Inhibition of IκB Kinase Activity by Sodium Salicylate in Vitro Does Not Reflect Its Inhibitory Mechanism in Intact Cells

Sodium salicylate inhibits activation of the transcription factor NF-κB by blocking the phosphorylation and degradation of the NF-κB inhibitor IκBα. We previously demonstrated that salicylate inhibits IκBα degradation induced by tumor necrosis factor (TNF) but not by interleukin-1 (IL-1) and implicated p38 mitogen-activated protein kinase activation by salicylate in the inhibition of TNF-induced IκBα phosphorylation. Both TNF and IL-1 rapidly activate the IκB kinase (IKK) complex, containing the catalytic subunits IKKα and IKKβ, which directly phosphorylates IκB proteins. Others have recently suggested that salicylate inhibits NF-κB activation by directly binding to IKKβ. To clarify the mechanism whereby salicylate inhibits IKK activity, we examined its effects upon cytokine-induced IKK activity in intact cells and in vitro. Treatment of intact cells with salicylate inhibited TNF-induced but not IL-1-induced IKK activity, and this inhibition was prevented by the p38 inhibitor SB203580. In contrast, inhibition of IKK activity by salicylate in vitro was neither selective for TNF nor affected by SB203580. In vitro, salicylate treatment comparably inhibited the kinase activity of overexpressed IKKα and IKKβ and also decreased p38 kinase activity. Therefore, direct inhibition of IKK activity in vitro does not reflect the inhibitory mechanism of salicylate in intact cells, which involves interference with TNF signaling.