Yang Ruhan, Yu Weijun, Lin Lu, Cui Zhurong, Tang Jiaqi, Li Guanglong, Jin Min, Gu Yuting, Lu Eryi
Department of Stomatology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, 160 Pujian Road, Shanghai, 200127, China.
Department of Stomatology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, 160 Pujian Road, Shanghai, 200127, China.
J Adv Res. 2025 Jun;72:303-317. doi: 10.1016/j.jare.2024.07.031. Epub 2024 Jul 31.
Excessive osteoclastogenesis is a key driver of inflammatory bone loss. Suppressing osteoclastogenesis has always been considered essential for the treatment of inflammatory bone loss. N-acetyltransferase 10 (NAT10) is the sole enzyme responsible for N4-acetylcytidine (ac4C) modification of mRNA, and is involved in cell development. However, its role in osteoclastogenesis and inflammatory bone loss remained elusive.
We aimed to clarify the regulatory mechanism of NAT10 and ac4C modification in osteoclastogenesis and inflammatory bone loss.
NAT10 expression and ac4C modification during osteoclastogenesis were determined by quantitative real-time PCR (qPCR), western blotting, dot blot and immunofluorescent staining, and the effect of NAT10 inhibition on osteoclast differentiation in vitro was measured by the tartrate-resistant acid phosphatase staining, podosome belts staining assay and bone resorption pit assay. Then, acRIP-qPCR and NAT10RIP-qPCR, ac4C site prediction, mRNA decay assay and luciferase reporter assay were performed to further study the underlying mechanisms. At last, mice models of inflammatory bone loss were applied to verify the therapeutic effect of NAT10 inhibition in vivo.
NAT10 expression was upregulated during osteoclast differentiation and highly expressed in alveolar bone osteoclasts from periodontitis mice. Inhibition of NAT10 notably reduced osteoclast differentiation in vitro, as indicated by great reduction of tartrated resistant acid phosphatse positive multinuclear cells, osteoclast-specific gene expression, F-actin ring formation and bone resorption capacity. Mechanistically, NAT10 catalyzed ac4C modification of Fos (encoding AP-1 component c-Fos) mRNA and maintained its stabilization. Besides, NAT10 promoted MAPK signaling pathway and thereby activated AP-1 (c-Fos/c-Jun) transcription for osteoclastogenesis. Therapeutically, administration of Remodelin, the specific inhibitor of NAT10, remarkably impeded the ligature-induced alveolar bone loss and lipopolysaccharide-induced inflammatory calvarial osteolysis.
Our study demonstrated that NAT10-mediated ac4C modification is an important epigenetic regulation of osteoclast differentiation and proposed a promising therapeutic target for inflammatory bone loss.
破骨细胞生成过多是炎症性骨丢失的关键驱动因素。抑制破骨细胞生成一直被认为是治疗炎症性骨丢失的关键。N-乙酰转移酶10（NAT10）是唯一负责mRNA的N4-乙酰胞苷（ac4C）修饰的酶，并参与细胞发育。然而，其在破骨细胞生成和炎症性骨丢失中的作用仍不清楚。
我们旨在阐明NAT10和ac4C修饰在破骨细胞生成和炎症性骨丢失中的调控机制。
通过定量实时PCR（qPCR）、蛋白质免疫印迹法、斑点印迹法和免疫荧光染色法检测破骨细胞生成过程中NAT10的表达和ac4C修饰，并通过抗酒石酸酸性磷酸酶染色、足体带染色试验和骨吸收陷窝试验检测NAT10抑制对体外破骨细胞分化的影响。然后，进行acRIP-qPCR和NAT10RIP-qPCR、ac4C位点预测、mRNA降解试验和荧光素酶报告基因试验以进一步研究潜在机制。最后，应用炎症性骨丢失小鼠模型验证NAT10抑制在体内的治疗效果。
在破骨细胞分化过程中，NAT10表达上调，且在牙周炎小鼠的牙槽骨破骨细胞中高表达。抑制NAT10可显著降低体外破骨细胞分化，表现为抗酒石酸酸性磷酸酶阳性多核细胞、破骨细胞特异性基因表达、F-肌动蛋白环形成和骨吸收能力显著降低。机制上，NAT10催化Fos（编码AP-1成分c-Fos）mRNA的ac4C修饰并维持其稳定性。此外，NAT10促进丝裂原活化蛋白激酶信号通路，从而激活AP-1（c-Fos/c-Jun）转录以促进破骨细胞生成。在治疗方面，给予NAT10特异性抑制剂Remodelin可显著抑制结扎诱导的牙槽骨丢失和脂多糖诱导的炎性颅骨骨溶解。
我们的研究表明，NAT10介导的ac4C修饰是破骨细胞分化的重要表观遗传调控，并为炎症性骨丢失提出了一个有前景的治疗靶点。
