Figure 7. Acetylation increases the transactivation activity of Osx and deacetylation of Osx impairs osteoblast differentiation.

A. HEK 293T cells were transfected with a hOc-Luc reporter, β-gal expression vector and together with or without Flag-Osx, HA-Osx^K307R-K312R and Flag-CBP-HA expression plasmids. B. HEK 293T cells were transfected with the hOc-Luc reporter, β-gal expression vector, and together with or without Flag-Osx, HA-Osx^K307R-K312R and Flag-HDAC4 expression plasmids. Relative luciferase activities of A and B were measured 36 h after transfection and normalized to the β-gal activity. C. MC3T3 E1 cells were transfected with Flag-Osx and Flag-CBP-HA expression plasmids alone or together. 36 h after transfection, ALP, BSP, Col1a1 and OC mRNAs were measured using real-time PCR. β-Actin was used as the internal control. D. MC3T3 cells were treated with rhBMP-2 (100 ng/ml) for 24 h followed by TSA (20 nM) for 24 h or not. ChIP assay were performed to examine the levels of Osx on the promoter of ALP, BSP, Col1a1, and OC. Data are presented as a percentage of input after subtracting control IgG values. E. C2C12 cells were transfected with HA-Osx(WT) or HA-Osx^K307R-K312R expression plasmids. 36 h after transfection, ALP, BSP, Col1a1, and OC mRNAs were measured using real-time PCR. β-Actin was used as the internal control. F. C2C12 cells were transiently transfected with empty RK2 vector, the HA-Osx(WT) plasmid or HA-Osx^K307R-K312R mutants. 24 h after transfection, the cells were incubated with BMP2 (100 ng/ml). ALP activity was examined by ALP staining 4–5 days later, or mineralization was assessed using Alizarin Red staining 10-12 days later. Representative images of three independent experiments are shown (left panel). Quantification of ALP activity and mineralization is shown in the right panel. Results are the mean ± S.D. of three independent experiments; *P <0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.