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. 1997 Apr;17(4):1938–1946. doi: 10.1128/mcb.17.4.1938

Suppression of autocrine and paracrine functions of basic fibroblast growth factor by stable expression of perlecan antisense cDNA.

D Aviezer 1, R V Iozzo 1, D M Noonan 1, A Yayon 1
PMCID: PMC232040  PMID: 9121441

Abstract

Heparan sulfate proteoglycans (HSPG) play a critical role in the formation of distinct fibroblast growth factor (FGF)-HS complexes, augmenting high-affinity binding and receptor activation. Perlecan, a secreted HSPG abundant in proliferating cells, is capable of inducing FGF-receptor interactions in vitro and angiogenesis in vivo. Stable and specific reduction of perlecan levels in mouse NIH 3T3 fibroblasts and human metastatic melanoma cells has been achieved by expression of antisense cDNA corresponding to the N-terminal and HS attachment domains of perlecan. Long-term perlecan downregulation is evidenced by reduced levels of perlecan mRNA and core protein as indicated by Northern blot analysis, immunoblots, and immunohistochemistry, using DNA probes and antibodies specific to mouse or human perlecan. The response of antisense perlecan-expressing cells to increasing concentrations of basic FGF (bFGF) is dramatically reduced in comparison to that in wild-type or vector-transfected cells, as measured by thymidine incorporation and rate of proliferation. Furthermore, receptor binding and affinity labeling of antisense perlecan-transfected cells with 125I-bFGF is markedly inhibited, indicating that eliminating perlecan expression results in reduced high-affinity bFGF binding. Both the binding and mitogenic response of antisense-perlecan-expressing clones to bFGF can be rescued by exogenous heparin or perlecan. These results support the notion that perlecan is a major accessory receptor for bFGF in mouse fibroblasts and human melanomas and point to the possible use of perlecan antisense constructs as specific modulators of bFGF-mediated responses.

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Selected References

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  1. Aviezer D., Hecht D., Safran M., Eisinger M., David G., Yayon A. Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and angiogenesis. Cell. 1994 Dec 16;79(6):1005–1013. doi: 10.1016/0092-8674(94)90031-0. [DOI] [PubMed] [Google Scholar]
  2. Aviezer D., Levy E., Safran M., Svahn C., Buddecke E., Schmidt A., David G., Vlodavsky I., Yayon A. Differential structural requirements of heparin and heparan sulfate proteoglycans that promote binding of basic fibroblast growth factor to its receptor. J Biol Chem. 1994 Jan 7;269(1):114–121. [PubMed] [Google Scholar]
  3. Basilico C., Moscatelli D. The FGF family of growth factors and oncogenes. Adv Cancer Res. 1992;59:115–165. doi: 10.1016/s0065-230x(08)60305-x. [DOI] [PubMed] [Google Scholar]
  4. Becker D., Meier C. B., Herlyn M. Proliferation of human malignant melanomas is inhibited by antisense oligodeoxynucleotides targeted against basic fibroblast growth factor. EMBO J. 1989 Dec 1;8(12):3685–3691. doi: 10.1002/j.1460-2075.1989.tb08543.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Brickman Y. G., Ford M. D., Small D. H., Bartlett P. F., Nurcombe V. Heparan sulfates mediate the binding of basic fibroblast growth factor to a specific receptor on neural precursor cells. J Biol Chem. 1995 Oct 20;270(42):24941–24948. doi: 10.1074/jbc.270.42.24941. [DOI] [PubMed] [Google Scholar]
  6. Chakravarti S., Horchar T., Jefferson B., Laurie G. W., Hassell J. R. Recombinant domain III of perlecan promotes cell attachment through its RGDS sequence. J Biol Chem. 1995 Jan 6;270(1):404–409. doi: 10.1074/jbc.270.1.404. [DOI] [PubMed] [Google Scholar]
  7. Cohen I. R., Murdoch A. D., Naso M. F., Marchetti D., Berd D., Iozzo R. V. Abnormal expression of perlecan proteoglycan in metastatic melanomas. Cancer Res. 1994 Nov 15;54(22):5771–5774. [PubMed] [Google Scholar]
  8. Dotto G. P., Moellmann G., Ghosh S., Edwards M., Halaban R. Transformation of murine melanocytes by basic fibroblast growth factor cDNA and oncogenes and selective suppression of the transformed phenotype in a reconstituted cutaneous environment. J Cell Biol. 1989 Dec;109(6 Pt 1):3115–3128. doi: 10.1083/jcb.109.6.3115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Folkman J., Shing Y. Control of angiogenesis by heparin and other sulfated polysaccharides. Adv Exp Med Biol. 1992;313:355–364. doi: 10.1007/978-1-4899-2444-5_34. [DOI] [PubMed] [Google Scholar]
  10. Gallagher J. T. Heparan sulphates as membrane receptors for the fibroblast growth factors. Eur J Clin Chem Clin Biochem. 1994 Apr;32(4):239–247. [PubMed] [Google Scholar]
  11. Guelstein V. I., Tchypysheva T. A., Ermilova V. D., Ljubimov A. V. Myoepithelial and basement membrane antigens in benign and malignant human breast tumors. Int J Cancer. 1993 Jan 21;53(2):269–277. doi: 10.1002/ijc.2910530217. [DOI] [PubMed] [Google Scholar]
  12. Guillonneau X., Tassin J., Berrou E., Bryckaert M., Courtois Y., Mascarelli F. In vitro changes in plasma membrane heparan sulfate proteoglycans and in perlecan expression participate in the regulation of fibroblast growth factor 2 mitogenic activity. J Cell Physiol. 1996 Jan;166(1):170–187. doi: 10.1002/(SICI)1097-4652(199601)166:1<170::AID-JCP19>3.0.CO;2-J. [DOI] [PubMed] [Google Scholar]
  13. Guimond S., Maccarana M., Olwin B. B., Lindahl U., Rapraeger A. C. Activating and inhibitory heparin sequences for FGF-2 (basic FGF). Distinct requirements for FGF-1, FGF-2, and FGF-4. J Biol Chem. 1993 Nov 15;268(32):23906–23914. [PubMed] [Google Scholar]
  14. Gunning P., Leavitt J., Muscat G., Ng S. Y., Kedes L. A human beta-actin expression vector system directs high-level accumulation of antisense transcripts. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4831–4835. doi: 10.1073/pnas.84.14.4831. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Halaban R., Kwon B. S., Ghosh S., Delli Bovi P., Baird A. bFGF as an autocrine growth factor for human melanomas. Oncogene Res. 1988 Sep;3(2):177–186. [PubMed] [Google Scholar]
  16. Iozzo R. V. Biosynthesis of heparan sulfate proteoglycan by human colon carcinoma cells and its localization at the cell surface. J Cell Biol. 1984 Aug;99(2):403–417. doi: 10.1083/jcb.99.2.403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Iozzo R. V., Cohen I. R., Grässel S., Murdoch A. D. The biology of perlecan: the multifaceted heparan sulphate proteoglycan of basement membranes and pericellular matrices. Biochem J. 1994 Sep 15;302(Pt 3):625–639. doi: 10.1042/bj3020625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ishihara M. Structural requirements in heparin for binding and activation of FGF-1 and FGF-4 are different from that for FGF-2. Glycobiology. 1994 Dec;4(6):817–824. doi: 10.1093/glycob/4.6.817. [DOI] [PubMed] [Google Scholar]
  19. Klagsbrun M. The fibroblast growth factor family: structural and biological properties. Prog Growth Factor Res. 1989;1(4):207–235. doi: 10.1016/0955-2235(89)90012-4. [DOI] [PubMed] [Google Scholar]
  20. Kokenyesi R., Silbert J. E. Formation of heparan sulfate or chondroitin/dermatan sulfate on recombinant domain I of mouse perlecan expressed in Chinese hamster ovary cells. Biochem Biophys Res Commun. 1995 Jun 6;211(1):262–267. doi: 10.1006/bbrc.1995.1805. [DOI] [PubMed] [Google Scholar]
  21. Kovalszky I., Schaff Z., Jeney A. Potential markers (enzymes, proteoglycans) for human liver tumors. Acta Biomed Ateneo Parmense. 1993;64(5-6):157–163. [PubMed] [Google Scholar]
  22. Laslett A. L., McFarlane J. R., Hearn M. T., Risbridger G. P. Requirement for heparan sulphate proteoglycans to mediate basic fibroblast growth factor (FGF-2)-induced stimulation of Leydig cell steroidogenesis. J Steroid Biochem Mol Biol. 1995 Sep;54(5-6):245–250. doi: 10.1016/0960-0760(95)00138-p. [DOI] [PubMed] [Google Scholar]
  23. Maccarana M., Casu B., Lindahl U. Minimal sequence in heparin/heparan sulfate required for binding of basic fibroblast growth factor. J Biol Chem. 1993 Nov 15;268(32):23898–23905. [PubMed] [Google Scholar]
  24. Mali M., Elenius K., Miettinen H. M., Jalkanen M. Inhibition of basic fibroblast growth factor-induced growth promotion by overexpression of syndecan-1. J Biol Chem. 1993 Nov 15;268(32):24215–24222. [PubMed] [Google Scholar]
  25. Mansukhani A., Dell'Era P., Moscatelli D., Kornbluth S., Hanafusa H., Basilico C. Characterization of the murine BEK fibroblast growth factor (FGF) receptor: activation by three members of the FGF family and requirement for heparin. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3305–3309. doi: 10.1073/pnas.89.8.3305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mathieu M., Chatelain E., Ornitz D., Bresnick J., Mason I., Kiefer P., Dickson C. Receptor binding and mitogenic properties of mouse fibroblast growth factor 3. Modulation of response by heparin. J Biol Chem. 1995 Oct 13;270(41):24197–24203. doi: 10.1074/jbc.270.41.24197. [DOI] [PubMed] [Google Scholar]
  27. Murdoch A. D., Dodge G. R., Cohen I., Tuan R. S., Iozzo R. V. Primary structure of the human heparan sulfate proteoglycan from basement membrane (HSPG2/perlecan). A chimeric molecule with multiple domains homologous to the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor. J Biol Chem. 1992 Apr 25;267(12):8544–8557. [PubMed] [Google Scholar]
  28. Noonan D. M., Fulle A., Valente P., Cai S., Horigan E., Sasaki M., Yamada Y., Hassell J. R. The complete sequence of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive similarity with laminin A chain, low density lipoprotein-receptor, and the neural cell adhesion molecule. J Biol Chem. 1991 Dec 5;266(34):22939–22947. [PubMed] [Google Scholar]
  29. Nurcombe V., Ford M. D., Wildschut J. A., Bartlett P. F. Developmental regulation of neural response to FGF-1 and FGF-2 by heparan sulfate proteoglycan. Science. 1993 Apr 2;260(5104):103–106. doi: 10.1126/science.7682010. [DOI] [PubMed] [Google Scholar]
  30. Ornitz D. M., Yayon A., Flanagan J. G., Svahn C. M., Levi E., Leder P. Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble receptor and for mitogenesis in whole cells. Mol Cell Biol. 1992 Jan;12(1):240–247. doi: 10.1128/mcb.12.1.240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pantoliano M. W., Horlick R. A., Springer B. A., Van Dyk D. E., Tobery T., Wetmore D. R., Lear J. D., Nahapetian A. T., Bradley J. D., Sisk W. P. Multivalent ligand-receptor binding interactions in the fibroblast growth factor system produce a cooperative growth factor and heparin mechanism for receptor dimerization. Biochemistry. 1994 Aug 30;33(34):10229–10248. doi: 10.1021/bi00200a003. [DOI] [PubMed] [Google Scholar]
  32. Rapraeger A. C., Guimond S., Krufka A., Olwin B. B. Regulation by heparan sulfate in fibroblast growth factor signaling. Methods Enzymol. 1994;245:219–240. doi: 10.1016/0076-6879(94)45013-7. [DOI] [PubMed] [Google Scholar]
  33. Rapraeger A. C., Krufka A., Olwin B. B. Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation. Science. 1991 Jun 21;252(5013):1705–1708. doi: 10.1126/science.1646484. [DOI] [PubMed] [Google Scholar]
  34. Rescan P. Y., Loréal O., Hassell J. R., Yamada Y., Guillouzo A., Clément B. Distribution and origin of the basement membrane component perlecan in rat liver and primary hepatocyte culture. Am J Pathol. 1993 Jan;142(1):199–208. [PMC free article] [PubMed] [Google Scholar]
  35. Saunders S., Jalkanen M., O'Farrell S., Bernfield M. Molecular cloning of syndecan, an integral membrane proteoglycan. J Cell Biol. 1989 Apr;108(4):1547–1556. doi: 10.1083/jcb.108.4.1547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Spivak-Kroizman T., Lemmon M. A., Dikic I., Ladbury J. E., Pinchasi D., Huang J., Jaye M., Crumley G., Schlessinger J., Lax I. Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation. Cell. 1994 Dec 16;79(6):1015–1024. doi: 10.1016/0092-8674(94)90032-9. [DOI] [PubMed] [Google Scholar]
  37. Walker A., Turnbull J. E., Gallagher J. T. Specific heparan sulfate saccharides mediate the activity of basic fibroblast growth factor. J Biol Chem. 1994 Jan 14;269(2):931–935. [PubMed] [Google Scholar]
  38. Whitelock J. M., Murdoch A. D., Iozzo R. V., Underwood P. A. The degradation of human endothelial cell-derived perlecan and release of bound basic fibroblast growth factor by stromelysin, collagenase, plasmin, and heparanases. J Biol Chem. 1996 Apr 26;271(17):10079–10086. doi: 10.1074/jbc.271.17.10079. [DOI] [PubMed] [Google Scholar]
  39. Yayon A., Klagsbrun M. Autocrine regulation of cell growth and transformation by basic fibroblast growth factor. Cancer Metastasis Rev. 1990 Nov;9(3):191–202. doi: 10.1007/BF00046360. [DOI] [PubMed] [Google Scholar]
  40. Yayon A., Klagsbrun M., Esko J. D., Leder P., Ornitz D. M. Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell. 1991 Feb 22;64(4):841–848. doi: 10.1016/0092-8674(91)90512-w. [DOI] [PubMed] [Google Scholar]
  41. Zakut R., Perlis R., Eliyahu S., Yarden Y., Givol D., Lyman S. D., Halaban R. KIT ligand (mast cell growth factor) inhibits the growth of KIT-expressing melanoma cells. Oncogene. 1993 Aug;8(8):2221–2229. [PubMed] [Google Scholar]

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