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. Author manuscript; available in PMC: 2011 Feb 1.
Published in final edited form as: Exp Gerontol. 2009 Oct 17;45(2):97. doi: 10.1016/j.exger.2009.10.009

Drosophila foxo acts in males to cause sexual-dimorphism in tissue-specific p53 life span effects

Jie Shen 1, John Tower 1,*
PMCID: PMC2814947  NIHMSID: NIHMS153594  PMID: 19840842

Abstract

Sex-specific selective pressures are hypothesized to lead to sexually antagonistic gene functions that contribute to phenotypes such as aging and cancer. However, relatively little is known about the identity of such genes and possible mechanisms. Here we report that nervous system-specific over-expression of wild-type p53 in Drosophila caused decreased life span in males and increased life span in females. In contrast, tissue-general over-expression produced the opposite pattern: increased life span in males and decreased life span in females. In a foxo null background, p53 life span effects in males were reversed, becoming similar to the effects in females. In contrast, a Sir2 null background tended to reduce the magnitude of p53 effects. The data demonstrate that wild-type p53 over-expression can regulate life span independent of foxo, and suggest that foxo acts in males to produce sexually antagonistic life span effects of p53.

Keywords: antagonistic pleiotropy, Sir2, aging

1. Introduction

The mammalian p53 transcription factor is a multi-functional tumor suppressor that regulates apoptosis, cell senescence, oxidative stress responses, and mitochondrial metabolism (Green and Kroemer, 2009), and it is preferentially required in females for neural tube closure, embryonic viability and adult fecundity (Chen et al., 2008; Hu et al., 2008; Kang et al., 2009). Several lines of evidence implicate p53 in aging. Truncated forms of p53 protein in mice can produce a premature aging-like phenotype, apparently by causing a state of p53 hyperactivation (Maier et al., 2004; Moore et al., 2007), whereas increasing the dose of wild-type p53 along with the p53 activator p19ARF could delay aging (Matheu et al., 2007). These data suggest that increased activity of wild-type p53 can promote longevity, whereas mis-regulated and activated p53 forms can promote aging. Taken together, the results from mammals suggest that p53 exhibits antagonistic pleiotropy, in that it favors development, fecundity and cancer resistance in young animals, but may promote aging in old animals (Hu et al., 2008; Kang et al., 2009; Rodier et al., 2007; Ungewitter and Scrable, 2009).

Inhibition of the insulin/IGF1-like signaling (IIS) pathway increases life span in C. elegans, Drosophila and mice, and in C. elegans this has been shown to be dependent upon the forkhead-family transcription factor Daf16 (Kenyon, 2005; Murphy et al., 2007; Samuelson et al., 2007). The most closely related transcription factor in Drosophila is Foxo, which can increase life span when over-expressed in fly fat-body tissue (Giannakou et al., 2004; Hwangbo et al., 2004). It has been suggested that one way p53 might affect life span in mammals and other species is by interacting with the IIS pathway (Scrable et al., 2009). For example, in C. elegans, inactivation of the p53 homolog cep-1 causes increased life span, and this was dependent upon function of Daf16 (Arum and Johnson, 2007). The phenotype of a mutation, which will affect the animal throughout development and adulthood, can sometimes differ from the effects of transgenic and RNAi manipulations, which may be targeted to specific tissues and life-cycle stages; indeed, both IIS (Broughton and Partridge, 2009) and p53 (this study; (Waskar et al., 2009)) manipulations can have contrasting effects on life span depending upon tissue, developmental stage and gender. Mutations in the p53 DNA binding domain can result in dominant mutant forms of the protein, that sometimes antagonize normal p53 function (Brodsky et al., 2000). Expression of a dominant mutant form of p53 in adult Drosophila has been shown to increase life span in females (Bauer et al., 2005; Shen et al., 2009), and this was found to correlate with decreased IIS (Bauer et al., 2007); however, it has not been determined if Drosophila life span regulation by p53 requires Foxo.

One potential problem in interpreting the effects of dominant mutant p53 transgenes is that the phenotypes might be neomorphic, i.e., not necessarily representative of the function(s) of wild-type p53. For example, dominant mutant forms of p53 containing a disruption of the DNA binding domain are expected to antagonize the transcriptional activity of p53, but may also promote one or more of the transactivation-independent effects of p53, in a tissue-specific manner (Green and Kroemer, 2009). We have recently shown that, in Drosophila, wild-type p53 can cause increased life span when over-expressed during development, and that in adults, wild-type p53 has sex-specific effects on life span: Over-expression of wild-type p53 in a tissue-general pattern in adult flies caused decreased life span in females, and increased life span in males (Waskar et al., 2009); and consistent with these observations, null mutation of Drosophila p53 caused increased life span preferentially in females.

Developmental stage-specific and sex-specific selective pressures are hypothesized to produce antagonistically-pleiotropic gene functions, that in turn affect phenotypes such as reproductive fitness, behavior and life span (Hughes and Reynolds, 2005; Leips et al., 2006; Long and Rice, 2007; Tower, 2006). Here we determine that the sexually antagonistic life span effects of p53 are tissue-specific, and that these effects are modulated in a sex-specific way by foxo and Sir2.

2. Materials and Methods

2.1 Drosophila culture and strains

Drosophila melanogaster culture, life span assays, Geneswitch driver strains, and the multi-copy UAS-eGFP strain (“UAS-ultraGFP”) are as previously described (Shen et al., 2009). Flies were cultured at 25°C until eclosion, and then adults were maintained at 29°C, or at 25°C as indicated for specific experiments; additional details are provided in Supplementary materials. UAS-p53 transgenic lines and chromosomal deficiency lines that uncover the foxo and Sir2 loci were obtained from Bloomington Drosophila Stock Center. “p53WT1” is P{GUS-p53}2.1, “p53WT2” is P{UAS-p53.Ex}2, and “p53WT3” is P{UAS-p53.Ex}3. The foxo[21rec7A] and foxo[w24] null mutation lines were provided by M. Tatar (Min et al., 2008), and the Sir2[4.5] and Sir2[5.26] null mutation lines were provided by S.L. Helfand (Rogina and Helfand, 2004). Certain genotypes were generated by chromosomal recombination and/or crosses to double-balancer strains. Details of strain construction and characterization are provided in Supplementary materials; all strains and genotypes are listed in Supplementary Table S1.

2.2 Quantitative real-time RT-PCR

Total RNA was isolated from 15 male or female flies, using TRIzol reagent (Invitrogen). Flies were 12 days of age, and had been cultured for 10 days at 29oC on RU486 food, or on control (ethanol-only) food. Quantitative real-time RT-PCR was performed using the Bio-RAD MyiQ™ Real-time PCR detection system and SYBR green dye, according to the manufacturer's instructions, and Rp49 gene expression was used as control. Values are plotted as mean ± SD of triplicate biological replicates. Additional details of methods and primer sequences are presented in Supplemental materials.

2.3 Western blot assay

Total protein was isolated by homogenizing 15 male or female flies in Laemmli sample buffer (Bio-Rad). Flies were 12 days of age, and had been cultured for 10 days at 29oC on RU486 food, or on control (ethanol-only) food. Antibodies were specific for the phosphorylated form of Akt (#4054, Cell Signaling Technology; 1:1,000 dilution), or for total Akt (#4691, Cell Signaling Technology; 1:1,000 dilution). Anti-beta-actin antibody (#4967, Cell Signaling Technology; 1:5,000 dilution) was used as a loading control. Additional details are provided in Supplemental materials.

2.4 Statistical analyses

For life span assays, mean, standard deviation, median, percent change in mean, percent change in median, and log rank (Breslow) p value were calculated using R 2.6.2 (RDevelopmentCoreTeam, 2006). Unpaired, two-sided t-tests were used to determine the significance of differences in mRNA levels between the RU486-treated and control groups; statistically significant differences (p<0.05) are indicated along with the fold change in mean above the bar graphs.

3. Results

3.1 Conditional over-expression of transgenes using the Geneswitch system

To over-express p53 in adult flies, the Geneswitch system was utilized, where transgene expression is induced by feeding the flies the drug RU486 (Mifepristone) (Nicholson et al., 2008). Characterization of the system using UAS-LacZ and UAS-GFP reporter strains demonstrated that the Act-GS-255B driver yields target gene expression throughout all the tissues of male and female flies, and that the Elav-GS driver produces expression that is strictly limited to the central nervous system (Ford et al., 2007; Shen et al., 2009)(Supplemental Fig. S1). To control for any potential effects on life span of the drug, the driver lines were crossed to the w[1118] injection strain and to Oregon-R wild-type strain to produce progeny containing only the drivers and no target transgene (“Control” flies). In the first set of control experiments with the Elav-GS driver, the drug-treated flies exhibited a small increase in life span in males (+4%) and no significant change in females (Fig. 1 A, B). For both the Elav-GS driver and the Act-GS-255B driver the change in life span observed in the absence of a target transgene was on average approximately +3%, which we interpret as the background variability of the life span assay (results summarized in Fig. 4; details of statistical analyses for all life span data are presented in Table 1 and Supplemental Table S2).

Fig. 1.

Fig. 1

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Effect of adult-specific p53 over-expression on life span. The indicated wild-type p53 transgenes (p53WT1 or p53WT2) were over-expressed using the nervous system-specific driver Elav-GS (“Elav”) and the tissue-general driver Act-GS-255B (“255B”), as indicated. p53 over-expression was carried out in wild-type background (“+/+”), foxo null background (“foxo−/−”) and Sir2 null background (“Sir2−/−”) as indicated. The life span assays were performed at 29°C. Solid diamonds represent adults treated with drug (“+”, ~125 flies), open circles represent the no-drug controls (“−“, ~125 flies). Survival curves are plotted as percent survival versus adult age in days. Mean life span for each cohort is presented along with the p value for log rank test (in parentheses). Details of statistical analyses are presented in Supplementary Table 2. (A, C, E, G, I, K) male flies. (B, D, F, H, J, L) female flies. (A, B) Control flies containing the Elav-GS driver and no target transgene, genotype w[1118]/yw ; +; Elav/+. (C, D) Elav-GS driver plus p53WT1, genotype w[1118]/yw ; p53WT1/+; Elav/+. (E, F) Elav-GS driver plus p53WT1, in the foxo null background, genotype w[1118]; p53WT1/+; Elav, Df(3R)Exel8159/foxo[21]. (G, H) Elav-GS driver plus p53WT1 in the Sir2 null background, genotype w[1118]; p53WT1, Df(2L)BSC344/ Sir2[4.5]; Elav/+. Survival curves for Elav-GS plus p53WT2, in wild-type, foxo null and Sir2 null background are presented in Supplementary Figure S2. (I, J) Act-GS-255B driver plus p53WT2, genotype w[1118]; 255B/p53WT2; +. (K, L) Act-GS-255B driver plus p53WT1 in the foxo null background, genotype w[1118];255B/p53WT1; Df(3R)Exel8159/foxo[21]. Survival curves for Act-GS-255B driver plus p53WT1 and p53WT3 in wild-type background, and p53WT2 in foxo null bankground, and p53WT3 in Sir2 null background are presented in Supplementary Figure S3.

Fig. 4.

Fig. 4

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Summary of effects of wild-type p53 over-expression. The life span data for nervous system-specific over-expression of p53 using the Elav-GS driver (“NS”), and tissue-general over-expression of p53 using the Act-GS-255B driver (“general”), are summarized for each sex, as indicated. Numbers in black type indicate the percent change in median life span obtained for replicate experiments using independent wild-type p53 transgenes; ns = not significant. The summary includes all data for the wild-type background (“+/+”), the foxo null background (“foxo−/−”) and the Sir2 null background (“Sir2−/−”). “Control (no p53)” indicates the changes in life span observed in the absence of a p53 target gene (background of the assay). Net positive effects are indicated with a blue or pink arrow, net negative effects are indicated with a blue or pink T-bar. Dotted lines indicate no effect, or reduced effect, relative to the wild-type background. The changes in life span caused by p53 over-expression in the wild-type (“+/+”) background are indicated in blue for males, and pink for females. In the foxo null background the effects in males are reversed in sign, becoming like the female pattern, and are therefore indicated in pink. Changes in phosphorylated Akt level (“AKT-P”) are indicated by up and down black arrows, and changes were consistently observed only for nervous system-specific expression of p53 in the wild-type background. Somatic sexual differentiation in Drosophila is controlled by the on/off status of the Sex lethal (Sxl) gene, as indicated.

Table 1.

Life span data with means, standard deviations, medians, percent change in mean and median, and log rank p value.

Cross
MxF		 RU486 Genotype Sex N Meana Median Δ Mean Δ Median Log Rank
p Value

Exp1 Life span assay with GS255B driver at 29°C
3-1 − w/Y; 255B/+; + M 126 52.52±10.68 56 -------- --------- ---------
3-1 + w/Y; 255B/+; + M 132 56.02±7.79 57 6.68 1.79 1.30E-05
3-1 − w/+ ;255B/+; + F 126 57.96±10.65 61 --------- --------- ---------
3-1 + w/+ ;255B/+; + F 124 60.33±9.89 62 4.09 1.64 5.92E-05
4-1 − w/Y; 255B/+; + M 130 48.11±8.56 48 --------- --------- ---------
4-1 + w/Y; 255B/+; + M 131 50.52±9.15 50 5.01 4.17 0.008
4-1 − w ;255B/+; + F 131 52.82±5.31 53 --------- --------- ---------
4-1 + w ;255B/+; + F 136 54.9±5.02 55.5 3.94 4.72 0.001
5-1 − w/Y; 255B/p53WT1; + M 128 47.01±4.97 46 --------- --------- ---------
5-1 + w/Y; 255B/p53WT1; + M 130 51.85±7.44 53 10.29 15.22 1.02E-12
5-1 − w; 255B/p53WT1; + F 134 49.95±5.15 49 --------- --------- ---------
5-1 + w; 255B/p53WT1; + F 134 46.61±5.53 47 −6.68 −4.08 1.22E-08
6-1 − w/Y; 255B/p53WT2; + M 125 43.59±8.45 44 --------- --------- ---------
6-1 + w/Y; 255B/p53WT2; + M 137 49.58±8.39 52 13.73 18.18 3.43E-09
6-1 − w; 255B/p53WT2; + F 129 49.32±6 50 --------- --------- ---------
6-1 + w; 255B/p53WT2; + F 129 46.29±7.86 47 −6.13 −6.00 9.42E-05
7-1 − w/Y; 255B/+; p53WT3/+ M 134 46.84±9 48 --------- --------- ---------
7-1 + w/Y; 255B/+; p53WT3/+ M 125 51.61±9.04 53 10.19 10.42 6.986E-07
7-1 − w/yw; 255B/+; p53WT3/+ F 130 51.72±9.32 54 --------- --------- ---------
7-1 + w/yw; 255B/+; p53WT3/+ F 136 50.43±6.06 52 −2.48 −3.70 5.64E-08
9-8 − w/Y;255B/p53WT1; foxo −/− M 118 35.7±11.26 36 --------- --------- ---------
9-8 + w/Y;255B/p53WT1; foxo −/− M 115 33.65±8.07 33 −5.75 −8.33 0.003
9-8 − w;255B/p53WT1; foxo −/− F 129 42.02±8.3 43 --------- --------- ---------
9-8 + w;255B/p53WT1; foxo −/− F 123 40.3±5.45 40 −4.10 −6.98 3.48E-04
10-8 − w/Y; 255B/p53WT2; foxo −/− M 104 30.05±12.47 33 --------- --------- ---------
10-8 + w/Y; 255B/p53WT2; foxo −/− M 104 16.59±6.55 18 −44.80 −45.45 0
10-8 − w; 255B/p53WT2; foxo −/− F 128 36.98±7.6 37 --------- --------- ---------
10-8 + w; 255B/p53WT2; foxo −/− F 128 33.83±10.91 36 −8.51 −2.70 0.579
12-11 − w/Y;255B Sir2 −/−;p53WT3/+ M 131 43.02±7.16 45 --------- --------- ---------
12-11 + w/Y;255B Sir2 −/−;p53WT3/+ M 122 43.37±10.12 45 0.80 0.00 0.003
12-11 − w/yw;255B Sir2 −/−;p53WT3/+ F 127 47.25±12.11 50 --------- --------- ---------
12-11 + w/yw;255B Sir2 −/−;p53WT3/+ F 131 47.86±12.45 52 1.28 4.00 0.064

Exp2 Life span assay with Elav-GS driver at 29°C
3-2 − yw/Y; +; Elav/+ M 126 48.59±5.02 50 --------- --------- ---------
3-2 + yw/Y; +; Elav/+ M 127 49.61±7.56 50 2.10 0.00 0.004
3-2 − yw/+; +; Elav/+ F 118 58.2±5.28 58 --------- --------- ---------
3-2 + yw/+; +; Elav/+ F 125 60.82±4.8 62 4.49 6.90 5.94E-05
4-2 − yw/Y; +; Elav/+ M 124 42.07±7.61 44 --------- --------- ---------
4-2 + yw/Y; +; Elav/+ M 127 43.29±8.52 46 2.90 4.55 0.002
4-2 − yw/w; +; Elav/+ F 127 43.99±10.55 47 --------- --------- ---------
4-2 + yw/w; +; Elav/+ F 129 46.58±8.29 48 5.89 2.13 0.076
5-2 − yw/Y; p53WT1/+; Elav/+ M 122 43.94±5.46 45 --------- --------- ---------
5-2 + yw/Y; p53WT1/+; Elav/+ M 123 39.56±8.4 41 −9.97 −8.89 0.036
5-2 − yw/w; p53WT1/+; Elav/+ F 125 50.78±9.47 55 --------- --------- ---------
5-2 + yw/w; p53WT1/+; Elav/+ F 120 60.21±6.33 61 18.56 10.91 0
6-2 − yw/Y; p53WT2/+; Elav/+ M 122 43.27±5.86 43.5 --------- --------- ---------
6-2 + yw/Y; p53WT2/+; Elav/+ M 129 39.37±8.01 38 −9.01 −12.64 0.016
6-2 − yw/w; p53WT2/+; Elav/+ F 125 44.34±13.5 49 --------- --------- ---------
6-2 + yw/w; p53WT2/+; Elav/+ F 122 53.89±10.37 55 21.54 12.24 1.49E-13
9-13 − w/Y;p53WT1/+; Elav foxo −/− M 127 33.46±12.42 35 --------- --------- ---------
9-13 + w/Y;p53WT1/+; Elav foxo −/− M 124 44.91±12.96 47 34.21 34.29 6.84E-14
9-13 − w;p53WT1/+;Elav foxo −/− F 125 46.17±7.55 48 --------- --------- ---------
9-13 + w;p53WT1/+;Elav foxo −/− F 127 52.94±8.82 55 14.68 14.58 0
10-13 − w/Y;p53WT2/+; Elav foxo −/− M 123 41.8±9.99 45 --------- --------- ---------
10-13 + w/Y;p53WT2/+; Elav foxo −/− M 123 45.56±13.58 48 9.01 6.67 7.21E-08
10-13 − w; p53WT2/+; Elav foxo −/− F 120 40.49±8.19 42 --------- --------- ---------
10-13 + w; p53WT2/+; Elav foxo −/− F 130 43.71±11.14 46 7.94 9.52 1.10E-05
16-14 − w/Y; p53WT1 Sir2 −/−;Elav/+ M 132 40.83±9.21 44 --------- --------- ---------
16-14 + w/Y; p53WT1 Sir2 −/−;Elav/+ M 133 34.8±9 34 −14.75 −22.73 5.20E-07
16-14 − w; p53WT1 Sir2 −/−;Elav/+ F 126 45.83±7.43 47.5 --------- --------- ---------
16-14 + w; p53WT1 Sir2 −/−;Elav/+ F 125 50.35±7.13 51 9.86 7.37 9.96E-10
16-15 − w/Y; p53WT2 Sir2 −/−; Elav/+ M 121 35.26±7.3 36 --------- --------- ---------
16-15 + w/Y; p53WT2 Sir2 −/−; Elav/+ M 131 31.99±8.08 32 −9.28 −11.11 0.017
16-15 − w;p53WT2 Sir2 −/−; Elav/+ F 126 46.19±7.97 47 --------- --------- ---------
16-15 + w;p53WT2 Sir2 −/−; Elav/+ F 132 47.61±8.86 49 3.06 4.26 0.084
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a

Mean life span, days +/− SD. All cross directions are presented with the male parent strain first, female parent strain second; details on strains and strain numbers are presented in Supplemental Table S1; additional life span data is presented in Supplemental Table S2.

3.2 Sexually antagonistic effects of p53 on adult life Span

When p53 was over-expressed specifically in adult nervous tissue using the Elav-GS driver, and either one of two independent p53-WT transgenes, this produced decreased life span in males (−9% and −13%, respectively), and increased life span in females (+11% and +12%, respectively)(Fig. 1C, D; Supplemental Fig. S2C, D; data summarized in Fig. 4 and Table 1). This result is in striking contrast to our previous observation that tissue-general over-expression of p53-WT3 using the Act-GS-255B driver produces the opposite effect: increased life span in males and decreased life span in females (Waskar et al., 2009). That result was confirmed here using three independent p53-WT transgenes, and life span was increased in males by +10 to +18%, and decreased in females by −4 to −6% (Fig. 1I, J; Supplemental Fig. S3C-F; data summarized in Fig. 4 and Table 1). Because tissue-general over-expression includes nervous-system expression, the opposite effects produced by nervous system-specific expression versus tissue-general expression suggests that the effect of p53 in peripheral tissues dominates over the effect in nervous system, and/or that signaling between tissues is involved in producing the effects on adult life span. Quantitative real-time RT-PCR (qPCR) assays confirmed that p53 RNA was being efficiently over-expressed in both males and females, using both the tissue-general driver (19-fold induction in males and 13-fold induction in females) and the nervous system-specific driver (~2-fold induction in both males and females; Supplemental Fig. S4).

3.3 Interaction of foxo and Sir2 mutations with p53 life span effects

To determine if the life span effects of wild-type p53 expression observed here might be due to altered IIS, several experiments were conducted. First, it was asked whether the life span changes were dependent upon the IIS target transcription factor gene foxo. When p53 was over-expressed in a foxo null background, the pattern and magnitude of effects in females remained essentially the same, i.e., increased life span with nervous system-specific expression (Fig. 1F; Supplemental Fig. S2F), and decreased life span with tissue-general over-expression (Fig. 1L; data summarized in Fig. 4 and Table 1). However, the foxo null background caused a reversal of the pattern in males, such that p53 effects in males now had a pattern similar to females, i.e., increased life span with nervous system-specific over-expression (Fig. 1E; Supplemental Fig. S2E) and decreased life span with tissue-general over-expression (Fig. 1K; Supplemental Fig. S3G; data summarized in Fig. 4 and Table 1). These data demonstrate that wild-type p53 over-expression can both positively and negatively regulate life span independent of foxo, while at the same time they suggest that foxo acts in males to modulate (reverse) the tissue-specific pattern of p53 life span effects, thereby producing sexually antagonistic effects of p53.

Previously adult-specific over-expression of foxo in the fat-body (FB) tissue of the head using the S1-32-GS driver (Hwangbo et al., 2004), or in the FB tissue of the thorax and abdomen using the S1-106-GS driver (Giannakou et al., 2004) was found to cause increased life span. Here these FB drivers were used together to drive expression of the p53-WT1 transgene throughout the FB tissues of adult male and female flies. No life span increases were observed upon FB expression of p53 in either males or females (Supplemental explanatory text; Supplemental Table S2). This suggests that FB is not likely to be the relevant tissue for the life span increase observed upon tissue-general p53 over-expression.

The effects of adult-specific p53 over-expression were also assayed in a Sir2 null mutant background. Sir2 encodes a conserved protein deacetylase known to regulate life span in several species (Finkel et al., 2009). For nervous system-specific over-expression of p53 in the Sir2 null background, the negative effect of p53 on life span remained intact in males (Fig. 1G; Supplemental Fig. S2G), whereas the positive effect in females appeared to be reduced (Fig. 1H; Supplemental Fig. S2H; data summarized in Fig. 4 and Table 1). For tissue-general over-expression of p53 in the Sir2 null background, the effect of p53 on life span was reduced in both males and females, such that no significant changes were observed (Supplemental Fig. S3I, J; data summarized in Fig. 4 and Table 1). Therefore, taken together, the data suggest that the Sir2 gene is required for the magnitude of some, but not all, of the observed effects of p53 over-expression on life span.

3.4 Effect of p53 over-expression on phosphorylated Akt levels

The activity of the Foxo transcription factor is regulated by IIS, whereby the phosphorylated and activated form of the protein kinase Akt acts to phosphorylate and inactivate Foxo. Phosphorylated Akt levels therefore provide one read-out of signaling through the IIS pathway. Phosphorylated Akt levels were assayed by western blot for control flies, and flies in which p53 was over-expressed, in the wild-type background as well as in foxo null and Sir2 null backgrounds. One striking result was that the foxo null mutation caused greatly reduced phosphorylated Akt levels in both males and females (Fig. 2A-D), whereas total Akt levels were unchanged (Fig. 2E; Supplemental Fig. S5A). This indicates that, in Drosophila, foxo positively regulates Akt phosphorylation level. This is consistent with a previous report that in cultured mammalian myocytes activation of either FoxO1 or FoxO3 caused increased Akt phosphorylation levels (Ni et al., 2007), as well as reports that in Drosophila foxo regulates expression of the upstream IIS gene InR (Puig et al., 2003), and that in C. elegans the foxo homolog Daf16 regulates IIS (Murphy et al., 2007).

Fig. 2.

Fig. 2

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Effect of p53 over-expression on levels of Akt and phosphorylated Akt. Total protein was isolated from 15 adult flies for each genotype and sex. “+” and “−“ indicates 10 days treatment with drug, and no-drug controls, respectively. 2X and 1X volume from the same sample were loaded next to each other, as indicated, as controls for quantification. Samples were analyzed by western blot, using antibodies specific for phosphorylated Akt (panels A-D; two Drosophila-specific isoforms are recognized), and for total Akt (panel E), and for the beta-actin loading control, as indicated. Signals were quantified by phosphorimager, normalized to the loading control, and the results are plotted in relative units as bar graphs to the right of each western blot. (A, B) Nervous system-specific over-expression of p53WT2 using the Elav-GS driver. Genotypes are indicated above the panels. “+/+” indicates w[1118]/yw ; p53WT2/+; Elav/+. “foxo−/−” indicates w[1118] ; p53WT2/+; Elav, Df(3R)Exel8159/foxo[21]. “Sir2−/−” indicates w[1118] ; p53WT2, Df(2L)BSC344/ Sir2[4.5]; Elav/+. “control” indicates w[1118]/yw ; +; Elav/+. (C, D) Tissue-general over-expression of p53WT using the Act-GS-255B driver. “+/+” indicates w[1118]; 255B/p53WT2; +. “foxo−/−” indicates w[1118];255B/p53WT2; Df(3R)Exel8159/foxo[21]. “Sir2−/−” indicates w[1118]/y[1]w[1118];255B, Df(2L)BSC344/ Sir2[4.5]; p53WT3/+. “control” indicates w[1118]; 255B/+; +. (E) Nervous system-specific over-expression of p53 in males using the Elav-GS driver, assay of total Akt protein levels; genotypes as in (A, B).

Interestingly, nervous system-specific over-expression of p53 in adult flies in the wild-type background caused decreased Akt phosphorylation in males (Fig. 2A), and increased Akt phosphorylation in females (Fig. 2B), indicating that the sexually antagonistic effects of p53 on life span can correlate with sexually opposite effects on physiology (data summarized in Fig. 4). However, the changes in Akt phosphorylation could be uncoupled from the regulation of life span by p53 in several ways. First, the effects on life span produced by tissue-general p53 expression did not correlate with any consistent alteration in Akt phosphorylation in males or females (Fig. 2C, D). Second, in the foxo null background, the increased life span produced by nervous system-specific p53 expression and the decreased life span produced by tissue-general p53 expression did not correlate with any consistent alteration in Akt phosphorylation in males or females (Fig. 2A, B); and this result was confirmed using two different foxo-null genetic backgrounds, as well as greater amounts of protein to increase the sensitivity of the assay (Supplemental Fig. S5B, C). Finally, the life span decrease produced in males by nervous system-specific p53 over-expression in the Sir2 null background also did not correlate with any consistent alterations in Akt phosphorylation (Fig. 2A, B). Note that the apparent alterations in Akt phosphorylation upon tissue-general p53 over-expression in Sir2 null background (Fig. 2C, D) were not observed in replicate experiments (Supplemental Fig. S5D, E). Therefore we conclude that the sexually opposite effects on Akt phosphorylation caused by nervous-system expression of p53 are dependent upon foxo and Sir2, but are not required for p53 to either positively or negatively regulate life span.

3.5 Effect of p53 on IIS pathway gene expression

Foxo is a transcription factor, and several positively-regulated Foxo target genes have been identified for Drosophila, including the small heat shock protein gene l(2)efl and the translation repressor gene 4E-BP (Junger et al., 2003; Puig et al., 2003; Teleman et al., 2008; Wang et al., 2005). Over-expression of l(2)efl is reported to increase life span, and induction of l(2)efl has been suggested to mediate part of the life-span promoting effects of foxo (Wang et al., 2005). The 4E-BP (or Thor) gene is induced by foxo, and is also a target of the TOR pathway that regulates translation, growth, and life span in response to diet in Drosophila (Kapahi et al., 2004; Teleman et al., 2008). qPCR was used to assay l(2)efl transcript levels upon p53 over-expression in wild-type, foxo null and Sir2 null mutant backgrounds (Fig. 3). Expression of l(2)efl was reduced in the foxo null background, particularly in males, consistent with the regulation of this gene by foxo (Fig. 3A-D). In addition, expression of l(2)efl was greater in males than females, suggesting the possibility of greater foxo activity in males; a similar set of results were obtained for 4E-BP (Supplemental Fig. S6). Conditional over-expression of p53 in either the tissue-general or nervous system-specific manner in a wild-type background had no detectable effect on either l(2)efl or 4E-BP expression levels, in males or females, consistent with the conclusion that p53 regulates life span by a mechanism independent of foxo and the expression of l(2)efl and 4E-BP.

Fig. 3.

Fig. 3

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Effect of p53 over-expression on endogenous 4E-BP and Dilp2 gene expression. Quantitative real-time RT-PCR assay was used to measure RNA levels for the 4E-BP and Dilp2 genes, as indicated. Mean ± SD of triplicate assays (biological replicates) is plotted as bar graphs. Statistically significant differences (p<0.05) were determined using unpaired, two-sided t-tests, and are indicated above the bars along with the fold change in mean. (A, B, E, F) Nervous system-specific over-expression of p53WT2 using the Elav-GS driver. (C, D, G, H) Tissue-general over-expression of p53WT2 using the Act-GS-255B driver. Specific genotypes are the same as in Figure 3. (A-D) Assay of l(2)efl RNA levels. (E-G) Assay of Dilp2 RNA levels. (A, C, E, G) Males. (B, D, F, H) Females.

The expression of two genes that act upstream in the IIS pathway, Dilp2 and InR, were also assayed, in wild-type, foxo null and Sir2 null mutant backgrounds. Dilp2 encodes a Drosophila insulin-like peptide, and Dilp2 expression is down-regulated in flies that are long-lived due to foxo over-expression in the head FB using driver S1-32-GS (Hwangbo et al., 2004). InR encodes the Drosophila insulin-like receptor, and InR expression was reported to be positively regulated by foxo (Puig et al., 2003). p53 over-expression had no consistent effects on the expression of either Dilp2 (Fig. 3E-H) or InR (Supplemental Fig. S7), supporting the conclusion that p53 over-expression affects life span through some mechanism other than altered IIS. Finally, expression of the Sir2 gene was assayed upon p53 over-expression in wild-type, foxo null and Sir2 null mutant backgrounds, and no consistent changes were detected (Supplemental Fig. S8), suggesting that p53 life span effects are not mediated by alterations in expression of Sir2.

4. Discussion

Here over-expression of wild-type p53 in adult flies was found to regulate life span in a tissue-specific and sexually-dimorphic manner. Nervous system-specific over-expression of p53 caused decreased life span in males and increased life span in females, whereas tissue-general over-expression produced the opposite pattern: decreased life span in females and increased life span in males (summarized in Fig. 4). In a foxo null background, p53 life span effects in males were reversed, becoming similar to the effects in females. In contrast, a Sir2 null background tended to reduce the magnitude of p53 effects. Because life span alteration did not correlate with changes in expression of IIS pathway genes, did not require foxo, and could be uncoupled from changes in Akt phosphorylation levels, the data suggest that wild-type p53 can both positively and negatively regulate life span independent of alterations in IIS. The fact that female life span was increased by adult nervous system-specific expression of both wild-type p53 (this study) and dominant mutant p53 transgenes (Bauer et al., 2007; Bauer et al., 2005) may indicate a transactivation-independent mechanism in that tissue.

Taken together, the present data demonstrate that wild-type p53 over-expression can both positively and negatively regulate life span independent of foxo, and suggest that foxo acts in males to modulate (reverse) the tissue-specific pattern of p53 life span effects, thereby producing sexually antagonistic effects of p53. This situation is somewhat reminiscent of the relationship of foxo to the DR response: foxo was not required for life span increase in response to DR, but was found to modulate the response (Giannakou et al., 2008; Min et al., 2008).

One relevant issue to consider is how any differences in the level of p53 expression achieved might affect the present results. The qPCR assay used to confirm p53 over-expression was performed on whole-body RNA, and therefore the overall p53 level observed is much greater with tissue-general over-expression (using driver Act-GS-255B) compared to the more limited nervous system-specific expression (using driver Elav-GS) (Supplemental Fig. S4). The expression level in the female brain is expected to be similar with these two drivers, as indicated by control experiments using GFP reporters (Shen et al., 2009). Therefore, at least for females, the opposite effect of p53 produced using these two drivers is most likely due to the site of over-expression rather than any difference in the magnitude of over-expression. However, in males, the ActGS-255B driver produced a lower level of GFP reporter expression (Supplemental Fig. S1; and (Shen et al., 2009)). Therefore, conceivably, the opposite result obtained upon tissue-general p53 over-expression in males (positive effect) compared to females (negative effect) could result from a threshold effect of p53 expression, where moderate over-expression is positive and higher-level expression is negative. Indeed a recent study of C. elegans hermaphrodites is consistent with a threshold in p53 effects: low levels of mitochondrial stress (suggesting modest p53 activation) caused increased life span, whereas high levels of mitochondrial stress (suggesting strong p53 activation) caused decreased life span (Ventura et al., 2009). However, such a threshold cannot explain the opposite effect of nervous-system expression observed here between male flies (negative effect) and females (positive effect), as that is in the wrong direction. The level of p53 over-expression achieved in wild-type versus foxo null and Sir2 null backgrounds was similar for male and female nervous-system expression, and for tissue-general expression in females (Supplemental Fig. S4), indicating that the different effects on life span observed in those instances are not simply due to variation in the level of p53 over-expression. In contrast, for tissue-general over-expression of p53 in males, the level of over-expression in wild-type background was moderate and produced increased life span, whereas over-expression in the foxo null background was greater and produced decreased life span, again conceivably consistent with a threshold effect. However, tissue-general over-expression of dominant-negative p53 transgenes produced the opposite effect relative to wild-type p53 transgenes, in both males and females (Shen et al., 2009; Waskar et al., 2009). This indicates that the opposite effects of p53 transgenes on male and female life span are not likely to be due to some difference in the efficiency of transgene expression in males versus females, or to some differential toxicity of the encoded proteins in males versus females. Therefore, in summary, while differences in the magnitude of p53 over-expression achieved may be contributing to the effects on life span, at least for males, they are not sufficient to explain the pattern of opposite effects on life span observed in males versus females, nor the opposite effects on life span observed with the two different drivers, nor the opposite effects observed with wild-type versus dominant-mutant p53 transgenes; these results appear to require truly opposite effects of p53 in the two sexes and in different tissues.

One way the present results might be interpreted is if foxo is activated in males to promote the differentiation of a male-specific metabolic state. This hypothesis is consistent with our observation that the Foxo target genes l(2)efl and 4E-BP were expressed at higher levels in males than females in a foxo dependent manner. There is ample precedent for a role for IIS in Drosophila sexual differentiation. For example, disruption of IIS and mutations in the InR gene caused a feminization of male locomotor behaviors (Belgacem and Martin, 2006), and IIS is required for both male and female gametogenesis and fecundity (Hsu and Drummond-Barbosa, 2009; Toivonen and Partridge, 2008; Ueishi et al., 2009). In mammals there are four FOXO factors (FoxO1-4) that may be partially redundant in function, and among these, FoxO3 appears most related to Drosophila Foxo. FoxO3-null mice exhibit abnormal ovarian follicular development and age-dependent infertility (Hosaka et al., 2004), consistent with a role for FoxO3 in mammalian sexual differentiation. Finally, dramatically increased life span in the Ames dwarf mouse correlates with a loss of sexually dimorphic gene expression patterns in the liver (Amador-Noguez et al., 2005). Therefore, one way that IIS and foxo might regulate life span in Drosophila and other species is by promoting sexual differentiation, thereby revealing the sexually-antagonistic effects of genes such as p53.

An interesting question is what are the mechanism(s) by which p53 is able to positively and negatively regulate life span independent of foxo. There are a number of possibilities, because p53 is known to regulate several processes that might in turn affect life span, including autophagy, mitochondrial metabolism, apoptosis, and cell senescence (Green and Kroemer, 2009). Indeed, in C. elegans, the p53 homolog cep-1 mediates the life span promoting effects of mild mitochondrial stress (Ventura et al., 2009), and the life span increase due to cep-1 mutation requires autophagy gene function (Tavernarakis et al., 2008). One possibility is if Drosophila p53 over-expression caused alterations in feeding behavior or nutrient utilization, this might regulate life span through the DR pathway. The Sirtuin gene family is implicated in mediating DR responses in several species (Finkel et al., 2009), and over-expression of Sir2 is reported to increase life span in Drosophila females (Rogina and Helfand, 2004). Moreover, the life span increases caused by nervous system-specific expression of a dominant mutation p53 transgene and a Sir2 transgene in female flies were not additive, leading to the suggestion that these effects might occur through a common pathway, perhaps one related to DR (Bauer et al., 2009). Our data indicate that some, but not all, of the tissue-specific effects on life span of wild-type p53 required Sir2. The positive effects of p53 over-expression on adult fly life span were eliminated or reduced by Sir2 mutation, consistent with a possible link to the DR pathway, whereas nervous-system-specific expression of p53 in males decreased life span independent of Sir2 (summarized in Fig. 4). This suggests the possibility that wild-type p53 may be acting through more than one pathway to affect adult fly life span, perhaps by acting through different pathways in different tissues and/or in the two sexes.

Taken together, the data are consistent with the conclusion that p53 exhibits antagonistic pleiotropy, and in particular, that Drosophila p53 and foxo exhibit sexual antagonistic pleiotropy with regard to life span. These results suggest the potential usefulness of gender-specific manipulation of p53 and IIS in future interventions in aging and cancer.

Supplementary Material

01

Acknowledgements

We thank Michelle Arbeitman for comments. This work was supported by a grant from the Department of Health and Human Services to JT (AG011833) and by a pilot project award to JT from the USC ADRC (1P50 AG05142)

Footnotes

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