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• W1997931429 abstract "Helicobacter pylori interacts with the apical membrane of the gastric epithelium and induces a number of proinflammatory cytokines/chemokines. The subsequent infiltration of macrophages and granulocytes into the mucosa leads to gastric inflammation accompanied by epithelial degeneration. Gastric diseases,e.g. peptic ulcer or gastric adenocarcinoma, are more common among people infected with H. pylori strains producing VacA (vacuolating cytotoxin A) and possessing acag (cytotoxin-associated antigen A) pathogenicity island. For the induction of the cytokine/chemokine genes in response toH. pylori, we studied the signaling leading to the nuclear activation of the early response transcription factor activator protein 1 (AP-1). We found that H. pylori strains carrying the pathogenicity island induce activation of AP-1 and nuclear factor κB. In contrast to the wild type or an isogenic strain without thevacA gene, isogenic H. pylori strains with mutations in certain cag genes revealed only weak AP-1 and nuclear factor κB activation. In respect to the molecular components that direct AP-1 activity, our results indicate a cascade of the cellular stress response kinases c-Jun N-terminal kinase, MAP kinase kinase 4, and p21-activated kinase, and small Rho-GTPases including Rac1 and Cdc42, which contributes to the activation of proinflammatory cytokines/chemokines induced by H. pylori encoding thecag pathogenicity island. Helicobacter pylori interacts with the apical membrane of the gastric epithelium and induces a number of proinflammatory cytokines/chemokines. The subsequent infiltration of macrophages and granulocytes into the mucosa leads to gastric inflammation accompanied by epithelial degeneration. Gastric diseases,e.g. peptic ulcer or gastric adenocarcinoma, are more common among people infected with H. pylori strains producing VacA (vacuolating cytotoxin A) and possessing acag (cytotoxin-associated antigen A) pathogenicity island. For the induction of the cytokine/chemokine genes in response toH. pylori, we studied the signaling leading to the nuclear activation of the early response transcription factor activator protein 1 (AP-1). We found that H. pylori strains carrying the pathogenicity island induce activation of AP-1 and nuclear factor κB. In contrast to the wild type or an isogenic strain without thevacA gene, isogenic H. pylori strains with mutations in certain cag genes revealed only weak AP-1 and nuclear factor κB activation. In respect to the molecular components that direct AP-1 activity, our results indicate a cascade of the cellular stress response kinases c-Jun N-terminal kinase, MAP kinase kinase 4, and p21-activated kinase, and small Rho-GTPases including Rac1 and Cdc42, which contributes to the activation of proinflammatory cytokines/chemokines induced by H. pylori encoding thecag pathogenicity island. interleukin nuclear factor κB activator protein 1 mitogen-activated protein kinase MAPK/extracellular signal-regulated kinase MEK kinase stress-activated protein kinase c-Jun N-terminal kinase MAP kinase kinase p21-activated kinase multiplicity of infection phorbol 12-myristate 13-acetate glutathioneS-transferase The immune response to Helicobacter pylori infection is initiated by a number of inflammatory mediators including cytokines and chemokines, which are produced from the gastric epithelium. In vitro and in vivo studies have shown that H. pylori induces chemokines IL-8,1 RANTES, GRO-α, MIP-1α, ENA-78 and MCP-1, and cytokines IL-1, IL-6, and tumor necrosis factor α (1Bodger K. Crabtree J.E. Br. Med. Bull. 1998; 54: 139-150Crossref PubMed Scopus (158) Google Scholar). The epithelial cytokine/chemokine response may be particularly important in the early stages of H. pylori-induced inflammation, wherein the epithelium represents the crucial first barrier of defense against pathogen infection. Inflammatory mediators produced from infiltrated polymorphonuclear leukocytes and mononuclear phagocytes could directly damage the surface epithelial layer leading to loss of microvilli, irregularity of the luminal border, and vacuolation (2Goodwin C.S. Armstrong J.A. Marshall B.J. J. Clin. Pathol. 1986; 39: 353-365Crossref PubMed Scopus (474) Google Scholar). The events that commonly follow the infection consist of gastritis, peptic ulcer (3Blaser M.J. Gastroenterology. 1987; 93: 371-383Abstract Full Text PDF PubMed Google Scholar, 4Graham D.Y. Gastroenterology. 1989; 96: 615-625Abstract Full Text PDF PubMed Scopus (681) Google Scholar), rarely gastric cancer, and low grade B-cell mucosal-associated lymphoid tissue-associated gastric lymphoma (5Parsonnet J. Friedman G.D. Vandersteen D.P. Chang Y. Vogelman J.H. Orentreich N. Sibley R.K. N. Engl. J. Med. 1991; 325: 1127-1131Crossref PubMed Scopus (3565) Google Scholar, 6Parsonnet J. Hansen S. Rodriguez L. Gelb A.B. Warnke R.A. Jellum E. Orentreich N. Vogelman J.H. Friedman G.D. N. Engl. J. Med. 1994; 330: 1267-1271Crossref PubMed Scopus (1712) Google Scholar). The inflammatory response and gastric diseases are more common in patients infected with H. pylori strains carrying thecagA gene (cytotoxin-associated gene A). These strains also produce the toxin VacA (vacuolating toxin A), which is responsible for cytopathic effects (7Telford J.L. Ghiara P. Dell'Orco M. Comanducci M. Burroni D. Bugnoli Tecce M.F. Censini S. Covacci A. Xiang Z. J. Exp. Med. 1994; 179: 1653-1658Crossref PubMed Scopus (519) Google Scholar, 8Marchetti M. Arico B. Burroni D. Figura N. Rappuoli R. Ghiara P. Science. 1995; 267: 1655-1658Crossref PubMed Scopus (531) Google Scholar). The analysis of the genomic region containing the cagA gene revealed a 40-kilobase DNA region that is present only in H. pylori strains inducing the production of the active form of the toxin and severe gastroduodenal diseases. The 40-kilobase region represents a pathogenicity island and codes for approximately 30 genes (9Censini S. Lange C. Xiang Z. Crabtree J.E. Ghiara P. Borodovsky M. Rappuoli R. Covacci A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14648-14653Crossref PubMed Scopus (1633) Google Scholar, 10Covacci A. Rappuoli R. Curr. Opin. Microbiol. 1998; 1: 96-102Crossref PubMed Scopus (59) Google Scholar). Comparison of the genes encoded from the pathogenicity island with genes in other species suggests that the cag region encodes a specialized secretion system that exports or allows surface expression of proteins that interact with epithelial cells (10Covacci A. Rappuoli R. Curr. Opin. Microbiol. 1998; 1: 96-102Crossref PubMed Scopus (59) Google Scholar). Knockouts of certaincag genes suppressed or reduced the production of IL-8 in epithelial cells (11Atherton J.C. Br. Med. Bull. 1998; 54: 105-120Crossref PubMed Scopus (165) Google Scholar), affected activation of the immediate early response transcription factor nuclear factor κB (NF-κB) (12Glocker E. Lange C. Covacci A. Bereswill S. Kist M. Pahl H.L. Infect. Immun. 1998; 66: 2346-2348Crossref PubMed Google Scholar), and blocked tyrosine phosphorylation of a 145-kDa host protein (13Segal E.D. Lange C. Covacci A. Tompkins L.S. Falkow S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7595-7599Crossref PubMed Scopus (165) Google Scholar), suggesting that the integrity of the whole pathogenicity island contributes in chronic inflammation. The inflammatory reaction requires de novo synthesis of defined proteins, which include chemokines attracting macrophages and inflammatory cytokines that serve to amplify and spread the primary pathogenic signal. The mechanism by which these proteins are newly synthesized involves an inducible transcriptional initiation of their respective genes. This is governed by several transcription factors playing a role in regulating immune response genes including the early response transcription factor AP-1 (14Karin M. Liu Z.G. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2267) Google Scholar). Very little is known about the nature of the H. pylori-induced proinflammatory signals and the intracellular signals directing the activation of immediate early response transcription factors. Previous results showed that infection of epithelial cells with H. pylori induced the activation of the transcription factor NF-κB (15Keates S. Hitti Y.S. Upton M. Kelly C.P. Gastroenterology. 1997; 113: 1099-1109Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 16Münzenmaier A. Lange C. Glocker E. Covacci A. Moran A. Bereswill S. Baeuerle P.A. Kist M. Pahl H.L. J. Immunol. 1997; 159: 6140-6147PubMed Google Scholar, 17Sharma S.A. Tummuru M.K.R. Blaser M.J. Kerr L.D. J. Immunol. 1998; 160: 2401-2407PubMed Google Scholar). In line with the known immunostimulatory function of NF-κB and AP-1 (18Baeuerle P.A. Baichwal V.R. Adv. Immunol. 1997; 65: 111-137Crossref PubMed Google Scholar, 19Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1369) Google Scholar) epithelial cells infected with H. pylori produced increased amounts of numerous proinflammatory cytokines/chemokines (11Atherton J.C. Br. Med. Bull. 1998; 54: 105-120Crossref PubMed Scopus (165) Google Scholar). The activation of the early response transcription factor AP-1 and the signaling pathways involved in the production of cytokines/chemokines in H. pylori-colonized gastric cells have not been studied so far. A common mechanism by which eucaryotic cells respond to extracellular signals involves the activation of AP-1 and a family of mitogen-activated protein kinases (MAPK) that consecutively activate their members by phosphorylation. MAPK cascades or modules are composed of a MAPK, a MAPK kinase, or mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK), and a MAPK kinase kinase or MEK kinase (MAPK kinase kinase kinase or MEKK) (20Wilkinson M.G. Millar J.B.A. Genes Dev. 1998; 12: 1391-1397Crossref PubMed Scopus (71) Google Scholar). Subfamiles of MAPKs, the stress-activated protein kinases (SAPK) are activated by multiple environmental stresses and target the transcription factors c-Jun and ATF2, which are components of AP-1 (14Karin M. Liu Z.G. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2267) Google Scholar). Phosphorylation and activation of c-Jun occur by c-Jun N-terminal kinases (JNKs) in N-terminally located transactivation domains and results in increased transcriptional activity (21Kallunki T. Su B. Tsigelny I. Sluss H.K. Derijard B. Moore G. Davis Karin M. Genes Dev. 1994; 8: 2996-3007Crossref PubMed Scopus (591) Google Scholar, 22Gupta S. Barrett T. Whitmarsh A.J. Cavanagh J. Sluss H.K. Derijard B. Davis R.J. EMBO J. 1996; 15: 2760-2770Crossref PubMed Scopus (1166) Google Scholar). JNKs themselves are activated by MAP kinase kinases 4 and 7 (MKK4 and MKK7) (23Sanchez I. Hughes R.T. Mayer B.J. Yee K. Woodgett J.R. Avruch J. Kyriakis J.M. Zon L.I. Nature. 1994; 372: 794-798Crossref PubMed Scopus (912) Google Scholar, 24Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (706) Google Scholar, 25Holland P.M. Suzanne M. Campbell J.S. Noselli S. Cooper J.A. J. Biol. Chem. 1997; 272: 24994-24998Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 26Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (1989) Google Scholar, 27Tournier C. Whitmarsh A.J. Cavanagh J. Barrett T. Davis R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7337-7342Crossref PubMed Scopus (337) Google Scholar, 28Wu Z. Wu J. Jacinto E. Karin M. Mol. Cell. Biol. 1997; 17: 7407-7416Crossref PubMed Scopus (94) Google Scholar), and MKK4 could be activated by MEKK1 (29Minden A. Lin A. Mcmahon M. Lange-Carter C. Derijard B. Davis R.J. Johnson G.L. Karin M. Science. 1994; 266: 1719-1723Crossref PubMed Scopus (1009) Google Scholar). Numerous MAPK kinase kinases in addition to MEKK1 have recently been identified and are all able to activate the JNK pathway (24Lin A. Minden A. Martinetto H. Claret F.X. Lange-Carter C. Mercurio F. Johnson G.L. Karin M. Science. 1995; 268: 286-290Crossref PubMed Scopus (706) Google Scholar, 30Bagrodia S. Derijard B. Davis R. Cerione R. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar, 31Pombo C.M. Kehrl J.H. Sanchez I. Katz P. Avruch J. Zon L.I. Woodgett JR Force T. Kyriakis J.M. Nature. 1995; 377: 750-754Crossref PubMed Scopus (203) Google Scholar, 32Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1166) Google Scholar, 33Rana A. Gallo K. Godowski P. Hirai S. Ohno S. Zon L. Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 19025-19028Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 34Salmeron A. Ahmad T.B. Carlile G.W. Pappin D. Narsimhan R.P. Ley S.C. EMBO J. 1996; 15: 817-826Crossref PubMed Scopus (268) Google Scholar). The p21-activated kinases (PAKs) have been identified as upstream regulators of the JNK kinase cascade (30Bagrodia S. Derijard B. Davis R. Cerione R. J. Biol. Chem. 1995; 270: 27995-27998Abstract Full Text Full Text PDF PubMed Scopus (597) Google Scholar, 35Coso O.A. Chiariello M., Yu, J.C. Teramoto H. Crespo P. Xu N. Miki T. Gutkind J.S. Cell. 1995; 81: 1137-1146Abstract Full Text PDF PubMed Scopus (1555) Google Scholar, 36Minden A. Lin A. Claret F.X. Abo A. Karin M. Cell. 1995; 81: 1147-1157Abstract Full Text PDF PubMed Scopus (1442) Google Scholar) and were the first kinases identified as direct effectors for the active small Rho-GTPases Rac1 and Cdc42 (37Manser E. Leung T. Salihuddin H. Zhao Z.S. Lim L. Nature. 1994; 367: 40-46Crossref PubMed Scopus (1280) Google Scholar). Similar to PAK, the MEK kinases 1 and 4 have also been shown to interact with Rac and Cdc42 (38Fanger G.R. Johnson N.L. Johnson G.L. EMBO J. 1997; 16: 4961-4972Crossref PubMed Scopus (253) Google Scholar). Despite the potential for extensive cross-talk, it is generally observed that individual MAPKs are activated in response to distinct sets of environmental stimuli. Here we show that colonization of gastric cells by H. pyloriinduces the activation of the AP-1 transcription factor by a distinct SAPK cascade involving JNK, MKK4, PAK, and Rho-GTPases but not p38 kinase. Activation of AP-1 and NF-κB is substantially reduced in cells colonized by certain cag mutant strains. The identification of H. pylori-specific signaling pathways to inflammatory cytokine production casts a light on ways for drug intervention. Gastric epithelial cells (AGS) and HeLa cells were grown in RPMI 1640 containing 4 mm glutamine (Life Technologies, Inc.), 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% fetal calf serum (Life Technologies, Inc.) in a humidified 5% CO2 atmosphere. The cells were seeded in tissue culture plates for 48 h prior to infection. 16 h before infection, the medium was replaced by fresh RPMI 1640 medium supplemented either with 0.1% fetal calf serum (AGS cells) or 5% fetal calf serum (HeLa cells). H. pyloristrains were cultured for 48–72 h on agar plates containing 10% horse serum in a microaerophilic atmosphere (generated by Campy Gen, Oxoid, Basingstoke, UK) at 37 °C. For the infection the bacteria were harvested in phosphate-buffered saline, pH 7.4, diluted corresponding to the multiplicity of infection (MOI) as indicated and incubated together with the epithelial cell monolayer for different periods of time. Infection with H. pylori was routinely monitored by light microscopy. Stimulation of the cells with 100 nmphorbol 12-myristate 13-acetate (PMA; Sigma) was performed for the indicated periods of time. In the experiments using 10 ng/ml Toxin B (F. Hofmann and K. Aktories, Freiburg, Germany), the cells were preincubated for 15 min before the bacteria were added. Different H. pylori strains were used for colonization of human epithelial cell lines. The isogenicP12 strains, wild type,cagA*− (mutation of cagA with a probable polar effect), and vacA − (39Schmitt W. Haas R. Mol. Microbiol. 1994; 12: 307-319Crossref PubMed Scopus (280) Google Scholar) and the isogenic G27 strains, wild type,cagF −, and cagI − (9Censini S. Lange C. Xiang Z. Crabtree J.E. Ghiara P. Borodovsky M. Rappuoli R. Covacci A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14648-14653Crossref PubMed Scopus (1633) Google Scholar) have been described previously. For cultivation the bacteria were resuspended in brain heart infusion (Difco, Detroit, MI) medium, and 103 bacteria were seeded per plate. For stock culturesH. pylori was resuspended in brain heart infusion and additionally supplemented with 10% fetal calf serum and 20% glycerol and maintained at −70 °C. Nuclear extracts were prepared by using a nonionic detergent method as described previously (40Naumann M. Scheidereit C. EMBO J. 1994; 13: 4597-4607Crossref PubMed Scopus (325) Google Scholar). Electrophoretic mobility shift assay for the detection of AP-1 activity in nuclear extracts was performed using oligonucleotides containing the AP-1 binding site: 5′-GATCTTCTAGACCGGATGAGTCATAGCTTG-3′ and 5′-CAAGCTATGACTCATCCGGTCTAGAAGATC-3′. The AP-1 DNA-binding oligonucleotide was labeled using T4 kinase (Roche Molecular Biochemicals) in the presence of [γ-32P]ATP. DNA binding reactions were performed using a binding buffer containing 10 mm Tris, pH 7.5, 2 μg of poly(dI-dC), 1 μg of bovine serum albumin, 10 mm MgCl2, 100 mmKCl, 1 mm EDTA, 1 mm dithiothreitol, 0.5 ng of unlabeled double-stranded oligonucleotide, and 10% glycerol. Supershift analysis was performed using c-Jun (sc-45) and ATF2 (sc-187) antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) that were preincubated together with nuclear extracts before the32P-labeled oligonucleotide was added. Electrophoretic mobility shift assay for the detection of NF-κB were performed with an Igκ oligo probe as described previously (41Naumann M. Wulczyn F.G. Scheidereit C. EMBO J. 1993; 12: 213-222Crossref PubMed Scopus (124) Google Scholar). The oligonucleotide containing the NF-κB recognition site was labeled using the large fragment DNA polymerase (Klenow; Roche Molecular Biochemicals) in the presence of [α-32P]dATP. The DNA binding reactions were performed with 20 μl of binding buffer (2 μg of poly(dI-dC), 1 μg of bovine serum albumin, 5 mm dithiothreitol, 20 mm HEPES, pH 8.4, 60 mm KCl, and 10% glycerol) for 20 min at 30 °C. Supershift analysis was performed using anti-p50, anti-p65, and anti-c-Rel antibodies as described previously (40Naumann M. Scheidereit C. EMBO J. 1994; 13: 4597-4607Crossref PubMed Scopus (325) Google Scholar). For competition experiments cold oligonucleotides were used. The reaction products were analyzed by electrophoresis in a 5% polyacrylamide gel using 12.5 mm Tris, 12.5 mmboric acid, and 0.25 mm EDTA, pH 8.3. The gels were dried and exposed to Amersham Pharmacia Biotech TM film at −70 °C using an intensifying screen. Quantitation of gel shift assays was performed by scanning the autoradiographs and densitometric analysis using the software program TINA (Raytest, Isotopen Messgeräte GmbH, München, Germany). The fold activity against the control is indicated below the gel shifts. IL-8 secretion was assayed from 100-μl supernatants of H. pylori-colonized AGS cells or HeLa cells by enzyme-linked immunosorbent assay. The IL-8 enzyme-linked immunosorbent assay was performed as described by the manufacturer's instructions (Pharmingen, Torreyana, CA). Transactivating activity of AP-1 was measured in AGS or HeLa cells at 50–70% confluence after cotransfection of the pSV-β-galactosidase construct (Promega, Heidelberg, Germany), 1 μg of a luciferase expression plasmid containing three repeats of the AP-1 binding site as an enhancer element and dominant negative expression constructs: DNPAK2(K278R), DNPAK1(K299R), or DNPAK1(H83L, H86L, K299R), DNMKK4 (K116R, DNJNKK), GFPDNRac1(T17N), and GFPDNCdc42(T17N) using cationic liposomes (DAC-30, Eurogentec, Sart Tilman, Belgium). 16 h after transfection cells were infected with H. pylori strains, treated with 100 nm PMA, preincubated with Toxin B, or left untreated. Luciferase assays were performed 3–4 h after treatment as recommended by the manufacturer's instructions (Promega). The results were recorded on a Wallac 409 β-counter (Berthold-Wallac, Bad Wildbad, Germany). The data represent the means ± S.D. calculated from more than three independent experiments as fold induction compared with the control. A portion of the cell lysates that was normalized for equivalent β-galactosidase activity was used for the luciferase assay. Activities varied <10% between transfection experiments. Immunoblots below the graphs indicate the expression of the dominant negative expression constructs DNMKK4, DNPAK1(Myc-tag), GFPDNRac1, and GFPDNCdc42 using the antibodies anti-MKK4 sc-837 (Santa Cruz), anti-Myc 14851A (Pharmingen), and anti-GFP sc-8334 (Santa Cruz), respectively. To analyze the kinase activity of JNK and p38, cells were lysed in RIPA buffer containing 20 mm Tris, pH 8.0, 150 mm NaCl, 0.5% Nonidet P-40, 0.05% SDS, 5% glycerol, 1 mm EGTA, 10 mm NaF, 10 mm K2HPO4, 1 mm Na3VO4, 100 μmphenylmethylsulfonyl fluoride, 10 μm pepstatin A, and 4 μm aprotinin. For PAK, cells were lysed in 50 mm Tris, pH 7.5, 1 mm EGTA, 5 mmMgCl2, 1% Nonidet P-40, 2,5% glycerol, 150 mmNaCl, 1 mm Na3VO4, 10 mm Na4P2O7, 50 mm NaF, 100 μm phenylmethylsulfonyl fluoride, 10 μm pepstatin A, and 4 μm aprotinin. For immunoprecipitation RIPA buffer lysed cells were disrupted and incubated with anti-JNK1 (sc-474, Santa Cruz) antibody, and anti-αPAK (sc-881, Santa Cruz) antibody detecting PAK1 and partially PAK2 as described previously (42Naumann M. Rudel T. Wieland B. Bartsch C. Meyer T.E. J. Exp. Med. 1998; 188: 1277-1286Crossref PubMed Scopus (38) Google Scholar). Immunocomplexes were recovered and washed, and immunoprecipitates were used for in vitro kinase reactions using substrates (1 μg of GST-c-Jun (Santa Cruz) for JNK; 1 μg of GST-ATF2 (Santa Cruz) for p38; 2.5 μg of myelin basic protein (Upstate Biotechnology Inc.) for PAK). The samples were separated in SDS-polyacrylamide gel electrophoresis and dried, and substrate phosphorylation was visualized by autoradiography (42Naumann M. Rudel T. Wieland B. Bartsch C. Meyer T.E. J. Exp. Med. 1998; 188: 1277-1286Crossref PubMed Scopus (38) Google Scholar). Quantitation of the in vitro kinase activity was performed by scanning the autoradiographs and densitometric analysis using the software program TINA. The fold activity against the control is indicated below the gels. Equal amounts of each sample were used for immunoblot analysis, as described previously (41Naumann M. Wulczyn F.G. Scheidereit C. EMBO J. 1993; 12: 213-222Crossref PubMed Scopus (124) Google Scholar) using anti-JNK or anti-PAK antibodies to indicate equivalent protein amounts in all lanes. H. pylori colonization of epithelial cells suggests the induction of intracellular signal transduction pathways that modulate cellular transcription factors. Therefore, we investigated whether H. pylori infection induces the transcription factor AP-1, which coordinately induces inflammatory cytokine/chemokine gene expression together with NF-κB. Subconfluent monolayers of AGS cells or HeLa cells were infected with different H. pylori strains. At different time points post-challenge, nuclear protein extracts were prepared and analyzed for the levels of cellular AP-1 DNA binding activity by using a radiolabeled oligonucleotide corresponding to the AP-1 DNA-binding site. As shown in Fig. 1 an enhanced binding of AP-1 was observed in AGS cells within 45 min post infection with the P12 and G27 wild type strains (Fig. 1,A, lanes 1–4, and B, lanes 1–4). The DNA binding activity induced to similar extents by bothH. pylori strains was further increased within 180 min (Fig.1, A, lanes 5 and 6, and B,lanes 5 and 6), indicating that members of the c-Jun/c-Fos family were activated. Whether c-Jun or ATF2 represent components that bind to the AP-1 binding site was analyzed in a supershift assay. Nuclear extracts from H. pylori-colonized AGS cells, preincubated with an anti-c-Jun antibody before addition of32P-labeled oligonucleotide, revealed a significantly reduced DNA binding activity of AP-1, whereas the anti-ATF2 antibody did not affect AP-1 DNA binding activity (Fig. 1 D,lanes 5–7). Further, the specificity of the AP-1 DNA-binding capability induced by H. pylori was determined using nonlabeled double-stranded oligonucleotide for competition (Fig.1 D, lanes 1–4). H. pylori(G27)-induced AP-1 DNA binding activity was slightly weaker than AP-1 activation in response to PMA, which induces AP-1 within 30 min (Fig. 1, B, lanes 3–6 versus C, lanes 3–6). In contrast to wild type strains, AP-1 activity was reduced by isogenic H. pylori strains carrying mutations in certain cag genes localized in the pathogenicity island (Fig. 1, A, lanes 4–6 versus lanes 14–16, and B,lanes 4–6 versus lanes 9–11 or lanes 14–16). An isogenic mutant of wild type vacuolating cytotoxin-producingH. pylori (P12) strain carrying a knockout of thevacA gene does not affect AP-1 activation (Fig.1 A, lanes 4–6 versus lanes 9–11). The activation of AP-1 in wild type H. pylori-colonized gastric cells was inducible at a MOI of 100, whereas the isogeniccag mutant strains showed a strongly reduced AP-1 activity in the following order: cagA*−,cagF −, cagI −. This indicates highly specific H. pylori-induced signaling, and the critical role of cag gene expression in the downstream activation of AP-1. A similar activation of AP-1 was obtained with allH. pylori strains used in colonized HeLa cells (data not shown). Coordinate activation of proinflammatory cytokines involves the activity of the immediate early transcription factors AP-1 and NF-κB. We observed enhanced binding of NF-κB using the Igκ binding site inH. pylori (P12 and G27)-colonized AGS cells within 45 min at a MOI of 50 (Fig.2, A and B). In contrast to wild type and vacA − strains, NF-κB activation was reduced by isogenic H. pylori strains carrying mutations in certain cag genes. PMA-treated cells exhibit strong NF-κB activation within 10 min (Fig. 2 C). The components that bind to the NF-κB binding site were analyzed in a supershift assay, which revealed a supershift and significantly reduced DNA binding activity of NF-κB using anti-p50 or anti-p65 antibodies, whereas the anti-c-Rel antibody did not affect NF-κB DNA binding activity (Fig. 2 D, lanes 1–4). Similar activation of NF-κB was obtained in H. pylori-colonized HeLa cells (data not shown). The specificity of the DNA binding activity was examined by adding nonlabeled double-stranded Igκ oligonucleotide for competition (Fig. 2 D, lanes 4–6). Consistent with previous data (15Keates S. Hitti Y.S. Upton M. Kelly C.P. Gastroenterology. 1997; 113: 1099-1109Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 17Sharma S.A. Tummuru M.K.R. Blaser M.J. Kerr L.D. J. Immunol. 1998; 160: 2401-2407PubMed Google Scholar) we observed activation of NF-κB in AGS cells colonized with H. pyloriwild type and weak NF-κB activation in cells colonized with the isogenic cag mutants. This indicates a highly specificH. pylori-induced signaling leading to downstream activation of NF-κB. Generally, the integrity of the cagpathogenicity island seems to be a prerequiste for an efficient activation of the immediate early response transcription factors NF-κB and AP-1. To demonstrate that AP-1 and NF-κB activation actually lead to increased proinflammatory cytokine release, we analyzed the IL-8 release by AGS cells and HeLa cells in response to H. pyloriwild type and the isogenic cagA*− strain.H. pylori (P12) induces IL-8 release in both cell lines within 6 h, whereas IL-8 secretion was reduced by 50% in cells colonized with the cagA*− strain (data not shown). The reduced IL-8 release fromcagA*− infected AGS cells corroborates the observation that cag mutant strains (16Münzenmaier A. Lange C. Glocker E. Covacci A. Moran A. Bereswill S. Baeuerle P.A. Kist M. Pahl H.L. J. Immunol. 1997; 159: 6140-6147PubMed Google Scholar) induce weak activation of transcription factors. The dimeric sequence specific enhancer factor AP-1 becomes activated through phosphorylation of c-Jun by members of the stress activated MAPK (SAPK) family in response to environmental stress (14Karin M. Liu Z.G. Zandi E. Curr. Opin. Cell Biol. 1997; 9: 240-246Crossref PubMed Scopus (2267) Google Scholar). Therefore, we examined whether the SAPK/JNK and/or p38 are involved in the signaling leading to H. pylori-induced AP-1 activation. Cellular extracts from H. pylori-colonized epithelial cells were used at different time points post infection for immunoprecipitation of the endogenous kinases, and in vitrokinase assays using appropriate substrates were performed. Wild type H. pylori strains at a MOI of 50 induce severalfold JNK1 activation (c-Jun substrate phosphorylation) in subconfluent monolayers of AGS cells (Fig.3) and HeLa cells (data not shown) within 30 min after infection (Fig. 3, A, lanes 1–3, and B, lanes 1–3). The immunoblots, probed with an anti-JNK1 antibody, in the lower panels of Fig. 3 is to demonstrate similar JNK protein amounts in all lanes. The immediate JNK1 induction was followed by a sustained JNK1 kinase activation for at least 90 min (Fig. 3, A, lanes 4 and5, and B, lanes 4 and 5). The activation of JNK1 in response to H. pylori was delayed compared with the JNK1 induction in PMA-treated cells (Fig. 3, compareA and B, lanes 1–5 with C,lanes 1–4) but showed a similar potential to induce JNK1 activity. To test whether cellular JNK1 activation is at variance in epithelial cells colonized with different H. pylori strains, we compared JNK1 kinase activity in AGS cells and HeLa cells colonized eithe" @default.
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