Figure 3.

YAP suppresses Runx2-mediated activation of the rat osteocalcin promoter. (A) The rOC −208 CAT reporter was transfected in HeLa cells along with the indicated Runx2 constructs (250 ng) and increasing concentrations (0 (first bar of each group), 250, 500 ng and 1 μg) of full-length YAP. Expression of YAP alone does not affect basal promoter activity. Runx2 activates osteocalcin promoter activity, which is suppressed in a dose-dependent manner by increasing concentrations of YAP. The Runx2 Y433A mutant, that does not interact with YAP, shows a 3–4-fold higher activity compared to wild-type Runx2. (B) YAP suppresses the activity of Runx2 in osteoblastic ROS 17/2.8 cells. In contrast to HeLa cells, wild-type YAP slightly enhances basal promoter activity (first group), and the Runx2 Y433A mutant moderately activates rOC −208 due to endogenous Runx2 in ROS 17/2.8 cells. The graphs represent at least three independent experiments (n=6 each); error bars=standard error of mean. (C) The rOC −208/CAT reporter was transfected with 250 ng each of Runx2 and YAP. Runx2 activates osteocalcin promoter activity, which is suppressed by YAP in osteoblastic MC3T3 cells, premyoblast C2C12 cells and fibroblast NIH3T3 cells. The Runx2 Y433A mutant which does not interact with YAP shows higher activity compared to wild-type Runx2 in C2C12 and NIH 3T3 cells. This effect, however, is not apparent in MC3T3 cells, probably because of higher levels of endogenous Runx2 protein. (D) Runx2–YAP complex regulates the activity of multiple Runx target promoters. ROS 17/2.8 cells were transfected with the indicated reporter and expression constructs. Cells were harvested 30 h after the transfection and subjected to luciferase reporter assay. The luciferase activity is expressed as fold activation (6 × OSE-Luc and TGFβRI-Luc promoter) or fold suppression (in case of Runx2-Luc and p21-Luc promoters). The open squares in promoter constructs represent functional Runx sites.