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.     

Expression of cyclooxygenase-2 and nitrotyrosine in human gastric mucosa before and after Helicobacter pylori eradication

To determine whether cure of Helicobacter pylori infection influences the expression of COX-2 and nitrotyrosine in the distal stomach of humans, biopsy specimens were examined immunohistochemically. H. pylori infection was determined using a rapid urease test, culture and histology. Positive staining of COX-2/nitrotyrosine in the epithelium was expressed as the percentage of stained cells to the total epithelial cells.There was a significant increase in COX-2/nitrotyrosine staining in H. pylori -positive subjects compared with H. pylori -negative subjects. Cure of the infection resulted in a significant decrease in both COX-2/nitrotyrosine staining in all patients (52.1+/-12.1% vs 15. 4+/-7.2%, P<0.001; and 57.3+/-13.6% vs 36.1+/-18.0%, P<0.01, respectively). However, immunoreactivity of COX-2/nitrotyrosine was observed in all cases with intestinal metaplasia even after the cure of H. pylori infection.Thus, cure of H. pylori infection may decrease the risk of gastric carcinogenesis due to COX-2 and NO-related compounds in gastric mucosa but not in those patients with intestinal metaplasia.     

The effect of ascorbic acid on Helicobacter pylori induced cyclooxygenase 2 expression and prostaglandin E2 production by gastric epithelial cells in vitro

BACKGROUND: Cyclooxygenase 2 (COX-2) is induced by the presence of Helicobacter pylori (H. pylori) on the gastric mucosa as part of the inflammatory response; this results in the synthesis of prostaglandins that amplify the local inflammatory response. The presence of H. pylori inhibits the secretion of ascorbate into the gastric lumen. Interestingly, ascorbate inhibits the growth of H. pylori and low dietary levels are associated with an increased risk of gastric adenocarcinoma. We therefore investigated the effect of ascorbate on H. pylori mediated COX-2 induction and prostaglandin production in vitro. METHODS: H. pylori was cocultured with gastric epithelial cells in the presence of ascorbate at physiological concentrations. The expression of COX-2 was assessed by Western blotting and prostaglandin E(2) (PGE(2)) was assessed by ELISA. RESULTS: Ascorbate inhibited gastric cell PGE(2) synthesis but not in COX-2 expression in response to H. pylori. In the absence of the organism, ascorbate also reduced PGE(2) expression in cells that constitutively express COX-2, again with no reduction of COX-2 protein expression. CONCLUSIONS: Physiological concentrations of ascorbate inhibit PGE(2) but not COX-2 expression in response to H. pylori in gastric epithelial cells.     

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.