Identification of novel Runx2 targets in osteoblasts:: Cell type-specific BMP-dependent regulation of Tram2

Runx2 is an osteoblast master transcription factor and a target for bone morphogenetic protein (BMP) signaling, but our knowledge of events downstream of Runx2 is limited. In this study, we used ChIP Display to discover seven novel genomic regions occupied by Runx2 in living MC3T3-E1 osteoblastic cells. Six of these regions are found within or up to 1 -kb away from annotated genes, but only two are found within 5'-gene flanking sequences. One of the newly identified Runx2 target genes is Tram2, whose product facilitates proper folding of type I collagen. We demonstrate that Tram2 mRNA is suppressed in non-osteoblasts when Runx2 is over-expressed, and that this suppression is alleviated upon treatment with BMP-2. Moreover, we show that BMP-induced Runx2 expression in the C3H1OT1/2, ST2, C2C12, and MC3T3-E1 cell lines coincides with an increase in Tram2 mRNA levels. Thus, Runx2 may regulate Tram2 expression in a BMP-dependent manner, and Tram2 may participate in the overall osteogenic function of Runx2. Among the other Runx2 target genes discovered in this study are Lnx2, an intracellular scaffolding protein that may play a role in Notch signaling, and Tnfrsf12a, a Tumor Necrosis Factor receptor family member that influences both osteoblast and osteoclast differentiation. Expanding our knowledge of Runx2 target genes, and manipulation of these genes, are warranted to better understand the regulation of osteoblast function and to provide opportunities for the development of new bone anabolics. J. Cell. Biochem. 102: 1458-1471, 2007. (c) 2007 Wiley-Liss, Inc.