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• W2002125536 abstract "Helicobacter pylori interacts with gastric epithelial cells, activating signaling pathways important for carcinogenesis. In this study we examined the role of H. pylori on cell invasion and the molecular mechanisms underlying this process. The relevance of H. pylori cag pathogenicity island-encoded type IV secretion system (T4SS), CagA, and VacA for cell invasion was also investigated. We found that H. pylori induces AGS cell invasion in collagen type I and in Matrigel invasion assays. H. pylori-induced cell invasion requires the direct contact between bacteria and cancer cells. H. pylori-mediated cell invasion was dependent on the activation of the c-Met receptor and on increased MMP-2 and MMP-9 activity. The abrogation of the c-Met receptor using the specific NK4 inhibitor or the silencing of c-Met expression with small interference RNA suppressed both cell invasion and MMP activity. Studies with different H. pylori strains revealed that cell invasion, c-Met tyrosine phosphorylation, and increased MMP-2 and MMP-9 activity were all dependent on the presence of a functional bacterial T4SS, but not on VacA cytotoxicity. Our findings demonstrate that H. pylori strains with a functional T4SS stimulate gastric epithelial cell invasion through a c-Met-dependent signaling pathway that comprises an increase in MMP-2 and MMP-9 activity. Helicobacter pylori interacts with gastric epithelial cells, activating signaling pathways important for carcinogenesis. In this study we examined the role of H. pylori on cell invasion and the molecular mechanisms underlying this process. The relevance of H. pylori cag pathogenicity island-encoded type IV secretion system (T4SS), CagA, and VacA for cell invasion was also investigated. We found that H. pylori induces AGS cell invasion in collagen type I and in Matrigel invasion assays. H. pylori-induced cell invasion requires the direct contact between bacteria and cancer cells. H. pylori-mediated cell invasion was dependent on the activation of the c-Met receptor and on increased MMP-2 and MMP-9 activity. The abrogation of the c-Met receptor using the specific NK4 inhibitor or the silencing of c-Met expression with small interference RNA suppressed both cell invasion and MMP activity. Studies with different H. pylori strains revealed that cell invasion, c-Met tyrosine phosphorylation, and increased MMP-2 and MMP-9 activity were all dependent on the presence of a functional bacterial T4SS, but not on VacA cytotoxicity. Our findings demonstrate that H. pylori strains with a functional T4SS stimulate gastric epithelial cell invasion through a c-Met-dependent signaling pathway that comprises an increase in MMP-2 and MMP-9 activity. Helicobacter pylori is a bacterial pathogen that colonizes the gastric mucosa of more than half of the human population (1Covacci A. Telford J.L. Del Giudice G. Parsonnet J. Rappuoli R. Science. 1999; 284: 1328-1333Crossref PubMed Scopus (951) Google Scholar). In most individuals the infection induces chronic superficial gastritis, a condition that will remain throughout life. However, in some individuals, more severe outcomes of the infection may develop, such as peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric carcinoma (2Peek R.M. Blaser M.J. Nat. Rev. Cancer. 2002; 2: 28-37Crossref PubMed Scopus (1457) Google Scholar). This diversity of clinical outcomes associated with H. pylori infection is probably a result of the interactions among host, environmental, and bacterial virulence factors (2Peek R.M. Blaser M.J. Nat. Rev. Cancer. 2002; 2: 28-37Crossref PubMed Scopus (1457) Google Scholar, 3Figueiredo C. Machado J.C. Pharoah P. Seruca R. Sousa S. Carvalho R. Capelinha A.F. Quint W. Caldas C. van Dorn L.J. Carneiro F. Sobrinho-Simões M. J. Natl. Cancer Inst. 2002; 22: 1680-1687Crossref Scopus (555) Google Scholar).Numerous studies have shown that H. pylori is able to interact with gastric epithelial cells, activating signaling pathways, modifying host cellular functions, and inducing cell phenotypes important for carcinogenesis (4Peek Jr., R.M. Wirth H.P. Moss S.F. Yang M. Abdalla A.M. Tham K.T. Zhang T. Tang L.H. Modlin I.M. Blaser M.J. Gastroenterology. 2000; 118: 48-59Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar, 5Amieva M.R. Vogelmann R. Covacci A. Tompkins L.S. Nelson W.J. Falkow S. Science. 2003; 300: 1430-1434Crossref PubMed Scopus (613) Google Scholar, 6Selbach M. Moese S. Hurwitz R. Hauck C.R. Meyer T.F. Backert S. EMBO J. 2003; 22: 515-528Crossref PubMed Scopus (186) Google Scholar, 7Viala J. Chaput C. Boneca I.G. Cardona A. Girardin S.E. Moran A.P. Athman R. Memet S. Huerre M.R. Coyle A.J. DiStefano P.S. Sansonetti P.J. Labigne A. Bertin J. Philpott D.J. Ferrero R.L. Nat. Immunol. 2004; 5: 1166-1174Crossref PubMed Scopus (982) Google Scholar). One of the less explored cell phenotypes induced by H. pylori is cellular invasion. Although little is known about the mechanisms involved in this process, H. pylori was shown to activate tyrosine kinase receptors frequently involved in invasion-related pathways, such as the epidermal growth factor receptor (EGFR), 4The abbreviations used are: EGFR, epidermal growth factor receptor; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; siRNA, short interfering RNA; MOI, multiplicity of infection. 4The abbreviations used are: EGFR, epidermal growth factor receptor; MMP, matrix metalloproteinase; PBS, phosphate-buffered saline; siRNA, short interfering RNA; MOI, multiplicity of infection. Her2/Neu (ErbB-2), and c-Met (8Keates S. Sougioultzis S. Keates A.C. Zhao D. Peek R.M. Shaw L.M. Kelly C.P. J. Biol. Chem. 2001; 276: 48127-48134Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 9Wallasch C. Crabtree J.E. Bevec D. Robinson P.A. Wagner H. Ullrich A. Biochem. Biophys. Res. Commun. 2002; 295: 695-701Crossref PubMed Scopus (104) Google Scholar, 10Churin Y. Al-Ghoul L. Kepp O. Meyer T.F. Birchmeier W. Naumann M. J. Cell Biol. 2003; 161: 249-255Crossref PubMed Scopus (308) Google Scholar).Another group of molecules associated with cancer cell invasion and influenced by H. pylori are matrix metalloproteinases (MMPs) (11Mareel M. Leroy A. Physiol. Rev. 2003; 83: 337-376Crossref PubMed Scopus (444) Google Scholar). MMP expression and activity are frequently enhanced in tumors as compared with normal tissue (11Mareel M. Leroy A. Physiol. Rev. 2003; 83: 337-376Crossref PubMed Scopus (444) Google Scholar, 12Egeblad M. Werb Z. Nat. Rev. Cancer. 2002; 2: 161-174Crossref PubMed Scopus (5079) Google Scholar). It has been shown that H. pylori up-regulates the expression and activity of several MMPs, both in gastric epithelial cell lines and in the gastric mucosa (13Göoz M. Göoz P. Smolka A.J. Am. J. Physiol. Gastrointest. Liver. Physiol. 2001; 281: G823-G832Crossref PubMed Google Scholar, 14Bebb J.R. Letley D.P. Thomas R.J. Aviles F. Collins H.M. Watson S.A. Hand N.M. Zaitoun A. Atherton J.C. Gut. 2003; 52: 1408-1413Crossref PubMed Scopus (77) Google Scholar, 15Crawford H.C. Krishna U.S. Israel D.A. Matrisian L.M. Washington M.K. Peek R.M. Gastroenterology. 2003; 125: 1125-1136Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar).H. pylori virulence factors differentially interfere with signaling pathways in gastric epithelial cells (16Guillemin K. Salama N.R. Tompkins L.S. Falkow S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15136-15141Crossref PubMed Scopus (191) Google Scholar). One well established H. pylori virulence factor is the presence of a cluster of about 30 genes, known as the cag pathogenicity island (cag PAI). The cag PAI encodes a type IV secretion system (T4SS), a multimolecular complex that mediates the translocation of bacterial factors into the host cell (17Segal E.D. Cha J. Lo J. Falkow S. Tompkins L.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14559-14564Crossref PubMed Scopus (664) Google Scholar, 18Odenbreit S. Puls J. Sedlmaier B. Gerland E. Fischer W. Haas R. Science. 2000; 287: 1497-1500Crossref PubMed Scopus (1064) Google Scholar). The T4SS translocates the CagA protein into the host cell cytoplasm, where it can stimulate cell signaling through interaction with several host proteins (6Selbach M. Moese S. Hurwitz R. Hauck C.R. Meyer T.F. Backert S. EMBO J. 2003; 22: 515-528Crossref PubMed Scopus (186) Google Scholar, 18Odenbreit S. Puls J. Sedlmaier B. Gerland E. Fischer W. Haas R. Science. 2000; 287: 1497-1500Crossref PubMed Scopus (1064) Google Scholar, 19Higashi H. Tsutsumi R. Muto S. Sugiyama T. Azuma T. Asaka M. Hatakeyama M. Science. 2002; 295: 683-686Crossref PubMed Scopus (842) Google Scholar), such as the tyrosine kinase c-Met receptor. The intracellular interaction between CagA and c-Met induces a motogenic response in gastric epithelial cells (10Churin Y. Al-Ghoul L. Kepp O. Meyer T.F. Birchmeier W. Naumann M. J. Cell Biol. 2003; 161: 249-255Crossref PubMed Scopus (308) Google Scholar).VacA is another H. pylori virulence factor. This bacterial toxin with multiple activities is inserted in the host cell membrane, inducing cytoplasmic vacuolation (20Cover T.L. Blaser M.J. J. Biol. Chem. 1992; 267: 10570-10575Abstract Full Text PDF PubMed Google Scholar, 21Cover T.L. Blanke S.R. Nat. Rev. Microbiol. 2005; 3: 320-332Crossref PubMed Scopus (412) Google Scholar). In Western populations, the presence of a functional T4SS, of CagA, and VacA cytotoxicity are frequently associated with severe gastric inflammation, ulceration, and increased risk of gastric carcinoma (22Blaser M.J. Perez-Perez G.I. Kleanthous H. Cover T.L. Peek R.M. Chyou P.H. Stemmermann G.N. Nomura A. Cancer Res. 1995; 55: 2111-2115PubMed Google Scholar, 23Atherton J.C. Peek R.M. Tham K.T. Cover T.L. Blaser M.J. Gastroenterology. 1997; 112: 92-99Abstract Full Text PDF PubMed Scopus (580) Google Scholar, 24van Doorn L.J. Figueiredo C. Sanna R. Plaisier A. Schneeberger P. de Boer W. Quint W. Gastroenterology. 1998; 115: 58-66Abstract Full Text Full Text PDF PubMed Scopus (544) Google Scholar, 25Nogueira C. Figueiredo C. Carneiro F. Taveira-Gomes A. Barreira R. Figueira P. Salgado C. Belo L. Peixoto A. Bravo J.C. Bravo L.E. Realpe J.L. Plaisier A.P. Quint W.G. Ruiz B. Correa P. van Doorn L.J. Am. J. Pathol. 2001; 158: 647-654Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar), although the precise molecular mechanisms underlying these associations are poorly understood.In this study our first goal was to examine the influence of H. pylori on epithelial gastric cancer cell invasion. After demonstrating that H. pylori is able to induce cell invasion in this model, we examined the molecular mechanisms underlying this process. Finally, we also evaluated whether there were differences between strains in their ability to stimulate cell invasion, and assessed the role of the cag PAI-encoded T4SS, CagA, and VacA in this process.EXPERIMENTAL PROCEDURESCell Culture and Reagents—AGS cells, derived from a human gastric carcinoma, were maintained in RPMI 1640 medium (Invitrogen), supplemented with 10% fetal bovine serum, 2.5 μg/ml fungizone (Bristol-Myers Squibb, Brussels, Belgium), 200 μg/ml streptomycin, and 200 international units/ml penicillin (Invitrogen) at 37 °C, under a 5% CO2 humidified atmosphere.Pharmacological inhibitors PD168393, ZD1839, and LY294002, were obtained from Calbiochem (Darmstadt, Germany), AstraZeneca (Macclesfield, UK), and Sigma, respectively. NK4 was kindly provided by W. Jiang (Dept. of Surgery, University of Wales College of Medicine, Cardiff, UK). The concentrations of inhibitors used were 2 μm, 1 μm, 100 mm, and 100 ng/ml, for PD168393, ZD1839, LY294002, and NK4, respectively. These concentrations were previously shown to inhibit cell invasion induced by stimulators, such as heregulin-β1, EGF, lithocholic acid, and HGF in AGS cells and in other cell lines (26Debruyne P.R. Bruyneel E.A. Karaguni I.M. Li X. Flatau G. Muller O. Zimber A. Gespach C. Mareel M.M. Oncogene. 2002; 21: 6740-6750Crossref PubMed Scopus (77) Google Scholar, 27Oliveira M.J. Lauwaet T. De Bruyne G. Mareel M. Leroy A.J Cancer Res. Clin. Oncol. 2005; 131: 49-59Crossref PubMed Scopus (5) Google Scholar). Drug toxicity was controlled by measuring cell viability with the trypan blue dye exclusion test.Bacterial Strains and Growth Conditions—Bacteria were grown in tryptic soy agar (TSA) supplemented with 5% sheep blood (BioMérieux, Marcy l'Étoile, France) and incubated for 48 h at 37 °C under a microaerophilic atmosphere. Bacterial density was estimated by the absorbance measurement at 600 nm. Heat-killed H. pylori was obtained by boiling during 30 min at 56 °C followed by an incubation for 10 min at 80 °C. Unless otherwise stated, experiments were performed with H. pylori strain 26695 (ATCC 700392, cag PAI+, vacA s1/m1), obtained from the American Type Culture Collection (ATCC). H. pylori insertion mutants with inactivation of the cagA (60190ΔcagA), cagE (60190ΔcagE) or vacA (60190ΔvacA) genes (14Bebb J.R. Letley D.P. Thomas R.J. Aviles F. Collins H.M. Watson S.A. Hand N.M. Zaitoun A. Atherton J.C. Gut. 2003; 52: 1408-1413Crossref PubMed Scopus (77) Google Scholar, 28Bebb J.R. Letley D.P. Rhead J.L. Atherton J.C. Inf. Immun. 2003; 71: 3623-3627Crossref PubMed Scopus (21) Google Scholar) were studied together with their parental wild type strain 60190 (ATCC 49503, cag PAI+, vacA s1/m1). In parallel experiments, H. pylori strain Tx30a (ATCC 51932, cag PAI–, vacA s2/m2) was also used.Preparation of Conditioned Medium—Conditioned medium from H. pylori (CM 26695) was prepared by washing three times in serum-free medium, 1 × 108 bacteria growing in TSA. Bacteria were added to 1.5 ml of RPMI 1640 serum and antibiotic-free medium, and incubated on Matrigel-coated filters in the absence of cells at 37 °C, under a microaerophilic atmosphere. Control-conditioned medium (CM control) was prepared similarly, in the absence of bacteria. Conditioned media were collected after 48 h, centrifuged at 3220 × g for 2 min and passed through 0.2-μm pore-size filters (Schleicher & Schüell, Dassel, Germany) prior to test on invasion assays.Infection of Gastric Cells—Prior to infection, 80% confluent AGS monolayers were washed twice in PBS and incubated overnight in serum and antibiotic free medium (Invitrogen). For infection, 48 h colonies of H. pylori were collected and added to cells at, unless otherwise stated, a multiplicity of infection (MOI) of 100. Cultures were maintained at 37 °C under a 5% CO2 humidified atmosphere. Control monolayers were processed similarly in the absence of bacteria.Collagen Invasion Assay—Collagen invasion assays were performed as previously described (29Oliveira M.J. Van Damme J. Lauwaet T. De Corte V. De Bruyne G. Verschraegen G. Vaneechoutte M. Goethals M. Ahmadian M.R. Muller O. Vandekerckhove J. Mareel M. Leroy A. EMBO J. 2003; 22: 6161-6173Crossref PubMed Scopus (20) Google Scholar). Briefly, collagen gels were prepared in 6-well plates (Becton and Dickinson, Bedford, MA), using a collagen type I solution (Upstate Biotechnology, Lake Placid, NY), and polymerized overnight at 37 °C. AGS cells (1 × 105) were incubated on top of the gels for 24 h at 37 °C, in the presence or absence (control) of H. pylori, and in some experiments with pharmacological inhibitors. Invasion was scored as the ratio between the number of invasive cells inside the gel and the total number of cells, counted in at least 12 microscopic fields with a computer-assisted inverted microscope. Cell viability was evaluated by trypan blue dye exclusion test at the end of each assay.Matrigel Invasion Assay—Prior to each experiment, 24-well Matrigel-coated invasion inserts of 8-μm pore size filters (Becton and Dickinson) were introduced into 24-well plates. For re-hydration, the inner and outer compartments of the system were filled with RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotic-free, and incubated for 60 min at 37 °C. After rehydration, 5 × 104 cells were incubated for 24 h at 37 °C, in the presence or absence (control) of H. pylori or of H. pylori-conditioned medium, and pharmacological inhibitors. Filters were washed in PBS, fixed in 4% paraformaldehyde, removed from the insert, and mounted in Vectashield with 4′,6-diamidino-2-phenylindole (DAPI, Vector Laboratories, Burlingame, CA). Invasive cells were scored in at least 25 microscopic fields (×20 objective), when DAPI-counterstained nuclei passed through the pores of the filter.Preparation of Cell Lysates and Immunoprecipitation—At the end of the infection period, cells were lysed in cold lysis buffer (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1% Nonidet P-40, 3 mm sodium vanadate, 20 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 10 μg/ml leupeptin). To immunoprecipitate tyrosine-phosphorylated molecules, 750 μg of proteins were incubated for 2 h at 4 °C with a mouse monoclonal anti-phosphotyrosine antibody (PY-20, BD Biosciences-Transduction Laboratories, San Jose, CA). Immunocomplexes were incubated for 60 min with protein G-Sepharose beads (Amersham Biosciences, Buckinghamshire, UK), washed and eluted in sample buffer. Proteins were separated by SDS-PAGE and visualized by immunoblot analysis, using antibodies directed to the phosphorylated molecules of interest.Immunoblot Analysis—After electrophoresis, proteins were transferred onto Hybond nitrocellulose membranes (Amersham Biosciences). Membranes were blocked with 4% bovine serum albumin (Sigma) in PBS + 0.5% Tween-20 (for detection of phosphorylated proteins) or with 5% nonfat milk in PBS + 0.5% Tween-20 (for overall protein detection) and incubated for 60 min with a rabbit polyclonal anti-c-Met antibody (Santa Cruz Biotechnology), a mouse monoclonal anti-α-tubulin antibody (Sigma), a rabbit polyclonal anti-MMP2 antibody (Labvision Neomarkers, Fremont, CA) or a mouse monoclonal anti-MMP-9 antibody (Calbiochem). A goat anti-rabbit (Santa Cruz Biotechnology) or a rabbit anti-mouse (Amersham Biosciences) horseradish peroxidase-conjugated secondary antibodies were used, followed by ECL detection (Amersham Biosciences). Immunoblots were quantified with the Quantity One Software (Bio-Rad).Small Interference RNA (siRNA) Transfection—siRNAs targeting c-Met, MMP-2, or MMP-9 mRNA, previously tested for knockdown efficiency by quantitative RT-PCR, were obtained from Qiagen (Valencia, CA), and prepared according to manufacturer's instructions. In parallel, non-silencing siRNA duplexes (Sense: 5′-UUCUCCGAACGUGUCACGU-3′ and antisense: 5′-ACGUGACACGUUCGGAGAA-3′) were used as negative control. Prior to transfection, 50% confluent AGS monolayers platted onto 6-well plates were washed with PBS and incubated in serum and antibiotic-free medium. For transfection with siRNAs targeting MMP-2 and MMP-9, cells were grown, also until 50% of confluency, on 6-well plates previously coated with a collagen type I solution. Cells were transiently transfected with 80 nm (for c-Met or non-silencing siRNA) or with 50 nm (for MMP-2 and MMP-9) of siRNA, using the Lipofectamine 2000 transfection reagent (Invitrogen). At the end of each transfection, putative cytotoxic effects were evaluated, analyzing cell viability by trypan blue dye exclusion test.Zymography—To detect MMP enzymatic activity, transfected, or non-transfected AGS cells, were cultured for 48 h on top of collagen type I gels, in the presence or absence of H. pylori. 12 μg of protein from conditioned medium of such cultures were loaded on 10% SDS-PAGE containing 1 mg/ml gelatin (MMP-2 and MMP-9) or 1 mg/ml β-casein (MMP-3 and MMP-9) as substrates. Zymograms were run in Tris/glycine SDS running buffer under nondenaturing conditions. After electrophoresis, gels were washed twice in 2% Triton X-100, to remove SDS. Zymograms were subsequently incubated for 20 h at 37 °C in the appropriated MMP substrate buffer (10 mm CaCl2 in 50 mm Tris-HCl, pH 7.5 for MMP-2 and MMP-9; 0.2 m NaCl, 5 mm CaCl2, 1% Triton X-100 in 50 mm Tris-HCl, pH 7.4 for MMP-3 and MMP-9). Proteolytic activity was visualized as the presence of clear bands against a blue background of Coomassie Blue-stained gelatin or β-casein substrates.Statistical Analysis—Data were analyzed with Student's t test and were expressed as mean values of at least three independent experiments ± S.D. Differences in data values were considered significant at a p value of less than 0.05.RESULTSH. pylori Stimulates AGS Cell Invasion—To investigate whether H. pylori was capable of inducing invasion of gastric epithelial cells, non-invasive AGS cells were infected with H. pylori and evaluated using two well established invasion assays. H. pylori significantly stimulated the invasion of these cells into both collagen type I gels (Fig. 1A) and Matrigel-coated filters (Fig. 1B).To test whether the proportion of bacteria:cell used in the experiments would influence the cellular phenotype, invasion was assessed using different MOIs (Fig. 1A). Because the highest MOI used (100) did not affect cellular viability, as assessed by the trypan blue dye exclusion test (data not shown), this proportion bacteria:cell was used in all further experiments.Stimulation of AGS Cell Invasion Requires Direct Contact with H. pylori—To investigate whether viable bacteria were necessary for AGS cell invasion, cells were cultured on collagen type I gels or Matrigel-coated filters, with intact or heat-killed H. pylori. On both substrates, heat-killed bacteria were no longer able to stimulate cancer cell invasion (Fig. 1, B and C). To determine whether stimulation of invasion occurred by direct contact between H. pylori and the cells or by release of soluble bacterial pro-invasive factors by H. pylori, conditioned medium of H. pylori incubated on Matrigel-coated filters without cells, was collected, filtered, and tested in the Matrigel assay. Conditioned medium from H. pylori induced significantly lower levels of cell invasion than those observed by bacterial direct stimulation (Fig. 1B), suggesting that the contact between H. pylori, and cells is necessary to induce an invasive phenotype.H. pylori-mediated AGS Cell Invasion Is Blocked by NK4, a c-Met Inhibitor—To assess the involvement of EGFR, ErbB-2, c-Met, and of the downstream signaling molecule phosphoinositide 3-kinase (PI3K) in H. pylori-mediated cell invasion, AGS cells were infected with H. pylori in the presence of specific inhibitors for each of these molecules, and assessed for invasion on collagen and Matrigel assays. On collagen type I gels, only NK4, an HGF antagonist that inhibits c-Met tyrosine phosphorylation and activity (30Date K. Matsumoto K. Shimura H. Tanaka M. Nakamura T. FEBS Lett. 1997; 420: 1-6Crossref PubMed Scopus (219) Google Scholar), was able to inhibit AGS cell invasion (Fig. 2A). The other inhibitors, although used at concentrations known to block invasion by specific stimulators, such as EGF, Heregulin β1 (Hrgβ1) (Fig. 2A), or lithocholic acid (27Oliveira M.J. Lauwaet T. De Bruyne G. Mareel M. Leroy A.J Cancer Res. Clin. Oncol. 2005; 131: 49-59Crossref PubMed Scopus (5) Google Scholar) had no effect on H. pylori-mediated cell invasion. On Matrigel-coated filters, although there was an inhibitory effect associated with PD168393, a dual EGFR/ErbB2 inhibitor, and ZD1839, a specific EGFR inhibitor, the most striking effect of inhibition of cell invasion was observed with NK4 (Fig. 2B). Identical results to those obtained with H. pylori strain 26695 were also observed with H. pylori strain 60190 (data not shown). In none of the assays LY294002, a specific PI3K inhibitor, was able to block cell invasion stimulated by H. pylori (Fig. 2, A and B). These results suggest that the c-Met receptor has an important role in H. pylori-mediated cell invasion.FIGURE 2H. pylori stimulation of AGS cell invasion is blocked by NK4, a c-Met inhibitor. Invasion assays of AGS cells infected for 24 h with H. pylori 26695, with EGF or with Heregulin β1 (Hrgβ1) and with a PI3K inhibitor (LY294002, 100 mm), an EGFR/ErbB2 inhibitor (PD168393, 2 μm), an EGFR inhibitor (ZD1839, 1 μm), or an HGF antagonist (NK4, 100 ng/ml) on collagen type I gels (A) and on Matrigel-coated filters (B). Data correspond to the mean value ± S.D. and are representative of three independent experiments. *, significantly different from untreated cells; **, significantly different from cells infected with H. pylori 26695.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Stimulation of Cell Invasion by H. pylori Occurs via a c-Met-dependent Mechanism—To further explore the relationship between c-Met inhibition and H. pylori-induced AGS cell invasion, we investigated the effect of H. pylori on the tyrosine phosphorylation status of the c-Met receptor. After infection with H. pylori, cells were lysed, immunoprecipitated with an antibody against tyrosine-phosphorylated residues (PY-20), and immunoblotted with an anti-c-Met antibody. As observed in Fig. 3A, H. pylori significantly increased the tyrosine phosphorylation level of c-Met. Similar results to those obtained with H. pylori strain 26695 were observed with H. pylori 60190.FIGURE 3H. pylori stimulation of AGS cell invasion occurs through a c-Met-dependent mechanism. AGS cells were infected with H. pylori 26695 for 0, 15, or 60 min (A) or with H. pylori 26695 and NK4 (100 ng/ml) for 60 min (B). AGS cells or AGS cells transfected with non-silencing siRNA (non-silenc. siRNA) or with siRNA to c-Met (siRNA c-Met) were infected with H. pylori 26695 or 60190 for 60 min (C). After infection, cells were lysed, immunoprecipitated with an antibody against tyrosine-phosphorylated residues (PY-20), and immunoblotted with an anti-c-Met antibody. In parallel, total cell lysates were immunoblotted with an anti c-Met and reblotted with an anti-α-tubulin antibody. Cultures with c-Met siRNA-transfected cells were incubated on Matrigel filters and assessed for invasion (D). Graphics represent the variation on c-Met tyrosine phosphorylation in comparison to the endogenous phosphorylation levels of untreated cells. Data correspond to the mean value ± S.D. and are representative of three independent experiments. *, significantly different from untreated cells or cells transfected with non-silencing siRNA; **, significantly different from cells infected with H. pylori 26695.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because NK4 suppressed the invasion of AGS cells stimulated by H. pylori, we also examined the effect of this inhibitor on H. pylori-induced c-Met tyrosine phosphorylation. For that, AGS cells were cultured for 60 min with H. pylori and with NK4. NK4 blocked not only H. pylori-mediated AGS cell invasion, but also c-Met tyrosine phosphorylation (Figs. 2, A and B and 3B), suggesting that H. pylori-mediated AGS cell invasion requires phosphorylation of the c-Met receptor. Neither H. pylori nor NK4 affected the expression levels of c-Met.To further confirm that c-Met is directly involved in the stimulation of host cell invasion by H. pylori, we transiently transfected AGS cells with siRNA abrogating c-Met expression (Fig. 3C). c-Met silencing was maximal 48 h after transfection, as confirmed by immunoblot analysis (data not shown). Therefore, c-Met phosphorylation levels were investigated 48 h after transfection, and invasion assays started 24 h after transfection by incubating AGS cells with H. pylori strains 26695 and 60190 on Matrigel filters for an additional 24-h period. AGS cells transfected with siRNA to c-Met were resistant to both H. pylori-induced c-Met tyrosine phosphorylation and invasion (Fig. 3, C and D). These findings demonstrate that invasion of AGS cells is stimulated by H. pylori through a c-Met-dependent mechanism.H. pylori-mediated Cell Invasion Requires MMP-2 and MMP-9 Activity—To investigate the participation of MMPs in H. pylori-mediated cell invasion, cells were infected with H. pylori strain 60190 for 48 h on collagen type I gels. Supernatants of such cultures were assessed for proteolytic activity on gelatin or β-casein substrates. We observed that AGS cells constitutively secrete MMP-2 and MMP-9, and that supernatants of AGS cells infected with H. pylori showed increased MMP-2, MMP-9, and de novo MMP-3 activity (Fig. 4, A and B).FIGURE 4H. pylori-mediated AGS cell invasion requires MMP-2 and MMP-9 activity, downstream c-Met receptor activation. AGS cells transfected or not with siRNA to c-Met (siRNA c-Met) were infected for 48 h with H. pylori 60190, 60190ΔcagA or 60190ΔcagE, on collagen type I gels. Filtered culture supernatants were run on gelatin (A) or β-casein (B) zymograms. AGS cells transfected or not with siRNA to MMP-2 (siRNA MMP-2) or to MMP-9 (siRNA MMP-9) were infected for 48 h with H. pylori 60190. Filtered culture supernatants were run on a gelatin zymogram to detect MMP activity (C). The invasion ability of the transfected cells was tested on Matrigel assays (D). Proteolytic bands were revealed in white on a Coomassie Blue-stained background. Data correspond to the mean value ± S.D. and are representative of three independent experiments. *, significantly different from untreated cells; **, significantly different from cells infected with H. pylori 60190.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To determine whether the induction of MMP-2 and MMP-9 activity was essential for H. pylori-mediated invasi" @default.
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• W2002125536 date "2006-11-01" @default.
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• W2002125536 title "Helicobacter pylori Induces Gastric Epithelial Cell Invasion in a c-Met and Type IV Secretion System-dependent Manner" @default.
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