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

Abstract

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.

Figure 1

Time course of LPS-stimulated NFκB-DNA binding and IκB degradation in RASMC. Serum starved RASMC were exposed to vehicle (C) or 100 μg ml⁻¹ LPS (L) for the times indicated. NF-κB-DNA binding (A), IκB-α, (B), IκB-β (C) and IκB-ε (D) protein expression were measured as described in the Methods section. The autoradiographs and blots shown are representative of three individual experiments.

Figure 2

LPS-stimulated kinase activity of IKK-α and -β in RASMC. Serum starved cells were incubated with vehicle (C) or 100 μg ml⁻¹ LPS (L) for the times indicated. IKK-α kinase activity (A) or IKK-β kinase activity (B) was assayed as described in the Methods section. The autoradiographs shown are representative of three individual experiments.

Figure 3

Effect of DN-IKK-α and -β and DN-NIK on LPS-stimulated NF-κB reporter activity. RASMC were co-transfected with 1 μg luciferase reporter plasmid with increasing concentrations of DN-IKK-α or DN-IKK-β (A) or DN-NIK (B) made up to 10 μg total DNA with pRK plasmid as described in the Methods section. Serum starved cells were then incubated with vehicle (C), 100 μg ml⁻¹ LPS (L) or 30 ng ml⁻¹ TNF-α for 8 h. and NF-κB reporter activity measured as described in the Methods section. Data is representative of three individual experiments±s.e.mean. ^*Statistically significant from stimulation with no DN-DNA P<0.05.

Figure 4

Effect of pervanadate and catalase on LPS-stimulated NF-κB-DNA binding in RASMC. Serum starved cells were incubated with vehicle (C), 100 μM catalase (cat), pervanadate (P) or pervanadate in combination with 100 μg ml⁻¹ catalase (P+cat) alone or 30 min prior to stimulation with 100 μg ml⁻¹ LPS (L). NF-κB-DNA binding was measured as described in the Methods section. The autoradiograph shown represents three separate experiments.

Figure 5

Effect of H₂O₂ on LPS-stimulated IKK/NF-κB signalling Serum starved cells were pretreated with vehicle (C) or increasing concentrations (as indicated) of H₂O₂ (H) for 30 min prior to exposure to 100 μg ml⁻¹ LPS (L). NF-κB-DNA binding (A), protein expression of IκB-α (B), and IκB-ε (C) and kinase activity of IKK-α (D) and IKK-β (E) were measured as described in the Methods section. The autoradiographs and blots shown are representative of three individual experiments.

Figure 6

Effect of H₂O₂ on p38, JNK and p42/44 MAP kinases Serum-starved RASMC were exposed to vehicle (C), FCS (10% v v⁻¹, 5 min) or 1 mM H₂O₂ for the times indicated. p38 MAP kinase (A) and JNK (B) kinase activities and phosphorylation of p42/44 MAP kinases (C) were determined as outlined in the Methods section. The autoradiographs and blots shown represent three individual experiments.

Figure 7

Effect of SB203580 on H₂O₂-stimulated MAPKAP kinase-2 activity. Serum-starved cells were pre-incubated with vehicle (C) or increasing concentrations of SB203580 (SB) (as indicated in μM) for 30 min prior to exposure to vehicle (C) or 1 mM H₂O₂. MAPKAP kinase-2 kinase activity was then determined as described in the Methods section. Data shown is representative of three individual experiments and expressed as mean±s.e.mean ^*Statistically significant from control P<0.05.

Figure 8

Effect of SB203580 on H₂O₂ inhibition of LPS-stimulated NF-κB signalling Serum starved cells were exposed to vehicle, (C), 20 μM SB203580, 1 mM hydrogen peroxide (H), 100 μg ml⁻¹ LPS (L) or pre-incubated for 30 min with SB203580 at the concentrations indicated (μM) prior to exposure to vehicle (C), 1 mM hydrogen peroxide (H), 100 μg ml⁻¹ LPS (L). NF-κB-DNA binding (A), IκB-α (B) and -ε (C) degradation and kinase activity of IKK-α (D) were determined as described in the Methods section. The autoradiographs shown are representative of three individual experiments.