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.

Keywords: animal model; cell differentiation; dental pulp stem cell(s); dentine; mRNA decay; odontoblast(s).