Gastric Epithelial Cell Line AGS Research Articles

Investigation of Campylobacter concisus gastric epithelial pathogenicity using AGS cells.

Campylobacter concisus is an oral bacterium. Recent studies suggest that C. concisus may be involved in human gastric diseases. The mechanisms, however, by which C. concisus causes human gastric diseases have not been investigated. Here we examined the gastric epithelial pathogenicity of C. concisus using a cell culture model. Six C. concisus strains and the human gastric epithelial cell line AGS cells were used. IL-8 produced by AGS cells after incubation with C. concisus was measured using enzyme-linked immunosorbent assay (ELISA), and AGS cell apoptosis was determined by caspase 3/7 activities. The effects of C. concisus on actin arrangement in AGS cells was determined using fluorescence staining. The effects of C. concisus on global gene expression in AGS cells was determined by transcriptomic analysis and quantitative real-time PCR (qRT-PCR). The role of the upregulated CYP1A1 gene in gastric cancer survival was assessed using the Kaplan-Meier method. C. concisus induced production of IL-8 by AGS cells with strain variation. Significantly increased caspase 3/7 activities were observed in AGS cells incubated with C. concisus strains when compared to AGS cells without bacteria. C. concisus induced actin re-arrangement in AGS cells. C. concisus upregulated 30 genes in AGS cells and the upregulation of CYP1A1 gene was confirmed by qRT-PCR. The Kaplan-Meier analysis showed that upregulation of CYP1A1 gene is associated with worse survival in gastric cancer patients. Our findings suggest that C. concisus may play a role in gastric inflammation and the progression of gastric cancer. Further investigation in clinical studies is warranted.

Herbal melanin modulates PGE2 and IL-6 gastroprotective markers through COX-2 and TLR4 signaling in the gastric cancer cell line AGS

We reported a gastric anti-ulcerogenic effect of the Nigella sativa (L.)-derived herbal melanin (HM) using rat models. However, the molecular mechanisms underlying this HM gastroprotective effect remain unknown. Cyclooxygenase-2 (COX-2)-catalyzed prostaglandin E2 (PGE2) and toll-like receptor 4 (TLR4)-mediated interleukin-6 (IL-6) production and secretion play major roles in gastric mucosal protection. In the current study, the human gastric carcinoma epithelial cell line AGS was used as a model to investigate the effect of HM on TLR4, COX-2, glycoprotein mucin 4 protein and gene expression using immuno-cyto-fluorescence staining, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Gastroprotective markers PGE2 and IL-6 production and secretion were also assessed using an enzyme-linked immunosorbent assay (ELISA). Bacterial lipopolysaccharides (LPS), well-known inducers of TLR4, COX-2, PGE2 and IL-6 expression, were used as a positive control. We showed that HM upregulated its main receptor TLR4 gene and protein expression in AGS cells. HM increased, in a dose- and time-dependent manner, the secretion of PGE2 and the expression of COX-2 mRNA and protein, which was detected in the nucleus, cytoplasm and predominantly at the intercellular junctions of the AGS cells. In addition, HM enhanced IL-6 production and secretion, and upregulated the mucin 4 gene expression, the hallmarks of gastroprotection. To check whether HM-induced PGE2 and IL-6 through TLR4 signaling and COX-2 generated, AGS cells were pre-treated with a TLR4 signaling inhibitor TAK242 and the COX-2 inhibitor NS-398. A loss of the stimulatory effects of HM on COX-2, PGE2 and IL-6 production and secretion was observed in TAK242 and NS-398-pre-treated AGS cells, confirming the role of TLR4 signaling and COX-2 generated in the HM gastroprotective effects. In conclusion, our results showed that HM enhances TLR4/COX-2-mediated secretion of gastroprotective markers PGE2 and IL-6, and upregulates mucin 4 gene expression in the human gastric epithelial cell line AGS, which may contribute to the promising beneficial gastroprotective effect of HM for human gastric prevention and treatment.

Inhibitory Effect of Korean Red Ginseng Extract on Interleukin-8 Expression in Helicobacter pylori-Infected Gastric Epithelial Cells

ObjectivesInfection of Helicobacter pylori (H. pylori), a gram-negative bacterium, leads to various gastric diseases, such as gastritis, peptic ulcer and gastric cancer. H. pylori increases cytokine release and activates inflammatory mediators in gastric mucosa. Particularly, H. pylori upregulates the inflammatory chemokine interleukin-8 (IL-8), which are activated by oxidative stress. IL-8 can cause severe inflammation of the stomach and gastric cancer. Korean red ginseng is the steamed root of 6-year-old Korean ginseng (Panax ginseng Meyer). Ginsenosides, triterpene glycosides, are the active components of Korean red ginseng. Ginsenosides have antioxidant, anti-inflammatory, and antitumor activities. The present study is aimed at determining whether Korean red ginseng extract inhibits H. pylori-induced IL-8 expression in gastric epithelial cells. MethodsThe human gastric epithelial cell line AGS was used. Gastric epithelial AGS cells were treated with Korean red ginseng extract, and infected with H. pylori (NCTC 11,637). Reactive oxygen species (ROS) levels were determined using dichlorofluorescein fluorescence. NADPH oxidase activity was measured using lucigenin chemiluminescence. IL-8 mRNA expression was measured by using real-time PCR. NADPH oxidase subunits were determined in cytosolic extract and membrane extract by using Western blotting. ResultsH. pylori increased NADPH oxidase activity, ROS levels, and upregulated IL-8 expression in gastric epithelial cells. Korean red ginseng extract inhibited IL-8 expression by suppressing NADPH oxidase activity and reducing ROS levels in gastric epithelial cells. H. pylori induced translocation of NADPH oxidase cytosolic subunits to membrane, which is a marker of NADPH oxidase activation, in AGS cells. Korean red ginseng extract inhibited translocation of cytosolic subunits of NADPH oxidase to membrane in AGS cells. ConclusionsKorean red ginseng may be beneficial for preventing H. pylori-associated gastric inflammation by inhibiting oxidative stress and IL-8 expression. Funding SourcesThis study was supported by a Brain Korea 21 FOUR Project, Yonsei University, Seoul, Republic of Korea.

Type IV secretion of Helicobacter pylori CagA into oral epithelial cells is prevented by the absence of CEACAM receptor expression

BackgroundHelicobacter pylori typically colonizes the human stomach, but it can occasionally be detected in the oral cavity of infected persons. Clinical outcome as a result of gastric colonization depends on presence of the pathogenicity island cagPAI that encodes a type-IV secretion system (T4SS) for translocation of the effector protein CagA and ADP-heptose. Upon injection into target cells, CagA is phosphorylated, which can be demonstrated by in vitro infection of the gastric epithelial cell line AGS, resulting in cell elongation. Here we investigated whether H. pylori can exert these responses during interaction with cells from the oral epithelium. To this purpose, three oral epithelial cell lines, HN, CAL-27 and BHY, were infected with various virulent wild-type H. pylori strains, and CagA delivery and ADP-heptose-mediated pro-inflammatory responses were monitored.ResultsAll three oral cell lines were resistant to elongation upon infection, despite similar bacterial binding capabilities. Moreover, T4SS-dependent CagA injection was absent. Resistance to CagA delivery was shown to be due to absence of CEACAM expression in these cell lines, while these surface molecules have recently been recognized as H. pylori T4SS receptors. Lack of CEACAM expression in HN, CAL-27 and BHY cells was overcome by genetic introduction of either CEACAM1, CEACAM5, or CEACAM6, which in each of the cell lines was proven sufficient to facilitate CagA delivery and phosphorylation upon H. pylori infection to levels similar to those observed with the gastric AGS cells. Pro-inflammatory responses, as measured by interleukin-8 ELISA, were induced to high levels in each cell line and CEACAM-independent.ConclusionsThese results show that lack of CEACAM receptors on the surface of the oral epithelial cells was responsible for resistance to H. pylori CagA-dependent pathogenic activities, and confirms the important role for the T4SS-dependent interaction of these receptors with H. pylori in the gastric epithelium.

Exopolysaccharide-producing Streptococcus thermophilus CRL1190 reduces the inflammatory response caused by Helicobacter pylori.

This work evaluated the ability of the probiotic Streptococcus thermophilus CRL1190 strain and its exopolysaccharides to adhere to gastric mucosa. Probiotic bacteria attachment to the human stomach epithelium was confirmed in human stomach tissue samples and the gastric epithelial cell line AGS. In addition, it was demonstrated that S. thermophilus CRL1190 strain reduced Helicobacter pylori adhesion and attenuated inflammatory response in AGS cells. This is the first demonstration of the capacity of S. thermophilus CRL1190 to adhere to the stomach gastric mucosa, and improve protection against H. pylori through the reduction of its adhesion and the modulation of the inflammatory response. Therefore, S. thermophilus CRL1190 fermented milk is a good candidate for further in vivo studying of the protective effect of functional food against H. pylori infection and gastric inflammatory damage.

Alteration of stomach microbiota compositions in the progression of gastritis induces nitric oxide in gastric cell.

Atrophic gastritis is considered to be an antecedent to intestinal metaplasia and gastric cancer. A previous study identified that Helicobacter pylori was absent at the severe atrophic gastritis stage, and alterations in the gastric microbial composition resembled those in gastric cancer. To explore the role of the bacteria absence of H. pylori in gastric carcinogenesis, in the current study, we compared the microbiota of clinically collected H. pylori-free gastric fluids from 30 patients with non-atrophic gastritis (N) and 22 patients with severe atrophic gastritis (S). We estimated the bacterial loads in the N and S groups by colony counting in culture agar as well as by measuring the concentration of the extracted DNA. The results showed a significant increase in bacterial load in patients with atrophic gastritis in comparison to non-atrophic gastritis. Then, we analyzed the microbial communities of the gastric fluids from all 52 patients using high-throughput sequencing of 16S rRNA amplicons. The Chao 1, Shannon and Simpson diversity indexes demonstrated that the bacterial richness and diversity were not significantly different between the N and S groups. Moreover, principal component analysis illustrated that the microbiomes from the S group were more scattered. Microbiota composition analysis showed that the entire dataset was clustered into 27 phyla, 61 classes, 106 orders, 177 families, 292 genera and 121 species. At the genus level, only the abundance of Prevotella was significantly different between the N and S groups. Further analysis showed that all the higher taxonomic categories were significantly different between the N and S groups. To assess the effects of the metabolic products of Prevotella spp. on gastric cell physiology, we treated the human gastric epithelial cell line AGS with acetic acid and monitored nitric oxide (NO) production. The results showed that acetic acid at low concentrations (0.5 and 5 µM) significantly inhibited AGS cells to secrete NO compared to phosphate buffer saline-treated control cells. These results suggest that the microbiota in non-atrophic gastritis may influence gastric epithelial cell physiology.

A Conserved Helicobacter pylori Gene, HP0102, Is Induced Upon Contact With Gastric Cells and Has Multiple Roles in Pathogenicity.

Contact with host cells is recognized as a signal capable of triggering expression of bacterial genes important for host pathogen interaction. Adherence of Helicobacter pylori to the gastric epithelial cell line AGS strongly upregulated expression of a gene, HP0102, in the adhered bacteria in all strains examined, including several Indian clinical isolates. The gene is highly conserved and ubiquitously present in all 69 sequenced H. pylori genomes at the same genomic locus, as well as in 15 Indian clinical isolates. The gene is associated with 2 distinct phenotypes related to pathogenicity. In AGS cell-adhered H. pylori, it has a role in upregulation of cagA expression from a specific σ(28)-RNAP promoter and consequent induction of the hummingbird phenotype in the infected AGS cells. Furthermore, HP0102 has a role in chemotaxis and a ΔHP0102 mutant exhibited low acid-escape response that might account for the poor colonization efficiency of the mutant.

Role of the Flagellar Hook-Length Control Protein FliK and σ28 in cagA Expression in Gastric Cell-Adhered Helicobacter pylori.

Adherence of Helicobacter pylori to the gastric epithelial cell line AGS strongly induces expression of fliK encoding a flagellar hook-length control protein. FliK has a role in triggering dissociation of the alternate sigma factor, σ(28), from a nonfunctional σ(28)-FlgM complex, releasing free, functional σ(28). The σ(28)-RNA polymerase initiates transcription of cagA, the major virulence gene, from a promoter identified in this study. Consequently, significant up-regulation of cagA was observed in AGS-adhered H. pylori. Direct binding of σ(28) to the cagA promoter was demonstrated by chromatin immunoprecipitation and the transcription start site was identified by 5' RACE (rapid amplification of complementary DNA ends). The σ(28)-dependent cagA promoter was active specifically in AGS-adhered H. pylori, and this motif might be associated with high cagA expression and severity of disease. These results also indicate that H. pylori has evolved to integrate expression of the major virulence gene cagA with the flagellar regulatory circuit, essential for colonization of the human host.

Host Cell Contact Induces Fur‐dependent Expression of Virulence Factors CagA and VacA in Helicobacter pylori

Helicobacter pylori, a gram negative bacterium, colonizes the stomach in a majority of the world population. The two major virulence factors of H.pylori VacA and CagA, thought to be associated with chronic inflammation and disease, have been extensively studied, but the regulation of the expression of these virulence genes in H.pylori remains poorly understood. qRT-PCR was performed to quantify gene expression in unadhered and AGS-adhered H.pylori. Δfur mutant was constructed by splicing by overlap extension PCR and allelic exchange. Adherence of H.pylori to the gastric epithelial cell line AGS strongly induces the expression of both cagA and vacA. Induction of cagA and vacA in the AGS cell-adhered H.pylori Δfur mutant strain was consistently lower than in the adhered parent strain. However, expression of the genes was similar between the wild-type and Δfur mutant strains in the unadhered state, suggesting that Fur has a role in the upregulation of cagA and vacA expression, especially in AGS-adhered H.pylori. Consistent with these results, microscopic observations revealed that infection of AGS cells with H.pylori Δfur mutant strain produced much less damage as compared to that produced by the wild-type H.pylori strain. These results suggested that cagA and vacA gene expression is upregulated in H.pylori, especially by host cell contact, and Fur has a role in the upregulation.

Effects of taraxerol and taraxeryl acetate on cell cycle and apoptosis of human gastric epithelial cell line AGS

To investigate the effects of taraxerol and taraxerol acetate on cell cycle and apoptosis of human gastric epithelial cell line AGS cells. The inhibitory effects of taraxerol and taraxerol acetate at different concentrations on AGS cell growth were measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) method and the concentrations of taraxerol and taraxerol acetate to be used in following experiments were decided. Then, cell cycle analysis was performed by FACScan flow cytometry after culture with taraxerol or taraxerol acetate. Annexin V-fluorescein isothiocyanate/propidium iodide staining was used to measure cell apoptosis. Taraxerol significantly inhibited AGS cell proliferation in a dose- and time-dependent manner. Taraxerol arrested the AGS cells at G(2)/M stage. 110 μmol/L taraxerol elevated the population of AGS cells arrested in G(2)/M phase compared with solvent (P<0.05). Taraxerol also promoted early cell apoptosis in AGS cells. 110 μmol/L taraxerol increased the early cell apoptosis rate from 4.45% to 10.29%, which was 1.31 times higher than that of the untreated cells. However, taraxerol acetate had a lower inhibitory effect than taraxerol, and it showed a tendency of G(2)/M arrest and apoptosis promotion but with no statistical significance (P>0.05). Taraxerol has inhibitory effects on AGS cell growth through inducing G(2)/M arrest and promotion of cell apoptosis. Taraxerol acetate has less effect on cell cycle arrest and apoptosis of AGS cells than taraxerol.

Biochemical and pathophysiological characterization of Helicobacter pylori asparaginase

Asparaginase was purified from Helicobacter pylori 26695 and its pathophysiological role explored. The K(m) value of asparagine was 9.75 ± 1.81 μM at pH 7.0, and the optimum pH range was broad and around a neutral pH. H. pylori asparaginase converted extracellular asparagine to aspartate. H. pylori cells were unable to take up extracellular asparagine directly. Instead, aspartate produced by the action of the asparaginase was transported into H. pylori cells, where it was partially converted to β-alanine. Asparaginase exhibited striking cytotoxic activity against histiocytic lymphoma cell line U937 cells via asparagine deprivation. The cytotoxic activity of live H. pylori cells against U937 cells was significantly diminished by deletion of the asparaginase gene, indicating that asparaginase functions as a cytotoxic agent of the bacterium. The cytotoxic effect was negligible for gastric epithelial cell line AGS cells, suggesting that the effect differs across host cell types. An asparaginase-deficient mutant strain was significantly less capable of colonizing Mongolian gerbils. Since asparagine depletion by exogenous asparaginase has been shown to suppress lymphocyte proliferation in vivo, the present results suggest that H. pylori asparaginase may be involved in inhibition of normal lymphocyte function at the gastric niche, allowing H. pylori to evade the host immune system.

In VitroInhibition ofHelicobacter pyloriGrowth and of Adherence ofcagA-Positive Strains to Gastric Epithelial Cells byLactobacillus paraplantarumKNUC25 Isolated from Kimchi

Lactobacillus paraplantarum KNUC25 strain was isolated from overfermented kimchi, a Korean traditional food. The strain had a broad antimicrobial activity spectrum, from gram-positive to gram-negative bacteria. The aim of this study was to evaluate the activity of L. paraplantarum KNUC25 against Helicobacter pylori strains. Judged by a disc agar diffusion method, the anti-H. pylori activity existed in the cell-free supernatants (CFSs) of KNUC25. The mean diameters of growth inhibition by 10, 30, and 60 microL of a 15-fold concentrated CFS per disc were 11.2, 17.7, and 23.7 mm, respectively. The neutralized CFS lost its anti-H. pylori activity, suggesting that acidic pH in CFS may be responsible for the anti-H. pylori activity. Adherence was determined by urease activity of H. pylori adhered to gastric epithelial cell line AGS cells after co-incubation of AGS cells with CFS and H. pylori strain ATCC43504 (s1m1vacA/cagA(+)), ATCC51932 (s2m2vacA/cagA(-)), or SS1 (s2m2vacA/cagA(+)) in vitro followed by three washes by means of centrifugation with saline. Adherence of ATCC43504 or SS1 to AGS cells was reduced by about 70% after a 30-minute incubation with 30 microL of a 15-fold concentrated KNUC25 CFS, whereas that of ATCC51932 to AGS cells was not. The results show KNUC25 CFS is effective in inhibiting the growth of H. pylori, which is related to pH and the adherence of cagA-positive H. pylori to gastric cells.

Secreted Trefoil Factor 2 Activates the CXCR4 Receptor in Epithelial and Lymphocytic Cancer Cell Lines

The secreted trefoil factor family 2 (TFF2) protein contributes to the protection of the gastrointestinal mucosa from injury by strengthening and stabilizing mucin gels, stimulating epithelial restitution, and restraining the associated inflammation. Although trefoil factors have been shown to activate signaling pathways, no cell surface receptor has been directly linked to trefoil peptide signaling. Here we demonstrate the ability of TFF2 peptide to activate signaling via the CXCR4 chemokine receptor in cancer cell lines. We found that both mouse and human TFF2 proteins (at approximately 0.5 microm) activate Ca2+ signaling in lymphoblastic Jurkat cells that could be abrogated by receptor desensitization (with SDF-1alpha) or pretreatment with the specific antagonist AMD3100 or an anti-CXCR4 antibody. TFF2 pretreatment of Jurkat cells decreased Ca2+ rise and chemotactic response to SDF-1alpha. In addition, the CXCR4-negative gastric epithelial cell line AGS became highly responsive to TFF2 treatment upon expression of the CXCR4 receptor. TFF2-induced activation of mitogen-activated protein kinases in gastric and pancreatic cancer cells, KATO III and AsPC-1, respectively, was also dependent on the presence of the CXCR4 receptor. Finally we demonstrate a distinct proliferative effect of TFF2 protein on an AGS gastric cancer cell line that expresses CXCR4. Overall these data identify CXCR4 as a bona fide signaling receptor for TFF2 and suggest a mechanism through which TFF2 may modulate immune and tumorigenic responses in vivo.

Caffeic acid phenethyl ester modulates Helicobacter pylori‐induced nuclear factor‐kappa B and activator protein‐1 expression in gastric epithelial cells

Caffeic acid phenethyl ester (CAPE), an active component of propolis from honeybee hives (honeybee resin), has anti-inflammatory, anti-carcinogenic and anti-bacterial properties. This study was designed to investigate the anti-inflammatory effects of CAPE on Helicobacter pylori-induced NF-kappaB and AP-1 in the gastric epithelial cell line AGS. Electrophoretic mobility shift assay was used to measure NF-kappaB- and AP-1-DNA binding activity. Western blotting was used to detect IkappaB-alpha and COX-2 expression in AGS cells cocultured with H. pylori. The antiproliferative effect of CAPE was measured by MTT assay. Our results showed that caffeic phenethyl ester inhibits H. pylori-induced NF-kappaB and AP-1 DNA-binding activity in a dose (0.1-25 microg ml(-1) approximately 0.35-88 microM) and time- (15-240 min) dependent manner in AGS cells. Maximum inhibition by CAPE was observed at concentrations of 25 microg ml(-1) ( approximately 88 microM) CAPE prevented H. pylori- and cytokine-induced degradation of IkappaB-alpha protein. Pretreatment of AGS cells with CAPE also blocked cytokine- and mitogen-induced NF-kappaB and AP-1 expression. Furthermore, CAPE suppressed H. pylori-induced cell proliferation and production of the cytokines TNF-alpha and IL-8. In addition, CAPE blocked H. pylori-induced COX-2 expression. The inhibition of such transcription by CAPE could result in suppression of many genes during H. pylori-induced inflammation, and also provide new insights into the anti-cancer and anti-inflammatory properties of CAPE.

Gastroprotective Effect and Cytotoxicity of Natural and Semisynthetic Labdane Diterpenes from Araucaria araucana Resin

The resin of the tree Araucaria araucana (Araucariaceae) is used by the Mapuche Amerindians in southern Chile and Argentina to treat ulcers and has been shown to display a gastroprotective effect in animal models. A study was undertaken to isolate, identify and assess the gastroprotective effect of the resin constituents and its semisynthetic derivatives as well as to evaluate the cytotoxicity of the products in cell cultures. Eleven diterpenes (ten labdane and a pimarane) were isolated from a resin sample collected in Chile. The labdane derivatives 15-acetoxylabd-8(17)-en-19-ol as well as 15,19-diacetoxylabd-8(17)-en are reported for the first time as natural products. Six diterpenes previously described from other plant sources are reported for the first time for the A. araucana resin. The structure of all compounds was elucidated by spectroscopic means. Some 24 diterpenes isolated/prepared in amounts over 10 mg were evaluated for gastroprotective effects in the ethanol/HCl-induced ulcer model in mice at 100 mg/kg. The highest gastroprotective activities were provided by 15-hydroxyimbricatolal, 15-acetoxyimbricatolal, 15-acetoxylabd-8(17)-en-19-oic acid methyl ester and 15-acetoxy-19-labdanoic acid, all of them being as active as the reference drug lansoprazole at 20 mg/kg. The cytotoxicity of 30 diterpenes as well as lansoprazole was assessed towards human lung fibroblasts (MRC-5) and 26 compounds were evaluated on the human gastric epithelial cell line AGS by means of the neutral red uptake assay. A concentration-dependent cell viability inhibition was found with IC50 values ranging from 27 up to > 1000 microM. The relationship between the cytotoxicity data and lipophilicity of the products is also discussed.

The Secreted Peptidyl Prolyl cis,trans-Isomerase HP0175 of Helicobacter pylori Induces Apoptosis of Gastric Epithelial Cells in a TLR4- and Apoptosis Signal-Regulating Kinase 1-Dependent Manner

Apoptosis contributes to the pathology of gastric epithelial cell damage that characterizes Helicobacter pylori infection. The secreted peptidyl prolyl cis, trans-isomerase of H. pylori, HP0175 executed apoptosis of the gastric epithelial cell line AGS in a dose- and time-dependent manner. The effect of HP0175 was confirmed by generating an isogenic mutant of H. pylori disrupted in the HP0175 gene. The apoptosis-inducing ability of this mutant was impaired compared with that of the wild type. The effect of HP0175 was mediated through TLR4. Preincubation of the gastric epithelial cell line AGS with anti-TLR4 mAb inhibited apoptosis induced by HP0175. Downstream of TLR4, apoptosis signal-regulating kinase 1 activated MAPK p38, leading to the caspase 8-dependent cleavage of Bid, its translocation to the mitochondria, mitochondrial pore formation, cytochrome c release, and activation of caspases 9 and 3. We show for the first time that a secreted bacterial Ag with peptidyl prolyl cis,trans-isomerase activity signals through TLR4, and that this Ag executes gastric epithelial cell apoptosis through a signaling pathway in which TLR4 and apoptosis signal-regulating kinase 1 are central players.

Involvement of Syntaxin 7 in Human Gastric Epithelial Cell Vacuolation Induced by the Helicobacter pylori-produced Cytotoxin VacA

The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. The formed vacuole is assumed to be a hybrid of late endosome and lysosome. To elucidate the molecular mechanism of VacA-induced vacuolation, we examined the participation of syntaxin 7 in the human gastric epithelial cell line AGS. Immunocytochemistry revealed that endogenous syntaxin 7 was localized to vacuoles induced by VacA. Northern and Western blotting demonstrated that VacA intoxication increased syntaxin 7 mRNA and protein expression, respectively, in a time-dependent manner. Transient transfection of dominant-negative mutant syntaxin 7, which lacks a carboxyl-terminal transmembrane domain, inhibited VacA-induced vacuolation. In contrast, transient transfection of wild-type syntaxin 7, dominant-negative mutant syntaxin 1a, or dominant-negative mutant syntaxin 4 did not alter VacA-induced vacuolation. Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with mutant syntaxin 7 but not in cells stably transfected with wild-type syntaxin 7, mutant syntaxin 1a, or mutant syntaxin 4. Sequential immunocytochemical observation confirmed that expression of mutant syntaxin 7 did not affect VacA attachment to or internalization into AGS cells. We suggest that syntaxin 7 is involved in the intracellular vacuolation induced by VacA.

Helicobacter pylori infection induces interleukin-8 receptor expression in the human gastric epithelium.

The gastric pathogen Helicobacter pylori is known to activate multiple proinflammatory signaling pathways in epithelial cells. In this study, we addressed the question of whether expression of the interleukin-8 receptors IL-8RA (CXCR1) and IL-8RB (CXCR2) is upregulated in H. pylori-infected human gastric biopsy samples. Biopsy samples from patients infected with H. pylori strains harboring the cag pathogenicity island (PAI) expressed larger amounts of both receptors. In addition, IL-8RB expression was induced in the gastric epithelial cell line AGS upon infection with a clinical isolate containing the cag PAI, while a strain lacking the cag PAI did not. Our finding suggests that gastric epithelial cells express IL-8R in response to H. pylori infection.

Involvement of syntaxin 7 in human gastric epithelial cell vacuolation induced by Helicobacter pylori-produced cytotoxin VacA

The Helicobacter pylori-produced cytotoxin VacA induces intracellular vacuolation. The formed vacuole is assumed to be a hybrid of late endosome and lysosome. To elucidate the molecular mechanism of VacA-induced vacuolation, we examined the participation of syntaxin 7 in the human gastric epithelial cell line AGS. Immunocytochemistry revealed that endogenous syntaxin 7 was localized to vacuoles induced by VacA. Northern and Western blotting demonstrated that VacA intoxication increased syntaxin 7 mRNA and protein expression, respectively, in a time-dependent manner. Transient transfection of dominant-negative mutant syntaxin 7, which lacks a carboxyl-terminal transmembrane domain, inhibited VacA-induced vacuolation. In contrast, transient transfection of wild-type syntaxin 7, dominant-negative mutant syntaxin 1a, or dominant-negative mutant syntaxin 4 did not alter VacA-induced vacuolation. Furthermore, under VacA treatment, neutral red dye uptake, a parameter of VacA-induced vacuolation, was inhibited in cells stably transfected with mutant syntaxin 7 but not in cells stably transfected with wild-type syntaxin 7, mutant syntaxin 1a, or mutant syntaxin 4. Sequential immunocytochemical observation confirmed that expression of mutant syntaxin 7 did not affect VacA attachment to or internalization into AGS cells. We suggest that syntaxin 7 is involved in the intracellular vacuolation induced by VacA.

Vacuolating cytotoxin of Helicobacter pylori induces apoptosis in the human gastric epithelial cell line AGS.

Helicobacter pylori induces cell death by apoptosis. However, the apoptosis-inducing factor is still unknown. The virulence factor vacuolating cytotoxin A (VacA) is a potential candidate, and thus its role in apoptosis induction was investigated in the human gastric epithelial cell line AGS. The supernatant from the vacA wild-type strain P12 was able to induce apoptotic cell death, whereas the supernatant from its isogenic mutant strain P14 could not. That VacA was indeed the apoptosis-inducing factor was demonstrated further by substantial reduction of apoptosis upon treatment of AGS cells with a supernatant specifically depleted of native VacA. Furthermore, a recombinant VacA produced in Escherichia coli was also able to induce apoptosis in AGS cells but failed to induce cellular vacuolation. These findings demonstrate that the vacuolating cytototoxin of H. pylori is a bacterial factor capable of inducing apoptosis in gastric epithelial cells.