Human cartilage was acquired from the femoral heads of OA patients who underwent hip-replacement surgery at the Notre-Dame Hospital. Normal bovine cartilage was obtained from the knee and hip joints of adult animals from a local abattoir. Chondrocytes were released by 90 min pronase and 9 hours digestion with collagenase (Sigma type IA). The cells were washed with PBS and grown in DMEM containing 10% FCS as high-density primary monolayer cultures until confluent growth. Cells were distributed in six-well plates, grown to confluence, washed with PBS and kept in serum-free DMEM for 24 hours; mithramycin (from Sigma-Aldrich Canada Ltd, Oakville, Ontario; dissolved in water as a 10 mM solution) was then added without medium change at final concentrations of 100 and 150 nM (doses known to inhibit Sp1 binding 20) for 30 min before treatment for 24 hours with human recombinant IL-1β (10 ng/ml), TNF-α (20 ng/ml) and IL-17 (20 ng/ml) (R&D systems, Minneapolis, MN). The human chondrosarcoma cell line SW1353 was obtained from the American Type Culture Collection (ATCC, Manassas, VA) and treated as described for primary chondrocytes.

Total cellular RNA was extracted by the guanidinium procedure 21 and aliquots of 3 to 5 μg were analyzed by electrophoretic fractionation in 1.2% formaldehyde-agarose gels. The integrity and quantity of RNA were verified by ethidium bromide staining of the 28S and 18S ribosomal RNA bands. The RNA was transferred onto Zeta-probe nylon membrane with a Bio-Rad Transblot in the presence of 0.5 × TAE (Tris-acetate-EDTA) buffer at a current of 500 mA for 12 hours. Northern blots were hybridized with a human stromelysin cDNA probe generously provided by Dr Richard Breathnach (Nantes, France). This probe was a 1.6-kilobase EcoRI cDNA fragment cloned in the plasmid pGEM-4Z (Promega Biotech, Madison, WI) and the vector was linearized with NarI. A 491-base-pair RT-PCR-generated 22 and cloned collagenase-3 cDNA was linearized with EcoRI. The human 28S ribosomal RNA plasmid (ATCC) was digested with XbaI. All antisense RNA probes were synthesized with T7 polymerase in accordance with the protocols of Promega Biotech and were labeled to high specific radioactivity (10⁸ c.p.m./μg) with [α-³²P]CTP (3,000 Ci/mmol; Perkin Elmer Life Sciences Inc., Boston, MA).

Total secreted proteins from the 2 to 3 ml of conditioned medium of the chondrocytes or SW1353 cells were concentrated by precipitation with trichloroacetic acid, quantified with the Bio-Rad protein assay system and different amounts of protein aliquots adjusted to 15 μl with 4 × sample buffer comprising 62.5 mM Tris-HCl, 20% glycerol, 0.032% bromophenol blue, 5% mercaptoethanol and 2% SDS. Along with the prestained broad-range molecular mass standards (Bio-Rad), samples were fractionated by a 4% stacking and 10% SDS-PAGE mini gel (Bio-Rad, Mississauga, ON) and transferred to nitrocellulose membrane by electroblotting at 200 mA in a buffer comprising 25 mM Tris-HCl, 192 mM glycine, 0.04% SDS and 20% ethanol. The membranes were rinsed with distilled water, incubated for 1 hour in PBS pH 7.4 with 5% Carnation non-fat milk to block non-specific interactions, and washed five times (twice for 5 min, once for 15 min and twice for 5 min) with PBS containing 0.1% Tween. They were then reacted overnight sequentially in the same buffer at 4°C with 1 to 2 μg/ml anti-human MMP-3 (developed in mouse) and MMP-13 (hinge region, developed in rabbit) antibodies (from Sigma-Aldrich). Subsequently, membranes were washed at 22°C five times with PBS containing 0.1% Tween, incubated with the anti-rabbit or anti-mouse secondary peroxidase-conjugated IgG (300 mU/ml), and washed seven times with PBS containing 0.1% Tween. To reveal the MMP-3 and MMP-13 bands, membranes were incubated with 10 μl of solution A and 990 μl of solution B of the chemiluminescence detection system of Roche Biochemicals (Laval, Québec) and exposed to film for 2 to 15 min.

For Western blots of MAPKs, human femoral-head chondrocytes were pretreated with mithramycin for 30 min and then stimulated with IL-1β for 20 min; total cellular protein extracts (20 μg) in lysis buffer (62.5 mM Tris-HCl pH 6.8, 10% glycerol, 1% Triton X-100, 50 mM dithiothreitol, 2% SDS, 0.01% bromophenol blue) were resolved by 10% SDS-PAGE, transferred to nitrocellulose membranes by electroblotting and incubated overnight at 4°C with primary phosphorylation-state-specific antibodies for phosphorylated extracellular signal-regulated kinase (p-ERK), phospho-p38 and phosphorylated c-Jun N-terminal kinase (p-JNK) (from Cell Signaling Technology Inc., Beverley, MA) at 1:1,000 dilution in 5% BSA, 1 × Tris-buffered saline and 0.1% Tween. Proteins were detected with the enhanced chemiluminescence system from Pharmacia-Amersham. Subsequently, the membranes were stripped with a buffer (containing 100 mM 2-mercaptoethanl, 2% SDS and 62.5 mM Tris-HCl, pH 6.8) at 55°C and reprobed with the antibodies detecting total ERK, p38 and JNK.

Human or bovine femoral head cartilage explants were maintained for 1 week in DMEM with 10% FCS, medium was then changed with 0.01% serum-containing DMEM for 3 days until treatments. Explants were treated with mithramycin and IL-1 vehicles as control (water and PBS-0.1% BSA) or exposed to mithramycin (150 nM) and IL-1 (33 ng/ml) for 15 days with replacement of the fresh reagents every 2 days; the secreted media were concentrated by precipitation with 10% trichloroacetic acid and equal amounts of protein (16 μg per lane for human explants and 20 μg per lane for bovine explants) were subjected to Western immunoblotting as described above. All the experiments described in this paper were performed at least two (primary human chondrocytes and cartilage) or three (SW1353 and bovine chondrocytes) times and the results were reproducible.

IL-1β potently induced expression of the genes encoding MMP-3 and MMP-13 in the human chondrocytic cell line SW1353 and in primary human femoral head chondrocytes. Mithramycin, a hypocalcemic antibiotic, potently blocked the induction of MMP-3 and MMP-13 mRNA by IL-1β without affecting the control 28S rRNA levels (Fig. 1a,b). The two MMP-13 mRNA bands correspond to transcripts produced by differential use of polyadenylation sites at the 3' end of the gene, the upper band being the longest transcript as reported previously 23. Induction of MMP-13 protein was also similarly inhibited (Fig. 1a,b). To examine whether mithramycin could also affect MMP gene expression in articular chondrocytes from other species, adult bovine chondrocytes (an important model system in cartilage research) were exposed to different concentrations of mithramycin and then stimulated with IL-1β. This cytokine induced MMP-3 and MMP-13 mRNA expression above basal levels, and pretreatment with mithramycin reduced both constitutive and induced expression in a fashion similar to that of human cells (Fig. 1c). The induction of MMP-13 protein (the main collagen-degrading MMP) by IL-1 was also inhibited. The double MMP-13 protein bands are due to a better resolution of the upper proenzyme and lower active MMP-13 forms.

IL-17 is a major proinflammatory cytokine and an inducer of MMP expression in chondrocytes and macrophages 1124. As hown in Fig. 2, IL-17 stimulated the basal MMP-3 and MMP-13 mRNA and MMP-13 protein expression in bovine and human OA chondrocytes. Mithramycin dose-dependently diminished these inductions.

TNF-α is another prominent inflammatory cytokine that increased the constitutive MMP-3 and MMP-13 mRNA expression in chondrocytic SW1353 cells, primary human chondrocytes and bovine chondrocytes. Exposure to mithramycin followed by stimulation with TNF-α resulted in decreased constitutive and induced MMP-3 and MMP-13 gene expression (Fig. 3). In some cases, a concentration of 100 nM caused maximal inhibition; a 150 nM dose did not have any additional effect (e. g. mRNA in 3B).

To study the impact of mithramycin on the production of MMP-13 by chondrocytes in their native cartilage matrix, human and bovine cartilage explants were maintained in low-serum (0.01%) medium and exposed to mithramycin (150 nM) and IL-1β for 15 days with changes of reagents every 2 days. Human cartilage had somewhat elevated constitutive levels of MMP-3 and MMP-13. Mithramycin drastically reduced the secreted basal and IL-1-induced MMP-3 and MMP-13 protein levels in human cartilage (Fig. 4a) and MMP-13 in bovine cartilage (Fig. 4b) as measured by Western blotting of the conditioned media. MMP-3 levels were too low to be measurable in bovine explants. Therefore mithramycin diminishes IL-1-stimulated MMP production in cartilage explants.

Because MAPKs are important mediators of proinflammatory cytokine signal transduction 25, we investigated whether mithramycin affected these signaling cascades. As reported previously 25, TNF-α induced the phosphorylation of the ERK, p38 and JNK subclasses of MAPKs without affecting their total protein levels. Mithramycin did not significantly influence their phosphorylation levels (Fig. 5).