Figure 6

ET-1-stimulated COX-2 promoter activity is mediated through AP-1-dependent pathway. (A) Time dependence of ET-1-enhanced AP-1 transcription activity; cells were transfected with an AP-1-luciferase reporter gene and then exposed to ET-1 for the indicated time intervals. (B) After transfection with AP-1-luciferase reporter gene, the cells were pretreated with PP1 PP1 (100 nM), AG1478 (AG, 1 μM), LY294002 (LY, 300 nM), SH-5 (10 nM), U0126 (U0, 1 μM), SB202190 (SB, 300 nM), SP600125 (SP, 300 nM), or (A) TSIIA (100 nM) for 1 h and then incubated with ET-1 (10 nM) for 90 min. (C) Cells were pretreated without or with TSIIA, U0126 (U0), SB202190 (SB), SP600125 (SP), or BQ788 (BQ) for 1 h and then incubated with ET-1 (10 nM) for the indicated time intervals (upper panel) or 90 min (lower panel). The c-Jun/AP-1 binding activity was analyzed by chromatin-IP (ChIP)-PCR assay. (D) For COX-2 promoter activity, cells were pretreated with PP1, AG1478 (AG), LY294002 (LY), SH-5, U0126 (U0), SB202190 (SB), or SP600125 (SP), or TSIIA for 1 h and then incubated with ET-1 (10 nM) for 6 h. (E) Schematic representation of a 5′-promoter regions of the mouse different COX-2 promoter constructs, either wild-type (WT) or mutated by single-point mutation of the AP-1 binding site (mu-AP-1) cloned to the pGL-luciferase reporter gene; the translational start site (+1) of the luciferase reporter gene is indicated by an arrow. Cells were transfected with WT COX-2 promoter reporter gene (WT) or AP-1 mutated COX-2 promoter reporter gene (mu-AP-1) and then incubated with or without ET-1 (10 nM) for 6 h. The promoter reporter activity was determined. Data are expressed as mean ± SEM of at least three individual experiments (n = 3). ^* P < 0.05, ^# P < 0.01 as compared with ET-1 alone.