Dnmt3a-mediated DNA Methylation Regulates P. gingivalis-suppressed Cementoblast Mineralization Partially Via Mitochondria-dependent Apoptosis Pathway

Background: DNA Methyltransferase 3A (Dnmt3a) is an Enzyme that catalyzes the de novo methylation of DNA, and plays essential roles in a wide range of physiological and pathological processes. However, it remains unclear whether Porphyromonas gingivalis affects cementoblasts, the cells responsible for cementum formation, through Dnmt3a.

Methods: The samples were collected from models of mouse periapical lesions and mice of different ages, and the expression of Dnmt3a was detected through immunofluorescence. Porphyromonas gingivalis was co-cultured with cementoblasts that simultaneously overexpressed Dnmt3a. Additionally, cementoblasts were subjected to either Dnmt3a knockout or DNA methylation inhibition. Changes in global DNA methylation were analyzed, and quantitative PCR, western blotting, Alkaline Phosphatase (ALP) activity assays, and Alizarin Red staining were employed to evaluate alterations in the mineralization capacity of cementoblasts.RNA Sequencing further showed the mechanisms by which Dnmt3a regulated mineralization. Flow cytometry, MitoSox, and TRMR staining were used to verify the participation of mitochondria-dependent Apoptosis.

Results: The effect of P. gingivalis on Dnmt3a and global DNA methylation in cementoblasts was first verified. Dnmt3a expression and global DNA methylation were upregulated during cementoblast mineralization. Samples with periapical inflammation exhibited reduced Dnmt3a expression. P. gingivalis stimulation reduced the global DNA methylation and the mineralization ability of cementoblasts. Both the knockdown of Dnmt3a and using DNA methylation inhibitors suppressed cementoblast mineralization. In addition, Dnm3a depletion was significantly correlated with the mitochondria-dependent Apoptosis pathway in cementoblasts.

Conclusions: P. gingivalis blocks DNA methylation by silencing Dnmt3a in cementoblasts, thereby inducing mitochondrial-dependent Apoptosis and, ultimately, impaired cementogenesis.

Apoptosis; Cell mineralization; Cementum; DNA methylation; DNA methyltransferase; Porphyromonas gingivalis.