• W2061369977 hasConcept C185592680 @default.
• W2061369977 hasConcept C2780114586 @default.
• W2061369977 hasConcept C2780723820 @default.
• W2061369977 hasConcept C54757728 @default.
• W2061369977 hasConcept C86803240 @default.
• W2061369977 hasConcept C95444343 @default.
• W2061369977 hasConceptScore W2061369977C159047783 @default.
• W2061369977 hasConceptScore W2061369977C185592680 @default.
• W2061369977 hasConceptScore W2061369977C2780114586 @default.
• W2061369977 hasConceptScore W2061369977C2780723820 @default.
• W2061369977 hasConceptScore W2061369977C54757728 @default.
• W2061369977 hasConceptScore W2061369977C86803240 @default.
• W2061369977 hasConceptScore W2061369977C95444343 @default.
• W2061369977 hasIssue "25" @default.

• next