The glutaredoxin/glutathione system modulates NF-κB activity by glutathionylation of p65 in cinnamaldehyde-treated endothelial cells

Abstract

Reversible protein glutathionylation is an important post-translational modification that provides protection against oxidation. In endothelial cells (ECs), cinnamaldehyde is an electrophilic compound that can increase the intracellular glutathione (GSH) levels or reactive oxygen species (ROS) production depending on the treatment duration. ECs treated with GSH and H2O2 show increased sulfhydryl modifications of the p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), which are responsible for NF-κB inactivation, and also a block in TNF-α-induced p65 nuclear translocation and inter-cellular adhesion molecule-1 (ICAM-1) expression. In our current study, we find that cinnamaldehyde induces p65 glutathionylation and inhibits TNF-α-induced p65 nuclear translocation and ICAM-1 expression within 12 h of treatment. Our analyses also reveal that p65 glutathionylation is suppressed by a GSH synthesis inhibitor, buthionine sulfoximine (BSO), and we further observed that the inhibitory effects of p65 nuclear translocation and ICAM-1 expression are also suppressed by BSO. NF-E2-related factor-2 small interfering RNA (siRNA) molecules not only inhibit glutamate-cysteine ligase catalytic subunit (GCLC) and glutamate-cysteine ligase modifier subunit (GCLM) induction and increases in GSH but also abolish cinnamaldehyde-induced p65 glutathionylation and its inhibitory effects. The gene expression and activity of glutaredoxin-1 (Grx-1), which catalyzes the formation of protein-glutathione mixed disulfides (protein-SSG), were also found to be increased after cinnamaldehyde treatment. A knock down of endogenous Grx-1 by siRNA or pretreatment with an inhibitor of Grx-1 activity, CdCl2, abolishes p65-SSG formation. In addition, Grx-1 siRNA blocks the inhibition of p65 nuclear translocation and ICAM-1 expression, suggesting that this enzyme is involved in the cinnamaldehyde-mediated NF-κB inhibition. Our current results thus indicate that the GSH/Grx-1-dependent glutathionylation of p65islikelytoberesponsiblefor cinnamaldehyde-mediated NF-κB inactivation and for the enhanced inhibitory effects of cinnamaldehyde upon TNF-a-treated ECs. © The Author 2010. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org.