Enamel matrix derivative stimulates core binding factor alpha1/Runt-related transcription factor-2 expression via activation of Smad1 in C2C12 cells.

Background: Although enamel matrix derivative (EMD) can promote osteogenic differentiation of the pluripotent mesenchymal precursor cell line, C2C12, the molecular mechanism that underlies this phenomenon is unclear. The purpose of this study was to determine which molecules in EMD stimulate osteogenic differentiation.

Methods: C2C12 cells were cultured in 5% serum-containing medium to induce differentiation, either with or without the addition of EMD. The expression of core binding factor alpha1/runtrelated transcription factor-2 (Cbfa1/Runx2) was measured using Northern blot, Western blot, and/or real-time polymerase chain reaction (R-PCR) analysis. Phosphorylation of mothers against decapentaplegic homolog 1 (Smad1) and bone morphogenetic protein (BMP)-like molecules in EMD was determined by Western blot.

Results: EMD increased Cbfa1/Runx2 mRNA and protein expression substantially. EMD also induced phosphorylation of Smad1. Noggin inhibited the EMD-induced phosphorylation of Smad1 markedly, and also partially blocked EMD-induced Cbfa1/ Runx2 mRNA expression. In the Western blot analysis, single bands that corresponded to approximately 15 and approximately 17.5 kDa proteins were recognized in EMD by anti-BMP-2/4 and anti-BMP-7 antibodies, respectively.

Conclusions: Our study demonstrates that EMD stimulates Cbfa1/Runx2 expression and the phosphorylation of Smad1, and that both of these processes can be blocked by noggin. Therefore, the osteogenic activity of EMD may be mediated by BMPlike molecules in EMD.