Chintapalli Janaki, Yang Shuo, Opawumi David, Goyal Sunita Ray, Shamsuddin Nazia, Malhotra Ashwani, Reiss Krzysztof, Meggs Leonard G
Center for Neurovirology and Cancer Biology, Temple University, Philadelphia, Pennsylvania, USA.
Am J Physiol Renal Physiol. 2007 Feb;292(2):F523-30. doi: 10.1152/ajprenal.00215.2006. Epub 2006 Oct 31.
Hyperglycemia triggers an exponential increase in reactive oxygen species (ROS) at the cellular level. Here, we demonstrate induction of the oxidant-resistant phenotype in mesangial cells by silencing the wild-type (WT) p66ShcA gene. Two approaches were employed to inhibit WTp66ShcA in SV40 murine mesangial cells and normal human mesangial cells: transient transfection with isoform-specific p66ShcA short-intervening RNA and stable transfection with mutant 36 p66ShcA expression vector. At high ambient glucose (HG), p66ShcA-deficient cells exhibit resistance to HG-induced ROS generation and attenuation in the amplitude of the kinetic curves for intracellular ROS metabolism, indicative of the pivotal role of WTp66ShcA in the generation of HG oxidant stress. We next examined phosphorylation and subcellular distribution of FKHRL1 (FOXO3a), a potent stress response regulator and downstream target of WTp66ShcA redox function. At HG, cell extracts of p66ShcA-deficient cells analyzed by immunoblotting show attenuation of FOXO3a phosphorylation at Thr-32, and indirect immunofluorescence of p66ShcA-deficient cells, cotransfected with HA-FOXO3a, show predominant HA-FOXO3a nuclear localization. Conversely, parental cells at HG show upregulation of phos-Thr-32 and nuclear export of HA-FOXO3a. To determine whether inhibition of cross talk between WTp66ShcA and FOXO3a confers protection against oxidant-induced DNA damage, DNA strand breaks (DSB) and apoptosis were examined. At HG, p66ShcA-deficient cells exhibit increased resistance to DSB and apoptosis, while parental cells show a striking increase in both parameters. We conclude that knockdown of WTp66ShcA redox function prevents HG-dependent FOXO3a regulation and promotes the survival phenotype.
高血糖在细胞水平上引发活性氧（ROS）呈指数级增加。在此，我们通过沉默野生型（WT）p66ShcA基因，证明了在系膜细胞中诱导抗氧化表型。采用了两种方法来抑制SV40小鼠系膜细胞和正常人系膜细胞中的WTp66ShcA：用异构体特异性p66ShcA短干扰RNA进行瞬时转染，以及用突变型36 p66ShcA表达载体进行稳定转染。在高环境葡萄糖（HG）条件下，缺乏p66ShcA的细胞对HG诱导的ROS生成具有抗性，并且细胞内ROS代谢动力学曲线的幅度减弱，这表明WTp66ShcA在HG氧化应激的产生中起关键作用。接下来，我们研究了FKHRL1（FOXO3a）的磷酸化和亚细胞分布，FKHRL1是一种强大的应激反应调节因子，也是WTp66ShcA氧化还原功能的下游靶点。在HG条件下，通过免疫印迹分析缺乏p66ShcA的细胞提取物，显示Thr-32处FOXO3a磷酸化减弱，并且与HA-FOXO3a共转染的缺乏p66ShcA的细胞的间接免疫荧光显示HA-FOXO3a主要定位于细胞核。相反，HG条件下的亲本细胞显示磷酸化Thr-32上调以及HA-FOXO3a的核输出。为了确定WTp66ShcA与FOXO3a之间的相互作用抑制是否赋予对氧化诱导的DNA损伤的保护作用，检测了DNA链断裂（DSB）和细胞凋亡。在HG条件下，缺乏p66ShcA的细胞对DSB和细胞凋亡的抗性增加，而亲本细胞在这两个参数上均显著增加。我们得出结论，WTp66ShcA氧化还原功能的敲低可防止HG依赖性FOXO3a调节并促进存活表型。
