Figure 3.

Runx2 activation and functional interaction with TCF-4 in osteoblasts. In panels A–D and F, fetal rat osteoblasts were transfected for 24 h with GAL4 DBD fusion plasmid devoid of Runx2 (M1), or fused to full-length Runx2 (M1-Runx2 or M1Rx2), or fragments of Runx2 encompassing amino-terminal amino acids 1-92 (M1ad); amino acids 93-228 encompassing the runt domain (M1rd); or carboxyl terminal amino acids 229-513 (M1cd) at 50 ng/2 cm², in combination with reporter plasmid driven by five GAL4 RE (5XGAL4) at 150 ng/2 cm²; or in panel E with TOP-Flash reporter at 150 ng/2 cm². In panel B the cells were cotransfected to express complementary amounts of empty vector (0) or Wnt1 at the concentrations indicated. In panel C and the middle and right panels of F the cells were cotransfected to express empty vector (MVN) encoding VP16 gene transactivation domain, or MVN fused to TCF-4 lacking a β-catenin-binding domain (MVN-TCFt) at 50 ng/2 cm². In panels D and E the cells were cotransfected to express empty vector (0) or TCFt lacking the MVN transactivation domain (pcTCFt) at 75 ng/2 cm². The cells were then treated with vehicle (0) or 1 μm PGE₂ alone or in combination with 20 mm LiCl, 10 μm WAg, or both agents (Li+W) as indicated in panels A, B, E, and F. Rat kidney fibroblasts were also used in panel E, or COS-7 monkey kidney fibroblasts were also used in panel F, as indicated. Reporter activity was measured after 24 h of treatment. WAg alone or Li+W significantly enhanced Runx2-dependent gene activation in panel A; PGE₂ significantly enhanced Runx2-dependent gene activation alone in panel B, designated by asterisk, or in combination with Li+W in the left and middle panels of F, designated by two stacked asterisks; coexpression of MVN-TCFt significantly enhanced Runx2-dependent gene expression in panel C, designated by asterisk, and further increased the stimulatory effects of Li+W without and with PGE₂ in the middle and right panels of F, designated by two stacked asterisks; pcTFCt had no effect on Runx2-dependent gene expression in panel D and significantly enhanced TOP-Flash activity in LiCl- or WAg-activated osteoblasts and significantly suppressed TOP-Flash activity in rat fibroblasts in E, designated by two stacked asterisks; in contrast to osteoblasts, MVN-TCFt significantly suppressed Runx2-dependent gene expression in Li+W-treated COS-7 fibroblasts in the right panel of F, designated by two stacked asterisks (P < 0.05). Fibs, Fibroblasts; Obs, osteoblasts.