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 lipo polysaccharid e (LPS) resulted in NF-kappaB-DNA binding, degradation of I kappaB-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-KB-induci ng kinase (NIK) abolished LPS-stimulated NF-KB reporter activity, suggestin g that activation of a NIK/IKK-dependent pathway is indispensable for NF-KB 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 sho wn to be mediated by excess hydrogen peroxide (H2O2) present in the reactio n mix. Preincubation of RASMC with H2O2 inhibited LPS-stimulated IKK kinase activity and downstream NF-KB-DNA binding activity. 4 H2O2 also strongly stimulated p38 MAP kinase activity in RASMCs. Effectiv e inhibition of this pathway using SB203580 did not reverse the effects of H2O2 on LPS-stimulated IKK/NF-kappaB signalling. 5 These studies show that hydrogen peroxide-mediated inhibition of LPS-stim ulated NF-KB activation in RASMC occurs upstream of IKK. The inhibitory eff ect of H2O2 is not due to tyrosine phosphatase inhibition, it is mediated b y H2O2 through a mechanism which is independent of any cross-talk involving MAP kinase homologues.