RESUMO
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.
Assuntos
Proteínas de Transporte de Fosfato/metabolismo , Calcificação de Dente/genética , Animais , Feminino , França , Humanos , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase em Tempo Real , Germe de Dente/embriologia , Germe de Dente/metabolismo , Microtomografia por Raio-X , Receptor do Retrovírus Politrópico e XenotrópicoRESUMO
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.
Assuntos
Amelogênese/genética , Esmalte Dentário/anormalidades , Proteínas de Ligação a TGF-beta Latente/genética , Ameloblastos/metabolismo , Amelogênese Imperfeita/genética , Animais , Esmalte Dentário/ultraestrutura , Genótipo , Masculino , Camundongos , Camundongos Mutantes , Microscopia Eletrônica de Varredura , Mutação , Osteocondrodisplasias/genética , Fenótipo , Calcificação de Dente/genética , Fator de Crescimento Transformador beta/genética , Microtomografia por Raio-XRESUMO
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.
Assuntos
Doenças da Aorta/genética , Hipoplasia do Esmalte Dentário/genética , Polpa Dentária/citologia , Metacarpo/anormalidades , Doenças Musculares/genética , Odontodisplasia/genética , Osteogênese/genética , Osteoporose/genética , Calcificação de Dente/genética , Calcificação Vascular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Proteínas da Matriz Extracelular/genética , Humanos , Metacarpo/citologiaRESUMO
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.
Assuntos
Proteína da Polipose Adenomatosa do Colo/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Odontoblastos/citologia , Odontoblastos/metabolismo , Via de Sinalização Wnt , beta Catenina/metabolismo , Animais , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Expressão Gênica , Camundongos , Odontogênese/genética , Odontogênese/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Calcificação de Dente/genética , Calcificação de Dente/fisiologiaRESUMO
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.
Assuntos
Cemento Dentário/metabolismo , Polpa Dentária/citologia , Polpa Dentária/metabolismo , Dentina/metabolismo , Proteínas Hedgehog/genética , Adenoviridae/genética , Animais , Fosfatos de Cálcio/química , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Diferenciação Celular/genética , Proliferação de Células/efeitos dos fármacos , Cemento Dentário/química , Cemento Dentário/ultraestrutura , Expressão Gênica , Vetores Genéticos/genética , Regeneração Tecidual Guiada , Proteínas Hedgehog/metabolismo , Humanos , Camundongos , Odontoblastos/citologia , Odontoblastos/efeitos dos fármacos , Fatores de Tempo , Calcificação de Dente/efeitos dos fármacos , Calcificação de Dente/genética , Transdução Genética , Transfecção , Alcaloides de Veratrum/farmacologiaRESUMO
Tooth enamel has the highest degree of biomineralization of all vertebrate hard tissues. During the secretory stage of enamel formation, ameloblasts deposit an extracellular matrix that is in direct contact with the ameloblast plasma membrane. Although it is known that integrins mediate cell-matrix adhesion and regulate cell signaling in most cell types, the receptors that regulate ameloblast adhesion and matrix production are not well characterized. We hypothesized that αvß6 integrin is expressed in ameloblasts where it regulates biomineralization of enamel. Human and mouse ameloblasts were found to express both ß6 integrin mRNA and protein. The maxillary incisors of Itgb6(-/-) mice lacked yellow pigment and their mandibular incisors appeared chalky and rounded. Molars of Itgb6(-/-) mice showed signs of reduced mineralization and severe attrition. The mineral-to-protein ratio in the incisors was significantly reduced in Itgb6(-/-) enamel, mimicking hypomineralized amelogenesis imperfecta. Interestingly, amelogenin-rich extracellular matrix abnormally accumulated between the ameloblast layer of Itgb6(-/-) mouse incisors and the forming enamel surface, and also between ameloblasts. This accumulation was related to increased synthesis of amelogenin, rather than to reduced removal of the matrix proteins. This was confirmed in cultured ameloblast-like cells, in which αvß6 integrin was not an endocytosis receptor for amelogenins, although it participated in cell adhesion on this matrix indirectly via endogenously produced matrix proteins. In summary, integrin αvß6 is expressed by ameloblasts and it plays a crucial role in regulating amelogenin deposition and/or turnover and subsequent enamel biomineralization.
Assuntos
Ameloblastos/metabolismo , Amelogênese Imperfeita/metabolismo , Antígenos de Neoplasias/metabolismo , Esmalte Dentário/metabolismo , Integrinas/metabolismo , Atrito Dentário/prevenção & controle , Ameloblastos/patologia , Amelogênese Imperfeita/complicações , Amelogênese Imperfeita/genética , Amelogenina/metabolismo , Animais , Antígenos de Neoplasias/genética , Adesão Celular/genética , Células Cultivadas , Esmalte Dentário/patologia , Matriz Extracelular/metabolismo , Integrinas/genética , Camundongos , Camundongos Knockout , Atrito Dentário/etiologia , Calcificação de Dente/genética , Desmineralização do DenteRESUMO
Bone sialoprotein (BSP) is an extracellular matrix protein found in mineralized tissues of the skeleton and dentition. BSP is multifunctional, affecting cell attachment and signaling through an RGD integrin-binding region, and acting as a positive regulator for mineral precipitation by nucleating hydroxyapatite crystals. BSP is present in cementum, the hard tissue covering the tooth root that anchors periodontal ligament (PDL) attachment. To test our hypothesis that BSP plays an important role in cementogenesis, we analyzed tooth development in a Bsp null ((-/-)) mouse model. Developmental analysis by histology, histochemistry, and SEM revealed a significant reduction in acellular cementum formation on Bsp (-/-) mouse molar and incisor roots, and the cementum deposited appeared hypomineralized. Structural defects in cementum-PDL interfaces in Bsp (-/-) mice caused PDL detachment, likely contributing to the high incidence of incisor malocclusion. Loss of BSP caused progressively disorganized PDL and significantly increased epithelial down-growth with aging. Bsp (-/-) mice displayed extensive root and alveolar bone resorption, mediated by increased RANKL and the presence of osteoclasts. Results collected here suggest that BSP plays a non-redundant role in acellular cementum formation, likely involved in initiating mineralization on the root surface. Through its importance to cementum integrity, BSP is essential for periodontal function.
Assuntos
Cementogênese/fisiologia , Cemento Dentário/patologia , Sialoproteína de Ligação à Integrina/fisiologia , Fosfatase Alcalina/análise , Perda do Osso Alveolar/patologia , Animais , Dentina/ultraestrutura , Epitélio/patologia , Incisivo/ultraestrutura , Sialoproteína de Ligação à Integrina/genética , Queratinas/análise , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Varredura , Dente Molar/ultraestrutura , Odontogênese/genética , Odontogênese/fisiologia , Osteoclastos/patologia , Osteopontina/análise , Perda da Inserção Periodontal/patologia , Ligamento Periodontal/patologia , Ligante RANK/análise , Reabsorção da Raiz/patologia , Calcificação de Dente/genética , Calcificação de Dente/fisiologia , Colo do Dente/ultraestrutura , Microtomografia por Raio-XRESUMO
The Transforming growth factor ß (Tgf-ß) pathway, by signaling via the activation of Smad transcription factors, induces the expression of many diverse downstream target genes thereby regulating a vast array of cellular events essential for proper development and homeostasis. In order for a specific cell type to properly interpret the Tgf-ß signal and elicit a specific cellular response, cell-specific transcriptional co-factors often cooperate with the Smads to activate a discrete set of genes in the appropriate temporal and spatial manner. Here, via a conditional knockout approach, we show that mice mutant for Forkhead Box O transcription factor FoxO1 exhibit an enamel hypomaturation defect which phenocopies that of the Smad3 mutant mice. Furthermore, we determined that both the FoxO1 and Smad3 mutant teeth exhibit changes in the expression of similar cohort of genes encoding enamel matrix proteins required for proper enamel development. These data raise the possibility that FoxO1 and Smad3 act in concert to regulate a common repertoire of genes necessary for complete enamel maturation. This study is the first to define an essential role for the FoxO family of transcription factors in tooth development and provides a new molecular entry point which will allow researchers to delineate novel genetic pathways regulating the process of biomineralization which may also have significance for studies of human tooth diseases such as amelogenesis imperfecta.
Assuntos
Amelogênese/genética , Esmalte Dentário/metabolismo , Fatores de Transcrição Forkhead/fisiologia , Calcificação de Dente/genética , Animais , Calcificação Fisiológica/genética , Calcificação Fisiológica/fisiologia , Esmalte Dentário/crescimento & desenvolvimento , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Testes de Dureza , Integrases/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Smad3/genética , Proteína Smad3/metabolismo , Proteína Smad3/fisiologia , Doenças Dentárias/genética , Doenças Dentárias/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
Dickkopf-related protein 1 (DKK1) is a potent inhibitor of Wnt/ß-catenin signaling. Dkk1-null mutant embryos display severe defects in head induction. Conversely, targeted expression of Dkk1 in dental epithelial cells leads to the formation of dysfunctional enamel knots and subsequent tooth defects during embryonic development. However, its role in post-natal dentinogenesis is largely unknown. To address this issue, we studied the role of DKK1 in post-natal dentin development using 2.3-kb Col1a1-Dkk1 transgenic mice, with the following key findings: (1) The Dkk1 transgene was highly expressed in pulp and odontoblast cells during post-natal developmental stages; (2) the 1(st) molar displayed short roots, an enlarged pulp/root canal region, and a decrease in the dentin formation rate; (3) a small malformed second molar and an absent third molar; (4) an increase of immature odontoblasts, few mature odontoblasts, and sharply reduced dentinal tubules; and (5) a dramatic change in Osx and nestin expression. We propose that DKK1 controls post-natal mandibular molar dentin formation either directly or indirectly via the inhibition of Wnt signaling at the following aspects: (i) post-natal dentin formation, (ii) formation and/or maintenance of the dentin tubular system, (iii) mineralization of the dentin, and (iv) regulation of molecules such as Osx and nestin.
Assuntos
Dentinogênese/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Dente Molar/crescimento & desenvolvimento , Anormalidades Dentárias/genética , Animais , Polpa Dentária/metabolismo , Dentina/anormalidades , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Proteínas de Filamentos Intermediários/biossíntese , Mandíbula/diagnóstico por imagem , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/biossíntese , Nestina , Odontoblastos/metabolismo , Ligamento Periodontal/crescimento & desenvolvimento , Transdução de Sinais , Fator de Transcrição Sp7 , Calcificação de Dente/genética , Fatores de Transcrição/biossíntese , Transgenes , Regulação para Cima , Proteínas Wnt/metabolismo , Microtomografia por Raio-XRESUMO
Inorganic phosphate (Pi) is required in many biological processes, including signaling cascades, skeletal development, tooth mineralization, and nucleic acid synthesis. Recently, we showed that Pi transport in osteoblasts, mediated by Slc20a1, a member of the type III sodium-dependent phosphate transporter family, is indispensable for osteoid mineralization in rapidly growing rat bone. In addition, we found that bone mineral density decreased slightly with dysfunction of Pi homeostasis in aged transgenic rats overexpressing mouse Slc20a1 (Slc20a1-Tg). Bone and tooth share certain common molecular features, and thus, we focused on tooth development in Slc20a1-Tg mandibular incisors in order to determine the role of Slc20a1 in tooth mineralization. Around the time of weaning, there were no significant differences in serologic parameters between wild-type and Slc20a1-Tg rats. However, histological analysis showed that Slc20a1-Tg ameloblasts formed clusters in the papillary layer during the maturation stage as early as 4 weeks of age. These pathologies became more severe with age and included the formation of cyst-like or multilayer ameloblast structures, accompanied by a chalky white appearance with abnormal attrition and fracture. Hyperphosphatemia was also observed in aging Slc20a1-Tg rats. Micro-computed tomography and electron probe microanalysis revealed impairments in enamel, such as delayed mineralization and hypomineralization. Our results suggest that enamel formation is sensitive to imbalances in Pit1-mediated cellular function as seen in bone, although these processes are under the control of systemic Pi homeostasis.
Assuntos
Esmalte Dentário/metabolismo , Incisivo/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Animais , Esmalte Dentário/crescimento & desenvolvimento , Esmalte Dentário/ultraestrutura , Hipoplasia do Esmalte Dentário/genética , Expressão Gênica , Incisivo/crescimento & desenvolvimento , Masculino , Camundongos , Ratos , Ratos Transgênicos , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo , Calcificação de Dente/genética , Calcificação de Dente/fisiologia , Transfecção , Regulação para Cima/genética , Regulação para Cima/fisiologiaRESUMO
Mutations of the matrix metalloproteinase 20 (MMP20, enamelysin) gene cause autosomal-recessive amelogenesis imperfecta, and Mmp20 ablated mice also have malformed dental enamel. Here we showed that Mmp20 null mouse secretory-stage ameloblasts maintain a columnar shape and are present as a single layer of cells. However, the maturation-stage ameloblasts from null mouse cover extraneous nodules of ectopic calcified material formed at the enamel surface. Remarkably, nodule formation occurs in null mouse enamel when MMP20 is normally no longer expressed. The malformed enamel in Mmp20 null teeth was loosely attached to the dentin and the entire enamel layer tended to separate from the dentin, indicative of a faulty dentino-enamel junction (DEJ). The enamel rod pattern was also altered in Mmp20 null mice. Each enamel rod is formed by a single ameloblast and is a mineralized record of the migration path of the ameloblast that formed it. The enamel rods in Mmp20 null mice were grossly malformed or absent, indicating that the ameloblasts do not migrate properly when backing away from the DEJ. Thus, MMP20 is required for ameloblast cell movement necessary to form the decussating enamel rod patterns, for the prevention of ectopic mineral formation, and to maintain a functional DEJ.
Assuntos
Ameloblastos/enzimologia , Amelogênese/genética , Esmalte Dentário/anormalidades , Esmalte Dentário/ultraestrutura , Dentina/anatomia & histologia , Metaloproteinase 20 da Matriz/genética , Metaloproteinase 20 da Matriz/fisiologia , Ameloblastos/citologia , Ameloblastos/fisiologia , Animais , Calcinose/genética , Movimento Celular , Esmalte Dentário/enzimologia , Órgão do Esmalte/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Calcificação de Dente/genéticaRESUMO
Enamelin is a secreted glycoprotein that is critical for dental enamel formation. Ameloblasts in enamelin (Enam) null mice develop atypical features that include the absence of a Tomes' process, expanded endoplasmic reticulum, apparent loss of polarity, and pooling of extracellular matrix in all directions, including between ameloblasts and the stratum intermedium. We hypothesized that ameloblast pathological changes may be associated with increased cell apoptosis. Our objective was to assess apoptotic activity in maxillary first molars of wild-type, Enam(+/-), and Enam(-/-) mice at postnatal days 5, 7, 9, 14, and 17. Mouse maxillae were characterized by light microscopy after terminal deoxynucleotidyl transferase (TdT)-mediated biotin-dUTP nick-end labelling (TUNEL) or 5-bromo-2'-deoxyuridine (BrdU) staining. Following the initial deposition of dentin matrix, ameloblasts became highly dysplastic and no enamel crystal ribbons were deposited. Ameloblast apoptosis was observed in the Enam null mice starting in the secretory stage and with no apparent alteration in cell proliferation. We conclude that in the absence of enamelin and subsequent shutdown of enamel formation, ameloblasts undergo pathological changes early in the secretory stage that are evident as radically altered cell morphology, detachment from the tooth surface, apoptosis, and formation of ectopic calcifications both outside and inside the dystrophic enamel organ.
Assuntos
Ameloblastos/patologia , Amelogênese Imperfeita/genética , Amelogênese Imperfeita/patologia , Proteínas do Esmalte Dentário/genética , Animais , Apoptose , Bromodesoxiuridina/metabolismo , Calcinose , Proliferação de Células , Cristalização , Marcação In Situ das Extremidades Cortadas , Camundongos , Camundongos Knockout , Dente Molar/patologia , Calcificação de Dente/genética , beta-Galactosidase/genéticaRESUMO
Hypophosphatasia is caused by mutations of the tissue-non-specific alkaline phosphatase (TNSALP) gene with deficiency of dentin structure. The aim of this study was to examine whether TNSALP mutation in dental pulp cells contributes to dentin dysplasia in hypophosphatasia. Mutation analysis showed that compound heterozygous mutations of TNSALP were identified in three hypophosphatasia patients, including 3 novel mutation sites. Exfoliated teeth from the patients showed abnormal dentin mineralization and loss of cementum, as assessed by ground sections and scanning electron microscope analysis. Dental pulp cells isolated from one of the patients showed a significantly reduced TNSALP activity and mineralization capacity when compared with those in dental pulp cells from the unaffected individuals. Our results suggested that dentin dysplasia in hypophosphatasia may be associated with the decreased mineralization ability of dental pulp cells.
Assuntos
Fosfatase Alcalina/genética , Polpa Dentária/patologia , Hipofosfatasia/genética , Adenina , Células Cultivadas , Criança , Pré-Escolar , Cemento Dentário/anormalidades , Esmalte Dentário/patologia , Dentina/anormalidades , Displasia da Dentina/genética , Éxons/genética , Variação Genética/genética , Guanina , Heterozigoto , Histidina/genética , Humanos , Íntrons/genética , Microscopia Eletrônica de Varredura , Mutação/genética , Mutação de Sentido Incorreto/genética , Fenótipo , Sítios de Splice de RNA/genética , Timina , Calcificação de Dente/genética , Esfoliação de Dente/genética , Tirosina/genéticaRESUMO
AIM: To investigate the role of Wnt5a in the process of differentiation of human dental papilla cells (HDPCs). METHODOLOGY: Recombinant adenovirus encoding full-length Wnt5a cDNA was constructed to investigate the biological role of Wnt5a on the differentiation of HDPCs. The effect of Wnt5a on HDPCs differentiation was determined by ALP activity assay, ALP staining and mineral induction assay. Mineralization-related gene expressions were assessed by RT-PCR. RESULTS: Immunostaining revealed Wnt5a expression in the odontoblast layer and dental papilla tissue. Over-expression of Wnt5a by transfecting HDPCs with an Wnt5a-carrying construct increased ALPase activity and the formation of mineralized nodules of HDPCs. RT-PCR analysis showed that the expressions of mineralization-related genes, such as bone sialoprotein, collagen type I, osteonectin, osteopontin (OCN), dentine matrix protein-1 were up-regulated by Wnt5a. CONCLUSIONS: Wnt5a promoted differentiation of HDPCs.
Assuntos
Papila Dentária/citologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Wnt/fisiologia , Adenoviridae/genética , Fosfatase Alcalina/análise , Fosfatase Alcalina/genética , Técnicas de Cultura de Células , Diferenciação Celular/fisiologia , Colágeno Tipo I/análise , Colágeno Tipo I/genética , DNA Complementar/genética , Proteínas da Matriz Extracelular/análise , Proteínas da Matriz Extracelular/genética , Regulação da Expressão Gênica/genética , Humanos , Sialoproteína de Ligação à Integrina , Odontoblastos/citologia , Osteonectina/análise , Osteonectina/genética , Osteopontina/análise , Osteopontina/genética , Fosfoproteínas/análise , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes , Sialoglicoproteínas/análise , Sialoglicoproteínas/genética , Calcificação de Dente/genética , Transfecção , Proteínas Wnt/genética , Proteína Wnt-5aRESUMO
Important factors involved in odontogenesis in mouse dental papillae disappear between the pre- and post-natal stages of development. Therefore, we hypothesized that certain genes involved in odontogenesis in dental papillae were subject to pre-/post-natal down-regulation. Our goal was to identify, by microarray analysis, which genes were down-regulated. Dental papillae were isolated from embryonic 16-day-, 18-day- (E16, E18), and post-natal 3-day-old (P3) murine first mandibular molar germs and analyzed by microarray. The number of down-regulated genes was 2269 between E16 and E18, and 3130 between E18 and P3. Drastic down-regulation (fold change > 10.0) of Adamts4, Aldha1a2, and Lef1 was observed at both E16 and E18, and quantitative RT-PCR revealed a post-natal reduction in their expression (Adamts4, 1/3; Aldh1a2, 1/13; and Lef1, 1/37). These results suggest that down-regulation of these three genes is an important factor in normal odontogenesis in dental papillae.
Assuntos
Papila Dentária/citologia , Polpa Dentária/citologia , Regulação para Baixo/genética , Odontogênese/genética , Proteínas ADAM/análise , Proteínas ADAM/genética , Proteína ADAMTS4 , Aldeído Desidrogenase/análise , Aldeído Desidrogenase/genética , Família Aldeído Desidrogenase 1 , Animais , Morte Celular/genética , Papila Dentária/embriologia , Polpa Dentária/embriologia , Células Epiteliais/citologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Hibridização In Situ , Fator 1 de Ligação ao Facilitador Linfoide/análise , Fator 1 de Ligação ao Facilitador Linfoide/genética , Camundongos , Camundongos Endogâmicos ICR , Odontoblastos/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Pró-Colágeno N-Endopeptidase/análise , Pró-Colágeno N-Endopeptidase/genética , Retinal Desidrogenase , Calcificação de Dente/genética , Germe de Dente/citologia , Germe de Dente/embriologiaRESUMO
Recent studies showed that local injection or upregulation of OPG gene would result in early temporal retardation of tooth development. It was assumed that this retardation might cause defective tooth mineralization and pulp formation as the long-term effects. However, since those OPG treatments were transient, any possible long-term effects of OPG addition could not be assessed previously. In the present study, a high-resolution microCT was used to evaluate the long-term effect of OPG gene deprivation on the mineralization and morphology of mouse tooth. Our results showed that the mineralization of alveolar bone in OPG(-/-) mouse tooth was decreased while those of enamel and dentin were increased, compared with the wild-type (WT) group. The labial and lingual dentin thicknesses of OPG(-/-) group were significantly higher and with larger area in enamel and dentin than those of WT group. The size of pulp chamber was also substantially decreased in OPG(-/-) mouse incisor. Different responses in mineralization and morphogenesis to OPG gene deprivation were found between bone and tooth. These effects may be independent of the early odontogenesis, and further studies are warranted to investigate the molecular mechanism of the effect of OPG gene expression on bone formation and later tooth development.
Assuntos
Perda do Osso Alveolar/metabolismo , Esmalte Dentário/metabolismo , Displasia da Dentina/metabolismo , Dentina/metabolismo , Calcificação de Dente/genética , Perda do Osso Alveolar/diagnóstico por imagem , Perda do Osso Alveolar/genética , Perda do Osso Alveolar/patologia , Animais , Densidade Óssea , Displasia da Dentina/genética , Displasia da Dentina/patologia , Expressão Gênica , Genótipo , Camundongos , Camundongos Knockout , Osteoprotegerina , Fenótipo , Microtomografia por Raio-XRESUMO
INTRODUCTION: The Wnt signaling pathway plays an important role in tissue development by acting on proliferation, differentiation, and cell fate decisions. Because the role of Wnt6 in tooth development was still unknown, the purpose of this study was to investigate the role of Wnt6 in tooth morphogenesis and dental tissue mineralization by elucidating its effect on human dental papilla cells (hDPCs) in vitro. METHODS: Human dental papilla cells were enzymatically separated from tooth germs. Recombinant adenovirus encoding full-length Wnt6 cDNA was constructed to overexpress Wnt6, and the biologic effects of Wnt6 on hDPCs were investigated. Wnt6-transduced changes in hDPC proliferation were examined by means of a 5-bromodeoxyuridine (BrdU) incorporation assay and cell cycle analysis. Wnt6-transduced changes in hDPC differentiation were investigated by evaluating alkaline phosphatase (ALPase) activity, by a mineralization assay, and analysis of mineralization-related gene expression including ALP, type I collagen (Col I), osteonectin (ON), osteopontin (OPN), bone sialoprotein (BSP), and dentin matrix protein-1 (DMP-1). RESULTS: Wnt6 overexpression had no significant effect on the proliferation of hDPCs by BrdU incorporation assay and flow cytometric analysis. Wnt6 enhanced differentiation of hDPCs into functional odontoblast-like cells with up-regulated activity of ALPase and the expression of mineralization-related genes such as ALP, Col I, ON, OPN, BSP, and DMP-1. Wnt6 overexpression also promoted the mineralization of hDPCs. CONCLUSIONS: Our findings verified that Wnt6 plays an important role in tooth development by promoting hDPC differentiation, without significant effects on hDPC proliferation.
Assuntos
Proliferação de Células , Papila Dentária/fisiologia , Odontogênese/fisiologia , Calcificação de Dente/fisiologia , Proteínas Wnt/fisiologia , Fosfatase Alcalina/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , DNA Complementar , Papila Dentária/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Odontogênese/genética , Proteínas Recombinantes , Calcificação de Dente/genética , Germe de Dente/citologia , Transdução Genética , Proteínas Wnt/genéticaRESUMO
BACKGROUND: Cementogenesis is sensitive to altered local phosphate levels; thus, we hypothesized a cementum phenotype, likely of decreased formation, would be present in the teeth of X-linked hypophosphatemic (Hyp) mice. Mutations in the phosphate-regulating gene with homologies to endopeptidases on the X chromosome (Phex) cause X-linked hypophosphatemia, characterized by rickets, osteomalacia, and hypomineralized dentin formation, a phenotype recapitulated in the Hyp mouse homolog. Here, we report a developmental study of tooth root formation in Hyp mouse molars, focusing on dentin and cementum. METHODS: Light and transmission electron microscopy were used to study molar tissues from wild-type (WT) and Hyp mice. Demineralized and hematoxylin and eosin-stained tissues at developmental stages 23 to 96 days postcoital (dpc) were examined by light microscopy. Immunohistochemistry methods were used to detect bone sialoprotein (BSP) distribution in Hyp and WT mouse molar tissues, and transmission electron microscopy was used to study similar molar tissues in the non-demineralized state. RESULTS: Dentin in Hyp mice exhibited mineralization defects by 33 dpc, as expected, but this defect was partially corrected by 96 dpc. In support of our hypothesis, a cementum phenotype was detected using a combination of immunohistochemistry and transmission electron microscopy, which included thinner BSP-positive staining within the cementum, discontinuous mineralization, and a globular appearance compared to WT controls. CONCLUSION: Mutations in the phosphate-regulating Phex gene of the Hyp mouse resulted in defective cementum development.
Assuntos
Cementogênese/genética , Cemento Dentário/anormalidades , Raquitismo Hipofosfatêmico Familiar/patologia , Doenças Genéticas Ligadas ao Cromossomo X , Animais , Cemento Dentário/patologia , Dentina/anormalidades , Dentina/patologia , Dentinogênese/genética , Feminino , Idade Gestacional , Imuno-Histoquímica , Sialoproteína de Ligação à Integrina , Masculino , Camundongos , Camundongos Mutantes , Microscopia Eletrônica de Transmissão , Dente Molar/anormalidades , Dente Molar/patologia , Mutação/genética , Odontogênese/genética , Endopeptidase Neutra Reguladora de Fosfato PHEX/genética , Fenótipo , Sialoglicoproteínas/análise , Calcificação de Dente/genética , Germe de Dente/anormalidades , Germe de Dente/patologia , Raiz Dentária/anormalidades , Raiz Dentária/patologiaRESUMO
Kallikrein-4 (KLK4) is a serine protease expressed during enamel maturation, and proteolytic processing of the enamel matrix by KLK4 is critical for proper enamel formation. KLK4 is secreted as an inactive zymogen (pro-KLK4), and identification of its activator remains elusive. Dipeptidyl peptidase I (DPPI) is a cysteine aminopeptidase that can activate several serine proteases. In this study, we sought to examine DPPI expression in mouse enamel organ and determine if DPPI could activate KLK4. Real-time PCR showed DPPI expression throughout amelogenesis, with highest expression at maturation, and immunohistochemical staining of mouse incisors confirmed DPPI expression by ameloblasts. We demonstrate in vitro that DPPI activates pro-KLK4 to cleave a fluorogenic peptide containing a KLK4 cleavage site. Examination of mature enamel from DPPI null mice by FTIR showed no significant accumulation of protein; however, microhardness testing revealed that loss of DPPI expression significantly reduced enamel hardness.
Assuntos
Amelogênese/fisiologia , Catepsina C/metabolismo , Esmalte Dentário/enzimologia , Calicreínas/metabolismo , Calcificação de Dente/fisiologia , Amelogênese/genética , Animais , Catepsina C/genética , Esmalte Dentário/ultraestrutura , Proteínas do Esmalte Dentário/genética , Proteínas do Esmalte Dentário/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Calicreínas/genética , Mandíbula , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Camundongos Mutantes , Dente Molar/ultraestrutura , Proteínas Recombinantes , Especificidade da Espécie , Calcificação de Dente/genéticaRESUMO
OBJECTIVE: KLF4 and KLF5, members of the Krüppel-like factor (KLF) family, play key roles in proliferation, differentiation and apoptosis during development. In order to determine if these transcription factors are associated with tooth development, we examined the expression pattern of KLF4 and KLF5 during murine tooth development. DESIGN: In situ hybridization and immunohistochemistry were performed to detect the expression pattern of KLF4 and KLF5 from E12.5 to PN3 during murine tooth development. RESULTS: In situ hybridization analysis revealed that Klf4 was specifically expressed in polarizing odontoblasts from E16.5 (incisor) or E18.5 (first molar) to PN3. Immunohistochemistry staining showed that KLF4 was specifically expressed in both polarizing odontoblasts and ameloblasts at the same stages. KLF5 was mainly expressed from E18.5 to PN3 in secretory ameloblasts when enamel mineralization occurs and in secretory odontoblasts. However, an expression of KLF5 was also observed at earlier stages (E14.5 and E16.5) mainly in proliferating epithelial cells. CONCLUSIONS: These results suggest that the expression of KLF4 is closely correlated to the growth-arrest and the first step of odontoblast and ameloblast differentiation. Furthermore, KLF5 maybe involved in proliferation at the early stages of tooth development and related to mineralization of both enamel and dentin matrices at later stages.