VacA-induced Akt phosphorylation leading to GSK3β phosphorylation at Ser9. A, AZ-521 cells were incubated with 120 nm VacA or heat-inactivated VacA (iVacA) at 37 °C for the indicated times. After incubation, cell lysates were analyzed by SDS-PAGE (10% gels), followed by Western blot analyses using anti-phospho-Thr-308 Akt, phospho-Ser-473 Akt, and Akt antibodies. Results are representative of three independent experiments. Quantification of phospho-Thr-308 Akt and phospho-Ser-473 Akt obtained with VacA (filled bars) and iVacA (open bars) was performed by densitometry. Data are means ± S.E. of values from triplicate experiments, with an n = 3 per experiment. Statistical significance: ^*, p < 0.01. B, AZ-521 cells were incubated with 120 nm VacA (filled bars) or iVacA (open bars) at 37 °C for indicated times. Cell lysates were prepared at indicated incubation times and subjected to Western blot analyses using anti-phospho-Ser-9 GSK3β and GSK3β antibodies. Results are representative of three independent experiments. Quantification of phospho-Ser-9 GSK3β obtained with VacA (filled bars) and iVacA (open bars) was determined by densitometry. Data are means ± S.E. of values from triplicate experiments, with an n = 3 per experiment. Statistical significance: ^*, p < 0.05.

Effect of PI-PLC, NPPB, or Bafilomycin A1 on VacA-induced Akt phosphorylation in AZ-521 cells. AZ-521 cells were pretreated with PI-PLC (1 unit/ml, 60 min), NPPB (50 μm, 30 min), or Bafilomycin A1 (5 nm, 30 min) at the indicated concentrations. After treatment, the cells were incubated with 120 nm VacA or iVacA at 37 °C for 30 and 60 min. Following incubation, the cell lysates were subjected to SDS-PAGE in 10% gels and transferred to PVDF membranes for Western blotting with anti-phospho-Ser-473 Akt and Akt antibodies. Quantification of phospho-Ser-473 Akt obtained with VacA (filled bars) and iVacA (open bars) was determined by densitometry. Data are means ± S.E. of values from triplicate experiments, with an n = 3 per experiment. Statistical significance: ^*, p < 0.05.

GSK3β/β-catenin complexes are dissociated by VacA in AZ-521 cells. AZ-521 cells were incubated with 120 nm VacA or iVacA for 0–240 min in the presence or absence of 50 μm LY294002. After incubation, the cells were solubilized with lysis buffer and incubated on ice for 30 min. The lysates were available for immunoprecipitation using anti-β-catenin mouse monoclonal antibody (Transduction Laboratories, BD). The precipitates were subjected to SDS-PAGE in 8% gels and transferred to PVDF membranes. Then, GSK3β and β-catenin were quantified by Western blot analysis using anti-GSK3β rabbit monoclonal antibody (1:1,000; Cell Signaling) and anti-β-catenin rabbit polyclonal antibody (1:1,000; H-102, Santa Cruz Biotechnology), respectively. Quantification of phospho-Ser-9 GSK3β obtained with VacA (filled bars) and iVacA (open bars) was determined by densitometry. Data are means ± S.E. of values from triplicate experiments, with an n = 3 per experiment. Statistical significance: ^*, p < 0.01.

Effect of VacA on β-catenin-Tcf-mediated transcription and cyclin D1 promoter activity. AZ-521 cells were transfected with the luciferase reporter plasmid TOPtkLuciferase (filled bars) or FOPtkLuciferase (open bars), and internal control pRL-TK, followed by incubation with 120 nm VacA (A) or iVacA (B) for indicated times. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System. Relative luciferase activities were compared with activities obtained for TOPtkLuciferase at 0 min in the presence of toxin. Data are means ± S.E. of values from triplicate experiments, with an n = 3 per experiment. Statistical significance: ^*, p < 0.01. C, AZ-521 cells were transfected with a reporter plasmid containing the cyclin D1 promoter-luciferase gene or a control empty vector. After transfection, cells were incubated with VacA (filled bars) or iVacA (open bars). Relative luciferase activities were compared with activities obtained at 0 min in the presence of iVacA. Data are means ± S.E. of values from triplicate experiments, with an n = 3 per experiment. Statistical significance: ^*, p < 0.01.

VacA or an H. pylori vacA-positive strain induced activation of PI3K p110α/Akt pathway leading to inactivation of GSK3β and enhanced transcriptional activity of β-catenin. VacA or an H. pylori vacA-positive strain activated the PI3K/Akt signaling pathway, resulting in phosphorylation and inhibition of GSK3β, and subsequent translocation of β-catenin to the nucleus, consistent with effects of VacA on β-catenin-regulated transcriptional activity.

Effect of various inhibitors on VacA-induced phosphorylation of Akt in AZ-521 cells. AZ-521 cells were pretreated with LY294002 for 30 min (A) or MCD for 60 min (B) at the indicated concentrations prior to incubation with or without 120 nm VacA. After incubation for 30 min, cell lysates were prepared and subjected to Western blot analyses using anti-phospho-Ser-473 Akt and Akt antibodies. Results are representative of three independent experiments. Quantification of phospho-Ser-473 Akt obtained with (filled bars) or without VacA (open bars) was determined by densitometry. Data are means ± S.E. of values from triplicate experiments, with an n = 3 per experiment. Statistical significance: ^*, p < 0.05.

Effect of PI3K p110α siRNA on VacA-induced Akt phosphorylation and effects of LY294002 and silencing of the PI3K gene on VacA-induced phosphorylation at Ser-9 of GSK3β. A, AZ-521 cells were transiently transfected with siRNA for PI3K p110α or negative siRNA as a control as described under “Experimental Procedures.” The transfected cells were incubated with 120 nm VacA at 37 °C for up to 60 min. After incubation, cell lysates were subjected to SDS-PAGE in 10% gels, and then transferred to PVDF membranes for Western blot analysis with the antibodies against phospho-Ser-473 Akt, Akt, and PI3K p110α. Quantification of phospho-Ser-473 Akt obtained with VacA (filled bars) and iVacA (open bars) was determined by densitometry scan analysis. Data are means ± S.E. of values from triplicate experiments, with an n = 3 per experiment. Statistical significance: ^*, p < 0.05. B, AZ-521 cells were pretreated with 50 μm LY294002 for 30 min before incubation with 120 nm VacA or iVacA at 37 °C. After incubation with VacA or iVacA for indicated times, the cell lysates were applied to SDS-PAGE in 10% gels. The separated proteins were transferred to PVDF membranes, and subjected to Western blot analysis with anti-phospho-Ser-9 GSK3β and GSK3β antibodies. Quantification of phospho-Ser-9 GSK3β was determined by densitometry for VacA (filled bars) and iVacA (open bars). Data are means ± S.E. of values from triplicate experiments, with an n = 3 per experiment. Statistical significance: ^*, p < 0.05. C, AZ-521 cells were transiently transfected with 200 pmol of siRNA for PI3K p110α or negative siRNA as negative control as described under “Experimental Procedures.” The transfected cells were incubated with 120 nm VacA or iVacA at 37 °C for indicated times. After incubation, the cell lysates were subjected to SDS-PAGE in 10% gels, and then transferred to PVDF membranes for Western blot analysis with anti-phospho-Ser-9 GSK3β, GSK3β, and PI3K p110α antibodies. Quantification of phospho-Ser-9 GSK3β obtained with VacA (filled bars) and iVacA (open bars) was determined by densitometry. Data are means ± S.E. of values from triplicate experiments, with an n = 3 per experiment. Statistical significance: ^*, p < 0.05.

Confocal microscopy analysis of β-catenin translocation to nucleus from cytosol. AZ-521 cells were incubated with 120 nm VacA (A) or iVacA (B) at 37 °C for 0, 30 and 60 min. After incubation, the cells were fixed using 2% paraformaldehyde at room temperature, treated with 0.1% Triton X-100, and blocked with Block Ace. The fixed cells were stained with DAPI for 5 min, and were subjected to triple immunostaining using anti-β-catenin monoclonal antibodies and anti-GSK3β monoclonal antibodies as primary antibodies. After treatment with the respective primary antibodies, cells were incubated with the secondary anti-mouse antibodies, conjugated with Alexa Fluor 488, and anti-rabbit antibodies conjugated with Alexa Fluor 546, respectively. Data are representative of three experiments. Scale bar, 5 μm.

Phosphorylation of Akt and GSK3β and dissociation of GSK3β/β-catenin complexes in AZ-521 cells infected with H. pylori, but not an isogenic VacA-knock-out mutant strain. AZ-521 cells were infected for 6 h with H. pylori ATCC43504 (WT strain) or its isogenic VacA-knock-out mutant strain (ΔVacA strain). Cells incubated without infection (uninfected cells) were used as a negative control. A, after incubation, phosphorylation of Akt and GSK3β in cells was determined by Western blot analysis with anti-phospho-Ser-473 Akt, Akt phospho-Ser-9 GSK3β, and GSK3β antibodies. Quantification of phospho-Ser-473 Akt and phospho-Ser-9 GSK3β obtained with control (open bars), WT strain (black bars), and ΔVacA strain (gray bars) was determined by densitometry. Data are means ± S.E. of values from triplicate experiments, with an n = 3 per experiment. Statistical significance: ^*, p < 0.05. B, after incubation, proteins from cell lysates were immunoprecipitated using anti-β-catenin antibody, followed by SDS-PAGE in 8% gels and Western blotting with anti-β-catenin and GSK3β antibodies. Relative densities of GSK3β, as determined by densitometry, were compared with densities obtained at 0 min. Data are means ± S.E. of values from triplicate experiments, with an n = 3 per experiment. Statistical significance: ^*, p < 0.05.