Amelogenin Phosphorylation Affects Acid Phosphatase 4 Expression

Objectives: Amelogenin (Amelx) phosphorylation is critical for development of enamel. Mutations in kinases are known to cause amelogenesis imperfecta (AI). Amelx has a sole phosphorylation site at Serine 16 (S16). AMELX^S16A KI mice lacking Amelx phosphorylation exhibit severely compromised enamel¹.
Recent studies have also demonstrated that ACP4 is expressed in Tomes’ processes of secretory ameloblasts². Mutations in ACP4 cause severe AI. The mouse ACP4 KI model carrying the human mutation presents with hypoplastic prism-less enamel with ectopic calcifications, an enamel phenotype very similar to that of the AMELX^S16A KI.
Our findings have led to the hypotheses that interactions of phosphorylated Amelx with ACP4 are essential for proper enamel formation and that changes in pH can affect these interactions. To test this hypothesis, we have decided to establish an in vitro model. Specifically, literature reports show that a MDPC-23 dental papilla cell line express ACP4³.

1. Shin et al., JBC. 2020.
2. Liang et al., Sci Rep. 2022.
3. Choi et al., Cell Tissue Res. 364, 95-103. 2016.
Methods: Using single cell RNA sequencing (scRNA-seq) we found acid phosphatase 4 (ACP4) to be significantly upregulated in the enamel organ of KI mice and that this upregulation was confirmed by RT-qPCR. Furthermore, immunohistochemistry revealed that ACP4 expression is higher in distal ends of KI secretory ameloblasts vs WT. Moreover, recent studies conducted in our lab revealed that secretory enamel in AMELX^S16A KI mice is acidified.
Results: We have confirmed these findings using RT-qPCR, western blot, and immunohistochemistry. Based on these studies we have selected MDPC-23 cell line for future studies of the effects of Amelx phosphorylation and pH on ACP4 expression and cell biology.
Conclusions: We anticipate that these studies will provide new insights on the relationships between Amelx and ACP4.
This study was supported by NIH grants #R01 DE029211, #R01 DE029211-S1 (H.C.M and E.B.)