Helicobacter pylori-induced histone modification, associated gene expression in gastric epithelial cells, and its implication in pathogenesis

Abstract

Histone modifications are critical in regulating gene expression, cell cycle, cell proliferation, and development. Relatively few studies have investigated whether Helicobacter pylori, the major cause of human gastric diseases, affects histone modification. We therefore investigated the effects of H. pylori infection on histone modifications in a global and promoter-specific manner in gastric epithelial cells. Infection of gastric epithelial cells by wild-type H. pylori induced time- and dose-dependent dephosphorylation of histone H3 at serine 10 (H3 Ser10) and decreased acetylation of H3 lysine 23, but had no effects on seven other specific modifications. Different cag pathogenicity island (PAI)-containing-clinical isolates showed similar abilities to induce H3 Ser10 dephosphorylation. Mutation of cagA, vacA, nonphosphorylateable CagA mutant cagA(EPISA), or disruption of the flagella showed no effects, while deletion of the entire cagPAI restored the H3 Ser10 phosphorylation to control levels. Analysis of 27 cagPAI mutants indicated that the genes that caused H3 Ser10 dephosphorylation were similar to those that were previously found to induce interleukin-8, irrespective of CagA translocation. This effect was independent of ERK or p38 pathways and type I interferon signaling. Additionally, c-Jun and hsp70 gene expression was associated with this histone modification. These results demonstrate that H. pylori alters histone modification and host response via a cagA-, vacA-independent, but cagPAI-dependent mechanisms, which contribute to its persistent infection and pathogenesis.

Figure 1. H. pylori-induced histone H3 Ser10 dephosphorylation is cagPAI-dependent in AGS cells.

AGS cells (5×10⁵) were treated with medium alone, wild-type H. pylori 26695 (HP 26695), or an isogenic cag deletion strain (8-1) (HP 8-1) in antibiotic-free Ham's F-12 medium plus 5% FBS at MOI of 100:1 for various periods of time. The cells were washed, lysed and proteins were separated on a 15% SDS-polyacrylamide gel and transferred to nitrocellulose membrane, which was probed with rabbit anti-phospho-histone H3 Ser10 antibodies (p-H3S10). The original membrane was then stripped and re-probed with anti-total histone H3 antibodies to monitor protein loading. Blots are representative of three separate experiments with similar results (Panel A). Data are mean±SEM from three densitometry scans, adjusted with total histone H3, and expressed as fold changes over the appropriate control, *P<0.01 when compared with controls (Panel B).

Figure 2. H. pylori-induced histone H3 Ser10 dephosphorylation is cagPAI-dependent in MKN45 cells.

MKN45 cells (1×10⁶) were treated with medium alone, wild-type H. pylori 26695 (HP 26695), or an isogenic cag deletion strain (8-1) (HP 8-1) in RPMI-1640 medium plus 5% FBS at MOI of 100:1 for various periods of time. The cells were washed, lysed and proteins were separated on a 15% SDS-polyacrylamide gel and transferred to nitrocellulose membrane, which was probed with rabbit anti-phospho-histone H3 Ser10 antibodies (p-H3S10). The original membrane was then stripped and re-probed with anti-total histone H3 antibodies to monitor protein loading. Blots are representative of three separate experiments with similar results (Panel A). Data are mean±SEM from three densitometry scans, adjusted with total histone H3, and expressed as fold changes over the appropriate control, *P<0.01 when compared with controls (Panel B).

Figure 3. H. pylori-induced histone H3 Ser10 dephosphorylation is cagPAI-dependent in gastric epithelial cells.

H. pylori 26695 was added at MOI of 30:1 to 300:1 MOI to AGS (5×10⁵) or MKN45 (1×10⁶) in antibiotic-free Hams F-12 or RPMI-1640 medium with 5% FBS, respectively, for 6 hours. The cells were washed, lysed and proteins were separated on a 15% SDS-polyacrylamide gel and transferred to nitrocellulose membrane, which was probed with rabbit anti-phospho-histone H3 Ser10 antibodies (p-H3S10). The original membrane was then stripped and re-probed with anti-total histone H3 antibodies to monitor protein loading. Blots are representative of three separate experiments with similar results (Panels A and C). Data are mean±SEM from three densitometry scans, adjusted with total histone H3, and expressed as fold changes over the appropriate control, *P<0.01 when compared with controls (Panels B and D).

Figure 4. Effects of different H. pylori isolates on histone H3 Ser10 dephosphorylation in AGS cells.

AGS cells (5×10⁵) were treated with medium alone or with 9 different cag+ clinical isolates at MOI of 150:1 for 6 hours. The cells were subsequently lysed and proteins were separated on 15% SDS-polyacrylamide gel, transferred to nitrocellulose membrane and probed with rabbit anti-phosphohistone H3 Ser10 (p-H3S10). Anti-total histone H3 antibodies (H3 total) were re-probed to monitor protein loading. Blots are representative of three separate experiments with similar results (Panel A). Data are mean±SEM from three densitometry scans, adjusted with total histone H3, and expressed as fold changes over the appropriate control, *P<0.01 when compared with controls (Panel B).

Figure 5. Effects of different H. pylori isolates on histone H3 Ser10 dephosphorylation in MKN45 cells.

MKN45 cells (1×10⁶) were treated with medium alone or with 9 different cag+ clinical isolates at MOI of 150:1 for 6 hours. The cells were subsequently lysed and proteins were separated on 15% SDS-polyacrylamide gel, transferred to nitrocellulose membrane and probed with rabbit anti-phosphohistone H3 Ser10 (p-H3S10). Anti-total histone H3 antibodies (H3 total) were re-probed to monitor protein loading. Blots are representative of three separate experiments with similar results (Panel A). Data are mean±SEM from three densitometry scans, adjusted with total histone H3, and expressed as fold changes over the appropriate control, *P<0.01 when compared with controls (Panel B).

Figure 6. Effects of H. pylori mutants on histone H3 Ser10 dephosphorylation in AGS cells.

AGS cells (5×10⁵) were treated with H. pylori G27-MA (G27) or its isogenic mutation strains in antibiotic-free medium without FBS for 6 hours at MOIs of 150:1. Control cells were treated with medium alone. The cells were washed, lysed and proteins were separated on a 15% SDS-polyacrylamide gel and transferred to nitrocellulose membrane, which was probed with rabbit anti-phospho-histone H3 Ser10 antibodies (p-H3S10). The original membrane was then stripped and re-probed with anti-total histone H3 antibodies to monitor protein loading. Photos are representative of two to four separate experiments with similar results (Panel A). Data are mean±SEM from densitometry scans, and expressed as fold changes over control without bacteria treatment, *P<0.01 when compared with controls (Panel B).

Figure 7. Effects of H. pylori mutations in flaA or vacA on histone H3 Ser10 dephosphorylation in AGS cells.

AGS cells were treated with wild-type H. pylori strain 60190 (HP 60190) and its isogenic flagella mutant (HP 60190 FlaA/KO), 60190 and its isogenic vacA mutant (HP 60190 VacA/KO) for 6 hours. Control cells were treated with medium alone. The cells were washed, lysed and proteins were separated on a 15% SDS-polyacrylamide gel and transferred to nitrocellulose membrane, which was probed with rabbit anti-phospho-histone H3 Ser10 antibodies (p-H3S10). The original membrane was then stripped and re-probed with anti-total histone H3 antibodies to monitor protein loading. Photos are representative of two to four separate experiments with similar results (Panel A). Data are mean±SEM from densitometry scans, and expressed as fold changes over control without bacteria treatment, *P<0.01 when compared with controls (Panel B).

Figure 8. H. pylori-induced other histone modifications in AGS cells.

AGS cells (5×10⁵) were treated with medium alone, wild-type H. pylori 26695 (HP 26695), an isogenic cag deletion strain 8-1 (HP 8-1) in antibiotic-free medium, plus 5% FBS at MOI of 100:1 (Panel A) or different MOIs (Panel B) for 6 hours. Cells were subsequently lysed and proteins were separated on 15% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Various rabbit anti-histone modification antibodies were used for immunoblotting. Anti-total histone H3 antibodies were used to re-probe the membrane and monitor protein loading. Blots are representative of three separate experiments with similar results.

Figure 9. H. pylori-induced other histone modifications in gastric epithelial cells.

AGS cells were treated as described in Figure 8 using various rabbit anti-histone modification antibodies. Data are mean±SEM from three assays, and expressed as fold changes over the appropriate control, *P<0.01 when compared with controls.

Figure 10. H. pylori-induced other histone modifications in gastric epithelial cells.

MKN45 cells were stimulated with H. pylori 26695 or 8-1 as described in Figure 8 in the presence or absence of triscostatin A (TSA). Cells were collected for RNA extraction for subsequent quantitative RT-PCR assay using primers to il-8 (Panel A) and c-fos (Panel B). Data are mean±SEM from three assays, and expressed as fold changes over the appropriate control, *P<0.01 when compared with controls.

Figure 11. Gene expression associated with phospho-histone H3 Ser10 during H. pylori infection in AGS cells.

AGS cells (5×10⁶) were treated with H. pylori G27-MA (G27) strains in antibiotic-free medium with 5% FBS at MOIs of 150:1. The cells are collected either for ChIP assay (Panel A) or quantitative RT-PCR analysis (Panel B) as described in Materials and Methods. For ChIP assay, cells were collected at 6 hours, and rabbit anti-phospho-histone H3 Ser10 (p-H3-Ser10) antibodies were used for immunoprecipitation (IP). Anti-total histone H3 or irrelevant IgG was used as monitoring control. Data represent the IP/total input ratio and expressed as fold changes over control without H. pylori treatment (Panel A). For quantitative RT-PCR, cells were collected at 6 hours RNA was extracted for PCR analysis. Data represent the relative mRNA expression, adjusted with HPRT and expressed as fold changes over control without H. pylori treatment (Panel B). Results are mean±SEM from 2-4 separate experiments, *P<0.01 when compared with controls.

Figure 12. Effects of MAPK inhibition on phospho-histone H3 Ser10 expression during H. pylori infection in AGS cells.

AGS cells (5×10⁵) were treated with H. pylori G27-MA (HP) strains in antibiotic-free medium with 5% FBS at MOIs of 100:1 for four hours. MAPK inhibitors including MEK1/2 inhibitor U0126 (5 µM), p38 inhibitor SB203580 (5 µM, SB) and JNK inhibitor SP600125 (25 µM, SP) were treated 30 minutes prior to bacteria stimulation. The cells were lysed and proteins were separated on 15% SDS-polyacrylamide gel for detection of phospho-histone H3 Ser10 (p-H3S10). Anti-total H3 antibodies were used to re-probe the membrane and monitor protein loading (H3 total). Blots are representative of three separate experiments with similar results (Panel A). Data are mean±SEM from densitometry scans, and expressed as fold changes over control without bacteria treatment (Panel B), *P<0.01 when compared with controls.