The Glutaredoxin/Glutathione System Modulates NF-κB Activity by Glutathionylation of p65 in Cinnamaldehyde-Treated Endothelial Cells

Abstract

Reversible protein glutathionylation is an important posttranslational 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 H(2)O(2) show increased sulfhydryl modifications of the p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB), which are responsible for NF-kappaB inactivation, and also a block in TNF-alpha-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-alpha-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, CdCl(2), 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-kappaB inhibition. Our current results thus indicate that the GSH/Grx-1-dependent glutathionylation of p65 is likely to be responsible for cinnamaldehyde-mediated NF-kappaB inactivation and for the enhanced inhibitory effects of cinnamaldehyde upon TNF-alpha-treated ECs.

IκBα glutathionylation and reduced histone H3 phosphorylation inhibit eotaxin and RANTES

“…treated endothelial cells, leading to an inhibition of NF-kB p65 nuclear translocation [12]. In vitro, NF-kB p50-SSG inhibited the capacity of NF-kB to bind DNA [13].…”

“…One such reversible modification is protein glutathionylation (protein-SSG), whereby a cysteine-thiol of a protein (protein-SH), forms a disulfide bond with the cysteine-thiol of glutathione (GSH). Protein-SSG is inducible by reduction of the AFFILIATIONS *Respiratory Research Group, Faculty of Pharmacy, intracellular GSH level [11] and has been shown to result in an inhibition of NF-kB nuclear entry and binding to DNA [12,13]. Dimethylfumarate (DMF) reduces cellular glutathione in many different cell types [14][15][16], and has also been shown to inhibit the nuclear entry of NF-kB and subsequent binding to DNA in ASMCs [7].…”

Changes in Mouse Liver Protein Glutathionylation after Acetaminophen Exposure

“…Glutathionylation has been shown to play a regulatory role in the activity of various proteins, in a manner similar to the regulatory changes induced by protein phosphorylation (Beer et al, 2004;Chen et al, 2007;Mueller et al, 2008;Townsend et al, 2009;Bundgaard et al, 2010;Hawkins et al, 2010;Liao et al, 2010;Yang et al, 2011). Therefore, alteration of the glutathionylation status of hepatocellular proteins after hepatotoxicant insult could lead to altered function of key proteins and subsequent cellular injury.…”

Dehydroepiandrosterone, Over-studied but Under-used in the Treatment of Vascular Remodeling Diseases

“…Glutathionylation depends on Glutaredoxin (GRX) and thioredoxin levels (deglutathionylation enzymes) and on the increase in the cellular GSH/GSSG ratio, which also exert a reversible action on protein S-glutathionylation. Interestingly Akt [96], NFκB [97], and other protein like eNOS or MEKK1 [98,99] are also subject to glutathionylation. This mechanism of regulation may be a masterpiece in the understanding of DHEA effect as a peroxysome proliferator.…”