FOXO1 and FOXO3 transcription factors have unique functions in meniscus development and homeostasis during aging and osteoarthritis.

The objective of this study was to examine FoxO expression and FoxO function in meniscus. In menisci from human knee joints with osteoarthritis (OA), FoxO1 and 3 expression were significantly reduced compared with normal menisci from young and old normal donors. The expression of FoxO1 and 3 was also significantly reduced in mouse menisci during aging and OA induced by surgical meniscus destabilization or mechanical overuse. Deletion of FoxO1 and combined FoxO1, 3, and 4 deletions induced abnormal postnatal meniscus development in mice and these mutant mice spontaneously displayed meniscus pathology at 6 mo. Mice with Col2Cre-mediated deletion of FoxO3 or FoxO4 had normal meniscus development but had more severe aging-related damage. In mature AcanCreERT2 mice, the deletion of FoxO1, 3, and 4 aggravated meniscus lesions in all experimental OA models. FoxO deletion suppressed autophagy and antioxidant defense genes and altered several meniscus-specific genes. Expression of these genes was modulated by adenoviral FoxO1 in cultured human meniscus cells. These results suggest that FoxO1 plays a key role in meniscus development and maturation, and both FoxO1 and 3 support homeostasis and protect against meniscus damage in response to mechanical overuse and during aging and OA.

The protective effects of resveratrol pretreatment in cyclophosphamide-induced rat ovarian injury: an vivo study.

OBJECTIVES: To explore whether resveratrol (Res) pretreatment could exert a protective effect on cyclophosphamide (Cy) induced ovarian toxicity in a rat model. METHODS: Twenty-four female 7-week old Sprague-Dawley rats were randomly divided into four groups: Con, administered with vehicle solutions; Cy, treated with Cy; Res + Cy, treated with Cy + Res combined; Res, treated with Res. After 21 d of treatments, the rats were euthanized and blood samples were collected to evaluate the levels of anti-Müllerian hormone (AMH). The Ovaries were processed for immunohistochemical and western blotting. RESULTS: Cy-treat caused the decrease of body weights and ovarian weight. AMH was lower in Cy group, whereas AMH levels were similar among other groups. Histomorphology showed a large number of primordial follicles were activated in Cy groups, whereas the primordial follicles were inhibited in the Res and Res + Cy groups. The expressions of Sirt1, Foxo3a were up-regulated and p53, Caspase-3, and Bax were down-regulated in Res + Cy and Res groups (p < .05). CONCLUSIONS: Res can prevent the primordial follicle activation and decrease apoptosis induced by Cy. Res may be an effective protection for ovarian function during chemotherapy, which means a new nonsurgical application for protection of ovarian reserve.

Phosphofructokinase-M inhibits cell growth via modulating the FOXO3 pathway in renal cell carcinoma cells.

Phosphofructokinase-M (PFKM) is a key enzyme in glycolysis. The expression and activity of PFKM is closely related to the occurrence and development of malignant tumors, but its role in the regulation of renal cell carcinoma (RCC) is still unknown. We found that the expression of PFKM was lower in RCC tumor tissue than in adjacent normal tissues, and that low expression of PFKM was related to the poor overall survival of RCC patients. In addition, our results showed that FOXO3 mediated PFKM inhibited the growth, migration and invasion of RCC cells, suggesting that PFKM is a protective factor for RCC.

FOXOs in the impaired heart: New therapeutic targets for cardiac diseases.

Cardiac diseases have a high morbidity and mortality and affect the global population. Based on recent accumulating evidence, Forkhead box O (FOXOs) play important roles in cardiac diseases. Therefore, a summary of the current literature on the molecular mechanisms and roles of FOXOs in the heart will provide valuable information. In this review, we first briefly introduce the molecular features of FOXOs. Then, we discuss the regulation and cardiac actions of the FOXO pathways. Based on this background, we expand our discussion to the roles of FOXOs in several major cardiac diseases, such as ischemic cardiac diseases, diabetic cardiomyopathy and myocardial hypertrophy. Then, we describe some methodological problems associated with the FOXO gene-modified animal models. Finally, we discuss potential future directions. The information reviewed here may be significant for the design of future studies and may increase the potential of FOXOs as therapeutic targets.

Evaluation of the p-AKT, p-JNK and FoxO3a function in oral epithelial dysplasia.

OBJECTIVES: To evaluate the expression of p-AKT, p-JNK, FoxO3a, and Ki-67 in samples of oral squamous cell carcinoma (OSCC) and oral epithelial dysplasias (OEDs) to understand their possible involvement in the malignant transformation process of oral lesions. MATERIALS AND METHODS: Tissue samples of 20 cases of OSCCs, 20 OEDs, and normal oral mucosa were subjected to immunohistochemistry reactions for anti-p-AKT, anti-p-JNK, anti-FoxO3a, and anti-Ki-67 antibodies. It was analyzed using quantitative (number of immunostained cells) and qualitative (immunostaining intensity) parameters in different cell immunostaining sublocations. RESULTS: Nuclear p-AKT was observed significantly greater immunostaining in OSCC (21.2 ± 19.0) than in dysplasias (7.9 ± 8.1) and controls (1.8 ± 4.7) (P = 0.002). Immunostaining of strong nuclear p-JNK was greater in controls (48.3 ± 13.7) than in OEDs (11.0 ± 10.3) and OSCCs (1.1 ± 1.3) (P < 0.001). Strong nuclear immunostaining of FoxO3a proved to be absent in OSCCs (0.0 ± 0.1) with little staining on dysplasias (3.2 ± 5.4) and increased expression in controls (13.5 ± 4.8) (P < 0.001). Immunostaining of strong nuclear Ki-67 was grater in OSCCs (48.1 ± 49.6) than in OED (11.8 ± 10.6) and controls (1.9 ± 2.0) (P < 0.001). CONCLUSIONS: Malignant process of OEDs in this research may involve the same mechanisms of established malignant lesions.

Inhibitory effect of Astragalus polysaccharide on osteoporosis in ovariectomized rats by regulating FoxO3a /Wnt signaling pathway.

PURPOSE: To investigate inhibitory effect of Astragalus polysaccharide (APS) on osteoporosis in ovariectomized rats by regulating FoxO3a/Wnt2 signaling pathway. METHODS: Postmenopausal osteoporosis (PMOP) animal model was developed by excising the bilateral ovaries of rats. The model rats were administered with APS (200 mg/kg, 400 mg/kg, 800 mg/kg) by intragastric administration once daily for 12 weeks. Bone density, bone metabolism index and oxidative stress index were measured in all groups. Furthermore, the regulation of APS of FoxO3a / Wnt2 signaling pathway was observed. RESULTS: APS has an estrogen-like effect, which can increase bone mass, lower serum ALP and BGP values, increase blood calcium content, and increase bone density of the femur and vertebrae in rats. At the same time, APS can increase the bone mineral content of the femur, increase the maximum stress, maximum load and elastic modulus of the ovariectomized rats, improve oxidative stress in rats by increasing the gene expression of ß-catenin and Wnt2 mRNA and inhibiting the gene expression of FoxO3a mRNA. CONCLUSION: Astragalus polysaccharide can effectively alleviate oxidative stress-mediated osteoporosis in ovariectomized rats, which may be related to its regulation of FoxO3a/Wnt2/ß-catenin pathway.

Forkhead box protein O3 suppresses uveal melanoma development by increasing the expression of Bcl­2­like protein 11 and cyclin­dependent kinase inhibitor 1B.

Forkhead box protein O3 (FoxO3a) is a forkhead box family transcription factor which serves an important role in a number of biological functions, including tumor growth. A previous study indicated that FoxO3a serves a role in insulin like growth factor­induced growth, migration and invasion of uveal melanoma (UM) cells; however, whether FoxO3a is associated with the development and formation of UM remains unknown. In the present study, the role of FoxO3a in UM development and formation was investigated by modulating the expression of FoxO3a in a human UM cell line. The results of the present study demonstrated that FoxO3a overexpression in UM cells inhibited cell proliferation and promoted cellular apoptosis, leading to an accumulation of cells at the G1 cell cycle phase. Western blot analysis demonstrated that FoxO3a overexpression increased the transcription and protein expression of Bcl­2­like protein 11 and cyclin­dependent kinase inhibitor 1B, and inhibited cyclin D1 transcription and expression. The opposite effects were observed when FoxO3a was knocked down in UM cells. The results of the present study indicated that FoxO3a may exhibit a negative role in UM development and formation, which is consistent with its role as a tumor suppressor.

The PI3K/Akt/FOXO3a pathway regulates regeneration following spinal cord injury in adult rats through TNF-α and p27kip1 expression.

The aim of the present study was to elucidate the expression and role of the phosphatidylinositol 3­kinase (PI3K)/Akt/forkhead box O3 (FOXO3a) pathway in the regeneration of the spinal cord following spinal cord injury (SCI), and its regulatory effect on tumor necrosis factor (TNF)-α and cyclin-dependent kinase inhibitor 1B (p27kip1) expression. Firstly, in a Sprague-Dawley rat model of SCI, western blot analysis revealed that the protein levels of PI3K, phosphorylated Akt and FOXO3a were markedly inhibited compared with those in the sham control group. In vitro experiments were also conducted, in which primary dissociated cultures of rat dorsal spinal cord cells were induced with lipopolysaccharide (LPS; 4 µg/ml). The downregulation of PI3K using LY294002 markedly suppressed cell viability, reduced the protein levels of FOXO3a and p27kip1, and increased TNF-α protein production in the LPS-induced spinal cord cells. In addition, when the LPS-induced spinal cord cells were infected with FOXO3a adenoviral vectors, the overexpression of FOXO3 markedly promoted cell proliferation, activated p27kip1 protein levels and inhibited TNF-α protein production in the spinal cord cells. These results suggest that the PI3K/Akt/FOXO3a pathway regulates regeneration following SCI in adult rats via its modulatory effects on TNF-α and p27kip1 expression.

FOXO3, IRF4, and xIAP Are Correlated with Immune Activation in HIV-1-Infected Men Who Have Sex with Men During Early HIV Infection.

Forkhead box O (FOXO)1, FOXO3, interferon regulatory factor (IRF)4, X-linked inhibitor of apoptosis protein (xIAP), and E74-like factor (ELF)4 have been described as important regulators of T cell functions and differentiation. However, whether these molecules are associated with HIV-1 disease progression is still unknown. In this study, we showed that the levels of FOXO3, IRF4, and xIAP mRNA in rapid progressors (RPs) were significantly higher than in HIV-negative healthy controls (HCs). Moreover, FOXO3 expression was positively correlated with HIV-1 viral load and CD4+ T cell activation. Remarkably, increased CD4+ and CD8+ T cell activation was apparent in RPs compared with typical progressors and HCs. In addition, a profile of higher apoptosis, more CD8+ TEM cells, and fewer CD4+ and CD8+ Naive T cells were observed in early HIV infection patients with low CD4+ T cell counts. Furthermore, in vitro, IRF4 and xIAP expression was enhanced in peripheral blood mononuclear cells from healthy people following T cell receptor stimulation. T cell activation was decreased by treatment with siRNA inhibiting FOXO3, IRF4, and xIAP. Our results show that significantly increased levels of FOXO3, IRF4, and xIAP mRNA in Chinese HIV-1-infected patients were related to T cell immune activation, implicating them as potential targets for developing new therapeutic avenues to slow down HIV-1 disease progression.

SIRT1 reduction is associated with sex-specific dysregulation of renal lipid metabolism and stress responses in offspring by maternal high-fat diet.

Rodent models of maternal obesity have been associated with kidney damage and dysfunction in offspring. However, the underlying mechanisms are yet to be elucidated. In this study, female rats were fed a high-fat diet (HFD) for 6 weeks prior to mating, throughout gestation and lactation; both male and female offspring were examined at weaning. Our results demonstrate that renal lipid deposition was increased in male offspring only, which is associated with reduced protein expression of Sirtuin (SIRT) 1, an essential regulator of lipid metabolism and stress response. Other components in its signalling network including phosphorylated 5'-AMP-activated protein kinase (pAMPKα), Forkhead box FOXO3a and Peroxisome proliferator-activated receptor (PPAR)γ coactivator 1-alpha (PGC-1α) were also downregulated. By contrast, in female offspring, renal fat/lipid distribution was unchanged in coupling with normal SIRT1 regulation. Specific autophagy and antioxidant markers were suppressed in both sexes. On the other hand, fibronectin and Collagen type IV protein expression was significantly higher in the offspring born HFD-fed dams, particularly in the males. Collectively, these findings suggest that maternal HFD consumption can induce sex-specific changes in offspring kidney lipid metabolism and stress responses at early ages, which may underpin the risk of kidney diseases later in life.

The mechanism of miR-23a in regulating myocardial cell apoptosis through targeting FoxO3.

OBJECTIVE: Myocardial cell apoptosis represents important pathologic basis of ischemia-reperfusion injury (I/R). MiR-23a is related to myocardial hypertrophy and cardiac remodeling by regulating myocardial cell growth and apoptosis. This study intended to observe the regulating effect of miR-23a in myocardial cell and related target, and investigate its clinical significance to I/R injury. MATERIALS AND METHODS: The rats were divided into sham group and myocardial I/R group. Myocardial cell cycle and miR-23a expression were tested. H2O2 was applied to treat H9c2 rat myocardial cell to simulate oxidative stress during I/R. The cells were divided into blank group, NC group, miR-23a mimic group, H2O2 group, and miR-23a + H2O2 group. ROS content and cell apoptosis were detected by flow cytometry. MiR-23a, FoxO3a, and BIM gene expression were determined by qRT-PCR. FoxO3a and BIM protein levels were measured by Western blot. RESULTS: Compared with sham group, myocardial apoptosis increased, while miR-23a expression was significantly downregulated in I/R group. H2O2 treatment markedly increased ROS levels in H9c2 cells and elevated apoptosis. The overexpression of mMiR-23a effectively reduced cell apoptosis induced by H2O2 treatment. H2O2 treatment significantly decreased miR-23a expression, while markedly elevated the levels of FoxO3a and BIM. The overexpression of miR-23a apparently impeded the induction of FoxO3a and BIM by H2O2. CONCLUSIONS: The downregulation of miR-23a plays a negative role in oxidative stress and cell apoptosis induced by I/R. The overexpression of miR-23a is of significance to alleviate cell apoptosis through inhibiting FoxO3a and downstream target BIM expression.

Correlations of Foxo3 and Foxo4 expressions with clinicopathological features and prognosis of bladder cancer.

OBJECTIVE: The study is performed to explore the correlations of forkhead box O3 (FoxO3) and forkhead box O4 (FoxO4) expressions with clinicopathological features and prognosis of bladder cancer. METHODS: Bladder cancer tissues and adjacent normal tissues from the recruited 222 patients were collected. Quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting and immunohistochemistry were applied to determine the expressions of FoxO3 and FoxO4. Spearman correlation analysis was conducted to examine the correlation between the expressions of FoxO3 and FoxO4. All patients were followed up and overall survival (OS) and disease-free survival (DFS) were recorded. Kaplan-Meier survival curve was drawn to determine the associations of FoxO3 and FoxO4 expressions and postoperative survival. Cox proportional hazards model was conducted to analyze the risk factors for poor prognosis of bladder cancer. RESULTS: The mRNA and expressions of FoxO3 and FoxO4 proteins in the bladder cancer tissues were lower than that in the adjacent normal tissues (both P<0.05). The positive rates of FoxO3 and FoxO4 were lower in the patients with lymph node metastasis than that in the patients without lymph node metastasis (P<0.05), and significantly lower in the patients with non-muscle invasive bladder cancer (Tis-T1) than in those with non-muscle invasive bladder cancer (T2-T3) in TNM staging, and remarkably lower in the patients with high grade than in those with low grade in the histological type (P<0.05). Furthermore, the expressions of FoxO3 and FoxO4 were positively correlated in the bladder cancer tissues (P<0.05). Negative expressions of FoxO3 and FoxO4 and lymph node metastasis were the risk factors for the poor prognosis of bladder cancer. CONCLUSIONS: The FoxO3 and FoxO4 expressions may potentially associate with the clinicopathological features and prognosis of bladder cancer.

Inhibitory effect of Astragalus polysaccharide on osteoporosis in ovariectomized rats by regulating FoxO3a /Wnt signaling pathway

Abstract Purpose: To investigate inhibitory effect of Astragalus polysaccharide (APS) on osteoporosis in ovariectomized rats by regulating FoxO3a/Wnt2 signaling pathway. Methods: Postmenopausal osteoporosis (PMOP) animal model was developed by excising the bilateral ovaries of rats. The model rats were administered with APS (200 mg/kg, 400 mg/kg, 800 mg/kg) by intragastric administration once daily for 12 weeks. Bone density, bone metabolism index and oxidative stress index were measured in all groups. Furthermore, the regulation of APS of FoxO3a / Wnt2 signaling pathway was observed. Results: APS has an estrogen-like effect, which can increase bone mass, lower serum ALP and BGP values, increase blood calcium content, and increase bone density of the femur and vertebrae in rats. At the same time, APS can increase the bone mineral content of the femur, increase the maximum stress, maximum load and elastic modulus of the ovariectomized rats, improve oxidative stress in rats by increasing the gene expression of β-catenin and Wnt2 mRNA and inhibiting the gene expression of FoxO3a mRNA. Conclusion: Astragalus polysaccharide can effectively alleviate oxidative stress-mediated osteoporosis in ovariectomized rats, which may be related to its regulation of FoxO3a/Wnt2/β-catenin pathway.