Cyclophilin C-associated protein regulation of phagocytic functions via NFAT activation in macrophages
Cyclophilin C-associated protein regulation of phagocytic functions via NFAT activation in macrophages
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亲环蛋白 C 相关蛋白通过巨噬细胞中 NFAT 激活调节吞噬功能
DOI：
10.1016/j.brainres.2011.03.036
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发表时间：
2011
期刊：
影响因子：
2.9
通讯作者：
Takeuchi T.
中科院分区：
文献类型：
--
作者：
Yamaguchi R;Hosaka M;Torii S;Hou N;Saito N;Yoshimoto Y;Imai H;Takeuchi T.
免疫抑制剂环孢菌素A（CsA）可减轻实验性脑缺血。亲环素C相关蛋白（Cyclophilin C-associated protein，CyCAP）被认为是CsA的内源性等价物，以CsA和CyCAP为靶点的亲环素C在缓解缺血方面的作用机制引起了广泛关注。本研究采用CyCAP特异性抗体，观察了CyCAP在大鼠大脑中动脉闭塞后缺血脑内的分布。在大鼠脑缺血恢复过程中，CyCAP在缺血损伤区活化的小胶质细胞/巨噬细胞中高表达。然而，它仍然是未知的CyCAP在激活巨噬细胞吞噬作用中发挥的作用。因此，我们使用RAW 264.7巨噬细胞系研究了CyCAP功能。当我们在RAW264.7细胞中表达CyCAP-GFP和亲环素C-FLAG时，我们发现CyCAP和亲环素C形成复合物，该复合物被CsA竞争性抑制。在抗钙调神经磷酸酶抗体免疫沉淀物中检测到亲环素C和CyCAP，并且CyCAP拉低NFATc 1，表明CyCAP和亲环素C与钙调神经磷酸酶和NFATc 1形成复合物。当CyCAP在RAW细胞中腺病毒过表达时，NFAT染色在细胞核上增加。此外，钙调神经磷酸酶和IL-2随时间增加。因此，CyCAP似乎通过激活NFAT和所得IL-2产生来控制巨噬细胞功能。用蛋白磷酸酶抑制剂PhoSTOP，NFAT更多地定位于细胞质，吞噬作用显著降低。因此，我们认为，在CyCAP-亲环蛋白C途径的巨噬细胞活化，钙调磷酸酶活性是必不可少的吞噬活性，通过去磷酸化的NFATc 1。
Experimental cerebral ischemia has been reportedly alleviated by the immunosuppressive agent cyclosporin A (CsA). Cyclophilin C-associated protein (CyCAP) was proposed to be an endogenous equivalent of CsA; CsA- and CyCAP-targeting protein cyclophilin C have attracted extensive attention regarding their ischemia-alleviating mechanisms. In this study we have introduced the specific CyCAP antibody for evaluating its distribution in the rat ischemic brain after middle cerebral artery occlusion. During the recovery of cerebral ischemia in rats, CyCAP was highly expressed in the activated microglia/macrophages in the ischemic lesion. However, it remains unknown what roles CyCAP plays in the activation of macrophage phagocytosis. Thus, we studied CyCAP function using a RAW264.7 macrophage cell line. When we expressed CyCAP-GFP and cyclophilin C-FLAG in RAW264.7 cells, we found that CyCAP and cyclophilin C make a complex, which is competitively inhibited by CsA. Consistently, in immunoprecipitates by anti-calcineurin antibody, cyclophilin C and CyCAP were detected, and CyCAP pulled down NFATc1, suggesting that both CyCAP and cyclophilin C form a complex with calcineurin and NFATc1. When CyCAP was adenovirally overexpressed in RAW cells, NFAT staining increased over the nucleus. Furthermore, calcineurin and IL-2 were increased with time. Thus, CyCAP appears to control macrophage functions by activating NFAT and the resultant IL-2 production. With a protein phosphatase inhibitor PhoSTOP, NFAT was localized more to the cytoplasm, and phagocytosis was decreased strikingly. Thus, we suggest that in a CyCAP-cyclophilin C pathway for macrophage activation, calcineurin phosphatase activity is essential for the phagocytosis activity via dephosphorylation of NFATc1.