AP-1, NF-κB, and C/EBP-β are not involved in hyperosmotic induction of COX-2.
A, luciferase assay using AP-1 reporter shows that AP-1 activity is up-regulated by hyperosmolarity. B, COX-2 promoter activity under hyperosmotic condition is significantly decreased by DN–AP-1. C and D, AP-1 inhibition by a specific inhibitor, SR11302, does not block hyperosmotic induction of COX-2. E, COX-2 promoter activity is decreased by DN–NF-κB, but unaltered by DN–C/EBP-β. F, unlike the promoter activities, COX-2 mRNA levels are further up-regulated by NF-κB inhibitor, SM7368, under hyperosmotic condition. G–I, NF-κB inhibition resulted in further up-regulation of COX-2 protein levels under hyperosmotic condition, but had no effect when the cells were treated with ionomycin/PMA. All the quantitative data are represented as mean ± S.D. from at least three independent experiments (three biological replicates). Promoter activity experiments were done with three technical replicates per independent experiment. NS: nonsignificant; *, p < 0.05. SR: SR11302 (AP-1 inhibitor); SM: SM7368 (NF-κB inhibitor); I + P: ionomycin/PMA.