Abstract
ABSTRACTHelicobacter pylori, a major cause of gastroduodenal diseases, produces vacuolating cytotoxin (VacA) and cytotoxin-associated gene A (CagA), which seem to be involved in virulence. VacA exhibits pleiotropic actions in gastroduodenal disorders via its specific receptors. Recently, we found that VacA induced the phosphorylation of cellular Src kinase (Src) at Tyr418 in AZ-521 cells. Silencing of receptor protein tyrosine phosphatase (RPTP)α, a VacA receptor, reduced VacA-induced Src phosphorylation. Src is responsible for tyrosine phosphorylation of CagA at its Glu-Pro-Ile-Tyr-Ala (EPIYA) variant C (EPIYA-C) motif in Helicobacter pylori-infected gastric epithelial cells, resulting in binding of CagA to SHP-2 phosphatase. Challenging AZ-521 cells with wild-type H. pylori induced phosphorylation of CagA, but this did not occur when challenged with a vacA gene-disrupted mutant strain. CagA phosphorylation was observed in cells infected with a vacA gene-disrupted mutant strain after addition of purified VacA, suggesting that VacA is required for H. pylori-induced CagA phosphorylation. Following siRNA-mediated RPTPα knockdown in AZ-521 cells, infection with wild-type H. pylori and treatment with VacA did not induce CagA phosphorylation. Taken together, these results support our conclusion that VacA mediates CagA phosphorylation through RPTPα in AZ-521 cells. These data indicate the possibility that Src phosphorylation induced by VacA is mediated through RPTPα, resulting in activation of Src, leading to CagA phosphorylation at Tyr972 in AZ-521 cells.
Highlights
Helicobacter pylori is a major causative agent for the development of gastroduodenal diseases, including chronic gastritis, peptic ulcer and gastric cancers (Blaser and Atherton, 2004; Peek and Blaser, 2002)
We found that vacuolating cytotoxin (VacA) did not enhance phosphorylation at Tyr418 in Src in RPTPα constitutive-knockdown AZ-521 cells (Fig. 3), consistent with the results using the small-interfering RNA (siRNA)-mediated RPTPα knockdown AZ-521 cells (Fig. 2A)
When we examined the effect of β1-integrin gene silencing on VacA-induced cytotoxin-associated gene A (CagA) phosphorylation in cells infected with wild-type H. pylori, we found that silencing of the β1-integrin gene mediated by siRNA did not result in differences of VacA-induced CagA phosphorylation compared to control siRNA-transfected cells (Fig. 6A), suggesting that RPTPα is responsible for CagA phosphorylation induced by VacA in AZ-521 cells
Summary
Helicobacter pylori is a major causative agent for the development of gastroduodenal diseases, including chronic gastritis, peptic ulcer and gastric cancers (Blaser and Atherton, 2004; Peek and Blaser, 2002). VacA is a potent cytotoxin secreted by most clinical isolates of H. pylori, and shows pleiotropic actions in cultured gastric epithelial cells, including generation of vacuoles in the cytoplasm, mitochondrial damage leading to apoptosis, and modulation of signal transduction pathways associated with immune responses (Boncristiano et al, 2003; Hisatsune et al, 2007; Isomoto et al, 2010; Nakayama et al, 2004, 2009; Yamasaki et al, 2006) To facilitate their biological actions in host cells, VacA binds to specific surface receptors. Other molecules, including sphingomyelin, have been reported to serve as VacA receptors (Gupta et al, 2010) Of these VacA receptors, during H. pylori infection, RPTPβ is associated with the development of gastric ulcers in experimental animal models and LRP1 is involved in VacA-dependent autophagy, followed by CagA degradation in infected host cells (Fujikawa et al, 2003; Tsugawa et al, 2012; Yahiro et al, 2012). The role of RPTPα in intoxication with VacA is unclear
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