Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1982 Feb 1;92(2):485–492. doi: 10.1083/jcb.92.2.485

Role of fibronectin in collagen deposition: Fab' to the gelatin-binding domain of fibronectin inhibits both fibronectin and collagen organization in fibroblast extracellular matrix

PMCID: PMC2112086  PMID: 7061591

Abstract

We report the effect of Fab' (anti-60k) to a 60,000 mol wt gelatin binding domain of fibronectin (1981, J. Biol. Chem. 256:5583) on diploid fibroblast (IMR-90) extracellular fibronectin and collagen organization. Anti-60k Fab' did not inhibit IMR-90 attachment or proliferation in fibronectin-depleted medium. Fibroblasts cultured with preimmune Fab' deposited a dense extracellular network of fibronectin and collagen detectable by immunofluorescence, while anti-60k Fab' prevented extracellular collagen and fibronectin fibril deposition. Matrix fibronectin and collagen deposition remained decreased in cultures containing anti-60k Fab' until cells became bilayered or more dense, when fibronectin and collagen began to appear in lower cell layers. Anti-60k Fab' added to confluent cultures 24 h before fixation and staining had no effect on matrix fibronectin or collagen, so anti- 60k Fab' did not simply block immunostaining. Confluent cultures grown in anti-60k Fab' and labeled for 24 h with [3H]proline incorporated identical amounts of [3H]proline and [3H]hydroxyproline, but [3H]hydroxyproline deposition in the cell layer was significantly decreased by anti-60k Fab' (P less than 0.01). Extracellular matrix collagen does not appear to form a scaffold for fibronectin deposition, as neither gelatin nor a gelatin-binding fragment of plasma fibronectin inhibited deposition of matrix fibronectin. Our results suggest that interstitial collagens and fibronectin interact to form a fibrillar component of the extracellular matrix, and that fibronectin is required for normal collagen organization and deposition by fibroblasts in vitro. Domain-specific antibodies to fibronectin are powerful tools to study the biological role of fibronectin in extracellular matrix organization and other processes.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

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

  1. Baum B. J., McDonald J. A., Crystal R. G. Metabolic fate of the major cell surface protein of normal human fibroblasts. Biochem Biophys Res Commun. 1977 Nov 7;79(1):8–15. doi: 10.1016/0006-291x(77)90053-5. [DOI] [PubMed] [Google Scholar]
  2. Booth B. A., Polak K. L., Uitto J. Collagen biosynthesis by human skin fibroblasts. I. Optimization of the culture conditions for synthesis of type I and type III procollagens. Biochim Biophys Acta. 1980 Mar 28;607(1):145–160. doi: 10.1016/0005-2787(80)90228-2. [DOI] [PubMed] [Google Scholar]
  3. Bornstein P., Ash J. F. Cell surface-associated structural proteins in connective tissue cells. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2480–2484. doi: 10.1073/pnas.74.6.2480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Byers P. H., McKenney K. H., Lichtenstein J. R., Martin G. R. Preparation of type III procollagen and collagen from rat skin. Biochemistry. 1974 Dec 3;13(25):5243–5248. doi: 10.1021/bi00722a030. [DOI] [PubMed] [Google Scholar]
  5. Chen L. B., Murray A., Segal R. A., Bushnell A., Walsh M. L. Studies on intercellular LETS glycoprotein matrices. Cell. 1978 Jun;14(2):377–391. doi: 10.1016/0092-8674(78)90123-x. [DOI] [PubMed] [Google Scholar]
  6. Ehrismann R., Chiquet M., Turner D. C. Mode of action of fibronectin in promoting chicken myoblast attachment. Mr = 60,000 gelatin-binding fragment binds native fibronectin. J Biol Chem. 1981 Apr 25;256(8):4056–4062. [PubMed] [Google Scholar]
  7. Emmerling M. R., Johnson C. D., Mosher D. F., Lipton B. H., Lilien J. E. Cross-linking and binding of fibronectin with asymmetric acetylcholinesterase. Biochemistry. 1981 May 26;20(11):3242–3247. doi: 10.1021/bi00514a040. [DOI] [PubMed] [Google Scholar]
  8. Engvall E., Ruoslahti E., Miller E. J. Affinity of fibronectin to collagens of different genetic types and to fibrinogen. J Exp Med. 1978 Jun 1;147(6):1584–1595. doi: 10.1084/jem.147.6.1584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Epstein E. H., Jr, Scott R. D., Miller E. J., Piez K. A. Isolation and characterization of the peptides derived from soluble human and baboon skin collagen after cyanogen bromide cleavage. J Biol Chem. 1971 Mar 25;246(6):1718–1724. [PubMed] [Google Scholar]
  10. Faris B., Snider R., Levine A., Moscaritolo R., Salcedo L., Franzblau C. Effect of ascorbate on collagen synthesis by lung embryonic fibroblasts. In Vitro. 1978 Dec;14(12):1022–1027. doi: 10.1007/BF02616217. [DOI] [PubMed] [Google Scholar]
  11. Furcht L. T., Wendelschafer-Crabb G., Mosher D. F., Foidart J. M. An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts. J Supramol Struct. 1980;13(1):15–33. doi: 10.1002/jss.400130103. [DOI] [PubMed] [Google Scholar]
  12. GOLDBERG B., GREEN H. AN ANALYSIS OF COLLAGEN SECRETION BY ESTABLISHED MOUSE FIBROBLAST LINES. J Cell Biol. 1964 Jul;22:227–258. doi: 10.1083/jcb.22.1.227. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Hedman K., Kurkinen M., Alitalo K., Vaheri A., Johansson S., Hök M. Isolation of the pericellular matrix of human fibroblast cultures. J Cell Biol. 1979 Apr;81(1):83–91. doi: 10.1083/jcb.81.1.83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Heggeness M. H., Ash J. F. Use of the avidin-biotin complex for the localization of actin and myosin with fluorescence microscopy. J Cell Biol. 1977 Jun;73(3):783–788. doi: 10.1083/jcb.73.3.783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Heitzmann H., Richards F. M. Use of the avidin-biotin complex for specific staining of biological membranes in electron microscopy. Proc Natl Acad Sci U S A. 1974 Sep;71(9):3537–3541. doi: 10.1073/pnas.71.9.3537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Helseth D. L., Jr, Veis A. Collagen self-assembly in vitro. Differentiating specific telopeptide-dependent interactions using selective enzyme modification and the addition of free amino telopeptide. J Biol Chem. 1981 Jul 25;256(14):7118–7128. [PubMed] [Google Scholar]
  18. Jilek F., Hörmann H. Cold-insoluble globulin (fibronectin), IV[1-35 affinity to soluble collagen of various types. Hoppe Seylers Z Physiol Chem. 1978 Feb;359(2):247–250. doi: 10.1515/bchm.1978.359.1.247. [DOI] [PubMed] [Google Scholar]
  19. Jilek F., Hörmann H. Fibronectin (cold-insoluble globulin), VI. Influence of heparin and hyaluronic acid on the binding of native collagen. Hoppe Seylers Z Physiol Chem. 1979 Apr;360(4):597–603. doi: 10.1515/bchm2.1979.360.1.597. [DOI] [PubMed] [Google Scholar]
  20. Johansson S., Hök M. Heparin enhances the rate of binding of fibronectin to collagen. Biochem J. 1980 May 1;187(2):521–524. doi: 10.1042/bj1870521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kleinman H. K., Klebe R. J., Martin G. R. Role of collagenous matrices in the adhesion and growth of cells. J Cell Biol. 1981 Mar;88(3):473–485. doi: 10.1083/jcb.88.3.473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kleinman H. K., McGoodwin E. B. Localization of the cell attachment region in types I and II collagens. Biochem Biophys Res Commun. 1976 Sep 20;72(2):426–432. doi: 10.1016/s0006-291x(76)80060-5. [DOI] [PubMed] [Google Scholar]
  23. Kleinman H. K., McGoodwin E. B., Martin G. R., Klebe R. J., Fietzek P. P., Woolley D. E. Localization of the binding site for cell attachment in the alpha1(I) chain of collagen. J Biol Chem. 1978 Aug 25;253(16):5642–5646. [PubMed] [Google Scholar]
  24. Kleinman H. K., Wilkes C. M., Martin G. R. Interaction of fibronectin with collagen fibrils. Biochemistry. 1981 Apr 14;20(8):2325–2330. doi: 10.1021/bi00511a039. [DOI] [PubMed] [Google Scholar]
  25. Kurkinen M., Vaheri A. Fibronectin matrix: antibody-induced reorganization in human fibroblast cultures. Cell Biol Int Rep. 1977 Sep;1(5):469–475. doi: 10.1016/0309-1651(77)90082-0. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  28. Ledger P. W., Uchida N., Tanzer M. L. Immunocytochemical localization of procollagen and fibronectin in human fibroblasts: effects of the monovalent ionophore, monensin. J Cell Biol. 1980 Dec;87(3 Pt 1):663–671. doi: 10.1083/jcb.87.3.663. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. McDonald J. A., Broekelmann T. J., Kelley D. G., Villiger B. Gelatin-binding domain-specific anti-human plasma fibronectin Fab' inhibits fibronectin-mediated gelatin binding but not cell spreading. J Biol Chem. 1981 Jun 10;256(11):5583–5587. [PubMed] [Google Scholar]
  30. McDonald J. A., Kelley D. G. Degradation of fibronectin by human leukocyte elastase. Release of biologically active fragments. J Biol Chem. 1980 Sep 25;255(18):8848–8858. [PubMed] [Google Scholar]
  31. McLean I. W., Nakane P. K. Periodate-lysine-paraformaldehyde fixative. A new fixation for immunoelectron microscopy. J Histochem Cytochem. 1974 Dec;22(12):1077–1083. doi: 10.1177/22.12.1077. [DOI] [PubMed] [Google Scholar]
  32. Menzel E. J., Smolen J. S., Liotta L., Reid K. B. Interaction of fibronectin with C1q and its collagen-like fragment (CLF). FEBS Lett. 1981 Jun 29;129(1):188–192. doi: 10.1016/0014-5793(81)80787-9. [DOI] [PubMed] [Google Scholar]
  33. Mosher D. F. Fibronectin. Prog Hemost Thromb. 1980;5:111–151. [PubMed] [Google Scholar]
  34. Mosher D. F., Schad P. E. Cross-linking of fibronectin to collagen by blood coagulation Factor XIIIa. J Clin Invest. 1979 Sep;64(3):781–787. doi: 10.1172/JCI109524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. O'Neill C. H., Riddle P. N., Jordan P. W. The relation between surface area and anchorage dependence of growth in hamster and mouse fibroblasts. Cell. 1979 Apr;16(4):909–918. doi: 10.1016/0092-8674(79)90106-5. [DOI] [PubMed] [Google Scholar]
  36. Perkins M. E., Ji T. H., Hynes R. O. Cross-linking of fibronectin to sulfated proteoglycans at the cell surface. Cell. 1979 Apr;16(4):941–952. doi: 10.1016/0092-8674(79)90109-0. [DOI] [PubMed] [Google Scholar]
  37. Ruoslahti E., Engvall E. Complexing of fibronectin glycosaminoglycans and collagen. Biochim Biophys Acta. 1980 Aug 13;631(2):350–358. doi: 10.1016/0304-4165(80)90308-6. [DOI] [PubMed] [Google Scholar]
  38. Trelstad R. L., Hayashi K. Tendon collagen fibrillogenesis: intracellular subassemblies and cell surface changes associated with fibril growth. Dev Biol. 1979 Aug;71(2):228–242. doi: 10.1016/0012-1606(79)90166-0. [DOI] [PubMed] [Google Scholar]
  39. Vaheri A., Kurkinen M., Lehto V. P., Linder E., Timpl R. Codistribution of pericellular matrix proteins in cultured fibroblasts and loss in transformation: fibronectin and procollagen. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4944–4948. doi: 10.1073/pnas.75.10.4944. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Villiger B., Kelley D. G., Engleman W., Kuhn C., 3rd, McDonald J. A. Human alveolar macrophage fibronectin: synthesis, secretion, and ultrastructural localization during gelatin-coated latex particle binding. J Cell Biol. 1981 Sep;90(3):711–720. doi: 10.1083/jcb.90.3.711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Yamada K. M. Immunological characterization of a major transformation-sensitive fibroblast cell surface glycoprotein. Localization, redistribution, and role in cell shape. J Cell Biol. 1978 Aug;78(2):520–541. doi: 10.1083/jcb.78.2.520. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yamada K. M., Kennedy D. W., Kimata K., Pratt R. M. Characterization of fibronectin interactions with glycosaminoglycans and identification of active proteolytic fragments. J Biol Chem. 1980 Jul 10;255(13):6055–6063. [PubMed] [Google Scholar]

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

RESOURCES