Inhibition of wild-type p66ShcA in mesangial cells prevents glycooxidant-dependent FOXO3a regulation and promotes the survival phenotype
Inhibition of wild-type p66ShcA in mesangial cells prevents glycooxidant-dependent FOXO3a regulation and promotes the survival phenotype
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DOI：
10.1152/ajprenal.00215.2006
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发表时间：
2007-02-01
影响因子：
4.2
通讯作者：
Meggs, Leonard G.
中科院分区：
文献类型：
--
作者：
Chintapalli, Janaki;Yang, Shuo;Meggs, Leonard G.
高血糖会引发细胞水平的活性氧（ROS）呈指数增加。在这里，我们证明了通过沉默野生型 (WT) p66ShcA 基因诱导系膜细胞中的抗氧化剂表型。采用两种方法抑制 SV40 鼠系膜细胞和正常人系膜细胞中的 WTp66ShcA：用异构体特异性 p66ShcA 短间插 RNA 瞬时转染和用突变型 36 p66ShcA 表达载体稳定转染。在高环境葡萄糖 (HG) 下，p66ShcA 缺陷细胞表现出对 HG 诱导的 ROS 生成的抵抗力，并且细胞内 ROS 代谢动力学曲线振幅减弱，表明 WTp66ShcA 在 HG 氧化应激产生中的关键作用。接下来我们检查了 FKHRL1 (FOXO3a) 的磷酸化和亚细胞分布，FKHRL1 是一种有效的应激反应调节剂，也是 WTp66ShcA 氧化还原功能的下游靶标。在 HG，通过免疫印迹分析的 p66ShcA 缺陷细胞的细胞提取物显示 FOXO3a Thr-32 磷酸化减弱，并且与 HA-FOXO3a 共转染的 p66ShcA 缺陷细胞的间接免疫荧光显示主要的 HA-FOXO3a 核定位。相反，HG 的亲代细胞显示磷酸 Thr-32 的上调和 HA-FOXO3a 的核输出。为了确定抑制 WTp66ShcA 和 FOXO3a 之间的串扰是否可以提供针对氧化剂诱导的 DNA 损伤的保护，检查了 DNA 链断裂 (DSB) 和细胞凋亡。在 HG 中，p66ShcA 缺陷细胞表现出对 DSB 和细胞凋亡的抵抗力增强，而亲本细胞则在这两个参数上表现出显着增加。我们得出结论，WTp66ShcA 氧化还原功能的敲低可阻止 HG 依赖性 FOXO3a 调节并促进生存表型。
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.