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Herbimycin A Abrogates Nuclear Factor-B Activation by Interacting Preferentially with the IB Kinase Subunit

Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan

Received October 31, 2003; accepted March 1, 2004

	Abstract

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NF (nuclear factor)-B is known to be a critical transcription factor in inflammatory responses. We have reported that herbimycin A, a potent Src tyrosine kinase inhibitor, attenuates the NF-B activation triggered by cytokines, bacterial endotoxin, and hydrogen peroxide. Accompanying the suppression by this agent, NF-B-dependent gene expressions, such as cytokine, chemokine, and inducible-type nitric oxide, are specifically inhibited in glial cells. In the present study, we attempted to elucidate the possible target protein for herbimycin A on this pathway. We demonstrate here that herbimycin A preferentially inhibits IKK (IB kinase). Furthermore, substituting alanine for the cysteine at 59 (Cys59) in IKK resulted in the insensitivity to herbimycin A, suggesting that this compound may interact with the Cys59 residue located near the catalytic ATP binding site. Taken together, these results indicate that herbimycin A can be considered a novel candidate for an anti-inflammatory drug agent through its specific inhibition of IKK, which results in prevention of the expression of NF-B-dependent genes implicated in the pathogenesis of inflammatory responses.

We have reported previously that herbimycin A, a potent Src tyrosine kinase inhibitor, suppresses the NF-B activation and subsequent induction of inducible nitric oxide synthase and chemokines by treatment with several stimuli in C6 glioma cells (Uehara et al., 1989, 1998, 1999; Nishiya et al., 1995, 2000). However, the target molecule of herbimycin A in NF-B-activating pathways remains to be identified. In this study, we attempted to elucidate the target protein for herbimycin A. Our results show that herbimycin A selectively inhibits IKK through the possible interaction with cysteine 59, which is located near the catalytic site in the kinase domain in IKK. We concluded that herbimycin A is a novel potent inhibitor of IKK in NF-B signaling.

	Materials and Methods

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Materials. Recombinant human IL-1 and anti-Flag monoclonal antibody M2 were purchased from Sigma-Aldrich (St. Louis, MO). Anti-IKK (M-280), anti-IKK (H-4), anti-IB (C-21), and anti-NF-B (p65) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-phospho IB (Ser32) and phospho IKK (Ser180)/ (Ser181) antibodies were purchased from New England Biolabs (Beverly, MA). Herbimycin A was obtained from Wako Pure Chemicals (Osaka, Japan).

Expression Vectors and Site-Directed Mutagenesis. FLAG epitope-tagged NIK, NIK (K429A/K430A) mutant, IKK (K44A) mutant, and IKK (K44A) mutant were generous gifts from Dr. David V. Goeddel (Tularik Inc., South San Francisco, CA). NIK and IKKs were subcloned into pRK vector. Several mutants of NIK, IKK, and IKK were generated by the overlapping PCR method. To clarify the possible modification site with herbimycin A in IKK, variants of IKK and IKK were prepared by PCR. In addition, seven cysteine residues in the kinase domain of IKK were displaced with an alanine residue by the overlapping PCR method.

Transfection, Immunoprecipitation, and Immunoblotting Analyses. HEK 293 T cells were transiently transfected with expression plasmids (8 µg each) by the calcium phosphate method. After 36 h, cells were washed twice with cold PBS and lysed in lysis buffer [50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF, 1% NP40, and the Complete protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN)]. Immunoprecipitation and immunoblotting analyses were performed as described previously (Ko et al., 2002). In brief, the total cell lysate was centrifuged at 15,000 rpm for 30 min at 4°C. The supernatant was incubated with protein G-Sepharose for 1 h at 4°C and then centrifuged for 10 min. The resultant supernatant was incubated for 16 h with the first antibody, which had been precoupled with protein G-Sepharose. The immunoprecipitates were washed twice with lysis buffer and three times with washing buffer (50 mM Tris/HCl, pH 7.5, 137 mM NaCl, 1 mM EGTA, 1 mM vanadate, and 0.1% Triton X-100). Otherwise, cells were washed twice with ice-cold PBS and added to the SDS sample buffer. Protein (20 µg) from the lysate was fractionated by electrophoresis in 10% polyacrylamide gel and transferred onto a nitrocellulose membrane. Horseradish peroxidase-conjugated anti-mouse or -rabbit IgG (Amersham Biosciences Inc., Piscataway, NJ) was used as the secondary antibody. The antibody-reactive bands were revealed by chemiluminescent detection (enhanced chemiluminescence Western detection kit).

Reporter Assays. For the reporter assays, cells were transfected with 0.25 µg pNF-B Luc vector (BD Biosciences Clontech, Palo Alto, CA) and 1.25 ng of pRL-TK (internal control plasmid). Reporter gene activity was determined with the luciferase assay system (Promega, Madison, WI). In particular, to confirm the possible target site for herbimycin A, Hs683 glial cells were transfected with 5 µg of C59A-mutated IKK together with pNF-B Luc and pRL-TK vectors. The cells were treated with or without herbimycin A and then stimulated with 5.0 ng/ml IL-1 for 6 h.

Immunohistochemistry. To analyze the localization of NF-B (p65 subunit), we used human Hs683 glioma cells transiently transfected with several genes. Cells were fixed in 4% paraformaldehyde, permeabilized in 0.5% Triton X-100, and incubated with anti-NF-B (1:100 dilution) and anti-phospho IKK (1:100 dilution) antibodies for 1 h at 37°C. After reaction with the first antibody, cells were incubated with anti-mouse IgG conjugated with Alexa-488, anti-goat IgG conjugated with Alexa-488, and anti-rabbit IgG conjugated with Alexa-594 (Molecular Probes, Eugene, OR) for 1 h at 37°C. All images were taken on a laser-scanning confocal microscope (LSM510; Carl Zeiss Inc., Thornwood, NY) (Tanaka et al., 2000; Furuta et al., 2003).

In vitro Kinase Assay. HEK 293 cells were transfected with wild-type IKK and then incubated for 36 h. The cells were washed rapidly with PBS and lysed with ice-cold lysis buffer, after which IKK was immunoprecipitated. The immunoprecipitated IKK with protein G-Sepharose beads was collected by centrifugation, then washed three times with lysis buffer and once with kinase assay buffer without ATP. The immunoprecipitates were then suspended in 20 µl of kinase assay buffer (20 mM Tris-HCl, pH 7.6, 10 µM MgCl₂, 100 µM ATP, and 20 µM -glycerophosphate) containing 5 µCi [-³²P]ATP and 1 µgofIB as substrate with or without several concentrations of herbimycin A for 20 min at 25°C. The reaction was stopped by the addition of Laemmli buffer, the proteins were separated by electrophoresis on 10% SDS-PAGE, and phosphorylated IB was visualized by autoradiography or on a Fuji BAS2000 apparatus (Tokyo, Japan). Quantitative data were obtained from Fuji BAStation software.

	Results

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Herbimycin A Inhibited NF-B Activation. We initially examined the inhibitory effect of herbimycin A on IL-1-stimulated reporter gene expression and translocation of the NF-B p65 subunit. Herbimycin A alone (1 µg/ml) did not affect the NF-B activity in this system (data not shown). As shown in Fig. 1A, treatment of human Hs683 glioma cells with herbimycin A resulted in a dose-dependent decrease in the NF-B activity detected by luciferase assay. Immunohistochemical analysis revealed that the NF-B p65 subunit was localized mostly in the cytosol in the quiescent state and translocated to the nucleus at 15 to 30 min after IL-1 treatment. After 60 min, p65 returned to the cytosol (Fig. 1B). Herbimycin A inhibited completely the translocation of p65 induced by IL-1 treatment.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Herbimycin A suppressed the nuclear translocation of NF-B in glial cells. Human Hs683 glioma cells were transiently transfected with pNF-B and together with pRL-TK by Effectene transfection reagent and then treated with or without herbimycin A (A). After 12 h, the transfected cells were stimulated with IL-1 for 6 h. The activity was assayed as described under Materials and Methods. Human glioma cells were incubated with 1 µg/ml herbimycin A for 12 h before treatment with 2.5 ng/ml IL-1 (B). The cells were fixed and subjected to indirect immunofluorescence staining with anti-NF-B pAb. Propidium iodide was used to stain the nuclei.

Effects of Herbimycin A on Phosphorylation of IKK and IB To identify the target molecule for herbimycin A, we investigated the phosphorylation states of IB by overexpressing several genes, such as wild types and kinase-negative mutants of NIK, IKK, and IKK with or without herbimycin A. Transfection of the wild types of NIK and IKK, but not IKK, resulted in significant phosphorylation of IB in a herbimycin A-sensitive manner (Fig. 2A). These results suggest strongly that both NIK and IKK, but not IKK, play a crucial role in NF-B activation. It has been reported previously that transfection of IKK or IKK alone into cells results in the formation of a homodimer (Karin, 1999a,b). However, IKK and IKK exist as heterodimers in cells. Therefore, we examined the effect of herbimycin A on simultaneous cotransfection of IKK and IKK. Although no phosphorylation of IKK and IB was observed by transfection of IKK alone, cotransfection of IKK plus IKK resulted in a marked phosphorylation of IKK (Fig. 2B). Under this condition, the phosphorylation of IKK and IB was attenuated by herbimycin A in a concentration-dependent manner (Fig. 2B). NIK alone also induced the phosphorylation of IKK, IKK, and IB (Fig. 2C). It was interesting that IKK phosphorylation by NIK transfection was insensitive to herbimycin A. Specific phosphorylation of IB by overexpression of NIK was abrogated completely by kinase-negative IKK but not by kinase-negative IKK (Fig. 2D).

View larger version (39K): [in this window] [in a new window]   Fig. 2. Herbimycin A inhibited NIK- and IKK-induced phosphorylation of IB. HEK 293 T cells were transfected with several genes for 24 h and then treated with or without the indicated concentration of herbimycin A for an additional 12 h. The total cell lysates were prepared and then subjected to Western blot analysis using anti-phospho IB (Ser329), anti-phospho-IKKs, anti-IKKs, and anti-FLAG antibodies.

Effects of Herbimycin A on the Protein-Protein Interactions in NF-B Signaling. It is possible that the inhibitory effects described above were a result of the incomplete interaction between signal molecules because of herbimycin A. We therefore investigated whether this compound interferes with the interaction between NIK and IKK or TRAF2. However, neither of these interactions was abrogated by treatment with herbimycin A (Fig. 3, A and B). We further examined the effect of herbimycin A on the binding of recombinant GST-fused IKKs to IB. The significant interactions between GST-IKK or GST-IKK and IB were observed in a herbimycin A-sensitive manner (Fig. 3C).

View larger version (33K): [in this window] [in a new window]   Fig. 3. Effects of herbimycin A on protein-protein interaction. HEK 293 cells were transfected with FLAG-NIK and/or Myc-TRAF2 and then treated with or without herbimycin A (A and B). The lysates were prepared and immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were resolved by SDS-PAGE and then subjected to Western blot analysis with anti-IKK, anti-IKK, anti-FLAG, and anti-Myc antibodies. C, in vitro pulldown assay. GST-fused IKK or IKK precoupled with glutathione-Sepharose 4B was incubated with cell lysates prepared from glial cells for 12 h at 4°C with or without herbimycin A. The precipitated proteins were then resolved by SDS-PAGE and subjected to Western blot analysis with anti-IKKs and anti-IB antibodies.

Herbimycin A Blocked the Kinase Activity of IKK Because overexpression of IKK, but not IKK, resulted in the phosphorylation of IB (Fig. 2A), we studied the inhibitory effects of herbimycin A on IKK kinase activity in vitro. We performed an in vitro kinase assay using the immunoprecipitates with an anti-FLAG antibody from FLAG-IKK-expressing cell lysates and recombinant IB as a substrate.

Herbimycin A dose-dependently impaired the phosphorylation of IB (Fig. 4).

View larger version (13K): [in this window] [in a new window]   Fig. 4. IKK activation was sensitive to herbimycin A. HEK 293 T cells were transiently transfected with the FLAG-epitope tagged IKK. Cell extracts were prepared and immunoprecipitated with anti-FLAG antibody. Then, the immunoprecipitates were incubated with recombinant GST-fused IB as a substrate in vitro. The expression levels of IKK in total cell extracts were determined by immunoblotting with anti-FLAG antibody. Each intensity was quantified using the Fuji BAStation software program. Values are mean ± S.E. of four separate experiments.

Treatment with DTT Disrupted the Inhibitory Effects of Herbimycin A. Because the reducing agents, such as dithiothreitol (DTT), inactivate the reaction of herbimycin A with substrate protein (Senga et al., 2000), we investigated the effects of simultaneous treatment with herbimycin A and DTT on IL-1-induced phosphorylation of IB. Treatment with DTT alone did not affect the state of IB phosphorylation compared with that of nontreatment cells. On the other hand, the protein levels of IB detected with anti-IB antibody were decreased in samples in which the phosphorylation of IB was evident. Although herbimycin A completely disrupted the IB phosphorylation stimulated by IL-1, treatment with DTT recovered the inhibitory effect (Fig. 5).

View larger version (19K): [in this window] [in a new window]   Fig. 5. The inhibitory effect of herbimycin A on NF-B signaling was reversed by DTT treatment. Hs683 cells were pretreated with 10 µM DTT and/or herbimycin A for 12 h and then stimulated with or without 2.5 ng/ml IL-1 for the indicated periods. Cell extracts were prepared and subjected to Western blot analysis with anti-phospho IB (Ser32) and anti-IB antibody.

In addition, we examined the effects of N-acetyl cysteine (NAC) as an another thiol compound. NAC is known to be a potent inhibitor on the NF-B pathway (Schreck et al., 1991; Hayakawa et al., 2003). High concentrations of NAC (more than 10 mM) are usually required to inhibit this pathway, but this condition was cytotoxic to cells in our system (data not shown). Hence, we could not detect the significant inhibition of NF-B activation or the phosphorylation of IKKs and IB caused by challenge with IL-1 or overexpression of several genes.

Herbimycin A Selectively Affected IKK Cys59 Near the Catalytic Site. It is possible that herbimycin A interacts with or modifies IKK and thereby inhibits the NF-B pathway. In this study, we constructed variants of IKK to clarify the possible modification site(s). Each of the seven cysteine residues in the kinase domain of IKK was displaced with an alanine residue (Fig. 6A). Each mutant was transfected transiently, and we then investigated the change in sensitivity of herbimycin A on IKK and IB phosphorylation. None of the variants altered the intrinsic kinase activities (Fig. 6B). However, herbimycin A effectively inhibited the phosphorylation in all mutant-transfected cells, with the exception of the C59A mutant of IKK, which showed insensitivity for herbimycin A. Although IKK has a structure similar to that of IKK (Fig. 6A), significant phosphorylation of IKK and IB was not observed in IKK-transfected cells (Fig. 2A).

View larger version (36K): [in this window] [in a new window]   Fig. 6. Determination of the target site of herbimycin A in IKK . A, Schematic representation of Cys residues at the kinase domain (KD) of IKK and IKK. B, HEK 293 T cells were transiently transfected with the indicated mutant of IKK (B) and then incubated with or without herbimycin A. Cell extracts were prepared and subjected to Western blot analysis with anti-phospho IKK and anti-phospho IB antibodies. The expression levels of IKK in total cell extracts were determined by immunoblotting with anti-FLAG antibody.

The C59A Mutant of IKK Was Insensitive to Herbimycin A. Next, we attempted to characterize the herbimycin A-insensitive C59A IKK mutant. Immunohistochemical analysis revealed that the wild type or C59A IKK induced their own phosphorylation in a herbimycin A-sensitive or -insensitive manner, respectively (Fig. 7A). It has been well established that activation of IKK is dependent on phosphorylation of the IKK subunit and that IKK is not implicated in IKK activation (Delhase et al., 1999; Karin, 1999b; O'Mahony et al., 2000). However, endogenous IKK complexes are composed of IKK·IKK heterodimers. Therefore, we attempted to characterize the effect of the C59A mutant in heterodimers against herbimycin A. As shown in Fig. 2B, coexpression of IKK and IKK induced the phosphorylation of IKK, IKK, and IB in a herbimycin A-sensitive manner. Next, we checked the effect of herbimycin A on IKK and IKK phosphorylation in cells transfected by both IKK and C59A-mutated IKK. Treatment with herbimycin A disrupted the phosphorylation in wild-type IKK/IKK-transfected cells but did not alter the phosphorylation in wild-type IKK- and mutated C59A IKK-transfected cells (Fig. 7C). Last, we examined whether the IKK C59A mutant abrogates NF-B activation challenged with IL-1 in glioma cells. As shown in Fig. 7D, treatment with herbimycin A resulted in a decrease in NF-B activity as detected by the luciferase assay in mock-transfected cells. It is noteworthy that the cells overexpressed by the C59A mutant of IKK showed significant insensitivity to herbimycin A.

View larger version (25K): [in this window] [in a new window]   Fig. 7. The C59A IKK mutant was insensitive to herbimycin A. HEK 293 T cells were transfected with FLAG-tagged wild type (wt) or FLAG-tagged C59A-mutated IKK and then treated with several concentrations of herbimycin A (A). Cell lysates were prepared and subjected to Western blot analysis with anti-phospho IB, anti-phospho IKK, and anti-FLAG antibodies. Hs683 cells were transiently transfected with wild type or C59A-mutated IKK together with enhanced green fluorescent protein (GFP) and treated with herbimycin A (B). The cells were fixed and subjected to immunofluorescent analysis with anti-phospho IKKs and anti-GFP antibodies. HEK 293 T cells were cotransfected with FLAG-tagged IKK and FLAG-tagged wt or C59A-mutated IKK and then treated with the indicated dose of herbimycin A (C). Cell extracts were prepared and subjected to Western blot analysis with anti-phospho IKKs. The expression levels of IKK and IKK in total cell extracts were determined by immunoblotting with anti-FLAG antibody. IKK C59A mutant abrogates NF-B activation challenged with IL-1 in glioma cells (D). To confirm the possible target site for herbimycin A, Hs683 glial cells were transfected with mock or C59A-mutated IKK together with pNF-B Luc and pRL-TK vectors. The cells were treated with or without herbimycin A and then stimulated with 5.0 ng/ml IL-1 for 6 h. The activity was assayed as described under Materials and Methods.

	Discussion

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We have demonstrated previously that treatment with herbimycin A selectively attenuates the gene induction regulated by NF-B in glial cells (Nishiya et al., 1995, 2000; Uehara et al., 1998, 1999). In this report, we attempted to elucidate the possible target for herbimycin A. We focused on NIK and IKK, which regulate NF-B signaling upstream of IB. The IB phosphorylation by transfection of wild-type NIK or IKK, but not of wild-type IKK, was sensitive to herbimycin A (Fig. 2A). Moreover, we indicate here that 1) NIK alone stimulates phosphorylation of IKKs and subsequently, IB, but phosphorylation of IKK and IKK is inhibited completely and partially, respectively, by herbimycin A treatment (Fig. 2C); 2) transfection of IKK together with IKK stimulates phosphorylation of IB and IKKs themselves in a herbimycin A-sensitive manner (Fig. 2B); and 3) NIK-induced phosphorylation of IB occurs via IKK but not IKK (Fig. 2D). From these observations, we conclude that 1) NIK induces both IKK and IKK phosphorylation and the subsequent IB phosphorylation and 2) IKK triggers the phosphorylation of itself and IKK and subsequently, IB. Therefore, we assert that IKK phosphorylation by NIK is inhibited partially by herbimycin A, probably caused by blockage of the pathway from IKK to IKK but not from NIK to IKK (Fig. 2C). Thus, we speculate that herbimycin A may attenuate IKK activity at least and thereby impair such subsequent reactions as IB phosphorylation and NF-B activation, because NIK is known to activate the IKK upstream of the IKK complex (Woronicz et al., 1997). However, some problems remain in determining the effects of herbimycin A on the IKK molecule. It is difficult at present to determine the kinase activity of IKK in vivo. Because IKK could not stimulate the phosphorylation of IB (NF-B activation) in vivo, it is hard to characterize the inhibitory effects of herbimycin A on IKK in our system. Thus, IKK may also be another target for herbimycin A, although we could not detect the NF-B activation by overexpression of IKK. On the other hand, the inhibitory effect of herbimycin A on NF-B signaling may be based on suppression of the interaction between NIK and the IKK complex. We therefore attempted to confirm the effect of herbimycin A on the interaction between IKK and NIK. However, we could not observe a significant abrogation of these interactions (Fig. 3A). On the other hand, IB interacted with IKK more tightly than did IKK in vitro, and the interaction between IB and IKK was attenuated slightly by treatment with herbimycin A (Fig. 3C). These results suggested that herbimycin A achieved its effect through inhibition of both IKK kinase activity and the interaction between IKK and IB.

Herbimycin A has been isolated as a potent selective inhibitor of v-Src tyrosine kinase (Uehara et al., 1989). This inhibition occurs via the irreversible binding of herbimycin A to a particular Cys residue in the C-terminal of v-Src (Uehara et al., 1989; Fukazawa et al., 1994; Senga et al., 2000). We also observed that herbimycin A blocked IKK activity dose dependently both in vivo and in vitro, and this effect disappeared with simultaneous addition of DTT (Figs. 2 and 4). Based on the above findings, it seemed possible that IKK could be covalently modified by herbimycin A at Cys residue(s) localized in or near its kinase domain. We then constructed several variant IKKs with cysteine-to-alanine mutations in their kinase domains. It was surprising that only the C59A mutant showed remarkable resistance against herbimycin A, and the C46A mutant was also less sensitive to this inhibitor than other mutants (Fig. 6B). On the other hand, none of the mutants of IKK resulted in any change compared with the wild-type IKK (data not shown). These results suggested strongly that herbimycin A selectively interacted with or modified Cys59 and partly Cys46 in IKK. These results may have been related to the three-dimensional structure of IKK or to a disturbance of IKK by interacting proteins in the IKK complex, such as IKK or NF-B essential modulator. In addition, the inhibitory effect of herbimycin A on the IKK·IKK heterodimer also disappeared in the cells overexpressing the C59A mutant of IKK (Fig. 6C). The kinase activity of heterodimerized IKKs was dependent on that of IKK (Karin, 1999a,b). From these observations, it seems possible that herbimycin A binds specifically to IKK (Cys59 or Cys46) in the IKK complex and thereby inhibits IKK activity. It has been reported previously that prostaglandin A1 and arsenite inhibit the IKK activity caused by binding at Cys178 of IKK and Cys179 of IKK, sites that are both located near the kinase activity-regulatory (phosphorylation) sites (Kapahi et al., 2000; Rossi et al., 2000). In addition, aspirin, which is used widely as an anti-inflammatory agent, has the ability to inhibit selectively only IKK activity by interfering competitively with ATP binding (Yin et al., 1998). Although we cannot currently explain the detailed interactive mechanism of herbimycin A or other compounds on the IKK molecule, the differences between modification/interaction sites in IKK by these chemicals may be dependent on their own structure or size. To finish, we attempted to determine whether the IKK C59A mutant prevents herbimycin A-dependent inhibition of NF-B activation by IL-1. As shown in Fig. 7D, the effects of herbimycin A on NF-B activation triggered by IL-1 disappeared in the C59A mutant-overexpressed cells. From these results, we conclude that Cys59 in IKK is a target for herbimycin A and consequently, abrogates the NF-B pathway.

In summary, our results indicate that herbimycin A may interact mainly with the Cys59 of IKK located in or near the catalytic (ATP binding) site. Therefore, this modification may lead to the inhibition of ATP binding or an interaction with substrate proteins, such as IB. In fact, herbimycin A also attenuated the interaction between IKK and IB in vitro (Fig. 3C). Hence, we consider herbimycin A is a potent inhibitor that induces modification specifically at the Cys59 (or Cys46) of IKK. Based on these findings, herbimycin A may be a useful tool for analysis of NF-B signaling and could be a new candidate anti-inflammatory drug with a novel function.

	Acknowledgements

 
We thank Dr. David V. Goeddel (Tularik Inc.) for providing us with the NIK, IKK, and IKK constructs.

	Footnotes

 
This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan.

S.O. and K.T. contributed equally to this work.

ABBREVIATIONS: NF, nuclear factor; IL, interleukin; GST, glutathione S-transferase; IKK, IB kinase; LZ, leucine zipper; NIK, NF-B-inducing kinase; TNF, tumor necrosis factor; DTT, dithiothreitol; TRAF, TNF receptor-associated factor; PCR, polymerase chain reaction; HEK, human embryonic kidney; pRL-TK, Renilla reniformis luciferase reporter plasmid; PAGE, polyacrylamide gel electrophoresis; NAC, N-acetyl cysteine.

Address correspondence to: Prof. Yasuyuki Nomura, Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan. E-mail: nomura{at}pharm.hokudai.ac.jp

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