Abstract
Osteoclast differentiation and function are crucial for maintaining bone homeostasis and preserving skeletal integrity. N6-methyladenosine (m6A) is an abundant mRNA modification that has recently been shown to be important in regulating cell lineage differentiation. Nevertheless, the effect of m6A on osteoclast differentiation remains unknown. In the present study, we observed that the m6A level and methyltransferase METTL3 expression increased during osteoclast differentiation. Mettl3 knockdown resulted in an increased size but a decreased bone-resorbing ability of osteoclasts. The expression of osteoclast-specific genes (Nfatc1, c-Fos, Ctsk, Acp5 and Dcstamp) was inhibited by Mettl3 depletion, while the expression of the cellular fusion-specific gene Atp6v0d2 was upregulated. Mechanistically, Mettl3 knockdown elevated the mRNA stability of Atp6v0d2 and the same result was obtained when the m6A-binding protein YTHDF2 was silenced. Moreover, the phosphorylation levels of key molecules in the MAPK, NF-κB and PI3K-AKT signaling pathways were reduced upon Mettl3 deficiency. Depletion of Mettl3 maintained the retention of Traf6 mRNA in the nucleus and reduced the protein levels of TRAF6. Taken together, our data suggest that METTL3 regulates osteoclast differentiation and function through different mechanisms involving Atp6v0d2 mRNA degradation mediated by YTHDF2 and Traf6 mRNA nuclear export. These findings elucidate the molecular basis of RNA epigenetic regulation in osteoclast development.
Highlights
N6-methyladenosine (m6A) is produced by a methylation modification at the N6 position of adenosine residues in RNA
The results showed that no significant differences were observed between cells transfected with Mettl3-short hairpin RNAs (shRNAs) and Mettl3-shCtrl at any time point of the experiment (Figure 2D,E), indicating that methyltransferase-like 3 (METTL3) had no effect on the proliferation of osteoclast precursor cells
Mettl3-deficient RAW264.7 cells were cultured in the presence of RANKL and Mettl3-deficient bone marrow-derived macrophages (BMMs) were treated with RANKL and macrophage-colony stimulating factor (M-CSF)
Summary
N6-methyladenosine (m6A) is produced by a methylation modification at the N6 position of adenosine residues in RNA. The binding of RANKL to the receptor RANK induces the recruitment of adaptor molecules, among which TNF receptor-associated factor-6 (TRAF6) acts as the most pivotal factor to trigger downstream MAPK (ERK, JNK and p38), NF-κB and PI3K-AKT pathways [22]. Activation of these pathways further stimulates the expression of transcription factors such as nuclear factor of activated T cells 1 (NFATC1) and c-FOS. The results of our previous study demonstrated that attenuated m6A levels upon Mettl deficiency inhibits the osteogenic differentiation potential of bone mesenchymal stem cells (BMSCs) [31]. The aim of this study was to elucidate the function and underlying molecular mechanisms of METTL3-dependent m6A modification in osteoclast differentiation
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