Differential Effects of Lipopolysaccharide and Tumor Necrosis Factor on Monocytic IκB Kinase Signalsome Activation and IκB Proteolysis

Abstract The inflammatory mediators lipopolysaccharide (LPS) and tumor necrosis factor (TNF) are potent activators of NF-κB. This study compared the effect of these stimuli on endogenous IκB kinase (IKK) signalsome activation and IκB phosphorylation/proteolysis in human monocytic cells and investigated the role of the signalsome proteins IKK-α, IKK-β, NF-κB-inducing kinase (NIK), IKK-γ (NF-κB essential modulator), and IKK complex-associated protein. Kinase assays showed that TNF elicited a rapid but short-lived induction of IKK activity with a 3-fold greater effect on IKK-α than on IKK-β, peaking at 5 min. In contrast, LPS predominantly stimulated IKK-β activity, which slowly increased, peaking at 30 min. A second peak was observed at a later time point following LPS stimulation, which consisted of both IKK-α and -β activity. The endogenous levels of the signalsome components were unaffected by stimulation. Furthermore, our studies showed association of the IKK-α/β heterodimer with NIK, IκB-α and -e in unstimulated cells. Exposure to LPS or TNF led to differential patterns of IκB-α and IκB-e disappearance from and reassembly with the signalsome, whereas IKK-α, IKK-β, and NIK remained complex-associated. NIK cannot phosphorylate IκB-α directly, but it appears to be a functionally important subunit, because mutated NIK inhibited stimulus-induced κB-dependent transcription more effectively than mutated IKK-α or -β. Overexpression of IKK complex-associated protein inhibited stimulus-mediated transcription, whereas NF-κB essential modulator enhanced it. The understanding of LPS- and TNF-induced signaling may allow the development of specific strategies to treat sepsis-associated disease.