Figure 5.

Effect of the FGF9/ERK signaling pathway on the osteogenic differentiation of DPSCs in vitro. (A) Expression of ERK1/2 and p-ERK1/2 in treated monolayer DPSCs was detected by western blot analysis at the indicated time-points. Tubulin was used to demonstrate equal loading of all samples. FGF9 (20 ng/ml) significantly enhanced the phosphorylation of ERK1/2 in DPSCs compared with that in the CON and OI groups. (B) Protein levels of Runx2/COL1 12 h and 4 days after treatment in DPSCs were investigated by western blot analysis. (C) ALP staining at day 7 and alizarin red staining at day 21 of the FGF9/inhibitor-treated monolayer DPSCs. Western blot analysis for Runx2 and COL1, ALP and alizarin red staining revealed that the inhibition of the osteogenic capacity of DPSCs by FGF9 was reversed when ERK1/2 phosphorylation was inhibited. (D–G) mRNA expression levels of Runx2, COL1, OPN and OCN in treated DPSCs were investigated using reverse transcription quantitative polymerase chain reaction. The gene and protein expression levels of these four osteogenic transcription factors were downregulated by exogenous FGF9 (20 ng/ml) 12 h and 7, 14 and 21 days after treatment, respectively. U0126 (10 μM) reversed the inhibited expression of the four osteogenic marker genes. Data are expressed as the mean ± standard deviation of triplicate independent experiments. ^*P<0.05, ^**P<0.01. DPSCs, dental pulp stem cells; CON, Dulbecco’s modified Eagle’s medium; OI, osteogenic induction medium, FGF9, 20 ng/ml exogenous fibroblast growth factor 9; U, 10 μM U0126; ERK, extracellular signal-regulated kinase; p-ERK, phosphorylated ERK; Runx2, runt-related transcription factor 2; COL1, collagen type 1; OPN, osteopontin; OCN, osteocalcin; ALP, alanine phosphatase.