Exp Mol Med.  2012 Aug;44(8):513-520.

Transient exposure to hydrogen peroxide inhibits the ubiquitination of phosphorylated IkappaBalpha in TNFalpha-stimulated HEK293 cells

• Lee Y
• Choi J
• Ha KH
• Jue DM

Abstract

During ischemia-reperfusion injury, brief pre-exposure to oxidative stress renders organs resistant to subsequent severe damage. NF-kappaB is a transcription factor that is involved in reperfusion-induced inflammatory and immune responses. The activity of NF-kappaB has been shown to be modulated by oxidative stress in various cell types through different pathways. We studied the effect of pre-exposure to oxidative stress on subsequent NF-kappaB activation in TNFalpha-stimulated HEK293 cells. The cells were transiently exposed to 0.5 mM H2O2 for 20 min, prior to stimulation with TNFalpha, and the subsequent expression of NF-kappaB-dependent genes and the levels of NF-kappaB signaling molecules were measured. Pre-exposure to H2O2 significantly delayed the TNFalpha-induced expression of an NF-kappaB reporter gene and inflammatory proteins (intercellular adhesion molecule-1 and IL-1beta). The degradation of inhibitor of NF-kappaB alpha (IkappaBalpha) and the nuclear translocation of NF-kappaB were also delayed by H2O2 treatment, whereas IkappaBalpha phosphorylation and IkappaB kinase activity were not changed. When we examined the ubiquitin/proteosome pathway in H2O2-treated cells, we could not detect significant changes in proteosomal peptidase activities, but we were able to detect a delay of IkappaBalpha poly-ubiquitination. Our results suggest that transient exposure to oxidative stress temporally inhibits NF-kappaB-dependent gene expression by suppressing the poly-ubiquitination of phosphorylated IkappaBalpha in HEK293 cells.

Keyword

hydrogen peroxide; inflammation; NF-kappaB; proteasome endopeptidase complex; reactive oxygen species; reperfusion injury; ubiquitin

MeSH Terms

Active Transport, Cell Nucleus
Cell Nucleus/metabolism
Enzyme Activation/drug effects
HEK293 Cells
Humans
Hydrogen Peroxide/*pharmacology
I-kappa B Kinase/antagonists & inhibitors/*metabolism
Phosphorylation/drug effects
Protein Transport
Tumor Necrosis Factor-alpha/*pharmacology
Ubiquitination/*drug effects