A Resorcylic Acid Lactone, 5Z-7-Oxozeaenol, Prevents Inflammation by Inhibiting the Catalytic Activity of TAK1 MAPK Kinase Kinase
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-κB. 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, IκB kinases, and NF-κB, 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. 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-κB. 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, IκB kinases, and NF-κB, 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. MAPKK kinase MAPK kinase mitogen-activated protein kinase interleukin-1 tumor necrosis factor c-Jun N-terminal kinase IκB kinase epidermal growth factor extracellular signal-regulated kinase picryl chloride cyclooxgenase 2 glutathione S-transferase TAK1 is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK)1family that phosphorylates and activates MKK3, MKK4, MKK6, and MKK7 MAPKKs, which in turn activate the c-Jun N-terminal kinase (JNK) and p38 MAPKs (1Moriguchi T. Kuroyanagi N. Yamaguchi K. Gotoh Y. Irie K. Kano T. Shirakabe K. Muro Y. Shibuya H. Matsumoto K. Nishida E. Hagiwara M. J. Biol. Chem. 1996; 271: 13675-13679Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 2Shirakabe K. Yamaguchi K. Shibuya H. Irie K. Matsuda S. Moriguchi T. Gotoh Y. Matsumoto K. Nishida E. J. Biol. Chem. 1997; 272: 8141-8144Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 3Yamaguchi K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1178) Google Scholar). We have recently demonstrated that TAK1 also activates IκB kinases (IKKs), ultimately leading to activation of the transcription factor NF-κB (4Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1023) Google Scholar). 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 factor6), a molecule essential for IL-1 activation of both JNK/p38 and NF-κB. This interaction in turn leads to TAK1 activation. We have previously identified two TAK1-binding proteins, TAB1 and TAB2 (5Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (524) Google Scholar, 6Takaesu G. Kishida S. Hiyama A. Yamaguchi K. Shibuya H. Irie K. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. 2000; 5: 649-658Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar). When ectopically co-expressed, TAB1 augments the kinase activity of TAK1, indicating that TAB1 functions as an activator of TAK1 (5Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (524) Google Scholar, 7Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). TAB2 functions as an adaptor linking TAK1 to TRAF6 by directly binding to both, thereby mediating TAK1 activation in the IL-1 signaling pathway (6Takaesu G. Kishida S. Hiyama A. Yamaguchi K. Shibuya H. Irie K. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. 2000; 5: 649-658Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 8Takaesu G. Ninomiya-Tsuji J. Kishida S. Li X. Stark G.R. Matsumoto K. Mol. Cell. Biol. 2001; 21: 2475-2484Crossref PubMed Scopus (160) Google Scholar). 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 (4Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1023) Google Scholar, 9Irie T. Muta T. Takeshige K. FEBS Lett. 2000; 467: 160-164Crossref PubMed Scopus (166) Google Scholar, 10Sakurai H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Overexpression of kinase-dead TAK1 inhibits IL-1- and TNF-induced activation of both JNK/p38 and NF-κB (4Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1023) Google Scholar, 10Sakurai H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). The Drosophila homolog of TAK1 was recently identified as an essential molecule for host defense signaling in Drosophila(11Vidal S. Khush R.S. Leulier F. Tzou P. Nakamura M. Lemaitre B. Genes Dev. 2001; 15: 1900-1912Crossref PubMed Scopus (251) Google Scholar). 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-κB activation in mammalian cells (12Takaesu G. Surabhi R.M. Park K.J. Ninomiya-Tsuji J. Matsumoto K. Gaynor R.B. J. Mol. Biol. 2003; 326: 105-115Crossref PubMed Scopus (322) Google Scholar). 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-κB. 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. Zeaenol analog and radicicol were prepared from the culture broth of fungal strain f6024 and f6065, respectively. Recombinant human IL-1β (Roche Applied Science), recombinant human TNFα (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-IKKα 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 (4Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1023) Google Scholar) were also used to immunoprecipitate and/or detect endogenous TAK1 and TAB1 in 293-IL-1RI cells (13Cao Z. Henzel W.J. Gao X. Science. 1996; 271: 1128-1131Crossref PubMed Scopus (777) Google Scholar). Expression vectors for FLAG-TAK1, FLAG-MEKK1ΔN, FLAG-ASK1, NF-κB-interacting kinase, and FLAG-IKKβ were described previously (4Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1023) Google Scholar, 14Hirai S. Izawa M. Osada S. Spyrou G. Ohno S. Oncogene. 1996; 12: 641-650PubMed Google Scholar, 15Mochida Y. Takeda K. Saitoh M. Nishitoh H. Amagasa T. Ninomiya-Tsuji J. Matsumoto K. Ichijo H. J. Biol. Chem. 2000; 275: 32747-32752Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 16Woronicz J.D. Gao X. Cao Z. Rothe M. Goeddel D.V. Science. 1997; 278: 866-869Crossref PubMed Scopus (1068) Google Scholar). 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. 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 mmβ-glycerophosphate, 1.5 mm MgCl2, 2 mm EGTA, 10 mm NaF, 2 mmdithiothreitol, 1 mm sodium orthovanadate, 1 mmphenylmethylsulfonyl 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 mmMgCl2, 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). 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 [γ-32P]ATP (3,000 Ci/mmol) in 10 μl of the kinase buffer containing 10 mmHEPES (pH 7.4), 1 mm dithiothreitol, 5 mmMgCl2 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 [γ-32P]ATP (3,000 Ci/mmol) and 1 μg of bacterially expressed MKK6 or GST-IκBα-(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. Assays for reporter gene activity were performed as described (4Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1023) Google Scholar). An Ig-κ-luciferase reporter was used to measure NF-κB-dependent transcription. A plasmid containing the β-galactosidase gene under the control of the β-actin promoter (pAct-β-galactosidase) was used for normalizing transfection efficiency. 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. We have previously shown that TAK1 has no kinase activity when expressed alone but is activated when TAB1 is co-expressed (5Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (524) Google Scholar, 7Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). 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 of and structurally related compounds such as radicicol had with an of The compounds did not effective inhibition of TAK1. These kinase inhibitors and protein kinase inhibitors, the growth factor inhibitor and the compounds, and with very to 5Z-7-oxozeaenol were previously demonstrated to inhibit kinase activity T. A. H. PubMed Scopus Google Scholar, A. M. R.B. J. 1999; PubMed Scopus Google Scholar). We the effect of 5Z-7-oxozeaenol on purified MEK1 kinase activity 5Z-7-Oxozeaenol did inhibit MEK1 kinase the of 5Z-7-oxozeaenol to inhibit MEK1 is which is that for activity of compounds on from two or three in a from two or three To the for 5Z-7-oxozeaenol inhibition of TAK1, we 5Z-7-oxozeaenol is with We incubated of and 5Z-7-oxozeaenol with purified TAK1 and subsequently kinase activity of TAK1. We found that the of 5Z-7-oxozeaenol to inhibit TAK1 to with suggesting that 5Z-7-oxozeaenol is a inhibitor of binding to TAK1. When 5Z-7-oxozeaenol was with TAK1 for 30 before the of the of 5Z-7-oxozeaenol did not with suggesting that the binding of 5Z-7-oxozeaenol to TAK1 is or very Thus, 5Z-7-oxozeaenol is to the binding of TAK1, thereby inhibiting the catalytic activity of TAK1. To 5Z-7-oxozeaenol is a inhibitor of TAK1 or it inhibits the MAPKKK family, we tested the effect of 5Z-7-oxozeaenol on bacterially expressed MEKK1 kinase activity in vitro 5Z-7-Oxozeaenol had a effect on MEKK1 kinase activity. The of 5Z-7-oxozeaenol to inhibit MEKK1 was To the of 5Z-7-oxozeaenol on MAPKKKs, we ectopically expressed in cells. TAK1 is to be when it is co-expressed with TAB1 (5Shibuya H. Yamaguchi K. Shirakabe K. Tonegawa A. Gotoh Y. Ueno N. Irie K. Nishida E. Matsumoto K. Science. 1996; 272: 1179-1182Crossref PubMed Scopus (524) Google Scholar, 7Kishimoto K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). When N-terminal MEKK1 or the ASK1 is in H. Nishida E. Irie K. P. Saitoh M. Moriguchi T. M. Matsumoto K. K. Gotoh Y. Science. 1997; 275: PubMed Scopus Google Scholar, M. T. Nature. PubMed Scopus Google Scholar). TAK1 with FLAG-MEKK1ΔN, or was expressed in and each kinase was immunoprecipitated with anti-FLAG We measured both to and to phosphorylate MAPKK MKK6 In this 5Z-7-oxozeaenol inhibited of TAK1 and TAK1 activity to phosphorylate MKK6 at of no effect of 5Z-7-oxozeaenol was observed on MEKK1 or ASK1. The kinase activity of was also not inhibited by 5Z-7-oxozeaenol at as high as 500 not Thus, 5Z-7-oxozeaenol is potent and selective inhibitor of TAK1. We have previously demonstrated that TAK1 is in the IL-1 signaling pathway (4Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1023) Google Scholar). The that 5Z-7-oxozeaenol inhibits TAK1 activity the that this compound be an effective inhibitor of IL-1 of cells with IL-1 activates endogenous TAK1 activity and the MAPK and IKK, leading to the activation of JNK/p38 MAPKs and NF-κB, respectively. To 5Z-7-oxozeaenol can inhibit IL-1 we for the effect of 5Z-7-oxozeaenol on NF-κB-dependent activation induced by IL-1 We found that of cells with 5Z-7-oxozeaenol effectively inhibited IL-1-induced activation of NF-κB. NF-κB is activated pathways including human protein and pathways. TAK1 is in TNF-induced NF-κB activation H. Miyoshi H. Toriumi W. Sugita T. J. Biol. Chem. 1999; 274: 10641-10648Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, G. Surabhi R.M. Park K.J. Ninomiya-Tsuji J. Matsumoto K. Gaynor R.B. J. Mol. Biol. 2003; 326: 105-115Crossref PubMed Scopus (322) Google whereas MEKK1 is in NF-κB activation Y. S. F. M. Gaynor R.B. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). NF-κB can also be activated in the of extracellular by of TAK1 and TAB1 or by NF-κB-interacting kinase alone (4Ninomiya-Tsuji J. Kishimoto K. Hiyama A. Inoue J. Cao Z. Matsumoto K. Nature. 1999; 398: 252-256Crossref PubMed Scopus (1023) Google Scholar, Nature. 1997; PubMed Scopus Google Scholar). To the effect of 5Z-7-oxozeaenol is to NF-κB activation by TAK1, cells were with or transfected TAK1, or NF-κB-interacting kinase expression We found that 5Z-7-oxozeaenol effectively inhibited activation of NF-κB induced by and of TAK1 and whereas it had effect on activation of NF-κB induced by of or NF-κB-interacting kinase These results suggest that 5Z-7-oxozeaenol inhibits NF-κB activation by TAK1 activity. The of 5Z-7-oxozeaenol to inhibit NF-κB activation by of TAK1 and TAB1 was This is that to inhibit purified TAK1 in We 5Z-7-oxozeaenol inhibits IL-1-induced JNK/p38 activation. We 293-IL-1RI cells with of 5Z-7-oxozeaenol and the cells with IL-1 The activated and p38 were with anti-phospho-JNK and antibodies that the activated of and p38, JNK/p38 activation was with of 5Z-7-oxozeaenol in a The of 5Z-7-oxozeaenol to inhibit IL-1-induced JNK/p38 activation was in a to that for NF-κB To the of 5Z-7-oxozeaenol, we tested the effect of 5Z-7-oxozeaenol on MAPK activated by other is a of it activates TAK1 in 293-IL-1RI suggesting that TAK1 is not in this 293-IL-1RI cells were with 5Z-7-oxozeaenol for 30 by inhibition of or p38 activation was observed in cells. We also and activation. and activate the kinase in which TAK1 not 293-IL-1RI cells were with 5Z-7-oxozeaenol and with or The activated was with antibody that the activated of and ERK2 and 5Z-7-Oxozeaenol had effect on or activation at a of 500 These results suggest that 5Z-7-oxozeaenol selectively inhibits TAK1, thereby inhibiting IL-1-induced JNK/p38 activation in culture cells. We 5Z-7-oxozeaenol inhibits kinase activity of endogenous TAK1 IL-1 stimulation. We have previously observed that TAK1 is activated after IL-1 when 293-IL-1RI cells were with IL-1 G. Ninomiya-Tsuji J. Kishida S. Li X. Stark G.R. Matsumoto K. Mol. Cell. Biol. 2001; 21: 2475-2484Crossref PubMed Scopus (160) Google Scholar). We 293-IL-1RI cells with various of 5Z-7-oxozeaenol to IL-1 stimulation. At 5 after IL-1 cells were and endogenous TAK1 was immunoprecipitated with anti-TAK1 The catalytic activity of TAK1 was measured using MKK6 as a substrate of the cells with 5Z-7-oxozeaenol inhibited kinase activity of endogenous TAK1. The of 5Z-7-oxozeaenol to inhibit endogenous TAK1 was which is with the to inhibit NF-κB and JNK/p38 activation We tested the inhibition of TAK1 in culture cells is or We 293-IL-1RI cells with 5Z-7-oxozeaenol for 30 and incubated for an 30 The cells were subsequently with and the catalytic activity of endogenous TAK1 was measured 5Z-7-Oxozeaenol inhibited TAK1 kinase activity after 5Z-7-oxozeaenol was removed from the culture These results suggest that 5Z-7-oxozeaenol to and inhibits TAK1 in 293-IL-1RI with the that 5Z-7-oxozeaenol inhibits binding to vitro Thus, it is that 5Z-7-oxozeaenol, when into the culture inhibits TAK1 activity by inhibiting the binding of to TAK1. We also the of activation of TAK1, IKK, JNK, and p38 IL-1 stimulation. In this 293-IL-1RI cells were with 500 5Z-7-oxozeaenol for 30 to inhibit kinase activity of TAK1 and with IL-1 Cells were at and and the lysates were immunoprecipitated with anti-TAK1 by in assay 5Z-7-Oxozeaenol IL-1-induced activation of TAK1. We have previously shown that of TAK1 IL-1 is essential for activation K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). TAK1 can be on SDS-PAGE as TAK1 We observed that 5Z-7-oxozeaenol inhibited IL-1-induced of TAK1. We have also previously demonstrated that endogenous TAK1 with TAB1 K. Matsumoto K. Ninomiya-Tsuji J. J. Biol. Chem. 2000; 275: 7359-7364Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). The of TAB1 in TAK1 immunoprecipitates was not with of 5Z-7-oxozeaenol suggesting that 5Z-7-oxozeaenol did not with interaction of TAK1 with activity was measured using as a substrate 5Z-7-Oxozeaenol inhibited of the kinase activity of the IL-1-induced activity. 5Z-7-oxozeaenol had no effect on kinase activity of 5Z-7-oxozeaenol inhibits IL-1-induced activation of by inhibiting TAK1 activity. We also observed that 5Z-7-oxozeaenol IL-1-induced activation of and p38 and results that 5Z-7-oxozeaenol inhibits the IL-1 signaling pathways that to activation of both NF-κB and JNK/p38 by inhibiting TAK1. IL-1 is a proinflammatory that induces the expression of that inflammation 1996; PubMed Google Scholar). such gene is cyclooxgenase 2 which the production of R.M. 2000; PubMed Scopus Google Scholar). We tested the effect of 5Z-7-oxozeaenol on IL-1-induced production The of proteins was after IL-1 whereas no was when cells were with 5Z-7-oxozeaenol, in the of Thus, 5Z-7-oxozeaenol inhibits production of inflammation We 5Z-7-oxozeaenol inflammation in For this we used ear swelling as a for The ear swelling system has been used as a for and G. J. Full Text PDF PubMed Scopus Google Scholar, Google Scholar). it has been shown that inhibitors of ear swelling P. F. P. J. Full Text PDF PubMed Scopus Google Scholar). Furthermore, of IL-1 production have also been shown to ear swelling induced by PC Y. Inoue Y. M. M. Ueno T. H. M. H. 2000; PubMed Scopus Google suggesting that IL-1 signaling is in this When 5Z-7-oxozeaenol was to the ear swelling was by to of that of the control ear with vehicle Thus, 5Z-7-oxozeaenol is able to prevent inflammation, inhibiting TAK1 activity. 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 production of P. T. S. G. B. S. M. J. 1999; 21: PubMed Scopus Google Scholar). 5Z-7-Oxozeaenol is also able to inhibit JNK/p38 activation K. S. T. T. 1999; PubMed Scopus Google Scholar). the these had been we that 5Z-7-oxozeaenol inhibits the catalytic activity of TAK1. TAK1 is activated with various endotoxins and it is that 5Z-7-oxozeaenol inhibit TAK1 activity activated by and thereby production and JNK/p38 activation. TAK1 is a protein kinase not in the IL-1 signaling pathway but also in the growth family signaling pathway K. Shirakabe K. Shibuya H. Irie K. Oishi I. Ueno N. Taniguchi T. Nishida E. Matsumoto K. Science. 1995; 270: 2008-2011Crossref PubMed Scopus (1178) Google Scholar, H. H. N. Gotoh Y. Yamaguchi K. Irie K. Matsumoto K. Nishida E. Ueno N. J. PubMed Scopus Google Scholar). Furthermore, we have recently found that TAK1 is in a pathway that the signaling pathway T. Ninomiya-Tsuji J. S. M. M. N. M. B. H. Shibuya H. Matsumoto K. Nature. 1999; PubMed Scopus Google Scholar, T. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar, T. Kishida S. J. Ueno N. J. M. Shibuya H. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. Biol. 2003; PubMed Scopus Google Scholar). 5Z-7-oxozeaenol is a highly potent and selective inhibitor of TAK1, this compound be a for on these pathways. Furthermore, in this study, we that when applied 5Z-7-oxozeaenol the of ear swelling. These results suggest that 5Z-7-oxozeaenol be a for such as and We H. S. H. and E. Nishida for and M. for of the