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TNF induces FOXO3 translocation in HT-29 cells. (a) HT-29 cells, control and treated with TNF were fixed, immunofluorescent stained for FOXO3 and images were taken with matched exposures. In control cells, FOXO3 is localized in the nucleus. During TNF treatment, nuclear FOXO3 translocates to the cytosol, suggesting its inactivation ( 60 magnification). This experiment was repeated three independent times. (b) Nuclear (8 g) and cytosolic (40 g) proteins from HT-29 cells, control and TNF treated for 3 h, were separated on SDS-PAGE and immunoblotted for FOXO3. Immunoblots reveal decreased nuclear and increased cytosolic amounts of FOXO3 after TNF treatment. Immunoblots were reprobed with antibodies against Oct-1 for nuclear extracts and actin for cytosolic extracts as a control. Densitometric analysis shows a significant differences (*) between groups (P<0.05).
Source publication
Inflammatory bowel disease (IBD), including Crohn's disease and ulcerative colitis, is characterized by chronic mucosal injury and the infiltration of inflammatory cells. Tumor suppressor FOXO3 regulates gene expression and its translocation to the cytosol leads to the abrogation of its transcriptional function. We have previously shown that bacter...
Contexts in source publication
Context 1
... To address the effect of cytokines on this process, FOXO3 localization was examined in HT-29 cells treated with TNFα. FOXO3 translocated from the nucleus to the cytosol during the first 30 min of TNFα treatment ( Figure 1a). An immunoblot analysis of subcellular fractions confirmed that the amount of nuclear FOXO3 decreases, whereas the amount of cytosolic FOXO3 increases after TNFα stimulation ( Figure 1b). ...
Context 2
... translocated from the nucleus to the cytosol during the first 30 min of TNFα treatment ( Figure 1a). An immunoblot analysis of subcellular fractions confirmed that the amount of nuclear FOXO3 decreases, whereas the amount of cytosolic FOXO3 increases after TNFα stimulation ( Figure 1b). This data suggests that FOXO3 becomes inactive after TNFα treatment of the HT-29 cells. ...
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Citations
... 54 This, in turn, may lead to more severe colonic inflammation during UC. 55 Additionally, both cryptbottom [CD44 + ] and crypt-top [CD66a + ] cells of patients with active UC had increased expression of hsa-miR-221-3p and hsa-miR-21-5p, which target the SOCS1 gene. 22 SOCS1 is an important regulator of IL-4 signalling, and its forced expression was shown to inhibit IL-13 signalling in epithelial cells. ...
Background and aims:
Colonic epithelial barrier dysfunction is one of the early events in ulcerative colitis (UC) and microRNAs (miRNAs) participate in its regulation. However, cell type-specific miRNome during UC is still unknown. Thus, we aimed to explore miRNA expression patterns in colon tissue and epithelial cells at active and quiescent UC.
Methods:
Small RNA-sequencing in colon tissue, crypt-bottom (CD44+), and crypt-top (CD66a+) colonic epithelial cells from two cohorts of UC patients (n=74) and healthy individuals (n=50) was performed. Data analysis encompassed differential expression, weighted gene co-expression network, correlation, gene-set enrichment analyses.
Results:
Differentially expressed colonic tissue miRNAs showed potential involvement in regulation of interleukin-4 and interleukin-13 signalling during UC. As this pathway plays role in intestinal barrier regulation, consecutive analysis of spatially distinct colonic epithelial cell populations was performed. Cell-type (crypt-top and crypt-bottom) specific miRNA expression patterns were identified in both active and quiescent UC. Target genes of differentially expressed epithelial miRNAs at different disease activity were overrepresented in epithelial cell migration and therefore intestinal barrier integrity regulation. The pro-inflammatory miRNA co-expression module M1 correlated with endoscopic disease activity and successfully distinguished active and quiescent UC not only in both epithelial cell populations, but also in the colon tissue. The anti-inflammatory module M2 was specific to crypt-bottom cells and significantly enriched in the quiescent UC patients.
Conclusions:
miRNA expression was specific to colonic epithelial cell populations and UC state, reflecting endoscopic disease activity. Irrespective of the UC state, deregulated epithelial miRNAs were associated with regulation of intestinal barrier integrity.
... The diseases and functions associated with these DEGs include gastrointestinal disorders, metabolic diseases, lipid metabolism, immune responses, immune cell trafficking, inflammatory pathways, and cancer ( Figure 3A). To determine the contribution of loss of FOXO3 in PMNs to colonic pathobiology, their upstream regulators of DEGs were identified and compared to transcriptomes from the total colonic tissue of FOXO3-deficient mice, which has exacerbated inflammatory and tumorigenic processes [27,35,42]. We found substantial similarities in upstream regulators of DEGs associated with FOXO3 KO PMNs and total FOXO3 KO colon, which were related to lipid metabolism (eicosapentenoic acid, LEP), immune response (CD40LG, CpG oligonucleotide), inflammatory pathways (SIRT1, HNF-4α), and tumorigenesis (NUPR1, ID1, CREB1, CBFB) ( Figure 3B). ...
Inflammatory bowel disease (IBD), characterized by infiltration of polymorphonuclear neutrophils (PMNs), increases the risk of colon cancer. PMN activation corresponds to the accumulation of intracellular Lipid Droplets (LDs). As increased LDs are negatively regulated by transcription factor Forkhead Box O3 (FOXO3), we aim to determine the significance of this regulatory network in PMN-mediated IBD and tumorigenesis. Affected tissue of IBD and colon cancer patients, colonic and infiltrated immune cells, have increased LDs’ coat protein, PLIN2. Mouse peritoneal PMNs with stimulated LDs and FOXO3 deficiency have elevated transmigratory activity. Transcriptomic analysis of these FOXO3-deficient PMNs showed differentially expressed genes (DEGs; FDR < 0.05) involved in metabolism, inflammation, and tumorigenesis. Upstream regulators of these DEGs, similar to colonic inflammation and dysplasia in mice, were linked to IBD and human colon cancer. Additionally, a transcriptional signature representing FOXO3-deficient PMNs (PMN-FOXO3389) separated transcriptomes of affected tissue in IBD (p = 0.00018) and colon cancer (p = 0.0037) from control. Increased PMN-FOXO3389 presence predicted colon cancer invasion (lymphovascular p = 0.015; vascular p = 0.046; perineural p = 0.03) and poor survival. Validated DEGs from PMN-FOXO3389 (P2RX1, MGLL, MCAM, CDKN1A, RALBP1, CCPG1, PLA2G7) are involved in metabolism, inflammation, and tumorigenesis (p < 0.05). These findings highlight the significance of LDs and FOXO3-mediated PMN functions that promote colonic pathobiology.
... The FOXO family of transcription factors such as FOXO3 are implicated in IBD pathology 52 and the regulation of intestinal homoeostatic processes such as inflammation, autophagy, mucus secretion, microbe-host interactions and maintenance of the intestinal barrier integrity. [53][54][55][56] GWAS data from CD patients suggested that the minor allele (rs12212067) mapped to the FOXO3 locus, affects disease burden and outcome. 52 Besides, members of the FOXO pathway, which we found to be downregulated in UC patients, are also expressed in lower levels in UC patients compared with non-IBD controls. ...
Objective
To infer potential mechanisms driving disease subtypes among patients with inflammatory bowel disease (IBD), we profiled the transcriptome of purified circulating monocytes and CD4 T-cells.
Design
RNA extracted from purified monocytes and CD4 T-cells derived from the peripheral blood of 125 endoscopically active patients with IBD was sequenced using Illumina HiSeq 4000NGS. We used complementary supervised and unsupervised analytical methods to infer gene expression signatures associated with demographic/clinical features. Expression differences and specificity were validated by comparison with publicly available single cell datasets, tissue-specific expression and meta-analyses. Drug target information, druggability and adverse reaction records were used to prioritise disease subtype-specific therapeutic targets.
Results
Unsupervised/supervised methods identified significant differences in the expression profiles of CD4 T-cells between patients with ileal Crohn’s disease (CD) and ulcerative colitis (UC). Following a pathway-based classification (Area Under Receiver Operating Characteristic - AUROC=86%) between ileal-CD and UC patients, we identified MAPK and FOXO pathways to be downregulated in UC. Coexpression module/regulatory network analysis using systems-biology approaches revealed mediatory core transcription factors. We independently confirmed that a subset of the disease location-associated signature is characterised by T-cell-specific and location-specific expression. Integration of drug-target information resulted in the discovery of several new (BCL6, GPR183, TNFAIP3) and repurposable drug targets (TUBB2A, PRKCQ) for ileal CD as well as novel targets (NAPEPLD, SLC35A1) for UC.
Conclusions
Transcriptomic profiling of circulating CD4 T-cells in patients with IBD demonstrated marked molecular differences between the IBD-spectrum extremities (UC and predominantly ileal CD, sandwiching colonic CD), which could help in prioritising particular drug targets for IBD subtypes.
... These results were confirmed by a subsequent study that found decreased expression of forkhead box O3 (FOXO3a) in the colon tissue of active UC patients and in HT29 cells treated with TNF-α, demonstrating that miR-155 significantly decreases FOXO3a expression in human colon epithelial cells [68]. Another study showed that FOXO3a deficiency resulted in severe gut inflammation in vivo, demonstrating a TNF-α-dependent role of miR-155 in the gut [69]. ...
MicroRNAs (miRNAs) are a group of non-coding RNAs that play a critical role in regulating epigenetic mechanisms in inflammation-related diseases. Inflammatory bowel diseases (IBDs), which primarily include ulcerative colitis (UC) and Crohn’s disease (CD), are characterized by chronic recurrent inflammation of intestinal tissues. Due to the multifactorial etiology of these diseases, the development of innovative treatment strategies that can effectively maintain remission and alleviate disease symptoms is a major challenge. In recent years, evidence for the regulatory role of miRNAs in the pathogenetic mechanisms of various diseases, including IBD, has been accumulating. In light of these findings, miRNAs represent potential innovative candidates for therapeutic application in IBD. In this review, we discuss recent findings on the role of miRNAs in regulating inflammatory responses, maintaining intestinal barrier integrity, and developing fibrosis in clinical and experimental IBD. The focus is on the existing literature, indicating potential therapeutic application of miRNAs in both preclinical experimental IBD models and translational data in the context of clinical IBD. To date, a large and diverse data set, which is growing rapidly, supports the potential use of miRNA-based therapies in clinical practice, although many questions remain unanswered.
... Both expression levels of phosphorylated and total forms of IκB-α were downregulated with acetylshikonin treatment (McDade et al., 1999). In a previous study, it was also reported that FOXO3 knockdown led to the reduced inhibitory IκBα in U2OS cells (Snoeks et al., 2009). We overall detected the expression levels of proteins associated with acetylshikonin-induced cellular apoptosis and cell cycle arrest (Fig. 4). ...
Acetylshikonin a natural compound isolated from the root of Lithospermum erythrorhizon and one of the shikonin derivatives which possess promising anticarcinogenic ability. In this study, we attempted to investigate the anti-cancer potential of acetylshikonin towards osteosarcoma U2OS cells. The effects of acetylshikonin towards the treatment of U2OS cells showed that decreased cell proliferation and inhibited migration ability of cells which are experimentally assessed via wide range of assays including MTT, WST-1, cell counting, colony formation assays, wound healing assay and gelatin zymography assay. We also observed that early apoptosis and late apoptosis were increased through fluorescence-activated cell sorter (FACS) analysis. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) assay showed that acetylshikonin induced DNA fragmentation. Western blot analysis revealed the apoptotic effect of acetylshikonin by measuring of proteins such as cleaved caspase −9, −8, −3, −6, −7, and Bcl-2 family. We observed that ROS level and DNA damage were increased via DCF-DA assay and comet assay. In terms of the presence of ROS, induction of apoptosis was detected by measuring proteins such as cleaved caspase 3, PARP, Bcl-2 and Bax. We suggested that the reactions were related to the nuclear translocation of FOXO3 through western blot of cytoplasmic/nuclear protein fractionation. We finally demonstrated that the knockdown of the FOXO3 induced the decrease of the apoptosis-associated proteins via western blot of FOXO3 siRNA transfection. Taken together, these results suggested that acetylshikonin might induce ROS-mediated apoptosis in a FOXO3-dependent manner against osteosarcoma cells. Therefore, acetylshikonin may be elucidated as an effective candidate for the treatment of osteosarcoma.
... Several previous studies of polymorphism of FOXO3a have revealed an association between FOXO3a and other in ammatory diseases, such as rheumatoid arthritis, in ammatory bowel diseases, and chronic obstructive pulmonary disease. [30][31][32][33][34][35][36][37][38][39][40][41][42] Our study ndings raise a question about the molecular mechanism behind the association between FOXO3a gene polymorphism and the pathogenesis of psoriasis. Also, our study opens a future investigative window for possible therapeutic options based on the FOXO3a transcription factor and blocking of the PI3K-AKT-FOXO signaling axis. ...
.ABSTRACT:
Background. Psoriasis vulgaris is a chronic, relapsing, inflammatory disorder marked by an intensified immune response. The role of immunogenetics in psoriasis is still poorly understood; however, experts agree that its expression depends on proinflammatory cytokines. Forkhead box class O3A (FOXO3a), a transcription factor, plays a crucial role in intercellular regulation, oxidative stress, deoxyribonucleic acid (DNA) repair, and cell death.
Objective. The objective of this study was to investigate the role of FOXO3a genetic polymorphism as a risk factor for psoriasis vulgaris and assess its possible relationship with disease severity.
Methods. A comparative case-control study included 53 patients with psoriasis and 41 matched healthy controls. We measured serum FOXO3a levels and used the PCR-RFLEP technique to detect FOXO3a genetic polymorphism (rs13217795) in both groups.
Results. Our results revealed significantly higher serum FOXO3a levels in the psoriasis group compared to the control group (p≤0.001). Serum FOXO3a levels were significantly higher in patients with severe psoriasis than in those with mild-to-moderate disease. FOXO3a genotypes found homozygous mutant genotype (TT) was substantially more frequent in the psoriasis group than in the control group. Furthermore, the T allele was more frequent in the psoriasis group than in the control group.
Conclusion. The study indicates that rs13217795 polymorphism of the FOXO3a gene is strongly associated with susceptibility to psoriasis. Also, the serum level of FOXO3a is significantly higher in patients with severe psoriasis, compared to patients with mild-to-moderate psoriasis. This finding could be an area of future targeted therapy.
Keywords: Psoriasis vulgaris, Serum FOXO3a, FOXO3a gene.
... Since Akt and FOXO3a have been identified as direct substrates of PI3K [12][13][14] we further investigated whether Akt and FOXO3a are associated with the protective effect of PI3K activator against HR-induced GnRH reduction. The PI3K activator induced the phosphorylation of Akt and enhanced the phosphorylation and nuclear localization of FOXO3a in GT1-7 cells (P<0.01; ...
... In the presence of growth factors, FOXOs are phosphorylated by Akt, after which they translocate to the cytoplasm [19,20]. FOXO3a is believed to be the key target of the PI3K/AKT pathway [12][13][14]21]. A previous study discovered that the activation of the PI3K/Akt/FOXO3a the PI3K/Akt/FOXO3a pathway promotes cell apoptosis Fig. 3. Activation of PI3K/Akt/FOXO3a pathway abrogates HR-induced NF-κB activation. ...
... pathway stimulates spinal cord regeneration following injury in adult rats [21]. In contrast, the inactivation of [12][13][14]. In this study, we hypothesized that the activation of the PI3K/Akt pathway might positively affect HR-associated GnRH decline. ...
Recent studies have suggested that the hypothalamus has an important role in aging by regulating nuclear factor-?B (NF-?B)-directed gonadotropin-releasing hormone (GnRH) decline. Moreover, our previous study has shown that ischemia-reperfusion (IR) injury activates NF-?B to reduce hypothalamic GnRH release, thus suggesting that IR injury may facilitate hypothalamic programming of system aging. In this study, we further examined the role of phosphoinositide 3-kinase (PI3K)/Protein kinase B (Akt) pathway, a critical intracellular signal pathway involved in the repair process after IR, in hypoxia-reoxygenation (HR)-associated GnRH decline in vitro. We used GT1-7 cells and primarily-cultured mouse GnRH neurons as cell models for investigation. Our data revealed that the activation of the PI3K/Akt/Forkhead box protein O3a (FOXO3a) pathway protects GnRH neurons from HR-induced GnRH decline by preventing HR-induced gnrh1 gene inhibition and NF-?B activation. Our results further the understanding of the regulatory mechanisms of HR-associated hypothalamic GnRH decline.
... 77,78 In physiological conditions, it is mostly expressed in thymus and spleen, where it regulates several pathways to maintain immune cell homeostasis and enhance the suppression of oncogenesis. 79 The results of many studies identified miR-155 as one of the top potential therapeutic targets in 82 this study demonstrated that miR-155 has a TNF-α-dependent role in the intestine. Similar findings were also reported in a study on pediatric CD patients, where the expression of miR-155 was significantly higher in inflamed CD mucosa in comparison with the intact CD mucosa and healthy controls. ...
Micro-RNAs (miRNAs) are noncoding RNAs usually 24-30 nucleotides long that play a central role in epigenetic mechanisms of inflammatory diseases and cancers. Recently, several studies have assessed the involvement of miRNAs in the pathogenesis of inflammatory bowel disease (IBD) and colitis-associated neoplasia. Particularly, it has been shown that many members of miRNAs family are involved in the pathways of inflammation and fibrogenesis of IBD; therefore, their use as inflammatory and fibrosis biomarkers has been postulated. In light of these results, the role of miRNAs in IBD therapy has been proposed and is currently under investigation with many in vitro and in vivo studies, murine models, and a phase 2a trial. The accumulating data have pushed miRNA-based therapy closer to clinical practice, although many open questions remain.
With this systematic review, we discuss the current knowledge about the therapeutic effects of miRNAs mimicking and inhibition, and we explore the new potential targets of miRNA family for the treatment of inflammation and fibrosis in IBD.
... Moreover, as FOXO3 emerges as an important regulator of macrophage function [6,49] and controls metabolism in various cells [25,26], it is plausible that FOXO3 plays a central role in metabolic reprograming in macrophages. This critical immuno-metabolic function of FOXO3 is also seen in colonic cells [30,31]. Further, altered macrophages can impair barrier function, as seen in IBD, in addition to promoting the tumor microenvironment [16,50]. ...
... Protein extraction and immunoblots were performed as described previously [30,31,41]. The following specific antibodies against proteins were used: phosphorylated FOXO3 (Ser253) (Cell Signaling, Danvers, MA, USA) and β-actin (Cell Signaling). ...
Obesity, characterized by augmented inflammation and tumorigenesis, is linked to genetic predispositions, such as FOXO3 polymorphisms. As obesity is associated with aberrant macrophages infiltrating different tissues, including the colon, we aimed to identify FOXO3-dependent transcriptomic changes in macrophages that drive obesity-mediated colonic inflammation and tumorigenesis. We found that in mouse colon, high-fat-diet-(HFD)-related obesity led to diminished FOXO3 levels and increased macrophages. Transcriptomic analysis of mouse peritoneal FOXO3-deficient macrophages showed significant differentially expressed genes (DEGs; FDR < 0.05) similar to HFD obese colons. These DEG-related pathways, linked to mouse colonic inflammation and tumorigenesis, were similar to those in inflammatory bowel disease (IBD) and human colon cancer. Additionally, we identified a specific transcriptional signature for the macrophage-FOXO3 axis (MAC-FOXO382), which separated the transcriptome of affected tissue from control in both IBD (p = 5.2 × 10−8 and colon cancer (p = 1.9 × 10−11), revealing its significance in human colonic pathobiologies. Further, we identified (heatmap) and validated (qPCR) DEGs specific to FOXO3-deficient macrophages with established roles both in IBD and colon cancer (IL-1B, CXCR2, S100A8, S100A9, and TREM1) and those with unexamined roles in these colonic pathobiologies (STRA6, SERPINH1, LAMB1, NFE2L3, OLR1, DNAJC28 and VSIG10). These findings establish an important understanding of how HFD obesity and related metabolites promote colonic pathobiologies.
... In vivo studies in mice showed that FoxO3 knockdown increased cytokine production whereas increased level of FoxO3, specifically in T-cells, decreased cytokine production [29]. Infection caused due to bacteria blocked FoxO3 and over-stimulated cytokine production in the epithelial cells of intestine, whereas TNF-α-induced FoxO3 inactivation boosted IL-8 in HT-29 cells [30,31]. Moreover, we carried out luciferase assays to determine the effects of miR-23a on circRNA_FOXO3 and FOXO3 suppression. ...
The expression of circRNA_FOXO3 was found to be positively associated with the expression of Forkhead Box O3 (FOXO3), which is targeted and regulated by miR-23a. Polymorphisms in rs12196996 and rs2232365 have been reported in various diseases. In this study, we recruited intensive care unit (ICU)-acquired sepsis patients and grouped them according to their genotypes of rs12196996 and rs2232365. Quantitative real-time PCR was performed to analyze the expression of circRNA_FOXO3, FOXO3 mRNA, and miR-23a. ELISA was carried out to evaluate the abundance of cytokines and luciferase assay was used to explore the inhibitory role of miR-23a on circRNA_FOXO3 and FOXO3. Accordingly, we found that rs12196996 GG and rs2232365 AA were significantly correlated with prolonged survival of ICU-acquired sepsis patients. Rs12196996 GG and rs2232365 AA were also correlated with increased level of miR-23a, IL-10 and decreased level of TNF, IL-2, IFN, IL-6 and IL-1β in the peripheral blood cell samples of patients with ICU-acquired sepsis. The luciferase activity of wild-type (WT) circRNA_FOXO3 and FOXO3 were severely reduced by miR-23a. MiR-23a precursors could effectively suppress the expression of circRNA_FOXO3 and FOXO3 in the cells. Moreover, LPS-induced cell viability loss and dysregulation of cytokines were effectively restored by the knockdown of FOXO3 or circRNA_FOXO3 siRNA in the cells. This study revealed that the minor allele of rs12196996 polymorphism and rs2232365 polymorphism collaboratively contributed to the increased survival and suppressed severity of ICU-acquired sepsis.
