Volume 119, Issue 1, Pages 97-108 (July 2000) H. pylori activates NF-κB through a signaling pathway involving IκB kinases, NF-κB— inducing kinase, TRAF2, and TRAF6 in gastric cancer cells  Shin Maeda, Haruhiko Yoshida, Keiji Ogura, Yuzo Mitsuno, Yoshihiro Hirata, Yutaka Yamaji, Masao Akanuma, Yasushi Shiratori, Masao Omata  Gastroenterology  Volume 119, Issue 1, Pages 97-108 (July 2000) DOI: 10.1053/gast.2000.8540 Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 1 H. pylori–induced NF-κB activation was observed in gastric cancer cells. Gastric cancer cells were transfected with NF-κB-Luc expression vectors (0.2 μg) for 24 hours and were stimulated with live H. pylori (107 CFU/mL), TNF-α (10 ng/mL), and IL-1 (10 ng/mL) for 8 hours before luciferase assay. Luciferase activity is presented as a fold induction relative to the basal level measured in unstimulated cells. The values are the mean ± SD from 3 independent experiments. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 2 Cag PAI was required for activating NF-κB. MKN45 cells were transfected with NF-κB-Luc expression vectors for 24 hours and were stimulated with different strains for 8 hours. TN2 and 26695 were cag PAI intact and T-25 and Tx30a were partially or totally deleted strains, respectively. TN2-ΔcagE and TN2-ΔvacA indicate isogenic cagE and vacA mutants prepared by insertion of a kanamycin-resistant gene into cagE or vacA loci, respectively. Luciferase activity is presented as a fold induction relative to the basal level measured in unstimulated cells. The values are the mean ± SD from 3 independent experiments. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 3 H. pylori–induced IκBα degradation. MKN45 cells were cocultured with H. pylori (107 CFU/mL) or TNF-α (10 ng/mL), and whole-cell extracts were prepared at the indicated times. Proteins (10 μg) were separated by electrophoresis and immunoblotted with anti-IκBα. Cell extracts were also probed with antiactin antibody to confirm equal loading of cell protein. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 4 NF-κB activation by H. pylori was executed through site-specific phosphorylation of IκBα. MKN45 and KATO III cells were transfected with 0.2 μg of NF-κB-Luc and the indicated amount of expression vector encoding dominant negative IκBα (S32A/S36A). After 24 hours, cells were cocultured with H. pylori (TN2) or treated with TNF-α (10 ng/mL) for 8 hours. Luciferase activity is presented as a fold induction relative to the basal level measured in unstimulated cells. The values are the mean ± SD from 3 independent experiments. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 IKKs were activated by H. pylori in vivo. (A) MKN45 cells were transfected with expression vectors encoding FLAG-IKKα or Flag-IKKβ (1 μg/6-well plate). After 30 hours, the cells were either treated with H. pylori for 1, 2, 4, and 8 hours or left untreated. FLAG-IKK was immunoprecipitated using agarose-conjugated anti-FLAG antibody, and its activity was determined by immunocomplex kinase assay with GST-IκBα (2-317) as a substrate. An aliquot of each lysate was analyzed for its content of IKK by immunoblotting using an anti-FLAG antibody. (B) Endogenous IKKβ was activated by H. pylori. MKN45 and KATO III cells were either treated with H. pylori for 1, 2, and 4 hours and TNF-α for 15 minutes or left untreated. IKKβ was immunoprecipitated using IKKβ polyclonal antibody, and its activity was determined by immunocomplex kinase assay with GST-IκBα (2-317) as a substrate. An aliquot of each lysate was analyzed for its content of IKKβ by immunoblotting. Similar results were obtained in 3 independent experiments. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 5 IKKs were activated by H. pylori in vivo. (A) MKN45 cells were transfected with expression vectors encoding FLAG-IKKα or Flag-IKKβ (1 μg/6-well plate). After 30 hours, the cells were either treated with H. pylori for 1, 2, 4, and 8 hours or left untreated. FLAG-IKK was immunoprecipitated using agarose-conjugated anti-FLAG antibody, and its activity was determined by immunocomplex kinase assay with GST-IκBα (2-317) as a substrate. An aliquot of each lysate was analyzed for its content of IKK by immunoblotting using an anti-FLAG antibody. (B) Endogenous IKKβ was activated by H. pylori. MKN45 and KATO III cells were either treated with H. pylori for 1, 2, and 4 hours and TNF-α for 15 minutes or left untreated. IKKβ was immunoprecipitated using IKKβ polyclonal antibody, and its activity was determined by immunocomplex kinase assay with GST-IκBα (2-317) as a substrate. An aliquot of each lysate was analyzed for its content of IKKβ by immunoblotting. Similar results were obtained in 3 independent experiments. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 H. pylori–induced NF-κB activation was mediated by IKKs. MKN45 and KATO III cells were cotransfected with the NF-κB-Luc reporter plasmid (0.2 μg) and the indicated amount of expression vectors encoding catalytically inactive (A) IKKα (K44M) or (B) IKKβ (K44M). After 24 hours, cells were cocultured with H. pylori (TN2) or treated with TNF-α (10 ng/mL) for 8 hours. Luciferase activity is presented as a fold induction relative to the basal level measured in unstimulated cells. The values are the mean ± SD from 3 independent experiments. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 6 H. pylori–induced NF-κB activation was mediated by IKKs. MKN45 and KATO III cells were cotransfected with the NF-κB-Luc reporter plasmid (0.2 μg) and the indicated amount of expression vectors encoding catalytically inactive (A) IKKα (K44M) or (B) IKKβ (K44M). After 24 hours, cells were cocultured with H. pylori (TN2) or treated with TNF-α (10 ng/mL) for 8 hours. Luciferase activity is presented as a fold induction relative to the basal level measured in unstimulated cells. The values are the mean ± SD from 3 independent experiments. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) Aspirin inhibits NF-κB activation by H. pylori. MKN45 cells were transfected with the NF-κB-Luc reporter plasmid (0.2 μg). After 24 hours, cells were treated with the indicated concentration of aspirin or indomethacin for 1 hour. Next, cells were cocultured with H. pylori or treated with 10 ng/mL TNF-α for 8 hours. Luciferase activity is presented as a fold induction relative to the basal level measured in unstimulated cells. The values are the mean ± SD from 4 independent experiments. (B) Aspirin inhibits IL-8 secretion by H. pylori. MKN cells were treated with the indicated concentration of aspirin or indomethacin for 1 hour. The cells were cocultured with H. pylori or treated with 10 ng/mL TNF-α for 12 hours. IL-8 levels were measured by enzyme-linked immunosorbent assay in harvested conditioned media. The values are the mean ± SD from 4 independent experiments. (C) Aspirin but not indomethacin inhibits H. pylori–mediated IKKβ kinase activity. MKN45 cells were treated with 5 mmol/L aspirin or 25 μmol/L indomethacin for 2 hours and cocultured with H. pylori for 2 hours. IKKβ was immunoprecipitated using IKKβ polyclonal antibody, and its activity was determined by immunocomplex kinase assay with GST-IκBα (2-317) as a substrate. An aliquot of each lysate was analyzed for its content of IKKβ by immunoblotting. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) Aspirin inhibits NF-κB activation by H. pylori. MKN45 cells were transfected with the NF-κB-Luc reporter plasmid (0.2 μg). After 24 hours, cells were treated with the indicated concentration of aspirin or indomethacin for 1 hour. Next, cells were cocultured with H. pylori or treated with 10 ng/mL TNF-α for 8 hours. Luciferase activity is presented as a fold induction relative to the basal level measured in unstimulated cells. The values are the mean ± SD from 4 independent experiments. (B) Aspirin inhibits IL-8 secretion by H. pylori. MKN cells were treated with the indicated concentration of aspirin or indomethacin for 1 hour. The cells were cocultured with H. pylori or treated with 10 ng/mL TNF-α for 12 hours. IL-8 levels were measured by enzyme-linked immunosorbent assay in harvested conditioned media. The values are the mean ± SD from 4 independent experiments. (C) Aspirin but not indomethacin inhibits H. pylori–mediated IKKβ kinase activity. MKN45 cells were treated with 5 mmol/L aspirin or 25 μmol/L indomethacin for 2 hours and cocultured with H. pylori for 2 hours. IKKβ was immunoprecipitated using IKKβ polyclonal antibody, and its activity was determined by immunocomplex kinase assay with GST-IκBα (2-317) as a substrate. An aliquot of each lysate was analyzed for its content of IKKβ by immunoblotting. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 7 (A) Aspirin inhibits NF-κB activation by H. pylori. MKN45 cells were transfected with the NF-κB-Luc reporter plasmid (0.2 μg). After 24 hours, cells were treated with the indicated concentration of aspirin or indomethacin for 1 hour. Next, cells were cocultured with H. pylori or treated with 10 ng/mL TNF-α for 8 hours. Luciferase activity is presented as a fold induction relative to the basal level measured in unstimulated cells. The values are the mean ± SD from 4 independent experiments. (B) Aspirin inhibits IL-8 secretion by H. pylori. MKN cells were treated with the indicated concentration of aspirin or indomethacin for 1 hour. The cells were cocultured with H. pylori or treated with 10 ng/mL TNF-α for 12 hours. IL-8 levels were measured by enzyme-linked immunosorbent assay in harvested conditioned media. The values are the mean ± SD from 4 independent experiments. (C) Aspirin but not indomethacin inhibits H. pylori–mediated IKKβ kinase activity. MKN45 cells were treated with 5 mmol/L aspirin or 25 μmol/L indomethacin for 2 hours and cocultured with H. pylori for 2 hours. IKKβ was immunoprecipitated using IKKβ polyclonal antibody, and its activity was determined by immunocomplex kinase assay with GST-IκBα (2-317) as a substrate. An aliquot of each lysate was analyzed for its content of IKKβ by immunoblotting. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 8 H. pylori–induced NF-κB activation mediated by NIK. MKN45 and KATO III cells were cotransfected with the NF-κB-Luc reporter plasmid (0.2 μg) and the indicated amount of expression vectors encoding catalytically inactive NIK (KK 429/430 MM). After 24 hours, cells were cocultured with H. pylori (TN2) or treated with 10 ng/mL TNF-α for 8 hours. Luciferase activity is presented as a fold induction relative to the basal level measured in unstimulated cells. The values are the mean ± SD from 3 independent experiments. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 9 H. pylori–induced NF-κB activation mediated by TRAF2 and TRAF6. (A) MKN45 and TMK1 cells were cotransfected with the NF-κB-Luc reporter plasmid (0.2 μg) and the indicated amount of expression vectors encoding dominant negative TRAF2. After 24 hours, cells were cocultured with H. pylori (TN2) or treated with 10 ng/mL TNF-α for 8 hours. (B) Cells were cotransfected with the NF-κB-Luc reporter plasmid (0.2 μg) and the indicated amount of expression vectors encoding dominant negative TRAF6. After 24 hours, cells were cocultured with H. pylori (TN2) or treated with 10 ng/mL IL-1 for 8 hours. (C) Cells were cotransfected with the NF-κB-Luc reporter plasmid (0.2 μg) and the indicated amount of expression vectors encoding dominant negative TRAF2 and TRAF6. After 24 hours, cells were cocultured with H. pylori (TN2) for 8 hours. Luciferase activity is presented as a fold induction relative to the basal level measured in unstimulated cells. The values are the mean ± SD from 3 independent experiments. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 9 H. pylori–induced NF-κB activation mediated by TRAF2 and TRAF6. (A) MKN45 and TMK1 cells were cotransfected with the NF-κB-Luc reporter plasmid (0.2 μg) and the indicated amount of expression vectors encoding dominant negative TRAF2. After 24 hours, cells were cocultured with H. pylori (TN2) or treated with 10 ng/mL TNF-α for 8 hours. (B) Cells were cotransfected with the NF-κB-Luc reporter plasmid (0.2 μg) and the indicated amount of expression vectors encoding dominant negative TRAF6. After 24 hours, cells were cocultured with H. pylori (TN2) or treated with 10 ng/mL IL-1 for 8 hours. (C) Cells were cotransfected with the NF-κB-Luc reporter plasmid (0.2 μg) and the indicated amount of expression vectors encoding dominant negative TRAF2 and TRAF6. After 24 hours, cells were cocultured with H. pylori (TN2) for 8 hours. Luciferase activity is presented as a fold induction relative to the basal level measured in unstimulated cells. The values are the mean ± SD from 3 independent experiments. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 9 H. pylori–induced NF-κB activation mediated by TRAF2 and TRAF6. (A) MKN45 and TMK1 cells were cotransfected with the NF-κB-Luc reporter plasmid (0.2 μg) and the indicated amount of expression vectors encoding dominant negative TRAF2. After 24 hours, cells were cocultured with H. pylori (TN2) or treated with 10 ng/mL TNF-α for 8 hours. (B) Cells were cotransfected with the NF-κB-Luc reporter plasmid (0.2 μg) and the indicated amount of expression vectors encoding dominant negative TRAF6. After 24 hours, cells were cocultured with H. pylori (TN2) or treated with 10 ng/mL IL-1 for 8 hours. (C) Cells were cotransfected with the NF-κB-Luc reporter plasmid (0.2 μg) and the indicated amount of expression vectors encoding dominant negative TRAF2 and TRAF6. After 24 hours, cells were cocultured with H. pylori (TN2) for 8 hours. Luciferase activity is presented as a fold induction relative to the basal level measured in unstimulated cells. The values are the mean ± SD from 3 independent experiments. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 10 (A) Effect of anti–TNF-α antibody on H. pylori–induced NF-κB activation. Cells were transfected as described, and 1 μg/mL anti–TNF-α antibody was added before treatment with either H. pylori or 10 ng/mL TNF-α. (B) Effect of IL-1 receptor antagonist (IL-1 RA) on H. pylori–induced NF-κB activation. Cells were transfected as described, and 100 ng/mL IL-1 receptor antagonist (IL-1 RA) was added before treatment with either H. pylori or 10 ng/mL IL-1β. Luciferase activity is presented as a fold induction relative to the basal level measured in unstimulated cells. The values are the mean ± SD from 3 independent experiments. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions

Fig. 10 (A) Effect of anti–TNF-α antibody on H. pylori–induced NF-κB activation. Cells were transfected as described, and 1 μg/mL anti–TNF-α antibody was added before treatment with either H. pylori or 10 ng/mL TNF-α. (B) Effect of IL-1 receptor antagonist (IL-1 RA) on H. pylori–induced NF-κB activation. Cells were transfected as described, and 100 ng/mL IL-1 receptor antagonist (IL-1 RA) was added before treatment with either H. pylori or 10 ng/mL IL-1β. Luciferase activity is presented as a fold induction relative to the basal level measured in unstimulated cells. The values are the mean ± SD from 3 independent experiments. Gastroenterology 2000 119, 97-108DOI: (10.1053/gast.2000.8540) Copyright © 2000 American Gastroenterological Association Terms and Conditions