Abstract：
BACKGROUND: 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.
OBJECTIVE: 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.
OBJECTIVE: 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.
摘要：
背景：破骨细胞过度生成是炎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性骨丢失的关键。N-乙酰转移酶10（NAT10）是负责mRNA的N4-乙酰胞苷（ac4C）修饰的唯一酶，并参与细胞发育。然而,其在破骨细胞生成和炎性骨丢失中的作用仍然难以捉摸。
目的：我们旨在阐明NAT10和ac4C修饰在破骨细胞生成和炎性骨丢失中的调控机制。
方法：通过定量实时PCR（qPCR）测定破骨细胞形成过程中NAT10的表达和ac4C修饰，西方印迹,斑点印迹和免疫荧光染色，并通过抗酒石酸酸性磷酸酶染色检测NAT10抑制对体外破骨细胞分化的影响，足体带染色测定和骨吸收坑测定。然后，acRIP-qPCR和NAT10RIP-qPCR，AC4C站点预测，进行mRNA衰减测定和荧光素酶报告基因测定以进一步研究其潜在机制。最后,应用炎性骨丢失的小鼠模型来验证NAT10抑制在体内的治疗效果。
结果：NAT10表达在破骨细胞分化过程中上调，在牙周炎小鼠牙槽骨破骨细胞中高表达。抑制NAT10显著降低体外破骨细胞分化，如酒石酸抗性酸性磷酸阳性多核细胞的大量减少所示，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。机械上,NAT10催化ac4C修饰Fos(编码AP-1组分c-Fos)mRNA并维持其稳定性。此外,NAT10促进MAPK信号通路,从而激活AP-1(c-Fos/c-Jun)转录用于破骨细胞生成。治疗学上,雷德林的管理，NAT10的特异性抑制剂显着阻碍结扎诱导的牙槽骨丢失和脂多糖诱导的炎性颅骨骨溶解。
结论：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希望的治疗靶点。
目的：我们旨在阐明NAT10和ac4C修饰在破骨细胞生成和炎性骨丢失中的调控机制。
方法：通过定量实时PCR（qPCR）测定破骨细胞形成过程中NAT10的表达和ac4C修饰，西方印迹,斑点印迹和免疫荧光染色，并通过抗酒石酸酸性磷酸酶染色检测NAT10抑制对体外破骨细胞分化的影响，足体带染色测定和骨吸收坑测定。然后，acRIP-qPCR和NAT10RIP-qPCR，AC4C站点预测，进行mRNA衰减测定和荧光素酶报告基因测定以进一步研究其潜在机制。最后,应用炎性骨丢失的小鼠模型来验证NAT10抑制在体内的治疗效果。
结果：NAT10表达在破骨细胞分化过程中上调，在牙周炎小鼠牙槽骨破骨细胞中高表达。抑制NAT10显著降低体外破骨细胞分化，如酒石酸抗性酸性磷酸阳性多核细胞的大量减少所示，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。机械上,NAT10催化ac4C修饰Fos(编码AP-1组分c-Fos)mRNA并维持其稳定性。此外,NAT10促进MAPK信号通路,从而激活AP-1(c-Fos/c-Jun)转录用于破骨细胞生成。治疗学上,雷德林的管理，NAT10的特异性抑制剂显着阻碍结扎诱导的牙槽骨丢失和脂多糖诱导的炎性颅骨骨溶解。
结论：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希望的治疗靶点。
