Digestive Diseases and Sciences

, Volume 55, Issue 4, pp 988–996 | Cite as

Involvement of Ras and AP-1 in Helicobacter pylori-Induced Expression of COX-2 and iNOS in Gastric Epithelial AGS Cells

  • Soon Ok Cho
  • Joo Weon Lim
  • Kyung Hwan Kim
  • Hyeyoung Kim
Original Article


Helicobacter pylori (H. pylori) is an important risk factor for chronic gastritis, peptic ulcer, and gastric cancer. The genetic differences of H. pylori isolates play a role in the clinical outcome of the infection. Inflammatory genes including cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are involved in H. pylori gastritis. Transcription factor AP-1 is composed of c-Fos and c-Jun and mediates inflammation and carcinogenesis. Ras acts as a regulator for AP-1 activation in various cells. We investigated whether H. pylori in a Korean isolate (HP99), a cagA+, vacA+ strain, induces the expression of c-Fos and c-Jun for AP-1 activation to induce COX-2 and iNOS and whether HP99-induced expressions of COX-2 and iNOS are mediated by Ras and AP-1, determined by the expressions of c-Fos and c-Jun, in gastric epithelial AGS cells, using transfection with mutant genes for Ras (ras N-17) and c-Jun (TAM-67). As a result, HP99 induced the expression of c-Fos and c-Jun and the expressions of COX-2 and iNOS in AGS cells. Transfection with mutant genes for Ras or c-Jun suppressed HP99-induced expressions of COX-2 and iNOS in AGS cells. In conclusion, H. pylori in a Korean isolate induces the expression of COX-2 and iNOS via AP-1 activation, which may be mediated by Ras and the expression of c-Fos and c-Jun in gastric epithelial cells.


AP-1 Gastric epithelial cells Helicobacter pylori c-Fos c-Jun RAS 


  1. 1.
    Parsonnet J, Friedman GD, Vandersteen DP, et al. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med. 1991;325:1127–1131.PubMedGoogle Scholar
  2. 2.
    Kim H, Seo JY, Kim KH. Inhibition of lipid peroxidation, NF-κB activation and IL-8 production by rebamipide in Helicobacter pylori-stimulated gastric epithelial cells. Dig Dis Sci. 2000;45:621–628. doi:10.1023/A:1005474013988.CrossRefPubMedGoogle Scholar
  3. 3.
    Fu S, Ramanujam KS, Wong A, et al. Increased expression and cellular localization of inducible nitric oxide synthase and cyclooxygenase 2 in Helicobacter pylori gastritis. Gastroenterology. 1999;116:1319–1329. doi:10.1016/S0016-5085(99)70496-8.CrossRefPubMedGoogle Scholar
  4. 4.
    Tatsuguchi A, Sakamoto C, Wada K, et al. Localization of cyclooxygenase 1 and cyclooxygenase 2 in Helicobacter pylori related gastritis and gastric ulcer tissues in humans. Gut. 2000;46:782–789. doi:10.1136/gut.46.6.782.CrossRefPubMedGoogle Scholar
  5. 5.
    McCarthy CJ, Crofford LJ, Greenson J, Scheiman JM. Cyclooxygenase-2 expression in gastric antral mucosa before and after eradication of Helicobacter pylori infection. Am J Gastroenterol. 1999;94:1218–1223. doi:10.1111/j.1572-0241.1999.01070.x.CrossRefPubMedGoogle Scholar
  6. 6.
    Seibert K, Zhang Y, Leahy K. Pharmacological and biochemical demonstration of the role of cyclooxygenase 2 in inflammation and pain. Proc Natl Acad Sci USA. 1994;91:12013–12017. doi:10.1073/pnas.91.25.12013.CrossRefPubMedGoogle Scholar
  7. 7.
    Boolbol SK, Dannenberg AJ, Chadburn A, et al. Cyclooxygenase-2 overexpression and tumor formation are blocked by sulindac in a murine model of familial adenomatous polyposis. Cancer Res. 1996;56:2556–2560.PubMedGoogle Scholar
  8. 8.
    Son HJ, Rhee JC, Park DI, et al. Inducible nitric oxide synthase expression in gastroduodenal diseases infected with Helicobacter pylori. Helicobacter. 2001;6:37–43. doi:10.1046/j.1523-5378.2001.00004.x.CrossRefPubMedGoogle Scholar
  9. 9.
    Lechner M, Rieder J, Tilg H. Helicobacter pylori infection, iNOS, and gastric cancer: the impact of another possible link. J Surg Oncol. 2006;94:226–233. doi:10.1002/jso.20372.CrossRefGoogle Scholar
  10. 10.
    Muller JM, Rupec RA, Baeuerle PA. Study of gene regulation by NF-κB and AP-1 in response to reactive oxygen intermediates. Methods. 1997;11:301–312. doi:10.1006/meth.1996.0424.CrossRefPubMedGoogle Scholar
  11. 11.
    Chu SH, Kim H, Seo JY, Lim JW, Mukaida N, Kim KH. Role of NF-κB and AP-1 on Helicobater pylori-induced IL-8 expression in AGS cells. Dig Dis Sci. 2003;48:257–265. doi:10.1023/A:1021963007225.CrossRefPubMedGoogle Scholar
  12. 12.
    Seo JH, Kim H, Kim KH. Cyclooxygenase-2 expression by transcription factors in Helicobacter pylori–infected gastric epithelial cells: Comparison between HP99 and NCTC 11637. Ann N Y Acad Sci. 2002;937:477–480.CrossRefGoogle Scholar
  13. 13.
    Kim H, Seo JY, Kim KH. Effects of mannitol and dimethylthiourea on Helicobacter pylori -induced IL-8 production in gastric epithelial cells. Pharmacology. 1999;59:201–211. doi:10.1159/000028321.CrossRefPubMedGoogle Scholar
  14. 14.
    Seo JY, Kim H, Kim KH. Transcriptional regulation by thiol compounds in Helicobacter pylori -induced interleukin-8 production in human gastric epithelial cells. Ann N Y Acad Sci. 2002;973:541–545.CrossRefPubMedGoogle Scholar
  15. 15.
    Kim H, Seo JY, Kim KH. Effect of mannitol on Helicobacter pylori-induced cyclooxygenase-2 expression in gastric epithelial AGS cells. Pharmacology. 2002;66:182–189. doi:10.1159/000065532.CrossRefPubMedGoogle Scholar
  16. 16.
    Lim JW, Kim H, Kim KH. NF-κB, inducible nitric oxide synthase and apoptosis by Helicobacter pylori infection. Free Radic Biol Med. 2001;31:355–366. doi:10.1016/S0891-5849(01)00592-5.CrossRefPubMedGoogle Scholar
  17. 17.
    Seo JH, Lim JW, Kim H, Kim KH. Helicobacter pylori in a Korean isolate activates mitogen-activated protein kinases, AP-1, and NF-κB and induces chemokine expression in gastric epithelial AGS cells. Lab Invest. 2004;84:49–62. doi:10.1038/labinvest.3700010.CrossRefPubMedGoogle Scholar
  18. 18.
    Angel P, Karin M. The role of Jun, Fos and the AP-1 complex in cell proliferation and transformation. Biochim Biophys Acta. 1991;1072:129–139.PubMedGoogle Scholar
  19. 19.
    Wasylyk C, Imler JL, Wasylyk B. Transforming but not immortalizing oncogenes activate the transcription factor PEA1. EMBO J. 1988;7:2475–2483.PubMedGoogle Scholar
  20. 20.
    Schonthal A, Herrlich P, Rahmsdorf HJ, Ponta H. Requirement for fos gene expression in the transcriptional activation of collagenase by other oncogenes and phorbol esters. Cell. 1988;54:325–334. doi:10.1016/0092-8674(88)90195-X.CrossRefPubMedGoogle Scholar
  21. 21.
    Herrlich P, Ponta H. Nuclear oncogenes convert extracellular stimuli into changes in the genetic program. Trends Genet. 1989;5:112–115. doi:10.1016/0168-9525(89)90041-3.CrossRefPubMedGoogle Scholar
  22. 22.
    Sistomen L, Hotta E, Makella TP. Keski Oja, Alitalo K: The cellular response to induction of the p21 c-Ha-ras oncoprotein includes stimulation of jun gene expression. EMBO J. 1989;8:815–822.Google Scholar
  23. 23.
    Keates S, Sougioultzis S, Keates AC, et al. Cag+ Helicobacter pylori induces transactivation of the epidermal growth factor receptor in AGS gastric epithelial cells. J Biol Chem. 2001;276:48127–48134. doi:10.1074/jbc.M107838200.CrossRefPubMedGoogle Scholar
  24. 24.
    Saadat I, Higashi H, Obuse C, et al. Helicobacter pylori CagA targets PAR1/MAPK kinase to disrupt epithelial cell polarity. Nature. 2008;447:330–334. doi:10.1038/nature05765.CrossRefGoogle Scholar
  25. 25.
    Kwok T, Zabler D, Urman S, et al. Helicobacter exploits integrin for typeIV secretion and kinase activation. Nature. 2007;449:862–866. doi:10.1038/nature06187.CrossRefPubMedGoogle Scholar
  26. 26.
    Snider JL, Allison C, Bellaire BH, Ferrero RL, Cardelli JA. The beta1 integrin activates JNK independent of CgA, and JNK activation is required for Helicobacter pylori CagA+-induced motility of gastric cancer cells. J Biol Chem. 2008;283:13952–13963. doi:10.1074/jbc.M800289200.CrossRefPubMedGoogle Scholar
  27. 27.
    Pillinger MH, Marjanovic N, Kim SY, et al. Helicobacter pylori stimulates gastric epithelial cell MMP-1 secretion via CagA-dependent and–independent ERK activation. J Biol Chem. 2007;282:18722–18731. doi:10.1074/jbc.M703022200.CrossRefPubMedGoogle Scholar
  28. 28.
    Brown PH, Chen TK, Birrer MJ. Mechanism of action of a dominant-negative mutant of c-Jun. Oncogene. 1994;9:791–799.PubMedGoogle Scholar
  29. 29.
    Cai H, Szeberenyi J, Cooper GM. Effect of a dominant inhibitory Ha-ras mutation on mitogenic signal transduction in NIH 3T3 cells. Mol Cell Biol. 1990;10:5314–5323.PubMedGoogle Scholar
  30. 30.
    Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidine isothiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159. doi:10.1016/0003-2697(87)90021-2.CrossRefPubMedGoogle Scholar
  31. 31.
    Hla T, Neilson KO. Human cyclooxygenase-2 cDNA. Proc Natl Acad Sci USA. 1992;89:7384–7388. doi:10.1073/pnas.89.16.7384.CrossRefPubMedGoogle Scholar
  32. 32.
    Chartrain NA, Geller DA, Koty PP, Sitrin NF, Nussler AK, Hoffman EP. Molecular cloning, structure, and chromosomal localization of the human inducible nitric oxide synthase gene. J Biol Chem. 1994;269:6765–6772.PubMedGoogle Scholar
  33. 33.
    Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254. doi:10.1016/0003-2697(76)90527-3.CrossRefPubMedGoogle Scholar
  34. 34.
    Misko TP, Schilling RJ, Salvemini D, Moore WM, Currie MG. A fluorometic assay for the measurement of nitrite in biologic samples. Anal Biochem. 1993;214:11–16. doi:10.1006/abio.1993.1449.CrossRefPubMedGoogle Scholar
  35. 35.
    Zar BH. Biostatistical Analysis. 2nd ed. Englewood Cliffs, New Jersey: Prentice-Hall; 1984.Google Scholar
  36. 36.
    Zinck R, Cahill MA, Kracht M. Protein synthesis inhibitors reveal differential regulation of mitogen-activated protein kinase and streaa-activated protein kinase pathways that converge on Elk-1. Mol Cell Biol. 1995;15:4930–4938.PubMedGoogle Scholar
  37. 37.
    Rinegaud J, Whitmarsh AJ, Barrett T. MKK3- and MKK6-regulated gene expression is mediated by the p38 mitogen-activated protein kinase signal transduction pathway. Mol Cell Biol. 1996;16:1247–1255.Google Scholar
  38. 38.
    Hipskind RA, Rao VN, Mueller CG, Nordheim A. Ets-related Elk-1 is homologous to the c-fos regulatory factor p62TCF. Nature. 1991;354:531–534. doi:10.1038/354531a0.CrossRefPubMedGoogle Scholar
  39. 39.
    Derijard B, Hibi M, Wu IH. JNK1: A protein kinase stimulated by UV light and Ha-Ras that binds and phosphorylates the c-Jun activation domain. Cell. 1994;76:1025–1037. doi:10.1016/0092-8674(94)90380-8.CrossRefPubMedGoogle Scholar
  40. 40.
    Cichocki M, Paluszczak J, Szaefer H, Piechowiak A, Rimando AM, Baer-Dubowska W. Pterostibene is equally potent as reseveratrol in inhibiting 12-O-tetradecanoylphorbol-13-acetate activate NF-kappa B, AP-1, COX-2, and iNOS in mouse epidermis. Mol Nutr Food Res. 2008;52(Suppl 1):S62–S70. doi:10.1002/mnfr.200700395.PubMedGoogle Scholar
  41. 41.
    Chittezhath M, Deep G, Singh RP, Agarwal C, Agarwal R. Sillibinin inhibits cytokine-induced signaling cascade and down-regulates inducibe nitric oxide synthase in human lung carcinoma A549 cells. Mol Cancer Ther. 2008;7:1817826. doi:10.1158/1535-7163.MCT-08-0256.CrossRefGoogle Scholar
  42. 42.
    Chang YJ, Wu MS, Lin JT, Chen CC. Helicobacter pylori-induced invasion and angiogenesis of gastric cells is mediated by cyclooxygenase-2 induction through TLR2/TKR9 and promoter regulation. J Immunol. 2005;175:8242–8252.PubMedGoogle Scholar
  43. 43.
    Zhang X, Ruiz B, Correa P, Miller MJ. Cellular dissociation of NF-kappaB and inducible nitric oxide synthase in Helicobacter pylori infection. Free Radic Biol Med. 2000;29:730–735. doi:10.1016/S0891-5849(00)00375-0.CrossRefPubMedGoogle Scholar
  44. 44.
    Cho SO, Kim KH, Yoon JH, Kim H. Signaling for integrin alpha5/beta1 expression in Helicobacter pylori-infected gastric epithelial AGS cells. Ann N Y Acad Sci. 2006;1090:298–304. doi:10.1196/annals.1378.032.CrossRefPubMedGoogle Scholar
  45. 45.
    Brandt S, Shafikhani S, Balachandran P, et al. Use of a novel coinfection system reveals a role for Rac1, H-Ras, and CrkII phosphorylation in Helicobacter pylori-induced host cell actin cytoskeletal rearrangements. FEMS Immunol Med Microbiol. 2007;50:190–205. doi:10.1111/j.1574-695X.2007.00234.x.CrossRefPubMedGoogle Scholar
  46. 46.
    Brandt S, Kwok T, Hartig R, König W, Backert S. NF-kappaB activation and potentiation of proinflammatory responses by the Helicobacter pylori CagA protein. Proc Natl Acad Sci USA. 2005;102:9300–9305. doi:10.1073/pnas.0409873102.CrossRefPubMedGoogle Scholar
  47. 47.
    Lu H, Wu JY, Kudo T, Ohno T, Graham DY, Yamaoka Y. Regulation of interleukin-6 promoter activation in gastric epithelial cells infected with Helicobacter pylori. Mol Biol Cell. 2005;16:4954–4966. doi:10.1091/mbc.E05-05-0426.CrossRefPubMedGoogle Scholar
  48. 48.
    Zhu Y, Zhong X, Zheng S, Du Q, Xu W. Transformed immortalized gastric epithelial cells by virulence factor CagA of Helicobacter pylori through Erk mitogen-activated protein kinase pathway. Oncogene. 2005;24:3886–3895. doi:10.1038/sj.onc.1208551.CrossRefPubMedGoogle Scholar
  49. 49.
    Binetruy B, Smeal T, Karin M. Ha-Ras augments c-Jun activity and stimulates phosphorylation of its activation domain. Nature. 1991;351:122–127. doi:10.1038/351122a0.CrossRefPubMedGoogle Scholar
  50. 50.
    Takehara H, Iwamoto J, Mizokami Y, et al. Involvement of cyclooxygenase-2-prostaglandin E2 pathway in interleukin-8 production in gastric cancer cells. Dig Dis Sci. 2006;51:2188–2197. doi:10.1007/s10620-006-9436-2.CrossRefPubMedGoogle Scholar
  51. 51.
    Chang YJ, Wu MS, Lin JT, Pestell RG, Blaser MJ, Chen CC. Mechanisms of Helicobacter pylori CagA-induced cyclin D1 expression that affect cell cycle. Cell Microbiol. 2006;8:1740–1752. doi:10.1111/j.1462-5822.2006.00743.x.CrossRefPubMedGoogle Scholar
  52. 52.
    Cao X, Tsukamoto T, Seki T, et al. 4-Vinyl-2, 6-dimethoxyphenol (canolol) suppresses oxidative stress and gastric carcinogenesis in Helicobacter pylori-infected carcinogen-treated Mongolian gerbils. Int J Cancer. 2008;122:1445–1454. doi:10.1002/ijc.23245.CrossRefPubMedGoogle Scholar
  53. 53.
    Rachmilewitz D, Karmeli F, Eliakim R, et al. Enhanced gastric nitric oxide synthase activity in duodenal ulcer patients. Gut. 1994;35:1394–1397. doi:10.1136/gut.35.10.1394.CrossRefPubMedGoogle Scholar
  54. 54.
    Touati E, Michel V, Thiberge JM, Wuscher N, Huerre M, Labigne A. Chronic Helicobacter pylori infections induce gastric mutations in mice. Gastroenterology. 2003;124:1408–1419. doi:10.1016/S0016-5085(03)00266-X.CrossRefPubMedGoogle Scholar
  55. 55.
    Toyoda T, Tsukamoto T, Hirano N, et al. Synergistic upregulation of inducible nitric oxide synthase and cyclooxygenase-2 in gastric mucosa of Mongoliaan gerbils by a high-salt diet and Helicobacter pylori infection. Histol Histopathol. 2008;23:593–599.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Soon Ok Cho
    • 1
  • Joo Weon Lim
    • 2
  • Kyung Hwan Kim
    • 1
  • Hyeyoung Kim
    • 3
  1. 1.Department of PharmacologyBrain Korea 21 Project for Medical SciencesSeoulKorea
  2. 2.Institute of GastroenterologyYonsei University College of MedicineSeoulKorea
  3. 3.Department of Food and Nutrition, Brain Korea 21 ProjectYonsei University College of Human EcologySeoulKorea

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