Extension of Human Cell Lifespan by Nicotinamide Phosphoribosyltransferase

Extending the productive lifespan of human cells could have major implications for diseases of aging, such as atherosclerosis. We identified a relationship between aging of human vascular smooth muscle cells (SMCs) and nicotinamide phosphoribosyltransferase (Nampt/PBEF/Visfatin), the rate-limiting enzyme for NAD+ salvage from nicotinamide. Replicative senescence(definition) of SMCs was preceded by a marked decline in the expression and activity of Nampt. Furthermore, reducing Nampt activity with the antagonist FK866 induced premature senescence in SMCs, assessed by serial quantification of the proportion of cells with senescence-associated β-galactosidase activity. In contrast, introducing the Nampt gene into aging human SMCs delayed senescence and substantially lengthened cell lifespan, together with enhanced resistance to oxidative stress. Nampt-mediated SMC lifespan extension was associated with increased activity of the NAD+-dependent longevity enzyme SIRT1 and was abrogated in Nampt-overexpressing cells transduced with a dominant-negative form of SIRT1 (H363Y). Nampt overexpression also reduced the fraction of p53 that was acetylated on lysine 382, a target of SIRT1, suppressed an age-related increase in p53 expression, and increased the rate of p53 degradation. Moreover, add-back of p53 with recombinant adenovirus blocked the anti-aging effects of Nampt. These data indicate that Nampt is a longevity protein that can add stress-resistant life to human SMCs by optimizing SIRT1-mediated p53 degradation. Extending the productive lifespan of human cells could have major implications for diseases of aging, such as atherosclerosis. We identified a relationship between aging of human vascular smooth muscle cells (SMCs) and nicotinamide phosphoribosyltransferase (Nampt/PBEF/Visfatin), the rate-limiting enzyme for NAD+ salvage from nicotinamide. Replicative senescence of SMCs was preceded by a marked decline in the expression and activity of Nampt. Furthermore, reducing Nampt activity with the antagonist FK866 induced premature senescence in SMCs, assessed by serial quantification of the proportion of cells with senescence-associated β-galactosidase activity. In contrast, introducing the Nampt gene into aging human SMCs delayed senescence and substantially lengthened cell lifespan, together with enhanced resistance to oxidative stress. Nampt-mediated SMC lifespan extension was associated with increased activity of the NAD+-dependent longevity enzyme SIRT1 and was abrogated in Nampt-overexpressing cells transduced with a dominant-negative form of SIRT1 (H363Y). Nampt overexpression also reduced the fraction of p53 that was acetylated on lysine 382, a target of SIRT1, suppressed an age-related increase in p53 expression, and increased the rate of p53 degradation. Moreover, add-back of p53 with recombinant adenovirus blocked the anti-aging effects of Nampt. These data indicate that Nampt is a longevity protein that can add stress-resistant life to human SMCs by optimizing SIRT1-mediated p53 degradation. Age is the greatest risk factor for myocardial infarctions and strokes (1Lakatta E.G. Levy D. Circulation. 2003; 107: 139-146Crossref PubMed Scopus (1673) Google Scholar). This risk is partly attributable to an age-related decline in the ability of vascular cells to resist stress and effectively remodel the arterial wall. Vascular smooth muscle cells (SMCs) 3The abbreviations used are: SMC, smooth muscle cell; Nampt, nicotinamide phosphoribosyltransferase; TSA, trichostatin A; FBS, fetal bovine serum; SA β-Gal, senescence-associated β-galactosidase; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; ANOVA, analysis of variance. 3The abbreviations used are: SMC, smooth muscle cell; Nampt, nicotinamide phosphoribosyltransferase; TSA, trichostatin A; FBS, fetal bovine serum; SA β-Gal, senescence-associated β-galactosidase; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; ANOVA, analysis of variance. are especially important in this regard; the efficiency with which SMCs stabilize a developing atherosclerotic lesion determines whether the lesion will rupture, a potentially fatal event. Strategies to prevent the premature senescence of SMCs could be a promising approach for reducing vascular disease if molecular targets can be identified. Nicotinamide phosphoribosyltransferase (Nampt, also known as Pre-B-cell colony-enhancing factor and Visfatin (2Fukuhara A. Matsuda M. Nishizawa M. Segawa K. Tanaka M. Kishimoto K. Matsuki Y. Murakami M. Ichisaka T. Murakami H. Watanabe E. Takagi T. Akiyoshi M. Ohtsubo T. Kihara S. Yamashita S. Makishima M. Funahashi T. Yamanaka S. Hiramatsu R. Matsuzawa Y. Shimomura I. Science. 2005; 307: 426-430Crossref PubMed Scopus (1653) Google Scholar)) is the rate-limiting enzyme for NAD+ biosynthesis from nicotinamide. The intracellular levels of NAD+ and nicotinamide have recently been identified as important for certain cell survival reactions, including those linked to the sirtuin family of protein deacetylases (3Bitterman K.J. Anderson R.M. Cohen H.Y. Latorre-Esteves M. Sinclair D.A. J. Biol. Chem. 2002; 277: 45099-45107Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar, 4Araki T. Sasaki Y. Milbrandt J. Science. 2004; 305: 1010-1013Crossref PubMed Scopus (909) Google Scholar). Sirtuins, such as Sir2 and its mammalian homolog SIRT1, consume NAD+ and generate nicotinamide as they hydrolytically remove a targeted acetyl group (3Bitterman K.J. Anderson R.M. Cohen H.Y. Latorre-Esteves M. Sinclair D.A. J. Biol. Chem. 2002; 277: 45099-45107Abstract Full Text Full Text PDF PubMed Scopus (811) Google Scholar). Nicotinamide is a known inhibitor of NAD+-dependent deacetylation reactions. Therefore, pathways that both replenish NAD+ and clear nicotinamide could be vital to SIRT1 activity. Recently, we discovered that Nampt was substantially up-regulated when a uniquely long-lived human vascular SMC line was subjected to the stress of complete serum withdrawal (5van