Regulation Of FoxO Transcription Factors Research Articles

Diverse Roles of Akt in T cells.

Akt kinases translate various external cues into intracellular signals that control cell survival, proliferation, metabolism and differentiation. This review discusses the requirement for Akt and its targets in determining the fate and function of T cells. We discuss the importance of Akt at various stages of T cell development including β-selection during which Akt fulfills the energy requirements of highly proliferative DN3 cells. Akt also plays an integral role in CD8 T cell biology where its regulation of Foxo transcription factors and mTORC1 metabolic activity controls effector versus memory CD8 T cell differentiation. Finally, Akt promotes the differentiation of naïve CD4 T cells into Th1, Th17 and Tfh cells but inhibits the development of Treg cells. We also highlight how modulating Akt in T cells is a promising avenue for enhancing cell-based cancer immunotherapy.

Molecular regulation and function of FoxO3 in chronic kidney disease.

FOXOs are transcription factors that regulate downstream target genes to counteract to cell stress. Here we review the function and regulation of FOXO transcription factors, the mechanism of FOXO3 activation in the kidney, and the role of FOXO3 in delaying the development of chronic kidney disease (CKD). Progressive renal hypoxia from vascular dropout and metabolic perturbation is a pathogenic factor for the initiation and development of CKD. Hypoxia and low levels of α-ketoglutarate generated from the TCA cycle inhibit prolyl hydroxylase domain (PHD)-mediated prolyl hydroxylation of FoxO3, thus reducing FoxO3 protein degradation via the ubiquitin proteasomal pathway, similar to HIF stabilization under hypoxic conditions. FoxO3 accumulation and nuclear translocation activate two key cellular defense mechanisms, autophagy and antioxidative response in renal tubular cells, to reduce cell injury and promote cell survival. FoxO3 directly activates the expression of Atg proteins, which replenishes core components of the autophagic machinery to allow sustained autophagy in the chronically hypoxic kidney. FoxO3 protects mitochondria by stimulating the expression of superoxide dismutase 2 (SOD2), as tubular deletion of FoxO3 in mice results in reduced SOD2 levels and profound mitochondrial damage. Knowledge gained from animal studies may help understand the function of stress responsive transcription factors that could be targeted to prevent or treat CKD.

The regulation of Akt and FoxO transcription factors during dehydration in the African clawed frog (Xenopus laevis).

The African clawed frog (Xenopus laevis) naturally tolerates severe dehydration using biochemical adaptation, one of which is the elevation of antioxidant defenses during whole-body dehydration. The present study investigated the role and regulation of a pathway known to regulate oxidative stress response, the Akt-FoxO signaling pathway, in clawed frog skeletal muscle, responding to medium (15%) and high (30%) dehydration. Protein levels of total and phosphorylated Akt, FoxO1, and FoxO3 were assessed via immunoblotting, in addition to the levels of the E3 ubiquitin ligase known to be associated with muscle atrophy, MAFbx. Akt activity/phosphorylation in addition to its total protein levels were decreased in the skeletal muscle during dehydration, and this corresponded with decreases in the relative phosphorylation of FoxO1 and FoxO3 as well on several residues. Akt is an inhibitor of FoxO1 and FoxO3 activity via phosphorylation, suggesting that FoxO activities were increased during dehydration stress. Furthermore, MAFbx showed decreased protein expression during high dehydration as well, suggesting that the clawed frog may exhibit some natural resistance to skeletal muscle atrophy during severe dehydration conditions. In addition to identifying that the suppression of Akt could lead to an activation of FoxO transcription factors in X. laevis during dehydration, these investigations suggest that X. laevis dehydration may implicate FoxO1 and FoxO3 in controlling skeletal muscle atrophy in X. laevis exposed to dehydration. This study implicates the Akt signaling pathway, its regulation of FoxO transcription factors, and FoxO-controlled targets, in stress adaptation against dehydration.

Pyropia yezoensis Protein Prevents Dexamethasone-Induced Myotube Atrophy in C2C12 Myotubes.

Glucocorticoids (GCs), which are endocrine hormones released under stress conditions, can cause skeletal muscle atrophy. This study investigated whether Pyropia yezoensis crude protein (PYCP) inhibits synthetic GCs dexamethasone (DEX)-induced myotube atrophy associated with proteolytic systems. Mouse skeletal muscle C2C12 myotubes were treated with DEX in the presence or absence of PYCP. DEX exposure (100 μM) for 24 h significantly decreased myotube diameter and myogenin expression, which were all increased by treatment with 20 and 40 μg/mL PYCP. Additionally, PYCP significantly reduced the nuclear expression of the forkhead box transcription factors, FoxO1 and FoxO3a, and ubiquitin-proteasome pathway activation. Further mechanistic research revealed that PYCP inhibited the autophagy-lysosome pathway in DEX-induced C2C12 myotubes. These findings indicate that PYCP prevents DEX-induced myotube atrophy through the regulation of FoxO transcription factors, followed by the inhibition of the ubiquitin-proteasome and autophagy-lysosome pathways. Therefore, we suggest that inhibiting these two proteolytic processes with FoxO transcription factors is a promising strategy for preventing DEX-related myotube atrophy.

OP-06 - Redox regulation of FOXO transcription factors – role of glutathione

FOXO transcription factors are involved in the cellular adaptive response to oxidative stimuli. FOXO activity is affected by the modulation of thiol levels both in cultured mammalian cells and in the model organism Caenorhabditis elegans. Exposure of HepG2 cells to diethyl maleate (DEM) resulted in a nuclear accumulation of overexpressed FOXO1-GFP. Pre- or postincubation of cells with DEM prevented or reversed this effect, triggering nuclear accumulation of FOXO-GFP. Similarly, the FOXO ortholog in C. elegans, DAF-16, accumulated in nuclei upon exposure of worms to DEM. DAF-16 was also essential to DEM-induced life span extension. Moreover, the expression of cytoprotective DAF-16 target genes was increased in worms held on DEM. These beneficial effects were observed only at low concentrations of DEM (10-100 µM), whereas higher concentrations (1 mM) were detrimental. Downregulation of GSH levels in young adult worms through attenuation of gcs-1 expression increased both stress resistance and life expectancy of the worms. In contrast, gcs-1 knockdown right after hatching rendered young adult worms susceptible to vulval ruptures during egg-laying. Thus, modest GSH depletion in young adult worms may promote stress resistance and life span, whereas depletion of GSH may be detrimental to developing worms.

Effects of ageing on expression of the muscle-specific E3 ubiquitin ligases and Akt-dependent regulation of Foxo transcription factors in skeletal muscle.

Controversy exists as to whether the muscle-specific E3 ubiquitin ligases MAFbx and MuRF1 are transcriptionally upregulated in the process of sarcopenia. In the present study, we investigated the effects of ageing on mRNA/protein expression of muscle-specific E3 ubiquitin ligases and Akt/Foxo signalling in gastrocnemius muscles of female mice. Old mice exhibited a typical sarcopenic phenotype, characterized by loss of muscle mass and strength, decreased amount of myofibrillar proteins, incidence of aberrant muscle fibres, and genetic signature to sarcopenia. Activation levels of Akt were lower in adult and old mice than in young mice. Consequently, Akt-mediated phosphorylation levels of Foxo1 and Foxo3 proteins were decreased. Nuclear levels of Foxo1 and Foxo3 proteins showed an overall increasing trend in old mice. MAFbx mRNA expression was decreased in old mice relative to adult mice, whereas MuRF1 mRNA expression was less affected by ageing. At the protein level, MAFbx was less affected by ageing, whereas MuRF1 was increased in old mice relative to adult mice, with ubiquitin-protein conjugates being increased with ageing. In conclusion, we provided evidence for no mRNA upregulation of muscle-specific E3 ubiquitin ligases and disconnection between their expression and Akt/Foxo signalling in sarcopenic mice. Their different responsiveness to ageing may reflect different roles in sarcopenia.

Src tyrosine kinase signaling antagonizes nuclear localization of FOXO and inhibits its transcription factor activity

Biochemical experiments in mammalian cells have linked Src family kinase activity to the insulin signaling pathway. To explore the physiological link between Src and a central insulin pathway effector, we investigated the effect of different Src signaling levels on the Drosophila transcription factor dFOXO in vivo. Ectopic activation of Src42A in the starved larval fatbody was sufficient to drive dFOXO out of the nucleus. When Src signaling levels were lowered by means of loss-of-function mutations or pharmacological inhibition, dFOXO localization was shifted to the nucleus in growing animals, and transcription of the dFOXO target genes d4E-BP and dInR was induced. dFOXO loss-of-function mutations rescued the induction of dFOXO target gene expression and the body size reduction of Src42A mutant larvae, establishing dFOXO as a critical downstream effector of Src signaling. Furthermore, we provide evidence that the regulation of FOXO transcription factors by Src is evolutionarily conserved in mammalian cells.

Resveratrol Rescues SIRT1-Dependent Adult Stem Cell Decline and Alleviates Progeroid Features in Laminopathy-Based Progeria

Abnormal splicing of LMNA gene or aberrant processing of prelamin A results in progeroid syndrome. Here we show that lamin A interacts with and activates SIRT1. SIRT1 exhibits reduced association with nuclear matrix (NM) and decreased deacetylase activity in the presence of progerin or prelamin A,leading to rapid depletion of adult stem cells (ASCs) in Zmpste24(-/-) mice. Resveratrol enhances the binding between SIRT1 and A-type lamins to increases its deacetylase activity. Resveratrol treatment rescues ASC decline, slows down body weight loss, improves trabecular bone structure and mineral density, and significantly extends the life span in Zmpste24(-/-) mice. Our data demonstrate lamin A as an activator of SIRT1 and provide a mechanistic explanation for the activation of SIRT1 by resveratrol. The link between conserved SIRT1 longevity pathway and progeria suggests a stem cell-based and SIRT1 pathway-dependent therapeutic strategy for progeria.

Heat Shock and Caloric Restriction Have a Synergistic Effect on the Heat Shock Response in a sir2.1-dependent Manner in Caenorhabditis elegans

The heat shock response (HSR) is responsible for maintaining cellular and organismal health through the regulation of proteostasis. Recent data demonstrating that the mammalian HSR is regulated by SIRT1 suggest that this response may be under metabolic control. To test this hypothesis, we have determined the effect of caloric restriction in Caenorhabditis elegans on activation of the HSR and have found a synergistic effect on the induction of hsp70 gene expression. The homolog of mammalian SIRT1 in C. elegans is Sir2.1. Using a mutated C. elegans strain with a sir2.1 deletion, we show that heat shock and caloric restriction cooperate to promote increased survivability and fitness in a sir2.1-dependent manner. Finally, we show that caloric restriction increases the ability of heat shock to preserve movement in a polyglutamine toxicity neurodegenerative disease model and that this effect is dependent on sir2.1.

Lymphocyte signaling: regulation of FoxO transcription factors by microRNAs

The Forkhead box O (FoxO) family of transcription factors is important for the maintenance of immunological homeostasis and tolerance by controlling the development and function of B and T lymphocytes. Because dysregulation in FoxO activity can result in chronic inflammation and autoimmunity, the transcriptional activity of FoxO proteins is tightly controlled and generally dependent on complex posttranslational modifications that lead either to their nuclear entry and subsequent activation or, alternatively, to their nuclear export. The phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/Akt) axis represents the major pathway phosphorylating and thereby inactivating FoxO proteins. However, recent results have revealed an additional posttranscriptional mechanism of FoxO inactivation by microRNAs. The discovery of this molecular pathway may provide a new therapeutic avenue for the modulation of FoxO activity in immune-mediated diseases using either microRNA targeting antagomirs or synthetic microRNA mimics, a topic that is addressed in this review.

The E-box Binding Factors Max/Mnt, MITF, and USF1 Act Coordinately with FoxO to Regulate Expression of Proapoptotic and Cell Cycle Control Genes by Phosphatidylinositol 3-Kinase/Akt/Glycogen Synthase Kinase 3 Signaling

Phosphatidylinositol (PI) 3-kinase/Akt signaling plays a critical role in cell proliferation and survival, partly by regulation of FoxO transcription factors. Previous work using global expression profiling indicated that inhibition of PI 3-kinase in proliferating cells led to induction of genes that promote cell cycle arrest and apoptosis. The upstream regulatory regions of these genes had binding sites not only for FoxO, but also for Myc/Max transcription factors. In the present study, we have addressed the role of Myc family members and related E-box-binding proteins in the regulation of these genes. Chromatin immunoprecipitations and RNA interference indicated that transcription was repressed by Max-Mnt-Sin3a-histone deacetylase complexes in proliferating cells. Inhibition of PI 3-kinase led to a loss of Max/Mnt binding and transcriptional induction by MITF and USF1, as well as FoxO. Both MITF and USF1 were activated by glycogen synthase kinase (GSK) 3, with GSK3 phosphorylation sites on USF1 identified as the previously described activating site threonine 153 as well as serine 186. siRNA against MITF as well as against FoxO3a protected cells from apoptosis following PI 3-kinase inhibition. These results define a novel E-box-regulated network that functions coordinately with FoxO to regulate transcription of apoptotic and cell cycle regulatory genes downstream of PI 3-kinase/Akt/GSK3 signaling.

CREB and ChREBP oppositely regulate SIRT1 expression in response to energy availability.

The nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase SIRT1 is a major metabolic regulator activated by energy stresses such as fasting or calorie restriction. SIRT1 activation during fasting not only relies on the increase in the NAD(+)/NADH ratio caused by energy deprivation but also involves an upregulation of SIRT1 mRNA and protein levels in various metabolic tissues. We demonstrate that SIRT1 expression is controlled systemically by the activation of the cyclic AMP response-element-binding protein upon low nutrient availability. Conversely, in the absence of energetic stress, the carbohydrate response-element-binding protein represses the expression of SIRT1. Altogether, these results demonstrate that SIRT1 expression is tightly controlled at the transcriptional level by nutrient availability and further underscore that SIRT1 is a crucial metabolic checkpoint connecting the energetic status with transcriptional programmes.

Peptide Switch Is Essential for Sirt1 Deacetylase Activity

In mammals, the Sirtuins are composed of seven Sir2 orthologs (Sirt1-7) with a conserved deacetylase core that utilizes NAD(+) as a cofactor. Interestingly, the deacetylase core of Sirt1 by itself has no catalytic activity. We found within the C-terminal domain a 25 aa sequence that is essential for Sirt1 activity (ESA). Our results indicate that the ESA region interacts with and functions as an "on switch" for the deacetylase core. The endogenous Sirt1 inhibitor DBC1, which also binds to the deacetylase core, competes with and inhibits the ESA region from interacting with the deacetylase core. We discovered an ESA mutant peptide that can bind to the deacetylase core and inhibit Sirt1 in trans. By using this mutant peptide, we were able to inhibit Sirt1 activity and to increase the chemosensitivity of androgen-refractory prostate cancer cells. Therefore, the ESA region is a potential target for development of therapies to regulate Sirt1.

Complex Between GlcNAcylation & Phosphorylation is Extensive: Roles in Nutrient Sensing & Signaling

O‐linked beta‐N‐acetylglucosamine (O‐GlcNAc) serves as a nutrient/stress sensor to modulate metabolic processes and gene expression (for review, Nature 446, 1017–1022; Am J Physiol Endocrinol Metab 295: E17–E28). O‐GlcNAc plays a direct role in the etiology of diabetes, neurodegenerative disease, and cancer. Recent phospho‐proteomic and glycomic studies have shown that the crosstalk between GlcNAcylation and phosphorylation is extensive at the individual site level. This dynamic interplay not only occurs by competition at the same or proximal sites, but also by each modification regulating the other's cycling enzymes. For example, several kinases are regulated by GlcNAcylation, and phosphorylation regulates both O‐GlcNAc Transferase and O‐GlcNAcase. The elucidation of this extensive crosstalk between these two most abundant protein modifications will have a major impact on our view of signaling and transcriptional regulation. Examples of the importance of this dynamic interplay in the regulation of FOXO transcription factors, kinases, RNA polymerase II, and cytokinesis will be presented. It is now clear that GlcNAcylation plays an important role in glucose toxcity and in insulin resistance. Dr. Hart receives a share of royalty received by the university on sales of the CTD 110.6 antibody. Terms of this arrangement are managed by JHUSOM.

FOXO transcription factors and VEGF neutralizing antibody enhance antiangiogenic effects of resveratrol

Resveratrol (trans-3,5,4'-trihydroxystilbene), a compound found largely in the skins of red grapes and wines, possesses anti-cancer and anti-angiogenic properties and protects the cardiovascular system. However, the molecular mechanisms by which resveratrol inhibits angiogenesis are currently subjects of intense investigation. The purpose of this study was to examine whether FOXO transcription factors mediate anti-angiogenic effects of resveratrol, and whether vascular endothelial growth factor (VEGF) neutralizing antibody can enhance these effects of resveratrol. Inhibition of PI3 kinase (PI3K)/AKT and MEK/ERK pathways synergistically inhibited migration and capillary tube formation of Human Umbilical Vein Endothelial Cells (HUVECs) and further enhanced the anti-angiogenic effects of resveratrol. Inhibitors of AKT and MEK kinase synergistically inhibited cytoplasmic FOXO3a phosphorylation, which was accompanied by its nuclear translocation in HUVECs. Interestingly, inhibition of PI3K/AKT and MEK/ERK pathways synergistically induced FOXO transcriptional activity and inhibited cell migration and capillary tube formation. Antiangiogenic effects of resveratrol were enhanced by inhibitors of AKT and MEK. Phosphorylation-deficient mutants of FOXOs induced FOXO transcriptional activity, inhibited HUVEC cell migration, and capillary tube formation, and also enhanced antiangiogenic effects of resveratrol. Finally, VEGF neutralizing antibody enhanced the anti-proliferative and anti-angiogenic effects of resveratrol. In conclusion, regulation of FOXO transcription factors by resveratrol may play an important role in angiogenesis which is critical for cancer, diabetic retinopathy, rheumatoid arthritis, psoriasis, and cardiovascular disorders.

SysPTM: A Systematic Resource for Proteomic Research on Post-translational Modifications

With the rapid expansion of protein post-translational modification (PTM) research based on large-scale proteomic work, there is an increasing demand for a suitable repository to analyze PTM data. Here we present a curated, web-accessible PTM data base, SysPTM. SysPTM provides a systematic and sophisticated platform for proteomic PTM research equipped not only with a knowledge base of manually curated multi-type modification data but also with four fully developed, in-depth data mining tools. Currently, SysPTM contains data detailing 117,349 experimentally determined PTM sites on 33,421 proteins involving nearly 50 PTM types, curated from public resources including five data bases and four web servers and more than one hundred peer-reviewed mass spectrometry papers. Protein annotations including Pfam domains, KEGG pathways, GO functional classification, and ortholog groups are integrated into the data base. Four online tools have been developed and incorporated, including PTMBlast, to compare a user's PTM dataset with PTM data in SysPTM; PTMPathway, to map PTM proteins to KEGG pathways; PTMPhylog, to discover potentially conserved PTM sites; and PTMCluster, to find clusters of multi-site modifications. The workflow of SysPTM was demonstrated by analyzing an in-house phosphorylation dataset identified by MS/MS. It is shown that in SysPTM, the role of single-type and multi-type modifications can be systematically investigated in a full biological context. SysPTM could be an important contribution to modificomics research. SysPTM is freely available online at www.sysbio.ac.cn/SysPTM.

Structure/function relationships underlying regulation of FOXO transcription factors

The FOXO subgroup of forkhead transcription factors plays a central role in cell-cycle control, differentiation, metabolism control, stress response and apoptosis. Therefore, the function of these important molecules is tightly controlled by a wide range of protein-protein interactions and posttranslational modifications including phosphorylation, acetylation and ubiquitination. The mechanisms by which these processes regulate FOXO activity are mostly elusive. This review focuses on recent advances in structural studies of forkhead transcription factors and the insights they provide into the mechanism of DNA recognition. On the basis of these data, we discuss structural aspects of protein-protein interactions and posttranslational modifications that target the forkhead domain and the nuclear localization signal of FOXO proteins.

The FoxO code

The FoxO family of Forkhead transcription factors plays an important role in longevity and tumor suppression by upregulating target genes involved in stress resistance, metabolism, cell cycle arrest and apoptosis. FoxO transcription factors translate a variety of environmental stimuli, including insulin, growth factors, nutrients and oxidative stress, into specific gene-expression programs. These environmental stimuli control FoxO activity primarily by regulating their subcellular localization, but also by affecting their protein levels, DNA-binding properties and transcriptional activity. The precise regulation of FoxO transcription factors is enacted by an intricate combination of post-translational modifications (PTMs), including phosphorylation, acetylation and ubiquitination, and binding protein partners. An intriguing possibility is that FoxO PTMs may act as a 'molecular FoxO code' read by selective protein partners to rapidly regulate gene-expression programs. The effective control of FoxO activity in response to environmental stimuli is likely to be critical to prevent aging and age-dependent diseases, including cancer, neurodegenerative diseases and diabetes.

Inhibition of PI3K/AKT and MEK/ERK pathways act synergistically to enhance antiangiogenic effects of EGCG through activation of FOXO transcription factor

BackgroundWe have recently shown that epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, inhibits angiogenesis. However, the molecular mechanisms by which EGCG inhibits angiogenesis have never been investigated. In this study, we examined the interaction of PI3K/AKT and MEK/ERK pathways on the regulation of FOXO transcription factors, which ultimately control the antiangiogenic effects of EGCG.ResultsInhibition of PI3K/AKT and MEK/ERK pathways interact synergistically to inhibit migration and capillary tube formation of HUVEC cells and further enhanced the antiangiogenic effects of EGCG. Inhibition of AKT and MEK kinases synergistically induced FOXO transcriptional activity, which was further enhanced in the presence of EGCG. Phosphorylation deficient mutants of FOXO induced FOXO transcriptional activity, inhibited HUVEC cell migration and capillary tube formation. Inhibition of FOXO phosphorylation also enhanced antiangiogenic effects of EGCG through transcriptional activation of FOXO.ConclusionInhibition of PI3K/AKT and MEK/ERK pathways act synergistically to regulate antiangiogenic effects of EGCG through activation of FOXO transcription factors. The activation of FOXO transcription factors through inhibition of these two pathways may have physiological significance in management of diabetic retinopathy, rheumatoid arthritis, psoriasis, cardiovascular diseases, and cancer.

A Combination of Genomic Approaches Reveals the Role of FOXO1a in Regulating an Oxidative Stress Response Pathway

BackgroundWhile many of the phenotypic differences between human and chimpanzee may result from changes in gene regulation, only a handful of functionally important regulatory differences are currently known. As a first step towards identifying transcriptional pathways that have been remodeled in the human lineage, we focused on a transcription factor, FOXO1a, which we had previously found to be up-regulated in the human liver compared to that of three other primate species. We concentrated on this gene because of its known role in the regulation of metabolism and in longevity.MethodologyUsing a combination of expression profiling following siRNA knockdown and chromatin immunoprecipitation in a human liver cell line, we identified eight novel direct transcriptional targets of FOXO1a. This set includes the gene for thioredoxin-interacting protein (TXNIP), the expression of which is directly repressed by FOXO1a. The thioredoxin-interacting protein is known to inhibit the reducing activity of thioredoxin (TRX), thereby hindering the cellular response to oxidative stress and affecting life span.ConclusionsOur results provide an explanation for the repeated observations that differences in the regulation of FOXO transcription factors affect longevity. Moreover, we found that TXNIP is down-regulated in human compared to chimpanzee, consistent with the up-regulation of its direct repressor FOXO1a in humans, and with differences in longevity between the two species.