Gastroenterology

Gastroenterology

Volume 119, Issue 1, July 2000, Pages 97-108
Gastroenterology

Alimentary Tract
H. pylori activates NF-κB through a signaling pathway involving IκB kinases, NF-κB—inducing kinase, TRAF2, and TRAF6 in gastric cancer cells

https://doi.org/10.1053/gast.2000.8540Get rights and content

Abstract

Background & Aims: H. pylori infection on gastric epithelial cells has been shown to induce NF-κB activation, but the mechanism of intracellular signal conduction that leads to NF-κB activation is not clear. The aim of this study was to analyze the molecular mechanism responsible for H. pylori–mediated NF-κB activation on gastric cancer cells. Methods: NF-κB activation by H. pylori was tested by using luciferase reporter assay. IκBα degradation by H. pylori infection was assessed by immunoblotting. IKKα and IKKβ activation was analyzed by kinase assay. In transfection experiments, effects of dominant negative IκBα, IKKα, IKKβ, NF-κB–inducing kinase (NIK), TRAF2, and TRAF6 mutants were investigated. The effects of an IKKβ-specific inhibitor, aspirin, on NF-κB activation and IL-8 secretion were also analyzed. Results: H. pylori promotes degradation of IκBα, a cytoplasmic inhibitor of NF-κB. In kinase assay, H. pylori induced IKKα and IKKβ catalytic activity in gastric cancer cells. Transfection of kinase-deficient mutant of either IKK inhibited H. pylori–mediated NF-κB activation dose-dependently. Aspirin inhibited both NF-κB activation and IL-8 secretion induced by H. pylori. NF-κB activation was also inhibited by transfection of kinase-deficient NIK or a dominant negative mutant of upstream adapter protein TRAF2 or TRAF6. Conclusions: H. pylori induces NF-κB activation through an intracellular signaling pathway that involves IKKα, IKKβ, NIK, TRAF2, and TRAF6.

GASTROENTEROLOGY 2000;119:97-108

Section snippets

Bacterial strains

H. pylori isolates used in the study were as follows (Table 1).

. H. pylori strains used in this study

Empty Cellcag PAIVacuolating cytotoxin
TN2IntactPositive
26695IntactPositive
T-25Partially deletedNegative
TX30aTotally deletedNegative
TN2ΔcagEDisruptedPositive
TN2ΔvacAIntactDisrupted
TN2 is a strain positive for CagA and VacA (vacuolating cytotoxin) and was provided by Dr. Nakao (Takeda Chemical Industries, Osaka, Japan). We performed Southern blot analysis of 13 cag PAI genes, as described previously,40 and

NF-κB activation in gastric cancer cells induced by H. pylori

To assess whether NF-κB is activated in gastric cancer cells by H. pylori, TNF-α, or IL-1, H. pylori (107 CFU/mL), TNF-α (10 ng/mL), or IL-1 (10 ng/mL) was incubated for 8 hours before luciferase assay. NF-κB activation was increased 5.2-fold with H. pylori, 6.1-fold with TNF-α, and 5.2-fold with IL-1 in MKN45 cells. In KATO III cells, H. pylori and TNF-α activated NF-κB at 6.9 and 7.0, respectively. However, IL-1 did not activate NF-κB. In TMK1 cells, NF-κB activation was increased 3.4-fold by

Discussion

This study evaluated and characterized H. pylori–mediated activation of the transcriptional factor NF-κB. Activation was inhibited in gastric cancer cells by transfection of kinase-deficient NIK, indicating that NIK is involved in H. pylori–mediated NF-κB activation, as reported in cytokine-mediated pathways. We also demonstrated that the H. pylori–mediated signaling pathway downstream of NIK—activation of IKKα and IKKβ, phosphorylation and degradation of IκB, and the resulting release into the

Acknowledgements

The authors thank Dr. D Goeddel for providing the expression plasmids of IKKα, IKKβ, the dominant negative mutant of IKKα, IKKα (K44A), IKKβ, IKKβ (K44A), and the dominant negative mutant of TRAF2, TRAF2 (86-501) and TRAF6, TRAF6 (289-522); Dr. D Wallach for providing the dominant negative mutant of NIK, NIK (KK429-430AA); Dr. H Suzuki for providing the dominant negative mutant of IκBα, IκBα (SS32/36AA) and glutathione GST-tagged IκBα protein (2-317); and Mitsuko Tsubouchi for excellent

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    Address requests for reprints to: Shin Maeda, M.D., Department of Gastroenterology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan. e-mail: [email protected]; fax: (81) 3-3814-0021.

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