Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1993 Aug 2;122(4):923–932. doi: 10.1083/jcb.122.4.923

Thrombospondin causes activation of latent transforming growth factor- beta secreted by endothelial cells by a novel mechanism [published erratum appears in J Cell Biol 1993 Sep;122(5):following 1143]

PMCID: PMC2119591  PMID: 8349738

Abstract

Thrombospondin (TSP) forms specific complexes with transforming growth factor-beta (TGF-beta) in the alpha granule releasate of platelets and these TSP-TGF-beta complexes inhibit the growth of bovine aortic endothelial cells (BAE). In these studies, we report that TSP stripped of associated TGF-beta (sTSP) retained growth inhibitory activity which was partially reversed by a neutralizing antibody specific for TGF- beta. Since BAE cells secrete latent TGF-beta, we determined whether sTSP activates the latent TGF-beta secreted by BAE cells. Cells were cultured with or without sTSP and then the conditioned medium was tested for the ability to support TGF-beta-dependent normal rat kidney (NRK) colony formation in soft agar. Medium conditioned with sTSP showed a dose- and time-dependent ability to stimulate BAE-secreted TGF- beta activity, reaching maximal activation by 1-2 h with 0.4 micrograms/ml (0.9 nM) sTSP. The sTSP-mediated stimulation of TGF-beta activity is not dependent on serum factors and is not a general property of extracellular matrix molecules. The sTSP-mediated stimulation of TGF-beta activity was blocked by a mAb specific for sTSP and by neutralizing antibodies to TGF-beta. Activation of BAE cell secreted latent TGF-beta by sTSP can occur in the absence of cells and apparently does not require interactions with cell surface molecules, since in conditioned medium removed from cells and then incubated with sTSP, activation occurs with kinetics and at levels similar to what is seen when sTSP is incubated in the presence of cells. Serine proteases such as plasmin are not involved in sTSP-mediated activation of TGF- beta. Factors that regulate the conversion of latent to active TGF-beta are keys to controlling TGF-beta activity. These data suggest that TSP is a potent physiologic regulator of TGF-beta activation.

Full Text

The Full Text of this article is available as a PDF (1.6 MB).

Selected References

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

  1. Allen-Hoffmann B. L., Crankshaw C. L., Mosher D. F. Transforming growth factor beta increases cell surface binding and assembly of exogenous (plasma) fibronectin by normal human fibroblasts. Mol Cell Biol. 1988 Oct;8(10):4234–4242. doi: 10.1128/mcb.8.10.4234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bagavandoss P., Wilks J. W. Specific inhibition of endothelial cell proliferation by thrombospondin. Biochem Biophys Res Commun. 1990 Jul 31;170(2):867–872. doi: 10.1016/0006-291x(90)92171-u. [DOI] [PubMed] [Google Scholar]
  3. Bodmer S., Strommer K., Frei K., Siepl C., de Tribolet N., Heid I., Fontana A. Immunosuppression and transforming growth factor-beta in glioblastoma. Preferential production of transforming growth factor-beta 2. J Immunol. 1989 Nov 15;143(10):3222–3229. [PubMed] [Google Scholar]
  4. Bornstein P. Thrombospondins: structure and regulation of expression. FASEB J. 1992 Nov;6(14):3290–3299. doi: 10.1096/fasebj.6.14.1426766. [DOI] [PubMed] [Google Scholar]
  5. Brown P. D., Wakefield L. M., Levinson A. D., Sporn M. B. Physicochemical activation of recombinant latent transforming growth factor-beta's 1, 2, and 3. Growth Factors. 1990;3(1):35–43. doi: 10.3109/08977199009037500. [DOI] [PubMed] [Google Scholar]
  6. Browne P. C., Miller J. J., Detwiler T. C. Kinetics of the formation of thrombin-thrombospondin complexes: involvement of a 77-kDa intermediate. Arch Biochem Biophys. 1988 Sep;265(2):534–538. doi: 10.1016/0003-9861(88)90158-0. [DOI] [PubMed] [Google Scholar]
  7. Brunner A. M., Gentry L. E., Cooper J. A., Purchio A. F. Recombinant type 1 transforming growth factor beta precursor produced in Chinese hamster ovary cells is glycosylated and phosphorylated. Mol Cell Biol. 1988 May;8(5):2229–2232. doi: 10.1128/mcb.8.5.2229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brunner A. M., Marquardt H., Malacko A. R., Lioubin M. N., Purchio A. F. Site-directed mutagenesis of cysteine residues in the pro region of the transforming growth factor beta 1 precursor. Expression and characterization of mutant proteins. J Biol Chem. 1989 Aug 15;264(23):13660–13664. [PubMed] [Google Scholar]
  9. Cheifetz S., Massagué J. Transforming growth factor-beta (TGF-beta) receptor proteoglycan. Cell surface expression and ligand binding in the absence of glycosaminoglycan chains. J Biol Chem. 1989 Jul 15;264(20):12025–12028. [PubMed] [Google Scholar]
  10. Dardik R., Lahav J. Cell-binding domain of endothelial cell thrombospondin: localization to the 70-kDa core fragment and determination of binding characteristics. Biochemistry. 1991 Sep 24;30(38):9378–9386. doi: 10.1021/bi00102a035. [DOI] [PubMed] [Google Scholar]
  11. Dennis P. A., Rifkin D. B. Cellular activation of latent transforming growth factor beta requires binding to the cation-independent mannose 6-phosphate/insulin-like growth factor type II receptor. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):580–584. doi: 10.1073/pnas.88.2.580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Flaumenhaft R., Abe M., Mignatti P., Rifkin D. B. Basic fibroblast growth factor-induced activation of latent transforming growth factor beta in endothelial cells: regulation of plasminogen activator activity. J Cell Biol. 1992 Aug;118(4):901–909. doi: 10.1083/jcb.118.4.901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Frazier W. A. Thrombospondin: a modular adhesive glycoprotein of platelets and nucleated cells. J Cell Biol. 1987 Aug;105(2):625–632. doi: 10.1083/jcb.105.2.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gentry L. E., Lioubin M. N., Purchio A. F., Marquardt H. Molecular events in the processing of recombinant type 1 pre-pro-transforming growth factor beta to the mature polypeptide. Mol Cell Biol. 1988 Oct;8(10):4162–4168. doi: 10.1128/mcb.8.10.4162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gentry L. E., Nash B. W. The pro domain of pre-pro-transforming growth factor beta 1 when independently expressed is a functional binding protein for the mature growth factor. Biochemistry. 1990 Jul 24;29(29):6851–6857. doi: 10.1021/bi00481a014. [DOI] [PubMed] [Google Scholar]
  16. Gentry L. E., Webb N. R., Lim G. J., Brunner A. M., Ranchalis J. E., Twardzik D. R., Lioubin M. N., Marquardt H., Purchio A. F. Type 1 transforming growth factor beta: amplified expression and secretion of mature and precursor polypeptides in Chinese hamster ovary cells. Mol Cell Biol. 1987 Oct;7(10):3418–3427. doi: 10.1128/mcb.7.10.3418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hannan R. L., Kourembanas S., Flanders K. C., Rogelj S. J., Roberts A. B., Faller D. V., Klagsbrun M. Endothelial cells synthesize basic fibroblast growth factor and transforming growth factor beta. Growth Factors. 1988;1(1):7–17. doi: 10.3109/08977198809000242. [DOI] [PubMed] [Google Scholar]
  18. Huber D., Fontana A., Bodmer S. Activation of human platelet-derived latent transforming growth factor-beta 1 by human glioblastoma cells. Comparison with proteolytic and glycosidic enzymes. Biochem J. 1991 Jul 1;277(Pt 1):165–173. doi: 10.1042/bj2770165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jullien P., Berg T. M., Lawrence D. A. Acidic cellular environments: activation of latent TGF-beta and sensitization of cellular responses to TGF-beta and EGF. Int J Cancer. 1989 May 15;43(5):886–891. doi: 10.1002/ijc.2910430525. [DOI] [PubMed] [Google Scholar]
  20. Kovacina K. S., Steele-Perkins G., Purchio A. F., Lioubin M., Miyazono K., Heldin C. H., Roth R. A. Interactions of recombinant and platelet transforming growth factor-beta 1 precursor with the insulin-like growth factor II/mannose 6-phosphate receptor. Biochem Biophys Res Commun. 1989 Apr 14;160(1):393–403. doi: 10.1016/0006-291x(89)91669-0. [DOI] [PubMed] [Google Scholar]
  21. Lyons R. M., Gentry L. E., Purchio A. F., Moses H. L. Mechanism of activation of latent recombinant transforming growth factor beta 1 by plasmin. J Cell Biol. 1990 Apr;110(4):1361–1367. doi: 10.1083/jcb.110.4.1361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lyons R. M., Keski-Oja J., Moses H. L. Proteolytic activation of latent transforming growth factor-beta from fibroblast-conditioned medium. J Cell Biol. 1988 May;106(5):1659–1665. doi: 10.1083/jcb.106.5.1659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Majack R. A., Cook S. C., Bornstein P. Control of smooth muscle cell growth by components of the extracellular matrix: autocrine role for thrombospondin. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9050–9054. doi: 10.1073/pnas.83.23.9050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Massagué J., Cheifetz S., Endo T., Nadal-Ginard B. Type beta transforming growth factor is an inhibitor of myogenic differentiation. Proc Natl Acad Sci U S A. 1986 Nov;83(21):8206–8210. doi: 10.1073/pnas.83.21.8206. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Miyazono K., Heldin C. H. Role for carbohydrate structures in TGF-beta 1 latency. Nature. 1989 Mar 9;338(6211):158–160. doi: 10.1038/338158a0. [DOI] [PubMed] [Google Scholar]
  26. Miyazono K., Hellman U., Wernstedt C., Heldin C. H. Latent high molecular weight complex of transforming growth factor beta 1. Purification from human platelets and structural characterization. J Biol Chem. 1988 May 5;263(13):6407–6415. [PubMed] [Google Scholar]
  27. Mooradian D. L., Lucas R. C., Weatherbee J. A., Furcht L. T. Transforming growth factor-beta 1 binds to immobilized fibronectin. J Cell Biochem. 1989 Dec;41(4):189–200. doi: 10.1002/jcb.240410404. [DOI] [PubMed] [Google Scholar]
  28. Mosher D. F. Physiology of thrombospondin. Annu Rev Med. 1990;41:85–97. doi: 10.1146/annurev.me.41.020190.000505. [DOI] [PubMed] [Google Scholar]
  29. Murphy-Ullrich J. E., Hök M. Thrombospondin modulates focal adhesions in endothelial cells. J Cell Biol. 1989 Sep;109(3):1309–1319. doi: 10.1083/jcb.109.3.1309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Murphy-Ullrich J. E., Schultz-Cherry S., Hök M. Transforming growth factor-beta complexes with thrombospondin. Mol Biol Cell. 1992 Feb;3(2):181–188. doi: 10.1091/mbc.3.2.181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Murphy-Ullrich J. E., Westrick L. G., Esko J. D., Mosher D. F. Altered metabolism of thrombospondin by Chinese hamster ovary cells defective in glycosaminoglycan synthesis. J Biol Chem. 1988 May 5;263(13):6400–6406. [PubMed] [Google Scholar]
  32. Müller G., Behrens J., Nussbaumer U., Böhlen P., Birchmeier W. Inhibitory action of transforming growth factor beta on endothelial cells. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5600–5604. doi: 10.1073/pnas.84.16.5600. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Paralkar V. M., Vukicevic S., Reddi A. H. Transforming growth factor beta type 1 binds to collagen IV of basement membrane matrix: implications for development. Dev Biol. 1991 Feb;143(2):303–308. doi: 10.1016/0012-1606(91)90081-d. [DOI] [PubMed] [Google Scholar]
  34. Phan S. H., Dillon R. G., McGarry B. M., Dixit V. M. Stimulation of fibroblast proliferation by thrombospondin. Biochem Biophys Res Commun. 1989 Aug 30;163(1):56–63. doi: 10.1016/0006-291x(89)92098-6. [DOI] [PubMed] [Google Scholar]
  35. Pircher R., Jullien P., Lawrence D. A. Beta-transforming growth factor is stored in human blood platelets as a latent high molecular weight complex. Biochem Biophys Res Commun. 1986 Apr 14;136(1):30–37. doi: 10.1016/0006-291x(86)90872-7. [DOI] [PubMed] [Google Scholar]
  36. Purchio A. F., Cooper J. A., Brunner A. M., Lioubin M. N., Gentry L. E., Kovacina K. S., Roth R. A., Marquardt H. Identification of mannose 6-phosphate in two asparagine-linked sugar chains of recombinant transforming growth factor-beta 1 precursor. J Biol Chem. 1988 Oct 5;263(28):14211–14215. [PubMed] [Google Scholar]
  37. 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]
  38. Sato Y., Rifkin D. B. Inhibition of endothelial cell movement by pericytes and smooth muscle cells: activation of a latent transforming growth factor-beta 1-like molecule by plasmin during co-culture. J Cell Biol. 1989 Jul;109(1):309–315. doi: 10.1083/jcb.109.1.309. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sato Y., Tsuboi R., Lyons R., Moses H., Rifkin D. B. Characterization of the activation of latent TGF-beta by co-cultures of endothelial cells and pericytes or smooth muscle cells: a self-regulating system. J Cell Biol. 1990 Aug;111(2):757–763. doi: 10.1083/jcb.111.2.757. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Taraboletti G., Roberts D., Liotta L. A., Giavazzi R. Platelet thrombospondin modulates endothelial cell adhesion, motility, and growth: a potential angiogenesis regulatory factor. J Cell Biol. 1990 Aug;111(2):765–772. doi: 10.1083/jcb.111.2.765. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wakefield L. M., Winokur T. S., Hollands R. S., Christopherson K., Levinson A. D., Sporn M. B. Recombinant latent transforming growth factor beta 1 has a longer plasma half-life in rats than active transforming growth factor beta 1, and a different tissue distribution. J Clin Invest. 1990 Dec;86(6):1976–1984. doi: 10.1172/JCI114932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Yamaguchi Y., Mann D. M., Ruoslahti E. Negative regulation of transforming growth factor-beta by the proteoglycan decorin. Nature. 1990 Jul 19;346(6281):281–284. doi: 10.1038/346281a0. [DOI] [PubMed] [Google Scholar]
  43. Zentella A., Massagué J. Transforming growth factor beta induces myoblast differentiation in the presence of mitogens. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5176–5180. doi: 10.1073/pnas.89.11.5176. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES