Knockdown endogenous CypA with siRNA in U2OS cells results in disruption of F-actin structure and alters tumor phenotype
Knockdown endogenous CypA with siRNA in U2OS cells results in disruption of F-actin structure and alters tumor phenotype
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DOI：
10.1007/s11010-008-9896-0
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发表时间：
2009-01-01
影响因子：
4.3
通讯作者：
Chiu, Robert
中科院分区：
文献类型：
--
作者：
Calhoun, Colonya C.;Lu, Ying-Chun;Chiu, Robert
亲环蛋白A （CypA）最初被认为是一种具有肽基脯氨酸异构酶活性的细胞质蛋白。CypA已被证明在免疫应答中起关键作用，但对CypA介导的生物事件的其他分子机制知之甚少。在我们目前的研究中，我们证明了在U2OS细胞中使用RNAi敲低CypA的表达会导致F-actin结构的破坏，以及不依赖锚定的生长、增殖和迁移的减少。经肽基脯氨酸异构酶抑制剂环孢素A （cyclosporine A, CsA）处理的野生型U2OS细胞显示出与敲低CypA细胞相同的表型，这表明CypA异构酶活性是维持正常表型所必需的。体外和体内结合实验表明，CypA与N-WASP结合，N-WASP通过Arp2/3复合物在肌动蛋白成核中起作用。脉冲追踪标记研究表明，缺乏CypA的细胞中N-WASP的降解增强，表明CypA是稳定N-WASP形成N-WASP/Arp2/3复合物以形成F-actin聚合的必要条件。
Cyclophilin A (CypA) was originally identified as a cytosolic protein possessing peptidyl-prolyl isomerase activity. CypA has been shown to play a pivotal role in the immune response, but little is known about other molecular mechanisms of CypA-mediated biologic events. In our present study, we demonstrate that knockdown CypA expression using RNAi in U2OS cells resulted in disruption of the F-actin structure, as well as decreased anchorage-independent growth, proliferation, and migration. Wild-type U2OS cells treated with cyclosporine A (CsA), a peptidyl-prolyl isomerase inhibitor, displayed the same phenotype as knockdown CypA cells, suggesting that the isomerase activity of CypA is required to maintain a normal phenotype. In vitro and in vivo binding assays revealed that CypA binds to N-WASP, which functions in the nucleation of actin via the Arp2/3 complex. Pulse-chase labeling study indicated an enhanced degradation of N-WASP in cell lacking CypA, suggesting that CypA is required for stabilizing N-WASP to form a N-WASP/Arp2/3 complex for the nucleation/initiation of F-actin polymerization.