Inducible gene expression of IκB-kinase ε is dependent on nuclear factor-κB in human pulmonary epithelial cells.

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-1β (IL-1β) and tumour necrosis factor-α (TNFα).

Determination of steady-state transcriptome modifications associated with repeated homotypic stress in the rat rostral posterior hypothalamic region.

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.

Integrated genomics approaches identify transcriptional mediators and epigenetic responses to Afghan desert particulate matter in small airway epithelial cells.

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.

Colocalization of Gene Expression and DNA Methylation with Genetic Risk Variants Supports Functional Roles of and in Idiopathic Pulmonary Fibrosis.

Common genetic variants have been associated with idiopathic pulmonary fibrosis (IPF). To determine functional relevance of the 10 IPF-associated common genetic variants we previously identified. 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.

Macrophage programming is regulated by a cooperative interaction between fatty acid binding protein 5 and peroxisome proliferator-activated receptor γ.

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).

Interplay between nuclear factor-κB, p38 MAPK, and glucocorticoid receptor signaling synergistically induces functional TLR2 in lung epithelial cells.

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).

Deconvolution of multiplexed transcriptional responses to wood smoke particles defines rapid aryl hydrocarbon receptor signaling dynamics.

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.

Repression of transcription by the glucocorticoid receptor: A parsimonious model for the genomics era.

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.

The MUC5B-associated variant rs35705950 resides within an enhancer subject to lineage- and disease-dependent epigenetic remodeling.

The G/T transversion rs35705950, located approximately 3 kb upstream of the MUC5B start site, is the cardinal risk factor for idiopathic pulmonary fibrosis (IPF). Here, we investigate the function and chromatin structure of this -3 kb region and provide evidence that it functions as a classically defined enhancer subject to epigenetic programming. We use nascent transcript analysis to show that RNA polymerase II loads within 10 bp of the G/T transversion site, definitively establishing enhancer function for the region.

Genomic determinants implicated in the glucocorticoid-mediated induction of KLF9 in pulmonary epithelial cells.

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).

Nascent transcript analysis of glucocorticoid crosstalk with TNF defines primary and cooperative inflammatory repression.

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.

Glucocorticoid Receptor ChIP-Seq Identifies PLCD1 as a KLF15 Target that Represses Airway Smooth Muscle Hypertrophy.

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.

Regulation of MUC5B Expression in Idiopathic Pulmonary Fibrosis.

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.

Glucocorticoid and TNF signaling converge at A20 (TNFAIP3) to repress airway smooth muscle cytokine expression.

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).

Cistrome-based Cooperation between Airway Epithelial Glucocorticoid Receptor and NF-κB Orchestrates Anti-inflammatory Effects.

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.

Response Element Composition Governs Correlations between Binding Site Affinity and Transcription in Glucocorticoid Receptor Feed-forward Loops.

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.

Feed-forward transcriptional programming by nuclear receptors: regulatory principles and therapeutic implications.

Nuclear receptors (NRs) are widely targeted to treat a range of human diseases. Feed-forward loops are an ancient mechanism through which single cell organisms organize transcriptional programming and modulate gene expression dynamics, but they have not been systematically studied as a regulatory paradigm for NR-mediated transcriptional responses. Here, we provide an overview of the basic properties of feed-forward loops as predicted by mathematical models and validated experimentally in single cell organisms.

Context-dependent cooperation between nuclear factor κB (NF-κB) and the glucocorticoid receptor at a TNFAIP3 intronic enhancer: a mechanism to maintain negative feedback control of inflammation.

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 IL1β, 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.

Central gene expression changes associated with enhanced neuroendocrine and autonomic response habituation to repeated noise stress after voluntary wheel running in rats.

Accumulating evidence indicates that regular physical exercise benefits health in part by counteracting some of the negative physiological impacts of stress. While some studies identified reductions in some measures of acute stress responses with prior exercise, limited data were available concerning effects on cardiovascular function, and reported effects on hypothalamic-pituitary-adrenocortical (HPA) axis responses were largely inconsistent. Given that exposure to repeated or prolonged stress is strongly implicated in the precipitation and exacerbation of illness, we proposed the novel hypothesis that physical exercise might facilitate adaptation to repeated stress, and subsequently demonstrated significant enhancement of both HPA axis (glucocorticoid) and cardiovascular (tachycardia) response habituation to repeated noise stress in rats with long-term access to running wheels compared to sedentary controls.

The glucocorticoid receptor and KLF15 regulate gene expression dynamics and integrate signals through feed-forward circuitry.

The glucocorticoid receptor (GR) regulates adaptive transcriptional programs that alter metabolism in response to stress. Network properties that allow GR to tune gene expression to match specific physiologic demands are poorly understood. We analyzed the transcriptional consequences of GR activation in murine lungs deficient for KLF15, a transcriptional regulator of amino acid metabolism that is induced by glucocorticoids and fasting.

Effects of voluntary wheel running on heart rate, body temperature, and locomotor activity in response to acute and repeated stressor exposures in rats.

Stress often negatively impacts physical and mental health but it has been suggested that voluntary physical activity may benefit health by reducing some of the effects of stress. The present experiments tested whether voluntary exercise can reduce heart rate, core body temperature and locomotor activity responses to acute (novelty or loud noise) or repeated stress (loud noise). After 6 weeks of running-wheel access, rats exposed to a novel environment had reduced heart rate, core body temperature, and locomotor activity responses compared to rats housed under sedentary conditions.