Fibroblast growth factor 2 (FGF2) plays important roles in tissue development and repair. Using heparan sulfates (HS)/heparin as a cofactor, FGF2 binds to FGF receptor (FGFR) and induces downstream signaling pathways, such as ERK pathway, that regulate cellular behavior. In most cell lines, FGF2 signaling displays biphasic dose-response profile, reaching maximal response to intermediate concentrations, but weak response to high levels of FGF2. Recent reports demonstrated that the biphasic cellular response results from competition between binding of FGF2 to HS and FGFR that impinge upon ERK signaling dynamics. However, the role of HS/heparin in FGF signaling has been controversial. Several studies suggested that heparin is not required for FGF-FGFR complex formation and that the main role of heparin is to protect FGF from degradation. In this study, we investigated the relationship between FGF2 stability, heparin dependence and ERK signaling dynamics using FGF2 variants with increased thermal stability (FGF2-STABs). FGF2-STABs showed higher efficiency in induction of FGFR-mediated proliferation, lower affinity to heparin and were less dependent on heparin than wild-type FGF2 (FGF2-wt) for induction of FGFR-mediated mitogenic response. Interestingly, in primary mammary fibroblasts, FGF2-wt displayed a sigmoidal dose-response profile, while FGF2-STABs showed a biphasic response. Moreover, at low concentrations, FGF2-STABs induced ERK signaling more potently and displayed a faster dynamics of full ERK activation and higher amplitudes of ERK signaling than FGF2-wt. Our results suggest that FGF2 stability and heparin dependence are important factors in FGF-FGFR signaling complex assembly and ERK signaling dynamics.

Department of Histology and Embryology Faculty of Medicine Masaryk University Brno Czechia

Enantis Brno Czechia

International Clinical Research Center St Anne's University Hospital Brno Czechia

Loschmidt Laboratories RECETOX and Department of Experimental Biology Faculty of Science Masaryk University Brno Czechia

See more in PubMed

Ameri J., Ståhlberg A., Pedersen J., Johansson J. K., Johannesson M. M., Artner I., et al. (2010). FGF2 specifies hESC-derived definitive endoderm into foregut/midgut cell lineages in a concentration-dependent manner. Stem Cells. 28 45–56. 10.1002/stem.249 PubMed DOI

Bishop J. R., Schuksz M., Esko J. D. (2007). Heparan sulphate proteoglycans fine-tune mammalian physiology. Nature 446 1030–1037. 10.1038/nature05817 PubMed DOI

Buchtova M., Chaloupkova R., Zakrzewska M., Vesela I., Cela P., Barathova J., et al. (2015). Instability restricts signaling of multiple fibroblast growth factors. Cell. Mol. Life Sci. 72 2445–2459. 10.1007/s00018-015-1856-8 PubMed DOI PMC

Bugaj L. J., Sabnis A. J., Mitchell A., Garbarino J. E., Toettcher J. E., Bivona T. G., et al. (2018). Cancer mutations and targeted drugs can disrupt dynamic signal encoding by the Ras-Erk pathway. Science 361:eaao3048. 10.1126/science.aao3048 PubMed DOI PMC

Caldwell M. A., Garcion E., terBorg M. G., He X., Svendsen C. N. (2004). Heparin stabilizes FGF-2 and modulates striatal precursor cell behavior in response to EGF. Exp. Neurol. 188 408–420. 10.1016/j.expneurol.2004.05.007 PubMed DOI

Cernek J. (2019). Western Blot densitometry analysis - Macro Tool for ImageJ 1.x [Online]. Available at: https://github.com/cernekj/WBGelDensitometryTool

Delehedde M., Lyon M., Gallagher J. T., Rudland P. S., Fernig D. G. (2002). Fibroblast growth factor-2 binds to small heparin-derived oligosaccharides and stimulates a sustained phosphorylation of p42/44 mitogen-activated protein kinase and proliferation of rat mammary fibroblasts. Biochem. J. 366 235–244. 10.1042/BJ20011718 PubMed DOI PMC

Delehedde M., Seve M., Sergeant N., Wartelle I., Lyon M., Rudland P. S., et al. (2000). Fibroblast growth factor-2 stimulation of p42/44MAPK phosphorylation and IkappaB degradation is regulated by heparan sulfate/heparin in rat mammary fibroblasts. J. Biol. Chem. 275 33905–33910. 10.1074/jbc.M005949200 PubMed DOI

Dvorak P., Bednar D., Vanacek P., Balek L., Eiselleova L., Stepankova V., et al. (2018). Computer-assisted engineering of hyperstable fibroblast growth factor 2. Biotechnol. Bioeng. 115 850–862. 10.1002/bit.26531 PubMed DOI

Fannon M., Nugent M. A. (1996). Basic fibroblast growth factor binds its receptors, is internalized, and stimulates DNA synthesis in Balb/c3T3 cells in the absence of heparan sulfate. J. Biol. Chem. 271 17949–17956. 10.1074/jbc.271.30.17949 PubMed DOI

Fox G. M., Schiffer S. G., Rohde M. F., Tsai L. B., Banks A. R., Arakawa T. (1988). Production, biological activity, and structure of recombinant basic fibroblast growth factor and an analog with cysteine replaced by serine. J. Biol. Chem. 263 18452–18458. PubMed

Gospodarowicz D., Cheng J. (1986). Heparin protects basic and acidic FGF from inactivation. J. Cell. Physiol. 128 475–484. 10.1002/jcp.1041280317 PubMed DOI

Ishihara M., Shaklee P. N., Yang Z., Liang W., Wei Z., Stack R. J., et al. (1994). Structural features in heparin which modulate specific biological activities mediated by basic fibroblast growth factor. Glycobiology 4 451–458. 10.1093/glycob/4.4.451 PubMed DOI

Kanodia J., Chai D., Vollmer J., Kim J., Raue A., Finn G., et al. (2014). Deciphering the mechanism behind Fibroblast Growth Factor (FGF) induced biphasic signal-response profiles. Cell Commun. Signal. 12:34. 10.1186/1478-811X-12-34 PubMed DOI PMC

Ke Y. Q., Fernig D. G., Smith J. A., Wilkinson M. C., Anandappa S. Y., Rudland P. S., et al. (1990). High-level production of human acidic fibroblast growth factor in E. coli cells: inhibition of DNA synthesis in rat mammary fibroblasts at high concentrations of growth factor. Biochem. Biophys. Res. Commun. 171 963–971. 10.1016/0006-291x(90)90778-l PubMed DOI

Koledova Z. (2017). 3D Coculture of Mammary Organoids with Fibrospheres: A Model for Studying Epithelial-Stromal Interactions During Mammary Branching Morphogenesis. Methods Mol. Biol. 1612 107–124. 10.1007/978-1-4939-7021-6_8 PubMed DOI

Lai J.-P., Sandhu D. S., Yu C., Han T., Moser C. D., Jackson K. K., et al. (2008). Sulfatase 2 up-regulates glypican 3, promotes fibroblast growth factor signaling, and decreases survival in hepatocellular carcinoma. Hepatology 47 1211–1222. 10.1002/hep.22202 PubMed DOI PMC

Lotz S., Goderie S., Tokas N., Hirsch S. E., Ahmad F., Corneo B., et al. (2013). Sustained levels of FGF2 maintain undifferentiated stem cell cultures with biweekly feeding. PLoS One 8:e56289. 10.1371/journal.pone.0056289 PubMed DOI PMC

Lundin L., Rönnstrand L., Cross M., Hellberg C., Lindahl U., Claesson-Welsh L. (2003). Differential tyrosine phosphorylation of fibroblast growth factor (FGF) receptor-1 and receptor proximal signal transduction in response to FGF-2 and heparin. Exp. Cell Res. 287 190–198. 10.1016/s0014-4827(03)00125-3 PubMed DOI

Matsumoto S., Tanaka R., Okada K., Arita K., Hyakusoku H., Miyamoto M., et al. (2013). The Effect of Control-released Basic Fibroblast Growth Factor in Wound Healing: Histological Analyses and Clinical Application. Plast. Reconstr. Surg. Glob. Open 1:e44. 10.1097/GOX.0b013e3182a88787 PubMed DOI PMC

Mohammadi M., Olsen S. K., Ibrahimi O. A. (2005). Structural basis for fibroblast growth factor receptor activation. Cytokine Growth Factor Rev. 16 107–137. 10.1016/j.cytogfr.2005.01.008 PubMed DOI

Nugent M. A., Edelman E. R. (1992). Kinetics of basic fibroblast growth factor binding to its receptor and heparan sulfate proteoglycan: a mechanism for cooperactivity. Biochemistry 31 8876–8883. 10.1021/bi00152a026 PubMed DOI

Ornitz D. M., Itoh N. (2015). The Fibroblast Growth Factor signaling pathway. Wiley Interdiscip. Rev. Dev. Biol. 4 215–266. 10.1002/wdev.176 PubMed DOI PMC

Ornitz D. M., Xu J., Colvin J. S., McEwen D. G., MacArthur C. A., Coulier F., et al. (1996). Receptor specificity of the fibroblast growth factor family. J. Biol. Chem. 271 15292–15297. PubMed

Ornitz D. M., Yayon A., Flanagan J. G., Svahn C. M., Levi E., Leder P. (1992). 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. 12 240–247. 10.1128/mcb.12.1.240 PubMed DOI PMC

Roghani M., Mansukhani A., Dell’Era P., Bellosta P., Basilico C., Rifkin D. B., et al. (1994). Heparin increases the affinity of basic fibroblast growth factor for its receptor but is not required for binding. J. Biol. Chem. 269 3976–3984. PubMed

Roszell B., Mondrinos M. J., Seaton A., Simons D. M., Koutzaki S. H., Fong G.-H., et al. (2009). Efficient derivation of alveolar type II cells from embryonic stem cells for in vivo application. Tissue Eng. Part A 15 3351–3365. 10.1089/ten.TEA.2008.0664 PubMed DOI PMC

Serls A. E., Doherty S., Parvatiyar P., Wells J. M., Deutsch G. H. (2005). Different thresholds of fibroblast growth factors pattern the ventral foregut into liver and lung. Dev. Camb. Engl. 132 35–47. 10.1242/dev.01570 PubMed DOI

Spivak-Kroizman T., Lemmon M. A., Dikic I., Ladbury J. E., Pinchasi D., Huang J., et al. (1994). Heparin-induced oligomerization of FGF molecules is responsible for FGF receptor dimerization, activation, and cell proliferation. Cell 79 1015–1024. 10.1016/0092-8674(94)90032-9 PubMed DOI

Sumbal J., Koledova Z. (2019). FGF signaling in mammary gland fibroblasts regulates multiple fibroblast functions and mammary epithelial morphogenesis. Development 2019:dev.185306. 10.1242/dev.185306 PubMed DOI

Turnbull J. E., Fernig D. G., Ke Y., Wilkinson M. C., Gallagher J. T. (1992). Identification of the basic fibroblast growth factor binding sequence in fibroblast heparan sulfate. J. Biol. Chem. 267 10337–10341. PubMed

Waksman G., Herr A. B. (1998). New insights into heparin-induced FGF oligomerization. Nat. Struct. Biol. 5 527–530. 10.1038/778 PubMed DOI

Wolzt M., Weltermann A., Nieszpaur-Los M., Schneider B., Fassolt A., Lechner K., et al. (1995). Studies on the neutralizing effects of protamine on unfractionated and low molecular weight heparin (Fragmin) at the site of activation of the coagulation system in man. Thromb. Haemost. 73 439–443. 10.1055/s-0038-1653794 PubMed DOI

Wu W., Tholozan F. M., Goldberg M. W., Bowen L., Wu J., Quinlan R. A. (2014). A gradient of matrix-bound FGF-2 and perlecan is available to lens epithelial cells. Exp. Eye Res. 120 10–14. 10.1016/j.exer.2013.12.004 PubMed DOI PMC

Wu Z. L., Zhang L., Yabe T., Kuberan B., Beeler D. L., Love A., et al. (2003). The involvement of heparan sulfate (HS) in FGF1/HS/FGFR1 signaling complex. J. Biol. Chem. 278 17121–17129. 10.1074/jbc.M212590200 PubMed DOI

Yayon A., Klagsbrun M., Esko J. D., Leder P., Ornitz D. M. (1991). Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 64 841–848. 10.1016/0092-8674(91)90512-w PubMed DOI

Zakrzewska M., Wiedlocha A., Szlachcic A., Krowarsch D., Otlewski J., Olsnes S. (2009). Increased protein stability of FGF1 can compensate for its reduced affinity for heparin. J. Biol. Chem. 284 25388–25403. 10.1074/jbc.M109.001289 PubMed DOI PMC

Zhu H., Duchesne L., Rudland P. S., Fernig D. G. (2010). The heparan sulfate co-receptor and the concentration of fibroblast growth factor-2 independently elicit different signalling patterns from the fibroblast growth factor receptor. Cell Commun. Signal. 8:14. 10.1186/1478-811X-8-14 PubMed DOI PMC

Structural analysis of the stable form of fibroblast growth factor 2 - FGF2-STAB

Fibroblast-induced mammary epithelial branching depends on fibroblast contractility

Computer-assisted stabilization of fibroblast growth factor FGF-18

Lumbar Interbody Fusion Conducted on a Porcine Model with a Bioresorbable Ceramic/Biopolymer Hybrid Implant Enriched with Hyperstable Fibroblast Growth Factor 2

Healing and Angiogenic Properties of Collagen/Chitosan Scaffolds Enriched with Hyperstable FGF2-STAB® Protein: In Vitro, Ex Ovo and In Vivo Comprehensive Evaluation

3D Cell Culture Models Demonstrate a Role for FGF and WNT Signaling in Regulation of Lung Epithelial Cell Fate and Morphogenesis