RESUMEN
The intestinal epithelial layer is susceptible to damage by chemical, physiological and mechanical stress. While it is essential to maintain the integrity of epithelium, the biochemical pathways that contribute to the barrier function have not been completely investigated. Here we demonstrate an aryl hydrocarbon receptor (AHR)-dependent mechanism facilitating the production of the antimicrobial peptide AMP regenerating islet-derived protein 3 gamma (REG3G), which is essential for intestinal homeostasis. Genetic ablation of AHR in mice impairs pSTAT3-mediated REG3G expression and increases bacterial numbers of Segmented filamentous bacteria (SFB) and Akkermansia muciniphila in the small intestine. Studies with tissue-specific conditional knockout mice revealed that the presence of AHR in the epithelial cells of the small intestine is not required for the production of REG3G through the phosphorylated STAT3-mediated pathway. However, immune-cell-specific AHR activity is necessary for normal expression of REG3G in all regions of the small intestine. A diet rich in broccoli, capable of inducing AHR activity, increases REG3G production when compared to a semi-purified diet that is devoid of ligands that can potentially activate the AHR, thus highlighting the importance of AHR in antimicrobial function. Overall, these data suggest that homeostatic antimicrobial REG3G production is increased by an AHR pathway intrinsic to the immune cells in the small intestine.
Asunto(s)
Antiinfecciosos , Receptores de Hidrocarburo de Aril , Animales , Ratones , Citoesqueleto , Células Epiteliales , Intestino Delgado , Ratones Noqueados , Receptores de Hidrocarburo de Aril/genéticaRESUMEN
The aryl hydrocarbon receptor (AHR) mediates intestinal barrier homeostasis. Many AHR ligands are also CYP1A1/1B1 substrates, which can result in rapid clearance within the intestinal tract, limiting systemic exposure and subsequent AHR activation. This led us to the hypothesis that there are dietary substrates of CYP1A1/1B1 that functionally increase the half-life of potent AHR ligands. We examined the potential of urolithin A (UroA), a gut bacterial metabolite of ellagitannins, as a CYP1A1/1B1 substrate to enhance AHR activity in vivo. UroA is a competitive substrate for CYP1A1/1B1 in an in vitro competition assay. A broccoli-containing diet promotes the gastric formation of the potent hydrophobic AHR ligand and CYP1A1/1B1 substrate, 5,11-dihydroindolo[3,2-b]carbazole (ICZ). In mice, dietary exposure to UroA in a 10% broccoli diet led to a coordinated increase in duodenal, cardiac, and pulmonary AHR activity, but no increase in activity in the liver. Thus, CYP1A1 dietary competitive substrates can lead to enhanced systemic AHR ligand distribution from the gut, likely through the lymphatic system, increasing AHR activation in key barrier tissues. Finally, this report will lead to a reassessment of the dynamics of distribution of other hydrophobic chemicals present in the diet.
Asunto(s)
Citocromo P-450 CYP1A1 , Tracto Gastrointestinal , Pulmón , Receptores de Hidrocarburo de Aril , Animales , Ratones , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Ligandos , Hígado/metabolismo , Pulmón/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Dieta , Tracto Gastrointestinal/metabolismoRESUMEN
In the face of mechanical, chemical, microbial, and immunologic pressure, intestinal homeostasis is maintained through balanced cellular turnover, proliferation, differentiation, and self-renewal. Here, we present evidence supporting the role of the aryl hydrocarbon receptor (AHR) in the adaptive reprogramming of small intestinal gene expression, leading to altered proliferation, lineage commitment, and remodeling of the cellular repertoire that comprises the intestinal epithelium to promote intestinal resilience. Ahr gene/protein expression and transcriptional activity exhibit marked proximalHI to distalLO and cryptHI to villiLO gradients. Genetic ablation of Ahr impairs commitment/differentiation of the secretory Paneth and goblet cell lineages and associated mucin production, restricts expression of secretory/enterocyte differentiation markers, and increases crypt-associated proliferation and villi-associated enterocyte luminal exfoliation. Ahr-/- mice display a decrease in intestinal barrier function. Ahr+/+ mice that maintain a diet devoid of AHR ligands intestinally phenocopy Ahr-/- mice. In contrast, Ahr+/+ mice exposed to AHR ligands reverse these phenotypes. Ligand-induced AHR transcriptional activity positively correlates with gene expression (Math1, Klf4, Tff3) associated with differentiation of the goblet cell secretory lineage. Math1 was identified as a direct target gene of AHR, a transcription factor critical to the development of goblet cells. These data suggest that dietary cues, relayed through the transcriptional activity of AHR, can reshape the cellular repertoire of the gastrointestinal tract.
Asunto(s)
Células Epiteliales , Receptores de Hidrocarburo de Aril , Animales , Ratones , Diferenciación Celular , Células Epiteliales/metabolismo , Mucosa Intestinal/metabolismo , Intestinos , Ligandos , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
BACKGROUND: The quinoline-3-carboxamide, Tasquinimod (TasQ), is orally active as a maintenance therapy with an on-target mechanism-of-action via allosteric binding to HDAC4. This prevents formation of the HDAC4/NCoR1/HDAC3 complex, disrupting HIF-1α transcriptional activation and repressing MEF-2 target genes needed for adaptive survival signaling in the compromised tumor micro environment. In phase 3 clinical testing against metastatic castration-resistant prostate cancer(mCRPC), TasQ (1 mg/day) increased time-to-progression, but not overall survival. METHODS: TasQ analogs were chemically synthesized and tested for activity compared to the parental compound. These included HDAC4 enzymatic assays, qRT-PCR and western blot analyses of gene and protein expression following treatment, in vitro and in vivo efficacy against multiple prostate cancer models including PDXs, pharmacokinetic analyses,AHR binding and agonist assays, SPR analyses of binding to HDAC4 and NCoR1, RNAseq analysis of in vivo tumors, 3D endothelial sprouting assays, and a targeted kinase screen. Genetic knockout or knockdown controls were used when appropriate. RESULTS: Here, we document that, on this regimen (1 mg/day), TasQ blood levels are 10-fold lower than the optimal concentration (≥2 µM) needed for anticancer activity, suggesting higher daily doses are needed. Unfortunately, we also demonstrate that TasQ is an arylhydrocarbon receptor (AHR) agonist, which binds with an EC50 of 1 µM to produce unwanted off-target side effects. Therefore, we screened a library of TasQ analogsto maximize on-target versus off-target activity. Using this approach, we identified ESATA-20, which has ~10-fold lower AHR agonism and 5-fold greater potency against prostate cancer patient-derived xenografts. CONCLUSION: This increased therapeuticindex nominates ESATA-20 as a lead candidate forclinical development as an orally active third generation quinoline-3-carboxamide analog thatretains its on-target ability to disrupt HDAC4/HIF-1α/MEF-2-dependent adaptive survival signaling in the compromisedtumor microenvironment found in mCRPC.
Asunto(s)
Neoplasias de la Próstata Resistentes a la Castración , Masculino , Humanos , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Neoplasias de la Próstata Resistentes a la Castración/genética , Neoplasias de la Próstata Resistentes a la Castración/patología , Western Blotting , Línea Celular Tumoral , Microambiente Tumoral , Histona Desacetilasas/metabolismo , Proteínas Represoras/metabolismoRESUMEN
Analysis of genomic DNA from pathogenic strains of Burkholderia cenocepacia J2315 and Escherichia coli O104:H4 revealed the presence of two unusual MTase genes. Both are plasmid-borne ORFs, carried by pBCA072 for B. cenocepacia J2315 and pESBL for E. coli O104:H4. Pacific Biosciences SMRT sequencing was used to investigate DNA methyltransferases M.BceJIII and M.EcoGIX, using artificial constructs. Mating properties of engineered pESBL derivatives were also investigated. Both MTases yield promiscuous m6A modification of single strands, in the context SAY (where S = C or G and Y = C or T). Strikingly, this methylation is asymmetric in vivo, detected almost exclusively on one DNA strand, and is incomplete: typically, around 40% of susceptible motifs are modified. Genetic and biochemical studies suggest that enzyme action depends on replication mode: DNA Polymerase I (PolI)-dependent ColE1 and p15A origins support asymmetric modification, while the PolI-independent pSC101 origin does not. An MTase-PolI complex may enable discrimination of PolI-dependent and independent plasmid origins. M.EcoGIX helps to establish pESBL in new hosts by blocking the action of restriction enzymes, in an orientation-dependent fashion. Expression and action appear to occur on the entering single strand in the recipient, early in conjugal transfer, until lagging-strand replication creates the double-stranded form.
Asunto(s)
Metilación de ADN/genética , ADN Polimerasa I/genética , ADN de Cadena Simple/genética , Metiltransferasas/genética , Proteínas Bacterianas/genética , Burkholderia cenocepacia/genética , Replicación del ADN/genética , Escherichia coli O104/genética , Proteínas de Escherichia coli/genética , Genoma Bacteriano/genética , Plásmidos/genética , Proteínas Ribosómicas/genéticaRESUMEN
OBJECTIVES: Existing scores are not accurate at predicting mortality in upper (UGIB) and lower (LGIB) gastrointestinal bleeding. We aimed to develop and validate a new pre-endoscopy score for predicting mortality in both UGIB and LGIB. DESIGN AND SETTING: International cohort study. Patients presenting to hospital with UGIB at six international centres were used to develop a risk score for predicting mortality using regression analyses. The score's performance in UGIB and LGIB was externally validated and compared with existing scores using four international datasets. We calculated areas under receiver operating characteristics curves (AUROCs), sensitivities, specificities and outcome among patients classified as low risk and high risk. PARTICIPANTS AND RESULTS: We included 3012 UGIB patients in the development cohort, and 4019 UGIB and 2336 LGIB patients in the validation cohorts. Age, Blood tests and Comorbidities (ABC) score was closer associated with mortality in UGIB and LGIB (AUROCs: 0.81-84) than existing scores (AUROCs: 0.65-0.75; p≤0.02). In UGIB, patients with low ABC score (≤3), medium ABC score (4-7) and high ABC score (≥8) had 30-day mortality rates of 1.0%, 7.0% and 25%, respectively. Patients classified low risk using ABC score had lower mortality than those classified low risk with AIMS65 (threshold ≤1) (1.0 vs 4.5%; p<0.001). In LGIB, patients with low, medium and high ABC scores had in-hospital mortality rates of 0.6%, 6.3% and 18%, respectively. CONCLUSIONS: In contrast to previous scores, ABC score has good performance for predicting mortality in both UGIB and LGIB, allowing early identification and targeted management of patients at high or low risk of death.
Asunto(s)
Hemorragia Gastrointestinal/mortalidad , Medición de Riesgo/métodos , Factores de Edad , Anciano , Comorbilidad , Femenino , Pruebas Hematológicas , Humanos , Masculino , Valor Predictivo de las Pruebas , Estudios Prospectivos , Factores de Riesgo , Sensibilidad y EspecificidadRESUMEN
The ability of the aryl hydrocarbon receptor (AHR) to alter hepatic expression of cholesterol synthesis genes in a DRE-independent manner in mice and humans has been reported. We have examined the influence of functionally distinct classes of AHR ligands on the levels of Niemann-Pick C1-like intracellular cholesterol transporter (NPC1L1) and enzymes involved in the cholesterol synthesis pathway. NPC1L1 is known to mediate the intestinal absorption of dietary cholesterol and is clinically targeted. AHR ligands were capable of attenuating cholesterol uptake through repression of NPC1L1 expression. Through mutagenesis experiments targeting the two DRE sequences present in the promoter region of the NPC1L1 gene, we provide evidence that the repression does not require functional DRE sequences; while knockdown experiments demonstrated that this regulation is dependent on AHR and sterol-regulatory element-binding protein-2 (SREBP-2). Furthermore, upon ligand activation of AHR, the human intestinal Caco-2 cell line revealed coordinate repression of both mRNA and protein levels for a number of the cholesterol biosynthetic enzymes. Transcription of NPC1L1 and genes of the cholesterol synthesis pathway is predominantly regulated by SREBP-2, especially after treatment with a statin. Immunoblot analyses revealed a significant decrease in transcriptionally active SREBP-2 levels upon ligand treatment, whereas the precursor form of SREBP-2 was modestly increased by AHR activation. Mechanistic insights indicate that AHR induces proteolytic degradation of mature SREBP-2 in a calcium-dependent manner, which correlates with the AHR ligand-mediated upregulation of the transient receptor potential cation channel subfamily V member 6 (TRPV6) gene encoding for a membrane calcium channel. These observations emphasize a role for AHR in the systemic homeostatic regulation of cholesterol synthesis and absorption, indicating the potential use of this receptor as a target for the treatment of hyperlipidosis-associated metabolic diseases.
Asunto(s)
Colesterol/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Proteína 2 de Unión a Elementos Reguladores de Esteroles/metabolismo , Células CACO-2 , Inhibidores Enzimáticos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Silenciador del Gen , Humanos , Regiones Promotoras Genéticas , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/genética , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genéticaRESUMEN
We describe the cloning, expression and characterization of the first truly non-specific adenine DNA methyltransferase, M.EcoGII. It is encoded in the genome of the pathogenic strain Escherichia coli O104:H4 C227-11, where it appears to reside on a cryptic prophage, but is not expressed. However, when the gene encoding M.EcoGII is expressed in vivo - using a high copy pRRS plasmid vector and a methylation-deficient E. coli host-extensive in vivo adenine methylation activity is revealed. M.EcoGII methylates adenine residues in any DNA sequence context and this activity extends to dA and rA bases in either strand of a DNA:RNA-hybrid oligonucleotide duplex and to rA bases in RNAs prepared by in vitro transcription. Using oligonucleotide and bacteriophage M13mp18 virion DNA substrates, we find that M.EcoGII also methylates single-stranded DNA in vitro and that this activity is only slightly less robust than that observed using equivalent double-stranded DNAs. In vitro assays, using purified recombinant M.EcoGII enzyme, demonstrate that up to 99% of dA bases in duplex DNA substrates can be methylated thereby rendering them insensitive to cleavage by multiple restriction endonucleases. These properties suggest that the enzyme could also be used for high resolution mapping of protein binding sites in DNA and RNA substrates.
Asunto(s)
Enzimas de Restricción del ADN/metabolismo , Escherichia coli/genética , Profagos/enzimología , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Adenina/metabolismo , Secuencia de Bases , Metilación de ADN , Enzimas de Restricción del ADN/genética , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/virología , Profagos/genética , Unión Proteica , ARN Bicatenario/genética , ARN Bicatenario/metabolismo , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/genética , Especificidad por SustratoRESUMEN
Increasingly, the aryl hydrocarbon receptor (AHR) is being recognized as a sensor for endogenous and pseudo-endogenous metabolites, and in particular microbiota and host generated tryptophan metabolites. One proposed explanation for this is the role of the AHR in innate immune signaling within barrier tissues in response to the presence of microorganisms. A number of cytokine/chemokine genes exhibit a combinatorial increase in transcription upon toll-like receptors and AHR activation, supporting this concept. The AHR also plays a role in the enhanced differentiation of intestinal and dermal epithelium leading to improved barrier function. Importantly, from an evolutionary perspective many of these tryptophan metabolites exhibit greater activation potential for the human AHR when compared to the rodent AHR. These observations underscore the importance of the AHR in barrier tissues and may lead to pharmacologic therapeutic intervention.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Microbioma Gastrointestinal/fisiología , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/agonistas , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/antagonistas & inhibidores , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Citocromo P-450 CYP1A1/metabolismo , Citocinas/metabolismo , Humanos , Ligandos , Ratones , Ratones Noqueados , Receptores de Hidrocarburo de Aril/agonistas , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/genética , Triptófano/metabolismoRESUMEN
The aryl hydrocarbon receptor (AHR) is a major regulator of immune function within the gastrointestinal tract. Resident microbiota are capable of influencing AHR-dependent signaling pathways via production of an array of bioactive molecules that act as AHR agonists, such as indole or indole-3-aldehyde. Bacteria produce a number of quinoline derivatives, of which some function as quorum-sensing molecules. Thus, we screened relevant hydroxyquinoline derivatives for AHR activity using AHR responsive reporter cell lines. 2,8-Dihydroxyquinoline (2,8-DHQ) was identified as a species-specific AHR agonist that exhibits full AHR agonist activity in human cell lines, but only induces modest AHR activity in mouse cells. Additional dihydroxylated quinolines tested failed to activate the human AHR. Nanomolar concentrations of 2,8-DHQ significantly induced CYP1A1 expression and, upon cotreatment with cytokines, synergistically induced IL6 expression. Ligand binding competition studies subsequently confirmed 2,8-DHQ to be a human AHR ligand. Several dihydroxyquinolines were detected in human fecal samples, with concentrations of 2,8-DHQ ranging between 0 and 3.4 pmol/mg feces. Additionally, in mice the microbiota was necessary for the presence of DHQ in cecal contents. These results suggest that microbiota-derived 2,8-DHQ would contribute to AHR activation in the human gut, and thus participate in the protective and homeostatic effects observed with gastrointestinal AHR activation.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/agonistas , Microbioma Gastrointestinal/fisiología , Oxiquinolina/análogos & derivados , Receptores de Hidrocarburo de Aril/agonistas , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células CACO-2 , Heces/microbiología , Humanos , Ratones , Oxiquinolina/metabolismo , Oxiquinolina/farmacología , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Triptófano/metabolismoRESUMEN
BACKGROUND & AIMS: Anti-thrombotic agents are risk factors for upper gastrointestinal bleeding (UGIB). However, few studies have evaluated their effects on patient outcomes. We assessed the effects of anti-thrombotic agents on outcomes of patients with high-risk UGIB. METHODS: We performed a prospective study of 619 patients with acute UGIB (defined by hematemesis, coffee-ground vomit or melena) who required intervention and underwent endoscopy at 8 centers in North America, Asia, and Europe, from March 2014 through March 2015. We collected data recorded on use of anti-thrombotic agents, clinical features, and laboratory test results to calculate AIMS65, Glasgow-Blatchford Score, and full Rockall scores. We also collected and analyzed data on co-morbidities, endoscopic findings, blood transfusion, interventional radiology results, surgeries, length of hospital stay, rebleeding, and mortality. RESULTS: Of the 619 patients who required endoscopic therapy, data on use of anti-thrombotic agents was available for 568; 253 of these patients (44%) used anti-thrombotic agents. Compared to patients not taking anti-thrombotic agents, patients treated with anti-thrombotics were older (P < .001), had a higher mean American Society of Anesthesiologists classification score (P < .0001), had a higher mean Rockall score (P < .0001), a higher mean AIMS65 score (P < .0001), and more frequently bled from ulcers (P < .001). There were no differences between groups in sex, systolic blood pressure, level of hemoglobin at hospital admission, frequency of malignancies, Glasgow-Blatchford Score, need for surgery or interventional radiology, number of rebleeding events, or requirement for transfusion. All-cause mortality was lower in patients who took anti-thrombotic drugs (11 deaths, 4%) than in patients who did not (37 deaths, 12%) (P = .002); this was due to lower bleeding-related mortality in patients taking anti-thrombotic drugs (3 deaths, 1%) than in patients who were not (19 deaths, 6%) (P = .003). Patients taking anti-thrombotic drugs had mean hospital stays of 6.9 days (95% CI, 2-23 days) compared to 7.9 days for non-users of anti-thrombotic agents (95% CI, 2-26 days) (P = .04). CONCLUSIONS: Despite being older, with higher American Society of Anesthesiologists classification, AIMS65, and Rockall scores, patients who have UGIB that requires endoscopic therapy and take anti-thrombotic drugs have lower mortality due to GI bleeding and shorter hospital stays, with similar rates of rebleeding, surgery, and transfusions, compared with those not taking anti-thrombotic drugs.
Asunto(s)
Fibrinolíticos/efectos adversos , Fibrinolíticos/uso terapéutico , Hemorragia Gastrointestinal/mortalidad , Tiempo de Internación/estadística & datos numéricos , Adulto , Anciano , Anciano de 80 o más Años , Asia , Europa (Continente) , Femenino , Humanos , Masculino , Persona de Mediana Edad , América del Norte , Estudios Prospectivos , Factores de Riesgo , Análisis de SupervivenciaRESUMEN
OBJECTIVE: There are little data on the prevalence of reduced bone mineral density (BMD) in young adult patients with coeliac disease; guidelines do not support routine investigation of these patients. We assessed the prevalence of reduced BMD in our patients by age. PATIENTS AND METHODS: Prospective observational study of 260 coeliac patients having DXA one year after commencing gluten-free diet. Nonparametric tests and regression were used. RESULTS: Median age was 51years, BMI 24 and 85 (32.7%) were male. Reduced BMD was associated with increasing age (p < .001), female sex (p = .005), low BMI (p < .001) and previous fracture (p < .01); 49% of all patients and all patients under 20 years old had reduced BMD. The median age of patients with BMI <20 kgm2 was 56 (27, 70) years with the majority of younger patients having normal BMI. CONCLUSIONS: Low BMD is a common finding in young patients with coeliac disease, yet routine assessment of BMD is not currently supported by national guidelines. Early identification may improve motivation to comply with GFD and allow adequate calcium and vitamin D supplementation to reduce risk of fracture later in life.
Asunto(s)
Enfermedades Óseas Metabólicas/complicaciones , Enfermedad Celíaca/complicaciones , Suplementos Dietéticos , Fracturas Óseas/prevención & control , Absorciometría de Fotón , Adulto , Anciano , Densidad Ósea , Enfermedades Óseas Metabólicas/diagnóstico por imagen , Calcio/administración & dosificación , Enfermedad Celíaca/dietoterapia , Dieta Sin Gluten , Inglaterra , Femenino , Humanos , Modelos Logísticos , Masculino , Persona de Mediana Edad , Prevalencia , Estudios Prospectivos , Factores Sexuales , Vitamina D/administración & dosificaciónRESUMEN
The Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in many physiological processes. Several studies indicate that AHR is also involved in energy homeostasis. Fibroblast growth factor 21 (FGF21) is an important regulator of the fasting and feeding responses. When administered to various genetic and diet-induced mouse models of obesity, FGF21 can attenuate obesity-associated morbidities. Here, we explore the role of AHR in hepatic Fgf21 expression through the use of a conditional, hepatocyte-targeted AHR knock-out mouse model (Cre(Alb)Ahr(Fx/Fx)). Compared with the congenic parental strain (Ahr(Fx/Fx)), non-fasted Cre(Alb)Ahr(Fx/Fx) mice exhibit a 4-fold increase in hepatic Fgf21 expression, as well as elevated expression of the FGF21-target gene Igfbp1 Furthermore, in vivo agonist activation of AHR reduces hepatic Fgf21 expression during a fast. The Fgf21 promoter contains several putative dioxin response elements (DREs). Using EMSA, we demonstrate that the AHR-ARNT heterodimer binds to a specific DRE that overlaps binding sequences for peroxisome proliferator-activated receptor α (PPARα), carbohydrate response element-binding protein (ChREBP), and cAMP response element-binding protein, hepatocyte specific (CREBH). In addition, we reveal that agonist-activated AHR impairs PPARα-, ChREBP-, and CREBH-mediated promoter activity in Hepa-1 cells. Accordingly, agonist treatment in Hepa-1 cells ablates potent ER stress-driven Fgf21 expression, and pre-treatment with AHR antagonist blocks this effect. Finally, we show that pre-treatment of primary human hepatocytes with AHR agonist diminishes PPARα-, glucose-, and ER stress-driven induction of FGF21 expression, indicating the effect is not mouse-specific. Together, our data show that AHR contributes to hepatic energy homeostasis, partly through the regulation of FGF21 expression and signaling.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Crecimiento de Fibroblastos/biosíntesis , Regulación de la Expresión Génica/fisiología , Hepatocitos/metabolismo , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal/fisiología , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Línea Celular , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Metabolismo Energético/fisiología , Factores de Crecimiento de Fibroblastos/genética , Hepatocitos/citología , Humanos , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Proteína 1 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , Elementos de Respuesta/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Alcoholic liver disease is a pathological condition caused by overconsumption of alcohol. Because of the high morbidity and mortality associated with this disease, there remains a need to elucidate the molecular mechanisms underlying its etiology and to develop new treatments. Because peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) modulates ethanol-induced hepatic effects, the present study examined alterations in gene expression that may contribute to this disease. Chronic ethanol treatment causes increased hepatic CYP2B10 expression inPparß/δ+/+ mice but not in Pparß/δ-/- mice. Nuclear and cytosolic localization of the constitutive androstane receptor (CAR), a transcription factor known to regulate Cyp2b10 expression, was not different between genotypes. PPARγ co-activator 1α, a co-activator of both CAR and PPARß/δ, was up-regulated in Pparß/δ+/+ liver following ethanol exposure, but not in Pparß/δ-/- liver. Functional mapping of the Cyp2b10 promoter and ChIP assays revealed that PPARß/δ-dependent modulation of SP1 promoter occupancy up-regulated Cyp2b10 expression in response to ethanol. These results suggest that PPARß/δ regulates Cyp2b10 expression indirectly by modulating SP1 and PPARγ co-activator 1α expression and/or activity independent of CAR activity. Ligand activation of PPARß/δ attenuates ethanol-induced Cyp2b10 expression in Pparß/δ+/+ liver but not in Pparß/δ-/- liver. Strikingly, Cyp2b10 suppression by ligand activation of PPARß/δ following ethanol treatment occurred in hepatocytes and was mediated by paracrine signaling from Kupffer cells. Combined, results from the present study demonstrate a novel regulatory role of PPARß/δ in modulating CYP2B10 that may contribute to the etiology of alcoholic liver disease.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/biosíntesis , Familia 2 del Citocromo P450/biosíntesis , Regulación Enzimológica de la Expresión Génica , Hepatopatías Alcohólicas/metabolismo , Hígado/metabolismo , PPAR delta/metabolismo , PPAR-beta/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Esteroide Hidroxilasas/biosíntesis , Animales , Hidrocarburo de Aril Hidroxilasas/genética , Familia 2 del Citocromo P450/genética , Etanol/toxicidad , Hepatocitos/metabolismo , Hepatocitos/patología , Macrófagos del Hígado/metabolismo , Macrófagos del Hígado/patología , Hígado/patología , Hepatopatías Alcohólicas/genética , Hepatopatías Alcohólicas/patología , Ratones , Ratones Noqueados , PPAR delta/genética , PPAR-beta/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factor de Transcripción Sp1/genética , Esteroide Hidroxilasas/genéticaRESUMEN
The Ah receptor (AHR) has been shown to exhibit both inflammatory and anti-inflammatory activity in a context-specific manner. In vivo macrophage-driven acute inflammation models were utilized here to test whether the selective Ah receptor modulator 1-allyl-7-trifluoromethyl-1H-indazol-3-yl]-4-methoxyphenol (SGA360) would reduce inflammation. Exposure to SGA360 was capable of significantly inhibiting lipopolysaccharide (LPS)-mediated endotoxic shock in a mouse model, both in terms of lethality and attenuating inflammatory signaling in tissues. Topical exposure to SGA360 was also able to mitigate joint edema in a monosodium urate (MSU) crystal gout mouse model. Inhibition was dependent on the expression of the high-affinity allelic AHR variant in both acute inflammation models. Upon peritoneal MSU crystal exposure SGA360 pretreatment inhibited neutrophil and macrophage migration into the peritoneum. RNA-seq analysis revealed that SGA360 attenuated the expression of numerous inflammatory genes and genes known to be directly regulated by AHR in thioglycolate-elicited primary peritoneal macrophages treated with LPS. In addition, expression of the high-affinity allelic AHR variant in cultured macrophages was necessary for SGA360-mediated repression of inflammatory gene expression. Mechanistic studies revealed that SGA360 failed to induce nuclear translocation of the AHR and actually enhanced cytoplasmic localization. LPS treatment of macrophages enhanced the occupancy of the AHR and p65 to the Ptgs2 promoter, whereas SGA360 attenuated occupancy. AHR ligand activity was detected in peritoneal exudates isolated from MSU-treated mice, thus suggesting that the anti-inflammatory activity of SGA360 is mediated at least in part through AHR antagonism of endogenous agonist activity. These results underscore an important role of the AHR in participating in acute inflammatory signaling and warrants further investigations into possible clinical applications.
Asunto(s)
Citoplasma/metabolismo , Inflamación/metabolismo , Macrófagos Peritoneales , Receptores de Hidrocarburo de Aril/metabolismo , Compuestos Alílicos/farmacología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Gota/metabolismo , Indazoles/farmacología , Lipopolisacáridos , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Ratones NoqueadosRESUMEN
We have identified a fixed nonsynonymous sequence difference between humans (Val381; derived variant) and Neandertals (Ala381; ancestral variant) in the ligand-binding domain of the aryl hydrocarbon receptor (AHR) gene. In an exome sequence analysis of four Neandertal and Denisovan individuals compared with nine modern humans, there are only 90 total nucleotide sites genome-wide for which archaic hominins are fixed for the ancestral nonsynonymous variant and the modern humans are fixed for the derived variant. Of those sites, only 27, including Val381 in the AHR, also have no reported variability in the human dbSNP database, further suggesting that this highly conserved functional variant is a rare event. Functional analysis of the amino acid variant Ala381 within the AHR carried by Neandertals and nonhuman primates indicate enhanced polycyclic aromatic hydrocarbon (PAH) binding, DNA binding capacity, and AHR mediated transcriptional activity compared with the human AHR. Also relative to human AHR, the Neandertal AHR exhibited 150-1000 times greater sensitivity to induction of Cyp1a1 and Cyp1b1 expression by PAHs (e.g., benzo(a)pyrene). The resulting CYP1A1/CYP1B1 enzymes are responsible for PAH first pass metabolism, which can result in the generation of toxic intermediates and perhaps AHR-associated toxicities. In contrast, the human AHR retains the ancestral sensitivity observed in primates to nontoxic endogenous AHR ligands (e.g., indole, indoxyl sulfate). Our findings reveal that a functionally significant change in the AHR occurred uniquely in humans, relative to other primates, that would attenuate the response to many environmental pollutants, including chemicals present in smoke from fire use during cooking.
Asunto(s)
Hominidae/genética , Hombre de Neandertal/genética , Receptores de Hidrocarburo de Aril/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Benzo(a)pireno , Evolución Biológica , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1B1/genética , ADN/metabolismo , Evolución Molecular , Humanos , Ligandos , Hidrocarburos Policíclicos Aromáticos/metabolismoRESUMEN
Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in the immune system by regulating tryptophan levels and T cell differentiation. Several tumor types overexpress IDO1 to avoid immune surveillance making IDO1 of interest as a target for therapeutic intervention. As a result, several IDO1 inhibitors are currently being tested in clinical trials for cancer treatment as well as several other diseases. Many of the IDO1 inhibitors in clinical trials naturally bear structural similarities to the IDO1 substrate tryptophan, as such, they fulfill many of the structural and functional criteria as potential AHR ligands. Using mouse and human cell-based luciferase gene reporter assays, qPCR confirmation experiments, and CYP1A1 enzyme activity assays, we report that some of the promising clinical IDO1 inhibitors also act as agonists for the aryl hydrocarbon receptor (AHR), best known for its roles in xenobiotic metabolism and as another key regulator of the immune response. The dual role as IDO antagonist and AHR agonist for many of these IDO target drugs should be considered for full interrogation of their biological mechanisms and clinical outcomes.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/agonistas , Inhibidores Enzimáticos/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/agonistas , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1A1/genética , Inducción Enzimática , Inhibidores Enzimáticos/toxicidad , Genes Reporteros , Células Hep G2 , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Ligandos , Ratones , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Medición de Riesgo , Transcripción Genética , TransfecciónRESUMEN
BACKGROUND & AIMS: Upper gastrointestinal hemorrhage (UGIH) is a common cause of hospital admission. The Glasgow Blatchford score (GBS) is an accurate determinant of patients' risk for hospital-based intervention or death. Patients with a GBS of 0 are at low risk for poor outcome and could be managed as outpatients. Some investigators therefore have proposed extending the definition of low-risk patients by using a higher GBS cut-off value, possibly with an age adjustment. We compared 3 thresholds of the GBS and 2 age-adjusted modifications to identify the optimal cut-off value or modification. METHODS: We performed an observational study of 2305 consecutive patients presenting with UGIH at 4 centers (Scotland, England, Denmark, and New Zealand). The performance of each threshold and modification was evaluated based on sensitivity and specificity analyses, the proportion of low-risk patients identified, and outcomes of patients classified as low risk. RESULTS: There were differences in age (P = .0001), need for intervention (P < .0001), mortality (P < .015), and GBS (P = .0001) among sites. All systems identified low-risk patients with high levels of sensitivity (>97%). The GBS at cut-off values of ≤1 and ≤2, and both modifications, identified low-risk patients with higher levels of specificity (40%-49%) than the GBS with a cut-off value of 0 (22% specificity; P < .001). The GBS at a cut-off value of ≤2 had the highest specificity, but 3% of patients classified as low-risk patients had adverse outcomes. All GBS cut-off values, and score modifications, had low levels of specificity when tested in New Zealand (2.5%-11%). CONCLUSIONS: A GBS cut-off value of ≤1 and both GBS modifications identify almost twice as many low-risk patients with UGIH as a GBS at a cut-off value of 0. Implementing a protocol for outpatient management, based on one of these scores, could reduce hospital admissions by 15% to 20%.
Asunto(s)
Técnicas de Apoyo para la Decisión , Hemorragia Gastrointestinal/diagnóstico , Hemorragia Gastrointestinal/terapia , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Dinamarca , Femenino , Hemorragia Gastrointestinal/patología , Humanos , Masculino , Persona de Mediana Edad , Nueva Zelanda , Pronóstico , Estudios Prospectivos , Estudios Retrospectivos , Medición de Riesgo , Sensibilidad y Especificidad , Reino Unido , Adulto JovenRESUMEN
The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor recognized for its role in xenobiotic metabolism. The physiologic function of AHR has expanded to include roles in immune regulation, organogenesis, mucosal barrier function, and the cell cycle. These functions are likely dependent upon ligand-mediated activation of the receptor. High-affinity ligands of AHR have been classically defined as xenobiotics, such as polychlorinated biphenyls and dioxins. Identification of endogenous AHR ligands is key to understanding the physiologic functions of this enigmatic receptor. Metabolic pathways targeting the amino acid tryptophan and indole can lead to a myriad of metabolites, some of which are AHR ligands. Many of these ligands exhibit species selective preferential binding to AHR. The discovery of specific tryptophan metabolites as AHR ligands may provide insight concerning where AHR is activated in an organism, such as at the site of inflammation and within the intestinal tract.
Asunto(s)
Suplementos Dietéticos , Indoles/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Triptófano/metabolismo , Animales , Frutas/metabolismo , Tracto Gastrointestinal/metabolismo , Humanos , Verduras/metabolismoRESUMEN
Rheumatoid arthritis (RA) is a chronic autoimmune disease with high morbidity and mortality. Within the inflammatory milieu, resident fibroblast-like synoviocytes (FLS) in the synovial tissue undergo hyperplasia, which leads to joint destruction. Epidemiologic studies and our previous research suggest that activation of the aryl hydrocarbon receptor (AHR) pathway plays an instrumental role in the inflammatory and destructive RA phenotype. In addition, our recent studies implicate the AHR in the regulation of the expression of several growth factors in established tumor cell lines. Thus, under inflammatory conditions, we hypothesized that the AHR is involved in the constitutive and inducible expression of several growth factors, FLS proliferation and migration, along with protease-dependent invasion in FLS from patients with RA (RA-FLS). Treatment with the AHR antagonist GNF351 inhibits cytokine-induced expression of vascular endothelial growth factor-A (VEGF-A), epiregulin, amphiregulin, and basic fibroblast growth factor mRNA through an AHR-dependent mechanism in both RA-FLS and FLS. Secretion of VEGF-A and epiregulin from RA-FLS was also inhibited upon GNF351 treatment. RA-FLS cell migration, along with cytokine-induced RA-FLS cell proliferation, was significantly attenuated by GNF351 exposure. Treatment of RA-FLS with GNF351 mitigated cytokine-mediated expression of matrix metalloproteinase-2 and -9 mRNA and diminished the RA-FLS invasive phenotype. These findings indicate that inhibition of AHR activity may be a viable therapeutic target in amelioration of disease progression in RA by attenuating growth factor release; FLS proliferation, migration, and invasion; and inflammatory activity.