RESUMO
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.
Assuntos
Células Epiteliais , Quinase I-kappa B , Humanos , Quinase I-kappa B/metabolismo , Quinase I-kappa B/genética , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Células A549 , Fator de Transcrição RelA/metabolismo , Fator de Transcrição RelA/genética , Interleucina-1beta/farmacologia , Interleucina-1beta/metabolismo , Interleucina-1beta/genética , NF-kappa B/metabolismo , NF-kappa B/genética , Fator de Necrose Tumoral alfa/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/genética , Pulmão/metabolismo , Pulmão/citologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/citologia , Regulação da Expressão Gênica/efeitos dos fármacosRESUMO
Although substantial progress has been made in our understanding of asthma pathogenesis and phenotypes over the nearly 60-year history of the Aspen Lung Conferences on asthma, many ongoing challenges exist in our understanding of the clinical and molecular heterogeneity of the disease and an individual patient's response to therapy. This report summarizes the proceedings of the 2023 Aspen Lung Conference, which was organized to review the clinical and molecular heterogeneity of asthma and to better understand the impact of genetic, environmental, cellular, and molecular influences on disease susceptibility, heterogeneity, and severity. The goals of the conference were to review new information about asthma phenotypes, cellular processes, and cellular signatures underlying disease heterogeneity and treatment response. The report concludes with ongoing gaps in our understanding of asthma pathobiology and provides some recommendations for future research to better understand the clinical and basic mechanisms underlying disease heterogeneity in asthma and to advance the development of new treatments for this growing public health problem.
Assuntos
Asma , Fenótipo , Humanos , Asma/terapia , Asma/genética , Pulmão/patologia , Pulmão/metabolismo , Congressos como AssuntoRESUMO
α1-Antitrypsin (AAT), a serine protease inhibitor, is the third most abundant protein in plasma. Although the best-known function of AAT is irreversible inhibition of elastase, AAT is an acute-phase reactant and is increasingly recognized to have a panoply of other functions, including as an anti-inflammatory mediator and a host-protective molecule against various pathogens. Although a canonical receptor for AAT has not been identified, AAT can be internalized into the cytoplasm and is known to affect gene regulation. Because AAT has anti-inflammatory properties, we examined whether AAT binds the cytoplasmic glucocorticoid receptor (GR) in human macrophages. We report the finding that AAT binds to GR using several approaches, including coimmunoprecipitation, mass spectrometry, and microscale thermophoresis. We also performed in silico molecular modeling and found that binding between AAT and GR has a plausible stereochemical basis. The significance of this interaction in macrophages is evinced by AAT inhibition of LPS-induced NF-κB activation and IL-8 production as well as AAT induction of angiopoietin-like 4 protein, which are, in part, dependent on GR. Furthermore, this AAT-GR interaction contributes to a host-protective role against mycobacteria in macrophages. In summary, this study identifies a new mechanism for the gene regulation, anti-inflammatory, and host-defense properties of AAT.
Assuntos
Receptores de Glucocorticoides , alfa 1-Antitripsina , Humanos , alfa 1-Antitripsina/metabolismo , Deficiência de alfa 1-Antitripsina , Angiopoietinas/metabolismo , Angiopoietinas/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Interleucina-8/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , NF-kappa B/metabolismo , Elastase Pancreática/metabolismo , Receptores de Glucocorticoides/metabolismo , Inibidores de Serina ProteinaseRESUMO
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.
Assuntos
Asma , NF-kappa B , Receptores de Glucocorticoides , Receptor 2 Toll-Like , Proteínas Quinases p38 Ativadas por Mitógeno , Asma/fisiopatologia , Budesonida/farmacologia , Citocinas/metabolismo , Dexametasona/farmacologia , Células Epiteliais/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glucocorticoides/farmacologia , Humanos , Pulmão/citologia , Pulmão/metabolismo , NF-kappa B/metabolismo , Receptores de Glucocorticoides/metabolismo , Receptor 2 Toll-Like/genética , Receptor 2 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
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.
Assuntos
PPAR gama , Doença Pulmonar Obstrutiva Crônica , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Regulação da Expressão Gênica , Humanos , Inflamação/metabolismo , Leucócitos Mononucleares/metabolismo , Macrófagos/metabolismo , PPAR gama/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismoRESUMO
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.
Assuntos
Fibrose Pulmonar Idiopática , Humanos , DNA , Metilação de DNA/genética , Expressão Gênica , Predisposição Genética para Doença/genética , Fibrose Pulmonar Idiopática/genética , Mucina-5B/genética , Locos de Características Quantitativas/genética , RNARESUMO
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.
Assuntos
Células Epiteliais Alveolares , Material Particulado , Fatores de Transcrição , Afeganistão , Células Epiteliais Alveolares/metabolismo , Cromatina/metabolismo , Epigênese Genética , Genômica/métodos , Destacamento Militar , Material Particulado/toxicidade , Doenças Respiratórias/epidemiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transposases/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
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.
Assuntos
Anti-Inflamatórios/uso terapêutico , Tratamento Farmacológico da COVID-19 , Dexametasona/uso terapêutico , Glucocorticoides/uso terapêutico , NF-kappa B/genética , Receptores de Glucocorticoides/genética , Animais , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Regulação da Expressão Gênica , Genômica/métodos , Humanos , Inflamação/prevenção & controle , Modelos Genéticos , NF-kappa B/antagonistas & inibidores , NF-kappa B/imunologia , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/imunologia , SARS-CoV-2/crescimento & desenvolvimento , SARS-CoV-2/imunologia , SARS-CoV-2/patogenicidade , Transdução de Sinais , Transcrição Gênica/efeitos dos fármacos , Transcrição Gênica/imunologiaRESUMO
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.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais , Fumaça/efeitos adversos , Transcrição Gênica , Madeira , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular Transformada , Montagem e Desmontagem da Cromatina , Citocromo P-450 CYP1A1/biossíntese , Citocromo P-450 CYP1A1/genética , DNA (Citosina-5-)-Metiltransferases/biossíntese , DNA (Citosina-5-)-Metiltransferases/genética , Humanos , Camundongos , NF-kappa B/genética , NF-kappa B/metabolismo , Células NIH 3T3 , Receptores de Hidrocarboneto Arílico/genética , Proteínas Repressoras/genéticaRESUMO
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.
Assuntos
Dexametasona/farmacologia , Genômica , Glucocorticoides/farmacologia , Fatores de Transcrição Kruppel-Like/biossíntese , Pulmão/efeitos dos fármacos , Células A549 , Elementos Facilitadores Genéticos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Pulmão/citologia , Pulmão/metabolismo , Ligação Proteica , RNA Mensageiro/genética , Receptores de Glucocorticoides/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
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.
Assuntos
Dexametasona/farmacologia , Inflamação/metabolismo , RNA Mensageiro/genética , Receptores de Glucocorticoides/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Cromatina/metabolismo , Dexametasona/metabolismo , Elementos Facilitadores Genéticos , Células HEK293 , Humanos , NF-kappa B/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Corticosteroides/farmacologia , Brônquios/imunologia , Resistência a Medicamentos/efeitos dos fármacos , Fluticasona/farmacologia , Interferon gama/imunologia , Miócitos de Músculo Liso/imunologia , Traqueia/imunologia , Fator de Necrose Tumoral alfa/imunologia , Brônquios/crescimento & desenvolvimento , Criança , Pré-Escolar , Resistência a Medicamentos/imunologia , Feminino , Humanos , Masculino , Células Th1/imunologia , Traqueia/crescimento & desenvolvimentoRESUMO
BACKGROUND: Histamine is a critical mediator of IgE/mast cell-mediated anaphylaxis. Histamine is synthesized by decarboxylating the amino acid histidine, a reaction catalyzed by the histidine decarboxylase (Hdc) gene-encoded enzyme HDC. However, regulation of the Hdc gene in mast cells is poorly understood. OBJECTIVE: We sought to investigate the in vivo regulation of IgE/mast cell-mediated anaphylaxis by the transcription factors GATA2 and microphthalmia-associated transcription factor (MITF) and the mechanisms by which GATA2 and MITF regulate Hdc gene expression in mouse and human mast cells. METHODS: Mice deficient in the transcription factors Gata2, aryl hydrocarbon receptor (Ahr), aryl hydrocarbon receptor repressor (Ahrr), or basic helix-loop-helix family member E40 (Bhlhe40) were assessed for anaphylactic reactions. Chromatin immunoprecipitation sequencing analysis identified putative Hdc enhancers. Luciferase reporter transcription assay confirmed enhancer activities of putative enhancers in the Hdc gene. The short hairpin RNA knockdown approach was used to determine the role of MITF in regulating mouse and human HDC gene expression. RESULTS: Connective tissue mast cell-specific Gata2-deficient mice did not have IgE/mast cell-mediated anaphylaxis. GATA2 induced the expression of Mitf, Ahr, Ahrr, and Bhlhe40 in mast cells. MITF, but not AHR, AHRR, or BHLHE40, was required for anaphylaxis. MITF bound to an enhancer located 8.8 kb upstream of the transcription start site of the Hdc gene and directed enhancer activity. MITF overexpression largely restored Hdc gene expression in the Gata2-deficient mast cells. In the human mast cell line LAD2, MITF was required for the HDC gene expression and histamine synthesis. CONCLUSION: The transcription factors GATA2 and MITF regulate Hdc gene expression in mast cells and are required for IgE/mast cell-mediated anaphylaxis.
Assuntos
Anafilaxia/genética , Fator de Transcrição GATA2/genética , Regulação da Expressão Gênica , Histidina Descarboxilase/genética , Mastócitos/imunologia , Fator de Transcrição Associado à Microftalmia/genética , Anafilaxia/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Homeodomínio/genética , Imunoglobulina E/imunologia , Camundongos Knockout , Receptores de Hidrocarboneto Arílico/genética , Proteínas Repressoras/genéticaRESUMO
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.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2/metabolismo , Receptores de Glucocorticoides/agonistas , Receptores de Glucocorticoides/metabolismo , Mucosa Respiratória/metabolismo , Transcrição Gênica/fisiologia , Agonistas de Receptores Adrenérgicos beta 2/farmacologia , Células Cultivadas , Preparações de Ação Retardada , Relação Dose-Resposta a Droga , Humanos , Receptores de Glucocorticoides/genética , Mucosa Respiratória/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Classic physiology studies dating to the 1930s demonstrate that moderate or transient glucocorticoid (GC) exposure improves muscle performance. The ergogenic properties of GCs are further evidenced by their surreptitious use as doping agents by endurance athletes and poorly understood efficacy in Duchenne muscular dystrophy (DMD), a genetic muscle-wasting disease. A defined molecular basis underlying these performance-enhancing properties of GCs in skeletal muscle remains obscure. Here, we demonstrate that ergogenic effects of GCs are mediated by direct induction of the metabolic transcription factor KLF15, defining a downstream pathway distinct from that resulting in GC-related muscle atrophy. Furthermore, we establish that KLF15 deficiency exacerbates dystrophic severity and muscle GC-KLF15 signaling mediates salutary therapeutic effects in the mdx mouse model of DMD. Thus, although glucocorticoid receptor (GR)-mediated transactivation is often associated with muscle atrophy and other adverse effects of pharmacologic GC administration, our data define a distinct GR-induced gene regulatory pathway that contributes to therapeutic effects of GCs in DMD through proergogenic metabolic programming.
Assuntos
Glucocorticoides/farmacologia , Músculo Esquelético/efeitos dos fármacos , Distrofia Muscular de Duchenne/tratamento farmacológico , Animais , Feminino , Glucocorticoides/uso terapêutico , Humanos , Fatores de Transcrição Kruppel-Like/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/fisiopatologia , Proteínas Nucleares/fisiologia , Receptores de Glucocorticoides/fisiologiaRESUMO
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.
Assuntos
Remodelação das Vias Aéreas/genética , Regulação da Expressão Gênica/fisiologia , Fatores de Transcrição Kruppel-Like/fisiologia , Músculo Liso/patologia , Proteínas Nucleares/fisiologia , Fosfolipase C delta/fisiologia , Receptores de Glucocorticoides/fisiologia , Sistema Respiratório/citologia , Adenoviridae/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Hipertrofia , Músculo Liso/metabolismo , Fosfolipase C delta/genética , Cultura Primária de Células , RNA Polimerase II/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de RNA , Transcriptoma , Transdução Genética , Fator de Crescimento Transformador beta/farmacologiaRESUMO
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.
Assuntos
Fibrose Pulmonar Idiopática/genética , Mucina-5B/genética , Sequência de Bases , Sítios de Ligação , Imunoprecipitação da Cromatina , Ilhas de CpG/genética , Metilação de DNA/genética , Técnicas de Silenciamento de Genes , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Pulmão/metabolismo , Pulmão/patologia , Mucina-5B/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas , Ligação Proteica/genética , RNA Polimerase II/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
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.