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• W1987515540 abstract "Background & Aims: Transforming growth factor-β (TGF-β) has been implicated in the pathogenesis of liver disease. TGF-β is involved in liver regeneration and in the fibrotic and cirrhotic transformation with hepatitis viral infection. Hepatitis delta virus (HDV) infection causes fulminant hepatitis and liver cirrhosis. To elucidate the molecular mechanism of HDV pathogenesis, we examined the effects of HDV-encoded–only protein, the small hepatitis delta antigen (SHDAg), and the large hepatitis delta antigen (LHDAg), on TGF-β– and c-Jun–induced signaling cascades. Methods: The effects of either SHDAg or LHDAg on TGF-β– and c-Jun–induced signaling cascades in Huh7 and Cos7 cells were investigated by luciferase reporter gene assay, immunoprecipitation assay, electrophoretic mobility shift assay, Western blot analysis, and confocal microscopy analysis. Results: The LHDAg, but not the SHDAg, potentiated TGF-β– and c-Jun–induced signal activation, and the isoprenylation of LHDAg played a major role in signaling cascades. LHDAg synergistically activated hepatitis B virus X protein–mediated TGF-β and AP-1 signaling cascades. In addition, LHDAg enhanced the protein expression level of TGF-β–induced plasminogen activator inhibitor-1. Conclusions: LHDAg may induce liver fibrosis through the regulation of TGF-β–induced signal transductions. This regulation of TGF-β–mediated signaling is accomplished by the isoprenylation of LHDAg, which is a novel mechanism involved in HDV pathogenesis. Background & Aims: Transforming growth factor-β (TGF-β) has been implicated in the pathogenesis of liver disease. TGF-β is involved in liver regeneration and in the fibrotic and cirrhotic transformation with hepatitis viral infection. Hepatitis delta virus (HDV) infection causes fulminant hepatitis and liver cirrhosis. To elucidate the molecular mechanism of HDV pathogenesis, we examined the effects of HDV-encoded–only protein, the small hepatitis delta antigen (SHDAg), and the large hepatitis delta antigen (LHDAg), on TGF-β– and c-Jun–induced signaling cascades. Methods: The effects of either SHDAg or LHDAg on TGF-β– and c-Jun–induced signaling cascades in Huh7 and Cos7 cells were investigated by luciferase reporter gene assay, immunoprecipitation assay, electrophoretic mobility shift assay, Western blot analysis, and confocal microscopy analysis. Results: The LHDAg, but not the SHDAg, potentiated TGF-β– and c-Jun–induced signal activation, and the isoprenylation of LHDAg played a major role in signaling cascades. LHDAg synergistically activated hepatitis B virus X protein–mediated TGF-β and AP-1 signaling cascades. In addition, LHDAg enhanced the protein expression level of TGF-β–induced plasminogen activator inhibitor-1. Conclusions: LHDAg may induce liver fibrosis through the regulation of TGF-β–induced signal transductions. This regulation of TGF-β–mediated signaling is accomplished by the isoprenylation of LHDAg, which is a novel mechanism involved in HDV pathogenesis. Hepatitis delta virus (HDV) is a hepatotropic virus that frequently causes severe or fulminant hepatitis.1Rizzetto M. Canese M.G. Arico S. Crivelli O. Trepo C. Bonino F. Verme G. Immunofluorescence detection of new antigen-antibody system (delta/anti-delta) associated to hepatitis B virus in liver and in serum of HBsAg carriers.Gut. 1977; 18: 997-1003Crossref PubMed Scopus (657) Google Scholar, 2Niro G.A. Rosina F. Rizzetto M. Treatment of hepatitis D.J Viral Hepat. 2005; 12: 2-9Crossref PubMed Scopus (106) Google Scholar HDV is a satellite virus that requires hepatitis B virus surface antigen (HBsAg) for viral assembly.3Rizzetto M. Hoyer B. Canes M.G. Shih W.K. Purcell R.H. Gerin J.L. Delta agent: association of δ antigen with hepatitis B surface antigen and RNA in serum of δ-infected chimpanzees.Proc Natl Acad Sci U S A. 1980; 77: 6124-6128Crossref PubMed Scopus (344) Google Scholar HDV contains an approximately 1.7-kb single-stranded circular RNA genome.4Kos A. Dijkema R. Arnberg A.C. Van der Meide P.H. Schellekens H. The hepatitis delta (δ) virus possesses a circular RNA.Nature. 1986; 323: 558-560Crossref PubMed Scopus (465) Google Scholar, 5Wang K.S. Choo Q.L. Weiner A.J. Ou H.J. Najarian R.C. Thayer R.M. Mullenbach G.T. Denniston K.J. Gerin J.L. Houghton M. Structure, sequence and expression of the hepatitis delta (δ) viral genome.Nature. 1986; 323: 508-514Crossref PubMed Scopus (516) Google Scholar, 6Makino S. Chang M.F. Shieh C.K. Kamahora T. Vannier D.M. Govindarajan S. Lai M.M.C. Molecular cloning and sequencing of a human hepatitis delta (δ) virus RNA.Nature. 1987; 329: 343-346Crossref PubMed Scopus (245) Google Scholar HDV replicates its genome through a double rolling-circle mechanism by using host transcriptional machinery.7Branch A.D. Robertson H.D. A replication cycle for viroids and other small infectious RNA’s.Science. 1984; 223: 450-455Crossref PubMed Scopus (333) Google Scholar HDV RNA encodes only a single-protein, hepatitis delta antigen (HDAg), from the antigenomic strand. HDAg has 2 isoforms, a small HDAg (SHDAg; 195 amino acids, 24 kDa) and a large HDAg (LHDAg; 214 amino acids, 27 kDa). The LHDAg contains an additional 19 amino acids at its C terminus by an RNA editing event of the termination codon of SHDAg during RNA replication.8Luo G. Chao M. Hsih S.Y. Sureau C. Nishikura K. Taylor J.M. A specific base transition occurs on replicating hepatitis delta virus RNA.J Virol. 1990; 64: 1021-1027Crossref PubMed Google Scholar Both HDAgs are nuclear phosphoprotein.9Chang M.F. Baker S.C. Soe L.H. Kamahora T. Keck J.G. Makino S. Govindarajan S. Lai M.M.C. Human hepatitis delta antigen is a nuclear phosphoprotein with RNA-binding activity.J Virol. 1988; 62: 2403-2410Crossref PubMed Google Scholar, 10Hwang S.B. Lee C.Z. Lai M.M.C. Hepatitis delta antigen expressed by recombinant baculoviruses: comparison of biochemical properties and post-translational modifications between the large and small forms.Virology. 1992; 190: 413-422Crossref PubMed Scopus (54) Google Scholar Although these 2 antigens are identical in sequence except C-terminal 19 amino acids in LHDAg (Figure 1A),11Lai M.M.C. The molecular biology of hepatitis delta virus.Annu Rev Biochem. 1995; 64: 259-286Crossref PubMed Scopus (262) Google Scholar, 12Weiner A.J. Choo Q.L. Wang K.S. Govindarajans S. Redeker A.G. Gerin J.L. Houghton M. A single antigenomic open reading frame of the hepatitis delta virus encodes the epitope(s) of both hepatitis delta antigen polypeptides p24 delta and p27 delta.J Virol. 1998; 62: 594-599Google Scholar they have entirely different functions in viral replication and assembly. The SHDAg is required for HDV RNA replication,13Kuo M.Y. Chao M. Taylor J. Initiation of replication of the human hepatitis delta virus genome from cloned DNA: role of delta antigen.J Virol. 1989; 63: 1945-1950Crossref PubMed Google Scholar whereas the LHDAg suppresses viral RNA replication and is required for HDV assembly.14Chao M. Hsieh S.Y. Taylor J.M. Role of two forms of hepatitis delta virus antigen: evidence for a mechanism of self-limiting genome replication.J Virol. 1990; 64: 5066-5069Crossref PubMed Google Scholar, 15Chang F.L. Chen P.J. Tu S.J. Wang C.J. Chen D.S. The large form of hepatitis delta antigen is crucial for assembly of hepatitis delta virus.Proc Natl Acad Sci U S A. 1991; 88: 8490-8494Crossref PubMed Scopus (261) Google Scholar, 16Ryu W.S. Bayer M. Taylor J.M. Assembly of hepatitis delta virus particles.J Virol. 1992; 66: 2310-2315Crossref PubMed Google Scholar The LHDAg contains a CaXX box at its C terminus, in which the cysteine residue is isoprenylated.10Hwang S.B. Lee C.Z. Lai M.M.C. Hepatitis delta antigen expressed by recombinant baculoviruses: comparison of biochemical properties and post-translational modifications between the large and small forms.Virology. 1992; 190: 413-422Crossref PubMed Scopus (54) Google Scholar, 17Glenn J.S. Watson J.A. Havel C.M. White J.M. Identification of a prenylation site in delta virus large antigen.Science. 1992; 256: 1331-1333Crossref PubMed Scopus (237) Google Scholar Isoprenylation of LHDAg is required for virion assembly.18Lee C.Z. Chen P.J. Lai M.M. Chen D.S. Isoprenylation of large hepatitis delta antigen is necessary but not sufficient for hepatitis delta virus assembly.Virology. 1994; 199: 169-175Crossref PubMed Scopus (59) Google ScholarTransforming growth factor-β (TGF-β) controls cell growth, differentiation, and apoptosis of cells, and it has an important function during embryonic developmental process.19Massagué J. TGF-beta signal transduction.Annu Rev Biochem. 1998; 67: 753-791Crossref PubMed Scopus (3964) Google Scholar, 20Derynck R. Akhurst R.J. Balmain A. TGF-beta signaling in tumor suppression and cancer progression.Nat Genet. 2001; 29: 117-129Crossref PubMed Scopus (1939) Google Scholar TGF-β has a major regulatory role in hepatic fibrosis and cirrhosis.21Castilla A. Prieto J. Fausto N. Transforming growth factors beta 1 and alpha in chronic liver disease Effects of interferon alfa therapy.N Engl J Med. 1991; 324: 933-940Crossref PubMed Scopus (636) Google Scholar TGF-β is also a potent growth inhibitor in many cell types, including carcinoma cells, endothelial cells, hepatocytes, and lymphocytes.22Coffey Jr, R.J. Bascom C.C. Sipes N.J. Graves-Deal R. Weissman B.E. Moses H.L. Selective inhibition of growth-related gene expression in murine keratinocytes by transforming growth factor beta.Mol Cell Biol. 1988; 8: 3088-3093Crossref PubMed Scopus (248) Google Scholar, 23Laiho M. DeCaprio J.A. Ludlow J.W. Livingston D.M. Massagué J. Growth inhibition by TGF-beta linked to suppression of retinoblastoma protein phosphorylation.Cell. 1990; 62: 175-185Abstract Full Text PDF PubMed Scopus (676) Google Scholar, 24Moses H.L. Yang E.Y. Pietenpol J.A. TGF-beta stimulation and inhibition of cell proliferation: new mechanistic insights.Cell. 1990; 63: 245-247Abstract Full Text PDF PubMed Scopus (876) Google Scholar, 25Newman M.J. Transforming growth factor beta and the cell surface in tumor progression.Cancer Metastasis Rev. 1993; 12: 239-254Crossref PubMed Scopus (49) Google Scholar, 26Ewen M.E. The cell cycle and the retinoblastoma protein family.Cancer Metastasis Rev. 1994; 13: 45-66Crossref PubMed Scopus (178) Google Scholar TGF-β–dependent signal pathway involves 2 transmembrane serine–threonine kinase receptors, known as type I and type II TGF-β receptors (TβR-I and TβR-II).27Attisano L. Wrana J.L. Lopez-Casillas F. Massagué J. TGF-beta receptors and actions.Biochim Biophys Acta. 1994; 1222: 71-80Crossref PubMed Scopus (311) Google Scholar, 28Derynck R. TGF-beta-receptor-mediated signaling.Trends Biochem Sci. 1994; 19: 548-553Abstract Full Text PDF PubMed Scopus (277) Google Scholar Ligand binding to TβR-II mediates receptor interaction between TβR-I and TβR-II. This interaction induces phosphorylation of TβR-I at its glycine and serine-rich domain (GS domain), thereby resulting in phosphorylation of Smad2 and Smad3. The phosphorylation of Smad2 and Smad3 causes conformational change and leads to formation of hetero-oligomeric complex with Smad4, which then translocates to the nucleus and regulates the transcription of target genes, either positively or negatively.29Deryneck R. Zhang Y. Feng X.H. Smads: transcriptional activators of TGF-beta responses.Cell. 1998; 95: 737-740Abstract Full Text Full Text PDF PubMed Scopus (945) Google Scholar, 30Attisano L. Wrana J.L. Smads as transcriptional co-modulators.Curr Opin Cell Biol. 2000; 12: 235-243Crossref PubMed Scopus (475) Google Scholar, 31Massagué J. Chen Y.G. Controlling TGF-beta signaling.Genes Dev. 2000; 14: 627-644PubMed Google Scholar, 32Shi Y. Massagué J. Mechanisms of TGF-beta signaling from cell membrane to the nucleus.Cell. 2003; 113: 685-700Abstract Full Text Full Text PDF PubMed Scopus (4739) Google ScholarThe activator protein-1 (AP-1) family of transcription factors are DNA-binding proteins consisting of Jun (c-Jun, JunB, and JunD), Fos (c-Fos, FosB, Fra-1, and Fra-2), Maf, and ATF2.33Eferl R. Wagner E.F. AP-1: a double-edged sword in tumorigenesis.Nat Rev Cancer. 2003; 3: 859-868Crossref PubMed Scopus (1600) Google Scholar, 34Karin M. Gallagher E. From JNK to pay dirt: Jun kinases, their biochemistry, physiology and clinical importance.IUBMB Life. 2005; 57: 283-295Crossref PubMed Scopus (352) Google Scholar These proteins belong to the basic-zipper (bZIP) family, and dimerization of AP-1 is achieved by the leucine zipper region.35Angel P. Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation.Biochim Biophys Acta. 1991; 1072: 129-157Crossref PubMed Scopus (3249) Google Scholar The activity of AP-1 can be regulated by various stimuli, including cytokines, growth factors, stress, and viral infection, and it controls the expression of target genes through binding to their specific promoters.34Karin M. Gallagher E. From JNK to pay dirt: Jun kinases, their biochemistry, physiology and clinical importance.IUBMB Life. 2005; 57: 283-295Crossref PubMed Scopus (352) Google Scholar c-Jun protein is the core component of AP-1 complexes.36Karin M. The regulation of AP-1 activity by mitogen-activated protein kinases.J Biol Chem. 1995; 270: 16483-16486Crossref PubMed Scopus (2245) Google Scholar The heteromeric complex of c-Jun–c-Fos, AP-1, regulates the cellular growth, proliferation, and transformation by the expression of cell cycle–related genes.37Hess J. Angel P. Schorpp-Kistner M. AP-1 subunits: quarrel and harmony among siblings.J Cell Sci. 2004; 117: 5965-5973Crossref PubMed Scopus (935) Google ScholarIn this study, we investigated the effects of HDAg on transcriptional regulation induced by TGF-β and AP-1. We demonstrated that the LHDAg, but not the SHDAg, up-regulated TGF-β– and c-Jun–dependent signaling cascades. These effects were mediated by isoprenylation of LHDAg, and hence, isoprenylation played a pivotal role in modulation of TGF-β and AP-1 signal transductions. Furthermore, we demonstrated that LHDAg synergistically activated hepatitis B virus X (HBx)–mediated TGF-β and c-Jun signaling cascades. Taken together, this is a newly identified novel function of LHDAg that may play an important role in HDV pathogenesis.Materials and MethodsPlasmid ConstructioncDNA encoding either SHDAg or LHDAg of HDV was amplified by polymerase chain reaction (PCR) by using the American isolate38Hwang S.B. Park K.J. Kim Y.S. Overexpression of hepatitis delta antigen protects insect cells from baculovirus–induced cytolysis.Biochem Biophys Res Commun. 1998; 244: 652-658Crossref PubMed Scopus (6) Google Scholar as a template and was subcloned into the pcDNA3 (Invitrogen, Carlsbad, CA), pGEX-4T-1 (Amersham Bioscience, Piscatawy, NJ), pEF6B/His-Myc (Invitrogen), and pGFP-C1 (Clontech, Mountain View, CA) vectors. HA-TβR-I (T204D), Flag-Smad2, Flag-Smad3, and Flag-Smad4 expression vectors were described elsewhere.39Choi S.H. Hwang S.B. Modulation of the transforming growth factor-β signal transduction pathway by hepatitis C virus nonstructural 5A protein.J Biol Chem. 2006; 281: 7468-7478Crossref PubMed Scopus (76) Google Scholar Flag-c-Jun, HA-c-Fos, and Flag-JNK1 expression plasmids were provided by Dr. Eui-Ju Choi (Korea University, Seoul, Korea). SHDAg-Myc, LHDAg-Myc, and Smad4-Myc expression plasmids were subcloned into the pEF6B/His-Myc vector. Deletion mutants of HDAg, Smad3, and c-Jun were generated by PCR and subcloned into the pGFP-C1, pEF6B/His-Myc, and Flag-pcDNA3 expression vectors. The gene encoding HBx protein (adr type) was subcloned into the pcDNA3 expression vector.Cell Culture and Transfection ExperimentHuh7 cells and Cos7 cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 100 IU/mL of penicillin–streptomycin. For transfection, ∼5 × 105 cells plated on 60-mm dishes were transfected with plasmid DNA by using Lipofectamine (Invitrogen).Luciferase Reporter Gene AssayHuh7 cells were transfected with expression plasmid SBE4-Luc, consisting of 4 Smad-binding elements in tandem,40Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Human Smad3 and Smad4 are sequence-specific transcription activators.Mol Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar 3TP-Lux reporter plasmid consisting of TGF-β–inducible elements in the promoter of the human plasminogen activator inhibitor–type I gene,41Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene.EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1573) Google Scholar AP-1-Luc (Stratagene, La Jolla, CA), and pCH110 (Amersham Biosciences) reference plasmid. The total DNA amount in each transfection was kept constant by adjustment with empty vector. At 36 hours after transfection, luciferase and β-galactosidase assays were performed as described elsewhere.42Kim B.C. Lee M.N. Kim J.Y. Lee S.S. Chang J.D. Kim S.S. Lee S.Y. Kim J.H. Roles of phosphatidylinositol 3-kinase and Rac in the nuclear signaling by tumor necrosis factor-alpha in rat-2 fibroblasts.J Biol Chem. 1999; 274: 24372-24377Crossref PubMed Scopus (55) Google ScholarGlutathione S-Transferase Pull-Down Assay and CoimmunoprecipitationGlutathione S-transferase (GST)–fusion proteins were expressed in Escherichia coli BL21 (DE3) cells (Novagen, San Diego, CA) and purified with glutathione–Sepharose 4B beads (Amersham Bioscience) according to the manufacturer’s instructions. At 36 hours after transfection, cells were lysed in buffer A (20 mmol/L Tris-HCl [pH 7.4], 150 mmol/L NaCl, 1 mmol/L ethylenediaminetetraacetic acid [EDTA], 1 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L β-glycerolphosphate, 1 mmol/L Na3VO4, 1 μg/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride). The cell lysates were processed by centrifuge at 15,000 rpm for 10 minutes, and the protein concentration was determined with a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA).For the in vitro binding assay, the cell lysates were incubated with 2 μg of either GST or GST-fusion proteins for 2 hours at 4°C in cell lysis buffer A. Samples were washed four times in cell lysis buffer A, and the bound proteins were detected by Western blot analysis. Alternatively, Flag-c-jun (224–331 aa) and HDAgs were translated in vitro by using a TNT quick-coupled transcription–translation system (Promega, Madison, WI) for 90 minutes at 30°C. In vitro–translated [35S] methionine-labeled Flag-c-Jun and HDAgs were incubated with either GST or GST-Smad3 protein. The bound proteins were subjected to 15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and visualized by autoradiography.For the coimmunoprecipitation assay, Cos7 cells were infected with the recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase.43Fuerst T.R. Niles E.G. Studier F.W. Moss B. Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase.Proc Natl Acad Sci U S A. 1986; 83: 8122-8126Crossref PubMed Scopus (1864) Google Scholar At 2 hours after infection, cells were transfected with 5 μg of the corresponding plasmids. After incubation at 37°C for 12 hours, cells were harvested and lysed in buffer B (50 mmol/L HEPES-KOH [pH 7.5], 150 mmol/L NaCl, 10% glycerol, 1 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 1% Triton X-100, 1.5 mmol/L MgCl2, 10 mmol/L sodium pyrophosphate, 100 mmol/L NaF, 1 mmol/L Na3VO4, and 1 mmol/L phenylmethylsulfonyl fluoride). The cell lysates were processed by centrifuge at 15,000 rpm for 10 minutes, and the supernatant was incubated at 4°C for 2 hours with the corresponding antibodies. The samples were further incubated with protein-A beads (Zymed, San Francisco, CA) for 1 hour. The beads were washed 5 times with cell lysis buffer B, and the bound proteins were detected by Western blot analysis.Confocal MicroscopyEither Huh7 or Cos7 cells grown on chamber slides (Nunc, Rochester, NY) were transfected with the corresponding plasmids. At 36 hours after transfection, cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde and 0.1% Triton X-100 for 20 minutes at 37°C. Cells were incubated in 5% bovine serum albumin for 20 minutes and then incubated with anti-Flag monoclonal antibody for 1 hour at 37°C. After being washed three times with PBS, cells were further incubated with tetramethylrhodamine isothiocyanate-conjugated goat anti-mouse IgG (American Qualex) for 30 minutes at 37°C. After 2 washes with 0.1% Triton X-100 in PBS and 3 washes with PBS, cells were analyzed by using the LSM 510 laser confocal microscopy system (Carl Zeiss, Dublin, CA).Coimmunoprecipitation of the Smad3–Smad4 ComplexApproximately 5 × 105 cells plated on 60-mm dishes were transfected with the Flag-Smad3, Smad4-Myc, HDAgs, and HA-TβR-I (T204D) expression plasmids by using Lipofectamine. At 36 hours after transfection, cells were lysed in buffer B and subjected to immunoprecipitation with anti-Flag monoclonal antibody. Bound Smad4 protein was detected by Western blot analysis by using anti-Myc monoclonal antibody.Electrophoretic Mobility Shift AssayHuh7 cells were transfected with the corresponding plasmids. At 24 hours, cells were treated with human TGF-β (5 ng/mL) for 24 hours, and then nuclear fractions were prepared as described elsewhere.39Choi S.H. Hwang S.B. Modulation of the transforming growth factor-β signal transduction pathway by hepatitis C virus nonstructural 5A protein.J Biol Chem. 2006; 281: 7468-7478Crossref PubMed Scopus (76) Google Scholar Briefly, cells were harvested by scraping in PBS and were incubated in buffer C (10 mmol/L HEPES-KOH [pH 7.9], 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.1% Nonidet P-40, 0.5 mmol/L dithiothreitol, and 0.5 mmol/L phenylmethylsulfonyl fluoride) for 5 minutes on ice. Crude nuclei were collected by centrifugation at 10,000 rpm for 2 minutes at 4°C. The pellets were rinsed once in buffer C and resuspended in buffer D (20 mmol/L HEPES-KOH [pH 7.9], 1.5 mmol/L MgCl2, 420 mmol/L KCl, 25% glycerol, 0.2 mmol/L EDTA, 0.5 mmol/L dithiothreitol, 0.5 mmol/L phenylmethylsulfonyl fluoride). The sample was further incubated with agitation in a cold room for 30 minutes and processed by centrifuge at 15,000 rpm for 10 minutes at 4°C. The protein concentration in nuclear extracts was determined by the Bradford method (Bio-Rad). Nuclear extract (10 μg) was incubated with an oligonucleotide probe44Lee D.K. Kim B.C. Kim I.Y. Cho E.A. Satterwhite D.J. Kim S.J. The human papilloma virus E7 oncoprotein inhibits transforming growth factor-beta signaling by blocking binding of the Smad complex to its target sequence.J Biol Chem. 2002; 277: 38557-38564Crossref PubMed Scopus (75) Google Scholar, 45Park K.J. Choi S.H. Choi D.H. Park J.M. Yie S.W. Lee S.Y. Hwang S.B. Hepatitis C virus NS5A protein modulates c-Jun N-terminal kinase through interaction with tumor necrosis factor receptor-associated factor 2.J Biol Chem. 2003; 278: 30711-30718Crossref PubMed Scopus (63) Google Scholar labeled with 32P (1 × 105 cpm) in 20 μL of binding buffer (20% glycerol, 5 mmol/L MgCl2, 2.5 mmol/L EDTA, 2.5 mmol/L dithiothreitol, 250 mmol/L NaCl, 50 mmol/L Tris-HCl [pH 7.5], and 0.25 mg/mL poly [dI-dC]) at room temperature for 20 minutes. The protein-DNA complexes were separated by electrophoresis on a 5% native polyacrylamide gel using 0.25× Tris borate–EDTA buffer and detected by autoradiography. For the competition assay, 100-fold molar excess of unlabeled oligonucleotide was incubated with nuclear extract in binding buffer for 20 minutes before the addition of radiolabeled oligonucleotide.Western-Blot AnalysisHuh7 cells were transfected with the corresponding expression plasmids. At 24 hours after transfection, cells were treated with human TGF-β (5 ng/mL) for the indicated times and were incubated in buffer B. Equal amounts of proteins were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred to a nitrocellulose membrane. The membrane was blocked in PBS containing 5% nonfat dry milk for 1 hour and then incubated overnight at 37°C with primary antibody in TTBS (20 mmol/L Tris-HCl [pH 7.5], 500 mmol/L NaCl, 0.05% Tween-20). After 2 washes in TTBS, the membrane was incubated with either horseradish peroxidase–conjugated goat anti-rabbit antibody or goat anti-mouse antibody (Jakson Immunoresearch, Suffolk, UK) in TTBS for 90 minutes at room temperature. Proteins were detected with an enhanced chemiluminescence kit (Amersham Biosciences).ResultsLHDAg Activates TGF-β–Induced Transcriptional ActivityTGF-β exerts a pivotal role in hepatic fibrosis and cirrhosis in chronic liver disease.21Castilla A. Prieto J. Fausto N. Transforming growth factors beta 1 and alpha in chronic liver disease Effects of interferon alfa therapy.N Engl J Med. 1991; 324: 933-940Crossref PubMed Scopus (636) Google Scholar HDV infection leads to chronic hepatitis and liver cirrhosis.1Rizzetto M. Canese M.G. Arico S. Crivelli O. Trepo C. Bonino F. Verme G. Immunofluorescence detection of new antigen-antibody system (delta/anti-delta) associated to hepatitis B virus in liver and in serum of HBsAg carriers.Gut. 1977; 18: 997-1003Crossref PubMed Scopus (657) Google Scholar, 2Niro G.A. Rosina F. Rizzetto M. Treatment of hepatitis D.J Viral Hepat. 2005; 12: 2-9Crossref PubMed Scopus (106) Google Scholar Because HDAg is the only protein encoded by HDV RNA, we first examined the role of HDAg in the TGF-β–dependent transcriptional activation by using a luciferase reporter gene assay. Huh7 cells were transiently cotransfected with the HDAg expression plasmid and either the SBE4-Luc reporter plasmid that contains four Smad-binding elements40Zawel L. Dai J.L. Buckhaults P. Zhou S. Kinzler K.W. Vogelstein B. Kern S.E. Human Smad3 and Smad4 are sequence-specific transcription activators.Mol Cell. 1998; 1: 611-617Abstract Full Text Full Text PDF PubMed Scopus (887) Google Scholar or the TGF-β–responsive 3TP-Lux reporter plasmid.41Dennler S. Itoh S. Vivien D. ten Dijke P. Huet S. Gauthier J.M. Direct binding of Smad3 and Smad4 to critical TGF beta-inducible elements in the promoter of human plasminogen activator inhibitor-type 1 gene.EMBO J. 1998; 17: 3091-3100Crossref PubMed Scopus (1573) Google Scholar At 24 hours after transfection, cells either were left untreated or were treated with human TGF-β (5 ng/mL) for 24 hours, and then luciferase activities were measured. As shown in Figure 1B and C, TGF-β induced a 15- to 20-fold transactivation of the reporters in vector-transfected Huh7 cells. Overexpression of LHDAg elevated TGF-β–induced transcriptional activation ∼10–13-fold of the vector control in both reporter plasmids. However, overexpression of SHDAg had no effect on TGF-β–induced transcriptional activation. It is noteworthy that LHDAg in the absence of TGF-β stimulation activated ∼10-fold of the reporter gene activity. We further investigated the dosage effect of HDAgs on TGF-β–induced transcriptional activation. Huh7 cells were transfected with constitutively active TβR-I (T204D), together with increased amounts (2 and 5 μg) of either SHDAg or LHDAg. Overexpression of LHDAg, but not SHDAg, elevated TGF-β–induced transcriptional activation in a dose-dependent manner (Figure 1D). We found that overexpression of either green fluorescence protein (GFP) or ras protein as a control showed no effect on TGF-β–induced transcriptional activation (data not shown). These results suggest that LHDAg specifically activates the TGF-β–induced signal-transduction pathway at the transcriptional level in Huh7 cells. To further explore the effect of LHDAg on TGF-β–induced Smad-DNA complex, we performed electrophoretic mobility gel shift assay (EMSA). Huh7 cells were transiently transfected individually with empty vector, SHDAg, or LHDAg expression plasmids. At 24 hours after transfection, cells were stimulated with TGF-β for 24 hours, and EMSA was performed by using the TGF-β–responsive element in the plasminogen activator inhibitor (PAI)-1 promoter (−586 to −551) as a probe.44Lee D.K. Kim B.C. Kim I.Y. Cho E.A. Satterwhite D.J. Kim S.J. The human papilloma virus E7 oncoprotein inhibits transforming growth factor-beta signaling by blocking binding of the Smad complex to its target sequence.J Biol Chem. 2002; 277: 38557-38564Crossref PubMed Scopus (75) Google Scholar Upon TGF-β treatment, shifted bands of Smad-DNA complexes appeared in the vector control cells (Figure 1E, lane 2), and these complexes were markedly increased in cells expressing LHDAg but not SHDAg (Figure 1E, lane 5 versus lane 8). It is noteworthy that TGF-β–induced Smad-DNA complexes form distinctive triple bands, as reported elsewhere.39Choi S.H. Hwang S.B. Modulation of the transforming growth factor-β signal transduction pathway by hepatitis C virus nonstructural 5A protein.J Biol Chem. 2006; 281: 7468-7478Crossref PubMed Scopus (76) Google Scholar, 44Lee D.K. Kim B.C. Kim I.Y. Cho E.A. Satterwhite D.J. Kim S.J. The human papilloma virus E7 oncoprotein inhibits transforming growth factor-beta signaling by blocking binding of the Smad complex to its target sequence.J Biol Chem. 2002; 277: 38557-38564Crossref PubMed Scopus (75) Google Scholar The disappearance of TGF-β–responsive complexes when an unlabeled probe was used as a competitor further suggests the specificity of Smad-DNA binding (Figure 1E, lanes 3, 6, and 9). These results suggest that LHDAg stimulates the TGF-β signal-transduction cascade through activation of Smad-DNA binding activity.Both LHDAg and SHDAg Interact With Smad3 In Vitro and In VivoBecause LHDAg, but not SHDAg, activated the TGF-β signal-tran" @default.
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• W1987515540 creator A5087995943 @default.
• W1987515540 date "2007-01-01" @default.
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• W1987515540 title "Large Hepatitis Delta Antigen Modulates Transforming Growth Factor-β Signaling Cascades: Implication of Hepatitis Delta Virus–Induced Liver Fibrosis" @default.
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• W1987515540 doi "https://doi.org/10.1053/j.gastro.2006.10.038" @default.
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