Nerve growth factor potentiates bone morphogenetic protein-9-induced osteogenic differentiation in mouse embryonic fibroblasts

Abstract

Objective To investigate the effect of nerve growth factor (NGF) on osteogenesis induced by bone morphogenetic protein-9 (BMP-9) in mouse embryonic fibroblasts (MEFs). Methods MEFs were respectively transfected with adenovirus-mediated NGF (NGF group), BMP-9 (BMP-9 group) and NGF + BMP-9 (combined group) and green fluorescence protein (GFP) (control group). Cytochemical staining was used to test the activity of alkaline phosphatase (ALP) 3 d and 5 d after treatment. Level of osteopontin (OPN) mRNA was detected by RT-PCR 9 d after treatment. Level of OPN protein was assayed by Western blot and immunocytochemistry 9 d after treatment. Mineralization was detected by Alizarin red staining 14 d after treatment. Results ALP activity in MEFs was elevated in BMP-9 group rather than in NGF group, but a significant increase in ALP activity was noted in combined group. In control group, BMP-9 group, NGF group and combined group, level of OPN mRNA was 0.92±0.03, 1.28±0.04, 0.94±0.03 and 1.62±0.04 respectively (F=214.60, P<0.01); level of OPN protein was 0.60±0.05, 0.84±0.03, 0.53±0.05 and 1.27±0.05 respectively(F=162.5, P<0.01). In comparison, OPN mRNA and protein were significantly up-regulated in combined group than in BMP-9 group (t=10.569 and 11.778, P<0.05). In control group, BMP-9 group, NGF group and combined group, relative density of OPN protein was 3.63±0.17, 6.27±0.30, 3.86±0.18 and 10.16±0.18 respectively (F=602.6, P<0.01), with a significant higher level in combined group than in BMP-9 group (t=22.280, P<0.05). Level of mineralization was significantly higher in combined group than in BMP-9 or NGF group. Conclusion NGF can potentiate the osteogenesis induced by BMP-9 in MEFs. Key words: Nerve growth factor; Bone morphogenetic proteins; Osteogenesis; Mesenchymal stem cells