Gastric Epithelial AGS Cells Research Articles

Functional analysis of the cag pathogenicity island in Helicobacter pylori isolates from patients with gastritis, peptic ulcer, and gastric cancer.

Helicobacter pylori is the causative agent of a variety of gastric diseases, but the clinical relevance of bacterial virulence factors is still controversial. Virulent strains carrying the cag pathogenicity island (cagPAI) are thought to be key players in disease development. Here, we have compared cagPAI-dependent in vitro responses in H. pylori isolates obtained from 75 patients with gastritis, peptic ulcer, and gastric cancer (n = 25 in each group). AGS gastric epithelial cells were infected with each strain and assayed for (i) CagA expression, (ii) translocation and tyrosine phosphorylation of CagA, (iii) c-Src inactivation, (iv) cortactin dephosphorylation, (v) induction of actin cytoskeletal rearrangements associated with cell elongation, (vi) induction of cellular motility, and (vii) secretion of interleukin-8. Interestingly, we found high but similar prevalences of all of these cagPAI-dependent host cell responses (ranging from 56 to 80%) among the various groups of patients. This study revealed CagA proteins with unique features, CagA subspecies of various sizes, and new functional properties for the phenotypic outcomes. We further showed that induction of AGS cell motility and elongation are two independent processes. Our data corroborate epidemiological studies, which indicate a significant association of cagPAI presence and functionality with histopathological findings in gastritis, peptic ulcer, and gastric cancer patients, thus emphasizing the importance of the cagPAI for the pathogenicity of H. pylori. Nevertheless, we found no significant association of the specific H. pylori-induced responses with any particular patient group. This may indicate that the determination of disease development is highly complex and involves multiple bacterial and/or host factors.

Sphingomyelinase of Helicobacter pylori-induced cytotoxicity in AGS gastric epithelial cells via activation of JNK kinase

The aim of this study was to determine whether the Helicobacter pylori-derived sphigomyelinase (SMase) affects the sphingomyelin pathway and growth in AGS epithelial cells. We showed that the exogenous SMase increased the intracellular level of ceramide in AGS cells and led to rapid stimulation of extracellular signal-regulated kinase (ERK) and c-Jun kinase (JNK) activities. Incubation of AGS cells with H. pylori-derived SMase also resulted in suppression of cell growth and a concomitant induction of apoptosis. Data showed that PD98059 (up to 50 μM), an ERK inhibitor, did not affect the cell viability, whereas the cytotoxicity of exogenous SMase was completely blocked by SP600125, a JNK inhibitor at a concentration of 210 nM. We conclude that the activation of the mitogen-activated protein (MAP) kinases in AGS cells by exogenous H. pylori SMase is a major pathway to mediate the cytotoxicity.

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.

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

Oxidant-sensitive transcription factors, nuclear factor-kappaB (NF-kappaB), 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-kappaB 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-kappaB 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 IkappaBalpha (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-kappaB. Transfection with mutant genes for Ras, c-Jun, and IkappaBalpha and treatment with MAPK inhibitors suppressed H. pylori-induced activation of transcription factors (NF-kappaB, 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-kappaB, which induce chemokine expression in H. pylori-infected AGS cells. Specific targeting of the activation of NF-kappaB and AP-1 may be effective for the prevention or treatment of gastric inflammation associated with H. pylori infection.

Cell adhesion-related gene expression by Helicobacter pylori in gastric epithelial AGS cells

Helicobacter pylori ( H. pylori) infection leads to gastroduodenal inflammation, peptic ulceration and gastric carcinoma. H. pylori may induce disease-specific gene expression in gastric epithelial cells. cDNA microarray for 352 cancer-related genes was used to identify the genes altered by H. pylori ( cagA positive) in gastric epithelial AGS cells. Expressions of the genes identified on the microarray and other genes closely associated with these genes were determined by reverse transcriptase-polymerase chain reaction (RT-PCR). Western blot analysis and cell adhesion assay were performed to confirm the protein levels of the genes and the role of the genes on cell adhesion in H. pylori-infected AGS cells. As a result, the expression of four genes (galectin 1, aldolase A, integrin α5, LIM domain only 7 (LMO7)) were up-regulated by H. pylori in AGS cells, determined by cDNA microarray. RT-PCR analysis showed that the genes up-regulated by H. pylori were the genes regulating cell–cell adhesion and cell–extracellular matrix interaction, such as galectin-1 and galectin-3, integrin α5, and LIM domain only 7 (LMO7), and cancer-related glycolytic enzyme aldolase A and C. Cell adhesion to extracellular matrix proteins such as poly- l-lysine and fibronectin was mediated by H. pylori-induced expression of integrin α5. RT-PCR and Western blot analysis showed that E-cadherin, regulating cell adhesion and contact cell inhibition, was decreased by H. pylori in AGS cells. In conclusion, the increased expression of cell adhesion molecules and decrease in E-cadherin expression by H. pylori might contribute to cell adhesion, invasion and possibly cell proliferation in gastric epithelial cells.

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

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-kappaB, 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 F(1alpha) (6-keto-PGF(1alpha)), 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-kappaB, not C/EBP, and increased the level of 6-keto-PGF(1alpha). HP 99 showed much higher activation of AP-1 compared to NCTC 11637. NF-kappaB might play an important role in COX-2 expression in H. pylori-induced gastric inflammation as compared to other transcription factors.

Reduced activation of inflammatory responses in host cells by mouse-adapted Helicobacter pylory isolates.

Helicobacter pylori strains that harbour the Cag pathogenicity island (Cag PAI) induce interleukin (IL)-8 secretion in gastric epithelial cells, via the activation of NF- kappa B, and are associated with severe inflammation in humans. To investigate the influence of Cag PAI-mediated inflammatory responses on H. pylori adaptation to mice, a selection of H. pylori clinical isolates (n = 12) was cag PAI genotyped and tested in co-culture assays with AGS gastric epithelial cells, and in mouse colonization studies. Six isolates were shown to harbour a complete cag PAI and to induce NF- kappa B activation and IL-8 secretion in AGS cells. Of the eight isolates that spontaneously colonized mice, six had a cag PAI(-) genotype and did not induce pro-inflammatory responses in these cells. Mouse-to-mouse passage of the two cag PAI(+) -colonizing strains yielded host-adapted variants that infected mice with bacterial loads 100-fold higher than those of the respective parental strains (P= 0.001). These mouse-adapted variants were affected in their capacity to induce pro-inflammatory responses in host cells, yet no changes in cag PAI gene content were detected between the strains by DNA microarray analysis. This work provides evidence for in vivo selection of H. pylori bacteria with a reduced capacity to induce inflammatory responses and suggests that such bacteria are better adapted to colonize mice.

H. pylori activates the EGF receptor to regulate IL-8 gene expression in AGS gastric epithelial cells

H. pylori activates the EGF receptor to regulate IL-8 gene expression in AGS gastric epithelial cells

H. pylori activates the EGF receptor to regulate IL-8 gene expression in AGS gastric epithelial cells

H. pylori activates the EGF receptor to regulate IL-8 gene expression in AGS gastric epithelial cells

Characterization of virulence factors of mouse-adapted Helicobacter pylori strain SS1 and effects on gastric hydrophobicity.

Gastric infection with Helicobacter pylori results in chronic active gastritis and in some individuals is associated with complications such as peptic ulceration and gastric cancers. A balance between bacterial factors and host responses may determine disease outcome. The mouse-adapted H. pylori strain SS1 has been utilized as a model to study disease pathogenesis. Although chronic gastritis is observed in this murine model of H. pylori infection, other complications of disease seen in the human host (such as peptic ulceration) are not identified. The objectives of this study were to characterize virulence factors of the mouse-adapted H. pylori strain SS1 and determine host responses to infection. Vacuolating cytotoxin activity of H. pylori strain SS1 was determined after incubation of HEp-2 cells with culture supernatant for 24 hr. Polymerase chain reaction was performed to detect the presence of the cagA and cagE genes. Chemokine responses from human gastric epithelial cells infected with H. pylori SS1 were assessed by measurement of the concentration of interleukin-8 in cell-free supernatants. C57BL/6 and gld mice were infected with strain SS1 or sham-infected. Eight weeks following infection, gastric tissues were obtained for histological analysis and surface hydrophobicity was measured by axisymmetric drop-shape analysis. H. pylori strain SS1 was cytotoxin negative, cagA positive, and cagE positive, but induced only a modest interleukin-8 response (684 +/- 140 pg/ml) from AGS gastric epithelial cells in comparison to a clinical isolate (4170 +/- 410 pg/ml, P < 0.0005). Increased inflammation was observed in the stomachs of H. pylori strain SS1-infected animals compared to uninfected controls. Gastritis was not associated with any disease complications. Despite mucosal inflammation, infected mice did not demonstrate alterations in gastric surface hydrophobicity (42.2 degrees +/- 2.2 degrees and 41.4 degrees +/- 3.2 degrees for C57BL/6 and gld, respectively) compared to uninfected mice (43.2 degrees +/- 2.3 degrees and 39.5 degrees +/- 1.6 degrees, respectively). In conclusion, murine infection with H. pylori SS1, which contains putative bacterial virulence factors, results in gastric inflammation. However, the mucosal changes are not associated with alterations in surface hydrophobicity. Therefore, the mouse model of infection with H. pylori, strain SS1 may not serve as an entirely appropriate model to study host factors associated with disease complications.

Extracellular signal-regulated kinases enhances NF-KB activation in ags gastric epithelial cells infected by Helicobacter pylori

Extracellular signal-regulated kinases enhances NF-KB activation in ags gastric epithelial cells infected by Helicobacter pylori

H. Pylori produces a soluble factor which inhibits ENA-78 chemokine production by AGS gastric epithelial cells

H. Pylori produces a soluble factor which inhibits ENA-78 chemokine production by AGS gastric epithelial cells

Activation of IL-8 Gene Expression byHelicobacter pyloriIs Regulated by Transcription Factor Nuclear Factor-κB in Gastric Epithelial Cells

AbstractIn vivo, gastric infection with Helicobacter pylori leads to substantial production of the inflammatory cytokines IL-1, IL-6, TNF-α, and IL-8. H. pylori strains that contain the cag pathogenicity island (cag+) and are associated with ulceration and gastric carcinoma induce greater cytokine production than cag− strains. Expression of these cytokines is often regulated by the transcription factor complex, nuclear factor-κB (NF-κB) through κB-binding elements in the enhancer/promoter regions of their genes. We report that more virulent cag+ H. pylori strains induce increased NF-κB-DNA binding activity, which elevates IL-8 expression in AGS gastric epithelial cells. The cag+ H. pylori strains induce significant stimulation of IL-8 promoter-driven reporter activity, while cag− strains do not. Furthermore, mutation of specific genes within the cag island (picA1 and picB) ablates enhanced NF-κB activation and IL-8 transcription. Increased IL-8 expression is inhibited by mutation in either the NF-κB or NF-IL-6 binding element. The cag+ strains, compared with the cag− strains, induce enhanced nuclear localization of a RelA-containing NF-κB binding complex, but no increase in NF-IL-6 binding activity. These studies demonstrate that the ability of different types of H. pylori strains to activate NF-κB correlates with their ability to induce IL-8 transcription and indicate a mechanism for the heightened inflammatory response seen in subjects infected with cag+ H. pylori strains.

STIMULATION OF IL-8 PRODUCTION IN HUMAN GASTRIC EPITHELIAL CELLS BY HELICOBACTER PYLORI, IL-1β AND TNF-α REQUIRES TYROSINE KINASE ACTIVITY, BUT NOT PROTEIN KINASE C

Production of interleukin 8 (IL-8) is believed to be important in the pathogenesis of the gastritis seen in Helicobacter pyloriinfection. The aim of this study was to investigate the roles of protein kinase A (PKA), protein kinase C (PKC), protein tyrosine kinase (PTK) and intracellular calcium in the induction of IL-8 production by gastric epithelial cells. AGS gastric epithelial cells were stimulated with H. pylori, tumour necrosis factor α or interleukin 1β together with activators or inhibitors of the relevant kinases. IL-8 production was measured by enzyme-linked immunosorbent assay. Helicobacter pylori, tumour necrosis factor α and interleukin 1β produced a dose-dependent increase in IL-8 production. The increase with all three was significantly reduced by the tyrosine kinase inhibitors herbimycin A and genistein. Activation of PKC by phorbol myristate acetate was also an effective stimulus to IL-8 production and this was blocked by PKC depletion or inhibitors. Protein kinase C inhibition did not reduce the stimulation produced by H. pylorior the cytokines. Stimulation of PKA with forskolin or dibutyryl cyclic adenosine monophosphate or inhibition with H89 had no effect on IL-8 production. The calcium ionophore A23187 was a weak, PKC dependent, stimulant of IL-8 production. The production of IL-8 in AGS cells is stimulated via tyrosine kinase and protein kinase C dependent pathways. Stimulation by H. pylori, tumour necrosis factor α and interleukin 1β requires tyrosine kinase activity.