G protein–coupled receptor Gpr115 (Adgrf4) is required for enamel mineralization mediated by ameloblasts
Yuta ChibaKeigo YoshizakiKan SaitoT IkeuchiTsutomu IwamotoCraig RhodesTakashi NakamuraSusana de VegaRobert J. MorellErich T. BogerDaniel Martı́nRyoko HinoHiroyuki InuzukaChristopher K. E. BleckAya YamadaYoshihiko YamadaSatoshi Fukumoto
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Dental enamel, the hardest tissue in the human body, is derived from dental epithelial cell ameloblast-secreted enamel matrices. Enamel mineralization occurs in a strictly synchronized manner along with ameloblast maturation in association with ion transport and pH balance, and any disruption of these processes results in enamel hypomineralization. G protein-coupled receptors (GPCRs) function as transducers of external signals by activating associated G proteins and regulate cellular physiology. Tissue-specific GPCRs play important roles in organ development, although their activities in tooth development remain poorly understood. The present results show that the adhesion GPCR Gpr115 (Adgrf4) is highly and preferentially expressed in mature ameloblasts and plays a crucial role during enamel mineralization. To investigate the in vivo function of Gpr115, knockout (Gpr115-KO) mice were created and found to develop hypomineralized enamel, with a larger acidic area because of the dysregulation of ion composition. Transcriptomic analysis also revealed that deletion of Gpr115 disrupted pH homeostasis and ion transport processes in enamel formation. In addition, in vitro analyses using the dental epithelial cell line cervical loop-derived dental epithelial (CLDE) cell demonstrated that Gpr115 is indispensable for the expression of carbonic anhydrase 6 (Car6), which has a critical role in enamel mineralization. Furthermore, an acidic condition induced Car6 expression under the regulation of Gpr115 in CLDE cells. Thus, we concluded that Gpr115 plays an important role in enamel mineralization via regulation of Car6 expression in ameloblasts. The present findings indicate a novel function of Gpr115 in ectodermal organ development and clarify the molecular mechanism of enamel formation.Keywords:
Amelogenesis
Enamel organ
Amelogenin
The formation of dental enamel is a precisely regulated and dynamic developmental process. The forming enamel starts as a soft, protein-rich tissue and ends as a hard tissue that is is over 95% mineral by weight. Intact amelogenin and its proteolytic cleavage products are the most abundant proteins present within the developing enamel. Proteinases are also present within the enamel matrix and are thought to help regulate enamel development and to expedite the removal of proteins prior to enamel maturation. Recently, a novel matrix metalloproteinase named enamelysin was cloned from the porcine enamel organ. Enamelysin transcripts have previously been observed in the enamel organ and dental papillae of the developing tooth. Here, we show that the sources of the enamelysin transcripts are the ameloblasts of the enamel organ and the odontoblasts of the dental papilla. Furthermore, we show that enamelysin is present within the forming enamel and that it is transported in secretory vesicles prior to its secretion from the ameloblasts. We also characterize the ability of recombinant enamelysin (rMMP-20) to degrade amelogenin under conditions of various pHs and calcium ion concentrations. Enamelysin displayed the greatest activity at neutral pH (7.2) and high calcium ion concentration (10 mM). During the initial stages of enamel formation, the enamel matrix maintains a. neutral pH of between 7.0 and 7.4. Thus, enamelysin may play a role in enamel and dentin formation by cleaving proteins that are also present during these initial developmental stages.
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Mutations in the human enamelin gene cause autosomal dominant hypoplastic amelogenesis imperfecta in which the affected enamel is thin or absent. Study of enamelin knockout NLS-lacZ knockin mice revealed that mineralization along the distal membrane of ameloblast is deficient, resulting in no true enamel formation. To determine the function of enamelin during enamel formation, we characterized the developing teeth of the Enam−/− mice, generated amelogenin-driven enamelin transgenic mouse models, and then introduced enamelin transgenes into the Enam−/− mice to rescue enamel defects. Mice at specific stages of development were subjected to morphologic and structural analysis using β-galactosidase staining, immunohistochemistry, and transmission and scanning electron microscopy. Enamelin expression was ameloblast-specific. In the absence of enamelin, ameloblasts pathology became evident at the onset of the secretory stage. Although the aggregated ameloblasts generated matrix-containing amelogenin, they were not able to create a well-defined enamel space or produce normal enamel crystals. When enamelin is present at half of the normal quantity, enamel was thinner with enamel rods not as tightly arranged as in wild type suggesting that a specific quantity of enamelin is critical for normal enamel formation. Enamelin dosage effect was further demonstrated in transgenic mouse lines over expressing enamelin. Introducing enamelin transgene at various expression levels into the Enam−/− background did not fully recover enamel formation while a medium expresser in the Enam+/− background did. Too much or too little enamelin abolishes the production of enamel crystals and prism structure. Enamelin is essential for ameloblast integrity and enamel formation.
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In Monodelphis domestica, although both processes from odontoblasts and projections from ameloblasts were found in developing enamel, the majority of the contents of enamel tubules were probably processes that originated from odontoblasts. Processes from odontoblasts penetrating into enamel touched part of the ameloblasts in the stage of enamel formation. No specialised cell junctions were seen at the adherence between the two. There were no enamel tubules in the aprismatic and pseudoprismatic enamel layer. It is likely that enamel tubules exist only in prismatic enamel. The majority of enamel tubules seemed to contain no cell processes in the stage of enamel maturation, indicating that the cell processes in the tubules probably degenerate after the stage of enamel formation.
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It was the purpose of this article to analyze the (micro) morphological structure of enamel at different stages of development in order to deduce movement patterns of ameloblasts during formation of the human dental primordium. Developing enamel and overlying ameloblasts were dried and fractured for scanning electron microscopy (SEM) and sectioned for transmission electron microscopy (TEM). Specimens of human permanent enamel were either fractured and/or ground and etched to visualize the enamel rods. All specimens were viewed by SEM. Moreover, three‐dimensional reconstructions were made from serial ground sections of enamel blocks to follow the enamel rods for a longer distance. In addition, the outline of the dentino–enamel junction was analyzed under the SEM after removal (using nitric acid) of the enamel cap, and in serial histological sections. Two basic movements of the inner enamel epithelium can be derived from the micromorphological features: (i) the scalloped dentino–enamel junction may be a consequence of a bulged inner enamel epithelium owing to initial spatial impediment; and (ii) the undulating path of the enamel rods may be a consequence of unequal growth of the cells in the cervical loop.
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Mutations in both the human amelogenin and human matrix metalloproteinase‐20 ( MMP20 , enamelysin) genes cause amelogenesis imperfecta . Both genes have also been individually deleted from the mouse and each deletion results in defective dental enamel. Here, we compare the stage‐specific progression of enamel development in continuously erupting mouse incisors from amelogenin null and MMP‐20 null mice. Our goal was to closely examine differences in enamel and enamel organ structure between these mice that would allow a better understanding of each protein's function. The predominant feature of the amelogenin null incisors was the late onset of mineral deposition, with little or no protein present within the forming mineral. Conversely, the developing MMP‐20 null incisors had a layer of protein between the apical surface of the ameloblasts and the forming enamel. Furthermore, the protein present within the enamel matrix was disorganized. An analysis of crystal structure demonstrated that the thin amelogenin null enamel was plate‐like, while the MMP‐20 null enamel had a disrupted prism pattern. These results suggest that amelogenin is essential for appositional crystal growth during the early to mid‐secretory stage and for the maintenance of the crystal ribbon structure. They also suggest that MMP‐20 is responsible for enamel matrix organization and for subsequent efficient reabsorption of enamel matrix proteins. Both genes are essential for the generation of full‐thickness enamel containing the characteristic decussating prism pattern.
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The progressive maturation of the ameloblast epithelium, formation of enamel matrix, and the mineralization of that matrix characterize normal amelogenesis. This process is regulated, in part, by the amelogenins. The expression by ameloblasts of amelogenin proteins as well as calcium transport proteins parallels matrix formation and mineralization. Odontogenic tumors reproduce, to varying degrees, the morphological and inductive relationships found among various parts of normal tooth germ. In normal amelogenesis, amelogenin, plasma membrane Ca(superscript ++)-pump PMCA , and calbindin-28KDa calbindin proteins are minimally expressed in presecretory ameloblasts, but progressively increase in expression later as the enamel matrix forms and begins to mineralize. Ameloblastomas, however, do not form matrix or mineralize. In the present study, we demonstrate by immunohistochemistry, that amelogenin, PMCA and calbindin proteins are all expressed in the ameloblast-like palisading peripheral epithelial cells of ten cases of ameloblastoma. In serial sections processed by the von Kossa technique, we also found diffuse calcium in all tumors studied. Our study suggests that ameloblastomas recapitulate a stage of differentiation beyond the presecretory stage and that the mechanisms of extracellular matrix formation and mineralization associated with normal amelogenesis may be uncoupled in ameloblastomas.
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Abstract The amelogenins are the predominant matrix proteins in developing enamel and are crucial for proper enamel mineralization. Transgenic mice were constructed in order to identify the segment of the amelogenin gene required for specific expression in enamel organ cells. A 3.5 kb fragment of the bovine X‐chromosomal amelogenin gene that includes a TATA box, the transcription initiation site, and 32 bp of exon 1 was linked to the β galactosidase gene and injected into fertilized mouse eggs. Newborn transgene positive mice expressed βgalactosidase activity in developing teeth treated with the chromogenic substrate Xgal. Foci of ameloblasts were positive in newborn mice; stain intensity and number of positive ameloblasts increased in 1‐day and 2‐day postnatal mice. Some of the adjacent stratum intermedium cells also were positive in the later stages. Targeting of the transgene to the enamel organ was specific; the only other cells observed to be positive were macrophages, which have endogenous βgalactosidase activity. © 1994 Wiley‐Liss, Inc.
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Bartlett JD, Skobe Z, Nanci A, Smith CE . Matrix metalloproteinase 20 promotes a smooth enamel surface, a strong dentino–enamel junction, and a decussating enamel rod pattern. Eur J Oral Sci 2011; 119 (Suppl. 1): 199–205. © 2011 Eur J Oral Sci 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.
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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 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. Thus, we hypothesized that αvβ6 integrin is expressed in ameloblasts where it regulates biomineralization of enamel. Human and mouse ameloblasts were found to express both β6 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, which did not use αvβ6 integrin as an endocytosis receptor for amelogenins, although it participated in cell adhesion on this matrix indirectly via endogenously produced matrix proteins. In summary, integrin αvβ6 is expressed by ameloblasts and it plays a crucial role in regulating amelogenin deposition/turnover and subsequent enamel biomineralization.
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