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. 2001 Jul;49(1):18–22. doi: 10.1136/gut.49.1.18

Helicobacter pylori induced transactivation of SRE and AP-1 through the ERK signalling pathway in gastric cancer cells

Y Mitsuno 1, H Yoshida 1, S Maeda 1, K Ogura 1, Y Hirata 1, T Kawabe 1, Y Shiratori 1, M Omata 1
PMCID: PMC1728350  PMID: 11413105

Abstract

BACKGROUND AND AIMS—Helicobacter pylori infection induces expression of proinflammatory cytokines such as interleukin (IL)-8 and tumour necrosis factor α (TNF-α) in gastric mucosa, and their genes have AP-1 binding sites in the promoter region. c-Fos is important for transactivation of AP-1 which has SRE in the promoter region. We conducted this study to confirm H pylori induced transactivation of these binding sites.
METHODS—Transactivation of SRE and AP-1 was evaluated in human gastric cancer cells TMK1 and MKN45 by luciferase reporter assay in transient transfection. We compared the effects of coculture with four H pylori strains, a cag pathogenicity island (PAI) positive strain TN2, its isogenic vacA negative (TN2-ΔvacA) or cagE negative (TN2-ΔcagE) mutants, and a cag PAI negative clinical isolate T68. Phosphorylation of ERK1/2, JNK, and c-Jun was measured by immunoblot, induction of IL-8 secretion by ELISA, and the effects of MEK by inhibitor U0126.
RESULTS—Both SRE and AP-1 were transactivated by coculture with TN2. Although TN2-ΔvacA induced comparable transactivation, TN2-ΔcagE and T68 showed decreased transactivation of SRE (65% and 51%) and AP-1 (71% and 54%, respectively, of TN2). Heat killed TN2 or indirect contact using a permeable membrane inhibited transactivation. Levels of phosphorylated ERK1/2, JNK, and c-Jun were increased by coculture with TN2. MEK inhibitor U0126 reduced TN2 induced transactivation of SRE and AP1, as well as secretion of IL-8, by 83%, 87%, and 53%, respectively, of TN2.
CONCLUSIONS—Transactivation of SRE and AP-1, through ERK/MAPK and JNK/SAPK cascades, respectively, was found in gastric cancer cells cocultured with H pylori. Direct contact with viable bacteria possessing intact cag PAI is a prerequisite for the onset of intracellular signalling leading to AP-1 transactivation.


Keywords: Helicobacter pylori; SRE; AP-1; cag pathogenicity island PAI; gastric cancer

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Figure 1  .

Figure 1  

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Transactivation of SRE by Helicobacter pylori. Transactivation of SRE in TMK1 (A) and MKN45 (B) cells induced by H pylori was measured by luciferase assay using pSRE-Luc. The TN2 strain was positive for two known virulence factors, CagA and VacA, and had intact cag pathogenicity island (PAI). The T68 strain, isolated from a Japanese patient at our institution, was negative for the above two virulence factors and lacked cag PAI. An isogenic cagE negative mutant (TN2-ΔcagE) was constructed by inserting a kanamycin resistant gene cassette into the cagE locus of cag PAI of TN2. The isogenic vacA negative mutant (TN2-ΔvacA) was constructed by disrupting the vacA gene of TN2. Luciferase activity is presented as a fold induction relative to basal levels measured in untreated cells. Mean (SD) values of four independent experiments are shown. *p<0.05 compared with TN2 by Dunnett's multiple comparison.

Figure 2  .

Figure 2  

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Transactivation of AP-1 induced by Helicobacter pylori. Transactivation of AP-1 in TMK1 (A) and MKN45 (B) cells induced by H pylori was measured by luciferase assay using pAP-1-Luc. The TN2 strain was positive for two known virulence factors, CagA and VacA, and had intact cag pathogenicity island (PAI). The T68 strain, isolated from a Japanese patient at our institution, was negative for the above two virulence factors and lacked cag PAI. An isogenic cagE negative mutant (TN2-ΔcagE) was constructed by inserting a kanamycin resistant gene cassette into the cagE locus of cag PAI of TN2. The isogenic vacA negative mutant (TN2-ΔvacA) was constructed by disrupting the vacA gene of TN2. Luciferase activity is presented as a fold induction relative to basal levels measured in untreated cells. Mean (SD) values of four independent experiments are shown. *p<0.05 compared with TN2 by Dunnett's multiple comparison.

Figure 3  .

Figure 3  

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Phosphorylation of ERK1/2 by Helicobacter pylori (HP). Immunoblot analysis showing phospho-ERK1/2 (top panel) and total ERK1/2 (bottom panel) in TMK1 cells cocultured with H pylori. Antibodies used react with both ERK1 and ERK2. Stimulation by epidermal growth factor (EGF 10 ng/ml for 60 minutes) was used as a positive control.

Figure 4  .

Figure 4  

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Phosphorylation of JNK/SAPK by Helicobacter pylori (HP). Immunoblot analysis showing phospho JNK (top panel) and total JNK (bottom panel) in TMK1 cells cocultured with H pylori. Stimulation by interleukin 1β (IL-1β 10 ng/ml for 60 minutes) was used as a positive control.

Figure 5  .

Figure 5  

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Phosphorylation of c-Jun by Helicobacter pylori (HP). Immunoblot analysis showing phospho c-JUN (top panel) and total c-JUN (bottom panel) in TMK1 cells cocultured with H pylori. Stimulation by interleukin 1β (IL-1β 10 ng/ml for 60 min) was used as a positive control.

Figure 6  .

Figure 6  

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Effects of MEK inhibitor on Helicobacter pylori induced transactivation of SRE and AP-1. Transactivation of SRE and AP-1 in TMK1 cells induced by H pylori was measured by luciferase assay using pSRE-Luc and pAP-1-Luc with (+) or without (−) the MEK inhibitor U0126 (10 µM). Luciferase activity is presented as a fold induction relative to basal levels measured in untreated cells. Mean (SD) values of three independent experiments are shown. *p<0.05 by unpaired Student's t test.

Figure 7  .

Figure 7  

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Effects of MEK inhibitor on Helicobacter pylori induced interleukin 8 (IL-8) secretion. TMK1 cells were cultured alone as control or were cocultured with H pylori with (+) or without (−) the MEK inhibitor U0126 (10 µM). Supernatant IL-8 concentrations are shown. Mean (SD) values of three independent experiments. *p<0.05 by unpaired Student's t test.

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