RESUMEN
The identification and targeting of the molecular pathways regulating amelogenesis is an ongoing challenge in dental research, and progress has been restricted by the limited number of genetic tools available to study gene function in ameloblasts. Here, we generated 4 transgenic Cre-driver mouse lines that express improved Cre (iCre)-recombinase from the locus of the mouse ameloblast-specific gene amelogenin X (Amelx-iCre) with a large (250-kb) bacterial artificial chromosome DNA vector. All 4 Amelx-iCre transgenic lines were bred with ROSA26 reporter mice to characterize the iCre developmental pattern with the LacZ gene encoding ß-galactosidase enzyme activity assay and Cre protein immunohistochemistry. From the 4 generated transgenic lines, 2 were selected for further analysis because they expressed a high amount of Cre recombinase exclusively in ameloblasts and showed developmental stage- and cell-specific ß-galactosidase activity mimicking the endogenous amelogenin expression. To test the functionality of the selected transgenic models, we bred the 2 Amelx-iCre mice lines with stromal interaction molecule 1 (Stim1) floxed mice to generate ameloblast-specific Stim1 conditional knockout mice (Stim1 cKO). STIM1 protein serves as one of the main calcium sensors in ameloblasts and plays a major role in enamel mineralization and ameloblast differentiation. Amelx-iCre mice displayed exclusive CRE-mediated recombination in incisor and molar ameloblasts. Stim1 cKO mice showed a severely defected enamel phenotype, including reduced structural integrity concomitant with increased attrition and smaller teeth. The phenotype and genotype of the Amelx-iCre/Stim1 cKO showed significant differences with the previously reported Ker14-Cre/Stim1 cKO, highlighting the need for cell- and stage-specific Cre lines for an accurate phenotype-genotype comparison. Furthermore, our model has the advantage of carrying the entire Amelx gene locus rather than being limited to an Amelx partial promoter construct, which greatly enhances the stability and the specificity of our Cre expression. As such, the Amelx-iCre transgenic lines that we developed may serve as a powerful tool for targeting ameloblast-specific gene expression in future investigations.
Asunto(s)
Ameloblastos/fisiología , Amelogénesis , Molécula de Interacción Estromal 1/fisiología , Amelogenina , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Noqueados , Ratones TransgénicosRESUMEN
We report a novel method for the isolation of adult human epithelial stem cells (hEpiSCs) from the epithelial component of the periodontal ligament-the human epithelial cell rests of Malassez (hERM). hEpiSC-rich integrin-α6(+ve) hERM cells derived by fluorometry can be clonally expanded, can grow organoids, and express the markers of pluripotency (OCT4, NANOG, SOX2), polycomb protein RING1B, and the hEpiSC supermarker LGR5. They maintain the growth profile of their originating hERM in vitro. Subcutaneous cotransplantation with mesenchymal stem cells from the dental pulp on poly-l-lactic acid scaffolds in nude mice gave rise to perfect heterotopic ossicles in vivo with ultrastructure of dentin, enamel, cementum, and bone. These remarkable fully mineralized ossicles underscore the importance of epithelial-mesenchymal crosstalk in tissue regeneration using human progenitor stem cells, which may have already committed to lineage despite maintaining hallmarks of pluripotency. In addition, we report the clonal expansion and isolation of human LGR5(+ve) cells from the hERM in xeno-free culture conditions. The genetic profile of LGR5(+ve) cells includes both markers of pluripotency and genes important for secretory epithelial and dental epithelial cell differentiation, giving us a first insight into periodontal ligament-derived hEpiSCs.
Asunto(s)
Células Madre Adultas/fisiología , Células Epiteliales/citología , Ligamento Periodontal/citología , Animales , Células Epiteliales/fisiología , Proteínas de Homeodominio/fisiología , Humanos , Ratones , Ratones Desnudos , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/fisiología , Ligamento Periodontal/fisiología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Complejo Represivo Polycomb 1/fisiología , Receptores Acoplados a Proteínas G/fisiología , Factores de Transcripción SOXB1/fisiología , Trasplante de Células Madre , Andamios del Tejido , Calcificación de Dientes/fisiologíaRESUMEN
The aim of this study was the fabrication and evaluation of a novel bioactive and bactericidal material, which could have applications in dentistry by supporting tissue regeneration and killing oral bacteria. Our hypothesis was that a new scaffold for pulp-dentin tissue engineering with enhanced antibacterial activity could be obtained by associating extracellular matrix derived from porcine bladder with an antibacterial bioactive glass. Our study combines in vitro approaches and ectopic implantation in scid mice. The novel material was fabricated by incorporating a sol-gel derived silver (Ag)-doped bioactive glass (BG) in a natural extracellular matrix (ECM) hydrogel in ratio 1:1 in weight % (Ag-BG/ECM). The biological properties of the Ag-BG/ECM were evaluated in culture with dental pulp stem cells (DPSCs). In particular, cell proliferation, cell apoptosis, stem cells markers profile, and cell differentiation potential were studied. Furthermore, the antibacterial activity against Streptococcus mutans and Lactobacillus casei was measured. Moreover, the capability of the material to enhance pulp/dentin regeneration in vivo was also evaluated. Our data show that Ag-BG/ECM significantly enhances DPSCs' proliferation, it does not affect cell morphology and stem cells markers profile, protects cells from apoptosis, and enhances in vitro cell differentiation and mineralisation potential as well as in vivo dentin formation. Furthermore, Ag-BG/ECM strongly inhibits S. mutans and L. casei growth suggesting that the new material has also anti-bacterial properties. This study provides foundation for future clinical applications in dentistry. It could potentially advance the currently available options of dental regenerative materials.
Asunto(s)
Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Matriz Extracelular/química , Vidrio/química , Plata/química , Células Madre/efectos de los fármacos , 5'-Nucleotidasa/metabolismo , Animales , Antibacterianos/química , Antibacterianos/farmacología , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Pulpa Dental/citología , Odontología/métodos , Expresión Génica/efectos de los fármacos , Hidrogel de Polietilenoglicol-Dimetacrilato , Ratones Desnudos , Ratones SCID , Microscopía Fluorescente , Odontogénesis/efectos de los fármacos , Odontogénesis/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Madre/metabolismo , Streptococcus mutans/efectos de los fármacos , Streptococcus mutans/crecimiento & desarrollo , Porcinos , Ingeniería de Tejidos/métodos , Andamios del Tejido/químicaRESUMEN
Cancer stem cells possess the qualities of self-renewal, tumorigenesis and the ability to recapitulate a heterogeneous tumor. Our group was the first to isolate head and neck squamous cell carcinoma (HNSCC) stem cells using the cell surface marker CD44. CD44 is a trans-membrane glycoprotein with a multitude of key-functions that regulate cancer cell proliferation and metastasis. The variety of CD44 functions is due to tissue-specific patterns of glycosylation of the extracellular portion, and to the multiple protein isoforms (CD44 variants, CD44v) generated by alternative splicing. This study investigates the expression pattern of CD44 variants in HNSCC. Ten cell lines from the most common HNSCC locations and representative of various clinical outcomes were assayed by quantitative realtime PCR, flow cytometry and immunofluorescence comparatively with normal oral keratinocytes. The CD44 v4 and v6 were exclusively abundant in HNSCC while the isoform v1,2 was expressed in normal oral keratinocytes. Of interest, the highest level of CD44v6 expression was detected in advanced metastatic HNSCC, suggesting a link between CD44v6 expression and HNSCC metastasis, while the highest CD44v4 was detected in a stage IV HNSCC refractory to chemotherapy which developed recurrence. Oral-derived HNSCC expressed the highest CD44v4 and v6, and levels corresponded with staging, showing also an increasing tendency with recurrence and metastasis. CD44v were detected predominantly in smaller cells (a characteristic that has been associated with stem cell properties) or cells with mesenchymal morphology (a characteristic that has been associated with the migratory and invasive potential of epithelial tumor cells), suggesting that CD44v differential expression in HNSCC may be representative of the morphological changes inherent during tumor progression towards a more aggressive potential, and thus contributing to the individual tumor biology. The mechanism of CD44 variant involvement in HNSCC progression and metastasis is under investigation.
Asunto(s)
Carcinoma de Células Escamosas/inmunología , Neoplasias de Cabeza y Cuello/inmunología , Receptores de Hialuranos/análisis , Neoplasias de la Boca/inmunología , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Técnica del Anticuerpo Fluorescente , Neoplasias de Cabeza y Cuello/patología , Humanos , Receptores de Hialuranos/fisiología , Neoplasias de la Boca/patología , Isoformas de Proteínas , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
Neural crest cells, delaminating from the neural tube during migration, undergo an epithelial-mesenchymal transition and differentiate into several cell types strongly reinforcing the mesoderm of the craniofacial body area - giving rise to bone, cartilage and other tissues and cells of this human body area. Recent studies on craniomaxillofacial neural crest-derived cells have provided evidence for the tremendous plasticity of these cells. Actually, neural crest cells can respond and adapt to the environment in which they migrate and the cranial mesoderm plays an important role toward patterning the identity of the migrating neural crest cells. In our experience, neural crest-derived stem cells, such as dental pulp stem cells, can actively proliferate, repair bone and give rise to other tissues and cytotypes, including blood vessels, smooth muscle, adipocytes and melanocytes, highlighting that their use in tissue engineering is successful. In this review, we provide an overview of the main pathways involved in neural crest formation, delamination, migration and differentiation; and, in particular, we concentrate our attention on the translatability of the latest scientific progress. Here we try to suggest new ideas and strategies that are needed to fully develop the clinical use of these cells. This effort should involve both researchers/clinicians and improvements in good manufacturing practice procedures. It is important to address studies towards clinical application or take into consideration that studies must have an effective therapeutic prospect for humans. New approaches and ideas must be concentrated also toward stem cell recruitment and activation within the human body, overcoming the classical grafting.
Asunto(s)
Regeneración Ósea , Células Madre Embrionarias/trasplante , Desarrollo Maxilofacial , Cresta Neural/citología , Osteogénesis , Animales , Anomalías Craneofaciales/terapia , Células Madre Embrionarias/citología , Humanos , Cresta Neural/embriologíaRESUMEN
BACKGROUND: The prognosis of the oral squamous cell carcinoma (OSCC) patients remains very poor, mainly due to their high propensity to invade and metastasize. E-cadherin reduced expression occurs in the primary step of oral tumour progression and gene methylation is a mode by which the expression of this protein is regulated in cancers. In this perspective, we investigated E-cadherin gene (CDH1) promoter methylation status in OSCC and its correlation with Ecadherin protein expression, clinicopathological characteristics and patient outcome. METHODS: Histologically proven OSCC and paired normal mucosa were analyzed for CDH1 promoter methylation status and E-cadherin protein expression by methylation-specific polymerase chain reaction and immunohistochemistry. Colocalization of E-cadherin with epidermal growth factor (EGF) receptor (EGFR) was evidenced by confocal microscopy and by immunoprecipitation analyses. RESULTS: This study indicated E-cadherin protein down-regulation in OSCC associated with protein delocalization from membrane to cytoplasm. Low E-cadherin expression correlated to aggressive, poorly differentiated, high grade carcinomas and low patient survival. Moreover, protein down-regulation appeared to be due to E-cadherin mRNA downregulation and CDH1 promoter hypermethylation. In an in vitro model of OSCC the treatment with EGF caused internalization and co-localization of E-cadherin with EGFR and the addition of demethylating agents increased E-cadherin expression. CONCLUSION: Low E-Cadherin expression is a negative prognostic factor of OSCC and is likely due to the hypermethylation of CDH1 promoter. The delocalization of E-cadherin from membrane to cytoplasm could be also due to the increased expression of EGFR in OSCC and the consequent increase of E-cadherin co-internalization with EGFR.
Asunto(s)
Biomarcadores de Tumor/genética , Cadherinas/genética , Carcinoma de Células Escamosas/genética , Metilación de ADN , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Neoplasias de Cabeza y Cuello/genética , Neoplasias de la Boca/genética , Adulto , Anciano , Anciano de 80 o más Años , Antígenos CD , Antimetabolitos Antineoplásicos/farmacología , Biomarcadores de Tumor/metabolismo , Cadherinas/metabolismo , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/mortalidad , Carcinoma de Células Escamosas/patología , Estudios de Casos y Controles , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Metilasas de Modificación del ADN/antagonistas & inhibidores , Metilasas de Modificación del ADN/metabolismo , Regulación hacia Abajo , Inhibidores Enzimáticos/farmacología , Epigénesis Genética/efectos de los fármacos , Receptores ErbB/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Predisposición Genética a la Enfermedad , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/mortalidad , Neoplasias de Cabeza y Cuello/patología , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Neoplasias de la Boca/metabolismo , Neoplasias de la Boca/mortalidad , Neoplasias de la Boca/patología , Pronóstico , Regiones Promotoras Genéticas , Transporte de Proteínas , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Carcinoma de Células Escamosas de Cabeza y Cuello , Factores de TiempoRESUMEN
Most physiological processes in mammals display circadian rhythms that are driven by the endogenous circadian clock. This clock is comprised of a central component located in the hypothalamic suprachiasmatic nucleus and subordinate clocks in peripheral tissues. Circadian rhythms sustain 24-hour oscillations of a large number of master genes controlling the correct timing and synchronization of diverse physiological and metabolic processes within our bodies. This complex regulatory network provides an important communication link between our brain and several peripheral organs and tissues. At the molecular level, circadian oscillations of gene expression are regulated by a family of transcription factors called "clock genes". Dysregulation of clock gene expression results in diverse human pathological conditions, including autoimmune diseases and cancer. There is increasing evidence that the circadian clock affects tooth development, salivary gland and oral epithelium homeostasis, and saliva production. This review summarizes current knowledge of the roles of clock genes in the formation and maintenance of oral tissues, and discusses potential links between "oral clocks" and diseases such as head and neck cancer and Sjögren's syndrome.
Asunto(s)
Relojes Circadianos/fisiología , Enfermedades de la Boca/fisiopatología , Salud Bucal , Enfermedades Autoinmunes/genética , Proteínas CLOCK/genética , Relojes Circadianos/genética , Ritmo Circadiano/fisiología , Neoplasias de Cabeza y Cuello/genética , Humanos , Enfermedades de la Boca/genética , Síndrome de Sjögren/genéticaRESUMEN
Pathological acid reflux is a common event in patients afflicted with head and neck squamous cell carcinomas (HNSCCs), known to play a role in HNSCC etiology and contribute to complications after surgery or during radiation and chemotherapy. Antacid medications are commonly prescribed in HNSCC patients as part of their cancer treatment, and consist of two classes: histamine 2 receptor antagonist class (H2RA, with cimetidine as its prototypical drug) and proton pump inhibitors class (PPI, with omeprazole as its prototypical drug). Clinical evidence revealed a significant survival benefit of antacid usage in a large cohort of HNSCC patients treated in our Otolaryngology Department, with a median follow-up of over 5 years. Therefore, we postulate that one mechanism by which antacid intake enhances patient survival could involve modulation of tumor cell adhesion to endothelium, critical in the initiation of the metastatic dissemination. This study investigates the potential physical interactions between cimetidine and omeprazole with the endothelial E-selection (E-sel) and its ligand sialyl Lewis X (sLe(x)) using a molecular visualization energy-based program (AutoDock). Docking results were further analyzed with the PyMOL program, which allowed for measurements of the distances between the drugs and the closest interacting atoms or residues on E-sel and sLe(x) molecules. Our model predicts that omeprazole displays a stronger interaction with E-sel than cimetidine, as extrapolated from the calculated overall binding energies. However, the shorter distances existing between interacting atoms in the proposed E-sel/cimetidine complex are suggestive of more stable interactions. Neither antacid/E-sel complex overcame the stronger Autodock-calculated sLe(x)/E-sel interaction, suggesting competitive inhibition was not involved. This study provides the first in silico evidence of omeprazole and cimetidine ability to bind to adhesion molecules involved in tumor dissemination, underlining their therapeutic potential in the HNSCC clinical management.
Asunto(s)
Antiácidos/química , Carcinoma de Células Escamosas/tratamiento farmacológico , Cimetidina/química , Selectina E/química , Endotelio/efectos de los fármacos , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Omeprazol/química , Antiácidos/uso terapéutico , Adhesión Celular/efectos de los fármacos , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
Circadian rhythms are endogenous self-sustained oscillations with 24-hour periods that regulate diverse physiological and metabolic processes through complex gene regulation by "clock" transcription factors. The oral cavity is bathed by saliva, and its amount and content are modified within regular daily intervals. The clock mechanisms that control salivary production remain unclear. Our objective was to evaluate the expression and periodicity of clock genes in salivary glands. Real-time quantitative RT-PCR, in situ hybridization, and immunohistochemistry were performed to show circadian mRNA and protein expression and localization of key clock genes (Bmal1, Clock, Per1, and Per2), ion and aqua channel genes (Ae2a, Car2, and Aqp5), and salivary gland markers. Clock gene mRNAs and clock proteins were found differentially expressed in the serous acini and duct cells of all major salivary glands. The expression levels of clock genes and Aqp5 showed regular oscillatory patterns under both light/dark and complete-dark conditions. Bmla1 overexpression resulted in increased Aqp5 expression levels. Analysis of our data suggests that salivary glands have a peripheral clock mechanism that functions both in normal light/dark conditions and in the absence of light. This finding may increase our understanding of the control mechanisms of salivary content and flow.
Asunto(s)
Relojes Circadianos/genética , Ritmo Circadiano/genética , Glándulas Salivales/metabolismo , Factores de Transcripción ARNTL/análisis , Animales , Proteínas de Transporte de Anión/análisis , Antiportadores/análisis , Acuaporina 5/análisis , Acuaporinas/análisis , Proteínas CLOCK/análisis , Anhidrasas Carbónicas/análisis , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Mucinas/análisis , Proteínas Circadianas Period/análisis , Proteínas SLC4A , Saliva/química , Simportadores de Sodio-Bicarbonato/análisis , Factores de Transcripción/análisisRESUMEN
Beta-catenin, normally expressed on the epithelial cell surface, plays a crucial role in cadherin-mediated cell adhesion. Recent evidence suggests that beta-catenin is also involved in other functions such as intracellular signaling via the Wnt pathway by creating a nuclear complex with members of the Lymphoid-Enhancer-Factor/T-Cell-Factor (LEF/TCF) family of transcription factors, and gene regulation that it is implicated in the development of several tumors. Little information is available on beta-catenin expression and its main partner in the Wnt signaling pathway, LEF1, in oropharyngeal squamous cell carcinomas (OP-SCCs). The aim of this study is to investigate the expression of beta-catenin and LEF1 expression in human primary OP-SCCs and to evaluate their clinical and prognostic significance. OP-SCCs and normal peritumoral areas were analyzed by immunohistochemistry, Western-blot and RT-PCR. Beta-catenin was overexpressed in tumors in comparison to normal peritumoral areas and displayed predominantly intracellular (cytosolic/nuclear) localization in 62% of the tumors. Immunoreactivity was correlated with clinicopathological parameters and long-term follow-up, and a significant association was found between protein expression and development of local recurrences (P =0.03). The OP-SCCs with poor clinical outcome, which displayed intracellular beta-catenin expression, were also strongly positive for LEF1, with their co-expression statistically significant (P = 0.040). All (100%) advanced (stages 3+4) SCCs, 66.7% of the SCCs with positive lymph nodes and 80% of the SSCs that developed local recurrences were LEF1 positive. Cox regression analysis confirmed a poorer overall survival in cases with high expression of beta-catenin and LEF1. Our results suggest that assessing intracellular beta-catenin and LEF1 expression might help in patient risk stratification and outcome prediction, and serve as novel therapeutic targets in advanced OP-SCC.
Asunto(s)
Carcinoma de Células Escamosas/química , Factor de Unión 1 al Potenciador Linfoide/análisis , Neoplasias Orofaríngeas/química , beta Catenina/análisis , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Células Escamosas/patología , Estudios de Cohortes , Femenino , Humanos , Inmunohistoquímica , Factor de Unión 1 al Potenciador Linfoide/fisiología , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Neoplasias Orofaríngeas/patología , Estudios Prospectivos , beta Catenina/fisiologíaAsunto(s)
Ligamento Periodontal/citología , Células Madre/citología , Ameloblastos/fisiología , Animales , Antígenos CD34/análisis , Antígenos de Neoplasias/análisis , Moléculas de Adhesión Celular/análisis , Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Linaje de la Célula , Separación Celular , Células Clonales/citología , Técnicas de Cocultivo , Pulpa Dental/citología , Molécula de Adhesión Celular Epitelial , Células Epiteliales/citología , Citometría de Flujo , Humanos , Integrina alfa6/análisis , Ratones , Ratones Desnudos , Receptor Notch1/análisis , Receptores Acoplados a Proteínas G/análisis , Andamios del TejidoRESUMEN
Epithelial-mesenchymal interactions guide tooth development through its early stages and establish the morphology of the dentin surface upon which enamel will be deposited. Starting with the onset of amelogenesis beneath the future cusp tips, the shape of the enamel layer covering the crown is determined by five growth parameters: the (1) appositional growth rate, (2) duration of appositional growth (at the cusp tip), (3) ameloblast extension rate, (4) duration of ameloblast extension, and (5) spreading rate of appositional termination. Appositional growth occurs at a mineralization front along the ameloblast distal membrane in which amorphous calcium phosphate (ACP) ribbons form and lengthen. The ACP ribbons convert into hydroxyapatite crystallites as the ribbons elongate. Appositional growth involves a secretory cycle that is reflected in a series of incremental lines. A potentially important function of enamel proteins is to ensure alignment of successive mineral increments on the tips of enamel ribbons deposited in the previous cycle, causing the crystallites to lengthen with each cycle. Enamel hardens in a maturation process that involves mineral deposition onto the sides of existing crystallites until they interlock with adjacent crystallites. Neutralization of acidity generated by hydroxyapatite formation is a key part of the mechanism. Here we review the growth parameters that determine the shape of the enamel crown as well as the mechanisms of enamel appositional growth and maturation.
Asunto(s)
Amelogénesis/fisiología , Esmalte Dental/embriología , Ameloblastos/fisiología , Animales , Fosfatos de Calcio/metabolismo , Proteínas del Esmalte Dental/metabolismo , Durapatita/metabolismo , Órgano del Esmalte/fisiología , Humanos , Calcificación de Dientes/fisiología , Corona del Diente/embriologíaRESUMEN
Proteolytic enzymes are necessary for the mineralization of dental enamel during development, and mutations in the kallikrein 4 (KLK4) and enamelysin (MMP20) genes cause autosomal-recessive amelogenesis imperfecta (ARAI). So far, only one KLK4 and two MMP20 mutations have been reported. We have identified an ARAI-causing point mutation (c.102G>A, g.102G>A, and p.W34X) in exon 1 of MMP20 in a proband with autosomal-recessive hypoplastic-hypomaturation amelogenesis imperfecta. The G to A transition changes the tryptophan (W) codon (TGG) at amino acid position 34 into a translation termination (X) codon (TGA). No disease-causing sequence variations were detected in KLK4. The affected enamel is thin, with mild spacing in the anterior dentition. The enamel layer is hypomineralized, does not contrast with dentin on radiographs, and tends to chip away from the underlying dentin. An intrinsic yellowish pigmentation is evident, even during eruption. The phenotype supports current ideas concerning the function of enamelysin.
Asunto(s)
Amelogénesis Imperfecta/genética , Codón sin Sentido/genética , Metaloproteinasa 20 de la Matriz/genética , Mutación Puntual/genética , Adenina , Niño , Esmalte Dental/patología , Exones/genética , Femenino , Guanina , Humanos , Análisis de Secuencia de ADN , Triptófano/genéticaRESUMEN
Amelogenin is the major protein of the developing enamel. Two additional exons, termed 8 and 9, have been characterized in the rat. Our aim was: to identify the mouse amelogenin exons 8/9 sequences; to investigate the potential presence of the alternative spliced isoforms of amelogenin exons 8/9; and to immunolocalize proteins containing sequences encoded by exons 8/9 during odontogenesis. RT-PCR analysis with exon 9 anti-sense primer generated 2 major amplicons with the use of a mouse tooth cDNA library and dental cell lines. DNA sequence analysis showed 93% identify with the rat exons 8/9 sequence. Alternative splicing of exon 3 was also found, but only in cDNAs lacking exons 8 and 9. Immunohistochemistry localized exons 8/9-encoded proteins in ameloblasts, young odontoblasts, and stratum intermedium cells. Analysis of our data supports the hypothesis that: (1) AMELX contains 2 additional exons; (2) ameloblasts and odontoblasts synthesize amelogenin 8/9; and (3) amelogenin splice variants may have unique functions during tooth formation.
Asunto(s)
Ameloblastos/metabolismo , Proteínas del Esmalte Dental/genética , Proteínas del Esmalte Dental/metabolismo , Odontoblastos/metabolismo , Germen Dentario/metabolismo , Empalme Alternativo/genética , Amelogenina , Animales , Secuencia de Bases , ADN/análisis , Exones/genética , Biblioteca de Genes , Inmunohistoquímica , Ratones , Datos de Secuencia Molecular , ARN/análisis , Análisis de Secuencia de ADN , Distribución Tisular , Germen Dentario/citologíaRESUMEN
The calciotropic hormone, 1,25(OH)2vitamin D3[1,25(OH)2D3], controls the formation of dental and bone mineralized tissues. The role of nuclear 1,25(OH)2D3 receptor has been extensively studied in the diverse secretory cells, i.e., osteoblasts, chondrocytes, ameloblasts, and odontoblasts. A nongenomic pathway also has been characterized and related to the interactions of 1,25(OH)2D3 ligand with a putative cell membrane receptor. This recognition moiety called 1,25(OH)2vitamin D3 membrane-associated, rapid-response steroid-binding [1,25D3-MARRS] protein is investigated here in the craniofacial skeleton of human embryos and fetuses. Immunolocalization using a specific Ab099 against chick intestinal basolateral 1,25D3-MARRS protein was performed. The data show a complementary expression pattern of the membrane receptor when compared with published data on the nuclear receptor, notably during amelogenesis. In mandible, membrane receptors for 1,25(OH)2D3 were identified in the heterogenous bone cell population, including osteoblasts and osteoclasts. Differential 1,25D3-MARRS protein levels were observed in distinct developmental stages and anatomical sites of tooth and bone, suggesting the existence of cross-talk between local factors and 1,25D3-MARRS protein expression.
Asunto(s)
Mandíbula/metabolismo , Proteínas de la Membrana/metabolismo , Organogénesis/fisiología , Receptores de Calcitriol/metabolismo , Calcificación de Dientes/fisiología , Germen Dentario/metabolismo , Edad Gestacional , Humanos , Técnicas para Inmunoenzimas , Mandíbula/embriología , Germen Dentario/embriologíaRESUMEN
Amelogenin is the major enamel protein produced by ameloblasts. Its expression has been shown to be down-regulated in ameloblasts of vitamin-D-deficient (-D) rats. The potential expression and localization of amelogenin in odontoblasts and its regulation by vitamin D were investigated in this study. RT-PCR and semi-quantitative Northern blot analyses were performed using the odontoblast cell line MO6-G3 and microdissected dental pulp mesenchyme. Both in vitro and in vivo odontoblasts expressed various alternatively spliced amelogenin transcripts. In situ hybridization studies showed that amelogenin expression was restricted to young odontoblasts during mantle dentin deposition. Electron microscopy studies localized the amelogenin protein in the odontoblast cell process cytoplasm and mantle dentin. Amelogenin immunolabeling was stronger in -D rats, suggesting an inverse regulation by vitamin D in odontoblasts. Furthermore, amelogenin mRNA steady-state levels were significantly increased in -D dental pulp mesenchyme. In addition, a temporal-spatial lengthening of the mantle dentin stage was observed in -D animals, suggesting that developmental perturbations occur in relation to the vitamin D status and/or amelogenin expression. These data show that amelogenin is expressed by odontoblasts selectively during mantle dentin deposition. This developmental regulated expression pattern is enhanced under vitamin-D-deficiency status and in a broader context may play an important role during ameloblast and odontoblast differentiation and function.
Asunto(s)
Proteínas del Esmalte Dental/genética , Odontoblastos/fisiología , Germen Dentario/citología , Amelogenina , Animales , Calcitriol/deficiencia , Comunicación Celular/fisiología , Células Cultivadas , Células Epiteliales/citología , Células Epiteliales/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Mesodermo/citología , Mesodermo/fisiología , Ratones , Microscopía Electrónica , Odontoblastos/ultraestructura , ARN Mensajero/análisis , Ratas , Ratas Sprague-Dawley , Germen Dentario/embriologíaRESUMEN
Our laboratory has determined the DNA sequence and transcriptional expression pattern of a mouse cDNA clone termed Nma/BAMBI. This clone encodes a highly conserved protein with 89% identity to the human homologue (termed Nma) and 78% similarity to the Xenopus homologue (termed BAMBI) at the predicted amino acid level. Nma/BAMBI encodes a 260-amino-acid transmembrane protein that has homology to the transforming growth factor (TGF) beta type I receptor family. This protein contains an extracellular ligand binding domain, a 24-amino-acid transmembrane domain, and a short intracellular domain that lacks a functional serine/threonine kinase domain. It is believed that Nma/BAMBI is important in the negative regulation of TGF beta signal transduction pathways during development and has implications in tumor progression. We have determined the genomic organization of the mouse Nma/BAMBI gene and confirmed the chromosomal mapping to human chromosome 10 and mouse chromosome 2. Furthermore, we report the production and utilization of an anti-peptide antibody in preliminary immunohistochemical analysis of an ameloblastoma.
Asunto(s)
Genoma , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ameloblastoma/etiología , Ameloblastoma/metabolismo , Animales , Mapeo Cromosómico , Exones , Humanos , Inmunohistoquímica , Intrones , Neoplasias Maxilomandibulares/etiología , Neoplasias Maxilomandibulares/metabolismo , RatonesRESUMEN
Enamel defects have been reported in rickets and related to disturbed expression of amelogenin in ameloblasts. The present study is devoted to amelogenin, enamelin, ameloblastin, and dentin sialophosphoprotein (DSPP) expression in both the epithelium and mesenchyme of vitamin D-deficient rat incisors. Quantitative Northern blotting analysis (relatively to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA steady-state levels) was performed on microdissected cells of rachitic (-D) and control (+D) 56 day old rats. Steady-state levels of amelogenin and enamelin mRNA were significantly reduced in the -D epithelium versus the +D epithelium ones. In contrast, ameloblastin expression was slightly increased in -D epithelium. In the same samples, DSPP mRNA levels remained unchanged in -D dental mesenchyme. Comparative electron microscopy studies between +D and -D animals showed a dramatic decrease of intraprismatic enamel (amelogenin and enamelin immunoreactive) consistent with our molecular results. In conclusion, tooth formation results from the coordinated expression of several matrix proteins that may be controlled by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3].
Asunto(s)
Calcitriol/fisiología , Proteínas de la Matriz Extracelular/metabolismo , Incisivo/metabolismo , Mesodermo/fisiología , Animales , Esmalte Dental/ultraestructura , Proteínas del Esmalte Dental/genética , Epitelio/fisiología , Masculino , Microscopía Electrónica , Fosfoproteínas/genética , Precursores de Proteínas , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Sialoglicoproteínas/genética , Deficiencia de Vitamina D/metabolismo , Deficiencia de Vitamina D/patologíaRESUMEN
Rickets is associated with site-specific disorders of enamel and dentin formation, which may reflect the impact of vitamin D on a morphogenetic pathway. This study is devoted to potential cross-talk between vitamin D and Msx/Dlx transcription factors. We raised the question of a potential link between tooth defects seen in mice with rickets and Msx2 gene misexpression, using mutant mice lacking the nuclear vitamin D receptor as an animal model. Our data showed a modulation of Msx2 expression. In order to search for a functional impact of this Msx2 misexpression secondary to rickets, we focused our attention on osteocalcin as a target gene for both vitamin D and Msx2. Combining Msx2 overexpression and vitamin D addition in vitro, we showed an inhibitory effect on osteocalcin expression in immortalized MO6-G3 odontoblasts. Finally, in the same cells, such combinations appeared to modulate VDR expression outlining the existence of complex cross-regulations between vitamin D and Msx/Dix pathways.
Asunto(s)
Proteínas de Unión al ADN/genética , Genes Homeobox/fisiología , Proteínas de Homeodominio/genética , Incisivo/fisiopatología , Minerales/metabolismo , Raquitismo/fisiopatología , Factores de Transcripción/genética , Vitamina D/metabolismo , Animales , Línea Celular , Proteínas de Unión al ADN/metabolismo , Expresión Génica , Mesodermo/metabolismo , Ratones , Ratones Noqueados/genética , Ratones Transgénicos , Diente Molar/embriología , Odontoblastos/metabolismo , Osteocalcina/genética , ARN Mensajero/metabolismo , Receptores de Calcitriol/genéticaRESUMEN
Biochemical investigations in rodents have shown that numerous mineralized matrix proteins share expression in bone, dentin, and cementum. Little information is available regarding the expression pattern of these proteins in human tissues, particularly during tooth formation. The aim of this study was to identify the expression pattern of the two major noncollagenous proteins of bone and dentin, osteocalcin (OC) and osteonectin (ON), in comparison to the dentin-specific protein, dentin sialophosphoprotein (DSPP). Mandibles from fetuses (5-26 weeks), neonate autopsies, forming teeth from 10-12-year-old patients, third molars extracted for orthodontic reasons, and bone tumors were collected with approval from the National Ethics Committee. Human OC, ON, and DSPP mRNAs were detected by reverse transcription-polymerase chain reaction (RT-PCR) in fetal mandibles (5-11 weeks) and in primary cell cultures of dental pulp. In addition, OC, ON, and DSPP proteins were localized in forming human mineralized tissues using immunohistochemistry. In vivo, DSPP expression was associated with tooth terminal epithelial-mesenchymal interaction events, amelogenesis and dentinogenesis. Transient DSPP expression was seen in the presecretory ameloblasts with continuous expression in the odontoblasts. In contrast, both osteoblasts and odontoblasts showed a temporal gap between OC and ON expression in early development. ON was expressed in the initial stages of cytodifferentiation, whereas OC was expressed only during the later stages, especially in the teeth. At the maturation stage of enamel formation, both proteins were detected in odontoblasts and their processes within the extracellular matrix. In contrast to bone, OC was not localized extracellularly within the collagen-rich dentin matrix (predentin or intertubular dentin), but was found in the mature enamel. ON was present mostly in the nonmineralized predentin. These results demonstrate for the first time that both OC and ON are produced by human odontoblasts and determine the expression pattern of DSPP in human teeth, and suggest that OC and ON move inside the canalicule via odontoblast cell processes becoming localized to specific extracellular compartments during dentin and enamel formation. These distinct extracellular patterns may be related to the nature of DSPP, OC, and ON interactions with other matrix-specific macromolecules (i.e., amelogenin, dentin matrix protein-1) and/or to the polarized organization of odontoblast secretion as compared with osteoblasts.