Cellular stress-related protein expression in Helicobacter pylori-infected gastric epithelial AGS cells

Helicobacter pylori infection leads to gastroduodenal inflammation, peptic ulceration, and gastric carcinoma. Moreover, H. pylori may induce disease-specific protein expression in gastric epithelial cells. The present study was aimed at determining differentially expressed proteins in H. pylori-infected gastric epithelial AGS cells. AGS cells were treated with H. pylori at a bacterium/cell ratio of 300:1 for 12 h. Altered protein patterns as separated by two-dimensional electrophoresis using pH gradients of 4-7 were conclusively identified by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the peptide digests. Four differentially expressed proteins, whose expression levels were increased by more than two-fold in H. pylori-infected cells, were analyzed. These proteins (14-3-3 protein alpha/beta, cullin homolog 3, alpha-enolase, ezrin) are known to be related to cell proliferation, cell adhesion, and carcinogenesis, and may be mediated by cellular stress, such as reactive oxygen species. In conclusion, the identification of these differentially expressed proteins provide valuable information for the understanding of the pathophysiologic mechanisms of H. pylori-induced gastric diseases, and may be useful as prognostic indices of H. pylori-related gastric disorders.     

Helicobacter pylori encoding the pathogenicity island activates matrix metalloproteinase 1 in gastric epithelial cells via JNK and ERK

Helicobacter pylori colonizes the human gastric epithelium and induces an inflammatory response that is a trigger for gastric carcinogenesis. Matrix metalloproteinases (MMPs) have recently been shown to be up-regulated in gastric epithelial cells infected with H. pylori and might contribute to the pathogenesis of peptic ulcer. The aim of this study was to extend the knowledge about the effect of H. pylori infection on MMP-1 expression by gastric epithelial cells, the kinetics of induction, the pathogenetic properties of the bacterium, and the intracellular signaling pathways required for MMP-1 up-regulation. Expression of MMP-1 was induced more than 10-fold by co-culture of AGS+cells with H. pylori strains carrying the pathogenicity island (PAI). H. pylori strains with mutations in the PAI and a defective type IV secretion system had no effect on MMP-1. Double immunofluorescence revealed strong MMP-1 staining in epithelial cells of gastric biopsies at sites of bacterial attachment. In vitro, MMP-1 is up-regulated by interleukin-1beta and tumor necrosis factor-alpha, but these regulatory mechanisms are not operating in H. pylori infection as shown by inhibitory antibodies. Specific inhibitors of JNK kinase and ERK1/2 kinase were found to suppress the H. pylori-induced MMP-1 expression and activity. AGS cells treated with antisense MMP-1 showed a significantly reduced potential to degrade reconstituted basement membrane. Our results suggest that in gastric epithelial cells, H. pylori up-regulates MMP-1 in a type IV secretion system-dependent manner via JNK and ERK1/2. Induction of MMP-1 is further implicated in complex processes induced by H. pylori, resulting in tissue degradation and remodeling of the gastric mucosa.     

Helicobacter pylori infection activates NF-kappaB signaling pathway to induce iNOS and protect human gastric epithelial cells from apoptosis

Helicobacter pylori infection induces apoptosis and inducible nitric oxide synthase (iNOS) expression in gastric epithelial cells. In this study, we investigated the effects of NF-kappaB activation and iNOS expression on apoptosis in H. pylori-infected gastric epithelial cells. The suppression of NF-kappaB significantly increased caspase-3 activity and apoptosis in H. pylori-infected MKN-45 and Hs746T gastric epithelial cell lines as well as primary gastric epithelial cells. An NF-kappaB signaling pathway via NF-kappaB-inducing kinase and IkappaB kinase-beta activation was found to be involved in the inhibition of apoptosis in H. pylori-infected gastric epithelial cells. In gastric epithelial cells transfected with retrovirus containing IkappaBalpha superrepressor, iNOS mRNA and protein levels were reduced, indicating that H. pylori infection induced the expression of iNOS by activating NF-kappaB. Moreover, a NO donor, S-nitroso-N-acetylpenicillamine (100 microM), decreased caspase-3 activity and apoptosis in NF-kappaB-suppressed cells infected with H. pylori. These results suggest that NF-kappaB activation may play a role in protecting gastric epithelial cells from H. pylori-induced apoptosis by upregulating endogenous iNOS.     

Phosphorylation of the gastric tumor suppressor RUNX3 following H. pylori infection results in its localization to the cytoplasm

As H. pylori infection progresses, intestinal metaplasia (IM), a key event in gastric carcinogenesis, develops in the stomach. The mechanism by which H. pylori infection causes the trans-differentiation of gastric cells to intestinal-type cells remains an important question. In the current study, we found that RUNX3 is deregulated in all human IM specimens examined by either down regulation or mislocalization; Aberrant localization of a gastric tumor suppressor RUNX3 is observed in most human cases of IM with concurrent H. pylori infection, and RUNX3 is down-regulated in most cases of IM without H. pylori-infection. The cytoplasmic mislocalization of a RUNX3 was associated with H. pylori-induced c-Src activation and RUNX tyrosine phosphorylation. Moreover, gastric epithelial cells of Runx3(-/-) mice expressed the intestinal markers Muc2 and Li-Cadherin, which suggests that the deregulation of Runx3 is a key event in the intestinalization of the gastric epithelium. Collectively, the results of the current study suggest that RUNX3 deregulation is associated with H. pylori-induced pathogenesis and the development of IM.     

Restoration of heat shock protein70 suppresses gastric mucosal inducible nitric oxide synthase expression induced by Helicobacter pylori

Heat shock proteins (HSPs) are crucial for the maintenance of cell integrity during normal cellular growth as well as during pathophysiological conditions. While functioning mainly as molecular chaperones, HSPs also appear to be involved in diverse biological activities, such as apoptosis, carcinogenesis, and cytoprotection from cytotoxic damage. Infection with Helicobacter pylori causes inflammation in the gastric mucosa, leading to gastritis, gastric ulcers, duodenal ulcer disease, and even gastric cancer, but the role of HSPs in H. pylori-associated gastropathy is not known. Using two-dimensional electrophoretic analysis, we have observed significant shifts in HSP profiles after H. pylori infection in RGM-1 cells. We therefore evaluated the effect of treatments that induce HSPs on H. pylori-induced inducible nitric oxide synthase (iNOS) expression. We found that H. pylori infection significantly attenuated the expression of HSP70, whereas exposure of cells to noncytotoxic heat shock or geranylgeranylacetone restored HSP70 expression, as well as suppressing the expression of iNOS, a major cause of H. pylori-induced gastric tissue damage. Our results suggest that induction of HSP70 confers cytoprotection against H. pylori infection by inhibiting the expression of iNOS. In conclusion, these results provide important insights into the flux in HSPs profiles in response to H. pylori infection and highlight the cytoprotective role of HSP70 in H. pylori infection.     

Helicobacter pylori environmental interactions: effect of acidic conditions on H. pylori-induced gastric mucosal interleukin-8 production

To explore the interactions between the host, environment and bacterium responsible for the different manifestations of Helicobacter pylori infection, we examined the effect of acidic conditions on H. pylori-induced interleukin (IL)-8 expression. AGS gastric epithelial cells were exposed to acidic pH and infected with H. pylori[wild-type strain, its isogenic cag pathogenicity island (PAI) mutant or its oipA mutant]. Exposure of AGS cells to acidic pH alone did not enhance IL-8 production. However, following exposure to acidic conditions, H. pylori infection resulted in marked enhancement of IL-8 production which was independent of the presence of the cag PAI and OipA, indicating that H. pylori and acidic conditions act synergistically to induce gastric mucosal IL-8 production. In neutral pH environments H. pylori-induced IL-8 induction involved the NF-kappaB pathways, the extracellular signal-regulated kinase (ERK)-->c-Fos/c-Jun-->activating protein (AP-1) pathways, JNK-->c-Jun-->AP-1 pathways and the p38 pathways. At acidic pH H. pylori-induced augmentation of IL-8 production involved markedly upregulated the NF-kappaB pathways and the ERK-->c-Fos-->AP-1 pathways. In contrast, activation of the JNK-->c-Jun-->AP-1 pathways and p38 pathways were pH independent. These results might explain the clinical studies in which patients with duodenal ulcers had higher levels of IL-8 in the antral gastric mucosa than patients with simple H. pylori gastritis.     

Anti-inflammatory effect of two isoforms of COX in H. pylori-induced gastritis in mice: possible involvement of PGE2

Neutrophil infiltration mediated by TNF-alpha is associated with various types of gastric injury, whereas PGs play a crucial role in gastric defense. We examined roles of two isoforms of cyclooxygenase (COX) and PGE2 in Helicobacter pylori-induced gastritis in mice. Mice infected with H. pylori were given selective COX-1 inhibitor SC-560 (10 mg/kg), selective COX-2 inhibitor NS-398 (10 mg/kg), or nonselective COX inhibitor indomethacin (2 mg/kg) with or without 16,16-dimethyl PGE2 for 1 wk. H. pylori infection increased levels of mRNA for COX-1 and -2 in gastric tissue by 1.2-fold and 3.3-fold, respectively, accompanied by a significant increase in PGE2 production by gastric tissue. H. pylori infection significantly elevated MPO activity, a marker of neutrophil infiltration, and epithelial cell apoptosis in the stomach. SC-560 augmented MPO activity and epithelial cell apoptosis with associated reduction in PGE2 production, whereas NS-398 had the same effects without affecting PGE2 production. Inhibition of both COX-1 and -2 by indomethacin or concurrent treatment with SC-560 and NS-398 resulted in a stronger increase in MPO activity and apoptosis than inhibition of either COX-1 or -2 alone. H. pylori infection elevated TNF-alpha mRNA expression in the stomach, which was further increased by indomethacin. Effects of COX inhibitors on neutrophil infiltration, apoptosis, and TNF-alpha expression in H. pylori-infected mice were abolished by exogenous 16,16-dimethyl PGE2. In conclusion, PGE2 derived from either COX-1 or -2 is involved in regulation of gastric mucosal inflammation and contributes to maintenance of mucosal integrity during H. pylori infection via inhibition of TNF-alpha expression.     

Helicobacter pylori-downregulated tumor necrosis factor receptor-associated protein 1 mediates apoptosis of human gastric epithelial cells

Heat shock proteins (HSPs) are crucial proteins in maintaining the homeostasis of human gastric epithelial cells. Tumor necrosis factor receptor-associated protein 1 (TRAP1), a member of the HSP90 family, has been shown to be involved in various crucial physiological processes, particularly against apoptosis. However, the regulation and function of TRAP1 in Helicobacter pylori infection is still unknown. Here, we found that TRAP1 expression was downregulated on human gastric epithelial cells during H. pylori infection by real-time polymerase chain reaction (PCR) and western blot analysis. Through virulence factors mutant H. pylori strains infection and inhibitors screening, we found that H. pylori vacuolating cytotoxin A ( vacA), but not cytotoxin-associated gene A ( cagA) protein, induced human gastric epithelial cells to downregulate TRAP1 via P38MAPK pathway by real-time PCR and western blot analysis. Furthermore, downregulation of TRAP1 with lentivirus carrying TRAP1 short hairpin RNA constructs impairs mitochondrial function, and increases apoptosis of gastric epithelial cells. The results indicate that H. pylori vacA downregulated TRAP1 is involved in the regulation of gastric epithelial cell apoptosis.     

Helicobacter pylori-induced prostaglandin E(2) synthesis involves activation of cytosolic phospholipase A(2) in epithelial cells

Helicobacter pylori initiates an inflammatory response and gastric diseases, which are more common in patients infected with H. pylori strains carrying the pathogenicity island, by colonizing the gastric epithelium. In the present study we investigated the mechanism of prostaglandin E(2) (PGE(2)) synthesis in response to H. pylori infection. We demonstrate that H. pylori induces the synthesis of PGE(2) via release of arachidonic acid predominately from phosphatidylinositol. In contrast to H. pylori wild type, an isogenic H. pylori strain with a mutation in the pathogenicity island exerts only weak arachidonic acid and PGE(2) synthesis. The H. pylori-induced arachidonic acid release was abolished by phospholipase A(2) (PLA(2)) inhibitors and by pertussis toxin (affects the activity of G alpha(i)/G alpha(o)). The role of phospholipase C, diacylglycerol lipase, or phospholipase D was excluded by using specific inhibitors. An inhibitor of the stress-activated p38 kinase (SB202190), but neither inhibitors of protein kinase C nor an inhibitor of the extracellular-regulated kinase pathway (PD98059), decreased the H. pylori-induced arachidonic acid release. H. pylori-induced phosphorylation of p38 kinase and cytosolic PLA(2) was blocked by SB202190. These results indicate that H. pylori induces the release of PGE(2) from epithelial cells by cytosolic PLA(2) activation via G alpha(i)/G alpha(o) proteins and the p38 kinase pathway.     

Helicobacter pylori inhibits gastric cell cycle progression

Helicobacter pylori infection of the gastric mucosa is associated with changes in gastric epithelial cell proliferation. In vitro studies have shown that exposure to H. pylori inhibits proliferation of gastric cells. This study sought to investigate the cell cycle progression of gastric epithelial cell lines in the presence and absence of H. pylori. Unsynchronized and synchronized gastric epithelial cell lines AGS and KatoIII were exposed to H. pylori over a 24-h period. Cell cycle progression was determined by flow cytometry using propidium iodide (PI), and by analysis of cyclin E, p21, and p53 protein expression using Western blots. In the absence of H. pylori 40, 45, and 15% of unsynchronized AGS cells were in G(0)-G(1), S, and G(2)-M phases, respectively, by flow cytometry analysis. When AGS cells were cultured in the presence of H. pylori, the S phase decreased 10% and the G(0)-G(1) phase increased 17% after 24 h compared with the controls. KatoIII cells, which have a deleted p53 gene, showed little or no response to H. pylori. When G1/S synchronized AGS cells were incubated with media containing H. pylori, the G(1) phase increased significantly (25%, P < 0.05) compared with controls after 24 h. In contrast, the control cells were able to pass through S phase. The inhibitory effects of H. pylori on the cell cycle of AGS cells were associated with a significant increase in p53 and p21 expression after 24 h. The expression of cyclin E was downregulated in AGS cells following exposure of AGS cells to H. pylori for 24 h. This study shows that H. pylori-induced growth inhibition in vitro is predominantly at the G(0)-G(1) checkpoint. Our results suggest that p53 may be important in H. pylori-induced cell cycle arrest. These results support a role for cyclin-dependent kinase inhibitors in the G(1) cell cycle arrest exerted by H. pylori and its involvement in changing the regulatory proteins, p53, p21, and cyclin E in the cell cycle.     

Upregulation of progranulin by Helicobacter pylori in human gastric epithelial cells via p38MAPK and MEK1/2 signaling pathway: role in epithelial cell proliferation and migration

Helicobacter pylori is a major human pathogen associated with gastric diseases such as chronic active gastritis, peptic ulcer, and gastric carcinoma. The growth factor progranulin (PGRN) is a secreted glycoprotein that functions as an important regulator of cell growth, migration, and transformation. We aimed to determine the molecular mechanisms by which H. pylori upregulates the expression of PGRN and the relationship between H. pylori infection and production of PGRN in controlling cell proliferation and migration. Levels of PGRN were examined in gastric tissues from patients and in vitro in gastric epithelial cells. Cell proliferation was measured by colony formation assay. Cell migration was monitored by wound healing migration assay. PGRN protein levels were increased in patients with gastritis and gastric cancer tissue. Infection of gastric epithelial cells with H. pylori significantly increased PGRN expression in a time-dependent manner. Blockade of the p38 and MEK1/2 pathway by inhibitor inhibited H. pylori-mediated PGRN upregulation. Activation of p38 and MEK1/2 pathway by H. pylori was also identified. Knockdown of PGRN attenuated the H. pylori-induced proliferative activity and migration of cancer cells. These findings suggest that the upregulation of PGRN in H. pylori-infected gastric epithelial cells may contribute to the carcinogenic process.     

The beta1 integrin activates JNK independent of CagA, and JNK activation is required for Helicobacter pylori CagA+-induced motility of gastric cancer cells

The Helicobacter pylori CagA protein is translocated into gastric epithelial cells through a type IV secretion system (TFSS), and published studies suggest CagA is critical for H. pylori-associated carcinogenesis. CagA is thought to be necessary and sufficient to induce the motogenic response observed in response to CagA+ strains, as CagA interacts with proteins involved in adhesion and motility. We report that H. pylori strain 60190 stimulated AGS cell motility through a CagA- and TFSS-dependent mechanism, because strains 60190DeltacagA or 60190DeltacagE (TFSS-defective) did not increase motility. The JNK pathway is critical for H. pylori-dependent cell motility, as inhibition using SP600125 (JNK1/2/3 inhibitor) or a JNK2/3-specific inhibitor blocked motility. JNK mediates H. pylori-induced cell motility by activating paxillin, because JNK inhibition blocked paxillinTyr-118 phosphorylation, and paxillin expression knockdown completely abrogated bacteria-induced motility. Furthermore, JNK and paxillinTyr-118 were activated by 60190DeltacagA but not 60190DeltacagE, demonstrating CagA-independent signaling critical for cell motility. A beta1 integrin-blocking antibody significantly inhibited JNK and paxillinTyr-118 phosphorylation and cell scattering, demonstrating that CagA-independent signaling required for cell motility occurs through beta1. The requirement of both Src and focal adhesion kinase for signaling and motility further suggests the importance of integrin signaling in H. pylori-induced cell motility. Finally, we show that JNK activation occurs independent of known upstream kinases and signaling molecules, including Nod1, Cdc42, Rac1, MKK4, and MKK7, which demonstrates novel signaling leading to JNK activation. We report for the first time that H. pylori mediates CagA-independent signaling that promotes cell motility through the beta1 integrin pathway.     

Differential phosphoproteome profiling reveals a functional role for VASP in Helicobacter pylori-induced cytoskeleton turnover in gastric epithelial cells

Infection with Helicobacter pylori induces various gastric diseases, including ulceration, gastritis and neoplasia. As H. pylori-induced cellular mechanisms leading to these disease states are widely unclear, we analysed the phosphoproteome of H. pylori-infected gastric epithelial cells. Phosphoproteins from infected cells were enriched using affinity columns and analysed by two-dimensional gel electrophoresis and mass spectrometry. Eleven novel phosphoproteins that showed differentially regulated phosphorylation levels during H. pylori infection were identified. Interestingly, the identified proteins were actin-binding, transport and folding, RNA/DNA-binding or cancer-associated proteins. We analysed functions of one identified H. pylori-regulated candidate, the vasodilator-stimulated phosphoprotein (VASP). H. pylori induced VASP phosphorylation at residues Ser157, Ser239 and Thr278, which was enhanced by the bacterial oncogene cytotoxin-associated gene A. Overexpression of a phosphorylation-resistant VASP mutant efficiently blocked host cell elongation. We identified cGMP-dependent protein kinase G-mediated Ser239 and Thr278 phosphorylation of VASP as a crucial event in H. pylori-dependent host cell elongation. These results suggest that phosphorylated VASP could be a novel target candidate for therapeutic intervention in H. pylori-related gastric diseases.