| Aim In this study,we investigated the possible effect of COX-2 on the BMP9-induced activation of PI3K/Akt signal in C2C12 cells.Methods The alkaline phosphatase activities assay,Western blot,RT-PCR and Alizarin Red S staining were employed in this study.The detailed methods were as following:1.Alkaline phosphatase activities assay and Alizarin Red S staining were employed to determine the effect of BMP9 on osteogenic differentiation in C2C12 cells.2.Western blot assay was introduced to evaluate the effect of BMP9 on the expression of osteogenic markers and phosphorylation of Akt1/2/3 in C2C12 cells.3.PI3K/Akt inhibitor LY294002 was used to determine the role of PI3K/Akt signal in the BMP9-induced osteogenic differentiation in C2C12 cells.4.Western blot and RT-PCR assay were introduced to detect the effect of BMP9 on the expression of COX-2 in C2C12 cells.5.COX-2 inhibitor NS-398 was used to determine the effect of COX-2on the BMP9-induced activation of PI3K/Akt signal in C2C12 cells.Results1.The ALP activity was increased by BMP9,as well as the protein level of OPN and OCN.The minerilization was also induced by BMP9 in C2C12 cells.2.The phosphorylation level of Akt1/2/3 was increased by BMP9 concentration dependently.The BMP9-induced ALP activity was decreased by the inhibitor of PI3K/Akt in C2C12 cells.3.The m RNA and protein level of COX-2 were both up-regulated by BMP9 in C2C12 cells.4.The BMP9-induced ALP activity was increased by exogenous COX-2,but it was decreased by the inhibitor of COX-2.5.The BMP9-induced phosphorylation level of Akt1/2/3 was increased by exogenous COX-2,but it was decreased by the inhibitor of COX-2.COX-2 or the inhibitor of COX-2 had no substantial effect on total level of Akt1/2.Conclusion The BMP9-induced osteogenic differentiation in C2C12 cells is partly mediated by the activation of PI3K/Akt signal pathway,which maybe regulated by the BMP9-induced expression of COX-2 in C2C12 cells. |
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