Interleukin-1-induced NF-κB Activation Is NEMO-dependent but Does Not Require IKKβ*

Activation of NF-κB by the pro-inflammatory cytokines tumor necrosis factor (TNF) and interleukin-1 (IL-1) requires the IκB kinase (IKK) complex, which contains two kinases named IKKα and IKKβ and a critical regulatory subunit named NEMO. Although we have previously demonstrated that NEMO associates with both IKKs, genetic studies reveal that only its interaction with IKKβ is required for TNF-induced NF-κB activation. To determine whether NEMO and IKKα can form a functional IKK complex capable of activating the classical NF-κB pathway in the absence of IKKβ, we utilized a panel of mouse embryonic fibroblasts (MEFs) lacking each of the IKK complex subunits. This confirmed that TNF-induced IκBα degradation absolutely requires NEMO and IKKβ. In contrast, we consistently observed intact IκBα degradation and NF-κB activation in response to IL-1 in two separate cell lines lacking IKKβ. Furthermore, exogenously expressed, catalytically inactive IKKβ blocked TNF- but not IL-1-induced IκBα degradation in wild-type MEFs, and reconstitution of IKKα/β double knockout cells with IKKα rescued IL-1- but not TNF-induced NF-κB activation. Finally, we have shown that incubation of IKKβ-deficient MEFs with a cell-permeable peptide that blocks the interaction of NEMO with the IKKs inhibits IL-1-induced NF-κB activation. Our results therefore demonstrate that NEMO and IKKα can form a functional IKK complex that activates the classical NF-κB pathway in response to IL-1 but not TNF. These findings further suggest NEMO differentially regulates the fidelity of the IKK subunits activated by distinct upstream signaling pathways.

mation, and cell survival (1,2). The NF-B family consists of five members named p50 and p52 (the NH 2 -terminal fragments of the longer NF-B1/p105 and NF-B2/p100 proteins respectively), p65 (RelA), c-Rel, and RelB. These proteins homo-or heterodimerize to form either transcriptionally active (e.g. p50: p65) or repressive (e.g. p50:p50) NF-B dimers (1,2). NF-B is maintained inactive in the cytosol of resting cells by members of the IB family of inhibitory proteins that include IB␣, IB␤, IB⑀, and the COOH termini of p105 and p100. In response to a wide range of stimuli, including pro-inflammatory cytokines (e.g. TNF and IL-1), bacterial products (e.g. lipopolysaccharide (LPS), CpG DNA), and the engagement of antigen receptors on T-and B-lymphocytes, IB proteins are rapidly phosphorylated, ubiquitinated, and degraded by the 26 S proteasome, thereby enabling NF-B dimers to localize to the nucleus and regulate target gene transcription (2).
Signal-induced IB phosphorylation is mediated by the high molecular weight IB kinase (IKK) complex that contains two catalytic subunits named IKK␣ (IKK1) and IKK␤ (IKK2) and a non-catalytic regulatory subunit named NF-B essential modulator (NEMO) or IKK␥ (3). IKK␣ and IKK␤ share significant structural identity, and each contains an NH 2 -terminal catalytic domain, a central leucine zipper motif through which they heterodimerize, and a COOH-terminal helix-loop-helix domain (2,3). IKK␤ also contains a novel ubiquitin-like domain, although the function of this region is not yet known (4). We have previously demonstrated that identical hexapeptide sequences (i.e. Leu-Asp-Trp-Ser-Trp-Leu) within the extreme COOH termini of both IKK␣ and IKK␤ facilitate their association with NEMO, and we named this region the NEMO binding domain (NBD) (5,6). Dissociation of NEMO from the IKK complex using a cell-permeable peptide spanning the NBD effectively blocks pro-inflammatory NF-B activation, thereby supporting the critical role of NEMO in regulating signal-induced activity of the intact IKK complex (5,7).
Despite their significant similarities, IKK␣ and IKK␤ play distinct roles within the overall NF-B signaling paradigm (1,2). In this regard, rapid and transient TNF-induced IB␣ degradation occurs through a pathway that depends upon IKK␤ and NEMO (1). This was definitively established in mice lacking either of these subunits that die during development from massive TNF-induced hepatocyte apoptosis due to the inability to mount an NF-B-dependent anti-apoptotic response (8 -12). This "classical" NF-B signaling pathway is now defined as NEMO-and IKK␤-dependent IB phosphorylation and degradation liberating canonical NF-B complexes typified by the ubiquitous p50:p65 heterodimer. All stimuli that induce IB␣ degradation, including IL-1, LPS, and antigen-receptor engagement, are considered to be activators of the NEMO-and IKK␤-dependent classical NF-B pathway (1,2).
Analysis of mice harboring inactive IKK␣ revealed an unanticipated role for this kinase in NIK (NF-B-inducing kinase)dependent processing of NF-B2/p100 to generate p52 (13,14). This processing occurs only in response to ligation of a subset of receptors, including the lymphotoxin-␤ receptor (LT␤R), CD40, and BAFF-R (13)(14)(15)(16)(17). The NF-B2/p100 targeted by IKK␣ is one-half of a heterodimer with RelB, thereby resulting after processing in the generation of p52:RelB NF-B complexes (14,17). These complexes regulate several genes encoding lymphoid chemokines (i.e. CCL19, CCL21, CXCL12, and CXCL13) and BAFF (13), and reflecting this, the major functions of this pathway are in lymphoid organogenesis and B-cell maturation. This pathway is termed the "non-canonical" or "alternative" NF-B pathway, and studies using IKK␤-and NEMO-deficient cells have demonstrated that it functions in the absence of each of these IKK complex subunits (1).
The function of IKK␣ in the non-canonical pathway does not therefore require its ability to interact with NEMO via its COOH-terminal NBD (13,(15)(16)(17). Furthermore, classical NF-B activation in response to TNF occurs in the absence of IKK␣, suggesting that the interaction of IKK␣ with NEMO plays no role in regulating this pathway (13, 14, 18 -20). We therefore sought to determine whether the ability of IKK␣ to interact with NEMO via its NBD plays any functional role in classical NF-B signaling. To address this question, we examined IB␣ degradation induced by TNF and IL-1 in murine embryonic fibroblasts (MEFs) lacking each of the IKK complex subunits. Remarkably we found that, although TNF-induced IB␣ degradation was absolutely dependent upon NEMO and IKK␤, IL-1-induced degradation and classical NF-B activation remained intact in cells lacking IKK␤. Furthermore, IL-1-induced NF-B was blocked by the cell-permeable NBD peptide in IKK␤ Ϫ/Ϫ MEFs, demonstrating that an IKK complex consisting of only IKK␣ and NEMO is sufficient for IL-1-but not TNF-induced classical NF-B activation. Our findings therefore identify differences in the absolute requirements for the separate IKK subunits activated in a NEMO-dependent manner by distinct upstream stimuli.
Wild-type (WT), IKK␣ Ϫ/Ϫ , and IKK␤ Ϫ/Ϫ murine embryonic fibroblasts were generously provided by Dr. Inder Verma (The Salk Institute for Biological Studies, La Jolla, CA; MEFs 1), who also provided the IKK␣/␤ double-deficient cells, and Dr. Michael Karin (University of California San Diego School of Medicine, La Jolla, CA; MEFs 2) who also provided the NEMOdeficient MEFs. Plat-E cells were cultured and used as previously described (21). All cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum, 2 mM L-glutamine, penicillin (50 units/ml), and streptomycin (50g/ml). For all experiments, unless otherwise indicated, cells were cultured in either 6-well tissue culture trays or 100-mm dishes and were stimulated with IL-1 (10 ng/ml) or TNF (10 ng/ml) when they reached 80% confluence.
Immunoblotting and Immunoprecipitation-Cells were washed once with phosphate-buffered saline and then incubated for 10 min at 4°C in 100 l of TNT lysis buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, and 1% Triton X-100) and a complete miniprotease inhibitor mixture (Roche Applied Science). Samples were then scraped and harvested into 1.5-ml microcentrifuge tubes, vortexed for 30 s, and then centrifuged (425 ϫ g for 10 min). Protein levels in the supernatants were determined using a Coomassie protein assay kit (Bio-Rad), and 20 g of protein from each sample was separated by SDS-PAGE (10%) and then transferred to a polyvinylidene difluoride membrane (Millipore, Milford, MA) and immunoblotted with primary and horseradish peroxidase-conjugated secondary antibodies. Detection of the bound antibody by enhanced chemiluminescence was performed according to the manufacturer's instructions (Pierce). For immunoprecipitations, cell extracts were incubated with 2 g of primary antibody for 1 h at 4°C followed by incubation (1 h at 4°C) with 30 l of protein G-Sepharose beads (50% slurry). A portion of each sample preimmunoprecipitation (5%) was retained for analysis. The beads were washed three times with lysis buffer, and then the samples were analyzed by SDS-PAGE (10%) followed by immunoblotting as described above.
Densitometry-Densitometry was performed using a Gel-Doc EQ gel documentation system and the QuantityOne software package (Bio-Rad). Pixel intensity was measured in identical rectangular volumes around each band on immunoblots, and a background value in an equal rectangular volume separate from the bands was subtracted to obtain the mean pixel intensity/mm 2 . Statistical analysis was performed using a twotailed paired Student's t test.
Transfections and Luciferase Reporter Assays-WT, IKK␣ Ϫ/Ϫ , IKK␤ Ϫ/Ϫ , and NEMO-deficient MEFs grown in 12-well plates (2.5 ϫ 10 5 /well) were transiently transfected using FuGENE 6 (Roche Applied Science) following the manufacturer's protocol. Cells were transfected with a total of 1.0 g of DNA/well consisting of the NF-B-dependent firefly luciferase reporter construct pBIIx-firefly luciferase (0.2 g/well) and a ␤-actin promoter Renilla luciferase reporter (0.02 g/well) together with either control vector alone or the test DNA. Cells were stimulated with IL-1 for 5 h and then lysed in passive lysis buffer (Promega) 24 -36 h after transfection. Samples were assayed using a Luminoscan 96-well automated luminometer (Thermo Labsystems, Franklin, MA), and FF:RL ratios were calculated using Ascent software (Thermo LabSystems).
Electrophoretic Mobility Shift Assays (EMSAs)-MEFs were stimulated with IL-1 (10 ng/ml) for the appropriate times and then scraped into phosphate-buffered saline at 4°C and pelleted (425 g, 10 min). Pellets were resuspended and swollen for 30 min on ice in 100 l of NarA buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 2 mM NaF, 2 mM ␤-glycerolphosphate, and complete miniprotease inhibitors), incubated a further 5 min on ice in 0.1% Nonidet P-40, and then vortexed and centrifuged (3800 ϫ g) for 1 min. Supernatants (cytoplasmic fraction) were centrifuged (20,000 ϫ g) for 1 h at 4°C, and the resulting supernatants were snap frozen and retained for analysis. Pelleted nuclei were washed once with 100 l of NarA buffer before being vortexed in 30 l of NarC buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, pH 8, 1 mM EDTA, pH 8, 2 mM NaF, 2 mM ␤-glycerolphosphate, and complete miniprotease inhibitors) for 1 h at 4°C. Nuclear lysates were then centrifuged for 20 min (20,000 ϫ g) at room temperature and then either used immediately or snap frozen and stored at Ϫ80°C.
Single-stranded complimentary oligonucleotides encompassing a consensus NF-B site (upper strand, 5Ј-AGTT-GAGGGGACTTTCCCAGGC-3Ј) or the Oct-1 probe (Santa Cruz Biotechnology) were annealed and then labeled with [␥-32 P]ATP using T4 polynucleotide kinase (New England Biolabs, Beverly, MA). The labeled probe was purified using mini-Quick Spin columns (Roche Applied Science) according to the manufacturer's instructions. For EMSA, 2-5 g of nuclear extracts supplemented with 1 g of poly(dI⅐dC) (Roche Applied Science) were incubated with an equal volume of 2ϫ binding buffer (40 mM Tris-HCl, pH 7.9, 100 mM NaCl, 10 mM MgCl 2 , 2 mM EDTA, 20% glycerol, 0.2% Nonidet P-40, 2 mM dithiothreitol, 100 g/ml bovine serum albumin) on ice for 10 min. After incubation, 1 l of labeled probe was added, and then samples were incubated at room temperature for 20 min. The resulting DNA⅐NF-B complexes were separated on 5% polyacrylamide non-denaturing gels by electrophoresis, and then gels were dried and visualized by autoradiography. Supershift analysis was performed following the same protocol, except that samples were incubated with antibodies (anti-p65 or anti-p50) for 2 h at 4°C prior to the addition of labeled probe.
NBD Peptides-Small scale Fmoc (N-(9-fluorenyl)methoxycarbonyl) synthesis of the peptides was carried out on a Rainin Symphony instrument (Rainin Instruments, LLC, Oakland, CA) at the Howard Hughes Medical Institute Biopolymer-Keck Foundation Biotechnology Resource Laboratory at Yale University (New Haven, CT). Peptides were characterized by matrix-assisted laser desorption ionization mass spectrometry and analytical reverse-phase high-performance liquid chromatography analysis. Immediately prior to use, the peptides were dissolved in dimethyl sulfoxide to a stock of 50 mM. The sequences of the wild-type and mutant NBD peptides have been described previously (5,6). The NBD peptide contains the region of IKK␤ from T735 to E745 synthesized in tandem with a membrane permeabilization sequence from the Drosophila antennapedia homeodomain protein. The mutant peptide is identical, except that Trp-739 and -741 are replaced by alanines to render it biologically inactive (5,6).
Preparation of Stable Cell Lines-All cloning procedures were performed by PCR using cloned Pfu DNA polymerase (Stratagene, La Jolla, CA). A cDNA encoding IKK␤ (K44M) was subcloned into the HindIII and NotI sites of LZRS-pBMN-lacZ retroviral vector (kindly provided by Garry Nolan, Stanford University, Stanford, CA). Resulting LZRS ϪIKK␤(K44M) was transiently transfected using FuGENE 6 into Plat-E cells and selected for gene expression 24 h after transfection using puromycin (1 g/ml). Puromycin-resistant cells were used to derive conditioned medium to provide a retroviral stock for MEF transduction. For cell transduction, MEFs were washed and incubated for 8 h with a retrovirus-conditioned medium containing Polybrene (8 g/ml, Sigma). After incubation, the retrovirus was removed and replaced with normal growth medium. The transduction process was repeated a further three times until the cells became Ͼ90% GFP-positive as determined by fluorescence-activated cell sorter (FACS) analysis using Cell Quest software (FACSort, BD Biosciences, San Jose, CA).
To generate double knock-out (DKO) MEFs stably transduced with IKK␣, full-length IKK␣ cDNA was cloned into pCR-Blunt II-TOPO vector (Invitrogen) and then subcloned into the EcoRI restriction site of retroviral GFP-MIGR1 vector (kindly provided by Dr. Warren Pear, University of Pennsylvania, Philadelphia, PA). The resulting MIG ϪIKK␣ was transiently transfected using FuGENE 6 into Plat-E cells to produce ecotropic virus that was derived from conditioned medium containing Polybrene (8 g/ml).
For cell sorting, transduced cells were trypsinized and washed in FACS buffer (sterile phosphate-buffered saline, 0.5 mM EDTA, and 0.5% bovine serum albumin). Evaluation of GFP was performed on a three-laser (argon (488 nm), krypton (407 nm), and dye laser (tuned to 600 nm)), 10-parameter FACSVantage TM cell sorter from BD Biosciences Immunocytometry Systems. Compensation and data analyses were performed using FlowJo software (Tree Star, Ashland, OR).
Densitometric analysis of data from eleven separate time course experiments confirmed that IL-1 (but not TNF) consistently induced IB␣ degradation in MEFs lacking IKK␤ (Fig. 1E, white bars). Significant differences between WT and IKK␤ Ϫ/Ϫ cells were only observed at the 15-and 30-min time points in the TNFtreated samples (60 min of TNF, p ϭ 0.17) (indicated on Fig. 1D). No significant differences were observed between WT and IKK␤ Ϫ/Ϫ MEFs treated with IL-1 for any time point tested (15 min, p ϭ 0.17; 30 min, p ϭ 0.42; 60 min, p ϭ 0.91). Collectively therefore, the data in panels C-E of Fig. 1 demonstrate that IL-1 induces the phosphorylation and degradation of IB␣ in cells lacking IKK␤ with similar kinetics to that in WT cells.
Activation of the classical NF-B pathway by LPS occurs via ligation of the Toll-like receptor TLR4 (22). As some of the signaling intermediates downstream of TLR4 are shared with the IL-1 signaling pathway (i.e. MyD88, IRAK, and TRAF6), we questioned whether LPS could also activate classical NF-B signaling in cells lacking IKK␤. As shown in Fig.  1F, LPS induced IB␣ degradation in WT and IKK␣ Ϫ/Ϫ cells (lanes 1-4 and 9 -11) but not in either NEMO-deficient or IKK␤ Ϫ/Ϫ MEFs (lanes 5-8 and 13-16). These findings therefore confirm that LPSand TNF-induced IB␣ degradation require NEMO and IKK␤ and occur in the absence of IKK␣. In contrast, our data clearly demonstrate that, although IL-1-induced IB␣ degradation does depend upon NEMO, it has no absolute requirement for either kinase and occurs in both IKK␣and IKK␤-deficient MEFs.
IL-1 Activates Classical NF-B in IKK␤ Ϫ/Ϫ MEFs-Consistent with its inability to stimulate IB␣ degradation, previous studies have demonstrated that TNF cannot induce NF-B DNA binding or transcriptional activity in cells lacking IKK␤ (8,9). We therefore questioned whether the IL-1-induced IB␣ degradation we observed in IKK␤ Ϫ/Ϫ MEFs precedes NF-B activation, nuclear translocation, and transcriptional activity. Nuclear lysates from IL-1-stimulated WT, NEMO-deficient, IKK␣ Ϫ/Ϫ , and IKK␤ Ϫ/Ϫ MEFs were subjected to EMSAs using an NF-B-specific probe, and as shown in Fig. 2A, NF-B DNA binding was maximal in WT cells after 15 min and returned to basal levels after 60 min (lanes 1-4). As expected, DNA binding was absent in NEMO-deficient cells ( Fig. 2A, lanes 5-9) but occurred in IKK␣ Ϫ/Ϫ MEFs (lanes 9 -12), although this was less robust than the levels seen in WT cells. Consistent with our findings in Fig.  1, C and D, NF-B DNA binding occurred in IKK␤ Ϫ/Ϫ cells to the same level and with identical kinetics to that observed in WT MEFs ( Fig. 2A, lanes 13-16).
To identify the NF-B complexes activated by IL-1 in the distinct cell types, we performed supershift analysis using antibodies against the classical NF-B subunits p65 and p50. This analysis revealed that both basal and IL-1-induced DNA-bound NF-B in WT, IKK␣-, and IKK␤-deficient MEFS was completely shifted using anti-p65 (Fig. 2B, lanes 2, 5, 8, 11, 14, and  17). In addition, anti-p50 up-shifted approximately half of the NF-B complexes (Fig. 2B, lanes 3, 6, 9, 12, 15, and 18), demonstrating that IL-1-induced IB␣ degradation leads to nuclear translocation and DNA binding of the classical NF-B p50:p65 heterodimer and a separate p65-containing dimer in the absence of either IKK␣ or IKK␤. Our data strongly suggest that IKK␣ can activate the classical NF-B pathway in response to IL-1 in the absence of IKK␤. As the well described role of IKK␣ is the kinase critical for noncanonical NF-B signaling (13,14), we questioned whether IL-1 activates the non-canonical NF-B pathway in IKK␤ Ϫ/Ϫ MEFs. As shown in Fig. 2C, incubation of both WT and IKK␤ Ϫ/Ϫ MEFs with anti-LT␤R for 8 h induced p100 processing to generate p52 (Fig. 2C, lanes 2 and 5) as previously described (13,17,23). In contrast, IL-1 did not induce any increase in p52 levels above basal in either cell type (Fig. 2C, lanes 3 and 6), confirming that IL-1 does not activate the non-canonical pathway in the absence of IKK␤.
We next performed NF-B-dependent luciferase reporter assays to determine whether IL-1 can induce NF-B transcriptional activity in cells lacking IKK␤. As expected, IL-1 induced NF-B transcriptional activity in WT MEFs but not in the cells lacking NEMO (Fig. 2D), again confirming the key role for NEMO in classical NF-B signaling. Surprisingly, IL-1 stimulation did not lead to NF-B transcriptional activity in IKK␣deficient MEFs, despite inducing IB␣ phosphorylation and degradation (Fig. 1, C-E) and NF-B DNA binding (Fig. 2, A  and B). This observation therefore supports the model proposed in several previous reports that IKK␣ plays a crucial downstream or subsidiary role in maintaining the full classical NF-B transcriptional response that is independent of its function as an IB kinase (24 -29). Consistent with our IB␣ phosphorylation, degradation, and NF-B DNA binding data (Fig. 1,   C-E; Fig. 2, A and B), IL-1-induced NF-B transcriptional activity was intact in IKK␤ Ϫ/Ϫ cells (Fig. 2D), leading us to surmise that IKK␣ can facilitate IL-1-induced classical NF-B signaling and transcriptional activation in the absence of IKK␤. In contrast, our data suggest that IKK␤ is unable to support the full NF-B transcriptional response to this cytokine in the absence of IKK␣.
To confirm that these findings were not unique to one line of IKK␤ Ϫ/Ϫ MEFS, we repeated our experiments using a separate IKK␤deficient cell line and appropriate WT controls (MEFs 2). We first confirmed that these IKK␤ Ϫ/Ϫ cells lack IKK␤ but contain IKK␣ and NEMO (Fig. 3A), and this was verified for each experiment performed. As shown in Fig. 3B, TNF was unable to induce IB␣ degradation in these IKK␤ Ϫ/Ϫ MEFs (Fig. 3B,  lanes 6 -8), whereas IL-1 stimulation induced transient degradation that was maximal at 15 min and returned to unstimulated levels after 60 min (Fig. 3B, lanes 9 and 10).
To determine whether IL-1 could activate NF-B in these cells, we performed EMSA analysis, and as shown in Fig. 3C, IL-1 induced significant mobility shift indicating NF-B activation in both lines of IKK␤ Ϫ/Ϫ cells. Luciferase reporter assays confirmed that, although the basal level of NF-B activity was reduced in these IKK␤ Ϫ/Ϫ cells compared with WT MEFs, incubation with IL-1 induced NF-B transcriptional activity in the absence of IKK␤ (Fig. 3D).
Dominant Negative IKK␤ Blocks TNF-but Not IL-1-induced IB␣ Degradation in MEFs-The data described above were generated using cells lacking distinct components of the IKK complex. We therefore wished to determine whether exogenous inhibition of IKK␤ could block IL-1-induced classical NF-B signaling in cells expressing an intact IKK complex. To accomplish this, we stably transduced WT MEFs with a retroviral construct encoding a catalytically inactive dominant negative version of IKK␤ (K44M). Concomitant introduction of an EGFP-tagged version of the vector (LZRS ϪEGFP ) confirmed that Ͼ96% of cells were transduced using our approach (Fig.  4A). As shown in Fig. 4B, TNF-induced IB␣ degradation was completely blocked in cells expressing IKK␤(K44M) compared with cells transduced with the LZRS ϪEGFP vector alone (Fig. 4B,  compare lanes 2 and 6). In contrast, IL-1-induced IB␣ degradation remained detectable in IKK␤(K44M)-expressing cells (Fig. 4C). These data therefore demonstrate that exogenous expression of a dominant negative inhibitor of IKK␤ blocks TNF-but not IL-1-induced IB␣ degradation in WT MEFs.

Reconstitution of IKK␣/␤ Doubledeficient Cells with IKK␣ Re-establishes IL-1 but Not TNF-induced IB␣ Degradation-Previous studies have clearly demonstrated that IB␣ degradation does not occur in
MEFs lacking both IKK␣ and IKK␤ in response to either TNF or IL-1 (30). Our findings to date strongly suggest that IKK␣ is sufficient to transduce IL-1-but not TNF-induced signaling to NF-B in the absence of IKK␤. To further explore this hypothesis, we therefore questioned whether reintroduction of IKK␣ into IKK DKO MEFs could reestablish IL-1-induced IB␣ degradation in these cells. We first verified that the DKO cells lacked both IKK␣ and IKK␤ but contained NEMO (Fig. 5A), and then the cells were retrovirally transduced with IKK␣ in the MIGR1 vector expressing GFP (Fig. 5B), and positive cells were sorted for use in experiments. As expected, incubation of DKO cells with either TNF or IL-1 did not induce IB␣ degradation (Fig. 5, C and D, lanes 5-8). Similarly, IB␣ degradation was absent in response to TNF in DKO cells stably transduced with IKK␣ (Fig. 5C, lanes 9 -12). Consistent with our findings in IKK␤-deficient cells, however, re-introduction of IKK␣ into DKO MEFs re-established IL-1-induced IB␣ degradation in these cells (Fig. 5D, lanes 9 -12).
We conclude from our accumulated data that, in the absence of IKK␤, IKK␣ can facilitate IL-1-induced IB␣ degradation and NF-B activation. Our findings using NEMO-deficient cells (Figs. 1 and 2) also lead us to surmise that NEMO is necessary for IKK␣-dependent signaling. However, a direct role for NEMO in regulating the function of IKK␣ in the classical NF-B pathway has not yet been described. We previously demonstrated that NEMO associates with IKK␣ via the NEMO binding domain (NBD) within the COOH terminus of the kinase (5, 6), and we verified that NEMO and IKK␣ formed a complex in our DKO-IKK␣ cells (Fig. 5E, lane 6). Our results therefore suggest that NEMO and IKK␣ form a complex in cells lacking IKK␤ that responds to IL-1 (but not TNF) to phosphorylate IB␣, leading to its degradation.
The Interaction of NEMO with IKK␣ Is Required for IL-1induced NF-B Activation in IKK␤ Ϫ/Ϫ MEFs-To definitively determine whether the interaction of NEMO with IKK␣ is required for IL-1-induced NF-B activation in the absence of IKK␤, we first verified that NEMO and IKK␣ associate in IKK␤ Ϫ/Ϫ MEFs. As shown in Fig. 6A, IKK␣ was present in immune complexes precipitated from both WT and IKK␤ Ϫ/Ϫ MEFs using anti-NEMO, confirming that these proteins do form a complex in IKK␤-deficient cells. We have previously reported that the interaction of IKK␤ and IKK␣ with NEMO can be blocked using a cell-permeable peptide encompassing  2) were either untreated or incubated for the times indicated with TNF (10 ng/ml) or IL-1 (10 ng/ml), and then lysates were immunoblotted using either anti-IB␣ or anti-tubulin. C, WT and IKK␤ Ϫ/Ϫ MEFs (MEFs 1 and 2) were incubated with IL-1 (10 ng/ml) for the times indicated, and then nuclear extracts were prepared for EMSA. Assays were performed using either a consensus NF-B probe (upper panel) or an Oct1 probe as a loading control (lower panel). D, MEFs 2 were transiently transfected with the NF-B-dependent reporter pBIIx-firefly luciferase together with ␤-actin Renilla luciferase. Twenty-four hours later, the cells were either untreated or incubated for a further 5 h with IL-1 (10 ng/ml), and then NF-B activity was determined by dual luciferase assay. The data are expressed as the mean ratio Ϯ S.E. of the firefly:Renilla (FFL:RL) luciferase activity from three separate experiments, each performed in triplicate. the NEMO binding domain (NBD) present in each of the kinases (5, 6). We therefore questioned whether the NBD peptide would inhibit IL-1-induced NF-B activation in IKK␤ Ϫ/Ϫ MEFs, and as shown in Fig. 6B, the WT (NBD WT , lanes 3-5) but not the inactive mutant peptide (NBD MUT , lanes 6 and 7) dosedependently blocked IL-1-induced NF-B activity measured by EMSA. These results therefore demonstrate that disruption of the NEMO-IKK␣ interaction blocks IL-1-induced NF-B activation in cells lacking IKK␤.

DISCUSSION
Genetic studies have established that TNF-induced classical NF-B activation depends upon NEMO and IKK␤, whereas IKK␣ is considered to play no role in the upstream activation events of this pathway (1,2). Consistent with this, we found that TNF-, IL-1-and LPS-induced IB␣ degradation is intact in IKK␣ Ϫ/Ϫ fibroblasts but absent in NEMO-deficient cells. However, we found that, although TNF-and LPS-induced IB␣ degradation absolutely required IKK␤, IL-1-induced classical NF-B activation remained intact in two separate IKK␤-deficient fibroblast cell lines. We provide comprehensive evidence that IL-1-induced IB␣ phosphorylation, degradation, p50:p65 nuclear translocation and NF-B-dependent luciferase reporter expression occurs in IKK␤ Ϫ/Ϫ fibroblasts, and we have demonstrated that reconstitution of IKK␣/b double knock-out MEFs with IKK␣ is sufficient to re-establish IL-1-but not TNFinduced IB␣ degradation. We have also shown that this activation requires the association of NEMO with IKK␣, as the cell-permeable NBD peptide blocks IL-1-induced NF-B in IKK␤ Ϫ/Ϫ MEFs. Our results therefore demonstrate that IL-1induced IB␣ degradation can occur via either NEMO⅐IKK␤ or NEMO⅐IKK␣ complexes, whereas TNF signaling is non-pro- C and D, WT, DKO, and DKO ϪIKK␣ MEFs were either untreated or incubated TNF (C) or IL-1 (10 ng/ml each) (D) for the times indicated. Lysates were prepared and immunoblotted using antibodies against IB␣, IKK␣, or ␣-tubulin as shown. E, lysates of DKO or DKO ϪIKK␣ MEFS were incubated with either protein G-Sepharose alone (PGS), a nonspecific antibody (NS Ab), or anti-NEMO, and immunoprecipitation (IP) was performed as described under "Experimental Procedures." Immunoprecipitated material was immunoblotted using anti-IKK␣, and samples of lysates retained prior to immunoprecipitation (Pre-IP) were probed using anti-IKK␣ and NEMO. , and then cytoplasmic and nuclear extracts were prepared for immunoblotting (IB) and EMSA, respectively. EMSAs were performed as described under "Experimental Procedures" using NF-B and Oct-1 consensus binding site probes, and cytoplasmic extracts were immunoblotted using anti-IKK␣ and -IKK␤ as indicated.
miscuous and depends solely on NEMO and IKK␤ for transduction (see model in Fig. 7). This leads us to conclude that, although classical NF-B activation by all stimuli critically depends upon NEMO, separate upstream signaling pathways differ in their categorical requirements for the effector subunits of the IKK complex.
Although our findings appear to conflict with the current model of classical NF-B activation, they are consistent with a number of previous studies. In the original report of mice lacking IKK␤, TNF-induced IB␣ degradation and NF-B activity were significantly reduced in IKK␤ Ϫ/Ϫ fibroblasts, whereas IL-1 signaling remained comparatively unaffected (8). Using different IKK␤-deficient MEFs, Schmidt-Supprian et al. (31) later reported profoundly defective TNF signaling, although they demonstrated a less dramatic effect on IL-1-induced NF-B activation. Tang et al. (32) have also observed that JNK inactivation, which requires NF-B-dependent gene expression in response to TNF, was intact in IKK␤ Ϫ/Ϫ MEFs stimulated with IL-1. Furthermore Schmidt-Supprian et al. (31) have demonstrated that neither TNF nor LPS could induce IL-6 expression in IKK␤ Ϫ/Ϫ MEFS, whereas IL-1 did so in the absence of IKK␤. Tanaka et al. (33) also demonstrated that IL-1 induced low level IL-6 expression in IKK␤ Ϫ/Ϫ MEFs; however, contrary to our study, they did not observe any difference between IL-1-and TNF-induced NF-B in their cells. Similarly, Li et al. (9) have reported no differences between the lack of TNF and IL-1 signaling in IKK␤-deficient MEFs. We per-formed our studies using two separate IKK␤ Ϫ/Ϫ MEF cell lines and found that IL-1 (but not TNF) induced IB degradation and NF-B activation in each. The reasons for the discrepancies among the separate reports are therefore unclear; however, our data coupled with that of previous studies (8,31,32) lead us to question the overall conclusion that IKK␤ is absolutely necessary for all classical NF-B-inducing stimuli.
It has been proposed that NF-B activity previously observed in IKK␤-deficient MEFs occurs because IKK␣ compensates for IKK␤ in the IKK complex (31). Our data argue against this interpretation, as we consistently observed complete inhibition of TNF signaling in the absence of IKK␤, whereas IL-1-induced IKK activity remained intact. A more accurate conclusion from these combined studies is therefore that IL-1 and TNF signaling to NF-B have different absolute requirements for the IKK subunits necessary for phosphorylating IB␣. A consistent finding between our study and many previous reports is that IB␣ degradation induced by both cytokines requires NEMO (10 -12, 34). We therefore speculate that the molecular components of the separate upstream signaling cascades differentially interface with NEMO, resulting in distinct downstream IKK activation profiles. This hypothesis is supported by the fact that specific mutations in the COOHterminal zinc finger domain of NEMO prevent TNF-induced NF-B activation but have no effect on IL-1-stimulated IKK activation (34,35). Furthermore, the COP9 signalosome component CSN3 has been identified as a NEMO-interacting protein that specifically blocks TNF-but not IL-1-induced NF-B activation (36). It is therefore possible that the terminal signaling components of the TNF and IL-1 pathways associate with distinct regions of NEMO and that this association directs the activation of either IKK␤ alone in response to TNF or, in the case of IL-1, either IKK␤ or IKK␣. Further studies exploring the relationship between the distinct domains of NEMO and the downstream kinases activated by separate signaling cascades are therefore required and may identify novel mechanisms to selectively disrupt pro-inflammatory signaling via the IKK complex.
Our results suggest that classical NF-B-inducing stimuli belong to two separate categories, those that that depend solely upon IKK␤ (e.g. TNF) and stimuli, such as IL-1, that can utilize either kinase in the IKK complex. As LPS signaling via TLR4 shares several adapter molecules with the IL-1 receptor-induced pathway (i.e. MyD88, IRAK, and TRAF6), we therefore expected LPS to mirror IL-1 and activate NF-B in IKK␤-deficient MEFs. However, LPS did not activate NF-B in IKK␤ Ϫ/Ϫ cells and behaved identically to TNF in all of our experiments. Despite sharing several components, closer examination of IL-1-and LPS-induced signaling pathways reveals differences in the other adapter proteins required for each. Unlike IL-1 signaling, which requires only MyD88, TLR4 engagement recruits additional adapters, including TIRAP/MAL, TRAM, and TRIF (22). It is therefore possible that other TLRs, such as TLRs 5,7,8,9,and 11, which signal solely through MyD88, may more closely resemble IL-1 and activate NF-B via either IKK␣ or IKK␤. Although further work will be required to address this hypothesis, it is interesting to note that the same mutations in NEMO that block TNF-but not IL-1-induced NF-B activation FIGURE 7. Distinct modes of IKK complex activity transduce TNF and IL-1 signaling to NF-B. Our findings verify that TNF-induced IB␣ degradation and classical NF-B activation is critically dependent upon NEMO and IKK␤ (left). Similarly, IL-1 signaling absolutely requires intact NEMO (right). However, IL-1-induced IB␣ degradation, NF-B nuclear translocation, and NF-B transcriptional activity occurs in the absence of IKK␤, demonstrating that NEMO and IKK␣ can form a signaling complex capable of activating the classical NF-B pathway in response to certain pro-inflammatory stimuli. also inhibit LPS signaling (34,35). This therefore strengthens the hypothesis that the differential IKK requirements of distinct stimuli involves separate domains of NEMO and, together with our findings, places LPS in the same category of stimuli as TNF that depends completely on IKK␤ for downstream NF-B activation. Intriguingly, Schmidt-Supprian et al. (31) report that T-cell receptor-induced NF-B activation and proliferation is intact in T-cells lacking IKK␤, suggesting that similar to IL-1 in our study, TCR signaling can utilize either IKK␣ or IKK␤.
A major goal of this study was to explore the physiological function of the interaction of IKK␣ with NEMO. In pursuing this, we have demonstrated that NEMO and IKK␣ can form a signaling unit capable of transducing a subset of classical NF-B signals. However, IKK␣ has been ascribed a number of separate roles in classical NF-B signaling that are distinct from IB␣ phosphorylation. In this regard, it has been proposed that IKK␣ regulates NF-B-dependent transcription by selectively phosphorylating histones associated with NF-B target genes (24,29) or by directly phosphorylating NF-B proteins on residues required for transcriptional competency (28). Our data support a transcriptional regulatory role for IKK␣, as we did not observe NF-B-dependent luciferase activity in IKK␣ Ϫ/Ϫ MEFs (Fig.  2D), although IB␣ degradation and NF-B DNA binding occurred in these cells (Fig. 1, B, C, and F; Fig. 2, A and B). As histone and possibly NF-B phosphorylation are nuclear events and NEMO has been shown to localize to the nucleus (37-39), it will be intriguing to determine whether the association with NEMO plays a role in any of these transcriptional regulatory functions of IKK␣.
Intact IKK␣ is absolutely critical for non-canonical NF-B activation in response to a range of stimuli, including ligation of the LT␤R (13,14). Because our data indicated that IL-1 stimulation leads to IKK␣ activation in the absence of IKK␤, it was therefore conceivable that IL-1 could induce the non-canonical pathway in IKK␤ Ϫ/Ϫ MEFs. However, as shown in Fig. 2C, we found that, similar to TNF (13,14,17,23), IL-1 did not activate the non-canonical pathway in either WT or IKK␤-deficient cells. This finding therefore broadens our understanding of the range of activities of IKK␣ in NF-B signaling and separates the roles of IKK␣ activated in response to both classical and noncanonical stimuli.
IKK␣ has also been reported to negatively regulate the classical NF-B pathway, and two separate studies have demonstrated that IB␣ degradation is prolonged in IKK␣-deficient cells following stimulation (26,27). We also observed protracted IB␣ degradation in IKK␣ Ϫ/Ϫ cells treated with TNF, IL-1, or LPS (Fig. 1, A, B, and F). The mechanisms proposed for this include hyperactivity of IKK␤, because of the absence of direct negative regulation by IKK␣ (27) or by lack of IKK␣-dependent NF-B protein turnover and removal from pro-inflammatory gene promoters (26). Although it remains to be determined whether either or both of these mechanisms fully account for the negatively regulatory role of IKK␣, it is exciting to speculate on the potential requirement of the IKK␣-NEMO interaction for this function. Notably, we had demonstrated previously that, although the NBD peptide blocks pro-inflammatory NF-B activation by disrupting the association of IKK␣ and IKK␤ with NEMO, it also modestly increases basal NF-B activity in unstimulated cells (5). Consistent with this, versions of zebrafish IKK␣ that lack the NBD do not negatively regulate NF-B activity when stably expressed in IKK␣ Ϫ/Ϫ MEFs (25). These findings therefore suggest that the suppressive function of IKK␣ in the classical NF-B pathway requires its ability to interact with NEMO; however, further direct studies of the IKK␣ NBD in mammalian systems are clearly required.
In conclusion, we have demonstrated that, although IL-1 and TNF both require NEMO for classical NF-B activation in MEFs, only TNF absolutely requires IKK␤. Our finding that IL-1-induced IB␣ phosphorylation and NF-B activation are intact in IKK␤ Ϫ/Ϫ MEFs leads us to propose a model in which NEMO "interprets" and differentially relays distinct upstream signals to the downstream IKK subunits (Fig. 7). These results also suggest that drugs targeting IKK␤ alone may not completely block pro-inflammatory NF-B activity in diseases in which IL-1 plays a major pathological role. However, a deeper understanding of the precise mechanisms by which the IKK complex responds to distinct upstream signals and activates the separate IKK subunits may identify more useful pathway-specific targets for effective therapeutic drugs.