FoxO1 Pathway Research Articles

Hypoxia-Inducible Factor 1-α-AA-Modified Bone Marrow Stem Cells Protect PC12 Cells from Hypoxia-Induced Apoptosis, Partially Through VEGF/PI3K/Akt/FoxO1 Pathway

Bone marrow stem cells (BMSCs) have been shown to improve neurological function recovery in cerebral ischemia. Hypoxia-inducible factor-1 (HIF-1) α-AA is a more stable mutant form of HIF-1α, which is a crucial oxygen-sensitive regulator. To investigate the protective effects of HIF-1α-AA-modified BMSCs on neuron survival in cerebral ischemia models, we co-cultured HIF-1α-AA-modified BMSCs with neuron-like cells (PC12 cells) and observed a significant increase in the release of vascular endothelial growth factor (VEGF) from BMSCs, the decreased PC12 cell apoptosis, and the upregulation of Survivin expression reduced by hypoxia in PC12 cells compared to enhanced green fluorescent protein (EGFP) BMSCs. In addition, to explore whether VEGF secreted by HIF-1α-AA-modified BMSCs plays an important role in preventing hypoxia-induced apoptosis and the possible mechanism involved, exogenous VEGF were applied and the similar protective effects on PC12 cells were observed in vitro. Furthermore, hypoxia reduced the expression of phosphorylated Akt and phosphorylated FoxO1, whereas the administration of VEGF reversed these changes. Transfection of FoxO1 H215R, a DNA-binding mutant, abrogated the inhibitory ability on Survivin promoter activity, whereas FoxO1 AAA, the active form of FoxO1, presented further repression on Survivin promoter, indicating that FoxO1 directly binds on Survivin promoter as a transcriptional repressor and that phosphorylation status of FoxO1 affects its inhibition on the Survivin promoter. Transplantation of HIF-1α-AA-modified BMSCs after cerebral ischemia in vivo sufficiently reduced neurons apoptosis, decreased cerebral infarction volume, and induced a significant improvement on the modified neurological severity score compared to the EGFP BMSCs group. In conclusion, HIF-1α-AA-modified MSCs showed an obvious protective effect on neuron-like cells or neuron after ischemia in vitro and in vivo, at least in part, through the VEGF/PI3K/Akt/FoxO1 pathway.

Overlapping and divergent signaling pathways of N-cadherin and VE-cadherin in endothelial cells

AbstractEndothelial cells (ECs) express 2 members of the cadherin family, VE and N-cadherin. Although VE-cadherin induces EC homotypic adhesion, N-cadherin function in ECs remains largely unknown. EC-specific inactivation of either VE or N-cadherin leads to early fetal lethality suggesting that these cadherins play a nonredundant role in vascular development. We report here that VE-cadherin negatively controls junctional localization and expression of N-cadherin by limiting p120-catenin availability and reducing β-catenin transcriptional activity. Using EC lines expressing either VE or N-cadherin we found that both cadherins inhibit cell proliferation and apoptosis. Both trigger the phosphatidylinositol-3-OH-kinase (PI3K)–AKT-Forkhead-box protein-O1 (FoxO1) pathway and reduce β-catenin transcriptional activity. The extent of signaling correlates with the total level of cadherins regardless of the type of cadherin expressed. In contrast, basal and fibroblast growth factor (FGF)–induced cell motility is promoted by N-cadherin and strongly inhibited by VE-cadherin. This opposite effect is partly because of the ability of VE-cadherin to associate with FGF receptor and the density-enhanced phosphatase-1 (Dep-1) which, in turn, inhibits receptor signaling. We conclude that VE and N-cadherin have both additive and divergent effects on ECs. Differences in signaling are due, in part, to cadherin association with growth factor receptors and modulation of their downstream signaling.

Acute exercise reduces hepatic glucose production through inhibition of the Foxo1/HNF‐4α pathway in insulin resistant mice

Protein hepatocyte nuclear factor 4alpha (HNF-4alpha) is atypically activated in the liver of diabetic rodents and contributes to hepatic glucose production. HNF-4alpha and Foxo1 can physically interact with each other and represent an important signal transduction pathway that regulates the synthesis of glucose in the liver. Foxo1 and HNF-4alpha interact with their own binding sites in the phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) promoters, and this binding is required for their effects on those promoters. However, the effect of physical activity on the HNF-4alpha/Foxo1 pathway is currently unknown. Here, we investigate the protein levels of HNF-4alpha and the HNF-4alpha/Foxo1 pathway in the liver of leptin-deficient (ob/ob) and diet-induced obese Swiss (DIO) mice after acute exercise. The ob/ob and DIO mice swam for four 30 min periods, with 5 min rest intervals for a total swimming time of 2h. Eight hours after the acute exercise protocol, the mice were submitted to an insulin tolerance test (ITT) and determination of biochemical and molecular parameters. Acute exercise improved insulin signalling, increasing insulin-stimulated Akt and Foxo1 phosphorylation and decreasing HNF-4alpha protein levels in the liver of DIO and ob/ob mice under fasting conditions. These phenomena were accompanied by a reduction in the expression of gluconeogenesis genes, such as PEPCK and G6Pase. Importantly, the PI3K inhibitor LY292004 reversed the acute effect of exercise on fasting hyperglycaemia, confirming the involvement of the PI3K pathway. The present study shows that exercise acutely improves the action of insulin in the liver of animal models of obesity and diabetes, resulting in increased phosphorylation and nuclear exclusion of Foxo1, and a reduction in the Foxo1/HNF-4alpha pathway. Since nuclear localization and the association of these proteins is involved in the activation of PEPCK and G6Pase, we believe that the regulation of Foxo1 and HNF-4alpha activities are important mechanisms involved in exercise-induced improvement of glucose homeostasis in insulin resistant states.

PDK-1/FoxO1 pathway in POMC neurons regulatesPomcexpression and food intake

Both insulin and leptin signaling converge on phosphatidylinositol 3-OH kinase [PI(3)K]/3-phosphoinositide-dependent protein kinase-1 (PDK-1)/protein kinase B (PKB, also known as Akt) in proopiomelanocortin (POMC) neurons. Forkhead box-containing protein-O1 (FoxO1) is inactivated in a PI(3)K-dependent manner. However, the interrelationship between PI(3)K/PDK-1/Akt and FoxO1, and the chronic effects of the overexpression of FoxO1 in POMC neurons on energy homeostasis has not been elucidated. To determine the extent to which PDK-1 and FoxO1 signaling in POMC neurons was responsible for energy homeostasis, we generated POMC neuron-specific Pdk1 knockout mice (POMCPdk1(-/-)) and mice selectively expressing a constitutively nuclear (CN)FoxO1 or transactivation-defective (Delta256)FoxO1 in POMC neurons (CNFoxO1(POMC) or Delta256FoxO1(POMC)). POMCPdk1(-/-) mice showed increased food intake and body weight accompanied by decreased expression of Pomc gene. The CNFoxO1(POMC) mice exhibited mild obesity and hyperphagia compared with POMCPdk1(-/-) mice. Although expression of the CNFoxO1 made POMCPdk1(-/-) mice more obese due to excessive suppression of Pomc gene, overexpression of Delta256FoxO1 in POMC neurons had no effects on metabolic phenotypes and Pomc expression levels of POMCPdk1(-/-) mice. These data suggest a requirement for PDK-1 and FoxO1 in transcriptional regulation of Pomc and food intake.

Resveratrol protects cardiomyocytes from hypoxia-induced apoptosis through the SIRT1–FoxO1 pathway

Loss of cardiomyocytes through apoptosis has been proposed as a cause of ventricular remodeling and heart failure. Ischemia- and hypoxia-induced apoptosis of cardiomyocytes reportedly plays an important role in many cardiac pathologies. We investigated whether resveratrol (Res) has direct cytoprotective effects against ischemia/hypoxia for cardiomyocytes. Exposure of H9c2 embryonic rat heart-derived cells to hypoxia for 24 h caused a significant increase in apoptosis, as evaluated by TUNEL and flow cytometry, while treatment with 20 μM Res greatly decreased hypoxia-induced apoptosis in these cells. Exposure of the cells to Res (20 μM) caused rapid activation of SIRT1, which had a dual effect on FoxO1 function: SIRT1 increased FoxO1’s ability to induce cell cycle arrest, but inhibited FoxO1’s ability to induce cell death. This effect could be reversed by SIRT1 inhibition. Results of our study indicate that Res inhibits hypoxia-induced apoptosis via the SIRT1–FoxO1 pathway in H9c2 cells. This polyphenol may have potential in preventing cardiovascular disease, especially in coronary artery disease (CAD) patients.

Disruption of Sarcolemmal ATP-Sensitive Potassium Channel Activity Impairs the Cardiac Response to Systolic Overload

Sarcolemmal ATP-sensitive potassium channels (K(ATP)) act as metabolic sensors that facilitate adaptation of the left ventricle to changes in energy requirements. This study examined the mechanism by which K(ATP) dysfunction impairs the left ventricular response to stress using transgenic mouse strains with cardiac-specific disruption of K(ATP) activity (SUR1-tg mice) or Kir6.2 gene deficiency (Kir6.2 KO). Both SUR1-tg and Kir6.2 KO mice had normal left ventricular mass and function under unstressed conditions. Following chronic transverse aortic constriction, both SUR1-tg and Kir6.2 KO mice developed more severe left ventricular hypertrophy and dysfunction as compared with their corresponding WT controls. Both SUR1-tg and Kir6.2 KO mice had significantly decreased expression of peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha and a group of energy metabolism related genes at both protein and mRNA levels. Furthermore, disruption of K(ATP) repressed expression and promoter activity of PGC-1alpha in cultured rat neonatal cardiac myocytes in response to hypoxia, indicating that K(ATP) activity is required to maintain PGC-1alpha expression under stress conditions. PGC-1alpha gene deficiency also exacerbated chronic transverse aortic constriction-induced ventricular hypertrophy and dysfunction, suggesting that depletion of PGC-1alpha can worsen systolic overload induced ventricular dysfunction. Both SUR1-tg and Kir6.2 KO mice had decreased FOXO1 after transverse aortic constriction, in agreement with the reports that a decrease of FOXO1 can repress PGC-1alpha expression. Furthermore, inhibition of K(ATP) caused a decrease of FOXO1 associated with PGC-1alpha promoter. These data indicate that K(ATP) channels facilitate the cardiac response to stress by regulating PGC-1alpha and its target genes, at least partially through the FOXO1 pathway.

Role of FoxO1 in FFA-induced oxidative stress in adipocytes

Reactive oxygen species (ROS) production has recently been established as an essential contributor in the pathogenesis of obesity-associated insulin resistance. The FoxO1 pathway plays a role not only in nutrient sensing but also in regulating ROS production. We exposed adipocytes to free fatty acids (FFA) and demonstrated that FoxO1 protein levels decrease in a dose-dependent manner. The FoxO1 downregulation correlated with an increase in the production of ROS and a proinflammatory adipokine pattern characterized by a decrease in adiponectin and an increase in IL-6, plasminogen activator inhibitor-1, and monocyte chemotactic protein-1 mRNA expression levels. Similarly, a decrease in FoxO1 protein levels was seen in adipocytes of db/db mice compared with controls. Treatment with the sirtuin agonist resveratrol, which translocates FoxO1 to the nucleus, increased FoxO1 protein levels in adipocytes exposed to FFA. This correlated with a decrease in the generation of ROS and a partial reversal of the proinflammatory adipokine pattern. Together these results indicate that the insulin-resistant adipocyte produced by the exposure to a high concentration of fatty acids is characterized by decreased levels of FoxO1. These data also suggest that modulation of the Sirt1/FoxO1 pathway is a potentially useful therapeutic target for the obesity-induced dysfunctional adipocyte.

Effect of estrogen and inhibition of phosphatidylinositol-3 kinase on Akt and FOXO1 in rat uterus

The importance of FOXO transcription factors in regulating different aspects of cellular homeostasis and apoptosis has become apparent. Akt/protein kinase B has been shown to phosphorylate and inactivate members of FOXO family of transcription factors. Akt and its upstream regulator, phosphatidylinositol-3 kinase (PI3K) are involved in rapid action of estrogen (E2) in different cells and tissues. The aim of the present study was to analyze the E2/PI3K/Akt/FOXO pathway in rat uterus. In response to E2, phosphorylation of Akt/PKB on Ser473 and FOXO1 on Ser256 and Thr24 residues increased but with distinct kinetics, regulating the activation and inactivation of Akt and FOXO1 proteins, respectively. The antiestrogen ICI 182,780 prevented E2 induced Akt activation suggesting that estrogen receptors mediate this effect of E2. Intrauterine injection of Wortmannin caused a decrease in the phosphorylation of Ser473 of Akt, and attenuated phosphorylation of its downstream target FOXO1 at Ser256 and at Thr24. However, the effect of E2 on phosphorylation of Thr24 showed a kinetic pattern distinct from that of Ser256. Our results suggest that the E2/PI3K/Akt/FOXO1 pathway in rat uterus is functioning even at the lack of ovarian hormones and responses to E2 treatment. Estradiol increases Akt phosphorylation through a Wortmannin sensitive way, presumably involving PI3K. The present work shows that PI3K plays a crucial role in the phosphorylation and inactivation of FOXO1 in vivo, indicating that the regulation of this transcription factor is a more complex event in uterine cells requiring further investigations.

Regulation of Cbl-associated protein/Cbl pathway in muscle and adipose tissues of two animal models of insulin resistance.

The phosphatidylinositol 3-kinase-independent pathway to induce glucose transport may involve the tyrosine phosphorylation of the protooncogene c-Cbl. In the present study, we examined whether acute exposure to insulin stimulates the tyrosine phosphorylation of Cbl and its association with Cbl-associated protein (CAP) in muscle and adipose tissue of rats in vivo. We report herein that insulin induces Cbl tyrosine phosphorylation and association with CAP in adipose tissue but not in muscle. We also examined the expression and tyrosyl-phosphorylation state of Cbl and CAP/Cbl association in adipose tissue of rats submitted to prolonged fasting and in monosodium glutamate (MSG)-insulin-resistant rats. An increase in Cbl phosphorylation is observed in the fat of MSG rats, parallel with an increase in association of CAP-Cbl as well as an augment in CAP and Cbl protein expression in the adipose tissue of these animals. These events are accompanied by a decrease in insulin-stimulated insulin receptor/ insulin receptor substrate (IRS)-1 tyrosine phosphorylation and an increase in the IRS-2/phosphatidylinositol 3-kinase/Akt/Foxo1 pathway. In adipocytes of fasted rats, there is a decrease in CAP and Cbl protein expression, insulin-induced Cbl phosphorylation, and the association with CAP. In parallel, there is also a decrease in the insulin receptor/IRSs/Akt/Foxo1 pathway. Thus, insulin is able to induce Cbl tyrosine phosphorylation and its association with CAP in the adipose tissue of normal rats. In addition, our data provide evidence that the CAP-Cbl pathway may have a role in the modulation of adiposity in fasting and in MSG-treated rats.