FOXO1 Knockdown Research Articles

Insulin Resistance Is a Modifying Factor for Parkinson's Disease.

Parkinson's disease (PD) is the second most common, and the fastest-growing neurodegenerative disorder with unclear etiology in most cases. Therefore, the identification of non-genetic risk factors for PD pathology is crucial to develop effective preventative or therapeutic strategies. An increasing number of evidence suggests that central insulin resistance might have an essential role in PD pathology. Nevertheless, it is not clear whether insulin resistance arises from external factors/lifestyle, comorbidities such as type 2 diabetes or it can occur in a PD patient's brain independently from peripheral insulin resistance. We aimed to investigate insulin resistance and its role in GBA1mutation-associated PD pathogenesis and phenotype severity. Midbrain organoids, generated from induced pluripotent stem cells (iPSCs) of PD patients carrying the GBA1-N370S heterozygous mutation (GBA-PD) and healthy donors, were exposed to different insulin concentrations to modify insulin signaling function. Transcriptomics analysis was performed to explore insulin signaling gene expression patterns in GBA-PD and to find a potential target for GBA-PD-associated phenotype rescue. The insulin signaling pathway genes show dysregulation in GBA-PD. Particularly, we highlight that a knockdown of FOXO1 mitigates the loss of dopaminergic neurons and cellular death in GBA-PD. Additionally, our findings suggest a promising therapeutic potential of the anti-diabetic drug Pioglitazone in decreasing dopaminergic neuron loss associated with GBA-PD. Local insulin signaling dysfunction plays a substantial role in GBA-PD pathogenesis, exacerbating dopaminergic neuron death. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Knock-down of FOXO3, GATA2, NFE2L2 and AHR promotes doxorubicin-induced cardiotoxicity in human cardiomyocytes

Recent advances in cancer therapy have substantially increased survival rates among patients, yet the prolonged effect of current treatment regimens with anthracyclines (ACs) often include severe long-term complications, notably in the form of anthracycline-induced cardiotoxicity (AIC). Despite known associations between AC treatment and AIC, a comprehensive understanding of the underlying molecular pathways remains elusive. This gap is highlighted by the scarcity of reliable therapeutic interventions, with dexrazoxane being the sole FDA-approved drug to mitigate AIC risks. This study aims at elucidating the transcriptional response of human cardiomyocytes (hCMs) to AC exposure by analyzing a previously generated RNA-sequencing dataset of cardiac spheroids subjected to clinically relevant doses of ACs. The analysis revealed a robust transcriptional response identified across various time points. We aimed at identifying important transcription factors (TFs) mediating AIC by employing predictive algorithms to highlight key TFs for further experimental validation. Using shRNA constructs, we further assessed the impact of these TFs on hCM response to doxorubicin (DOX) and revealed that these TFs had a notable impact on hCM survival upon DOX exposure. TFs FOXO3, GATA2, AHR and NFE2L2 were further investigated for their role in AIC including cell viability, DOX uptake, DNA damage repair and induction of apoptosis through Cleaved-Caspase 3. Our study demonstrated that eliminating FOXO3 and GATA2 made hCMs more vulnerable to DOX and the lack of GATA2, NFE2L2 and AHR led to significantly higher intracellular levels of DOX. Additionally, FOXO3 played a role in the repair of hCM DNA damage as we observed markedly enhanced levels of CDKN1A. We also noted significant increases in DNA damage through COMET-assays when FOXO3, GATA2, NFE2L2 and AHR were absent. Furthermore, we investigated the clinical relevance by comparing our results with those from a study based on hiPSC-CMs derived from patients with doxorubicin-induced cardiotoxicity, identifying overlapping TFs and their regulatory roles in critical cellular processes like the cell cycle and DNA repair. This approach not only advances the understanding of the molecular mechanisms behind AIC but also opens possible windows for new therapeutic approaches to mitigate the negative side-effects from patient AC treatment.

Transcription and post-translational mechanisms: dual regulation of adiponectin-mediated Occludin expression in diabetes

BackgroundOccludin, a crucial component of tight junctions, has emerged as a promising biomarker for the diagnosis of acute ischemic disease, highlighting its significant potential in clinical applications. In the diabetes, Occludin serves as a downstream target gene intricately regulated by the adiponectin (APN) signaling pathway. However, the specific mechanism by which adiponectin regulates Occludin expression remains unclear.Methods and resultsEndothelial-specific Ocln knockdown reduced APN-mediated blood flow recovery after femoral artery ligation and nullified APN's protection against high-fat diet (HFD)-triggered apoptosis and angiogenesis inhibition in vivo. Mechanically, we have meticulously elucidated APN's regulatory role in Occludin expression through a comprehensive analysis spanning transcriptional and post-translational dimensions. Foxo1 has been elucidated as a crucial transcriptional regulator of Occludin that is modulated by the APN/APPL1 signaling axis, as evidenced by validation through ChIP-qPCR assays and Western blot analysis. APN hindered Occludin degradation via the ubiquitin–proteasome pathway. Mass spectrometry analysis has recently uncovered a novel phosphorylation site, Tyr467, on Occludin. This site responds to APN, playing a crucial role in inhibiting Occludin ubiquitination by APN. The anti-apoptotic and pro-angiogenic effects of APN were attenuated in vitro and in vivo following Foxo1 knockdown or expression of a non-phosphorylatable mutant, OccludinY467A. Clinically, elevated plasma concentrations of Occludin were observed in patients with diabetes. A significant negative correlation was found between Occludin levels and APN concentrations.ConclusionOur study proposes that APN modulates Occludin expression through mechanisms involving both transcriptional and post-translational interactions, thereby conferring a protective effect on endothelial integrity within diabetic vasculature.Graphical abstract

Deciphering MARCH5’s impact on multiple myeloma: insights into autophagy regulation and AKT-FOXO3 signaling

AbstractMultiple myeloma (MM) stands as a formidable blood malignancy, necessitating innovative therapeutic approaches. Excessive immunoglobulin production within myeloma cells leads to a buildup of toxic proteins, and autophagy plays a crucial role in their survival by degrading toxic aggregates and generating energy. Membrane-associated RING finger protein 5 (MARCH5) is an E3-ligase positioned at the outer mitochondrial membrane and has been shown to regulate autophagy by competing for MIR30A. Given the fundamental significance of autophagy in promoting the survival of myeloma cells, coupled with the regulatory role of MARCH5 in autophagic activity, we hypothesized that MARCH5 plays an essential function in MM and holds a pivotal position in the pathogenesis and progression of MM. We identified MARCH5’s unique dependencies in MM cells by analyzing the Cancer Dependency Map, thereby establishing its significance in MM biology. Examining various data sets, including CoMMpass and HOVON, demonstrated a correlation between MARCH5 expression and patient outcomes. Knockdown of MARCH5 revealed a substantial reduction in MM cell viability, which was associated with a decrease in autophagic activity. Mechanistically, we unraveled a novel MARCH5/AKT/FOXO3 axis, wherein MARCH5 regulates autophagy through the AKT-mediated degradation of FOXO3. Compromised MM cell viability observed with MARCH5 knockdown was recapitulated in FOXO3 knockdown experiments, validating the pivotal role of FOXO3 in mediating MARCH5’s effects. In conclusion, this research highlights the crucial role of MARCH5 in MM, and the identified MARCH5/AKT/FOXO3 axis enhances our understanding of MM biology and provides a foundation for developing targeted therapies.

Forkhead box O-1 regulates the biological behavior of BMP-2-induced human bone mesenchymal stem cells through mitochondrial dynamics and autophagy.

This study explored the mechanism by which forkhead box O-1 (FoxO1) modulates the biological behaviors of bone mesenchymal stem cell (BMSC). Human BMSCs were cultured for 7 days in DMEM containing bone morphogenetic protein-2 (BMP-2) and treated with a short hairpin-FoxO1 plasmid. The study assessed cell proliferation, migration, apoptosis, adenosine triphosphate (ATP) levels, mitochondrial DNA levels, membrane potential, autophagy, and the levels of FoxO1, apoptosis-associated proteins, osteogenic differentiation-associated proteins, mitochondrial fusion and fission proteins and mitochondrial autophagy-related proteins. The cells were also treated with the mitochondrial fusion activator MASM7 and the mitochondrial autophagy activator carbonyl cyanide 3-chlorophenylhydrazone. The study evaluated whether mitochondrial dynamics and autophagy activation could rescue the FoxO1 knockdown-induced changes in BMSC biological behaviors, mitochondrial dynamics, and mitochondrial autophagy. BMP-2-induced BMSCs exhibited up-regulated FoxO1 expression, enhanced proliferation and migration, and induced osteogenic differentiation, while FoxO1 knockdown inhibited BMP-2-induced BMSC proliferation, migration and osteogenic differentiation, increased apoptosis, and affected mitochondrial dynamics and autophagy. Promoting mitochondrial fusion partially reversed the regulatory effects of FoxO1 downregulation on mitochondrial autophagy and the inhibitory effects of FoxO1 silencing on BMP-2-induced BMSC biological behaviors. Activated mitochondrial autophagy facilitated the homeostasis of mitochondrial dynamics and partially counteracted the inhibitory effects of FoxO1 knockdown on BMP-2-induced BMSC biological behaviors. In conclusion, FoxO1 regulates mitochondrial dynamics and autophagy to modulate the osteogenic differentiation of BMP-2-induced human BMSCs.

P14-05 Knock-down of FOXO3, GATA2, NFE2L2 and AHR promotes doxorubicin-induced cardiotoxicity in human cardiomyocytes

P14-05 Knock-down of FOXO3, GATA2, NFE2L2 and AHR promotes doxorubicin-induced cardiotoxicity in human cardiomyocytes

FoxO1-modulated macrophage polarization regulates osteogenesis via PPAR-γ signaling

Periodontitis, a common chronic inflammatory disease, epitomizes a significant impairment in the host immune system and an imbalance of bone metabolism. Macrophage polarization, a dynamic process dictated by the microenvironment, intricately contributes to the interplay between the immune system and bone remodeling, namely the osteoimmune system. Forkhead box protein O1 (FoxO1) has been shown to play a dramatic role in mediating oxidative stress, bone mass, as well as cellular metabolism. Nevertheless, the function and underlying mechanisms of FoxO1 in regulating macrophage polarization-mediated osteogenesis in periodontitis remain to be further elucidated. Here, we found that FoxO1 expression was closely linked to periodontitis, accompanied by aggravated inflammation. Notably, FoxO1 knockdown skewed macrophage polarization from M1 to the antiinflammatory M2 phenotype under inflammatory conditions, which rescued the impaired osteogenic potential. Mechanistically, we revealed that the enhancement of the transcription of peroxisome proliferator-activated receptor (PPAR) signaling in FoxO1-knockdown macrophages. In agreement with this contention, GW9662, a specific inhibitor of PPAR-γ signaling, greatly aggravated macrophage polarization from M2 to the M1 phenotype and attenuated osteogenic potential under inflammatory conditions. Additionally, PPAR-γ signaling agonist rosiglitazone (RSG) was applied to address ligature-induced periodontitis with attenuated inflammation. Our data lend conceptual credence to the function of FoxO1 in mediating macrophage polarization-regulated osteogenesis which serves as a novel therapeutic target for periodontitis.

MiR-486-5p predicted adverse outcomes of SCAP and regulated K. pneumonia infection via FOXO1

PurposeSevere community-acquired pneumonia (SCAP) is a common respiratory system disease with rapid development and high mortality. Exploring effective biomarkers for early detection and development prediction of SCAP is of urgent need. The function of miR-486-5p in SCAP diagnosis and prognosis was evaluated to identify a promising biomarker for SCAP.Patients and methodsThe serum miR-486-5p in 83 patients with SCAP, 52 healthy individuals, and 68 patients with mild CAP (MCAP) patients were analyzed by PCR. ROC analysis estimated miR-486-5p in screening SCAP, and the Kaplan-Meier and Cox regression analyses evaluated the predictive value of miR-486-5p. The risk factors for MCAP patients developing SCAP were assessed by logistic analysis. The alveolar epithelial cell was treated with Klebsiella pneumonia to mimic the occurrence of SCAP. The targeting mechanism underlying miR-486-5p was evaluated by luciferase reporter assay.ResultsUpregulated serum miR-486-5p screened SCAP from healthy individuals and MCAP patients with high sensitivity and specificity. Increasing serum miR-486-5p predicted the poor outcomes of SCAP and served as a risk factor for MCAP developing into SCAP. K. pneumonia induced suppressed proliferation, significant inflammation and oxidative stress in alveolar epithelial cells, and silencing miR-486-5p attenuated it. miR-486-5p negatively regulated FOXO1, and the knockdown of FOXO1 reversed the effect of miR-486-5p in K. pneumonia-treated alveolar epithelial cells.ConclusionmiR-486-5p acted as a biomarker for the screening and monitoring of SCAP and predicting the malignancy of MCAP. Silencing miR-486-5p alleviated inflammation and oxidative stress induced by K. pneumonia via negatively modulating FOXO1.

FoxO1 promotes ovarian cancer by increasing transcription and METTL14-mediated m6A modification of SMC4.

The transcription factor forkhead box protein O1 (FoxO1) is closely related to the occurrence and development of ovarian cancer (OC), however its role and molecular mechanisms remain unclear. Herein, we found that FoxO1 was highly expressed in clinical samples of OC patients and was significantly correlated with poor prognosis. FoxO1 knockdown inhibited the proliferation of OC cells invitro and invivo. ChIP-seq combined with GEPIA2 and Kaplan-Meier database analysis showed that structural maintenance of chromosome 4 (SMC4) is a downstream target of FoxO1, and FoxO1 promotes SMC4 transcription by binding to its -1400/-1390 bp promoter. The high expression of SMC4 significantly blocked the tumor inhibition effect of FoxO1 knockdown. Furtherly, FoxO1 increased SMC4 mRNA abundance by transcriptionally activating methyltransferase-like 14 (METTL14) and increasing SMC4 m6A methylation on its coding sequence region. The Cancer Genome Atlas dataset analysis confirmed a significant positive correlation between FoxO1, SMC4, and METTL14 expression in OC. In summary, this study revealed the molecular mechanisms of FoxO1 regulating SMC4 and established a clinical link between the expression of FoxO1/METTL14/SMC4 in the occurrence of OC, thus providing a potential diagnostic target and therapeutic strategy.

Inhibition of FoxO1 alleviates polycystic ovarian syndrome by reducing inflammation and the immune response.

The aim of this study was to explore the role of forkhead box transcription Factor O1 (FoxO1) in chronic inflammation in polycystic ovary syndrome (PCOS). A PCOS rat model was constructed as an in vivo model by letrozole induction, and granulosa cells (GCs) from PCOS rats were isolated and cultured as an in vitro cellular model. FoxO1 was knocked down by shRNA and siRNA in the PCOS rat model and GCs model, respectively. H&E staining was conducted to evaluate the effect of FoxO1 inhibition on ovarian pathology and dysfunction in PCOS rats. The levels of inflammatory cytokines in the ovaries and uterus of PCOS rats and in GCs were assessed by ELISA. Flow cytometry was used to evaluate the changes in the contents of neutrophils and macrophages in the peripheral blood and spleen of PCOS rats. CCK-8 assays and Annexin V-FITC/PI staining were performed to evaluate the proliferation and apoptosis of GCs. The expression of genes and proteins related to the TLR4/NF-κB/NLRP3 pathway in GCs was determined by RT-qPCR and Western blotting. The results indicated that FoxO1 was highly expressed in PCOS rat model. Inhibition of FoxO1 significantly mitigated the pathological changes and dysfunction in the ovaries of PCOS rats while also suppressing inflammation and fibrosis in the ovaries and uterus. Moreover, knocking down FoxO1 facilitated the restoration of the normal ratio of neutrophils and macrophages in the peripheral blood and spleen of PCOS rats and promoted M2 polarization of macrophages. Additionally, inhibition of FoxO1 promoted the proliferation of GCs and inhibited the inflammatory response in GCs. Furthermore, FoxO1 knockdown inhibited the activation of the NF-κB pathway and the formation of the NLRP3 inflammasome in GCs. In conclusion, inhibition of FoxO1 can alleviate PCOS by inhibiting the TLR4/NF-κB/NLRP3 pathway to reduce inflammation and the immune response.

Prolyl isomerase Pin1 promotes autophagy and cancer cell viability through activating FoxO3 signalling.

Pin1-directed prolyl isomerization is a central common oncogenic mechanism to drive tumorigenic processes. However, the role of Pin1 in cellular autophagy is still poorly understood. Here we report that pharmacological inhibition of Pin1 decreased the formation of autophagosome/autolysosomes upon nutrient starvation. Inhibition of Pin1 reduced, whereas forced expression of Pin1 increased, the level of LC3 and viability of U2OS and PANC-1 cells. Pin1 could augment the accumulation of LC3 upon chloroquine treatment, while chloroquine also disturbed its function on cell viability. RNA-Seq and qPCR identified altered autophagic pathway upon Pin1 silencing. Mechanistically, FoxO3 was identified critical for Pin1-mediated autophagy. Knockdown of FoxO3 could rescue the changes of LC3 level and cellular viability caused by Pin1 overexpression. In xenograft mouse model, Pin1 reduced the sensitivity of PANC-1 to chloroquine while FoxO3 silencing could inhibit Pin1's function. Moreover, Pin1 could bind FoxO3 via its pS284-P motif, reduce its phosphorylation at T32, facilitate its nuclear retention, and therefore increased its transcriptional activity. S284A mutation of FoxO3 interfered with its T32 phosphorylation, reduced its nuclear localization and disrupted its function to support cell viability upon nutrient starvation. Furthermore, the protein level of Pin1 positively correlated with FoxO3 nuclear localization and LC3 level in pancreatic adenocarcinoma and osteosarcoma samples. Together, this study highlights an important role for Pin1-FoxO3 axis in regulating autophagy and cancer cell viability. Intervening in the Pin1-FoxO3 interaction would serve as an effective therapeutic strategy and the pS284-P motif of FoxO3 provides a potential target for drug design.

Interferon-tau protects bovine endometrial epithelial cells against inflammatory injury by regulating the PI3K/AKT/β-catenin/FoxO1 signaling axis

Endometritis is one of the most common causes of infertility in dairy cows, and is histopathologically characterized by inflammation and damage of endometrial epithelium. Interferon-tau (IFN-τ) is a novel type I interferon secreted by ruminant trophoblast cells with low cytotoxicity even at high doses. Previous studies suggested that IFN-τ plays an important role in inflammation. However, the mechanisms whereby IFN-τ may modulate the inflammatory responses in the bovine endometrium are unknown. In the present study, primary bovine endometrial epithelial cells (BEEC) isolated from fresh and healthy uterine horns were used for in vitro studies. The integrity of BEEC was assessed by immunofluorescence staining for cytokeratin 18 (CK-18, a known epithelial marker). For the experiments, BEEC were stimulated with different concentrations of lipopolysaccharide (LPS; 0-20 µg/mL) for different times (0-24 h). Cell viability and apoptosis were assessed via CCK-8 and flow cytometry. In a preliminary study, we observed that compared with the control group without LPS, 10 µg/mL of LPS stimulation for 24 h induced apoptosis. In a subsequent study, 20 or 40 ng/mL of IFN-τ alleviated LPS-induced apoptosis. Relative to the LPS group, western blotting further revealed that IFN-τ inhibited the protein abundance of TLR4 and phosphorylated (p-) p65 (p-p65) and Bax/Bcl-2 ratio, suggesting that IFN-τ can protect BEEC against inflammatory injury. Furthermore, the protein abundance of p-phosphoinositide 3-kinase (p-PI3K), p-protein kinase B (p-AKT), p-glycogen synthase kinase-3β (p-GSK3β), β-catenin, and p-forkhead box O1 (p-FoxO1) was lower in the LPS group, whereas IFN-τ upregulated their abundance. The use of LY294002, a specific inhibitor of PI3K/AKT, attenuated the upregulation of p-PI3K, p-AKT p-GSK3β, β-catenin, and p-FoxO1 induced by IFN-τ, and also blocked the downregulation of TLR4, p-p65, and Bax/Bcl-2 ratio. This suggested that the inhibition of TLR4 signaling by IFN-τ was mediated by the PI3K/AKT pathway. Furthermore, compared with the LPS group, the β-catenin agonist SB216763 led to greater p-FoxO1 and lower p-p65 and cell apoptosis. In contrast, knockdown of β-catenin using small interfering RNA had the opposite effects. To explore the role of FoxO1 on the inhibition of TLR4 by IFN-τ, we employed LY294002 to inhibit the PI3K/AKT while FoxO1 was knocked down. Results revealed that the knockdown of FoxO1 blocked the upregulation of TLR4 and p-p65 induced by LY294002, and enhanced the inhibition of IFN-τ on TLR4, p-p65, and cell apoptosis. Overall, these findings confirmed that IFN-τ can protect endometrial epithelial cells against inflammatory injury via suppressing TLR4 activation through the regulation of the PI3K/AKT/β-catenin/FoxO1 axis. These represent new insights into the molecular mechanisms underlying the anti-inflammatory function of IFN-τ in BEEC, and also provide a theoretical basis for further studies on the in vivo application of IFN-τ to help prevent negative effects of endometritis.

M6A demethylase ALKBH5 regulates FOXO1 mRNA stability and chemoresistance in triple-negative breast cancer

Resistance to chemotherapy is the main reason for treatment failure and poor prognosis in patients with triple-negative breast cancer (TNBC). Although the association of RNA N6-methyladenosine (m6A) modifications with therapy resistance is noticed, its role in the development of therapeutic resistance in TNBC is not well documented. This study aimed to investigate the potential mechanisms underlying reactive oxygen species (ROS) regulation in doxorubicin (DOX)-resistant TNBC. Here, we found that DOX-resistant TNBC cells displayed low ROS levels because of increased expression of superoxide dismutase (SOD2), thus maintaining cancer stem cells (CSCs) characteristics and DOX resistance. FOXO1 is a master regulator that reduces cellular ROS in DOX-resistant TNBC cells, and knockdown of FOXO1 significantly increased ROS levels by inhibiting SOD2 expression. Moreover, the m6A demethylase ALKBH5 promoted m6A demethylation of FOXO1 mRNA and increased FOXO1 mRNA stability in DOX-resistant TNBC cells. The analysis of clinical samples revealed that the increased expression levels of ALKBH5, FOXO1, and SOD2 were significantly positively correlated with chemoresistance and poor prognosis in patients with TNBC. To our knowledge, this is the first study to highlight that ALKBH5-mediated FOXO1 mRNA demethylation contributes to CSCs characteristics and DOX resistance in TNBC cells. Furthermore, pharmacological targeting of FOXO1 profoundly restored the response of DOX-resistant TNBC cells, both in vitro and in vivo. In conclusion, we demonstrated a critical function of ALKBH5-mediated m6A demethylation of FOXO1 mRNA in restoring redox balance, which in turn promoting CSCs characteristics and DOX resistance in TNBC, and suggested that targeting the ALKBH5/FOXO1 axis has therapeutic potential for patients with TNBC refractory to chemotherapy.

Therapeutic Consequences of Targeting the IGF-1/PI3K/AKT/FOXO3 Axis in Sarcopenia: A Narrative Review.

The high prevalence of sarcopenia in an aging population has an underestimated impact on quality of life by increasing the risk of falls and subsequent hospitalization. Unfortunately, the application of the major established key therapeutic-physical activity-is challenging in the immobile and injured sarcopenic patient. Consequently, novel therapeutic directions are needed. The transcription factor Forkhead-Box-Protein O3 (FOXO3) may be an option, as it and its targets have been observed to be more highly expressed in sarcopenic muscle. In such catabolic situations, Foxo3 induces the expression of two muscle specific ubiquitin ligases (Atrogin-1 and Murf-1) via the PI3K/AKT pathway. In this review, we particularly evaluate the potential of Foxo3-targeted gene therapy. Foxo3 knockdown has been shown to lead to increased muscle cross sectional area, through both the AKT-dependent and -independent pathways and the reduced impact on the two major downstream targets Atrogin-1 and Murf-1. Moreover, a Foxo3 reduction suppresses apoptosis, activates satellite cells, and initiates their differentiation into muscle cells. While this indicates a critical role in muscle regeneration, this mechanism might exhaust the stem cell pool, limiting its clinical applicability. As systemic Foxo3 knockdown has also been associated with risks of inflammation and cancer progression, a muscle-specific approach would be necessary. In this review, we summarize the current knowledge on Foxo3 and conceptualize a specific and targeted therapy that may circumvent the drawbacks of systemic Foxo3 knockdown. This approach presumably would limit the side effects and enable an activity-independent positive impact on skeletal muscle.

Effective-components combination alleviates PM2.5-induced inflammation by evoking macrophage autophagy in COPD

Ethnopharmacological relevanceBufei Yishen formula (BYF) is clinically used to treat chronic obstructive pulmonary disease (COPD). Effective-component compatibility (ECC) is a combination of five active components derived from BYF, which has an equal effect on COPD to BYF. Our previous study has also demonstrated that ECC can protect COPD rats against PM2.5 exposure. However, the precise mechanisms remain to be elucidated. Aim of the studyTo explore the mechanism underlying the anti-inflammatory effects of ECC-BYF against PM2.5-accelerated COPD. Materials and methodsMH-S macrophages were stimulated by PM2.5 suspension to establish an in vitro model. Western blotting and immunofluorescent staining were used to measure the protein levels of autophagy markers. ELISA and quantitative PCR were used to detect the levels of inflammatory cytokines. In vivo, an established PM2.5-accelerated COPD rat model was used to determine the protective effect of ECC-BYF. Lung function, pathology, autophagy, and inflammatory mediators were detected. ResultsFirstly, we observed a significantly increased number of macrophages in the lungs upon PM2.5 exposure. Then, decreased autophagy flux while elevated inflammation was detected in PM2.5-exposed rats and MH-S cells. In MH-S cells, ECC-BYF significantly suppressed the PM2.5-increased inflammatory cytokines production, which was accompanied by the enhancement of autophagy flux. An autophagy inhibitor counteracted the anti-inflammatory effect elicited by ECC-BYF. In addition, ECC-BYF stimulated Foxo3 nuclear translocation and upregulated Foxo3 expression, whereas Foxo3 knockdown abrogated the inhibitory effect of ECC-BYF on inflammation. In PM2.5-accelerated COPD rats, ECC-BYF also attenuated the autophagy disruption and increased Foxo3 in the lungs, finally resulting in a suppression of pulmonary inflammation and an enhancement of lung function. ConclusionECC-BYF can ameliorate PM2.5-aggravated inflammation in COPD, which might be associated with the enhancement of autophagy flux in alveolar macrophages through the activation of Foxo3 signals.

Abstract 18545: GSK-3β Prevents Lipotoxicity in Obesity Cardiomyopathy Through Upregulation of ATGL

Introduction: Obesity induces accumulation of lipids in cardiomyocytes, leading to cardiac dysfunction, termed lipotoxic cardiomyopathy. We have shown previously that GSK-3α promotes fatty acid uptake in the heart. In contrast, GSK-3β may mediate lipid catabolism. In this study, we aimed to elucidate the role of GSK-3β in lipolysis, primarily focusing on the post-translational regulation of FoxO1 and its association with ATGL, which is the first lipase to hydrolyze lipids. Methods: Wild-type (WT) and homozygous cardiac-specific GSK-3β knock-out (GSK-3β-cKO) mice were fed a normal diet (ND) or a high fat diet (HFD) for 3 months. Results: HFD consumption in WT mice activated GSK-3β in the heart, as indicated by decreases in Ser9-phosphorylated GSK-3β/total GSK-3β (0.5-fold, p<0.05 compared to ND consumption), and upregulated ATGL (2.4-fold, p<0.05) in the heart. Echocardiographic analyses showed preserved systolic and diastolic function in WT mice at this time point (LVEF: 56.3 ± 1.5 %, E/A: 2.06 ± 0.22, n = 13). However, ATGL was not increased (1.6-fold) and both systolic and diastolic dysfunction were observed (LVEF: 45.8 ± 1.5 %, p<0.01 compared to WT, E/A: 1.43 ± 0.20, p<0.05, n =12) in GSK-3β-cKO mice fed a HFD. GSK-3β overexpression in neonatal rat ventricular cardiomyocytes increased ATGL in a dose-dependent manner (p<0.05). FoxO1 knock-down on this background inhibited upregulation of ATGL, suggesting that GSK-3β-induced upregulation of ATGL is mediated through a FoxO1-dependent mechanism. Immunoprecipitation showed that GSK-3β interacts with FoxO1, while mass spectrometry analyses revealed that GSK-3β phosphorylates FoxO1 at 8 Serine/Threonine residues. A FoxO1 mutant in which Serine/Threonine residues at the GSK-3β phosphorylation sites are mutated to Alanine was excluded from the nucleus, and its overexpression prevented upregulation of ATGL in the presence of GSK-3β overexpression. Conclusions: GSK-3β is activated in the presence of HFD consumption in the heart, which may, in turn, prevent lipotoxic cardiomyopathy through phosphorylation of FoxO1 and upregulation of ATGL.

Abstract 12253: Occludin as a Key Mediator in Adiponectin-Mediated Diabetic Vascular Protection

Background: APN exerts its vascular protective effects through APPL1. However, the downstream target genes that mediate the protective effects of this signaling axis on diabetic vascular endothelium remain unclear. Methods and Results: APPL1knock-out mice were fed normal or high-fat-diet (HFD) for 10 weeks. 8 weeks after HFD, mice were randomized to receive vehicle or APN treatment for another two weeks. Aortic vessels were prepared for transcriptome analysis. Bioinformatics analysis indicates that the Tight Junction (TJ) pathway exhibited the most prominent changes. Using a global signaling transduction network showed that TJ-related genes had the highest degree values. Among these genes, PCR results indicated that Occludin was the sole gene inhibited in endothelial cells treated by HG/HL and promoted by APN treatment in an APPL1-dependent manner. The protein level of Occludin was reduced in diabetic condition, and APN treatment upregulated the expression of Occludin in APPL1-mediated manner in vivo and vitro. Endothelial specific OCLN knockdown blunted APN-mediated blood flow recovery after femoral artery ligation in vivo , and abolished APN’s effects upon HG/HL-induced permeability, and apoptosis in vitro . Mechanically, transcription factor expression and activity assay kits, and mass spectrometry techniques were employed to identify transcription factors involved in mediating the APN/APPL1 signaling axis. Through a comprehensive analysis, we discovered MYB, HDAC1, and FOXO1 play a role in the regulation of Occludin. This finding was validated by CHIP-QPCR. Western blot analysis revealed that the APN-induced increase in Occludin protein level was abolished after knockdown of MYB and FOXO1. However, knockdown of HDAC1 in endothelial cells did not alter the expression of Occludin mediated by APN. Finally, CHIP-qPCR showed that both MYB and FOXO1 participate in the enrichment of Occludin promoters mediated by APN. Conclusion: Our findings firstly indicate that the APN/APPL1 signaling axis promotes the expression of Occludin through MYB and FOXO1, ultimately exerting a protective effect on vascular endothelium.

FoxO1 knockdown inhibits RANKL-induced osteoclastogenesis by blocking NLRP3 inflammasome activation.

This study aimed to elucidate the connection between osteoclastic forkhead transcription factor O1 (FoxO1) and periodontitis and explore the underlying mechanism by which FoxO1 knockdown regulates osteoclast formation. A conventional ligature-induced periodontitis model was constructed to reveal the alterations in the proportion of osteoclastic FoxO1 in periodontitis via immunofluorescence staining. Additionally, RNA sequencing (RNA-seq) was performed to explore the underlying mechanisms of FoxO1 knockdown-mediated osteoclastogenesis, followed by western blotting, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. FoxO1+ osteoclasts were enriched in the alveolar bone in experimental periodontitis. Moreover, FoxO1 knockdown led to impaired osteoclastogenesis with low expression of osteoclast differentiation-related genes, accompanied by an insufficient osteoclast maturation phenotype. Mechanistically, RNA-seq revealed that the nuclear factor kappa B (NF-κB) and nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome signaling pathways were inhibited in FoxO1-knockdown osteoclasts. Consistent with this, MCC950, an effective inhibitor of the NLRP3 inflammasome, substantially attenuated osteoclast formation. FoxO1 knockdown contributed to the inhibition of osteoclastogenesis by effectively suppressing NF-κB signaling and NLRP3 inflammasome activation. This prospective study reveals the role of FoxO1 in mediating osteoclastogenesis and provides a viable therapeutic target for periodontitis treatment.

Foxo3 Knockdown Mediates Decline of Myod1 and Myog Reducing Myoblast Conversion to Myotubes.

Sarcopenia has a high prevalence among the aging population. Sarcopenia is of tremendous socioeconomic importance because it can lead to falls and hospitalization, subsequently increasing healthcare costs while limiting quality of life. In sarcopenic muscle fibers, the E3 ubiquitin ligase F-Box Protein 32 (Fbxo32) is expressed at substantially higher levels, driving ubiquitin-proteasomal muscle protein degradation. As one of the key regulators of muscular equilibrium, the transcription factor Forkhead Box O3 (FOXO3) can increase the expression of Fbxo32, making it a possible target for the regulation of this detrimental pathway. To test this hypothesis, murine C2C12 myoblasts were transduced with AAVs carrying a plasmid for four specific siRNAs against Foxo3. Successfully transduced myoblasts were selected via FACS cell sorting to establish single clone cell lines. Sorted myoblasts were further differentiated into myotubes and stained for myosin heavy chain (MHC) by immunofluorescence. The resulting area was calculated. Myotube contractions were induced by electrical stimulation and quantified. We found an increased Foxo3 expression in satellite cells in human skeletal muscle and an age-related increase in Foxo3 expression in older mice in silico. We established an in vitro AAV-mediated FOXO3 knockdown on protein level. Surprisingly, the myotubes with FOXO3 knockdown displayed a smaller myotube size and a lower number of nuclei per myotube compared to the control myotubes (AAV-transduced with a functionless control plasmid). During differentiation, a lower level of FOXO3 reduced the expression Fbxo32 within the first three days. Moreover, the expression of Myod1 and Myog via ATM and Tp53 was reduced. Functionally, the Foxo3 knockdown myotubes showed a higher contraction duration and time to peak. Early Foxo3 knockdown seems to terminate the initiation of differentiation due to lack of Myod1 expression, and mediates the inhibition of Myog. Subsequently, the myotube size is reduced and the excitability to electrical stimulation is altered.

Targeting FoxO proteins induces lytic reactivation of KSHV for treating herpesviral primary effusion lymphoma.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic virus consisting of both latent and lytic life cycles. Primary effusion lymphoma (PEL) is an aggressive B-cell lineage lymphoma, dominantly latently infected by KSHV. The latent infection of KSHV is persistent and poses an obstacle to killing tumor cells. Like the "shock and kill" strategy designed to eliminate latent HIV reservoir, methods that induce viral lytic reactivation in tumor latently infected by viruses represent a unique antineoplastic strategy, as it could potentially increase the specificity of cytotoxicity in cancer. Inspired by this conception, we proposed that the induction of KSHV lytic reactivation from latency could be a potential therapeutic stratagem for KSHV-associated cancers. Oxidative stress, the clinical hallmark of PEL, is one of the most prominent inducers for KSHV reactivation. Paradoxically, we found that hydrogen peroxide (H2O2) triggers robust cytotoxic effects on KSHV-negative rather than KSHV-positive B lymphoma cells in a dose-dependent manner. Mechanistically, we identified forkhead box protein O1 (FoxO1) and FoxO3 as irrevocable antioxidant defense genes and both of them are upregulated by KSHV latent infection, which is essential for the promoted ROS scavenging in KSHV-positive B lymphoma cells. Pharmacological inhibition or functional knockdown of either FoxO1 or FoxO3 is sufficient to ablate the antioxidant ability and therefore increases the intracellular ROS level that further reverses KSHV from latency to active lytic replication in PEL cells, resulting in tremendous cell death both in vitro and in vivo. Additionally, the elevated level of ROS by inhibiting FoxO proteins further sensitizes PEL cells to ROS-induced apoptosis. Our study therefore demonstrated that the lytic reactivation of KSHV by inhibiting FoxO proteins is a promising therapeutic approach for PEL, which could be further extended to other virus-associated diseases.