Volume 124, Issue 4, Pages 983-992 (April 2003) Helicobacter pylori induces matrix metalloproteinase-9 through activation of nuclear factor κB  Naoki Mori, Hiroshi Sato, Toshihisa Hayashibara, Masachika Senba, Romas Geleziunas, Akihiro Wada, Toshiya Hirayama, Naoki Yamamoto  Gastroenterology  Volume 124, Issue 4, Pages 983-992 (April 2003) DOI: 10.1053/gast.2003.50152 Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 1 H. pylori-induced MMP-9 mRNA expression in gastric epithelial cells. (A and B) Time course of H. pylori-induced MMP-9 mRNA expression. Total RNA was extracted from (A) MKN45 and (B) MKN28 cells infected with H. pylori for the indicated time intervals and used for RT-PCR. (C) The cag PAI of H. pylori is required for induction of MMP-9 expression in MNK45 cells. Total RNA was extracted from the cells infected with H. pylori for 6 hours and used for RT-PCR. β-actin expression served as control. Gastroenterology 2003 124, 983-992DOI: (10.1053/gast.2003.50152) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 2 Zymographic analysis of serum-free culture media conditioned by MKN45 cells alone and by MKN45 cells infected with H. pylori for the indicated time intervals. A total of 12 μL conditioned medium was subjected to electrophoresis on 10% sodium dodecyl sulfate/polyacrylamide gels containing 1 mg/mL gelatin. Sodium dodecyl sulfate was removed following electrophoresis, and gels were incubated to restore gelatinase activity. Lane 1, conditioned media from peripheral blood mononuclear cells activated by phorbol myristate acetate (positive control for MMP-2 and MMP-9). Major gelatinolytic activities corresponding to pro-MMP-9 (92 kilodaltons) and pro-MMP-2 (72 kilodaltons) are indicated. Gastroenterology 2003 124, 983-992DOI: (10.1053/gast.2003.50152) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 3 H. pylori infection activates the MMP-9 promoter in gastric epithelial cells. (A) Deletion and mutational analysis of the cis elements required for H. pylori-induced MMP-9 promoter activity. The arrow indicates the transcription start site, and × indicates the sites of mutation in the MMP-9 5′ flanking sequence inserted upstream of the CAT gene. These constructs were transfected into MKN45 cells, and the cells were subsequently infected with H. pylori for 24 hours. The activities are expressed relative to that of cells transfected with −73 CAT without further treatment, which was defined as 1. Data are also expressed as a fold induction relative to the basal level measured in uninfected cells. (B) Functional effects of IκBα and IκBβ dominant interfering mutants and kinase-deficient IKKα, IKKβ, and NIK mutants on H. pylori-induced activation of the MMP-9 promoter. MKN45 cells were transfected with 10 μg of −670 CAT and 10 μg of the IκBα mutant IκBαΔN, the IκBβ mutant IκBβΔN, the K44M mutant of IKKα, the K44A mutant of IKKβ, the kinase-deficient KK429/430AA mutant of NIK, or empty vector (pCMV4) and then infected with H. pylori for 24 hours. □, CAT activity of empty vector without H. pylori infection. All values were first calculated as a fold induction relative to the basal level measured in uninfected cells. Data are mean ± SD values of 3 independent experiments. Gastroenterology 2003 124, 983-992DOI: (10.1053/gast.2003.50152) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 4 H. pylori infection induced NF-κB but not SP-1 binding activity. (A) Time course of NF-κB activation in MKN45 cells infected with H. pylori, evaluated by electrophoretic mobility shift assay. Nuclear extracts from MKN45 cells infected with H. pylori for the indicated time periods were mixed with either NF-κB or SP-1 32P-labeled probes. (B) Sequence specificity of NF-κB binding activity and characterization of NF-κB proteins that bound to the NF-κB binding site of the MMP-9 gene. Competition assays were performed with nuclear extracts from MKN45 cells infected with H. pylori for 60 minutes. Where indicated, 100-fold excess amounts of each specific competitor oligonucleotide were added to the reaction mixture with labeled probes NF-κB (lanes 7-11). Supershift assay of NF-κB DNA-binding complexes in the same nuclear extracts was also performed. Where indicated, appropriate antibodies were added to the reaction mixture before the addition of 32P-labeled probes (lanes 1-6). Arrowheads show the DNA-binding complexes supershifted by antibodies. (C) Sequence specificity of SP-1 binding activity in MKN45 cells. The radiolabeled SP-1 probe derived from the MMP-9 promoter was incubated with nuclear extracts from H. pylori-infected MKN45 cells without (lane 1) or with (lanes 2-4) a 100-fold excess amount of each specific competitor oligonucleotide. (D) cag PAI products of H. pylori are required for induction of NF-κB binding activity in MKN45 cells. Nuclear extracts from uninfected MKN45 cells (lane 1) and cells cocultured with the indicated H. pylori strains (lanes 2-5) were analyzed for NF-κB. Gastroenterology 2003 124, 983-992DOI: (10.1053/gast.2003.50152) Copyright © 2003 American Gastroenterological Association Terms and Conditions

Fig. 5 Expression of MMP-9 in H. pylori-infected gastric mucosa. (A) RT-PCR analysis of MMPs in human gastric tissues. Lanes 1-3, normal mucosa; lanes 4-6, H. pylori-positive gastritis. β-actin expression served as control. (B-E) Immunohistochemical detection of MMP-9 in tissues from patients with gastric diseases. The serial sections of gastric biopsy specimens were stained with 56-2A4 monoclonal antibody. Sections were counterstained with methyl green. Tissues in B and C are H. pylori-positive gastritis; other tissues are (D) H. pylori-positive and (E) -negative gastric ulcers. In H. pylori-positive gastritis and gastric ulcers, there was MMP-9 staining of (B-D) epithelial cells and (C, arrows) macrophages. MMP-9 protein in H. pylori-negative gastric ulcers was stained primarily in macrophages (E, arrows). H. pylori-negative gastric ulcer tissues showed MMP-9 staining in fibroblasts (E), but this staining was less intense than that of H. pylori-positive gastric diseases (B-D). (Original magnification 400×.) Gastroenterology 2003 124, 983-992DOI: (10.1053/gast.2003.50152) Copyright © 2003 American Gastroenterological Association Terms and Conditions