Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2001 Jun 1;356(Pt 2):531–537. doi: 10.1042/0264-6021:3560531

Rat embryo fibroblasts require both the cell-binding and the heparin-binding domains of fibronectin for survival.

J Jeong 1, I Han 1, Y Lim 1, J Kim 1, I Park 1, A Woods 1, J R Couchman 1, E S Oh 1
PMCID: PMC1221866  PMID: 11368782

Abstract

Fibronectin (FN) is known to transduce signal(s) to rescue cells from detachment-induced apoptosis (anoikis) through an integrin-mediated survival pathway. However, the functions of individual FN domains have not been studied in detail. In the present study we investigated whether the interaction of the cell-binding domain of FN with integrin is sufficient to rescue rat embryo fibroblasts (REFs) from detachment-induced apoptosis. REFs attached and spread normally after plating on substrates coated with either intact FN or a FN fragment, FN120, that contains the cell-binding domain but lacks the C-terminal heparin-binding domain, HepII. REFs on FN maintained a well-spread fibroblastic shape and even proliferated in serum-free medium at 20 h after plating. In contrast, previously well-spread REFs on FN120 started losing fibroblastic shape with time and detached from FN120-coated plates after approx. 8 h. Nuclear condensation indicated apototic cell death. This was due to the decreased activity/stability of focal adhesion kinase (pp125FAK) in the absence of HepII domain. A peptide in the HepII domain [peptide V, WQPPRARI (single-letter amino acid codes)], which has previously been implicated in cytoskeletal organization, rescued apoptotic changes. Consistently, pp125FAK phosphorylation was increased, and both cleavage of pp125FAK and activation of caspase 3 on FN120 were partly blocked by peptide V. Thus the interaction of the cell-binding domain with integrin has a major role in cell survival but is itself not sufficient for cell survival. One or more additional survival signals come from the HepII domain to regulate pp125FAK activity/stability.

Full Text

The Full Text of this article is available as a PDF (294.4 KB).

Selected References

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

  1. Bachelder R. E., Ribick M. J., Marchetti A., Falcioni R., Soddu S., Davis K. R., Mercurio A. M. p53 inhibits alpha 6 beta 4 integrin survival signaling by promoting the caspase 3-dependent cleavage of AKT/PKB. J Cell Biol. 1999 Nov 29;147(5):1063–1072. doi: 10.1083/jcb.147.5.1063. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bernfield M., Kokenyesi R., Kato M., Hinkes M. T., Spring J., Gallo R. L., Lose E. J. Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans. Annu Rev Cell Biol. 1992;8:365–393. doi: 10.1146/annurev.cb.08.110192.002053. [DOI] [PubMed] [Google Scholar]
  3. Boudreau N. J., Jones P. L. Extracellular matrix and integrin signalling: the shape of things to come. Biochem J. 1999 May 1;339(Pt 3):481–488. [PMC free article] [PubMed] [Google Scholar]
  4. Buckley C. D., Pilling D., Henriquez N. V., Parsonage G., Threlfall K., Scheel-Toellner D., Simmons D. L., Akbar A. N., Lord J. M., Salmon M. RGD peptides induce apoptosis by direct caspase-3 activation. Nature. 1999 Feb 11;397(6719):534–539. doi: 10.1038/17409. [DOI] [PubMed] [Google Scholar]
  5. Cardarelli P. M., Cobb R. R., Nowlin D. M., Scholz W., Gorcsan F., Moscinski M., Yasuhara M., Chiang S. L., Lobl T. J. Cyclic RGD peptide inhibits alpha 4 beta 1 interaction with connecting segment 1 and vascular cell adhesion molecule. J Biol Chem. 1994 Jul 15;269(28):18668–18673. [PubMed] [Google Scholar]
  6. Cardone M. H., Salvesen G. S., Widmann C., Johnson G., Frisch S. M. The regulation of anoikis: MEKK-1 activation requires cleavage by caspases. Cell. 1997 Jul 25;90(2):315–323. doi: 10.1016/s0092-8674(00)80339-6. [DOI] [PubMed] [Google Scholar]
  7. Chen X., Wang J., Fu B., Yu L. RGD-containing peptides trigger apoptosis in glomerular mesangial cells of adult human kidneys. Biochem Biophys Res Commun. 1997 May 29;234(3):594–599. doi: 10.1006/bbrc.1997.6593. [DOI] [PubMed] [Google Scholar]
  8. Couchman J. R., Woods A. Syndecan-4 and integrins: combinatorial signaling in cell adhesion. J Cell Sci. 1999 Oct;112(Pt 20):3415–3420. doi: 10.1242/jcs.112.20.3415. [DOI] [PubMed] [Google Scholar]
  9. Day M. L., Foster R. G., Day K. C., Zhao X., Humphrey P., Swanson P., Postigo A. A., Zhang S. H., Dean D. C. Cell anchorage regulates apoptosis through the retinoblastoma tumor suppressor/E2F pathway. J Biol Chem. 1997 Mar 28;272(13):8125–8128. doi: 10.1074/jbc.272.13.8125. [DOI] [PubMed] [Google Scholar]
  10. Frisch S. M. Evidence for a function of death-receptor-related, death-domain-containing proteins in anoikis. Curr Biol. 1999 Sep 23;9(18):1047–1049. doi: 10.1016/s0960-9822(99)80455-2. [DOI] [PubMed] [Google Scholar]
  11. Frisch S. M., Ruoslahti E. Integrins and anoikis. Curr Opin Cell Biol. 1997 Oct;9(5):701–706. doi: 10.1016/s0955-0674(97)80124-x. [DOI] [PubMed] [Google Scholar]
  12. Frisch S. M., Vuori K., Ruoslahti E., Chan-Hui P. Y. Control of adhesion-dependent cell survival by focal adhesion kinase. J Cell Biol. 1996 Aug;134(3):793–799. doi: 10.1083/jcb.134.3.793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fukai F., Mashimo M., Akiyama K., Goto T., Tanuma S., Katayama T. Modulation of apoptotic cell death by extracellular matrix proteins and a fibronectin-derived antiadhesive peptide. Exp Cell Res. 1998 Jul 10;242(1):92–99. doi: 10.1006/excr.1998.4076. [DOI] [PubMed] [Google Scholar]
  14. Gervais F. G., Thornberry N. A., Ruffolo S. C., Nicholson D. W., Roy S. Caspases cleave focal adhesion kinase during apoptosis to generate a FRNK-like polypeptide. J Biol Chem. 1998 Jul 3;273(27):17102–17108. doi: 10.1074/jbc.273.27.17102. [DOI] [PubMed] [Google Scholar]
  15. Giancotti F. G., Ruoslahti E. Integrin signaling. Science. 1999 Aug 13;285(5430):1028–1032. doi: 10.1126/science.285.5430.1028. [DOI] [PubMed] [Google Scholar]
  16. Hanks S. K., Polte T. R. Signaling through focal adhesion kinase. Bioessays. 1997 Feb;19(2):137–145. doi: 10.1002/bies.950190208. [DOI] [PubMed] [Google Scholar]
  17. Horowitz A., Simons M. Phosphorylation of the cytoplasmic tail of syndecan-4 regulates activation of protein kinase Calpha. J Biol Chem. 1998 Oct 2;273(40):25548–25551. doi: 10.1074/jbc.273.40.25548. [DOI] [PubMed] [Google Scholar]
  18. Hungerford J. E., Compton M. T., Matter M. L., Hoffstrom B. G., Otey C. A. Inhibition of pp125FAK in cultured fibroblasts results in apoptosis. J Cell Biol. 1996 Dec;135(5):1383–1390. doi: 10.1083/jcb.135.5.1383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Ilić D., Almeida E. A., Schlaepfer D. D., Dazin P., Aizawa S., Damsky C. H. Extracellular matrix survival signals transduced by focal adhesion kinase suppress p53-mediated apoptosis. J Cell Biol. 1998 Oct 19;143(2):547–560. doi: 10.1083/jcb.143.2.547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kapila Y. L., Wang S., Johnson P. W. Mutations in the heparin binding domain of fibronectin in cooperation with the V region induce decreases in pp125(FAK) levels plus proteoglycan-mediated apoptosis via caspases. J Biol Chem. 1999 Oct 22;274(43):30906–30913. doi: 10.1074/jbc.274.43.30906. [DOI] [PubMed] [Google Scholar]
  21. Khwaja A., Rodriguez-Viciana P., Wennström S., Warne P. H., Downward J. Matrix adhesion and Ras transformation both activate a phosphoinositide 3-OH kinase and protein kinase B/Akt cellular survival pathway. EMBO J. 1997 May 15;16(10):2783–2793. doi: 10.1093/emboj/16.10.2783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lewis J. M., Cheresh D. A., Schwartz M. A. Protein kinase C regulates alpha v beta 5-dependent cytoskeletal associations and focal adhesion kinase phosphorylation. J Cell Biol. 1996 Sep;134(5):1323–1332. doi: 10.1083/jcb.134.5.1323. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. McGill G., Shimamura A., Bates R. C., Savage R. E., Fisher D. E. Loss of matrix adhesion triggers rapid transformation-selective apoptosis in fibroblasts. J Cell Biol. 1997 Aug 25;138(4):901–911. doi: 10.1083/jcb.138.4.901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Oh E. S., Woods A., Couchman J. R. Syndecan-4 proteoglycan regulates the distribution and activity of protein kinase C. J Biol Chem. 1997 Mar 28;272(13):8133–8136. doi: 10.1074/jbc.272.13.8133. [DOI] [PubMed] [Google Scholar]
  25. Re F., Zanetti A., Sironi M., Polentarutti N., Lanfrancone L., Dejana E., Colotta F. Inhibition of anchorage-dependent cell spreading triggers apoptosis in cultured human endothelial cells. J Cell Biol. 1994 Oct;127(2):537–546. doi: 10.1083/jcb.127.2.537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Rytömaa M., Martins L. M., Downward J. Involvement of FADD and caspase-8 signalling in detachment-induced apoptosis. Curr Biol. 1999 Sep 23;9(18):1043–1046. doi: 10.1016/s0960-9822(99)80454-0. [DOI] [PubMed] [Google Scholar]
  27. Saoncella S., Echtermeyer F., Denhez F., Nowlen J. K., Mosher D. F., Robinson S. D., Hynes R. O., Goetinck P. F. Syndecan-4 signals cooperatively with integrins in a Rho-dependent manner in the assembly of focal adhesions and actin stress fibers. Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2805–2810. doi: 10.1073/pnas.96.6.2805. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Sharma A., Askari J. A., Humphries M. J., Jones E. Y., Stuart D. I. Crystal structure of a heparin- and integrin-binding segment of human fibronectin. EMBO J. 1999 Mar 15;18(6):1468–1479. doi: 10.1093/emboj/18.6.1468. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Streuli C. Extracellular matrix remodelling and cellular differentiation. Curr Opin Cell Biol. 1999 Oct;11(5):634–640. doi: 10.1016/s0955-0674(99)00026-5. [DOI] [PubMed] [Google Scholar]
  30. Tang D. G., Tarrien M., Dobrzynski P., Honn K. V. Melanoma cell spreading on fibronectin induced by 12(S)-HETE involves both protein kinase C- and protein tyrosine kinase-dependent focal adhesion formation and tyrosine phosphorylation of focal adhesion kinase (pp125FAK). J Cell Physiol. 1995 Nov;165(2):291–306. doi: 10.1002/jcp.1041650210. [DOI] [PubMed] [Google Scholar]
  31. Tsuchida M., Manthei E. R., Alam T., Knechtle S. J., Hamawy M. M. Regulation of T cell receptor- and CD28-induced tyrosine phosphorylation of the focal adhesion tyrosine kinases Pyk2 and Fak by protein kinase C. A role for protein tyrosine phosphatases. J Biol Chem. 2000 Jan 14;275(2):1344–1350. doi: 10.1074/jbc.275.2.1344. [DOI] [PubMed] [Google Scholar]
  32. Tumova S., Woods A., Couchman J. R. Heparan sulfate chains from glypican and syndecans bind the Hep II domain of fibronectin similarly despite minor structural differences. J Biol Chem. 2000 Mar 31;275(13):9410–9417. doi: 10.1074/jbc.275.13.9410. [DOI] [PubMed] [Google Scholar]
  33. Vitale M., Di Matola T., Fenzi G., Illario M., Rossi G. Fibronectin is required to prevent thyroid cell apoptosis through an integrin-mediated adhesion mechanism. J Clin Endocrinol Metab. 1998 Oct;83(10):3673–3680. doi: 10.1210/jcem.83.10.5175. [DOI] [PubMed] [Google Scholar]
  34. Wen L. P., Fahrni J. A., Troie S., Guan J. L., Orth K., Rosen G. D. Cleavage of focal adhesion kinase by caspases during apoptosis. J Biol Chem. 1997 Oct 10;272(41):26056–26061. doi: 10.1074/jbc.272.41.26056. [DOI] [PubMed] [Google Scholar]
  35. Woods A., Couchman J. R., Johansson S., Hök M. Adhesion and cytoskeletal organisation of fibroblasts in response to fibronectin fragments. EMBO J. 1986 Apr;5(4):665–670. doi: 10.1002/j.1460-2075.1986.tb04265.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Woods A., McCarthy J. B., Furcht L. T., Couchman J. R. A synthetic peptide from the COOH-terminal heparin-binding domain of fibronectin promotes focal adhesion formation. Mol Biol Cell. 1993 Jun;4(6):605–613. doi: 10.1091/mbc.4.6.605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Zhang Z., Vuori K., Reed J. C., Ruoslahti E. The alpha 5 beta 1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):6161–6165. doi: 10.1073/pnas.92.13.6161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zimmermann P., David G. The syndecans, tuners of transmembrane signaling. FASEB J. 1999;13 (Suppl):S91–S100. doi: 10.1096/fasebj.13.9001.s91. [DOI] [PubMed] [Google Scholar]
  39. van de Water B., Nagelkerke J. F., Stevens J. L. Dephosphorylation of focal adhesion kinase (FAK) and loss of focal contacts precede caspase-mediated cleavage of FAK during apoptosis in renal epithelial cells. J Biol Chem. 1999 May 7;274(19):13328–13337. doi: 10.1074/jbc.274.19.13328. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES