Yap Methylation Modulates Ameloblast Mineralization Via Mitophagy

The regulatory role of Yap methylation in ameloblast-mediated enamel mineralization remains poorly understood, particularly its connection to mitochondrial autophagy. This study aims to elucidate how Yap methylation governs ameloblast function and enamel mineralization by dissecting its mechanistic interplay with mitophagy, a process critical for cellular homeostasis during biomineralization. Yap K327M transgenic mice (C57BL/6 background) were genotyped through gene-specific PCR to validate the methylation-modified allele. Ameloblast- lineage cells (ALC) isolated from postnatal molars were cultured in mineralization-inducing medium for 7 days. Experimental groups included wild-type ALC and Yap methylated ALC cells. RNA sequencing (RNA-Seq) was employed to profile mitophagy-related pathways, while transmission electron microscopy (TEM) assessed autophagosome ultrastructure. Protein/gene expression of mitophagy markers (LC3-II, PINK1) was analyzed via western blot and RT-qPCR. Yap K327M transgenic mice displayed distinct dental phenotypes, including chalky-white enamel and elongated tooth crowns, with markedly excessive mineralization compared to wild-type littermates. In vitro, Yap methylation group exhibited accelerated amelogenesis, evidenced by increased enamel matrix deposition, and enhanced mineralization capacity. RNA sequencing identified significant upregulation of mitophagy-associated genes (e.g., Parl, Bcl2l1), corroborated by TEM observations of abundant autophagosomes and mitochondrial remnants in Yap methylated cells. Western blot and RT-qPCR confirmed elevated LC3-II and PINK1 levels, indicative of activated mitophagy. This study demonstrates that Yap methylation serves as a pivotal regulator of ameloblast mineralization by orchestrating mitophagy activation.