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LPS induces Foxo3a translocation and degradation in human HT-29 intestinal epithelial cells. (A) HT-29 cells treated with LPS were fixed and immunofluorescently stained for Foxo3a (magnification, ×60). This experiment was repeated three independent times in triplicate. (B) Total proteins from HT-29 cells (control cells and cells treated with LPS) were separated by SDS-PAGE and immunoblotted for Foxo3a and actin. Each experiment was performed three times, and three samples were used per experimental group. Densitometric analysis shows significant (*) degradation of Foxo3a during the course of LPS treatment compared to untreated cells (P < 0.05).
Source publication
Enteric bacteria and their products play an important role in intestinal inflammation; however, the complete mechanisms are not elucidated yet. Tumor suppressor Foxo3a regulates gene expression in the nucleus, and its translocation to the cytosol leads to inactivation. Proximally, Foxo3a is regulated by different pathways including the phosphoinosi...
Citations
... Previously, we demonstrated that BFT exposure in IECs provokes specific signaling pathways that eventually result in the activation of transcription factors such as NF-κB and AP-1 [14][15][16]. Since early work revealed that lipopolysaccharide (LPS) and infection with Citrobacter rodentium target the FoxO3a transcription factor in intestinal epithelia [38], we hypothesized that BFT-induced autophagy requires FoxO3a in IECs. In the present study, the increased expression of FoxO3a by BFT treatment was observed in the nuclear fraction of HCT-116 cells. ...
Macroautophagy/autophagy is essential for preserving cellular homeostasis by recycling nutrients and removing spoiled or aged proteins and organelles. It also has an essential role in defense mechanisms against microbial infections. However, the role of autophagy in enterotoxigenic Bacteroides fragilis infection remains largely unknown. In this study, we explored the role of B. fragilis enterotoxin (BFT) in the autophagic process of intestinal epithelial cells (IECs). The LC3-I of human HCT-116 IECs was converted to LC3-II by BFT stimulation. In addition, BFT-exposed cells showed the decreased expression of p62 in a time-dependent manner and increased levels of ATG5 and ATG12 gradually. Evidence of an enhanced autophagic process was supported by autophagosomes co-localized with LC3-lysosome-associated protein 2 in BFT-stimulated cells. The AMP-activated protein kinase (AMPK) and Forkhead box O3 (FoxO3a) axis were required for BFT-induced autophagy activation. In contrast with the activation of autophagy at 3–6 h after BFT exposure, IECs induced apoptosis-related signals at 12–48 h. HCT-116 IECs suppressing the formation of autophagosomes significantly activated apoptosis signals instead of autophagy early after BFT exposure. These data suggest that BFT can activate autophagy through the AMPK-FoxO3a pathway and the autophagy may suppress apoptosis during early exposure of IECs to BFT.
... Dysregulated FOXO3a activity has been detected in multiple pathologies including various types of cancers, neurodegenerative diseases, and muscle dystrophy (29,30). Moreover, FOXO3a activity has been associated with either host protective or pathogenic roles during viral and bacterial infections (e.g., LCMV, HIV, rhinovirus, Mycobacterium tuberculosis, and Citrobacter rodentium) (31)(32)(33)(34)(35). For instance, FOXO3a promotes type I IFN antiviral innate immune responses while limiting tissue damage during rhinovirus infection (31). ...
... In line with these observations, we provide evidence that T. gondii actively forces FOXO3a out of the nucleus via AKT to hamper transcriptional programs involved in host defense responses in HFF. Similarly, the bacterium C. rodentium was shown to induce the translocation of nuclear FOXO3a into the cytosol of infected human HT-29 and mouse CMT-93 epithelial cell lines and colonic epithelium of infected mice (34). Even though no experimental evidence was provided on the alteration of transcriptional programs downstream of FOXO3a, the observed phenotype was associated with exacerbated pro-inflammatory cytokine production and disease pathogenesis during C. rodentium infection (34). ...
... Similarly, the bacterium C. rodentium was shown to induce the translocation of nuclear FOXO3a into the cytosol of infected human HT-29 and mouse CMT-93 epithelial cell lines and colonic epithelium of infected mice (34). Even though no experimental evidence was provided on the alteration of transcriptional programs downstream of FOXO3a, the observed phenotype was associated with exacerbated pro-inflammatory cytokine production and disease pathogenesis during C. rodentium infection (34). We observed a dramatic increase in AKT-sensitive phosphoryla tion and nuclear exclusion of FOXO3a in T. gondii-infected HFF. ...
The intracellular parasite Toxoplasma gondii induces host AKT activation to prevent autophagy-mediated clearance; however, the molecular underpinnings are not fully understood. Autophagy can be negatively regulated through AKT-sensitive phosphorylation and nuclear export of the transcription factor Forkhead box O3a (FOXO3a). Using a combination of pharmacological and genetic approaches, herein we investigated whether T. gondii hinders host autophagy through AKT-dependent inactivation of FOXO3a. We found that infection by type I and II strains of T. gondii promotes gradual and sustained AKT-dependent phosphorylation of FOXO3a at residues S253 and T32 in human foreskin fibroblasts (HFF) and murine 3T3 fibroblasts. Mechanistically, AKT-sensitive phosphorylation of FOXO3a by T. gondii required live infection and the activity of PI3K but was independent of the plasma membrane receptor EGFR and the kinase PKCα. Phosphorylation of FOXO3a at AKT-sensitive residues was paralleled by its nuclear exclusion in T. gondii -infected HFF. Importantly, the parasite was unable to drive cytoplasmic localization of FOXO3a upon pharmacological blockade of AKT or overexpression of an AKT-insensitive mutant form of FOXO3a. Transcription of a subset of bona fide autophagy-related targets of FOXO3a was reduced during T. gondii infection in an AKT-dependent fashion. However, parasite-directed repression of autophagy-related genes was AKT-resistant in cells deficient in FOXO3a. Consistent with this, T. gondii failed to inhibit the recruitment of acidic organelles and LC3, an autophagy marker, to the parasitophorous vacuole upon chemically or genetically induced nuclear retention of FOXO3a. In all, we provide evidence that T. gondii suppresses FOXO3a-regulated transcriptional programs to prevent autophagy-mediated killing.
IMPORTANCE
The parasite Toxoplasma gondii is the etiological agent of toxoplasmosis, an opportunistic infection commonly transmitted by ingestion of contaminated food or water. To date, no effective vaccines in humans have been developed and no promising drugs are available to treat chronic infection or prevent congenital infection. T. gondii targets numerous host cell processes to establish a favorable replicative niche. Of note, T. gondii activates the host AKT signaling pathway to prevent autophagy-mediated killing. Herein, we report that T. gondii inhibits FOXO3a, a transcription factor that regulates the expression of autophagy-related genes, through AKT-dependent phosphorylation. The parasite’s ability to block the recruitment of the autophagy machinery to the parasitophorous vacuole is impeded upon pharmacological inhibition of AKT or overexpression of an AKT-insensitive form of FOXO3a. Thus, our study provides greater granularity in the role of FOXO3a during infection and reinforces the potential of targeting autophagy as a therapeutic strategy against T. gondii .
... 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.
... In the absence of environment signals or growth factors, FoxO members localize to the nucleus. After PI3K mediated activation of the serine/threonine kinase Akt, FoxO3 are rapidly phosphorylated at T32, S253 and S315, and then bind to 14-3-3 chaperone protein, which sequester FoxO3 within the cytoplasm, leading to its functional inactivation [22,28,29]. However, in some cell types such as acute myeloid leukemia cells, FoxO3 is in constant inactive state due to cytoplasmic localization and ubiquitin-dependent degradation mediated by Iκκβ induced phosphorylation at S644. ...
... However, in some cell types such as acute myeloid leukemia cells, FoxO3 is in constant inactive state due to cytoplasmic localization and ubiquitin-dependent degradation mediated by Iκκβ induced phosphorylation at S644. In HE-29 intestinal epithelial cells, LPS was found to induce FoxO3 degradation by activating PI3K [29]. However, in this study, LPS up-regulated FoxO3 instantly in human monocytes with Iκκα/β phosphorylation at post-transcription level, without increased FoxO3 degradation, indicating LPS might affect FoxO3 translational efficiency or FoxO3 mRNA stability in human monocytes. ...
Objective
Previous studies have found that forkhead box o3 S574 phosphorylation status can regulate inflammation by inducing monocytes/macrophages apoptosis, and whether it directly affects the inflammatory response of monocytes has not been demonstrated. The aim of this study was to investigate the role of forkhead box o3 in inflammatory response of monocytes against lipopolysaccharide.
Methods
THP-1 cells were used to knock down or overexpress forkhead box o3 and its mutants, and then detect the activation of inflammatory cytokines expression and activation of nuclear factor kappa B after lipopolysaccharide treatment.
Results
The present study demonstrated that lipopolysaccharide can up-regulate forkhead box o3 protein expression, especially the non-phosphorylated form at S574, in a post-transcriptional way. Knockdown of forkhead box o3 attenuated lipopolysaccharide mediated nuclear factor kappa B activation and downstream inflammatory cytokines expression. When overexpressing forkhead box o3, only non-phosphorylated S574A forkhead box o3 mutant enhanced lipopolysaccharide induced nuclear factor kappa B activation and inflammatory cytokines expression. Further studies have found that S574A forkhead box o3 may promote toll like receptor 4 expression through binding and accelerating its transcriptional activity from promoter.
Conclusion
There might be a positive feedback loop between lipopolysaccharide and forkhead box o3 in monocytes to promote the lipopolysaccharide mediated inflammatory response.
... 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 and FOXO3 was significantly downregulated by miR-23a. FOXOs regulates number of genes and interacts with several different elements involved in cell fate process whereas GSK-3β in turn stimulates expression of FoxO3A [30,32]. Even though the significance of FOXO3A in myocardial injury was unclear, FOXO3A had been shown to protect the cardiac function from hypertrophy pathology [33]. ...
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.
... However, little is known about FOXO3a function during the immune response in epithelial cells. Previous research reported that FOXO3a negatively regulated cytokine expression in colonic epithelial cells [42]. In our study, FOXO3a plays a different function during IAV infection. ...
Accumulating evidence has shown that long noncoding RNAs (lncRNAs) are involved in several biological processes, including immune responses. However, the role of lncRNAs in antiviral innate immune responses remains largely elusive. Here, we identify an uncharacterized human lncRNA AVAN from influenza A virus (IAV) infected patients, that is significantly upregulated following RNA virus infection. During IAV infection, AVAN play an indispensable role in antiviral immune responses. In vivo, we enforced the expression of AVAN in transgenic mice or adeno-associated virus encoding AVAN delivery system and found that AVAN significantly alleviated IAV virulence and virus replication. Mechanistically, nuclear AVAN positively regulates the transcription of forkhead box O3A (FOXO3a) by associating with its promoter and inducing chromatin remodeling to promote neutrophil chemotaxis. Meanwhile, cytoplasmic AVAN binds directly to the E3 ligase TRIM25 and enhances TRIM25-mediated K63-linked ubiquitination of RIG-I, thereby promoting TRIM25- and RIG-I-mediated antiviral innate immune responses, including the induction of type I interferon and ISGs. Moreover, AVAN binds to the B Box/CCD domain of TRIM25 and 1–200nt of AVAN were the functional moieties. Collectively, our findings highlight the potential clinical implications of human lncRNA AVAN as a key positive regulator of the antiviral innate immune response and a promising target for developing broad antiviral therapeutics.
... Various miRNAs related to cardiac disease could directly target FOXO3A, for example, miR-30d in diabetic cardiomyopathy [25], miR-223 in myocardial infarction (MI) [26], miR-23a in cardiac hypertrophy [27]. Given the role of FOXO3A is in inflammatory response [28][29][30][31], it is crucial to reveal its relationship with miRNAs in the septic cardiac dysfunction. ...
Cardiac dysfunction represents a main component of death induced by sepsis in critical care units. And microRNAs (miRNAs) have been reported as important modulators or biomarkers of sepsis. However, the molecular detail of miRNAs involved in septic cardiac dysfunction remains unclear. Here we showed that endotoxin (lipopolysaccharide, LPS) significantly down-regulated expression of miR-29b-3p in heart. Increased expression of miR-29b-3p by lentivirus improved cardiac function and attenuated damage of cardiac induced by LPS in mice. Furthermore, overexpression or knockdown of miR-29b-3p showed its crucial roles on regulation of apoptosis and production of pro-inflammatory cytokines in NRCMs through directly targeting FOXO3A. miR-29b-3p ameliorates inflammatory damage likely via reducing activation of MAPKs and nuclear-translocation of NF-κB to block LPS-activated NF-κB signaling. Notably, miR-29b is also down-regulated in septic patients' plasma compared with normal subjects, indicating a potential clinical relevance of miR-29b. Taken together, our findings demonstrate that upregulation of miR-29b-3p can attenuate myocardial injury induced by sepsis via regulating FOXO3A, which provide a potential therapy target for interference of septic cardiac dysfunction.
... CXCL8-CXCR1/2 signaling may be targeted not only directly by regulation of CXCL8/CXCR1/CXCR2 expression and signaling but also indirectly by the regulation of significant signaling pathways and transcription factors, including PI3K/Akt, MEK/ERK, and NF-κB, which are responsible for the regulation of CXCL8 and CXCR1/CXCR2 expression. It has been reported that CXCL8 was downregulated in the HT-29 CRC cell line after treatment with the PI3K/Akt inhibitor LY294002 [62]. Furthermore, neurotensin-(NT-) induced CXCL8 mRNA expression and protein secretion was inhibited when treated with both U0126 and PD98059, which blocked ERK activation in HCT116 cells [63], indicating that the MEK/ERK pathway was involved in CXCL8 regulation by NT. ...
CXCL8 (also known as IL-8) can produce different biological effects by binding to its receptors: CXCR1, CXCR2, and the Duffy antigen receptor for chemokines (DARC). CXCL8 and its receptors are associated with the development of various tumor types, especially colorectal cancer and its liver metastases. In addition to promoting angiogenesis, proliferation, invasion, migration, and the survival of colorectal cancer (CRC) cells, CXCL8 and its receptors have also been known to induce the epithelial-mesenchymal transition (EMT) of CRC cells, to help them to escape host immunosurveillance as well as to enhance resistance to anoikis, which promotes the formation of circulating tumor cells (CTCs) and their colonization of distant organs. In this paper, we will review the established roles of CXCL8 signaling in CRC and discuss the possible strategies of targeting CXCL8 signaling for overcoming CRC drug resistance and cancer progression, including direct targeting of CXCL8/CXCR1/2 or indirect targeting through the inhibition of CXCL8-CXCR1/2 signaling.
... In response to various stresses, such as oxidative stress, FOXO3a can activate or repress multiple target genes, such as Bim (Sunters et al., 2003) to induce apoptosis and p21 (Seoane et al., 2004) and cyclin D (Schmidt et al., 2002) to induce cell cycle arrest. The inhibition of FOXO3a expression and activity is critical for the promotion of cell transformation and tumor progression, suggesting that FOXO3a has a tumor-suppressive role (Snoeks et al., 2008;Liu et al., 2018). The inactivation of FOXO3a is also correlated with poor prognosis of a variety of cancers, including breast cancer (Jiang et al., 2013), lung cancer (Liu et al., 2012(Liu et al., , 2014, and leukemia (Sakoe et al., 2010). ...
Cardamonin (CD), a naturally occurring chalcone isolated from large black cardamom, was previously reported to suppress the proliferation of breast cancer cells. However, its precise molecular antitumor mechanisms have not been well elucidated. In this study, we found that CD markedly inhibited the proliferation of MDA‐MB 231 and MCF‐7 breast cancer cells through the induction of G2/M arrest and apoptosis. Reactive oxygen species (ROS) plays a pivotal role in the inhibition of CD‐induced cell proliferation. Treatment with N‐acetyl‐cysteine (NAC), an ROS scavenger, blocked CD‐induced G2/M arrest and apoptosis in this study. Quenching of ROS by overexpression of catalase also blocked CD‐induced cell cycle arrest and apoptosis. We showed that CD enhanced the expression and nuclear translocation of Forkhead box O3 (FOXO3a) via upstream c‐Jun N‐terminal kinase, inducing the expression of FOXO3a and its target genes, including p21, p27, and Bim. This process led to the reduction of cyclin D1 and enhancement of activated caspase‐3 expression. The addition of NAC markedly reversed these effects, knockdown of FOXO3a using small interfering RNA also decreased CD‐induced G2/M arrest and apoptosis. In vivo, CD efficiently suppressed the growth of MDA‐MB 231 breast cancer xenograft tumors. Taken together, our data provide a molecular mechanistic rationale for CD‐induced cell cycle arrest and apoptosis in breast cancer cells. This article is protected by copyright. All rights reserved.
