In mammals, insulin signalling regulates glucose transport together with the expression and activity of various metabolic enzymes. In the nematode Caenorhabditis elegans, a related pathway regulates metabolism, development and longevity. Wild-type animals enter the developmentally arrested dauer stage in response to high levels of a secreted pheromone, accumulating large amounts of fat in their intestines and hypodermis. Mutants in DAF-2 (a homolog of the mammalian insulin receptor) and AGE-1 (a homolog of the catalytic subunit of mammalian phosphatidylinositol 3-OH kinase) arrest development at the dauer stage. Moreover, animals bearing weak or temperature-sensitive mutations in daf-2 and age-1 can develop reproductively, but nevertheless show increased energy storage and longevity. Null mutations in daf-16 suppress the effects of mutations in daf-2 or age-1; lack of daf-16 bypasses the need for this insulin receptor-like signalling pathway. The principal role of DAF-2/AGE-1 signalling is thus to antagonize DAF-16. daf-16 is widely expressed and encodes three members of the Fork head family of transcription factors. The DAF-2 pathway acts synergistically with the pathway activated by a nematode TGF-beta-type signal, DAF-7, suggesting that DAF-16 cooperates with nematode SMAD proteins in regulating the transcription of key metabolic and developmental control genes. The probable human orthologs of DAF-16, FKHR and AFX, may also act downstream of insulin signalling and cooperate with TGF-beta effectors in mediating metabolic regulation. These genes may be dysregulated in diabetes (Ogg, 1997).
The wild-type Caenorhabditis elegans nematode ages rapidly, undergoing development, senescence, and death in less than 3 weeks. In contrast, mutants with reduced activity of the gene daf-2, a homolog of the insulin and insulin-like growth factor receptors, age more slowly than normal and live more than twice as long. These mutants are active and fully fertile and have normal metabolic rates. The life-span extension caused by daf-2 mutations requires the activity of the gene daf-16. daf-16 appears to play a unique role in life-span regulation and encodes a member of the hepatocyte nuclear factor 3 (HNF-3)/forkhead family of transcriptional regulators. In humans, insulin down-regulates the expression of certain genes by antagonizing the activity of HNF-3, raising the possibility that aspects of this regulatory system have been conserved (Lin, 1997).
A neurosecretory pathway regulates a reversible developmental arrest and metabolic shift at the Caenorhabditis elegans dauer larval stage. Defects in an insulin-like signaling pathway cause arrest at the dauer stage. Two C. elegans Akt/PKB homologs, akt-1 and akt-2, transduce insulin receptor-like signals that inhibit dauer arrest and AKT-1 and AKT-2 signaling are indispensable for insulin receptor-like signaling in C. elegans. A loss-of-function mutation in the Fork head transcription factor DAF-16 relieves the requirement for Akt/PKB signaling, which indicates that AKT-1 and AKT-2 function primarily to antagonize DAF-16. This is the first evidence that the major target of Akt/PKB signaling is a transcription factor. An activating mutation in akt-1, revealed by a genetic screen, as well as increased dosage of wild-type akt-1 relieves the requirement for signaling from AGE-1 PI3K, which acts downstream of the DAF-2 insulin/IGF-1 receptor homolog. This demonstrates that Akt/PKB activity is not necessarily dependent on AGE-1 PI3K activity. akt-1 and akt-2 are expressed in overlapping patterns in the nervous system and in tissues that are remodeled during dauer formation (Paradis, 1998).
C. elegans insulin-like signaling regulates metabolism, development, and life span. This signaling pathway negatively regulates the activity of the forkhead transcription factor DAF-16. daf-16 encodes multiple isoforms that are expressed in distinct tissue types and are probable orthologs of human FKHRL1, FKHR, and AFX. Human FKHRL1 can partially replace DAF-16, proving the orthology. In mammalian cells, insulin and insulin-like growth factor signaling activate AKT/PKB kinase to negatively regulate the nuclear localization of DAF-16 homologs. The absence of AKT consensus sites on DAF-16 is sufficient to cause dauer arrest in daf-2 plus animals, proving that daf-16 is the major output of insulin signaling in C. elegans. FKHR, FKRHL1, and AFX may similarly be the major outputs of mammalian insulin signaling. daf-2 insulin signaling, via AKT kinases, negatively regulates DAF-16 by controlling its nuclear localization. Surprisingly, daf-7 TGF-beta signaling also regulates DAF-16 nuclear localization specifically at the time when the animal makes the commitment between diapause and reproductive development. daf-16 function is supported by the combined action of two distinct promoter/enhancer elements, whereas the coding sequences of two major DAF-16 isoforms are interchangeable. Together, these observations suggest that the combined effects of transcriptional and posttranslational regulation of daf-16 transduce insulin-like signals in C. elegans and perhaps more generally (Lee, 2001).
Evolutionary models of aging propose that a trade-off exists between the resources an organism devotes to reproduction and growth and those devoted to cellular maintenance and repair, such that an optimal life history always entails an imperfect ability to resist stress. Yet, since environmental stressors, such as caloric restriction or exposure to mild stress, can increase stress resistance and life span, it is possible that a common genetic mechanism could regulate the allocation of resources in response to a changing environment. Consistent with predictions of evolutionary trade-off models, nematodes carrying an integrated DAF-16::GFP transgene grow and reproduce more slowly yet are more stress resistant and longer lived than controls carrying the integration marker alone. The nuclear localization of the DAF-16::GFP fusion protein responds to environmental inputs as well as genetic. Environmental stresses, such as starvation, heat, and oxidative stress, cause rapid nuclear localization of DAF-16. In conditions rich in food, DAF-16::GFP is inhibited from entry into the nucleus by daf-2 and akt-1/akt-2, both components of insulin-like signaling in nematodes. It is suggested that changes in the subcellular localization of DAF-16 by environmental cues allows for rapid reallocation of resources in response to a changing environment at all stages of life (Henderson, 2001).
The lifespan of Caenorhabditis elegans is regulated by the insulin/insulin-like growth factor (IGF)-1 receptor homolog DAF-2, which signals through a conserved phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathway. Mutants in this pathway remain youthful and active much longer than normal animals and can live more than twice as long. This lifespan extension requires DAF-16, a forkhead/winged-helix transcription factor. DAF-16 is thought to be the main target of the DAF-2 pathway. Insulin/IGF-1 signaling is thought to lead to phosphorylation of DAF-16 by AKT activity, which in turn shortens lifespan. The DAF-2 pathway prevents DAF-16 accumulation in nuclei. Disrupting Akt-consensus phosphorylation sites in DAF-16 causes nuclear accumulation in wild-type animals, but, surprisingly, has little effect on lifespan. Thus the DAF-2 pathway must have additional outputs. Lifespan in C. elegans can be extended by perturbing sensory neurons or germ cells. In both cases, lifespan extension requires DAF-16. Both sensory neurons and germline activity regulate DAF-16 accumulation in nuclei, but the nuclear localization patterns are different. Together these findings reveal unexpected complexity in the DAF-16-dependent pathways that regulate aging (Lin, 2001).
Aging and limited life span are fundamental biological phenomena observed in a variety of species. Approximately 55 genes have been identified that can extend longevity when altered in Caenorhabditis elegans. These genes include an insulin-like receptor (daf-2) and a phosphatidylinositol 3-OH kinase (age-1) regulating a forkhead transcription factor (daf-16), as well as genes mediating metabolic throughput, sensory perception, and reproduction. Moreover, these mutant alleles both extend life span and increase resistance to ultraviolet (UV) radiation, heat, and oxidative stress, though the stress resistance of clk-1 is controversial. With the exception of old-1 and perhaps some other genes, all of the life-extension alleles are hypomorphic or nullomorphic. The OLD-1 transmembrane tyrosine kinase (formerly TKR-1) is expressed in a variety of tissues, is stress inducible, and is a positive regulator of longevity and stress resistance. The transcription of old-1 is upregulated in long-lived age-1 and daf-2 mutants and is upregulated in response to heat, UV light, and starvation. Both RT-PCR and analysis of an OLD-1::GFP tag suggest that old-1 expression is dependent on daf-16. Importantly, old-1 is required for the life extension of age-1 and daf-2 mutants. This study reveals a new system for specifying longevity and stress resistance and suggests possible mechanisms for mediating life extension by dietary restriction and hormesis (Murakami, 2001).
The daf-2 insulin-like receptor pathway regulates development and life-span in Caenorhabditis elegans. Reduced DAF-2 signaling leads to changes in downstream targets via the daf-16 gene, a fork-head transcription factor which is regulated by DAF-2, and results in extended life-span. This paper describes the identification of genes whose expression is controlled by the DAF-2 signaling cascade. dao-1, dao-2, dao-3, dao-4, dao-8 and dao-9 are down-regulated in daf-2 mutant adults compared to wild-type adults, whereas dao-5, dao-6 and dao-7 are up-regulated. The latter genes are negatively regulated by DAF-2 signaling and positively regulated by DAF-16. Positive regulation by DAF-2 on dao-1, dao-4 and dao-8 is mediated by DAF-16, whereas daf-16 mediates only part of DAF-2 signaling for dao-2 and dao-9. Regulation by DAF-2 is most likely DAF-16 independent for dao-3 and hsp-90. RNA levels of dao-5 and dao-6 show elevated expression in daf-2 adults, as well as being strongly expressed in dauer larvae. In contrast, hsp-90 transcript levels are low in daf-2 mutant adults though they are enriched in dauer larvae, indicating overlapping but not identical mechanisms of efficient life maintenance in stress-resistant dauer larvae and long-lived daf-2 mutant adults. dao-1, dao-8 and dao-9 are homologs of the FK506 binding proteins that interact with the mammalian insulin pathway. dao-3 encodes a putative methylenetetrahydrofolate dehydrogenase. DAO-5 shows 33 % identity with human nucleolar phosphoprotein P130. dao-7 is similar to the mammalian ZFP36 protein. Distinct regulatory patterns of dao genes implicate their diverse positions within the signaling network of DAF-2 pathway, and suggest they have unique contributions to development, metabolism and longevity (Yu, 2001).
Oxidative damage shortens the life span of the nematode Caenorhabditis elegans, even in an age-1 mutant that is characterized by a long life and oxygen resistance. Daily short-term exposure (3 h) to hyperoxia further extends the life span of age-1, a phenomenon known as an adaptive response. age-1 also shows resistance to paraquat and heat. Acute hyperoxic treatment does not extend the life spans of wild type, daf-16 or mev-1. daf-16 mutants have a slightly shorter life span compared to wild type and are sensitive to heat and paraquat. The daf-16 phenotype resembles that of mev-1 showing a short life and oxygen sensitivity. mRNA levels were measured of superoxide dismutase genes (sod-1 through 4), catalase genes (clt-1 and ctl-2), known to encode anti-oxidant enzymes, and found that these levels were elevated in age-1 young adults. However, in daf-16 and mev-1, the expression of sod-1, sod-2 and sod-3 genes was lower rather than in wild type. Conversely, ctl-1 and ctl-2 genes expression was significantly elevated in daf-16 and mev-1. This suggests that DAF-16, a forkhead/winged-helix transcription factor, whose expression is suppressed by AGE-1, phosphoinositide 3-kinase (PI3-kinase), regulates anti-oxidant genes as well as energy metabolism under atmospheric conditions. However, the level of gene expression of SOD and catalase was not elevated by short-term exposure to 90% oxygen in wild type, mev-1, daf-16 and even age-1. This suggests that SOD and catalase do not play a role in the adaptive response against oxidative stress under hyperoxia, at least under these experimental conditions (Yanase, 2002).
Signaling from the DAF-2/insulin receptor to the DAF-16/FOXO transcription factor controls longevity, metabolism, and development in disparate phyla. To identify genes that mediate the conserved biological outputs of daf-2/insulin-like signaling, comparative genomics were used to identify 17 orthologous genes from Caenorhabditis and Drosophila, each of which bears a DAF-16 binding site in the promoter region. One-third of these DAF-16 downstream candidate genes are regulated by daf-2/insulin-like signaling in C. elegans, and RNA interference inactivation of the candidates show that many of these genes mediate distinct aspects of daf-16 function, including longevity, metabolism, and development (Lee, 2003).
Ageing is a fundamental, unsolved mystery in biology. DAF-16, a FOXO-family transcription factor, influences the rate of ageing of Caenorhabditis elegans in response to insulin/insulin-like growth factor 1 (IGF-I) signalling. Using DNA microarray analysis, DAF-16 has been found to affect expression of a set of genes during early adulthood, the time at which this pathway is known to control ageing. Many of these genes influence the ageing process. The insulin/IGF-I pathway functions cell non-autonomously to regulate lifespan, and these findings suggest that this pathway signals other cells, at least in part, by feedback regulation of an insulin/IGF-I homolog. Furthermore, these findings suggest that the insulin/IGF-I pathway ultimately exerts its effect on lifespan by upregulating a wide variety of genes, including cellular stress-response, antimicrobial and metabolic genes, and by downregulating specific life-shortening genes (Murphy, 2003).
The Caenorhabditis elegans transcripion factor HSF-1, which regulates the heat-shock response, also influences aging. Reducing hsf-1 activity accelerates tissue aging and shortens life-span, and hsf-1 overexpression extends lifespan. HSF-1, like the transcription factor DAF-16, is required for daf-2-insulin/IGF-1 receptor mutations to extend life-span. This is because HSF-1 and DAF-16 together activate expression of specific genes, including genes encoding small heat-shock proteins, which in turn promote longevity. The small heat-shock proteins also delay the onset of polyglutamine-expansion protein aggregation, suggesting that these proteins couple the normal aging process to this type of age-related disease (Hsu, 2003).
In Caenorhabditis elegans, an insulin-like signaling pathway, which includes the daf-2 and age-1 genes, controls longevity and stress resistance. Downregulation of this pathway activates the forkhead transcription factor DAF-16, whose transcriptional targets are suggested to play an essential role in controlling the phenotypes governed by this pathway. The genes that have the DAF-16 consensus binding element (DBE) within putative regulatory regions have been surveyed. One such gene, termed scl-1, is a positive regulator of longevity and stress resistance. Expression of scl-1 is upregulated in long-lived daf-2 and age-1 mutants and is undetectable in a short-lived daf-16 mutant. SCL-1 is a putative secretory protein with an SCP domain and is homologous to the mammalian cysteine-rich secretory protein (CRISP) family. scl-1 is required for the extension of the life span of daf-2 and age-1 mutants, and downregulation of scl-1 reduces both life span and stress resistance of this animal. SCL-1, whose expression is dependent on DAF-16, is the first example of a putative secretory protein that positively regulates longevity and stress resistance (Ookuma, 2003).
The highly conserved target-of-rapamycin (TOR) protein kinases control cell growth in response to nutrients and growth factors. In mammals, TOR has been shown to interact with raptor (regulatory associated protein of mTOR: potential Drosophila homolog CG4320) to relay nutrient signals to downstream translation machinery. Raptor associates in a near stoichiometric ratio with mTOR to form a complex that functions as the nutrient sensor. It was proposed that raptor acts as a scaffold to bridge TOR with its putative phosphorylation targets. In C. elegans, mutations in the genes encoding CeTOR and raptor result in dauer-like larval arrest, implying that CeTOR regulates dauer diapause. The daf-15 (raptor) and let-363 (CeTOR) mutants shift metabolism to accumulate fat, and raptor mutations extend adult life span. daf-15 transcription is regulated by DAF-16, a FOXO transcription factor that is in turn regulated by daf-2 insulin/IGF signaling. This is a new mechanism that regulates the TOR pathway. Thus, DAF-2 insulin/IGF signaling and nutrient signaling converge on DAF-15 (raptor) to regulate C. elegans larval development, metabolism and life span (Jia, 2004).
Insulin/IGF-1 signaling (IIS) regulates aging in worms, flies, and mice through a well-characterized, highly conserved core set of components. IIS also regulates early developmental decisions, the reproductive status of the animal, innate immunity, and stress-resistance functions. In C. elegans, the sole insulin/IGF-1 receptor, DAF-2, negatively regulates the FOXO transcription factor, DAF-16. A new component of the IIS longevity pathway, SMK-1, specifically influences DAF-16-dependent regulation of the aging process in C. elegans by regulating the transcriptional specificity of DAF-16 activity. Localization analysis of DAF-16 places SMK-1 downstream of DAF-16's phosphorylation-dependent relocation to the nucleus. Physiological and transcription analyses indicate that smk-1 is required for the innate immune, UV, and oxidative stress but not the thermal stress functions of DAF-16. SMK-1 therefore plays a role in longevity by modulating DAF-16 transcriptional specificity without affecting other processes regulated by IIS (Wolff, 2006).
In C. elegans, removing the germ cells extends life span by triggering the nuclear localization and activation of the DAF-16/FOXO transcription factor in the intestine. This study identifies and analyzes genes required to extend life span as a consequence of germline ablation. The reproductive system communicates with the intestine through lipophilic-hormone signaling and a gene called kri-1 is likely to act in the intestine to promote DAF-16 nuclear localization in response to this signal. This lipophilic-signaling pathway and kri-1 are not required for DAF-16's nuclear localization and life-span extension in animals with decreased insulin/IGF-1 signaling. Thus, this pathway specifically enables the integration of cues from the reproductive system with central DAF-16-activation pathways to influence the aging of the animal (Berman, 2006).
The kri-1 gene encodes a conserved protein with ankyrin repeats that is constitutively expressed in the pharynx and intestine throughout postembryonic stages. Like daf-12, an orphan nuclear receptor that regulates dauer diapause, reproductive development, fat metabolism, and life span, kri-1 mutations suppress the increase in life span associated with germline loss but do not affect the life span of wild-type animals. In addition, kri-1 mutants have no significant effect on the life span of germline-deprived daf-2 mutants (daf-2 encodes an insulin receptor-like gene that regulates longevity and diapause in Caenorhabditis elegans) or on their Daf-c (dauer constitutive) phenotype, indicating that kri-1 acts independently of DAF-2 in the reproductive-signaling pathway. Upon germline ablation, DAF-16 in the intestine translocates from the cytoplasm to the nucleus, where its activity accounts for the entire increase in life span. By using a DAF-16::GFP fusion protein, kri-1 is shown to be required for DAF-16 nuclear localization in the intestinal cells of germline-deficient animals. A less dramatic but significant reduction in nuclear DAF-16::GFP was also seen in daf-9 or daf-12 mutants. The nuclear localization of DAF-16 seen in daf-2 mutants, however, is not affected by kri-1, daf-9 (encoding a CYP2 cytochrome P450 enzyme involved in metabolizing steroid hormones), or daf-12 mutations. This indicates that the roles of kri-1 and lipophilic hormone production on DAF-16 nuclear localization are specific to the reproductive pathway and act largely independently of insulin signaling (Berman, 2006; Beckstead, 2006).
A constitutively nuclear active DAF-16 protein was used to perform epistasis tests with kri-1, daf-9, and daf-12 mutations to address the long-standing question of how DAF-9 and DAF-12 contribute to the longevity of germline-deficient animals. As expected, nuclear DAF-16 extends the life span of daf-16 mutants that lack a germline. Similarly, nuclear DAF-16 rescues the increase in longevity seen upon germline ablation of kri-1 mutants, demonstrating that a key function for KRI-1 is to facilitate this localization of DAF-16 in the intestine. Interestingly, a daf-12 null mutation strongly blocked the longevity of germline-deficient animals that express nuclear DAF-16, indicating that DAF-12 can control longevity independently of DAF-16. Even more remarkable, a strong daf-9 allele has no effect on the longevity of germline-deficient animals that express nuclear DAF-16, indicating that daf-9 acts upstream from DAF-16 and that DAF-12 has functions in the germline-longevity pathway that are independent of lipophilic hormone signaling. The identification of dafachronic-acid ligands for DAF-12 provides new directions for these studies. It will be interesting to determine how the hormone affects DAF-16 nuclear localization and adult life span in both wild-type and mutant worms (Berman, 2006; Beckstead, 2006).
Development is typically studied as a continuous process under laboratory conditions, but wild animals often develop in variable and stressful environments. C. elegans larvae hatch in a developmentally arrested state (L1 arrest) and initiate post-embryonic development only in the presence of food (E. coli in lab). In contrast to the well-studied dauer arrest, L1 arrest occurs without morphological modification, although larvae in L1 arrest are more resistant to environmental stress than developing larvae. Consistent with its role in dauer formation and aging, insulin/insulin-like growth factor (IGF) signaling is shown to regulate L1 arrest. daf-2 insulin/IGF receptor mutants have a constitutive-L1-arrest phenotype when fed and extended survival of L1 arrest when starved. Conversely, daf-16/FOXO mutants have a defective-arrest phenotype, failing to arrest development and dying rapidly when starved. DAF-16 is required for transcription of the cyclin-dependent kinase inhibitor cki-1 in stem cells in response to starvation, accounting for the failure of daf-16/FOXO mutants to arrest cell division during L1 arrest. Other developmental events such as cell migration, cell fusion, and expression of the microRNA lin-4, a temporal regulator of post-embryonic development, are also observed in starved daf-16/FOXO mutants. These results suggest that DAF-16/FOXO promotes developmental arrest via transcriptional regulation of numerous target genes that control various aspects of development (Baugh, 2006).
Dietary restriction (DR) is the most effective environmental intervention to extend lifespan in a wide range of species. However, the molecular mechanisms underlying the benefits of DR on longevity are still poorly characterized. AMP-activated protein kinase (AMPK; see Drosophila SNF1A/AMP-activated protein kinase) is activated by a decrease in energy levels, raising the possibility that AMPK might mediate lifespan extension by DR. By using a novel DR assay that was developed and validated in C. elegans, it was found that AMPK is required for this DR method to extend lifespan and delay age-dependent decline. It was found that AMPK exerts its effects in part via the FOXO transcription factor DAF-16. FOXO/DAF-16 is necessary for the beneficial effects of this DR method on lifespan. Expression of an active version of AMPK in worms increases stress resistance and extends longevity in a FOXO/DAF-16-dependent manner. Lastly, it was found that AMPK activates FOXO/DAF-16-dependent transcription and phosphorylates FOXO/DAF-16 at previously unidentified sites, suggesting a possible direct mechanism of regulation of FOXO/DAF-16 by AMPK. This study shows that an energy-sensing AMPK-FOXO pathway mediates the lifespan extension induced by a novel method of dietary restriction in C. elegans (Greer, 2007).
Previous genetic evidence suggested that the C. elegans TGF-β Dauer pathway is responsible solely for the regulation of dauer formation, with no role in longevity regulation, whereas the insulin/IGF-1 signaling (IIS) pathway regulates both dauer formation and longevity. A significant longevity-regulating activity by the TGF-β Dauer pathway has been discovered that is masked by an egg-laying (Egl) phenotype; mutants in the pathway display up to 2-fold increases in life span. The expression profiles of adult TGF-β mutants overlap significantly with IIS pathway profiles: Adult TGF-β mutants regulate the transcription of many DAF-16-regulated genes, including genes that regulate life span, the two pathways share enriched Gene Ontology categories, and a motif previously associated with DAF-16-regulated transcription (the DAE, or DAF-16-associated element) is overrepresented in the promoters of TGF-β regulated genes. The TGF-β Dauer pathway's regulation of longevity appears to be mediated at least in part through insulin interactions with the IIS pathway and the regulation of DAF-16 localization. Together, these results suggest there are TGF-β-specific downstream targets and functions, but that the TGF-β and IIS pathways might be more tightly linked in the regulation of longevity than has been previously appreciated (Shaw, 2007).
Genetic and RNA interference (RNAi) screens for life span regulatory genes have revealed that the daf-2 insulin-like signaling pathway plays a major role in Caenorhabditis elegans longevity. This pathway converges on the DAF-16 transcription factor and may regulate life span by controlling the expression of a large number of genes, including free-radical detoxifying genes, stress resistance genes, and pathogen resistance genes. A genome-wide RNAi screen was conducted to identify genes necessary for the extended life span of daf-2 mutants and ~200 gene inactivations were identified that shorten daf-2 life span. Some of these gene inactivations dramatically shorten daf-2 mutant life span but less dramatically shorten daf-2; daf-16 mutant or wild-type life span. Molecular and behavioral markers for normal aging and for extended life span in low insulin/IGF1 (insulin-like growth factor 1) signaling were assayed to distinguish accelerated aging from general sickness and to examine age-related phenotypes. Detailed demographic analysis, molecular markers of aging, and insulin signaling mutant test strains were used to filter progeric gene inactivations for specific acceleration of aging. Highly represented in the genes that mediate life span extension in the daf-2 mutant are components of endocytotic trafficking of membrane proteins to lysosomes. These gene inactivations disrupt the increased expression of the DAF-16 downstream gene superoxide dismutase sod-3 in a daf-2 mutant, suggesting trafficking between the insulin-like receptor and DAF-16. The activities of these genes may normally decline during aging (Samuelson, 2007).
Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/threonine kinase Akt, which then phosphorylates and inactivates components of the apoptotic machinery, including BAD and Caspase 9. Akt also regulates the activity of FKHRL1, a member of the Forkhead family of transcription factors. In the presence of survival factors, Akt phosphorylates FKHRL1, leading to FKHRL1's association with 14-3-3 proteins and FKHRL1's retention in the cytoplasm. Survival factor withdrawal leads to FKHRL1 dephosphorylation, nuclear translocation, and target gene activation. Within the nucleus, FKHRL1 triggers apoptosis most likely by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene (Brunet, 1999).
Although genetic analysis has demonstrated that members of the winged helix, or forkhead, family of transcription factors play pivotal roles in the regulation of cellular differentiation and proliferation, both during development and in the adult, little is known of the mechanisms underlying their regulation. The activation of phosphatidylinositol 3 (PI3) kinase by extracellular growth factors induces phosphorylation, nuclear export, and transcriptional inactivation of FKHR1, a member of the FKHR subclass of the forkhead family of transcription factors. Protein kinase B (PKB)/Akt, a key mediator of PI3 kinase signal transduction, phosphorylates recombinant FKHR1 in vitro at threonine-24 and serine-253. Mutants FKHR1(T24A), FKHR1(S253A), and FKHR1(T24A/S253A) are resistant to both PKB/Akt-mediated phosphorylation and PI3 kinase-stimulated nuclear export. These results indicate that phosphorylation by PKB/Akt negatively regulates FKHR1 by promoting export from the nucleus (Biggs, 1999).
The phosphatidylinositol-3-OH-kinase (PI(3)K) effector protein kinase B regulates certain insulin-responsive genes, but the transcription factors regulated by protein kinase B have yet to be identified. Genetic analysis in Caenorhabditis elegans has shown that the Forkhead transcription factor daf-16 is regulated by a pathway consisting of insulin-receptor-like daf-2 and PI(3)K-like age-1. Protein kinase B phosphorylates AFX, a human ortholog of daf-16, both in vitro and in vivo. Inhibition of endogenous PI(3)K and protein kinase B activity prevents protein kinase B-dependent phosphorylation of AFX and reveals residual protein kinase B-independent phosphorylation that requires Ras signalling towards the Ral GTPase. In addition, phosphorylation of AFX by protein kinase B inhibits its transcriptional activity. Together, these results delineate a pathway for PI(3)K-dependent signalling to the nucleus (Kops, 1999).
The regulation of intracellular localization of AFX, a human Forkhead transcription factor, was studied. AFX was recovered as a phosphoprotein from transfected COS-7 cells growing in the presence of FBS, and the phosphorylation was eliminated by wortmannin, a potent inhibitor of phosphatidylinositol (PI) 3-kinase. AFX is phosphorylated in vitro by protein kinase B (PKB), a downstream target of PI 3-kinase, but a mutant protein in which three putative phosphorylation sites of PKB have been replaced by Ala is not recognized by PKB. In Chinese hamster ovary cells (CHO-K1) cultured with serum, the AFX protein fused with green fluorescence protein (AFX-GFP) is localized mainly in the cytoplasm, and wortmannin induces transient nuclear translocation of the fusion protein. The AFX-GFP mutant in which all three phosphorylation sites have been replaced by Ala is detected exclusively in the cell nucleus. AFX-GFP is in the nucleus when the cells are infected with an adenovirus vector encoding a dominant-negative form of either PI 3-kinase or PKB, whereas the fusion protein stays in the cytoplasm when the cells express constitutively active PKB. In CHO-K1 cells expressing AFX-GFP, DNA fragmentation is induced by the stable PI 3-kinase inhibitor LY294002, and the expression of the active form of PKB suppresses this DNA fragmentation. The phosphorylation site mutant of AFX-GFP enhances DNA fragmentation irrespective of the presence and absence of PI 3-kinase inhibitor. These results indicate that the nuclear translocation of AFX is negatively regulated through its phosphorylation by PKB (Takaishi, 1999).
AFX belongs to a subfamily of Forkhead transcription factors that are phosphorylated by protein kinase B (PKB), also known as Akt. Phosphorylation inhibits the transcriptional activity of AFX and changes the steady-state localization of the protein from the nucleus to the cytoplasm. The goal of this study was threefold: (1) to identify the cellular compartment in which PKB phosphorylates AFX, (2) to determine whether the nuclear localization of AFX plays a role in regulating its transcriptional activity, and (3) to elucidate the mechanism by which phosphorylation alters the localization of AFX. Phosphorylation of AFX by PKB is shown to occur in the nucleus. In addition, nuclear export mediated by the export receptor, Crm1, is required for the inhibition of AFX transcriptional activity. Both phosphorylated and unphosphorylated AFX, however, bind Crm1 and can be exported from the nucleus. These results suggest that export is unregulated and that phosphorylation by PKB is not required for the nuclear export of AFX. AFX enters the nucleus by an active, Ran-dependent mechanism. Amino acids 180 to 221 of AFX comprise a nonclassical nuclear localization signal (NLS). S193, contained within this atypical NLS, is a PKB-dependent phosphorylation site on AFX. Addition of a negative charge at S193 by mutating the residue to glutamate reduces nuclear accumulation. PKB-mediated phosphorylation of AFX, therefore, attenuates the import of the transcription factor, which shifts the localization of the protein from the nucleus to the cytoplasm and results in the inhibition of AFX transcriptional activity (Brownawell, 2001).
In Caenorhabditis elegans, an insulin-like signaling pathway to phosphatidylinositol 3-kinase (PI 3-kinase) and AKT negatively regulates the activity of DAF-16, a Forkhead transcription factor. In mammalian cells, C. elegans DAF-16 is a direct target of AKT and AKT phosphorylation generates 14-3-3 binding sites and regulates the nuclear/cytoplasmic distribution of DAF-16 as previously shown for its mammalian homologs FKHR and FKHRL1. In vitro, interaction of AKT- phosphorylated DAF-16 with 14-3-3 prevents DAF-16 binding to its target site in the insulin-like growth factor binding protein-1 gene, the insulin response element. In HepG2 cells, insulin signaling to PI 3-kinase/AKT inhibits the ability of a GAL4 DNA binding domain/DAF-16 fusion protein to activate transcription via the insulin-like growth factor binding protein-1-insulin response element, but not the GAL4 DNA binding site, which suggests that insulin inhibits the interaction of DAF-16 with its cognate DNA site. Elimination of the DAF-16/14-3-3 association by mutation of the AKT/14-3-3 sites in DAF-16, prevents 14-3-3 inhibition of DAF-16 DNA binding and insulin inhibition of DAF-16 function. Similarly, inhibition of the DAF-16/14-3-3 association by exposure of cells to the PI 3-kinase inhibitor LY294002, enhances DAF-16 DNA binding and transcription activity. Surprisingly constitutively nuclear DAF-16 mutants that lack AKT/14-3-3 binding sites also show enhanced DNA binding and transcription activity in response to LY294002, pointing to a 14-3-3-independent mode of regulation. Thus, these results demonstrate at least two mechanisms, one 14-3-3-dependent and the other 14-3-3-independent, whereby PI 3-kinase signaling regulates DAF-16 DNA binding and transcription function (Cahill, 2001).
FKHR is a member of the FOXO subfamily of Forkhead transcription factors, which are important targets for insulin and growth factor signaling. FKHR contains three predicted protein kinase B phosphorylation sites (Thr-24, Ser-256, and Ser-319) that are conserved in other FOXO proteins. Phosphorylation of Ser-256 is critical for the ability of insulin and insulin-like growth factors to suppress transactivation by FKHR and for its exclusion from the nucleus. Ser-256 is located in a basic region of the FKHR DNA binding domain where phosphorylation may have direct effects on DNA binding and/or nuclear targeting. Phosphorylation of Ser-256 may also be required for the phosphorylation of Thr-24 and Ser-319. Evidence is provided that basic residues in the FKHR DNA binding domain are critical for DNA binding and that Ser-256 phosphorylation alters binding activity. Ser-256 phosphorylation also is critical for regulating nuclear/cytoplasmic trafficking; however, this effect requires Thr-24/Ser-319 phosphorylation. Transient transfection studies with reporter gene constructs in 293 cells reveal that the phosphorylation of Ser-256 can inhibit the function of FKHR independent of Thr-24/Ser-319 phosphorylation. Studies with GFP(1) fusion proteins indicate that Ser-256 phosphorylation is critical for nuclear exclusion of FKHR. However, this effect is disrupted when Thr-24 and Ser-319 are replaced by alanine, indicating that nuclear exclusion of FKHR also requires Thr-24/Ser-319 phosphorylation. Gel shift and fluorescence anisotropy studies reveal that basic residues at the C-terminal end of the FKHR DBD are important for DNA binding, and the introduction of a negative charge at the site of Ser-256 limits binding activity. Binding is rapid and reversible, providing an opportunity for the phosphorylation of Ser-256 and subsequent phosphorylation of Thr-24 and Ser-319 and nuclear exclusion of FKHR (Zhang, 2002).
Forkhead transcription factors of the FoxO-group are associated with cellular processes like cell cycle progression and DNA-repair. FoxO function is regulated by protein kinase B (PKB) via the phosphatidylinositol 3-kinase/PKB survival pathway. Phosphorylation of serine and threonine residues in specific PKB phosphorylation motifs leads to exclusion of FoxO-proteins from the nucleus, which excludes them from exerting transactivating activity. Members of the FoxO-group have three highly conserved regions containing a PKB phosphorylation motif. This study describes the cloning and characterization of a novel forkhead domain gene from mouse that appeared to be highly related to the FoxO group of transcription factors and was therefore designated FoxO6. The FoxO6 gene was mapped in region D1 on mouse chromosome 4. In humans, FOXO6 is located on chromosomal region 1p34.1. Embryonic expression of FoxO6 is most apparent in the developing brain, and FoxO6 is expressed in a specific temporal and spatial pattern. Therefore it is probably involved in regulation of specific cellular differentiation. In the adult animal FoxO6 expression is maintained in areas of the nucleus accumbens, cingulate cortex, parts of the amygdala, and in the hippocampus. Structure function analysis of FoxO6 compared with its group members shows that the overall homology is high, but surprisingly a highly conserved region containing multiple phosphorylation sites is lacking. In transfection studies, FoxO6 coupled to GFP showed an unexpected high nuclear localization after stimulation with growth factors, in contrast to the predominant cytosolic localization of FoxO1 and FoxO3. Nuclear export of FoxO6 is mediated through the phosphatidylinositol 3-kinase/PKB pathway. Using a chimeric approach it has been shown that the ability of FoxO6 to shuttle between nucleus and cytosol can be fully restored. In conclusion, the data presented here gives a new view on regulation of FoxO-function through multiple phosphorylation events and other mechanisms involved in the nuclear exclusion of FoxO-proteins (Jacobs, 2003).
Forkhead transcription factor FKHR (Foxo1) is a key regulator of glucose homeostasis, cell-cycle progression, and apoptosis. FKHR is phosphorylated via insulin or growth factor signaling cascades, resulting in its cytoplasmic retention and the repression of target gene expression. The fate has been investigated of FKHR after cells are stimulated by insulin. Insulin treatment is shown to decrease endogenous FKHR proteins in HepG2 cells; this decrease is inhibited by proteasome inhibitors. FKHR is ubiquitinated in vivo and in vitro, and insulin enhances the ubiquitination in the cells. In addition, the signal to FKHR degradation from insulin is mediated by the phosphatidylinositol 3-kinase pathway, and mutation of FKHR at the serine or threonine residues phosphorylated by protein kinase B, a downstream target of phosphatidylinositol 3-kinase, inhibits the ubiquitination in vivo and in vitro. Finally, efficient ubiquitination of FKHR requires both phosphorylation and cytoplasmic retention in the cells. These results demonstrate that the insulin-induced phosphorylation of FKHR leads to the multistep negative regulation, not only by the nuclear exclusion but also the ubiquitination-mediated degradation (Matsuzaki, 2003).
Growth factor receptors promote cell growth and survival by stimulating the activities of phosphatidylinositol 3-kinase and Akt/PKB. Akt activation causes proteasomal degradation of substrates that control cell growth and survival. Expression of activated Akt triggers proteasome-dependent declines in the protein levels of the Akt substrates tuberin, FOXO1, and FOXO3a. The addition of proteasome inhibitors stabilizes the phosphorylated forms of multiple Akt substrates, including tuberin and FOXO proteins. Activation of Akt triggers the ubiquitination of several proteins containing phosphorylated Akt substrate motifs. Together the data indicate that activated Akt stimulates proteasomal degradation of its substrates and suggest that Akt-dependent cell growth and survival are induced through the degradation of negative regulators of these processes (Plas, 2003).
The life span of C. elegans is extended by mutations that inhibit the function of sensory neurons. In this study, specific subsets of sensory neurons are shown to influence longevity. Certain gustatory neurons inhibit longevity, whereas others promote longevity, most likely by influencing insulin/IGF-1 signaling. Olfactory neurons also influence life span, and they act in a distinct pathway that involves the reproductive system. In addition, a putative chemosensory G protein-coupled receptor expressed in some of these sensory neurons inhibits longevity. Together these findings imply that the life span of C. elegans is regulated by environmental cues and that these cues are perceived and integrated in a complex and sophisticated fashion by specific chemosensory neurons (Alcedo, 2004).
These findings suggest that gustatory neurons are likely to influence life span by perturbing the insulin/IGF-1 pathway. One possibility is that these neurons sense cues that regulate the release of insulin/IGF-1-like hormones that influence the insulin/IGF-1 receptor DAF-2 activity. The C. elegans genome contains more than 30 insulin/IGF-1 homologs, and several of these are expressed in gustatory neurons. The model is favored that longevity-inhibiting ASI and ASG neurons exert their effects on life span by inhibiting the activities of the ASJ and ASK neurons. Thus, one possibility is that the ASI and ASG neurons prevent the longevity-promoting ASJ and ASK neurons from producing a DAF-2 antagonist. It is intriguing that double mutants that have defects in proteins thought to be required for neuronal insulin secretion and in daf-2 activity have an intermediate life span between those of the corresponding neurosecretory single mutants and daf-2 hypomorphic single mutants (Ailion et al., 1999). This finding is consistent with the idea that some sensory neurons might secrete DAF-2 antagonists (Alcedo, 2004).
A number of insulin-like peptides have now been implicated in the regulation of aging. One such candidate DAF-2 antagonist is ins-1, but this gene is expressed not only in longevity-promoting neurons but also in longevity-inhibiting neurons. At this point, the information available about specific insulin-like peptides does not suggest simple models that explain the data. However, this may change as more is learned about the functions of these proteins. For example, it is possible that other insulin/IGF-1-like peptides function as antagonists in the ASJ and ASK neurons but not in the ASI and ASG neurons, since other insulin/IGF-1-like peptides are expressed in ASJ (Alcedo, 2004).
These observations suggest that olfactory neurons act in a regulatory pathway distinct from gustatory neurons to affect life span. (1) The combined ablation of the gustatory ASI and olfactory AWA and AWC neurons increases life span more than does ablation of either ASI or of AWA and AWC neurons alone. (2) Killing ASJ and ASK suppresses the longevity of ASI-ablated animals but not that of olfactory neuron-ablated animals. (3) The life span extension produced by killing gustatory neurons is completely daf-16 dependent, whereas the life span extension produced by killing olfactory neurons is only partially daf-16 dependent (Alcedo, 2004).
Olfactory neurons may influence life span by perturbing an endocrine signaling pathway that involves the reproductive system. Previous findings have suggested that the germline of C. elegans generates a signal that inhibits longevity and is counterbalanced by a signal from the somatic gonad that promotes longevity. Like the olfactory neurons characterized, the somatic gonad of C. elegans affects life span, at least in part, in a daf-16-independent fashion. In addition, olfactory neurons are required for the somatic gonad to influence life span. In wild-type animals, killing the somatic gonad precursors completely prevents germline ablation from extending life span, but in animals lacking olfactory neurons, it does not. One possibility is that olfactory neurons regulate the release of a hormone that allows the somatic gonad to influence longevity. If this model is correct, then it implies that, under some environmental conditions, the somatic gonad signal is silenced and may no longer be able to counterbalance the signals from the animal's germline. Alternatively, these olfactory neurons could produce a longevity signal in response to a different signal from the somatic gonad. The somatic gonad appears to regulate a pathway that involves DAF-2. Thus, as with the gustatory neurons, it is possible that the olfactory neurons influence longevity by regulating the release of insulin-like peptides (Alcedo, 2004).
Why might sensory neurons influence longevity? One environmental condition, food limitation, is known to have a dramatic effect on life span in many organisms. Caloric restriction extends life span and also delays reproduction. When ample food is restored to calorically-restricted rats, they can reproduce, even at a time when the age-matched controls are post-reproductive or dead. Thus, this response to caloric restriction has obvious survival value, since it allows animals to postpone reproduction until conditions improve. Dauer formation, which is regulated, at least in part, by sensory cues, serves the same function in C. elegans -- it allows animals to postpone reproduction under harsh environmental conditions. No obvious changes were observed in the timing of reproduction in the neuron-ablated animals; however, it is possible that the environmental cues that influence the activities of these neurons in nature also influence other neurons that control reproduction. In this way, sensory cues could affect life span and reproduction coordinately. Alternatively, certain environmental conditions could favor a shorter post-reproductive life span to prevent the aging animals from competing for resources with their progeny. A population of worms that lacks parental competition for resources should, over time, develop a significant advantage relative to populations in which such competition takes place (Alcedo, 2004).
The odr-10 gene encodes an olfactory G protein-coupled receptor that senses diacetyl, an odorant sensed by AWA neurons. odr-10 null mutants are not long-lived, implying that neither diacetyl nor its receptor regulates life span. In contrast, decreasing the mRNA levels of the putative chemosensory G protein-coupled receptor str-2, through RNA-mediated interference, extends life span. This suggests that C. elegans' life span is influenced by its perception of an environmental cue -- as yet unidentified -- that is sensed by STR-2. The identification of sensory cues that influence life span, such as those sensed by STR-2, should make it possible to address this interesting question experimentally (Alcedo, 2004).
Despite genetic evidence establishing angiopoietin-1 (Ang-1) as an essential regulator of vascular development, the molecular mechanisms underlying Ang-1 function are almost completely uncharacterized. This study demonstrates that Ang-1, via Akt activation, is a potent inhibitor of the forkhead transcription factor FKHR (FOXO1), identifying a nuclear signaling pathway through which Ang-1 modulates gene expression. Microarray analysis was used to show that FKHR, whose function in endothelial cells has not previously been elucidated, regulates many genes associated with vascular destabilization and remodeling (including angiopoietin-2, an Ang-1 antagonist) and endothelial cell apoptosis (e.g., survivin, TRAIL). Ang-1 inhibits FKHR-mediated changes in gene expression and FKHR-induced apoptosis. Analysis of gene expression changes induced by an activated version of Akt confirms that FKHR is a major target through which Akt regulates transcription in endothelial cells. RNA interference was used to demonstrate that FKHR is required for the expression of genes (including Ang-2) that have important vascular functions. These data suggest a novel, tissue-specific role for the Akt/FKHR pathway in the vasculature and suggest a mechanistic basis for the previously described actions of Ang-1 as a regulator of endothelial cell survival and blood vessel stability (Daly, 2004).
AFX is a Forkhead transcription factor that induces a G(1) cell cycle arrest via upregulation of the cell cycle inhibitor p27(Kip1). Protein kinase B (PKB) phosphorylates AFX causing inhibition of AFX by nuclear exclusion. In addition, Ras, through the activation of the RalGEF-Ral pathway, induces phosphorylation of AFX. The Ras-Ral pathway provokes phosphorylation of threonines 447 and 451 in the C terminus of AFX. A mutant protein in which both threonines are substituted for alanines (T447A/T451A) still responds to PKB-regulated nuclear-cytoplasmic shuttling, but transcriptional activity and consequent G(1) cell cycle arrest are greatly impaired. Furthermore, inhibition of the Ral signaling pathway abolishes both AFX-mediated transcription and regulation of p27(Kip1), while activation of Ral augments AFX activity. From these results it is concluded that Ral-mediated phosphorylation of threonines 447 and 451 is required for proper activity of AFX-WT. Interestingly, the T447A/T451A mutation did not affect the induction of transcription and G(1) cell cycle arrest by the PKB-insensitive AFX-A3 mutant, suggesting that Ral-mediated phosphorylation plays a role in the regulation of AFX by PKB (De Ruiter, 2001).
The cytokine IL-2 plays a very important role in the proliferation and survival of activated T cells. These effects of IL-2 are dependent on signaling through the phosphatidylinositol 3-kinase (PI3K) pathway. PI3K, through activation of protein kinase B/Akt, inhibits transcriptional activation by a number of forkhead transcription factors (FoxO1, FoxO3, and FoxO4). The role of these forkhead transcription factors in the IL-2-induced T cell proliferation and survival has been investigated. IL-2 regulates phosphorylation of FoxO3 in a PI3K-dependent fashion. Phosphorylation and inactivation of FoxO3 appears to play an important role in IL-2-mediated T cell survival, because mere activation of FoxO3 is sufficient to trigger apoptosis in T cells. Indeed, active FoxO3 can induce expression of IL-2-regulated genes, such as the cdk inhibitor p27(Kip1) and the proapoptotic Bcl-2 family member Bim. Furthermore, IL-2 triggers a rapid, PI3K-dependent, phosphorylation of FoxO1a in primary T cells. Thus, it is proposed that inactivation of FoxO transcription factors by IL-2 plays a critical role in T cell proliferation and survival (Stahl, 2002).
Myc synergizes with Ras and PI3-kinase in cell transformation, yet the molecular basis for this behavior is poorly understood. Myc is shown to recruit TFIIH, P-TEFb and Mediator to the cyclin D2 and other target promoters, while the PI3-kinase pathway controls formation of the preinitiation complex and loading of RNA polymerase II. The PI3-kinase pathway involves Akt-mediated phosphorylation of FoxO transcription factors. In a nonphosphorylated state, FoxO factors inhibit induction of multiple Myc target genes, Myc-induced cell proliferation and transformation by Myc and Ras. Abrogation of FoxO function enables Myc to activate target genes in the absence of PI3-kinase activity and to induce foci formation in primary cells in the absence of oncogenic Ras. It is suggested that the cooperativity between Myc and Ras is at least in part due to the fact that Myc and FoxO proteins control distinct steps in the activation of an overlapping set of critical target genes (Bouchard, 2004).
Forkhead transcription factors of the FOXO class are negatively regulated by PKB/c-Akt in response to insulin/IGF signalling, and are involved in regulating cell cycle progression and cell death. In contrast to insulin signalling, low levels of oxidative stress generated by treatment with H2O2 induce the activation of FOXO4. Upon treatment of cells with H2O2, the small GTPase Ral is activated and this results in a JNK-dependent phosphorylation of FOXO4 on threonine 447 and threonine 451. This Ral-mediated, JNK-dependent phosphorylation is involved in the nuclear translocation and transcriptional activation of FOXO4 after H2O2 treatment. In addition, it is shown that this signalling pathway is also employed by tumor necrosis factor alpha to activate FOXO4 transcriptional activity. FOXO members have been implicated in cellular protection against oxidative stress via the transcriptional regulation of manganese superoxide dismutase and catalase gene expression. The results reported here, therefore, outline a homeostasis mechanism for sustaining cellular reactive oxygen species that is controlled by signalling pathways that can convey both negative (PI-3K/PKB) and positive (Ras/Ral) inputs (Essers, 2004).
Forkhead box class O (FOXO) proteins are transcription factors that function downstream of the PTEN tumor suppressor and directly control the expression of genes involved in apoptosis, cell cycle progression, and stress responses. In the present study, FOXO1 is shown to interact with four and a half LIM 2 (FHL2) in prostate cancer cells. This interaction occurs in the nucleus and is enhanced by lysophosphatic acid. FHL2 decreases the transcriptional activity of FOXO1 and the expression of known FOXO target genes and inhibits FOXO1-induced apoptosis. Interestingly, SIRT1, a mammalian homolog of yeast Sir2, binds to and deacetylates FOXO1 and inhibits its transcriptional activity. FHL2 enhances the interaction of FOXO1 and SIRT1 and the deacetylation of FOXO1 by Sirtuin-1 (SIRT1). Overall, these data show that FHL2 inhibits FOXO1 activity in prostate cancer cells by promoting the deacetylation of FOXO1 by SIRT1 (Yang, 2005 ).
Several studies indicate that FKHR and AFX, mammalian homologs of the Caenorhabditis elegans forkhead transcription factor DAF-16, function in the insulin signaling pathway. A novel AFX isoform, designated AFX zeta, has been discoved in which the first 16 amino acids of the forkhead domain are not present. PCR analysis shows that this isoform is most abundant in the liver, kidney, and pancreas. In HepG2 cells, overexpressed AFX zeta induces reporter gene activity through the insulin-responsive sequences of the phosphoenolpyruvate carboxykinase (PEPCK), IGFBP-1, and G6Pase promoters. AFX zeta-mediated stimulation was repressed by insulin treatment, by bisperoxovanadate treatment, and by overexpression of constitutively active protein kinase B (PKB). Insulin treatment and PKB overexpression result in phosphorylation of AFX zeta. Furthermore, an AMP-activated protein kinase activator, represses AFX zeta-dependent reporter activation. Taken together, these findings suggest that AFX zeta is a downstream target of both the phosphatidylinositol 3-kinase/PKB insulin signaling pathway and an AMP-activated protein kinase-dependent pathway (Yang, 2002).
Because overexpression of the glucose-6-phosphatase catalytic subunit (G-6-Pase) in both type 1 and type 2 diabetes may contribute to the characteristic increased rate of hepatic glucose production, whether the insulin response unit (IRU) identified in the mouse G-6-Pase promoter is conserved in the human promoter was investigated. A series of human G-6-Pase-chloramphenicol acetyltransferase (CAT) fusion genes was transiently transfected into human HepG2 hepatoma cells, and the effect of insulin on basal CAT expression was analyzed. The results suggest that the IRU identified in the mouse promoter is conserved in the human promoter, but that an upstream multimerized insulin response sequence (IRS) motif that is only found in the human promoter appears to be functionally inactive. The G-6-Pase IRU comprises two distinct promoter regions, designated A and B. Region B contains an IRS, whereas region A acts as an accessory element to enhance the effect of insulin, mediated through region B, on basal G-6-Pase gene transcription. The accessory factor binding region A is hepatocyte nuclear factor-1, and the forkhead protein FKHR is a candidate for the insulin-responsive transcription factor binding region B (Ayala, 1999).
Cell death is regulated mainly through apoptosis. Deregulation of apoptosis has been associated with cancer, autoimmune diseases and degenerative disorders. Many cells, particularly those of the hematopoietic system, have a default program of cell death and survival that is dependent on the constant supply of survival signals. The Bcl-2 family, which has both pro- and anti-apoptotic members, plays a critical role in regulating cell survival. One family member, the Bcl-2 interacting mediator of cell death (Bim), contains only a protein-interaction motif known as the BH3 domain, allowing it to bind pro-survival Bcl-2 molecules, neutralizing their function. Disruption of the bim gene results in resistance to apoptosis following cytokine withdrawal in leukocytes, indicating that regulation of the pro-apoptotic activity of Bim is critical for maintenance of the default apoptotic program. Withdrawal of cytokine results in upregulation of Bim expression concomitant with induction of the apoptotic program in lymphocytes. Activation of the forkhead transcription factor FKHR-L1, implicated in regulation of apoptosis in T lymphocytes, is sufficient to induce Bim expression. A mechanism is proposed by which cytokines promote lymphocyte survival by inhibition of FKHR-L1, preventing Bim expression (Dijkers, 2000).
Insulin negatively regulates expression of the insulin-like growth factor binding protein 1 (IGFBP-1) gene by means of an insulin-responsive element (IRE) that also contributes to glucocorticoid stimulation of this gene. The Caenorhabditis elegans protein DAF-16 binds the IGFBP-1-IRE with specificity similar to that of the forkhead (FKH) factor(s) that act both to enhance glucocorticoid responsiveness and to mediate the negative effect of insulin at this site. In HepG2 cells, DAF-16 and its mammalian homologs, FKHR, FKHRL1, and AFX, activate transcription through the IGFBP-1.IRE; this effect is inhibited by the viral oncoprotein E1A, but not by mutants of E1A that fail to interact with the coactivator p300/CREB-binding protein (CBP). DAF-16 and FKHR can interact with both the KIX and E1A/SRC interaction domains of p300/CBP, as well as the steroid receptor coactivator (SRC). A C-terminal deletion mutant of DAF-16 that is nonfunctional in C. elegans fails to bind the KIX domain of CBP, fails to activate transcription through the IGFBP-1.IRE, and inhibits activation of the IGFBP-1 promoter by glucocorticoids. Thus, the interaction of DAF-16 homologs with the KIX domain of CBP is essential to basal and glucocorticoid-stimulated transactivation. Although AFX interacts with the KIX domain of CBP, it does not interact with SRC and does not respond to glucocorticoids or insulin. Thus, it is concluded that DAF-16 and FKHR act as accessory factors to the glucocorticoid response, by recruiting the p300/CBP/SRC coactivator complex to an FKH factor site in the IGFBP-1 promoter, which allows the cell to integrate the effects of glucocorticoids and insulin on genes that carry this site (Nasrin, 2000).
Paclitaxel is used to treat breast cancers, but the mechanisms by which it induces apoptosis are poorly understood. Consequently, the role of the FoxO transcription factors in determining cellular response to paclitaxel has been examined. Western blotting has revealed that in a panel of 9 breast cancer cell lines expression of FoxO1a and FoxO3a correlates with the expression of the pro-apoptotic FoxO target Bim, which is associated with paclitaxel-induced apoptosis. In paclitaxel sensitive MCF-7 cells, the already high basal levels of FoxO3a and Bim protein increases dramatically after drug treatment, as does Bim mRNA; this increase correlates with apoptosis induction. This was not observed in MDA-231 cells which expressed low levels of FoxOs and Bim. In MCF-7 cells, maximal induction of Bim promoter is dependent on a FoxO binding site, suggesting that FoxO3a is responsible for the transcriptional upregulation of Bim. Gene silencing experiments show that siRNA specific for FoxO3a reduces the levels of FoxO3a and Bim protein as well as inhibits apoptosis in paclitaxel-treated MCF-7 cells. Furthermore, siRNA specific for Bim reduces the levels of Bim protein and inhibits apoptosis in paclitaxel-treated MCF-7 cells. This is the first demonstration that upregulation of FoxO3a by paclitaxel can result in increased levels of Bim mRNA and protein which can be a direct cause of apoptosis in breast cancer cells (Sunters, 2003).
Insulin inhibits the expression of multiple genes in the liver containing an insulin response sequence (IRS) (CAAAA(C/T)AA), and protein kinase B (PKB) mediates this effect of insulin. Genetic studies in Caenorhabditis elegans indicate that daf-16, a forkhead/winged-helix transcription factor, is a major target of the insulin receptor-PKB signaling pathway. FKHR, a human homolog of daf-16, contains three PKB sites and is expressed in the liver. Reporter gene studies in HepG2 hepatoma cells show that FKHR stimulates insulin-like growth factor-binding protein-1 promoter activity through an IRS, and introduction of IRSs confers this effect on a heterologous promoter. Insulin disrupts IRS-dependent transactivation by FKHR, and phosphorylation of Ser-256 by PKB is necessary and sufficient to mediate this effect. Antisense studies indicate that FKHR contributes to basal promoter function and is required to mediate effects of insulin and PKB on promoter activity via an IRS. These results provide the first report that FKHR stimulates promoter activity through an IRS and that phosphorylation of FKHR by PKB mediates effects of insulin on gene expression. Signaling to FKHR-related forkhead proteins via PKB may provide an evolutionarily conserved mechanism by which insulin and related factors regulate gene expression (Guo, 1999).
The insulin response element (IRE) in the IGFBP-1 promoter, and in other gene promoters, contains a T(A/G)TTT motif essential for insulin inhibition of transcription. Studies presented here test whether FKHR may be the transcription factor that confers insulin inhibition through this IRE motif. Immunoblots using antiserum to the synthetic peptide FKHR413-430, RNase protection, and Northerns blots show that FKHR is expressed in HEP G2 human hepatoma cells. Southwestern blots, electromobility shift assays, and DNase I protection assays show that Escherichia coli-expressed GST-FKHR binds specifically to IREs from the IGFBP-1, PEPCK and TAT genes; however, unlike HNF3beta, another protein proposed to be the insulin regulated factor, GST-FKHR does not bind the insulin unresponsive G/C-A/C mutation of the IGFBP-1 IRE. When HEP G2 cells were cotransfected with FKHR expression vectors and with IGFBP-1 promoter plasmids containing either native or mutant IREs, FKHR expression induced a 5-fold increase in activity of the native IGFBP-1 promoter but no increase in activity of promoter constructs containing insulin unresponsive IRE mutants. These data suggest that FKHR, and/or a related family member, is the important T(G/A)TTT binding protein that confers the inhibitory effect of insulin on gene transcription (Durham, 1999).
Winged helix/forkhead (Fox) transcription factors have been implicated in the regulation of a number of insulin-responsive genes. The insulin response elements (IREs) of the phosphoenolpyruvate carboxykinase (PEPCK) and insulin-like growth factor-binding protein-1 (IGFBP-1) genes bind members of the FKHR and HNF3 subclasses of Fox proteins. Mutational analyses of the PEPCK and IGFBP-1 IREs reveal mutations that do not affect the binding of HNF3 proteins to these elements but do eliminate the ability of the IREs to mediate an insulin response. This dissociation of binding and function provides compelling evidence that HNF3 proteins, per se, are not insulin response proteins. The same approach was used here to determine if FKHRL1, a member of the FKHR subclass of Fox proteins, binds to the PEPCK and IGFBP-1 IREs in a manner that correlates with the ability of these elements to mediate an insulin response. Overexpression of FKHRL1 stimulates transcription from transfected reporter constructs that contain a multimerized PEPCK IRE or an IGFBP-1 IRE and this stimulation is repressed by insulin. There is a direct correlation between the ability of mutant versions of the PEPCK and IGFBP-1 IREs to bind FKHRL1 and their ability to mediate FKHRL1-induced transcription when FKHRL1 is overexpressed. However, under conditions where FKHRL1 is not overexpressed, there is a lack of correlation between FKHRL1 binding to mutant versions of the PEPCK and IGFBP-1 IREs and the ability of these elements to mediate an insulin response. Therefore, the PEPCK and IGFBP-1 IREs mediate FKHRL1-induced transcription and its inhibition by insulin when this protein is overexpressed, but at the normal cellular concentration of FKHRL1 the insulin response mediated by these elements must involve another protein (Hall, 2000).
The forkhead rhabdomyosarcoma transcription factor (FKHR) is a promising candidate to be the transcription factor that binds to the insulin response element of the insulin-like growth factor-binding protein-1 (IGFBP-1) promoter and mediates insulin inhibition of IGFBP-1 promoter activity. Cotransfection of mouse FKHR increases IGFBP-1 promoter activity 2-3-fold in H4IIE rat hepatoma cells; insulin inhibits FKHR-stimulated promoter activity approximately 70%. A C-terminal fragment of mouse FKHR (residues 208-652) that contains the transcription activation domain fused to a Gal4 DNA binding domain potently stimulated Gal4 promoter activity. Insulin inhibits FKHR fragment-stimulated promoter activity by approximately 70%. Inhibition is abolished by coincubation with the phosphatidylinositol-3 kinase inhibitor, LY294002. The FKHR 208-652 fragment contains two consensus sites for phosphorylation by protein kinase B (PKB)/Akt, Ser-253 and Ser-316. Neither site is required for insulin inhibition of promoter activity stimulated by the FKHR fragment, and overexpression of Akt does not inhibit FKHR fragment-stimulated Gal4 promoter activity. These results suggest that insulin- and phosphatidylinositol-3 kinase-dependent phosphorylation of another site in the fragment by a kinase different from PKB/Akt inhibits transcription activation by the fragment. Phosphorylation of this site also may be involved in insulin inhibition of transcription activation by full-length FKHR, but only after phosphorylation of Ser-253 by PKB/Akt (Tomizawa, 2000).
Glucose-6-phosphatase plays an important role in the regulation of hepatic glucose production, and insulin suppresses glucose-6-phosphatase gene expression. Recent studies indicate that protein kinase B and Forkhead proteins contribute to insulin-regulated gene expression in the liver. The role has been examined of protein kinase B and Forkhead proteins in mediating effects of insulin on glucose-6-phosphatase promoter activity. Transient transfection studies with reporter gene constructs demonstrate that insulin suppresses both basal and dexamethasone/cAMP-induced activity of the glucose-6-phosphatase promoter in H4IIE hepatoma cells. Both effects are partially mimicked by coexpression of protein kinase Balpha. Coexpression of the Forkhead transcription factor FKHR stimulates the glucose-6-phosphatase promoter activity via interaction with an insulin response unit (IRU), and this activation is suppressed by protein kinase B. Coexpression of a mutated form of FKHR that cannot be phosphorylated by protein kinase B abolishes the regulation of the glucose-6-phosphatase promoter by protein kinase B and disrupts the ability of insulin to regulate the glucose-6-phosphatase promoter via the IRU. Mutation of the insulin response unit of the glucose-6-phosphatase promoter also prevents the regulation of promoter activity by FKHR and protein kinase B but only partially impairs the ability of insulin to suppress both basal and dexamethasone/cAMP-stimulated promoter function. Taken together, these results indicate that signaling by protein kinase B to Forkhead proteins can account for the ability of insulin to regulate glucose-6-phosphatase promoter activity via the IRU and that other mechanisms that are independent of the IRU, protein kinase B, and Forkhead proteins also are important in mediating effects of insulin on glucose-6-phosphatase gene expression (Schmoll, 2000).
The transcription factor Foxo1 controls the expression of genes involved in fundamental cellular processes. In keeping with its important physiological roles, Foxo1 activity is negatively regulated in response to growth factors and cytokines that activate a phosphatidylinositol 3-kinase (PI 3-kinase) protein kinase B (PKB)/Akt pathway. PKB/Akt-mediated phosphorylation of Foxo1 has been shown to result in the inhibition of target gene transcription and to trigger the export of Foxo1 from the nucleus, which is generally believed to explain the subsequent decrease of transcription. In the present study, using a chimeric protein in which a C-terminal fragment of Foxo1 (amino acids 208-652) containing the transactivation domain is fused to the yeast Gal4 DNA binding domain, evidence is presented showing that insulin can directly regulate transactivation by Foxo1 in H4IIE rat hepatoma cells. Insulin inhibition of Foxo1-(208-652)-stimulated transactivation is mediated by PI 3-kinase but in contrast to full-length Foxo1, does not require either of the two PKB/Akt phosphorylation sites (Ser253 and Ser316) present in the protein fragment. Using mutational and deletion studies, two potential phosphorylation sites, Ser319 and Ser499, as well as a 15-amino acid region located between residues 350 and 364, are identified that are critical for insulin inhibition of transactivation by Foxo1-(208-652). It is concluded that the transcriptional activity of Foxo1 is regulated at different levels by insulin: transactivation, as well as DNA binding and nuclear exclusion. These different regulatory mechanisms allow the precise control of transcription of Foxo1 target genes by insulin (Perrot, 2003).
Peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1) plays a major role in mediating hepatic gluconeogenesis in response to starvation, during which PGC-1 is induced by the cyclic AMP response element binding protein. Although it is observed that insulin counteracts PGC-1 transcription, the mechanism by which insulin suppresses the transcription of PGC-1 is still unclear. Forkhead transcription factor FKHR is shown to contribute to mediating the effects of insulin on PGC-1 promoter activity. Reporter assays demonstrate that insulin suppresses the basal PGC-1 promoter activity and coexpression of PKB mimics the effect of insulin in HepG2 cells. Insulin response sequences (IRSs) are addressed in the PGC-1 promoter as the direct target for FKHR in vivo. Coexpression of FKHR stimulates the PGC-1 promoter activity via interaction with the IRSs, while coexpression of FKHR (3A), in which the three putative PKB sites in FKHR are mutated, mainly abolishes the suppressive effect of PKB. Whereas deletion of the IRSs prevents the promoter stimulation by FKHR, that activity is still partially inhibited by insulin. These results indicate that signaling via PKB to FKHR can partly account for the effect of insulin to regulate the PGC-1 promoter activity via the IRSs (Daitoku, 2003).
The FOXO family of forkhead transcription factors stimulates the transcription of target genes involved in many fundamental cell processes, including cell survival, cell cycle progression, DNA repair, and insulin sensitivity. The activity of FOXO proteins is principally regulated by activation of protein kinase B (PKB)/Akt by insulin and other cytokines. PKB/Akt phosphorylates three consensus sites in FOXO proteins, leading to their export from the nucleus and the inhibition of FOXO-stimulated transcription. It has been widely accepted that the decreased transcription results from reduced abundance of FOXO proteins in the nucleus. In the present study Leu375 was mutated to alanine in the nuclear export signal of Foxo1 (mouse FOXO1), so that Foxo1 would remain in the nucleus of H4IIE rat hepatoma cells after insulin treatment. Would insulin still inhibit transcription stimulated by the Foxo1 mutant? Despite the retention of the Foxo1 mutant in the nucleus, insulin inhibits L375A-Foxo1-stimulated transcription to the same extent as transcription stimulated by wild-type Foxo1. Similar results were obtained using reporter plasmids containing the rat IGF-binding protein-1 promoter or a minimal promoter with three copies of the insulin response element to which FOXO proteins bind. It was concluded that insulin can inhibit Foxo1-stimulated transcription even when nuclear export of Foxo1 is prevented, indicating that insulin inhibition can occur by direct mechanisms that do not depend on altering the subcellular distribution of the transcription factor (Tsai, 2003).
The forkhead factor Foxo1 (or FKHR) was identified in a yeast two-hybrid screen as a peroxisome proliferator-activated receptor (PPAR) gamma-interacting protein. Foxo1 antagonized PPARgamma activity and vice versa indicating that these transcription factors functionally interact in a reciprocal antagonistic manner. One mechanism by which Foxo1 antagonizes PPARgamma activity is through disruption of DNA binding; Foxo1 inhibits the DNA binding activity of a PPARgamma/retinoid X receptor alpha heterodimeric complex. The Caenorhabditis elegans nuclear hormone receptor, DAF-12, interacts with the C. elegans forkhead factor, DAF-16, paralleling the interaction between PPARgamma and Foxo1. daf-12 and daf-16 have been implicated in C. elegans insulin-like signaling pathways, and PPARgamma and Foxo1 likewise have been linked to mammalian insulin signaling pathways. These results suggest a convergence of PPARgamma and Foxo1 signaling that may play a role in insulin action and the insulinomimetic properties of PPARgamma ligands. A more general convergence of nuclear hormone receptor and forkhead factor pathways may be important for multiple biological processes and this convergence may be evolutionarily conserved (Dowell, 2003).
Recent studies have suggested that the protection of cell apoptosis by AKT involves phosphorylation and inhibition of FKHR and related FOXO forkhead transcription factors and that androgens provide an AKT-independent cell survival signal in prostate cancer cells. This study reports receptor-dependent repression of FKHR function by androgens in prostate cancer cells. Transcriptional analysis demonstrates that activation of the androgen receptor caused an inhibition of both wild-type FKHR and a mutant in which all three known AKT sites were mutated to alanines, showing that the repression is AKT independent. In vivo and in vitro coprecipitation studies demonstrate that the repression is mediated through protein-protein interaction between FKHR and the androgen receptor. Mapping analysis localized the interacting domains to the carboxyl terminus between amino acids 350 and 655 of FKHR and to the amino-terminal A/B region and the ligand binding domain of the receptor. Further analysis demonstrates that the activated androgen receptor blocks FKHR's DNA binding activity and impairs its ability to induce Fas ligand expression and prostate cancer cell apoptosis and cell cycle arrest. These studies identify a new mechanism for androgen-mediated prostate cancer cell survival that appears to be independent of the activity of the receptor on androgen response element-mediated transcription and establish FKHR and related FOXO forkhead proteins as important nuclear targets for both AKT-dependent and -independent survival signals in prostate cancer cells (Li, 2003).
Smooth muscle cells (SMCs) modulate their phenotype between proliferative and differentiated states in response to physiological and pathological cues. Insulin-like growth factor-I stimulates differentiation of SMCs by activating phosphoinositide-3-kinase (PI3K)-Akt signaling. Foxo forkhead transcription factors act as downstream targets of Akt and are inactivated through phosphorylation by Akt. Foxo4 represses SMC differentiation by interacting with and inhibiting the activity of myocardin, a transcriptional coactivator of smooth muscle genes. PI3K/Akt signaling promotes SMC differentiation, at least in part, by stimulating nuclear export of Foxo4, thereby releasing myocardin from its inhibitory influence. Accordingly, reduction of Foxo4 expression in SMCs by siRNA enhances myocardin activity and SMC differentiation. It is concluded that signal-dependent interaction of Foxo4 with myocardin couples extracellular signals with the transcriptional program for SMC differentiation (Liu, 2005).
The Forkhead transcription factors AFX, FKHR and FKHR-L1 are orthologs of DAF-16, a Forkhead factor that regulates longevity in Caenorhabditis elegans. Overexpression of these Forkhead transcription factors causes growth suppression in a variety of cell lines, including a Ras-transformed cell line and a cell line lacking the tumor suppressor PTEN. Expression of AFX blocks cell-cycle progression at phase G1, independent of functional retinoblastoma protein (pRb) but dependent on the cell-cycle inhibitor p27kip1. Indeed, AFX transcriptionally activates p27kip1, resulting in increased protein levels. It is concluded that AFX-like proteins are involved in cell-cycle regulation and that inactivation of these proteins is an important step in oncogenic transformation (Medema, 2000).
Cell cycle progression is a process that is tightly controlled by internal and external signals. Environmental cues, such as those provided by growth factors, activate early signals that promote cell cycle entry. Cells that have progressed past the restriction point become independent of growth factors, and cell cycle progression is then controlled endogenously. The phosphatidylinositol 3OH kinase (PI(3)K)/protein kinase B (PKB) pathway must be activated in G1 to inactivate forkhead transcription factors (FKH-TFs) and allow cell cycle entry. Subsequent attenuation of the PI(3)K/PKB pathway is required to allow transcriptional activation of FKH-TF in G2. FKH-TF activity in G2 controls mammalian cell cycle termination, since interference with FKH transcriptional activation by disrupting PI(3)K/PKB downregulation, or by expressing a transcriptionally inactive FKH mutant, induces cell accumulation in G2/M, defective cytokinesis, and delayed transition from M to G1 of the cell cycle. FKH-TFs regulate expression of mitotic genes such as cyclin B and polo-like kinase (Plk). These results support the important role of forkhead in the control of mammalian cell cycle completion, and suggest that efficient execution of the mitotic program depends on downregulation of PI(3)K/PKB and consequent induction of FKH transcriptional activity (Alvarez, 2001).
The Forkhead factors AFX (FOXO4) and FKHR-L1 (FOXO3a) directly control transcription of the retinoblastoma-like p130 protein and cause upregulation of p130 protein expression. Detailed analysis of p130 regulation demonstrates that following Forkhead-induced cell cycle arrest, cells enter G0 and become quiescent. This is shown by a change in phosphorylation of p130 to G0-specific forms and increased p130/E2F-4 complex formation. Most importantly, long-term Forkhead activation causes a sustained but reversible inhibition of proliferation without a marked increase in apoptosis. As for the activity of the Forkheads, protein levels of p130 are controlled by endogenous PI3K/PKB signaling upon cell cycle reentry. Surprisingly, not only nontransformed cells, but also cancer cells such as human colon carcinoma cells, are forced into quiescence by Forkhead activation. It is therefore proposed that Forkhead inactivation by PKB signaling in quiescent cells is a crucial step in cell cycle reentry and contributes to the processes of transformation and regeneration (Kops, 2002a).
The FoxO forkhead transcription factors FoxO4 (AFX), FoxO3a (FKHR.L1), and FoxO1a (FKHR) represent important physiological targets of phosphatidylinositol-3 kinase (PI3K)/protein kinase B (PKB) signaling. Overexpression or conditional activation of FoxO factors is able to antagonize many responses to constitutive PI3K/PKB activation including its effect on cellular proliferation. The FoxO-induced cell cycle arrest is partially mediated by enhanced transcription and protein expression of the cyclin-dependent kinase inhibitor p27(kip1). A p27(kip1)-independent mechanism has been identified that plays an important role in the antiproliferative effect of FoxO factors. Forced expression or conditional activation of FoxO factors leads to reduced protein expression of the D-type cyclins D1 and D2 and is associated with an impaired capacity of CDK4 to phosphorylate and inactivate the S-phase repressor pRb. Downregulation of D-type cyclins involves a transcriptional repression mechanism and does not require p27(kip1) function. Ectopic expression of cyclin D1 can partially overcome FoxO factor-induced cell cycle arrest, demonstrating that downregulation of D-type cyclins represents a physiologically relevant mechanism of FoxO-induced cell cycle inhibition (Schmidt, 2002).
The insulin-like growth factor I (IGF-I) stimulates muscle satellite cell proliferation. IGF-I-stimulated proliferation of primary satellite cells is associated with the activation of phosphatidylinositol 3'-kinase (PI3K)/Akt and the downregulation of a cell-cycle inhibitor p27Kip1. To understand mechanisms by which IGF-I signals the downregulation of p27Kip1 in rat skeletal satellite cells, the role of Forkhead transcription factor FoxO1 in transcriptional activity of p27Kip1 was examined. When primary rat satellite cells are transfected with a p27Kip1 promoter-reporter gene construct, IGF-I inhibits specific p27Kip1 promoter activity. Addition of LY294002, an inhibitor of PI3K, reverses the IGF-I-mediated downregulation of p27Kip1 promoter activity. Co-transfection of wild type (WT) FoxO1 into satellite cells increases p27Kip1 promoter activity in the absence of IGF-I supplementation. Addition of IGF-I reverses the induction of p27Kip1 promoter activity by WT FoxO1. When a mutated FoxO1 (without Thr24, Ser256, and Ser316 Akt phosphorylation sites) is used, IGF-I is no longer able to reverse the FoxO1 induced stimulation of p27Kip1 promoter activity that is seen when WT FoxO1 is present. When the satellite cells are treated with IGF-I, phosphorylation of Akt-Ser473 and FoxO1-Ser256 is increased. In addition, when the cells are pre-incubated with LY294002 before IGF-I stimulation, the phosphorylation of Akt-Ser473 and FoxO1-Ser256 is inhibited, implying that phosphorylation of Akt and FoxO1 is downstream of IGF-I-induced PI3K signaling. However, IGF-I does not induce phosphorylation of FoxO1 on residues Thr24 and Ser316. These results suggest that IGF-I induces the phosphorylation of Ser256 and inactivates FoxO1, thereby downregulating the activation of the p27Kip1 promoter. Thus, inactivation of FoxO1 by IGF-I plays a critical role in rat skeletal satellite cell proliferation through regulation of p27Kip1 expression (Machida, 2003).
Cytochrome P450-derived epoxyeicosatrienoic acids (EETs) stimulate endothelial cell proliferation and angiogenesis. The involvement of the FOXO family of transcription factors and their downstream target p27Kip1 has been investigated in EET-induced endothelial cell proliferation. Incubation of human umbilical vein endothelial cells with 11,12-EET induces a time- and dose-dependent decrease in p27Kip1 protein expression, whereas p21Cip1 is not significantly affected. This effect on p27Kip1 protein is associated with decreased mRNA levels as well as p27Kip1 promoter activity. 11,12-EET also stimulates the time-dependent phosphorylation of Akt and of the forkhead factors FOXO1 and FOXO3a, effects prevented by the phosphatidylinositol 3-kinase inhibitor LY 294002. Transfection of endothelial cells with either a dominant-negative or an 'Akt-resistant'/constitutively active FOXO3a mutant reverses the 11,12-EET-induced down-regulation of p27Kip1, whereas transfection of a constitutive active Akt decreases p27Kip1 expression independent of the presence or absence of 11,12-EET. To determine whether these effects are involved in EET-induced proliferation, endothelial cells were transfected with the 11,12-EET-generating epoxygenase CYP2C9. Transfection of CYP2C9 elicits endothelial cell proliferation and this effect is inhibited in cells co-transfected with CYP2C9 and either a dominant-negative Akt or constitutively active FOXO3a. However, reducing FOXO expression using RNA interference attenuates p27Kip1 expression and stimulates endothelial cell proliferation. These results indicate that EET-induced endothelial cell proliferation is associated with the phosphatidylinositol 3-kinase/Akt-dependent phosphorylation and inactivation of FOXO factors and the subsequent decrease in expression of the cyclin-dependent kinase inhibitor p27Kip1 (Potente, 2003).
FoxO Forkhead transcription factors have been shown to act as signal transducers at the confluence of Smad, PI3K, and FoxG1 pathways. Smad proteins activated by TGF-ß form a complex with FoxO proteins to turn on the growth inhibitory gene p21Cip1. This process is negatively controlled by the PI3K pathway, a known inhibitor of FoxO localization in the nucleus, and by the telencephalic development factor FoxG1, which binds to FoxO-Smad complexes and blocks p21Cip1 expression. It is suggested that the activity of this network confers resistance to TGF-ß-mediated cytostasis during the development of the telencephalic neuroepithelium and in glioblastoma brain tumor cells (Seoane, 2004).
Activation of the transcription factor FKHR in various established cell lines induces cell cycle arrest followed by apoptosis. These effects are inhibited through activation of the phosphatidylinositol 3-kinase/Akt pathway, resulting in FKHR phosphorylation and its export from the nucleus, thus blocking its pro-apoptotic activity. FKHR regulates fusion of differentiating primary myoblasts. FKHR is localized in the cytoplasm of proliferating myoblasts, yet translocates to the nucleus by a phosphorylation-independent pathway following serum starvation, a condition that induces myoblast differentiation. FKHR phosphorylation during terminal differentiation appears to downregulate its fusion activity; a dominant-active non-phosphorylatable FKHR mutant dramatically augments the rate and extent of myotube fusion. However, this FKHR mutant exerts its effects only after other events initiated the differentiation process. Conversely, enforced expression of a dominant-negative FKHR mutant blocks myotube formation whereas wild-type FKHR has no effect. It is concluded that in addition to the role of FoxO proteins in regulating cell cycle progress and apoptosis, FKHR controls the rate of myotube fusion during myogenic differentiation (Bois, 2003).
Insulin-like growth factors promote myoblast differentiation through phosphoinositol 3-kinase and Akt signaling. Akt substrates required for myogenic differentiation are unknown. Forkhead transcription factors of the forkhead box gene, group O (Foxo) subfamily are phosphorylated in an insulin-responsive manner by phosphatidylinositol 3-kinase-dependent kinases. Phosphorylation leads to nuclear exclusion and inactivation. A constitutively active Foxo1 mutant inhibits differentiation of C2C12 cells and prevents myotube differentiation induced by constitutively active Akt. In contrast, a transcriptionally inactive mutant Foxo1 partially rescues inhibition of C2C12 differentiation mediated by wortmannin, but not by rapamycin, and is able to induce aggregation-independent myogenic conversion of teratocarcinoma cells. Inhibition of Foxo expression by siRNA results in more efficient differentiation, associated with increased myosin expression. These observations indicate that Foxo proteins are key effectors of Akt-dependent myogenesis (Hribal, 2003).
Foxo transcription factors have been implicated in diverse biological processes, including metabolism, cellular stress responses, and aging. Foxo3a minus female mice exhibit a distinctive ovarian phenotype of global follicular activation leading to oocyte death, early depletion of functional ovarian follicles, and secondary infertility. Foxo3a thus functions at the earliest stages of follicular growth as a suppressor of follicular activation. In addition to providing a molecular entry point for studying the regulation of follicular growth, these results raise the possibility that accelerated follicular initiation plays a role in premature ovarian failure, a common cause of infertility and premature aging in women (Castrillon, 2003).
Activated phosphoinositide 3-kinase (PI3K)-AKT signaling appears to be an obligate event in the development of cancer. The highly related members of the mammalian FoxO transcription factor family, FoxO1, FoxO3, and FoxO4, represent one of several effector arms of PI3K-AKT signaling, prompting genetic analysis of the role of FoxOs in the neoplastic phenotypes linked to PI3K-AKT activation. While germline or somatic deletion of up to five FoxO alleles produced remarkably modest neoplastic phenotypes, broad somatic deletion of all FoxOs engendered a progressive cancer-prone condition characterized by thymic lymphomas and hemangiomas, demonstrating that the mammalian FoxOs are indeed bona fide tumor suppressors. Transcriptome and promoter analyses of differentially affected endothelium identified direct FoxO targets and revealed that FoxO regulation of these targets in vivo is highly context-specific, even in the same cell type. Functional studies validated Sprouty2 and PBX1, among others, as FoxO-regulated mediators of endothelial cell morphogenesis and vascular homeostasis (Paik, 2007).
Phenotypic characterization shows that FoxO family functions in normal tissue homeostasis and cancer suppression are not only lineage-restricted, but also organ-specific. To gain additional insights into the mechanistic basis for such specificity, comparative transcriptome analyses was conducted of purified lung and liver ECs following pI-pC treatment of age-matched Mx-Cre+ and Mx-Cre− mice (The Mx-Cre transgene achieves widespread somatic FoxO deletion in adult tissues). It was hypothesized that normalization against phenotypically unaffected lung ECs would provide an effective biological filter for identifying physiologically relevant FoxO targets versus secondary/bystander transcriptional events—the latter would include genes whose expression responds to FoxO regulation but who do not play a rate-limiting role in the observed phenotypes, as well as those whose expression might be altered by activation of Cre expression during FoxO deletion. Transcriptome profiles of liver ECs with and without functional FoxOs were compared with those of lung ECs to generate a list of 138 significantly differentially expressed genes—89 of which were upregulated and 49 downregulated in liver, but not in lung, ECs upon documented FoxO . Consistent with the observed in vivo phenotypes, several differentially expressed genes have validated roles in EC biology, angiogenesis, and tissue morphogenesis, such as XLKD1 (LYVE-1), VCAM1, angiopoietin-like 4 (ANGPTL4), adrenomedullin (ADM), thrombospondin1 (THBS1), and ID1, and extracellular matrix proteins such as fibrillin (FBN1) (Paik, 2007).
Next, it was reasoned that the identification of FoxO binding elements (BEs) in differentially expressed genes would provide more direct insights into FoxO's actions in the observed phenotypes. To that end, a systematic in silico analysis was conducted of the regulatory regions of these 138 genes to ascertain the presence of evolutionarily conserved FoxO consensus BEs. For each gene, the −8 kb to +2 kb region surrounding the transcription start site and the 0 to +5 kb region downstream of the transcription end site in the mouse genome was surveyed. A position-specific weight matrix (PWM) based on evolutionary conservation of canonical insulin-regulated FoxO targets (IGFBP1, G6PD, PEPCK) was constucted to characterize the FoxO binding motif (consensus = BBTRTTTTD). Potential FoxO BEs were filtered further by cross-species conservation with two independent methodologies. BEs that could be identified in mouse, human, and at least one other species were designated as a “3-species conserved” BE. By requiring at least one evolutionary conserved FoxO BE predicted by both methods, 21 putative direct targets of FoxO family were identified in liver ECs, 9 of which were downmodulated and 12 upmodulated upon FoxO deletion. Interestingly, several of these genes are highly relevant to cancer (e.g., TCF4), vascular biology (e.g., CTGF), or both, such as ID1 and ADM, two factors known to play critical roles in EC survival and to promote angiogenesis during development and tumorigenesis (Paik, 2007).
To validate the computational approach, attempts were made to document direct binding of FoxOs on predicted BEs by chromatin immunoprecipitation (ChIP) and to confirm expression modulation by quantitative RT-PCR and RISH in vitro and in vivo, respectively. Sprouty2 was verified as a direct FoxO target. First, using a mixture of anti-FoxO1/3/4 antibodies, DNA fragments spanning FoxO BEs from both the proximal and distal regions of Sprouty2 gene were more efficiently coimmunoprecipitated in the FoxO-expressing liver ECs versus those deficient for the FoxOs. Next, it was documented that Sprouty2 was significantly and reproducibly downregulated in independently derived Mx-Cre+ liver ECs, but not in Mx-Cre+ lung ECs. Mindful of the potential artifact introduced by culturing EC in vitro, RISH was performed in tissue sections to confirm that Sprouty2 mRNA levels were comparable in Mx-Cre− and Mx-Cre+ lung EC but were significantly downmodulated in vascular beds of affected tissues, including liver, skeletal muscle, and uterus, in Mx-Cre+ mice 3 weeks after treatment with pI-pC (Paik, 2007).
For the other targets, similar validation studies were constructed. By RISH, 12 of 14 randomly selected putative targets exhibited transcriptional regulation by FoxO in vivo. Of note, Meis1 and Klf6, which did not confirm by RISH, were shown to be regulated by quantitative RT-PCR, pointing to the detection limits of RISH. Moreover, all 17 randomly selected putative targets (8 downmodulated and 9 upmodulated) showed expression modulation by quantitative RT-PCR in response to FoxO deletion in culture. Finally, all eight randomly selected putative targets (six downmodulated and two upmodulated) were validated to be true direct targets of FoxO by ChIP. Thus, eight of eight putative targets satisfied both ChIP and expression validation, indicating the robustness of the integrated computational-biological approach in the identification of direct targets of FoxOs in ECs in vivo (Paik, 2007).
The signaling pathway from phosphoinositide 3-kinase to the protein kinase Akt controls organismal life-span in invertebrates and cell survival and proliferation in mammals by inhibiting the activity of members of the FOXO family of transcription factors. Mammalian FOXO3a also functions at the G2 to M checkpoint in the cell cycle and triggers the repair of damaged DNA. By gene array analysis, FOXO3a was found to modulate the expression of several genes that regulate the cellular response to stress at the G2-M checkpoint. The growth arrest and DNA damage response gene Gadd45a appears to be a direct target of FOXO3a; Gadd45a mediates part of FOXO3a's effects on DNA repair. These findings indicate that in mammals FOXO3a regulates the resistance of cells to stress by inducing DNA repair and thereby may also affect organismal life-span (Tran, 2002).
Reactive oxygen species are required for cell proliferation but can also induce apoptosis. In proliferating cells this paradox is solved by the activation of protein kinase B (PKB; also known as c-Akt), which protects cells from apoptosis. By contrast, it is unknown how quiescent cells that lack PKB activity are protected against cell death induced by reactive oxygen species. The PKB-regulated Forkhead transcription factor FOXO3a (also known as FKHR-L1) protects quiescent cells from oxidative stress by directly increasing their quantities of manganese superoxide dismutase (MnSOD) messenger RNA and protein. This increase in protection from reactive oxygen species antagonizes apoptosis caused by glucose deprivation. In quiescent cells that lack the protective mechanism of PKB-mediated signalling, an alternative mechanism is induced as a consequence of PKB inactivity. This mechanism entails the activation of Forkhead transcription factors, the transcriptional activation of MnSOD and the subsequent reduction of reactive oxygen species. Increased resistance to oxidative stress is associated with longevity. The model of Forkhead involvement in regulating longevity stems from genetic analysis in Caenorhabditis elegans, and it is concluded that this model also extends to mammalian systems (Kops, 2002b).
Survival signals elicited by cytokines include the activation of phosphatidylinositol 3-kinase (PI3K), which in turn promotes the activation of protein kinase B (PKB). Recently, PKB has been demonstrated to phosphorylate and inactivate forkhead transcription factor FKHR-L1, a potent inducer of apoptosis. To explore the mechanisms underlying the induction of apoptosis after cytokine withdrawal or FKHR-L1 activation, a cell line was used in which FKHR-L1 activity could be specifically induced. Both cytokine withdrawal and FKHR-L1 activation induce apoptosis, which is preceded by an upregulation in p27KIP1 and a concomitant decrease in cells entering the cell cycle. Induction of apoptosis by both cytokine withdrawal and activation of FKHR-L1 correlates with the disruption of mitochondrial membrane integrity and cytochrome c release. This is preceded by upregulation of the pro-apoptotic Bcl-2 family member Bim. Ectopic expression of an inhibitory mutant of FKHR-L1 substantially reduces the levels of apoptosis observed after cytokine withdrawal. Activation of PKB alone is sufficient to promote cell survival, as measured by maintenance of mitochondrial integrity and the resultant inhibition of effector caspases. Furthermore, hematopoietic stem cells isolated from Bim-/- mice exhibit reduced levels of apoptosis upon inhibition of PI3K/PKB signaling. These data demonstrate that activation of FKHR-L1 alone can recapitulate all known elements of the apoptotic program normally induced by cytokine withdrawal. Thus PI3K/PKB--mediated inhibition of this transcription factor likely provides an important mechanism by which survival factors act to prevent programmed cell death (Dijkers, 2002).
Members of the FOXO family of mammalian forkhead transcription factors, including AFX, FKHRL1, and FKHR, are homologs of DAF-16, which regulates genes that contribute both to longevity and to resistance to various stresses (including oxidative stress) in Caenorhabditis elegans. Mouse myoblastic C2C12 cell lines have been generated in which expression of a constitutively active form of AFX (AFX-TM) is inducible by Cre-mediated recombination at loxP sites. Forced expression of AFX-TM is shown to block cell cycle progression at the G(1) and G(2) phases, and FOXO family members regulate the expression of stress-inducible genes such as GADD45. AFX and FKHRL1 each directly activate the GADD45 promoter through interaction with FOXO binding motifs. Oxidative stress activates the GADD45 promoter in a FOXO-dependent manner, resulting in an increased abundance of GADD45 mRNA and protein as well as G(2) arrest. These responses were evident in cells in which the tumor suppressor protein p53 is inactivated. These results suggest that the FOXO family of transcription factors plays an important role in the regulation of GADD45 in response to oxidative stress and thereby contributes to G(2)-M checkpoint (Furukawa-Hibi, 2002).
Developing sympathetic neurons die by apoptosis when deprived of NGF. BIM, a BH3-only member of the BCL-2 family, is induced after NGF withdrawal in these cells and contributes to NGF withdrawal-induced death. The involvement of the Forkhead box, class O (FOXO) subfamily of Forkhead transcription factors was examined in the regulation of BIM expression by NGF. Overexpression of FOXO transcription factors induces BIM expression and promotes death of sympathetic neurons in a BIM-dependent manner. In addition, FKHRL1 (FOXO3a) directly activates the bim promoter via two conserved FOXO binding sites and that mutation of these sites abolishes bim promoter activation after NGF withdrawal. Finally, it has been shown that FOXO activity contributes to the NGF deprivation-induced death of sympathetic neurons (Gilley, 2003).
A forkhead-type transcription factor, DAF-16, is located in the most downstream part of the insulin signalling pathway via PI3K (phosphoinositide 3-kinase). It is essential for the extension of life-span and is also involved in dauer formation induced by food deprivation in Caenorhabditis elegans. The present study addresses whether or not FOXO members AFX, FKHR (forkhead homolog in rhabdomyosarcoma) and FKHRL1 (FKHR-like protein 1), mammalian counterparts of DAF-16, are involved in starvation stress. A remarkable selective induction of FKHR and FKHRL1 transcripts was found in skeletal muscle of mice during starvation. The induction of FKHR gene expression was observed at 6 h after food deprivation, peaked at 12 h, and returned to the basal level by 24 h of refeeding. The induction was also found in skeletal muscle of mice with glucocorticoid treatment. Moreover, the levels of PDK4 (pyruvate dehydrogenase kinase 4) gene expression were up-regulated through the direct binding of FKHR to the promoter region of the gene in C2C12 cells. These results suggest that FKHR has an important role in the regulation of energy metabolism, at least in part, through the up-regulation of PDK4 gene expression in skeletal muscle during starvation (Furuyama, 2003).
In C. elegans, the transcription factor DAF-16 promotes longevity in response to reduced insulin/IGF-1 signaling or germline ablation. This study asks how different tissues interact to specify the lifespan of the animal. Several tissues act as signaling centers. In particular, DAF-16 activity in the intestine, which also comprises the animal's adipose tissue, completely restores the longevity of daf-16 minus germline-deficient animals, and increases the lifespans of daf-16 minus insulin/IGF-1-pathway mutants substantially. These findings indicate that DAF-16 may control two types of downstream signals: DAF-16 activity in signaling cells upregulates DAF-16 in specific responding tissues, possibly via regulation of insulin-like peptides, and also evokes DAF-16-independent responses. It is suggested that this network of tissue interactions and feedback regulation allows the tissues to equilibrate and fine-tune their expression of downstream genes, which, in turn, coordinates the rate of tissue aging within the animal (Libina, 2003).
The signaling pathway of insulin/insulin-like growth factor-1/phosphatidylinositol-3 kinase/Akt is known to regulate longevity as well as resistance to oxidative stress in the nematode Caenorhabditis elegans. This regulatory process involves the activity of DAF-16, a forkhead transcription factor. Although reduction-of-function mutations in components of this pathway have been shown to extend the lifespan in organisms ranging from yeast to mice, activation of Akt has been reported to promote proliferation and survival of mammalian cells. Akt activity has been shown to increase along with cellular senescence; inhibition of Akt extends the lifespan of primary cultured human endothelial cells. Constitutive activation of Akt promotes senescence-like arrest of cell growth via a p53/p21-dependent pathway, and inhibition of forkhead transcription factor FOXO3a by Akt is essential for this growth arrest to occur. FOXO3a influences p53 activity by regulating the level of reactive oxygen species. These findings reveal a novel role of Akt in regulating the cellular lifespan and suggest that the mechanism of longevity is conserved in primary cultured human cells and that Akt-induced senescence may be involved in vascular pathophysiology (Miyauchi, 2004).
To understand the role of FoxO family members in hematopoiesis, FoxO1, FoxO3, and FoxO4 were conditionally deleted in the adult hematopoietic system. FoxO-deficient mice exhibited myeloid lineage expansion, lymphoid developmental abnormalities, and a marked decrease of the lineage-negative Sca-1+, c-Kit+ (LSK) compartment that contains the short- and long-term hematopoietic stem cell (HSC) populations. FoxO-deficient bone marrow had defective long-term repopulating activity that correlated with increased cell cycling and apoptosis of HSC. Notably, there was a marked context-dependent increase in reactive oxygen species (ROS) in FoxO-deficient HSC compared with wild-type HSC that correlated with changes in expression of genes that regulate ROS. Furthermore, in vivo treatment with the antioxidative agent N-acetyl-L-cysteine resulted in reversion of the FoxO-deficient HSC phenotype. Thus, FoxO proteins play essential roles in the response to physiologic oxidative stress and thereby mediate quiescence and enhanced survival in the HSC compartment, a function that is required for its long-term regenerative potential (Tothova, 2007).
FoxO transcription factors play critical roles in cell cycle control and cellular stress responses, and abrogation of FoxO function promotes focus formation by Myc in vitro. Stable introduction of a dominant-negative FoxO moiety (dnFoxO) into Eµ-myc transgenic hematopoietic stem cells accelerates lymphoma development in recipient mice by attenuating Myc-induced apoptosis. When expressed in Eµ-myc; p53+/- progenitor cells, dnFoxO alleviates the pressure to inactivate the remaining p53 allele in upcoming lymphomas. Expression of the p53 upstream regulator p19Arf (alternative reading frame of p16INK, also called p14arf in humans and p19arf in mice) is virtually undetectable in most dnFoxO-positive Myc-driven lymphomas. It was found that FoxO proteins bind to a distinct site within the Ink4a/Arf locus and activate Arf expression. Moreover, constitutive Myc signaling induces a marked increase in nuclear FoxO levels and stimulates binding of FoxO proteins to the Arf locus. These data demonstrate that FoxO factors mediate Myc-induced Arf expression and provide direct genetic evidence for their tumor-suppressive capacity (Bouchard, 2007).
The FoxO subclass of forkhead-box transcription factors (consisting of FoxO1 (FKHR), FoxO3a (FKHRL1), FoxO4 (AFX), and FoxO6) regulates numerous cellular functions including proliferation, stress sensitivity, and survival; it has also been implicated in the regulation of organism life span. The members of this family activate gene expression via interaction with a specific DNA sequence, and known targets include the cell cycle regulating Kip1, the proapoptotic Bim, the DNA damage-responsive Gadd45a, and the oxidative stress-protective manganese superoxide dismutase genes. In addition, FoxO proteins can repress several cell cycle promoting genes (e.g., cyclin D1 and cyclin D2) in a manner that might be independent of direct DNA binding (Bouchard, 2007 and references therein).
In response to growth factor signaling and to oxidative stress, FoxO proteins are post-translationally modified by phosphorylation, acetylation, and ubiquitination; collectively, these modifications regulate FoxOs’ subcellular localization, transcriptional activity, and stability. Notably, all FoxO proteins are inhibited by protein kinase B/Akt-mediated phosphorylation that promotes their nuclear export and subsequent proteolytic degradation via ubiquitination by the SCFSkp2 complex. As a consequence, FoxO proteins mediate the induction of p27Kip1 and Bim expression in response to inhibition of the phosphatidylinositol-3-OH (PI3)-kinase/Akt pathway (Bouchard, 2007 and references therein).
Conditional codeletion of the FoxO1, FoxO3, and FoxO4 alleles uncovers a context-dependent cancer-prone phenotype characterized by thymic lymphomas forming in some and hemangiomas developing in most animals after a long latency, suggesting that FoxO proteins exert their tumor-suppressive capability in the presence of additional oncogenic mutations. In support of this view, Akt-mediated phosphorylation of FoxO proteins has been identified as the critical PI3-kinase signaling component that substitutes for oncogenic Ras in Myc-induced proliferation and focus formation in vitro. Furthermore, constitutive Akt signaling cooperates with Myc to accelerate B-cell lymphomagenesis; however, it remains unclear whether Akt-mediated phosphorylation of FoxO proteins contributes to Eµ-myc transgenic lymphoma formation in this setting (Bouchard, 2007).
Proapoptotic Arf/p53 signaling is known as the pivotal Myc-induced tumor-suppressive barrier. Eµ-myc transgenic mice lacking one p53 allele develop lymphomas that inactivate the remaining wild-type allele. Likewise, Eµ-myc; Arf+/- or Eµ-myc; Ink4a/Arf+/- mice produce tumors that lack expression of p19Arf. Primary Arf deletions protect cells from acquiring p53 mutations during lymphoma development. Similarly, introduction of strictly anti-apoptotic genes such as bcl2 or a dominant-negative form of caspase 9 into Eµ-myc; p53+/- hematopoietic stem cells alleviates the pressure to inactivate p53, thereby underscoring apoptosis as the critical p53-governed tumor suppressor function in Myc-driven lymphomagenesis (Bouchard, 2007).
Previous work has shown that p53 and FoxO3a share target genes and that FoxO3a can activate transcription via p53 sites, suggesting a potential collaboration of FoxO3a and p53 in tumor suppression. Although a direct interaction between FoxO3a and p53 proteins has been demonstrated under conditions of overexpression, the observed collaboration would be consistent with an as-yet-unidentified FoxO target acting upstream of p53. This study reports that FoxO factors elicit their tumor-suppressive potential as critical inducers of Arf during Myc-driven lymphomagenesis, providing further evidence for a close link between the FoxO and p53 tumor suppressor pathways (Bouchard, 2007).
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