FIGURE 6.

Foxo1 regulates Runx2 promoter activity, binds to the promoter of Runx2, ALP, and Osteocalcin genes, and directly interacts with Runx2 protein. Shown are the schematic of potential Foxo1 binding sites in the Runx2 promoter, position of synthetic oligonucleotide probes, PCR primer location, and mutations in three Foxo1 binding sites (A). C3H10T1/2 cells were co-transfected for 48 h with a plasmid containing either Foxo1 or control plasmid, and Runx2 promoter luciferase plasmid (containing a 0.9- or 1.3-kb promoter fragment), in the absence of any osteogenic stimulant (B). Data are presented as a ratio of experimental/control luciferase. Values relative to control vector are shown. *, significantly different from control (p < 0.05). Activation of Foxo1 was measured by EMSA after C3H10T1/2 stimulation with BMP2 (100 ng/ml) for 24 h, using competitive DNA probes A, B, and C. C, unlabeled Foxo1 in excess was used as a competitive inhibitor. A probe with a nonspecific sequence was used as non-competitive probe. Runx2 promoter luciferase constructs carrying a four-base mutation in Foxo1 binding sites (M1, M2, and M3) were used to measure promoter activity in response to Foxo1 overexpression (D). Data are presented as a ratio of experimental/control luciferase. Values relative to control vector are shown. Data are expressed as means ± S.D. of three experiments. *, significantly different from wild-type (WT) construct (p < 0.05). Foxo1 protein binding to the Runx2, ALP, and osteocalcin promoter was evaluated using ChIP assay. C3H10T1/2 cells were treated with BMP2 (100 ng/ml) for 48 h. Chromatin was immunoprecipitated with either anti-Foxo1 antibody or normal rabbit IgG as control. Western analysis of immunoprecipitates was performed using anti-Foxo1 antibody (E). DNA from each immunoprecipitation reaction was examined by real-time PCR using specific primers flanking the putative Foxo1 binding site in the Runx2 (F) or ALP (G) or Osteocalcin (H) promoters. Results are presented as amplification levels in relation to IgG control precipitates. Data are expressed as means ± S.D. of three experiments. *, significantly different from IgG control (p < 0.05). Nuclear extracts of C3H10T1/2 cells that have been transfected with pcDNA3 FLAG Foxo1 and treated with BMP2 (100 ng/ml) were immunoprecipitated (IP) with an anti-FLAG, anti-Runx2, or with nonspecific IgG antibodies. Immunoprecipitated complexes were analyzed by Western blotting with anti-Runx2 (left panel) or anti-Foxo1 (right panel) antibodies (I). In similar experiments, MC3T3-E1 cells were treated with BMP2 (100 ng/ml) and nuclear extracts were immunoprecipitated with anti-Foxo1 or with nonspecific IgG antibodies. Immunoprecipitated complexes were analyzed by Western blotting with anti-Runx2 antibody (J). C3H10T1/2 cells were transfected for 48 h with a plasmid containing Foxo1, Runx2, or control plasmid. Some cells were co-transfected with a Foxo1 and Runx2 plasmid. Expression of osteocalcin was measured by semiquantitative RT-PCR (K). Values relative to control vector are shown. Data are expressed as means ± S.D. of three experiments. *, significantly different from Foxo1 or Runx2 alone (p < 0.05) (I).