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Helicobacter pylori VacA induces apoptosis by accumulation of connexin 43 in autophagic vesicles via a Rac1/ERK-dependent pathway

View Article: PubMed Central - PubMed

ABSTRACT

Helicobacter pylori (H. pylori) produces vacuolating cytotoxin (VacA), a potent protein toxin, which is associated with gastric inflammation and ulceration. Recent studies demonstrated that connexins (Cxs), which are responsible for intracellular communication at gap junctions (GJs) as well as cell homeostasis, participate in VacA-induced cell death. We now demonstrate in AZ-521 cells that VacA increased cytoplasmic Cx43, accompanied by LC3-II generation in a time- and dose-dependent manner without induction of Cx43 mRNA expression. Inhibition of VacA-induced Rac1 activity prevented ERK phosphorylation and the increase in Cx43. Suppression of ERK activity and addition of N-acetyl-cysteine inhibited VacA-dependent increase in Cx43 and LC3-II. DIDS, an anion-selective inhibitor, suppressed VacA-dependent increase in Cx43, suggesting that VacA channel activity was involved in this pathway. By confocal microscopy, Cx43 increased by VacA was predominately localized in cholesterol-rich, detergent-resistant membranes including GJs, and a fraction of Cx43 was incorporated in endocytotic vesicles and autophagolysosomes. Accumulation of Cx43 was also observed in gastric mucosa from H. pylori-infected patients compared with healthy controls, suggesting that the pathogen caused a similar effect in vivo. Our findings show that VacA-mediated effects on autophagy inhibits turnover of Cx43, resulting in increased levels in the cytoplasm, leading eventually to apoptotic cell death.

No MeSH data available.


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H. pylori infection is associated with increased Cx43 expression in human gut tissues. Cx43 was detected (i.e., brown staining) in H. pylori-negative (a and c) and -positive (b and d) gastric mucosa. Blue color indicated nuclear staining by hematoxylin. The black square shows low magnification views of the structures in c and d, respectively (a and b: ×20 ; c and d: ×80). The figure shows one experimental result representative of 5 H. pylori-negative and 11 of H. pylori-positive samples. Statistically significant difference between H. pylori-negative and -positive mucosa was observed (P=0.0256). Fisher’s exact test, H. pylori-positive versus -negative mucosa. Bars represent 50 μm.
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fig7: H. pylori infection is associated with increased Cx43 expression in human gut tissues. Cx43 was detected (i.e., brown staining) in H. pylori-negative (a and c) and -positive (b and d) gastric mucosa. Blue color indicated nuclear staining by hematoxylin. The black square shows low magnification views of the structures in c and d, respectively (a and b: ×20 ; c and d: ×80). The figure shows one experimental result representative of 5 H. pylori-negative and 11 of H. pylori-positive samples. Statistically significant difference between H. pylori-negative and -positive mucosa was observed (P=0.0256). Fisher’s exact test, H. pylori-positive versus -negative mucosa. Bars represent 50 μm.

Mentions: To assess the above findings in H. pylori-infected human mucosa, we investigated Cx43 protein level in the biopsy samples. The demographic characteristic of the patients is shown in Supplementary Table 1. Eleven H. pylori-positive and five H. pylori-negative biopsy samples from gastric antrum were examined. Interestingly, Cx43 content was not significantly increased in gastric mucosa of H. pylori-negative patients. In contrast, Cx43 was clearly observed in the gastric epithelium region in 8 out of 11 H. pylori-positive biopsy specimens (Figure 7), which was a statistically significant difference compared with H. pylori-negative mucosa (P=0.0256, Fisher’s exact test, H. pylori-positive versus -negative mucosa). These results suggested that Cx43 significantly accumulated in H. pylori-infected human gastric mucosa, with a potential role in the pathogenesis of human peptic diseases caused by H. pylori.


Helicobacter pylori VacA induces apoptosis by accumulation of connexin 43 in autophagic vesicles via a Rac1/ERK-dependent pathway
H. pylori infection is associated with increased Cx43 expression in human gut tissues. Cx43 was detected (i.e., brown staining) in H. pylori-negative (a and c) and -positive (b and d) gastric mucosa. Blue color indicated nuclear staining by hematoxylin. The black square shows low magnification views of the structures in c and d, respectively (a and b: ×20 ; c and d: ×80). The figure shows one experimental result representative of 5 H. pylori-negative and 11 of H. pylori-positive samples. Statistically significant difference between H. pylori-negative and -positive mucosa was observed (P=0.0256). Fisher’s exact test, H. pylori-positive versus -negative mucosa. Bars represent 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4979424&req=5

fig7: H. pylori infection is associated with increased Cx43 expression in human gut tissues. Cx43 was detected (i.e., brown staining) in H. pylori-negative (a and c) and -positive (b and d) gastric mucosa. Blue color indicated nuclear staining by hematoxylin. The black square shows low magnification views of the structures in c and d, respectively (a and b: ×20 ; c and d: ×80). The figure shows one experimental result representative of 5 H. pylori-negative and 11 of H. pylori-positive samples. Statistically significant difference between H. pylori-negative and -positive mucosa was observed (P=0.0256). Fisher’s exact test, H. pylori-positive versus -negative mucosa. Bars represent 50 μm.
Mentions: To assess the above findings in H. pylori-infected human mucosa, we investigated Cx43 protein level in the biopsy samples. The demographic characteristic of the patients is shown in Supplementary Table 1. Eleven H. pylori-positive and five H. pylori-negative biopsy samples from gastric antrum were examined. Interestingly, Cx43 content was not significantly increased in gastric mucosa of H. pylori-negative patients. In contrast, Cx43 was clearly observed in the gastric epithelium region in 8 out of 11 H. pylori-positive biopsy specimens (Figure 7), which was a statistically significant difference compared with H. pylori-negative mucosa (P=0.0256, Fisher’s exact test, H. pylori-positive versus -negative mucosa). These results suggested that Cx43 significantly accumulated in H. pylori-infected human gastric mucosa, with a potential role in the pathogenesis of human peptic diseases caused by H. pylori.

View Article: PubMed Central - PubMed

ABSTRACT

Helicobacter pylori (H. pylori) produces vacuolating cytotoxin (VacA), a potent protein toxin, which is associated with gastric inflammation and ulceration. Recent studies demonstrated that connexins (Cxs), which are responsible for intracellular communication at gap junctions (GJs) as well as cell homeostasis, participate in VacA-induced cell death. We now demonstrate in AZ-521 cells that VacA increased cytoplasmic Cx43, accompanied by LC3-II generation in a time- and dose-dependent manner without induction of Cx43 mRNA expression. Inhibition of VacA-induced Rac1 activity prevented ERK phosphorylation and the increase in Cx43. Suppression of ERK activity and addition of N-acetyl-cysteine inhibited VacA-dependent increase in Cx43 and LC3-II. DIDS, an anion-selective inhibitor, suppressed VacA-dependent increase in Cx43, suggesting that VacA channel activity was involved in this pathway. By confocal microscopy, Cx43 increased by VacA was predominately localized in cholesterol-rich, detergent-resistant membranes including GJs, and a fraction of Cx43 was incorporated in endocytotic vesicles and autophagolysosomes. Accumulation of Cx43 was also observed in gastric mucosa from H. pylori-infected patients compared with healthy controls, suggesting that the pathogen caused a similar effect in vivo. Our findings show that VacA-mediated effects on autophagy inhibits turnover of Cx43, resulting in increased levels in the cytoplasm, leading eventually to apoptotic cell death.

No MeSH data available.


Related in: MedlinePlus