Colocalization of dentin matrix protein 1 and dentin sialoprotein at late stages of rat molar development.

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are acidic proteins found in the extracellular matrices of bones and teeth. Recent data from gene knockouts, along with those of gene mutations, indicate that these two phosphoproteins are critical for bone and tooth development and/or maintenance. However, the precise functions of the two proteins have not been elucidated. In order to gain insights into their functions in tooth formation, we performed systematic, comparative investigations on the immunolocalization of DMP1 and dentin sialoprotein (DSP, a cleaved fragment of DSPP), using the rat first molar at different developmental stages as a model. Immunohistochemistry (IHC) was performed with specific, monoclonal antibodies against the COOH-terminal fragments of DMP1 and against DSP. In 1-day- and 1-week-old rats, weak immunoreactions for DMP1 were observed in dentinal tubules while stronger reactions for DSP were seen in the tubules and predentin. In rats older than 2 weeks, immunoreactions for DMP1 were found in dentinal tubules, predentin and odontoblasts. In 5-week- and 8-week-old rats, strong immunoreactions for DMP1 were widely distributed in odontoblasts and predentin. The distribution pattern of DSP was strikingly similar to that of DMP1 after 2 weeks and the localization of each was distinctly different from that of bone sialoprotein (BSP). The unique colocalization of DMP1 and DSPP in tooth development suggests that the two proteins play complementary and/or synergistic roles in formation and maintenance of healthy teeth.     

The Rescue of Dentin Matrix Protein 1 (DMP1)-deficient Tooth Defects by the Transgenic Expression of Dentin Sialophosphoprotein (DSPP) Indicates That DSPP Is a Downstream Effector Molecule of DMP1 in Dentinogenesis

Dentin matrix protein 1 (DMP1) and dentin sialophosphoprotein (DSPP) are essential for the formation of dentin. Previous in vitro studies have indicated that DMP1 might regulate the expression of DSPP during dentinogenesis. To examine whether DMP1 controls dentinogenesis through the regulation of DSPP in vivo, we cross-bred transgenic mice expressing normal DSPP driven by a 3.6-kb rat Col1a1 promoter with Dmp1 KO mice to generate mice expressing the DSPP transgene in the Dmp1 KO genetic background (referred to as “Dmp1 KO/DSPP Tg mice”). We used morphological, histological, and biochemical techniques to characterize the dentin and alveolar bone of Dmp1 KO/DSPP Tg mice compared with Dmp1 KO and wild-type mice. Our analyses showed that the expression of endogenous DSPP was remarkably reduced in the Dmp1 KO mice. Furthermore, the transgenic expression of DSPP rescued the tooth and alveolar bone defects of the Dmp1 KO mice. In addition, our in vitro analyses showed that DMP1 and its 57-kDa C-terminal fragment significantly up-regulated the Dspp promoter activities in a mesenchymal cell line. In contrast, the expression of DMP1 was not altered in the Dspp KO mice. These results provide strong evidence that DSPP is a downstream effector molecule that mediates the roles of DMP1 in dentinogenesis.     

Dentin sialoprotein and dentin phosphoprotein have distinct roles in dentin mineralization

Dentin sialophosphoprotein (DSPP), a major non-collagenous matrix protein of odontoblasts, is proteolytically cleaved into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Our previous studies revealed that DSPP null mice display a phenotype similar to human autosomal dominant dentinogenesis imperfecta, in which teeth have widened predentin and irregular dentin mineralization resulting in sporadic unmineralized areas in dentin and frequent pulp exposure. Earlier in vitro studies suggested that DPP, but not DSP, plays a significant role in initiation and maturation of dentin mineralization. However, the precise in vivo roles of DSP and DPP are far from clear. Here we report the generation of DPPcKO mice, in which only DSP is expressed in a DSPP null background, resulting in a conditional DPP knockout. DPPcKO teeth show a partial rescue of the DSPP null phenotype with the restored predentin width, an absence of irregular unmineralized areas in dentin, and less frequent pulp exposure. Micro-computed tomography (micro-CT) analysis of DPPcKO molars further confirmed this partial rescue with a significant recovery in the dentin volume, but not in the dentin mineral density. These results indicate distinct roles of DSP and DPP in dentin mineralization, with DSP regulating initiation of dentin mineralization, and DPP being involved in the maturation of mineralized dentin.     

Dentin sialophosphoprotein is processed by MMP-2 and MMP-20 in vitro and in vivo

Dentin sialophosphoprotein (DSPP) is a major secretory product of odontoblasts and is critical for proper tooth dentin formation. During dentinogenesis, DSPP is proteolytically cleaved into smaller subunits. These cleavages are proposed activation steps, and failure to make these cleavages is a potential cause of developmental tooth defects. We tested the hypothesis that dentin-resident matrix metalloproteinases catalyze the cleavages that process DSPP. We defined the exact DSPP cleavages that are catalyzed by proteases during crown formation by isolating DSPP-derived proteins from developing porcine molars and characterizing their N-terminal sequences and apparent size on SDS-PAGE and Western blots. The in vivo DSPP cleavage sites were on the N-terminal sides of Thr(200), Ser(330), Val(353), Leu(360), Ile(362), Ser(377), Ser(408), and Asp(458). The initial DSPP cleavage is between dentin glycoprotein (DGP) and dentin phosphoprotein (DPP), generating dentin sialoprotein (DSP)/DGP and DPP. Gelatin and casein zymograms identified MMP-2, MMP-20, and KLK4 in the dentin extracts. MMP-2 and MMP-20 were purified from over 150 g of porcine dentin powder and incubated with DSP-DGP and DPP. These enzymes show no activity in further cleaving DPP. MMP-20 cleaves DSP-DGP to generate DSP and DGP. MMP-20 also cleaves DSP at multiple sites, releasing N-terminal DSP cleavage products ranging in size from 25 to 38 kDa. MMP-2 makes multiple cleavages near the DSP C terminus, releasing larger forms of DGP, or "extended DGPs." Exact correspondence between DSPP cleavage sites that occur in vivo and those generated in vitro demonstrates that MMP-2 and MMP-20 process DSPP into smaller subunits in the dentin matrix during odontogenesis.     

Transgenic Expression of Dentin Phosphoprotein Inhibits Skeletal Development

Dentin sialophosphoprotein (DSPP) is proteolytically processed into an NH₂-terminal fragment called dentin sialoprotein (DSP) and a COOH-terminal fragment known as dentin phosphoprotein (DPP). These two fragments are believed to perform distinct roles in formation of bone and dentin. To investigate the functions of DPP in skeletal development, we generated transgenic mice to overexpress hemagglutinin (HA)-tagged DPP under the control of a 3.6 kb type I collagen (Col1a1) promoter (designated as Col1a1-HA-DPP). The Col1a1-HA-DPP transgenic mice were significantly smaller by weight, had smaller skeletons and shorter long bones than their wild type littermates, as demonstrated by X-ray radiography. They displayed reduced trabecular bone formation and narrower zones of proliferative and hypertrophic chondrocytes in the growth plates of the long bones. Histological analyses showed that the transgenic mice had reduced cell proliferation in the proliferating zone, but lacked obvious defects in the chondrocyte differentiation. In addition, the transgenic mice with a high level of transgene expression developed spontaneous long bone fractures. In conclusion, overexpressing DPP inhibited skeletal development, suggesting that the balanced actions between the NH₂- and COOH-terminal fragments of DSPP may be required for normal skeletal development.Key words: Dentin sialophosphoprotein, dentin phosphoprotein, development, bone, transgenic mice, growth plate     

Dentin sialophosphoprotein (DSPP) is cleaved into its two natural dentin matrix products by three isoforms of bone morphogenetic protein-1 (BMP1)

The protease that cleaves the most abundant non-collagenous protein of dentin matrix, dentin sialophosphoprotein (DSPP), into its two final dentin matrix products, dentin sialoprotein (DSP) and dentin phosphoprotein (DPP), has not been directly identified. In this study, full-length recombinant mouse DSPP was made for the first time in furin-deficient mammalian LoVo cells and used to test the ability of three different isoforms of one candidate protease, bone morphogenetic protein-1 (BMP1) to cleave DSPP at the appropriate site. Furthermore, two reported enhancers of BMP1/mTLD activity (procollagen C-endopeptidase enhancer-1, PCPE-1, and secreted frizzled-related protein-2, sFRP2) were tested for their abilities to modulate BMP1-mediated processing of both DSPP and another SIBLING family member with a similar cleavage motif, dentin matrix protein-1 (DMP1). Three splice variants of BMP1 (classic BMP1, the full-length mTolloid (mTLD), and the shorter isoform lacking the CUB3 domain, BMP1-5) were all shown to cleave the recombinant DSPP in vitro although mTLD was relatively inefficient at processing both DSPP and DMP1. Mutation of the MQGDD peptide motif to IEGDD completely eliminated the ability of all three recombinant isoforms to process full-length recombinant DSPP in vitro thereby verifying the single predicted cleavage site. Furthermore when human bone marrow stromal cells (which naturally express furin-activated BMP1) were transduced with the adenovirus-encoding either wild-type or mutant DSPP, they were observed to fully cleave wild-type DSPP but failed to process the mutant DSPP^MQΔIE during biogenesis. All three BMP1 isoforms were shown to process type I procollagen as well as DSPP and DMP1 much more efficiently in low-salt buffer (≤ 50 mM NaCl) compared to commonly used normal saline buffers (150 mM NaCl). Neither PCPE-1 nor sFRP2 were able to enhance any of the three BMP1 isoforms in cleaving either DSPP or DMP1 under either low or normal saline conditions. Interestingly, we were unable to reproduce sFRP2's reported ability to enhance the processing of type I procollagen by BMP1/mTLD. In summary, three isoforms of BMP1 process both DSPP and DMP1 at the MQX/DDP motif, but the identity of a protein that can enhance the cleavage of the two SIBLING proteins remains elusive.     

Regulation of the Cell Type-specific dentin sialophosphoprotein gene expression in mouse odontoblasts by a novel transcription repressor and an activator CCAAT-binding factor

Dentin sialophosphoprotein (DSPP) is an extracellular matrix protein that is cleaved into dentin sialoprotein (DSP) and dentin phosphoprotein (DPP) with a highly restricted expression pattern in tooth and bone. Mutations of the DSPP gene are associated with dentin genetic diseases. Regulation of tissue-specific DSPP expression has not been described. To define the molecular basis of this cell-specific expression, we characterized the promoter responsible for the cell-specific expression of the DSPP gene in odontoblasts. Within this region, DNase I footprinting and electrophoretic mobility shift assays delineated one element that contains an inverted CCAAT-binding factor site and a protein-DNA binding site using nuclear extracts from odontoblasts. A series of competitive electrophoretic mobility shift assay analyses showed that the protein-DNA binding core sequence, ACCCCCA, is a novel site sufficient for protein binding. These two protein-DNA binding sequences are conserved at the same proximal position in the mouse, rat, and human DSPP gene promoters and are ubiquitously present in the promoters of other tooth/bone genes. Mutations of the CCAAT-binding factor binding site resulted in a 5-fold decrease in promoter activity, whereas abolishment of the novel protein-DNA binding site increased promoter activity by about 4.6-fold. In contrast to DSPP, expression levels of the novel protein were significantly reduced during odontoblastic differentiation and dentin mineralization. The novel protein was shown to have a molecular mass of 72 kDa. This study shows that expression of the cell type-specific DSPP gene is mediated by the combination of inhibitory and activating mechanisms.     

In Vitro Reparative Dentin: a Biochemical and Morphological Study

In this study, starting from human dental pulp cells cultured in vitro, we simulated reparative dentinogenesis using a medium supplemented with different odontogenic inductors. The differentiation of dental pulp cells in odontoblast-like cells was evaluated by means of staining, and ultramorphological, biochemical and biomolecular methods. Alizarin red staining showed mineral deposition while transmission electron microscopy revealed a synthesis of extracellular matrix fibers during the differentiation process. Biochemical assays demonstrated that the differentiated phenotype expressed odontoblast markers, such as Dentin Matrix Protein 1 (DMP1) and Dentin Sialoprotein (DSP), as well as type I collagen. Quantitative data regarding the mRNA expression of DMP1, DSP and type I collagen were obtained by Real Time PCR. Immunofluorescence data demonstrated the various localizations of DSP and DMP1 during odontoblast differentiation. Based on our results, we obtained odontoblast-like cells which simulated the reparative dentin processes in order to better investigate the mechanism of odontoblast differentiation, and dentin extracellular matrix deposition and mineralization.Key words: dental tissue, in vitro differentiation, DMP1, DSP, type I collagen     

Dentin glycoprotein: the protein in the middle of the dentin sialophosphoprotein chimera

Dentin sialophosphoprotein (DSPP) is a major secretory product of odontoblasts and is critical for proper dentin formation. DSPP is believed to be processed into only two structural/functional domains: dentin sialoprotein (DSP) and dentin phosphoprotein (DPP). Here we report the isolation and characterization of a third domain of DSPP, designated dentin glycoprotein (DGP). DGP was isolated from a guanidine/EDTA extract of porcine tooth dentin by ion exchange, hydroxyapatite affinity, size exclusion, and RP-HPL chromatography. Endoproteinase lysine C digestion products of DGP were characterized by Edman sequencing and mass spectrometry. The porcine DGP backbone is the 81-amino acid segment of DSPP (Ser392 to Gly472) between the DSP and DPP domains. DGP has four phosphorylated serine residues (Ser453, Ser455, Ser457, and Ser462) and one glycosylated asparagine (Asn397). There are no other post-translational modifications. DGP is a stains-all positive protein with an apparent molecular mass on SDS-PAGE of 19 kDa, which is reduced by glycopeptidase A digestion to 16 kDa. A variety of glycans can be linked to Asn397. All are complex biantennary structures with a common N-linked pentasaccharide core (mannose3-N-acetylglucosamine2), most with a fucosyl residue on the innermost N-acetylglucosamine. The alpha1-3 and alpha1-6 arms are always galactose beta1-4 N-acetylglucosamine beta1-2 mannose, and either or both arms can be unsialidated or monosialidated. The calculated monoisotopic molecular masses of the different glycosylated forms of the DGP phosphoprotein are: unsialidated 10,523 and 10,670, monosialidated 10,815 and 10,961, and disialidated 11,106, and 11,252 Da, with the disialidated forms being the most abundant.     

Rescue of odontogenesis in Dmp1-deficient mice by targeted re-expression of DMP1 reveals roles for DMP1 in early odontogenesis and dentin apposition in vivo

Dentin matrix protein 1 (DMP1) is expressed in both pulp and odontoblast cells and deletion of the Dmp1 gene leads to defects in odontogenesis and mineralization. The goals of this study were to examine how DMP1 controls dentin mineralization and odontogenesis in vivo. Fluorochrome labeling of dentin in Dmp1-null mice showed a diffuse labeling pattern with a 3-fold reduction in dentin appositional rate compared to controls. Deletion of DMP1 was also associated with abnormalities in the dentinal tubule system and delayed formation of the third molar. Unlike the mineralization defect in Vitamin D receptor-null mice, the mineralization defect in Dmp1-null mice was not rescued by a high calcium and phosphate diet, suggesting a different effect of DMP1 on mineralization. Re-expression of Dmp1 in early and late odontoblasts under control of the Col1a1 promoter rescued the defects in mineralization as well as the defects in the dentinal tubules and third molar development. In contrast, re-expression of Dmp1 in mature odontoblasts, using the Dspp promoter, produced only a partial rescue of the mineralization defects. These data suggest that DMP1 is a key regulator of odontoblast differentiation, formation of the dentin tubular system and mineralization and its expression is required in both early and late odontoblasts for normal odontogenesis to proceed.     

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

Bone and dentin are mineralized extracellular matrices produced by osteoblasts and odontoblasts, respectively, and their major organic portion is type I collagen. Dentinogenesis Imperfecta (DGI) is one of the most common clinically- and genetically-based disturbances of dentin formation, causing irreversible dentin defects. Among several types of DGI, patients with DGI type II exhibit opalescent dentin with partial or complete pulp obliteration. It has been previously reported that the non-sense mutation (c.133C>T) in Dentin Sialophosphoprotein (DSPP) was identified in DGI type II patients at glutamine residue 45, resulting in the premature stop codon (p.Q45X). DSPP is known to be synthesized as a single gene product and further processed at Gly⁴⁶²-Asp⁴⁶³, resulting in the production of Dentin Sialoprotein (DSP) and Dentin Phosphoprotein (DPP). We hypothesized that the shorter form (Q45X) of N-terminal Dentin Sialoprotein (N-DSP) may cause over-production of type I collagen protein as obliterated pulp is occupied by dentin. To test this hypothesis, we generated mouse recombinant Glutathione-S-Transferase (GST)-N-DSP fusion protein, and the effect of GST-N-DSP was investigated in calvarial bone explant culture and MC3T3-E1 osteoblastic culture systems. Here we show that a significant increase in calvarial bone formation is observed by GST-N-DSP. GST-N-DSP accelerates MC3T3-E1 osteoblast cell growth and proliferation and subsequent osteoblast differentiation by inducing the expression of certain osteogenic markers such as type I collagen, Runx2, Osterix and ATF4. Interestingly, GST-N-DSP significantly enhances dentinogenesis marker gene expression including Dspp and Dmp1 gene expression in non-odontogenic MC3T3-E1 cells. To rule out any artificial effect of GST-tag, we also used the synthetic peptide of N-DSP and confirmed the results of N-DSP peptide were essentially similar to those of GST-N-DSP. Taken together, our data suggest that N-DSP promotes bone formation by accelerating osteoblast cell proliferation and subsequent osteoblast differentiation accompanied by marked up-regulation of the dentin matrix markers, such as Dspp and Dmp1 genes.     

牙本质基质蛋白1及其对骨形成的调控作用

牙本质基质蛋白1(dentin matrix protein 1,DMP1)是一种高度磷酸化的偏酸性非胶原蛋白, 属于小整合素结合配体N端连接糖蛋白（small integrin-binding ligand, N-linked glycoprotein, SIBLINGs）家族.和SIBLINGs家族其它成员一样,DMP1基因定位于人类染色体4q21除存在于牙组织外,该蛋白还普遍分布于骨组织中.在骨组织与细胞中已发现4种DMP1的主要存在形式,即全长DMP1、57 kD C-DMP1、37 kD N-DMP1、DMP1-PG.它们的分布与功能均不相同,但对骨的正常形成均有重要意义. DMP1的氨基酸序列拥有大量的酸性结构域,携带负电荷,与钙离子有较强的结合能力.它在体外能够促进羟基磷灰石形成,并调控细胞分化,在体内参与硬组织的矿化过程.另外,DMP1的水解过程对其调控矿化的功能十分关键.人体内DMP1基因的突变可导致常染色体隐性低血磷性佝偻病.本文就近几年对DMP1基因结构与调控、蛋白结构与代谢、在骨组织与细胞中的分布及其对骨形成调控作用的研究进展作一综述.     

Spatial and temporal activity of the dentin sialophosphoprotein gene promoter: differential regulation in odontoblasts and ameloblasts

Dentin sialoprotein and dentin phosphoprotein are non-collagenous proteins that are cleavage products of dentin sialophosphoprotein (DSPP). Although these two protein products are believed to have a crucial role in the process of tooth mineralization, their precise biological functions and the molecular mechanisms of gene regulation are not clearly understood. To understand such functions, we have developed a transgenic mouse model expressing a reporter gene (lacZ) under the control of approximately 6 kb upstream sequences of Dspp. The transgenic fusion protein was designed to reside within the cells to facilitate the precise identification of cell type and developmental stages at which the Dspp-lacZ gene is expressed. The results presented in this report demonstrate: (a) the 6 kb upstream sequences of Dspp have the necessary regulatory elements to direct the tissue specific expression of the transgene similar to endogenous Dspp, (b) both odontoblasts and ameloblasts exhibit transgene expression in a differentiation dependent manner, and (c) a differential regulation of the transgene in odontoblasts and ameloblasts occurs during tooth development and mineralization.     

Dentin sialophosphoprotein knockout mouse teeth display widened predentin zone and develop defective dentin mineralization similar to human dentinogenesis imperfecta type III

Dentin sialophosphoprotein (Dspp) is mainly expressed in teeth by the odontoblasts and preameloblasts. The Dspp mRNA is translated into a single protein, Dspp, and cleaved into two peptides, dentin sialoprotein and dentin phosphoprotein, that are localized within the dentin matrix. Recently, mutations in this gene were identified in human dentinogenesis imperfecta II (Online Mendelian Inheritance in Man (OMIM) accession number 125490) and in dentin dysplasia II (OMIM accession number 125420) syndromes. Herein, we report the generation of Dspp-null mice that develop tooth defects similar to human dentinogenesis imperfecta III with enlarged pulp chambers, increased width of predentin zone, hypomineralization, and pulp exposure. Electron microscopy revealed an irregular mineralization front and a lack of calcospherites coalescence in the dentin. Interestingly, the levels of biglycan and decorin, small leucine-rich proteoglycans, were increased in the widened predentin zone and in void spaces among the calcospherites in the dentin of null teeth. These enhanced levels correlate well with the defective regions in mineralization and further indicate that these molecules may adversely affect the dentin mineralization process by interfering with coalescence of calcospherites. Overall, our results identify a crucial role for Dspp in orchestrating the events essential during dentin mineralization, including potential regulation of proteoglycan levels.     

Dentin matrix protein 1 immobilized on type I collagen fibrils facilitates apatite deposition in vitro

During bone and dentin mineralization, the crystal nucleation and growth processes are considered to be matrix regulated. Osteoblasts and odontoblasts synthesize a polymeric collagenous matrix, which forms a template for apatite initiation and elongation. Coordinated and controlled reaction between type I collagen and bone/dentin-specific noncollagenous proteins are necessary for well defined biogenic crystal formation. However, the process by which collagen surfaces become mineralized is not understood. Dentin matrix protein 1 (DMP1) is an acidic noncollagenous protein expressed during the initial stages of mineralized matrix formation in bone and dentin. Here we show that DMP1 bound specifically to type I collagen, with the binding region located at the N-telopeptide region of type I collagen. Peptide mapping identified two acidic clusters in DMP1 responsible for interacting with type I collagen. The collagen binding property of these domains was further confirmed by site-directed mutagenesis. Transmission electron microscopy analyses have localized DMP1 in the gap region of the collagen fibrils. Fibrillogenesis assays further demonstrated that DMP1 accelerated the assembly of the collagen fibrils in vitro and also increased the diameter of the reconstituted collagen fibrils. In vitro mineralization studies in the presence of calcium and phosphate ions demonstrated apatite deposition only at the collagen-bound DMP1 sites. Thus specific binding of DMP1 and possibly other noncollagenous proteins on the collagen fibril might be a key step in collagen matrix organization and mineralization.