Function Of FoxO Proteins Research Articles

Regulation of mitochondrial function by FOXOs in ischemic stroke and Alzheimer’s disease

Transcriptional control is a pivotal mechanism governing various cellular processes. FOXO proteins, a subgroup of the forkhead family of transcription factors, play a key role in determining cell fate. The localization and function of FOXO proteins are regulated by post-translational modifications to control target gene expression, with a pronounced impact on various aspects of mitochondrial function, including mitochondrial dynamics, biogenesis, and quality control. Mitochondria stand out as the primary target of FOXO transcription factors, which recruit downstream signaling factors to govern mitochondrial processes. Essential signaling pathways are modulated by FOXOs, exemplified by their regulation of mitochondrial biogenesis through SIRT1-Pgc1a and NRF1-TFAM, as well as their influence on mitochondrial dynamics involving Mfn1, Mfn2, Drp1, and Fis1. Furthermore, FOXOs demonstrate the ability to upregulate and downregulate genes that serve as modulators in oxidative and apoptosis cascades. The functional role of FOXO proteins is highly context-dependent, varying with cell type, organ, and specific FOXO isoform. Notably, FOXOs emerge as prominent players in various pathological conditions, including ischemic conditions, neurodegenerative diseases, cancer, and metabolic disorders. Unraveling the complex role of FOXOs in mammalian cell pathology positions them as promising therapeutic targets receptive to pharmacological treatment. This review aims to provide insights into the intricate roles of FOXOs in mitochondria, illuminating their potential as therapeutic targets amenable to pharmacological intervention in diverse pathological contexts, particularly in ischemic stroke and Alzheimer’s disease.

MiR-128-3p regulates chicken granulosa cell function via 14-3-3β/FoxO and PPAR-γ/LPL signaling pathways

MicroRNAs (miRNAs) are class of 22 nt short RNA sequences which inhibit protein translation through binding to the 3’UTR of its target genes. The continuous ovulatory property of chicken follicle makes it a perfect model for studying granulosa cell (GC) functions. In this study, we found that large number of miRNAs including miR-128-3p, were differentially expressed in the GCs of F1 and F5 follicles of chicken. Subsequently, the results revealed that miR-128-3p inhibited proliferation, the formation of lipid droplets, and hormone secretion in chicken primary GCs through directly targeting YWHAB and PPAR-γ genes. To determine the effects of 14-3-3β (encoded by YWHAB) protein on GCs functions, we overexpressed or inhibited the expression of YWHAB, and the results showed that YWHAB inhibited the function of FoxO proteins. Collectively, we found that miR-128-3p was highly expressed in the chicken F1 follicles compared to the F5 follicles. In addition, the results indicated that miR-128-3p promoted GC apoptosis through 14-3-3β/FoxO pathway via repressing YWHAB, and inhibited lipid synthesis by impeding the PPAR-γ/LPL pathway, as well as reduced the secretion of progesterone and estrogen. Taken together, the results showed that miR-128-3p plays a regulatory role in chicken granulosa cell function via 14-3-3β/FoxO and PPAR-γ/LPL signaling pathways.

Forkhead Domains of FOXO Transcription Factors Differ in both Overall Conformation and Dynamics.

FOXO transcription factors regulate cellular homeostasis, longevity and response to stress. FOXO1 (also known as FKHR) is a key regulator of hepatic glucose production and lipid metabolism, and its specific inhibition may have beneficial effects on diabetic hyperglycemia by reducing hepatic glucose production. Moreover, all FOXO proteins are considered potential drug targets for drug resistance prevention in cancer therapy. However, the development of specific FOXO inhibitors requires a detailed understanding of structural differences between individual FOXO DNA-binding domains. The high-resolution structure of the DNA-binding domain of FOXO1 reported in this study and its comparison with structures of other FOXO proteins revealed differences in both their conformation and flexibility. These differences are encoded by variations in protein sequences and account for the distinct functions of FOXO proteins. In particular, the positions of the helices H1, H2 and H3, whose interface form the hydrophobic core of the Forkhead domain, and the interactions between hydrophobic residues located on the interface between the N-terminal segment, the H2-H3 loop, and the recognition helix H3 differ among apo FOXO1, FOXO3 and FOXO4 proteins. Therefore, the availability of apo structures of DNA-binding domains of all three major FOXO proteins will support the development of FOXO-type-specific inhibitors.

The functions of FoxO transcription factors in epithelial wound healing.

Re-epithelialisation is a complex process that involves the migration and proliferation of keratinocytes, as well as the production of cytokines and growth factors that affect wound healing. The precise mechanisms that control the tissue repair process remain poorly understood. Recent evidence indicates that FoxO transcription factors play a crucial role in wound healing. In mammals, different isoforms of FoxO, namely, FoxO1, FoxO3, FoxO4 and FoxO6, are present; however, FoxO1 and FoxO3 primarily function in epithelial wound healing. The functions of FoxO proteins in normal wound healing are opposite of those in diabetic wound healing. On the one hand, FoxO transcription factors promote the migration of keratinocytes through up-regulating the expression of transforming growth factor-beta and protecting keratinocytes from oxidative stress. On the other hand, FoxO transcription factors negatively regulate some genes that are needed for re-epithelialisation and keratinocyte migration. This review provides a summary of the functions of FoxO proteins in re-epithelialisation.

Mechanisms controlling the anti-neoplastic functions of FoxO proteins.

The Forkhead box O (FoxO) proteins comprise a family of evolutionarily conserved transcription factors that predominantly function as tumor suppressors. These proteins assume diverse roles in the cellular anti-neoplastic response, including regulation of apoptosis and autophagy, cancer metabolism, cell-cycle arrest, oxidative stress and the DNA damage response. More recently, FoxO proteins have been implicated in cancer immunity and cancer stem-cell (CSC) homeostasis. Interestingly, in some sporadic sub-populations, FoxO protein function may also be manipulated by factors such as β-catenin whereby they instead can facilitate cancer progression via maintenance of CSC properties or promoting drug resistance or metastasis and invasion. This review highlights the essential biological functions of FoxOs and explores the areas that may be exploited in FoxO protein signaling pathways in the development of novel cancer therapeutic agents.

Abstract A121: Suppression of orthotopic pancreatic cancer growth by sulforaphane is associated with activation of FOXO transcription factors

Abstract FoxO (forkhead box O) transcription factors are crucial regulators of cell fate. This function of FoxO proteins relies on their ability to control diverse functions such as proliferation, differentiation, DNA repair and apoptosis. Constitutively active AKT and ERK in cancer cells have been shown to phosphorylate FoxO transcription factors (FKHR, FKHRL1 and AFX), which bind to protein 14-3-3 and sequester in the cytoplasm. In the absence of survival signals, FoxO proteins dephosphorylate, translocate to the nucleus and upregulate a series of target genes, thereby promoting cell cycle arrest and apoptosis. Sulforaphane a naturally occurring isothiocyanate, exerts anti cancer effects through diverse mechanisms. In this study we have shown that sulforaphane acts both by suppressing cell proliferation and inducing apoptosis in pancreatic tumors orthotopically implanted in nude mice. Sulforaphane regulates cell survival and cell death by modulating the expression of two interacting survival signaling pathways. The inhibition of PI3K/AKT and MEK/ERK pathways by sulforaphane resulted in the inhibition of cytoplasmic phosphorylation of FOXOs, and enhanced their nuclear translocation and transcriptional activities leading to apoptosis. Furthermore, deletion of FOXO1, FOXO3 and FOXO4 genes by shRNA abrogated sulforaphane-induced cell cycle arrest and apoptosis whereas the phosphorylation deficient triple mutants of FOXOs enhanced sulforaphane-induced FOXO transcriptional activity and apoptosis by nuclear translocation of FOXO. Sulforaphane-treated mice showed significant inhibition in tumor growth which was associated with reduced phosphorylation of ERK, PI3K, AKT, FOXO1 and FOXO3a, cyclin D1 and induction of FOXO’s target genes Bim, p21/CIP1, and p27/KIP1. These data suggest that inhibition of ERK and AKT pathways act together to activate FOXO transcription factors which are involved in sulforaphane-mediated pancreatic tumor growth suppression, thus supporting the use of sulforaphane as a chemopreventive agent for the treatment of pancreatic cancer. Citation Format: Wei Yu, Rakesh Srivastava, Sharmila Shankar. Suppression of orthotopic pancreatic cancer growth by sulforaphane is associated with activation of FOXO transcription factors. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr A121.

FoxO3a Serves as a Biomarker of Oxidative Stress in Human Lens Epithelial Cells under Conditions of Hyperglycemia

BackgroundForkhead box ‘O’ transcription factors (FoxOs) are implicated in the pathogenesis of type2 diabetes and other metabolic diseases. Abnormal activity of FoxOs was reported in the glucose and insulin metabolism. Expression of FoxO proteins was reported in ocular tissues; however their function under hyperglycemic conditions was not examined.MethodsHuman lens epithelial cell line was used to study the function of FoxO proteins. Immunofluorescence, flow cytometry and Western blotting were employed to detect the FoxO proteins under the conditions of hyperglycemia.ResultsIn this study we examined the role of FoxO3a in hyperglycemia-induced oxidative stress in human lens epithelial cells. FoxO3a protein expression was elevated in a dose- and time-dependent fashion after high glucose treatment. Anti-oxidant defense mechanisms of the lens epithelial cells were diminished as evidenced from loss of mitochondrial membrane integrity and lowered MnSOD after 72 h treatment with high glucose. Taken together, FoxO3a acts as a sensitive indicator of oxidative stress and cell homeostasis in human lens epithelial cells during diabetic conditions.ConclusionFoxO3a is an early stress response protein to glucose toxicity in diabetic conditions.

Abstract 3058: miRNA networks mediate the tumor suppressive function of FOXO proteins.

Abstract FOXO transcription factors, as regulators of a variety of cellular functions, including promotion of apoptotic cell death, are negatively regulated by various oncogenic signaling cascades and have emerged as bona fide tumor suppressors. Indeed, studies by others and us have shown that the suppression of FOXO function is critical in promoting evasion of apoptosis and is a significant occurrence in several hematological malignancies. Consequently, FOXO proteins signify an important therapeutic target in hematopoietic malignancies as well as other types of cancer, where repression of FOXO plays a key role in malignancy. Since transcription factors are not yet easily “druggable," it is vital to identify mediators of FOXO function that can be readily targeted. It is now apparent that aberrant miRNA expression plays a major role in cell and tissue-specific manner in the pathogenesis of various types of cancers including hematological malignancies. Both miRNA loss of function and miRNA overexpression have been shown to target key cellular proteins and contribute to cancer pathogenesis. Conversely, miRNA genes themselves have been shown to be direct targets of transcription factors that are known as important tumor suppressors or oncogenic proteins. Through a systematic screening approach, we have identified members of three miRNA clusters that are dependent on FOXO transcription factors for expression. We have observed that the level of these miRNAs is dependent on BCR-ABL status of hematopoeitc cells and they their levels are changed in specimens from Leukemia patients. Our studies indicates a mechanism of action for FOXO tumor suppressors whereby members of two miRNA families are direct transcriptional targets of FOXO3. Moreover, these miRNAs target and reduce the expression of two key oncogenic factors, the serine/threonine kinase mTOR, and proto-onocogene c-myc, to keep cell growth at bay and prevent tumorigenesis. Our investigations have unraveled the miRNA arm of the FOXO tumor suppression network. Citation Format: Monica Nadler, Robin Zabolian, Kenneth Ndebele, thierry bertomeu, Octavian Bucur, Bodvael Pennarun, Roya Khosravi-Far. miRNA networks mediate the tumor suppressive function of FOXO proteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3058. doi:10.1158/1538-7445.AM2013-3058 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Haplotypes of bovine FoxO1 gene sequence variants and association with growth traits in Qinchuan cattle

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).

FoxO3 hastens autophagy and shrinks the heart but does not curtail pathological hypertrophy in adult mice

This editorial refers to ‘FoxO3 induces reversible cardiac atrophy and autophagy in a transgenic mouse model’ by T.G. Schips et al ., doi:10.1093/cvr/cvr144 The FoxO subfamily of Forkhead box transcription factors regulate many target genes that are involved in diverse cellular processes. FoxO proteins show partial functional redundancy due to the conserved DNA-binding domain, but also exhibit isoform specificity with regard to cellular function.1 Three of the four FoxO isoforms—FoxO1, FoxO3, and FoxO4, but not FoxO6—are expressed in the heart. The biological functions of FoxO proteins in the heart have been mainly revealed by studies on FoxO1 and FoxO3 using either cultured cardiomyocytes or loss-of-function animal models. Combined loss of FoxO1 and FoxO3 in the heart does not affect cardiac development,1 suggesting functional redundancy of FoxO proteins in regulating cardiac development. Indeed, loss of all three FoxO isoforms is required for an oncogenic phenotype in mice.2 So far, a viable animal model of FoxO gain-of-function is lacking, hindering a better understanding on the pathophysiological significance of FoxO proteins in adult hearts. Schips et al .3 report the first mouse model of cardiac-specific and temporally controllable …

Abstract 859: EGCG inhibits pancreatic cancer growth and stem cell characteristics in xenografts and KrasG12D transgenic mice models

Abstract FoxO (forkhead box O) transcription factors are crucial regulators of cell fate. This function of FoxO proteins relies on their ability to control diverse functions such as proliferation, differentiation, DNA repair and apoptosis. Constitutively active AKT and ERK in cancer cells have been shown to phosphorylate FoxO transcription factors (FKHR, FKHRL1 and AFX), which bind to protein 14-3-3 and sequester in the cytoplasm. In the absence of survival signals, FoxO proteins dephosphorylate, translocate to the nucleus and upregulate a series of target genes, thereby promoting cell cycle arrest and apoptosis. EGCG, a component of green tea, exerts antiproliferative and proapoptotic actions on a number of different cancer cell lines through diverse mechanisms. However, the mechanisms by which EGCG inhibits cell proliferation and induces apoptosis are poorly understood. EGCG inhibited viability and induced apoptosis in human pancreatic cancer cell lines. EGCG induces PTEN expression and inhibits Akt phosphorylation in pancreatic cancer Hs766T, AsPC-1, MIA PaCa-2 and PANC-1 cells. Treatment of PANC-1 cells with EGCG caused a decrease in ERK phosphorylation, and an increase in JNK phosphorylation. Overexpression of FOXO1, FOXO3a, and FOXO4 inhibited cell viability in PANC-1 cells. The inhibitory effects of EGCG on cell viability were further enhanced when cells were transfected with FOXO1, FOXO3a, and FOXO4. EGCG enhanced FOXO-DNA binding and transcriptional activities. Interestingly, EGCG inhibited pancreatic cancer growth and/or development in xenograft, orthotopic and KrasG12D transgenic mice models. EGCG also inhibited pancreatic cancer stem cell characteristics. In conclusion, our data demonstrate that EGCG can inhibit pancreatic cancer growth and/or development through multiple mechanisms and thus can be used for the management of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 859. doi:10.1158/1538-7445.AM2011-859

Linking Alzheimer's disease to insulin resistance: the FoxO response to oxidative stress

Oxidative stress is an important determinant not only in the pathogenesis of Alzheimer's disease (AD), but also in insulin resistance (InsRes) and diabetic complications. Forkhead box class O (FoxO) transcription factors are involved in both insulin action and the cellular response to oxidative stress, thereby providing a potential integrative link between AD and InsRes. For example, the expression of intra- and extracellular antioxidant enzymes, such as manganese-superoxide dismutase and selenoprotein P, is regulated by FoxO proteins, as is the expression of important hepatic enzymes of gluconeogenesis. Here, we review the molecular mechanisms involved in the pathogenesis of AD and InsRes and discuss the function of FoxO proteins in these processes. Both InsRes and oxidative stress may promote the transcriptional activity of FoxO proteins, resulting in hyperglycaemia and a further increased production of reactive oxygen species (ROS). The consecutive activation of c-Jun N-terminal kinases and inhibition of Wingless (Wnt) signalling may result in the formation of β-amyloid plaques and τ protein phosphorylation. Wnt inhibition may also result in a sustained activation of FoxO proteins with induction of apoptosis and neuronal loss, thereby completing a vicious circle from oxidative stress, InsRes and hyperglycaemia back to the formation of ROS and consecutive neurodegeneration. In view of their central function in this model, FoxO proteins may provide a potential molecular target for the treatment of both InsRes and AD.

Stimulation of Sirt1-Regulated FoxO Protein Function by the Ligand-Bound Vitamin D Receptor

Hormonal vitamin D, 1,25-dihydroxyvitamin D (1,25D), signals through the nuclear vitamin D receptor (VDR). 1,25D regulates cell proliferation and differentiation and has been identified as a cancer chemopreventive agent. FoxO proteins are transcription factors that control cell proliferation and survival. They function as tumor suppressors and are associated with longevity in several organisms. Accumulating data have revealed that 1,25D and FoxO proteins regulate similarly common target genes. We show here that the ligand-bound VDR regulates the posttranslational modification and function of FoxO proteins. 1,25D treatment enhances binding of FoxO3a and FoxO4 within 4 h to promoters of FoxO target genes and blocks mitogen-induced FoxO protein nuclear export. The VDR associates directly with FoxO proteins and regulators, the sirtuin 1 (Sirt1) class III histone deacetylase (HDAC), and protein phosphatase 1. In addition, phosphatase activity and trichostatin A-resistant HDAC activity coimmunoprecipitate with the VDR. 1,25D treatment rapidly (in <4 h) induces FoxO deacetylation and dephosphorylation, consistent with activation. In contrast, ablation of VDR expression enhances FoxO3a phosphorylation, as does knockdown of Sirt1, consistent with the coupling of FoxO acetylation and phosphorylation. 1,25D regulation of common VDR/FoxO target genes is attenuated by blockade of phosphatase activity or by small interfering RNA (siRNA)-mediated knockdown of Sirt1 or FoxO protein expression. Finally, 1,25D-dependent cell cycle arrest is blocked in FoxO3a-deficient cells, indicating that FoxO proteins are key downstream mediators of the antiproliferative actions of 1,25D. These studies link 1,25D signaling through the VDR directly to Sirt1 and FoxO function and provide a molecular basis for the cancer chemopreventive actions of 1,25D.

Abstract 2868: EGCG inhibits pancreatic cancer growth in xenografts and KrasG12D transgenic mice models

Abstract FoxO (forkhead box O) transcription factors are crucial regulators of cell fate. This function of FoxO proteins relies on their ability to control diverse functions such as proliferation, differentiation, DNA repair and apoptosis. Constitutively active AKT and ERK in cancer cells have been shown to phosphorylate FoxO transcription factors (FKHR, FKHRL1 and AFX), which bind to protein 14-3-3 and sequester in the cytoplasm. In the absence of survival signals, FoxO proteins dephosphorylate, translocate to the nucleus and upregulate a series of target genes, thereby promoting cell cycle arrest and apoptosis. EGCG, a component of green tea, exerts antiproliferative and proapoptotic actions on a number of different cancer cell lines through diverse mechanisms. However, the mechanisms by which EGCG inhibits cell proliferation and induces apoptosis are poorly understood. EGCG inhibited viability and induced apoptosis in human pancreatic cancer cell lines. EGCG induces PTEN expression and inhibits Akt phosphorylation in pancreatic cancer Hs766T, AsPC-1, MIA PaCa-2 and PANC-1 cells. Treatment of PANC-1 cells with EGCG caused a decrease in ERK phosphorylation, and an increase in JNK phosphorylation. Overexpression of FOXO1, FOXO3a, and FOXO4 inhibited cell viability in PANC-1 cells. The inhibitory effects of EGCG on cell viability were further enhanced when cells were transfected with FOXO1, FOXO3a, and FOXO4. EGCG enhances FOXO-DNA binding activity. EGCG-induced FOXO transcriptional activity was further enhanced in the presence of FOXO1-TM, FOXO3a-TM, and FOXO4-TM. Interestingly, EGCG inhibited pancreatic cancer growth in xenograft, orthotopic and KrasG12D transgenic mice models. In conclusion, our data demonstrate that EGCG can inhibit pancreatic cancer growth through multiple mechanisms and thus can be used for the pancreatic cancer prevention. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2868.

FoxO Proteins Mediate Hypoxic Induction of Connective Tissue Growth Factor in Endothelial Cells

Hypoxia, a driving force in neovascularization, promotes alterations in gene expression mediated by hypoxia-inducible factor (HIF)-1alpha. Connective tissue growth factor (CTGF, CCN2) is a modulator of endothelial cell growth and migration, but its regulation by hypoxia is poorly understood. Therefore, we analyzed signaling pathways involved in the regulation of CTGF by hypoxia in endothelial cells. Exposure to low oxygen tension or treatment with the hypoxia-mimetic dimethyloxalyl glycine (DMOG) stabilized HIF-1alpha and up-regulated CTGF in human umbilical vein endothelial cells and in a murine microvascular endothelial cell line. Induction of CTGF correlated with a HIF-dependent increase in protein and mRNA levels, and nuclear accumulation of the transcription factor FoxO3a. By contrast, gene expression and cellular localization of FoxO1 were not significantly altered by hypoxia. Expression of CTGF was strongly reduced by siRNA silencing of FoxO1 or FoxO3a. Furthermore, nuclear exclusion of FoxO1/3a transcription factors by inhibition of serine/threonine protein phosphatases by okadaic acid inhibited CTGF expression, providing evidence for both FoxO proteins as regulators of CTGF expression. The DMOG-stimulated induction of CTGF was further increased when endothelial cells were co-incubated with transforming growth factor-beta, an activator of Smad signaling. Activation of RhoA-Rho kinase signaling by the microtubule-disrupting drug combretastatin A4 also enhanced the DMOG-induced CTGF expression, thus placing CTGF induction by hypoxia in a network of interacting signaling pathways. Our findings provide evidence that FoxO1, hypoxia-stimulated expression of FoxO3a and its nuclear accumulation are required for the induction of CTGF by hypoxia in endothelial cells.

Cyclin D1 promotes anchorage-independent cell survival by inhibiting FOXO-mediated anoikis

O-class forkhead box (FOXO) transcription factors are critical regulators of diverse cellular processes, including apoptosis, cell-cycle arrest, DNA damage repair and oxidative stress resistance. Here, we show that FOXO1 and FOXO3a have an essential function in promoting cell detachment-induced anoikis, resistance to which is implicated in cancer development and metastasis. In contrast, the oncoprotein cyclin D1 inhibits anoikis. We further show that cyclin D1 interacts with FOXO proteins and impedes their transcriptional regulatory and anoikis-promoting functions. This effect of cyclin D1 requires its transcription repression domain but is independent of cyclin-dependent kinases CDK4 and CDK6. Moreover, we show that cancer-derived mutants of cyclin D1 are much more stable than wild-type cyclin D1 under anchorage-independent conditions and possess a greater antagonistic effect on FOXO-regulated anoikis and anchorage-independent growth of cancer cells. These data suggest that cyclin D1 may have a critical function in tumorigenesis and cancer metastasis by inhibiting the anoikis-promoting function of FOXO proteins.

Expression of Foxo transcription factors during osteoblast differentiation and cell death

Foxo transcription factors regulate the differentiation of adipocytes and myoblasts. However less is known regarding their role in the differentiation of other mesenchyme‐derived cell types such as osteoblasts and chondrocytes. Here we examined the expression levels of Foxo protein and mRNA during osteo‐ and chondrogenesis in vitro. Levels of Foxo1a and Foxo4 protein remained relatively unchanged throughout osteoblast differentiation of MC3T3 cells. Low levels of Foxo3a protein were detected in undifferentiated MC3T3 cells however, Foxo3a increased 1 day after the onset of differentiation. Foxo3a mRNA and protein levels also increased following BMP2 treatment of mouse primary limb bud micromass cultures. Since Foxo proteins play critical roles in mediating cell survival following oxidative stress, we assayed the expression of Foxo proteins following H2O2 treatment. MC3T3 apoptosis was detected by caspase 3 cleavage in cells treated H2O2 and resulted in a decrease in Foxo1a. Finally the affect of modulating Sirt1 activity and Foxo protein function, on survivial of MC3T3‐E1 cells was examined. H2O2‐mediated apoptosis increased in MC3T3‐E1 cells when treated with the Sirt1 agonists, while treatment with Sirt1 inhibitors decreased apoptosis. Together these findings indicate the Foxo3a and Foxo1a participate specific processes governing differentiation and apoptosis of osteoblast cells in vitro.

FoxO proteins: cunning concepts and considerations for the cardiovascular system

Dysfunction in the cardiovascular system can lead to the progression of a number of disease entities that can involve cancer, diabetes, cardiac ischaemia, neurodegeneration and immune system dysfunction. In order for new therapeutic avenues to overcome some of the limitations of present clinical treatments for these disorders, future investigations must focus upon novel cellular processes that control cellular development, proliferation, metabolism and inflammation. In this respect, members of the mammalian forkhead transcription factors of the O class (FoxOs) have increasingly become recognized as important and exciting targets for disorders of the cardiovascular system. In the present review, we describe the role of these transcription factors in the cardiovascular system during processes that involve angiogenesis, cardiovascular development, hypertension, cellular metabolism, oxidative stress, stem cell proliferation, immune system regulation and cancer. Current knowledge of FoxO protein function combined with future studies should continue to lay the foundation for the successful translation of these transcription factors into novel and robust clinical therapies.

Dynamic FoxO transcription factors

Forkhead box O (FoxO) transcription factors FoxO1, FoxO3a, FoxO4 and FoxO6, the mammalian orthologs of Caenorhabditis elegans DAF-16, are emerging as an important family of proteins that modulate the expression of genes involved in apoptosis, the cell cycle, DNA damage repair, oxidative stress, cell differentiation, glucose metabolism and other cellular functions. FoxO proteins are regulated by multiple mechanisms. They undergo inhibitory phosphorylation by protein kinases such as Akt, SGK, IKK and CDK2 in response to external and internal stimuli. By contrast, they are activated by upstream regulators such as JNK and MST1 under stress conditions. Their activities are counterbalanced by the acetylases CBP and p300 and the deacetylase SIRT1. Also, whereas polyubiquitylation of FoxO1 and FoxO3a leads to their degradation by the proteasome, monoubiquitylation of FoxO4 facilitates its nuclear localization and augments its transcriptional activity. Thus, the potent functions of FoxO proteins are tightly controlled by complex signaling pathways under physiological conditions; dysregulation of these proteins may ultimately lead to disease such as cancer.