nuDMP1 regulates Dspp expression during dentinogenesis

Objectives: Both dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are highly expressed in the odontoblasts. We have previously shown that DMP1 regulates Dspp expression and that the transcript encoding the secretory DMP1 also codes for a nuclear isoform of DMP1 (referred to as “nuDMP1”), through translation initiation from an alternative internal in-frame start codon. The goal of this study was to determine the function of nuDMP1 in regulating Dspp expression.
Methods: Transgenic mice were generated to express the nuDMP1 under the control of a 3.6-kb type I collagen promoter (designated as “Col1a1-nuDMP1”). Quantitative real-time PCR, immunohistochemistry and immunofluorescent staining were performed to determine the expression of the Col1a1-nuDMP1 transgene and the endogenous Dspp gene. Promoter-luciferase assay was used to analyze the effects of nuDMP1 on a 5.7 kb Dspp regulatory sequence, containing the 2.4 kb promoter, exon 1 and 3.2 kb intron 1 of the Dspp gene.
Results: Immunohistochemical staining showed that DMP1 signals were extremely weak or absent in the odontoblasts and dentin matrix in the wild-type mice, whereas strong DMP1 signals were restricted within the odontoblasts, but not in the dentin matrix in the Col1a1-nuDMP1 transgenic mice. Immunofluorescent staining with an anti-HA antibody further confirmed the nuclear localization of the nuDMP1 in the primary dental mesenchymal cells. Quantitative real-time PCR showed that the transgenic expression of nuDMP1 upregulated Dspp expression in a dose-dependent manner. Consistently, the promoter-luciferase assay showed that the nuDMP1 stimulated the activity of the 5.7 kb Dspp regulatory sequence by about 2-fold, but had no effect on the 2.4 kb Dspp promoter only.
Conclusions: These results suggest that nuDMP1 regulates Dspp expression and that it might exert its regulatory effects via the intron 1 of the Dspp gene.