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Abdominal aortic aneurysm (AAA) is a vascular degenerative disease. Macrophage polarization and the balance between classically activated macrophages (M1) and alternatively activated macrophages (M2) are crucial for AAA pathogenesis. The present study aims to investigate the roles of macrophage SIRT1 in AAA formation and macrophage polarization. We found that in mouse peritoneal macrophages, SIRT1 expression was decreased after M1 stimulation, but was enhanced after M2 stimulation. Results from SIRT1 flox/flox mice and macrophage specific SIRT1 knockout mice with treatment of angiotensin II (Ang II) for 4 weeks showed that macrophage specific deficiency of SIRT1 increased the incidence of AAA and exacerbated the severity, including more severe aneurysm types, enlarged diameter of the aneurysm and increased degradation of elastin. In mouse aortas, SIRT1 deficiency increased the pro-inflammatory M1 molecule inducible nitric oxide synthase (iNOS), and decreased M2 molecules such as arginase 1 (Arg1) and mannose receptor (MR). Furthermore, in peritoneal macrophages, SIRT1 deficiency increased the expression of M1 inflammatory molecules, but decreased the expression of M2 molecules. Overexpression of SIRT1 had the opposite effects. Thus, macrophage specific knockout of SIRT1 influences macrophage polarization and accelerates Ang II-induced AAA formation., (Copyright © 2018 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

The homeodomain transcription factor Nkx2.5/Csx is critically essential for heart specification, morphogenesis, and homeostasis. Acetylation/deacetylation is important for the localization, stability and activation of transcription factors. It remains unknown how Nkx2.5 is deacetylated and how Nkx2.5 acetylation determines its activity. In this study, we provide evidence that the NAD + -dependent class III protein deacetylase SIRT1 deacetylates Nkx2.5 in cardiomyocytes and represses the transcriptional activity of Nkx2.5. We show that SIRT1 interacts with the C-terminus of Nkx2.5 and deacetylates Nkx2.5 at lysine 182 in the homeodomain. The mutation of Nkx2.5 at lysine 182 reduces its transcriptional activity. Furthermore, SIRT1 inhibits the transcriptional activity of Nkx2.5 and represses the expression of its target genes partly by reducing Nkx2.5 binding to its co-factors, including SRF and TBX5. Taken together, these findings demonstrate that SIRT1 deacetylates Nkx2.5 and inhibits the transcriptional activity of Nkx2.5.

Rationale: Uncontrolled growth of abdominal aortic aneurysms (AAAs) is a life-threatening vascular disease without an effective pharmaceutical treatment. AAA incidence dramatically increases with advancing age in men. However, the molecular mechanisms by which aging predisposes individuals to AAAs remain unknown., Objective: In this study, we investigated the role of SIRT1 (Sirtuin 1), a class III histone deacetylase, in AAA formation and the underlying mechanisms linking vascular senescence and inflammation., Methods and Results: The expression and activity of SIRT1 were significantly decreased in human AAA samples. SIRT1 in vascular smooth muscle cells was remarkably downregulated in the suprarenal aortas of aged mice, in which AAAs induced by angiotensin II infusion were significantly elevated. Moreover, vascular smooth muscle cell-specific knockout of SIRT1 accelerated angiotensin II-induced formation and rupture of AAAs and AAA-related pathological changes, whereas vascular smooth muscle cell-specific overexpression of SIRT1 suppressed angiotensin II-induced AAA formation and progression in Apoe - /- mice. Furthermore, the inhibitory effect of SIRT1 on AAA formation was also proved in a calcium chloride (CaCl 2 )-induced AAA model. Mechanistically, the reduction of SIRT1 was shown to increase vascular cell senescence and upregulate p21 expression, as well as enhance vascular inflammation. Notably, inhibition of p21-dependent vascular cell senescence by SIRT1 blocked angiotensin II-induced nuclear factor-κB binding on the promoter of monocyte chemoattractant protein-1 and inhibited its expression., Conclusions: These findings provide evidence that SIRT1 reduction links vascular senescence and inflammation to AAAs and that SIRT1 in vascular smooth muscle cells provides a therapeutic target for the prevention of AAA formation., (© 2016 American Heart Association, Inc.)

Mouse somatic cells can be reprogrammed into induced pluripotent stem cells by defined factors known to regulate pluripotency, including Oct4, Sox2, Klf4, and c-Myc. Together with Oct4, Sox2 plays a major role as a master endogenous pluripotent genes trigger in reprogramming. It has been reported that Sirtuin 1 (Sirt1), a member of the Sirtuin family of NAD(+) -dependent protein deacetylases, is involved in embryonic stem cell antioxidation, differentiation, and individual development. However, as a deacetylation enzyme, whether Sirt1 influences reprogramming through its post-translational modification function remains unknown. In this study, we provide evidence that deacetylation of Sox2 by Sirt1 is required for reprogramming. We found that a low level of Sox2 acetylation could significantly increase reprogramming efficiency. Furthermore, we found that Sox2 can be deacetylated by Sirt1 in an Oct4-mediated manner. Compared with wild-type cells, Sirt1-null mouse embryonic fibroblasts exhibit decreased reprogramming efficiency, and overexpression of Sirt1 rescues this defect. In addition, Sirt1 functions in the regulation of reprogramming through deacetylating Sox2. Taken together, we have identified a new regulatory role of Sirt1 in reprogramming and provided a link between deacetylation events and somatic cell reprogramming. Stem Cells 2015;33:2135-2147., (© 2015 AlphaMed Press.)

Pulmonary arterial hypertension (PAH) is a major cause of morbidity and mortality in rheumatic diseases. Vascular remodeling due to the proliferation of pulmonary arterial smooth muscle cells (PASMCs) is central to the development of PAH. To date, it is still unclear if Silence Information Regulator 1 (SIRT1) regulates cell cycle regulators in the proliferation of PASMCs and contributes to prevention of PAH by resveratrol. In this study, we found that a significant decrease of SIRT1 expression levels in platelet-derived growth factor BB (PDGF-BB) treated human PASMCs (HPASMCs) and in monocrotaline (MCT) induced PAH rat. Overexpression of SIRT1 induced G1 phase arrest and increased p21 expression but decreased cyclin D1 expression in PDGF-BB treated HPASMCs. Moreover, resveratrol attenuated pulmonary arterial remodeling, decreased pulmonary arterial pressure, and upregulated SIRT1 and p21 expression but downregulated cyclin D1 expression in MCT induced PAH rat. Notably, knockdown of SIRT1 eliminated the regulation of resveratrol on p21 and cyclin D1 expression in PDGF-BB treated HPASMCs. These results demonstrated that SIRT1 mediated the regulation of resveratrol on the expression of cell cycle regulatory molecules. It suggests that SIRT1 exerts a protective role in PAH associated with rheumatic diseases and can be a potential treatment target.

The inactivation of plasminogen activator inhibitor-1 (PAI-1) has been shown to exert beneficial effects in age-related vascular diseases. Limited information is available on the molecular mechanisms regarding the negatively regulated expression of PAI-1 in the vascular system. In this study, we observed an inverse correlation between SIRT1, a class III histone deacetylase, and PAI-1 expression in human atherosclerotic plaques and the aortas of old mice, suggesting that internal negative regulation exists between SIRT1 and PAI-1. SIRT1 overexpression reversed the increased PAI-1 expression in senescent human umbilical vein endothelial cells (HUVECs) and aortas of old mice, accompanied by decreased SA-β-gal activity in vitro and improved endothelial function and reduced arterial stiffness in vivo. Moreover, the SIRT1-mediated inhibition of PAI-1 expression exerted an antisenescence effect in HUVECs. Furthermore, we demonstrated that SIRT1 is able to bind to the PAI-1 promoter, resulting in a decrease in the acetylation of histone H4 lysine 16 (H4K16) on the PAI-1 promoter region. Thus, our findings suggest that the SIRT1-mediated epigenetic inhibition of PAI-1 expression exerts a protective effect in vascular endothelial senescence., (© 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

SIRT1, a class III histone deacetylase, acts as a negative regulator for many transcription factors, and plays protective roles in inflammation and atherosclerosis. Transcription factor nuclear factor of activated T cells (NFAT) has been previously shown to play pro-inflammatory roles in endothelial cells. Inhibition of NFAT signaling may be an attractive target to regulate inflammation in atherosclerosis. However, whether NFAT transcriptional activity is suppressed by SIRT1 remains unknown. In this study, we found that SIRT1 suppressed NFAT-mediated transcriptional activity. SIRT1 interacted with NFAT, and the NHR and RHR domains of NFAT mediated the interaction with SIRT1. Moreover, we found that SIRT1 primarily deacetylated NFATc3. Adenoviral over-expression of SIRT1 suppressed PMA and calcium ionophore Ionomycin (PMA/Io)-induced COX-2 expression in human umbilical vein endothelial cells (HUVECs), while SIRT1 RNAi reversed the effects in HUVECs. Moreover, inhibition of COX-2 expression by SIRT1 in PMA/Io-treated HUVECs was largely abrogated by inhibiting NFAT activation. Furthermore, SIRT1 inhibited NFAT-induced COX-2 promoter activity, and reduced NFAT binding to the COX-2 promoter in PMA/Io-treated HUVECs. These results suggest that suppression of NFAT transcriptional activity is involved in SIRT1-mediated inhibition of COX-2 expression induced by PMA/Io, and that the negative regulatory mechanisms of NFAT by SIRT1 may contribute to its anti-inflammatory effects in atherosclerosis.

Unlabelled: Angiotensin II (AngII) induces the development of vascular hypertrophy and hypertension. We have shown previously that overexpression of class III deacetylase SIRT1 inhibits AngII-induced hypertrophy in vascular smooth muscle cells (VSMCs). However, the direct role of SIRT1 in VSMCs in response to AngII infusion in vivo remains unclear. Here, we found that the expression and activity of SIRT1 in mouse aortas was decreased significantly by AngII infusion. VSMC-specific SIRT1 transgene (SV-Tg) prevented the increase in systolic blood pressure (SBP) caused by AngII infusion without affecting heart function in mice. SIRT1 overexpression alleviated vascular remodeling in mouse thoracic and renal aortas induced by AngII infusion, and significantly inhibited reactive oxygen species (ROS) generation, vascular inflammation, and collagen synthesis in arterial walls. Reduced expression of transforming growth factor-β 1 (TGF-β1) was also observed in the aortas of AngII-infused SV-Tg mice. Moreover, SIRT1 overexpression decreased AngII-increased binding of nuclear factor-κB on its specific binding sites on TGF-β1 promoter. Taken together, these data demonstrate that SIRT1 overexpression in VSMCs reduces SBP and inhibits AngII-induced vascular remodeling in mice. The inhibition of vascular remodeling contributes, at least in part, to the antihypertensive effect of SIRT1., Key Message: SIRT1 is reduced in aortas of AngII-infused hypertensive mice. SIRT1 VSMC transgene alleviates AngII-increased systolic blood pressure. SIRT1 VSMC transgene attenuates AngII-induced vascular remodeling. VSMC SIRT1 overexpression inhibits remodeling-related pathological changes. VSMC SIRT1 overexpression reduces AngII-induced TGF-β1 expression.

Accumulating evidence indicates that oxidative stress can occur through overproduction of reactive oxygen species (ROS) and/or reduced anti-oxidant potentials under pathophysiological conditions and plays an important role in the development of cardiovascular diseases (CVDs). Adapter protein p66Shc has the property to directly stimulate mitochondrial ROS generation by an oxidoreductase activity. A growing body of evidence implies that p66Shc plays a critical role in the pathophysiology of age-related vascular diseases. Silent mating type information regulator 2 homolog 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD+)-dependent class III histone deacetylase (HDAC), has also been implicated in protection against vascular aging and age-related vascular diseases. Recently, we demonstrated that SIRT1 protects blood vessels from hyperglycemia-induced endothelial dysfunction through a novel mechanism involving the downregulation of p66Shc expression. In this review, we discuss the cross-talk between these two longevity genes as a mechanism of preventing vascular diseases by involving anti-oxidative stress responses and inhibiting endothelial senescence., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Nkx2.5 plays protective roles in cardiac homeostasis and survival in the postnatal hearts. However, the underlying molecular mechanisms that mediate the protective functions of Nkx2.5 remain unknown. Here, we showed that Nkx2.5 was downregulated in response to various stresses and was required for protection against the stress-induced apoptosis of cardiomyocytes. SIRT1, a member of the sirtuin family of proteins, was found to be a direct transcriptional target of Nkx2.5 and was required for the Nkx2.5-mediated protection of cardiomyocytes from doxorubicin (DOX)-induced apoptosis. Moreover, using chromatin immunoprecipitation assays, we found that Nkx2.5 was able to bind to the SIRT1 promoter and that this binding was significantly decreased in DOX-treated mouse hearts. Furthermore, the cardiac-specific overexpression of SIRT1 decreased the DOX-induced apoptosis of cardiomyocytes in SIRT1 transgenic mouse hearts compared with the hearts of their wild-type littermates. These findings demonstrate that SIRT1 acts as a direct transcriptional target of Nkx2.5 that maintains cardiomyocyte homeostasis and survival.

Objective: To study the role of sirtuin 1 (SIRT1) in Fas ligand (FasL) expression regulation during vascular lesion formation and to elucidate the potential mechanisms., Methods: SIRT1 and FasL protein levels were detected by Western blotting in either mouse arteries extract or the whole rat aortic vascular smooth muscle cell (VSMC) lysate. Smooth muscle cell (SMC)-specific human SIRT1 transgenic (Tg) C57BL/6 mice and their littermate wild-type (WT) controls underwent complete carotid artery ligation (ligation groups) or the ligation-excluded operation (sham groups). The carotid arteries were collected 1 day after operation. Reverse transcription-polymerase chain reaction was performed to detect the mRNA levels of SIRT1 and FasL. Luciferase reporter assays were performed to detect the effect of WT-SIRT1, a dominant-negative form of SIRT1 (SIRT1H363Y), and GATA-6 on the promoter activity of FasL. Flow cytometry assay was applied to measure the hypodiploid DNA content of VSMC so as to monitor cellular apoptosis., Results: SIRT1 was expressed in both rat aortic VSMCs and mouse arteries. Forced SIRT1 expression increased FasL expression both in injured mouse carotid arteries 1 day after ligation (P<0.001) and VSMCs treated with serum (P<0.05 at the transcriptional level, P<0.001 at the protein level). No notable apoptosis was observed. Furthermore, transcription factor GATA-6 increased the promoter activity of FasL (P<0.001). The induction of FasL promoter activity by GATA-6 was enhanced by WT-SIRT1 (P<0.001), while SIRT1H363Y significantly relieved the enhancing effect of WT-SIRT1 on GATA-6 (P<0.001)., Conclusions: Overexpression of SIRT1 up-regulates FasL expression in both flow-restricted mouse carotid arteries and serum-stimulated VSMCs. The transcription factor GATA-6 participates in the transcriptional regulation of FasL expression by SIRT1.

The higher order chromatin structure has recently been revealed as a critical new layer of gene transcriptional control. Changes in higher order chromatin structures were shown to correlate with the availability of transcriptional factors and/or MAR (matrix attachment region) binding proteins, which tether genomic DNA to the nuclear matrix. How posttranslational modification to these protein organizers may affect higher order chromatin structure still pending experimental investigation. The type III histone deacetylase silent mating type information regulator 2, S. cerevisiae, homolog 1 (SIRT1) participates in many physiological processes through targeting both histone and transcriptional factors. We show that MAR binding protein SATB1, which mediates chromatin looping in cytokine, MHC-I and β-globin gene loci, as a new type of SIRT1 substrate. SIRT1 expression increased accompanying erythroid differentiation and the strengthening of β-globin cluster higher order chromatin structure, while knockdown of SIRT1 in erythroid k562 cells weakened the long-range interaction between two SATB1 binding sites in the β-globin locus, MAR(HS2) and MAR(ε). We also show that SIRT1 activity significantly affects ε-globin gene expression in a SATB1-dependent manner and that knockdown of SIRT1 largely blocks ε-globin gene activation during erythroid differentiation. Our work proposes that SIRT1 orchestrates changes in higher order chromatin structure during erythropoiesis, and reveals the dynamic higher order chromatin structure regulation at posttranslational modification level.

The c-Myc oncoprotein plays critical roles in multiple biological processes by controlling cell proliferation, apoptosis, differentiation, and metabolism. Especially, c-Myc is frequently overexpressed in many human cancers and widely involved in tumorigenesis. However, how the post-translational modifications, especially acetylation of c-Myc, contribute to its activity in the leukemia cells remains largely unknown. Sirt1, a NAD-dependent class III histone deacetylase, has a paradoxical role in tumorigenesis by deacetylating several transcription factors, including p53, E2F1 and forkhead proteins. In this study, we show that Sirt1 interacts physically with the C-terminus of c-Myc and deacetylates c-Myc both in vitro and in vivo. Moreover, the deacetylation of c-Myc by Sirt1 promotes its association with Max, a partner essential for its activation, thereby facilitating c-Myc transactivation activity on hTERT promoter. Finally, inhibition of endogenous Sirt1 in K562 cells by either RNAi or its inhibitor NAM causes the overall decrease of c-Myc target genes expression, including hTERT, cyclinD2 and LDHA, which further suppress cell proliferation and arrest cell cycle at G1/S phase. Thus, our results demonstrate the positive effect of Sirt1 on c-Myc activity by efficiently enhancing c-Myc/Max association in human leukemia cell line K562, suggesting a potential role of Sirt1 in tumorigenesis., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Rationale: Inactivation of the p66Shc adaptor protein confers resistance to oxidative stress and protects mice from aging-associated vascular diseases. However, there is limited information about the negative regulating mechanisms of p66Shc expression in the vascular system., Objective: In this study, we investigated the role of SIRT1, a class III histone deacetylase, in the regulation of p66Shc expression and hyperglycemia-induced endothelial dysfunction., Methods and Results: Expressions of p66Shc gene transcript and protein were significantly increased by different kinds of class III histone deacetylase (sirtuin) inhibitors in human umbilical vein endothelial cells and 293A cells. Adenoviral overexpression of SIRT1 inhibited high-glucose-induced p66Shc upregulation in human umbilical vein endothelial cells. Knockdown of SIRT1 increased p66Shc expression and also increased the expression levels of plasminogen activator inhibitor-1 expression, but decreased manganese superoxide dismutase expression in high-glucose conditions. However, knockdown of p66Shc significantly reversed the effects of SIRT1 knockdown. In addition, p66Shc overexpression significantly decreased manganese superoxide dismutase expression and increased plasminogen activator inhibitor-1 expression in high-glucose conditions, which were recovered by SIRT1 overexpression. Moreover, compared to streptozotocin-induced wild-type diabetic mice, endothelium-specific SIRT1 transgenic diabetic mice had decreased p66Shc expression at both the mRNA and the protein levels, improved endothelial function, and reduced accumulation of nitrotyrosine and 8-OHdG (markers of oxidative stress). We further found that SIRT1 was able to bind to the p66Shc promoter (-508 bp to -250 bp), resulting in a decrease in the acetylation of histone H3 bound to the p66Shc promoter region., Conclusion: Our findings indicate that repression of p66Shc expression by SIRT1 contributes to the protection of hyperglycemia-induced endothelial dysfunction.

A wide variety of nuclear regulators and enzymes are subjected to acetylation of the lysine residue, which regulates different aspects of protein functions. The MYST family histone acetyltransferase, human ortholog of MOF (hMOF), plays critical roles in transcription activation by acetylating nucleosomal H4K16. In this study, we found that hMOF acetylates itself in vitro and in vivo, and the acetylation is restricted to the conserved MYST domain (C2HC zinc finger and HAT), of which the K274 residue is the major autoacetylation site. Furthermore, the class III histone deacetylase SIRT1 was found to interact with the MYST domain of hMOF through the deacetylase catalytic region and deacetylate autoacetylated hMOF. In vitro binding assays showed that non-acetylated hMOF robustly binds to nucleosomes while acetylation decreases the binding ability. In HeLa cells, the recruitment of hMOF to the chromatin increases in response to SIRT1 overexpression and decreases after knockdown of SIRT1. The acetylation mimic mutation K274Q apparently decreases the chromatin recruitment of hMOF as well as the global H4K16Ac level in HeLa cells. Finally, upon SIRT1 knockdown, hMOF recruitment to the gene body region of its target gene HoxA9 decreases, accompanied with decrease of H4K16Ac at the same region and repression of HoxA9 transcription. These results suggest a dynamic interplay between SIRT1 and hMOF in regulating H4K16 acetylation.

Objective: To study the regulatory rolesof SIRT1 on EZH2 expression and the further effects on EZH 2’ s repression of target gene expression., Methods: The stable SIRT1 RNAi and Control RNAi HeLa cells were established by infection with retroviruses expressing shSIRT1 and shLuc respectively followed by puromycin selection. EZH2 protein level was detected by Western blot in either whole cell lysate or the fractional cell extract. Reverse transcription-polymerase chain reaction was performed to detect the mRNA level of EZH2. Cycloheximide was used to treat SIRT1 RNAi and Control RNAi cells for protein stability assay. Chromatin immunoprecipitation(ChIP) assay was applied to measure enrichment of SIRT1, EZH2, and trimethylated H3K27 (H3K27me3) at SATB1 promoter in SIRT1 RNAi and Control RNAi cells., Results: Western blot results showed that EZH2 protein level increased upon SIRT1 depletion. Fractional extraction results showed unchanged cytoplasmic fraction and increased chromatin fraction of EZH2 protein in SIRT1 RNAi cells. The mRNA level of EZH2 was not affected by knockdown of SIRT1. SIRT1 recruitment was not detected at the promoter regionof EZH2 gene locus. The protein stability assay showed that the protein stability of EZH2 increases upon SIRT1 knockdown. Upon SIRT1 depletion, EZH2 and H3K27me3 recruitment at SATB1 promoter increases and the mRNA level of SATB1 decreases., Conclusions: Depletion of SIRT1 increases the protein stability of EZH2. The regulation of EZH2 protein level by SIRT1 affects the repressive effects of EZH2 on the target gene expression.

Rationale: Vascular smooth muscle cell (VSMC) proliferation and migration are crucial events involved in the pathophysiology of vascular diseases. Sirtuin 1 (SIRT1), a class III histone deacetylase (HDAC), has been reported to have the function of antiatherosclerosis, but its role in neointima formation remains unknown., Objective: The present study was designed to investigate the role of SIRT1 in the regulation of neointima formation and to elucidate the underlying mechanisms., Methods and Results: A decrease in SIRT1 expression was observed following carotid artery ligation. smooth muscle cell (SMC)-specific human SIRT1 transgenic (Tg) mice were generated. SIRT1 overexpression substantially inhibited neointima formation after carotid artery ligation or carotid artery wire injury. In the intima of injured carotid arteries, VSMC proliferation (proliferating cell nuclear antigen (PCNA)-positive cells) was significantly reduced. SIRT1 overexpression markedly inhibited VSMC proliferation and migration and induced cell cycle arrest at G1/S transition in vitro. Accordingly, SIRT1 overexpression decreased the induction of cyclin D1 and matrix metalloproteinase-9 (MMP-9) expression by treatment with serum and TNF-α, respectively, whereas RNAi knockdown of SIRT1 resulted in the opposite effect. Decreased cyclin D1 and MMP-9 expression/activity were also observed in injured carotid arteries from SMC-SIRT1 Tg mice. Furthermore, 2 targets of SIRT1, c-Fos and c-Jun, were involved in the downregulation of cyclin D1 and MMP-9 expression., Conclusions: Our findings demonstrate the inhibitory effect of SIRT1 on the VSMC proliferation and migration that underlie neointima formation and implicate SIRT1 as a potential target for intervention in vascular diseases.

The proinflammatory cytokine TNF-alpha plays an important role in stimulating inflammatory responses of vascular smooth muscle cells (VSMCs). The anti-inflammatory function of Sirtuin 1 (SIRT1), a NAD-dependent class III histone/protein deacetylase, has been well documented, but how SIRT1 is regulated under inflammatory conditions is largely unknown. In the present research, we showed that levels of SIRT1 mRNA and protein expression increased in TNF-alpha-treated VSMCs. Overexpression of the p65/RelA subunit of NF-kappaB, a TNF-alpha-activated inflammatory transcription factor, in A7r5 cells, upregulated SIRT1 mRNA and protein expression as well as SIRT1 promoter activity, while knockdown of endogenous p65/RelA expression by RNAi not only led to a decrease in SIRT1's basal protein expression and promoter activity, but almost abolished the TNF-alpha-induced elevation of SIRT1 protein expression and SIRT1 promoter activity. Furthermore, using promoter deletion analysis and chromatin immunoprecipitation assays, we found that p65/RelA bound to the SIRT1 promoter at a consensus NF-kappaB binding site. Our study indicates that p65/RelA mediates the TNF-alpha-induced elevated expression of SIRT1 in VSMCs, shedding new light on the regulation of SIRT1 under inflammatory conditions.

SIRT1 (Sirtuin type 1), a mammalian orthologue of yeast SIR2 (silent information regulator 2), has been shown to mediate a variety of calorie restriction (CR)-induced physiological events, such as cell fate regulation via deacetylation of the substrate proteins. However, whether SIRT1 deacetylates activator protein-1 (AP-1) to influence its transcriptional activity and target gene expression is still unknown. Here we demonstrate that SIRT1 directly interacts with the basic leucine zipper domains of c-Fos and c-Jun, the major components of AP-1, by which SIRT1 suppressed the transcriptional activity of AP-1. This process requires the deacetylase activity of SIRT1. Notably, SIRT1 reduced the expression of COX-2, a typical AP-1 target gene, and decreased prostaglandin E(2) (PGE(2)) production of peritoneal macrophages (pMPhis). pMPhis with SIRT1 overexpression displayed improved phagocytosis and tumoricidal functions, which are associated with depressed PGE(2). Furthermore, SIRT1 protein level was up-regulated in CR mouse pMPhis, whereas elevated SIRT1 decreased COX-2 expression and improved PGE(2)-related macrophage functions that were reversed following inhibition of SIRT1 deacetylase activity. Thus, our results indicate that SIRT1 may be a mediator of CR-induced macrophage regulation, and its deacetylase activity contributes to the inhibition of AP-1 transcriptional activity and COX-2 expression leading to amelioration of macrophage function.

The silent information regulator protein 2 (Sir2) and its homologues play an important role in the regulation of cellular physiological processes such as survival, apoptosis, and aging. SIRT1, the mammalian Sir 2 homologue, has been shown to deacetylate a wide range of non-histone substrates and histone substrates. It has been constantly reported that SIRT1 may be associated with the occurrence of metabolic syndrome, genomic homeostasis, tumors, and neurodegenerative diseases. Calorie restriction may mitigate many major diseases in rodent models by SIRT1-mediated deacetylase activity and prolong the life expectancies in these animals. Therefore, SIRT1 may be emphasized as a new therapy target for many different diseases.