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. 1998 Mar 15;101(6):1310–1317.

Increased calvaria cell differentiation and bone matrix formation induced by fibroblast growth factor receptor 2 mutations in Apert syndrome.

A Lomri 1, J Lemonnier 1, M Hott 1, N de Parseval 1, E Lajeunie 1, A Munnich 1, D Renier 1, P J Marie 1
PMCID: PMC508685  PMID: 9502772

Abstract

Apert syndrome, associated with fibroblast growth factor receptor (FGFR) 2 mutations, is characterized by premature fusion of cranial sutures. We analyzed proliferation and differentiation of calvaria cells derived from Apert infants and fetuses with FGFR-2 mutations. Histological analysis revealed premature ossification, increased extent of subperiosteal bone formation, and alkaline phosphatase- positive preosteoblastic cells in Apert fetal calvaria compared with age-matched controls. Preosteoblastic calvaria cells isolated from Apert infants and fetuses showed normal cell growth in basal conditions or in response to exogenous FGF-2. In contrast, the number of alkaline phosphatase- positive calvaria cells was fourfold higher than normal in mutant fetal calvaria cells with the most frequent Apert FGFR-2 mutation (Ser252Trp), suggesting increased maturation rate of cells in the osteoblastic lineage. Biochemical and Northern blot analyses also showed that the expression of alkaline phosphatase and type 1 collagen were 2-10-fold greater than normal in mutant fetal calvaria cells. The in vitro production of mineralized matrix formed by immortalized mutant fetal calvaria cells cultured in aggregates was also increased markedly compared with control immortalized fetal calvaria cells. The results show that Apert FGFR-2 mutations lead to an increase in the number of precursor cells that enter the osteogenic pathway, leading ultimately to increased subperiosteal bone matrix formation and premature calvaria ossification during fetal development, which establishes a connection between the altered genotype and cellular phenotype in Apert syndromic craniosynostosis.

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Selected References

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  1. Canalis E., Centrella M., McCarthy T. Effects of basic fibroblast growth factor on bone formation in vitro. J Clin Invest. 1988 May;81(5):1572–1577. doi: 10.1172/JCI113490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chentoufi J., Hott M., Lamblin D., Buc-Caron M. H., Marie P. J., Kellermann O. Kinetics of in vitro mineralization by an osteogenic clonal cell line (C1) derived from mouse teratocarcinoma. Differentiation. 1993 Jul;53(3):181–189. doi: 10.1111/j.1432-0436.1993.tb00707.x. [DOI] [PubMed] [Google Scholar]
  3. De Pollack C., Renier D., Hott M., Marie P. J. Increased bone formation and osteoblastic cell phenotype in premature cranial suture ossification (craniosynostosis). J Bone Miner Res. 1996 Mar;11(3):401–407. doi: 10.1002/jbmr.5650110314. [DOI] [PubMed] [Google Scholar]
  4. Deng C., Wynshaw-Boris A., Zhou F., Kuo A., Leder P. Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell. 1996 Mar 22;84(6):911–921. doi: 10.1016/s0092-8674(00)81069-7. [DOI] [PubMed] [Google Scholar]
  5. Galvin B. D., Hart K. C., Meyer A. N., Webster M. K., Donoghue D. J. Constitutive receptor activation by Crouzon syndrome mutations in fibroblast growth factor receptor (FGFR)2 and FGFR2/Neu chimeras. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7894–7899. doi: 10.1073/pnas.93.15.7894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Globus R. K., Patterson-Buckendahl P., Gospodarowicz D. Regulation of bovine bone cell proliferation by fibroblast growth factor and transforming growth factor beta. Endocrinology. 1988 Jul;123(1):98–105. doi: 10.1210/endo-123-1-98. [DOI] [PubMed] [Google Scholar]
  7. Hanada K., Dennis J. E., Caplan A. I. Stimulatory effects of basic fibroblast growth factor and bone morphogenetic protein-2 on osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells. J Bone Miner Res. 1997 Oct;12(10):1606–1614. doi: 10.1359/jbmr.1997.12.10.1606. [DOI] [PubMed] [Google Scholar]
  8. Hott M., Marie P. J. Glycol methacrylate as an embedding medium for bone. Stain Technol. 1987 Jan;62(1):51–57. doi: 10.3109/10520298709107965. [DOI] [PubMed] [Google Scholar]
  9. Hurley M. M., Abreu C., Harrison J. R., Lichtler A. C., Raisz L. G., Kream B. E. Basic fibroblast growth factor inhibits type I collagen gene expression in osteoblastic MC3T3-E1 cells. J Biol Chem. 1993 Mar 15;268(8):5588–5593. [PubMed] [Google Scholar]
  10. Ip Y. T. Transcriptional regulation. Converting an activator into a repressor. Curr Biol. 1995 Jan 1;5(1):1–3. doi: 10.1016/s0960-9822(95)00001-7. [DOI] [PubMed] [Google Scholar]
  11. Jabs E. W., Li X., Scott A. F., Meyers G., Chen W., Eccles M., Mao J. I., Charnas L. R., Jackson C. E., Jaye M. Jackson-Weiss and Crouzon syndromes are allelic with mutations in fibroblast growth factor receptor 2. Nat Genet. 1994 Nov;8(3):275–279. doi: 10.1038/ng1194-275. [DOI] [PubMed] [Google Scholar]
  12. Jaye M., Schlessinger J., Dionne C. A. Fibroblast growth factor receptor tyrosine kinases: molecular analysis and signal transduction. Biochim Biophys Acta. 1992 Jun 10;1135(2):185–199. doi: 10.1016/0167-4889(92)90136-y. [DOI] [PubMed] [Google Scholar]
  13. Johnson D. E., Williams L. T. Structural and functional diversity in the FGF receptor multigene family. Adv Cancer Res. 1993;60:1–41. doi: 10.1016/s0065-230x(08)60821-0. [DOI] [PubMed] [Google Scholar]
  14. Kimelman D., Kirschner M. Synergistic induction of mesoderm by FGF and TGF-beta and the identification of an mRNA coding for FGF in the early Xenopus embryo. Cell. 1987 Dec 4;51(5):869–877. doi: 10.1016/0092-8674(87)90110-3. [DOI] [PubMed] [Google Scholar]
  15. Lajeunie E., Ma H. W., Bonaventure J., Munnich A., Le Merrer M., Renier D. FGFR2 mutations in Pfeiffer syndrome. Nat Genet. 1995 Feb;9(2):108–108. doi: 10.1038/ng0295-108. [DOI] [PubMed] [Google Scholar]
  16. Maher P. A. Nuclear Translocation of fibroblast growth factor (FGF) receptors in response to FGF-2. J Cell Biol. 1996 Jul;134(2):529–536. doi: 10.1083/jcb.134.2.529. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Marie P. J., de Vernejoul M. C., Connes D., Hott M. Decreased DNA synthesis by cultured osteoblastic cells in eugonadal osteoporotic men with defective bone formation. J Clin Invest. 1991 Oct;88(4):1167–1172. doi: 10.1172/JCI115418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Martin I., Muraglia A., Campanile G., Cancedda R., Quarto R. Fibroblast growth factor-2 supports ex vivo expansion and maintenance of osteogenic precursors from human bone marrow. Endocrinology. 1997 Oct;138(10):4456–4462. doi: 10.1210/endo.138.10.5425. [DOI] [PubMed] [Google Scholar]
  19. Mayahara H., Ito T., Nagai H., Miyajima H., Tsukuda R., Taketomi S., Mizoguchi J., Kato K. In vivo stimulation of endosteal bone formation by basic fibroblast growth factor in rats. Growth Factors. 1993;9(1):73–80. doi: 10.3109/08977199308991583. [DOI] [PubMed] [Google Scholar]
  20. McCarthy T. L., Centrella M., Canalis E. Effects of fibroblast growth factors on deoxyribonucleic acid and collagen synthesis in rat parietal bone cells. Endocrinology. 1989 Oct;125(4):2118–2126. doi: 10.1210/endo-125-4-2118. [DOI] [PubMed] [Google Scholar]
  21. Muenke M., Schell U., Hehr A., Robin N. H., Losken H. W., Schinzel A., Pulleyn L. J., Rutland P., Reardon W., Malcolm S. A common mutation in the fibroblast growth factor receptor 1 gene in Pfeiffer syndrome. Nat Genet. 1994 Nov;8(3):269–274. doi: 10.1038/ng1194-269. [DOI] [PubMed] [Google Scholar]
  22. Nakamura T., Hanada K., Tamura M., Shibanushi T., Nigi H., Tagawa M., Fukumoto S., Matsumoto T. Stimulation of endosteal bone formation by systemic injections of recombinant basic fibroblast growth factor in rats. Endocrinology. 1995 Mar;136(3):1276–1284. doi: 10.1210/endo.136.3.7867582. [DOI] [PubMed] [Google Scholar]
  23. Naski M. C., Wang Q., Xu J., Ornitz D. M. Graded activation of fibroblast growth factor receptor 3 by mutations causing achondroplasia and thanatophoric dysplasia. Nat Genet. 1996 Jun;13(2):233–237. doi: 10.1038/ng0696-233. [DOI] [PubMed] [Google Scholar]
  24. Neilson K. M., Friesel R. E. Constitutive activation of fibroblast growth factor receptor-2 by a point mutation associated with Crouzon syndrome. J Biol Chem. 1995 Nov 3;270(44):26037–26040. doi: 10.1074/jbc.270.44.26037. [DOI] [PubMed] [Google Scholar]
  25. Newberry E. P., Boudreaux J. M., Towler D. A. The rat osteocalcin fibroblast growth factor (FGF)-responsive element: an okadaic acid-sensitive, FGF-selective transcriptional response motif. Mol Endocrinol. 1996 Aug;10(8):1029–1040. doi: 10.1210/mend.10.8.8843419. [DOI] [PubMed] [Google Scholar]
  26. Noff D., Pitaru S., Savion N. Basic fibroblast growth factor enhances the capacity of bone marrow cells to form bone-like nodules in vitro. FEBS Lett. 1989 Jul 3;250(2):619–621. doi: 10.1016/0014-5793(89)80808-7. [DOI] [PubMed] [Google Scholar]
  27. Ornitz D. M., Leder P. Ligand specificity and heparin dependence of fibroblast growth factor receptors 1 and 3. J Biol Chem. 1992 Aug 15;267(23):16305–16311. [PubMed] [Google Scholar]
  28. Parfitt A. M., Drezner M. K., Glorieux F. H., Kanis J. A., Malluche H., Meunier P. J., Ott S. M., Recker R. R. Bone histomorphometry: standardization of nomenclature, symbols, and units. Report of the ASBMR Histomorphometry Nomenclature Committee. J Bone Miner Res. 1987 Dec;2(6):595–610. doi: 10.1002/jbmr.5650020617. [DOI] [PubMed] [Google Scholar]
  29. Park W. J., Theda C., Maestri N. E., Meyers G. A., Fryburg J. S., Dufresne C., Cohen M. M., Jr, Jabs E. W. Analysis of phenotypic features and FGFR2 mutations in Apert syndrome. Am J Hum Genet. 1995 Aug;57(2):321–328. [PMC free article] [PubMed] [Google Scholar]
  30. Pitaru S., Kotev-Emeth S., Noff D., Kaffuler S., Savion N. Effect of basic fibroblast growth factor on the growth and differentiation of adult stromal bone marrow cells: enhanced development of mineralized bone-like tissue in culture. J Bone Miner Res. 1993 Aug;8(8):919–929. doi: 10.1002/jbmr.5650080804. [DOI] [PubMed] [Google Scholar]
  31. Reardon W., Winter R. M., Rutland P., Pulleyn L. J., Jones B. M., Malcolm S. Mutations in the fibroblast growth factor receptor 2 gene cause Crouzon syndrome. Nat Genet. 1994 Sep;8(1):98–103. doi: 10.1038/ng0994-98. [DOI] [PubMed] [Google Scholar]
  32. Rodan S. B., Wesolowski G., Thomas K. A., Yoon K., Rodan G. A. Effects of acidic and basic fibroblast growth factors on osteoblastic cells. Connect Tissue Res. 1989;20(1-4):283–288. doi: 10.3109/03008208909023898. [DOI] [PubMed] [Google Scholar]
  33. Rutland P., Pulleyn L. J., Reardon W., Baraitser M., Hayward R., Jones B., Malcolm S., Winter R. M., Oldridge M., Slaney S. F. Identical mutations in the FGFR2 gene cause both Pfeiffer and Crouzon syndrome phenotypes. Nat Genet. 1995 Feb;9(2):173–176. doi: 10.1038/ng0295-173. [DOI] [PubMed] [Google Scholar]
  34. Stachowiak M. K., Maher P. A., Joy A., Mordechai E., Stachowiak E. K. Nuclear accumulation of fibroblast growth factor receptors is regulated by multiple signals in adrenal medullary cells. Mol Biol Cell. 1996 Aug;7(8):1299–1317. doi: 10.1091/mbc.7.8.1299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Turksen K., Aubin J. E. Positive and negative immunoselection for enrichment of two classes of osteoprogenitor cells. J Cell Biol. 1991 Jul;114(2):373–384. doi: 10.1083/jcb.114.2.373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Webster M. K., D'Avis P. Y., Robertson S. C., Donoghue D. J. Profound ligand-independent kinase activation of fibroblast growth factor receptor 3 by the activation loop mutation responsible for a lethal skeletal dysplasia, thanatophoric dysplasia type II. Mol Cell Biol. 1996 Aug;16(8):4081–4087. doi: 10.1128/mcb.16.8.4081. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Webster M. K., Donoghue D. J. Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia. EMBO J. 1996 Feb 1;15(3):520–527. [PMC free article] [PubMed] [Google Scholar]
  38. Wilkie A. O., Slaney S. F., Oldridge M., Poole M. D., Ashworth G. J., Hockley A. D., Hayward R. D., David D. J., Pulleyn L. J., Rutland P. Apert syndrome results from localized mutations of FGFR2 and is allelic with Crouzon syndrome. Nat Genet. 1995 Feb;9(2):165–172. doi: 10.1038/ng0295-165. [DOI] [PubMed] [Google Scholar]
  39. de Pollak C., Arnaud E., Renier D., Marie P. J. Age-related changes in bone formation, osteoblastic cell proliferation, and differentiation during postnatal osteogenesis in human calvaria. J Cell Biochem. 1997 Jan;64(1):128–139. [PubMed] [Google Scholar]

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