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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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Rac1 involvement in VacA-increased Cx43. (a) AZ-521 cells were pretreated with 50 μM Rac1 inhibitor, NSC27366, for 30 min and then incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05. Experiments were repeated three times with similar results. (b) Control (NC) or Rac1 siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from four experiments and significance is *P<0.01. (c) Rac1 inhibitor-treated cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 1 h and lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced phospho-ERK (P-ERK) and ERK in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.01. (d) The indicated siRNA transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 1 h and lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced phospho-ERK (P-ERK) and ERK in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.01. (e) AZ-521 cells were incubated with 120 nM wild-type VacA for 0, 5, 15, 30 min. After pull-down (PD) with PAK-PBD beads, active Rac1 was determined by immunoblotting with anti-Rac1 antibodies. The amounts of Rac1 and GAPDH in total cell lysate (TCL) were determined by immunoblotting with anti-Rac1 and anti-GAPDH antibodies. Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05.
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fig5: Rac1 involvement in VacA-increased Cx43. (a) AZ-521 cells were pretreated with 50 μM Rac1 inhibitor, NSC27366, for 30 min and then incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05. Experiments were repeated three times with similar results. (b) Control (NC) or Rac1 siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from four experiments and significance is *P<0.01. (c) Rac1 inhibitor-treated cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 1 h and lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced phospho-ERK (P-ERK) and ERK in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.01. (d) The indicated siRNA transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 1 h and lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced phospho-ERK (P-ERK) and ERK in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.01. (e) AZ-521 cells were incubated with 120 nM wild-type VacA for 0, 5, 15, 30 min. After pull-down (PD) with PAK-PBD beads, active Rac1 was determined by immunoblotting with anti-Rac1 antibodies. The amounts of Rac1 and GAPDH in total cell lysate (TCL) were determined by immunoblotting with anti-Rac1 and anti-GAPDH antibodies. Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05.

Mentions: Hotchin et al.46 showed that VacA activity is regulated by Rac1, a small GTP-binding protein that controls actin cytoskeleton reorganization and signal transduction. Furthermore, cytoskeletal reorganization is regulated by ROS, important mediators acting downstream of Rac1.47 We investigated here whether Rac1 is involved in the VacA-induced Cx43 increase. AZ-521 cells treated with the Rac1 inhibitor, NSC23766, in the presence of VacA, showed a significant decrease in the levels of Cx43 compared with treatment with VacA alone (Figure 5a). Furthermore, Rac1 knockdown inhibited VacA-induced Cx43 increase (Figure 5b). We further examined the effect of Rac1 on VacA-induced ERK phosphorylation. In the presence of the Rac1 inhibitor or following Rac1 knockdown, VacA-induced ERK phosphorylation was suppressed compared with treatment with VacA alone (Figures 5c and d). We next investigated whether VacA induced Rac1 activation. AZ-521 cells were incubated with VacA in the presence or absence of NAC. In control cells, VacA enhanced formation of Rac1-GTP in a time-dependent manner, which was suppressed in the presence of NAC (Figure 5e).


Helicobacter pylori VacA induces apoptosis by accumulation of connexin 43 in autophagic vesicles via a Rac1/ERK-dependent pathway
Rac1 involvement in VacA-increased Cx43. (a) AZ-521 cells were pretreated with 50 μM Rac1 inhibitor, NSC27366, for 30 min and then incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05. Experiments were repeated three times with similar results. (b) Control (NC) or Rac1 siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from four experiments and significance is *P<0.01. (c) Rac1 inhibitor-treated cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 1 h and lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced phospho-ERK (P-ERK) and ERK in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.01. (d) The indicated siRNA transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 1 h and lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced phospho-ERK (P-ERK) and ERK in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.01. (e) AZ-521 cells were incubated with 120 nM wild-type VacA for 0, 5, 15, 30 min. After pull-down (PD) with PAK-PBD beads, active Rac1 was determined by immunoblotting with anti-Rac1 antibodies. The amounts of Rac1 and GAPDH in total cell lysate (TCL) were determined by immunoblotting with anti-Rac1 and anti-GAPDH antibodies. Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05.
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fig5: Rac1 involvement in VacA-increased Cx43. (a) AZ-521 cells were pretreated with 50 μM Rac1 inhibitor, NSC27366, for 30 min and then incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05. Experiments were repeated three times with similar results. (b) Control (NC) or Rac1 siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from four experiments and significance is *P<0.01. (c) Rac1 inhibitor-treated cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 1 h and lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced phospho-ERK (P-ERK) and ERK in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.01. (d) The indicated siRNA transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 1 h and lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced phospho-ERK (P-ERK) and ERK in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.01. (e) AZ-521 cells were incubated with 120 nM wild-type VacA for 0, 5, 15, 30 min. After pull-down (PD) with PAK-PBD beads, active Rac1 was determined by immunoblotting with anti-Rac1 antibodies. The amounts of Rac1 and GAPDH in total cell lysate (TCL) were determined by immunoblotting with anti-Rac1 and anti-GAPDH antibodies. Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05.
Mentions: Hotchin et al.46 showed that VacA activity is regulated by Rac1, a small GTP-binding protein that controls actin cytoskeleton reorganization and signal transduction. Furthermore, cytoskeletal reorganization is regulated by ROS, important mediators acting downstream of Rac1.47 We investigated here whether Rac1 is involved in the VacA-induced Cx43 increase. AZ-521 cells treated with the Rac1 inhibitor, NSC23766, in the presence of VacA, showed a significant decrease in the levels of Cx43 compared with treatment with VacA alone (Figure 5a). Furthermore, Rac1 knockdown inhibited VacA-induced Cx43 increase (Figure 5b). We further examined the effect of Rac1 on VacA-induced ERK phosphorylation. In the presence of the Rac1 inhibitor or following Rac1 knockdown, VacA-induced ERK phosphorylation was suppressed compared with treatment with VacA alone (Figures 5c and d). We next investigated whether VacA induced Rac1 activation. AZ-521 cells were incubated with VacA in the presence or absence of NAC. In control cells, VacA enhanced formation of Rac1-GTP in a time-dependent manner, which was suppressed in the presence of NAC (Figure 5e).

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