All animals coordinate growth and maturation to reach their final size and shape. In insects, insulin family molecules control growth and metabolism, whereas pulses of the steroid 20-hydroxyecdysone (20E) initiate major developmental transitions. 20E signaling also negatively controls animal growth rates by impeding general insulin signaling involving localization of the transcription factor dFOXO and transcription of the translation inhibitor 4E-BP. The larval fat body, equivalent to the vertebrate liver, is a key relay element for ecdysone-dependent growth inhibition. Hence, ecdysone counteracts the growth-promoting action of insulins, thus forming a humoral regulatory loop that determines organismal size (Colombani, 2005).
In metazoans, the insulin/IGF signaling pathway (IIS) plays a key role in regulating growth and metabolism. In Drosophila, a family of insulin-like molecules called Dilps activates a unique insulin receptor (InR) and a conserved downstream kinase cascade that includes PI3-kinase (PI3K) and Akt/PKB. Recent genetic experiments have established that this pathway integrates extrinsic signals such as nutrition with the control of tissue growth during larval stages. The larval period is critical for the control of animal growth, since it establishes the size at which maturation occurs and, consequently, the final adult size. Maturation is itself a complex process that is controlled by the steroid 20-hydroxyecdysone (20E). Peaks of 20E determine the timing of all developmental transitions, from embryo to larva, larva to pupa, and pupa to adult. Ecdysteroids are mainly produced by the prothoracic gland (PG), a part of a composite endocrine tissue called the ring gland. Final adult size thus mainly depends on two parameters: the speed of growth (or growth rate), which is primarily controlled by IIS, and the overall duration of the growth period, which is limited by the onset of the larval-pupal transition and timed by peaks of ecdysone secretion. Very little is known concerning the mechanisms that coordinate these two parameters during larval development (Colombani, 2005).
To investigate the function of ecdysone in controlling organismal growth, a genetic approach was developed that allowed modulation basal levels of ecdysteroids in Drosophila. The initial rationale was to modify the mass of the ring gland in order to change the level of ecdysteroid production. For this goal, the levels of PI3- kinase activity were manipulated in the PG by crossing P0206-Gal4 (P0206>), a line with specific Gal4 expression in the PG and corpora allata (CA), with flies carrying UAS constructs allowing expression of either wild-type (PI3K) or dominant-negative (PI3KDN) PI3-kinase. As expected, these crosses produced dramatic autonomous growth effects in the ring gland, and particularly in the PG: tissue size was increased upon PI3K activation and decreased upon inhibition. Surprisingly, the changes in ring gland growth were accompanied by opposite effects at the organismal level. P0206>PI3K animals (with large ring glands) showed reduced growth at all stages of development and produced emerging adults with reduced size and body weight (78% of wt). Conversely, reducing PI3K activity in the ring gland of P0206>PI3KDN animals led to increased growth and produced adults with 17% greater weight on average. Adult size increase was attributable to an increase in cell number in the wing and the eye. Adult size reduction was accompanied by a decrease in cell number in the wing and in cell size in the eye (Colombani, 2005).
Importantly, the timing of embryonic and larval development of these animals was comparable to control. Both the L2 to L3 transition as well as the cessation of feeding (wandering) occurred at identical times. Further, animals entered pupal development at the same time, except for P0206>PI3KDN animals, which showed a 1-2 hrs delay intrinsic to the UAS-PI3KDN line itself. The duration of pupal development was slightly modified, however, as adult emergence was delayed in P0206>PI3K animals and advanced in P0206>PI3KDN animals, albeit by less than 4 hours following 10 days of development. In contrast, the speed of larval growth was found to be increased in P0206>PI3KDN animals and decreased in the P0206>PI3K animals background at the earliest stage that could be measured (early L2 instar). Because none of these effects were observed when PI3K levels were modified specifically in the CA using the Aug21- Gal4 driver, it was concluded that the observed phenotypes are solely due to PI3K modulation in the PG. Together, these results demonstrate that manipulating PI3K levels in the PG induces non-autonomous changes in the speed of larval growth (growth rate effects), without changing the timing of larval development (Colombani, 2005).
To investigate whether these effects could be attributed to changes in 20E levels, ecdysteroid titers were measured in third instar larvae of the different genotypes. Early after ecdysis into third instar (74hrs AED) ecdysteroids are present at basal level. They accumulate to an intermediate plateau around 90hrs AED and reach peak levels before pupariation (120hrs AED). Because early L3 levels are below the detection limit of the EIA assay, ecdysteroid titers were measured at the intermediate plateau (90hrs AED). In these conditions, a very modest increase of ecdysteroids was observed in P0206>PI3K animals larvae and a small but significant decrease in P0206>PI3KDN animals animals. This was further confirmed by measuring the transcript levels of a direct target of 20E, E74B, which responds to low/moderate levels of 20E. However, in early L3 larvae with basal ecdysteroid levels (74hrs AED), differences in E74B transcripts were clearly visible, with a 1.9-fold increase seen for P0206>PI3K animals and a 1.7-fold decrease for P0206>PI3KDN animals. This establishes that basal circulating levels of 20E are modified in response to manipulation of PI3K levels in the PG. It also suggests that the differences observed on basal 20E level off with the strong global increase of ecdysteroids in mid/late L3 (Colombani, 2005).
Several related lines of evidence strengthen these results: (1) the increase in growth rate and size observed in P0206>PI3KDN animals can be efficiently reverted by adding 20E to their food; (2) feeding wild-type larvae 20E recapitulates the effects observed in P0206>PI3K animals animals; (3) ubiquitous silencing of EcR using an inducible EcR RNAi construct results in a growth increase similar to that observed in P0206>PI3KDN larvae. Finally, the phantom (phm) and disembodied (dib) genes, which are specifically expressed in the PG and encode hydroxylases required for ecdysteroid biosynthesis, showed 1.65- and 2.2- fold increased expression, respectively, upon PI3K activation in the ring gland. This supports the notion that 20E biosynthesis is mildly induced in these experimental conditions. In line with previous results, neither 20E treatment nor EcR silencing has any effect on developmental timing. Overall, the results indicate that manipulating basal levels of 20E signaling in various ways modifies the larval growth rate without affecting the timing of the larval transitions (Colombani, 2005).
Variations in ecdysone levels in animals with different sized ring glands could be due to changes in the PG tissue mass or, alternatively, to a specific effect of PI3K signaling in the secreting tissue. To distinguish between these two possibilities, extra growth was induced in the PG using either dMyc or CyclinD/Cdk4, two potent growth inducers in Drosophila. Although the size of the larval ring gland was markedly increased under these conditions, no effect on pupal or adult size was observed, implying that the size of the ring gland is not the critical factor in the control of body size. Instead, it is likely that the InR/PI3K signaling pathway can specifically activate ecdysone production from the PG (Colombani, 2005).
The possibility was tested that ecdysone signaling opposes the growth-promoting effects of IIS in the larva. To test this, larvae were fed 20E and xPI3K activity was assessed in vivo using a GFP-PH fusion (tGPH) as a marker. Membrane tGPH localization shows a marked decrease in the fat body of 20E-fed animals, and this effect can be reverted by specifically silencing EcR in the fat body. This indicates that ecdysone-induced growth inhibition correlates with decreased IIS, and is mediated through the nuclear receptor EcR. Conversely, larvae with PI3KDN expression in the PG show a 4.2-fold increase in the global levels of dPKB/Akt activity, as measured by the phosphorylation levels of serine 505. In Drosophila cells (as in other metazoan cells) high levels of PI3K/AKT activity cause the transcription factor dFOXO to be retained in the cytoplasm, while low PI3K/AKT activity allows dFOXO to enter the nucleus where it promotes 4E-BP transcription. In larvae with ectopic PI3K expression in the PG, a strong increase is observed in nuclear dFOXO in fat body cells. Similar results were obtained by feeding larvae with 20E. Conversely, inactivation of EcR signaling in fat body cells was carried out using the clonal over-expression of a dominant-negative form of EcR (EcRF645A). In these conditions, a reduction was observed in the accumulation of dFOXO in the nuclei of EcRF645A-expressing cells. As an expected consequence of the increased nuclear dFOXO, global accumulation of 4E-BP transcripts was consistently higher in P0206>PI3K animals as well as in 20E-fed early L3 larvae as compared to control animals, and reduced in arm>EcR-RNAi animals. Together, these results indicate that ecdysone-dependent inhibition of larval growth correlates with a general alteration of insulin/IGF signaling, and a relocalization of dFOXO into the cell nuclei. To more directly test the role of dFOXO in the growth-inhibitory function of ecdysone signaling, the effects of increasing ecdysone levels were examined in a dFOXOmutant genetic background. Although homozygous dFOXO21 animals do not display a detectable growth phenotype, introducing the dFOXO21 mutation was sufficient to totally revert the growth defects of P0206>PI3K animals animals, either when homozygous or heterozygous. This data establishes that dFOXO is required for 20E-mediated growth inhibition (Colombani, 2005).
The endocrine activities of the brain and the fat body have previously been implicated in the humoral control of larval growth. In order to test for possible roles of these two organs in mediating the systemic growth effects of ecdysone, EcR expression was silenced specifically in the brain cells that produce insulins (IPCs) or in the fat body. While specific expression of EcR RNAi in the IPCs fails to reproduce the overgrowth observed in armGal4>EcR-RNAi animals, EcR silencing in the fat body elicits an acceleration of larval growth and a remarkable increase in pupal size. Moreover, no detectable delay in the larval timing was observed in pplGal4>EcR-RNAi animals. Thus, specifically reducing 20E signaling in the fat body is sufficient to recapitulate the systemic effects of global EcR silencing. This demonstrates that the fat body is a major target for ecdysone, and that this tissue can act to relay the 20E growth-inhibitory signal to all larval tissues (Colombani, 2005).
In summary, these results establish an additional role for 20E in modulating animal growth rates. This function is mediated by an antagonistic interaction with IIS that ultimately targets dFOXO function. A similar antagonistic interaction between 20E and insulin signaling controls developmentally-regulated autophagy in Drosophila larva (Colombani, 2005).
Although a direct effect of ecdysone on the cellular growth rate of all larval tissues cannot be ruled out, the experiments reveal a key role for the fat body in relaying ecdysone-dependent growth control signals. Together with previous work, these data suggest that various inputs such as nutrition and ecdysone converge on this important regulatory organ, which then controls the general IIS to modulate organismal growth (Colombani, 2005).
How then is growth connected to developmental timing? The finding that 20E can modulate growth rates in addition to developmental transitions places this hormone in a central position for coordinating these two key processes and controlling organismal size (Colombani, 2005).
Aging of a eukaryotic organism is affected by its nutrition state and by its ability to prevent or repair oxidative damage. Consequently, signal transduction systems that control metabolism and oxidative stress responses influence life span. When nutrients are abundant, the insulin/IGF signaling pathway promotes growth and energy storage but shortens life span. The transcription factor Foxo, which is inhibited by insulin/IGF signaling, extends life span in conditions of low insulin/IGF signaling activity. Life span can also be increased by activating the stress-responsive Jun-N-terminal kinase (JNK) pathway. JNK requires Foxo to extend life span in Drosophila. JNK antagonizes insulin/IGF signaling, causing nuclear localization of Foxo and inducing its targets, including growth control and stress defense genes. JNK and Foxo also restrict insulin/IGF signaling activity systemically by repressing insulin/IGF ligand expression in neuroendocrine cells. The convergence of JNK signaling and IIS on Foxo provides a model to explain the effects of stress and nutrition on longevity (Wang, 2005).
These data suggest Foxo is a convergence point for insulin/IGF signaling (IIS) and JNK signaling. Through its responsiveness to these two pathways, Foxo is well positioned to integrate information about environmental stress and nutrient availability and to elicit appropriate biological responses. Such a system would ensure that growth could proceed in an unrestrained manner when energy resources are available and the cell is not exposed to external insults (IIS is active, JNK is off, and Foxo is repressed). However, in situations of low food availability or an adverse environment, IIS would cease to signal, or JNK would be activated, resulting in translocation of Foxo to the nucleus. The ensuing Foxo-induced gene expression has several effects at the cell as well as the organism level and is likely to counteract premature senescence. The induction of genes such as thor can reduce cell growth, presumably to limit the cell’s anabolic expenses in adverse situations. Other target genes, such as the small heat shock protein l(2)efl, are expected to have a direct role in allaying damage inflicted by environmental insults and may prevent the accumulation of toxic protein aggregates. The suppression of dilp2 expression by JNK and Foxo in insulin-producing cells, in contrast, is likely to control growth, metabolism, and stress responses systemically by downregulating IIS in all responsive tissues in a coordinated fashion (Wang, 2005).
The interaction between JNK and Foxo is thus expected to influence stress tolerance and life span at two levels. In peripheral tissues, JNK activates Foxo and prevents senescence cell-autonomously. Such a mechanism is exemplified by the recent finding that Foxo overexpression prevents age-dependent decline of cardiac performance. Systemic control of IIS by JNK-mediated activation of Foxo in IPCs, in contrast, would serve to coordinate cellular responses to changes in the environment throughout the organism. These data indicate that this latter mechanism plays a significant role in the regulation of life span by JNK and Foxo. The identification of this endocrine function of JNK/Foxo signaling supports and extends the proposed role of JNK signaling on longevity and demonstrates a role for IPCs in life span regulation. In addition to controlling growth and metabolism, IPCs may thus act as a coordination point for the organism’s stress response by downregulating Dilp production in response to oxidative stress and JNK activation. In target tissues, such a mechanism would induce protective gene expression by the second, cell-autonomous tier of Foxo signaling. Interestingly, the effects of IPC-specific JNK activation on longevity and growth are separable. Life span can be extended by moderately increased JNK activity in IPCs when growth effects are yet not evident. This finding is consistent with observations by others who showed that the extension of life span in IIS loss-of-function situations is not a mere consequence of small body size (Wang, 2005).
How did such a multilayered regulation of IIS activity by JNK evolve? It is tempting to speculate that localized activation of Foxo is required to prevent cellular damage and ultimately senescence in conditions in which stressful insults are confined to specific tissues. Such localized insults could, for example, be inflicted by reactive oxygen species that are produced in the environment of amyloid deposits in Alzheimer’s disease as well as by mechanical and oxidative stress experienced by particularly active tissues such as the heart. Systemic regulation of Foxo activity, in contrast, is expected to be an important response mechanism to coordinate metabolism and stress defenses throughout the organism upon changes in the environment. A good example for such a mechanism is the induction of diapause in invertebrates in response to environmental stress or food deprivation. Accordingly, sensory neurons expressing the insulin-like peptide DAF-28 are required for the induction of the dauer larval stage in response to environmental cues in C. elegans (Wang, 2005).
Systemic and tissue-autonomous effects of JNK/Foxo signaling may be connected in multiple ways. The data indicate that JNK and Foxo interact in IPCs to repress dilp2 expression, ultimately activating Foxo in Dilp2 target tissues in a coordinated fashion. Since JNK is be activated in IPCs even under normal culture conditions, it is likely that this systemic control of IIS activity by JNK and Foxo plays a critical role in life span regulation. It is, however, also possible that the cell-autonomous protective function of JNK/Foxo signaling is most critical for the survival of specific tissues as the organism ages, thus extending life of the organism by preventing the loss of indispensable cells or tissues. In addition, stress and the JNK-mediated activation of Foxo in peripheral tissues may signal back to IPCs to initiate a systemic response. In Drosophila, such a mechanism has been documented in the case of the fatbody. Activation of Foxo in this tissue relays a signal to the IPCs, causing them to curb Dilp2 production, a process that has been proposed to require Foxo activity. The exact nature of this feedback signaling mechanism in flies is unclear, but it is reminiscent of the complex signaling interactions between β cells and insulin target tissues in mammals. Further studies are required to shed light on the relative contributions of JNK/Foxo signaling in IPCs or Dilp target tissues to life span regulation (Wang, 2005).
JNK-mediated modulation of IIS activity is likely to be evolutionarily conserved. Inhibitory crosstalk from JNK to IIS in mammalian cells has been found to occur by JNK-mediated phosphorylation and inhibition of IRS-1. This interaction is responsible for obesity-induced insulin resistance in mice. Whether mammalian homologs of Foxo take part in this pathology remains to be determined. A second possible mechanism for JNK/IIS pathway interaction is the direct phosphorylation and activation of Foxo by JNK. Such a mechanism is supported by the observation that in mouse cells JNK can phosphorylate the DFoxo homolog Foxo4 in response to oxidative stress. The physiological relevance of this phosphorylation event has not yet been addressed. The JNK target residues on IRS-1 and Foxo4 are not conserved in the Drosophila homologs Chico and DFoxo, and further studies are thus required to determine whether JNK-Foxo crosstalk in Drosophila is mediated via homologous mechanisms (Wang, 2005).
The systemic regulation of IIS activity by JNK and Foxo appears to be conserved as well. It has been suggested that C. elegans Daf16/Foxo regulates life span (at least in part) by reducing the expression of insulin-like peptides. In mammals, pancreatic β cells (the counterparts of IPCs) reduce their production of insulin in response to oxidative stress-mediated JNK activation. Conversely, dephosphorylation of JNK by MAPK phosphatase 1 can induce insulin expression in these cells. Reducing circulating insulin levels by JNK-mediated Foxo activation may thus be a general mechanism that balances growth and metabolism with stress defense and damage repair (Wang, 2005).
Foxo is a key regulator of the insulin signaling pathway. As expected, Foxo activity is inhibited via the InR/PI3K/Akt pathway. It was also found that Foxo activates transcription of a major downstream target (4EBP) of this pathway. In Drosophila, this pathway has been linked to cell size and cell number regulation. It was of interest to discover whether Foxo expression in vivo would affect these same parameters. To address this point, transgenic flies that overexpressed Foxo were generated by using the UAS/GAL4 system. Foxo expression was directed to the eye by using eyeless-GAL4 earller than wild type. Interestingly, the reduction in eye size (35%) was caused by a reduction in cell number (673 ± 24 ommatidia in control eyes vs. 438 ± 50 ommatidia in ey-GAL4/UAS-Foxo), but no significant change in cell size was observed (85.8 ± 5.9 area units/ommatidia in control eyes vs. 91.2 ± 4.8 area units/ommatidia in ey-GAL4/UAS-Foxo). When GMR-GAL4, which directs expression in cells posterior to the morphogenetic furrow was used to drive Foxo expression, a more severe phenotype was observed. Many ommatidia were lost, and the remaining ommatidia lacked bristles and appeared disorganized, altering the general structure of the eye. These results suggest that Foxo overexpression can severely affect normal development of the eye (Puig, 2003).
The effect of Foxo overexpression was tested in an organ other than the eye. dpp-GAL4 was used to direct expression of Foxo in the wing region encompassed by the third and fourth longitudinal veins. Foxo overexpression results in a significant reduction of compartment size. This reduction in size is caused by a reduction in cell number but not in cell size (71 ± 7.5 cells/area unit in the control vs. 71 ± 5.5 cells/area unit in dpp-GAL4/UAS-Foxo). Ectopic expression of Foxo in the wing using MS1096-GAL4 produces a more striking reduction in wing size, again because of loss of cell number with no significant variation in cell size (70.6 ± 6.5 cells/area unit in the control vs. 74.8 ± 11.8 cells/area unit in MS1096-GAL4/UAS-Foxo) (Puig, 2003).
Akt can phosphorylate and inactivate Foxo in S2 cells. It was therefore important to know whether Akt also inhibits the phenotypic effects of Foxo in flies expressing Foxo, Akt, or both. Indeed, Akt expression partially rescues the eye phenotype observed with Foxo, showing that both these proteins interact genetically. These results provide further evidence of both proteins acting in the same pathway (Puig, 2003).
Forkhead transcription factors belonging to the FOXO subfamily are negatively regulated by protein kinase B (PKB) in response to signaling by insulin and insulin-like growth factor in Caenorhabditis elegans and mammals. In Drosophila, the insulin-signaling pathway regulates the size of cells, organs, and the entire body in response to nutrient availability, by controlling both cell size and cell number. A genetic characterization has been reported for foxo, the only Drosophila FOXO ortholog. Ectopic expression of foxo and human FOXO3a induces organ-size reduction and cell death in a manner dependent on phosphoinositide (PI) 3-kinase and nutrient levels. Surprisingly, flies homozygous for foxo null alleles are viable and of normal size. They are, however, more sensitive to oxidative stress. Furthermore, Foxo function is required for growth inhibition associated with reduced insulin signaling. Loss of Foxo suppresses the reduction in cell number but not the cell-size reduction elicited by mutations in the insulin-signaling pathway. By microarray analysis and subsequent genetic validation, 4EBP, which encodes a translation inhibitor, has been identified as a relevant Foxo target gene. These results show that Foxo is a crucial mediator of insulin signaling in Drosophila, mediating the reduction in cell number in insulin-signaling mutants. It is proposed that in response to cellular stresses, such as nutrient deprivation or increased levels of reactive oxygen species, Foxo is activated and inhibits growth through the action of target genes such as 4EBP (Jünger, 2003).
To assess whether Foxo has a key function in insulin signaling like that of DAF-16 in C. elegans, tests were performed to see whether overexpression of wild-type or mutant forms of hFOXO3a and foxo could antagonize insulin signaling. Elimination of the three PKB consensus phosphorylation sites in mammalian FOXO3a prevents its phosphorylation, subsequent binding to 14-3-3 proteins, and sequestration in the cytoplasm. This leads to constitutive nuclear localization of the mutant FOXO3a and transcriptional activation of its target genes. Assuming that blocking the PKB signal would have the same activating effect on Drosophila Foxo, wild-type and triple PKB-phosphorylation-mutant (TM) variants of both foxo and human FOXO3a were expressed. Furthermore, an EP transposable element insertion was identified in the second foxo intron, which permits the GAL4-induced overexpression of endogenous foxo. The GMR-Gal4 construct was used to drive UAS-dependent expression in postmitotic cells in the eye imaginal disc. While expression of wild-type hFOXO3a in the developing eye did not result in a visible phenotype, triple mutant constitutively active hFOXO3a-TM expression causes pupal lethality. Few escaper flies eclosed and displayed a strong necrotic eye phenotype. A block of cell differentiation and necrosis was also observed when hFOXO3a-TM was expressed in cell clones in the developing eye (Jünger, 2003).
Assuming that the lack of a phenotype observed upon UAS-hFOXO3a expression is due to hFOXO3a inactivation by endogenous Drosophila Inr signaling in the eye disc, the same experiment was performed in a background of reduced insulin signaling. Indeed, in the presence of a dominant-negative (DN) form of Dp110 (encoding the PI 3-kinase catalytic subunit), hFOXO3a expression induces a necrotic phenotype similar to the one observed with the hyperactive phosphorylation mutant. To confirm that hFOXO3a is responsive to Drosophila insulin signaling and rule out artificial coexpression effects, hFOXO3a was expressed in flies mutant for either dPKB or Dp110, and similar phenotypes were observed as those seen upon coexpression of Dp110DN. Drosophila FOXO has qualitatively similar, but stronger effects. Expressing the wild-type form of foxo causes a weak eye-size reduction and disruption of the ommatidial pattern even in a wild-type background, and the phenotype is strongly affected by Dp110DN as well. The UAS-Foxo-TM transgene appears to cause lethality even in the absence of a Gal4 driver, as no viable transgenic lines were obtained with this construct. Furthermore, the effects of nutrient deprivation on FOXO-expressing tissues was examined. If nutrient availability is limited, FOXO should be more active in response to lowered insulin signaling. Indeed, the overexpression phenotypes of both hFOXO3a and foxo are enhanced under conditions of starvation. Drosophila larvae that are starved until 70 h after egg laying (AEL) die within a few days. But if the onset of nutrient deprivation occurs after they have surpassed the metabolic '70 h change', they survive and develop into small adult flies. Therefore larvae expressing hFOXO3a or foxo (under GMR control) were subjected to either protein starvation (sugar as the only energy source) or complete starvation, starting 80-90 h AEL, and the effect on the adult's eyes was analyzed. Both phenotypes are progressively exacerbated by protein starvation and complete starvation, the latter condition being accompanied by early adult or larval lethality, in the case of hFOXO3a or foxo, respectively. The resulting phenotypes are due to the foxo transgenes, because wild-type control flies that have been starved during development display only a body-size reduction while maintaining normal proportions and normal eye structure (Jünger, 2003).
The foxo overexpression phenotype does not appear to be caused by the activation of any of the known cell-death pathways. Expression of the caspase inhibitors p35 or DIAP1, or of p21, an inhibitor of p53-induced apoptosis, and loss of eiger, which encodes the Drosophila homolog of tumor necrosis factor (TNF), did not suppress the eye phenotype. In agreement with these results, it was observed in a parallel study that the GMR-Foxo overexpression phenotype is insensitive to caspase inhibitors, and is not accompanied by increased acridine-orange-detectable apoptosis in the imaginal disc (Kramer, 2003). It therefore remains unclear whether high levels of nuclear Foxo induce a specific caspase-independent cell-death program or whether nuclear accumulation of overexpressed Foxo leads to secondary necrosis in a rather nonspecific fashion. Furthermore, the necrotic eye phenotype does not reflect the phenotype observed following a complete block in insulin signaling. Loss-of-function mutations in insulin-signaling components reduce cell size and cell number but do not increase cell death in larval tissues. In summary, these overexpression experiments are consistent with a model in which, under normal conditions, excess Foxo transcription factor is phosphorylated by PKB and kept inactive in the cytoplasm. Under conditions of reduced insulin-signaling activity or nutrient deprivation, Foxo or hFOXO3a protein translocates to the nucleus and induces growth arrest and necrosis (Jünger, 2003).
foxo loss-of-function mutants are viable, have no overgrowth phenotype and are hypersensitive to oxidative stress. Although these overexpression experiments did not reveal the physiological function of Foxo, they provided an entry point for isolation of loss-of-function mutations. Use of the EP35-147 element permits the generation of the necrotic eye phenotype by driving expression of endogenous foxo in the presence of Dp110DN. Homozygous EP males were mutagenized, mated to homozygous GMR-Gal4 UAS-Dp110DN females and then the F1 generation was screened for reversion of the strong gain-of-function phenotype and its associated semilethality. Several loss-of-function alleles of foxo were isolated and molecularly characterized. Two such revertants were characterized. In Foxo21 and Foxo25, the codons for W95 and W124 within the forkhead domain are mutated to stop codons, respectively, so they are assumed to be null alleles of Foxo (Jünger, 2003).
Because FOXO transcription factors have been proposed to be the primary effectors of insulin signaling, on the basis of epistasis of daf-16 over daf-2 in C. elegans, it seemed reasonable to expect an overgrowth phenotype in Foxo-/- flies as is observed in dPTEN loss-of-function mutants. Surprisingly, foxo loss-of-function mutants are homozygous-viable and display no obvious phenotype under normal culturing conditions. Thus, foxo is not essential for development. Only close inspection of the foxo mutants reveals that their wing size is significantly reduced. But cellular and organismal growth are unaffected by foxo mutations. To assess whether Foxo-mutant tissue grows to a different size than wild-type tissue, the Foxo21 and Foxo25 alleles were recombined onto the FRT82 chromosome and genetic mosaic flies were induced with the ey-Flp/FRT system. When the eye and head capsule were composed almost exclusively of Foxo-/- tissue, no head-size difference was observed compared to the control fly with a head homozygous for the FRT82 chromosome without the foxo mutation. This is consistent with experience from extensive genetic screens carried out for recessive growth mutations. An ey-Flp-screen on the right arm of chromosome 3 did not reveal any mutations in foxo based on an altered head-size phenotype (Jünger, 2003).
It was next asked whether cell size, like organ size, is not affected by the loss of Foxo. For this purpose, a heat shock-inducible Flp construct was used to generate clones of homozygous Foxo-/-photoreceptor cells and wild-type cells within one adult eye. The cells lacking Foxo are marked by the absence of pigment granules. Consistent with the absence of a 'bighead' phenotype, Foxo-/- cells and wild-type cells have the same size. Similarly, no significant difference in the body weight of mutant and control flies was observed. In contrast, flies with a viable heteroallelic combination of dPTEN loss-of-function alleles are significantly bigger than wild-type flies. Taken together, these results argue that with the exception of the slight wing-size reduction, Foxo is not required to control cellular, tissue, or organismal growth in a wild-type background (Jünger, 2003).
A critical role has been reported for mammalian and C. elegans FOXO proteins in resistance against various cellular stresses, in particular oxidative stress, DNA damage and cytokine withdrawal. The stress resistance of adult foxo mutant flies was tested by measuring survival time following different challenges. Among starvation on water, oxidative-stress challenge, bacterial infection, heat shock, and heavy-metal stress, the only condition for which hypersensitivity was observed is oxidative stress. When placed on hydrogen-peroxide-containing food, foxo mutant flies display a significantly reduced survival time compared to control flies. A very similar effect is elicited by paraquat feeding. These observations are consistent with the paraquat hypersensitivity of daf-16 mutants in C. elegans, suggesting that a role for FOXO proteins in protecting against oxidative stress is conserved across species (Jünger, 2003).
The growth-deficient phenotypes of Inr, chico, Dp110 and PKB mutants are significantly suppressed by loss of Foxo. Genetic epistasis experiments were performed to examine whether the growth phenotypes of Inr-signaling mutants are dependent on Foxo function. For this purpose, either double-mutant flies were generated or the double-mutant effect only in the head was investigated using the ey-Flp/FRT system. In contrast to the absence of a growth phenotype in single foxo mutant flies, lack of Foxo significantly suppresses the growth-deficient phenotype observed in flies mutant for the insulin receptor substrate (IRS) homolog chico. Flies mutant for chico are smaller because they have fewer and smaller cells. Loss of one foxo copy dominantly suppresses the cell-number reduction in chico mutant flies without affecting cell size. The suppression is more pronounced when both copies of foxo are removed in a chico mutant background. In this situation, the chico small body-size phenotype is partially suppressed. Homozygous chico-Foxo double-mutant flies have more, and even slightly smaller, cells than homozygous chico single mutants. It seems that removal of foxo accelerates the cell cycle at the expense of cell size in a chico background (Jünger, 2003).
It was next asked whether foxo interacts with other components of the Drosophila insulin-signaling pathway. The ey-Flp/FRT system was used to generate heterozygous insulin-signaling mutant flies with heads homozygous for each mutation. Removal of Inr, Dp110 or PKB leads to a characteristic 'pinhead' phenotype, which is substantially suppressed by the presence of a foxo loss-of-function allele on the same FRT chromosome as the insulin-signaling mutation. In all three cases, a partial rather than a complete rescue of the tissue growth repression was observed, consistent with the finding that foxo mutations affect only the cell-number aspect of the chico phenotype. Surprisingly, loss of Foxo dramatically delays lethality in PKB mutants. Complete loss of PKB leads to larval lethality in the early third instar, but homozygous PKB-Foxo double mutants are able to develop into pharate adults of reduced size, most of which fail to eclose. The lethality associated with the complete loss of PKB is therefore largely due to hyperactivation of Foxo (Jünger, 2003).
It was also observed that foxo interacts with the tumor suppressors TSC1 and PTEN. Tissue-specific removal of either gene from the head leads to a bighead phenotype. The TSC1-/- bighead phenotype is enhanced by loss of Foxo. This observation is consistent with the recently reported negative feedback loop between Drosophila S6K and PKB. Mutant TSC1 larvae have elevated levels of S6K activity, which in turn downregulates PKB activity. This reduction in PKB activity probably leads to enhanced activation of Foxo, which in turn partially mitigates the overgrowth phenotype by slowing down proliferation. The TSC1 phenotype can therefore be enhanced by loss of the inhibitory function of Foxo. Unexpectedly, the PTEN-/- bighead phenotype is slightly suppressed by foxo mutations. From the current model, it would be expected that in a PTEN mutant PKB activity is high and Foxo is to a large extent inactive in the cytoplasm. Thus, removal of Foxo function should have no effect on the PTEN phenotype. At present, it is possible only to speculate about possible explanations for this observation. In a parallel study, it has been shown that Foxo can induce transcription of DInr (Puig, 2003). It may be that in a dPTEN-mutant background Foxo activates Inr expression in a negative-feedback loop. In this model, concomitant loss of Foxo would alleviate the PTEN overgrowth phenotype by lowering Inr levels. Another possible explanation is that Foxo has additional functions when localized to the cytoplasm or during its nuclear export, such as interacting with other proteins. Loss of Foxo might affect the function of interaction partners that have a role in PTEN signaling (Jünger, 2003).
In summary, this epistasis analysis provides strong genetic evidence that Foxo is required to mediate the organismal growth arrest that is elicited in insulin-signaling mutants (Jünger, 2003).
Studies with several components of the Drosophila InR/PI3K/Akt pathway have shown that insulin promotes growth and proliferation by activating PI3K and Akt. Foxo activity is inhibited by Akt upon insulin treatment. It was therefore interesting to ask whether induction of Foxo would inhibit growth in S2 cells. To do so, S2 cells stably transfected with either Foxo or mutant dFoxoA3 (Constitutively active Drosophila Foxo in which all three putative Akt phosphorylation sites were mutated to alanine) were cultured in the presence of serum and insulin for 48 h. Then exogenous Foxo and dFoxoA3 expression was induced with CuSO4 for a period of 8 h. Subsequently, Cu2+ was removed, and cells were then allowed to divide for 1 wk with samples taken every 12 h to measure cell numbers. Cells stably transfected with the wild-type protein proliferate at the same rate irrespective of Foxo induction. In contrast, cells stably transfected with the mutant protein, dFoxoA3, display a significantly slower growth rate when compared with the same cells grown without dFoxoA3 induction. Because insulin was present throughout these experiments (inactivating Foxo but not dFoxoA3), these findings suggest that the constitutively active dFoxoA3 can induce cell arrest. Importantly, cells expressing dFoxoA3 arrest growth during the first 44 h after induction when dFoxoA3 is present. After 44 h, when dFoxoA3 has apparently been turned over, cells recover and start dividing normally again. FACS analysis of samples taken during the different time points indicates that S2 cells arrest their growth at G2/M. These results indicate that activation of Foxo can induce cell cycle arrest and this effect is mitigated by insulin (Puig, 2003).
Components of the insulin signaling pathway are important regulators of growth. The FOXO transcription factors regulate cellular processes under conditions of low levels of insulin signaling. Expression of Drosophila Foxo during early larval development causes inhibition of larval growth and alterations in feeding behavior. Inhibition of larval growth is reversible upon discontinuation of Foxo expression. Expression of Foxo during the third larval instar or at low levels during development leads to the generation of adults that are reduced in size. Analysis of the wings and eyes of these small flies indicates that the reduction in size is due to decreases in cell size and cell number. Overexpression of Foxo in the developing eye leads to a characteristic phenotype with reductions in cell size and cell number. This phenotype can be rescued by co-expression of upstream insulin signaling components, PI3K and Akt, however, this rescue is not seen when Foxo is mutated to a constitutively active form. It is concluded that Drosophila Foxo is conserved in both sequence and regulatory mechanisms when compared with other FOXO homologs. The establishment of Drosophila as a model for the study of FOXO transcription factors should prove beneficial in determining the biological role of these signaling molecules. The alterations in larval development seen upon overexpression of Foxo closely mimic the phenotypic effects of starvation, suggesting a role for Foxo in the response to nutritional adversity. This work has implications in the understanding of cancer and insulin related disorders, such as diabetes and obesity (Kramer, 2003).
Ectopic expression of Foxo during development phenocopies starvation and alters feeding behavior. Drosophila larvae feed continuously for about 5 days after egg laying (AEL). During this time the appetite and growth rate of the larvae is enormous. If young larvae are deprived of food, they do not grow and tend to disperse randomly. When the food supply is replenished, the larvae immediately move towards it and continue eating until they are close to pupation. If the food supply is depleted, the larvae will disperse again. The UAS/Gal4 ectopic expression system was used to overexpress Foxo in the developing larvae under the control of the ActGal4 driver. This results in complete developmental arrest of the larvae, which remain as first instar for up to 7 days, similar to the life expectancy of starved larvae. This trend was also seen using a constitutively active version of murine Foxo1 (mFoxo1) containing an alanine substitution at the T1 (T24A), and S1 (S253A) Akt phosphorylation sites (mFoxo1-AA). In addition, larvae expressing Foxo and mFoxo1-AA are often found to be wandering far from their food supply. Feeding behavior was monitored by assessing the number of larvae away from their food at 48 and 72 hours after egg laying (AEL). Larvae expressing Foxo and mFoxo1-AA show a 3-4 fold increase in wandering over larvae expressing Gal4 alone. Thus, Foxo expression drastically alters feeding behavior and is able to induce a starvation type response in larvae that have an adequate food supply (Kramer, 2003).
In Drosophila, PI3K consists of an adaptor subunit, dp60, and a catalytic subunit, dp110. Unexpectedly, expression of an inhibitory or 'dominant negative' version of dp110 (UAS-dPI3K-DN) under the control of the ActGal4 does not lead to increased larval wandering. Expression of this construct also does not appear to inhibit larval growth, whereas other negative regulators of insulin signaling do. It is possible that the level of expression of this construct is not high enough under the control of the ActGal4 driver to have a complete dominant negative effect (Kramer, 2003).
Starved larvae that are developmentally arrested are able to resume growth upon acquisition of food. Whether larvae that are expressing Foxo could resume growth upon termination of Foxo expression was analyzed. To do this the hsGal4 driver was used. Foxo was expressed in the larvae by heat shock treatment (HST) for 10 minutes every 24 hours. This treatment is sufficient to inhibit growth while allowing controls to survive to adulthood with a 48 hour delay in the time to pupation. When Foxo expression was discontinued after 2, 4, and 6 days of HST, developmentally arrested larvae were able to recover with decreased levels of survival as time progressed. Significant lethality was observed in controls as well suggesting that low survival rate is partially due to the expression of Gal4, which can induce apoptosis, or the HST itself. Nevertheless, developmental arrest caused by Foxo is clearly reversible since these individuals could be returned to their normal path of development (Kramer, 2003).
The formation of dauer larvae in C. elegans is a developmental response to nutrient limitation. The dauer larvae provides a temporary defense mechanism allowing the nematode to persevere until nutrients are available, at which point development can continue. Interestingly, constitutive activation of Daf-16 by mutation of its Akt phosphorylation sites to alanine residues causes obligatory dauer larvae formation. A similar result was found in the Drosophila larvae using the constitutively active mFoxo1-AA. This construct has an effect similar to that of Foxo when expressed under the control of ActGal4, and hsGal4. Upon removal from HST, larvae expressing mFoxo1-AA do not resume growth but remain in a state of developmental arrest until death. Although a few larvae did survive to adulthood after 2 days of HST, none of the larvae were able to continue development after 4, or 6 days of HST. Out of 450 larvae examined at all time points, only 10 expressing mFoxo1-AA survived, when compared to 110 and 180 for larvae expressing Foxo, and Gal4 alone, respectively. Presumably this occurs because Akt is unable to deactivate mFoxo1-AA, allowing it to continue functioning long after expression is induced. Taken together, this data suggests that Foxo is evolutionarily conserved in function, possibly playing a role in the response to nutritional adversity, as seen in the formation of dauer larvae in C. elegans (Kramer, 2003).
Expression of Foxo in the third instar larvae causes significant lethality, however, rare flies that do survive are much smaller than control flies, showing a phenotype similar to that caused by mutations in chico, Akt and Inr. Expression of Foxo under the control of the ubiquitous low level Gal4 drivers, armadillo-Gal4, and hsGal4 (raised at 25°C with no heat shock) had very little effect on growth. In contrast, increasing expression of Foxo using the hsGal4 driver in flies raised at 29°C leads to the development of small adults, which are approximately half the weight of control flies. Analysis of the wings of these flies shows that the wing area was reduced by nearly one third and that this reduction was due to a decrease in both cell size and cell number. SEM analysis of the eyes reveals reductions in both ommatidia number and ommatidia area, which reflect cell number and cell size, respectively. These results implicate Foxo in the control of body size through alterations in cell size and cell number (Kramer, 2003).
When Foxo is expressed in the developing eye under the control of the GMR-Gal4 driver, the eye is smaller, lacking many ommatidia and nearly all of the mechanosensory bristles. The remaining ommatidia are arranged in the typical hexahedral array and cross sectional analysis reveals that all of the normal photoreceptor cells are present. Thus, it appears that Foxo expression causes a reduction in the number of cells but does not interfere with cellular differentiation and the organization of the ommatidia themselves. This eye phenotype was used to test for interactions between Foxo and other components of the insulin signaling pathway (Kramer, 2003).
Expression of PI3K-DN under the control of GMR-Gal4 leads to the formation of relatively normal eyes with fewer and smaller cells. When Foxo is co-expressed in the developing eye with PI3K-DN, the eye is nearly obliterated. In contrast, co-expression of Akt, and wild type PI3K with Foxo causes nearly complete rescue of the phenotype, restoring the ommatidia and nearly all of the mechanosensory bristles. Thus, diminishing insulin signaling (through overexpression of PI3K-DN) allows for greater activity of Foxo, and enhancing insulin signaling (through overexpression of Akt or PI3K) leads to inhibition of Foxo activity. Similar results were obtained using a Murine Foxo1 (mFoxo1) construct, indicating that the regulatory mechanisms between these two proteins is conserved and that they are functionally interchangeable (Kramer, 2003).
The constitutively active mFoxo1-AA construct was also expressed in the developing eye. Expression of this construct causes a phenotype similar to that of Foxo and mFoxo1, with characteristic lack of ommatidia and mechanosensory bristles. When mFoxo1-AA is co-expressed with Drosophila PI3K-DN, the eye is nearly obliterated, as seen with Foxo and mFoxo1. Co-expression of mFoxo1-AA with PI3K leads to a partial rescue of the phenotype, with still an obvious lack of ommatidia and mechanosensory bristles. In contrast, co-expression of mFoxo1-AA with Drosophila Akt does not cause rescue of the ommatidia or mechanosensory bristles, indicating that this construct is not responsive to Akt signaling. The partial rescue of the Foxo phenotype by PI3K appears to be mediated through alterations in cell size rather than cell number, since there is still an obvious lack of ommatidia and mechanosensory bristles. This data indicates that inactivation of Foxo is required for the full effects of growth mediated by PI3K and Akt (Kramer, 2003).
To examine the effect of Foxo overexpression on cell size, the area of the ommatidia was measured. Expression of Foxo, mFoxo1, and mFoxo1-AA causea a significant reduction in the area of the ommatidia. Expression of PI3K causes a significant increase in ommatidia size over wild type. This result is consistent with previous studies showing that PI3K affects cell size in a cell autonomous manner. Co-expression of Foxo, mFoxo1, and mFoxo1-AA with dPI3K has no significant effect on the enlarged ommatidia. Thus, it appears that FOXO proteins have a very minimal effect on cell size in the presence of high levels of PI3K. Surprisingly, this is the case even with the mFoxo1-AA construct, which is only partially responsive to PI3K signaling. This indicates that the Drosophila PI3K mediated increase in cell size can occur through Akt independent mechanisms (Kramer, 2003).
Expression of Akt in the developing eye causes a significant increase in ommatidia size, similar to that seen with PI3K. Co-expression of Drosophila Akt with either Foxo or mFoxo1, cause a slight, but insignificant decrease in the size of the enlarged ommatidia. However, co-expression of Drosophila Akt with mFoxo1-AA results in ommatidia that are approximately the same size as the ommatidia in eyes expressing Gal4 alone, and significantly smaller than the ommatidia in eyes expressing Akt alone. This indicates that the deactivation of FOXO by Akt is essential for Akt to induce an increase in cell size (Kramer, 2003).
The lack of ommatidia and mechanosensory bristles caused by Foxo expression suggests a reduction in cell number during eye development. Reduction of cell number can occur through either increased cell death, or decreased of cell proliferation. The Drosophila inhibitors of apoptosis, Diap1 and Diap2, and the baculovirus inhibitor of apoptosis, p35, were unable to rescue the phenotype caused by Foxo expression. In addition, acridine orange staining of eye imaginal discs expressing Foxo showed no increase in apoptosis when compared to controls. Drosophila Epidermal Growth Factor Receptor (Egfr) signaling acts to protect differentiated cells from death during eye development. It was thought that the pro-survival effects of Egfr may be sufficient to suppress the phenotype caused by Foxo overexpression. Co-expression of Egfr with Foxo, however, does not rescue the Foxo phenotype since ommatidia and bristles are clearly still missing. Conversely, Foxo does not appear to affect the phenotype of Egfr overexpression since the general disorganization of the ommatidia appears to be the same. Thus, it appears that these two mechanisms are acting independently. Taken together, these results suggest that Foxo overexpression does not cause cell death during eye development because direct inhibitors of the apoptotic machinery (p35 and Diap1/2) and a known cell survival factor (Egfr) are unable to rescue the Foxo phenotype (Kramer, 2003).
Since inhibition of apoptosis could not rescue the phenotype caused by Foxo overexpression in the eye, an examination was performed to see if activating the cell cycle could inhibit the phenotype. Expression of the E2F and Dp transcription factors has been shown to promote cell proliferation in the wing imaginal disc. Co-expression of E2F and Dp with Foxo is not sufficient to rescue the Foxo phenotype. Overexpression of constitutively active Drosophila Ras1 (dRas1V12) has been shown to induce ectopic cell proliferation and G1/S progression in the Drosophila wing disc. Co-expression of dRas1V12 with Foxo is lethal, a constitutively active version of Drosophila Ras2 (dRas2V14) was used. Although Ras2 has not been characterized for its role in cell cycle control, it is possible that it has a similar function to Ras1. Expression of UAS-dRas2V14 under the control of GMR-Gal4 led to extreme overgrowth of the eye, lack of ommatidial organization, and the formation of huge ommatidia. Co-expression of dRas2V14 with Foxo is sufficient to restore many of the ommatidia and mechanosensory bristles lost through overexpression of Foxo alone. A similar effect was observed upon co-expression of dRas2V14 with mFoxo1. In contrast, the loss of ommatidia and bristles seen upon over expression of mFoxo1-AA is not rescued by dRas2V14 . This suggests that dRas2V14 inhibits Foxo via a Akt phosphorylation dependent mechanism (Kramer, 2003).
Thus, Foxo as a negative controller of growth and organism size, which is regulated by components of the Drosophila insulin signaling pathway, PI3K and Akt. Through overexpression studies in the developing eye, it has been shown that Foxo is regulated by PI3K and Akt in a manner that is consistent with the regulatory mechanisms deduced through studies in C. elegans and mammalian cell culture. In addition, overexpression of Foxo in the larvae reduces larval growth, phenocopies the effects of nutritional stress, and causes alterations in feeding behavior. With this in mind, it is proposed that Foxo is involved in the response of Drosophila larvae to nutritional stress (Kramer, 2003).
The FOXO homologs appear to play an evolutionarily conserved role in the control of cellular processes under conditions of low levels of insulin signaling. These experiments have provided three lines of evidence supporting the conservation of this mechanism in Drosophila. (1) Foxo shows strong sequence homology to Daf-16 and the human FOXO homologs. One significant characteristic is the high conservation of the three consensus Akt phosphorylation sites, suggesting that Drosophila Akt is most likely able to phosphorylate Foxo in vivo, as shown biochemically with the mammalian FOXO homologs. (2) These experiments show that Foxo and mFoxo1 cause nearly identical phenotypic responses when overexpressed in the developing Drosophila eye. This suggests that the activity of these proteins is highly conserved as is observed when the C. elegans FOXO homolog, Daf-16, is expressed in mammalian cell culture. (3) The phenotypic effects of FOXO overexpression can be modulated by alterations in the insulin signaling pathway. Reduced insulin signaling leads to a drastic enhancement of the phenotype that results from expression of FOXO factors. In contrast, increased insulin signaling tends to mask these phenotypes, in a manner that is dependent on the integrity of the Akt phosphorylation sites. As a result, it is believed that regulation of FOXO is conserved in Drosophila, and that this will be a very useful system in elucidating the function of FOXO transcription factors in a model organism (Kramer, 2003).
Results show that ectopic Foxo expression can mediate reduction in cell size and cell number. However, the mechanisms by which these reductions occur are still unclear. Net reduction in cell number may occur through decreased cell proliferation or increased apoptosis. Insulin and other growth factors that activate PI3K and Akt have been implicated as potent survival factors in mammalian cell culture. They prevent cell death, in part, by inhibition of FOXO factors and it has been shown that FOXO3a can upregulate expression of the pro-apoptotic protein Bim. In Drosophila, reduction of insulin signaling can lead to apoptosis in the developing embryo. It is possible that this increase in apoptosis is a result of Foxo activation, however, when Foxo is expressed in the developing eye there is no apparent increase in apoptosis, nor is the phenotype suppressed by inhibition of caspases, or by co-expression of a known cell survival factor, Egfr. These apparent discrepancies may be the result of tissue specific differences. In mammalian cell culture, induction of cell death by FOXO factors seems to be limited to non-transformed hematopoietic cell lineages. In Drosophila, loss of Akt function, inhibition of PI3K, or overexpression of PTEN, all induce cell death in the embryo. However, in imaginal disc cells lacking PI3K function, there is no increase in apoptosis. Thus, the cells in the embryo and imaginal discs may react differently to reduced levels of insulin signaling. Although no induction of apoptosis upon Foxo expression is observed, it is possible that increased levels of Foxo activity (e.g., through dominant negative inhibition of PI3K) do cause apoptosis (Kramer, 2003).
Studies in mammalian cell culture have implicated FOXO factors in control of the cell cycle through increased expression of the cyclin dependent kinase inhibitor p27Kip1. It is possible that the reduction of cell number seen upon Foxo expression is a result of cell cycle inhibition. Co-expression of an activated version of Drosophila Ras2 (dRas2V14) is sufficient to increase cell number in the presence of Foxo. Ras1 has been shown to induce growth in Drosophila imaginal discs through activation of PI3K and the transcription factor Myc. Although there is very little information available about Ras2, it is possible that the function of Ras2 overlaps with that of Ras1. Expression of dRas2V14 in the developing eye does cause a phenotype that suggests overgrowth of cells, and the dRas2V14 interaction with Foxo appears to be dependent on Akt signaling. This is not surprising considering that Ras1 and mammalian Ras have been shown to activate PI3K signaling. Interestingly, increasing the cell cycle through overexpression of the transcription factors E2F and Dp does not rescue the cell number deficit seen upon overexpression of Foxo. This suggests the possibility that activation of Foxo may override the function of other growth promoting factors, such as dMyc, which mediates Ras1 induced G1/S progression. Supporting this, it has been observed that increased growth as mediated by Akt is entirely dependent on its ability to inactivate Foxo. Furthermore, increased growth mediated by PI3K appears to be dependent on Foxo inactivation with respect to increased cell number, but not cell size. In humans, inactivation of FOXO factors may play an important role in tumor suppression by down regulating expression of D-type cyclins, thus inhibiting cell cycle progression and transformation. It will be interesting to test the interactions between Foxo and other cell cycle promoters to determine the extent of Foxo dominance over cell proliferation (Kramer, 2003).
In addition to its effect on cell number, Foxo is able to control cell size. The ability of Akt to increase cell size is dependent on Foxo inactivation, however, Drosophila PI3K does not need to inactivate Foxo to increase cell size. The difference between dPI3K and Akt might be attributed to greater activity of the UAS-dPI3K transgene. However, expression of these constructs individually yields very similar results indicating that this is probably not the case. This suggests that PI3K may control size through Akt-independent mechanisms. One possibility is through the positive growth regulator, S6k. Akt appears to increase growth through inhibition of a TSC1/TSC2 (tuberous sclerosis) complex. This complex acts through inhibition of TOR (target of rampamycin), which promotes growth through activation of S6K. Although it appears that Akt can upregulate growth through S6K, S6K activity is not reduced in larvae lacking Akt or PI3K. These results do not necessarily suggest that PI3K and Akt can not activate S6K, since S6K levels may be maintained through amino acid signals. S6K activity is dependent on phosphoinositide dependent kinase (PDK1), which interacts genetically with Akt, PI3K, PTEN, and Inr. Thus, it is possible that PI3K can modulate S6K activity through PDK1, independently of Akt (Kramer, 2003).
Studies in C. elegans indicate that insulin signaling is a critical mediator of longevity and stress resistance. One of the most well-studied stress responses is the Daf-16 mediated formation of the dauer larvae under conditions of starvation and/or crowding. Several lines of evidence indicate that Foxo may play a similar role in Drosophila larvae. (1) Drosophila larvae are deprived of food prior to 70 hours AEL, they live in a state of developmental arrest for several days before death. However, when starved after 70 hours AEL, the larvae are able to develop into adults that are reduced in size. This alteration in developmental response has been termed the '70 hour change'. and is likely determined by the minimum size required for a Drosophila larvae to enter pupation. The '70 hour change' can be mimicked through overexpression of Foxo at different stages of larval development, in the presence of ample food. For example, ubiquitous high level expression of Foxo in the early larvae (i.e., before 70 hours AEL) leads to developmental arrest, whereas heat shock induced expression of Foxo during the third instar (i.e. ,after 70 hours AEL) leads to the development of small adults. (2) The normal development of starved larvae can resume upon the acquisition of food. Similarly, developmental arrest caused by expression of Foxo prior to the '70 hour change' can be reversed if Foxo expression is discontinued. Developmental arrest caused by expression of mFoxo1-AA before the '70 hour change' is not reversible, suggesting a constitutive starvation type response as seen in C. elegans when Daf-16 phosphorylation sites are mutated. Interestingly, the reversibility of FOXO induced arrest has also been observed in mammalian cell culture. (3) Under conditions of poor nutrition or crowding larval development does not cease, but the larval period is extended and small adults are produced. This effect has been replicated through low level expression of Foxo during the course of development. (4) Feeding behavior is drastically altered in larvae expressing Foxo, causing them to wander away from their food. These larvae are often found crawling on the sides and lids of Petri dishes. This response may provide a selective advantage in the search for food as seen in C. elegans dauer larvae, which often crawl up to the highest point possible in hopes of attaching to passing organisms that could move the larvae to new locations with better food supply. Taken together, these results suggest that Foxo activity may act to promote survival during times of nutritional stress in a manner that recapitulates the formation of dauer larvae in C. elegans. It is tempting to speculate that Foxo plays a role in response to other forms of stress, as observed with Daf-16. Mammalian FOXO factors have been implicated in the protective response to oxidative stress and FOXO factors are upregulated in response to caloric restriction in rat skeletal muscle. Thus, it is possible that FOXO factors provide an evolutionarily conserved switch, by which an organism can alter its developmental program in order to promote survival under harsh conditions (Kramer, 2003).
Activation of insulin signaling causes larvae to wander away from their food. A similar effect was observed through overexpression of Foxo, which acts in opposition to insulin signaling. It is possible that hyperactivation of insulin signaling may lead to depletion of the hemolymph by increasing the cellular uptake of nutrients. This would lead to increased hunger and cause the larvae to wander in search of food. Since PI3K activity is lost under conditions of starvation, it stands to reason that Foxo would be active under these conditions. Being a transcription factor, endogenous Foxo could activate a host of genes under conditions of starvation leading to a 'genetic starvation profile'. Indeed gene expression is drastically altered upon starvation. Thus, Foxo may induce larval wandering through expression of a sub-set of genes which are normally active during starvation, whereas activation of insulin signaling may induce larval wandering by causing physiological changes that lead to a false sense of starvation (Kramer, 2003).
Insulin-IGF receptor (InR) signaling has a conserved role in regulating lifespan, but little is known about the genetic control of declining organ function. This study describes progressive changes of heart function in aging fruit flies: from one to seven weeks of a fly's age, the resting heart rate decreases and the rate of stress-induced heart failure increases. These age-related changes are minimized or absent in long-lived flies when systemic levels of insulin-like peptides are reduced and by mutations of the only receptor, InR, or its substrate, Chico. Moreover, interfering with InR signaling exclusively in the heart, by overexpressing the phosphatase PTEN or the forkhead transcription factor FOXO, prevents the decline in cardiac performance with age. Thus, insulin-IGF signaling influences age-dependent organ physiology and senescence directly and autonomously, in addition to its systemic effect on lifespan. The aging fly heart is a model for studying the genetics of age-sensitive organ-specific pathology (Wessells, 2004).
In Drosophila ageing is slowed when insulin-like signalling is reduced: life expectancy is extended by more than 50% when the insulin-like receptor (InR) or its receptor substrate (Chico) are mutated, or when insulin-producing cells are ablated. But it has yet to be resolved whether insulin affects ageing, or whether insulin signals regulate ageing directly or indirectly through secondary hormones. C. elegans lifespan is also extended when insulin signalling is inhibited in certain tissues, or when repressed in adult worms, and this requires the forkhead transcription factor (FOXO) encoded by daf-16. The Drosophila insulin-like receptor mediates phosphorylation of Foxo, the equivalent of nematode Daf-16 and mammalian FOXO3a. Drosophila Foxo regulates ageing when activated in the adult pericerebral fat body. It is further shown that this limited activation of Foxo reduces expression of the Drosophila insulin-like peptide dilp-2 synthesized in neurons, and represses endogenous insulin-dependent signalling in peripheral fat body. These findings suggest that autonomous and non-autonomous roles of insulin signalling combine to control ageing (Hwangbo, 2004).
To investigate whether activated Foxo affects ageing in Drosophila, foxo was conditionally expressed in specific adult tissues. Without ligand binding at the insulin-like receptor, Foxo remains unphosphorylated and is transported to the nucleus where it promotes factors that retard cell growth and proliferation. Drosophila was transformed with UAS-constructs, containing either a wild-type full-length complementary DNA of foxo (UAS-foxo) or Foxo with the three protein kinase B (PKB) phosphorylation sites mutated to permit insulin-insensitive nuclear transport (UAS-foxo-TM). Expression of these constructs in the eye disc reduced growth, as has previously been reported for independent transformants of UAS-foxo and for a phosphorylation-site mutant of human FOXO3a. The constitutive expression of UAS-foxo or UAS-foxo-TM killed larvae when promoted from actin-GAL4, or when expressed from fat body (adh-GAL4) or neurons (ELAV-GAL4). Therefore, conditional expression of foxo is required to bypass developmental lethality as well as to study its impact on ageing exclusively in the adult stage (Hwangbo, 2004).
The mifepristone inducible-GAL4 system (annotated P{Switch} and GeneSwitch) was used to drive the expression of UAS constructs in defined adult tissues. Ingested mifepristone strongly induces reporter expression at all ages, and the compound alone has no effect on adult survival. Adult survival is not improved when UAS-foxoTM is induced by a pan-neuronal driver (ELAV-GeneSwitch), or in glial cells (P{Switch} MB221) or neurolemma (P{Switch} S113). Thus, broadly activated dFOXO in neuron-associated cells is not sufficient to slow ageing; however, it may do so if expressed in subsets of cells within these tissues. Similarly, expression of UAS-foxo-TM or UAS-foxo did not affect survival when induced with the P{Switch} strain S1106, an efficient promoter in the fat body. In contrast, survival is significantly increased in both sexes when foxo is induced with the P{Switch} strain S132, which is also expressed in fat body. Multiple independent inserts of UAS-foxo-TM and of wild-type UAS-foxo increase median lifespan by as much as 35% when induced with 25microg/ml mifepristone and 56% when induced by 50microg/ml mifepristone; averaged across trials, lifespan was increased by 15.5% in males and 19.4% in females. Because the phosphatase and tensin homologue protein (PTEN) antagonizes phosphatidylinositol-3-OH kinase activity, which promotes nuclear localization of endogenous Foxo and inhibits Tor function, UAS-Pten was induced with S1106 and S132. Survival was unaffected when Pten was expressed from S1106 but increased by about 20% when expressed from SS13232. Together, these data demonstrate that Foxo activated in a specific tissue can regulate lifespan in adult flies (Hwangbo, 2004).
To understand how S132 but not S1106 can improve lifespan, their patterns of expression were compared in adult tissue. S1106 is expressed in fat body of the thorax, abdomen and occasionally in the cavity surrounding the mouthparts. In contrast, S132 is expressed in fat body of the head and not in the abdomen or thorax; S32 uniquely appears in the pericerebral fat body located above the brain. The insertion site of S132 maps to the first intron of bunched. Although bunched was identified in egg follicles, S132 does not express in these cells, perhaps because it is inserted near two intronic open reading frames. It is concluded that specifically activated foxo in the adult head fat body is sufficient to slow ageing. Surprisingly, although UAS-foxo-TM (expressing activated Foxo) induced by S132 increases longevity it does not affect fecundity. In contrast, when adults are challenged with an acute oxidative stress agent (paraquat), survival is improved when UAS-foxo-TM is induced by S132 (in the head fat body) but not by S1106, in agreement with long-lived insulin signalling mutants of C. elegans and Drosophila that are often stress resistant. Similarly, lipids are frequently elevated in C. elegans and Drosophila insulin-signalling mutants and as anticipated, when foxo-TM is expressed in the head fat body, lipid aggregates appear in this tissue. Remarkably, in the same animals, lipids also accumulated in the peripheral fat tissue even though this construct is not expressed outside the head (Hwangbo, 2004).
To understand how Foxo-TM that is expressed exclusively in the head fat body can regulate integrated physiological traits such as ageing, stress resistance and lipid metabolism, the cellular location of the Foxo protein was followed in the head and peripheral fat body. Without transgene induction, endogenous Foxo is distributed throughout the cytoplasm in all cells. On expression of UAS-foxo-TM, antibody-labelled Foxo was increased in both the cytoplasm and nuclei of the targeted tissue. Notably, activated Foxo induced by S132 in head fat body also increases endogenous Foxo nuclear localization of peripheral fat body, in agreement with the pattern of lipid accumulation. In contrast, Foxo-TM expressed in peripheral fat body does not affect endogenous Foxo in the head. Since endogenous Foxo in peripheral tissue can become localized in the nucleus in response to decreased insulin signalling, these results suggest that Foxo that is activated in the head fat body retards systemic levels of the insulin ligand (Hwangbo, 2004).
Cells in the pars intercebralis of the adult brain synthesize insulin peptides. To test whether activated Foxo in the head fat body influences insulin production messenger RNA levels of the seven Drosophila insulin-like peptides (dilp) was measured. Complementary DNA was prepared from the heads of S132 /UAS-foxo-TM adults fed mifepristone or treated as controls. In a preliminary screen, multiple independent samples were specifically analysed for dilp message abundance using microarrays: dilp-2 alone is reduced in response to activated Foxo in the head fat body. These samples were used to perform quantitative polymerase chain reaction with reverse transcription (RT-PCR) to determine robustly the relative abundance of dilp message originating in the adult brain: the dilp-2 message decreased nearly threefold whereas dilp-3 and dilp-5 were unchanged. Therefore, insulin signalling within the head fat body influences transcription of one specific dilp of the neuronal insulin-producing cells (Hwangbo, 2004).
Studies across model systems have established that insulin-like signalling can control lifespan non-autonomously from a limited set of cells or a specific tissue. In C. elegans, these cells may occur primarily in the intestine and secondarily in neurons. In mice, a disrupted insulin receptor in the adipose tissue across all life stages alters adult adipose morphology, decreases fasted insulin levels and modestly increases adult survival. This study shows with the fly that activated Foxo in the head fat body is sufficient to increase both male and female lifespan, to increase resistance to oxidative challenge and to alter whole-animal lipid metabolism. Therefore, in Drosophila systemic secondary signals must function downstream of Foxo, activated in the head fat body. Both juvenile hormone and 20-hydroxyecdysone are reduced in Drosophila mutants of InR, and both these hormones have the potential to regulate lifespan. Candidates for secondary hormonal signals have yet to be identified in C. elegans but these may involve sterols because Daf-9, a cytochrome P450 related to mammalian steroidogenic hydroxylases, functions downstream of Daf-2 but upstream of Daf-12, which encodes a putative nuclear hormone receptor (Hwangbo, 2004).
The data suggest that an insulin peptide itself may function as one secondary messenger of insulin-regulated ageing in D. melanogaster. A similar model is emerging for C. elegans: the peptide encoded by ins-7 accelerates ageing and is a systemic agonist of the daf-2 encoded receptor, whereas functional DAF-16 in the intestine non-autonomously activates DAF-16 in distant tissues and is sufficient to increase lifespan. This study finds that Dilp-2 is uniquely reduced when Foxo is activated in the head fat body of Drosophila; this transcriptional change will decrease the amount of circulating insulin peptide released from insulin-producing neurons. Notably, ablation of neuronal cells expressing Dilp-2 is sufficient to retard demographic ageing and to retard functional decline of the adult heart. Ageing of the heart, however, can also be delayed when insulin signalling is inhibited exclusively within the cardiac tissue itself, including heart-specific expression of activated Foxo. If senescence of tissue and systems throughout the adult is regulated autonomously by insulin, decreased circulation of this peptide downstream of regulatory insulin action within the head fat body could extend lifespan by reducing the mortality risk associated with degeneration of the soma as a whole (Hwangbo, 2004).
Aging of a eukaryotic organism is affected by its nutrition state and by its ability to prevent or repair oxidative damage. Consequently, signal transduction systems that control metabolism and oxidative stress responses influence life span. When nutrients are abundant, the insulin/IGF signaling (IIS) pathway promotes growth and energy storage but shortens life span. The transcription factor Foxo, which is inhibited by IIS, extends life span in conditions of low IIS activity. Life span can also be increased by activating the stress-responsive Jun-N-terminal kinase (JNK) pathway. This study shows that JNK requires Foxo to extend life span in Drosophila. JNK antagonizes IIS, causing nuclear localization of Foxo and inducing its targets, including growth control and stress defense genes. JNK and Foxo also restrict IIS activity systemically by repressing IIS ligand expression in neuroendocrine cells. The convergence of JNK signaling and IIS on Foxo provides a model to explain the effects of stress and nutrition on longevity (Wang, 2005).
The data suggest Foxo as a convergence point for IIS and JNK signaling. Through its responsiveness to these two pathways, Foxo is well positioned to integrate information about environmental stress and nutrient availability and to elicit appropriate biological responses. Such a system would ensure that growth could proceed in an unrestrained manner when energy resources are available and the cell is not exposed to external insults (IIS is active, JNK is off, and Foxo is repressed). However, in situations of low food availability or an adverse environment, IIS would cease to signal, or JNK would be activated, resulting in translocation of Foxo to the nucleus. The ensuing Foxo-induced gene expression has several effects at the cell as well as the organism level and is likely to counteract premature senescence. The induction of genes such as thor can reduce cell growth, presumably to limit the cell's anabolic expenses in adverse situations. Other target genes, such as the small heat shock protein l(2)efl, are expected to have a direct role in allaying damage inflicted by environmental insults and may prevent the accumulation of toxic protein aggregates. The suppression of dilp2 expression by JNK and Foxo in insulin-producing cells (IPCs), in contrast, is likely to control growth, metabolism, and stress responses systemically by downregulating IIS in all responsive tissues in a coordinated fashion (Wang, 2005).
The interaction between JNK and Foxo is thus expected to influence stress tolerance and life span at two levels. In peripheral tissues, JNK activates Foxo and prevents senescence cell-autonomously. Such a mechanism is exemplified by the recent finding that Foxo overexpression prevents age-dependent decline of cardiac performance (Wessells, 2004). Systemic control of IIS by JNK-mediated activation of Foxo in IPCs, in contrast, would serve to coordinate cellular responses to changes in the environment throughout the organism. The data indicate that this latter mechanism plays a significant role in the regulation of life span by JNK and Foxo. The identification of this endocrine function of JNK/Foxo signaling supports and extends the proposed role of JNK signaling on longevity and demonstrates a role for IPCs in life span regulation. In addition to controlling growth and metabolism, IPCs may thus act as a coordination point for the organism's stress response by downregulating Dilp production in response to oxidative stress and JNK activation. In target tissues, such a mechanism would induce protective gene expression by the second, cell-autonomous tier of Foxo signaling. Interestingly, the effects of IPC-specific JNK activation on longevity and growth are separable. Life span can be extended by moderately increased JNK activity in IPCs when growth effects are yet not evident. This finding is consistent with observations that the extension of life span in IIS loss-of-function situations is not a mere consequence of small body size (Wang, 2005).
How did such a multilayered regulation of IIS activity by JNK evolve? It is tempting to speculate that localized activation of Foxo is required to prevent cellular damage and ultimately senescence in conditions in which stressful insults are confined to specific tissues. Such localized insults could, for example, be inflicted by reactive oxygen species that are produced in the environment of amyloid deposits in Alzheimer's disease as well as by mechanical and oxidative stress experienced by particularly active tissues such as the heart. Systemic regulation of Foxo activity, in contrast, is expected to be an important response mechanism to coordinate metabolism and stress defenses throughout the organism upon changes in the environment. A good example for such a mechanism is the induction of diapause in invertebrates in response to environmental stress or food deprivation. Accordingly, sensory neurons expressing the insulin-like peptide DAF-28 are required for the induction of the dauer larval stage in response to environmental cues in C. elegans (Wang, 2005).
Systemic and tissue-autonomous effects of JNK/Foxo signaling may be connected in multiple ways. The data indicate that JNK and Foxo interact in IPCs to repress dilp2 expression, ultimately activating Foxo in Dilp2 target tissues in a coordinated fashion. Since JNK was found to be activated in IPCs even under normal culture conditions, it is likely that this systemic control of IIS activity by JNK and Foxo plays a critical role in life span regulation. It is, however, also possible that the cell-autonomous protective function of JNK/Foxo signaling is most critical for the survival of specific tissues as the organism ages, thus extending life of the organism by preventing the loss of indispensable cells or tissues. In addition, stress and the JNK-mediated activation of Foxo in peripheral tissues may signal back to IPCs to initiate a systemic response. In Drosophila, such a mechanism has been documented in the case of the fatbody. Activation of Foxo in this tissue relays a signal to the IPCs, causing them to curb Dilp2 production, a process that has been proposed to require Foxo activity (Hwangbo, 2004). The exact nature of this feedback signaling mechanism in flies is unclear, but it is reminiscent of the complex signaling interactions between β cells and insulin target tissues in mammals. Further studies are required to shed light on the relative contributions of JNK/Foxo signaling in IPCs or Dilp target tissues to life span regulation (Wang, 2005).
JNK-mediated modulation of IIS activity is likely to be evolutionarily conserved. Inhibitory crosstalk from JNK to IIS in mammalian cells has been found to occur by JNK-mediated phosphorylation and inhibition of IRS-1. This interaction is responsible for obesity-induced insulin resistance in mice. Whether mammalian homologs of Foxo take part in this pathology remains to be determined. A second possible mechanism for JNK/IIS pathway interaction is the direct phosphorylation and activation of Foxo by JNK. A recent study supports such a mechanism, showing that in mouse cells JNK can phosphorylate the DFoxo homolog Foxo4 in response to oxidative stress. The physiological relevance of this phosphorylation event has not yet been addressed. The JNK target residues on IRS-1 and Foxo4 are not conserved in the Drosophila homologs Chico and DFoxo, and further studies are thus required to determine whether JNK-Foxo crosstalk in Drosophila is mediated via homologous mechanisms (Wang, 2005).
The systemic regulation of IIS activity by JNK and Foxo appears to be conserved as well. It has been suggested that C. elegans Daf16/Foxo regulates life span (at least in part) by reducing the expression of insulin-like peptides. In mammals, pancreatic β cells (the counterparts of IPCs) reduce their production of insulin in response to oxidative stress-mediated JNK activation. Conversely, dephosphorylation of JNK by MAPK phosphatase 1 can induce insulin expression in these cells. Reducing circulating insulin levels by JNK-mediated Foxo activation may thus be a general mechanism that balances growth and metabolism with stress defense and damage repair (Wang, 2005).
One of the most consistent behavioral changes that occurs with age in humans is the loss of sleep consolidation. This can be quite disruptive and yet little is known about its underlying basis. To better understand the effects of aging on sleep:wake cycles, this problem was studied in Drosophila. By assaying flies of different ages as well as monitoring individual flies constantly over the course of their lifetime, it was found that the strength of sleep:wake cycles decreased and that sleep became more fragmented with age in Drosophila. These changes in sleep:wake cycles became faster or slower with manipulations of ambient temperature that decreased or increased lifespan, respectively, demonstrating that they are a function of physiological rather than chronological age. The effect of temperature on lifespan was not mediated by changes in overall activity level or sleep amount. Flies treated with the oxidative stress-producing reagent paraquat showed a breakdown of sleep:wak cycles similar to that seen with aging, leading to a proposal that the accumulation of oxidative damage with age contributes to the changes in rhythm and sleep. Together, these findings establish Drosophila as a valuable model for studying age-associated sleep fragmentation and breakdown of rhythm strength, and indicate that these changes in sleep:wake cycles are an integral part of the physiological aging process (Koh, 2006).
Aging is associated with an accumulation of oxidative damage. Young flies lacking the FOXO protein, which protects cells from oxidative stress in mammalian tissue culture, show rhythm phenotypes. In particular, foxo mutant flies are unable to sustain sleep:wake rhythms in the presence of the oxidative stress-producing agent paraquat. These data suggested that an increase in oxidative damage can cause deterioration of sleep:wake cycles. To determine whether paraquat can produce similar effects in wild-type flies, flies were maintained on food containing 1 mM paraquat throughout life. At this low dosage, wild-type flies could live on average for ~1 month, which is approximately half of their normal lifespan. For the first 3 weeks of treatment, paraquat had little effect on sleep:wake cycles. At ~3 weeks of age, however, compared with control flies, flies treated with paraquat showed a faster rate of decrease in rhythm strength and average duration of sleep bouts and a faster rate of increase in sleep bout numbers. Paraquat treatment also promoted faster changes in overall activity and total sleep amount in females. These measures showed little change in males after ~3 weeks and were relatively unaffected by paraquat treatment. These results resemble those found in flies whose aging process was accelerated with high ambient temperature and suggest that the effects of increased oxidative stress on sleep:wake cycles are similar to those that occur with aging (Koh, 2006).
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date revised: 30 May 2008
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