Hospital of Stomatology, Guanghua School of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou 510055, China.
Department of Stomatology, Shenzhen University General Hospital, Shenzhen University Clinical Medical Academy, Shenzhen 518055, China.
Cell Signal. 2022 Jul;95:110335. doi: 10.1016/j.cellsig.2022.110335. Epub 2022 Apr 21.
Osteoblast apoptosis is a prominent factor for disrupting skeletal homeostasis in multiple inflammatory bone diseases. METTL3, a key methyltransferase that catalyzes the N6-methyladenosine (mA) 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 mA 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.
成骨细胞凋亡是多种炎症性骨病破坏骨骼动态平衡的一个突出因素。METTL3 是一种关键的甲基转移酶，可催化 mRNA 的 N6-甲基腺苷（m6A）修饰，最近被证明在成骨分化中发挥关键作用。然而，METTL3 在炎症条件下成骨细胞凋亡中的作用仍不清楚。在本研究中，我们观察到分化的成骨细胞中总 m6A 水平和 METTL3 表达上调，LPS 刺激后下调。METTL3 敲低可诱导 LPS 处理的成骨细胞凋亡率升高。抗凋亡蛋白 BCL-2 的表达降低，而凋亡蛋白 cleaved Caspase-3、cleaved PARP-1 和 cleaved Caspase-12 的表达增加。同时，METTL3 沉默抑制成骨细胞增殖，降低成骨标志物表达、ALP 活性和矿化结节。RNA-seq 分析显示，METTL3 缺陷细胞中差异表达基因显著富集于未折叠蛋白反应途径。METTL3 耗竭上调了 ER 应激相关标志物的表达，包括 p-PERK、p-eIF2α、p-IRE1α、GRP78、ATF4、CHOP 和 ATF6。用 4-PBA 抑制 ER 应激可显著挽救 METTL3 敲低诱导的凋亡，并促进成骨细胞增殖和分化。机制上，METTL3 耗竭增强了 Grp78 的表达和 mRNA 稳定性，YTHDF2 敲低后也观察到类似结果。RIP-qPCR 显示 YTHDF2 直接与 Grp78 mRNA 相互作用，这种相互作用依赖于 METTL3。综上所述，我们的研究表明，METTL3 敲低通过 YTHDF2 介导的 RNA 降解增强了 Grp78 的表达，引发 ER 应激，从而促进 LPS 诱导的炎症条件下成骨细胞凋亡，并抑制细胞增殖和分化。
