Human recombinant IL-1 was obtained from Genzyme (Cambridge, MA, USA). Aprotinin, leupeptin, pepstatin, phenylmethylsulphonyl fluoride (PMSF), cycloheximide (CHX), and sodium orthovanadate (Na₃VO₄) were from Sigma-Aldrich Canada (Oakville, ON, Canada). Dulbecco's modified Eagle's medium (DMEM), penicillin and streptomycin, fetal calf serum (FCS), and Trizol reagents were supplied by Invitrogen (Burlington, ON, Canada). Antibodies against Egr-1, Sp1, and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The antibody against PPARγ was from Cayman Chemical Company (Ann Arbor, MI, USA). Polyclonal goat anti-rabbit immunoglobulin G (IgG) with horseradish peroxidase (HRP) was from Pierce (Rockford, IL, USA).

Human normal cartilage (from femoral condyles) was obtained at necropsy, within 12 hours of death, from donors with no history of arthritic diseases (n = 12, mean ± standard deviation (SD) age: 61 ± 14 years). To ensure that only normal tissue was used, cartilage specimens were thoroughly examined both macroscopically and microscopically. Only those with neither alteration were further processed. Human OA cartilage was obtained from patients undergoing total knee replacement (n = 31, mean ± SD age: 66 ± 15 years). In all patients, OA was diagnosed on the basis of criteria developed by the American College of Rheumatology Diagnostic Subcommittee for OA 26 . At the time of surgery, the patients had symptomatic disease requiring medical treatment in the form of non-steroidal anti-inflammatory drugs or selective COX-2 inhibitors. Patients who had received intra-articular injections of steroids were excluded. The Clinical Research Ethics Committee of Notre-Dame Hospital approved the study protocol and the use of human articular tissues. Informed consent was obtained from each donor or from an authorized third party.

Chondrocytes were released from cartilage by sequential enzymatic digestion as previously described 25 . Cells were seeded at 3.5 × 10⁵ cells per well in 12-well culture plates (Costar, Corning, NY, USA) or at 6 to 7 × 10⁵ cells per well in six-well culture plates in DMEM supplemented with 10% FCS and were cultivated at 37°C for 48 hours. Cells were washed and incubated for an additional 24 hours in DMEM containing 0.5% FCS before stimulation with IL-1.

Chondrocytes were lysed in ice-cold lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM ethylenediaminetetraacetic acid (EDTA), 1 mM PMSF, 10 μg/mL each of aprotinin, leupeptin, and pepstatin, 1% NP-40, 1 mM Na₃VO₄, and 1 mM NaF). Lysates were sonicated on ice and centrifuged at 12,000 revolutions per minute for 15 minutes. The protein concentration of the supernatant was determined by using the bicinchoninic acid method (Pierce). Twenty micrograms of total cell lysate was subjected to SDS-polyacrylamide gel electrophoresis and electrotransferred to a nitrocellulose membrane (Bio-Rad, Mississauga, ON, Canada). After blocking in 20 mM Tris-HCl pH 7.5 containing 150 mM NaCl, 0.1% Tween 20, and 5% (wt/vol) non-fat dry milk, blots were incubated overnight at 4°C with the primary antibody and washed with a Tris buffer (Tris-buffered saline (TBS) pH 7.5 with 0.1% Tween 20). The blots were then incubated with HRP-conjugated secondary antibody (Pierce), washed again, incubated with SuperSignal Ultra Chemiluminescent reagent (Pierce), and exposed to Kodak X-Omat film (Eastman Kodak Company, Rochester, NY, USA).

Total RNA from cultured chondrocytes or cartilage was isolated by using the TRIzol reagent (Invitrogen) in accordance with the instructions of the manufacturer. To remove contaminating DNA, isolated RNA was treated with RNase-free DNase I (Ambion, Austin, TX, USA). The RNA was quantitated by using a RiboGreen RNA quantitation kit (Molecular Probes Inc., now part of Invitrogen Corporation, Carlsbad, CA, USA), dissolved in diethylpyrocarbonate-treated H₂O, and stored at -80°C until use. One microgram of total RNA was reverse-transcribed by using Moloney murine leukemia virus reverse transcriptase (Fermentas, Burlington, ON, Canada) as detailed in the guidelines of the manufacturer. One fiftieth of the reverse transcriptase reaction was analyzed by real-time polymerase chain reaction (PCR) as described below. The following primers were used: PPARγ, sense, 5'-AAAGAAGCCAACACTAAACC-3' and antisense, 5'-CTTCCATTACGGAGAGATCC-3'; Egr-1, sense, 5'-CTGACCGCAGAGTCTTTTCCTG-3' and antisense, 5'-TGGGTGCCGCTGAGTAAATG-3'; Sp1, sense 5'-AAACATATCAAAGACCCACCAGAAT-3' and antisense 5'-ATATTGGTGGTAATAAGGGCTGAA-3'; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sense 5'-CAGAACATCATCCCTGCCTCT-3' and antisense 5'-GCTTGACAAAGTGGTCGTTGAG -3'.

Real-time PCR analysis was performed in a total volume of 50 μL containing template DNA, 200 nM of sense and antisense primers, 25 μL of SYBR^® Green master mix (Qiagen, Mississauga, ON, Canada) and uracil-N-glycosylase (UNG) (0.5 Units; Epicentre Technologies, Madison, WI, USA). After incubation at 50°C for 2 minutes (UNG reaction) and at 95°C for 10 minutes (UNG inactivation and activation of the AmpliTaq Gold enzyme), the mixtures were subjected to 40 amplification cycles (15 seconds at 95°C for denaturation and 1 minute for annealing and extension at 60°C). Incorporation of SYBR^® Green dye into PCR products was monitored in real time by using a GeneAmp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and allowing determination of the threshold cycle (C_T) at which exponential amplification of PCR products begins. After PCR, dissociation curves were generated with one peak, indicating the specificity of the amplification. A C_T value was obtained from each amplification curve by using the software provided by the manufacturer (Applied Biosystems).

Relative mRNA expression in chondrocytes was determined by using the ΔΔC_T method, as detailed in the guidelines of the manufacturer (Applied Biosystems). A ΔC_T value was first calculated by subtracting the C_T value for the housekeeping gene GAPDH from the C_T value for the gene of interest. A ΔΔC_T value was then calculated by subtracting the ΔC_T value of the control (unstimulated cells) from the ΔC_T value of each treatment. Fold changes compared with the control were then determined by raising 2 to the -ΔΔC_T power. Each PCR generated only the expected specific amplicon as shown by the melting-temperature profiles of the final product and by gel electrophoresis of test PCRs. Each PCR was performed in triplicate on two separate occasions for each independent experiment.

The chromatin immunoprecipitation (ChIP) experiments were performed according to the ChIP protocol provided by Upstate/Millipore Biotechnology Inc. (Lake Placid, NY, USA) and previously published protocols 27 . After treatment, the cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature. The fixed cells were washed twice with ice-cold phosphate-buffered saline containing protease inhibitors and then lysed for 10 minutes at 1 × 10⁶ cells per 200 μL of SDS lysis buffer (50 mM Tris-Cl (pH 8.0), 0.5% SDS, 100 mM NaCl, and 5 mM EDTA) plus protease inhibitors. The chromatin samples were sonicated to reduce DNA length to 200 to 500 base pairs (bp). Twenty microliters of the supernatant was saved as the input DNA, and the remainder was diluted 1:10 in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, and 16.7 mM Tris-Cl) containing protease inhibitors. The chromatin samples were precleared with a salmon sperm DNA/protein A-agarose 50% gel slurry for 3 hours. The samples were then immunoprecipitated overnight at 4°C with antibodies specific for either Sp1 and Egr-1. As negative controls, cross-linked chromatin was incubated overnight with control Ig or in the absence of antibody. Immune complexes were recovered by addition of salmon sperm DNA/protein A-agarose slurry for 2 hours at 4°C. The immune complexes were sequentially washed three times each (5 minutes on a rotating platform), with low salt, high salt, lithium chloride, and Tris/EDTA buffers, and eluted twice with 250 μL of 1% SDS and 0.1 M NaHCO₃ for 15 minutes. The eluted material and the DNA input samples were heated for 4 hours at 65°C to reverse cross-linking. The samples were treated with 40 μg/mL DNase-free proteinase K for 1 hour at 45°C, extracted with phenol-chloroform-isoamyl alcohol and chloroform, and ethanol-precipitated in the presence of 20 μg of glycogen. Pellets were suspended in 25 to 30 μL of H₂O and subjected to PCR analysis. The primer sequences used were PPARγ sense, 5'-TCGGATCCCTCCTCGGAAATGG-3' and antisense, 5'-GCGCGACTGGGAGGGA-3'.

The luciferase reporter construct pGL3-PPARγ1p3000, containing a 3,000-bp fragment of the human PPARγ1 gene promoter, was kindly provided by Johan Auwerx (Institut de Génétique et de Biologie moléculaire et Cellulaire, Illkirch, France). Egr-1 expression vector (pcDNA3) was donated by Yuqing Chen (Morehouse School of Medicine, Atlanta, GA, USA) 28 . The β-galactosidase reporter vector under the control of SV40 promoter (pSV40-β-gal) was from Promega Corporation (Madison, WI, USA). Transient transfection experiments were performed by using the FuGene-6 transfection reagent in accordance with the recommended protocol of the manufacturer (Roche Applied Science, Indianapolis, IN, USA). Briefly, chondrocytes were seeded 24 hours prior to transfection at a density of 3 × 10⁵ cells per well in 12-well plates and transiently transfected with 1 μg of the PPARγ promoter construct and 0.5 μg of the internal control pSV40-β-gal. Six hours later, the medium was replaced with DMEM containing 1% FCS. At 1 day after transfection, the cells were left untreated or treated with IL-1 (100 pg/mL) for 20 hours. In the overexpression experiments, the amount of transfected DNA was kept constant by using the corresponding empty vector. At the end of the indicated treatment, the cells were washed twice in ice-cold phosphate-buffered saline (PBS) and extracts were prepared for firefly luciferase reporter assay (Promega Corporation). Luciferase activity was normalized for transfection efficiency by using the corresponding β-galactosidase activity.

Specific small interfering RNA (siRNA) for Sp1, Egr-1, or scrambled control was obtained from Dharmacon Inc. (Lafayette, CO, USA). Chondrocytes were seeded in six-well plates at 6 × 10⁵ cells per well and incubated for 24 hours. Cells were transfected with 100 nM of siRNA by using the HiPerFect Transfection Reagent (Qiagen) in accordance with the recommendations of the manufacturer. The medium was changed 24 hours later, and the cells were incubated for an additional 24 hours before stimulation with 100 pg/mL IL-1 for 1 or 20 hours.

Cartilage specimens were processed for immunohistochemistry as previously described 25 . The specimens were fixed in 4% paraformaldehyde and embedded in paraffin. Sections (5 μm) of paraffin-embedded specimens were deparaffinized in toluene and dehydrated in a graded series of ethanol. The specimens were then preincubated with chondroitinase ABC (0.25 U/mL in PBS pH 8.0) for 60 minutes at 37°C followed by a 30-minute incubation with Triton X-100 (0.3%) at room temperature. Slides were then washed in PBS followed by incubation with 2% hydrogen peroxide/methanol for 15 minutes. They were further incubated for 60 minutes with 2% normal serum (Vector Laboratories, Burlingame, CA, USA) and overlaid with an anti-Egr-1 antibody (Santa Cruz Biotechnology, Inc.) for 18 hours at 4°C in a humidified chamber. Each slide was washed three times in PBS pH 7.4 and stained by using the avidin-biotin complex method (Vectastain ABC kit; Vector Laboratories). The color was developed with 3,3'-diaminobenzidine (Vector Laboratories) containing hydrogen peroxide. The slides were counterstained with eosin. The specificity of staining was evaluated by using an antibody that had been preadsorbed (1 hour at 37°C) with a 20-fold molar excess of the protein fragment corresponding to amino acids 500 to 550 of human Set1A (Santa Cruz Biotechnology, Inc.) and by substituting the primary antibody with non-immune rabbit IgG (Chemicon, Temecula, CA, USA), used at the same concentration as the primary antibody. The evaluation of positive-staining chondrocytes was performed by using our previously published method 25 . For each specimen, six microscopic fields were examined under 40× magnification. The total number of chondrocytes and the number of chondrocytes staining positive were evaluated, and results were expressed as the percentage of chondrocytes staining positive (cell score).

Data are expressed as the mean ± SD. Statistical significance was assessed by the two-tailed Student t test. P values of less than 0.05 were considered significant.

First, we investigated whether IL-1-mediated downregulation of PPARγ expression in chondrocytes requires de novo protein synthesis. Chondrocytes were incubated with cycloheximide (10 μg/mL) for 30 minutes prior to stimulation with 100 pg/mL IL-1 for 18 hours, and the levels of PPARγ mRNA were analyzed by real-time PCR. Changes in PPARγ mRNA gene expression were evaluated as percentage over control (untreated cells) after normalization to the internal control gene, GAPDH. As shown in Figure 1, stimulation with IL-1 down-regulated PPARγ mRNA expression to approximately 80% of control (bar 2 versus bar 1), confirming our earlier findings 25 . Treatment with CHX prevented IL-1-mediated suppression of PPARγ mRNA expression (bar 4 versus bar 2), suggesting that the suppressive effect of IL-1 on PPARγ was an indirect effect and was dependent on de novo protein synthesis.

Analysis of the PPARγ promoter identified a putative Egr-1-binding site, which overlaps with a Sp1-binding site, between nucleotide-184 and -173 relative to the transcription start site. To evaluate the role of these transcription factors in the suppressive effect of IL-1 on PPARγ expression, we first examined the effect of IL-1 on their expression in chondrocytes. Cells were treated with IL-1 for different time periods, and the levels of Egr-1 mRNA were quantified by using real-time reverse transcriptase-PCR (RT-PCR). IL-1-induced changes in gene expression were evaluated as fold over control (untreated cells) after normalization to the internal control gene, GAPDH. As shown in Figure 2a, treatment with IL-1 enhanced Egr-1 mRNA expression in a time-dependent manner. Egr-1 mRNA was rapidly and significantly induced at 0.5 hours post-stimulation with IL-1, reached the maximum at 1 hour, and started to decrease at 2 hours. Next, we performed Western blot analysis to determine whether changes in mRNA levels were paralleled by changes in Egr-1 protein levels. Consistent with its effects on Egr-1 mRNA, IL-1 induced Egr-1 protein expression in a time-dependent manner (Figure 2d). Egr-1 protein levels were significantly increased by 0.5 hours post-stimulation, further increased up to 1 hour, then gradually declined starting at 2 hours, and reached basal levels at 8 hours. The induction of Egr-1 mRNA by IL-1 was also dose-dependent (data not shown). These results indicated that IL-1 is a potent inducer of Egr-1 mRNA and protein expression in human chondrocytes. In contrast, IL-1 had no significant effect on the expression levels of Sp1 mRNA and protein (Figure 2b,e). Importantly, the induction of Egr-1 protein expression by IL-1 preceded the suppression of PPARγ expression (Figure 2c,f). The correlation between the downregulation of PPARγ expression and the induction of Egr-1 suggests a link between these two events.

To determine whether Egr-1 and Sp1 proteins physically interact with the PPARγ promoter in vivo, we performed ChIP assays. Chondrocytes were stimulated with IL-1 for various time periods, and formaldehyde-cross-linked DNA-proteins were immunoprecipitated by using antibodies specific to Egr-1 and Sp1. Control Ig and no antibody were used as controls. DNA isolated from the immunoprecipitates was analyzed by real-time PCR by using primers amplifying the PPARγ promoter region (bp -322 to -139) that harbors the overlapping Sp1/Egr-1 site.

As shown in Figure 3b, treatment with IL-1 enhanced the levels of Egr-1 at the PPARγ promoter in a time-dependent manner. It started to increase significantly at 1 hour after IL-1 stimulation, reached a maximum at 2 hours, and returned to a near basal level by 8 hours. In contrast, the levels of Sp1 at the PPARγ promoter decreased after IL-1 stimulation (Figure 3c). Sp1 levels were significantly decreased by 2 hours after stimulation, with a further decrease at 4 hours, and remained downregulated until the 18-hour time point. No immunoprecipitable PPARγ promoter DNA was detected with the control Ig and no antibody controls (data not shown).

The recruitment of Egr-1 and reduced occupancy of Sp1 at the PPARγ promoter preceded the suppression of PPARγ transcription by IL-1, suggesting that the recruitment of Egr-1 mediates PPARγ downregulation. Taken together, these results strongly suggest that IL-1-mediated downregulation of PPARγ involves the recruitment of Egr-1 and reduced occupancy of Sp1.

To further characterize the functional roles of Sp1 and Egr-1 in IL-1-mediated downregulation of PPARγ expression, we performed transient transfection experiments in which we examined the effects of Egr-1 and Sp1 on PPARγ promoter activity. Chondrocytes were transiently co-transfected with the PPARγ promoter and increasing concentrations of expression vectors that encode Sp1 or Egr-1, and at 18 hours post-transfection, the cells were left untreated or stimulated with IL-1 for an additional 18 hours.

As shown in Figure 4a, treatment with IL-1 suppressed PPARγ promoter activity (bar 5 versus bar 1), consistent with previous data 25 . Overexpression of Egr-1 had no significant effect on the basal PPARγ promoter activity (bars 2 to 4) but dose-dependently potentiated the suppressive effect of IL-1 (bars 6 to 8). These data suggest that Egr-1 mediates the suppressive effect of IL-1 on PPARγ expression. In contrast, overexpression of Sp1 slightly enhanced (bars 2 to 4) the basal activity of the PPARγ promoter and prevented (bars 6 to 8) the suppressive effect of IL-1 on the PPARγ promoter activity (Figure 4b). These data corroborate the ChIP data, suggesting that Egr-1 mediates the suppressive effect of IL-1 on PPARγ expression, likely by competing with endogenous Sp1.

To further confirm the role of Egr-1, we examined the impact of its silencing by siRNA on IL-1-mediated downregulation of PPARγ protein expression. Chondrocytes were transfected with the scrambled control siRNA, siRNA for Sp1, or siRNA for Egr-1, and after 48 hours of transfection, the cells were stimulated or not with IL-1 for 1 or 18 hours. As shown in Figure 5, transfection with Egr-1 siRNA reversed the suppressive effect of IL-1 on PPARγ. In contrast, transfection with Sp1 siRNA or with scrambled control siRNA had no effect. Sp1 protein levels were reduced by as much as 70% to 75%, and Egr-1 protein levels were almost completely suppressed, confirming silencing of both genes (Figure 5). Together, these data clearly show that Egr-1 is required for IL-1-mediated downregulation of PPARγ protein expression.

To determine whether Egr-1 levels were altered under OA conditions, we analyzed the levels of Egr-1 mRNA in total cartilage from normal (n = 9) and OA (n = 9) donors by using real-time quantitative RT-PCR. As shown in Figure 6a, the level of Egr-1 mRNA was approximately 3.5-fold higher in OA cartilage compared with normal cartilage. Next, we used immunohistochemistry to analyze the localization and the expression level of Egr-1 protein in normal and OA cartilage. As shown in Figure 6c and 6d, Egr-1 was expressed primarily in chondrocytes of the superficial and upper intermediate zones of the cartilage. Statistical evaluation for the cell score revealed that the percentage of cells expressing Egr-1 was approximately 3-fold higher in OA cartilage (n = 9) compared with normal cartilage (n = 9). The specificity of the staining was confirmed by using an antibody that had been preadsorbed (1 hour at 37°C) with a 20-fold molar excess of the peptide antigen or non-immune control IgG (data not shown). Together, these data indicate that the expression level of Egr-1 is increased in OA cartilage.