Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, 2000 Rosario, Argentina.
Toxicol Appl Pharmacol. 2012 Oct 15;264(2):246-54. doi: 10.1016/j.taap.2012.08.005. Epub 2012 Aug 15.
Human aquaporin-8 (AQP8) channels facilitate the diffusional transport of H(2)O(2) across membranes. Since AQP8 is expressed in hepatic inner mitochondrial membranes, we studied whether mitochondrial AQP8 (mtAQP8) knockdown in human hepatoma HepG2 cells impairs mitochondrial H(2)O(2) release, which may lead to organelle dysfunction and cell death. We confirmed AQP8 expression in HepG2 inner mitochondrial membranes and found that 72h after cell transfection with siRNAs targeting two different regions of the human AQP8 molecule, mtAQP8 protein specifically decreased by around 60% (p<0.05). Studies in isolated mtAQP8-knockdown mitochondria showed that H(2)O(2) release, assessed by Amplex Red, was reduced by about 45% (p<0.05), an effect not observed in digitonin-permeabilized mitochondria. mtAQP8-knockdown cells showed an increase in mitochondrial ROS, assessed by dichlorodihydrofluorescein diacetate (+120%, p<0.05) and loss of mitochondrial membrane potential (-80%, p<0.05), assessed by tetramethylrhodamine-coupled quantitative fluorescence microscopy. The mitochondria-targeted antioxidant MitoTempol prevented ROS accumulation and dissipation of mitochondrial membrane potential. Cyclosporin A, a mitochondrial permeability transition pore blocker, also abolished the mtAQP8 knockdown-induced mitochondrial depolarization. Besides, the loss of viability in mtAQP8 knockdown cells verified by MTT assay, LDH leakage, and trypan blue exclusion test could be prevented by cyclosporin A. Our data on human hepatoma HepG2 cells suggest that mtAQP8 facilitates mitochondrial H(2)O(2) release and that its defective expression causes ROS-induced mitochondrial depolarization via the mitochondrial permeability transition mechanism, and cell death.
人水通道蛋白 8（AQP8）通道促进 H（2）O（2）穿过膜的扩散运输。由于 AQP8 在肝内线粒体膜上表达，我们研究了人肝癌 HepG2 细胞中线粒体 AQP8（mtAQP8）敲低是否会损害线粒体 H（2）O（2）的释放，这可能导致细胞器功能障碍和细胞死亡。我们在 HepG2 内线粒体膜中证实了 AQP8 的表达，并发现在用靶向人 AQP8 分子两个不同区域的 siRNA 转染细胞 72 小时后，mtAQP8 蛋白特异性降低约 60%（p<0.05）。在分离的 mtAQP8 敲低线粒体中进行的研究表明，通过 Amplex Red 评估的 H（2）O（2）释放减少了约 45%（p<0.05），而在二辛可宁酸通透的线粒体中未观察到这种作用。mtAQP8 敲低细胞的线粒体 ROS 增加，通过二氯二氢荧光素二乙酸酯（+120%，p<0.05）评估，线粒体膜电位丧失（-80%，p<0.05），通过四甲基罗丹明耦联定量荧光显微镜评估。线粒体靶向抗氧化剂 MitoTempol 可防止 ROS 积累和线粒体膜电位的耗散。线粒体通透性转换孔阻滞剂环孢菌素 A 也消除了 mtAQP8 敲低诱导的线粒体去极化。此外，MTT 测定、LDH 漏出和台盼蓝排除试验证实的 mtAQP8 敲低细胞活力丧失可通过环孢菌素 A 来预防。我们在人肝癌 HepG2 细胞上的数据表明，mtAQP8 促进线粒体 H（2）O（2）的释放，其表达缺陷通过线粒体通透性转换机制引起 ROS 诱导的线粒体去极化，并导致细胞死亡。
