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 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 der Veer E. Nong Z. O'Neil C. Urquhart B. Freeman D. Pickering J.G. Circ. Res. 2005; 97: 25-34Crossref PubMed Scopus (174) Google Scholar). Here, we report that Nampt is a longevity protein that extends the lifespan of human SMCs by activating SIRT1 and restraining the accumulation of p53. Cell Culture—Experiments were performed using primary human vascular SMCs derived by outgrowth from fragments of internal thoracic artery and the HITC6 SMC clonal line, also originally generated from the human internal thoracic artery (6Li S. Fan Y.S. Chow L.H. Van Den Diepstraten C. van Der Veer E. Sims S.M. Pickering J.G. Circ. Res. 2001; 89: 517-525Crossref PubMed Scopus (125) Google Scholar). Dermal fibroblasts from an individual with Hutchison-Gilford progeria syndrome were obtained from the Coriell Cell Repository. To quantify replication, cells were plated at 4,500 cells/cm2, and growth medium with 10% FBS was changed every 2 days until cells reached 90–95% confluence. Harvested cells were counted from triplicate plates, and the number of population doublings was calculated based on: log10 [(number of cells harvested) – log10 (number of cells seeded)]/log10 (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). Population growth curves were compared using nonlinear regression. Recombinant Retrovirus and Adenovirus Infection—A retroviral gene delivery system was used to generate human cells stably overexpressing Nampt, using methods described previously (7Rocnik E.F. Van Der Veer E. Cao H. Hegele R.A. Pickering J.G. J. Biol. Chem. 2002; 277: 38571-38578Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Retrovirus containing pQCXIP-Nampt-IRES-PURO or pQCXIP-IRES-PURO (Clontech Laboratories) was generated by calcium phosphate-mediated transfection of the Phoenix-amphotropic retrovirus packaging cell line (ATCC, Manassas, VA). Stable transductants were selected with puromycin (3 μg/ml). Dominant-negative SIRT1 (H363Y) was similarly transduced using the pBABE retroviral expression vector. Recombinant adenoviral vectors carrying human p53 or enhanced green fluorescent protein expression cassettes were constructed, purified, and titered as described previously (8Cregan S.P. MacLaurin J. Gendron T.F. Callaghan S.M. Park D.S. Parks R.J. Graham F.L. Morley P. Slack R.S. Gene Ther. 2000; 7: 1200-1209Crossref PubMed Scopus (39) Google Scholar). Experiments were performed at a multiplicity of infection of 100 plaque-forming units/cell. Western Blot and Immunoprecipitation Analysis—Protein expression was assessed by Western blot analysis with chemiluminescence detection, as described (7Rocnik E.F. Van Der Veer E. Cao H. Hegele R.A. Pickering J.G. J. Biol. Chem. 2002; 277: 38571-38578Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Nampt was detected using a rabbit polyclonal anti-human Nampt/Pre-B-cell colony-enhancing factor antibody (Bethyl Laboratories, Montgomery, TX). SIRT1 was detected using a rabbit polyclonal anti-Sirt1 antibody (Abcam, Cambridge, MA), and p53 and α-tubulin were detected with monoclonal antibodies (DO-1 from Santa Cruz Biotechnology, Santa Cruz, CA and Clone B-5-1-1 from Sigma, respectively). Cytoplasmic p53 was similarly assessed following cell fractionation (NucBuster, Novagen, Minneapolis, MN). Identification of p53 that underwent NAD+-dependent removal of an acetyl group on residue Lys-382 was determined by pretreating SMCs for 2 h with 5 μm trichostatin A (TSA), immunoprecipitating p53 with a goat polyclonal antibody (R&D Systems, Minneapolis, MN), and immunoblotting using a rabbit polyclonal anti-acetylated p53 (Lys-382) antibody (American Proteomics, Carlsbad, CA) and horseradish peroxidase-labeled donkey anti-rabbit IgG antibody (Amersham Biosciences). Nampt Activity—Whole cell lysates were reacted with 5 μm [carbonyl-14C]nicotinamide (Sigma) and 0.5 mm phosphoribosylpyrophosphate in 10 mm NaH2PO4/Na2HPO4 buffer, pH 8.8 (9Rongvaux A. Shea R.J. Mulks M.H. Gigot D. Urbain J. Leo O. Andris F. Eur. J. Immunol. 2002; 32: 3225-3234Crossref PubMed Scopus (476) Google Scholar). Labeled acetone-precipitable nicotinamide mononucleotide was quantified by scintillation counting. Senescence-associated β-Galactosidase Activity Assay—SMCs at ∼70% confluence were fixed in 2% formaldehyde/0.2% glutaraldehyde in phosphate-buffered saline for 3 min and incubated with X-gal-containing reaction mixture, as described (10Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley C. Medrano E.E. Linskens M. Rubelj I. Pereira-Smith O. Peacocke M. Campisi J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9363-9367Crossref PubMed Scopus (5707) Google Scholar). Cells were stained with Hoechst 33258 (2.5 μg/ml), and the proportion of senescence-associated β-galactosidase (SA β-Gal)-positive cells was quantified (Olympus BX51, ×20 objective, ∼1200 cells). Senescence-free survival was determined using Kaplan-Meier analysis of survival versus replicative age. In Vivo Assessment of SIRT1 DeacetylaseActivity—Cell-basedassessment of SIRT1 enzymatic activity was performed using the Fluor de Lys-SIRT1 substrate (Biomol, Plymouth Meeting, PA), as described (11de Boer V.C. de Goffau M.C. Arts I.C. Hollman P.C. Keijer J. Mech. Ageing Dev. 2006; 127: 618-627Crossref PubMed Scopus (145) Google Scholar). SMCs in phenol red-free M199 with 5% FBS were incubated for 2 h with 5 μm TSA followed by the addition of the fluorogenic substrate for 4 h. Signal was quantified by spectrofluorometry (Wallac, Wellesley, MA) and normalized to total protein content. Time-lapse Analysis of SMC Response to Oxidative Stress—The morphologic response to oxidative stress was dynamically assessed by digital time-lapse microscopy, using methods previously described (12Fera E. O'Neil C. Lee W. Li S. Pickering J.G. J. Biol. Chem. 2004; 279: 35573-35582Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Hoffman-modulated contrast images (Axiovert S100; Carl Zeiss, Inc. Thornwood, NY) were digitally acquired every 5 min over 3 h, beginning immediately after the addition of 150 μm H2O2 to SMCs in M199 containing 1% FBS. Nampt Expression and Activity Decline as Human SMCs Undergo Replicative Aging—To track Nampt expression as SMCs undergo replicative aging, two human SMC preparations with different in vitro lifespans were studied: 1) primary cultures of SMCs, initiated by outgrowth from the internal thoracic artery harvested from adult patients undergoing coronary artery bypass surgery; and 2) a clonal line of human SMCs (HITC6) that also originated from the internal thoracic artery but displayed enhanced longevity in culture (6Li S. Fan Y.S. Chow L.H. Van Den Diepstraten C. van Der Veer E. Sims S.M. Pickering J.G. Circ. Res. 2001; 89: 517-525Crossref PubMed Scopus (125) Google Scholar). SMCs were serially subcultured until they reached senescence, indicated by cessation of proliferation, an enlarged flattened morphology, and cytoplasmic β-galactosidase activity at pH 6.0 (Fig. 1, A and B). In both SMC preparations, Nampt protein expression declined significantly as SMCs approached senescence (Fig. 1, C and D). Nampt enzyme activity, assessed by quantifying the conversion of nicotinamide to nicotinamide mononucleotide (5van der Veer E. Nong Z. O'Neil C. Urquhart B. Freeman D. Pickering J.G. Circ. Res. 2005; 97: 25-34Crossref PubMed Scopus (174) Google Scholar), fell in presenescent SMCs even more strikingly than Nampt expression, reaching 14 ± 3% (mean ± S.D., n = 3) of basal activity (Fig. 1C). These findings identify the regeneration of NAD+ from nicotinamide as a metabolic pathway that becomes exhausted as SMCs approach senescence. Nampt Regulates and Extends Human Cellular Lifespan—To determine whether human SMC lifespan could be extended by overriding this innate decline in Nampt expression and activity, we augmented Nampt gene dosage by introducing human Nampt cDNA into SMCs using retrovirus. This yielded a 7.1 ± 3.1-fold increase in Nampt activity in stably transduced SMCs. Primary adult SMCs overexpressing Nampt surpassed the maximal lifespan of vector-infected SMCs by an additional 2.1 ± 0.3 population doublings, which, given their otherwise short in vitro lifespan, constituted a 34 ± 4% prolongation of lifespan (Fig. 2A). Equally notably, this extension proceeded in cells that were already well advanced along their path to replicative senescence. In the longer lived HITC6 SMCs, lifespan extension by Nampt was even more striking, with an additional 6.3 ± 0.3 population doublings or a 71 ± 7% extension of lifespan (Fig. 2A). Cell lifespan extension by Nampt was not limited to SMCs and was also seen with human fibroblasts derived from a subject with Hutchinson-Gilford progeria syndrome, a condition associated with markedly premature atherosclerosis (13Al-Shali K.Z. Hegele R.A. Arterioscler. Thromb. Vasc. Biol. 2004; 24: 1591-1595Crossref PubMed Scopus (42) Google Scholar) (Fig. 2A). To determine the role of endogenous Nampt in SMC lifespan and senescence, SMCs were incubated with the specific Nampt antagonist FK866 (14Hasmann M. Schemainda I. Cancer Res. 2003; 63: 7436-7442PubMed Google Scholar). FK866 is a long, almost linear molecule that binds Nampt from within a narrow tunnel at the Nampt dimer interface, an unusual structural relationship that accounts for its specificity (15Khan J.A. Tao X. Tong L. Nat. Struct. Mol. Biol. 2006; 13: 582-588Crossref PubMed Scopus (203) Google Scholar). We quantified the proportion of senescent SMCs in successive subcultures incubated with 10 nm FK866, a concentration we determined reduced Nampt activity in SMCs to 22 ± 2% of baseline. Kaplan-Meier survival analysis revealed a substantially shortened senescence-free survival of Nampt-inhibited cells SMCs (p < 0.0001) (Fig. 2B). In contrast, there was markedly extended senescence-free survival in Nampt-overexpressing SMCs versus vector-infected cells (p < 0.0001) (Fig. 2C). Therefore, a direct relationship exists between Nampt activity and the number of replication cycles a SMC can undergo before becoming senescent. Together with the age-related decline in Nampt activity, these data firmly establish Nampt as a longevity enzyme for human SMCs. Nampt Postpones Senescence by Activating SIRT1—To explore the mechanism by which Nampt regulates SMC lifespan, we considered that Nampt both stimulates NAD+ production and consumes nicotinamide, positioning this enzyme as a potential regulator of the NAD+-dependent deacetylase, SIRT1 (16Revollo J.R. Grimm A.A. Imai S. J. Biol. Chem. 2004; 279: 50754-50763Abstract Full Text Full Text PDF PubMed Scopus (768) Google Scholar). SIRT1 consumes NAD+, is inhibited by nicotinamide, and mediates lifespan extension of caloric restricted animals (17Cohen H.Y. Lavu S. Bitterman K.J. Hekking B. Imahiyerobo T.A. Miller C. Frye R. Ploegh H. Kessler B.M. Sinclair D.A. Mol. Cell. 2004; 13: 627-638Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar). To test whether Nampt could stimulate SIRT1 activity in human SMCs, TSA-independent deacetylation of a fluorogenic SIRT1 substrate (Biomol) was quantified. As shown in Fig. 3A, SMCs overexpressing Nampt had a 86 ± 4% (p = 0.03, n = 4) increase in SIRT1 activity. We were surprised to find that Nampt overexpression also modestly increased abundance of SIRT1 protein (1.3 ± 0.3-fold, p = 0.02), although not enough to fully account for the increased deacetylase activity (Fig. 3B). To determine whether the lifespan extending actions of Nampt were mediated by the increased SIRT1 expression and activity, human SMCs were double-transduced to express Nampt and a dominant-negative form of SIRT1 (H363Y) (18Vaziri H. Dessain S.K. Ng Eaton E. Imai S.I. Frye R.A. Pandita T.K. Guarente L. Weinberg R.A. Cell. 2001; 107: 149-159Abstract Full Text Full Text PDF PubMed Scopus (2288) Google Scholar). This revealed that the extended lifespan conferred by Nampt was abrogated when the SIRT1 H363Y allele was co-expressed (5.4 ± 0.5 versus 0.3 ± 0.3 of additional population doublings, p = 0.003). Furthermore, the reduction in senescent SMCs in late passage (37th Nampt-overexpressing SMCs ± p = was longer when dominant-negative SIRT1 was (p = with a increase in senescence that was than for vector-infected SMCs SIRT1 H363Y ± versus ± p = Fig. Nampt p53 and p53 Nampt-mediated of is by of and this p53 is a known target of SIRT1-mediated deacetylation S. J.A. T. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar). p53 aging Cell. 2006; PubMed Scopus Google Scholar), and in with we that p53 abundance increased as human SMCs approached replicative senescence (Fig. this age-related increase in p53 was in cultures of SMCs overexpressing Nampt. Furthermore, the fraction of p53 that was acetylated on Lys-382 was substantially in Nampt-overexpressing SMCs than in cells (Fig. This p53 was associated with a significantly increased rate of p53 in Nampt-overexpressing SMCs, as assessed in SMCs incubated with (Fig. To determine whether p53 levels the in senescence induced by Nampt, p53 was to SMCs using recombinant adenovirus (9Rongvaux A. Shea R.J. Mulks M.H. Gigot D. Urbain J. Leo O. Andris F. Eur. J. Immunol. 2002; 32: 3225-3234Crossref PubMed Scopus (476) Google Scholar). As shown in Fig. add-back of p53 to Nampt-overexpressing SMCs abrogated the reduction in senescent SMCs by augmented Nampt activity. these findings indicate that Nampt senescence by and of p53. Nampt Oxidative Cell using time-lapse (12Fera E. O'Neil C. Lee W. Li S. Pickering J.G. J. Biol. Chem. 2004; 279: 35573-35582Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar), we that the additional replicative life conferred by Nampt was associated with a stress-resistant vector-infected SMCs to 150 μm H2O2 by of Nampt-overexpressing SMCs, for population their and ability to (Fig. and H2O2 induced a increase in cytoplasmic p53 in SMCs, the response was in Nampt-overexpressing SMCs (Fig. that a in the to NAD+ from nicotinamide is a of human SMC senescence. Moreover, by Nampt activity, lifespan can be a that we in human primary SMCs, human clonal SMCs, and fibroblasts derived from a with Hutchinson-Gilford progeria We that this anti-aging is mediated by enhanced SIRT1 deacetylase activity in p53 levels those which senescence. These findings a as a of human cell The role of Nampt as a of stress resistance and longevity is in the of its expression Nampt is up-regulated by including infection and Li Y. J. A. Fan L. J. 2004; PubMed Scopus Google Scholar). In human SMCs, Nampt expression increased substantially in response to the stress of complete serum removal (5van der Veer E. Nong Z. O'Neil C. Urquhart B. Freeman D. Pickering J.G. Circ. Res. 2005; 97: 25-34Crossref PubMed Scopus (174) Google Scholar). This response is of the of in subjected to caloric H. Lavu S. Sinclair D.A. 2006; PubMed Scopus Google Scholar). a nicotinamide and the of nicotinamide is considered to be a in the stress of caloric into extended longevity R.M. Bitterman K.J. J.G. O. Sinclair D.A. 2003; PubMed Scopus Google Scholar). The ability of Nampt to resistance to stress and longevity in a that Nampt an longevity in human data also that such a response to stress be on age. the endogenous decline in Nampt activity that we in presenescent cells a in the response to stress from lifespan extension to Cell senescence is in age-related as well as the decline in potential with T.A. 2006; PubMed Scopus Google Scholar). Vascular SMC senescence, a of atherosclerotic C. I. Scott S. P. A. M. M. Circ. Res. 2006; PubMed Scopus Google Scholar), can be the and can lesion and vascular The findings identify Nampt as an aging pathway in SMCs, with potential to atherosclerosis and diseases of

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