Figure 4

The JNK and Akt signaling pathways are involved in peripheral blood-macrophage-conditioned medium (PB-MCM)-induced urokinase-type PA (uPA) promoter activity. Chondrocytes were maintained as controls (CL) or pretreated with vehicle (DMSO), with SP600125 (SP) and LY294002 (LY) individually or in combination for 1 hour, or transfected with control siRNA (si-CL), control pcDNA3 vector (vec), JNK-siRNA (si-JNK), or DN-Akt plasmid, and then stimulated with PB-MCM. (A) uPA-2350-Luc activity was determined with luciferase assay after stimulation with PB-MCM for 2 hours. (B) NF-κB p65 activation was determined by TF ELISA after stimulation with PB-MCM for 1 hour. (C) NF-κB p65 binding to uPA promoter in chondrocytes after 1 hour PB-MCM stimulation was analyzed with ChIP assay. Cells were pretreated with vehicle (DMSO), or with SP600125 (SP) and LY294002 (LY) individually or in combination for 1 hour. The ChIP assay was performed by using p65 antibody. All bar graphs indicate the fold changes compared with CL chondrocytes, mean ± SEM. *P < 0.05 versus CL. ^#P < 0.05 versus DMSO control, si-CL, or vec-transfected cells with PB-MCM stimulation. **P < 0.05 versus SP- or LY-pretreated cells under PB-MCM stimulation.