Inhibition Of FOXO1 Research Articles

Cytochrome P450 2E1-dependent hepatic ethanol metabolism induces fatty acid-binding protein 4 and steatosis.

Hepatic steatosis is an early pathology of alcohol-associated liver disease (ALD). Fatty acid-binding protein-4 (FABP4, a FABP not normally produced in the liver) is secreted by hepatocytes in ALD and stimulates hepatoma proliferation and migration. This study sought to investigate the mechanism[s] by which hepatic ethanol metabolism regulates FABP4 and steatosis. Human hepatoma cells (HepG2/HuH7) and cells stably transfected to express cytochrome P450 2E1 (CYP2E1), were exposed to ethanol in the absence or presence of chlormethiazole (a CYP2E1-inhibitor; CMZ) and/or EX-527 (a sirtuin-1 [SIRT1] inhibitor). The culture medium was analyzed for ethanol metabolism and FABP4 protein abundance. Cells were analyzed for FABP4mRNA expression, SIRT1 protein abundance, and neutral lipid accumulation. In parallel, cells were analyzed for forkhead box O1 [FOXO1], β-catenin, peroxisome proliferator-activated receptor-α [PPARα], and lipin-1α protein abundance in the absence or presence of ethanol and pharmacological inhibitors of the respective target proteins. CYP2E1-dependent ethanol metabolism inhibited the amount of SIRT1 protein detected, concomitant with increased FABP4mRNA expression, FABP4 protein secretion, and neutral lipid accumulation, effects abolished by CMZ. Analysis of pathways associated with lipid oxidation revealed increased FOXO1 nuclear localization and decreased β-catenin, PPARα, and lipin-1α protein levels in CYP2E1-expressing cells in the presence of ethanol. Pharmacological inhibition of SIRT1mimicked the effects of ethanol, while inhibition of FOXO1 abrogated the effect of ethanol on FABP4mRNA expression, FABP4 protein secretion, and neutral lipid accumulation in CYP2E1-expressing cells. Pharmacological inhibition of β-catenin, PPARα, or lipin-1α failed to alter the effects of ethanol on FABP4 or neutral lipid accumulation. CYP2E1-dependent ethanol metabolism inhibits SIRT1-FOXO1signaling, which leads to increased FABP4mRNA expression, FABP4 protein secretion, and neutral lipid accumulation. These data suggest that FABP4 released from steatotic hepatocytes could play a role in promoting tumor cell expansion in the setting of ALD and represents a potential target for therapeutic intervention.

Serpina3c deficiency induced necroptosis promotes non-alcoholic fattyliverdisease through β-catenin/Foxo1/TLR4 signaling.

Non-alcoholic fatty liver disease (NAFLD) is a public health challenge and an increasing cause of chronic liver disease worldwide. However, the underlying molecular mechanism remains unclear. The aim of this study was to determine the precise role of serpina3c in the process of NAFLD. MaleApoe-/- /serpina3c-/- double knockout (DKO) and Apoe-/- mice were fed a high-fat diet (HFD) for 12weeks. Several markers of steatosis and inflammation were evaluated. In vitro cell models induced by palmitic acid (PA) treatment were used to evaluate the beneficial effect of serpina3c on necroptosis and the underlying molecular mechanism. Compared with Apoe-/- mice, DKO mice exhibited a significantly exacerbated hepatic steatosis, increased hepatic triglyceride content and expression of genes involved in lipid metabolism (SREBP1c and SCD1), promoted hepatic inflammation and fibrosis, promoted necroptosis by increasing expression of receptor-interacting protein 3 (RIP3), phosphorylated mixed lineage kinase domain-like (MLKL) and high mobility group box 1 (HMGB1). Notably, serpina3c deficiency increased β-catenin, Foxo1, and Toll-like receptor 4 (TLR4) protein expression. In vitro , serpina3c knockdown promoted necroptosis and lipid droplet formation under condition of lipotoxicity. However, these phenomena were reversed by the overexpression of serpina3c. Mechanistically, downregulation of serpina3c expression promoted Foxo1 and β-catenin colocalized in the nucleus under condition of lipotoxicity, consequently upregulating the expression of TLR4. Conversely, disruption of Foxo1-β/catenin by Foxo1 and β-catenin inhibitors decreased TLR4 expression and ameliorated hepatic necroptosis in vitro. This study highlights a novel mechanism that serpina3c modulates NAFLD development by inhibiting necroptosis via β-catenin/Foxo1/TLR4.

Targeting Forkhead box O1-aquaporin 5 axis mitigates neuropathic pain in a CCI rat model through inhibiting astrocytic and microglial activation

ABSTRACT Forkhead box O1 (FoxO1) is a critical molecule in modulating cell growth, differentiation and metabolism, acting as a vital transcription factor. This study explored the role of FoxO1 in chronic constriction injury (CCI)-induced neuropathic pain (NP). Microglial and astrocyte activation was achieved with lipopolysaccharide (LPS, 100 ng/mL) to establish an in-vitro NP model. Morphological alterations in LPS-induced microglia and astrocytes were assayed by light microscopy. The levels of inflammatory cytokines and proteins in microglia and astrocytes were gauged by enzyme-linked immunosorbent assay (ELISA), and Western blot (WB). The CCI-induced NP rat model was constructed for investigating the FoxO1-AQP5 axis in NP. LPS markedly expanded the expression of inflammatory factors and boosted the expression of FoxO1 and AQP5 in microglia and astrocytes. Inhibition of FoxO1 or AQP5 dramatically decreased the LPS-induced inflammation in microglia and astrocytes. In vivo, CCI exacerbated the inflammatory response and NP symptoms and substantially raised the contents of FoxO1 and AQP5 in rats’ spinal cord tissues. Intrathecal administration of the Sirt1 agonist Resveratrol abated CCI-induced activation of FoxO1 and AQP5, abrogated CCI-induced mechanical hyperalgesia and thermal hyperalgesia, depressed microglial and astrocyte activation, and declined the generation of pro-inflammatory mediators in spinal cord tissues. Mechanistically, blocking the FoxO1-AQP5 pathway inactivated the ERK and p38 MAPK pathways. Suppressing the FoxO1-AQP5 axis alleviated CCI-induced NP and inflammatory responses by modulating the ERK and p38 MAPK signaling pathways.

Rosmarinic acid downregulates the oxLDL‑induced interaction between monocytes and endothelial cells, in addition to monocyte diapedesis, under high glucose conditions.

Endothelial dysfunction during diabetes has been previously reported to be at least in part attributed to increased oxidized low-density lipoprotein (oxLDL) levels mediated by high glucose (HG) levels. Endothelial inflammation increases the adhesiveness of monocytes to the endothelium in addition to increasing vascular permeability, promoting diabetic atherogenesis. In a previous study, it was reported that oxLDL treatment induced nucleotide-binding domain and leucine-rich repeat containing family, pyrin domain-containing 3 inflammasome activation in endothelial cells (ECs) under HG conditions, in a manner that could be effectively reversed by rosmarinic acid. However, it remains unclear whether oxLDL-mediated inflammasome activation can regulate the interaction between monocytes and ECs. The effects of oxLDL-mediated inflammasome activation on endothelial permeability under HG conditions, in addition to the effects of rosmarinic acid on these oxLDL-mediated processes, also remain poorly understood. Therefore, the present study aimed to elucidate the mechanisms involved in oxLDL-induced endothelial permeability and monocyte diapedesis under HG conditions, in addition to the potential effects of rosmarinic acid. ECs were treated with oxLDL under HG conditions in the presence or absence of ROS scavengers mitoTEMPO and NAC, p38 inhibitor SB203580, FOXO1 inhibitor AS1842856 or transfected with the TXNIP siRNA, before protein expression levels of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), phosphorylated vascular endothelial-cadherin (VE-cadhedrin), VE-cadherin and zonula occludens-1 (ZO-1) were measured by western blotting. In addition, adhesion assay and Transwell assays were performed. oxLDL was found to significantly increase the expression of ICAM-1 and VCAM-1 in ECs under HG conditions whilst also enhancing the adhesion of monocytes to ECs. This was found to be dependent on the reactive oxygen species (ROS)/p38 MAPK/forkhead box O1 (FOXO1)/thioredoxin interacting protein (TXNIP) signaling pathway. In addition, oxLDL-stimulated ECs under HG conditions exhibited increased phosphorylated VE-cadherin protein levels and decreased ZO-1 protein expression levels compared with those in untreated ECs, suggesting increased endothelial permeability. Furthermore, monocyte transmigration through the endothelial monolayer was significantly increased by oxLDL treatment under HG conditions. These oxLDL-mediated effects under HG conditions were also demonstrated to be dependent on this ROS/p38 MAPK/FOXO1/TXNIP signaling pathway. Subsequently, rosmarinic acid treatment significantly reversed oxLDL-induced overexpression of adhesion molecules and monocyte-EC adhesion, oxLDL-induced endothelial junction hyperpermeability and monocyte transmigration through the endothelial monolayer under HG conditions, in a dose-dependent manner. These results suggest that rosmarinic acid can exert a protective effect against oxLDL-mediated endothelial dysfunction under HG conditions by reducing the interaction between monocytes and ECs in addition to preventing monocyte diapedesis.

Downregulation of MEG3 and upregulation of EZH2 cooperatively promote neuroblastoma progression.

Neuroblastoma (NB), an embryonic tumour originating from sympathetic crest cells, is the most common extracranial solid tumour type in children with poor overall prognosis. Accumulating evidence has demonstrated the involvement of long non‐coding RNA (lncRNA) in numerous biological processes and their associations with embryonic development and multiple diseases. Ectopic lncRNA expression is linked to malignant tumours. Previous studies by our team indicate that MEG3 attenuates NB autophagy through inhibition of FOXO1 and epithelial‐mesenchymal transition via the mTOR pathway in vitro. Moreover, MEG3 and EZH2 negatively regulate each other. In present study, we first collected 60 NB tissues and 20 adjacent tissues for Quantitative real‐time polymerase chain reaction (Q‐PCR) experiments and performed clinical correlation analysis of the results. At the same time, nude mice were used for subcutaneous tumour formation to detect the effect of MEG3 in vivo. Two NB cell lines, SK‐N‐AS and SK‐N‐BE(2)C, were overexpressed MEG3 and rescued with EZH2 and then were subjected to proliferation, migration, invasion, apoptosis and autophagy experiments. RNA‐binding protein immunoprecipitation (RIP) and Co‐Immunoprecipitation (Co‐IP) experiments were performed to explore the molecular mechanism of MEG3 and EZH2 interaction. Q‐PCR revealed that MEG3 expression was negatively correlated with INSS stage and risk grade of NB. Moreover, MEG3 overexpression was associated with inhibition of NB growth in vivo. MEG3 exerted an anti‐cancer effect via stimulatory effects on EZH2 ubiquitination leading to its degradation. Conversely, EZH2 interacted with DNMT1 and HDAC1 to induce silencing of MEG3. The EZH2 inhibitor, DZNep, and HDAC inhibitor, SAHA, displayed synergistic activity against NB. Combined treatment with DZNep and SAHA inhibited proliferation, migration and invasion of NB through suppression of the PI3K/AKT/mTOR/FOXO1 pathway. In conclusion, downregulation of MEG3 and upregulation of EZH2 forms a feedback loop that concertedly promotes the development of NB. Combined blockage of EZH2 and HDAC1 with the appropriate inhibitors may therefore present an effective treatment strategy for NB cases with low MEG3 and high EZH2 expression.

Silent Information Regulator 1 (SIRT1) Promotes Pancreatic β-Cell Mitochondrial Autophagy and Pyrin Domains-Containing Protein 3 (NLRP3) Activation Under High Glucose Environment

Mitochondrial autophagy and inflammatory response involves in diabetes. This study mainly explores the role of Silent Information Regulator (SIRT1) in pancreatic β-cells under high glucose conditions and related mechanism. Pancreatic β cells was cultured in a high-glucose environment with SRT1720 and EX527 respectively to define activation group and inhibition group followed by analysis of SIRT1, P-FOXO1, FOXO1, LC3, ATG5, PINK, Parkin, Mfn1, Mfn2, Fis1, IL-6, TNF-α, NLRP3 protein and mRNA expression by qRT-PCR, Western blot and fluorescent probe technology. Compared with control group, SIRT1 protein and mRNA expression in the high glucose group was significantly reduced. Activation group had highest protein and mRNA expression of SIRT1 P-FOXO1, FOXO1, Mfn1, Mfn2, Fis1, PINK, Parkin and mitochondrial membrane potential followed by blank group and inhibition group.SIRT1 secretion by pancreatic β-cells under high glucose environment is reduced. After activating SIRT1, mitochondrial autophagy decreased significantly and inflammatory response is significantly alleviated, indicating that SIRT1 might be used as a therapeutic target.

Protective Effect of Grape Seed Proanthocyanidins on Oxidative Damage of Chicken Follicular Granulosa Cells by Inhibiting FoxO1-Mediated Autophagy.

A significant decrease in poultry egg production occurs due to ovarian aging and autophagy is one of the important factors of ovarian aging that is induced predominantly by oxidative stress. Increasing evidence showed potential roles of plant-derived grape seed proanthocyanidin (GSPs) in protecting ovarian granulosa cells (GCs) from oxidative damage, although the underlying mechanism is still unclear. Here we investigated the possible functions of autophagy involved in the preventive effect of GSPs on oxidative stress in the GCs of ovarian hierarchical follicles of laying chickens. The results showed that increased autophagy was observed in the aging hens (580-day-old, D580) compared with the peak-lay hens (D280). Treatment of GSPs significantly restored the elevated autophagy and decreased viability of cultured D280 chicken GCs that were elicited by hydrogen peroxide. GSPs also suppressed the increased autophagy in the natural aging hens. Similar to the effect of GSPs on GC viability, inhibition of autophagy also showed a protective effect on the decreased viability of GCs under oxidative damage. However, GSPs were not able to provide further protection in GCs that were pretreated with 3-methyladenine (an autophagy inhibitor). In addition to its promoting action on antioxidant capacity, treatment with GSPs increased survival of GCs from autophagy that was caused by oxidative stress through the FoxO1-related pathway. Inhibition of FoxO1 or activation of PI3K-Akt pathway by GSPs increased the confrontation of GCs to oxidative damage and decreased autophagy in GCs. In addition, activation of the SIRT1 signal inhibited the GCs autophagy that was caused by oxidative stress via GSPs-induced deacetylation of FoxO1. These results revealed a new mechanism of GSPs against oxidative stress of GCs via inhibiting FoxO1, which was probably a possible target for alleviating ovarian aging in laying poultry.

Laminaria japonica Polysaccharide Suppresses Atherosclerosis via Regulating Autophagy-Mediated Macrophage Polarization.

The present work aimed to explore the effect and underlying mechanism of a homogeneous Laminaria japonica polysaccharide (LJP61A) on macrophage polarization in high-fat-diet-fed LDLr-/- mice and Ox-LDL-induced macrophages. Results showed that LJP61A remarkably reduced the lesion burden in atherosclerotic mice, alleviated lipid deposition in Ox-LDL-stimulated macrophages, decreased the expression of M1 macrophage markers, and increased the expression of M2 macrophage markers, thus reducing the M1/M2 macrophage phenotype ratio. Meanwhile, the autophagic flux of macrophages was enhanced by LJP61A treatment in vitro and in vivo. 3-Methyladenine is an autophagic inhibitor. As expected, this inhibitor blocked the effects of LJP61A on macrophage polarization. SIRT1 and FoxO1 are two key upstream genes that control the autophagy behavior. We also found that LJP61A significantly up-regulated the expression of SIRT1 and FoxO1. However, these effects of LJP61A were abolished by the SIRT1 siRNA and FoxO1 inhibitor AS1842856. These results suggested that LJP61A reduced atherosclerosis in HFD-induced LDLr-/- mice via regulating autophagy-mediated macrophage polarization.

Exosomal MicroRNA-325 Enhances Trophoblast Migration and Invasion Through Downregulation of Lethal-7b and Upregulation of Forkhead Box Protein O1 Expression in Preeclampsia

We aimed to explore the mechanism by how microRNA (miRNA)-325 derived from marrow mesenchymal stem cell exosomes (MSC-exos) affects the trophoblast progression in preeclampsia (PE). RT-qPCR detected the level of miRNA let-7b and FOXO1 in the placenta tissue of PE patients. Functional experiment was performed to analyze the effect of FOXO1 inhibitor and let-7b mimics on cell migration, invasion and apoptosis through Transwell assay and TUNEL staining. The trophoblast cell was co-cultured with overexpressed-miR-325 MSC-exos to measure gene expression and cell progression. let-7b was highly and FOXO1 was lowly expressed in PE placenta tissue. let-7b directly targeted and inhibited FOXO1 expression. Importantly, as miR-325 was internalized by trophoblast cells through MSC-exos, MSC-exos overexpressing miR-325 inhibited let-7b expression in trophoblasts, up-regulated FOXO1 and activated AKT signaling pathway. Further, MSC-exos treatment promoted invasion and migration of trophoblast cell and inhibited apoptosis. In conclusion, miR-325 derived from MSC-exos promotes the invasion and migration of trophoblast cells in PE through inhibition of let7b and upregulation of FOXO1.

Insulin-like Growth Factor 1 Promotes Cell Proliferation by Downregulation of G-Protein-Coupled Receptor 17 Expression via PI3K/Akt/FoxO1 Signaling in SK-N-SH Cells.

Insulin-like growth factor 1 (IGF-1) not only regulates neuronal function and development but also is neuroprotective in the setting of acute ischemic stroke. G-protein-coupled receptor 17 (GPR17) expression in brain tissue serves as an indicator of brain damage. As whether IGF-1 regulates GPR17 expression remains unknown, the aim of this study is to investigate how IGF-1 regulates GPR17 expression in vitro. Human neuroblastoma SK-N-SH cells were used. Lentivirus-mediated short hairpin RNA (shRNA) was constructed to mediate the silencing of FoxO1, while adenoviral vectors were used for its overexpression. Verification of the relevant signaling cascade was performed using a FoxO1 inhibitor (AS1842856), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), and a GPR17 antagonist (cangrelor). Cell proliferation was analyzed using EdU staining; immunofluorescence staining was used to detect the expression and subcellular localization of FoxO1. Chromatin immunoprecipitation was used to analyze the binding of FoxO1 to the GPR17 promoter in SK-N-SH cells. The expression of FoxO1, GPR17, and protein kinase B (also known as Akt) mRNA and protein as well as the levels of FoxO1 and Akt phosphorylation were investigated in this study. IGF-1 was found to downregulate FoxO1 and GPR17 expression in SK-N-SH cells while promoting cell viability and proliferation. Inhibition of FoxO1 and antagonism of GPR17 were found to play a role similar to that of IGF-1. Silencing of FoxO1 by lentivirus-mediated shRNA resulted in the downregulation of FoxO1 and GPR17 expression. The overexpression of FoxO1 via adenoviral vectors resulted in the upregulation of FoxO1 and GPR17 expression. Blocking of PI3K signaling by LY294002 inhibited the effect of IGF-1 on GPR17 suppression. Results from chromatin immunoprecipitation revealed that IGF-1 promotes FoxO1 nuclear export and reduces FoxO1 binding to the GPR17 promoter in SK-N-SH cells. Here, we conclude that IGF-1 enhances cell viability and proliferation in SK-N-SH cells via the promotion of FoxO1 nuclear export and reduction of FoxO1 binding to the GPR17 promoter via PI3K/Akt signaling. Our findings suggest that the enhancement of IGF-1 signaling to antagonize GPR17 serves as a potential therapeutic strategy in the management of acute ischemic stroke.

FoxO4 controls sGCβ transcription in vascular smooth muscle.

Nitric oxide (NO) binds soluble guanylyl cyclase β (sGCβ) to produce cGMP and relax vascular smooth muscle cells (SMCs) needed for vasodilation. Although the regulation of NO-stimulated sGC activity has been well characterized at the posttranslational level, the mechanisms that govern sGC transcription remain incompletely understood. Recently, we identified Forkhead box subclass O (FoxO) transcription factors as essential for expression of sGC; however, the specific FoxO family member responsible for the expression of sGCβ in SMC remains unknown. Using FoxO shRNA knockdown adenovirus treatment in rat aortic SMCs, we show that FoxO1 or FoxO3 knockdown causes greater than twofold increases in Gucy1a3 and Gucy1b3 mRNA expression, without changes in NO-dependent cGMP production or cGMP-dependent phosphorylation. FoxO4 knockdown produced a 50% decrease in Gucy1a3 and Gucy1b3 mRNA with 70% loss of sGCα and 50% loss of sGCβ protein expression. Knockdown of FoxO4 expression decreased cGMP production and downstream protein kinase G-dependent phosphorylation more than 50%. Triple FoxO knockdown exacerbated loss of sGC-dependent function, phenocopying previous FoxO inhibition studies. Using promoter luciferase and chromatin immunoprecipitation assays, we find that FoxO4 acts as a transcriptional activator by directly binding several FoxO DNA motifs in the promoter regions of GUCY1B3 in human aortic SMCs. Collectively, our data show FoxO4 is a critical transcriptional regulator of sGCβ expression in SMC.NEW & NOTEWORTHY One of the key mechanisms of vascular smooth muscle cell (SMC) dilation occurs through nitric oxide (NO)-dependent induction of soluble guanylyl cyclase (sGC) by means of its β-subunit. Herein, we are the first to identify Forkhead box subclass O protein 4 (FoxO4) as a key transcriptional regulator of GUCY1B3 expression, which codes for sGCβ protein in human and animal SMCs. This discovery will likely have important implications for the future usage of antihypertensive and vasodilatory therapies which target NO production, sGC, or FoxO transcription factors.

MiR-486 attenuates cardiac ischemia/reperfusion injury and mediates the beneficial effect of exercise for myocardial protection

Exercise and its regulated molecules have myocardial protective effects against cardiac ischemia/reperfusion (I/R) injury. The muscle-enriched miR-486 was previously identified to be upregulated in the exercised heart, which prompted us to investigate the functional roles of miR-486 in cardiac I/R injury and to further explore its potential in contributing to exercise-induced protection against I/R injury. Our data showed that miR-486 was significantly downregulated in the heart upon cardiac I/R injury. Both preventive and therapeutic interventions of adeno-associated virus 9 (AAV9)-mediated miR-486 overexpression could reduce cardiac I/R injury. Using AAV9 expressing miR-486 with a cTnT promoter, we further demonstrated that cardiac muscle cell-targeted miR-486 overexpression was also sufficient to protect against cardiac I/R injury. Consistently, miR-486 was downregulated in oxygen-glucose deprivation/reperfusion (OGDR)-stressed cardiomyocytes, while upregulating miR-486 inhibited cardiomyocyte apoptosis through PTEN and FoxO1 inhibition and AKT/mTOR activation. Finally, we observed that miR-486 was necessary for exercise-induced protection against cardiac I/R injury. In conclusion, miR-486 is protective against cardiac I/R injury and myocardial apoptosis through targeting of PTEN and FoxO1 and activation of the AKT/mTOR pathway, and mediates the beneficial effect of exercise for myocardial protection. Increasing miR-486 might be a promising therapeutic strategy for myocardial protection.

Microvascular Barrier Protection by microRNA-183 via FoxO1 Repression: A Pathway Disturbed in Neuropathy and Complex Regional Pain Syndrome

Blood nerve barrier disruption and edema are common in neuropathic pain as well as in complex regional pain syndrome (CRPS). MicroRNAs (miRNA) are epigenetic multitarget switches controlling neuronal and non-neuronal cells in pain. The miR-183 complex attenuates hyperexcitability in nociceptors, but additional non-neuronal effects via transcription factors could contribute as well. This study explored exosomal miR-183 in CRPS and murine neuropathy, its effect on the microvascular barrier via transcription factor FoxO1 and tight junction protein claudin-5, and its antihyperalgesic potential. Sciatic miR-183 decreased after CCI. Substitution with perineural miR-183 mimic attenuated mechanical hypersensitivity and restored blood nerve barrier function. In vitro, serum from CCI mice und CRPS patients weakened the microvascular barrier of murine cerebellar endothelial cells, increased active FoxO1 and reduced claudin-5, concomitant with a lack of exosomal miR-183 in CRPS patients. Cellular stress also compromised the microvascular barrier which was rescued either by miR-183 mimic via FoxO1 repression or by prior silencing of Foxo1. PerspectiveLow miR-183 leading to barrier impairment via FoxO1 and subsequent claudin-5 suppression is a new aspect in the pathophysiology of CRPS and neuropathic pain. This pathway might help untangle the wide symptomatic range of CRPS and nurture further research into miRNA mimics or FoxO1 inhibitors.

Bevacizumab Increases Endothelin-1 Production via Forkhead Box Protein O1 in Human Glomerular Microvascular Endothelial Cells In Vitro.

Molecular mechanisms underlying the nephrotoxicity associated with bevacizumab are unclear. Endothelin-1 (ET-1) is involved in podocyte injury and proteinuria, and its level increases in most cases of kidney disorders. Forkhead box protein O1 (FoxO1), a transcription factor, is a major determinant of ET-1 promoter activation and is regulated by protein kinase B (Akt) phosphorylation-dependent nuclear exclusion. We evaluated the effect of bevacizumab on ET-1 production in human glomerular microvascular endothelial cells (hGECs). We analyzed the changes in the mRNA and protein levels of ET-1 in hGECs treated with bevacizumab using real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Changes in the protein levels and phosphorylation status of Akt and FoxO1 in hGECs treated with bevacizumab were analyzed by western blotting. After cell lysis, FoxO1 protein was isolated from the cytoplasmic and nuclear fractions. We also investigated the effects of AS1842856 (a FoxO1 inhibitor) on bevacizumab-induced ET-1 production. Bevacizumab significantly and dose-dependently increased the mRNA and protein levels of ET-1 in hGECs (p < 0.05). Bevacizumab treatment also led to a decrease in phosphorylated Akt protein levels. Inhibition of Akt activity by LY294002 promoted ET-1 production. Bevacizumab also induced an increase in FoxO1 protein levels in the nucleus. Inhibition of FoxO1 activity by AS1842856 resulted in decreased ET-1 levels in bevacizumab-treated hGECs. ET-1 axis activation, Akt inactivation, and FoxO1 nuclear localization are the molecular mechanisms underlying bevacizumab-induced nephrotoxicity. Therefore, inhibition of renal ET-1 production could be a promising approach to protect against or treat bevacizumab-induced nephrotoxicity.

FoxO1 Regulates Neuropeptide Y and Pro-opiomelanocortin in the Hypothalamus of Rat Offspring Small for Gestational Age.

Adulthood obesity, diabetes, and metabolic diseases are associated with small for gestational age (SGA) newborns. This association could be related to abnormal appetite signaling pathways in the hypothalamus. This study investigated the appetite regulation by the hypothalamus of SGA newborns by establishing an SGA rat model and culturing SGA neural progenitor cells (NPCs) in vitro. Models of SGA were established by maternal food restriction embryonic day 10 (E10). At E18, postpartum day 1 (P1), and P5, hypothalamic neural precursor cells (NPCs) of offspring were cultured in vitro. Immunofluorescence, Western blot (WB), and qRT-PCR were used to assess NPY, POMC, and FoxO1 expression levels. The effects on mRNA expression of the FoxO1-specific inhibitor AS1842856 were examined. The results indicated that compared with controls, NPY was higher, and POMC was lower at embryonic day 18 (E18), postpartum day 1 (P1), and P5. The proliferation and migration of NPCs in the third ventricle of SGA hypothalami were lower than in controls. After treatment with the FoxO1 inhibitor AS1842856, the differences in the mRNA expression of NPY and POMC between the two groups disappeared. NPY and POMC mRNA levels in the SGA group treated with AS1842856 were not significantly different compared with the control group without AS1842856 treatment. In conclusion, SGA pups showed an increase in appetite-promoting NPY and a decrease in appetite-reducing POMC, probably contributing to adulthood weight gain, obesity, and endocrine disorders.

AKT controls protein synthesis and oxidative metabolism via combined mTORC1 and FOXO1 signalling to govern muscle physiology.

BackgroundSkeletomuscular diseases result in significant muscle loss and decreased performance, paralleled by a loss in mitochondrial and oxidative capacity. Insulin and insulin‐like growth factor‐1 (IGF‐1) are two potent anabolic hormones that activate a host of signalling intermediates including the serine/threonine kinase AKT to influence skeletal muscle physiology. Defective AKT signalling is associated with muscle pathology, including cachexia, sarcopenia, and disuse; however, the mechanistic underpinnings remain unresolved.MethodsTo elucidate the role of AKT signalling in muscle mass and physiology, we generated both congenital and inducible mouse models of skeletal muscle‐specific AKT deficiency. To understand the downstream mechanisms mediating AKT's effects on muscle biology, we generated mice lacking AKT1/2 and FOXO1 (M‐AKTFOXO1TKO and M‐indAKTFOXO1TKO) to inhibit downstream FOXO1 signalling, AKT1/2 and TSC1 (M‐AKTTSCTKO and M‐indAKTTSCTKO) to activate mTORC1, and AKT1/2, FOXO1, and TSC1 (M‐QKO and M‐indQKO) to simultaneously activate mTORC1 and inhibit FOXO1 in AKT‐deficient skeletal muscle. Muscle proteostasis and physiology were assessed using multiple assays including metabolic labelling, mitochondrial function, fibre typing, ex vivo physiology, and exercise performance.ResultsHere, we show that genetic ablation of skeletal muscle AKT signalling resulted in decreased muscle mass and a loss of oxidative metabolism and muscle performance. Specifically, deletion of muscle AKT activity during development or in adult mice resulted in a significant reduction in muscle growth by 30–40% (P < 0.0001; n = 12–20) and 15% (P < 0.01 and P < 0.0001; n = 20–30), respectively. Interestingly, this reduction in muscle mass was primarily due to an ~40% reduction in protein synthesis in both M‐AKTDKO and M‐indAKTDKO muscles (P < 0.05 and P < 0.01; n = 12–20) without significant changes in proteolysis or autophagy. Moreover, a significant reduction in oxidative capacity was observed in both M‐AKTDKO (P < 0.05, P < 0.01 and P < 0.001; n = 5–12) and M‐indAKTDKO (P < 0.05 and P < 0.01; n = 4). Mechanistically, activation and inhibition of mTORC1/FOXO1, respectively, but neither alone, were sufficient to restore protein synthesis, muscle oxidative capacity, and muscle function in the absence of AKT in vivo. In a mouse model of disuse‐induced muscle loss, simultaneous activation of mTORC1 and inhibition of FOXO1 preserved muscle mass following immobilization (~5–10% reduction in casted M‐indFOXO1TSCDKO muscles vs. ~30–40% casted M‐indControl muscles, P < 0.05 and P < 0.0001; n = 8–16).ConclusionsCollectively, this study provides novel insights into the AKT‐dependent mechanisms that underlie muscle protein homeostasis, function, and metabolism in both normal physiology and disuse‐induced muscle wasting.

PI3K Pathway Inhibition with NVP-BEZ235 Hinders Glycolytic Metabolism in Glioblastoma Multiforme Cells.

Glioblastoma (GBM) is the most lethal primary brain cancer that lacks effective molecular targeted therapies. The PI3K/AKT/mTOR pathway is activated in 90% of all Glioblastoma multiforme (GBM) tumors. To gain insight into the impact of the PI3K pathway on GBM metabolism, we treated U87MG GBM cells with NVP-BEZ235 (PI3K and mTOR a dual inhibitor) and identified differentially expressed genes with RNA-seq analysis. RNA-seq identified 7803 differentially regulated genes in response to NVP-BEZ235. Gene Set Enrichment Analysis (GSEA) identified two glycolysis-related gene sets that were significantly enriched (p < 0.05) in control samples compared to NVP-BEZ235-treated samples. We validated the inhibition of glycolytic genes by NVP-BEZ235 and examined the impact of the FOXO1 inhibitor (AS1842856) on these genes in a set of GBM cell lines. FOXO1 inhibition alone was associated with reduced LDHA expression, but not ENO1 or PKM2. Bioinformatics analyses revealed that PI3K-impacted glycolytic genes were over-expressed and co-expressed in GBM clinical samples. The elevated expression of PI3K-impacted glycolytic genes was associated with poor prognosis in GBM based on Kaplan–Meier survival analyses. Our results suggest novel insights into hallmark metabolic reprogramming associated with the PI3K-mTOR dual inhibition.

NORHA, a novel follicular atresia-related lncRNA, promotes porcine granulosa cell apoptosis via the miR-183-96-182 cluster and FoxO1 axis

BackgroundFollicular atresia has been shown to be strongly associated with a low follicle utilization rate and female infertility, which are regulated by many factors such as microRNAs (miRNAs), which constitute a class of noncoding RNAs (ncRNAs). However, little is known about long noncoding RNAs (lncRNAs), which constitute another ncRNA family that regulate follicular atresia.ResultsA total of 77 differentially expressed lncRNAs, including 67 upregulated and 10 downregulated lncRNAs, were identified in early atretic follicles compared to healthy follicles by RNA-Sequencing. We characterized a noncoding RNA that was highly expressed in atretic follicles (NORHA). As an intergenic lncRNA, NORHA was one of the upregulated lncRNAs identified in the atretic follicles. To determine NORHA function, RT-PCR, flow cytometry and western blotting were performed, and the results showed that NORHA was involved in follicular atresia by influencing GC apoptosis with or without oxidative stress. To determine the mechanism of action, bioinformatics analysis, luciferase reporter assay and RNA immunoprecipitation assay were performed, and the results showed that NORHA acted as a ‘sponge’, that directly bound to the miR-183-96-182 cluster, and thus prevented its targeted inhibition of FoxO1, a major sensor and effector of oxidative stress.ConclusionsWe provide a comprehensive perspective of lncRNA regulation of follicular atresia, and demonstrate that NORHA, a novel lncRNA related to follicular atresia, induces GC apoptosis by influencing the activities of the miR-183-96-182 cluster and FoxO1 axis.

Clinical Evaluation of FOXO1 as a Tumor Suppressor in Prostate Cancer.

Objective Prostate cancer (PCa) is considered the most serious cancer in the world. Nevertheless, the accuracy of current biomarkers, such as pathological staging, Gleason's score, and serum prostate-specific antigen (PSA) levels, is limited. FOXO1 is a key downstream effector of PTEN and a tumor suppressor in PCA, which has been reported extensively. However, the clinical relevance of FOXO1 in PCa remains unclear. Methods In this study, we first detected its expression in four public databases to explore the clinical role of FOXO1. Verification of the knockdown effect of FOXO1 siRNA was performed by real-time PCR analysis. Changes in cell viability were assessed using cell counting kit-8 (CCK-8) assays. In addition, we verified the effect of FOXO1 on the PCa cell cycle using a cell cycle assay. Results Herein, we found that FOXO1 was significantly downregulated in PCa tissues and was significantly associated with Gleason's score, age, biochemical recurrence (BCR), and lymph node (LN) status, while FOXO1 expression was independent of pathological staging and preoperative PSA levels. The Kaplan-Meier survival analysis showed that PCA patients with high FOXO1 expression were less likely to develop BCR compared with patients with low FOXO1 expression. In terms of function, FOXO1 inhibition significantly promoted the proliferation and cell cycle progression of PCa cells. Conclusions In summary, our study suggests that FOXO1 may be one of the prognostic factors that describe the risk of PCa for BCR. These results suggest that FOXO1 may be a therapeutic target for PCa.

The Mutual Inhibition of FoxO1 and SREBP-1c Regulated the Progression of Hepatoblastoma by Regulating Fatty Acid Metabolism.

Background Hepatoblastoma (HB) is the most common liver malignancy in pediatrics, but the treatment for this disease is minimal. This study is aimed at exploring the effect of FoxO1 and SREBP-1c on HB and their mechanism. Methods FoxO1, SREBP-1c, FASN, ACLY, ACC, and MAGL expressions in tissue samples were detected by RT-qPCR and WB. IHC was utilized to measure FASN content. Overexpression and knockdown of FoxO1 and sSREBP-1c were performed on Huh-6 cells. Cell proliferation, migration, and invasion were examined by CCK8, scratch, and transwell assay. ELISA was performed to test the ATP, FAO, NEFA, and Acetyl-CoA contents. ChIP was used to detect the interaction between SREBP-1c protein and the FoxO1 gene. In vivo tumorigenesis was conducted on mice. The morphology of tumor tissue sections was observed by HE staining. Results FoxO1 expression was downregulated in HB tissue, while the expressions of SREBP-1c, FASN, ACLY, ACC, and MAGL were upregulated. In Huh-6 cells and mouse tumor tissues, FoxO1 knockdown resulted in increased cell proliferation, migration, and invasion and active fatty acid metabolism. On the contrary, after the knockdown of SREBP-1c, cell proliferation, migration, and invasion were weakened, and fatty acid metabolism was significantly reduced. SREBP-1c interacted with the promoter of the FoxO1 gene. When FoxO1 was knocked down, the tumor tissue was more closely packed. After the knockdown of the SREBP-1c gene, the structure of tumor cells was deformed. Conclusion FoxO1 and SREBP-1c inhibited each other in HB, leading to the increase of intracellular fatty acid metabolism, and ultimately facilitated the development of HB.