TGF-ß Signaling Regulates Cementum Formation through Osterix Expression.

TGF-ß/BMPs have widely recognized roles in mammalian development, including in bone and tooth formation. To define the functional relevance of the autonomous requirement for TGF-ß signaling in mouse tooth development, we analyzed osteocalcin-Cre mediated Tgfbr2 (OC(Cre)Tgfbr2(fl/fl)) conditional knockout mice, which lacks functional TGF-ß receptor II (TßRII) in differentiating cementoblasts and cementocytes. Strikingly, OC(Cre)Tgfbr2(fl/fl) mutant mice exhibited a sharp reduction in cellular cementum mass with reduced matrix secretion and mineral apposition rates. To explore the molecular mechanisms underlying the roles of TGF-ß signaling through TßRII in cementogenesis, we established a mouse cementoblast model with decreased TßRII expression using OCCM-30 cells. Interestingly, the expression of osterix (Osx), one of the major regulators of cellular cementum formation, was largely decreased in OCCM-30 cells lacking TßRII. Consequently, in those cells, functional ALP activity and the expression of genes associated with cementogenesis were reduced and the cells were partially rescued by Osx transduction. We also found that TGF-ß signaling directly regulates Osx expression through a Smad-dependent pathway. These findings strongly suggest that TGF-ß signaling plays a major role as one of the upstream regulators of Osx in cementoblast differentiation and cementum formation.

Expression of Caveolin-1 in Periodontal Tissue and Its Role in Osteoblastic and Cementoblastic Differentiation In Vitro.

It has been previously reported that caveolin-1 (Cav-1) knockout mice exhibit increased bone size and stiffness. However, the expression and role of Cav-1 on periodontal tissue is poorly understood. The aim of this study was to investigate the immunohistochemical expression of Cav-1 in the mouse periodontium and explore the role of Cav-1 on osteoblastic and cementoblastic differentiation in human periodontal ligament cells (hPDLCs), cementoblasts, and osteoblasts. To reveal the molecular mechanisms of Cav-1 activity, associated signaling pathways were also examined. Immunolocalization of Cav-1 was studied in mice periodontal tissue. Differentiation was evaluated by ALP activity, alizarin red S staining, and RT-PCR for marker genes. Signal transduction was analyzed using Western blotting and confocal microscopy. Cav-1 expression was observed in hPDLCs, cementoblasts, and osteoblasts of the periodontium both in vivo and in vitro. Inhibition of Cav-1 expression by methyl-ß-cyclodextrin (MßCD) and knockdown of Cav-1 by siRNA promoted osteoblastic and cementoblastic differentiation by increasing ALP activity, calcium nodule formation, and mRNA expression of differentiation markers in hPDLCs, cementoblasts, and osteoblasts. Osteogenic medium-induced BMP-2 and BMP-7 expression, and phosphorylation of Smad1/5/8 were enhanced by MßCD and siRNA knockdown of Cav-1, which was reversed by BMP inhibitor noggin. MßCD and Cav-1 siRNA knockdown increased OM-induced AMPK, Akt, GSK3ß, and CREB phosphorylation, which were reversed by Ara-A, a specific AMPK inhibitor. Moreover, OM-induced activation of p38, ERK, JNK, and NF-κB was enhanced by Cav-1 inhibition. This study demonstrates, for the first time, that Cav-1 is expressed in developing periodontal tissue and in vitro in periodontal-related cells. Cav-1 inhibition positively regulates osteoblastic differentiation in hPDLCs, cementoblasts, and osteoblasts via BMP, AMPK, MAPK, and NF-κB pathway. Thus, Cav-1 inhibition may be a novel molecular target for therapeutic approaches in periodontitis or osteolytic disease.

Expression of thymosin beta-4 in human periodontal ligament cells and mouse periodontal tissue and its role in osteoblastic/cementoblastic differentiation.

A recent report showed that thymosin beta-4 (Tß4) is expressed during the development of tooth germ, but its effect on osteoblastic/cementoblastic differentiation is a controversial topic. Furthermore, the precise expression and function of Tß4 in periodontal tissue remains unclear. Therefore, the purpose of this study was to investigate the immunolocalization of Tß4 in the developing periodontium of mouse, the function of Tß4 in osteoblastic/cementoblastic differentiation, and the underlying mechanism regulating periodontal regeneration in human periodontal ligament cells (hPDLCs), cementoblasts, and osteoblasts. Tß4 expression was observed in differentiating hPDLCs, osteoblasts of the periodontium during development, as well as in mature tissue. Higher Tß4 expression was observed in hPDLCs than in cementoblasts and osteoblasts in the developing periodontium. The expression of Tß4 mRNA and protein gradually increased during PDL cell differentiation. The downregulation of Tß4 expression by Tß4 siRNA transfection inhibited osteoblastic differentiation by decreasing calcium nodule formation, alkaline phosphatase (ALP) activity, and mRNA expression of differentiation markers in hPDLCs, cementoblasts, and osteoblasts. In contrast, Tß4 activation using a Tß4 peptide, promoted these processes by activation of Akt, p38, ERK MAPKs, and the NF-κB pathway. The expression of nuclear NFATc1 was upregulated by Tß4 peptide in hPDLCs. Inhibition of the calcineurin/NFATc1 pathway by cyclosporin A and FK506, attenuated Tß4-induced osteoblastic differentiation and activation of Wnt-related genes, as well as nuclear ß-catenin in hPDLCs. In conclusion, this study demonstrates, for the first time, that Tß4 is expressed in developing periodontal tissue and that its expression is associated with osteoblastic/cementoblastic differentiation. These results suggests that Tß4 is a potential therapeutic target for periodontal regeneration or bone disease.

Nfic regulates tooth root patterning and growth.

Molecular interactions between epithelium and mesenchyme are important for root formation. Nuclear factor I-C (Nfic) has been identified as a key regulator of root formation. However, the mechanisms of root formation and their interactions between Hertwig's epithelial root sheath (HERS) and mesenchyme remain unclear. In this study, we investigated the role of Nfic in root patterning and growth during molar root development. The molars of Nfic knockout mice exhibited an enlarged pulp chamber and apical displacement of the pulpal floor, characteristic features of taurodontism, due to delayed furcation formation. In developing molar roots of mutant mice at P14, BrdU positive cells decreased in the apical mesenchyme of the elongation region whereas those cells increased in the dental papilla of the furcation region. Whereas cytokeratin 14 and laminin were localized in HERS cells of mutant molars, Smoothened (Smo) and Gli1 were downregulated in preodontoblasts. In contrast, cytokeratin 14 and Smo were localized in the cells of the furcation region of mutant molars. These results indicate that Nfic regulates cell proliferation in the dental mesenchyme and affects the fate of HERS cells in a site-specific manner. From the results, it is suggested that Nfic is required for root patterning and growth during root morphogenesis.

Temporo-spatial requirement of Smad4 in dentin formation.

The TGF-ß/BMP family plays an important role in multiple stages of tooth development. TGF-ß/BMP signaling is required for odontoblast differentiation and dentin formation; however, the precise molecular mechanisms underlying dentin formation remain unclear. To address the role of TGF-ß/BMP signaling in dentin formation, we analyzed mice in which Smad4, a key intracellular mediator of TGF-ß/BMP signaling, was subjected to tissue-specific ablation under the control of Dspp, OC, or Col1a1 promoters. Three independent Smad4 conditional knockout mice exhibited various dentin defects in the crowns and roots of their molars depending on the transactivator. In all mutant molars, crown dentin thickness was thinner than that of the control. In addition, impaired dentin was found in the cervical region and root furcation area. Although the initial differentiation of odontoblasts was normal, odontoblast polarity abruptly decreased and the expression of Col1a1, OC, and Dspp was reduced in the odontoblasts of mutant molars. In Dspp-Cre-mediated Smad4 disruption mice, primary dentin formation was slightly delayed, while secondary dentin formation was severely affected in the cervical region of the molars. These results indicate that TGF-ß/BMP signaling is required for odontoblast maturation and dentin formation in a stage- and site-dependent manner.

COMP-angiopoietin 1 increases proliferation, differentiation, and migration of stem-like cells through Tie-2-mediated activation of p38 MAPK and PI3K/Akt signal transduction pathways.

Recombinant COMP-Ang1, a chimera of angiopoietin-1 (Ang1) and a short coiled-coil domain of cartilage oligomeric matrix protein (COMP), is under consideration as a therapeutic agent capable of inducing the homing of cells with increased angiogenesis. However, the potentials of COMP-Ang1 to stimulate migration of mesenchymal stem cells (MSCs) and the associated mechanisms are not completely understood. We examined the potential of COMP-Ang1 on bone marrow (BM)-MSCs, human periodontal ligament stem cells (PDLSCs), and calvarial osteoblasts. COMP-Ang1 augmented Tie-2 induction at protein and mRNA levels and increased proliferation and expression of runt-related transcription factor 2 (Runx2), osterix, and CXCR4 in BMMSCs, but not in osteoblasts. The COMP-Ang1-mediated increases were inhibited by Tie-2 knockdown and by treating inhibitors of phosphoinositide 3-kinase (PI3K), LY294002, or p38 mitogen-activated protein kinase (MAPK), SB203580. Phosphorylation of p38 MAPK and Akt was prevented by siRNA-mediated silencing of Tie-2. COMP-Ang1 also induced in vitro migration of BMMSCs and PDLSCs. The induced migration was suppressed by Tie-2 knockdown and by CXCR4-specific peptide antagonist or LY294002, but not by SB203580. Furthermore, COMP-Ang1 stimulated the migration of PDLSCs into calvarial defect site of rats. Collectively, our results demonstrate that COMP-Ang1-stimulated proliferation, differentiation, and migration of progenitor cells may involve the Tie-2-mediated activation of p38 MAPK and PI3K/Akt pathways.

New population of odontoblasts responsible for tooth root formation.

Root formation is initiated with the extension of Hertwig's epithelial root sheath (HERS) after crown morphogenesis. To date, little is known about the molecular and cellular mechanisms controlling root formation. Recently we found rootless molars are formed in the dental mesenchyme-specific ß-catenin conditional knockout mice. The striking root phenotypes of these mutant mice result from the disrupted differentiation of differentiating odontoblasts, caused by ablation of ß-catenin during initiation of root formation. Here we show the cellular and molecular characteristics of differentiating odontoblasts using histochemistry and immunohistochemistry. These cells were not found in crown formation, but appeared only in the apical end of developing tooth, thus we have named these cells "apical odontoblasts" (AOds). AOds appeared immediately after HERS formation and were always present on the apical side of developing roots until root formation was complete. These findings indicate that AOds are closely associated with the transition from crown to root and with root elongation. In AOds, several transcription factors, including Nfic, Creb3l1, and Osx, as well as ß-catenin and alkaline phosphatase were expressed but Phex and Dspp were not expressed. Taken together, our results indicate that AOds are the principal cells responsible for tooth root formation. These findings may contribute to the further understanding of the mechanisms underlying tooth root formation and root regeneration.

Col1a1-cre mediated activation of ß-catenin leads to aberrant dento-alveolar complex formation.

Wnt/ß-catenin signaling plays a critical role in bone formation and regeneration. Dentin and cementum share many similarities with bone in their biochemical compositions and biomechanical properties. Whether Wnt/ß-catenin signaling is involved in the dento-alveolar complex formation is unknown. To understand the roles of Wnt/ß-catenin signaling in the dento-alveolar complex formation, we generated conditional ß-catenin activation mice through intercross of Catnb(+/lox(ex3)) mice with Col1a1-cre mice. In mutant mice, tooth formation and eruption was disturbed. Lower incisors and molars did not erupt. Bone formation was increased in the mandible but tooth formation was severely disturbed. Hypomineralized dentin was deposited in the crown but roots of molars were extremely short and distorted. In the odontoblasts of mutant molars, expression of dentin matrix proteins was obviously downregulated following the activation of ß-catenin whereas that of mineralization inhibitor was increased. Cementum and periodontal ligament were hypoplastic but periodontal space was narrow due to increased alveolar bone formation. While cementum matrix proteins were decreased, bone matrix proteins were increased in the cementum and alveolar bone of mutant mice. These results indicate that local activation of ß-catenin in the osteoblasts and odontoblasts leads to aberrant dento-alveolar complex formation. Therefore, appropriate inhibition of Wnt/ß-catenin signaling is important for the dento-alveolar complex formation.