Dentin sialophosphoprotein (Dspp) as a differentiation marker of odontoblasts is regulated by BMP-2. However, the intimate mechanism is still unknown. Transcription factors Dlx3 and Osx are essential for odontoblasts differentiation. We hypothesized that BMP-2 regulation of Dspp transcription was mediated by Dlx3 and/or Osx in odontoblasts. In the present investigation, we found that BMP-2 stimulated expression and nuclear translocation of Dlx3 and Osx in odontoblasts both in vitro and in vivo. Osx was a downstream target of Dlx3 and both of them stimulated Dsp expression. Both Dlx3 and Osx were able to activate Dspp promoter from nucleotides (nt)-318 to +54 by transfections of luciferase reports containing different lengths of mouse Dspp promoters. The binding of Dlx3 and Osx with nt-318 to +54 of Dspp promoter was verified by chromatin immunoprecipitation in vivo. Two Dlx3 binding sites and one Osx binding site on Dspp promoter were found by EMSA. Furthermore, the exact biological function of these binding sites was confirmed by site-directed mutagenesis. At last, the protein-protein interaction between Dlx3 and Osx in odontoblasts was detected by co-immunoprecipitation. In conclusion, in this study we found a novel signaling pathway in which BMP-2 activates Dspp gene transcription via Dlx3/Osx pathway.
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