A Soluble Form of Fibroblast Growth Factor Receptor 2 (FGFR2) with S252W Mutation Acts as an Efficient Inhibitor for the Enhanced Osteoblastic Differentiation Caused by FGFR2 Activation in Apert Syndrome

Abstract

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 ...

Fibroblast growth factor signaling in skeletal development and disease

“…At a later stage, activating FGFR2 mutations were widely found to cause increased osteoblast marker gene expression and accelerated osteoblast maturation in cranial sutures (Eswarakumar et al 2004;Yang et al 2008;Yin et al 2008;Holmes et al 2009;Yeh et al 2012;Liu et al 2013a,b;Morita et al 2014). This finding in mice supports the original observation in humans that Apert and Crouzon FGFR2 mutations cause increased osteoblast maturation and function in postnatal human suture development (Lomri et al 1998;Lemonnier et al 2001b;Tanimoto et al 2004;Baroni et al 2005;Marie 2015). FGFR2 mutations are also associated with increased osteoblast apoptosis in fused sutures in mice Chen et al 2014) and humans (Lemonnier et al 2001a;Lomri et al 2001;Kaabeche et al 2005); however, this appears to be a secondary event that occurs subsequent to osteoblast maturation and not the primary cause of the synostosis (Holmes et al 2009).…”

“…In vitro, overactivated FGFR2 signaling by Apert and Crouzon mutations can be attenuated by specific glycosaminoglycans (McDowell et al 2006), FGFR or tyrosine kinase inhibitors (Perlyn et al 2006), or a soluble mutant form of FGFR2 that exerts a dominant-negative effect on FGFR2 (Tanimoto et al 2004). In vivo, selective uncoupling of the docking protein FRS2α and the Crouzon-like activated FGFR2c mutant was found to attenuate FGFR signaling and prevent premature suture fusion in mice (Eswarakumar et al 2006).…”

“…In human craniosynostosis, increased osteoblast gene expression and cranial osteogenesis induced by Apert FGFR2(S252W) mutation are related to PLCγ and PKCα activation (Lemonnier et al 2001b). The consequence is increased expression of Runx2, which is associated with premature fusion of cranial sutures in mice and humans (Zhou et al 2000;Eswarakumar et al 2002;Tanimoto et al 2004;Baroni et al 2005;Guenou et al 2005). In both mice and humans, FGFR2 levels are decreased in response to the Apert FGFR2(S252W) mutation as a result of c-CBL-mediated FGFR2 ubiquitination and degradation (Kaabeche et al 2004;Holmes et al 2009).…”

Calcium sensing receptor forms complex with and is up-regulated by caveolin-1 in cultured human osteosarcoma (Saos-2) cells

“…In the bone, osteoblast cells play an important role in bone remodeling. They have many functional proteins related to bone metabolism ( Lisanti et al, 1994;Hikiji et al, 2004;Kondo and Togari, 2004;Tanimoto et al, 2004;Urano et al, 2004). In this connection, osteoblast cells are known to contain many receptors and membrane associated signal molecules like the G-protein, protein kinase C and protein kinase A, among others.…”

The molecular and cellular basis of Apert syndrome

“…Longterm cultured human fetal calvarial osteoblasts from two Apert (S252W) fetuses increased expression of osteoblast markers alkaline phosphatase (ALP), type 1 collagen (COLIA1) and osteocalcin (OC) compared to normal osteoblasts (34). Tanimoto et al compared differentiation of osteoblasts from the digital bone of two Apert patients with the FGFR2 S252W mutation and two independent non-syndromic polydactyly patients, and revealed that the Apert osteoblasts showed more prominent ALP, OC and osteopontin mRNA expression and mineralized nodule formation (35). In 2012, Yeh et al found both that the coronal suture periosteal fibroblasts and MSCs from three unrelated AS patients showed enhanced osteogenic differentiation compared to cells from age-and sexmatched control subjects in vivo and vitro (30).…”