Inhibition Of Forkhead Box O1 Research Articles

FOXO1 regulates RUNX2 ubiquitination through SMURF2 in calcific aortic valve disease

The prevalence of calcific aortic valve disease (CAVD) remains substantial while there is currently no medical therapy available. Forkhead box O1 (FOXO1) is known to be involved in the pathogenesis of cardiovascular diseases, including vascular calcification and atherosclerosis; however, its specific role in calcific aortic valve disease remains to be elucidated. In this study, we identified FOXO1 significantly down-regulated in the aortic valve interstitial cells (VICs) of calcified aortic valves by investigating clinical specimens and GEO database analysis. FOXO1 silencing or inhibition promoted VICs osteogenic differentiation in vitro and aortic valve calcification in Apoe−/− mice, respectively. We identified that FOXO1 facilitated the ubiquitination and degradation of RUNX2, which process was mainly mediated by SMAD-specific E3 ubiquitin ligase 2 (SMURF2). Our discoveries unveil a heretofore unacknowledged mechanism involving the FOXO1/SMURF2/RUNX2 axis in CAVD, thereby proposing the potential therapeutic utility of FOXO1 or SMURF2 as viable strategies to impede the progression of CAVD.

FOXO1 regulates wound-healing responses in human gingival fibroblasts.

Forkhead box-O 1 (FOXO1) is a transcription factor actively involved in oral wound healing at the epithelial barrier. However, less is known regarding the role of FOXO1 during the tissue repair response in the connective tissue compartment. This study explored the involvement of FOXO1 in the modulation of fibroblast activity related to wound healing. Primary cultures of human gingival fibroblasts were obtained from four healthy young donors. Myofibroblastic differentiation, collagen gel contraction, cell migration, cell spreading, and integrin activation were evaluated in the presence or absence of a FOXO1 inhibitor (AS1842856). Variations in mRNA and proteins of interest were evaluated through qRT-PCR and western blot, respectively. Distribution of actin, α-smooth muscle actin, and β1 integrin was evaluated using immunofluorescence. FOXO1 and TGF-β1 expression in gingival wound healing was assessed by immunohistochemistry in gingival wounds performed in C57BL/6 mice. Images were analyzed using ImageJ/Fiji. ANOVA or Kruskal-Wallis test followed by Tukey's or Dunn's post-hoc test was performed. All data are expressed as mean ± SD. p < .05 was considered statistically significant. FOXO1 inhibition caused a decrease in the expression of the myofibroblastic marker α-SMA along with a reduction in fibronectin, type I collagen, TGF-β1, and β1 integrin mRNA level. The FOXO1 inhibitor also caused decreases in cell migration, cell spreading, collagen gel contraction, and β1 integrin activation. FOXO1 and TGF-β1 were prominently expressed in gingival wounds in fibroblastic cells located at the wound bed. The present study indicates that FOXO1 plays an important role in the modulation of several wound-healing functions in gingival fibroblast. Moreover, our findings reveal an important regulatory role for FOXO1 on the differentiation of gingival myofibroblasts, the regulation of cell migration, and collagen contraction, all these functions being critical during tissue repair and fibrosis.

Upregulation of FOXO1 contributes to lipopolysaccharide-induced pulmonary endothelial injury by induction of autophagy.

BackgroundAutophagy is activated during the pathogenesis of endothelial dysfunction and sepsis-associated acute lung injury (ALI). This study aimed to investigate whether autophagy affected endothelial barrier dysfunction and lung injury in a murine model of lipopolysaccharide (LPS)-induced ALI, and then further clarify whether forkhead box O1 (FOXO1), an autophagy-related transcriptional factor, contributed to autophagy activation and ALI induced by LPS.MethodsMale C57BL/6 mice were treated with LPS (30 mg/kg), and then were allocated to a control group and an LPS group with or without FOXO1 inhibitor (AS1842856) treatment, respectively. Primary cultured mouse lung vascular endothelial cells (MLVECs) were treated with LPS, autophagy inhibitor 3-methyladenine (3-MA), AS1842856, and small interfering RNA (siRNA) targeting autophagy-related gene 5 (ATG5) or FOXO1. Endothelial autophagic flux was assessed by transfection of MLVECs with red fluorescent protein (RFP)-green fluorescent protein (GFP) tandem fluorescent-tagged LC3 (RFP-GFP-LC3) adenovirus. Endothelial permeability was analyzed by the diffusion of fluorescein isothiocyanate-carboxymethyl (FITC)-dextran through the endothelial monolayer. Evans blue albumin tracer was used to measure the pulmonary transvascular permeability, and hematoxylin and eosin (H&E) staining was used to observe pathological changes in the lung tissues. Immunofluorescence staining was also used to detect the expression of zonula occludens-1 (ZO-1) and FOXO1.ResultsThis study found autophagy induction in lung tissues of endotoxemic mice and LPS-treated MLVECs, as evidenced by elevated expression of light chain 3 II (LC3-II) and Unc-51-like kinase (ULK1) and autophagic flux. LPS treatment decreased vascular endothelial (VE)-cadherin and ZO-1 expression and increased endothelial permeability in MLVECs, which were significantly alleviated by autophagy inhibitor 3-MA and ATG5 siRNA. It was found that both phosphorylated FOXO1 and FOXO1 were upregulated in the lung tissues of endotoxemic mice and LPS-treated MLVECs. Both FOXO1 inhibitor AS1842856 and FOXO1 siRNA suppressed LPS-induced autophagy and endothelial cell injury in MLVECs. Moreover, FOXO1 inhibition profoundly alleviated autophagy, lung endothelial hyperpermeability, and ALI in endotoxemic mice.ConclusionsThis work demonstrated that FOXO1 upregulation is an important contributor to LPS-induced autophagy in pulmonary VE cells. The detrimental effects of FOXO1 in endotoxemia-associated endothelial dysfunction and ALI are partly due to its potent pro-autophagic property. Inhibition of FOXO1 may be a potential therapeutic option for the treatment of ALI.

Downregulation of ROR2 promotes dental pulp stem cell senescence by inhibiting STK4-FOXO1/SMS1 axis in sphingomyelin biosynthesis.

Dental pulp stem cells (DPSCs) play a vital role in tooth restoration, regeneration, and homeostasis. The link between DPSC senescence and tooth aging has been well‐recognized. ROR2 plays an important role in aging‐related gene expression. However, the expression and function of ROR2 in DPSC aging remain largely unknown. In this study, we found that ROR2 expression was significantly decreased in aged pulp tissues and DPSCs. The depletion of ROR2 in young DPSCs inhibits their self‐renewal capacity, while its overexpression in aged DPSCs restores their self‐renewal capacity. Interestingly, we found that sphingomyelin (SM) is involved in the senescence of DPSCs regulated by ROR2. Mechanistically, we confirmed that ROR2 inhibited the phosphorylation of STK4, which promoted the translocation of Forkhead Box O1 (FOXO1) to the nucleus. STK4 inhibition or knockdown of FOXO1 markedly increased the proliferation of DPSCs and upregulated the expression of SMS1, which catalyzed SM biogenesis. Moreover, FOXO1 directly bound to the SMS1 promoter, repressing its transcription. Our findings demonstrated the critical role of the ROR2/STK4‐FOXO1/SMS1 axis in the regulation of SM biogenesis and DPSC senescence, providing a novel target for antagonizing tooth aging.

Hepatic AKT orchestrates adipose tissue thermogenesis via FGF21-dependent and -independent mechanisms.

SUMMARYOrganismal stressors such as cold exposure require a systemic response to maintain body temperature. Brown adipose tissue (BAT) is a key thermogenic tissue in mammals that protects against hypothermia in response to cold exposure. Defining the complex interplay of multiple organ systems in this response is fundamental to our understanding of adipose tissue thermogenesis. In this study, we identify a role for hepatic insulin signaling via AKT in the adaptive response to cold stress and show that liver AKT is an essential cell-nonautonomous regulator of adipocyte lipolysis and BAT function. Mechanistically, inhibition of forkhead box O1 (FOXO1) by AKT controls BAT thermogenesis by enhancing catecholamine-induced lipolysis in the white adipose tissue (WAT) and increasing circulating fibroblast growth factor 21 (FGF21). Our data identify a role for hepatic insulin signaling via the AKT-FOXO1 axis in regulating WAT lipolysis, promoting BAT thermogenic capacity, and ensuring a proper thermogenic response to acute cold exposure.

Antineoplastic Activity of an Old Natural Antidiabetic Biguanide on the Human Thyroid Carcinoma Cell Line.

In the last decades, metformin (Met), an herbal anti-diabetic medicine, has been proposed as an anti-cancer agent. Thyroid cancers are the most common malignancy of the endocrine system. Therefore, the current study was performed to assess the effects of Met on cell proliferation and activation of the Phosphoinositide 3- Kinase (PI3K)/Protein kinase B (AKT)/Forkhead Box O1 (FOXO1) signaling pathway in the Medullary Thyroid Carcinoma (MTC) cells. The effects of Met on the expression of REarranged during Transfection (RET) proto-oncogene were also investigated. MTC cell line (TT) was treated with 0, 2.5, 5, 10, 20, 30, 40, 50, and 60 mM concentrations of Met for 24, 48, and 72h. The viability and apoptosis of the treated cells were measured by the 3-(4,5-Dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Annexin V- Propidium Iodide (PI) assays. The expression level of PI3K, AKT, FOXO1, and RET genes was investigated by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), and phosphorylation of their proteins was determined by the Enzyme-Linked Immunosorbent Assay (ELISA). Results showed that Met significantly decreased the viability of the MTC cells. Met also reduced the expression level of PI3K, AKT, and FOXO1 genes (P<0.05), whereas it elevated the expression level of RET proto-oncogene (P<0.05). It seems that the Met has a cytostatic effect on the TT cells. Our results showed that anti-tumoral effects of Met may be cell type-specific, and according to the induction of RET (as a proto-oncogene) and inhibition of FOXO1 (as a tumor suppressor gene), Met could not be an appropriate agent in the treatment of MTC. The antineoplastic activity of Met has been confirmed against several malignancies in "in vitro" and "in vivo" studies. However, its molecular mechanisms in the treatment of different carcinomas particularly in thyroid cancers are not clearly understood and more studies are required to confirm its exact effect on the MTC.

Hsa_circ_0030042 regulates abnormal autophagy and protects atherosclerotic plaque stability by targeting eIF4A3.

Rationale: Abnormal autophagic death of endothelial cells is detrimental to plaque structure as endothelial loss promotes lesional thrombosis. As emerging functional biomarkers, circular RNAs (circRNAs) are involved in various diseases, including cardiovascular. This study is aimed to determine the role of hsa_circ_0030042 in abnormal endothelial cell autophagy and plaque stability.Methods: circRNA sequencing and quantitative polymerase chain reaction were performed to detect hsa_circ_0030042 expression in coronary heart disease (CHD) and human umbilical vein endothelial cells (HUVECs). Transfection of stubRFP-sensGFP-LC3 adenovirus, flow cytometry, and electron microscopy were used to identify the role of hsa_circ_0030042 in ox-LDL‒induced abnormal autophagy in vitro. Bioinformatic analysis, RNA immunoprecipitation, immunofluorescence assay and other in vitro experiments were performed to elucidate the mechanism underlying hsa_circ_0030042-mediated regulation of autophagy. To evaluate the role of hsa_circ_0030042 in atherosclerotic plaques and endothelial function, we measured the carotid artery tension and performed histopathology and immunohistochemistry analysis.Results: hsa_circ_0030042 was significantly downregulated in CHD, while upon overexpression, it acted as an endogenous eukaryotic initiation factor 4A-III (eIF4A3) sponge to inhibit ox-LDL-induced abnormal autophagy of HUVECs and maintain plaque stability in vivo. Furthermore, hsa_circ_0030042 influenced autophagy by sponging eIF4A3 and blocking its recruitment to beclin1 and forkhead box O1 (FOXO1) mRNA, while hsa_circ_0030042-induced inhibition of beclin1 and FOXO1 was counteracted by eIF4A3 overexpression or decreased hsa_circ_0030042 binding. In high-fat-diet fed ApoE-/- mice, hsa_circ_0030042 also ameliorated plaque stability and counteracted eIF4A3-induced plaque instability.Conclusions: These results demonstrate a novel pathway involving hsa_circ_0030042, eIF4A3, FOXO1, and beclin1; hence, modulating their levels may be a potential therapeutic strategy against CHD.

Effect of forkhead box O1 in renal tubular epithelial cells on endotoxin-induced acute kidney injury.

Mitochondrial damage in renal tubular epithelial cells (RTECs) is a hallmark of endotoxin-induced acute kidney injury (AKI). Forkhead box O1 (FOXO1) is responsible for regulating mitochondrial function and is involved in several kidney diseases. Here, we investigated the effect of FOXO1 on endotoxin-induced AKI and the related mechanism. In vivo, FOXO1 downregulation in mouse RTECs and mitochondrial damage were found in endotoxin-induced AKI. Overexpression of FOXO1 by kidney focal adeno-associated virus (AAV) delivery improved renal function and reduced mitochondrial damage. Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1-α), a master regulator of mitochondrial biogenesis and function, was reduced in endotoxin-induced AKI, but the reduction was reversed by FOXO1 overexpression. In vitro, exposure to LPS led to a decline in HK-2 cell viability, mitochondrial fragmentation, and mitochondrial superoxide accumulation, as well as downregulation of FOXO1, PGC1-α, and mitochondrial complex I/V. Moreover, overexpression of FOXO1 in HK-2 cells increased HK-2 cell viability and PGC1-α expression, and it alleviated the mitochondrial injury and superoxide accumulation induced by LPS. Meanwhile, inhibition of FOXO1 in HK-2 cells by siRNA treatment decreased PGC1-α expression and HK-2 cell viability. Chromatin immunoprecipitation assays and PCR analysis confirmed that FOXO1 bound to the PGC1-α promoter in HK-2 cells. In conclusion, downregulation of FOXO1 in RTECs mediated endotoxin-induced AKI and mitochondrial damage. Overexpression of FOXO1 could improve renal injury and mitochondrial dysfunction, and this effect occurred at least in part as a result of PGC1-α signaling. FOXO1 might be a potential target for the prevention and treatment of endotoxin-induced AKI.

Abstract MP131: The CDK2-FOXO1 Axis Mediates Doxorubicin-induced Cardiomyocyte Apoptosis and Atrophy

Recent clinical investigations indicate that anthracycline-based chemotherapy induces early declines in heart mass in cancer patients. A decline in heart mass can be caused by a decrease in cardiac cell number due to exaggerated cell death, and/or a reduction in cell size via atrophy. Our previous study reveals that the anthracycline doxorubicin (DOX) induces apoptotic death of cardiomyocytes through activation of cyclin-dependent kinase 2 (CDK2). However, the signaling pathway downstream of CDK2 remains to be characterized. In addition, it is unclear whether the same pathway also mediates cardiac atrophy. Here we demonstrated that DOX exposure induced CDK2-dependent phosphorylation of forkhead box O1 (FOXO1) at S249, leading to transcription of its pro-apoptotic target gene Bim. In cultured cardiomyocytes, treatment with the small molecule FOXO1 inhibitor AS1842856, or transfection with FOXO1-specific siRNAs protected against DOX-induced apoptosis and mitochondrial damage. Oral administration of AS1842856 in mice hindered apoptosis and prevented DOX-induced decline in left ventricular systolic function. Intriguingly, pharmacologic inhibition of FOXO1 also attenuated DOX-induced cardiac atrophy, likely due to repression of MuRF1, a pro-atrophic FOXO1 target gene. In conclusion, DOX exposure induces CDK2-dependent FOXO1 activation, resulting in cardiomyocyte apoptosis and atrophy. Our results identify FOXO1 as a promising drug target for DOX-induced cardiotoxicity. FOXO1 inhibitors may represent a novel class of therapeutics for cardioprotection during chemotherapy.

Human Norovirus Replication in Temperature-Optimized MDCK Cells by Forkhead Box O1 Inhibition.

Human noroviruses (HuNoVs) are a leading cause of gastroenteritis outbreaks worldwide. However, the paucity of appropriate cell culture model for HuNoV replication has prevented developing effective anti-HuNoV therapy. In this study, first, the replication of the virus at various temperatures in different cells was compared, which showed that lowering the culture temperature from 37°C significantly increased virus replication in Madin-Darby canine kidney (MDCK) cells. Second, the expression levels of autophagy-, immune-, and apoptosis-related genes at 30°C and 37°C were compared to explore factors affecting HuNoV replication. HuNoV cultured at 37°C showed significantly increased autophagy- (ATG5 and ATG7) and immune- (IFNA, IFNB, ISG15, and NFKB) related genes compared to mock. However, the virus cultured at 30°C showed significantly decreased expression of autophagy- (ATG5 and ATG7) and not significantly different in major immune- (IFNA, ISG15, and NFKB) related genes compared to mock. Importantly, expression of the transcription factor FOXO1, which controls autophagy- and immune-related gene expression, was significantly lower at 30°C. Moreover, FOXO1 inhibition in temperature-optimized MDCK cells enhanced HuNoV replication, highlighting FOXO1 inhibition as an approach for successful virus replication. In the temperature-optimized cells, various HuNoV genotypes were successfully replicated, with GI.8 showing the highest replication levels followed by GII.1, GII.3, and GII.4. Furthermore, ultrastructural analysis of the infected cells revealed functional HuNoV replication at low temperature, with increased cellular apoptosis and decreased autophagic vacuoles. In conclusion, temperature-optimized MDCK cells can be used as a convenient culture model for HuNoV replication by inhibiting FOXO1, providing adaptability to different genotypes.

Investigating the role and mechanism of microRNA-196a in oral squamous cell carcinoma by targeting FOXO1.

Oral squamous cell carcinoma (OSCC) is one of the most prevalent malignancies worldwide. MicroRNAs (miRNAs or miRs) serve crucial roles in the development of OSCC. miR-196a is upregulated in various tumors; however, the role of miR-196a in OSCC remains unclear. This present study aimed to determine the role and underlying mechanism of miR-196a in OSCC cells. Reverse transcription-quantitative PCR (RT-qPCR) was used to measure miR-196a levels in OSCC cells. MTT assays were also performed to determine cell proliferation. Cell migration was detected using wound healing assays and transwell assays, and cell apoptosis was analyzed via flow cytometry. The results indicated that the expression of miR-196a was increased in OSCC cells compared with normal oral squamous cells. TargetScan and luciferase reporter assays also confirmed that forkhead box O1 (FOXO1) was a target gene of miR-196a. It was demonstrated that FOXO1 small interfering RNA significantly promoted SCC9 cell proliferation and migration, and inhibited cell apoptosis. Furthermore, inhibition of miR-196a suppressed SCC9 cell proliferation and migration, and induced cell apoptosis. However, all effects of the miR-196a inhibitor were reversed following FOXO1 inhibition. Western blotting and RT-qPCR were subsequently performed to determine the effect of miR-196a on the PI3K/Akt signaling pathway. In the present study, transfection of miR-196a inhibitor suppressed the expression of phosphorylated (p)-PI3K and p-Akt, and enhanced the levels of FOXO1, while inhibition of FOXO1 exerted the opposite effects. Furthermore, it was demonstrated that miR-196a mimic significantly enhanced SCC9 cell proliferation and migration, and inhibited cell apoptosis. In conclusion, the results indicated that miR-196a serve as an oncogene in OSCCs. Downregulation of miR-196a inhibited the malignant biological processes of OSCC cells by targeting FOXO1. The current results may provide a novel therapeutic strategy for the treatment of patients with OSCC.

MicroRNA Let-7f-1-3p attenuates smoke-induced apoptosis in bronchial and alveolar epithelial cells in vitro by targeting FOXO1

Bronchial and alveolar epithelial cell apoptosis is a vital step in smoke-induced lung injury. We investigated whether and how microRNA (miRNA) Let-7f-1-3p would regulate smoke-induced apoptosis in bronchial and alveolar epithelial cells. Human small airway epithelial cells (HSAEC) and human pulmonary alveolar epithelial cells (HPAEpiC) were cultured using an air-liquid interface cell culture system. These cells were treated with Let-7f-1-3p agomir or antagomir for 24 h before smoke exposure or sham operation, after which the cells were rinsed and cultured for 24 h before cell viability, apoptosis, cytolysis, Caspase-9/8/3 activity assays, quantitative real-time polymerase chain reaction and Western blot. Bioinformatic and luciferase reporter assays were performed to predict or verify the target gene of Let-7f-1-3p. We found that smoke exposure significantly reduced Let-7f-1-3p expression level in HSAEC and HPAEpiC. Let-7f-1-3p agomir significantly attenuated cell apoptosis, cytolysis and Caspase-3, -8 and -9 activation while rescuing cell viability of smoke-exposed HSAEC and HPAEpiC. Let-7f-1-3p agomir downregulated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), Fas ligand (FasL) and B-cell lymphoma-2 (Bcl2)-like protein 11 (Bim) protein level in HSAEC and HPAEpiC. Forkhead box-O1 (FOXO1) was verified as a putative regulatory target of Let-7f-1-3p. Smoke exposure increased FOXO1 mRNA and protein level in HSAEC and HPAEpiC, which was attenuated by Let-7f-1-3p agomir treatment. FOXO1 inhibition by small-molecule drug partially attenuated the increase in smoke-exposed HSAEC and HPAEpiC apoptosis, cytolysis and the decrease in cell viability caused by Let-7f-1-3p antagomir treatment. We concluded Let-7f-1-3p attenuated smoke-induced apoptosis in HSAEC and HPAEpiC by targeting FOXO1.

Forkhead box O1 (FOXO1) controls the migratory response of Toll-like receptor (TLR3)-stimulated human mesenchymal stromal cells

Mesenchymal stromal cells (MSCs) can potently regulate the functions of immune cells and are being investigated for the management of inflammatory diseases. Toll-like receptor 3 (TLR3)-stimulated human MSCs (hMSCs) exhibit increased migration and chemotaxis within and toward damaged tissues. However, the regulatory mechanisms underlying these migratory activities are unclear. Therefore, we analyzed the migration capability and gene expression profiles of TLR3-stimulated hMSCs using RNA-Seq, wound healing, and transwell cell migration assay. Along with increased cell migration, the TLR3 stimulation also increased the expression of cytokines, chemokines, and cell migration-related genes. The promoter regions of the latter showed an enrichment of putative motifs for binding the transcription factors forkhead box O1 (FOXO1), FOXO3, NF-κB (NF-κB1), and RELA proto-oncogene and NF-κB subunit. Of note, FOXO1 inhibition by the FOXO1-selective inhibitor AS1842856 significantly reduced both migration and the expression of migration-related genes. In summary, our results indicate that TLR3 stimulation induces hMSC migration through the expression of FOXO1-activated genes.

Resveratrol Promotes Diabetic Wound Healing via SIRT1-FOXO1-c-Myc Signaling Pathway-Mediated Angiogenesis.

Background/Aims: Diabetic non-healing skin ulcers represent a serious challenge in clinical practice, in which the hyperglycemia-induced disturbance of angiogenesis, and endothelial dysfunction play a crucial role. Resveratrol (RES), a silent information regulator 1 (SIRT1) agonist, can improve endothelial function and has strong pro-angiogenic properties, and has thus become a research focus for the treatment of diabetic non-healing skin ulcers; however, the underlying mechanism by which RES regulates these processes remains unclear. Therefore, the present study was intended to determine if RES exerts its observed protective role in diabetic wound healing by alleviating hyperglycemia-induced endothelial dysfunction and the disturbance of angiogenesis.Methods: We investigated the effects of RES on cell migration, cell proliferation, apoptosis, tube formation, and the underlying molecular mechanisms in 33 mM high glucose-stimulated human umbilical vein endothelial cells (HUVECs) by semi-quantitative RT-PCR, western blot analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining, and immunofluorescence in vitro. We further explored the role of RES on endothelial dysfunction and wound healing disturbance in db/db mice by TUNEL staining, immunofluorescence, and photography in vivo.Results: We observed an obvious inhibition of hyperglycemia-triggered endothelial dysfunction and a disturbance of angiogenesis, followed by the promotion of diabetic wound healing via RES, along with restoration of the activity of the hyperglycemia-impaired SIRT1 signaling pathway. Pretreatment with EX-527, a SIRT1 inhibitor, abolished the RES-mediated endothelial protection and pro-angiogenesis action, and then delayed diabetic wound healing. Furthermore, examination of the overexpression of forkhead box O1 (FOXO1), a transcription factor substrate of SIRT1, in HUVECs and db/db mice revealed that RES activated SIRT1 to restore hyperglycemia-triggered endothelial dysfunction and disturbance of angiogenesis, followed by the promotion of diabetic wound healing in a c-Myc-dependent manner. Pretreatment with 10058-F4, a c-Myc inhibitor, repressed RES-mediated endothelial protection, angiogenesis, and diabetic wound healing.Conclusion: Our findings indicate that the positive role of RES in diabetic wound healing via its SIRT1-dependent endothelial protection and pro-angiogenic effects involves the inhibition of FOXO1 and the de-repression of c-Myc expression.

Metformin Protects the Heart Against Hypertrophic and Apoptotic Remodeling After Myocardial Infarction.

Cardiovascular complications are the most prevalent cause of morbidity and mortality in diabetic patients. Metformin is currently the first-line blood glucose-lowering agent with potential relevance to cardiovascular diseases. However, the underpinning mechanisms of action remain elusive. Here, we report that metformin represses cardiac apoptosis at least in part through inhibition of Forkhead box O1 (FoxO1) pathway. In a mouse model of ischemia-reperfusion (I/R), treatment with metformin attenuated cardiac and hypertrophic remodeling after 14 days of post-reperfusion. Additionally, cardiac expression of brain-like natriuretic peptide (BNP) was significantly reduced in metformin-treated mice after 14 days of cardiac I/R. In cultured H9C2 cells, metformin counteracted hypertrophic and apoptotic responses to metabolic or hypoxic stress. FoxO1 silencing by siRNA abolished anti-apoptotic effect of metformin under hypoxic stress in H9C2 cells. Taken together, these results suggest that metformin protects the heart against hypertrophic and apoptotic remodeling after myocardial infarction.

Role of Overexpressed Transcription Factor FOXO1 in Fatal Cardiovascular Septal Defects in Patau Syndrome: Molecular and Therapeutic Strategies.

Patau Syndrome (PS), characterized as a lethal disease, allows less than 15% survival over the first year of life. Most deaths owe to brain and heart disorders, more so due to septal defects because of altered gene regulations. We ascertained the cytogenetic basis of PS first, followed by molecular analysis and docking studies. Thirty-seven PS cases were referred from the Department of Pediatrics, King Abdulaziz University Hospital to the Center of Excellence in Genomic Medicine Research, Jeddah during 2008 to 2018. Cytogenetic analyses were performed by standard G-band method and trisomy13 were found in all the PS cases. Studies have suggested that genes of chromosome 13 and other chromosomes are associated with PS. We, therefore, did molecular pathway analysis, gene interaction, and ontology studies to identify their associations. Genomic analysis revealed important chr13 genes such as FOXO1, Col4A1, HMGBB1, FLT1, EFNB2, EDNRB, GAS6, TNFSF1, STARD13, TRPC4, TUBA3C, and TUBA3D, and their regulatory partners on other chromosomes associated with cardiovascular disorders, atrial and ventricular septal defects. There is strong indication of involving FOXO1 (Forkhead Box O1) gene—a strong transcription factor present on chr13, interacting with many septal defects link genes. The study was extended using molecular docking to find a potential drug lead for overexpressed FOXO1 inhibition. The phenothiazine and trifluoperazine showed efficiency to inhibit overexpressed FOXO1 protein, and could be potential drugs for PS/trisomy13 after validation.

Gα12gep oncogene inhibits FOXO1 in hepatocellular carcinoma as a consequence of miR-135b and miR-194 dysregulation

The high mortality rate of hepatocellular carcinoma (HCC) is associated with its fast-growing malignancy. In tumor microenvironments, certain GPCRs are coupled to Gα12 for signal transduction. Given the role of forkhead box O1 (FOXO1) in the inhibition of various tumors, this study investigated whether increase of Gα12 in HCC causes FOXO1 repression, and if so, whether this event occurs through microRNA dysregulation. Overexpression of an active mutant of Gα12 (Gα12QL) decreased FOXO1 levels, whereas knockdown of Gα12 had the opposite effect. Of the microRNAs targeting FOXO1, miR-135b levels were markedly increased by Gα12 signaling, which led to FOXO1 repression as shown by the experiments using mimic, antisense oligonucleotide or siRNA. Gα12QL increased the primary form of miR-135b by activating JunB (or c-Jun)/AP-1. Consistently, knockdown of JunB (or c-Jun) decreased miR-135b levels, thereby increasing FOXO1. Moreover, Gα12QL induced MDM2, the deficiency of which facilitated FOXO1 accumulation. In addition, Gα12QL repressed miR-194 cluster gene products (194/192/215), which contributed to MDM2-mediated FOXO1 repression. In functional assays, Gα12QL facilitated tumor cell growth with alterations in cell cycle-associated protein levels, which was antagonized by enforced expression of FOXO1. In human HCCs, FOXO1 levels were decreased as compared with the surrounding liver tissue. Moreover, decrease of FOXO1 or miR-194 was statistically significant between stages T1 and T2, whereas increase of miR-135b discriminated tumor stage T3a versus T1/T2. In conclusion, Gα12gep oncogene inhibits FOXO1, which may result from the inhibition of FOXO1 de novo synthesis by miR-135b in conjunction with MDM2-mediated destabilization of FOXO1.

Inhibition of miR-96 expression reduces cell proliferation and clonogenicity of HepG2 hepatoma cells

microRNAs (miRNAs) are negative regulators of gene expression and can function as tumor suppressors or oncogenes. Several miRNAs are associated with the development of hepatocellular carcinoma (HCC). miR-96 has been closely associated with cell proliferation and clonogenicity. Upregulation of miR-96 has been observed in various types of cancer. However, the biological function of miR-96 in hepatocarcinogenesis remains largely unknown. In this study, we demonstrated that miR-96 was upregulated in HCC and inhibition of miR-96 significantly suppressed HCC cell proliferation and colony formation. The expression levels of forkhead box O1 (FOXO1) and forkhead box O3a (FOXO3a) were upregulated when miR-96 was inhibited in HCC cells and the inhibition of FOXO1 and FOXO3a promoted HCC cell proliferation and colony formation. Collectively, these data reveal an important contribution of miR-96 to hepatocarcinogenesis and suggest a role for FOXO1 and FOXO3a dysregulation in this process. Thus, the use of a synthetic inhibitor of miR-96 may be a promising approach for the treatment of HCC.

Mild Endoplasmic Reticulum Stress Augments the Proinflammatory Effect of IL-1β in Pancreatic Rat β-Cells via the IRE1α/XBP1s Pathway

The prevalence of obesity and type 1 diabetes in children is increasing worldwide. Insulin resistance and augmented circulating free fatty acids associated with obesity may cause pancreatic β-cell endoplasmic reticulum (ER) stress. We tested the hypothesis that mild ER stress predisposes β-cells to an exacerbated inflammatory response when exposed to IL-1β or TNF-α, cytokines that contribute to the pathogenesis of type 1 diabetes. INS-1E cells or primary rat β-cells were exposed to a low dose of the ER stressor cyclopiazonic acid (CPA) or free fatty acids, followed by low-dose IL-1β or TNF-α. ER stress signaling was inhibited by small interfering RNA. Cells were evaluated for proinflammatory gene expression by RT-PCR and ELISA, gene reporter activity, p65 activation by immunofluorescence, and apoptosis. CPA pretreatment enhanced IL-1β- induced, but not TNF-α-induced, expression of chemokine (C-C motif) ligand 2, chemokine (C-X-C motif) ligand 1, inducible nitric oxide synthase, and Fas via augmented nuclear factor κB (NF-κB) activation. X-box binding protein 1 (XBP1) and inositol-requiring enzyme 1, but not CCAAT/enhancer binding protein homologous protein, knockdown prevented the CPA-induced exacerbation of NF-κB-dependent genes and decreased IL-1β-induced NF-κB promoter activity. XBP1 modulated NF-κB activity via forkhead box O1 inhibition. In conclusion, rat β-cells facing mild ER stress are sensitized to IL-1β, generating a more intense and protracted inflammatory response through inositol-requiring enzyme 1/XBP1 activation. These observations link β-cell ER stress to the triggering of exacerbated local inflammation.

Inhibition of Forkhead Box O1 Protects Pancreatic β-Cells against Dexamethasone-Induced Dysfunction

Forkhead Box O1 (FoxO1) is a key transcription regulator of insulin/IGF-I signaling pathway, and its activity can be increased by dexamethasone (DEX) in several cell types. However, the role of FoxO1 in DEX-induced pancreatic beta-cell dysfunction has not been fully understood. Therefore, in this study, we investigated whether FoxO1 could mediate DEX-induced beta-cell dysfunction and the possible underlying mechanisms in pancreatic beta-cell line RINm5F cells and primary rat islet. We found that DEX markedly increased FoxO1 mRNA and protein expression and decreased FoxO1 phosphorylation through the Akt pathway, which resulted in an increase in active FoxO1 in RINm5F cells and isolated rat islets. Activated FoxO1 subsequently inhibited pancreatic duodenal homeobox-1 expression and induced nuclear exclusion of pancreatic duodenal homeobox-1. Knockdown of FoxO1 by RNA interference restored the expression of pancreatic duodenal homeobox-1 and prevented DEX-induced dysfunction of glucose-stimulated insulin secretion in rat islets. Together, the results of present study demonstrate that FoxO1 is integrally involved in DEX-induced inhibition of pancreatic duodenal homeobox-1 and glucose-stimulated insulin secretion dysfunction in pancreatic islet beta-cells. Inhibition of FoxO1 can effectively protect beta-cells against DEX-induced dysfunction.