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 non-collagenous proteins 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 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.

S U M M A R Y Mutations in FAM20C were recently identified as the cause of lethal osteosclerotic bone dysplasia, which highlighted the important role of this molecule in biomineralization. No systematic studies have been performed to evaluate the expression pattern of this relatively new molecule in the developmental processes of bone and tooth. In the present study, we analyzed in detail the expression profile of FAM20C during osteogenesis and odontogenesis using ISH and IHC approaches. The specimens analyzed were mouse tissues spanning embryonic day 13.5 (E13.5) to postnatal 8 weeks. The earliest presence of FAM20C was observed at E14.5. During osteogenesis, FAM20C mRNA was detected in the chondrocytes and osteoblasts of the long bone, whereas its protein was observed in the extracellular matrix (ECM) of bone and in the cytoplasm of the chondrocytes, osteoblasts, and osteocytes. During odontogenesis, FAM20C mRNA was detected in the ameloblasts, odontoblasts, cementoblasts, and periodontal ligament fibroblasts, whereas its protein was observed in the matrices of dentin, enamel, and alveolar bone and in the cytoplasm of the aforementioned cells. The temporospatial expression profile revealed in this study indicates that FAM20C is an ECM protein that may play an important role in controlling the mineralization of bone and tooth. (J Histochem Cytochem 58:957-967, 2010)

It is known that dentin sialophosphoprotein (DSPP) is processed into NH2- and COOH-terminal fragments, but its key cleavage site has not been identified, nor has its full-length form been discovered. The objectives of this study were to identify the key cleavage site during DSPP processing and to search for full-length DSPP in vivo. We generated a construct encoding DSPP, in which Asp452, a cleavage site residue, was replaced by Ala452. The pulp-odontoblast complex and dentin were extracted, chromatographically separated, and assessed by Stains-All staining, Western immunoblotting, and mass spectrometry. These studies showed that the substitution of Asp452 by Ala452 completely blocks the cleavage of mouse DSPP in the transfected cells, indicating that the NH2-terminal peptide bond of Asp452 is essential for the initiation of DSPP proteolytic processing. The results of this study revealed the presence of full-length DSPP and its processed fragments in extracts from the pulp/odontoblast and dentin.

Dentin matrix protein 1 (DMP1), an acidic protein important to the formation of bone and dentin, primarily exists as the processed NH 2 -terminal and COOH-terminal fragments in the extracellular matrix of the two tissues. Previous in vitro studies showed that the substitution of residue Asp 213 by Ala 213 (D213A) at a cleavage site blocked the processing of mouse DMP1 in cells. In this study, we generated transgenic mice expressing mutant D213A-DMP1 (WT/D213A-Tg mice) to test the hypothesis that the proteolytic processing of DMP1 is an activation step essential to osteogenesis. By crossbreeding WT/D213A-Tg mice with Dmp1 knock-out (Dmp1-KO) mice, we obtained mice expressing D213A-DMP1 in a Dmp1-KO background; these mice will be referred to as "Dmp1-KO/ D213A-Tg" mice. Biochemical, radiological, and morphological approaches were used to characterize the skeletal phenotypes of Dmp1-KO/D213A-Tg mice compared with wild-type mice, Dmp1-KO mice, and Dmp1-KO mice expressing the normal Dmp1 transgene. Protein chemistry analyses showed that DMP1 was barely cleaved in the bone of the Dmp1-KO/ D213A-Tg mice, indicating that D213A substitution effectively blocked the proteolytic processing of DMP1 in vivo. While the expression of the normal Dmp1 transgene completely rescued the phenotypic skeletal changes of the Dmp1-KO mice, the expression of the mutant D213A-Dmp1 transgene failed to do so. These results indicate that the fulllength form of DMP1 is an inactive precursor and its proteolytic processing is an activation step essential to the biological functions of this protein in osteogenesis.Dentin matrix protein 1 (DMP1), 2 first identified by cDNA cloning using a rat odontoblast mRNA library, was originally postulated to be dentin-specific (1). Several research groups later demonstrated that DMP1 is also expressed in bone (2, 3, 4) at more abundant levels than in teeth (5,6,7,8). Mouse and human genetic studies have demonstrated that the inactivation of DMP1 leads to osteomalacia/rickets and dentin hypomineralization, indicating that this protein plays crucial roles in osteogenesis and dentinogenesis (9, 10, 11, 12). The major histopathological changes resulting from DMP1-deficiency include an excess accumulation of osteoid in the bone and widening of predentin in the tooth. Although these data have established an association between DMP1 and the formation of healthy mineralized tissues, the exact mechanistic pathways by which this protein participates in vital steps leading to the formation of healthy bone and dentin are unclear.In the extracellular matrix (ECM) of bone and dentin, DMP1 mainly occurs as the proteolytically processed fragments originating from the NH 2 -terminal and COOH-terminal regions of the DMP1 amino acid sequence, respectively (7). The NH 2 -terminal fragment of DMP1 (designated as "DMP1-N") exists in two forms: the 37-kDa fragment (7) and the proteoglycan form referred to as "DMP1-PG" (13), while the COOH-terminal fragment (designated as "DMP1-C") is present as the 57-kDa fragment (7). DMP1-PG cont...

SummaryDentin sialophosphoprotein (DSPP) and its cleaved products, dentin phosphoprotein (DPP) and dentin sialoprotein (DSP), play important roles in biomineralization. Believed to be tooth specific, the authors' group revealed its expression in bone, and more recently, they and other groups also showed its expression in a few types of soft tissues. In this study, the authors systematically examined the expression of DSPP in a variety of non-mineralized tissues using reverse-transcription polymerase chain reaction (RT-PCR), real-time PCR, Western immunoblotting, and immunohistochemistry analyses in wildtype mice as well as β-galactosidase assays in the Dspp lacZ knock-in mice. These approaches showed the presence of DSPP in the salivary glands, cartilage, liver, kidney, and brain and its absence in the heart and spleen. Real-time PCR showed that the expression levels of DSPP mRNA in salivary glands, cartilage, liver, and kidney were higher than in the bone. Interestingly, DSPP was observed in the pericytes of blood vessels in the dental pulp, which are believed to be able to differentiate into odontoblasts. On the basis of these observations, the authors conclude that DSPP and/or its cleaved products may fulfill important functions in certain non-mineralized tissues in addition to its role in biomineralization. (J Histochem Cytochem 59:1009-1021, 2011