Hydrogen peroxide-mediated inhibition of lipopolysaccharide-stimulated inhibitory kappa B kinase activity in rat aortic smooth muscle cells.
1. In rat aortic smooth muscle cells (RASMC), exposure to lipopolysaccharide (LPS) resulted in NF-kappaB-DNA binding, degradation of IkappaB-alpha, -beta and -epsilon and increased activity of both alpha and beta isoforms of inhibitory kappa B kinase (IKK). 2. Expression of dominant-negative (DN)-IKK-alpha, IKK-beta and NF-kappaB-inducing kinase (NIK) abolished LPS-stimulated NF-kappaB reporter activity, suggesting that activation of a NIK/IKK-dependent pathway is indispensable for NF-kappaB activation by LPS in this cell type. 3. The tyrosine phosphatase inhibitor, pervanadate, abolished LPS-stimulated NF-kappaB-DNA-binding activity. However, the effect of pervanadate was shown to be mediated by excess hydrogen peroxide (H(2)O(2)) present in the reaction mix. Preincubation of RASMC with H(2)O(2) inhibited LPS-stimulated IKK kinase activity and downstream NF-kappaB-DNA binding activity. 4. H(2)O(2) also strongly stimulated p38 MAP kinase activity in RASMCs. Effective inhibition of this pathway using SB203580 did not reverse the effects of H(2)O(2) on LPS-stimulated IKK/NF-kappaB signalling. 5. These studies show that hydrogen peroxide-mediated inhibition of LPS-stimulated NF-kappaB activation in RASMC occurs upstream of IKK. The inhibitory effect of H(2)O(2) is not due to tyrosine phosphatase inhibition, it is mediated by H(2)O(2) through a mechanism which is independent of any cross-talk involving MAP kinase homologues.
Torrie
L J
LJ
Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow, Scotland, G4 0NR.
MacKenzie
C J
CJ
Paul
A
A
Plevin
R
R
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000187:drug effects / Q000201:enzymology; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D051550:I-kappa B Kinase; D048031:JNK Mitogen-Activated Protein Kinases; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008156:Luciferases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000187:drug effects / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D016328:NF-kappa B / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D016877:Oxidants / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014638:Vanadates / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases
D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D016328:NF-kappa B; D016877:Oxidants; D011993:Recombinant Fusion Proteins; C076715:pervanadate; D014638:Vanadates; D004247:DNA; D006861:Hydrogen Peroxide; D008156:Luciferases; D017346:Protein Serine-Threonine Kinases; D051550:I-kappa B Kinase; D048031:JNK Mitogen-Activated Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases