RESUMEN
While IκB-kinase-ε (IKKε) induces immunomodulatory genes following viral stimuli, its up-regulation by inflammatory cytokines remains under-explored. Since airway epithelial cells respond to airborne insults and potentiate inflammation, IKKε expression was characterized in pulmonary epithelial cell lines (A549, BEAS-2B) and primary human bronchial epithelial cells grown as submersion or differentiated air-liquid interface cultures. IKKε expression was up-regulated by the pro-inflammatory cytokines, interleukin-1ß (IL-1ß) and tumour necrosis factor-α (TNFα). Thus, mechanistic interrogations in A549 cells were used to demonstrate the NF-κB dependence of cytokine-induced IKKε. Furthermore, chromatin immunoprecipitation in A549 and BEAS-2B cells revealed robust recruitment of the NF-κB subunit, p65, to one 5' and two intronic regions within the IKKε locus (IKBKE). In addition, IL-1ß and TNFα induced strong RNA polymerase 2 recruitment to the 5' region, the first intron, and the transcription start site. Stable transfection of the p65-binding regions into A549 cells revealed IL-1ß- and TNFα-inducible reporter activity that required NF-κB, but was not repressed by glucocorticoid. While critical NF-κB motifs were identified in the 5' and downstream intronic regions, the first intronic region did not contain functional NF-κB motifs. Thus, IL-1ß- and TNFα-induced IKKε expression involves three NF-κB-binding regions, containing multiple functional NF-κB motifs, and potentially other mechanisms of p65 binding through non-classical NF-κB binding motifs. By enhancing IKKε expression, IL-1ß may prime, or potentiate, responses to alternative stimuli, as modelled by IKKε phosphorylation induced by phorbol 12-myristate 13-acetate. However, since IKKε expression was only partially repressed by glucocorticoid, IKKε-dependent responses could contribute to glucocorticoid-resistant disease.
Asunto(s)
Células Epiteliales , Quinasa I-kappa B , Humanos , Quinasa I-kappa B/metabolismo , Quinasa I-kappa B/genética , Células Epiteliales/metabolismo , Células Epiteliales/efectos de los fármacos , Células A549 , Factor de Transcripción ReIA/metabolismo , Factor de Transcripción ReIA/genética , Interleucina-1beta/farmacología , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , FN-kappa B/metabolismo , FN-kappa B/genética , Factor de Necrosis Tumoral alfa/farmacología , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/genética , Pulmón/metabolismo , Pulmón/citología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/citología , Regulación de la Expresión Génica/efectos de los fármacosRESUMEN
While glucocorticoids act via the glucocorticoid receptor (GR; NR3C1) to reduce the expression of many inflammatory genes, repression is not an invariable outcome. Here, we explore synergy occurring between synthetic glucocorticoids (dexamethasone and budesonide) and proinflammatory cytokines (IL1B and TNF) on the expression of the toll-like receptor 2 (TLR2). This effect is observed in epithelial cell lines and both undifferentiated and differentiated primary human bronchial epithelial cells (pHBECs). In A549 cells, IL1B-plus-glucocorticoid-induced TLR2 expression required nuclear factor (NF)-κB and GR. Likewise, in A549 cells, BEAS-2B cells, and pHBECs, chromatin immunoprecipitation identified GR- and NF-κB/p65-binding regions â¼32 kb (R1) and â¼7.3 kb (R2) upstream of the TLR2 gene. Treatment of BEAS-2B cells with TNF or/and dexamethasone followed by global run-on sequencing confirmed transcriptional activity at these regions. Furthermore, cloning R1 or R2 into luciferase reporters revealed transcriptional activation by budesonide or IL1B, respectively, while R1+R2 juxtaposition enabled synergistic activation by IL1B and budesonide. In addition, small-molecule inhibitors and siRNA knockdown showed p38α MAPK to negatively regulate both IL1B-induced TLR2 expression and R1+R2 reporter activity. Finally, agonism of IL1B-plus-dexamethasone-induced TLR2 in A549 cells and pHBECs stimulated NF-κB- and interferon regulatory factor-dependent reporter activity and chemokine release. We conclude that glucocorticoid-plus-cytokine-driven synergy at TLR2 involves GR and NF-κB acting via specific enhancer regions, which combined with the inhibition of p38α MAPK promotes TLR2 expression. Subsequent inflammatory effects that occur following TLR2 agonism may be pertinent in severe neutrophilic asthma or chronic obstructive pulmonary disease, where glucocorticoid-based therapies are less efficacious.
Asunto(s)
Asma , FN-kappa B , Receptores de Glucocorticoides , Receptor Toll-Like 2 , Proteínas Quinasas p38 Activadas por Mitógenos , Asma/fisiopatología , Budesonida/farmacología , Citocinas/metabolismo , Dexametasona/farmacología , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Humanos , Pulmón/citología , Pulmón/metabolismo , FN-kappa B/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Resolution of inflammation is an active process that is tightly regulated to achieve repair and tissue homeostasis. In the absence of resolution, persistent inflammation underlies the pathogenesis of chronic lung disease such as chronic obstructive pulmonary disease (COPD) with recurrent exacerbations. Over the course of inflammation, macrophage programming transitions from pro-inflammatory to pro-resolving, which is in part regulated by the nuclear receptor Peroxisome Proliferator-Activated Receptor γ (PPARγ). Our previous work demonstrated an association between Fatty Acid Binding Protein 5 (FABP5) expression and PPARγ activity in peripheral blood mononuclear cells of healthy and COPD patients. However, a role for FABP5 in macrophage programming has not been examined. Here, using a combination of in vitro and in vivo approaches, we demonstrate that FABP5 is necessary for PPARγ activation. In turn, PPARγ acts directly to increase FABP5 expression in primary human alveolar macrophages. We further illustrate that lack of FABP5 expression promotes a pro-inflammatory macrophage programming with increased secretion of pro-inflammatory cytokines and increased chromatin accessibility for pro-inflammatory transcription factors (e.g., NF-κB and MAPK). And finally, real-time cell metabolic analysis using the Seahorse technology shows an inhibition of oxidative phosphorylation in FABP5-deficient macrophages. Taken together, our data indicate that FABP5 and PPARγ reciprocally regulate each other's expression and function, consistent with a novel positive feedback loop between the two factors that mediates macrophage pro-resolving programming. Our studies highlight the importance of defining targets and regulatory mechanisms that control the resolution of inflammation and may serve to inform novel interventional strategies directed towards COPD.
Asunto(s)
PPAR gamma , Enfermedad Pulmonar Obstructiva Crónica , Proteínas de Unión a Ácidos Grasos/genética , Proteínas de Unión a Ácidos Grasos/metabolismo , Regulación de la Expresión Génica , Humanos , Inflamación/metabolismo , Leucocitos Mononucleares/metabolismo , Macrófagos/metabolismo , PPAR gamma/metabolismo , Enfermedad Pulmonar Obstructiva Crónica/metabolismoRESUMEN
Rationale: Common genetic variants have been associated with idiopathic pulmonary fibrosis (IPF). Objectives: To determine functional relevance of the 10 IPF-associated common genetic variants we previously identified. Methods: We performed expression quantitative trait loci (eQTL) and methylation quantitative trait loci (mQTL) mapping, followed by co-localization of eQTL and mQTL with genetic association signals and functional validation by luciferase reporter assays. Illumina multi-ethnic genotyping arrays, mRNA sequencing, and Illumina 850k methylation arrays were performed on lung tissue of participants with IPF (234 RNA and 345 DNA samples) and non-diseased controls (188 RNA and 202 DNA samples). Measurements and Main Results: Focusing on genetic variants within 10 IPF-associated genetic loci, we identified 27 eQTLs in controls and 24 eQTLs in cases (false-discovery-rate-adjusted P < 0.05). Among these signals, we identified associations of lead variants rs35705950 with expression of MUC5B and rs2076295 with expression of DSP in both cases and controls. mQTL analysis identified CpGs in gene bodies of MUC5B (cg17589883) and DSP (cg08964675) associated with the lead variants in these two loci. We also demonstrated strong co-localization of eQTL/mQTL and genetic signal in MUC5B (rs35705950) and DSP (rs2076295). Functional validation of the mQTL in MUC5B using luciferase reporter assays demonstrates that the CpG resides within a putative internal repressor element. Conclusions: We have established a relationship of the common IPF genetic risk variants rs35705950 and rs2076295 with respective changes in MUC5B and DSP expression and methylation. These results provide additional evidence that both MUC5B and DSP are involved in the etiology of IPF.
Asunto(s)
Fibrosis Pulmonar Idiopática , Humanos , ADN , Metilación de ADN/genética , Expresión Génica , Predisposición Genética a la Enfermedad/genética , Fibrosis Pulmonar Idiopática/genética , Mucina 5B/genética , Sitios de Carácter Cuantitativo/genética , ARNRESUMEN
Military Deployment to Southwest Asia and Afghanistan and exposure to toxic airborne particulates have been associated with an increased risk of developing respiratory disease, collectively termed deployment-related respiratory diseases (DRRDs). Our knowledge about how particulates mediate respiratory disease is limited, precluding the appropriate recognition or management. Central to this limitation is the lack of understanding of how exposures translate into dysregulated cell identity with dysregulated transcriptional programs. The small airway epithelium is involved in both the pathobiology of DRRD and fine particulate matter deposition. To characterize small airway epithelial cell epigenetic and transcriptional responses to Afghan desert particulate matter (APM) and investigate the functional interactions of transcription factors that mediate these responses, we applied two genomics assays, the assay for transposase accessible chromatin with sequencing (ATAC-seq) and Precision Run-on sequencing (PRO-seq). We identified activity changes in a series of transcriptional pathways as candidate regulators of susceptibility to subsequent insults, including signal-dependent pathways, such as loss of cytochrome P450 or P53/P63, and lineage-determining transcription factors, such as GRHL2 loss or TEAD3 activation. We further demonstrated that TEAD3 activation was unique to APM exposure despite similar inflammatory responses when compared with wood smoke particle exposure and that P53/P63 program loss was uniquely positioned at the intersection of signal-dependent and lineage-determining transcriptional programs. Our results establish the utility of an integrated genomics approach in characterizing responses to exposures and identifying genomic targets for the advanced investigation of the pathogenesis of DRRD.
Asunto(s)
Células Epiteliales Alveolares , Material Particulado , Factores de Transcripción , Afganistán , Células Epiteliales Alveolares/metabolismo , Cromatina/metabolismo , Epigénesis Genética , Genómica/métodos , Despliegue Militar , Material Particulado/toxicidad , Enfermedades Respiratorias/epidemiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transposasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Glucocorticoids are potent anti-inflammatory drugs that are used to treat an extraordinary range of human disease, including COVID-19, underscoring the ongoing importance of understanding their molecular mechanisms. Early studies of GR signaling led to broad acceptance of models in which glucocorticoid receptor (GR) monomers tether repressively to inflammatory transcription factors, thus abrogating inflammatory gene expression. However, newer data challenge this core concept and present an exciting opportunity to reframe our understanding of GR signaling. Here, we present an alternate, two-part model for transcriptional repression by glucocorticoids. First, widespread GR-mediated induction of transcription results in rapid, primary repression of inflammatory gene transcription and associated enhancers through competition-based mechanisms. Second, a subset of GR-induced genes, including targets that are regulated in coordination with inflammatory transcription factors such as NF-κB, exerts secondary repressive effects on inflammatory gene expression. Within this framework, emerging data indicate that the gene set regulated through the cooperative convergence of GR and NF-κB signaling is central to the broad clinical effectiveness of glucocorticoids in terminating inflammation and promoting tissue repair.
Asunto(s)
Antiinflamatorios/uso terapéutico , Tratamiento Farmacológico de COVID-19 , Dexametasona/uso terapéutico , Glucocorticoides/uso terapéutico , FN-kappa B/genética , Receptores de Glucocorticoides/genética , Animales , COVID-19/inmunología , COVID-19/patología , COVID-19/virología , Regulación de la Expresión Génica , Genómica/métodos , Humanos , Inflamación/prevención & control , Modelos Genéticos , FN-kappa B/antagonistas & inhibidores , FN-kappa B/inmunología , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/inmunología , SARS-CoV-2/crecimiento & desarrollo , SARS-CoV-2/inmunología , SARS-CoV-2/patogenicidad , Transducción de Señal , Transcripción Genética/efectos de los fármacos , Transcripción Genética/inmunologíaRESUMEN
The heterogeneity of respirable particulates and compounds complicates our understanding of transcriptional responses to air pollution. Here, we address this by applying precision nuclear run-on sequencing and the assay for transposase-accessible chromatin sequencing to measure nascent transcription and chromatin accessibility in airway epithelial cells after wood smoke particle (WSP) exposure. We used transcription factor enrichment analysis to identify temporally distinct roles for ternary response factor-serum response factor complexes, the aryl hydrocarbon receptor (AHR), and NFκB in regulating transcriptional changes induced by WSP. Transcription of canonical targets of the AHR, such as CYP1A1 and AHRR, was robustly increased after just 30 min of WSP exposure, and we discovered novel AHR-regulated pathways and targets including the DNA methyltransferase, DNMT3L. Transcription of these genes and associated enhancers rapidly returned to near baseline by 120 min after exposure. The kinetics of AHR- and NFκB-regulated responses to WSP were distinguishable based on the timing of both transcriptional responses and chromatin remodeling, with induction of several cytokines implicated in maintaining NFκB-mediated responses through 120 min of exposure. In aggregate, our data establish a direct and primary role for AHR in mediating airway epithelial responses to WSP and identify crosstalk between AHR and NFκB signaling in controlling proinflammatory gene expression. This work also defines an integrated genomics-based strategy for deconvoluting multiplexed transcriptional responses to heterogeneous environmental exposures.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Humo/efectos adversos , Transcripción Genética , Madera , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Línea Celular Transformada , Ensamble y Desensamble de Cromatina , Citocromo P-450 CYP1A1/biosíntesis , Citocromo P-450 CYP1A1/genética , ADN (Citosina-5-)-Metiltransferasas/biosíntesis , ADN (Citosina-5-)-Metiltransferasas/genética , Humanos , Ratones , FN-kappa B/genética , FN-kappa B/metabolismo , Células 3T3 NIH , Receptores de Hidrocarburo de Aril/genética , Proteínas Represoras/genéticaRESUMEN
Ligand-activated glucocorticoid receptor (GR) elicits variable glucocorticoid-modulated transcriptomes in different cell types. However, some genes, including Krüppel-like factor 9 (KLF9), a putative transcriptional repressor, demonstrate conserved responses. We show that glucocorticoids induce KLF9 expression in the human airways in vivo and in differentiated human bronchial epithelial (HBE) cells grown at air-liquid interface (ALI). In A549 and BEAS-2B pulmonary epithelial cells, glucocorticoids induce KLF9 expression with similar kinetics to primary HBE cells in submersion culture. A549 and BEAS-2B ChIP-seq data reveal four common glucocorticoid-induced GR binding sites (GBSs). Two GBSs mapped to the 5'-proximal region relative to KLF9 transcription start site (TSS) and two occurred at distal sites. These were all confirmed in primary HBE cells. Global run-on (GRO) sequencing indicated robust enhancer RNA (eRNA) production from three of these GBSs in BEAS-2B cells. This was confirmed in A549 cells, plus submersion, and ALI culture of HBE cells. Cloning each GBS into luciferase reporters revealed glucocorticoid-induced activity requiring a glucocorticoid response element (GRE) within each distal GBS. While the proximal GBSs drove modest reporter induction by glucocorticoids, this region exhibited basal eRNA production, RNA polymerase II enrichment, and looping to the TSS, plausibly underlying constitutive KLF9 expression. Post glucocorticoid treatment, interactions between distal and proximal GBSs and the TSS correlated with KLF9 induction. CBP/P300 silencing reduced proximal GBS activity, but negligibly affected KLF9 expression. Overall, a model for glucocorticoid-mediated regulation of KLF9 involving multiple GBSs is depicted. This work unequivocally demonstrates that mechanistic insights gained from cell lines can translate to physiologically relevant systems.
Asunto(s)
Dexametasona/farmacología , Genómica , Glucocorticoides/farmacología , Factores de Transcripción de Tipo Kruppel/biosíntesis , Pulmón/efectos de los fármacos , Células A549 , Elementos de Facilitación Genéticos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Pulmón/citología , Pulmón/metabolismo , Unión Proteica , ARN Mensajero/genética , Receptores de Glucocorticoides/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
The glucocorticoid receptor (NR3C1, also known as GR) binds to specific DNA sequences and directly induces transcription of anti-inflammatory genes that contribute to cytokine repression, frequently in cooperation with NF-kB. Whether inflammatory repression also occurs through local interactions between GR and inflammatory gene regulatory elements has been controversial. Here, using global run-on sequencing (GRO-seq) in human airway epithelial cells, we show that glucocorticoid signaling represses transcription within 10 min. Many repressed regulatory regions reside within "hyper-ChIPable" genomic regions that are subject to dynamic, yet nonspecific, interactions with some antibodies. When this artifact was accounted for, we determined that transcriptional repression does not require local GR occupancy. Instead, widespread transcriptional induction through canonical GR binding sites is associated with reciprocal repression of distal TNF-regulated enhancers through a chromatin-dependent process, as evidenced by chromatin accessibility and motif displacement analysis. Simultaneously, transcriptional induction of key anti-inflammatory effectors is decoupled from primary repression through cooperation between GR and NF-kB at a subset of regulatory regions. Thus, glucocorticoids exert bimodal restraints on inflammation characterized by rapid primary transcriptional repression without local GR occupancy and secondary anti-inflammatory effects resulting from transcriptional cooperation between GR and NF-kB.
Asunto(s)
Dexametasona/farmacología , Inflamación/metabolismo , ARN Mensajero/genética , Receptores de Glucocorticoides/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Cromatina/metabolismo , Dexametasona/metabolismo , Elementos de Facilitación Genéticos , Células HEK293 , Humanos , FN-kappa B/metabolismo , Transducción de SeñalRESUMEN
Corticosteroids (CSs) are commonly used to manage wheezing and asthma in pediatric populations. Although corticosteroids are effective in alleviating airway diseases, some children with more moderate-severe asthma phenotypes show CS resistance and exhibit significant airflow obstruction, persistent inflammation, and more frequent exacerbations. Previous studies have demonstrated that Th1 cytokines, such as TNF-α and IFN-γ, promote CS resistance in adult human airway smooth muscle (ASM). In the present study, using a human fetal ASM cell model, we tested the hypothesis that TNF-α/IFN-γ induces CS resistance. In contrast to TNF-α or IFN-γ alone, the combination of TNF-α/IFN-γ blunted the ability of fluticasone propionate (FP) to reduce expression of the chemokines CCL5 and CXCL10 despite expression of key anti-inflammatory glucocorticoid receptor target genes being largely unaffected by TNF-α/IFN-γ. Expression of the NF-κB subunit p65 and phosphorylation of Stat1 were elevated in cells treated with TNF-α/IFN-γ, an effect that remained in the presence of FP. siRNA knockdown studies demonstrated the effects of TNF-α/IFN-γ on increased p65 are mediated by Stat1, a transcription factor activated by IFN-γ. Expression of TNFAIP3, a negative regulator of NF-κB activity, was not altered by TNF-α/IFN-γ. However, the effects of TNF-α/IFN-γ were partially reduced by overexpression of TNFAIP3 but did not influence p65 expression. Together, these data suggest that IFN-γ augments the effects of TNF-α on chemokines by enhancing expression of key inflammatory pathways in the presence of CS. Interactions between TNF-α- and IFN-γ-mediated pathways may promote inflammation in asthmatic children resistant to CSs.
Asunto(s)
Corticoesteroides/farmacología , Bronquios/inmunología , Resistencia a Medicamentos/efectos de los fármacos , Fluticasona/farmacología , Interferón gamma/inmunología , Miocitos del Músculo Liso/inmunología , Tráquea/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Bronquios/crecimiento & desarrollo , Niño , Preescolar , Resistencia a Medicamentos/inmunología , Femenino , Humanos , Masculino , Células TH1/inmunología , Tráquea/crecimiento & desarrolloRESUMEN
In asthma, the clinical efficacy of inhaled corticosteroids (ICSs) is enhanced by long-acting ß2-adrenoceptor agonists (LABAs). ICSs, or more accurately, glucocorticoids, promote therapeutically relevant changes in gene expression, and, in primary human bronchial epithelial cells (pHBECs) and airway smooth muscle cells, this genomic effect can be enhanced by a LABA. Modeling this interaction in human bronchial airway epithelial BEAS-2B cells transfected with a 2× glucocorticoid response element (2×GRE)-driven luciferase reporter showed glucocorticoid-induced transcription to be enhanced 2- to 3-fold by LABA. This glucocorticoid receptor (GR; NR3C1)-dependent effect occurred rapidly, was insensitive to protein synthesis inhibition, and was maximal when glucocorticoid and LABA were added concurrently. The ability of LABA to enhance GR-mediated transcription was not associated with changes in GR expression, serine (Ser203, Ser211, Ser226) phosphorylation, ligand affinity, or nuclear translocation. Chromatin immunoprecipitation demonstrated that glucocorticoid-induced recruitment of GR to the integrated 2×GRE reporter and multiple gene loci, whose mRNAs were unaffected or enhanced by LABA, was also unchanged by LABA. Transcriptomic analysis revealed glucocorticoid-induced mRNAs were variably enhanced, unaffected, or repressed by LABA. Thus, events leading to GR binding at target genes are not the primary explanation for how LABAs modulate GR-mediated transcription. As many glucocorticoid-induced genes are independently induced by LABA, gene-specific control by GR- and LABA-activated transcription factors may explain these observations. Because LABAs promote similar effects in pHBECs, therapeutic relevance is likely. These data illustrate the need to understand gene function(s), and the mechanisms leading to gene-specific induction, if existing ICS/LABA combination therapies are to be improved.
Asunto(s)
Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/metabolismo , Mucosa Respiratoria/metabolismo , Transcripción Genética/fisiología , Agonistas de Receptores Adrenérgicos beta 2/farmacología , Células Cultivadas , Preparaciones de Acción Retardada , Relación Dosis-Respuesta a Droga , Humanos , Receptores de Glucocorticoides/genética , Mucosa Respiratoria/efectos de los fármacos , Transcripción Genética/efectos de los fármacosRESUMEN
Glucocorticoids exert important therapeutic effects on airway smooth muscle (ASM), yet few direct targets of glucocorticoid signaling in ASM have been definitively identified. Here, we show that the transcription factor, Krüppel-like factor 15 (KLF15), is directly induced by glucocorticoids in primary human ASM, and that KLF15 represses ASM hypertrophy. We integrated transcriptome data from KLF15 overexpression with genome-wide analysis of RNA polymerase (RNAP) II and glucocorticoid receptor (GR) occupancy to identify phospholipase C delta 1 as both a KLF15-regulated gene and a novel repressor of ASM hypertrophy. Our chromatin immunoprecipitation sequencing data also allowed us to establish numerous direct transcriptional targets of GR in ASM. Genes with inducible GR occupancy and putative antiinflammatory properties included IRS2, APPL2, RAMP1, and MFGE8. Surprisingly, we also observed GR occupancy in the absence of supplemental ligand, including robust GR binding peaks within the IL11 and LIF loci. Detection of antibody-GR complexes at these areas was abrogated by dexamethasone treatment in association with reduced RNA polymerase II occupancy, suggesting that noncanonical pathways contribute to cytokine repression by glucocorticoids in ASM. Through defining GR interactions with chromatin on a genome-wide basis in ASM, our data also provide an important resource for future studies of GR in this therapeutically relevant cell type.
Asunto(s)
Remodelación de las Vías Aéreas (Respiratorias)/genética , Regulación de la Expresión Génica/fisiología , Factores de Transcripción de Tipo Kruppel/fisiología , Músculo Liso/patología , Proteínas Nucleares/fisiología , Fosfolipasa C delta/fisiología , Receptores de Glucocorticoides/fisiología , Sistema Respiratorio/citología , Adenoviridae/genética , Células Cultivadas , Inmunoprecipitación de Cromatina , Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Hipertrofia , Músculo Liso/metabolismo , Fosfolipasa C delta/genética , Cultivo Primario de Células , ARN Polimerasa II/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Análisis de Secuencia de ARN , Transcriptoma , Transducción Genética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The gain-of-function mucin 5B (MUC5B) promoter variant, rs35705950, confers the largest risk, genetic or otherwise, for the development of idiopathic pulmonary fibrosis; however, the mechanisms underlying the regulation of MUC5B expression have yet to be elucidated. Here, we identify a critical regulatory domain that contains the MUC5B promoter variant and has a highly conserved forkhead box protein A2 (FOXA2) binding motif. This region is differentially methylated in association with idiopathic pulmonary fibrosis, MUC5B expression, and rs35705950. In addition, we show that this locus binds FOXA2 dynamically, and that binding of FOXA2 is necessary for enhanced expression of MUC5B. In aggregate, our findings identify novel targets to regulate the expression of MUC5B.
Asunto(s)
Fibrosis Pulmonar Idiopática/genética , Mucina 5B/genética , Secuencia de Bases , Sitios de Unión , Inmunoprecipitación de Cromatina , Islas de CpG/genética , Metilación de ADN/genética , Técnicas de Silenciamiento del Gen , Factor Nuclear 3-beta del Hepatocito/metabolismo , Humanos , Pulmón/metabolismo , Pulmón/patología , Mucina 5B/metabolismo , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas , Unión Proteica/genética , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño/metabolismoRESUMEN
Antagonism of pro-inflammatory transcription factors by monomeric glucocorticoid receptor (GR) has long been viewed as central to glucocorticoid (GC) efficacy. However, the mechanisms and targets through which GCs exert therapeutic effects in diseases such as asthma remain incompletely understood. We previously defined a surprising cooperative interaction between GR and NF-κB that enhanced expression of A20 (TNFAIP3), a potent inhibitor of NF-κB. Here we extend this observation to establish that A20 is required for maximal cytokine repression by GCs. To ascertain the global extent of GR and NF-κB cooperation, we determined genome-wide occupancy of GR, the p65 subunit of NF-κB, and RNA polymerase II in airway epithelial cells treated with dexamethasone, TNF, or both using chromatin immunoprecipitation followed by deep sequencing. We found that GR recruits p65 to dimeric GR binding sites across the genome and discovered additional regulatory elements in which GR-p65 cooperation augments gene expression. GR targets regulated by this mechanism include key anti-inflammatory and injury response genes such as SERPINA1, which encodes α1 antitrypsin, and FOXP4, an inhibitor of mucus production. Although dexamethasone treatment reduced RNA polymerase II occupancy of TNF targets such as IL8 and TNFAIP2, we were unable to correlate specific binding sequences for GR or occupancy patterns with repressive effects on transcription. Our results suggest that cooperative anti-inflammatory gene regulation by GR and p65 contributes to GC efficacy, whereas tethering interactions between GR and p65 are not universally required for GC-based gene repression.
Asunto(s)
Antiinflamatorios/farmacología , Células Epiteliales/efectos de los fármacos , Receptores de Glucocorticoides/metabolismo , Factor de Transcripción ReIA/metabolismo , Western Blotting , Línea Celular , Células Cultivadas , Dexametasona/farmacología , Células Epiteliales/metabolismo , Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Humanos , Unión Proteica/efectos de los fármacos , Interferencia de ARN , ARN Polimerasa II/metabolismo , Receptores de Glucocorticoides/genética , Sistema Respiratorio/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción ReIA/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/genética , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
PURPOSE OF REVIEW: The recent developments and clinical applications of natural orifice translumenal endoscopic surgery (NOTES)-procedures and technologies are going to be presented. RECENT FINDINGS: In experimental as well as clinical settings, NOTES-procedures are predominantly performed in hybrid technique. Current experimental studies focus on the implementation of new surgical approaches as well as on the training of procedures. One emphasis in the clinical application is transrectal and transanal interventions. Transanal total mesorectal excision is equivalent to laparoscopic procedures but with the benefit of an even less invasive access. Transvaginal cholecystectomy can achieve results that are comparable to surgeries that are performed with laparoscopic techniques alone. An analysis of the German NOTES-Register concerning appendectomies as well as the national performance of NOTES-interventions in Switzerland is presented. Apart from intraabdominal approaches, several centers proclaim transoral thyroidectomies and transoral mediastinoscopies. SUMMARY: NOTES-procedures are performed in animal experiments as well as in clinical setting although with less frequency. At this time, hybrid techniques using rigid instruments are mainly applied.
Asunto(s)
Colecistectomía/métodos , Laparoscopía , Cirugía Endoscópica por Orificios Naturales , Tiroidectomía/métodos , Colecistectomía/instrumentación , Colecistectomía/tendencias , Humanos , Laparoscopía/tendencias , Cirugía Endoscópica por Orificios Naturales/tendencias , Tempo Operativo , Selección de Paciente , Tiroidectomía/instrumentación , Tiroidectomía/tendenciasRESUMEN
Combinatorial gene regulation through feed-forward loops (FFLs) can bestow specificity and temporal control to client gene expression; however, characteristics of binding sites that mediate these effects are not established. We previously showed that the glucocorticoid receptor (GR) and KLF15 form coherent FFLs that cooperatively induce targets such as the amino acid-metabolizing enzymes AASS and PRODH and incoherent FFLs exemplified by repression of MT2A by KLF15. Here, we demonstrate that GR and KLF15 physically interact and identify low affinity GR binding sites within glucocorticoid response elements (GREs) for PRODH and AASS that contribute to combinatorial regulation with KLF15. We used deep sequencing and electrophoretic mobility shift assays to derive in vitro GR binding affinities across sequence space. We applied these data to show that AASS GRE activity correlated (r(2) = 0.73) with predicted GR binding affinities across a 50-fold affinity range in transfection assays; however, the slope of the linear relationship more than doubled when KLF15 was expressed. Whereas activity of the MT2A GRE was even more strongly (r(2) = 0.89) correlated with GR binding site affinity, the slope of the linear relationship was sharply reduced by KLF15, consistent with incoherent FFL logic. Thus, GRE architecture and co-regulator expression together determine the functional parameters that relate GR binding site affinity to hormone-induced transcriptional responses. Utilization of specific affinity response functions and GR binding sites by FFLs may contribute to the diversity of gene expression patterns within GR-regulated transcriptomes.
Asunto(s)
Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas Nucleares/metabolismo , Prolina Oxidasa/metabolismo , Receptores de Glucocorticoides/metabolismo , Elementos de Respuesta , Sacaropina Deshidrogenasas/metabolismo , Transcripción Genética , Animales , Secuencia de Bases , Sitios de Unión , Bronquios/citología , Bronquios/efectos de los fármacos , Bronquios/metabolismo , Línea Celular , Dexametasona/farmacología , Ensayo de Cambio de Movilidad Electroforética , Células Epiteliales/citología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Factores de Transcripción de Tipo Kruppel/química , Factores de Transcripción de Tipo Kruppel/genética , Ratones , Datos de Secuencia Molecular , Proteínas Nucleares/química , Proteínas Nucleares/genética , Prolina Oxidasa/química , Prolina Oxidasa/genética , Regiones Promotoras Genéticas , Unión Proteica , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Sacaropina Deshidrogenasas/química , Sacaropina Deshidrogenasas/genética , Transducción de SeñalRESUMEN
Airway smooth muscle is a major target tissue for glucocorticoid (GC)-based asthma therapies, however, molecular mechanisms through which the GC receptor (GR) exerts therapeutic effects in this key airway cell type have not been fully elucidated. We previously identified the nuclear factor-κB (NF-κB) inhibitor, A20 (TNFAIP3), as a mediator of cytokine repression by glucocorticoids (GCs) in airway epithelial cells and defined cooperative regulation of anti-inflammatory genes by GR and NF-κB as a key mechanistic underpinning of airway epithelial GR function. Here, we expand on these findings to determine whether a similar mechanism is operational in human airway smooth muscle (HASM). Using HASM cells derived from normal and fatal asthma samples as an in vitro model, we demonstrate that GCs spare or augment TNF-mediated induction of A20 (TNFAIP3), TNIP1, and NFKBIA, all implicated in negative feedback control of NF-κB-driven inflammatory processes. We applied chromatin immunoprecipitation and reporter analysis to show that GR and NF-κB directly regulate A20 expression in HASM through cooperative induction of an intronic enhancer. Using overexpression, we show for the first time that A20 and its interacting partner, TNIP1, repress TNF signaling in HASM cells. Moreover, we applied small interfering RNA-based gene knockdown to demonstrate that A20 is required for maximal cytokine repression by GCs in HASM. Taken together, our data suggest that inductive regulation of A20 by GR and NF-κB contributes to cytokine repression in HASM.
Asunto(s)
Citocinas/biosíntesis , Dexametasona/farmacología , Glucocorticoides/farmacología , Músculo Liso/metabolismo , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Adolescente , Asma/metabolismo , Sitios de Unión , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Elementos de Facilitación Genéticos , Femenino , Silenciador del Gen , Humanos , Masculino , Persona de Mediana Edad , Músculo Liso/efectos de los fármacos , FN-kappa B/metabolismo , Unión Proteica , Receptores de Glucocorticoides/metabolismo , Transducción de Señal , Transcripción Genética , Activación Transcripcional , Adulto JovenRESUMEN
TNF expression is elevated in asthma and other inflammatory airway diseases that are commonly treated with glucocorticoid-based therapies, but the impact of glucocorticoids on negative feedback control of TNF is not well understood. We analyzed the effect of dexamethasone, a potent synthetic glucocorticoid, on TNF-regulated gene expression in cultured airway epithelial cells. Although dexamethasone-mediated activation of the glucocorticoid receptor (GR) potently repressed expression of IL1ß, IL8, and several other pro-inflammatory TNF targets, the expression of anti-inflammatory TNF targets such as TNFAIP3 (A20) and NFKBIA was selectively spared or augmented by dexamethasone treatment. Despite divergent effects on gene expression, GR and NF-κB occupancy at the TNFAIP3 locus and GR-repressed targets was similar. A co-occupied intronic TNFAIP3 regulatory element mediated cooperative enhancement of transcription by GR and NF-κB that required the presence of a functional GR binding site (GBS). GBS exchanges between reporters for TNFAIP3 and FKBP5, a canonical GR-induced target, revealed substantial latitude in the GBS sequence requirements for GR/NF-κB cooperation, suggesting that the TNFAIP3 GBS acts primarily as a docking site in this context. Supporting this notion, a selective GR ligand with only weak agonist activity for induction of FKBP5 enabled robust GR/NF-κB cooperative induction of a mutant TNFAIP3 reporter harboring the FKBP5 GBS. Taken together, our data support a model in which the expression of anti-inflammatory targets of TNF is maintained during treatment with glucocorticoids through context-dependent cooperation between GR and NF-κB.
Asunto(s)
Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Inflamación/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Intrones , FN-kappa B/inmunología , Proteínas Nucleares/genética , Receptores de Glucocorticoides/inmunología , Secuencia de Bases , Línea Celular , Proteínas de Unión al ADN/inmunología , Humanos , Inflamación/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Proteínas Nucleares/inmunología , Activación Transcripcional , Proteína 3 Inducida por el Factor de Necrosis Tumoral alfa , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Chronic stress is epidemiologically correlated with physical and psychiatric disorders. Whereas many animal models of chronic stress induce symptoms of psychopathology, repeated homotypic stressors to moderate intensity stimuli typically reduce stress-related responses with fewer, if any, pathological symptoms. Recent results indicate that the rostral posterior hypothalamic (rPH) region is a significant component of the brain circuitry underlying response reductions (habituation) associated with repeated homotypic stress. To test whether posterior hypothalamic transcriptional regulation associates with the neuroendocrine modifications induced by repeated homotypic stress, RNA-seq was performed in the rPH dissected from adult male rats that experienced either no stress, 1, 3, or 7 stressful loud noise exposures. Plasma samples displayed reliable increases of corticosterone in all stressed groups, with the smallest increase in the group exposed to 7 loud noises, indicating significant habituation compared to the other stressed groups. While few or no differentially expressed genes were detected 24-h after one or three loud noise exposures, relatively large numbers of transcripts were differentially expressed between the group exposed to 7 loud noises when compared to the control or 3-stress groups, respectively, which correlated with the corticosterone response habituation observed. Gene ontology analyses indicated multiple significant functional terms related to neuron differentiation, neural membrane potential, pre- and post-synaptic elements, chemical synaptic transmission, vesicles, axon guidance and projection, glutamatergic and GABAergic neurotransmission. Some of the differentially expressed genes (Myt1l, Zmat4, Dlx6, Csrnp3) encode transcription factors that were independently predicted by transcription factor enrichment analysis to target other differentially regulated genes in this study. A similar experiment employing in situ hybridization histochemical analysis in additional animals validated the direction of change of the 5 transcripts investigated (Camk4, Gabrb2, Gad1, Grin2a and Slc32a) with a high level of temporal and regional specificity for the rPH. In aggregate, the results suggest that distinct patterns of gene regulation are obtained in response to a repeated homotypic stress regimen; they also point to a significant reorganization of the rPH region that may critically contribute to the phenotypic modifications associated with repeated homotypic stress habituation.