Thiol-Reactive Metal Compounds Inhibit NF-κB Activation by Blocking IκB Kinase

Abstract

Gold compounds are used in the treatment of rheumatoid arthritis. NF-κB is a transcription factor implicated in the expression of many inflammatory genes. NF-κB is activated by signal-induced phosphorylation and subsequent degradation of inhibitory IκB (inhibitory protein that dissociates from NF-κB) proteins, and a multisubunit IκB kinase (IKK) has been identified previously. We tested the effect of various gold compounds on the activation of NF-κB and IKK in LPS-stimulated RAW 264.7 mouse macrophages. A lipophilic gold compound, auranofin, suppressed the LPS-induced increase of nuclear κB-binding activity, degradation of IκB proteins, and IKK activation. Auranofin also blocked IKK activation induced by TNF and PMA/ionomycin, suggesting that the target of auranofin action is common among these diverse signal pathways. In vitro IKK activity was suppressed by addition of hydrophilic gold compounds, such as aurothiomalate, aurothioglucose, and AuCl3. Other thiol-reactive metal ions such as zinc and copper also inhibited IKK activity in vitro, and induction of IKK in LPS-stimulated macrophages. In vitro IKK activity required the presence of reducing agent and was blocked by addition of thiol group-reactive agents. Two catalytic subunits of IKK complex, IKKα and IKKβ, were both inhibited by these thiol-modifying agents, suggesting the presence of a cysteine sulfhydryl group in these subunits, which is critical for enzyme activity. The antiinflammatory activity of gold compounds in the treatment of rheumatoid arthritis may depend on modification of this thiol group by gold.

Zinc supplementation attenuates ethanol- and acetaldehyde-induced liver stellate cell activation by inhibiting reactive oxygen species (ROS) production and by influencing intracellular signaling

“…Together these data indicated that if zinc inhibits NFB activity, such inhibition occurs via blocking of IB kinase (IK) activity. Indeed, recent studies have demonstrated that thiol-reactive metal compounds inhibit Page 22 of 57 A c c e p t e d M a n u s c r i p t 22 NFB activation by blocking IK [60]. In our study, we also detected that zinc supplementation significantly inhibited phosphorylation of IB while increasing its total level.…”

Auranofin induces apoptosis and when combined with retinoic acid enhances differentiation of acute promyelocytic leukaemia cells in vitro

“…AF is an antirheumatic drug with immunosuppressive and anti-inflammatory properties and it is known to inhibit NF-kB activation by blocking IkB-kinase (Jeon et al, 2000). NF-kB is important in cell survival, acting as a transcription factor to induce the expression of antiapoptotic IAP genes (Lee & Collins, 2001).…”

Cysteine-179 of IκB kinase β plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines

“…Recombinant glutathione S-transferase (GST)-IκBα containing N-terminal 54 residues of IκBα and recombinant human TNF were prepared by expression in Escherichia coli as described previously (Jeon et al, 2000). Expression vectors for FLAG-tagged IKKβ and IKKβ (S177/181E) were kindly provided by Dr. F. Mercurio (Signal Pharmaceuticals, San Diego, CA).…”

“…Cells were transfected with expression vectors for IKKβ and their mutants using Fugene 6 (Roche Molecular Biochemicals, Mannheim, Germany) and incubated for 48 h. Preparation of cytoplasmic extracts and immunoprecipitation were performed as described (Byun et al, 2002). Kinase activity was measured in reaction mixtures containing 10 µM ATP, [γ-32 P]ATP (2-5 µCi) and GST-IκBα (1 µg) (Jeon et al, 2000). Reaction products were analyzed by SDS-PAGE on a 12.5% gel and electrophoretically transferred to nitrocellulose membrane.…”

“…Our previous study showed that thiol-reactive metal compounds such as gold, zinc, and copper inhibit NF-κB activation by blocking IKK in LPSstimulated macrophages (Jeon et al, 2000). Other thiol-reactive agents such as cyclopentenone PGs [PGA1 and 15-deoxy-∆ 12-14 -PGJ2 (15dPGJ2)] (Rossi et al, 2000;Straus et al, 2000), arsenite anion (AsO3 3-) (Kapahi et al, 2000), parthenolide (Kwok et al, 2001), and epoxyquinone A (Liang et al, 2006) were also shown to inhibit NF-κB and IKK activation in cells stimulated with TNF, IL-1, and phorbol esters.…”