Nuclear factor-κB (NF-κB) is involved in the pathophysiology of rheumatoid arthritis (RA) and is considered to be a feasible molecular target in treating patients. In the RA joint tissues, activation of NF-κB is often observed together with high amounts of the proinflammatory cytokines tumor necrosis factor (TNF)α and interleukin (IL)-1β. TNFα and IL-1β are known to stimulate NF-κB signaling and are produced as the effect of NF-κB signaling, thus forming a vicious cycle leading to a self-perpetuating nature of rheumatoid inflammation and expansion of such inflammatory response to other joints. Because a kinase called IκB kinase complex (IKK) is involved in the NF-κB activation cascade, we examined the effect of a novel IKK inhibitor, (7-[2-(cyclopropyl-methoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one hydrochloride; CHPD), on the production of inflammatory cytokines from rheumatoid synovial fibroblasts (RSF). TNFα stimulation induced production of inflammatory cytokines such as IL-6 and IL-8 in RSF, and the extent of IL-6 and IL-8 induction was dramatically reduced by CHPD under noncytotoxic concentrations. Likewise, expression of il-6 and il-8 genes was significantly reduced by CHPD. In addition, chromatin immunoprecipitation assays revealed that the DNA binding of NF-κB (p65) to il-8 promoter in RSF was induced after TNFα stimulation and that, upon CHPD treatment to RSF for 1 h, the NF-κB binding to il-8 promoter was significantly decreased. Here, we have demonstrated that an IKKβ inhibitor, CHPD, acts as an effective inhibitor for the production of inflammatory cytokines in response to proinflammatory cytokines. These findings indicate that such a IKKβ inhibitor could be a feasible candidate for an antirheumatic drug.
Rheumatoid arthritis (RA) is a chronic inflammatory disease that affects systemic synovial joints (Firestein, 2003). In RA, proliferation of synovial cells and infiltration of activated immunoinflammatory cells, including T cells, macrophages, and plasma cells (Firestein, 2003), leads to progressive destruction of cartilage and bone. Various cytokines, including interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, IL-8, IL-17, and macrophage–colony-stimulating factor, are present in the synovial fluid and tissue of RA patients (Okamoto, 2006).
Synovial hyperplasia in RA is considered to be due to the impairment of apoptosis (Huber et al., 2006). Most of the above-mentioned pathophysiological features of RA can be explained by activation of a transcription factor nuclear factor-κB (NF-κB) (Feldmann, 2001; Okamoto, 2005, 2006), which is highly activated in the synovial lining cells of RA joint tissue (Sakurada et al., 1996; Huber et al., 2006). NF-κB induces both TNFα and IL-1β gene expression, whereas TNFα and IL-1β stimulate NF-κB signaling, forming a vicious cycle that can perpetuate and expand the inflammatory responses. Thus, blocking this cascade by inhibiting NF-κB signaling is considered feasible for the treatment of RA.
It is interesting to note that some of the drugs for RA were shown to block either the NF-κB activation cascade or its action (Yang et al., 1995; Yamamoto and Gaynor, 2001; Okamoto, 2006). For example, monovalent gold compounds, often used for RA treatment, could inhibit the DNA-binding activity of NF-κB through oxidation of the cysteines associated with zinc (Yang et al., 1995; Yoshida et al., 1999b). Similarly, methotrexate is known to suppress NF-κB activation (Majumdar and Aggarwal, 2001). In addition, intervention therapies using anti-TNFα antibody and soluble TNFα receptor exhibited dramatic therapeutic efficacies by blocking the vicious cycle of NF-κB activation cascade mentioned above (Elliott et al., 1993; Moreland et al., 1997). However, these drugs are expensive and have to be administered by injection, and various adverse effects were reported, thus warranting necessity of effective small-molecule compounds.
NF-κB is a heterodimer or homodimer consisting of Rel family proteins, p65 (RelA), RelB, c-Rel, p50/p105, and p52/p100, and it is normally present in the cytoplasm in association with its inhibitor, IκB (Zabel and Baeuerle, 1990). Stimulation by the proinflammatory cytokines such as TNFα and IL-1β results in the activation of IκB kinase complex (IKK) (Nakano et al., 1998), which consists of three subunits, IKKα, IKKβ, and IKKγ/NEMO. Activated IKK, mainly through IKKβ activity (Zandi et al., 1997; Li et al., 1999b; Karin and Delhase, 2000), phosphorylates IκBα, leading to ubiquitination and degradation of IκB. Then, NF-κB, translocates to nucleus and binds to the κB site of target genes. In this regard, IKKβ is a reasonable molecular target for blocking NF-κB signaling upon inflammatory stimuli.
NF-κB is highly activated not only in the synovial tissue of patients with RA but also in some types of neoplasm, such as multiple myeloma and adult T-cell leukemia (Mori et al., 1999; Sanda et al., 2005, 2006). In these cells, NF-κB is constitutively activated as evidenced by 1) the continuous phosphorylation of IκBα and p65 subunit of NF-κB, 2) activation of NF-κB DNA binding, and 3) up-regulation of various target genes that are responsible for inhibition of apoptosis. Moreover, a specific IKK inhibitor, 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin- 4-yl nicotinonitrile (ACHP; Murata et al., 2004), could inhibit cell growth and induce apoptosis of multiple myeloma and adult T-cell leukemia cells (Sanda et al., 2005, 2006) by inhibiting the phosphorylation of IκBα and p65.
In this study, we examined the effects of a novel IKK inhibitor, 7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one hydrochloride (CHPD; Ziegelbauer et al., 2005), a chemical derivative of ACHP, on the cytokine production of rheumatoid synovial cell. ACHP was initially synthesized by Murata et al. (2004) based on a massive screening, whereas CHPD was identified among the synthesized derivatives of ACHP with highest selectivity for IKKβ and IKKα (IC50 values for IKKβ and IKKα are 2 and 135 nM, respectively, by in vitro kinase assays) over other kinases (Ziegelbauer et al., 2005). In addition, CHPD showed good aqueous solubility and cell permeability, thus demonstrating a very high oral bioavailability in mice and rats (Ziegelbauer et al., 2005). However, effects of CHPD on the production of inflammatory cytokines have never been examined. Here, we show that CHPD could effectively block NF-κB pathway in rheumatoid synovial fibroblasts (RSFs) and inhibit the production of IL-6 and IL-8 from these cells upon induction of NF-κB by TNFα. Future perspective of this compound in the treatment of RA is discussed.
Materials and Methods
A relatively selective IKKβ inhibitor, CHPD (Ziegelbauer et al., 2005), was a kind gift from Dr. T. Murata (Bayer Yakuhin Inc., Kyoto, Japan). The chemical structure of CHPD is shown in Fig. 1a. Human recombinant TNFα was purchased from Roche Diagnostics (Mannheim, Germany) and used at 1.0 ng/ml for NF-κB stimulation. Antibodies for IκBα, p65, and α-tubulin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), whereas the antibody for phospho-IκBα (Ser32), JNK, phospho-JNK (Thr183/Tyr185), ERK1/2, and phospho-ERK1/2 (Thr202/Tyr204) was purchased from Cell Signaling Technology Inc. (Danvers, MA). Murine monoclonal TNFα antibody used in the neutralization assay (Fig. 1b) was obtained from R&D Systems (Minneapolis, MN) and used at 10 μg/ml. Horseradish peroxidase-conjugated secondary antibodies were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK).
RSF cultures were isolated from fresh synovial tissue biopsy samples from six RA patients at total knee arthroplasty or arthroscopic synovectomy as reported previously (Sakurada et al., 1996; Yoshida et al., 1999a,b). Diagnosis of RA was made according to the clinical criteria of the American College of Rheumatology (Arnett et al., 1988). These RA patients included five females and one male, from 37 to 63 years old, and all patients had active RA at various clinical stages and classes. The mean disease duration was 7.7 ± 6.7 years, with a range of 1.4 to 18.0 years. Clinical characteristics of each donor are shown in Table 1. None of the patients had been treated with any biologic agents such as infliximab and etanercept. Informed consents were obtained from each patient in conformity with the requirements of the Ethics Committee of the Nagoya City University Graduate School of Medical Sciences.
RSF cultures were performed as reported previously (Sakurada et al., 1996; Yoshida et al., 1999a,b). In brief, fresh synovial tissue biopsy samples were minced into small pieces and treated with 1 mg/ml collagenase/dispase (Roche Diagnostics) for 10 to 20 min at 37°C. The cells were cultured in Ham's F-12 medium (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.5 mM mercaptoethanol. The culture medium was changed every 3 to 5 days, and nonadherent lymphoid cells were removed during the initial passages. All the experiments were conducted using synoviocyte cultures during the third to eighth passages. To characterize the cytological phenotype, the cells were stained with mouse monoclonal antibodies against human HLA-DR, von Willebrand factor, desmin, smooth muscle actin, CD1a, CD68, and 5B5 (Dako Denmark A/S, Glostrup, Denmark). Only 5B5 was positive for RSF, indicating their fibroblast-like phenotype, consistent with our previous findings (Sakurada et al., 1996; Yoshida et al., 1999a,b). There was some heterogeneity in cell growth property of each RSF preparation; however, there was no quantitative difference that warrant aggressive nature of synovial cells. Human embryonic kidney 293 cells were grown at 37°C in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO) with 10% heat-inactivated fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin.
The cytokine concentrations were determined using cytokine-specific ELISA kits for IL-6 and IL-8 (Quantikine ELISA kits; R&D Systems) in RSF and 293 cells culture supernatant with experimental procedures recommended by the manufacturer. Triplicates were used for each test condition in the three independent cultures.
Cell Proliferation Assay.
To examine the cytotoxicity of CHPD, the cell proliferation of RSF upon treatment with CHPD at various concentrations was determined using WST-1 (Roche Diagnostics) according to the manufacturer's protocol. In brief, RSF cultures were incubated with CHPD in a 96-well plate for 24 h, incubated further for 4 h in the presence of WST-1, and the dissolved formazan was measured at 450 nm by spectrophotometry.
Reverse Transcription-Polymerase Chain Reaction.
To measure mRNA expression of various genes, 2.0 × 105 RSF cells were cultured at 37°C in a CO2 incubator, washed once with phosphate-buffered saline (PBS), and homogenized with QIAshredder (QIAGEN Operon, Alameda, CA), and total RNA was purified using RNeasy (QIAGEN, Valencia, CA) according to the manufacturer's protocol. After incubation with DNase I (Invitrogen), 1.0 μg of total RNA was reverse-transcribed using SuperScript first-strand synthesis system (Invitrogen). The cDNA was then amplified from each RNA sample with HotStarTaq Master Mix kit and gene-specific primers. The number of cycles was selected to allow linear amplification of the cDNA under study. The PCR products were analyzed by agarose gel electrophoresis. The oligonucleotide primers were as follows: il-6, sense 5′-TCT CAG CCC TGA GAA AGG AGA C-3′ and antisense 5′-GAA GAG CCC TCA GGC TGG ACT G-3′; il-8, sense 5′-GCA GCT CTG TGT GAA GGT GC-3′ and antisense 5′-TCC TTG GGG TCC AGA CAG AG-3′; β-actin, sense 5′-CCA GGC ACC AGG GCG TGA TG-3′ and antisense 5′-CGG CCA GCC AGG TCC AGA CG-3′; matrix metalloproteinase (mmp)-3, sense 5′-GGA GGA AAA CCC ACC TTA CAT AC-3′ and antisense 5′-AGT GTT GGC TGA GTG AAA GAG AC-3′; and vascular cell adhesion molecule (vcam)-1, sense 5′-GTC TGC ATC CTC CAG AAA TTC C-3′ and antisense 5′-TAA AAT CGA GAC CAC CCC-3′. The relative amount of each PCR product was quantified by densitometric scanner using ImageJ software (National Institutes of Health, Bethesda, MD; http://rsbweb.nih.gov/ij/download.html).
For detection and analyses of various proteins, RSF cells were maintained with or without CHPD at 37°C. These cells were washed once with ice-cold PBS and resuspended in 50 μl of lysis buffer containing 20 mM HEPES-NaOH, pH 7.9, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 5 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 0.2% Triton X-100, and Protease Inhibitors Cocktail (Roche Diagnostics). After 15 min of incubation on ice, the samples were centrifuged at 15,000 rpm for 10 min, and the supernatant was collected as “whole cell extract.” Protein concentration was measured using detergent-compatible protein assay (Bio-Rad Laboratories, Hercules, CA). Equal amounts of the proteins were electrophoresed on 10% SDS-polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA). The membranes were blocked with Tris-buffered saline with Tween 20 (TBS-T: 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween 20) containing 5% nonfat milk for 1 h at room temperature and then incubated with TBS-T containing 5% nonfat milk and 1:1000 diluted specific antibodies overnight at 4°C. After incubation, the membranes were rinsed three times with TBS-T and further incubated with horseradish peroxidase-conjugated secondary antibodies in TBS-T with 5% nonfat milk at room temperature for 1 h. Each protein was detected by chemiluminescence using SuperSignal chemiluminescent substrate (Pierce Chemical, Rockford, IL).
Transfection and Luciferase Assay.
The 293 cells (1.0 × 105/well) were transfected with reporter plasmids using FuGENE 6 transfection reagent (Roche Applied Science, Indianapolis, IN) according to the manufacturer's instructions. For each transfection, 0.03 μg of 4xκB-luc, where luciferase gene expression is under the control of NF-κB, and 0.01 μg of the internal control plasmid pRL-TK, expressing Renilla reniformis luciferase under the control of TK promoter, were used (Yang et al., 1999). Twenty-four hours after transfection, the cells were treated with CHPD for 30 min and stimulated with TNFα (1.0 ng/ml) for 24 h. The transfected cells were then harvested, and the extracts were subjected to luciferase assay using the Luciferase Assay system (Promega, Madison, WI). The luciferase activity was normalized with R. reniformis luciferase activity as an internal control to assess the transfection efficiency.
Chromatin immunoprecipitation (ChIP) assays were performed using ChIP assay kits (ChIP-IT Express; Active Motif Inc., Carlsbad CA) according to the protocol reported previously (Imai and Okamoto, 2006), with minor modifications. In brief, 1.0 × 106 RSF cells either with or without CHPD treatment or TNFα stimulation were cross-linked by adding formaldehyde to the medium (final concentration, 1%) and incubated at room temperature for 10 min. The cells were then washed with ice-cold PBS containing protease inhibitors, and the fixation reaction was stopped by adding 10 ml of “glycine stop-fix” solution. Samples were lysed for 30 min in lysis buffer on ice, and the chromatin was sheared by sonication 40 times for 30 s each time at the maximal power with 30 s of cooling on ice between each pulse with a sonicator (Bioruptor; COSMO Bio, Tokyo, Japan). Cross-linked and released chromatin fractions were immunoprecipitated with magnetic beads and specific antibodies on a rolling shaker overnight at 4°C. Cross-linking of the immunoprecipitates containing fragmented DNA was chemically reversed. Then, PCR was performed with a HotStarTaq Master Mix kit (QIAGEN). The PCR primers used for amplifying promoters containing the NF-κB binding sites included human il-8 promoter (−121 to +61), 5′-GGG CCA TCA GTT GCA AAT C-3′ and 5′-TTC CTT CCG GTG GTT TCT TC-3′; and human β-actin promoter (−980 to −915), 5′-TGC ACT GTG CGG CGA AGC-3′ and 5′-TCG AGC CAT AAA AGG CAA-3′. The relative amount of il-8 promoter DNA bound to p65 was quantified by densitometric scanner using ImageJ software that was downloadable from http://rsbweb.nih.gov/ij/download.html.
The statistical significances of difference were evaluated by the Steel-Dwass's test, with p < 0.05 considered statistically significant.
CHPD Inhibited the Spontaneous IL-6 Production from RSF.
To investigate the inhibitory effect of CHPD on the production of IL-6 upon stimulation of RSF with TNFα (1.0 ng/ml), involving NF-κB activation, cytokine concentration was measured in the cell culture supernatant 24 h after stimulation. As shown in Fig. 1b, the extent of spontaneous production of IL-6 from RSF was much higher than that from 293 cells (data not shown), a human epithelial cell line and down-regulated by CHPD but not by neutralizing antibody to TNFα. The extent of inhibition of the spontaneous production of IL-6 by 1.0 μM CHPD was 41%. In addition, a neutralizing antibody against IL-1β did not change the levels of IL-6 spontaneously produced from RSF (data not shown). The representative data with RSF1 are shown in Fig. 1b, and similar results were observed with other RSF cultures (data not shown). These data suggest that this spontaneous production of cytokines might be due to other factors involving IKKβ. In addition, we did not detect detectable levels of TNFα or IL-1β production in this study (data not shown), which we have reported previously (Sakurada et al., 1996).
CHPD Inhibited the IL-6 and IL-8 Production Induced by TNFα from RSF.
In Table 2, we have measured the levels of IL-6 and IL-8 production in the culture supernatant of RSF cultures obtained from six individual patients with RA. As shown, the extent of augmentation of production of inflammatory cytokines after 24-h TNFα stimulation were 1.8- to 7.1-fold and 19.6- to 35.7-fold for IL-6 and IL-8, respectively. The concentrations of spontaneous production of IL-6 without TNFα stimulation in the absence and the presence of 1 μM CHPD were 2.3 ± 0.9 and 1.8 ± 0.6 ng/ml, respectively. Similarly, we measured the extents of inhibition of production of these cytokines upon TNFα stimulation by CHPD (as indicated by IC50 values). We noted a slight heterogeneity in the responsiveness to the TNFα treatment and CHPD in these synovial cell cultures. It is interesting that when RSF cultures from six RA patients were pretreated with CHPD for 1 h before TNFα treatment, the extents of IL-6 and IL-8 induction were significantly reduced by CHPD in a concentration-dependent manner (Fig. 2, a and b). Although there was heterogeneity in the responsiveness to CHPD, a concentration-dependent suppression of cytokine production by CHPD was noted. IL-8 appeared to be preferentially inhibited by CHPD. The extents of inhibition by 1.0 μM CHPD of the TNFα-induced production of IL-6 or IL-8 were 55 (Fig. 2a) and 90% (Fig. 2b), on average, for IL-6 and IL-8, respectively. CHPD at these concentrations did not show significant cytotoxicity (Fig. 2c). These results indicate that CHPD suppressed the IL-6 and IL-8 cytokine levels that were induced by TNFα without significant inhibition of cell proliferation or cytotoxicity.
CHPD Inhibited TNFα-Induced il-6 and il-8 mRNA Expression.
To further examine whether CHPD suppresses the TNFα-induced gene expression of il-6 and il-8, we semiquantitatively detected the mRNA levels of il-6 and il-8 using reverse transcription-PCR. As demonstrated in Fig. 3a, the il-6 and il-8 mRNA expression levels in RSF were increased as early as 1 h until 16 to 24 h after TNFα stimulation (at 1.0 ng/ml) and then gradually decreased. When RSF was pretreated with 1.0 μM CHPD for 1 h before the treatment with TNFα, the extent of induction of il-6 and il-8 mRNA was significantly reduced by CHPD (Fig. 3b). These results indicate that CHPD could inhibit il-6 and il-8 mRNA synthesis that was induced by TNFα. In addition to il-6 and il-8, gene expressions of vcam-1 (Iademarco et al., 1992) and mmp-3 (Borghaei et al., 2004), also under the control of NF-κB, were induced by TNFα and effectively inhibited by CHPD.
CHPD Inhibited TNFα-Induced IκBα Phosphorylation and Degradation.
TNFα stimulation activates IKK complex and the activated IKKs induce IκBα phosphorylation, leading to ubiquitination and subsequent degradation of IκBα by the 26S proteasome (Okamoto, 2006). We then examined the inhibitory effect of CHPD on TNFα-induced IκBα phosphorylation and degradation by immunoblot analysis. As shown in Fig. 4a, TNFα stimulation caused IκBα phosphorylation (P-IκBα in Fig. 4), which was observed at 5 min after stimulation of RSF. However, treatment with CHPD reduced IκBα phosphorylation in a concentration-dependent manner (Fig. 4b). CHPD at 1.0 μM appeared to prevent the phosphorylation of IκBα in all of the RSF cultures tested (data not shown). We then proceeded to examine whether CHPD could block IκBα degradation as well. As also shown in Fig. 4, the IκBα protein committed nearly complete degradation in RSFs 15 min after TNFα stimulation and blocked by 1.0 μM CHPD. In contrast, α-tubulin levels were unchanged, indicating equal loading of proteins in the gel. Furthermore, although mitogen-activated protein kinase family members, including JNK and ERK, were reported to be activated by TNFα stimulation (Firestein, 2003), CHPD failed to inhibit these kinases (Fig. 4, c and d). In addition, although there are two JNK molecular species, p54 and p46, we could detect only p46 protein. The representative results with RSF3 are shown here, and similar results were observed with other RSF cultures (data not shown). These results suggest that CHPD exhibited abrogation in the TNFα-induced NF-κB activation through inhibition of IKKβ without inhibiting MAPK activities.
CHPD Inhibited Gene Expression Driven by TNFα and Analysis with ChIP Assay.
We then examined the effect of CHPD on NF-κB-dependent gene expression using luciferase assay with the 4xκB-luc reporter plasmid, where luc gene expression depends on NF-κB (Yang et al., 1999). Because RSF cultures were hardly susceptible to gene transfection even using various modifications of lipofection, a human cell line derived from embryonic kidney, 293 cells, that is more susceptible for gene transfection was used instead. As shown in Fig. 5a, TNFα stimulated expression of NF-κB-dependent gene such as 4xκB-luc by 17.0 ± 3.8-fold, which was similarly observed in TNFα-induced IL6/IL-8 production (Fig. 2; Table 2). When cells were pretreated with CHPD, however, the gene expression was inhibited in a concentration-dependent manner (Fig. 5a). Approximately 80% of this activity was inhibited at 2.0 μM CHPD.
To further confirm the nuclear translocation and promoter binding of NF-κB, we have adopted ChIP assay and examined the inhibitory effect of CHPD on the TNFα-induced activation of NF-κB-DNA binding. As shown in Fig. 5b, ChIP assays revealed that the DNA-binding of the p65 subunit of NF-κB in il-8 promoter was induced after 60 min of TNFα stimulation. When RSF was pretreated with CHPD for 1 h, the recruitment of NF-κB to the il-8 promoter was significantly inhibited. No amplification in the absence of p65 antibody nor NF-κB binding to β-actin promoter (internal control) was observed, confirming the specificity of the DNA immunoprecipitation and ChIP assays.
The intervention therapy using blocker of TNFα, IL-1β, and IL-6 has been developed and demonstrated remarkable therapeutic efficacies (Elliott et al., 1993; Moreland et al., 1997; Bresnihan et al., 1998; Nishimoto et al., 2004). These findings indicate that these proinflammatory cytokines, which eventually lead to activation of NF-κB, play crucial roles in the pathogenesis of RA among other signaling cascades (Firestein, 2003; Okamoto, 2006). However, these cytokine blockers currently used are considered “biologic agents” requiring intradermal or intravenous injections, inducing allergic reactions as well as adverse effects and consuming substantial medical resources. Thus, development of small-molecular-weight chemical compounds that share a similar molecular target is desperately needed.
The biological cascade involving NF-κB forms a positive feedback loop, or “vicious cycle,” that can perpetuate by itself and expand the inflammatory responses to other joints and tissues (Okamoto, 2006). Thus, inhibiting NF-κB signaling by blocking this cycle is considered to be a feasible treatment strategy for RA (Feldmann, 2001; Okamoto, 2006). There are multiple steps by which NF-κB is activated by extracellular signals: 1) binding of proinflammatory cytokines to their receptors; 2) signal transduction near the cytoplasmic membrane through signal transducers such as TNF receptor-associated death domain, TNF receptor-associated factor 2, and receptor-interacting protein; 3) upstream kinases including PI3K, Akt, and p38 mitogen-activated protein kinase; 4) the enzymatic activation of IKK; 5) proteasome-mediated IκB degradation; 6) nuclear transport; and 7) the DNA binding of the liberated NF-κB (Okamoto, 2006). In contrast, several specific NF-κB inhibitors have been developed with these signaling steps as targets. For example, dehydroxymethylepoxyquinomicin appears to inhibit the nuclear translocation of NF-κB (Wakamatsu et al., 2005), and bortezomib (originally named PS-341) was identified to inhibit proteasome (Adams et al., 1999). Among these target molecules, however, IKK appears to be specific for NF-κB activation and is the converging molecule of several distinct NF-κB-activating agents such as TNFα, IL-1β, B-cell-activating factor, lymphotoxin β, CD40, and Toll-like receptor signaling (Okamoto, 2006). In addition, there are several compounds exhibiting the IKK inhibition activity. As reported previously, ACHP, CHPD, and IMD-0560 inhibited IKKβ (Murata et al., 2004; Okazaki et al., 2005; Ziegelbauer et al., 2005) and the NF-κB essential modulator-binding peptide could dissociate IKKγ from the IKKα-IKKβ complex, thus inhibiting IKK activity (Jimi et al., 2004). Among these IKK inhibitors, CHPD appears to have higher specificity to IKKβ (in vitro IC50 values for IKKβ and IKKα were 2 and 135 nM, respectively, whereas other kinases, including IKKγ, mitogen-activated protein kinase kinase 4, mitogen-activated protein kinase kinase 7, ERK-1, Syk, Lck, Fyn, PI3Kγ, PKA, and PKC were not inhibited at >10 μM; Ziegelbauer et al., 2005). Furthermore, CHPD inhibited the TNFα-induced NF-κB activation in 293 cell cultures without significant inhibition of cell proliferation.
In cell culture experiments, IC50 and CC50 values were 0.27 to 0.51 and 47.5 μM, respectively, with a therapeutic window of 93 to 176. However, considering the significant difference from the IC50 value of IKKβ inhibition in vitro and in vivo (Ziegelbauer et al., 2005), we suggest that this compound needs further modification for efficient incorporation into human cells. It was also shown that CHPD could inhibit the lipopolysaccharide-induced production of TNFα in mice when administered orally without any systemic side effects such as weight loss and lethargy (Ziegelbauer et al., 2005). In addition, CHPD did not exert any inhibitory effect on JNK and ERK in RSF (Fig. 4d) and activator protein-1 in normal human lung fibroblast MRC5 (Ziegelbauer et al., 2005). These characteristics indicate that CHPD is one of the feasible candidates for therapeutic IKKβ inhibitor, although further improvements are needed as therapeutic compounds. Because the inhibitory effects of CHPD were observed with synovial cell cultures that showed relatively rapid cell growth ex vivo, further studies in this area are warranted.
As discussed previously, IKKβ represents the major effector kinase in the canonical pathway of NF-κB activation, IKKα on the other hand is primarily involved in the noncanonical pathway (Okamoto, 2006). Although IKKβ knockout mice exhibited early embryonic death because of the massive apoptosis in liver (Li et al., 1999a), gene knockout of IKKα showed only impairment in skin and digit abnormality such as syndactyly (Hu et al., 1999). Moreover, recent experimental evidences (Enzler et al., 2006; Okamoto, 2006) have indicated the crucial importance of IKKα in autoimmunity. However, considering the cross-talk between the canonical and noncanonical pathways, such as phosphorylation of p65 at Ser536 and subsequent induction of the transcriptional activity of NF-κB (Jiang et al., 2003a,b), application of IKKβ inhibitor for the treatment of rheumatic diseases should be considered.
We noticed the production of inflammatory cytokines such as IL-6 from rheumatoid synovial cell cultures without stimulation with TNFα (Fig. 1b). Because neutralizing antibodies to TNFα and IL-1β did not block but CHPD did block the production of these cytokines, other factors that can stimulate the NF-κB cascade involving IKKβ, such as oxidative or environment stress and irradiation, are considered to be involved in such background activity. In addition, we could not find any correlation between the clinical characteristics and the extent of TNFα-mediated cytokine production or the responsiveness to CHPD. Similar findings were reported by Miyazawa et al. (1998) where constitutive IL-6 production was observed without any stimulation in rheumatoid synoviocytes from 11 RA patients, whereas no such background production of inflammatory cytokines was detected in dermal fibroblasts and synoviocytes from osteoarthritis.
In conclusion, NF-κB activation in synovial cells and production of inflammatory cytokines by proinflammatory cytokines such as TNFα and IL-1β play a crucial role in rheumatoid arthritis. Our observations clearly indicate that the IKK inhibitors such as CHPD have therapeutic efficacy in the inflammatory processes associated with rheumatoid arthritis. Further development of IKK inhibitors are needed for the development of feasible and affordable drug therapy against RA.
We thank Marni Cueno for critical reading of the manuscript.
- Received July 15, 2009.
- Accepted January 5, 2010.
This work was supported in part by the Aichi D.R.G. Foundation.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- rheumatoid arthritis
- tumor necrosis factor
- nuclear factorκB
- IκB kinase complex
- 2-amino-6-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile
- 7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one hydrochloride
- rheumatoid synovial fibroblast
- c-Jun NH2-terminal kinase
- extracellular signal-regulated kinase
- enzyme-linked immunosorbent assay
- 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate
- phosphate-buffered saline
- polymerase chain reaction
- matrix metalloproteinase
- vascular cell adhesion molecule
- Tris-buffered saline with Tween 20
- chromatin immunoprecipitation
- mitogen-activated protein kinase kinase
- phosphatidylinositol 3-kinase
- protein kinase
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics