Epidermal growth factor receptor activation protects gastric epithelial cells from Helicobacter pylori-induced apoptosis

Abstract

Background & aims: Helicobacter pylori infection disrupts the balance between gastric epithelial cell proliferation and apoptosis, which is likely to lower the threshold for the development of gastric adenocarcinoma. H pylori infection is associated with epidermal growth factor (EGF) receptor (EGFR) activation through metalloproteinase-dependent release of EGFR ligands in gastric epithelial cells. Because EGFR signaling regulates cell survival, we investigated whether activation of EGFR following H pylori infection promotes gastric epithelial survival.

Methods: Mouse conditionally immortalized stomach epithelial cells (ImSt) and a human gastric epithelial cell line, AGS cells, as well as wild-type and kinase-defective EGFR (EGFRwa2) mice, were infected with the H pylori cag+ strain 7.13. Apoptosis, caspase activity, EGFR activation (phosphorylation), and EGFR downstream targets were analyzed.

Results: Inhibiting EGFR kinase activity or decreasing EGFR expression significantly increased H pylori-induced apoptosis in ImSt. Blocking H pylori-induced EGFR activation with a heparin-binding (HB)-EGF neutralizing antibody or abrogating a disintegrin and matrix metalloproteinase-17 (ADAM-17) expression increased apoptosis of H pylori-infected AGS and ImSt, respectively. Conversely, pretreatment of ImSt with HB-EGF completely blocked H pylori-induced apoptosis. H pylori infection stimulated gastric epithelial cell apoptosis in EGFRwa2 but not in wild-type mice. Furthermore, H pylori-induced EGFR phosphorylation stimulated phosphotidylinositol-3'-kinase-dependent activation of the antiapoptotic factor Akt, increased expression of the antiapoptotic factor Bcl-2, and decreased expression of the proapoptotic factor Bax.

Conclusions: EGFR activation by H pylori infection has an antiapoptotic effect in gastric epithelial cells that appears to involve Akt signaling and Bcl family members. These findings provide important insights into the mechanisms of H pylori-associated tumorigenesis.

Figure 1. EGFR kinase activity is required for surv ival of gastric epithelial cells infected with H. pylori

ImSt were infected with the wt H. pyloricagA⁺ strain 7.13 (bacteria:cells = 100:1) for 90 min (A) or 24 h (B–E) pretreated for 30 min with AG1478 (150 nM) or not pretreated. A: EGFR Y1068 phosphorylation was detected by Western blot analysis of cellular lysates using an anti-EGFR-phospho (P)-Y1068 antibody. An anti-EGFR antibody was used as a loading control. Band density was quantified using ImageJ software. The relative density was calculated by comparing density of EGFR-P-Y1068 to the density of EGFR. The relative density in the control group (no AG1478 and no H. pylori) was set as 1.0. The relative densities in the treated groups were compared to that of control. B and C: Cells were stained with Annexin V-FITC and PI, and flow cytometry was performed. The percentage of cells in each quadrant is shown (C). The upper right quadrant represents late apoptosis, and the lower right quadrant represents early apoptosis. D and E: Cells were fixed for TUNEL assay, and apoptotic nuclei were labeled with FITC (green) and total nuclei were labeled with DAPI (blue). FITC and DAPI labeled images were taken from the same field. Apoptosis was determined by counting at least 500 cells. The percentage of cells undergoing apoptosis is shown (E). Data in this and subsequent figures are representative of at least three separate experiments. * p < 0.05 vs cells alone, ** and *** p < 0.001 vs cells alone, § and §§ p < 0.001 vs cells infected with H. pylori, + p < 0.001 vs cells treated with AG1478.

Figure 2. Suppression of EGFR expression promotes apoptosis in H. pylori-infected gastric epithelial cells

ImSt tansfected with EGFR siRNA or non-targeting siRNA were infected with H. pylori for 24 h. A: EGFR expression levels were analyzed by Western blot analysis of cellular lysates using an anti-EGFR antibody. An anti-ERK1/2 antibody was used as a loading control. B. Apoptosis was detected using TUNEL assay as described in Figure 1. Apoptosis was determined by counting at least 500 cells. The percentage of cells undergoing apoptosis is shown. *, **, and ***p < 0.001 vs uninfected cells in each cell line, § p < 0.001 vs non-transfected cells infected with H. pylori.

Figure 3. Loss of EGFR kinase activity promotes gastric epithelial cell apoptosis in mice infected with H. pylori.

Wt and EGFR ^wa2 (EGFR kinase defective) mice on a C57BL/6 background were infected with H. pylori (10⁹ cfu/mouse) for 5 or 24 h. Paraffin-embedded stomach tissues (A, C–D) and gastric mucosal lysates (B) were prepared. A. Immunostaining with anti-H. pylori antiserum was performed to detect the presence of H. pylori (arrowheads). B: EGFR Y1068 phosphorylation was detected by Western blot analysis of gastric mucosal lysates as described in Figure 1. C: EGFR Y1068 phosphorylation was detected using immunostaining with anti-EGFR Tyr1068 and FITC-labeled secondary antibody. Green represents positive labeling (L: Luminal side). D: Apoptosis was detected by ISOL staining and active caspase-3 immunostaining using an anti-active caspase-3 antibody. Apoptotic nuclei were labeled with peroxidase and developed with DAB and observed using DIC microscopy. Brown nuclei (arrows) represent ISOL or activated caspase-3 positive staining cells. n = 5–7 mice for each condition.

Figure 4. ADAM-17 mediates H. pylori-induced activation of EGFR and is required for an anti-apoptotic effects

Cells were infected with H. pylori for 90 min (A) or 24 h (B and C). EGFR Y1068 phosphorylation was analyzed by Western blot analysis of cellular lysates (as described in Figure 1), activation of Akt using anti-phospho (P)-Ser 473 Akt, ERK1/2 MAPK activation using anti-phospho (P)-Thr183/Tyr185 ERK1/2, and p38 using anti-phospho (P)-Tyr180/182 p38 antibodies. B–C: Cells were fixed for TUNEL assay and apoptotic nuclei were labeled with peroxidase and developed with DAB and observed using DIC microscopy. Brown staining represents apoptotic nuclei (B). Apoptotic cells were enumerated by counting at least 500 cells. The percentage of cells undergoing apoptosis is shown (C). *, **, *** and ****p < 0.001 vs uninfected cells in each cell line. § and # p < 0.001 vs ImSt and wt ADAM-17 transfected cells infected with H. pylori.

Figure 5. H. pylori -induced gastric epithelial cell apoptosis is enhanced by blocking HB-EGF binding toEGFR, but is prevented by HB-EGF pretreatment

AGS cells were pretreated for 30 min with an anti-HB-EGF neutralizing antibody (3 μg/ml) or HB-EGF (10 ng/ml), as indicated, and then infected with H. pylori for 90 min (A) or 24 h (B) A: Cellular lysates were analyzed by Western blot analysis to detect EGFR, Akt, ERK1/2 MAPK and p38 activation as described in Figures 1 and 4. B. Apoptosis was detected using TUNEL assay as described in Figure 4. Apoptotic cells were enumerated by counting at least 500 cells. The percentage of cells undergoing apoptosis is shown. * p < 0.001 vs cells alone, ** p < 0.001 vs cells treated with HB-EGF antibody only. § p < 0.001 vs cells infected with H. pylori.

Figure 6. Akt is a target of EGFR for survival of gastric epithelial cells infected with H. pylori.

ImSt were infected with H. pylori 90 min (A) or 24 h (B) in the presence or absence of 30 min pretreatment with AG1478 (150 nM), a PI3K inhibitor, wortmannin (100 nM), or MEK inhibitor, PD98059 (10 μM). A: Cellular lysates were collected for detecting EGFR, Akt and ERK1/2 MAPK activation by Western blot analysis as described in Figures 1 and 4. B: Cells were fixed for TUNEL assay as described in Figure 4. Apoptosis was determined by counting at least 500 cells. The percentage of cells undergoing apoptosis is shown. *, **, and *** p < 0.001 vs uninfected cells in each inhibitor treatment group. § p < 0.001 vs ImSt infected with H. pylori.

Figure 7. Role of EGFR in regulating anti-apoptotic and pro-apoptotic proteins in gastric epithelial cells

A and B: ImSt were pretreated for 30 min with a EGFR kinase inhibitor, AG1478 (150 nM) or not pretreated, and then infected with H. pylori 24 h. Cells were fixed and stained using antibodies against active caspase-3 and Bcl-2 and flow cytometry was performed. Representative dot plots (A) show the presence of a population of cells that are both low Bcl-2 and active caspase-3 (left lower quadrant), high Bcl-2 and low active caspase-3 (right lower quadrant), low Bcl-2 and high active caspase-3 (left upper quadrant), and high Bcl-2 and active caspase-3 (right upper quadrant). Percentage of Bcl-2 or active caspase-3 cells are shown (B). C and D: Mice were infected with H. pylori (10⁹ cfu/mouse) for 24 h. C: Gastric mucosal lysates were prepared for Western Blot analysis to detect relative Bax and Bcl-2 protein expression levels using anti-Bax and anti-Bcl-2 antibodies, respectively, and EGFR phosphorylation and actin levels were determined as described in Figure 1. D: The relative densities of Bax and Bcl-2 protein bands on Western blots were compared to determine the protein expression ratio of Bax to Bcl-2 in each group. The ratio in broth treated mice was set as 100%, and fold change of the ratios in H. pylori-infected mice were compared to the broth treated group. N = 5–7 mice for each condition. * p < 0.05 vs cells alone, ** p < 0.001 vs cells alone, *** p < 0.001 vs cells treated with AG1478, § p < 0.001 vs cells infected with H. pylori. @ p < 0.001 vs broth treatment in wt mice. # p < 0.001 vs broth treatment in EGFR^wa2 mice.

Figure 8

A schematic model of mechanisms for determining the fate of gastric epithelial cells infected with H. pylori.