Reduced Protein Expression of the Na+/Ca2++K+-Exchanger (SLC24A4) in Apical Plasma Membranes of Maturation Ameloblasts of Fluorotic Mice

Abstract

Exposure of forming enamel to fluoride results into formation of hypomineralized enamel. We tested whether enamel hypomineralization was caused by lower expression of the NCKX4/SLC24A4 Ca²⁺-transporter by ameloblasts. Three commercial antibodies against NCKX4 were tested on enamel organs of wild-type and Nckx4-null mice, one of which (a mouse monoclonal) was specific. This antibody gave a prominent staining of the apical plasma membranes of maturation ameloblasts, starting at early maturation. The layer of immuno-positive ameloblasts contained narrow gaps without immunostaining or with reduced staining. In fluorotic mouse incisors, the quantity of NCKX4 protein in ameloblasts as assessed by western blotting was not different from that in non-fluorotic ameloblasts. However, immunostaining of the apical plasma membranes of fluorotic ameloblasts was strongly reduced or absent suggesting that trafficking of NCKX4 to the apical membrane was strongly reduced. Exposure to fluoride may reduce NCKX4-mediated transport of Ca²⁺ by maturation stage ameloblasts which delays ameloblast modulation and reduces enamel mineralization.

Keywords: Calcium; Enamel fluorosis; Mechanism; Null mutation; Transport.

Fig. 1

Western blots of enamel organ from Nckx4-null mice immunostained with three different antibodies to NCKX4. Tissues were stained with antibodies to NCKX4 from a Protein Tech (NCKX4 in green, ß-actin in red). b Abcam (NCKX4 in green, ß-actin in red) and c NeuroMab (NCKX4 in red, ß-actin in green). d Western blots of fluorotic and non-fluorotic wild-type ameloblasts stained with anti-NCKX4 (NeuroMab, NCKX4 in red; ß-actin in green) and e quantified after normalization to ß-actin. The 50 kD band contained almost fourfold more protein than the 60 kD band (Color figure online)

Fig. 2

Validation of the antibodies to NCKX4 (a–d), developmental expression in wild-type incisors (a, e, f, g for NeuroMab antibody) and expression in fluorotic wild-type enamel organ (i, j NeuroMab antibody). a Apical membranes of wild-type maturation ameloblasts (ma) but not membranes of the papillary layer (pl) stain positive with mouse anti-NCKX4 (NeuroMab). b–d Nckx4-null maturation ameloblasts stained with anti-NCKX4 from NeuroMab (b), Protein Tec (c) and Abcam (d). All three antibodies stained to various degrees the extracellular enamel matrix when matrix was retained in the enamel space in Nckx4-null teeth. Similar variable stainings of forming enamel are also seen with other non-related antibodies in undecalcified (or partly decalcified) enamel sections or when primary antibodies are replaced by non-immuno-IgG, rabbit or mouse normal serum. This staining was considered non-specific. e Developmental expression of NCKX4 protein starts at late secretory stage ameloblasts (sa), continues in transitional ameloblasts (ta) and soon locates prominent in apical membranes of all early maturation ameloblasts (ma). Large arrow at the bottom points incisally.Pl papillary layer. f Detail of mid-maturation ameloblasts with strongly stained apical membranes. g Small group of cells underlined by a dotted line (likely SE ameloblasts) without pronounced apical staining in contrast to strong apical staining in neighbor cells (arrows). Es enamel space. h Shows negative staining in Nckx4-null ameloblasts stained with NeuroMab antibody, also showing periodic changes in attachment of the apical membrane to the enamel matrix (em). The black arrows point at local detachment of the apical membrane from the enamel; the arrows with white arrow heads point at plaque-like focal adhesions of the apical membrane to the enamel. Note these plaques stain blue with hematoxylin. i Shows fluorotic wild-type mouse ameloblasts without apical staining; j shows strong apical staining in non-fluorotic wild-type control ameloblasts