2012 Fiscal Year Final Research Report
Identification of the cell group participating in the onset of craniosynostosis and study on the differentiation control
| Project/Area Number |
23792418
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| Research Category |
Grant-in-Aid for Young Scientists (B)
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| Allocation Type | Multi-year Fund |
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| Research Field |
Orthodontic/Pediatric dentistry
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| Research Institution | Tokyo Medical and Dental University |
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Principal Investigator |
KOBAYASHI Yukiho 東京医科歯科大学, 硬組織疾患ゲノムセン ター, 特任助教 (20596233)
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| Project Period (FY) |
2011 – 2012
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| Keywords | craniosynostosis / Apert syndrome / osteoblast |
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| Research Abstract |
Apert syndrome (AS), an autosomal dominant inherited disorder caused by missense mutation resulting from amino acid substitutions in the fibroblast growth factor receptor (FGFR) 2, is characterized by the major clinical features of craniosynostosis, exophthalmus, midface deficiency, and symmetric syndactyly of the hands and feet. Ligand-dependent constitutive activation of FGFR2 has been reported to play a causative role in the pathogenesis of AS, however the precise mechanism remains to be elucidated. Surgical procedures are frequently required to treat the AS, thedevelopment of non-invasive procedures for treating AS are keenly awaited. Objectives: To determine etiological mechanisms of craniosynostosis in AS, and verify the therapeutic effects of the purified soluble form of FGFR2S252W (sFGFR2S252W) complexed with polysaccharide nanogel in vitro. Methods: Recombinant sFGFR2S252W was purified by affinity chromatography and was complexed with nanogel. Calvarial tissues were obtained f
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rom Fgfr2+/S252Wmice (AS mice) and wild-type mice (control mice), and were cultured for 4 days in the presence of either sFGFR2S252W/nanogel complex or vehicle nanogel on either side of the coronal suture. MC3T3-E1 cells overexpressing FGFR2IIIcS252W (MC3T3-E1-Ap) were established. The mRNA expression in MC3T3-E1 cells was analyzed by real-time PCR or semi-quantitative RT-PCR, and protein expression and phosphorylation were analyzed by Western blotting. Results: The coronal suture of the AS mice exhibited increased Runx2 and Osteopontin mRNA expression, as well as accelerated phosphorylation of ERK and MEK, unlike that observed in the control mice. The ectopic expression of Fgf10 and Fgfr2IIIb, which are probably indispensable for epidermal development, were observed in the coronal suture of the AS mice, whereas Fgfr2IIIc expression was detected in both the AS and the control mice. Administration of sFGFR2S252W inhibited FGF2-stimulated proliferation; phosphorylation of ERK, p38, MEK, SAPK/JNK, and Akt; and the mineralization of MC3T3-E1-Ap in vitro. The sFGFR2S252W/nanogel complex maintained the patency of the coronal sutures in the AS mice (n = 4/4); however, synostosis was observed to occur on the side where only nanogel was applied (n = 4/4) in the organ culture. Conclusion: The ectopic expression of Fgf10 and Fgfr2IIIb probably induce the onset of craniosynostosis in AS, and anappropriate delivery of purified sFGFR2S252W could be an effective method for treating AS. Less
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