RESUMO
Tooth enamel is the hardest tissue in the body. The organic matrix configuration is provided by the main proteins amelogenin, ameloblastin and enamelysin (MMP20), an enzyme that helps to shape the matrix. The aim of this study was to determine by histochemistry the expression of amelogenin and enamelysin through the rough endoplasmic reticulum in the late stages of amelogenesis, and its expression in the Complexus golgiensis (Golgi complex / Golgi apparatus) in the early stages in human fetuses. In early stages a colocalization of both proteins inside the Golgi apparatus was found, being more evident the relationship between Golgi and amelogenin (99.92 %). In the late stage, a colocalization of both proteins and rugged endoplasmic reticulum was found. With enamelysin being more evident in relation with rough endoplasmic reticulum (99.95 %). Our findings demonstrated the presence of amelogenin and enamelysin in odontoblast and ameloblast. However, the presence of these two proteins in odontoblast remains unknown.
El esmalte dental es el tejido más duro del cuerpo. La configuración de la matriz orgánica es proporcionada por las proteínas principales amelogenina, ameloblastina y enamelisina (MMP20), una enzima que ayuda a dar forma a la matriz. El objetivo de este estudio fue determinar mediante histoquímica la expresión de amelogenina y enamelisina a través del retículo endoplasmático rugoso en las últimas etapas de la amelogénesis , y su expresión en el Complexo golgiensis en las primeras etapas de formación en fetos humanos. En las primeras etapas se observó colocalización de ambas proteínas en el interior del Complexo golgiensis, siendo más evidente la relación entre Golgi y amelogenina (99,92 %). En la última etapa, se identificó una colocalización de ambas proteínas y retículo endoplásmico rugoso. Resulto más evidente la enamelisina en relación con el retículo endoplasmático rugoso (99,95 %). Nuestros resultados demostraron la presencia de amelogenina y enamelisina en odontoblastos y ameloblastos, sin embargo se desconoce la presencia de estas dos proteínas en odontoblastos.
Assuntos
Humanos , Amelogenina/metabolismo , Proteínas do Esmalte Dentário , Retículo Endoplasmático Rugoso , Complexo de Golgi , Metaloproteinase 20 da Matriz/metabolismo , Amelogênese , ImunofluorescênciaRESUMO
Amelogenesis imperfecta is a group of rare inherited disorders that affect tooth enamel formation, quantitatively and/or qualitatively. The aim of this study was to identify the genetic etiologies of two families presenting with hypomaturation amelogenesis imperfecta. DNA was isolated from peripheral blood samples obtained from participating family members. Whole exome sequencing was performed using DNA samples from the two probands. Sequencing data was aligned to the NCBI human reference genome (NCBI build 37.2, hg19) and sequence variations were annotated with the dbSNP build 138. Mutations in MMP20 were identified in both probands. A homozygous missense mutation (c.678T>A; p.His226Gln) was identified in the consanguineous Family 1. Compound heterozygous MMP20 mutations (c.540T>A, p.Tyr180* and c.389C>T, p.Thr130Ile) were identified in the non-consanguineous Family 2. Affected persons in Family 1 showed hypomaturation AI with dark brown discoloration, which is similar to the clinical phenotype in a previous report with the same mutation. However, the dentition of the Family 2 proband exhibited slight yellowish discoloration with reduced transparency. Functional analysis showed that the p.Thr130Ile mutant protein had reduced activity of MMP20, while there was no functional MMP20 in the Family 1 proband. These results expand the mutational spectrum of the MMP20 and broaden our understanding of genotype-phenotype correlations in amelogenesis imperfecta.
RESUMO
BACKGROUND/AIM: Matrix metalloproteinase 20 (MMP20) is a member of the family of matrix metalloproteinases. Under normal conditions the expression of MMP20 is restricted to ameloblasts and odontoblasts. In order to identify a possible expression of MMP20 under pathological conditions, we investigated three major human tumor entities, i.e. colon, breast and lung tumors, on the mRNA and protein level. MATERIALS AND METHODS: Real-time RT-PCR and immunocytochemical analyses of established human tumor cell lines were employed for our study; immunohistochemical analysis was performed on both primary tumors and normal control tissues. RESULTS: MMP20 was identified on both the mRNA and the protein level in breast MCF-7, colon HT-29, and lung A549 cell lines. MMP20 was also detected in primary tumor tissue by immunohistochemistry. CONCLUSION: MMP20 is a new potential candidate for tumor diagnosis or therapy.
Assuntos
Metaloproteinase 20 da Matriz/análise , Neoplasias/enzimologia , Humanos , Imuno-Histoquímica , Células MCF-7 , Metaloproteinase 20 da Matriz/genética , Metaloproteinase 20 da Matriz/fisiologiaRESUMO
Matrix metalloproteinase 20 (MMP20) and kallikrein-related peptidase 4 (KLK4) are secreted proteinases that are essential for proper dental enamel formation. We characterized and compared enamel formed in wild-type, Mmp20 (-/-), Klk4 (-/-), Mmp20 (+/-) Klk4 (+/-), and Mmp20 (-/-) Klk4 (-/-) mice using dissecting and light microscopy, backscattered scanning electron microscopy (bSEM), SEM, microcomputed tomography (µCT), and energy-dispersive X-ray analysis (EDX). Following eruption, fractures were observed on Mmp20 (-/-), Klk4 (-/-), Mmp20 (+/-) Klk4 (+/-), and Mmp20 (-/-) Klk4 (-/-) molars. Failure of the enamel in the Mmp20 (+/-) Klk4 (+/-) molars was unexpected and suggested that digenic effects could contribute to the etiology of amelogenesis imperfecta in humans. Micro-CT analyses of hemimandibles demonstrated significantly reduced high-density enamel volume in the Mmp20 (-/-) and Klk4 (-/-) mice relative to the wild-type, which was further reduced in Mmp20 (-/-) Klk4 (-/-) mice. bSEM images of 7-week Mmp20 (-/-) and Mmp20 (-/-) Klk4 (-/-) mandibular incisors showed rough, pitted enamel surfaces with numerous indentations and protruding nodules. The Mmp20 (+/-) and Mmp20 (+/-) Klk4 (+/-) incisors showed prominent, evenly spaced, horizontal ridges that were more distinct in Mmp20 (+/-) Klk4 (+/-) incisors relative to Mmp20 (+/-) incisors due to the darkening of the valleys between the ridges. In cross sections, the Mmp20 (-/-) and Mmp20 (-/-) Klk4 (-/-) exhibited three distinct layers. The outer layer exhibited a disturbed elemental composition and an irregular enamel surface covered with nodules. The Mmp20 null enamel was apparently unable to withstand the sheer forces associated with eruption and separated from dentin during development. Cells invaded the cracks and interposed between the dentin and enamel layers. MMP20 and KLK4 serve overlapping and complementary functions to harden enamel by removing protein, but MMP20 potentially serves multiple additional functions necessary for the adherence of enamel to dentin, the release of intercellular protein stores into the enamel matrix, the retreat of ameloblasts to facilitate thickening of the enamel layer, and the timely transition of ameloblasts to maturation.
RESUMO
ABSTRACT. Introduction: ameloblasts are cells responsible for the production and mineralization of the organic matrix of enamel through several stages: pre-secretory, secretory, transition, and maturation. The organic matrix components are produced in the secretory phase. In the maturation phase, the organic component is removed and the mineralization process starts. This process requires the involvement of matrix metalloproteinase 20 (MMP-20), also called enamelysin. Several studies have shown the presence of MMP-20 in tooth development and its relationship to alterations in enamel formation. The objective was: to classify the different studies and laboratory techniques used to demonstrate the involvement of enamelysin in tooth development and its relation to pathologies during enamel formation. Methods: a systematic review was conducted with the following bibliographic databases: PubMed, Science-Direct, Hinari, and SciELO, in order to classify the different studies related to the involvement of MMP-20 in tooth development and the methods to detect its expression, between the years of 2009 and 2014. Results and conclusions: 11 in vitro models show that MMP-20 has specific cleavage sites for enamel matrix proteins. This process is altered by chemical composition, ions, and the presence of hydroxyapatite. Enamel morphology is altered in the knockout models. In human studies, MMP-20 has been associated with increased susceptibility to dental caries, enamel thickness, and dental agenesis.
RESUMEN. Introducción: el ameloblasto es la célula encargada de la producción y mineralización de la matriz orgánica del esmalte. Atraviesa varias etapas: la fase pre-secretora, secretora, de transición y maduración. En la fase secretora se producen los componentes de la matriz orgánica. En la fase de maduración se elimina el componente orgánico y se inicia el proceso de mineralización. Este proceso requiere de la participación de la metaloproteinasa de matriz 20 (MMP-20) o también llamada enamelisina. Diversos estudios demuestran la presencia de MMP-20 en el desarrollo dentario y su relación con alteraciones en la formación del esmalte. El objeto fue clasificar los diferentes estudios y técnicas de laboratorio empleadas que demuestren la participación de enamelisina en el desarrollo dentario y su relación con patologías en la formación del esmalte. Métodos: se realizó una revisión sistemática de la literatura con las siguientes bases bibliográficas: PubMed, Science-Direct, Hinari y SciELO, con el fin de clasificar los diferentes estudios relacionados con la participación de MMP-20 en el desarrollo dental y los métodos utilizados para detectar su expresión, entre los años de 2009 a 2014. Resultados y conclusiones: los modelos in vitro evidencian que MMP-20 tiene sitios específicos de escisión para las proteínas de matriz de esmalte. Este proceso se ve alterado por la composición química, iones, y la presencia de hidroxiapatita. En los modelos knockout la morfología del esmalte está alterada. En los estudios en humanos, se ha relacionado la MMP-20 con una mayor susceptibilidad de caries dental, el grosor completo de esmalte y agenesias dentales.
Assuntos
Restauração Dentária Permanente , Resinas Sintéticas , Materiais DentáriosRESUMO
ABSTRACT. Introduction: ameloblasts are cells responsible for the production and mineralization of the organic matrix of enamel through several stages: pre-secretory, secretory, transition, and maturation. The organic matrix components are produced in the secretory phase. In the maturation phase, the organic component is removed and the mineralization process starts. This process requires the involvement of matrix metalloproteinase 20 (MMP-20), also called enamelysin. Several studies have shown the presence of MMP-20 in tooth development and its relationship to alterations in enamel formation. The objective was: to classify the different studies and laboratory techniques used to demonstrate the involvement of enamelysin in tooth development and its relation to pathologies during enamel formation. Methods: a systematic review was conducted with the following bibliographic databases: PubMed, Science-Direct, Hinari, and SciELO, in order to classify the different studies related to the involvement of MMP-20 in tooth development and the methods to detect its expression, between the years of 2009 and 2014. Results and conclusions: 11 in vitro models show that MMP-20 has specific cleavage sites for enamel matrix proteins. This process is altered by chemical composition, ions, and the presence of hydroxyapatite. Enamel morphology is altered in the knockout models. In human studies, MMP-20 has been associated with increased susceptibility to dental caries, enamel thickness, and dental agenesis.
RESUMEN. Introducción: el ameloblasto es la célula encargada de la producción y mineralización de la matriz orgánica del esmalte. Atraviesa varias etapas: la fase pre-secretora, secretora, de transición y maduración. En la fase secretora se producen los componentes de la matriz orgánica. En la fase de maduración se elimina el componente orgánico y se inicia el proceso de mineralización. Este proceso requiere de la participación de la metaloproteinasa de matriz 20 (MMP-20) o también llamada enamelisina. Diversos estudios demuestran la presencia de MMP-20 en el desarrollo dentario y su relación con alteraciones en la formación del esmalte. El objeto fue clasificar los diferentes estudios y técnicas de laboratorio empleadas que demuestren la participación de enamelisina en el desarrollo dentario y su relación con patologías en la formación del esmalte. Métodos: se realizó una revisión sistemática de la literatura con las siguientes bases bibliográficas: PubMed, Science-Direct, Hinari y SciELO, con el fin de clasificar los diferentes estudios relacionados con la participación de MMP-20 en el desarrollo dental y los métodos utilizados para detectar su expresión, entre los años de 2009 a 2014. Resultados y conclusiones: los modelos in vitro evidencian que MMP-20 tiene sitios específicos de escisión para las proteínas de matriz de esmalte. Este proceso se ve alterado por la composición química, iones, y la presencia de hidroxiapatita. En los modelos knockout la morfología del esmalte está alterada. En los estudios en humanos, se ha relacionado la MMP-20 con una mayor susceptibilidad de caries dental, el grosor completo de esmalte y agenesias dentales.
Assuntos
Anormalidades Dentárias , Esmalte Dentário , Metaloproteinase 20 da Matriz , Amelogênese , Amelogênese ImperfeitaRESUMO
In order to achieve highly mineralized tooth enamel, enamel proteinases serve the important function of removing the remaining organic matrix in the mineralization and maturation of the enamel matrix. Mutations in the kallikrein 4 (KLK4), enamelysin (MMP20), and WDR72 genes have been identified as causing hypomaturation enamel defects in an autosomal-recessive hereditary pattern. In this report, 2 consanguineous families with a hypomaturation-type enamel defect were recruited, and mutational analysis was performed to determine the molecular genetic etiology of the disease. Whole exome sequencing and autozygosity mapping identified novel homozygous mutations in the KLK4 (c.620_621delCT, p.Ser207Trpfs*38) and MMP20 (c.1054G>A, p.Glu352Lys) genes. Further analysis on the effect of the mutations on the translation, secretion, and function of KLK4 and MMP20 revealed that mutant KLK4 was degraded intracellularly and became inactive while mutant MMP20 was expressed at a normal level but secreted only minimally with proteolytic function.
Assuntos
Amelogênese Imperfeita/genética , Calicreínas/genética , Metaloproteinase 20 da Matriz/genética , Mutação , Amelogênese Imperfeita/diagnóstico por imagem , Western Blotting , Criança , Consanguinidade , Feminino , Genótipo , Homozigoto , Humanos , Linhagem , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Radiografia Panorâmica , Análise de Sequência de DNARESUMO
During experiments in animal studies, it has been observed that enamelysin (MMP-20) is expressed during tooth development in the late secretory stage of amelogenesis but not in the mature enamel.The aim of this research was to determine the location of MMP-20 in human tooth germs in the different structures of the enamel organ.The detection of MMP-20 was performed by immunohistochemistry in 20 specimens obtained from human fetuses. Immunostaining of MMP-20 was observed from the presecretor stadium in stellate reticulum and intermediate stratum and in the basal portion of ameloblasts in the secretory stage in stellate reticulum, stratum intermedium, secretory ameloblasts, odontoblasts and dental papilla. The results of this research show the location of MMP-20 in tooth germ development in humans and provides the foundation for future research about the process of dental organ formation.
En estudios realizados en animales de experimentación se ha observado que la enamelisina (MMP-20) se expresa durante el desarrollo dental durante el estadio de secreción tardío de la amelogénesis pero no en el esmalte maduro. El objetivo de la presente investigación fue determinar la localización de MMP-20 en gérmenes dentarios humanos en las diferentes estructuras del órgano del esmalte. Se analizaron 20 especímenes obtenidos de fetos humanos, efectuando la detección de MMP-20 por Inmunohistoquímica. Se observó inmunolocalización de MMP-20 desde el estadio presecretor en retículo estrellado y estrato intermedio, así como en porción basal de ameloblastos; en el estadio secretor en retículo estrellado, estrato intermedio, ameloblastos secretores, odontoblastos y papila dental. Los resultados de la presente investigación muestran la localización de la MMP-20 en el desarrollo del germen dentario en humanos y aporta las bases para futuras investigaciones acerca del proceso de formación de los órganos dentales.