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

The novel oleanane triterpenoid 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid (CDDO) and the C-28 methyl ester (CDDO-Me) induce apoptosis of human tumor cells by disruption of redox balance and are currently in clinical trials. The present studies show that CDDO and CDDO-Me block tumor necrosis factorα-induced targeting of NF-κB p65 to the nucleus. CDDO-Me also blocked tumor necrosis factor α-induced phosphorylation of IκBα. In concert with these results, we found that CDDO-Me inhibits IκBα kinaseβ (IKKβ) activity in cells. In support of a direct mechanism, CDDO-Me inhibited recombinant IKKβ activity in vitro. The results also demonstrate that (i) CDDO and CDDO-Me form adducts with IKKβ, but not IKKβ with mutation of Cys-179 to Ala, and (ii) CDDO-Me inhibits IKKβ by a mechanism dependent on oxidation of Cys-179. These findings indicate that CDDO and CDDO-Me directly block IKKβ activity and thereby the NF-κB pathway by interacting with Cys-179 in the IKKβ activation loop.

NF-B activates the transcription of diverse genes that regulate cell proliferation and survival (18). In the absence of stimulation, the NF-B proteins (RelA/p65, RelB, c-Rel, NF-B1/ p50, and NF-B1/p52) localize to the cytoplasm in complexes with members of the IB family of inhibitor proteins (19). Phosphorylation of IB␣ induces ubiquitination and degradation of IB␣ and release of NF-B p65 to the nucleus. In the classical NF-B pathway, the IB kinase ␤ (IKK␤) in a complex with the regulatory IKK␥ subunit is the major kinase responsible for phosphorylation of IB␣ (20). Previous work indicated that CDDO inhibits activation of the NF-B pathway by a mechanism after translocation of NF-B to the nucleus (5). The present results demonstrate that CDDO and CDDO-Me block the 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.
Immunoprecipitation and Immunoblot Analysis-Lysates from subconfluent cells were prepared as described (22). Soluble proteins were incubated with anti-IKK␤ (Cell Signaling Technology) or anti-FLAG (Sigma) and precipitated with protein A/G beads. Immune complexes or cell lysates were subjected to immunoblotting with anti-NF-B p65 (Santa Cruz Biotechnology), anti-lamin B (Calbiochem), anti-IB␣ (Santa Cruz Biotechnology), anti-␣-tubulin (Santa Cruz Biotechnology), anti-phospho-IB␣ (Cell Signaling Technology), anti-␤actin (Sigma), anti-IKK␤, anti-phospho-IKK␤ (Cell Signaling Technology), anti-Bcl-2, anti-Bcl-xL (Santa Cruz Biotechnology), or anti-FLAG (Sigma). The immune complexes were detected with horseradish peroxidase-conjugated second antibodies and enhanced chemiluminescence (ECL; Amersham Biosciences). * This work was supported by NCI, National Institutes of Health Grants CA42802, CA100707, and CA98628. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed. Generation of IKK␤(C179A) Mutant-Mutation of IKK␤ Cys-179 to Ala was generated by site-directed mutagenesis (Stratagene) using pGEX-IKK␤ as the template and confirmed by DNA sequencing. IKK␤ and IKK␤(C179A) were purified after cleavage with thrombin to remove the GST moiety.

CDDO-Me Inhibits NF-B p65
Activation by Blocking IB␣ Phosphorylation-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 translocation of NF-B p65 to the nucleus (Fig.  1A). Treatment of the TNF-␣-stimulated cells with CDDO-Me was associated with a concentration-dependent decrease in nuclear translocation of p65 (Fig. 1A). Equal loading and purity of the nuclear lysates was confirmed by immunoblotting with antibodies against nuclear lamin B, cytosolic IB␣, and cytosolic ␣-tubulin (Fig. 1A). In concert with these results, TNF-␣-induced expression of Bcl-2 and Bcl-xL, which is activated by NF-B (18), was attenuated by CDDO-Me treatment, a response delayed compared with that for nuclear translocation of p65 (Fig. 1B). Similar findings were obtained when the TNF-␣-stimulated cells were treated with the parent compound, CDDO (data not shown), indicating that this effect is not selective for the methyl ester. NF-B p65 is released from cytosolic IB␣ and targeted to the nucleus in response to phosphorylation and ubiquitination of IB␣ (24). To determine whether CDDO-Me affects IB␣ phosphorylation, cytosolic lysates from TNF-␣-stimulated cells were immunoblotted with anti-phospho-IB␣. The results demonstrate that CDDO-Me inhibits TNF-␣induced phosphorylation of IB␣ (Fig. 1C). In concert with these results, CDDO and CDDO-Me also inhibited TNF-␣-induced degradation of IB␣ (Fig. 1C). These findings indicate that CDDO and CDDO-Me act upstream to IB␣ in the NF-B pathway. 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 prepared from cells pretreated with CDDO-Me and then stimulated with TNF-␣. Incubation of the precipitates in kinase reactions with GST-IB␣ and [␥-32 P]ATP demonstrated that CDDO-Me treatment is associated with inhibition of IKK␤ activity ( Fig. 2A). Consistent with these results, CDDO-Me inhibited TNF-␣-induced autophosphorylation of IKK␤ on Ser-181 (Fig. 2B). To determine whether CDDO-Me inhibits IKK␤ activity in vitro, anti-IKK␤ precipitates from TNF-␣-stimulated cells were incubated with GST-IB␣ in the absence and presence of CDDO-Me. The results 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 carbonyl moiety (16). To determine whether the effects of CDDO-Me are direct, we preincubated recombinant 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 contrast, the inhibitory effect of CDDO-Me was blocked in the presence of DTT (Fig. 2D, right). Taken together with the finding that DTT abolishes CDDO-Me-mediated inhibition of IKK␤, these results indicate that CDDO-Me directly inhibits IKK␤ activity.

CDDO-Me Inhibition of IKK␤ Is Reversed by Mutation of Cys-179-
IKK␤ contains a cysteine at position 179 in its activation loop. To determine whether this cysteine is involved in inhibition by CDDO-Me, we transfected 293 cells to express wild-type FLAG-IKK␤ or  FLAG-IKK␤ with a C179A mutation. Analysis of anti-FLAG precipitates for phosphorylation of GST-IB␣ demonstrated similar levels of activity for FLAG-IKK␤ and FLAG-IKK␤(C179A) (Fig. 3A). CDDO-Me treatment was associated 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 little effect on IKK␤(C179) activity when added directly to in vitro kinase assays (Fig. 3B). Moreover, CDDO-Me-induced inhibition of NF-B-mediated transcription was substantially 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 reacting with Cys-179.

CDDO-Me Inhibits IKK␤ by Oxidizing
Cys-179-CDDO forms reversible adducts with DTT and cysteine-rich protein targets (16,17). To determine whether CDDO-Me interacts 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). Immunoblot analysis of lysates from cells expressing FLAG-IKK␤ demonstrated that CDDO-Me treatment is associated with the induction of a higher molecular mass species that reacts with anti-IKK␤ (Fig. 4C). The absence of this high molecular mass species in cells expressing FLAG-IKK␤(C179A) (Fig. 4C) suggests that CDDO-Me may induce the formation of IKK␤ dimers by a mechanism dependent on the interaction with Cys-179. Cells were also pretreated with DTT to block the interaction between CDDO-Me and IKK␤. The results demonstrate that DTT reverses CDDO-Me-induced inhibition of IB␣ phosphoryla- tion and degradation (Fig. 4D). Consistent with these results, DTT also reversed CDDO-Me-induced inhibition of NF-B p65 targeting to the nucleus (Fig. 4E). These findings indicate that CDDO-Me inhibits IKK␤ by direct oxidation of Cys-179.
CDDO-Me Functions as an Electrophile in Inhibiting IKK␤-CDDO contains an ␣,␤-unsaturated carbonyl in the A-ring that forms reversible adducts with thiol nucleophiles (16). Other studies have shown that Cys-179 in the IKK␤ activation loop is sensitive to modification by cyclopentenone prostaglandins that also contain an ␣,␤-unsaturated carbonyl moiety (28). The present 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 inhibition of IKK␤ in vitro and in cells was reversed by DTT, which forms reversible adducts with CDDO, indicating that, like the cyclopentenone prostaglandins, CDDO-Me inhibits IKK␤ by oxidation of a reactive cysteine moiety. Similar results were obtained with CDDO, indicating that the presence of the methyl ester is not required for direct interaction with the IKK␤ Cys-179 residue. Moreover, the findings that (i) IKK␤ with a C179A mutation is insensitive to the effects of CDDO-Me and (ii) CDDO-Me and CDDO bind directly to IKK␤, but not IKK␤(C179A), in vitro and in cells support oxidation of the thiol group on Cys-179 as the inhibitory mechanism. Thus, the anti-inflammatory effects of CDDO and its derivatives (1) may, like the cyclopentenone prostaglandins, be mediated by inhibiting IKK␤. The CDDO triterpenoids are also potent inducers of apoptosis by a mechanism involving in part the activation of JNK (15). In this context, inhibition of NF-B sensitizes cells to the induction of apoptosis by sustained activation of JNK (30). Direct inhibition of IKK␤ and thereby the NF-B pathway by CDDO or its derivatives could therefore contribute to the apoptotic response of tumor cells to treatment with these agents.