BMP-2-induced Runx2 expression is mediated by Dlx5, and TGF-β1 opposes the BMP-2-induced osteoblast differentiation by suppression of Dlx5 expression

Intramuscular injection of BMP-2 induces ectopic bone formation in vivo. Similarly, BMP-2 treatment blocks myogenic differentiation and induces osteoblastic transdifferentiation of premyoblastic C2C12 cells. Previous reports suggested that BMP-2-stimulated Runx2 expression could play a pivotal role in transdifferentiation. However, increased Runx2 expression by TGF-beta1 did not support osteoblast differentiation in vitro. These results indicate that the induction of Runx2 is not sufficient to explain the BMP-induced transdifferentiation. We found that Dlx5 is specifically expressed in osteogenic cells, and is specifically induced by BMP-2 or -4 signaling but not by other osteotrophic signals or other TGF-beta superfamily members. Cycloheximide treatment indicated that Dlx5 was immediately induced by BMP signaling, while Runx2 required de novo protein synthesis. In addition, blocking or overexpressing each transcription factor indicated that Dlx5 is an indispensable mediator of BMP-2-induced Runx2 expression but is not involved in TGF-beta1-induced Runx2 expression. Moreover, TGF-beta1 opposed BMP-2-induced osteogenic transdifferentiation through Dlx5 suppression by de novo induction of AP-1. Taken together, these results indicate that Dlx5 is an indispensable regulator of BMP-2-induced osteoblast differentiation as well as the counteraction point of the opposing TGF-beta1 action.