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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References
Ornitz, D.M. & Itoh, N. Fibroblast growth factors. Genome Biol. 2, reviews 3005 (2001).
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).
Su, N., Du, X. & Chen, L. FGF signaling: its role in bone development and human skeleton diseases. Front. Biosci. 13, 2842–2865 (2008).
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).
Martin, G.R. The roles of FGFs in the early development of vertebrate limbs. Genes Dev. 12, 1571–1586 (1998).
Ornitz, D.M. FGF signaling in the developing endochondral skeleton. Cytokine Growth Factor Rev. 16, 205–213 (2005).
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).
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).
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).
Ohbayashi, N. et al. FGF18 is required for normal cell proliferation and differentiation during osteogenesis and chondrogenesis. Genes Dev. 16, 870–879 (2002).
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).
Ornitz, D.M. FGFs, heparan sulfate and FGFRs: complex interactions essential for development. Bioessays 22, 108–112 (2000).
Nybakken, K. & Perrimon, N. Heparan sulfate proteoglycan modulation of developmental signaling in Drosophila. Biochim. Biophys. Acta 1573, 280–291 (2002).
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).
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).
Murakami, H. et al. Elbow knee synostosis (Eks): a new mutation on mouse Chromosome 14. Mamm. Genome 13, 341–344 (2002).
Ornitz, D.M. et al. Receptor specificity of the fibroblast growth factor family. J. Biol. Chem. 271, 15292–15297 (1996).
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).
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).
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).
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).
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).
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).
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).
Yoshida, T. et al. Twist is required for establishment of the mouse coronal suture. J. Anat. 206, 437–444 (2005).
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).
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).
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).
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).
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).
Tsang, M. & Dawid, I.B. Promotion and attenuation of FGF signaling through the Ras–MAPK pathway. Sci. STKE 2004, pe17 (2004).
Mohammadi, M., Olsen, S.K. & Ibrahimi, O.A. Structural basis for fibroblast growth factor receptor activation. Cytokine Growth Factor Rev. 16, 107–137 (2005).
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).
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).
Kessel, M. & Gruss, P. Murine developmental control genes. Science 249, 374–379 (1990).
Kessel, M. & Gruss, P. Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid. Cell 67, 89–104 (1991).
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).
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).
Case, D.A. et al. AMBER 8 (University of California, San Francisco, 2004).
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).
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).
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).
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).
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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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.
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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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DOI: https://doi.org/10.1038/ng.316
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