Effect of PKD inhibition on collagenase expression in human chondrocytes.

<p>Primary human articular chondrocytes were stimulated with IL-1 (0.2 ng/ml) alone, OSM (10 ng/ml) alone or IL-1+OSM as detailed in the Methods for 20 min (A-C). Cells were either (A) pre-treated with Gӧ6983 (20 μM; PKCi) for 1 h or (B and C) transfected with siRNA for non-targeting siCon (white bars), <i>PRKCZ</i> (grey bars) or <i>PRKCI</i> (black bars) (all 100 nM) for 48 h prior to stimulation. Cell lysates were prepared as described in Methods and subjected to SDS-PAGE and immunoblotting (using the antibodies indicated). Relative densitometry of some blots was performed, scans combined and plotted graphically (C), where ^††, p≤0.01 versus siCon-treated IL-1+OSM. (D) Human chondrocytes were pre-treated with NB142-70 (5 μM; PKDi), or a DMSO vehicle control for 1 h prior to stimulation with IL-1 (0.05 ng/ml) + OSM (10 ng/ml) for 24 h. Real-time RT-PCR was performed on isolated RNA to determine relative levels of <i>MMP1</i> (black bars) and <i>MMP13</i> (hatched bars), normalised to <i>18S rRNA</i> housekeeping gene and presented as a percentage of the IL-1+OSM induction (n = 6; mean ± S.E.), where ***, p≤0.001 versus the relevant DMSO-treated vehicle control. Data are representative of at least 3 separate chondrocyte populations.