Enterotoxigenic Escherichia coli infection promotes enteric defensin expression via FOXO6-METTL3-m6A-GPR161 signalling axis.

The production of natural antimicrobial peptides has emerged as an important mechanism of innate immunity in animals. Defensins, members of a large family of antimicrobial peptides, have been suggested as effector molecules in host defence against bacteria, fungi, protozoa and enveloped viruses. However, the molecular mechanism underlying defensin upregulation in bacterial infection remains poorly understood. The modification of mRNA by N6-adenosine methylation (m6A) on internal bases influences gene expression in eukaryotes. Here, we show that ß-defensin production triggered by Enterotoxigenic Escherichia coli K88 (E. coli K88) infection is controlled by the cellular m6A methyltransferase METTL3. Adding back with METTL3 robustly stimulated the re-expression of defensin, which further supports the conclusion. Furthermore, using a MeRIP-seq approach, we identified a functional connection between m6A dependent GPR161 signalling and the expression of defensins. Mechanistically, we found that the transcription factor FOXO6 interacted with METTL3 to trigger the transcription of GPR161 and the subsequent regulation of ß-defensin expression. The study has shed light on the mechanisms by which enterotoxigenic Escherichia coli infection promotes enteric defensin expression.

LncRNA HOXC-AS1 promotes nasopharyngeal carcinoma (NPC) progression by sponging miR-4651 and subsequently upregulating FOXO6.

The incidence rate of nasopharyngeal carcinoma (NPC) is the highest among the malignant tumors of otorhinolaryngology, posing a huge burden to public health. Long noncoding RNAs (lncRNAs) exert an important role in tumorigenesis and the progression of various cancers. The present study found that HOXC-AS1 was highly expressed in NPC and in NPC cell lines, suggesting a critical role of HOXC-AS1 in NPC progression. In addition, the abundance of HOXC-AS1 was negatively correlated with the prognosis of NPC. To molecularly dissect the mechanism of HOXC-AS1 in NPC progression, we knocked down the expression of HOXC-AS1 in HNE1 and C666-1 cells. Then, we employed CCK8, colony-formation experiment and Transwell to investigate how the cell performed when HOXC-AS1 was knocked down. It could be observed that HOXC-AS1 knockdown decreases cell proliferation, migration and invasion, but induces cell apoptosis in NPC. We found that HOXC-AS1 could sponge miR-4651 subsequently binding FOXO6 and inhibiting its expression. Therefore, HOXC-AS1/miR-4651/FOXO6 may form a competing endogenous RNA (ceRNA) network that promotes NPC progression. In conclusion, our study demonstrates that HOXC-AS1 promotes NPC progression by sponging miR-4651 and regulating FOXO6 expression, thus providing potential pharmaceutical targets for developing new NPC treatments.

Downregulation of FOXO6 alleviates hypoxia-induced apoptosis and oxidative stress in cardiomyocytes by enhancing Nrf2 activation via upregulation of SIRT6.

Forkhead box protein O6 (FOXO6) has been recently identified as a novel regulator of oxidative stress in multiple pathological processes. However, whether FOXO6 participates in the regulation of oxidative stress of myocardial infarction is unclear. The present study was performed to evaluate the potential role of FOXO6 in regulating hypoxia-induced apoptosis and oxidative stress in cardiomyocytes in vitro. Our results demonstrated that FOXO6 expression was highly elevated in cardiomyocytes exposed to hypoxia. Downregulation of FOXO6 expression by the siRNA-mediated gene knockdown in hypoxia-exposed cardiomyocytes increased cell viability, while repressing apoptosis and reactive oxygen species (ROS) production. In contrast, overexpression of FOXO6 enhanced the sensitivity of cardiomyocytes to hypoxia-induced injury. Further, in-depth research revealed that knockdown of FOXO6 promoted the expression of sirtuin6 (SIRT6) and enhanced the activation of nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant signaling. Moreover, SIRT6 inhibition markedly blocked the FOXO6 knockdown-induced promotion effect on Nrf2 activation. In addition, Nrf2 inhibition partially reversed the FOXO6 knockdown-mediated protective effect against hypoxia-induced cardiomyocyte injury. Taken together, the findings of our study demonstrate that knockdown of FOXO6 is capable of protecting cardiomyocytes from hypoxia-induced apoptosis and oxidative stress by enhancing Nrf2 activation via upregulation of SIRT6. Our study highlights a potential role of FOXO6 in myocardial infarction and suggests it as an attractive target for cardioprotection.

Interaction between CHOP and FoxO6 promotes hepatic lipid accumulation.

BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress is one of the major causes of hepatic insulin resistance through increasing de novo lipogenesis. Forkhead box O6 (FoxO6) is a transcription factor mediating insulin signalling to glucose and lipid metabolism, therefore, dysregulated FoxO6 is involved in hepatic insulin resistance. In this study, we elucidated the role of FoxO6 in ER stress-induced hepatic lipogenesis. METHODS: Hepatic ER stress responses and lipogenesis were monitored in mice overexpressed with constitutively active FoxO6 allele and FoxO6-null mice. In the in vitro study, HepG2 cells overexpressing constitutively active FoxO6 were treated with palmitate, and then alterations in ER stress and lipid metabolism were measured. RESULTS: FoxO6 activation induced hepatic lipogenesis and the expression of ER stress-inducible genes. The expression and transcriptional activity of peroxisome proliferator-activated receptor γ (PPARγ) were significantly increased in constitutively active FoxO6 allele. Interestingly, we found that the active FoxO6 physically interacted with C/EBP homologous protein (CHOP), an ER stress-inducible transcription factor, which was responsible for PPARγ expression. Palmitate treatment caused the expression of ER stress-inducible genes, which was deteriorated by FoxO6 activation in HepG2 cells. Palmitate-induced ER stress led to PPARγ expression through interactions between CHOP and FoxO6 corresponding to findings in the in vivo study. On the other hand, the expression of PPARα and ß-oxidation were decreased in constitutively active FoxO6 allele which implied that lipid catabolism is also regulated by FoxO6. CONCLUSION: Our data present significant evidence demonstrating that CHOP and FoxO6 interact to induce hepatic lipid accumulation through PPARγ expression during ER stress.

[Effects of FoxO6 on proliferation and invasion of colorectal cancer cells].

Objective: To investigate the effects and the mechanism of FoxO6 on the proliferation and invasion of colorectal cancer cells. Methods: FoxO6 siRNA was transfected into colorectal cancer cell HCT116 and SW480. The overexpression vector pcDNA.3.1-c-Myc was constructed and co-transfected into HCT116 and SW480 cells with FoxO6 siRNA. Real-time fluorescent quantitative PCR (RT-qPCR) and western blot were used to detect the mRNA and protein expressions of FoxO6, c-Myc, and p21 in HCT116 and SW480 cells. Bromodeoxyuridine (BrdU) was used to detect cell proliferation and Transwell assay was performed to detect the invasion ability of these cells. SW480 cells transfected with FoxO6 shRNA lentivirus (LV-FoxO6) and were injected into the right armpit of BAL b/c nude mice to construct a tumor-bearing mode and the tumor volumes were measured on the days of 10, 13, 16, 19, 22, and 25 after injection. Results: The FoxO6 mRNA were 0.91±0.04, 1.72±0.07, and 2.03±0.06, and protein expression were 0.70±0.04, 1.35±0.08, and 1.56±0.07 in normal colon cell FHC, colorectal cancer cells HT116 and SW480, respectively. The protein and mRNA levels of FoxO6 in HCT116 and SW480 were significantly higher than those in FHC (both P<0.05). Knockdown of FoxO6 in HCT116 and SW480 cells decreased the mRNA and protein expressions of FoxO6 (both P<0.05), the cell proliferation ability (absorbances were 0.26±0.07 and 0.27±0.06, both P<0.05), cell invasion ability (the invaded cell numbers were 42.3±3.3 and 45.7±4.1, both P<0.05), and the mRNA and protein expressions of c-Myc, while increased the mRNA and protein expressions of p21 (both P<0.01). Overexpression of Myc in FoxO6 silenced HCT116 and SW480 cells decreased the expression of p21, while increased the cell proliferation ability (absorbances were 0.54±0.09 and 0.58±0.07, both P<0.01) and invasion ability (the invaded cell numbers were 79.2±5.9 and 80.5±6.4, both P<0.01). On the 25th day after cell inoculation in nude mice, the tumor volume of LV-FoxO6 group was (190.6±36.2) mm(3), significantly lower than (437.8.6±69.2) mm(3) of LV-NC group (P<0.05). Conclusion: FoxO6 promotes the proliferation and invasion of colorectal cancer cells through facilitating c-Myc mediated p21 expression inhibition.

FoxO6 affects Plxna4-mediated neuronal migration during mouse cortical development.

The forkhead transcription factor FoxO6 is prominently expressed during development of the murine neocortex. However, its function in cortical development is as yet unknown. We now demonstrate that cortical development is altered in FoxO6+/- and FoxO6-/- mice, showing migrating neurons halted in the intermediate zone. Using a FoxO6-directed siRNA approach, we substantiate the requirement of FoxO6 for a correct radial migration in the developing neocortex. Subsequent genome-wide transcriptome analysis reveals altered expression of genes involved in cell adhesion, axon guidance, and gliogenesis upon silencing of FoxO6 We then show that FoxO6 binds to DAF-16-binding elements in the Plexin A4 (Plxna4) promoter region and affects Plxna4 expression. Finally, ectopic Plxna4 expression restores radial migration in FoxO6+/- and siRNA-mediated knockdown models. In conclusion, the presented data provide insights into the molecular mechanisms whereby transcriptional programs drive cortical development.

Forkhead Box O6 (FoxO6) Depletion Attenuates Hepatic Gluconeogenesis and Protects against Fat-induced Glucose Disorder in Mice.

Excessive endogenous glucose production contributes to fasting hyperglycemia in diabetes. FoxO6 is a distinct member of the FoxO subfamily. To elucidate the role of FoxO6 in hepatic gluconeogenesis and assess its contribution to the pathogenesis of fasting hyperglycemia in diabetes, we generated FoxO6 knock-out (FoxO6-KO) mice followed by determining the effect of FoxO6 loss-of-function on hepatic gluconeogenesis under physiological and pathological conditions. FoxO6 depletion attenuated hepatic gluconeogenesis and lowered fasting glycemia in FoxO6-KO mice. FoxO6-deficient primary hepatocytes were associated with reduced capacities to produce glucose in response to glucagon. When fed a high fat diet, FoxO6-KO mice exhibited significantly enhanced glucose tolerance and reduced blood glucose levels accompanied by improved insulin sensitivity. These effects correlated with attenuated hepatic gluconeogenesis in FoxO6-KO mice. In contrast, wild-type littermates developed fat-induced glucose intolerance with a concomitant induction of fasting hyperinsulinemia and hyperglycemia. Furthermore, FoxO6-KO mice displayed significantly diminished macrophage infiltration into liver and adipose tissues, correlating with the reduction of macrophage expression of C-C chemokine receptor 2 (CCR2), a factor that is critical for regulating macrophage recruitment in peripheral tissues. Our data indicate that FoxO6 depletion protected against diet-induced glucose intolerance and insulin resistance by attenuating hepatic gluconeogenesis and curbing macrophage infiltration in liver and adipose tissues in mice.

Thyroid hormones differentially regulate phosphorylation of ERK and Akt via integrin αvß3 receptor in undifferentiated and differentiated PC-12 cells.

The effects of 3,5,3'-triiodo-l-thyronine (T3) and l-thyroxine (T4) on the integrin αvß3 receptor of thyroid hormones (TH) were investigated in pheochromocytoma PC-12 cells. Differentiation was induced by treatment of PC-12 cells with fisetin and the levels of phosphorylated extracellular signal-regulated kinase (ERK) and Akt in cytoplasm, as well as the content of FoxO6 transcription factor in nuclei was analysed in undifferentiated and differentiated conditions. We have found that in undifferentiated PC-12 cells, tetraiodothyroacetic acid (TETRAC), a known inhibitor of binding of T4 and T3 to plasma membrane integrin αvß3 receptor inhibits T4-dependent phosphorylation of ERK, whereas in differentiated PC-12 cells, TETRAC abolishes the effect of T3. In undifferentiated PC-12 cells, both TH increase the level of p-Akt, and this enhancement is not sensitive to TETRAC. In differentiated PC-12 cells, both TH increase the level of p-Akt; however, only T3-dependent activation of Akt is sensitive to the TETRAC. Furthermore, our results have shown that in differentiated PC-12 cells, the expression of FoxO6 was higher than in undifferentiated PC-12 cells, and this elevation has not changed under the action of TH. Only in undifferentiated PC-12 cells the T3-dependent expression of FoxO6 was sensitive to the TETRAC. We propose that PC-12 cells contain integrin αvß3 receptor, which T3 and T3/T4 sites are differentially regulated by TH in undifferentiated and differentiated conditions.

Endoplasmic reticulum stress induces hepatic steatosis through interaction between PPARα and FoxO6 in vivo and in vitro.

Endoplasmic reticulum (ER) stress is a major cause of hepatic steatosis through increasing de novo lipogenesis. Forkhead box O6 (FoxO6) is a transcription factor mediating insulin signaling to glucose and lipid metabolism. Therefore, dysregulated FoxO6 is involved in hepatic lipogenesis. This study elucidated the role of FoxO6 in ER stress-induced hepatic steatosis in vivo and in vitro. Hepatic ER stress responses and ß-oxidation were monitored in mice overexpressed with constitutively active FoxO6 allele and FoxO6-null mice. For the in vitro study, liver cells overexpressing constitutively active FoxO6 and FoxO6-siRNA were treated with high glucose, and lipid metabolism alterations were measured. ER stress-induced FoxO6 activation suppressed hepatic ß-oxidation in vivo. The expression and transcriptional activity of peroxisome proliferator-activated receptor α (PPARα) were significantly decreased in the constitutively active FoxO6 allele. Otherwise, inhibiting ß-oxidation genes were reduced in the FoxO6-siRNA and FoxO6-KO mice. Our data showed that the FoxO6-induced hepatic lipid accumulation was negatively regulated by insulin signaling. High glucose treatment as a hyperglycemia condition caused the expression of ER stress-inducible genes, which was deteriorated by FoxO6 activation in liver cells. However, high glucose-mediated ER stress suppressed ß-oxidation gene expression through interactions between PPARα and FoxO6 corresponding to findings in the in vivo study-lipid catabolism is also regulated by FoxO6. Furthermore, insulin resistance suppressed b-oxidation through the interaction between FoxO6 and PPARα promotes hepatic steatosis, which, due to hyperglycemia-induced ER stress, impairs insulin signaling. KEY MESSAGES: Our original aims were to delineate the interrelation between the regulation of PPARα and the transcription factor FoxO6 pathway in relation to lipid metabolism at molecular levels. Evidence on high glucose promoted FoxO6 activation induced lipid accumulation in liver cells. The effect of PPARα activation of the insulin signaling. FoxO6 plays a pivotal role in hepatic lipid accumulation through inactivation of PPARα in FoxO6-overexpression mice.

Betaine Suppresses Lipid Accumulation in Liver: Inhibition of FoxO6 and PPARγ Interaction.

Betaine is the major water-soluble component of Lycium chinensis. Although there are reports of a protective effect of betaine on fatty liver disease, the underlying mechanisms are unclear. We attempted to elucidate the molecular regulation of betaine on hyperglycemia-induced hepatic lipid accumulation via Forkhead box O (FoxO)6 activation. HepG2 cells and liver tissue isolated from db/db mice treated with betaine were used. The present study investigated whether betaine ameliorates hepatic steatosis by inhibiting FoxO6/peroxisome proliferator-activated receptor gamma (PPARγ) signaling in liver cells. Interestingly, betaine notably decreased lipid accumulation in tissues with FoxO6-induced mRNA expression of lipogenesis-related genes. Furthermore, betaine inhibited the FoxO6 interaction with PPARγ and cellular triglycerides in high-glucose- or FoxO6-overexpression-treated liver cells. In addition, we confirmed that betaine administration via oral gavage significantly ameliorated hepatic steatosis in db/db mice. We conclude that betaine ameliorates hepatic steatosis, at least in part, by inhibiting the interaction between FoxO6 and PPARγ, thereby suppressing lipogenic gene transcription.

FoxO6-Mediated TXNIP Induces Lipid Accumulation in the Liver through NLRP3 Inflammasome Activation.

BACKGRUOUND: Hepatic steatosis, which involves the excessive accumulation of lipid droplets in hepatocytes, presents a significant global health concern due to its association with obesity and metabolic disorders. Inflammation plays a crucial role in the progression of hepatic steatosis; however, the precise molecular mechanisms responsible for this process remain unknown. METHODS: This study investigated the involvement of the nucleotide-binding oligomerization domain-like receptor pyrin domain-containing-3 (NLRP3) inflammasome and the forkhead box O6 (FoxO6) transcription factor in the pathogenesis of hepatic steatosis. We monitored the NLRP3 inflammasome and lipogenesis in mice overexpressing the constitutively active (CA)-FoxO6 allele and FoxO6-null mice. In an in vitro study, we administered palmitate to liver cells overexpressing CA-FoxO6 and measured changes in lipid metabolism. RESULTS: We administered palmitate treatment to clarify the mechanisms through which FoxO6 activates cytokine interleukin (IL)-1ß through the NLRP3 inflammasome. The initial experiments revealed that dephosphorylation led to palmitate-induced FoxO6 transcriptional activity. Further palmitate experiments showed increased expression of IL-1ß and the hepatic NLRP3 inflammasome complex, including adaptor protein apoptotic speck protein containing a caspase recruitment domain (ASC) and pro-caspase-1. Furthermore, thioredoxin-interacting protein (TXNIP), a key regulator of cellular redox conditions upstream of the NLRP3 inflammasome, was induced by FoxO6 in the liver and HepG2 cells. CONCLUSION: The findings of this study shed light on the molecular mechanisms underpinning the FoxO6-NLRP3 inflammasome axis in promoting inflammation and lipid accumulation in the liver.

FOXO6 transcription inhibition of CTRP3 promotes OGD/R-triggered cardiac microvascular endothelial barrier disruption via SIRT1/Nrf2 signalling.

BACKGROUND: C1q/TNF-related protein 3 (CTRP3) has been clarified to display its protective roles in cardiac function. The current study is concentrated on exploring the impacts of CTRP3 on myocardial ischaemia. MATERIALS AND METHODS: Oxygen and glucose hypoxia/reoxygenation (OGD/R) model was constructed in human cardiac microvascular endothelial cells (HCMECs). Reverse transcription-quantitative polymerase chain reaction and western blot analysis of CTRP3 expression were conducted. CCK-8 assay was to estimate cell activity and lactate dehydrogenase (LDH) assay kit was to test LDH release. TUNEL assay and western blot were to judge apoptosis. Endothelial barrier function was detected by in vitro vascular permeability assay kit. Zonula occludens-1 (ZO-1) expression was evaluated by immunofluorescence assay. The interaction between CTRP3 promoter and Forkhead Box O6 (FOXO6) was predicted by JASPAR database and verified by chromatin immunoprecipitation and luciferase reporter assays. After OGD/R-induced HCMECs were co-transfected with CTRP3 overexpression and FOXO6 overexpression plasmids, the above functional experiments above were conducted again. Lastly, the expression of sirtuin 1 (SIRT1)/nuclear factor erythroid 2-related factor 2 (Nrf2) signalling-related proteins was examined by western blot. RESULTS: CTRP3 was down-regulated in OGD/R-induced HCMECs. CTRP3 enhanced the viability and barrier integrity while reduced the apoptosis and permeability of OGD/R-insulted HCMECs. This process may be regulated by FOXO6 transcription. Also, FOXO6 inhibition-mediated CTRP3 up-regulation activated the SIRT1/Nrf2 signalling. CONCLUSIONS: FOXO6 transcription inhibition of CTRP3 promotes OGD/R-triggered cardiac microvascular endothelial barrier disruption via SIRT1/Nrf2 signalling.

Forkhead box O6 (FoxO6) promotes cardiac pathological remodeling and dysfunction by activating Kif15-TGF-ß1 under aggravated afterload.

Pathological cardiac hypertrophy exhibits complex and abnormal gene expression patterns and progresses to heart failure. Forkhead box protein O6 (FoxO6) is a key transcription factor involved in many biological processes. This study aimed to explore the role of FoxO6 in cardiac hypertrophy. Three groups of mice were established: wild-type, FoxO6 knockout, and FoxO6-overexpressing. The mice received daily administration of angiotensin-II (Ang-II) or saline for 4 weeks, after which they were examined for cardiac hypertrophy, fibrosis, and function. Elevated cardiac expression of FoxO6 was observed in Ang-II-treated mice. FoxO6 deficiency attenuated contractile dysfunction and cardiac remodeling, including cardiomyocyte hypertrophy and fibroblast proliferation and differentiation. Conversely, FoxO6 overexpression aggravated the cardiomyopathy and heart dysfunction. Further studies identified kinesin family member 15 (Kif15) as downstream molecule of FoxO6. Kif15 inhibition attenuated the aggravating effect of FoxO6 overexpression. In vitro, FoxO6 overexpression increased Kif15 expression in cardiomyocytes and elevated the concentration of transforming growth factor-ß1 (TGF-ß1) in the medium where fibroblasts were grown, exhibiting increased proliferation and differentiation, while FoxO6 knockdown attenuated this effect. Cardiac-derived FoxO6 promoted pathological cardiac remodeling induced by aggravated afterload largely by activating the Kif15/TGF-ß1 axis. This result further complements the mechanisms of communication among different cells in the heart, providing novel therapeutic targets for heart failure.

Homology Modeling, Screening, and Identification of Potential FOXO6 Inhibitors Curtail Gastric Cancer Progression: an In Silico Drug Repurposing Approach.

Gastric cancer is the world's second leading cause of cancer-related fatalities, with the epidemiology changing over the previous several decades. FOXOs are the O subfamily of the forkhead box (FOX) transcription factor family, which consists of four members: FOXO1, FOXO3, FOXO4, and FOXO6. FOXO6 mRNA and protein levels are increased in gastric cancer tissues. FOXO6 forced overexpression enhances gastric cancer cell growth, while knockdown decreases proliferation. In our study, the GEPIA, Kaplan-Meier, KEGG, and STRING databases were used to determine FOXO6 mRNA expression, overall survival ratio, interactive pathways, and top 10 associated proteins in gastric cancer respectively. Due to the lack of a solved structure for FOXO6, homology modeling was performed to obtain a 3D structure model, and we used anti-cancer drugs and small molecules to target FOXO6 for identifying a potential selective FOXO6 inhibitor. The chemical composition of the proteins and ligands has a significant impact on docking procedure performance. With this in mind, a critical evaluation of the performance of three regularly used docking routines was carried out: MVD, AutoDock Vina in PyRx, and ArgusLab. The binding affinities, docking scores, and intermolecular interactions were used as assessment criteria. In the study, the porfimer sodium showed excellent binding affinity to the FOXO6 protein. The major three docking software packages were used to analyze the scoring/H-bonding energy and intermolecular interactions. Based on the results, we concluded that FOXO6 was upregulated in gastric cancer and the ligand porfimer sodium emerges as a promising potential FOXO6 inhibitor to curtail gastric cancer progression.

Overexpression of FTO inhibits excessive proliferation and promotes the apoptosis of human glomerular mesangial cells by alleviating FOXO6 m6A modification via YTHDF3-dependent mechanisms.

Background: N6-methyladenosine (m6A) is a prevalent post-transcriptional modification presented in messenger RNA (mRNA) of eukaryotic organisms. Chronic glomerulonephritis (CGN) is characterised by excessive proliferation and insufficient apoptosis of human glomerular mesangial cells (HGMCs) but its underlying pathogenesis remains undefined. Moreover, the role of m6A in CGN is poorly understood. Methods: The total level of m6A modification was detected using the m6A quantification assay (Colorimetric). Cell proliferation was assessed by EdU cell proliferation assay, and cell apoptosis was detected by flow cytometry. RNA sequencing was performed to screen the downstream target of fat mass and obesity-associated protein (FTO). MeRIP-qPCR was conducted to detect the m6A level of forkhead box o6 (FOXO6) in HGMCs. RIP assay was utilized to indicate the targeting relationship between YTH domain family 3 (YTHDF3) and FOXO6. Actinomycin D assay was used to investigate the stability of FOXO6 in HGMCs. Results: The study found that the expression of FTO was significantly reduced in lipopolysaccharide (LPS)-induced HGMCs and renal biopsy samples of patients with CGN. Moreover, FTO overexpression and knockdown could regulate the proliferation and apoptosis of HGMCs. Furthermore, RNA sequencing and cellular experiments revealed FOXO6 as a downstream target of FTO in regulating the proliferation and apoptosis of HGMCs. Mechanistically, FTO overexpression decreases the level of FOXO6 m6A modification and reduces the stability of FOXO6 mRNA in a YTHDF3-dependent manner. Additionally, the decreased expression of FOXO6 inhibits the PI3K/AKT signaling pathway, thereby inhibiting the proliferation and promoting apoptosis of HGMCs. Conclusion: This study offers insights into the mechanism through which FTO regulates the proliferation and apoptosis of HGMCs by mediating m6A modification of FOXO6 mRNA. These findings also suggest FTO as a potential diagnostic marker and therapeutic target for CGN.

Biphenotypic Sinonasal Sarcoma with a Novel PAX3::FOXO6 Fusion: A Case Report and Review of the Literature.

BACKGROUND: Biphenotypic sinonasal sarcoma (BSS) is a low-grade, locally aggressive sarcoma unique to the sinonasal region. BSS is most common in middle aged patients and affects women more frequently than men. It is characterized by a bland spindled cell proliferation with neural and myogenic differentiation. BSS are usually associated with rearrangement t(2;4)(q35;q31.1) resulting in a PAX3::MAML3 fusion. Less commonly, other genes are found in combination with PAX3 and some cases reported in the literature have an unknown fusion partner. METHODS: A 54-year-old man presented with nasal mass. Endoscopic resection showed a low-grade spindle cell neoplasm with morphologic features of BSS and immunohistochemical and next generation sequencing were performed to confirm the diagnosis. RESULTS: The tumor was positive for S100 and smooth muscle actin but negative for SOX10. Next generation sequencing demonstrated a novel PAX3::FOXO6 gene fusion. CONCLUSIONS: Although a PAX3::FOXO6 gene fusion has never been reported, this finding combined with the morphologic and immunophenotypic features supports the diagnosis of supports the diagnosis of BSS.

Control of preadipocyte proliferation, apoptosis and early adipogenesis by the forkhead transcription factor FoxO6.

AIMS: Previous studies have shown that the forkhead transcription factor FoxO6 involved in memory consolidation and hepatic glucose homeostasis. Here we asked whether chicken FoxO6 may regulate preadipocyte proliferation, apoptosis and early adipogenesis. MAIN METHODS: Overexpression and knockdown of FoxO6 were performed and evaluated through cell proliferation methods, Oil-Red-O staining, and specific marker expression. Chromatin immunoprecipitation (ChIP) assay was performed to confirm cyclin G2 (CCNG2) as a direct target gene of FoxO6. KEY FINDINGS: FoxO6 is ubiquitously expressed in different chicken tissues and highly expressed in liver, abdominal fat, and preadipocytes in cultured cell. FoxO6 overexpression decreased preadipocyte proliferation by causing G1-phase cell-cycle arrest, whereas inhibition of FoxO6 showed the opposite effects. Overexpression or knockdown of FoxO6 significantly altered the mRNA and protein levels of cell-cycle related markers, such as CCNG2, cyclin dependent kinase inhibitor 1B (CDKN1B), cyclin dependent kinase inhibitor 1A (CDKN1A) and cyclin D2 (CCND2). During preadipocyte proliferation, FoxO6 targets and induces expression of CCNG2, as confirmed by ChIP assay and qPCR. In addition, FoxO6 induces preadipocyte apoptosis through increasing the protein expression levels of cleaved caspase-3 and cleaved caspase-8. Moreover, FoxO6 at the early stage of adipogenesis suppressed mRNA and protein levels of the key early regulators of adipogenesis, such as PPARγ and C/EBPα. SIGNIFICANCE: The results demonstrate that FoxO6 controls preadipocyte proliferation, apoptosis and early adipogenesis, and point to new approaches for further studies related to obesity.

Protease-activated receptor 2 induces ROS-mediated inflammation through Akt-mediated NF-κB and FoxO6 modulation during skin photoaging.

Long-term exposure to ultraviolet irradiation to skin leads to deleterious intracellular effects, including reactive oxygen species (ROS) production and inflammatory responses, causing accelerated skin aging. Previous studies have demonstrated that increased expression and activation of protease-activated receptor 2 (PAR2) and Akt is observed in keratinocyte proliferation, suggesting their potential regulatory role in skin photoaging. However, the specific underlying molecular mechanism of PAR2 and the Akt/NF-κB/FoxO6-mediated signaling pathway is not clearly defined. In this study, we first used the UVB-irradiated photoaged skin of hairless mice and observed an increase in PAR2 and Gαq expression and PI3-kinase/Akt, NF-κB, and suppressed FoxO6. Consequently, increased levels of proinflammatory cytokines and decreased levels of antioxidant MnSOD was observed. Next, to investigate PAR2-specific roles in inflammation and oxidative stress, we used photoaged hairless mice topically applied with PAR2 antagonist GB83 and photoaged PAR2 knockout mice. PAR2 inhibition and deletion significantly suppressed inflammatory and oxidative stress levels, which were associated with decreased IL-6 and IL-1ß levels and increased MnSOD levels, respectively. Furthermore, NF-κB phosphorylation and decreased FoxO6 was reduced by PAR2 inhibition and deletion in vivo. To confirm the in vivo results, we conducted PAR2 knockdown and overexpression in UVB-irradiated HaCaT cells. In PAR2 knockdown cells by si-PAR2 treatment, it suppressed Akt/NF-κB and increased FoxO6, whereas PAR2 overexpression reversed these effects and subsequently modulated proinflammatory target genes. Collectively, our data define that PAR2 induces oxidative stress and inflammation through Akt-mediated phosphorylation of NF-κB (Ser536) and FoxO6 (Ser184), which could be a critical upstream regulatory mechanism in ROS-mediated inflammatory response.

Upregulation of deubiquitinase USP7 by transcription factor FOXO6 promotes EC progression via targeting the JMJD3/CLU axis.

Esophageal carcinoma (EC) is recognized as one of the most frequently occurring malignancies worldwide, and its high morbidity rate motivates efforts to identify potential therapeutic targets. Notably, forkhead box (FOX) family genes are highlighted as possible biomarkers for diagnostics, prognostics, and therapeutics of various malignancies, including EC. Our present study was performed to investigate the underlying mechanism of FOXO6 on the development of EC. We observed a significant upregulation of FOXO6 in EC tissues, contributing to the migration and proliferation in EC cells through gain- and loss-of-function assays. FOXO6 directly interacted with the ubiquitin-specific processing protease 7 (USP7) gene promoter and enhanced its transcriptional activity, which resulted in suppressed cancer cell apoptosis as revealed by chromatin immunoprecipitation (ChIP)-qPCR. USP7 enhanced the ubiquitination of Jumonji domain-containing protein D3 (JMJD3), elevated JMJD3-promoted growth of EC cells, and transcriptionally activated clusterin (CLU) expression at the promoter region via histone H3 lysine 27 tri-methyl (H3K27me3) demethylation, according to immunoprecipitation and ubiquitination assays. Finally, we verified that FOXO6 mediated effects on the USP7/JMJD3/CLU axis to exert an oncogenic role in vivo, which was blocked by USP7 and JMJD3 inhibitor. Our findings demonstrate an important role of the FOXO6/USP7/JMJD3/CLU pathway in EC progression and thus provide attractive potential therapeutic targets for EC patients.

FoxO6 inhibits melanogenesis partly by elevating intracellular antioxidant capacity.

Of the various transcription factors that play a role in controlling oxidative stress, the role of FoxO proteins in skin aging has recently become of interest. Unlike other FoxOs, FoxO6 remains in the nucleus due to the lack of nuclear export signal, so that it may respond sensitively to intracellular stimuli for the induction of target genes. However, the role of FoxO6 in melanogenesis and its related signaling pathways are unclear. We used UV exposed and intrinsically aged mice that exhibited skin aging. Our data showed that FoxO6 activation was markedly decreased in the skin of aging mice and UVB-exposed hairless mice that exhibited an increase in melanogenesis. The reduced FoxO6 activity was closely associated with the elevation of oxidative stress in the skin of these animal models. To our interest, siRNA-mediated FoxO6 knockdown markedly increased melanin content and related signaling pathways in B16F10 cells even without any stimulation. On the contrary, adenovirus-mediated FoxO6 activation significantly reduced melanin content in UVB-exposed B16F10 cells, which is closely associated with the induction of antioxidant genes including MnSOD and catalase, leading to a decrease in oxidative stress. Furthermore, vitamin C treatment reversed the elevated melanogenesis by the FoxO6 knockdown, indicating that the decreased antioxidant capacity greatly contributes to increased melanogenesis in the FoxO6 knockdown condition. For the upstream of a FoxO6 signaling pathway in melanocytes, FoxO6 phosphorylation by Akt appears to be essential evidenced by the reduction of FoxO6 activity and the increase in melanogenesis by PI3K/AKT inhibitor treatment. Our study suggests that FoxO6 is an antioxidant gene that prevents oxidative stress-induced melanogenesis.