Abstract: Objective To analysis the role of Notch signaling pathway in bone morphogentic protein 9 (BMP9) induced osteogenic diffierentiaion of C2C12 cells through MAPK signaling. Methods C2C12 cells were infected by recombinant adenovirus expressing BMP9,then the total protein level and phosphorylated form of p38,Erk1/2,JNK MAPKs were determined by RT-PCR and Western blot. After treatment C2C12 cells with DAPT,the early osterogenic marker,ALP activity were detected by quantitative and staining assay, later ostergenic marker calcium deposition was determined by Alizarin Red S staining. The expression of ostergenic marker genes Runx2 and OCN, BMPs target genes Id1,Id2,Id3 were analysed by RT-PCR and Western Blot.The total protein level and phosphorylated form of p38, Erk1/2 and JNK MAPKs were determined by Western blot. Results BMP9 increased Hey1 and Notch ligands and receptors mRNA level and the protein expression of Hey1 and phosphorylated form of p38,Erk1/2 and JNK MAPKs, not the total protein level. The Notch specific inhibitor DAPT dose-dependently decreased ALP activity and calcium deposition. Furthermore, DAPT inhibited the RNA expression of BMPs target genes Id1, Id2, Id3. Moreover, The BMP9-induced phosphorylated form of p38, Erk1/2 were reduced by treatment with DAPT. Conclusion Notch signaling may involve in BMP9 induced osteoblast commitment of C2C12 mesenchymal stem cells through MAPK signaling pathway.

Key words: Notch signaling pathway, bone morphogentic protein 9, MAPK signaling , ostegenic differentiation