Helicobacter pylori CagL activates ADAM17 to induce repression of the gastric H, K-ATPase alpha subunit

Abstract

Background & aims: Infection with Helicobacter pylori represses expression of the gastric H, K-adenosine triphosphatase alpha-subunit (HKalpha), which could contribute to transient hypochlorhydria. CagL, a pilus protein component of the H pylori type IV secretion system, binds to the integrin alpha(5)beta1 to mediate translocation of virulence factors into the host cell and initiate signaling. alpha(5)beta1 binds a disintegrin and metalloprotease (ADAM) 17, a metalloenzyme that catalyzes ectodomain shedding of receptor tyrosine kinase ligands. We investigated whether H pylori-induced repression of HKalpha is mediated by CagL activation of ADAM17 and release of heparin-binding epidermal growth factor (HB-EGF).

Methods: HKalpha promoter and ADAM17 activity were measured in AGS gastric epithelial cells transfected with HKalpha promoter-reporter constructs or ADAM17-specific small interfering RNAs and infected with H pylori. HB-EGF secretion was measured by enzyme-linked immunosorbent assay analysis, and ADAM17 interaction with integrins was investigated by coimmunoprecipitation analyses.

Results: Infection of AGS cells with wild-type H pylori or an H pylori cagL-deficient isogenic mutant that also contained a wild-type version of cagL (P12DeltacagL/cagL) repressed HKalpha promoter-Luc reporter activity and stimulated ADAM17 activity. Both responses were inhibited by point mutations in the nuclear factor-kappaB binding site of HKalpha or by infection with P12DeltacagL. Small interfering RNA-mediated silencing of ADAM17 in AGS cells inhibited the repression of wild-type HKalpha promoter and reduced ADAM17 activity and HB-EGF production, compared to controls. Coimmunoprecipitation studies of AGS lysates showed that wild-type H pylori disrupted ADAM17-alpha5beta1 complexes.

Conclusions: During acute H pylori infection, CagL dissociates ADAM17 from the integrin alpha(5)beta1 and activates ADAM17-dependent, nuclear factor-kappaB-mediated repression of HKalpha. This might contribute to transient hypochlorhydria in patients with H pylori infection.

Figure 1

(A) CagL mediates H. pylori-induced repression of HKα promoter. AGS cells transfected with HKα206 or HKαΔ206 promoter-Luc reporter constructs were infected (MOI=50, 8h) with H. pylori WT P12, P12ΔcagL, or P12ΔcagL/cagL strains. HKα promoter activities in cell lysates were then measured as RLU of luciferin luminescence normalized to co-transfected GFP fluorescence (means, SD, n=3; **P<0.01). (B) Immunoblot of H. pylori WT P12, P12ΔcagL, and P12ΔcagL/cagL strain lysates probed with CagL-specific or CagA-specific (loading control) antibodies.

Figure 2

H. pylori activates ADAM17 in AGS cells. (A) AGS cells transfected with ADAM17-specific siRNA or non-targeting siRNA for 72 h were infected (MOI=50, 1 h) with WT H. pylori strains P12 or 7.13. After medium replacement, metalloproteinase activity was measured using a quenched fluorogenic oligopeptide substrate. Mock siRNA-transfected AGS cells served as a control (mean, SD, n=8; ***P<0.001). (B) ADAM17 siRNA-tranfected cells were subsequently transfected with HKα promoter-Luc reporter construct (HKα2179) and were then processed to extract RNA or lysed for immunoblot analysis. ADAM17 mRNA was measured 72 h after siRNA transfection using RT-PCR and β microglobin as the internal control (mean, SD, n=3; *P <0.05). (B insert) Immunoblot of ADAM17 protein in cell lysates 72 h after siRNA transfection (total ERK is the gel loading control. (C) Immunoblot of total cell ADAM17 expression over an 8 h time-course of H. pylori P12 infection.

Figure 3

CagL mediates H. pylori-induced ADAM17 activation in AGS cells. AGS cells (3 × 10⁴ cells/well) were incubated overnight in untreated, human recombinant (r) CagL-treated (5 μg/ml), or fibronectin-treated (5 μg/ml) 96-well plates, infected (MOI=50, 8 h) with WT H. pylori strain P12, P12 ΔcagL or genetically-complemented P12 ΔcagL/cagL, and after medium replacement cellular ADAM17 activity was measured using a quenched fluorogenic oligopeptide substrate (means, SD, n=8; *P<0.05, **P<0.01).

Figure 4

ADAM17 down-regulation abrogates H. pylori-induced HKα promoter repression. AGS cells were first transfected with ADAM17-specific pooled siRNAs and then with HKα2197 promoter-Luc constructs. The cells were then infected (MOI=50, 8 h) with WT H. pylori strain P12 or P12 ΔcagL. HKα promoter activity was measured as RLU of luciferin luminescence normalized to co-transfected GFP fluorescence (means, SD, n=3; **P<0.01, ***P<0.001).

Figure 5

CagL mediates dissociation of ADAM17 from β₁ integrin. AGS cells were infected (MOI=50, 1 h) with WT H. pylori strain P12 or P12 ΔcagL. Cell lysates were immunoprecipitated with (A) ADAM17-specific antibody followed by immunoblotting (IB) of IP protein with β₁ integrin-specific antibody; ADAM17 IB served as loading control. Reverse IP was performed by immunoprecipitating the lysates with (B) β₁ integrin-specific antibody followed by IB with ADAM17-specific antibody; β₁ integrin IB served as loading control.. (C) Direct ADAM17 and β₁ integrin IB of cell lysates served as a gel loading control and a positive control of antibody specificity respectively. Protein G beads (pG) and non-immune immunoglobulin Gs (IgG)-precipitated lysates served as negative controls. Mock-infected AGS cells are shown in lanes “C”.

Figure 6

CagL mediates H. pylori-induced shedding of HB-EGF from AGS cells. AGS cells were infected (MOI=50, 1 h) with WT H. pylori strain P12 or P12 ΔcagL with or without previously silencing ADAM17 expression by siRNA. Culture medium supernatants were concentrated by centrifugal filtration and HB-EGF in the retentates was measured by ELISA (mean, SD, n=2; ***P<0.001).

Figure 7

RGD peptide, rCagL and rhADAM17 independently repress HKα promoter activity. AGS cells were transfected with a WT HKα promoter-Luc reporter construct (HKα206) or an HKα206 construct with two point mutations in the NF-κB binding site (HKαΔ206). Transfected AGS cells were incubated for 8 h with (A) synthetic RGD or RAD-containing peptides; (B) recombinant CagL (rCagL); or (C) recombinant human ADAM17 (rhADAM17) and HKα promoter activity was measured as RLU of luciferin luminescence normalized to co-transfected GFP fluorescence (means, SD, n=3).

Figure 7

RGD peptide, rCagL and rhADAM17 independently repress HKα promoter activity. AGS cells were transfected with a WT HKα promoter-Luc reporter construct (HKα206) or an HKα206 construct with two point mutations in the NF-κB binding site (HKαΔ206). Transfected AGS cells were incubated for 8 h with (A) synthetic RGD or RAD-containing peptides; (B) recombinant CagL (rCagL); or (C) recombinant human ADAM17 (rhADAM17) and HKα promoter activity was measured as RLU of luciferin luminescence normalized to co-transfected GFP fluorescence (means, SD, n=3).

Figure 7

RGD peptide, rCagL and rhADAM17 independently repress HKα promoter activity. AGS cells were transfected with a WT HKα promoter-Luc reporter construct (HKα206) or an HKα206 construct with two point mutations in the NF-κB binding site (HKαΔ206). Transfected AGS cells were incubated for 8 h with (A) synthetic RGD or RAD-containing peptides; (B) recombinant CagL (rCagL); or (C) recombinant human ADAM17 (rhADAM17) and HKα promoter activity was measured as RLU of luciferin luminescence normalized to co-transfected GFP fluorescence (means, SD, n=3).