FIGURE 7.

ASPP 049-induced preosteoblastic cell differentiation is mediated by Wnt/β-catenin signaling. A, ASPP 049 induces ALP expression. MC3T3-E1 cells were cultured in differentiation medium and treated with various concentrations of ASPP 049 or E2 for 21 days. The ALP expression was stained with Naphthol AS-MX phosphate. DMSO, dimethyl sulfoxide. B, ASPP 049 induces ALP activity. MC3T3-E1 cells were cultured in differentiation medium and treated with indicated concentrations of ASPP 049 or E2 for 7 days. Cells were harvested, and ALP activity was measured at 405 nm and normalized with total protein. C, DKK-1 blocks ASPP 049-induced ALP activity. MC3T3-E1 cells were cultured in differentiation medium and treated with 1 μm ASPP 049 or 0.1 μm E2 for 7 days with or without pretreatment with DKK-1 (0.2 μg/ml) for 3 h, as indicated. Cells were harvested, and ALP activity was measured at 405 nm and normalized with total protein. Data are expressed as fold change compared with the cells treated with vehicle control and are mean ± S.E. (n = 4). **, p < 0.01; ***, p < 0.001 compared with treatments (ANOVA). ##, p < 0.01; ###, p < 0.001 compared with the presence and absence of DKK-1 (Student's t test).