FOXO Transcriptional Activity Research Articles

Progesterone generates cancer stem cells through membrane progesterone receptor-triggered signaling in basal-like human mammary cells

Ionizing radiation and cumulative exposure to steroid hormones are known risk factors for breast cancer. There is increasing evidence that breast tumors are driven by a subpopulation of tumor-initiating cancer stem cells (CSCs). In MCF10A non-cancerous basal-like PR− cells, progesterone treatment and X-rays generated ALDH+ and CD44+/CD24− CSCs. Here, we report that in irradiated MCF10A cells, progesterone activated the PI3K/Akt pathway via membrane progesterone receptor (mPR). Inhibition of the PI3K/Akt pathway counteracted the generation of CSCs by progesterone and irradiation. The stimulation of PI3K/Akt via mPR resulted in the inactivation of FOXO transcriptional activity, the upregulation of snail and slug expression and a downregulation of miR-29 expression, which led to increased levels of KLF4, a transcription factor required for breast CSC maintenance. Stabilization of miR-29 expression impeded the generation of CSCs, while its inhibition alone was sufficient to generate CSCs. This study provides a new mechanistic basis for progesterone and radiation-induced breast cancer risk in basal cells. In addition, the elucidation of new pathways and miRNA regulations involved in CSC generation and maintenance may open the door to potential novel anti-CSC strategies.

The roles of Cdk5-mediated subcellular localization of FOXO1 in neuronal death.

Deficiency of cyclin-dependent kinase 5 (Cdk5) has been linked to the death of postmitotic cortical neurons during brain development. We now report that, in mouse cortical neurons, Cdk5 is capable of phosphorylating the transcription factor FOXO1 at Ser249 in vitro and in vivo. Cellular stresses resulting from extracellular stimulation by H2O2 or β-amyloid promote hyperactivation of Cdk5, FOXO1 nuclear export and inhibition of its downstream transcriptional activity. In contrast, a loss of Cdk5 leads to FOXO1 translocation into the nucleus: a shift due to decreased AKT activity but independent of S249 phosphorylation. Nuclear FOXO1 upregulates transcription of the proapoptotic gene, BIM, leading to neuronal death, which can be rescued when endogenous FOXO1 was replaced by the cytoplasmically localized form of FOXO1, FOXO1-S249D. Cytoplasmic, but not nuclear, Cdk5 attenuates neuronal death by inhibiting FOXO1 transcriptional activity and BIM expression. Together, our findings suggest that Cdk5 plays a novel and unexpected role in the degeneration of postmitotic neurons through modulation of the cellular location of FOXO1, which constitutes an alternative pathway through which Cdk5 deficiency leads to neuronal death.

β-Hydroxy-β-methylbutyrate (HMB) normalizes dexamethasone-induced autophagy-lysosomal pathway in skeletal muscle.

Dexamethasone-induced muscle atrophy is due to an increase in protein breakdown and a decrease in protein synthesis, associated with an over-stimulation of the autophagy-lysosomal pathway. These effects are mediated by alterations in IGF-1 and PI3K/Akt signaling. In this study, we have investigated the effects of β-Hydroxy-β-methylbutyrate (HMB) on the regulation of autophagy and proteosomal systems. Rats were treated during 21 days with dexamethasone as a model of muscle atrophy. Co-administration of HMB attenuated the effects promoted by dexamethasone. HMB ameliorated the loss in body weight, lean mass and the reduction of the muscle fiber cross-sectional area (shrinkage) in gastrocnemius muscle. Consequently, HMB produced an improvement in muscle strength in the dexamethasone-treated rats. To elucidate the molecular mechanisms responsible for these effects, rat L6 myotubes were used. In these cells, HMB significantly attenuated lysosomal proteolysis induced by dexamethasone by normalizing the changes observed in autophagosome formation, LC3 II, p62 and Bnip3 expression after dexamethasone treatment. HMB effects were mediated by an increase in FoxO3a phosphorylation and concomitant decrease in FoxO transcriptional activity. The HMB effect was due to the restoration of Akt signaling diminished by dexamethasone treatment. Moreover, HMB was also involved in the regulation of the activity of ubiquitin and expression of MurF1 and Atrogin-1, components of the proteasome system that are activated or up-regulated by dexamethasone. In conclusion, in vivo and in vitro studies suggest that HMB exerts protective effects against dexamethasone-induced muscle atrophy by normalizing the Akt/FoxO axis that controls autophagy and ubiquitin proteolysis.

FoxO transcription factors and endurance training: a role for FoxO1 and FoxO3 in exercise-induced angiogenesis.

FoxO transcription factors and endurance training: a role for FoxO1 and FoxO3 in exercise-induced angiogenesis.

Monitoring and Modeling Effects of IGF1, Insulin and Green Tea Compound EGCG on Nuclear-Cytoplasmic Distribution of Foxo1-GFP in Skeletal Muscle Fibers

The transcription factor Foxo1 promotes transcription of the ubiquitin ligases MuRF1 and MAFbx/atrogin1, which contribute to muscle fiber protein breakdown, leading to muscle atrophy and wasting. Foxo1 phosphorylated at its protein kinase B (Akt) sites is transported out of nuclei, and Foxo1 dephosphorylated at these sites is transported into nuclei. Foxo1 net nuclear efflux decreases Foxo1 transcriptional activity, thus opposing muscle protein breakdown and atrophy. Here we use quantitative fluorescence confocal imaging of the time course of Foxo1-GFP net nuclear fluxes together with mathematical modeling of Foxo1-GFP nuclear movements to examine the effects of upstream signaling on nuclear Foxo1. IGF1 and insulin, which activate Akt via the PI3k/PDK1/Akt signaling pathway, caused concentration dependent decreases of nuclear Foxo1-GFP. High IGF1 or insulin caused similar rapid and marked Foxo1-GFP net nuclear efflux. ECGC caused a more delayed net loss of nuclear Foxo1-GFP, possibly indicative of need for EGCG to enter the fiber to initiate its effect, whereas IGF1 and insulin work at cell surface receptors. Excluding the delay, the final rate and extent of decay of nuclear Foxo1-GFP was similar for EGCG, IGF1 and insulin. Analysis using our recently presented reduced (2 state)model for Foxo nuclear fluxes (Wimmer et al, 2014) showed that IGF1, insulin and EGCG all markedly and similarly increased the apparent unidirectional rate constant for Foxo1-GFP nuclear efflux, as well as markedly decreasing the apparent unidirectional rate constant for nuclear influx, indicative of increased Foxo1 phosphorylation in both cytoplasm and nuclei, both of which contribute to the observed decrease in nuclear Foxo1-GFP. Thus, IGF1, insulin and EGCG all activate Akt, both in the cytoplasm and within the nuclei. Supported by NIH R01AR056477 and by UMB-UMBC Seed Grant

Thioredoxin 1 upregulates FOXO1 transcriptional activity in drug resistance in ovarian cancer cells

Drug resistance is the major cause of failure of cancer chemotherapy in ovarian cancer. However, the molecular mechanisms on the regulation of drug resistance are not fully understood. Here we showed that Trx1 and FOXO1 were involved in paclitaxel (PTX)-induced drug resistance in ovarian cancer A2780 cells. PTX induced reactive oxygen species (ROS) and resulted in Trx1 and FOXO1 nuclear translocation. We further found that Trx1 bound to FOXO1 and enhanced FOXO1 transcriptional activity; however Trx1 C69S mutant which is barely detected in the nucleus downregulated Trx1–FOXO1 interaction and Trx1-induced FOXO1 transcriptional activation. Silencing of FOXO1 abrogated Trx1-induced drug resistance. Trx1 increased FOXO1-induced drug resistance, while Trx1 C69S mutant completely abolished the regulation of FOXO1-mediated drug resistance by Trx1. These findings provided a novel mechanism on Trx1/FOXO1 signaling in drug resistance in ovarian cancer cells.

Pelota mediates gonocyte maturation and maintenance of spermatogonial stem cells in mouse testes.

Pelota (Pelo) is an evolutionarily conserved gene, and its deficiency in Drosophila affects both male and female fertility. In mice, genetic ablation of Pelo leads to embryonic lethality at the early implantation stage as a result of the impaired development of extra-embryonic endoderm (ExEn). To define the consequences of Pelo deletion on male germ cells, we temporally induced deletion of the gene at both embryonic and postnatal stages. Deletion of Pelo in adult mice resulted in a complete loss of whole-germ cell lineages after 45 days of deletion. The absence of newly emerging spermatogenic cycles in mutants confirmed that spermatogonial stem cells (SSCs) were unable to maintain spermatogenesis in the absence of PELO protein. However, germ cells beyond the undifferentiated SSC stage were capable of completing spermatogenesis and producing spermatozoa, even in the absence of PELO. Following the deletion of Pelo during embryonic development, we found that although PELO is dispensable for maintaining gonocytes, it is necessary for the transition of gonocytes to SSCs. Immunohistological and protein analyses revealed the attenuation of FOXO1 transcriptional activity, which induces the expression of many SSC self-renewal genes. The decreased transcriptional activity of FOXO1 in mutant testes was due to enhanced activity of the PI3K/AKT signaling pathway, which led to phosphorylation and cytoplasmic sequestration of FOXO1. These results suggest that PELO negatively regulates the PI3K/AKT pathway and that the enhanced activity of PI3K/AKT and subsequent FOXO1 inhibition are responsible for the impaired development of SSCs in mutant testes.

Abstract 3392: Thromboxane-A2 receptor β-isoform regulates the activity of the tumor suppressor FOXO3 via phosphorylation by ERK in urothelial cancer

Abstract The tumor suppressor FOXO3 is a transcription factor whose activity and stability are regulated by phosphorylation on S294 via extracellular regulated kinases (ERK1/2). Previous data by our group has demonstrated overexpressing the thromboxane A2 isoform-β receptor (TPβ) increased ERK activation and induced malignant transformation of immortalized bladder cells. These data describe a novel mechanism of TPβ mediated regulation of FOXO3 via phosphorylation by ERK. Immunohistochemical studies revealed increased expression of nuclear pFOXO3-S294 in late stage bladder tumors, whereas total FOXO3 nuclear expression was reduced in high grade tumors. Both cytoplasmic and nuclear pFOXO3-S294 staining scores positively correlated with TPβ staining scores. Overexpressing TPβ in non-transformed urothelial cell lines, UROsta and SV-HUC, induced phosphorylation of FOXO3 at -S294. Knockdown of FOXO3 expression by shRNA increased cell migration and invasion. Conversely, overexpression of mutated FOXO3 that is resistant to ERK phosphorylation reduced the overall and TP agonist (U46619) mediated increase in malignant UMUC3 cell migration and invasion. Stimulation of UMUC3 cells with U46619 increased pFOXO3-S294 expression which could be attenuated with TP antagonist (PTXA2) pre-treatment. A dual luciferase assay demonstrated that U46619 stimulation decreased overall FOXO3 transcriptional activity. Additionally, U46619 stimulation initially caused nuclear accumulation of pFOXO3-S294, but prolonged stimulation increased FOXO3 cytoplasmic localization. In summary, the data describe a novel mechanism of TPβ signaling in bladder cancer that results in the ERK mediated regulation of FOXO3 activity, which could contribute to tumor progression. Citation Format: Philip M. Sobolesky, Perry V. Halushka, Elizabeth Garrett-Mayer, Omar Moussa. Thromboxane-A2 receptor β-isoform regulates the activity of the tumor suppressor FOXO3 via phosphorylation by ERK in urothelial cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3392. doi:10.1158/1538-7445.AM2014-3392

Transmembrane Inhibitor of RICTOR/mTORC2 in Hematopoietic Progenitors

SummaryCentral to cellular proliferative, survival, and metabolic responses is the serine/threonine kinase mTOR, which is activated in many human cancers. mTOR is present in distinct complexes that are either modulated by AKT (mTORC1) or are upstream and regulatory of it (mTORC2). Governance of mTORC2 activity is poorly understood. Here, we report a transmembrane molecule in hematopoietic progenitor cells that physically interacts with and inhibits RICTOR, an essential component of mTORC2. Upstream of mTORC2 (UT2) negatively regulates mTORC2 enzymatic activity, reducing AKTS473, PKCα, and NDRG1 phosphorylation and increasing FOXO transcriptional activity in an mTORC2-dependent manner. Modulating UT2 levels altered animal survival in a T cell acute lymphoid leukemia (T-ALL) model that is known to be mTORC2 sensitive. These studies identify an inhibitory component upstream of mTORC2 in hematopoietic cells that can reduce mortality from NOTCH-induced T-ALL. A transmembrane inhibitor of mTORC2 may provide an attractive target to affect this critical cell regulatory pathway.

Regulation of the tumor suppressor FOXO3 by the thromboxane-A2 receptors in urothelial cancer.

The transcription factor FOXO3 is a well-established tumor suppressor whose activity, stability, and localization are regulated by phosphorylation and acetylation. Previous data by our laboratory demonstrated amplified thromboxane-A2 signaling was associated with poor prognoses in bladder cancer patients and overexpression of the thromboxane-A2 isoform-β receptor (TPβ), but not TPα, induced malignant transformation of immortalized bladder cells in vivo. Here, we describe a mechanism of TP mediated modulation of FOXO3 activity and localization by phosphorylation and deacetylation in a bladder cancer cell model. In vitro gain and loss of function studies performed in non-transformed cell lines, UROsta and SV-HUC, revealed knockdown of FOXO3 expression by shRNA increased cell migration and invasion, while exogenously overexpressing TPβ raised basal phosphorylated (p)FOXO3-S294 levels. Conversely, overexpression of ERK-resistant, mutant FOXO3 reduced increases in UMUC3 cell migration and invasion, including that mediated by TP agonist (U46619). Additionally, stimulation of UMUC3 cells with U46619 increased pFOXO3-S294 expression, which could be attenuated by treatment with a TP antagonist (PTXA2) or ERK inhibitor (U0126). Initially U46619 caused nuclear accumulation of pFOXO3-S294; however, prolonged stimulation increased FOXO3 cytoplasmic localization. U46619 stimulation decreased overall FOXO3 transcriptional activity, but was associated with increased expression of its pro-survival target, manganese superoxide dismutase. The data also shows that TP stimulation increased the expression of the histone deacetylase, SIRT1, and corresponded with decreased acetylated-FOXO3. Collectively, the data suggest a role for TP signaling in the regulation of FOXO3 activity, mediated in part through phosphorylation and deacetylation.

AMP-activated protein kinase mediates the antioxidant effects of resveratrol through regulation of the transcription factor FoxO1.

Numerous physiological functions are controlled by redox-responsive signaling pathways. Disruption of redox balance by oxidative stress is recognized as a major cause of many pathological conditions, including aging, highlighting the importance of investigating how antioxidants maintain redox homeostasis. AMP-activated protein kinase (AMPK) is activated in response to cellular conditions that accompany energy depletion and plays a central role in the regulation of energy homeostasis, tumorigenesis and longevity. Recently, several antioxidants have been reported to activate AMPK, although the mechanisms by which AMPK acts to adjust the levels of cellular reactive oxygen species are not fully characterized. In the present study, we investigated the role of AMPK in mediating resveratrol-induced antioxidant effects and the molecular mechanisms underlying its actions. We demonstrate that AMPK activity plays an indispensable role in the operation of the ROS defense system by inducing the expression of the antioxidant enzymes, manganese superoxide dismutase and catalase, in response to resveratrol or the AMPK agonist 5-aminoimidazole-4-carboxamide-1-β-d-ribonucleotide. In addition, we identified the mechanism involved in the antioxidant function of AMPK, demonstrating that AMPK directly phosphorylates human FoxO1 (forkhead box O1) at Thr(649) in vitro and increases FoxO1-dependent transcription of manganese superoxide dismutase and catalase. Mutagenesis studies showed that this AMPK-mediated phosphorylation of FoxO1 is critical for FoxO1 stability and nuclear localization, establishing the molecular basis for the induction of FoxO1 transcriptional activity. Our results reveal a novel FoxO1-dependent mechanism by which AMPK controls the expression of antioxidant enzymes and suggest that AMPK has an important role in maintaining redox homeostasis.

Effects of over-expressed forkhead transcription factor O1 on high glucose induced extracellular matrix secretion in rat mesangial cells

To explore the role and molecular mechanism of forkhead transcription factor O1 (FoxO1) for extracellular matrix (ECM) protein accumulation in rat mesangial cells (MCs) cultured under high-glucose conditions. The MCs were transfected with either lentiviral vectors containing the sequences of constitutively active FoxO1 (LV-CA-FoxO1) or empty vectors (LV-NC-GFP). The MCs were divided into 4 groups of normal glucose (5.6 mmol/L) (NG), high glucose (25 mmol/L) (HG), high glucose plus LV-CA-FoxO1 (LV-CA) and high-glucose plus empty lentiviral vectors (LV-NC). After 72 h treatment, the mRNA levels of MCs were measured through real-time polymerase chain reaction (PCR) in each group, including FoxO1, fibronectin (FN), collagen I(Col I), collagen IV (Col IV), transforming growth factor-β1 (TGF-β1), TGF-β type Iand type II receptor (TGF-βRI/II). The protein levels of FoxO1, phosphorylation FoxO1 (p-FoxO1), TGF-β1 and TGF-βRI/II were assessed by Western blot. And the distributions of TGF-βRI in MCs were detected by immunofluorescence. Expression and bioactivity changes of FoxO1: Compared with NG group, there was no significant difference in either mRNA or protein levels of FoxO1 in HG group (P > 0.05) whereas the protein levels of p-FoxO1 markedly increased (P < 0.05). The levels of FoxO1 mRNA and protein increased in LV-CA group versus HG group (mRNA: 17.36 ± 1.13 vs 1.01 ± 0.12, protein: 4.38 ± 0.09 vs 0.93 ± 0.10, both P < 0.05). And there was a significant decrease of p-FoxO1/FoxO1 ratio (P < 0.05). Thus it suggested an enhancement in FoxO1 expression and transcriptional activity. Changes of ECM protein expression. The mRNA expression levels of FN, Col Iand Col IV in HG group were elevated compared with NG group (all P < 0.05) while the expressions of these indices in LV-CA group became attenuated compared with HG group (all P < 0.05). Changes of TGF-β pathway activation: the expressions of TGF-β1 and TGF-βRI/IIboth increased in HG group (all P < 0.05), but decreased in LV-CA group (all P < 0.05). The results of immunofluorescence suggested that the staining of TGF-βRIdecreased in MCs in LV-CA group versus HG group. All indices of LV-NC group had no statistical difference with those of HG group (all P > 0.05). FoxO1 plays a pivotal role in down-regulating the activation of TGF-β pathway and thereby inhibiting the secretion of ECM.

The Wnt receptor Ryk reduces neuronal and cell survival capacity by repressing FOXO activity during the early phases of mutant huntingtin pathogenicity.

Author SummaryNeuronal cell decline in neurodegenerative disease can be caused by inherited mutations and involves neuronal dysfunction followed by neuronal death. The ability of neurons to cope with the chronic stress induced by mutant protein expression may determine the course of their decline and eventual demise. Although the pathophysiological importance of these stress responses has been previously shown, very little is known about the signaling networks that regulate neuronal homeostasis during the early presymptomatic—but pathogenic—phases of a neurodegenerative disorder such as Huntington's disease (HD). In particular, it remains unclear whether neuronal differentiation factors regulate stress response pathways during neurodegenerative disease and how this might impact the overall capacity of neurons to cope with stress and maintain their function. Here, we show that the Wnt receptor Ryk, a protein known to be important for neurogenesis, is increased in different animal models of HD, before or during the early phases of the disease process. Interestingly, increased levels of Ryk repress activity of the FOXO proteins—a family of transcription factors that play a role in cell survival/longevity and in neuronal homeostasis and protection. Ryk represses FOXO protective activity, possibly directly, through its intracellular domain, a product of γ-secretase–mediated cleavage previously implicated in the birth of new cortical neurons. This highlights the regulation of HD neuron survival by a Ryk-dependent pathway that is distinct from canonical Wnt/Ryk signaling. From our findings, we postulate that neurons are unable to develop an efficient FOXO-mediated survival response during the very early, pathogenic phases of HD.

SMAD3 augments FoxO3-induced MuRF-1 promoter activity in a DNA-binding-dependent manner.

Muscle-specific RING finger-1 (MuRF-1), a ubiquitin ligase and key regulator of proteasome-dependent protein degradation, is highly expressed during skeletal muscle atrophy. The transcription factor forkhead box O3 (FoxO3) induces MuRF-1 expression, but the direct role of other major atrophy-related transcription factors, such as SMAD3, is largely unknown. The goal of this study was to determine whether SMAD3 individually regulates, or with FoxO3 coordinately regulates, MuRF-1 expression. In cultured myotubes or human embryonic kidney cells, MuRF-1 mRNA content and promoter activity were increased by FoxO3 but not by SMAD3 overexpression. However, FoxO3 and SMAD3 coexpression synergistically increased MuRF-1 mRNA and promoter activity. Mutation of the SMAD-binding element (SBE) in the proximal MuRF-1 promoter or overexpression of a SMAD3 DNA-binding mutant attenuated FoxO3-dependent MuRF-1 promoter activation, showing that SMAD binding to DNA is required for optimal activation of FoxO3-induced transcription of MuRF-1. Using chromatin immunoprecipitation, SMAD3 DNA binding increased FoxO3 abundance and SBE mutation reduced FoxO3 abundance on the MuRF-1 promoter. Furthermore, SMAD3 overexpression dose-dependently increased FoxO3 protein content, and coexpression of FoxO3 and SMAD3 synergistically increased FoxO-dependent gene transcription [assessed with a FoxO response element (FRE)-driven reporter]. Collectively, these results show that SMAD3 regulates transcription of MuRF-1 by increasing FoxO3 binding at a conserved FRE-SBE motif within the proximal promoter region, and by increasing FoxO3 protein content and transcriptional activity. These data are the first to indicate that two major transcription factors regulating protein degradation, FoxO3 and SMAD3, converge to coordinately and directly regulate transcription of MuRF-1.

CAR-mediated repression of Foxo1 transcriptional activity regulates the cell cycle inhibitor p21 in mouse livers

1,4-Bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), an agonist of constitutive androstane receptor (CAR), is a well-known strong primary chemical mitogen for the mouse liver. Despite extensive investigation of the role of CAR in the regulation of cell proliferation, our knowledge of the intricate mediating mechanism is incomplete. In this study, we demonstrated that long-term CAR activation by TCPOBOP increased liver-to-body weight ratio and decreased tumour suppressor Foxo1 expression and transcriptional activity, which were correlated with reduced expression of genes regulated by Foxo1, including the cell-cycle inhibitor Cdkn1a(p21), and upregulation of the cell-cycle regulator Cyclin D1. Moreover, we demonstrated the negative regulatory effect of TCPOBOP-activated CAR on the association of Foxo1 with the target Foxo1 itself and Cdkn1a(p21) promoters. Thus, we identified CAR-mediated repression of cell cycle inhibitor p21, as mediated by repression of FOXO1 expression and transcriptional activity. CAR-FOXO1 cross-talk may provide new opportunities for understanding liver diseases and developing more effective therapeutic approaches to better drug treatments.

USP7 Attenuates Hepatic Gluconeogenesis Through Modulation of FoxO1 Gene Promoter Occupancy

Hepatic forkhead protein FoxO1 is a key component of systemic glucose homeostasis via its ability to regulate the transcription of rate-limiting enzymes in gluconeogenesis. Important in the regulation of FoxO1 transcriptional activity are the modifying/demodifying enzymes that lead to posttranslational modification. Here, we demonstrate the functional interaction and regulation of FoxO1 by herpesvirus-associated ubiquitin-specific protease 7 (USP7; also known as herpesvirus-associated ubiquitin-specific protease, HAUSP), a deubiquitinating enzyme. We show that USP7-mediated mono-deubiquitination of FoxO1 results in suppression of FoxO1 transcriptional activity through decreased FoxO1 occupancy on the promoters of gluconeogenic genes. Knockdown of USP7 in primary hepatocytes leads to increased expression of FoxO1-target gluconeogenic genes and elevated glucose production. Consistent with this, USP7 gain-of-function suppresses the fasting/cAMP-induced activation of gluconeogenic genes in hepatocyte cells and in mouse liver, resulting in decreased hepatic glucose production. Notably, we show that the effects of USP7 on hepatic glucose metabolism depend on FoxO1. Together, these results place FoxO1 under the intimate regulation of deubiquitination and glucose metabolic control with important implication in diseases such as diabetes.

Insulin‐like growth factor I regulation of FOXO1 in gonadotropes (1012.7)

The transcription factor FOXO1 is important for several aspects of reproductive function, including endometrial decidualization, ovarian follicular maturation, and spermatogenesis. We have previously reported that in pituitary gonadotropes FOXO1 functions as a repressor of both luteinizing hormone beta (Lhb) and follicle stimulating hormone beta (Fshb) transcription, which are critical for reproduction. Insulin‐like growth factor I (IGFI) is a negative regulator of FOXO1 transcriptional activity. Given that FOXO1 can repress Lhb and Fshb transcription and that IGFI stimulation of gonadotrope cells enhances Lhb and Fshb synthesis, we investigated how IGFI regulates FOXO1 activity in gonadotropes in the current study. In LβT2 gonadotrope cells, we determined by western analyses that IGFI induced Ser256 FOXO1 phosphorylation, causing FOXO1 inhibition and cytoplasmic localization as identified by immunofluorescence microscopy. In addition, inhibition of components of the IGFI signaling pathway, such as PI3K or AKT, blocked IGFI‐induced phosphorylation of FOXO1. As the in vivo gonadotrope hormone milieu includes GnRH along with IGFI, we next investigated the effect of GnRH on FOXO1 phosphorylation and subcellular localization. Stimulation of LβT2 cells with GnRH alone induced moderate cytoplasmic localization of FOXO1, although GnRH did not induce phosphorylation of FOXO1 Ser256. Moreover, the GnRH‐induced shuttling of FOXO1 to the cytoplasm was not blocked by inhibition of PI3K activity. We are currently investigating the effects of IGFI and GnRH co‐treatment.Grant Funding Source: Supported by NIH Grants T32 HD007203 and F32 HD074414 to DVS; R01 HD067448 and U54 HD012303 to VGT.

PKA turnover by the REGγ-proteasome modulates FoxO1 cellular activity and VEGF-induced angiogenesis

The REGγ-proteasome serves as a short-cut for the destruction of certain intact mammalian proteins in the absence of ubiquitin- and ATP. The biological roles of the proteasome activator REGγ are not completely understood. Here we demonstrate that REGγ controls degradation of protein kinase A catalytic subunit-α (PKAca) both in primary human umbilical vein endothelial cells (HUVECs) and mouse embryonic fibroblast cells (MEFs). Accumulation of PKAca in REGγ-deficient HUVECs or MEFs results in phosphorylation and nuclear exclusion of the transcription factor FoxO1, indicating that REGγ is involved in preserving FoxO1 transcriptional activity. Consequently, VEGF-induced expression of the FoxO1 responsive genes, VCAM-1 and E-Selectin, was tightly controlled by REGγ in a PKA dependent manner. Functionally, REGγ is crucial for the migration of HUVECs. REGγ−/− mice display compromised VEGF-instigated neovascularization in cornea and aortic ring models. Implanted matrigel plugs containing VEGF in REGγ−/− mice induced fewer capillaries than in REGγ+/+ littermates. Taken together, our study identifies REGγ as a novel angiogenic factor that plays an important role in VEGF-induced expression of VCAM-1 and E-Selectin by antagonizing PKA signaling. Identification of the REGγ–PKA–FoxO1 pathway in endothelial cells (ECs) provides another potential target for therapeutic intervention in vascular diseases.

FOXO1-dependent DNA damage repair is regulated by JNK in lung cancer cells

DNA damage or mutation in cells contributes to tumorigenesis. The transcription factor FOXO1 modulates the expression of genes involved in DNA damage repair, cell cycle arrest and apoptosis. The transcriptional activity of FOXO1 is fundamentally regulated by post-translational modification and subcellular localization. H1299 lung cancer cells were treated with the alkylating agent MNNG, and the cell viability and DNA damage were separately determined by MTT and comet assay. Using immunofluorescence and western blotting, we observed the subcellular localization of FOXO1 and measured the relevant protein expression levels, respectively. To examine cell cycle arrest and apoptosis, flow cytometry analysis was preformed. The interaction between FOXO1 and JNK was analyzed through immunoprecipitation. Our results showed that cell viability was reduced at 24 h after MNNG treatment, and appeared to recover to some degree at 48 h. The increased expression and nuclear export of FOXO1 emerged at 4 h after the treatment. Nuclear FOXO1 played a pivotal role in cell cycle arrest, apoptosis and DNA damage repair by upregulating p27(Kip1), Bim and GADD45 gene expression, respectively. AKT-dependent S256 phosphorylation of FOXO1 and the S473 phosphorylation of AKT were both enhanced following DNA damage. Moreover, our studies revealed that FOXO1 directly interacted with JNK, and the inhibition of the JNK activity led to decreased expression of FOXO1 target genes. These findings suggest for the first time that FOXO1 is a promising candidate substrate for JNK, and the FOXO1-dependent DNA damage repair may be regulated positively by the JNK pathway in H1299 lung cancer cells.

Hepatitis C and Alcohol Exacerbate Liver Injury by Suppression of FOXO3

Hepatitis C virus (HCV) infection exacerbates alcoholic liver injury by mechanisms that include enhanced oxidative stress. The forkhead box transcription factor FOXO3 is an important component of the antioxidant stress response that can be altered by HCV. To test whether FOXO3 is protective for alcoholic liver injury, we fed alcohol to FOXO3(-/-) mice. After 3 weeks, one third of these mice developed severe hepatic steatosis, neutrophilic infiltration, and >10-fold alanine aminotransferase (ALT) elevations. In cell culture, either alcohol or HCV infection alone increased FOXO3 transcriptional activity and expression of target genes, but the combination of HCV and alcohol together caused loss of nuclear FOXO3 and decreased its transcriptional activity. This was accompanied by increased phosphorylation of FOXO3. Mice expressing HCV structural proteins on a background of reduced expression of superoxide dismutase 2 (SOD2; Sod2(+/-)) also had increased liver sensitivity to alcohol, with elevated ALT, steatosis, and lobular inflammation. Elevated ALT was associated with an alcohol-induced decrease in SOD2 and redistribution of FOXO3 to the cytosol. These results demonstrate that FOXO3 functions as a protective factor preventing alcoholic liver injury. The combination of HCV and alcohol, but not either condition alone, inactivates FOXO3, causing a decrease in expression of its target genes and an increase in liver injury. Modulation of the FOXO3 pathway is a potential therapeutic approach for HCV-alcohol-induced liver injury.