Identification, characterization, and expression of dentin matrix protein 1 gene in Xenopus laevis.

Dentin matrix protein 1 (DMP1) is an acidic extracellular matrix protein expressed mainly in bone and dentin, and is a member of the small integrin-binding ligand N-linked glycoprotein (SIBLING) family. The DMP1 gene, however, appears to evolve rapidly in comparison with other SIBLING genes, even though such functionally important molecules usually evolve more slowly than less important ones. The purpose of this study was to identify and characterize an ortholog of the DMP1 gene in an amphibian (Xenopus laevis; X. laevis) to clarify molecular evolutionary alterations in DMP1 associated with calcified tissues in tetrapods. Furthermore, we analyzed the mRNA expression of this gene to elucidate its functional change in bone and developing tooth germ in comparison with amniote DMP1s. The similarities of the deduced amino acid sequence of X. laevis DMP1 to that of the corresponding amniote proteins were low, although they did share several unique features specific to DMP1 and have similar properties. Expression of X. laevis DMP1 mRNA was predominant in osteocytes and odontoblasts, but only transiently observed in ameloblasts, as in amniotes. These results suggest that DMP1 has conserved several functions during tetrapod evolution. This indicates that continuity of biochemical properties has been more important in maintaining DMP1 functionality than that of the sequence of amino acid residues, which has undergone change over the course of molecular evolution.

Immunolocalization of TAK1, TAB1, and p38 in the developing rat molar.

In tooth development, transforming growth factor beta (TGF-ß) and bone morphogenetic protein (BMP) are involved in cell differentiation and matrix protein production. TGF-ß and BMP have two signaling pathways: the Smad pathway and the non-Smad pathway. However, only a few studies have focused on the non-Smad pathway in tooth development. TGF-ß-activated kinase 1 (TAK1) is activated by TGF-ß or BMP and binds to TAK1-binding protein (TAB1), activating p38 or c-Jun N-terminal kinase (JNK), forming the non-Smad signaling pathway. In this study, we examined the distribution of these kinases, TGF-ß receptor 1 (TGF-ß-R1), BMP receptor-1B (BMPR-1B) and Smad4 in cells of the rat molar germ histochemically, in order to investigate the signaling pathway in each type of cell. Immunostaining for TGF-ß-R1, BMPR-1B, Smad4, TAK1, TAB1 and phosphorylated-p38 (p-p38) showed similar reactions. In the cervical loop, reactions were clearer than in other enamel epithelium. In the inner enamel epithelium, signal increased with differentiation into ameloblasts, became strongest in the secretory stage, and decreased rapidly in the maturation stage. Signal also increased upon differentiation from preodontoblasts to odontoblasts. In Hertwig's epithelial sheath, with the exception of BMPR-1B, reactions were stronger in the later stage, showing more enamel protein secretion than in the early stage. However, no clear reaction corresponding to phosphorylated-JNK was observed in any type of cell. These results suggest that TGF-ß or BMP is involved in the induction of differentiation of inner enamel epithelium cells into ameloblasts, and preodontoblast differentiation into odontoblasts, the regulation of cervical loop cell proliferation, the elongation or regulation of the epithelial sheath, and the secretion of enamel protein and dentin matrix protein through the non-Smad signaling pathway via TAK1, TAB1 and p38 as well as Smad signaling pathways in the rat molar germ.

Transforming growth factor ß inducible apoptotic cascade in epithelial cells during rat molar tooth eruptions.

In tooth eruptions, the presence of apoptotic epithelial cells at the eruption site has been reported, but the factors that induce apoptosis in these cells remain to be elucidated, as do the induction pathways. In this study, we focused our attention on transforming growth factor beta (TGF-beta), which is known to induce apoptosis during embryonic development. Oral epithelium and dental lamina of maxillary first molars in 8- and 15-day-old rats were used to investigate the induction pathway of apoptosis by performing the immunohistochemical tests outlined below and assessing the characteristics of cells that undergo apoptosis by transmission electron microscopy in rats 8 and 15 days after birth. We examined TGF-beta-receptor 1, TGF-beta inducible transcription factor 1 (TIEG1), NADPHoxidase 4 (Nox4), cytochrome c, caspase-3 (active form and pro-enzyme), apoptosis-inducing protein Daxx, apoptosis signal-regulating kinase 1 (ASK1), glycogen synthase kinase-3 beta phosphorylated on serine 9 (p-GSK-3beta), and beta-catenin. We also performed periodic acid Schiff (PAS) reaction and terminal deoxynucleotidyl transferase-mediated dUTD nick end labeling (TUNEL) staining. At eruption sites 8 days after birth, reactions to TGF-beta-receptor 1, TIEG1, Nox4, cytochrome c, caspase-3, p-GSK-3beta, and beta-catenin, and PAS-positive cells were observed in areas close to the basal layer of oral epithelium through to the center of the dental lamina, but no reaction to Daxx or ASK1 was noted at these sites. Electron microscopy revealed the accumulation of glycogen granules in the cells that showed reactions to the above-mentioned markers as well as in the spaces among them. In the rats 15 days after birth (immediately before tooth eruption), the PAS-positive cells that showed reactions to the above antibodies remained on the buccal side of the epithelium, and high-electron-density apoptotic bodies and TUNEL-positive bodies were noted. Therefore, during tooth eruption, TGF-beta may induce apoptosis of cells rich in glycogen granules, and cytochrome c and caspase-3 may function to induce apoptosis. In addition, reactive oxygen species may be involved in this induction pathway via TIEG1 and Nox4 without involvement of Daxx and ASK1. Moreover, overexpression of p-GSK-3beta and beta-catenin may also contribute to apoptosis of oral epithelium at the eruption site and dental lamina cells. Glycogen storage mediated by p-GSK-3beta and crosstalk between the TGF-beta and Wnt signaling pathways may participate in the formation of tooth eruption passage.

Immunocytochemistry of keratan sulfate proteoglycan and dermatan sulfate proteoglycan in porcine tooth-germ dentin.

Keratan sulfate proteoglycan and dermatan sulfate proteoglycan have been reported to inhibit collagen fibrillogenesis. We investigated their distribution in order to evaluate the role of proteoglycan in dentinogenesis. Specimens of porcine tooth-germ dentin and erupted teeth were the materials on which antibodies to keratin sulfate and dermatan sulfate proteoglycan were used. Predentin was found to be positive for both antibodies and the reaction ceased in the calcification front. Uniformly thick collagen fibrils (30-70 nm in diameter) were distributed in the predentin matrix, which would become intertubular dentin in the future. Both antibodies reacted positively along these fibrils. In contrast, along the surface layer of dentin in the tooth germ and that in erupted teeth, collagen fibrils of 10-300 nm in diameter were noted occasionally in dentinal tubules whose odontoblastic processes had disappeared and these heterogeneous fibrils were negative for both antibodies. Our findings suggest that keratan sulfate proteoglycan and dermatan sulfate proteoglycan distributed in the predentin inhibit calcification of collagen fibrils in the uncalcified matrix and disappear in the calcification front. It is further suggested that keratan sulfate proteoglycan and dermatan sulfate proteoglycan distributed along collagen fibrils in the predentin matrix maintain uniform thickness, whereas collagen fibrils in dentinal tubules varied in thickness because of the absence of involvement of both proteoglycans. Therefore, keratan sulfate proteoglycan and dermatan sulfate proteoglycan were thought to be involved in both calcification and matrix formation.