| Objective: To elucidate and validate the effect of RUNX3 on BMP9-induced osteogenic differentiation of mesenchymal stem cells.Method: MSCs were infected with Ad-BMP9 and then the gene expression of endogenous Runx3 was determined by RT-qPCR. The protein expression of endogenous RUNX3 was determined by Western Blot. Contruct Ad-RUNX3 and Ad-siRUNX3 and identify the function of them. MSCs were infected with Ad-RUNX3 and Ad-siRUNX3. The early osteogenic differentiation marker, alkaline phosphatase( ALP), was detected by staining and quantitative aassay. The late osteogenic marker, matrix mineralization, was detected by alizarin red s staining. The gene expression levels of Id1, Id2, Dlx5 and Runx2 were determined by RT-qPCR and the protein expression level of DLX5 and RUNX2 were determined by Western Blot. The phosphorylation levels of Smad1/5/8, p38 and ERK1/2 were determined by Western Blot.Results: BMP9 increased the endogenous gene and protein expression of RUNX3 in MSCs. Overexpression or/and knocking down of RUNX3 both can increase BMP9-induced early osteogenic differentiation marker, alkaline phosphatase(ALP), promote the number of cells in s phase and upregulate the gene expression of Id1 and Id2. Nevertheless, BMP9-induced late osteogenic marker matrix mineralization was enhanced by overexpression of RUNX3, whereas inhibited by knocking down of RUNX3. BMP9-induced expression levels of DLX5 and RUNX2 were increased by overexpression of RUNX3, and yet reduced by knocking down of RUNX3. Ad-RUNX3 increased BMP9-induced phosphorylation of Smad1/5/8 and Ad-siRUNX3 decreased BMP9-induced phosphorylation of Smad1/5/8. However, the expression of total Smad1/5/8 was not affected by RUNX3. The BMP9- induced phosphorylation of p38 and ERK1/2 was not affected by RUNX3.Conclusion: Our findings suggest that RUNX3 is an essential modulator in BMP9-induced osteoblast lineage differentiation of MSCs, which was mainly via BMPs/Smad signaling pathway. |
