Zinc balance is critical for NFI-C mediated regulation of odontoblast differentiation.

Zinc is trace element essential for diverse metabolic and cellular signaling pathways for the growth, development, and maintenance. Zinc deficiency is involved in bone malformations and oral disease. Mice deficient in zinc transporter Zip13 show connective tissue and skeletal disorders, abnormal incisor teeth, and reduced root dentin formation in the molar teeth and share a morphologically similar phenotype to nuclear factor I-C (NFI-C)-deficient mice. However, the precise function of zinc in NFI-C signaling-mediated odontoblast differentiation and dentin formation remains unclear. Here, we show that zinc stimulated the expression of metal transcription factor-1, but decreased NFI-C expression in odontoblastic MDPC-23 cells. Zinc also enhanced the phosphorylation of Smad2/3 (p-Smad2/3) and increased the binding efficiency of NFI-C and p-Smad2/3 in the cytoplasm. In contrast, zinc deficiency resulted in the accumulation of NFI-C into nucleus. Consequently, NFI-C had the biologic properties of a transcription factor, including DNA binding affinity for metallothionein-1 and the dentin sialophosphoprotein (DSPP) promoter, and transcriptional activation of the DSPP gene. Furthermore, zinc deficiency condition promoted DSPP expression in odontoblasts and dentin mineralization, while zinc sufficiency condition decreased DSPP expression and slightly delayed dentin mineralization. These data suggest that zinc equilibrium is required for odontoblast differentiation and dentin formation during dentinogenesis through the nuclear accumulation and modulation of NFI-C.

Odontogenic ameloblasts-associated protein (ODAM), via phosphorylation by bone morphogenetic protein receptor type IB (BMPR-IB), is implicated in ameloblast differentiation.

To elucidate the function of the odontogenic ameloblast-associated protein (ODAM) in ameloblasts, we identified more than 74 proteins that interact with ODAM using protoarray. Of the identified proteins, bone morphogenetic protein receptor type-IB (BMPR-IB) was physiologically relevant in differentiating ameloblasts. ODAM and BMPR-IB exhibited similar patterns of expression in vitro, during ameloblast differentiation. ODAM and BMPR-IB interacted through the C-terminus of ODAM, which resulted in increased ODAM phosphorylation in the presence of bone morphogenetic protein 2 (BMP-2). Immunoprecipitation assays using Ser-Xaa-Glu (SXE) mutants of ODAM demonstrated that the phosphorylation of ODAM by BMPR-IB occurs at this motif, and this phosphorylation is required for the activation of MAPKs. ODAM phosphorylation was detected in ameloblasts during ameloblast differentiation and enamel mineralization in vitro and involved in the activation of downstream factors of MAPKs. Therefore, the BMP-2-BMPR-IB-ODAM-MAPK signaling cascade has important roles in ameloblast differentiation and enamel mineralization. Our data suggest that ODAM facilitates the progression of tooth development in cooperation with BMPR-IB through distinct domains of ODAM.

Crosstalk between nuclear factor I-C and transforming growth factor-ß1 signaling regulates odontoblast differentiation and homeostasis.

Transforming growth factor-ß1 (TGF-ß1) signaling plays a key role in vertebrate development, homeostasis, and disease. Nuclear factor I-C (NFI-C) has been implicated in TGF-ß1 signaling, extracellular matrix gene transcription, and tooth root development. However, the functional relationship between NFI-C and TGF-ß1 signaling remains uncharacterized. The purpose of this study was to identify the molecular interactions between NFI-C and TGF-ß1 signaling in mouse odontoblasts. Real-time polymerase chain reaction and western analysis demonstrated that NFI-C expression levels were inversely proportional to levels of TGF-ß1 signaling molecules during in vitro odontoblast differentiation. Western blot and immunofluorescence results showed that NFI-C was significantly degraded after TGF-ß1 addition in odontoblasts, and the formation of the Smad3 complex was essential for NFI-C degradation. Additionally, ubiquitination assay results showed that Smurf1 and Smurf2 induced NFI-C degradation and polyubiquitination in a TGF-ß1-dependent manner. Both kinase and in vitro binding assays revealed that the interaction between NFI-C and Smurf1/Smurf2 requires the activation of the mitogen-activated protein kinase pathway by TGF-ß1. Moreover, degradation of NFI-C induced by TGF-ß1 occurred generally in cell types other than odontoblasts in normal human breast epithelial cells. In contrast, NFI-C induced dephosphorylation of p-Smad2/3. These results show that crosstalk between NFI-C and TGF-ß1 signaling regulates cell differentiation and homeostatic processes in odontoblasts, which might constitute a common cellular mechanism.

The odontogenic ameloblast-associated protein (ODAM) cooperates with RUNX2 and modulates enamel mineralization via regulation of MMP-20.

We have previously reported that the odontogenic ameloblast-associated protein (ODAM) plays important roles in enamel mineralization through the regulation of matrix metalloproteinase-20 (MMP-20). However, the precise function of ODAM in MMP-20 regulation remains largely unknown. The aim of the present study was to uncover the molecular mechanisms responsible for MMP-20 regulation. The subcellular localization of ODAM varies in a stage-specific fashion during ameloblast differentiation. During the secretory stage of amelogenesis ODAM was localized to both the nucleus and cytoplasm of ameloblasts. However, during the maturation stage of amelogenesis, ODAM was observed in the cytoplasm and at the interface between ameloblasts and the enamel layer, but not in the nucleus. Secreted ODAM was detected in the conditioned medium of ameloblast-lineage cell line (ALC) from days 14 to 21, which coincided with the maturation stage of amelogenesis. Interestingly, the expression of Runx2 and nuclear ODAM correlated with MMP-20 expression in ALC. We therefore examined whether ODAM cooperates with Runx2 to regulate MMP-20 and modulate enamel mineralization. Increased expression of ODAM and Runx2 augmented MMP-20 expression, and Runx2 expression enhanced expression of ODAM, although overexpression of ODAM did not influence Runx2 expression. Conversely, loss of Runx2 in ALC decreased ODAM expression, resulting in down-regulation of MMP-20 expression. Increased MMP-20 expression accelerated amelogenin processing during enamel mineralization. Our data suggest that Runx2 regulates the expression of ODAM and that nuclear ODAM serves an important regulatory function in the mineralization of enamel through the regulation of MMP-20 apart from a different, currently unidentified, function of extracellular ODAM.

Nuclear factor I-C is essential for odontogenic cell proliferation and odontoblast differentiation during tooth root development.

Our previous studies have demonstrated that nuclear factor I-C (NFI-C) null mice developed short molar roots that contain aberrant odontoblasts and abnormal dentin formation. Based on these findings, we performed studies to elucidate the function of NFI-C in odontoblasts. Initial studies demonstrated that aberrant odontoblasts become dissociated and trapped in an osteodentin-like mineralized tissue. Abnormal odontoblasts exhibit strong bone sialoprotein expression but a decreased level of dentin sialophosphoprotein expression when compared with wild type odontoblasts. Loss of Nfic results in an increase in p-Smad2/3 expression in aberrant odontoblasts and pulp cells in the subodontoblastic layer in vivo and primary pulp cells from Nfic-deficient mice in vitro. Cell proliferation analysis of both cervical loop and ectomesenchymal cells of the Nfic-deficient mice revealed significantly decreased proliferative activity compared with wild type mice. In addition, Nfic-deficient primary pulp cells showed increased expression of p21 and p16 but decreased expression of cyclin D1 and cyclin B1, strongly suggesting cell growth arrest caused by a lack of Nfic activity. Analysis of the pulp and abnormal dentin in Nfic-deficient mice revealed an increase in apoptotic activity. Further, Nfic-deficient primary pulp cells exhibited an increase in caspase-8 and -3 activation, whereas the cleaved form of Bid was hardly detected. These results indicate that the loss of Nfic leads to the suppression of odontogenic cell proliferation and differentiation and induces apoptosis of aberrant odontoblasts during root formation, thereby contributing to the formation of short roots.

Disruption of Nfic causes dissociation of odontoblasts by interfering with the formation of intercellular junctions and aberrant odontoblast differentiation.

We reported previously that Nfic-deficient mice exhibit short and abnormal molar roots and severely deformed incisors. The objective of this study is to address the mechanisms responsible for these changes using morphological, IHC, and RT-PCR analysis. Nfic-deficient mice exhibited aberrant odontoblasts and abnormal dentin formation in molar roots and the labial crown analog of incisors. The most striking changes observed in these aberrant odontoblasts were the loss of intercellular junctions and the decreased expression of ZO-1 and occludin. As a result, they became dissociated, had a round shape, and lost their cellular polarity and arrangement as a sheet of cells. Furthermore, the dissociated odontoblasts became trapped in dentin-like mineralized tissue, resembling osteodentin in the overall morphology. These findings suggest that loss of the Nfic gene interferes with the formation of intercellular junctions that causes aberrant odontoblast differentiation and abnormal dentin formation. Collectively, these changes in odontoblasts contributed to development of molars with short and abnormal roots in Nfic-deficient mice.

The effect of odontoblast conditioned media and dentin non-collagenous proteins on the differentiation and mineralization of cementoblasts in vitro.

OBJECTIVE: Cementum is an important mineralized tissue in root formation, however, the precise mechanism of cementum formation remains undetermined. The purpose of this study was to evaluate the effect of odontoblast conditioned media (CM) and dentin non-collagenous proteins (dNCPs) on the differentiation and mineralization of cementoblastic OCCM-30 cells. METHODS: The CM of ameloblastic ALCs, odontoblastic MDPC-23 and OD-11, osteoblastic MG-63, and fibroblastic NIH3T3 cells were transferred to OCCM-30 cells. dNCPs were extracted directly from porcine and human dentin and applied to OCCM-30 cells. The results were evaluated through the analysis of the morphologic appearance, expression of cementum matrix genes, and the formation of mineralized nodules in vitro. RESULTS: dNCPs hardly influenced proliferation, cell cycle modification, and chemotaxis of cementoblasts. Mineralization of cementoblasts was accelerated with dNCPs and CM from odontoblastic MDPC-23 and OD-11. RT-PCR analysis revealed the earlier and stronger expression of bone sialoprotein (BSP), alkaline phosphatase (ALP), and osteocalcin (OC) mRNAs in the MDPC23- and OD11-CM-treated OCCM-30 cells than those in the control OCCM-30 cells. The level of gene expression was also significantly higher in the dNCP-treated group than the control group. CONCLUSION: These results suggest that dentin matrix proteins, or the secreted products of odontoblasts, induced cementoblast differentiation and mineralization. These findings may contribute to the development of a periodontal treatment that includes cementum regeneration.

Nfic gene disruption inhibits differentiation of odontoblasts responsible for root formation and results in formation of short and abnormal roots in mice.

BACKGROUND: Nuclear factor I genes play an important role in the development of the brain, lung, and roots of teeth. We had reported that Nfic-deficient mice form normal crowns, but abnormal roots of molar teeth. However, the mechanism by which the disruption of Nfic gene causes abnormal root formation remains unknown. METHODS: To understand this mechanism, the root formation in Nfic-deficient mice was examined and compared to that of wild-type mice by morphological, immunohistochemical, and in situ hybridization analyses. RESULTS: Nfic-deficient mice formed normal Hertwig's epithelial root sheath (HERS) but severely disrupted odontoblast differentiation, leading to the formation of aberrant odontoblasts in the early stage of root formation. They became dissociated and polygonal in shape, lost their orientation and polarity, and did not express dentin sialophosphoprotein. The abnormal roots contained trapped aberrant odontoblasts, thereby resembling osteodentin in overall morphology. No osteoclasts were associated with abnormal roots. Further, the abnormal roots exhibited a decreased number of cementoblasts and cementum formation on the root surface. CONCLUSIONS: The loss of Nfic did not interfere with the formation of HERS, but it caused disrupted odontoblast differentiation, which resulted in the formation of short and abnormal roots, and decreased cementum. This finding suggests that root dentin is required for normal cementum formation. Therefore, Nfic may be a key regulator of root odontoblast differentiation and root formation.

The amyloid protein APin is highly expressed during enamel mineralization and maturation in rat incisors.

This study investigated the expression and localization of APin (which was previously identified and cloned from a rat odontoblast cDNA library), during ameloblast differentiation in rat incisors, by using in situ hybridization and immunohistochemistry. The subcellular localization of APin varied during ameloblast differentiation, but was stage-specific. APin mRNA was not expressed in pre-ameloblasts, was weakly expressed in secretory ameloblasts, and was strongly expressed in maturation-stage ameloblasts as well as in the junctional epithelium attached to the enamel of erupted molars. In the maturation-stage ameloblasts, APin protein was conspicuous in the supranuclear area (Golgi complex) of smooth-ended ameloblasts as well as in both the supranuclear area and the ruffle end of ruffle-ended ameloblasts. During ameloblast-lineage cell culture, APin was expressed at a low level in the early stages of culture, but at a high level in the late stage of culture, which was equivalent to the maturation stage. APin protein was efficiently secreted from transfected cells in culture. Furthermore, its overexpression and inactivation caused an increase and decrease in matrix metalloproteinase-20 (MMP-20) and tuftelin expression, respectively. These findings indicate a functional role for APin in the mineralization and maturation of enamel that is mediated by the expression of MMP-20 and tuftelin.

Effect of dexamethasone on root resorption after delayed replantation of rat tooth.

The purpose of this study was to evaluate the effect of dexamethasone after delayed tooth replantation with specific regard to root resorption and ankylosis. In addition, the study was planned to elucidate further the usefulness of the model. Fifty-two maxillary first molar teeth were extracted from 26 Sprague-Dawley white female rats fed 0.4% beta-aminoproprionitrile for 3 days to facilitate the extraction. After extraction, the mesiobuccal root canals were endodontically treated under a microscope to prevent subsequent inflammatory resorption of pulpal origin and were assigned to three groups. Teeth in group 1, the dexamethasone group (n = 22), were demineralized in citric acid (1 min), washed, soaked in 1000 nM dexamethasone solution (3 min), air-dried, and replanted in the original sockets. Total extraoral treatment time for each tooth was controlled to 30 min. Teeth in group 2, the dried-only group (n = 22), were air-dried for 30 min after obturation without any surface treatment and replanted. Teeth in group 3, the immediate group (n = 8), were extracted, not root-filled and replanted immediately into their sockets. All experimental animals were killed at 3 weeks after replantation and evaluated histologically. Forty-three of the 52 teeth were available for histological interpretation. They consisted of six immediate, 18 dried-only, and 19 dexamethasone-treated teeth. The degree of progressive root resorption was significantly less in the dexamethasone-treated group than in the dried-only group (p < 0.05). The dexamethasone-treated group exhibited significantly more bone ankylosis than the dried-only group (p < 0.05). These results indicate that the topical use of dexamethasone may be of value in reducing the degree or rate of progressive root resorption secondary to traumatic avulsion and that the rat is a reasonable model for tooth replantation.

Essential role for NFI-C/CTF transcription-replication factor in tooth root development.

The mammalian tooth forms by a series of reciprocal epithelial-mesenchymal interactions. Although several signaling pathways and transcription factors have been implicated in regulating molar crown development, relatively little is known about the regulation of root development. Four genes encoding nuclear factor I (NFI) transcription-replication proteins are present in the mouse genome: Nfia, Nfib, Nfic, and NFIX: In order to elucidate its physiological role(s), we disrupted the Nfic gene in mice. Heterozygous animals appear normal, whereas Nfic(-/-) mice have unique tooth pathologies: molars lacking roots, thin and brittle mandibular incisors, and weakened abnormal maxillary incisors. Feeding in Nfic(-/-) mice is impaired, resulting in severe runting and premature death of mice reared on standard laboratory chow. However, a soft-dough diet mitigates the feeding impairment and maintains viability. Although Nfic is expressed in many organ systems, including the developing tooth, the tooth root development defects were the prominent phenotype. Indeed, molar crown development is normal, and well-nourished Nfic(-/-) animals are fertile and can live as long as their wild-type littermates. The Nfic mutation is the first mutation described that affects primarily tooth root formation and should greatly aid our understanding of postnatal tooth development.