The N6‐methyladenosine demethylase FTO is required for odontoblast differentiation in vitro and dentine formation in mice by promoting RUNX2 exon 5 inclusion through RBM4

Abstract Aim Fat mass and obesity‐associated (FTO) protein, the first discovered N6‐methyladenine (m6A) demethylase, played positive roles in bone formation. In this study, the aim was to investigate the function and potential mechanism of Fto in dentine formation. Methodology In vivo model, postnatal 12‐day (PN12), 4‐week‐old (4 wk), 6‐week‐old (6 wk) healthy male C57BL/6J were randomly divided into Fto knockout (Fto −/− ) mice and wild‐type (WT) littermates according to their genotypes, with 3–5 mice in each group. The mandibles of Fto −/− mice and WT control littermates were isolated for analysis by micro–computed tomography (micro‐CT), 3‐dimensional reconstruction and Haematoxylin–eosin (HE) staining. In vitro , mouse dental papilla cells (mDPCs) and human dental stem pulp cells (hDPSCs) were cultured with odontogenetic medium to evaluate differentiation capacity; expression levels of odontoblastic related genes were evaluated using quantitative real‐time polymerase chain reaction (qRT‐PCR). The inclusion levels of Runt‐related transcription factor 2 (RUNX2) exon 5 in mDPCs and hDPSCs were detected by semiquantitative real‐time polymerase chain reaction (RT‐PCR). The RNA binding motif protein 4 (RBM4) m6A site was verified through m6A methylated RNA immunoprecipitation (MeRIP) and the stability of RBM4 mRNA influenced by FTO knockdown was measured by mRNA stability assay. Differences with p values < .05 were regarded as statistically significant. Results We discovered that Fto −/− mice showed significant dentine formation defects characterized by widened pulp cavity, enlarged pulp‐tooth volume ratio, thinned dentine and pre‐dentine layer of root ( p < .05). Fto −/− mDPCs and FTO‐silencing hDPSCs not only exhibited insufficient mineralization ability and decreased expression levels of odontoblastic mineralization related genes ( p < .05), but showed significantly reduced Runx2 exon 5 inclusion level ( p < .05). FTO knockdown increased the m6A level of RBM4 and destabilized the mRNA of RBM4, thus contributing to the reduced RBM4 expression level. Moreover, Rbm4 overexpression in Fto −/− mDPCs can partly restore Runx2 exon 5 inclusion level and the differentiation ability disrupted by Fto knockout. Conclusion Thus, within the limitations of this study, the data suggest that FTO promotes odontoblastic differentiation during dentine formation by stabilizing RBM4 mRNA to promote RUNX2 exon 5 inclusion.