NAT10 通过催化 Fos mRNA ac4C 修饰和上调 MAPK 信号通路,促进炎症性骨质流失中的破骨细胞生成。

Ruhan Yang, Weijun Yu, Lu Lin, Zhurong Cui, Jiaqi Tang, Guanglong Li, Min Jin, Yuting Gu, Eryi Lu
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引用次数: 0

摘要

简介过度的破骨细胞生成是炎症性骨质流失的主要驱动因素。抑制破骨细胞生成一直被认为是治疗炎症性骨质流失的关键。N-acetyltransferase 10(NAT10)是负责对 mRNA 进行 N4-乙酰胞苷(ac4C)修饰的唯一酶,参与细胞发育。然而,它在破骨细胞生成和炎症性骨质流失中的作用仍然难以捉摸:我们旨在阐明 NAT10 和 ac4C 修饰在破骨细胞生成和炎性骨质流失中的调控机制:方法:通过实时定量 PCR(qPCR)、Western 印迹、点印迹和免疫荧光染色等方法检测破骨细胞发生过程中 NAT10 的表达和 ac4C 的修饰,并通过耐酒石酸磷酸酶染色、荚膜带染色和骨吸收坑实验检测 NAT10 抑制对体外破骨细胞分化的影响。然后,通过acRIP-qPCR和NAT10RIP-qPCR、ac4C位点预测、mRNA衰变实验和荧光素酶报告实验进一步研究其潜在机制。最后,应用炎性骨质流失小鼠模型验证了抑制 NAT10 在体内的治疗效果:结果:NAT10在破骨细胞分化过程中表达上调,并在牙周炎小鼠牙槽骨破骨细胞中高表达。抑制 NAT10 可显著降低破骨细胞的体外分化,表现为酒石酸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的大幅降低。从机理上讲,NAT10 可催化 Fos(编码 AP-1 成分 c-Fos)mRNA 的 ac4C 修饰并保持其稳定。此外,NAT10 还能促进 MAPK 信号通路,从而激活 AP-1(c-Fos/c-Jun)转录,促进破骨细胞生成。在治疗上,服用 NAT10 的特异性抑制剂 Remodelin 能显著抑制结扎诱导的牙槽骨流失和脂多糖诱导的炎性钙质溶骨:我们的研究表明,NAT10介导的ac4C修饰是破骨细胞分化的一个重要表观遗传调控因子,并为炎症性骨质流失提出了一个很有前景的治疗靶点。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
NAT10 promotes osteoclastogenesis in inflammatory bone loss by catalyzing Fos mRNA ac4C modification and upregulating MAPK signaling pathway.

Introduction: 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.

Objectives: We aimed to clarify the regulatory mechanism of NAT10 and ac4C modification in osteoclastogenesis and inflammatory bone loss.

Methods: 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.

Results: 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.

Conclusions: 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.

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