Phenotypes of calvarial coronal sutures isolated from Apert syndrome (AS) mice.

<p>(A) Embryonic calvarial coronal sutures (E15.5) were dissected from AS mice and control littermates. The mRNA expression of Runt-related transcription factor 2 <i>(Runx2)</i> and Osteopontin in calvarial coronal sutures of AS mice and littermate controls were determined by real-time PCR. Expression levels are shown as the mean ± SEM of three replicates, relative to a control #1 for each gene. (B) The mRNA expression levels of fibroblast growth factor receptor 2 (<i>Fgfr2</i>) <i>IIIb</i> and <i>IIIc</i> isoforms, <i>Fgf10</i>, and <i>Esrp1</i> were determined by real-time PCR. Increased expression of <i>Fgfr2IIIb, Fgf10, and Espr1</i> mRNA was observed in AS mice, whereas <i>Fgfr2IIIc</i> was expressed equally in both AS and control mice. The protein expression of Fgf10 and epithelial splicing regulatory protein 1 (Esrp1) in calvarial coronal sutures of AS mice and littermate controls was determined by western blot analysis. Increased expression of Fgf10 and Esrp1 was observed only in AS mice. *<i>p</i><0.01, **<i>p</i><0.005. (C) Phosphorylation of intracellular signaling molecules in calvarial coronal sutures was determined by western blot analysis. In AS mice, phosphorylation of Erk1/2, MEK, and SAPK/JNK were significantly enhanced, and the expression of Bax was increased.</p

Purification of soluble forms of FGFR2 and their biological activities <i>in vitro</i>.

<p>(A, B) The expression levels of purified sFGFR2IIIc and sFGFR2IIIc^S252W proteins (A), as well as that of Fgf2-His (B), were analyzed by western blot analysis. (B) Pull-down assay for the binding of Fgf2-His with sFGFR2IIIc-FLAG and sFGFR2IIIc^S252W-FLAG. Fgf2 interacted with sFGFR2IIIc-FLAG and sFGFR2IIIc^S252W-FLAG. (C) Dimerization between sFGFR2IIIc^S252W and FGFR2IIIc, FGFR2IIIc^S252W, or FGFR2IIIb^S252W was determined by immunoprecipitation assays using anti-FGFR2 antibodies that recognized the cytoplasmic region of Fgfr2, followed by western blot analysis using anti-FLAG antibodies. The formation of heterodimers between sFGFR2IIIc^S252W-FLAG and membrane-bound FGFR2IIIc-FLAG, FGFR2IIIc-FLAG, FGFR2IIIc^S252W-FLAG, or FGFR2IIIb^S252W-FLAG was confirmed.</p

Analysis of the inhibitory effects of sFGFR2IIIc^S252W on the proliferation of MC3T3-E1 cells.

<p>(A) Protein expression of FGFR2IIIc^S252W-FLAG in the established MC3T3-E1 cell clone (MC3T3-Ap) was confirmed by western blot analysis using anti-FLAG antibodies. Morphological examination of MC3T3-E1-MOCK (left) and MC3T3-Ap (right) indicated that MC3T3-Ap had a somewhat cuboidal shape, unlike the controls. (B) MTT assays were used to measure the proliferation of parental MC3T3-E1 and MC3T3-Ap cells upon administration of FGF2 (25 ng/mL) in the presence of U0126 (20 µM), SB203580 (20 µM), or sFGFR2IIIc^S252W (60 ng/mL). FGF2 was found to promote the proliferation of parental MC3T3-E1 cells, but not MC3T3-Ap cells. Basal proliferation levels of MC3T3-Ap cells were lower than those of parental MC3T3-E1 cells. Additionally, U0126 and SB203580 (ERK and p38 inhibitors, respectively) inhibited the proliferation of MC3T3-E1 cells. The proliferation of all cell lines was significantly decreased by addition of sFGFR2IIIc^S252W. Statistical analysis was performed using ANOVA and the Student-Newman-Keuls test. *<i>p</i><0.01.</p

Analysis of the inhibitory effects of sFGFR2IIIc^S252W on intracellular signaling and <i>in vitro</i> mineralization.

<p>(A) Phosphorylation of Erk1/2, p38, MEK, SAPK/JNK, and Akt in parental MC3T3-E1 or MC3T3-Ap cells upon addition of FGF and sFGFR2IIIc^S252W was determined by western blot analysis. FGF2 stimulated the phosphorylation of Erk1/2, MEK, SAPK/JNK, p38, and Akt in parental MC3T3-E1 cells. MC3T3-Ap cells exhibited increased phosphorylation of all proteins except Akt. U0126 was found to inhibit the phosphorylation of Erk1/2, MEK, p38, and Akt, whereas SB203580 inhibited p38 and Akt, but not Erk1/2, MEK, or SAPK/JNK. Treatment with either sFGFR2IIIc or sFGFR2IIIc^S252W inhibited the phosphorylation of all of these molecules; in particular, sFGFR2IIIc^S252W had stronger inhibitory effects than sFGFR2IIIc on Erk1/2, SAPK/JNK, and p38. (B) Mineralization of control MC3T3-E1 cells and MC3T3-Ap cells in the presence or absence of sFGFR2IIIc^S252W, U0126, or SB203580 was visualized by Alizarin Red S staining. MC3T3-Ap cells showed enhanced mineralization at 1 week, compared to control MC3T3-E1 cells, and administration of U0126, SB203580, or sFGFR2IIIc^S252W inhibited mineralization.</p

Analysis of the inhibitory effects of sFGFR2IIIc^S252W on the premature fusion of coronal sutures.

<p>(A) The effects of nanogel-crosslinked hydrogels incorporating sFGFR2IIIc^S252W on the coronal sutures of AS mice were determined using a tissue culture system. Mature bones are shown in purple, resulting from <i>in situ</i> hybridization using the bone sialoprotein (<i>Bsp</i>) probe. Yellow lines on HE-stained images show the contours of the parietal and frontal bony edges, whereas a black arrowhead shows the existence of FLAG-tagged proteins in the calvarial tissue, as determined by immunohistochemistry using anti-FLAG antibodies. The images of blue fluorescence (4′, 6-diamidino-2-phenylindole [DAPI], staining the nucleic acids), green fluorescence (bromodeoxyuridine [BrdU], incorporated into cellular DNA during proliferation), and red fluorescence (rhodamine-stained nanogels) were merged. Administration of sFGFR2IIIc^S252W/nanogel complex maintained the patency of the coronal sutures in AS mice (n = 4/4; AS mice Nos. 9 and 10 are shown as representative examples); however, synostosis was observed on the side where only the nanogel was applied (n = 4/4). Control mice (n = 2/2; control mouse No. 8 is shown as a representative example) did not show any fusion between the frontal and parietal bones. Scale bar = 200 µm. f, frontal bone; p, parietal bone. (B) The ratios of BrdU-positive cells in AS mice and littermate controls are shown. Although no significant difference was observed, the ratio tended to be decreased in the suture tissue of AS mice, compared to the control littermates. Statistical analysis was performed using ANOVA and the Student-Newman-Keuls test.</p