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

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 (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‐κB‐DNA binding activity. 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‐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 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.British Journal of Pharmacology (2001) 134 , 393–401; doi: 10.1038/sj.bjp.0704259