Helicobacter pylori and H2O2 increase AP endonuclease-1/redox factor-1 expression in human gastric epithelial cells
2004; Elsevier BV; Volume: 127; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2004.06.017
ISSN1528-0012
AutoresSong-Ze Ding, Ann M. O’Hara, Timothy L. Denning, Bernadette Dirden-Kramer, Randy C. Mifflin, Victor E. Reyes, Kieran A. Ryan, Susan N. Elliott, Tadahide Izumi, István Boldogh, Sankar Mitra, Peter B. Ernst, Sheila E. Crowe,
Tópico(s)Veterinary medicine and infectious diseases
ResumoBackground & Aims: Helicobacter pylori infection causes inflammation, accumulation of reactive oxygen species, and oxidative DNA damage in the gastric mucosa. Apurinic/apyrimidinic endonuclease-1 (APE-1)/redox factor-1 (Ref-1) repairs damaged DNA and reductively activates transcription factors, including activator protein-1. Considering that H. pylori generate reactive oxygen species and that reactive oxygen species modulate APE-1/Ref-1 in other cell types, we examined the effect of H. pylori, oxidative stress, and antioxidants on APE-1/Ref-1 expression in human gastric epithelial cells.Methods: Human gastric epithelial cell lines or cells isolated from mucosal biopsy samples were stimulated with H. pylori, Campylobacter jejuni, and/or H2O2 in the presence or absence of antioxidants. APE-1/Ref-1 expression was assayed by Western blot or reverse-transcription polymerase chain reaction, and its cellular distribution was determined by using indirect conventional and confocal immunofluorescence. New protein synthesis was detected by [S35]methionine labeling. APE-1/Ref-1 function was assessed by using a luciferase-linked reporter construct containing 3 activator protein 1 binding sites.Results: APE-1/Ref-1 protein and messenger RNA were detected in resting gastric epithelial cells. APE-1/Ref-1 protein expression was increased after stimulation with H2O2 or live cag pathogenicity island-bearing H. pylori, but not cag pathogenicity island-negative H. pylori or C. jejuni. H. pylori- or reactive oxygen species-mediated increases in APE-1/Ref-1 expression involved de novo protein synthesis that was inhibited by antioxidants. H. pylori or H2O2 also induced nuclear accumulation of APE-1/Ref-1, and overexpression of APE-1/Ref-1 increased activator protein 1 binding activity.Conclusions: The data show that H. pylori or reactive oxygen species enhance APE-1/Ref-1 protein synthesis and nuclear accumulation in human gastric epithelial cells and implicate APE-1/Ref-1 in the modulation of the pathogenesis of H. pylori infection. Background & Aims: Helicobacter pylori infection causes inflammation, accumulation of reactive oxygen species, and oxidative DNA damage in the gastric mucosa. Apurinic/apyrimidinic endonuclease-1 (APE-1)/redox factor-1 (Ref-1) repairs damaged DNA and reductively activates transcription factors, including activator protein-1. Considering that H. pylori generate reactive oxygen species and that reactive oxygen species modulate APE-1/Ref-1 in other cell types, we examined the effect of H. pylori, oxidative stress, and antioxidants on APE-1/Ref-1 expression in human gastric epithelial cells. Methods: Human gastric epithelial cell lines or cells isolated from mucosal biopsy samples were stimulated with H. pylori, Campylobacter jejuni, and/or H2O2 in the presence or absence of antioxidants. APE-1/Ref-1 expression was assayed by Western blot or reverse-transcription polymerase chain reaction, and its cellular distribution was determined by using indirect conventional and confocal immunofluorescence. New protein synthesis was detected by [S35]methionine labeling. APE-1/Ref-1 function was assessed by using a luciferase-linked reporter construct containing 3 activator protein 1 binding sites. Results: APE-1/Ref-1 protein and messenger RNA were detected in resting gastric epithelial cells. APE-1/Ref-1 protein expression was increased after stimulation with H2O2 or live cag pathogenicity island-bearing H. pylori, but not cag pathogenicity island-negative H. pylori or C. jejuni. H. pylori- or reactive oxygen species-mediated increases in APE-1/Ref-1 expression involved de novo protein synthesis that was inhibited by antioxidants. H. pylori or H2O2 also induced nuclear accumulation of APE-1/Ref-1, and overexpression of APE-1/Ref-1 increased activator protein 1 binding activity. Conclusions: The data show that H. pylori or reactive oxygen species enhance APE-1/Ref-1 protein synthesis and nuclear accumulation in human gastric epithelial cells and implicate APE-1/Ref-1 in the modulation of the pathogenesis of H. pylori infection. The gram-negative flagellated bacterium Helicobacter pylori colonizes the human gastric mucosa. Infection with H. pylori leads to diverse clinical and pathologic outcomes, including chronic superficial gastritis, duodenal or gastric ulceration, and gastric adenocarcinoma.1Peterson W.L. Helicobacter pylori and peptic ulcer disease.N Engl J Med. 1991; 324: 1043-1048Crossref PubMed Scopus (0) Google Scholar, 2Blaser M.J. Perez-Perez G.I. 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Although studies show that H. pylori infection activates transcription factors NF-κB and AP-1,34Keates S. Hitti Y.S. Upton M. Kelly C.P. Helicobacter pylori infection activates NF-kappaB in gastric epithelial cells.Gastroenterology. 1997; 113: 1099-1109Abstract Full Text Full Text PDF PubMed Scopus (379) Google Scholar, 35Naumann M. Wessler S. Bartsch C. Wieland B. Covacci A. Haas R. Meyer T.F. Activation of activator protein 1 and stress response kinases in epithelial cells colonized by Helicobacter pylori encoding the cag pathogenicity island.J Biol Chem. 1999; 274: 31655-31662Crossref PubMed Scopus (155) Google Scholar which are known to be regulated by APE-1/Ref-1, and that oxidants also activate NF-κB,36Rokutan K. Teshima S. Miyoshi M. Nikawa T. Kishi K. Oxidant-induced activation of nuclear factor-kappa B in cultured guinea pig gastric epithelial cells.Dig Dis Sci. 1997; 42: 1880-1889Crossref PubMed Scopus (10) Google Scholar the effect of H. pylori or any other infection on APE-1/Ref-1 expression has not been reported. In this study, we investigated whether H. pylori or ROS affect APE-1/Ref-1 expression in human gastric epithelial cells. Our data indicate that APE-1/Ref-1 is constitutively expressed by human gastric epithelial cells, with enhanced expression and nuclear translocation after infection with H. pylori or stimulation with ROS, and that this effect is inhibited by antioxidants. Moreover, our results show that APE-1/Ref-1 plays a functional role in human gastric epithelial cells by enhancing AP-1-mediated transcription. The human gastric epithelial cell lines Kato III, NCI-N87 (N87), and AGS were obtained from the American Type Culture Collection (Rockville, MD). Kato III and N87 cell lines were cultured in RPMI 1640 (GIBCO-BRL, Grand Island, NY) supplemented with 10% heat-inactivated fetal calf serum (FCS; GIBCO-BRL), whereas the AGS cells were grown in Hams F12 (GIBCO-BRL) supplemented with 10% FCS. All cells were grown in a monolayer in 25- or 75-cm2 tissue culture flasks at 37°C in a humidified, 5% carbon dioxide incubator until they approached approximately 80%–90% confluency. Gastric biopsy specimens were collected from consenting adults undergoing gastroesophageal duodenoscopy for various clinical indications, according to a University of Texas Medical Branch Institutional Review Board-approved protocol. Patients were considered infected if H. pylori were detected by rapid urease testing or by histopathology of biopsy samples. Biopsy specimens (4–6 pinches) from the antral gastric mucosa of infected or uninfected individuals were collected in cold calcium- and magnesium-free Hank's balanced salt solution (HBSS; GIBCO-BRL) supplemented with 5% FCS, 100 U/mL penicillin (Sigma Chemical Company, St. Louis, MO), and 100 μg/mL streptomycin (Sigma). Epithelial cells were isolated from the specimens as described previously.7Fan X.J. Crowe S.E. Behar S. Gunasena H. Ye G. Haeberle H. Van Houten N. Gourley W.K. Ernst P.B. Reyes V.E. The effect of class II MHC expression on adherence of Helicobacter pylori and induction of apoptosis in gastric epithelial cells a mechanism for Th1 cell-mediated damage.J Exp Med. 1998; 187: 1659-1669Crossref PubMed Scopus (182) Google Scholar, 37Ye G. Barrera C. Fan X.J. Gourley W.K. Crowe S.E. Ernst P.B. Reyes V.E. Expression of B7-1 and B7-2 costimulatory molecules by human gastric epithelial cells. Potential role in CD4+ T cell activation during Helicobacter pylori infection.J Clin Invest. 1997; 99: 1628-1636Crossref PubMed Scopus (132) Google Scholar, 38Barrera C. Ye G. Espejo R. Gunasena S. Almanza R. Leary J. Crowe S. Ernst P. Reyes V.E. Expression of cathepsins B, L, S, and D by gastric epithelial cells implicates them as antigen presenting cells in local immune responses.Hum Immunol. 2001; 62: 1081-1091Crossref PubMed Scopus (42) Google Scholar Briefly, rinsed tissue was agitated at 37°C in HBSS containing 0.1 mmol/L dithiothreitol (DTT; Sigma) and 0.1 mmol/L ethylenediaminetetraacetic acid (EDTA; Sigma) for 15 minutes. The supernatant was decanted, replaced with fresh HBSS/EDTA/DTT, and incubated for another 15 minutes. Subsequently, the biopsy tissue was placed in a 2.4 U/mL dispase solution (Boehringer Mannheim, Mannheim, Germany), agitated at 37°C for 15 minutes, and then centrifuged at 200g for 5 minutes. Fresh dispase solution was added, and the procedure was repeated. The isolated cells were confirmed to be >90% epithelial cells according to morphology with May-Grunwald-Giemsa (Sigma) staining and immunofluorescence staining of epithelial anticytokeratin antigen, as described previously.38Barrera C. Ye G. Espejo R. Gunasena S. Almanza R. Leary J. Crowe S. Ernst P. Reyes V.E. Expression of cathepsins B, L, S, and D by gastric epithelial cells implicates them as antigen presenting cells in local immune responses.Hum Immunol. 2001; 62: 1081-1091Crossref PubMed Scopus (42) Google Scholar The viability of the isolated cells was determined by trypan blue exclusion, and cells were not used unless viability exceeded 90%. The freshly isolated gastric epithelial cells were maintained in RPMI 1640 containing 10% FCS and were used immediately in assays as described below. H. pylori 26695, a cag PAI-bearing strain,39Akopyants N.S. Clifton S.W. Kersulyte D. Crabtree J.E. Youree B.E. Reece C.A. Burkanov N.O. Drazek E.S. Roe B.A. Berg D.E. Analyses of the cag pathogenicity island of Helicobacter pylori.Mol Microbiol. 1998; 28: 37-53Crossref PubMed Scopus (480) Google Scholar and the isogenic cag PAI-deficient knockout strain, H. pylori 8-1, were provided by D. Berg (Washington University School of Medicine, St. Louis, MO). H. pylori LC-11, a cag PAI-bearing strain isolated from a clinical specimen,40Dytoc M. Gold B. Louie M. Heusca M. Fedorko L. Crowe S.E. Lingwood C. Brunton J. Sherman P.M. Comparison of Helicobacter pylori and attaching-effacing Escherichia coli adhesion to eukaryotic cells.Infect Immun. 1993; 61: 448-456Crossref PubMed Google Scholar was provided by P. Sherman (University of Toronto, Toronto, Ontario, Canada). H. pylori AH 244, a naturally occurring cagA- and cagE-deficient strain, was obtained from R. Alms (AstraZeneca, Boston, MA). A nongastric mucosal pathogen, Campylobacter jejuni, was provided by R. Guerrant (University of Virginia, Charlottesville, VA). All strains of bacteria were maintained on blood agar plates (Becton Dickinson, Cockeysville, MD). Before use in experiments, the bacteria were cultured overnight at 37°C in 25 mL of brucella broth (GIBCO-BRL) supplemented with 10% FCS under microaerophilic conditions. Subsequently, the bacteria were harvested, and the bacterial number was determined as described previously.7Fan X.J. Crowe S.E. Behar S. Gunasena H. Ye G. Haeberle H. Van Houten N. Gourley W.K. Ernst P.B. Reyes V.E. The effect of class II MHC expression on adherence of Helicobacter pylori and induction of apoptosis in gastric epithelial cells a mechanism for Th1 cell-mediated damage.J Exp Med. 1998; 187: 1659-1669Crossref PubMed Scopus (182) Google Scholar Before use, bacterial mobility was confirmed by phase-contrast microscopy. For all assays, gastric epithelial cell viability was determined by trypan blue exclusion, and a known number of AGS (5 × 105), N87 (1 × 106), or Kato III (5 × 105) cells were seeded into 9.5-cm2 6-well plates. Freshly isolated gastric epithelial cells (1–2 × 105) were seeded into 1.9-cm2 24-well plates. The gastric epithelial cell lines were incubated for 24 hours before stimulation, whereas the native gastric epithelial cells were used immediately after isolation. To study the induction of APE-1/Ref-1 expression, dose-response studies were performed to determine an optimal bacterial/epithelial cell ratio and the optimal concentrations of H2O2 (Sigma) to use for stimulation of gastric epithelial cells. Dose-response studies showed that infection with H. pylori 26695 at ratios of 50:1 to 1000:1 or stimulation with 200–400 μmol/L H2O2 significantly enhanced APE-1/Ref-1 expression in gastric epithelial cell lines compared with control cells. Increased levels of APE-1/Ref-1 were significantly and consistently detected with 200 μmol/L H2O2 and a bacterial/epithelial cell ratio of 300:1, a dose we have used in previous studies.7Fan X.J. Crowe S.E. Behar S. Gunasena H. Ye G. Haeberle H. Van Houten N. Gourley W.K. Ernst P.B. Reyes V.E. The effect of class II MHC expression on adherence of Helicobacter pylori and induction of apoptosis in gastric epithelial cells a mechanism for Th1 cell-mediated damage.J Exp Med. 1998; 187: 1659-1669Crossref PubMed Scopus (182) Google Scholar Stimulation with H. pylori or H2O2 at these doses resulted in a time-dependent up-regulation of APE-1/Ref-1 expression, with maximal levels detected after 3–6 hours. Consequently, in subsequent assays, epithelial cells were stimulated for 3 hours with 300:1 H. pylori or 200 μmol/L H2O2 to observe their effect on APE-1/Ref-1 expression. In some assays, epithelial cells were co-stimulated with 300:1 H. pylori and 200 μmol/L H2O2 to determine whether H. pylori and H2O2 together exert a synergistic effect on APE-1/Ref-1 expression. The effect of antioxidants on H. pylori- or ROS-induced APE-1/Ref-1 expression was examined by preincubation of the epithelial cells with 10 mmol/L N-acetylcysteine (NAC; Sigma), 10 mmol/L glutathione (GSH; Sigma), or 100 μmol/L butylated hydroxyanisole (BHA) for 30 minutes before stimulation. These concentrations were based on our observations in dose-response studies, and similar concentrations have been used in other studies of oxidative injury.41Quillet-Mary A. Jaffrezou J.P. Mansat V. Bordier C. Naval J. Laurent G. Implication of mitochondrial hydrogen peroxide generation in ceramide-induced apoptosis.J Biol Chem. 1997; 272: 21388-21395Crossref PubMed Scopus (446) Google Scholar, 42Seo J.Y. Kim H. Kim K.H. Transcriptional regulation by thiol compounds in Helicobacter pylori-induced interleukin-8 production in human gastric epithelial cells.Ann N Y Acad Sci. 2002; 973: 541-545Crossref PubMed Scopus (25) Google Scholar To evaluate whether live H. pylori were necessary to up-regulate APE-1/Ref-1 expression, formalin-killed H. pylori, prepared as previously described,43Crowe S.E. Alvarez L. Sherman P.M. Jin Y. Dytoc M. Hunt R.H. Patel J. Muller M.J. Ernst P.B. Expression of interleukin-8 and CD54 by human gastric epithelium after H. pylori infection in vitro.Gastroenterology. 1995; 108: 65-74Abstract Full Text PDF PubMed Scopus (249) Google Scholar were used to infect gastric epithelial cells at a ratio of 300:1. To compare the effect of another human gut mucosal pathogen, C. jejuni, a nongastric gram-negative pathogen was used to infect gastric epithelial cells, also at a ratio of 300:1. To examine APE-1/Ref-1 expression, whole cell lysates of untreated or stimulated gastric epithelial cells were prepared as previously described.44Wang J. Brooks E.G. Bamford K.B. Denning T.L. Pappo J. Ernst P.B. Negative selection of T cells by Helicobacter pylori as a model for bacterial strain selection by immune evasion.J Immunol. 2001; 167: 926-934PubMed Google Scholar Briefly, cells were lysed in cold lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.5% Nonidet P-40 [Sigma], 50 mmol/L NaF, 1 mmol/L NaVO3, 1 mmol/L phenylmethylsulfonyl fluoride, 25 μg/mL aprotinin, 25 μg/mL pepstatin A, and 25 μg/mL leupeptin) for 5 minutes on ice and centrifuged at 14,000 × g for 5 minutes at 4°C. The protein concentration of the resulting lysates was determined by Bradford assay (Bio-Rad, Hercules, CA), and the samples were stored at −80°C. To determine whether APE-1/Ref-1 was expressed in cell nuclei, nuclear proteins were extracted from H. pylori- or H2O2-treated N87 (1 × 107), Kato III (5 × 106), or AGS (5 × 106) cells. At different time points, the cells were harvested and were washed once with cold phosphate-buffered saline (PBS). Subsequently, nuclear pellets were prepared by resuspending cells in 400 μL of hypotonic lysis buffer [10 mmol/L HEPES, 10 mmol/L KCl, 1 mmol/L DTT, 0.1 mmol/L EDTA, 0.1 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N, N′,N′-tetraacetic acid, and 1% protein inhibitor cocktail; Sigma] at 4°C for 30 minutes. Then 25 μL of 10% Nonidet P-40 was added, and the suspension was mixed vigorously. After a 30-second centrifugation (14,000 × g; 4°C), the pelleted nuclei were resuspended in 100 μL of extraction buffer [20 mmol/L HEPES, 0.4 mol/L KCl, 1 mmol/L DTT, 0.1 mmol/L EDTA, 0.1 mmol/L ethylene glycol-bis(β-aminoethyl ether)-N,N, N′,N′-tetraacetic acid, and 1% protein inhibitor cocktail] and incubated on ice for 20 minutes. The protein concentration of the extracts was determined by Bradford assay, and the nuclear extracts were stored at −80°C until assayed. Immunoblotting was performed on whole cell lysates or nuclear proteins as previously described.44Wang J. Brooks E.G. Bamford K.B. Denning T.L. Pappo J. Ernst P.B. Negative selection of T cells by Helicobacter pylori as a model for bacterial strain selection by immune evasion.J Immunol. 2001; 167: 926-934PubMed Google Scholar Protein samples (10 μg) were fractionated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (12%) in a discontinuous buffer system. After electrophoresis, the separated proteins were transferred onto nitrocellulose membranes with the Trans-blot cell (Bio-Rad). Immunodetection analysis was performed by incubating the membrane with a specific rabbit anti-human APE-1/Ref-1 polyclonal antibody24Ramana C.V. Boldogh I. Izumi T. Mitra S. Activation of apurinic/apyrimidinic endonuclease in human cells by reactive oxygen species and its correlation with their adaptive response to genotoxicity of free radicals.Proc Natl Acad Sci U S A. 1998; 95: 5061-5065Crossref PubMed Scopus (383) Google Scholar at a 1:2000 dilution, followed by goat anti-rabbit immunoglobulin G horseradish peroxidase conjugate (Santa Cruz Biotechnology, Santa Cruz, CA) as a second antibody. Immunoreactions were visualized by enhanced chemiluminescence (Amersham Biosciences, Buckinghamshire, UK) and autoradiographed. Quantitative changes were determined by densitometric analysis of the immunoreactive band by using an Alphaimager image-analysis system (Alpha Innotech, San Leandro, CA). Protein loading was normalized by stripping the membrane with stripping buffer (62.5 mmol/L T
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