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. 1997 Feb;150(2):747–760.

Intestinal epithelial restitution. Involvement of specific laminin isoforms and integrin laminin receptors in wound closure of a transformed model epithelium.

M M Lotz 1, A Nusrat 1, J L Madara 1, R Ezzell 1, U M Wewer 1, A M Mercurio 1
PMCID: PMC1858283  PMID: 9033287

Abstract

Disruptions in the mucosal lining of the gastrointestinal tract reseal by epithelial cell migration, a process termed restitution. We examined the involvement of laminin isoforms and their integrin receptors in restitution using the intestinal epithelial cell line T84. T84 cells express primarily laminins 5, 6, and 7 as indicated by immunostaining using laminin subunit-specific monoclonal antibodies (MAbs). A MAb (BM2) specific for the laminin alpha 3 subunit, a component of laminins 5, 6, and 7, completely inhibited the closure of mechanical wounds in T84 monolayers. Confocal microscopy using MAbs BM2 (laminin alpha 3 subunit) and 6F12 (laminin beta 3 subunit) revealed that laminin-5 is deposited in a basal matrix that extends into the wound. The MAbs 4E10 (laminin beta 1 subunit) and C4 (laminin beta 2 subunit) stained the lateral membranes between T84 cells. This staining was enhanced in cells adjoining wounds. Because T84 cells stained faintly with MAbs 4C7 (laminin alpha 1 subunit) and with MAbs 4F11 and 1B4 (laminin alpha 2 subunit), we suggest that expression of laminins 6 and 7 is enhanced in response to wounding. The alpha 3 beta 1 integrin and the alpha 6-containing integrins function in wound closure because MAbs specific for the beta 1 integrin subunit (MAb13), the alpha 3 subunit (IVA5), and the alpha 6 subunit (2B7) potently inhibited T84 migration into wounds. Immunofluorescence using UMA9, a beta 4-integrin-specific MAb, revealed that alpha 6 beta 4 integrin exists in a Triton-X-100-insoluble structure at the basal surface and that the staining of this structure is enhanced in cells adjoining wounds. In addition, a Triton-X-100-soluble pool of alpha 6 beta 4, as well as alpha 3 beta 1 and presumably alpha 6 beta 1, was found along lateral surfaces of T84 cells. On flattened cells adjoining wounds, staining for these integrins was distributed diffusely, suggesting a redistribution that accompanies cell migration. Taken together, these data suggest that wound-induced epithelial cell migration is a finely tuned process that is dependent upon the regulated function and localization of specific laminins and their integrin receptors.

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Selected References

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  1. Akiyama S. K., Yamada S. S., Chen W. T., Yamada K. M. Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization. J Cell Biol. 1989 Aug;109(2):863–875. doi: 10.1083/jcb.109.2.863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Basson M. D., Modlin I. M., Flynn S. D., Jena B. P., Madri J. A. Independent modulation of enterocyte migration and proliferation by growth factors, matrix proteins, and pharmacologic agents in an in vitro model of mucosal healing. Surgery. 1992 Aug;112(2):299–308. [PubMed] [Google Scholar]
  3. Basson M. D., Modlin I. M., Madri J. A. Human enterocyte (Caco-2) migration is modulated in vitro by extracellular matrix composition and epidermal growth factor. J Clin Invest. 1992 Jul;90(1):15–23. doi: 10.1172/JCI115828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Beaulieu J. F., Vachon P. H. Reciprocal expression of laminin A-chain isoforms along the crypt-villus axis in the human small intestine. Gastroenterology. 1994 Apr;106(4):829–839. doi: 10.1016/0016-5085(94)90740-4. [DOI] [PubMed] [Google Scholar]
  5. Cavani A., Zambruno G., Marconi A., Manca V., Marchetti M., Giannetti A. Distinctive integrin expression in the newly forming epidermis during wound healing in humans. J Invest Dermatol. 1993 Oct;101(4):600–604. doi: 10.1111/1523-1747.ep12366057. [DOI] [PubMed] [Google Scholar]
  6. Champliaud M. F., Lunstrum G. P., Rousselle P., Nishiyama T., Keene D. R., Burgeson R. E. Human amnion contains a novel laminin variant, laminin 7, which like laminin 6, covalently associates with laminin 5 to promote stable epithelial-stromal attachment. J Cell Biol. 1996 Mar;132(6):1189–1198. doi: 10.1083/jcb.132.6.1189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ciacci C., Lind S. E., Podolsky D. K. Transforming growth factor beta regulation of migration in wounded rat intestinal epithelial monolayers. Gastroenterology. 1993 Jul;105(1):93–101. doi: 10.1016/0016-5085(93)90014-4. [DOI] [PubMed] [Google Scholar]
  8. Clark R. A., Lanigan J. M., DellaPelle P., Manseau E., Dvorak H. F., Colvin R. B. Fibronectin and fibrin provide a provisional matrix for epidermal cell migration during wound reepithelialization. J Invest Dermatol. 1982 Nov;79(5):264–269. doi: 10.1111/1523-1747.ep12500075. [DOI] [PubMed] [Google Scholar]
  9. De Arcangelis A., Neuville P., Boukamel R., Lefebvre O., Kedinger M., Simon-Assmann P. Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation. J Cell Biol. 1996 Apr;133(2):417–430. doi: 10.1083/jcb.133.2.417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dean D. C., Newby R. F., Bourgeois S. Regulation of fibronectin biosynthesis by dexamethasone, transforming growth factor beta, and cAMP in human cell lines. J Cell Biol. 1988 Jun;106(6):2159–2170. doi: 10.1083/jcb.106.6.2159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dharmsathaphorn K., Madara J. L. Established intestinal cell lines as model systems for electrolyte transport studies. Methods Enzymol. 1990;192:354–389. doi: 10.1016/0076-6879(90)92082-o. [DOI] [PubMed] [Google Scholar]
  12. Dignass A. U., Lynch-Devaney K., Podolsky D. K. Hepatocyte growth factor/scatter factor modulates intestinal epithelial cell proliferation and migration. Biochem Biophys Res Commun. 1994 Jul 29;202(2):701–709. doi: 10.1006/bbrc.1994.1987. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Dignass A. U., Tsunekawa S., Podolsky D. K. Fibroblast growth factors modulate intestinal epithelial cell growth and migration. Gastroenterology. 1994 May;106(5):1254–1262. doi: 10.1016/0016-5085(94)90017-5. [DOI] [PubMed] [Google Scholar]
  15. Engvall E., Davis G. E., Dickerson K., Ruoslahti E., Varon S., Manthorpe M. Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site. J Cell Biol. 1986 Dec;103(6 Pt 1):2457–2465. doi: 10.1083/jcb.103.6.2457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Engvall E., Earwicker D., Haaparanta T., Ruoslahti E., Sanes J. R. Distribution and isolation of four laminin variants; tissue restricted distribution of heterotrimers assembled from five different subunits. Cell Regul. 1990 Sep;1(10):731–740. doi: 10.1091/mbc.1.10.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Feil W., Wenzl E., Vattay P., Starlinger M., Sogukoglu T., Schiessel R. Repair of rabbit duodenal mucosa after acid injury in vivo and in vitro. Gastroenterology. 1987 Jun;92(6):1973–1986. doi: 10.1016/0016-5085(87)90632-9. [DOI] [PubMed] [Google Scholar]
  18. Gailit J., Clark R. A. Wound repair in the context of extracellular matrix. Curr Opin Cell Biol. 1994 Oct;6(5):717–725. doi: 10.1016/0955-0674(94)90099-x. [DOI] [PubMed] [Google Scholar]
  19. Gipson I. K., Spurr-Michaud S., Tisdale A., Elwell J., Stepp M. A. Redistribution of the hemidesmosome components alpha 6 beta 4 integrin and bullous pemphigoid antigens during epithelial wound healing. Exp Cell Res. 1993 Jul;207(1):86–98. doi: 10.1006/excr.1993.1166. [DOI] [PubMed] [Google Scholar]
  20. Grinnell F. Fibronectin and wound healing. J Cell Biochem. 1984;26(2):107–116. doi: 10.1002/jcb.240260206. [DOI] [PubMed] [Google Scholar]
  21. Heino J., Massagué J. Transforming growth factor-beta switches the pattern of integrins expressed in MG-63 human osteosarcoma cells and causes a selective loss of cell adhesion to laminin. J Biol Chem. 1989 Dec 25;264(36):21806–21811. [PubMed] [Google Scholar]
  22. Jones J. C., Asmuth J., Baker S. E., Langhofer M., Roth S. I., Hopkinson S. B. Hemidesmosomes: extracellular matrix/intermediate filament connectors. Exp Cell Res. 1994 Jul;213(1):1–11. doi: 10.1006/excr.1994.1166. [DOI] [PubMed] [Google Scholar]
  23. Juhasz I., Murphy G. F., Yan H. C., Herlyn M., Albelda S. M. Regulation of extracellular matrix proteins and integrin cell substratum adhesion receptors on epithelium during cutaneous human wound healing in vivo. Am J Pathol. 1993 Nov;143(5):1458–1469. [PMC free article] [PubMed] [Google Scholar]
  24. Kikkawa Y., Umeda M., Miyazaki K. Marked stimulation of cell adhesion and motility by ladsin, a laminin-like scatter factor. J Biochem. 1994 Oct;116(4):862–869. doi: 10.1093/oxfordjournals.jbchem.a124608. [DOI] [PubMed] [Google Scholar]
  25. Klein G., Langegger M., Timpl R., Ekblom P. Role of laminin A chain in the development of epithelial cell polarity. Cell. 1988 Oct 21;55(2):331–341. doi: 10.1016/0092-8674(88)90056-6. [DOI] [PubMed] [Google Scholar]
  26. Kleinman H. K., Cannon F. B., Laurie G. W., Hassell J. R., Aumailley M., Terranova V. P., Martin G. R., DuBois-Dalcq M. Biological activities of laminin. J Cell Biochem. 1985;27(4):317–325. doi: 10.1002/jcb.240270402. [DOI] [PubMed] [Google Scholar]
  27. Kurpakus M. A., Quaranta V., Jones J. C. Surface relocation of alpha 6 beta 4 integrins and assembly of hemidesmosomes in an in vitro model of wound healing. J Cell Biol. 1991 Dec;115(6):1737–1750. doi: 10.1083/jcb.115.6.1737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lacy E. R., Ito S. Rapid epithelial restitution of the rat gastric mucosa after ethanol injury. Lab Invest. 1984 Nov;51(5):573–583. [PubMed] [Google Scholar]
  29. Lawson M. A., Maxfield F. R. Ca(2+)- and calcineurin-dependent recycling of an integrin to the front of migrating neutrophils. Nature. 1995 Sep 7;377(6544):75–79. doi: 10.1038/377075a0. [DOI] [PubMed] [Google Scholar]
  30. Leivo I., Engvall E. Merosin, a protein specific for basement membranes of Schwann cells, striated muscle, and trophoblast, is expressed late in nerve and muscle development. Proc Natl Acad Sci U S A. 1988 Mar;85(5):1544–1548. doi: 10.1073/pnas.85.5.1544. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Marinkovich M. P., Lunstrum G. P., Burgeson R. E. The anchoring filament protein kalinin is synthesized and secreted as a high molecular weight precursor. J Biol Chem. 1992 Sep 5;267(25):17900–17906. [PubMed] [Google Scholar]
  32. Mercurio A. M. Laminin receptors: achieving specificity through cooperation. Trends Cell Biol. 1995 Nov;5(11):419–423. doi: 10.1016/s0962-8924(00)89100-x. [DOI] [PubMed] [Google Scholar]
  33. Moore R., Carlson S., Madara J. L. Rapid barrier restitution in an in vitro model of intestinal epithelial injury. Lab Invest. 1989 Feb;60(2):237–244. [PubMed] [Google Scholar]
  34. Moore R., Madri J., Carlson S., Madara J. L. Collagens facilitate epithelial migration in restitution of native guinea pig intestinal epithelium. Gastroenterology. 1992 Jan;102(1):119–130. doi: 10.1016/0016-5085(92)91791-2. [DOI] [PubMed] [Google Scholar]
  35. Mustoe T. A., Pierce G. F., Morishima C., Deuel T. F. Growth factor-induced acceleration of tissue repair through direct and inductive activities in a rabbit dermal ulcer model. J Clin Invest. 1991 Feb;87(2):694–703. doi: 10.1172/JCI115048. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Nusrat A., Parkos C. A., Bacarra A. E., Godowski P. J., Delp-Archer C., Rosen E. M., Madara J. L. Hepatocyte growth factor/scatter factor effects on epithelia. Regulation of intercellular junctions in transformed and nontransformed cell lines, basolateral polarization of c-met receptor in transformed and natural intestinal epithelia, and induction of rapid wound repair in a transformed model epithelium. J Clin Invest. 1994 May;93(5):2056–2065. doi: 10.1172/JCI117200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Orian-Rousseau V., Aberdam D., Fontao L., Chevalier L., Meneguzzi G., Kedinger M., Simon-Assmann P. Developmental expression of laminin-5 and HD1 in the intestine: epithelial to mesenchymal shift for the laminin gamma-2 chain subunit deposition. Dev Dyn. 1996 May;206(1):12–23. doi: 10.1002/(SICI)1097-0177(199605)206:1<12::AID-AJA2>3.0.CO;2-3. [DOI] [PubMed] [Google Scholar]
  39. Paimela H., Goddard P. J., Carter K., Khakee R., McNeil P. L., Ito S., Silen W. Restitution of frog gastric mucosa in vitro: effect of basic fibroblast growth factor. Gastroenterology. 1993 May;104(5):1337–1345. doi: 10.1016/0016-5085(93)90342-a. [DOI] [PubMed] [Google Scholar]
  40. Panayotou G., End P., Aumailley M., Timpl R., Engel J. Domains of laminin with growth-factor activity. Cell. 1989 Jan 13;56(1):93–101. doi: 10.1016/0092-8674(89)90987-2. [DOI] [PubMed] [Google Scholar]
  41. Parkos C. A., Colgan S. P., Bacarra A. E., Nusrat A., Delp-Archer C., Carlson S., Su D. H., Madara J. L. Intestinal epithelia (T84) possess basolateral ligands for CD11b/CD18-mediated neutrophil adherence. Am J Physiol. 1995 Feb;268(2 Pt 1):C472–C479. doi: 10.1152/ajpcell.1995.268.2.C472. [DOI] [PubMed] [Google Scholar]
  42. Pulkkinen L., Christiano A. M., Airenne T., Haakana H., Tryggvason K., Uitto J. Mutations in the gamma 2 chain gene (LAMC2) of kalinin/laminin 5 in the junctional forms of epidermolysis bullosa. Nat Genet. 1994 Mar;6(3):293–297. doi: 10.1038/ng0394-293. [DOI] [PubMed] [Google Scholar]
  43. Pyke C., Rømer J., Kallunki P., Lund L. R., Ralfkiaer E., Danø K., Tryggvason K. The gamma 2 chain of kalinin/laminin 5 is preferentially expressed in invading malignant cells in human cancers. Am J Pathol. 1994 Oct;145(4):782–791. [PMC free article] [PubMed] [Google Scholar]
  44. Rousselle P., Lunstrum G. P., Keene D. R., Burgeson R. E. Kalinin: an epithelium-specific basement membrane adhesion molecule that is a component of anchoring filaments. J Cell Biol. 1991 Aug;114(3):567–576. doi: 10.1083/jcb.114.3.567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Rutten M. J., Ito S. Morphology and electrophysiology of guinea pig gastric mucosal repair in vitro. Am J Physiol. 1983 Feb;244(2):G171–G182. doi: 10.1152/ajpgi.1983.244.2.G171. [DOI] [PubMed] [Google Scholar]
  46. Sanes J. R., Engvall E., Butkowski R., Hunter D. D. Molecular heterogeneity of basal laminae: isoforms of laminin and collagen IV at the neuromuscular junction and elsewhere. J Cell Biol. 1990 Oct;111(4):1685–1699. doi: 10.1083/jcb.111.4.1685. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schmidt C. E., Horwitz A. F., Lauffenburger D. A., Sheetz M. P. Integrin-cytoskeletal interactions in migrating fibroblasts are dynamic, asymmetric, and regulated. J Cell Biol. 1993 Nov;123(4):977–991. doi: 10.1083/jcb.123.4.977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Shaw L. M., Lotz M. M., Mercurio A. M. Inside-out integrin signaling in macrophages. Analysis of the role of the alpha 6A beta 1 and alpha 6B beta 1 integrin variants in laminin adhesion by cDNA expression in an alpha 6 integrin-deficient macrophage cell line. J Biol Chem. 1993 May 25;268(15):11401–11408. [PubMed] [Google Scholar]
  49. Simo P., Bouziges F., Lissitzky J. C., Sorokin L., Kedinger M., Simon-Assmann P. Dual and asynchronous deposition of laminin chains at the epithelial-mesenchymal interface in the gut. Gastroenterology. 1992 Jun;102(6):1835–1845. doi: 10.1016/0016-5085(92)90303-g. [DOI] [PubMed] [Google Scholar]
  50. Simo P., Simon-Assmann P., Bouziges F., Leberquier C., Kedinger M., Ekblom P., Sorokin L. Changes in the expression of laminin during intestinal development. Development. 1991 Jun;112(2):477–487. doi: 10.1242/dev.112.2.477. [DOI] [PubMed] [Google Scholar]
  51. Simon-Assmann P., Bouziges F., Arnold C., Haffen K., Kedinger M. Epithelial-mesenchymal interactions in the production of basement membrane components in the gut. Development. 1988 Feb;102(2):339–347. doi: 10.1242/dev.102.2.339. [DOI] [PubMed] [Google Scholar]
  52. Simon-Assmann P., Bouziges F., Vigny M., Kedinger M. Origin and deposition of basement membrane heparan sulfate proteoglycan in the developing intestine. J Cell Biol. 1989 Oct;109(4 Pt 1):1837–1848. doi: 10.1083/jcb.109.4.1837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Simon-Assmann P., Duclos B., Orian-Rousseau V., Arnold C., Mathelin C., Engvall E., Kedinger M. Differential expression of laminin isoforms and alpha 6-beta 4 integrin subunits in the developing human and mouse intestine. Dev Dyn. 1994 Sep;201(1):71–85. doi: 10.1002/aja.1002010108. [DOI] [PubMed] [Google Scholar]
  54. Sonnenberg A., Calafat J., Janssen H., Daams H., van der Raaij-Helmer L. M., Falcioni R., Kennel S. J., Aplin J. D., Baker J., Loizidou M. Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion. J Cell Biol. 1991 May;113(4):907–917. doi: 10.1083/jcb.113.4.907. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Spinardi L., Einheber S., Cullen T., Milner T. A., Giancotti F. G. A recombinant tail-less integrin beta 4 subunit disrupts hemidesmosomes, but does not suppress alpha 6 beta 4-mediated cell adhesion to laminins. J Cell Biol. 1995 Apr;129(2):473–487. doi: 10.1083/jcb.129.2.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Stepp M. A., Spurr-Michaud S., Tisdale A., Elwell J., Gipson I. K. Alpha 6 beta 4 integrin heterodimer is a component of hemidesmosomes. Proc Natl Acad Sci U S A. 1990 Nov;87(22):8970–8974. doi: 10.1073/pnas.87.22.8970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Streuli C. H., Schmidhauser C., Bailey N., Yurchenco P., Skubitz A. P., Roskelley C., Bissell M. J. Laminin mediates tissue-specific gene expression in mammary epithelia. J Cell Biol. 1995 May;129(3):591–603. doi: 10.1083/jcb.129.3.591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Svanes K., Ito S., Takeuchi K., Silen W. Restitution of the surface epithelium of the in vitro frog gastric mucosa after damage with hyperosmolar sodium chloride. Morphologic and physiologic characteristics. Gastroenterology. 1982 Jun;82(6):1409–1426. [PubMed] [Google Scholar]
  59. Terranova V. P., Rohrbach D. H., Martin G. R. Role of laminin in the attachment of PAM 212 (epithelial) cells to basement membrane collagen. Cell. 1980 Dec;22(3):719–726. doi: 10.1016/0092-8674(80)90548-6. [DOI] [PubMed] [Google Scholar]
  60. Van Waes C., Kozarsky K. F., Warren A. B., Kidd L., Paugh D., Liebert M., Carey T. E. The A9 antigen associated with aggressive human squamous carcinoma is structurally and functionally similar to the newly defined integrin alpha 6 beta 4. Cancer Res. 1991 May 1;51(9):2395–2402. [PubMed] [Google Scholar]
  61. Wewer U. M., Gerecke D. R., Durkin M. E., Kurtz K. S., Mattei M. G., Champliaud M. F., Burgeson R. E., Albrechtsen R. Human beta 2 chain of laminin (formerly S chain): cDNA cloning, chromosomal localization, and expression in carcinomas. Genomics. 1994 Nov 15;24(2):243–252. doi: 10.1006/geno.1994.1612. [DOI] [PubMed] [Google Scholar]
  62. Wewer U., Albrechtsen R., Manthorpe M., Varon S., Engvall E., Ruoslahti E. Human laminin isolated in a nearly intact, biologically active form from placenta by limited proteolysis. J Biol Chem. 1983 Oct 25;258(20):12654–12660. [PubMed] [Google Scholar]
  63. Yurchenco P. D., Cheng Y. S. Self-assembly and calcium-binding sites in laminin. A three-arm interaction model. J Biol Chem. 1993 Aug 15;268(23):17286–17299. [PubMed] [Google Scholar]
  64. Zambruno G., Marchisio P. C., Marconi A., Vaschieri C., Melchiori A., Giannetti A., De Luca M. Transforming growth factor-beta 1 modulates beta 1 and beta 5 integrin receptors and induces the de novo expression of the alpha v beta 6 heterodimer in normal human keratinocytes: implications for wound healing. J Cell Biol. 1995 May;129(3):853–865. doi: 10.1083/jcb.129.3.853. [DOI] [PMC free article] [PubMed] [Google Scholar]

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