inhibition of inflammatory cytokine production from...

1 JPET#158899

Inhibition of Inflammatory Cytokine Production from Rheumatoid Synovial Fibroblasts

by a Novel IκB Kinase Inhibitor

Atsushi Tsuchiya, Kenichi Imai, Kaori Asamitsu, Yuko Waguri-Nagaya, Takanobu Otsuka

and Takashi Okamoto*

Departments of 1 Molecular and Cellular Biology (AT, KI, KA, TO), and 2 Orthopedic Surgery

(AT, YW-N, TO), Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi,

Mizuho-cho, Mizuho-ku, Nagoya, Aichi 467-8601, Japan

JPET Fast Forward. Published on January 6, 2010 as DOI:10.1124/jpet.109.158899

Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on January 6, 2010 as DOI: 10.1124/jpet.109.158899

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Running Title: A new IKK inhibitor inhibiting cytokine production

*Corresponding Author: Takashi Okamoto, M.D., Ph.D.

Address: Department of Molecular and Cellular Biology, Nagoya City University

Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya,

Aichi 467-8601, JAPAN

E-mail: [email protected]

Tel: +81-52-853-8204; FAX: +81-52-859-1235

Number of text pages: 20

Number of Tables: 2

Number of figures: 5

Number of references: 40

Number of words in abstract: 245

Number of words introduction: 744

Number of words in discussion: 916

List of non-standard abbreviations: ACHP, 2-amino-6-[2-(cyclopropylmethoxy)- 6-

hydroxyphenyl]-4-piperidin-4-yl nicotinonitrile; ATL, adult T-cell leukemia; ChIP, chromatin

immunoprecipitation; CHPD, (7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3S)-3-

piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one hydrochloride); ELISA, enzyme-

linked immunosorbent assay; IKK, IκB kinase; IL,interleukin; MKK, mitogen-activated

protein kinase kinase; MMP, matrix metalloproteinase; NF-κB, nuclear factor kappa B; PBS,

phosphate-buffered saline; PI3K, phosphatidylinositol 3-kinase; RA, rheumatoid arthritis;

RSF, rheumatoid synovial fibroblasts; RT, reverse transcription; TBS-T, Tris-buffered saline

with Tween 20; TNFα, tumor necrosis factor α; VCAM, vascular cell adhesion molecule.

Section option: Inflammation, Immunopharmacology


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Abstract

Nuclear factor kappa B (NF-κB) is involved in 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 proinflammatory

cytokines, TNFα and IL-1β. TNFα and IL-1β are known to stimulate NF-κB signalling and

produced as the effect of NF-κB signalling, thus forming a vicious cycle leading to a self-

perpetuating nature of rheumatoid inflammation and expansion of such inflammatory

response to other joints. Since a kinase called IκB kinase (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 extents of IL-6 and IL-8 induction were dramatically

reduced by CHPD under non-cytotoxic concentrations. Similarly, expression of il-6 and il-8

genes was significantly reduced by CHPD. In addition, ChIP assays revealed that the DNA-

binding of NF-κB (p65) to il-8 promoter in RSF was induced after TNFα stimulation, and

upon CHPD treatment to RSF for 1 h the NF-κB binding to il-8 promoter was significantly

decreased. In this paper, 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 IKKβ inhibitor could be a

feasible candidate of anti-rheumatic drug.


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Introduction

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 immuno-inflammatory cells including T cells, macrophages and plasma cells

(Firestein, 2003) leads to progressive destruction of cartilage and bone. Various cytokines,

including interleukin-1β (IL-1β), tumor necrosis factor α (TNFα), 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 kappa B (NF-κB) (Feldmann,

2001; Okamoto, 2005; Okamoto, 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 while TNFα and IL-1β stimulate NF-κB signalling, forming a vicious

cycle that can perpetuate and expand the inflammatory responses. Thus, blocking this

cascade by inhibiting NF-κB signalling is considered feasible for the treatment of RA.

Interestingly, 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 cysteins 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;


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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 molecular compounds.

NF-κB is a hetero- or homodimer consisting of Rel family proteins, p65 (RelA), RelB,

c-Rel, p50/p105 and p52/p100, and 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 (IKK) complex (Nakano et al.,

1998), which consists of three subunits, IKKα, IKKβ and IKKγ/NEMO. Activated IKK

complex, mainly through the IKKβ activity (Mercurio et al., 1997; Zandi et al., 1997; Li et al.,

1999b; Karin and Delhase, 2000), phosphorylates IκBα leading to ubiquitination and

degradation of IκB. NF-κB, then, 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 signalling

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 leukaemia (ATL)

(Mori et al., 1999; Sanda et al., 2005; Sanda et al., 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) upregulation of various

target genes that are responsible for inhibition of apoptosis. Moreover, a specific IKK

inhibitor, ACHP (2-amino-6-[2-(cyclopropylmethoxy)- 6-hydroxyphenyl]-4-piperidin-4-yl

nicotinonitrile) (Murata et al., 2004), could inhibit cell growth and induce apoptosis of

multiple myeloma and ATL cells (Sanda et al., 2005; Sanda et al., 2006) by inhibiting the

phosphorylation of IκBα and p65.

In this study, we examined the effects of a novel IKK inhibitor, CHPD (7-[2-

(cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-


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d][1,3]oxazin-2-one hydrochloride) (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. (Murata et al., 2004) based on a massive screening while 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 (RSF) 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.


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Methods

Reagents:

A relatively selective IKKβ inhibitor, CHPD (7-[2-(cyclopropylmethoxy)-6-

hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one

hydrochloride)(Ziegelbauer et al., 2005) was a kind gift from Dr. T. Murata of Bayer Yakuhin

Inc. (Kyoto, Japan). The chemical structure of CHPD is shown in Fig. 1a. Human

recombinant TNFα was purchased from Roche (Mannheim, Germany) and used at 1.0 ng/ml

for NF-κB stimulation. Antibodies for IκBα (sc-371), p65 (sc-372) and α-tubulin (sc-8035)

were obtained from Santa Cruz Biotechnology (Santa Cruz, CA), while the antibody for

phospho-IκBα (Ser32) (#9241L), JNK (#9252), phospho- JNK (Thr183/Tyr185) (#9251),

ERK1/2 (#9102) and phosphor-ERK1/2 (Thr202/Tyr204) (#9191) was purchased from Cell

Signaling Technology (Beverly, MA). Murine monoclonal TNFα antibody (MAB610) used

in the neutralization assay (Fig. 1b) was commercially obtained (R&D Systems, Minneapolis,

MN) and used at 10 μg/ml. Horseradish peroxidase-conjugated secondary antibodies were

obtained from Amersham Biosciences (Little Chalfont, United Kingdom).

Patients:

RSF cultures were isolated from fresh synovial tissue biopsy samples from six RA

patients at total knee arthroplasty or arthroscopic synovectomy as previously reported

(Sakurada et al., 1996; Yoshida et al., 1999a; Yoshida et al., 1999b). 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 63years old,

and all patients had active RA at various clinical stages and classes. The mean disease


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duration was 7.7 ± 6.7 year with a range of 1.4-18.0 years. Clinical characteristics of each

donor are shown in Table1. 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.

Cell culture:

RSF cultures were performed as previously reported (Sakurada et al., 1996; Yoshida et

al., 1999a; Yoshida et al., 1999b). Briefly, fresh synovial tissue biopsy samples were minced

into small pieces and treated with 1 mg/ml collagenase/dispase (Roche Diagnostics GmbH,

Mannheim, Germany) for 10-20 min at 37 °C. The cells were cultured in F-12 (HAM)

(Invitrogen Co.) supplemented with 10% heat-inactivated fetal bovine serum, 100 U/ml of

penicillin, 100 μg/ml of streptomycin and 0.5 mM mercaptoethanol. The culture medium was

changed every 3-5 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, 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; Yoshida et al., 1999b). 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) with

10% heat-inactivated fetal bovine serum, 100 U/ml of penicillin and 100 μg/ml of

streptomycin.


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Cytokine assays:

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:

In order 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.

RT-PCR:

To measure mRNA expression of various genes, 2.0 x 105 RSF cells were cultured at

37 °C in CO2 incubator, washed once with PBS, homogenized with QIAshredder (Qiagen,

Alameda, CA), and total RNA was purified using RNeasy (Qiagen) according to

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


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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’;

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 Image J software.

(http://rsbweb.nih.gov/ij/download.html).

Immunoblot analyses:

For detection and analyses of various proteins, RSF cells were maintained with or

without CHPD at 37 °C. These cells were washed once with cold phosphate-buffered saline

(PBS) and resuspended in 50 μl of lysis buffer containing 20 mM HEPES-NaOH (pH7.9),

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, Hercules, CA). Equal amounts of the proteins

were electrophoresed on 10% SDS-polyacrylamide gel and transferred onto a polyvinylidene

fluoride membrane (Millipore Corporation, MA). The membranes were blocked with Tris-

buffered saline with Tween 20 (TBS-T) (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.1%

Tween 20) containing 5% nonfat milk for 1 h at room temperature, and incubated with TBS-T

containing 5% nonfat milk and 1:1,000 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 chemi-luminescence using

SuperSignal (Pierce, Rockford, IL).


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Transfection and luciferase assay:

The 293 cells (1.0 x 105/well) were transfected with reporter plasmids using FUGENE

6 transfection reagent (Roche Applied Science) according to the manufacturer’s instructions.

For each transfection, 0.03 μg of 4κB-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

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). The luciferase

activity was normalized with Renilla luciferase activity as an internal control to assess the

transfection efficiency.

ChIP assays:

Chromatin immunoprecipitation (ChIP) assays were performed using ChIP assay kits

(ChIP-ITTM Express; Active Motif, Carlsbad CA) according to the protocol as previously

reported (Imai and Okamoto, 2006) with minor modifications. In brief, 1.0 x 106 RSF cells

either with or without CHPD treatment or TNFα stimulation were cross-linked by adding

formaldehyde to the medium (1% final concentration) and incubated at room temperature for

10 min. The cells were then washed with cold PBS containing protease inhibitors and the

fixation reaction was stopped by adding 10 ml “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 maximum 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 immumoprecipitated 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


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(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 Image J software that was downloadable from (http://rsbweb.nih.gov/ij/download.html).

Statistical analysis:

The statistical significances of difference were evaluated by the Steel-Dwass’s test with

p<0.05 considered statistically significant.


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Results

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

downregulated 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 6 individual patients with RA. As

shown, the extent of augmentation of production of inflammatory cytokines after 24 h

TNFα stimulation were 1.8-7.1 fold and 19.6-35.7 fold for IL-6 and IL-8, respectively.

The concentrations of spontaneous production of IL-6 without TNFα stimulation in


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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. Interestingly, when RSF cultures from 6 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.2a, 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) in 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 semi-quantitatively detected the mRNA levels of il-6

and il-8 using RT-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 were significantly reduced by CHPD (Fig. 3b). These results

indicate that CHPD could inhibit il-6 and il-8 mRNA synthesis that were induced by


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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”),

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 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 MAPK family members

including JNK and ERK were reported to be activated by TNFα stimulation (Firestein,

2003), CHPD failed to inhibit these kinases (Fig. 4c, 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.


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CHPD inhibited gene expression driven by TNFα and analysis with chromatin

immunoprecipitation (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). Since 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, were utilized 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 as in the case of 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 of 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 immuno-

precipitation and ChIP assays.


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Discussion

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 signalling 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 which 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 signalling by

blocking this cycle is considered to be a feasible treatment strategy of 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

TRADD, TRAF2 and RIP; (3) upstream kinases including Phosphatidylinositol 3-

kinase (PI3K), Akt and p38 mitogen-activated protein kinase; (4) the enzymatic

activation of IKK complex; (5) proteasome mediated IκB degradation; (6) nuclear

transport; and (7) the DNA-binding of the liberated NF-κB (Okamoto, 2006). On the

other hand, a number of specific NF-κB inhibitors have been developed with these


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signalling steps as targets. For example, dehydroxymethylepoxyquinomicin

(DHMEQ) appears to inhibit the nuclear translocation of NF-κB (Wakamatsu et al.,

2005) and PS341 (Bortezomib) was identified to inhibit proteasome (Adams et al.,

1999). Among these target molecules, however, IKK complex appears to be specific

for NF-κB activation and is the converging molecule of a number of distinct NF-κB-

activating agents such as TNFα, IL-1β, B-cell activating factor, lymphotoxin β, CD40

and Toll-like receptor signalling (Okamoto, 2006). In addition, there are a number of

compounds exhibiting the IKK inhibition activity. As previously reported, 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 nM and 135 nM, respectively,

whereas other kinases including IKKγ, MKK4, MKK7, ERK-1, Syk, Lck, Fyn, PI3Kγ,

PKA and PKC were not inhibited at over 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 0.27-0.51 μM and 47.5

μM, respectively, with a therapeutic window of 93-176. 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


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(Ziegelbauer et al., 2005). In addition, CHPD did not exert any inhibitory effect on

JNK and ERK in RSF (Fig. 4d) and AP-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. Since 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 previously discussed, IKKβ represents the major effector kinase in the

canonical pathway of NF-κB activation, IKKα on the other hand is primarily involved

in the non-canonical 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 crosstalk between the canonical

and non-canonical pathways, such as phosphorylation of p65 at Ser536 and

subsequent induction of the transcriptional activity of NF-κB (Jiang et al., 2003a;

Jiang et al., 2003b), 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). Since

neutralizing antibodies to TNFα and IL-1β did not 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


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20 JPET#158899

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. (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 plays

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.


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Acknowledgements

We acknowledge Mr. Marni Cueno for critical reading of the manuscript.


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Footnotes: This work was supported in part by Aichi D.R.G. Foundation (no grant number is

provided).


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Legends for Figures

Figure 1. Inhibition of the spontaneous IL-6 production by CHPD in rheumatoid

synovial fibroblasts.

(a) Structure of CHPD. IC50 values for various kinases are described. “Other kinases”

include IKKγ, MKK4, MKK7, ERK1, Syk, Lck, Fyn, PI3Kγ, PKA and PKC (28). Mw,

molecular weight (Daltons). (b) Amounts of IL-6 produced in the absence of TNFα. RSF

cells were cultured with or without 1.0 μM CHPD and 10 μg/ml TNFα antibody, and the

concentrations of IL-6 in the culture supernatant were measured using ELISA. The symbol

“-” indicates that only DMSO was added in all the experiments of Figs. 1, 2, 3, 4 and 5. Anti-

TNFα, anti-TNFα antibody.

Figure 2. Inhibition of the TNFα induced IL-8 and IL-6 production by CHPD in

rheumatoid synovial fibroblasts.

(a) Summary of IL-6 production by TNFα and its inhibition by CHPD. Amounts of IL-6

produced in the presence of TNFα as in Fig. 1b. RSF cell cultures from six patients with RA

were individually stimulated with 1.0 ng/ml TNFα in the presence or absence of CHPD and

the concentrations of IL-6 in the culture supernatant were measured using ELISA. (b)

Summary of IL-8 production by TNFα and its inhibition by CHPD. Amounts of IL-8

produced in the presence of TNFα. RSF cell cultures were stimulated with TNFα in the

presence or absence of CHPD. The concentrations of IL-8 in the culture supernatant were

measured. (c) Cell proliferation of CHPD on RSF cultures. Cell proliferation of three RSF

cultures in the presence of various concentrations of CHPD were determined by the WST-1

method. The mean 50% cytotoxic concentration (CC50 value) of 47.5 μM was extrapolated

from this measurement.


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Figure 3. Inhibitory effects of CHPD on NF-κB-driven gene expression.

(a) Time course of il-6 and il-8 mRNA expression induced by 1.0 ng/ml TNFα stimulation.

The total RNA was prepared from RSF cultures and examined for expression of the NF-κB-

dependent genes such as il-6 and il-8. The relative amounts of PCR products were quantified

by densitometric scanning using Image J software. (b) Effects of CHPD on the expression of

il-6, il-8, mmp-3, vcam-1 genes induced by 1.0 ng/ml TNFα. RSF cultures were stimulated

by TNFα for 16 h with or without pre-treatment of 1.0 μM CHPD that was added 1 h before.

The representative data with two independent RSF cultures are shown.

Figure 4. Inhibition of TNFα induced IκBα phosphorylation and degradation by CHPD.

(a) Time course of IκBα phosphorylation and its degradation induced by TNFα stimulation.

RSF cultures were stimulated by 1.0 ng/ml TNFα for indicated time. Whole cell extracts

were prepared and subjected to immunoblotting with the indicated antibodies. (b) Inhibition

of IκBα phosphorylation and its degradation by CHPD. RSF were stimulated by TNFα for

indicated time with or without pre-treatment of various concentrations of CHPD. Whole cell

extracts were prepared and subjected to immunoblotting. The IκBα proteins were visualized

by immunoblotting with anti-IκBα and anti-phospho-IκBα (Ser32) antibodies. P-IκBα, IκBα

proteins phosphorylated at Ser32. (c) Time course of JNK and ERK phosphorylation upon

stimulation with TNFα. RSF cultures were stimulated by 1.0 ng/ml TNFα for indicated time.

Whole cell extracts were prepared and subjected to immunoblotting with the indicated

antibodies. (d) Inhibition of JNK and ERK phosphorylation by CHPD. RSF were stimulated

by TNFα for 15 minutes with or without pre-treatment of various concentrations of CHPD.

Whole cell extracts were prepared and subjected to immunoblotting. The JNK and ERK


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30 JPET#158899

proteins were visualized by immunoblotting with specific antibodies. P-JNK, JNK proteins

phosphorylated at Thr183/Tyr185; P-ERK, ERK proteins phosphorylated at Thr202/Tyr204.

The representative data with RSF3 are shown in Fig. 4c and d and similar results were

observed with other RSF cultures (data not shown).

Figure 5. Luciferase and chromatin immunoprecipitation (ChIP) assays.

(a) Effects of CHPD on the NF-κB dependent gene expression. 293 cells were tansfected

with the 4κB-luc reporter plasmid together with the internal control plasmid, pRL-TK,

expressing Renilla luciferase. Stimulation with 1.0 ng/ml TNFα was carried out 24 h after the

transfection. CHPD was added 1 h before the treatment with TNFα. (b) RSF-1 culture was

stimulated with 1.0 ng/ml TNFα and the p65 bound to the κB site within the il-8 promoter

was detected by ChIP assay. It is noted that preincubation of cells with 1.0 μM CHPD 1 h

before the TNFα stimulation suppressed the p65 (NF-κB)-DNA binding. The experimental

details are described in Materials and Methods. Similar observations were obtained

reproducible at least for three times. Representative results are shown. The relative amount

of il-8 promoter DNA bound to p65 was quantified by densitometric scanner using Image J

software. n.d., not detectable.


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Table 1 - Clinical characteristics of six RSF donors.

Donor Sex Age Disease duration Class Stage Medication

RSF1 female 56 16y IV 4 PSL, SASP, DS

RSF2 female 51 3y8mo I 4 DS

RSF3 female 37 15y II 4 SASP, MTX

RSF4 female 63 2y5mo III 4 SASP

RSF5 male 57 1y3mo II 2 Bu

RSF6 female 54 14y II 4 Dex, Ind

RSF, rheumatoid synovial fibroblasts; PSL, prednisolone; SASP, salazosulfapyridine; DS,

diclofenac sodium; MTX, methotrexate; Bu, bucillamine; Dex, dexamethasone; Ind,

indomethacin.


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Table 2 - Effects of CHPD on production of inflammatory cytokines.

RSF cell cultures from six patients with RA were individually stimulated with 1.0 ng/ml

TNFα in the presence or absence of CHPD and the concentrations of IL-6 and IL-8 in the

culture supernatant were measured using ELISA (n=6).

IL-6 IL-8

Basal level (ng/ml) 2.3 ± 0.9 0.58 ± 0.25

Induced level (ng/ml) 8.8 ± 3.2 16 ± 6.1

Fold induction 4.2 ± 1.7 28 ± 5.3

CHPD IC50 (μM) 0.51 ± 0.20 0.27 ± 0.16


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Fig.1a, b

7-[2-(cyclopropylmethoxy)-6-hydroxyphenyl]-5-[(3S)-3-piperidinyl]-1,4-dihydro-2H-pyrido[2,3-d][1,3]oxazin-2-one hydrochloride (CHPD) (Mw.=431.92)

IC50 = 2 nM (IKKβ)IC50 = 135 nM (IKKα)IC50 > 10 μM (other kinases)

a

OH

O

O

O

N NH

NH HCl

b

CHPD - +- +-

-

anti-TNFα ++

RSF1

IL-6

(ng

/ml)

0

10

5


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CHPD (μM)

Cel

l pro

lifer

atio

n(%

)

RSF1RSF2RSF3 CC50= 47.5μM

0

20

40

60

80

100

120

0 0.5 1 2 4 8 16 32 64 128

Fig.2a, b, c

aIL

-6 (f

old

ind

uct

ion

)

CHPD (μM) - -TNFα - - + + +

1 0.2 1

p<0.05

p<0.05

0123

56

4

7

b

CHPD (μM) - -TNFα - - + + +

1 0.2 1

IL-8

(fo

ld in

du

ctio

n)

0

30

20

p<0.05

p<0.05

p<0.05n.s.

c

10

n.s.

p<0.05


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Fig. 3a, b

0 1 3 16 24 48

il-6

(1) (3.4) (7.6) (16.5) (20.3) (18.5)

(1) (4.8) (10.8) (14.6) (11.7) (5.3)

a

Time (h) afterTNFα stimulation

il-8

β-actin

(fold)

(fold)

TNFαCHPD

RSF1

il-6

b- - + +- + - +

RSF2

β-actin

il-8

- - + +- + - +

mmp-3

vcam-1

RSF1


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0 5 15 30 60 120TNFα (min) 0 5 15 30 60 120

RSF1 RSF3

IκBα

a

P-IκBα

α-tubulin

Fig. 4a, b, c, d

c d

TNFα - + + + + +

- - 0.01 0.1 1 10

RSF1

IκBα

P-IκBα

α-tubulin

α-tubulin

b

CHPD (μM)

5min

15min

RSF3

- + + + + +

- - 0.01 0.1 1 10

0 5 15 30 60 120p54

p46

TNFα (min)

P-JNK

P-ERK

- + + + +

- - 0.01 0.1 1 p54

p46

p54

p46

TNFα

P-ERK

P-JNK

ERK

JNK

CHPD (μM)

α-tubulin

α-tubulin


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Fig.5a, b

aκB-luc

Rel

ativ

e lu

c ac

tivi

ty (

fold

)

0

5

10

15

20

25

CHPD (μM) - 0.5 1 2TNFα - - - -

- 0.5 1 2+ + + +

TNFαCHPD

p65

- + +- - +

(1.0) (16) (8.3)

β-actinil-8

(fold)

No antibody

Input (1/10)

n.d. n.d. n.d.

- + +- - +

bRSF1


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