Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1989 Jul 1;109(1):441–448. doi: 10.1083/jcb.109.1.441

Transforming growth factor-beta activity is potentiated by heparin via dissociation of the transforming growth factor-beta/alpha 2- macroglobulin inactive complex

PMCID: PMC2115487  PMID: 2473082

Abstract

The control of smooth muscle cell (SMC) proliferation is determined by the combined actions of mitogens, such as platelet-derived growth factor, and the opposing action of growth inhibitory agents, such as heparin and transforming growth factor-beta (TGF-beta). The present studies identify an interaction between heparin and TGF-beta in which heparin potentiates the biological action of TGF-beta. Using a neutralizing antibody to TGF-beta, we observed that the short term antiproliferative effect of heparin depended upon the presence of biologically active TGF-beta. This effect was observed in rat and bovine aortic SMC and in CCL64 cells, but not in human saphenous vein SMC. Binding studies demonstrated that the addition of heparin (100 micrograms/ml) to medium containing 10% plasma-derived serum resulted in a 45% increase in the specific binding of 125I-TGF-beta to cells. Likewise, heparin induced a twofold increase in the growth inhibitory action of TGF-beta at concentrations of TGF-beta near its apparent dissociation constant. Using 125I-labeled TGF-beta, we demonstrated that TGF-beta complexes with the plasma component alpha 2- macroglobulin, but not with fibronectin. Heparin increases the electrophoretic mobility of TGF-beta apparently by freeing TGF-beta from its complex with alpha 2-macroglobulin. Dextran sulfate, another highly charged antiproliferative molecule, but not chondroitin sulfate or dermatan sulfate, similarly modified TGF-beta's mobility. Relatively high, antiproliferative concentrations of heparin (1-100 micrograms/ml) were required to dissociate the TGF-beta/alpha 2-macroglobulin complex. Thus, it appears that the antiproliferative effect of heparin may be partially attributed to its ability to potentiate the biological activity of TGF-beta by dissociating it from alpha 2-macroglobulin, which normally renders it inactive. We suggest that heparin-like agents may be important regulators of TGF-beta's biological activity.

Full Text

The Full Text of this article is available as a PDF (1.2 MB).

Selected References

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

  1. Assoian R. K., Sporn M. B. Type beta transforming growth factor in human platelets: release during platelet degranulation and action on vascular smooth muscle cells. J Cell Biol. 1986 Apr;102(4):1217–1223. doi: 10.1083/jcb.102.4.1217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Baird A., Durkin T. Inhibition of endothelial cell proliferation by type beta-transforming growth factor: interactions with acidic and basic fibroblast growth factors. Biochem Biophys Res Commun. 1986 Jul 16;138(1):476–482. doi: 10.1016/0006-291x(86)90305-0. [DOI] [PubMed] [Google Scholar]
  3. Bassols A., Massagué J. Transforming growth factor beta regulates the expression and structure of extracellular matrix chondroitin/dermatan sulfate proteoglycans. J Biol Chem. 1988 Feb 25;263(6):3039–3045. [PubMed] [Google Scholar]
  4. Benitz W. E., Lessler D. S., Coulson J. D., Bernfield M. Heparin inhibits proliferation of fetal vascular smooth muscle cells in the absence of platelet-derived growth factor. J Cell Physiol. 1986 Apr;127(1):1–7. doi: 10.1002/jcp.1041270102. [DOI] [PubMed] [Google Scholar]
  5. Chen J. K., Hoshi H., McKeehan W. L. Transforming growth factor type beta specifically stimulates synthesis of proteoglycan in human adult arterial smooth muscle cells. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5287–5291. doi: 10.1073/pnas.84.15.5287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chenu C., Pfeilschifter J., Mundy G. R., Roodman G. D. Transforming growth factor beta inhibits formation of osteoclast-like cells in long-term human marrow cultures. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5683–5687. doi: 10.1073/pnas.85.15.5683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Clark W. C., Bressler J. Transforming growth factor-beta-like activity in tumors of the central nervous system. J Neurosurg. 1988 Jun;68(6):920–924. doi: 10.3171/jns.1988.68.6.0920. [DOI] [PubMed] [Google Scholar]
  8. Clowes A. W., Karnowsky M. J. Suppression by heparin of smooth muscle cell proliferation in injured arteries. Nature. 1977 Feb 17;265(5595):625–626. doi: 10.1038/265625a0. [DOI] [PubMed] [Google Scholar]
  9. Coffey R. J., Jr, Kost L. J., Lyons R. M., Moses H. L., LaRusso N. F. Hepatic processing of transforming growth factor beta in the rat. Uptake, metabolism, and biliary excretion. J Clin Invest. 1987 Sep;80(3):750–757. doi: 10.1172/JCI113130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Del Vecchio P. J., Bizios R., Holleran L. A., Judge T. K., Pinto G. L. Inhibition of human scleral fibroblast proliferation with heparin. Invest Ophthalmol Vis Sci. 1988 Aug;29(8):1272–1276. [PubMed] [Google Scholar]
  11. Ewton D. Z., Spizz G., Olson E. N., Florini J. R. Decrease in transforming growth factor-beta binding and action during differentiation in muscle cells. J Biol Chem. 1988 Mar 15;263(8):4029–4032. [PubMed] [Google Scholar]
  12. Fager G., Hansson G. K., Ottosson P., Dahllöf B., Bondjers G. Human arterial smooth muscle cells in culture. Effects of platelet-derived growth factor and heparin on growth in vitro. Exp Cell Res. 1988 Jun;176(2):319–335. doi: 10.1016/0014-4827(88)90334-5. [DOI] [PubMed] [Google Scholar]
  13. Falanga V., Tiegs S. L., Alstadt S. P., Roberts A. B., Sporn M. B. Transforming growth factor-beta: selective increase in glycosaminoglycan synthesis by cultures of fibroblasts from patients with progressive systemic sclerosis. J Invest Dermatol. 1987 Jul;89(1):100–104. doi: 10.1111/1523-1747.ep12580445. [DOI] [PubMed] [Google Scholar]
  14. Falcone D. J., Salisbury B. G. Fibronectin stimulates macrophage uptake of low density lipoprotein-heparin-collagen complexes. Arteriosclerosis. 1988 May-Jun;8(3):263–273. doi: 10.1161/01.atv.8.3.263. [DOI] [PubMed] [Google Scholar]
  15. Fava R. A., McClure D. B. Fibronectin-associated transforming growth factor. J Cell Physiol. 1987 May;131(2):184–189. doi: 10.1002/jcp.1041310207. [DOI] [PubMed] [Google Scholar]
  16. Fritze L. M., Reilly C. F., Rosenberg R. D. An antiproliferative heparan sulfate species produced by postconfluent smooth muscle cells. J Cell Biol. 1985 Apr;100(4):1041–1049. doi: 10.1083/jcb.100.4.1041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Guyton J. R., Rosenberg R. D., Clowes A. W., Karnovsky M. J. Inhibition of rat arterial smooth muscle cell proliferation by heparin. In vivo studies with anticoagulant and nonanticoagulant heparin. Circ Res. 1980 May;46(5):625–634. doi: 10.1161/01.res.46.5.625. [DOI] [PubMed] [Google Scholar]
  18. Heimark R. L., Twardzik D. R., Schwartz S. M. Inhibition of endothelial regeneration by type-beta transforming growth factor from platelets. Science. 1986 Sep 5;233(4768):1078–1080. doi: 10.1126/science.3461562. [DOI] [PubMed] [Google Scholar]
  19. Hill D. J., Strain A. J., Elstow S. F., Swenne I., Milner R. D. Bi-functional action of transforming growth factor-beta on DNA synthesis in early passage human fetal fibroblasts. J Cell Physiol. 1986 Aug;128(2):322–328. doi: 10.1002/jcp.1041280226. [DOI] [PubMed] [Google Scholar]
  20. Hoover R. L., Rosenberg R., Haering W., Karnovsky M. J. Inhibition of rat arterial smooth muscle cell proliferation by heparin. II. In vitro studies. Circ Res. 1980 Oct;47(4):578–583. doi: 10.1161/01.res.47.4.578. [DOI] [PubMed] [Google Scholar]
  21. Hoshi H., Kan M., Chen J. K., Mckeehan W. L. Comparative endocrinology-paracrinology-autocrinology of human adult large vessel endothelial and smooth muscle cells. In Vitro Cell Dev Biol. 1988 Apr;24(4):309–320. doi: 10.1007/BF02628833. [DOI] [PubMed] [Google Scholar]
  22. Huang S. S., O'Grady P., Huang J. S. Human transforming growth factor beta.alpha 2-macroglobulin complex is a latent form of transforming growth factor beta. J Biol Chem. 1988 Jan 25;263(3):1535–1541. [PubMed] [Google Scholar]
  23. Kardami E., Spector D., Strohman R. C. Heparin inhibits skeletal muscle growth in vitro. Dev Biol. 1988 Mar;126(1):19–28. doi: 10.1016/0012-1606(88)90234-5. [DOI] [PubMed] [Google Scholar]
  24. Keski-Oja J., Lyons R. M., Moses H. L. Immunodetection and modulation of cellular growth with antibodies against native transforming growth factor-beta 1. Cancer Res. 1987 Dec 15;47(24 Pt 1):6451–6458. [PubMed] [Google Scholar]
  25. Kimchi A., Wang X. F., Weinberg R. A., Cheifetz S., Massagué J. Absence of TGF-beta receptors and growth inhibitory responses in retinoblastoma cells. Science. 1988 Apr 8;240(4849):196–199. doi: 10.1126/science.2895499. [DOI] [PubMed] [Google Scholar]
  26. Kimura A., Katoh O., Kuramoto A. Effects of platelet derived growth factor, epidermal growth factor and transforming growth factor-beta on the growth of human marrow fibroblasts. Br J Haematol. 1988 May;69(1):9–12. doi: 10.1111/j.1365-2141.1988.tb07595.x. [DOI] [PubMed] [Google Scholar]
  27. Klebe R. J., Escobedo L. V., Bentley K. L., Thompson L. K. Regulation of cell motility, morphology, and growth by sulfated glycosaminoglycans. Cell Motil Cytoskeleton. 1986;6(3):273–281. doi: 10.1002/cm.970060304. [DOI] [PubMed] [Google Scholar]
  28. Krycève-Martinerie C., Lawrence D. A., Crochet J., Jullien P., Vigier P. Further study of beta-TGFs released by virally transformed and non-transformed cells. Int J Cancer. 1985 Apr 15;35(4):553–558. doi: 10.1002/ijc.2910350421. [DOI] [PubMed] [Google Scholar]
  29. Lawrence D. A., Pircher R., Jullien P. Conversion of a high molecular weight latent beta-TGF from chicken embryo fibroblasts into a low molecular weight active beta-TGF under acidic conditions. Biochem Biophys Res Commun. 1985 Dec 31;133(3):1026–1034. doi: 10.1016/0006-291x(85)91239-2. [DOI] [PubMed] [Google Scholar]
  30. Lawrence D. A., Pircher R., Krycève-Martinerie C., Jullien P. Normal embryo fibroblasts release transforming growth factors in a latent form. J Cell Physiol. 1984 Oct;121(1):184–188. doi: 10.1002/jcp.1041210123. [DOI] [PubMed] [Google Scholar]
  31. Leof E. B., Proper J. A., Goustin A. S., Shipley G. D., DiCorleto P. E., Moses H. L. Induction of c-sis mRNA and activity similar to platelet-derived growth factor by transforming growth factor beta: a proposed model for indirect mitogenesis involving autocrine activity. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2453–2457. doi: 10.1073/pnas.83.8.2453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lippman M. M., Mathews M. B. Heparins: varying effects on cell proliferation in vitro and lack of correlation with anticoagulant activity. Fed Proc. 1977 Jan;36(1):55–59. [PubMed] [Google Scholar]
  33. 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]
  34. Majack R. A. Beta-type transforming growth factor specifies organizational behavior in vascular smooth muscle cell cultures. J Cell Biol. 1987 Jul;105(1):465–471. doi: 10.1083/jcb.105.1.465. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Massagué J., Like B. Cellular receptors for type beta transforming growth factor. Ligand binding and affinity labeling in human and rodent cell lines. J Biol Chem. 1985 Mar 10;260(5):2636–2645. [PubMed] [Google Scholar]
  36. McCaffrey T. A., Agarwal L. A., Weksler B. B. A rapid fluorometric DNA assay for the measurement of cell density and proliferation in vitro. In Vitro Cell Dev Biol. 1988 Mar;24(3):247–252. doi: 10.1007/BF02623555. [DOI] [PubMed] [Google Scholar]
  37. McCaffrey T. A., Nicholson A. C., Szabo P. E., Weksler M. E., Weksler B. B. Aging and arteriosclerosis. The increased proliferation of arterial smooth muscle cells isolated from old rats is associated with increased platelet-derived growth factor-like activity. J Exp Med. 1988 Jan 1;167(1):163–174. doi: 10.1084/jem.167.1.163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. 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]
  39. Nilsson J., Ksiazek T., Heldin C. H., Thyberg J. Demonstration of stimulatory effects of platelet-derived growth factor on cultivated rat arterial smooth muscle cells. Differences between cells from young and adult animals. Exp Cell Res. 1983 May;145(2):231–237. doi: 10.1016/0014-4827(83)90001-0. [DOI] [PubMed] [Google Scholar]
  40. Nilsson J., Ksiazek T., Thyberg J., Wasteson A. Cell surface components and growth regulation in cultivated arterial smooth muscle cells. J Cell Sci. 1983 Nov;64:107–121. doi: 10.1242/jcs.64.1.107. [DOI] [PubMed] [Google Scholar]
  41. O'Connor-McCourt M. D., Wakefield L. M. Latent transforming growth factor-beta in serum. A specific complex with alpha 2-macroglobulin. J Biol Chem. 1987 Oct 15;262(29):14090–14099. [PubMed] [Google Scholar]
  42. Penttinen R. P., Kobayashi S., Bornstein P. Transforming growth factor beta increases mRNA for matrix proteins both in the presence and in the absence of changes in mRNA stability. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1105–1108. doi: 10.1073/pnas.85.4.1105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pfeilschifter J., Seyedin S. M., Mundy G. R. Transforming growth factor beta inhibits bone resorption in fetal rat long bone cultures. J Clin Invest. 1988 Aug;82(2):680–685. doi: 10.1172/JCI113647. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. 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]
  45. Redini F., Galera P., Mauviel A., Loyau G., Pujol J. P. Transforming growth factor beta stimulates collagen and glycosaminoglycan biosynthesis in cultured rabbit articular chondrocytes. FEBS Lett. 1988 Jul 4;234(1):172–176. doi: 10.1016/0014-5793(88)81327-9. [DOI] [PubMed] [Google Scholar]
  46. Reilly C. F., Fritze L. M., Rosenberg R. D. Antiproliferative effects of heparin on vascular smooth muscle cells are reversed by epidermal growth factor. J Cell Physiol. 1987 May;131(2):149–157. doi: 10.1002/jcp.1041310203. [DOI] [PubMed] [Google Scholar]
  47. Reilly C. F., Fritze L. M., Rosenberg R. D. Heparin inhibition of smooth muscle cell proliferation: a cellular site of action. J Cell Physiol. 1986 Oct;129(1):11–19. doi: 10.1002/jcp.1041290103. [DOI] [PubMed] [Google Scholar]
  48. Rizzino A. Appearance of high affinity receptors for type beta transforming growth factor during differentiation of murine embryonal carcinoma cells. Cancer Res. 1987 Aug 15;47(16):4386–4390. [PubMed] [Google Scholar]
  49. Ruff E., Rizzino A. Preparation and binding of radioactively labeled porcine transforming growth factor type beta. Biochem Biophys Res Commun. 1986 Jul 31;138(2):714–719. doi: 10.1016/s0006-291x(86)80555-1. [DOI] [PubMed] [Google Scholar]
  50. Sackler J. P., Liu L. Heparin-induced osteoporosis. Br J Radiol. 1973 Jul;46(547):548–550. doi: 10.1259/0007-1285-46-547-548. [DOI] [PubMed] [Google Scholar]
  51. Segarini P. R., Seyedin S. M. The high molecular weight receptor to transforming growth factor-beta contains glycosaminoglycan chains. J Biol Chem. 1988 Jun 15;263(17):8366–8370. [PubMed] [Google Scholar]
  52. Smeland E. B., Blomhoff H. K., Holte H., Ruud E., Beiske K., Funderud S., Godal T., Ohlsson R. Transforming growth factor type beta (TGF beta) inhibits G1 to S transition, but not activation of human B lymphocytes. Exp Cell Res. 1987 Jul;171(1):213–222. doi: 10.1016/0014-4827(87)90264-3. [DOI] [PubMed] [Google Scholar]
  53. Sporn M. B., Roberts A. B., Wakefield L. M., de Crombrugghe B. Some recent advances in the chemistry and biology of transforming growth factor-beta. J Cell Biol. 1987 Sep;105(3):1039–1045. doi: 10.1083/jcb.105.3.1039. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Stemerman M. B., Weinstein R., Rowe J. W., Maciag T., Fuhro R., Gardner R. Vascular smooth muscle cell growth kinetics in vivo in aged rats. Proc Natl Acad Sci U S A. 1982 Jun;79(12):3863–3866. doi: 10.1073/pnas.79.12.3863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Söderdahl G., Betsholtz C., Johansson A., Nilsson K., Bergh J. Differential expression of platelet-derived growth factor and transforming growth factor genes in small- and non-small-cell human lung carcinoma lines. Int J Cancer. 1988 Apr 15;41(4):636–641. doi: 10.1002/ijc.2910410426. [DOI] [PubMed] [Google Scholar]
  56. Takehara K., LeRoy E. C., Grotendorst G. R. TGF-beta inhibition of endothelial cell proliferation: alteration of EGF binding and EGF-induced growth-regulatory (competence) gene expression. Cell. 1987 May 8;49(3):415–422. doi: 10.1016/0092-8674(87)90294-7. [DOI] [PubMed] [Google Scholar]
  57. Tashjian A. H., Jr, Voelkel E. F., Lazzaro M., Singer F. R., Roberts A. B., Derynck R., Winkler M. E., Levine L. Alpha and beta human transforming growth factors stimulate prostaglandin production and bone resorption in cultured mouse calvaria. Proc Natl Acad Sci U S A. 1985 Jul;82(13):4535–4538. doi: 10.1073/pnas.82.13.4535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Thornton S. C., Mueller S. N., Levine E. M. Human endothelial cells: use of heparin in cloning and long-term serial cultivation. Science. 1983 Nov 11;222(4624):623–625. doi: 10.1126/science.6635659. [DOI] [PubMed] [Google Scholar]
  59. Tsukada T., McNutt M. A., Ross R., Gown A. M. HHF35, a muscle actin-specific monoclonal antibody. II. Reactivity in normal, reactive, and neoplastic human tissues. Am J Pathol. 1987 May;127(2):389–402. [PMC free article] [PubMed] [Google Scholar]
  60. Van Obberghen-Schilling E., Roche N. S., Flanders K. C., Sporn M. B., Roberts A. B. Transforming growth factor beta 1 positively regulates its own expression in normal and transformed cells. J Biol Chem. 1988 Jun 5;263(16):7741–7746. [PubMed] [Google Scholar]
  61. Varga J., Rosenbloom J., Jimenez S. A. Transforming growth factor beta (TGF beta) causes a persistent increase in steady-state amounts of type I and type III collagen and fibronectin mRNAs in normal human dermal fibroblasts. Biochem J. 1987 Nov 1;247(3):597–604. doi: 10.1042/bj2470597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Wahl S. M., Hunt D. A., Wakefield L. M., McCartney-Francis N., Wahl L. M., Roberts A. B., Sporn M. B. Transforming growth factor type beta induces monocyte chemotaxis and growth factor production. Proc Natl Acad Sci U S A. 1987 Aug;84(16):5788–5792. doi: 10.1073/pnas.84.16.5788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Wakefield L. M., Smith D. M., Flanders K. C., Sporn M. B. Latent transforming growth factor-beta from human platelets. A high molecular weight complex containing precursor sequences. J Biol Chem. 1988 Jun 5;263(16):7646–7654. [PubMed] [Google Scholar]
  64. Wakefield L. M., Smith D. M., Masui T., Harris C. C., Sporn M. B. Distribution and modulation of the cellular receptor for transforming growth factor-beta. J Cell Biol. 1987 Aug;105(2):965–975. doi: 10.1083/jcb.105.2.965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Wrana J. L., Maeno M., Hawrylyshyn B., Yao K. L., Domenicucci C., Sodek J. Differential effects of transforming growth factor-beta on the synthesis of extracellular matrix proteins by normal fetal rat calvarial bone cell populations. J Cell Biol. 1988 Mar;106(3):915–924. doi: 10.1083/jcb.106.3.915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Yahalom J., Eldor A., Fuks Z., Vlodavsky I. Degradation of sulfated proteoglycans in the subendothelial extracellular matrix by human platelet heparitinase. J Clin Invest. 1984 Nov;74(5):1842–1849. doi: 10.1172/JCI111603. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES