Role of oxidant stress in cytokine-induced activation of NF-κB in human aortic smooth muscle cells

The transcription factor nuclear factor-kappa B (NF-kappa B) has been implicated in inflammatory and proliferative vascular mechanisms. Activated NF-kappa B 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-kappa B 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-kappa B activation. Western blot analysis revealed the presence of inhibitory protein I-kappa B alpha in resting human aortic SMC, which was rapidly phosphorylated and degraded on exposure to interleukin-1 beta (IL-1 beta) followed by NF-kappa B translocation to the nucleus. IL-1 beta 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 H2O2-induced DCF fluorescence, had no effect on cytokine-induced NF-kappa B activation. In contrast to NAG, the metal chelators pyrrolidine dithiocarbamate and diethyldithiocarbamate attenuated IL-1 beta-induced NF-kappa B activation but had no effect on intracellular sulfhydryl content. Treatment of the cells with the oxidant H2O2 caused an increase in DCF fluorescence and decreased intracellular sulfhydryl content but had no effect on I-kappa B alpha or NF-kappa B. In conclusion, this study suggests that oxidant stress may not play a major role in cytokine-induced activation of NF-kappa B in human aortic SMC and that oxidants may not be primary activators of NF-kappa B in these cells.