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Thiol-Reactive Metal Compounds Inhibit NFκB Activation by Blocking IκB Kinase Kye-Im Jeon, Jae-Yeon Jeong and Dae-Myung Jue This information is current as of June 18, 2017. Subscription Permissions Email Alerts This article cites 63 articles, 29 of which you can access for free at: http://www.jimmunol.org/content/164/11/5981.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 References J Immunol 2000; 164:5981-5989; ; doi: 10.4049/jimmunol.164.11.5981 http://www.jimmunol.org/content/164/11/5981 Thiol-Reactive Metal Compounds Inhibit NF-B Activation by Blocking IB Kinase1 Kye-Im Jeon, Jae-Yeon Jeong, and Dae-Myung Jue2 uclear factor-B is a transcription factor that plays a pivotal role in the expression of a wide range of genes involved in chronic inflammatory diseases, including TNF, IL-1, IL-6, IL-8, GM-CSF, inducible NO synthase, ICAM-1, E-selectin, and the MHC class I and II molecules (1, 2). In unstimulated cells, NF-B proteins are sequestered in the cytosol through interactions with a class of inhibitory proteins called IBs3 (IB␣, -, and -⑀). Many stimuli induce NF-B activity, including TNF, IL-1, activators of protein kinase C, viruses, bacterial LPS, ionizing radiation, and oxidants. These signals cause phosphorylation and subsequent degradation of IB proteins via the ubiquitination-proteasome pathway, and thereby, free NF-B can enter the nucleus and induce gene expression (2). In the synovium of rheumatoid arthritis (RA) patients, active forms of NF-B are detected in the nucleus of macrophages and endothelial cells, suggesting that NF-B is involved in the expression of inflammatory genes in these cells (3, 4). Macrophages have been recognized as playing important roles in the pathogenesis of RA, in that there is a relative abundance of macrophage-derived cytokines, such as TNF and IL-1, in rheumatoid synovium (5). In N Department of Biochemistry, College of Medicine, The Catholic University of Korea, Seoul, South Korea Received for publication October 12, 1999. Accepted for publication March 22, 2000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by grants from KOSEF (981-0704-031-2) and the Ministry of Health and Welfare, Republic of Korea (HMP-98-D-4-0055). 2 Address correspondence and reprint requests to Dr. Dae-Myung Jue, Department of Biochemistry, College of Medicine, The Catholic University of Korea, 505 BanpoDong, Socho-Ku, Seoul 137-701, South Korea. E-mail address: dmjue@cmc. cuk.ac.kr Abbreviations used in this paper: IB, inhibitory protein that dissociates from NFB; ATF, activating transcription factor; ERK, extracellular signal-regulated kinase; NEMO, NF-B essential modulator; HA, hemagglutinin; IKK, IB kinase; JNK/ SAPK, c-Jun N-terminal kinase/stress-activated protein kinase; NIK, NF-B-inducing kinase; RA, rheumatoid arthritis. 3 Copyright © 2000 by The American Association of Immunologists clinical trials of RA patients, administration of Ab or soluble receptors to TNF has led to significant reduction in disease severity, demonstrating a pivotal role of TNF in the maintenance of RA (6, 7). Recent studies suggested that activated NF-B is responsible for the overexpression of TNF in rheumatoid joints and RA disease activity. Blockade of NF-B activation by adenoviral transfer of IB␣ suppressed expression of TNF in cultured synoviocytes (8), and intraarticular injection of an oligodeoxynucleotide-containing B-binding sequence (NF-B decoy) inhibited development of joint inflammation in an animal model of arthritis (9). Phosphorylation of IB is likely to be the central point of control at which diverse stimuli converge to activate NF-B (1, 2). Previous studies have identified an IB kinase (IKK) complex with a molecular mass of 500 –900 kDa, which is induced by inflammatory signals and able to phosphorylate two conserved N-terminal serine residues of IB␣ and IB required to activate NF-B in vivo (10, 11). Two IKKs, designated IKK␣ (or IKK1) and IKK (IKK2), have been cloned and shown to be part of the multicomponent IKK complex, called IKK signalsome (11–15). Both IKK␣ and IKK are Ser/Thr kinases of similar structure and contain an N-terminal kinase domain, followed by a leucine zipper region and a C-terminal helix-loop-helix domain (2, 11–15). Overexpression of each kinase or transfection using catalytically inactive mutants demonstrated that both kinases are involved in IL-1 and TNFinduced activation of NF-B (11–15). Moreover, recombinant IKK␣ and IKK expressed independently in insect cells and purified to apparent homogeneity were able to phosphorylate IB␣ at specific serine residues in in vitro assays, indicating that each kinase can directly phosphorylate IB proteins (16, 17). Recently, other subunits of the IKK complex were also characterized by biochemical analysis and molecular cloning, and they include mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase kinase 1, NF-B-inducing kinase (NIK), NF-B essential modulator (NEMO)/IKK␥/IKKAP1, and IKK complex-associated protein (12, 18 –21). These subunits were shown to play roles in the transmission of upstream signals to 0022-1767/00/$02.00 Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Gold compounds are used in the treatment of rheumatoid arthritis. NF-B is a transcription factor implicated in the expression of many inflammatory genes. NF-B is activated by signal-induced phosphorylation and subsequent degradation of inhibitory IB (inhibitory protein that dissociates from NF-B) proteins, and a multisubunit IB kinase (IKK) has been identified previously. We tested the effect of various gold compounds on the activation of NF-B and IKK in LPS-stimulated RAW 264.7 mouse macrophages. A lipophilic gold compound, auranofin, suppressed the LPS-induced increase of nuclear B-binding activity, degradation of IB 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, IKK␣ and IKK, 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. The Journal of Immunology, 2000, 164: 5981– 5989. 5982 Materials and Methods Materials Polyclonal Ab to IB␣ (C-21), IB (C-20), IKK␣ (M-280), c-Jun Nterminal kinase/stress-activated protein kinase (JNK/SAPK) (C-17), p38 kinase (C-20), and hemagglutinin (HA) tag (Y-11) and anti-IKK mAb (H-4) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Ab to ERK1/2 were from Zymed Laboratories (South San Francisco, CA), and anti-Flag M2 mAb from Stratagene (La Jolla, CA). rATF-2 (aa 1–96 of human origin) in the form of GST fusion protein (overall size, 40 kDa) was obtained from Santa Cruz Biotechnology, and bovine brain myelin basic protein (18 –20 kDa) from Life Technologies (Rockville, MD). Aurothioglucose, sodium aurothiomalate, and other metal compounds were obtained from Sigma (St. Louis, MO). Auranofin (purity 99%) was a kind gift from Yukyung Medica (Seoul, South Korea) and dissolved in ethanol at a 100fold concentration before use. A synthetic peptide (Lys-Cys-Thr-Cys-CysAla) representing the C-terminal portion of mouse liver metallothionein-I was obtained from Bachem AG (Bubendorf, Switzerland). Preparation of recombinant proteins Human peripheral blood T cells were stimulated for 1 h with PMA (10 ng/ml) and ionomycin (500 ng/ml), and cDNA was prepared by reverse transcription. cDNA containing N-terminal 54 residues of IB␣ was amplified by PCR using primers 5⬘-TCTCTGGATCCCCATGTTCCAG GCGGCCGAG-3⬘ and 5⬘-AAGGGAATTCCCTCAGAGGCGGATCTC CTGCAG-3⬘ and cloned into BamHI/EcoRI site of pGEX-3X (Pharmacia Biotech, Uppsala, Sweden). After expression in Escherichia coli strain BL21, the GST-IB␣ (1–54) fusion protein was purified (32). Expression vectors encoding IB␣ mutants in which serines 32, 36, or both are substituted with alanine were constructed using a site-directed mutagenesis kit (Quikchange; Stratagene). Mutations were verified by DNA-sequencing analysis. A similar strategy was used to produce human TNF. Primers used for PCR, 5⬘-CGCGGATCCCCGTCAGATCATCTTCTCGA-3⬘ and 5⬘CGCGAATTCTTTCACAGGGCAATGATCCC-3⬘, contain the first and the last five codons of mature human TNF, respectively. GST-TNF fusion protein was purified and digested with coagulation factor Xa, and its biological activity was determined in L-929 cell cytotoxicity assay (32). Cell culture and analysis of TNF mRNA RAW 264.7 murine macrophages, HeLa human epithelial cells, and U937 human histiocytic lymphoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). RAW 264.7 cells were maintained in RPMI 1640 medium supplemented with 5% heat-inactivated FCS, 20 mM HEPES, and gentamicin (50 g/ml), as described previously (32). HeLa and U937 cells were grown in the same RPMI 1640 medium supplemented with 10% FCS. COS-7 monkey kidney cells (ATCC CRL-1651) were maintained in DMEM supplemented with 10% heat-inactivated FCS, penicillin (100 U/ml), and streptomycin (100 g/ml). Cell viability after incubation with various agents was measured by staining cells with MTT (33). Total cellular RNA prepared from RAW 264.7 cells with an RNA isolation kit (Ultraspec-II; Biotecx, Houston, TX) was used to determine TNF and GAPDH mRNA by Northern blot analysis (32). EMSA of NF-B and analysis of IB proteins Nuclear and cytoplasmic extracts were prepared from RAW 264.7 cells, as described previously (32). The oligonucleotide probe (5⬘-CAAA CAGGGGGCTTTCCCTCCTCA-3⬘) contained the B site (underlined) in the murine TNF promoter (B enhancer 3) (32). Binding reaction was performed with 5 g of nuclear extract and radiolabeled B probe. The reaction mixtures were analyzed by electrophoresis on a 4% polyacrylamide gel in 0.25⫻ TBE buffer and autoradiography. Immunoblot analyses of IB␣ and IB in the cytoplasmic extracts were performed with corresponding Ab and visualized by ECL detection kit (Amersham, Buckinghamshire, U.K.). In vitro kinase assays RAW 264.7, HeLa, or U937 cells grown in 100-mm plates were treated with various agents and washed three times with ice-cold PBS containing 1 mM Na3VO4 and 5 mM EDTA. The cells were scraped and resuspended in 0.75 ml of lysis buffer containing 20 mM Tris-HCl, 0.5 M NaCl, 0.25% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM -glycerophosphate, 10 mM NaF, 10 mM 4-nitrophenylphosphate, 300 M Na3VO4, 1 mM benzamidine, 2 M PMSF, aprotinin (10 g/ml), leupeptin (1 g/ml), pepstatin (1 g/ml), and 1 mM DTT (12). After incubation for 20 min on ice, the lysate was cleared by centrifugation at 20,000 ⫻ g for 15 min. The supernatant fraction was analyzed for protein with bicinchoninic acid reagent (Pierce, Rockford, IL). An aliquot (120 g protein) of the lysate was incubated on ice for 1–2 h with 2– 4 g of anti-IKK␣ Ab, then protein A (Pharmacia Biotech) or protein G beads (Calbiochem, La Jolla, CA) (5 l) were added, and the samples were incubated for another 1–2 h at 4°C. The immunoprecipitate was washed twice with the lysis buffer containing 0.1% Nonidet P-40 instead of Triton X-100 and once with kinase buffer without ATP. In vitro kinase assay was performed with immune complexes and bacterially synthesized GST-IB␣ proteins (2 g) in 15 l of kinase buffer containing 20 mM HEPES (pH 7.7), 2 mM MgCl2, 2 mM MnCl2, 10 M ATP, 5 Ci of [␥-32P]ATP, 10 mM -glycerophosphate, 10 mM NaF, 300 M Na3VO4, 1 mM benzamidine, 2 M PMSF, aprotinin (10 g/ml), leupeptin (1 g/ml), pepstatin (1 g/ml), and 1 mM DTT at 30°C for 30 – 60 min (12). Samples were analyzed by 12.5% SDS-PAGE and autoradiography. Phosphorylation of GST-IB␣ was quantitated in a phosphor image analyzer (BAS-2500; Fujifilm, Tokyo, Japan). To measure activities of ERK1/2, JNK/SAPK, and p38 kinase, RAW 264.7 cells were incubated in medium containing 0.5% FCS for 24 h. The cells were stimulated with LPS for 30 min, and lysed as described before for IKK. ERK1/2 and JNK/ SAPK were isolated by immunoprecipitation with specific Ab from an aliquot of cell lysate containing 150 g protein, and p38 kinase from cell lysate of 1.2 mg protein. The kinase reaction was performed in 25 l mixture, as described for IKK, with myelin basic protein (10 g) as a substrate protein for ERK1/2, and ATF-2 (1 g) for JNK/SAPK and p38 kinase. Expression of IKK␣ and IKK in COS-7 cells The expression plasmids pRcActin-3⫻HA-IKK-1 and pFlagCMV2IKK-2 encoding wild-type IKK␣-HA and IKK-Flag, respectively, were kindly provided by Dr. F. Mercurio (Signal Pharmaceuticals, San Diego, CA). Wild-type NIK construct was a gift from Dr. J.-H. Kim (Kwang-Ju Institute of Science and Technology, Kwang-Ju, South Korea). COS-7 cells grown in six-well plates were transfected with either IKK␣-HA (0.5 g) Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 IKK␣ and IKK by directly phosphorylating them or by acting as regulatory and scaffold proteins that control enzyme activity or aid association of various IKK components (18 –21). Gold compounds, comprised of elemental Au(I) and a sulfurcontaining ligand, have been used for the treatment of RA, inducing improvement of clinical conditions in a majority of patients (22, 23). Although its mode of antirheumatic action is not clearly understood, gold is selectively concentrated within inflamed synovial tissues, and gold-rich deposits are formed in synovial macrophages during chrysotherapy (24 –27). A previous immunohistochemical study showed that administration of aurothiomalate results in reduced accumulation of inflammatory monocytes and macrophages in the RA synovial membranes, and significant inhibition of IL-1, IL-6, and TNF expression in these cells (28). In cultured human monocytes and mouse macrophages, a lipid-soluble gold compound, auranofin ((1-thio--D-glucopyranose 2,3,4,6tetraaceto-S)-(triethylphosphine)gold(I)), but not water-soluble aurothiomalate, inhibited LPS-induced production of IL-1 and TNF (29). The inhibitory effect of auranofin appeared by reducing mRNA level of these cytokines, suggesting that it blocks some common step in the signal pathways for the transcriptional activation of IL-1 and TNF genes (30). In conjunction with these results, various metal compounds, including gold thiolates, were shown to inhibit the in vitro binding of NF-B to DNA, albeit only in relatively high concentrations (31). In this study, we tested the effect of various gold compounds on the production of TNF and activation of NF-B in LPS-stimulated macrophages. Our results show that auranofin suppresses TNF expression by blocking NF-B activation, which in turn is caused by inhibition of IKK activation. Gold compounds were able to suppress IKK activity when added directly to an in vitro kinase assay, a property shared with other thiol-binding metal ions such as zinc, copper, and mercury and thiol-reactive agents. Our data imply that IKK contains a metal-sensitive cysteine residue and is one of the major targets of gold and related metal compounds in the treatment of RA. INHIBITION OF IB KINASE BY THIOL-REACTIVE METALS The Journal of Immunology 5983 Since induction of TNF gene expression in LPS-activated macrophages is known to be mediated by NF-B (34, 35), we tested the effect of gold compounds on LPS-induced NF-B activation. Nuclear B-binding activity was measured by EMSA with a probe-containing B sequence of TNF promoter (Fig. 1C). LPS stimulation of macrophages resulted in a remarkable increase in NF-B activity. In cells treated with auranofin, the nuclear level of NF-B was reduced in a dose-dependent manner at 5–10 M concentrations. High concentrations of aurothioglucose and AuCl3 did not significantly block induction of NF-B. Immunoblot analysis of IB proteins in the cytosol of RAW 264.7 cells indicated that the auranofin-induced inhibition of NF-B activation was associated with suppression of IB degradation (Fig. 1, D and E). Stimulation of cells with LPS induced significant degradation of IB␣ and IB within 20 min. However, addition of auranofin blocked the degradation of both IB proteins, suggesting that auranofin inhibits an intermediate step in the signal pathway between the LPS-receptor binding and degradation of IB␣ and IB. and NIK (0.5 g) or IKK-Flag (0.5 g) expression vectors using Fugene 6 (Roche Molecular Biochemicals, Mannheim, Germany), according to a procedure recommended by manufacturer. Empty pFlagCMV2 vector was used to equalize total amount of DNA. After 48 h, cells were resuspended in 0.25 ml of lysis buffer, and aliquots of lysates were immunoprecipitated with anti-HA or anti-Flag Ab. The immunoprecipitates were incubated in kinase buffer with [␥-32P]ATP and GST-IB␣ to measure IKK activity in the presence or absence of inhibitors. To analyze the immune complexes, IKK␣ and IKK immunoprecipitates were mixed with an equal volume of 2⫻ SDS-PAGE sample buffer, and boiled for 3 min. Proteins were separated by SDS-PAGE in 8% gel and electrophoretically transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was sequentially probed with anti-IKK␣ and anti-IKK Ab to visualize each protein. Results Effects of gold compounds on the expression of TNF and NF-B activation We tested the effect of various gold compounds on TNF gene expression in RAW 264.7 mouse macrophages stimulated with LPS. TNF mRNA was measured by Northern blot analysis (Fig. 1A). TNF mRNA was not detected in nonstimulated control cells, whereas addition of LPS induced a remarkable increase of its level. Incubation of cells with auranofin resulted in a dose-dependent suppression of TNF mRNA induction, and partial and complete inhibition was observed at 5 M and 10 M concentrations, respectively. In contrast, hydrophilic gold compounds, such as aurothioglucose, AuCl3, and aurothiomalate, did not inhibit TNF mRNA induction even at 100 M concentrations (Fig. 1A, and data not shown). The mRNA level of control GAPDH did not change significantly by auranofin (Fig. 1B). The cell viability was about 70% after incubation with 10 M auranofin, while in other cells it was ⬎80%. Inhibition of IKK activation by auranofin Degradation of IB proteins was shown to occur after signal-induced phosphorylation of IB proteins at specific serine residues by IKK (1, 2). To determine whether auranofin inhibits signal pathway leading to IB phosphorylation, we measured IKK activity in LPS-stimulated RAW 264.7 cells. The cell lysate was immunoprecipitated with an anti-IKK␣ Ab and incubated with GSTIB␣ and [␥-32P]ATP. IKK activity was barely detectable in nonstimulated cells, whereas incubation with LPS induced a remarkable increase in IKK activity, which was detectable within 5 min, peaked at 10 –20 min, then gradually declined over a 1-h period (Fig. 2A). As shown with other IKK enzymes (10 –15), the IKK immune complex isolated from RAW 264.7 cells was able to phosphorylate rIB␣ proteins with either Ser32 or Ser36, but not a mutant protein in which both of the serine residues were substituted with alanine (Fig. 2B). When we measured induction of IKK activity in cells treated with various gold compounds, it was shown to be blocked by auranofin, but not by aurothioglucose or AuCl3 (Fig. 2C, and data not shown). The inhibitory effect of auranofin appeared at concentration ranges that inhibit NF-B activation. To determine whether auranofin also blocks IKK activation induced by other signals and in other types of cells, IKK activity was measured in HeLa and U937 cells stimulated with TNF and PMA/ ionomycin, respectively. In HeLa cells, IKK activity was rapidly induced by addition of TNF, and maximal induction was observed within 5 min (data not shown). This TNF-induced increase in IKK activity was blocked almost completely by addition of 2.5 M auranofin (Fig. 2C). In contrast to HeLa cells, U937 cells showed a significant level of basal IKK activity even when they were not stimulated. Treatment of U937 cells with PMA/ionomycin induced about a 2-fold increase in IKK activity over the basal level that could be detected after 1 h (data not shown). Addition of auranofin to U937 cells also suppressed IKK activity, reducing it below the basal level when a concentration of 10 M was applied (Fig. 2C). The viability of HeLa and U937 cells incubated with 10 M auranofin was ⬎80%. Our results demonstrate that auranofin inhibits IKK activation elicited by various stimuli in different cell types and suggest that the target of auranofin action is common among these diverse signal pathways for NF-B activation. Gold and other thiol-reactive metal compounds inhibit in vitro IKK activity We examined whether auranofin and other gold compounds could directly inhibit IKK activity by carrying out the kinase reaction in Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 1. Auranofin inhibits TNF gene expression by blocking NF-B activation. A and B, RAW 264.7 cells were incubated with or without various doses of auranofin, 100 M aurothioglucose (ATG), or 100 M AuCl3 for 2 h. After addition of LPS (1 g/ml), the cells were further incubated for 2 h and total cellular RNA was prepared. Cells in the first lane were not treated with LPS. Northern blot analysis of TNF (A) and GAPDH (B) mRNA was conducted with digoxigenin-labeled probes, and the bands were visualized by chemiluminescence reaction. C, Nuclear extracts prepared from RAW 264.7 cells treated as described in A were incubated with 32P-labeled B sequence, and analyzed by EMSA. D and E, Cells were treated as described in A and cytosolic extracts were obtained 20 min after LPS stimulation. Immunoblot analysis of IB␣ and IB was performed with specific Ab by chemiluminescence reaction. The data represent three independent experiments. 5984 the presence of various gold compounds (Fig. 3A). Our result revealed a dose-dependent inhibition of IKK activity by hydrophilic gold compounds such as aurothiomalate, aurothioglucose, and AuCl3. Inhibitory effects of these compounds appeared at 10 –100 M concentrations. Auranofin was far less effective in blocking in vitro IKK activity compared with other gold compounds; only partial inhibition was detected at 100 M concentration. This result suggested that insolubility of auranofin in water limits its interaction with IKK. Because treatment of cells with auranofin blocked IKK induction, it seemed likely that the inhibitory effect of gold in auranofin on IKK appears after auranofin is taken up by cells and gold is transferred to other water-soluble ligands. To test this hypothesis, we prepared cell lysates from auranofin-treated RAW 264.7 cells, and measured whether they could inhibit exogenous IKK. EDTA and EGTA were omitted from the cell lysis buffer because they could chelate gold in the lysate. As shown in Fig. 3B, incubation of cells with auranofin did result in a dose-dependent formation of IKK-inhibitory activity. Lysates of cells incubated with 10 and 20 M auranofin reduced the activity of exogenous enzyme by 30% and 50%, respectively. The specificity of IKK inhibition by gold compounds was examined by determining the effect of aurothiomalate on the activity of other stress-induced protein kinases, ERK1/2, JNK/SAPK, and p38 kinase. The protein kinases were isolated from LPS-stimulated RAW 264.7 cells using specific Ab, and their activity was mea- FIGURE 3. Inhibition of in vitro IKK activity by gold compounds. A, IKK immune complex was obtained from RAW 264.7 cells 15 min after LPS stimulation. In vitro kinase reactions were conducted with GST-IB␣ and [␥-32P]ATP in the presence or absence of various doses of auranofin, aurothiomalate (ATM), aurothioglucose (ATG), or AuCl3. B, Suppression of in vitro IKK activity by lysates of auranofin-treated cells. RAW 264.7 cells were incubated with indicated doses of auranofin for 2 h, washed three times with cold PBS, and lysed in cell lysis buffer in which EDTA and EGTA were omitted. After removal of IKK complex by immunoprecipitation with anti-IKK␣ Ab, the cell lysates were incubated for 2 h with exogenous IKK immune complex prepared from LPS-stimulated RAW 264.7 cells. In control assay, the IKK complex was incubated with the cell lysate prepared from cells not pretreated with auranofin. The IKK complex was washed again and used in in vitro kinase reactions with GST-IB␣ and [␥-32P]ATP. Radioactivity of phosphorylated GST-IB␣ was measured by phosphor image analysis. The values shown are mean (⫾SD) of three experiments done in duplicate samples. The statistical significance of differences in the kinase activity was determined by Student’s t test using the SAS program (SAS Institute, Cary, NC). ⴱ, p ⬍ 0.05; ⴱⴱⴱ, p ⬍ 0.001. C, RAW 264.7 cells were incubated in low serum medium for 18 h and stimulated with LPS for 30 min. Mitogen-activated protein kinases, ERK1/2, JNK/SAPK, and p38 kinase, were isolated from cell lysates by immunoprecipitation. In vitro kinase activities were measured in the presence of various doses of aurothiomalate and substrates, myelin basic protein for ERK1/2, and ATF-2 for JNK/SAPK and p38 kinase. The arrowheads indicate positions of substrate protein. The data represent three independent experiments. sured in the absence or presence of various doses of aurothiomalate (Fig. 3C). Aurothiomalate added up to 100 M concentration did not inhibit phosphorylation of substrate proteins by either of these protein kinases. To understand the inhibitory mode of gold compounds, we tested various metal compounds for their effects on in vitro IKK activity (Table I). Our result revealed a potent inhibition of IKK by Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 2. Auranofin inhibits signal-induced activation of IKK. A, RAW 264.7 cells were incubated with LPS for times indicated and the cells were lysed. IKK was immunoprecipitated with anti-IKK␣ Ab and used in in vitro kinase reactions with GST-IB␣ (aa 1–54) and [␥-32P]ATP. Phosphorylated GST-IB␣ was visualized by SDS-PAGE and autoradiography. B, IKK immune complex was obtained from RAW 264.7 cells stimulated for 15 min with LPS. In vitro phosphorylation reactions were conducted using wild-type GST-IB␣ (SS), and mutant proteins, in which serine 32 (AS), 36 (SA), or both serines were substituted with alanine (AA) as substrates. Coomassie blue (CB)-stained recombinant proteins are shown below. C, RAW 264.7 cells were incubated in the presence or absence of various doses of auranofin for 2 h and stimulated with LPS for 15 min. HeLa and U937 cells, treated with various doses of auranofin for 2 h, were stimulated with TNF (200 ng/ml) for 5 min or PMA (25 ng/ml)/ionomycin (500 ng/ml) for 60 min, respectively. IKK in the cell lysate was immunopurified and used in in vitro kinase assays with GSTIB␣ and [␥-32P]ATP. The data represent three independent experiments. INHIBITION OF IB KINASE BY THIOL-REACTIVE METALS The Journal of Immunology 5985 Table I. Metal compounds that inhibit IKK activity in vitroa Metal Compounds ZnSO4 Aurothiomalate HgCl2 Aurothioglucose AuCl3 CuSO4 CoCl2 Na2Cr2O7 ID50 (M) 8.7 10.9 12.3 19.6 24.1 26.9 55.7 69.2 a RAW 264.7 cells were stimulated with LPS and the cell lysate was immunoprecipitated with anti-IKK␣ Ab. The IKK immune complex was incubated with GSTIB␣ and [␥-32P]ATP in the absence or presence of various concentrations of metal compounds. The reaction mixture was resolved by SDS-PAGE, and phosphorylation of GST-IB␣ was measured by phosphor image analysis. The ID50 value was calculated using a Pharm/PCS program (MicroComputer Specialists, Philadelphia, PA). The values are the mean of three determinations. Zn2⫹ and Cu2⫹ inhibit LPS-induced activation of NF-B and IKK Our results suggest that auranofin inhibits NF-B activation in RAW 264.7 cells by blocking IKK, and the incubation of cells with metal compounds containing zinc, copper, or mercury can also inhibit NF-B activation. We thus examined the effect of these metal ions on the expression of TNF mRNA, NF-B activation, and IKK induction in RAW 264.7 cells. As shown in Fig. 4A, while addition of ZnSO4 up to 60 M concentration did not inhibit induction of TNF mRNA, it was reduced to basal levels at 120 M ZnSO4. LPS induction of TNF mRNA was also blocked by CuSO4, although inhibition was observed at the 500-1000 M range. The effect of HgCl2 could not be determined because of its extreme toxicity to RAW 264.7 cells. The viability of cells incubated with 1 mM ZnSO4 or 1 mM CuSO4 was ⬎80%. As observed in cells treated with auranofin (Fig. 1), ZnSO4 and CuSO4 suppressed LPS-induced increase in nuclear B-binding activity, and degradation of IB␣ and IB proteins (Fig. 4, C–E). Induction of IKK activity was also blocked by Zn2⫹ and Cu2⫹ at the same concentration ranges that inhibit TNF gene expression and NF-B activation (Fig. 4F). Modulation of IKK activity by thiol-reactive agents Our results that metals such as gold, zinc, and copper inhibit IKK activity in vitro and induction of IKK in LPS-stimulated macrophages suggest that a metal-sensitive thiol group exists in IKK complex and plays a critical role in the regulation of enzyme activity. Therefore, we tested whether IKK activity is affected by other thiol-modifying agents. Reducing agent DTT was routinely added to the reaction mixture at 1 mM concentration. When we varied the amount of DTT added to the IKK reaction mixture in the 0 –10 mM range, enzyme activity changed accordingly, indicating that reduced state of IKK is required for an optimal activity (Fig. 5A). In the absence of added DTT, the enzyme activity was 36% of 1 mM DTT control. Aurothiomalate added in submaximal inhibitory dose of 10 M decreased IKK activity depending on the FIGURE 4. Zn2⫹ and Cu2⫹ inhibit TNF synthesis by blocking IKK activation. RAW 264.7 cells were incubated for 2 h with or without various doses of ZnSO4 or CuSO4 and stimulated with LPS, as described in Fig. 1. A and B, Northern blot analysis of TNF and GAPDH mRNA. C, EMSA of NF-B in nuclear extract. D and E, Western blot analysis of IB␣ and IB in the cytosol. n.s., Nonspecific band. F, RAW 264.7 cells treated with various doses of ZnSO4 or CuSO4 were stimulated with LPS for 15 min. IKK immunopurified from the cell lysate was used in in vitro kinase reactions performed with GST-IB␣ and [␥-32P]ATP. The data represent two independent experiments. concentration of DTT in the reaction mixture. Aurothiomalate blocked IKK activity almost completely in the absence of DTT, while its inhibitory effect was no longer evident in the presence of 10 mM DTT. In contrast to DTT, addition of thiol-blocking agents, N-ethylmaleimide and p-hydroxymercuribenzoate, reduced in vitro IKK activity in a dose-dependent way (Fig. 5, B and C). Both agents inhibited enzyme activity by 60% at 0.3 mM concentration and almost completely at 1 mM concentration. The modes of inhibition, however, were different between the two agents; while low dose of N-ethylmaleimide reduced enzyme activity by 45%, inhibitory effect of p-hydroxymercuribenzoate appeared more gradually. Aurothiomalate added along with thiol-blocking agents showed a partial additive inhibitory effect at low doses of thiolblocking agents, while no such effect could be observed at 0.3 mM concentration (Fig. 5, B and C). In vitro activities of other stressactivated protein kinases, ERK1/2, JNK/SAPK, and p38 kinase, were not dependent on the presence of reducing agent in the reaction buffer, and they were active in the absence of DTT (data not shown). However, addition of sulfhydryl-reactive N-ethylmaleimide at 1 mM concentration blocked activities of ERK 1/2 and JNK/ SAPK, but not of p38 kinase. Effect of thiol-modifying agents on the activity of overexpressed IKK␣ and  IKK signalsome is known to exist in cells predominantly in IKK␣IKK heterodimeric form (11, 12, 15, 18), although a minor portion of IKK complex containing IKK homodimer was also identified in HeLa cells (20). To determine which subunit is involved in the inhibition of IKK by metal and thiol-reactive agents, COS-7 cells were transfected independently with IKK␣-HA and IKKFlag expression plasmids, and overexpressed IKK enzyme was Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 metal ions such as Zn2⫹, Hg2⫹, and Cu2⫹, known to have properties similar to gold in binding to thiol and imidazole groups on a protein (36). These metal ions and gold compounds blocked IKK activity with a half-maximal inhibitory dose (ID50) of 8 –27 M, whereas inhibition by CoCl2 and Na2Cr2O7 appeared at higher concentrations. The following compounds tested were not effective in blocking IKK activity or at most inhibitory at ⬎100 M concentrations: CaCl2, FeSO4, FeCl3, NiCl2, (NH4)6Mo7O24, CdCl2, cis-Pt(NH3)2Cl2 (cisplatin), and lead acetate. 5986 INHIBITION OF IB KINASE BY THIOL-REACTIVE METALS isolated by immunoprecipitation using corresponding anti-tag Ab. The immune complexes were used to measure kinase activity with GST-IB␣ as a substrate (Fig. 6A). Because IKK␣ expressed alone FIGURE 6. Inhibition of homodimeric IKK␣ and IKK by aurothiomalate and other thiol-modifying agents. A, COS-7 cells were transfected either with vector control (lane 1), or expression vectors for IKK␣-HA and NIK (lane 2) or IKK-Flag (lane 3). After 48 h, cells were lysed and aliquots of lysate were immunoprecipitated with anti-HA (lanes 1 and 2) or anti-Flag Ab (lane 3). The amounts of cell lysate protein used for immunoprecipitation were 120 g (lanes 1 and 2) and 2 g (lane 3). Immune complexes were assayed for kinase activity with GST-IB␣ and [␥-32P]ATP. B, Cell extract (lane 1) and immunoprecipitated complex (lanes 2 and 3) obtained from cells transfected as in A were analyzed by immunoblotting using Ab to IKK␣ and IKK. Aliquots of cell lysate containing 40 g protein were used in all lanes. C, IKK␣-HA and IKK-Flag immune complex obtained as described in A were subjected to in vitro IKK assay in the presence (⫹) or absence (⫺) of aurothiomalate (ATM), DTT, N-ethylmaleimide (NEM), or p-hydroxymercuribenzoate (pHMB). The reaction products were separated by SDS-PAGE, and phosphorylated GSTIB␣ was measured by autoradiography and phosphor imager analysis. The data represent three experiments. in the absence of stimuli exhibited diminished IB kinase activity, we coexpressed one of the IKK-activating upstream kinase, NIK, along with IKK␣-HA (37– 41). In constrast, IKK-Flag overexpressed in COS-7 cells showed high constitutive activity. In our result, activity of IKK-Flag was 140-fold higher than IKK␣-HA isolated from cells cotransfected with NIK on the basis of cellular protein level. Immunoblotting analysis of IKK␣-HA and IKKFlag immune complexes with anti-IKK␣ and IKK Ab revealed that the isolated IKK complexes are composed mainly of the homodimeric forms of expressed subunits (Fig. 6B). Endogenous IKK subunits that might have formed heterodimeric complex with the expressed enzyme were not detected by immunoblotting analysis. Polyclonal Ab to IKK␣ used for immunoblotting analysis cross-reacted with IKK, while anti-IKK mAb did not show cross-reactivity to IKK␣. We tested the effect of aurothiomalate and other thiol-modifying agents on the kinase activity of immunoprecipitated IKK␣-HA and IKK-Flag (Fig. 6C). Phosphor imager analysis revealed that 60 M aurothiomalate blocked IKK␣-HA activity by 68%, while IKK-Flag was only slightly inhibited at the same concentration of aurothiomalate. In contrast, while removal of reducing agent DTT from the reaction mixture reduced IKK␣-HA activity moderately to 47% of control, it suppressed the activity of IKK-Flag to 19% of 1 mM DTT control. N-ethylmaleimide and p-hydroxymercuribenzoate (1 mM each) suppressed activity of both kinases almost completely. Discussion We have shown in this study that an antirheumatic gold compound, auranofin, inhibits TNF gene expression in RAW 264.7 macrophages by blocking NF-B activation. The inhibitory effect of auranofin was associated with suppression of IB␣ and IB degradation, as well as the suppression of IKK activation. In a previous study, auranofin was shown to inhibit induction of TNF and IL-1 mRNA in LPS-stimulated mouse peritoneal macrophages (30). In view of the critical role of NF-B in the transcriptional activation of both TNF and IL-1 genes, these results indicate that auranofin inhibits expression of these cytokines by blocking LPS-induced activation of IKK, and thereby NF-B activation. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 5. Reducing and thiol-reactive agents modulate IKK activity in vitro. IKK immune complex was obtained from RAW 264.7 cells 15 min after LPS stimulation. In vitro IKK reaction was conducted with GST-IB␣ and [␥-32P]ATP in the presence or absence of various concentrations of DTT (A, DTT), N-ethylmaleimide (B, NEM), or p-hydroxymercuribenzoate (C, pHMB). In another set of samples, aurothiomalate (ATM, 10 M) was added along with the thiol-modifying agents. A, The IKK immune complex was washed in kinase buffer containing corresponding concentrations of DTT before enzyme reaction. B and C, Dithiothreitol (1 mM) was added to buffers for IKK immune complex washing and enzyme reaction. Enzyme activity calculated from the radioactivity of phosphorylated GST-IB␣ is presented as percentage of the control assay containing 1 mM DTT. Each value represents the mean ⫾ SD of three independent measurements. The Journal of Immunology questering the metal ions (data not shown). These results suggest that there is a sulfhydryl group in IKK that is critical for the enzyme activity, and the metal ions inhibit IKK activity by binding to this site. Structural data of IKK subunits reveal that cysteine residues are present in the kinase domain of IKK␣ and IKK and some of them located at functionally important sites such as activation T loop and the catalytic site (11–15). Oxidation of this sulfhydryl group to disulfide, binding to metal ions, or blocking with thiol-reactive agents seems to inactivate the enzyme. On the other hand, the presence of large amounts of thiol compound such as DTT could probably prevent metal-induced enzyme inactivation by sequestering the metal ions. After submission of the manuscript, Rossi et al. (44) reported that cyclopentenone PGs (PGA1 and 15-deoxy-⌬12–14-PGJ2) inhibit NF-B and IKK activation in cells stimulated with TNF, IL-1, or PMA, and block activity of IKK complex in vitro. They showed that expression of mutant IKK, in which Cys179 located within the activation loop was replaced with alanine, rendered cells to become resistant to inhibition by 15-deoxy-⌬12–14-PGJ2, suggesting that cyclopentenone PGs inhibit IKK activity by modifying this cysteine residue. This finding further supports our conclusion that gold and other thiolreactive agents may inhibit IKK by directly modifying cysteine thiols in IKK␣ and  molecules. Our result that in vitro IKK activity requires the presence of reducing agent in the reaction mixture suggests that alteration in cellular oxidation/reduction (redox) status can modulate signalinduced activation of NF-B. In view that other stress-activated protein kinases were fully active in the absence of reducing agent in the reaction buffer, this redox sensitivity of IKK seems to be unique among protein kinases. It seems plausible that certain critical cysteine residues in IKK may act as a redox-sensitive sulfhydryl switch to control the enzyme activity. Previous studies suggested that NF-B activity is regulated by the intracellular reactive oxygen species levels, in that H2O2 can induce NF-B activation in a number of cell types (45– 47). Moreover, NF-B activation by diverse signals was shown to be blocked by various structurally unrelated antioxidants (48), and by overexpression of antioxidant enzymes (47–50). The source of oxygen radicals in the signaling pathways of NF-B activation by IL-1 was suggested to be either 5-lipoxygenase or NADPH oxidase pathways in lymphoid and monocytic cells, respectively (51). These results suggest that a shift in the cellular redox equilibrium to an oxidized state should induce NF-B activation, while alteration to more reduced status should inhibit NF-B activation. However, it was also shown that depletion of cellular glutathione in Molt-4 T cells caused inhibition of TNF-induced NF-B activation and its nuclear translocation (52). In addition, chronic exposure of human T cells to oxidative stress suppressed NF-B-dependent transcriptional activity by blocking phosphorylation and degradation of IB␣ (53, 54). These results and our result of in vitro IKK assays suggest that, although reactive oxygen species are involved in NF-B activation, oxidation and depletion of cellular thiols lead to inactivation of IKK. Our result also suggests that IKK does not act as a sensor molecule of reactive oxygen species in the signaling pathway that leads to NF-B activation following cellular stimulation. It rather indicates that oxygen radicals generated by NF-B-inducing signals may negatively regulate the IKK activity by shifting the cellular redox equilibrium to more oxidized status and contribute to its transient activation in cells stimulated with various stress signals. The IKK immune complex that we used in the kinase reaction should contain subunits such as IKK␣ and IKK, which catalyze Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Our results showed that auranofin inhibits activation of IKK in various cell types (RAW 264.7, HeLa, and U937 cells) induced by different stimuli (LPS, TNF, and PMA/ionomycin, respectively). These results suggested that there is a common auranofin-susceptible step in these diverse signal pathways for NF-B activation. When we examined the effect of auranofin and other gold compounds on in vitro IKK activity, hydrophilic gold compounds, such as aurothiomalate, aurothioglucose, and AuCl3, suppressed enzyme activity, whereas lipophilic auranofin was relatively ineffective. Because these results suggested that IKK is susceptible to gold, we speculated that inhibition of IKK induction in auranofintreated cells is due to blocking of IKK activity by a reactive gold compound derived from auranofin after its cellular uptake. Our experiment with lysates of auranofin-treated cells supported this hypothesis in that they significantly suppressed exogenous IKK activity. The lysate obtained from cells incubated with 10 M auranofin reduced the activity of exogenous IKK by 30%. However, at this concentration of auranofin, the innate enzyme should be completely suppressed. This discrepancy is most likely due to dilution (about 5- to 10-fold) of cellular contents that occurs during the preparation of cell lysates. In our test with other stress-induced protein kinases, ERK1/2, JNK/SAPK, and p38 kinase, aurothiomalate had no effect on the enzyme activities. These results suggest that the inhibitory effect of auranofin on IKK induction appears by gold-induced inactivation of IKK and the inhibition of IKK activity by gold is relatively specific. The inhibitory effect of auranofin was observed at 5–10 M range in our result with RAW 264.7 cells, while auranofin blocked induction of TNF and IL-1 mRNA over 0.1–1 M concentration range in murine peritoneal macrophages (30). This discrepancy is likely to have been caused by the presence of 5% FCS in the medium that we used to add auranofin to the cells, while serumfree medium was used in the previous study. Sulfhydryl group of albumin in FCS was shown to significantly interfere with cellular association of auranofin gold, and only a fraction of the auranofin gold is taken up by the cells in the presence of FCS (42). Considering that whole blood levels of gold in RA patients treated with auranofin are 0.5–7.5 M (43), the concentrations of auranofin used in this study should be in the range that is relevant to its in vivo effect. However, it is not clear whether the gold in the blood or synovial tissues in auranofin-treated patients is in the form that can be readily taken up by the immune cells. Therapeutic effectiveness of auranofin and other gold compounds may depend on various parameters that control absorption and metabolism of these compounds, transport to the inflamed tissues, uptake by inflammatory cells, and the status of metal-binding ligands in those cells that might compete with IKK for binding of metals. In vitro IKK assay revealed that metal compounds containing gold, zinc, copper, and mercury block IKK activity at 1–100 M concentration ranges. These metal ions are commonly known to interact with sulfur- or nitrogen-containing groups of an enzyme such as thiol and imidazole groups (36). When we measured enzyme activity in reaction buffer containing various concentrations of a reducing agent, DTT, the IKK activity was shown to change according to the doses of added DTT. Moreover, thiol groupblocking agents, N-ethylmaleimide and p-hydroxymercuribenzoate, inhibited IKK activity, suppressing the enzyme activity completely at 1 mM concentrations. Aurothiomalate added in the absence of DTT further reduced IKK activity, whereas excess DTT significantly attenuated the inhibitory effect of aurothiomalate. In another experiment, addition of cysteine-rich metallothionein-I peptide, which binds metal ions with high affinity, could abrogate the inhibitory effect of aurothiomalate and zinc, probably by se- 5987 5988 Acknowledgments We thank Dr. B.-S. Shim and W. Kim for critically reading the manuscript, Yuhan Medica for auranofin, Dr. F. Mercurio for pRcActin-3⫻HAIKK-1 and pFlagCMV2-IKK-2, and Dr. J.-H. Kim for pFlagCMV-NIK. References 1. Barnes, P. J., and M. Karin. 1997. Nuclear factor-B: a pivotal transcription factor in chronic inflammatory diseases. N. Engl. J. Med. 336:1066. 2. May, M. J., and S. Ghosh. 1998. Signal transduction through NF-B. Immunol. Today 19:80. 3. Handel, M. L., L. B. McMorrow, and E. M. Gravallese. 1995. Nuclear factor-B in rheumatoid synovium: localization of p50 and p65. Arthritis Rheum. 38:1762. 4. Marok, R., P. G. Winyard, A. Coumbe, M. L. Kus, K. Gaffney, S. Blades, P. I. Mapp, C. J. Morris, D. R. Blake, C. Kaltschmidt, and P. A. Baeuerle. 1996. Activation of the transcription factor nuclear factor-B in human inflamed synovial tissue. Arthritis Rheum. 39:583. 5. Firestein, G. S., J. M Alvaro-Gracia, R. Maki, and J. M. Alvaro-Gracia. 1990. Quantitative analysis of cytokine gene expression in rheumatoid arthritis. J. Immunol. 144:3347. 6. Elliott, M. J., R. N. Maini, M. Feldmann, J. R. 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When we transfected COS-7 cells with IKK␣ and IKK expression plasmids and isolated IKK complex by immunoprecipitation with anti-tag Ab, only expressed IKK subunit was detected in each immune complex upon immunoblotting analysis, suggesting that they contain IKK enzyme of mainly homodimeric form of IKK␣ or IKK. Incubation of these immune complexes with aurothiomalate and other thiol-modifying agents resulted in suppression of both IKK␣ and  activities. This result suggests that thiolmodifying agents target both IKK␣ and IKK in IKK signalsomes, and similar sulfhydryl groups in IKK␣ and  are involved in regulation of enzyme activity. However, the precise inhibition mode seems different between the two IKK subunits in that IKK complex containing IKK␣ was more susceptible to inhibition by aurothiomalate than that of IKK, while depletion of reducing agent DTT in the reaction mixture preferentially inactivated IKK complex containing IKK. Our results show that zinc and copper, which block IKK in vitro, also inhibit TNF expression and induction of NF-B and IKK in LPS-stimulated RAW 264.7 macrophages. These results demonstrate that zinc and copper inhibit NF-B activation by blocking IKK and provide a rationale for the reported inhibitory effects of these metal ions on NF-B activation and immune responses. In previous studies, zinc was shown to inhibit NF-B activation in endothelial cells (55), and pyrrolidine dithiocarbamate-induced inhibition of NF-B activation in cerebral endothelial cells was also shown to depend on the presence of zinc in the medium (56). In Jurkat T cells, Cu2⫹ was shown to inhibit NF-B activation through blocking signal-induced phosphorylation and degradation of IB␣ (57). Antiinflammatory and antirheumatic actions of zinc and copper are well documented (58), and administration of high doses of zinc was associated with a reduced immune response in healthy persons (59). Chronic inflammatory processes such as RA are known to promote the redistribution of Zn2⫹ and Cu2⫹ in body compartments, and accumulation of these metal ions in inflamed sites (60), suggesting their possible role as a physiologic regulator of inflammatory responses. However, the physiological relevance of inhibition of NF-B activation by these metal ions is not clear, because the level of zinc and copper in serum and synovial fluid (5- 20 M) (61) is far lower than the doses of these metal ions that we observed to block NF-B activation in cultured cells (100 – 1000 M). Previous studies showed that glucocorticoids inhibit NF-B activation by the binding of glucocorticoid-receptor complexes with NF-B and also by promoting transcription of IB␣ gene (1). This effect of glucocorticoids should account for their suppressive activity in the expression of genes involved in inflammatory responses. 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