Apert syndrome mutant FGFR2 and its soluble form reciprocally alter osteogenesis of primary calvarial osteoblasts

Abstract

Apert syndrome is characterized by craniosynostosis and syndactyly, and is predominantly caused by mutation of either S252W or P253W in the fibroblast growth factor receptor (FGFR) 2 gene. In this study, we characterized the effects of one of the mutations (S252W) using primary calvarial osteoblasts derived from transgenic mice, Ap-Tg and sAp-Tg, that expressed an Apert-type mutant FGFR2 (FGFR2IIIc-S252W; FGFR2IIIc-Ap), and the soluble form (extracellular domain only) of the mutant FGFR2 (sFGFR2IIIc-Ap), respectively. Compared to WT-derived osteoblasts, osteoblasts from Ap-Tg mouse showed a higher proliferative activity and enhanced differentiation, while those from sAp-Tg mouse exhibited reduced potential for proliferation and osteogenic differentiation. When transplanted with β-tricalcium phosphate (β-TCP) granules into immunodeficient mice, Ap-Tg-derived osteoblasts showed a higher bone forming capacity, whereas sAp-Tg-derived osteoblasts were completely deficient for this phenotype. Phosphorylation of extracellular signal-regulated kinase (ERK), MEK, PLCγ, and p38 was increased in Ap-Tg-derived osteoblasts, whereas phosphorylation of these signaling molecules was reduced in sAp-Tg-derived osteoblasts. Interestingly, when these experiments were carried out using osteoblasts from the mice generated by crossing Ap-Tg and sAp-Tg (Ap/sAp-Tg), which co-expressed FGFR2IIIc-Ap and sFGFR2IIIc-Ap, the results were comparable to those obtained from WT-derived osteoblasts. Taken together, these results indicate that osteoblasts expressing FGFR2IIIc-Ap proliferate and differentiate via highly activated MEK, ERK, and p38 pathways, while these pathways are suppressed in osteoblasts expressing sFGFR2IIIc-Ap. Our findings also suggest that altered FGFR2IIIc signaling in osteoblasts is mostly responsible for the phenotypes seen in Apert syndrome, therefore these osteoblast cell lines are useful tools for investigating the pathogenesis of Apert syndrome.

Fibroblast growth factor signaling in skeletal development and disease

“…In several mouse models, Apert FGFR2(S252W) mutations activate ERK1/2, p38 MAPK, AKT, β-catenin, and PLCγ in osteogenic cells, which may contribute to premature cranial ossification (Chen et al 2003;Kim et al 2003;Shukla et al 2007;Yin et al 2008;Wang et al 2010;Suzuki et al 2012). 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).…”

Combinatorial Therapy Using Dovitinib and ICI182.780 (Fulvestrant) Blocks Tumoral Activity of Endometrial Cancer Cells

“…FGFs compromise a large group of heparin-binding growth factors that include 22 ligands that exert their function through 4 high-affinity tyrosine kinase receptors (FGFR1, FGFR2, FGFR3, and FGFR4; ref. 14). Binding of FGFs to the extracellular domains of FGFRs results in dimerization and conformational shift in receptor structure, leading to activation of the intracellular kinase domain.…”

The molecular and cellular basis of Apert syndrome

“…Lomri et al analyzed differentiation of calvarial cells by histological analysis, which revealed an increased extent of subperiosteal bone formation and alkaline phosphatase-positive preosteoblastic cells in Apert (S252W) fetal calvaria compared with age-matched controls in vivo, and both the expression of alkaline phosphatase and type 1 collagen, and production of the mineralized matrix of Apert cells were higher than controls in vitro (27 P253R Apert mouse models, phosphorylation of ERK was increased less than 1.5-fold in the neurocranium compared to WT controls (39). In another study activation of ERK1/2 was higher in primary calvarial osteoblasts from the FGFR2 S252W mouse than normal osteoblasts (8). The FGFR2 E731K mutation which was found in a Korean AS patient enhanced phosphorylation and activation of ERK1/2 (26).…”

“…A study by Miraoui et al also demonstrated that in mesenchymal C3H10T1/2 cells and calvarial preosteoblast MC3T3-E1 cells stably transfected with WT and S252W mutated FGFR2 respectively, both cell types expressing MT FGFR2 presented enhanced osteodifferentitation ability by analysis of mineralization, ALP activity and expression of the osteoblast differentiation markers compared with those expressing WT FGFR2 (38). A recent study stated primary calvarial osteoblasts derived from FGFR2IIIc S252W transgenic mice showed enhanced mineralization, higher ALP activity and greater expression of the differentiation markers than cells from WT mice (8).…”

“…The disturbance of the FGFs/FGFR2 signal, which changes the balance of proliferation, differentiation and apoptosis of osteoblasts, is ascribed to the development of AS (7). Signal pathways which play a vital role in bone formation are also involved in this process (8). In this review, we summarize recent…”