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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 -
Journal of Dental Research Aug 2020Ameloblastin (Ambn) has the potential to regulate cell-matrix adhesion through familiar cell-binding domains, but the proposed sequence motifs are not highly conserved...
Ameloblastin (Ambn) has the potential to regulate cell-matrix adhesion through familiar cell-binding domains, but the proposed sequence motifs are not highly conserved across species. Here, we report that Ambn binds to ameloblast-like cell membranes through a highly evolutionary conserved amphipathic helix-forming (AH) motif encoded by exon 5. We applied high-resolution confocal microscopy to show colocalization of Ambn with ameloblast membrane surfaces in developing mouse incisors. Using a series of Ambn-derived peptides and Ambn variants, we showed that Ambn binds to cell membranes through a motif within the sequence encoded by exon 5. Using peptides derived from the N- or C-termini of this sequence, as well as Ambn variants that lacked or had a disrupted AH motif, we demonstrated that the AH motif located at the N-terminus of the sequence is involved in cell-Ambn adhesion. Sequence analysis revealed that this highly conserved AH motif is absent from other enamel matrix proteins, including amelogenin, enamelin, and amelotin. Collectively, these data suggest that Ambn binds to the cell surface membrane via a helix-forming motif and provide insight into the molecular mechanism and function of Ambn in enamel cell-matrix interaction.
Topics: Ameloblasts; Amelogenin; Animals; Cell Communication; Dental Enamel; Dental Enamel Proteins; Incisor; Mice
PubMed: 32401578
DOI: 10.1177/0022034520918521 -
Experimental and Therapeutic Medicine Nov 2018Amelogenesis is a complicated process that concerns the interaction between growing hydroxyapatite crystals and extracellular proteins, which requires the tight... (Review)
Review
Amelogenesis is a complicated process that concerns the interaction between growing hydroxyapatite crystals and extracellular proteins, which requires the tight regulation of pH. In dental fluorosis, the balance of pH regulation is broken, leading to abnormal mineralization. The current review focuses on the electrolyte transport processes associated with pH homeostasis, particularly regarding the changes in ion transporters that occur during amelogenesis, following exposure to excessive fluoride. Furthermore, the possible mechanism of fluorosis is discussed on the basis of acid hypothesis. There are two main methods by which F accelerates crystal formation in ameloblasts. Firstly, it induces the release of protons, lowering the pH of the cell microenvironment. The decreased pH stimulates the upregulation of ion transporters, which attenuates further declines in the pH. Secondly, F triggers an unknown signaling pathway, causing changes in the transcription of ion transporters and upregulating the expression of bicarbonate transporters. This results in the release of a large amount of bicarbonate from ameloblasts, which may neutralize the pH to form a microenvironment that favors crystal nucleation. The decreased pH stimulates the diffusion of F into the cytoplasm of amelobalsts along the concentration gradient formed by the release of protons. The retention of F causes a series of pathological changes, including oxidative and endoplasmic reticulum stress. If the buffering capacity of ameloblasts facing F toxicity holds, normal mineralization occurs; however, if F levels are high enough to overwhelm the buffering capacity of ameloblasts, abnormal mineralization occurs, leading to dental fluorosis.
PubMed: 30402142
DOI: 10.3892/etm.2018.6728 -
International Journal of Molecular... Jul 2021Amelogenin comprises ~90% of enamel proteins; however, the involvement of transcriptional activation in regulating ameloblast differentiation from induced pluripotent...
Amelogenin comprises ~90% of enamel proteins; however, the involvement of transcriptional activation in regulating ameloblast differentiation from induced pluripotent stem cells (iPSCs) remains unknown. In this study, we generated doxycycline-inducible -expressing mouse iPSCs (Amelx-iPSCs). We then established a three-stage ameloblast induction strategy from Amelx-iPSCs, including induction of surface ectoderm (stage 1), dental epithelial cells (DECs; stage 2), and ameloblast lineage (stage 3) in sequence, by manipulating several signaling molecules. We found that adjunctive use of lithium chloride (LiCl) in addition to bone morphogenetic protein 4 and retinoic acid promoted concentration-dependent differentiation of DECs. The resulting cells had a cobblestone appearance and keratin14 positivity. Attenuation of LiCl at stage 3 together with transforming growth factor β1 and epidermal growth factor resulted in an ameloblast lineage with elongated cell morphology, positivity for ameloblast markers, and calcium deposition. Although stage-specific activation of did not produce noticeable phenotypic changes in ameloblast differentiation, activation at stage 3 significantly enhanced cell adhesion as well as decreased proliferation and migration. These results suggest that the combination of inducible transcription and stage-specific ameloblast induction for iPSCs represents a powerful tool to highlight underlying mechanisms in ameloblast differentiation and function in association with expression.
Topics: Ameloblasts; Amelogenin; Animals; Cell Adhesion; Cell Differentiation; Doxycycline; Epithelial Cells; Induced Pluripotent Stem Cells; Mice; Signal Transduction; Transcriptional Activation
PubMed: 34281250
DOI: 10.3390/ijms22137195 -
Biochemical and Biophysical Research... Dec 2021Tooth development involves the coordinated transcriptional regulation of extracellular matrix proteins produced by ameloblasts and odontoblasts. In this study,...
Tooth development involves the coordinated transcriptional regulation of extracellular matrix proteins produced by ameloblasts and odontoblasts. In this study, whole-genome ChIP-seq analysis was applied to identify the transcriptional regulatory gene targets of Sp6 in mesenchymal cells of the developing tooth. Bioinformatic analysis of a pool of Sp6 target peaks identified the consensus nine nucleotide binding DNA motif CTg/aTAATTA. Consistent with these findings, a number of enamel and dentin matrix genes including amelogenin (Amelx), ameloblastin (Ambn), enamelin (Enam) and dental sialophosphoprotein (Dspp), were identified to contain Sp6 target sequences. Sp6 peaks were also found in other important tooth genes including transcription factors (Dlx2, Dlx3, Dlx4, Dlx5, Sp6, Sp7, Pitx2, and Msx2) and extracellular matrix-related proteins (Col1a2, Col11a2, Halpn1). Unsupervised UMAP clustering of tooth single cell RNA-seq data confirmed the presence of Sp6 transcripts co-expressed with many of the identified target genes within ameloblasts and odontoblasts. Lastly, transcriptional reporter assays using promoter fragments from the Hapln1 and Sp6 gene itself revealed that Sp6 co-expression enhanced gene transcriptional activity. Taken together these results highlight that Sp6 is a major regulator of multiple extracellular matrix genes in the developing tooth.
Topics: Ameloblasts; Amelogenin; Animals; Animals, Newborn; Collagen Type I; Dental Enamel Proteins; Extracellular Matrix Proteins; Gene Expression Regulation, Developmental; Gene Regulatory Networks; Kruppel-Like Transcription Factors; Mice; Mice, Inbred C57BL; Molar; Odontoblasts; Odontogenesis; Promoter Regions, Genetic; Proteoglycans; RNA, Messenger; Sequence Analysis, RNA; Signal Transduction; Single-Cell Analysis; Sp7 Transcription Factor
PubMed: 34662808
DOI: 10.1016/j.bbrc.2021.10.017 -
Scientific Reports May 2022Melatonin plays a critical role in promoting the proliferation of osteoblasts and the growth and development of dental papilla cells. However, the effect and mechanism...
Melatonin plays a critical role in promoting the proliferation of osteoblasts and the growth and development of dental papilla cells. However, the effect and mechanism of melatonin on the growth and development of ALCs still need to be explored. CCK8 assay was used for the evaluation of cell numbers. qRT-PCR was used to identify the differentially expressed genes in ALCs after melatonin treatment. The number and morphology of ALCs were investigated by confocal microscopy. Alkaline phosphatase assay and Alizarin red S staining were used for measuring mineralization. Then, we focused on observing the crucial factors of the signaling pathway by RNA-seq and qRT-PCR. Melatonin limited the cell number of ALCs in a dose-dependent manner and promoted the production of actin fibers. A high concentration of melatonin significantly promoted the mRNA levels of enamel matrix proteins and the formation of mineralized nodules. RNA-seq data showed that Wnt signaling pathway may be involved in the differentiation of ALCs under the influence of melatonin. This study suggests that melatonin plays a regulatory role in the cell number, differentiation, and mineralization of the ALCs, and then shows the relationship between the Wnt signaling pathway with the ALCs under melatonin.
Topics: Ameloblasts; Animals; Cell Differentiation; Cell Line; Cell Proliferation; Melatonin; Mice; Osteoblasts; Osteogenesis; Wnt Signaling Pathway
PubMed: 35581244
DOI: 10.1038/s41598-022-11912-3 -
Developmental Dynamics : An Official... Oct 2015Orai1 is a plasma membrane protein that forms the pore of the calcium release activated calcium channel. Humans with mutated Orai1 present with hereditary combined...
BACKGROUND
Orai1 is a plasma membrane protein that forms the pore of the calcium release activated calcium channel. Humans with mutated Orai1 present with hereditary combined immunodeficiency, congenital myopathy and anhidrotic ectodermal dysplasia. Consistent with the ectodermal dysplasia phenotype, enamel formation and mineralization is also abnormal in Orai1 deficient patients. The expression pattern and potential functions of Orai1 in enamel formation remains unclear. To contribute toward understanding the role of Orai1 in amelogenesis we characterized ORAI1 protein developmental pattern in comparison with other ectodermal organs. We also examined the effects of Orai1 down-regulation in ameloblast cell proliferation and differentiation.
RESULTS
Our data show strong expression of ORAI1 protein during the ameloblast secretory stage, which weans at the end of the maturation stage. In salivary glands, ORAI1 is expressed mainly in acini cells. ORAI1 expression is also found in hair follicle and oral epithelium. Knockdown of Orai1 expression decreases cell proliferation and results in RNA expression levels changes of key ameloblast genes regulating enamel thickness and mineralization.
CONCLUSIONS
This study provides insights in the anhidrotic ectodermal dysplasia phenotype due to Orai1 mutation and highlights the importance of calcium signaling in controlling ameloblast differentiation and maturation during tooth development.
Topics: Ameloblasts; Animals; Calcium Channels; Calcium Signaling; Cell Differentiation; Cell Proliferation; Ectodermal Dysplasia; Gene Expression; Gene Knockdown Techniques; Hair Follicle; Mice, Inbred C57BL; Mouth Mucosa; ORAI1 Protein; ORAI2 Protein; Organogenesis; Salivary Glands; Tooth
PubMed: 26178077
DOI: 10.1002/dvdy.24307 -
Proceedings of the National Academy of... Mar 2009The transcription factor Ctip2/Bcl11b plays essential roles in developmental processes of the immune and central nervous systems and skin. Here we show that Ctip2 also...
The transcription factor Ctip2/Bcl11b plays essential roles in developmental processes of the immune and central nervous systems and skin. Here we show that Ctip2 also plays a key role in tooth development. Ctip2 is highly expressed in the ectodermal components of the developing tooth, including inner and outer enamel epithelia, stellate reticulum, stratum intermedium, and the ameloblast cell lineage. In Ctip2(-/-) mice, tooth morphogenesis appeared to proceed normally through the cap stage but developed multiple defects at the bell stage. Mutant incisors and molars were reduced in size and exhibited hypoplasticity of the stellate reticulum. An ameloblast-like cell population developed ectopically on the lingual aspect of mutant lower incisors, and the morphology, polarization, and adhesion properties of ameloblasts on the labial side of these teeth were severely disrupted. Perturbations of gene expression were also observed in the mandible of Ctip2(-/-) mice: expression of the ameloblast markers amelogenin, ameloblastin, and enamelin was down-regulated, as was expression of Msx2 and epiprofin, transcription factors implicated in the tooth development and ameloblast differentiation. These results suggest that Ctip2 functions as a critical regulator of epithelial cell fate and differentiation during tooth morphogenesis.
Topics: Ameloblasts; Animals; Cell Differentiation; DNA-Binding Proteins; Down-Regulation; Embryonic Development; Epithelial Cells; Mandible; Mice; Mice, Knockout; Odontogenesis; Repressor Proteins; Tooth; Transcription Factors; Tumor Suppressor Proteins
PubMed: 19251658
DOI: 10.1073/pnas.0900568106 -
Importance of bicarbonate transport in pH control during amelogenesis - need for functional studies.Oral Diseases Sep 2018Dental enamel, the hardest mammalian tissue, is produced by ameloblasts. Ameloblasts show many similarities to other transporting epithelia although their secretory... (Review)
Review
Dental enamel, the hardest mammalian tissue, is produced by ameloblasts. Ameloblasts show many similarities to other transporting epithelia although their secretory product, the enamel matrix, is quite different. Ameloblasts direct the formation of hydroxyapatite crystals, which liberate large quantities of protons that then need to be buffered to allow mineralization to proceed. Buffering requires a tight pH regulation and secretion of bicarbonate by ameloblasts. Many investigations have used immunohistochemical and knockout studies to determine the effects of these genes on enamel formation, but up till recently very little functional data were available for mineral ion transport. To address this, we developed a novel 2D in vitro model using HAT-7 ameloblast cells. HAT-7 cells can be polarized and develop functional tight junctions. Furthermore, they are able to accumulate bicarbonate ions from the basolateral to the apical fluid spaces. We propose that in the future, the HAT-7 2D system along with similar cellular models will be useful to functionally model ion transport processes during amelogenesis. Additionally, we also suggest that similar approaches will allow a better understanding of the regulation of the cycling process in maturation-stage ameloblasts, and the pH sensory mechanisms, which are required to develop sound, healthy enamel.
Topics: Ameloblasts; Amelogenesis; Bicarbonates; Biological Transport; Cell Line; Humans; Hydrogen-Ion Concentration; Receptors, G-Protein-Coupled
PubMed: 28834043
DOI: 10.1111/odi.12738 -
Scientific Reports Mar 2019Dental enamel is the highly mineralized tissue covering the tooth surface and is formed by ameloblasts. Ameloblasts have been known to be impossible to detect in adult...
Dental enamel is the highly mineralized tissue covering the tooth surface and is formed by ameloblasts. Ameloblasts have been known to be impossible to detect in adult tooth because they are shed by apoptosis during enamel maturation and tooth eruption. Owing to these, little was known about appropriate cell surface markers to isolate ameloblast-like cells in tissues. To overcome these problems, epithelial cells were selectively cultivated from the gingival tissues and used as a stem cell source for ameloblastic differentiation. When gingival epithelial cells were treated with a specified concentration of BMP2, BMP4, and TGFβ-1, the expression of ameloblast-specific markers was increased, and both the MAPK and Smad signaling pathways were activated. Gingival epithelial cells differentiated into ameloblast-like cells through epithelial-mesenchymal transition. By RNA-Seq analysis, we reported 20 ameloblast-specific genes associated with cell surface, cell adhesion, and extracellular matrix function. These cell surface markers might be useful for the detection and isolation of ameloblast-like cells from dental tissues.
Topics: Adult; Ameloblasts; Amelogenesis; Amelogenin; Cell Differentiation; Cells, Cultured; Epithelial Cells; Epithelial-Mesenchymal Transition; Gene Expression Profiling; Gingiva; Humans; Sequence Analysis, RNA; Signal Transduction; Young Adult
PubMed: 30842534
DOI: 10.1038/s41598-019-40091-x