Role Of FOXO Transcription Factors Research Articles

Acupuncture and cellular mechanisms: the role of foxo transcription factors in autophagy and senescence.

The current narrative review was planned to elucidate the interplay between acupuncture, a cornerstone of Traditional Chinese Medicine, and the modulation of cellular mechanisms via Forkhead box O transcription factors, focussing on autophagy and cellular senescence. Autophagy, essential for cellular health, and cellular senescence, associated with aging and age-related diseases, are both significantly influenced by Forkhead box O transcription factors among other regulatory factors and pathways. These factors, known to diminish with age, play a pivotal role in promoting longevity. The current review analysed experimental models demonstrating acupuncture's capacity to activate Forkhead box O transcription factors, thereby inducing autophagy and reducing cellular senescence. Evidence from studies on human fibroblasts, ageing animal models, obesity-induced insulin resistance, and osteoporosis corroborates the beneficial effects of acupuncture. Despite these promising findings, the complexities of Forkhead box O transcription factors' activation by acupuncture and their precise impact on cellular processes warrant further investigation with a particular focus on the distinct roles of Forkhead box O isoforms.

Role of FOXO transcription factors in the tolerance of whole-body freezing in the wood frog, Rana sylvatica

The wood frog (Rana Sylvatica) can endure the sub-zero temperatures of winter by freezing up to 65% of total body water as extracellular ice and retreating into a prolonged hypometabolic state. Freeze survival requires the coordination of various adaptations, including a global suppression of metabolic functions and select activation of pro-survival genes. Transcription factors playing roles in metabolism, stress tolerance, and cell proliferation may assist in making survival in a frozen state possible. In this study, the role of Forkhead box ‘other’ (FOXO) transcription factors in freeze tolerance, and related changes to the insulin pathway, are investigated. Immunoblotting was used to assess total and phosphorylated amounts of FOXO proteins in wood frogs subjected to freezing for 24 h and thawed recovery for 8 h. Levels of active FOXO3 increased in brain, kidney, and liver during freezing and thawing, suggesting a need to maintain or enhance antioxidant defenses under these stresses. Results implicate FOXO involvement in the metabolic regulation of natural freeze tolerance.

Differential roles of FOXO transcription factors on insulin action in brown and white adipose tissue.

Insulin and IGF-1 are essential for adipocyte differentiation and function. Mice lacking insulin and IGF-1 receptors in fat (FIGIR-KO, fat-specific IGF-1 receptor and insulin receptor-KO) exhibit complete loss of white and brown adipose tissue (WAT and BAT), glucose intolerance, insulin resistance, hepatosteatosis, and cold intolerance. To determine the role of FOXO transcription factors in the altered adipose phenotype, we generated FIGIR-KO mice with fat-specific KO of fat-expressed Foxos [Foxo1, Foxo3, Foxo4] (F-Quint-KO). Unlike FIGIR-KO mice, F-Quint-KO mice had normal BAT, glucose tolerance, insulin-regulated hepatic glucose production, and cold tolerance. However, loss of FOXOs only partially rescued subcutaneous WAT and hepatosteatosis, did not rescue perigonadal WAT or systemic insulin resistance, and led to even more marked hyperinsulinemia. Thus, FOXOs play different roles in insulin/IGF-1 action in different adipose depots, being most important in BAT, followed by subcutaneous WAT and then by visceral WAT. Disruption of FOXOs in fat also led to a reversal of insulin resistance in liver, but not in skeletal muscle, and an exacerbation of hyperinsulinemia. Thus, adipose FOXOs play a unique role in regulating crosstalk between adipose depots, liver, and β cells.

Role of FoxO transcription factors in aging-associated cardiovascular diseases.

Aging constitutes a major risk factor toward the development of cardiovascular diseases (CVDs). The aging heart undergoes several changes at the molecular, cellular and physiological levels, which diminishes its contractile function and weakens stress tolerance. Further, old age increases the exposure to risk factors such as hypertension, diabetes and hypercholesterolemia. Notably, research in the past decades have identified FoxO subfamily of the forkhead transcription factors as key players in regulating diverse cellular processes linked to cardiac aging and diseases. In the present chapter, we discuss the important role of FoxO in the development of various aging-associated cardiovascular complications such as cardiac hypertrophy, cardiac fibrosis, heart failure, vascular dysfunction, atherosclerosis, hypertension and myocardial ischemia. Besides, we will also discuss the role of FoxO in cardiometabolic alterations, autophagy and proteasomal degradation, which are implicated in aging-associated cardiac dysfunction.

FoxO transcription factors 1 regulate mouse preimplantation embryo development.

The aim of the present study is to investigate role of FoxO transcription factors in preimplantation embryo development by knocking down FoxO1, FoxO3, and FoxO4 genes and also to assess cell cycle arrest related proteins, p53 and p21, and apoptosis-related proteins, fas ligand (FASL), and cleaved caspase 3. Knockdown of FoxOs using siRNA was confirmed utilizing RT-PCR and qRT-PCR in gene level and using immunofluorescence in protein level. Following knockdown of FoxO1, FoxO3, and FoxO4 in two-cell mouse embryos with or without resveratrol treatment; developmental competence of embryos and expression patterns of SIRT1, p53, p21, FASL, and CLEAVED CASPASE 3 proteins in embryos by immunofluorescence were assessed after 48h. ROS levels were measured in knockdown embryos. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay was used to determine resveratrol dose. Successful knockdown of FoxO genes in mouse embryos utilizing a non-invasive siRNA method was achieved. Significantly, knockdown of FoxO genes impaired preimplantation embryo development which cannot be prevented by resveratrol treatment. Immunofluorescence results showed that resveratrol could protect embryos from cell cycle arrest and apoptosis. FOXO proteins regulate apoptosis and cell cycle related proteins in mouse preimplantation embryos. Moreover, there might be an autofeedback mechanism where FOXO1, FOXO3, and FOXO4 regulate SIRT1 protein expression. These results suggest that FOXO transcription factors could contribute to mouse preimplantation embryo development, and it remains to investigate whether they have crucial roles in human preimplantation embryo and infertility.

Role of Forkhead Box O Transcription Factors in Oxidative Stress-Induced Chondrocyte Dysfunction: Possible Therapeutic Target for Osteoarthritis?

Chondrocyte dysfunction occurs during the development of osteoarthritis (OA), typically resulting from a deleterious increase in oxidative stress. Accordingly, strategies for arresting oxidative stress-induced chondrocyte dysfunction may lead to new potential therapeutic targets for OA treatment. Forkhead box O (FoxO) transcription factors have recently been shown to play a protective role in chondrocyte dysfunction through the regulation of inflammation, autophagy, aging, and oxidative stress. They also regulate growth, maturation, and matrix synthesis in chondrocytes. In this review, we discuss the recent progress made in the field of oxidative stress-induced chondrocyte dysfunction. We also discuss the protective role of FoxO transcription factors as potential molecular targets for the treatment of OA. Understanding the function of FoxO transcription factors in the OA pathology may provide new insights that will facilitate the development of next-generation therapies to prevent OA development and to slow OA progression.

WNT-Mediated Regulation of FOXO1 Constitutes a Critical Axis Maintaining Pubertal Mammary Stem Cell Homeostasis

Puberty is characterized by dynamic tissue remodeling in the mammary gland involving ductal elongation, resolution into the mature epithelial bilayer, and lumen formation. To decipher the cellular mechanisms underlying these processes, we studied the fate of putative stem cells, termed cap cells, present in terminal end buds of pubertal mice. Employing a p63CreERT2-based lineage-tracing strategy, we identified a unipotent fate for proliferative cap cells that only generated cells with basal features. Furthermore, we observed that dislocated "cap-in-body" cells underwent apoptosis, which aided lumen formation during ductal development. Basal lineage-specific profiling and genetic loss-of-function experiments revealed a critical role for FOXO transcription factors in mediating these proliferative versus apoptotic fates. Importantly, these studies revealed a mode of WNT signaling-mediated FOXO1 inhibition, potentially mediated through AKT. Together, these data suggest that the WNT pathway confers proliferative and survival advantages on cap cells via regulation of FOXO1 localization.

FOXO in B-cell lymphopoiesis and B cell neoplasia.

FOX O family transcription factors are important for differentiation and function of multiple cell types. In B lymphocytes they play a critical role. The activity of FOXOs is directly regulated both by signaling from B cell receptor (BCR) and cytokine receptors. FOXO1 action controls the transition between differentiation stages of B cell development. In comparison to other FOXO family members, FOXO1 plays a superior role in the regulation of early stages of B-cell differentiation. Although being known as a negative regulator of cell proliferation and therefore potential tumor suppressor, FOXO1 is downregulated only in Hodgkin lymphoma (HL) subtypes. In non-Hodgkin lymphoma (NHL) entities its expression is maintained at significant levels, raising the question on the role of FOXO-transcription factors in the proliferation and survival programs in the process of B cell differentiation as well as their contribution to the oncogenic programs of B-cell lymphomas. In particular, we discuss molecular mechanisms that might determine the switch between pro-apoptotic and pro-survival effects of FOXO1 and their interplay with specific differentiation programs.

Role of FOXO transcription factors in crosstalk between mitochondria and the nucleus.

FOXO transcription factors are evolutionally conserved regulators of organismal life span downstream of insulin signaling. After integrating cellular signals from various stimuli such as growth factors, oxidative stress, and energy deprivation, FOXO factors induce expression of a specific set of genes that regulate various cellular processes to maintain homeostasis at a cellular or organismal level. In this review, we discuss roles of FOXO proteins in the maintenance of mitochondria, organelles critical for cellular quality control. FOXO factors protect mitochondria by activating mitochondrial antioxidant enzymes and they help remodel damaged mitochondria by inducing remodeling processes such as mitophagy. Furthermore, we also review the recently identified FOXO-dependent retrograde signaling from stressed mitochondria to the nucleus, which suggest that FOXO mediates the crosstalk between these two important organelles to maintain cell homeostasis. In addition, we introduce a mitohormetic role of gamitrinib-triphenylphosphonium (G-TPP), a mitochondrial heat shock protein (Hsp) inhibitor that can induce mild mitochondrial stress to protect cells from future insults in a FOXO-dependent manner.

Toll-tally tubular: A newly identified Toll-like receptor-FoxO pathway regulates dynamics of the neuronal microtubule network.

Recent studies reveal a conserved role for FoxO transcription factors in establishing neuronal structure and circuit function. In this issue, McLaughlin et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201601014) identify a novel Toll-like receptor–FoxO pathway that represses the mitotic kinesin Pavarotti/MKLP1 to promote dynamic microtubules required for axonal transport and activity-dependent remodeling of presynaptic terminals.

FoxO transcription factors and endurance training: a role for FoxO1 and FoxO3 in exercise-induced angiogenesis.

FoxO transcription factors and endurance training: a role for FoxO1 and FoxO3 in exercise-induced angiogenesis.

FoxO transcription factors support oxidative stress resistance in human chondrocytes.

A major signaling pathway that regulates cellular aging is the insulin/insulin-like growth factor 1 (IGF-1)/phosphatidylinositol 3-kinase (PI3K)/Akt/FoxO transcription factor axis. We previously observed that FoxO transcription factors are dysregulated in aged and OA cartilage. The objective of this study was to investigate the impact of down-regulated FoxO transcription factors on chondrocytes. Small interfering RNAs (siRNAs) targeting FOXO1 (siFOXO1) and FOXO3 (siFOXO3) were transfected into human articular chondrocytes. Cell viability following treatment with the oxidant tert-butyl-hydroperoxide (tBHP) was measured by MTT assay. Caspase 3/7 activation and apoptotic cells were examined. Gene and protein expression of antioxidant proteins and autophagy-related proteins and changes in inflammatory mediators following treatment with interleukin-1β were assessed. Cells transfected with FOXO plasmids were also analyzed. Cell viability was significantly reduced by siFOXO after treatment with tBHP. Apoptosis accompanied by caspase activation was significantly increased in siFOXO-transfected chondrocytes. Knockdown of FOXO1 and FOXO1+3 resulted in significant reductions in levels of glutathione peroxidase 1 (GPX-1), catalase, light chain 3 (LC3), Beclin1, and sirtuin 1 (SIRT-1) proteins following treatment with tBHP. In contrast, the constitutive active form of FOXO3 increased cell viability while inducing GPX-1, Beclin1, and LC3 in response to tBHP. Expression and production of ADAMTS-4 and chemerin were significantly increased in siFOXO-transfected chondrocytes. Reduced expression of FoxO transcription factors in chondrocytes increased susceptibility to cell death induced by oxidative stress. This was associated with reduced levels of antioxidant proteins and autophagy-related proteins. Our data provide evidence for a key role of FoxO transcription factors as regulators of chondrocyte oxidative stress resistance and tissue homeostasis.

FOXO1 repression contributes to block of plasma cell differentiation in classical Hodgkin lymphoma

AbstractThe survival of classical Hodgkin lymphoma (cHL) cells depends on activation of NF-κB, JAK/STAT, and IRF4. Whereas these factors typically induce the master regulator of plasma cell (PC) differentiation PRDM1/BLIMP-1, levels of PRDM1 remain low in cHL. FOXO1, playing a critical role in normal B-cell development, acts as a tumor suppressor in cHL, but has never been associated with induction of PC differentiation. Here we show that FOXO1 directly upregulates the full-length isoform PRDM1α in cHL cell lines. We also observed a positive correlation between FOXO1 and PRDM1 expression levels in primary Hodgkin-Reed-Sternberg cells. Further, we show that PRDM1α acts as a tumor suppressor in cHL at least partially by blocking MYC. Here we provide a link between FOXO1 repression and PRDM1α downregulation in cHL and identify PRDM1α as a tumor suppressor in cHL. The data support a potential role for FOXO transcription factors in normal PC differentiation.

Role of FOXO Transcription Factors in the Protective Mechanism by Resveratrol Against Cardiomyopathy in the Dystrophin-deficient Mdx Mouse

Purpose: FOXO transcription factors (FOXO) transcriptionally regulate anti-oxidant and autophagy-related genes and elicit cellular stress resistance. We previously reported that treatment with resveratrol (RSV), an activator of the deacetylase SIRT1, ameliorates cardiomyopathy in the dystrophin-deficient mdx mouse (MDX), a model of Duchenne muscular dystrophy. Since SIRT1 is known to activate FOXO under stress conditions, we hypothesized that FOXO play roles in protection by RSV in the mdx heart. Methods and Results: In Experiment 1, we analyzed cardiac phenotypes in MDX. At 42-week-old, MDX showed cardiac hypertrophy evaluated by heart weight and echocardiography compared with age-matched C57BL10 mice. Left ventricular (LV) systolic function was preserved in MDX at this age. Dihydroethidium (DHE) staining for analysis of cardiac superoxide anion showed that fluorescence intensity was 4.5-fold higher in MDX than control C57BL10. Immunoblot showed that myocardial p62 and LC3-II levels were increased in MDX at 24-week-old, and this was associated with increased phosphor-S6 level, suggesting impaired autophagy due to mTORC1 activation. In Experiment 2, MDX were divided into untreated (RSV0) and RSV-treated (400 mg/kg chow, RSV400) groups. RSV administration was started at 8-week-old, and mice were sacrificed at 65-week-old. Echocardiography at 62-week-old demonstrated that LV fractional shortening was higher (38±2% vs. 34±1%, P Conclusions: RSV ameliorated cardiomyopathy in the mdx mouse probably by attenuating oxidative stress and restoring autophagy via FOXO3a activation.

Transcription Factor Binding Site Analysis Identifies FOXO Transcription Factors as Regulators of the Cutaneous Wound Healing Process

The search for significantly overrepresented and co-occurring transcription factor binding sites in the promoter regions of the most differentially expressed genes in microarray data sets could be a powerful approach for finding key regulators of complex biological processes. To test this concept, two previously published independent data sets on wounded human epidermis were re-analyzed. The presence of co-occurring transcription factor binding sites for FOXO1, FOXO3 and FOXO4 in the majority of the promoter regions of the most significantly differentially expressed genes between non-wounded and wounded epidermis implied an important role for FOXO transcription factors during wound healing. Expression levels of FOXO transcription factors during wound healing in vivo in both human and mouse skin were analyzed and a decrease for all FOXOs in human wounded skin was observed, with FOXO3 having the highest expression level in non wounded skin. Impaired re-epithelialization was found in cultures of primary human keratinocytes expressing a constitutively active variant of FOXO3. Conversely knockdown of FOXO3 in keratinocytes had the opposite effect and in an in vivo mouse model with FOXO3 knockout mice we detected significantly accelerated wound healing. This article illustrates that the proposed approach is a viable method for identifying important regulators of complex biological processes using in vivo samples. FOXO3 has not previously been implicated as an important regulator of wound healing and its exact function in this process calls for further investigation.

Deregulation of FoxO3a accelerates prostate cancer progression in TRAMP mice

Forkhead box, class "O" (FoxO) transcription factors are involved in multiple signaling pathways and possess tumor suppressor functions. Loss of PTEN and activation of PI3K/Akt is frequently observed in prostate cancer, which may potentially inactivate FoxO activity. We therefore investigated the role of FoxO transcription factors in prostate cancer progression, in particular FoxO3a, in transgenic adenocarcinoma of the mouse prostate (TRAMP) mice, which mimics progressive forms of human disease. Prostate cancer progression in TRAMP mice was followed from 8 to 28 weeks. Expression patterns of Akt, FoxO1a, FoxO3a, FoxO4, and their phosphorylated form, DNA binding activity and downstream signaling molecules during different stages of disease progression were examined by immunoblotting, immunoprecipitation, enzyme-linked immunoabsorbant assay (ELISA), and immunohistochemistry. Inhibition of FoxO3a activity was attained by using FoxO3a peptide treatment to TRAMP mice. In TRAMP mice, FoxO3a activity is negatively regulated by Akt/PKB through post-translational modification. Progressive increase in Akt activation during prostate cancer progression led to increase phosphorylation of FoxO3a and binding with 14-3-3, which potentially affected its transcriptional activity in age-specific manner. Furthermore, blocking FoxO3a activity resulted in accelerated prostate cancer progression in these mice, which was associated with the loss of cell cycle control and increased proliferation and survival markers. Restoration of FoxO3a activity represents an attractive therapeutic target in the chemoprevention and possibly in inhibition of progression of prostate cancer.

Modulation of glutamine metabolism by the PI(3)K–PKB–FOXO network regulates autophagy

The PI(3)K-PKB-FOXO signalling network provides a major intracellular hub for the regulation of cell proliferation, survival and stress resistance. Here we report an unexpected role for FOXO transcription factors in regulating autophagy by modulating intracellular glutamine levels. To identify transcriptional targets of this network, we performed global transcriptional analyses after conditional activation of the key components PI(3)K, PKB/Akt, FOXO3 and FOXO4. Using this pathway approach, we identified glutamine synthetase as being transcriptionally regulated by PI(3)K-PKB-FOXO signalling. Conditional activation of FOXO also led to an increased level of glutamine production. FOXO activation resulted in mTOR inhibition by preventing the translocation of mTOR to lysosomal membranes in a glutamine-synthetase-dependent manner. This resulted in an increased level of autophagy as measured by LC3 lipidation, p62 degradation and fluorescent imaging of multiple autophagosomal markers. Inhibition of FOXO3-mediated autophagy increased the level of apoptosis, suggesting that the induction of autophagy by FOXO3-mediated glutamine synthetase expression is important for cellular survival. These findings reveal a growth-factor-responsive network that can directly modulate autophagy through the regulation of glutamine metabolism.

The role of Forkhead Box O 3a (FoxO3a) Transcription Factors in the Pathogenesis of Pulmonary Fibrosis

Idiopathic Pulmonary Fibrosis (IPF) is a progressive and fatal interstitial pulmonary disease with no proven drug therapy and still unclear signaling mechanisms. Forkhead box O-3a (FoxO3a) transcription factor plays a major role in regulation of cell proliferation and migration. The objectives of this study are to examine how the pro-fibrotic mediators and growth factors regulate FoxO3a protein in primary human lung fibroblasts, as well as to study the role of FoxO transcription factors in the pathogenesis of IPF. We found an increase of phospho-FoxO3a (inactive FoxO3a) protein in IPF lung homogenate compared to control. Under in vitro conditions, qRT-PCR has revealed down regulation of FoxO3 in IPF fibroblasts compared to donor fibroblasts. Pro-fibrotic mediators such as transforming growth factor-ß (TGF-ß), platelet derived growth factor (PDGF)-BB and insulin-like growth factor (IGF)-1 stimulated phosphorylation of FoxO3 in human lung fibroblasts. In addition, PDGF-BB and IGF-1 treatment resulted in nuclear exclusion of FoxO3a. Wortmannin, an inhibitor of PI3K/Akt pathway, blocked PDGF-induced FoxO3a phosphorylation and nuclear exclusion of FoxO3. Paclitaxel, a FoxO3a activator, inhibited TGF-ß, PDGF-BB and IGF-1 induced-down regulation of FoxO3a expression. As well, Paclitaxel inhibited proliferation of donor and IPF fibroblast in a dose-dependent manner. Our primary data suggest that TGF-ß, PDGF-BB and IGF-1 lead to inhibition of FoxO3a transcriptional activity, thereby increasing fibroblast proliferation. FoxO activator drugs, such as Paclitaxel, inhibit fibroblast proliferation and might be used as an anti-fibrotic agent.

Role of Forkhead Transcription Factors in Diabetes-Induced Oxidative Stress

Diabetes is a chronic metabolic disorder, characterized by hyperglycemia resulting from insulin deficiency and/or insulin resistance. Recent evidence suggests that high levels of reactive oxygen species (ROS) and subsequent oxidative stress are key contributors in the development of diabetic complications. The FOXO family of forkhead transcription factors including FOXO1, FOXO3, FOXO4, and FOXO6 play important roles in the regulation of many cellular and biological processes and are critical regulators of cellular oxidative stress response pathways. FOXO1 transcription factors can affect a number of different tissues including liver, retina, bone, and cell types ranging from hepatocytes to microvascular endothelial cells and pericytes to osteoblasts. They are induced by oxidative stress and contribute to ROS-induced cell damage and apoptosis. In this paper, we discuss the role of FOXO transcription factors in mediating oxidative stress-induced cellular response.

Hepatic Suppression of Foxo1 and Foxo3 Causes Hypoglycemia and Hyperlipidemia in Mice

Dysregulation of blood glucose and triglycerides are the major characteristics of type 2 diabetes mellitus. We sought to identify the mechanisms regulating blood glucose and lipid homeostasis. Cell-based studies established that the Foxo forkhead transcription factors Forkhead box O (Foxo)-1, Foxo3, and Foxo4 are inactivated by insulin via a phosphatidylinositol 3-kinase/Akt-dependent pathway, but the role of Foxo transcription factors in the liver in regulating nutrient metabolism is incompletely understood. In this study, we used the Cre/LoxP genetic approach to delete the Foxo1, Foxo3, and Foxo4 genes individually or a combination of two or all in the liver of lean or db/db mice and assessed the role of Foxo inactivation in regulating glucose and lipid homeostasis in vivo. In the lean mice or db/db mice, hepatic deletion of Foxo1, rather than Foxo3 or Foxo4, caused a modest reduction in blood glucose concentrations and barely affected lipid homeostasis. Combined deletion of Foxo1 and Foxo3 decreased blood glucose levels, elevated serum triglyceride and cholesterol concentrations, and increased hepatic lipid secretion and caused hepatosteatosis. Analysis of the liver transcripts established a prominent role of Foxo1 in regulating gene expression of gluconeogenic enzymes and Foxo3 in the expression of lipogenic enzymes. Our findings indicate that Foxo1 and Foxo3 inactivation serves as a potential mechanism by which insulin reduces hepatic glucose production and increases hepatic lipid synthesis and secretion in healthy and diabetic states.