Transcription Factor Forkhead Box O1 Research Articles

Photobiomodulation therapy decreases free fatty acid generation and release in adipocytes to ameliorate insulin resistance in type 2 diabetes

Excessive circulating free fatty acids (FFA) cause insulin resistance in peripheral tissues by inhibiting the proximal insulin signaling pathway. White adipose tissue (WAT) is a primary source of FFA generation and release through triglyceride (TG) hydrolysis. Thus, reducing excessive lipolysis in adipocytes ameliorates whole-body insulin resistance in type 2 diabetes. Here, we found that a noninvasive photobiomodulation therapy (PBMT), decreased FFA generation and release in WATs from high-fat diet (HFD)-fed mice and diabetic db/db mice. Meanwhile, plasma FFA and TG levels were reduced in two mouse models after PBMT. PBMT promoted mitochondrial reactive oxygen species (ROS) generation, which inhibited phosphatase and tensin homologue (PTEN) and promoted protein kinase B (AKT) activation. Photoactivation of AKT inhibited the transcriptional activity of Forkhead box transcription factor O1 (FoxO1), reducing expression of lipolytic enzymes and FFA generation and release. Eliminating ROS elimination or inhibiting AKT blocked the effects of the laser therapy in vivo and in vitro. Taken together, PBMT suppresses FFA generation and release in insulin-resistant adipocytes, contributing to improvement of insulin resistance in mouse models of type 2 diabetes.

MiR-223-3p promotes autoreactive Th17 cell responses in experimental autoimmune uveitis (EAU) by inhibiting transcription factor FOXO3 expression.

Pathogenic T helper (Th)17 cells are key mediators of autoimmune diseases such as uveitis and its animal model, experimental autoimmune uveitis (EAU). However, the contribution of microRNAs (miRs) to the intrinsic control of pathogenic Th17 cells in EAU remains largely unknown. Here, we have reported that miR-223-3p was significantly up-regulated in interphotoreceptor retinoid-binding protein-specific Th17 cells, and its expression was enhanced by IL-23-signal transducer and activator of transcription 3 signaling. Knockdown of miR-223-3p decreased the pathogenicity of Th17 cells in a T-cell transfer model of EAU. Mechanistic studies showed that miR-223-3p directly repressed the expression of forkhead box O3 (FOXO3), and FOXO3 negatively regulated pathogenic Th17 cell responses partially via suppression of IL-23 receptor expression. Thus, our results reveal an important role for miR-223-3p in autoreactive Th17 cell responses and suggest a potential therapeutic avenue for uveitis.-Wei, Y., Chen, S., Sun, D., Li, X., Wei, R., Li, X., Nian, H. miR-223-3p promotes autoreactive Th17 cell responses in experimental autoimmune uveitis (EAU) by inhibiting transcription factor FOXO3 expression.

Exogenous miR-26a suppresses muscle wasting and renal fibrosis in obstructive kidney disease.

Kidney fibrosis occurs in almost every type of chronic kidney disease. We found that microRNA (miR)-26a was decreased in the kidney, muscle, and exosomes of unilateral ureteral obstruction (UUO) mice. We hypothesized that exogenous miR-26 could suppresses renal fibrosis and muscle wasting in obstructive kidney disease. For this purpose, we generated exosomes that encapsulated miR-26, then injected these into skeletal muscle of UUO mice. The expression of miR-26a was elevated in serum exosomes from UUO mice following exosome-miR-26a injection. In these mice, muscle wasting has been ameliorated as evidenced by increased muscle weights. In addition, a muscle atrophy marker, myostatin, is increased in UUO muscle; provision of miR-26a abolished this increase. We detected a remote effect of exosomes containing miR-26a in UUO-induced renal fibrosis. The intervention of miR-26a attenuated UUO-induced renal fibrosis as determined by immunohistological assessment of α-smooth muscle actin and Masson's trichrome staining. Furthermore, exogenous miR-26a decreased the protein levels of 2 profibrosis proteins, connective tissue growth factor (CTGF) and TGF-β1, in UUO kidney. Our data showed that exosomes containing miR-26a prevented muscle atrophy by inhibiting the transcription factor forkhead box O1. Likewise, the exosome-carried miR-26a limited renal fibrosis by directly suppressing CTGF. Our findings provide an experimental basis for exosome-mediated therapy of muscle atrophy and renal fibrosis.-Zhang, A., Wang, H., Wang, B., Yuan, Y., Klein, J. D., Wang, X. H. Exogenous miR-26a suppresses muscle wasting and renal fibrosis in obstructive kidney disease.

Skeletal Muscle Atrophy Was Alleviated by Salidroside Through Suppressing Oxidative Stress and Inflammation During Denervation

Skeletal muscle atrophy is a common and debilitating condition that lacks an effective therapy. Oxidative stress and inflammation are two main molecular mechanisms involved in muscle atrophy. In the current study, we want to explore whether and how salidroside, with antioxidant and anti-inflammatory properties, protects against skeletal muscle atrophy induced by denervation. First, oxidative stress and inflammatory response were examined during myotube atrophy induced by nutrition deprivation. The results demonstrated that oxidative stress and inflammatory response were induced in cultured myotubes suffered from nutrition deprivation, and salidroside not only inhibited oxidative stress and inflammatory response but also attenuated nutrition deprivation-induced myotube atrophy, as evidenced by an increased myotube diameter. The antioxidant, anti-inflammatory, and antiatrophic properties of salidroside in cultured myotubes were confirmed in denervated mouse models. The mice treated with salidroside showed less oxidative stress and less inflammatory cytokines, as well as higher skeletal muscle wet weight ratio and larger average cross sectional areas of myofibers compared with those treated with saline only during denervation-induced skeletal muscle atrophy. Moreover, salidroside treatment of denervated mice resulted in an inhibition of the activation of mitophagy in skeletal muscle. Furthermore, salidroside reduced the expression of atrophic genes, including MuRF1 and MAFbx, autophagy genes, including PINK1, BNIP3, LC3B, ATG7, and Beclin1, and transcription factor forkhead box O3 A (Foxo3A), and improved the expression of myosin heavy chain and transcriptional factor phosphorylated Foxo3A. Taken together, these results suggested that salidroside alleviated denervation-induced muscle atrophy by suppressing oxidative stress and inflammation.

LIPUS promotes FOXO1 accumulation by downregulating miR-182 to enhance osteogenic differentiation in hPDLCs

Low intensity pulsed ultrasound (LIPUS) promotes bone fracture healing in clinical therapy. Transcription factor Forkhead box O1 (FOXO1) is crucial for bone differentiation. But whether FOXO1 is involved in LIPUS-promoted bone differentiation is largely unknown. In the current study, treatment of human primary periodontal ligament cells (hPDLCs) with LIPUS promoted total and nucleus FOXO1 protein accumulation. LIPUS-induced activation of FOXO1 further lead to higher alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2) expression and matrix mineralization. LIPUS inhibited miR-182 expression, which functioned as a repressor of FOXO1 through post-transcriptional regulation. Overexpression of miR-182 reversed the LIPUS-enhanced FOXO1 expression and osteogenic differentiation. Moreover, LIPUS enhanced Akt phosphorylation, which functioned in preventing active FOXO1 excessive accumulation via inducing the cytoplasm translocation of nucleus FOXO1. In conclusion, our study revealed that FOXO1, which was a target gene of miR-182, played an essential role in LIPUS-promoted osteogenic differentiation. This new molecular insight throws light upon the application of LIPUS therapy on periodontal bone defect.

Long-term c-Kit overexpression in beta cells compromises their function in ageing mice.

c-Kit signalling regulates intracellular pathways that enhance beta cell proliferation, insulin secretion and islet vascularisation in mice up to 28weeks of age and on short-term high-fat diet. However, long-term c-Kit activation in ageing mouse islets has yet to be examined. This study utilises beta cell-specific c-Kit-overexpressing transgenic (c-KitβTg) ageing mice (~60weeks) to determine the effect of its activation on beta cell dysfunction and insulin secretion. Wild-type and c-KitβTg mice, aged 60weeks, were examined using metabolic tests to determine glucose tolerance and insulin secretion. Pancreas histology and proteins in isolated islets were examined to determine the expression of beta cell transcription factors, proliferation and intracellular signalling. To determine the role of insulin receptor signalling in ageing c-KitβTg mice, we generated beta cell-specific inducible insulin receptor knockout in ageing c-KitβTg mice (c-KitβTg;βIRKO mice) and examined the ageing mice for glucose tolerance and islet histology. Ageing c-KitβTg mice progressively developed glucose intolerance, compared with age-matched wild-type littermates, due to impaired insulin secretion. Increased beta cell mass, proliferation and nuclear forkhead box transcription factor O1 (FOXO1) expression and reduced exocytotic protein levels were detected in ageing c-KitβTg mouse islets. Protein analyses of isolated islets showed increased insulin receptor, phosphorylated IRS-1Ser612 and cleaved poly(ADP-ribose) polymerase levels in ageing c-KitβTg mice. Ageing c-KitβTg mouse islets treated ex vivo with insulin demonstrated reduced Akt phosphorylation, indicating that prolonged c-Kit induced beta cell insulin insensitivity. Ageing c-KitβTg;βIRKO mice displayed improved glucose tolerance and beta cell function compared with ageing c-KitβTg mice. These findings indicate that long-term c-Kit overexpression in beta cells has a negative impact on insulin exocytosis and that temporally dependent regulation of c-Kit-insulin receptor signalling is important for optimal beta cell function.

Imatinib mesylate does not counteract ovarian tissue fibrosis in postnatal rat ovary

Chemotherapy may result in ovarian atrophy, a depletion of the primordial follicle pool, diminished ovarian weight, cortical and stromal fibrosis. Imatinib mesylate is an anticancer agent that inhibits competitively several receptor tyrosine kinases (RTKs). RTKs play important roles in cell metabolism, proliferation, and apoptosis. In clinic, imatinib mesylate is also known as an anti-fibrotic medicine. In the present study, the impact of imatinib on the ovarian tissue was investigated by assessing ovarian tissue fibrosis in postnatal rat administered with or without imatinib for three days. Fibrosis in the ovarian tissue was determined by histology (Picrosirius and Masson’s trichrome staining) and the protein expression of vimentin and alpha-smooth muscle actin (α-SMA). Furthermore, mRNA expression of Forkhead box transcription factor O1 and O3 (FOXO1 and FOXO3), which are markers of cell proliferation was quantified. A short-term exposure to imatinib showed to increase tissue fibrosis in ovaries. This was observed by Masson’s trichrome staining. Exposure to imatinib led also to a down-regulation of vimentin protein expression and up-regulation mRNA expression of FOXO3. This may indicate a role of FOXO3 in ovarian tissue fibrosis in postnatal rat ovaries.

The CtBP1-p300-FOXO3a transcriptional complex represses the expression of the apoptotic regulators Bax and Bim in human osteosarcoma cells.

C-terminal binding protein 1 (CtBP1), a well-known transcriptional corepressor, functions as an oncogene in multiple cancer types, including osteosarcoma, by modulating the transcription of many tumor suppressors, such as cadherin 1 (CDH1), phosphatase and tensin homolog (PTEN), Bcl2-associated X (Bax), Bcl-2-interacting mediator (Bim), and cyclin-dependent kinase inhibitor 1A (CDKN1A). However, it is still unclear how CtBP1 regulates the expression of these downstream targets. Here, we identified that CtBP1 is overexpressed in osteosarcoma cells and found that CtBP1 directly interacts with the transcription factor forkhead box O3 (FOXO3a) and the histone acetyltransferase p300 in vivo and in vitro. Through microarray analysis, we found that CtBP1 negatively regulates FOXO3a levels. In contrast to the CtBP1 level, the FOXO3a expression level was found to be significantly reduced in osteosarcoma cells. Knockdown of CtBP1 or overexpression of FOXO3a in U2OS cells resulted in different gene expression patterns, and the former caused upregulation of CtBP1 downstream target genes such as CDH1, PTEN, Bax, Bim, and CDKN1A, whereas the latter caused upregulation of Bax and Bim, but not CDH1, PTEN, and CDKN1A. Further analysis indicated that the CtBP1-p300-FOXO3a transcriptional complex specifically binds to the promoters of Bax and Bim. Inhibition of CtBP1 by the constitutive expression of Pep1-E1AWT peptide in U2OS and OSA cells reversed oncogenic phenotypes, including colony formation, cellular proliferation, and migration, and limited tumor growth in vivo. Together our results demonstrated that the CtBP1-p300-FOXO3a transcriptional complex represses the expression of the apoptotic regulators Bax and Bim in human osteosarcoma cells and that targeting CtBP1-mediated transcriptional events might be a potential therapeutic strategy for the osteosarcoma treatment.

Phosphorylation of Forkhead Protein FoxO1 at S253 Regulates Glucose Homeostasis in Mice.

The transcription factor forkhead box O1 (FoxO1) is a key mediator in the insulin signaling pathway and controls multiple physiological functions, including hepatic glucose production (HGP) and pancreatic β-cell function. We previously demonstrated that S256 in human FOXO1 (FOXO1-S256), equivalent to S253 in mouse FoxO1 (FoxO1-S253), is a key phosphorylation site mediating the effect of insulin as a target of protein kinase B on suppression of FOXO1 activity and expression of target genes responsible for gluconeogenesis. Here, we investigated the role of FoxO1-S253 phosphorylation in control of glucose homeostasis in vivo by generating global FoxO1-S253A/A knockin mice, in which FoxO1-S253 alleles were replaced with alanine (A substitution) blocking FoxO1-S253 phosphorylation. FoxO1-S253A/A mice displayed mild increases in feeding blood glucose and insulin levels but decreases in fasting blood glucose and glucagon concentrations, as well as a reduction in the ratio of pancreatic α-cells/β-cells per islet. FoxO1-S253A/A mice exhibited a slight increase in energy expenditure but barely altered food intake and glucose uptake among tissues. Further analyses revealed that FoxO1-S253A/A enhances FoxO1 nuclear localization and promotes the effect of glucagon on HGP. We conclude that dephosphorylation of S253 in FoxO1 may reflect a molecular basis of pancreatic plasticity during the development of insulin resistance.

FoxO1 regulates beige adipogenesis and adiposity via autophagy

The transcription factor forkhead box O1 (FoxO1) plays an important role in autophagy and metabolic homeostasis. Previous studies from our lab and others have shown that FoxO1 is required for autophagy and white adipocyte differentiation. However, it remains largely unknown whether and how FoxO1 mediates beige adipogenesis, a cellular event that promotes energy expenditure through mitochondrial uncoupling and thermogenesis. To address this question, we modulated FoxO1 activity in primary white adipocytes in vitro with FoxO1 antagonist (AS1842856). AS1842856 increased mitochondrial uncoupling protein UCP1, concomitant with upregulation of mitochondrial respiration chain proteins. These changes were associated with suppressed autophagy as evaluated by LC3 and p62. Interestingly, treatment of white adipocytes with classic autophagy inhibitor bafilomycin A1 also increased UCP1 and mitochondrial respiration chain protein, suggesting that FoxO1 may regulate beiging of white adipocytes via autophagy. Indeed, knockout (KO) of FoxO1 in mouse adipose tissue dampens autophagy and promotes beiging phenotype (i.e., upregulation of UCP1 and other mitochondrial contents). In line with beige adipogenesis priming energy expenditure through thermogenesis, the FoxO1‐KO mice showed significantly higher body temperature and lower adiposity. Together, our data reveals a role for FoxO1 to mediate beiging of white adipocytes by regulating autophagy, and suggests that targeting adipocyte FoxO1 may lead to new strategies to treat or prevent obesity.Support or Funding InformationUSDA National Institute of Food and Agriculture Hatch Project 1007334 (Z.C.) and American Heart Association Grant 18TPA34230082 (Z.C.)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Down-regulation of MicroRNA-592 in obesity contributes to hyperglycemia and insulin resistance.

BackgroundMany studies have demonstrated that microRNAs, a class of small and non-coding RNA molecules, play an important role in the regulation of glucose and lipid homeostasis. In the present study, we sought to investigate the function of miR-592 in the development of obesity-associated metabolic disorders, including hyperglycemia andinsulin resistance.MethodsThe expression levels of miR-592 were measured in the liver of obese mice and humans by quantitative reverse transcription PCR. Loss- and gain-of function experiments were employed to explore the metabolic function of miR-592 using locked nucleic acids and adenovirus in lean and obese mice, respectively. The molecular target of miR-592 was determined by western blotting and luciferase reporter assays.FindingsWe found a significant decreased expression of miR-592 in the liver of obese mice and humans. Inhibition of miR-592 led to elevated blood glucose levels, enhanced gluconeogenesis and reduced insulin sensitivity in lean mice. In contrast, adenovirus-mediated overexpression of hepatic miR-592 improved metabolic disorders in obese mice. Mechanistically, we found that the transcription factor forkhead box O1 (FOXO1) is a direct target gene of miR-592 to mediate its metabolic functions. miR-592 was able to inhibit the mRNA and protein expression of FOXO1 by binding to its 3′-untranslated region.InterpretationsOur findings demonstrate that obesity-associated down-regulation of miR-592 plays an important role in the progression of metabolic diseases. Restoration of hepatic miR-592 could improve glucose and lipid metabolism in obese mice.FundThis work is supported by the National Key Research and Development Program of China (No. 2016YFC1304805 to Dr. Chen), Natural Science Foundation of China (No. 81771574 to Dr. Wu), Shanghai Science Foundation (No. 18ZR1437800 to Dr. Li), Science and Technology Commission of Shanghai Municipality (Nos.18dz2304400 and 15,411,970,700 to Dr. Yang).

Quxie Capsule Inhibits Colon Tumor Growth Partially Through Foxo1-Mediated Apoptosis and Immune Modulation

Quxie capsule (QX), a herbal remedy used in traditional Chinese medicine, is routinely used in advanced colorectal cancer treatment in Xiyuan Hospital in Beijing, China. However, the mechanism(s) underlying the effect of QX in colorectal cancer remain unclear, which hampers the optimal use of QX for the treatment of the disease. The transcription factor forkhead box O1 (Foxo1) plays important roles in regulation of cell cycle, apoptosis, and immune response in various cancers. In this study, we examined the antitumor efficacy of QX in a mouse model of colorectal cancer and further investigated the mechanism by which QX regulated Foxo1 protein-mediated pathways. QX administered via gavage daily for 2 weeks in mice carrying CT26 mouse colon tumors resulted in significantly lower mean tumor weight (0.93 ± 0.32 g) compared with that in vehicle control-treated mice (1.57 ± 0.57 g, P <.05). Foxo1 protein expression in tumors was also higher in the QX group than that in the vehicle control group. Furthermore, QX treatment upregulated apoptotic proteins such as Fas, Bim, and cleaved caspase-3 in tumor tissue compared with those in the vehicle control group. Intriguingly, the ratios of Th1/Th2 and Th17/Treg cells and levels of T-bet protein (the key regulator of Th1 and Th2 cells) were higher while the level of Foxp3 (the key regulator of Treg cells) was lower in QX-treated mice compared to vehicle control mice, revealing that Foxo1 upregulated T-bet and downregulated Foxp3 and induced a shift in immune balance. This shift could be critical in the antitumor efficacy of QX. Furthermore, knocking down Foxo1 in human colon cancer HCT116 cells partially blocked the effect of QX-elicited antiproliferative activity. Together, these results suggest that QX exerts antitumor activity in CT26 mouse colon cancer model partially mediated by Foxo1-induced apoptosis and antitumor immune response.

Endothelial-specific FoxO1 depletion prevents obesity-related disorders by increasing vascular metabolism and growth.

Impaired angiogenesis is a hallmark of metabolically dysfunctional adipose tissue in obesity. However, the underlying mechanisms restricting angiogenesis within this context remain ill-defined. Here, we demonstrate that induced endothelial-specific depletion of the transcription factor Forkhead Box O1 (FoxO1) in male mice led to increased vascular density in adipose tissue. Upon high-fat diet feeding, endothelial cell FoxO1-deficient mice exhibited even greater vascular remodeling in the visceral adipose depot, which was paralleled with a healthier adipose tissue expansion, higher glucose tolerance and lower fasting glycemia concomitant with enhanced lactate levels. Mechanistically, FoxO1 depletion increased endothelial proliferative and glycolytic capacities by upregulating the expression of glycolytic markers, which may account for the improvements at the tissue level ultimately impacting whole-body glucose metabolism. Altogether, these findings reveal the pivotal role of FoxO1 in controlling endothelial metabolic and angiogenic adaptations in response to high-fat diet and a contribution of the endothelium to whole-body energy homeostasis.

FOXO1 regulates uterine epithelial integrity and progesterone receptor expression critical for embryo implantation.

Successful embryo implantation requires a receptive endometrium. Poor uterine receptivity can account for implantation failure in women who experience recurrent pregnancy loss or multiple rounds of unsuccessful in vitro fertilization cycles. Here, we demonstrate that the transcription factor Forkhead Box O1 (FOXO1) is a critical regulator of endometrial receptivity in vivo. Uterine ablation of Foxo1 using the progesterone receptor Cre (PgrCre) mouse model resulted in infertility due to altered epithelial cell polarity and apoptosis, preventing the embryo from penetrating the luminal epithelium. Analysis of the uterine transcriptome after Foxo1 ablation identified alterations in gene expression for transcripts involved in the activation of cell invasion, molecular transport, apoptosis, β-catenin (CTNNB1) signaling pathway, and an increase in PGR signaling. The increase of PGR signaling was due to PGR expression being retained in the uterine epithelium during the window of receptivity. Constitutive expression of epithelial PGR during this receptive period inhibited expression of FOXO1 in the nucleus of the uterine epithelium. The reciprocal expression of PGR and FOXO1 was conserved in human endometrial samples during the proliferative and secretory phase. This demonstrates that expression of FOXO1 and the loss of PGR during the window of receptivity are interrelated and critical for embryo implantation.

SGK1-FoxO1 Signaling Pathway Mediates Th17/Treg Imbalance and Target Organ Inflammation in Angiotensin II-Induced Hypertension.

It has been demonstrated that serum/glucocorticoid regulated kinase 1 (SGK1) and the downstream transcription factor forkhead box O1 (FoxO1) plays a critical role in the differentiation of T helper 17 cells/regulatory T cells (Th17/Treg). In the present study, we hypothesized that this SGK1-FoxO1 signaling pathway is involved in Th17/Treg imbalance and target organ damage in angiotensin II (AngII)-induced hypertensive mice. Results show that SGK1 inhibitor EMD638683 significantly reversed renal dysfunction and cardiac dysfunction in echocardiography as indicated by decreased blood urine nitrogen and serum creatinine in AngII-infused mice. Flow cytometric assay shows that there was significant Th17/Treg imbalance in spleen and in renal/cardiac infiltrating lymphocytes as indicated by the increased Th17 cells (CD4+-IL17A+ cells) and decreased Treg cells (CD4+-Foxp3+). Consistently, real-time PCR shows that Th17-related cytokines including IL-17A, IL-23, and tumor necrosis factor α (TNF-α) was increased and Treg-related cytokine IL-10 was decreased in renal and cardiac infiltrating lymphocytes in AngII-infused mice. Meanwhile, SGK1 protein level, as well as its phosphorylation and activity, was significantly increased in spleen in AngII-infused rats. Furthermore, it was found that splenic phosphorylated FoxO1 was significantly increased, whereas total FoxO1 in nuclear preparation was significantly decreased in AngII-infused mice, suggesting that increased FoxO1 phosphorylation initiate its translocation from cytoplasm to nucleus. Notably, all changes were significantly inhibited by the treatment of EMD638683. These results suggest that SGK1 was involved in Th17/Treg imbalance and target organ damage in AngII-induced hypertension.

Hyperglycaemia correlates with skeletal muscle capillary regression and is associated with alterations in the murine double minute-2/forkhead box O1/thrombospondin-1 pathway in type 1 diabetic BioBreeding rats.

Type 1 diabetes can have deleterious effects on skeletal muscle and its microvasculature. Our laboratory has recently identified murine double minute-2 as a master regulator of muscle microvasculature by controlling expression levels of two key molecular actors of the angio-adaptive process: the pro-angiogenic vascular endothelial growth factor-A and the anti-angiogenic thrombospondin-1. Here, we show for the first time that in the soleus and plantaris muscles of the diabetes-prone BioBreeding rats, a rodent model of autoimmune type 1 diabetes, murine double minute-2 protein levels are significantly decreased, coinciding with elevated protein levels of thrombospondin-1 and its transcription factor forkhead box O1. Significant capillary regression was observed to similar extent in soleus and plantaris muscles of type 1 diabetic rats. Elevated blood glucose levels were correlated with the loss of capillaries, the reduction in murine double minute-2 expression and with the elevations in thrombospondin-1. Vascular endothelial growth factor-A protein levels were unaltered or even increased in diabetic animals, yet type 1 diabetic animals had less vascular endothelial growth factor receptor-2 abundance. The vascular endothelial growth factor-A/thrombospondin-1 ratio, a good indicator of skeletal muscle angio-adaptive environment, was decreased in type 1 diabetic muscle. Our results suggest that the murine double minute-2-forkhead box O1-thrombospondin-1 pathway plays an important role in angio-regulation of the skeletal muscle in the pathophysiological context of type 1 diabetes.

Forkhead box O1 as an indicator of prognosis is inactivated in urothelial carcinoma of the upper urinary tract.

The transcription factor forkhead box O1 (FOXO1) can be inactivated via its phosphorylation, resulting in suppression of apoptosis. Using immunohistochemistry, the expression of a phosphorylated form of FOXO1 was assessed in upper urinary tract urothelial carcinoma (UUTUC) specimens. Overall, phospho-FOXO1 (p-FOXO1) was immunoreactive in all 99 UUTUC specimens [12 (12.1%) weak (1+), 46 (46.5%) moderate (2+) and 41 (41.4%) strong (3+)], which was significantly (P=0.018) increased, compared with benign urothelium specimens [77/82 (93.9%): 18 (22.0%) 1+, 41 (50.0%) 2+ and 18 (22.0%) 3+]. Muscle invasion (P=0.031) and lymphovascular invasion (P=0.025) were observed more frequently in p-FOXO1(2+/3+) tumor samples compared with p-FOXO1(1+) tumor samples. No statistically significant associations between p-FOXO1 expression and tumor grade or presence of concurrent carcinoma in situ, hydronephrosis or lymph node metastasis were observed. Furthermore, the levels of p-FOXO1 and estrogen receptor-β expression were significantly (P<0.05) correlated in UUTUC samples [correlation coefficient (CC)=0.244], particularly in tumor samples from male patients (CC=0.330). Additionally, patients with p-FOXO1(3+) tumors had a significantly increased risk of cancer-specific mortality (P=0.043), compared with those with p-FOXO1(1+/2+) tumors. Multivariate analysis further demonstrated a notable, albeit not significant, association between p-FOXO1 expression and cancer-specific survival (hazard ratio=2.204; P=0.053). These findings indicate that FOXO1 is inactivated in UUTUC specimens and p-FOXO1 overexpression may serve as a predictor of poor patient outcomes.

HDL-apoA-I induces the expression of angiopoietin like 4 (ANGPTL4) in endothelial cells via a PI3K/AKT/FOXO1 signaling pathway

BackgroundHigh Density Lipoprotein (HDL) and its main protein component, apolipoprotein A-I (apoA-I), have numerous atheroprotective functions on various tissues including the endothelium. Therapies based on reconstituted HDL containing apoA-I (rHDL-apoA-I) have been used successfully in patients with acute coronary syndrome, peripheral vascular disease or diabetes but very little is known about the genomic effects of rHDL-apoA-I and how they could contribute to atheroprotection. ObjectiveThe present study aimed to understand the endothelial signaling pathways and the genes that may contribute to rHDL-apoA-I-mediated atheroprotection. MethodsHuman aortic endothelial cells (HAECs) were treated with rHDL-apoA-I and their total RNA was analyzed with whole genome microarrays. Validation of microarray data was performed using multiplex RT-qPCR. The expression of ANGPTL4 in EA.hy926 endothelial cells was determined by RT-qPCR and Western blotting. The contribution of signaling kinases and transcription factors in ANGPTL4 gene regulation by HDL-apoA-I was assessed by RT-qPCR, Western blotting and immunofluorescence using chemical inhibitors or siRNA-mediated gene silencing. ResultsIt was found that 410 transcripts were significantly changed in the presence of rHDL-apoA-I and that angiopoietin like 4 (ANGPTL4) was one of the most upregulated and biologically relevant molecules. In validation experiments rHDL-apoA-I, as well as natural HDL from human healthy donors or from transgenic mice overexpressing human apoA-I (TgHDL-apoA-I), increased ANGPTL4 mRNA and protein levels. ANGPTL4 gene induction by HDL was direct and was blocked in the presence of inhibitors for the AKT or the p38 MAP kinases. TgHDL-apoA-I caused phosphorylation of the transcription factor forkhead box O1 (FOXO1) and its translocation from the nucleus to the cytoplasm. Importantly, a FOXO1 inhibitor or a FOXO1-specific siRNA enhanced ANGPTL4 expression, whereas administration of TgHDL-apoA-I in the presence of the FOXO1 inhibitor or the FOXO1-specific siRNA did not induce further ANGPTL4 expression. These data suggest that FOXO1 functions as an inhibitor of ANGPTL4, while HDL-apoA-I blocks FOXO1 activity and induces ANGPTL4 through the activation of AKT. ConclusionOur data provide novel insights into the global molecular effects of HDL-apoA-I on endothelial cells and identify ANGPTL4 as a putative mediator of the atheroprotective functions of HDL-apoA-I on the artery wall, with notable therapeutic potential.

Foxo1 Promotes Th9 Cell Differentiation and Airway Allergy

T helper 9 (Th9) cells are effector CD4+ T cells that are characterized by the production of interleukin-9 (IL-9) and have been associated with allergic responses. Here, we found that the expression of the transcription factor forkhead box O1 (Foxo1) was induced in Th9 and Foxo1 plays a crucial role in the differentiation of Th9 cells. Pharmacological inhibition of Foxo1 or genetic disruption of Foxo1 in CD4+ T cells caused a reduction in IL-9 expression while upregulating IL-17A and IFNγ production. Furthermore, chromatin immunoprecipitation (ChIP) followed by luciferase assays revealed direct binding of Foxo1 to both the Il9 and Irf4 promoters and induces their transactivation. Lastly, adoptive transfer of Th9 cells into lungs induced asthma-like symptoms that were ameliorated by Foxo1 inhibitor, AS1842856. Together, our findings demonstrate a novel regulator of Th9 cells with a direct implication in allergic inflammation.

The relationship between FoxO1 factor and beta cells dedifferentiation

Type 2 diabetes is a chronic metabolic disorder caused by a deficiency of beta cell function leading to varying degrees of insulin deficiency and insulin resistance. The decline of beta cell function is the central part in the development of type 2 diabetes. The dedifferentiation of pancreatic beta cells is one of the important mechanisms of islet dysfunction. In recent years, it has been shown that the decrease of the activity of the transcription factor forkhead box O1 (FoxO1) is closely related to the dedifferentiation of beta cells. The study of the specific mechanism of FoxO1 involved in the dedifferentiation of beta cells and the redifferentiation of the differentiated cells will provide new ideas for the prevention and treatment of type 2 diabetes. Key words: Forkhead transcription factors/ME; Islets of langerhans/CY/PA/ME; Review