Abstract
Dentin sialophosphoprotein (DSPP), a typical dentin-specific protein, is mainly expressed in the dentin extracellular matrix and plays a role in dentin mineralization. BMP-2 provides a strong signal for differentiation and mineralization of odontoblasts and osteoblasts. Previously, BMP-2 treatment is reported to stimulate Dspp expression in the MD10-F2 pre-odontoblast cells through activation of the heterotrimeric transcription factor Y (NF-Y). The canonical BMP signaling pathway is known to contribute greatly to biomineralization, however, it is not known whether it is involved in Dspp expression. Here, we investigated this question. Activation of the canonical BMP-2 signaling pathway in MDPC-23, preodontoblast cell, by overexpression of constitutively active Smad1/5 or downstream transcription factors Dlx5 and Runx2 stimulated Dspp expression. Conversely, knockdown of each element with siRNA significantly blocked the BMP-2-induced Dspp expression. To test whether these transcription factors downstream of BMP-2 are directly involved in regulating Dspp, we analyzed the mouse Dspp promoter. There are 5 well conserved homeodomain binding elements, H1 to H5, in Dspp proximal promoter regions (-791 to +54). A serial deletion of H1 and H2 greatly changed basal promoter activity and responsiveness to Dlx5 or Msx2. However, further deletions did not change the responsiveness to Dlx5 or Msx2. H1 and H2 sites can be suggested as specific response elements of Dlx5 and Msx2, respectively, based on their promoter activity modulation. Thus, the canonical BMP-2 signaling pathway plays a crucial part in the regulation of Dspp expression through the action of Smads, Dlx5, Runx2, and Msx2.
Highlights
Health and Welfare Project numbers A090913 and A010252. 1 To whom correspondence should be addressed: 28 Yeongeon-dong, mutations of the Dentin sialophosphoprotein (DSPP) gene have been identified in human families with dentinogenesis imperfecta II and III [5, 6], in whom dentin mineralization is defective
Dspp Expression Is Stimulated by the Canonical bone morphogenetic protein (BMP)-2 Signaling Pathway—Treatment of MDPC-23 pre-odontoblast cells with BMP-2 (100 ng/ml) stimulated Dspp expression (Fig. 1A)
DSPP is a marker of odontoblast differentiation and is associated with dentin mineralization
Summary
Cell Culture—C2C12 mouse myogenic cells and MDPC-23 mouse pre-odontoblast cells were maintained in Dulbecco’s modified Eagle’s medium (Hyclone, Logan, UT) supplemented with 10% fetal bovine serum (FBS). The Dspp promoter deletion constructs, D-610 (Ϫ610 to ϩ54 bp), D-426 (Ϫ426 to ϩ54 bp), D-249 (Ϫ249 to ϩ54 bp), D-216 (Ϫ216 to ϩ54 bp), D-94 (Ϫ94 to ϩ54 bp), and D-15 (Ϫ15 to ϩ54 bp) were subcloned into the pGL3-enhancer vector (Promega, Madison, WI) for the luciferase reporter assay. Site-directed Mutagenesis of Homeodomain Binding Sites— To produce constructs bearing mutations in the putative homeodomain binding sites, a site-directed mutagenic PCR was performed with the Ϫ433 Mut and Ϫ415 Mut primers and universal RVprimer (RV3) and GLprimer (GL2) listed in Table 1 establishing mutant promoters M-433 and M-415 (see Table 1 for the primer sequence). The lowercase letters designate the substitution of nucleotide for site-directed mutagenesis
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