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

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 AD 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) messenger RNA (mRNA) and protein levels. This was confirmed in vivo by the abundant immunoreactive N- and E-cadherins in preosteoblasts in the Ap suture whereas N-CAM and alpha- and beta -catenins were unaffected. Neutralizing anti-N-cadherin antibody or N-cadherin antisense (AS) oligonucleotides but not anti-E-cadherin antibody or AS reduced ALP activity as well as ALP, COLIA1, and OC mRNA overexpression in mutant osteoblasts. Analysis of signal transduction revealed increased phospholipase C gamma (PLC gamma) and protein kinase C alpha (PLC alpha) phosphorylation and increased PKC activity in mutant cells in basal conditions. Inhibition of PKC by calphostin C or the PKC alpha -specific inhibitor Go6976 suppressed the increased N-cadherin mRNA and protein levels as well as the overexpression of ALP COLIA1, and OC mRNA in mutant cells. Thus, N-cadherin plays a role in the activation of osteoblast differentiation marker genes in mutant osteoblasts and PKC alpha signaling appears to be involved in the increased N-cadherin and osteoblast gene expression induced by the S252W FGFR-2 mutation in human osteoblasts.