Expression Of Enamel Matrix Proteins Research Articles

Tmem2 Deficiency Leads to Enamel Hypoplasia and Soft Enamel in Mouse

Teeth consist of 3 mineralized tissues: enamel, dentin, and cementum. Tooth malformation, the most common craniofacial anomaly, arises from complex genetic and environmental factors affecting enamel structure, size, shape, and tooth eruption. Hyaluronic acid (HA), a primary extracellular matrix component, contributes to structural and physiological functions in periodontal tissue. Transmembrane protein 2 (TMEM2), a novel cell surface hyaluronidase, has been shown to play a critical role during embryogenesis. In this study, we demonstrate Tmem2 messenger RNA expression in inner enamel epithelium and presecretory, secretory, and mature ameloblasts. Tmem2 knock-in reporter mice reveal TMEM2 protein localization at the apical and basal ends of secretory ameloblasts. Micro–computed tomography analysis of epithelial-specific Tmem2 conditional knockout (Tmem2-CKO) mice shows a significant reduction in enamel layer thickness and severe enamel deficiency. Enamel matrix protein expression was remarkably downregulated in Tmem2-CKO mice. Scanning electron microscopy of enamel from Tmem2-CKO mice revealed an irregular enamel prism structure, while the microhardness and density of enamel were significantly reduced, indicating impaired ameloblast differentiation and enamel matrix mineralization. Histological evaluation indicated weak adhesion between cells and the basement membrane in Tmem2-CKO mice. The reduced and irregular expressions of vinculin and integrin β1 suggest that Tmem2 deficiency attenuated focal adhesion formation. In addition, abnormal HA accumulation in the ameloblast layer and weak claudin 1 immunoreactivity in Tmem2-CKO mice indicate impaired tight junction gate function. Irregular actin filament assembly was also observed at the apical and basal ends of secretory ameloblasts. Last, we demonstrated that Tmem2-deficient mHAT9d mouse ameloblasts exhibit defective adhesion to HA-containing substrates in vitro. Collectively, our data highlight the importance of TMEM2 in adhesion to HA-rich extracellular matrix, cell-to-cell adhesion, ameloblast differentiation, and enamel matrix mineralization.

Evolution of dental tissue mineralization: an analysis of the jawed vertebrate SPARC and SPARC-L families

BackgroundThe molecular bases explaining the diversity of dental tissue mineralization across gnathostomes are still poorly understood. Odontodes, such as teeth and body denticles, are serial structures that develop through deployment of a gene regulatory network shared between all gnathostomes. Dentin, the inner odontode mineralized tissue, is produced by odontoblasts and appears well-conserved through evolution. In contrast, the odontode hypermineralized external layer (enamel or enameloid) produced by ameloblasts of epithelial origin, shows extensive structural variations. As EMP (Enamel Matrix Protein) genes are as yet only found in osteichthyans where they play a major role in the mineralization of teeth and others skeletal organs, our understanding of the molecular mechanisms leading to the mineralized odontode matrices in chondrichthyans remains virtually unknown.ResultsWe undertook a phylogenetic analysis of the SPARC/SPARC-L gene family, from which the EMPs are supposed to have arisen, and examined the expression patterns of its members and of major fibrillar collagens in the spotted catshark Scyliorhinus canicula, the thornback ray Raja clavata, and the clawed frog Xenopus tropicalis. Our phylogenetic analyses reveal that the single chondrichthyan SPARC-L gene is co-orthologous to the osteichthyan SPARC-L1 and SPARC-L2 paralogues. In all three species, odontoblasts co-express SPARC and collagens. In contrast, ameloblasts do not strongly express collagen genes but exhibit strikingly similar SPARC-L and EMP expression patterns at their maturation stage, in the examined chondrichthyan and osteichthyan species, respectively.ConclusionsA well-conserved odontoblastic collagen/SPARC module across gnathostomes further confirms dentin homology. Members of the SPARC-L clade evolved faster than their SPARC paralogues, both in terms of protein sequence and gene duplication. We uncover an osteichthyan-specific duplication that produced SPARC-L1 (subsequently lost in pipidae frogs) and SPARC-L2 (independently lost in teleosts and tetrapods).Our results suggest the ameloblastic expression of the single chondrichthyan SPARC-L gene at the maturation stage reflects the ancestral gnathostome situation, and provide new evidence in favor of the homology of enamel and enameloids in all gnathostomes.

Deletion of epithelial cell-specific Cdc42 leads to enamel hypermaturation in a conditional knockout mouse model

Recent evidence suggests that GTPases Rho family plays an important role in tooth development; however, the role of Cdc42 in tooth development remains unclear. We aimed to investigate the function of Cdc42 in tooth development and amelogenesis. We generated an epithelial cell-specific K5-Cdc42 knockout (KO) mouse to evaluate post-eruption dental phenotypes using a K5-Cre driver line. This model overcomes the previously reported perinatal lethality. Tooth phenotypes were analyzed by micro X-ray, micro-computed tomography (CT), scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDX), wear rate, shear strength, and a microhardness test. Enamel matrix protein expression was determined by immunohistochemistry.KO mice displayed a hypomaturation phenotype, including incisors that lacked yellow pigmentation and were abnormally white, rapid attrition of molars following eruption, and decreased micro-hardness and shearing strength. Micro-CT data revealed that of incisor and molar enamel volumes were smaller in the KO than in wild-type (WT) mice. SEM analysis showed that the enamel prism structure was disordered. In addition, HE staining indicated a remarkable difference in the ameloblast morphology and function between KO and WT mice, and immunohistochemistry showed increased expression of amelogenin, ameloblastin, matrix metallopeptidase 20, kallikrein-related peptidase 4 and amelotin in the KO mice teeth.Our results suggest epithelium cell-specific Cdc42 deletion leads to tooth hypomaturation and transformation of the enamel prism structure that is likely due to altered ameloblast morphology and the secretion of enamel matrix proteins and proteases. This is the first in vivo evidence suggesting that Cdc42 is essential for proper tooth development and amelogenesis.

V-ATPases Containing a3 Subunit Play a Direct Role in Enamel Development in Mice.

Vacuolar H+ -ATPases (V-ATPases) are ubiquitous multisubunit proton pumps responsible for organellar pH maintenance. Mutations in the a3 subunit of V-ATPases cause autosomal recessive osteopetrosis, a rare disease due to impaired bone resorption. Patients with osteopetrosis also display dental anomalies, such as enamel defects; however, it is not clear whether these enamel abnormalities are a direct consequence of the a3 mutations. We investigated enamel mineralization, spatiotemporal expression of enamel matrix proteins and the a3 protein during tooth development using an osteopetrotic mouse model with a R740S point mutation in the V-ATPase a3 subunit. Histology revealed aberrations in both crown and root development, whereas SEM analysis demonstrated delayed enamel mineralization in homozygous animals. Enamel thickness and mineralization were significantly decreased in homozygous mice as determined by μCT analysis. The expression patterns of the enamel matrix proteins amelogenin, amelotin, and odontogenic ameloblast-associated protein (ODAM) suggested a delay in transition to the maturation stage in homozygous animals. Protein expression of the a3 subunit was detected in ameloblasts in all three genotypes, suggesting that a3-containing V-ATPases play a direct role in amelogenesis, and mutations in a3 delay transition from the secretory to the maturation stage, resulting in hypomineralized and hypoplastic enamel. J. Cell. Biochem. 118: 3328-3340, 2017. © 2017 Wiley Periodicals, Inc.

Bcl11b regulates enamel matrix protein expression and dental epithelial cell differentiation during rat tooth development.

Amelogenesis, beginning with thickened epithelial aggregation and ending with highly mineralized enamel formation, is a process mediated by a complex signaling network that involves several molecules, including growth and transcription factors. During early tooth development, the transcription factor B‑cellCLL/lymphoma11B (Bcl11b) participates in dental epithelial cell proliferation and differentiation. However, whether it affects the postnatal regulation of enamel matrix protein expression and ameloblast differentiation remains unclear. To clarify the role of Bcl11b in enamel development, the present study initially detected the protein expression levels of Bcl11b during tooth development using immunohistochemistry, from the embryonic lamina stage to the postnatal period, and demonstrated that Bcl11b is predominantly restricted to cervical loop epithelial cells at the cap and bell stages, whereas expression is reduced in ameloblasts. Notably, the expression pattern of Bcl11b during tooth development differed between rats and mice. Knockdown of Bcl11b by specific small interfering RNA attenuated the expression of enamel‑associated genes, including amelogenin, X‑linked (Amelx), ameloblastin (Ambn), enamelin (Enam), kallikrein related peptidase4 (Klk4), matrix metallopeptidase20 and Msh homeobox2 (Msx2). Chromatin immunoprecipitation assay verified that Msx2 was a transcriptional target of Bcl11b. However, overexpression of Msx2 resulted in downregulation of enamel‑associated genes, including Ambn, Amelx, Enam and Klk4. The present study suggested that Bcl11b serves a potentially important role in the regulation of ameloblast differentiation and enamel matrix protein expression. In addition, a complex feedback regulatory network may exist between Bcl11b and Msx2.

Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation.

Bone morphogenetic proteins (BMPs) regulate hard tissue formation, including bone and tooth. Growth differentiation factor 5 (GDF5), a known BMP, is expressed in cartilage and regulates chondrogenesis, and mutations have been shown to cause osteoarthritis. Notably, GDF5 is also expressed in periodontal ligament tissue; however, its role during tooth development is unclear. Here, we used cell culture and in vivo analyses to determine the role of GDF5 during tooth development. GDF5 and its associated BMP receptors are expressed at the protein and mRNA levels during postnatal tooth development, particularly at a stage associated with enamel formation. Furthermore, whereas BMP2 was observed to induce evidently the differentiation of enamel-forming ameloblasts, excess GDF5 induce mildly this differentiation. A mouse model harbouring a mutation in GDF5 (W408R) showed enhanced enamel formation in both the incisors and molars, but not in the tooth roots. Overexpression of the W408R GDF5 mutant protein was shown to induce BMP2-mediated mRNA expression of enamel matrix proteins and downstream phosphorylation of Smad1/5/8. These results suggest that mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-signalling.

The Impact of R740s Mutation of TCIRG1 on Dental Development in Osteopetrotic Mice

Osteopetrosis is a rare disease characterized by sclerotic bone due to impaired osteoclast function. Over 50% of the cases of the most severe, autosomal recessive form of the disease are associated with a defect in the TCIRG1 gene encoding the a3 subunit of vacuolar-type H + -ATPase (V-ATPase). These patients have bone resorption defects, delayed tooth eruption and variable enamel defects; however, little is known about amelogenesis in this condition. We studied an osteopetrotic mouse with a point mutation (R740S) in the V-ATPase a3 subunit, resulting in severe osteopetrosis, hypocalcemia, and dental abnormalities. To elucidate the role of V-ATPases in amelogenesis, we investigated the cellular distribution of a3, spatiotemporal expression of enamel matrix proteins, and measured thickness and mineral content of the enamel in the R740S mouse. Micro CT analysis demonstrated a statistically significant decrease in mineralization and thickness of the enamel in the homozygote relative to the heterozygote and wild type mice. SEM analysis showed that enamel mineralization was slightly delayed in the homozygote, although minimal crystallization was noted in all genotypes. To assess spatiotemporal protein expression of the enamel proteins, mouse mandibles were collected on days 1, 5 and 9 postnatal, fixed, decalcified, and analyzed using immunohistochemistry (IHC). IHC demonstrated that amelogenin and amelotin expression patterns were similar among all genotypes. Expression of the a3 subunit was not detected in the ameloblasts, suggesting that the hypomineralized and hypoplastic enamel found in the R740S mouse may be due to underlying systemic conditions affecting the enamel microenvironment.

Transcriptional factor DLX3 promotes the gene expression of enamel matrix proteins during amelogenesis.

Mutation of distal-less homeobox 3 (DLX3) is responsible for human tricho-dento-osseous syndrome (TDO) with amelogenesis imperfecta, indicating a crucial role of DLX3 in amelogenesis. However, the expression pattern of DLX3 and its specific function in amelogenesis remain largely unknown. The aim of this study was to investigate the effects of DLX3 on enamel matrix protein (EMP) genes. By immunohistochemistry assays of mouse tooth germs, stronger immunostaining of DLX3 protein was identified in ameloblasts in the secretory stage than in the pre-secretory and maturation stages, and the same pattern was found for Dlx3 mRNA using Realtime PCR. In a mouse ameloblast cell lineage, forced expression of DLX3 up-regulated the expression of the EMP genes Amelx, Enam, Klk4, and Odam, whereas knockdown of DLX3 down-regulated these four EMP genes. Further, bioinformatics, chromatin immunoprecipitation, and luciferase assays revealed that DLX3 transactivated Enam, Amelx, and Odam through direct binding to their enhancer regions. Particularly, over-expression of mutant-DLX3 (c.571_574delGGGG, responsible for TDO) inhibited the activation function of DLX3 on expression levels and promoter activities of the Enam, Amelx, and Odam genes. Together, our data show that DLX3 promotes the expression of the EMP genes Amelx, Enam, Klk4, and Odam in amelogenesis, while mutant-DLX3 disrupts this regulatory function, thus providing insights into the molecular mechanisms underlying the enamel defects of TDO disease.

Establishment and characterization of a primary calcifying epithelial odontogenic tumor cell population

Calcifying epithelial odontogenic tumors (CEOTs) are rare neoplasms derived from dental tissue with the unique characteristic of calcifying amyloid-like material. To establish primary CEOT epithelial-derived cell populations, investigate the expression of enamel matrix proteins (EMPs), and identify potential ameloblastin (AMBN) and patched 1 (PTCH1) gene alterations. A 28-year-old patient with a lesion of the posterior maxilla, radiographically characterized by a radiolucency with well-defined borders containing mixed radiopacities, agreed to participate with informed consent. The patient's biopsy confirmed the diagnosis of CEOT, and a small representative tumor fragment was ascertained for cell culture. Explant cultures were established and used to establish primary cell populations. These were analyzed for morphology, cell proliferation, mineralization activity, expression of epithelial-associated markers (qRT-PCR and immunocytochemistry), and gene mutations of AMBN or PTCH1. DNA was extracted from tumor cells and gene coding and exon-intron boundaries overlapping fragments amplified. PCR products were bidirectional DNA sequenced and compared against reference sequence. A CEOT cell population was established and proliferated in culture and could be maintained for several passages. Expression of EMPs, cytokeratin 14 and 17, and patched (PTCH1), as well as ALP activity, was detected. These cells also had the ability to mineralize, similar to the primary tumor. Two AMBN alterations were identified in the sample: c.1323G>A/A441A (rs7680880) and c.1344*+111delA. Two single-nucleotide polymorphisms were identified in the PTCH1 gene. Our data support the establishment of a CEOT-derived cell population, which expresses known epithelial-associated proteins.

Epithelial‐specific knockout of the Rac1 gene leads to enamel defects

The Ras-related C3 botulinum toxin substrate 1 (Rac1) gene encodes a 21-kDa GTP-binding protein belonging to the RAS superfamily. RAS members play important roles in controlling focal adhesion complex formation and cytoskeleton contraction, activities with consequences for cell growth, adhesion, migration, and differentiation. To examine the role(s) played by RAC1 protein in cell-matrix interactions and enamel matrix biomineralization, we used the Cre/loxP binary recombination system to characterize the expression of enamel matrix proteins and enamel formation in Rac1 knockout mice (Rac1(-/-)). Mating between mice bearing the floxed Rac1 allele and mice bearing a cytokeratin 14-Cre transgene generated mice in which Rac1 was absent from epithelial organs. Enamel of the Rac1 conditional knockout mouse was characterized by light microscopy, backscattered electron imaging in the scanning electron microscope, microcomputed tomography, and histochemistry. Enamel matrix protein expression was analyzed by western blotting. Major findings showed that the Tomes' processes of Rac1(-/-) ameloblasts lose contact with the forming enamel matrix in unerupted teeth, the amounts of amelogenin and ameloblastin are reduced in Rac1(-/-) ameloblasts, and after eruption, the enamel from Rac1(-/-) mice displays severe structural defects with a complete loss of enamel. These results support an essential role for RAC1 in the dental epithelium involving cell-matrix interactions and matrix biomineralization.

Functional role of rho‐kinase in ameloblast differentiation

During tooth development, inner enamel epithelial (IEE) cells differentiate into enamel-secreting ameloblasts, a polarized and elongated cellular population. The molecular underpinnings of this morphogenesis and cytodifferentiation, however, are not well understood. Here, we show that Rho-associated coiled-coil-containing protein kinase (ROCK) regulates ameloblast differentiation and enamel formation. In mouse incisor organ cultures, inhibition of ROCK, hindered IEE cell elongation and disrupted polarization of differentiated ameloblasts. Expression of enamel matrix proteins, such as amelogenin and ameloblastin, and formation of the terminal band structure of actin and E-cadherin were also perturbed. Cultures of dental epithelial cells revealed that ROCK regulates cell morphology and cell adhesion through localization of actin bundles, E-cadherin, and β-catenin to cell membranes. Moreover, inhibition of ROCK promoted cell proliferation. Small interfering RNA specific for ROCK1 and ROCK2 demonstrated that the ROCK isoforms performed complementary functions in the regulation of actin organization and E-cadherin-mediated cell-cell adhesion. Thus, our results have uncovered a novel role for ROCK in amelogenesis.

Induction of enamel matrix protein expression in an ameloblast cell line co-cultured with a mesenchymal cell line in vitro

Interactions between epithelium and mesenchyme are important for organ and tissue development. In this study, in order to mimic interactions between epithelium and mesenchyme during native tooth development, we constructed three-dimensional culture systems in vitro using a collagen membrane. Two types of collagen membrane-based in vitro culture systems were constructed in which dental epithelial and dental follicle cell lines were cultured. One co-culture method involved inoculation of one cell line into one side of the collagen membrane, and the other cell line into the opposite side of the membrane (sandwich co-culture). As a control, the second method involved culture of one of the cell lines on a culture dish and the second cell line on a collagen membrane, facing away from the first cell line (separate co-culture). The HAT-7 cells were also grown as a monolayer culture on collagen. Ameloblast differentiation in these cultures was investigated by analysis of the mRNA and/or protein expression of ameloblastin and amelogenin. Our results suggest that interaction of epithelial and mesenchymal cells via the extracellular matrix is important for tooth differentiation in vitro. Our culture system should be a useful method for investigation of epithelial-mesenchymal interactions.

Effect of BMP-2 on Gene Expression of Enamel Matrix Proteins at the Dental Epithelial Cell Line

Effect of BMP-2 on Gene Expression of Enamel Matrix Proteins at the Dental Epithelial Cell Line

Effect of BMP-2 on Gene Expression of Enamel Matrix Proteins at the Dental Epithelial Cell Line

Epithelial-mesenchymal interactions play an important role in the control of ameloblasts and odentoblasts differentiation, and the bone morphogenetic proteins (BMPs) are known factors that regulate the differentiation of ameloblasts. We examined the effect of BMP-2 on the expression of the enamel matrix protein genes at the dental epithelial cell line. BMP-2 induced a 3- to 4-fold increase in amelogenin and ameloblastin mRNA expression, suggesting that BMP-2 is important for ameloblast differentiation. This finding has potential application in the tissue engineering of tooth re-constructions.

Characterization of human primary enamel organ epithelial cells in vitro

Tooth enamel is formed by ameloblasts, which are derived from the epithelial cells of the enamel organ. The purpose of this study was to grow human ameloblast-like epithelial cells in culture. Human fetal tooth organs were isolated, and the cells were separated by digestion in collagenase/dispase. The cells were cultured in KGM-2 media with and without serum and at different calcium concentrations. The expression of enamel matrix proteins was analyzed by RT-PCR and cytokeratin 14 was detected by immunohistochemistry. The cells were further characterized by osteogenesis/odontogenesis-related DNA array. Cells isolated from the tooth organs grown in KGM-2 media containing 2-10% serum, were mixture of cobblestone and spindle shaped cells. Culturing these cells in KGM-2 with 0.05 mM calcium was selective for cobblestone ameloblasts-like cells (CAB), which were immunopositive for cytokeratin 14. Amelogenin, ameloblastin, enamelin, MMP-20 and KLK-4 were detected in CAB cells by RT-PCR. Osteogenesis SuperArray analyses could not detect the presence of typical molecules related to mesenchymal odontoblast or osteoblast lineage cells in these cultures. These studies showed that cobblestone-shaped ameloblast-like cells are selected from the tooth organ cells, by culture in KGM-2 media with 0.05 mM calcium.

Laminin alpha2 is essential for odontoblast differentiation regulating dentin sialoprotein expression.

Laminin alpha2 is subunit of laminin-2 (alpha2beta1gamma1), which is a major component of the muscle basement membrane. Although the laminin alpha2 chain is expressed in the early stage of dental mesenchyme development and localized in the tooth germ basement membrane, its expression pattern in the late stage of tooth germ development and molecular roles are not clearly understood. We analyzed the role of laminin alpha2 in tooth development by using targeted mice with a disrupted lama2 gene. Laminin alpha2 is expressed in dental mesenchymal cells, especially in odontoblasts and during the maturation stage of ameloblasts, but not in the pre-secretory or secretory stages of ameloblasts. Lama2 mutant mice have thin dentin and a widely opened dentinal tube, as compared with wild-type and heterozygote mice, which is similar to the phenotype of dentinogenesis imperfecta. During dentin formation, the expression of dentin sialoprotein, a marker of odontoblast differentiation, was found to be decreased in odontoblasts from mutant mice. Furthermore, in primary cultures of dental mesenchymal cells, dentin matrix protein, and dentin sialophosphoprotein, mRNA expression was increased in laminin-2 coated dishes but not in those coated with other matrices, fibronectin, or type I collagen. Our results suggest that laminin alpha2 is essential for odontoblast differentiation and regulates the expression of dentin matrix proteins.