CAPN2 promotes apalutamide resistance in metastatic hormone-sensitive prostate cancer by activating protective autophagy.

Apalutamide, a novel endocrine therapy agent, has been shown to significantly improve the prognosis of patients with metastatic hormone-sensitive prostate cancer (mHSPC). However, resistance to apalutamide has also been reported, and the underlying mechanism for this response has yet to be clearly elucidated. First, this study established apalutamide-resistant prostate cancer (PCa) cells, and confirmed that apalutamide activated the release of calcium ions (Ca2+) and endoplasmic reticulum stress (ERS) to enhance autophagy. Second, RNA sequencing, western blotting, and immunohistochemistry revealed significantly decreased Calpain 2 (CAPN2) expression in the apalutamide-resistant PCa cells and tissues. Furthermore, immunofluorescence and transmission electron microscopy (TEM) showed that CAPN2 promoted apalutamide resistance by activating protective autophagy. CAPN2 promoted autophagy by reducing Forkhead Box O1 (FOXO1) degradation while increasing nuclear translocation via nucleoplasmic protein isolation and immunofluorescence. In addition, FOXO1 promoted protective autophagy through the transcriptional regulation of autophagy-related gene 5 (ATG5). Furthermore, a dual-fluorescence assay confirmed that transcription factor 3 (ATF3) stimulation promoted CAPN2-mediated autophagy activation via transcriptional regulation. In summary, CAPN2 activated protective autophagy by inhibiting FOXO1 degradation and promoting its nuclear translocation via transcriptional ATG5 regulation. ATF3 activation and transcriptional CAPN2 regulation jointly promoted this bioeffect. Thus, our findings have not only revealed the mechanism underlying apalutamide resistance, but also provided a promising new target for the treatment of metastatic PCa.

FOXO1 regulates RUNX2 ubiquitination through SMURF2 in calcific aortic valve disease.

The prevalence of calcific aortic valve disease (CAVD) remains substantial while there is currently no medical therapy available. Forkhead box O1 (FOXO1) is known to be involved in the pathogenesis of cardiovascular diseases, including vascular calcification and atherosclerosis; however, its specific role in calcific aortic valve disease remains to be elucidated. In this study, we identified FOXO1 significantly down-regulated in the aortic valve interstitial cells (VICs) of calcified aortic valves by investigating clinical specimens and GEO database analysis. FOXO1 silencing or inhibition promoted VICs osteogenic differentiation in vitro and aortic valve calcification in Apoe-/- mice, respectively. We identified that FOXO1 facilitated the ubiquitination and degradation of RUNX2, which process was mainly mediated by SMAD-specific E3 ubiquitin ligase 2 (SMURF2). Our discoveries unveil a heretofore unacknowledged mechanism involving the FOXO1/SMURF2/RUNX2 axis in CAVD, thereby proposing the potential therapeutic utility of FOXO1 or SMURF2 as viable strategies to impede the progression of CAVD.

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.

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.

Effect of South African Herb Hoodia gordonii on Liver Glucolipid Metabolism and PI3K/Akt/FoxO1 Signalling Pathway in db/db Mice / 中国实验方剂学杂志

ObjectiveTo observe the effects of the South African herb Hoodia gordonii （HG） on glucolipid metabolism in diabetic db/db mice and explore the possible mechanisms of HG on the liver of db/db mice based on the phosphoinositide-3 kinase （PI3K）/protein kinase B （Akt）/factor forkhead protein O1 （FoxO1） signaling pathway. MethodA total of 30 db/db mice were randomly divided into five groups according to fasting blood glucose： model group， metformin group （0.195 g·kg-1）， and low dose （0.39 g·kg-1）， medium dose （0.78 g·kg-1）， and high dose （1.56 g·kg-1） HG groups， with six m/m mice in each group， and another six m/m mice were set as normal group. The mice in the normal and model groups were given saline of 9 mL·kg-1 by gavage. Body weight， water intake， and fasting blood glucose of the mice in each group were measured weekly. After six weeks of continuous administration， serum insulin （FINS）， low-density lipoprotein cholesterol （LDL）， total cholesterol （TC）， triglyceride （TG）， alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， urea， and creatinine （CREA） were measured， and liver sections were embedded and stained with hematoxylin-eosin （HE）， periodic acid-Schiff （PAS）， and oil red O. Protein expression of PI3K p85， p-Akt， and p-FoxO1 in liver was detected by immunohistochemistry. The mRNA expression of PI3K， Akt， and FoxO1 in liver tissue was detected by real-time polymerase chain reaction （Real-time PCR）. ResultAfter six weeks of administration intervention， it was found that fasting blood glucose was significantly downregulated in mice in the three HG groups （P<0.05）. The level of islet resistance index was significantly reduced in both the low and medium dose HG groups （P<0.05）. The expression levels of TC， TG， and LDL were reduced in all HG groups （P<0.05， P<0.01）. Pathologically， HG could alleviate hepatocyte steatosis， reduce the volume and content of lipid droplets in liver， and increase the distribution of glycogen granules in liver to some extent in mice. Immunohistochemical assays revealed that PI3K p85 protein expression was significantly increased in the low， medium， and high dose HG groups compared with the model group （P<0.01）. p-Akt protein expression was significantly increased in the medium and high dose HG groups （P<0.05， P<0.01）. p-FoxO1 protein expression was significantly increased in the low， medium， and high dose HG groups （P<0.05， P<0.01）. Compared with the model group， PI3K mRNA was increased in low dose， medium dose， and high dose HG groups （P<0.05）， and Akt mRNA was increased in high dose HG group （P<0.05）. FoxO1 mRNA was decreased in low dose， medium dose， and high dose HG groups （P<0.05）. ConclusionHG can ameliorate the disorder of glucolipid metabolism in db/db mice， which may be related to its activation of the hepatic PI3K/Akt/FoxO1 signaling pathway.

METTL3-regulated m6A modification impairs the decidualization of endometrial stromal cells by regulating YTHDF2-mediated degradation of FOXO1 mRNA in endometriosis-related infertility.

BACKGROUND: Endometriosis-related infertility is a common worldwide reproductive health concern. Despite ongoing research, the causes of infertility remain unclear. Evidence suggests that epigenetic regulation is crucial in reproduction. However, the role of N6-methyladenosine (m6A) modification of RNA in endometriosis-related infertility requires further investigation. METHODS: We examined the expression of m6A and methyltransferase-like 3 (METTL3) in endometrial samples taken from normal fertile women in the proliferative phase (the NP group) or the mid-secretory phase (the NS group) or from women with endometriosis-related infertility at the mid-secretory phase (the ES group). We treated primary endometrial stromal cells (ESCs) with medroxyprogesterone acetate and 8-Bromo-cyclic adenosine monophosphate for in vitro decidualization and detected the expression of m6A, METTL3, and decidual markers. We analyzed the expression of m6A, METTL3, and forkhead box O1 (FOXO1) in ESCs from normal fertile women (the ND group) or women with endometriosis-related infertility (the ED group). We also assessed the expression of m6A, METTL3, and decidual markers, as well as the embryo adhesion rate, upon METTL3 overexpression or knockdown. Additionally, we investigated the role of METTL3 in embryo implantation in vivo by applying mice with endometriosis. Furthermore, we performed RNA stability assays, RNA immunoprecipitation (RIP), and methylated RIP assays to explore the mechanisms underlying the regulation of FOXO1 by METTL3-mediated m6A. RESULTS: The expression of m6A and METTL3 was reduced only in the NS group; the NP and ES groups demonstrated increased m6A and METTL3 levels. m6A and METTL3 levels decreased in ESCs with prolonged decidual treatment. Compared to the ND group, m6A and METTL3 levels in the ED group increased after decidual treatment, whereas the expression of FOXO1 decreased. METTL3 overexpression suppressed the expression of decidual markers and embryo implantation in vitro; METTL3 knockdown exhibited the opposite effect. Inhibition of METTL3 promoted embryo implantation in vivo. Furthermore, we observed that METTL3-mediated m6A regulated the degradation of FOXO1 mRNA through YTHDF2, a m6A binding protein. CONCLUSIONS: METTL3-regulated m6A promotes YTHDF2-mediated decay of FOXO1 mRNA, thereby affecting cellular decidualization and embryo implantation. These findings provide novel insights into the development of therapies for women with endometriosis-related infertility.

HDAC3 deficiency protects against acute lung injury by maintaining epithelial barrier integrity through preserving mitochondrial quality control.

Sepsis is one common cause of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), which is closely associated with high mortality in intensive care units (ICU). Histone deacetylase 3 (HDAC3) serves as an important epigenetic modifying enzyme which could affect chromatin structure and transcriptional regulation. Here, we explored the effects of HDAC3 in type II alveolar epithelial cells (AT2) on lipopolysaccharide (LPS)-induced ALI and shed light on potential molecular mechanisms. We generated ALI mouse model with HDAC3 conditional knockout mice (Sftpc-cre; Hdac3f/f) in AT2 and the roles of HDAC3 in ALI and epithelial barrier integrity were investigated in LPS-treated AT2. The levels of HDAC3 were significantly upregulated in lung tissues from mice with sepsis and in LPS-treated AT2. HDAC3 deficiency in AT2 not only decreased inflammation, apoptosis, and oxidative stress, but also maintained epithelial barrier integrity. Meanwhile, HDAC3 deficiency in LPS-treated AT2 preserved mitochondrial quality control (MQC), evidenced by the shift of mitochondria from fission into fusion, decreased mitophagy, and improved fatty acid oxidation (FAO). Mechanically, HDAC3 promoted the transcription of Rho-associated protein kinase 1 (ROCK1) in AT2. In the context of LPS stimulation, the upregulated ROCK1 elicited by HDAC3 could be phosphorylated by Rho-associated (RhoA), thus disturbing MQC and triggering ALI. Furthermore, we found that forkhead box O1 (FOXO1) was one of transcription factors of ROCK1. HDAC3 directly decreased the acetylation of FOXO1 and promoted its nuclear translocation in LPS-treated AT2. Finally, HDAC3 inhibitor RGFP966 alleviated epithelial damage and improved MQC in LPS-treated AT2. Altogether, HDAC3 deficiency in AT2 alleviated sepsis-induced ALI by preserving mitochondrial quality control via FOXO1-ROCK1 axis, which provided a potential strategy for the treatment of sepsis and ALI.

Insulin resistance in adipose tissue and metabolic diseases.

Adipose tissue regulates systemic energy metabolism through adipokine production as well as energy storage and energy supply to other organs in response to changes in energy status. Adipose tissue dysfunction is therefore thought to be a key contributor to the pathogenesis of a variety of metabolic disorders including nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Given that insulin plays a central role in the regulation of many aspects of adipocyte function, insulin resistance in adipose tissue is implicated in the pathogenesis of metabolic disorders as a cause of adipose tissue dysfunction. The concept of metabolic dysfunction-associated fatty liver disease (MAFLD) has recently been proposed for liver disease associated with metabolic disorders in both obese and nonobese individuals, with insulin resistance in adipose tissue likely being an important factor in its pathogenesis. This review outlines the relation between insulin resistance in adipose tissue and metabolic disorders, with a focus on the physiological relevance and mechanism of action of 3'-phosphoinositide-dependent kinase 1 (PDK1), a key kinase in insulin signaling, and its downstream transcription factor FoxO1 in adipocytes.

Anticancer Activity of Methyl Protodioscin against Prostate Cancer by Modulation of Cholesterol-Associated MAPK Signaling Pathway via FOXO1 Induction.

Methyl protodioscin (MPD), a furostanol saponin found in the rhizomes of Dioscoreaceae, has lipid-lowering and broad anticancer properties. However, the efficacy of MPD in treating prostate cancer remains unexplored. Therefore, the present study aimed to evaluate the anticancer activity and action mechanism of MPD in prostate cancer. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), wound healing, transwell, and flow cytometer assays revealed that MPD suppressed proliferation, migration, cell cycle, and invasion and induced apoptosis of DU145 cells. Mechanistically, MPD decreased cholesterol concentration in the cholesterol oxidase, peroxidase and 4-aminoantipyrine phenol (COD-PAP) assay, disrupting the lipid rafts as detected using immunofluorescence and immunoblot analyses after sucrose density gradient centrifugation. Further, it reduced the associated mitogen-activated protein kinase (MAPK) signaling pathway protein P-extracellular regulated protein kinase (ERK), detected using immunoblot analysis. Forkhead box O (FOXO)1, a tumor suppressor and critical factor controlling cholesterol metabolism, was predicted to be a direct target of MPD and induced by MPD. Notably, in vivo studies demonstrated that MPD significantly reduced tumor size, suppressed cholesterol concentration and the MAPK signaling pathway, and induced FOXO1 expression and apoptosis in tumor tissue in a subcutaneous mouse model. These results suggest that MPD displays anti-prostate cancer activity by inducing FOXO1 protein, reducing cholesterol concentration, and disrupting lipid rafts. Consequently, the reduced MAPK signaling pathway suppresses proliferation, migration, invasion, and cell cycle and induces apoptosis of prostate cancer cells.

METTL3 Mediates Epithelial-Mesenchymal Transition by Modulating FOXO1 mRNA N6 -Methyladenosine-Dependent YTHDF2 Binding: A Novel Mechanism of Radiation-Induced Lung Injury.

The biological roles of epithelial-mesenchymal transition (EMT) in the pathogenesis of radiation-induced lung injury (RILI) have been widely demonstrated, but the mechanisms involved have been incompletely elucidated. N6 -methyladenosine (m6 A) modification, the most abundant reversible methylation modification in eukaryotic mRNAs, plays vital roles in multiple biological processes. Whether and how m6 A modification participates in ionizing radiation (IR)-induced EMT and RILI remain unclear. Here, significantly increased m6 A levels upon IR-induced EMT are detected both in vivo and in vitro. Furthermore, upregulated methyltransferase-like 3 (METTL3) expression and downregulated α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5) expression are detected. In addition, blocking METTL3-mediated m6 A modification suppresses IR-induced EMT both in vivo and in vitro. Mechanistically, forkhead box O1 (FOXO1) is identified as a key target of METTL3 by a methylated RNA immunoprecipitation (MeRIP) assay. FOXO1 expression is downregulated by METTL3-mediated mRNA m6 A modification in a YTH-domain family 2 (YTHDF2)-dependent manner, which subsequently activates the AKT and ERK signaling pathways. Overall, the present study shows that IR-responsive METTL3 is involved in IR-induced EMT, probably by activating the AKT and ERK signaling pathways via YTHDF2-dependent FOXO1 m6 A modification, which may be a novel mechanism involved in the occurrence and development of RILI.

Knockdown of lncRNA SNHG16 attenuates myocardial ischemia­reoxygenation injury via targeting miR­183/FOXO1 axis.

Accumulating evidence shows that long non-coding RNAs (lncRNAs) are widely involved in cellular processes of myocardial ischemia/reperfusion (I/R). The present study investigated the functions of lncRNA SNHG16 in myocardial I/R and the mechanism mediated by SNHG16. The myocardial I/R rat and cell model and hypoxia/reoxygenation injury (H/R) models of H9C2 cardiomyocytes were established to detect the expression of SNHG16. Cell Counting Kit-8, flow cytometric and western blot assays were conducted to detect cell viability, apoptosis and protein expression. Myocardial cell apoptosis was assessed by TUNEL staining. Dual-luciferase gene reporter was applied to determine the interaction between the molecules. The expressions of SNHG16 were upregulated in myocardial I/R injury models. Inhibition of SNHG16 relieved myocardial I/R injury in vivo and in vitro silencing of SNHG16 alleviated H/R induced cardiomyocyte apoptosis. To explore the regulatory mechanism, it was discovered that SNHG16 directly interacted with miR-183, while forkhead box O1 (FoxO1) was a target of microRNA (miR)-183. Findings from rescue assays revealed that miR-183 inhibitor and upregulation of FOXO1 can rescue the effect of sh-SNHG16 on H/R-induced cardiomyocyte apoptosis. The results indicated that the lncRNA SNHG16/miR-183/FOXO1 axis exacerbated myocardial cell apoptosis in myocardial I/R injury, suggesting SNHG16 as a potential therapeutic target for myocardial I/R injury.

Rehmannioside D mitigates disease progression in rats with experimental-induced diminished ovarian reserve via Forkhead Box O1/KLOTHO axis.

This study aims to explore the impact of Rehmannioside D (RD) on ovarian functions of rats with diminished ovarian reserve (DOR) and its underlying mechanisms of action. A single injection of cyclophosphamide was performed to establish a DOR rat model, and fourteen days after the injection, the rats were intragastrically administrated with RD for two weeks. Rat estrus cycles were tested using vaginal smears. Ovarian tissues were histologically evaluated, the number of primordial, mature, and atretic follicles was calculated, and the apoptotic rate of granulosa cells. Follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) levels were determined by ELISA assays. Protein levels of Forkhead Box O1 (FOXO1), KLOTHO, Bcl-2, and Bax were investigated in ovarian tissues of DOR rats. The binding between FOXO1 and KLOTHO was verified by ChIP assay. High-dose administration of RD into DOR rats improved their estrus cycles, increased ovarian index, enhanced the number of primordial and mature follicles, reduced the number of atretic follicle number, and ovarian granulosa cell apoptosis in addition to inhibiting FSH and LH levels and upregulating E2 expression. FOXO1 and KLOTHO were significantly suppressed in DOR rats. FOXO1 knockdown partially suppressed the protective effects of RD on DOR rats, and KLOTHO overexpression could restore RD-induced blockade of DOR development despite knocking down FOXO1. FOXO1 antibody enriched KLOTHO promoter, and the binding between them was reduced in DOR group compared to that in sham group. RD improved ovarian functions in DOR rats and diminished granulosa cell apoptosis via the FOXO1/KLOTHO axis.

Extracellular PPM1A promotes mineralization of osteoblasts differentiation in ankylosing spondylitis via the FOXO1A-RUNX2 pathway.

Protein phosphatase magnesium-dependent 1A (PPM1A), serine/threonine protein phosphatase, in sera level was increased in patients with ankylosing spondylitis (AS). Preosteoblasts were differentiated actively to matured osteoblasts by intracellular PPM1A overexpression. However, it was unclear whether extracellular PPM1A contributes to the excessive bone-forming activity in AS. Here, we confirmed that PPM1A and runt-related transcription factor 2 (RUNX2) were increased in facet joints of AS. During osteoblasts differentiation, exogenous PPM1A treatment showed increased matrix mineralization in AS-osteoprogenitor cells accompanied by induction of RUNX2 and factor forkhead box O1A (FOXO1A) protein expressions. Moreover, upon growth condition, exogenous PPM1A treatment showed an increase in RUNX2 and FOXO1A protein expression and a decrease in phosphorylation at ser256 of FOXO1A protein in AS-osteoprogenitor cells, and positively regulated promoter activity of RUNX2 protein-binding motif. Mechanically, exogenous PPM1A treatment induced the dephosphorylation of transcription factor FOXO1A protein and translocation of FOXO1A protein into the nucleus for RUNX2 upregulation. Taken together, our results suggest that high PPM1A concentration promotes matrix mineralization in AS via the FOXO1A-RUNX2 pathway.

First Report of FARSA in the Regulation of Cell Cycle and Survival in Mantle Cell Lymphoma Cells via PI3K-AKT and FOXO1-RAG1 Axes.

Cancer-associated factors have been largely identified in the understanding of tumorigenesis and progression. However, aminoacyl-transfer RNA (tRNA) synthetases (aaRSs) have so far been neglected in cancer research due to their canonical activities in protein translation and synthesis. FARSA, the alpha subunit of the phenylalanyl-tRNA synthetase is elevated across many cancer types, but its function in mantle cell lymphoma (MCL) remains undetermined. Herein, we found the lowest levels of FARSA in patients with MCL compared with other subtypes of lymphomas, and the same lower levels of FARSA were observed in chemoresistant MCL cell lines. Unexpectedly, despite the essential catalytic roles of FARSA, knockdown of FARSA in MCL cells did not lead to cell death but resulted in accelerated cell proliferation and cell cycle, whereas overexpression of FARSA induced remarkable cell-cycle arrest and overwhelming apoptosis. Further RNA sequencing (RNA-seq) analysis and validation experiments confirmed a strong connection between FARSA and cell cycle in MCL cells. Importantly, FARSA leads to the alteration of cell cycle and survival via both PI3K-AKT and FOXO1-RAG1 axes, highlighting a FARSA-mediated regulatory network in MCL cells. Our findings, for the first time, reveal the noncanonical roles of FARSA in MCL cells, and provide novel insights into understanding the pathogenesis and progression of B-cell malignancies.

HYOU1 promotes cell proliferation, migration, and invasion via the PI3K/AKT/FOXO1 feedback loop in bladder cancer.

BACKGROUND: Hypoxia up-regulated 1 (HYOU1) was identified as a proto-oncogene and involved in tumorigenesis and progression in several cancer. Nonetheless, the biological function and mechanism of HYOU1 in bladder cancer (BCa) remian unclear. METHODS: The HYOU1 level in BCa tissues and cells was examined using RT-qPCR and western blot methods. The relationship between HYOU1 expression and clinicopathologic characteristics of BCa was analyzed. The biological role of HYOU1 on BCa cell proliferation, apoptosis, migration and invasion were analyzed via counting kit-8 (CCK-8), flow cytometry, wound healing and Transwell assays, respectively. The association between HYOU1 and the PI3K/AKT/Forkhead box O1 (FOXO1) signalling was assessed via western blot assay, meanwhile the the association of FOXO1 with HYOU1 was also investigated. RESULTS: HYOU1 was up-regulated in BCa tissues and cell lines, and the high level of HYOU1 was associated with bladder cancer histological grade and pathologic stage. Moreover, patients with high expression of HYOU1 showed poor overall survival from Kaplan-Meier Plotter. HYOU1 depletion impeded cell proliferation, migration and invasion, and induced cell apoptosis, while HYOU1 overexpression promoted cell proliferation, migration and invasion. Mechanically, our results showed that HYOU1 knockdown repressed PI3K/AKT/FOXO1 pathway and HYOU1 was negative regulated by FOXO1 in BCa. Significantly, we confirmed that the HYOU1/PI3K-AKT/FOXO1 negative feedback loop was involved in BCa cell proliferation, migration and invasion. CONCLUSION: These findings revealed that HYOU1 acted as a pro-oncogene on BCa progression, and it will be a possible target for BCa treatment.

Effects of Ginkgo biloba extract on renal injury in rats with experimental renal failure through miR-145/FOXO1 axis / 中国临床药理学与治疗学

ABSTARCT AIM: To investigate the effect of Ginkgo biloba extract (GBE) on kidney injury in rats with chronic renal failure (CRF) and its potential molecular mechanism. METHODS: SD rats were given 5/6 nephrectomy to construct CRF models and divided into model group, GBE group (100 mg /kg), GBE+ Agomir-NC group, and GBE+Agomir-145 group, 12 per group; another 12 were selected as the sham group, with only the kidney exposed and no nephrectomy. Rats in the GBE group were given GBE 100 mg/kg gavage daily, once a day, for 4 consecutive weeks; rats in the GBE+Agomir-NC group and GBE+Agomir-145 group were given GBE 100 mg/kg gavage daily, and then Agomir-NC and Agomir-145 were injected via the tail vein every 3 days for 4 weeks; the sham group and the model group were given the same amount of normal saline by gavage and injection through the tail vein respectively. The general state of the rat was observed, and the renal function indicators [24 h urine microalbumin (24 h UAlb), blood urea nitrogen (BUN), blood creatinine (SCr)] and oxidative stress indicators [malonaldehyde (MDA), Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px)] were detected, Masson staining was used to observe the fibrosis of kidney tissue, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the mRNA expression levels of microRNA-145 (miR-145), transforming growth factor - β1 (TGF - β1) and forkhead box O1 (FOXO1) in renal tissue, Western blot was used to detect the protein levels of TGF - β1 and FOXO1 in kidney tissue. RESULTS: The general state of CRF rats improved significantly after GBE intervention, the body weight, renal tissue SOD and GSH-Px activities, and FOXO1 mRNA and protein levels were significantly higher than those in the model group (P<0.05); the 24 h UAlb, serum BUN, SCr and renal tissue MDA levels, the relative area of renal interstitial fibrosis, and renal tissue miR-145, TGF - β1 mRNA and protein levels were significantly lower than those in the model group (P<0.05); and on the basis of GBE intervention, up-regulating the expression of miR-145 could significantly weaken the protective effect of GBE on renal injury in CRF rats (P<0.05). CONCLUSION: GBE can alleviate kidney damage in CRF rats, and its mechanism of action may be related to down-regulation of miR-145, up-regulation of FOXO1 expression, and inhibition of renal fibrosis.

Exenatide regulates Th17/Treg balance via PI3K/Akt/FoxO1 pathway in db/db mice.

BACKGROUND: The T helper 17 (Th17)/T regulatory (Treg) cell imbalance is involved in the course of obesity and type 2 diabetes mellitus (T2DM). In the current study, the exact role of glucagon-like peptide-1 receptor agonist (GLP-1RA) exenatide on regulating the Th17/Treg balance and the underlying molecular mechanisms are investigated in obese diabetic mice model. METHODS: Metabolic parameters were monitored in db/db mice treated with/without exenatide during 8-week study period. The frequencies of Th17 and Treg cells from peripheral blood and pancreas in db/db mice were assessed. The phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/Forkhead box O1 (FoxO1) pathway in Th17 and Treg cells from the spleens of male C57BL/6J mice was detected by western blotting. In addition, the expression of glucagon-like peptide-1 receptor (GLP-1R) in peripheral blood mononuclear cells (PBMCs) of male C57BL/6J mice was analyzed. RESULTS: Exenatide treatment improved ß-cell function and insulitis in addition to glucose, insulin sensitivity and weight. Increased Th17 and decreased Treg cells in peripheral blood were present as diabetes progressed while exenatide corrected this imbalance. Progressive IL-17 + T cell infiltration of pancreatic islets was alleviated by exenatide intervention. In vitro study showed no significant difference in the level of GLP-1R expression in PBMCs between control and palmitate (PA) groups. In addition, PA could promote Th17 but suppress Treg differentiation along with down-regulating the phosphorylation of PI3K/Akt/FoxO1, which was reversed by exenatide intervention. FoxO1 inhibitor AS1842856 could abrogate all these effects of exenatide against lipid stress. CONCLUSIONS: Exenatide could restore systemic Th17/Treg balance via regulating FoxO1 pathway with the progression of diabetes in db/db mice. The protection of pancreatic ß-cell function may be partially mediated by inhibiting Th17 cell infiltration into pancreatic islets, and the resultant alleviation of islet inflammation.

MDSCs participate in the pathogenesis of diffuse pulmonary hemorrhage in murine lupus through mTOR-FoxO1 signaling.

Diffuse pulmonary hemorrhage (DPH) is a common respiratory complication in patients with systemic lupus erythematosus (SLE). Our previous study found that myeloid-derived suppressor cells (MDSCs) have an important role in SLE pathogenesis. In this study, we further examined the role of MDSCs in the DPH mice model. We first observed an increased proportion of MDSCs and impaired immunosuppressive function in bronchoalveolar lavage fluid (BALF) and peritoneal cavity in the DPH mice model induced by pristane. By injecting anti-Gr-1 antibody, we found that MDSCs clearance can significantly alleviate DPH symptoms. The detection of downstream molecules proved that the mTOR signaling pathway was obviously activated in purified DPH MDSCs. After treatment of DPH model mice with AMPK agonist metformin, mammalian target of rapamycin (mTOR) inhibitor INK128, and rapamycin, respectively, we observed that inhibition of the mTOR signal alleviated DPH symptoms, inhibited the expansion of mononuclear MDSCs (M-MDSCs) and the differentiation of pro-inflammatory M1 macrophages (M1), which, in turn, promoted the expansion of granulocytes MDSCs (G-MDSCs) and differentiation of anti-inflammatory M2 macrophages (M2). We then demonstrated that inhibition of the mTOR signal increased the expansion of G-MDSCs, promoted M-MDSCs differentiation into M2 and inhibited their differentiation into M1 by administering TLR7 agonist R848 in vitro to simulate lupus environment. In addition, we also observed increased forkhead box-O1 (FoxO1) expression in M-MDSCs and macrophages after mTOR signal inhibition, both in vivo and in vitro. After down-regulation of FoxO1 by siRNA transfection, the regulatory effects of mTOR signal inhibition on M-MDSCs, M1 and M2 were reversed. Taken together, inhibition of the AMPK/mTOR signal could alleviate lupus-like diffuse lung injury by inducing M-MDSCs to differentiate into M2 by up-regulating FoxO1.

Gaining insight into the role of FoxO1 in the progression of disuse-induced skeletal muscle atrophy.

Expression of FoxO transcription factors increases during certain forms of atrophy. In a dephosphorylated state, FoxOs participate in ubiquitin-mediated proteasomal degradation through the transcriptional activation of E3-ubiquitin ligases such as MAFbx/atrogin-1 and MuRF1. There is exhaustive research demonstrating that FoxO3a is sufficient to induce MAFbx/atrogin-1 and MuRF-1 expressions. In contrast, the data are conflicting on the requirement of FoxO1 signaling in the activation of the E3-ubiquitin ligases. Moreover, no reports currently exist on the particular role of FoxO1 in the molecular mechanisms involved in the progression of physiological muscle wasting. Here, we have applied the most extensively used rodent model of microgravity/functional unloading to stimulate disuse-induced skeletal muscle atrophy such as rat hindlimb suspension (HS). We showed that inhibition of FoxO1 activity by a selective inhibitor AS1842856 completely reversed an increase in expression of MuRF-1, but not MAFbx/atrogin-1, observed upon HS. Furthermore, we demonstrated that FoxO1 induced upregulation of another E3-ubiquitin-ligase of a MuRF protein family MuRF-2 in skeletal muscle subjected to disuse. Prevention of the MuRF increase upon HS impeded upregulation of transcript expression of a negative regulator of NFATc1 pathway calsarcin-2, which was associated with a partial reversion of MyHC-IId/x and MyHC-IIb mRNA expressions. Importantly, FoxO1 inhibition induced a marked increase in p70S6k phosphorylation, an important stage in the initiation of protein translation, concomitant with the restoration of global protein synthesis in the skeletal muscle of the HS rats. Examination of eIF3f expression and the eEF2k/eEF2 pathway, other factors controlling translation initiation and elongation respectively, did not reveal any impact of FoxO1 on their activity. Lastly, we observed a decrease in transcript levels of Sesn3, but not Sesn1 and Sesn2, upon disuse, which was completely reversed by FoxO1 inhibition. These data demonstrate that FoxO1 signaling contributes to the development of disuse-induced skeletal muscle atrophy, including slow to fast MyHC isoform shift, mostly through upregulation of MuRF-1 and MuRF-2 expression. Furthermore, FoxO1 inhibition is required to recover Sesn3 mRNA expression in atrophic conditions, which likely contributes to the enhanced p70S6k activity and restoration of the protein synthesis rate.

Upregulation of FOXO1 contributes to lipopolysaccharide-induced pulmonary endothelial injury by induction of autophagy.

Background: Autophagy is activated during the pathogenesis of endothelial dysfunction and sepsis-associated acute lung injury (ALI). This study aimed to investigate whether autophagy affected endothelial barrier dysfunction and lung injury in a murine model of lipopolysaccharide (LPS)-induced ALI, and then further clarify whether forkhead box O1 (FOXO1), an autophagy-related transcriptional factor, contributed to autophagy activation and ALI induced by LPS. Methods: Male C57BL/6 mice were treated with LPS (30 mg/kg), and then were allocated to a control group and an LPS group with or without FOXO1 inhibitor (AS1842856) treatment, respectively. Primary cultured mouse lung vascular endothelial cells (MLVECs) were treated with LPS, autophagy inhibitor 3-methyladenine (3-MA), AS1842856, and small interfering RNA (siRNA) targeting autophagy-related gene 5 (ATG5) or FOXO1. Endothelial autophagic flux was assessed by transfection of MLVECs with red fluorescent protein (RFP)-green fluorescent protein (GFP) tandem fluorescent-tagged LC3 (RFP-GFP-LC3) adenovirus. Endothelial permeability was analyzed by the diffusion of fluorescein isothiocyanate-carboxymethyl (FITC)-dextran through the endothelial monolayer. Evans blue albumin tracer was used to measure the pulmonary transvascular permeability, and hematoxylin and eosin (H&E) staining was used to observe pathological changes in the lung tissues. Immunofluorescence staining was also used to detect the expression of zonula occludens-1 (ZO-1) and FOXO1. Results: This study found autophagy induction in lung tissues of endotoxemic mice and LPS-treated MLVECs, as evidenced by elevated expression of light chain 3 II (LC3-II) and Unc-51-like kinase (ULK1) and autophagic flux. LPS treatment decreased vascular endothelial (VE)-cadherin and ZO-1 expression and increased endothelial permeability in MLVECs, which were significantly alleviated by autophagy inhibitor 3-MA and ATG5 siRNA. It was found that both phosphorylated FOXO1 and FOXO1 were upregulated in the lung tissues of endotoxemic mice and LPS-treated MLVECs. Both FOXO1 inhibitor AS1842856 and FOXO1 siRNA suppressed LPS-induced autophagy and endothelial cell injury in MLVECs. Moreover, FOXO1 inhibition profoundly alleviated autophagy, lung endothelial hyperpermeability, and ALI in endotoxemic mice. Conclusions: This work demonstrated that FOXO1 upregulation is an important contributor to LPS-induced autophagy in pulmonary VE cells. The detrimental effects of FOXO1 in endotoxemia-associated endothelial dysfunction and ALI are partly due to its potent pro-autophagic property. Inhibition of FOXO1 may be a potential therapeutic option for the treatment of ALI.