Impaired breakdown of Herwig's epithelial root sheath disturbs tooth root development.

BACKGROUND: Wnt/ß-catenin signaling plays a variety of roles in both the dental epithelium and mesenchyme at most stages of tooth development. In this study, we verified the roles of Hertwig's epithelial root sheath (HERS) breakdown in tooth root development. This breakdown results in formation of epithelial cell rests of Malassez (ERM). RESULTS: Following induction of ß-catenin stabilization in the epithelium of developing tooth at the moment of HERS breakdown, HERS failed to break down for ERM formation. HERS with stabilized ß-catenin was altered into a multicellular layer enveloping elongated root dentin with higher expression of junctional proteins such as Zo-1 and E-cadherin. Importantly, this impairment of HERS breakdown led to arrest of further root elongation. In addition, the portion of root dentin enveloped by the undissociated HERS remained in a hypomineralized state. The odontoblasts showed ectopically higher expression of pyrophosphate regulators including Ank and Npp1, whereas Tnap expression was unchanged. CONCLUSIONS: Our data suggest that Wnt/ß-catenin signaling is decreased in HERS for ERM formation during root development. Furthermore, ERM formation is important for further elongation and dentin mineralization of the tooth roots. These findings may provide new insight to understand the contribution of ERM to root formation.

Cell dynamics in Hertwig's epithelial root sheath are regulated by ß-catenin activity during tooth root development.

ß-catenin, a key mediator of Wnt signaling, plays multiple roles in tooth development. However, the role of ß-catenin in Hertwig's epithelial root sheath (HERS) during root formation remains unclear. In this study, we generated inducible tissue-specific ß-catenin conditional knockout mice (Ctnnb1i∆shh ) to investigate how ß-catenin in HERS affects tooth root development. The inactivation of ß-catenin in HERS led to interrupted root elongation due to premature disruption of HERS. This phenotype was accompanied by reduced cell-cell adhesion and decreased expression of junctional proteins, as well as increased epithelial-to-mesenchymal transition of HERS cells upon ß-catenin depletion. Accordingly, stabilization of ß-catenin in HERS (Catnbi∆shh ) led to the formation of unfragmented HERS and resulted in the failure of HERS dissociation, with increased expression of junctional proteins. Our results suggest that fine control of ß-catenin is important for HERS to guide root formation through regulating its structural integrity.

Recessive Mutations in ACPT, Encoding Testicular Acid Phosphatase, Cause Hypoplastic Amelogenesis Imperfecta.

Amelogenesis imperfecta (AI) is a heterogeneous group of genetic disorders affecting tooth enamel. The affected enamel can be hypoplastic and/or hypomineralized. In this study, we identified ACPT (testicular acid phosphatase) biallelic mutations causing non-syndromic, generalized hypoplastic autosomal-recessive amelogenesis imperfecta (AI) in individuals from six apparently unrelated Turkish families. Families 1, 4, and 5 were affected by the homozygous ACPT mutation c.713C>T (p.Ser238Leu), family 2 by the homozygous ACPT mutation c.331C>T (p.Arg111Cys), family 3 by the homozygous ACPT mutation c.226C>T (p.Arg76Cys), and family 6 by the compound heterozygous ACPT mutations c.382G>C (p.Ala128Pro) and 397G>A (p.Glu133Lys). Analysis of the ACPT crystal structure suggests that these mutations damaged the activity of ACPT by altering the sizes and charges of key amino acid side chains, limiting accessibility of the catalytic core, and interfering with homodimerization. Immunohistochemical analysis confirmed localization of ACPT in secretory-stage ameloblasts. The study results provide evidence for the crucial function of ACPT during amelogenesis.

Testicular acid phosphatase induces odontoblast differentiation and mineralization.

Odontoblasts differentiate from dental mesenchyme during dentin formation and mineralization. However, the molecular mechanisms controlling odontoblast differentiation remain poorly understood. Here, we show that expression of testicular acid phosphatase (ACPT) is restricted in the early stage of odontoblast differentiation in proliferating dental mesenchymal cells and secretory odontoblasts. ACPT is expressed earlier than tissue-nonspecific alkaline phosphatase (TNAP) and partly overlaps with TNAP in differentiating odontoblasts. In MDPC-23 odontoblastic cells, expression of ACPT appears simultaneously with a decrease in ß-catenin activity and is abolished with the expression of Phex and Dsp. Knockdown of ACPT in MDPC-23 cells stimulates cell proliferation together with an increase in active ß-catenin and cyclin D1. In contrast, the overexpression of ACPT suppresses cell proliferation with a decrease in active ß-catenin and cyclin D1. Expression of TNAP, Osx, Phex and Dsp is reduced by knockdown of ACPT but is enhanced by ACPT overexpression. When ACPT is blocked with IgG, alkaline phosphatase activity is inhibited but cell proliferation is unchanged regardless of ACPT expression. These findings suggest that ACPT inhibits cell proliferation through ß-catenin-mediated signaling in dental mesenchyme but elicits odontoblast differentiation and mineralization by supplying phosphate during dentin formation. Thus, ACPT might be a novel candidate for inducing odontoblast differentiation and mineralization for dentin regeneration.

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.

Constitutive stabilization of ß-catenin in the dental mesenchyme leads to excessive dentin and cementum formation.

Wnt/ß-catenin signaling plays an important role in morphogenesis and cellular differentiation during development. Essential roles of Wnt/ß-catenin signaling in tooth morphogenesis have been well known, but the involvement of Wnt/ß-catenin signaling in the dental hard tissue formation remains undefined. To understand roles of Wnt/ß-catenin signaling in dentin and cementum formation, we generated and analyzed the conditional ß-catenin stabilized mice in the dental mesenchyme. The OC-Cre;Catnb(lox(ex3)/+) mice exhibited malformed teeth characterized by aberrantly formed dentin and excessively deposited cementum. Large amount of dentin was rapidly formed with widened predentin and numerous globular calcifications in the crown. Whereas roots of molars were short and covered with the excessively formed cellular cementum. With age, the coronal pulp chamber and periodontal space were narrowed by the excessively formed dentin and cementum, respectively. To compare the changes of gene expression in the mutant mice, Col1a1 expression was increased but that of Dspp was decreased in the odontoblasts. However, both of Col1a1 and Bsp expression was increased in the cementoblasts. The gene expression changes were consistent with the localization of matrix proteins. Biglycan and PC-1 was increased but Phex was decreased in the odontoblasts and dentin matrix, respectively. TNAP was increased but Dmp1 and FGF23 was decreased in the cementoblasts and cementum matrix, respectively. Our results indicate that persistent stabilization of ß-catenin in the dental mesenchyme leads to premature differentiation of odontoblasts and differentiation of cementoblasts, and induces excessive dentin and cementum formation in vivo. These results suggest that temporospatial regulation of Wnt/ß-catenin signaling plays critical roles in the differentiation of odontoblasts and cementoblasts, and that inhibition of Wnt/ß-catenin signaling may be important for the formation of dentin and cementum during tooth development. Local modulation of Wnt/ß-catenin signaling has therapeutic potential to improve the regeneration of dentin and periodontium.

Antagonistic interactions between osterix and pyrophosphate during cementum formation.

During cementum formation, the key roles of osterix (Osx) and inorganic pyrophosphate (PPi), mainly controlled by nucleotide pyrophosphatase 1 (Npp1; encoded by the Enpp1 gene) and progressive ankylosis protein (Ank), have been demonstrated by animal models displaying altered cementum formation. In this study, we analyzed the relationship of Osx and local PPi during cementum formation using compound mutant mice with their wildtype and corresponding single gene mutants. Importantly, functional defects in PPi regulation led to the induction of Osx expression at the cervical cementum as demonstrated by Enpp1 mutant mice and cementoblasts with the retroviral transduction of small hairpin RNA for Enpp1 or Ank. Conversely, cementoblasts exposed to inorganic PPi or with the enforced expression of Enpp1 or Ank reduced Osx expression in a concentration-dependent manner. Furthermore, the loss of Osx induced the higher expression of Npp1 and Ank at the apical region of the developing tooth root as observed in Osx-deficient mice. The activity of PPi-generating ectoenzymes (nucleoside triphosphate pyrophosphohydrolase, NTPPPHase) and the level of extracellular PPi were significantly increased in Osx-knockdown cementoblasts. However, the formation of ectopic cervical cementum was not completely diminished by inactivation of Osx in Enpp1 mutant mice. In addition, fibroblast growth factor (FGF) receptor 1 (Fgfr1) was strongly localized in cementoblasts lining the acellular cementum and involved in the inhibitory regulation of matrix accumulation and further mineralization by supporting PPi production. Taken together, these results suggest that local PPi suppresses matrix accumulation and further mineralization through an antagonistic interaction with Osx under the synergistic influence of FGF signaling during cementum formation.

Expression of the Hutchinson-Gilford Progeria Mutation Leads to Aberrant Dentin Formation.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare accelerated senescence disease, manifesting dental abnormalities and several symptoms suggestive of premature aging. Although irregular secondary dentin formation in HGPS patients has been reported, pathological mechanisms underlying aberrant dentin formation remain undefined. In this study, we analyzed the mandibular molars of a tissue-specific mouse model that overexpresses the most common HGPS mutation (LMNA, c.1824C > T, p.G608G) in odontoblasts. In the molars of HGPS mutant mice at postnatal week 13, targeted expression of the HGPS mutation in odontoblasts results in excessive dentin formation and pulp obliteration. Circumpulpal dentin of HGPS mutants was clearly distinguished from secondary dentin of wild-type (WT) littermates and its mantle dentin by considering the irregular porous structure and loss of dentinal tubules. However, the dentin was significantly thinner in the molars of HGPS mutants at postnatal weeks 3 and 5 than in those of WT mice. In vitro analyses using MDPC-23, a mouse odontoblastic cell line, showed cellular senescence, defects of signaling pathways and consequential downregulation of matrix protein expression in progerin-expressing odontoblasts. These results indicate that expression of the HGPS mutation in odontoblasts disturbs physiological secondary dentin formation. In addition, progerin-expressing odontoblasts secrete paracrine factors that can stimulate odontogenic differentiation of dental pulp cells. Taken together, our results suggest that the aberrant circumpulpal dentin of HGPS mutants results from defects in physiological secondary dentin formation and consequential pathologic response stimulated by paracrine factors from neighboring progerin-expressing odontoblasts.

Temporospatial localization of acetylcholinesterase activity in the dental epithelium during mouse tooth development.

Acetylcholinesterase (AChE), a principal modulator of cholinergic neurotransmission, also has been demonstrated to be involved in the morphogenetic processes of neuronal and non-neuronal tissues. This study shows that AChE exhibits temporospatial activity in the dental epithelium of the developing mouse tooth. To identify the AChE activity in the mouse tooth during development, we performed enzyme histochemistry on the mouse embryos from embryonic day 13 (E13) to E18 and on the incisors and molars of the neonatal mouse at 10 days after birth (P10). In the developing molars of mouse embryos, AChE activity was not found in the dental epithelium at E13 (bud stage). AChE activity first appeared in the developing cervical loops of the enamel organ at E14 (cap stage), but was not found in the enamel knot. At E18 (bell stage), AChE activity was localized in the inner enamel epithelium except the cervical-loop area. In the incisors and molars of neonatal mice (P10), AChE activity was localized in the inner enamel epithelium of the cervical-loop and enamel-free area. Overall, AChE activity was localized in the differentiating dental epithelium while the activity of butyrylcholinesterse, another cholinesterase, was located primarily in the cells of the dental follicle. The results suggest that AChE may play a role in the histo- and cytodifferentiation of dental epithelium during tooth development.

A Reciprocal Interaction between ß-Catenin and Osterix in Cementogenesis.

Although accumulating evidence indicates that both ß-catenin and osterix (Osx) are essential for bone and tooth development, few studies have investigated the interaction of these two key proteins in the context of cementogenesis. In this study, we used transgenic mice with constitutively active ß-catenin and inactive Osx in the dental mesenchyme to address this question. We found that cementoblasts with constitutively active ß-catenin require Osx to produce excessive cellular cementum, and that ablation of Osx prevents this abnormal accumulation. Importantly, cementoblasts transduced with retrovirus expressing constitutively active ß-catenin exhibited upregulation of Osx expression through direct binding to the promoter region of Osx. Osx regulates Lef1 expression and consequently could regulate T-cell factor/lymphoid enhancer factor (Tcf/Lef) binding activity in Wnt/ß-catenin signaling. However, the loss of Tcf/Lef binding activity by Osx ablation was not rescued by transduction of retrovirus expressing constitutively active ß-catenin or ectopic Lef1 overexpression. These results suggest that the Tcf/Lef binding activity of Wnt/ß-catenin signaling is Osx-dependent during cementogenesis. Moreover, Osx differentially regulates the expression of various Tcf family members, suggesting that Osx regulates cementogenesis by utilizing various Tcf/Lef-dependent mechanisms. This is the first report to show that downstream Osx signaling through Tcf/Lefs is critical for cementogenesis.

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.

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.

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.