FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury.

BACKGROUND: Neutrophils are traditionally viewed as first responders but have a short onset of action in response to traumatic brain injury (TBI). However, the heterogeneity, multifunctionality, and time-dependent modulation of brain damage and outcome mediated by neutrophils after TBI remain poorly understood. METHODS: Using the combined single-cell transcriptomics, metabolomics, and proteomics analysis from TBI patients and the TBI mouse model, we investigate a novel neutrophil phenotype and its associated effects on TBI outcome by neurological deficit scoring and behavioral tests. We also characterized the underlying mechanisms both in vitro and in vivo through molecular simulations, signaling detections, gene expression regulation assessments [including dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays], primary cultures or co-cultures of neutrophils and oligodendrocytes, intracellular iron, and lipid hydroperoxide concentration measurements, as well as forkhead box protein O1 (FOXO1) conditional knockout mice. RESULTS: We identified that high expression of the FOXO1 protein was induced in neutrophils after TBI both in TBI patients and the TBI mouse model. Infiltration of these FOXO1high neutrophils in the brain was detected not only in the acute phase but also in the chronic phase post-TBI, aggravating acute brain inflammatory damage and promoting late TBI-induced depression. In the acute stage, FOXO1 upregulated cytoplasmic Versican (VCAN) to interact with the apoptosis regulator B-cell lymphoma-2 (BCL-2)-associated X protein (BAX), suppressing the mitochondrial translocation of BAX, which mediated the antiapoptotic effect companied with enhancing interleukin-6 (IL-6) production of FOXO1high neutrophils. In the chronic stage, the "FOXO1-transferrin receptor (TFRC)" mechanism contributes to FOXO1high neutrophil ferroptosis, disturbing the iron homeostasis of oligodendrocytes and inducing a reduction in myelin basic protein, which contributes to the progression of late depression after TBI. CONCLUSIONS: FOXO1high neutrophils represent a novel neutrophil phenotype that emerges in response to acute and chronic TBI, which provides insight into the heterogeneity, reprogramming activity, and versatility of neutrophils in TBI.

Cold-induced FOXO1 nuclear transport aids cold survival and tissue storage.

Cold-induced injuries severely limit opportunities and outcomes of hypothermic therapies and organ preservation, calling for better understanding of cold adaptation. Here, by surveying cold-altered chromatin accessibility and integrated CUT&Tag/RNA-seq analyses in human stem cells, we reveal forkhead box O1 (FOXO1) as a key transcription factor for autonomous cold adaptation. Accordingly, we find a nonconventional, temperature-sensitive FOXO1 transport mechanism involving the nuclear pore complex protein RANBP2, SUMO-modification of transporter proteins Importin-7 and Exportin-1, and a SUMO-interacting motif on FOXO1. Our conclusions are supported by cold survival experiments with human cell models and zebrafish larvae. Promoting FOXO1 nuclear entry by the Exportin-1 inhibitor KPT-330 enhances cold tolerance in pre-diabetic obese mice, and greatly prolongs the shelf-life of human and mouse pancreatic tissues and islets. Transplantation of mouse islets cold-stored for 14 days reestablishes normoglycemia in diabetic mice. Our findings uncover a regulatory network and potential therapeutic targets to boost spontaneous cold adaptation.

Oxidative Stress and FOXO-1 Relationship in Stage III Periodontitis.

OBJECTIVES: 8-Hydroxideoxyguanosine (8-OHdG) is a marker of oxidative stress, and Forkhead Box-O1 (FOXO1) is a transcription factor and signaling integrator in cell and tissue homeostasis. This study aims to determine FOXO1 and 8-OHdG levels in serum and saliva samples of periodontitis patients and to evaluate their relationship with clinical periodontal parameters. MATERIALS AND METHODS: Twenty healthy individuals, twenty generalized Stage III Grade B periodontitis patients, and nineteen generalized Stage III Grade C periodontitis patients were included in the study. Clinical periodontal parameters (plaque index (PI), probing depth (PD), bleeding on probing (BOP), and clinical attachment level (CAL)) were recorded. Salivary and serum 8-OHdG and FOX-O1 levels were analyzed by enzyme-linked immunosorbent assay (ELISA). RESULTS: Clinical periodontal parameters showed a statistically significant increase in periodontitis groups compared to the control group (p < 0.05). 8-OHdG salivary levels were significantly higher in both periodontitis groups compared to the control group. The salivary FOXO1 levels were significantly lower in both periodontitis groups compared to the control group. Salivary FOXO1 level had a low-grade negative correlation with BOP and salivary 8-OHdG level. CONCLUSIONS: While reactive oxygen species increase in periodontal inflammation, low expression of FOXO1, an important transcription factor for antioxidant enzymes, supports that this molecule plays a vital role in tissue destruction, and FOXO1 can be seen as a potential immune modulator. CLINICAL RELEVANCE: The role of FOXO1 in supporting antioxidant defense may suggest that FOXO1 is a candidate target for periodontitis treatment.

Polystyrene nanoplastics with different functional groups and charges have different impacts on type 2 diabetes.

BACKGROUND: Increasing attention is being paid to the environmental and health impacts of nanoplastics (NPs) pollution. Exposure to nanoplastics (NPs) with different charges and functional groups may have different adverse effects after ingestion by organisms, yet the potential ramifications on mammalian blood glucose levels, and the risk of diabetes remain unexplored. RESULTS: Mice were exposed to PS-NPs/COOH/NH2 at a dose of 5 mg/kg/day for nine weeks, either alone or in a T2DM model. The findings demonstrated that exposure to PS-NPs modified by different functional groups caused a notable rise in fasting blood glucose (FBG) levels, glucose intolerance, and insulin resistance in a mouse model of T2DM. Exposure to PS-NPs-NH2 alone can also lead the above effects to a certain degree. PS-NPs exposure could induce glycogen accumulation and hepatocellular edema, as well as injury to the pancreas. Comparing the effect of different functional groups or charges on T2DM, the PS-NPs-NH2 group exhibited the most significant FBG elevation, glycogen accumulation, and insulin resistance. The phosphorylation of AKT and FoxO1 was found to be inhibited by PS-NPs exposure. Treatment with SC79, the selective AKT activator was shown to effectively rescue this process and attenuate T2DM like lesions. CONCLUSIONS: Exposure to PS-NPs with different functional groups (charges) induced T2DM-like lesions. Amino-modified PS-NPs cause more serious T2DM-like lesions than pristine PS-NPs or carboxyl functionalized PS-NPs. The underlying mechanisms involved the inhibition of P-AKT/P-FoxO1. This study highlights the potential risk of NPs pollution on T2DM, and provides a new perspective for evaluating the impact of plastics aging.

Temporal coordination of the transcription factor response to H2O2 stress.

Oxidative stress from excess H2O2 activates transcription factors that restore redox balance and repair oxidative damage. Although many transcription factors are activated by H2O2, it is unclear whether they are activated at the same H2O2 concentration, or time. Dose-dependent activation is likely as oxidative stress is not a singular state and exhibits dose-dependent outcomes including cell-cycle arrest and cell death. Here, we show that transcription factor activation is both dose-dependent and coordinated over time. Low levels of H2O2 activate p53, NRF2 and JUN. Yet under high H2O2, these transcription factors are repressed, and FOXO1, NF-κB, and NFAT1 are activated. Time-lapse imaging revealed that the order in which these two groups of transcription factors are activated depends on whether H2O2 is administered acutely by bolus addition, or continuously through the glucose oxidase enzyme. Finally, we provide evidence that 2-Cys peroxiredoxins control which group of transcription factors are activated.

Linoleic acid induces human ovarian granulosa cell inflammation and apoptosis through the ER-FOXO1-ROS-NFκB pathway.

Polycystic ovary syndrome (PCOS) is a complex reproductive endocrinological disorder influenced by a combination of genetic and environmental factors. Linoleic acid (LA) is a widely consumed ω-6 polyunsaturated fatty acid, accounting for approximately 80% of daily fatty acid intake. Building upon the prior investigations of our team, which established a connection between LA levels in the follicular fluid and PCOS, this study deeply examined the specific impact of LA using a granulosa cell line. Our findings revealed that LA exerts its influence on granulosa cells (GCs) by binding to the estrogen receptor (ER). Activated ER triggers the transcription of the FOXO1 gene. Reactive oxygen species (ROS)-related oxidative stress (OS) and inflammation occur downstream of LA-induced FOXO1 activation. Increased OS and inflammation ultimately culminate in GC apoptosis. In summary, LA modulates the apoptosis and inflammation phenotypes of GCs through the ER-FOXO1-ROS-NF-κB pathway. Our study provides additional experimental evidence to comprehend the pathophysiology of PCOS and provides novel insights into the dietary management of individuals with PCOS.

Silencing of forkhead box protein O-1 (FOXO-1) enhances insulin-producing cell generation from adipose mesenchymal stem cells for diabetes therapy.

AIMS: Generation of mature ß-cells from MSCs has been a challenge in the field of stem cell therapy of diabetes. Adipose tissue-derived mesenchymal stem cells (Ad-MSCs) have made their mark in regenerative medicine, and provide several advantages compared to other MSCs sources. Forkhead box protein O-1 (FOXO-1) is an important transcription factor for normal development of ß-cells, yet its over expression in ß-cells may cause glucose intolerance. In this study, we isolated, characterized Ad-MSCs from rat epididymal fat pads, differentiated these MSCs into insulin producing cells (IPCs) and studied the role of FOXO-1 in such differentiation. MATERIALS AND METHODS: We examined the expression of FOXO-1 and its nuclear cytoplasmic localization in the generated IPCs. Afterwards we knocked down FOXO-1 using siRNA targeting FOXO-1 (siFOXO-1). The differentiated siFOXO-1 IPCs were compared to non-targeting siRNA (siNT) IPCs regarding expression of ß-cell markers by qRT-PCR and western blotting, dithizone (DTZ) staining and glucose stimulated insulin secretion (GSIS). KEY FINDINGS: Isolated Ad-MSCs exhibited all characteristics of MSCs and can generate IPCs. FOXO-1 was initially elevated during differentiation followed by a decline towards end of differentiation. FOXO-1 was dephosphorylated and localized to the nucleus upon differentiation into IPCs. Knock down of FOXO-1 improved the expression of ß-cell markers in final differentiated IPCs, improved DTZ uptake and showed increased insulin secretion upon challenging with increased glucose concentration. SIGNIFICANCE: These results portray FOXO-1 as a hindering factor of generation of IPCs whose down-regulation can generate more mature IPCs for MSCs therapy of diabetes mellitus.

KDM3B inhibitors disrupt the oncogenic activity of PAX3-FOXO1 in fusion-positive rhabdomyosarcoma.

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.

Type 1 interferons and Foxo1 down-regulation play a key role in age-related T-cell exhaustion in mice.

Foxo family transcription factors are critically involved in multiple processes, such as metabolism, quiescence, cell survival and cell differentiation. Although continuous, high activity of Foxo transcription factors extends the life span of some species, the involvement of Foxo proteins in mammalian aging remains to be determined. Here, we show that Foxo1 is down-regulated with age in mouse T cells. This down-regulation of Foxo1 in T cells may contribute to the disruption of naive T-cell homeostasis with age, leading to an increase in the number of memory T cells. Foxo1 down-regulation is also associated with the up-regulation of co-inhibitory receptors by memory T cells and exhaustion in aged mice. Using adoptive transfer experiments, we show that the age-dependent down-regulation of Foxo1 in T cells is mediated by T-cell-extrinsic cues, including type 1 interferons. Taken together, our data suggest that type 1 interferon-induced Foxo1 down-regulation is likely to contribute significantly to T-cell dysfunction in aged mice.

IGF-1 Regulates Skeletal Muscle Degradation and Remolding in Ventilator-Induced Diaphragmatic Dysfunction by Mediating FOXO1 Expression.

BACKGROUND: Mechanical ventilation (MV) sustains life in critically ill patients by providing adequate alveolar ventilation. However, prolonged MV could induce inspiratory muscle atrophy known as ventilator-induced diaphragmatic dysfunction (VIDD). Insulin-like growth factor (IGF)-1 has been proven to play crucial roles in regulating skeletal muscle size and function. Meanwhile, the forkhead box protein O1 (FOXO1) has been linked to muscle atrophy. This study aimed to explore the effect of IGF-1 on muscle degradation and remodeling in VIDD and delved into the association of the underlying mechanism involving FOXO1. METHODS: VIDD models were established by treating rats with MV. Adeno-associated virus (AAV) was used for transfection to construct IGF-1 and/or FOXO1 overexpressed rats. There were four groups in this study: normal rats (NC), normal rats with MV treatment (MV), IGF-1-overexpressed rats with MV treatment (MV+IGF-1), and rats overexpressing both IGF-1 and FOXO1 with MV treatment (MV+IGF-1+FOXO1). Protein levels were measured by western blot or enzyme-linked immunosorbent assay (ELISA), and mRNA levels were detected by real-time reverse transcriptase-polymerase chain reaction (RT-qPCR). IGF-1 and FOXO1 expression were validated by detecting mRNA and protein levels. Diaphragmatic muscle contractility and morphometry were tested using stimulating electrodes in conjunction with hematoxylin and eosin (H&E) staining. Interleukin (IL)-6 and carbonylated protein were used for evaluating muscle atrophy and oxidation, respectively. Protein degradation was determined by troponin-I level and tyrosine release. Apoptosis was assessed using the terminal deoxynucleotidyl transferase-mediated uridine 5'-triphosphate (UTP) nick-end labeling (TUNEL) assay, alongside markers like Bax, B-cell lymphoma 2 (BCL-2), and Cleaved Caspase-3. Atrogin-1, muscle RING finger 1 (MURF1), neuronally expressed developmentally downregulated 4 (NEDD4), muscle ubiquitin ligase of SCF complex in atrophy-1 (MUSA1), and ubiquitinated protein was used to determine proteolysis. Additionally, protein synthesis was measured by assessing the rates of mixed muscle protein (MMP) and myosin heavy chain (MHC). RESULTS: MV treatment caused IGF-1 downregulation (p < 0.01) and FOXO1 upregulation (p < 0.01). The IGF-1 upregulation downregulated FOXO1 in the MV+IGF-1 group (p < 0.001) while IGF-1 and FOXO1 were both upregulated in the MV+IGF-1+FOXO1 group (p < 0.001). The treatment of MV decreased muscle contractility and cross-sectional areas of diaphragm muscle fibers (p < 0.01). Additionally, IL-6, troponin-1, tyrosine release, carbonylated protein, TUNEL positive nuclei, Bax, Cleaved Caspase-3, Atrogin-1, MURF1, neuronally expressed developmentally downregulated 4 (NEDD4), MUSA1, and ubiquitinated protein levels increased significantly in MV group (p < 0.001) while levels of BCL-2, fractional synthetic rate of MMP and MHC, and type I and type II MHC protein mRNA expression decreased in MV group (p < 0.001). All of these alterations were reversed in the MV+IGF-1 group (p < 0.01), while the IGF-1-induced reversion was disrupted in the MV+IGF-1+FOXO1 group (p < 0.01). CONCLUSIONS: IGF-1 may protect diaphragmatic muscles from VIDD-induced structural damage and function loss by downregulating FOXO1. This action suppresses muscle breakdown and facilitates muscle remodeling in diaphragmatic muscles affected by VIDD.

The Roles and Regulatory Mechanisms of Tight Junction Protein Cingulin and Transcription Factor Forkhead Box Protein O1 in Human Lung Adenocarcinoma A549 Cells and Normal Lung Epithelial Cells.

Tight junction (TJ) protein cingulin (CGN) and transcription factor forkhead box protein O1 (FOXO1) contribute to the development of various cancers. Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for some cancers. HDAC inhibitors affect the expression of both CGN and FOXO1. However, the roles and regulatory mechanisms of CGN and FOXO1 are unknown in non-small cell lung cancer (NSCLC) and normal human lung epithelial (HLE) cells. In the present study, to investigate the effects of CGN and FOXO1 on the malignancy of NSCLC, we used A549 cells as human lung adenocarcinoma and primary human lung epithelial (HLE) cells as normal lung tissues and performed the knockdown of CGN and FOXO1 by siRNAs. Furthermore, to investigate the detailed mechanisms in the antitumor effects of HDAC inhibitors for NSCLC via CGN and FOXO1, A549 cells and HLE cells were treated with the HDAC inhibitors trichostatin A (TSA) and Quisinostat (JNJ-2648158). In A549 cells, the knockdown of CGN increased bicellular TJ protein claudin-2 (CLDN-2) via mitogen-activated protein kinase/adenosine monophosphate-activated protein kinase (MAPK/AMPK) pathways and induced cell migration, while the knockdown of FOXO1 increased claudin-4 (CLDN-4), decreased CGN, and induced cell proliferation. The knockdown of CGN and FOXO1 induced cell metabolism in A549 cells. TSA and Quisinostat increased CGN and tricellular TJ protein angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) in A549. In normal HLE cells, the knockdown of CGN and FOXO1 increased CLDN-4, while HDAC inhibitors increased CGN and CLDN-4. In conclusion, the knockdown of CGN via FOXO1 contributes to the malignancy of NSCLC. Both HDAC inhibitors, TSA and Quisinostat, may have potential for use in therapy for lung adenocarcinoma via changes in the expression of CGN and FOXO1.

Baicalin Maintains Articular Cartilage Homeostasis and Alleviates Osteoarthritis by Activating FOXO1.

Baicalin has been acknowledged for its anti-inflammatory properties. However, its potential impact on osteoarthritis (OA) has not yet been explored. Therefore, our study aimed to examine the effects of Baicalin on OA, both in laboratory and animal models. To evaluate its efficacy, human chondrocytes affected by OA were treated with interleukin-1ß and/or Baicalin. The effects were then assessed through viability tests using the cell counting kit-8 (CCK-8) method and flow cytometry. In addition, we analyzed the expressions of various factors such as FOXO1, autophagy, apoptosis, and cartilage synthesis and breakdown to corroborate the effects of Baicalin. We also assessed the severity of OA through analysis of tissue samples. Our findings demonstrate that Baicalin effectively suppresses inflammatory cytokines and MMP-13 levels caused by collagenase-induced osteoarthritis, while simultaneously preserving the levels of Aggrecan and Col2. Furthermore, Baicalin has been shown to enhance autophagy. Through the use of FOXO1 inhibitors, lentivirus-mediated knockdown, and chromatin immunoprecipitation, we verified that Baicalin exerts its protective effects by activating FOXO1, which binds to the Beclin-1 promoter, thereby promoting autophagy. In conclusion, our results show that Baicalin has potential as a therapeutic agent for treating OA (Clinical Trial Registration number: 2023-61).

Oleic and linoleic acids promote chondrocyte apoptosis by inhibiting autophagy via downregulation of SIRT1/FOXO1 signaling.

Osteoarthritis (OA) is a complex joint disease that currently has no cure. OA involves metabolic disorders in chondrocytes and an imbalance between autophagy and apoptosis. As a common risk factor for OA, obesity induces changes in the fatty acid composition of synovial fluid, thereby disturbing chondrocyte homeostasis. However, whether unsaturated fatty acids affect the development of OA by regulating chondrocyte autophagy remains unclear. This study aimed to determine the effects of oleic and linoleic acids on chondrocyte autophagy and related mechanisms. Based on the mass spectrometry results, the levels of multiple unsaturated fatty acids, including oleic and linoleic acids, in the synovial fluid of patients with OA and obesity were significantly higher than those in patients with OA only. Moreover, we found that FOXO1 and SIRT1 were downregulated after oleic and linoleic acids treatment of chondrocytes, which inhibited chondrocyte autophagy. Importantly, the upregulation of SIRT1 and FOXO1 expression not only increased the level of autophagy but also improved the expression of chondrocyte extracellular matrix proteins. Furthermore, upregulated SIRT1 and FOXO1 expression alleviated the destruction of the articular cartilage in an OA rat model. Our results suggest that SIRT1/FOXO1 signaling can alleviate oleic acid- and linoleic acid-induced cartilage degradation both in vitro and in vivo and that the SIRT1/FOXO1 pathway may serve as an effective treatment target for inhibiting OA progression.

FoxO1 promotes ovarian cancer by increasing transcription and METTL14-mediated m6A modification of SMC4.

The transcription factor forkhead box protein O1 (FoxO1) is closely related to the occurrence and development of ovarian cancer (OC), however its role and molecular mechanisms remain unclear. Herein, we found that FoxO1 was highly expressed in clinical samples of OC patients and was significantly correlated with poor prognosis. FoxO1 knockdown inhibited the proliferation of OC cells in vitro and in vivo. ChIP-seq combined with GEPIA2 and Kaplan-Meier database analysis showed that structural maintenance of chromosome 4 (SMC4) is a downstream target of FoxO1, and FoxO1 promotes SMC4 transcription by binding to its -1400/-1390 bp promoter. The high expression of SMC4 significantly blocked the tumor inhibition effect of FoxO1 knockdown. Furtherly, FoxO1 increased SMC4 mRNA abundance by transcriptionally activating methyltransferase-like 14 (METTL14) and increasing SMC4 m6A methylation on its coding sequence region. The Cancer Genome Atlas dataset analysis confirmed a significant positive correlation between FoxO1, SMC4, and METTL14 expression in OC. In summary, this study revealed the molecular mechanisms of FoxO1 regulating SMC4 and established a clinical link between the expression of FoxO1/METTL14/SMC4 in the occurrence of OC, thus providing a potential diagnostic target and therapeutic strategy.

FOXO1 regulates bovine skeletal muscle cells differentiation by targeting MYH3.

The growth and development of bovine skeletal muscle and beef yield is closely intertwined. Our previous research found that forkhead box O1 (FOXO1) plays an important role in the regulation of beef muscle formation, but its specific mechanism is still unknown. In this study, we aimed to clarify the regulatory mechanism of FOXO1 in proliferation and differentiation of bovine skeletal muscle cells (BSMCs). The results showed that interfering with FOXO1 can promote proliferation and the cell G1/S phase of BSMCs by up-regulating the expression of PCNA, CDK1, CDK2, CCNA2, CCNB1, CCND1 and CCNE2. Besides, interfering with FOXO1 inhibited the apoptosis of BSMCs by up-regulating the expression of anti-apoptosis gene BCL2, while simultaneously down-regulating the expression of the pro-apoptosis genes BAD and BAX. Inversely, interfering with FOXO1 can promote the differentiation of BSMCs by up-regulating the expression of myogenic differentiation marker genes MYOD, MYOG, MYF5, MYF6 and MYHC. Furthermore, RNA-seq combined with western bolt, immunofluorescence and chromatin immunoprecipitation analysis showed that FOXO1 could regulate BSMCs differentiation process by influencing PI3K-Akt, Relaxin and TGF-beta signaling pathways, and target MYH3 for transcriptional inhibition. In conclusion, this study provides a basis for studying the role and molecular mechanism of FOXO1 in BSMCs.

O-GlcNAcylation mediates H2O2-induced apoptosis through regulation of STAT3 and FOXO1.

The O-linked-ß-N-acetylglucosamine (O-GlcNAc) glycosylation (O-GlcNAcylation) is a critical post-translational modification that couples the external stimuli to intracellular signal transduction networks. However, the critical protein targets of O-GlcNAcylation in oxidative stress-induced apoptosis remain to be elucidated. Here, we show that treatment with H2O2 inhibited O-GlcNAcylation, impaired cell viability, increased the cleaved caspase 3 and accelerated apoptosis of neuroblastoma N2a cells. The O-GlcNAc transferase (OGT) inhibitor OSMI-1 or the O-GlcNAcase (OGA) inhibitor Thiamet-G enhanced or inhibited H2O2-induced apoptosis, respectively. The total and phosphorylated protein levels, as well as the promoter activities of signal transducer and activator of transcription factor 3 (STAT3) and Forkhead box protein O 1 (FOXO1) were suppressed by OSMI-1. In contrast, overexpressing OGT or treating with Thiamet-G increased the total protein levels of STAT3 and FOXO1. Overexpression of STAT3 or FOXO1 abolished OSMI-1-induced apoptosis. Whereas the anti-apoptotic effect of OGT and Thiamet-G in H2O2-treated cells was abolished by either downregulating the expression or activity of endogenous STAT3 or FOXO1. These results suggest that STAT3 or FOXO1 are the potential targets of O-GlcNAcylation involved in the H2O2-induced apoptosis of N2a cells.

METTL14-mediated lncRNA XIST silencing alleviates GDM progression by facilitating trophoblast cell proliferation and migration via the miR-497-5p/FOXO1 axis.

Gestational diabetes mellitus (GDM), a prevalent complication during the gestation period, has been linked to impaired proliferation and migration of trophoblasts causing placental maldevelopment. We previously found that lncRNA X-inactive specific transcript (XIST) played an essential role in GDM progression. Here, we investigated the precise biological functions as well as the upstream and downstream regulatory mechanisms of XIST in GDM. We found that XIST and forkhead box O1 (FOXO1) were conspicuously upregulated and miR-497-5p and methyltransferase-like 14 (METTL14) were downregulated in the placentas of GDM patients. XIST silencing facilitated proliferation and migration and inhibited cell apoptosis and cell cycle arrest in HG-cultured HTR8/SVneo cells. METTL14 inhibited XIST expression through m6A methylation modification. XIST overexpression abrogated the positive effect of METTL14 overexpression on HG-cultured HTR8/SVneo cell progression. MiR-497-5p and FOXO1 are downstream regulatory genes of XIST in HTR8/SVneo cells. Reverse experiments illustrated that XIST mediated HTR8/SVneo cell functions by regulating the miR-497-5p/FOXO1 axis. Additionally, XIST silencing augmented glucose tolerance and alleviated fetal detrimental changes in GDM rats. To conclude, METTL14-mediated XIST silencing facilitated proliferation and migration and inhibited cell apoptosis and cell cycle arrest in HG-cultured HTR8/SVneo cells via the miR-497-5p/FOXO1 axis, thereby alleviating GDM progression in rats.

Brg1/PRMT5 nuclear complex epigenetically regulates FOXO1 in IPF mesenchymal progenitor cells.

We have discovered intrinsically fibrogenic mesenchymal progenitor cells (MPCs) in the human idiopathic pulmonary fibrosis (IPF) lung. IPF MPCs display a durably distinct transcriptome, suggesting that they have undergone epigenetic modifications. Prior studies indicate that the chromatin remodeler Brg1 associates with the arginine methyltransferase PRMT5 to epigenetically regulate transcription factors. We hypothesize that a Brg1/PRMT5 nuclear complex epigenetically regulates critical nodes in IPF MPC self-renewal signaling networks. IPF and control MPCs were isolated from primary mesenchymal cell lines established from IPF and control patients. RNA-sequencing identified increased expression of the FOXO1 transcription factor in IPF MPCs compared with controls, a result we confirmed by Q-PCR and Western blot analysis. Immunoprecipitation identified a CD44/Brg1/PRMT5 nuclear complex in IPF MPCs. Chromatin immunoprecipitation assays showed that PRMT5 and its methylation mark H3R2me2 are enriched on the FOXO1 promoter. We show that loss of Brg1 and PRMT5 function decreases FOXO1 expression and impairs IPF MPC self-renewal, and that loss of FOXO1 function decreases IPF MPC self-renewal and expression of the SOX2 and OCT4 stemness markers. Our findings indicate that the FOXO1 gene is overexpressed in IPF MPCs in a CD44/Brg1/PRMT5 nuclear complex-dependent manner. Our data suggest that Brg1 alters chromatin accessibility, enriching PRMT5 occupancy on the FOXO1 promoter, and PRMT5 methylates histone H3 arginine 2 (H3R2) on the FOXO1 promoter, increasing its expression. Our data are in accord with the concept that this coordinated interplay is responsible for promoting IPF MPC self-renewal and maintaining a critical pool of fibrogenic MPCs that drive IPF progression.NEW & NOTEWORTHY Our research offers valuable understanding regarding the epigenetic control of IPF MPC. The data we obtained strongly support the idea that the coordination between chromatin remodeling and histone methylation plays a key role in regulating transcription factors. Specifically, our findings indicate that FOXO1, an essential transcription factor, likely governs the self-renewal of IPF MPC, which is crucial for maintaining a critical pool of fibrogenic MPCs. This interplay could be an important therapeutic target.

The FOXO1/G6PC axis promotes gastric cancer progression and mediates 5-fluorouracil resistance by targeting the PI3K/AKT/mTOR signaling pathway.

Gastric cancer (GC) is a prevalent malignancy of the digestive system. Distant metastasis and chemotherapy resistance are the crucial obstacles to prognosis in GC. Recent research has discovered that the glucose-6-phosphatase catalytic subunit (G6PC) plays an important role in tumor malignant development. However, little evidence has highlighted its role in GC. Herein, through a comprehensive analysis including profiling of tissue samples and functional validation in vivo and in vitro, we identify G6PC as a crucial factor in GC tumorigenesis. Importantly, we found that the FOXO1/G6PC axis could accelerate GC cell proliferation, metastasis, and 5-Fluorouracil (5-FU) resistance by targeting the PI3K/AKT/mTOR signaling pathway, implicating that as a prospective therapeutic approach in GC.

Enhanced anticancer synergy of LOM612 in combination with selinexor: FOXO1 nuclear translocation-mediated inhibition of Wnt/ß-catenin signaling pathway in breast cancer.

BACKGROUND: The intricate relationship between Forkhead box O1 (FOXO1), a well-established tumor suppressor, and breast cancer (BC) remains partially elucidated. This study aims to investigate the mechanistic role of FOXO1 nuclear localization in the context of BC. METHODS: In vitro experiments employed BC cell lines MCF-7 and MDA-MB-175 treated with LOM612, a small molecule activator of FOXO nuclear-cytoplasmic shuttling, and selinexor, an exportin 1 inhibitor. Nuclear accumulation of FOXO1, its interaction with ß-catenin, and expressions of key proteins like V-Myc avian myelocytomatosis viral oncogene homolog (c-Myc), cyclin D1 and apoptosis markers were assessed. In vivo, the effects of LOM612 and selinexor were studied using MCF-7 cell-derived xenografts (CDX). RESULTS: Treatment with LOM612 exhibited a significant enhancement in nuclear accumulation of FOXO1 within BC cells. This effect coincided with suppressed migratory behavior and heightened apoptosis susceptibility in these cells. Mechanistically, LOM612 orchestrated FOXO1 to compete with transcription factors (TCF) for binding to ß-catenin in the nucleus, leading to reduced c-Myc and cyclin D1 expressions, along with elevated levels of apoptosis-related proteins. Similar trends were observed in CDX models, where LOM612 effectively suppressed tumor growth, increased FOXO1 nuclear localization, and downregulated c-Myc and cyclin D1 expressions. Importantly, selinexor synergistically reinforced the therapeutic effects of LOM612 both in vitro and in vivo. CONCLUSIONS: Collectively, this study underscores the potential of combining LOM612 and selinexor as an efficacious anti-BC strategy. The underlying mechanism involves FOXO1's nuclear translocation, which disrupts TCF-ß-catenin interactions, thus indirectly inhibiting the Wnt/ß-catenin signaling pathway.