UCSF

PURPOSE: G protein-coupled receptor 155 (GPR155), which is significantly upregulated in maturation stage ameloblasts as compared to secretory stage ameloblasts, has not been previously explored in the context of amelogenesis. This study aims to determine the role of GPR155 in enamel maturation, and specifically its involvement in trans-ameloblast calcium transport.

METHODS: To profile the expression pattern of Gpr155 in the progressively advancing ameloblasts, we collected enamel organ cells from P5, P9, P11, P12, and P13 wild type mouse first molars, providing ameloblasts at the secretory stage, transition stage, early maturation and late maturation stage of development. RNA was extracted from these ameloblasts and the expression of Gpr155 was quantified via RT-qPCR. The alteration of GPR155 expression in Nckx4-/- ameloblasts compared with Nckx4+/+ ameloblasts was analyzed by qPCR and immunostaining. Mice with loss-of-function of NCKX4 (potassium dependent sodium/calcium exchanger 4) developed hypocalcified and hypomature enamel, which resembles the defective enamel seen in humans harboring NCKX4 gene mutations. We further used RT-qPCR to characterize the expression of Orai2 and Trpm7, which have been previously characterized to contribute to the trans-ameloblast calcium transport. The correlation between the expression of GPR155 and enamel matrix calcification was also analyzed in the lactating mouse enamel organs.

RESULTS: Among these progressively differentiating ameloblasts, Gpr155 expression started to increase in the transition stage, then reached to its climax in the middle of maturation stage, and slightly reduced at the end of maturation stage. There was a significant increase (p<0.05) of Gpr155 expression in Nckx4-/- maturation stage ameloblasts as compared with Nckx4+/+ maturation stage ameloblasts. Furthermore, calcium transport associated genes, Trpm7 and Orai2 which are co-upregulated with Gpr155 in the wild type maturation ameloblasts, were also synchronically upregulated with Gpr155 in the Nckx4-/- late stage of maturation ameloblasts. Finally, immunostaining showed increased signals of GPR155 protein in maturation ameloblasts of lactating mice compared to the ameloblasts of age-matched virgin mice.

CONCLUSION: Results suggest that GPR155, coordinating with NCKX4, ORAI2, and TRPM7, participates in the active calcium transport function in maturation stage ameloblasts. This less-characterized gene has shown great promise in the realm of calcium signaling and control during the later stage of amelogenesis, contributing to our understanding of the interactions between the enamel matrix and ameloblasts and how these interactions direct the biomineralization of the hardest tissue in our bodies.