Role of oxidant stress in cytokine-induced activation of NF-kappaB in human aortic smooth muscle cells.
The transcription factor nuclear factor-kappaB (NF-kappaB) has been implicated in inflammatory and proliferative vascular mechanisms. Activated NF-kappaB has been documented in human atherosclerotic lesions, and its activation in human vascular smooth muscle cells (SMC) by cytokines has been reported. However, intracellular mechanisms mediating NF-kappaB activation in human SMC are poorly understood. The aim of this study was to explore the potential role of reactive oxygen species and oxidant stress as signaling events in cytokine-induced NF-kappaB activation. Western blot analysis revealed the presence of inhibitory protein I-kappaBalpha in resting human aortic SMC, which was rapidly phosphorylated and degraded on exposure to interleukin-1beta (IL-1beta) followed by NF-kappaB translocation to the nucleus. IL-1beta had no effect on two measures of intracellular oxidant stress, fluorescence generated by the oxidation of 2',7'-dichlorodihydrofluorescin to dichlorofluorescein (DCF) or changes in intracellular sulfhydryl content. N-acetylcysteine (NAC) a membrane-permeant antioxidant, which augmented intracellular sulfhydryl content and inhibited H(2)O(2)-induced DCF fluorescence, had no effect on cytokine-induced NF-kappaB activation. In contrast to NAC, the metal chelators pyrrolidine dithiocarbamate and diethyldithiocarbamate attenuated IL-1beta-induced NF-kappaB activation but had no effect on intracellular sulfhydryl content. Treatment of the cells with the oxidant H(2)O(2) caused an increase in DCF fluorescence and decreased intracellular sulfhydryl content but had no effect on I-kappaBalpha or NF-kappaB. In conclusion, this study suggests that oxidant stress may not play a major role in cytokine-induced activation of NF-kappaB in human aortic SMC and that oxidants may not be primary activators of NF-kappaB in these cells.
Hoare
G S
GS
Department of Cardiothoracic Surgery, National Heart and Lung Institute, Imperial College of Science, Harefield, Middlesex UB9 6JH, UK.
Marczin
N
N
Chester
A H
AH
Yacoub
M H
MH
D000975:Antioxidants / Q000494:pharmacology; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D005434:Flow Cytometry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000378:metabolism; D008670:Metals / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000502:physiology*; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress / Q000502:physiology*; D013438:Sulfhydryl Compounds / Q000378:metabolism
D000975:Antioxidants; D002614:Chelating Agents; D007375:Interleukin-1; D008670:Metals; D016328:NF-kappa B; D016877:Oxidants; D013438:Sulfhydryl Compounds; D006861:Hydrogen Peroxide