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Helicobacter pylori protein JHP0290 exhibits proliferative and anti-apoptotic effects in gastric epithelial cells.

Tavares R, Pathak SK - PLoS ONE (2015)

Bottom Line: JHP0290 existed in monomeric and dimeric forms in H. pylori cell extracts and culture broth.Recombinant purified JHP0290 (rJHP0290) also showed monomeric and dimeric forms, whereas the rJHP0290 C162A mutant exhibited only a monomeric form.CPT-induced caspase 3 activation was significantly reduced in the presence of rJHP0290.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

ABSTRACT
The influence of Helicobacter pylori infection on gastric epithelial cell proliferation, apoptosis and signaling pathways contributes to the development of infection-associated diseases. Here we report that JHP0290, which is a poorly functionally characterized protein from H. pylori, regulates multiple responses in human gastric epithelial cells. The differential expression and release of JHP0290 homologues was observed among H. pylori strains. JHP0290 existed in monomeric and dimeric forms in H. pylori cell extracts and culture broth. Recombinant purified JHP0290 (rJHP0290) also showed monomeric and dimeric forms, whereas the rJHP0290 C162A mutant exhibited only a monomeric form. The dimeric form of the protein was found to bind more efficiently to gastric epithelial cells than the monomeric form. The exposure of gastric epithelial cells to rJHP0290 induced proliferation in a dose-dependent manner. Faster progression into the cell cycle was observed in rJHP0290-challenged gastric epithelial cells. Furthermore, we detected an anti-apoptotic effect of rJHP0290 in gastric epithelial cells when the cells were treated with rJHP0290 in combination with Camptothecin (CPT), which is an inducer of apoptosis. CPT-induced caspase 3 activation was significantly reduced in the presence of rJHP0290. In addition, the activation of ERK MAPK and the transcription factor NFκB was observed in rJHP0290-challenged gastric epithelial cells lines. Our results suggest that JHP0290 may affect H. pylori-induced gastric diseases via the regulation of gastric epithelial cell proliferation and anti-apoptotic pathways.

No MeSH data available.


Related in: MedlinePlus

Anti-apoptotic effect of rJHP0290 on epithelial cells.(A) AGS cells were incubated either with protein storage buffer (Ctrl) or rJHP0290 (100 ng/ml) for 2 h followed by addition of corresponding volume of vehicle control or Camptothecin (CPT, 10 μM) for 20 h. Cells were washed, stained with FITC-conjugated Annexin V antibody and propidium iodide (PI). Apoptotic cells were analyzed by flow cytometry. Result shown is the representative of results obtained in three independent experiments. (B) AGS cells were pre-treated with rJHP0290 (100 ng/ml) followed by addition of CPT (10 μM). Another set of cells were simultaneous treated with rJHP0290 (100 ng /ml) and CPT (10 μM). Percentage of apoptotic cells was determined. Values indicate mean ± SD of three independent experiments. (C) AGS, MKN45 and PHSEC cells were incubated either with protein storage buffer (Ctrl) or rJHP0290 (100 ng/ml) for 2 h followed by addition of corresponding volume of vehicle control or Camptothecin (CPT, 10 μM) for 14 h. Caspase 3 activity in the samples was determined as described in material and methods. Values indicate mean ± SD of three independent experiments. Statistically significant differences are indicated by * (p < 0.05).
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pone.0124407.g005: Anti-apoptotic effect of rJHP0290 on epithelial cells.(A) AGS cells were incubated either with protein storage buffer (Ctrl) or rJHP0290 (100 ng/ml) for 2 h followed by addition of corresponding volume of vehicle control or Camptothecin (CPT, 10 μM) for 20 h. Cells were washed, stained with FITC-conjugated Annexin V antibody and propidium iodide (PI). Apoptotic cells were analyzed by flow cytometry. Result shown is the representative of results obtained in three independent experiments. (B) AGS cells were pre-treated with rJHP0290 (100 ng/ml) followed by addition of CPT (10 μM). Another set of cells were simultaneous treated with rJHP0290 (100 ng /ml) and CPT (10 μM). Percentage of apoptotic cells was determined. Values indicate mean ± SD of three independent experiments. (C) AGS, MKN45 and PHSEC cells were incubated either with protein storage buffer (Ctrl) or rJHP0290 (100 ng/ml) for 2 h followed by addition of corresponding volume of vehicle control or Camptothecin (CPT, 10 μM) for 14 h. Caspase 3 activity in the samples was determined as described in material and methods. Values indicate mean ± SD of three independent experiments. Statistically significant differences are indicated by * (p < 0.05).

Mentions: The H. pylori-dependent regulation of apoptosis and proliferation in gastric epithelial cells has a major impact on ultimate disease outcome. H. pylori proteins, such as CagA and SlyD, have been shown to promote proliferation and exert an anti-apoptotic effect on gastric epithelial cells [15, 44]. Considering the induction of proliferation by rJHP0290, we further explored the rJHP0290-dependent regulation of apoptosis in AGS cells. CPT was used to induce apoptosis in AGS cells, and the induction of apoptosis was determined using FACS after staining with Annexin V and PI. A representative FACS analysis is shown in Fig 5A. At a concentration of 10 μM CPT, 26±3.5% of the cells were in early apoptosis (i.e., Annexin+ and PI-). Pretreatment of AGS cells with rJHP0290 for 2 h significantly reduced CPT-induced apoptosis (Fig 5B). However, the anti-apoptotic effect of rJHP0290 was markedly reduced when rJHP0290 and CPT were added to cells simultaneously (Fig 5B). rJHP0290 alone had no effect on AGS cell apoptosis at concentrations up to 10 μg/ml (data not shown).


Helicobacter pylori protein JHP0290 exhibits proliferative and anti-apoptotic effects in gastric epithelial cells.

Tavares R, Pathak SK - PLoS ONE (2015)

Anti-apoptotic effect of rJHP0290 on epithelial cells.(A) AGS cells were incubated either with protein storage buffer (Ctrl) or rJHP0290 (100 ng/ml) for 2 h followed by addition of corresponding volume of vehicle control or Camptothecin (CPT, 10 μM) for 20 h. Cells were washed, stained with FITC-conjugated Annexin V antibody and propidium iodide (PI). Apoptotic cells were analyzed by flow cytometry. Result shown is the representative of results obtained in three independent experiments. (B) AGS cells were pre-treated with rJHP0290 (100 ng/ml) followed by addition of CPT (10 μM). Another set of cells were simultaneous treated with rJHP0290 (100 ng /ml) and CPT (10 μM). Percentage of apoptotic cells was determined. Values indicate mean ± SD of three independent experiments. (C) AGS, MKN45 and PHSEC cells were incubated either with protein storage buffer (Ctrl) or rJHP0290 (100 ng/ml) for 2 h followed by addition of corresponding volume of vehicle control or Camptothecin (CPT, 10 μM) for 14 h. Caspase 3 activity in the samples was determined as described in material and methods. Values indicate mean ± SD of three independent experiments. Statistically significant differences are indicated by * (p < 0.05).
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pone.0124407.g005: Anti-apoptotic effect of rJHP0290 on epithelial cells.(A) AGS cells were incubated either with protein storage buffer (Ctrl) or rJHP0290 (100 ng/ml) for 2 h followed by addition of corresponding volume of vehicle control or Camptothecin (CPT, 10 μM) for 20 h. Cells were washed, stained with FITC-conjugated Annexin V antibody and propidium iodide (PI). Apoptotic cells were analyzed by flow cytometry. Result shown is the representative of results obtained in three independent experiments. (B) AGS cells were pre-treated with rJHP0290 (100 ng/ml) followed by addition of CPT (10 μM). Another set of cells were simultaneous treated with rJHP0290 (100 ng /ml) and CPT (10 μM). Percentage of apoptotic cells was determined. Values indicate mean ± SD of three independent experiments. (C) AGS, MKN45 and PHSEC cells were incubated either with protein storage buffer (Ctrl) or rJHP0290 (100 ng/ml) for 2 h followed by addition of corresponding volume of vehicle control or Camptothecin (CPT, 10 μM) for 14 h. Caspase 3 activity in the samples was determined as described in material and methods. Values indicate mean ± SD of three independent experiments. Statistically significant differences are indicated by * (p < 0.05).
Mentions: The H. pylori-dependent regulation of apoptosis and proliferation in gastric epithelial cells has a major impact on ultimate disease outcome. H. pylori proteins, such as CagA and SlyD, have been shown to promote proliferation and exert an anti-apoptotic effect on gastric epithelial cells [15, 44]. Considering the induction of proliferation by rJHP0290, we further explored the rJHP0290-dependent regulation of apoptosis in AGS cells. CPT was used to induce apoptosis in AGS cells, and the induction of apoptosis was determined using FACS after staining with Annexin V and PI. A representative FACS analysis is shown in Fig 5A. At a concentration of 10 μM CPT, 26±3.5% of the cells were in early apoptosis (i.e., Annexin+ and PI-). Pretreatment of AGS cells with rJHP0290 for 2 h significantly reduced CPT-induced apoptosis (Fig 5B). However, the anti-apoptotic effect of rJHP0290 was markedly reduced when rJHP0290 and CPT were added to cells simultaneously (Fig 5B). rJHP0290 alone had no effect on AGS cell apoptosis at concentrations up to 10 μg/ml (data not shown).

Bottom Line: JHP0290 existed in monomeric and dimeric forms in H. pylori cell extracts and culture broth.Recombinant purified JHP0290 (rJHP0290) also showed monomeric and dimeric forms, whereas the rJHP0290 C162A mutant exhibited only a monomeric form.CPT-induced caspase 3 activation was significantly reduced in the presence of rJHP0290.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, Stockholm, Sweden.

ABSTRACT
The influence of Helicobacter pylori infection on gastric epithelial cell proliferation, apoptosis and signaling pathways contributes to the development of infection-associated diseases. Here we report that JHP0290, which is a poorly functionally characterized protein from H. pylori, regulates multiple responses in human gastric epithelial cells. The differential expression and release of JHP0290 homologues was observed among H. pylori strains. JHP0290 existed in monomeric and dimeric forms in H. pylori cell extracts and culture broth. Recombinant purified JHP0290 (rJHP0290) also showed monomeric and dimeric forms, whereas the rJHP0290 C162A mutant exhibited only a monomeric form. The dimeric form of the protein was found to bind more efficiently to gastric epithelial cells than the monomeric form. The exposure of gastric epithelial cells to rJHP0290 induced proliferation in a dose-dependent manner. Faster progression into the cell cycle was observed in rJHP0290-challenged gastric epithelial cells. Furthermore, we detected an anti-apoptotic effect of rJHP0290 in gastric epithelial cells when the cells were treated with rJHP0290 in combination with Camptothecin (CPT), which is an inducer of apoptosis. CPT-induced caspase 3 activation was significantly reduced in the presence of rJHP0290. In addition, the activation of ERK MAPK and the transcription factor NFκB was observed in rJHP0290-challenged gastric epithelial cells lines. Our results suggest that JHP0290 may affect H. pylori-induced gastric diseases via the regulation of gastric epithelial cell proliferation and anti-apoptotic pathways.

No MeSH data available.


Related in: MedlinePlus