Reduced caloric intake decreases arterial blood pressure in healthy individuals and improves endothelium-dependent vasodilation in obese and overweight individuals. The SIRT1 protein deacetylase mediates many of the effects of calorie restriction (CR) on organismal lifespan and metabolic pathways. However, the role of SIRT1 in regulating endothelium-dependent vasomotor tone is not known. Here we show that SIRT1 promotes endotheliumdependent vasodilation by targeting endothelial nitric oxide synthase (eNOS) for deacetylation. SIRT1 and eNOS colocalize and coprecipitate in endothelial cells, and SIRT1 deacetylates eNOS, stimulating eNOS activity and increasing endothelial nitric oxide (NO). SIRT1-induced increase in endothelial NO is mediated through lysines 496 and 506 in the calmodulin-binding domain of eNOS. Inhibition of SIRT1 in the endothelium of arteries inhibits endothelium-dependent vasodilation and decreases bioavailable NO. Finally, CR of mice leads to deacetylation of eNOS. Our results demonstrate that SIRT1 plays a fundamental role in regulating endothelial NO and endothelium-dependent vascular tone by deacetylating eNOS. Furthermore, our results provide a possible molecular mechanism connecting the effects of CR on the endothelium and vascular tone to SIRT1-mediated deacetylation of eNOS.calorie restriction ͉ vasorelaxation ͉ silent information regulator 2 ͉ resveratrol ͉ deacetylation C aloric restriction (CR) is a well recognized nonpharmacological approach to reducing arterial blood pressure. CR not only is capable of independently controlling blood pressure of patients with mild hypertension, but also allows a reduction in the number and dosage of medications used to treat hypertension (1, 2). CR and weight loss resulting from CR also improve endothelium-dependent vascular relaxation in obese and overweight individuals with hypertension (3, 4).In addition, CR prolongs organismal lifespan. In the budding yeast, Saccharomyces cerevisiae, aging of replicating cells is determined by the SIR2 gene (5). Retardation of yeast aging by CR depends on the product of this gene, Sir2 (silent information regulator 2), a class III NAD-dependent histone deacetylase. The mammalian ortholog of Sir2, SIRTUIN 1 (SIRT1), targets histones and many nonhistone proteins (6-10). Resveratrol, a plant polyphenol that stimulates SIRT1 activity (11), activates endothelial nitric oxide synthase (eNOS) (12), improves endothelial function, prevents elevation in blood pressure, and restores vascular eNOS activity in animal models of endothelial dysfunction (13). Hypothesizing that the effects of CR and resveratrol on vascular function are mediated, in part, by SIRT1, we investigated the role of SIRT1 in regulating eNOS activity and endothelium-dependent vascular tone. Results and DiscussionTo determine whether SIRT1 plays an important role in regulating endothelium-dependent vascular tone, vasomotor function of rat aortic rings, in which wild-type SIRT1 or a dominant negative SIRT1 mutant (to inhibit endogenous SIRT1) was adenovi...

Apurinic/apyrimidinic endonuclease-1 (APE1) is an essential enzyme in the base excision repair (BER) pathway. Here, we show that APE1 is a target of the SIRTUIN1 (SIRT1) protein deacetylase. SIRT1 associates with APE1, and this association is increased with genotoxic stress. SIRT1 deacetylates APE1 in vitro and in vivo targeting lysines 6 and 7. Genotoxic insults stimulate lysine acetylation of APE1 which is antagonized by transcriptional upregulation of SIRT1. Knockdown of SIRT1 increases cellular abasic DNA content, sensitizing cells to death induced by genotoxic stress, and this vulnerability is rescued by overexpression of APE1. Activation of SIRT1 with resveratrol promotes binding of APE1 to the BER protein X-ray cross-complementing-1 (XRCC1), while inhibition of SIRT1 with nicotinamide (NAM) decreases this interaction. Genotoxic insult also increases binding of APE1 to XRCC1, and this increase is suppressed by NAM or knockdown of SIRT1. Finally, resveratrol increases APE activity in XRCC1-associated protein complexes, while NAM or knockdown of SIRT1 suppresses this DNA repair activity. These findings identify APE1 as a novel protein target of SIRT1, and suggest that SIRT1 plays a vital role in maintaining genomic integrity through regulation of the BER pathway.

The Son of Sevenless 1 protein (sos1) is a guanine nucleotide exchange factor (GEF) for either the ras or rac1 GTPase. We show that p66shc, an adaptor protein that promotes oxidative stress, increases the rac1-specific GEF activity of sos1, resulting in rac1 activation. P66shc decreases sos1 bound to the growth factor receptor bound protein (grb2) and increases the formation of the sos1–eps8–e3b1 tricomplex. The NH2-terminal proline-rich collagen homology 2 (CH2) domain of p66shc associates with full-length grb2 in vitro via the COOH-terminal src homology 3 (C-SH3) domain of grb2. A proline-rich motif (PPLP) in the CH2 domain mediates this association. The CH2 domain competes with the proline-rich COOH-terminal region of sos1 for the C-SH3 domain of grb2. P66shc-induced dissociation of sos1 from grb2, formation of the sos1–eps8–e3b1 complex, rac1-specific GEF activity of sos1, rac1 activation, and oxidative stress are also mediated by the PPLP motif in the CH2 domain. This relationship between p66shc, grb2, and sos1 provides a novel mechanism for the activation of rac1.

Abstract-The transcription factor, p53, and the adaptor protein, p66shc, both play essential roles in promoting oxidative stress in the vascular system. However, the relationship between the two in the context of endothelium-dependent vascular tone is unknown. Here, we report a novel, evolutionarily conserved, p53-mediated transcriptional mechanism that regulates p66shc expression and identify p53 as an important determinant of endothelium-dependent vasomotor function. We provide evidence of a p53 response element in the promoter of p66shc and show that angiotensin II-induced upregulation of p66shc in endothelial cells is dependent on p53.In addition, we demonstrate that downregulation of p66shc expression, as well as inhibition of p53 function in mice, mitigates angiotensin II-induced impairment of endothelium-dependent vasorelaxation, decrease in bioavailable nitric oxide, and hypertension. These findings reveal a novel p53-dependent transcriptional mechanism for the regulation of p66shc expression that is operative in the vascular endothelium and suggest that this mechanism is important in impairing endothelium-dependent vascular relaxation. Key Words: tumor suppressor p53 Ⅲ p66shc angiotensin II Ⅲ endothelial dysfunction P 66shc belongs to the shcA family of adaptor proteins. P66shc is structurally and functionally distinct from p52shc and p46shc, the other 2 members of this family. It has a unique N-terminal collagen homology (CH2) domain that is important in governing its activity, and it functions as a protein that promotes oxidative stress within cells and tissues. Cells lacking p66shc have reduced levels of oxidants, and mice deficient for p66shc are resistant to oxidative stresses, and have an extended lifespan. 1,2 In addition to living longer, p66shc-deficient mice are protected against age-associated and hyperglycemia-induced endothelial dysfunction and oxidative stress. 3,4 Similarly, downregulation of p66shc in normal blood vessels and cells increases endothelial NO synthase activity and improves endothelium-dependent vasorelaxation. 5 P66shc also promotes atherogenesis. P66shc-deficient mice have reduced susceptibility to atheroma formation when fed a high-fat diet. 6 Despite this growing body of evidence implicating p66shc in many pathophysiological states of the cardiovascular system, little information exists about the mechanisms that govern p66shc expression in the cardiovascular system and the relevance of these mechanisms to human cardiovascular disease states.The role that the tumor suppressor protein p53 plays in different cell types comprising the vascular wall, and in the overall pathogenesis of atherosclerosis, as well as in other vascular disorders that are not necessarily associated with atherosclerosis, is highly controversial. P53 is undetectable in normal vascular specimens, but is expressed in endothelial cells, smooth muscle cells, and macrophages of advanced human atherosclerosis. 7 In animal models of high-fat dietinduced atheroma formation, knockout of p53 results in an incre...

Objective-To evaluate if p53 decreases Kruppel-Like Factor 2 (KLF2) expression and determine whether p53-mediated suppression of KLF2 plays a role in p53-induced endothelial dysfunction. Methods and Results-Endothelial KLF2 mediates endothelium-dependent vascular homeostasis by differentially regulating endothelial genes, leading to an anti-inflammatory and antithrombotic endothelial surface with normal vasodilatory function. In contrast, the tumor suppressor p53 leads to inflammatory gene expression and impairs endothelium-dependent vasodilatation, thus promoting endothelial dysfunction. The effect of p53 on KLF2 expression was determined. p53 inhibited KLF2 transcription in a histone deacetylase-dependent and a histone acetyltransferaseindependent fashion. KLF2 expression was suppressed by p53 via a conserved p53-binding repressor sequence in its promoter. p53 bound to, and stimulated, deacetylation of Histone H3 at the KLF2 promoter. The effect of p53 on endothelial KLF2 target genes was examined. Downregulation of p53 increased expression of endothelial NO synthase and thrombomodulin and inhibited expression of plasminogen activator inhibitor 1. Conversely, overexpression of p53 suppressed endothelial NO synthase and thrombomodulin expression and stimulated plasminogen activator inhibitor 1 and endothelin-1 expression. Knockdown of KLF2 abolished the p53-induced decrease in thrombomodulin and increase in endothelin-1. Both, overexpression of p53 and knockdown of KLF2 in endothelial cells increased blood coagulation on an endothelial cell monolayer. The p53-induced increase in coagulation was rescued by forced expression of KLF2. p53 also impaired endothelium-dependent vasodilatation and decreased bioavailable vascular NO, both of which were rescued by forced KLF2 expression. Conclusion-These findings illustrate a novel p53-dependent mechanism for the regulation of endothelial KLF2expression. In addition, they show that downregulation of KLF2, in part, mediates a p53-stimulated dysfunctional endothelium. (Arterioscler Thromb Vasc Biol. 2011;31:133-141.)Key Words: endothelial function Ⅲ endothelium Ⅲ gene expression Ⅲ NO synthase Ⅲ thrombosis Ⅲ vascular biology T he Kruppel-Like factor (KLF)/Sp is a subclass of the zinc finger family of DNA-binding transcriptional factors. There are 17 KLF Factors (KLF1-KLF17) and 4 Sp factors (S1-S4). These family members are characterized by DNA binding domains containing the conserved sequence CX2CX3FX5LX2HX3H (X is any amino acid; underlined cysteine and histidine residues coordinate zinc binding). 1 Members of this family can bind to the consensus DNA sequence CACCC. One member of this family, lung KLF/ KLF2, is highly expressed in vascular endothelium and serves as a molecular switch to regulate a range of endothelial functions. KLF2 expression in the endothelium is upregulated by laminar shear stress and inhibited by proinflammatory cytokines. 2,3 Forced expression of KLF2 in endothelial cells results in induction of endothelial-specific NO synthase (eNOS) and thrombomo...