Figure 3

The role of nuclear factor (NF)-κB in peripheral blood-macrophage-conditioned medium (PB-MCM)-induced urokinase-type PA (uPA) mRNA expression and promoter activity. (A) Left panel, uPA promoter p2350-Luc plasmid, deletion, and mutant promoter constructs. Right panel, PromoCell HCs were cotransfected with uPA promoter constructs and stimulated with PB-MCM for 2 hours. Promoter activities were measured by using a luciferase assay normalized to β-galactosidase activity. They are shown relative to the activities in cells transfected with p2350-Luc (set to 100%). *P < 0.05 versus p2350-Luc. ^#P < 0.01 versus p2350-Luc. (B) uPA mRNA and uPA p2350-Luc activity in chondrocytes wase determined in cells pretreated with vehicle (DMSO), different doses of NF-kB inhibitor SN50, or AP-1 inhibitor Tanshinone IIA (TIIA), and then stimulated with PB-MCM for 2 hours. *P < 0.05 versus CL. ^#P < 0.01 versus DMSO control with PB-MCM stimulation. **P < 0.05 versus DMSO control with PB-MCM stimulation. (C) NF-κB p65 activation determined with TF ELISA. All bar graphs are indicative of fold-changes compared with control chondrocytes (CL). *P < 0.05 versus CL. (D) ChIP analysis of NF-κB by using a p65 antibody.