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Fig. 8. MyoD affects transcriptional activity of Runx2/Cbfa1. (A) The effect of Runx2/Cbfa1 on MyoD transcriptional activity was determined by using the 4Rtk-luc reporter construct transiently transfected into C3H10T1/2 cells. Luciferase activity in extracts of cultures incubated for 48 hours with or without BMP-7 (400 ng/ml) was normalized to ß-Gal activity to correct for transfection efficiency. The results are expressed relative to the activity in cultures transfected with empty vectors and incubated without BMP-7. Each transfection was done in triplicate. In the absence of BMP-7, Runx2/Cbfa1 has little effect on MyoD transactivation. However in the presence of BMP-7 the transactivation was decreased, consistent with the inhibition of myogenesis. (B) The effect of bHLH-factors on Runx2/Cbfa1 transcriptional activity was determined using the p6OSE-luc reporter construct as described in A. MyoD transfection increased the Runx2/Cbfa1 transactivation and the effect was markedly increased by incubating the transfected cultures with BMP-7. None of the other bHLH factors could synergize with Runx2/Cbfa1 to increase luciferase expression. (C) The effect of MyoD on transactivation of a reporter construct containing the -147/+13 fragment of the osteocalcin promoter.
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