Gastric Epithelial AGS Cells Research Articles

695 H. pylori cagY Rearrangements Modify Function of the cag Type 4 Secretion System in Response to Oncogenic Changes in the Gastric Microenvironment

The H. pylori cag type 4 secretion system (T4SS) translocates CagA into host cells which can lead to NF-κβ activation. The T4SS component CagY is essential for CagA translocation. cagY harbors two repeat motifs which engender considerable genetic diversity to this locus. We used Mongolian gerbils to select for Hp derivative strains that differed in intensity of host interactions to define the biology of long-term colonization and genetic alteration of virulence factors within the gastric niche. Gerbils were initially challenged with the carcinogenic Hp strain 7.13 for 12 weeks; two derivative isolates, 7.13-1 and 7.13-2, were then obtained from 2 independent gerbils. In vitro, strain 7.13-1 induced significantly higher levels of NF-κβ activation and CagA translocation in AGS gastric epithelial cells compared to strain 7.13-2 (fold/uninfected: 100±7 vs. 26±7, respectively; p<0.001) .We next infected new populations of gerbils with parental 7.13 and the two polar 7.13 Hp derivatives for up to 16 weeks. There were no differences in colonization among the groups, although all animals that developed adenocarcinoma harbored decreased bacterial burdens. Strain 7.131 induced premalignant lesions in 25%, 50%, 62.5%, and 75% at 4, 8, 12, and 16 weeks respectively. In contrast, the incidence of dysplasia and adenocarcinoma was only 25% and 12.5% with the 7.13 parental strain or the 7.13-2 derivative 16 weeks post-infection. In vivo-readapted strains from animals infected with parental 7.13 and 7.13-1 maintained their ability to induce high levels of NFκβ activation similar to the input strains, except for strains isolated from animals that developed cancer, which were attenuated in the ability to activate NF-κβ and translocate CagA. In contrast, readapted isolates from animals infected with strain 7.13-2 induced increased levels of NFκβ activation and CagA translocation in vitro, which was concordant with increased levels of inflammation in the corresponding gerbils. To define mechanisms that mediate these phenotypes, we examined cagY rearrangements by RFLP. We identified two distinct patterns, one specific for parental 7.13 and 7.131 and the other for strain 7.13-2. Readaptation revealed that a proportion of parental 7.13 (25%) and 7.13-1 (37.5%) isolate derivatives altered their cagY pattern, developing a 7.132-type pattern in the presence of cancer which was accompanied by decreases in NFκβ activation and CagA translocation. In contrast, 62.5% of Hp readapted isolates from animals infected with strain 7.13-2 changed their cagY pattern to one that mirrored the original 7.13 parental strain and those changes were linked to increases in inflammation, NFκβactivation and CagA translocation. Collectively, these results suggest Hp can modify function of the T4SS by alternating cagY genotype in response to the neoplastic landscape within gastric mucosa.

696 Whole-Genome Sequencing Detects Novel Virulence Variants of Helicobacter pylori cagA Gene and Its Relationship With Oncogenic cagA

The H. pylori cag type 4 secretion system (T4SS) translocates CagA into host cells which can lead to NF-κβ activation. The T4SS component CagY is essential for CagA translocation. cagY harbors two repeat motifs which engender considerable genetic diversity to this locus. We used Mongolian gerbils to select for Hp derivative strains that differed in intensity of host interactions to define the biology of long-term colonization and genetic alteration of virulence factors within the gastric niche. Gerbils were initially challenged with the carcinogenic Hp strain 7.13 for 12 weeks; two derivative isolates, 7.13-1 and 7.13-2, were then obtained from 2 independent gerbils. In vitro, strain 7.13-1 induced significantly higher levels of NF-κβ activation and CagA translocation in AGS gastric epithelial cells compared to strain 7.13-2 (fold/uninfected: 100±7 vs. 26±7, respectively; p<0.001) .We next infected new populations of gerbils with parental 7.13 and the two polar 7.13 Hp derivatives for up to 16 weeks. There were no differences in colonization among the groups, although all animals that developed adenocarcinoma harbored decreased bacterial burdens. Strain 7.131 induced premalignant lesions in 25%, 50%, 62.5%, and 75% at 4, 8, 12, and 16 weeks respectively. In contrast, the incidence of dysplasia and adenocarcinoma was only 25% and 12.5% with the 7.13 parental strain or the 7.13-2 derivative 16 weeks post-infection. In vivo-readapted strains from animals infected with parental 7.13 and 7.13-1 maintained their ability to induce high levels of NFκβ activation similar to the input strains, except for strains isolated from animals that developed cancer, which were attenuated in the ability to activate NF-κβ and translocate CagA. In contrast, readapted isolates from animals infected with strain 7.13-2 induced increased levels of NFκβ activation and CagA translocation in vitro, which was concordant with increased levels of inflammation in the corresponding gerbils. To define mechanisms that mediate these phenotypes, we examined cagY rearrangements by RFLP. We identified two distinct patterns, one specific for parental 7.13 and 7.131 and the other for strain 7.13-2. Readaptation revealed that a proportion of parental 7.13 (25%) and 7.13-1 (37.5%) isolate derivatives altered their cagY pattern, developing a 7.132-type pattern in the presence of cancer which was accompanied by decreases in NFκβ activation and CagA translocation. In contrast, 62.5% of Hp readapted isolates from animals infected with strain 7.13-2 changed their cagY pattern to one that mirrored the original 7.13 parental strain and those changes were linked to increases in inflammation, NFκβactivation and CagA translocation. Collectively, these results suggest Hp can modify function of the T4SS by alternating cagY genotype in response to the neoplastic landscape within gastric mucosa.

Physiological implications of biased signaling at histamine H2 receptors.

Histamine mediates numerous functions acting through its four receptor subtypes all belonging to the large family of seven transmembrane G-protein coupled receptors. In particular, histamine H2 receptor (H2R) is mainly involved in gastric acid production, becoming a classic pharmacological target to treat Zollinger–Ellison disease and gastric and duodenal ulcers. H2 ligands rank among the most widely prescribed and over the counter-sold drugs in the world. Recent evidence indicate that some H2R ligands display biased agonism, selecting and triggering some, but not all, of the signaling pathways associated to the H2R. The aim of the present work is to study whether famotidine, clinically widespread used ligand acting at H2R, exerts biased signaling. Our findings indicate that while famotidine acts as inverse agonist diminishing cAMP basal levels, it mimics the effects of histamine and the agonist amthamine concerning receptor desensitization and internalization. Moreover, the treatment of HEK293T transfected cells with any of the three ligands lead to a concentration dependent pERK increment. Similarly in AGS gastric epithelial cells, famotidine treatment led to both, the reduction in cAMP levels as well as the increment in ERK phosphorylation, suggesting that this behavior could have pharmacological relevant implications. Based on that, histidine decarboxylase expression was studied by quantitative PCR in AGS cells and its levels were increased by famotidine as well as by histamine and amthamine. In all cases, the positive regulation was impeded by the MEK inhibitor PD98059, indicating that biased signaling toward ERK1/2 pathway is the responsible of such enzyme regulation. These results support that ligand bias is not only a pharmacological curiosity but has physiological and pharmacological implications on cell metabolism.

Jak1/Stat3 is an upstream signaling of NF-κB activation in Helicobacter pylori-induced IL-8 production in gastric epithelial AGS cells.

Helicobacter pylori (H. pylori) induces the activation of nuclear factor-kB (NF-κB) and cytokine expression in gastric epithelial cells. The Janus kinase/signal transducers and activators of transcription (Jak/Stat) cascade is the inflammatory signaling in various cells. The purpose of the present study is to determine whether H. pylori-induced activation of NF-κB and the expression of interleukin-8 (IL-8) are mediated by the activation of Jak1/Stat3 in gastric epithelial (AGS) cells. Thus, gastric epithelial AGS cells were infected with H. pylori in Korean isolates (HP99) at bacterium/cell ratio of 300:1, and the level of IL-8 in the medium was determined by enzyme-linked immonosorbent assay. Phospho-specific and total forms of Jak1/Stat3 and IκBα were assessed by Western blot analysis, and NF-κB activation was determined by electrophoretic mobility shift assay. The results showed that H. pylori induced the activation of Jak1/Stat3 and IL-8 production, which was inhibited by a Jak/Stat3 specific inhibitor AG490 in AGS cells in a dose-dependent manner. H. pylori-induced activation of NF-κB, determined by phosphorylation of IκBα and NF-κB-DNA binding activity, were inhibited by AG490. In conclusion, Jak1/Stat3 activation may mediate the activation of NF-κB and the expression of IL-8 in H. pylori-infected AGS cells. Inhibition of Jak1/Stat3 may be beneficial for the treatment of H. pylori-induced gastric inflammation, since the activation of NF-κB is inhibited and inflammatory cytokine expression is suppressed.

Epithelial cell ADAM17 activation by Helicobacter pylori: role of ADAM17 C-terminus and Threonine-735 phosphorylation

Helicobacter pylori transactivates the epidermal growth factor receptor (EGFR) on gastric epithelial cells via a signalling cascade involving a disintegrin and metalloprotease 17 (ADAM17) cleavage of membrane bound heparin binding-epidermal growth factor (HB-EGF). The effects of H. pylori on ADAM17 C-terminus in epithelial cells have been examined. Total cellular ADAM17 and surface expression of ADAM17 were significantly increased by H. pylori in AGS gastric epithelial cells. These changes were associated with ADAM17 C-terminal phosphorylation at T375 and S791. AGS cells lacking the ADAM17 C-terminal domain induced significantly attenuated cleavage of HB-EGF and were also unable to upregulate HB-EGF and EGFR transcripts to the same extent as cells expressing full length ADAM17. In mitotic unstimulated AGS and ADAM17 over-expressing AGS cells, ADAM17 was highly T735 phosphorylated indicating ADAM17 T735 phosphorylation is modified during the cell cycle. In conclusion, H. pylori induced ADAM17 C-terminal T735 and/or S791 phosphorylation in gastric epithelial cells are likely to be an important trigger inducing ADAM17 activation and shedding of HB-EGF leading to EGFR transactivation. ADAM17 over-expression in gastric cancer represents a potential target for therapeutic intervention.

The emerging role of dendritic cells in the host immune response against Helicobacter pylori.

The emerging role of dendritic cells in the host immune response against Helicobacter pylori.

Anti‐inflammatory Properties of Gastric‐derived Lactobacillus plantarum XB7 in the Context of Helicobacter pylori Infection

Helicobacter pylori colonization of the gastric epithelium induces interleukin-8 (IL-8) production and inflammation leading to host cell damage. We searched for gastric-derived Lactobacillus with the ability to suppress H. pylori-induced inflammation. Conditioned media from gastric-derived Lactobacillus spp. were tested for the ability to suppress H. pylori-induced IL-8 production in AGS gastric epithelial cells. IL-8 protein and mRNA levels were measured by ELISA and qPCR, respectively. The changes on host cell signaling pathway were analyzed by Western blotting and the anti-inflammatory effect was tested in a Sprague-Dawley rat model. Conditioned media from L. salivarius B101, L. rhamnosus B103, and L. plantarum XB7 suppressed IL-8 production and IL-8 mRNA expression in H. pylori-induced AGS cells without inhibiting H. pylori growth. Conditioned media from LS-B101, LR-B103, and LP-XB7 suppressed the activation of NF-κB in AGS cells, while strain LP-XB7 also suppressed c-Jun activation. The anti-inflammatory effect of LP-XB7 was further assessed in vivo using a H. pylori-infected Sprague-Dawley rat model. Strain LP-XB7 contributed to a delay in the detection and colonization of H. pylori in rat stomachs, attenuated gastric inflammation, and ameliorated gastric histopathology. Additionally, the administration of LP-XB7 correlated with the suppression of TNF-α and CINC-1 in sera, and suppression of CINC-1 in the gastric mucosa of H. pylori-infected rats. These results suggest that L. plantarum XB7 produces secreted factors capable of modulating inflammation during H. pylori infection, and this probiotic Lactobacillus strain shows promise as an adjunctive therapy for treating H. pylori-associated disease.

α-Lipoic Acid InhibitsHelicobacter pylori-Induced Oncogene Expression and Hyperproliferation by Suppressing the Activation of NADPH Oxidase in Gastric Epithelial Cells

Hyperproliferation and oncogene expression are observed in the mucosa of Helicobacter pylori- (H. pylori-) infected patients with gastritis or adenocarcinoma. Expression of oncogenes such as β-catenin and c-myc is related to oxidative stress. α-Lipoic acid (α-LA), a naturally occurring thiol compound, acts as an antioxidant and has an anticancer effect. The aim of this study is to investigate the effect of α-LA on H. pylori-induced hyperproliferation and oncogene expression in gastric epithelial AGS cells by determining cell proliferation (viable cell numbers, thymidine incorporation), levels of reactive oxygen species (ROS), NADPH oxidase activation (enzyme activity, subcellular levels of NADPH oxidase subunits), activation of redox-sensitive transcription factors (NF-κB, AP-1), expression of oncogenes (β-catenin, c-myc), and nuclear localization of β-catenin. Furthermore, we examined whether NADPH oxidase mediates oncogene expression and hyperproliferation in H. pylori-infected AGS cells using treatment of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase. As a result, α-LA inhibited the activation of NADPH oxidase and, thus, reduced ROS production, resulting in inhibition on activation of NF-κB and AP-1, induction of oncogenes, nuclear translocation of β-catenin, and hyperproliferation in H. pylori-infected AGS cells. DPI inhibited H. pylori-induced activation of NF-κB and AP-1, oncogene expression and hyperproliferation by reducing ROS levels in AGS cells. In conclusion, we propose that inhibiting NADPH oxidase by α-LA could prevent oncogene expression and hyperproliferation occurring in H. pylori-infected gastric epithelial cells.

Proteomics Analysis of Gastric Epithelial AGS Cells Infected with Epstein-Barr Virus

Effects of the Epstein-Barr virus (EBV) on cellular protein expression are essential for viral pathogenesis. To characterize the cellular response to EBV infection, differential proteomes of gastric epithelial AGS cells were analyzed with two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization- time of flight (MALDI-TOF) and liquid chromatography electrospray/ionization ion trap (LC-ESI-IT) mass spectrometry identification. Mass spectrometry identified 9 altered cellular proteins, including 5 up-regulated and 4 down-regulated proteins after EBV infection. Notably 2-DE analysis revealed that EBV infection induced increased expression of heat shock cognate 71 kDa protein, actin cytoplasmic 1, pyridoxine-5'-phosphate oxidase, caspase 9, and t-complex protein 1 subunit alpha. In addition, EBV infection considerably suppressed those cellular proteins of zinc finger protein 2, cyclin-dependent kinase 2, macrophage-capping protein, and growth/ differentiation factor 11. Furthermore, the differential expressional levels of partial proteins (cyclin-dependent kinase 2 and caspase 9) were confirmed by Western blot analysis.Thus, this work effectively provided useful protein-related information to facilitate further investigation of the mechanisms underlying EBV infection and pathogenesis.

CagA EPIYA polymorphisms in Colombian Helicobacter pylori strains and their influence on disease-associated cellular responses.

To investigate the influence of the CagA diversity in Helicobacter pylori (H. pylori) strains from Colombia on the host cell biology. Eighty-four H. pylori-cagA positive strains with different Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs patterns, isolated from patients with gastritis (n = 17), atrophic gastritis (n = 17), duodenal ulcer (n = 16), intestinal metaplasia (n = 16) and gastric cancer (n = 18), were included. To determine the integrity of the cag pathogenicity island (cagPAI) we evaluated the presence of cagA, cagT, cagE, and cag10 genes by polymerase chain reaction. AGS gastric epithelial cells were infected with each strain and assayed for translocation and tyrosine phosphorylation of CagA by western blot, secretion of interleukin-8 (IL-8) by enzyme-linked immuno sorbent assay after taking supernatants from cocultures and cell elongation induction. For cell elongation quantification, coculture photographs were taken and the proportion of "hummingbird" cells (> 15 μm) was determined. Overall 72% (60/84) of the strains were found to harbor a functional cagPAI. Levels of phosphorylated CagA were significantly higher for isolates from duodenal ulcer than the ones in strains from gastritis, atrophic gastritis, intestinal metaplasia and gastric cancer (49.1% ± 23.1% vs 21.1% ± 19.5%, P < 0.02; 49.1% ± 23.1% vs 26.2% ± 14.8%, P < 0.045; 49.1% ± 23.1% vs 21.5% ± 19.5%, P < 0.043 and 49.1% ± 23.1% vs 29.5% ± 27.1%, P < 0.047 respectively). We observed variable IL-8 expression levels ranging from 0 to 810 pg/mL and from 8.8 to 1442 pg/mL at 6 h and 30 h post-infection, respectively. cagPAI-defective strains did not induce detectable levels of IL-8 at 6 h post-infection. At 30 h post-infection all strains induced IL-8 expression in AGS cells, although cagPAI-defective strains induced significantly lower levels of IL-8 than strains with a functional cagPAI (57.1 ± 56.6 pg/mL vs 513.6 ± 338.6 pg/mL, P < 0.0001). We did not observe differences in the extent of cell elongation induction between strains with a functional or a defective cagPAI in 6 h cocultures. At 24 h post infection strains with functional cagPAI showed high diversity in the extent of hummingbird phenotype induction ranging from 7% to 34%. cagPAI defective strains induced significantly lower levels of elongation than strains with functional cagPAI with one or more than one EPIYA-C motif (15.1% ± 5.2% vs 18.9% ± 4.7%, P < 0.03; and 15.1% ± 5.2% vs 20.0% ± 5.1%, P < 0.003 respectively). No differences were observed in cellular elongation induction or IL-8 expression among H. pylori strains bearing one and more than one EPIYA-C motifs, neither at 6 h nor at 24 h of coculture. There were no associations between the levels of induction of cell elongation or IL-8 expression and number of EPIYA motifs or pathology. The present work describes a lack of association between H. pylori CagA protein EPIYA motifs variations from Colombian isolates and disease-associated cellular responses.

Human gastric biopsy-derived lactobacilli suppress Helicobacter pylori-induced interleukin-8 production from gastric epithelial cells in vitro

In this report we evaluate the anti-inflammatory effect of a variety of Lactobacillus spp. isolated from human gastric biopsies of dyspeptic patients. These lactobacilli were previously shown to secrete factors that inhibit tumor necrosis factor production from human monocytoid cells in vitro. Conditioned media from these lactobacilli were tested for the ability to modulate Helicobacter pylori-induced interleukin-8 (IL-8) production in AGS gastric epithelial cells. Out of 26 Lactobacillus spp. isolated from urease-negative patients thought not to harbor H. pylori, five significantly inhibited IL-8 production. The IL-8-inhibitory lactobacilli were identified as L. casei or L. paracasei, L. plantarum, L. rhamnosus, and L. salivarius and had no antagonistic effects on H. pylori growth. Our findings suggest potential candidates of Lactobacillus for further investigation of their beneficial effects on H. pylori-infected patients.

β-Carotene and Lutein Inhibit Hydrogen Peroxide-Induced Activation of NF-κB and IL-8 Expression in Gastric Epithelial AGS Cells

Reactive oxygen species (ROS) including hydrogen peroxide (H(2)O(2)) are involved in the pathogenesis of gastric inflammation. Interleukin-8 (IL-8) is a potent mediator of the inflammatory response by activating and recruiting neutrophils to the site of infection. Oxidant-sensitive transcription factor NF-κB regulates the expression of IL-8 in the immune and inflammatory events. Carotenoids (carotenes and oxygenated carotenoids) show antioxidant and anti-inflammatory activities. Low intake of β-carotene leads to high risk of gastric cancer. Oxygenated carotenoid lutein inhibited NF-κB activation in experimental uveitis. The present study aims to investigate whether β-carotene and lutein inhibit H(2)O(2)-induced activation of NF-κB and expression of IL-8 in gastric epithelial AGS cells. The cells were treated with carotenoids 2 h prior to the treatment of H(2)O(2). mRNA expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and real time RT-PCR analyses. IL-8 level in the medium was determined by enzyme-linked immunosorbent assay. NF-κB activation was assessed by electrophoretic mobility shift assay. ROS levels of the cells were detected by confocal microscopic analysis for fluorescent dichlorofluorescein. As a result, H(2)O(2 )induced the activation of NF-κB and expression of IL-8 in AGS cells time-dependently. β-Carotene and lutein showed inhibitory effects on H(2)O(2)-induced increase in intracellular ROS levels, activation of NF-κB, and IL-8 expression in AGS cells. In conclusion, supplementation of carotenoids such as β-carotene and lutein may be beneficial for the treatment of oxidative stress-mediated gastric inflammation.

Effect of Pertussis Toxin and Herbimycin A on Proteinase-Activated Receptor 2-Mediated Cyclooxygenase 2 Expression inHelicobacter pylori-Infected Gastric Epithelial AGS Cells

Helicobacter pylori (H. pylori) is an important risk factor for chronic gastritis, peptic ulcer, and gastric cancer. Proteinase-activated receptor 2 (PAR2), subgroup of G-protein coupled receptor family, is highly expressed in gastric cancer, and chronic expression of cyclooxygenase-2 (COX-2) plays an important role in H. pylori-associated gastric carcinogenesis and inflammation. We previously demonstrated that H. pylori induced the expression of PAR2 and COX-2 in gastric epithelial cells. Present study aims to investigate whether COX-2 expression induced by H. pylori in Korean isolates is mediated by PAR2 via activation of Gi protein and Src kinase in gastric epithelial AGS cells. Results showed that H. pylori-induced COX-2 expression was inhibited in the cells transfected with antisense oligonucleotide for PAR2 or treated with Gi protein blocker pertussis toxin, Src kinase inhibitor herbimycin A and soybean trypsin inbitor, indicating that COX-2 expression is mediated by PAR2 through activation of Gi protein and Src kinase in gastric epithelial cells infected with H. pylori in Korean isolates. Thus, targeting the activation of PAR2 may be beneficial for prevention or treatment of gastric inflammation and carcinogenesis associated with H. pylori infection.

Diphenyleneiodonium inhibits the activation of mitogen-activated protein kinases and the expression of monocyte chemoattractant protein-1 in Helicobacter pylori-infected gastric epithelial AGS cells

To investigate whether NADPH oxidase induces MCP-1 expression and the activation of mitogen-activated protein kinases (MAPKs) in H. pylori-infected gastric epithelial cells. H. pylori in Korean isolates, human gastric epithelial AGS cells AGS cells pretreated with or without an NADPH oxidase inhibitor diphenyleneiodonium (DPI) are cultured in the presence of H. pylori at a bacterium/cell ratio of 300:1. Reactive oxygen species (ROS) and MCP-1 were determined by confocal microscopy and enzyme-linked immonosorbent assay. NADPH oxidase activity was measured by lucigenin assay. mRNA expression of MCP-1 was analyzed by reverse transcription-polymerase chain reaction. Levels of MAPKs were assessed by Western blot analysis. H. pylori induced increase in ROS, NADPH oxidase activity, MCP-1 expression, and the activation of MAPKs including extracellular signal-regulated kinases, p38, and jun N-terminal kinases in AGS cells, which was inhibited by DPI. Inhibiting NADPH oxidase by DPI suppresses H. pylori-induced activation of MAPKs and MCP-1 expression in AGS cells.

Helicobacter pylori represses proton pump expression and inhibits acid secretion in human gastric mucosa

Background and aimsHelicobacter pylori infection of gastric mucosa causes gastritis and transient hypochlorhydria, which may provoke emergence of a mucosal precancer phenotype; H pylori strains containing a cag pathogenicity island...

Protective Effect of Rebamipide Against Helicobacter pylori-CagA-Induced Effects on Gastric Epithelial Cells

Helicobacter pylori CagA dysregulates cell signaling pathways and leads to targeted transcriptional up-regulation of genes implicated in gastric cell injury. The aim of this study was to determine the effects of rebamipide on CagA-induced effects on gastric epithelial cells. We investigated the effects of rebamipide treatment (pre- or post-treatment before or after CagA transfection) on CagA-induced gastric cell injury. We evaluated the morphologic changes (hummingbird phenotype) associated with ZO-1 mislocalization by confocal microscopy, IL-8 production by ELISA, and NF-κB activation by luciferase assay in AGS gastric epithelial cells and MDCK cells. Transfection of CagA into gastric epithelial cells induced morphologic changes (hummingbird phenotype), ZO-1 mislocalization, and IL-8 production in gastric epithelial cells. Pre-treatment with rebamipide inhibits CagA-induced effects on gastric epithelial cells, including morphologic changes (hummingbird phenotype) associated with ZO-1 mislocalization, IL-8 production, and NF-κB activity. These results suggest that rebamipide might have a potential role in the protection of H. pylori CagA-induced effects on gastric epithelial cells.

HSP90β interacts with Rac1 to activate NADPH oxidase in Helicobacter pylori-infected gastric epithelial cells

Reactive oxygen species (ROS) are involved in the pathogenesis of Helicobacter pylori ( H. pylori)-induced gastric disorders including inflammation. NADPH oxidase has been considered as a major source of ROS in phagocytic and non-phagocytic cells. Small G protein Rac1 is one components of NADPH oxidase complex. Heat shock protein 90 kDa (HSP90) modulates cytokine production in H. pylori-infected gastric epithelial cells. The present study aims to investigate the relation of HSP90β and Rac1 on the activation of NADPH oxidase in H. pylori-infected gastric epithelial cells. As a result, H. pylori-induced translocation of HSP90β from the cytosol to the membrane and activated Rac1 in gastric epithelial AGS cells. HSP90β physically interacted with Rac1, which resulted in the activation of NADPH oxidase in H. pylori-infected AGS cells. Down-regulation of HSP90β by transfection of HSP90β siRNA suppressed the activation of Rac1, activity of NADPH oxidase and the production of H 2O 2 in H. pylori-infected AGS cells. In conclusion, H. pylori induces the translocation of HSP90β from the cytosol to the membrane and interaction of HSP90β and Rac1, which leads to the activation of NADPH oxidase and production of ROS in gastric epithelial cells. HSP90β may be the target molecule for treatment of H. pylori-induced gastric injury.

Helicobacter pylori CagA Phosphorylation Status Determines the gp130-activated SHP2/ERK and JAK/STAT Signal Transduction Pathways in Gastric Epithelial Cells

The Helicobacter pylori protein CagA may undergo tyrosine phosphorylation following its entry into human gastric epithelial cells with downstream effects on signal transduction. Disruption of the gp130 receptor that modulates the balance of the SHP2/ERK and JAK/STAT pathways enhanced peptic ulceration and gastric cancer in gp130 knock-out mice. In this study, we evaluated the effect of translocated CagA in relation to its tyrosine phosphorylation status on the gp130-mediated signal switch between the SHP2/ERK and JAK/STAT3 pathways. We showed that in the presence of CagA, SHP2 was recruited to gp130. Phosphorylated CagA showed enhanced SHP2 binding activity and ERK1/2 phosphorylation, whereas unphosphorylated CagA showed preferential STAT3 activation. These findings indicate that the phosphorylation status of CagA affects the signal switch between the SHP2/ERK and JAK/STAT3 pathways through gp130, providing a novel mechanism to explain H. pylori signaling.

Involvement of Ras and AP-1 in Helicobacter pylori-Induced Expression of COX-2 and iNOS in Gastric Epithelial AGS Cells

Helicobacter pylori (H. pylori) is an important risk factor for chronic gastritis, peptic ulcer, and gastric cancer. The genetic differences of H. pylori isolates play a role in the clinical outcome of the infection. Inflammatory genes including cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are involved in H. pylori gastritis. Transcription factor AP-1 is composed of c-Fos and c-Jun and mediates inflammation and carcinogenesis. Ras acts as a regulator for AP-1 activation in various cells. We investigated whether H. pylori in a Korean isolate (HP99), a cagA ( + ), vacA ( + ) strain, induces the expression of c-Fos and c-Jun for AP-1 activation to induce COX-2 and iNOS and whether HP99-induced expressions of COX-2 and iNOS are mediated by Ras and AP-1, determined by the expressions of c-Fos and c-Jun, in gastric epithelial AGS cells, using transfection with mutant genes for Ras (ras N-17) and c-Jun (TAM-67). As a result, HP99 induced the expression of c-Fos and c-Jun and the expressions of COX-2 and iNOS in AGS cells. Transfection with mutant genes for Ras or c-Jun suppressed HP99-induced expressions of COX-2 and iNOS in AGS cells. In conclusion, H. pylori in a Korean isolate induces the expression of COX-2 and iNOS via AP-1 activation, which may be mediated by Ras and the expression of c-Fos and c-Jun in gastric epithelial cells.

Proteinase-Activated Receptor-2 Mediates the Expression of Integrin α5 and β1 in Helicobacter pylori-Infected Gastric Epithelial AGS Cells

Background/Aim: Proteinase-activated receptor-2 (PAR2), which is activated by trypsin, is known to be associated with expression of adhesion molecule integrins. We previously demonstrated that Helicobacter pylori induced the expression of integrin α₅ and β₁ in human gastric epithelial cells. The present study aims to investigate whether H. pylori in a Korean isolate (HP99) induces the expression of PAR2, which mediates the expression of integrin α₅ and β₁ and thus cell adhesion to fibronectin in gastric epithelial AGS cells. Methods and Results: mRNA expressions of PAR2, trypsinogen 1 and 2, and integrin α₅ and β₁ were assessed by RT-PCR analysis while protein levels of PAR2, trypsin as well as integrin α₅ and β₁ were determined by Western blot analysis. The activity of trypsin in the medium was determined by fluorometric analysis. Cell adhesion assay was performed colorimetrically. H. pylori induced the expressions of PAR2 and integrin α₅ and β₁ of the cells. H. pylori increased mRNA expression of trypsinogen 1 and 2 as well as the level and activity of trypsin in the medium. H. pylori induced cell adhesion to fibronectin. H. pylori-induced expression of integrin α₅ and β₁ and adhesion of the cells to fibronectin were inhibited in the cells transfected with PAR2 antisense oligonucleotide or treated with a soybean trypsin inhibitor, anti-integrin α₅ antibody or β₁ antibody. Conclusion:H. pylori induces the expression of integrin α₅ and β₁ and adhesion of the cells to fibronectin through PAR2 which is induced and may be activated by trypsin in H. pylori-infected gastric epithelial cells. PAR2 may have an important role in gastric cell adhesion and possibly carcinogenesis associated with H. pylori.