<i>Helicobacter pylori</i> Protein JHP0290 Exhibits Proliferative and Anti-Apoptotic Effects in Gastric Epithelial Cells

<div><p>The influence of <i>Helicobacter pylori</i> 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 <i>H</i>. <i>pylori</i>, regulates multiple responses in human gastric epithelial cells. The differential expression and release of JHP0290 homologues was observed among <i>H</i>. <i>pylori</i> strains. JHP0290 existed in monomeric and dimeric forms in <i>H</i>. <i>pylori</i> 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 <i>H</i>. <i>pylori</i>-induced gastric diseases via the regulation of gastric epithelial cell proliferation and anti-apoptotic pathways.</p></div

rJHP0290 activates ERK MAPK in gastric epithelial cells.

<p>AGS <b>(A)</b> and MKN45 <b>(B)</b> cells were treated with rJHP0290 for various time points (min) as indicated in the figure legends. Cell lysates were prepared and immunoblotted with anti-phospho-ERK antibody followed by reprobing with anti-ERK and anti-actin antibody to confirm equal loading. Blot shown is representative of results obtained in five independent experiments. The graph shows the western blot band intensities normalized to the actin control in five experiments. Statistically significant differences are indicated by * (p < 0.05).</p

Primers used in this study.

<p>Primers used in this study.</p

Expression of JHP0290 homologues in different <i>H</i>. <i>pylori</i> strains.

<p>Whole cell lysate <b>(A)</b> and culture broth (Supernatant) from equal number of cells <b>(B)</b> of various <i>H</i>. <i>pylori</i> strains as indicated in figure legends were immunoblotted with anti-JHP0290 antibody. Blot shown is representative of results obtained in more than five independent experiments. The graph shows western blot band intensities quantified by the ImageJ software. <b>(C)</b> Sequence homology analysis of JHP0290 homologues in <i>H</i>. <i>pylori</i> strains J99 (JHP0290), 26695 (HP0305), P12 (HPP12_HP0304) and HPAG1 (HPAG1_HP0307) using Clustal Omega software (EMBL-EBI). N-terminal 1–17 amino acids were identified as signal peptide by the SignalP 4.1 software (ExpPASy Bioinformatics Resource Portal). Conserved Cysteine at position 162 is marked by an arrow.</p

Effect of rJHP0290 on gastric epithelial cell proliferation.

<p>AGS <b>(A)</b>, MKN45 <b>(B)</b> and PHSEC <b>(C)</b> 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. <b>(D)</b> 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).</p

rJHP0290 modulates cell cycle progression in gastric epithelial cells.

<p>AGS cells were incubated either with protein storage buffer (Ctrl) or rJHP0290 (100 ng/ml) for 24 h. Cells were washed with PBS, fixed in ice-cold 70% ethanol at 4°C and stained with PI and RNAse A in PBS for 30 min. The DNA profile was generated by FACS. One representative profile (A) and mean ± SD of six independent experiments (B) are shown. Statistically significant differences are indicated by * (p < 0.05).</p

<i>Helicobacter pylori</i> Protein JHP0290 Binds to Multiple Cell Types and Induces Macrophage Apoptosis via Tumor Necrosis Factor (TNF)-Dependent and Independent Pathways

<div><p>Activated macrophages at the sub-mucosal space play a major role in generating innate immune responses during <i>H. pylori</i> infection. Final disease outcome largely depends on how <i>H. pylori</i> and bacterium-derived products modulate macrophage responses. Here, we report that JHP0290, a functionally unknown protein from <i>H. pylori</i>, regulates macrophage functions. Recombinant purified JHP0290 (rJHP0290) had the ability to bind to several cell types including macrophages, human gastric epithelial cell lines, human monocyte-derived dendritic cells (MoDC) and human neutrophils. Exposure to rJHP0290 induced apoptosis in macrophages concurrent with release of proinflammatory cytokine tumor necrosis factor (TNF). A mutant strain of <i>H. pylori</i> disrupted in the <i>jhp0290</i> gene was significantly impaired in its ability to induce apoptosis and TNF in macrophages confirming the role of endogenous protein in regulating macrophage responses. Intracellular signaling involving Src family of tyrosine kinases (SFKs) and ERK MAPK were required for rJHP0290-induced TNF release and apoptosis in macrophages. Furthermore, rJHP0290-induced TNF release was partly dependent on activation of nuclear transcription factor-κB (NF-κB). Neutralizing antibodies against TNF partially blocked rJHP0290-induced macrophage apoptosis indicating TNF-independent pathways were also involved. These results provide mechanistic insight into the potential role of the protein JHP0290 during <i>H. pylori</i>-associated disease development. By virtue of its ability to induce TNF, an acid suppressive proinflammatory cytokine and induction of macrophage apoptosis, JHP0290 possibly helps in persistent survival of the bacterium inside the stomach.</p></div

The role of NF-κB and AP-1 transcription factors in rJHP0290 mediated responses in macrophages.

<p>RAW264.7 cells (<b>A</b>) or MDM (<b>B</b>) were treated with rJHP0290 for various time points (min) as indicated in figure legends. Positive controls cells were treated with <i>E. coli</i> LPS for 30 min. Cell lysates were prepared and immunoblotted with anti-IκBα antibody followed by reprobing with anti-actin antibody to confirm equal loading. Blot shown are representative of results obtained in three independent experiments. RAW264.7 cells were treated either with DMSO or IKK inhibitor Wedelolactone (50 µM) or AP-1 inhibitor SR11302 (1 µM) for 1 h followed by treatment with rJHP0290. TNF release in culture supernatant (<b>C</b>) or percentage of apoptotic cells (<b>D</b>) was determined. Values indicate mean ± SD of three independent experiments. Statistically significant differences are indicated by *(p<0.05).</p

TNF mRNA expression in RAW264.7 cells and apoptosis and TNF induction in primary MDM after rJHP0290 treatment.

<p>(<b>A</b>) Real-time PCR was used to determine fold changes (RQ) in mRNA of TNF after treatment of RAW264.7 cells with rJHP0290 (2 µg/ml) for 6 h. Monocyte derived macrophages (MDM) were treated either with protein storage buffer (Ctrl) or rJHP0290. Percentage of apoptotic cells (<b>B</b>) or TNF release in culture supernatant (<b>C</b>) was determined. Values indicate mean ± SD of three independent experiments. Statistically significant differences are indicated by *(p<0.05).</p

The role of endogenous JHP0290 on macrophage apoptosis and TNF release.

<p>(<b>A</b>) Equal amount of cell lysates from <i>H. pylori</i> wild-type strain (J99 Wt) or mutant strains (J99 Δ<i>jhp0290</i>) were immunoblotted with anti-JHP0290 antibody. Blots were reprobed with antibody raised against another <i>H. pylori</i> protein, alkyl hydroperoxide-reductase (AhpC), to confirm equal loading. Blot shown is representative of results obtained in three independent experiments. MDM and RAW264.7 cells were infected either with <i>H. pylori</i> wild-type strain J99 (Wt) or mutant strain (Δ<i>jhp0290</i>) at MOI100. Percentage of apoptotic cells (<b>B</b>) or TNF release in culture supernatant (<b>C</b>) was determined. (<b>D</b>) RAW264.7 cells were infected either with J99 Wt or J99 Δ<i>jhp0290</i> mutant strain at MOI100 for 6 h. RT-PCR was performed to assess the fold changes (RQ) in mRNA level of TNF. Values indicate mean ± SD of three independent experiments. Statistically significant differences are indicated by *(p<0.05).</p