Fig. 5
cAMP, IGF-2 and 15d-PGJ2 are critical for MMP-12 expression via PI3-K-dependent NF-κB-, c-Jun-, and PPARγ-activating mechanisms in human chondrocytes. T/C-28a2 chondrocytes were treated with (a, c) forskolin (10 μM) or (b, d) IGF-2 (10 ng/ml) in the absence or presence of the PI3-K inhibitor LY294002 (10 μM), NF-κB inhibitor QNZ (1 μM) or JNK inhibitor SP600125 (10 μM) for 48 h. In select experiments, (e) T/C-28a2 chondrocytes were treated with 15d-PGJ2 (500 nM) in the absence or presence of the PPARγ antagonist GW9662 (1 μM) for 48 h. f In separate experiments, T/C-28a2 chondrocytes were subjected to fluid shear stress (20 dyn/cm2) or static conditions (0 dyn/cm2) in the absence or presence of the PPARγ antagonist GW9662 (1 μM) for 48 h. a and b Phosphorylated Akt (Ser473), p65 (Ser 536), c-Jun (Ser 63), total Akt, p65, and c-Jun protein expressions are shown by immunoblotting using specific Abs. Equal loading in each lane is ensured by the similar intensities of β-actin. c–f MMP-12 mRNA and protein levels were determined by qRT-PCR and western blots, respectively. GAPDH and MMP-11 served as internal controls in qRT-PCR and western blots, respectively. Data represent the mean ± SE of at least three independent experiments. *p < 0.05 with respect to vehicle-treated control. # p < 0.05 with respect to forskolin, IGF-2 or 15d-PGJ2 treatment
