Hydrogen peroxide-mediated inhibition of lipopolysaccharide-stimulated inhibitory kappa b kinase activity in rat aortic smooth muscle cells

L.J. Torrie, C. MacKenzie, A. Paul, R.J. Plevin

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Abstract

In rat aortic smooth muscle cells (RASMC), exposure to lipopolysaccharide (LPS) resulted in NF-κB-DNA binding, degradation of IκB-α, -β and -ε and increased activity of both α and β isoforms of inhibitory kappa B kinase (IKK). Expression of dominant-negative (DN)-IKK-α, IKK-β and NF-κB-inducing kinase (NIK) abolished LPS-stimulated NF-κB reporter activity, suggesting that activation of a NIK/IKK-dependent pathway is indispensable for NF-κB activation by LPS in this cell type. The tyrosine phosphatase inhibitor, pervanadate, abolished LPS-stimulated NF-κB-DNA-binding activity. However, the effect of pervanadate was shown to be mediated by excess hydrogen peroxide (H2O2) present in the reaction mix. Preincubation of RASMC with H2O2 inhibited LPS-stimulated IKK kinase activity and downstream NF-κB-DNA binding activity. H2O2 also strongly stimulated p38 MAP kinase activity in RASMCs. Effective inhibition of this pathway using SB203580 did not reverse the effects of H2O2 on LPS-stimulated IKK/NF-κB signalling. These studies show that hydrogen peroxide-mediated inhibition of LPS-stimulated NF-κB activation in RASMC occurs upstream of IKK. The inhibitory effect of H2O2 is not due to tyrosine phosphatase inhibition, it is mediated by H2O2 through a mechanism which is independent of any cross-talk involving MAP kinase homologues.
LanguageEnglish
Pages393-401
Number of pages9
JournalBritish Journal of Pharmacology
Volume134
Issue number2
DOIs
Publication statusPublished - 2001

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Hydrogen Peroxide
Smooth Muscle Myocytes
Lipopolysaccharides
Phosphotransferases
Phosphoric Monoester Hydrolases
Tyrosine
DNA
p38 Mitogen-Activated Protein Kinases
Protein Isoforms

Keywords

  • hydrogen peroxide
  • rat aortic smooth muscle cells
  • lipopolysaccharide
  • inhibitory kappa B kinase

Cite this

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title = "Hydrogen peroxide-mediated inhibition of lipopolysaccharide-stimulated inhibitory kappa b kinase activity in rat aortic smooth muscle cells",
abstract = "In rat aortic smooth muscle cells (RASMC), exposure to lipopolysaccharide (LPS) resulted in NF-κB-DNA binding, degradation of IκB-α, -β and -ε and increased activity of both α and β isoforms of inhibitory kappa B kinase (IKK). Expression of dominant-negative (DN)-IKK-α, IKK-β and NF-κB-inducing kinase (NIK) abolished LPS-stimulated NF-κB reporter activity, suggesting that activation of a NIK/IKK-dependent pathway is indispensable for NF-κB activation by LPS in this cell type. The tyrosine phosphatase inhibitor, pervanadate, abolished LPS-stimulated NF-κB-DNA-binding activity. However, the effect of pervanadate was shown to be mediated by excess hydrogen peroxide (H2O2) present in the reaction mix. Preincubation of RASMC with H2O2 inhibited LPS-stimulated IKK kinase activity and downstream NF-κB-DNA binding activity. H2O2 also strongly stimulated p38 MAP kinase activity in RASMCs. Effective inhibition of this pathway using SB203580 did not reverse the effects of H2O2 on LPS-stimulated IKK/NF-κB signalling. These studies show that hydrogen peroxide-mediated inhibition of LPS-stimulated NF-κB activation in RASMC occurs upstream of IKK. The inhibitory effect of H2O2 is not due to tyrosine phosphatase inhibition, it is mediated by H2O2 through a mechanism which is independent of any cross-talk involving MAP kinase homologues.",
keywords = "hydrogen peroxide, rat aortic smooth muscle cells , lipopolysaccharide, inhibitory kappa B kinase",
author = "L.J. Torrie and C. MacKenzie and A. Paul and R.J. Plevin",
year = "2001",
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T1 - Hydrogen peroxide-mediated inhibition of lipopolysaccharide-stimulated inhibitory kappa b kinase activity in rat aortic smooth muscle cells

AU - Torrie, L.J.

AU - MacKenzie, C.

AU - Paul, A.

AU - Plevin, R.J.

PY - 2001

Y1 - 2001

N2 - In rat aortic smooth muscle cells (RASMC), exposure to lipopolysaccharide (LPS) resulted in NF-κB-DNA binding, degradation of IκB-α, -β and -ε and increased activity of both α and β isoforms of inhibitory kappa B kinase (IKK). Expression of dominant-negative (DN)-IKK-α, IKK-β and NF-κB-inducing kinase (NIK) abolished LPS-stimulated NF-κB reporter activity, suggesting that activation of a NIK/IKK-dependent pathway is indispensable for NF-κB activation by LPS in this cell type. The tyrosine phosphatase inhibitor, pervanadate, abolished LPS-stimulated NF-κB-DNA-binding activity. However, the effect of pervanadate was shown to be mediated by excess hydrogen peroxide (H2O2) present in the reaction mix. Preincubation of RASMC with H2O2 inhibited LPS-stimulated IKK kinase activity and downstream NF-κB-DNA binding activity. H2O2 also strongly stimulated p38 MAP kinase activity in RASMCs. Effective inhibition of this pathway using SB203580 did not reverse the effects of H2O2 on LPS-stimulated IKK/NF-κB signalling. These studies show that hydrogen peroxide-mediated inhibition of LPS-stimulated NF-κB activation in RASMC occurs upstream of IKK. The inhibitory effect of H2O2 is not due to tyrosine phosphatase inhibition, it is mediated by H2O2 through a mechanism which is independent of any cross-talk involving MAP kinase homologues.

AB - In rat aortic smooth muscle cells (RASMC), exposure to lipopolysaccharide (LPS) resulted in NF-κB-DNA binding, degradation of IκB-α, -β and -ε and increased activity of both α and β isoforms of inhibitory kappa B kinase (IKK). Expression of dominant-negative (DN)-IKK-α, IKK-β and NF-κB-inducing kinase (NIK) abolished LPS-stimulated NF-κB reporter activity, suggesting that activation of a NIK/IKK-dependent pathway is indispensable for NF-κB activation by LPS in this cell type. The tyrosine phosphatase inhibitor, pervanadate, abolished LPS-stimulated NF-κB-DNA-binding activity. However, the effect of pervanadate was shown to be mediated by excess hydrogen peroxide (H2O2) present in the reaction mix. Preincubation of RASMC with H2O2 inhibited LPS-stimulated IKK kinase activity and downstream NF-κB-DNA binding activity. H2O2 also strongly stimulated p38 MAP kinase activity in RASMCs. Effective inhibition of this pathway using SB203580 did not reverse the effects of H2O2 on LPS-stimulated IKK/NF-κB signalling. These studies show that hydrogen peroxide-mediated inhibition of LPS-stimulated NF-κB activation in RASMC occurs upstream of IKK. The inhibitory effect of H2O2 is not due to tyrosine phosphatase inhibition, it is mediated by H2O2 through a mechanism which is independent of any cross-talk involving MAP kinase homologues.

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