PURPOSE: Activating mutations of Fibroblast growth factor receptor (FGFR) 2 (e.g. P253R, C278F) lead to premature fusion of cranial sutures such as Apert (P253R) and Crouzon (C278F) syndromes. The dura mater plays a critical role in the formation and maintenance of cranial sutures; however, its role in syndromic craniosynostosis remains unclear. We have previously reported that dura mediates suture patency by secreting soluble factors, inhibiting osteodifferentiation of mesenchymal stem cells (MSCs) in a co-culture system. This study examines the influence of FGFR2 mutations (P253R and C278F) in dural cells on undifferentiated MSCs in co-culture.
METHODS: Dural cells were harvested from N6 mice and grown to near confluence. Bone marrow-derived MSCs were prepared from 6 week old mouse femurs. Mutant (P253R and C278F) and wild-type FGFR2 constructs were subcloned into adenoviral vectors. Dural cells were infected with adenovirus and protein expression was confirmed by immunostaining. Infected dural cells were co-cultured with MSCs using a transwell system. MSCs co-cultured with empty transwell inserts served as a negative control. MSC RNA was extracted on days 7 and 14, and RNA expression of Runx2 (early marker of osteodifferentiation), osteopontin (OP), alkaline phosphatase (AP), and bone sialoprotein (BSP, a late marker) was determined by Northern blotting.
RESULTS: MSCs co-cultured with P253R- and C278F-FGFR2 expressing dural cells showed an increase in Runx2, AP, and OP expression by day 7, versus controls. By day 14, there was a further increase in AP, while Runx2 and OP levels decreased. BSP expression did not increase for all groups. Higher AP and Runx2 expression in MSCs co-cultured with P253R-FGFR2 dural cells than MSCs co-cultured with C278F-FGFR2 dural cells indicated increased osteodifferentiation.
CONCLUSION: P253R- and C278F-FGFR2 mutations in neonatal dural cells promote osteodifferentiation of MSCs in co-culture. P253R-FGFR2 expressing dural cells were more effective in stimulating osteodifferentiation, suggesting an increased maturation rate of preosteoblastic cells in Apert syndromic craniosynostosis.