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SIRT1 Functionally Interacts with the Metabolic Regulator and Transcriptional Coactivator PGC-1α *

SIRT1 Functionally Interacts with the Metabolic Regulator and Transcriptional Coactivator PGC-1α * THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 16, Issue of April 22, pp. 16456–16460, 2005 Printed in U.S.A. SIRT1 Functionally Interacts with the Metabolic Regulator and Transcriptional Coactivator PGC-1* Received for publication, February 8, 2005 Published, JBC Papers in Press, February 16, 2005, DOI 10.1074/jbc.M501485200 Shino Nemoto, Maria M. Fergusson, and Toren Finkel‡ From the Cardiovascular Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1454 olism of NAD. These two activities are coupled, because the In lower organisms, increased expression of the NAD- dependent deacetylase Sir2 augments lifespan. The acetyl group from the target protein is transferred to the ADP- mechanism through which this life extension is medi- ribose moiety of NAD. Although acetylation and deacetylation ated remains incompletely understood. Here we have have been clearly recognized as reversible modulators of pro- examined the cellular effects of overexpression of tein activity, the role, if any, of the novel O-acetyl-ADP-ribose SIRT1, the closest mammalian ortholog of Sir2. In PC12 moiety remains unclear. Interestingly, a recent report sug- cells, increased expression of the NAD-dependent gested that direct injection of O-acetyl-ADP-ribose into oocytes deacetylase SIRT1 reduces cellular oxygen consumption retards subsequent maturation (9). by 25%. We further demonstrate that SIRT1 expression The requirement for NAD in the deacetylase activity of Sir2 can alter the transcriptional activity of the mitochon- has led to the suggestion that enzymatic activity could be drial biogenesis coactivator PGC-1. In addition, SIRT1 regulated by the concentration of NAD, the ratio of NAD/ and PGC-1 directly interact and can be co-immunopre- NADH, or by the intracellular concentration of nicotinamide cipitated as a molecular complex. A single amino acid (10–13). In particular, caloric restriction appears to augment mutation in the putative ADP-ribosyltransferase do- lifespan in a wide range of species, and evidence suggests that main of SIRT1 inhibits the interaction of SIRT1 with the life extension induced by nutritional deprivation involves, PGC-1 but does not effect the interaction of SIRT1 with and in some cases requires, Sir2 (11, 14, 15). Because the level either p53 or Foxo3a. We further show that PGC-1 is of oxidized and reduced NAD may be altered under starved acetylated in vivo. This acetylation is augmented by conditions, this has led to the hypothesis that Sir2 activity is treatment with the SIRT1 inhibitor nicotinamide or by augmented during caloric restriction by alterations in the expression of the transcriptional coactivator p300. Fi- NAD/NADH ratio (11). Nonetheless, considerable controversy nally we demonstrate that SIRT1 catalyzes PGC-1 exists as to whether starvation significantly alters the NAD/ deacetylation both in vitro and in vivo. These results provide a direct link between the sirtuins, a family of NADH ratio and whether or not this ratio does or does not proteins linked to lifespan determination and PGC-1,a physiologically regulate Sir2 activity (12, 16). In this report, we coactivator that regulates cellular metabolism. provide evidence for another direct link between sirtuins and metabolism. In particular we demonstrate a molecular inter- action between SIRT1 and the transcriptional coactivator The most widely held theory of aging suggests that mitochon- PGC-1, a master regulator of metabolism and mitochondrial drial oxygen consumption and resulting reactive oxygen spe- biogenesis (17). cies generation fuel the aging process (1). In lower organisms EXPERIMENTAL PROCEDURES such as yeast and worms, increased expression of the enzyme Cell Lines and Plasmids—Clonal cell lines were established from Sir2, an NAD-dependent deacetylase, results in an increased PC12 cells obtained from ATCC (Rockville, MD). Cells were maintained life span (2, 3). Sir2 belongs to a growing family of enzymes in a basal growth medium consisting of Dulbecco’s modified Eagle’s collectively known as the sirtuins (4–7). The mechanism medium (Invitrogen) supplemented with 5% fetal calf serum. This me- through which Sir2 extends lifespan is unclear, although its dia formulation contains high glucose (4.5 g/liter) but no exogenous role in gene silencing has been commonly implicated (2, 8). pyruvate. HeLa cells (ATCC) were grown in the same medium. The wild Nonetheless, the relationship, if any, between the sirtuins and type mouse SIRT1 cDNA was purchased from Upstate Biotechnology and subcloned into a HA -tagged cDNA cloning vector (pCruzHA, Santa mitochondrial metabolism remains largely unexplored. Cruz Biotechnology). A glycine to alanine site ADP-ribosyltransferase Analysis of Sir2 enzymatic function has revealed that it mutant at position 261 of SIRT1 was constructed by standard methods, functions differently from previously described histone and mutant and wild type constructs were confirmed by direct sequenc- deacetylases. In particular, studies with purified Sir2 protein ing. This amino acid substitution has been previously demonstrated to revealed that, for every acetyl lysine group that is removed abrogate essentially all of the ADP-ribosyltransferase activity in yeast from a presumed substrate, one molecule of NAD is cleaved Sir2 protein (8). Clonal cell lines expressing wild type SIRT1 (WT), SIRT1 , or the empty HA-vector alone (Neo) were obtained by (4–7). The products produced by this Sir2 enzymatic deactey- G261A transfection and subsequent isolation by limiting dilutions in G418 lation include both nicotinamide and O-acetyl-ADP-ribose. selection. In stable clones that expressed wild type or mutant SIRT1, Therefore, Sir2 appears to possess two enzymatic activities, transgene expression was determined by Western blot analysis using including the deacetylation of a target protein and the metab- an antibody recognizing the HA epitope (Santa Cruz Biotechnology). Total SIRT1 expression was determined by Western blot using an SIRT1-specific antibody (Upstate). Levels of SIRT1 mRNA were deter- * The costs of publication of this article were defrayed in part by the mined by reverse transcription-PCR analysis using the following payment of page charges. This article must therefore be hereby marked SIRT1-specific primers: 5-CCTGACTTCAGATCAAGAGACGGTA-3 “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed: National Institutes of Health, Bldg. 10/CRC, Rm. 5–3330, Bethesda, MD 20892-1454. Tel.: The abbreviations used are: HA, hemagglutinin; WT, wild type; 301-402-4081; Fax: 301-402-9311; E-mail: [email protected]. GST, glutathione S-transferase. 16456 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. SIRT1 Regulates PGC-1 16457 and 5-CTGATTAAAAATGTCTCCACGAACAG-3. Primers for -actin were provided by the manufacturer (Clontech). Measurement of cyto- chrome oxidase subunit II expression was also determined by Western blot expression using a commercially available antibody (Santa Cruz Biotechnology). Human SIRT3 was cloned by standard PCR-based methods and verified by direct sequencing. Oxygen Consumption—Oxygen consumption was measured using a fiber optic oxygen monitor (Instech). All measurements of oxygen con- sumption were performed in intact cells. Cells were plated in 15-cm dishes and allowed to grow for 7 days prior to trypsinization and subsequent oxygen determination. Oxygen consumption was performed in basal growth medium (see above), because we observed that meas- ured oxygen consumption varied substantially depending on the extra- cellular supply of glucose, pyruvate, and serum. For analysis we used 20 individual control cell lines (Neo), 23 SIRT1-overexpressing cells, and 7 clones of SIRT1 . Each cell line was measured by either duplicate or G261A triplicate determinations. PGC-1 Transcriptional Activity and Protein-Protein Interaction— For determination of PGC-1 transcriptional activity we transfected PC12 cells with a previously described construct encoding full-length PGC-1 fused to the GAL-4 DNA binding domain (18). This construct (0.4 g) was co-transfected with increasing amount of wild type SIRT1 (0.1–0.4 g) or a similar amount of the ADP- ribosyltransferase-inac- tive mutant. A GAL-4-dependent luciferase reporter (0.2 g) and an internal Renilla control (0.05 g) were also transfected into each dish. FIG.1. SIRT1 regulates oxygen consumption. A, reverse tran- Twenty-four hours after transfection, cells were harvested and PGC-1 scription-PCR analysis of SIRT1 mRNA expression in four control cell transcriptional activity was analyzed using the dual luciferase reporter lines (Neo) or four random cell lines expressing wild type SIRT1 (WT). assay (Promega). Results are expressed in arbitrary units from a single Levels of -actin are included as a control. B, measurement of oxygen experiment performed in triplicate and are representative of at least consumption in clonal PC12 cells expressing G418 resistance alone three similar experiments. (Neo, n  20 individual clones), wild type SIRT1 (WT, n  23 individual To assess the interaction of PGC-1 and SIRT1, cells were transfected clones), or an ADP-ribosyltransferase domain mutant of SIRT1 (G261A, with wild type HA-SIRT1 and Myc-PGC-1 alone or in combination. n  7 individual clones). Measurements of individual clones were made in duplicate or triplicate and mean  S.E. of all clones are shown. C, These experiments were performed in HeLa cells, because these cells oxygen consumption of control PC12 cells before or after the addition of resulted in substantially higher transgene expression. Twenty-four hours the electron transport inhibitor antimycin A. The arrow indicates the after transfection, extracts were prepared in Lysis buffer (19), and 1 mg of addition of 80 nM antimycin A and demonstrates that essentially all total cell extract was immunoprecipitated with an anti-HA antibody measured oxygen consumption derives from mitochondrial metabolism. (BabCO). The immunoprecipitates were resolved by SDS-PAGE, and the Similar results were obtained in SIRT1-overexpressing cells. D, expres- level of co-immunoprecipitated Myc-PGC-1 was determined by Western sion of subunit II of cytochrome oxidase (COX II) in four random control blot analysis using an anti-Myc antibody (Santa Cruz Biotechnology). cell lines (Neo) and four random SIRT1 wild type overexpressing cell Reciprocal immunoprecipitations were also performed in an analogous lines (WT). fashion by first immunoprecipitating 1 mg of protein lysate with a Myc- epitope antibody followed by Western blotting with an anti-HA antibody. To analyze the interaction of wild type SIRT1 and SIRT1 with acid position 261 of the protein (SIRT1 ). This amino acid G261A G261A Foxo3a and p53, we immunoprecipitated cell lysates as above except that is in a highly conserved domain required for the ADP-ribosyl- cells were previously transfected with expression vectors encoding either transferase activity of yeast Sir2 but not its deacetylase activ- wild type p53 or epitope-tagged Myc-Foxo3a. These constructs have been ity (8). To avoid the confounding issue of clonal variation, we previously described (20). Co-immunoprecipitated p53 was visualized fol- established 20 individual control clones expressing G418 resist- lowing Western blot analysis using an anti-p53 antibody (FL-393; Santa ance alone (Neo) and compared them to a similar number of Cruz Biotechnology). PGC-1 Acetylation—To assess the level of PGC-1 acetylation, clones transfected with a vector encoding both G418 resistance HeLa cells were transfected with a Myc-PGC-1 expression vector and and an epitope-tagged wild type SIRT1 construct (n  23). As where indicated, an expression vector encoding p300. Acetylation status demonstrated in Fig. 1B, increased SIRT1 expression led to a was determined by immunoprecipitation of 2 mg of protein lysate in roughly 25% reduction in oxygen consumption (p  0.001). In lysis buffer (19) using an antibody directed against the Myc-epitope tag. contrast, cell lines expressing the mutant in the ADP-ribosyl- Levels of acetylated PGC-1 were subsequently assessed with an anti- transferase domain had oxygen consumption that was 10% acetyl lysine antibody (Cell Signaling). Where indicated, 10 mM nico- tinamide was added for the last 15 h prior to harvest. For the in vitro higher than control cells (p  0.01). Treatment of control or deacetylation reaction, immunoprecipitated PGC-1 was incubated SIRT1-overexpressing clonal cell lines with the electron trans- with 1 g of GST-SIRT1. The deacteylation reaction was performed in port inhibitor antimycin A demonstrated that 90% of the 20 mM Tris, pH 8.0, for1hat30 °Cinthe presence or absence of the measured oxygen consumption derived from mitochondrial indicated concentration of NAD or nicotinamide. After incubation with metabolism (Fig. 1C). GST-SIRT1 the level of acetylated PGC-1 was determined as above by One simple explanation for the decrease in oxygen consump- Western blot analysis followed by detection using an anti-acetyl lysine antibody. tion following increased wild type SIRT1 expression is that these cells contain less mitochondria. Consistent with this hy- RESULTS pothesis, levels of subunit II of the mitochondrial enzyme cy- To assess whether SIRT1 influenced cellular metabolic rate, tochrome oxidase (COX II) were reduced in cell lines that we sought to mimic the conditions previously demonstrated to overexpressed SIRT1 (Fig. 1D). Analysis of several random extend life span in lower organisms. In both yeast and worms, control and SIRT1-overexpressing cell lines demonstrated that simple overexpression of Sir2 results in an increase in life span relative COX II expression was reduced 45% in SIRT1-ex- (2, 3). We therefore established stable PC12 cell lines that pressing cells (n  4 random wild type and control clones; p constitutively expressed increased levels of SIRT1. On average 0.05). SIRT1 mRNA expression and protein (data not shown) were Recent evidence suggests that mitochondrial biogenesis is increased 3-fold in these cell lines (Fig. 1A). We also simul- regulated by the transcriptional coactivator PGC-1 (17). We taneously established cell lines that expressed a mutant of therefore next tested whether SIRT1 might regulate PGC-1 SIRT1 harboring a glycine to alanine substitution at amino activity. To assess the intrinsic transcriptional activity of 16458 SIRT1 Regulates PGC-1 FIG.2. SIRT1 regulates PGC-1 transcriptional activity. The activity of full-length PGC-1 fused to the DNA binding domain of GAL4 was assessed in the presence of increasing amounts of trans- fected wild type SIRT1 (WT) or a position 261 mutant of SIRT1 (G261A). Results are normalized to an internal Renilla control and are expressed as mean  S.D. of triplicate determination. SIRT1 had no effect on the Renilla control. PGC-1 we employed a previously described fusion construct of FIG.3. PGC-1 and SIRT1 interact. A, detection of wild type SIRT1 and PGC-1 interaction. Cells were transfected with epitope-tagged con- full-length PGC-1 fused to the DNA binding domain of GAL4 structs encoding Myc-PGC-1 or HA-SIRT1 and immunoprecipitated (IP) (18). This construct was transfected into PC12 cells along with first with an HA-specific antibody followed by Western blotting (WB) with a luciferase reporter construct under the control of tandem a Myc-specific antibody. B, similar analysis comparing the interaction of GAL4 DNA binding elements. As noted in Fig. 2, expression of wild type SIRT1 (WT) and SIRT1 (GA) with Myc-PGC-1 demon- G261A strating significantly reduced interaction with the ADP-ribosyltrans- wild type SIRT1 but not the SIRT1 mutant reduced the G261A ferase domain mutant. C, reciprocal interaction analysis by first immu- transcriptional activity of a GAL4-PGC-1 fusion protein. noprecipitation (IP) of lysates with a Myc-epitope antibody followed by Based on these observations, we next sought to determine Western blotting (WB) with an HA-specific antibody again demonstrating whether both PGC-1 and SIRT1 were capable of direct inter- reduced affinity of the SIRT1 (GA) for PGC-1. G261A action. We transiently expressed epitope-tagged forms of wild type SIRT1 or SIRT1 along with PGC-1 in HeLa cells. As G261A demonstrated in Fig. 3A, immunoprecipitation of wild type HA-tagged SIRT1 co-immunoprecipitated PGC-1. In contrast to the association of wild type protein with PGC-1, similar analysis of SIRT1 resulted in significantly reduced levels G261A of co-immunoprecipitated PGC-1 (Fig. 3B). This disparity was also evident when the reciprocal immunoprecipitation was per- formed. As shown in Fig. 3C, immunoprecipitation of PGC-1 from lysates revealed an association with wild type SIRT1 but not with SIRT1 . G261A Previous results have demonstrated that SIRT1 can interact with both p53 (21–23) and with members of the Forkhead transcription factor family, including Foxo3a (24–27). Interest- ingly, both of these protein partners of SIRT1 have been impli- FIG.4. SIRT1 interaction with p53 and Foxo3a is pre- G261A cated in the aging process (28, 29). To date the region of SIRT1 served. A, comparison of the strength of interaction between wild type required for these various interactions has not been defined. SIRT1 (WT) and SIRT1 (GA) with p53. B, comparison of the G261A We therefore sought to test whether the reduced strength of strength of interaction between wild type SIRT1 (WT) and SIRT1 G261A (GA) with Foxo3a. In both case wild type or mutant SIRT1 was immu- interaction between PGC-1 and the SIRT1 mutant was G261A noprecipitated from lysates using the HA-epitope and the amount of also observed with other known protein partners. As shown in co-immunoprecipitated p53 or Foxo3a determined by Western blot. Fig. 4, the interaction of the SIRT1 mutant with both p53 G261A (Fig. 4A) and with Foxo3a (Fig. 4B) was qualitatively very similar to wild type SIRT1. These results suggest the SIRT1/ activity (32). Given that p300 also possesses histone acetyl- PGC-1 interaction differs structurally from the interaction transferase activity, we wondered whether co-expression with with either the tumor suppressor p53 or with the transcription PGC-1 would alter acetylation. As noted in Fig. 5B, the ex- factor Foxo3a. pression of p300 dramatically increased PGC-1 acetylation. Given that SIRT1 has been shown to deacetylate numerous Indeed, the level of PGC-1 acetylation showed a mark corre- transcriptional regulators, including p53 (21–23), forkhead lation to the amount of transfected p300 (Fig. 5C). proteins (24–27), and NF-B (30), we next wondered whether To assess whether PGC-1 could be deacetylated by SIRT1 PGC-1 might also be a deacetylase target of SIRT1. Using an in vitro, we immunoprecipitated PGC-1 from lysates of cells antibody that recognized acetyl-lysine, we were able to detect co-transfected with both a PGC-1 and p300 expression vector. low levels of acetylated PGC-1 in cells (Fig. 5A). Treatment To these immunoprecipitates we added purified recombinant with nicotinamide, a known inhibitor of sirtuins (13, 31), re- SIRT1. As demonstrated in Fig. 6, recombinant SIRT1 in the sulted in a substantial increase in PGC-1 acetylation. Previ- presence of NAD but not nicotinamide deacetylated PGC-1. ous studies have demonstrated that the transcriptional co- Addition of NAD did not reduce PGC-1 acetylation in the activator p300 can directly bind PGC-1 a (32). The interaction absence of SIRT1 (data not shown). of PGC-1 and p300 has been documented to result in a con- Finally, we asked whether SIRT1 could stimulate similar formational change in PGC-1 that increases transcriptional deacetylation in vivo. Cells were therefore transfected with SIRT1 Regulates PGC-1 16459 FIG.7. SIRT1 deacetylates PGC-1 in vivo. A, levels of acetylated PGC-1 in the presence or absence of increasing amounts of wild type FIG.5. In vivo acetylation of PGC-1 is stimulated by nicotin- amide or expression of p300. A, levels of acetylated PGC-1 are SIRT1 or the position 261 (G261A) mutant. As noted previously, ex- pression of p300 dramatically increases PGC-1 acetylation. Both wild stimulated by the sirtuin inhibitor nicotinamide. Where indicated, cells were treated for 15 h with 10 mM nicotinamide. PGC-1 was immuno- type and SIRT1 reduced acetylation of PGC-1, although the wild G261A type protein is more efficient. Transfections were performed with 2 g precipitated (IP) from protein lysates using the Myc-epitope tag, and levels of acetylated PGC-1 were determined by Western blot (WB) of Myc-PGC-1, 0.5 g of p300, and increasing amounts of wild type or mutant SIRT1 (0.02, 0.1, or 0.5 g). Empty vector DNA was used to analysis using an antibody that recognizes acetyl-lysine (AcLys) resi- dues. The amount of total PGC-1 immunoprecipitated was assessed maintain equal (3 g) DNA per well. B, SIRT3, a sirtuin family member that localizes to the mitochondria, does not affect PGC-1 acetylation. using the Myc-epitope antibody. B, expression of p300 stimulates PGC-1 acetylation. Levels of acetylated PGC-1 were assessed in the presence or absence of co-transfected p300. C, levels of PGC-1 acety- drial gene expression (33). Given that PGC-1 plays such a lation as a function of increasing amounts of transfected p300. central role in mitochondrial gene expression, we hypothesized that PGC-1 and SIRT1 might physically or functionally inter- act. Our results support this conjecture. It should be noted how- ever that recent evidence suggests that SIRT1 and p300 can also interact (24, 34). In addition, as previously mentioned, PGC-1 and p300 also bind to each other (32). This situation is analogous to the interactions described between SIRT1 and other protein targets. For instance, SIRT1 had been shown to separately bind to both p53 and Foxo3a (21–27). In addition, evidence suggests that p53 and Foxo3a can bind to each other (20, 25). Thus as described above, SIRT1 may be an important aspect for a number FIG.6. SIRT1 deacetylates PGC-1 in vitro in an NAD-depend- of distinct transcriptional complexes potentially acting as a scaf- ent fashion. Cells were transfected with expression plasmids encoding fold to tether various members of the complex together. In addi- Myc-PGC-1 and p300, and PGC-1 was immunoprecipitated from tion, given that NAD regulates the activity of SIRT1, it is possible protein lysates using the Myc-epitope. Recombinant GST-SIRT1 (1 g) was added to the immunoprecipitated PGC-1 for1hinthe presence or that SIRT1 functions as bridge coordinating metabolic status absence of indicated concentrations of either NAD or nicotinamide. with transcription of key target genes. Levels of acetylated PGC-1 were subsequently assessed by Western We cannot at this point determine whether SIRT1 deacety- blot (WB) analysis using an antibody that recognizes acetyl-lysine res- lation of PGC-1 is the sole means by which SIRT1 overexpres- idues (AcLys). sion regulates oxygen consumption in cells. Clearly, as de- scribed above, SIRT1 has numerous cellular targets, including PGC-1 and p300 along with either wild type or SIRT1 G261A other potential regulators of metabolism such as the Forkhead expression vectors. As noted in Fig. 7A, expression of wild type family of transcription factors. In addition, two other observa- SIRT1 dramatically reduced the steady-state level of acety- tions suggest that the relationship between SIRT1 and oxygen lated PGC-1. The expression of SIRT1 also reduced G261A consumption is complex. First, although SIRT1 is not as PGC-1 acetylation, albeit slightly less efficiently than wild G261A efficient in deacetylating PGC-1 as wild type SIRT1, the mu- type SIRT1. In contrast to the effects of SIRT1, expression of tant still appears relatively robust in this activity (see Fig. 7A). the related SIRT3 had no effect on PGC-1 acetylation As such, one might have expected that if the level of PGC-1 (Fig. 7B). acetylation was the only factor regulating oxygen consumption, DISCUSSION than the increased expression of SIRT1 would also lower G261A Our results demonstrate that SIRT1 and PGC-1 physically respiration. As noted in Fig. 1 this does not appear to be the interact and that SIRT1 can regulate PGC-1 acetylation both case. These results suggest that there is not a perfect concord- in vitro and in vivo. In the context of a GAL4 fusion protein, ance between PGC-1 acetylation status and measured oxygen SIRT1 appears to inhibit the transcriptional activity of PGC- consumption. It should be noted that some of the observed 1. Similarly, the effect of stable overexpression of SIRT1 is to differences between Fig. 1B and 7A might relate to specific reduce the level of oxygen consumption. Given that oxygen experimental variables such as the presence or absence of consumption is linked to the generation of reactive oxygen coexpressed p300 or the difference between stable and tran- species and reactive oxygen species levels correlate with lifes- sient expression of SIRT1. Alternatively, these results perhaps pan (1), these results may have important implications for how suggest that a stable SIRT1-PGC-1 interaction may have sirtuins regulate aging. important cellular effects that are independent of the deacety- Our original impetus for these experiments was based on an lation reaction. 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SIRT1 Functionally Interacts with the Metabolic Regulator and Transcriptional Coactivator PGC-1α *

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American Society for Biochemistry and Molecular Biology
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 280, No. 16, Issue of April 22, pp. 16456–16460, 2005 Printed in U.S.A. SIRT1 Functionally Interacts with the Metabolic Regulator and Transcriptional Coactivator PGC-1* Received for publication, February 8, 2005 Published, JBC Papers in Press, February 16, 2005, DOI 10.1074/jbc.M501485200 Shino Nemoto, Maria M. Fergusson, and Toren Finkel‡ From the Cardiovascular Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892-1454 olism of NAD. These two activities are coupled, because the In lower organisms, increased expression of the NAD- dependent deacetylase Sir2 augments lifespan. The acetyl group from the target protein is transferred to the ADP- mechanism through which this life extension is medi- ribose moiety of NAD. Although acetylation and deacetylation ated remains incompletely understood. Here we have have been clearly recognized as reversible modulators of pro- examined the cellular effects of overexpression of tein activity, the role, if any, of the novel O-acetyl-ADP-ribose SIRT1, the closest mammalian ortholog of Sir2. In PC12 moiety remains unclear. Interestingly, a recent report sug- cells, increased expression of the NAD-dependent gested that direct injection of O-acetyl-ADP-ribose into oocytes deacetylase SIRT1 reduces cellular oxygen consumption retards subsequent maturation (9). by 25%. We further demonstrate that SIRT1 expression The requirement for NAD in the deacetylase activity of Sir2 can alter the transcriptional activity of the mitochon- has led to the suggestion that enzymatic activity could be drial biogenesis coactivator PGC-1. In addition, SIRT1 regulated by the concentration of NAD, the ratio of NAD/ and PGC-1 directly interact and can be co-immunopre- NADH, or by the intracellular concentration of nicotinamide cipitated as a molecular complex. A single amino acid (10–13). In particular, caloric restriction appears to augment mutation in the putative ADP-ribosyltransferase do- lifespan in a wide range of species, and evidence suggests that main of SIRT1 inhibits the interaction of SIRT1 with the life extension induced by nutritional deprivation involves, PGC-1 but does not effect the interaction of SIRT1 with and in some cases requires, Sir2 (11, 14, 15). Because the level either p53 or Foxo3a. We further show that PGC-1 is of oxidized and reduced NAD may be altered under starved acetylated in vivo. This acetylation is augmented by conditions, this has led to the hypothesis that Sir2 activity is treatment with the SIRT1 inhibitor nicotinamide or by augmented during caloric restriction by alterations in the expression of the transcriptional coactivator p300. Fi- NAD/NADH ratio (11). Nonetheless, considerable controversy nally we demonstrate that SIRT1 catalyzes PGC-1 exists as to whether starvation significantly alters the NAD/ deacetylation both in vitro and in vivo. These results provide a direct link between the sirtuins, a family of NADH ratio and whether or not this ratio does or does not proteins linked to lifespan determination and PGC-1,a physiologically regulate Sir2 activity (12, 16). In this report, we coactivator that regulates cellular metabolism. provide evidence for another direct link between sirtuins and metabolism. In particular we demonstrate a molecular inter- action between SIRT1 and the transcriptional coactivator The most widely held theory of aging suggests that mitochon- PGC-1, a master regulator of metabolism and mitochondrial drial oxygen consumption and resulting reactive oxygen spe- biogenesis (17). cies generation fuel the aging process (1). In lower organisms EXPERIMENTAL PROCEDURES such as yeast and worms, increased expression of the enzyme Cell Lines and Plasmids—Clonal cell lines were established from Sir2, an NAD-dependent deacetylase, results in an increased PC12 cells obtained from ATCC (Rockville, MD). Cells were maintained life span (2, 3). Sir2 belongs to a growing family of enzymes in a basal growth medium consisting of Dulbecco’s modified Eagle’s collectively known as the sirtuins (4–7). The mechanism medium (Invitrogen) supplemented with 5% fetal calf serum. This me- through which Sir2 extends lifespan is unclear, although its dia formulation contains high glucose (4.5 g/liter) but no exogenous role in gene silencing has been commonly implicated (2, 8). pyruvate. HeLa cells (ATCC) were grown in the same medium. The wild Nonetheless, the relationship, if any, between the sirtuins and type mouse SIRT1 cDNA was purchased from Upstate Biotechnology and subcloned into a HA -tagged cDNA cloning vector (pCruzHA, Santa mitochondrial metabolism remains largely unexplored. Cruz Biotechnology). A glycine to alanine site ADP-ribosyltransferase Analysis of Sir2 enzymatic function has revealed that it mutant at position 261 of SIRT1 was constructed by standard methods, functions differently from previously described histone and mutant and wild type constructs were confirmed by direct sequenc- deacetylases. In particular, studies with purified Sir2 protein ing. This amino acid substitution has been previously demonstrated to revealed that, for every acetyl lysine group that is removed abrogate essentially all of the ADP-ribosyltransferase activity in yeast from a presumed substrate, one molecule of NAD is cleaved Sir2 protein (8). Clonal cell lines expressing wild type SIRT1 (WT), SIRT1 , or the empty HA-vector alone (Neo) were obtained by (4–7). The products produced by this Sir2 enzymatic deactey- G261A transfection and subsequent isolation by limiting dilutions in G418 lation include both nicotinamide and O-acetyl-ADP-ribose. selection. In stable clones that expressed wild type or mutant SIRT1, Therefore, Sir2 appears to possess two enzymatic activities, transgene expression was determined by Western blot analysis using including the deacetylation of a target protein and the metab- an antibody recognizing the HA epitope (Santa Cruz Biotechnology). Total SIRT1 expression was determined by Western blot using an SIRT1-specific antibody (Upstate). Levels of SIRT1 mRNA were deter- * The costs of publication of this article were defrayed in part by the mined by reverse transcription-PCR analysis using the following payment of page charges. This article must therefore be hereby marked SIRT1-specific primers: 5-CCTGACTTCAGATCAAGAGACGGTA-3 “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ To whom correspondence should be addressed: National Institutes of Health, Bldg. 10/CRC, Rm. 5–3330, Bethesda, MD 20892-1454. Tel.: The abbreviations used are: HA, hemagglutinin; WT, wild type; 301-402-4081; Fax: 301-402-9311; E-mail: [email protected]. GST, glutathione S-transferase. 16456 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. SIRT1 Regulates PGC-1 16457 and 5-CTGATTAAAAATGTCTCCACGAACAG-3. Primers for -actin were provided by the manufacturer (Clontech). Measurement of cyto- chrome oxidase subunit II expression was also determined by Western blot expression using a commercially available antibody (Santa Cruz Biotechnology). Human SIRT3 was cloned by standard PCR-based methods and verified by direct sequencing. Oxygen Consumption—Oxygen consumption was measured using a fiber optic oxygen monitor (Instech). All measurements of oxygen con- sumption were performed in intact cells. Cells were plated in 15-cm dishes and allowed to grow for 7 days prior to trypsinization and subsequent oxygen determination. Oxygen consumption was performed in basal growth medium (see above), because we observed that meas- ured oxygen consumption varied substantially depending on the extra- cellular supply of glucose, pyruvate, and serum. For analysis we used 20 individual control cell lines (Neo), 23 SIRT1-overexpressing cells, and 7 clones of SIRT1 . Each cell line was measured by either duplicate or G261A triplicate determinations. PGC-1 Transcriptional Activity and Protein-Protein Interaction— For determination of PGC-1 transcriptional activity we transfected PC12 cells with a previously described construct encoding full-length PGC-1 fused to the GAL-4 DNA binding domain (18). This construct (0.4 g) was co-transfected with increasing amount of wild type SIRT1 (0.1–0.4 g) or a similar amount of the ADP- ribosyltransferase-inac- tive mutant. A GAL-4-dependent luciferase reporter (0.2 g) and an internal Renilla control (0.05 g) were also transfected into each dish. FIG.1. SIRT1 regulates oxygen consumption. A, reverse tran- Twenty-four hours after transfection, cells were harvested and PGC-1 scription-PCR analysis of SIRT1 mRNA expression in four control cell transcriptional activity was analyzed using the dual luciferase reporter lines (Neo) or four random cell lines expressing wild type SIRT1 (WT). assay (Promega). Results are expressed in arbitrary units from a single Levels of -actin are included as a control. B, measurement of oxygen experiment performed in triplicate and are representative of at least consumption in clonal PC12 cells expressing G418 resistance alone three similar experiments. (Neo, n  20 individual clones), wild type SIRT1 (WT, n  23 individual To assess the interaction of PGC-1 and SIRT1, cells were transfected clones), or an ADP-ribosyltransferase domain mutant of SIRT1 (G261A, with wild type HA-SIRT1 and Myc-PGC-1 alone or in combination. n  7 individual clones). Measurements of individual clones were made in duplicate or triplicate and mean  S.E. of all clones are shown. C, These experiments were performed in HeLa cells, because these cells oxygen consumption of control PC12 cells before or after the addition of resulted in substantially higher transgene expression. Twenty-four hours the electron transport inhibitor antimycin A. The arrow indicates the after transfection, extracts were prepared in Lysis buffer (19), and 1 mg of addition of 80 nM antimycin A and demonstrates that essentially all total cell extract was immunoprecipitated with an anti-HA antibody measured oxygen consumption derives from mitochondrial metabolism. (BabCO). The immunoprecipitates were resolved by SDS-PAGE, and the Similar results were obtained in SIRT1-overexpressing cells. D, expres- level of co-immunoprecipitated Myc-PGC-1 was determined by Western sion of subunit II of cytochrome oxidase (COX II) in four random control blot analysis using an anti-Myc antibody (Santa Cruz Biotechnology). cell lines (Neo) and four random SIRT1 wild type overexpressing cell Reciprocal immunoprecipitations were also performed in an analogous lines (WT). fashion by first immunoprecipitating 1 mg of protein lysate with a Myc- epitope antibody followed by Western blotting with an anti-HA antibody. To analyze the interaction of wild type SIRT1 and SIRT1 with acid position 261 of the protein (SIRT1 ). This amino acid G261A G261A Foxo3a and p53, we immunoprecipitated cell lysates as above except that is in a highly conserved domain required for the ADP-ribosyl- cells were previously transfected with expression vectors encoding either transferase activity of yeast Sir2 but not its deacetylase activ- wild type p53 or epitope-tagged Myc-Foxo3a. These constructs have been ity (8). To avoid the confounding issue of clonal variation, we previously described (20). Co-immunoprecipitated p53 was visualized fol- established 20 individual control clones expressing G418 resist- lowing Western blot analysis using an anti-p53 antibody (FL-393; Santa ance alone (Neo) and compared them to a similar number of Cruz Biotechnology). PGC-1 Acetylation—To assess the level of PGC-1 acetylation, clones transfected with a vector encoding both G418 resistance HeLa cells were transfected with a Myc-PGC-1 expression vector and and an epitope-tagged wild type SIRT1 construct (n  23). As where indicated, an expression vector encoding p300. Acetylation status demonstrated in Fig. 1B, increased SIRT1 expression led to a was determined by immunoprecipitation of 2 mg of protein lysate in roughly 25% reduction in oxygen consumption (p  0.001). In lysis buffer (19) using an antibody directed against the Myc-epitope tag. contrast, cell lines expressing the mutant in the ADP-ribosyl- Levels of acetylated PGC-1 were subsequently assessed with an anti- transferase domain had oxygen consumption that was 10% acetyl lysine antibody (Cell Signaling). Where indicated, 10 mM nico- tinamide was added for the last 15 h prior to harvest. For the in vitro higher than control cells (p  0.01). Treatment of control or deacetylation reaction, immunoprecipitated PGC-1 was incubated SIRT1-overexpressing clonal cell lines with the electron trans- with 1 g of GST-SIRT1. The deacteylation reaction was performed in port inhibitor antimycin A demonstrated that 90% of the 20 mM Tris, pH 8.0, for1hat30 °Cinthe presence or absence of the measured oxygen consumption derived from mitochondrial indicated concentration of NAD or nicotinamide. After incubation with metabolism (Fig. 1C). GST-SIRT1 the level of acetylated PGC-1 was determined as above by One simple explanation for the decrease in oxygen consump- Western blot analysis followed by detection using an anti-acetyl lysine antibody. tion following increased wild type SIRT1 expression is that these cells contain less mitochondria. Consistent with this hy- RESULTS pothesis, levels of subunit II of the mitochondrial enzyme cy- To assess whether SIRT1 influenced cellular metabolic rate, tochrome oxidase (COX II) were reduced in cell lines that we sought to mimic the conditions previously demonstrated to overexpressed SIRT1 (Fig. 1D). Analysis of several random extend life span in lower organisms. In both yeast and worms, control and SIRT1-overexpressing cell lines demonstrated that simple overexpression of Sir2 results in an increase in life span relative COX II expression was reduced 45% in SIRT1-ex- (2, 3). We therefore established stable PC12 cell lines that pressing cells (n  4 random wild type and control clones; p constitutively expressed increased levels of SIRT1. On average 0.05). SIRT1 mRNA expression and protein (data not shown) were Recent evidence suggests that mitochondrial biogenesis is increased 3-fold in these cell lines (Fig. 1A). We also simul- regulated by the transcriptional coactivator PGC-1 (17). We taneously established cell lines that expressed a mutant of therefore next tested whether SIRT1 might regulate PGC-1 SIRT1 harboring a glycine to alanine substitution at amino activity. To assess the intrinsic transcriptional activity of 16458 SIRT1 Regulates PGC-1 FIG.2. SIRT1 regulates PGC-1 transcriptional activity. The activity of full-length PGC-1 fused to the DNA binding domain of GAL4 was assessed in the presence of increasing amounts of trans- fected wild type SIRT1 (WT) or a position 261 mutant of SIRT1 (G261A). Results are normalized to an internal Renilla control and are expressed as mean  S.D. of triplicate determination. SIRT1 had no effect on the Renilla control. PGC-1 we employed a previously described fusion construct of FIG.3. PGC-1 and SIRT1 interact. A, detection of wild type SIRT1 and PGC-1 interaction. Cells were transfected with epitope-tagged con- full-length PGC-1 fused to the DNA binding domain of GAL4 structs encoding Myc-PGC-1 or HA-SIRT1 and immunoprecipitated (IP) (18). This construct was transfected into PC12 cells along with first with an HA-specific antibody followed by Western blotting (WB) with a luciferase reporter construct under the control of tandem a Myc-specific antibody. B, similar analysis comparing the interaction of GAL4 DNA binding elements. As noted in Fig. 2, expression of wild type SIRT1 (WT) and SIRT1 (GA) with Myc-PGC-1 demon- G261A strating significantly reduced interaction with the ADP-ribosyltrans- wild type SIRT1 but not the SIRT1 mutant reduced the G261A ferase domain mutant. C, reciprocal interaction analysis by first immu- transcriptional activity of a GAL4-PGC-1 fusion protein. noprecipitation (IP) of lysates with a Myc-epitope antibody followed by Based on these observations, we next sought to determine Western blotting (WB) with an HA-specific antibody again demonstrating whether both PGC-1 and SIRT1 were capable of direct inter- reduced affinity of the SIRT1 (GA) for PGC-1. G261A action. We transiently expressed epitope-tagged forms of wild type SIRT1 or SIRT1 along with PGC-1 in HeLa cells. As G261A demonstrated in Fig. 3A, immunoprecipitation of wild type HA-tagged SIRT1 co-immunoprecipitated PGC-1. In contrast to the association of wild type protein with PGC-1, similar analysis of SIRT1 resulted in significantly reduced levels G261A of co-immunoprecipitated PGC-1 (Fig. 3B). This disparity was also evident when the reciprocal immunoprecipitation was per- formed. As shown in Fig. 3C, immunoprecipitation of PGC-1 from lysates revealed an association with wild type SIRT1 but not with SIRT1 . G261A Previous results have demonstrated that SIRT1 can interact with both p53 (21–23) and with members of the Forkhead transcription factor family, including Foxo3a (24–27). Interest- ingly, both of these protein partners of SIRT1 have been impli- FIG.4. SIRT1 interaction with p53 and Foxo3a is pre- G261A cated in the aging process (28, 29). To date the region of SIRT1 served. A, comparison of the strength of interaction between wild type required for these various interactions has not been defined. SIRT1 (WT) and SIRT1 (GA) with p53. B, comparison of the G261A We therefore sought to test whether the reduced strength of strength of interaction between wild type SIRT1 (WT) and SIRT1 G261A (GA) with Foxo3a. In both case wild type or mutant SIRT1 was immu- interaction between PGC-1 and the SIRT1 mutant was G261A noprecipitated from lysates using the HA-epitope and the amount of also observed with other known protein partners. As shown in co-immunoprecipitated p53 or Foxo3a determined by Western blot. Fig. 4, the interaction of the SIRT1 mutant with both p53 G261A (Fig. 4A) and with Foxo3a (Fig. 4B) was qualitatively very similar to wild type SIRT1. These results suggest the SIRT1/ activity (32). Given that p300 also possesses histone acetyl- PGC-1 interaction differs structurally from the interaction transferase activity, we wondered whether co-expression with with either the tumor suppressor p53 or with the transcription PGC-1 would alter acetylation. As noted in Fig. 5B, the ex- factor Foxo3a. pression of p300 dramatically increased PGC-1 acetylation. Given that SIRT1 has been shown to deacetylate numerous Indeed, the level of PGC-1 acetylation showed a mark corre- transcriptional regulators, including p53 (21–23), forkhead lation to the amount of transfected p300 (Fig. 5C). proteins (24–27), and NF-B (30), we next wondered whether To assess whether PGC-1 could be deacetylated by SIRT1 PGC-1 might also be a deacetylase target of SIRT1. Using an in vitro, we immunoprecipitated PGC-1 from lysates of cells antibody that recognized acetyl-lysine, we were able to detect co-transfected with both a PGC-1 and p300 expression vector. low levels of acetylated PGC-1 in cells (Fig. 5A). Treatment To these immunoprecipitates we added purified recombinant with nicotinamide, a known inhibitor of sirtuins (13, 31), re- SIRT1. As demonstrated in Fig. 6, recombinant SIRT1 in the sulted in a substantial increase in PGC-1 acetylation. Previ- presence of NAD but not nicotinamide deacetylated PGC-1. ous studies have demonstrated that the transcriptional co- Addition of NAD did not reduce PGC-1 acetylation in the activator p300 can directly bind PGC-1 a (32). The interaction absence of SIRT1 (data not shown). of PGC-1 and p300 has been documented to result in a con- Finally, we asked whether SIRT1 could stimulate similar formational change in PGC-1 that increases transcriptional deacetylation in vivo. Cells were therefore transfected with SIRT1 Regulates PGC-1 16459 FIG.7. SIRT1 deacetylates PGC-1 in vivo. A, levels of acetylated PGC-1 in the presence or absence of increasing amounts of wild type FIG.5. In vivo acetylation of PGC-1 is stimulated by nicotin- amide or expression of p300. A, levels of acetylated PGC-1 are SIRT1 or the position 261 (G261A) mutant. As noted previously, ex- pression of p300 dramatically increases PGC-1 acetylation. Both wild stimulated by the sirtuin inhibitor nicotinamide. Where indicated, cells were treated for 15 h with 10 mM nicotinamide. PGC-1 was immuno- type and SIRT1 reduced acetylation of PGC-1, although the wild G261A type protein is more efficient. Transfections were performed with 2 g precipitated (IP) from protein lysates using the Myc-epitope tag, and levels of acetylated PGC-1 were determined by Western blot (WB) of Myc-PGC-1, 0.5 g of p300, and increasing amounts of wild type or mutant SIRT1 (0.02, 0.1, or 0.5 g). Empty vector DNA was used to analysis using an antibody that recognizes acetyl-lysine (AcLys) resi- dues. The amount of total PGC-1 immunoprecipitated was assessed maintain equal (3 g) DNA per well. B, SIRT3, a sirtuin family member that localizes to the mitochondria, does not affect PGC-1 acetylation. using the Myc-epitope antibody. B, expression of p300 stimulates PGC-1 acetylation. Levels of acetylated PGC-1 were assessed in the presence or absence of co-transfected p300. C, levels of PGC-1 acety- drial gene expression (33). Given that PGC-1 plays such a lation as a function of increasing amounts of transfected p300. central role in mitochondrial gene expression, we hypothesized that PGC-1 and SIRT1 might physically or functionally inter- act. Our results support this conjecture. It should be noted how- ever that recent evidence suggests that SIRT1 and p300 can also interact (24, 34). In addition, as previously mentioned, PGC-1 and p300 also bind to each other (32). This situation is analogous to the interactions described between SIRT1 and other protein targets. For instance, SIRT1 had been shown to separately bind to both p53 and Foxo3a (21–27). In addition, evidence suggests that p53 and Foxo3a can bind to each other (20, 25). Thus as described above, SIRT1 may be an important aspect for a number FIG.6. SIRT1 deacetylates PGC-1 in vitro in an NAD-depend- of distinct transcriptional complexes potentially acting as a scaf- ent fashion. Cells were transfected with expression plasmids encoding fold to tether various members of the complex together. In addi- Myc-PGC-1 and p300, and PGC-1 was immunoprecipitated from tion, given that NAD regulates the activity of SIRT1, it is possible protein lysates using the Myc-epitope. Recombinant GST-SIRT1 (1 g) was added to the immunoprecipitated PGC-1 for1hinthe presence or that SIRT1 functions as bridge coordinating metabolic status absence of indicated concentrations of either NAD or nicotinamide. with transcription of key target genes. Levels of acetylated PGC-1 were subsequently assessed by Western We cannot at this point determine whether SIRT1 deacety- blot (WB) analysis using an antibody that recognizes acetyl-lysine res- lation of PGC-1 is the sole means by which SIRT1 overexpres- idues (AcLys). sion regulates oxygen consumption in cells. Clearly, as de- scribed above, SIRT1 has numerous cellular targets, including PGC-1 and p300 along with either wild type or SIRT1 G261A other potential regulators of metabolism such as the Forkhead expression vectors. As noted in Fig. 7A, expression of wild type family of transcription factors. In addition, two other observa- SIRT1 dramatically reduced the steady-state level of acety- tions suggest that the relationship between SIRT1 and oxygen lated PGC-1. The expression of SIRT1 also reduced G261A consumption is complex. First, although SIRT1 is not as PGC-1 acetylation, albeit slightly less efficiently than wild G261A efficient in deacetylating PGC-1 as wild type SIRT1, the mu- type SIRT1. In contrast to the effects of SIRT1, expression of tant still appears relatively robust in this activity (see Fig. 7A). the related SIRT3 had no effect on PGC-1 acetylation As such, one might have expected that if the level of PGC-1 (Fig. 7B). acetylation was the only factor regulating oxygen consumption, DISCUSSION than the increased expression of SIRT1 would also lower G261A Our results demonstrate that SIRT1 and PGC-1 physically respiration. As noted in Fig. 1 this does not appear to be the interact and that SIRT1 can regulate PGC-1 acetylation both case. These results suggest that there is not a perfect concord- in vitro and in vivo. In the context of a GAL4 fusion protein, ance between PGC-1 acetylation status and measured oxygen SIRT1 appears to inhibit the transcriptional activity of PGC- consumption. It should be noted that some of the observed 1. Similarly, the effect of stable overexpression of SIRT1 is to differences between Fig. 1B and 7A might relate to specific reduce the level of oxygen consumption. Given that oxygen experimental variables such as the presence or absence of consumption is linked to the generation of reactive oxygen coexpressed p300 or the difference between stable and tran- species and reactive oxygen species levels correlate with lifes- sient expression of SIRT1. Alternatively, these results perhaps pan (1), these results may have important implications for how suggest that a stable SIRT1-PGC-1 interaction may have sirtuins regulate aging. important cellular effects that are independent of the deacety- Our original impetus for these experiments was based on an lation reaction. The second caveat concerning the effects of observation derived from mitochondrial proteomics analysis that SIRT1 and oxygen consumption is that our preliminary results demonstrated a coordinate regulation of SIRT1 and mitochon- suggest that knockdown of SIRT1 by siRNA also leads to a 16460 SIRT1 Regulates PGC-1 7. North, B. J., and Verdin, E. (2004) Genome Biology http://genomebiology. reduction of oxygen consumption. These results suggest that com/2004/5/1/reviews/0224 the relationship between SIRT1 expression and oxygen con- 8. Imai, S. I., Armstrong, C. M., Kaeberlein, M., and Guarente, L. (2000) Nature 403, 795–799 sumption may be more bell-shaped than linear. 9. Borra, M. T., O’Neill, F. J., Jackson, M. D., Marshall, B., Verdin, E., Foltz, The role of PGC-1 in oxygen consumption is undoubtedly K. R., and Denu, J. M. (2002) J. Biol. Chem. 277, 12632–12641 complex. Part of this complexity may result from the realiza- 10. Revollo, J. R., Grimm, A. A., and Imai, S. (2004) J. Biol. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Apr 22, 2005

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