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Biochemical and Molecular Actions of Nutrients

Curcumin Blocks Interleukin-1 (IL-1) Signaling by Inhibiting the Recruitment of the IL-1 Receptor–Associated Kinase IRAK in Murine Thymoma EL-4 Cells¹

Nadine Jurrmann^*,, Regina Brigelius-Flohé^*, and Gaby-Fleur Böl^*,2

^* German Institute of Human Nutrition, Potsdam-Rehbruecke, Biochemistry of Micronutrients, and Institute of Nutritional Science, University of Potsdam, 14558 Nuthetal, Germany

²To whom correspondence should be addressed. E-mail: boel{at}mail.dife.de.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Curcumin is a dietary compound with diverse anti-inflammatory and anticarcinogenic effects in several experimental models. A mechanism by which curcumin exerts these actions might be the direct modification of protein thiols, thereby altering the activity of the affected proteins. An early event in inflammatory signaling cascades is the recruitment of the interleukin-1 (IL-1) receptor–associated kinase (IRAK) to the IL-1 receptor (IL-1RI) upon stimulation with IL-1. IRAK recruitment was shown recently to be inhibited by agents that modify thiols of IRAK. We asked, therefore, whether IRAK is also a target for curcumin. Curcumin indeed blocked IRAK thiols in a murine T-cell line stably overexpressing IRAK (EL-4^IRAK), which resulted in the inhibition of IRAK recruitment to the IL-1RI and phosphorylation of IRAK and IL-1RI-associated proteins. Inhibitory effects were not reversible by thiol-reducing agents. Thus, modification by curcumin did not occur by oxidation but rather by alkylation, as is typical for electrophilic compounds reacting as Michael addition acceptors. The block in one of the earliest events in the IL-1 signaling cascade can explain the often observed inhibition of IL-1-mediated signaling steps by curcumin further downstream. Hence, thiol modification might be a crucial step in the anti-inflammatory functions of curcumin.

KEY WORDS: • curcumin • interleukin-1 receptor-associated kinase • redox regulation • thiol modification

Various plant-derived chemicals have been described as anti-inflammatory and anticarcinogenic. Curcumin (diferuloylmethane) is such a substance (Fig. 1); it has been of interest for several years. Curcumin is the yellow pigment and active component of turmeric (Curcuma longa) and gives the specific flavor and yellow color to curry.

View larger version (6K): [in this window] [in a new window]   FIGURE 1 The chemical structure of curcumin.

In cell culture studies, curcumin inhibited the proliferation of human peripheral blood mononuclear cells (3) and suppressed the phorbol 12-myristate 13-acetate–induced activation of c-jun/activator protein 1 (AP-1) in mouse fibroblasts (4), a key event in tumor promotion. Activation of the c-jun N-terminal kinase by various agonists was inhibited by curcumin in Jurkat T cells (5). It also reduced AK-5 tumor (a rat histiocytoma) growth by activation of caspase-3, thereby inducing apoptosis (6). Curcumin-mediated induction of apoptosis was also observed in tumor cell lines by the release of cytochrome c, activation of caspases (7,8), fragmentation of DNA (9), and inhibition of Akt (10).

Curcumin dose dependently reduced the mRNA expression of inducible nitric oxide synthase (11). The enzymatic activity of COX-2 was only weakly diminished by curcumin (12), whereas the catalytic activity of lipoxygenase was significantly inhibited in mouse epidermal cells, and the synthesis of leukotrienes and 5-hydroxyeicosatetraenoic acid was reduced (13). Studies on intracellular signaling showed that curcumin inhibits late events in the tumor necrosis factor- (TNF-)– and interleukin-1 (IL-1)–mediated signaling cascade, e.g., inhibitor of B (IB) kinase (IKK) activation and, thus, nuclear factor-B (NF-B) activation (14,15), finally blocking expression of the intercellular adhesion molecule-1 (15,16). It abolished the cytokine-mediated phosphorylation of IB by inhibiting the activity of IKK, thereby preventing the induction of COX-2 (17).

The effects of curcumin were first attributed to its antioxidant properties in vitro. Curcumin was reported to scavenge O₂^–·, thereby inhibiting the generation of H₂O₂ in rat liver mitochondria (18). Other effects of curcumin cannot easily be explained by an antioxidant function. The curcumin molecule has the structural feature of a Michael reaction acceptor (an ,ß-unsaturated carbonyl group) (19). As such, it elevates the expression of phase-2 detoxification enzymes such as -glutamylcysteine synthetase, glutathione-S-transferase, and NAD(P)H:quinone oxidoreductase (20). The underlying mechanism is the activation of the nuclear factor erythroid 2-related factor 2/Kelch-like ECH-associated protein 1 (Nrf2/Keap1) system (21,22). Nrf2 is sequestered in the cytosol by Keap1. Keap1 contains 25 cysteine residues, from which C273 and C288 are required for the Keap1-mediated retention of Nrf2 under basal culture conditions. C151 becomes modified by sulforaphane or oxidative stress (23). Upon thiol modification, the conformation of Keap1 is changed; Nrf2 is released, translocates into the nucleus, and activates genes by binding to the antioxidant-responsive element (21). By this mechanism, curcumin activates Nrf2, leading to the induction of heme oxygenase-1, which has been classified as antioxidant enzyme (22). Thus, curcumin acts as a thiol-modifying agent rather than as an antioxidant.

To further elucidate the mechanisms of the anti-inflammatory effects of curcumin, we studied early signaling events mediated by IL-1. IL-1 is a potent inflammatory cytokine with various biological activities regulating host defense and immune response (24). It exerts its action via the IL-1 receptor type I (IL-1RI) (25). Upon binding of IL-1, adapter molecules, such as the myeloid differentiation marker MyD88 (26) and the Toll-interacting protein Tollip (27), and kinases, such as IL-1 receptor-associated kinase (IRAK) (28,29) and IRAK-4 (30), are recruited to the receptor. IRAK becomes phosphorylated (31), presumably first by IRAK-4 (30), then autonomously. Activation of this cascade finally leads to the activation of the transcription factor NF-B. We showed recently that IRAK is a target for thiol modification (32). Modification of IRAK thiols by diamide, menadione, or phenylarsine oxide (PAO) prevented the recruitment of IRAK to the receptor, one of the earliest steps in the IL-1 signaling cascade (32). Downstream events such as activation of IL-1RI-associated kinases or NF-B or the expression of adhesion molecules were also inhibited (32–34).

We asked, therefore, whether curcumin can similarly block the IL-1 signaling cascade. We show here that curcumin modifies thiol groups in IRAK and prevents the recruitment of IRAK to the IL-1RI. Hence, curcumin can block the IL-1 signaling pathway at the most early step, explaining its inhibitory effect on further downstream events in proinflammatory pathways.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
    Cell culture and treatment. The murine thymoma cell line EL-4 6.1 stably overexpressing IRAK (EL-4^IRAK) was a kind gift of J. Knop and M. U. Martin, Hannover Medical School. Cells were cultured as described by Böl et al. (32). Curcumin (Sigma) was dissolved in 100% ethanol to a concentration of 20 mmol/L; 2 x 10⁷ cells were pretreated for 15 min at 37°C in cell culture medium with the indicated doses of curcumin (0–50 µmol/L) or with ethanol vehicle (0.25%), washed once with medium lacking additives, and then left untreated or stimulated with 5 µg/L recombinant human IL-1ß (GBF) for 5 min at 37°C. PBS-washed cells were lysed in 1 mL lysis buffer containing 1% Brij97 (Sigma); 50 mmol/L NaCl; 50 mmol/L Tris, pH 7.4; 5 mmol/L EDTA; and the protease inhibitors pepstatin (1 mg/L, Calbiochem) and pefabloc® (1 mmol/L, Roth) at 4°C for 30 min.

    Immunoprecipitation (IP) and in vitro kinase assay. Lysates were incubated with 0.35 µg anti-IRAK antibody (UBI) or 1 µg anti-IL-1RI antibody (Pharmingen) and 30 µL of a protein G-Sepharose slurry at 4°C overnight. Immunoprecipitates were washed and centrifuged (1000 x g for 2 min) 3 times in lysis buffer (see above) and 3 times in kinase buffer consisting of 20 mmol/L Hepes, 100 mmol/L NaCl, 5 mmol/L MnCl₂, and 5 mmol/L MgCl₂, pH 6.5. Pellets were resuspended in 45 µL kinase buffer, and an in vitro kinase assay was performed by adding 37 kBq [-³²P]-ATP for 10 min at room temperature. The reaction was stopped by the addition of kinase buffer containing 40 mmol/L EDTA followed by 2 washing steps with the same buffer. The samples were boiled in Laemmli buffer for 5 min, and the supernatants were separated on 7.5% SDS-PAGE. Phosphorylated IRAK was made visible by autoradiography using Kodak X-Omat films.

    Western blot (WB) analysis. Washed immunoprecipitates were separated on 7.5% SDS-polyacrylamide gels and blotted to nitrocellulose (2 h, 1.2 mA/cm², 4°C). The blots were blocked in Tris-buffered saline containing 0.1% Tween 20 (TTBS) with 5% nonfat dry milk at room temperature for 1 h and subsequently incubated with an anti-IRAK antibody in TTBS (1 mg/L, Santa Cruz Biotechnology). Immunoblot analyses were performed with horseradish peroxidase–conjugated goat anti-rabbit secondary antibody (1:50000) and proteins were visualized by chemiluminescence imaging (using Supersignal West Dura, Perbio) with the Fuji LAS 1000-CCD-camera system.

    Labeling of free sulfhydryl (SH) groups with iodo-acetyl-[¹²⁵I]-iodotyrosine ([¹²⁵I]-IAIT). [¹²⁵I]-IAIT was synthesized as described by the method of Gitler (35). Immunoprecipitates from 1 x 10⁸ EL-4^IRAK cells were washed 3 times in lysis buffer and 3 times in washing buffer consisting of 50 mmol/L Hepes, pH 7.4; 100 mmol/L NaCl; and 1 mmol/L EDTA. The immunoprecipitates were incubated with 185 kBq [¹²⁵I]-IAIT in washing buffer (50 mmol/L NaCl instead of 100 mmol/L) for 1 h at 37°C and subsequently washed 3 times. The samples were boiled in Laemmli buffer for 5 min and proteins were separated on 10% SDS-PAGE. [¹²⁵I]-IAIT-labeled SH groups were detected by autoradiography on Kodak BioMax MS films.

    Quantification of [¹²⁵I]-IL-1 binding to the IL-1RI. IL-1 was labeled with [¹²⁵I] using the chloramine T method (36); 2 x 10⁷ cells were pretreated for 15 min at 37°C with the indicated doses of curcumin (0–50 µmol/L) and incubated in 1 mL medium containing 0.1% sodium azide, 1% bovine serum albumin, and 20 mmol/L Hepes, pH 7.4, for 30 min at 4°C without or with a 300-fold molar excess of unlabeled IL-1 or IL-1ß. Then, 7.4 kBq [¹²⁵I]-IL-1 (106 fmol) was added to each sample for 3 h at 4°C. Labeled cells were separated from excess [¹²⁵I]-IL-1 by centrifugation through phthalic acid-bis-2-ethylhexyl ester:phthalic acid-dibutyl ester (1:1.5, Fluka) for 2 min at 8800 x g. Bound [¹²⁵I]-IL-1 was measured using a counter.

    Statistical analysis. The WBs shown and autoradiographies are representative of at least 3 independent experiments. The data of Figure 6 were analyzed by 1-way ANOVA with Dunnett’s post-hoc test.

View larger version (14K): [in this window] [in a new window]   FIGURE 6 Curcumin only slightly affects the interaction of IL-1 and the IL-1RI in EL-4IRAK cells. EL-4IRAK cells were pretreated with ethanol vehicle or with the indicated concentrations of curcumin and incubated with [125I]-IL-1. For competition analysis, unlabeled IL-1 or IL-1ß was added before incubation with the radioligand. [125I]-IL-1 bound to the IL-1 receptor was measured. The relative radioactivity (routinely >303 Bq) of the sample without curcumin (ethanol vehicle) was set as 100%. Data are means ± SD of at least 3 independent experiments. **Different from the sample without curcumin (ethanol vehicle), P < 0.01.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

View larger version (52K): [in this window] [in a new window]   FIGURE 2 Recruitment of IRAK to the IL-1RI is enhanced in EL-4IRAK cells. EL-4 and EL-4IRAK cells were stimulated with IL-1ß or left untreated as indicated. Lysates were incubated either with an anti-IRAK antibody or with an anti-IL-1RI antibody (IP) and IRAK was detected by WB. Phosphorylated IRAK is marked with a line and the heavy chain of the antibody with "H." Molecular masses are indicated in kDa on the left.

    Curcumin blocks IL-1-mediated recruitment of IRAK to the IL-1RI. The effects of curcumin on IRAK recruitment were analyzed by treatment of the cells with curcumin and subsequent stimulation with IL-1ß (Fig. 3A). As shown before (Fig. 2), IL-1RI-associated IRAK was detectable only upon IL-1 stimulation and characterized by the shifted band of 80–100 kDa, indicating phosphorylation (Fig. 3A, lane 2). Preincubation of cells with curcumin dose dependently inhibited IL-1–induced recruitment of IRAK to the IL-1RI (Fig. 3A, lanes 3–8).

View larger version (67K): [in this window] [in a new window]   FIGURE 3 Curcumin inhibits the recruitment of IRAK to the IL-1RI in EL-4IRAK cells. EL-4IRAK cells were preincubated with the indicated concentrations of curcumin. Cells were stimulated with IL-1ß or left untreated. (A) The IL-1RI was immunoprecipitated (IP) and IRAK was detected by WB. (B) IL-1RI-IP was applied to an in vitro kinase assay. Phosphorylated IRAK is marked with a line and p60 with an arrow. Ethanol used to dissolve curcumin did not affect the IL-1–induced recruitment of IRAK (A and B, lane 2). Molecular masses are indicated in kDa on the left.

    Curcumin inhibits IL-1-induced phosphorylation of IRAK. Phosphorylation of IRAK at the IL-1RI could have been catalyzed by IRAK itself or by another kinase that was also associated at the IL-1RI and, thus, coprecipitated with the anti-IL-1RI-antibody. Therefore, IRAK was precipitated directly (Fig. 4A). Without IL-1-stimulation, IRAK was present at a molecular weight of 80 kDa in accordance with its unphosphorylated state (Fig. 4A, lane 1). Treatment with IL-1 led to the precipitation of phosphorylated IRAK, as indicated by the shift to 80–100 kDa (Fig. 4A, lane 2). The phosphorylation of IRAK was dose dependently inhibited by curcumin (Fig. 4A, lanes 3–8).

View larger version (50K): [in this window] [in a new window]   FIGURE 4 Curcumin inhibits IL-1–induced phosphorylation of IRAK in EL-4IRAK cells. EL-4IRAK cells were treated as described in Figure 3. (A) IRAK was immunoprecipitated (IP) and detected by WB. (B) IRAK-IP was applied to an in vitro kinase assay. Phosphorylated IRAK is marked with a line and the heavy chain of the antibody with "H." Ethanol used as a solvent did not affect IL-1–induced phosphorylation of IRAK (A and B, lane 2). Molecular masses are indicated in kDa on the left.

    Curcumin modifies thiols of IRAK. To investigate whether curcumin modifies IRAK thiols, free SH groups in IRAK were labeled with [¹²⁵I]-IAIT. Thiols of IRAK were labeled with [¹²⁵I]-IAIT only in IL-1-stimulated cells (Fig. 5, compare lane 1 and 2). This might indicate either that IL-1 mediates conformational changes in IRAK in such a way that SH groups become exposed at the surface or that IRAK may be released from high-molecular-weight protein complexes (37) in which thiols are not accessible. Whatever the mechanism, it was inhibited by increasing doses of curcumin (Fig. 5, lanes 3–9). Notably, only unphosphorylated IRAK (80 kDa) was detectable, suggesting that phosphorylation might interfere with the labeling of thiols.

View larger version (25K): [in this window] [in a new window]   FIGURE 5 Curcumin modifies thiols of IRAK in EL-4IRAK cells. EL-4IRAK cells were preincubated with curcumin and stimulated with IL-1ß or left untreated. IRAK-IP was incubated with [125I]-IAIT to label accessible thiols. [125I]-IAIT-labeled IRAK is marked with an arrow. Molecular masses are indicated in kDa on the left.

    Reducing agents do not reverse the effects of curcumin. The inhibitory effect of PAO on NF-B activation could be reversed by strong reductants containing vicinal thiols such as dithiothreitol (DTT) and dimercaptopropanol (33,38). A possible reversibility of the inhibitory effect of curcumin on IRAK phosphorylation by reducing agents was measured by incubation of cells with curcumin and subsequent treatment with DTT, ß-mercaptoethanol (ß-ME), or Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). Phosphorylation of IRAK, detectable at 80–100 kDa, was not altered by the reducing agents (Fig. 7, lane 2 compared with lanes 3–5). The inhibition of IRAK-phosphorylation caused by curcumin (Fig. 7, lane 6 compared with lane 2) was not reversible by incubation of the washed cells for 1 h in medium (Fig. 7, lane 7), incubation with DTT (Fig. 7, lane 8), ß-ME (Fig. 7, lane 9), or TCEP (Fig. 7, lane 10). This shows that PAO and curcumin modify proteins differently.

View larger version (25K): [in this window] [in a new window]   FIGURE 7 Inhibition of IRAK phosphorylation in EL-4IRAK cells by curcumin is not reversed by reducing agents. EL-4IRAK cells were left untreated or preincubated with curcumin. Thereafter, cells were either stimulated with IL-1ß and harvested directly (lane 6) or incubated for 1 h in medium alone (1 h) or with 100 µmol/L DTT (D), 50 µmol/L ß-ME (M), or 100 µmol/L TCEP (T). Cells were then stimulated with IL-1ß as indicated or left untreated. IRAK-IP was applied to an in vitro kinase assay. Phosphorylated IRAK is marked with a line. Molecular masses are indicated in kDa on the left.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Curcumin does not interfere only with IL-1-mediated signals. In brain microglia, cells activated with LPS, interferon- (IFN-), or gangliosides, curcumin inhibited the phosphorylation of the signal transducers and activators of transcription (STATs) STAT1 and STAT3 as well as the Janus kinases (JAKs) JAK1 and JAK2 (42). IRAK was also identified as a conserved component in signal cascades activated by IL-18 (43), TNF- (44), and p75-neurotrophin (45). Toll-like receptors (TLRs) of which 11 members have been cloned to date, recently emerged as triggers of innate and adaptive immune reactions [reviewed in (46)]. They recognize distinct pathogen-associated molecular patterns such as bacterial endotoxin LPS (TLR4), bacterial lipoproteins (TLR2), flagellin (TLR5), unmethylated CpG DNA of bacteria and viruses (TLR9), single-stranded viral RNA (TLR7), and double-stranded RNA (TLR3). TLRs also use IRAK for activation of the IKK complex or mitogen-activated protein kinases, which leads to an NF-B– or AP-1–dependent transcriptional response. Hence, inhibition of IRAK by curcumin might be a general mechanism for interference with different inflammatory signaling cascades.

After recruitment and phosphorylation, IRAK dissociates from the receptor complex and translocates into the nucleus (47). Translocation requires an intact kinase activity. Because IL-1 is still able to activate NF-B in cells transfected with a kinase-defective IRAK (48,49), the function of the kinase activity of IRAK, especially in the nucleus, remains unknown. In a recent publication, however, Huang et al. (50) reported on the phosphorylation of STAT3 by IRAK in the nucleus of peripheral blood mononuclear cells of atherosclerotic patients. This was taken as an explanation for the elevated serum IL-10 concentrations in these patients. Using chromatin IP assays, it could indeed be demonstrated that IRAK binds to the endogenous IL-10 promotor region in THP-1 cells upon LPS stimulation (50). This indicates that IRAK may have still unidentified targets in the nucleus and that it is not restricted to the IL-1 signaling cascade (see above). Thus, prevention of IRAK phosphorylation and subsequent translocation into the nucleus would also suppress a putative gene regulatory function of IRAK.

The mechanism by which curcumin exerts the inhibition of IL-1 signaling is not due to an interference with the binding of IL-1 to its receptor. Up to a concentration of 25 µmol/L, curcumin did not affect the interaction of IL-1 with the IL-1RI, as was also shown in rat primary microglia, in which 10 µmol/L curcumin did not displace IFN- from its receptor (42). Rather, it modifies protein thiols especially of IRAK, as shown by the prevention of thiol labeling with [¹²⁵I]-IAIT. Curcumin affects IRAK activation similarly to other thiol modifying agents, such as diamide, menadione, and PAO (32). The inhibitory effects of PAO were reversible by thiol reductants such as DTT or dimercaptopropanol (33,38), whereas inhibition caused by curcumin was not. Thus, curcumin does not oxidize protein thiols; rather, it alkylates thiols via a Michael addition (20). The reactive structural motif is the ,ß-unsaturated carbonyl group, which makes curcumin a Michael acceptor. OH-radical scavenging would be possible by the very same motif and by the hydroxyl:methoxy moiety, but is unlikely to be physiologically relevant. In particular, proteins containing highly reactive vicinal SH groups (with low pK_a values) are expected to be sensitive to Michael acceptors. This sensitivity of distinct cysteines is increased by flanking basic amino acids as reported for Keap1 (19,22). Because 5 of the 15 cysteine residues in the murine IRAK protein are vicinal thiols and 4 of them are located close to arginine or histidine, this kinase is a likely candidate for easy modification by curcumin.

We report here on the general mechanism of thiol modification for regulating protein activities. It appears also to be relevant for the curcumin-mediated inhibition of the formation of the IL-1 signaling complex. Thiol modification may explain anti-inflammatory (inhibition of IRAK) properties of curcumin, as was suggested for its anticarcinogenic effects (induction of phase 2 detoxification enzymes via Nrf2 activation). It remains to be established whether the common denominator of the diverse functions of curcumin may be the modification of the thiols that are pivotal to signaling cascades.

	ACKNOWLEDGMENTS

 
We thank Jacqueline Scheel, Gerlinde Aust, and Jörg-Uwe Bittner for expert technical assistance.

	FOOTNOTES

 
¹ Supported by the Deutsche Forschungsgemeinschaft (Bo 1174/4–3) and the Sonnenfeld-Stiftung (Berlin, Germany).

³ Abbreviations used: AP-1, activator protein-1; ß-ME, ß-mercaptoethanol; COX-2, cyclooxygenase-2; DTT, dithiothreitol; EL-4^IRAK, EL-4 cells stably overexpressing IRAK; [¹²⁵I]-IAIT, iodo-acetyl-[¹²⁵I]-iodotyrosine; IFN-, interferon-; IB, inhibitor of B; IKK, inhibitor of B kinase; IL-1, interleukin-1; IL-1RI, interleukin-1 receptor type I; IP, immunoprecipitation; IRAK, interleukin-1 receptor-associated kinase; JAK, Janus kinase; Keap1, Kelch-like ECH-associated protein 1; NF-B, nuclear factor-B; Nrf2, nuclear factor erythroid 2-related factor 2; PAO, phenylarsine oxide; SH, sulfhydryl; STAT, signal transducer and activator of transcription; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride; TLR, Toll-like receptor; TNF-, tumor necrosis factor-; WB, Western blot.

Manuscript received 21 January 2005. Initial review completed 16 March 2005. Revision accepted 23 May 2005.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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