FIGURE 5.

The PERK-eIF2α-ATF4 pathway is involved in osteoblast differentiation. A, Western blotting of primary osteoblasts infected with adenovirus expressing ATF4. The expression levels of OCN and BSP were restored by the introduction of ATF4 to Perk^−/− osteoblasts. mock is an empty vector. B, mineralization nodule formation in WT and Perk^−/− osteoblasts. WT and Perk^−/− primary osteoblasts were treated with BMP2 for 0 or 4 days, and cells were stained with alizarin red for visualizing mineralization. The delayed mineralized nodule formation in Perk^−/− osteoblasts was restored by the infection with an adenovirus expressing ATF4. The lower panel shows the quantitative analysis of alizarin red staining (mean ± S.D., n = 4, *, p < 0.05, **, p < 0.01; Student's t test). C, alkaline phosphatase activity in WT and Perk^−/− primary osteoblasts. Cells were stained with 5-bromo-4-chloro-3-indolyl phosphate for measuring ALP activities. Although ALP activities were reduced in Perk^−/− osteoblasts, they were restored by the introduction of ATF4 into Perk^−/− osteoblasts. The lower panel shows the quantitative analysis of ALP activities (mean ± S.D., n = 4, **, p < 0.01, ***, p < 0.001; Student's t test). D, proposed model for the induction of ATF4 target osteoblast markers mediated by the PERK-eIF2α-ATF4 pathway during osteoblast differentiation.