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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

rJHP0290 activates NF-κB in AGS and MKN45 cells.(A) NF-κB-inducible SEAP reporter transfected AGS and MKN45 cells were treated with various concentrations of rJHP0290 as indicated in figure legends and activation of NF-κB was assessed using Quanti-Blue as substrate. Lipoteichoic acid (LTA) was used as positive control. Values indicate mean ± SD of three independent experiments. Statistically significant differences are indicated by * (p < 0.05). AGS (B) and MKN45 (C) cells were treated with rJHP0290 (100 ng/ml) for various time points (min) as indicated in figure legends. Cell lysates were prepared and immunoblotted with IκBα antibody followed by reprobing with anti-actin antibody to confirm equal loading. Blot shown is representative of results obtained in three independent experiments. The graph shows the densitometric analysis of western blot band intensities normalized to the actin control in three experiments. Statistically significant differences are indicated by * (p < 0.05).
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pone.0124407.g006: rJHP0290 activates NF-κB in AGS and MKN45 cells.(A) NF-κB-inducible SEAP reporter transfected AGS and MKN45 cells were treated with various concentrations of rJHP0290 as indicated in figure legends and activation of NF-κB was assessed using Quanti-Blue as substrate. Lipoteichoic acid (LTA) was used as positive control. Values indicate mean ± SD of three independent experiments. Statistically significant differences are indicated by * (p < 0.05). AGS (B) and MKN45 (C) cells were treated with rJHP0290 (100 ng/ml) for various time points (min) as indicated in figure legends. Cell lysates were prepared and immunoblotted with IκBα antibody followed by reprobing with anti-actin antibody to confirm equal loading. Blot shown is representative of results obtained in three independent experiments. The graph shows the densitometric analysis of western blot band intensities normalized to the actin control in three experiments. Statistically significant differences are indicated by * (p < 0.05).

Mentions: NF-κB is a key transcription factor, which is involved in the regulation of various cellular responses against H. pylori [36]. We further assessed the effect of rJHP0290 on NF-κB using two approaches. AGS and MKN45 cells were transfected with the pNiFty-SEAP reporter plasmid, which is composed of three key elements: an ELAM proximal promoter, five NF-κB repeated transcription factor binding sites (TFBS) and a secreted alkaline phosphatase (SEAP) reporter gene. The cells were then treated with rJHP0290. SEAP activity in the culture supernatant was detected using the QUANTI-Blue detection system (InvivoGen). As shown in Fig 6A, rJHP0290 significantly induced SEAP activity in AGS and MKN45 cells in a dose-dependent manner. Using another approach, we determined the levels of IκBα in AGS and MKN45 cells after treatment with JHP0290. Treatment with rJHP0290 reduced the level of IκBα (Fig 6B and 6C), indicating that NF-κB was activated. IκBα began degrading 30 minutes post-treatment, and the basal IκBα level was restored after 90–120 min.


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

Tavares R, Pathak SK - PLoS ONE (2015)

rJHP0290 activates NF-κB in AGS and MKN45 cells.(A) NF-κB-inducible SEAP reporter transfected AGS and MKN45 cells were treated with various concentrations of rJHP0290 as indicated in figure legends and activation of NF-κB was assessed using Quanti-Blue as substrate. Lipoteichoic acid (LTA) was used as positive control. Values indicate mean ± SD of three independent experiments. Statistically significant differences are indicated by * (p < 0.05). AGS (B) and MKN45 (C) cells were treated with rJHP0290 (100 ng/ml) for various time points (min) as indicated in figure legends. Cell lysates were prepared and immunoblotted with IκBα antibody followed by reprobing with anti-actin antibody to confirm equal loading. Blot shown is representative of results obtained in three independent experiments. The graph shows the densitometric analysis of western blot band intensities normalized to the actin control in three experiments. Statistically significant differences are indicated by * (p < 0.05).
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Related In: Results  -  Collection

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pone.0124407.g006: rJHP0290 activates NF-κB in AGS and MKN45 cells.(A) NF-κB-inducible SEAP reporter transfected AGS and MKN45 cells were treated with various concentrations of rJHP0290 as indicated in figure legends and activation of NF-κB was assessed using Quanti-Blue as substrate. Lipoteichoic acid (LTA) was used as positive control. Values indicate mean ± SD of three independent experiments. Statistically significant differences are indicated by * (p < 0.05). AGS (B) and MKN45 (C) cells were treated with rJHP0290 (100 ng/ml) for various time points (min) as indicated in figure legends. Cell lysates were prepared and immunoblotted with IκBα antibody followed by reprobing with anti-actin antibody to confirm equal loading. Blot shown is representative of results obtained in three independent experiments. The graph shows the densitometric analysis of western blot band intensities normalized to the actin control in three experiments. Statistically significant differences are indicated by * (p < 0.05).
Mentions: NF-κB is a key transcription factor, which is involved in the regulation of various cellular responses against H. pylori [36]. We further assessed the effect of rJHP0290 on NF-κB using two approaches. AGS and MKN45 cells were transfected with the pNiFty-SEAP reporter plasmid, which is composed of three key elements: an ELAM proximal promoter, five NF-κB repeated transcription factor binding sites (TFBS) and a secreted alkaline phosphatase (SEAP) reporter gene. The cells were then treated with rJHP0290. SEAP activity in the culture supernatant was detected using the QUANTI-Blue detection system (InvivoGen). As shown in Fig 6A, rJHP0290 significantly induced SEAP activity in AGS and MKN45 cells in a dose-dependent manner. Using another approach, we determined the levels of IκBα in AGS and MKN45 cells after treatment with JHP0290. Treatment with rJHP0290 reduced the level of IκBα (Fig 6B and 6C), indicating that NF-κB was activated. IκBα began degrading 30 minutes post-treatment, and the basal IκBα level was restored after 90–120 min.

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