BMP-2 stimulates the expression of three osteogenic master transcription factors: Runx2, D1x5, and Osterix (Osx). However, the hierarchical regulatory relationships among them are not yet clearly understood. Osx was commonly stimulated in osteogenic and non-osteogenic cells in response to BMP-signaling, as D1x5 was in our previous report. A cycloheximide experiment indicated that Osx expression by BMP-2 requires new protein synthesis. Even if Osx has been suggested as a downstream target of Runx2, the results of this study indicated that Osx expression was still induced by BMP-2 treatment in Runx2 null cells, but not induced by Runx2 overexpression in myogenic C2C12 cells. Instead, Osx expression by BMP-2 was completely abrogated by the antisense blocking of D1x5. Depending upon the coincident expression pattern of Osx and D1x5, and the blocking of Osx expression by the antisense D1x5, BMP-2-induced Osx expression is mainly mediated not by Runx2, but by D1x5. (C) 2003 Elsevier Inc. All rights reserved.