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

FASEB J. 2023 Jun;37(6):e22950. doi: 10.1096/fj.202201417RRR.

Abstract

Fracture 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.

Keywords: MFG-E8; Wnt/beta-catenin; hBMSCs; osteogenesis; osteogenic differentiation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bone Marrow Cells / metabolism
  • Cell Differentiation / physiology
  • Cells, Cultured
  • Factor VIII / metabolism
  • Factor VIII / pharmacology
  • Glycogen Synthase Kinase 3 beta / genetics
  • Glycogen Synthase Kinase 3 beta / metabolism
  • Glycoproteins / metabolism
  • Humans
  • Mesenchymal Stem Cells* / metabolism
  • Osteogenesis* / physiology
  • Rats
  • Signal Transduction / physiology
  • Wnt Signaling Pathway
  • beta Catenin / genetics
  • beta Catenin / metabolism

Substances

  • milk fat globule
  • beta Catenin
  • Factor VIII
  • Glycogen Synthase Kinase 3 beta
  • Glycoproteins