FIGURE 6.

STAT3 gene silencing and ERK pathway inhibition abrogate collagenase induction. A, primary human articular chondrocytes were transfected with siRNA (100 nm) specific to STAT1, STAT3, or siCon non-targeting control, prior to stimulation with OSM (10 ng/ml) for 20 min. Cells were lysed and proteins resolved by SDS-PAGE. Immunoblots were probed using the antibodies indicated. B, chondrocytes were transfected as above, but subsequently stimulated with IL-1 (0.02 ng/ml) and OSM (10 ng/ml) for 24 h. Real time reverse transcription-PCR from the isolated RNA was performed for MMP1 and MMP13, 72 h after transfection. C, bovine cartilage explant cultures were stimulated with IL-1 (1 ng/ml) and OSM (10 ng/ml) for 14 days, with fresh medium and cytokines at day 7, in the presence of DMSO vehicle control or MEK inhibitor UO126 (10 μm). Medium assayed for cumulative collagen release by day 14 were expressed as a percentage of the total for each treatment. D, chondrocytes were incubated with UO126 (3 μm) or DMSO vehicle for 1 h prior to stimulation with IL-1 (0.02 ng/ml) and OSM (10 ng/ml) for 24 h. Real time reverse transcription-PCR of the isolated RNA was performed for MMP1 and MMP13. Data are representative of (A) or pooled from (B-D) three separate chondrocyte populations (each assayed in at least quadruplicate) compared with stimulated control (mean ± S.E.; ***, p < 0.001).