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Journal of Dental Research Jul 2018Ameloblasts responsible for enamel formation express matrix metalloproteinase 20 (MMP20), an enzyme that cleaves enamel matrix proteins, including amelogenin (AMELX) and...
Ameloblasts responsible for enamel formation express matrix metalloproteinase 20 (MMP20), an enzyme that cleaves enamel matrix proteins, including amelogenin (AMELX) and ameloblastin (AMBN). Previously, we showed that continuously erupting incisors from transgenic mice overexpressing active MMP20 had a massive cell infiltrate present within their enamel space, leading to enamel mineralization defects. However, effects of MMP20 overexpression on mouse molars were not analyzed, although these teeth more accurately represent human odontogenesis. Therefore, MMP20-overexpressing mice ( Mmp20Tg) were assessed by multiscale analyses, combining several approaches from high-resolution micro-computed tomography to enamel organ immunoblots. During the secretory stage at postnatal day 6 (P6), Mmp20Tg mice had a discontinuous ameloblast layer and, unlike incisors, molar P12 maturation stage ameloblasts abnormally migrated away from the enamel layer into the stratum intermedium/stellate reticulum. TOPflash assays performed in vitro demonstrated that MMP20 expression promoted β-catenin nuclear localization and that MMP20 expression promoted invasion through Matrigel-coated filters. However, for both assays, significant differences were eliminated in the presence of the β-catenin inhibitor ICG-001. This suggests that MMP20 activity promotes cell migration via the Wnt pathway. In vivo, the unique molar migration of amelogenin-expressing ameloblasts was associated with abnormal deposition of ectopic calcified nodules surrounding the adherent enamel layer. Enamel content was assessed just prior to eruption at P15. Compared to wild-type, Mmp20Tg molars exhibited significant reductions in enamel thickness (70%), volume (60%), and mineral density (40%), and MMP20 overexpression resulted in premature cleavage of AMBN, which likely contributed to the severe defects in enamel mineralization. In addition, Mmp20Tg mouse molar enamel organs had increased levels of inactive p-cofilin, a protein that regulates cell polarity. These data demonstrate that increased MMP20 activity in molars causes premature degradation of ameloblastin and inactivation of cofilin, which may contribute to pathological Wnt-mediated cell migration away from the enamel layer.
Topics: Actin Depolymerizing Factors; Ameloblasts; Amelogenesis; Amelogenin; Animals; Animals, Newborn; Bridged Bicyclo Compounds, Heterocyclic; Cell Movement; Dental Enamel Proteins; Immunoblotting; Matrix Metalloproteinase 20; Mice; Mice, Transgenic; Molar; Pyrimidinones; Wnt Signaling Pathway; X-Ray Microtomography
PubMed: 29481294
DOI: 10.1177/0022034518758657 -
Current Pharmaceutical Design 2018The pancreas, the salivary glands and the dental enamel producing ameloblasts have marked developmental, structural and functional similarities. One of the most striking... (Review)
Review
The pancreas, the salivary glands and the dental enamel producing ameloblasts have marked developmental, structural and functional similarities. One of the most striking similarities is their bicarbonate-rich secretory product, serving acid neutralization. An important difference between them is that while pancreatic juice and saliva are delivered into a lumen where they can be collected and analyzed, ameloblasts produce locally precipitating hydroxyapatite which cannot be easily studied. Interestingly, the ion and protein secretion by the pancreas, the salivary glands, and maturation ameloblasts are all two-step processes, of course with significant differences too. As they all have to defend against acid exposure by producing extremely large quantities of bicarbonate, the failure of this function leads to deteriorating consequences. The aim of the present review is to describe and characterize the defense mechanisms of the pancreas, the salivary glands and enamel-producing ameloblasts against acid exposure and to compare their functional capabilities to do this by producing bicarbonate.
Topics: Animals; Dental Enamel; Humans; Pancreatic Juice; Salivary Glands
PubMed: 29769002
DOI: 10.2174/1381612824666180515125654 -
European Cells & Materials Dec 2010The junctional epithelium (JE) adheres to the tooth surface, and seals off periodontal tissues from the oral environment. This incompletely differentiated epithelium is...
The junctional epithelium (JE) adheres to the tooth surface, and seals off periodontal tissues from the oral environment. This incompletely differentiated epithelium is formed initially by the fusion of the reduced enamel organ with the oral epithelium (OE). Two proteins, odontogenic ameloblast-associated (ODAM) and amelotin (AMTN), have been identified in the JE. The objective of this study was to evaluate their expression pattern during formation and regeneration of the JE. Cytokeratin 14 was used as a differentiation marker for oral epithelial cells, and Ki67 for cell proliferation. Immunohistochemistry was carried out on erupting rat molars, and in regenerating JE following gingivectomy. In the reducing enamel organ and in established JE, ODAM and AMTN were present at the cell-tooth interface while only ODAM and CK14 were found throughout the JE. Both were also conspicuously present in cell clusters situated between the erupting tooth and OE. During JE regeneration, ODAM was detected first at the leading wound edge and then in the regenerating JE. Some cell clusters in the subjacent connective tissue were also positive for ODAM. AMTN appeared later and both AMTN and ODAM accumulated at the interface with the tooth. Cytokeratin 14 gradually appeared in the regenerating JE but the cell clusters showed variable labeling. Cells associated with JE formation and regeneration exhibited higher division activity than adjacent epithelial cells. These findings suggest that ODAM and AMTN have a role at the cell-tooth interface, and that ODAM is likely also implicated in cellular events during formation and regeneration of the JE.
Topics: Ameloblasts; Animals; Calcium-Binding Proteins; Cell Proliferation; Dental Enamel Proteins; Epithelial Attachment; Keratin-14; Male; Microscopy, Fluorescence; Rats; Rats, Wistar; Regeneration; Tooth Eruption
PubMed: 21154245
DOI: 10.22203/ecm.v020a32 -
Australian Dental Journal Jun 2014During four days of prenatal development in the mouse, the morphology of the first lower molar moves from the early cap to the bell stage. Five phenomena characterize... (Review)
Review
During four days of prenatal development in the mouse, the morphology of the first lower molar moves from the early cap to the bell stage. Five phenomena characterize this period: growth of the tooth germ; development of the cervical loop; histogenesis of the enamel organ; folding of the epithelial-mesenchymal junction associated with cusp formation; and change in cellular heterogeneity in the mesenchyme. All these processes are controlled by epithelial-mesenchymal interactions. These complex histo-morphogenetic events have been documented using histological sections and 3D reconstructions. When combined with functional tests in vitro, this approach allowed searching for possible relationships between simultaneous changes occurring in both the epithelial and ecto-mesenchymal compartments. Parallel changes that occur in the two tissues could result from different mechanisms, as illustrated by the increasing number of pre-odontoblasts and pre-ameloblasts during crown growth. Cell division was involved mainly in the ecto-mesenchyme, while proliferation and cell re-organization occurred in the inner dental epithelium. 3D reconstructions also raised still unsolved questions, such as the possible relationship between cusp size and spatial specification of cell kinetic parameters, changes in cell position within the inner dental epithelium, and tracing cell migration in the mesenchyme during development.
Topics: Ameloblasts; Animals; Cell Differentiation; Dentinogenesis; Epithelium; Imaging, Three-Dimensional; Mesoderm; Mice; Molar; Odontoblasts; Odontogenesis; Tooth Cervix; Tooth Crown; Tooth Migration
PubMed: 24495111
DOI: 10.1111/adj.12132 -
Cellular and Molecular Life Sciences :... Jan 2003The amelogenins, the major proteins of the developing tooth enamel matrix, are highly conserved throughout most species studied. The gene structure is similar, with a... (Review)
Review
The amelogenins, the major proteins of the developing tooth enamel matrix, are highly conserved throughout most species studied. The gene structure is similar, with a set of seven exons and intervening introns, and remarkable conservation of particular exon sizes over divergent species. Studies of exon skipping and consequent alternative gene splicing suggest that, in vertebrates, exon definition is crucial. In this mechanism, exon size is important. If too small, an exon can be readily skipped, if too large, internal cryptic splice sites may be utilized. Other factors, such as intron length and specific nucleotide sequences at the splice boundaries also modulate splicing efficiency, but amelogenin gene splicing conforms well to the generalized exon length model. Exons 1, 2 and 7 are not subject to splicing that affects the secreted protein product, but exons 3, 4 and 5 are at the lower boundary of exon size, rendering them, 4 and 5 especially, subject to skipping. On the other hand, exon 6 is very long and has cryptic splicing sites that can be used. In the mouse, nine distinct splice product proteins have been detected. The question now is the functions of these products. The larger forms, those that contain the intact proline-rich, hydrophobic exon 6 domains, are important for enamel mineralization. Recent work suggests that the small proteins resulting from deletion of a major part of amelogenin gene exon 6 via utilization of a cryptic site may have signal transduction functions during tooth development. Furthermore, new work also suggests that odontoblasts transiently express the small amelogenins during the period that epithelial-mesenchymal signaling between preodontoblasts and preameloblasts determines the course of tooth development. The same peptides have been demonstrated to act on non-odontogenic cells and effect their phenotypic expression patterns in vitro, and to induce bone formation in implants in vivo.
Topics: Alternative Splicing; Ameloblasts; Amelogenin; Amino Acid Sequence; Animals; Base Sequence; Cattle; Cell Differentiation; Conserved Sequence; Dental Enamel Proteins; Exons; Humans; Mice; Odontoblasts; RNA, Messenger; Signal Transduction; Swine
PubMed: 12613657
DOI: 10.1007/s000180300003 -
Bone Jan 2022Amelogenesis consists of secretory, transition, maturation, and post-maturation stages, and the morphological changes of ameloblasts at each stage are closely related to...
Amelogenesis consists of secretory, transition, maturation, and post-maturation stages, and the morphological changes of ameloblasts at each stage are closely related to their function. p130 Crk-associated substrate (Cas) is a scaffold protein that modulates essential cellular processes, including cell adhesion, cytoskeletal changes, and polarization. The expression of p130Cas was observed from the secretory stage to the maturation stage in ameloblasts. Epithelial cell-specific p130Cas-deficient (p130Cas) mice exhibited enamel hypomineralization with chalk-like white mandibular incisors in young mice and attrition in aged mouse molars. A micro-computed tomography analysis and Vickers micro-hardness testing showed thinner enamel, lower enamel mineral density and hardness in p130Cas mice in comparison to p130Cas mice. Scanning electron microscopy, and an energy dispersive X-ray spectroscopy analysis indicated the disturbance of the enamel rod structure and lower Ca and P contents in p130Cas mice, respectively. The disorganized arrangement of ameloblasts, especially in the maturation stage, was observed in p130Cas mice. Furthermore, expression levels of enamel matrix proteins, such as amelogenin and ameloblastin in the secretory stage, and functional markers, such as alkaline phosphatase and iron accumulation, and Na/Ca+K-exchanger in the maturation stage were reduced in p130Cas mice. These findings suggest that p130Cas plays important roles in amelogenesis (197 words).
Topics: Ameloblasts; Amelogenesis; Animals; Crk-Associated Substrate Protein; Dental Enamel Proteins; Epithelial Cells; Mice; X-Ray Microtomography
PubMed: 34592494
DOI: 10.1016/j.bone.2021.116210 -
Frontiers in Physiology 2018The Leucine Rich Amelogenin Peptide (LRAP) is a product of alternative splicing of the gene. As full length amelogenin, LRAP has been shown, in precipitation...
The Leucine Rich Amelogenin Peptide (LRAP) is a product of alternative splicing of the gene. As full length amelogenin, LRAP has been shown, in precipitation experiments, to regulate hydroxyapatite (HAP) crystal formation depending on its phosphorylation status. However, very few studies have questioned the impact of its phosphorylation status on enamel mineralization in biological models. Therefore, we have analyzed the effect of phosphorylated (+P) or non-phosphorylated (-P) LRAP on enamel formation in ameloblast-like cell lines and cultures of murine postnatal day 1 molar germs. To this end, the mineral formed was analyzed by micro-computed tomography, Field Emission Scanning Electron Microscopy, Transmission Electron Microscopy, Selected Area Electon Diffraction imaging. gene transcription was evaluated by qPCR analysis. Our data show that, in both cells and germ cultures, LRAP is able to induce an up-regulation of transcription independently of its phosphorylation status. Mineral formation is promoted by LRAP(+P) in all models, while LRAP(-P) essentially affects HAP crystal formation through an increase in crystal length and organization in ameloblast-like cells. Altogether, these data suggest a differential effect of LRAP depending on its phosphorylation status and on the ameloblast stage at the time of treatment. Therefore, LRAP isoforms can be envisioned as potential candidates for treatment of enamel lesions or defects and their action should be further evaluated in pathological models.
PubMed: 29472869
DOI: 10.3389/fphys.2018.00055 -
Matrix Biology : Journal of the... Jan 2022Studies on animal models with mutations in ameloblastin gene have suggested that the extracellular matrix protein ameloblastin (AMBN) plays important roles in...
Studies on animal models with mutations in ameloblastin gene have suggested that the extracellular matrix protein ameloblastin (AMBN) plays important roles in controlling cell-matrix adhesion and ameloblast polarization during amelogenesis. In order to examine the function of AMBN in cell polarization and morphology, we developed an in vitro 3D cell culture model to examine the effect of AMBN and amelogenin (AMEL) addition on ameloblast cell lines. We further used high resolution confocal microscopy to detect expression of polarization markers in response to AMBN addition. Addition of AMBN to the 3D culture matrix resulted in the clustering and elongation (higher aspect ratio) of ALC in a dose dependent manner. The molar concentration of AMEL required to exact this response from ALC was 2.75- times greater than that of AMBN. This polarization effect of ameloblastin was attributable directly to an evolutionary conserved domain within its exon 5-encoded region. The lack of exon 6-encoded region also influenced AMBN-cell interactions but to a lesser extent. The clusters formed with AMBN were polarized with expression of E-cadherin, Par3 and Cldn1 assembly at the nascent cell-cell junctions. The elongation effect was specific to epithelial cells of ameloblastic lineage ALC and LS8 cells. Our data suggest that AMBN may play critical signaling roles in the initiation of cell polarity by acting as a communicator between cell-cell and cell-matrix interactions. Our investigation has important implications for understanding the function of ameloblastin in enamel-cell matrix adhesion and the outcomes may contribute to efforts to develop strategies for enamel tissue regeneration.
Topics: Ameloblasts; Amelogenesis; Amelogenin; Animals; Cell Culture Techniques, Three Dimensional; Cell Line; Dental Enamel Proteins
PubMed: 34813898
DOI: 10.1016/j.matbio.2021.11.002 -
Medecine Sciences : M/S May 2015Enamel is a unique tissue in vertebrates, acellular, formed on a labile scaffolding matrix and hypermineralized. The ameloblasts are epithelial cells in charge of... (Review)
Review
Enamel is a unique tissue in vertebrates, acellular, formed on a labile scaffolding matrix and hypermineralized. The ameloblasts are epithelial cells in charge of amelogenesis. They secrete a number of matrix proteins degraded by enzymes during enamel mineralization. This ordered cellular and extracellular events imply that any genetic or environmental perturbation will produce indelible and recognizable defects. The specificity of defects will indicate the affected cellular process. Thus, depending on the specificity of alterations, the teratogenic event can be retrospectively established. Advances in the field allow to use enamel defects as diagnostic tools for molecular disorders. The multifunctionality of enamel peptides is presently identified from their chemical roles in mineralization to cell signaling, constituting a source of concrete innovations in regenerative medicine.
Topics: Ameloblasts; Amelogenesis; Animals; Dental Enamel; Dental Enamel Hypoplasia; Dental Enamel Proteins; Durapatite; Enamel Organ; Fluorosis, Dental; Humans; Molecular Diagnostic Techniques; Nanospheres; Peptide Hydrolases; Teratogens; Tooth Calcification
PubMed: 26059302
DOI: 10.1051/medsci/20153105013 -
Frontiers in Pharmacology 2021Amelogenesis, the formation of dental enamel, is well understood at the histomorphological level but the underlying molecular mechanisms are poorly characterized....
Amelogenesis, the formation of dental enamel, is well understood at the histomorphological level but the underlying molecular mechanisms are poorly characterized. Ameloblasts secrete enamel matrix proteins and Ca, and also regulate extracellular pH as the formation of hydroxyapatite crystals generates large quantities of protons. Genetic or environmental impairment of transport and regulatory processes (e.g. dental fluorosis) leads to the development of enamel defects such as hypomineralization. Our aims were to optimize the culture conditions for the three-dimensional growth of ameloblast-derived HAT-7 cells and to test the effects of fluoride exposure on HAT-7 spheroid formation. To generate 3D HAT-7 structures, cells were dispersed and plated within a Matrigel extracellular matrix scaffold and incubated in three different culture media. Spheroid formation was then monitored over a two-week period. Ion transporter and tight-junction protein expression was investigated by RT-qPCR. Intracellular Ca and pH changes were measured by microfluorometry using the fluorescent dyes fura-2 and BCECF. A combination of Hepato-STIM epithelial cell differentiation medium and Matrigel induced the expansion and formation of 3D HAT-7 spheroids. The cells retained their epithelial cell morphology and continued to express both ameloblast-specific and ion transport-specific marker genes. Furthermore, like two-dimensional HAT-7 monolayers, the HAT-7 spheroids were able to regulate their intracellular pH and to show intracellular calcium responses to extracellular stimulation. Finally, we demonstrated that HAT-7 spheroids may serve as a disease model for studying the effects of fluoride exposure during amelogenesis. In conclusion, HAT-7 cells cultivated within a Matrigel extracellular matrix form three-dimensional, multi-cellular, spheroidal structures that retain their functional capacity for pH regulation and intracellular Ca signaling. This new 3D model will allow us to gain a better understanding of the molecular mechanisms involved in amelogenesis, not only in health but also in disorders of enamel formation, such as those resulting from fluoride exposure.
PubMed: 34149428
DOI: 10.3389/fphar.2021.682654