GPR155 and NCKX4 Regulate Calcium Homeostasis in Maturation Amelogenesis

Objectives: GPR155 is a significantly upregulated G-protein coupled receptor within maturation stage ameloblasts (MABs). However, its role in amelogenesis has yet to be elucidated. This study aims to characterize GPR155’s expression and involvement in transepithelial calcium transport as well as its relationship to NCKX4, the principal calcium transporter in MABs.
Methods: RNA-seq analysis was employed to unveil the whole transcriptome of mouse enamel organs (EOs) at secretory (postnatal day 5, P5) and maturation stages (P9 and P13). Gpr155’s temporal expression profile was further verified through RT-qPCR, utilizing mouse first molar EOs undergoing differentiation progressing from P5, 7, 8, 9, 11, to P13. The assessment of GPR155 protein abundance and cellular localization was characterized by western blotting and immunohistochemistry. To study the role of GPR155 in trans-ameloblast calcium transport, the expression levels of Gpr155 and other MAB calcium transporters (Trpm7 and Orai2) were quantitated by RT-qPCR of Nckx4^+/+ and Nckx4^-/- EOs. Inhibition of histone deacetylase by trichostatin A (TSA) and sodium butyrate (NaB) followed by RT-qPCR and western blot was used to study the epigenetic regulation of Gpr155 in an ameloblast cell line. Finally, a gross examination for enamel malformations was performed in a Gpr155^-/- mouse model.
Results: RNA-seq analysis revealed a notably high expression of Gpr155 in maturation ameloblasts (MABs) in contrast to secretory ameloblasts (SABs). RT-qPCR determined that Gpr155 expression peaked at the onset of the maturation stage, a nearly 40-fold increase compared to SABs. GPR155 immunolocalized to the apical border of MABs and western blotting confirmed its stage-specific distribution. Disrupted calcium transport due to null NCKX4 function appeared to synchronously upregulate Gpr155 along with Trpm7 and Orai2. Our in vitro study suggested that histone acetylation could transcriptionally activate the Gpr155 gene. Mice lacking functional GPR155 exhibited white and chalky enamel.
Conclusions: Taken together, GPR155 emerges as an indispensable player in enamel formation given its robust expression in MABs, potential role as a regulator of calcium transport, as well as clear enamel phenotype upon genetic ablation.