METTL3 mediates osteoblast apoptosis by regulating endoplasmic reticulum stress during LPS-induced inflammation

Osteoblast apoptosis is a prominent factor for disrupting skeletal homeostasis in multiple inflammatory bone diseases. METTL3, a key methyltransferase that catalyzes the N6-methyladenosine (m6A) modification of mRNA, has recently been shown to exert a critical role in osteogenic differentiation. However, the function of METTL3 in osteoblast apoptosis under inflammatory conditions remains elusive. In the present study, we observed that the total m6A level and METTL3 expression were upregulated in differentiated osteoblasts and downregulated after LPS stimulation. METTL3 knockdown induced a higher apoptotic rate in LPS-treated osteoblasts. The expression of the antiapoptotic protein BCL-2 decreased, and the apoptotic proteins cleaved Caspase-3, cleaved PARP-1 and cleaved Caspase-12 increased following METTL3 knockdown. Meanwhile, METTL3 silencing inhibited osteoblast proliferation and decreased osteogenic marker expression, ALP activity and mineralized nodules. RNA-seq analysis revealed that differentially expressed genes were significantly enriched in unfolded protein response pathways in METTL3-deficient cells. METTL3 depletion upregulated the expression of the ER stress-related markers, including p-PERK, p-eIF2α, p-IRE1α, GRP78, ATF4, CHOP and ATF6. Inhibition of ER stress by 4-PBA remarkably rescued METTL3 knockdown-induced apoptosis and promoted osteoblast proliferation and differentiation. Mechanistically, METTL3 depletion enhanced the expression and mRNA stability of Grp78, and similar results were observed after YTHDF2 knockdown. RIP-qPCR revealed that YTHDF2 directly interacted with Grp78 mRNA and that the interaction relied on METTL3. Taken together, our study demonstrated that METTL3 knockdown enhanced Grp78 expression through YTHDF2-mediated RNA degradation, which elicited ER stress, thereby promoting osteoblast apoptosis and inhibiting cell proliferation and differentiation under LPS-induced inflammatory condition.