| Target-To elucidate the transcriptional repression function of the histone deacetylase Sirtl on AP-1, and the effects of Sirtl on macrophage COX-2 expression and macrophage inflammatory response and functions.Background-The silent information regulator 2 (Sir2) protein family (sirtuins or SIRTs) belongs to classⅢhistone/protein deacetylases (HDACs). Unlike the other classⅠandⅡHDACs, which require zinc for deacetylation, Sirtuins require nicotinamide adenosine dinucleotide (NAD+) as a cofactor. Sirtl, the mammalian homolog of Sir2 also mediate a variety of physiological processes such as life span, fat mobilization. Sirtl has been reported to deacetylate a broad array of targets including histones (H3, H4), transcription factors (p53, FOXO, NFκB) and transcriptional integrators (p300, NcoR, PGC-1α). AP-1, a transcriptional factor composed of homodimers or heterodimers of Jun, Fos, Maf and ATF families, is known to be proto-oncogenes. In response to growth factors, cytokines, oxidative stress and PMA, AP-1 could bind to the various promoters and alter the expression of genes, which are involved in cell proliferation, inflammation and so on. The c-Jun and c-Fos are the major component of cellular AP-1. The activity of AP-1 is regulated at many levels: the transcription of c-Fos and c-Jun genes, the post-translational modification of Fos and Jun proteins. Among AP-1 modifications, phosphorylation has been the most extensively studied. Sumoylation of c-Fos and c-Jun has been shown as another covalent modification of AP-1, which may down-regulate AP-1 transcriptional activity. In addition, p300-mediated acetylation of AP-1 has been reported recently. As for all bZIP transcription factors, AP-1 activity is also regulated by the composition of dimerization that is required for binding to DNA. The leucine zipper domain of c-Fos/c-Jun is required for dimerization and the basic region functions to bind to DNA. Cyclooxygenase-2(COX-2), the rate-limiting enzyme for Prostaglandins (PGs) production, has been one of the most intensively studied AP-1 target gene.Methods-To examine the ability of Sirtl to repress AP-1 transcriptional activity, we tranniently transfected HEK293 cells with AP-1 luciferase plasmid and pcDNA3.1 or pcDNA3.1 Sirtl. Using immunoprecipitation, we identified the domain of c-Fos, c-Jun that mediated the association between AP-1 and Sirtl. We detect the AP-1 DNA binding activity by EMSA. Using luciferase assay, ChIP, western blotting and PGE2 measurement, we examined the effect of Sirtl on COX-2, one of classic AP-1 target genes. We further detected the effects of Sirtl on macrophage function including phagocytosis and tumoricidal function. Using calorie restriction moedel to mimic the overexpression of Sirtl in mice, we detect the effects of calorie restriction on macrophage COX-2 expression and macrophage function.Results-We show here that Sirtl depresses the transcriptional activity of activator protein-1(AP-1) and directly interacts with basic DNA-binding region of c-Fos and c-Jun, which are the major components of AP-1 in most cells. c-Fos and c-Jun can be acetylated by p300 and the acetylation is reduced by the overexpression of Sirtl. Unexpectedly, the dominant-negative mutant Sirtl HY could also bind to c-Fos and c-Jun and suppress AP-1 transcriptional activity. Sirtl reduces COX-2, a typical AP-1 target gene, and improves the phagocytosis and tumoricidal function of peritoneal macrophages (MΦ). Calorie restriction (CR) could mimic the effects of Sirtl overexpression in MΦ.Conclusion-To our knowledge, this report demonstrates for the first time that Sirtl could interact with and deacetylate AP-1 to regulate its transcriptional activity. The results of this study may provide additional insight into the mechanism underlying the effects of CR and Sirt1. We hypothesize that PMA induce COX-2 expression by AP-1 and meantime PMA still increase the Sirt1, which could counteractive the AP-1 transcriptional activity and abrogate COX-2 transcriptional expression as a feedback mechanism.
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