Forkhead Box O3a Phosphorylation Research Articles

MO629XANTHINE OXIDASE INHIBITOR ATTENUATES RENAL OXIDATIVE STRESS AND ENDOTHELIAL DYSFUNCTION THROUGH THE INHIBITION OF VEGF-NADPH OXIDASES IN DIABETIC NEPHROPATHY

Abstract Background and Aims Xanthine oxidase (XO) is one of major source of reactive oxygen species, and a XO inhibitor, febuxostat has been reported to the protection of kidney diseases. We investigated whether febuxostat exerts renoprotective effects against diabetic nephropathy (DN). Method Eight-week Male C57BL/6 mice were divided into four groups: Control group (Cont), Febuxostat control group (FEB), streptozotocin treated group (STZ) and a febuxostat and STZ-treated diabetes group (STZ+FEB). STZ was used to induce diabetes (50 mg/kg/day, 5 days), and 5 mg/kg of febuxostat was treated to experimental mice for 8 weeks. Results STZ-treated diabetic mice were significantly decreased in serum and kidney XO levels, serum cystatin C and albuminuria by febuxostat treatment. Febuxostat treatment decreased renal hypertrophy and mesangial matrix expansion in STZ-treated diabetic mice. Febuxostat treatment suppressed the expression of vascular endothelial growth factor (VEGF)1 and 3, NADPH oxidase (NOX)1, 2, and 4, and the levels of their catalytic subunit mRNA in in STZ-treated diabetic mice. Febuxostat treatment was accompanied by the downregulation of Akt phosphorylation, followed by the suppression of transcription factor forkhead box O3a phosphorylation and the enhancement of endothelial nitric oxide synthase. Finally, febuxostat improved oxidative stress and resulted in decreased 8-hydroxy-2'-deoxyguanosine and kidney malondialdehyde levels, and increased superoxide dismutase activity in STZ-treated diabetic mice. Conclusions Febuxostat attenuated DN by modulating oxidative stress and endothelial function through VEGF–NADPH oxidase signaling pathway.

Effects of Shenfu Qiangxin Drink on H2O2-induced oxidative stress, inflammation and apoptosis in neonatal rat cardiomyocytes and possible underlying mechanisms.

The aim of the present study was to investigate the effects of Shenfu Qiangxin Drink (SFQXD) on acute myocardial infarction (AMI) and identify the possible underlying mechanisms. Levels of reactive oxygen species (ROS) and inflammatory factors, including interleukin (IL)-6, IL-1β and tumor necrosis factor-α (TNF-α) in the blood samples of patients with AMI were measured using commercially available kits by visible spectrophotometry after SFQXD administration. The contents of phosphorylated (p-) forkhead box O3a (FOXO3a) was examined using an ELISA kit. In addition, a hydrogen peroxide (H2O2)-induced myocardial injury model was established in vitro using neonatal rat cardiomyocytes. Following treatment with SFQXD, the levels of intracellular ROS, cell apoptosis, oxidative stress- and inflammation-related markers were measured using commercially available kits by visible spectrophotometry. Additionally, western blot analysis was used to measure the expression of sirtuin-4 (SIRT4), p-FOXO3a, acetylated FOXO3a (ace-FOXO3a) and apoptosis-related genes (Bcl-2, Bax, BIM and cleaved caspase-3). Subsequently, to investigate the possible underlying regulatory mechanisms, SIRT4 expression was silenced by transfection with small hairpin RNA against SIRT4, following which changes in the extent of oxidative stress, inflammation and apoptosis were assessed. The levels of ROS and interleukin (IL)-1β were found to be significantly reduced, whilst FOXO3a phosphorylation was markedly increased following administration with SFQXD. In vitro, SFQXD dose-dependently inhibited H2O2-induced oxidative stress, inflammation and apoptosis in neonatal rat cardiomyocytes. In addition, FOXO3a phosphorylation was markedly upregulated whilst FOXO3a acetylation was downregulated following treatment of H2O2-induced primary neonatal cardiomyocytes with SFQXD. SIRT4 knockdown also markedly reversed the effects of SFQXD on oxidative stress, inflammation and apoptosis in neonatal rat cardiomyocytes. In conclusion, these findings demonstrated that SFQXD may alleviate oxidative stress-induced myocardial injury by potentially regulating SIRT4/FOXO3a signaling, suggesting that SFQXD may be of clinical value for the treatment of AMI.

Rutin prevents cisplatin-induced ovarian damage via antioxidant activity and regulation of PTEN and FOXO3a phosphorylation in mouse model

The aims of the present study were to evaluate the protective effects of rutin during cisplatin-induced ovarian toxicity in mice and to verify the possible involvement of the phosphatase and tension homolog (PTEN)/Forkhead box O3a (FOXO3a) pathway in the rutin actions. Mice received saline solution (control, 0.15 M, i.p.) or cisplatin (5 mg/Kg body weight, i.p.) or they were pretreated with N-acetylcysteine (positive control; 150 mg/Kg of body weight [p.o.]) or with rutin (10, 30 or 50 mg/Kg body weight, p.o.) before cisplatin (5 mg/Kg body weight, i.p.) once daily for 3 days. Next, the ovaries were harvested and destined to histological (follicular morphology and activation), immunohistochemical (cell proliferation and apoptosis) and fluorescence (reactive oxygen species [ROS], glutathione [GSH] and mitochondrial activity) analyses. Moreover, the expression of phosphorylated PTEN (p-PTEN) and FOXO3a (p-FOXO3a) were evaluated to investigate a molecular mechanism by which rutin would prevent the cisplatin-induced ovarian damage. The results showed that pretreatment with N-acetylcysteine or 10 mg/Kg rutin before cisplatin preserved the percentage of normal follicles and cell proliferation, reduced apoptosis and ROS levels and increased active mitochondria and GSH levels compared to the cisplatin treatment (P < 0.05). Cisplatin treatment increased p-PTEN and decreased p-FOXO3a expression in follicles, which was prevented by 10 mg/kg rutin. In conclusion, treatment with 10 mg/Kg rutin has the potential to protect the ovarian follicles against cisplatin-induced toxicity through its antioxidant effects and PTEN/FOXO3a pathway.

P2Y2R contributes to the development of diabetic nephropathy by inhibiting autophagy response

ObjectiveDiabetic nephropathy (DN) is one of the most common complications of diabetes and a critical risk factor for developing end-stage renal disease. Activation of purinergic receptors, including P2Y2R has been associated with the pathogenesis of renal diseases, such as polycystic kidney and glomerulonephritis. However, the role of P2Y2R and its precise mechanisms in DN remain unknown. We hypothesised that P2Y2R deficiency may play a protective role in DN by modulating the autophagy signalling pathway. MethodsWe used a mouse model of DN by combining a treatment of high-fat diet and streptozotocin after unilateral nephrectomy in wild-type or P2Y2R knockout mice. We measured renal functional parameter in plasma, examined renal histology, and analysed expression of autophagy regulatory proteins. ResultsHyperglycaemia and ATP release were induced in wild type-DN mice and positively correlated with renal dysfunction. Conversely, P2Y2R knockout markedly attenuates albuminuria, podocyte loss, development of glomerulopathy, renal tubular injury, apoptosis and interstitial fibrosis induced by DN. These protective effects were associated with inhibition of AKT-mediated FOXO3a (forkhead box O3a) phosphorylation and induction of FOXO3a-induced autophagy gene transcription. Furthermore, inhibitory phosphorylation of ULK-1 was decreased, and the downstream Beclin-1 autophagy signalling was activated in P2Y2R deficiency. Increased SIRT-1 (sirtuin-1) and FOXO3a expression in P2Y2R deficiency also enhanced autophagy response, thereby ameliorating renal dysfunction in DN. ConclusionsP2Y2R contributes to the pathogenesis of DN by impairing autophagy and serves as a therapeutic target for treating DN.

Antitumor activity of luteolin in human colon cancer SW620 cells is mediated by the ERK/FOXO3a signaling pathway

The aim of this study was to investigate the mechanism of the anticancer activity of luteolin in metastatic human colon cancer SW620 cells. Luteolin dose-dependently reduced the viability and proliferation of SW620 cells and increased the expression of antioxidant enzymes. The expression of antiapoptotic protein Bcl-2 decreased whereas the expression of proapoptotic proteins Bax and caspase-3 increased by luteolin treatment, resulting in increased poly (ADP-ribose) polymerase (PARP) cleavage and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity. Luteolin also increased the expression of autophagic proteins Beclin-1, autophagy-related protein 5 (Atg5) and microtubule-associated protein 1A/1B-light chain 3 beta-I/II (LC3B-I/II), while the usage of 3-methyladenine suggested a prosurvival role of autophagy. Moreover, treatment with luteolin induced reversal of the epithelial-mesenchymal transition process through the suppression of the wingless-related integration site protein (Wnt)/β-catenin pathway. The cytotoxic activity of luteolin coincided with the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and forkhead box O3a (FOXO3a). Treatment with the mitogen-activated protein kinase kinase (MEK) inhibitor PD0325901 inhibited ERK-dependent FOXO3a phosphorylation, resulting in increased FOXO3a expression and apoptosis, with the suppression of autophagy. The results of the current study suggest the antitumor activity of luteolin in SW620 cells through the ERK/FOXO3a-dependent mechanism, as well as its antimetastatic potential.

Applied electric fields suppress osimertinib-induced cytotoxicity via inhibiting FOXO3a nuclear translocation through AKT activation.

Osimertinib is a third-generation epidermal growth factor receptor tyrosine kinase inhibitor against T790M-mutant non-small cell lung cancer (NSCLC). Acquired resistance to osimertinib is a growing clinical challenge that is not fully understood. Endogenous electric fields (EFs), components of the tumor microenvironment, are associated with cancer cell migration and proliferation. However, the impact of EFs on drug efficiency has not been studied. In this study, we observed that EFs counteracted the effects of osimertinib. EFs of 100 mV/mm suppressed osimertinib-induced cell death and promoted cell proliferation. Transcriptional analysis revealed that the expression pattern induced by osimertinib was altered by EFs stimulation. KEGG analysis showed that differential expression genes were mostly enriched in PI3K-AKT pathway. Then, we found that osimertinib inhibited AKT phosphorylation, while EFs stimulation resulted in significant activation of AKT, which could override the effects generated by osimertinib. Importantly, pharmacological inhibition of PI3K/AKT by LY294002 diminished EF-induced activation of AKT and restored the cytotoxicity of osimertinib suppressed by EFs, which proved that AKT activation was essential for EFs to attenuate the efficacy of osimertinib. Furthermore, activation of AKT by EFs led to phosphorylation of forkhead box O3a (FOXO3a), and reduction in nuclear translocation of FOXO3a induced by osimertinib, resulting in decreased expression of Bim and attenuated cytotoxicity of osimertinib. Taken together, we demonstrated that EFs suppressed the antitumor activity of osimertinib through AKT/FOXO3a/Bim pathway, and combination of PI3K/AKT inhibitor with osimertinib counteracted the effects of EFs. Our findings provided preliminary data for therapeutic strategies to enhance osimertinib efficacy in NSCLC patients.

Scutellarin exerts protective effects against atherosclerosis in rats by regulating the Hippo-FOXO3A and PI3K/AKT signaling pathways.

Atherosclerosis (AS), a progressive disorder, is one of the tough challenges in the clinic. Scutellarin, an extract from Herba Erigerontis, is found to have oxygen-free radicals scavenging effects and antioxidant effects. In this study, we aimed to investigate the anti-AS effects of scutellarin is related to controlling the Hippo-FOXO3A and PI3K/AKT signal pathway. To establish an AS model, the rats in the scutellarin and model groups were intraperitoneally injected with vitamin D 3 and then fed a high-fat diet for 12 weeks. In addition, in vitro angiotensin II-induced apoptosis of human aortic endothelial cells (HAECs) were used to establish models. Scutellarin significantly reduced blood lipid levels and increased antioxidase levels in both models. Additionally, scutellarin inhibited reactive oxygen species generation and apoptosis in HAECs. The impaired vascular barrier function was restored by using scutellarin in AS rats and in HAECs cells characterized by inhibiting mammalian sterile-20-like kinases 1 (Mst1) phosphorylation, Yes-associated protein (YAP) phosphorylation, forkhead box O3A (FOXO3A) phosphorylation at serine 207, nuclear translocation of FOXO3A, and upregulating protein expression of AKT and FOXO3A phosphorylation at serine 253. Scutellarin significantly reduced Bcl-2 interacting mediator of cell death (Bim), caspase-3, APO-1, CD95 (Fas), and Bax: Bcl-2-associated X (Bax) levels and activated Bcl-2: B-cell lymphoma-2 (Bcl-2). Scutellarin also significantly inhibited the expression of Mst1, YAP, FOXO3A at the messenger RNA level. When Mst1 was overexpressed or phosphoinositide 3-kinases suppressed, the effects of scutellarin were significantly blocked. In conclusion, the results of the present study suggest that scutellarin exerts protective effects against AS by inhibiting endothelial cell injury and apoptosis by regulating the Hippo-FOXO3A and PI3K/AKT signal pathways.

FoxO3a plays a key role in the protective effects of pomegranate peel extract against amikacin-induced ototoxicity.

The use of amikacin (AMK) in present treatment strategies results in severe ototoxicity; however, the underlying molecular mechanisms of this toxicity remain unclear. In this study, we investigated the effectiveness of orally administered pomegranate peel extract (PPE), a strong antioxidant, as a protective agent against AMK-induced ototoxicity. To this end, PPE was orally administered to adult BALB/c mice for 5 days, and the mice were then concurrently treated with AMK (500 mg/kg/day for 15 consecutive days). Auditory threshold shifts induced by AMK were significantly attenuated. The results of immunohistochemical staining and western blot analysis revealed that PPE exerted its protective effects by by downregulating the phosphorylation of Forkhead box O3a (FoxO3a), an important transcription factor which is involved in the responses to oxidative stress. The results also showed that PPE treatment inhibited mitogen-activated protein kinase phosphorylation, prevented the activation of pro-apoptotic protein caspase-3, decreased the levels of apoptosis-inducing Bax protein, and increased the levels of the anti-apoptotic mediator, Bcl-2, induced by AMK in the mouse cochlea. Taken together, our experimental findings suggest that phosphorylated FoxO3a mediates AMK-induced apoptosis in BALB/c mice cochlea. PPE effectively attenuated oxidative stress and ototoxicity by regulating FoxO3a, and may thus prove to be beneficial in protecting auditory cells from ototoxic drugs.

AMPK Mediates Glucocorticoids Stress-Induced Downregulation of the Glucocorticoid Receptor in Cultured Rat Prefrontal Cortical Astrocytes

Chronic stress induces altered energy metabolism and plays important roles in the etiology of depression, in which the glucocorticoid negative feedback is disrupted due to imbalanced glucocorticoid receptor (GR) functions. The mechanism underlying the dysregulation of GR by chronic stress remains elusive. In this study, we investigated the role of AMP-activated protein kinase (AMPK), the key enzyme regulating cellular energy metabolism, and related signaling pathways in chronic stress-induced GR dysregulation. In cultured rat cortical astrocytes, glucocorticoid treatment decreased the level, which was accompanied by the decreased expression of liver kinase B1 (LKB1) and reduced phosphorylation of AMPK. Glucocorticoid-induced effects were attenuated by glucocorticoid-inducible kinase 1 (SGK1) inhibitor GSK650394, which also inhibited glucocorticoid induced phosphorylation of Forkhead box O3a (FOXO3a). Furthermore, glucocorticoid-induced down-regulation of GR was mimicked by the inhibition of AMPK and abolished by the AMPK activators or the histone deacetylase 5 (HDAC5) inhibitors. In line with the role of AMPK in GR expression, AMPK activator metformin reversed glucocorticoid-induced reduction of AMPK phosphorylation and GR expression as well as behavioral alteration of rats. Taken together, these results suggest that chronic stress activates SGK1 and suppresses the expression of LKB1 via inhibitory phosphorylation of FOXO3a. Downregulated LKB1 contributes to reduced activation of AMPK, leading to the dephosphorylation of HDAC5 and the suppression of transcription of GR.

Cdc2-like kinase 2 is a key regulator of the cell cycle via FOXO3a/p27 in glioblastoma.

Cdc2-like kinase 2 (CLK2) is known as a regulator of RNA splicing that ultimately controls multiple physiological processes. However, the function of CLK2 in glioblastoma progression has not been described. Reverse-phase protein array (RPPA) was performed to identify proteins differentially expressed in CLK2 knockdown cells compared to controls. The RPPA results indicated that CLK2 knockdown influenced the expression of survival-, proliferation-, and cell cycle-related proteins in GSCs. Thus, knockdown of CLK2 expression arrested the cell cycle at the G1 and S checkpoints in multiple GSC lines. Depletion of CLK2 regulated the dephosphorylation of AKT and decreased phosphorylation of Forkhead box O3a (FOXO3a), which not only translocated to the nucleus but also increased p27 expression. In two glioblastoma xenograft models, the survival duration of mice with CLK2-knockdown GSCs was significantly longer than mice with control tumors. Additionally, tumor volumes were significantly smaller in CLK2-knockdown mice than in controls. Knockdown of CLK2 expression reduced the phosphorylation of FOXO3a and decreased Ki-67 in vivo. Finally, high expression of CLK2 protien was significantly associated with worse patient survival. These findings suggest that CLK2 plays a critical role in controlling the cell cycle and survival of glioblastoma via FOXO3a/p27.

Apelin induces vascular smooth muscle cells migration via a PI3K/Akt/FoxO3a/MMP-2 pathway

Apelin is an adipokine that has a critical role in the development of atherosclerosis, which may offer potential for therapy. Because migration of vascular smooth muscle cells (VSMCs) is a key event in the development of atherosclerosis, understanding its effect on the atherosclerotic vasculature is needed. Here we investigated the effect of apelin on VSMC migration and the possible signaling mechanism. In cultured rat VSMCs, apelin dose- and time-dependently promoted VSMC migration. Apelin increased the phosphorylation of Akt, whereas LY294002, an inhibitor of phosphatidylinositol 3-kinase (PI3K), and an Akt1/2 kinase inhibitor blocked the apelin-induced VSMC migration. Apelin dose-dependently induced phosphorylation of Forkhead box O3a (FoxO3a) and promoted its translocation from the nucleus to cytoplasm, which were blocked by LY294002 and Akt1/2 kinase inhibitor. Furthermore, apelin increased matrix metalloproteinase 2 (MMP-2) expression and gelatinolytic activity. Overexpression of a constitutively active, phosphorylation-resistant mutant, TM-FoxO3a, in VSMCs abrogated the effect of apelin on MMP-2 expression and VSMC migration. ARP101, an inhibitor of MMP-2, suppressed apelin-induced VSMC migration. Moreover, the levels of apelin, phosphorylated Akt, FoxO3a, and MMP-2 were higher in human carotid-artery atherosclerotic plaque than in adjacent normal vessels. We demonstrate that PI3K/Akt/FoxO3a signaling may be involved in apelin inducing VSMC migration. Phosphorylation of FoxO3a plays a central role in mediating the apelin-induced MMP-2 activation and VSMC migration.

Paclitaxel suppresses the viability of breast tumor MCF7 cells through the regulation of EF1α and FOXO3a by AMPK signaling.

Paclitaxel (Taxol), a potent drug of natural origin isolated from the bark of the Pacific yew, is widely used for treating ovarian, lung and breast cancers. Currently, there is little information regarding the specific mechanism underlying the anticancer activity of paclitaxel. In the present study, we found that 5-amino-1-β-D-ribofuranosyl-imidazole-4-carboxamide (AICAR), a well-known activator of adenosine monophosphate (AMP)-activated protein kinase (AMPK), downregulated the protein and mRNA levels of elongation factor 1 α (EF1α) in breast cancer MCF7 cells. Paclitaxel increased the phosphorylation of AMPK and also downregulated the expression of EF1α in MCF7 cells. In addition, paclitaxel increased the expression, as well as the phosphorylation of forkhead box O3a (FOXO3a). Phosphorylation of FOXO3a was suppressed in the presence of compound C, a specific AMPK inhibitor, suggesting the involvement of AMPK in paclitaxel-induced FOXO3a phosphorylation. The induction and phosphorylation of FOXO3a by paclitaxel were not observed in EF1α and AMPK knockdown cells. Co-treatment with AICAR resulted in increased susceptibility of cancer cells to paclitaxel-induced suppression of their viability and further enhanced paclitaxel-induced FOXO3a phosphorylation. These results suggest that the antitumor effects of paclitaxel in breast cancer are mediated by activation of the AMPK/EF1α/FOXO3a signaling pathway.

Markers of autophagy are adapted to hyperglycaemia in skeletal muscle in type 2 diabetes.

Autophagy is a catabolic process that maintains cellular homeostasis by degradation of protein aggregates and selective removal of damaged organelles, e.g. mitochondria (mitophagy). Insulin resistance in skeletal muscle has been linked to mitochondrial dysfunction and altered protein metabolism. Here, we investigated whether abnormalities in autophagy are present in human muscle in obesity and type 2 diabetes. Using a case-control design, skeletal muscle biopsies obtained in the basal and insulin-stimulated states from patients with type 2 diabetes during both euglycaemia and hyperglycaemia, and from glucose-tolerant lean and obese individuals during euglycaemia, were used for analysis of mRNA levels, protein abundance and phosphorylation of autophagy-related proteins. Muscle transcript levels of autophagy-related genes (ULK1, BECN1, PIK3C3, ATG5, ATG7, ATG12, GABARAPL1, MAP1LC3B, SQSTM1, TP53INP2 and FOXO3A [also known as FOXO3]), including some specific for mitophagy (BNIP3, BNIP3L and MUL1), and protein abundance of autophagy-related gene (ATG)7 and Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNIP3), as well as content and phosphorylation of forkhead box O3A (FOXO3A) were similar among the groups. Insulin reduced lipidation of microtubule-associated protein light chain 3 (LC3)B-I to LC3B-II, a marker of autophagosome formation, with no effect on p62/sequestosome 1 (SQSTM1) content in muscle of lean and obese individuals. In diabetic patients, insulin action on LC3B was absent and p62/SQSTM1 content increased when studied under euglycaemia, whereas the responses of LC3B and p62/SQSTM1 to insulin were normalised during hyperglycaemia. Our results demonstrate that the levels of autophagy-related genes and proteins in muscle are normal in obesity and type 2 diabetes. This suggests that muscle autophagy in type 2 diabetes has adapted to hyperglycaemia, which may contribute to preserve muscle mass.

Differential Activation of Signaling Pathways by Low‐Osmolar and Iso‐Osmolar Radiocontrast Agents in Human Renal Tubular Cells

Radiocontrast media (RCM)-induced nephrotoxicity (CIN) is a major clinical problem accounting for 12% of all hospital-acquired cases of acute kidney injury (AKI). The pathophysiology of AKI due to RCM is not well understood, but direct toxic effects on renal cells have been postulated as contributing to CIN. It is believed that iso-osmolar RCM (IOCM) are less nephrotoxic than low-osmolar RCM (LOCM) but clinical data have been controversial. We have investigated the intracellular signaling pathways that may be affected by the LOCM iomeprol (IOM) and the IOCM iodixanol (IOD). Both IOM and IOD caused a dramatic decrease in phosphorylation of the kinase Akt at Ser473 and Thr308 in human renal tubular (HK-2) cells, with IOM having a greater effect; IOM also caused a greater decrease in cell viability. IOM also had a greater effect on phosphorylation of p38 MAP kinases, JNKs, and NF-kB (Ser276), and caused a marked decrease in the phosphorylation of forkhead box O3a (FOXO3a) and signal transducer and activator of transcription 3 (STAT3). However, IOD caused a greater decrease in the phosphorylation of mTOR (Ser2448) and phospho-ERK 1/2 while both RCM caused a similar decrease in the phosphorylation of phospho-p70S6 kinase (Ser371). In vivo studies showed that both IOM and IOD caused a significant decrease in both pAkt (Ser473) and pERK 1/2 in rat kidneys. Our study gives an insight into the possible mechanism of toxicity of RCM via their action on intracellular signaling pathways and may help in developing pharmacological interventions for their side-effects.

IGF-1-Induced Enhancement of PRNP Expression Depends on the Negative Regulation of Transcription Factor FOXO3a

The conformational conversion of the cellular prion protein (PrPC) into its β-sheet-rich scrapie isoform (PrPSc) causes fatal prion diseases, which are also called transmissible spongiform encephalopathies (TSEs). Recent studies suggest that the expression of PrPC by the PRNP gene is crucial for the development of TSEs. Therefore, the identification of the exogenous and endogenous stimulating factors that regulate PRNP expression would help to understand the pathogenesis of TSEs. Here, we demonstrate that forkhead box O3a (FOXO3a) negatively regulates PRNP expression by binding to the PRNP promoter, which is negatively regulated by insulin-like growth factor 1 (IGF-1). Our results show that the IGF-1-induced enhancement of PRNP mRNA and protein levels is due to the activation of the PI3K-Akt signaling pathway. The activation of Akt then induces the phosphorylation of FOXO3a, leading to its translocation from the nucleus to the cytoplasm and preventing its binding to the PRNP promoter. Treatment with the PI3K-Akt inhibitor LY294002 induces the nuclear retention of FOXO3a, which leads to a decrease in PRNP expression. We present a new IGF-1-PI3K-Akt-FOXO3a pathway, which influences PRNP expression. The results of this work are vital for understanding the function of PrPC and for future therapeutic approaches to human TSEs.

Aldose Reductase Inhibition Prevents Colon Cancer Growth by Restoring Phosphatase and Tensin Homolog Through Modulation of miR-21 and FOXO3a

We have shown earlier that inhibition of aldose reductase (AR), an oxidative stress-response protein, prevents colon cancer cell growth in vitro and in vivo. Changes in microribonucleic acid (miR) expression can contribute to cancer by modulating the functional expression of critical genes involved in cancer growth and metastasis. However, the molecular mechanisms by which AR regulates miR expression and their dependent mitogenic effects in cancer cells are not known. Therefore, we investigated how AR regulates growth factor-induced expression of miRs and growth of colon cancer cells. Inhibition of AR significantly downregulated growth factor-induced miR-21 expression in human colon cancer cells, HT29, SW480, and Caco-2. Further, AR inhibition also increased phosphatase and tensin homolog (PTEN) (a direct target of miR-21) and forkhead box O3A (FOXO3a) in colon cancer cells. Our results obtained with HT29 cells ablated with FOXO3a siRNA showed increased activator protein-1 (AP-1) activation and miR-21 expression, indicating that FOXO3a represses miR-21 via AP-1 inactivation. Inhibition of AR also prevented the epidermal growth factor-induced phosphorylation of phosphatidylinositol 3-kinase (PI3K), serine/threonine kinase (AKT), c-Jun, c-Fos, PTEN, and FOXO3a, and deoxyribonucleic acid (DNA)-binding activity of AP-1. More importantly, in human colon adenocarcinoma xenograft tissues, miR-21 expression was lower, and PTEN and FOXO3a levels were significantly higher in AR inhibitor-treated mice compared to controls. These findings demonstrate a novel role of AR in the regulation of miR-21 and its target PTEN in growth factor-induced colon cancer cell growth. Collectively, these results show a novel role of AR in mediation of growth factor-induced colon cancer growth by modulating miR-21, PTEN, and FOXO3a expression through reactive oxygen species (ROS)/PI3K/AKT/AP-1.

Regulatory effects of curcumin on FOXO3a in THP‐1 monocytes/macrophages

Forkhead box O3a (FOXO3a) is a transcription factor centrally involved in regulating several stress resistance and lipid transport genes. Here we investigated whether the dietary curcumin, a polyphenol in turmeric spice, can modulate FOXO3a activity and the accumulation of lipids in monocytes/macrophages, a process important in atherogenesis. Curcumin in vitro increased the expression of two lipid transport genes, the fatty acids transporter CD36/FAT and the fatty acids binding protein 4 (FABP4/aP2), leading to increased lipid levels in THP‐1 monocytes and macrophages. Curcumin increased FOXO3a activity by 2‐fold, as measured using a FOXO3a‐luciferase reporter gene, possibly as a result of influencing FOXO3a phosphorylation and nuclear translocation. In contrast to the in vitro results, supplementing the high fat diet of LDL receptor knockout mice with curcumin for four months reduced the expression of CD36, aP2, and ATP binding cassette transporter A1 (ABCA1) expression, and lowered the lipid levels in peritoneal macrophages, suggesting involvement of additional regulatory mechanisms in vivo. Thus, the modulation of FOXO3a activity by curcumin could be a mechanism to modulate lipid‐induced stress in the inflammatory cells of the vascular system and reduce the risk for cardiovascular disease. Supported by USDA Contract #58‐1950‐7‐707, and by NIFA/USDA Contract #2010‐65200‐20395.

Regulatory effects of curcumin on lipid accumulation in monocytes/macrophages

Recent evidence suggests potential benefits from phytochemicals and micronutrients in protecting against atherosclerosis and inflammation, but the molecular mechanisms of these actions are still unclear. Here, we investigated whether the dietary polyphenol curcumin can modulate the accumulation of lipids in monocytes/macrophages. Curcumin increased the expression of two lipid transport genes, the fatty acids transporter CD36/FAT and the fatty acids binding protein 4 (FABP4/aP2; P < 0.05), leading to increased lipid levels in THP-1 and RAW264.7 monocytes and macrophages (P < 0.05). To investigate the molecular mechanisms involved, we assessed the activity of Forkhead box O3a (FOXO3a), a transcription factor centrally involved in regulating several stress resistance and lipid transport genes. Curcumin increased FOXO3a-mediated gene expression by twofold (P < 0.05), possibly as a result of influencing FOXO3a phosphorylation and nuclear translocation. The curcumin derivative, tetrahydrocurcumin (THC), with similar chemical antioxidant activity as curcumin, did not show any measurable effects. In contrast to the in vitro results, curcumin showed a trend for reduction of lipid levels in peritoneal macrophages in LDL receptor knockout mice fed a high fat diet for 4 months, suggesting additional regulatory mechanisms in vivo. Thus, the up-regulation of FOXO3a activity by curcumin could be a mechanism to protect against oxidant- and lipid-induced damage in the inflammatory cells of the vascular system.

Mitochondrial Oxidative Stress in Aortic Stiffening With Age

Age-related aortic stiffness is an independent risk factor for cardiovascular diseases. Although oxidative stress is implicated in aortic stiffness, the underlying molecular mechanisms remain unelucidated. Here, we examined the source of oxidative stress in aging and its effect on smooth muscle cell (SMC) function and aortic compliance using mutant mouse models. Pulse wave velocity, determined using Doppler, increased with age in superoxide dismutase 2 (SOD2)+/- but not in wild-type, p47phox-/- and SOD1+/- mice. Echocardiography showed impaired cardiac function in these mice. Increased collagen I expression, impaired elastic lamellae integrity, and increased medial SMC apoptosis were observed in the aortic wall of aged SOD2+/- versus wild-type (16-month-old) mice. Aortic SMCs from aged SOD2+/- mice showed increased collagen I and decreased elastin expression, increased matrix metalloproteinase-2 expression and activity, and increased sensitivity to staurosporine-induced apoptosis versus aged wild-type and young (4-month-old) SOD2+/- mice. Smooth muscle α-actin levels were increased with age in SOD2+/- versus wild-type SMCs. Aged SOD2+/- SMCs had attenuated insulin-like growth factor-1-induced Akt and Forkhead box O3a phosphorylation and prolonged tumor necrosis factor-α-induced Jun N-terminal kinase 1 activation. Aged SOD2+/- SMCs had increased mitochondrial superoxide but decreased hydrogen peroxide levels. Finally, dominant-negative Forkhead box O3a overexpression attenuated staurosporine-induced apoptosis in aged SOD2+/- SMCs. Mitochondrial oxidative stress over a lifetime causes aortic stiffening, in part by inducing vascular wall remodeling, intrinsic changes in SMC stiffness, and aortic SMC apoptosis.

Alleviating allergic airway diseases by means of short-term administration of IL-2 and dexamethasone

IL-2 combined with dexamethasone can upregulate regulatory T (Treg) cells, but the mechanism is still under exploration. Although previous studies focused on upregulating Treg cells in normal mice, here we investigated whether the IL-2 and dexamethasone combination treatment can upregulate Treg cells in pathological conditions, specifically in alleviating allergic airway disease. We also examined the potential pathway involved in Treg cell upregulation by IL-2 and dexamethasone. We evaluated the dose of IL-2 and dexamethasone required to upregulate Treg cells in vivo and in vitro. We also tested IL-2 and dexamethasone in the intervention of allergic airway disease in a murine model. We found that administration of 400,000 IU of IL-2 and 0.1 mg of dexamethasone per mouse was effective in upregulating Treg cells, as well as in alleviating allergic airway disease in an established animal model, but this phenomenon disappeared after anti-CD25 antibody administration. We discovered that an in vitro low dose of IL-2 can protect Treg cells did not protect CD4(+)CD25(-) cells from dexamethasone-induced apoptosis by affecting forkhead box O3a phosphorylation through the Akt and serum and glucocorticoid-induced protein kinase pathways. IL-2/dexamethasone treatment can alleviate existing allergic airway diseases by upregulating Treg cells in vivo. Alow dose of IL-2 (10(-9) to 10(-11) mol/L) can protect Treg cells but not CD4(+)CD25(-) cells from dexamethasone-induced apoptosis in vitro, thereby explaining a possible mechanism of increased proportion of Treg cells.