Abstract
The inflammatory mediators lipopolysaccharide (LPS) and tumor necrosis factor (TNF) are potent activators of NF-kappaB. This study compared the effect of these stimuli on endogenous IkappaB kinase (IKK) signalsome activation and IkappaB phosphorylation/proteolysis in human monocytic cells and investigated the role of the signalsome proteins IKK-alpha, IKK-beta, NF-kappaB-inducing kinase (NIK), IKK-gamma (NF-kappaB 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, IkappaB-alpha and -epsilon in unstimulated cells. Exposure to LPS or TNF led to differential patterns of IkappaB-alpha and IkappaB-epsilon disappearance from and reassembly with the signalsome, whereas IKK-alpha, IKK-beta, and NIK remained complex-associated. NIK cannot phosphorylate IkappaB-alpha directly, but it appears to be a functionally important subunit, because mutated NIK inhibited stimulus-induced kappaB-dependent transcription more effectively than mutated IKK-alpha or -beta. Overexpression of IKK complex-associated protein inhibited stimulus-mediated transcription, whereas NF-kappaB essential modulator enhanced it. The understanding of LPS- and TNF-induced signaling may allow the development of specific strategies to treat sepsis-associated disease.
Highlights
Bacterial lipopolysaccharide (LPS),1 a glycolipid membrane component, accounts for many of the cellular responses to Gram-negative infections, including sepsis and the development of septic shock [1]
The interaction of LPS with cells of, for example, the monocytic lineage appears to be especially important because subsequent cellular activation results in the release of highly active proinflammatory molecules, such as tumor necrosis factor (TNF) and interleukin (IL)-1, which in turn mediate systemic effects [2, 3]
Overexpression of Wild Type or Mutated Kinase-active Signalsome Components—we examined the effect of the kinase-active proteins IKK-␣ and -, as well as NF-B-inducing kinase (NIK), on LPSand TNF-induced B-dependent transcription
Summary
Bacterial lipopolysaccharide (LPS),1 a glycolipid membrane component, accounts for many of the cellular responses to Gram-negative infections, including sepsis and the development of septic shock [1]. The present study was designed to compare the effect of TNF and LPS on endogenous IKK signalsome activation and IB phosphorylation/ proteolysis in monocytic cells.
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