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Decreased oxidant buffering impairs NF-kappaB activation and ICAM-1 transcription in endothelial cells
1Department of Pharmacology, The University of Illinois College of Medicine, Chicago 60612, USA.
Insights
Endothelial cell redox state impacts NF-kappaB activity. Glutathione depletion impairs TNFalpha-induced NF-kappaB binding and ICAM-1 expression, highlighting the role of oxidative modification in regulating this pathway.
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- Nuclear factor kappa B (NF-kappaB) is a transcription factor regulated by its inhibitor, IkappaB.
- Redox reactions, specifically involving cysteine residues, are known post-translational modifications affecting NF-kappaB activity.
- Endothelial cell redox status is crucial for cellular signaling pathways.
Purpose of the Study:
- To investigate the role of endothelial cell redox state in modulating tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activity.
- To examine the impact of reduced intracellular glutathione (GSH) on NF-kappaB DNA-binding and ICAM-1 gene expression.
Main Methods:
- Human pulmonary artery endothelial (HPAE) cells were treated with buthionine sulfoximine (BSO) to deplete GSH.
- TNFalpha was used to stimulate NF-kappaB activity and ICAM-1 mRNA expression in both control and GSH-depleted cells.
- The effect of the reducing agent dithiothreitol (DTT) on NF-kappaB activity was assessed.
Main Results:
- TNFalpha stimulation in control cells induced reactive oxygen species (ROS) generation, NF-kappaB binding, and ICAM-1 transcription.
- GSH depletion led to ROS accumulation and significantly impaired TNFalpha-induced NF-kappaB binding and ICAM-1 mRNA expression.
- DTT treatment restored or augmented NF-kappaB binding and ICAM-1 expression in GSH-depleted cells.
Conclusions:
- The redox state of endothelial cells, influenced by GSH levels, critically regulates TNFalpha-induced NF-kappaB activation.
- Oxidative modification of NF-kappaB, resulting from compromised ROS buffering, plays a key role in modulating its DNA-binding to the ICAM-1 promoter.
- These findings suggest a mechanism by which cellular redox balance controls inflammatory gene transcription in endothelial cells.
Abstract:
The DNA binding activity of the transcription factor, NF-kappaB, is regulated by the phosphorylation and degradation of its inhibitory protein, IkappaB, and post-translational modification involving redox reaction of a cysteine residue (Cys62) of NF-kappaB. We addressed the role of the redox state of endothelial cells in modulating TNFalpha-induced NF-kappaB activity. The effects of TNFalpha on DNA-binding activity of NF-kappaB and expression of mRNA encoding ICAM-1 (an NF-kappaB-activated gene) were studied in human pulmonary artery endothelial (HPAE) cells under basal conditions and after decreasing the intracellular glutathione (GSH) concentration. HPAE cells were treated with buthionine sulfoximine (BSO) (16 h), an inhibitor of GSH synthesis, which caused concentration-dependent decreases in GSH concentration. Stimulation of control cells with TNFalpha resulted in reactive oxygen species (ROS) generation and activation of NF-kappaB binding to the ICAM-1 promoter and ICAM-1 transcription. However, stimulation of GSH-depleted cells with TNFalpha resulted in ROS accumulation secondary to the decreased ROS buffering capacity, and marked impairment of NF-kappaB-binding activity and ICAM-1 mRNA expression. Exposure of BSO-treated cells to the reducing agent dithiothreitol (DTT) before TNFalpha treatment or supplementation of nuclear extract (isolated after TNFalpha challenge of BSO-treated cells) with DTT significantly augmented the effect of TNFalpha on NF-kappaB-binding activity and ICAM-1 mRNA expression. Thus the oxidative modification of NF-kappaB secondary to the loss of ROS buffering capacity may regulate NF-kappaB binding to ICAM-1 promoter, and thereby ICAM-1 transcription in endothelial cells.