Fig. 1.

The BMP-6-Smad1 pathway directly induces Atoh8 expression in osteoblasts. a–e ST-2 stromal cells or MC3T3-E1 osteoblasts were stimulated with 100 ng·mL^–1 or the indicated concentrations of BMP-6 for 48 h and subjected to RT-qPCR for Id1, Atoh8, Rankl, and Opg. Values were normalized to Hprt1 (n = 3). f ST-2 cells were stimulated with 100 ng·mL^–1 BMP-6 with or without 1 μmol·L^–1 LDN193189 for 48 h and subjected to RT-qPCR for Atoh8. Values were normalized to Hprt1 (n = 3). g Structure of murine Atoh8, including Smad1/5-binding regions (SBRs), is illustrated. Blue boxes, untranslated regions; orange boxes, protein-coding regions. ChIP was performed using an anti-Smad1 antibody and lysate of mouse primary osteoblasts (n = 1) (h) or MC3T3-E1 osteoblasts (n = 3) (i), and purified DNA fragments were subjected to qPCR with primer sets against the indicated regions. The Hprt1 gene served as a negative control. MC3T3-E1 (j) or ST-2 (k) cells were transfected with the indicated reporter constructs, stimulated with 100 ng·mL^–1 BMP-6, and subjected to a luciferase assay (n = 3). Data are shown as the mean ± SD. n.s., not significant; *P < 0.05; **P < 0.01; ***P < 0.001