The Secreted Peptidyl Prolyl cis,trans-Isomerase HP0175 of Helicobacter pylori Induces Apoptosis of Gastric Epithelial Cells in a TLR4- and Apoptosis Signal-Regulating Kinase 1-Dependent Manner

HP0175 signals through TLR4 to induce apoptosis in AGS cells. A, The ability of HP0175 to interact directly with TLR4 was examined by a pull-down assay. AGS cell lysate was incubated with HP0175 immobilized on Ni²⁺-NTA agarose in the absence (lane b) or the presence (lane e) of polymyxin B or with Ni²⁺-NTA agarose alone (lane c) or combined with an irrelevant His-tagged protein (lane d) immobilized on Ni²⁺-NTA agarose for 2 h; the agarose-bound proteins were separated on SDS-PAGE; and coprecipitated agarose-bound TLR4 was detected by Western blotting using anti-TLR4 Ab. Lane a represents immunoprecipitated TLR4 only. The arrow indicates the band corresponding to TLR4. The blot shown is representative of three separate experiments. B, AGS cells were transfected with pcDNA-TLR4 (lanes b, d, and e) or with empty vector (lane c), Cell lysates from these cells were incubated with His-HP0175 (lanes b, c, and e) or irrelevant His-tagged protein (lane d), followed by immunoprecipitation with anti-TLR4 Ab (lanes b–d) or with anti-isotype IgG (lane e). Western blotting was performed with anti-His Ab to detect coprecipitated His-tagged protein. Lane a represents His-HP0175 only. The arrow indicates the band corresponding to HP0175. C, AGS cells were transfected with empty vector or TLR4(dn), followed by incubation without (□) or with (▪) HP0175 (100 ng/ml) for 24 h. ∗, p < 0.05 vs empty vector-transfected AGS challenged with HP0175 (by t test). D, AGS cells were incubated without or with 25 μg/ml human TLR4-specific mAb (HTA125) or an isotype-matched irrelevant Ab (i/c) for 1 h. Cells were washed and incubated without or with HP0175 in fresh medium as described in C. ∗, p < 0.005 vs HP0175-treated cells only (by ANOVA). C and D, Cell death was measured as described in Fig. 1. Values are the mean ± SD of three different experiments.

HP0175-induced cell death depends on ASK1, MAPKs, and caspases. AGS cells were left untreated or were treated with the cell-permeable MAPK inhibitors U0126 and SB203580 at the indicated concentrations (A) for 60 min, followed by removal of the inhibitors and incubation without or with HP0175 (100 ng/ml) for 24 h. ∗, p < 0.001 vs control treated with HP0175 (by ANOVA). B, AGS cells were transfected with different constructs as indicated in the figure, followed by treatment without or with HP0175 (100 ng/ml) for 60 min. Cell death was measured as described in Fig. 1. ∗, p < 0.001 vs WT (by ANOVA). Values are the mean ± SD of three different experiments. □, Without HP0175; ▪, with HP0175.

HP0175 activates ASK1 and p38 MAPK. AGS cells were incubated with HP0175 (100 ng/ml) for various periods of time (A), or cells were transfected with empty vector or TLR4(dn), followed by treatment with HP0175 (100 ng/ml) for 60 min (B). Whole-cell lysates were prepared, ASK1 was immunoprecipitated, and in vitro ASK1 kinase activity was determined using MBP as substrate as described in Materials and Methods. The bottom panel is a representative Western blot with anti-ASK1 to show that the same amount of ASK1 was present in each sample. The bar diagrams represent densitometric scans of the phospho-MBP bands obtained in three different experiments (mean ± SD). C, AGS cells were transfected with ASK1(WT) or a catalytically inactive mutant of ASK1(KM), followed by incubation without or with HP0175 (100 ng/ml) for 2 h. Whole-cell lysates were prepared, followed by immunoblotting with anti-phospho-p38 MAPK Abs. The lower blots show that the same amount of p38 MAPK was present in each sample. The right panel represents the densitometric analysis of the phospho-p38 MAPK bands (error bars represent the SD from three independent experiments).

HP0175-induced caspase activation in AGS cells. AGS cells were left untreated or were treated with the cell-permeable irreversible caspase inhibitors (50 μM): z-IETD-FMK (for caspase 8), z-DEVD-FMK (for caspase 3), or z-LEHD-FMK (for caspase 9; A); or were transfected with different constructs as indicated in Fig. lB, followed by treatment without or with HP0175 (100 ng/ml) for 60 min. Cell death was measured as described in Fig. 1. Results are expressed as the fold increase in the release of histone compared with that in the control (untreated, nontransfected AGS). Values are the mean ± SD of three different experiments. □, Without HP0175; ▪, with HP0175. A: ∗, p < 0.001 vs no inhibitor (by ANOVA). B: ∗, p < 0.05 vs empty vector-transfected AGS cells treated with HP0175 (by t test). AGS cells were incubated with HP0175 (100 ng/ml) for various periods of time (C and D) or were treated with z-DEVD-FMK (50 μM; lanes 5 and 6) or z-LEHD-FMK (50 μM; lanes 7 and 8) for 60 min (C) before incubation without (lanes 5 and 7) or with (lanes 6 and 8) HP0175 (100 ng/ml) for 14 h. Cell lysates were prepared as described in Materials and Methods. Cleaved caspase 3 (17 kDa) and PARP (89 kDa) were detected by Western blotting using Abs against cleaved caspase 3 (C) or PARP (D), respectively. E, AGS cells were incubated with HP0175 (100 ng/ml) for various periods of time or were treated with z-IETD-FMK (50 μM) for 60 min (last two lanes) before incubation without or with HP0175 (100 ng/ml) for 9 h. Cell lysates were prepared as described in Materials and Methods. F, AGS cells were transfected with p38(WT) or p38(agf), followed by incubation without or with HP0175 (100 ng/ml) for 9 h. E and F, Cleaved caspase 9 (37 kDa) was detected by Western blotting using Abs against caspase 9. G, AGS cells were treated with HP0175 (100 ng/ml) for various periods of time or were treated with z-IETD-FMK (50 μM) for 60 min (last two lanes) before incubation without or with HP0175 (100 ng/ml) for 5 h. Cell lysates were prepared as described in Materials and Methods. H, AGS cells were transfected with p38(WT) or p38(agf), followed by incubation without or with HP0175 (100 ng/ml) for 5 h. Cleaved caspase 8 (20 kDa) was detected by Western blotting using Abs against caspase 8 (G and H). Each blot is representative of results obtained in three separate experiments. Equal protein loading was verified in all cases by analyzing samples for β-actin expression.

HP0175-induced loss of mitochondrial membrane permeability transition, cleavage of Bid, and release of cyt c from the mitochondria. AGS cells were treated with HP0175 for various periods of time (A). In separate experiments, AGS cells were transfected with p38(WT), p38(agf), ASK1(WT), or ASK1(KM) before incubation without or with HP0175 (100 ng/ml) for 8 h (B). Cells were washed, and the fluorescence of DiOC₆ was determined at excitation and emission wavelengths of 488 and 500 nm, respectively. Values for de-energized mitochondria were determined by simultaneous treatment of cells with 10 μM CCCP and DiOC6 (A). ▪, Without HP0175; □, with HP0175. Values are the mean ± SD of three different experiments. Cells were left untreated or were treated with the caspase 8 inhibitor z-IETD-FMK (50 μM; C and D) for 1 h before treatment with HP0175 (100 ng/ml) for 6 h (C) or for various periods of time (D). In a separate set of experiments, cells were transfected with p38(WT), p38(agf), ASK1(WT), or ASK1(KM) before incubation without or with HP0175 (100 ng/ml) for 8 h (E). Cells were processed to obtain mitochondrial (C) or cytosolic (D and E) fractions as described in Materials and Methods. Truncated Bid (15 kDa; C) or cyt c (12 kDa) and actin (D and E) were detected by SDS-PAGE, followed by Western blotting. Blots are representative of results obtained from three separate experiments.