Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Aug 1;109(2):903–914. doi: 10.1083/jcb.109.2.903

Reappearance of an embryonic pattern of fibronectin splicing during wound healing in the adult rat

PMCID: PMC2115730  PMID: 2760116

Abstract

The adhesive extracellular matrix glycoprotein fibronectin (FN) is thought to play an important role in the cell migration associated with wound healing. Immunolocalization studies show abundant FN in healing wounds; however, these studies cannot define the cellular site(s) of FN synthesis, nor do they distinguish the different and potentially functionally distinct forms of FN that can arise from alternative splicing of the primary gene transcript. To examine these questions of FN synthesis and splicing during wound healing, we have performed in situ hybridization with segment-specific probes on healing wounds in adult rat skin. We find that the FN gene is expressed at increased levels after wounding both in the cells at the base of the wound and in subjacent muscle and dermis lateral to the wound. Interestingly, however, the pattern of splicing of FN mRNA was different in these areas. In adjacent dermis and muscle, the splicing pattern remains identical with that seen in normal adult rat skin, with two of the three spliced segments (EIIIA and EIIIB) excluded from FN mRNA. In contrast, these two segments are included in the FN mRNA present in the cells at the base of the wound. As a result, the mRNA in this region is spliced in a pattern identical with that found during early embryogenesis. The finding that the pattern of FN splicing during wound healing resembles an embryonic pattern suggests that alternative splicing may be used during wound healing as a mechanism to generate forms of FN that may be functionally more appropriate for the cell migration and proliferation associated with tissue repair.

Full Text

The Full Text of this article is available as a PDF (4.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ali I. U., Hynes R. O. Effects of LETS glycoprotein on cell motility. Cell. 1978 Jun;14(2):439–446. doi: 10.1016/0092-8674(78)90129-0. [DOI] [PubMed] [Google Scholar]
  2. Alitalo K., Kuismanen E., Myllylä R., Kiistala U., Asko-Seljavaara S., Vaheri A. Extracellular matrix proteins of human epidermal keratinocytes and feeder 3T3 cells. J Cell Biol. 1982 Sep;94(3):497–505. doi: 10.1083/jcb.94.3.497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Angerer L. M., Cox K. H., Angerer R. C. Demonstration of tissue-specific gene expression by in situ hybridization. Methods Enzymol. 1987;152:649–661. doi: 10.1016/0076-6879(87)52071-7. [DOI] [PubMed] [Google Scholar]
  4. Assoian R. K., Komoriya A., Meyers C. A., Miller D. M., Sporn M. B. Transforming growth factor-beta in human platelets. Identification of a major storage site, purification, and characterization. J Biol Chem. 1983 Jun 10;258(11):7155–7160. [PubMed] [Google Scholar]
  5. Balza E., Borsi L., Allemanni G., Zardi L. Transforming growth factor beta regulates the levels of different fibronectin isoforms in normal human cultured fibroblasts. FEBS Lett. 1988 Feb 8;228(1):42–44. doi: 10.1016/0014-5793(88)80580-5. [DOI] [PubMed] [Google Scholar]
  6. Berger C. N. In situ hybridization of immunoglobulin-specific RNA in single cells of the B lymphocyte lineage with radiolabelled DNA probes. EMBO J. 1986 Jan;5(1):85–93. doi: 10.1002/j.1460-2075.1986.tb04181.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Boucaut J. C., Darribère T., Boulekbache H., Thiery J. P. Prevention of gastrulation but not neurulation by antibodies to fibronectin in amphibian embryos. 1984 Jan 26-Feb 1Nature. 307(5949):364–367. doi: 10.1038/307364a0. [DOI] [PubMed] [Google Scholar]
  8. Bowersox J. C., Sorgente N. Chemotaxis of aortic endothelial cells in response to fibronectin. Cancer Res. 1982 Jul;42(7):2547–2551. [PubMed] [Google Scholar]
  9. Bronner-Fraser M. Alterations in neural crest migration by a monoclonal antibody that affects cell adhesion. J Cell Biol. 1985 Aug;101(2):610–617. doi: 10.1083/jcb.101.2.610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bronner-Fraser M. An antibody to a receptor for fibronectin and laminin perturbs cranial neural crest development in vivo. Dev Biol. 1986 Oct;117(2):528–536. doi: 10.1016/0012-1606(86)90320-9. [DOI] [PubMed] [Google Scholar]
  11. Clark R. A., DellaPelle P., Manseau E., Lanigan J. M., Dvorak H. F., Colvin R. B. Blood vessel fibronectin increases in conjunction with endothelial cell proliferation and capillary ingrowth during wound healing. J Invest Dermatol. 1982 Nov;79(5):269–276. doi: 10.1111/1523-1747.ep12500076. [DOI] [PubMed] [Google Scholar]
  12. 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]
  13. Clark R. A., Nielsen L. D., Howell S. E., Folkvord J. M. Human keratinocytes that have not terminally differentiated synthesize laminin and fibronectin but deposit only fibronectin in the pericellular matrix. J Cell Biochem. 1985;28(2):127–141. doi: 10.1002/jcb.240280206. [DOI] [PubMed] [Google Scholar]
  14. Clark R. A. Potential roles of fibronectin in cutaneous wound repair. Arch Dermatol. 1988 Feb;124(2):201–206. [PubMed] [Google Scholar]
  15. Clark R. A., Wikner N. E., Doherty D. E., Norris D. A. Cryptic chemotactic activity of fibronectin for human monocytes resides in the 120-kDa fibroblastic cell-binding fragment. J Biol Chem. 1988 Aug 25;263(24):12115–12123. [PubMed] [Google Scholar]
  16. Clark R. A., Winn H. J., Dvorak H. F., Colvin R. B. Fibronectin beneath reepithelializing epidermis in vivo: sources and significance. J Invest Dermatol. 1983 Jun;80 (Suppl):26s–30s. [PubMed] [Google Scholar]
  17. Couchman J. R., Gibson W. T., Thom D., Weaver A. C., Rees D. A., Parish W. E. Fibronectin distribution in epithelial and associated tissues of the rat. Arch Dermatol Res. 1979 Nov;266(3):295–310. doi: 10.1007/BF00418575. [DOI] [PubMed] [Google Scholar]
  18. Critchley D. R., England M. A., Wakely J., Hynes R. O. Distribution of fibronectin in the ectoderm of gastrulating chick embryos. Nature. 1979 Aug 9;280(5722):498–500. doi: 10.1038/280498a0. [DOI] [PubMed] [Google Scholar]
  19. Donaldson D. J., Mahan J. T. Fibrinogen and fibronectin as substrates for epidermal cell migration during wound closure. J Cell Sci. 1983 Jul;62:117–127. doi: 10.1242/jcs.62.1.117. [DOI] [PubMed] [Google Scholar]
  20. Donaldson D. J., Mahan J. T., Smith G. N., Jr Newt epidermal cell migration in vitro and in vivo appears to involve Arg-Gly-Asp-Ser receptors. J Cell Sci. 1987 May;87(Pt 4):525–534. doi: 10.1242/jcs.87.4.525. [DOI] [PubMed] [Google Scholar]
  21. Duband J. L., Thiery J. P. Distribution of fibronectin in the early phase of avian cephalic neural crest cell migration. Dev Biol. 1982 Oct;93(2):308–323. doi: 10.1016/0012-1606(82)90120-8. [DOI] [PubMed] [Google Scholar]
  22. Dufour S., Duband J. L., Kornblihtt A. R., Thiery J. P. The role of fibronectins in embryonic cell migrations. Trends Genet. 1988 Jul;4(7):198–203. doi: 10.1016/0168-9525(88)90076-5. [DOI] [PubMed] [Google Scholar]
  23. Dvorak H. F., Form D. M., Manseau E. J., Smith B. D. Pathogenesis of desmoplasia. I. Immunofluorescence identification and localization of some structural proteins of line 1 and line 10 guinea pig tumors and of healing wounds. J Natl Cancer Inst. 1984 Nov;73(5):1195–1205. [PubMed] [Google Scholar]
  24. Ffrench-Constant C., Hynes R. O. Alternative splicing of fibronectin is temporally and spatially regulated in the chicken embryo. Development. 1989 Jun;106(2):375–388. doi: 10.1242/dev.106.2.375. [DOI] [PubMed] [Google Scholar]
  25. Ffrench-Constant C., Hynes R. O. Patterns of fibronectin gene expression and splicing during cell migration in chicken embryos. Development. 1988 Nov;104(3):369–382. doi: 10.1242/dev.104.3.369. [DOI] [PubMed] [Google Scholar]
  26. Grinnell F., Billingham R. E., Burgess L. Distribution of fibronectin during wound healing in vivo. J Invest Dermatol. 1981 Mar;76(3):181–189. doi: 10.1111/1523-1747.ep12525694. [DOI] [PubMed] [Google Scholar]
  27. Grinnell F., Toda K., Takashima A. Activation of keratinocyte fibronectin receptor function during cutaneous wound healing. J Cell Sci Suppl. 1987;8:199–209. doi: 10.1242/jcs.1987.supplement_8.11. [DOI] [PubMed] [Google Scholar]
  28. Grinnell F., Toda K., Takashima A. Role of fibronectin in epithelialization and wound healing. Prog Clin Biol Res. 1988;266:259–272. [PubMed] [Google Scholar]
  29. Gutman A., Kornblihtt A. R. Identification of a third region of cell-specific alternative splicing in human fibronectin mRNA. Proc Natl Acad Sci U S A. 1987 Oct;84(20):7179–7182. doi: 10.1073/pnas.84.20.7179. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Heasman J., Hynes R. O., Swan A. P., Thomas V., Wylie C. C. Primordial germ cells of Xenopus embryos: the role of fibronectin in their adhesion during migration. Cell. 1981 Dec;27(3 Pt 2):437–447. doi: 10.1016/0092-8674(81)90385-8. [DOI] [PubMed] [Google Scholar]
  31. Humphries M. J., Akiyama S. K., Komoriya A., Olden K., Yamada K. M. Identification of an alternatively spliced site in human plasma fibronectin that mediates cell type-specific adhesion. J Cell Biol. 1986 Dec;103(6 Pt 2):2637–2647. doi: 10.1083/jcb.103.6.2637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Humphries M. J., Akiyama S. K., Komoriya A., Olden K., Yamada K. M. Neurite extension of chicken peripheral nervous system neurons on fibronectin: relative importance of specific adhesion sites in the central cell-binding domain and the alternatively spliced type III connecting segment. J Cell Biol. 1988 Apr;106(4):1289–1297. doi: 10.1083/jcb.106.4.1289. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Humphries M. J., Komoriya A., Akiyama S. K., Olden K., Yamada K. M. Identification of two distinct regions of the type III connecting segment of human plasma fibronectin that promote cell type-specific adhesion. J Biol Chem. 1987 May 15;262(14):6886–6892. [PubMed] [Google Scholar]
  34. Hynes R. Molecular biology of fibronectin. Annu Rev Cell Biol. 1985;1:67–90. doi: 10.1146/annurev.cb.01.110185.000435. [DOI] [PubMed] [Google Scholar]
  35. Ignotz R. A., Endo T., Massagué J. Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-beta. J Biol Chem. 1987 May 15;262(14):6443–6446. [PubMed] [Google Scholar]
  36. Ignotz R. A., Massagué J. Cell adhesion protein receptors as targets for transforming growth factor-beta action. Cell. 1987 Oct 23;51(2):189–197. doi: 10.1016/0092-8674(87)90146-2. [DOI] [PubMed] [Google Scholar]
  37. Kariniemi A. L., Lehto V. P., Vartio T., Virtanen I. Cytoskeleton and pericellular matrix organization of pure adult human keratinocytes cultured from suction-blister roof epidermis. J Cell Sci. 1982 Dec;58:49–61. doi: 10.1242/jcs.58.1.49. [DOI] [PubMed] [Google Scholar]
  38. Kitten G. T., Markwald R. R., Bolender D. L. Distribution of basement membrane antigens in cryopreserved early embryonic hearts. Anat Rec. 1987 Apr;217(4):379–390. doi: 10.1002/ar.1092170409. [DOI] [PubMed] [Google Scholar]
  39. Kornblihtt A. R., Umezawa K., Vibe-Pedersen K., Baralle F. E. Primary structure of human fibronectin: differential splicing may generate at least 10 polypeptides from a single gene. EMBO J. 1985 Jul;4(7):1755–1759. doi: 10.1002/j.1460-2075.1985.tb03847.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Kubo M., Kan M., Isemura M., Yamane I., Tagami H. Effects of extracellular matrices on human keratinocyte adhesion and growth and on its secretion and deposition of fibronectin in culture. J Invest Dermatol. 1987 May;88(5):594–601. doi: 10.1111/1523-1747.ep12470207. [DOI] [PubMed] [Google Scholar]
  41. Kurkinen M., Alitalo K., Vaheri A., Stenman S., Saxén L. Fibronectin in the development of embryonic chick eye. Dev Biol. 1979 Apr;69(2):589–600. doi: 10.1016/0012-1606(79)90313-0. [DOI] [PubMed] [Google Scholar]
  42. Kurkinen M., Vaheri A., Roberts P. J., Stenman S. Sequential appearance of fibronectin and collagen in experimental granulation tissue. Lab Invest. 1980 Jul;43(1):47–51. [PubMed] [Google Scholar]
  43. Linask K. K., Lash J. W. Precardiac cell migration: fibronectin localization at mesoderm-endoderm interface during directional movement. Dev Biol. 1986 Mar;114(1):87–101. doi: 10.1016/0012-1606(86)90385-4. [DOI] [PubMed] [Google Scholar]
  44. Mayer B. W., Jr, Hay E. D., Hynes R. O. Immunocytochemical localization of fibronectin in embryonic chick trunk and area vasculosa. Dev Biol. 1981 Mar;82(2):267–286. doi: 10.1016/0012-1606(81)90451-6. [DOI] [PubMed] [Google Scholar]
  45. Newgreen D., Thiery J. P. Fibronectin in early avian embryos: synthesis and distribution along the migration pathways of neural crest cells. Cell Tissue Res. 1980;211(2):269–291. doi: 10.1007/BF00236449. [DOI] [PubMed] [Google Scholar]
  46. Norris D. A., Clark R. A., Swigart L. M., Huff J. C., Weston W. L., Howell S. E. Fibronectin fragment(s) are chemotactic for human peripheral blood monocytes. J Immunol. 1982 Oct;129(4):1612–1618. [PubMed] [Google Scholar]
  47. Norton P. A., Hynes R. O. Alternative splicing of chicken fibronectin in embryos and in normal and transformed cells. Mol Cell Biol. 1987 Dec;7(12):4297–4307. doi: 10.1128/mcb.7.12.4297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. O'Keefe E. J., Woodley D. T., Falk R. J., Gammon W. R., Briggaman R. A. Production of fibronectin by epithelium in a skin equivalent. J Invest Dermatol. 1987 May;88(5):634–639. doi: 10.1111/1523-1747.ep12470246. [DOI] [PubMed] [Google Scholar]
  49. O'Keefe E. J., Woodley D., Castillo G., Russell N., Payne R. E., Jr Production of soluble and cell-associated fibronectin by cultured keratinocytes. J Invest Dermatol. 1984 Feb;82(2):150–155. doi: 10.1111/1523-1747.ep12259708. [DOI] [PubMed] [Google Scholar]
  50. Paul J. I., Hynes R. O. Multiple fibronectin subunits and their post-translational modifications. J Biol Chem. 1984 Nov 10;259(21):13477–13487. [PubMed] [Google Scholar]
  51. Paul J. I., Schwarzbauer J. E., Tamkun J. W., Hynes R. O. Cell-type-specific fibronectin subunits generated by alternative splicing. J Biol Chem. 1986 Sep 15;261(26):12258–12265. [PubMed] [Google Scholar]
  52. Poole T. J., Thiery J. P. Antibodies and a synthetic peptide that block cell-fibronectin adhesion arrest neural crest cell migration in vivo. Prog Clin Biol Res. 1986;217B:235–238. [PubMed] [Google Scholar]
  53. Price J., Hynes R. O. Astrocytes in culture synthesize and secrete a variant form of fibronectin. J Neurosci. 1985 Aug;5(8):2205–2211. doi: 10.1523/JNEUROSCI.05-08-02205.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Rappolee D. A., Mark D., Banda M. J., Werb Z. Wound macrophages express TGF-alpha and other growth factors in vivo: analysis by mRNA phenotyping. Science. 1988 Aug 5;241(4866):708–712. doi: 10.1126/science.3041594. [DOI] [PubMed] [Google Scholar]
  55. Roberts A. B., Sporn M. B., Assoian R. K., Smith J. M., Roche N. S., Wakefield L. M., Heine U. I., Liotta L. A., Falanga V., Kehrl J. H. Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4167–4171. doi: 10.1073/pnas.83.12.4167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Roberts C. J., Birkenmeier T. M., McQuillan J. J., Akiyama S. K., Yamada S. S., Chen W. T., Yamada K. M., McDonald J. A. Transforming growth factor beta stimulates the expression of fibronectin and of both subunits of the human fibronectin receptor by cultured human lung fibroblasts. J Biol Chem. 1988 Apr 5;263(10):4586–4592. [PubMed] [Google Scholar]
  57. Rovasio R. A., Delouvee A., Yamada K. M., Timpl R., Thiery J. P. Neural crest cell migration: requirements for exogenous fibronectin and high cell density. J Cell Biol. 1983 Feb;96(2):462–473. doi: 10.1083/jcb.96.2.462. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Ruoslahti E., Pierschbacher M. D. New perspectives in cell adhesion: RGD and integrins. Science. 1987 Oct 23;238(4826):491–497. doi: 10.1126/science.2821619. [DOI] [PubMed] [Google Scholar]
  59. Schwarzbauer J. E., Patel R. S., Fonda D., Hynes R. O. Multiple sites of alternative splicing of the rat fibronectin gene transcript. EMBO J. 1987 Sep;6(9):2573–2580. doi: 10.1002/j.1460-2075.1987.tb02547.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Schwarzbauer J. E., Tamkun J. W., Lemischka I. R., Hynes R. O. Three different fibronectin mRNAs arise by alternative splicing within the coding region. Cell. 1983 Dec;35(2 Pt 1):421–431. doi: 10.1016/0092-8674(83)90175-7. [DOI] [PubMed] [Google Scholar]
  61. Sekiguchi K., Klos A. M., Kurachi K., Yoshitake S., Hakomori S. Human liver fibronectin complementary DNAs: identification of two different messenger RNAs possibly encoding the alpha and beta subunits of plasma fibronectin. Biochemistry. 1986 Aug 26;25(17):4936–4941. doi: 10.1021/bi00365a032. [DOI] [PubMed] [Google Scholar]
  62. Takashima A., Billingham R. E., Grinnell F. Activation of rabbit keratinocyte fibronectin receptor function in vivo during wound healing. J Invest Dermatol. 1986 May;86(5):585–590. doi: 10.1111/1523-1747.ep12355243. [DOI] [PubMed] [Google Scholar]
  63. Tamkun J. W., Hynes R. O. Plasma fibronectin is synthesized and secreted by hepatocytes. J Biol Chem. 1983 Apr 10;258(7):4641–4647. [PubMed] [Google Scholar]
  64. Thiery J. P., Duband J. L., Delouvée A. Pathways and mechanisms of avian trunk neural crest cell migration and localization. Dev Biol. 1982 Oct;93(2):324–343. doi: 10.1016/0012-1606(82)90121-x. [DOI] [PubMed] [Google Scholar]
  65. Toda K., Grinnell F. Activation of human keratinocyte fibronectin receptor function in relation to other ligand-receptor interactions. J Invest Dermatol. 1987 Apr;88(4):412–417. doi: 10.1111/1523-1747.ep12469745. [DOI] [PubMed] [Google Scholar]
  66. Zardi L., Carnemolla B., Siri A., Petersen T. E., Paolella G., Sebastio G., Baralle F. E. Transformed human cells produce a new fibronectin isoform by preferential alternative splicing of a previously unobserved exon. EMBO J. 1987 Aug;6(8):2337–2342. doi: 10.1002/j.1460-2075.1987.tb02509.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES