Exosome-mediated small interfering RNA delivery inhibits aberrant osteoblast differentiation in Apert syndrome model mice.

OBJECTIVE: Apert syndrome, an autosomal dominant congenital disorder characterized by craniosynostosis, is caused by a missense mutation (S252W or P253R) in fibroblast growth factor receptor 2 (FGFR2). Exosomes are naturally occurring carriers that deliver nucleic acids, including small interfering RNA (siRNA), to induce gene silencing. This study aimed to develop siRNA-loaded exosomes (Ex-siRNAFgfr2S252W) to silence the Fgfr2S252W gain-of-function mutation, thereby inhibiting the increased osteoblastic differentiation caused by the constitutive activation of FGFR2 signaling in calvarial osteoblastic cells isolated from Apert syndrome model mice. DESIGN: Primary calvarial osteoblast-like cells were isolated from the embryonic calvarial sutures of the Apert syndrome model (Fgfr2S252W/+) and littermate wild-type mice (Ap-Ob and Wt-Ob, respectively). Exosomes were extracted from the serum of wild-type mice, validated using biomarkers, and used to encapsulate siRNAs. After exosome-mediated siRNA transfection, cells were analyzed under a fluorescence microscope to validate the delivery of Ex-siRNAFgfr2S252W, followed by western blot and real-time reverse transcription polymerase chain reaction analyses. RESULTS: After 24 h of Ex-siRNAFgfr2S252W delivery in both Ap-Ob and Wt-Ob, siRNA-loaded exosome delivery was validated. Moreover, p44/42 mitogen-activated protein kinase (MAPK) phosphorylation, runt-related transcription factor 2 (Runx2), and collagen type 1 alpha 1 (Col1a1) mRNA expression, and alkaline phosphatase (ALP) activity were significantly increased in Ap-Ob. The levels of phospho-p44/42 protein, Runx2, Col1a1, and ALP were significantly decreased after Ex-siRNAFgfr2S252W transfection but did not affect Wt-Ob. CONCLUSIONS: These results indicate that exosome-mediated delivery of siRNA targeting Fgfr2S252W is a potential non-invasive treatment for aberrant FGF/FGFR signaling.

Changes in superoxide dismutase 3 (SOD3) expression in periodontal tissue during orthodontic tooth movement of rat molars and the effect of SOD3 on in vitro hypoxia-exposed rat periodontal ligament cells.

BACKGROUND/OBJECTIVES: Hypoxia during orthodontic tooth movement (OTM) induces reactive oxygen species (ROS) production in periodontal tissues. Superoxide dismutase 3 (SOD3) is an anti-inflammatory enzyme that protects cells from ROS. This study investigated the expression and function of SOD3 during rat OTM and in hypoxia-exposed rat periodontal ligament (PDL) cells. MATERIALS/METHODS: OTM of right maxillary first molars were performed in 8-week-old male Sprague-Dawley rats using closed-coil spring for 1 and 14 days (n = 6 per group). SOD3 and hypoxia-inducible factor 1-alpha (HIF-1α) protein expression was evaluated by immunohistochemistry. The effects of SOD3 on cell viability and proliferation, ROS production, and mRNA expression of Hif1-α, receptor activator of nuclear factor kappa-Β ligand (Rankl), and osteoprotegerin (Opg) in PDL cells and osteoclast differentiation were investigated under normal and hypoxic conditions. RESULTS: SOD3 expression in PDL tissues significantly decreased on the compression side on day 1 and on both sides on day 14 of OTM. HIF-1α levels significantly increased on the compression side on day 14. Cell viability, cell proliferation, and Opg mRNA expression decreased, whereas ROS production and Hif1-α and Rankl mRNA expression increased in the PDL cells upon SOD3 silencing. Hypoxia reduced Sod3 and Opg mRNA expression and increased ROS, Rankl mRNA expression, and osteoclast formation; SOD3 treatment attenuated these effects. CONCLUSION/IMPLICATIONS: SOD3 plays a role in periodontal tissue remodelling during OTM and in hypoxia-exposed PDL cells through ROS, HIF-1α, and RANKL/OPG pathways. Moreover, SOD3 treatment could attenuate the negative effects of hypoxia on the PDL cells.

Three-dimensional analysis of the palatal morphology in growing patients with Apert syndrome and Crouzon syndrome.

Patients with Apert syndrome or Crouzon syndrome present with severe defects in oral-maxillofacial growth and development. In this study, we conducted a quantitative three-dimensional (3D) analysis of the palatal morphology of patients with Apert syndrome and Crouzon syndrome. Four patients with Apert syndrome (average age, 11.0 ± 0.8 years) and five with Crouzon syndrome (average age, 10.1 ± 1.6 years) were investigated. The participants' maxillary dental casts were scanned and analyzed using 3D imaging. Palatal width, depth, cross-sectional area, and palatal angle (PW, PD, PCA, and PA, respectively) were measured, and standard scores were calculated based on sex- and age-matched Japanese standard values; the actual palatal surface areas (PSA) and palatal volumes (PV) were also measured. Our results show that patients with Apert syndrome and Crouzon syndrome had a very narrow PW (standard score: -3.79 and - 0.47, respectively). 3D analysis revealed that patients with Apert syndrome had a significantly shallower PD (standard score: -1.35) than those with Crouzon syndrome (standard score: 2.47), resulting in a smaller PCA (standard score: -5.13), PSA (5.49 cm2 ), and PV (1.11 cm3 ) and larger PA (standard score: -0.12) than those in patients with Crouzon syndrome. This might be due to the former having a narrower and shallower palate caused by the predominant swelling of the palatal mucosa. These findings improve our understanding of the differences in palatal morphology between Apert syndrome and Crouzon syndrome patients.

Expression pattern of transcriptional enhanced associate domain family member 1 (Tead1) in developing mouse molar tooth.

The Hippo pathway is essential for determining organ size by regulating cell proliferation. Previous reports have shown that impairing this pathway causes abnormal tooth development. However, the precise expression profile of the members of the transcriptional enhanced associate domain family (Tead), which are key transcription factors mediating Yap function, during tooth development is unclear. In this study, among the four isoforms of Tead (Tead1 - 4), only the expression of Tead1 mRNA was observed using semiquantitative RT- PCR in murine developing tooth germ at E16.5. The expression level of Tead1 mRNA in the excised murine mandibular molar tooth germ was significantly higher at E16.5 than at other developmental stages, as determined using quantitative PCR. We found that the mRNA expression of connective tissue growth factor (Ctgf), which is one of the Yap target genes directly controlling cell growth, changed consistently with that of Tead1 in developing molars. Fluorescent immunostaining revealed that Tead1 protein was expressed in both epithelial cells and mesenchymal cells of the dental lamina and dental epithelium, including the primary enamel knot during the cap stage. During the early bell stage (E16.5), Tead1 was expressed intensely in the inner and outer enamel epithelium, including the secondary enamel knot and the neighboring mesenchymal cells. Tead1 then specifically localized to the inner and outer enamel epithelium, which is responsible for enamel formation during the bell stage. These expression patterns were consistent with those of Yap, Taz, and Ctgf protein in developing molars. These results suggest that Tead1 acts as a mediator of the biological functions of Yap, such as the morphogenesis of cusp formation, during tooth development.

Mkx regulates the orthodontic tooth movement via osteoclast induction.

INTRODUCTION: The periodontal ligament (PDL) plays an important role in orthodontic tooth movement; however, the underlying molecular mechanism remains unclear. We have previously reported that the Mohawk homeobox (Mkx), a tendon-specific transcription factor, is expressed in the PDL and regulates its homeostasis. MATERIALS AND METHODS: In the present study, we examined the role of Mkx in orthodontic tooth movement via bone remodeling induced by mechanical stimulation in Mkx-deficient rats, which are widely used as experimental animals for orthodontic force application. Orthodontic tooth movement of the maxillary first molar was performed in 7-week-old male Mkx-deficient rats (n = 4) and wild-type Wistar rats (n = 4) using coil springs for 14 days. Hematoxylin and eosin (H&E) staining and tartrate-resistant acid phosphatase (TRAP) staining were performed to evaluate morphological changes and osteoclasts. Furthermore, changes in the expression of receptor activator nuclear factor-kappa B ligand (RANKL) were demonstrated using immunostaining. RESULTS: The amount of tooth movement was significantly lower in Mkx-deficient rats than in wild-type rats. The number of TRAP-positive cells was suppressed in Mkx-deficient rats on the compression side. CONCLUSION: Orthodontic tooth movement experiments in Mkx-deficient rats suggested that Mkx is involved in osteoclast induction at the alveolar bone surface on the compression side. This study reveals the possibility that Mkx plays a mechanosensory role in orthodontic tooth movement by inducing RANKL expression and osteoclastogenesis.

Aberrantly activated Wnt/ß-catenin pathway co-receptors LRP5 and LRP6 regulate osteoblast differentiation in the developing coronal sutures of an Apert syndrome (Fgfr2S252W/+ ) mouse model.

BACKGROUND: Apert syndrome is an autosomal, dominant inherited disorder characterized by craniosynostosis and syndactyly caused by gain-of-function mutations in the fibroblast growth factor receptor 2 (FGFR2) gene. Wnt/ß-catenin signaling plays critical roles in regulating the skeletal development. Here, we analyzed the role of this pathway in the developing coronal sutures (CS) of a murine Apert syndrome model (Fgfr2S252W/+ ). RESULTS: We observed aberrantly increased mRNA expression of Lrp5 and Lrp6 in CS of Fgfr2S252W/+ mice, whereas both wild type (WT) and Fgfr2S252W/+ mice showed similar expression of other Wnt/ß-catenin-related genes, such as Wnt3, Wnt3a, Fzd4, Fzd6, Axin2, and Dkk1 as evidenced by in situ hybridization. Significantly increased Lrp5 and Lrp6 mRNA expression was observed by quantitative PCR analysis of cultured cells isolated from CS of Fgfr2S252W/+ mice. Phospho-LRP5, phospho-LRP6, and non-phospho-ß-catenin were upregulated in Fgfr2S252W/+ CS compared with that in WT CS. Short-interfering RNA targeting Lrp5 and Lrp6 significantly reduced runt-related transcription factor 2, collagen type 1 alpha 1, and osteocalcin mRNA expression, and alkaline phosphatase activity in cultured cells. CONCLUSIONS: The Wnt/ß-catenin pathway was activated in the CS of Fgfr2S252W/+ mice during craniofacial development, suggesting the involvement of the Wnt/ß-catenin pathway in the pathogenesis of CS synostosis in Fgfr2S252W/+ mice.

Craniofacial, oral, and cervical morphological characteristics in Japanese patients with Apert syndrome or Crouzon syndrome.

BACKGROUND AND OBJECTIVES: Mutations in the fibroblast growth factor receptor 2 (FGFR2) gene are responsible for both Apert syndrome (AS) and Crouzon syndrome (CS). These diseases share phenotypic characteristics, including midfacial hypoplasia and premature fusion of the calvarial suture(s). Given the extensive range of craniofacial growth and developmental abnormalities, management of these patients requires a multidisciplinary approach. This study aimed to compare craniofacial, oral, and cervical morphological characteristics in Japanese orthodontic patients with AS or CS. SUBJECTS AND METHODS: Lateral cephalograms, orthopantomograms, dental casts, medical interview records, facial photographs, and intraoral photographs of 7 AS patients and 12 CS patients on initial visits were used in this study. Cephalometric analyses were performed, and standard scores were calculated based on age- and sex-matched Japanese standard values. RESULTS: Cephalometric analysis revealed that AS patients had significantly more severe maxillary hypoplasia in two dimensions and increased clockwise mandibular rotation. Additionally, cleft of the soft palate, anterior open bite, severe crowding in the maxillary dental arch, and congenitally missing teeth occurred more frequently among AS patients. Multiple fusions between cervical vertebrae C2, C3, C5, and C6 were observed in the AS patients. LIMITATIONS: Small sample size. CONCLUSIONS/IMPLICATIONS: Our study shows that AS patients have more severe craniofacial and maxillofacial deformities than CS patients.

Relaxin 2 carried by magnetically directed liposomes accelerates rat midpalatal suture expansion and subsequent new bone formation.

Relaxin (RLN) is an insulin-like peptide hormone that enables softening and lengthening of the pubic symphysis and uterine cervix. Here, we analyzed the effects of RLN2 on the expansion of rat midpalatal suture (MPS) using a magnetically directed liposome-based drug delivery system. Thirty-six male rats were divided into three groups: control (MPS was not expanded), lipo (expanded for 1 week with vehicle liposomes encapsulating ferric oxide and Cy5.5), and RLN-lipo (expanded for 1 week with the liposomes coated with RLN2). Rats were sacrificed after 1 week of expansion or after 2 weeks of retention. To accumulate RLN2-liposomes, a magnetic sheet was fixed to the palatal mucosa of the MPS. In vivo imaging showed magnetically controlled accumulation of liposomes in the MPS for 72 h. Immunohistochemistry revealed RLN2 expression in the MPS after expansion and relaxin receptor (RXFP) 2 expression at the osteogenic front (OF) in the RLN-lipo group; all groups expressed RXFP1 in the MPS. MPS expansion and bone formation were significantly accelerated at the OF in RLN-lipo group compared with the other groups. In the RLN-lipo group, significantly accelerated serrate bone deposition and elevated periostin (POSTN), iNOS, and MMP-1 levels were observed in the MPS. Sclerostin (SOST) expression was significantly reduced in newly formed bone in the RLN-lipo group. Our data revealed that RLN2 enhanced suture expansion via MMP-1 and iNOS secretion in the sutural fibroblasts and new bone formation via POSTN expression in osteoblasts at the OF. These properties may be useful for developing a new less-invasive orthopedic treatment aiming at sutural modification of cranio- and maxillofacial deformity patients.

Regulation of Calvarial Osteogenesis by Concomitant De-repression of GLI3 and Activation of IHH Targets.

Loss-of-function mutations in GLI3 and IHH cause craniosynostosis and reduced osteogenesis, respectively. In this study, we show that Ihh ligand, the receptor Ptch1 and Gli transcription factors are differentially expressed in embryonic mouse calvaria osteogenic condensations. We show that in both Ihh-/- and Gli3Xt-J/Xt-J embryonic mice, the normal gene expression architecture is lost and this results in disorganized calvarial bone development. RUNX2 is a master regulatory transcription factor controlling osteogenesis. In the absence of Gli3, RUNX2 isoform II and IHH are upregulated, and RUNX2 isoform I downregulated. This is consistent with the expanded and aberrant osteogenesis observed in Gli3Xt-J/Xt-J mice, and consistent with Runx2-I expression by relatively immature osteoprogenitors. Ihh-/- mice exhibited small calvarial bones and HH target genes, Ptch1 and Gli1, were absent. This indicates that IHH is the functional HH ligand, and that it is not compensated by another HH ligand. To decipher the roles and potential interaction of Gli3 and Ihh, we generated Ihh-/-;Gli3Xt-J/Xt-J compound mutant mice. Even in the absence of Ihh, Gli3 deletion was sufficient to induce aberrant precocious ossification across the developing suture, indicating that the craniosynostosis phenotype of Gli3Xt-J/Xt-J mice is not dependent on IHH ligand. Also, we found that Ihh was not required for Runx2 expression as the expression of RUNX2 target genes was unaffected by deletion of Ihh. To test whether RUNX2 has a role upstream of IHH, we performed RUNX2 siRNA knock down experiments in WT calvarial osteoblasts and explants and found that Ihh expression is suppressed. Our results show that IHH is the functional HH ligand in the embryonic mouse calvaria osteogenic condensations, where it regulates the progression of osteoblastic differentiation. As GLI3 represses the expression of Runx2-II and Ihh, and also elevates the Runx2-I expression, and as IHH may be regulated by RUNX2 these results raise the possibility of a regulatory feedback circuit to control calvarial osteogenesis and suture patency. Taken together, RUNX2-controlled osteoblastic cell fate is regulated by IHH through concomitant inhibition of GLI3-repressor formation and activation of downstream targets.

A preliminary investigation of the effect of relaxin on bone remodelling in suture expansion.

Background and objectives: Relaxin (RLN) is an insulin-like hormone associated with extracellular matrix degradation, osteoclastogenesis, and osteoblast differentiation. This study aimed to assess the effect of RLN during and after lateral expansion of murine calvarial sagittal sutures. Materials and methods: RLN was injected topically using a nano-sized liposome carrier into the sagittal sutures of 8- to 10-week-old wild type mice just before lateral expansion. Suture morphology, bone mineral density (BMD), and bone volume were analysed by micro-computed tomography. Collagen deposition and osteoclast differentiation were observed by Verhoeff-Van Gieson (VVG) and tartrate-resistant acid phosphatase (TRAP) staining, respectively. Results: Less collagen staining and higher tissue-specific relaxin/insulin-like family peptide receptor (Rxfp)-1 and -2 expression were observed in the RLN-treated samples after 48 hours. Increased BMD and volume, and thick well-organised osteoid tissue, with multinucleated TRAP-positive cells, were observed in RLN-treated samples after 1 week. Increased Rxfp-1 expression was observed in the sagittal sutures in the mid-suture fibrous tissue following RLN treatment. Rxfp-2 was only expressed in the calvarial bone under tensile stimulation and RLN treatment further increased its expression. Limitations: RLN-liposomes were not detected at any instance under the current experimental conditions. This is a preliminary study and the sample number limits the power of its results. VVG staining cannot quantify collagen contents but can provide preliminary information on the presence of collagen fibres. Conclusions: RLN treatment may modify bone remodelling and collagen metabolism during and after suture expansion.

Therapeutic effect of nanogel-based delivery of soluble FGFR2 with S252W mutation on craniosynostosis.

Apert syndrome is an autosomal dominantly inherited disorder caused by missense mutations in fibroblast growth factor receptor 2 (FGFR2). Surgical procedures are frequently required to reduce morphological and functional defects in patients with Apert syndrome; therefore, the development of noninvasive procedures to treat Apert syndrome is critical. Here we aimed to clarify the etiological mechanisms of craniosynostosis in mouse models of Apert syndrome and verify the effects of purified soluble FGFR2 harboring the S252W mutation (sFGFR2IIIcS252W) on calvarial sutures in Apert syndrome mice in vitro. We observed increased expression of Fgf10, Esrp1, and Fgfr2IIIb, which are indispensable for epidermal development, in coronal sutures in Apert syndrome mice. Purified sFGFR2IIIcS252W exhibited binding affinity for fibroblast growth factor (Fgf) 2 but also formed heterodimers with FGFR2IIIc, FGFR2IIIcS252W, and FGFR2IIIbS252W. Administration of sFGFR2IIIcS252W also inhibited Fgf2-dependent proliferation, phosphorylation of intracellular signaling molecules, and mineralization of FGFR2S252W-overexpressing MC3T3-E1 osteoblasts. sFGFR2IIIcS252W complexed with nanogels maintained the patency of coronal sutures, whereas synostosis was observed where the nanogel without sFGFR2S252W was applied. Thus, based on our current data, we suggest that increased Fgf10 and Fgfr2IIIb expression may induce the onset of craniosynostosis in patients with Apert syndrome and that the appropriate delivery of purified sFGFR2IIIcS252W could be effective for treating this disorder.

RELAXIN enhances differentiation and matrix mineralization through Relaxin/insulin-like family peptide receptor 2 (Rxfp2) in MC3T3-E1 cells in vitro.

RELAXIN (RLN) is a polypeptide hormone of the insulin-like hormone family; it facilitates birth by softening and widening the pubic symphysis and cervix in many mammals, including humans. The role of RLN in bone metabolism was recently suggested by its ability to induce osteoclastogenesis and activate osteoclast function. RLN binds to RELAXIN/INSULIN-LIKE FAMILY PEPTIDE 1 (RXFP1) and 2 (RXFP2), with varying species-specific affinities. Young men with mutated RXFP2 are at high risk for osteoporosis, as RXFP2 influences osteoblast metabolism by binding to INSULIN-LIKE PEPTIDE 3 (INSL3). However, there have been no reports on RLN function in osteoblast differentiation and mineralization or on the functionally dominant receptors for RLN in osteoblasts. We previously described Rxfp1 and 2 expression patterns in developing mouse oral components, including the maxillary and mandibular bones, Meckel's cartilage, tongue, and tooth primordia. We hypothesized that Rln/Rxfp signaling is a key mediator of skeletal development and metabolism. Here, we present the gene expression patterns of Rxfp1 and 2 in developing mouse calvarial frontal bones as determined by in situ hybridization. In addition, RLN enhanced osteoblastic differentiation and caused abnormal mineralization and extracellular matrix metabolism through Rxfp2, which was predominant over Rxfp1 in MC3T3-E1 mouse calvarial osteoblasts. Our data suggest a novel role for Rln in craniofacial skeletal development and metabolism through Rxfp2.

Long-term orthodontic and surgical treatment and stability of a patient with Beckwith-Wiedemann syndrome.

Beckwith-Wiedemann syndrome (BWS) is a congenital growth disorder. Children born with BWS develop enlarged organs, including the tongue, a large body, and other signs. A woman with BWS was treated and followed for 30 years. Treatment consisted of tongue reduction, orthopedic and orthodontic treatment, orthognathic surgery, and retention. The patient was first treated when she was 5 years old. Her original orthodontic problems included macroglossia, anterior open bite, anterior crossbite, and a skeletal Class III jaw relationship caused by significant mandibular protrusion. The jaw-base relationships did not improve in the early preadolescent period after phase 1 of orthodontic treatment with a vertical chincap. With the growth spurt accompanying puberty, she developed a severe skeletal Class III jaw relationship and a constricted maxillary arch. Surgically assisted rapid maxillary expansion was performed at 23 years of age to correct the severe discrepancy between the maxillary and mandibular dental arch widths. Then, at 26 years, a LeFort I osteotomy, a horseshoe osteotomy, a bilateral sagittal split ramus osteotomy, and genioplasty were performed after presurgical orthodontic treatment with extraction of the mandibular first molars. Both the facial profile and the occlusion were stable after 6 years of retention. This case report discusses the result of long-term observation of a patient with BWS who underwent tongue reduction, early orthodontic treatment, and surgical-orthodontic treatment.

Soluble form of FGFR2 with S252W partially prevents craniosynostosis of the apert mouse model.

BACKGROUND: Apert syndrome (AS) is characterized by craniosynostosis, midfacial hypoplasia, and bony syndactyly. It is an autosomal dominantly inherited disease caused by point mutations (S252W or P253R) in fibroblast growth factor receptor (FGFR) 2. These mutations cause activation of FGFR2 depending on ligand binding. Recently, an AS mouse model, Fgfr2(+/) (S252W) , showed phenotypes similar to those of AS patients. We previously reported that the soluble form of FGFR2(S252W) (sFGFR2IIIc(S252W) ) efficiently inhibits enhanced osteoblastic differentiation caused by FGFR2 activation in AS in vitro, presumably because FGFs binding to FGFRs is interrupted. In this study, we developed Fgfr2(+/) (S252W) (Ap) mice expressing the sFGFR2IIIc(S252W) protein, and we investigated the effects of sFGFR2IIIc(S252W) on AS-like phenotypes. RESULTS: In Ap mice, the coronal suture (CS) was fused prematurely at P1. In addition, the mice exhibited a widened interfrontal suture (IFS) with ectopic bone and thickened cartilage formation. In Fgfr2(+/) (S252W) sFGFR2IIIc(S252W) (Ap/Sol) mice, the CS was similar to that of wild-type mice. Ap/Sol mice did not show any ectopic bone or cartilage formation in the IFS, but showed a wider IFS than that of the wild-type mice. CONCLUSIONS: sFGFR2IIIc(S252W) may partially prevent craniosynostosis in the Apert mouse model by affecting the CS and IFS in vivo.

Relaxin receptors 1 and 2 and nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor) mRNAs are expressed in oral components of developing mice.

OBJECTIVE: Relaxin is a pleiotropic hormone of the insulin-like peptide hormone family that plays an important role in reproductive physiology as well as in fibrosis, angiogenesis, and bone remodelling. It binds to the relaxin family peptide receptors 1 and 2 (Rxfp1 and Rxfp2) and can, in addition and independently, bind and activate the glucocorticoid receptor Nr3c1. Despite the wide-ranging effect of relaxin, the expression patterns of Rxfp1 and 2 during facial development have not been examined. In this study, we aimed to identify the mRNA expression patterns of Rxfp1, Rxfp2, and Nr3c1 in oral tissues during late mouse facial development in order to pinpoint the structures that could be sensitive to relaxin signalling during this period. DESIGN: Rxfp1, Rxfp2, and Nr3c1 mRNAs were identified by in situ hybridization using digoxigenin-labelled riboprobes on coronal sections of mouse heads from embryonic days 13.5 to 18.5. RESULTS: We found that Rxfp1, Rxfp2, and Nr3c1 mRNAs were expressed on the developing maxilla and mandible, Meckel's cartilage, tongue, and tooth primordia between embryonic days 13.5-18.5. CONCLUSIONS: Receptors that bind relaxin were present in developing oral tissues of mice. This finding suggests that relaxin may be involved in the prenatal development of the face.

Apert syndrome mutant FGFR2 and its soluble form reciprocally alter osteogenesis of primary calvarial osteoblasts.

Apert syndrome is characterized by craniosynostosis and syndactyly, and is predominantly caused by mutation of either S252W or P253W in the fibroblast growth factor receptor (FGFR) 2 gene. In this study, we characterized the effects of one of the mutations (S252W) using primary calvarial osteoblasts derived from transgenic mice, Ap-Tg and sAp-Tg, that expressed an Apert-type mutant FGFR2 (FGFR2IIIc-S252W; FGFR2IIIc-Ap), and the soluble form (extracellular domain only) of the mutant FGFR2 (sFGFR2IIIc-Ap), respectively. Compared to WT-derived osteoblasts, osteoblasts from Ap-Tg mouse showed a higher proliferative activity and enhanced differentiation, while those from sAp-Tg mouse exhibited reduced potential for proliferation and osteogenic differentiation. When transplanted with ß-tricalcium phosphate (ß-TCP) granules into immunodeficient mice, Ap-Tg-derived osteoblasts showed a higher bone forming capacity, whereas sAp-Tg-derived osteoblasts were completely deficient for this phenotype. Phosphorylation of extracellular signal-regulated kinase (ERK), MEK, PLCγ, and p38 was increased in Ap-Tg-derived osteoblasts, whereas phosphorylation of these signaling molecules was reduced in sAp-Tg-derived osteoblasts. Interestingly, when these experiments were carried out using osteoblasts from the mice generated by crossing Ap-Tg and sAp-Tg (Ap/sAp-Tg), which co-expressed FGFR2IIIc-Ap and sFGFR2IIIc-Ap, the results were comparable to those obtained from WT-derived osteoblasts. Taken together, these results indicate that osteoblasts expressing FGFR2IIIc-Ap proliferate and differentiate via highly activated MEK, ERK, and p38 pathways, while these pathways are suppressed in osteoblasts expressing sFGFR2IIIc-Ap. Our findings also suggest that altered FGFR2IIIc signaling in osteoblasts is mostly responsible for the phenotypes seen in Apert syndrome, therefore these osteoblast cell lines are useful tools for investigating the pathogenesis of Apert syndrome.