Abstract Amelogenin (AMELX), the predominant extracellular enamel matrix protein, contains a single phosphorylation site at Serine16 (S16). To investigate the functional significance of AMELX phosphorylation, we generated a knock-in (KI) mouse model (AmelxS16A) in which S16 is substituted with Alanine, preventing AMELX phosphorylation. These KI mice exhibited hypoplastic, hypomineralized enamel lacking the characteristic rod structure, demonstrating that AMELX phosphorylation is critical for dental enamel formation. Mineralization analysis revealed accelerated formation of carbonated hydroxyapatite (CHA) and rapid transformation of amorphous calcium phosphate (ACP) to CHA during the secretory stage, resulting in enamel acidification. Based on these findings, we hypothesized that excessive acidity disrupts pH regulation in KI ameloblasts. To test this hypothesis, we examined mRNA and protein expression of three carbonic anhydrases involved in pH regulation: intracellular CA2, transmembrane CA9, and extracellular CA6, localized their distribution; and measured total CA enzymatic activity in enamel organs and matrix lysates. Additionally, we assessed mRNA expression of four ion transporters (Slc4a4, Slc24a3, Slc24a4, Cftr) involved in in pH regulation. Our results revealed significant downregulation of all studied genes during the secretory stage in KI versus wild-type (WT) mice, with a general trend of reduced expression during maturation. Consistent with these findings, CA activity was markedly lower in KI enamel organs. Collectively, these data indicate that AMELX phosphorylation supports ameloblast function by stabilizing enamel pH during secretory stage amelogenesis through its capacity to slow the rate of enamel mineral formation and prevent enamel acidification. In the absence of AMELX phosphorylation, excessive acidity overwhelms ameloblast pH regulation, leading to dysregulation and defective enamel. This study provides evidence that loss of biological control over extracellular mineralization can lead to cellular disruption, highlighting a critical link between protein phosphorylation, mineralization kinetics and pH regulation.
Bui et al. (Fri,) studied this question.