Mesenchymal stem cells (MSCs)3 have the potential to differentiate into different lineages, including osteoblasts, chondroblasts, and adipocytes (1-7). The osteoblast differentiation program of MSCs is characterized by cell recruitment, which is followed by timely expressed genes including Runx2, alkaline phosphatase (ALP), type I collagen (ColA1), and osteocalcin (OC), which is associated with extracellular matrix mineralization (8 -10). The program of MSC osteogenic differentiation can be induced by soluble molecules such as bone morphogenetic proteins (BMPs) or Wnt proteins that activate several signaling pathways to trigger osteoblast differentiation (11-15). Although various downstream signals are known to promote osteogenic differentiation (16 -20), the molecular mechanisms that control the early stages of MSC osteoblast differentiation are not fully elucidated.Fibroblast growth factors (FGFs) play an important major role in the control of cell proliferation, differentiation, and survival in several tissues including bone (21-24). Notably, FGF2 was found to promote cell growth and osteoblast differentiation in bone marrow-derived mesenchymal cells (25,26). Consistent with an important role of FGF signaling in the control of osteoprogenitor cells, deletion of FGF2 in mice results in decreased bone marrow stromal cell osteogenic differentiation and altered bone formation (27). The actions of FGFs are highly dependent on high affinity FGF receptors (FGFRs) (28). FGF binding to FGFRs leads to receptor dimerization and phosphorylation of intrinsic tyrosine residues, which leads to activation of several signal transduction pathways including phospholipase C␥ (PLC␥), mitogen-activated protein kinases (MAPK), and phosphatidylinositol 3-kinase (PI3K) (29,30). In bone, activation of extracellular-related kinase (ERK1/2) MAPK and protein kinase C (PKC) was found to enhance osteoblast gene expression (31, 32). The important role of FGFR signaling in bone formation is highlighted by the finding that gain-of-function mutations in FGFRs results in premature cranial osteogenesis (33, 34). FGFR1 was recently shown to be an important transducer of FGF signals in proliferating osteoblasts (35). In contrast, activated FGFR2 was shown to enhance osteoblast differentiation in Apert syndromic craniosynostosis (36 -41). However, the role of FGFR2 signaling in osteogenic differentiation of mesenchymal stem cells is yet to be elucidated.In the present study, we investigated the specific role of FGFR2 signaling on osteoblast commitment and differentiation

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Gli3 is a zinc-finger transcription factor whose activity is dependent on the level of hedgehog (Hh) ligand. Hh signaling has key roles during endochondral ossification; however, its role in intramembranous ossification is still unclear. In this study, we show that Gli3 performs a dual role in regulating both osteoprogenitor proliferation and osteoblast differentiation during intramembranous ossification. We discovered that Gli3Xt-J/Xt-J mice, which represent a Gli3-null allele, exhibit craniosynostosis of the lambdoid sutures and that this is accompanied by increased osteoprogenitor proliferation and differentiation. These cellular changes are preceded by ectopic expression of the Hh receptor Patched1 and reduced expression of the transcription factor Twist1 in the sutural mesenchyme. Twist1 is known to delay osteogenesis by binding to and inhibiting the transcription factor Runx2. We found that Runx2 expression in the lambdoid suture was altered in a pattern complimentary to that of Twist1. We therefore propose that loss of Gli3 results in a Twist1-, Runx2-dependent expansion of the sutural osteoprogenitor population as well as enhanced osteoblastic differentiation which results in a bony bridge forming between the parietal and interparietal bones. We show that FGF2 will induce Twist1, normalize osteoprogenitor proliferation and differentiation and rescue the lambdoid suture synostosis in Gli3Xt-J/Xt-J mice. Taken together, we define a novel role for Gli3 in osteoblast development; we describe the first mouse model of lambdoid suture craniosynostosis and show how craniosynostosis can be rescued in this model.

Apert syndrome is an autosomal dominant disease characterized by craniosynostosis and bony syndactyly associated with point mutations (S252W and P253R) in the fibroblast growth factor receptor (FGFR) 2 that cause FGFR2 activation. Here we investigated the role of the S252W mutation of FGFR2 on osteoblastic differentiation. Osteoblastic cells derived from digital bone in two Apert patients with the S252W mutation showed more prominent alkaline phosphatase activity, osteocalcin and osteopontin mRNA expression, and mineralized nodule formation compared with the control osteoblastic cells derived from two independent non-syndromic polydactyly patients. Stable clones of the human MG63 osteosarcoma cells (MG63-Ap and MG63-IIIc) overexpressing a splice variant form of FGFR2 with or without the S252W mutation (FGFR2IIIcS252W and FGFR2IIIc) showed a higher RUNX2 mRNA expression than parental MG63 cells. Furthermore MG63-Ap exhibited a higher osteopontin mRNA expression than did MG63-IIIc. The enhanced osteoblastic marker gene expression and mineralized nodule formation of the MG63-Ap was inhibited by the conditioned medium from the COS-1 cells overexpressing the soluble FGFR2IIIcS252W. Furthermore the FGF2-induced osteogenic response in the mouse calvarial organ culture system was blocked by the soluble FGFR2IIIcS252W. These results show that the S252W mutation in the FGFR2 gene enhances the osteoblast phenotype in human osteoblasts and that a soluble FGFR2 with the S252W mutation controls osteoblast differentiation induced by the S252W mutation through a dominant negative effect on FGFR2 signaling in Apert syndrome.Apert syndrome is an autosomal dominantly inherited syndrome characterized by craniosynostosis, which results in skull deformity, and symmetric bony syndactyly of the hands and feet. Its prevalence is ϳ15.5/1,000,000 newborns (1) and accounts for about 4.5% of all cases of craniosynostosis (2). Mutations of the human fibroblast growth factor receptors (FGFRs) 1 have been identified to be the cause of a number of craniosynostosis syndromes such as Crouzon, Pfeiffer, JacksonWeiss, Apert, Beare-Stevenson, and Muenke syndromes (3-8).With rare exceptions, Apert syndrome is caused by one of the two missense mutations of the FGFR2 gene involving an amino acid substitution, S252W or P253R, in the linker region between the second and third extracellular Ig domains (4, 9, 10). S252W results from a C755G missense mutation and is more common than P253R caused by C758G in Apert patients (11), and each mutation shows differential effects on the phenotype of syndactyly and cleft palate in this syndrome (12, 13). Most of the Apert patients are sporadic cases and are exclusively affected by mutations arising in the paternal germ line (14). Apert mutation in the FGFR2 gene serves as a gain-offunction mutation by decreasing the dissociation rate of FGFs from FGFR2 (15, 16) as well as by evoking the liganddependent receptor activation. In addition to the retained ligand dependence for the receptor activation, the loss of ligand ...

Background: Gli3-deficient mice (Gli3Xt-J/Xt-J ) show premature suture closure (craniosynostosis). Results: Gli3Xt-J/Xt-J mice have aberrant cell proliferation and osteogenic differentiation in the sutures. Reducing the dosage of Runx2 (Gli3 Xt-J/Xt-J ;Runx2 ϩ/Ϫ mice) rescues the abnormality through canonical Bmp-Smad signaling.