Figure 3
PGE2 and cAMP induced by COX-2 regulate the expression and enzymatic activity of MMP-1 via PI3-K/AKT- and p38-dependent NF-κB and c-Jun activating pathways in OA development.
PGE2 (2 μg/5 μl) or forskolin (2 μg/5 μl) was injected to the cavity of articular cartilage of wild type mice (a).In select experiments, T/C-28a2 cells were treated with LY294002 (10 μM) or SB203580 (10 μM) in the absence or presence of PGE2 (10 μM) or forskolin (10 μM) for 48 h (b-d). In seperate experiments, T/C-28a2 cells were incubated with QNZ (1 μM) or SP600125 (5 μM) in the absence or presence of PGE2 (10 μM) or forskolin (10 μM) for 48 h (e). In distinct experiments, T/C-28a2 chondrocytes were exposed to shear stress (20 dyn/cm2) in the absence or presence of LY294002 (10 μM), SB203580 (10 μM) (f) QNZ (1 μM) or SP600125 (5 μM) for 48 h (g). (a) The tissues of articular cartilage were immunostained with MMP-1 antibody. Phosphorylated AKT, p38, NF-κB and c-Jun were detected by immunoblotting using specific Abs. Equal lane loading is demonstrated by the similar intensities of total AKT, p38, NF-κB, c-Jun and β-actin. These western blots are representative of three independent experiments, all revealing similar results (b,d,f). The MMP-1 mRNA and protein levels were determined by qRT-PCR, western blots and zymography, respectively. The total amounts of GAPDH and MMP-11 served as internal controls in the qRT-PCR and zymography assays, respectively (c,e,g). *, p < 0.05 with respect to the static- or vehicle-treated control. #, p < 0.05 compared with the fluid shear stress, PGE2 or forskolin treatment.
