A Runx2-deficient cell line shows osteoblast differentiation induced by BMP-2

Objectives: It has been verified that Runx2 is an essential transcription factor for osteoblast differentiation. Although Runx2 plays crucial roles in differentiation of osteoblasts, it has been reported that BMP-2 induced ALP activity and osteocalcin synthesis in Runx2-deficient mouse calvarial cells, suggesting that BMP-2 exerts osteoblast differentiation by Runx2-independent pathway. Therefore, out study tried to investigate the Runx2-independent pathway. Methods: we established a clonal cell line, named RD-C2, from calvarial region of Runx2-deficient mouse embryos. Northern blot analysis confirmed no expression of Runx2 mRNA in the cell line with or without recombinant human BMP-2 (rhBMP-2) treatment. We induced RD-C2 cells differentiation using rhBMP-2 or transducing BMP-2 and Runx2 by adenovirus. Results: The cell line expressed undetectable levels of mRNA relating to osteoblast differentiation at basal condition, whereas rhBMP-2 treatment increased ALP activity about 2.3-fold compared with nontreated RD-C2 cells. Using real time-based RT-PCR, we demonstrate high expression of ALP and osteocalcin, which are markers of osteoblast differentiation. Furthermore, osteoblast differentiation-related transcription factors, including osterix and Msx2, were upregulated by rhBMP-2 treatment. These results suggested that RD-C2 cell differentiated towards osteoblast induced by BMP-2. To investigate whether exogenous overexpression of BMP-2 or Runx2 could rescue the differentiation of osteoblast in RD-C2 cells, we transduced the two genes by adenovirus and compared the effects with GFP transduction. Both BMP-2 and Runx2 transduction induced a greater number of ALP-positive cells than GFP transduction, suggesting both BMP-2 and Runx2 could induce osteoblast differentiation in RD-C2 cells. Real time based RT-PCR demonstrated both BMP-2 and Runx2 transduction induced high expression of osteoblast markers, such as ALP and osteocalcin. Conclusions: These results suggest that BMP-2 exerts similar effect with Runx2 in RD-C2 cell differentiation towards osteoblasts. We hope that RD-C2 cell will serve a useful tool in searching Runx2-independent pathway regulating osteoblast differentiation.