SIRT1 suppresses NFAT-induced COX-2 promoter activity and reduces NFAT binding to the promoter of COX-2.

<p>(A) HEK293 cells were transfected with 0.1 µg COX-2 luciferase reporter (COX-2 -luc), 30 ng pRL-TK, and 0.3 µg SIRT1 expression vector or control (pcDNA3.1) vectors for 24 h and were treated with PMA (10 ng/ml) and Io (0.25 µM), or vehicle DMSO for another 3 h. Luciferase activities are presented as the means ± standard deviation (S.D.) of triplicate samples and are representative of three independent experiments. **, p≤0.01 versus control. *, p≤0.05 versus control. (B–D) HEK293 cells were transfected with 0.1 µg COX-2 luciferase reporter (COX-2 -luc), 30 ng pRL-TK, 0.3 µg SIRT1 expression vector or control (pcDNA3.1) vector, and 0.3 µg NFATc1 (B), or NFATc2 (C), or NFATc3 (D) for 24 h. Luciferase activities are presented as the means ± standard deviation (S.D.) of triplicate samples and are representative of three independent experiments.**, p≤0.01 versus control. *, p≤0.05 versus control. (E) Diagrammatic drawing shows the binding sites of NFAT and NF-κB on human COX-2 promoter. There are two NFAT cis-acting elements named NFAT distal (dNFAT, −102 to −96 bp) and NFAT proximal (pNFAT, −73 to −67 bp) elements locating upstream of the transcription starting site of COX-2. There is a NF-κB cis-acting element (−447 to −439 bp) locating upstream of the transcription starting site of COX-2. And the designed primers covered these sites are shown. (F) HUVECs were infected with Ad-SIRT1 or control Ad-GFP for 24 h, and then were treated with PMA (10 ng/ml) and Io (0.25 µM) for 3 h. ChIP assays were performed with chromatin prepared from HUVECs. Chromatin was immunoprecipitated with normal rabbit IgG, antibody against NFATc3 or NF-κB p65, and precipitated genomic DNA was analyzed by real-time PCR using primers for the NFAT or NF-κB p65 binding site on COX-2 promoter or 3′-UTR region of COX-2 gene. Data was shown as the mean ± standard deviation (S.D.) for three independent experiments. **, p≤0.01 versus indicated group.*, p≤0.05 versus indicated group.