Background

Exogenous 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.

Methods

MC3T3-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.

Results

We 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.

Conclusion

NGF 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.

中文翻译：

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神经生长因子通过BMP-2/Smads通路促进MC3T3-E1细胞成骨分化

背景

外源性神经生长因子（NGF）可诱导成骨前体细胞分化，促进骨折愈合。然而，NGF诱导成骨的分子机制尚不清楚。BMP-2 具有良好的成骨功效，是已知的最具成骨诱导作用的生长因子之一。因此，本研究旨在确定NGF是否通过BMP-2诱导小鼠胚胎成骨前体细胞系MC3T3-E1的成骨分化，并进一步探索NGF对BMP-2的作用机制。

方法

MC3T3-E1 细胞在浓度梯度下用 NGF 处理指定时间，之后通过 CCK-8 试剂盒测量细胞活力。通过碱性磷酸酶 (ALP) 活性的定量检测成骨分化，也通过 ALP 染色可视化。分别通过qPCR和western blotting检测相关基因的转录和表达。用 qPCR 和荧光素酶报告基因分析研究了 NGF 对 BMP2 的影响。通过蛋白质印迹用特异性抗体探测Smads的磷酸化，并通过免疫荧光观察Smads的位置。

结果

我们发现NGF促进了MC3T3-E1的增殖和成骨分化，增加了BMP-2的表达水平，以及Smad1/5/8的磷酸化和核转位。然而，用 si-BMP-2 或 BMP-2 信号抑制剂中和 BMP-2 可逆转 NGF 诱导的 Smad1/5/8 磷酸化和核转位，以及 Runx2、I 型胶原蛋白、骨钙素和骨桥蛋白的表达。此外，si-BMP-2 消除了 NGF 诱导的 ALP 活性。

结论

NGF通过BMP-2/Smads通路和Runx2诱导诱导MC3T3-E1细胞成骨分化。本研究将为NGF临床治疗骨折提供理论依据。