Role of Notch Signaling in FGF2- Regulated Condylar Cartilage Proliferation

Notch1 is recognized as a modulator of chondrogenic/osteogenic differentiation

in progenitor cell populations in many tissues. We have previously localized Notch to the prechondroblastic and chondroblastic layers of the mandibular condylar cartilage (MCC) and shown that treatment of MCC explants with exogenous FGF2 upregulates Notch gene expression in a dose-dependent fashion (Capps et al., 2006). More recently, we have demonstrated that proliferation is reduced in MCC explants treated with an inhibitor of Notch signaling (So et al., 2007). Nakanishi and co-workers (2007) have suggested that FGF2 activates Notch signaling in mouse neural crest cells and that this activation is accompanied by increased proliferation and adoption of a chondrogenic phenotype. Objective: The goal of this study was to investigate whether Notch is downstream of FGF2 in the regulation of proliferation in MCC. Methods: To establish whether FGF2 affects proliferation, mandibular condyles with ramus bone harvested from E17 mice pups were placed in serum-free explant culture containing 0, 30, or 100 ng/mL of FGF2. Using the concentration of FGF2 with the stronger effect (30 ng/mL), explants were placed in serum-free medium containing 0 ng/mL FGF2 + DMSO vehicle, 30 ng/mL FGF2 + DMSO vehicle, or 30 ng/mL FGF2 + 50nM DAPT (inhibitor of Notch signaling) in 0.1% DMSO. After 3d, the MCC was dissected from the adjacent bone and the RNA extracted for real-time RT-PCR. Results: Explants incubated in either 30 or 100 ng/mL FGF2 showed a large increase in cyclin B expression, with the 30 ng/mL treatment the more potent. Incubation of explants in 30 ng/mL FGF2 plus DAPT reduced FGF2-stimulated cyclin B expression by 60%. Conclusion: These data suggest that Notch is a downstream regulator of FGF2-induced proliferation in the MCC. This study was supported by NIDCR grant DE015401 to RJH.