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Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues
Abstract
daf-16 is a forkhead-type transcription factor, functioning downstream of insulin-like signals, and is known to be critical to the regulation of life span in Caenorhabditis elegans. Mammalian DAF-16 homologues include AFX, FKHR and FKHRL1, which contain a conserved forkhead domain and three putative phosphorylation sites for the Ser/Thr kinase Akt/protein kinase B (PKB), as well as for DAF-16. To assess the function of the homologues, we examined tissue distribution patterns of mRNAs for DAF-16 homologues in mice. In the embryos, expressions of AFX, FKHR and FKHRL1 mRNAs were complementary to each other and were highest in muscle, adipose tissue and embryonic liver. The characteristic expression pattern remained in the adult, except that signals of FKHRL1 became evident in more tissues, including the brain. In order to clarify whether each DAF-16 homologue had different target genes, we determined the consensus sequences for the binding of DAF-16 and the mouse homologues. The binding sequences for all four proteins shared a core sequence, TTGTTTAC, daf-16 family protein-binding element (DBE) binding protein. However, electrophoretic mobility shift assay showed that the binding affinity of DAF-16 homologues to the core sequence was stronger than that to the insulin-responsive element in the insulin-like growth factor binding protein-1 promoter region, which has been identified as a binding sequence for them. We identified one copy of the DBE upstream of the first exon of sod-3 by searching the genomic database of C. elegans. Taken together, DAF-16 homologues can fundamentally regulate the common target genes in insulin-responsive tissues and the specificity to target genes of each protein is partially determined by the differences in their expression patterns.
... The Forkhead box, a 110 a.a. region located in the N-terminal part of FOXO protein, can bind to DNA containing two of the consensus recognition DNA sequences: DAF-16 family member-binding element (5′-GTAA(C/T)A-3′) [28] or insulin-responsive element (5′-(C/A)(A/C)AAA(C/T)AA-3′). All of the members in the FOXO family can recognize the core DNA sequence (5′-(A/C)AA(C/T)A-3′) [29]. ...
... The Forkhead box, a 110 a.a. region located in the N-terminal part of FOXO protein, can bind to DNA containing two of the consensus recognition DNA sequences: DAF-16 family member-binding element (5 -GTAA(C/T)A-3 ) [28] or insulin-responsive element (5 -(C/A)(A/C)AAA(C/T)AA-3 ). All of the members in the FOXO family can recognize the core DNA sequence (5 -(A/C)AA(C/T)A-3 ) [29]. ...
The prevalence of nonalcoholic fatty liver disease (NAFLD) worldwide is on the rise and NAFLD is becoming the most common cause of chronic liver disease. In the USA, NAFLD affects over 30% of the population, with similar occurrence rates reported from Europe and Asia. This is due to the global increase in obesity and type 2 diabetes mellitus (T2DM) because patients with obesity and T2DM commonly have NAFLD, and patients with NAFLD are often obese and have T2DM with insulin resistance and dyslipidemia as well as hypertriglyceridemia. Excessive accumulation of triglycerides is a hallmark of NAFLD and NAFLD is now recognized as the liver disease component of metabolic syndrome. Liver glucose and lipid metabolisms are intertwined and carbon flux can be used to generate glucose or lipids; therefore, in this review we discuss the important transcription factors and coactivators that regulate glucose and lipid metabolism.
... The functions of p53 are closely intertwined with the activity of Forkhead box O (FOXO) transcription factors. FOXO proteins (FOXO1, FOXO3, FOXO4, and FOXO6) regulate cellular homeostasis, longevity and stress response by controlling the transcription of target genes when binding to the consensus recognition motif 5 0 -GTAAA(T/C)AA-3 0 , also known as the Daf-16 family member-binding element (DBE), 18,19 and to the 5 0 -(C/A)(A/C)AAA(C/T)AA-3 0 motif, located in the IGFBP-1 promoter region and known as the insulin-responsive element (IRE). 20 FOXO1 and FOXO3 proteins have similar lengths of 655 and 673 amino acid residues, whereas FOXO4 and FOXO6 sequences are shorter and contain 505 and 492 residues, respectively ( Figure S1). ...
... This motif contains two 5 0 -AACA-3 0 repeats, and this sequence is also present in motifs recognized by FOXO proteins. [18][19][20] As a result, FOXO4 1-505 and p53 1-393 bind to the motif with similar affinity. ...
Transcription factor p53 protects cells against tumorigenesis when subjected to various cellular stresses. Under these conditions, p53 interacts with transcription factor Forkhead box O (FOXO) 4, thereby inducing cellular senescence by upregulating the transcription of senescence‐associated protein p21. However, the structural details of this interaction remain unclear. Here, we characterize the interaction between p53 and FOXO4 by NMR, chemical cross‐linking, and analytical ultracentrifugation. Our results reveal that the interaction between p53 TAD and the FOXO4 Forkhead domain is essential for the overall stability of the p53:FOXO4 complex. Furthermore, contacts involving the N‐terminal segment of FOXO4, the C‐terminal negative regulatory domain of p53 and the DNA‐binding domains of both proteins stabilize the complex, whose formation blocks p53 binding to DNA but without affecting the DNA‐binding properties of FOXO4. Therefore, our structural findings may help to understand the intertwined functions of p53 and FOXO4 in cellular homeostasis, longevity, and stress response.
... Because one of the candidate regions for TRRAP binding comprised the C-terminal TA domain, we next analyzed whether TRRAP overexpression may influence FoxO transactivation. To this end, we performed reporter assays using an established FoxO-responsive 6xDBE-luc reporter that consists of six tandem repeats of the consensus binding sequence for DAF-16 cloned in front of a Firefly luciferase reporter (26). Figure 3A illustrates that 4-OHT treatment expectedly resulted in a strong induction of the 6xDBE-luc reporter in HUVECs transfected with a combination of FoxO3.A3.ER and empty vector. ...
... The transient expression construct for FLAG-tagged TRRAP protein and the corresponding empty vector plasmid were a kind gift from Michael Cole, Geisel School for Medicine (Addgene plasmids: pCbS-FLAG [catalog no.: 32104] and pCbS-FLAG-TRRAP [catalog no.: 32103]) and have been described previously (32). Similarly, the pMT2-HA.FoxO4.A3, pMT2-HA.FoxO4.DB, pMT2-HA.FoxO4.ΔDB, pCDNA3-HA.FoxO3.A3.ER, pECE-HA.FoxO3 wt, and the 6xDBE-luc reporter constructs for transfections have been described previously (9,10,14,26). ...
Forkhead Box O (FoxO) transcription factors are conserved proteins involved in the regulation of life span and age-related diseases, such as diabetes and cancer. Stress stimuli or growth factor deprivation promotes nuclear localization and activation of FoxO proteins, which - depending on the cellular context - can lead to cell cycle arrest or apoptosis. In endothelial cells (ECs), they additionally regulate angiogenesis and may promote inflammation and vessel destabilization implicating a role of FoxOs in vascular diseases. In several cancers, FoxO transcription factors exert a tumor-suppressive function, due to their critical role in regulating proliferation and survival. We and others have previously shown that FoxOs can regulate these processes via two different mechanisms: by direct binding to FoxO-responsive elements (FRE) at the promoter of target genes, or by a poorly understood alternative process that does not require direct DNA binding and regulates key targets in primary human ECs. Here we performed an interaction study in ECs to identify new nuclear FoxO3 interaction partners that might contribute to FoxO-dependent gene regulation. Mass spectrometry analysis of FoxO3- interacting proteins revealed Transformation/Transcription Domain-Associated Protein (TRRAP), a member of multiple histone acetyltransferase (HAT) complexes, as a novel binding partner of FoxO family proteins. We demonstrate that TRRAP is required to support FoxO3 transactivation and FoxO3-dependent G1 arrest and apoptosis in ECs via transcriptional activation of cyclin-dependent kinase inhibitor p27kip1 and the proapoptotic Bcl-2 family member BIM. Moreover, FoxO-TRRAP interaction could explain FoxO-induced alternative gene regulation via TRRAP-dependent recruitment to target promoters lacking FRE sequences.
... The CR3 domain is also known as the transactivation domain (TAD). Only the FHD, which binds to two conserved DNA sequences, 5' -TTGTTTAC −3', named the Daf-16 family member-binding element [21,22], and 5' -(C/A)(A/C) AAA(C/T)AA −3', named the insulin-responsive sequence [22], has a defined three-dimensional structure. A previous study suggested that FHD and CR3 interact to form an intramolecular complex, and CR3 kinetically affects selective DNA recognition of FHD [23]. ...
... The CR3 domain is also known as the transactivation domain (TAD). Only the FHD, which binds to two conserved DNA sequences, 5' -TTGTTTAC −3', named the Daf-16 family member-binding element [21,22], and 5' -(C/A)(A/C) AAA(C/T)AA −3', named the insulin-responsive sequence [22], has a defined three-dimensional structure. A previous study suggested that FHD and CR3 interact to form an intramolecular complex, and CR3 kinetically affects selective DNA recognition of FHD [23]. ...
Cellular senescence is protective against external oncogenic stress, but its accumulation causes aging‐related diseases. Forkhead box O4 (FOXO4) and p53 are human transcription factors known to promote senescence by interacting with each other and activating p21 transcription. Inhibition of the interaction is a strategy for inducing apoptosis of senescent cells, but the binding surfaces that mediate the FOXO4–p53 interaction remain elusive. Here, we investigated two binding sites involved in the interaction between FOXO4 and p53 by NMR spectroscopy. NMR chemical shift perturbation analysis showed that the binding between FOXO4’s forkhead domain (FHD) and p53’s transactivation domain (TAD), and between FOXO4’s C‐terminal transactivation domain (CR3) and p53’s DNA‐binding domain (DBD), mediate the FOXO4–p53 interaction. Isothermal titration calorimetry data showed that both interactions have micromolar Kd values, and FOXO4 FHD–p53 TAD interaction has a higher binding affinity. We also showed that the intramolecular CR3‐binding surface of FOXO4 FHD interacts with p53 TAD2, and FOXO4 CR3 interacts with the DNA/p53 TAD‐binding surface of p53 DBD, suggesting a network of potentially competitive and/or coordinated interactions. Based on these results, we propose that a network of intramolecular and intermolecular interactions contributes to the two transcription factors’ proper localisation on the p21 promoter and consequently promotes p21 transcription and cell senescence. This work provides structural information at the molecular level that is key to understanding the interplay of two proteins responsible for cellular senescence.
... FOXO3, a member of the FOXO family located downstream of the PI3K/AKT pathway, binds to the DNA consensus sequence as a transcription factor [34]. FOXO3 promotes apoptosis by upregulating the pro-apoptotic factors-BIM and PUMA, and downregulating the anti-apoptotic factors-FLIP and BCL2 [35][36][37]. ...
Pancreatic ductal adenocarcinoma (PDAC) is highly malignant, with a 5-year survival rate of less than 10%. Furthermore, the acquisition of anticancer drug resistance makes PDAC treatment difficult. We established MIA-GEM cells, a PDAC cell line resistant to gemcitabine (GEM), a first-line anticancer drug, using the human PDAC cell line—MIA-PaCa-2. Microtubule-associated serine/threonine kinase-4 (MAST4) expression was increased in MIA-GEM cells compared with the parent cell line. Through inhibitor screening, dysregulated AKT signaling was identified in MIA-GEM cells with overexpression of AKT3. MAST4 knockdown effectively suppressed AKT3 overexpression, and both MAST4 and AKT3 translocation into the nucleus, phosphorylating forkhead box O3a (FOXO3) in MIA-GEM cells. Modulating FOXO3 target gene expression in these cells inhibited apoptosis while promoting stemness and proliferation. Notably, nuclear MAST4 demonstrated higher expression in GEM-resistant PDAC cases compared with that in the GEM-sensitive cases. Elevated MAST4 expression correlated with a poorer prognosis in PDAC. Consequently, nuclear MAST4 emerges as a potential marker for GEM resistance and poor prognosis, representing a novel therapeutic target for PDAC.
... Alternatively, γ-ray irradiation directly effects DNA molecules and organelles, and directly disrupts the molecular structure (Bacq and Alexander, 1961). However, reduced expression of several assayed genes such as sod-3 and sod-5, which are target genes of the DAF-16 transcription factor via the ins/IGF-1 signaling pathway, and sir-2.1 and exo-3, was restored during aging (Furuyama et al., 2000;Yanase et al., 2020). In particular, expression of some genes, such as sir-2.1 and exo-3, showed IR-dependent recovery under hyperoxia throughout the course of aging. ...
Human astronauts follow a fixed prebreathing protocol consisting of 100% hyperoxia exposure repeated several times weekly before conducting the extravehicular activities in interplanetary space. However, despite astronaut exposure to both space radiation and hyperoxia, which have different biological effects on cells, the combinatorial effects on aging and longevity is unknown. Here, we evaluated the interactive effects on aging using Caenorhabditis elegans exposed to short-term ⁶⁰Co γ-ray irradiation and hyperoxia. Thus, combined treatment extended the lifespan by approximately 10-15% in wild-type worms compared with irradiation only. Moreover, we found that the interaction induced not only the expression of antioxidant genes via insulin/IGF-1 signaling and the reduction of mitochondrial ROS, but also the activation of DNA damage repair system and sirtuin homologue genes during aging. Despite short-term irradiation of low linear energy transfer (LET) radiation such as γ-ray inducing transiently transcriptional inactivation of most genes, the levels of genes expression were recovered under hyperoxia during aging, at least partially. These findings in the C. elegans, showing that genetic transcriptional and mitochondrial dynamics depend on the interaction of low LET radiation with hyperoxia to consequently boost longevity, suggest some intracellular molecular targets to protect astronauts in space.
... As such, FOXOs are vital for the regulation of a plethora of cellular processes, from cell cycle arrest and apoptosis to metabolism and oxidative damage modulation (7). Structurally, FOXOs differ from the rest of the FOX superfamily, containing a specific amino acid sequence flanking the DNA-binding domain (DBD) (Gly-Asp-Ser-Asn-Ser) enabling interaction with the FHRE (forkhead response element; 5'-GTAAACAA-3') (8)(9)(10). Within the family, FOXOs have shared DBD homology, however the structure of their transactivation domains (TAD) differ; it is this difference in TAD structure that determines the nature of FOXO interactors to define role specificity (11). ...
Forkhead box (FOX) class O (FOXO) proteins are a dynamic family of transcription factors composed of four family members: FOXO1, FOXO3, FOXO4 and FOXO6. As context-dependent transcriptional activators and repressors, the FOXO family regulates diverse cellular processes including cell cycle arrest, apoptosis, metabolism, longevity and cell fate determination. A central pathway responsible for negative regulation of FOXO activity is the phosphatidylinositol-3-kinase (PI3K)-AKT signalling pathway, enabling cell survival and proliferation. FOXO family members can be further regulated by distinct kinases, both positively (e.g., JNK, AMPK) and negatively (e.g., ERK-MAPK, CDK2), with additional post-translational modifications further impacting on FOXO activity. Evidence has suggested that FOXOs behave as ‘bona fide’ tumour suppressors, through transcriptional programmes regulating several cellular behaviours including cell cycle arrest and apoptosis. However, an alternative paradigm has emerged which indicates that FOXOs operate as mediators of cellular homeostasis and/or resistance in both ‘normal’ and pathophysiological scenarios. Distinct FOXO family members fulfil discrete roles during normal B cell maturation and function, and it is now clear that FOXOs are aberrantly expressed and mutated in discrete B-cell malignancies. While active FOXO function is generally associated with disease suppression in chronic lymphocytic leukemia for example, FOXO expression is associated with disease progression in diffuse large B cell lymphoma, an observation also seen in other cancers. The opposing functions of the FOXO family drives the debate about the circumstances in which FOXOs favour or hinder disease progression, and whether targeting FOXO-mediated processes would be effective in the treatment of B-cell malignancies. Here, we discuss the disparate roles of FOXO family members in B lineage cells, the regulatory events that influence FOXO function focusing mainly on post-translational modifications, and consider the potential for future development of therapies that target FOXO activity.
... The FOXO subfamily that includes FOXO1, FOXO3a, FOXO4, and FOXO6 is the most evolutionarily divergent clade of FOX TFs [101]. These TFs are differentially expressed in various tissues with diverse regulatory functions [102,103]. In vivo, these proteins display redundant tumor-suppressive functions, but, interestingly, have distinct lineage and organspecific effects arising from the differential expressions of both unique and overlapping target genes [104]. ...
Polo-like kinase 1 (PLK1) is a serine/threonine kinase with more than 600 phosphorylation substrates through which it regulates many biological processes, including mitosis, apoptosis, metabolism, RNA processing, vesicle transport, and G2 DNA-damage checkpoint recovery, among others. Among the many PLK1 targets are members of the FOX family of transcription factors (FOX TFs), including FOXM1, FOXO1, FOXO3, and FOXK1. FOXM1 and FOXK1 have critical oncogenic roles in cancer through their antagonism of apoptotic signals and their promotion of cell proliferation, metastasis, angiogenesis, and therapeutic resistance. In contrast, FOXO1 and FOXO3 have been identified to have broad functions in maintaining cellular homeostasis. In this review, we discuss PLK1-mediated regulation of FOX TFs, highlighting the effects of PLK1 on the activity and stability of these proteins. In addition, we review the prognostic and clinical significance of these proteins in human cancers and, more importantly, the different approaches that have been used to disrupt PLK1 and FOX TF-mediated signaling networks. Furthermore, we discuss the therapeutic potential of targeting PLK1-regulated FOX TFs in human cancers.
... When IIS is reduced, such as in cases of stress or mutations in daf-2, DAF-16 translocates to the nucleus and regulates genes involved in longevity, metabolism, autophagy and cellular stress response (Li and Zhang, 2016;Murphy et al., 2003). DAF-16 upregulates target genes through the DAF-16 binding element (DBE) (Furuyama et al., 2000;Murphy et al., 2003;Schuster et al., 2010), and downregulates target genes through the DAF-16 associated element (DAE) (McElwee et al., 2004;Murphy et al., 2003). ...
While aging was traditionally viewed as a stochastic process of damage accumulation, it is now clear that aging is strongly influenced by genetics. The identification and characterization of long-lived genetic mutants in model organisms has provided insights into the genetic pathways and molecular mechanisms involved in extending longevity. Long-lived genetic mutants exhibit activation of multiple stress response pathways leading to enhanced resistance to exogenous stressors. As a result, lifespan exhibits a significant, positive correlation with resistance to stress. Disruption of stress response pathways inhibits lifespan extension in multiple long-lived mutants representing different pathways of lifespan extension and can also reduce the lifespan of wild-type animals. Combined, this suggests that activation of stress response pathways is a key mechanism by which long-lived mutants achieve their extended longevity and that many of these pathways are also required for normal lifespan. These results highlight an important role for stress response pathways in determining the lifespan of an organism.
... In total, 60% (23/38) of the DEGs had either a potential DAF-16 regulatory motif through our analysis or experimental evidence of DAF-16 regulation [42,43]; this enrichment was significant compared to the entire genome (p < 0.001). The promoter sequences we identified for DAF-16 transcriptional regulation consisted of either the DAF-16-binding element (DBE) or the DAF-16-associated element (DAE) [34,44,45]. We detected four DEGs with DBE sequences (5 -TTGTTTAC-3 ), which suggests these targets could be directly regulated by DAF-16 (Table 4), but this would need to be validated experimentally. ...
Oxidative stress is a contributing factor to Parkinson’s disease (PD). Considering the prevalence of sporadic PD, environmental exposures are postulated to increase reactive oxygen species and either incite or exacerbate neurodegeneration. We previously determined that exposure to the common soil bacterium, Streptomyces venezuelae (S. ven), enhanced oxidative stress and mitochondrial dysfunction in Caenorhabditis elegans, leading to dopaminergic (DA) neurodegeneration. Here, S. ven metabolite exposure in C. elegans was followed by RNA-Seq analysis. Half of the differentially identified genes (DEGs) were associated with the transcription factor DAF-16 (FOXO), which is a key node in regulating stress response. Our DEGs were enriched for Phase I (CYP) and Phase II (UGT) detoxification genes and non-CYP Phase I enzymes associated with oxidative metabolism, including the downregulated xanthine dehydrogenase gene, xdh-1. The XDH-1 enzyme exhibits reversible interconversion to xanthine oxidase (XO) in response to calcium. S. ven metabolite exposure enhanced XO activity in C. elegans. The chelation of calcium diminishes the conversion of XDH-1 to XO and results in neuroprotection from S. ven exposure, whereas CaCl2 supplementation enhanced neurodegeneration. These results suggest a defense mechanism that delimits the pool of XDH-1 available for interconversion to XO, and associated ROS production, in response to metabolite exposure.
... Research demonstrates that FoxO has a vital role in many developmental and physiological processes, including cell proliferation, metabolism, reproduction, longevity, and oxidative stress, by regulating target genes involved in cell cycle arrest, apoptosis, energy metabolism, and oxidative stress resistance (Furuyama et al., 2000, Xuan & Zhang, 2005Tsai et al., 2007;Arden, 2008;Burgering, 2008;Calnan & Brunet, 2008;Zhang et al., 2021). In the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster, overexpression of FoxO caused extreme longevity, whereas depletion of FoxO resulted in reduced oxidative stress and longevity, which was in part mediated by the interaction between FoxO and β-Catenin (Jünger et al., 2003;Hwangbo et al., 2004;Essers et al., 2005;Lim et al., 2020). ...
The forkhead box O (FoxO) protein is the main transcriptional effector downstream of the insulin/insulin‐like signaling pathway and regulates many developmental and physiological processes. Holometabolous insects with loss‐of‐function mutations in FoxO exhibit phenotypes distinct from those of hemimetabolous insects in which RNA interference was used. Despite the functional importance of FoxO, whether hemimetabolous insects share an evolutionally conserved function of FoxO with holometabolous insects remains to be clarified. We used the clustered regularly interspaced short palindromic repeats/CRISPR‐associated 9 (CRISPR/Cas9) genome editing‐system to establish a homozygous FoxO‐null mutant (NlFoxO4E) of the wing‐dimorphic brown planthopper (BPH) Nilaparvata lugens, an economically important insect pest of rice fields. The phenotypes of NlFoxO4E mutants included extended nymphal duration, shortened lifespan, reduced reproduction, and decreased stress resistance. In addition, depletion of NlFoxO promoted cell proliferation in wing buds and led to 100% long‐winged morphs, in stark contrast to short‐winged wild‐type BPHs. These findings indicate that NlFoxO is highly functionally conserved with its counterpart in holometabolous insects, and is required for optimal fitness of N. lugens. The insights from FoxO studies may facilitate the identification of potential target genes for BPH control applications. This article is protected by copyright. All rights reserved
... Human FOXO3a is ubiquitously expressed in the body, but varies in different types of cells, predominantly expressed in the liver [19]. The transactivation activity of FOXO3a is fine-tuned by four main domains necessary for the key locations of the PTMs, containing a nuclear localization signal (NLS), a nuclear export sequence (NES), an amino-terminal forkhead DNA-binding domain (DBD), and a C-terminal transactivation domain (TAD) (Figure 2). ...
Oxidative stress has been identified as a key mechanism in liver damage caused by various chemicals. The transcription factor FOXO3a has emerged as a critical regulator of redox imbalance. Multiple post-translational changes and epigenetic processes closely regulate the activity of FOXO3a, resulting in synergistic or competing impacts on its subcellular localization, stability, protein–protein interactions, DNA binding affinity, and transcriptional programs. Depending on the chemical nature and subcellular context, the oxidative-stress-mediated activation of FOXO3a can induce multiple transcriptional programs that play crucial roles in oxidative injury to the liver by chemicals. Here, we mainly review the role of FOXO3a in coordinating programs of genes that are essential for cellular homeostasis, with an emphasis on exploring the regulatory mechanisms and potential application of FOXO3a as a therapeutic target to prevent and treat liver oxidative injury.
... The foxo1 mRNA was identified with length of 2176 bp and the full-length ORF of foxo1 was 2025 bp, encoding peptides of 675 amino acids. Tissue distribution of foxo1 in turbot showed that the expression of foxo1 gene was high in the liver and was similar to the results in human (Anderson et al., 1998), mouse (Furuyama et al., 2000), pig (Pang et al., 2009) and grass carp (Sun et al., 2017). Because liver is the key organ of glucose metabolism and foxo1 has high expression level in the liver of turbot, the research can lay the foundation for future research about the signals of foxo1 that mediate glucose metabolism in turbot liver. ...
Forkhead box O1 (foxo1) is a transcription factor and plays important roles in glucose metabolism. In the present study, foxo1 in turbot Scophthalmus maximus was cloned and characterized. The siRNA of foxo1 was used to investigate the functions of foxo1 in turbot hepatocytes glucose metabolism. After that, a 10-week feeding trial with two different dietary carbohydrate levels (15% and 21%, respectively) was conducted to analyze the function of foxo1 in glucose metabolism in vivo. Results showed that the foxo1 was identified as 2176 bp (base pair) with a 2025 bp open reading frame, which encoded 675 amino acids. Sequence analysis showed that foxo1 of turbot was highly homologous to most of fishes. Tissue distribution analysis revealed that the highest expression of foxo1 was in liver. After in vitro analysis, foxo1-specific small interfering RNA (sifoxo1) treatment significantly decreased the expressions of cytosolic phosphoenolpyruvate carboxykinase (cpepck) and glucose-6-phosphatase1(g6pase1) in primary hepatocytes. Expression of mitochondrial phosphoenolpyruvate carbox-ykinase (mpepck) was not significantly inhibited. In contrast, the expression of glucose-6-phosphatase2 (g6pase2) increased significantly. After the in vivo study (feeding trial), with the decreased expression of foxo1 in turbot due to high dietary carbohydrate level (21%), the expression of g6pase2 was significantly upregulated. However, the expression of glucokinase (gk) was not changed significantly. These increased the level of blood glucose and hepatic glycogen. In conclusion, data from both in vitro (primary hepatocytes) and in vivo (feeding trial) showed that downregulated foxo1 in turbot could not result in significant depression of gluconeogenesis and activation of glycolysis. This could be one of the reasons why intake of high level of carbohydrate resulted in prolonged hyperglycemia in turbot.
... The solution structure of the FOXO4 FH-CR3 complex has been solved as described previously (Bourgeois et al., 2021). In the simulations, all proteins were described by the AMBER ff19SB force field (Kim et al., 2021)) and the solvent molecules were represented as OPC water molecules (Furuyama et al., 2000).Periodic boundary conditions were employed with a cutoff of 10 Å for non-bonded interactions and the Particle Mesh Ewald method for long-range electrostatics was used. All the MD simulations were run using the Amber20 software package (Case HMA et al., 2021). ...
Transcription factors play key roles in orchestrating a plethora of cellular mechanisms and controlling cellular homeostasis. Transcription factors share distinct DNA binding domains, which allows to group them into protein families. Among them, the Forkhead box O (FOXO) family contains transcription factors crucial for cellular homeostasis, longevity and response to stress. The dysregulation of FOXO signaling is linked to drug resistance in cancer therapy or cellular senescence, however, selective drugs targeting FOXOs are limited, thus knowledge about structure and dynamics of FOXO proteins is essential. Here, we provide an extensive study of structure and dynamics of all FOXO family members. We identify residues accounting for different dynamic and structural features. Furthermore, we show that the auto-inhibition of FOXO proteins by their C-terminal trans-activation domain is conserved throughout the family and that these interactions are not only possible intra-, but also inter-molecularly. This indicates a model in which FOXO transcription factors would co-regulate their activities.
... The expression of Ad-Myc-FoxO1 was verified ( Figure 4A). As shown in Figure 4B, IRS (insulin response sequence) is one cis-element consisting of TTGTTTAC repeated binding motif of FOXO [17,18]. So, the activity of IRS-Luc reporter represent the activity of FOXO1. ...
Endometrial receptivity damage caused by impaired decidualization may be one of the mechanisms of infertility in endometriosis (EMs). Our previous study demonstrated that Calpain-7 (CAPN7) is abnormally overexpressed in EMs. Whether CAPN7 affects the regulation of decidualization and by what mechanism CAPN7 regulates decidualization remains to be determined. In this study, we found CAPN7 expression decreased during human endometrial stromal cell (HESC) decidualization in vitro. CAPN7 negatively regulated decidualization in vitro and in vivo. We also identified one conserved potential PEST sequence in the AKT1 protein and found that CAPN7 was able to hydrolyse AKT1 and enhance AKT1’s phosphorylation. Correspondingly, CAPN7 notably promoted the phosphorylation of Forkhead Box O1 (FoxO1), the downstream of AKT1 protein, at Ser319, leading to increased FoxO1 exclusion from nuclei and attenuated FoxO1 transcriptional activity in decidualized HESC. In addition, we detected endometrium CAPN7, p-AKT1 and p-FoxO1 expressions were increased in EMs. These data demonstrate that CAPN7 negatively regulates HESC decidualization in EMs probably by promoting FoxO1’s phosphorylation and FoxO1 nuclear exclusion via hydrolyzing AKT1. The dysregulation of CAPN7 may be a novel cause of EMs.
... Finally, we investigated whether novel FOXO1 target genes identified in this study could be direct transcriptional targets of FOXO1. FOXO1 has been reported to directly bind to two consensus FOXO1-binding sites [Daf-16 binding element (DBE): TT(G/A)TTTA(C/T) 69 and insulin response element: CAAAA(C/T)AA 70 ] to regulate the promoter activity of its target genes. Therefore, we searched for two consensus FOXO1-binding sites in the putative promoter regions upstream of 2 kb from the transcription start sites and found at least 1 DBE or insulin response element in murine C/EBPδ, ATF4, Redd1, Sestrin1, Castor2, Chac1, Depp1, and Lat3 promoter regions, suggesting these genes are direct targets of FOXO1. ...
Catabolic conditions, such as starvation, inactivity, and cancer cachexia, induce Forkhead box O (FOXO) transcription factor(s) expression and severe muscle atrophy via the induction of ubiquitin–proteasome system‐mediated muscle proteolysis, resulting in frailty and poor quality of life. Although FOXOs are clearly essential for the induction of muscle atrophy, it is unclear whether there are other factors involved in the FOXO‐mediated transcriptional regulation. As such, we identified FOXO–CCAAT/enhancer‐binding protein δ (C/EBPδ) signaling pathway as a novel proteolytic pathway. By comparing the gene expression profiles of FOXO1‐transgenic (gain‐of‐function model) and FOXO1,3a,4–/– (loss‐of‐function model) mice, we identified several novel FOXO1‐target genes in skeletal muscle including Redd1, Sestrin1, Castor2, Chac1, Depp1, Lat3, as well as C/EBPδ. During starvation, C/EBPδ abundance was increased in a FOXOs‐dependent manner. Notably, knockdown of C/EBPδ prevented the induction of the ubiquitin–proteasome system and decrease of myofibers in FOXO1‐activated myotubes. Conversely, C/EBPδ overexpression in primary myotubes induced myotube atrophy. Furthermore, we demonstrated that FOXO1 enhances the promoter activity of target genes in cooperation with C/EBPδ and ATF4. This research comprehensively identifies novel FOXO1 target genes in skeletal muscle and clarifies the pathophysiological role of FOXO1, a master regulator of skeletal muscle atrophy.
... Importantly, the protein expressions of FBXO-9, FBXO-32, and MuRF1 are unregulated in the arteries of STZ-induced T1DM animals and in primary human coronary arterial SMCs cultured with high glucose (Zhang et al., 2010a(Zhang et al., , 2020Lu et al., 2012;Yi et al., 2014). Such upregulation of FBXO expression is mediated through the suppression of PI3K/AKT-dependent phosphorylation in FOXO-3a, thereby promoting FOXO-3a nuclear translocation and binding to the consensus sequence [GTAAA(C/T)A] in the promoter of Fbxo gene, activating its transcription (Furuyama et al., 2000). However, activation of MuRF1 is due to an increase of NF-κB-mediated Trim63 (encoding MuRF1) transcription (Wu et al., 2014). ...
Diabetes mellitus (DM) is an independent risk of macrovascular and microvascular complications, while cardiovascular diseases remain a leading cause of death in both men and women with diabetes. Large conductance Ca²⁺-activated K⁺ (BK) channels are abundantly expressed in arteries and are the key ionic determinant of vascular tone and organ perfusion. It is well established that the downregulation of vascular BK channel function with reduced BK channel protein expression and altered intrinsic BK channel biophysical properties is associated with diabetic vasculopathy. Recent efforts also showed that diabetes-associated changes in signaling pathways and transcriptional factors contribute to the downregulation of BK channel expression. This manuscript will review our current understandings on the molecular, physiological, and biophysical mechanisms that underlie coronary BK channelopathy in diabetes mellitus.
... Therefore, although we observed impaired intestinal barrier integrity in both Vil1 Cre Foxo1 fl/fl Foxo1 AAA/+ and Vil1 Cre Foxo1 fl/fl mice, it is possible that enforced nuclear Foxo1 expression in Foxo1 AAA mice led to disruption of the intestinal barrier not only by affecting goblet cell autophagy but also by interfering with IEC survival and division. Moreover, while it has been reported that Foxo family members have certain redundancies (Furuyama et al., 2000;Paik et al., 2007), our mRNA-seq data showed no up-regulated expression of other Foxo family members in the Foxo1-deficient IECs. Also, considering that both Foxo3-and Foxo4-deficient mice exhibit enhanced susceptibility to DSS-induced colitis (Snoeks et al., 2009;Zhou et al., 2009), there is unlikely to be a compensatory effect of Foxo3 or Foxo4 in the absence of Foxo1 for IEC homeostasis. ...
Mucus produced by goblet cells in the gastrointestinal tract forms a biological barrier that protects the intestine from invasion by commensals and pathogens. However, the host-derived regulatory network that controls mucus secretion and thereby changes gut microbiota has not been well studied. Here, we identify that Forkhead box protein O1 (Foxo1) regulates mucus secretion by goblet cells and determines intestinal homeostasis. Loss of Foxo1 in intestinal epithelial cells (IECs) results in defects in goblet cell autophagy and mucus secretion, leading to an impaired gut microenvironment and dysbiosis. Subsequently, due to changes in microbiota and disruption in microbiome metabolites of short-chain fatty acids, Foxo1 deficiency results in altered organization of tight junction proteins and enhanced susceptibility to intestinal inflammation. Our study demonstrates that Foxo1 is crucial for IECs to establish commensalism and maintain intestinal barrier integrity by regulating goblet cell function.
Transgenerational phenotypic plasticity (TPP) refers to the phenomenon that environmental conditions experienced by one generation can influence the phenotype of subsequent generations to adapt to the environment without modification of their DNA sequences. Aphid wing dimorphism is a textbook example of TPP by which a maternal aphid perceives the environmental cues to decide the wing morph of her offspring. However, the signaling mechanism from mother to daughter remains unclear. In this study, we showed that the population density and physical contact caused high proportion of winged offspring in the pea aphid Acyrthosiphon pisum. Its vesicular glutamate transporter 2 (ApvGluT2) and metabotropic glutamate receptor 3 (ApmGluR3) were identified by tissue-specific RNA-seq as differentially expressed genes in the head and embryo respectively between solitary and more densely housed maternal aphids. Elevated expression of brain ApvGluT2 and embryonic ApmGluR3 led to increases in the winged proportion. Knockdown of either gene inhibited phosphorylation of ApFoxO in embryos. Furthermore, EMSA showed that dephosphorylated ApFoxO directly bound to the promotor of hedgehog (ApHh), a morphogen gene for wing development, to repress its transcription in stage 20 embryos, causing a lower winged proportion. Our results demonstrated that brain vGluT2 and embryonic mGluR3 coordinately relayed the maternal physical contact signals and control wing development in offspring, showcasing a novel regulatory mechanism underlying physical contact-dependent, transgenerational wing dimorphism in aphids.
The insulin/insulin-like signaling (IIS) pathway regulates many of C. elegans’ adult functions, including learning and memory ¹ . While whole-worm and tissue-specific transcriptomic analyses have identified IIS targets 2,3 , a higher-resolution single-cell approach is required to identify changes that confer neuron-specific improvements in the long-lived insulin receptor mutant, daf-2 . To understand how behaviors that are controlled by a small number of neurons change in daf-2 mutants, we used the deep resolution of single-nucleus RNA sequencing to define each neuron type’s transcriptome in adult wild-type and daf-2 mutants. First, we found surprising differences between wild-type L4 larval neurons and young adult neurons in chemoreceptor expression, synaptic genes, and learning and memory genes. These Day 1 adult neuron transcriptomes allowed us to identify adult AWC-specific regulators of chemosensory function and to predict neuron-to-neuron peptide/receptor pairs. We then identified gene expression changes that correlate with daf-2’s improved cognitive functions, particularly in the AWC sensory neuron that controls learning and associative memory ⁴ , and used behavioral assays to test their roles in cognitive function. Combining deep single-neuron transcriptomics, genetic manipulation, and behavioral analyses enabled us to identify genes that may function in a single adult neuron to control behavior, including conserved genes that function in learning and memory.
One-Sentence Summary
Single-nucleus sequencing of adult wild-type and daf-2 C. elegans neurons reveals functionally relevant transcriptional changes, including regulators of chemosensation, learning, and memory.
Tumors in dogs and humans share many similar molecular and genetic features, incentivizing a better understanding of canine neoplasms not only for the purpose of treating companion animals, but also to facilitate research of spontaneously developing tumors with similar biologic behavior and treatment approaches in an immunologically competent animal model. Multiple tumor types of both species have similar dysregulation of signal transduction through phosphatidylinositol 3-kinase (PI3K), protein kinase B (PKB; AKT), and mechanistic target of rapamycin (mTOR), collectively known as the PI3K-AKT-mTOR pathway. This review aims to delineate the pertinent aspects of the PI3K-AKT-mTOR signaling pathway in health and in tumor development. It will then present a synopsis of current understanding of PI3K-AKT-mTOR signaling in important canine cancers and advancements in targeted inhibitors of this pathway.
In C. elegans mechanisms by which peripheral organs relay internal state information to the nervous system remain unknown, although strong evidence suggests that such signals do exist. Here we report the discovery of a peptide of the ancestral insulin superfamily called INS-7 that functions as an enteroendocrine peptide and is secreted from specialized cells of the intestine. INS-7 secretion increases during fasting, and acts as a bona fide gut-to-brain homeostatic signal that attenuates neuronally induced fat loss during food shortage. INS-7 functions as an antagonist at the canonical DAF-2 receptor in the nervous system, and phylogenetic analysis suggests that INS-7 bears greater resemblance to members of the broad insulin/relaxin superfamily than to conventional mammalian insulin and IGF peptides. The discovery of an endogenous insulin antagonist secreted by specialized intestinal cell with enteroendocrine functions suggests that much remains to be learned about the intestine and its role in directing neuronal functions.
The forkhead box protein O (FOXO, consisting of FOXO1, FOXO3, FOXO4 and FOXO6) transcription factors are the mammalian orthologues of Caenorhabditis elegans DAF-16, which gained notoriety for its capability to double lifespan in the absence of daf-2 (the gene encoding the worm insulin receptor homologue). Since then, research has provided many mechanistic details on FOXO regulation and FOXO activity. Furthermore, conditional knockout experiments have provided a wealth of data as to how FOXOs control development and homeostasis at the organ and organism levels. The lifespan-extending capabilities of DAF-16/FOXO are highly correlated with their ability to induce stress response pathways. Exogenous and endogenous stress, such as cellular redox stress, are considered the main drivers of the functional decline that characterizes ageing. Functional decline often manifests as disease, and decrease in FOXO activity indeed negatively impacts on major age-related diseases such as cancer and diabetes. In this context, the main function of FOXOs is considered to preserve cellular and organismal homeostasis, through regulation of stress response pathways. Paradoxically, the same FOXO-mediated responses can also aid the survival of dysfunctional cells once these eventually emerge. This general property to control stress responses may underlie the complex and less-evident roles of FOXOs in human lifespan as opposed to model organisms such as C. elegans.
FOXO family of proteins are transcription factors involved in many physiological and pathological processes including cellular homeostasis, stem cell maintenance, cancer, metabolic and cardiovascular diseases. Genetic evidence has been accumulating to suggest a prominent role of FOXOs in lifespan regulation in animal systems from hydra, C elegans, Drosophila and mice. Together with the observation that FOXO3 is the second most replicated gene associated with extreme human longevity suggests that pharmacological targeting of FOXO proteins can be a promising approach to treat cancer and other age-related diseases and extend life and health span. However, due to the broad range of cellular functions of the FOXO family members FOXO1, 3, 4 and 6, isoform-specific targeting of FOXOs might lead to greater benefit and cause less side effects. Therefore, a deeper understanding of the common and specific features of these proteins as well as their redundant and specific functions in our cells represents the basis of specific targeting strategies. In this review, we provide an overview on the evolution, structure, function and disease-relevance of each of the FOXO family members.
The FOX family of transcription factors was originally identified in 1989, comprising the FOXA to FOXS subfamilies. FOXO3, a well-known member of the FOXO subfamily, is widely expressed in various human organs and tissues, with higher expression levels in the ovary, skeletal muscle, heart, and spleen. The biological effects of FOXO3 are mostly determined by its phosphorylation, which occurs in the nucleus or cytoplasm. Phosphorylation of FOXO3 in the nucleus can promote its translocation into the cytoplasm and inhibit its transcriptional activity. In contrast, phosphorylation of FOXO3 in the cytoplasm leads to its translocation into the nucleus and exerts regulatory effects on biological processes, such as inflammation, aerobic glycolysis, autophagy, apoptosis, oxidative stress, cell cycle arrest and DNA damage repair. Additionally, FOXO3 isoform 2 acts as an important suppressor of osteoclast differentiation. FOXO3 can also interfere with the development of various diseases, including inhibiting the proliferation and invasion of tumor cells, blocking the production of inflammatory factors in autoimmune diseases, and inhibiting β-amyloid deposition in Alzheimer's disease. Furthermore, FOXO3 slows down the aging process and exerts anti-aging effects by delaying telomere attrition, promoting cell self-renewal, and maintaining genomic stability. This review suggests that changes in the levels and post-translational modifications of FOXO3 protein can maintain organismal homeostasis and improve age-related diseases, thus counteracting aging. Moreover, this may indicate that alterations in FOXO3 protein levels are also crucial for longevity, offering new perspectives for therapeutic strategies targeting FOXO3.
High concentrations of antioxidants can exert pro-oxidative effects, elevate the level of intracellular reactive oxygen species (ROS), and cause oxidative stress in cells. We previously found that high concentrations of curcumin, a natural polyphenol antioxidant, elevated ROS levels and upregulated the expression of histone deacetylase 1 (HDAC1) in human gastric cancer cells (hGCCs); however, its potential mechanisms and subsequent functions have not been elucidated. In the present study, we treated hGCCs with high concentrations of curcumin, detected several indicators of oxidative stress, and investigated the mechanism of curcumin-treatment-mediated HDAC1 upregulation and its effect on histone acetylation. The results showed that curcumin treatment caused oxidative stress in hGCCs and upregulated HDAC1/2 expression via the forkhead box O (FOXO) signaling pathway, ultimately leading to the deacetylation of histones in hGCCs. Moreover, HDAC1/2 mediates the deacetylation of FOXOs and promotes their transcription activities, implying a positive feedback loop between FOXOs and HDAC1/2. These findings present a mechanism by which oxidative stress induces histone deacetylation in hGCCs.
MicroRNAs are critical regulators of the plethora of genes, including FOXO "forkhead" dependent transcription factors, which are bonafide tumour suppressors. The FOXO family members modulate a hub of cellular processes like apoptosis, cell cycle arrest, differentiation, ROS detoxification, and longevity. Aberrant expression of FOXOs in human cancers has been observed due to their down-regulation by diverse microRNAs, which are predominantly involved in tumour initiation, chemo-resistance and tumour progression. Chemo-resistance is a major obstacle in cancer treatment. Over 90 % of casualties in cancer patients are reportedly associated with chemo-resistance. Here, we have primarily discussed the structure, functions of FOXO and also their post-translational modifications which influence the activities of these FOXO family members. Further, we have addressed the role of microRNAs in carcinogenesis by regulating the FOXOs at post-transcriptional level. Therefore, microRNAs-FOXO axis can be exploited as a novel cancer therapy. The administration of microRNA-based cancer therapy is likely to be beneficial to curb chemo-resistance in cancers.
Increased FOXO3 nuclear localization is involved in neuroblastoma chemoresistance and tumor angiogenesis. Accordingly, FOXO3 inhibition is a promising strategy for boosting antitumor immune responses and suppressing FOXO3-mediated therapy resistance in cancer cells. However, no FOXO3 inhibitors are currently available for clinical use. Nevertheless, we have recently identified (4-propoxy)phenylpyrimidinylguanidine as a FOXO3 inhibitor in cancer cells in the low micromolar range. Here, we report the synthesis and structure−activity relationship study of a small library of its derivatives, some of which inhibit FOXO3-induced gene transcription in cancer cells in a submicromolar range and are thus 1 order of magnitude more potent than their parent compound. By NMR and molecular docking, we showed that these compounds differ in their interactions with the DNA-binding domain of FOXO3. These results may provide a foundation for further optimizing (4-propoxy)phenylpyrimidinylguanidine and developing therapeutics for inhibiting the activity of forkhead box (FOX) transcription factors and their interactions with other binding partners. ■ INTRODUCTION Forkhead box (FOX) transcription factors display high functional diversity and participate in development, proliferation , differentiation, stress resistance, apoptosis, and metabolic control processes. 1 This diverse group of transcriptional regulators shares a conserved, 110-amino-acid-long, DNA-binding domain (DBD) known as the Forkhead domain. 2,3 The O subclass of FOX transcription factors consists of four members, namely, FOXO1, FOXO3, FOXO4, and FOXO6, which are key regulators of cellular homeostasis, longevity, and stress responses. 4−7 All FOXO proteins recognize the consensus DNA sequence 5′-TTGTTTAC-3′, and their transcriptional activity is negatively regulated by the proproliferative phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB, also known as Akt) signaling pathway. 5,8 PKB/Akt phosphorylates three Ser/Thr residues and induces the nuclear exclusion of FOXO proteins in a process involving binding to the scaffolding 14-3-3 protein. 9−11 In addition to phosphorylation by PKB/Akt, FOXO proteins are further regulated through phosphorylation by other kinases, acetylation, ubiquitination, and methyl-ation. 12 Phosphorylation by stress-induced signaling kinases, such as JNK and MST1, can override the effect of the PKB/ Akt-mediated phosphorylation of FOXO3 and induce its nuclear localization and activation, thus promoting FOXO3-triggered therapy resistance in cancer cells. 13−15 In cancer, FOXO3 stands out for its dual role. On the one hand, FOXO3 induces cell cycle arrest and apoptosis, thus functioning as a typical tumor suppressor. 16 On the other hand, FOXO3 activity can also promote tumor development and progression by inducing drug resistance, 7,17 cyclin transcription upregulation, 18 cellular detoxification, 19,20 and cancer stem cell maintenance 13 and by inhibiting apoptosis inducers such as the transcription factor p53. 14 Moreover, all FOXO proteins also regulate T cell differentiation, especially the pathway that leads to the development and function of regulatory T cells. 21,22 Therefore, pharmacological inhibition of FOXO3 transcriptional activity is considered a highly promising approach to boosting antitumor immune responses and suppressing FOXO3-mediated therapy resistance in cancer cells.
To test whether homologous recombination repair (HRR) depends on FOXO3a, a cellular aging model of human dermal fibroblast (HDF) and tet-on flag-h-FOXO3a transgenic mice were studied. HDF cells transfected with over-expression of wt-h-FOXO3a increased the protein levels of MRE11, BRCA1, BRIP1, and RAD50, while knock-down with siFOXO3a decreased them. The protein levels of MRE11, BRCA1, BRIP1, RAD50, and RAD51 decreased during cellular aging. Chromatin immunoprecipitation (ChIP) assay was performed on FOXO3a binding accessibility to FOXO consensus sites in human MRE11, BRCA1, BRIP1, and RAD50 promoters; the results showed FOXO3a binding decreased during cellular aging. When the tet-on flag-h-FOXO3a mice were administered doxycycline orally, the protein and mRNA levels of flag-h-FOXO3a, MRE11, BRCA1, BRIP1, and RAD50 increased in a doxycycline-dose-dependent manner. In vitro HRR assays were performed by transfection with an HR vector and I-SceI vector. The mRNA levels of the recombined GFP increased after doxycycline treatment in MEF but not in wt-MEF, and increased in young HDF comparing to old HDF, indicating that FOXO3a activates HRR. Overall, these results demonstrate that MRE11, BRCA1, BRIP1, and RAD50 are transcriptional target genes for FOXO3a, and HRR activity is increased via transcriptional activation of MRE11, BRCA1, BRIP1, and RAD50 by FOXO3a.
T-cell acute lymphoblastic leukemia (T-ALL) is a T-cell malignancy, characterized by cell subsets, enriched with leukemia-initiating cells (LIC). β-Catenin modulates LIC activity in T-ALL. However, its role in maintaining established leukemia stem cells remains largely unknown. To identify functionally relevant protein interactions of -Catenin in T-ALL, we performed co-Immunoprecipitation (Co-IP) followed by liquid-chromatography mass spectrometry. Here, we report that a non-canonical functional interaction of β-Catenin with the Forkhead-Box-O3 (FOXO3) transcription factor positively regulates LIC related genes including the Cyclin-dependent-kinase-4 (CDK4), which is a crucial modulator of cell cycle and tumor maintenance. We also confirm the relevance of these findings using stably integrated fluorescent reporters of β-Catenin and FOXO3 activity in patient-derived xenografts, which identify minor subpopulations with enriched LIC activity. Additionally, gene expression data at the single-cell level of leukemic cells of primary patients at the diagnosis and minimal residual disease (MRD) up to 30 days from the standard treatments reveal that the expression of β-Catenin and FOXO3 dependent genes is present in the CD82+CD117+ cell fraction, which is substantially enriched with LICs in MRD as well as in early T-cell precursor acute lymphoblastic leukemia (ETP-ALL). These findings highlight key functional roles for β-Catenin and FOXO3 and suggest novel therapeutic strategies to eradicate aggressive cell subsets in T-ALL.
Gastrointestinal (GI) tumors are among the most common causes of cancer related mortalities in the world. Despite various treatment modalities, there are still significant high mortality rates in these patients. Late diagnosis in advanced tumor stages is one of the major reasons for the high mortality rate that results in treatment failure and tumor relapse among these patients. Therefore, it is required to assess the molecular mechanisms involved in GI tumor progression to introduce novel efficient early detection markers. Forkhead box (FOX) is a family of transcription factors that have critical roles in regulation of cellular processes. MicroRNAs (miRNAs) are pivotal regulators of FOX gene expressions during tumor progressions. In the present review, we discussed the role of miRNAs-FOX axis during GI tumor progression and metastasis. This review paves the way to introduce the miRNAs-FOX axis as a novel candidate for the early detection and targeted therapy in GI tumors.
Increased FOXO3 nuclear localization is involved in neuroblastoma chemoresistance and tumor angiogenesis. Accordingly, FOXO3 inhibition is a promising strategy for boosting antitumor immune responses and suppressing FOXO3-mediated therapy resistance in cancer cells. However, no FOXO3 inhibitors are currently available for clinical use. Nevertheless, we have recently identified (4-propoxy)phenylpyrimidinylguanidine as a FOXO3 inhibitor in cancer cells in the low micromolar range. Here, we report the synthesis and structure-activity relationship study of a small library of its derivatives, some of which inhibit FOXO3-induced gene transcription in cancer cells in a submicromolar range and are thus 1 order of magnitude more potent than their parent compound. By NMR and molecular docking, we showed that these compounds differ in their interactions with the DNA-binding domain of FOXO3. These results may provide a foundation for further optimizing (4-propoxy)phenylpyrimidinylguanidine and developing therapeutics for inhibiting the activity of forkhead box (FOX) transcription factors and their interactions with other binding partners.
Aims
In this paper, the authors have predicted Forkhead box O (FOXO) using the Ensemble learning algorithm. When FOXO is in excess in the human body it leads to LNCap prostate cancer cells leads and if deficit leads to neurodegenerative diseases.
Objective
Neurodegenerative diseases like Alzheimer's and Parkinson's are neurological illnesses that are caused by damaged brain cells. For prediction of FOXO protein, Gradient Boosted Machine (GBM) and Random forest (RF) techniques are used.
Method
The main idea of using GBM is its non-linear nature but it is difficult for any single decision tree to fit all training. To overcome this, an RF algorithm is used. RF combines the results at the end of the process by average or majority rules, while the GBM algorithm combines the results along the way.
Results
29.16% improvement has been observed by RF over GBM. Average square error is also evaluated to check the testing and training of data for 100 trees on 100 tree sizes.
Conclusion
In this paper, a computational model for the prediction of FOXO protein using Ensemble learning techniques (Random Forest and GBM) has been proposed. If the dataset has many variable features and the prediction accuracy is not as important then RF can be considered. On the other hand, GBMs are better suited for datasets that have very few or fewer input features and where high accuracy predictions are required. However, there are instances when either GBM or RF can perform equally well depending on how they are tuned.
Sleep is an essential state that allows for recuperation and survival processes. Disturbing sleep triggers stress responses that promote protective gene expression. Sleep and its deprivation grossly impact gene expression, but little is known about how normal or disturbed sleep control gene expression. Central to the induction of sleep are sleep-active neurons, which inhibit wakefulness and promote survival. Sleep and sleep-active neurons are highly conserved. In Caenorhabditis elegans, the sleep-active RIS neuron is crucial for sleep and survival. Here, we show that RIS depolarization promotes the protective gene expression response that occurs during developmental arrest. This response includes the activation of FOXO/DAF-16 and expression of DAF-16 target genes such as HSP-12.6, a small heat-shock protein that is required for starvation survival. Disturbing sleep by mechanical stimulation increases RIS depolarization. RIS activation in turn activates DAF-16 and other genes required for survival. Hence, during normal sleep, RIS depolarization promotes protective gene expression. When sleep is disturbed, protective gene expression gets further increased by raised RIS depolarization. We thus link sleep-active neuron depolarization to protective gene expression changes and suggest that the cellular stress response following sleep deprivation could be understood as a safeguarding process that is caused by the overactivation of sleep-active neurons.
β‐Amyloid toxicity (Aβ) is an important pathological factor of Alzheimer's disease (AD). Studies have shown that genistein can reduce the toxicity of Aβ to a certain extent; however, the specific mechanism is still uncertain. In the study, we applied Caenorhabditis elegans strains expressing Aβ peptides to evaluate the role of genistein inhibiting Aβ toxicity and the undying mechanism. Genistein influencing the sterol metabolism pathway, the HSP‐16.2 pathway, and lipofuscin in different strains of C. elegans were studied using reverse transcription‐polymerase chain reaction, fluorescence labeling, RNA interference (RNAi), and so forth. Our results showed that genistein alleviated the paralysis of transgenic C. elegans strains. Furthermore, in AD C. elegans, genistein reduced the fluorescence of lipofuscin, downregulated the messenger RNA (mRNA) level of vit‐3 and vit‐6 which were related to the sterol metabolism pathway, significantly increased the mRNA level and protein level of HSP‐16.2, increased the nuclear translocation of the DAF‐16 transcription factor and increased the survival rate after heat stress, which was closely associated with HSP‐16.2 levels. However, the paralysis‐alleviating effect of genistein was greatly reduced because of RNAi‐mediated inhibition of hsp‐16.2, indicating that the anti‐Aβ toxicity effect of genistein was greatly dependent on HSP‐16.2. The above results suggest that genistein inhibiting the toxicity of Aβ in C. elegans, is involved in the modulation of the sterol metabolism pathway by promoting transcription factor DAF‐16 translocation into the nucleus, increasing the expression level of HSP‐16.2, and reducing the levels of lipofuscin through its antioxidant activity. Relative signal pathways of genistein enhancing resistance to β‐amyloid in Caenorhabditis elegans.
Rationale: Triple-negative breast cancer (TNBC) is characterized by its unique molecular profile, aggressive nature and lack of targeted therapy. Chemotherapy induces expression of pluripotency factors and mediates an active induction of breast cancer stem cells (BCSCs) in TNBC, which potentiates the risk of tumor recurrence and metastasis and increases patient mortality. Adenosine receptor 2B (A2BR) expression and activation of its downstream signaling pathway has been implied to promote breast cancer metastasis. This study is to investigate the role of A2BR in the regulation of chemotherapy-induced BCSC enrichment.
Methods: We generated shRNA-mediated A2BR knockdown subclones in TNBC cell lines and evaluated the effect on the BCSC phenotype by Aldefluor and mammosphere assays in vitro. We performed chromatin immunoprecipitation (ChIP) assay to investigate recruitment of transcription factor FOXO3 and histone modification enzymes KDM6A and p300 to the regulatory regions of pluripotency factors, as well as levels of histone modification marks H3K27ac and H3K27me3 on these regions. We employed both xenograft model and genetically engineered, autochthonous breast cancer model to evaluate the effect of A2BR on chemotherapy-induced BCSC enrichment in vivo.
Results: We demonstrated that chemotherapy increased protein level of A2BR, which contributed to chemotherapy-induced pluripotency factor expression and BCSC enrichment in TNBC. A2BR mediated activation of p38 MAPK and nuclear translocation of chromatin remodeling factor SMARCD3, which interacted and recruited histone demethylase KDM6A and histone acetyltransferase p300 specifically to the pluripotency factor genes NANOG, SOX2 and KLF4. Recruitment of KDM6A and p300 decreased histone H3K27me3 and increases H3K27ac marks, and increased transcription factor FOXO3 binding to NANOG, SOX2 and KLF4 genes, leading to transcriptional activation of these genes and BCSC specification. Genetic or pharmacological inhibition of A2BR blocked chemotherapy-mediated epigenetic activation of pluripotency factor genes and BCSC enrichment in vitro and in vivo, and delayed tumor recurrence after chemotherapy was discontinued.
Conclusion: Chemotherapy-induced A2BR expression mediates epigenetic activation of pluripotency factors and promotes breast cancer stemness. Targeting A2BR in combination with chemotherapy may block BCSC enrichment and improve outcome in TNBC.
BACKGROUND
Both forkhead box O (FOXO) and nuclear factor erythroid‐derived 2‐like‐2 (Nrf2) are key transcription factors related to stress responses. Whereas limited studies in mammals and Caenorhabditis elegans have revealed the interaction between FoxO/DAF‐16 and Nrf2/SKN‐1, the role of FOXO in metabolic detoxification and regulation of the Nrf2–Keap1 signaling pathway are poorly understood in insects.
RESULTS
Using Tribolium castaneum as a model organism, we found that RNA interference‐mediated knockdown of FOXO enhanced deltamethrin‐induced lethality by affecting the messenger RNA (mRNA) levels of CYP6BQ cluster genes. We further demonstrated that injection of dsFOXO into the beetle larvae decreased expression of CncC and KEAP1 at both the mRNA and protein level. Notably, dual‐luciferase and electrophoretic mobility shift assays both confirmed direct regulation of CncC by FOXO, whereas Keap1 was directly regulated by CncC.
CONCLUSION
FOXO can directly regulate the expression of CncC and indirectly regulate the expression of Keap1 through CncC. The data provide insights into the regulatory mechanisms of the Nrf2–Keap1 signaling pathway in insects.
We design a “wisdom-of-the-crowds” GRN inference pipeline, and couple it to complex network analysis, to understand the organisational principles governing gene regulation in long-lived glp-1/Notch C. elegans. The GRN has three layers (input, core, output) and is topologically equivalent to bow-tie/hourglass structures prevalent among metabolic networks. To assess the functional importance of structural layers, we screened 80% of regulators and discovered 50 new ageing genes, 86% with human orthologues. Genes essential for longevity—including ones involved in insulin-like signalling (ILS)—are at the core, indicating that GRN’s structure is predictive of functionality. We used in vivo reporters, and a novel functional network covering 5,497 genetic interactions to make mechanistic predictions. We used genetic epistasis to test some of these predictions, uncovering a novel transcriptional regulator, sup-37 that works alongside DAF-16/FOXO. We present a framework with predictive power that can accelerate discovery in C. elegans and potentially humans.
Many tissue-specific stem cells maintain the ability to produce multiple cell types during long periods of non-division, or quiescence. FOXO transcription factors promote quiescence and stem cell maintenance, but the mechanisms by which FOXO proteins promote multipotency during quiescence are still emerging. The single FOXO ortholog in C . elegans , daf-16 , promotes entry into a quiescent and stress-resistant larval stage called dauer in response to adverse environmental cues. During dauer, stem and progenitor cells maintain or re-establish multipotency to allow normal development to resume after dauer. We find that during dauer, daf-16 /FOXO prevents epidermal stem cells (seam cells) from prematurely adopting differentiated, adult characteristics. In particular, dauer larvae that lack daf-16 misexpress collagens that are normally adult-enriched. Using col-19p :: gfp as an adult cell fate marker, we find that all major daf-16 isoforms contribute to opposing col-19p :: gfp expression during dauer. By contrast, daf-16(0) larvae that undergo non-dauer development do not misexpress col-19p :: gfp . Adult cell fate and the timing of col-19p :: gfp expression are regulated by the heterochronic gene network, including lin-41 and lin-29 . lin-41 encodes an RNA-binding protein orthologous to LIN41/TRIM71 in mammals, and lin-29 encodes a conserved zinc finger transcription factor. In non-dauer development, lin-41 opposes adult cell fate by inhibiting the translation of lin-29 , which directly activates col-19 transcription and promotes adult cell fate. We find that during dauer, lin-41 blocks col-19p :: gfp expression, but surprisingly, lin-29 is not required in this context. Additionally, daf-16 promotes the expression of lin-41 in dauer larvae. The col-19p :: gfp misexpression phenotype observed in dauer larvae with reduced daf-16 requires the downregulation of lin-41 , but does not require lin-29 . Taken together, this work demonstrates a novel role for daf-16 /FOXO as a heterochronic gene that promotes expression of lin-41/ TRIM71 to contribute to multipotent cell fate in a quiescent stem cell model.
Understanding the mechanisms underlying malignancy in myeloma cells is important for targeted treatment and drug development. Histone deacetylases (HDACs) can regulate the progression of various cancer types; however, their roles in myeloma are not well known. In the present study, the expression of class I HDACs in myeloma cells and tissues was evaluated. Furthermore, the effects of HDAC1 on the migration of myeloma cells and the associated mechanisms were investigated. Among the class I HDACs evaluated, HDAC1 was upregulated in both myeloma cells and tissues. Targeted inhibition of HDAC1 suppressed the migration of myeloma cells. Of the assessed transcription factors, small interfering (si)‑HDAC1 decreased the expression of Slug. Overexpression of Slug reversed the si‑HDAC1‑mediated suppressed migration of myeloma cells. Mechanistically, the results revealed that HDAC1 regulated the mRNA stability of Slug, while it had no effect on its transcription or nuclear export. Furthermore, HDAC1 negatively regulated the expression of long non‑coding RNA (lncRNA) NONHSAT113026, which could bind with the 3'‑untranslated region of Slug mRNA to facilitate its degradation. The present study demonstrated that HDAC1 promoted the migration of human myeloma cells via regulation of lncRNA/Slug signaling.
Aging happens to all of us as we live. Thanks to the improved living standard and discovery of life-saving medicines, our life expectancy has increased substantially across the world in the past century. However, the rise in lifespan leads to unprecedented increases in both the number and the percentage of individuals 65 years and older, accompanied by the increased incidences of age-related diseases such as type 2 diabetes mellitus and Alzheimer’s disease. FoxO transcription factors are evolutionarily conserved molecules that play critical roles in diverse biological processes, in particular aging and metabolism. Their dysfunction is often found in the pathogenesis of many age-related diseases. Here, we summarize the signaling pathways and cellular functions of FoxO proteins. We also review the complex role of FoxO in aging and age-related diseases, with focus on type 2 diabetes and Alzheimer’s disease and discuss the possibility of FoxO as a molecular link between aging and disease risks.
Forkhead box O (FOXO) transcription factors (TFs) are a subclass of the larger family of forkhead TFs. Mammalians express four members FOXO1, FOXO3, FOXO4, and FOXO6. The interest in FOXO function stems mostly from their observed role in determining lifespan, where in model organisms, increased FOXO activity results in extended lifespan. FOXOs act as downstream of several signaling pathway and are extensively regulated through post‐translational modifications. The transcriptional program activated by FOXOs in various cell types, organisms, and under various conditions has been described and has shed some light on what the critical transcriptional targets are in mediating FOXO function. At the cellular level, these studies have revealed a role for FOXOs in cell metabolism, cellular redox, cell proliferation, DNA repair, autophagy, and many more. The general picture that emerges hereof is that FOXOs act to preserve equilibrium, and they are important for cellular homeostasis. Here, we will first briefly summarize the general knowledge of FOXO regulation and possible functions. We will use genomic stability to illustrate how FOXOs ensure homeostasis. Genomic stability is critical for maintaining genetic integrity, and therefore preventing disease. However, genomic mutations need to occur during lifetime to enable evolution, yet their accumulation is believed to be causative to aging. Therefore, the role of FOXO in genomic stability may underlie its role in lifespan and aging. Finally, we will come up with questions on some of the unknowns in FOXO function, the answer(s) to which we believe will further our understanding of FOXO function and ultimately may help to understand lifespan and its consequences.
Uterine natural killer cells are regulated via surface inhibitory receptors for IL15 and galectin-9 (LGALS9) secreted by endometrial stromal cells (ESCs). However, the mechanism that regulates LGALS9 mRNA levels in ESCs is unclear. The aim of this study is to clarify the transcriptional regulation of LGALS9 in ESCs. Here, LGALS9 mRNA expression levels significantly decreased in the endometrial tissue in the early- to mid-secretory phase, and recovered in the mid- to late-secretory phase, compared to that in the proliferative phase. In ESCs, LGALS9 mRNA expression significantly decreased following estradiol + medroxyprogesterone acetate treatment for 1 day and increased after 12 days compared to that in the control. The transcriptional activity of the LGALS9 upstream region was up-regulated by heart and neural crest derivatives expressed 2 (HAND2) and down-regulated by forkhead box O1 (FOXO1). In ESCs, HAND2 expression significantly increased throughout the 12 days treatment with steroid hormones, whereas FOXO1 expression significantly increased on day 1, reached a plateau, and significantly increased again after 6 days of treatment. Levels of FOXO1 phosphorylation (pFOXO1) remained unchanged after 3-day treatment of ESCs with steroid hormones, but significantly increased following a 12-day treatment. pFOXO1 could not bind to the DNA and was thus unable to directly suppress LGALS9 transcription. Therefore, expression level of HAND2 and phosphorylation status of FOXO1 may determine LGALS9 mRNA expression. This study provides a novel molecular mechanism underlying the transcriptional regulation of LGALS9 mRNA in ESCs, which could be valuable in the treatment of diseases associated with decidualization failure.
Several chemical compounds including natural products have been suggested as being effective against age-related diseases or as beneficial for a healthy life. On the other hand, forkhead box O (FOXO) proteins are emerging as key cellular components associated with extreme human longevity. FOXO proteins are mainly regulated by posttranslational modifications and as these modifications are reversible, activation and inactivation of FOXO are attainable through pharmacological treatment. Here, we questioned whether a panel of compounds with known health-beneficial properties has the capacity to induce the activity of FOXO factors. We show that resveratrol, a phytoalexin present in grapes and other food products, the amide alkaloid piperlongumine found in the fruit of the long pepper, and the plant-derived β-carboline compound harmine induced nuclear translocation of FOXO3. We also show that piperlongumine and harmine but not resveratrol activate FOXO-dependent transcription. We determined the half maximal effective concentration (EC50) values for resveratrol, piperlongumine, and harmine for FOXO translocation, and analyzed their inhibitory impact on chromosomal maintenance 1 (CRM1)-mediated nuclear export and the production of reactive oxygen species (ROS). We also used chemical biology approach and Western blot analysis to explore the underlying molecular mechanisms. We show that harmine, piperlongumine, and resveratrol activate FOXO3 independently of phosphoinositide 3-kinase (PI3K)/AKT signaling and the CRM1-mediated nuclear export. The effect of harmine on FOXO3 activity is at least partially mediated through the inhibition of dual-specificity tyrosine (Y) phosphorylationregulated kinase 1A (DYRK1A) and can be reverted by the inhibition of sirtuins (SIRTs).
BACKGROUND
Thallium (Tl) is a toxic metalloid and an emerging pollutant due to electronic devices and dispersal nearby base-metal mining. Therefore, Tl poses a threat to human health and especially the long-term impact on younger individuals exposed is still unknown. This study aimed to evaluate the toxic effects of thallium acetate in C. elegans in early larval stages, considering physiological and behavioral endpoints, as well as the Tl absorption and bioaccumulation.
METHODS
Caenorhabditis elegans (C. elegans) was exposed to Thallium acetate (50, 100, 150, 200, 250, 500, and 1000 μM) in the L1 larval stage, with the purpose to observe the toxic effects invoked until adulthood. Transgenic worms strains were transported GFP, reporters to DAF-16 and were used to verify the antioxidant response. ICP-MS quantified total Tl⁺ concentration to evidence Tl uptake and bioaccumulation.
RESULTS
Thallium acetate caused a significant reduction in the number of living worms (p < 0.0001 in 100 - 1000 μM), a delay in larval development (p < 0.01; p < 0.001 and p < 0.0001 in 100 - 1000 μM) through the larval stages, and egg production in the worm's uterus was reduced. Thallium acetate also induced behavioral changes. Additionally, thallium acetate activated antioxidant pathway responses in C. elegans by translocating the DAF-16 transcription factor and activation of SOD-3::GFP expression. The Tl⁺ quantification in worms showed its absorption in the L1 larval stage and bioaccumulation in the body after development.
CONCLUSIONS
Thallium acetate reduced survival, delayed development, caused behavioral changes, induced responses inherent to oxidative stress, and serious damage to the worm's reproduction. In addition, C. elegans absorbed and bioaccumulated Tl⁺. Together, our results highlight the impacts of Tl⁺ exposure in the early stages of life, even for a short period.
The transcription factor forkhead box O1 (FOXO1), which instructs the dark zone program to direct germinal center (GC) polarity, is typically inactivated by phosphatidylinositol 3-kinase (PI3K) signals. Here, we investigated how FOXO1 mutations targeting this regulatory axis in GC-derived B cell non-Hodgkin lymphomas (B-NHLs) contribute to lymphomagenesis. Examination of primary B-NHL tissues revealed that FOXO1 mutations and PI3K pathway activity were not directly correlated. Human B cell lines bearing FOXO1 mutations exhibited hyperactivation of PI3K and Stress-activated protein kinase (SAPK)/Jun amino-terminal kinase (JNK) signaling, and increased cell survival under stress conditions as a result of alterations in FOXO1 transcriptional affinities and activation of transcriptional programs characteristic of GC-positive selection. When modeled in mice, FOXO1 mutations conferred competitive advantage to B cells in response to key T-dependent immune signals, disrupting GC homeostasis. FOXO1 mutant transcriptional signatures were prevalent in human B-NHL and predicted poor clinical outcomes. Thus, rather than enforcing FOXO1 constitutive activity, FOXO1 mutations enable co-option of GC-positive selection programs during the pathogenesis of GC-derived lymphomas.
Carbon monoxide releasing molecule-3 (CORM-3) has been shown to protect inflammatory diseases via the upregulation of heme oxygenases-1 (HO-1). However, in rat brain astrocytes (RBA-1), the mechanisms underlying CORM-3-induced HO-1 remain poorly defined. This study used western blot, real-time PCR, and promoter activity assays to determine the levels of HO-1 expression and 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) and dihydroethidium (DHE) to measure reactive oxygen species (ROS). We found that CORM-3-induced HO-1 expression was mediated through ROS generation by Nox or mitochondria. The signaling components were differentiated by pharmacological inhibitors and small interfering RNA (siRNA). Subcellular fractions, immunofluorescent staining, and chromatin immunoprecipitation assay were used to evaluate the nuclear translocation and promoter binding activity of Nrf2 induced by CORM-3. The roles of mTOR and FoxO1 in CORM-3-stimulated responses are still unknown in RBA-1 cells. Our results demonstrated that transfection with siRNAs or pretreatment with pharmacological inhibitors attenuated the levels of HO-1 and phosphorylation of signaling components including Akt, mTOR, FoxO1, and Nrf2 stimulated by CORM-3. Moreover, pretreatment with N-acetyl-L-cysteine, diphenyleneiodonium chloride, apocynin, or rotenone blocked nuclear translocation and promoter binding activity of Nrf2 induced by CORM-3. The present study concluded that in RBA-1 cells, CORM-3-induced HO-1 expression is, at least partially, mediated through Nox and mitochondria/ROS-dependent PI3K/Akt/mTOR cascade to activate FoxO1 or ROS leading to activation of Nrf2 activity.
Previous studies have revealed the importance of inter-tissue communications for lifespan regulation. However, the inter-tissue network responsible for lifespan regulation is not well understood, even in a simple organism Caenorhabditis elegans. To understand the mechanisms underlying systemic lifespan regulation, we focused on lifespan regulation by the insulin/IGF-1 signaling(IIS) pathway; IIS reduction activates the DAF-16/FOXO transcription factor, which results in lifespan extension. Our tissue-specific knockdown and knockout analyses demonstrated that IIS reduction in neurons and the intestine markedly extended lifespan. DAF-16 activation in neurons resulted in DAF-16 activation in the intestine, and vice versa. Our dual gene manipulation method revealed that intestinal and neuronal DAF-16 mediate longevity induced by daf-2 knockout in neurons and the intestine, respectively. In addition, the systemic regulation of intestinal DAF-16 required the IIS pathway in intestinal and neurons. Collectively, these results highlight the importance of the neuronal DAF-16-to-intestinal DAF-16 communication for organismal lifespan regulation.
Genetic screening technologies to identify and validate macromolecular interactions (MMIs) essential for complex pathways remain an important unmet need for systems biology and therapeutics development. Here, we use a library of peptides from diverse prokaryal genomes to screen MMIs promoting the nuclear relocalization of Forkhead Box O3 (FOXO3a), a tumor suppressor more frequently inactivated by post-translational modification than mutation. A hit peptide engages the 14-3-3 family of signal regulators through a phosphorylation-dependent interaction, modulates FOXO3a-mediated transcription, and suppresses cancer cell growth. In a crystal structure, the hit peptide occupies the phosphopeptide-binding groove of 14-3-3ε in a conformation distinct from its natural peptide substrates. A biophysical screen identifies drug-like small molecules that displace the hit peptide from 14-3-3ε, providing starting points for structure-guided development. Our findings exemplify “protein interference,” an approach using evolutionarily diverse, natural peptides to rapidly identify, validate, and develop chemical probes against MMIs essential for complex cellular phenotypes.
In mammals, insulin signalling regulates glucose transport together with the expression and activity of various metabolic enzymes. In the nematode Caenorhabditis elegans, a related pathway regulates metabolism, development and longevity. Wild-type animals enter the developmentally arrested dauer stage in response to high levels of a secreted pheromone, accumulating large amounts of fat in their intestines and hypodermis. Mutants in DAF-2 (a homologue of the mammalian insulin receptor) and AGE-1 (a homologue of the catalytic subunit of mammalian phosphatidylinositol 3-OH kinase) arrest development at the dauer stage. Moreover, animals bearing weak or temperature-sensitive mutations in daf-2 and age-1 can develop reproductively, but nevertheless show increased energy storage and longevity. Here we show that null mutations in daf-16 suppress the effects of mutations in daf-2 or age-1; lack of daf-16 bypasses the need for this insulin receptor-like signalling pathway. The principal role of DAF-2/AGE-1 signalling is thus to antagonize DAF-16. daf-16 is widely expressed and encodes three members of the Fork head family of transcription factors. The DAF-2 pathway acts synergistically with the pathway activated by a nematode TGF-β-type signal, DAF-7, suggesting that DAF-16 cooperates with nematode SMAD proteins in regulating the transcription of key metabolic and developmental control genes. The probable human ortholognes of DAF-16, FKHR and AFX, may also act downstream of insulin signalling and cooperate with TGF-β effectors in mediating metabolic regulation. These genes may be dysregulated in diabetes.
The forkhead domain is a monomeric DNA binding motif that defines a rapidly growing family of eukaryotic transcriptional regulators. Genetic and biochemical data suggest a central role in embryonic development for genes encoding forkhead proteins. We have used PCR and low stringency hybridization to isolate clones from human cDNA and genomic libraries that represent seven novel forkhead genes, freac-1 to freac-7. The spatial patterns of expression for the seven freac genes range from specific for a single tissue to nearly ubiquitous. The DNA binding specificities of four of the FREAC proteins were determined by selection of binding sites from random sequence oligonucleotides. The binding sites for all four FREAC proteins share a core sequence, RTAAAYA, but differ in the positions flanking the core. Domain swaps between two FREAC proteins identified two subregions within the forkhead domain as responsible for creating differences in DNA binding specificity. Applying a circular permutation assay, we show that binding of FREAC proteins to their cognate sites results in bending of the DNA at an angle of 80-90 degrees.
age-1(hx546) is a recessive mutant allele in Caenorhabditis elegans that results in an increase in mean life span averaging 40% and in maximal life span averaging 60% at 20 degrees; at 25 degrees age-1(hx546) averages a 65% increase in mean life span (25.3 days vs. 15.0 days) and a 110% increase in maximum life span (46.2 days vs. 22.0 days for wild-type hermaphrodites). Mutant males also show extended life spans. age-1(hx546) is associated with a 75% decrease in hermaphrodite self-fertility as compared to the age-1+ allele at 20 degrees. Using two novel strategies for following the segregation of age-1, we present evidence that longer life results from a mutation in a single gene that increases the probability of survival at all chronological ages. The long-life and reduced-fertility phenotypes cosegregate and are tightly linked to fer-15, a locus on linkage group II. age-1(hx546) does not affect the timing of larval molts, the length of embryogenesis, food uptake, movement, or behavior in any way tested. Although age-1(hx546) lowers hermaphrodite self-fertility, it does not markedly affect the length of the reproductive period with all the increase in life expectancy due to an increase in the length of postreproductive life. In so far as we are aware, this mutant in age-1 is the only instance of a well-characterized genetic locus in which the mutant form results in lengthened fife. It is likely that the action of age-1 in lengthening life results not from eliminating a programmed aging function but rather from reduced hermaphrodite self-fertility or from some other unknown metabolic or physiologic alteration.
We report the cloning and characterization of the entire AFX gene which fuses to MLL in acute leukemias with a t(X;ll)(q13;q23). AFX consists of two exons and encodes for a protein of 501 amino acids. We found that normal B- and T-cells contain similar levels of AFX mRNA and that both the MLL/AFX as well as the AFX/MLL fusion transcripts are present in the cell line and the ANLL sample with a t(X;11)(q13;q23). The single intron of the AFX gene consists of 3706 nucleotides. It contains five simple sequence repeats with lengths of at least 12 bps, a chi-like octamer sequence (GCA/TGGA/TGG) and several immunoglobulin heptamer-like sequences (GATAGTG) that are distributed throughout the entire AFX intron sequence. In the KARPAS 45 cell line the breakpoints occur at nucleotides 2913/2914 of the AFX intron and at nucleotides 4900/4901 of the breakpoint cluster region of the MLL gene. The AFX protein belongs to the forkhead protein family. It is highly homologous to the human FKHR protein, the gene of which is disrupted by the t(2;13)(q35;q14), a chromosome rearrangement characteristic of alveolar rhabdomyosarcomas. It is noteworthy that the t(X;11)(q13;q23) in the KARPAS 45 cell line and in one acute nonlymphoblastic leukemia (ANLL) disrupts the forkhead domain of the AFX protein exactly at the same amino acids as does the t(2;13)(q35;q14) in case of the FKHR protein. In addition, the 5'-part of the AFX protein contains a conserved hexapeptide motif (QIYEWM) that is homologous to the functionally important conserved hexapeptide QIYPWM upstream of the homeobox domain in Hox proteins. This motif mediates the co-operative DNA binding of Pbx family members and Hox proteins and, therefore, plays an important role in physiologic and oncogenic processes. In acute leukemias with a t(X;11)(q13;q23), this hexapeptide motif is separated from the remaining forkhead domain within the AFX protein. The predicted amino acid sequence of AFX differs significantly from the partial AFX protein sequence published previously (Genes, Chromosomes and Cancer, 1994, 11, 79-84). This discrepancy can be explained by the occurrence of two sequencing errors in the earlier work at nucleotide number 783 and 844 (loss of a cytosine residue or guanosine residue, respectively) that lead to two reading frame shifts.
Akt2 encodes a protein-serine/threonine kinase containing a pleckstrin homology domain characteristic of many signaling molecules. Although there has been extensive interest in the mechanism by which the closely-related Akt kinase participates in phosphatidylinositol 3-kinase-mediated signaling, comparatively little is known regarding the expression and function of Akt2. This manuscript is the first to describe Akt2 mRNA expression in the developing mouse and the activation of AKT2 by insulin. These studies demonstrate that Akt2 is especially abundant in brown fat and, to a lesser extent, skeletal muscle and liver, tissues which are highly insulin-responsive and play a role in glucose metabolism. Endogenous Akt2 expression also is upregulated in fully-differentiated C2C12 myotubes and 3T3-L1 adipocytes, suggesting that these murine cell lines represent useful in vitro models for studies of Akt2 function. We show that HA-tagged AKT2 is activated in response to insulin stimulation in vitro and that activation of AKT2 is not induced in cells pretreated with wortmannin, an inhibitor of phosphatidylinositol 3-kinase. These data suggest that Akt2 expression is fundamental to the differentiated state of fat and muscle cells and that activation of AKT2 kinase by insulin is mediated through the phosphatidylinositol 3-kinase signaling pathway.
Survival factors can suppress apoptosis in a transcription-independent manner by activating the serine/ threonine kinase Akt, which then phosphorylates and inactivates components of the apoptotic machinery, including BAD and Caspase 9. In this study, we demonstrate that Akt also regulates the activity of FKHRL1, a member of the Forkhead family of transcription factors. In the presence of survival factors, Akt phosphorylates FKHRL1, leading to FKHRL1's association with 14-3-3 proteins and FKHRL1's retention in the cytoplasm. Survival factor withdrawal leads to FKHRL1 dephosphorylation, nuclear translocation, and target gene activation. Within the nucleus, FKHRL1 triggers apoptosis most likely by inducing the expression of genes that are critical for cell death, such as the Fas ligand gene.
In the nematode Caenorhabditis elegans, mutations of the insulin/insulin-like growth factor-1 receptor homologue Daf-2 gene cause developmental arrest at the dauer stage. The effect of Daf-2 mutations is counteracted by mutations in the Daf-16 gene, suggesting that Daf-16 is required for signaling by Daf-2. Daf-16 encodes a forkhead transcription factor. Based on sequence similarity, the FKHR genes are the likeliest mammalian Daf-16 homologues. FKHR proteins contain potential sites for phosphorylation by the serine/threonine kinase Akt. Because Akt is phosphorylated in response to insulin and has been implicated in a variety of insulin effects, we investigated whether insulin affects phosphorylation of FKHR. Insulin stimulated phosphorylation of endogenous FKHR and of a recombinant c-Myc/FKHR fusion protein transiently expressed in murine SV40-transformed hepatocytes. The effect of insulin was inhibited by wortmannin treatment, suggesting that PI 3-kinase activity is required for FKHR phosphorylation. Mutation of serine 253, located in a consensus Akt phosphorylation site at the carboxyl-terminal end of the forkhead domain, abolished the effect of insulin on FKHR phosphorylation. In contrast, mutation of two additional Akt phosphorylation sites, at amino acids threonine 24 or serine 316, did not abolish insulin-induced phosphorylation. These data indicate that FKHR may represent a distal effector of insulin action.
Insulin inhibits the expression of multiple genes in the liver containing an insulin response sequence (IRS) (CAAAA(C/T)AA), and we have reported that protein kinase B (PKB) mediates this effect of insulin. Genetic studies in Caenorhabditis elegans indicate that daf-16, a forkhead/winged-helix transcription factor, is a major target of the insulin receptor-PKB signaling pathway. FKHR, a human homologue of daf-16, contains three PKB sites and is expressed in the liver. Reporter gene studies in HepG2 hepatoma cells show that FKHR stimulates insulin-like growth factor-binding protein-1 promoter activity through an IRS, and introduction of IRSs confers this effect on a heterologous promoter. Insulin disrupts IRS-dependent transactivation by FKHR, and phosphorylation of Ser-256 by PKB is necessary and sufficient to mediate this effect. Antisense studies indicate that FKHR contributes to basal promoter function and is required to mediate effects of insulin and PKB on promoter activity via an IRS. To our knowledge, these results provide the first report that FKHR stimulates promoter activity through an IRS and that phosphorylation of FKHR by PKB mediates effects of insulin on gene expression. Signaling to FKHR-related forkhead proteins via PKB may provide an evolutionarily conserved mechanism by which insulin and related factors regulate gene expression.
Protein kinase B lies "downstream" of phosphatidylinositide (PtdIns) 3-kinase and is thought to mediate many of the intracellular actions of insulin and other growth factors. Here we show that FKHR, a human homologue of the DAF16 transcription factor in Caenorhabditis elegans, is rapidly phosphorylated by human protein kinase Balpha (PKBalpha) at Thr-24, Ser-256, and Ser-319 in vitro and at a much faster rate than BAD, which is thought to be a physiological substrate for PKB. The same three sites, which all lie in the canonical PKB consensus sequences (Arg-Xaa-Arg-Xaa-Xaa-(Ser/Thr)), became phosphorylated when FKHR was cotransfected with either PKB or PDK1 (an upstream activator of PKB). All three residues became phosphorylated when 293 cells were stimulated with insulin-like growth factor 1 (IGF-1). The IGF-1-induced phosphorylation was abolished by the PtdIns 3-kinase inhibitor wortmannin but not by PD 98059 (an inhibitor of the mitogen-activated protein kinase cascade) or by rapamycin. These results indicate that FKHR is a physiological substrate of PKB and that it may mediate some of the physiological effects of PKB on gene expression. DAF16 is known to be a component of a signaling pathway that has been partially dissected genetically and includes homologues of the insulin/IGF-1 receptor, PtdIns 3-kinase and PKB. The conservation of Thr-24, Ser-256, and Ser-319 and the sequences surrounding them in DAF16 therefore suggests that DAF16 is also a direct substrate for PKB in C. elegans.
While insulin has long been known to modulate intracellular metabolism by altering the activity or intracellular location of various enzymes, it is only in the past 10 years that the regulation of gene expression by insulin has been recognized as a major action of this hormone. This review principally focuses on the regulation of gene transcription by insulin, although recent progress in the understanding of insulin-regulated mRNA stability and translation is also summarized. The identification of cis-acting elements and associated trans-acting factors through which insulin either increases or decreases the transcription of specific genes is reviewed in detail. Recent advances in the understanding of the mechanisms of insulin signaling are discussed in the context of insulin-regulated gene transcription, and emphasis is placed on the gaps that remain between the upstream signaling molecules and the downstream trans-acting factors whose binding/transactivation potential is ultimately regulated. Finally, potential gene expression defects that may contribute to the pathophysiology of non-insulin-dependent diabetes mellitus and hypertriglyceridemia are considered.
Alveolar rhabdomyosarcomas are associated with unique chromosomal translocations t(2;13) and t(1;13), which arise from fusion of the genes for the paired box proteins PAX3 and PAX7, respectively, to the FKHR (forkhead in rhabdomyosarcoma) gene on chromosome 13q14. Here we report the identification and characterization of three novel human forkhead genes with similarity to FKHR. The three genes (HGMW-approved symbols FKHRP1, FKHRL1, and FKHRL1P1) map to chromosomal regions 5q35.2–q35.3, 6q21, and 17p11, respectively. Based on amino acid sequence comparisons of their forkhead domains, FKHRL1, FKHRL1P1, and FKHRP1 share 86, 84, and 68% identity, respectively, with FKHR. While FKHR and FKHRL1 are expressed in every human adult tissue examined, FKHRP1 mRNA expression could not be detected, and FKHRL1P1 expression was present only at low levels. FKHR and FKHRL1 share a similar genomic organization, each having a very large intron 1 (FKHR ∼130 kb and FKHRL1 >90 kb), which bisects their respective forkhead domains at identical positions, as well as a second intron just downstream of each stop codon. FKHRP1 and FKHRL1P1 lack introns and contain stop codons that prevent them from yielding full-length proteins. Thus, while FKHR and FKHRL1 represent functional genes, FKHRP1 and FKHRL1P1 probably are processed pseudogenes. These results suggest that these four genes represent an FKHR-like gene subfamily within the larger human forkhead gene family.
A pheromone-induced neurosecretory pathway in Caenorhabditis elegans triggers developmental arrest and an increase in longevity at the dauer diapause stage. The gene age-1 is required for non-dauer development and normal senescence. age-1 encodes a homologue of mammalian phosphatidylinositol-3-OH kinase (PI(3)K) catalytic subunits. Lack of both maternal and zygotic age-1 activity causes dauer formation, whereas animals with maternal but not zygotic age-1 activity develop as non-dauers that live more than twice as long as normal. These data suggest that phosphatidylinositol signalling mediated by AGE-1 protein controls lifespan and the dauer diapause decision.
The insulin/IGF receptor homolog DAF-2 regulates the aging in C. elegans. Decreasing daf-2 activity causes fertile adults to remain active much longer than normal and to live more than twice as long. A more severe decrease in daf-2 function causes young larvae to enter a state of diapause rather than progressing to adulthood. We have asked which cells require daf-2 gene activity in order for the animal to develop to adulthood and to age normally. We found that daf-2 functions cell nonautonomously in both processes. Our findings imply that the life span of C. elegans is determined by a signaling cascade in which the DAF-2 receptor acts in multiple cell lineages to regulate the production or activity of a secondary signal (or signals), which, in turn, controls the growth and longevity of individual tissues in the animal.
Copper homeostasis in the brain must be strictly maintained, since copper is an essential trace element and is potentially toxic. To understand the mechanism of copper homeostasis in the brain, we cloned several mouse homologues of copper trafficking genes and performed in situ hybridization histochemistry. mCTR1, mATX1, and mATP7a were highly expressed in the choroid plexus, indicating that the choroid plexus uses the trafficking pathway from uptake to efflux to transport copper to the cerebrospinal fluids. We suggest that these genes may regulate copper concentration in the brain through the choroid plexus.
Longevity is regulated by the daf-2 gene network in Caenorhabditis elegans. Mutations in the daf-2 gene, which encodes a member of the insulin receptor family, confer the life extension (Age) phenotype and the constitutive dauer (a growth-arrested larval form specialized for dispersal) formation phenotype. The Age phenotype is mutually potentiated by two life extension mutations in the daf-2 gene and the clk-1 gene, a homologue of yeast CAT5/COQ7 known to regulate ubiquinone biosynthesis. In this study, we demonstrated that the daf-2 mutation also conferred an oxidative stress resistance (Oxr) phenotype, which was also enhanced by the clk-1 mutation. Similar to the Age phenotype, the Oxr phenotype was regulated by the genetic pathway of insulin-like signaling from daf-2 to the daf-16 gene, a homologue of the HNF-3/forkhead transcription factor. These findings led us to examine whether the insulin-like signaling pathway regulates the gene expression of antioxidant defense enzymes. We found that the mRNA level of the sod-3 gene, which encodes Mn-superoxide dismutase (SOD), was much higher in daf-2 mutants than in the wild type. Moreover, the increased sod-3 gene expression phenotype is regulated by the insulin-like signaling pathway. Although the clk-1 mutant itself did not display Oxr and the increased sod-3 expression phenotypes, the clk-1 mutation enhanced them in the daf-2 mutant, suggesting that clk-1 is involved in longevity in two ways: clk-1 composes the original clk-1 longevity program and the daf-2 longevity program. These observations suggest that the daf-2 gene network controls longevity by regulating the Mn-SOD-associated antioxidant defense system. This system appears to play a role in efficient life maintenance at the dauer stage.
is a pre-doctoral fellow at Tohoku University School of Medicine, Japan. This work was supported, in part, by a grant for Longevity Sciences from the Ministry of Health
- T N Yamashita
Yamashita for technical advice. T.N. is a pre-doctoral fellow at Tohoku University
School of Medicine, Japan. This work was supported, in part, by a grant for Longevity
Sciences from the Ministry of Health and Welfare to N.M.
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