| Abstrakt: |
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 -ε in unstimulated cells. Exposure to LPS or TNF led to differential patterns of IκB-α and IκB-ε 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. |
