The Role Of Notch In BMP2-induced Osteogenic Differentiation Of MSCs

Bone morphogenetic protein 2(BMP2) has been found to be the strongger mediator that can induce MSCs into osteoblasts, and has been appled in the treatment of diseases in clinical orthopaedics so far. But the clinical application effect inferior to early aboratoratory research, so the osteogenetic mechanism of BMP2 needs to research furtherly. Notch pathway also plays an important role in osteogenic differentiation in MSCs. Then the Notch signal can promote BMP2-induced osteogenesis? What’s the possible mechanism? The main objectives of this study lie in exploring the role of Notch in BMP2-induced osteogenesis of MSCs and its related mechanisms for offering solution to improve clinical therapeutic effect of BMP2.In the first part of this study lie in exploring the role of Notch in BMP2-induced osteogenesis in mesenchymal stem cells(MSCs). Methods:The specific γ-secretase inhibitors DAPT, the dominant negative extracellular domain of Notch1(dnNotch1) and over-express DLL1 adenoviruses were used to up-regulate and down-regulate Notch pathway. Cytochemical staining and qRT-PCR were used to evaluate early-stage index alkaline phosphatase(ALP), late-stage indicator calcium deposits and osteogenesis-related gene. Results : Compared with control group, osteogenic indicators ALP and calcium deposits were inhibited by DAPT in a dose-dependent manner in BMP2-induced osteogenic differentiation. dnNotch1 was inhibited early-stage index alkaline phosphatase(ALP),late-stage indicator calcium deposits and osteogenesis-related gene. But DLL1 obviously enchanced the early-stage indicator ALP, calcium deposits and osteogenesis-related gene(p<0.05). Conclusion:Notch remarkablely enhances BMP2-induced osteogenesis in MSCs.In the second part of this study lie in investigating the possible mechanisms of Notch in BMP2-induced osteogenesis in mesenchymal stem cells(MSCs). Methods:the specific γ-secretase inhibitors DAPT, the dominant negative extracellular domain of Notch1(dnNotch1) and over-express DLL1 adenoviruses were used to up-regulate and down-regulate Notch pathway.qRT-PCR, Western Blotting and luciferase reporter assay were conducted to detect the expression level of BMP receptors, p-Smad1/5/8 and Smad-binding element(SBE) activity. Analysis of its effects on cell cycle and apoptosis with Crystal violet staining and flow cytometry. Results:Compared with control group,ALK2,ALK3,and BMPRⅡin BMP signal pathway were significantly decreased by DAPT,but on change was seen in the level of ALK1,ALK6,ActRⅡ and ActRⅡ β. ALK2 was down-regulated by DAPT in a dose-dependent manner in BMP2-induced osteogenic differentiation. dnNotch1 treatment obviously deseased only ALK2 expression level. DLL1 elevated the expression level of ALK2,ALK3 and BMPRⅡ.There were also no obvious changes in the total protein level of Smad1/5/8, but those of phosphorylated Smad1/5/8 and SBE activity were decreased by dnNotch1. But DLL1 increased protein level of phosphorylated Smad1/5/8 and SBE activity. Crystal violet and flow cytometric presented that dn Notch1 can inhibit cell proliferation induced by BMP2,and the G2+S phase with BMP2 treatment were significantly deduced,but not apoptosis.Under DLL1 treatment, the cell proliferation and G2+S phase were higher than control group in BMP2-induced osteogenesis in oMEFs, but not apoptosis(p<0.05).Conclusion:Notch remarkablely enhances BMP2-induced osteogenesis in MSCs and it might exert the effect though the activation BMP-Smad pathway and cell proliferation.