Osteoporosis (OP) is a common and fracture-prone skeletal disease characterized by deteriorated trabecular microstructure and pathologically involving various forms of regulated bone cell death. However, the exact role, cellular nature and regulatory mechanisms of ferroptosis in OP are not fully understood. Here, we reported that OP femurs from ovariectomized (Ovx) mice exhibited pronounced iron deposition, ferroptosis, and transcriptional suppression of a key anti-ferroptotic factor GPX4 (glutathione peroxidase 4). GPX4 suppression was accompanied by hypermethylation of the Gpx4 promoter and an increase in DNA methyltransferases DNMT1/3a/3b and was transcriptionally promoted by repressive KLF5 and the transcriptional corepressors NCoR and SnoN. Conversely, DNMT inhibition with SGI-1027 reversed promoter hypermethylation, GPX4 suppression and ferroptotic osteoporosis. In cultured primary bone cells, ferric ammonium citrate (FAC) mimicking iron loading similarly induced GPX4 suppression and ferroptosis in osteoblasts but not in osteoclasts, which were rescued by siRNA-mediated individual knockdown of DNMT 1/3a/3b. Intriguingly, SGI-1027 alleviated the ferroptotic changes caused by FAC, but not by a GPX4 inactivator RSL3. More importantly, we generated a strain of osteoblast-specific Gpx4 haplo-deficient mice Gpx4^Ob+/− that developed spontaneous and more severe ferroptotic OP alterations after Ovx operation, and showed that GPX4 inactivation by RSL3 or semi-knockout in osteoblasts largely abolished the anti-ferroptotic and osteoprotective effects of SGI-1027. Taken together, our data suggest that GPX4 epigenetic suppression caused by DNMT aberration and the resulting osteoblastic ferroptosis contribute significantly to OP pathogenesis, and that the strategies preserving GPX4 by DNMT intervention are potentially effective to treat OP and related bone disorders.

骨质疏松症 （OP） 是一种常见且易发生骨折的骨骼疾病，其特征是小梁微观结构恶化，病理上涉及各种形式的调节性骨细胞死亡。然而，铁死亡在 OP 中的确切作用、细胞性质和调节机制尚不完全清楚。在这里，我们报道了来自卵巢切除 （Ovx） 小鼠的 OP 股骨表现出明显的铁沉积、铁死亡和关键抗铁死亡因子 GPX4 （谷胱甘肽过氧化物酶 4） 的转录抑制。GPX4 抑制伴有 Gpx4 启动子的高甲基化和 DNA 甲基转移酶 DNMT1/3a/3b 的增加，并被抑制性 KLF5 和转录辅阻遏蛋白 NCoR 和 SnoN 转录促进。相反，SGI-1027 的 DNMT 抑制可逆转启动子高甲基化、GPX4 抑制和铁死亡性骨质疏松症。在培养的原代骨细胞中，模拟铁负荷的柠檬酸铁铵 （FAC） 同样诱导成骨细胞中的 GPX4 抑制和铁死亡，但在破骨细胞中未诱导，这是通过 siRNA 介导的 DNMT 1/3a/3b 的个体敲低来挽救的。有趣的是，SGI-1027 减轻了由 FAC 引起的铁死亡变化，但没有减轻由 GPX4 灭活剂 RSL3 引起的铁死亡变化。更重要的是，我们生成了一株成骨细胞特异性 Gpx4 单倍体缺陷小鼠 Gpx4^{Ob + /-} ，该品系在 Ovx 手术后出现自发且更严重的铁死亡 OP 改变，并表明成骨细胞中 RSL3 或半敲除的 GPX4 失活在很大程度上消除了 SGI-1027 的抗铁死亡和骨保护作用。 综上所述，我们的数据表明，由 DNMT 畸变引起的 GPX4 表观遗传抑制和由此产生的成骨细胞铁死亡对 OP 发病机制有显着贡献，并且通过 DNMT 干预保留 GPX4 的策略可能有效治疗 OP 和相关骨骼疾病。