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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.

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Atg16L1, ROS and ERK regulate Cx43 in the presence of VacA. (a) Control (NC) or Atg16L1 siRNA-transfected cells were 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, LC3-II generation and Atg16L1 in AZ-521 cells was performed by densitometry (right panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05, **P<0.01. Experiments were repeated three times with similar results. (b) The indicated siRNA-transfected cells were treated with toxin as shown above. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced cPARP, Atg16L1 and GAPDH in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from two experiments. Experiments were repeated two times with similar results. (c and d) AZ-521 cells were pretreated with or without 10 mM NAC (left panel) or 10 mM GSH (right panel) 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 and LC3-II generation in AZ-521 cells was performed by densitometry. Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05 and **P<0.02. Experiments were repeated three times with similar results. (e) AZ-521 cells were pretreated with control (DMSO) or 10 mM NAC and then 120 nM heat-inactivated (iV) or wild-type VacA (V) for the indicated times. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Experiments were repeated three times with similar results. (f) AZ-521 cells were pretreated with 10 μM U0126 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 and LC3-II generation in AZ-521 cells was performed by densitometry (right 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. (g) Control (NC) or ERK 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 for immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 and LC3-II generation and ERK levels in AZ-521 cells was performed by densitometry (right panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05, **P<0.01. Experiments were repeated three times with similar results. (h) The indicated siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 4 h and total RNA was extracted as described in Materials and Methods. Cx43 mRNA was measured by real-time qPCR. Data are shown as mean±S.D. of values from two experiments. Results are shown as fold increase of GAPDH as an internal control. Experiments were repeated two times with similar results. (i) The indicated siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and then reacted with anti-Cx43 antibodies (red) and incubated with DAPI (cyan). Bars represent 20 μm. Experiments were repeated three times with similar results. (j) The indicated siRNA-transfected AZ-521 cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and lysed with 1× SDS sample buffer for immunoblotting with the indicated antibodies. Quantification of VacA-induced cPARP and cCas9 was performed by densitometry (right 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.
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fig4: Atg16L1, ROS and ERK regulate Cx43 in the presence of VacA. (a) Control (NC) or Atg16L1 siRNA-transfected cells were 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, LC3-II generation and Atg16L1 in AZ-521 cells was performed by densitometry (right panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05, **P<0.01. Experiments were repeated three times with similar results. (b) The indicated siRNA-transfected cells were treated with toxin as shown above. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced cPARP, Atg16L1 and GAPDH in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from two experiments. Experiments were repeated two times with similar results. (c and d) AZ-521 cells were pretreated with or without 10 mM NAC (left panel) or 10 mM GSH (right panel) 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 and LC3-II generation in AZ-521 cells was performed by densitometry. Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05 and **P<0.02. Experiments were repeated three times with similar results. (e) AZ-521 cells were pretreated with control (DMSO) or 10 mM NAC and then 120 nM heat-inactivated (iV) or wild-type VacA (V) for the indicated times. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Experiments were repeated three times with similar results. (f) AZ-521 cells were pretreated with 10 μM U0126 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 and LC3-II generation in AZ-521 cells was performed by densitometry (right 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. (g) Control (NC) or ERK 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 for immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 and LC3-II generation and ERK levels in AZ-521 cells was performed by densitometry (right panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05, **P<0.01. Experiments were repeated three times with similar results. (h) The indicated siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 4 h and total RNA was extracted as described in Materials and Methods. Cx43 mRNA was measured by real-time qPCR. Data are shown as mean±S.D. of values from two experiments. Results are shown as fold increase of GAPDH as an internal control. Experiments were repeated two times with similar results. (i) The indicated siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and then reacted with anti-Cx43 antibodies (red) and incubated with DAPI (cyan). Bars represent 20 μm. Experiments were repeated three times with similar results. (j) The indicated siRNA-transfected AZ-521 cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and lysed with 1× SDS sample buffer for immunoblotting with the indicated antibodies. Quantification of VacA-induced cPARP and cCas9 was performed by densitometry (right 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.

Mentions: We next examined whether autophagy regulates VacA-dependent increase in Cx43. VacA induced LC3-II generation independent of the increase in Cx43 (Figures 3b and c). Silencing of the Atg16L1 gene with Atg16L1 siRNA resulted in suppression of both Cx43 increase and LC3-II generation in VacA-treated cells as compared with control siRNA-transfected cells (Figure 4a). Thus, our results indicate that Atg16L1, which has an essential role in autophagy,40 participated in VacA-induced Cx43 increase and LC3-II generation. In addition, depletion of Atg16L1 significantly suppressed VacA-induced PARP cleavage (Figure 4b).


Helicobacter pylori VacA induces apoptosis by accumulation of connexin 43 in autophagic vesicles via a Rac1/ERK-dependent pathway
Atg16L1, ROS and ERK regulate Cx43 in the presence of VacA. (a) Control (NC) or Atg16L1 siRNA-transfected cells were 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, LC3-II generation and Atg16L1 in AZ-521 cells was performed by densitometry (right panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05, **P<0.01. Experiments were repeated three times with similar results. (b) The indicated siRNA-transfected cells were treated with toxin as shown above. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced cPARP, Atg16L1 and GAPDH in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from two experiments. Experiments were repeated two times with similar results. (c and d) AZ-521 cells were pretreated with or without 10 mM NAC (left panel) or 10 mM GSH (right panel) 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 and LC3-II generation in AZ-521 cells was performed by densitometry. Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05 and **P<0.02. Experiments were repeated three times with similar results. (e) AZ-521 cells were pretreated with control (DMSO) or 10 mM NAC and then 120 nM heat-inactivated (iV) or wild-type VacA (V) for the indicated times. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Experiments were repeated three times with similar results. (f) AZ-521 cells were pretreated with 10 μM U0126 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 and LC3-II generation in AZ-521 cells was performed by densitometry (right 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. (g) Control (NC) or ERK 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 for immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 and LC3-II generation and ERK levels in AZ-521 cells was performed by densitometry (right panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05, **P<0.01. Experiments were repeated three times with similar results. (h) The indicated siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 4 h and total RNA was extracted as described in Materials and Methods. Cx43 mRNA was measured by real-time qPCR. Data are shown as mean±S.D. of values from two experiments. Results are shown as fold increase of GAPDH as an internal control. Experiments were repeated two times with similar results. (i) The indicated siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and then reacted with anti-Cx43 antibodies (red) and incubated with DAPI (cyan). Bars represent 20 μm. Experiments were repeated three times with similar results. (j) The indicated siRNA-transfected AZ-521 cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and lysed with 1× SDS sample buffer for immunoblotting with the indicated antibodies. Quantification of VacA-induced cPARP and cCas9 was performed by densitometry (right 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.
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fig4: Atg16L1, ROS and ERK regulate Cx43 in the presence of VacA. (a) Control (NC) or Atg16L1 siRNA-transfected cells were 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, LC3-II generation and Atg16L1 in AZ-521 cells was performed by densitometry (right panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05, **P<0.01. Experiments were repeated three times with similar results. (b) The indicated siRNA-transfected cells were treated with toxin as shown above. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Quantification of VacA-induced cPARP, Atg16L1 and GAPDH in AZ-521 cells was performed by densitometry (bottom panel). Data are presented as mean±S.D. of values from two experiments. Experiments were repeated two times with similar results. (c and d) AZ-521 cells were pretreated with or without 10 mM NAC (left panel) or 10 mM GSH (right panel) 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 and LC3-II generation in AZ-521 cells was performed by densitometry. Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05 and **P<0.02. Experiments were repeated three times with similar results. (e) AZ-521 cells were pretreated with control (DMSO) or 10 mM NAC and then 120 nM heat-inactivated (iV) or wild-type VacA (V) for the indicated times. Cells were lysed with 1× SDS sample buffer and analyzed by immunoblotting with the indicated antibodies. Experiments were repeated three times with similar results. (f) AZ-521 cells were pretreated with 10 μM U0126 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 and LC3-II generation in AZ-521 cells was performed by densitometry (right 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. (g) Control (NC) or ERK 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 for immunoblotting with the indicated antibodies. Quantification of VacA-induced Cx43 and LC3-II generation and ERK levels in AZ-521 cells was performed by densitometry (right panel). Data are presented as mean±S.D. of values from three experiments and significance is *P<0.05, **P<0.01. Experiments were repeated three times with similar results. (h) The indicated siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 4 h and total RNA was extracted as described in Materials and Methods. Cx43 mRNA was measured by real-time qPCR. Data are shown as mean±S.D. of values from two experiments. Results are shown as fold increase of GAPDH as an internal control. Experiments were repeated two times with similar results. (i) The indicated siRNA-transfected cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and then reacted with anti-Cx43 antibodies (red) and incubated with DAPI (cyan). Bars represent 20 μm. Experiments were repeated three times with similar results. (j) The indicated siRNA-transfected AZ-521 cells were incubated with 120 nM heat-inactivated (iV) or wild-type VacA (V) for 10 h and lysed with 1× SDS sample buffer for immunoblotting with the indicated antibodies. Quantification of VacA-induced cPARP and cCas9 was performed by densitometry (right 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.
Mentions: We next examined whether autophagy regulates VacA-dependent increase in Cx43. VacA induced LC3-II generation independent of the increase in Cx43 (Figures 3b and c). Silencing of the Atg16L1 gene with Atg16L1 siRNA resulted in suppression of both Cx43 increase and LC3-II generation in VacA-treated cells as compared with control siRNA-transfected cells (Figure 4a). Thus, our results indicate that Atg16L1, which has an essential role in autophagy,40 participated in VacA-induced Cx43 increase and LC3-II generation. In addition, depletion of Atg16L1 significantly suppressed VacA-induced PARP cleavage (Figure 4b).

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