Cellular and Molecular Life Sciences

, Volume 72, Issue 12, pp 2445–2459 | Cite as

Instability restricts signaling of multiple fibroblast growth factors

  • Marcela Buchtova
  • Radka Chaloupkova
  • Malgorzata Zakrzewska
  • Iva Vesela
  • Petra Cela
  • Jana Barathova
  • Iva Gudernova
  • Renata Zajickova
  • Lukas Trantirek
  • Jorge Martin
  • Michal Kostas
  • Jacek Otlewski
  • Jiri Damborsky
  • Alois Kozubik
  • Antoni Wiedlocha
  • Pavel Krejci
Research Article


Fibroblast growth factors (FGFs) deliver extracellular signals that govern many developmental and regenerative processes, but the mechanisms regulating FGF signaling remain incompletely understood. Here, we explored the relationship between intrinsic stability of FGF proteins and their biological activity for all 18 members of the FGF family. We report that FGF1, FGF3, FGF4, FGF6, FGF8, FGF9, FGF10, FGF16, FGF17, FGF18, FGF20, and FGF22 exist as unstable proteins, which are rapidly degraded in cell cultivation media. Biological activity of FGF1, FGF3, FGF4, FGF6, FGF8, FGF10, FGF16, FGF17, and FGF20 is limited by their instability, manifesting as failure to activate FGF receptor signal transduction over long periods of time, and influence specific cell behavior in vitro and in vivo. Stabilization via exogenous heparin binding, introduction of stabilizing mutations or lowering the cell cultivation temperature rescues signaling of unstable FGFs. Thus, the intrinsic ligand instability is an important elementary level of regulation in the FGF signaling system.


Fibroblast growth factor FGF Unstable Proteoglycan Regulation 



We thank M. Pokorna and K. Tvaruzkova for assistance with CD measurements, J. Medalova and T. Spoustova for assistance with manuscript preparation, and P. B. Mekikian for technical support. The study was supported by the Ministry of Education, Youth and Sports of the Czech Republic (KONTAKT LH12004, CZ.1.07/2.3.00/30.0053, LO1214), Grant Agency of Masaryk University (0071-2013), Czech Science Foundation (14-31540S, P207/12/0775, GBP302/12/G157), European Regional Development Fund (FNUSA-ICRC No.CZ.1.05/1.1.00/02.0123), European Union (ICRC-ERA-HumanBridge No.316345), Netherlands Organization for Scientific Research (700.59.426) and Polish National Science Centre (NCN, 2011/02/A/NZ1/00066).

Supplementary material

18_2015_1856_MOESM1_ESM.pdf (3.4 mb)
Supplementary material 1 (PDF 3468 kb)


  1. 1.
    Itoh N, Ornitz DM (2008) Functional evolutionary history of the mouse Fgf gene family. Dev Dyn 237:18–27CrossRefPubMedGoogle Scholar
  2. 2.
    Itoh N (2010) Hormone-like (endocrine) Fgfs: their evolutionary history and roles in development, metabolism, and disease. Cell Tissue Res 342:1–11CrossRefPubMedCentralPubMedGoogle Scholar
  3. 3.
    Copeland RA, Ji H, Halfpenny AJ, Williams RW, Thompson KC, Herber WK, Thomas KA, Bruner MW, Ryan JA, Marquis-Omer D (1991) The structure of human acidic fibroblast growth factor and its interaction with heparin. Arch Biochem Biophys 289(1):53–61CrossRefPubMedGoogle Scholar
  4. 4.
    Culajay JF, Blaber SI, Khurana A, Blaber M (2000) Thermodynamic characterization of mutants of human fibroblast growth factor 1 with an increased physiological half-life. Biochemistry 39:7153–7158CrossRefPubMedGoogle Scholar
  5. 5.
    Zakrzewska M, Krowarsch D, Wiedlocha A, Otlewski J (2004) Design of fully active FGF-1 variants with increased stability. Protein Eng Des Sel 17:603–611CrossRefPubMedGoogle Scholar
  6. 6.
    Buczek O, Krowarsch D, Otlewski J (2002) Thermodynamics of single peptide bond cleavage in bovine pancreatic trypsin inhibitor (BPTI). Protein Sci 11:924–932CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Zakrzewska M, Krowarsch D, Wiedlocha A, Olsnes S, Otlewski J (2005) Highly stable mutants of human fibroblast growth factor-1 exhibit prolonged biological action. J Mol Biol 352:860–875CrossRefPubMedGoogle Scholar
  8. 8.
    Damon DH, Lobb RR, D’Amore PA, Wagner JA (1989) Heparin potentiates the action of acidic fibroblast growth factor by prolonging its biological half-life. J Cell Physiol 138:221–226CrossRefPubMedGoogle Scholar
  9. 9.
    Derrick T, Grillo AO, Vitharana SN, Jones L, Rexroad J, Shah A, Perkins, Spitznagel TM, Middaugh CR (2007) Effect of polyanions on the structure and stability of repifermin (keratinocyte growth factor-2). J Pharm Sci 96:761–776CrossRefPubMedGoogle Scholar
  10. 10.
    Chen G, Gulbranson DR, Yu P, Hou Z, Thomson JA (2012) Thermal stability of fibroblast growth factor protein is a determinant factor in regulating self-renewal, differentiation, and reprogramming in human pluripotent stem cells. Stem Cells 30:623–630CrossRefPubMedCentralPubMedGoogle Scholar
  11. 11.
    Econs MJ, McEnery PT (1997) Autosomal dominant hypophosphatemic rickets/osteomalacia: clinical characterization of a novel renal phosphate-wasting disorder. J Clin Endocrinol Metab 82(2):674–681CrossRefPubMedGoogle Scholar
  12. 12.
    White KE, Carn G, Lorenz-Depiereux B, Benet-Pages A, Strom TM, Econs MJ (2001) Autosomal-dominant hypophosphatemic rickets (ADHR) mutations stabilize FGF-23. Kidney Int 60(6):2079–2086CrossRefPubMedGoogle Scholar
  13. 13.
    Kato K, Jeanneau C, Tarp MA, Benet-Pagès A, Lorenz-Depiereux B, Bennett EP, Mandel U, Strom TM, Clausen H (2006) Polypeptide GalNAc-transferase T3 and familial tumoral calcinosis. Secretion of fibroblast growth factor 23 requires O-glycosylation. J Biol Chem 281(27):18370–18377CrossRefPubMedGoogle Scholar
  14. 14.
    Fukumoto S, Yamashita T (2007) FGF23 is a hormone-regulating phosphate metabolism-unique biological characteristics of FGF23. Bone 40(5):1190–1195CrossRefPubMedGoogle Scholar
  15. 15.
    Colvin JS, White AC, Pratt SJ, Ornitz DM (2001) Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme. Development 128(11):2095–2106PubMedGoogle Scholar
  16. 16.
    Ohbayashi N, Shibayama M, Kurotaki Y, Imanishi M, Fujimori T, Itoh N, Takada S (2002) FGF18 is required for normal cell proliferation and differentiation during osteogenesis and chondrogenesis. Genes Dev 16(7):870–879CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Usui H, Shibayama M, Ohbayashi N, Konishi M, Takada S, Itoh N (2004) Fgf18 is required for embryonic lung alveolar development. Biochem Biophys Res Commun 322(3):887–892CrossRefPubMedGoogle Scholar
  18. 18.
    Vincentz JW, McWhirter JR, Murre C, Baldini A, Furuta Y (2005) Fgf15 is required for proper morphogenesis of the mouse cardiac outflow tract. Genesis 41(4):192–201CrossRefPubMedGoogle Scholar
  19. 19.
    Lu SY, Sheikh F, Sheppard PC, Fresnoza A, Duckworth ML, Detillieux KA, Cattini PA (2008) FGF-16 is required for embryonic heart development. Biochem Biophys Res Commun 373(2):270–274CrossRefPubMedGoogle Scholar
  20. 20.
    Cholfin JA, Rubenstein JL (2007) Patterning of frontal cortex subdivisions by Fgf17. Proc Natl Acad Sci USA 104(18):7652–7657CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Sekine K, Ohuchi H, Fujiwara M, Yamasaki M, Yoshizawa T, Sato T, Yagishita N, Matsui D, Koga Y, Itoh N, Kato S (1999) Fgf10 is essential for limb and lung formation. Nat Genet 21(1):138–141CrossRefPubMedGoogle Scholar
  22. 22.
    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 284:25388–25403CrossRefPubMedCentralPubMedGoogle Scholar
  23. 23.
    Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88:49–92CrossRefPubMedGoogle Scholar
  24. 24.
    Plant MR, MacDonald ME, Grad LI, Ritchie SJ, Richman JM (2000) Locally released retinoic acid repatterns the first branchial arch cartilages in vivo. Dev Biol 222:12–26CrossRefPubMedGoogle Scholar
  25. 25.
    Krejci P, Prochazkova J, Smutny J, Chlebova K, Lin P, Aklian A, Bryja V, Kozubik A, Wilcox WR (2010) FGFR3 signaling induces a reversible senescence phenotype in chondrocytes similar to oncogene-induced premature senescence. Bone 47:102–110CrossRefPubMedCentralPubMedGoogle Scholar
  26. 26.
    Krejci P, Salazar L, Goodridge HS, Kashiwada TA, Schibler MJ, Jelinkova P, Thompson LM, Wilcox WR (2008) STAT1 and STAT3 do not participate in FGF-mediated growth arrest in chondrocytes. J Cell Sci 121:272–281CrossRefPubMedGoogle Scholar
  27. 27.
    Krejci P, Bryja V, Pachernik J, Hampl A, Pogue R, Mekikian P, Wilcox WR (2004) FGF2 inhibits proliferation and alters the cartilage-like phenotype of RCS cells. Exp Cell Res 297:152–164CrossRefPubMedGoogle Scholar
  28. 28.
    Ornitz DM, Xu J, Colvin JS, McEwen DG, MacArthur CA, Coulier F, Gao G, Goldfarb M (1996) Receptor specificity of the fibroblast growth factor family. J Biol Chem 27:15292–15297Google Scholar
  29. 29.
    Zhang X, Ibrahimi OA, Olsen SK, Umemori H, Mohammadi M, Ornitz DM (2006) Receptor specificity of the fibroblast growth factor family. The complete mammalian FGF family. J Biol Chem 281:15694–15700CrossRefPubMedCentralPubMedGoogle Scholar
  30. 30.
    Wu X, Ge H, Gupte J, Weiszmann J, Shimamoto G, Stevens J, Hawkins N, Lemon B, Shen W, Xu J, Veniant MM, Li YS, Lindberg R, Chen JL, Tian H, Li Y (2007) Co-receptor requirements for fibroblast growth factor-19 signaling. J Biol Chem 282:29069–29072CrossRefPubMedGoogle Scholar
  31. 31.
    Goetz R, Beenken A, Ibrahimi OA, Kalinina J, Olsen SK, Eliseenkova AV, Xu C, Neubert TA, Zhang F, Linhardt RJ, Yu X, White KE, Inagaki T, Kliewer SA, Yamamoto M, Kurosu H, Ogawa Y, Kuro-o M, Lanske B, Razzaque MS, Mohammadi M (2007) Molecular insights into the klotho-dependent, endocrine mode of action of fibroblast growth factor 19 subfamily members. Mol Cell Biol 27:3417–3428CrossRefPubMedCentralPubMedGoogle Scholar
  32. 32.
    Krejci P, Krakow D, Mekikian PB, Wilcox WR (2007) Fibroblast growth factors 1, 2, 17, and 19 are the predominant FGF ligands expressed in human fetal growth plate cartilage. Pediatr Res 61:267–272CrossRefPubMedGoogle Scholar
  33. 33.
    Raucci A, Laplantine E, Mansukhani A, Basilico C (2004) Activation of the ERK1/2 and p38 mitogen-activated protein kinase pathways mediates fibroblast growth factor-induced growth arrest of chondrocytes. J Biol Chem 279:1747–1756CrossRefPubMedGoogle Scholar
  34. 34.
    Krejci P, Masri B, Fontaine V, Mekikian PB, Weis M, Prats H, Wilcox WR (2005) Interaction of fibroblast growth factor and C-natriuretic peptide signaling in regulation of chondrocyte proliferation and extracellular matrix homeostasis. J Cell Sci 118:5089–5100CrossRefPubMedGoogle Scholar
  35. 35.
    Yayon A, Klagsbrun M, Esko JD, Leder P, Ornitz DM (1991) Cell surface, heparin-like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell 64:841–848CrossRefPubMedGoogle Scholar
  36. 36.
    Angelin B, Larsson TE, Rudling M (2012) Circulating fibroblast growth factors as metabolic regulators-a critical appraisal. Cell Metab 16:693–705CrossRefPubMedGoogle Scholar
  37. 37.
    Burke CJ, Volkin DB, Mach H, Middaugh CR (1993) Effect of polyanions on the unfolding of acidic fibroblast growth factor. Biochemistry 32:6419–6426CrossRefPubMedGoogle Scholar
  38. 38.
    Lavinder JJ, Hari SB, Sullivan BJ, Magliery TJ (2009) High-throughput thermal scanning: a general, rapid dye-binding thermal shift screen for protein engineering. J Am Chem Soc 131:3794–3795CrossRefPubMedCentralPubMedGoogle Scholar
  39. 39.
    Fasman GD (1996) Circular dichroism and conformational analysis of biomolecules. Plenum Press, New YorkCrossRefGoogle Scholar
  40. 40.
    Foldynova-Trantirkova S, Wilcox WR, Krejci P (2012) Sixteen years and counting: the current understanding of fibroblast growth factor receptor 3 (FGFR3) signaling in skeletal dysplasias. Hum Mutat 33:29–41CrossRefPubMedCentralPubMedGoogle Scholar
  41. 41.
    Ornitz DM, Itoh N (2001) Fibroblast growth factors. Genome Biol 2:REVIEWS3005CrossRefPubMedCentralPubMedGoogle Scholar
  42. 42.
    Faham S, Lindardt RJ, Rees DC (1998) Diversity does make a difference: fibroblast growth factor-heparin interactions. Curr Opin Struct Biol 8:578–586CrossRefPubMedGoogle Scholar
  43. 43.
    Raman R, Venkataraman G, Ernst S, Sasisekharan V, Sasisekharan R (2003) Structural specifity of heparin binding in the fibroblast growth factor family of proteins. Proc Natl Acad Sci USA 100:2357–2362CrossRefPubMedCentralPubMedGoogle Scholar
  44. 44.
    Jayasundar R, Singh VP (2002) In vivo temperature measurements in brain tumors using proton MR spectroscopy. Neurol India 50:436–439PubMedGoogle Scholar
  45. 45.
    Chambers CD, Johnson KA, Dick LM, Felix RJ, Jones KL (1998) Maternal fever and birth outcome: a prospective study. Teratology 58:251–257CrossRefPubMedGoogle Scholar

Copyright information

© Springer Basel 2015

Authors and Affiliations

  • Marcela Buchtova
    • 1
    • 2
    • 3
  • Radka Chaloupkova
    • 4
  • Malgorzata Zakrzewska
    • 5
  • Iva Vesela
    • 1
    • 3
  • Petra Cela
    • 2
    • 3
  • Jana Barathova
    • 6
  • Iva Gudernova
    • 6
  • Renata Zajickova
    • 2
  • Lukas Trantirek
    • 7
    • 8
  • Jorge Martin
    • 9
  • Michal Kostas
    • 10
  • Jacek Otlewski
    • 5
  • Jiri Damborsky
    • 4
    • 11
  • Alois Kozubik
    • 3
    • 12
  • Antoni Wiedlocha
    • 13
    • 14
  • Pavel Krejci
    • 6
    • 11
    • 15
  1. 1.Department of Anatomy, Histology and EmbryologyUniversity of Veterinary and Pharmaceutical SciencesBrnoCzech Republic
  2. 2.Institute of Animal Physiology and GeneticsAcademy of Sciences of the Czech RepublicBrnoCzech Republic
  3. 3.Department of Experimental BiologyMasaryk UniversityBrnoCzech Republic
  4. 4.Loschmidt Laboratories, Department of Experimental Biology and Research Centre for Toxic Compounds in the Environment RECETOXMasaryk UniversityBrnoCzech Republic
  5. 5.Department of Protein EngineeringUniversity of WroclawWroclawPoland
  6. 6.Department of Biology, Faculty of MedicineMasaryk UniversityBrnoCzech Republic
  7. 7.Central European Institute of TechnologyMasaryk UniversityBrnoCzech Republic
  8. 8.Bijvoet Center for Biomolecular ResearchUtrecht UniversityUtrechtThe Netherlands
  9. 9.Medical Genetics InstituteCedars-Sinai Medical CenterLos AngelesUSA
  10. 10.Department of Protein BiotechnologyUniversity of WroclawWroclawPoland
  11. 11.International Clinical Research CenterSt. Anne’s University HospitalBrnoCzech Republic
  12. 12.Department of Cytokinetics, Institute of BiophysicsAcademy of Sciences of the Czech RepublicBrnoCzech Republic
  13. 13.Centre for Cancer BiomedicineUniversity of OsloOsloNorway
  14. 14.Department of Biochemistry, Institute for Cancer ResearchOslo University HospitalOsloNorway
  15. 15.Department of Orthopaedic SurgeryDavid Geffen School of Medicine at UCLALos AngelesUSA

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