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ATM gene regulates oxygen-glucose deprivation-induced nuclear factor-kappaB DNA-binding activity and downstream
Ke-jie Yin1, Shang-Der Chen, Jin-Moo Lee
1Department of Neurology and Center for the Study of Nervous System Injury, Washington University School of Medicine, St Louis, Mo 63110, USA.
Background And Purpose:
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.
Insights
Ataxia telangectasia mutated (ATM) protects cerebral endothelial cells from oxygen-glucose deprivation (OGD) damage. ATM regulates nuclear factor-kappaB (NF-kappaB) signaling, which is crucial for cell survival during oxidative stress.
Area of Science:
- Neuroscience
- Molecular Biology
- Cell Biology
Background:
- Cells lacking ATM are sensitive to DNA damage.
- ATM may regulate NF-kappaB signaling in response to oxidative stress.
- The interaction between ATM and NF-kappaB in cerebral endothelial cells (CECs) during oxygen-glucose deprivation (OGD) is not well understood.
Purpose of the Study:
- To investigate the interaction between ATM and NF-kappaB in CECs following OGD.
- To determine the role of ATM in OGD-induced cell death pathways.
Main Methods:
- Primary mouse CECs were subjected to OGD with or without ATM antisense oligonucleotides or NF-kappaB inhibitor SN50.
- ATM expression and NF-kappaB activity were assessed using molecular biology techniques.
- Mitochondrial DNA damage, caspase activation, and cell death were measured.
Main Results:
- OGD increased ATM gene and protein expression in CECs.
- ATM knockdown attenuated NF-kappaB activation and downstream gene expression, including antiapoptotic c-IAP2.
- ATM knockdown exacerbated OGD-induced mitochondrial DNA damage, caspase activation, and cell death, similar to NF-kappaB inhibition.
Conclusions:
- ATM plays a cytoprotective role in OGD-induced CEC death.
- This protective effect is mediated through an NF-kappaB-dependent signaling pathway.