Figure 5. TNF-induced, NF-κB-dependent COX-2 mRNA expression and PGE₂ secretion can be repressed by FTY720 and FTY720-P.

(A) To demonstrate that unlike S1P, TNF induced IκB-α degradation (a surrogate measure of NF-κB activation), A549 cells were stimulated with TNF (4 ng/ml) or S1P (1 μM) for 0, 10, 30, and 60 min. Cells were then lysed and IκB-α analyzed by Western blotting, compared to α-tubulin as a loading control. (B,C) A549 cells were pretreated with 2.5 μM FTY720 or FTY720-P for 6 h, compared to vehicle-treated controls. Cells were treated with vehicle or TNF (4 ng/ml) and then (B) COX-2 mRNA expression measured (results expressed as % TNF-induced COX-2 mRNA expression (at 2 h)) and (C) secreted PGE₂ at 24 h measured by enzyme immunoassay. Statistical analysis was performed using the Student’s unpaired t test (where * denotes a significant effect of TNF compared to vehicle-treated cells, and § denotes significant repression by FTY720 or FTY720-P (P < 0.05)). Data are mean + SEM values from (A) n = 3 and (B,C) n = 4 independent experiments.