FIGURE 6.

Inhibition of BMP9-mediated osteogenic differentiation due to Hey1 deficiency can be partially rescued by Runx2. A, Hey1 regulates expression of the late osteogenic marker. C3H10-Hey1^KD and control lines were infected with AdBMP9 or AdGFP. Total RNA was collected at the indicated time points and subjected to qPCR analysis of osteocalcin (OC) expression. B, Runx2 and BMP9 act synergistically in the C3H10-Hey1^KD line to increase osteogenic differentiation. Subconfluent C3H10-Hey1^KD cells were infected with a low titer of AdBMP9 and increasing doses of AdRunx2. At indicated time points, cells were collected for colorimetric assays of ALP activity. C, Runx2 rescue on Hey1 knockdown was demonstrated in vivo by MicroCT imaging. D, Runx2 rescues the Hey1 knockdown phenotype. MicroCT imaging and volumetric data acquisition were carried out as described in Fig. 5B. E, histologic evidence of the Runx2 rescue effect on the Hey1^KD MSCs. MM, mineralized matrix; OC, osteocyte; OB, osteoblast; UD, undifferentiated MSCs; magnification, 200×. F, regulation of Runx2 expression by Hey1. Total RNA was isolated from C3H10-Hey1^KD and C3H10T1/2 control lines, and subjected to qPCR analysis for mouse Runx2 expression. See text for details.