YTHDF1 regulates endoplasmic reticulum stress, NF-ΚB, MAPK and PI3K-AKT signaling pathways in inflammatory osteoclastogenesis

Abnormal increases in osteoclast differentiation and activity contribute to excessive bone resorption in inflam-matory bone diseases. The specific m6A-binding protein YT521-B homology domain family 1 (YTHDF1) par-ticipates in many physiopathological processes by regulating mRNA stability or translation. However, whether YTHDF1 is involved in the regulation of inflammatory osteoclastogenesis remains a mystery. This study revealed that YTHDF1 expression was upregulated during lipopolysaccharide (LPS)-stimulated osteoclast differentiation. Knockdown of Ythdf1 inhibited osteoclast formation, bone resorption and the expression of osteoclast-related genes (Tnfrsf11a, Traf6, Mmp9 and Acp5). Analysis of RNA sequencing data showed that the genes down -regulated by Ythdf1 knockdown were closely associated with endoplasmic reticulum (ER) stress and osteoclast differentiation. Western blotting confirmed that Ythdf1 depletion suppressed activation of the ER stress-related PERK, IRE1 alpha and ATF6 signaling pathways. The ER stress activator tunicamycin (Tm) partially rescued the decreased expression of Mmp9 and Acp5 caused by Ythdf1 deficiency. Meanwhile, Ythdf1 depletion inhibited the phosphorylation levels of key proteins in the NF-kappa B, MAPK and PI3K-AKT signaling pathways and decreased the mRNA stability of Tnfrsf11a, which is the major upstream signaling molecule that mediates the activation of these pathways during osteoclast differentiation. In conclusion, our findings suggest that Ythdf1 knockdown inhibits inflammatory osteoclast differentiation and function by suppressing ER stress signaling pathways. Ythdf1 knockdown also inactivates the signaling pathways involved in osteoclast differentiation by inhibiting Tnfrsf11a mRNA stability. These findings will help shed light on the molecular mechanisms of m6A-mediated epigenetic regulation in inflammatory osteoclastogenesis.