NBCe1 (SLC4A4) a potential pH regulator in enamel organ cells during enamel development in the mouse

Abstract

During formation of dental enamel maturation-stage ameloblasts express ion-transporting transmembrane proteins. The SLC4 family of ion-transporters regulates intra- and extracellular pH in eukaryotic cells by co-transporting HCO3− with Na+. Mutation in SLC4A4 (coding for the Na+ bicarbonate co-transporter NBCe1) induces developmental defects in human and murine enamel. We hypothesized that NBCe1 in dental epithelium is engaged in neutralizing protons released during crystal formation in the enamel space. We immunolocalized NBCe1 protein in mouse wild-type dental epithelium and examined the effect of NBCe1-null mutation on enamel formation in mice. Ameloblasts expressed gene transcripts for NBCe1 isoforms B/D/C/E. In wild-type mice weak to moderate immunostaining for NBCe1 with antibodies that recognize isoforms A/B/D/E and isoform C was seen in ameloblasts in secretory stage, no or very low staining in early maturation-stage but moderately to high staining in late maturation-stage. The papillary layer showed the opposite pattern and immunostained prominently at early maturation-stage but gradually showed less staining at mid- and late maturation-stage. In NBCe1−/− mice ameloblasts were disorganized, the enamel thin and severely hypomineralized. Enamel organs of CFTR−/− and AE2a,b−/− mice (believed to be pH regulators in ameloblasts) contained higher levels of NBCe1 protein than wild-type mice. Our data show that expression of NBCe1 in ameloblast and papillary layer cell depends on developmental stage and possibly responds to pH changes.

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

“…HAT‐7 cells are functionally polarized with high apical membrane CO 2 permeability and vigorous basolateral uptake (Bori et al., ). This is similar to other ‐secreting epithelia, such as guinea pig pancreatic duct (Ishiguro et al., ) and the human CFPAC pancreatic cell line (Rakonczay et al., ), and is consistent with the assumption that maturation‐stage ameloblasts are equipped with transporters to secrete to neutralize the acidity generated during hydroxyapatite formation (Jalali et al., ; Lacruz et al., ; Smith, ). We also detected AE2 exchanger expression in HAT‐7 cells by immunocytochemistry, and its function was confirmed in Cl − substitution experiments by the inhibitory effect of 4,4′‐diisothiocyano‐2,2′‐stilbenedisulfonic acid) DIDS to block anion exchanger activity (Bori et al., ).…”

“…The two most likely pathways for basolateral uptake in ameloblasts are by Na + ‐ cotransport and by CO 2 diffusion into the cells with carbonic anhydrase catalyzing its conversion into ions and protons (Jalali et al., ; Lacruz et al., ). In HAT‐7 cells in the absence of /CO 2 , we identified a basolateral Na + dependent, amiloride sensitive proton extruding entity suggesting the presence of Na + /H + exchanger activity at this membrane (Racz et al., ).…”

MicroRNA 224 Regulates Ion Transporter Expression in Ameloblasts To Coordinate Enamel Mineralization

“…Located on the apical or basolateral membrane of polarized ameloblasts, NBCe1 and AE2 regulate intra-and extracellular pH by mediating the electrogenic transport of Na ϩ and bases (HCO 3 Ϫ and CO 3 2Ϫ ) (5). More recently, NBCe1 has been found in mouse dental epithelium and might be engaged in neutralizing protons released during crystal formation in the enamel space, thus regulating pH (53). In the current study, SLC4A4 and CFTR expression levels were significantly increased during human ameloblast differentiation, inversely linearly correlating with miR-224 expression.…”

Claudin-16 Deficiency Impairs Tight Junction Function in Ameloblasts, Leading to Abnormal Enamel Formation

“…The importance of a tight control of the extracellular pH on enamel mineralization has been previously emphasized . Mutations of SLC4 family bicarbonate transporters expressed by ameloblasts (eg, AE2 and NBCe1) result in altered enamel structure in mice and humans . Recent analyses have demonstrated that repeated pH cycling during the maturation stage allowed the progressive formation of enamel made of near‐perfect hydroxyapatite crystals .…”