SIRT1 Suppresses The Expression Of NFAT-dependent Inflammatory Genes In Activated Endothelial Cells

Aim-To elucidate the role of histone deacetylase SIRT1 on NFAT transcriptional activity, and the effects of SIRT1 on inflammatory gene expression and inflammatory response by inhibiting NFAT transcriptional activity in HUVEC.Background-The silent information regulator 2 (Sir2) protein family (sirtuins or SIRTs) belongs to class III histone/protein deacetylases (HDACs). SIRT1, the mammalian homolog of Sir2 also mediates a variety of physiological processes such as life span, fat mobilization. SIRT1 has been reported to deacetylate a broad array of targets including histones (H3, H4), transcription factors (p53, FOXO, NFkB) and transcriptional cofactors (p300, NcoR, PGC-1?). In addition, recent studies point to SIRTl as a key regulator of vascular endothelial homeostasis controlling angiogenesis, vascular tone and endothelial dysfunction, especially probably for anti-inflammation. However, the role of SIRT1 in anti-inflammation in vascular endothelial cell and its underlying mechanism still remains unclear.The classical members of NFAT family proteins include NFATc1, NFATc2, NFATc3, NFATc4 and NFAT5. Four of NFAT members including NFATc3 are regulated by Ca2+ signal pathway. In response to growth factors, cytokines, oxidative stress, NFAT binds to the promoters of inducible genes and alters their expression, which are involved in cell proliferation, inflammation and so on. NFAT plays an important role in angiogenesis and pro-inflammation in vascular system. NFAT targeted genes, such as COX-2, ICAM-1, play an important role in endothelial inflammation. However, limited information is available regarding the regulating mechanisms of that NFAT is involved in inflammation in vascular endothelial cell.Methods-Using Real-time PCR and Western Blotting (WB), we detected the inflammatory genes expression in PMA/Ionomycin (Io) induced endothelial cells. To determine whether NFATc3 was activated by PMA/Io, we examined the nuclear translocation of NFATc3 induced by PMA/Io through immunofluorescence. To further confirm that NFATc3 mediates the expression of inflammatory genes induced by PMA/Io in endothelial cells, we used NFAT inhibitor CsA to pre-treat endothelial cells, and detected the effect of CsA on the expression of inflammatory genes induced by PMA/Io. Using Real-time PCR and WB, we detected the effects of SIRT1 overexpression, SIRT1 activator resveratrol and inhibitor Sirtinol on NFAT targeting inflammatory genes expression in PMA/Io induced endothelial cells. To examine whether SIRT1 represses NFATc3 transcriptional activity, we tranfected with NFAT luciferase plasmid and pcDNA3.1 or pcDNA3.1 SIRT1 plasmid in HEK293 cells. Using immunoprecipitation, we detected whether NFATc3 interacted with SIRT1, which domain of NFATc3 mediated the interaction between NFATc3 and SIRT1, and whether SIRT1 deacetylated NFATc3.Results-We show here that inflammatory genes expression are upregulated in PMA/Io induced endothelial cells. NFATc3 mediates the expression of inflammatory gene induced by PMA/Io. SIRT1 expression is also upregulated in PMA/Io induced endothelial cells. SIRT1 inhibits NFAT targeting inflammatory genes expression in PMA/Io induced endothelial cells. SIRT1 inhibits NFATc3 transcriptional activity basically and PMA/Io induced. NFATc3 interacts with SIRT1. NHR and RHR domains of NFATc3 mediate the interaction between NFATc3 and SIRT1. Finally, NFATc3 is acetylated by p300 and deacetylatd by SIRT1.Conclusion-This report demonstrates for the first time that NFAT is a new target of SIRT1. SIRT1 inhibits NFATc3 transcriptional activity by deacetylating NFATc3. And, SIRT1 suppresses PMA/Io induced NFATc3 dependent inflammatory genes expression in endothelial cells, leading to inflammation inhibition. PMA/Io induces inflammatory genes expression as well as upregulates the SIRT1 expression, which counteracts the NFATc3 transcriptional activity and abrogates inflammatory genes expression as a negative feedback mechanism.