METTL3 Modulates Osteoclast Differentiation and Function by Controlling RNA Stability and Nuclear Export

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. <i>Mettl3</i> knockdown resulted in an increased size but a decreased bone-resorbing ability of osteoclasts. The expression of osteoclast-specific genes (<i>Nfatc1</i>, <i>c-</i><i>Fos</i>, <i>Ctsk</i>, <i>Acp5</i> and <i>Dcstamp</i>) was inhibited by <i>Mettl3</i> depletion, while the expression of the cellular fusion-specific gene <i>Atp6v0d2</i> was upregulated. Mechanistically, <i>Mettl3</i> knockdown elevated the mRNA stability of <i>Atp6v0d2</i> 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 <i>Mettl3</i> deficiency. Depletion of <i>Mettl3</i> maintained the retention of <i>Traf6</i> 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 <i>Atp6v0d2</i> mRNA degradation mediated by YTHDF2 and <i>Traf6</i> mRNA nuclear export. These findings elucidate the molecular basis of RNA epigenetic regulation in osteoclast development.