Role of N-Cadherin and Protein Kinase C in Osteoblast Gene Activation Induced by the S252W Fibroblast Growth Factor Receptor 2 Mutation in Apert Craniosynostosis

Abstract

Apert (Ap) syndrome is characterized by premature cranial suture ossification caused by fibroblast growth factor receptor 2 (FGFR-2) mutations. We studied the role of cadherins and signaling events in the phenotypic alterations induced by the Ap FGFR-2 S252W mutation in mutant immortalized fetal human calvaria osteoblasts. The FGFR-2 mutation caused increased expression of the osteoblast markers alkaline phosphatase (ALP), type 1 collagen (COLIA1), and osteocalcin (OC) in long-term culture. The mutation also increased cell-cell aggregation, which was suppressed by specific neutralizing anti-N-and anti-E-cadherin antibodies. Mutant osteoblasts showed increased N-and E-cadherin, but not N-cell adhesion molecule (N-CAM

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).…”

“…Interestingly, reduced dosage of ERF, an inhibitory ETS transcription factor directly bound by ERK1/2 signaling, was found to cause craniosynostosis in humans and mice, implying a role of ERF downstream from ERK signaling in cranial suture ossification (Twigg et al 2013). 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).…”

Cbl-mediated Degradation of Lyn and Fyn Induced by Constitutive Fibroblast Growth Factor Receptor-2 Activation Supports Osteoblast Differentiation

“…Apert and control cells were treated for 24 h, and then total Src kinase activity was determined as described below. After 24 h of treatment, ALP activity, an early marker of osteoblast differentiation that is constitutively increased in FGFR2 human mutant osteoblasts compared with control cells (11)(12)(13)(14), was determined as described (11).…”

“…Gain-of-function mutations in FGFR2 were found to induce changes in osteoblast proliferation, differentiation, and survival in mice and humans (8 -11). In human osteoblasts, we have shown that single missense point mutations (S252W and P253R) located in the linker region between the second and third extracellular immunoglobulin domains of FGFR2 activate the expression of early and late osteoblast differentiation genes, including alkaline phosphatase (ALP), type I collagen (COLIA1), and osteocalcin in vitro and in vivo (11,12), a phenotype that is mediated in part through protein kinase C activation (13,14). The role of other FGFR signaling pathways in the osteoblast phenotype induced by overactive FGFR2 mutations remains unknown.…”

FGF signaling pathways in endochondral and intramembranous bone development and human genetic disease

“…In human osteoblasts, Apert syndrome mutations do not increase cell proliferation (Lomri et al 1998;Fragale et al 1999;Lemonnier et al 2000) but alternatively, increase the expression of type 1 collagen, osteocalcin, and osteopontin, and enhance osteogenesis (Lomri et al 1998;Lemonnier et al 2000). This premature osteogenic cell differentiation induced by Apert Fgfr2 mutations is associated with increased N-cadherin expression and cell-cell adhesion (Lemonnier et al 2001b), which is reproduced by application of FGF2 (Debiais et al 2001). The increased osteoblast differentiation and bone formation induced by activating mutations in Fgfr2 is consistent with the phenotype of stenotic sutures in human nonsyndromic craniosynostosis (De Pollak et al 1996;Shevde et al 2001).…”