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NF-B Regulation by IB Kinase in Primary Fibroblast-Like Synoviocytes¹

Karlfried R. Aupperle^2,*, Brydon L. Bennett^2,, David L. Boyle^*, Paul-Peter Tak^*, Anthony M. Manning and Gary S. Firestein^3,*

^* Division of Rheumatology, Allergy, and Immunology, University of California at San Diego School of Medicine, La Jolla, CA 92093; and Signal Pharmaceuticals, 5555 Oberlin Drive, San Diego, CA 92121

	Abstract

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NF-B is a key regulator of inflammatory gene transcription and is activated in the rheumatoid arthritis (RA) synovium. In resting cells, NF-B is retained as an inactive cytoplasmic complex by its inhibitor, IB. Phosphorylation of IB targets it for proteolytic degradation, thereby releasing NF-B for nuclear translocation. Recently, two related IB kinases (IKK-1 and IKK-2) were identified in immortalized cell lines that regulate NF-B activation by initiating IB degradation. To determine whether IKK regulates NF-B in primary cells isolated from a site of human disease, we characterized IKK in cultured fibroblast-like synoviocytes (FLS) isolated from synovium of patients with RA or osteoarthritis. Immunoreactive IKK protein was found to be abundant in both RA and osteoarthritis FLS by Western blot analysis. Northern blot analysis showed that IKK-1 and IKK-2 genes were constitutively expressed in all FLS lines. IKK function in FLS extracts was determined by measuring phosphorylation of recombinant IB in vitro. IKK activity in both RA and osteoarthritis FLS was strongly induced by TNF- and IL-1 in a concentration-dependent manner. Activity was significantly increased within 10 min of stimulation and declined to near basal levels within 80 min. Activation of IKK in FLS was accompanied by phosphorylation and degradation of endogenous IB as determined by Western blot analysis. Concomitant activation and nuclear translocation of NF-B was documented by EMSA and immunohistochemistry. Transfection with a dominant negative IKK-2 mutant prevented TNF--mediated NF-B nuclear translocation, whereas a dominant negative IKK-1 mutant had no effect. This is the first demonstration that IKK-2 is a pivotal regulator of NF-B in primary human cells.

	Introduction

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Rheumatoid arthritis (RA)⁴ is a chronic inflammatory disease marked by synovial hyperplasia, leukocyte infiltration, and proinflammatory cytokine production (1). NF-B is one of the key transcription factors implicated in RA and regulates expression of over 70 genes identified in inflammatory conditions (2). A variety of mediators can activate NF-B, including inflammatory cytokines known to be involved in rheumatoid synovitis (e.g., IL-1ß and TNF-), bacterial LPS, viral proteins (tax, HIV), receptor binding (CD28, CD95), UV irradiation, and cellular stress (3, 4). While cytokines like IL-1 and TNF- are thought to be key activators of NF-B in RA synovium, the signal transduction process in the synovial lining fibroblast-like synoviocytes (FLS) has not been defined.

NF-B normally resides in the cytoplasm, where it is retained by the association with IB proteins (, ß, ) that mask the nuclear localization signal (3). Activation of NF-B is dependent on the phosphorylation and degradation of IB, an endogenous inhibitor that binds to NF-B in the cytoplasm. Two recently discovered IB kinases (IKK) appear to regulate this process in immortalized cell lines and tumor cells (5, 6, 7, 8). The two kinases are relatively conserved but, surprisingly, are encoded on separate chromosomes (9). The IKK complex represents a potential convergence point for multiple signaling stimuli that activate NF-B and contains a 300- to 900-kDa assembly, IKK-1 and IKK-2 (also called IKK and IKKß), a scaffold protein IKKAP-1 (10), and other unidentified proteins. IKK is activated by upstream kinases such as mitogen-activated protein (MAP)/extracellular signal-related kinase kinase 1 and NF-B-inducing kinase) (11, 12). IKK then phosphorylates IB on two N-terminal serine residues. This forms a recognition motif for a second enzyme system, the ubiquitin ligases, that bind and assemble a chain of covalently linked ubiquitin residues affixed to lysine 21 and 23 of IB. The ubiquitin tagged NF-B is a target for the 26S proteasome and is rapidly degraded, thereby releasing NF-B for translocation to the nucleus (13, 14).

To assess the role of IKK and NF-B in RA, we evaluated the expression and function of IKK in primary synovial FLS. These studies showed that the IKK complex is expressed in FLS and is activated by IL-1 and TNF-. In addition, IKK activation is required for TNF--mediated translocation of NF-B to the nucleus in FLS. This is the first study demonstrating that the IKK complex plays a pivotal role in cytokine-mediated NF-B activation in primary human cells isolated from the site of disease.

	Materials and Methods

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Fibroblast-like synoviocytes

FLS were isolated from RA and osteoarthritis (OA) synovial tissues obtained at joint replacement surgery as previously described (15). The diagnoses conformed to the 1987 revised American Council of Rheumatology criteria (16). Briefly, the tissues were minced and incubated with 1 mg/ml collagenase in serum free DMEM (Life Technologies, Grand Island, NY) for 2 h at 37°C, filtered through a nylon mesh, extensively washed, and cultured in DMEM supplemented with 10% FCS (Life Technologies, endotoxin content <0.006 ng/ml), penicillin, streptomycin, and L-glutamine in a humidified 5% CO₂ atmosphere. After overnight culture, nonadherent cells were removed, and adherent cells were cultivated in DMEM plus 10% FCS. At confluence, cells were trypsinized, split at a 1:3 ratio, and recultured in medium. Synoviocytes were used from passages 3 through 6 in these experiments, during which time they comprised a homogeneous population of FLS (<1% CD11b, <1% phagocytic, and <1% FcRII positive). All studies were approved by the University of California, San Diego Institutional Review Board.

Abs and reagents

Affinity-purified rabbit polyclonal Ab to IKK (IKK2 CT) was raised against a peptide encoding the carboxyl terminus of IKK-2 (Signal Pharmaceuticals, San Diego, CA). Rabbit polyclonal anti-IKKß, anti-IKB, and anti-Rel A (Santa Cruz Biotechnology, Santa Cruz, CA), mAb to IKK (PharMingen, San Diego, CA), donkey anti-rabbit IgG peroxidase-conjugated Ab (Amersham, Arlington Heights, IL), sheep anti-mouse IgG peroxidase-conjugated Ab (Amersham), TNF-, and IL-1ß were purchased from R&D Systems (Minneapolis, MN).

Immunohistochemistry

FLS were cultured in chamber slides (Nalge Nunc International, Naperville, IL) and fixed in cold acetone for 10 min. The specimens were incubated with 75 µl anti-Rel A (1:1000) (Santa Cruz Biotechnology) or irrelevant control Ab for 1 h at room temperature. The slides were then washed three times in PBS and incubated with biotinylated goat anti-rabbit Ab (Bio-Rad, Hercules, CA). The slides were washed, incubated with avidin-biotin complex (Vector Laboratories, Burlingame, CA), and the peroxidase was developed with diaminobenzidine and hydrogen peroxide (Vector Laboratories).

Northern blot analysis

Total RNA was isolated using RNA STAT-60 (Tel-Test, Friendswood, TX). RNA was fractionated in a 1.2% agarose gel containing 5.5% formaldehyde. The RNA was transferred to nylon membrane using the turbo blotter (Schleicher and Schuell, Keene, NH) and cross-linked at 80°C for 45 min. The blots were prehybridized in 50% formamide, 5x saline-sodium phosphate-EDTA, 1x Denhardt’s solution, 1% SDS, 200 µg/ml ssDNA, and 50 µg/ml tRNA. cDNA probes were denatured and labeled by random primed incorporation of [-³²P]dATP (Ambion, Austin, TX). The probes were denatured at 100°C, and the blots were hybridized overnight at 42°C. The membrane was then washed in 2x saline-sodium phosphate-EDTA and 0.1% SDS at 37°C and autoradiographed with Kodak X-OMAT AR film (Rochester, NY) with an intensifying screen for 18–24 h at 80°C.

Western blot analysis

Whole cell lysate (100 µg) was fractionated on Tris-glycine-buffered 10% SDS-polyacrylamide gels (Novex, San Diego, CA) and transferred to nitrocellulose membrane (Amersham, Cleveland, OH). Membranes were blocked with 5% nonfat milk powder (Bio-Rad) and probed with primary Ab to IB, IKK-2, or IKK-1 and then with donkey anti-rabbit HRP-conjugated Ab (1:2500) or sheep anti-mouse IgG peroxidase-conjugated Ab (1:2500) in PBS with 0.1% Tween-20 and 5% nonfat milk powder. Immunoreactive proteins were detected with chemiluminescence and autoradiography (Amersham). For immunoprecipitation experiments, IKK-1 and IKK-2 precipitates were prepared as described in IKK kinase assay below.

IKK kinase assay

IKK activity was detected by immunoprecipitating of IKK and addition of radiolabeled phosphate to recombinant IB as previously described (5). Briefly, cells (3 x 10⁶) were rotated for 1 h at 4°C in lysis buffer (20 mM HEPES, pH 7.9, 0.5 M NaCl, 0.25% Triton X-100, 1 mM EDTA, 1 mM EGTA, 1 mM DTT with phosphatase and protease inhibitors). Phosphatase and protease inhibitors consisted of: 20 mM ß-glycerophosphate, 10 mM NaF, 0.3 mM Na₃VO₄, 1 mM benzamidine, 10 mM p-nitrophenyl phosphate, and complete protease inhibitor mixture (Boehringer Mannheim, Indianapolis, IN). Anti-IKK Ab was added to the lysis buffer and mixed at 4°C for 2 h. Then, 35 µl of washed protein A agarose (Calbiochem, San Diego, CA) was added for an additional 1 h. Immunoprecipitated material was washed four times in wash buffer (40 mM Tris, pH 8.0, 0.5 M NaCl, 0.1% Nonidet P-40, 6 mM EDTA, 6 mM EGTA, 1 mM DTT with phosphatase and protease inhibitors) and once with kinase buffer (20 mM HEPES, pH 7.9, 1 mM MgCl₂, 1 mM MnCl₂, 1 mM DTT with phosphatase and protease inhibitors). Kinase activity was assayed in 40 µl of kinase buffer containing 10 µM [-³²P]dATP and 3 µg GST-IB 1–54(1–54) for 30 min at 30°C. The reaction was stopped by the addition of SDS gel sample buffer and analyzed by SDS-Page and autoradiography.

EMSA

Nuclear protein was extracted from FLS (1 x 10⁶ cells per treatment) and assayed for DNA binding of NF-B. After washing the cells in ice-cold PBS, the cell pellet was resuspended in 1 ml buffer A (10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KCl, and 1 mM DTT) containing 0.1% Triton X-100. After incubating for 10 min on ice, the lysate was centrifuged and the nuclei resuspended in 20–40 µl of buffer C (20 mM HEPES, pH 7.9, 25% [v/v] glycerol, 420 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 1 mM DTT). This suspension was incubated for 30 min on ice followed by centrifugation at 10,000 x g for 20 min. The supernatant was stored at -80°C as nuclear extract after protein concentrations were determined by the Bradford method using BSA as standard. Double-stranded oligonucleotides containing a consensus NF-B or AP-1 recognition sequence (Promega, Madison, WI) were end-labeled with T4 polynucleotide kinase in the presence of [-³²P]dATP. The DNA binding reaction was performed at room temperature for 30 min in a final volume of 15 µl, which contained 3–5 µg nuclear extract, oligonucleotide probe (40 fmol), binding buffer (10 mM Tris-HCl, pH 7.5, 4% (v/v) glycerol, 50 mM NaCl, 1 mM MgCl, 0.5 mM EDTA, and 0.5 mM DTT, 100 µg/ml poly dI-dC). Reactions were subjected to electrophoresis on nondenaturing 5% polyacrylamide gels in 0.5x TBE (90 mM Tris, 64.6 mM boric acid, 2.5 mM EDTA, pH 8.3) at 125 mA for 4 h at 4°C. The gels were dried under vacuum, and exposed to Hyperfilm MP (Amersham) with an intensifying screen at -70°C.

FLS transfection

FLS were transfected with IKK constructs that have been previously described and characterized (5). FLS cultured at 8000 cells/ml/well in four-chambered glass slides (Falcon, Becton Dickinson, Franklin Lakes, NJ) were transfected with 0.5 µg of plasmid DNA and 1.5 µl of Superfect transfection reagent (Qiagen, Hilden, Germany) in 0.5 ml DMEM for 3 h. Media was then changed to DMEM plus 10% FCS and the cells cultured for 24 h before analysis.

Immunofluorescence

RA FLS were cultured at 8000 cells/ml into four-chambered glass slides (Falcon) at 1 ml per chamber and allowed to adhere and culture. After 16 h, cells were transiently transfected with anti-HA-tagged IKK-1 or FLAG-tagged IKK-2 constructs. Cells were treated as described in Results, washed with PBS, fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Ft. Washington, PA) for 30 min, and permeabilized with wash buffer (0.5% Triton X-100, 0.01% sodium azide in PBS). Cells were blocked with wash buffer containing 5% donkey serum (Jackson ImmunoResearch, West Grove, PA) for 30 min and probed with primary Ab; anti-Rel A at 1:1000 dilution (Santa Cruz Biotechnology), anti-FLAG mAb at 1:1,000 (Kodak), or anti-HA mAb 1:1,000 (Boehringer Mannheim, Indianapolis, IN). After washing, cells were incubated with secondary Ab donkey anti-rabbit FITC conjugate at 1:100 or donkey anti-mouse Texas Red conjugate at 1:100 (Jackson ImmunoResearch). Following extensive washing, coverslips were placed with polyvinyl alcohol/DABCO (Sigma) mounting medium and allowed to dry. Slides were viewed under fluorescence with a Nikon Microphot-FXA microscope.

	Results

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TNF- induces nuclear translocation of NF-B

Initial experiments were performed to establish conditions under which NF-B was quiescent and subsequently activated by TNF- in cultured FLS. Cells were grown in chamber slides and cultured in 0.5% FCS for 24 h. Medium or 100 ng/ml of TNF- was added to the cells for 1 h, and immunohistochemistry was performed to localize Rel A protein. Fig. 1A shows a representative experiment in which cytoplasmic staining was observed in resting cells. When FLS were stimulated with TNF-, immunoreactive NF-B localized to nuclei in the vast majority of cells (Fig. 1B) (10.6 ± 3.4% nuclear staining in resting cells and 84.5 ± 2.0% in stimulated cells; p < 0.001; n = 3).

View larger version (58K): [in this window] [in a new window]   FIGURE 1. Immunohistochemistry for Rel A in FLS. Cultured FLS were stimulated with (A) medium or (B) TNF- (100 ng/ml) for 1 h and immunostained for the p65 subunit of NF-B as described in Material and Methods. In contrast to cells cultured with medium alone, TNF--stimulated cells contained immunoreactive p65 in the nuclei.

Northern blot assay was performed to determine steady-state mRNA levels for IKK-1 and IKK-2 in three OA and three RA FLS as well as one normal FLS line (Fig. 2A). All cells expressed IKK-1 and IKK-2 mRNA, and no differences were observed between normal, OA, or RA FLS.

View larger version (69K): [in this window] [in a new window]   FIGURE 2. A, IKK mRNA expression in RA and OA FLS. Northern blot analysis of FLS revealed IKK-1 and IKK-2 mRNA in RA, OA, and normal synovial fibroblasts (NF). Jurkat cells served as a positive control. G3PDH-normalized signal intensities are: IKK-1 (OA = 93 ± 11; RA 122 ± 17; NF = 96); IKK-2 (OA = 82 ± 8; RA 84 ± 21; NF = 63). p > 0.10 for RA compared with OA. B, Expression of IKK protein in RA and OA FLS. Total protein from RA and OA FLS was extracted and IKK was immunoprecipitated as described in Material and Methods. Western blot analysis demonstrated the presence of both IKK-1 and IKK-2 protein in three OA and three RA cell lines.

IKK protein levels in FLS from OA and RA were then determined by immunoprecipitation and Western blot analysis (Fig. 2B). Studies in transformed and immortalized cell lines have suggested that IKK-1 and IKK-2 are bound as a heterodimer or multimer in a higher order complex of 400–900 kDa containing multiple heterodimers and associated scaffold proteins (5). Whole-cell lysates were immunoprecipitated with Ab to either IKK-1 or IKK-2, fractionated on SDS-PAGE, blotted, and then probed with the reciprocal Ab. Immunoprecipitation with anti-IKK-1 copurified IKK-2, while immunoprecipitation with anti-IKK-2 copurified IKK-1, confirming the presence of both IKK-1 and IKK-2 in FLS kinase complex. All OA and RA isolates contained IKK-1 and IKK-2 immunoreactive protein, and no differences were observed between OA and RA FLS (n = 3 each).

Regulation of IKK activity in FLS by cytokines

IL-1ß and TNF- are known to induce NF-B activation and inflammatory gene expression in FLS (see Fig. 1). Therefore, the ability of these cytokines to activate IKK in RA and OA FLS was evaluated. FLS were incubated with increasing concentrations of IL-1ß or TNF- for 10 min, lysed, and the IKK complex was immunoprecipitated in the presence of protease and phosphatase inhibitors. The precipitated complex was then assayed for kinase activity in the presence of substrate (GST-IB 1–54) and [³²P]dATP. IKK activity significantly increased in the presence of IL-1ß and TNF- in a concentration-dependent manner (n = 3 RA and 3 OA cell line; see Fig. 3 for one representative experiment). The dose responses for RA and OA FLS were not significantly different (data not shown).

View larger version (30K): [in this window] [in a new window]   FIGURE 3. IL-1- and TNF--mediated activation of IKK. RA FLS were stimulated for 10 min with increasing concentrations of IL-1 (0.1–10 ng/ml) or TNF- (1–100 ng/ml). Cells were then lysed in presence of protease and phosphatase inhibitors, IKK precipitated by anti-IKK-2 Ab, and kinase function was determined as described in Material and Methods. IKK activity is demonstrated by detection of phosphorylated recombinant IKK-substrate (IB).

Correlation of IKK activation, IB degradation, and NF-B activation in FLS

The temporal correlation of IKK induction with IB degradation and NF-B DNA binding activity in OA and RA FLS was subsequently examined. IKK activity rapidly increased after treatment with TNF- and reached peak activity after 5 to 10 min (n = 3 RA and 3 OA FLS). There were minor differences in the peak time of IKK activation between individual experiments and FLS lines, although the peak was always 5–10 min after cytokine stimulation. No statistical differences in the kinetics or level of IKK activity were observed between OA and RA cell lines (peak activity = 8.3 ± 2.9 min for RA and 6.7 ± 2.9 for OA; p > 0.10) (Fig. 4, A and B). The kinetics of IKK activation in FLS for the combined OA and RA data is shown in Fig. 5. A similar profile was observed in one normal FLS line, with peak IKK activity observed after 10 min of cytokine stimulation (data not shown). Peak IKK activity correlated with the appearance of a higher molecular mass species of IB (see Western blots in Fig. 4, A and B). This identifies the hyperphosphorylated form IB, which is then rapidly degraded by the ubiquitin-proteasome system. Degradation of IB was, in turn, temporally correlated with the appearance of NF-B binding as determined by EMSA. Finally, immunoreactive IB reappeared after 40–80 min, confirming that it also serves as a NF-B-regulated early response gene.

View larger version (52K): [in this window] [in a new window]   FIGURE 4. A, Time course of IKK activity in OA FLS. The kinetics of IKK activation in response to TNF- (100 ng/ml) was determined in OA FLS. IKK functional activity (kinase assay), IB protein levels (Western blot), and NF-B binding (EMSA) in FLS were determined at various time points. B, Time course of IKK activity in RA FLS. The kinetics of IKK activation in response to TNF- (100 ng/ml) was determined in RA FLS. IKK functional activity (kinase assay), IB protein levels (Western blot), and NF-B binding (EMSA) in FLS were determined at various time points. No consistent differences were noted between OA and RA FLS.

View larger version (11K): [in this window] [in a new window]   FIGURE 5. Kinetics of IKK activation. IKK activity was determined in three RA and three OA FLS cell lines that had been stimulated with 100 ng/ml TNF-. Pooled data are shown as the percent increase over basal IKK activity. IKK is rapidly activated in response to TNF-, peaking after 10 min and declining to near basal levels after 80 min.

Role of IKK-2 in TNF-mediated NF-B activation in FLS

To determine whether IKK activation is necessary and sufficient for NF-B activation in FLS, cells were transfected with tagged IKK constructs and assessed for both NF-B translocation and transgene expression. RA FLS were transfected with wild-type (wt) IKK, dominant negative IKK in which the catalytic lysine was mutated to methionine (K>M), or constitutive active IKK in which two serine residues in the MAP kinase kinase kinase activation loop were mutated to glutamate residues (S>E). Rel A was spontaneously translocated to the nucleus in unstimulated FLS expressing constitutive active IKK-2 S>E, indicating that activation of IKK was sufficient for NF-B nuclear translocation (see Fig. 6A for a representative field and Table I for a summary of the data). FLS that had been transduced with the dominant negative K>M mutant were then stimulated with TNF-. Stimulated cells that did not express the tagged transgene exhibited Rel A activation as shown by intense nuclear staining (see Fig. 6B for a representative field). However, FLS expressing IKK-2 K>M showed complete abrogation of Rel A translocation. These data indicate that IKK-2 is a key NF-B regulatory protein in primary FLS. The dominant negative IKK-1 transfectant did not block activation of NF-B by TNF-, although the constitutive active construct was able to induce nuclear translocation (see Table I). This suggests that IKK-2 is the major functional isoform involved in cytokine-induced NF-B activation in FLS.

View larger version (136K): [in this window] [in a new window]   FIGURE 6. A, Constitutive activate IKK mutant initiates nuclear NF-B accumulation. FLS were transfected with a HA-tagged constitutive active IKK-2 mutant (S>E). The figure shows a representative field in which one of three cells was successfully transfected with the mutant as confirmed by immunostaining with HA-specific Ab (see red cell in right panel). Only the cell containing the constitutive activate with the IKK mutant demonstrates accumulation of NF-B (p65) in the nucleus (left panel). B, Dominant negative IKK mutant prevents nuclear NF-B accumulation. FLS were transfected with a FLAG-tagged IKK-2 dominant negative mutant (K>M). The figure shows a representative field in which one of four cells was successfully transfected with the mutant as confirmed by immunostaining with FLAG-specific Ab (see red cell in right panel). Stimulation with TNF- (100 ng/ml) induced nuclear accumulation of NF-B (p65) in FLS with functional IKK, whereas cells containing the dominant negative mutant did not demonstrate nuclear NF-B staining.

View this table: [in this window] [in a new window]   Table I. Effect of IKK transfection on NF-B translocation in FLS1

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

NF-B regulates many genes that are central to synovial inflammatory diseases (4, 5). For instance, NF-B activation increases expression of the endothelial cell adhesion molecules E-selectin, VCAM-1, and ICAM-1, and NF-B inhibition reduces leukocyte adhesion and transmigration (17, 18). NF-B is involved in the regulation of IL-1ß in monocytes (19), as well as ICAM-1, TNF-, and IL-6 in rheumatoid synoviocytes (20, 21). Some anti-rheumatic drugs, including aspirin, gold sodium thiomalate, sulfasalazine, and corticosteroids might act through their ability to inhibit NF-B (22, 23, 24).

NF-B function is regulated through rapid degradation of its endogenous inhibitory molecule IB. Inflammatory stimuli, such as cytokines, initiate a signaling cascade that can lead to activation of two recently identified IB kinases, IKK-1 and IKK-2, that phosphorylate IB at two N-terminal serine residues (5, 6, 7, 8). The IB kinases IKK-1 and IKK-2 are members of a new family of intracellular signal transduction enzymes, containing an amino-terminal kinase domain and a C-terminal region with two protein interaction motifs, a leucine zipper and a helix-loop-helix motif. IKK-1 and IKK-2 may be activated by one or more upstream activating kinases, including members of the MAP kinase kinase kinase family (11). Subsequently, phosphorylated IB is ubiquitinated and degraded by the 26S proteasome complex (13, 14). NF-B is then transported to the nucleus where it binds its target genes to initiate transcription.

NF-B expression and activation in RA synovium has been extensively documented. Immunohistochemistry studies identified nuclear Rel A and p50 staining in rheumatoid synovium, especially in the intimal lining where FLS reside (25, 26, 27, 28). Although NF-B proteins were also detected in OA synovium, EMSA experiments demonstrate significantly higher NF-B binding activity in RA synovial tissue extracts. It was not determined if this difference was a result of the inflammatory cytokine milieu in RA or whether the RA cells were intrinsically different. The ability of the synovial inflammation to activate NF-B has also been demonstrated in several animal models of arthritis. NF-B expression is increased in the synovial intimal lining of rats with adjuvant arthritis early in the course of the disease (29), and inhibitors of IB phosphorylation suppress clinical arthritis (30). In collagen-induced arthritis in mice, synovial NF-B activation occurs well before clinical evidence of synovitis (28).

Although NF-B is activated in RA synovium, many of the specific pathways involved in this process are not known. Presumably, this involves proinflammatory cytokines like IL-1 and TNF-, both of which are abundant in the rheumatoid joint (31, 32). We hypothesized that IKK is a key signal transduction kinase that coordinates this process. Previous studies on the role of IKK in NF-B regulation have been limited to immortalized cell lines, and there is little information on their function in normal cells or cells derived from the site of human inflammatory diseases. Therefore, we determined whether IKK is expressed in primary FLS derived from RA and OA synovium and whether these kinases are key regulatory enzymes in the regulation of NF-B.

Initial studies demonstrated that both IKK-1 and IKK-2 are constitutively expressed in FLS, regardless of the source, and are rapidly activated after cells are stimulated by IL-1 or TNF-. Additionally, coprecipitation studies indicated that IKK-1 and IKK-2 proteins are physically associated in FLS. Kinetics experiments showed a rapid increase in functional kinase activity, with subsequent degradation of phosphorylated IB. No differences were observed with regard to the extent or time course of IKK activation in RA, OA, or normal FLS. This suggests that the machinery for NF-B activation is present in all synoviocytes and that the ability to activate this transcription factor is not due to intrinsic differences between FLS isolated from OA and RA joint samples. More likely, the increased levels of NF-B activation in RA synovium likely represents a response to the local cytokine milieu. NF-B binding activity and nuclear translocation were temporally associated with IKK activation and the loss of IB protein. Ultimately, IB protein levels increased and NF-B binding gradually decreased.

Subsequent studies using IKK-2 mutants demonstrated that constitutively activated IKK leads to NF-B activation even in the absence of exogenous cytokines. Hence, IKK alone is capable of initiating the NF-B cascade in FLS. More important, transduction with a dominant negative IKK-2 clone prevented NF-B translocation when cells were stimulated with TNF-. However, dominant negative IKK-1 did not block NF-B activation. Therefore, the IKK complex, especially IKK-2, is a key convergence site for cytokine-mediated NF-B activation. It is both necessary and sufficient for NF-B translocation induced in primary synoviocytes.

Given the proinflammatory functions of NF-B, IKK represents a potential therapeutic target for RA. In fact, the efficacy of some antiinflammatory agents like aspirin might be mediated in part by the ability to inhibit IKK (33). Our study indicates that it is a key regulatory site that mediates NF-B activation by TNF- in FLS, which are major sources of cytokines and matrix metalloproteinases in arthritis. TNF- inhibitors demonstrate remarkable clinical efficacy in RA, and it is possible that decreased NF-B activation is one of the primary mechanisms of action. Because IKK is the key pathway through which TNF- activates NF-B in FLS, this might be an alternative approach to cytokine inhibition in RA.

	Footnotes

 
¹ This work was supported in part by grants from Signal Pharmaceuticals, the Arthritis Foundation, and the Deutsche Forschungsgemeinschaft. P.P.T. is supported by a North Atlantic Treaty Organization Science Fellowship and the Dutch Arthritis Foundation (Nationaal Reumafonds).

² K.R.A. and B.L.B. contributed equally to this work.

³ Address correspondence and reprint requests to Dr. Gary S. Firestein, Division of Rheumatology, Allergy, and Immunology, Mail code 0656, University of California, San Diego School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0656. E-mail address:

⁴ Abbreviations used in this paper: RA, rheumatoid arthritis; FLS, fibroblast-like synoviocytes; IKK, IB kinase, OA, osteoarthritis; MAP, mitogen-activated protein.

Received for publication December 15, 1998. Accepted for publication April 15, 1999.

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