Abstract
Calcium export is a key function for the enamel organ during all stages of amelogenesis. Expression of a number of ATPase calcium transporting, plasma membrane genes (ATP2B1-4/PMCA1-4), solute carrier SLC8A genes (sodium/calcium exchanger or NCX1-3), and SLC24A gene family members (sodium/potassium/calcium exchanger or NCKX1-6) have been investigated in the developing enamel organ in earlier studies. This paper reviews the calcium export pathways that have been described and adds novel insights to the spatiotemporal expression patterns of PMCA1, PMCA4, and NCKX3 during amelogenesis. New data are presented to show the mRNA expression profiles for the four Atp2b1-4 gene family members (PMCA1-4) in secretory-stage and maturation-stage rat enamel organs. These data are compared to expression profiles for all Slc8a and Slc24a gene family members. PMCA1, PMCA4, and NCKX3 immunolocalization data is also presented. Gene expression profiles quantitated by real time PCR show that: (1) PMCA1, 3, and 4, and NCKX3 are most highly expressed during secretory-stage amelogenesis; (2) NCX1 and 3, and NCKX6 are expressed during secretory and maturation stages; (3) NCKX4 is most highly expressed during maturation-stage amelogenesis; and (4) expression levels of PMCA2, NCX2, NCKX1, NCKX2, and NCKX5 are negligible throughout amelogenesis. In the enamel organ PMCA1 localizes to the basolateral membrane of both secretory and maturation ameloblasts; PMCA4 expression is seen in the basolateral membrane of secretory and maturation ameloblasts, and also cells of the stratum intermedium and papillary layer; while NCKX3 expression is limited to Tomes' processes, and the apical membrane of maturation-stage ameloblasts. These new findings are discussed in the perspective of data already present in the literature, and highlight the multiplicity of calcium export systems in the enamel organ needed to regulate biomineralization.
Highlights
Enamel is the hardest and most calcified tissue in mammals, and understanding enamel formation is crucial for developing strategies to repair or regenerate it (Smith, 1998; Hubbard, 2000; Lacruz et al, 2013)
Quantitative PCR comparing mRNA expression levels in secretory and maturation enamel organ cells for all PMCA (Atp2b), NCX (Slc8) and NCKX (Slc24) gene family members indicate that: (1) PMCA1 (Atp2b1), 3 (Atp2b3), and 4 (Atp2b4), and NCKX3 (Slc24a3) expression is highest during secretory-stage amelogenesis; (2) NCX1 (Slc8a1) and 3 (Slc8a3), and NCKX6 (Slc24a6) were expressed during secretory and maturation stages; and (3) NCKX4 (Slc24a4) is most highly expressed during maturation-stage amelogenesis (Figure 1)
Of the four unique genes coding the PMCAs, PMCA1, and PMCA4 are highly expressed on the basolateral membranes of polarized ameloblasts; and both are expressed during secretory- and maturationstage amelogenesis
Summary
Enamel is the hardest and most calcified tissue in mammals, and understanding enamel formation is crucial for developing strategies to repair or regenerate it (Smith, 1998; Hubbard, 2000; Lacruz et al, 2013). Epithelial-derived enamel-forming cells (ameloblasts) differentiate from the inner enamel epithelium (IEE) during the pre-secretory stage (Orrenius et al, 2015) These amelobasts are highly polarized with an apical end that faces the enamel area and a basal end that faces the blood circulation. Ameloblasts migrate away from the dentin while synthesizing and secreting enamel matrix proteins (EMPs) such as amelogenin, ameloblastin, and enamelin into the enamel area from Tomes’ processes at their apical ends. These EMPs serve as a scaffold for the orientation and elongation of enamel hydroxyapatite (Hap) crystals (Smith, 1998). While general concepts of ion transport throughout amelogenesis have been well-studied and discussed elsewhere (Arquitt et al, 2002; Paine et al, 2007; Lyaruu et al, 2008; Bronckers et al, 2010, 2015; Josephsen et al, 2010; Yin et al, 2015), and in particular the transcellular calcium ion (Ca2+) transport (reviewed in Nurbaeva et al, 2015b), in this paper we focus primarily on Ca2+ export
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