RESUMEN
Much information is currently available for molecules in early odontogenesis, but there is limited knowledge regarding terminal cytodifferentiation of ameloblasts and odontoblasts for the determination of normal crown morphology. The present differential display PCR (DD-PCR) revealed that insulin-like growth factor-binding protein 5 (IGFBP5) was differentially expressed in molar tooth germs between the cap (before crown mineralization) and root formation (after crown mineralization) stages. Real-time PCR confirmed that the expression levels of IGFBP1-4 were not significantly changed but those of IGFBP5-7 were upregulated in a time-dependent manner. Immunoreactivities for IGFBP5-7 were hardly seen in molar germs at the cap/early bell stage and protective-stage ameloblasts at the root formation stage. However, the reactivity was strong in odontoblasts and maturation-stage ameloblasts, which are morphologically and functionally characterized by wide intercellular space and active enamel matrix mineralization. The localization of each IGFBP was temporospatial. IGFBP5 was localized in the nuclei of fully differentiated odontoblasts and ameloblasts, while IGFBP6 was localized in the apical cytoplasm of ameloblasts and odontoblasts with dentinal tubules, and IGFBP7 was mainly found in the whole cytoplasm of odontoblasts and the intercellular space of ameloblasts. IGFBP silencing using specific siRNAs upregulated representative genes for dentinogenesis and amelogenesis, such as DMP1 and amelogenin, respectively, and augmented the differentiation media-induced mineralization, which was confirmed by alizarin red s and alkaline phosphatase staining. These results suggest that IGFBP5-7 may play independent and redundant regulatory roles in late-stage odontogenesis by modulating the functional differentiation of ameloblasts and odontoblasts.
Asunto(s)
Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Odontogénesis , Calcificación de Dientes , Amelogénesis/genética , Animales , Esmalte Dental/metabolismo , Dentina/metabolismo , Regulación de la Expresión Génica , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/genética , Diente Molar/metabolismo , Odontoblastos/metabolismo , Odontogénesis/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Calcificación de Dientes/genética , Germen Dentario/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
OBJECTIVES: The purpose of this study was to determine if shear bond resistance of orthodontic brackets bonded to enamel is associated with genes implicated in the enamel mineralization process. METHODS: Ninety-two permanent, caries-free premolars extracted for orthodontic purposes and their associated saliva samples were obtained. Eighteen single nucleotide polymorphisms (SNPs) were studied for association with shear bond resistance. The genes of interest in this study were those previously associated with dental caries by our group. All tooth samples were bonded on the buccal surface with metallic lower lateral brackets, and then subjected to physical debonding. The force required to debond the bracket was recorded in Newtons (N) and converted to a shear bond resistance value in Megapascals (N/mm2). The data were analyzed for statistical significance as compared with the mean shear bond resistance value via PLINK whole genome analysis software. RESULTS: Associations were found between the SNPs for tuftelin (rs7526319, P = 0.004) and amelogenin (rs17878486, P = 0.04) and a higher shear bond resistance. CONCLUSION: The collected data support the proposed hypothesis that genes involved in the mineralization process affect the bonding of orthodontic brackets, and such an association is of value for the field of orthodontics, particularly in evaluating the efficacy of enamel-resin bond strength for patients receiving treatment.
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Esmalte Dental/fisiología , Soportes Ortodóncicos/efectos adversos , Polimorfismo de Nucleótido Simple , Calcificación de Dientes/genética , Adolescente , Estudios Transversales , Femenino , Humanos , Masculino , Factores de Riesgo , Resistencia al Corte , Desmineralización Dental/genéticaRESUMEN
FAM83H was identified as the major causative gene for autosomal dominant hypocalcified amelogenesis imperfect (ADHCAI). The pathogenic mechanism of FAM83H in ADHCAI remains elusive. The present study aims to investigate the effect of Fam83h mutation on the mineralization of mouse ameloblast cell line LS8 and to explore the possible pathogenesis of ADHCAI. Lentivirus package was performed for the plasmids with mouse Fam83h mutant cDNA (c.1186Câ¯>â¯T, M3) and empty vector (Control) and transfected into LS8, which were divided into M3-FLAG and Control groups. Immunoprecipitation, western-blot and immunofluorescence were performed to detect the expression and subcellular localization of Fam83â¯h, CK1α and ß-catenin. ALP activity, ALP staining, expression of the mineralization factors were detected in two groups during mineralization induction. Expression of the mineralization factors was also detected in M3-FLAG and LS8 exposing to pyrvinium pamoate. Compared with the Control, the Fam83h mutation altered the expression and localization of Fam83â¯h, CK1α and ß-catenin in LS8, inhibited the mineralization and down-regulated the expression of mineralization factors in M3-FLAG. Pyrvinium pamoate, an inhibitor of the Wnt/ß-catenin signaling pathway, up-regulated expression of mineralization factors in LS8 and rescued the inhibited mineralization in M3-FLAG. The results indicated that the Fam83h mutation could inhibit the mineralization in ameloblasts by activating Wnt/ß-catenin signaling pathway.
Asunto(s)
Ameloblastos/metabolismo , Proteínas/genética , Proteínas/metabolismo , Amelogénesis/genética , Amelogénesis/fisiología , Amelogénesis Imperfecta/etiología , Amelogénesis Imperfecta/genética , Amelogénesis Imperfecta/metabolismo , Animales , Quinasa de la Caseína I/genética , Quinasa de la Caseína I/metabolismo , Línea Celular , Humanos , Ratones , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Calcificación de Dientes/genética , Calcificación de Dientes/fisiología , Transfección , Vía de Señalización Wnt , beta Catenina/metabolismoRESUMEN
The importance of phosphate (Pi) as an essential component of hydroxyapatite crystals suggests a key role for membrane proteins controlling Pi uptake during mineralization in the tooth. To clarify the involvement of the currently known Pi transporters (Slc17a1, Slc34a1, Slc34a2, Slc34a3, Slc20a1, Slc20a2, and Xpr1) during tooth development and mineralization, we determined their spatiotemporal expression in murine tooth germs from embryonic day 14.5 to postnatal day 15 and in human dental samples from Nolla stages 6 to 9. Using real-time polymerase chain reaction, in situ hybridization, immunohistochemistry, and X-gal staining, we showed that the expression of Slc17a1, Slc34a1, and Slc34a3 in tooth germs from C57BL/6 mice were very low. In contrast, Slc34a2, Slc20a1, Slc20a2, and Xpr1 were highly expressed, mostly during the postnatal stages. The expression of Slc20a2 was 2- to 10-fold higher than the other transporters. Comparable results were obtained in human tooth germs. In mice, Slc34a2 and Slc20a1 were predominantly expressed in ameloblasts but not odontoblasts, while Slc20a2 was detected neither in ameloblasts nor in odontoblasts. Rather, Slc20a2 was highly expressed in the stratum intermedium and the subodontoblastic cell layer. Although Slc20a2 knockout mice did not show enamel defects, mutant mice showed a disrupted dentin mineralization, displaying unmerged calcospherites at the mineralization front. This latter phenotypical finding raises the possibility that Slc20a2 may play an indirect role in regulating the extracellular Pi availability for mineralizing cells rather than a direct role in mediating Pi transport through mineralizing plasma cell membranes. By documenting the spatiotemporal expression of Pi transporters in the tooth, our data support the possibility that the currently known Pi transporters may be dispensable for the initiation of dental mineralization and may rather be involved later during the tooth mineralization scheme.
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Proteínas de Transporte de Fosfato/metabolismo , Calcificación de Dientes/genética , Animales , Femenino , Francia , Humanos , Inmunohistoquímica , Hibridación in Situ , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la Polimerasa , Germen Dentario/embriología , Germen Dentario/metabolismo , Microtomografía por Rayos X , Receptor de Retrovirus Xenotrópico y PolitrópicoRESUMEN
Latent-transforming growth factor beta-binding protein 3 (LTBP-3) is important for craniofacial morphogenesis and hard tissue mineralization, as it is essential for activation of transforming growth factor-ß (TGF-ß). To investigate the role of LTBP-3 in tooth formation we performed micro-computed tomography (micro-CT), histology, and scanning electron microscopy analyses of adult Ltbp3-/- mice. The Ltbp3-/- mutants presented with unique craniofacial malformations and reductions in enamel formation that began at the matrix formation stage. Organization of maturation-stage ameloblasts was severely disrupted. The lateral side of the incisor was affected most. Reduced enamel mineralization, modification of the enamel prism pattern, and enamel nodules were observed throughout the incisors, as revealed by scanning electron microscopy. Molar roots had internal irregular bulbous-like formations. The cementum thickness was reduced, and microscopic dentinal tubules showed minor nanostructural changes. Thus, LTBP-3 is required for ameloblast differentiation and for the formation of decussating enamel prisms, to prevent enamel nodule formation, and for proper root morphogenesis. Also, and consistent with the role of TGF-ß signaling during mineralization, almost all craniofacial bone components were affected in Ltbp3-/- mice, especially those involving the upper jaw and snout. This mouse model demonstrates phenotypic overlap with Verloes Bourguignon syndrome, also caused by mutation of LTBP3, which is hallmarked by craniofacial anomalies and amelogenesis imperfecta phenotypes.
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Amelogénesis/genética , Esmalte Dental/anomalías , Proteínas de Unión a TGF-beta Latente/genética , Ameloblastos/metabolismo , Amelogénesis Imperfecta/genética , Animales , Esmalte Dental/ultraestructura , Genotipo , Masculino , Ratones , Ratones Mutantes , Microscopía Electrónica de Rastreo , Mutación , Osteocondrodisplasias/genética , Fenotipo , Calcificación de Dientes/genética , Factor de Crecimiento Transformador beta/genética , Microtomografía por Rayos XRESUMEN
OBJECTIVES: Incremental markings in dental enamel suggest that the circadian clock may influence the molecular underpinnings orchestrating enamel formation. The aim of this study was to investigate whether the genes and microRNAs (miRNAs) oscillate in a circadian pattern during tooth and enamel development. MATERIAL AND METHODS: Comparative gene and miRNA expression profiling of the first mandibular molar tooth germ isolated at different time-points during the light and night period was performed using microarrays and validated using real-time RT-PCR. Bioinformatic analysis was carried out using Ingenuity Pathway Analysis (IPA), and TargetScan software was used in order to identify computationally predicted miRNA-mRNA target relationships. RESULTS: In total, 439 genes and 32 miRNAs exhibited significantly different (p < 0.05) levels of expression in the light phase compared with the night phase tooth germs. Genes involved in enamel formation, i.e. Amelx, Ambn, Amtn, and Odam, oscillated in a circadian pattern. Furthermore, the circadian clock genes, in particular Clock and Bmal1, oscillated in mouse molar tooth germ during 24-h intervals. The expression of Clock and Bmal1 was inversely correlated with the expression of miR-182 and miR-141, respectively. CONCLUSIONS: MiRNAs, including miR-182 and miR-141, are involved in the control of peripheral circadian rhythms in the developing tooth by regulating the expression of genes coding for circadian transcription factors such as CLOCK and BMAL1. Regulation of circadian rhythms may be important for enamel phenotype, and the morphology of dental enamel may vary between individuals due to differences in circadian profiles.
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Ritmo Circadiano , Regulación del Desarrollo de la Expresión Génica , Diente Molar/crecimiento & desarrollo , Calcificación de Dientes/genética , Germen Dentario/crecimiento & desarrollo , Amelogénesis , Animales , Esmalte Dental/crecimiento & desarrollo , Ratones , MicroARNs , Diente Molar/química , Odontogénesis/fisiología , ARN Mensajero/análisisRESUMEN
INTRODUCTION: This study determined the gene expression profiles of the human coronal pulp (CP) and apical pulp complex (APC) with the aim of explaining differences in their functions. METHODS: Total RNA was isolated from the CP and APC, and gene expression was analyzed using complementary DNA microarray technology. Gene ontology analysis was used to classify the biological function. Quantitative reverse-transcription polymerase chain reaction and immunohistochemical staining were performed to verify microarray data. RESULTS: In the microarray analyses, expression increases of at least 2-fold were present in 125 genes in the APC and 139 genes in the CP out of a total of 33,297 genes. Gene ontology class processes found more genes related to immune responses, cell growth and maintenance, and cell adhesion in the APC, whereas transport and neurogenesis genes predominated in the CP. Quantitative reverse-transcription polymerase chain reaction and immunohistochemical staining confirmed the microarray results, with DMP1, CALB1, and GABRB1 strongly expressed in the CP, whereas SMOC2, SHH, BARX1, CX3CR1, SPP1, COL XII, and LAMC2 were strongly expressed in the APC. CONCLUSIONS: The expression levels of genes related to dentin mineralization, neurogenesis, and neurotransmission are higher in the CP in human immature teeth, whereas those of immune-related and tooth development-related genes are higher in the APC.
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Pulpa Dental/crecimiento & desarrollo , Expresión Génica , Odontogénesis/genética , Ápice del Diente/crecimiento & desarrollo , Adolescente , Receptor 1 de Quimiocinas CX3C , Calbindina 1/genética , Proteínas de Unión al Calcio/genética , Adhesión Celular/genética , Niño , Preescolar , Colágeno Tipo XII/genética , Pulpa Dental/anatomía & histología , Pulpa Dental/citología , Pulpa Dental/diagnóstico por imagen , Proteínas de la Matriz Extracelular/genética , Femenino , Perfilación de la Expresión Génica , Proteínas Hedgehog/genética , Proteínas de Homeodominio/genética , Humanos , Inmunohistoquímica , Laminina/genética , Masculino , Análisis por Micromatrices/métodos , Neurogénesis/genética , Osteopontina/genética , Fosfoproteínas/genética , ARN/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Receptores de Quimiocina/genética , Receptores de GABA-A/genética , República de Corea , Transmisión Sináptica/genética , Ápice del Diente/anatomía & histología , Ápice del Diente/citología , Ápice del Diente/diagnóstico por imagen , Calcificación de Dientes/genética , Factores de Transcripción/genética , Adulto JovenRESUMEN
The molar tooth of the crayfish Cherax quadricarinatus is part of the mandible, and is covered by a layer of apatite (calcium phosphate). This tooth sheds and is regenerated during each molting cycle together with the rest of the exoskeleton. We discovered that molar calcification occurs at the pre-molt stage, unlike calcification of the rest of the new exoskeleton. We further identified a novel molar protein from C. quadricarinatus and cloned its transcript from the molar-forming epithelium. We termed this protein Cq-M13. The temporal level of transcription of Cq-M13 in an NGS library of molar-forming epithelium at different molt stages coincides with the assembly and mineralization pattern of the molar tooth. The predicted protein was found to be related to the pro-resilin family of cuticular proteins. Functionally, in vivo silencing of the transcript caused molt cycle delay and a recombinant version of the protein was found to bind chitin and exhibited elastic properties.
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Proteínas de Artrópodos/metabolismo , Astacoidea/metabolismo , Quitina/metabolismo , Muda/fisiología , Diente/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/genética , Astacoidea/crecimiento & desarrollo , Clonación Molecular , Elasticidad , Epitelio/metabolismo , Expresión Génica , Datos de Secuencia Molecular , Muda/genética , Filogenia , Unión Proteica , Diente/crecimiento & desarrollo , Calcificación de Dientes/genética , Calcificación de Dientes/fisiologíaRESUMEN
Amelogenins are proteins formed by alternative splicing of the amelogenin gene, and are essential for tooth enamel formation. However, the unique functions of various alternatively spliced amelogenins in enamel formation are not well understood. In this study, we determined the spatiotemporal location of amelogenins derived from transcripts containing exon4 (AMG+4) in the enamel matrix, and the relative binding of recombinant AMG+4 to hydroxyapatite (HAP). Immunohistochemistry and mass spectrometry analyses showed that AMG+4 proteins were secreted into the enamel matrix at the early maturation stage. A stage-specific increase in the synthesis of AMG+4 was further supported by our observation that in mice overexpressing leucine-rich amelogenin peptide (TgLRAP), in which ameloblasts differentiate earlier, AMG+4 transcripts were also upregulated earlier. In vitro binding studies, supported by in silico modeling of protein binding to calcium and phosphate, showed that more recombinant AMG+4 bound to hydroxyapatite (HAP) as compared with recombinant AMG-4. The temporal and spatial localization of amelogenins containing exon4 peptide, and their functional differences in HAP binding, suggests that the unique properties of amelogenins containing exon4 cause a specific enhancement of biomineralization related to stabilization of early-formed HAP at the maturation stage.
Asunto(s)
Amelogenina/genética , Exones/genética , Calcificación de Dientes/genética , Empalme Alternativo/genética , Ameloblastos/fisiología , Amelogénesis/genética , Animales , Calcio/metabolismo , Diferenciación Celular/fisiología , Proteínas del Esmalte Dental/genética , Durapatita/metabolismo , Femenino , Espectrometría de Masas , Ratones , Fosfatos/metabolismo , Unión Proteica , Ratas , Ratas Wistar , Proteínas Recombinantes , Factores de Tiempo , Regulación hacia ArribaRESUMEN
Amelotin (AMTN) is a relatively recently discovered enamel protein that is predominantly expressed by ameloblasts during the maturation stage of amelogenesis and is present at lower levels in the junctional epithelium of erupted teeth. Previous studies have suggested a function of this protein in enamel mineralization and cell attachment. Genetic mouse models have been instrumental in defining the role of many enamel-related proteins, but a genetic mouse model lacking the Amtn gene has not been reported. Here, we describe the generation of amelotin-deficient mice and the analysis of their enamel phenotype in comparison with that of wild-type animals. Ablation of AMTN expression resulted in mechanically inferior enamel of mandibular incisors that showed chipping and fractures at the incisal edge. Enamel mineralization was delayed, resulting in hypomineralized inner enamel and structural defects in the outer enamel. Erupted enamel close to the gingival margin showed increased surface roughness. The expression levels of the enamel matrix proteins AMEL, AMBN, ENAM, and ODAM and the enamel proteases MMP-20 and KLK-4 were not significantly altered, although the expression of KLK-4 was delayed. The morphology of ameloblasts showing prominent Tomes' processes during the secretory stage was not altered, and there was no indication of disruption of cell structures or activities, but a residual layer, presumably consisting of organic material, remained at the enamel surface close to the gingival margin. The integrity of the dentogingival attachment at the junctional epithelium appeared unaffected by AMTN deficiency. These observations indicate that AMTN plays a subtle yet critical role in enamel biomineralization, particularly during the establishment of the outer and surface enamel layers. This role appears to be largely independent of other enamel proteins.
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Hipoplasia del Esmalte Dental/genética , Proteínas del Esmalte Dental/genética , Ameloblastos/patología , Amelogénesis/genética , Amelogenina/análisis , Animales , Adhesión Celular/fisiología , Esmalte Dental/ultraestructura , Hipoplasia del Esmalte Dental/patología , Proteínas del Esmalte Dental/análisis , Inserción Epitelial/patología , Encía/patología , Péptidos y Proteínas de Señalización Intracelular , Calicreínas/análisis , Metaloproteinasa 20 de la Matriz/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Fenotipo , Proteínas/análisis , Calcificación de Dientes/genéticaRESUMEN
The coelacanth is the basal-most extant sarcopterygian that has teeth and tooth-like structures, comprising bone, dentin, and enamel or enameloid. Formation of these tissues involves many members of the secretory calcium-binding protein (SCPP) family. In tetrapods, acidic-residue-rich SCPPs are used in mineralization of bone and dentin, whereas Pro/Gln-rich SCPPs participate in enamel formation. Teleosts also employ many SCPPs for tissue mineralization. Nevertheless, the repertoire of SCPPs is largely different in teleosts and tetrapods; hence, filling this gap would be critical to elucidate early evolution of mineralized tissues in osteichthyans. In the present study, we searched for SCPP genes in the coelacanth genome and identified 11, of which two have clear orthologs in both tetrapods and teleosts, seven only in tetrapods, and two in neither of them. Given the divergence times of these vertebrate lineages, our discovery of this many SCPP genes shared between the coelacanth and tetrapods, but not with teleosts, suggests a complicated evolutionary scheme of SCPP genes in early osteichthyans. Our investigation also revealed both conserved and derived characteristics of SCPPs in the coelacanth and other vertebrates. Notably, acidic SCPPs independently evolved various acidic repeats in different lineages, while maintaining high acidity, presumably important for interactions with calcium. Furthermore, the three Pro/Gln-rich SCPP genes, required for mineralizing enamel matrix and confirmed only in tetrapods, were all identified in the coelacanth, strongly suggesting that enamel is equivalent in the coelacanth and tetrapods. This finding corroborates the previous proposition that true enamel evolved much earlier than the origin of tetrapods.
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Proteínas de Unión al Calcio/genética , Evolución Molecular , Peces/genética , Amelogénesis/genética , Animales , Evolución Biológica , Calcificación Fisiológica/genética , Proteínas del Esmalte Dental/genética , Dentina/química , Filogenia , Calcificación de Dientes/genética , Vertebrados/genéticaRESUMEN
Singleton-Merten syndrome (SMS) is a rare disease with a phenotype of dental dysplasia. Currently, the underlying mechanism of this disease is unknown. In order to investigate the functional mechanism of the SMS tooth phenotypes, we isolated dental pulp tissue and established SMS primary pulp cells. These cells exhibited normal morphology and could be maintained in culture. Their ability to express alkaline phosphatase and mineralize was confirmed by in vitro staining. A comparative osteogenesis polymerase chain reaction array analysis was performed revealing 22 genes up-regulated and 8 genes down-regulated greater than 2-fold in SMS versus unaffected pulp cells. Down-regulated genes included ALP, IGF2, TGFBR2 and COL1A1. Collagen type I was reduced in SMS cells as shown by Western blot analysis. Furthermore, matrix metallopeptidase 13 was found to be dramatically increased in SMS pulp cells. Our findings suggest that dentin mineralization is dysregulated in SMS and may contribute to the root phenotype found in this disease.
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Enfermedades de la Aorta/genética , Hipoplasia del Esmalte Dental/genética , Pulpa Dental/citología , Metacarpo/anomalías , Enfermedades Musculares/genética , Odontodisplasia/genética , Osteogénesis/genética , Osteoporosis/genética , Calcificación de Dientes/genética , Calcificación Vascular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Proteínas de la Matriz Extracelular/genética , Humanos , Metacarpo/citologíaRESUMEN
MicroRNAs (miRNAs) play an essential role in regulating cell differentiation either by inhibiting mRNA translation or by inducing its degradation. However, the role of miRNAs in odontoblastic cell differentaion is largely unknown. In the present study, we demonstrate that the expression of miR-27 was significantly increased during MDPC-23 odontoblastic cell differentiation. Furthermore, the up-regulation of miR-27 promotes the differentiation of MDPC-23 odontoblastic cells and accelerates mineralization without cell proliferation. In addition, our results of target gene prediction revealed that the mRNA of adenomatous polyposis coli (APC) associated with Wnt/ß-catenin signaling pathway has miR-27 binding site in the its 3' UTR and is suppressed by miR-27. Subsequentially, the down-regulated APC by miR-27 triggered the activation of Wnt/ß-catenin signaling through accumulation of ß-catenin in the nucleus. Our data suggest that miR-27 promotes MDPC-23 odontoblastic cell differentiation by targeting APC and activating Wnt/ß-catenin signaling. Therefore, miR-27 might be considered a critical candidate as an odontoblastic differentiation molecular target for the development of miRNA based therapeutic agents in the dental medicine.
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Proteína de la Poliposis Adenomatosa del Colon/genética , MicroARNs/genética , MicroARNs/metabolismo , Odontoblastos/citología , Odontoblastos/metabolismo , Vía de Señalización Wnt , beta Catenina/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Línea Celular , Expresión Génica , Ratones , Odontogénesis/genética , Odontogénesis/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Calcificación de Dientes/genética , Calcificación de Dientes/fisiologíaRESUMEN
Enamel fluorosis is an irreversible structural enamel defect following exposure to supraoptimal levels of fluoride during amelogenesis. We hypothesized that fluorosis is associated with excess release of protons during formation of hypermineralized lines in the mineralizing enamel matrix. We tested this concept by analyzing fluorotic enamel defects in wild-type mice and mice deficient in anion exchanger-2a,b (Ae2a,b), a transmembrane protein in maturation ameloblasts that exchanges extracellular Cl(-) for bicarbonate. Defects were more pronounced in fluorotic Ae2a,b (-/-) mice than in fluorotic heterozygous or wild-type mice. Phenotypes included a hypermineralized surface, extensive subsurface hypomineralization, and multiple hypermineralized lines in deeper enamel. Mineral content decreased in all fluoride-exposed and Ae2a,b(-/-) mice and was strongly correlated with Cl(-). Exposure of enamel surfaces underlying maturation-stage ameloblasts to pH indicator dyes suggested the presence of diffusion barriers in fluorotic enamel. These results support the concept that fluoride stimulates hypermineralization at the mineralization front. This causes increased release of protons, which ameloblasts respond to by secreting more bicarbonates at the expense of Cl(-) levels in enamel. The fluoride-induced hypermineralized lines may form barriers that impede diffusion of proteins and mineral ions into the subsurface layers, thereby delaying biomineralization and causing retention of enamel matrix proteins.
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Antiportadores de Cloruro-Bicarbonato/efectos de los fármacos , Fluoruros/efectos adversos , Fluorosis Dental/etiología , Ameloblastos/efectos de los fármacos , Ameloblastos/patología , Amelogénesis/efectos de los fármacos , Amelogénesis/genética , Animales , Bicarbonatos/análisis , Antiportadores de Cloruro-Bicarbonato/análisis , Antiportadores de Cloruro-Bicarbonato/genética , Cloruros/análisis , Colorantes , Esmalte Dental/química , Esmalte Dental/efectos de los fármacos , Esmalte Dental/patología , Proteínas del Esmalte Dental/análisis , Difusión , Femenino , Fluorosis Dental/genética , Fluorosis Dental/patología , Heterocigoto , Homocigoto , Concentración de Iones de Hidrógeno , Indicadores y Reactivos , Ratones , Ratones Noqueados , Minerales/análisis , Fenotipo , Ratas , Ratas Wistar , Calcificación de Dientes/efectos de los fármacos , Calcificación de Dientes/genéticaRESUMEN
Expression of clusterin (Clu) in the murine first molar tooth germ was markedly increased at postnatal developmental stages. The time-course of expression of this gene paralleled those of other genes encoding proteins involved during the secretory phase of odontogenesis, as described previously. Immunohistochemical studies of clusterin in murine molar tooth germs suggested this protein to be located in outer enamel epithelium, regressing enamel organ, secretory ameloblasts, and the dental epithelium connecting the tooth to the oral epithelium at an early eruptive stage. Immunolabelling of transforming growth factor beta-1 (TGF-ß1) revealed it to be located close to clusterin. The levels of expression of Clu and Tgfb1 were markedly decreased following in-vivo transfection with anti-miR-214. In contrast, the expression of several genes associated with regulation of growth and development were increased by this treatment. We suggest that clusterin has functions during secretory odontogenesis and the early eruptive phase. Bioinformatic analysis after treatment with anti-miR-214 suggested that, whilst cellular activities associated with tooth mineralization and eruption were inhibited, activities associated with an alternative developmental activity (i.e. biosynthesis of contractile proteins) appeared to be stimulated. These changes probably occur through regulation mediated by a common cluster of transcription factors and support suggestions that microRNAs (miRNAs) are highly significant as regulators of differentiation during odontogenesis.
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Clusterina/genética , Regulación del Desarrollo de la Expresión Génica , MicroARNs/antagonistas & inhibidores , Diente Molar/crecimiento & desarrollo , Odontogénesis/genética , Calcificación de Dientes/genética , Germen Dentario/crecimiento & desarrollo , Factor de Crecimiento Transformador beta1/genética , Animales , Animales Recién Nacidos , Clusterina/análisis , Perfilación de la Expresión Génica , Inmunohistoquímica , Ratones , Diente Molar/embriología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Germen Dentario/embriología , Germen Dentario/metabolismo , Transfección , Factor de Crecimiento Transformador beta1/análisisRESUMEN
Phosphatases are involved in bone and tooth mineralization, but their mechanisms of action are not completely understood. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) regulates inhibitory extracellular pyrophosphate through its pyrophosphatase activity to control mineral propagation in the matrix; mice without TNAP lack acellular cementum, and have mineralization defects in dentin, enamel, and bone. PHOSPHO1 is a phosphatase found within membrane-bounded matrix vesicles in mineralized tissues, and double ablation of Alpl and Phospho1 in mice leads to a complete absence of skeletal mineralization. Here, we describe mineralization abnormalities in the teeth of Phospho1(-/-) mice, and in compound knockout mice lacking Phospho1 and one allele of Alpl (Phospho1(-/-);Alpl(+/-) ). In wild-type mice, PHOSPHO1 and TNAP co-localized to odontoblasts at early stages of dentinogenesis, coincident with the early mineralization of mantle dentin. In Phospho1 knockout mice, radiography, micro-computed tomography, histology, and transmission electron microscopy all demonstrated mineralization abnormalities of incisor dentin, with the most remarkable findings being reduced overall mineralization coincident with decreased matrix vesicle mineralization in the Phospho1(-/-) mice, and the almost complete absence of matrix vesicles in the Phospho1(-/-);Alpl(+/-) mice, whose incisors showed a further reduction in mineralization. Results from this study support prominent non-redundant roles for both PHOSPHO1 and TNAP in dentin mineralization.
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Fosfatasa Alcalina/genética , Dentina/enzimología , Monoéster Fosfórico Hidrolasas/genética , Calcificación de Dientes/genética , Alelos , Proceso Alveolar/enzimología , Ameloblastos/enzimología , Animales , Apatitas/análisis , Calcificación Fisiológica/genética , Dentinogénesis/genética , Órgano del Esmalte/enzimología , Matriz Extracelular/enzimología , Inmunohistoquímica , Incisivo/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Ratones Mutantes , Microscopía Electrónica de Transmisión , Diente Molar/enzimología , Odontoblastos/enzimología , Osteoblastos/enzimología , Intensificación de Imagen Radiográfica , Germen Dentario/enzimología , Microtomografía por Rayos XRESUMEN
The aim of the present study was to investigate the effect of Sonic hedgehog (Shh) on human dental pulp cells (hDPCs) and the potential of complexes with Shh gene modified hDPCs and porous calcium phosphate cement (CPC) for mineralized tissue formation. hDPCs were cultured and transfected with adenoviral mediated human Shh gene (AdShh). Overexpression of Shh and cell proliferation was tested by real-time PCR analysis, western blotting analysis, and MTT analysis, respectively. The odontoblastic differentiation was assessed by alkaline phosphatase (ALP) activity and real-time PCR analysis on markers of Patched-1 (Ptc-1), Smoothened (Smo), Gli 1, Gli 2, Gli 3, osteocalcin (OCN), dentin matrix protein-1 (DMP-1), and dentin sialophosphoprotein (DSPP). Finally, AdShh-transfected hDPCs were combined with porous CPC and placed subcutaneously in nude mice for 8 and 12 weeks, while AdEGFP-transfected and untransfected hDPCs were treated as control groups. Results indicated that Shh could promote proliferation and odontoblastic differentiation of hDPCs, while Shh/Gli 1 signaling pathway played a key role in this process. Importantly, more mineralized tissue formation was observed in combination with AdShh transfected hDPCs and porous CPC, moreover, the mineralized tissue exhibited dentin-like features such as structures similar to dentin-pulp complex and the positive staining for DSPP protein similar to the tooth tissue. These results suggested that the constructs with AdShh-transfected hDPCs and porous CPC might be a better alternative for dental tissue regeneration.
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Cemento Dental/metabolismo , Pulpa Dental/citología , Pulpa Dental/metabolismo , Dentina/metabolismo , Proteínas Hedgehog/genética , Adenoviridae/genética , Animales , Fosfatos de Calcio/química , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Diferenciación Celular/genética , Proliferación Celular/efectos de los fármacos , Cemento Dental/química , Cemento Dental/ultraestructura , Expresión Génica , Vectores Genéticos/genética , Regeneración Tisular Dirigida , Proteínas Hedgehog/metabolismo , Humanos , Ratones , Odontoblastos/citología , Odontoblastos/efectos de los fármacos , Factores de Tiempo , Calcificación de Dientes/efectos de los fármacos , Calcificación de Dientes/genética , Transducción Genética , Transfección , Alcaloides de Veratrum/farmacologíaRESUMEN
INTRODUCTION: The molecular mechanisms behind odontoblast differentiation remain obscure. Lymphoid enhancer-binding factor 1 (Lef1) is a transcription factor that mediates Wnt signaling and has been suggested to regulate dentin sialophosphoprotein (Dspp) expression in vitro. This study aimed to clarify their precise relationship in the process of odontoblast differentiation in vivo. METHODS: The detailed spatiotemporal expression patterns of Lef1 and Dspp together with other known and putative odontoblast differentiation markers such as P21 and heat-shock protein 25 (Hsp25) were examined by in situ hybridization and immunohistochemistry on paraffin sections of rat incisors and developing molars at postnatal days 1-100. To observe odontoblast regeneration following tooth injury, a cavity was prepared on the upper first molar of 10-week-old rats and the expressions of Lef1 and Dspp were investigated. RESULTS: Following undifferentiated state expressing none of these examined markers, preodontoblasts begun to express P21, Lef1 and Hsp25 according to their progress of differentiation, although Dspp was undetectable. Immature odontoblasts commenced transcribing Dspp simultaneously with dentin calcification. Lef1, Dspp and Hsp25 were co-expressed in mature odontoblasts. In contrast to continuously growing incisors, Lef1, Dspp and P21 were down-regulated in the resting odontoblasts in molars when primary dentin formation was completed. Remarkably, Lef1 expression also preceded Dspp expression in newly differentiated odontoblast-like cells during the pulpal healing process after tooth injury. CONCLUSIONS: Lef1 expression precedes Dspp expression without exception in both primary and reparative dentinogeneses. Our results suggest that Lef1 might play a key role in odontoblast differentiation through regulating Dspp expression.
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Proteínas de la Matriz Extracelular/genética , Factor de Unión 1 al Potenciador Linfoide/genética , Odontoblastos/fisiología , Fosfoproteínas/genética , Sialoglicoproteínas/genética , Animales , Diferenciación Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Dentina/crecimiento & desarrollo , Dentinogénesis/genética , Proteínas de Choque Térmico HSP27/genética , Incisivo/crecimiento & desarrollo , Diente Molar/citología , Diente Molar/crecimiento & desarrollo , Diente Molar/lesiones , Inhibidores de Proteínas Quinasas/análisis , Ratas , Ratas Wistar , Regeneración/genética , Calcificación de Dientes/genética , Transcripción Genética/genética , Vía de Señalización Wnt/genéticaRESUMEN
Dentin sialophosphoprotein (DSPP) is a large precursor protein that is proteolytically processed into a NH2 -terminal fragment [composed of dentin sialoprotein (DSP) and a proteoglycan form (DSP-PG)] and a COOH-terminal fragment [dentin phosphoprotein (DPP)]. In vitro studies indicate that DPP is a strong initiator and regulator of hydroxyapatite crystal formation and growth, but the role(s) of the NH2 -terminal fragment of DSPP (i.e., DSP and DSP-PG) in dentinogenesis remain unclear. This study focuses on the function of the NH2 -terminal fragment of DSPP in dentinogenesis. Here, transgenic (Tg) mouse lines expressing the NH2 -terminal fragment of DSPP driven by a 3.6-kb type I collagen promoter (Col 1a1) were generated and cross-bred with Dspp null mice to obtain mice that express the transgene but lack the endogenous Dspp (Dspp KO/DSP Tg). We found that dentin from the Dspp KO/DSP Tg mice was much thinner, more poorly mineralized, and remarkably disorganized compared with dentin from the Dspp KO mice. The fact that Dspp KO/DSP Tg mice exhibited more severe dentin defects than did the Dspp null mice indicates that the NH2 -terminal fragment of DSPP may inhibit dentin mineralization or may serve as an antagonist against the accelerating action of DPP and serve to prevent predentin from being mineralized too rapidly during dentinogenesis.
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Dentina/metabolismo , Dentinogénesis/fisiología , Proteínas de la Matriz Extracelular/metabolismo , Fosfoproteínas/metabolismo , Sialoglicoproteínas/metabolismo , Calcificación de Dientes/fisiología , Animales , Dentina/química , Dentinogénesis/genética , Proteínas de la Matriz Extracelular/química , Proteínas de la Matriz Extracelular/genética , Inmunohistoquímica , Ratones , Ratones Noqueados , Microscopía Electrónica de Rastreo , Fosfoproteínas/química , Fosfoproteínas/genética , ARN Mensajero , Reacción en Cadena en Tiempo Real de la Polimerasa , Sialoglicoproteínas/química , Sialoglicoproteínas/genética , Calcificación de Dientes/genética , Microtomografía por Rayos XRESUMEN
Sphingomyelin phosphodiesterase 3 (Smpd3) encodes a membrane-bound enzyme that cleaves sphingomyelin to generate several bioactive metabolites. A recessive mutation called fragilitas ossium (fro) in the Smpd3 gene leads to impaired mineralization of bone and tooth extracellular matrix (ECM) in fro/fro mice. In teeth from fro/fro mice at various neonatal ages, radiography and light and electron microscopy showed delayed mantle dentin mineralization and a consequent delay in enamel formation as compared with that in control +/fro mice. These tooth abnormalities progressively improved with time. Immunohistochemistry showed expression of SMPD3 by dentin-forming odontoblasts. SMPD3 deficiency, however, did not affect the differentiation of these cells, as shown by osterix and dentin sialophosphoprotein expression. Using a transgenic mouse rescue model (fro/fro; Col1a1-Smpd3) in which Smpd3 expression is driven by a murine Col1a1 promoter fragment active in osteoblasts and odontoblasts, we demonstrate a complete correction of the tooth mineralization delays. In conclusion, analysis of these data demonstrates that Smpd3 expression in odontoblasts is required for tooth mineralization.