FIGURE 4.

IL-1β induces MMP-9 synthesis in human T/C-28a2 chondrocytes via PI3K-, ERK1/2-, and JNK- dependent pathways. T/C-28a2 cells were treated with IL-1β (100 ng/ml) for the indicated time intervals (A). In selected experiments, T/C-28a2 cells were incubated with the ERK1/2 inhibitor PD98059 (20 μm) or U0126 (10 μm), the PI3K/Akt inhibitor LY294002 (10 μm) or wortmannin (1 μm), the JNK inhibitor SP600125 (10 μm or 20 μm), or the p38 inhibitor SB203580 (5 μm or 10 μm) in the absence or presence of IL-1β (100 ng/ml) for 48 h (B–D). In distinct experiments, cells were transfected with a siRNA oligonucleotide sequence specific for Akt, ERK1/2, p38, or c-Jun in the absence or presence of IL-1β (100 ng/ml) for 48 h (E and F). Phosphorylated Akt (Ser-473), ERK1/2 (Thr-202/Tyr-204), and c-Jun (Ser-63) and total Akt, ERK1/2, and c-Jun protein expression are shown by immunoblotting using specific Abs. Equal loading in each lane is ensured by the similar intensities of β-actin (A, B, and E). The intensity of bands was quantified relative to β-actin for each treatment using the Bio-Rad gel image system. MMP-9 mRNA expression and activity levels were determined by qRT-PCR and zymography, respectively (C, D, and F). GAPDH and MMP-2 served as internal controls in qRT-PCR and zymography assays, respectively. These gels are representative of three independent experiments, all revealing similar results. Data represent the mean ± S.E. of three independent experiments. *, p < 0.05 as compared with the control; ▴, p < 0.05 with respect to IL-1β treatment.