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Triterpenoid CDDO-Me Blocks the NF-κB Pathway by Direct Inhibition of IKKβ on Cys-179 *

Triterpenoid CDDO-Me Blocks the NF-κB Pathway by Direct Inhibition of IKKβ on Cys-179 * THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 47, pp. 35764 –35769, November 24, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Triterpenoid CDDO-Me Blocks the NF-B Pathway by Direct Inhibition of IKK on Cys-179 Received for publication, July 27, 2006, and in revised form, September 14, 2006 Published, JBC Papers in Press, September 24, 2006, DOI 10.1074/jbc.M607160200 ‡ ‡ § ‡ ‡1 Rehan Ahmad , Deepak Raina , Colin Meyer , Surender Kharbanda , and Donald Kufe ‡ § From the Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 and Reata Pharmaceuticals, Inc., Dallas, Texas 75207 The novel oleanane triterpenoid 2-cyano-3,12-dioxooleana- gen species and induce apoptosis by oxidizing critical cysteines 1,9,-dien-28-oic acid (CDDO) and the C-28 methyl ester (CDDO- in proteins that regulate redox balance and survival. Me) induce apoptosis of human tumor cells by disruption of redox NF-B activates the transcription of diverse genes that regu- balance and are currently in clinical trials. The present studies late cell proliferation and survival (18). In the absence of stim- showthatCDDOandCDDO-Meblocktumornecrosisfactor-in- ulation, the NF-B proteins (RelA/p65, RelB, c-Rel, NF-B1/ duced targeting of NF-B p65 to the nucleus. CDDO-Me also p50, and NF-B1/p52) localize to the cytoplasm in complexes blocked tumor necrosis factor -induced phosphorylation of with members of the IB family of inhibitor proteins (19). Phos- IB. In concert with these results, we found that CDDO-Me phorylation of IB induces ubiquitination and degradation of inhibits IB kinase (IKK) activity in cells. In support of a direct IB and release of NF-B p65 to the nucleus. In the classical mechanism, CDDO-Me inhibited recombinant IKK activity in NF-B pathway, the IB kinase  (IKK) in a complex with the vitro. The results also demonstrate that (i) CDDO and CDDO-Me regulatory IKK subunit is the major kinase responsible for form adducts with IKK, but not IKK with mutation of Cys-179 phosphorylation of IB (20). Previous work indicated that to Ala, and (ii) CDDO-Me inhibits IKK by a mechanism depend- CDDO inhibits activation of the NF-B pathway by a mecha- ent on oxidation of Cys-179. These findings indicate that CDDO nism after translocation of NF-B to the nucleus (5). The pres- andCDDO-MedirectlyblockIKKactivityandtherebytheNF-B ent results demonstrate that CDDO and CDDO-Me block the pathway by interacting with Cys-179 in the IKK activation loop. NF-B pathway by inhibiting IKK. The results also indicate that CDDO and CDDO-Me directly inhibit IKK by interacting with Cys-179 in the IKK activation loop. The synthetic triterpenoid CDDO induces differentiation of EXPERIMENTAL PROCEDURES human myeloid leukemia cells and mouse 3T3-Li fibroblasts Cell Culture—Human U-937 myeloid leukemia cells were (1). CDDO also inhibits cytokine-mediated induction of nitric- grown in RPMI 1640 medium containing 10% heat-inactivated oxide synthase and functions as a ligand for peroxisome prolif- fetal bovine serum, 100 units/ml penicillin, 100 g/ml strepto- erator-activated receptor  (1, 2). Other studies have demon- mycin, and 2 mML-glutamine. 293 cells were grown in Dulbec- strated that CDDO and its derivatives at the C-28 position co’s modified Eagle’s medium containing 10% heat-inactivated induce apoptosis of human myeloid leukemia (3–7), osteosar- fetal bovine serum, antibiotics, and L-glutamine. Cells were coma (8), multiple myeloma (9), lung cancer (10, 11), breast treated with CDDO or CDDO-Me (provided by Reata Pharma- cancer (12, 13), and pancreatic cancer (14) cells. CDDO, the ceuticals), human TNF- (20 ng/ml; BD Biosciences), the C-28 methyl ester (CDDO-Me), and the C-28 imidazolide ester proteasome inhibitor MG-132 (25 M; Calbiochem), or DTT induce apoptosis by increasing reactive oxygen species and (300 M; Sigma). decreasing intracellular glutathione (6, 9, 14, 15). How the Subcellular Fractionation—Nuclear and cytosolic fractions CDDO triterpenoids disrupt redox balance is not known. How- were prepared as described (21). ever, the A-ring of these triterpenoids contains an ,-unsat- Immunoprecipitation and Immunoblot Analysis—Lysates urated carbonyl moiety that can form reversible adducts with from subconfluent cells were prepared as described (22). Solu- reactive thiol groups in dithiothreitol (DTT) (16) or with spe- ble proteins were incubated with anti-IKK (Cell Signaling cific cysteine-rich protein targets (17). These findings have Technology) or anti-FLAG (Sigma) and precipitated with pro- indicated that the CDDO triterpenoids increase reactive oxy- tein A/G beads. Immune complexes or cell lysates were sub- jected to immunoblotting with anti-NF-B p65 (Santa Cruz Biotechnology), anti-lamin B (Calbiochem), anti-IB (Santa * This work was supported by NCI, National Institutes of Health Grants Cruz Biotechnology), anti--tubulin (Santa Cruz Biotechnol- CA42802, CA100707, and CA98628. The costs of publication of this article were defrayed in part by the payment of page charges. This article must ogy), anti-phospho-IB (Cell Signaling Technology), anti-- therefore be hereby marked “advertisement” in accordance with 18 U.S.C. actin (Sigma), anti-IKK, anti-phospho-IKK (Cell Signaling Section 1734 solely to indicate this fact. Technology), anti-Bcl-2, anti-Bcl-xL (Santa Cruz Biotechnol- To whom correspondence should be addressed. Tel.: 617-632-3141; Fax: 617-632-2934; E-mail: [email protected]. ogy), or anti-FLAG (Sigma). The immune complexes were The abbreviations used are: CDDO, 2-cyano-3,12-dioxooleana-1,9,-dien-28- detected with horseradish peroxidase-conjugated second anti- oic acid; CDDO-Me, CDDO methyl ester; IKK, IB kinase; DTT, dithiothrei- bodies and enhanced chemiluminescence (ECL; Amersham tol; TNF-, tumor necrosis factor ; GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase. Biosciences). 35764 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 47 •NOVEMBER 24, 2006 This is an Open Access article under the CC BY license. CDDO-Me Inhibits IKK Binding of CDDO-Me-biotin and CDDO-biotin to IKK—CDDO and CDDO-Me were biotinylated as described (23). For in vitro binding studies, (i) anti-FLAG precipitates from 293 cells expressing FLAG- IKK or FLAG-IKK(C179A) were incubated with 5 M CDDO-biotin, and (ii) recombinant IKK or IKK(C179A) was incubated with 1 M CDDO-Me-biotin or 5 M CDDO-biotin. For in vivo studies, 293 cells expressing FLAG-IKK or FLAG-IKK(C179A) were cultured with 5 M CDDO-biotin. Lysates were then precipitated with anti- FLAG. Proteins were separated by SDS-PAGE and transferred to nitro- cellulose membranes. After washing, the membranes were incubated with streptavidin horseradish peroxidase (Amersham Biosciences) and devel- oped with enhanced chemilumines- cence (ECL; Amersham Biosciences). RESULTS AND DISCUSSION CDDO-Me Inhibits NF-B p65 Activation by Blocking IB Phos- phorylation—To assess the effects of CDDO-Me on regulation of the NF-B pathway, we stimulated human U-937 myeloid leukemia cells with TNF- to induce transloca- tion of NF-B p65 to the nucleus (Fig. FIGURE 1. CDDO-Me inhibits NF-B activation by attenuating IB phosphorylation. A, U-937 cells were pretreated with 0.25, 0.5, or 1.0 M CDDO-Me for 6 h and then stimulated with TNF- for 15 min. Nuclear lysates 1A). Treatment of the TNF--stimu- were immunoblotted with anti-NF-B p65 and, as controls for equal loading and purity, with antibodies lated cells with CDDO-Me was asso- against nuclear lamin B, cytosolic IB, and cytosolic-tubulin. WCL, whole cell lysate. B, cells were treated with 1.0 M CDDO-Me for 2 h, and then TNF- was added for the indicated times. Whole cell lysates were immuno- ciated with a concentration-depend- blotted with antibodies against Bcl-xL, Bcl-2, and -actin. C, cells were treated with 0.25, 0.5, or 1.0 M ent decrease in nuclear translocation CDDO-Me for 6 h and with 25 M MG-132 for1hto inhibit the proteasome. The cells were then stimulated with of p65 (Fig. 1A). Equal loading and TNF- for 15 min. Cytosolic lysates were immunoblotted with anti-phospho-IB, anti-IB, and anti--actin. purity of the nuclear lysates was con- firmed by immunoblotting with antibodies against nuclear lamin Luciferase Assays—Cells were transfected with pNF-B-Luc B, cytosolic IB, and cytosolic-tubulin (Fig. 1A). In concert with (Stratagene) and SV-40-Renilla-Luc (Promega) in the presence of Lipofectamine 2000 (Invitrogen). After 24 h, lysates prepared these results, TNF--induced expression of Bcl-2 and Bcl-xL, in passive lysis buffer were analyzed using the dual luciferase which is activated by NF-B (18), was attenuated by CDDO-Me assay kit (Promega). treatment, a response delayed compared with that for nuclear translocation of p65 (Fig. 1B). Similar findings were obtained when IKK Kinase Assays—Anti-IKK precipitates or recombi- nant His-IKK (Upstate Cell Signaling Solutions) were incu- the TNF--stimulated cells were treated with the parent com- bated in kinase buffer (50 mM HEPES, pH 7.4, 10 mM MgCl ,10 pound, CDDO (data not shown), indicating that this effect is not mM MnCl , 0.1 mM sodium vanadate, 10 M ATP, and 1 mM selective for the methyl ester. NF-B p65 is released from cytosolic IB and targeted to the nucleus in response to phosphorylation DTT) with GST-IB and [- P]ATP (PerkinElmer Life Sci- ences) for 30 min at 30 °C. DTT was omitted from the reactions and ubiquitination of IB (24). To determine whether where indicated. The reaction products were analyzed by SDS- CDDO-Me affects IB phosphorylation, cytosolic lysates from PAGE and autoradiography. TNF--stimulated cells were immunoblotted with anti-phospho- Generation of IKK(C179A) Mutant—Mutation of IKK IB. The results demonstrate that CDDO-Me inhibits TNF-- Cys-179 to Ala was generated by site-directed mutagenesis induced phosphorylation of IB (Fig. 1C). In concert with these (Stratagene) using pGEX-IKK as the template and confirmed results, CDDO and CDDO-Me also inhibited TNF--induced by DNA sequencing. IKK and IKK(C179A) were purified degradation of IB (Fig. 1C). These findings indicate that CDDO after cleavage with thrombin to remove the GST moiety. and CDDO-Me act upstream to IB in the NF-B pathway. NOVEMBER 24, 2006• VOLUME 281 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 35765 CDDO-Me Inhibits IKK CDDO-Me Directly Inhibits IKK—The IKK kinase function is necessary and sufficient for phosphorylation of IB (25). To determine whether CDDO-Me inhibits IKK activity, anti-IKK immunoprecipitates were pre- pared from cells pretreated with CDDO-Me and then stimulated with TNF-. Incubation of the precipitates in kinase reactions with GST-IB and [- P]ATP demonstrated that CDDO-Me treatment is associated with inhi- bition of IKK activity (Fig. 2A). Consistent with these results, CDDO-Me inhibited TNF--in- FIGURE 2. CDDO-Me inhibits IKK kinase activity. A and B, cells were pretreated with 0.25 and 0.5 M duced autophosphorylation of CDDO-Me for 6 h and then stimulated with TNF- for 15 min. A, anti-IKK precipitates were incubated in kinase IKK on Ser-181 (Fig. 2B). To deter- reactions with GST-IB and [- P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiog- mine whether CDDO-Me inhibits raphy. The precipitates were also immunoblotted with anti-IKK. B, lysates were immunoblotted with anti- phospho-IKK-Ser-181 and anti-IKK. C, anti-IKK precipitates from control and TNF--treated cells were incu- IKK activity in vitro, anti-IKK bated with 0.25 and 0.5 M CDDO-Me in kinase buffer (without and with 1 mM DTT) containing GST-IB and 32 precipitates from TNF--stimu- [- P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiography. The precipitates were lated cells were incubated with also immunoblotted with anti-IKK. D, kinase-active His-IKK was incubated with 0.25 and 0.5M CDDO-Me for 10 min at 30 °C. IKK activity was then assayed in kinase buffer without (left) and with 1 mM DTT (right) GST-IB in the absence and pres- containing GST-IB and [- P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiography. ence of CDDO-Me. The results Equal loading of His-IKK and GST-IB was determined by immunoblotting. show that IKK activity is also inhibited by CDDO-Me in vitro (Fig. 2C, left). Notably, addition of DTT to the kinase reactions blocked CDDO-Me-mediated inhibition of IKK activity (Fig. 2C, right). In this regard, DTT contains thiol groups that form reversible adducts with the CDDO ,-unsaturated car- bonyl moiety (16). To determine whether the effects of CDDO-Me are direct, we preincubated recom- binant kinase-active His-IKK with CDDO-Me and then assayed for phosphorylation of GST-IB. His- IKK activity was inhibited by CDDO-Me (Fig. 2D, left). By con- trast, the inhibitory effect of CDDO-Me was blocked in the pres- ence of DTT (Fig. 2D, right). Taken together with the finding that DTT abolishes CDDO-Me-medi- ated inhibition of IKK, these results indicate that CDDO-Me FIGURE 3. CDDO-Me inhibits IKK by modification of Cys-179. A, 293 cells were transfected with FLAG-IKK directly inhibits IKK activity. or FLAG-IKK(C179A). At 48 h after transfection, the cells were treated with 0.25 and 0.5 M CDDO-Me for 6 h. CDDO-Me Inhibition of IKK Is Anti-FLAG precipitates were incubated in kinase reactions containing GST-IB and [- P]ATP. The reaction Reversed by Mutation of Cys-179— products were analyzed by SDS-PAGE and autoradiography. The precipitates were also immunoblotted with anti-IKK. B, 293 cells were transfected with FLAG-IKK or FLAG-IKK(C179A). At 48 h after transfection, lysates IKK contains a cysteine at position were immunoprecipitated with anti-FLAG. The precipitates were incubated with 0.25 and 0.5 M CDDO-Me in 179 in its activation loop. To deter- kinase buffer (no DTT) containing GST-IB and [- P]ATP. The reaction products and precipitates were mine whether this cysteine is analyzed as described in panel A. C, 293 cells were cotransfected with pNF-B-Luc, SV-40-Renilla-Luc, and FLAG-IKK or FLAG-IKK(C179A). At 24 h after transfection, cells were treated with 0.25 and 0.5 M involved in inhibition by CDDO- CDDO-Me for 6 h and then assayed for luciferase activity. The results are expressed as the fold activation Me, we transfected 293 cells to (mean  S.D. of three separate experiments) relative to that obtained with the FLAG-IKK control (lane 1, assigned a value of 1). express wild-type FLAG-IKK or 35766 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 47 •NOVEMBER 24, 2006 CDDO-Me Inhibits IKK with inhibition of wild-type IKK (Fig. 3A). By contrast, CDDO-Me had no apparent effect on IKK(C179A) activity (Fig. 3A). In concert with these results, CDDO-Me also had lit- tle effect on IKK(C179) activity when added directly to in vitro kinase assays (Fig. 3B). Moreover, CDDO- Me-induced inhibition of NF-B-me- diated transcription was substan- tially attenuated in cells expressing IKK(C179A) as compared with that obtained with wild-type IKK (Fig. 3C). These findings indicate that CDDO-Me inhibits IKK by react- ing with Cys-179. CDDO-Me Inhibits IKK by Oxi- dizing Cys-179—CDDO forms reversible adducts with DTT and cys- teine-rich protein targets (16, 17). To determine whether CDDO-Me inter- acts directly with IKK in vitro, recombinant IKK was incubated with CDDO-Me conjugated to biotin (CDDO-Me-biotin). Analysis of the reaction products demonstrated the formation of IKK-CDDO adducts (Fig. 4A). By contrast, the interaction was substantially blocked when recombinant IKK(C179A) was incubated with CDDO-Me-biotin (Fig. 4A). Unlabeled CDDO-Me competed with CDDO-Me-biotin for binding to IKK, indicating that the interaction with IKK is not due to the biotin moiety (Fig. 4B, left). In addition, unlabeled CDDO-Me and CDDO-Me-biotin were similarly effective in inhibiting IKK (Fig. 4B, right). Previous studies of certain direct chemical inhibitors of IKK have demonstrated the induction of IKK dimerization (26, 27). Immu- FIGURE 4. CDDO-Me inhibits IKK by oxidizing Cys-179. A, recombinant IKK or IKK(C179A) was incubated noblot analysis of lysates from cells with 1 M CDDO-Me-biotin for 1 h. The reaction products were analyzed by SDS-PAGE, transfer of proteins to a expressing FLAG-IKK demon- nitrocellulose membrane, and detection with streptavidin horseradish peroxidase. B, left, recombinant IKK strated that CDDO-Me treatment is was incubated with 1 M CDDO-Me-biotin and 0 (),1(), or 5 () M unlabeled CDDO-Me. The reaction products were analyzed as described in panel A. Right, kinase-active His-IKK was incubated with 1 M associated with the induction of a CDDO-Me or 1 M CDDO-Me-biotin for 10 min at 30 °C. IKK activity was then assayed in kinase buffer (without higher molecular mass species that DTT) containing GST-IB and [- P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiog- reacts with anti-IKK (Fig. 4C). The raphy. Equal loading of His-IKK and GST-IB was determined by immunoblotting. C, 293 cells expressing IKK or IKK(C179A) were treated with CDDO-Me for 6 h. Lysates were immunoblotted with anti-IKK and absence of this high molecular mass anti--actin. The asterisk (*) identifies the position of the higher molecular mass species that reacts with species in cells expressing FLAG- anti-IKK. D and E, U-937 cells were pretreated with 300 M DTT for 1 h and/or 1 M CDDO-Me for an additional 6 h before stimulation with TNF- for 15 min. Cytosolic lysates (D) were immunoblotted with anti-phospho- IKK(C179A) (Fig. 4C) suggests IB, anti-IB, and anti--actin. Nuclear lysates (E) were immunoblotted with the indicated antibodies. that CDDO-Me may induce the for- mation of IKK dimers by a mecha- FLAG-IKK with a C179A mutation. Analysis of anti-FLAG nism dependent on the interaction with Cys-179. Cells were precipitates for phosphorylation of GST-IB demonstrated also pretreated with DTT to block the interaction between similar levels of activity for FLAG-IKK and FLAG- CDDO-Me and IKK. The results demonstrate that DTT IKK(C179A) (Fig. 3A). CDDO-Me treatment was associated reverses CDDO-Me-induced inhibition of IB phosphoryla- NOVEMBER 24, 2006• VOLUME 281 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 35767 CDDO-Me Inhibits IKK that also contain an ,-unsatur- ated carbonyl moiety (28). The pres- ent studies demonstrate that CDDO-Me directly inhibits IKK activity and thereby the NF-B pathway (Fig. 5D). Previous work has indicated that CDDO inhibits the NF-B pathway following nuclear translocation of p65 (5). In addition, since submission of the present work, another study has reported that CDDO-Me inhibits the NF-B pathway and that CDDO-Me is not a direct inhibitor of IKK (29). By contrast, our results clearly demonstrate that CDDO and CDDO-Me interact directly with IKK. CDDO-Me-induced inhibi- tion of IKK in vitro and in cells was reversed by DTT, which forms reversible adducts with CDDO, indicating that, like the cyclopen- tenone prostaglandins, CDDO-Me inhibits IKK by oxidation of a reac- tive cysteine moiety. Similar results FIGURE 5. CDDO also interacts directly with IKK. A, anti-FLAG precipitates from 293 cells expressing were obtained with CDDO, indicat- FLAG-IKK or FLAG-IKK(C179A) were incubated with 5 M CDDO-biotin for 1 h. The reaction products ing that the presence of the methyl were analyzed by SDS-PAGE, transfer of proteins to a nitrocellulose membrane, and detection with ester is not required for direct inter- streptavidin horseradish peroxidase. B, 293 cells expressing FLAG-IKK or FLAG-IKK(C179A) were cul- tured with 5 M CDDO-biotin for 6 h. Anti-FLAG precipitates were analyzed by SDS-PAGE, transfer to a action with the IKK Cys-179 resi- nitrocellulose membrane, and detection with streptavidin horseradish peroxidase. C, recombinant IKK due. Moreover, the findings that (i) or IKK(C179A) was incubated with 5 M CDDO-biotin for 1 h. The reaction products were analyzed by IKK with a C179A mutation is SDS-PAGE, transfer of proteins to a nitrocellulose membrane, and detection with streptavidin horseradish peroxidase. D, schema depicting CDDO/CDDO-Me inhibition of IKK and the NF-B pathway. insensitive to the effects of CDDO-Me and (ii) CDDO-Me and tion and degradation (Fig. 4D). Consistent with these results, CDDO bind directly to IKK, but not IKK(C179A), in vitro DTT also reversed CDDO-Me-induced inhibition of NF-B and in cells support oxidation of the thiol group on Cys-179 as p65 targeting to the nucleus (Fig. 4E). These findings indicate the inhibitory mechanism. Thus, the anti-inflammatory effects that CDDO-Me inhibits IKK by direct oxidation of Cys-179. of CDDO and its derivatives (1) may, like the cyclopentenone CDDO Also Forms Adducts with IKK Cys-179—To deter- prostaglandins, be mediated by inhibiting IKK. The CDDO mine whether CDDO also interacts with IKK by oxidation of triterpenoids are also potent inducers of apoptosis by a mech- Cys-179, FLAG-IKK and FLAG-IKK(C179A) immunopre- anism involving in part the activation of JNK (15). In this con- cipitated from 293 cells were incubated with CDDO conjugated text, inhibition of NF-B sensitizes cells to the induction of to biotin (CDDO-biotin). The results demonstrate that CDDO apoptosis by sustained activation of JNK (30). Direct inhibition forms adducts with IKK and not IKK(C179A) (Fig. 5A). To of IKK and thereby the NF-B pathway by CDDO or its deriv- determine whether CDDO forms adducts with IKK in vivo, atives could therefore contribute to the apoptotic response of cells expressing FLAG-IKK or FLAG-IKK(C179A) were cul- tumor cells to treatment with these agents. tured with CDDO-biotin. In concert with the in vitro results, analysis of anti-FLAG precipitates demonstrated that CDDO Acknowledgments—We thank Dr. Thomas Gilmore, Boston Univer- binds to IKK and not IKK(C179A) (Fig. 5B). Moreover, incu- sity, for the FLAG-IKK and FLAG-IKK(C179A) vectors and Dr. Michael Karin for GST-IKK. Kamal Chauhan is acknowledged for bation of recombinant IKK and IKK(C179A) with CDDO- technical support. biotin confirmed that CDDO directly forms adducts with the IKK Cys-179 residue (Fig. 5C). These findings demonstrate that, like CDDO-Me, CDDO interacts with IKK by oxidizing REFERENCES Cys-179. 1. Suh, N., Wang, Y., Honda, T., Gribble, G. 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(2004) Cancer Res. 64, 1902–1905 NOVEMBER 24, 2006• VOLUME 281 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 35769 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Triterpenoid CDDO-Me Blocks the NF-κB Pathway by Direct Inhibition of IKKβ on Cys-179 *

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American Society for Biochemistry and Molecular Biology
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Copyright © 2006 Elsevier Inc.
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0021-9258
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DOI
10.1074/jbc.m607160200
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO. 47, pp. 35764 –35769, November 24, 2006 © 2006 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Triterpenoid CDDO-Me Blocks the NF-B Pathway by Direct Inhibition of IKK on Cys-179 Received for publication, July 27, 2006, and in revised form, September 14, 2006 Published, JBC Papers in Press, September 24, 2006, DOI 10.1074/jbc.M607160200 ‡ ‡ § ‡ ‡1 Rehan Ahmad , Deepak Raina , Colin Meyer , Surender Kharbanda , and Donald Kufe ‡ § From the Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 and Reata Pharmaceuticals, Inc., Dallas, Texas 75207 The novel oleanane triterpenoid 2-cyano-3,12-dioxooleana- gen species and induce apoptosis by oxidizing critical cysteines 1,9,-dien-28-oic acid (CDDO) and the C-28 methyl ester (CDDO- in proteins that regulate redox balance and survival. Me) induce apoptosis of human tumor cells by disruption of redox NF-B activates the transcription of diverse genes that regu- balance and are currently in clinical trials. The present studies late cell proliferation and survival (18). In the absence of stim- showthatCDDOandCDDO-Meblocktumornecrosisfactor-in- ulation, the NF-B proteins (RelA/p65, RelB, c-Rel, NF-B1/ duced targeting of NF-B p65 to the nucleus. CDDO-Me also p50, and NF-B1/p52) localize to the cytoplasm in complexes blocked tumor necrosis factor -induced phosphorylation of with members of the IB family of inhibitor proteins (19). Phos- IB. In concert with these results, we found that CDDO-Me phorylation of IB induces ubiquitination and degradation of inhibits IB kinase (IKK) activity in cells. In support of a direct IB and release of NF-B p65 to the nucleus. In the classical mechanism, CDDO-Me inhibited recombinant IKK activity in NF-B pathway, the IB kinase  (IKK) in a complex with the vitro. The results also demonstrate that (i) CDDO and CDDO-Me regulatory IKK subunit is the major kinase responsible for form adducts with IKK, but not IKK with mutation of Cys-179 phosphorylation of IB (20). Previous work indicated that to Ala, and (ii) CDDO-Me inhibits IKK by a mechanism depend- CDDO inhibits activation of the NF-B pathway by a mecha- ent on oxidation of Cys-179. These findings indicate that CDDO nism after translocation of NF-B to the nucleus (5). The pres- andCDDO-MedirectlyblockIKKactivityandtherebytheNF-B ent results demonstrate that CDDO and CDDO-Me block the pathway by interacting with Cys-179 in the IKK activation loop. NF-B pathway by inhibiting IKK. The results also indicate that CDDO and CDDO-Me directly inhibit IKK by interacting with Cys-179 in the IKK activation loop. The synthetic triterpenoid CDDO induces differentiation of EXPERIMENTAL PROCEDURES human myeloid leukemia cells and mouse 3T3-Li fibroblasts Cell Culture—Human U-937 myeloid leukemia cells were (1). CDDO also inhibits cytokine-mediated induction of nitric- grown in RPMI 1640 medium containing 10% heat-inactivated oxide synthase and functions as a ligand for peroxisome prolif- fetal bovine serum, 100 units/ml penicillin, 100 g/ml strepto- erator-activated receptor  (1, 2). Other studies have demon- mycin, and 2 mML-glutamine. 293 cells were grown in Dulbec- strated that CDDO and its derivatives at the C-28 position co’s modified Eagle’s medium containing 10% heat-inactivated induce apoptosis of human myeloid leukemia (3–7), osteosar- fetal bovine serum, antibiotics, and L-glutamine. Cells were coma (8), multiple myeloma (9), lung cancer (10, 11), breast treated with CDDO or CDDO-Me (provided by Reata Pharma- cancer (12, 13), and pancreatic cancer (14) cells. CDDO, the ceuticals), human TNF- (20 ng/ml; BD Biosciences), the C-28 methyl ester (CDDO-Me), and the C-28 imidazolide ester proteasome inhibitor MG-132 (25 M; Calbiochem), or DTT induce apoptosis by increasing reactive oxygen species and (300 M; Sigma). decreasing intracellular glutathione (6, 9, 14, 15). How the Subcellular Fractionation—Nuclear and cytosolic fractions CDDO triterpenoids disrupt redox balance is not known. How- were prepared as described (21). ever, the A-ring of these triterpenoids contains an ,-unsat- Immunoprecipitation and Immunoblot Analysis—Lysates urated carbonyl moiety that can form reversible adducts with from subconfluent cells were prepared as described (22). Solu- reactive thiol groups in dithiothreitol (DTT) (16) or with spe- ble proteins were incubated with anti-IKK (Cell Signaling cific cysteine-rich protein targets (17). These findings have Technology) or anti-FLAG (Sigma) and precipitated with pro- indicated that the CDDO triterpenoids increase reactive oxy- tein A/G beads. Immune complexes or cell lysates were sub- jected to immunoblotting with anti-NF-B p65 (Santa Cruz Biotechnology), anti-lamin B (Calbiochem), anti-IB (Santa * This work was supported by NCI, National Institutes of Health Grants Cruz Biotechnology), anti--tubulin (Santa Cruz Biotechnol- CA42802, CA100707, and CA98628. The costs of publication of this article were defrayed in part by the payment of page charges. This article must ogy), anti-phospho-IB (Cell Signaling Technology), anti-- therefore be hereby marked “advertisement” in accordance with 18 U.S.C. actin (Sigma), anti-IKK, anti-phospho-IKK (Cell Signaling Section 1734 solely to indicate this fact. Technology), anti-Bcl-2, anti-Bcl-xL (Santa Cruz Biotechnol- To whom correspondence should be addressed. Tel.: 617-632-3141; Fax: 617-632-2934; E-mail: [email protected]. ogy), or anti-FLAG (Sigma). The immune complexes were The abbreviations used are: CDDO, 2-cyano-3,12-dioxooleana-1,9,-dien-28- detected with horseradish peroxidase-conjugated second anti- oic acid; CDDO-Me, CDDO methyl ester; IKK, IB kinase; DTT, dithiothrei- bodies and enhanced chemiluminescence (ECL; Amersham tol; TNF-, tumor necrosis factor ; GST, glutathione S-transferase; JNK, c-Jun N-terminal kinase. Biosciences). 35764 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 47 •NOVEMBER 24, 2006 This is an Open Access article under the CC BY license. CDDO-Me Inhibits IKK Binding of CDDO-Me-biotin and CDDO-biotin to IKK—CDDO and CDDO-Me were biotinylated as described (23). For in vitro binding studies, (i) anti-FLAG precipitates from 293 cells expressing FLAG- IKK or FLAG-IKK(C179A) were incubated with 5 M CDDO-biotin, and (ii) recombinant IKK or IKK(C179A) was incubated with 1 M CDDO-Me-biotin or 5 M CDDO-biotin. For in vivo studies, 293 cells expressing FLAG-IKK or FLAG-IKK(C179A) were cultured with 5 M CDDO-biotin. Lysates were then precipitated with anti- FLAG. Proteins were separated by SDS-PAGE and transferred to nitro- cellulose membranes. After washing, the membranes were incubated with streptavidin horseradish peroxidase (Amersham Biosciences) and devel- oped with enhanced chemilumines- cence (ECL; Amersham Biosciences). RESULTS AND DISCUSSION CDDO-Me Inhibits NF-B p65 Activation by Blocking IB Phos- phorylation—To assess the effects of CDDO-Me on regulation of the NF-B pathway, we stimulated human U-937 myeloid leukemia cells with TNF- to induce transloca- tion of NF-B p65 to the nucleus (Fig. FIGURE 1. CDDO-Me inhibits NF-B activation by attenuating IB phosphorylation. A, U-937 cells were pretreated with 0.25, 0.5, or 1.0 M CDDO-Me for 6 h and then stimulated with TNF- for 15 min. Nuclear lysates 1A). Treatment of the TNF--stimu- were immunoblotted with anti-NF-B p65 and, as controls for equal loading and purity, with antibodies lated cells with CDDO-Me was asso- against nuclear lamin B, cytosolic IB, and cytosolic-tubulin. WCL, whole cell lysate. B, cells were treated with 1.0 M CDDO-Me for 2 h, and then TNF- was added for the indicated times. Whole cell lysates were immuno- ciated with a concentration-depend- blotted with antibodies against Bcl-xL, Bcl-2, and -actin. C, cells were treated with 0.25, 0.5, or 1.0 M ent decrease in nuclear translocation CDDO-Me for 6 h and with 25 M MG-132 for1hto inhibit the proteasome. The cells were then stimulated with of p65 (Fig. 1A). Equal loading and TNF- for 15 min. Cytosolic lysates were immunoblotted with anti-phospho-IB, anti-IB, and anti--actin. purity of the nuclear lysates was con- firmed by immunoblotting with antibodies against nuclear lamin Luciferase Assays—Cells were transfected with pNF-B-Luc B, cytosolic IB, and cytosolic-tubulin (Fig. 1A). In concert with (Stratagene) and SV-40-Renilla-Luc (Promega) in the presence of Lipofectamine 2000 (Invitrogen). After 24 h, lysates prepared these results, TNF--induced expression of Bcl-2 and Bcl-xL, in passive lysis buffer were analyzed using the dual luciferase which is activated by NF-B (18), was attenuated by CDDO-Me assay kit (Promega). treatment, a response delayed compared with that for nuclear translocation of p65 (Fig. 1B). Similar findings were obtained when IKK Kinase Assays—Anti-IKK precipitates or recombi- nant His-IKK (Upstate Cell Signaling Solutions) were incu- the TNF--stimulated cells were treated with the parent com- bated in kinase buffer (50 mM HEPES, pH 7.4, 10 mM MgCl ,10 pound, CDDO (data not shown), indicating that this effect is not mM MnCl , 0.1 mM sodium vanadate, 10 M ATP, and 1 mM selective for the methyl ester. NF-B p65 is released from cytosolic IB and targeted to the nucleus in response to phosphorylation DTT) with GST-IB and [- P]ATP (PerkinElmer Life Sci- ences) for 30 min at 30 °C. DTT was omitted from the reactions and ubiquitination of IB (24). To determine whether where indicated. The reaction products were analyzed by SDS- CDDO-Me affects IB phosphorylation, cytosolic lysates from PAGE and autoradiography. TNF--stimulated cells were immunoblotted with anti-phospho- Generation of IKK(C179A) Mutant—Mutation of IKK IB. The results demonstrate that CDDO-Me inhibits TNF-- Cys-179 to Ala was generated by site-directed mutagenesis induced phosphorylation of IB (Fig. 1C). In concert with these (Stratagene) using pGEX-IKK as the template and confirmed results, CDDO and CDDO-Me also inhibited TNF--induced by DNA sequencing. IKK and IKK(C179A) were purified degradation of IB (Fig. 1C). These findings indicate that CDDO after cleavage with thrombin to remove the GST moiety. and CDDO-Me act upstream to IB in the NF-B pathway. NOVEMBER 24, 2006• VOLUME 281 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 35765 CDDO-Me Inhibits IKK CDDO-Me Directly Inhibits IKK—The IKK kinase function is necessary and sufficient for phosphorylation of IB (25). To determine whether CDDO-Me inhibits IKK activity, anti-IKK immunoprecipitates were pre- pared from cells pretreated with CDDO-Me and then stimulated with TNF-. Incubation of the precipitates in kinase reactions with GST-IB and [- P]ATP demonstrated that CDDO-Me treatment is associated with inhi- bition of IKK activity (Fig. 2A). Consistent with these results, CDDO-Me inhibited TNF--in- FIGURE 2. CDDO-Me inhibits IKK kinase activity. A and B, cells were pretreated with 0.25 and 0.5 M duced autophosphorylation of CDDO-Me for 6 h and then stimulated with TNF- for 15 min. A, anti-IKK precipitates were incubated in kinase IKK on Ser-181 (Fig. 2B). To deter- reactions with GST-IB and [- P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiog- mine whether CDDO-Me inhibits raphy. The precipitates were also immunoblotted with anti-IKK. B, lysates were immunoblotted with anti- phospho-IKK-Ser-181 and anti-IKK. C, anti-IKK precipitates from control and TNF--treated cells were incu- IKK activity in vitro, anti-IKK bated with 0.25 and 0.5 M CDDO-Me in kinase buffer (without and with 1 mM DTT) containing GST-IB and 32 precipitates from TNF--stimu- [- P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiography. The precipitates were lated cells were incubated with also immunoblotted with anti-IKK. D, kinase-active His-IKK was incubated with 0.25 and 0.5M CDDO-Me for 10 min at 30 °C. IKK activity was then assayed in kinase buffer without (left) and with 1 mM DTT (right) GST-IB in the absence and pres- containing GST-IB and [- P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiography. ence of CDDO-Me. The results Equal loading of His-IKK and GST-IB was determined by immunoblotting. show that IKK activity is also inhibited by CDDO-Me in vitro (Fig. 2C, left). Notably, addition of DTT to the kinase reactions blocked CDDO-Me-mediated inhibition of IKK activity (Fig. 2C, right). In this regard, DTT contains thiol groups that form reversible adducts with the CDDO ,-unsaturated car- bonyl moiety (16). To determine whether the effects of CDDO-Me are direct, we preincubated recom- binant kinase-active His-IKK with CDDO-Me and then assayed for phosphorylation of GST-IB. His- IKK activity was inhibited by CDDO-Me (Fig. 2D, left). By con- trast, the inhibitory effect of CDDO-Me was blocked in the pres- ence of DTT (Fig. 2D, right). Taken together with the finding that DTT abolishes CDDO-Me-medi- ated inhibition of IKK, these results indicate that CDDO-Me FIGURE 3. CDDO-Me inhibits IKK by modification of Cys-179. A, 293 cells were transfected with FLAG-IKK directly inhibits IKK activity. or FLAG-IKK(C179A). At 48 h after transfection, the cells were treated with 0.25 and 0.5 M CDDO-Me for 6 h. CDDO-Me Inhibition of IKK Is Anti-FLAG precipitates were incubated in kinase reactions containing GST-IB and [- P]ATP. The reaction Reversed by Mutation of Cys-179— products were analyzed by SDS-PAGE and autoradiography. The precipitates were also immunoblotted with anti-IKK. B, 293 cells were transfected with FLAG-IKK or FLAG-IKK(C179A). At 48 h after transfection, lysates IKK contains a cysteine at position were immunoprecipitated with anti-FLAG. The precipitates were incubated with 0.25 and 0.5 M CDDO-Me in 179 in its activation loop. To deter- kinase buffer (no DTT) containing GST-IB and [- P]ATP. The reaction products and precipitates were mine whether this cysteine is analyzed as described in panel A. C, 293 cells were cotransfected with pNF-B-Luc, SV-40-Renilla-Luc, and FLAG-IKK or FLAG-IKK(C179A). At 24 h after transfection, cells were treated with 0.25 and 0.5 M involved in inhibition by CDDO- CDDO-Me for 6 h and then assayed for luciferase activity. The results are expressed as the fold activation Me, we transfected 293 cells to (mean  S.D. of three separate experiments) relative to that obtained with the FLAG-IKK control (lane 1, assigned a value of 1). express wild-type FLAG-IKK or 35766 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 281 • NUMBER 47 •NOVEMBER 24, 2006 CDDO-Me Inhibits IKK with inhibition of wild-type IKK (Fig. 3A). By contrast, CDDO-Me had no apparent effect on IKK(C179A) activity (Fig. 3A). In concert with these results, CDDO-Me also had lit- tle effect on IKK(C179) activity when added directly to in vitro kinase assays (Fig. 3B). Moreover, CDDO- Me-induced inhibition of NF-B-me- diated transcription was substan- tially attenuated in cells expressing IKK(C179A) as compared with that obtained with wild-type IKK (Fig. 3C). These findings indicate that CDDO-Me inhibits IKK by react- ing with Cys-179. CDDO-Me Inhibits IKK by Oxi- dizing Cys-179—CDDO forms reversible adducts with DTT and cys- teine-rich protein targets (16, 17). To determine whether CDDO-Me inter- acts directly with IKK in vitro, recombinant IKK was incubated with CDDO-Me conjugated to biotin (CDDO-Me-biotin). Analysis of the reaction products demonstrated the formation of IKK-CDDO adducts (Fig. 4A). By contrast, the interaction was substantially blocked when recombinant IKK(C179A) was incubated with CDDO-Me-biotin (Fig. 4A). Unlabeled CDDO-Me competed with CDDO-Me-biotin for binding to IKK, indicating that the interaction with IKK is not due to the biotin moiety (Fig. 4B, left). In addition, unlabeled CDDO-Me and CDDO-Me-biotin were similarly effective in inhibiting IKK (Fig. 4B, right). Previous studies of certain direct chemical inhibitors of IKK have demonstrated the induction of IKK dimerization (26, 27). Immu- FIGURE 4. CDDO-Me inhibits IKK by oxidizing Cys-179. A, recombinant IKK or IKK(C179A) was incubated noblot analysis of lysates from cells with 1 M CDDO-Me-biotin for 1 h. The reaction products were analyzed by SDS-PAGE, transfer of proteins to a expressing FLAG-IKK demon- nitrocellulose membrane, and detection with streptavidin horseradish peroxidase. B, left, recombinant IKK strated that CDDO-Me treatment is was incubated with 1 M CDDO-Me-biotin and 0 (),1(), or 5 () M unlabeled CDDO-Me. The reaction products were analyzed as described in panel A. Right, kinase-active His-IKK was incubated with 1 M associated with the induction of a CDDO-Me or 1 M CDDO-Me-biotin for 10 min at 30 °C. IKK activity was then assayed in kinase buffer (without higher molecular mass species that DTT) containing GST-IB and [- P]ATP. The reaction products were analyzed by SDS-PAGE and autoradiog- reacts with anti-IKK (Fig. 4C). The raphy. Equal loading of His-IKK and GST-IB was determined by immunoblotting. C, 293 cells expressing IKK or IKK(C179A) were treated with CDDO-Me for 6 h. Lysates were immunoblotted with anti-IKK and absence of this high molecular mass anti--actin. The asterisk (*) identifies the position of the higher molecular mass species that reacts with species in cells expressing FLAG- anti-IKK. D and E, U-937 cells were pretreated with 300 M DTT for 1 h and/or 1 M CDDO-Me for an additional 6 h before stimulation with TNF- for 15 min. Cytosolic lysates (D) were immunoblotted with anti-phospho- IKK(C179A) (Fig. 4C) suggests IB, anti-IB, and anti--actin. Nuclear lysates (E) were immunoblotted with the indicated antibodies. that CDDO-Me may induce the for- mation of IKK dimers by a mecha- FLAG-IKK with a C179A mutation. Analysis of anti-FLAG nism dependent on the interaction with Cys-179. Cells were precipitates for phosphorylation of GST-IB demonstrated also pretreated with DTT to block the interaction between similar levels of activity for FLAG-IKK and FLAG- CDDO-Me and IKK. The results demonstrate that DTT IKK(C179A) (Fig. 3A). CDDO-Me treatment was associated reverses CDDO-Me-induced inhibition of IB phosphoryla- NOVEMBER 24, 2006• VOLUME 281 • NUMBER 47 JOURNAL OF BIOLOGICAL CHEMISTRY 35767 CDDO-Me Inhibits IKK that also contain an ,-unsatur- ated carbonyl moiety (28). The pres- ent studies demonstrate that CDDO-Me directly inhibits IKK activity and thereby the NF-B pathway (Fig. 5D). Previous work has indicated that CDDO inhibits the NF-B pathway following nuclear translocation of p65 (5). In addition, since submission of the present work, another study has reported that CDDO-Me inhibits the NF-B pathway and that CDDO-Me is not a direct inhibitor of IKK (29). By contrast, our results clearly demonstrate that CDDO and CDDO-Me interact directly with IKK. CDDO-Me-induced inhibi- tion of IKK in vitro and in cells was reversed by DTT, which forms reversible adducts with CDDO, indicating that, like the cyclopen- tenone prostaglandins, CDDO-Me inhibits IKK by oxidation of a reac- tive cysteine moiety. Similar results FIGURE 5. CDDO also interacts directly with IKK. A, anti-FLAG precipitates from 293 cells expressing were obtained with CDDO, indicat- FLAG-IKK or FLAG-IKK(C179A) were incubated with 5 M CDDO-biotin for 1 h. The reaction products ing that the presence of the methyl were analyzed by SDS-PAGE, transfer of proteins to a nitrocellulose membrane, and detection with ester is not required for direct inter- streptavidin horseradish peroxidase. B, 293 cells expressing FLAG-IKK or FLAG-IKK(C179A) were cul- tured with 5 M CDDO-biotin for 6 h. Anti-FLAG precipitates were analyzed by SDS-PAGE, transfer to a action with the IKK Cys-179 resi- nitrocellulose membrane, and detection with streptavidin horseradish peroxidase. C, recombinant IKK due. Moreover, the findings that (i) or IKK(C179A) was incubated with 5 M CDDO-biotin for 1 h. The reaction products were analyzed by IKK with a C179A mutation is SDS-PAGE, transfer of proteins to a nitrocellulose membrane, and detection with streptavidin horseradish peroxidase. D, schema depicting CDDO/CDDO-Me inhibition of IKK and the NF-B pathway. insensitive to the effects of CDDO-Me and (ii) CDDO-Me and tion and degradation (Fig. 4D). Consistent with these results, CDDO bind directly to IKK, but not IKK(C179A), in vitro DTT also reversed CDDO-Me-induced inhibition of NF-B and in cells support oxidation of the thiol group on Cys-179 as p65 targeting to the nucleus (Fig. 4E). These findings indicate the inhibitory mechanism. Thus, the anti-inflammatory effects that CDDO-Me inhibits IKK by direct oxidation of Cys-179. of CDDO and its derivatives (1) may, like the cyclopentenone CDDO Also Forms Adducts with IKK Cys-179—To deter- prostaglandins, be mediated by inhibiting IKK. The CDDO mine whether CDDO also interacts with IKK by oxidation of triterpenoids are also potent inducers of apoptosis by a mech- Cys-179, FLAG-IKK and FLAG-IKK(C179A) immunopre- anism involving in part the activation of JNK (15). In this con- cipitated from 293 cells were incubated with CDDO conjugated text, inhibition of NF-B sensitizes cells to the induction of to biotin (CDDO-biotin). The results demonstrate that CDDO apoptosis by sustained activation of JNK (30). Direct inhibition forms adducts with IKK and not IKK(C179A) (Fig. 5A). To of IKK and thereby the NF-B pathway by CDDO or its deriv- determine whether CDDO forms adducts with IKK in vivo, atives could therefore contribute to the apoptotic response of cells expressing FLAG-IKK or FLAG-IKK(C179A) were cul- tumor cells to treatment with these agents. tured with CDDO-biotin. In concert with the in vitro results, analysis of anti-FLAG precipitates demonstrated that CDDO Acknowledgments—We thank Dr. Thomas Gilmore, Boston Univer- binds to IKK and not IKK(C179A) (Fig. 5B). Moreover, incu- sity, for the FLAG-IKK and FLAG-IKK(C179A) vectors and Dr. Michael Karin for GST-IKK. Kamal Chauhan is acknowledged for bation of recombinant IKK and IKK(C179A) with CDDO- technical support. biotin confirmed that CDDO directly forms adducts with the IKK Cys-179 residue (Fig. 5C). These findings demonstrate that, like CDDO-Me, CDDO interacts with IKK by oxidizing REFERENCES Cys-179. 1. Suh, N., Wang, Y., Honda, T., Gribble, G. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Nov 24, 2006

References