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. 1999 Mar 15;338(Pt 3):745–751.

Intestinal trefoil factor binds to intestinal epithelial cells and induces nitric oxide production: priming and enhancing effects of mucin.

X D Tan 1, Q P Liu 1, W Hsueh 1, Y H Chen 1, H Chang 1, F Gonzalez-Crussi 1
PMCID: PMC1220112  PMID: 10051448

Abstract

Intestinal trefoil factor (ITF or TFF3), NO and epithelium-associated mucin have important roles in sustaining mucosal integrity in the gastrointestinal tract. In the present study we examined ITF-binding molecules on IEC-18 cells (an intestinal epithelial cell line) with the use of flow cytometry and localized these molecules on the cell surface by confocal microscopy. Furthermore, we studied the interaction of mucin and ITF and their co-operative effect on NO production by the epithelium. Stimulation of cells with mucin (5 mg/ml) for 90 min resulted in a 5-fold increase in ITF binding. Treatment of IEC-18 cells with actinomycin D or cycloheximide attenuated mucin-enhanced ITF binding. Ligand blot analysis confirmed the induction of ITF-binding protein in IEC-18 cells by mucin. These results indicate that transcriptional and translational mechanisms are involved in the effect of mucin. Treatment with ITF overnight resulted in a low level of nitrite production by the cells, a 5-fold increase over control, in a concentration-dependent manner. ITF-induced NO production was attenuated by 1400W, a selective type II nitric oxide synthase (NOS2) inhibitor. By immunoblotting we found that NOS2 was up-regulated by ITF treatment. Priming IEC-18 cells with mucin for 90 min enhanced the effect of ITF on NO production, suggesting that the up-regulation of ITF-binding molecules by mucin might be physiologically relevant. Taken together, these observations indicate (1) that ITF-binding molecules that are up-regulated by mucin exist on the intestinal epithelial surface, and (2) that ITF modulates epithelial NO production via the NOS2 pathway, which is enhanced by mucin.

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Selected References

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  1. Babyatsky M. W., deBeaumont M., Thim L., Podolsky D. K. Oral trefoil peptides protect against ethanol- and indomethacin-induced gastric injury in rats. Gastroenterology. 1996 Feb;110(2):489–497. doi: 10.1053/gast.1996.v110.pm8566596. [DOI] [PubMed] [Google Scholar]
  2. Beckman J. S., Koppenol W. H. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am J Physiol. 1996 Nov;271(5 Pt 1):C1424–C1437. doi: 10.1152/ajpcell.1996.271.5.C1424. [DOI] [PubMed] [Google Scholar]
  3. Chinery R., Cox H. M. Immunoprecipitation and characterization of a binding protein specific for the peptide, intestinal trefoil factor. Peptides. 1995;16(4):749–755. doi: 10.1016/0196-9781(95)00045-l. [DOI] [PubMed] [Google Scholar]
  4. Chinery R., Cox H. M. Modulation of epidermal growth factor effects on epithelial ion transport by intestinal trefoil factor. Br J Pharmacol. 1995 May;115(1):77–80. doi: 10.1111/j.1476-5381.1995.tb16322.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chinery R., Poulsom R., Elia G., Hanby A. M., Wright N. A. Expression and purification of a trefoil peptide motif in a beta-galactosidase fusion protein and its use to search for trefoil-binding sites. Eur J Biochem. 1993 Mar 1;212(2):557–563. doi: 10.1111/j.1432-1033.1993.tb17693.x. [DOI] [PubMed] [Google Scholar]
  6. Chinery R., Poulsom R., Rogers L. A., Jeffery R. E., Longcroft J. M., Hanby A. M., Wright N. A. Localization of intestinal trefoil-factor mRNA in rat stomach and intestine by hybridization in situ. Biochem J. 1992 Jul 1;285(Pt 1):5–8. doi: 10.1042/bj2850005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Dignass A. U., Podolsky D. K., Rachmilewitz D. NO chi generation by cultured small intestinal epithelial cells. Dig Dis Sci. 1995 Sep;40(9):1859–1865. doi: 10.1007/BF02208647. [DOI] [PubMed] [Google Scholar]
  9. Dignass A., Lynch-Devaney K., Kindon H., Thim L., Podolsky D. K. Trefoil peptides promote epithelial migration through a transforming growth factor beta-independent pathway. J Clin Invest. 1994 Jul;94(1):376–383. doi: 10.1172/JCI117332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Efstathiou J. A., Noda M., Rowan A., Dixon C., Chinery R., Jawhari A., Hattori T., Wright N. A., Bodmer W. F., Pignatelli M. Intestinal trefoil factor controls the expression of the adenomatous polyposis coli-catenin and the E-cadherin-catenin complexes in human colon carcinoma cells. Proc Natl Acad Sci U S A. 1998 Mar 17;95(6):3122–3127. doi: 10.1073/pnas.95.6.3122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hauser F., Poulsom R., Chinery R., Rogers L. A., Hanby A. M., Wright N. A., Hoffmann W. hP1.B, a human P-domain peptide homologous with rat intestinal trefoil factor, is expressed also in the ulcer-associated cell lineage and the uterus. Proc Natl Acad Sci U S A. 1993 Aug 1;90(15):6961–6965. doi: 10.1073/pnas.90.15.6961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Jakowlew S. B., Breathnach R., Jeltsch J. M., Masiakowski P., Chambon P. Sequence of the pS2 mRNA induced by estrogen in the human breast cancer cell line MCF-7. Nucleic Acids Res. 1984 Mar 26;12(6):2861–2878. doi: 10.1093/nar/12.6.2861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jørgensen K. H., Thim L., Jacobsen H. E. Pancreatic spasmolytic polypeptide (PSP): I. Preparation and initial chemical characterization of a new polypeptide from porcine pancreas. Regul Pept. 1982 Mar;3(3-4):207–219. doi: 10.1016/0167-0115(82)90126-4. [DOI] [PubMed] [Google Scholar]
  14. Kanai M., Mullen C., Podolsky D. K. Intestinal trefoil factor induces inactivation of extracellular signal-regulated protein kinase in intestinal epithelial cells. Proc Natl Acad Sci U S A. 1998 Jan 6;95(1):178–182. doi: 10.1073/pnas.95.1.178. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kindon H., Pothoulakis C., Thim L., Lynch-Devaney K., Podolsky D. K. Trefoil peptide protection of intestinal epithelial barrier function: cooperative interaction with mucin glycoprotein. Gastroenterology. 1995 Aug;109(2):516–523. doi: 10.1016/0016-5085(95)90340-2. [DOI] [PubMed] [Google Scholar]
  16. Király A., Sütö G., Taché Y. Role of nitric oxide in the gastric cytoprotection induced by central vagal stimulation. Eur J Pharmacol. 1993 Aug 24;240(2-3):299–301. doi: 10.1016/0014-2999(93)90913-3. [DOI] [PubMed] [Google Scholar]
  17. Liu D., el-Hariry I., Karayiannakis A. J., Wilding J., Chinery R., Kmiot W., McCrea P. D., Gullick W. J., Pignatelli M. Phosphorylation of beta-catenin and epidermal growth factor receptor by intestinal trefoil factor. Lab Invest. 1997 Dec;77(6):557–563. [PubMed] [Google Scholar]
  18. Mashimo H., Podolsky D. K., Fishman M. C. Structure and expression of murine intestinal trefoil factor: high evolutionary conservation and postnatal expression. Biochem Biophys Res Commun. 1995 May 5;210(1):31–37. doi: 10.1006/bbrc.1995.1623. [DOI] [PubMed] [Google Scholar]
  19. Mashimo H., Wu D. C., Podolsky D. K., Fishman M. C. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor. Science. 1996 Oct 11;274(5285):262–265. doi: 10.1126/science.274.5285.262. [DOI] [PubMed] [Google Scholar]
  20. McCormick D. A., Horton L. W., Mee A. S. Mucin depletion in inflammatory bowel disease. J Clin Pathol. 1990 Feb;43(2):143–146. doi: 10.1136/jcp.43.2.143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Meyer T. A., Tiao G. M., James J. H., Noguchi Y., Ogle C. K., Fischer J. E., Hasselgren P. O. Nitric oxide inhibits LPS-induced IL-6 production in enterocytes. J Surg Res. 1995 Jun;58(6):570–575. doi: 10.1006/jsre.1995.1090. [DOI] [PubMed] [Google Scholar]
  22. Misko T. P., Schilling R. J., Salvemini D., Moore W. M., Currie M. G. A fluorometric assay for the measurement of nitrite in biological samples. Anal Biochem. 1993 Oct;214(1):11–16. doi: 10.1006/abio.1993.1449. [DOI] [PubMed] [Google Scholar]
  23. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992 Sep;6(12):3051–3064. [PubMed] [Google Scholar]
  24. Noiri E., Peresleni T., Srivastava N., Weber P., Bahou W. F., Peunova N., Goligorsky M. S. Nitric oxide is necessary for a switch from stationary to locomoting phenotype in epithelial cells. Am J Physiol. 1996 Mar;270(3 Pt 1):C794–C802. doi: 10.1152/ajpcell.1996.270.3.C794. [DOI] [PubMed] [Google Scholar]
  25. Noiri E., Peresleni T., Srivastava N., Weber P., Bahou W. F., Peunova N., Goligorsky M. S. Nitric oxide is necessary for a switch from stationary to locomoting phenotype in epithelial cells. Am J Physiol. 1996 Mar;270(3 Pt 1):C794–C802. doi: 10.1152/ajpcell.1996.270.3.C794. [DOI] [PubMed] [Google Scholar]
  26. Nusrat A., Delp C., Madara J. L. Intestinal epithelial restitution. Characterization of a cell culture model and mapping of cytoskeletal elements in migrating cells. J Clin Invest. 1992 May;89(5):1501–1511. doi: 10.1172/JCI115741. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. O'Toole T. E., Katagiri Y., Faull R. J., Peter K., Tamura R., Quaranta V., Loftus J. C., Shattil S. J., Ginsberg M. H. Integrin cytoplasmic domains mediate inside-out signal transduction. J Cell Biol. 1994 Mar;124(6):1047–1059. doi: 10.1083/jcb.124.6.1047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Playford R. J., Marchbank T., Goodlad R. A., Chinery R. A., Poulsom R., Hanby A. M. Transgenic mice that overexpress the human trefoil peptide pS2 have an increased resistance to intestinal damage. Proc Natl Acad Sci U S A. 1996 Mar 5;93(5):2137–2142. doi: 10.1073/pnas.93.5.2137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Podolsky D. K., Isselbacher K. J. Composition of human colonic mucin. Selective alteration in inflammatory bowel disease. J Clin Invest. 1983 Jul;72(1):142–153. doi: 10.1172/JCI110952. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Poulsom R., Wright N. A. Trefoil peptides: a newly recognized family of epithelial mucin-associated molecules. Am J Physiol. 1993 Aug;265(2 Pt 1):G205–G213. doi: 10.1152/ajpgi.1993.265.2.G205. [DOI] [PubMed] [Google Scholar]
  31. Rio M. C., Bellocq J. P., Daniel J. Y., Tomasetto C., Lathe R., Chenard M. P., Batzenschlager A., Chambon P. Breast cancer-associated pS2 protein: synthesis and secretion by normal stomach mucosa. Science. 1988 Aug 5;241(4866):705–708. doi: 10.1126/science.3041593. [DOI] [PubMed] [Google Scholar]
  32. Rio M. C., Chenard M. P., Wolf C., Marcellin L., Tomasetto C., Lathe R., Bellocq J. P., Chambon P. Induction of pS2 and hSP genes as markers of mucosal ulceration of the digestive tract. Gastroenterology. 1991 Feb;100(2):375–379. doi: 10.1016/0016-5085(91)90205-y. [DOI] [PubMed] [Google Scholar]
  33. Salzman A. L., Eaves-Pyles T., Linn S. C., Denenberg A. G., Szabó C. Bacterial induction of inducible nitric oxide synthase in cultured human intestinal epithelial cells. Gastroenterology. 1998 Jan;114(1):93–102. doi: 10.1016/s0016-5085(98)70637-7. [DOI] [PubMed] [Google Scholar]
  34. Sands B. E., Podolsky D. K. The trefoil peptide family. Annu Rev Physiol. 1996;58:253–273. doi: 10.1146/annurev.ph.58.030196.001345. [DOI] [PubMed] [Google Scholar]
  35. Suemori S., Lynch-Devaney K., Podolsky D. K. Identification and characterization of rat intestinal trefoil factor: tissue- and cell-specific member of the trefoil protein family. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11017–11021. doi: 10.1073/pnas.88.24.11017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tan X. D., Hsueh W., Chang H., Wei K. R., Gonzalez-Crussi F. Characterization of a putative receptor for intestinal trefoil factor in rat small intestine: identification by in situ binding and ligand blotting. Biochem Biophys Res Commun. 1997 Aug 28;237(3):673–677. doi: 10.1006/bbrc.1997.7144. [DOI] [PubMed] [Google Scholar]
  37. Thim L. Trefoil peptides: from structure to function. Cell Mol Life Sci. 1997 Dec;53(11-12):888–903. doi: 10.1007/s000180050108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tysk C., Riedesel H., Lindberg E., Panzini B., Podolsky D., Järnerot G. Colonic glycoproteins in monozygotic twins with inflammatory bowel disease. Gastroenterology. 1991 Feb;100(2):419–423. doi: 10.1016/0016-5085(91)90211-3. [DOI] [PubMed] [Google Scholar]
  39. Van Klinken B. J., Dekker J., Büller H. A., Einerhand A. W. Mucin gene structure and expression: protection vs. adhesion. Am J Physiol. 1995 Nov;269(5 Pt 1):G613–G627. doi: 10.1152/ajpgi.1995.269.5.G613. [DOI] [PubMed] [Google Scholar]
  40. Wright N. A., Poulsom R., Stamp G., Van Noorden S., Sarraf C., Elia G., Ahnen D., Jeffery R., Longcroft J., Pike C. Trefoil peptide gene expression in gastrointestinal epithelial cells in inflammatory bowel disease. Gastroenterology. 1993 Jan;104(1):12–20. doi: 10.1016/0016-5085(93)90830-6. [DOI] [PubMed] [Google Scholar]
  41. Yamasaki K., Edington H. D., McClosky C., Tzeng E., Lizonova A., Kovesdi I., Steed D. L., Billiar T. R. Reversal of impaired wound repair in iNOS-deficient mice by topical adenoviral-mediated iNOS gene transfer. J Clin Invest. 1998 Mar 1;101(5):967–971. doi: 10.1172/JCI2067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yanaka A., Muto H., Fukutomi H., Ito S., Silen W. Role of nitric oxide in restitution of injured guinea pig gastric mucosa in vitro. Am J Physiol. 1995 Jun;268(6 Pt 1):G933–G942. doi: 10.1152/ajpgi.1995.268.6.G933. [DOI] [PubMed] [Google Scholar]
  43. Yanaka A., Muto H., Fukutomi H., Ito S., Silen W. Role of nitric oxide in restitution of injured guinea pig gastric mucosa in vitro. Am J Physiol. 1995 Jun;268(6 Pt 1):G933–G942. doi: 10.1152/ajpgi.1995.268.6.G933. [DOI] [PubMed] [Google Scholar]

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