Localization Of FOXO3 Research Articles

FOXO3 and PTEN expression in the ovary of girls with extra-gonadal cancer with or without chemotherapy treatment prior to cryopreservation

BackgroundFOXO3/pFOXO3 and PTEN expression is known to regulate the dormancy/activation of ovarian primordial follicles. How chemotherapy could influence the expression of FOXO3 and PTEN in pre- and post-menarcheal girls with extra-gonadal cancer remains unexplored.MethodsOvarian samples were collected from 27 girls suffering from extra-gonadal cancer. Of these, 8 patients had received chemotherapy before the time of sample collection. Ovarian tissue collected at the time of surgery was fixed in 10% formaldehyde for FOXO3/pFOXO3 and PTEN immunohistochemistry or immunofluorescence, or stored at -80 °C for Western blot, or preserved in RNA later for RT-PCR.ResultsPTEN was detected in a limited number of primordial follicle-enclosed oocytes in approximately fifty percent of the patients, regardless of whether they had received anti-cancer treatment or not. However, there was a significant decrease in PTEN detection in patients who underwent chemotherapy treatment prior to the retrieval of the sample. Both primordial follicle-enclosed oocytes that expressed FOXO3 and those that did not were identified in patients who were treated with chemotherapy and those who were not. FOXO3-positive primordial follicles exhibited either nuclear FOXO3 localization or cytoplasmic pFOXO3 localization. Furthermore, transitional primordial follicles that expressed nuclear FOXO3 and cytoplasmic pFOXO3 were also observed. Primary follicle-enclosed oocytes displayed cytoplasmic pFOXO3 localization, whereas in more advanced stages of folliculogenesis, the expression moved to the somatic stratum. No significant statistical differences were identified in the detection of FOXO3 and pFOXO3 in patients who had or had not received chemotherapy prior to sample collection.ConclusionPrimordial follicles expressing and not expressing FOXO3 were equally present in both the ovaries of patients who underwent chemotherapy and those who did not. The expression of FOXO3 remained unaltered in response to chemotherapy treatment. Notably, the detection of PTEN was significantly reduced in the treated patients, thereby warranting in-depth investigation, given the limited sample size examined in the present study.

Cellular Localization of FOXO3 Determines Its Role in Cataractogenesis

The transcription factor FOXO3 is a core regulator of cellular homeostasis, stress response, and longevity. The cellular localization of FOXO3 is closely related to its function. Here, we explored the role of FOXO3 in cataract formation. FOXO3 showed nuclear translocation in lens epithelial cells arranged in a single layer on lens capsule tissues from both human cataract and N-methyl-N-nitrosourea (MNU)-induced rat cataract, also in MNU-injured human lens epithelial cell lines (HLECs). FOXO3 knockdown inhibited the MNU-induced increase in expression of genes related to cell cycle arrest (GADD45α and CCNG2) and apoptosis (Bak and TP53). H2 is highly effective in reducing oxidative impairments in nuclear DNA and mitochondria. When we applied H2 to MNU-injured HLECs, FOXO3 underwent cleavage by MAPK1 and translocated into mitochondria, thereby increasing the transcription of oxidative phosphorylation-related genes (MTCO1, MTCO2, MTND1, and MTND6) in HLECs. Furthermore, H2 mediated the translocation of FOXO3 from the nucleus to the mitochondria within the lens epithelial cells of cataract capsule tissues of rats exposed to N-methyl-N-nitrosourea (MNU). This intervention ameliorated MNU-induced cataracts in the rat model. In conclusion, we demonstrated the correlation between the localization of FOXO3 and its function in cataract formation. We also determined that H2 protects HLECs from injury by leading FOXO3 mitochondrial translocation via MAPK1 activation. Mitochondrial FOXO3 can increase mtDNA transcription, stabilize mitochondrial function in HLECs.

MTORC2 Is the Major Second Layer Kinase Negatively Regulating FOXO3 Activity.

Forkhead box O (FOXO) proteins are transcription factors involved in cancer and aging and their pharmacological manipulation could be beneficial for the treatment of cancer and healthy aging. FOXO proteins are mainly regulated by post-translational modifications including phosphorylation, acetylation and ubiquitination. As these modifications are reversible, activation and inactivation of FOXO factors is attainable through pharmacological treatment. One major regulatory input of FOXO signaling is mediated by protein kinases. Here, we use specific inhibitors against different kinases including PI3K, mTOR, MEK and ALK, and other receptor tyrosine kinases (RTKs) to determine their effect on FOXO3 activity. While we show that inhibition of PI3K efficiently drives FOXO3 into the cell nucleus, the dual PI3K/mTOR inhibitors dactolisib and PI-103 induce nuclear FOXO translocation more potently than the PI3Kδ inhibitor idelalisib. Furthermore, specific inhibition of mTOR kinase activity affecting both mTORC1 and mTORC2 potently induced nuclear translocation of FOXO3, while rapamycin, which specifically inhibits the mTORC1, failed to affect FOXO3. Interestingly, inhibition of the MAPK pathway had no effect on the localization of FOXO3 and upstream RTK inhibition only weakly induced nuclear FOXO3. We also measured the effect of the test compounds on the phosphorylation status of AKT, FOXO3 and ERK, on FOXO-dependent transcriptional activity and on the subcellular localization of other FOXO isoforms. We conclude that mTORC2 is the most important second layer kinase negatively regulating FOXO activity.

Piceatannol Induces Fetal Hemoglobin in Erythroid Progenitor Cells from Patients with Sickle Cell Disease

Background: We have identified the FOXO3-AMPK-AKT pathway as important to HbF regulation through unbiased analysis of whole exome sequencing from patients with sickle cell disease (SCD). Both AMPK and AKT are activated by phosphorylation; AMPK is a FOXO3 activator; AKT inhibits AMPK and FOXO3. We have also treated hematopoietic stem and progenitor cells (HSPCs) from patients with SCD with metformin, a known AMPK/FOXO3 activator, and produced a significant rise in HbF from 17.6% at baseline, to 44.9%. This data is the foundation for an IRB approved clinical trial of metformin in patients with hemoglobinopathies, enrolling patients now.Objectives: We seek to investigate other drugs which act on the FOXO3-AMPK-AKT pathway as possible HbF inducing agents. Resveratrol and piceatannol are both known activators of the pathway; however, resveratrol is not bioavailable. We will investigate piceatannol as a HbF inducing agent, assess toxicity, and verify its action is via the FOXO3-AMPK-AKT pathway.Methods: HSPCs from 5 patients with SCD were treated with serial doses of piceatannol at 5, 12.5, 25, 50 and 100 µM on day 7 of two phase erythroid culture. Cell lysate was collected on day 14, and hemoglobin profile analyzed by HPLC. 2 µM Compound C was added with piceatannol to block AMPK activity, and the effect on HbF measured by HPLC. The effect of piceatannol on the amount of phosphorylated AMPK and AKT in HSPCs was measured by quantitative western blot in triplicate, and the effect of piceatannol on FOXO3 cellular localization was determined by fluorescent microscopy and fractionation of the cells into nuclear and cytoplasmic compartments, followed by quantitative western blot analysis.Results: Erythroid progenitor cells from 5 unique patients with SCD treated with 25 µM piceatannol all exhibited a 3-4 fold rise in HbF as measured by HPLC. Toxicity studies showed that 12.5-50 µM of piceatannol induced HbF without significantly altering CFU generation of human CD34+ HSPCs; treatment with 100 µM piceatannol showed a reduction in CFU. In the presence of Compound C, piceatannol induction of HbF was attenuated. 5-25 µM piceatannol markedly increased the amount of phosphorylated AMPK present in the cell, caused an increase in FOXO3 localization in the nucleus, and effectively inhibited AKT phosphorylation.Conclusions: Piceatannol induces HbF in SCD patient derived HSPCs. While piceatannol has been reported to have HDAC inhibitor activity, the fact that pharmacologic blockage of AMPK activity with Compound C results in reduction of HbF induction by piceatannol supports the hypothesis that piceatannol acts through the FOXO3-AMPK-AKT pathway. Additionally, piceatannol reduced phosphorylation of a known FOXO3 inhibitor, AKT, and increased phosphorylation of AMPK, a FOXO3 activator, thereby increasing FOXO3 activity and localization to the nucleus. With further safety studies, piceatannol could be a novel HbF inducing agent for patients with hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

Abstract 17034: The AKT/AMPK/FOXO3 Axis in Pulmonary Arterial Hypertension

Like cancer, pulmonary arterial hypertension (PAH) is characterized by exaggerated proliferation and resistance to apoptosis related to metabolic alterations (Warburg effect) of pulmonary smooth muscle cells (PASMCs). These anomalies result in a progressive narrowing of the pulmonary arteries, increasing pulmonary resistance and leading to right heart failure and premature death. In cancer cells, unphosphorylated and nuclear FOXO3 has been extensively studied as a crucial protein that functions as a tumor suppressor by regulating expression of genes involved in apoptosis and cell cycle arrest. These functions combined with other FOXO3 attributes, including its key role in communicating mitochondrial-nuclear signals, make the FOXO3 a suitable candidate for controlling the cancer-like phenotype of PAH-PASMCs. Interestingly, AKT and AMPK known to be implicated in PAH exert antagonistic effects on FOXO3; AKT promoting its nuclear exclusion while AMPK favors its nuclear and mitochondrial accumulation. The thus made the hypothesis that FOXO3’s nuclear exclusion (secondary to AKT/AMPK imbalance) promotes metabolic reprogramming towards glycolysis leading to enhanced proliferation/resistance to apoptosis of PAH-PASMCs and vascular remodeling. Using Western blot and immunofluorescence in isolated PASMCs from both PAH and control patients (n=10), we found that nuclear and mitochondrial exclusion of FOXO3 due to its phosphorylation is a feature of PAH-PASMCs. In vitro, we demonstrated that nuclear localization of FOXO3 using an adenovirus expressing a constitutively active, non-phosphorylable form of FOXO3 or trifluoperazine (TFP) resulted in reduced PAH-PASMC proliferation (Ki67 labeling, p<0,0005) and resistance to apoptosis (Annexin V assay, p<0,05). These effects were accompanied by increased expression of P27 and SOD2 and diminished expression of Survivin (p<0,05). In vivo, we showed that FOXO3 activation using TPF improved established PAH in the monocrotaline rats (reduced RVSP and increased Sv and CO, by right catheterization, p<0,01, n=29) without any sign of toxicity. We showed that FOXO3 is implicated in pulmonary vascular remodeling. Pharmacological activation of FOXO3 may represent a novel avenue to improve PAH.

PTEN and FOXO3 expression in the prenatal and postnatal human ovary

The objective of this study was to analyse the expression and cellular localization of FOXO3, pFOXO3 and PTEN throughout human ovary development both before and after birth. Foetal, pubertal and adult paraffin-embedded ovarian samples were analysed by immunohistochemistry for cellular localization of FOXO3, pFOXO3 and PTEN proteins. Protein and mRNA expression were analysed by western blot and real time PCR, respectively, from fresh biopsies. PTEN was not detected by immunohistochemistry in germ cells and follicles of foetal, pubertal and adult ovaries. Occasional PTEN immunoreactive granulosa cells were found in atretic antral follicles in the adult ovary. Western blot analysis showed low levels of PTEN protein. Nuclear FOXO3-expressing primordial follicles represented a variable proportion of the ovarian reserve. The presence of FOXO3-expressing primordial follicles was very low in foetal ovary; although always represented in a low proportion, prevalence increased during pubertal and adult life. Our results seem to indicate that two subpopulations of primordial follicles, i.e. nuclear FOXO3-expressing and no FOXO3-expressing primordial follicles are found in the postnatal human ovary. This scenario suggests that FOXO3 could be acting as in the mouse model, preventing primordial follicle activation. However, the strategy would not be an "all or nothing" system as in mouse ovary but rather a selected subpopulation of primordial follicles preserved to ensure long-term fertility.

Torin2 Exploits Replication and Checkpoint Vulnerabilities to Cause Death of PI3K-Activated Triple-Negative Breast Cancer Cells.

Frequent mutation of PI3K/AKT/mTOR signaling pathway genes in human cancers has stimulated large investments in targeted drugs but clinical successes are rare. As a result, many cancers with high PI3K pathway activity, such as triple-negative breast cancer (TNBC), are treated primarily with chemotherapy. By systematically analyzing responses of TNBC cells to a diverse collection of PI3K pathway inhibitors, we find that one drug, Torin2, is unusually effective because it inhibits both mTOR and other PI3K-like kinases (PIKKs). In contrast to mTOR-selective inhibitors, Torin2 exploits dependencies on several kinases for S-phase progression and cell-cycle checkpoints, thereby causing accumulation of single-stranded DNA and death by replication catastrophe or mitotic failure. Thus, Torin2 and its chemical analogs represent a mechanistically distinct class of PI3K pathway inhibitors that are uniquely cytotoxic to TNBC cells. This insight could be translated therapeutically by further developing Torin2 analogs or combinations of existing mTOR and PIKK inhibitors.

Akt Inhibition Differently Controls PRC2 Components and Synergizes with Dual EZH2/1 Inhibitor in the Treatment of Multiple Myeloma

PI3K/Akt pathway is constitutively activated in multiple myeloma (MM). A plethora of studies extensively investigated Akt inhibitors, alone or in combination; however, the outcomes in hematological malignancies were largely unsatisfactory, emphasizing the need for critical preclinical evaluations. Polycomb repressive complex 2 (PRC2) components, EZH2 and its related homolog EZH1, induce H3K27me3 to silence the transcription of target genes. Recent studies ensured that EZH2 inhibition alone is not sufficient to completely disrupt the oncogenic functions of PRC2. With the importance of PRC2 as a therapeutic target in MM, we aimed to investigate the mechanisms by which Akt inhibition may impact PRC2 function, and test whether targeting both EZH2 and EZH1 together with Akt inhibition is a promising treatment strategy for MM. We herein evaluated the cytotoxic effect of TAS-117, a potent and selective non-competitive Akt inhibitor, against different MM cell lines and found that responsive cell lines tended to have significant levels of activated Akt, coupled with low/deleted PTEN. Then, we examined signaling-epigenetic crosstalk on EZH2 level. TAS-117 significantly down-regulated EZH2 mRNA and protein in dose- and time-dependent manners, while H3K27me3 levels were rather maintained or elevated, suggesting compensation by EZH1. As EZH2 is a direct target for E2F1, we focused on Rb-E2F pathway as a regulatory mechanism for EZH2. TAS-117 induced marked down-regulation of E2F1 and E2F2. Moreover, TAS-117 induced the up-regulation of CDKN1B, in addition to the inactivation of cyclins and cyclin dependent kinases, hence, hypo-phosphorylated Rb, thereby stabilizing Rb-E2F1 complex and diminishing free E2F1 available for binding to its target genes, including EZH2 promoter. This prompted us to examine the impact of TAS-117 combination with either dual EZH2/1 inhibitor, UNC1999, or selective EZH2 inhibitor, GSK126. In agreement, UNC1999, but not GSK126, synergistically enhanced TAS-117-induced cytotoxicity, confirmed by combination index calculation, and provoked MM cell apoptosis. As we observed an increase in H3K27me3 levels after TAS-117 treatment, we hypothesized that EZH1 function was augmented. Consistently, we found that EZH1 was markedly up-regulated after TAS-117 treatment in dose- and time-dependent manners. Importantly, EZH1 knockdown significantly enhanced the sensitivity of myeloma cells to TAS-117-induced cytotoxicity. To clarify the molecular mechanisms underlying EZH1 up-regulation, we performed RNA-seq followed by KEGG pathway analysis for up-regulated genes in TAS-117-treated group. We focused on FOXO pathway enrichment as it is a crucial target in MM treatment using Akt inhibitors. We then focused on FOXO3 as it was the main FOXO family gene expressed in MM cells according to our RNA-seq data. We examined the nuclear localization of FOXO3 following TAS-117 treatment. We found that TAS-117 significantly enhanced the nuclear accumulation of FOXO3, as depicted by both the immunostaining images and the digital calculations of the nuclear subset of FOXO3. Murine Ezh1 promoter was shown to be bound by Foxo transcription factors (TFs) in neuronal progenitors, T-regulatory cells, CD8+ cells, and pre-B cells. More than 80% of FOXO3-binding sites share the common binding motif, GTAAACAA, which was found both in human EZH1 (+48 from the TSS) and mouse Ezh1 (+77 from the TSS) promoter regions. So, we hypothesized that FOXO3 may be a regulatory partner for human EZH1 gene in myeloma cells in response to TAS-117 treatment. To this end, we performed ChIP-qPCR analysis for TAS-117-treated and -untreated cells. TAS-117 promoted the binding of FOXO3 to EZH1 promoter, in addition to one of the canonical FOXO3 targets, BIM promoter. To further confirm our results, we expressed shRNA against FOXO3 (shFOXO3) in MM cells which, interestingly, induced the down-regulation of EZH1 mRNA. In conclusion, the present results defined novel signaling-epigenetic crosstalk between PI3K/Akt pathway and PRC2 components, EZH2 and EZH1, and demonstrated that Akt inhibition can differently modulates EZH2 and EZH1 levels via Akt downstream effectors, E2F1 and FOXO3, respectively. Therefore, targeting both EZH2 and EZH1 in addition to Akt inhibition may be a promising rationale to eradicate MM, leading to significant advances in treatment. Disclosures No relevant conflicts of interest to declare.

FOXO3 Expression in the Human Ovary [6OP

INTRODUCTION: To assess FOXO3 gene expression levels and members of the KL-PI3K-AKT-FOXO3 pathway in pre- and postmenopausal human ovaries. To evaluate in a temporal manner protein localization of FOXO3 in pre- and postmenopausal human ovaries by immunohistochemistry (IHC). METHODS: Total RNA was isolated from human ovary collected from pre- and postmenopausal patients undergoing oophorectomy and used for real-time qPCR for members of the KL-PTEN-PI3K-FOXO3 pathway, as well as other genes. IHC was performed on archived paraffin embedded ovaries to visualize the presence and localization of FOXO3 in human ovaries ranging from fetal to menopausal. RESULTS: FOXO3 is expressed in premenopausal human ovary and to a significantly reduced level in postmenopausal ovary. Nine additional genes in the KL-PTEN-PI3K-FOXO3 pathway were also expressed in premenopausal ovaries but like FOXO3 displayed significantly decreased levels in the postmenopausal cohort. IHC analysis revealed strong nuclear FOXO3 staining in primordial follicles from pre-pubertal ovaries through menarche/post-pubertal. A dominant population of primordial follicles were identified in older post-pubertal ovaries and in the reproductive/premenopausal groups that displayed light to negative nuclear staining for FOXO3, with a second much less abundant population of primordial follicles retaining strong positive nuclear FOXO3 staining. Postmenopausal ovaries were devoid of follicles but had strong stromal FOXO3 staining. CONCLUSION: As predicted from mouse genetic models, FOXO3 mRNA and protein is present in premenopausal human ovary as are nine members of the KL- PI3K-AKT-FOXO3 pathway. FOXO3 localization in the human ovary undergoes an age-dependent transition from strongly positive nuclei in young ovaries to predominantly light to negative staining in reproductive/premenopausal. Given FOXO3's importance to genome stability, primordial follicles with high nuclear FOXO3 levels may define high quality oocytes lacking detrimental levels of DNA damage, so that the ratio of FOXO3 positive to negative oocytes may explain the consequences of decreasing ovarian reserve.

Carbocysteine counteracts the effects of cigarette smoke on cell growth and on the SIRT1/FoxO3 axis in bronchial epithelial cells

BackgroundCigarette smoke may accelerate cellular senescence by increasing oxidative stress. Altered proliferation and altered expression of anti-aging factors, including SIRT1 and FoxO3, characterise cellular senescence. The effects of carbocysteine on the SIRT1/FoxO3 axis and on downstream molecular mechanisms in human bronchial epithelial cells exposed to cigarette smoke are largely unknown. AimsAim of this study was to explore whether carbocysteine modulated SIRT1/FoxO3 axis, and downstream molecular mechanisms associated to cellular senescence, in a bronchial epithelial cell line (16-HBE) exposed to cigarette smoke. Methods16HBE cells were stimulated with/without cigarette smoke extracts (CSE) and carbocysteine. Flow cytometry and clonogenic assay were used to assess cell proliferation; western blot analysis was used for assessing nuclear expression of SIRT1 and FoxO3. The nuclear co-localization of SIRT1 and FoxO3 was assessed by fluorescence microscopy. Beta galactosidase (a senescence marker) and SIRT1 activity were assessed by specific staining and colorimetric assays, respectively. ChiP Assay and flow cytometry were used for assessing survivin gene regulation and protein expression, respectively. ResultsCSE decreased cell proliferation, the nuclear expression of SIRT1 and FoxO3 and increased beta galactosidase staining. CSE, reduced SIRT1 activity and FoxO3 localization on survivin promoter thus increasing survivin expression. In CSE stimulated bronchial epithelial cells carbocysteine reverted these phenomena by increasing cell proliferation, and SIRT1 and FoxO3 nuclear expression, and by reducing beta galactosidase staining and survivin expression. ConclusionsThe study shows for the first time that carbocysteine may revert some senescence processes induced by oxidative stress due to cigarette smoke exposure.

Regulation of FOXO3 subcellular localization by Kit ligand in the neonatal mouse ovary.

Foxo3 protein is required in the oocyte nucleus for the maintenance of primordial follicles in a dormant state. PI3K/AKT-dependent phosphorylation of Foxo3 leads to its relocalization to the cytoplasm and subsequent follicular activation. However, the nature of the upstream signals controlling Foxo3 activity and subcellular localization remains unknown. We aimed to study the in vitro effects of Kit ligand (stem cell factor) on the subcellular localization of Foxo3 in primordial follicles within the postnatal mouse ovary. This was an in vitro study using explants of intact neonatal mouse ovaries. The study was performed in laboratory animal facility and basic science research laboratory at a University Hospital. The animals used for this study were FVB mice. Neonatal FVB mice ovaries at postnatal day 7 (PD7) were harvested and incubated in culture medium (DMEM) at 37 °C and 5 % CO(2) for 60-90 min with (n = 3) or without (n = 3) Kit ligand at 150 ng/mL (8 nM). Similar experimental conditions were used to establish a dose-response curve for the effects of Kit ligand and assess the effects of imatinib (small molecule inhibitor of the Kit receptor). Immunofluorescence was used to identify the subcellular location of Foxo3 in oocytes. Proportions of cytoplasmic versus nuclear Foxo3 in primordial follicles were determined. Kit ligand treatment increased the cytoplasmic localization of Foxo3 from 40 % in the untreated ovaries to 74 % in the treated group (p = 0.007 in paired samples and p = 0.03 in unpaired samples). Furthermore, this effect was reversible with imatinib (p = 0.005). A dose-response curve for Kit ligand treatment showed that maximum effect was seen at 150 ng/mL. Kit ligand treatment in vitro increases the proportion of cytoplasmic Foxo3 in primordial follicles at PD7, lending support to the idea that Kit receptor/ligand controls Foxo3 activity in the context of primordial follicle activation.

The Role of Foxo3 in Leydig Cells.

PurposeFoxo3 in female reproduction has been reported to regulate proliferation of granulose cells that form follicles. There are no reports so far that discuss on the role of Foxo3 in males. This study was designed to outline the role of Foxo3 in the testes.Materials and MethodsTestes from mice at birth to postpartum week (PPW) 5 were isolated and examined for the expression of Foxo3 using immunostaining. To elucidate role of Foxo3 in Leydig cells, R2C cells were treated with luteinizing hormone (LH) and the phosphorylation of Foxo3. Testosterone and steroidogenic acute regulatory (StAR) protein levels were measured after constitutive active [triple mutant (TM)] human FOXO3 adenovirus was transduced and StAR promoter assay was performed.ResultsFoxo3 expression in the testicles started from birth and lasted until PPW 3. After PPW 3, most Foxo3 expression occurred in the nuclei of Leydig cells; however, at PPW 5, Foxo3 was expressed in both the nucleus and cytoplasm. When R2C cells were treated with luteinizing hormone, Foxo3 phosphorylation levels by AKT increased. After blocking the PI3K pathway, LH-induced phosphorylated Foxo3 levels decreased, indicating that LH signaling regulates Foxo3 localization. When active FOXO3-TM adenovirus was introduced into a Leydig tumor cell line, the concentrations of testosterone and StAR protein decreased. When FOXO3 and a StAR promoter vector were co-transfected into HEK293 cells for a reporter assay, FOXO3 inhibited the StAR promoter.ConclusionFOXO3 affects testosterone synthesis by inhibiting the formation of StAR protein. LH hormone, meanwhile, influences Foxo3 localization, mediating its function.

Regulation of foxo3 subcellular localization by kit ligand in the neonatal mouse ovary

Foxo3 protein in the oocyte nucleus is required for the maintenance of primordial follicles in a dormant state. PI3K/AKT-dependent phosphorylation of Foxo3 leads to its relocalization to the cytoplasm and subsequent follicular activation. However, the nature of the upstream signals controlling Foxo3 activity and subcellular localization remain unknown. We aimed to study the in vitro effects of Kit ligand (SCF) on the subcellular localization of Foxo3 in primordial follicles within the postnatal mouse ovary.

Saturated free fatty acids induce cholangiocyte lipoapoptosis.

Recent studies have identified a cholestatic variant of nonalcoholic fatty liver disease (NAFLD) with portal inflammation and ductular reaction. Based on reports of biliary damage, as well as increased circulating free fatty acids (FFAs) in NAFLD, we hypothesized the involvement of cholangiocyte lipoapoptosis as a mechanism of cellular injury. Here, we demonstrate that the saturated FFAs palmitate and stearate induced robust and rapid cell death in cholangiocytes. Palmitate and stearate induced cholangiocyte lipoapoptosis in a concentration-dependent manner in multiple cholangiocyte-derived cell lines. The mechanism of lipoapoptosis relied on the activation of caspase 3/7 activity. There was also a significant up-regulation of the proapoptotic BH3-containing protein, PUMA. In addition, palmitate-induced cholangiocyte lipoapoptosis involved a time-dependent increase in the nuclear localization of forkhead family of transcription factor 3 (FoxO3). We show evidence for posttranslational modification of FoxO3, including early (6 hours) deacetylation and dephosphorylation that coincide with localization of FoxO3 in the nuclear compartment. By 16 hours, nuclear FoxO3 is both phosphorylated and acetylated. Knockdown studies confirmed that FoxO3 and its downstream target, PUMA, were critical for palmitate- and stearate-induced cholangiocyte lipoapoptosis. Interestingly, cultured cholangiocyte-derived cells did not accumulate appreciable amounts of neutral lipid upon FFA treatment. Our data show that the saturated FFAs palmitate and stearate induced cholangiocyte lipoapoptosis by way of caspase activation, nuclear translocation of FoxO3, and increased proapoptotic PUMA expression. These results suggest that cholangiocyte injury may occur through lipoapoptosis in NAFLD and nonalcoholic steatohepatitis patients.

Docosahexaenoic acid prevents palmitate-induced activation of proteolytic systems in C2C12 myotubes

Saturated fatty acids like palmitate contribute to muscle atrophy in a number of conditions (e.g., type II diabetes) by altering insulin signaling. Akt is a key modulator of protein balance that inhibits the FoxO transcription factors (e.g., FoxO3) which selectively induce the expression of atrophy-inducing genes (atrogenes) in the ubiquitin–proteasome and autophagy–lysosome systems. Conversely, omega-3 polyunsaturated fatty acids have beneficial effects on insulin signaling and may preserve muscle mass. In an earlier report, the omega-3 fatty acid docosahexaenoic acid (DHA) protected myotubes from palmitate-induced atrophy; the mechanisms underlying the alterations in protein metabolism were not identified. This study investigated whether DHA prevents a palmitate-induced increase in proteolysis by restoring Akt/FoxO signaling. Palmitate increased the rate of protein degradation, while cotreatment with DHA prevented the response. Palmitate reduced the activation state of Akt and increased nuclear FoxO3 protein while decreasing its cytosolic level. Palmitate also increased the messenger RNAs (mRNAs) of two FoxO3 atrogene targets, the E3 ubiquitin ligase atrogin-1/MAFbx and the autophagy mediator Bnip3. DHA attenuated the effects of palmitate on Akt activation, FoxO3 localization and atrogene mRNAs. DHA, alone or in combination with palmitate and decreased the ratio of LC3B-II:LC3B-I protein as well as the rate of autophagosome formation, as indicated by reduced LC3B-II protein in the presence of 10 mmol/L methylamine, suggesting an independent effect of DHA on the macroautophagy pathway. These data indicate that palmitate induces myotube atrophy, at least in part, by activating multiple proteolytic systems and that DHA counters the catabolic effects of palmitate by restoring Akt/FoxO signaling.

Therapy-resistant acute lymphoblastic leukemia (ALL) cells inactivate FOXO3 to escape apoptosis induction by TRAIL and Noxa

Forkhead transcription factors (FOXO) are downstream targets of the phosphoinositol-3-kinase (PI3K) protein kinase B (PKB) signaling cascade and play a pivotal role in cell differentiation, cell cycle and apoptosis. We found that cells from prednisone-resistant T-acute lymphoblastic leukemia (T-ALL) patients showed cytoplasmic localization of FOXO3 in comparison to prednisone-sensitive patients suggesting its inactivation. To determine the impact of FOXO3, T-ALL cells were infected with a 4OH-tamoxifen-regulated, phosphorylation-independent FOXO3(A3)ERtm allele. After FOXO3-activation these cells undergo caspase-dependent apoptosis. FOXO3 induces the death ligand TRAIL and the BH3-only protein Noxa implicating extrinsic as well as intrinsic death signaling. Whereas dnFADD partially inhibited cell death, CrmA and dnBID efficiently rescued ALL cells after FOXO3 activation, suggesting a caspase-8 amplifying feedback loop downstream of FADD. Knockdown of TRAIL and Noxa reduced FOXO3-induced apoptosis, implicating that mitochondrial destabilization amplifies TRAIL-signaling. The-reconstitution of the cell cycle inhibitor p16INK4A, which sensitizes ALL cells to mitochondria-induced cell death, represses FOXO3 protein levels and reduces the dependency of these leukemia cells on PI3K-PKB signaling. This suggests that if p16INK4A is deleted during leukemia development, FOXO3 levels elevate and FOXO3 has to be inactivated by deregulation of the PI3K-PKB pathway to prevent FOXO3-induced cell death.

Lineage-specific growth inhibition of NK cell lines by FOXO3 in association with Akt activation status

FOXO3 and PRDM1 are located on 6q21, one of the most frequently deleted regions among natural killer (NK) cell neoplasms. We previously demonstrated that forced expression of each gene suppresses the proliferation of NK cell lines with the 6q deletion. In this study, the forced expression of FOXO3 or PRDM1 was performed in various cell lines to clarify these suppressive effects. Forced expression of PRDM1 suppressed the proliferation of not only NK cell lines, but also other broad lineage cell lines. On the other hand, forced expression of FOXO3 was only effective on NK cell lines. FOXO3 functions as a transcriptional factor when it is localized in nuclei. Akt is known to induce cytoplasmic localization of FOXO3 as a result of phosphorylation. Transduced FOXO3 was predominantly localized in nuclei of NK cell lines, while it was localized in the cytoplasm of all non-NK cell lines. NK cell lines showed significantly lower Akt activity compared to other lineage cell lines. The low Akt activity and nucleic localization of FOXO3 in NK cell neoplasms seemed to cause NK cell-specific suppression. These findings indicate the "functional lineage specificity" of FOXO3 and the possibility for NK cell-specific gene therapy with minimal unexpected effects.

Abstract P324: Unhealthy Diet and Air Pollution Compromise Human Cardiovascular Cell Functions: A First Mechanistic Analysis

Diet and pollution are environmental factors known to compromise ″healthy cardiovascular aging″. The molecular consequences of the permanent burden for aging of the cardiovascular system are unknown, since they have never been examined in primary, adult human cells. Therefore, this study investigates the impact of unhealthy diet on aging-related signaling pathways of human, primary cardiovascular cells and of airborne particles on human endothelial cells, as several studies demonstrated that ultrafine particles can enter the circulation and thus may interact with endothelial cells directly. Nutrition health reports have shown that the diet in industrialized countries contains more than 100 mg/dl low density lipoprotein (LDL) and a too high fraction of monosaccharides, especially fructose, which is metabolized insulin-independently. Both components have been shown to increase the risk for cardiovascular diseases. To simulate unhealthy diet we supplemented cell culture media of human, primary endothelial cells (EC), smooth muscle cells (SMC) and cardiomyocytes (CM) with 100 mg/dl LDL and replaced 1/3 of the glucose with fructose for one week. This treatment did not induce cell death in any of the cell types. However, we observed increased senescence, loss of endothelial nitric oxide synthase and increased nuclear localization of Foxo3 in EC, increased proliferation in SMC and hypertrophy in CM. With respect to pollution we have used ultrafine carbon black particles (ufCB), one of the major constituents of industrial and exhaust emissions, in concentrations our vessels are constantly exposed to. These concentrations of ufCB are non-toxic and non-inflammatory for EC. Despite these missing immediate effects, ufCB dramatically reduced the S-NO content, a marker for NO-bioavailability in EC and increased reactive oxgen species formation. As a consequence, ufCB dramatically increased senescence of EC after two weeks. Thus, unhealthy diet and a high burden of ultrafine carbon black nanoparticles, to which we are exposed every day, seem to induce a ″cardiovascular aging″ phenotype and can lead to severe cardiovascular diseases.

Ectopic localization of FOXO3a protein in Lewy bodies in Lewy body dementia and Parkinson's disease

Lewy bodies and Lewy neurites constitute the cardinal neuropathological features of both Parkinson's disease (PD) and Lewy body dementia (LBD). Whereas α-synuclein has been found to be the major component of the Lewy body, the mechanisms by which neurons degenerate, as well as basic mechanisms involved in the formation of α-synuclein-related inclusions, remain obscure. We have suggested previously that potential mechanisms are likely to leave a "molecular signature" or protein adduct within the Lewy body, and have found examples of such signatures in previous studies. In this study, we demonstrate increased FOXO3 in association with Lewy bodies and Lewy neurites in LBD and PD brain tissue. Since FOXO proteins are involved in several pathways responsible for the regulation of cell death, cell proliferation, and cell metabolism, the ectopic localization of FOXO3 to Lewy bodies provides evidence that aberrations in basic cellular biochemistry may contribute to inclusion formation, which is likely more complex than a simple "gain of function" toxicity as is commonly opined. In light of the known interaction of FOXO3 and 14-3-3, basic protein-protein interaction between these proteins and α-synuclein may be key.

Kit signaling via PI3K promotes ovarian follicle maturation but is dispensable for primordial follicle activation

In mammals, primordial follicles are generated early in life and remain dormant for prolonged intervals. Their growth resumes via a process known as primordial follicle activation. Recent genetic studies have demonstrated that phosphoinositide 3-kinase (PI3K) is the essential signaling pathway controlling this process throughout life, acting via Akt to regulate nucleocytoplasmic shuttling of Foxo3, which functions as a downstream molecular switch. The receptor tyrosine kinase Kit has been implicated by numerous studies as the critical upstream regulator of primordial follicle activation via PI3K/Akt. Here we present a genetic analysis of the contribution of Kit in regulating primordial follicle activation and early follicle growth, employing a knock-in mutation (KitY719F) that completely abrogates signaling via PI3K. Surprisingly, homozygous KitY719F female mice undergo primordial follicle activation and are fertile, demonstrating that Kit signaling via PI3K is dispensable for this process. However, other abnormalities were identified in KitY719F ovaries, including accelerated primordial follicle depletion and accumulation of morphologically abnormal primary/secondary follicles with persistent nuclear Foxo3 localization. These findings reveal specific roles of Kit in the maintenance of the primordial follicle reserve and in the primary to secondary follicle transition, but argue that Kit is dispensable in primordial follicle activation.