Hormone-induced enhancer assembly requires an optimal level of hormone receptor multivalent interactions.

Transcription factors (TFs) activate enhancers to drive cell-specific gene programs in response to signals, but our understanding of enhancer assembly during signaling events is incomplete. Here, we show that androgen receptor (AR) forms condensates through multivalent interactions mediated by its N-terminal intrinsically disordered region (IDR) to orchestrate enhancer assembly in response to androgen signaling. AR IDR can be substituted by IDRs from selective proteins for AR condensation capacity and its function on enhancers. Expansion of the poly(Q) track within AR IDR results in a higher AR condensation propensity as measured by multiple methods, including live-cell single-molecule microscopy. Either weakening or strengthening AR condensation propensity impairs its heterotypic multivalent interactions with other enhancer components and diminishes its transcriptional activity. Our work reveals the requirement of an optimal level of AR condensation in mediating enhancer assembly and suggests that alteration of the fine-tuned multivalent IDR-IDR interactions might underlie AR-related human pathologies.

Axon Injury-Induced Autophagy Activation Is Impaired in a C. elegans Model of Tauopathy.

Autophagy is a conserved pathway that plays a key role in cell homeostasis in normal settings, as well as abnormal and stress conditions. Autophagy dysfunction is found in various neurodegenerative diseases, although it remains unclear whether autophagy impairment is a contributor or consequence of neurodegeneration. Axonal injury is an acute neuronal stress that triggers autophagic responses in an age-dependent manner. In this study, we investigate the injury-triggered autophagy response in a C. elegans model of tauopathy. We found that transgenic expression of pro-aggregant Tau, but not the anti-aggregant Tau, abolished axon injury-induced autophagy activation, resulting in a reduced axon regeneration capacity. Furthermore, axonal trafficking of autophagic vesicles were significantly reduced in the animals expressing pro-aggregant F3ΔK280 Tau, indicating that Tau aggregation impairs autophagy regulation. Importantly, the reduced number of total or trafficking autophagic vesicles in the tauopathy model was not restored by the autophagy activator rapamycin. Loss of PTL-1, the sole Tau homologue in C. elegans, also led to impaired injury-induced autophagy activation, but with an increased basal level of autophagic vesicles. Therefore, we have demonstrated that Tau aggregation as well as Tau depletion both lead to disruption of injury-induced autophagy responses, suggesting that aberrant protein aggregation or microtubule dysfunction can modulate autophagy regulation in neurons after injury.

Intestinal Epithelial Cells Exposed to Bacteroides fragilis Enterotoxin Regulate NF-κB Activation and Inflammatory Responses through ß-Catenin Expression.

The Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), interacts with intestinal epithelial cells and can provoke signals that induce mucosal inflammation. Although ß-catenin signaling is reported to be associated with inflammatory responses and BFT is known to cleave E-cadherin linked with ß-catenin, little is known about the ß-catenin-mediated regulation of inflammation in ETBF infection. This study was conducted to investigate the role of ß-catenin as a cellular signaling intermediate in the induction of proinflammatory responses to stimulation of intestinal epithelial cells with BFT. Expression of ß-catenin in intestinal epithelial cells was reduced relatively early after stimulation with BFT and then recovered to normal levels relatively late after stimulation. In contrast, phosphorylation of ß-catenin in BFT-exposed cells occurred at high levels early in stimulation and decreased as time passed. Concurrently, late after stimulation the nuclear levels of ß-catenin were relatively higher than those early after stimulation. Suppression of ß-catenin resulted in increased NF-κB activity and interleukin-8 (IL-8) expression in BFT-stimulated cells. However, suppression or enhancement of ß-catenin expression neither altered the phosphorylated IκB kinase α/ß complex nor activated activator protein 1 signals. Furthermore, inhibition of glycogen synthase kinase 3ß was associated with increased ß-catenin expression and attenuated NF-κB activity and IL-8 expression in BFT-exposed cells. These findings suggest the negative regulation of NF-κB-mediated inflammatory responses by ß-catenin in intestinal epithelial cells stimulated with BFT, resulting in attenuation of acute inflammation in ETBF infection.

Bacteroides fragilis Enterotoxin Induces Formation of Autophagosomes in Endothelial Cells but Interferes with Fusion with Lysosomes for Complete Autophagic Flux through a Mitogen-Activated Protein Kinase-, AP-1-, and C/EBP Homologous Protein-Dependent Pathway.

Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), plays an essential role in mucosal inflammation. Although autophagy contributes to the pathogenesis of diverse infectious diseases, little is known about autophagy in ETBF infection. This study was conducted to investigate the role of BFT in the autophagic process in endothelial cells (ECs). Stimulation of human umbilical vein ECs (HUVECs) with BFT increased light chain 3 protein II (LC3-II) conversion from LC3-I and protein expression of p62, Atg5, and Atg12. In addition, BFT-exposed ECs showed increased indices of autophagosomal fusion with lysosomes such as LC3-lysosome-associated protein 2 (LAMP2) colocalization and the percentage of red vesicles monitored by the expression of dual-tagged LC3B. BFT also upregulated expression of C/EBP homologous protein (CHOP), and inhibition of CHOP significantly increased indices of autophagosomal fusion with lysosomes. BFT activated an AP-1 transcription factor, in which suppression of AP-1 activity significantly downregulated CHOP and augmented autophagosomal fusion with lysosomes. Furthermore, suppression of Jun N-terminal protein kinase (JNK) mitogen-activated protein kinase (MAPK) significantly inhibited the AP-1 and CHOP signals, leading to an increase in autophagosomal fusion with lysosomes in BFT-stimulated ECs. These results suggest that BFT induced accumulation of autophagosomes in ECs, but activation of a signaling pathway involving JNK, AP-1, and CHOP may interfere with complete autophagy.

Bacteroides fragilis Enterotoxin Upregulates Heme Oxygenase-1 in Intestinal Epithelial Cells via a Mitogen-Activated Protein Kinase- and NF-κB-Dependent Pathway, Leading to Modulation of Apoptosis.

The Bacteroides fragilis enterotoxin (BFT), a virulence factor of enterotoxigenic B. fragilis (ETBF), interacts with intestinal epithelial cells and can provoke signals that induce mucosal inflammation. Although expression of heme oxygenase-1 (HO-1) is associated with regulation of inflammatory responses, little is known about HO-1 induction in ETBF infection. This study was conducted to investigate the effect of BFT on HO-1 expression in intestinal epithelial cells. Stimulation of intestinal epithelial cells with BFT resulted in upregulated expression of HO-1. BFT activated transcription factors such as NF-κB, AP-1, and Nrf2 in intestinal epithelial cells. Upregulation of HO-1 in intestinal epithelial cells was dependent on activated IκB kinase (IKK)-NF-κB signals. However, suppression of Nrf2 or AP-1 signals in intestinal epithelial cells did not result in significant attenuation of BFT-induced HO-1 expression. HO-1 induction via IKK-NF-κB in intestinal epithelial cells was regulated by p38 mitogen-activated protein kinases (MAPKs). Furthermore, suppression of HO-1 activity led to increased apoptosis in BFT-stimulated epithelial cells. These results suggest that a signaling pathway involving p38 MAPK-IKK-NF-κB in intestinal epithelial cells is required for HO-1 induction during exposure to BFT. Following this induction, increased HO-1 expression may regulate the apoptotic process in responses to BFT stimulation.

Crude Preparations of Helicobacter pylori Outer Membrane Vesicles Induce Upregulation of Heme Oxygenase-1 via Activating Akt-Nrf2 and mTOR-IκB Kinase-NF-κB Pathways in Dendritic Cells.

Helicobacter pylori sheds outer membrane vesicles (OMVs) that contain many surface elements of bacteria. Dendritic cells (DCs) play a major role in directing the nature of adaptive immune responses against H. pylori, and heme oxygenase-1 (HO-1) has been implicated in regulating function of DCs. In addition, HO-1 is important for adaptive immunity and the stress response. Although H. pylori-derived OMVs may contribute to the pathogenesis of H. pylori infection, responses of DCs to OMVs have not been elucidated. In the present study, we investigated the role of H. pylori-derived crude OMVs in modulating the expression of HO-1 in DCs. Exposure of DCs to crude H. pylori OMVs upregulated HO-1 expression. Crude OMVs obtained from a cagA-negative isogenic mutant strain induced less HO-1 expression than OMVs obtained from a wild-type strain. Crude H. pylori OMVs activated signals of transcription factors such as NF-κB, AP-1, and Nrf2. Suppression of NF-κB or Nrf2 resulted in significant attenuation of crude OMV-induced HO-1 expression. Crude OMVs increased the phosphorylation of Akt and downstream target molecules of mammalian target of rapamycin (mTOR), such as S6 kinase 1 (S6K1). Suppression of Akt resulted in inhibition of crude OMV-induced Nrf2-dependent HO-1 expression. Furthermore, suppression of mTOR was associated with inhibition of IκB kinase (IKK), NF-κB, and HO-1 expression in crude OMV-exposed DCs. These results suggest that H. pylori-derived OMVs regulate HO-1 expression through two different pathways in DCs, Akt-Nrf2 and mTOR-IKK-NF-κB signaling. Following this induction, increased HO-1 expression in DCs may modulate inflammatory responses in H. pylori infection.

Helicobacter pylori vacuolating cytotoxin induces apoptosis via activation of endoplasmic reticulum stress in dendritic cells.

BACKGROUND AND AIM: Dendritic cells (DCs) are observed on the Helicobacter pylori-infected gastric mucosa. DCs generally play an important role in the regulation of inflammation. Although stimulation of gastric epithelial cells with H. pylori vacuolating cytotoxin (VacA) has been reported to induce apoptosis and endoplasmic reticulum (ER) stress, the effects of VacA on the DC apoptotic response have not been well elucidated. This study was conducted to investigate the role of H. pylori VacA on the apoptotic process and ER stress in DCs. METHODS: Murine and human DCs were generated from specific pathogen-free C57BL/6 mice and human peripheral blood mononuclear cells, respectively. DCs were incubated with purified VacA, after which Bax activation, cytochrome c release, and DNA fragmentation for apoptosis were measured by fluorescent microscopy, immunoblot, and ELISA. ER stress-related molecules such as GRP78 and CHOP were analyzed by immunoblot. RESULTS: Treatment of DCs with purified H. pylori VacA resulted in the induction of apoptosis. DC stimulation with VacA led to the translocation of cytoplasmic Bax to mitochondria and cytochrome c release from mitochondria. H. pylori VacA induced signals for ER stress early during the stimulation process in DCs. Furthermore, suppression of ER stress resulted in a significant inhibition of the VacA-induced apoptosis in DCs. CONCLUSION: These results suggest that ER stress is critical for regulation of DC apoptotic process in response to VacA stimulation.

Helicobacter pylori outer membrane vesicle proteins induce human eosinophil degranulation via a ß2 Integrin CD11/CD18- and ICAM-1-dependent mechanism.

Eosinophil cationic protein (ECP), a cytotoxic protein contained in eosinophils granules, can contribute to various inflammatory responses. Although Helicobacter pylori infection increases infiltration of eosinophils, the mechanisms of eosinophil degranulation by H. pylori infection are largely unknown. The goal of this study was to investigate the role of H. pylori outer membrane vesicles (OMVs) in modulating eosinophil degranulation. We found that eosinophils treated with H. pylori OMVs released significantly more ECP compared with untreated controls. In addition, eosinophils cocultured with OMV-preexposed primary gastric epithelial cells exhibited significantly increased ECP release. Similarly, eosinophils cocultured with culture supernatant (CM) from primary gastric epithelial cells exposed to OMVs (OMV-CM) released significantly higher amounts of ECP compared with eosinophils cocultured with CM from unexposed control cells. Furthermore, OMVs and OMV-CM both induced the upregulation of ICAM-1 on gastric epithelial cells and ß2 integrin CD11b on eosinophils. In addition, both transduction of ICAM-1 shRNA into gastric epithelial cells and treatment with neutralizing mAbs to CD18 significantly decreased OMV-mediated or OMV-CM-mediated release of ECP. These results suggest that the eosinophil degranulation response to H. pylori OMVs occurs via a mechanism that is dependent on both ß2 integrin CD11/CD18 and ICAM-1.

Anti-inflammatory mechanism of metformin and its effects in intestinal inflammation and colitis-associated colon cancer.

BACKGROUND AND AIM: The aim of this study is to evaluate the effect of metformin on intestinal inflammation. METHODS: COLO205 cells were pretreated with metformin and stimulated with tumor necrosis factor (TNF)-α. Expression of interleukin (IL)-8 was determined by luciferase assay and real-time PCR. Inhibitor of kappaB (IκB) phosphorylation/degradation and adenosine monohosphate-activated protein kinase (AMPK) activity were evaluated by Western blotting. DNA-binding activity of transcription factor nuclear factor-kappaB (NF-κB) was assessed by electrophoretic mobility shift assay. In an acute colitis model, mice were given 4% dextran sulfate sodium (DSS) for 5 days. IL-10−/− mice were used to evaluate the effect of metformin on chronic colitis. In an inflamation-associated tumor model, mice were given a single intraperitoneal injection of azoxymethane followed by three cycles of 2% DSS for 5 days and 2 weeks of free water consumption. RESULTS: Metformin significantly inhibited IL-8 induction in COLO 205 cells stimulated with TNF-α. Metformin attenuated IκBα phosphorylation and NF-κB DNA-binding activity. Administration of metformin significantly reduced the severity of DSS-induced colitis. In addition, DSS-induced IκB kinase (IKK) activation was significantly reduced in mice treated with metformin. Metformin significantly attenuated the severity of colitis in IL-10−/− mice, induced AMPK activity in intestinal epithelial cells, and inhibited the development of colitic cancer in mice. CONCLUSIONS: These results indicate that metformin suppresses NF-κB activation in intestinal epithelial cells and ameliorates murine colitis and colitis-associated tumorigenesis in mice, suggesting that metformin could be a potential therapeutic agent for the treatment of inflammatory bowel disease.

Bacteroides fragilis enterotoxin upregulates lipocalin-2 expression in intestinal epithelial cells.

Enterotoxigenic Bacteroides fragilis (ETBF) produces an ≈ 20 kDa B. fragilis enterotoxin (BFT), which plays an essential role in mucosal inflammation. Lipocalin (Lcn)-2, a siderophore-binding antimicrobial protein, is critical for control of bacterial infection; however, expression of Lcn-2 in BFT-exposed intestinal epithelial cells has not been elucidated. In the present study, stimulation of human intestinal epithelial cells with BFT resulted in the upregulation of Lcn-2 expression that was a relatively late response of intestinal epithelial cells compared with human ß-defensin (hBD)-2 expression. The upregulation of Lcn-2 was dependent on AP-1 but not on NF-κB signaling. Lcn-2 induction via AP-1 was regulated by mitogen-activated protein kinases (MAPKs) including ERK and p38. Lcn-2 was secreted from the apical and basolateral surfaces in BFT-treated cells. These results suggest that a signaling pathway involving MAPKs and AP-1 is required for Lcn-2 induction in intestinal epithelial cells exposed to BFT, after which the secreted Lcn-2 may facilitate antimicrobial activity within ETBF-infected mucosa.

The guggulsterone derivative GG-52 inhibits NF-κB signaling in gastric epithelial cells and ameliorates ethanol-induced gastric mucosal lesions in mice.

Gastric mucosal inflammation can develop after challenge with noxious stimuli such as alcohol. Specially, alcohol stimulates the release of inflammatory cytokines but does not increase gastric acid secretion, leading to gastric mucosal damage. The plant sterol guggulsterone and its novel derivative GG-52 have been reported to inhibit nuclear factor-κB (NF-κB) signaling in intestinal epithelial cells and experimental colitis. In the present study, we investigated the anti-inflammatory effects of GG-52 on gastric epithelial cells and on ethanol-induced gastric mucosal inflammation in mice. GG-52 inhibited the expression of interleukin-8 (IL-8) in gastric epithelial AGS and MKN-45 cell lines stimulated with tumor necrosis factor (TNF)-α in a dose-dependent manner. Pretreatment with GG-52 suppressed TNF-α-induced activation of IκB kinase (IKK) and NF-κB signaling in MKN-45 cells. In contrast, the inactive analog GG-46 did not produce significant changes in IL-8 expression or NF-κB activation. In a model of ethanol-induced murine gastritis, administration of GG-52 significantly reduced the severity of gastritis, as assessed by macroscopic and histological evaluation of gastric mucosal damage. In addition, the ethanol-induced upregulation of chemokine KC, a mouse homolog of IL-8, and phosphorylated p65 NF-κB signals were significantly inhibited in murine gastric mucosa pretreated with GG-52. These results indicate that GG-52 suppresses NF-κB activation in gastric epithelial cells and ameliorates ethanol-induced gastric mucosal lesions in mice, suggesting that GG-52 may be a potential gastroprotective agent.

Bacteroides fragilis enterotoxin upregulates intercellular adhesion molecule-1 in endothelial cells via an aldose reductase-, MAPK-, and NF-κB-dependent pathway, leading to monocyte adhesion to endothelial cells.

Enterotoxigenic Bacteroides fragilis (ETBF) produces a ∼ 20-kDa heat-labile enterotoxin (BFT) that plays an essential role in mucosal inflammation. Although a variety of inflammatory cells is found at ETBF-infected sites, little is known about leukocyte adhesion in response to BFT stimulation. We investigated whether BFT affected the expression of ICAM-1 and monocytic adhesion to endothelial cells (ECs). Stimulation of HUVECs and rat aortic ECs with BFT resulted in the induction of ICAM-1 expression. Upregulation of ICAM-1 was dependent on the activation of IκB kinase (IKK) and NF-κB signaling. In contrast, suppression of AP-1 did not affect ICAM-1 expression in BFT-stimulated cells. Suppression of NF-κB activity in HUVECs significantly reduced monocytic adhesion, indicating that ICAM-1 expression is indispensable for BFT-induced adhesion of monocytes to the endothelium. Inhibition of JNK resulted in a significant attenuation of BFT-induced ICAM-1 expression in ECs. Moreover, inhibition of aldose reductase significantly reduced JNK-dependent IKK/NF-κB activation, ICAM-1 expression, and adhesion of monocytes to HUVECs. These results suggest that a signaling pathway involving aldose reductase, JNK, IKK, and NF-κB is required for ICAM-1 induction in ECs exposed to BFT, and may be involved in the leukocyte-adhesion cascade following infection with ETBF.

Bacteroides fragilis Enterotoxin Induces Autophagy through an AMPK and FoxO3-Pathway, Leading to the Inhibition of Apoptosis in Intestinal Epithelial 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.

A novel endoplasmic reticulum adaptation is critical for the long-lived Caenorhabditis elegans rpn-10 proteasomal mutant.

The loss of proteostasis due to reduced efficiency of protein degradation pathways plays a key role in multiple age-related diseases and is a hallmark of the aging process. Paradoxically, we have previously reported that the Caenorhabditis elegans rpn-10(ok1865) mutant, which lacks the RPN-10/RPN10/PSMD4 subunit of the 19S regulatory particle of the 26S proteasome, exhibits enhanced cytosolic proteostasis, elevated stress resistance and extended lifespan, despite possessing reduced proteasome function. However, the response of this mutant against threats to endoplasmic reticulum (ER) homeostasis and proteostasis was unknown. Here, we find that the rpn-10 mutant is highly ER stress resistant compared to the wildtype. Under unstressed conditions, the ER unfolded protein response (UPR) is activated in the rpn-10 mutant as signified by increased xbp-1 splicing. This primed response appears to alter ER homeostasis through the upregulated expression of genes involved in ER protein quality control (ERQC), including those in the ER-associated protein degradation (ERAD) pathway. Pertinently, we find that ERQC is critical for the rpn-10 mutant longevity. These changes also alter ER proteostasis, as studied using the C. elegans alpha-1 antitrypsin (AAT) deficiency model, which comprises an intestinal ER-localised transgenic reporter of an aggregation-prone form of AAT called ATZ. The rpn-10 mutant shows a significant reduction in the accumulation of the ATZ reporter, thus indicating that its ER proteostasis is augmented. Via a genetic screen for suppressors of decreased ATZ aggregation in the rpn-10 mutant, we then identified ecps-2/H04D03.3, a novel ortholog of the proteasome-associated adaptor and scaffold protein ECM29/ECPAS. We further show that ecps-2 is required for improved ER proteostasis as well as lifespan extension of the rpn-10 mutant. Thus, we propose that ECPS-2-proteasome functional interactions, alongside additional putative molecular processes, contribute to a novel ERQC adaptation which underlies the superior proteostasis and longevity of the rpn-10 mutant.

Age-dependent autophagy induction after injury promotes axon regeneration by limiting NOTCH.

Macroautophagy/autophagy is essential for maintaining cellular homeostasis through the degradation of organelles and proteins. It also has a prominent role in modulating aging. However, the role of autophagy in the neuronal response to axon injury and axon regeneration, particularly in the context of aging, remains largely unknown. Our candidate genetic screen for axon regeneration regulators has identified genes in the autophagy pathway. Using a reporter that monitors autophagosomes and autolysosomes, we were able to monitor the dynamics of autophagy during axon regeneration. In response to axon injury, there was a significant increase in the number of autophagic vesicles. Injury-triggered autophagy activation and axon regeneration capacity undergo an age-dependent decline, and autophagy-activating agents partially rescued these declines. We found that DLK-1 was both required and sufficient for injury-induced autophagy activation. Autophagic vesicles co-localized with the NOTCH4 ortholog, LIN-12 receptor, a previously identified inhibitor of axon regeneration. Epistasis analyses indicate that LIN-12 might be a target of autophagy in axon regeneration. Together, our data suggest that DLK-mediated injury signaling can activate autophagy, which might limit the level of LIN-12 and NOTCH proteins to promote axon regeneration. Our findings reveal that autophagy activation can promote axon regeneration in neurons that lack maximal regrowth capacity, providing a promising therapeutic strategy for axon injury. Abbreviations: 3-MA: 3-methyladenine; ALs: autolysosomes; APs: autophagosomes; ARF-6: ADP-Ribosylation Factor related 6; ATG-9: AuTophaGy (yeast Atg homolog) 9; ATG9A: autophagy related 9A; BA1: bafilomycin A1; BEC-1: BEClin (human autophagy) homolog; BECN1: beclin 1; C. elegans: Caenorhabditis elegans; CEBP-1: C/EBP (CCAAT/enhancer-binding protein) homolog; CNS: central nervous system; DLK-1: Dual-Leucine zipper Kinase; DMSO: dimethyl sulfoxide; DRG: dorsal root ganglion; FOS: Fos proto-oncogene, AP-1 transcription factor subunit; GABA: gamma-aminobutyric acid; GFP: green fluorescent protein; HDA-3: Histone DeAcetylase; IP3: inositol trisphosphate; ITR-1: Inositol Triphosphate Receptor; KLF-2: Kruppel-Like Factor (zinc finger protein) 2; LGG-1: LC3, GABARAP and GATE-16 family; MAK-2: MAP kinase Activated protein Kinase; MAP kinase: mitogen-activated protein kinase; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MKK-4: mitogen activated protein kinase kinase 4; MTOR: mechanistic target of rapamycin kinase; NGM: nematode growth medium; NICD: Notch intracellular domain; NOTCH: notch receptor; PLM: posterior lateral microtubule; PMK-3: P38 Map kinase family; PNS: peripheral nervous system; SCG10: superior cervical ganglion protein 10; SCI: spinal cord injury; UNC-51: UNCoordinated 51; ULK1: unc-51 like autophagy activating kinase 1; wnd: wallenda.

Bacteroides fragilis enterotoxin upregulates heme oxygenase-1 in dendritic cells via reactive oxygen species-, mitogen-activated protein kinase-, and Nrf2-dependent pathway.

BACKGROUND: Enterotoxigenic Bacteroides fragilis (ETBF) causes colitis and diarrhea, and is considered a candidate pathogen in inflammatory bowel diseases as well as colorectal cancers. These diseases are dependent on ETBF-secreted toxin (BFT). Dendritic cells (DCs) play an important role in directing the nature of adaptive immune responses to bacterial infection and heme oxygenase-1 (HO-1) is involved in the regulation of DC function. AIM: To investigate the role of BFT in HO-1 expression in DCs. METHODS: Murine DCs were generated from specific pathogen-free C57BL/6 and Nrf2-/- knockout mice. DCs were exposed to BFT, after which HO-1 expression and the related signaling factor activation were measured by quantitative RT-PCR, EMSA, fluorescent microscopy, immunoblot, and ELISA. RESULTS: HO-1 expression was upregulated in DCs stimulated with BFT. Although BFT activated transcription factors such as NF-κB, AP-1, and Nrf2, activation of NF-κB and AP-1 was not involved in the induction of HO-1 expression in BFT-exposed DCs. Instead, upregulation of HO-1 expression was dependent on Nrf2 activation in DCs. Moreover, HO-1 expression via Nrf2 in DCs was regulated by mitogen-activated protein kinases such as ERK and p38. Furthermore, BFT enhanced the production of reactive oxygen species (ROS) and inhibition of ROS production resulted in a significant decrease of phospho-ERK, phospho-p38, Nrf2, and HO-1 expression. CONCLUSION: These results suggest that signaling pathways involving ROS-mediated ERK and p38 mitogen-activated protein kinases-Nrf2 activation in DCs are required for HO-1 induction during exposure to ETBF-produced BFT.

Microtubule regulators act in the nervous system to modulate fat metabolism and longevity through DAF-16 in C. elegans.

Microtubule (MT) regulation is involved in both neuronal function and the maintenance of neuronal structure, and MT dysregulation appears to be a general downstream indicator and effector of age-related neurodegeneration. But the role of MTs in natural aging is largely unknown. Here, we demonstrate a role of MT regulators in regulating longevity. We find that loss of EFA-6, a modulator of MT dynamics, can delay both neuronal aging and extend the lifespan of C. elegans. Through the use of genetic mutants affecting other MT-regulating genes in C. elegans, we find that loss of MT stabilizing genes (including ptrn-1 and ptl-1) shortens lifespan, while loss of MT destabilizing gene hdac-6 extends lifespan. Via the use of tissue-specific transgenes, we further show that these MT regulators can act in the nervous system to modulate lifespan. Through RNA-seq analyses, we found that genes involved in lipid metabolism were differentially expressed in MT regulator mutants, and via the use of Nile Red and Oil Red O staining, we show that the MT regulator mutants have altered fat storage. We further find that the increased fat storage and extended lifespan of the long-lived MT regulator mutants are dependent on the DAF-16/FOXO transcription factor. Our results suggest that neuronal MT status might affect organismal aging through DAF-16-regulated changes in fat metabolism, and therefore, MT-based therapies might represent a novel intervention to promote healthy aging.

Mitogen-activated protein kinase/IκB kinase/NF-κB-dependent and AP-1-independent CX3CL1 expression in intestinal epithelial cells stimulated with Clostridium difficile toxin A.

UNLABELLED: Clostridium difficile toxin A causes acute colitis associated with inflammatory cell infiltration and increased production of proinflammatory mediators. Although CX3CL1 (fractalkine) plays a role in chemoattracting monocytes/macrophages, NK cells, and T cells, little information is available on the regulated expression of CX3CL1 in response to toxin A stimulation. In this study, we investigated the role of C. difficile toxin A on CX3CL1 induction in intestinal epithelial cells. Stimulation of murine intestinal epithelial cells with toxin A resulted in the upregulation of CX3CL1. Expression of CX3CL1 was dependent on nuclear factor-kappaB (NF-κB) and IκB kinase (IKK) activation, while the suppression of activator protein-1 (AP-1) did not affect toxin A-induced CX3CL1 expression. Suppression of p38 mitogen-activated protein kinase (MAPK) significantly inhibited IKK-NF-κB signaling leading to CX3CL1 induction in C. difficile toxin A-stimulated cells. CX3CL1 was mainly secreted from the basolateral surfaces in toxin A-treated cells. Furthermore, inhibition of p38 activity attenuated the toxin A-induced upregulation of CX3CL1 in the mouse ileum in vivo. These results suggest that a pathway, including p38 MAPK, IKK, and NF-κB activation, is required for CX3CL1 induction in intestinal epithelial cells exposed to C. difficile toxin A and may regulate the development of intestinal inflammation induced by infection with toxigenic C. difficile. KEY MESSAGE: C. difficile toxin A causes colitis with inflammatory cell infiltration. CX3CL1 plays a role in chemoattracting immune cells. MAPK-NF-κB signaling is required for CX3CL1 induction in toxin A-exposed cells. CX3CL1 is mainly secreted from the basolateral surfaces. CX3CL1 may contribute to the regulation of toxigenic C. difficile infection.