Differential effects of lipopolysaccharide and tumor necrosis factor on monocytic IκB kinase signalsome activation and IκB proteolysis

The inflammatory mediators lipopolysaccharide (LPS) and tumor necrosis factor (TNF) are potent activators of NF-kappa B, This study compared the effect of these stimuli on endogenous I kappa B kinase (IKK) signalsome activation and I kappa B phosphorylation/proteolysis in human monocytic cells and investigated the role of the signal-some proteins IKK-alpha, IKK-beta, NF-kappa B-inducing kinase (NIK), IKK-gamma (NF-kappa B 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-alpha than on IKK-beta, peaking at 5 min. In contrast, LPS predominantly stimulated IKK-beta 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-alpha and -beta activity. The endogenous levels of the signalsome components were unaffected by stimulation. Furthermore, our studies showed association of the IKK-alpha/beta heterodimer with NIK, I kappa B-alpha and -epsilon in unstimulated cells. Exposure to LPS or TNF led to differential patterns of I kappa B-alpha and I kappa B-epsilon disappearance from and reassembly with the signalsome, whereas IKK-alpha, IKK-beta, and NIK remained complex-associated. NIK cannot phosphorylate I kappa B-alpha directly, but it appears to be a functionally important subunit, because mutated NIK inhibited stimulus-induced kappa B-dependent transcription more effectively than mutated IKK-alpha or -beta, Overexpression of IKK complex-associated protein inhibited stimulus-mediated transcription, whereas NF-kappa B essential modulator enhanced it. The understanding of LPS- and TNF-induced signaling may allow the development of specific strategies to treat sepsis-associated disease.