Specific Effects Of PparΓ2Silencing On Bmp9Induced Osteogenic Differentiation Of C2C12Cells

AimIn this study, we investigate the specifc effects of PPARy silencing on BMP9induced osteogenic differentiation of C2C12cells.MethodsChemiluminescence, histo-chemical staining, PCR, Western blotting and reporter plasmid assay were employed to demonstrate the effects of PPARy silencing on BMP9induced osteogenic differentiation of C2C12cells.1. Chemiluminescence and histo-chemical staining were carried out to determine the ALP activity of C2C12treated with AdsimPPARy2and/or AdBMP9.2. Early osteogenic markers were detected on expression levels of gene and protein in C2C12cells treated with AdsimPPARy2and/or AdBMP9.3. PCR and Western Blotting were employed to determine the expression of a adipogenic factor C/EBPa.4. Alizarin Red S Staining was employed to detect the matrix mineralization of C2C12cells treated with AdsimPPARy2and/or AdBMP9and protein levels of OCN in late stage was determined with Western Blotting.5. Luciferase reporter assay and Western Blotting were used to detect the transcriptional activities of BMPR-Smad signaling pathway induced by AdsimPPARy2and/or AdBMP9.6. Western Blotting was used to check the effect on PTEN signaling pathway induced by AdsimPPARy2and/or AdBMP9.7. RT-PCR was employed to check the effect on IGF1signaling pathway, and compare the effect of IGF1and PPARy silencing on ALP activities with IGF1conditioned medium.Results1. We found that PPARy2silencing could effectively promote the early osteogenic marker ALP activity in C2C12cells induced by BMP9in early phase.2. AdsimPPARy2promoted early osteogenic markers induced by AdBMP9on gene and protein levels in C2C12cells.3. PPARy2silencing surppressed the expression of C/EBPa induced by BMP9.4. The matrix mineralization and OCN expression in late stage were diminished by combined treatment with AdsimPPARy2and AdBMP9compared with AdBMP9alone.5. The combination of AdsimPPARy2and AdBMP9promoted the transcription activity of BMPR-Smad Signaling.6. The combination of AdsimPPARy2and AdBMP9blocked the increase of PTEN induced by BMP9.7. AdsimPPARy2increased the expression level of IGF1mRNA in early stage, and enhanced the early osteogenic differentiation of C2C12cells induced by BMP9.ConclusionsPPARy2silencing can facilitate the osteogenic differentiation induced by BMP9in early stage, but it can restrain the osteogenesis in late phase. Incease of transcription activity of BMPR-Smad Signaling, surppression of PTEN sinaling and up-regulating of IGF1signaling may facilitate the early osteogenesis, the suppression in late phase may linked with attenuating activities of Smad.