The Role of DSPP in Dentine Formation and Hereditary Dentine Defects.

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.

Two of the proteins found in significant quantity in the extracellular matrix (ECM) of dentin are dentin phosphoprotein (DPP) and dentin sialoprotein (DSP). DPP, the most abundant of the noncollagenous proteins (NCPs) in dentin is an unusually polyanionic protein, containing a large number of aspartic acids (Asp) and phosphoserines (Pse) in the repeating sequences of (Asp–Pse)n. and (Asp–Pse–Pse)n. The many negatively charged regions of DPP are thought to promote mineralization by binding calcium and presenting it to collagen fibers at the mineralization front during the formation of dentin. This purported role of DPP is supported by a sizeable pool of in vitro mineralization data showing that DPP is an important initiator and modulator for the formation and growth of hydroxyapatite (HA) crystals. Quite differently, DSP is a glycoprotein, with little or no phosphate. DPP and DSP are the cleavage products of dentin sialophosphoprotein (DSPP). Human and mouse genetic studies have demonstrated that mutations in, or knockout of, the Dspp gene result in mineralization defects in dentin and/or bone. The discoveries in the past 40 years with regard to DPP, DSP, and DSPP have greatly enhanced our understanding of biomineralization and set a new stage for future studies. In this review, we summarize the important and new developments made in the past four decades regarding the structure and regulation of the Dspp gene, the biochemical characteristics of DSPP, DPP, and DSP as well as the cell/tissue localizations and functions of these molecules.

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.

The dentin sialophosphoprotein (DSPP) gene (4q21.3) encodes two major noncollagenous dentin matrix proteins: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Defects in the human gene encoding DSPP cause inherited dentin defects, and these defects can be associated with bilateral progressive high-frequency sensorineural hearing loss. Clinically, five different patterns of inherited dentin defects are distinguished and are classified as dentinogenesis imperfecta (DGI) types I, II, and III, and dentin dysplasia types I and II. The genetic basis for this clinical heterogeneity is unknown. Among the 11 members recruited from the studied kindred, five were affected with autosomal dominant DGI type II. The mutation (g.1188C-->G, IVS2-3C-->G) lay in the third from the last nucleotide of intron 2 and changed its sequence from CAG to GAG. The mutation was correlated with the affection status and was absent in 104 unaffected individuals (208 alleles) with the same ethnic and geological background. The proband was in the primary dentition stage and presented with multiple pulp exposures. The occlusal surface of his dental enamel was generally abraded, and the dentin was heavily worn and uniformly shaded brown. The dental pulp chambers appeared originally to be within normal limits without any sign of obliteration, but over time (by age 4), the pulp chambers became partially or completely obliterated. The oldest affected member (age 59) showed mild hearing loss at high-frequency (8 kHz). Permanent dentition was severely affected in the adults, who had advanced dental attrition, premature loss of teeth, and extensive dental reconstruction.

The revolution in genetics disclosed the types of malformations that occur when expression of a particular gene is lost. In the case of tooth dentin, mutations in the two genes encoding type I collagen cause osteogenesis imperfecta, a bone condition that often includes dentin malformations. Besides collagen, there are a number of non-collagenous proteins in dentin. Among the genes encoding the dentin non-collagenous proteins, only mutations in DSPP (dentin sialophosphoprotein) cause inherited dental malformations. DSPP mutations cause dentinogenesis imperfecta types II and III, and dentin dysplasia type II. DSPP is the most abundant non-collagenous protein in dentin. DSPP protein is necessary for proper dentin formation, and understanding its structure and function should yield important insights into how dentin forms and biomineralization is controlled. DSPP is expressed and secreted by odontoblasts, the cells that make tooth dentin and that also maintain cell processes extending into the mineralized tissue. Following its secretion, DSPP is cleaved into smaller pieces by multiple extracellular proteases. For the last five years I have devoted myself to characterizing DSPP-derived proteins. DSPP is cleaved by proteases into three main parts : dentin sialoprotein (DSP), dentin glycoprotein (DGP), and dentin phosphoprotein (DPP). We have learned that DSP is a proteoglycan that forms covalent dimers, DGP is a phosphorylated glycoprotein, and DPP is a highly phosphorylated intrinsically disordered protein that shows extensive length polymorphisms due to the genetic heterogeneity of its coding region.

Dentin Sialoprotein (DSP) and phosphophoryn (PP) are two most dominant non-collagenous proteins in dentin, which are the cleavage products of the DSPP (dentin sialophosphoprotein) precursor protein. The absence of the DSPP gene in DSPP knock-out (KO) mice results in characteristics that are consistent with dentinogenesis imperfecta type III in humans. Symptoms include thin dentin, bigger pulp chamber with frequent pulp exposure as well as abnormal epithelial-mesenchymal interactions, and the appearance of chondrocyte-like cells in dental pulp. To better understand how DSPP influences tooth development and dentin formation, we used a bacterial artificial chromosome transgene construct (BAC-DSPP) that contained the complete DSPP gene and promoter to generate BAC-DSPP transgenic mice directly in a mouse DSPP KO background. Two BAC-DSPP transgenic mouse strains were generated and characterized. DSPP mRNA expression in BAC-DSPP Strain A incisors was similar to that from wild-type (wt) mice. DSPP mRNA expression in BAC-DSPP Strain B animals was only 10% that of wt mice. PP protein content in Strain A incisors was 25% of that found in wt mice, which was sufficient to completely rescue the DSPP KO defect in mineral density, since microCT dentin mineral density analysis in 21-day postnatal animal molars showed essentially identical mineral density in both strain A and wt mice. Strain B mouse incisors, with 5% PP expression, only partially rescued the DSPP KO defect in mineral density, as microCT scans of 21-day postnatal animal molars indicated a reduced dentin mineral density compared to wt mice, though the mineral density was still increased over that of DSPP KO. Furthermore, our findings showed that DSPP dosage in Strain A was sufficient to rescue the DSPP KO defect in terms of epithelial-mesenchymal interactions, odontoblast lineage maintenance, along with normal dentin thickness and normal mineral density while DSPP gene dosage in Strain B only partially rescued the aforementioned DSPP KO defect.

Dentinogenesis imperfecta (DGI) type II (OMIM # 125490) is an inherited disorder affecting dentin. Defective dentin formation results in discolored teeth that are prone to attrition and fracture. To date, several mutations have been described in the dentin sialophosphoprotein (DSPP) gene, causing DGI types II and III and dentin dysplasia type II. DSPP encodes two proteins: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Here, we describe a mutational analysis of DSPP in seven Finnish families with DGI type II. We report two mutations and five single nucleotide polymorphisms. In one family we found a mutation that has been described earlier in families with different ethnicity, while in six families we found a novel g.1194C>A (IVS2-3) transversion. Bioinformatic analysis of known DSPP mutations suggests that DGI type II is usually caused by aberration of normal splicing.

Dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) are expressed as a single mRNA transcript. This transcript codes for a large precursor protein termed dentin sialophosphoprotein (DSPP). DSP, DPP, and DSPP have been considered to be tooth-specific. Recently, we found out that the dspp gene was expressed in osteoblasts and bone. With Western immunoblots, we detected DSP in the Gdm/EDTA extracts of rat long bone, at a level of about 1/400 of that in dentin. Using reverse transcription polymerase chain reaction (RT-PCR) techniques with primers specific to the 5'DSP portion (termed DSP, 1432 bp), 3'DPP sequence (DPP, 2135 bp), and the region covering portions of both the DSP and DPP (DSPP, 3471 bp), we detected DSPP mRNA in MC3T3-E1 cells, ROS 17/2.8 osteoblast-like cells, and mouse calvaria. The results from PCR show that this gene is expressed at a much lower level in osteoblasts than in odontoblasts. The data indicate that DSPP is not a tooth-specific protein and that dramatically different regulatory mechanisms governing DSPP expression are involved in tooth and bone.

Dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) are expressed as a single mRNA transcript. This transcript codes for a large precursor protein termed dentin sialophosphoprotein (DSPP). DSP, DPP, and DSPP have been considered to be tooth-specific. Recently, we found out that the dspp gene was expressed in osteoblasts and bone. With Western immunoblots, we detected DSP in the Gdm/EDTA extracts of rat long bone, at a level of about 1/400 of that in dentin. Using reverse transcription polymerase chain reaction (RT-PCR) techniques with primers specific to the 5'DSP portion (termed DSP, 1432 bp), 3'DPP sequence (DPP, 2135 bp), and the region covering portions of both the DSP and DPP (DSPP, 3471 bp), we detected DSPP mRNA in MC3T3-E1 cells, ROS 17/2.8 osteoblast-like cells, and mouse calvaria. The results from PCR show that this gene is expressed at a much lower level in osteoblasts than in odontoblasts. The data indicate that DSPP is not a tooth-specific protein and that dramatically different regulatory mechanisms governing DSPP expression are involved in tooth and bone.

N-terminal Dentin Sialoprotein fragment induces type I collagen production and upregulates dentinogenesis marker expression in osteoblasts

The current system for the classification of hereditary defects of tooth dentin is based upon clinical and radiographic findings and consists of two types of dentin dysplasia (DD) and three types of dentinogenesis imperfecta (DGI). However, whether DGI type III should be considered a distinct phenotype or a variation of DGI type II is debatable. In the 30 years since the classification system was first proposed, significant advances have been made regarding the genetic etiologies of inherited dentin defects. DGI type II is recognized as an autosomal dominant disorder with almost complete penetrance and a low frequency of de novo mutations. We have identified a mutation (c.52G-->T, p.V18F) at the first nucleotide of exon 3 of the DSPP (dentin sialophosphoprotein) gene in a Korean family (de novo) and a Caucasian family. This mutation has previously been reported as causing DGI type II in a Chinese family. These findings suggest that this mutation site represents a mutational "hot spot" in the DSPP gene. The clinical and radiographic features of these two families include the classic phenotypes associated with both DGI type II and type III. Finding that a single mutation causes both phenotypic patterns strongly supports the conclusion that DGI type II and DGI type III are not separate diseases but rather the phenotypic variation of a single disease. We propose a modification of the current classification system such that the designation "hereditary opalescent dentin" or "DGI type II" should be used to describe both the DGI type II and type III phenotypes.

Our laboratory has reported that two major noncollagenous dentin proteins, dentin sialoprotein and dentin phosphoprotein, are specific cleavage products of a larger precursor protein termed dentin sialophosphoprotein (MacDougall, M., Simmons, D., Luan, X., Nydegger, J., Feng, J. Q., and Gu, T. T. (1997) J. Biol. Chem. 272:835-842). To confirm our single gene hypothesis and initiate in vitro promoter studies, we have characterized the structural organization of the mouse dentin sialophosphoprotein gene. This gene has a transcription unit of approximately 9.4 kilobase pairs and is organized into 5 exons and 4 introns. Exon 1 contains a noncoding 5' sequence, and exon 2 contains the transcriptional start site, signal peptide, and first two amino acids of the NH2 terminus. Exons 3 and 4 contain coding information for 29 and 314 amino acids, respectively. The remainder of the coding information and the untranslated 3' region are contained in exon 5. Chromosomal mapping localized the gene to mouse chromosome 5q21 in close proximity to other dentin/bone matrix genes. Computer analysis of the promoter proximal 1.6-kilobase pair sequence revealed a number of potentially important cis-regulatory sequences; these include the recognition elements of AP-1, AP-2, Msx-1, serum response elements, SP-1, and TCF-1. In vitro studies showed that the DSPP promoter is active in an odontoblast cell line, MO6-G3, with basal activity mapped to -95 bp. Two potential enhancer and suppresser elements were identified in the regions between -1447 and -791 bp and -791 and -95 bp, respectively. The structural organization of the dentin sialophosphoprotein gene confirms our finding that both dentin sialoprotein and dentin phosphoprotein are encoded by a single gene with a continuous open reading frame.

Mutations in the dentin sialophosphoprotein (DSPP) gene cause dentinogenesis imperfecta. After synthesis, DSPP is proteolytically processed into NH2- and COOH-terminal fragments. The NH2-terminal fragment of DSPP is highly glycosylated but not phosphorylated, whereas the COOH-terminal fragment (named “dentin phosphoprotein” or “DPP”) is highly phosphorylated but not glycosylated. These two fragments are believed to perform distinct roles in dentin formation. To analyze the functions of DPP in dentinogenesis, we created “Dspp-/-;DPP Tg mice”, which expressed transgenic DPP driven by a Type I collagen promoter but lacked the endogenous Dspp gene. We characterized the dentin of the Dspp-/-;DPP Tg mice using X-ray radiography, histology, scanning electron microscopy, double fluorochrome labeling, immunohistochemistry and in situ hybridization. Micro-computed tomography analyses revealed that at postnatal 6 months, the transgenic expression of DPP increased the dentin thickness of the Dspp-null mice by 97.1% and restored the dentin material density by 29.5%. Histological analyses showed that the Dspp-null mice manifested an abnormal widening of the predentin while the predentin in Dspp-/-;DPP Tg mice was narrower than in the Dspp-null mice. Scanning electron microscopy analyses showed that the dentinal tubules in the Dspp-/-;DPP Tg mice were better organized than in the Dspp-null mice. The double fluorochrome labeling analyses demonstrated that the dentin mineral deposition rate in the Dspp-/-;DPP Tg mice was significantly improved compared to that in the Dspp-null mice. These findings indicate that the transgenic expression of DPP partially rescued the dentin defects of the DSPP-null mice, suggesting that DPP may promote dentin formation and that the coordinated actions between DPP and the NH2-terminal fragment of DSPP may be necessary for dentinogenesis.

Structural features, processing mechanism and gene splice variants of dentin sialophosphoprotein.

The dentin sialophosphoprotein (DSPP) gene encodes the most abundant non-collagenous protein in tooth dentin and DSPP protein is cleaved into several segments including the highly phosphorylated dentin phosphoprotein (DPP). Mutations in the DSPP gene have been solely related to non-syndromic form of hereditary dentin defects. We recruited three Korean families with dentinogenesis imperfecta (DGI) type II and sequenced the exons and exon-intron boundaries of the DSPP gene based on the candidate gene approach. Direct sequencing of PCR products and allele-specific cloning of the highly repetitive exon 5 revealed novel single base pair (bp) deletional mutations (c.2688delT and c.3560delG) introducing hydrophobic amino acids in the hydrophilic repeat domain of the DPP coding region. All affected members of the three families showed exceptionally rapid pulp chambers obliteration, even before tooth eruption. Individuals with the c.3560delG mutation showed only mild, yellowish tooth discoloration, in contrast to the affected individuals from two families with c.2688delT mutation. We believe that these results will help us to understand the molecular pathogenesis of DGI type II as well as the normal process of dentin biomineralization.