Forkhead Box Class O Research Articles

Moderate embryonic delay of paternal mitochondrial elimination impairs mating and cognition and alters behaviors of adult animals.

Rapid elimination of paternal mitochondria following fertilization is a conserved event in most animals, but its physiological significance remains unclear. We find that modest delay of paternal mitochondrial elimination (PME) in Caenorhabditis elegans embryos unexpectedly impairs mating and cognition of adult animals and alters their locomotion behaviors. Delayed PME causes decreased adenosine triphosphate (ATP) levels in early embryos, which lead to impaired physiological functions of adult animals through an energy-sensing pathway mediated by an adenosine monophosphate (AMP)-activated protein kinase, AAK-2, and a forkhead box class O (FOXO) transcription factor, DAF-16. Treatment of PME-delayed animals with MK-4, a subtype of vitamin K2 that can improve mitochondrial ATP production, restores ATP levels in early embryos, and rescues physiological defects of adult animals. Our results suggest that moderate PME delay during embryo development adversely affects crucial physiological functions in adults, which could be evolutionarily disadvantageous. These observations provide mechanistic explanations for the need to swiftly remove paternal mitochondria early during embryo development.

Effects of molting on the expression of ecdysteroid responsive genes in the crustacean molting gland (Y-organ)

Ecdysteroid molting hormones coordinate arthropod growth and development. Binding of 20-hydroxyecdysone (20E) to ecdysteroid receptor EcR/RXR activates a cascade of nuclear receptor transcription factors that mediate tissue responses to hormone. Insect ecdysteroid responsive and Forkhead box class O (FOXO) transcription factor gene sequences were used to extract orthologs from blackback land crab (Gecarcinus lateralis) Y-organ (YO) transcriptome: Gl-Ecdysone Receptor (EcR), Gl-Broad Complex (Br-C), Gl-E74, Gl-Hormone Receptor 3 (HR3), Gl-Hormone Receptor 4 (HR4), Gl-FOXO, and Gl-Fushi tarazu factor-1 (Ftz-f1). Quantitative polymerase chain reaction quantified mRNA levels in tissues from intermolt animals and in YO of animals induced to molt by multiple limb autotomy (MLA) or eyestalk ablation (ESA). Gl-EcR, Gl-Retinoid X Receptor (RXR), Gl-Br-C, Gl-HR3, Gl-HR4, Gl-E74, Gl-E75, Gl-Ftz-f1, and Gl-FOXO were expressed in all 10 tissues, with Gl-Br-C, Gl-E74, Gl-E75, and Gl-HR4 mRNA levels in the YO lower than those in most of the other tissues. In MLA animals, molting had no effect on Gl-Br-C, Gl-E74, and Gl-Ftz-f1 mRNA levels and little effect on Gl-EcR, Gl-E75, and Gl-HR4 mRNA levels. Gl-HR3 and Gl-FOXO mRNA levels were increased during premolt stages, while Gl-RXR mRNA level was highest during intermolt and premolt stages and lowest at postmolt stage. In ESA animals, YO mRNA levels were not correlated with hemolymph ecdysteroid titers. ESA had no effect on Gl-EcR, Gl-E74, Gl-HR3, Gl-HR4, Gl-Ftz-f1, and Gl-FOXO mRNA levels, while Gl-RXR, Gl-Br-C, and Gl-E75 mRNA levels were decreased at 3 days post-ESA. These data suggest that transcriptional up-regulation of Gl-FOXO and Gl-HR3 contributes to increased YO ecdysteroidogenesis during premolt. By contrast, transcriptional regulation of ecdysteroid responsive genes and ecdysteroidogenesis were uncoupled in the YO of ESA animals.

Trichostatin C Synergistically Interacts with DNMT Inhibitor to Induce Antineoplastic Effect via Inhibition of Axl in Bladder and Lung Cancer Cells.

Aberrant epigenetic modifications are fundamental contributors to the pathogenesis of various cancers. Consequently, targeting these aberrations with small molecules, such as histone deacetylase (HDAC) inhibitors and DNA methyltransferase (DNMT) inhibitors, presents a viable strategy for cancer therapy. The objective of this study is to assess the anti-cancer efficacy of trichostatin C (TSC), an analogue of trichostatin A sourced from the fermentation of Streptomyces sp. CPCC 203909. Our investigations reveal that TSC demonstrates potent activity against both human lung cancer and urothelial bladder cancer cell lines, with IC50 values in the low micromolar range. Moreover, TSC induces apoptosis mediated by caspase 3/7 and arrests the cell cycle at the G2/M phase. When combined with the DNMT inhibitor decitabine, TSC exhibits a synergistic anti-cancer effect. Additionally, protein analysis elucidates a significant reduction in the expression of the tyrosine kinase receptor Axl. Notably, elevated concentrations of TSC correlate with the up-regulation of the transcription factor forkhead box class O1 (FoxO1) and increased levels of the proapoptotic proteins Bim and p21. In conclusion, our findings suggest TSC as a promising anti-cancer agent with HDAC inhibitory activity. Furthermore, our results highlight the potential utility of TSC in combination with DNMT inhibitors for cancer treatment.

Lineage-specific multifunctional double-layer scaffold accelerates the integrated regeneration of cartilage and subchondral bone

Repairing cartilage/subchondral bone defects that involve subchondral bone is a major challenge in clinical practice. Overall, the integrated repair of the structure and function of the osteochondral (OC) unit is very important. Some studies have demonstrated that the differentiation of cartilage is significantly enhanced by reducing the intake of nutrients such as lipids. This study demonstrates that using starvation can effectively optimize the therapeutic effect of bone marrow mesenchymal stem cells (BMSCs)-derived extracellular vesicles (EVs). A hyaluronic acid (HA)-based hydrogel containing starved BMSCs-EVs displayed continuous release for more than 3 weeks and significantly promoted the proliferation and biosynthesis of chondrocytes around the defect regulated by the forkhead-box class O (FOXO) pathway. When combined with vascular inhibitors, the hydrogel inhibited cartilage hypertrophy and facilitated the regeneration of hyaline cartilage. A porous methacrylate gelatine (GelMA)-based hydrogel containing calcium salt loaded with thrombin rapidly promoted haematoma formation upon contact with the bone marrow cavity to quickly block the pores and prevent the blood vessels in the bone marrow cavity from invading the cartilage layer. Furthermore, the haematoma could be used as nutrients to accelerate bone survival. The in vivo experiments demonstrated that the multifunctional lineage-specific hydrogel promoted the integrated regeneration of cartilage/subchondral bone. Thus, this hydrogel may represent a new strategy for osteochondral regeneration and repair.

The effects of aerobic exercise on liver function, insulin resistance, and lipid profiles in prediabetic and type 2 diabetic mice

Background and aimsThe purpose of the current study was to evaluate the expression of gluconeogenesis and insulin resistance key genes; including insulin receptor substrate 1 (Irs1), a serine/threonine protein kinase (Akt), forkhead box class-O 1 (FoxO1) and phosphoenolpyruvate carboxykinase (Pepck) genes, and lipid profiles following either a standard or a high-fat diet (HFD) and either an aerobic exercise or non-exercise intervention in prediabetic and type 2 diabetic (T2DM) mice. Methods24 male mice were randomly assigned to two groups fed with a normal diet (ND) or a HFD for 12 weeks. The mice in each group were again randomly assigned to two groups to create four groups in total: 1. Prediabetes-exercised (Prediabetes-Exe), 2. Prediabetes-non exercised (Prediabetes-Non exe), 3. Healthy-exercised (Healthy-Exe), and 4. Healthy-non exercised (Healthy-Non exe). Eighteen additional male mice were fed with the HFD for 8 weeks, after which streptozotocin (STZ) was administered. The mice were then fed the HFD for an additional 4 weeks. These T2DM mice were then randomly divided into two groups: 1. Diabetes-exercised (Diabetic-Exe), and 2. Diabetes-Non exe. The three Exe groups all exercised on a treadmill for 8 weeks for 5 sessions/week. After the last training session, liver tissue was extracted, and the expression of Irs1, Akt, FoxO1, and Pepck genes was measured using real time quantitative Polymerase chain reaction tests. Lipid profiles were measured in serum and in the liver. ResultsThe expression of both Irs1 and Akt was significantly increased in the Healthy-Exe, Prediabetes-Exe, and Diabetes-Exe groups as compared to the Healthy-Non exe, Prediabetes-Non exe, and Diabetes-Non exe groups (p < 0.001). Additionally, the expression of FoxO1 (p < 0.05) and Pepck (p < 0.001) decreased significantly in the Prediabetes-Exe, and Diabetes-Exe groups as compared to the Prediabetes-non exe, and Diabetes-Non exe groups. Aerobic exercise did not lead to reductions in FoxO1 or Pepck expression in the Healthy-Exe mice. ConclusionsEight weeks of aerobic exercise (5 sessions/week) significantly increased the expression of key genes that are important for maintaining glucose homeostasis and improving insulin resistance (Irs1 and Akt), and decreased expression of genes that are important for decreasing gluconeogenesis in the liver (FoxO1 and Pepck) in healthy, prediabetic, and T2DM mice. The lipid profiles improved in healthy, prediabetic, and T2DM mice.

The discrete roles of individual FOXO transcription factor family members in B-cell malignancies.

Forkhead box (FOX) class O (FOXO) proteins are a dynamic family of transcription factors composed of four family members: FOXO1, FOXO3, FOXO4 and FOXO6. As context-dependent transcriptional activators and repressors, the FOXO family regulates diverse cellular processes including cell cycle arrest, apoptosis, metabolism, longevity and cell fate determination. A central pathway responsible for negative regulation of FOXO activity is the phosphatidylinositol-3-kinase (PI3K)-AKT signalling pathway, enabling cell survival and proliferation. FOXO family members can be further regulated by distinct kinases, both positively (e.g., JNK, AMPK) and negatively (e.g., ERK-MAPK, CDK2), with additional post-translational modifications further impacting on FOXO activity. Evidence has suggested that FOXOs behave as 'bona fide' tumour suppressors, through transcriptional programmes regulating several cellular behaviours including cell cycle arrest and apoptosis. However, an alternative paradigm has emerged which indicates that FOXOs operate as mediators of cellular homeostasis and/or resistance in both 'normal' and pathophysiological scenarios. Distinct FOXO family members fulfil discrete roles during normal B cell maturation and function, and it is now clear that FOXOs are aberrantly expressed and mutated in discrete B-cell malignancies. While active FOXO function is generally associated with disease suppression in chronic lymphocytic leukemia for example, FOXO expression is associated with disease progression in diffuse large B cell lymphoma, an observation also seen in other cancers. The opposing functions of the FOXO family drives the debate about the circumstances in which FOXOs favour or hinder disease progression, and whether targeting FOXO-mediated processes would be effective in the treatment of B-cell malignancies. Here, we discuss the disparate roles of FOXO family members in B lineage cells, the regulatory events that influence FOXO function focusing mainly on post-translational modifications, and consider the potential for future development of therapies that target FOXO activity.

The Expression of FOXO3a as a Forensic Diagnostic Tool in Cases of Traumatic Brain Injury: An Immunohistochemical Study.

Traumatic brain injury (TBI) is one of the most well-known causes of neurological impairment and disability in the world. The Forkhead Box class O (FOXO) 3a is a transcription factor that is involved in different molecular processes, such as cell apoptosis regulation, neuroinflammation and the response to oxidative stress. This study is the first to evaluate the post-mortem immunohistochemical (IHC) positivity of FOXO3a expression in human cases of TBI deaths. The autopsy databases of the Legal Medicine and Forensic Institutes of the "Sapienza" University of Roma and the University of Pisa were retrospectively reviewed. After analyzing autopsy reports, 15 cases of TBI deaths were selected as the study group, while the other 15 cases were chosen among non-traumatic brain deaths as the control group. Decomposed bodies and those with initial signs of putrefaction were excluded. Routine histopathological studies were performed using hematoxylin-eosin (H&E) staining. Furthermore, an IHC investigation on cerebral samples was performed. To evaluate FOXO3a expression, anti-FOXO3a antibodies (GTX100277) were utilized. Concerning the IHC analysis, all 15 samples of TBI cases showed positivity for FOXO3a in the cerebral parenchyma. All control cerebral specimens showed FOXO3a negativity. In addition, the longer the survival time, the greater the positivity to the reaction with FOXO3a was. This study shows the important role of FOXO3a in neuronal autophagy and apoptosis regulation and suggests FOXO3a as a possible potential pharmacological target.

Melatonin Attenuates Ischemic-like Cell Injury by Promoting Autophagosome Maturation via the Sirt1/FoxO1/Rab7 Axis in Hippocampal HT22 Cells and in Organotypic Cultures.

Dysfunctional autophagy is linked to neuronal damage in ischemia/reperfusion injury. The Ras-related protein 7 (Rab7), a member of the Rab family of small GTPases, appears crucial for the progression of the autophagic flux, and its activity is strictly interconnected with the histone deacetylase Silent information regulator 1 (Sirt1) and transcription factor Forkhead box class O1 (FoxO1). The present study assessed the neuroprotective role of melatonin in the modulation of the Sirt1/FoxO1/Rab7 axis in HT22 cells and organotypic hippocampal cultures exposed to oxygen-glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin re-established physiological levels of autophagy and reduced propidium iodide-positive cells, speeding up autophagosome (AP) maturation and increasing lysosomal activity. Our study revealed that melatonin modulates autophagic pathways, increasing the expression of both Rab7 and FoxO1 and restoring the Sirt1 expression affected by OGD/R. In addition, the Sirt1 inhibitor EX-527 significantly reduced Rab7, Sirt1, and FoxO1 expression, as well as autolysosomes formation, and blocked the neuroprotective effect of melatonin. Overall, our findings provide, for the first time, new insights into the neuroprotective role of melatonin against ischemic injury through the activation of the Sirt1/FoxO1/Rab7 axis.

Neddylation inactivation affects cell cycle and apoptosis in sheep follicular granulosa cells.

Protein neddylation inactivation is a novel topic in cancer research. However, there are few studies on the mechanism of neddylation underlying the development of sheep follicular granulosa cells (GCs). In this study, the development of follicular GCs in sheep was inactivated by MLN4924, a neddylation-specific inhibitor, which significantly attenuated the proliferation and cell index of sheep follicular GCs. Further, the inactivation of neddylation by MLN4924 caused the accumulation of the cullin ring ligase (CRLs) substrates Wee1 and c-Myc, which could upregulate NOXA protein expression. Meanwhile, the B-cell lymphoma/leukemia 2 (BCL2) family members Bcl-2 and MCL-1 were downregulated, subsequently inducing apoptosis in follicular GCs of sheep. Increasing Wee1 levels caused G2/M-phase arrest. The effects of neddylation inactivation on Akt, the JAK2/STAT3 signaling pathway, and Forkhead box class O(FOXO) family members were evaluated. Neddylation inactivation by MLN4924 increased the levels of phospho-Akt, JAK2, phospho-STAT3, and FOXO1 (p < 0.05) and decreased the levels of phospho-FOXO3a and STAT3 (p < 0.05). In addition, MLN4924 could alter the mitochondrial morphology of GCs, increase cellular glucose utilization and lactate production, increase reactive oxygen species (ROS) generation, and promote sheep follicular GCs glycolysis, thus causing changes in mitochondrial functions. Together, these findings point to an unrecognized role of neddylation in regulating follicular GCs proliferation in sheep.

C10orf10/DEPP activates mitochondrial autophagy and maintains chondrocyte viability in the pathogenesis of osteoarthritis.

Osteoarthritis (OA), the most prevalent joint disease, is characterized by the progressive loss of articular cartilage. Autophagy, a lysosomal degradation pathway, maintains cellular homeostasis, and autophagic dysfunction in chondrocytes is a hallmark of OA pathogenesis. However, the cause of autophagic dysfunction in OA chondrocytes remains incompletely understood. Recent studies have reported that decidual protein induced by progesterone (C10orf10/DEPP) positively regulates autophagic functions. In this study, we found that DEPP was involved in mitochondrial autophagic functions of chondrocytes, as well as in OA pathogenesis. DEPP expression decreased in human OA chondrocytes in the absence or presence of pro-inflammatory cytokines, and was induced by starvation, hydrogen peroxide (H2 O2 ), and hypoxia (cobalt chloride). For functional studies, DEPP knockdown decreased autophagic flux induced by H2 O2 , whereas DEPP overexpression increased autophagic flux and maintained cell viability following H2 O2 treatment. DEPP was downregulated by knockdown of forkhead box class O (FOXO) transcription factors and modulated the autophagic function regulated by FOXO3. In an OA mouse model by destabilization of the medial meniscus, DEPP-knockout mice exacerbated the progression of cartilage degradation with TUNEL-positive cells, and chondrocytes isolated from knockout mice were decreased autophagic flux and increased cell death following H2 O2 treatment. Subcellular fractionation analysis revealed that mitochondria-located DEPP activated mitochondrial autophagy via BCL2 interacting protein 3. Taken together, our data demonstrate that DEPP is a major stress-inducible gene involved in the activation of mitochondrial autophagy in chondrocytes, and maintains chondrocyte viability during OA pathogenesis. DEPP represents a potential therapeutic target for enhancing autophagy in patients with OA.

Differential Dose- and Tissue-Dependent Effects of foxo on Aging, Metabolic and Proteostatic Pathways.

Aging is the gradual deterioration of physiological functions that culminates in death. Several studies across a wide range of model organisms have revealed the involvement of FOXO (forkhead box, class O) transcription factors in orchestrating metabolic homeostasis, as well as in regulating longevity. To study possible dose- or tissue-dependent effects of sustained foxo overexpression, we utilized two different Drosophila transgenic lines expressing high and relatively low foxo levels and overexpressed foxo, either ubiquitously or in a tissue-specific manner. We found that ubiquitous foxo overexpression (OE) accelerated aging, induced the early onset of age-related phenotypes, increased sensitivity to thermal stress, and deregulated metabolic and proteostatic pathways; these phenotypes were more intense in transgenic flies expressing high levels of foxo. Interestingly, there is a defined dosage of foxo OE in muscles and cardiomyocytes that shifts energy resources into longevity pathways and thus ameliorates not only tissue but also organismal age-related defects. Further, we found that foxo OE stimulates in an Nrf2/cncC dependent-manner, counteracting proteostatic pathways, e.g., the ubiquitin-proteasome pathway, which is central in ameliorating the aberrant foxo OE-mediated toxicity. These findings highlight the differential dose- and tissue-dependent effects of foxo on aging, metabolic and proteostatic pathways, along with the foxo-Nrf2/cncC functional crosstalk.

Resveratrol Promotes Angiogenesis in a FoxO1-Dependent Manner in Hind Limb Ischemia in Mice.

Critical limb ischemia (CLI) is a severe form of peripheral artery diseases (PAD) and seriously endangers the health of people. Therapeutic angiogenesis represents an important treatment strategy for CLI; various methods have been applied to enhance collateral circulation. However, the current development drug therapy to promote angiogenesis is limited. Resveratrol (RSV), a polyphenol compound extracted from plants, has various properties such as anti-oxidative, anti-inflammatory and anti-cancer effects. Whether RSV exerts protective effects on CLI remains elusive. In the current study, we demonstrated that oral intake of RSV significantly improved hind limb ischemia in mice, and increased the expression of phosphorylated Forkhead box class-O1 (FoxO1). RSV treatment in human umbilical vein endothelial cells (HUVECs) could increase the phosphorylation of FoxO1 and its cytoplasmic re-localization to promote angiogenesis. Then we manipulated FoxO1 in HUVECs to further verify that the effect of RSV on angiogenesis is in a FoxO1-dependent manner. Furthermore, we performed metabolomics to screen the metabolic pathways altered upon RSV intervention. We found that the pathways of pyrimidine metabolism, purine metabolism, as well as alanine, aspartate and glutamate metabolism, were highly correlated with the beneficial effects of RSV on the ischemic muscle. This study provides a novel direction for the medical therapy to CLI.

The role of forkhead box class O1 during implant osseointegration.

FOXO1, a member of the forkhead family of transcription factors, plays a vital role in the osteogenic lineage commitment of mesenchymal stem cells, and affects multiple cellular functions of osteogenic cells. However, prior studies have focused on mesenchymal stem cells but not on differentiated osteoblasts. In addition, studies about the role of FOXO1 during osseointegration are lacking. In this present study, we constructed osteoblast conditional FOXO1 knock-out mice and lentivirus-mediated FoxO1 overexpression to investigate maxillary titanium implant osseointegration. After 4wk post implant placement, micro-computed tomography, histomorphometric analyses, and RT-qPCR assays were performed. Results showed that compared with the control group, overexpression of FOXO1 significantly enhanced bone formation around implant and bone-implant contact ratio, while loss of FOXO1 impaired peri-implant osteogenesis and osseointegration. Moreover, overexpression of FoxO1 enhanced expression of osteogenesis-related genes, such as Runx2, Alp1, Col1a1, and Bglap. Whereas, knock-out of Foxo1 reduced the expression of osteogenesis-related genes. Taken together, our results suggested that FOXO1 in osteoblasts could enhance osteogenesis-related gene expression to improve osseointegration.

Activation of angiotensin-converting enzyme 2/angiotensin (1-7)/mas receptor axis triggers autophagy and suppresses microglia proinflammatory polarization via forkhead box class O1 signaling.

Brain renin‐angiotensin (Ang) system (RAS) is implicated in neuroinflammation, a major characteristic of aging process. Angiotensin (Ang) II, produced by angiotensin‐converting enzyme (ACE), activates immune system via angiotensin type 1 receptor (AT1), whereas Ang(1–7), generated by ACE2, binds with Mas receptor (MasR) to restrain excessive inflammatory response. Therefore, the present study aims to explore the relationship between RAS and neuroinflammation. We found that repeated lipopolysaccharide (LPS) treatment shifted the balance between ACE/Ang II/AT1 and ACE2/Ang(1–7)/MasR axis to the deleterious side and treatment with either MasR agonist, AVE0991 (AVE) or ACE2 activator, diminazene aceturate, exhibited strong neuroprotective actions. Mechanically, activation of ACE2/Ang(1–7)/MasR axis triggered the Forkhead box class O1 (FOXO1)‐autophagy pathway and induced superoxide dismutase (SOD) and catalase (CAT), the FOXO1‐targeted antioxidant enzymes. Meanwhile, knockdown of MasR or FOXO1 in BV2 cells, or using the selective FOXO1 inhibitor, AS1842856, in animals, suppressed FOXO1 translocation and compromised the autophagic process induced by MasR activation. We further used chloroquine (CQ) to block autophagy and showed that suppressing either FOXO1 or autophagy abrogated the anti‐inflammatory action of AVE. Likewise, Ang(1–7) also induced FOXO1 signaling and autophagic flux following LPS treatment in BV2 cells. Cotreatment with AS1842856 or CQ all led to autophagic inhibition and thereby abolished Ang(1–7)‐induced remission on NLRP3 inflammasome activation caused by LPS exposure, shifting the microglial polarization from M1 to M2 phenotype. Collectively, these results firstly illustrated the mechanism of ACE2/Ang(1–7)/MasR axis in neuroinflammation, strongly indicating the involvement of FOXO1‐mediated autophagy in the neuroimmune‐modulating effects triggered by MasR activation.

FOXO transcription factors in antioxidant defense.

Forkhead box, class O (FOXO) family proteins are widely expressed and highly conserved transcriptional regulators that modulate cellular fuel metabolism, stress resistance and cell death. FOXO target genes include genes encoding antioxidant proteins, thus likely contributing to the key role FOXOs play in the cellular response to oxidative stress and supporting the cellular strategies of antioxidant defense, that is, prevention (of the formation of reactive oxygen species), interception (of reactive species prior to their reaction with cellular components), repair (of damaged biomolecules), and adaptation (i.e., the stimulation of signaling pathways allowing for the expression of protective proteins). FOXOs themselves are regulated by redox processes at several levels, including expression of FOXO genes and enzymatic as well as nonenzymatic posttranslational modifications of FOXO proteins. The latter include modifications of FOXO cysteine residues. Here, an overview is provided on (i) the contribution of FOXO target genes to cellular antioxidative strategies, and (ii) on the impact of thiol homeostasis and thiol modification on FOXO activity.

Association of genetic variants of FBXO32 and FOXO6 in the FOXO pathway with breast cancer risk.

Forkhead box class O (FOXO) transcription factors play a pivotal role in regulating a variety of biological processes, including organismal development, cell signaling, cell metabolism, and tumorigenesis. Therefore, we hypothesize that genetic variants in FOXO pathway genes are associated with breast cancer (BC) risk. To test this hypothesis, we conducted a large meta-analysis using 14 published genome-wide association study (GWAS) data sets in the Discovery, Biology, and Risk of Inherited Variants in Breast Cancer (DRIVE) study. We assessed associations between 5214 (365 genotyped in DRIVE and 4849 imputed) common single-nucleotide polymorphisms (SNPs) in 55 FOXO pathway genes and BC risk. After multiple comparison corrections by the Bayesian false-discovery probability method, we found five SNPs to be significantly associated with BC risk. In stepwise multivariate logistic regression analysis with adjustment for age, principal components, and previously published SNPs in the same data set, three independent SNPs (i.e., FBXO32 rs10093411 A>G, FOXO6 rs61229336 C>T, and FBXO32 rs62521280 C>T) remained to be significantly associated with BC risk (p = 0.0008, 0.0011, and 0.0017, respectively). Additional expression quantitative trait loci analysis revealed that the FBXO32 rs62521280 T allele was associated with decreased messenger RNA (mRNA) expression levels in breast tissue, while the FOXO6 rs61229336 T allele was found to be associated with decreased mRNA expression levels in the whole blood cells. Once replicated by other investigators, these genetic variants may serve as new biomarkers for BC risk.

Rola czynników transkrypcyjnych FoxO w rozwoju cukrzycy i potencjalne możliwości terapeutyczne

Forkhead box class O (FOXO) family member proteins are key transcription factors for maintaining the intracellular homeostasis in response to changes in the internal and external environment. They participate in the control of such cellular processes as proliferation, cell cycle, apoptosis, glucose and lipid metabolism, and oxidative stress response. Altered expression and activity of these factors are associated with development of metabolic disturbances, primarily type 2 diabetes. Understanding of the role of FOXO in the pathophysiology of these abnormalities will enable appropriate steps to prevent their development and to create therapies targeted at the disturbances underlying type 2 diabetes and metabolic syndrome. In the present article, we summarized the current knowledge about the physiology and pathophysiology of these transcription factors and described their role in the development of diabetes and functioning of various organs. We focused on their role in progression of diabetes and indicated potential targets for future therapeutic interventions.

Resveratrol prevents steroid-induced osteonecrosis of the femoral head via miR-146a modulation.

The purpose of this study was to investigate the possible use of resveratrol (Res) to reverse abnormal osteogenesis/osteoclastogenesis activity that occurs during femoral head osteonecrosis and to explore the detailed mechanisms. Application of Res to bone marrow-derived mesenchymal stem cells in vitro promoted survival, inhibited apoptosis, and downregulated expression of reactive oxygen species expression. Moreover, Res application was associated with elevated microRNA-146a (miR-146a) expression, osteogenic differentiation, and suppressed osteoclastic differentiation, which were markedly reversed by miR-146a inhibitor. Histopathological observations and micro-computed tomography scanning results indicated that the Res-treated group had lower incidence of osteonecrosis and better bone microstructure than the untreated group. Res inhibited osteoclastogenesis through altering the levels of sirtuin1 (Sirt1), nuclear transcription factor-κB (NF-κB), and receptor activator of NF-κB ligand (RANKL). Simultaneously, Res treatment improved bone formation and increased β-catenin and runt-related transcription factor 2 (Runt2) expression levels, while reducing forkhead box class O (FOXO) family protein levels. The results of our study suggest that Res prevents steroid-induced osteonecrosis by upregulating miR-146a, and thereby stabilizes osteogenesis/osteoclastogenesis homeostasis via Wnt/FOXO and Sirt1/NF-κB pathways.

In vitro study of the role of FOXO transcription factors in regulating cigarette smoke extract-induced autophagy.

Cigarette smoking is the chief etiological factor for chronic obstructive pulmonary disease (COPD). Oxidative stress induced by cigarette smoke (CS) causes protein degradation, DNA damage, and cell death, thereby resulting in acute lung injury (ALI). In this regard, autophagy plays a critical role in regulating inflammatory responses by maintaining protein and organelle homeostasis and cellular viability. Expression of autophagy-related proteins (ARPs) isregulated by the fork head box class O (FOXO) transcription factors. In the current study, we examined the role of FOXO family proteins-FOXO1 and FOXO3a-in regulating CS extract (CSE)-induced autophagy. Using human lung adenocarcinoma cells with type II alveolar epithelial characteristics (A549), we observed CSE-mediated downregulation of FOXO3a. In contrast, there was a pronounced increase in the expression of FOXO1 at both the transcriptional and translational levels in the CSE-challenged cells compared with controls. Interestingly, knockdown of FOXO3a heightened the CSE-mediated increase in expression of cytokines/chemokines (IL-6, IL-8, andMCP-1), ARPs, and the FOXO1 transcription factor. Moreover, FOXO1 knockdown rescued CSE-mediated upregulation of ARPs in A549 cells. In addition, using the ROS inhibitor N-acetyl-L-cysteine (NAC), we observed abrogated mRNA expression of several ARPs and production of inflammatory cytokines/chemokines (IL-6, IL-8, MCP-1, and CCL-5) in the CSE-challenged cells suggesting an important role of ROS in regulating CSE-induced autophagy. Chromatin immunoprecipitation of FOXO1 and FOXO3a demonstrated increased binding of theformer to promoter regions of autophagy genes- BECLIN1, ATG5, ATG12, ATG16, and LC3 in CSE challenged cells. These findings suggest the role of FOXO1 in regulating the expression of these genesduring CSE exposure. Overall, our findings provide evidence for FOXO3a-dependent FOXO1-mediated regulation of autophagy in the CSE-challenged cells. Graphical abstract.

1723-P: Transcription Factor Foxo1 Mediates TGF-ß1-Induced Apoptosis in Primary Hepatocytes

Dysregulation of hepatocyte apoptosis or failure is associated with several types of chronic liver diseases. Transforming growth factor β1 (TGF- β1) is a well-known pro-apoptotic factor in the liver, which is constituted by a receptor complex composed of TGFβ receptor type I (TβRI) and TβRII, along with transcriptional factors Smad proteins. As a member of the forkhead box O-class transcription factors, Foxo1 is a predominant regulator for hepatic glucose production. While the role of Foxo1 in functions beyond glucose metabolism is gradually recognized, since there has been increasing evidence showing the potential relationship between TGF-β1 signaling and Foxo1. Here, we investigated whether Foxo1 mediates the pro-apoptotic effect of TGF-β1 in primary hepatocytes. By using hepatocytes isolated from both of wild type (Foxo1fl/fl) and liver specific Foxo1 knockout (LFKO, albumin-Cre+/-:: Foxo1fl/fl) mice, it was found that TGF-β1 induces hepatocytes apoptosis in a Foxo1-dependent manner. We also explored the associated molecular mechanisms and demonstrated that TGF-β1 activates protein kinase A (PKA) through TβRI-Smad3, followed by phosphorylation of Foxo1 at Ser273 to promote apoptosis of hepatocytes. The significant role of pFoxo1-S273 in mediating the pro-apoptotic effect of TGF-β1 was validated by using hepatocytes isolated from Foxo1-S273A mice, which mimics the dephosphorylation of Foxo1-Ser273. The establishment of TGF-β1-PKA-Foxo1axis in this study reveals a novel role of Foxo1 in hepatic functions. Disclosure W. Ai: None. W. Liao: None. Q. Pan: None. W. Yang: None. Z. Shen: None. S. Guo: None. Y. Chen: None. Funding American Diabetes Association (1-15-CD-09 to S.G., Sr.); National Institutes of Health (R01DK095118, R56DK11833401)