The Effects Of Gli1 And Gli2 On BMP9-induced Osteogenic Differentiation Of Mesenchymal Stem Cells

Objective: To investigate the effect of Gli1 and Gli2 on BMP9-induced osteogenic differentiation of mesenchymal stem cells(MSCs).Methods: C3H10T1/2 cells were infected with adenovirus expression exogenous BMP9(Ad-BMP9),the endogenous expression of Gli1 and Gli2 was detected by quantitative PCR at 1,2,and 3 days.Subsequently,the effects of Gli1 and Gli2 inhibitors(GANT61)on BMP9-induced osteogenic differentiation of mesenchymal stem cells(MSCs)were detected by the stain of alkaline phosphatase(ALP).After that,C3H10T1/2cells,C2C12 cells and MEFs were treated with Ad-BMP9 or Ad-GFP in the presence of GANT61(4?M).Alkaline phosphatase(ALP),an early osteogenic differentiation markers,was measured by cytochemical staining and chemiluminescent quantitative assays.Calcium deposition were detected by Alizarin Red S staining.The gene expression of osteogenic Dlx5,Runx2,Osterix and Ocn were detected by RT-q PCR.The protein expression of RUNX2,OPN and OCN were detected by Western Blot.Finally,total amount and phosphorylated forms of Smad1/5/8,p38 andERK1/2 were analyzed by western blot.Results: BMP9 promote the expression of Gli1 and Gli2.Treatment of BMP9-induced mesenchymal stem cells with 4 ?M GANT61 inhibit BMP9-induced alkaline phosphatase activity,calcium deposition,and restrain the expression of related transcription factors Dlx5,Runx2,Osterix and Ocn in the mRNA Level,as well as suppress the protein expression of RUNX2,OPN and OCN.GANT61 had no effect on the phosphorylation of ERK1/2 induced by BMP9,but weaken BMP9-induced phosphorylation of Smad1/5/8 and p38.Conclusion: Gli1 and Gli2 can regulate BMP9-induced osteogenic differentiation of mesenchymal stem cell.