Elevated expression of calcineurin subunits during active mineralization of developing mouse molar teeth

Calcineurin is a Ca²⁺/calmodulin‐dependent protein phosphatase consisting of two subunits – catalytic subunit A (CnA) and regulatory subunit B (CnB) – and plays a critical role in transducing Ca²⁺ signals into cellular responses. In this study, we investigated the expression of calcineurin in the mouse developing tooth. In‐situ hybridization detected mRNAs for the CnAα and CnAβ isoforms of CnA and for the CnB1 isoform of CnB in the upper molar tooth germ at embryonic day 15. Immunohistochemistry with antibodies specific for CnAα, CnAβ, and CnB1 showed strong immunoreactivity of these proteins in secretory‐stage ameloblasts and in odontoblasts during dentin formation. CnAβ and CnB1 were strongly immunoreactive in ruffle‐ended ameloblasts at the enamel‐maturation stage. In ameloblasts and odontoblasts, we also noted different subcellular distributions of CnAα and CnAβ. From these data, temporal profiles of calcineurin expression appear to correlate with active mineralization in tooth development. Furthermore, the distinct subcellular distribution of the two CnA subunits may reflect their distinct substrates or responsive sites within single cells, thus contributing to the diversity of calcineurin‐dependent cellular responses during active tooth mineralization.     

Enamel and dental anomalies in latent‐transforming growth factor beta‐binding protein 3 mutant mice

Latent‐transforming growth factor beta‐binding protein 3 (LTBP‐3) is important for craniofacial morphogenesis and hard tissue mineralization, as it is essential for activation of transforming growth factor‐β (TGF‐β). To investigate the role of LTBP‐3 in tooth formation we performed micro‐computed tomography (micro‐CT), histology, and scanning electron microscopy analyses of adult Ltbp3‐/‐ mice. The Ltbp3‐/‐ mutants presented with unique craniofacial malformations and reductions in enamel formation that began at the matrix formation stage. Organization of maturation‐stage ameloblasts was severely disrupted. The lateral side of the incisor was affected most. Reduced enamel mineralization, modification of the enamel prism pattern, and enamel nodules were observed throughout the incisors, as revealed by scanning electron microscopy. Molar roots had internal irregular bulbous‐like formations. The cementum thickness was reduced, and microscopic dentinal tubules showed minor nanostructural changes. Thus, LTBP‐3 is required for ameloblast differentiation and for the formation of decussating enamel prisms, to prevent enamel nodule formation, and for proper root morphogenesis. Also, and consistent with the role of TGF‐β signaling during mineralization, almost all craniofacial bone components were affected in Ltbp3‐/‐ mice, especially those involving the upper jaw and snout. This mouse model demonstrates phenotypic overlap with Verloes Bourguignon syndrome, also caused by mutation of LTBP3, which is hallmarked by craniofacial anomalies and amelogenesis imperfecta phenotypes.     

Cholinergic autoantibodies from primary Sjögren’s syndrome modulate submandibular gland Na+/K+‐ATPase activity via prostaglandin E2 and cyclic AMP

Passafaro D, Reina S, Sterin‐Borda L, Borda E. Cholinergic autoantibodies from primary Sjögren’s syndrome modulate submandibular gland Na ⁺ /K ⁺ ‐ATPase activity via prostaglandin E ₂ and cyclic AMP. Eur J Oral Sci 2010; 118: 131–138. © 2010 The Authors. Journal compilation © 2010 Eur J Oral Sci We demonstrate that patients with primary Sjögren’s syndrome (pSS) produce functional IgG autoantibodies that interact with the glandular M₃ muscarinic acetylcholine receptors (mAChRs). These autoantibodies act as a partial muscarinic agonist, increasing prostaglandin E₂ (PGE₂) and cyclic AMP production through modifying Na⁺/K⁺‐ATPase activity, but also interfere with the secretory effect of the parasympathetic neurotransmitter. The IgG from patients with pSS has two effects on the submandibular gland. On the one hand, it may act as an inducer of the proinflammatory molecule (PGE₂) that, in turn, inhibits Na⁺/K⁺‐ATPase activity. On the other hand, it plays a role in the pathogenesis of dry mouth, abolishing the Na⁺/K⁺‐ATPase inhibition and the net K⁺ efflux stimulation of the salivary gland in response to the authentic agonist pilocarpine, decreasing salivary fluid production.     

Conditional knockout of transient receptor potential melastatin 7 in the enamel epithelium: Effects on enamel formation

Transient receptor potential melastatin 7 (TRPM7) is a unique ion channel connected to a kinase domain. We previously demonstrated that Trpm7 expression is high in mouse ameloblasts and odontoblasts, and that amelogenesis is impaired in TRPM7 kinase-dead mice. Here, we analyzed TRPM7 function during amelogenesis in Keratin 14-Cre;Trpm7^fl/fl conditional knockout (cKO) mice and Trpm7 knockdown cell lines. cKO mice showed lesser tooth pigmentation than control mice and broken incisor tips. Enamel calcification and microhardness were lower in cKO mice. Electron probe microanalysis (EPMA) showed that the calcium and phosphorus contents in the enamel were lower in cKO mouse than in control mice. The ameloblast layer in cKO mice showed ameloblast dysplasia at the maturation stage. The morphological defects were observed in rat SF2 cells with Trpm7 knockdown. Compared with mock transfectants, the Trpm7 knockdown cell lines showed lower levels of calcification with Alizarin Red-positive staining and an impaired intercellular adhesion structures. These findings suggest that TRPM7 is a critical ion channel in enamel calcification for the effective morphogenesis of ameloblasts during amelogenesis.     

Composition of mineralizing incisor enamel in cystic fibrosis transmembrane conductance regulator‐deficient mice

Formation of crystals in the enamel space releases protons that need to be buffered to sustain mineral accretion. We hypothesized that apical cystic fibrosis transmembrane conductance regulator (CFTR) in maturation ameloblasts transduces chloride into forming enamel as a critical step to secrete bicarbonates. We tested this by determining the calcium, chloride, and fluoride levels in developing enamel of Cftr‐null mice by quantitative electron probe microanalysis. Maturation‐stage enamel from Cftr‐null mice contained less chloride and calcium than did wild‐type enamel, was more acidic when stained with pH dyes ex vivo, and formed no fluorescent modulation bands after in vivo injection of the mice with calcein. To acidify the enamel further we exposed Cftr‐null mice to fluoride in drinking water to stimulate proton release during formation of hypermineralized lines. In Cftr‐deficient mice, fluoride further lowered enamel calcium without further reducing chloride levels. The data support the view that apical CFTR in maturation ameloblasts tranduces chloride into developing enamel as part of the machinery to buffer protons released during mineral accretion.     

Buffering of protons released by mineral formation during amelogenesis in mice

Regulation of pH by ameloblasts during amelogenesis is critical for enamel mineralization. We examined the effects of reduced bicarbonate secretion and the presence or absence of amelogenins on ameloblast modulation and enamel mineralization. To that end, the composition of fluorotic and non‐fluorotic enamel of several different mouse mutants, including enamel of cystic fibrosis transmembrane conductance regulator‐deficient (Cftr null), anion exchanger‐2‐deficient (Ae2a,b null), and amelogenin‐deficient (Amelx null) mice, was determined by quantitative X‐ray microanalysis. Correlation analysis was carried out to compare the effects of changes in the levels of sulfated‐matrix (S) and chlorine (Cl; for bicarbonate secretion) on mineralization and modulation. The chloride (Cl^?) levels in forming enamel determined the ability of ameloblasts to modulate, remove matrix, and mineralize enamel. In general, the lower the Cl^? content, the stronger the negative effects. In Amelx‐null mice, modulation was essentially normal and the calcium content was reduced least. Retention of amelogenins in enamel of kallikrein‐4‐deficient (Klk4‐null) mice resulted in decreased mineralization and reduced the length of the first acid modulation band without changing the total length of all acidic bands. These data suggest that buffering by bicarbonates is critical for modulation, matrix removal and enamel mineralization. Amelogenins also act as a buffer but are not critical for modulation.     

Localization of EMSP1 expression during tooth formation and cloning of mouse cDNA

Enamel matrix serine proteinase 1 (EMSP1) is a proteolytic enzyme that has been isolated from the developing enamel of pig teeth. Its apparent function is to degrade the organic matrix in preparation for enamel maturation. The expression of EMSP1 has never been investigated in another organism besides the pig, and EMSP1 expression in the enamel organ has never been specifically demonstrated in ameloblasts. Here we report the expression of recombinant pig EMSP 1 (rpEMSP 1), the generation of rabbit polyclonal antibodies against rpEMSP1, the characterization of the antibodies and EMSP1 expression by Western blot and immunohistochemical analyses, the cloning and characterization of a full-length cDNA encoding mouse EMSP1, and the localization of EMSP1 expression in ameloblasts in mouse day 14 first and second molars by in situ hybridization. The full-length mouse EMSP1 cDNA clone has 1,237 nucleotides, excluding the poly(A+) tail, and encodes a preproprotein of 255 amino acids. Mouse EMSP1 shares 75% amino acid identity with pig EMSP1 and has three potential N-linked glycosylation sites, two of which are conserved in the pig homologue. Western blot analysis shows that the polyclonal antibodies are specific for EMSP1 and do not cross-react with trypsin. Immunohistochemistry of pig incisors shows discrete staining in the surface enamel at the earliest part of the maturation stage. In mouse molars, in situ hybridization gives a distinct and specific signal in maturation-stage ameloblasts, and in the junctional epithelium following tooth eruption. We conclude that EMSP1 is expressed by pig and mouse ameloblasts during the early maturation stage of amelogenesis.     

Fate of fluoride-induced subameloblastic cysts in developing hamster molar tooth germs

White opacities and pits are developmental defects in enamel caused by high intake of fluoride (F) during amelogenesis. We tested the hypothesis that these enamel pits develop at locations where F induces the formation of sub-ameloblastic cysts. We followed the fate of these cysts during molar development over time. Mandibles from hamster pups injected with 20 mg NaF/kg at postnatal day 4 were excised from 1 h after injection till shortly after tooth eruption, 8 days later. Tissues were histologically processed and cysts located and measured. Cysts were formed at early secretory stage and transitional stage of amelogenesis and detected as early 1 h after injection. The number of cysts increased from 1 to almost 4 per molar during the first 16 h post-injection. The size of the cysts was about the same, i.e., 0.46 ± 0.29 × 10⁶ μm³ at 2 h and 0.50 ± 0.35 × 10⁷ μm³ at 16 h post-injection. By detachment of the ameloblasts the forming enamel surface below the cyst was cell-free for the first 16 h post-injection. With time new ameloblasts repopulated and covered the enamel surface in the cystic area. Three days after injection all cysts had disappeared and the integrity of the ameloblastic layer restored. After eruption, white opaque areas with intact enamel surface were found occlusally at similar anatomical locations as late secretory stage cysts were seen pre-eruptively. We conclude that at this moderate F dose, the opaque sub-surface defects with intact surface enamel (white spots) are the consequence of the fluoride-induced cystic lesions formed earlier under the late secretory–transitional stage ameloblasts.     

Overexpression of constitutively active MAP3K7 in ameloblasts causes enamel defects of mouse teeth

ObjectiveCompelling evidence suggests that mitogen-activated protein kinases (Mapks) play an important role in amelogenesis. However, the role of transforming growth factor (TGF)-β-activating kinase 1 (Tak1, Map3k7), which is a known upstream kinase of Mapks, during amelogenesis remains to be determined. The aim of this study was to investigate the possible involvement of Map3k7 in amelogenesis.DesignWe generated transgenic mice that produced constitutively active human MAP3K7 (caMAP3K7) under the control of amelogenin (Amelx) gene promoter. Radiography and micro-computed tomography (μCT) analysis was used to detect the radio-opacity and density of the teeth. The enamel microstructure was observed with a scanning electron microscope. Histological analysis was used to observe the adhesion between ameloblasts and residual organic matrix of the enamel. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the expression of enamel matrix protein.ResultsThe enamel of mandibular molars in caMAP3K7-overexpressing mice displayed pigmentation and a highly irregular structure compared with the wild type littermates. Teeth of transgenic animals underwent rapid attrition due to the brittleness of the enamel layer. The microstructure of enamel, normally a highly ordered arrangement of hydroxyapatite crystals, was completely disorganized. The gross histological appearances of ameloblasts and supporting cellular structures, as well as the expression of the enamel protein amelotin (Amtn) were altered by the overexpression of caMAP3K7.ConclusionsOur data demonstrated that protein expression, processing and secretion occurred abnormally in transgenic mice overexpressing caMAP3K7. The overexpression of caMAP3K7 had a profound effect on enamel structure by disrupting the orderly growth of enamel prisms.     

Organic anion transport during rat enamel formation

ObjectiveThe C-terminal end of nascent amelogenin is dissociated immediately after secretion and rapidly re-absorbed by ameloblasts, presumably by endocytosis. The purpose of this study was to test whether organic anion transporters (OATs) are also involved in the re-absorption process of enamel matrix proteins via non-endocytotic pathways.Materials and methodsLocalization of OAT1, OAT2, and OAT3 in rat tooth germs was examined by immunohistochemistry using specific antibodies. Actual translocation of organic anions through the ameloblast layer was further tested by systemic tracer experiments in rats in which Lucifer Yellow (LY), a fluorescent organic anion, was used as a tracer.ResultsIn rat tooth germs, OAT2 was associated exclusively with the distal cell membranes of secretory ameloblasts where Tomes' processes were developed and disappeared when matrix formation was terminated. On the other hand, OAT1 was absent in secretory ameloblasts and was colocalized with the ruffled border of ruffle-ended ameloblasts in the maturation stage. OAT3 was undetectable in ameloblasts and located instead only in the stratum intermedium cells. Systemic administration of LY resulted in intense labeling of immature enamel and also a transient labeling of the cytosol of secretory ameloblasts immunopositive for OAT2. In the maturation stage, cytosolic labeling of LY was negligible in all cells of the enamel organs, including ameloblasts.ConclusionsThese data suggest the existence of OATs in rat tooth germs and their possible involvement in matrix re-absorption at least in the secretory stage of amelogenesis.     

Ameloblasts require active RhoA to generate normal dental enamel

RhoA plays a fundamental role in regulation of the actin cytoskeleton, intercellular attachment, and cell proliferation. During amelogenesis, ameloblasts (which produce the enamel proteins) undergo dramatic cytoskeletal changes and the RhoA protein level is up‐regulated. Transgenic mice were generated that express a dominant‐negative RhoA transgene in ameloblasts using amelogenin gene‐regulatory sequences. Transgenic and wild‐type (WT) molar tooth germs were incubated with sodium fluoride (NaF) or sodium chloride (NaCl) in organ culture. Filamentous actin (F‐actin) stained with phalloidin was elevated significantly in WT ameloblasts treated with NaF compared with WT ameloblasts treated with NaCl or with transgenic ameloblasts treated with NaF, thereby confirming a block in the RhoA/Rho‐associated protein kinase (ROCK) pathway in the transgenic mice. Little difference in quantitative fluorescence (an estimation of fluorosis) was observed between WT and transgenic incisors from mice provided with drinking water containing NaF. We subsequently found reduced transgene expression in incisors compared with molars. Transgenic molar teeth had reduced amelogenin, E‐cadherin, and Ki67 compared with WT molar teeth. Hypoplastic enamel in transgenic mice correlates with reduced expression of the enamel protein, amelogenin, and E‐cadherin and cell proliferation are regulated by RhoA in other tissues. Together these findings reveal deficits in molar ameloblast function when RhoA activity is inhibited.     

Molecular insights into hypomineralized enamel

Hypomineralized enamel may be found in connection with the condition molar incisor hypomineralization (MIH), which has a prevalence of around 15% in most parts of the world. Molar incisor hypomineralization is associated with extensive objective and subjective problems, such as hypersensitivity of the affected teeth, enamel breakdown, and problems with retention of restorations. The etiology behind MIH has not yet been elucidated, but a number of possible factors, which affect the same or different functions of ameloblasts during their different stages of maturation, have been suggested. The aim of this study was to utilize multi‐nuclear, solid‐state nuclear magnetic resonance (ss‐NMR) and time‐of‐flight secondary ion mass spectroscopy (ToF‐SIMS) to elucidate any differences, at a molecular level, between enamel powder prepared from normal, healthy teeth and enamel powder prepared from teeth diagnosed with MIH. ³¹P and ²³Na ss‐NMR confirmed the presence of and two different Na⁺ sites in hypomineralized enamel, suggesting a heterogeneous chemical composition. The content of organic components was higher in hypomineralized enamel, as shown by both ¹³C ss‐NMR and ToF‐SIMS, indicating the presence of higher numbers of proteins and phospholipids. The interplay between both is necessary for the formation and mineralization of enamel, which might be disturbed or halted in hypomineralized enamel.     

TGF-β autocrine signaling at secretory-stage enamel

Background

In recent years, transforming growth factor (TGF)-β has been found in the enamel matrix, and along with enamel protein, is thought to play an important role in the process of calcification of tooth enamel. The purpose of this study was to investigate the dynamics of TGF-β and its interactions with enamel proteins, through experiments at the genetic and protein levels.

Autoradiographic study with [35S]-sodium sulphate of loss of sulphated glycosaminoglycans during amelogenesis in the guinea pig.

The fate of sulphated organic compounds, probably glycosaminoglycans (GAG), during amelogenesis was studied by autoradiography in molar teeth of guinea pigs that had received a single dose of 2.5 μCi/g of body weight of [³⁵S]-sodium sulphate and killed from 10 min to 168 hr after injection. The sulphation site was the Golgi region of ameloblasts, from where the sulphated compounds migrated into the young enamel matrix along the entire zone lined by secretory ameloblasts. After forming an ill-defined band along the Tomes processes and the adjacent matrix, the ³⁵S diffused through the whole thickness of the matrix, reaching the dentine-enamel junction.By correlating the silver grain concentration over matrix with the growth of the tooth. it was shown that the radioactive reaction, after attaining a maximum of intensity at 24 hr. decreased at 48 hr when the matrix moved from one region to the next, which was still related to the secretory ameloblasts. After this sharp decrease, the concentration of silver grains tended to be relatively constant and the remaining labelled compound seemed to be stable.The results were interpreted as indicating that the removal of organic material in enamel maturation is related at least in part to the secretory ameloblasts.     

Cyclical incorporation of 33P into rat incisor enamel in vivo as visualized by whole-mount radioautography

Phosphorus uptake during amelogenesis was investigated in the continuously erupting rat incisor. Five minutes after intravenous injection of ³³P-labelled orthophosphoric acid, whole-mount radioautography of entire incisors revealed heavy labelling in the form of bands and narrow parallel stripes at the surface of the enamel in the maturation zone. There was relatively little labelling over enamel in the secretion zone and over pigmented enamel. Thus ³³P is incorporated cyclically into maturing enamel and is visualized as (1) a banded pattern that reflects the modulation of ruffle-ended and smooth-ended maturation ameloblasts and (2) a striped pattern that reflects the distribution of newly-formed protein secreted by maturation ameloblasts. Presumably these P incorporation patterns are closely related to other cyclical events known to occur during enamel maturation.     

Odontogenic ameloblast-associated protein (ODAM) and amelotin: Major players in hypermineralization of enamel and enameloid

Odontogenic ameloblast-associated protein (ODAM) and amelotin (AMTN) both belong to the secretory calcium-binding phosphoprotein family, which is critical to biomineralization in vertebrates. In mammals, both ODAM and AMTN are expressed by ameloblasts in the maturation stage, when immature enamel grows into a hypermineralized inorganic tissue. At the onset of this stage, ameloblasts produce a specialized basal lamina (BL), over which both ODAM and AMTN are distributed. Enameloid is a different hypermineralized tissue that is found on the tooth surface of most ray-finned fish. Unlike amelogenesis, no such BL is produced during the maturation of enameloid. Nevertheless, ODAM is also found in ray-finned fish, and the expression of this gene has been detected in inner dental epithelial cells, which correspond to ameloblasts, after the enameloid is considerably mineralized. This specific gene expression suggests that ODAM is not a constituent of the BL but is still involved in the hypermineralization of enameloid. Both ODAM and AMTN are unusually rich in Pro and Gln, and they have 1 or 2 clusters of phospho-Ser residues. These characteristics suggest that ODAM and AMTN associate with weak interactions between relatively hydrophobic regions and further bind calcium phosphate via phospho-Ser clusters, similar to milk caseins that are evolutionary descendants of ODAM. Based on these considerations, I hypothesized that ODAM and AMTN generate and maintain the interface between unmineralized and hypermineralizing domains through weak protein–protein interactions and associations with calcium phosphate. This interface presumably facilitates hypermineralization, efficient removal of degraded proteins from the matrix, and the transfer of calcium phosphate to the matrix.     

Importance of bicarbonate transport in pH control during amelogenesis — need for functional studies

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.