Original article
Sirt1 modulates premature senescence-like phenotype in human endothelial cells

https://doi.org/10.1016/j.yjmcc.2007.08.008Get rights and content

Abstract

Yeast Sir2 plays critical roles in gene silencing, stress resistance and longevity. Mammalian Sirt1 NAD+-dependent protein deacetylase, the closest homolog of Sir2, regulates cell cycle, cellular senescence, apoptosis and metabolism, by functional interactions with a number of biological molecules such as p53. To investigate a role of Sirt1 in endothelial dysfunction and premature senescence, we examined the effects of Sirt1 inhibition in human umbilical vein endothelial cells (HUVEC). Sirt1 inhibition by sirtinol, which is a 2-hydroxy-1-napthaldehyde derivative, or siRNA for Sirt1-induced premature senescence-like phenotype, as judged by increased senescence-associated β-galactosidase (SA-β-gal) activity, sustained growth arrest and enlarged and flattened cell morphology at 10 days after the treatment. Sixty-four percent of sirtinol (60 μmol/L)-treated HUVEC was SA-β-gal-positive, whereas only 17% of vehicle-treated cells were positive. Sirt1 inhibition by sirtinol or Sirt1 siRNA increased PAI-1 expression and decreased both protein expression and activity of eNOS. Treatment with sirtinol or Sirt1 siRNA increased acetylation of p53, while p53 expression was unaltered. Impaired epidermal growth factor-induced activation of mitogen-activated protein kinases was associated with Sirt1 inhibition-induced senescence-like growth arrest. Conversely, overexpression of Sirt1 prevented hydrogen peroxide-induced SA-β-gal activity, morphological changes and deranged expression of PAI-1 and eNOS. These results showed that Sirt1 inhibition increased p53 acetylation and induced premature senescence-like phenotype in parallel with increased PAI-1 and decreased eNOS expression. Our data suggest that Sirt1 may exert protective effects against endothelial dysfunction by preventing stress-induced premature senescence and deranged expression of PAI-1 and eNOS.

Introduction

Replicative senescence is the limited ability of mammalian cells to divide when cultured in vitro [1]. Cessation of cell division after extended propagation in weeks – or months – culture results from the attrition of telomeres and is termed replicative senescence. In contrast to replicative senescence, stressors such as oxidative stress elicit irreversible growth arrest within just a few days, which is referred to as stress-induced premature senescence (SIPS) [2], [3]. Although cellular phenotypes of replicative senescence and SIPS are quite similar or indistinguishable, unlike replicative senescence, SIPS can be induced independent of telomere attrition. In either type of cellular senescence, it is accompanied by a specific set of changes in cell function, morphology and gene expression [4], [5]. Well-established biomarkers of cellular senescence include staining for senescence-associated β-galactosidase (SA-β-gal) at pH 6.0 as opposed to endogenous lysosomal enzyme detected at pH 4.0 in normal cells.

Recently, premature senescence of endothelial cells has been proposed to be involved in endothelial dysfunction and atherogenesis [6]. Increased plasminogen activator inhibitor-1 (PAI-1) expression and reduced endothelial nitric oxide synthase (eNOS) activity, which play key roles in endothelial dysfunction and atherogenesis [7], are associated with premature senescence of vascular endothelial cells. Indeed, previous studies have shown that senescence of endothelial cells leads to endothelial dysfunction and may contribute to the progression of atherosclerosis [8], [9]. Human aortae with atherosclerotic lesion exhibited premature senescence-like phenotype, including SA-β-gal activity and increased expression of PAI-1 [10], in association with decreased production of nitric oxide (NO) in endothelial cells [6]. In addition to the role as a prothrombotic factor, the induction of PAI-1 is part of cellular senescence program and has been used as an indicator of cellular senescence in various cell types, including endothelial cells [11], [12]. Moreover, NO production [13] and eNOS expression [14], [15] are markedly attenuated in senescent endothelial cells. However, the molecular mechanisms underlying premature senescence and endothelial dysfunction remain to be clarified.

Sir2 (silent information regulator-2), an NAD+-dependent histone deacetylase, is highly conserved in organisms ranging from archaea to humans [16], [17]. In yeast, Sir2 is responsible for silencing at repeated DNA sequences in mating-type loci, telomeres and rDNA and plays critical roles in DNA repair, stress resistance and longevity [18], [19], [20]. Mammalian Sirt1 NAD+-dependent protein deacetylase, the closest homolog of Sir2, regulates cell cycle, premature senescence, apoptosis and metabolism by interacting with a number of molecules, including p53, PML, Foxo, Ku70 and PPAR-γ [21], [22], [23], [24], [25], [26]. A previous study has shown that Sirt1 antagonizes p53-mediated premature senescence in mouse embryo fibroblasts [25]. Recently, we have demonstrated that Sirt1 inhibition induces premature senescence-like growth arrest in human cancer cells [28].

In the present study, to test the hypothesis that Sirt1 plays a role in endothelial premature senescence and dysfunction in vitro, we evaluated the effects of inhibition and overexpression of Sirt1. Here, we demonstrate in human vascular endothelial cells that inhibition of Sirt1-induced premature senescence-like phenotype in parallel with acetylation of p53, and that Sirt1 overexpression reverted premature senescence induced by hydrogen peroxide.

Section snippets

Cell culture

Human umbilical vein endothelial cells (HUVEC) were purchased from CAMBREX (Walkersville, MD) and maintained in endothelial growth medium (EGM-2, EGM-2 singleQuots, CAMBREX). Population doubling levels (PDL) were calculated as described previously [29], and all experiments were performed at PDL of 8–9, unless otherwise stated.

Sirt1 inhibition and overexpression

Exponentially proliferating cells were washed three times with growth medium and exposed for 24 h to the indicated concentrations of sirtinol (Calbiochem, San Diego, CA),

Inhibition of Sirt1 induces premature senescence-like phenotype in HUVEC

To investigate whether Sirt1 modulates stress-induced premature senescence-like phenotype in HUVEC, we first examined the effect of Sirt1 inhibition. Sirt1 was inhibited by sirtinol, a specific chemical inhibitor of sirtuins, and Sirt1 siRNA. Sirtinol increased the acetylation of histone H3 (Lys14) and H4 (Lys16), endogenous substrates for Sirt1 (Fig. 1A), as expected. However, the protein expression of Sirt1 was not affected. Knockdown of Sirt1 with siRNA was confirmed by Western blotting (

Discussion

We found that Sirt1 inhibition by a specific chemical inhibitor of sirtuins, sirtinol or gene knockdown by Sirt1 siRNA induced premature senescence-like phenotype, as judged by representative markers for premature senescence, SA-β-gal activity, enlarged and flattened cell morphology, sustained growth arrest, increased PAI-1 expression and suppressed activation of MAPKs in response to a growth factor, EGF. Another chemical inhibitor of sirtuins, splitomicin, also elicited similar effects in

Acknowledgments

We thank Dr. M. Takata (Department of Immunology and Molecular Genetics, Kawasaki Medical School, Okayama, Japan) and R.A. Weinberg for pIRES-Sirt1 plasmid. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan (18590801).

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