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. 1998 Apr 15;101(8):1604–1613. doi: 10.1172/JCI1174

Helicobacter pylori upregulates expression of epidermal growth factor-related peptides, but inhibits their proliferative effect in MKN 28 gastric mucosal cells.

M Romano 1, V Ricci 1, A Di Popolo 1, P Sommi 1, C Del Vecchio Blanco 1, C B Bruni 1, U Ventura 1, T L Cover 1, M J Blaser 1, R J Coffey 1, R Zarrilli 1
PMCID: PMC508741  PMID: 9541490

Abstract

Acute exposure to Helicobacter pylori causes cell damage and impairs the processes of cell migration and proliferation in cultured gastric mucosal cells in vitro. EGF-related growth factors play a major role in protecting gastric mucosa against injury, and are involved in the process of gastric mucosal healing. We therefore studied the acute effect of H. pylori on expression of EGF-related growth factors and the proliferative response to these factors in gastric mucosal cells (MKN 28) derived from gastric adenocarcinoma. Exposure of MKN 28 cells to H. pylori suspensions or broth culture filtrates upregulated mRNA expression of amphiregulin (AR) and heparin-binding EGF-like growth factor (HB-EGF), but not TGFalpha. This effect was specifically related to H. pylori since it was not observed with E. coli, and was independent of VacA, CagA, PicA, PicB, or ammonia. Moreover, H. pylori broth culture filtrates stimulated extracellular release of AR and HB-EGF protein by MKN 28 cells. AR and HB-EGF dose-dependently and significantly stimulated proliferation of MKN 28 cells in the absence of H. pylori filtrate, but had no effect in the presence of H. pylori broth culture filtrates. Inhibition of AR- or HB-EGF- induced stimulation of cell growth was not mediated by downregulation of the EGF receptor since EGF receptor protein levels, EGF binding affinity, number of specific binding sites for EGF, or HB-EGF- or AR-dependent tyrosine phosphorylation of the EGF receptor were not significantly altered by incubation with H. pylori broth culture filtrates. Increased expression of AR and HB-EGF were mediated by an H. pylori factor > 12 kD in size, whereas antiproliferative effects were mediated by both VacA and a factor < 12 kD in size. We conclude that H. pylori increases mucosal generation of EGF-related peptides, but in this acute experimental model, this event is not able to counteract the inhibitory effect of H. pylori on cell growth. The inhibitory effect of H. pylori on the reparative events mediated by EGF-related growth factors might play a role in the pathogenesis of H. pylori-induced gastroduodenal injury.

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Selected References

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  1. Aaronson S. A. Growth factors and cancer. Science. 1991 Nov 22;254(5035):1146–1153. doi: 10.1126/science.1659742. [DOI] [PubMed] [Google Scholar]
  2. Auricchio F., Di Domenico M., Migliaccio A., Castoria G., Bilancio A. The role of estradiol receptor in the proliferative activity of vanadate on MCF-7 cells. Cell Growth Differ. 1995 Feb;6(2):105–113. [PubMed] [Google Scholar]
  3. Barnard J. A., Beauchamp R. D., Russell W. E., Dubois R. N., Coffey R. J. Epidermal growth factor-related peptides and their relevance to gastrointestinal pathophysiology. Gastroenterology. 1995 Feb;108(2):564–580. doi: 10.1016/0016-5085(95)90087-x. [DOI] [PubMed] [Google Scholar]
  4. Blaser M. J. Hypotheses on the pathogenesis and natural history of Helicobacter pylori-induced inflammation. Gastroenterology. 1992 Feb;102(2):720–727. doi: 10.1016/0016-5085(92)90126-j. [DOI] [PubMed] [Google Scholar]
  5. Blaser M. J., Perez-Perez G. I., Kleanthous H., Cover T. L., Peek R. M., Chyou P. H., Stemmermann G. N., Nomura A. Infection with Helicobacter pylori strains possessing cagA is associated with an increased risk of developing adenocarcinoma of the stomach. Cancer Res. 1995 May 15;55(10):2111–2115. [PubMed] [Google Scholar]
  6. Censini S., Lange C., Xiang Z., Crabtree J. E., Ghiara P., Borodovsky M., Rappuoli R., Covacci A. cag, a pathogenicity island of Helicobacter pylori, encodes type I-specific and disease-associated virulence factors. Proc Natl Acad Sci U S A. 1996 Dec 10;93(25):14648–14653. doi: 10.1073/pnas.93.25.14648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen M. C., Lee A. T., Soll A. H. Mitogenic response of canine fundic epithelial cells in short-term culture to transforming growth factor alpha and insulinlike growth factor I. J Clin Invest. 1991 May;87(5):1716–1723. doi: 10.1172/JCI115189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  9. Ciacci C., Zarrilli R., Ricci V., De Luca A., Mazzacca G., Del Vecchio Blanco C., Romano M. Histamine H2-receptor antagonists stimulate proliferation but not migration of human gastric mucosal cells in vitro. Dig Dis Sci. 1996 May;41(5):972–978. doi: 10.1007/BF02091539. [DOI] [PubMed] [Google Scholar]
  10. Cook P. W., Pittelkow M. R., Keeble W. W., Graves-Deal R., Coffey R. J., Jr, Shipley G. D. Amphiregulin messenger RNA is elevated in psoriatic epidermis and gastrointestinal carcinomas. Cancer Res. 1992 Jun 1;52(11):3224–3227. [PubMed] [Google Scholar]
  11. Correa P., Shiao Y. H. Phenotypic and genotypic events in gastric carcinogenesis. Cancer Res. 1994 Apr 1;54(7 Suppl):1941s–1943s. [PubMed] [Google Scholar]
  12. Coso O. A., Chiariello M., Yu J. C., Teramoto H., Crespo P., Xu N., Miki T., Gutkind J. S. The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway. Cell. 1995 Jun 30;81(7):1137–1146. doi: 10.1016/s0092-8674(05)80018-2. [DOI] [PubMed] [Google Scholar]
  13. Covacci A., Censini S., Bugnoli M., Petracca R., Burroni D., Macchia G., Massone A., Papini E., Xiang Z., Figura N. Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer. Proc Natl Acad Sci U S A. 1993 Jun 15;90(12):5791–5795. doi: 10.1073/pnas.90.12.5791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cover T. L., Blaser M. J. Purification and characterization of the vacuolating toxin from Helicobacter pylori. J Biol Chem. 1992 May 25;267(15):10570–10575. [PubMed] [Google Scholar]
  15. Cover T. L., Glupczynski Y., Lage A. P., Burette A., Tummuru M. K., Perez-Perez G. I., Blaser M. J. Serologic detection of infection with cagA+ Helicobacter pylori strains. J Clin Microbiol. 1995 Jun;33(6):1496–1500. doi: 10.1128/jcm.33.6.1496-1500.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cover T. L., Tummuru M. K., Cao P., Thompson S. A., Blaser M. J. Divergence of genetic sequences for the vacuolating cytotoxin among Helicobacter pylori strains. J Biol Chem. 1994 Apr 8;269(14):10566–10573. [PubMed] [Google Scholar]
  17. Derynck R., Roberts A. B., Winkler M. E., Chen E. Y., Goeddel D. V. Human transforming growth factor-alpha: precursor structure and expression in E. coli. Cell. 1984 Aug;38(1):287–297. doi: 10.1016/0092-8674(84)90550-6. [DOI] [PubMed] [Google Scholar]
  18. Dignass A. U., Podolsky D. K. Cytokine modulation of intestinal epithelial cell restitution: central role of transforming growth factor beta. Gastroenterology. 1993 Nov;105(5):1323–1332. doi: 10.1016/0016-5085(93)90136-z. [DOI] [PubMed] [Google Scholar]
  19. Dluz S. M., Higashiyama S., Damm D., Abraham J. A., Klagsbrun M. Heparin-binding epidermal growth factor-like growth factor expression in cultured fetal human vascular smooth muscle cells. Induction of mRNA levels and secretion of active mitogen. J Biol Chem. 1993 Aug 25;268(24):18330–18334. [PubMed] [Google Scholar]
  20. Dunn B. E. Pathogenic mechanisms of Helicobacter pylori. Gastroenterol Clin North Am. 1993 Mar;22(1):43–57. [PubMed] [Google Scholar]
  21. Ghiara P., Marchetti M., Blaser M. J., Tummuru M. K., Cover T. L., Segal E. D., Tompkins L. S., Rappuoli R. Role of the Helicobacter pylori virulence factors vacuolating cytotoxin, CagA, and urease in a mouse model of disease. Infect Immun. 1995 Oct;63(10):4154–4160. doi: 10.1128/iai.63.10.4154-4160.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hashimoto K., Higashiyama S., Asada H., Hashimura E., Kobayashi T., Sudo K., Nakagawa T., Damm D., Yoshikawa K., Taniguchi N. Heparin-binding epidermal growth factor-like growth factor is an autocrine growth factor for human keratinocytes. J Biol Chem. 1994 Aug 5;269(31):20060–20066. [PubMed] [Google Scholar]
  23. Higashiyama S., Abraham J. A., Miller J., Fiddes J. C., Klagsbrun M. A heparin-binding growth factor secreted by macrophage-like cells that is related to EGF. Science. 1991 Feb 22;251(4996):936–939. doi: 10.1126/science.1840698. [DOI] [PubMed] [Google Scholar]
  24. Homma T., Sakai M., Cheng H. F., Yasuda T., Coffey R. J., Jr, Harris R. C. Induction of heparin-binding epidermal growth factor-like growth factor mRNA in rat kidney after acute injury. J Clin Invest. 1995 Aug;96(2):1018–1025. doi: 10.1172/JCI118087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Johnson G. R., Prigent S. A., Gullick W. J., Stromberg K. Characterization of high and low molecular weight forms of amphiregulin that differ in glycosylation and peptide core length. Evidence that the NH2-terminal region is not critical for bioactivity. J Biol Chem. 1993 Sep 5;268(25):18835–18843. [PubMed] [Google Scholar]
  26. Konturek P. C., Brzozowski T., Konturek S. J., Ernst H., Drozdowicz D., Pajdo R., Hahn E. G. Expression of epidermal growth factor and transforming growth factor alpha during ulcer healing. Time sequence study. Scand J Gastroenterol. 1997 Jan;32(1):6–15. doi: 10.3109/00365529709025056. [DOI] [PubMed] [Google Scholar]
  27. Konturek S. J., Brzozowski T., Majka J., Dembinski A., Slomiany A., Slomiany B. L. Transforming growth factor alpha and epidermal growth factor in protection and healing of gastric mucosal injury. Scand J Gastroenterol. 1992 Aug;27(8):649–655. doi: 10.3109/00365529209000134. [DOI] [PubMed] [Google Scholar]
  28. Lee A., Fox J., Hazell S. Pathogenicity of Helicobacter pylori: a perspective. Infect Immun. 1993 May;61(5):1601–1610. doi: 10.1128/iai.61.5.1601-1610.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lynch D. A., Mapstone N. P., Clarke A. M., Sobala G. M., Jackson P., Morrison L., Dixon M. F., Quirke P., Axon A. T. Cell proliferation in Helicobacter pylori associated gastritis and the effect of eradication therapy. Gut. 1995 Mar;36(3):346–350. doi: 10.1136/gut.36.3.346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McPherson G. A. Analysis of radioligand binding experiments. A collection of computer programs for the IBM PC. J Pharmacol Methods. 1985 Nov;14(3):213–228. doi: 10.1016/0160-5402(85)90034-8. [DOI] [PubMed] [Google Scholar]
  31. Murayama Y., Miyagawa J., Higashiyama S., Kondo S., Yabu M., Isozaki K., Kayanoki Y., Kanayama S., Shinomura Y., Taniguchi N. Localization of heparin-binding epidermal growth factor-like growth factor in human gastric mucosa. Gastroenterology. 1995 Oct;109(4):1051–1059. doi: 10.1016/0016-5085(95)90562-6. [DOI] [PubMed] [Google Scholar]
  32. Nomura A., Stemmermann G. N., Chyou P. H., Kato I., Perez-Perez G. I., Blaser M. J. Helicobacter pylori infection and gastric carcinoma among Japanese Americans in Hawaii. N Engl J Med. 1991 Oct 17;325(16):1132–1136. doi: 10.1056/NEJM199110173251604. [DOI] [PubMed] [Google Scholar]
  33. Parsonnet J., Friedman G. D., Vandersteen D. P., Chang Y., Vogelman J. H., Orentreich N., Sibley R. K. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med. 1991 Oct 17;325(16):1127–1131. doi: 10.1056/NEJM199110173251603. [DOI] [PubMed] [Google Scholar]
  34. Playford R. J., Wright N. A. Why is epidermal growth factor present in the gut lumen? Gut. 1996 Mar;38(3):303–305. doi: 10.1136/gut.38.3.303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Polk W. H., Jr, Dempsey P. J., Russell W. E., Brown P. I., Beauchamp R. D., Barnard J. A., Coffey R. J., Jr Increased production of transforming growth factor alpha following acute gastric injury. Gastroenterology. 1992 May;102(5):1467–1474. doi: 10.1016/0016-5085(92)91703-7. [DOI] [PubMed] [Google Scholar]
  36. Ricci V., Ciacci C., Zarrilli R., Sommi P., Tummuru M. K., Del Vecchio Blanco C., Bruni C. B., Cover T. L., Blaser M. J., Romano M. Effect of Helicobacter pylori on gastric epithelial cell migration and proliferation in vitro: role of VacA and CagA. Infect Immun. 1996 Jul;64(7):2829–2833. doi: 10.1128/iai.64.7.2829-2833.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Riese D. J., Kim E. D., Elenius K., Buckley S., Klagsbrun M., Plowman G. D., Stern D. F. The epidermal growth factor receptor couples transforming growth factor-alpha, heparin-binding epidermal growth factor-like factor, and amphiregulin to Neu, ErbB-3, and ErbB-4. J Biol Chem. 1996 Aug 16;271(33):20047–20052. doi: 10.1074/jbc.271.33.20047. [DOI] [PubMed] [Google Scholar]
  38. Romano M., Lesch C. A., Meise K. S., Veljaca M., Sanchez B., Kraus E. R., Boland C. R., Guglietta A., Coffey R. J. Increased gastroduodenal concentrations of transforming growth factor alpha in adaptation to aspirin in monkeys and rats. Gastroenterology. 1996 May;110(5):1448–1455. doi: 10.1053/gast.1996.v110.pm8613050. [DOI] [PubMed] [Google Scholar]
  39. Romano M., Polk W. H., Awad J. A., Arteaga C. L., Nanney L. B., Wargovich M. J., Kraus E. R., Boland C. R., Coffey R. J. Transforming growth factor alpha protection against drug-induced injury to the rat gastric mucosa in vivo. J Clin Invest. 1992 Dec;90(6):2409–2421. doi: 10.1172/JCI116132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Romano M., Razandi M., Sekhon S., Krause W. J., Ivey K. J. Human cell line for study of damage to gastric epithelial cells in vitro. J Lab Clin Med. 1988 Apr;111(4):430–440. [PubMed] [Google Scholar]
  41. Rutten M. J., Dempsey P. J., Luttropp C. A., Hawkey M. A., Sheppard B. C., Crass R. A., Deveney C. W., Coffey R. J., Jr Identification of an EGF/TGF-alpha receptor in primary cultures of guinea pig gastric mucous epithelial cells. Am J Physiol. 1996 Apr;270(4 Pt 1):G604–G612. doi: 10.1152/ajpgi.1996.270.4.G604. [DOI] [PubMed] [Google Scholar]
  42. Shoyab M., McDonald V. L., Bradley J. G., Todaro G. J. Amphiregulin: a bifunctional growth-modulating glycoprotein produced by the phorbol 12-myristate 13-acetate-treated human breast adenocarcinoma cell line MCF-7. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6528–6532. doi: 10.1073/pnas.85.17.6528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tarnawski A., Stachura J., Durbin T., Sarfeh I. J., Gergely H. Increased expression of epidermal growth factor receptor during gastric ulcer healing in rats. Gastroenterology. 1992 Feb;102(2):695–698. doi: 10.1016/0016-5085(92)90123-g. [DOI] [PubMed] [Google Scholar]
  44. Tarnawski A., Tanoue K., Santos A. M., Sarfeh I. J. Cellular and molecular mechanisms of gastric ulcer healing. Is the quality of mucosal scar affected by treatment? Scand J Gastroenterol Suppl. 1995;210:9–14. doi: 10.3109/00365529509090261. [DOI] [PubMed] [Google Scholar]
  45. Telford J. L., Covacci A., Ghiara P., Montecucco C., Rappuoli R. Unravelling the pathogenic role of Helicobacter pylori in peptic ulcer: potential new therapies and vaccines. Trends Biotechnol. 1994 Oct;12(10):420–426. doi: 10.1016/0167-7799(94)90031-0. [DOI] [PubMed] [Google Scholar]
  46. Tso J. Y., Sun X. H., Kao T. H., Reece K. S., Wu R. Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene. Nucleic Acids Res. 1985 Apr 11;13(7):2485–2502. doi: 10.1093/nar/13.7.2485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tummuru M. K., Cover T. L., Blaser M. J. Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production. Infect Immun. 1993 May;61(5):1799–1809. doi: 10.1128/iai.61.5.1799-1809.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Tummuru M. K., Cover T. L., Blaser M. J. Mutation of the cytotoxin-associated cagA gene does not affect the vacuolating cytotoxin activity of Helicobacter pylori. Infect Immun. 1994 Jun;62(6):2609–2613. doi: 10.1128/iai.62.6.2609-2613.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Tummuru M. K., Sharma S. A., Blaser M. J. Helicobacter pylori picB, a homologue of the Bordetella pertussis toxin secretion protein, is required for induction of IL-8 in gastric epithelial cells. Mol Microbiol. 1995 Dec;18(5):867–876. doi: 10.1111/j.1365-2958.1995.18050867.x. [DOI] [PubMed] [Google Scholar]
  50. Uribe J. M., Barrett K. E. Nonmitogenic actions of growth factors: an integrated view of their role in intestinal physiology and pathophysiology. Gastroenterology. 1997 Jan;112(1):255–268. [PubMed] [Google Scholar]
  51. Xiang Z., Censini S., Bayeli P. F., Telford J. L., Figura N., Rappuoli R., Covacci A. Analysis of expression of CagA and VacA virulence factors in 43 strains of Helicobacter pylori reveals that clinical isolates can be divided into two major types and that CagA is not necessary for expression of the vacuolating cytotoxin. Infect Immun. 1995 Jan;63(1):94–98. doi: 10.1128/iai.63.1.94-98.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Zarrilli R., Pignata S., Romano M., Gravina A., Casola S., Bruni C. B., Acquaviva A. M. Expression of insulin-like growth factor (IGF)-II and IGF-I receptor during proliferation and differentiation of CaCo-2 human colon carcinoma cells. Cell Growth Differ. 1994 Oct;5(10):1085–1091. [PubMed] [Google Scholar]
  53. Zarrilli R., Romano M., Pignata S., Casola S., Bruni C. B., Acquaviva A. M. Constitutive insulin-like growth factor-II expression interferes with the enterocyte-like differentiation of CaCo-2 cells. J Biol Chem. 1996 Apr 5;271(14):8108–8114. doi: 10.1074/jbc.271.14.8108. [DOI] [PubMed] [Google Scholar]

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