title: Differential effects of lipopolysaccharide and tumor necrosis factor on monocytic IkappaB kinase signalsome activation and IkappaB proteolysis.
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.
authors.affiliation: Institute of Clinical Chemistry and Pathobiochemistry, Klinikumrechts der Isar, Technical University Munich, Ismaninger Strasse 22, 81675 Munich, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Fischer
authors.forename: S
authors.initials: S
authors.lastname: Page
authors.forename: M
authors.initials: M
authors.lastname: Weber
authors.forename: T
authors.initials: T
authors.lastname: Eisele
authors.forename: D
authors.initials: D
authors.lastname: Neumeier
authors.forename: K
authors.initials: K
authors.lastname: Brand
mesh_terms: D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D051550:I-kappa B Kinase; D008070:Lipopolysaccharides / Q000494:pharmacology*; D009000:Monocytes; D016328:NF-kappa B / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D000097782:NF-kappaB-Inducing Kinase
chemical_list: D051550:I-kappa B Kinase; D008070:Lipopolysaccharides; D016328:NF-kappa B; D017346:Protein Serine-Threonine Kinases; D014409:Tumor Necrosis Factor-alpha; D000097782:NF-kappaB-Inducing Kinase; C496559:CHUK protein, human; C496560:IKBKB protein, human; C496565:IKBKE protein, human