Differential effects of lipopolysaccharide and tumor necrosis factor on monocytic I kappa B kinase signalsome activation and I kappa B proteolysis

The inflammatory mediators lipopolysaccharide (LPS) and tumor necrosis fact or (TNF) are potent activators of NF-kappa B, This study compared the effec t of these stimuli on endogenous I kappa B kinase (IKK) signalsome activati on and I kappa B phosphorylation/proteolysis in human monocytic cells and i nvestigated the role of the signal-some proteins IKK-alpha, IKK-beta, NF-ka ppa B-inducing kinase (NIK), IKK-gamma (NF-kappa B essential modulator), an d IKK complex-associated protein. Kinase assays showed that TNF elicited a rapid but short-lived induction of IKK activity with a 3-fold greater effec t on IKK-alpha than on IKK-beta, peaking at 5 min. In contrast, LPS predomi nantly stimulated IKK-beta activity, which slowly increased, peaking at 30 min. A second peak was observed at a later time point following LPS stimula tion, which consisted of both IKK-alpha and -beta activity. The endogenous levels of the signalsome components were unaffected by stimulation. Further more, our studies showed association of the IKK-alpha/beta heterodimer with NIK, I kappa B-alpha and -epsilon in unstimulated cells. Exposure to LPS o r 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 k appa B-alpha directly, but it appears to be a functionally important subuni t, because mutated NIK inhibited stimulus-induced kappa B-dependent transcr iption more effectively than mutated IKK-alpha or -beta, Overexpression of IKK complex-associated protein inhibited stimulus-mediated transcription, w hereas NF-kappa B essential modulator enhanced it. The understanding of LPS - and TNF-induced signaling may allow the development of specific strategie s to treat sepsis-associated disease.