MFG‐E8 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells through GSK3β/β‐catenin signaling pathway

Abstract

AbstractFracture nonunion and bone defects are challenging for orthopedic surgeons. Milk fat globule‐epidermal growth factor 8 (MFG‐E8), a glycoprotein possibly secreted by macrophages in a fracture hematoma, participates in bone development. However, the role of MFG‐E8 in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unclear. We investigated the osteogenic effect of MFG‐E8 in vitro and in vivo. The CCK‐8 assay was used to assess the effect of recombinant human MFG‐E8 (rhMFG‐E8) on the viability of hBMSCs. Osteogenesis was investigated using RT‐PCR, Western blotting, and immunofluorescence. Alkaline phosphatase (ALP) and Alizarin red staining were used to evaluate ALP activity and mineralization, respectively. An enzyme‐linked immunosorbent assay was conducted to evaluate the secretory MFG‐E8 concentration. Knockdown and overexpression of MFG‐E8 in hBMSCs were established via siRNA and lentivirus vector transfection, respectively. Exogenous rhMFG‐E8 was used to verify the in vivo therapeutic effect in a tibia bone defect model based on radiographic analysis and histological evaluation. Endogenous and secretory MFG‐E8 levels increased significantly during the early osteogenic differentiation of hBMSCs. Knockdown of MFG‐E8 inhibited the osteogenic differentiation of hBMSCs. Overexpression of MFG‐E8 and rhMFG‐E8 protein increased the expression of osteogenesis‐related genes and proteins and enhanced calcium deposition. The active β‐catenin to total β‐catenin ratio and the p‐GSK3β protein level were increased by MFG‐E8. The MFG‐E8‐induced enhanced osteogenic differentiation of hBMSCs was partially attenuated by a GSK3β/β‐catenin signaling inhibitor. Recombinant MFG‐E8 accelerated bone healing in a rat tibial‐defect model. In conclusion, MFG‐E8 promotes the osteogenic differentiation of hBMSCs by regulating the GSK3β/β‐catenin signaling pathway and so, is a potential therapeutic target.