FOXO1 reshapes neutrophils to aggravate acute brain damage and promote late depression after traumatic brain injury.

BACKGROUND: Neutrophils are traditionally viewed as first responders but have a short onset of action in response to traumatic brain injury (TBI). However, the heterogeneity, multifunctionality, and time-dependent modulation of brain damage and outcome mediated by neutrophils after TBI remain poorly understood. METHODS: Using the combined single-cell transcriptomics, metabolomics, and proteomics analysis from TBI patients and the TBI mouse model, we investigate a novel neutrophil phenotype and its associated effects on TBI outcome by neurological deficit scoring and behavioral tests. We also characterized the underlying mechanisms both in vitro and in vivo through molecular simulations, signaling detections, gene expression regulation assessments [including dual-luciferase reporter and chromatin immunoprecipitation (ChIP) assays], primary cultures or co-cultures of neutrophils and oligodendrocytes, intracellular iron, and lipid hydroperoxide concentration measurements, as well as forkhead box protein O1 (FOXO1) conditional knockout mice. RESULTS: We identified that high expression of the FOXO1 protein was induced in neutrophils after TBI both in TBI patients and the TBI mouse model. Infiltration of these FOXO1high neutrophils in the brain was detected not only in the acute phase but also in the chronic phase post-TBI, aggravating acute brain inflammatory damage and promoting late TBI-induced depression. In the acute stage, FOXO1 upregulated cytoplasmic Versican (VCAN) to interact with the apoptosis regulator B-cell lymphoma-2 (BCL-2)-associated X protein (BAX), suppressing the mitochondrial translocation of BAX, which mediated the antiapoptotic effect companied with enhancing interleukin-6 (IL-6) production of FOXO1high neutrophils. In the chronic stage, the "FOXO1-transferrin receptor (TFRC)" mechanism contributes to FOXO1high neutrophil ferroptosis, disturbing the iron homeostasis of oligodendrocytes and inducing a reduction in myelin basic protein, which contributes to the progression of late depression after TBI. CONCLUSIONS: FOXO1high neutrophils represent a novel neutrophil phenotype that emerges in response to acute and chronic TBI, which provides insight into the heterogeneity, reprogramming activity, and versatility of neutrophils in TBI.

The Roles and Regulatory Mechanisms of Tight Junction Protein Cingulin and Transcription Factor Forkhead Box Protein O1 in Human Lung Adenocarcinoma A549 Cells and Normal Lung Epithelial Cells.

Tight junction (TJ) protein cingulin (CGN) and transcription factor forkhead box protein O1 (FOXO1) contribute to the development of various cancers. Histone deacetylase (HDAC) inhibitors have a potential therapeutic role for some cancers. HDAC inhibitors affect the expression of both CGN and FOXO1. However, the roles and regulatory mechanisms of CGN and FOXO1 are unknown in non-small cell lung cancer (NSCLC) and normal human lung epithelial (HLE) cells. In the present study, to investigate the effects of CGN and FOXO1 on the malignancy of NSCLC, we used A549 cells as human lung adenocarcinoma and primary human lung epithelial (HLE) cells as normal lung tissues and performed the knockdown of CGN and FOXO1 by siRNAs. Furthermore, to investigate the detailed mechanisms in the antitumor effects of HDAC inhibitors for NSCLC via CGN and FOXO1, A549 cells and HLE cells were treated with the HDAC inhibitors trichostatin A (TSA) and Quisinostat (JNJ-2648158). In A549 cells, the knockdown of CGN increased bicellular TJ protein claudin-2 (CLDN-2) via mitogen-activated protein kinase/adenosine monophosphate-activated protein kinase (MAPK/AMPK) pathways and induced cell migration, while the knockdown of FOXO1 increased claudin-4 (CLDN-4), decreased CGN, and induced cell proliferation. The knockdown of CGN and FOXO1 induced cell metabolism in A549 cells. TSA and Quisinostat increased CGN and tricellular TJ protein angulin-1/lipolysis-stimulated lipoprotein receptor (LSR) in A549. In normal HLE cells, the knockdown of CGN and FOXO1 increased CLDN-4, while HDAC inhibitors increased CGN and CLDN-4. In conclusion, the knockdown of CGN via FOXO1 contributes to the malignancy of NSCLC. Both HDAC inhibitors, TSA and Quisinostat, may have potential for use in therapy for lung adenocarcinoma via changes in the expression of CGN and FOXO1.

Regulatory action of PGRMC1 on cyclic AMP-mediated COX2 expression in human endometrial cells.

Human endometrial stromal cells (ESCs) undergo differentiation, known as decidualization, and endometrial epithelial cells mature around the embryo implantation stage. In the uterus, cyclooxygenase 2 (COX2), the rate-limiting enzyme that produces prostaglandin E2, is expressed in endometrial stromal and epithelial cells, and promotes decidualization of the former cells. Our recent study demonstrated that progesterone receptor membrane component 1 (PGRMC1) is downregulated during decidualization and may be involved in cellular senescence associated with decidualization via the transcription factor forkhead box protein O1 (FOXO1). Therefore, we investigated the role of PGRMC1 in COX2 expression during differentiation and maturation of endometrial stromal and epithelial cells. Inhibition or knockdown of PGRMC1 significantly enhanced differentiation stimuli-induced COX2 expression in both cell types. However, this COX2 expression was suppressed by FOXO1 knockdown or nuclear factor-kappa B (NF-κB) inhibition. Silencing of COX2 expression inhibited PGRMC1 knockdown-induced expression of decidual markers in ESCs. Thus, PGRMC1 may be linked to FOXO1- and NF-κB-mediated COX2 expression in endometrial cells. Taken together, our data suggest that downregulation of PGRMC1 expression facilitates differentiation of endometrial cells, i.e., decidualization and glandular maturation, via upregulation of COX2 expression.

FOXO1-miR-506 axis promotes chemosensitivity to temozolomide and suppresses invasiveness in glioblastoma through a feedback loop of FOXO1/miR-506/ETS1/FOXO1.

To explore the role of forkhead box protein O1 (FOXO1) in the progression of glioblastoma multiforme (GBM) and related drug resistance, we deciphered the roles of FOXO1 and miR-506 in proliferation, apoptosis, migration, invasion, autophagy, and temozolomide (TMZ) sensitivity in the U251 cell line using in vitro and in vivo experiments. Cell viability was tested by a cell counting kit-8 (CCK8) kit; migration and invasion were checked by the scratching assay; apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and flow cytometry. The construction of plasmids and dual-luciferase reporter experiment were carried out to find the interaction site between FOXO1 and miR-506. Immunohistochemistry was done to check the protein level in tumors after the in vivo experiment. We found that the FOXO1-miR-506 axis suppresses GBM cell invasion and migration and promotes GBM chemosensitivity to TMZ, which was mediated by autophagy. FOXO1 upregulates miR-506 by binding to its promoter to enhance transcriptional activation. MiR-506 could downregulate E26 transformation-specific 1 (ETS1) expression by targeting its 3'-untranslated region (UTR). Interestingly, ETS1 promoted FOXO1 translocation from the nucleus to the cytosol and further suppressed the FOXO1-miR-506 axis in GBM cells. Consistently, both miR-506 inhibition and ETS1 overexpression could rescue FOXO1 overactivation-mediated TMZ chemosensitivity in mouse models. Our study demonstrated a negative feedback loop of FOXO1/miR-506/ETS1/FOXO1 in GBM in regulating invasiveness and chemosensitivity. Thus, the above axis might be a promising therapeutic target for GBM.

The role of FOXO1-mediated autophagy in the regulation of bone formation.

Autophagy is essential for the maintenance of intracellular homeostasis, implicated in various biological processes. Forkhead box protein O1 (FOXO1) is regarded as a key mediator regulating skeletal development. Recent studies indicate that FOXO1 has a multifaceted role in autophagy regulation and dysregulation. Here, we aimed to elucidate the role of FOXO1-autophagy axis in osteogenesis. Osteoblast conditional Foxo1-knockout mice (Foxo1OB-/-, KO) and FOXO1 lentivirus overexpression (Len-FoxO1) model were constructed in vivo. Primary osteoblasts were isolated from KO and their wild-type (WT) littermates. And we also applied overexpression lentivirus to investigate the effects of FOXO1 in vitro. Using Micro-CT, fluorescence labeling detection, real-time qPCR and western blot analyses, we found that bone formation was promoted in Len-FOXO1 mice, which was impaired in KO group. Similarly, FOXO1 overexpression enhanced proliferation, migration and differentiation of osteoblasts, while FOXO1 ablation resulted in poor biological functions of osteoblasts. Through the investigation of autophagic process using mRFP-GFP-LC3 fluorescence labeling and co-immunoprecipitation, we observed that overexpression of FOXO1 initiated autophagy induction, with enhanced FOXO1 interaction with autophagy-related protein 7 (ATG7). On the contrary, FOXO1 knockout in osteoblasts impeded FOXO1-ATG7 conjugation, leading to impaired autophagic activity. Furthermore, inhibition of autophagy by chloroquine (CQ) could reverse favorable influences in bone formation induced by FOXO1 overexpression. Our findings confirmed that FOXO1 was an important regulator of bone formation and autophagy might be part of the underlying mechanisms, offering a significant avenue for the potential strategy in the treatment of bone-related disorders.

FOXO1-miR-506 axis promotes chemosensitivity to temozolomide and suppresses invasiveness in glioblastoma through a feedback loop of FOXO1/miR-506/ETS1/FOXO1 / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

To explore the role of forkhead box protein O1 (FOXO1) in the progression of glioblastoma multiforme (GBM) and related drug resistance, we deciphered the roles of FOXO1 and miR-506 in proliferation, apoptosis, migration, invasion, autophagy, and temozolomide (TMZ) sensitivity in the U251 cell line using in vitro and in vivo experiments. Cell viability was tested by a cell counting kit-8 (CCK8) kit; migration and invasion were checked by the scratching assay; apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining and flow cytometry. The construction of plasmids and dual-luciferase reporter experiment were carried out to find the interaction site between FOXO1 and miR-506. Immunohistochemistry was done to check the protein level in tumors after the in vivo experiment. We found that the FOXO1-miR-506 axis suppresses GBM cell invasion and migration and promotes GBM chemosensitivity to TMZ, which was mediated by autophagy. FOXO1 upregulates miR-506 by binding to its promoter to enhance transcriptional activation. MiR-506 could downregulate E26 transformation-specific 1 (ETS1) expression by targeting its 3'-untranslated region (UTR). Interestingly, ETS1 promoted FOXO1 translocation from the nucleus to the cytosol and further suppressed the FOXO1-miR-506 axis in GBM cells. Consistently, both miR-506 inhibition and ETS1 overexpression could rescue FOXO1 overactivation-mediated TMZ chemosensitivity in mouse models. Our study demonstrated a negative feedback loop of FOXO1/miR-506/ETS1/FOXO1 in GBM in regulating invasiveness and chemosensitivity. Thus, the above axis might be a promising therapeutic target for GBM.

Proteolysis Pathways via FoxO1 Reduce the Diameter of Myotube under Hypoxia Exposure / 中国生物化学与分子生物学报

The high altitude/hypoxic environment induced skeletal muscle atrophy is considered to be the interaction of multi-system and multi-organ, but the direct mechanism of hypoxia on muscle cells in this process is not clear. This study intended to investigate the effects of hypoxia exposure on proteins in ubiquitin and autophagy pathways, and explored the possible mechanism of hypoxia induced change of myotube diameter. The expression of myosin, hypoxia inducible factor-1 α (HIF-1α), forkhead box protein O1 (FoxO1), and ubiquitin protease pathway (MuRF1 and Atrogin1) and autophagy lysosomal pathway (p62, Beclin1, LC3) related proteins were detected by Western blot; The integrated optical density (IOD) of Myosin and LC3 was detected by IF. The results showed that the diameters of myotube at 6 h and 12 h were significantly reduced, and the expression of myosin was significantly reduced at 6 h after hypoxia exposure (P<0. 05); the protein levels of HIF-1α and FoxO1 were significantly increased at 6 h (P<0. 05); The expression of MuRF1 in each time points of hypoxia was significantly higher than 0 h (P<0. 05), but no difference of Atrogin1 expression was detected; Compared with 0 h, the expression of p62 was reduced significantly in response to hypoxia. The protein expression of Beclin1 and the IOD of LC3 was increased significantly at 6 h, and the LC3Ⅱ/Ⅰ ratio was significantly higher at 6 h, but significantly lower at 12 h and 24 h (P<0. 05).The results above indicated that the reduction of the myotube diameter of L6 skeletal muscle cells was induced by hypoxia exposure (1% O

Role and mechanism of PI3K/AKT/FoxO1/PDX-1 signaling pathway in functional changes of pancreatic islets in rats after severe burns.

AIMS: Studies on diabetes mellitus have shown that the phosphoinositide 3-kinase (PI3K)/serine threonine kinase (AKT)/forkhead box protein O1 (FoxO1) signaling pathway can regulate insulin secretion by modulating the expression of pancreatic and duodenal homeobox-1 (PDX-1). Therefore, it was hypothesized that the pathway also played an important role in functional abnormalities of pancreatic islets after severe burns. This study aimed to explore the role and mechanism of the PI3K/AKT/FoxO1/PDX-1 signaling pathway in functional changes of pancreatic islets in rats post severe burns. MAIN METHODS: Rats were grouped, subjected to full thickness burn injuries involving 50% total body surface area (TBSA), and injected intraperitoneally with BPV (HOpic) (0.6 mg/kg) or DMSO (0.55 mg/kg) once a day for three days. Glucose metabolism related indexes were measured by the glucometer, transmission electron microscopy (TEM) and enzyme-linked immunosorbent assay (ELISA). PI3K/AKT/FoxO1/PDX-1 signaling pathway related indexes were detected through immunofluorescence, western blot and RT-qPCR analyses. KEY FINDINGS: Dysglycemia and impaired insulin secretion occurred in rats, the activity of the PI3K/AKT/FoxO1/PDX-1 signaling pathway in the islets fell, and PDX-1 was translocated from the nucleus to the cytoplasm post severe burns. When BPV (HOpic) was used, glucose metabolism and insulin secretion were improved, the activity of the PI3K/AKT/FoxO1/PDX-1 signaling pathway in the islets was up-regulated, and PDX-1 was redistributed from the cytoplasm to the nucleus. SIGNIFICANCE: The activity of the PI3K/AKT/FoxO1/PDX-1 signaling pathway declined following severe burns. When its activity was up-regulated, insulin secretion could be improved, thus ameliorating hyperglycemia.

The transcription factor HAND2 up-regulates transcription of the IL15 gene in human endometrial stromal cells.

Cyclic changes of the human endometrium, such as proliferation, secretion, and decidualization, occur during regular menstrual cycles. Heart- and neural crest derivatives-expressed transcript 2 (HAND2) is a key transcription factor in progestin-induced decidualization of human endometrial stromal cells (ESCs). It has been suggested that HAND2 regulates interleukin 15 (IL15), a key immune factor required for the activation and survival of uterine natural killer (uNK) cells. Activated uNK cells can promote spiral artery remodeling and secrete cytokines to induce immunotolerance. To date, no studies have evaluated the transcription factors that regulate IL15 expression in human ESCs. In the present study, we examined whether HAND2 controls IL15 transcriptional regulation in human ESCs. Quantitative RT-PCR and histological analyses revealed that HAND2 and IL15 levels increase considerably in the secretory phase of human endometrium tissues. Results from ChIP-quantitative PCR suggested that HAND2 binds to a putative HAND2 motif, which we identified in the upstream region of the human IL15 gene through in silico analysis. Using a luciferase reporter assay, we found that the upstream region of the human IL15 gene up-regulates reporter gene activities in response to estradiol and a progestin representative (medroxyprogesterone) in ESCs. The upstream region of the human IL15 gene also exhibited increasing responsiveness to transfection with a HAND2 expression vector. Of note, deletion and substitution variants of the putative HAND2 motif in the upstream region of IL15 did not respond to HAND2 transfection. These findings confirm that HAND2 directly up-regulates human IL15 transcription in ESCs.

Protease Activated Receptor 4 as a Novel Modulator of Regulatory T Cell Function.

Regulatory T cells (Tregs) are a subpopulation of T cells that maintain immunological tolerance. In inflammatory responses the function of Tregs is tightly controlled by several factors including signaling through innate receptors such as Toll like receptors and anaphylatoxin receptors allowing an effective immune response to be generated. Protease-activated receptors (PARs) are another family of innate receptors expressed on multiple cell types and involved in the pathogenesis of autoimmune disorders. Whether proteases are able to directly modulate Treg function is unknown. Here, we show using two complimentary approaches that signaling through PAR-4 influences the expression of CD25, CD62L, and CD73, the suppressive capacity, and the stability of Tregs, via phosphorylation of FoxO1 and negative regulation of PTEN and FoxP3. Taken together, our results demonstrate an important role of PAR4 in tuning the function of Tregs and open the possibility of targeting PAR4 to modulate immune responses.

FOXO1 associated with sensitivity to chemotherapy drugs and glial-mesenchymal transition in glioma.

Mesenchymal subtype of glioblastoma (GBM), identified as one of four clinically relevant molecular subtypes, has worst prognosis because of its close relation with the malignant biological properties induced by glial-mesenchymal transition (GMT). However, the molecular mechanism of GMT and its characterized molecule of GBM have not been studied. Forkhead box protein O1 (FOXO1) is at a convergence point of receptor tyrosine kinase signaling as one of the three core pathways implicated in GBM. Our previous study indicated that the inactivation of FOXO1 involved in the inhibition of GMT is an independent prognosis factor of GBM. In this study, we will further confirm the role of FOXO1 in GMT through cytological experiments to clarify how FOXO1 regulates GMT and its clinical significance. We established virus-infected FOXO1 overexpression and FOXO1 knockdown cells of U373 MG and U251 mediated by lentivirus, based on the effect of which FOXO1-correlated-GMT experiments were performed in vitro and in vivo. Our data suggested that FOXO1 played a crucial role in resistance to TMZ, BCNU, and CDDP; migration and invasion; and stem cell properties of glioma cells. FOXO1 may serve as a targeted biomarker for prediction of sensitivity to chemotherapy drugs, metastasis, and prognosis, which provides a new idea for mesenchymal GBM treatment.

Novel Transcriptional Mechanisms for Regulating Metabolism by Thyroid Hormone.

The thyroid hormone plays a key role in energy and nutrient metabolisms in many tissues and regulates the transcription of key genes in metabolic pathways. It has long been believed that thyroid hormones (THs) exerted their effects primarily by binding to nuclear TH receptors (THRs) that are associated with conserved thyroid hormone response elements (TREs) located on the promoters of target genes. However, recent transcriptome and ChIP-Seq studies have challenged this conventional view as discordance was observed between TH-responsive genes and THR binding to DNA. While THR association with other transcription factors bound to DNA, TH activation of THRs to mediate effects that do not involve DNA-binding, or TH binding to proteins other than THRs have been invoked as potential mechanisms to explain this discrepancy, it appears that additional novel mechanisms may enable TH to regulate the mRNA expression. These include activation of transcription factors by SIRT1 via metabolic actions by TH, the post-translational modification of THR, the THR co-regulation of transcription with other nuclear receptors and transcription factors, and the microRNA (miR) control of RNA transcript expression to encode proteins involved in the cellular metabolism. Together, these novel mechanisms enlarge and diversify the panoply of metabolic genes that can be regulated by TH.

Fusion status in patients with lymph node-positive (N1) alveolar rhabdomyosarcoma is a powerful predictor of prognosis: Experience of the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG).

BACKGROUND: Alveolar rhabdomyosarcoma (aRMS) with lymph node involvement (N1 classification) accounts for up to 10% of all cases of RMS. The prognosis is poor, and is comparable to that of distant metastatic disease. In the European Paediatric Soft Tissue Sarcoma Study Group (EpSSG) RMS2005 protocol, patients with a histologic diagnosis of aRMS/N1 received intensified chemotherapy with systematic locoregional treatment. METHODS: Patients with aRMS/N1 were enrolled prospectively after primary surgery/biopsy and fusion status was assessed in tumor samples. All patients received 9 cycles of induction chemotherapy and 6 months of maintenance therapy. Local treatment included radiotherapy to the primary site and lymph nodes with or without secondary surgical resection. RESULTS: A total of 103 patients were enrolled. The clinical characteristics of the patients were predominantly unfavorable: 90% had macroscopic residual disease after initial surgery/biopsy, 63% had locally invasive tumors, 77% had a tumor measuring >5 cm, and 81% had disease at unfavorable sites. Fusion genes involving forkhead box protein O1 (FOXO1) were detected in 56 of 84 patients. Events occurred in 52 patients: 43 developed disease recurrence, 7 had disease that was refractory to treatment, and 2 patients developed second neoplasms. On univariate analysis, unfavorable disease site, tumor invasiveness, Intergroup Rhabdomyosarcoma Study group III, and fusion-positive status correlated with worse prognosis. The 5-year event-free survival rate of patients with fusion-positive tumors was 43% compared with 74% in patients with fusion-negative tumors (P = .01). On multivariate analysis, fusion positivity and tumor invasiveness proved to be unfavorable prognostic markers. CONCLUSIONS: Fusion status and tumor invasiveness appear to have a strong impact on prognosis in patients with aRMS/N1. Fusion status will be used to stratify these patients in the next EpSSG RMS study, and treatment will be intensified in patients with fusion-positive tumors. Cancer 2018. © 2018 American Cancer Society.

MicroRNA and Adipogenesis.

In obesity, the process of adipogenesis largely determines the number of adipocytes in body fat depots. Adipogenesis is regulated by several adipocyte-selective microRNAs (miRNAs) and transcription factors that modulate adipocyte proliferation and differentiation. However, some miRNAs block expression of master regulators of adipogenesis. Additionally, specific miRNAs have been implicated in adipocyte differentiation and mature adipocyte functions. While, each miRNA targets multiple mRNAs, which may coordinate or antagonize each other's functions, several miRNAs are dysregulated in other tissues during obesity-related comorbidities. In this respect, development of lipid droplets, macrophage accumulation, macrophage polarization, tumor necrosis factor receptor-associated factor 6 activity, lipolysis, lipotoxicity and insulin resistance are effectively controlled by miRNAs.

Role of thyroid hormone in hepatic gene regulation, chromatin remodeling, and autophagy.

Thyroid hormone (TH) actions on development and metabolism have been studied ever since the discovery of thyroxine almost a century ago. Initial studies focused on the physiological and biochemical actions of TH. Later, the cloning of the thyroid hormone receptor (THR) isoforms and the development of techniques enabled the study of TH regulation of complex cellular processes (such as gene transcription). Recently we found that TH activates secondary transcription factors such as FOXO1, to amplify gene transcription; and also is a potent inducer of autophagy that was critical for fatty acid ß-oxidation in the liver. This review summarizes the recent advancements in our understanding of TH regulation of gene expression of metabolic genes (via co-regulators/transcription factors and epigenetic control) and autophagy in the liver. Our deeper understanding of TH action recently has led to the development of tissue- and THR isoform-specific TH mimetics that may be useful for the treatment of metabolic disorders.

Knockdown of GSK3ß increases basal autophagy and AMPK signalling in nutrient-laden human aortic endothelial cells.

High concentrations of glucose and palmitate increase endothelial cell inflammation and apoptosis, events that often precede atherogenesis. They may do so by decreasing basal autophagy and AMP-activated protein kinase (AMPK) activity, although the mechanisms by which this occurs are not clear. Decreased function of the lysosome, an organelle required for autophagy and AMPK, have been associated with hyperactivity of glycogen synthase kinase 3ß (GSK3ß). To determine whether GSK3ß affects nutrient-induced changes in autophagy and AMPK activity, we used a primary human aortic endothelial cell (HAEC) model of type 2 diabetes that we had previously characterized with impaired AMPK activity and autophagy [Weikel et al. (2015) Am. J. Phys. Cell Physiol. 308: , C249-C263]. Presently, we found that incubation of HAECs with excess nutrients (25 mM glucose and 0.4 mM palmitate) increased GSK3ß activity and impaired lysosome acidification. Suppression of GSK3ß in these cells by treatment with a chemical inhibitor or overexpression of kinase-dead GSK3ß attenuated these lysosomal changes. Under control and excess nutrient conditions, knockdown of GSK3ß increased autophagosome formation, forkhead box protein O1 (FOXO1) activity and AMPK signalling and decreased Akt signalling. Similar changes in autophagy, AMPK and Akt signalling were observed in aortas from mice treated with the GSK3ß inhibitor CHIR 99021. Thus, increasing basal autophagy and AMPK activity by inhibiting GSK3ß may be an effective strategy in the setting of hyperglycaemia and dyslipidaemia for restoring endothelial cell health and reducing atherogenesis.

Porphyromonas gingivalis attenuates the insulin-induced phosphorylation and translocation of forkhead box protein O1 in human hepatocytes.

OBJECTIVE: Porphyromonas gingivalis (P. gingivalis) is a pathogen involved in periodontal disease. Recently, periodontal disease has been demonstrated to increase the risk of developing diabetes mellitus, although the molecular mechanism is not fully understood. Forkhead box protein O1 (FoxO1) is a transcriptional factor that regulates gluconeogenesis in the liver. Gluconeogenesis is a key process in the induction of diabetes mellitus; however, little is known regarding the relationship between periodontal disease and gluconeogenesis. In this study, to investigate whether periodontal disease influences hepatic gluconeogenesis, we examined the effects of P. gingivalis on the phosphorylation and translocation of FoxO1 in insulin-induced human hepatocytes. DESIGN: The human hepatocyte HepG2 was treated with insulin and Akt and FoxO1 phosphorylation was detected by western blot analysis. The localization of phosphorylated FoxO1 was detected by immunocytochemistry and western blot analysis. HepG2 cells were treated with SNAP26b-tagged P. gingivalis (SNAP-P.g.) before insulin stimulation, and then the changes in Akt and FoxO1 were determined by western blot analysis and immunocytochemistry. RESULTS: Insulin (100nM) induced FoxO1 phosphorylation 60min after treatment in HepG2 cells. Phosphorylated FoxO1 translocated to the cytoplasm. SNAP-P.g. internalized into HepG2 cells and decreased Akt and FoxO1 phosphorylation induced by insulin. The effect of insulin on FoxO1 translocation was also attenuated by SNAP-P.g. CONCLUSIONS: Our study shows that P. gingivalis decreases the phosphorylation and translocation of FoxO induced by insulin in HepG2 cells. Our results suggest that periodontal disease may increase hepatic gluconeogenesis by reducing the effects of insulin on FoxO1.

The right ventricle in pulmonary arterial hypertension: disorders of metabolism, angiogenesis and adrenergic signaling in right ventricular failure.

The right ventricle (RV) is the major determinant of functional state and prognosis in pulmonary arterial hypertension. RV hypertrophy (RVH) triggered by pressure overload is initially compensatory but often leads to RV failure. Despite similar RV afterload and mass some patients develop adaptive RVH (concentric with retained RV function), while others develop maladaptive RVH, characterized by dilatation, fibrosis, and RV failure. The differentiation of adaptive versus maladaptive RVH is imprecise, but adaptive RVH is associated with better functional capacity and survival. At the molecular level, maladaptive RVH displays greater impairment of angiogenesis, adrenergic signaling, and metabolism than adaptive RVH, and these derangements often involve the left ventricle. Clinically, maladaptive RVH is characterized by increased N-terminal pro-brain natriuretic peptide levels, troponin release, elevated catecholamine levels, RV dilatation, and late gadolinium enhancement on MRI, increased (18)fluorodeoxyglucose uptake on positron emission tomography, and QTc prolongation on the ECG. In maladaptive RVH there is reduced inotrope responsiveness because of G-protein receptor kinase-mediated downregulation, desensitization, and uncoupling of ß-adrenoreceptors. RV ischemia may result from capillary rarefaction or decreased right coronary artery perfusion pressure. Maladaptive RVH shares metabolic abnormalities with cancer including aerobic glycolysis (resulting from a forkhead box protein O1-mediated transcriptional upregulation of pyruvate dehydrogenase kinase), and glutaminolysis (reflecting ischemia-induced cMyc activation). Augmentation of glucose oxidation is beneficial in experimental RVH and can be achieved by inhibition of pyruvate dehydrogenase kinase, fatty acid oxidation, or glutaminolysis. Therapeutic targets in RV failure include chamber-specific abnormalities of metabolism, angiogenesis, adrenergic signaling, and phosphodiesterase-5 expression. The ability to restore RV function in experimental models challenges the dogma that RV failure is irreversible without regression of pulmonary vascular disease.

MicroRNA-145 suppresses hepatocellular carcinoma by targeting IRS1 and its downstream Akt signaling.

Accumulating evidences have proved that dysregulation of microRNAs (miRNAs) is involved in cancer initiation and progression. In this study, we showed that miRNA-145 level was significantly decreased in hepatocellular cancer (HCC) tissues and cell lines, and its low expression was inversely associated with the abundance of insulin receptor substrate 1 (IRS1), a key mediator in oncogenic insulin-like growth factor (IGF) signaling. We verified IRS1 as a direct target of miR-145 using Western blotting and luciferase reporter assay. Further, the restoration of miR-145 in HCC cell lines suppressed cancer cell growth, owing to down-regulated IRS1 expression and its downstream Akt/FOXO1 signaling. Our results demonstrated that miR-145 could inhibit HCC through targeting IRS1 and its downstream signaling, implicating the loss of miR-145 regulation may be a potential molecular mechanism causing aberrant oncogenic signaling in HCC.

The role of FOXO1 in cyclic stretch induced-proliferation of vascular smooth muscle cells during hypertension / 医用生物力学

Objective To investigate the role of pathologically increased-cyclic stretch in proliferation of vascular smooth muscle cells (VSMCs) during hypertension, and the effect of Forkhead box protein O1 (FOXO1) during this process. Methods Coarctation of abdominal aorta above kidney artery of rat was used as hypertensive animal model, and sham-operated animal as control. FX-4000 cyclic stretch loading system was used to apply 5% physiologically cyclic stretch and 15% pathologically cyclic stretch during hypertension on VSMCs in vitro. Western blot was used to reveal the expressions of FOXO1 and phosphor-FOXO1 in VSMCs, and BrdU kit to detect the proliferation of VSMCs in vitro. By using RNA interference in static, the role of FOXO1 on cell proliferation was further detected. Results After abdominal aorta coarctation for 2 and 4 weeks, respectively, the blood pressure was significantly increased compared with the sham operated rats. The proliferation of vascular cells in aorta of hypertensive rat was significantly increased, and so did the expressions of FOXO1 and phosphor-FOXO1. In vitro experiment revealed that 15% cyclic stretch remarkably increased the proliferation and expressions of FOXO1 and phospho FOXO1 in VSMCs. Target siRNA transfection in static decreased the expression of FOXO1 and phosphor-FOXO1, as well as the proliferation of VSMCs. Conclusions Pathologically increased-cyclic stretch may increase the expression and phosphorylation of FOXO1, subsequently modulate VSMC proliferation during hypertension. Based on animal models, this study intends to reveal the role of FOXO1 in vascular reconstruction of hypertension and the involved biomechanical mechanism, so as to make the mechanobiological mechanism of hypertension explicit and discover new target in the prevention and treatment of vascular remodeling.