Signaling by Fibroblast Growth Factors (Fgf) and Fibroblast Growth Factor Receptor 2 (Fgfr2)–Activating Mutations Blocks Mineralization and Induces Apoptosis in Osteoblasts

Abstract

Fibroblast growth factors (FGF) play a critical role in bone growth and development affecting both chondrogenesis and osteogenesis. During the process of intramembranous ossification, which leads to the formation of the flat bones of the skull, unregulated FGF signaling can produce premature suture closure or craniosynostosis and other craniofacial deformities. Indeed, many human craniosynostosis disorders have been linked to activating mutations in FGF receptors (FGFR) 1 and 2, but the precise effects of FGF on the proliferation, maturation and differentiation of the target osteoblastic cells are still unclear. In this report, we studied the effects of FGF treatment on primary murine calvarial osteoblast, and on OB1, a newly established osteoblastic cell line. We show that FGF signaling has a dual effect on osteoblast proliferation and differentiation. FGFs activate the endogenous FGFRs leading to the formation of a Grb2/FRS2/Shp2 complex and activation of MAP kinase. However, immature osteoblasts respond to FGF treatment with increased proliferation, whereas in differentiating cells FGF does not induce DNA synthesis but causes apoptosis. When either primary or OB1 osteoblasts are induced to differentiate, FGF signaling inhibits expression of alkaline phosphatase, and blocks mineralization. To study the effect of craniosynostosis-linked mutations in osteoblasts, we introduced FGFR2 carrying either the C342Y (Crouzon syndrome) or the S252W (Apert syndrome) mutation in OB1 cells. Both mutations inhibited differentiation, while dramatically inducing apoptosis. Furthermore, we could also show that overexpression of FGF2 in transgenic mice leads to increased apoptosis in their calvaria. These data provide the first biochemical analysis of FGF signaling in osteoblasts, and show that FGF can act as a cell death inducer with distinct effects in proliferating and differentiating osteoblasts.

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

“…FGF2 reduces apoptosis in cultured calvarial cells (Hill et al 1997) but stimulates apoptosis in the developing coronal suture (Mathijssen et al 2001). In transgenic mice overexpressing FGF2, apoptosis is restricted to more differentiated calvarial osteoblasts at the osteogenic front (Mansukhani et al 2000). These observations and studies on proliferation and differentiation show that FGFs can control the balance between undifferentiated and differentiated osteogenic cells by increasing the proliferation of immature cells and by promoting the differentiation and apoptosis of more mature osteoblasts in developing calvaria (Fig.…”

“…Several of the mutations in Fgfr1 and Fgfr2 in CS, PS, and JWS constitutively activate the receptor by stabilizing intermolecular disulfide bonds, causing ligand-independent dimerization and signaling (Neilson and Friesel 1995;Wilkie et al 1995a;Galvin et al 1996;Robertson et al 1998). Other mutations are thought to prolong the duration of receptor signaling or alter ligand-binding specificity (Anderson et al 1998;Mansukhani et al 2000;Plotnikov et al 2000;Yu et al 2000;Ibrahimi et al 2001). For example, one of the CS mutations in Fgfr2 results in ligand-independent activation and dramatically decreased ligand binding .…”

“…In vitro studies show that FGF1, FGF2, FGF4, or FGF18 can potentiate growth of fetal or neonatal calvarial osteoblasts (Canalis et al 1988;Tang et al 1996;Hurley et al 2001;Shimoaka et al 2001) but not mature osteoblasts (Debiais et al 1998;Mansukhani et al 2000). In vivo, FGF2 increases the number of osteogenic cells and promotes calvarial osteogenesis (Mundy et al 1999).…”

Fibroblast growth factor signaling in skeletal development and disease

“…In osteoblast precursor cells, FGF2 induces osteoblast survival through activation of PI3K/AKT signaling (Debiais et al 1998) and FGFR1-mediated increased Bcl2/Bax ratio . In more differentiated osteoblasts, FGF treatment or overexpression of FGF2 in transgenic mice induces apoptosis in mouse calvaria (Mansukhani et al 2000;Ignelzi et al 2003), which limits the increase in the osteoblast population.…”

Ultraviolet light-induced DNA damage triggers apoptosis in nucleotide excision repair-deficient cells via Bcl-2 decline and caspase-3/-8 activation

“…Besides damaging DNA, UV-C light activates a variety of extranuclear cellular functions, including growth factor cytokine receptors, which were also discussed to be involved in cell killing (Mansukhani et al, 2000). Activation of growth factor receptors results in stimulation of MAP-kinase cascade covering ERKs, JNKs and p38 MAP kinase.…”