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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

Effect of rJHP0290 on gastric epithelial cell proliferation.AGS (A), MKN45 (B) and PHSEC (C) cells were incubated either with protein storage buffer (Ctrl) or different concentrations of rJHP0290 as indicated in the figure legends for 48 h and 72 h, followed by MTT assay to assess proliferation. Values indicate mean ± SD of six independent experiments performed at least in triplicate. (D) AGS and MKN45 cells were treated for 48h with buffer (ctrl) or rJHP0290 Wt (100 ng/ml) or rJHP0290 C162A (100 ng/ml) or an irrelevant His-tagged protein (Irr-His, 100 ng/ml). rJHP0290 was subjected to boiling for 30 min (heat) or was treated with polymyxin B (PMB) for 1 h before treatment of cells. The relative BrdU incorporation in cells was measured. Values indicate mean ± SD of four independent experiments performed in triplicate. Statistically significant differences are indicated by * (p < 0.05).
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pone.0124407.g003: Effect of rJHP0290 on gastric epithelial cell proliferation.AGS (A), MKN45 (B) and PHSEC (C) cells were incubated either with protein storage buffer (Ctrl) or different concentrations of rJHP0290 as indicated in the figure legends for 48 h and 72 h, followed by MTT assay to assess proliferation. Values indicate mean ± SD of six independent experiments performed at least in triplicate. (D) AGS and MKN45 cells were treated for 48h with buffer (ctrl) or rJHP0290 Wt (100 ng/ml) or rJHP0290 C162A (100 ng/ml) or an irrelevant His-tagged protein (Irr-His, 100 ng/ml). rJHP0290 was subjected to boiling for 30 min (heat) or was treated with polymyxin B (PMB) for 1 h before treatment of cells. The relative BrdU incorporation in cells was measured. Values indicate mean ± SD of four independent experiments performed in triplicate. Statistically significant differences are indicated by * (p < 0.05).

Mentions: H. pylori infection is associated with the enhanced proliferation of gastric epithelial cells, and several virulence factors that regulate proliferation have been identified [15, 23, 29]. Considering the ability of rJHP0290 to bind to epithelial cells, we further studied the effect of rJHP0290 on gastric epithelial cell proliferation. AGS, MKN45 and PHSEC cells were treated with various concentrations of rJHP0290 for various periods of time. An MTT assay revealed that rJHP0290 could induce gastric epithelial cell proliferation in a dose-dependent manner (Fig 3A–3C). A BrdU cell proliferation assay, which is an additional method for assessing cell proliferation, further confirmed the proliferation-promoting property of rJHP0290 (Fig 3D). To rule out the possibility that the His-Tag contributes to the observed effect, an irrelevant His-tagged protein (Irr-His) was used to treat both cell lines under similar conditions. Irr-His failed to induce proliferation in both cell lines (Fig 3D), suggesting that the His-tag was not involved. Heat treatment of rJHP0290 inhibited the proliferation-inducing ability of rJHP0290, supporting the view that proliferation induction occurred due to the protein rJHP0290 (Fig 3D). Further, treatment with the LPS antagonist Polymyxin B (PMB) had no effect on rJHP0290-induced proliferation (Fig 3D), ruling out the possibility that the observed induction of proliferation was due to LPS contamination. The rJHP0290 C162A mutant was significantly impaired in its ability to induce gastric epithelial cell proliferation, which was expected due to the poor binding of the mutant to gastric epithelial cells (Fig 3D).


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

Tavares R, Pathak SK - PLoS ONE (2015)

Effect of rJHP0290 on gastric epithelial cell proliferation.AGS (A), MKN45 (B) and PHSEC (C) cells were incubated either with protein storage buffer (Ctrl) or different concentrations of rJHP0290 as indicated in the figure legends for 48 h and 72 h, followed by MTT assay to assess proliferation. Values indicate mean ± SD of six independent experiments performed at least in triplicate. (D) AGS and MKN45 cells were treated for 48h with buffer (ctrl) or rJHP0290 Wt (100 ng/ml) or rJHP0290 C162A (100 ng/ml) or an irrelevant His-tagged protein (Irr-His, 100 ng/ml). rJHP0290 was subjected to boiling for 30 min (heat) or was treated with polymyxin B (PMB) for 1 h before treatment of cells. The relative BrdU incorporation in cells was measured. Values indicate mean ± SD of four independent experiments performed in triplicate. Statistically significant differences are indicated by * (p < 0.05).
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Related In: Results  -  Collection

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pone.0124407.g003: Effect of rJHP0290 on gastric epithelial cell proliferation.AGS (A), MKN45 (B) and PHSEC (C) cells were incubated either with protein storage buffer (Ctrl) or different concentrations of rJHP0290 as indicated in the figure legends for 48 h and 72 h, followed by MTT assay to assess proliferation. Values indicate mean ± SD of six independent experiments performed at least in triplicate. (D) AGS and MKN45 cells were treated for 48h with buffer (ctrl) or rJHP0290 Wt (100 ng/ml) or rJHP0290 C162A (100 ng/ml) or an irrelevant His-tagged protein (Irr-His, 100 ng/ml). rJHP0290 was subjected to boiling for 30 min (heat) or was treated with polymyxin B (PMB) for 1 h before treatment of cells. The relative BrdU incorporation in cells was measured. Values indicate mean ± SD of four independent experiments performed in triplicate. Statistically significant differences are indicated by * (p < 0.05).
Mentions: H. pylori infection is associated with the enhanced proliferation of gastric epithelial cells, and several virulence factors that regulate proliferation have been identified [15, 23, 29]. Considering the ability of rJHP0290 to bind to epithelial cells, we further studied the effect of rJHP0290 on gastric epithelial cell proliferation. AGS, MKN45 and PHSEC cells were treated with various concentrations of rJHP0290 for various periods of time. An MTT assay revealed that rJHP0290 could induce gastric epithelial cell proliferation in a dose-dependent manner (Fig 3A–3C). A BrdU cell proliferation assay, which is an additional method for assessing cell proliferation, further confirmed the proliferation-promoting property of rJHP0290 (Fig 3D). To rule out the possibility that the His-Tag contributes to the observed effect, an irrelevant His-tagged protein (Irr-His) was used to treat both cell lines under similar conditions. Irr-His failed to induce proliferation in both cell lines (Fig 3D), suggesting that the His-tag was not involved. Heat treatment of rJHP0290 inhibited the proliferation-inducing ability of rJHP0290, supporting the view that proliferation induction occurred due to the protein rJHP0290 (Fig 3D). Further, treatment with the LPS antagonist Polymyxin B (PMB) had no effect on rJHP0290-induced proliferation (Fig 3D), ruling out the possibility that the observed induction of proliferation was due to LPS contamination. The rJHP0290 C162A mutant was significantly impaired in its ability to induce gastric epithelial cell proliferation, which was expected due to the poor binding of the mutant to gastric epithelial cells (Fig 3D).

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