A Resorcylic Acid Lactone, 5Z-7-Oxozeaenol, Prevents Inflammation by Inhibiting the Catalytic Activity of TAK1 MAPK Kinase Kinase*

Jun Ninomiya-TsujiDagger §, Taisuke KajinoDagger , Koichiro Ono, Toshihiko Ohtomo, Masahiko Matsumoto, Masashi Shiina, Masahiko Mihara, Masayuki Tsuchiya, and Kunihiro MatsumotoDagger ||

From the Dagger  Department of Molecular Biology, Graduate School of Science, Nagoya University, and CREST, Japan Science and Technology Corporation, Chikusa-ku, Nagoya 464-8602, Japan,  Chugai Pharmaceutical Co., Ltd., Fuji-Gotemba Research Laboratories, 1-135 Komakado, Gotemba-shi, Shizuoka 412-8513, Japan, and the § Department of Environmental and Molecular Toxicology, North Carolina State University, Raleigh, North Carolina 27695-7633

Received for publication, July 24, 2002, and in revised form, February 24, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

TAK1, a member of the mitogen-activated kinase kinase kinase (MAPKKK) family, participates in proinflammatory cellular signaling pathways by activating JNK/p38 MAPKs and NF-kappa B. To identify drugs that prevent inflammation, we screened inhibitors of TAK1 catalytic activity. We identified a natural resorcylic lactone of fungal origin, 5Z-7-oxozeaenol, as a highly potent inhibitor of TAK1. This compound did not effectively inhibit the catalytic activities of the MEKK1 or ASK1 MAPKKKs, suggesting that 5Z-7-oxozeaenol is a selective inhibitor of TAK1. In cell culture, 5Z-7-oxozeaenol blocked interleukin-1-induced activation of TAK1, JNK/p38 MAPK, Ikappa B kinases, and NF-kappa B, resulting in inhibition of cyclooxgenase-2 production. Furthermore, in vivo 5Z-7-oxozeaenol was able to inhibit picryl chloride-induced ear swelling. Thus, 5Z-7-oxozeaenol blocks proinflammatory signaling by selectively inhibiting TAK1 MAPKKK.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

TAK1 is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK)1 family that phosphorylates and activates MKK3, MKK4, MKK6, and MKK7 MAPKKs, which in turn activate the c-Jun N-terminal kinase (JNK) and p38 MAPKs (1-3). We have recently demonstrated that TAK1 also activates Ikappa B kinases (IKKs), ultimately leading to activation of the transcription factor NF-kappa B (4). TAK1 participates in proinflammatory cellular signaling pathways such as the interleukin-1 (IL-1) pathway by activating both JNK/p38 MAPKs and IKKs. Exposure of cells to IL-1 induces the interaction between endogenous TAK1 and TRAF6 (tumor necrosis factor (TNF) receptor-associated factor 6), a molecule essential for IL-1 activation of both JNK/p38 and NF-kappa B. This interaction in turn leads to TAK1 activation. We have previously identified two TAK1-binding proteins, TAB1 and TAB2 (5, 6). When ectopically co-expressed, TAB1 augments the kinase activity of TAK1, indicating that TAB1 functions as an activator of TAK1 (5, 7). TAB2 functions as an adaptor linking TAK1 to TRAF6 by directly binding to both, thereby mediating TAK1 activation in the IL-1 signaling pathway (6, 8).

Several lines of evidence suggest that TAK1 is a key molecule in proinflammatory signaling pathways. Various proinflammatory cytokines and endotoxins activate the kinase activity of endogenous TAK1 (4, 9, 10). Overexpression of kinase-dead TAK1 inhibits IL-1- and TNF-induced activation of both JNK/p38 and NF-kappa B (4, 10). The Drosophila homolog of TAK1 was recently identified as an essential molecule for host defense signaling in Drosophila (11). Furthermore, the TAK1 gene-silencing study using the small interfering RNA method defined that TAK1 is essential for both IL-1- and TNF-induced NF-kappa B activation in mammalian cells (12). Therefore, it can be expected that inhibition of TAK1 activity may be effective in preventing inflammation and tissue destruction promoted by proinflammatory cytokines.

In this study, we screened for compounds that can inhibit TAK1 kinase activity. This strategy resulted in the isolation of one natural compound 5Z-7-oxozeaenol, a resorcylic lactone of fungal origin. We found that 5Z-7-oxozeaenol inhibited the kinase activity of purified TAK1, whereas no significant inhibition of TAK1 activity was observed with structurally related compounds including radicicol. 5Z-7-Oxozeaenol had no significant effect on the kinase activities of other members of the MAPKKK family such as MEKK1 and ASK1. Exposure of cells to 5Z-7-oxozeaenol blocked IL-1-induced activation of TAK1, IKK, JNK, p38, and NF-kappa B. Furthermore, 5Z-7-oxozeaenol inhibited IL-1-induced production of cyclooxygenase-2 and relieved ear swelling induced by picryl chloride. These results suggest that 5Z-7-oxozeaenol blocks proinflammatory signaling by selectively inhibiting TAK1 MAPKKK.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Compounds, Reagents, and Cell Culture-- Zeaenol analog and radicicol were prepared from the culture broth of fungal strain f6024 and f6065, respectively. Recombinant human IL-1beta (Roche Applied Science), recombinant human TNFalpha (Roche Applied Science), and epidermal growth factor (EGF) (BD Biosciences) were used. The following antibodies were used: anti-TAK1 polyclonal antibody M-17 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-FLAG monoclonal antibody M2 (Sigma), anti-phosphoextracellular signal-regulated kinase (ERK) (Thr-202/Tyr-204) polyclonal antibody (Cell Signaling), anti-ERK polyclonal antibody (Cell Signaling), anti-phospho-JNK (Thr-183/Tyr-185) monoclonal antibody (Cell Signaling), anti-JNK polyclonal antibody FL (Santa Cruz Biotechnology), anti-phospho-p38 (Thr-180/Tyr-182) polyclonal antibody (Cell Signaling), anti-p38 polyclonal antibody (Cell Signaling), anti-IKKalpha polyclonal antibody H-744 (Santa Cruz Biotechnology), and anti-cyclooxygenase-2 polyclonal antibody M-19 (Santa Cruz Biotechnology). The rabbit anti-TAK1 and anti-TAB1 polyclonal antibodies (4) were also used to immunoprecipitate and/or detect endogenous TAK1 and TAB1 in 293-IL-1RI cells (13). Expression vectors for FLAG-TAK1, FLAG-MEKK1Delta N, FLAG-ASK1, NF-kappa B-interacting kinase, and FLAG-IKKbeta were described previously (4, 14-16). Purified MEKK1 and MEK1 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). 293-IL-1RI cells and mouse embryonic fibroblast cells were maintained in high glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin G (100 units/ml) and streptomycin (100 µg/ml). For the transfection studies, cells (1 × 106) were plated in 10-cm dishes, transfected with a total of 10 µg of DNA containing various expression vectors by the calcium phosphate precipitate method, and incubated for 24-36 h before stimulation.

Immunoprecipitation and Immunoblotting-- Cells were washed once with ice-cold phosphate-buffered saline and lysed in 0.3 ml of 0.5% Triton X-100 lysis buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, 12.5 mM beta -glycerophosphate, 1.5 mM MgCl2, 2 mM EGTA, 10 mM NaF, 2 mM dithiothreitol, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and 20 µM aprotinin. Cellular debris was removed by centrifugation at 10,000 × g for 5 min. Proteins from cell lysates were immunoprecipitated with 1 µg of various antibodies and 20 µl of protein G-Sepharose (Amersham Biosciences). The immune complexes were washed three times with wash buffer containing 20 mM HEPES (pH 7.4), 500 mM NaCl, and 10 mM MgCl2, and once with rinse buffer containing 20 mM HEPES (pH 7.4), 150 mM NaCl, and 10 mM MgCl2 and suspended in 30 µl of rinse buffer. For immunoblotting, the immunoprecipitates or whole cell lysates were resolved on SDS-PAGE and transferred to Hybond-P membranes (Amersham Biosciences). The membranes were immunoblotted with various antibodies, and the bound antibodies were visualized with horseradish peroxidase-conjugated antibodies against rabbit or mouse IgG using the ECL Western blotting system (Amersham Biosciences).

Kinase Assay-- For screening TAK1 inhibitors, insect expression vectors for TAK1 and TAB1 were co-infected into Sf9 cells. After 2 days of incubation, cell lysates were immunoprecipitated with anti-TAK1 antibody (M-17). The immunoprecipitates were incubated with various compounds and subsequently incubated with 2 µg of myelin basic protein and 10 µCi of [gamma -32P]ATP (3,000 Ci/mmol) in 10 µl of the kinase buffer containing 10 mM HEPES (pH 7.4), 1 mM dithiothreitol, 5 mM MgCl2 at 30 °C for 5 min. Samples were separated by 10% SDS-PAGE, and 32P incorporated into myelin basic protein was quantified with a bioimage analyzer (FUJIX BAS2000). The catalytic activity of MEK1 was determined by activation of ERK2 (Upstate Biotechnology) to phosphorylate myelin basic protein according to the manufacturer's procedure. The catalytic activity of MEKK1 was measured with 2 µg of myelin basic protein as a substrate in the kinase buffer. For subsequent kinase assays, immunoprecipitates were incubated with 5 µCi of [gamma -32P]ATP (3,000 Ci/mmol) and 1 µg of bacterially expressed MKK6 or GST-Ikappa Balpha -(1-72) in 10 µl of the kinase buffer at 25 °C for 2 min. Samples were separated by 10% SDS-PAGE and visualized by autoradiography.

Reporter Gene Assay-- Assays for reporter gene activity were performed as described (4). An Ig-kappa -luciferase reporter was used to measure NF-kappa B-dependent transcription. A plasmid containing the beta -galactosidase gene under the control of the beta -actin promoter (pAct-beta -galactosidase) was used for normalizing transfection efficiency.

Ear Swelling Assay-- Female BALB/c mice (6 weeks old) were sensitized by applying 0.1 ml of picryl chloride (50 mg/ml) in an olive oil/acetone solution (1:5, v/v) to the shaved abdomen of the mice at day 0. Seven days later, 10 µl of picryl chloride solution (10 mg/ml) in olive oil was applied to each side of the right ear (PC challenge). At day 10, mice were resensitized with picryl chloride. At day 17, the PC challenge was repeated (second PC challenge). Ten µl of 1 mg/ml 5Z-7-oxozeaenol or vehicle alone (ethanol) were painted on each side of the right ear before and after the second PC challenge. The ear thickness was measured with calibrated digital thickness gauges before and 24 h after the second PC challenge, and the difference in thickness was calculated.

    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

Screening for Inhibitors of TAK1 MAPKKK-- We have previously shown that TAK1 has no kinase activity when expressed alone but is activated when TAB1 is co-expressed (5, 7). To identify inhibitors of TAK1, we developed an in vitro kinase assay system using purified TAK1 and TAB1 proteins expressed in insect cells. We tested 90 compounds, including 59 compounds that have been reported to inhibit protein kinases, 24 oxindole-related compounds, and 7 resorcylic acid lactone-related compounds. Of these compounds, one resorcylic acid lactone-related compound, 5Z-7-oxozeaenol, was found to be a very potent inhibitor of TAK1, with an IC50 of 8 nM (Fig. 1A and Table I). Other structurally related compounds such as radicicol had little inhibitory activity, with an IC50 of >10 µM. The remaining 89 compounds did not exhibit any effective inhibition of TAK1. These included oxindole protein-tyrosine kinase inhibitors (SU5402 and SU4984), staurosporine-related protein kinase C inhibitors, the platelet-derived growth factor receptor inhibitor AG1433, and the plant flavonoid apigenin. Two compounds, Ro092210 and L783277, with very similar structures to 5Z-7-oxozeaenol were previously demonstrated to inhibit MEK kinase activity (17, 18). We examined the effect of 5Z-7-oxozeaenol on purified rat MEK1 kinase activity (Fig. 1B). 5Z-7-Oxozeaenol did inhibit MEK1 kinase activity; however, the IC50 of 5Z-7-oxozeaenol required to inhibit MEK1 is 411 nM, which is 50-fold higher than that for TAK1.


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Fig. 1.   Inhibition of TAK1 activity by 5Z-7-oxozeaenol. A, TAK1 and TAB1 proteins purified from insect cells were used for in vitro kinase assays in the presence of 5Z-7-oxozeaenol or radicicol. The percentage inhibition of kinase activity is shown. B, purified rat MEK1 was used for in vitro kinase assays in the presence of 5Z-7-oxozeaenol. The percentage inhibition of kinase activity is shown. C, bacterially expressed MEKK1 was used for in vitro kinase assays in the presence of 5Z-7-oxozeaenol. The percentage inhibition of kinase activity is shown. D, TAK1 and TAB1 proteins purified from insect cells were incubated with various concentrations of ATP together with 5Z-7-oxozeaenol or incubated with 5Z-7-oxozeaenol for 30 min before the addition of ATP (preincubation). IC50 values shown are means from three independent experiments (n = 3). ND, not done.


                              
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Table I
Inhibitory activity of compounds on TAK1

To explore the mechanism for 5Z-7-oxozeaenol inhibition of TAK1, we examined whether 5Z-7-oxozeaenol is competitive with ATP. We incubated increasing concentrations of ATP and 5Z-7-oxozeaenol with purified TAK1 and subsequently assayed kinase activity of TAK1. We found that the IC50 of 5Z-7-oxozeaenol required to inhibit TAK1 shifted to higher values with increasing ATP concentrations (Fig. 1D), suggesting that 5Z-7-oxozeaenol is a competitive inhibitor of ATP binding to TAK1. When 5Z-7-oxozeaenol was preincubated with TAK1 for 30 min before the addition of ATP, the IC50 values of 5Z-7-oxozeaenol did not shift with increasing ATP concentrations (Fig. 1D), suggesting that the binding of 5Z-7-oxozeaenol to TAK1 is either irreversible or very slowly reversible. Thus, 5Z-7-oxozeaenol is likely to irreversibly interact within the ATP binding site of TAK1, thereby inhibiting the catalytic activity of TAK1.

5Z-7-Oxozeaenol Selectively Inhibits TAK1 Kinase Activity-- To evaluate whether 5Z-7-oxozeaenol is a specific inhibitor of TAK1 or if it more generally inhibits the MAPKKK family, we tested the effect of 5Z-7-oxozeaenol on bacterially expressed MEKK1 kinase activity in vitro (Fig. 1C). 5Z-7-Oxozeaenol had a weak effect on MEKK1 kinase activity. The IC50 of 5Z-7-oxozeaenol required to inhibit MEKK1 was 268 nM. To further verify the effects of 5Z-7-oxozeaenol on MAPKKKs, we utilized ectopically expressed MAPKKKs in 293 cells. TAK1 is known to be active when it is co-expressed together with TAB1 (5, 7). When N-terminal truncated MEKK1 (MEKK1Delta N) or the full-length ASK1 is overexpressed in 293 cells, they are catalytically active (19, 20). FLAG-tagged TAK1 together with TAB1, FLAG-MEKK1Delta N, or FLAG-ASK1 was expressed in 293 cells, and each kinase was immunoprecipitated with anti-FLAG antibody. We measured their abilities both to autophosphorylate themselves and to phosphorylate MAPKK MKK6 (Fig. 2). In this assay, 5Z-7-oxozeaenol inhibited autophosphorylation of TAK1 and TAK1 activity to phosphorylate MKK6 at concentrations of 30-300 nM. By contrast, no inhibitory effect of 5Z-7-oxozeaenol was observed on MEKK1 or ASK1. The kinase activity of another MAPKKK, MEKK4, was also not inhibited by 5Z-7-oxozeaenol at concentrations as high as 500 nM (data not shown). Thus, 5Z-7-oxozeaenol is potent and selective inhibitor of TAK1.


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Fig. 2.   Effect of 5Z-7-oxozeaenol on MAPKKKs. 293 cells were transfected with expression vectors for FLAG-TAK1, TAB1, FLAG-MEKK1Delta N, FLAG-ASK1, and FLAG-IKKbeta as indicated. Cell extracts were immunoprecipitated with anti-FLAG antibody. The immunoprecipitates were treated with various concentrations of 5Z-7-oxozeaenol and subjected to kinase reactions with bacterially expressed MKK6. The autophosphorylation of TAK1, MEKK1Delta N, and ASK1 and phosphorylation of MKK6 are shown as indicated. The kinase activity of IKKbeta was assayed using bacterial expressed GST-Ikappa B as a substrate.

5Z-7-Oxozeaenol Inhibits the IL-1 Signaling Pathway-- We have previously demonstrated that TAK1 is involved in the IL-1 signaling pathway (4). The observation that 5Z-7-oxozeaenol inhibits TAK1 activity raised the possibility that this compound might be an effective inhibitor of IL-1 signaling. Treatment of cells with IL-1 activates endogenous TAK1 activity and consequently stimulates the MAPK cascade and IKK, leading to the activation of JNK/p38 MAPKs and NF-kappa B, respectively. To verify if 5Z-7-oxozeaenol can inhibit IL-1 signaling, we test for the effect of 5Z-7-oxozeaenol on NF-kappa B-dependent transcriptional activation induced by IL-1 (Fig. 3A). We found that treatment of cells with 5Z-7-oxozeaenol effectively inhibited IL-1-induced activation of NF-kappa B.


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Fig. 3.   Effect of 5Z-7-oxozeaenol on NF-kappa B activation. A, 293 cells were transfected with an Ig-kappa -luciferase reporter and pAct-beta -galactosidase, together with empty vector or expression vectors encoding TAK1, TAB1, Tax, or NF-kappa B-interacting kinase (NIK) as indicated. The indicated amounts of 5Z-7-oxozeaenol or the same volumes of Me2SO were added to culture medium upon transfection and at 12 and 24 h after transfection. Cells transfected with empty vector were treated with IL-1 (5 ng/ml) or TNF (10 ng/ml). Following a 36-h incubation, luciferase activity was determined and normalized to the levels of beta -galactosidase activity. The percentage stimulation of 5Z-7-oxozeaenol-treated samples relative to the untreated controls is shown. Stimulation relative to control transfection without IL-1 treatment was as follows: IL-1, 79-fold; TNF, 241-fold; TAK1 plus TAB1, 160-fold; Tax, 51-fold; NF-kappa B-interacting kinase (NIK), 133-fold. B, 293 cells were transfected with an Ig-kappa -luciferase reporter and pAct-beta -galactosidase, together with expression vectors encoding TAK1 and TAB1. Luciferase activity was determined and normalized to the levels of beta -galactosidase activity. The percentage stimulation of 5Z-7-oxozeaenol-treated samples relative to the untreated controls is shown. The IC50 value of 5Z-7-oxozeaenol to inhibit NF-kappa B activation by overexpression of TAK1 and TAB1 is shown.

NF-kappa B is activated through several pathways including human T-cell leukemia virus Tax protein and TNF pathways. TAK1 is implicated in TNF-induced NF-kappa B activation (10, 12), whereas MEKK1 is involved in Tax-induced NF-kappa B activation (21). NF-kappa B can also be activated in the absence of extracellular signals by overexpression of TAK1 and TAB1 together or by NF-kappa B-interacting kinase alone (4, 22). To examine whether the effect of 5Z-7-oxozeaenol is specific to NF-kappa B activation mediated by TAK1, cells were treated with TNF or transfected TAK1, TAB1, Tax, or NF-kappa B-interacting kinase expression vectors. We found that 5Z-7-oxozeaenol treatment effectively inhibited activation of NF-kappa B induced by TNF and overexpression of TAK1 and TAB1, whereas it had marginal inhibitory effect on activation of NF-kappa B induced by overexpression of Tax or NF-kappa B-interacting kinase (Fig. 3A). These results suggest that 5Z-7-oxozeaenol specifically inhibits NF-kappa B activation by blocking TAK1 activity. The IC50 value of 5Z-7-oxozeaenol required to inhibit NF-kappa B activation by overexpression of TAK1 and TAB1 was 83 nM (Fig. 3B). This IC50 is 10-fold higher than that required to inhibit purified TAK1 in vitro (Fig. 1A).

We next examined whether 5Z-7-oxozeaenol inhibits IL-1-induced JNK/p38 activation. We pretreated 293-IL-1RI cells with increasing amounts of 5Z-7-oxozeaenol and stimulated the cells with IL-1 treatment. The activated JNK and p38 were detected with anti-phospho-JNK and -p38 antibodies that specifically recognize the dually phosphorylated activated forms of JNK1/JNK2 and p38, respectively (Fig. 4A). IL-1-dependent JNK/p38 activation was abrogated with treatment of 5Z-7-oxozeaenol in a dose-dependent manner. The amount of 5Z-7-oxozeaenol required to inhibit IL-1-induced JNK/p38 activation was in a similar range to that required for NF-kappa B inhibition.


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Fig. 4.   Effect of 5Z-7-oxozeaenol on activation of MAPKs. A, 293-IL-1RI cells were pretreated with increasing concentrations of 5Z-7-oxozeaenol for 30 min and stimulated with IL-1 (5 ng/ml) for 10 min. Cell lysates were immunoblotted (IB) with anti-phospho-JNK, anti-JNK, anti-phospho-p38, and anti-p38. B, 293-IL-1RI cells were pretreated with increasing concentrations of 5Z-7-oxozeaenol for 30 min and stimulated with hydrogen peroxide (H2O2) (500 µM) for 30 min. Cell lysates were immunoblotted with anti-phospho-JNK, anti-JNK, anti-phospho-p38, and anti-p38. C, 293-IL-1RI cells were pretreated with increasing concentrations of 5Z-7-oxozeaenol for 30 min, irradiated with UV-B (20 J/m2), and subsequently incubated for 30 min. Cell lysates were immunoblotted with anti-phospho-ERK and anti-ERK. D, 293-IL-1RI cells were pretreated with increasing concentrations of 5Z-7-oxozeaenol for 30 min and stimulated with EGF (50 ng/ml) for 10 min. Cell lysates were immunoblotted with anti-phospho-ERK and anti-ERK.

To further examine the specificity of 5Z-7-oxozeaenol, we tested the effect of 5Z-7-oxozeaenol on MAPK cascades activated by several other stimuli. Hydrogen peroxide is a strong stimulator of JNK/p38; however, it poorly activates TAK1 in 293-IL-1RI cells,2 suggesting that TAK1 is not involved in this pathway. 293-IL-1RI cells were treated with 5Z-7-oxozeaenol for 30 min followed by hydrogen peroxide simulation (Fig. 4B). No pronounced inhibition of either JNK or p38 activation was observed in 5Z-7-oxozeaenol-treated cells. We also assayed UV- and EGF-induced ERK activation. UV and EGF activate the MEK-ERK MAP kinase cascade, in which TAK1 does not participate. 293-IL-1RI cells were pretreated with 5Z-7-oxozeaenol and stimulated with UV or EGF. The activated ERK was detected with anti-phospho-ERK antibody that specifically recognizes the dually phosphorylated activated forms of ERK1 and ERK2 (Fig. 4, C and D). 5Z-7-Oxozeaenol had little effect on UV- or EGF-induced ERK activation even at a concentration of 500 nM. These results suggest that 5Z-7-oxozeaenol selectively inhibits TAK1, thereby inhibiting IL-1-induced JNK/p38 activation in culture cells.

5Z-7-Oxozeaenol Inhibits IL-1-induced TAK1 Activation in Culture Cells-- We then examined whether 5Z-7-oxozeaenol inhibits kinase activity of endogenous TAK1 upon IL-1 stimulation. We have previously observed that TAK1 is transiently activated around 2-5 min after IL-1 stimulation when 293-IL-1RI cells were treated with IL-1 (8). We treated 293-IL-1RI cells with various concentrations of 5Z-7-oxozeaenol prior to IL-1 stimulation. At 5 min after IL-1 stimulation, cells were lysed, and endogenous TAK1 was immunoprecipitated with anti-TAK1 antibody. The catalytic activity of TAK1 was measured using MKK6 as a substrate (Fig. 5A, upper panel). Treatment of the cells with 5Z-7-oxozeaenol inhibited kinase activity of endogenous TAK1. The IC50 of 5Z-7-oxozeaenol to inhibit endogenous TAK1 was 65 nM (Fig. 5A), which is correlated with the IC50 to inhibit NF-kappa B and JNK/p38 activation (Fig. 3 and 4).


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Fig. 5.   5Z-7-Oxozeaenol inhibits kinase activity of endogenous TAK1. A, 293-IL-1RI cells were pretreated with various concentrations of 5Z-7-oxozeaenol for 30 min and then stimulated with IL-1 (5 ng/ml) for 5 min. Cell lysates were immunoprecipitated with anti-TAK1. The immunoprecipitates were subjected to kinase assay with MKK6 as a substrate (upper panel). The amounts of immunoprecipitated TAK1 are shown in the lower panel. B, 293-IL-1RI cells were pretreated with 100 nM 5Z-7-oxozeaenol (Z) or the same volume of Me2SO (D) for 30 min (0-30) followed by additional preincubation with 100 nM 5Z-7-oxozeaenol (Z) or the same volume of Me2SO (D) for 30 min (30-60). The cells were subsequently treated with 5 ng/ml IL-1 for 5 min. Cell lysates were immunoprecipitated with anti-TAK1. The immunoprecipitates were subjected to a kinase assay with MKK6 (upper panel). The amounts of immunoprecipitated TAK1 are shown in the lower panel. IB, immunoblot.

We next tested whether the 5Z-oxozeaenol-mediated inhibition of TAK1 in culture cells is reversible or irreversible. We treated 293-IL-1RI cells with 100 nM 5Z-7-oxozeaenol for 30 min and then incubated for an additional 30 min without 5Z-7-oxozeaenol. The cells were subsequently stimulated with IL-1, and the catalytic activity of endogenous TAK1 was measured (Fig. 5B). 5Z-7-Oxozeaenol significantly inhibited TAK1 kinase activity even after 5Z-7-oxozeaenol was removed from the culture medium. These results suggest that 5Z-7-oxozeaenol irreversibly binds to and inhibits TAK1 in 293-IL-1RI cells, consistent with the result showing that 5Z-7-oxozeaenol irreversibly inhibits ATP binding to TAK1 in vitro (Fig. 1D). Thus, it is likely that 5Z-7-oxozeaenol, when added into the culture medium, inhibits TAK1 activity by irreversibly inhibiting the binding of ATP to TAK1.

We also investigated the time course of activation of TAK1, IKK, JNK, and p38 upon IL-1 stimulation. In this assay, 293-IL-1RI cells were treated with 500 nM 5Z-7-oxozeaenol for 30 min to completely inhibit kinase activity of TAK1 and then stimulated with IL-1 (Fig. 6). Cells were harvested at 3 and 12 min post-IL-1 stimulation, and the lysates were immunoprecipitated with anti-TAK1 followed by in vitro kinase assay (Fig. 6A). 5Z-7-Oxozeaenol treatment abolished IL-1-induced activation of TAK1. We have previously shown that autophosphorylation of TAK1 upon IL-1 stimulation is essential for its activation (7). TAK1 autophosphorylation can be detected on SDS-PAGE as slowly migrating TAK1 bands (Fig. 6A, lower panel). We observed that 5Z-7-oxozeaenol inhibited IL-1-induced autophosphorylation of TAK1. We have also previously demonstrated that endogenous TAK1 constitutively interacts with TAB1 (7). The amount of coprecipitated TAB1 in TAK1 immunoprecipitates was not changed with treatment of 5Z-7-oxozeaenol (Fig. 6A, lower panel), suggesting that 5Z-7-oxozeaenol did not interfere with interaction of TAK1 with TAB1. IKK activity was measured using GST-Ikappa B as a substrate (Fig. 6B). 5Z-7-Oxozeaenol treatment inhibited 70-80% of the kinase activity of the IL-1-induced IKK activity. Since 5Z-7-oxozeaenol had no inhibitory effect on kinase activity of IKK itself (Fig. 2), 5Z-7-oxozeaenol presumably inhibits IL-1-induced activation of IKK by inhibiting TAK1 activity. We also observed that 5Z-7-oxozeaenol abolished IL-1-induced activation of JNK and p38 (Fig. 6, C and D). Taken together, our results indicate that 5Z-7-oxozeaenol inhibits the IL-1 signaling pathways that normally lead to activation of both NF-kappa B and JNK/p38 by inhibiting TAK1.


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Fig. 6.   Effect of 5Z-7-oxozeaenol on IL-1-induced activation of endogenous TAK1, IKK, JNK, and p38. 293-IL-1RI cells were pretreated with 500 nM 5Z-7-oxozeaenol for 30 min and then stimulated with IL-1 (5 ng/ml) for the indicated times. A, cell lysates were immunoprecipitated with anti-TAK1. The immunoprecipitates were subjected to kinase assay with MKK6 as a substrate (upper panel). The immunoprecipitated TAK1 and coprecipitated TAB1 were immunoblotted (IB) with anti-TAK1 and anti-TAB1, respectively, in the lower panel. B, cell lysates were immunoprecipitated with anti-IKKalpha , and the in vitro kinase assay was performed with GST-Ikappa B (upper panel). The amounts of immunoprecipitated IKKalpha were detected by immunoblotting with anti-IKKalpha (lower panel). C, whole cell lysates were immunoblotted with anti-phospho-JNK and anti-JNK. D, whole cell lysates were immunoblotted with anti-phospho-p38 and anti-p38.

Effects of 5Z-7-Oxozeaenol on Inflammation-- IL-1 is a proinflammatory cytokine that induces the expression of many genes that up-regulate inflammation (23). One such gene product is cyclooxgenase 2 (COX-2), which catalyzes the production of prostaglandin (24). We tested the effect of 5Z-7-oxozeaenol on IL-1-induced COX-2 production (Fig. 7A). The level of COX-2 proteins was increased after IL-1 treatment, whereas no increase was detected when cells were pretreated with 5Z-7-oxozeaenol, even in the presence of IL-1. Thus, 5Z-7-oxozeaenol inhibits production of inflammation mediators.


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Fig. 7.   Effects of 5Z-7-oxozeaenol on inflammation. A, effect of 5Z-7-oxozeaenol on IL-1-induced COX-2 production. Mouse embryonic fibroblast cells were pretreated with 5Z-7-oxozeaenol (500 nM) for 30 min and stimulated with IL-1 (5 ng/ml). After a 17-h incubation, whole cell lysates were immunoblotted (IB) with anti-COX-2 (upper panel) and anti-TAK1 (lower panel). B, effect of 5Z-7-oxozeaenol on picryl chloride-induced ear swelling. Mice were repeatedly sensitized by picryl chloride and subsequently challenged with picryl chloride (PC challenge) on the ear twice. Ten µl of 5Z-7-oxozeaenol (1 mg/ml) or vehicle alone (ethanol) were painted on each ear 2 h before and 4 h after the second PC challenge (total of twice) (upper panel) or every hour three times before and 4 h after the second PC challenge (total of four times) (bottom panel). The ear thickness was measured 24 h after the second PC challenge. Results shown are means ± S.D. from five mice.

We next examined whether 5Z-7-oxozeaenol could suppress inflammation in vivo. For this experiment, we used PC-induced ear swelling as a model for COX-2-mediated inflammation. The ear swelling system has been widely used as a model for allergic cutaneous diseases and inflammatory skin disorders (25, 26). Indeed, it has been shown that inhibitors of COX-2 block PC-induced ear swelling (27). Furthermore, reduction of IL-1 production have also been shown to block ear swelling induced by PC (28), suggesting that IL-1 signaling is involved in this disorder. When 5Z-7-oxozeaenol was administrated to the PC-challenged ear, ear swelling was reduced by up to 50% of that of the control ear treated with vehicle (Fig. 7B). Thus, 5Z-7-oxozeaenol is able to prevent inflammation, probably through inhibiting TAK1 activity.

Our screening for a TAK1 kinase inhibitor identified a natural compound, 5Z-7-oxozeaenol, a fungal resorcylic acid lactone that has been previously reported to inhibit endotoxin-induced production of TNF (29). 5Z-7-Oxozeaenol is also able to inhibit anisomycin-induced JNK/p38 activation (30). However, the mechanisms underlying these inhibitory effects had been unclear. Here we demonstrate that 5Z-7-oxozeaenol specifically inhibits the catalytic activity of TAK1. Since TAK1 is activated upon treatment with various endotoxins and stresses, it is likely that 5Z-7-oxozeaenol might inhibit TAK1 activity activated by endotoxin and anisomycin, thereby reducing TNF production and JNK/p38 activation.

TAK1 is a multifunctional protein kinase involved not only in the IL-1 signaling pathway but also in the transforming growth factor-beta family signaling pathway (3, 31). Furthermore, we have recently found that TAK1 is involved in a MAP kinase-like pathway that negatively regulates the Wnt signaling pathway (32-34). Since 5Z-7-oxozeaenol is a highly potent and selective inhibitor of TAK1, this compound will be a useful tool for studies on these signal transduction pathways. Furthermore, in this study, we show that when applied topically, 5Z-7-oxozeaenol significantly reduces the level of PC-induced ear swelling. These results suggest that 5Z-7-oxozeaenol might be a useful therapeutic agent for allergic cutaneous disorders such as allergic contact dermatitis and atopic dermatitis.

    ACKNOWLEDGEMENTS

We thank H. Ichijo, S. Ohno, H. Saito, and E. Nishida for materials and M. Lamphier for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by a grant from Advanced Research on Cancer from the Ministry of Education, Culture, and Science of Japan; the Asahi Glass Foundation; Daiko Foundation; the Uehara Foundation; and the Yamanouchi Foundation for Research on Metabolic Disorders (to K. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed. Tel.: 81-52-789-3000; Fax: 81-52-789-2589 or 81-52-789-3001; E-mail: g44177a@nucc.cc.nagoya-u.ac.jp.

Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M207453200

2 J. Ninomiya-Tsuji, unpublished result.

    ABBREVIATIONS

The abbreviations used are: MAPKKK, MAPKK kinase; MAPKK, MAPK kinase; MAPK, mitogen-activated protein kinase; IL-1, interleukin-1; TNF, tumor necrosis factor; JNK, c-Jun N-terminal kinase; IKK, Ikappa B kinase; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; PC, picryl chloride; COX-2, cyclooxgenase 2; GST, glutathione S-transferase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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