Calcineurin/NFAT pathway mediates wear particle-induced TNF-α release and osteoclastogenesis from mice bone marrow macrophages in vitro
Calcineurin/NFAT pathway mediates wear particle-induced TNF-α release and osteoclastogenesis from mice bone marrow macrophages in vitro
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DOI：
10.1038/aps.2013.99
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发表时间：
2013-11-01
影响因子：
8.2
通讯作者：
Tang, Ting-ting
中科院分区：
文献类型：
--
作者：
Liu, Feng-xiang;Wu, Chuan-long;Tang, Ting-ting
目的：探讨钙调神经磷酸酶/活化 T 细胞核因子 (NFAT) 通路在体外调节磨损颗粒诱导的小鼠骨髓巨噬细胞细胞因子释放和破骨细胞生成中的作用。方法：在 100 ng/mL NF-κ B 配体受体激活剂 (RANKL) 存在下，诱导小鼠骨髓巨噬细胞 (BMM) 诱导破骨细胞。吖啶橙染色和MTT法检测细胞活力。使用TRAP染色和RT-PCR测定破骨细胞生成。骨坑吸收测定用于检查破骨细胞表型。使用 RT-PCR 和免疫荧光染色检查 NFATc1 的表达和细胞定位。用ELISA分析TNFα的产生。结果：钛（Ti）或聚甲基丙烯酸甲酯（PMMA）颗粒（0.1mg/mL）没有显着改变BMM的活力，但两次增加BMM向成熟破骨细胞的分化，并显着增加TNF-α的产生。 PMMA组的TNF-α水平显着高于Ti组（96小时）。在存在磨损颗粒和 RANKL 的情况下，在 BMM 中发现了 NFATc1 的表达。在骨坑吸收测定中，磨损颗粒显着增加了 BMM 种子象牙切片中的吸收面积和吸收坑总数。添加11R-VIVIT肽（钙调神经磷酸酶介导的NFAT激活的特异性抑制剂，2.0μmol/L）并没有显着影响BMM的活力，但消除了BMM中几乎所有磨损颗粒引起的改变。此外，VIVIT 在 PMMA 组中比 Ti 组更有效地减少 TNF-α 的产生（96 小时）。结论：钙调神经磷酸酶/NFAT 途径介导磨损颗粒诱导的 TNF-α 释放和 BMM 的破骨细胞生成。用 VIVIT 阻断该信号通路可能为假体周围骨溶解的治疗提供一种有前途的治疗方式。
Aim: To investigate the roles of the calcineurin/nuclear factor of activated T cells (NFAT) pathway in regulation of wear particles-induced cytokine release and osteoclastogenesis from mouse bone marrow macrophages in vitro.Methods: Osteoclasts were induced from mouse bone marrow macrophages (BMMs) in the presence of 100 ng/mL receptor activator of NF-kappa B ligand (RANKL). Acridine orange staining and MTT assay were used to detect the cell viability. Osteoclastogenesis was determined using TRAP staining and RT-PCR. Bone pit resorption assay was used to examine osteoclast phenotype. The expression and cellular localization of NFATc1 were examined using RT-PCR and immunofluorescent staining. The production of TNF alpha was analyzed with ELISA.Results: Titanium (Ti) or polymethylmethacrylate (PMMA) particles (0.1 mg/mL) did not significantly change the viability of BMMs, but twice increased the differentiation of BMMs into mature osteoclasts, and markedly increased TNF-alpha production. The TNF-alpha level in the PMMA group was significantly higher than in the Ti group (96 h). The expression of NFATc1 was found in BMMs in the presence of the wear particles and RANKL. In bone pit resorption assay, the wear particles significantly increased the resorption area and total number of resorption pits in BMMs-seeded ivory slices. Addition of 11R-VIVIT peptide (a specific inhibitor of calcineurin-mediated NFAT activation, 2.0 mu mol/L) did not significantly affect the viability of BMMs, but abolished almost all the wear particle-induced alterations in BMMs. Furthermore, VIVIT reduced TNF-alpha production much more efficiently in the PMMA group than in the Ti group (96 h).Conclusion: Calcineurin/NFAT pathway mediates wear particles-induced TNF-alpha release and osteoclastogenesis from BMMs. Blockade of this signaling pathway with VIVIT may provide a promising therapeutic modality for the treatment of periprosthetic osteolysis.