Yin Ke-jie, Chen Shang-Der, Lee Jin-Moo, Xu Jan, Hsu Chung Y
Department of Neurology and Center for the Study of Nervous System Injury, Washington University School of Medicine, St Louis, Mo 63110, USA.
Stroke. 2002 Oct;33(10):2471-7. doi: 10.1161/01.str.0000030316.79601.03.
Cells lacking the ATM (ataxia telangectasia mutated) gene are hypersensitive to DNA damage caused by a variety of insults. ATM may regulate oxidative stress-induced signaling cascades involving nuclear factor-kappaB (NF-kappaB), a transcription factor that is upstream of a wide variety of stress-responsive genes. We investigated the potential interaction of ATM and NF-kappaB after oxygen-glucose deprivation (OGD) in cerebral endothelial cells (CECs).
Primary cultures of mouse CECs were subjected to OGD in the absence or presence of ATM antisense oligonucleotides or the NF-kappaB inhibitor SN50. ATM expression was determined with the use of reverse transcription-polymerase chain reaction and Western blot, and NF-kappaB activity was assessed by electrophoretic mobility shift assay. Cells were assessed for mitochondrial DNA damage with the use of long polymerase chain reaction and were assessed for caspase-3 and caspase-8 activity with the use of fluorogenic substrates. Cell death was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide and LDH release.
OGD stimulated ATM gene expression at the mRNA and protein level in CECs as early as 1 hour after OGD initiation. ATM gene knockdown with the use of an antisense oligonucleotide suppressed OGD-induced ATM protein expression, which was accompanied by an attenuation of NF-kappaB activation and the subsequent expression of downstream genes, including the antiapoptotic gene c-IAP2. ATM knockdown also accentuated OGD-induced mitochondrial DNA damage and the activation of caspase-3 and caspase-8, leading to enhanced CEC death. The specific NF-kappaB inhibitor SN50 mimicked the effects of ATM knockdown.
We conclude that ATM may play a cytoprotective role in OGD-induced CEC death via a NF-kappaB-dependent signaling pathway.
缺乏ATM（共济失调毛细血管扩张症突变基因）的细胞对多种损伤所导致的DNA损伤高度敏感。ATM可能调节涉及核因子-κB（NF-κB）的氧化应激诱导信号级联反应，NF-κB是多种应激反应基因上游的一种转录因子。我们研究了脑内皮细胞（CECs）在氧糖剥夺（OGD）后ATM与NF-κB之间的潜在相互作用。
对原代培养的小鼠CECs在存在或不存在ATM反义寡核苷酸或NF-κB抑制剂SN50的情况下进行OGD处理。通过逆转录-聚合酶链反应和蛋白质印迹法测定ATM表达，通过电泳迁移率变动分析评估NF-κB活性。使用长聚合酶链反应评估细胞的线粒体DNA损伤，使用荧光底物评估caspase-3和caspase-8活性。通过3-(4,5-二甲基噻唑-2-基)-2,5-二苯基溴化四氮唑和乳酸脱氢酶释放来确定细胞死亡。
OGD最早在开始后1小时就刺激了CECs中ATM基因在mRNA和蛋白质水平的表达。使用反义寡核苷酸敲低ATM基因可抑制OGD诱导的ATM蛋白表达，这伴随着NF-κB激活的减弱以及包括抗凋亡基因c-IAP2在内的下游基因的随后表达。敲低ATM还加剧了OGD诱导的线粒体DNA损伤以及caspase-3和caspase-8的激活，导致CECs死亡增加。特异性NF-κB抑制剂SN50模拟了敲低ATM的作用。
我们得出结论，ATM可能通过NF-κB依赖性信号通路在OGD诱导的CECs死亡中发挥细胞保护作用。
