Molecular Characterization of Helicobacter pylori VacA Induction of IL-8 in U937 Cells Reveals a Prominent Role for p38MAPK in Activating Transcription Factor-2, cAMP Response Element Binding Protein, and NF-κB Activation

A, U937 cells were incubated with 120 nM VacA for the indicated times. Cell lysates were prepared at indicated incubation times and subjected to Western blot analyses using Abs recognizing MAPKs and phosphorylated MAPKs (upper panel). Data are representative of three independent experiments. Relative densities of phospho-p38 and phospho-ERK, as determined by densitometry scan analysis, were compared with densities obtained at 0 min (bottom panel). Data are mean ± SE of values from triplicate experiments. B, Confluent U937 cells were pretreated with SB203580 (10 μM), PD98059 (10 μM), or both inhibitors (10 μM) for 1 h before incubation with VacA or iVacA (120 nM) in serum-free medium. IL-8 production was measured by ELISA. The data are representative of at least three experiments. Statistical significance: ∗, p < 0.05; ∗∗, p < 0.01.

A, U937 cells were treated with 120 nM VacA or iVacA at 37°C for 60 min. The cells were collected by centrifugation and lysed in lysis buffer. Nuclear (N) and cytosolic (C) extracts were prepared, as described in Materials and Methods. Subsequently, NF-κBp65 in cytosolic and nuclear extracts was normalized to GAPDH after Western blotting. The percentage in the nucleus is based on densitometric quantification. The data are representative of at least three experiments. Statistical significance: ∗, p < 0.05. B, U937 cells were incubated with 120 nM VacA or iVacA at 37°C for 60 min. The cells were fixed using 2% paraformaldehyde, and treated for 5 min with 0.1% Triton X-100 for membrane permeabilization. The fixed cells were stained with 1 μg/ml DAPI for 5 min, followed by incubation with anti-NF-κB mAbs (1/100) in TBS containing 1% BSA. After treatment with the respective primary Abs, cells were incubated with secondary Ab in TBS containing 1% BSA and anti-mouse polyclonal Abs conjugated with Alexa fluor 546 (1/1000). Scale bar, 5 um. C, Confluent U937 cells were pretreated with BAY11-7082 (0, 1.25, 2.5, 5, and 10 μM) for 1 h before incubation with 120 nM VacA (▪) or iVacA (□) in serum-free medium. IL-8 production was measured by ELISA. The data are representative of at least three experiments. Statistical significance: ∗, p < 0.05; ∗∗, p < 0.01.

U937 cells were loaded with fura 2-AM (5 μM, 37°C) in RPMI 1640 serum-free medium. Cells were washed with medium, and then treated with 0, 30, 60, or 120 nM VacA, or iVacA (120 nM) in the medium. Changes in cytoplasmic free Ca²⁺ concentration were determined by confocal microscopy, as described in Materials and Methods. The data are representative of at least three experiments.

A, Confluent U937 cells were pretreated with BAPTA-AM (A) or BHA (B) for 1 h before incubation with VacA or iVacA (120 nM) in serum-free medium. IL-8 production was measured by ELISA. The data are representative of at least three experiments. C, U937 cells were preincubated with 50 μM BHA or 10 μM BAPTA-AM for 1 h before incubation with VacA or iVacA (120 nM) in serum-free medium. After incubation for 60 min, the cells were fixed using 2% paraformaldehyde, and treated for 5 min with 0.1% Triton X-100 for membrane permeabilization. The fixed cells were stained with 1 μg/ml DAPI for 5 min, followed by incubation with anti-NF-κB mAbs (1/100) in TBS containing 1% BSA. After treatment with the respective primary Abs, cells were incubated with secondary Ab in TBS containing 1% BSA and anti-mouse polyclonal Abs conjugated with Alexa fluor 546 (1/1000). Scale bar, 5 um. Statistical significance: ∗, p < 0.01.

Confluent U937 cells were loaded with 5 μM 5(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate for 30 min, washed with serum-free medium, and treated with 60, 120, or 240 nM VacA, 240 nM iVacA, or 2-AG (12.5, 25, 50, or 100 μM, as positive control). ROS formation was measured in a multiwell plate reader in triplicate. Data are mean ± SE of values from triplicate experiments, with n = 3 per experiment.

U937 cells were incubated with the indicated amounts of BAPTA-AM (A) or BHA (B) for 1 h before incubation with VacA or iVacA (120 nM) in serum-free medium. After incubation for 60 min, the cells were solubilized, followed by SDS-PAGE in 10% gels and Western blotting using anti-phospho-p38 Ab. Results are representative of three independent experiments. Relative densities of phospho-p38 as determined by densitometry scan analysis were compared with densities obtained without VacA and inhibitor. Data are mean ± SE of values from triplicate experiments, with n = 3 per experiment. Statistical significance: ∗, p < 0.05.

To examine whether thapsigargin (TG), an endoplasmic reticulum Ca²⁺-ATPase inhibitor, induces IL-8 expression in U937 cells via increasing cytosolic free Ca²⁺ concentration induced by emptying Ca²⁺ stores, confluent U937 cells were treated with the indicated amounts of TG for 12 h in serum-free medium (A). In parallel with this experiment, U937 cells were incubated with 120 nM VacA or iVacA. IL-8 production was measured by ELISA. The data are representative of at least three experiments. To examine the effects of dantrolene, an inhibitor of intracellular Ca²⁺ channels, or SKF-96365, a blocker of Ca²⁺ influx, on VacA-induced IL-8 expression, confluent U937 cells were treated with the indicated amounts of Dantrolene (B) or SKF-96365 (C) for 1 h before incubation with 120 nM VacA or iVacA for 12 h in serum-free medium. IL-8 production was measured by ELISA. The data are representative of at least three experiments, with n = 3 per experiment. Statistical significance: ∗, p < 0.05; ∗∗, p < 0.01.

U937 cells were incubated with 120 nM VacA for the indicated times. Cell lysates were prepared and subjected to Western blot analyses using anti-phospho-ATF-2 (A) or anti-phospho-CREB (B) Abs. Relative amounts of phospho-ATF-2 and phospho-CREB, as determined by densitometry scan analysis, were compared with densities obtained at 0-min incubation. Data are mean ± SE of values from triplicate experiments. C, U937 cells were incubated with 120 nM VacA or iVacA at 37°C for 60 min. The cells were fixed using 2% paraformaldehyde, and permeabilized by incubation with 0.1% Triton X-100 for 5 min. Fixed cells, stained with 1 μg/ml DAPI for 5 min, were incubated with anti-phospho ATF-2 polyclonal Abs (1/100) in TBS containing 1% BSA. After treatment with the respective primary Abs, cells were incubated with secondary Ab in TBS containing 1% BSA, either anti-rabbit polyclonal Abs conjugated with Alexa fluor 546(1/1000) or anti-mouse polyclonal Abs conjugated with Alexa fluor 546(1/1000). Data are representative of three experiments. D, U937 cells were incubated with 120 nM activated VacA or iVacA at 37°C for 60 min. The cells were fixed using 2% paraformaldehyde, and permeabilized for 5 min with 0.1% Triton X-100. The fixed cells were stained with 1 μg/ml DAPI for 5 min, then incubated with anti-phospho-CREB polyclonal Abs (1/100) as primary Ab in TBS containing 1% BSA, followed by incubation with secondary Ab in TBS containing 1% BSA, either anti-rabbit polyclonal Abs conjugated with Alexa fluor 546(1/1000) or anti-mouse polyclonal Abs conjugated with Alexa fluor 546(1/1000).

U937 cells were grown overnight, and silencing of CREB or ATF-2 gene was performed with CREB-siRNA, ATF-2-siRNA, NC-siRNA, or without siRNA, as described in Materials and Methods. After a 24-h transfection, cells were suspended in serum-free medium and treated with VacA or iVacA for 2 h. A, Reduction of CREB, ATF-2, or GAPDH protein level was confirmed by Western blotting with anti-CREB, anti-ATF-2, or anti-GAPDH Abs (left upper panel), and relative amounts determined by densitometry scan analysis (bottom panels and right upper panel) were compared with densities obtained by mock transfection (without siRNA) or NC-siRNA transfection. The data are representative of at least two experiments, with n = 3 plates per experiment. B, U937 cells were grown overnight, and silencing of CREB or ATF-2 gene was performed with CREB-siRNA, ATF-2-siRNA, or NC-siRNA, as described in Materials and Methods. After 24-h transfection, cells were suspended in serum-free medium and treated with VacA or iVacA for 2 h. IL-8 production was measured by ELISA. The data are representative of at least three experiments, with n = 3 per experiment. Statistical significance: ∗, p < 0.05.

A, Schematic representation of wild-type and mutant IL-8 reporter constructs. The AP-1 site (−126 to −120; TGACTCA), NF-IL-6-like site (−94 to −81; CAGTTGCAAATCGT), or κB-like site (−80 to −71; GGAATTTCCT) in the IL-8 promoter (−133 to +44), linked to a luciferase reporter gene, was mutated to TatCTCA, agcTTGCAAATCGT, and taAcTTTCCT, respectively. B, Effect of point mutations in the IL-8 promoter on the inducibility of luciferase activity. U937 cells were transiently transfected with IL-8 promoter-luciferase reporter plasmids with the −133/+44, −98, −50, NF-κB mut, NF-IL-6 mut, or AP-1 mut promoters, as well as the reference plasmid pRL-CMV. Cells were either treated with 120 nM VacA or iVacA (0 or 6 h) at 37°C. Relative changes in luciferase expression were measured. □, Represent incubations with iVacA, and ▪, with VacA. Luciferase activity was normalized for Renilla luciferase activity. Data are means ± SD of values from three independent experiments, with n = 3 per experiment. Statistical significance: ∗, p < 0.05; ∗∗, p < 0.01.

A, Flow cytometry of CD14⁺ monocytes. Monocytes were generated from PBMC by autoMACS; >90% of the isolated cells were CD14⁺. B, Isolated CD14⁺ cells were incubated with 120 nM VacA, iVacA, or as negative control in medium alone. After 24 h, the supernatants were analyzed in a human IL-8 ELISA. Data are means ± SD of values from three independent experiments with assays in duplicate. Statistical significance: ∗, p < 0.01.