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The Journal of Immunology, 2005, 174: 6424-6430.
Copyright © 2005 by The American Association of Immunologists

Regulation of c-Jun Phosphorylation by the I{kappa}B Kinase-{epsilon} Complex in Fibroblast-Like Synoviocytes1

Susan E. Sweeney, Deepa Hammaker, David L. Boyle and Gary S. Firestein2

Division of Rheumatology, Allergy, and Immunology, University of California-San Diego School of Medicine, La Jolla, CA 92093


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Rheumatoid arthritis (RA) causes a symmetric, inflammatory polyarthritis that results in joint destruction and significant disability. Signaling pathways that regulate the production of cytokines and destructive enzymes have been implicated in its pathogenesis and represent potential therapeutic targets. The I{kappa}B kinase (IKK)-related kinase, IKK{epsilon}/IKKi, which plays a pivotal role in regulating antiviral gene transcription, is constitutively expressed by cultured fibroblast-like synoviocytes (FLS) and could participate in the pathogenesis of RA. In the current studies we demonstrate that IKK{epsilon} protein is expressed in RA and osteoarthritis synovium and that the protein is found primarily in the synovial intimal lining. Functional studies in cultured FLS showed that IKK{epsilon} kinase activity is rapidly induced by cytokines, although I{kappa}B phosphorylation is significantly less compared with IKK2. Because NF-{kappa}B activation is similar in wild-type and IKK{epsilon} knockout murine FLS, studies were performed to identify an alternative substrate for IKK{epsilon}. Interestingly, c-Jun is a more efficient substrate for IKK{epsilon} immunocomplexes in human FLS and this activity appears to be independent of JNK. The functional relevance of IKK{epsilon} was examined using murine IKK{epsilon}–/– cultured FLS. IL-1-, TNF-{alpha}-, and LPS-mediated induction of matrix metalloproteinases, MMP3 and MMP13, is significantly decreased in the IKK{epsilon}–/– cells. These data suggest a novel role for the IKK{epsilon} complex in synovial inflammation, extracellular matrix destruction, and activation of the viral program and innate immune response in RA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Rheumatoid arthritis (RA)3 is a chronic inflammatory disease marked by symmetric polyarthritis and joint destruction. Signaling pathways that regulate the production of inflammatory mediators such as cytokines and degradative enzymes have been implicated in the pathogenesis of RA and represent potential therapeutic targets (1). Among these, the transcription factors NF-{kappa}B (2) and AP-1 (3) are especially important and regulate an array of proinflammatory genes that participate in synovitis (4, 5). Understanding the critical signal transduction networks that modulate these host responses has stimulated the development of small molecule inhibitors as promising agents to treat RA (6, 7, 8). For instance, NF-{kappa}B blockade in RA has focused on the I{kappa}B kinase (IKK) signal complex, which contains IKK2, as a key convergence point for rapid NF-{kappa}B activation (9, 10). However, systemic inhibition of IKK2 activity poses significant potential safety concerns due to the role of NF-{kappa}B in host defense and apoptosis.

The possibility that alternative pathways might regulate NF-{kappa}B was in part stimulated by the discovery of IKK{epsilon} (also known as IKKi). This kinase was originally identified as an inducible NF-{kappa}B-activating kinase that phosphorylates I{kappa}B (11, 12). IKK{epsilon} along with TRAF-associated NF-{kappa}B activator-binding kinase 1 (TBK1) form a family of IKK-related kinases with ~30% structural homology to IKK1 and IKK2. There are clear functional differences between the classical IKKs and the IKK-related kinases. For instance, IKK{epsilon} and TBK1 only phosphorylate serine 36 of I{kappa}B, but not serine 32, whereas IKK2 phosphorylates both residues (13). IKK{epsilon} also has distinct substrate specificity and kinetics compared with IKK1 and IKK2 (14). IKK{epsilon} can also phosphorylate other proteins involved in NF-{kappa}B signaling, including TRAF-interacting protein, and possibly IKK2 itself (15, 16). More recently, IKK{epsilon} and the related kinase, TBK-1, were implicated as key initiators of anti-viral gene expression by virtue of their ability to activate IFN regulatory factor 3 (IRF3) (17, 18).

To determine a possible role for IKK{epsilon} in RA, we previously studied its regulation in cultured fibroblast-like synoviocytes (FLS). We observed that this gene is constitutively expressed in FLS, in contrast to macrophages, but there is still little information on its function in these cells. Using in vitro kinase assays in human FLS, we now report that IKK{epsilon} kinase activity is rapidly induced by cytokines and the TLR agonist LPS. Surprisingly, c-Jun, rather than I{kappa}B, was identified as an efficient downstream target of the IKK{epsilon} immunoprecipitates. Gene expression studies in murine IKK{epsilon}–/– FLS demonstrate a novel role for IKK{epsilon} in regulation of matrix metalloproteinase (MMP) expression in human FLS. These data suggest diverse roles for IKK{epsilon} in synovial inflammation and in the innate immune response.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Preparation of human FLS and murine IKK{epsilon} knockout FLS

Synovial tissue and FLS were obtained from patients with osteoarthritis (OA) and RA at the time of total joint replacement or synovectomy as previously described (19). The diagnosis of RA conformed to ACR 1987 revised criteria (20). Synovium was minced and incubated with 1 mg/ml collagenase type VIII (Sigma-Aldrich) in serum-free RPMI 1640 (Invitrogen Life Technologies) for 1 h at 37°C, filtered, extensively washed, and cultured in DMEM (Invitrogen Life Technologies) supplemented with 10% FCS (Gemini BioProducts), penicillin, streptomycin, gentamicin, and glutamine in a humidified 5% CO2 atmosphere. Cells were allowed to adhere overnight, and FLS were grown in DMEM containing 10% FCS and split at 1:3 when the cells were 70–80% confluent. FLS were used from passages 3–8, during which time they are a homogeneous population of cells (<1% CD11b positive, <1% phagocytic, and <1% FcRII and FcRIII positive). In addition, multiple FLS lines were derived from IKK{epsilon} knockout and wild-type mice (Pharmacia) by microdissecting synovium and enzymatically dispersing the cells as previously described (21, 22). FLS were cultured in six-well plates or 100-mm dishes and used at 80% confluence. Cells were synchronized in 0.1% FCS for 24–48 h before the addition of cytokines or LPS.

Reagents

Affinity-purified goat polyclonal anti-IKKi (IKK{epsilon}) Ab (SC-5694), rabbit polyclonal anti-IKK1/2 Ab (SC-7607), mouse anti-JNK2 mAb (SC-7345), rabbit polyclonal anti-MAPK kinase 4 (anti-MKK-4) Ab (SC-964), goat polyclonal anti-c-Jun Ab (SC-44-G), and secondary Abs were purchased from Santa Cruz Biotechnology. GST-c-Jun, GST-I{kappa}B{alpha}, and the JNK inhibitor, SP600125 were obtained from Celgene. TNF-{alpha} and IL-1 were purchased from R&D Systems. LPS was obtained from Sigma-Aldrich. Dominant negative (dn) IKK{epsilon} adenovirus containing a K38A mutation (provided by Dr. C. Tripp, Pharmacia, St. Louis, MO) and dnIKK2 adenovirus (9) were also used.

Immunohistochemistry

Immunostaining was performed as previously described (23, 24). Briefly, cryosections (5 µm) of synovial tissue from RA and OA patients were fixed with cold acetone for 10 min, then incubated with anti-IKK{epsilon} Ab or isotype control Ab overnight at 4°C. Sections were washed, and endogenous peroxidase was depleted with 0.1% H2O2 and 0.1% NaN3. The sections were then incubated with secondary HRP-conjugated anti-goat Ab, washed, and developed with diaminobenzidine (Vector Laboratories).

Western blot analysis

Western blot was performed as described previously (25). FLS were cultured in DMEM with 10% FCS in 100-mm dishes at 80% confluence and synchronized in DMEM in 0.1% FCS for 48 h. The cells were then incubated with medium alone or TNF-{alpha} (100 ng/ml) for 15 min to 24 h. Cells were washed with PBS, and protein was extracted using lysis buffer (50 mM HEPES, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM MgCl2, 1.5 mM EDTA (pH 8.0), 20 mM {beta}-glycerophosphate, 50 mM NaF, 1 mM Na3VO4, 10 µg/ml aprotonin, 1 µM pepstatin A, and 1 mM PMSF). For experiments using synovial tissue, snap-frozen tissue was pulverized, and protein was extracted in lysis buffer. The protein concentrations of tissue and FLS were determined using the DC protein assay kit (Bio-Rad). Samples containing 50 µg of protein from cultured FLS or 100 µg of protein from synovial tissue were resolved via 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were blocked with TBS plus 0.1% Tween 20 and 5% dry milk for 1 h at room temperature, followed by incubation with the appropriate Ab at 4°C overnight. The membranes were washed three times and incubated with HRP-conjugated secondary Ab for 1 h. Proteins were visualized with chemiluminescence using Kodak X-AR film (Eastman Kodak).

Kinase assay

Control and stimulated FLS (100 ng/ml TNF-{alpha}, 1 ng/ml IL-1, and 1 µg/ml LPS) were washed with cold PBS, then incubated with lysis buffer for 30 min on ice as previously described (25). The homogenate was centrifuged for 20 min at 12,000 x g. Supernatants were incubated with primary Ab or isotype control Ab conjugated to 50 µl of a 50% protein G-agarose (Oncogene) slurry in lysis buffer and incubated overnight at 4°C. After centrifugation for 5 min at 1,000 x g, immune complexes were washed with lysis buffer and kinase buffer. The kinase reaction was started by adding the 30 µl of kinase buffer with substrate (GST-c-Jun or GST-I{kappa}B{alpha}) at 2 µg/reaction plus [{gamma}-32P]ATP. After 30 min, samples were heated for 5 min at 95°C, separated using 10% SDS-PAGE, and visualized by autoradiography. The density of target bands was analyzed using NIH Image version 1.61 (National Institutes of Health) in arbitrary absorption units. The background value for each lane was subtracted from each densitometry measurement. The fold increase in kinase activity was calculated by determining the ratio of phosphorylation of stimulated FLS and medium-treated samples.

Immunoprecipitation assay

FLS lysates were prepared as described above for kinase assay. The lysates were precleared with 150 µl of protein G-agarose slurry and 10 µg of isotype control Ab overnight at 4°C. The precleared lysates were then incubated with Ab at 5 µg/ml with 50 µl of a 1:1 slurry of protein G-agarose beads at 4°C overnight. The immunoprecipitates were washed five times with lysis buffer, incubated in nonreducing Laemmli sample buffer, and heated for 5 min at 95°C. The samples were subjected to SDS-PAGE, and Western blot analysis was performed as described above.

EMSA

After serum starvation for 48 h, IKK{epsilon} knockout and wild-type murine FLS were treated with medium, TNF-{alpha} (100 ng/ml), or LPS (1 µg/ml) for 1 h. Cells were then washed with cold PBS and nuclear extracts were prepared as previously described (9, 21). A nuclear extraction kit (Chemicon International) was used to isolate extracts, and EMSA was performed using the gel-shift assay system kit (Promega) with control HeLa nuclear extract and cold competition oligonucleotides to demonstrate specificity. The protein concentration was determined, and lane loading was normalized appropriately. The nuclear extract was mixed with the appropriate purified {alpha}-32P-labeled oligonucleotides, resolved by 4% PAGE, dried, and visualized by autoradiography.

Quantitative PCR

IKK{epsilon} wild-type and knockout murine FLS were cultured in six-well plates and incubated with TNF-{alpha} (100 ng/ml) or LPS (1 µg/ml) for 6 h. RNA isolation and RT-PCR were performed as previously described using TaqMan PCR analysis and the GeneAmp 5700 Sequence Detection System (Applied Biosystems) (25, 26). Forward and reverse primers for MMP3, MMP13, and IFN-inducing protein 10 (IP-10) as well as fluorogenic TaqMan FAM/TAMRA-labeled hybridization probes were used (Assays on Demand; Applied Biosystems). To control for sample cellularity, hypoxanthine guanine phosphoribosyl transferase (HGPRT) forward and reverse primers and a TaqMan-labeled probe were included in separate PCRs. Each 20 µl of PCR mix also included 1x TaqMan universal PCR Master Mix with AmpliTaq Gold DNA polymerase, uracil-N-glycosylase (AmpErase), dNTPs with dUTP, and a passive reference to minimize background fluorescence fluctuations. Thermal cycle conditions were 2 min at 50°C to allow activation of uracil-N-glycosylase, 10 min at 95°C to activate AmpliTaq polymerase, and 40 cycles of 95°C for 15 s at 60°C for 1 min. The threshold cycle was determined for each sample using GeneAmp software. Standard curves are generated by linear regression using log (threshold cycle) vs log (cell number). The cell equivalent (CE) number for samples was calculated using the standard curve. Data are expressed as the ratio between the gene of interest CE and the HGPRT CE, yielding the relative expression.

Statistical analysis

Statistics were generally performed using paired Student’s t test. A comparison was considered significant at p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IKK{epsilon} expression in synovial tissue and synoviocytes

To assess the potential role of IKK{epsilon} in synovial inflammation and arthritis, initial studies were performed to determine whether the IKK{epsilon} protein is expressed in RA and OA synovial tissue. Western blot analysis was performed on RA and OA synovial lysates using polyclonal anti-IKK{epsilon} Ab. Immunoreactive IKK{epsilon} protein was readily detected, with similar levels present in RA and OA samples (see Fig. 1A). As shown in Fig. 1B, immunohistochemical analysis confirmed tissue expression of IKK{epsilon}, with prominent staining in the synovial intimal lining of RA and OA tissue as well as scattered sublining mononuclear cells. Because FLS are located in the synovial intimal lining, we also evaluated cultured FLS for IKK{epsilon} expression. As previously described, IKK{epsilon} was constitutively expressed by both RA and OA FLS (Fig. 2) (27).



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  		FIGURE 1. Expression of IKK{epsilon} in RA and OA synovial tissue. Total protein from RA and OA synovial tissue was extracted, and Western blot analysis was performed on lysates of synovial tissue. A, Western blot analysis demonstrates similar levels of IKK{epsilon} expression in RA and OA synovial tissue. Expression of actin is included as a control for protein loading. This figure is representative of four experiments. B, Immunohistochemical analysis of IKK{epsilon} protein expression in RA and OA synovial tissue sections (n = 3). Prominent staining was observed in the synovial intimal lining of RA tissue as well as some sublining cells. OA synovium also contains immunoreactive IKK{epsilon} in a similar pattern of distribution.

 


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  		FIGURE 2. Expression of IKK{epsilon} protein in FLS derived from RA and OA synovium. FLS were incubated with medium or TNF-{alpha} for 15 min, followed by Western blot analysis of protein lysates as described in Materials and Methods. IKK{epsilon} is constitutively expressed in FLS, with minimal evidence of cytokine-induced alteration in protein level. No differences in IKK{epsilon} expression after normalization were noted between RA and OA FLS. A representative experiment is shown (n = 5).

 
IKK{epsilon} function in FLS

Earlier studies in FLS indicated that IKK{epsilon} mRNA expression is increased by cytokines, but that the effects on protein levels are less pronounced (27). These data suggested that IKK{epsilon} activity, like many other kinases, might be primarily regulated at a post-translational level. To evaluate this possibility, FLS were stimulated with TNF-{alpha} for 15 min or 24 h, and lysates were immunoprecipitated with anti-IKK{epsilon} or anti-IKK2 Ab. The samples were then tested for I{kappa}B phosphorylating activity by an in vitro kinase assay. As shown in Fig. 3, both IKK{epsilon} and IKK2 kinase activity were increased by TNF-{alpha} after 15 min; however, the increase from baseline of I{kappa}B phosphorylation by IKK2 was 13 ± 6-fold (n = 5; p < 0.005), but only increased 5.4 ± 1.1-fold (n = 5; p < 0.02) with IKK{epsilon}. The amount of phospho-I{kappa}B generated by IKK2 after 15 min of cytokine stimulation was significantly greater than that by IKK{epsilon} (n = 5; p < 0.01). Because I{kappa}B was not an efficient substrate, we evaluated the role of IKK{epsilon} in cytokine- or LPS-mediated NF-{kappa}B activation by nuclear localization. IKK{epsilon} knockout and wild-type murine FLS were stimulated with TNF-{alpha} or LPS, and EMSA was performed. As previously described in murine embryonic fibroblasts (MEF) (28), NF-{kappa}B activation was not lower in knockout FLS (Fig. 4).



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  		FIGURE 3. Phosphorylation of I{kappa}B{alpha} by IKK{epsilon} and IKK1/2. In vitro kinase assays were performed to evaluate IKK{epsilon} functional activity in TNF-{alpha}-stimulated FLS (left panel). Classical IKKs (IKK1/2) were used as a positive control (right panel). FLS were stimulated with TNF-{alpha} for 15 min or 24 h and immunoprecipitated with anti-IKK{epsilon} Ab, anti-IKK1/2 Ab, or control IgG. The immunocomplexes were evaluated in a kinase assay using GST-I{kappa}B{alpha} as the substrate. Both IKK{epsilon} and IKK1/2 kinase function were increased by TNF-{alpha} within 15 min, and activity persisted for up to 24 h. IKK{epsilon}-mediated phosphorylation of I{kappa}B was significantly less than IKK1/2-mediated phosphorylation. Isotype control IgG immunoprecipitates did not demonstrate I{kappa}B phosphorylating activity (data not shown). Each kinase assay was performed with four separate FLS cell lines, and one representative example is shown.

 


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  		FIGURE 4. NF-{kappa}B binding activity in IKK{epsilon} knockout (KO) synoviocytes. Murine FLS were stimulated with medium, TNF-{alpha}, or LPS for 1 h, followed by nuclear extraction and EMSA with labeled NF-{kappa}B oligonucleotides. HeLa nuclear extract was used as a positive control. Cold NF-{kappa}B oligo competition was used to demonstrate specificity. NF-{kappa}B nuclear activation was not lower in IKK{epsilon} knockout FLS and was, if anything, higher than that in wild-type (WT) cells.

 
c-Jun phosphorylation by IKK{epsilon} immunoprecipitates

Because I{kappa}B{alpha} appeared to be a relatively poor substrate for IKK{epsilon} and did not alter the canonical pathway of NF-KB activation, other substrates were evaluated. Among the potential candidates, we were surprised to find that IKK{epsilon} immunoprecipitates rapidly and efficiently phosphorylated c-Jun, which was evaluated because it plays a key role in the regulation of MMP gene expression as well as activation of the antiviral program (29, 30). Fig. 5A shows a representative experiment demonstrating that IL-1 and TNF-{alpha} increase c-Jun activation (10.4 ± 3.5-fold; p < 0.001; n = 4) with a return to baseline in 24 h. Phosphorylation of c-Jun was also observed when FLS were stimulated with the TLR4 ligand LPS for 15 min (see Fig. 5B for a representative experiment). Additional evidence that IKK{epsilon} directly contributes to c-Jun phosphorylation was provided in experiments using an adenoviral construct expressing dnIKK{epsilon}. As shown in Fig. 5C, human FLS were infected with adenoviruses encoding dnIKK{epsilon}, LacZ, or dnIKK2. After stimulating infected cells with TNF-{alpha} for 15 min, cell lysates were immunoprecipitated with anti-IKK{epsilon} Ab, and kinase assays were performed. The dnIKK{epsilon} blocked cytokine-stimulated c-Jun phosphorylation, whereas LacZ and dnIKK2 had no effect (n = 2).



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  		FIGURE 5. TNF-{alpha}-, IL-1-, and LPS-mediated activation of c-Jun phosphorylation by IKK{epsilon}. FLS were stimulated with cytokines or LPS, and IKK{epsilon} immunoprecipitates were evaluated for the ability to phosphorylate GST-c-Jun in vitro. A, GST-c-Jun phosphorylating activity increased within 15 min of IL-1 or TNF-{alpha} stimulation, with a return to baseline in 24 h (n = 4). IgG immunoprecipitates did not have I{kappa}B-phosphorylating activity (data not shown). B, LPS stimulation also results in IKK{epsilon}-mediated c-Jun phosphorylation (n = 2). GST-c-Jun phosphorylation by JNK is included as a positive control (right lane). C, IKK{epsilon}-mediated phosphorylation of c-Jun is inhibited by dnIKK{epsilon}. FLS were infected overnight with adenoviral vectors encoding LacZ or dnIKK{epsilon}, then stimulated with TNF-{alpha} for 15 min. The dnIKK2 adenoviral vector was included as an additional control. In vitro kinase assays were performed with IKK{epsilon} immunoprecipitates using GST-c-Jun substrate. DnIKK{epsilon}, but not dnIKK2, inhibited c-Jun phosphorylation by IKK{epsilon} immunoprecipitates. The medium controls for adenovirus-infected and uninfected FLS were similar in multiple experiments (data not shown).

 
Contribution of JNK to c-Jun phosphorylation by IKK{epsilon} immunoprecipitates

One potential explanation for the phosphorylation of c-Jun by IKK{epsilon} immunoprecipitates is that JNK might coprecipitate with the complex. To evaluate this possibility, in vitro kinase reactions were performed in the presence or the absence of the JNK inhibitor SP600125 (6). Because MKK4 and JNK are known to coprecipitate (25, 31), anti-MKK4 Ab was used as a positive control. As shown in Fig. 6A, phosphorylation of c-Jun by the IKK{epsilon} complex is not blocked by the JNK inhibitor, whereas activity of the MKK4-JNK complex is markedly decreased. As a second approach, we performed Western blot analysis to determine whether JNK is present in the complexes. Anti-IKK{epsilon} or control IgG immunoprecipitates were probed with anti-JNK2 mAb, but JNK was not detected in the complexes (Fig. 6B).



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  		FIGURE 6. IKK{epsilon}-mediated c-Jun phosphorylation is independent of JNK. A, TNF-{alpha}-mediated activation of c-Jun phosphorylation by IKK{epsilon} immunoprecipitates. In vitro kinase assays were performed on IKK{epsilon} immunoprecipitates of TNF-{alpha}-activated FLS using GST-c-Jun substrate. IKK{epsilon} immunoprecipitates were prepared from control and cytokine-stimulated FLS, and kinase assays were performed in the presence and the absence of the JNK inhibitor SP600125 (left). The JNK inhibitor did not block c-Jun phosphorylation mediated by IKK{epsilon} immunoprecipitates. As a positive control, SP600125 did block c-Jun phosphorylation by the MKK4 immunoprecipitate (which contains JNK) (right) (31 ). This figure is representative of three experiments. B, Western blot analysis of IKK{epsilon} immunoprecipitates. To determine whether JNK is physically associated with IKK{epsilon}, immunoprecipitation with either a control IgG Ab (left) or anti-IKK{epsilon} Ab (right) was performed. Western blot analysis of the complex using anti-JNK2 mAb indicated that JNK is not present in the IKK{epsilon} complex (the control is FLS lysates on the same blot). These data suggest that IKK{epsilon} complex phosphorylates c-Jun independent of JNK (n = 3).

 
Regulation of MMP expression by IKK{epsilon}

Because IKK{epsilon} immunoprecipitates phosphorylated c-Jun, which plays a key role in MMP gene expression and subsequent joint destruction in arthritis, we evaluated the role of IKK{epsilon} in MMP production (21, 32). Murine IKK{epsilon}–/– and IKK{epsilon}+/+ FLS lines were generated by synovial microdissection. Western blot analysis of murine FLS isolated from joints of IKK{epsilon}–/– and IKK{epsilon}+/+ mice confirmed the absence of the IKK{epsilon} protein in the knockout cells (Fig. 7). Because c-Jun is a key regulator of MMP gene expression, we focused our attention on MMP3 and MMP13. IKK{epsilon}+/+ and IKK{epsilon}–/– FLS were stimulated with LPS or TNF-{alpha} for 6 h, and MMP3 and MMP13 mRNA levels were quantified by real-time quantitative PCR. Fig. 8, A (MMP3) and B (MMP13), shows that MMP induction was significantly greater in the IKK{epsilon}+/+ FLS compared with IKK{epsilon}–/– FLS after either TNF-{alpha} or LPS stimulation (n = 4). Fig. 8C demonstrates that IP-10 (CXCL-10) expression is not attenuated in the murine IKK{epsilon}–/– FLS, indicating that the results of the MMP experiments are not due to nonspecific effects on cell viability or gene transcription. The lack of effect on IP-10 is surprising, but might reflect a more important role of TBK-1 for this gene in FLS.



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  		FIGURE 7. Western blot analysis of murine IKK{epsilon}–/– and IKK{epsilon}+/+ synoviocyte lines. IKK{epsilon} mice were sacrificed, and FLS lines were prepared from microdissected knees. Western blot analyses of murine FLS isolated from joints of IKK{epsilon}–/– and IKK{epsilon}+/+ mice demonstrated that the immunoreactive IKK{epsilon} protein is expressed only in wild-type murine FLS (n = 3 each).

 


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  		FIGURE 8. MMP gene expression in IKK{epsilon} knockout synoviocytes. Because c-Jun is a substrate for IKK{epsilon} and a key determinant of MMP gene transcription, we evaluated the possibility that IKK{epsilon} regulates MMP gene expression in FLS. IKK{epsilon}–/– and IKK{epsilon}+/+ FLS were stimulated with medium, TNF-{alpha}, or LPS for 6 h. MMP3 and MMP13 mRNA levels were quantified by real-time PCR and normalized to HGPRT (n = 3–5 separate experiments). A, MMP3 gene expression induction is decreased in IKK{epsilon} knockout synoviocytes. B, MMP13 gene induction is decreased in IKK{epsilon} knockout synoviocytes. C, IP-10 (CXCL-10) gene expression is similar in IKK{epsilon} knockout and wild-type synoviocytes, supporting the specificity of the MMP experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Therapy for RA has improved dramatically over the last decade with the advent of anti-cytokine therapy (33). However, many patients have persistent disease despite optimal treatment. The search for novel therapeutic targets to address this unmet medical need has focused attention on the intracellular machinery that regulates the production of pathogenic mediators. Transcription factors such as NF-{kappa}B and AP-1 are especially important because of their pivotal role in cytokine and MMP gene expression (34, 35). Kinase cascades that modulate these pathways could potentially be blocked by small molecule inhibitors and thereby target an array of genes involved in the disease pathogenesis (36).

One concern with inhibition of major pathways such as NF-{kappa}B is that many homeostatic and host defense mechanisms might also be affected (37). For instance, targeting NF-{kappa}B through IKK2 could have a major impact on normal immune responses and cell survival. This issue led us to evaluate alternative pathways that could alter pathogenic gene transcription while leaving basal expression intact. Hence, we considered whether a novel inducible IKK, known as IKK{epsilon}, might represent a safer way to modulate, rather than ablate, transcription factor activation. IKK{epsilon}/IKKi is an IKK-related kinase and was initially identified as an inducible protein that could phosphorylate I{kappa}B in LPS-stimulated macrophages (11, 13). However, NF-{kappa}B-independent functions were suggested by additional studies demonstrating that IKK{epsilon} can phosphorylate other proteins, including TRAF-interacting protein (15). This along with recent data demonstrating that MEFs lacking IKK{epsilon} retain normal activation of the canonical NF-{kappa}B pathway after TNF-{alpha} stimulation (28) suggested that IKK{epsilon} has many other effects.

Although IKK{epsilon} is usually considered an inducible gene, our previous studies of IKK{epsilon} in arthritis demonstrated constitutive mRNA and protein expression in FLS (27). IL-1 and TNF-{alpha} markedly induced IKK{epsilon} mRNA, but protein levels only modestly increased. These data raised the possibility that post-translational activation of IKK{epsilon} might have a more important role in FLS. Even though the cytokines known to induce IKK{epsilon} in vitro are present in RA tissues, IKK{epsilon} RNA transcript levels are similar in RA and OA. In the present study we extended these observations by demonstrating that IKK{epsilon} protein expression in inflammatory and noninflammatory synovial tissue is similar. Immunohistochemistry also showed abundant immunoreactive IKK{epsilon} in the synovial intimal lining, suggesting that FLS produce the protein in vivo. These protein studies are the first demonstration of IKK{epsilon} protein expression and distribution in human disease.

The similar levels of IKK{epsilon} protein in OA and RA led us to evaluate whether its functional status is modulated in FLS after cytokine or TLR ligand stimulation. In vitro kinase assays showed that IKK{epsilon} function is rapidly induced by IL-1 and TNF-{alpha} as well as LPS stimulation. Although I{kappa}B{alpha} could serve as a substrate, we were struck by the observation that IKK2 was significantly more efficient than IKK{epsilon} at generating phospho-I{kappa}B{alpha}. In addition, NF-{kappa}B activation was not altered by the absence of IKK{epsilon} in FLS. This raised the possibility that alternative substrates and signaling cascades might be important for IKK{epsilon}-mediated functions in FLS. Surprisingly, we found that c-Jun served as an excellent substrate for the IKK{epsilon} immunoprecipitate, with a 10-fold increase in activity after cytokine stimulation. As a component of the transcription factor AP-1, c-Jun is a key regulator of MMP gene expression and has been implicated as a major pathway responsible for extracellular matrix destruction in animal models of arthritis (32). These data suggest that rather than NF-{kappa}B, IKK{epsilon} regulates c-Jun-driven gene expression in FLS.

Although IKK{epsilon} might phosphorylate c-Jun directly, it is also possible that other kinases coprecipitate with IKK{epsilon}. One major concern was that JNK could associate with the IKK{epsilon} complex because it phosphorylates c-Jun while binding to many other kinases, including MEKK1, MEKK2, MKK4, and MKK7 (25, 31). Three lines of evidence suggest that this is not the case. First, phosphorylation of c-Jun by the IKK{epsilon} immunoprecipitate was not inhibited by a selective JNK inhibitor. Second, immunoreactive JNK was not detected in the IKK{epsilon} complex by Western blot analysis. Finally, a dominant negative IKK{epsilon} construct blocked c-Jun phosphorylation in vitro. Additional studies are needed to determine whether IKK{epsilon} or an undefined associated kinase is responsible for the kinase activity resulting in phosphorylation of c-Jun. For instance, preliminary studies suggest that Ser63 and Ser73 can be phosphorylated in cytokine-stimulated IKK{epsilon}–/– cells (data not shown), suggesting that the extent or kinetics of phosphorylation or the specific residues activated by the IKK{epsilon} complex might be important. Studies to identify the specific residues on c-Jun that are phosphorylated by IKK{epsilon} immunoprecipitates are in progress.

Because c-Jun is known to regulate MMP gene expression, we evaluated the functional role of IKK{epsilon} by assessing MMP3 and MMP13 gene expression in cultured FLS. Small molecule inhibitors of IKK{epsilon} are not currently available; therefore, we focused our attention on stimulated FLS isolated from wild-type and IKK{epsilon} knockout mice. We noted that MMP induction was strikingly lower in the IKK{epsilon} knockout FLS compared with wild-type cells after either cytokine or LPS stimulation even though basal expression was similar. Hence, our original search for alternative pathways to pathogenic NF-{kappa}B activation ultimately led us to a novel mechanism that regulates c-Jun and MMP production. Because IKK{epsilon} is expressed in the rheumatoid intimal lining, which is the primary source of MMPs and proinflammatory cytokines, this could represent an interesting therapeutic target. Based on studies with IKK{epsilon}–/– embryonic fibroblasts and our own studies in FLS, however, IKK{epsilon} blockade will probably not have a major impact on the canonical NF-{kappa}B pathway (28).

As noted above, our studies of the function of IKK{epsilon} in RA focused initially on its participation in NF-{kappa}B signaling and subsequently the alternative activation pathways. However, we were also intrigued by the data indicating that the IKK-related kinases play an unanticipated role in innate immunity (17, 18). For instance, TLR3 ligation and formation of a virus-activated kinase complex leads to IKK{epsilon} and TBK-1 activation, followed by IRF phosphorylation. In fact, TLRs have also been implicated in the pathogenesis of RA, and many TLR ligands, such as peptidoglycans, are present in the rheumatoid joint and activate synovial fibroblasts (38). TLR and cytokine signaling pathways are convergent, and the clinical efficacy of cytokine defects can be bypassed by the innate immune system to induce arthritis (39). Our results demonstrating that LPS induces IKK{epsilon} kinase activity in FLS and that MMP expression in IKK{epsilon} knockout synoviocytes is suppressed suggest that these pathways might be operative in human cells as well. TLR signaling pathways are emerging as small molecule targets for the development of new therapeutics that modify the innate immune response (40).

After phosphorylation by the IKK{epsilon}/TBK-1 complex, the IRFs bind to IFN-stimulated response elements in promoters of various antiviral genes, including IFN-stimulated genes, IFN-{beta}, and RANTES. Of interest, c-Jun as a heterodimer with ATF-2 has also been implicated in activation of these genes as a component of the IFN enhanceosome (29, 30). The presence of c-Jun and IRF3 in the enhanceosome provides a link among IKK{epsilon}, c-Jun, the antiviral program, and MMP expression. By participating in the IRF signaling cascade as a result of TLR ligation, IKK{epsilon} can potentially regulate IFN-{beta} and RANTES expression (17, 18). The impact of these observations on RA is still not certain. However, IFN-{beta} might have a protective role in synovitis and represent a counter-regulatory pathway that suppresses synovial inflammation in animal models of arthritis (41, 42). IFN-{beta} is expressed in RA synovium; however, it is not clear whether the beneficial effects of IKK{epsilon} blockade and proinflammatory cytokine and MMP suppression will be offset by the decreased IFN-{beta} production.

Yet another potential proinflammatory link between IKK{epsilon} and RA was suggested in IKK{epsilon} knockout MEFs. Interaction between NF-{kappa}B and C/EBP signaling pathways activated by LPS was altered in IKK{epsilon} knockout MEFs (28). Cells deficient in IKK{epsilon} retained normal activation of the canonical NF-{kappa}B pathway, but failed to induce C/EBP{delta}-specific DNA binding and transcription of C/EBP and NF-{kappa}B target genes after LPS stimulation. In MEFs, IKK{epsilon} deficiency resulted in a reduction of LPS-induced mRNA expression of cyclooxygenase-2, IL-1, IL-6, TNF-{alpha}, IP-10, and RANTES (28). C/EBP{beta} has also been implicated in MMP expression, and inhibition of MMP production in IKK{epsilon}-deficient FLS might also involve C/EBP-mediated effects on transcription (43). These pathways are obviously of interest and will be the subject of future studies.

Based on these intriguing data, we propose that IKK{epsilon} could participate in multiple novel signaling pathways that are nearly unexplored in chronic inflammation. IKK{epsilon} also represents a potential therapeutic target in RA with interesting pharmacology that could link innate immunity, extracellular matrix destruction, and cell recruitment. It is activated by factors known to be present in the rheumatoid joint and initiates a cascade that modulates innate and adaptive immune responses. Multiple transcription factor families, including NF-{kappa}B, AP-1, C/EBP, and IRF, interact with the IKK-related kinase IKK{epsilon}. These data indicate that IKK{epsilon} and other related kinases could serve as therapeutic targets in RA.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 This work was supported by grants from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Gary S. Firestein, University of California-San Diego School of Medicine, Mail Code 0656, 9500 Gilman Drive, La Jolla, CA 92093-0656. E-mail address: gfirestein{at}ucsd.edu Back

3 Abbreviations used in this paper: RA, rheumatoid arthritis; CE, cell equivalent; dn, dominant negative; FLS, fibroblast-like synoviocyte; IKK, I{kappa}B kinase; IKKi, inducible I{kappa}B kinase; IP-10, IFN-{gamma}-inducing protein 10; IRF, IFN regulatory factor; MEF, murine embryonic fibroblast; MKK, MAPK kinase; MMP, matrix metalloproteinase; OA, osteoarthritis; TBK, TRAF-associated NF-{kappa}B activator-binding kinase; TRAF, TNF receptor-activating factor; HGPRT, hypoxanthine guanine phosphoribosyl transferase. Back

Received for publication December 15, 2004. Accepted for publication March 8, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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