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The importance of FOXO transcription factors in regulating different aspects of cellular homeostasis and apoptosis has become apparent. Akt/protein kinase B has been shown to phosphorylate and inactivate members of FOXO family of transcription factors. Akt and its upstream regulator, phosphatidylinositol-3 kinase (PI3K) are involved in rapid action of estrogen (E2) in different cells and tissues. The aim of the present study was to analyze the E2/PI3K/Akt/FOXO pathway in rat uterus. In response to E2, phosphorylation of Akt/PKB on Ser473 and FOXO1 on Ser256 and Thr24 residues increased but with distinct kinetics, regulating the activation and inactivation of Akt and FOXO1 proteins, respectively. The antiestrogen ICI 182,780 prevented E2 induced Akt activation suggesting that estrogen receptors mediate this effect of E2. Intrauterine injection of Wortmannin caused a decrease in the phosphorylation of Ser473 of Akt, and attenuated phosphorylation of its downstream target FOXO1 at Ser256 and at Thr24. However, the effect of E2 on phosphorylation of Thr24 showed a kinetic pattern distinct from that of Ser256. Our results suggest that the E2/PI3K/Akt/FOXO1 pathway in rat uterus is functioning even at the lack of ovarian hormones and responses to E2 treatment. Estradiol increases Akt phosphorylation through a Wortmannin sensitive way, presumably involving PI3K. The present work shows that PI3K plays a crucial role in the phosphorylation and inactivation of FOXO1 in vivo, indicating that the regulation of this transcription factor is a more complex event in uterine cells requiring further investigations.

The life span of the nematode Caenorhabditis elegans is under control of sensory signals detected by the amphid neurons. In these neurons, C. elegans expresses at least 13 Galpha subunits and a Ggamma subunit, which are involved in the transduction and modulation of sensory signals. Here, we show that loss-of-function mutations in the Galpha subunits odr-3, gpa-1 and gpa-9, in the Ggamma subunit gpc-1 and the introduction of extra copies of the Galpha subunit gpa-11 extend the life span of C. elegans. Loss-of-function of odr-3 and extra copies of gpa-11 act synergistically and can together extend life span more than two-fold, indicating that sensory signals play an important role in regulating life span. We show that gpa-1, gpa-11, odr-3 and gpc-1 all signal via the daf-16 FOXO family transcription factor. In addition, odr-3 and gpa-11 might suppress life span extension partially independent of the insulin/IGF-1 like receptor homologue daf-2. Our results suggest that the previously unanticipated nociceptive ASH and/or ADL neurons regulate longevity. We expect that the implication of specific G proteins will eventually contribute to the identification of the sensory cues that determine the rate of aging in C. elegans.

Summary To understand the role of FoxO family members in hematopoiesis, we conditionally deleted FoxO1 , FoxO3 , and FoxO4 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.

SummaryActivated 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.

Forkhead transcription factors of the FoxO family have important roles in cellular proliferation, apoptosis, differentiation and stress resistance. FoxO proteins also play important roles in metabolism of complex organisms. FoxO1 regulates glucose and lipid metabolism in liver, as well as preadipocyte, myoblast and vascular endothelial cell differentiation. In the hypothalamus, FoxO controls food intake. In this chapter, we review the role of FoxO in pancreatic beta cells. Pancreatic beta cells secrete insulin to maintain the plasma glucose levels in a strict physiological range. Defects of beta cell function cause diabetes. The expression pattern of FoxO1 during pancreatic organogenesis is similar to that of Pdx1, Nkx2.2 and Pax4, transcription factors known to be critical for beta cell development. FoxO1 is expressed in a subset of pancreatic duct cells, in which insulin and/or Pdx1 are occasionally expressed. FoxO1 inhibits beta cell proliferation through suppression of Pdx1 by competing with FoxA2 and protects against beta cell failure induced by oxidative stress through NeuroD and MafA induction. Thus, a series of FoxO1 studies in pancreas suggested that FoxO1 plays important roles in pancreatic beta cell differentiation, neogenesis, proliferation and stress resistance. Genetic or pharmacological manipulation of FoxO can be used to prevent beta cell failure or aid in the differentiation of uncommitted endocrine progenitors into beta cells for transplantation.

The Foxo subfamily of forkhead (Fox) transcription factors are mammalian homologues of the Caenorhabditis elegans DAF-16 longevity gene, and play key roles in cellular and organism survival, death, proliferation and metabolism. A growing body of evidence indicates that Foxo proteins furthermore play critical roles in immune cell homeostasis, modulating inflammation in some disease states such as inflammatory arthritis and systemic lupus erythematosus (SLE), via fundamental roles in T cells, B cells, neurophils and other myeloid lineages. This review summarizes current knowledge of the Foxo family members in general and in immunity, including their potential use as therapeutic targets.

Asthma is a chronic inflammatory disorder delineated by a heightened immunological response due to environmental or genetic factors. Single nucleotide polymorphism studies have shown that FOXO3a plays a pivotal role in maintaining immunoregulation. Polymorphism in FOXO3a has been linked to inflammatory diseases such as chronic obstructive pulmonary disease (COPD), Rheumatoid Arthritis, and Crohn’s disease suggesting that FOXO3a may be associated with asthma. Airway inflammation in asthma is characterized by activation of T helper type 2 (Th2) T cells and Foxo family members are reported to play critical roles in the suppression of T cell activation. Thus this study was undertaken to investigate an association between single nucleotide polymorphism of the FOXO3a (rs13217795, C>T transition) gene and asthma in Indian population. To our knowledge we are the first ones reporting an association between FOXO3a and asthma.

Comment on: Zou P, et al. Targeting FoxO1 with AS1842856 suppresses adipogenesis. Cell Cycle 2014; 13(23):3759–67; PMID: 25483084; http://dx.doi.org/10.4161/15384101.2014.965977 Adipose tissue is increasingly being recognized as a key regulator of whole-body energy homeostasis and consequently as a prime therapeutic target for metabolic syndrome. The differentiation and function of white adipocytes, which can store and release lipids and release hormones (adipokines), and brown adipocytes, which can “burn off” energy, are therefore under intensive study. FOXO1, a member of the Forkhead Box O (FOXO) family of transcription factors, plays an important role in both types of adipocytes. FoxO1+/− mice have smaller, “healthier” white adipocytes when fed a high-fat diet and are protected from developing type 2 diabetes, possibly through de-repression of the adipocyte transcription factor PPARγ1 In brown adipose tissue a dominant-negative form of FoxO1 enhances energy expenditure in vivo1 suggesting that inhibition of FoxO1 in white and/or brown adipose tissue may present an attractive therapeutic approach to maintain a healthy energy status. In a recent issue of Cell Cycle, Zou et al.2 now show in a series of in vitro experiments that FoxO1 phosphorylation status – which is inversely associated with its activity – displays a complex, wave-like pattern during adipocyte differentiation, and that inhibition of FoxO1 with the selective inhibitor AS1842856 can suppress differentiation, depending on the timing of addition (Fig. 1) Figure 1. FoxO1 phoshorylation and activity during 3T3-L1 adipogenesis, as described by Zou et al., indicated are the 3 stages of 3T3-L1 adipogenesis: growing cells to confluency, mitotic clonal expansion (MCE) and terminal differentiation. Active FoxO1 ... Short-term treatment of diabetic db/db mice with AS1842856 ameliorates fasting glycemia, possibly through inhibition of gluconeogenic genes in the liver2 Whether systemic inhibition of FoxO1 in vivo is a realistic therapeutic option warrants further studies, specifically to address the following questions. Firstly, expansion of white adipose tissue may prevent lipid storage in non-adipose organs (liver, muscle, pancreas) and adipose tissue inflammation, processes associated with insulin resistance and type 2 diabetes3 It therefore needs to be established whether AS1842856 suppresses white adipose tissue expansion and harms non-adipose organs. Secondly, FoxO1 inhibition could in principle result in reduced energy expenditure in brown adipose tissue (BAT), and cause weight gain and ultimately type 2 diabetes4,5 Thirdly, FoxO1 inhibition potentially reduces the expression of adiponectin, an adipokine implicated in maintaining a healthy energy balance6 Fourthly, although it has been suggested that AS1842856 has some specificity for inhibition of FoxO1 and not the other FoxO family members FoxO3 and FoxO4, this has to be further established in vivo. The latter is of high importance, as FOXO transcription factors play crucial roles in controlling fundamental cellular processes (e.g. cell cycle, cell death) and development, and both their negative and positive deregulation has been implicated in several pathologies including cancer, diabetes and stem cell depletion7 Because of these mixed effects of FOXO1 inactivation, both on metabolism and may be on other biological processes, the prime value of selective inhibitors like AS1842856 probably lies in their use as research tools. To dissect the exact role(s) of FoxO1 in whole body energy metabolism, WAT- and BAT-specific FoxO1 knock out mouse models will need to be developed to dissect BAT/WAT phenotypes from systemic metabolic responses evoked by other organs like liver.

Pro-proliferative and inflammatory signaling converge on FoxO1 transcription factor in pulmonary hypertension Savai et al Nat Medicine . 2014;20:1289–1300. > “…We think Android is very fragmented and becoming more fragmented by the day. We think integrated will trump fragmented every time…” > > Steve Jobs (on iPhone) Pulmonary arterial hypertension (PAH) is a mysterious killer that, like cancer, is characterized by tremendous complexity. A myriad of apparently unrelated molecular abnormalities have been described but, perhaps because of this complexity, the progress in PAH drug development suffers from poor bench-to-bedside translation.1 Savai et al,2 attacked the master transcription factor forkhead box O1 (FoxO1), arguing that targeting more proximal hubs that integrate many mechanisms in the PAH pathogenesis cascade will be more effective than attacking individual distal targets. It is not the first time that this has been attempted in PAH. Master transcription factors like nuclear factor of activated T-cell c2 (NFATc2),3 hypoxia inducible factor 1α (HIF-1α),4 and signal transducer and activator of transcription 3 (STAT3)5 have effectively been targeted in PAH models. Attempts to integrate this fragmented field, like the metabolic6 and inflammatory7 theories of PAH, have emerged. The Fox-containing proteins are a diverse family of ≥100 transcription factors that can integrate many cellular signals.8 Their diversity ensures their ability to fine tune gene transcription in health, development, and common diseases like cancer. The most studied, that is, the FoxO family, are bona fide tumor suppressors and are almost ubiquitously expressed.8 FoxO factors mediate G1 phase cell-cycle arrest and G2/M cell-cycle phase transition; they are also important in inflammation, metabolism, and vascular homeostasis (Figure). FoxOs are inhibited by phosphorylation that creates a binding motif for the 14-3-3 scaffolding proteins, causing FoxO1 exclusion from the nucleus, promoting their cytoplasmic ubiquination and degradation.8 Acetylation also attenuates …

Drosophila melanogaster accumulate large and small heat shock protein transcripts in response to oxidative damage. The large Hsp70 and the small Hsps are two different classes of molecular chaperones that protect against proteotoxicity by preventing the accumulation of mis-folded, non-native proteins using two separate mechanisms. Here we establish the small Hsps and Hsp70 are targets of Foxo in Drosophila. The Foxo family of pioneer transcription factors promotes cellular stress resistance in response to many conditions including oxidative stress. In response to acute oxidative stress caused by paraquat, Foxo and HSF collaborate by increasing expression of these chaperones. These data indicate a mechanism where overlapping pathways transcriptionally upregulate protective genes to promote survival.

The FOXO family of transcription factors has been implicated in several cellular processes including cell cycle arrest, cell death and protection from stress stimuli. FOXO function is influenced by multiple signaling pathways. Many of these pathways are known to be misregulated in cancer. Perturbation of FOXO function leads to uncontrolled cell proliferation and accumulation of DNA damage. It is becoming clear that active FOXO proteins play an important role in keeping cells in check and inactivation of FOXO proteins is associated with characteristics of cancer cells. FOXO proteins may represent new therapeutic targets for a broad spectrum of cancers.

Experiments utilizing RNA interference technology were performed to determine whether the forkhead transcription factor FOXO1A, a member of the FOXO family of proteins, plays a critical role in the induction of human uterine decidualization. Human decidual fibroblast cells were decidualized in vitro for 6 days with medroxyprogester-one, estradiol, and dibutyryl cAMP in the presence or absence of a highly specific FOXO1A small interfering RNA (siRNA) that inhibits FOXO1A mRNA and protein expression by more than 80%. RNA and proteins were extracted from the cells at 0, 2, 4, and 6 days. FOXO1A and IGFBP-1 proteins were determined by immunoblotting; and intracellular mRNA levels for several decidualization marker genes were determined by real-time PCR. Exposure of the cells to FOXO1A siRNA in five separate experiments resulted in a 40–75% inhibition of prolactin, IGFBP-1, tissue inhibitor of metalloproteinase 3 (TIMP3), somatostatin and endometrial bleeding-associated factor (EBAF) mRNAs, all of which are markedly induced during the decidualization process. In contrast, actin and GAPDH mRNA levels did not change during decidualization. The inhibition of mRNA levels was first noted at day 2 and persisted for the remainder of each experiment. Western blot analysis indicated that the FOXO1A siRNA inhibited IGFBP-1 protein expression by 60–80%. Decidual fibroblast cells exposed in an identical manner to a control RNA that had no effect on FOXO1A expression caused only a 0–15% inhibition of the marker genes and IGFBP-1 protein. Taken together, these findings strongly suggest a critical role for FOXO1A in the induction of human decidualization.

A wide range of human diseases, including cancer, has a striking age-dependent onset. However, the molecular mechanisms that connect aging and cancer are just beginning to be unraveled. FOXO transcription factors are promising candidates to serve as molecular links between longevity and tumor suppression. These factors are major substrates of the protein kinase Akt. In the presence of insulin and growth factors, FOXO proteins are relocalized from the nucleus to the cytoplasm and degraded via the ubiquitin-proteasome pathway. In the absence of growth factors, FOXO proteins translocate to the nucleus and upregulate a series of target genes, thereby promoting cell cycle arrest, stress resistance, or apoptosis. Stress stimuli also trigger the relocalization of FOXO factors into the nucleus, thus allowing an adaptive response to stress stimuli. Consistent with the notion that stress resistance is highly coupled with lifespan extension, activation of FOXO transcription factors in worms and flies increases longevity. Emerging evidence also suggests that FOXO factors play a tumor suppressor role in a variety of cancers. Thus, FOXO proteins translate environmental stimuli into changes in gene expression programs that may coordinate organismal longevity and tumor suppression.

Forkhead box O (Foxo) transcription factors are emerging as critical transcriptional integrators among pathways regulating differentiation, proliferation, and survival, yet the role of the distinct Foxo family members in angiogenic activity of endothelial cells and postnatal vessel formation has not been studied. Here, we show that Foxo1 and Foxo3a are the most abundant Foxo isoforms in mature endothelial cells and that overexpression of constitutively active Foxo1 or Foxo3a, but not Foxo4, significantly inhibits endothelial cell migration and tube formation in vitro. Silencing of either Foxo1 or Foxo3a gene expression led to a profound increase in the migratory and sprout-forming capacity of endothelial cells. Gene expression profiling showed that Foxo1 and Foxo3a specifically regulate a nonredundant but overlapping set of angiogenesis- and vascular remodeling–related genes. Whereas angiopoietin 2 (Ang2) was exclusively regulated by Foxo1, eNOS, which is essential for postnatal neovascularization, was regulated by Foxo1 and Foxo3a. Consistent with these findings, constitutively active Foxo1 and Foxo3a repressed eNOS protein expression and bound to the eNOS promoter. In vivo, Foxo3a deficiency increased eNOS expression and enhanced postnatal vessel formation and maturation. Thus, our data suggest an important role for Foxo transcription factors in the regulation of vessel formation in the adult.

Vascular smooth muscle cell (VSMC) proliferation and migration contribute significantly to atherosclerosis, postangioplasty restenosis, and transplant vasculopathy. Forkhead transcription factors belonging to the FoxO subfamily have been shown to inhibit growth and cell cycle progression in a variety of cell types. We hypothesized that forkhead proteins may play a role in VSMC biology. Under in vitro conditions, platelet-derived growth factor (PDGF)-BB, tumor necrosis factor-alpha, and insulin-like growth factor 1 stimulated phosphorylation of FoxO in human coronary artery smooth muscle cells via MEK1/2 and/or phosphatidylinositol 3-kinase-dependent signaling pathways. PDGF-BB, tumor necrosis factor-alpha, and insulin-like growth factor 1 treatment resulted in the nuclear exclusion of FoxO, whereas PDGF-BB alone down-regulated the FoxO target gene, p27(kip1), and enhanced cell survival and progression through the cell cycle. These effects were abrogated by overexpression of a constitutively active, phosphorylation-resistant mutant of the FoxO family member, TM-FKHRL1. The anti-proliferative effect of TM-FKHRL1 was partially reversed by small interfering RNA against p27(kip1). In a rat balloon carotid arterial injury model, adenovirus-mediated gene transfer of FKHRL1 caused an increase in the expression of p27(kip1) in the VSMC and inhibition of neointimal hyperplasia. These data suggest that FoxO activity inhibits VSMC proliferation and activation and that this signaling axis may represent a therapeutic target in vasculopathic disease states.

β-Catenin is a multifunctional protein that mediates Wnt signaling by binding to members of the T cell factor (TCF) family of transcription factors. Here, we report an evolutionarily conserved interaction of β-catenin with FOXO transcription factors, which are regulated by insulin and oxidative stress signaling. β-Catenin binds directly to FOXO and enhances FOXO transcriptional activity in mammalian cells. In Caenorhabditis elegans , loss of the β-catenin BAR-1 reduces the activity of the FOXO ortholog DAF-16 in dauer formation and life span. Association of β-catenin with FOXO was enhanced in cells exposed to oxidative stress. Furthermore, BAR-1 was required for the oxidative stress–induced expression of the DAF-16 target gene sod-3 and for resistance to oxidative damage. These results demonstrate a role for β-catenin in regulating FOXO function that is particularly important under conditions of oxidative stress.

The signaling pathway of insulin/insulin-like growth factor/phosphatidylinositol-3 kinase/Akt/forkhead transcription factors is known to control life span and senescence in organisms ranging from yeast to mice. The FOXO family of forkhead transcription factors, FOXO1, FOXO3a, and FOXO4, play a critical role in this signal transduction pathway. However, the impact of FOXO3a activation on life span of primary cultured human dermal fibroblasts (HDFs) is unknown. To investigate the role of FOXO3a in the regulation of cellular senescence, we prepared FOXO3a-siRNA stable HDFs. We found that the down-regulation of FOXO3a RNA and protein in HDFs induced many senescent phenotypes, including changes in cell morphology, increases in population doubling times, senescence-associated beta-galactosidase staining and the cellular reactive oxygen species, and up-regulation of p53/p21 protein expression. Our data provide evidence of the key role of FOXO3a transcription factor as a mediator of cellular senescence in HDFs, and suggest that the mechanism of senescence is conserved in HDFs.

Members of the Foxo family, Foxo1 (Fkhr), Foxo3 (Fkhrl1), and Foxo4 (Afx), are mammalian homologs of daf-16, which influences life span and energy metabolism in Caenorhabditis elegans. Mammalian FOXO proteins also play important roles in cell cycle arrest, apoptosis, stress resistance, and energy metabolism. In this study, we generated Foxo1-deficient mice to investigate the physiological role of FOXO1. The Foxo1-deficient mice died around embryonic day 11 because of defects in the branchial arches and remarkably impaired vascular development of embryos and yolk sacs. In vitro differentiation of embryonic stem cells demonstrated that endothelial cells derived from wild-type and Foxo1-deficient embryonic stem cells were able to produce comparable numbers of colonies supported by a layer of OP9 stromal cells. Although the morphology of the endothelial cell colonies was identical in both genotypes in the absence of exogenous vascular endothelial growth factor (VEGF), Foxo1-deficient endothelial cells showed a markedly different morphological response compared with wild-type endothelial cells in the presence of exogenous VEGF. These results suggest that Foxo1 is essential to the ability of endothelial cells to respond properly to a high dose of VEGF, thereby playing a critical role in normal vascular development.

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. We also show that nuclear export of FoxO6 is mediated through the phosphatidylinositol 3-kinase/PKB pathway. Furthermore, we show using a chimeric approach that we can fully restore the ability of FoxO6 to shuttle between nucleus and cytosol. 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.

An outstanding question in adipocyte biology is how hormonal cues are relayed to the nucleus to activate the transcriptional program that promotes adipogenesis. The forkhead transcription factor Foxo1 is regulated by insulin via Akt-dependent phosphorylation and nuclear exclusion. We show that Foxo1 is induced in the early stages of adipocyte differentiation but that its activation is delayed until the end of the clonal expansion phase. Constitutively active Foxo1 prevents the differentiation of preadipocytes, while dominant-negative Foxo1 restores adipocyte differentiation of fibroblasts from insulin receptor-deficient mice. Further, Foxo1 haploinsufficiency protects from diet-induced diabetes in mice. We propose that Foxo1 plays an important role in the integration of hormone-activated signaling pathways with the complex transcriptional cascade that promotes adipocyte differentiation.

High forkhead box protein 01 (Foxo1) activity in liver increases gluconeogenesis and hepatic glucose output; thus, Foxo1 is accused of contributing to the pathology of diabetes (for review, see Ref. 1). Until recently, other closely related members of the forkhead/wingedhelix family of transcriptional regulators, Foxo3 and Foxo4, have received little attention because of negligible knockout phenotypes in mice (2, 3). In the current issue of Endocrinology, Zhang et al. (4) reveal new metabolic functions for these seemingly redundant family members through analysis of overlapping, liver-specific deletions of the three Foxo proteins. This elegant new study illustrates the increasing complexity of the transcriptional networks controlling metabolism and emphasizes the importance of considering these interactions when devising new therapeutic strategies. The Fox transcriptional regulators (including Foxa, Foxo, Foxm, and Foxl proteins) have well-known roles in liver organogenesis (for review, see Ref. 5). However, in terms of liver metabolism, Foxo1 has gained the most notoriety due to its potent regulation of glucose homeostasis. Since the discovery of Foxo1 as a key regulator of gluconeogenic gene expression, there has been great interest in developing therapies to block hepatic Foxo1 activity in diabetic patients to reduce glycemia. Toward this end, Zhang et al. (4) show that a liver-specific knockout of Foxo1 can reduce blood glucose levels, and that concurrent reduction of Foxo3 further improves glycemia and whole-body insulin sensitivity. These particular data are not surprising, as another recent study reports similar findings (6). Although they differ in their conclusions concerning the role of Foxo4 in glycemia, these papers and others provide strong evidence that reducing Foxo activity in liver may indeed be useful to treat diabetes and metabolic syndrome. However, pertinent information contradicting this dogma is later revealed as Zhang et al. (4) further characterize the knockout metabolic phenotypes. Zhang et al. observe that loss of both Foxo1 and Foxo3 also results in significant hypertriglyceridemia and hypercholesterolemia due to increased hepatic lipid secretion and mild steatosis. These new data not only reveal previously unrecognized metabolic functions for the mostly ignored Foxo3 but also demonstrate that Foxo-regulated metabolic networks may be far more complex than originally thought. By dissecting both global and target gene expression, Zhang et al. (4) suggest that Foxo1 and Foxo3 synergistically suppress key lipogenic pathways, in addition to enhancing genes controlling glucose metabolism. This has significant therapeutic relevance, as it implies that targeting Foxo proteins for the treatment of hyperglycemia may also have unforeseen negative consequences on cardiovascular health. The metabolic outcome of reducing Foxo expression in diabetic models has been of great interest for a number of years (see Table 1 for summary). Thus, evidence of hyperlipidemia in mice on a Foxo-null background can be found in other reports. An independent triple Foxo1/3/4 hepatic Foxo-null mouse line also displays hypertriglyceridemia and increased lipogenesis, but only after exposure to a high-fat diet (7). Unfortunately, this earlier study presents data only for the triple knockout and does not address the relative contributions of individual Foxo proteins. In contrast, Zhang et al. (4) systematically analyze the detailed phenotype of singleand double-knockout mice, shedding light on the role of each family member in liver metabolism. Interestingly, the new study does not include detailed phenotypic or expression data for the triple knockout, with the authors citing lack of an additive effect. These

PAX3-FOXO1 (PAX3-FKHR) is the fusion protein produced by the genomic translocation that characterizes the alveolar subtype of Rhabdomyosarcoma, a pediatric sarcoma with myogenic phenotype. PAX3-FOXO1 is an aberrant but functional transcription factor. It retains PAX3-DNA-binding activity and functionally overlaps PAX3 function while also disturbing it, in particular its role in myogenic differentiation. We herein show that PAX3-FOXO1 interferes with normal FOXO function. PAX3-FOXO1 affects FOXO-family member trans-activation capability and the FOXO-dependent TGF-β response. PAX3-FOXO1 may contribute to tumor formation by inhibiting the tumor suppressor activities which are characteristic of both FOXO family members and TGF-β pathways. The recognition of this mechanism raises new questions about how FOXO family members function.

Mutations in PTEN occur in 60–80% of prostate cancers and lead to a constitutive activation of the phosphatidylinositol 3-kinase pathway and a resultant loss of activity of the FOXO family of forkhead transcription factors FKHRL1 and FKHR. To provide insight into the role of PTENmutations in prostate cancer, we used microarrays to identify genes regulated by FKHRL1 and FKHR in LAPC4 prostate carcinoma cells. These studies revealed that adenoviral overexpression of FKHRL1 and FKHR in the LAPC4 prostate cancer cell line resulted in apoptosis and induced the expression of many genes that affect cellular proliferation or survival. The expression of one of these FOXO-regulated genes,TRAIL, a pro-apoptotic member of the tumor necrosis factor family, was decreased in human metastatic prostate tumors. The altered expression of TRAIL in these tumors correlated directly with decreasedPTEN expression and the resultant loss of FKHRL1 and FKHR activity. Analysis of the effects of FOXO proteins on theTRAIL promoter localized the FKHRL1 responsive element of the TRAIL promoter to nucleotides −138 to −121 and demonstrated that TRAIL is a direct target of FKHRL1. These findings suggest that the decreased activity of FKHRL1 and FKHR in prostate cancers resulting from loss of PTEN leads to a decrease in TRAIL expression that may contribute to increased survival of the tumor cells.

Reactive oxygen species are required for cell proliferation but can also induce apoptosis1. In proliferating cells this paradox is solved by the activation of protein kinase B (PKB; also known as c-Akt), which protects cells from apoptosis2. By contrast, it is unknown how quiescent cells that lack PKB activity are protected against cell death induced by reactive oxygen species. Here we show that 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 elegans3,4,5,6, and we conclude that this model also extends to mammalian systems.

The mammalian DAF-16-like transcription factors, FKHR, FKHRL1, and AFX, function as key regulators of insulin signaling, cell cycle progression, and apoptosis downstream of phosphoinositide 3-kinase. Gene activation through binding to insulin response sequences (IRS) has been thought to be essential for mediating these functions. However, using transcriptional profiling, chromatin immunoprecipitation, and functional experiments, we demonstrate that rather than activation of IRS regulated genes (Class I transcripts), transcriptional repression of D-type cyclins (in Class III) is required for FKHR mediated inhibition of cell cycle progression and transformation. These data suggest that a novel mechanism of FKHR-mediated gene regulation is linked to its activity as a suppressor of tumor growth.

The FOXO family of Forkhead transcription factors, FKHR (FOXO1), FKHR-L1 (FOXO3a) and AFX (FOXO4), are regulated by the phosphoinositide-3-kinase-protein-kinase-B (PI3K-PKB/c-Akt) pathway. Direct phosphorylation by PKB results in cytoplasmic retention and inactivation, inhibiting the expression of FOXO-regulated genes, which control the cell cycle, cell death, cell metabolism and oxidative stress. This pathway appears to be well conserved throughout evolution. In the nematode Caenorhabditis elegans, it affects lifespan and controls dauer formation. Recent discoveries about FOXO regulation by PI3K-PKB signalling suggest that the PI3K-PKB-FOXO pathway might participate in similar processes in higher eukaryotes.

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. We and others have shown that PI3K, through activation of protein kinase B/Akt, inhibits transcriptional activation by a number of forkhead transcription factors (FoxO1, FoxO3, and FoxO4). In this study we have investigated the role of these forkhead transcription factors in the IL-2-induced T cell proliferation and survival. We show that 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 p27Kip1 and the proapoptotic Bcl-2 family member Bim. Furthermore, we show that IL-2 triggers a rapid, PI3K-dependent, phosphorylation of FoxO1a in primary T cells. Thus, we propose that inactivation of FoxO transcription factors by IL-2 plays a critical role in T cell proliferation and survival.

In mammals deprived of food, induction of hepatic glucose production (HGP) is important to ensure energy homeostasis, including prevention of hypoglycemia in response to the energy demands [1], and is especially critical in sites that predominantly utilize glucose as an energy source, such as the brain, red blood cells, and renal medulla. Glucose homeostasis is maintained through tight regulation of glucose production (i.e., glycogenolysis and gluconeogenesis) in the liver during the state of fasting and uptake of glucose in peripheral tissues under the fed condition. The pancreatic hormones glucagon and insulin are central to this regulation. During fasting, glucagon enhances HGP to maintain euglycemia. Conversely, when nutrients are increased after a meal, insulin blunts HGP and increases glucose uptake in the skeletal muscle and adipose tissue to prevent hyperglycemia. However, these processes are dysregulated in type 2 diabetes, resulting in the development of hyperglycemia [2]. The aberrant increase of HGP during the postabsorptive state in diabetic patients is primarily due to suppression failure by insulin, resulting primarily from excessive gluconeogenesis rather than glycogenolysis [3]. Gluconeogenesis is mainly regulated at the transcriptional level of rate-limiting gluconeogenic enzymes, such as glucose-6-phosphatase (G6pc; G6Pase) and phosphoenolpyruvate carboxykinase (Pck1; PEPCK) through hormone-dependent modulation of transcriptional regulators, including the transcription factors for FoxO (forkhead box O), CREB (cAMP response element binding protein), HNF4ahepatocytes nuclear factor 4a and glucocorticoid receptor, and transcriptional coactivators, such as PGC-1a (peroxisome proliferator-activated receptor gamma coactivator-1 alpha), CRTC2 (CREB-regulated transcription coactivator 2), and the histone acetyltransferase CBP (CREB-binding protein) [4]. Glucagon stimulates the induction of gluconeogenic genes through the following signaling pathway: cAMP, a second messenger of the glucagon receptor, leads cAMPdependent protein kinase (PKA)-mediated activation of the CRTC2/CREB complex to induce PGC-1a, which in turn coactivates the FoxO and HNF4a transcription factors on the G6pc and Pck1 promoters (Fig. 1). Insulin blunts gluconeogenic gene induction by affecting the same pathway. This is mediated mainly through the serine/threonine kinase Akt, which inactivates FoxO and PGC-1a via direct phosphorylation, as well as CRTC2 via SIK (salt-inducible kinase) 1/2 [5] (Fig. 1). The FoxO family of transcription factors, mainly FoxO1 and FoxO3 in mammalian liver [6], plays pivotal roles in regulating gluconeogenesis, as shown by studies on in vivo loss of and gain of function study; FoxO1 loss of function in the liver reduces HGP by inhibiting gluconeogenesis and glycogenolysis, resulting in neonatal and fasting-induced hypoglycemia [6], whereas its gain of function impairs hormonal regulation of HGP, including gluconeogenesis [7]. FoxO is regulated by insulin; Akt-dependent phosphorylation promotes its nuclear exclusion and inactivates FoxO1, leading to the reduction of gluconeogenic genes [8]. FoxO is also controlled by cAMP, which promotes its dephosphorylation and nuclear retention in cultured hepatocytes [8]. In vivo, FoxO1 loss of function in the liver attenuates gluconeogenic gene induction and HGP during fasting [6]. Although these findings indicate that FoxO is indispensable for cAMP-dependent induction of gluconeogenic genes, the M. Matsumoto (&) Department of Molecular Metabolic Regulation, Diabetes Research Center, National Center for Global Health and Medicine, 1-21-1 Toyama, Shinjuku-ku, Tokyo 162-8655, Japan e-mail: mmatsumoto@ri.ncgm.go.jp

One of the major determinants of aging in organisms ranging from worms to man are FOXO family transcription factors, which are downstream effectors of Insulin/IGF-1 signaling (IIS). The molecular mechanisms that actively promote DAF16/FOXO stability and function are unknown. Here we identify the deubiquitylating enzyme MATH-33 as an essential DAF-16 regulator in IIS, which stabilizes active DAF-16 protein levels and, as a consequence, influences DAF-16 functions, such as metabolism, stress response and longevity in C. elegans. MATH-33 associates with DAF-16 in cellulo and in vitro. MATH-33 functions as a deubiquitylase by actively removing ubiquitin moieties from DAF-16, thus counteracting the action of the RLE-1 E3-ubiquitin ligase. Our findings support a model in which MATH-33 promotes DAF-16 stability in response to decreased IIS by directly modulating its ubiquitylation state, suggesting that regulated oscillations in the stability of DAF-16 protein play an integral role in controlling processes such as metabolism and longevity.

The Forkhead box O (FoxO) family has recently been highlighted as an important transcriptional regulator of crucial proteins associated with the many diverse functions of cells. So far, FoxO1, FoxO3a, FoxO4 and FoxO6 proteins have been identified in humans. Although each FoxO family member has its own role, unlike the other FoxO families, FoxO3a has been extensively studied because of its rather unique and pivotal regulation of cell proliferation, apoptosis, metabolism, stress management and longevity. FoxO3a alteration is closely linked to the progression of several types of cancers, fibrosis and other types of diseases. In this review, we will examine the function of FoxO3a in disease progression and also explore FoxO3a’s regulatory mechanisms. We will also discuss FoxO3a as a potential target for the treatment of several types of disease.

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. Our goal was threefold: to identify the cellular compartment in which PKB phosphorylates AFX, to determine whether the nuclear localization of AFX plays a role in regulating its transcriptional activity, and to elucidate the mechanism by which phosphorylation alters the localization of AFX. We show that phosphorylation of AFX by PKB occurs 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. We show that 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.

Foxo family transcription factors contribute to muscle atrophy by promoting transcription of the ubiquitin ligases muscle-specific RING finger protein and muscle atrophy F-box/atrogin-1. Foxo transcriptional effectiveness is largely determined by its nuclear-cytoplasmic distribution, with unphosphorylated Foxo1 transported into nuclei and phosphorylated Foxo1 transported out of nuclei. We expressed the fluorescent fusion protein Foxo1-green fluorescent protein (GFP) in cultured adult mouse flexor digitorum brevis muscle fibers and tracked the time course of the nuclear-to-cytoplasmic Foxo1-GFP mean pixel fluorescence ratio (N/C) in living fibers by confocal imaging. We previously showed that IGF-I, which activates the Foxo kinase Akt/PKB, caused a rapid marked decline in N/C, whereas inhibition of Akt caused a modest increase in N/C. Here we develop a two-state mathematical model for Foxo1 nuclear-cytoplasmic redistribution, where Foxo phosphorylation/dephosphorylation is assumed to be fast compared with nuclear influx and efflux. Cytoplasmic Foxo1-GFP mean pixel fluorescence is constant due to the much larger cytoplasmic than nuclear volume. Analysis of N/C time courses reveals that IGF-I strongly increased unidirectional nuclear efflux, indicating similarly increased fractional phosphorylation of Foxo1 within nuclei, and decreased unidirectional nuclear influx, indicating increased cytoplasmic fractional phosphorylation of Foxo1. Inhibition of Akt increased Foxo1 unidirectional nuclear influx, consistent with block of Foxo1 cytoplasmic phosphorylation, but did not decrease Foxo1 unidirectional nuclear efflux, indicating that Akt may not be involved in Foxo1 nuclear efflux under control conditions. New media change experiments show that cultured fibers release IGF-I-like factors, which maintain low nuclear Foxo1 in the medium. This study demonstrates the power of quantitative modeling of observed nuclear fluxes.

Early B-cell development is an ordered and highly regulated process with alternating phases of cell proliferation and differentiation leading to B cells with the ability to recognize an extraordinarily large repertoire of different antigens. Here, we discuss what is currently known about the receptors in B-cell progenitors and how their signaling pathways influence immunoglobulin (Ig) gene rearrangement and the transcriptional program of early B cells. In particular, we address the interplay of the interleukin-7 receptor and the pre-B-cell receptor (preBCR) in shaping the survival, proliferation, and differentiation of early B cells. Each receptor addresses a unique set of signaling components but they also share signaling pathways, most prominently the MAPK/Erk and phosphoinositide-3 kinase pathways. The latter pathway regulates transcription factors of the FoxO family that play a central role in the proliferation to differentiation switch of pre-B cells. Interestingly, these two alternative cellular programs (proliferation and differentiation) are both controlled by the preBCR. Finally, we discuss how mutations or alterations of these pathways result in deregulated pre-B-cell expansion and leukemia.

Antimicrobial peptides (AMPs) are conserved cationic peptides which act both as defense molecules of the host immune system and as regulators of the commensal microbiome. Expression of AMPs is induced in response to infection by the Toll and Imd pathway. Under non-infected conditions, the transcription factor dFOXO directly regulates a set of AMP expression at low levels when nutrients are limited. Here we have analyzed whether target of rapamycin (TOR), another major regulator of growth and metabolism, also modulates AMP responses in Drosophila. We found that downregulation of TOR by feeding the drug rapamycin or by overexpressing the negative TOR regulators TSC1/TSC2, resulted in a specific induction of the AMPs Diptericin (Dpt) and Metchnikowin (Mtk). In contrast, overexpression of Rheb, which positively regulates TOR led to a repression of the two AMPs. Genetic and pharmacological experiments indicate that Dpt and Mtk activation is controlled by the transcription factor Forkhead (FKH), the founding member of the FoxO family. Shuttling of FKH from the cytoplasm to the nucleus is induced in the fat body and in the posterior midgut in response to TOR downregulation. The FKH-dependent induction of Dpt and Mtk can be triggered in dFOXO null mutants and in immune-compromised Toll and IMD pathway mutants indicating that FKH acts in parallel to these regulators. Together, we have discovered that FKH is the second conserved member of the FoxO family cross-regulating metabolism and innate immunity. dFOXO and FKH, which are activated upon downregulation of insulin or TOR activities, respectively, act in parallel to induce different sets of AMPs, thereby modulating the immune status of metabolic tissues such as the fat body or the gut in response to the oscillating energy status of the organism.

p21(Cip1) protein inhibits the activity of cyclins at the G(1) checkpoint and influences transition of cells from the G(1) to the S phase of the cell cycle. Moreover, expression of members of the FOXO family (active form of forkhead transcription factors of the O class) in dividing cells promotes cell-cycle arrest at the G(1)/S boundary via regulation of p21(Cip1). Recently, the exocyst complex, including Sec8, has been implicated in various roles independent of its role in secretion, such as cell migration, invadopodia formation, cytokinesis, glucose uptake and neural development. Given the essential roles of the exocyst complex in cellular and developmental processes, disruption of its function may be involved in various diseases such as cancer, diabetes and neuronal disorders. However, the relationship between Sec8 and the cell cycle remains to be elucidated. In this study, knockdown of Sec8 inhibited cell growth and promoted cell-cycle arrest at the G(1)/S phase by control of p21 expression and retinoblastoma protein phosphorylation. Furthermore, Sec8 regulated FOXO family proteins via ubiquitin-proteasome degradation by regulating the expression of the murine double minute 2 (Mdm2) protein but not S-phase kinase-associated protein 2 (Skp2).

Transcription factors of the FoxO (forkhead box O) family regulate a wide range of cellular physiological processes, including metabolic adaptation and myogenic differentiation. The transcriptional activity of most FoxO members is inhibitory to myogenic differentiation and overexpression of FoxO1 inhibits the development of oxidative type I fibres in vivo. In this study, we found that FoxO6, the last discovered FoxO family member, is expressed ubiquitously in various tissues but with higher expression levels in oxidative tissues, such as brain and oxidative muscles. Both the expression level and promoter activity of FoxO6 were found to be enhanced by PGC-1α (peroxisome-proliferator-activated receptor γ co-activator 1α), thus explained its enriched expression in oxidative tissues. We further demonstrated that FoxO6 represses the expression of PGC-1α via direct binding to an upstream A/T-rich element (AAGATATCAAAACA,−2228–2215) in the PGC-1α promoter. Oxidative low-intensity exercise induced PGC-1α but reduced FoxO6 expression levels in hind leg muscles, and the binding of FoxO6 to PGC-1α promoter was also prevented by exercise. As FoxO6 promoter can be co-activated by PGC-1α and its promoter in turn can be repressed by FoxO6, it suggests that FoxO6 and PGC-1α form a regulatory loop for setting oxidative metabolism level in the skeletal muscle, which can be entrained by exercise.

The winged helix or forkhead box (Fox) class of transcription factors constitutes a family of structurally related transcriptional activators that have been identified in species ranging from yeast to human. The first member of this transcription factor class was identified as a nuclear homeotic gene involved in embryonic development in Drosophila melanogaster (Weigel et al. 1989). The FoxO family of forkhead transcription factors represents a subfamily within the larger group of Fox transcription factors. Mammalian FoxO proteins (FoxO1, FoxO3a, FoxO4 and FoxO6), which are homologous to Caenorhabditis elegans protein DAF-16, belong to the O (‘other’) class of the Fox superfamily (Kaestner et al. 2000; Barthel et al. 2005). As transcription factors in the nucleus, the primary function of FoxO proteins is to bind to their cognate DNA target sequences as monomers. The cocrystal structure of another Fox protein, HNF-3γ , with DNA shows that there are 14 protein–DNA contacts distributed throughout the forkhead domain, but the third α-helix (H3) plays the most important role in a winged helix/forkhead protein’s DNA-binding specificity (Clark et al. 1993). In addition, both winged loops also make important interactions with DNA (Clark et al. 1993; Boura et al. 2007). Although the molecular basis of the DNA-binding specificity of FoxO transcription factors is poorly understood, high-affinity DNA-binding studies have identified a consensus FoxOrecognized element (FRE), (G/C) (T/A)AA(C/T)AA (Biggs Iii et al. 1999; Furuyama et al. 2000; Gilley et al. 2003).

The FOXO family of transcription factors is central to the regulation of organismal longevity and cellular survival. Several studies have indicated that FOXO factors lie at the center of a complex network of upstream pathways, cofactors and downstream targets (FOXO-interaction networks), which may have developmental and post-developmental roles in the regulation of chronic-stress response in normal and diseased cells. Noticeably, FOXO factors are important for the regulation of proteotoxicity and neuron survival in several models of neurodegenerative disease, suggesting that FOXO-interaction networks may have therapeutic potential. However, the status of FOXO-interaction networks in neurodegenerative disease remains largely unknown. Systems modeling is anticipated to provide a comprehensive assessment of this question. In particular, interrogating the context-dependent variability of FOXO-interaction networks could predict the clinical potential of cellular-stress response genes and aging regulators for tackling brain and peripheral pathology in neurodegenerative disease. Using published transcriptomic data obtained from murine models of Huntington’s disease and post-mortem brains, blood samples and induced-pluripotent-stem cells from Huntington’s disease carriers as a case example, this review briefly highlights how the biological status and clinical potential of FOXO-interaction networks for Huntington’s disease may be decoded by developing network and entropy based feature selection across heterogeneous datasets.

Human forkhead box class O (FoxO) transcription factors, activated in response to a wide range of external stimuli, like growth factors, insulin, nutrient levels and oxidative stress, are able to control several specific geneexpression programs. Besides their clear implication in metabolic processes, they appear to play a relevant role in tumour suppression by upregulation of genes involved in cell cycle arrest or apoptosis. Recent research efforts provide new insights into the molecular modulation of FoxO in liver cancer and disclose potential opportunities for developing new antitumor drugs. Through an intricate regulatory model, achieved via several post-translational modifications, including phosphorylation, acetylation, and ubiquitination, which control their subcellular localization and DNA binding activity, FoxO factors act as tumour suppressors. Low levels of FoxOs are associated with poor prognosis in cancer patients, and seem to confer chemotherapy resistance. Within FoxO members, FoxO3a appears to present anti-tumour properties in hepatocellular carcinoma, inducing the expression of pro-apoptotic genes, or interfering with signaling cascades commonly altered in this disease such as Wnt/β-catenin, PI3K/AKT/mTOR or MAPKs pathways. Here, we describe the main mechanisms of FoxO proteins regulation, and their cross-link with altered pathways in liver cancer. Moreover, based on the current knowledge of FoxO modulation, emphasis is placed on the development of novel agents which specifically activate FoxO family members and could be useful in the treatment of hepatocarcinoma.

Diabetes, characterized by hyperglycemia resulting from either insulin deficiency or insulin resistance, is a chronic metabolic disorder. Hearts of subjects with diabetes are more sensitive to myocardial ischemia/reperfusion injury but the underlying mechanism is largely unclear. Recent evidence suggests that alteration in cardiac metabolism is a key contributor to the increased vulnerability of diabetic heart to ischemia/reperfusion injury. The FoxO family of forkhead transcription factors including FoxO1, 3 and 4 play important roles in the regulation of many cellular and biological processes and are critical regulators of cardiac metabolism and cellular oxidative stress in the heart. This brief review focuses on the role of FoxO in regulating cardiac metabolism and its association with myocardial ischemia/reperfusion injury, especially in diabetes.

The PI3K–PTEN–AKT signaling pathway is involved in various cellular activities, including proliferation, migration, cell growth, cell survival and differentiation during adult homeostasis as well as in tumorigenesis. It has been suggested that the constitutive activation of PI3K/AKT signaling with concurrent loss of function of the tumor suppressor molecule PTEN contributes to cancer formation. Members of the PI3K–PTEN–AKT pathway, including these proteins and mTOR, are altered in melanoma tumors and cell lines. A hallmark of activation of the pathway is the loss of function of PTEN. Indeed, loss of heterozygosity of PTEN has been observed in approximately 30% of human melanomas, implicating this signaling pathway in this cancer. PI3K signaling activation, via loss of PTEN function, can inhibit proapoptotic genes such as the FoxO family of transcription factors, while inducing cell growth- and cell survival-related elements such as p70S6K and AKT. Determining how the PI3K–PTEN–AKT signaling pathway, alone or in cooperation with other pathways, orchestrates the induction of target genes involved in a diverse range of activities is a major challenge in research into melanoma initiation and progression. Moreover, the acquisition of basic knowledge will help patient management with appropriate therapies that are already, or will shortly be, on the market.

The FoxO family of transcription factors plays an important role in longevity and tumor suppression by regulating the expression of a wide range of target genes. FoxO3 has recently been found to be associated with extreme longevity in humans and to regulate the homeostasis of adult stem cell pools in mammals, which may contribute to longevity. The activity of FoxO3 is controlled by a variety of post-translational modifications that have been proposed to form a 'code' affecting FoxO3 subcellular localization, DNA binding ability, protein-protein interactions and protein stability. Lysine methylation is a crucial post-translational modification on histones that regulates chromatin accessibility and is a key part of the 'histone code'. However, whether lysine methylation plays a role in modulating FoxO3 activity has never been examined. Here we show that the methyltransferase Set9 directly methylates FoxO3 in vitro and in cells. Using a combination of tandem mass spectrometry and methyl-specific antibodies, we find that Set9 methylates FoxO3 at a single residue, lysine 271, a site previously known to be deacetylated by Sirt1. Methylation of FoxO3 by Set9 decreases FoxO3 protein stability, while moderately increasing FoxO3 transcriptional activity. The modulation of FoxO3 stability and activity by methylation may be critical for fine-tuning cellular responses to stress stimuli, which may in turn affect FoxO3's ability to promote tumor suppression and longevity.

The generation of lipid products catalyzed by PI3K is critical for normal T cell homeostasis and a productive immune response. PI3K can be activated in response to antigen receptor, co-stimulatory, cytokine, and chemokine signals. Moreover, dysregulation of this pathway frequently occurs in T cell lymphomas and is implicated in lymphoproliferative autoimmune disease. Akt acts as a central mediator of PI3K signals, downstream of which is the mTOR pathway, controlling cell growth and metabolism. Members of the Foxo family of transcription factors are also regulated by Akt, thus linking control over homing and migration of T cells, as well cell cycle entry, apoptosis, and DNA damage and oxidative stress responses, to PI3K signaling. PTEN, first identified as a tumor suppressor gene, encodes a lipid phosphatase that, by catalyzing the reverse of the PI3K "reaction," directly opposes PI3K signaling. However, PTEN may have other functions as well, and recent reports have suggested roles for PTEN as a tumor suppressor independent of its effects on PI3K signaling. Through the use of models in which Pten is deleted specifically in T cells, it is becoming increasingly clear that control over autoimmunity and lymphomagenesis by PTEN involves multi-faceted functions of this molecule at multiple stages within the T cell compartment.

Uncontrolled oxidative stress impairs bone formation and induces age-related bone loss in humans. The FoxO family is widely accepted to play an important role in protecting diverse cells from reactive oxygen species (ROS). Activation of FoxO1, the main FoxO in bone, stimulates proliferation and differentiation as well as inhibits apoptosis of osteoblast lineage cells. Despite the important role of FoxO1, little is known about how FoxO1 expression in bone is regulated. Meanwhile, several recent studies reported that microRNAs (miRNAs) could play a role in osteoblast differentiation and bone formation by targeting various transcriptional factors. Here, we identified one additional crucial miRNA, miR-182, which regulates osteoblastogenesis by repressing FoxO1 and thereby negatively affecting osteogenesis. Overexpression of miR-182 in osteoblast lineage cells increased cell apoptosis and inhibited osteoblast differentiation, whereas in vivo overexpression of miR-182 in zebrafish impaired bone formation. From in silico analysis and validation experiments, FoxO1 was identified as the target of miR-182, and restoration of FoxO1 expression in miR-182-overexpressing osteoblasts rescued them from the inhibitory effects of miR-182. These results indicate that miR-182 functions as a FoxO1 inhibitor to antagonize osteoblast proliferation and differentiation, with a subsequent negative effect on osteogenesis. To treat bone aging, an antisense approach targeting miR-182 could be of therapeutic value.

Although PI3-kinase mutations are uncommon in renal cell carcinoma (RCC), the PI3-kinase/Akt signalingpathway is active in most RCC. The activation of PI3-kinase would be expected to drive protein and lipid synthesisthrough its effects on mTORC1 and SREBP1, respectively. PI3-kinase also activates numerous transcription factors (e.g.the FOXO family, c-myc, NF-κB) that regulate cell proliferation and viability. The consequences of blocking PI3-kinasein RCC are just now beginning to be elucidated and are expected to include effects on tumor cell proliferation,metabolism, and angiogenesis. Several PI3-kinase inhibitors currently undergoing clinical testing are active site inhibitorsof mTOR as well and it is likely that these agents will prove particularly useful in the treatment of RCC.

The leading cause of death in diabetic patients is cardiovascular disease; diabetic cardiomyopathy is typified by alterations in cardiac morphology and function, independent of hypertension or coronary disease. However, the molecular mechanism that links diabetes to cardiomyopathy is incompletely understood. Insulin resistance is a hallmark feature of diabetes, and the FoxO family of transcription factors, which regulate cell size, viability, and metabolism, are established targets of insulin and growth factor signaling. Here, we set out to evaluate a possible role of FoxO proteins in diabetic cardiomyopathy. We found that FoxO proteins were persistently activated in cardiac tissue in mice with diabetes induced either genetically or by high-fat diet (HFD). FoxO activity was critically linked with development of cardiomyopathy: cardiomyocyte-specific deletion of FoxO1 rescued HFD-induced declines in cardiac function and preserved cardiomyocyte insulin responsiveness. FoxO1-depleted cells displayed a shift in their metabolic substrate usage, from free fatty acids to glucose, associated with decreased accumulation of lipids in the heart. Furthermore, we found that FoxO1-dependent downregulation of IRS1 resulted in blunted Akt signaling and insulin resistance. Together, these data suggest that activation of FoxO1 is an important mediator of diabetic cardiomyopathy and is a promising therapeutic target for the disease.

The hallmark of cancer cells is deregulated growth, inhibition of differentiation, and delay or blockage of programmed cell death. Tumor cells that are independent of extraand/or intracellular regulatory mechanisms due to mutations in proto-oncogenes and tumor suppressors acquire the ability of uncontrolled proliferation and invasion into other tissues. FOXO (FOXO1, FOXO3, FOXO4, FOXO6) transcription factors control apoptosis, stress resistance and longevity in mammalian cells. Their activity and subcellular localization is regulated by phosphorylation on conserved serine/threonin residues via protein kinase B (PKB) and stress-induced kinases. Hyperactivation of PKB contributes to the inactivation of FOXO3 and predicts poor outcome for neuroblastoma patients. Depending on their posttranslational modifications and cellular context FOXO transcription factors exert diverse functions thereby either inducing cell death but also cell survival and resistance to environmental stress. Although FOXO transcription factors share the same DNA binding motive and seem to have overlapping functions, knock out animals for single FOXO family members show different defects: Whereas FOXO1 knock-out mice die during embryonic development due to defective vasculature, FOXO3 and FOXO4 knock out mice show a mild phenotype. However, conditional triple-knock-out mice provide evidence that FOXO1, FOXO3 and FOXO4 are critically involved in the maintenance of the hematopoietic stem cell population and the regulation of endothelial cell homeostasis. Consistent with their putative role as tumor suppressors, in cultured neuroblastoma cells the activation of FOXO3 triggers the intrinsic death pathway and induces programmed cell death via induction of the proapoptotic BH3-only proteins BCL2L11/Bim and PMAIP1/Noxa. Surprisingly, in neuroblastoma cells FOXO3-mediated cell death depends on the accumulation of reactive oxygen species (ROS). Activation of FOXO3 also causes the repression of the apoptosisinhibitor protein Survivin and determines sensitivity of neuroblastoma cells to DNAdamaging chemotherapeutic agents. More recently it was shown that beside its function as a tumor suppressor FOXO3 might also facilitate cancer cell survival under certain circumstances. In other tumor types FOXO3 also induces detoxification and stress resistance