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Figure 6 | Cell Communication and Signaling

Figure 6

From: Up-regulation of COX-2/PGE2 by endothelin-1 via MAPK-dependent NF-κB pathway in mouse brain microvascular endothelial cells

Figure 6

ET-1-stimulated COX-2 promoter activity is mediated through NF-κB-dependent pathway. (A) Time dependence of ET-1-enhanced NF-κB transcription activity, cells were transfected with a NF-κB-luciferase reporter gene and then exposed to ET-1 for the indicated times. (B) After transfection, the cells were pretreated with BQ-788 (1 μM), GPA2 (1 μM), GPA2A (1 μM), U0126 (1 μM), SB202190 (300 nM), SP600125 (300 nM), or (A) Bay11-7082 (10 nM) for 1 h and then incubated with ET-1 (10 nM) for 60 min. (C) Cells were pretreated with BQ-788, U0126, SB202190, or SP600125 for 1 h and then incubated with ET-1. The p65 NF-κB binding activity was analyzed by ChIP-PCR as described in Methods. (D) For COX-2 promoter activity, cells were transfected with a COX-2-promoter-luciferase reporter gene and then exposed to ET-1. After transfection, the cells were pretreated with BQ-788, GPA2, GPA2A, U0126, SB202190, SP600125, or Bay11-7082 for 1 h and then incubated with ET-1 for 6 h. (E) Schematic representation of a 5′-promoter regions of the mouse different COX-2 promoter constructs, either wild-type (WT) or mutation of the κB-binding site (mt-κB) fused to the pGL-luciferase reporter gene, the translational start site (+1) of the luciferase reporter gene was indicated by an arrow. Cells were transfected with WT COX-2 promoter reporter gene (WT-COX-2) or NF-κB mutated COX-2 promoter reporter gene (mt-κB-COX-2) and then incubated with or without ET-1 for 6 h. The promoter reporter assay was performed as described in Methods. (F) Cells were pretreated with BQ-123, BQ-788, GPA2, GPA2A, U0126, SB202190, SP600125, Bay11-7082, or transfected with p65 siRNA and then incubated with ET-1 for 6 h. The PGE2 levels were analyzed by EIA. Data are expressed as mean ± SEM of at least three individual experiments (n=3 in each group; #P<0.01 as compared with ET-1 alone).

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