Nerve growth factor promotes osteogenic differentiation of MC3T3-E1 cells via BMP-2/Smads pathway

Abstract

BackgroundExogenous nerve growth factor (NGF) can induce osteogenic precursor cell differentiation and promote fracture healing. However, the molecular mechanism by which NGF induces osteogenesis is not well understood. BMP-2 has good osteogenic efficacy and is one of the most osteogenic-inducing growth factors known. Therefore, this study aimed to determine whether NGF induces osteogenic differentiation of mouse embryonic osteogenic precursor cell line MC3T3-E1 by BMP-2 and search further mechanisms of NGF on BMP-2. MethodsMC3T3-E1 cells were treated with NGF at a concentration gradient for indicated times, after which the cell viability was measured by CCK-8 kit. Osteogenic differentiation was detected with quantification of alkaline phosphatase (ALP) activity also visualized with ALP staining. The transcription and expression of relevant genes were detected by qPCR and western blotting, respectively. NGF’s effect on BMP2 was studied with qPCR and luciferase reporter assay. The phosphorylation of Smads was probed with specific antibodies by western blotting, and the location of Smads was observed through immunofluorescence. ResultsWe found that NGF promoted proliferation and osteogenic differentiation of MC3T3-E1, increased the expression level of BMP-2, as well as the phosphorylation and nuclear translocation of Smad1/5/8. However, neutralization of BMP-2 with si-BMP-2 or BMP-2 signal inhibitors reversed NGF induced phosphorylation and nuclear translocation of Smad1/5/8, as well as the expression of Runx2, type I collagen, osteocalcin and osteopontin. In addition, si-BMP-2 abrogated NGF-induced ALP activity. ConclusionNGF induced osteogenic differentiation of MC3T3-E1 cells through BMP-2/Smads pathway and induction of Runx2. Our study would provide a theoretical basis for clinical treatment of fractures using NGF.