Helicobacter pylori VacA Activates the p38/Activating Transcription Factor 2-mediated Signal Pathway in AZ-521 Cells

Abstract

Persistent Helicobacter pylori colonization in the stomach induces gastritis and peptic ulcer and interferes with ulcer healing. Most strains of H. pylori produce a cytotoxin, VacA, that induces cytoplasmic vacuolation in epithelial cells with structural and functional changes, leading to gastric injury. VacA is known to cause cell death by mitochondrial damage. We hypothesized that VacA might disrupt other signaling pathways; to that end, we examined the effects of VacA on MAPKs to elucidate their role in the abnormalities seen in VacAtreated cells. VacA stimulated phosphorylation of p38 and Erk1/2, but not JNK, in AZ-521 cells. Both phosphorylation and kinase activation of p38 were maximal 10 -30 min after addition of VacA and declined thereafter. Treatment with anti-VacA antibody or the p38 inhibitor SB203580 blocked p38 phosphorylation caused by VacA and inhibited VacA-induced phosphorylation of activating transcription factor 2 (ATF-2), which is implicated in transcriptional control of stress-responsive genes. These data indicate that VacA stimulates a p38/ ATF-2-mediated signal pathway. However, 10 M SB203580, which is sufficient to decrease p38 phosphorylation, did not inhibit VacA-induced cellular vacuolation, decrease in mitochondrial membrane potential, or cytochrome c release from mitochondria. These results suggest that VacA-induced activation of p38/ATF-2-mediated signal pathway is independent of cellular vacuolation, decrease in mitochondrial membrane potential, or cytochrome c release from mitochondria caused by VacA. The cytotoxin may thus act independently on several cellular targets, leading to disruption of signaling, regulatory, and metabolic pathways.Persistent Helicobacter pylori infection results in the development of chronic gastritis, peptic ulcer, and gastric cancer. Pathogenic strains of H. pylori produce and secrete a potent vacuolating cytotoxin, VacA, that is capable of directly inducing progressive vacuolation (1), mitochondrial damage (2), cytochrome c release (3), and apoptotic death of epithelial cells (4). Both epidemiological study (5) and animal experiments (6 -10) have demonstrated that VacA is a major virulence factor associated with the damage to gastric mucosa. VacA is produced as a 140-kDa precursor and cleaved at the bacterial outer membranes, generating the 87-95-kDa mature toxin (11). The mature toxin contains two major domains, the N-terminal region of 37 kDa that exhibits vacuolating activity and the C-terminal region of 58 kDa responsible for toxin binding to target cells (12)(13)(14)(15).In gastric tissues infected with H. pylori, many apoptotic cells were observed due to inflammation resulting from overproduction of cytokines induced by H. pylori (9, 16 -19). Recent work on the gastric mucosal inflammatory response to persistent H. pylori infection suggested that the p38 mitogen-activated protein kinase (MAPK) 1 signal pathway plays a significant role (20,21). Activation of p38 MAPK in response to environmental stress and inflammatory cytokines can result in ap...

Dual effects of Helicobacter pylori vacuolating cytotoxin on human eosinophil apoptosis in early and late periods of stimulation

“…In contrast, Nakayama et al reported that p38 did not participate in VacA-induced cytochrome c release and Bax activation in gastric epithelial cells [38]. However, several studies have demonstrated that p38 MAPK activates Bax to induce mitochondrial cytochrome c release and apoptosis in neuroblastoma cells [39], keratinocytes [40], and lens epithelial cells [41].…”

Essential Domain of Receptor Tyrosine Phosphatase β (RPTPβ) for Interaction with Helicobacter pylori Vacuolating Cytotoxin

“…Recently, we established that RPTP␤ functions as a receptor for VacA (24,29,31,32) and that RPTP␤, expressed in gastric tissue, is responsible for gastric injury caused by VacA (9). Notably, as there was no difference in vacuole development between wild-type mice and RPTP␤-deficient mice, we concluded that RPTP␤ is not essential either for the internalization of VacA or for vacuolation in gastric epithelial cells.…”

“…Purification of VacA-VacA was purified from H. pylori ATCC49503 strain culture supernatant using an anti-VacA antibody column as described (24,29). VacA was precipitated from culture supernatant with 50% saturated ammonium sulfate.…”

“…Transfection was performed as described previously (24,29,31,32) with the following modifications. In brief, cells were seeded in 24-well culture plates (0.5 or 1.0 ϫ 10 5 cells in 1 ml of DMEM per well), incubated at 37°C for 24 h, and rinsed with phosphate-buffered saline (PBS).…”

“…Mitochondrial damage (17,18) and apoptosis (17)(18)(19)(20) appear to be vacuolation-independent effects of VacA. Other cytotoxic actions of VacA, such as inhibition of the invariant chain-dependent pathway of antigen presentation by the major histocompatibility complex class II (21), suppression of nuclear translocation of nuclear factor of activated T cells, NFAT, in Jurkat T cells (22,23), and ATF-2 activation in a gastric cell line (24) have been reported. More recently, Sundrud et al (25) reported that VacA inhibited the proliferation of primary human CD4ϩ T cells and that this inhibition was not attributable to VacA effects on NFAT activation or IL-2 expression.…”