Constitutive and Regulated Shedding of Soluble FGF Receptors Releases Biologically Active Inhibitors of FGF-2
Abstract
:1. Introduction
2. Results
2.1. Identification of a Shed FGFR-1 Ectodomain in the Conditioned Media of Transfected COS 7 Cells
2.2. Ligand-Induced Shedding Leads to a Decrease in Full-Length FGFR-1 Receptors
2.3. Purification of the Soluble FGFR-1 Ectodomains
2.4. The Shed FGFR-1 Ectodomain Functions as a Biologically Active Inhibitor of FGF-2
2.5. The Secreted FGFR-1 Receptor Inhibits Capillary Tube Formation in Collagen Gels
2.6. Inhibition of FGFR-1 Ectodomain Shedding by Metalloprotease Inhibitors
2.7. FGFR-1 Ectodomain Shedding Is Not Inhibited by Mutations Surrounding the Cleavage Site
2.8. Constitutive FGFR-1 Ectodomain Shedding Is Activated by TPA
2.9. FGF-2-Activated Shedding Is Blocked by a Specific Inhibitor of the FGF Receptor Tyrosine Kinase, But Not by a PKC Inhibitor
3. Discussion
4. Experimental Procedures
4.1. Plasmids and Reagents
4.2. Cell Culture and Transfections
4.3. Production of Stably Transfected FGFR-1 Expressing CHO Cell Lines
4.4. Cell Lysis, Immunoprecipitation, and Gel Electrophoresis
4.5. Immunoblotting
4.6. Analysis of the Cleavage and Release of Soluble FGFR-1
4.7. Purification of Recombinant Soluble FGFR-1 Ectodomains
4.8. Cell Proliferation Assays
4.9. Gel Invasion Angiogenesis Assays
4.10. 125I-FGF-2 Ligand Binding Assays
4.11. Measurement of the Stimulation of DNA Synthesis
4.12. Construction of FGFR-1 Mutants
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
FGF | fibroblast growth factor |
FGFR | fibroblast growth factor receptor |
PKC | protein kinase C |
MMP | matrix metalloprotease |
TNF | tumor necrosis factor |
CHO | Chinese hamster ovary |
ADAM | a disintegrin and metalloprotease |
MAP kinase | mitogen activated protein kinase |
ERK | extracellular signal regulated kinase |
EGF | epidermal growth factor |
TIMP | tissue inhibitor of metalloprotease |
TPA | 12-O-tetradecanoyl phorbol 13-acetate |
ABAE cells | adult bovine aortic endothelial cells |
DMEM | Dulbecco’s modified Eagle media |
IMDM | Iscoves modified Dulbecco’s media |
SDS-PAGE | sodium dodecyl sulfate-polyacrylamide gel electrophoresis |
WGA | wheat germ agglutinin |
CSF | colony stimulatory factor |
PCR | polymerase chain reaction |
References
- Lichtenthaler, S.F.; Lemberg, M.K.; Fluhrer, R. Proteolytic ectodomain shedding of membrane proteins in mammals-hardware, concepts, and recent developments. EMBO J. 2018, 37, e99456. [Google Scholar] [CrossRef] [PubMed]
- Schlondorff, J.; Blobel, C.P. Metalloprotease-disintegrins: Modular proteins capable of promoting cell-cell interactions and triggering signals by protein-ectodomain shedding. J. Cell Sci. 1999, 112, 3603–3617. [Google Scholar] [PubMed]
- Werb, Z.; Yan, Y. A cellular striptease act. Science 1998, 282, 1279–1280. [Google Scholar] [CrossRef]
- Werb, Z. ECM and cell surface proteolysis: Regulating cellular ecology. Cell 1997, 91, 439–442. [Google Scholar] [CrossRef] [Green Version]
- Hsia, H.E.; Tüshaus, J.; Brummer, T.; Zheng, Y.; Scilabra, S.D.; Lichtenthaler, S.F. Functions of ‘A disintegrin and metalloproteases (ADAMs)’ in the mammalian nervous system. Cell. Mol. Life Sci. 2019, 76, 3055–3081. [Google Scholar] [CrossRef] [PubMed]
- Peschon, J.J.; Slack, J.L.; Reddy, P.; Stocking, K.L.; Sunnarborg, S.W.; Lee, D.C.; Russell, W.E.; Castner, B.J.; Johnson, R.S.; Fitzner, J.M.; et al. An essential role for ectodomain shedding in mammalian development. Science 1998, 282, 281–284. [Google Scholar] [CrossRef] [PubMed]
- Kato, M.; Wang, H.; Kainulainen, V.; Fitzgerald, M.L.; Ledbetter, S.; Ornitz, D.M.; Bernfield, M. Physiological degradation converts the soluble syndecan-1 ectodomain from an inhibitor to a potent activator of FGF-2. Nature 1998, 4, 691–697. [Google Scholar] [CrossRef]
- Pan, D.; Rubin, G.M. Kuzbanian controls proteolytic processing of Notch and mediates lateral inhibition during Drosophila and vertebrate neurogenesis. Cell 1997, 90, 271–280. [Google Scholar] [CrossRef] [Green Version]
- Kajita, M.; Itoh, Y.; Chiba, T.; Mori, H.; Okada, A.; Kinoh, H.; Seiki, M. Membrane-type 1 metalloproteinase cleaves CD44 and promotes cell migration. J. Cell Biol. 2001, 153, 893–904. [Google Scholar] [CrossRef]
- Walcheck, B.; Kahn, J.; Fisher, J.; Wang, B.; Fisk, S.; Payan, D.; Feehan, C.; Betageri, R.; Darlak, K.; Spatola, A.; et al. Neutrophil rolling altered by inhibition of L-selectin shedding in vitro. Nature 1996, 380, 720–723. [Google Scholar] [CrossRef]
- Lochter, A.; Sybille, G.; Muschler, J.; Freedman, N.; Werb, Z.; Bissell, M. Matrix Metalloproteinase Stromelysin-1 Triggers a Cascade of Molecular Alterations That Leads to Stable Epithelial-to-Mesenchymal Conversion and a Premalignant Phenotype in Mammary Epithelial Cells. J. Cell Biol. 1997, 139, 1861–1872. [Google Scholar] [CrossRef] [PubMed]
- Zhao, J.; Chen, H.; Peschon, J.; Shi, W.; Zhang, Y.; Frank, S.; Warburton, D. Pulmonary Hypoplasia in Mice Lacking Tumor Necrosis Factor- Converting Enzyme Indicates an Indispensable Role for Cell Surface Protein Shedding during Embryonic Lung Branching Morphogenesis. Dev. Biol. 2001, 232, 204–218. [Google Scholar] [CrossRef] [Green Version]
- McDermott, M.F.; Aksentijevich, I.; Galon, J.; McDermott, E.M.; Ogunkolade, B.W.; Centola, M.; Mansfield, E.; Gadina, M.; Karenko, L.; Pettersson, T.; et al. Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes. Cell 1999, 97, 133–144. [Google Scholar] [CrossRef]
- Brown, D.M.; Schmidt-Erfurth, U.; Do, D.V.; Holz, F.G.; Boyer, D.S.; Midena, E.; Heier, J.S.; Terasaki, H.; Kaiser, P.K.; Marcus, D.M.; et al. Intravitreal Aflibercept for Diabetic Macular Edema: 100-Week Results From the VISTA and VIVID Studies. Ophthalmology 2015, 122, 2044–2052. [Google Scholar] [CrossRef] [PubMed]
- Moreland, L.W.; Baumgartner, S.W.; Schiff, M.H.; Tindall, E.A.; Fleischmann, R.M.; Weaver, A.L.; Ettlinger, R.E.; Cohen, S.; Koopman, W.J.; Mohler, K.; et al. Treatment of rheumatoid arthritis with a recombinant human tumor necrosis factor receptor (p75)-Fc fusion protein. N. Engl. J. Med. 1997, 337, 141–147. [Google Scholar] [CrossRef] [PubMed]
- Haak-Frendscho, M.; Marsters, S.A.; Mordenti, J.; Brady, S.; Gillett, N.A.; Chen, S.A.; Ashkenazi, A. Inhibition of TNF by a TNF receptor immunoadhesin. Comparison to an anti-TNF monoclonal antibody. J. Immunol. 1994, 152, 1347–1353. [Google Scholar]
- Arribas, J.; Coodly, L.; Vollmer, P.; Kishimoto, T.K.; Rose-John, S.; Massague, J. Diverse Cell Surface Protein Ectodomains Are Shed by a System Sensitive to Metalloprotease Inhibitors. J. Biol. Chem. 1996, 271, 11376–11382. [Google Scholar] [CrossRef] [Green Version]
- Nath, D.; Williamson, N.J.; Jarvis, R.; Murphy, G. Shedding of c-Met is regulated by crosstalk between a G-protein coupled receptor and the EGF receptor and is mediated by a TIMP-3 sensitive metalloproteinase. J. Cell Sci. 2001, 114, 1213–1220. [Google Scholar] [CrossRef] [Green Version]
- Fan, H.; Derynck, R. Ectodomain shedding of TGF-alpha and other transmembrane proteins is induced by receptor tyrosine kinase activation and MAP kinase signaling cascades. EMBO J. 1999, 18, 6962–6972. [Google Scholar] [CrossRef] [Green Version]
- Pandiella, A.; Massague, J. Multiple signals activate cleavage of the membrane transforming growth factor-alpha precursor. J. Biol. Chem. 1991, 266, 5769–5773. [Google Scholar] [CrossRef]
- Wolfsberg, T.G.; Straight, P.D.; Gerena, R.L.; Huovila, A.P.; Primakoff, P.; Myles, D.G.; White, J.M. ADAM, a widely distributed and developmentally regulated gene family encoding membrane proteins with a disintegrin and metalloprotease domain. Dev. Biol. 1995, 169, 378–383. [Google Scholar] [CrossRef] [Green Version]
- Black, R.A.; Rauch, C.T.; Kozlosky, C.J.; Peschon, J.J.; Slack, J.L.; Wolfson, M.F.; Castner, B.J.; Stocking, K.L.; Reddy, P.; Srinivasan, S.; et al. A metalloproteinase disintegrin that releases tumor-necrosis factor-alpha from cells. Nature 1997, 385, 729–732. [Google Scholar] [CrossRef] [PubMed]
- Moss, M.L.; Jin, S.-L.C.; Milla, M.E.; Burkhart, W.; Carter, H.L.; Chen, W.-J.; Clay, W.C.; Didsbury, J.R.; Hassler, D.; Hoffman, C.R.; et al. Cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha. Nature 1997, 385, 733–736. [Google Scholar] [CrossRef] [PubMed]
- Izumi, Y.; Hirata, M.; Hasumwa, H.; Iwanoto, R.; Umata, Y.; Miyado, K.; Tamai, Y.; Kurisaki, T.; Sehara-Fujisawa, A.; Ohno, S.; et al. A metalloprotease-disintegrin, MDC9/meltrin-gamma/ADAM9 and PKCdelta are involved in TPA-induced ectodomain shedding of membrane-anchored heparin-binding EGF-like growth factor. EMBO J. 1998, 17, 7260–7272. [Google Scholar] [CrossRef] [Green Version]
- Gechtman, Z.; Alonso, J.L.; Raab, G.; Ingber, D.E.; Klagsbrun, M. The shedding of membrane-anchored heparin-binding epidermal-like growth factor is regulated by the Raf/mitogen-activated protein kinase cascade and by cell adhesion and spreading. J. Biol. Chem. 1999, 274, 28828–28835. [Google Scholar] [CrossRef] [Green Version]
- Hanneken, A. Structural characterization of the circulating soluble FGF receptors reveals multiple isoforms generated by secretion and ectodomain shedding. FEBS Lett. 2001, 489, 176–181. [Google Scholar] [CrossRef] [Green Version]
- Hanneken, A.; Baird, A. Soluble forms of the high-affinity fibroblast growth factor receptor in human vitreous fluid. Investig. Ophthalmol. Vis. Sci. 1995, 36, 1192–1196. [Google Scholar]
- Hanneken, A.M.; Ying, W.; Ling, N.; Baird, A. Identification of soluble forms of the fibroblast growth factor receptor in blood. Proc. Natl. Acad. Sci. USA 1994, 91, 9170–9174. [Google Scholar] [CrossRef] [Green Version]
- Baird, A.; Böhlen, P. Fibroblast growth factors. In Peptide Growth Factors and Their Receptors, 1st ed.; Sporn, M.B., Roberts, A.B., Eds.; Springer: Berlin/Heidelberg, Germany, 1991; pp. 369–418. [Google Scholar]
- Kimelman, D.; Abraham, J.A.; Haaparanta, T.; Palisi, T.M.; Kirschner, M.W. The presence of fibroblast growth factor in the frog egg: Its role as a natural mesoderm inducer. Science 1988, 242, 1053–1056. [Google Scholar] [CrossRef] [PubMed]
- Slack, J.M.W.; Isaacs, H.V. Presence of basic fibroblast growth factor in the early Xenopus embryo. Development 1989, 105, 147–153. [Google Scholar]
- Amaya, E.; Musci, T.J.; Kirschner, M.W. Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. Cell 1991, 66, 257–270. [Google Scholar] [CrossRef]
- Dennis, P.A.; Saksela, O.; Harpel, P.; Rifkin, D.B. Alpha-2 macroglobulin is a binding protein for basic fibroblast growth factor. J. Biol. Chem. 1989, 264, 7210–7216. [Google Scholar] [CrossRef]
- Celli, G.; LaRochelle, W.J.; Mackem, S.; Sharp, R.; Merlino, G. Soluble dominant-negative receptor uncovers essential roles for fibroblast growth factors in multi-organ induction and patterning. EMBO J. 1998, 17, 1642–1655. [Google Scholar] [CrossRef] [Green Version]
- Hanneken, A.; Maher, P.A.; Baird, A. High affinity immunoreactive FGF receptors in the extracellular matrix of vascular endothelial cells--implications for the modulation of FGF-2. J. Cell Biol. 1995, 128, 1221–1228. [Google Scholar] [CrossRef] [Green Version]
- Baird, A.; Ueno, N.; Esch, F.; Ling, N. Distribution of fibroblast growth factors (FGFs) in tissues and structure-function studies with synthetic fragments of basic FGF. J. Cell. Physiol. 1987, 5, 101–106. [Google Scholar] [CrossRef]
- Vilgrain, I. Phosphorylation of basic fibroblast growth factor by a protein kinase associated with the outer surface of a target cell. Mol. Endocrinol. 1991, 5, 1003–1012. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Maher, P. p38 Mitogen-activated protein kinase activation is required for fibroblast growth factor-2-stimulated cell proliferation but not differentiation. J. Biol. Chem. 1999, 274, 17491–17498. [Google Scholar] [CrossRef] [Green Version]
- Patstone, G.; Pasquale, E.B.; Maher, P.A. Different members of the fibroblast growth factor receptor family are specific to distinct cell types in the developing chicken embryo. Dev. Biol. 1993, 155, 107–123. [Google Scholar] [CrossRef] [PubMed]
- Johnson, D.E.; Lee, P.L.; Lu, J.; Williams, L.T. Diverse forms of a receptor for acidic and basic fibroblast growth factors. Mol. Cell. Biol. 1990, 10, 4728–4736. [Google Scholar] [CrossRef]
- Johnson, D.E.; Lu, J.; Chen, H.; Werner, S.; Williams, L.T. The human fibroblast growth factor receptor genes: A common structural arrangement underlies the mechanisms for generating receptor forms that differ in their third immunoglobulin domain. Mol. Cell. Biol. 1991, 11, 4627–4634. [Google Scholar] [CrossRef]
- Werner, S.; Duan, D.R.; DeVries, C.; Peters, K.G.; Johnson, D.E.; Williams, L.T. Differential splicing in the extracellular region of fibroblast growth factor receptor 1 generates receptor variants with different ligand-binding specificities. Mol. Cell. Biol. 1992, 12, 82–88. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Kim, C.W.; Goldberger, O.A.; Gallo, R.L.; Bernfield, M. Members of the syndecan family of heparan sulfate proteoglycans are expressed in distinct cell-, tissue-, and development-specific patterns. Mol. Biol. Cell. 1994, 5, 797–805. [Google Scholar] [CrossRef] [Green Version]
- Subramanian, S.V.; Fitzgerald, M.L.; Bernfield, M. Regulated shedding of syndecan-1 and -4 ectodomains by thrombin and growth factor receptor activation. J. Biol. Chem. 1997, 272, 14713–14720. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Fitzgerald, M.L.; Wang, Z.; Park, P.W.; Murphy, G.; Bernfield, M. Shedding of syndecan-1 and -4 ectodomains is regulated by multiple signaling pathways and mediated by a TIMP-3 sensitive metalloproteinase. J. Cell. Biol. 2000, 148, 811–824. [Google Scholar] [CrossRef] [PubMed]
- Hanneken, A.; Frautschy, S.; Galasko, D.; Baird, A. A fibroblast growth factor binding protein in human cerebral spinal fluid. Neuroreports 1995, 6, 886–888. [Google Scholar] [CrossRef] [PubMed]
- Levi, E.; Fridman, R.; Miao, H.-Q.; Ma, Y.-C.; Yayon, A.; Vlodavsky, I. Matrix metalloproteinase 2 releases active soluble ectodomain of fibroblast growth factor receptor 1. Proc. Natl. Acad. Sci. USA 1996, 93, 7069–7074. [Google Scholar] [CrossRef] [Green Version]
- Kiefer, M.C.; Baird, A.; Nguyen, T.; George-Nascimento, C.; Mason, O.B.; Boley, L.J.; Valenzuela, P.; Barr, P.J. Molecular cloning of a human basic fibroblast growth factor receptor cDNA and expression of a biologically active extracellular domain in a baculovirus system. Growth Factors 1991, 5, 115–127. [Google Scholar] [CrossRef]
- Maher, P. Phorbol esters inhibit fibroblast growth factor-2-stimulated fibroblast proliferation by a p38 MAP kinase dependent pathway. Oncogene 2002, 21, 1978–1988. [Google Scholar] [CrossRef] [Green Version]
- Montesano, R.; Vassalli, J.D.; Baird, A.; Guillemin, R.; Orci, L. Basic fibroblast growth factor induces angiogenesis in vitro. Proc. Natl. Acad. Sci. USA 1986, 83, 7297–7301. [Google Scholar] [CrossRef] [Green Version]
- Zeidman, R.; Pettersson, L.; Sailaja, P.R.; Truedsson, E.; Fagerstrom, S.; Pahlman, S.; Larsson, C. Novel and classical protein kinase C isoforms have different functions in proliferation, survival and differentiation of neuroblastoma cells. Int. J. Cancer 1999, 81, 494–501. [Google Scholar] [CrossRef]
- Mohammadi, M.; McMahon, G.; Sun, L.; Tang, C.; Hirth, P.; Yeh, B.K.; Hubbard, S.R.; Schlessinger, J. Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors. Science 1997, 276, 955–960. [Google Scholar] [CrossRef]
- Lantz, M.; Gullberg, U.; Nilsson, E.; Olsson, I. Characterization in vitro of a human tumor necrosis factor binding protein: A soluble form of a tumor necrosis factor receptor. J. Clin. Investig. 1990, 86, 1396–1402. [Google Scholar] [CrossRef] [Green Version]
- Downing, J.R.; Roussel, M.F.; Sherr, C.J. Ligand and protein kinase C downmodulate the colony-stimulating factor 1 receptor by independent mechanisms. Mol. Cell. Biol. 1989, 9, 2890–2896. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lammich, S.; Kojro, E.; Postina, R.; Gilbert, S.; Pfeiffer, R.; Jasionowski, M.; Haass, C.; Fahrenholz, F. Constitutive and regulated alpha-secretase cleavage of Alzheimer’s amyloid precursor protein by a disintegrin metalloprotease. Proc. Natl. Adac. Sci. USA 1999, 96, 3922–3927. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Barlaam, B.; Bird, T.G.; Lambert-van der Brempt, C.; Campbell, D.; Foster, S.J.; Maciewicz, R. New alpha-substituted succinate-based hydroxamic acids as TNFalpha convertase inhibitors. J. Med. Chem. 1999, 42, 4890–4908. [Google Scholar] [CrossRef]
- Arribas, J.; Lopez-Casillas, F.; Massague, J. Role of the juxtamembrane domains of the transforming growth factor-alpha precursor and the beta-amyloid precursor protein in regulated ectodomain shedding. J. Biol. Chem. 1997, 272, 17160–17165. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Althoff, K.; Mullberg, J.; Aasland, D.; Voltz, N.; Kallen, K.-J.; Grotzinger, J.; Rose-John, S. Recognition sequences and structural elements contribute to shedding susceptibility of membrane proteins. Biochem. J. 2001, 353, 663–672. [Google Scholar] [CrossRef]
- Isacchi, A.; Statuto, M.; Chiesa, R.; Bergonzoni, L.; Rusnati, M.; Sarmientos, P.; Ragnotti, G.; Presta, M. A six-amino acid deletion in basic fibroblast growth factor dissociates its mitogenic activity from its plasminogen activator-inducing capacity. Proc. Natl. Acad. Sci. USA 1991, 88, 2628–2632. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Presta, M.; Moscatelli, D.; Joseph-Silverstein, J.; Rifkin, D. Purification of a human hepatoma cell line of a basic fibroblast growth factor-like molecule that stimulates capillary endothelial cell plasminogen activator production, DNA synthesis and migration. Mol. Cell Biol. 1986, 6, 4060–4066. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Isacchi, A.; Bergonzoni, L.; Sarmientos, P. Complete sequence of a human receptor for acidic and basic fibroblast growth factors. Nucl. Acids Res. 1990, 18, 1906. [Google Scholar] [CrossRef] [Green Version]
- Ausubel, F.M.; Brent, R.; Kingston, R.E.; Moore, D.D.; Seidman, J.G.; Smith, J.A.; Struhl, K. (Eds.) Current Protocols in Molecular Biology; John Wiley & Sons: New York, NY, USA, 1999; Volume 2, pp. 9.1.4–9.1.9. [Google Scholar]
- 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, 33, 10229–10248. [Google Scholar] [CrossRef] [PubMed]
- Maher, P. Inhibition of the tyrosine kinase activity of the fibroblast growth factor receptor by the methyltransferase inhibitor 5’-methylthioadenosine. J. Biol. Chem. 1993, 268, 4244–4249. [Google Scholar] [CrossRef]
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. |
© 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Hanneken, A.; Mercado, M.; Maher, P. Constitutive and Regulated Shedding of Soluble FGF Receptors Releases Biologically Active Inhibitors of FGF-2. Int. J. Mol. Sci. 2021, 22, 2712. https://doi.org/10.3390/ijms22052712
Hanneken A, Mercado M, Maher P. Constitutive and Regulated Shedding of Soluble FGF Receptors Releases Biologically Active Inhibitors of FGF-2. International Journal of Molecular Sciences. 2021; 22(5):2712. https://doi.org/10.3390/ijms22052712
Chicago/Turabian StyleHanneken, Anne, Maluz Mercado, and Pamela Maher. 2021. "Constitutive and Regulated Shedding of Soluble FGF Receptors Releases Biologically Active Inhibitors of FGF-2" International Journal of Molecular Sciences 22, no. 5: 2712. https://doi.org/10.3390/ijms22052712