FIGURE 9.

Collagenase expression and MMP1 promoter activity are dependent upon canonical AP-1 components. A, primary human articular chondrocytes were transfected with siRNA (100 nm) specific to c-fos, c-jun, or siCon non-targeting control, prior to stimulation with IL-1 (0.2 ng/ml) and OSM (10 ng/ml) for 3 h. Nuclear extracts were isolated, and proteins were resolved by SDS-PAGE. Immunoblots were probed using the antibodies indicated. Blots shown are representative of two experiments using chondrocytes from different donors. B and C, following transfection with siRNA specific to c-fos, c-jun, or siCon (100 nm), chondrocytes were stimulated with IL-1 (0.02 ng/ml) and OSM (10 ng/ml) for 24 h and real time reverse transcription-PCR of the isolated RNA was performed for MMP1 and MMP13, 72 h after transfection. Data are pooled from at least three separate chondrocyte populations (each assayed in hextuplicate) presented as a percentage of the cytokine-induced expression (specific siPKC transfected versus siCon-transfected; mean ± S.E.; ***, p < 0.001; **, p < 0.01; *, p < 0.05). D, T/C28a4 human chondrocytes were transiently co-transfected with pCMV2 plasmid alone or constructs for overexpression of c-fos, c-jun, or both c-fos and c-jun, together with a pGL3-derived construct harboring the proximal (−517/+63) region of the human MMP1 promoter controlling luciferase expression or empty vector control. Luciferase activity was determined and data are representative of two experiments conducted in quadruplicate; mean ± S.E.; ***, p < 0.001 compared with pCMV alone.