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FGF9 monomer–dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

Nature Genetics volume 41, pages 289–298 (2009)Cite this article

Abstract

The spontaneous dominant mouse mutant, Elbow knee synostosis (Eks), shows elbow and knee joint synosotsis, and premature fusion of cranial sutures. Here we identify a missense mutation in the Fgf9 gene that is responsible for the Eks mutation. Through investigation of the pathogenic mechanisms of joint and suture synostosis in Eks mice, we identify a key molecular mechanism that regulates FGF9 signaling in developing tissues. We show that the Eks mutation prevents homodimerization of the FGF9 protein and that monomeric FGF9 binds to heparin with a lower affinity than dimeric FGF9. These biochemical defects result in increased diffusion of the altered FGF9 protein (FGF9Eks) through developing tissues, leading to ectopic FGF9 signaling and repression of joint and suture development. We propose a mechanism in which the range of FGF9 signaling in developing tissues is limited by its ability to homodimerize and its affinity for extracellular matrix heparan sulfate proteoglycans.

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Figure 1: Missense mutation in the Fgf9 gene of Eks mice.
Figure 2: Synostotic phenotypes in Fgf9Eks/Eks mice.
Figure 3: The Eks mutation affects dimerization of FGF9.
Figure 4: The Eks mutation affects the mitogenic activity of FGF9.
Figure 5: The Eks mutation reduces FGF9 affinity for heparin by impairing its dimerization.
Figure 6: FGF9Eks can inhibit joint and suture development as well as FGF9WT.
Figure 7: Ectopic FGF9Eks signaling due to its hyperdiffusibility.
Figure 8: FGF9WT modulates FGF9Eks action by forming FGF9WT–FGF9Eks heterodimers.

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References

  1. Ornitz, D.M. & Itoh, N. Fibroblast growth factors. Genome Biol. 2, reviews 3005 (2001).

    Article  CAS  Google Scholar 

  2. Wilkie, A.O.M. Bad bone, absent smell, selfish testes: the pleiotropic consequences of human FGF receptor mutations. Cytokine Growth Factor Rev. 16, 187–203 (2005).

    Article  CAS  Google Scholar 

  3. Su, N., Du, X. & Chen, L. FGF signaling: its role in bone development and human skeleton diseases. Front. Biosci. 13, 2842–2865 (2008).

    Article  CAS  Google Scholar 

  4. Ornitz, D.M. & Marie, P.J. FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease. Genes Dev. 16, 1446–1465 (2002).

    Article  CAS  Google Scholar 

  5. Martin, G.R. The roles of FGFs in the early development of vertebrate limbs. Genes Dev. 12, 1571–1586 (1998).

    Article  CAS  Google Scholar 

  6. Ornitz, D.M. FGF signaling in the developing endochondral skeleton. Cytokine Growth Factor Rev. 16, 205–213 (2005).

    Article  CAS  Google Scholar 

  7. Montero, A. et al. Disruption of the fibroblast growth factor-2 gene results in decreased bone mass and bone formation. J. Clin. Invest. 105, 1085–1093 (2000).

    Article  CAS  Google Scholar 

  8. Hung, I.H., Yu, K., Lavine, K.J. & Ornitz, D.M. FGF9 regulates early hypertrophic chondrocyte differentiation and skeketal vascularization in the developing stylopod. Dev. Biol. 307, 300–313 (2007).

    Article  CAS  Google Scholar 

  9. Liu, Z., Xu, J., Colvin, J.S. & Ornitz, D.M. Coordination of chondrogenesis and osteogenesis by fibroblast growth factor 18. Genes Dev. 16, 859–869 (2002).

    Article  CAS  Google Scholar 

  10. Ohbayashi, N. et al. FGF18 is required for normal cell proliferation and differentiation during osteogenesis and chondrogenesis. Genes Dev. 16, 870–879 (2002).

    Article  CAS  Google Scholar 

  11. Garofalo, S. et al. Skeletal dysplasia and defective chondrocyte differentiation by targeted overexpression of fibroblast growth factor 9 in transgenic mice. J. Bone Miner. Res. 14, 1909–1915 (1999).

    Article  CAS  Google Scholar 

  12. Ornitz, D.M. FGFs, heparan sulfate and FGFRs: complex interactions essential for development. Bioessays 22, 108–112 (2000).

    Article  CAS  Google Scholar 

  13. Nybakken, K. & Perrimon, N. Heparan sulfate proteoglycan modulation of developmental signaling in Drosophila. Biochim. Biophys. Acta 1573, 280–291 (2002).

    Article  CAS  Google Scholar 

  14. Plotnikov, A.N. et al. Crystal structure of fibroblast growth factor 9 reveals regions implicated in dimerization and autoinhibition. J. Biol. Chem. 276, 4322–4329 (2001).

    Article  CAS  Google Scholar 

  15. Hecht, H.J. et al. Structure of fibroblast growth factor 9 shows a symmetric dimer with unique receptor- and heparin-binding interfaces. Acta Crystallogr. D Biol. Crystallogr. 57, 378–384 (2001).

    Article  CAS  Google Scholar 

  16. Murakami, H. et al. Elbow knee synostosis (Eks): a new mutation on mouse Chromosome 14. Mamm. Genome 13, 341–344 (2002).

    Article  Google Scholar 

  17. Ornitz, D.M. et al. Receptor specificity of the fibroblast growth factor family. J. Biol. Chem. 271, 15292–15297 (1996).

    Article  CAS  Google Scholar 

  18. Hajihosseini, M.K. & Heath, J.K. Expression patterns of fibroblast growth factors-18 and -20 in mouse embryos is suggestive of novel roles in calvarial and limb development. Mech. Dev. 113, 79–83 (2002).

    Article  CAS  Google Scholar 

  19. Colvin, J.S., Feldman, B., Nadeau, J.H., Goldfarb, M. & Ornitz, D.M. Genomic organization and embryonic expression of the mouse fibroblast growth factor 9 gene. Dev. Dyn. 216, 72–88 (1999).

    Article  CAS  Google Scholar 

  20. Wang, Q., Green, R.P., Zhao, G. & Ornitz, D.M. Differential regulation of endochondral bone growth and joint development by FGFR1 and FGFR3 tyrosine kinase domains. Development 128, 3867–3876 (2001).

    CAS  PubMed  Google Scholar 

  21. Eswarakumar, V.P., Horowitz, M.C., Locklin, R., Morriss-Kay, G.M. & Lonai, P. A gain-of-function mutation of Fgfr2c demonstrates the roles of this receptor variant in osteogenesis. Proc. Natl. Acad. Sci. USA 101, 12555–12560 (2004).

    Article  CAS  Google Scholar 

  22. Storm, E.E. & Kingsley, D.M. Joint patterning defects caused by single and double mutations in members of the bone morphogenetic protein (BMP) family. Development 122, 3969–3979 (1996).

    CAS  PubMed  Google Scholar 

  23. Nalin, A.M., Greenlee, T.K. & Sandell, L.J. Collagen gene expression during development of avian synovial joints: Transient expression of types II and XI collagen genes in the joint capsule. Dev. Dyn. 203, 352–362 (1995).

    Article  CAS  Google Scholar 

  24. Iseki, S. et al. Fgfr2 and osteopontin domains in the developing skull vault are mutually exclusive and can be altered by locally applied FGF2. Development 124, 3375–3384 (1997).

    CAS  PubMed  Google Scholar 

  25. Yoshida, T. et al. Twist is required for establishment of the mouse coronal suture. J. Anat. 206, 437–444 (2005).

    Article  CAS  Google Scholar 

  26. Flaumenhaft, R., Moscatelli, D. & Rifkin, D.B. Heparin and heparan sulfate increase the radius of diffusion and action of basic fibroblast growth factor. J. Cell Biol. 111, 1651–1659 (1990).

    Article  CAS  Google Scholar 

  27. Woo, H.J. & Roux, B. Calculation of absolute protein-ligand binding free energy from computer simulations. Proc. Natl. Acad. Sci. USA 102, 6825–6830 (2005).

    Article  CAS  Google Scholar 

  28. Schlessinger, J. et al. Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. Mol. Cell 6, 743–750 (2000).

    Article  CAS  Google Scholar 

  29. Dowd, C.J., Cooney, C.L. & Nugent, M.A. Heparan sulfate mediates bFGF transport through basement membrane by diffusion with rapid reversible binding. J. Biol. Chem. 274, 5236–5244 (1999).

    Article  CAS  Google Scholar 

  30. Johnson, D., Iseki, S., Wilkie, A.O.M. & Morriss-Kay, G.M. Expression patterns of Twist and Fgfr1, -2 and -3 in the developing mouse coronal suture suggest a key role for Twist in suture initiation and biogenesis. Mech. Dev. 91, 341–345 (2000).

    Article  CAS  Google Scholar 

  31. Tsang, M. & Dawid, I.B. Promotion and attenuation of FGF signaling through the Ras–MAPK pathway. Sci. STKE 2004, pe17 (2004).

    PubMed  Google Scholar 

  32. Mohammadi, M., Olsen, S.K. & Ibrahimi, O.A. Structural basis for fibroblast growth factor receptor activation. Cytokine Growth Factor Rev. 16, 107–137 (2005).

    Article  CAS  Google Scholar 

  33. Ornitz, D.M. et al. 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 (1992).

    Article  CAS  Google Scholar 

  34. Venkataraman, G., Shriver, Z., Davis, J.C. & Sasisekharan, R. Fibroblast growth factors 1 and 2 are distinct in oligomerization in the presence of heparin-like glycosaminoglycans. Proc. Natl. Acad. Sci. USA 96, 1892–1897 (1999).

    Article  CAS  Google Scholar 

  35. Kessel, M. & Gruss, P. Murine developmental control genes. Science 249, 374–379 (1990).

    Article  CAS  Google Scholar 

  36. Kessel, M. & Gruss, P. Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid. Cell 67, 89–104 (1991).

    Article  CAS  Google Scholar 

  37. Verdonk, M.L., Cole, J.C., Hartshorn, M.J., Murray, C.W. & Taylor, R.D. Improved protein-ligand docking using GOLD. Proteins 52, 609–623 (2003).

    Article  CAS  Google Scholar 

  38. Jorgensen, W.L., Chandrasekhar, J., Madura, J.D., Impey, R.W. & Klein, M.L. Comparison of simple potential functions for simulating liquid water. J. Chem. Phys. 79, 926–935 (1983).

    Article  CAS  Google Scholar 

  39. Case, D.A. et al. AMBER 8 (University of California, San Francisco, 2004).

    Google Scholar 

  40. Narumi, T. et al. A 55 Tflops simulation of amyloid-forming peptides from yeast prion Sup35 with the special-purpose computer System MD-GRAPE3. <http://doi.acm.org/10.1145/1188455.1188506> (2006).

  41. Duan, Y. et al. A point-charge force field for molecular mechanics simulations of proteins based on condensed-phase quantum mechanical calculations. J. Comput. Chem. 24, 1999–2012 (2003).

    Article  CAS  Google Scholar 

  42. Srinivasan, J., Miller, J., Kollma, P.A. & Case, D.A. Continuum solvent studies of the stability of RNA hairpin loops and helices. J. Biomol. Struct. Dyn. 16, 671–682 (1998).

    Article  CAS  Google Scholar 

  43. Hughes, S.H., Greenhouse, J.J., Petropoulos, C.J. & Sutrave, P. Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors. J. Virol. 61, 3004–3012 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This study was supported in part by the RIKEN Structural Genomics/Proteomics Initiative (RSGI) and the National Project on Protein Structural and Functional Analysis, Ministry of Education, Culture, Sports, Science and Technology of Japan (S.Y.) and US National Institutes of Health grant HD049808 (D.M.O.).

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Author notes

  1. Shuichi Hiraoka & Taka Nakahara

    Present address: Present addresses: Department of Systems Biomedicine, National Research Institute for Child Health and Development, 2-10-1 Okura, Setagaya-ku, Tokyo 157-8535, Japan (S.H.) and Section of Developmental and Regenerative Dentistry, School of Life Dentistry at Tokyo, The Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan (T.N.).,

  2. Hirotaka Murakami, Akihiko Okawa, Noriaki Okimoto, Shuichi Hiraoka, Taka Nakahara, Ryogo Akasaka and Yo-ichi Shiraishi: These authors contributed equally to this work.

Authors and Affiliations

  1. RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan

    Masayo Harada, Shuichi Hiraoka, Yoko Mizutani-Koseki & Haruhiko Koseki

  2. Department of Immunology and Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan

    Masayo Harada

  3. Department of Orthopaedic Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba, 260-8670, Japan

    Hirotaka Murakami & Akihiko Okawa

  4. RIKEN Advanced Science Institute, Computational Systems Biology Research Group, 61-1 Ono-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0046, Japan

    Noriaki Okimoto, Noriyuki Futatsugi & Makoto Taiji

  5. Section of Molecular Craniofacial Embryology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo, 113-8549, Japan

    Taka Nakahara & Sachiko Iseki

  6. RIKEN Systems and Structural Biology Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan

    Ryogo Akasaka, Mikako Shirouzu & Shigeyuki Yokoyama

  7. Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8602, Japan

    Yo-ichi Shiraishi & Atsushi Kuroiwa

  8. Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan

    Shigeyuki Yokoyama

  9. Department of Developmental Biology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, 63110, Missouri, USA

    David M Ornitz

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Contributions

M.H., D.M.O. and H.K. developed the project and wrote the manuscript. M.H., S.H. and H.K. contributed to the purification of FGF9 proteins, mitogenic assays, analytical gel filtration chromatography, analytical heparin affinity chromatography, surface plasmon resonance analysis, skeletal preparation, histological analyses and in situ hybridization of sections, implantation of FGF9 beads in mouse forelimb buds and immunoprecipitation and protein blot analysis. H.M., A.O. and H.K. contributed to the identification of the Eks mutation. N.O., N.F. and M.T. contributed to the molecular-dynamics simulation. T.N. and S.I. contributed to the implantation of FGF9 beads in mouse fetal skulls. R.A., M.S. and S.Y. contributed to the analytical ultracentrifugation. Y.S. and A.K. contributed to the retroviral misexpression. Y.M.-K. contributed to the in situ hybridization of sections.

Corresponding author

Correspondence to Haruhiko Koseki.

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Harada, M., Murakami, H., Okawa, A. et al. FGF9 monomer–dimer equilibrium regulates extracellular matrix affinity and tissue diffusion. Nat Genet 41, 289–298 (2009). https://doi.org/10.1038/ng.316

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