From the College of Pharmacy, Chung-Ang University, Seoul 156-756 and.
J Biol Chem. 2014 Jan 24;289(4):2469-81. doi: 10.1074/jbc.M113.450163. Epub 2013 Dec 6.
Annexin A5 belongs to a large family of calcium-binding and phospholipid-binding proteins and may act as an endogenous regulator of various pathophysiological processes. There is increasing evidence that annexin A5 is related to cytotoxicity, but the precise function of this protein has yet to be elucidated. In this study, we aimed to verify the function of annexin A5 in the apoptosis of renal epithelial cells. Real-time PCR and Western blot analysis, together with immunofluorescence analysis, showed that the expression of annexin A5 significantly increased in the presence of cisplatin in both human and rat renal epithelial cells. With regard to the mechanism of cisplatin-induced apoptosis, apoptosis-inducing factor (AIF) release into the cytosol was observed, and the underlying mechanism was identified as voltage-dependent anion channel (VDAC) oligomerization. Mitochondrial membrane potential (Δψm) was found to be greatly disrupted in cisplatin-treated cells. Moreover, cisplatin strongly induced translocation of annexin A5 into mitochondria. To understand the functional significance of annexin A5 in renal cell death, we used a siRNA-mediated approach to knock down annexin A5. Annexin A5 depletion by siRNA led to decreased annexin A5 translocation into mitochondria and significantly reduced VDAC oligomerization and AIF release. Annexin A5 siRNA also increased cell viability compared with the control. Moreover, expression of annexin A5 was induced by other nephrotoxicants such as CdCl2 and bacitracin. Taken together, our data suggest that annexin A5 may play a crucial role in cisplatin-induced toxicity by mediating the mitochondrial apoptotic pathway via the induction and oligomerization of VDAC.
annexin A5 属于钙结合和磷脂结合蛋白大家族，可能作为各种病理生理过程的内源性调节剂发挥作用。越来越多的证据表明 annexin A5 与细胞毒性有关，但该蛋白的确切功能仍有待阐明。在本研究中，我们旨在验证 annexin A5 在肾上皮细胞凋亡中的作用。实时 PCR 和 Western blot 分析以及免疫荧光分析表明， annexin A5 在人肾上皮细胞和大鼠肾上皮细胞中顺铂存在的情况下表达显著增加。关于顺铂诱导凋亡的机制，观察到凋亡诱导因子（AIF）释放到细胞质中，其潜在机制被鉴定为电压依赖性阴离子通道（VDAC）寡聚化。发现顺铂处理的细胞中线粒体膜电位（Δψm）严重破坏。此外，顺铂强烈诱导 annexin A5 向线粒体易位。为了了解 annexin A5 在肾细胞死亡中的功能意义，我们使用 siRNA 介导的方法敲低 annexin A5。siRNA 敲低 annexin A5 导致 annexin A5 向线粒体的易位减少，VDAC 寡聚化和 AIF 释放明显减少。与对照组相比，annexin A5 siRNA 还增加了细胞活力。此外，其他肾毒性物质如 CdCl2 和杆菌肽也诱导 annexin A5 的表达。总之，我们的数据表明，annexin A5 可能通过诱导和寡聚化 VDAC 介导线粒体凋亡途径，在顺铂诱导的毒性中发挥关键作用。
