Amelogenins as potential buffers during secretory-stage amelogenesis

Abstract

Amelogenins are the most abundant protein species in forming dental enamel, taken to regulate crystal shape and crystal growth. Unprotonated amelogenins can bind protons, suggesting that amelogenins could regulate the pH in enamel in situ. We hypothesized that without amelogenins the enamel would acidify unless ameloblasts were buffered by alternative ways. To investigate this, we measured the mineral and chloride content in incisor enamel of amelogenin-knockout (AmelX(-/-)) mice and determined the pH of enamel by staining with methyl-red. Ameloblasts were immunostained for anion exchanger-2 (Ae2), a transmembrane pH regulator sensitive for acid that secretes bicarbonate in exchange for chloride. The enamel of AmelX(-/-) mice was 10-fold thinner, mineralized in the secretory stage 1.8-fold more than wild-type enamel and containing less chloride (suggesting more bicarbonate secretion). Enamel of AmelX(-/-) mice stained with methyl-red contained no acidic bands in the maturation stage as seen in wild-type enamel. Secretory ameloblasts of AmelX(-/-) mice, but not wild-type mice, were immunopositive for Ae2, and stained more intensely in the maturation stage compared with wild-type mice. Exposure of AmelX(-/-) mice to fluoride enhanced the mineral content in the secretory stage, lowered chloride, and intensified Ae2 immunostaining in the enamel organ in comparison with non-fluorotic mutant teeth. The results suggest that unprotonated amelogenins may regulate the pH of forming enamel in situ. Without amelogenins, Ae2 could compensate for the pH drop associated with crystal formation.

Keywords: SLC4A2; alveolar bone; dentin; enamel; mineral density; pH control.

Figure 1.

The mineral density of lower incisor enamel from unexposed and fluoride (F)-exposed AmelX-/- mice measured by micro CT. (a, b) The density in the 4 groups was plotted against slice numbers (representing progressive stages of enamel formation with 300 μm intervals for (a), or 60 μm intervals for (b)). 0 in the X axis stands for the beginning of the maturation stage. Negative numbers along X axis represent the secretory stage. The boxed area (secretory stage) in (a) is shown in more detail in (b). In (b) the graphs with the same color (blue, red, green, or purple) represent measurements of separate mice from the same experimental group. (c) The average values of secretion-stage and maturation-stage enamel in comparison with the values for dentin and bone are shown for each group. WT, wild-type mice; WT+F, wild-type mice exposed to fluoride; AKO, AmelX-/- mice; AKO+F, AmelX-/- mice exposed to fluoride. Exposure of wild-type mice and AmelX-/- mice to fluoride had opposite effects on the enamel mineral density. ***P < 0.001; **0.001 < P < 0.01; *0.01 < P < 0.05.

Figure 2.

Backscattered (BSD) images and the composition of the enamel in late maturation-stage for different experimental groups measured by electron probe X-ray microanalysis (means and standard deviation). Late maturation enamel from wild-type (a), fluorotic wild-type (b), AmelX-/- (c) and fluorotic AmelX-/- (d) mandibular incisors. Disruption of amelogenin gene significantly reduced the enamel thickness (c, d). The white arrows in (b) show the hypermineralized lines in the superficial and deep fluorotic enamel layers. The white arrow in (d) shows the heterogeneous surface of the fluorotic AmelX-/- enamel. To highlight the changes in the enamel layer, the BSD images are presented with different dentin “densities.” CaO (e), fluoride (f), chlorine (g) and SO3 (h). ***P < 0.001; **0.001 < P <0.01; *0.01 < P < 0.05. WT, wild-type mice; WT+F: wild-type mice exposed to fluoride; AKO, AmelX-/- mice; AKO+F, AmelX-/- mice exposed to fluoride.

Figure 3.

Immunolocalization of amelogenin (a, b, e, f) and ameloblastin (c, d) in developing maxillary incisors. Secretory ameloblasts of wild-type mice are positive for amelogenin (a) and ameloblastin (c). Ameloblasts of AmelX^-/- mice are negative for amelogenin (b) but strongly positive for ameloblastin (d). Arrows point to the thin layer of enamel in null mice. In upper incisors of wild-type mice, amelogenin disappears by the mid-stage of maturation (e, yellow arrow), whereas intense amelogenin immunoreactions persist throughout the maturing and post-eruptive enamel layers in fluorotic wild-type incisors (f). a, b, rabbit anti-mouse amelogenins; e, f, rabbit anti-porcine amelogenins. Red arrows indicate transitional stage. P, dental pulp; *gingival margin; d, dentin; e, enamel.

Figure 4.

Staining for pH (a, b) and Ae2 protein (c–l) during amelogenesis in incisors from AmelX^-/- mice and wild-type mice. (a) Methyl-red-stained cell-free enamel of lower incisors from a wild-type mouse and (b) from an AmelX^-/- mouse. Methyl-red revealed 2 acidic bands, a dark-red one apically and a weaker pink (acidic) one more incisally in wild-type but not in AmelX^-/- enamel. M1 indicates the position of the first molar. (c) A low-power micrograph of Ae2 immunostaining in upper incisors of a non-fluorotic AmelX^-/- enamel organ. Ameloblasts, stratum intermedium (si), and papillary layer (pl) are strongly positive (f, h, j). In wild-type mouse incisors (d), staining for Ae2 is absent in secretory ameloblasts (sa) and gradually begins at early maturation, but staining is weaker. Boxed areas are magnified and restricted to maturation ameloblasts (ma; e, g, i). Asterisks in (c) and (d) indicate approximate onset of transitional stage. Exposure to fluoride strongly enhanced staining in fluorotic AmelX^-/- enamel organ in early (k) and latematuration (l).