The Macrophage-derived GPNMB Promotes MSC Osteoblast Differentiation By Activating STAT3 Signaling Pathway

Purpose:?1?In vitro,bone marrow-derived macrophages were polarized into M1 and M2 phenotype.and identified by flow cytometry and qPCR.To study the role of macrophages in osteogenic differentiation of mesenchymal stem cells,different polarization state of macrophages were cocultured with MSCs.?2?The diffenertial expression of GPNMB in M1 and M2 macrophages was investigated.The effect of GPNMB on osteogenic differentiation of mesenchymal stem cells and the molecular mechanism were examined by using Gpnmb mutant or overexpressed cells.?3?In vivo,a cranial bone defects mouse model was used to investigate the effect of macrophage derived GPNMB on the repair of bone defects.Methods:?1?Murine bone marrow-derived primary mononuclear macrophages were cultured in the medium contained 20ng/ml rmM-CSF.After 6 days,100ng/ml LPS,20ng/ml INF-?and20ng/ml IL-4 were added to induce M1 and M2 respectively.The expression of M1surface marker CD86 and M2 surface marker CD206 were identified by flow cytometry.The mRNA expression of M1 surface marker?iNOS,CD86?and M2 surface marker?Arg-1,CD206?were detected by qPCR.?2?M0,M1 and M2 macrophages were co-cultured with mesenchymal stem cells respectively in the osteogenic medium.At 14 days,,the mRNA expression level of osteogenic differentiation markers?ALP,Runx2,Collagen I,Osteocalcin?was examined by qPCR and calcium deposition was assessed with alizarin red staining at 21 days.?3?The level of GPNMB mRNA was detected by qPCR and the protein by ELISA.The M2macrophages with overexpressed or mutant GPNMB were co-cultured with MSCs to determine their effect on osteogenic differentiation.Different concentrations of recombinant GPNMB protein were added during the process of osteogenic induction of MSC to further verify its effect on osteogenesis of MSC.The effect of different concentration of GPNMB protein on STAT3 phosphorylation in MSC with either scrambled siRNA or STAT3 siRNA knockdown was explored by western-blot.?4?Gpnmb^-/-mices were used to establish the cranial bone defects model,and then Gpnmb^+/+and Gpnmb^-/-macrophages were transplanted.After 4 weeks,the bone defect healing in different treatment groups was evaluated by?-CT scan.HE staining was performed to evaluate the bone in growth.Results:?1?Macrophages isolated from the mouse bone marrow can be successfully induced into M1 by LPS/INF-?,and successfully induced into M2 by IL-4.M1 macrophages inhibited MSC osteogenic differentiation,while M2 macrophages promoted osteogenic differentiation of MSC.This effect is related to the high expression of GPNMB in M2macrophages.?2?Macrophages with overpression of GPNMB can promote the osteogenic differentiation of MSC,whereas GPNMB mutant macrophages can reverse this effect.?3?Exogenous GPNMB protein can promote osteogenic differentiation of MSC by activating phosphorylation of STAT3,which was confirmed by STAT3 knockdown.?4?In the mouse cranial bone defects model,the rate of bone healing of Gpnmb^+/+macrophages transplanted was significantly accelerated compared to Gpnmb^-/-counterpart.Conclusion:?1?In vitro,macrophages can be successfully polarized into M1,M2 phenotype.?2?M2 macrophages can promote the osteogenic differentiation of MSC,which is related to the high expression of GPNMB in M2 macrophages.This effect was abrogated in GPNMB mutant M2 macrophages.?3?Exogenous GPNMB protein can promote MSC osteoblast differentiation by activating STAT3 signaling pathway.?4?In the mouse cranial bone defects model,the bone healing ability of Gpnmb+/+macrophage transplantation was significantly greater than that of the Gpnmb-/-macrophage transplanted group.