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Proc Natl Acad Sci U S A. 2008 June 24; 105(25): 8673–8678. Published online 2008 June 17. doi: 10.1073/pnas.0803837105. | PMCID: PMC2438412 |
Copyright © 2008 by The National Academy of Sciences of the USA Genetics Sir2 mediates apoptosis through JNK-dependent pathways in Drosophila Anthony J. Griswold,*† Karen T. Chang,*‡ Alexander P. Runko,†‡ Melanie A. Knight,† and Kyung-Tai Min*§ *Department of Biology, Indiana University, Bloomington, IN 47405; and †Porter Neuroscience Research Center, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892 Received January 30, 2008. Increased expression of the histone deacetylase sir2 has been reported to extend the life span of diverse organisms including yeast, Caenorhabditis elegans, and Drosophila melanogaster. A small molecule activator of Sir2, resveratrol, has also been suggested to extend the fitness and survival of these simple model organisms as well as mice fed high calorie diets. However, other studies in yeast have shown that Sir2 itself may prevent life extension, and high expression levels of Sir2 can be toxic to yeast and mouse cells. This conflicting evidence highlights the importance of understanding the mechanisms by which Sir2 expression or activation affects survival of organisms. To investigate the downstream signaling pathways affected by Sir2 in Drosophila, we generated transgenic flies expressing sir2. Here, we show that overexpression of sir2 in Drosophila promotes caspase-dependent but p53-independent apoptosis that is mediated by the JNK and FOXO signaling pathways. Furthermore, we find that a loss-of-function sir2 mutant partially prevents apoptosis induced by UV irradiation in the eye. Together, these results suggest that Sir2 normally participates in the regulation of cell survival and death in Drosophila. Keywords: Foxo, cell death, deacetylase, dnaJ-H Sir2 is a NAD +-dependent class III histone deacetylase ( 1) that is highly conserved from bacteria to mammals ( 2). Increased expression of sir2 has been associated with life span extension of diverse organisms ranging from yeast to Drosophila melanogaster ( 3). However, the role of sir2 genes in life span regulation has recently been the subject of debate. An extra copy of sir2 increases, whereas a deletion of sir2 decreases the replicative life span of yeast, as measured by counting the number of daughter cells generated from mother cells ( 4). On the contrary, deletion of sir2 has been shown to have no effect on chronological life span, an indicator of nondividing yeast survival, whereas further extending chronological life span induced by calorie restriction ( 5). Moreover, high expression levels of Sir2 can be toxic to yeast ( 6) as well as cardiac cells of transgenic mice overexpressing high levels of Sir2 in heart tissue ( 7). Activation of Sir2 by resveratrol has been reported to extend the fitness and survival of these simple model organisms ( 8, 9) as well as mice fed high calorie diets ( 10, 11). Recently, the role of resveratrol in life span extension has also been disputed, and it is still in question whether resveratrol can activate Sir2 in vivo ( 12) or increase longevity in Drosophila and Caenorhabditis elegans ( 13). Hence, the underlying mechanisms by which sir2 expression or activation can modulate survival or death remain unresolved. One way to clarify these issues is to identify pathways that are altered by Sir2 overexpression and that have strong effects on cell death and survival. In Drosophila, the role of Sir2 in survival is also ambiguous because one report suggests that a deletion of sir2 shortens life span ( 14), whereas another deletion shows an extension of life span under stressful conditions ( 15). In this report, we determine that Sir2 is involved in mediating apoptotic cell death in Drosophila through activation of JNK and Foxo signaling pathways. Sir2 Expression Induces Rough Eye Phenotype in Drosophila. Drosophila has five sir2-like genes, with sir2 being most homologous to the yeast sir2, C. elegans sir2.1, and human sirt1 ( 16). Ubiquitous overexpression of sir2 in Drosophila by using EP lines has been reported to extend life span ( 3). To understand the consequences of Drosophila sir2 overexpression and to identify its downstream signaling pathways, we generated transgenic flies that can express sir2 in the Drosophila eye, a well established system for characterizing signaling pathways. A shows that the use of the gmr-Gal4 driver line to overexpress this gene in developing eyes caused a phenotype, specifically a lack of pigmentation and a rough, bristled appearance. The severity of this phenotype correlates well with dosage of sir2 ( A and B). Consistent with the known pattern of gmr-Gal4 expression ( 17, 18), Sir2 expression was seen in the developing eye imaginal disc in cells posterior to the morphogenic furrow ( C). The endogenous Sir2 is also found at a low level in whole heads of Drosophila as well as in photoreceptor cells posterior to the morphogenic furrow and regions of the antennal disc [ supporting information (SI) Fig. S1]. Also, we confirmed that three other independent UAS-sir2 insertion lines on two different chromosomes demonstrated the same defective eye phenotype when crossed to the gmr-Gal4 line, suggesting that the observed phenotype is not due to insertion of the transgene. Furthermore, sir2 expression in the eye using another driver, eyeless-Gal4, produced a small eye phenotype, and in wing discs, expression by the A9-Gal4 driver caused defective wings ( Fig. S2). Ubiquitous sir2 overexpression using the actin-5C-Gal4 or the pan-neuronal driver elav-gal4 resulted in premature death during development, suggesting that Sir2 affects survival in other cell types as well. Additionally, we found that the overexpressed Sir2 was enzymatically functional because it increased NAD +-dependent deacetylase activity in both larval eye imaginal discs and adult fly heads ( D). To verify whether the observed phenotype is Sir2-specific, we generated transgenic flies to express a Sir2 paralog, CG5085, which shares 43% identity and 63% similarity in amino acid sequence in the deacetylase domain ( Fig. S3A). CG5085 is indeed a functional Sir2 deacetylase family member because recombinant CG5085 demonstrates NAD +-dependent deacetylase activity ( Fig. S3B). However, although structurally and enzymatically similar, when overexpressed by using the gmr-Gal4 driver, CG5085 did not alter the phenotype of the eye ( Fig. S3C). Furthermore, overexpression of lacZ or the Drosophila G protein-coupled receptor methuselah had no effect on eye morphology ( Fig. S3C). This indicates that the eye phenotype in the transgenic flies overexpressing sir2 is indeed Sir2-specific and not due to either general overexpression of proteins or the deacetylase activity itself. Our finding that overexpression of sir2 results in a deleterious effect on various tissues of the fly contrasts with a previously reported effect of sir2 overexpression on longevity ( 3). To clarify this contradictory result, we first crossed a sir2 overexpression line (EP2300) used in the previous report with the gmr-Gal4 driver but found no defective eye phenotype ( A), although it highly expressed sir2 ( B). It is plausible that the insertion of EP2300 could affect genes neighboring sir2, thus leading to modification of the eye phenotype. EP2300 is inserted in a 500-bp region upstream of a chaperone gene dnaJ-H ( C), and we observed an increase in the transcription level of dnaJ-H in adult heads when EP2300 was crossed with the gmr-Gal4 driver ( D). Because overexpression of dnaJ-H can suppress the effects of toxic proteins in the eye ( 19, 20), we generated transgenic flies overexpressing UAS-sir2 and UAS-dnaJ-H together in the eye and found that up-regulation of dnaJ-H ameliorates the defective phenotype caused by sir2 overexpression ( E and Fig. S4). This result suggests an explanation for the lack of an eye phenotype in EP2300 line despite an increase in sir2 expression. Coexpression of sir2 and dnaJ-H by EP2300 raises the possibility that the reported effects of this line ( 3) may give a misleading picture of the role of sir2 in Drosophila aging. Sir2 Expression Induces Apoptosis. Based on the defective eye phenotype, we hypothesized that sir2 expression may cause cell death. Thus, we examined the developing eye in third-instar larval imaginal discs by using acridine orange staining, a vital dye that detects dying cells ( 21), and the TUNEL assay, which identifies cells undergoing programmed cell death ( 22). Staining with acridine orange showed an increase in dying cells in the posterior part of eye discs overexpressing sir2 ( A and Fig. S5), suggesting that sir2 overexpression causes cell death. Numerous TUNEL-positive cells in the imaginal discs with sir2 overexpression were also found, whereas the control showed few positive cells ( A and Fig. S5), indicating that the phenotype is mediated by apoptotic cell death in the developing eye. In addition, in vitro caspase-3 activity in the eye imaginal disc overexpressing sir2 increased 1.5-fold when compared with the control ( B). This increase in caspase-3 activity was also verified by immunostaining for active caspase-3 in the imaginal discs; overexpression of sir2 showed an increase in the number of more intensely stained cells ( A and Fig. S5). To further explore the involvement of caspase-dependent cell death in transgenic flies overexpressing sir2, we investigated whether the apoptosis inhibitors DIAP1, which inhibits initiator caspase activity ( 23), or p35, a viral protein that inhibits downstream effector caspases ( 24), can block the cell death induced by the overexpression of sir2. Overexpression of DIAP1 together with sir2 in the eyes of transgenic flies showed a significant rescue of the eye phenotype ( C). Similarly, coexpression of p35 with sir2 also restored the normal eye phenotype ( C). The rescue of the eye phenotype was not a result of reduced sir2 expression because the level of Sir2 was similar in the rescued flies and those expressing lacZ ( Fig. S6). Taken together, these results suggest that sir2 overexpression induces a caspase-dependent apoptotic cell death that can be blocked by DIAP1 or p35 overexpression. Sir2 Mediates Apoptosis Through JNK and Foxo Pathways. A previous report has characterized mammalian Sir2 as an apoptosis inhibitor through its deacetylation of p53 ( 25). In Drosophila, the role of p53 is still under investigation, although it has been shown to regulate cell death in response to stress ( 26), similar to its mammalian homolog. However, the relationship of p53 and Sir2 in Drosophila has not been explored. To determine whether p53 and Sir2 are in the same genetic pathway to cause apoptosis in the eye, we coexpressed p53 and sir2. The eyes of the flies overexpressing both p53 and sir2 were more severely affected than either p53 or sir2 overexpressed alone ( A). However, overexpression of dominant negative p53 constructs ( 27) did not rescue the sir2 phenotype ( A). This suggests that the sir2 overexpression effect is p53-independent and that p53 and sir2 overexpression work in parallel to induce cell death in Drosophila. Sir2 can alter the activity of mammalian FOXO3a by deacetylation ( 28, 29). Additionally, to increase life span in C. elegans, overexpression of sir2.1 requires DAF-16, the FOXO3a transcription factor homolog ( 30). No such direct link between Sir2 and FOXO has been established in Drosophila; however, overexpression of foxo in the Drosophila eye exhibits a defective eye phenotype ( 31, 32). Because our results show that sir2 overexpression induces apoptotic cell death in the Drosophila eye, we decided to investigate whether FOXO activity might be involved in the induction of apoptosis as a result of sir2 overexpression in Drosophila. We overexpressed sir2 in a foxo null mutant background and found a less severe eye phenotype ( B), suggesting a genetic interaction between Sir2 and FOXO in cell death pathways in Drosophila. Recently, it was shown that the foxo-induced defective eye phenotype can be modulated through the JNK signaling pathway ( 33). Furthermore, increased activation of JNK is associated with an apoptotic eye phenotype in Drosophila ( 34, 35). Because JNK signaling interacts with FOXO and influences apoptotic pathways, we examined whether the JNK pathway is also involved in the Sir2-induced eye phenotype. We found that the transcription level of the JNK phosphatase puc, a downstream target of the JNK signaling pathway ( 36), is increased in the heads of flies overexpressing sir2 ( C), suggesting an increase in JNK-dependent transcription. Inhibition of this signaling pathway by overexpression of bskDN, a dominant negative form of Drosophila JNK, resulted in a major improvement of the eye phenotype caused by sir2 overexpression ( D). Additionally, inhibition of JNK signaling by coexpression of puc with sir2 demonstrated a significant rescue in the eye ( D) and wing ( Fig. S2B), consistent with a report that constitutive overexpression of puc can rescue the eye phenotype caused by increased JNK activity ( 36). These rescue flies do not reduce the level of Sir2 below that expressed by coexpression of lacZ ( Fig. S6). Together, these results suggest that sir2 overexpression requires JNK signaling to induce cell death in the eye. Sir2 Overexpression Induces Proapoptotic Gene Expression. Active JNK signaling can induce caspase-dependent apoptotic cell death ( 35, 36). This is consistent with our results showing increased caspase-3 activity in the eye imaginal discs overexpressing sir2 ( A and Fig. S5). Because active JNK mediates the expression of the proapoptotic gene reaper ( 36, 37) and FOXO can induce the proapoptotic gene hid ( 38), we next determined the transcription levels of the proapoptotic genes reaper, grim, and hid in flies overexpressing sir2 by using quantitative real-time RT-PCR. The transcription levels of reaper, grim, and hid in fly heads overexpressing sir2 were increased, whereas the levels of deacetylases rpd3 and CG5085 were not significantly altered ( E). To verify the involvement of these genes in the apoptosis of flies overexpressing sir2, we used a deficiency line, Df(3L)H99, that reduces the level of reaper, grim, and hid. Overexpression of sir2 in the eye of Df(3L)H99/+ partially rescues the defective phenotype ( F), suggesting that these genes are indeed involved in the Sir2-induced apoptotic pathways, but other components may also be involved. Together, these results imply that sir2 overexpression activates JNK signaling and increases the expression of proapoptotic genes that leads to apoptotic cell death and consequently the abnormal eye phenotype. Endogenous Sir2 Enhances UV-Induced Apoptosis. The findings described above resulted from misexpression of Sir2 but leave open the question of whether this gene normally plays a role in survival decisions. We therefore studied apoptosis in a loss-of-function sir2 mutant ( 39). Because it has been reported that the JNK and FOXO pathways regulate the apoptotic response to UV irradiation ( 38), we irradiated one pupal retina of sir2 mutant and control flies with 5 mJ/cm 2 of UV. Upon eclosion to adult, the UV-irradiated eyes of sir22A-7-11, a targeted deletion of sir2 by homologous recombination ( 39), were larger and more morphologically normal than that of the control. Based on a calculation of the size ratio between the normal eye and the eye exposed to UV irradiation, the sir2 mutant flies shows 50% larger eyes than control ( G and H). To confirm that this effect reflected the loss of sir2 function and was not the result of an adventitious mutation in this strain, we performed a complementation analysis by using a deletion ( Df(2L)ED784) from the DrosDel collection ( 40) that removes a cluster of genes that surround and include sir2. Indeed, flies heterozygous for sir22A-7-11 and Df(2L)ED784 show less damage of the UV-irradiated eye than do flies heterozygous for sir22A-7-11 and an isogenic control deletion ( Df(2L)ED774) that removes a cluster of genes from a nearby region but leaves sir2 intact ( Fig. S7). Thus, the effect on apoptosis seen in the mutant line maps to the vicinity of the sir2 gene. Our evidence that endogenous Sir2 in the Drosophila eye plays a role in apoptosis is consistent with our finding that sir2 overexpression induces apoptotic cell death in the eye imaginal discs. An important issue is thus the identification of signaling pathways that mediate these effects. JNK signaling is implicated by phenotypic alleviation via the expression of a dominant negative JNK or an inhibitor JNK signaling. Also, loss of function of foxo can ameliorate the eye phenotype induced by sir2 overexpression, suggesting that sir2 can activate a proapoptotic function of FOXO. These pathways may intersect at JNK to induce the proapoptotic function of FOXO ( 38). The result of these pathways is the observed increase in proapoptotic gene expression of reaper, grim, and hid. This in turn leads to increased caspase activity and ultimately cell death ( Fig. S8). Our results show that sir2 overexpression in Drosophila does not necessarily promote longevity, and endogenous Sir2 plays a critical role in regulating cell survival and death in the animal. Hence, it will be of future interest to study the signaling pathways induced by sir2 expression that lead to JNK activation as well as the relationship between Sir2 and FOXO in modulating apoptotic and survival pathways in Drosophila. Together, these will determine pathways affected by sir2 expression and give insights as to how it can mediate both cell survival and cell death. Fly Stocks. The following fly stocks were obtained from the Bloomington Stock Center: gmr-Gal4, EP2300, ey-Gal4, A9-Gal4, UAS-lacZ, UAS-bskDN, UAS-DIAP1, UAS-p35, GUS-p53, GUS-p53.Ct, GUS-p53.259H, Df(3L)H99, sir22A-7-11, Df(2L)ED774, and Df(2L)ED784. The FOXO mutant foxo25 was a gift from E. Hafen. UAS-puc, UAS-danJ-H, and UAS-methuselah were gifts from D. McEwen (University of Texas Health Science Center, San Antonio), H. Tricoire (Institut Jacques Monod, Paris), L. Seroude (Queen's University, Kingston), respectively. All flies were maintained and crosses performed at 25°C. To create the transgenic UAS-sir2 and UAS-CG5085 lines, the coding region of each gene was subcloned from a pAc5.1 vector ( 41) into a pINDY6 vector. The transgenic fly lines were generated by standard germ-line transformation method (Genetic Services). Western Blotting. 50 μg of total fly head protein was run on an 8% SDS-acrylamide gel and transferred to nitrocellulose. The membrane was blocked in 5% milk and probed with a Sir2 polyclonal antibody [a gift from S. Parkhurst (Fred Hutchinson Cancer Research Center, Seattle)] at a 1:200 dilution, followed by donkey anti-mouse HRP-conjugated secondary antibody (Jackson ImmunoResearch) at 1:2,000 dilution. Blots were developed by using ECL reagents (Amersham). Immunohistochemistry and Detection of Cell Death. Imaginal discs were dissected from third-instar larvae in Drosophila Ringer's solution (182 mM KCl, 46 mM NaCl, 3 mM CaCl2, 10 mM Tris). For Sir2 and caspase-3 staining, discs were fixed in 4% paraformaldehyde for 20 min at room temperature and labeled with the anti-Sir2 antibody at 1:50 dilution or anticleaved caspase-3 antibody (Cell Signaling Technology) and a Cy3-labeled anti-mouse (Sir2) or rabbit (caspase-3) IgG secondary antibody (Jackson ImmunoResearch) at 1:200. For acridine orange staining, imaginal discs were incubated for 5 min at room temperature in a 1.6 × 10−6 M solution of acridine orange (Molecular Probes). TUNEL staining was performed with the ApopTag fluorescein in situ apoptosis detection kit (Chemicon) according to the manufacturer's instructions. Real-Time RT-PCR. One μg of total RNA from adult fly heads was isolated by using TRIzol reagent (Invitrogen), converted to cDNA by using SuperScriptII reverse transcriptase (Invitrogen), and then used for real-time PCR with SYBR Green reagent (Applied Biosystems). Primers were designed from mRNA sequence by using PrimerExpress software v1.0 (Applied Biosystems) to amplify ≈150-bp near the 3′ end of each mRNA. Thermocycling was conducted in the ABI 7000 sequence detection system (Applied Biosystems) according to the manufacturer's protocol. Sequence Detection System Version 1.7 software (Applied Biosystems) was used to analyze amplification plots. The relative quantity of amplified cDNA corresponding to each gene was calculated by using the ΔΔCt method and normalized for expression of actin5C in each sample. In Vitro Caspase and Deacetylase Activity. For caspase activity, 50 μg of imaginal disk protein lysate was used in a caspase-3/CPP32 fluorometric assay (Biovision) according to the manufacturer's protocol. For Sir2 deacetylase activity, 40 μg of adult head and 15 μg of imaginal disk protein lysates were used in SIRT1 fluorometric activity assay/drug discovery kit (Biomol) according to the manufacturer's protocol. Pupal Retina UV Irradiation. Retinal sensitivity to irradiation was performed basically as described previously ( 38). Briefly, pupae were collected 24 h after puparium formation, and the pupal shell surrounding one eye was removed. Pupae were immobilized, and the retina was exposed to 5 mJ/cm 2 in a UV cross-linker (UVP Laboratory Products HybriLinker 200). Pupae were maintained in the dark after irradiation until eclosion. The heads of eclosed flies were imaged from the top, and the number of pixels contained in each eye was determined by using Photoshop (Adobe). 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