Decreased Proliferation and Altered Differentiation in Osteoblasts from Genetically and Clinically Distinct Craniosynostotic Disorders

Abstract

Craniosynostoses are a heterogeneous group of disorders characterized by premature fusion of cranial sutures. Mutations in fibroblast growth factor receptors (FGFRs) have been associated with a number of such conditions. Nevertheless , the cellular mechanism(s) involved remain unknown. We analyzed cell proliferation and differentiation in osteoblasts obtained from patients with three genetically and clinically distinct craniosynostoses: Pfeiffer syndrome carrying the FGFR2 C342R substitution , Apert syndrome with FGFR2 P253R change , and a nonsyndromic craniosynostosis without FGFR canonic mutations , as compared with control osteoblasts. Osteoblasts from craniosynostotic patients exhibited a lower proliferation rate than control osteoblasts. P253R and nonsyndromic craniosynostosis osteoblasts showed a marked differentiated phenotype, characterized by high alkaline phosphatase activity, increased mineralization and expression of noncollagenous matrix proteins , associated with high expression and activation of protein kinase C␣ and protein kinase C⑀ isoenzymes. By contrast , the low proliferation rate of C342R osteoblasts was not associated with a differentiated phenotype. Although they showed higher alkaline phosphatase activity than control , C342R osteoblasts failed to mineralize and expressed low levels of osteopontin and osteonectin and high protein kinase C levels. Stimulation of proliferation and inhibition of differentiation were observed in all cultures on FGF2 treatment. Our results suggest that an anticipated proliferative/differentiative switch , associated with alterations of the FGFR transduction pathways , could be the causative com- Craniosynostosis, the premature ossification of one or more sutures of the flat bones of the developing skull, is a relatively common defect of the cranial morphogenetic program, with a prevalence at birth of approximately 1:3000. It results in a wide spectrum of craniofacial anomalies, including abnormal head shape, protruding eyes, and midface underdevelopment.

Fibroblast Growth Factor Receptor 2 Promotes Osteogenic Differentiation in Mesenchymal Cells via ERK1/2 and Protein Kinase C Signaling

“…We and others (37,38) previously found that FGF signaling activates PLC␥-PKC signaling in osteoblasts. To determine the potential role of PKC in osteoblast differentiation induced by FGFR2 activation in mesenchymal cells, we analyzed PKC activity in C3H10T1/2 cells stably expressing WT or MT Fig.…”

In Vivo Modulation of FGF Biological Activity Alters Cranial Suture Fate

“…Finally, osteoblasts derived from human synostotic sutures (ie, isolated and syndromic sutures) demonstrate greater basal levels of type I collagen and noncollagenous matrix molecules than control osteoblasts. [41][42][43] Taken together, these studies suggest that alterations in the expression of osteogenic cytokines (ie, FGF-2 and TGF-␤1) and extracellular matrix molecules likely determine the fate of the overlying cranial sutures. These conclusions are supported by our current study.…”

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

“…The protein kinase C (PKC) pathway is involved in the control of sodium-dependent phosphate transport (Suzuki et al 2000) and expression of N-cadherin in calvarial osteoblasts (Debiais et al 2001). Mutations in Fgfr2 that cause Apert syndrome induce constitutive activation of PKC in human calvarial osteoblasts (Fragale et al 1999;Lomri et al 2001). This signaling pathway is responsible for the increased differentiation and apoptosis in mutant osteo-blasts (Lemonnier et al , 2001a.…”

“…In murine calvarial cells, transfection of the Fgfr2 S252W (Apert mutation) gene inhibits cell differentiation and increases proliferation (Mansukhani et al 2000). 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).…”