FIGURE 4.

Shear stress induces phosphorylation of Akt, CREB, and p65 in human chondrocytes via a COX-2/EP2/EP3-dependent pathway. T/C-28a2 cells were subjected to shear stress (20 dyn/cm²) or static conditions (0 dyn/cm²) for 1 or 2 h in the absence or presence of the selective COX-2 inhibitor NS398 (30 μm) (A), the EP2 receptor antagonist AH6809 (3 μm) or the EP3 receptor agonist sulprostone (1 μm) (B). In select experiments, T/C-28a2 chondrocytes were transfected with an EP2 shRNA plasmid or scramble shRNA control before their exposure to fluid shear (C). In separate experiments, cells were transfected with either a plasmid containing the cDNA of EP3 receptor or the empty vector prior to shear exposure (C). Phosphorylated Akt (Ser-473), CREB (Ser-133), and p65 (Ser-536 and Ser-276) are showing by immunoblotting using specific Abs. Equal loading in each lane is ensured by the similar intensities of total Akt, CREB, p65, and β-actin. These Western blots are representative of three independent experiments, all revealing similar results.