Figure 4
NF-κB and c-Jun are key transcriptional factors involved in the synthesis of MMP-1 in sheared human T/C-28a2 chondrocytes.
In promoter assay experiments, T/C-28a2 cells were transiently transfected with a series of truncated or mutated pMMP-1-luc plasmid (a,b). The luciferase activities were measured using the Dual-Luciferase Reporter Assay Kit, which were normalized to the Renilla luciferase activities (a,b). In select experiments, nuclear extracts were isolated and c-Jun- and NF-κB-specific DNA-protein complex formation was determined by EMSA (c,d). In separate experiments, cross-linked chromatin was immunoprecipitated using an anti-c-Jun (e) or anti-p65 antibody (f). In the ChIP assays, the anti-RNA polymerase II antibody was used as a positive control. DNA purified from immunoprecipitated (IP) and preimmune (Input) specimens was subjected to qPCR amplification using primers for the mmp-1 promoter. All experiments are representative of three independent experiments, all revealing similar results (e,f). The data represent the means ± S.E. of three independent experiments. *, p < 0.05 with respect to the static or vehicle treatment control. #, p < 0.05 compared with fluid shear stress, IL-1β, FGF-2, PGE2 or forskolin treatment alone.
