After obtaining informed consent, synovial tissues were collected from RA patients who met the 1987 American College of Rheumatology criteria for the diagnosis of RA and who were undergoing therapeutic joint surgery. FLSs were isolated as follows. Tissues were digested with gentle shaking in 20 ml RPMI 1640 (Gibco-BRL, Grand Island, NY, USA) containing 1 mg/ml collagenase (Gibco-BRL) at 37°C for 90 min, filtered through a 70 μm cell strainer and cultured in 75 cm² culture flasks with Dulbecco's modified essential medium (Gibco-BRL) supplemented with 20% (vol/vol) foetal bovine serum (Gibco-BRL) and 1× antibiotic-antimycotic (Gibco-BRL). After the cells had grown to confluence, they were detached with 0.25% trypsin (Gibco-BRL) and split at a 1:4 ratio. FLS passages three to six were used for all experiments. Visual examination of cell morphology under light microscopy and fluorescence activated cell sorting analysis of cells stained with anti-CD11b antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) confirmed that FLSs accounted for more than 95% of the cells.

TauCl was synthesized by mixing equimolar amounts of sodium hypochlorite (Aldrich Chemical, Milwaukee, MI, USA) and taurine (Sigma, St. Louis, MO, USA). TauCl formation was verified by UV absorption (200 to 400 nM) 27. Endotoxin-free or low-endotoxin grade water and buffers were used. Stock solutions of taurine and TauCl were kept at 4°C and used within 3 days.

TRIzol^® reagent (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from arthritic FLSs (2.5 × 10⁵ cells/60-mm dish/2 ml serum-free media) that had been starved in serum-free media overnight and treated with IL-1β for 6 hours in the presence or absence of TauCl. Complementary DNA was synthesized from 1 μg total RNA in 20 μl reverse transcription reaction mixture containing 5 mmol/l MgCl₂, 1× RT buffer, 1 mmol/l dNTP, 1 U/μl RNase inhibitor, 0.25 U/μl AMV reverse transcriptase, and 2.5 μmol/l random 9-mers. For semi-quantitative PCR, aliquots of cDNA were amplified with the primers in a 25 μl PCR mixture containing 1× PCR buffer, 0.625 units of TaKaRa Ex Taq™ HS, and 0.2 μmol/l of specific upstream primers, in accordance with the manufacturer's protocol (TaKaRa Bio, Kyoto, Japan). The PCR conditions for the MMPs were as follows: 30 to 33 cycles at 95°C for 45 s, 55 to 60°C for 45 s, and 72°C for 45 s. PCR products were subjected to electrophoresis in 1.5% agarose gels containing ethidium bromide, and the bands were visualized under UV light. The primers were synthesized by Bioneer Co. Ltd (Seoul, Republic of Korea), and their sequences are listed in Table 1.

For real-time quantitative PCR analysis, the reaction was carried out using the LightCycler PCR system (Roche Diagnostics, Meylan, France), with the DNA-binding SYBR Green I dye used to detect the PCR products. A serial dilution was used to generate each standard curve. Each 20 μl reaction mixture contained 1× LightCycler-DNA Master SYBR Green I, a specific primer, along with 2 μl cDNA. After 2 min denaturation at 95°C, the MMPs and β-actin underwent 40 reaction cycles at 95°C for 5 s, 55 to 60°C for 10 s (annealing) and 72°C for 13 s. Product specificity was determined by melting curve analysis, as described in the LightCycler manual. The results are expressed as ratios of MMP transcripts to β-actin transcripts, with the quantity of transcripts in each sample expressed as a copy number. The ratio of MMP/β-actin mRNA was assigned a value of 100%, with the corresponding results calculated as relative percentages.

The levels of MMP-1 and MMP-13 secreted in the culture media by IL-1β stimulated FLSs (5 × 10⁵ cells/60-mm dish/2-ml serum-free media) in the presence or absence of TauCl were measured by ELISA (R&D Systems, Inc., Minneapolis, MN, USA).

FLSs (5 × 10⁵ cells) cultured in 60-mm dishes were serum starved overnight and stimulated by IL-1β (10 ng/ml) for 10 or 30 min in the presence or absence of TauCl. The cells were subsequently washed twice in phosphate-buffered saline (PBS) and treated with 50 μl lysis buffer (20 mmol/l Tris-Cl [pH 8.0], 150 mmol/l NaCl, 1 mmol/l EDTA, 1% Triton X-100, 20 μg/ml chymostatin, 2 mmol/l phenylmethylsuphonyl fluoride [PMSF], 10 μmol/l leupeptin, and 1 mmol/l 4-[2-aminoethyl]benzenesulfonyl fluoride). Cells were scraped using a rubber policeman before addition of another 50 μl lysis buffer. The cells were transferred to a microcentrifuge tube, incubated on ice for 30 min with occasional agitation every 5 min and centrifuged for 15 min at 12,000 rpm (16,090 g), and the supernatant was then analyzed for protein concentration using the Bio-Rad Protein Assay Kit (Bio-Rad, Hercules, CA, USA). Thirty micrograms of cytoplasmic protein extract were then boiled in 5× Laemmli sample buffer for 5 min. The samples were separated by 12% SDS-PAGE and transferred to a Hybond-ECL membrane (Amersham, Arlington Heights, IL, USA). The membranes were blocked with 6% nonfat milk dissolved in TBST buffer (10 mmol/l Tris-Cl [pH 8.0], 150 mmol/l NaCl, 0.05% Tween 20). The blots were probed with various rabbit polyclonal antibodies for phosphorylated extracellular signal regulated kinase-1/2 (phospho-ERK-1/2), phospharylated p38 (phospho-p38), phospharylated c-jun amino-terminal kinase (phospho-JNK), and inhibitor of nuclear factor-κB (IκB)α (Cell Signaling Technology, Beverly, MA, USA) diluted 1:1000 in Tris-buffered saline for 2 hours and incubated with 1:1000 dilutions of goat anti-rabbit IgG secondary antibody, coupled with horseradish peroxidase. The blots were developed using the ECL method (Amersham). For re-probing, the blots were incubated in the stripping buffer (100 mmol/l 2-mercaptoethanol, 2% SDS, 62.5 mmol/l Tris-HCl [pH 6.7]) at 50°C for 30 min with occasional agitation.

FLSs (2 × 10⁶ cells) were seeded in 100-mm dishes and cultured for 2 days. The cells were kept in serum-free medium overnight and pretreated with TauCl 30 min before IL-1β (10 ng/ml) stimulation for 90 min. The cells were then washed with cold PBS, and nuclear extracts were prepared by cell lysis followed by nuclear lysis. In brief, cells were suspended in 400 μl of buffer A (10 mmol/l HEPES [pH 7.9], 1.5 mmol/l MgCl₂, 10 mmol/l KCl, 0.5 mmol/l DTT, 1 μmol/l leupeptin and 0.2 mmol/l PMSF) and vortexed for 15 s. After incubation for 20 min at 4°C, the lysates were centrifuged at 10,000 g for 6 min. The unclear pellet was re-suspended in buffer B (20 mmol/l HEPES [pH 7.9], 25% glycerol, 420 mmol/l NaCl, 1.5 mmol/l MgCl₂, 0.2 mmol/l EDTA, 0.5 mmol/l DTT, 1 μmol/l leupeptin and 0.2 mmol/l PMSF), incubated on ice for 40 min and centrifuged at 10,000 g for 20 min. Protein concentrations were determined using the Bradford method (Bio-Rad).

The protein-DNA binding activity in nuclear factor-κB (NF-κB) was determined using electrophoretic mobility shift assay (EMSA). In brief, 10 μg nuclear protein was incubated with 0.25 μg of poly(dI-dC) (Amersham) and ³²P-labelled DNA probe (5,000 counts per minute) in binding buffer (10 mmol/l Tris-HCl [pH 7.5], 50 mmol/l NaCl, 1 mmol/l MgCl₂, 0.5 mmol/l EDTA, 5% glycerol and 0.5 mmol/l DTT) for 30 min at 30°C. The protein-DNA complexes were then analyzed on 5% native polyacrylamide gels. For the supershift experiment, antibodies were included in the above reaction mixture and incubated at 4°C for 3 hours before the addition of the ³²P-labelled DNA probe. The oligonucleotide sequences used to detect NF-κB activity were as follows: 5'-AGT TGA GGG GAC TTT CCC AGG-3' (sense) and 5'-GCC TGG GAA AGT CCC CTC AAC T-3' (antisense).

FLSs were cultured at 4 × 10⁴ cells/well in four-well Lab-Tek chamber slides (Falcon; Becton Dickinson Labware, Oxnard, CA, USA) in order to visualize the translocation of NF-κB to the nucleus under IL-1β stimulation. After serum starvation overnight, the cells were stimulated with IL-1β at 10 ng/ml for 90 min, washed with cold PBS, and fixed with 4% paraformaldehyde in PBS for 20 min. Cells were permeabilized with PBS and 0.5% Triton X-100 in PBS for 10 min, and were then incubated for 30 min with the blocking buffer, 5% goat serum, in order to prevent nonspecific binding. The cells were incubated with 5 μg/ml rabbit polyclonal anti-NF-κB p65 antibody (Santa Cruz Biotechnology) overnight, followed by incubation with 20 μg/ml cyan3-conjugated goat anti-rabbit antibody for 60 min at room temperature. After washing, the cells were counterstained with 0.1 mg/ml DAPI for 30 min at room temperature. The coverslips were fixed with mounting media (DakoCytomation, Carpinteria, CA, USA), and the slides were visualized using confocal microscopy (Carl Zeiss, Oberkochen, Germany).

TauCl cytotoxicity was assessed by a colorimetric assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). In brief, FLSs (2.5 × 10⁵ cells/2 ml) were seeded into six-well plates. After overnight incubation at 37°C, the medium was replaced with serum-free medium and treated with TauCl 30 min before stimulation with IL-1β (10 ng/ml). Cells were subsequently cultured for 24 hours, and MTT solution (5 mg/ml) was added to each well at a final concentration of 0.5 mg/ml. The plates were incubated for 4 hours at 37°C, and formazan crystals were dissolved by the addition of 1 ml isopropanol containing 0.04 M HCl. Finally, absorbance was measured at 595 nm.

All experiments were repeated three times, and the results are expressed as the mean ± standard deviation. Statistical evaluation was performed by means of a paired Student's t-test. Differences were considered statistically significant at P < 0.05.

The stimulation of arthritic FLSs using IL-1β greatly upregulated the expressions of the MMP-1 and MMP-13 collagenases, as determined by ELISA (Figure 1a), real time PCR (Figure 1b) and semi-quantitative RNA analysis (Figure 1c). However, the expressions of the gelatinases MMP-2 and MMP-9 remained unchanged (Figure 1). MMP-2 and MMP-9 remained unchanged at the mRNA level, even after 24 hours of stimulation, which indicates that IL-1β did not stimulate MMP-2 and MMP-9 (data not shown). Consistent with the mRNA levels of MMP-1 and MMP-13, ELISA analyses of culture supernatants at 24 hours revealed that IL-1β upregulated the expressions of MMP-1 and MMP-13 at the protein level by about 30-fold and 15-fold, respectively (Figure 1a). The protein levels of the MMP-2 remained unchanged, whereas the protein levels of the MMP-9 genes were below the ELISA detection limit (data not shown).

To identify whether TauCl inhibits the expression of MMPs, FLSs were treated with TauCl 30 min before 24 hours or 6 hours of IL-1β stimulation for protein analysis and RNA analysis, respectively. Treatment with TauCl at concentrations of 400 and 600 μmol/l differentially inhibited the expressions of MMP-1 and MMP-13. The expression of MMP-1 remained unchanged at TauCl 400 and 600 μmol/l, whereas MMP-13 levels were reduced to about 50% or 20% of that observed in the IL-1β treated group (with no TauCl treatment), respectively (Figure 1a). However, at a TauCl concentration of 800 μmol/l, the protein expressions of MMP-1 and MMP-13 diminished to 50% and 10%, respectively.

Consistent with the effects of TauCl on the protein levels of MMP-1 and MMP-13, RNA analyses revealed that the levels of MMP-13 were more sensitive to TauCl at a concentration of 600 μmol/l than were the levels of MMP-1. At a TauCl concentration of 800 μmol/l, the transcriptional expressions of the MMP-1 and MMP-13 genes diminished to 20% and 5%, respectively (Figure 1b,c).

IL-1β stimulates the signal transduction pathways of both mitogen-activated protein kinase (MAPK) and IκB kinase in chondrocytes and astrocytes 2829. To identify the pathways that are involved in enhancing MMP-1 and MMP-13 expression under IL-1β stimulation, the levels of phospho-ERK-1/2, phospho-JNK, phospho-p38MAPK and IκBα were measured according to the duration of stimulation. IL-1β treatment led to remarkable increases in the phosphorylation of ERK-1/2 and p38MAPK by 10 min, and the increased levels were maintained for 45 min. The level of phospho-JNK peaked at 30 min. IκBα was completely degraded at 30 min but recovered by 60 min, which indicates that IκBα was fully phosphorylated within 30 min of activation of IL-1β (Figure 2a).

We investigated the level of MAPK phosphorylation in an effort to clarify the inhibitory mechanism of TauCl on MMPs. As shown in Figure 2b, TauCl did not significantly inhibit the phosphorylation of ERK-1/2, p38, or JNK, even when the highest test concentration of 800 μmol/l was used. At 800 μmol/l, TauCl strongly blocked the IκB degradation that normally occurs upon IL-1β stimulation, which suggests that TauCl prevents NF-κB from migrating to the nucleus by inhibiting the degradation of IκB. The effectiveness of TauCl as an inhibitor of IκB was investigated by comparing it with MG132, which is an NF-κB inhibitor that slows IκB degradation by deactivating the proteasome 30. TauCl inhibited IκB degradation as potently as MG132, in this instance; however, the concentration of TauCl employed was greater than that of MG132 (Figure 3).

To further demonstrate that the effects of IκB degradation extended to the transnuclear migration of NF-κB, levels of NF-κB in the nucleus were assessed using EMSA (Figure 4) and immunohistochemistry (Figure 5). As shown in Figure 4, at a concentration of 800 μmol/l, TauCl completely blocked the nuclear binding of NF-κB; however, at 600 αmol/l, TauCl did not block binding activity. These results were confirmed by confocal microscopy. After 90 min of IL-1β stimulation, the majority of cytoplasmic NF-κB migrated into the nucleus, as indicated by strong nuclear NF-κB staining following stimulation and strong cytoplasmic staining before stimulation (Figure 5). Confirming previous findings, the migration of NF-κB into the nucleus was not inhibited at TauCl concentrations of up to 600 μmol/l. However, at a concentration of 800 μmol/l, TauCl blocked the transnuclear migration of NF-κB.