Store-operated Ca2+ entry controls ameloblast cell function and enamel development
Miriam EcksteinMartin VaethCinzia FornaiManikandan VinuTimothy G. BromageMeerim K. NurbaevaJessica L. SorgePaulo G. CoelhoYoussef IdaghdourStefan FeskeRodrigo S. Lacruz
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Abstract:
Loss-of-function mutations in stromal interaction molecule 1 (STIM1) impair the activation of Ca2+ release-activated Ca2+ (CRAC) channels and store-operated Ca2+ entry (SOCE), resulting in a disease syndrome called CRAC channelopathy that is characterized by severe dental enamel defects. The cause of these enamel defects has remained unclear given a lack of animal models. We generated Stim1/2K14cre mice to delete STIM1 and its homolog STIM2 in enamel cells. These mice showed impaired SOCE in enamel cells. Enamel in Stim1/2K14cre mice was hypomineralized with decreased Ca content, mechanically weak, and thinner. The morphology of SOCE-deficient ameloblasts was altered, showing loss of the typical ruffled border, resulting in mislocalized mitochondria. Global gene expression analysis of SOCE-deficient ameloblasts revealed strong dysregulation of several pathways. ER stress genes associated with the unfolded protein response were increased in Stim1/2-deficient cells, whereas the expression of components of the glutathione system were decreased. Consistent with increased oxidative stress, we found increased ROS production, decreased mitochondrial function, and abnormal mitochondrial morphology in ameloblasts of Stim1/2K14cre mice. Collectively, these data show that loss of SOCE in enamel cells has substantial detrimental effects on gene expression, cell function, and the mineralization of dental enamel.Keywords:
Orai1
The past five years have witnessed the discovery of the endoplasmic reticulum calcium (Ca(2+)) sensor STIM1 and the plasma membrane Ca(2+) channel Orai1 as the bona fide molecular components of the store-operated Ca(2+) entry (SOCE) and the Ca(2+) release-activated Ca(2+) current (I (CRAC)). It has been known for two decades that SOCE and I (CRAC) are required for lymphocyte activation as evidenced by severe immunodeficient phenotypes in patients lacking I (CRAC). In recent years however, studies have uncovered expression of STIM1 and Orai1 proteins in various tissues and described additional roles for these proteins in physiological functions and pathophysiological conditions. Here, we will summarize novel findings pertaining to the role of STIM1 and Orai1 in the vascular system and discuss their potential use as targets in the therapy of vascular disease.
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AbstractCalcium (Ca2+) entry into non-excitable cells is mainly carried by store-operated channels (SOCs), which serve essential functions ranging from regulation of transcription to cell growth. The best-characterised store-operated current, ICRAC, is the calcium release-activated calcium (CRAC) current initially discovered in T-lymphocytes and mast cells. The search for the molecular components of the CRAC channel lasted over 20 years. Recently STIM1 has been identified as the Ca2+ sensor in the endoplasmic reticulum (ER) that accumulates into punctae close to the plasma-membrane following store-depletion. The identification of STIM1 has been closely followed by the discovery of Orai1 as the CRAC channel pore in human T-cells. Upon punctae formation STIM1 activates Ca2+ influx via Orai1 channels. This review covers functional details concerning the activation cascade of the STIM1 / Orai1 complex from ER Ca2+ sensing to Ca2+ influx through Orai1. Furthermore, functional domains within STIM1 and Orai1 in comparison to their structural homologs STIM2 as well as Orai2 and Orai3, respectively, are displayed together with recent findings on the pore architecture and selectivity filter of Orai channels. A broad tissue expression of STIM and Orai proteins together with substantial effects in STIM1 / Orai1 knock-out mice suggests an essential physiological role in store-operated Ca2+ signaling in human health and disease.
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ABSTRACT – The electron microprobe technique was used to study the accumulation of iron in rat incisor ameloblasts as well as the subsequent release of iron from the cells and deposition into the outer layer of the enamel. Starting about 3 mm from the developing end, a gradual accumulation of iron occurred in the ameloblasts. At a stage where the iron content of the cells had reached a maximal level, and the calcium content of the adjacent hard tissue had reached the level of mature enamel, the initial incorporation of iron in the enamel was seen. In the iron incorporation zone the iron content of the enamel increased from less than 0.1 % to about 9 % and the iron content of the ameloblasts was gradually reduced. Concomitant with the increase of iron in the enamel, a decrease of the calcium content was observed in the same region, indicating a withdrawal of calcium from the enamel. Since the incorporation of iron occurs at a stage where the enamel is highly mineralized, the processes involved can hardly be explained as an interaction with the organic matrix. The key to the understanding of these processes should therefore be sought in the adjacent cell layer.
Enamel organ
Amelogenesis
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In Monodelphis domestica, although both processes from odontoblasts and projections from ameloblasts were found in developing enamel, the majority of the contents of enamel tubules were probably processes that originated from odontoblasts. Processes from odontoblasts penetrating into enamel touched part of the ameloblasts in the stage of enamel formation. No specialised cell junctions were seen at the adherence between the two. There were no enamel tubules in the aprismatic and pseudoprismatic enamel layer. It is likely that enamel tubules exist only in prismatic enamel. The majority of enamel tubules seemed to contain no cell processes in the stage of enamel maturation, indicating that the cell processes in the tubules probably degenerate after the stage of enamel formation.
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The large hydroxyapatite (HA) crystals in dental enamel results from a long lasting process called enamel maturation, which follows a stage of enamel matrix secretion. Thus, after the full thickness of enamel is reached, the enamel still requires about 60‐70 % mineral to be deposited by HA‐crystals growing in width and thickness. This is facilitated by the ameloblasts, which during maturation change morphology every 6‐8th hour. This study aims to test the role of the ameloblasts in maintaining enamel pH in the maturation zone in order to elucidate the role of the cyclic changes in ameloblast morphology for HA‐crystal growth. Colorimetric pH‐indicators and ratiometric fluorometry were used to measure surface pH in maturation zone enamel of rat incisors. Alternating acidic (down to pH 6.24±0.06) and alkaline zones (up to pH 7.34±0.08) corresponding to ruffled‐ended and smooth‐ended ameloblasts, respectively, were found along the tooth thus coinciding with ameloblast morphological cycles. Underlying the cyclic pattern, a gradual decrease in pH towards the incisal edge was seen. Vinblastine or FR167356 (H+‐ATPase‐inhibitor) disturbed ameloblast acid‐secretion, especially in the early parts of acidic zones. At the pH‐values observed, PO43‐ would be protonated (pKa >12) and HA dissolved. However, by molecular dynamics simulations we estimate the pKa of HPO42‐ at an ideal HA surface to be 4.3. The acidic pH measured at the enamel surface may thus only dissolve non‐perfect domains of HA crystals in which HPO42‐ is less electrostatically shielded. As a result of the repeated alkaline/acidic cycles induced by ameloblasts, near‐perfect domains will therefore gradually replace less perfect domains and large near‐perfect HA crystals will be produced. In conclusion, fluctuations in surface pH of maturing rat incisor enamel are a result of cycles of H+‐secretion by ameloblasts and variations in enamel buffer characteristics.
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In non-excitable cells, agonist-induced depletion of intracellular Ca(2+) stores triggers Ca(2+) influx via a process termed store-operated Ca(2+) entry (SOCE). In T-lymphocytes, stromal interaction molecule 1 (STIM1) acts as the intra-store Ca(2+) sensor and Orai1 functions as the Ca(2+)-permeable SOCE channel activated by STIM1 following store depletion. Two functionally distinct Ca(2+) entry pathways exist in skeletal muscle; one activated by store depletion (SOCE) and a second by sustained/repetitive depolarization that does not require store depletion (excitation-coupled Ca(2+) entry, ECCE). However, the role of STIM1 and Orai1 in coordinating SOCE and ECCE activity in skeletal muscle and whether these two Ca(2+) entry pathways represent distinct molecular entities or two different activation mechanisms of the same channel complex is unknown. Here we address these issues using siRNA-mediated STIM1 knockdown, dominant-negative Orai1, and permeation-defective Orai1 to determine the role of STIM1 and Orai1 in store-operated and excitation-coupled Ca(2+) entry in skeletal myotubes. SOCE and ECCE activity were quantified from both intracellular Ca(2+) measurements and Mn(2+) quench assays. We found that STIM1 siRNA reduced STIM1 protein by more than 90% and abolished SOCE activity, while expression of siRNA-resistant hSTIM1 fully restored SOCE. SOCE was also abolished by dominant-negative Orai1 (E106Q) and markedly reduced by expression of a permeation-defective Orai1 (E190Q). In contrast, ECCE was unaffected by STIM1 knockdown, E106Q expression or E190Q expression. These results are the first to demonstrate that SOCE in skeletal muscle requires both STIM1 and Orai1 and that SOCE and ECCE represent two distinct molecular entities.
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Endoplasmic reticulum calcium(Ca2+) sensor stromal interaction molecule 1(STIM1) and plasma membrane Ca2+ channel Orai1 are the bona fide molecular components of the store-operated Ca2+ entry(SOCE) and the Ca2+ release-activated Ca2+ current(ICRAC).We have uncovered the protein expression of STIM1 and Orai1 in various tissues and described many kinds of roles for these proteins under physiological and pathophysiological conditions.Here,we summarize the novel findings pertaining to the role of STIM1 and Orai1 in the vascular system and discuss their potential use as targets in the treatment of vascular diseases.
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Pathophysiology
Calcium Signaling
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Store-operated Ca(2+) entry (SOCE) is an ubiquitous mechanism for Ca(2+) entry in eukaryotic cells. This route for Ca(2+) influx is regulated by the filling state of the intracellular Ca(2+) stores communicated to the plasma membrane channels by the proteins of the Stromal Interaction Molecule (STIM) family, STIM1, and STIM2. Store-dependent, STIM1-modulated, channels include the Ca(2+) release-activated Ca(2+) channels, comprised of subunits of Orai proteins, as well as the store-operated Ca(2+) (SOC) channels, involving Orai1, and members of the canonical transient receptor potential family of proteins. Recent studies have revealed the expression of splice variants of STIM1, STIM2, and Orai1 in different cell types. While certain variants are ubiquitously expressed, others, such as STIM1L, show a more restricted expression. The splice variants for STIM and Orai1 proteins exhibit significant functional differences and reveal that alternative splicing enhance the functional diversity of STIM1, STIM2, and Orai1 genes to modulate the dynamics of Ca(2+) signals.
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Bartlett JD, Skobe Z, Nanci A, Smith CE . Matrix metalloproteinase 20 promotes a smooth enamel surface, a strong dentino–enamel junction, and a decussating enamel rod pattern. Eur J Oral Sci 2011; 119 (Suppl. 1): 199–205. © 2011 Eur J Oral Sci Mutations of the matrix metalloproteinase 20 ( MMP20 , enamelysin) gene cause autosomal‐recessive amelogenesis imperfecta, and Mmp20 ablated mice also have malformed dental enamel. Here we showed that Mmp20 null mouse secretory‐stage ameloblasts maintain a columnar shape and are present as a single layer of cells. However, the maturation‐stage ameloblasts from null mouse cover extraneous nodules of ectopic calcified material formed at the enamel surface. Remarkably, nodule formation occurs in null mouse enamel when MMP20 is normally no longer expressed. The malformed enamel in Mmp20 null teeth was loosely attached to the dentin and the entire enamel layer tended to separate from the dentin, indicative of a faulty dentino–enamel junction (DEJ). The enamel rod pattern was also altered in Mmp20 null mice. Each enamel rod is formed by a single ameloblast and is a mineralized record of the migration path of the ameloblast that formed it. The enamel rods in Mmp20 null mice were grossly malformed or absent, indicating that the ameloblasts do not migrate properly when backing away from the DEJ. Thus, MMP20 is required for ameloblast cell movement necessary to form the decussating enamel rod patterns, for the prevention of ectopic mineral formation, and to maintain a functional DEJ.
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Enamel organ
Amelogenesis imperfecta
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