Calmodulin blocker inhibits Ca++-ATPase activity in secretory ameloblast of rat incisor

Summary The effects of the calmodulin blocker, trifluoperazine (TEP), on membrane-bound Ca⁺⁺ -ATPase, Na⁺ -K⁺ -ATPase (EC 3.6.1.3.) and the ultrastructure of the enamel organ were investigated in the lower incisors of normal and TFP-injected rats. The rats, of about 100 g body weight, were given either 0.2 ml physiological saline or 100 g TFP dissolved in 0.2 ml physiological saline through a jugular vein and fixed by transcardiac perfusion with a formaldehyde-glutaraldehyde mixture at 1 and 2 h after TFP administration. Non-decalcified sections of the enamel organ less than 50 m in thickness, prepared from dissected lower incisors, were processed for the ultracytochemical demonstration of Ca⁺⁺-ATPase and Na⁺-K⁺ -ATPase by the one-step lead method at alkaline pH. In control saline-injected animals the most intense enzymatic reaction of Ca⁺⁺-ATPase was demonstrated along the plasma membranes of the entire cell surfaces of secretory ameloblasts. Moderate enzymatic reaction was also observed in the plasma membranes of the cells of stratum intermedium and papillary layer. Reaction precipitates of Na⁺-K⁺-ATPase activity were localized clearly along the plasma membranes of only the cells of stratum intermedium and papillary layer. The most drastic effect of TFP was a marked disappearance of enzymatic reaction of Ca⁺⁺-ATPase from the plasma membranes of secretory ameloblasts, except for a weak persistent reaction in the basolateral cell surfaces of the infranuclear region facing the stratum intermedium. The cells of stratum intermedium and papillary layer, however, continued to react for Ca⁺⁺-ATPase even after TFP treatment. Similarly, Na⁺-K⁺-ATPase activity in these cells was not inhibited by TFP administration. Ultrastructural examination of secretory ameloblasts revealed that administration of TFP caused no considerable cytological changes and did not act as a cytotoxic agent. These results suggest that secretory ameloblasts may have an active Ca⁺⁺ transport system, which is modulated by an endogenous calmodulin.     

CRAC channels in dental enamel cells

Enamel mineralization relies on Ca²⁺ availability provided by Ca²⁺ release activated Ca²⁺ (CRAC) channels. CRAC channels are modulated by the endoplasmic reticulum Ca²⁺ sensor STIM1 which gates the pore subunit of the channel known as ORAI1, found the in plasma membrane, to enable sustained Ca²⁺ influx. Mutations in the STIM1 and ORAI1 genes result in CRAC channelopathy, an ensemble of diseases including immunodeficiency, muscular hypotonia, ectodermal dysplasia with defects in sweat gland function and abnormal enamel mineralization similar to amelogenesis imperfecta (AI). In some reports, the chief medical complain has been the patient’s dental health, highlighting the direct and important link between CRAC channels and enamel. The reported enamel defects are apparent in both the deciduous and in permanent teeth and often require extensive dental treatment to provide the patient with a functional dentition. Among the dental phenotypes observed in the patients, discoloration, increased wear, hypoplasias (thinning of enamel) and chipping has been reported. These findings are not universal in all patients. Here we review the mutations in STIM1 and ORAI1 causing AI-like phenotype, and evaluate the enamel defects in CRAC channel deficient mice. We also provide a brief overview of the role of CRAC channels in other mineralizing systems such as dentine and bone.     

Constitutive activation of β-catenin in ameloblasts leads to incisor enamel hypomineralization

Enamel is the hardest tissue with the highest degree of mineralization protecting the dental pulp from injury in vertebrates. The ameloblasts differentiated from ectoderm-derived epithelial cells are a single cell layer and are important for the enamel formation and mineralization. Wnt/β-catenin signaling has been proven to exert an important role in the mineralization of bone, dentin and cementum. Little was known about the regulatory mechanism of Wnt/β-catenin signaling pathway in ameloblasts during amelogenesis, especially in the mineralization of enamel. To investigate the role of β-catenin in ameloblasts, we established Amelx-Cre; β-catenin^?ex3fl/fl (CA-β-catenin) mice, which could constitutive activate β-catenin in ameloblasts. It showed the delayed mineralization and eventual hypomineralization in the incisor enamel of CA-β-catenin mice. Meanwhile, the amelogenesis-related proteinases Mmp20 and Klk4 were decreased in the incisors of CA-β-catenin mice. These data indicated that β-catenin plays an essential role in differentiation and function of ameloblasts during amelogenesis.     

Fluorosed Mouse Ameloblasts Have Increased SATB1 Retention and Gαq Activity

Dental fluorosis is characterized by subsurface hypomineralization and increased porosity of enamel, associated with a delay in the removal of enamel matrix proteins. To investigate the effects of fluoride on ameloblasts, A/J mice were given 50 ppm sodium fluoride in drinking water for four weeks, resulting serum fluoride levels of 4.5 µM, a four-fold increase over control mice with no fluoride added to drinking water. MicroCT analyses showed delayed and incomplete mineralization of fluorosed incisor enamel as compared to control enamel. A microarray analysis of secretory and maturation stage ameloblasts microdissected from control and fluorosed mouse incisors showed that genes clustered with Mmp20 appeared to be less downregulated in maturation stage ameloblasts of fluorosed incisors as compared to control maturation ameloblasts. One of these Mmp20 co-regulated genes was the global chromatin organizer, special AT-rich sequence-binding protein-1 (SATB1). Immunohistochemical analysis showed increased SATB1 protein present in fluorosed ameloblasts compared to controls. In vitro, exposure of human ameloblast-lineage cells to micromolar levels of both NaF and AlF₃ led to a significantly increase in SATB1 protein content, but not levels of Satb1 mRNA, suggesting a fluoride-induced mechanism protecting SABT1 from degradation. Consistent with this possibility, we used immunohistochemistry and Western blot to show that fluoride exposed ameloblasts had increased phosphorylated PKCα both in vivo and in vitro. This kinase is known to phosphorylate SATB1, and phosphorylation is known to protect SATB1 from degradation by caspase-6. In addition, production of cellular diacylglycerol (DAG) was significantly increased in fluorosed ameloblasts, suggesting that the increased phosphorylation of SATB1 may be related to an effect of fluoride to enhance Gαq activity of secretory ameloblasts.     

Ameloblast differentiation in the human developing tooth: Effects of extracellular matrices

Tooth enamel is formed by epithelially-derived cells called ameloblasts, while the pulp dentin complex is formed by the dental mesenchyme. These tissues differentiate with reciprocal signaling interactions to form a mature tooth. In this study we have characterized ameloblast differentiation in human developing incisors, and have further investigated the role of extracellular matrix proteins on ameloblast differentiation. Histological and immunohistochemical analyses showed that in the human tooth, the basement membrane separating the early developing dental epithelium and mesenchyme was lost shortly before dentin deposition was initiated, prior to enamel matrix secretion. Presecretary ameloblasts elongated as they came into contact with the dentin matrix, and then shortened to become secretory ameloblasts. In situ hybridization showed that the presecretory stage of odontoblasts started to express type I collagen mRNA, and also briefly expressed amelogenin mRNA. This was followed by upregulation of amelogenin mRNA expression in secretory ameloblasts. In vitro, amelogenin expression was upregulated in ameloblast lineage cells cultured in Matrigel, and was further up-regulated when these cells/Matrigel were co-cultured with dental pulp cells. Co-culture also up-regulated type I collagen expression by the dental pulp cells. Type I collagen coated culture dishes promoted a more elongated ameloblast lineage cell morphology and enhanced cell adhesion via integrin α2β1. Taken together, these results suggest that the basement membrane proteins and signals from underlying mesenchymal cells coordinate to initiate differentiation of preameloblasts and regulate type I collagen expression by odontoblasts. Type I collagen in the dentin matrix then anchors the presecretary ameloblasts as they further differentiate to secretory cells. These studies show the critical roles of the extracellular matrix proteins in ameloblast differentiation.     

Interaction between Fibronectin and β1 Integrin Is Essential for Tooth Development

The dental epithelium and extracellular matrix interact to ensure that cell growth and differentiation lead to the formation of teeth of appropriate size and quality. To determine the role of fibronectin in differentiation of the dental epithelium and tooth formation, we analyzed its expression in developing incisors. Fibronectin mRNA was expressed during the presecretory stage in developing dental epithelium, decreased in the secretory and early maturation stages, and then reappeared during the late maturation stage. The binding of dental epithelial cells derived from postnatal day-1 molars to a fibronectin-coated dish was inhibited by the RGD but not RAD peptide, and by a β1 integrin-neutralizing antibody, suggesting that fibronectin-β1 integrin interactions contribute to dental epithelial-cell binding. Because fibronectin and β1 integrin are highly expressed in the dental mesenchyme, it is difficult to determine precisely how their interactions influence dental epithelial differentiation in vivo. Therefore, we analyzed β1 integrin conditional knockout mice (Intβ1^lox-/lox-/K14-Cre) and found that they exhibited partial enamel hypoplasia, and delayed eruption of molars and differentiation of ameloblasts, but not of odontoblasts. Furthermore, a cyst-like structure was observed during late ameloblast maturation. Dental epithelial cells from knockout mice did not bind to fibronectin, and induction of ameloblastin expression in these cells by neurotrophic factor-4 was inhibited by treatment with RGD peptide or a fibronectin siRNA, suggesting that the epithelial interaction between fibronectin and β1 integrin is important for ameloblast differentiation and enamel formation.     

Cytochemical localization of Ca2+-Mg2+ adenosine triphosphatase in rat incisor ameloblasts during enamel secretion and maturation

A modified Wachstein-Meisel medium containing lead or cerium as capturing ions was used to localize Ca2+-Mg2+ adenosine triphosphatase (ATPase; EC 3.6.1.3) in rat incisor ameloblasts during enamel formation. Sections representing different developmental stages were processed for electron microscopic cytochemistry. Distribution and intensity of the observed reaction product, which was almost exclusively associated with cell membranes, varied according to the stage of enamel formation. During the secretory stage, intense reaction product was evident along the entire plasma membrane of ameloblasts and papillary cells. The early transitional ameloblasts showed reaction product on their proximal and lateral cell membranes, but not distally. In late transitional (pre-absorptive) ameloblasts, distal cell membranes exhibited intense reaction product. During enamel maturation, smooth-ended ameloblasts showed reaction product proximally and laterally, but not distally. Ruffle-ended maturative ameloblasts exhibited intense reaction product along their lateral and distal membranes. The intensity of the latter was decreased but not eliminated by levamisole. In the transition from smooth-ended to ruffle-ended cells, the reaction product became evident distally, concomitant with the appearance of cell membrane invaginations. These data are consistent with a possible role for Ca2+-Mg2+ ATPase in controlling calcium availability at the enamel mineralization front.     

The absence of muscle segment homeobox 2 leads to the pyroptosis of ameloblasts by inducing squamous epithelial hyperplasia in the enamel organ

Muscle segment homeobox 2 (MSX2) has been confirmed to be involved in the regulation of early tooth development. However, the role of MSX2 has not been fully elucidated in enamel development. To research the functions of MSX2 in enamel formation, we used a Msx2^−/− (KO) mouse model with no full Msx2 gene. In the present study, the dental appearance and enamel microstructure were detected by scanning electron microscopy and micro-computed tomography. The results showed that the absence of Msx2 resulted in enamel defects, leading to severe tooth wear in KO mice. To further investigate the mechanism behind the phenotype, we performed detailed histological analyses of the enamel organ in KO mice. We discovered that ameloblasts without Msx2 could secrete a small amount of enamel matrix protein in the early stage. However, the enamel epithelium occurred squamous epithelial hyperplasia and partial keratinization in the enamel organ during subsequent developmental stages. Ameloblasts depolarized and underwent pyroptosis. Overall, during the development of enamel, MSX2 affects the formation of enamel by regulating the function of epithelial cells in the enamel organ.     

Rat forming incisor requires a rigorous ECM remodeling modulated by MMP/RECK balance

Reversion-inducing-cysteine-rich protein with Kazal motifs (RECK) is a single membrane-anchored MMP-regulator and regulates matrix metalloproteinases (MMP) 2, 9 and 14. In turn, MMPs are endopeptidases that play a pivotal role in remodeling ECM. In this work, we decided to evaluate expression pattern of RECK in growing rat incisor during, specifically focusing out amelogenesis process. Based on different kinds of ameloblasts, our results showed that RECK expression was conducted by secretory and post-secretory ameloblasts. At the secretory phase, RECK was localized in the infra-nuclear region of the ameloblast, outer epithelium, near blood vessels, and in the stellate reticulum. From the transition to the maturation phases, RECK was strongly expressed by non-epithelial immuno-competent cells (macrophages and/or dendritic-like cells) in the papillary layer. From the transition to the maturation stage, RECK expression was increased. RECK mRNA was amplified by RT-PCR from whole enamel organ. Here, we verified the presence of RECK mRNA during all stages of amelogenesis. These events were governed by ameloblasts and by non-epithelial cells residents in the enamel organ. Concluding, we found differential expression of MMPs-2, -9 and RECK in the different phases of amelogenesis, suggesting that the tissue remodeling is rigorously controlled during dental mineralization.     

Mutations in the Beta Propeller WDR72 Cause Autosomal-Recessive Hypomaturation Amelogenesis Imperfecta

Healthy dental enamel is the hardest and most highly mineralized human tissue. Though acellular, nonvital, and without capacity for turnover or repair, it can nevertheless last a lifetime. Amelogenesis imperfecta (AI) is a collective term for failure of normal enamel development, covering diverse clinical phenotypes that typically show Mendelian inheritance patterns. One subset, known as hypomaturation AI, is characterised by near-normal volumes of organic enamel matrix but with weak, creamy-brown opaque enamel that fails prematurely after tooth eruption. Mutations in genes critical to enamel matrix formation have been documented, but current understanding of other key events in enamel biomineralization is limited. We investigated autosomal-recessive hypomaturation AI in a consanguineous Pakistani family. A whole-genome SNP autozygosity screen identified a locus on chromosome 15q21.3. Sequencing candidate genes revealed a point mutation in the poorly characterized WDR72 gene. Screening of WDR72 in a panel of nine additional hypomaturation AI families revealed the same mutation in a second, apparently unrelated, Pakistani family and two further nonsense mutations in Omani families. Immunohistochemistry confirmed intracellular localization in maturation-stage ameloblasts. WDR72 function is unknown, but as a putative β propeller is expected to be a scaffold for protein-protein interactions. The nearest homolog, WDR7, is involved in vesicle mobilization and Ca²⁺-dependent exocytosis at synapses. Vesicle trafficking is important in maturation-stage ameloblasts with respect to secretion into immature enamel and removal of cleaved enamel matrix proteins via endocytosis. This raises the intriguing possibility that WDR72 is critical to ameloblast vesicle turnover during enamel maturation.     

Electron probe analysis of maturation ameloblasts of the rat incisor and calf molar

Summary Rapidly frozen upper incisor teeth of rats and molar teeth of calves were freeze fractured, freeze dried and dry dissected in preparation for energy dispersive x-ray emission microanalysis in the scanning electron microscope.Successive zones of ameloblasts adjacent to maturing rat incisor enamel were examined, beginning with cells adjacent to the least mature enamel and progressing to cells over increasingly more mature enamel. Pronounced K _{1, 2}, x-ray peaks were obtained for P, S, Cl, K and Fe but not for Ca. Ca levels were also very low compared with P, S, Cl and K in calf molar maturation ameloblasts, whereas they were high in the distal poles of the secretory odontoblasts in the same specimens.The findings indicate that both intra- and extracellular Ca levels are extremely low in maturation ameloblasts. It is concluded that Ca is neither stored nor concentrated in large amounts by the maturation ameloblasts prior to its entry into the enamel. The suggestion is made that the maturation ameloblasts might regulate entry of calcium into enamel by serving as a selective barrier.     

Calcium-induced patterns of calcium-oxalate crystals in isolated leaflets of Gleditsia triacanthos L. and Albizia julibrissin Durazz

For experimental induction of crystal cells (=crystal idioblasts) containing calcium-oxalate crystals, the lower epidermis was peeled from seedling leaflets of Gleditsia triacanthos L., exposing the crystal-free mesophyll and minor veins to the experimental solutions on which leaflets were floated for up to 10 d under continous light. On 0.3–2.0 mM Ca-acetate, increasing numbers of crystals, appearing 96 h after peeling, were induced. The pattern of crystal distribution changed with Ca²⁺-concentration ([Ca²⁺]): at low [Ca²⁺], crystals formed only in the non-green bundlesheath cells surrounding the veins, believed to have a relatively low Ca²⁺-extrusion capacity; at higher [Ca²⁺], crystals developed in up to 90% of the mesophyll cells, and at supraoptimal [Ca²⁺], large extracellular crystals formed on the tissue surface. By sequential treatments with solutions of different [Ca²⁺], the following three phases were identified in the induction of crystal cells: (1) during the initial 24-h period (adaptive aging), Ca²⁺ is not required and crystal induction is not possible; (2) during the following 48 h (induction period), exposure to 1–2 mM Ca-acetate induces the differentiation of mesophyll cells into crystal cells; (3) crystal growth begins 72 h after the start of induction. In intact leaflets of Albizia julibrissin Durazz., calcium-oxalate crystals are found exclusively in the bundle-sheath cells of the veins, but crystals were induced in the mesophyll of peeled leaflets floating on 1 mM Ca-acetate. Exposure to inductive [Ca²⁺] will thus trigger the differentiation of mature leaf cells into crystal cells; the spatial distribution of crystals is determined by the external [Ca²⁺] and by the structural and functional properties of the cells in the tissue.     

Cytochemical calcium distribution in secretory ameloblasts of the rat in relation to enamel mineralization

Calcium distribution in secretory ameloblasts was studied in rat incisor enamel in which mineralization was temporarily disturbed by injection of either fluoride or cobalt. Pyroantimonate precipitates of calcium were analysed morphometrically in regions of the cell membranes, mitochondria and secretory granules. The disturbances in mineralization were characterized by accumulations of unmineralized enamel matrix at the secretory regions of Tomes' process within 1 h after injection. Fluoride-induced disturbances in mineralization were not accompanied by marked changes in calcium concentration and distribution. It may be that fluoride causes alterations in the synthesis and secretion of the organic matrix which affects its ability to mineralize. Secretory ameloblasts treated with cobalt showed a broad basis for interference with calcium, in particular that which is associated with cell membranes and secretory granules. Secretory ameloblasts may be actively controlling the availability of calcium to enamel by mechanisms involving the cell membrane as well as the secretory granules.     

Ultrastructural morphometric analysis of ameloblasts exposed to fluoride during tooth development

Since a considerable amount of the world population is exposed to high doses of fluoride, it is of special concern to investigate its action mechanisms during dental enamel development. In this study, the toxicity of fluoride in ameloblasts during enamel development was evaluated by means of ultrastructural morphometric analysis. A total of 18 male Wistar rats were distributed into three groups. In Group I, the animals received deionized drinking water ad libitum (negative control) and in Groups II and III, they received sodium fluorided (NaF) drinking water at doses of 7 and 100 ppm ad libitum, respectively, for 6 weeks. Morphometric data were expressed as volume density of the most significant organelles present in the secretory and maturation phases of amelogenesis such as RER, granules, lysosomes, phagic vacuoles, microfilaments and mitochondria. The results showed that the volume density of mitochondria in the 100 ppm experimental group was 29% (P < 0.05) higher than the control group in secretory ameloblasts. No remarkable differences were found in maturation ameloblasts for all organelles evaluated. Taken together, these data indicate that NaF at high doses is able to induce cellular damage in secretory ameloblasts, whereas no noxious effect was observed during maturation stage of amelogenesis as depicted by ultrastructural analysis.     

WDR72 models of structure and function: A stage-specific regulator of enamel mineralization

Amelogenesis Imperfecta (AI) is a clinical diagnosis that encompasses a group of genetic mutations, each affecting processes involved in tooth enamel formation and thus, result in various enamel defects. The hypomaturation enamel phenotype has been described for mutations involved in the later stage of enamel formation, including Klk4, Mmp20, C4orf26, and Wdr72. Using a candidate gene approach we discovered a novel Wdr72 human mutation in association with AI to be a 5-base pair deletion (c.806_810delGGCAG; p.G255VfsX294). To gain insight into the function of WDR72, we used computer modeling of the full-length human WDR72 protein structure and found that the predicted N-terminal sequence forms two beta-propeller folds with an alpha-solenoid tail at the C-terminus. This domain iteration is characteristic of vesicle coat proteins, such as beta′-COP, suggesting a role for WDR72 in the formation of membrane deformation complexes to regulate intracellular trafficking. Our Wdr72 knockout mouse model (Wdr72^−/−), containing a LacZ reporter knock-in, exhibited hypomineralized enamel similar to the AI phenotype observed in humans with Wdr72 mutations. MicroCT scans of Wdr72^−/− mandibles affirmed the hypomineralized enamel phenotype occurring at the onset of the maturation stage. H&E staining revealed a shortened height phenotype in the Wdr72^−/− ameloblasts with retained proteins in the enamel matrix during maturation stage. H⁺/Cl⁻ exchange transporter 5 (CLC5), an early endosome acidifier, was co-localized with WDR72 in maturation-stage ameloblasts and decreased in Wdr72^−/− maturation-stage ameloblasts. There were no obvious differences in RAB4A and LAMP1 immunostaining of Wdr72^−/− mice as compared to wildtype controls. Moreover, Wdr72^−/− ameloblasts had reduced amelogenin immunoreactivity, suggesting defects in amelogenin fragment resorption from the matrix. These data demonstrate that WDR72 has a major role in enamel mineralization, most notably during the maturation stage, and suggest a function involving endocytic vesicle trafficking, possibly in the removal of amelogenin proteins.     

Amelogenesis: Transformation of a protein-mineral matrix into tooth enamel

During enamel formation, the organic enamel protein matrix interacts with calcium phosphate minerals to form elongated, parallel, and bundled enamel apatite crystals of extraordinary hardness and biomechanical resilience. The enamel protein matrix consists of unique enamel proteins such as amelogenin, ameloblastin, and enamelin, which are secreted by highly specialized cells called ameloblasts. The ameloblasts also facilitate calcium and phosphate ion transport toward the enamel layer. Within ameloblasts, enamel proteins are transported as a polygonal matrix with 5 nm subunits in secretory vesicles. Upon expulsion from the ameloblasts, the enamel protein matrix is re-organized into 20 nm subunit compartments. Enamel matrix subunit compartment assembly and expansion coincide with C-terminal cleavage by the MMP20 enamel protease and N-terminal amelogenin self-assembly. Upon enamel crystal precipitation, the enamel protein phase is reconfigured to surround the elongating enamel crystals and facilitate their elongation in C-axis direction. At this stage of development, and upon further amelogenin cleavage, central and polyproline-rich fragments of the amelogenin molecule associate with the growing mineral crystals through a process termed “shedding”, while hexagonal apatite crystals fuse in longitudinal direction. Enamel protein sheath-coated enamel “dahlite” crystals continue to elongate until a dense bundle of parallel apatite crystals is formed, while the enamel matrix is continuously degraded by proteolytic enzymes. Together, these insights portrait enamel mineral nucleation and growth as a complex and dynamic set of interactions between enamel proteins and mineral ions that facilitate regularly seeded apatite growth and parallel enamel crystal elongation.     

Hypomaturation Enamel Defects in Klk4 Knockout/LacZ Knockin Mice

Kallikrein 4 (Klk4) is believed to play an essential role in enamel biomineralization, because defects in KLK4 cause hypomaturation amelogenesis imperfecta. We used gene targeting to generate a knockin mouse that replaces the Klk4 gene sequence, starting at the translation initiation site, with a lacZ reporter gene. Correct targeting of the transgene was confirmed by Southern blot and PCR analyses. Histochemical X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) staining demonstrated expression of β-galactosidase in maturation stage ameloblasts. No X-gal staining was observed in secretory stage ameloblasts or in odontoblasts. Retained enamel proteins were observed in the maturation stage enamel of the Klk4 null mouse, but not in the Klk4 heterozygous or wild-type mice. The enamel layer in the Klk4 null mouse was normal in thickness and contained decussating enamel rods but was rapidly abraded following weaning, despite the mice being maintained on soft chow. In function the enamel readily fractured within the initial rod and interrod enamel above the parallel enamel covering the dentino-enamel junction. Despite the lack of Klk4 and the retention of enamel proteins, significant levels of crystal maturation occurred (although delayed), and the enamel achieved a mineral density in some places greater than that detected in bone and dentin. An important finding was that individual enamel crystallites of erupted teeth failed to grow together, interlock, and function as a unit. Instead, individual crystallites seemed to spill out of the enamel when fractured. These results demonstrate that Klk4 is essential for the removal of enamel proteins and the proper maturation of enamel crystals.Dental enamel is composed of highly ordered, very long crystals of calcium hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂). Mature enamel crystallites are about 70 nm wide and 30 nm thick, but are of unmeasurable length (1), probably extending all the way from the dentin layer to the surface of the tooth (2). Enamel crystallites are organized into bundles called rods, with about 10,000 parallel crystals in a rod (3). Each enamel rod is the product of a single ameloblast, the cell type that forms a continuous sheet over the developing enamel and orchestrates its formation. Dental enamel of erupted teeth is ∼95% mineral (by weight) (4), with most of the non-mineral component being water. Protein comprises <1% of its weight. Forming enamel, however, is over 30% protein (5). Much of the protein is reabsorbed by ameloblasts and degraded in lysosomes (6, 7), but extracellular proteases also play a role in matrix protein removal (8–10).Dental enamel formation is divided into secretory, transition, and maturation stages (11, 12). During the secretory stage, enamel crystals grow primarily in length. As the crystals extend, the enamel layer expands. Enamel crystallites lengthen along a mineralization front at the secretory surface of the ameloblast cell membrane. There, mineral deposits rapidly on the crystallite tips, and very slowly on their sides (3, 13, 14). By the end of the secretory stage the enamel crystals are full-length and the enamel layer as a whole is as thick as it will ever be, but it has only about 14% of the mineral as it will have when the tooth erupts (15). Following the secretory stage there is then a transition during which the ameloblasts greatly reduce their secretion of enamel proteins (16) and convert to maturation ameloblasts (17). During the maturation stage, mineral is deposited exclusively on the sides of pre-existing enamel crystallites (18), which grow in width and thickness until further growth is prevented by contact with adjacent crystals (19, 20). During early maturation the percentage protein by weight drops from 30 to 2% (5), and half of the total enamel mineral is deposited. The final 30–35% of mineral is deposited in the absence of significant protein and allows the crystals to grow firmly against one another and to mechanically interlock (15).The major secretory stage enamel proteins are amelogenin (21, 22), ameloblastin (23–25), and enamelin (26, 27). These proteins function specifically during enamel formation, and the disease phenotypes exhibited by mice lacking these genes are confined to the developing teeth and include enamel agenesis (28–30). These genes are often deleted or are reduced to pseudogenes in vertebrates such as birds or baleen whales that evolved alternatives to developing teeth (31, 32). Although the enamel extracellular matrix proteins are critical for growing enamel crystals, they are not part of the final enamel product. Prior to tooth eruption, enamel proteins are digested by proteases and reabsorbed by ameloblasts. Two extracellular matrix proteases are involved in the cleavage of enamel proteins: matrix metalloproteinase 20 (Mmp-20)² (33) and kallikrein 4 (Klk4) (34).Mmp-20 is secreted along with amelogenin, ameloblastin, and enamelin by secretory stage ameloblasts (35–37). Mmp-20 activity can account for the range of cleavages observed in secretory stage enamel proteins (38) and appears to be the only protease secreted by ameloblasts during the secretory stage. Mmp20-null mice have enamel that is thinner and softer than normal, lacks enamel rod organization, and tends to chip off the crown surface (39, 40). Like the other secretory stage enamel proteins, Mmp20 expression appears to be restricted to developing teeth (41), as is the diseased phenotype when the human gene is defective (42–44).Klk4 is a serine protease that is secreted by transition and maturation stage ameloblasts but is not expressed by secretory stage ameloblasts (45, 46). Klk4 might also be expressed by odontoblasts, the cells that form dentin (47). Klk4 has broad substrate specificity (48, 49) and is capable of activating other proteases (50–52) and protease activated receptors (53, 54). Unlike most proteins secreted by ameloblasts, Klk4 is expressed in other tissues, most notably the prostate (55) and endometrium (56). Much attention has been focused on the potential role of Klk4 in cancers. Klk4 is increased in breast cancer stromal cells (57), in prostate cancer cells (58–61), and ovarian cancer cells (62–65). Despite this focus on the potential role of Klk4 in tumors, very little is known about the normal expression and function of Klk4 in nondental tissues. A loss of function mutation in both human KLK4 alleles caused a hypomaturation enamel phenotype in the absence of any observable defects elsewhere in the body (66).To gain insights into the role of Klk4 in normal dental enamel formation, and to better characterize the normal temporal and spatial patterns of Klk4 expression, we have used gene targeting to knock out normal Klk4 expression, while replacing the Klk4 code with lacZ, the bacterial gene encoding β-galactosidase reporter in mice. We demonstrate that Klk4 is not expressed by secretory stage ameloblasts, but is specifically expressed by ameloblasts later in enamel formation and is necessary for the proper removal of enamel proteins, the final thickening of enamel crystals, and ultimately, for hardening of the enamel layer.     

Ca2+ as developmental signal in the formation of Ca-oxalate crystal spacing patterns during leaf development inCarya ovata

Changes in the spacing patterns of Ca-oxalate crystals during enlargement ofCarya ovata Mill. leaves were quantified by computerized image-analysis. Single Ca-oxalate crystals form in the vacuoles of young mesophyll cells transformed into crystal cells Crystals are very small in newly induced crystal cells and increase in size throughout leaf development. Crystal patterns thus reflect both induction and relative age of crystal cells. Shortly after the emergence of young leaves from the bud, very small crystals are formed in the mesophyll at high density. As leaves expand, these crystals grow larger and become separated by increasing distances. New small crystals appear in the gaps between the older, larger crystals. Later crystal patterns consist of widely spaced, larger crystals only. Finally, clusters of small crystals are formed again in the gaps between large crystals. No crystals were observed in young leaves expanding in a moist chamber, but large numbers of crystal cells were induced experimentally in sections of immature leaves floating on 4 mM Ca-acetate. The observations support the following mechanism of crystal-pattern formation: Ca²⁺ carried into leaves with the transpiration stream acts as the developmental signal inducing transdifferentiation of a few mesophyll cells into crystal cells when apoplastic [Ca²⁺] rises. Crystal cells precipitate absorbed Ca²⁺ as oxalate and, acting as Ca²⁺ sinks, inhibit crystal-cell induction in their vicinity by depleting apoplastic Ca²⁺. This prevents close spacing of crystal cells. New crystal cells form in the gaps between the depletion zones of older crystal cells when these move apart during leaf expansion. Later changes in crystal patterns result from increasing sink strength of crystal cells, lowered inducibility of mesophyll cells, and increased Ca²⁺ influx into leaves during intensive transpiration. Throughout leaf development, spacing of crystal cells permits rapid secretion of apoplastic Ca²⁺ as Ca-oxalate. Dedicated to Professor Erwin Bünning, University of Tübingen, Germany, who pioneered the analysis of spacing patterns