ATM gene regulates oxygen-glucose deprivation-induced nuclear factor-kappaB DNA-binding activity and downstream apoptotic cascade in mouse cerebrovascular endothelial cells.
OBJECTIVE
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).


METHODS
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.


RESULTS
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.


CONCLUSIONS
We conclude that ATM may play a cytoprotective role in OGD-induced CEC death via a NF-kappaB-dependent signaling pathway.
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D064007:Ataxia Telangiectasia Mutated Proteins; D001921:Brain / Q000098:blood supply*; D020169:Caspases / Q000378:metabolism; D018797:Cell Cycle Proteins; D015687:Cell Hypoxia; D002478:Cells, Cultured; D019610:Cytoprotection / Q000187:drug effects / Q000502:physiology; D004247:DNA / Q000378:metabolism; D004249:DNA Damage / Q000187:drug effects; D004272:DNA, Mitochondrial / Q000378:metabolism; D004268:DNA-Binding Proteins; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D005947:Glucose / Q000172:deficiency / Q000378:metabolism*; D002534:Hypoxia, Brain / Q000378:metabolism*; D051379:Mice; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D025521:Tumor Suppressor Proteins; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology
D018797:Cell Cycle Proteins; D004272:DNA, Mitochondrial; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D016376:Oligonucleotides, Antisense; D010455:Peptides; D011506:Proteins; D012333:RNA, Messenger; C118258:SN50 peptide; D025521:Tumor Suppressor Proteins; D004247:DNA; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases; D020169:Caspases; D005947:Glucose