Shi Hui, Williams Jennifer A E, Guo Li, Stampoulis Dimitrios, Francesca Cordeiro M, Moss Stephen E
Department of Cell Biology, UCL Institute of Ophthalmology, 11-43 Bath Street, London, EC1V 9EL, UK.
Apoptosis. 2015 Apr;20(4):433-43. doi: 10.1007/s10495-015-1091-7.
The loss of photoreceptors is the defining characteristic of many retinal degenerative diseases, but the mechanisms that regulate photoreceptor cell death are not fully understood. Here we have used the 661W cone photoreceptor cell line to ask whether exposure to the terminal complement complex C5b-9 induces cell death and/or modulates the sensitivity of these cells to other cellular stressors. 661W cone photoreceptors were exposed to complete normal human serum following antibody blockade of CD59. Apoptosis induction was assessed morphologically, by flow cytometry, and on western blotting by probing for cleaved PARP and activated caspase-3. Necroptosis was assessed by flow cytometry and Sirtuin 2 inhibition using 2-cyano-3-[5-(2,5-dichlorophenyl)-2-furyl]-N-5-quinolinylacrylamide (AGK2). The sensitivity of 661W cells to ionomycin, staurosporine, peroxide and chelerythrine was also investigated, with or without prior formation of C5b-9. 661W cells underwent apoptotic cell death following exposure to C5b-9, as judged by poly(ADP-ribose) polymerase 1 cleavage and activation of caspase-3. We also observed apoptotic cell death in response to staurosporine, but 661W cells were resistant to both ionomycin and peroxide. Interestingly, C5b-9 significantly increased 661W sensitivity to staurosporine-induced apoptosis and necroptosis. These studies show that low levels of C5b-9 on 661W cells can induce apoptosis, and that C5b-9 specifically sensitizes 661W cells to certain apoptotic and necroptotic pathways. Our observations provide new insight into the potential role of the complement system in photoreceptor loss, with implications for the molecular aetiology of retinal disease.
光感受器的丧失是许多视网膜退行性疾病的决定性特征，但调节光感受器细胞死亡的机制尚未完全明确。在此，我们利用661W视锥光感受器细胞系来研究暴露于终末补体复合物C5b - 9是否会诱导细胞死亡和/或调节这些细胞对其他细胞应激源的敏感性。在对CD59进行抗体阻断后，将661W视锥光感受器暴露于完全正常人血清中。通过形态学、流式细胞术以及蛋白质免疫印迹法检测裂解的PARP和活化的caspase - 3来评估凋亡诱导情况。通过流式细胞术和使用2 - 氰基 - 3 - [5 - (2,5 - 二氯苯基) - 2 - 呋喃基] - N - 5 - 喹啉基丙烯酰胺（AGK2）抑制沉默调节蛋白2来评估坏死性凋亡。还研究了661W细胞在有无预先形成C5b - 9的情况下对离子霉素、星形孢菌素、过氧化物和白屈菜红碱的敏感性。根据聚（ADP - 核糖）聚合酶1的裂解和caspase - 3的活化判断，661W细胞在暴露于C5b - 9后发生凋亡性细胞死亡。我们还观察到661W细胞对星形孢菌素也有凋亡性细胞死亡反应，但对离子霉素和过氧化物均有抗性。有趣的是，C5b - 9显著增加了661W细胞对星形孢菌素诱导的凋亡和坏死性凋亡的敏感性。这些研究表明，661W细胞上低水平的C5b - 9可诱导凋亡，并且C5b - 9特异性地使661W细胞对某些凋亡和坏死性凋亡途径敏感。我们的观察结果为补体系统在光感受器丧失中的潜在作用提供了新的见解，对视网膜疾病的分子病因学具有重要意义。
