Ji Hye Seo

Helicobacter pylori in a Korean isolate activates mitogen-activated protein kinases, AP-1 and NF-κB and induces chemokine expression in gastric epithelial AGS cells

Oxidant-sensitive transcription factors, nuclear factor-κB (NF-κB), and activator protein-1 (AP-1) have been considered as the regulators of inducible genes such as chemokines. Since oxygen radicals are considered as an important regulator in the pathogenesis of Helicobacter pylori (H. pylori)-induced gastric ulceration and carcinogenesis, chemokines such as interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) may be regulated by NF-κB and/or AP-1. Ras, the upstream activator for mitogen-activated protein kinase (MAPK) and MAPK cascade regulate AP-1 activation. The present study aims to investigate whether H. pylori in a Korean isolate (HP99) induces the expression of chemokines (IL-8, MCP-1), which is regulated by Ras, MAPK, AP-1, and NF-κB in gastric epithelial AGS cells, and whether these transcriptional regulations of chemokines are inhibited by transfection with mutant genes for Ras (ras N-17), c-Jun (TAM-67), and IκBα (MAD-3) or treatment with MAPK inhibitors (U0126 for extracellular signal-regulated kinase or SB203580 for p38 kinase). In addition, virulence factors of HP99 were characterized by PCR analysis for the isolated DNA. As a result, HP99 is identified as cagA+, vacA s1b, m2, iceA1 H. pylori strain. HP99 induced a time-dependent expression of mRNA and protein for IL-8 and MCP-1 via mediation of MAPK, AP-1, and NF-κB. Transfection with mutant genes for Ras, c-Jun, and IκBα and treatment with MAPK inhibitors suppressed H. pylori-induced activation of transcription factors (NF-κB, AP-1) and expression of chemokines (IL-8, MCP-1) in AGS cells. In conclusion, Ras and MAPK cascade may act as the upstream signaling for the activation of AP-1 and NF-κB, which induce chemokine expression in H. pylori-infected AGS cells. Specific targeting of the activation of NF-κB and AP-1 may be effective for the prevention or treatment of gastric inflammation associated with H. pylori infection.

Role of Proteinase-Activated Receptor-2 on Cyclooxygenase-2 Expression in H. pylori–Infected Gastric Epithelial Cells

Abstract:  Proteinase‐activated receptor‐2 (PAR‐2) belongs to a novel subfamily of G protein–coupled receptors with seven‐transmembrane domains. PAR‐2 is activated by serine proteases, such as trypsin, mast cell tryptase, and allergic or bacterial proteases. The presence of trypsin has been shown in human stomach. Cyclooxygenase‐2 (COX‐2) is induced by inflammatory cytokines, growth factors, gastrin, and reactive oxygen species in gastric epithelial cells, which may lead to mutagenesis and subsequent metaplasia, dysplasia, and cancer formation. We investigated whether PAR‐2 is activated in H. pylori (HP99)‐infected cells, which is related to COX‐2 induction in gastric epithelial cells. After treatment of H. pylori to AGS (gastric adenocarcinoma) cells at a bacteria/cell ratio of 100:1, we determine the expression and the activation of PAR‐2 and the expression of COX‐2. The same experiments were performed in the cells treated with PAR‐2 agonist peptide. mRNA and protein expression of PAR‐2 and COX‐2 were determined by reverse transcriptase‐polymerase chain reaction (RT‐PCR) and Western blotting. PAR‐2 activation was assessed by increase in intracellular calcium level. As a result, H. pylori induced the activation and expression of PAR‐2 as well as COX‐2 expression. PAR‐2 agonist peptide augmented H. pylori‐induced COX‐2 expression in AGS cells. H. pylori induces COX‐2 expression, which is mediated by both activation and expression of PAR‐2 in gastric epithelial cells.

The effect of p38 mitogen-activated protein kinase on mucin gene expression and apoptosis in Helicobacter pylori-infected gastric epithelial cells.

Abstract: The loss of mucus coat continuity and apoptosis have been shown in Helicobacter pylori (H. pylori)‐infected gastric tissues. Blockade of p38 mitogen‐activated kinase (MAPK) produced reversal in the LPS‐induced reduction in mucin synthesis and apoptosis in gastric epithelial cells. This study investigates whether H. pylori induces apoptosis, alterations in mucin gene (MUC) expression, and p38 MAPK activation in human gastric epithelial AGS cells. After treatment of AGS cells with H. pylori at the ratio of 1:300, apoptosis was determined by DNA fragmentation and DNA laddering. MUC expression was assessed by RT‐PCR. p38 MAPK activation and mucin protein level, using anti‐mucin antibody for MUC5/6, were determined by Western blot analysis. As a result, H. pylori induced apoptosis and loss of mucin, which was supported by reduced mRNA expression of MUC5AC by H. pylori in AGS cells. MUC7/8 expression and p38 MAPK activation were induced in H. pylori‐infected AGS cells. In conclusion, H. pylori induces p38 MAPK activation, wh3.ich may be the underlying mechanism of alterations in MUC expression and apoptosis in gastric epithelial cells.

Differential Role of ERK and p38 on NF-κB Activation in Helicobacter pylori-Infected Gastric Epithelial Cells

Gastric cancer, as well as inflammation, caused by Helicobacter pylori, activates the production of chemokines by activation of redox-sensitive transcription factor NF-κB in gastric epithelial cells. Mitogen-activated protein kinases including extracellular signal-regulated kinase (ERK) and p38 kinase (p38) are activated by Helicobacter pylori, which may regulate NF-κB activation in the infected cells. However the mechanisms how ERK and p38 induce NF-κB activation have not been investigated. Present study aims to investigate the role of ERK and p38 on the activation of NF-κB in Helicobacter pylori-infected AGS cells. Western blot analysis was performed for determining the levels of IκB, p105, p50 and p65 in gastric epithelial cells infected with Helicobacter pylori and treated with ERK inhibitor U0126 and p38 inhibitor SB203580. Helicobacter pylori induced the degradation of IκBα and upregulation of p105, p50 and p65 in the infected cells. U0126 inhibited the degradation of IκBα while SB203580 suppressed expression of p105, p50 and p65 in Helicobacter pylori-infected cells. ERK and p38 differentially activate NF-κB; ERK induces degradation of IκBα while p38 upregulates the expression of p50 and p65, subunits of NF-κB in Helicobacter pylori-infected gastric epithelial AGS cells.

Cyclooxygenase-2 expression by transcription factors in Helicobacter pylori-infected gastric epithelial cells: comparison between HP 99 and NCTC 11637.

Abstract: Helicobacter pylori, a gram‐negative spiral bacterium, has been associated with chronic gastritis and gastric cancer. HP 99, isolated from the Korean patients, and NCTC 11637, obtained from ATCC, have different genotypes. The present study aims to investigate whether these H. pylori strains show the discrepancy for activating transcription factors (NF‐κB, AP‐1, C/EBP) and induction of cyclooxygenase‐2 (COX‐2) gene in gastric epithelial AGS cells. After treatment of H. pylori to AGS cells at the ratio of 300:1, the activation of transcription factors was assessed by electrophoretic mobility shift assay. COX‐2 protein was determined by Western blot analysis. The level of 6‐keto‐prostaglandin F1α (6‐keto‐PGF1α), a COX‐2 product, was measured in the medium by enzyme‐linked immunosorbent assay. As a result, both H. pylori strains similarly induced COX‐2 expression via activation of NF‐κB, not C/EBP, and increased the level of 6‐keto‐PGF1α. HP 99 showed much higher activation of AP‐1 compared to NCTC 11637. NF‐κB might play an important role in COX‐2 expression in H. pylori‐induced gastric inflammation as compared to other transcription factors.

Effect of Pertussis Toxin and Herbimycin A on Proteinase-Activated Receptor 2-Mediated Cyclooxygenase 2 Expression in Helicobacter 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.

Membrane Proteome Analysis of Cerulein-Stimulated Pancreatic Acinar Cells: Implication for Early Event of Acute Pancreatitis

BACKGROUND/AIMS Cerulein pancreatitis is similar to human edematous pancreatitis with dysregulation of the production and secretion of digestive enzymes, edema formation, cytoplasmic vacuolization and the death of acinar cells. We hypothesized that membrane proteins may be altered as the early event during the induction of acute pancreatitis. Present study aims to determine the differentially expressed proteins in the membranes of cerulein-treated pancreatic acinar cells. METHODS Pancreatic acinar AR42J cells were treated with 10(-8) M cerulein for 1 hour. Membrane proteins were isolated from the cells and separated by two-dimensional electrophoresis using pH gradients of 5-8. Membrane proteins were identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. The differentially expressed proteins, whose expression levels were more or less than three-fold in cerulein-treated cells, were analyzed. RESULTS Two differentially expressed proteins (mannan-binding lectin-associated serine protease-2, heat shock protein 60) were up-regulated while four proteins (protein disulfide isomerase, gamma-actin, isocitrate dehydrogenase 3, seven in absentia homolog 1A) were down-regulated by cerulein treatment in pancreatic acinar cells. These proteins are related to cell signaling, oxidative stress, and cytoskeleton arrangement. CONCLUSIONS Oxidative stress may induce cerulein-induced cell injury and disturbances in defense mechanism in pancreatic acinar 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.