Bone Morphogenetic Protein 2 Mediates Dentin Sialophosphoprotein Expression and Odontoblast Differentiation via NF-Y Signaling

Lei Chen,Yimin Wu,Hui-Hsiu Chuang,Isabel Gay,Marcos Martinez,Shuo Chen,Mary MacDougall,Tong Li,Jelica Gluhak-Heinrich,Juan Dong

doi:10.1074/jbc.m709492200

Abstract

Dentin sialophosphoprotein (DSPP), an important odontoblast differentiation marker, is necessary for tooth development and mineralization. Bone morphogenetic protein 2 (BMP2) plays a vital role in odontoblast function via diverse signal transduction systems. We hypothesize that BMP2 regulates DSPP gene transcription and thus odontoblast differentiation. Here we report that expression of BMP2 and DSPP is detected during mouse odontogenesis by in situ hybridization assay, and BMP2 up-regulates DSPP mRNA and protein expression as well as DSPP-luciferase promoter activity in mouse preodontoblasts. By sequentially deleting fragments of the mouse DSPP promoter, we show that a BMP2-response element is located between nucleotides -97 and -72. By using antibody and oligonucleotide competition assays in electrophoretic mobility shift analysis and chromatin immunoprecipitation experiments, we show that the heterotrimeric transcription factor Y (NF-Y) complex physically interacts with the inverted CCAAT box within the BMP2-response element. BMP2 induces NF-Y accumulation into the nucleus increasing its recruitment to the mouse DSPP promoter in vivo. Furthermore, forced overexpression of NF-Y enhances promoter activity and increases endogenous DSPP protein levels. In contrast, mutations in the NF-Y-binding motif reduce BMP2-induced DSPP transcription. Moreover, inhibiting BMP2 signaling by Noggin, a BMP2 antagonist, results in significant inhibition of DSPP gene expression in preodontoblasts. Taken together, these results indicate that BMP2 mediates DSPP gene expression and odontoblast differentiation via NF-Y signaling during tooth development.

Highlights

Formation of dentin or dentinogenesis originates from the neural crest-derived mesenchymal cells and proceeds in a series of cytodifferentiation stages to form odontoblasts in a specific spatial and temporal pattern originating at the principal cusp tip and advancing toward the base of the tooth [1,2,3]
This study demonstrates for the first time that Bone morphogenetic protein 2 (BMP2) stimulated dentin sialophosphoprotein (DSPP) expression in mouse preodontoblasts and committed odontoblast differentiation through NF-Y signaling
DSPP has been characterized as a unique marker of odontoblast differentiation [4, 5]

Summary

EXPERIMENTAL PROCEDURES

Antibody supershift experiments were performed with specific antibodies to NF-YA, NF-YB, and NF-YC subunits (Santa Cruz Biotechnology Inc., Santa Cruz, CA) This antibody was added to the nuclear extracts 10 min prior to the addition of the radiolabeled probe. For quantitative detection of expression of DSPP and NF-Y subunit proteins in MD10-F2 cells, fluorescent immunohistochemistry was performed using antibodies directed against mouse DSP (Alpha Diagnostic International, San Antonio, TX). For the detection of mouse DSP protein, Western blotting assay was performed as described earlier [5]. Immunoprecipitations were performed with protein G beads and 5 ␮g of the indicated antibodies as follows: anti-NF-YB goat polyclonal and anti-TFIIB as a positive control (Santa Cruz Biotechnology Inc.). Analysis of variance with Student’s t test was used to determine significant differences in the control and treated groups

RESULTS

In situ hybridization studies showed

DISCUSSION

These observations suggest that

The A and C subunits associate through a subdomain that binds