Transcription Factor Forkhead Box Research Articles

Parkin-mediated mitophagy is negatively regulated by FOXO3A, which inhibits Plk3-mediated mitochondrial ROS generation in STZ diabetic stress-treated pancreatic β cells.

Diabetes mellitus (DM) is one of the most researched metabolic diseases worldwide. It leads to extensive complications such as cardiovascular disease, nephropathy, retinopathy, and peripheral and central nervous system through an inability to produce or respond to insulin. Although oxidative stress-mediated mitophagy has been reported to play an important role in the pathogenesis of DM, specific studies are still lacking as well as remain highly controversial. Here, we found that Parkin-mediated mitophagy in pancreatic β cells under streptozotocin (STZ)-diabetic stress was induced by Polo-like kinase 3 (Plk3) and inhibited by the transcription factor Forkhead Box O3A (FOXO3A). STZ stress induces mitochondrial recruitment of Parkin through Plk3-mediated mitochondrial reactive oxygen species (ROS) generation, which causes pancreatic cell damage. Conversely, FOXO3A acts as negative feedback to prevent diabetic stress by inhibiting Plk3. Meanwhile, antioxidants including N-acetylcysteine (NAC) and natural COA water scientifically block these mitochondrial ROS and mitochondrial recruitment of Parkin by inhibiting Plk3. Through a 3D organoid ex vivo model, we confirmed that not only ROS inhibitors but also mitophagy inhibitory factors such as 3-MA or Parkin deletion can compensate for pancreatic cell growth and insulin secretion under STZ diabetic stress. These findings suggest that the Plk3-mtROS-PINK1-Parkin axis is a novel mitophagy process that inhibits pancreatic β-cell growth and insulin secretion and FOXO3A and antioxidants may provide new alternatives for effective diabetes treatment strategies in the future.

FOXK1 promotes nonalcoholic fatty liver disease by mediating mTORC1-dependent inhibition of hepatic fatty acid oxidation

Nonalcoholic fatty liver disease (NAFLD) is a chronic metabolic disorder caused by overnutrition and can lead to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC). The transcription factor Forkhead box K1 (FOXK1) is implicated in regulation of lipid metabolism downstream of mechanistic target of rapamycin complex 1 (mTORC1), but its role in NAFLD-NASH pathogenesis is understudied. Here, we show that FOXK1 mediates nutrient-dependent suppression of lipid catabolism in the liver. Hepatocyte-specific deletion of Foxk1 in mice fed a NASH-inducing diet ameliorates not only hepatic steatosis but also associated inflammation, fibrosis, and tumorigenesis, resulting in improved survival. Genome-wide transcriptomic and chromatin immunoprecipitation analyses identify several lipid metabolism-related genes, including Ppara, as direct targets of FOXK1 in the liver. Our results suggest that FOXK1 plays a key role in the regulation of hepatic lipid metabolism and that its inhibition is a promising therapeutic strategy for NAFLD-NASH, as well as for HCC.

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.

Starvation-induced changes in somatic insulin/IGF-1R signaling drive metabolic programming across generations

Exposure to adverse nutritional and metabolic environments during critical periods of development can exert long-lasting effects on health outcomes of an individual and its descendants. Although such metabolic programming has been observed in multiple species and in response to distinct nutritional stressors, conclusive insights into signaling pathways and mechanisms responsible for initiating, mediating, and manifesting changes to metabolism and behavior across generations remain scarce. By using a starvation paradigm in Caenorhabditis elegans, we show that starvation-induced changes in dauer formation-16/forkhead box transcription factor class O (DAF-16/FoxO) activity, the main downstream target of insulin/insulin-like growth factor 1 (IGF-1) receptor signaling, are responsible for metabolic programming phenotypes. Tissue-specific depletion of DAF-16/FoxO during distinct developmental time points demonstrates that DAF-16/FoxO acts in somatic tissues, but not directly in the germline, to both initiate and manifest metabolic programming. In conclusion, our study deciphers multifaceted and critical roles of highly conserved insulin/IGF-1 receptor signaling in determining health outcomes and behavior across generations.

FOX transcription factors are common regulators of Wnt/β-catenin–dependent gene transcription

The Wnt/β-catenin pathway is a critical regulator of development and stem cell maintenance. Mounting evidence suggests that the outcome of Wnt signaling is determined by the collaborative action of multiple transcription factors, including members of the highly conserved forkhead box (FOX) protein family. However, the contribution of FOX transcription factors to Wnt signaling has not been investigated in a systematic manner. Here, we performed complementary screens of all 44 human FOX proteins to identify new Wnt pathway regulators. By combining β-catenin reporter assays with Wnt pathway-focused qPCR arrays and proximity proteomics of selected candidates, we determine that most FOX proteins are involved in the regulation of Wnt pathway activity. As proof-of-principle, we additionally characterize class D and I FOX transcription factors as physiologically relevant regulators of Wnt/β-catenin signaling. We conclude that FOX proteins are common regulators of Wnt/β-catenin-dependent gene transcription that may control Wnt pathway activity in a tissue-specific manner.

Ram sperm cryopreservation disrupts metabolism of unsaturated fatty acids

In ram sperm, metabolites are important components of the plasma membrane, energy metabolism cycle, and precursors for other membrane lipids, and they may have important roles in maintaining plasma membrane integrity, energy metabolism, and regulation of cryotolerance. In this study, the ejaculates from 6 Dorper rams were pooled and sperm were systematically investigated by metabolomics at various steps of cryopreservation (37 °C, fresh [F]; from 37 to 4 °C, cooling [C]; and from 4 to −196 to 37 °C, frozen-thawed [FT]) to identify differential metabolites (DM). There were 310 metabolites identified, of which 86 were considered DMs. Regarding the DMs, there were 23 (0 up and 23 down), 25 (12 up and 13 down), and 38 (7 up and 31 down) identified during cooling (C vs F), freezing (FT vs C), and cryopreservation (FT vs F), respectively. Furthermore, some key polyunsaturated fatty acids (FAs), particularly, linoleic acid (LA), docosahexaenoic acid (DHA), and arachidonic acid (AA) were down-regulated during cooling and cryopreservation. Significant DMs were enriched in several metabolic pathways including biosynthesis of unsaturated FAs, LA metabolism, mammalian target of rapamycin (mTOR), forkhead box transcription factors (FoxO), adenosine monophosphate-activated protein kinase (AMPK), phosphatidylinositol 3-kinase/protein kinase B (PI3K-Akt) signaling pathways, regulation of lipolysis in adipocytes, and FA biosynthesis. This was apparently the first report to compare metabolomics profiles of ram sperm during cryopreservation and provided new knowledge to improve this process.

Associations of CTCF and FOXA1 with androgen and IGF pathways in men with localized prostate cancer

AimsTo examine associations between the transcription factors CCCTC-binding factor (CTCF) and forkhead box protein A1 (FOXA1) and the androgen receptor (AR) and their association with components of the insulin-like growth factor (IGF)-pathway in a cohort of men with localized prostate cancer. MethodsUsing prostate tissue samples collected during the Prostate cancer: Evidence of Exercise and Nutrition Trial (PrEvENT) trial (N = 70 to 92, depending on section availability), we assessed the abundance of CTCF, FOXA1, AR, IGFIR, p-mTOR, PTEN and IGFBP-2 proteins using a modified version of the Allred scoring system. Validation studies were performed using large, publicly available datasets (TCGA) (N = 489). ResultsWe identified a strong correlation between CTCF and AR staining with benign prostate tissue. CTCF also strongly associated with the IGFIR, with PTEN and with phospho-mTOR. FOXA1 was also correlated with staining for the IGF-IR, with IGFBP-2 and with staining for activated phosphor-mTOR. The staining for the IGF-IR was strongly correlated with the AR. ConclusionOur findings emphasise the close and complex links between the endocrine controls, well known to play an important role in prostate cancer, and the transcription factors implicated by the recent genetic evidence.

Retrotransposon-mediated evolutionary rewiring of a pathogen response orchestrates a resistance phenotype in an insect host

Ongoing host-pathogen interactions can trigger a coevolutionary arms race, while genetic diversity within the host can facilitate its adaptation to pathogens. Here, we used the diamondback moth (Plutella xylostella) and its pathogen Bacillus thuringiensis (Bt) as a model for exploring an adaptive evolutionary mechanism. We found that insect host adaptation to the primary Bt virulence factors was tightly associated with a short interspersed nuclear element (SINE - named SE2) insertion into the promoter of the transcriptionally activated MAP4K4 gene. This retrotransposon insertion coopts and potentiates the effect of the transcription factor forkhead box O (FOXO) in inducing a hormone-modulated Mitogen-activated protein kinase (MAPK) signaling cascade, leading to an enhancement of a host defense mechanism against the pathogen. This work demonstrates that reconstructing a cis-trans interaction can escalate a host response mechanism into a more stringent resistance phenotype to resist pathogen infection, providing a new insight into the coevolutionary mechanism of host organisms and their microbial pathogens.

Study of FOXO1-interacting proteins using TurboID-based proximity labeling technology

BackgroundProtein‒protein interactions (PPIs) are the foundation of the life activities of cells. TurboID is a biotin ligase with higher catalytic efficiency than BioID or APEX that reduces the required labeling time from 18 h to 10 min. Since many proteins participate in binding and catalytic events that are very short-lived, it is theoretically possible to find relatively novel binding proteins using the TurboID technique. Cell proliferation, apoptosis, autophagy, oxidative stress and metabolic disorders underlie many diseases, and forkhead box transcription factor 1 (FOXO1) plays a key role in these physiological and pathological processes.ResultsThe FOXO1-TurboID fusion gene was transfected into U251 astrocytes, and a cell line stably expressing FOXO1 was constructed. While constructing the FOXO1 overexpression plasmid, we also added the gene sequence of TurboID to perform biotin labeling experiments in the successfully fabricated cell line to look for FOXO1 reciprocal proteins. Label-free mass spectrometry analysis was performed, and 325 interacting proteins were found. A total of 176 proteins were identified in the FOXO1 overexpression group, and 227 proteins were identified in the Lipopolysaccharide -treated group (Lipopolysaccharide, LPS). Wild-type U251 cells were used to exclude interference from nonspecific binding. The FOXO1-interacting proteins hnRNPK and RBM14 were selected for immunoprecipitation and immunofluorescence verification.ConclusionThe TurboID technique was used to select the FOXO1-interacting proteins, and after removing the proteins identified in the blank group, a large number of interacting proteins were found in both positive groups. This study lays a foundation for further study of the function of FOXO1 and the regulatory network in which it is involved.

Role of miR-300-3p in Leydig cell function and differentiation: A therapeutic target for obesity-related testosterone deficiency

MicroRNAs (miRNAs) regulate various cellular functions, but their specific roles in the regulation of Leydig cells (LCs) have yet to be fully understood. Here, we found that the expression of miR-300-3p varied significantly during the differentiation from progenitor LCs (PLCs) to adult LCs (ALCs). High expression of miR-300-3p in PLCs inhibited testosterone production and promoted PLC proliferation by targeting the steroidogenic factor-1 (Sf-1) and transcription factor forkhead box O1 (FoxO1) genes, respectively. As PLCs differentiated into ALCs, the miR-300-3p expression level significantly decreased, which promoted testosterone biosynthesis and suppressed proliferation of ALCs by upregulating SF-1 and FoxO1 expression. The LH/METTL3/SMURF2/SMAD2 cascade pathway controlled miR-300-3p expression, in which luteinizing hormone (LH) upregulated SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2) expression through methyltransferase like 3 (METTL3)-mediated Smurf2 N6-methyladenosine modification. The Smurf2 then suppressed miR-300 transcription by inhibiting SMAD family member 2 (SMAD2) binding to the promoter of miR-300. Notably, miR-300-3p was associated with an obesity-related testosterone deficiency in men and the inhibition of miR-300-3p effectively rescued testosterone deficiency in obese mice. These findings suggested that miR-300-3p plays a pivotal role in LC differentiation and function, and could be a promising diagnostic or therapeutic target for obesity-related testosterone deficiency.

Androgen promotes squamous differentiation of atypical cells in cervical intraepithelial neoplasia via an ELF3-dependent pathway.

Since the human papillomavirus vaccines do not eliminate preexisting infections, nonsurgical alternative approaches to cervical intraepithelial neoplasia (CIN) have been required. We previously reported that FOXP4 (forkhead box transcription factor P4) promoted proliferation and inhibited squamous differentiation of CIN1-derived W12 cells. Since it was reported that FOXP expressions were regulated by the androgen/androgen receptor (AR) complex and AR was expressed on the CIN lesions, in this study we examined the effects of androgen on CIN progression. Since AR expression was negative in W12 cells and HaCaT cells, a human male skin-derived keratinocyte cell line, we transfected AR to these cell lines and investigated the effects of dihydrotestosterone (DHT) on their proliferation and squamous differentiation. We also examined the immunohistochemical expression of AR in CIN lesions. DHT reduced the intranuclear expression of FOXP4, attenuating cell proliferation and promoting squamous differentiation in AR-transfected W12 cells. Si-RNA treatments showed that DHT induced the expression of squamous differentiation-related genes in AR-transfected W12 cells via an ELF3-dependent pathway. DHT also reduced FOXP4 expression in AR-transfected HaCaT cells. An immunohistochemical study showed that AR was expressed in the basal to parabasal layers of the normal cervical epithelium. In CIN1 and 2 lesions, AR was detected in atypical squamous cells, whereas AR expression had almost disappeared in the CIN3 lesion and was not detected in SCC, suggesting that androgens do not act to promote squamous differentiation in the late stages of CIN. Androgen is a novel factor that regulates squamous differentiation in the early stage of CIN, providing a new strategy for nonsurgical and hormone-induced differentiation therapy against CIN1 and CIN2.

Pharmacogenetic association of the NR1H3 promoter variant with antihypertensive response among patients with hypertension: A longitudinal study.

Background: The genetic factors in assessing therapeutic efficacy and predicting antihypertensive drug response are unclear. Therefore, this study aims to identify the associations between variants and antihypertensive drug response. Methods: A longitudinal study including 1837 hypertensive patients was conducted in Northern China and followed up for a median 2.24years. The associations of 11 candidate variants with blood pressure changes in response to antihypertensive drugs and with the risk of cardiovascular events during the follow-up were examined. The dual-luciferase assay was carried out to assess the effect of genetic variants on gene transcriptional activity. Results: The variant rs11039149A>G in the promoter of nuclear receptor subfamily 1 group H member 3 (NR1H3) was associated with the change in systolic blood pressure (ΔSBP) in response to calcium channel blockers (CCBs) monotherapy. Patients carrying rs11039149AG genotype showed a significant increase of systolic blood pressure (SBP) at follow-up compared with AA carriers, and the difference of ΔSBP between AG and AA carriers was 5.94mm Hg (95%CI: 2.09-9.78, p = 0.002). In 1,184 patients with CCBs therapy, SBP levels decreased in AA carriers, but increased in AG carriers, the difference of ΔSBP between AG and AA carriers was 8.04mm Hg (95%CI: 3.28-12.81, p = 0.001). Further analysis in 359 patients with CCBs monotherapy, the difference of ΔSBP between AG and AA carriers was 15.25mm Hg (95%CI: 6.48-24.02, p = 0.001). However, there was no significant difference in ΔSBP between AG and AA carriers with CCBs multitherapy. The rs11039149A>G was not associated with the cardiovascular events incidence during the follow-up. Additionally, transcriptional factor forkhead box C1 (FOXC1) bound to the NR1H3 promoter containing rs11039149A and significantly increased the transcriptional activity, while rs11039149 A to G change led to a loss-of-function and disabled FOXC1 binding. For the other 10 variants, associations with blood pressure changes or risk of cardiovascular events were not observed. Conclusion: Hypertensive patients with rs11039149AG genotype in the NR1H3 gene have a significant worse SBP control in response to CCBs monotherapy compared with AA carriers. Our findings suggest that the NR1H3 gene might act as a promising genetic factor to affect individual sensitivity to antihypertensive drugs.

Functional dissection of two amino acid substitutions unique to the human FOXP2 protein

The transcription factor forkhead box P2 (FOXP2) is involved in the development of language and speech in humans. Two amino acid substitutions (T303N, N325S) occurred in the human FOXP2 after the divergence from the chimpanzee lineage. It has previously been shown that when they are introduced into the FOXP2 protein of mice they alter striatal synaptic plasticity by increasing long-term depression in medium spiny neurons. Here we introduce each of these amino acid substitutions individually into mice and analyze their effects in the striatum. We find that long-term depression in medium spiny neurons is increased in mice carrying only the T303N substitution to the same extent as in mice carrying both amino acid substitutions. In contrast, the N325S substitution has no discernable effects.

Correlation of 18F-FDG PET/CT metabolic parameters with the expression of immune biomarkers in the tumour microenvironment in lung adenocarcinoma

To explore the association between metabolic parameters evaluated by integrated 2-[18F]-fluoro-2-deoxy-d-glucose (FDG) positron-emission tomography (PET)/computed tomography (CT) and the expression of immune biomarkers in the tumour microenvironment in lung adenocarcinoma. This study included 134 patients. Metabolic parameters were obtained by PET/CT. Immunohistochemistry analysis was used for FOXP3-TILs (transcription factor forkhead box protein 3 tumour-infiltrating lymphocytes), CD8-TILs, CD4-TILs, CD68-TAMs (tumour-associated macrophages) and galectin-1 (Gal-1) tumour expression. There were significant positive associations between FDG PET metabolic parameters and the median percentage of immune reactive areas (IRA%) covered by FOXP3-TILs and CD68-TAMs. Negative associations with the median IRA% covered by CD4-TILs and CD8-TILs were observed: maximal standardised uptake value (SUVmax), metabolic tumour volume (MTV), total lesion glycolysis (TLG), and IRA% for FOXP3-TILs (rho=0.437, 0.400, 0.414; p<0.0001 for all parameters); SUVmax, MTV, TLG, and IRA% for CD68-TAMs (rho=0.356, 0.355, 0.354; p<0.0001 for all parameters); SUVmax, MTV, TLG, and IRA% for CD4-TILs (rho=-0.164, -0.190, -0.191; p=0.059, 0.028, 0.027, respectively); SUVmax, MTV, TLG, and IRA% for CD8-TILs (rho=-0.305, -0.316, -0.322; p<0.0001 for all parameters). There were significant positive associations between tumour Gal-1 expression and the median IRA% covered by FOXP3-TILs and CD68-TAMs (rho=0.379; p<0.0001; rho=0.370; p<0.0001, respectively), and a significant negative association with the median IRA% covered by CD8-TILs (rho=-0.347; p<0.0001) was observed. Tumour stage (p=0.008), Gal-1 expression (p=0.008), and median IRA% covered by CD8-TILs (p=0.054) were independent risk factors for overall survival. FDG PET may facilitate a comprehensive evaluation of the tumour microenvironment and predict response to immunotherapy.

Effects of Cortical FoxP1 Knockdowns on Learned Song Preference in Female Zebra Finches.

The search for molecular underpinnings of human vocal communication has focused on genes encoding forkhead-box transcription factors, as rare disruptions of FOXP1, FOXP2, and FOXP4 have been linked to disorders involving speech and language deficits. In male songbirds, an animal model for vocal learning, experimentally altered expression levels of these transcription factors impair song production learning. The relative contributions of auditory processing, motor function or auditory-motor integration to the deficits observed after different FoxP manipulations in songbirds are unknown. To examine the potential effects on auditory learning and development, we focused on female zebra finches (Taeniopygia guttata) that do not sing but develop song memories, which can be assayed in operant preference tests. We tested whether the relatively high levels of FoxP1 expression in forebrain areas implicated in female song preference learning are crucial for the development and/or maintenance of this behavior. Juvenile and adult female zebra finches received FoxP1 knockdowns targeted to HVC (proper name) or to the caudomedial mesopallium (CMM). Irrespective of target site and whether the knockdown took place before (juveniles) or after (adults) the sensitive phase for song memorization, all groups preferred their tutor's song. However, adult females with FoxP1 knockdowns targeted at HVC showed weaker motivation to hear song and weaker song preferences than sham-treated controls, while no such differences were observed after knockdowns in CMM or in juveniles. In summary, FoxP1 knockdowns in the cortical song nucleus HVC were not associated with impaired tutor song memory but reduced motivation to actively request tutor songs.

P16 INK4A drives nonalcoholic fatty liver disease phenotypes in high fat diet fed mice through biliary E2F1/FOXO1/IGF-1 signaling.

NAFLD is characterized by steatosis, hepatic inflammation, and fibrosis, which can develop into NASH. Patients with NAFLD/NASH have increased ductular reaction (DR) and biliary senescence. High fat/high cholesterol diet feeding increases biliary senescence, DR, and biliary insulin-like growth factor-1 (IGF-1) expression in mice. p16/IGF-1 converges with fork-head box transcription factor O1 (FOXO1) through E2F1. We evaluated p16 inhibition on NAFLD phenotypes and biliary E2F1/FOXO1/IGF-1 signaling. 4-week wild-type (C57BL/6J) male mice were fed a control diet (CD) or high fat/high cholesterol diet and received either p16 or control Vivo Morpholino (VM) by tail vein injection 2× during the 16th week of feeding. We confirmed p16 knockdown and examined: (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling. Human normal, NAFLD, and NASH liver samples and isolated cholangiocytes treated with control or p16 VM were evaluated for p16/E2F1/FOXO1/IGF-1 signaling. p16 VM treatment reduced cholangiocyte and hepatocyte p16. In wild-type high fat/high cholesterol diet mice with control VM, there were increased (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling; however, p16 VM treatment reduced these parameters. Biliary E2F1/FOX-O1/IGF-1 signaling increased in human NAFLD/NASH but was blocked by p16 VM. In vitro , p16 VM reduced biliary E2f1 and Foxo1 transcription by inhibiting RNA pol II binding and E2F1 binding at the Foxo1 locus, respectively. Inhibition of E2F1 reduced biliary FOXO1 in vitro. Attenuating hepatic p16 expression may be a therapeutic approach for improving NAFLD/NASH phenotypes.

P38-mediated FOXN3 phosphorylation modulates lung inflammation and injury through the NF-κB signaling pathway.

NF-κB activates the primary inflammatory response pathway responsible for methicillin-resistant Staphylococcus aureus (MRSA)-induced lung inflammation and injury. Here, we report that the Forkhead box transcription factor FOXN3 ameliorates MRSA-induced pulmonary inflammatory injury by inactivating NF-κB signaling. FOXN3 competes with IκBα for binding to heterogeneous ribonucleoprotein-U (hnRNPU), thereby blocking β-TrCP-mediated IκBα degradation and leading to NF-κB inactivation. FOXN3 is directly phosphorylated by p38 at S83 and S85 residues, which induces its dissociation from hnRNPU, thus promoting NF-κB activation. After dissociation, the phosphorylated FOXN3 becomes unstable and undergoes proteasomal degradation. Additionally, hnRNPU is essential for p38-mediated FOXN3 phosphorylation and subsequent phosphorylation-dependent degradation. Functionally, genetic ablation of FOXN3 phosphorylation results in strong resistance to MRSA-induced pulmonary inflammatory injury. Importantly, FOXN3 phosphorylation is clinically positively correlated with pulmonary inflammatory disorders. This study uncovers a previously unknown regulatory mechanism underpinning the indispensable role of FOXN3 phosphorylation in the inflammatory response to pulmonary infection.

Identification of signalling downstream of the transcription factor forkhead box protein M1 that protects against endoplasmic reticulum stress in a diabetic foot ulcer model.

Diabetic foot ulcer (DFU) has a significant impact on the quality of life of diabetic mellitus (DM) patients. Here, we aimed to explore the molecules with aberrant expression and their regulatory mechanisms in DFU. The expression of gene and protein was examined using quantitative polymerase chain reaction (qPCR) and western blot. Pearson's correlation analysis was used to analyse interactions among FOXM1, GAS5 and SDF4. Immunofluorescence was used to detect PDI and GRP78 expression. Flow cytometry was used to assess cell apoptosis. Tube formation assay was used to determine angiogenic capacity. Fluorescence in situ hybridization (FISH) assay was employed to determine the cellular localization of GAS5 and SDF4 in human umbilical vein endothelial cells (HUVECs). The interactions among FOXM1, GAS5 and SDF4 were validated by chromatin immunoprecipitation (ChIP), luciferase, RNA pull-down and RNA immunoprecipitation (RIP) assays. FOXM1, GAS5 and SDF4 were decreased in the skin tissues of DFU patients. High glucose (HG) stimulation induced endoplasmic reticulum (ER) stress and cell apoptosis but suppressed angiogenesis in HUVECs, which were abolished by FOXM1 overexpression. FOXM1 promoted GAS5 transcriptional activity, resulting in increased GAS5 expression, and GAS5 knockdown reversed the effects of FOXM1 overexpression in HG-treated HUVECs. Moreover, GAS5 recruited TAF15 to promote SDF4 expression in HUVECs. GAS5 overexpression inhibited ER stress, cell apoptosis and induced angiogenesis in HG-treated HUVECs which could be reversed by silencing SDF4. Our results revealed that FOXM1 suppressed ER stress, cell apoptosis and promoted angiogenesis in HG-induced HUVECs via mediating GAS5/TAF15/SDF4 axis, providing a novel therapeutic molecule mechanism for DFU.

Inhibition of Xanthine Oxidase Protects against Diabetic Kidney Disease through the Amelioration of Oxidative Stress via VEGF/VEGFR Axis and NOX-FoxO3a-eNOS Signaling Pathway.

Xanthine oxidase (XO) is an important source of reactive oxygen species. This study investigated whether XO inhibition exerts renoprotective effects by inhibiting vascular endothelial growth factor (VEGF) and NADPH oxidase (NOX) in diabetic kidney disease (DKD). Febuxostat (5 mg/kg) was administered to streptozotocin (STZ)-treated 8-week-old male C57BL/6 mice via intraperitoneal injection for 8 weeks. The cytoprotective effects, its mechanism of XO inhibition, and usage of high-glucose (HG)-treated cultured human glomerular endothelial cells (GECs) were also investigated. Serum cystatin C, urine albumin/creatinine ratio, and mesangial area expansion were significantly improved in febuxostat-treated DKD mice. Febuxostat reduced serum uric acid, kidney XO levels, and xanthine dehydrogenase levels. Febuxostat suppressed the expression of VEGF mRNA, VEGF receptor (VEGFR)1 and VEGFR3, NOX1, NOX2, and NOX4, and mRNA levels of their catalytic subunits. Febuxostat caused downregulation of Akt phosphorylation, followed by the enhancement of dephosphorylation of transcription factor forkhead box O3a (FoxO3a) and the activation of endothelial nitric oxide synthase (eNOS). In an in vitro study, the antioxidant effects of febuxostat were abolished by a blockade of VEGFR1 or VEGFR3 via NOX-FoxO3a-eNOS signaling in HG-treated cultured human GECs. XO inhibition attenuated DKD by ameliorating oxidative stress through the inhibition of the VEGF/VEGFR axis. This was associated with NOX-FoxO3a-eNOS signaling.

LAPTM5 regulated by FOXP3 promotes the malignant phenotypes of breast cancer through activating the Wnt/β‑catenin pathway.

Breast cancer remains the most common malignancy and the leading cause of cancer‑associated mortality in women worldwide. Lysosomal protein transmembrane 5 (LAPTM5), a lysosomal membrane protein, plays an important role in several human malignancies. However, the biological functions and mechanism of LAPTM5 in breast cancer remain unclear. In the present study, the potential tumor‑promoting effect of LAPTM5 was predicted by bioinformatics analysis. LAPTM5 was highly expressed in breast cancer clinical specimens. Moreover, invitro studies demonstrated that cell proliferation, migration and invasion, as well as the process of epithelial‑mesenchymal transition (EMT) were promoted by LAPTM5 overexpression and were suppressed by LAPTM5 downregulation invitro. The tumor‑promoting effects of LAPTM5 were also confirmed by xenograft tumor assay invivo. It was found that the tumor‑promoting effects of LAPTM5 were partly dependent on the activation of the Wnt/β‑catenin signaling pathway. Furthermore, dual‑luciferase and chromatin immunoprecipitation assays verified that the transcription factor forkhead box protein 3 (FOXP3) directly bound to the promoter of LAPTM5 and negatively regulated its expression. Taken together, the present findings indicated that LAPTM5, negatively regulated by FOXP3, promoted the malignant phenotypes of breast cancer through activating the Wnt/β‑catenin signaling pathway.