Wu Zhihao, Rogers Brian, Kachi Shu, Hackett Sean F, Sick Anna, Campochiaro Peter A
Departments of Ophthalmology and Neuroscience, Johns Hopkins University School of Medicine, 600 N. Wolfe Street, Baltimore, MD 21287, USA.
J Cell Physiol. 2006 Dec;209(3):996-1005. doi: 10.1002/jcp.20819.
The largest isoform of the Shc adapter protein, p66Shc, has been implicated in oxidative damage-induced apoptosis in vital organs, because mice deficient in p66Shc have a 30% increase in life span and are resistant to the lethal effects of systemically administered paraquat, a source of severe oxidative damage. In this study, we utilized siRNA directed against the CH2 domain of Shc, to reduce p66Shc, but not p52Shc nor p46Shc in retinal pigmented epithelial (RPE) cells. RPE cells deficient in p66Shc had reduced susceptibility to oxidative stress-induced apoptosis. Compared to control cells, those with reduced p66Shc had increased basal and oxidative stress-induced NF-kappaB transcriptional activity, increased levels of antioxidant enzymes, and less generation of reactive oxygen species when challenged with H(2)O(2). The increase in oxidative stress-induced NF-kappaB activity was mediated by activation of ERK. Compared to eyes injected with GFP siRNA, those injected with p66Shc siRNA showed less loss of retinal function as assessed by electroretinograms from paraquat-induced oxidative stress. These data suggest that p66Shc and molecular signals involved in its regulation provide therapeutic targets for retinal degenerations in which oxidative-damage plays a major role, including age-related macular degeneration and cone cell death in retinitis pigmentosa.
Shc衔接蛋白的最大异构体p66Shc与重要器官中氧化损伤诱导的细胞凋亡有关，因为缺乏p66Shc的小鼠寿命延长30%，并且对全身给予百草枯（一种严重氧化损伤源）的致死作用具有抗性。在本研究中，我们利用针对Shc的CH2结构域的小干扰RNA（siRNA），以减少视网膜色素上皮（RPE）细胞中的p66Shc，但不减少p52Shc和p46Shc。缺乏p66Shc的RPE细胞对氧化应激诱导的细胞凋亡的敏感性降低。与对照细胞相比，p66Shc减少的细胞具有增加的基础和氧化应激诱导的核因子κB（NF-κB）转录活性、增加的抗氧化酶水平，并且在用过氧化氢（H₂O₂）刺激时产生的活性氧物种较少。氧化应激诱导的NF-κB活性增加是由细胞外信号调节激酶（ERK）的激活介导的。与注射绿色荧光蛋白（GFP）siRNA的眼睛相比，注射p66Shc siRNA的眼睛在百草枯诱导的氧化应激后通过视网膜电图评估显示出较少的视网膜功能丧失。这些数据表明，p66Shc及其调控所涉及的分子信号为视网膜变性提供了治疗靶点，其中氧化损伤起主要作用，包括年龄相关性黄斑变性和色素性视网膜炎中的视锥细胞死亡。
