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1.  Cytokines Alter Glucocorticoid Receptor Phosphorylation in Airway Cells 
Corticosteroid insensitivity (CSI) represents a profound challenge in managing patients with asthma. We recently demonstrated that short exposure of airway smooth muscle cells (ASMCs) to proasthmatic cytokines drastically reduced their responsiveness to glucocorticoids (GCs), an effect that was partially mediated via interferon regulatory factor-1, suggesting the involvement of additional mechanisms (Am J Respir Cell Mol Biol 2008;38:463–472). Although GC receptor (GR) can be phosphorylated at multiple serines in the N-terminal region, the major phosphorylation sites critical for GR transcriptional activity are serines 211 (Ser211) and 226 (Ser226). We tested the novel hypothesis that cytokine-induced CSI in ASMCs is due to an impaired GR phosphorylation. Cells were treated with TNF-α (10 ng/ml) and IFN-γ (500 UI/ml) for 6 hours and/or fluticasone (100 nm) added 2 hours before. GR was constitutively phosphorylated at Ser226 but not at Ser211 residues. Cytokines dramatically suppressed fluticasone-induced phosphorylation of GR on Ser211 but not on Ser226 residues while increasing the expression of Ser/Thr protein phosphatase (PP)5 but not that of PP1 or PP2A. Transfection studies using a reporter construct containing GC responsive elements showed that the specific small interfering RNA–induced mRNA knockdown of PP5, but not that of PP1 or PP2A, partially prevented the cytokine suppressive effects on GR-meditated transactivation activity. Similarly, cytokines failed to inhibit GC-induced GR-Ser211 phosphorylation when expression of PP5 was suppressed. We propose that the novel mechanism that proasthmatic cytokine-induced CSI in ASMCs is due, in part, to PP5-mediated impairment of GR-Ser211 phosphorylation.
PMCID: PMC3488623  PMID: 22592921
serine/threonine protein phosphatase; airway smooth muscle; asthma; corticosteroid insensitivity; airway remodeling
2.  Cyclin D1 in ASM Cells from Asthmatics Is Insensitive to Corticosteroid Inhibition 
Journal of Allergy  2012;2012:307838.
Hyperplasia of airway smooth muscle (ASM) is a feature of the remodelled airway in asthmatics. We examined the antiproliferative effectiveness of the corticosteroid dexamethasone on expression of the key regulator of G1 cell cycle progression—cyclin D1—in ASM cells from nonasthmatics and asthmatics stimulated with the mitogen platelet-derived growth factor BB. While cyclin D1 mRNA and protein expression were repressed in cells from nonasthmatics in contrast, cyclin D1 expression in asthmatics was resistant to inhibition by dexamethasone. This was independent of a repressive effect on glucocorticoid receptor translocation. Our results corroborate evidence demonstrating that corticosteroids inhibit mitogen-induced proliferation only in ASM cells from subjects without asthma and suggest that there are corticosteroid-insensitive proliferative pathways in asthmatics.
PMCID: PMC3303636  PMID: 22500182
3.  Cytokines Induce an Early Steroid Resistance in Airway Smooth Muscle Cells 
We have previously shown that long-term treatment of airway smooth muscle (ASM) cells with a combination of TNF-α and IFN-γ impaired steroid anti-inflammatory action through the up-regulation of glucocorticoid receptor beta isoform (GRβ) (Mol Pharmacol 2006;69:588–596). We here found that steroid actions could also be suppressed by short-term exposure of ASM cells to TNF-α and IFN-γ (6 h) as shown by the abrogated glucocorticoid responsive element (GRE)-dependent gene transcription; surprisingly, neither GRα nuclear translocation nor GRβ expression was affected by cytokine mixture. The earlier induction of CD38, a molecule recently involved in asthma, seen with TNF-α and IFN-γ combination but not with cytokine alone, was also completely insensitive to steroid pretreatment. Chromatin-immunoprecipitation (IP) and siRNA strategies revealed not only increased binding of interferon regulatory factor 1 (IRF-1) transcription factor to CD38 promoter, but also its implication in regulating CD38 gene transcription. Interestingly, the capacity of fluticasone to completely inhibit TNF-α–induced IRF-1 expression, IRF-1 DNA binding, and transactivation activities was completely lost in cells exposed to TNF-α and IFN-γ in combination. This early steroid dysfunction seen with cytokine combination could be reproduced by enhancing IRF-1 cellular levels using constitutively active IRF-1, which dose-dependently inhibited GRE-dependent gene transcription. Consistently, reducing IRF-1 cellular levels using siRNA approach significantly restored steroid transactivation activities. Collectively, our findings demonstrate for the first time that IRF-1 is a novel alternative GRβ-independent mechanism mediating steroid dysfunction induced by pro-asthmatic cytokines, in part via the suppression of GRα activities.
PMCID: PMC2274949  PMID: 17947510
transcription factor; glucocorticoid; inflammation; asthma; mesenchymal cells
4.  Inhibition of Histone Deacetylase 2 Expression by Elevated Glucocorticoid Receptor β in Steroid-resistant Asthma 
Rationale: Cross-talk between glucocorticoid receptors and histone deacetylases (HDACs) under steroid-insensitive conditions has not been explored.
Objectives: To evaluate expression and interaction of HDACs with glucocorticoid receptor isoforms in bronchoalveolar lavage and peripheral blood mononuclear cells from steroid-resistant versus steroid-sensitive patients with asthma.
Methods: Expression of HDACs 1 through 11 was measured by real-time polymerase chain reaction in primary cells and in the DO11.10 cell line, designed to overexpress glucocorticoid receptor β. Glucocorticoid receptor β expression was inhibited in bronchoalveolar lavage cells by small interfering RNA. Human HDAC2 promoter fragments were cloned into a luciferase reporter vector, and transiently transfected with glucocorticoid receptor α– and β–encoding plasmids into the cells. Luciferase activity was then assayed in response to glucocorticoids.
Measurements and Main Results: Levels of HDAC2 mRNA, but not other histone deacetylases, were significantly decreased in bronchoalveolar lavage cells but not in peripheral blood mononuclear cells from steroid-resistant patients with asthma. Overexpression of glucocorticoid receptor β in DO11.10 cells selectively reduced HDAC2 mRNA and protein levels. Silencing of glucocorticoid receptor β in bronchoalveolar lavage cells from patients with asthma significantly increased HDAC2 mRNA. Luciferase activity assays with HDAC2 promoter reporter constructs identified two glucocorticoid-inducible regions in the HDAC2 promoter. Promoter activity was increased more than fourfold in dexamethasone-treated cells cotransfected with glucocorticoid receptor α. Cotransfection of glucocorticoid receptor β abolished this effect in a dose-dependent manner.
Conclusions: Glucocorticoid receptor β controls expression of histone deacetylase 2 by inhibiting glucocorticoid response elements in its promoter. This highlights a novel mechanism by which glucocorticoid receptor β promotes steroid insensitivity (Li et al.: J Allergy Clin Immunol 2009;123:S146; and Li et al.: J Allergy Clin Immunol 2010;125:AB104).
PMCID: PMC2970859  PMID: 20538962
glucocorticoid receptor β; asthma; glucocorticoid response element; gene expression regulation
5.  Glucocorticoids: mechanisms of action and anti-inflammatory potential in asthma. 
Mediators of Inflammation  1998;7(4):229-237.
GLUCOCORTICOIDS are potent inhibitors of inflammatory processes and are widely used in the treatment of asthma. The anti-inflammatory effects are mediated either by direct binding of the glucocorticoid/glucocorticoid receptor complex to glucocorticoid responsive elements in the promoter region of genes, or by an interaction of this complex with other transcription factors, in particular activating protein-1 or nuclear factor-kappaB. Glucocorticoids inhibit many inflammation-associated molecules such as cytokines, chemokines, arachidonic acid metabolites, and adhesion molecules. In contrast, anti-inflammatory mediators often are up-regulated by glucocorticoids. In vivo studies have shown that treatment of asthmatic patients with inhaled glucocorticoids inhibits the bronchial inflammation and simultaneously improves their lung function. In this review, our current knowledge of the mechanism of action of glucocorticoids and their anti-inflammatory potential in asthma is described. Since bronchial epithelial cells may be important targets for glucocorticoid therapy in asthma, the effects of glucocorticoids on epithelial expressed inflammatory genes will be emphasized.
PMCID: PMC1781857  PMID: 9792333
6.  Repression of TNF-α-induced IL-8 expression by the glucocorticoid receptor-β involves inhibition of histone H4 acetylation 
Experimental & Molecular Medicine  2009;41(5):297-306.
Increased expression of a number of proinflammatory genes, including IL-8, is associated with inflammatory conditions such as asthma. Glucocorticoid receptor (GR)β, one of the GR isoforms, has been suggested to be upregulated in asthma associated with glucocorticoid insensitivity and to work as a dominant negative inhibitor of wild type GRα. However, recent data suggest that GRβ is not a dominant negative inhibitor of GRα in the transrepressive process and has its own functional role. We investigated the functional role of GRβ expression in the suppressive effect of glucocorticoids on tumor necrosis factor (TNF)-α-induced IL-8 release in an airway epithelial cell line. GRβ expression was induced by treatment of epithelial cells with either dexamethasone or TNF-α. GRβ was able to inhibit glucocorticoid-induced transcriptional activation mediated by binding to glucocorticoid response elements (GREs). The suppressive effect of dexamethasone on TNF-α-induced IL-8 transcription was not affected by GRβ overexpression, rather GRβ had its own weak suppressive activity on TNF-α-induced IL-8 expression. Overall histone deacetylase activity and histone acetyltransferase activity were not changed by GRβ overexpression, but TNF-α-induced histone H4 acetylation at the IL-8 promoter was decreased with GRβ overexpression. This study suggests that GRβ overexpression does not affect glucocorticoid-induced suppression of IL-8 expression in airway epithelial cells and GRβ induces its own histone deacetylase activity around IL-8 promoter site.
PMCID: PMC2701978  PMID: 19307749
asthma; glucocorticoids; histone acetyltransferases; histone deacetylases; interleukin-8; receptors, glucocorticoid; tumor necrosis factor-α
7.  Glucocorticoid- and Protein Kinase A–Dependent Transcriptome Regulation in Airway Smooth Muscle 
Glucocorticoids (GCs) and protein kinase A (PKA)–activating agents (β-adrenergic receptor agonists) are mainstream asthma therapies based on their ability to prevent or reverse excessive airway smooth muscle (ASM) constriction. Their abilities to regulate another important feature of asthma—excessive ASM growth—are poorly understood. Recent studies have suggested that GCs render agents of inflammation such as IL-1β and TNF-α mitogenic to ASM, via suppression of (antimitogenic) induced cyclooxygenase-2–dependent PKA activity. To further explore the mechanistic basis of these observations, we assessed the effects of epidermal growth factor and IL-1β stimulation, and the modulatory effects of GC treatment and PKA inhibition, on the ASM transcriptome by microarray analysis. Results demonstrate that ASM stimulated with IL-1β, in a manner that is often cooperative with stimulation with epidermal growth factor, exhibit a profound capacity to function as immunomodulatory cells. Moreover, results implicate an important role for induced autocrine/paracrine factors (many whose regulation was minimally affected by GCs or PKA inhibition) as regulators of both airway inflammation and ASM growth. Induction of numerous chemokines, in conjunction with regulation of proteases and agents of extracellular matrix remodeling, is suggested as an important mechanism promoting upregulated G protein–coupled receptor signaling capable of stimulating ASM growth. Additional functional assays suggest that intracellular PKA plays a critical role in suppressing the promitogenic effects of induced autocrine factors in ASM. Finally, identification and comparison of GC- and PKA-sensitive genes in ASM provide insight into the complementary effects of β-agonist/GC combination therapies, and suggest specific genes as important targets for guiding the development of new generations of GCs and adjunct asthma therapies.
PMCID: PMC2701960  PMID: 19059887
airway smooth muscle; protein kinase A; glucocorticoid; gene expression; G protein–coupled receptors
8.  Negative regulation of interleukin 2 transcription by the glucocorticoid receptor 
The Journal of Experimental Medicine  1992;175(5):1235-1245.
Glucocorticoid-dependent transcriptional enhancement is known to occur through the interaction of the glucocorticoid receptor (GR) with specific DNA response elements. In contrast, negative regulation of gene expression by this class of hormone is less well understood. Glucocorticoids are potent immunosuppressive agents acting primarily by inhibiting T lymphocyte activation and lymphokine production. Interleukin 2 (IL-2) gene expression, a critical early event during T lymphocyte activation, is inhibited in glucocorticoid-sensitive cells by hormone treatment. We have studied the mechanism of this inhibition. In transgenic mice carrying c-myc linked to the IL-2 enhancer, mitogen- induced expression of the transgene is inhibited by concurrent glucocorticoid treatment, while a similar transgene construct driven by three copies of the binding site for nuclear factor of activated T cells is not inhibited. Cotransfection experiments into glucocorticoid- insensitive jurkat cells show that the NH2 terminus of the glucocorticoid receptor is dispensable for inhibition of the IL-2 enhancer but that an intact DNA binding domain, although not necessarily binding to DNA, is required. Hybrid GRs containing the DNA binding domains of either the estrogen receptor (ER) or thyroid receptor, as well as the entire wild-type ER, all function as repressors of the IL-2 enhancer. We have localized the site of inhibition to two sequences located in the proximal half of the enhancer. These sequences bind a similar, if not identical, inducible nuclear factor that has biologic characteristics that distinguish it from AP-1. The mechanism of IL-2 inhibition likely involves direct interactions between the GR and this factor.
PMCID: PMC2119221  PMID: 1569395
9.  Functional KCa3.1 channels regulate steroid insensitivity in bronchial smooth muscle cells1 
Identifying the factors responsible for relative glucocorticosteroid (GC) resistance present in patients with severe asthma and finding tools to reverse it are of paramount importance. In asthma there is in vivo evidence of GC-resistant pathways in airway smooth muscle (ASM) bundles which can be modelled in vitro by exposing cultured ASM cells to TNFα/IFNγ. This drives GC insensitivity via protein phosphatase-5 (PP5)-dependent impairment of GC receptor (GR) phosphorylation. Here, we investigated whether KCa3.1 ion channels modulate the activity of GC-resistant pathways using our ASM model of GC insensitivity. Immunohistochemical staining of endobronchial biopsies revealed that KCa3.1 channels are localized to the plasma membrane and nucleus of ASM in both healthy controls and asthmatic patients, irrespective of disease severity. Western blot assays and immunofluorescence staining confirmed the nuclear localisation of KCa3.1 channels in ASM cells. The functional importance of KCa3.1 channels in the regulation of GC-resistant chemokines induced by TNFα/IFNγ was assessed using complementary inhibitory strategies including KCa3.1 blockers (TRAM-34 and ICA-17043) or KCa3.1-specific shRNA delivered by adenoviruses. KCa3.1 channel blockade led to a significant reduction of fluticasone-resistant CX3CL1, CCL5 and CCL11 gene and protein expression. KCa3.1 channel blockade also restored fluticasone-induced GRα phosphorylation at ser211 and transactivation properties via the suppression of cytokine-induced PP5 expression. The effect of KCa3.1 blockade was evident in ASM cells from both healthy controls and asthmatic subjects. In summary KCa3.1 channels contribute to the regulation of GC-resistant inflammatory pathways in ASM cells: blocking KCa3.1 channels may enhance corticosteroid activity in severe asthma.
PMCID: PMC3753579  PMID: 23904164
Corticosteroid insensitivity; chemokines; GR phosphorylation; TNFα; transactivation; transrepression; KCa3.1; severe asthma; airway smooth muscle; transcription factors
10.  Mechanism of glucocorticoid protection of airway smooth muscle from proasthmatic effects of long-acting β2-adrenoceptor agonist exposure 
Chronic use of long-acting β2-adrenergic receptor (β2AR) agonists (LABAs), resulting in β2AR desensitization, has been associated with increased asthma morbidity. When LABAs are used in combination with inhaled glucocorticoids (GCs), however, asthma control is improved, raising the question: Do GCs inhibit the proasthmatic mechanism that mediates altered contractility in LABA-exposed airway smooth muscle (ASM)?
This study aimed to identify the potential protective role and mechanism of action of GCs in mitigating the effects of prolonged LABA exposure on ASM constrictor and relaxation responsiveness.
Cultured human ASM (HASM) cells and isolated rabbit ASM tissues were examined for induced changes in agonist-mediated cAMP accumulation, constrictor and relaxation responsiveness, and expression of specific GC-regulated molecules following 24h exposure to the LABA, salmeterol, in the absence and presence of dexamethasone (DEX).
Salmeterol-exposed ASM exhibited impaired cAMP and relaxation responses to isoproterenol and increased acetylcholine-induced contractility. These pro-asthmatic effects of prolonged LABA exposure were attributed to upregulated phosphodiesterase 4 (PDE4) activity, and ablated by pretreatment with DEX. Further studies demonstrated that: 1) DEX suppressed activation of the mitogen-activated protein kinase (MAPK), ERK1/2, which upregulates PDE4 expression in salmeterol-exposed ASM; and 2) the inhibitory actions of DEX on salmeterol-induced ERK1/2 activation and resultant PDE4-mediated changes in ASM responsiveness were prevented by gene silencing or pharmacological inhibition of DEX-induced expression of MAPK phosphatase-1 (MKP-1), an endogenous deactivator of ERK1/2 signaling.
GCs prevent the adverse proasthmatic effects of prolonged LABA exposure on airway responsiveness due to GC-induced upregulation of MKP-1, which inhibits proasthmatic ERK1/2 signaling in the LABA-exposed ASM.
PMCID: PMC2866838  PMID: 20392484
asthma; salmeterol; homologous β2-adrenergic receptor desensitization; airway smooth muscle; phosphodiesterase; ERK1/2 phosphorylation; siRNA; MAPK phosphatase-1
11.  Molecular Mechanisms Regulating Glucocorticoid Sensitivity and Resistance 
Glucocorticoid receptor agonists are mainstays in the treatment of various malignancies of hematological origin. Glucocorticoids are included in therapeutic regimens for their ability to stimulate intracellular signal transduction cascades that culminate in alterations in the rate of transcription of genes involved in cell cycle progression and programmed cell death. Unfortunately, subpopulations of patients undergoing systemic glucocorticoid therapy for these diseases are or become insensitive to glucocorticoid-induced cell death, a phenomenon recognized as glucocorticoid resistance. Multiple factors contributing to glucocorticoid resistance have been identified. Here we summarize several of these mechanisms and describe the processes involved in generating a host of glucocorticoid receptor isoforms from one gene. The potential role of glucocorticoid receptor isoforms in determining cellular responsiveness to glucocorticoids is emphasized.
PMCID: PMC2674248  PMID: 19000736
glucocorticoid receptor; glucocorticoid resistance; hematological malignancy; alternative initiation of translation
12.  Expression Profiling Identifies Klf15 as a Glucocorticoid Target That Regulates Airway Hyperresponsiveness 
Glucocorticoids (GCs), which activate GC receptor (GR) signaling and thus modulate gene expression, are widely used to treat asthma. GCs exert their therapeutic effects in part through modulating airway smooth muscle (ASM) structure and function. However, the effects of genes that are regulated by GCs on airway function are not fully understood. We therefore used transcription profiling to study the effects of a potent GC, dexamethasone, on human ASM (HASM) gene expression at 4 and 24 hours. After 24 hours of dexamethasone treatment, nearly 7,500 genes had statistically distinguishable changes in expression; quantitative PCR validation of a 40-gene subset of putative GR-regulated genes in 6 HASM cell lines suggested that the early transcriptional targets of GR signaling are similar in independent HASM lines. Gene ontology analysis implicated GR targets in controlling multiple aspects of ASM function. One GR-regulated gene, the transcription factor, Kruppel-like factor 15 (Klf15), was already known to modulate vascular smooth and cardiac muscle function, but had no known role in the lung. We therefore analyzed the pulmonary phenotype of Klf15−/− mice after ovalbumin sensitization and challenge. We found diminished airway responses to acetylcholine in ovalbumin-challenged Klf15−/− mice without a significant change in the induction of asthmatic inflammation. In cultured cells, overexpression of Klf15 reduced proliferation of HASM cells, whereas apoptosis in Klf15−/− murine ASM cells was increased. Together, these results further characterize the GR-regulated gene network in ASM and establish a novel role for the GR target, Klf15, in modulating airway function.
PMCID: PMC3175579  PMID: 21257922
asthma; glucocorticoid; Klf15
13.  Vitamin D and glucocorticoids differentially modulate chemokine expression in human airway smooth muscle cells 
British Journal of Pharmacology  2008;155(1):84-92.
Background and purpose:
Chemokines play a critical role in the pathogenesis of asthma and facilitate the recruitment of inflammatory cells in the airways. Evidence now suggests that airway smooth muscle (ASM) may serve as a source of chemokines in inflamed airways. Although vitamin D has potent anti-inflammatory properties in vitro in some cell types, its effects on ASM cells remain unclear. Here, we investigated whether 1α, 25-dihydroxy vitamin D3 (calcitriol) modulated chemokine production in ASM.
Experimental approach:
Human ASM cell cultures were derived from tracheal samples taken during surgery. ASM cells were treated with tumour necrosis factor alpha (TNFα) and/or interferon gamma (IFNγ) for 24 h in the presence of calcitriol and/or the glucocorticoid fluticasone added 2 h before. RANTES (regulated upon activation, normal T-cell expressed and secreted), interferon-inducible protein 10 (IP-10) and fractalkine (FKN) levels in cell supernatants were measured by ELISA.
Key results:
In TNFα-treated cells, calcitriol inhibited RANTES and IP-10 secretion in a concentration-dependent manner. FKN levels were negligible. In TNFα/IFNγ-treated cells, whereas fluticasone or calcitriol alone partially inhibited RANTES secretion (by 38 and 20%, respectively), the combination of both drugs additively inhibited RANTES secretion (by 60%). No effect was observed on IP-10 secretion. Whereas fluticasone enhanced FKN secretion (by 50%), calcitriol significantly decreased FKN levels (by 50%). Interestingly, calcitriol blocked the stimulatory effect of fluticasone on FKN secretion, which was inhibited by 60% with the combination of calcitriol and fluticasone.
Conclusions and implications:
These findings suggest that vitamin D uniquely modulates human ASM expression of chemokines and may exert some beneficial effects in the treatment of steroid-resistant patients with asthma.
PMCID: PMC2440089  PMID: 18552877
chemokines; cytokines; glucocorticoids; steroid resistance; asthma; airway structural cells; inflammation; gene expression; drug; calcitriol
14.  IRF8 suppresses pathological cardiac remodelling by inhibiting calcineurin signalling 
Nature Communications  2014;5:3303.
Interferon regulatory factor 8 (IRF8) is known to affect the innate immune response, for example, by regulating the differentiation and function of immune cells. However, whether IRF8 can influence cardiac hypertrophy is unknown. Here we show that IRF8 levels are decreased in human dilated/hypertrophic cardiomyopathic hearts and in murine hypertrophic hearts. Mice overexpressing Irf8 specifically in the heart are resistant to aortic banding (AB)-induced cardiac hypertrophy, whereas mice lacking IRF8 either globally or specifically in cardiomyocytes develop an aggravated phenotype induced by pressure overload. Mechanistically, we show that IRF8 directly interacts with NFATc1 to prevent NFATc1 translocation and thus inhibits the hypertrophic response. Inhibition of NFATc1 ameliorates the cardiac abnormalities in IRF8−/− mice after AB. In contrast, constitutive activation of NFATc1 nullifies the protective effects of IRF8 on cardiac hypertrophy in IRF8-overexpressing mice. Our results indicate that IRF8 is a potential therapeutic target in pathological cardiac hypertrophy.
The transcription factor interferon regulatory factor 8 (IRF8) is known to regulate differentiation and function of immune cells. Here the authors show that IRF8 is upregulated in the hypertrophic heart in humans and mice, where it suppresses cardiac remodelling by inhibiting calcineurin signalling.
PMCID: PMC3929801  PMID: 24526256
15.  An RGS4-Mediated Phenotypic Switch of Bronchial Smooth Muscle Cells Promotes Fixed Airway Obstruction in Asthma 
PLoS ONE  2012;7(1):e28504.
In severe asthma, bronchodilator- and steroid-insensitive airflow obstruction develops through unknown mechanisms characterized by increased lung airway smooth muscle (ASM) mass and stiffness. We explored the role of a Regulator of G-protein Signaling protein (RGS4) in the ASM hyperplasia and reduced contractile capacity characteristic of advanced asthma. Using immunocytochemical staining, ASM expression of RGS4 was determined in endobronchial biopsies from healthy subjects and those from subjects with mild, moderate and severe asthma. Cell proliferation assays, agonist-induced calcium mobilization and bronchoconstriction were determined in cultured human ASM cells and in human precision cut lung slices. Using gain- and loss-of-function approaches, the precise role of RGS proteins was determined in stimulating human ASM proliferation and inhibiting bronchoconstriction. RGS4 expression was restricted to a subpopulation of ASM and was specifically upregulated by mitogens, which induced a hyperproliferative and hypocontractile ASM phenotype similar to that observed in recalcitrant asthma. RGS4 expression was markedly increased in bronchial smooth muscle of patients with severe asthma, and expression correlated significantly with reduced pulmonary function. Whereas RGS4 inhibited G protein-coupled receptor (GPCR)-mediated bronchoconstriction, unexpectedly RGS4 was required for PDGF-induced proliferation and sustained activation of PI3K, a mitogenic signaling molecule that regulates ASM proliferation. These studies indicate that increased RGS4 expression promotes a phenotypic switch of ASM, evoking irreversible airway obstruction in subjects with severe asthma.
PMCID: PMC3257220  PMID: 22253691
16.  Steroid-Resistant Neutrophilic Inflammation in a Mouse Model of an Acute Exacerbation of Asthma 
Neutrophilic inflammation in acute exacerbations of asthma tends to be resistant to treatment with glucocorticoids. This may be related to decreased activity and expression of histone deacetylase-2 (HDAC2), which down-regulates expression of proinflammatory genes via recruitment to the glucocorticoid receptor complex. We assessed airway inflammation and response to steroid treatment in a novel mouse model of an acute exacerbation of chronic asthma. Systemically sensitized mice received low-level challenge with aerosolized ovalbumin for 4 weeks, followed by a single moderate-level challenge to induce enhanced inflammation in distal airways. We assessed the effects of pre-treatment with dexamethasone on the accumulation of inflammatory cells in the airways, airway responsiveness to methacholine, expression and enzymatic activity of nuclear proteins including histone acetyl transferase (HAT) and HDAC2, and levels of transcripts for neutrophil chemoattractant and survival cytokines. Dexamethasone suppressed inflammation associated with eosinophil and T-lymphocyte recruitment, but did not prevent neutrophil accumulation or development of airway hyperresponsiveness. Increased activity of HAT was suppressed by steroid treatment, but the marked diminution of HDAC2 activity and increased activity of nuclear factor-κB were not reversed. Correspondingly, elevated expression of mRNA for TNF-α, granulocyte-macrophage colony-stimulating factor, IL-8, and p21waf were also not suppressed by dexamethasone. Levels of lipid peroxidation and protein nitration products were elevated in the acute exacerbation model. We conclude that impaired nuclear recruitment of HDAC2 could be an important mechanism of steroid resistance of the neutrophilic inflammation in exacerbations of asthma. Oxidative stress may contribute to decreased HDAC2 activity.
PMCID: PMC2643207  PMID: 18474669
airway inflammation; cytokines; dexamethasone; histone deacetylase-2
17.  Ciclesonide inhibits TNFα- and IL-1β-induced monocyte chemotactic protein-1 (MCP-1/CCL2) secretion from human airway smooth muscle cells 
Monocyte chemotactic protein-1 (MCP-1) is a member of the CC family of cytokines. It has monocyte and lymphocyte chemotactic activity and stimulates histamine release from basophils. MCP-1 is implicated in the pathogenesis of inflammatory diseases, including asthma. The airway smooth muscle (ASM) layer is thickened in asthma, and the growth factors and cytokines secreted by ASM cells play a role in the inflammatory response of the bronchial wall. Glucocorticoids and β2-agonists are first-line drug treatments for asthma. Little is known about the effect of asthma treatments on MCP-1 production from human ASM cells. Here, we determined the effect of ciclesonide (a glucocorticoid) and formoterol (a β2-agonist) on MCP-1 production from human ASM cells. TNFα and IL-1β induced MCP-1 secretion from human ASM cells. Formoterol had no effect on MCP-1 expression, while ciclesonide significantly inhibited IL-1β- and TNFα-induced MCP-1. Furthermore, ciclesonide inhibited IL-1β- and TNFα-induced MCP-1 mRNA and IL-1β- and TNFα-induced MCP-1 promoter and enhancer luciferase reporters. Western blots showed that ciclesonide had no effect on IκB degradation. Finally, ciclesonide inhibited an NF-κB luciferase reporter. Our data show that ciclesonide inhibits IL-1β- and TNFα-induced MCP-1 production from human ASM cells via a transcriptional mechanism involving inhibition of NF-κB binding.
PMCID: PMC3331580  PMID: 22246000
glucocorticoid; nuclear factor-κB; inflammation
18.  Antigen-specific CD4+ T cells drive airway smooth muscle remodeling in experimental asthma 
Journal of Clinical Investigation  2005;115(6):1580-1589.
Airway smooth muscle (ASM) growth contributes to the mechanism of airway hyperresponsiveness in asthma. Here we demonstrate that CD4+ T cells, central to chronic airway inflammation, drive ASM remodeling in experimental asthma. Adoptive transfer of CD4+ T cells from sensitized rats induced an increase in proliferation and inhibition of apoptosis of airway myocytes in naive recipients upon repeated antigen challenge, which resulted in an increase in ASM mass. Genetically modified CD4+ T cells expressing enhanced GFP (EGFP) were localized by confocal microscopy in juxtaposition to ASM cells, which suggests that CD4+ T cells may modulate ASM cell function through direct cell-cell interaction in vivo. Coculture of antigen-stimulated CD4+ T cells with cell cycle–arrested ASM cells induced myocyte proliferation, dependent on T cell activation and direct T cell–myocyte contact. Reciprocally, direct cell contact prevented postactivation T cell apoptosis, which suggests receptor-mediated T cell–myocyte crosstalk. Overall, our data demonstrate that activated CD4+ T cells drive ASM remodeling in experimental asthma and suggest that a direct cell-cell interaction participates in CD4+ T cell regulation of myocyte turnover and induction of remodeling.
PMCID: PMC1088014  PMID: 15902312
19.  TIF1α mediates physical interaction and functional synergy between the CARM1 and GRIP1 nuclear receptor coactivators 
In previous studies Transcriptional Intermediary Factor 1α (TIF1α) was identified as a direct binding partner and potential transcriptional coactivator for nuclear receptors (NR), but its over-expression inhibited rather than enhanced transcriptional activation by NRs. Here we show that TIF1α bound to and enhanced the function of the C-terminal activation domain of Coactivator Associated Arginine Methyltransferase 1 (CARM1) and the N-terminal activation domain of Glucocorticoid Receptor Interacting Protein 1 (GRIP1). Furthermore, although TIF1α had little or no NR coactivator activity by itself, it cooperated synergistically with GRIP1 and CARM1 to enhance NR-mediated transcription. Inhibition of endogenous TIF1α expression reduced transcriptional activation by the GRIP1 N-terminal domain but not by the CARM1 C-terminal domain, suggesting that TIF1α may be more important for mediating the activity of the former than the latter. Reduction of endogenous TIF1α levels also compromised the androgen-dependent induction of an endogenous target gene of the androgen receptor. Finally, TIF1α formed a ternary complex with the GRIP1 N-terminal and CARM1 C-terminal domains. Thus, we conclude that TIF1α cooperates with NR coactivators GRIP1 and CARM1 by forming a stable ternary complex with them and enhancing the activation domain function of one or both of them.
PMCID: PMC1626528  PMID: 16322096
20.  Glucocorticoid-Dependent Phosphorylation of the Transcriptional Coregulator GRIP1 
Molecular and Cellular Biology  2012;32(4):730-739.
Much of the regulatory diversity in eukaryotic transcription is provided by coregulators, which are recruited by DNA-binding factors to propagate signaling to basal machinery or chromatin. p160 family members, including the glucocorticoid receptor (GR)-interacting protein 1 (GRIP1), function as coactivators for GR, a ligand-dependent transcription factor of the nuclear receptor superfamily. Unlike other p160s, GRIP1 also potentiates GR-mediated repression of AP1 and NF-κB targets and, surprisingly, transcriptional activation by interferon regulatory factors. What enables GRIP1 activating or repressing properties or discrimination between physiologically antagonistic pathways is unknown. We found that endogenous GRIP1 in mammalian cells undergoes glucocorticoid-induced, GR interaction-dependent phosphorylation and identified one constitutive and six inducible phosphorylation sites and two putative GRIP1 kinases, casein kinase 2 and cyclin-dependent kinase 9. We raised phosphospecific antibodies to the four closely spaced sites in a previously uncharacterized part of GRIP1 which, combined with mutagenesis, revealed the conservation of GRIP1 phosphorylation across several cell types and species and its functional relevance to GR-activated transcription and to response element-specific recruitment of phospho-GRIP1 to native GR targets. We propose that cofactor engagement by GR is neither passive nor stochastic; rather, GR actively imparts modifications that dictate GRIP1 function in a subset of complexes, adding a layer of specificity to GR transcriptional control.
PMCID: PMC3272970  PMID: 22158970
21.  IRF4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating NOTCH2 expression and activity 
The Journal of Experimental Medicine  2013;210(13):2887-2902.
The transcription factor IRF4 limits the retention of B cells in the marginal zone by inhibiting NOTCH2 signaling.
The transcription factor interferon regulatory factor-4 (IRF4) is expressed in B cells at most developmental stages. In antigen-activated B cells, IRF4 controls germinal center formation, class-switch recombination, and the generation of plasma cells. Here we describe a novel function for IRF4 in the homeostasis of mature B cells. Inducible deletion of irf4 specifically in B cells in vivo led to the aberrant accumulation of irf4-deleted follicular B cells in the marginal zone (MZ) area. IRF4-deficient B cells showed elevated protein expression and activation of NOTCH2, a transmembrane receptor and transcriptional regulator known to be required for MZ B cell development. Administration of a NOTCH2-inhibitory antibody abolished nuclear translocation of NOTCH2 in B cells within 12 h and caused a rapid and progressive disintegration of the MZ that was virtually complete 48 h after injection. The disappearance of the MZ was accompanied by a transient increase of MZ-like B cells in the blood rather than increased B cell apoptosis, demonstrating that continued NOTCH2 activation is critical for the retention of B cells in the MZ. Our results suggest that IRF4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating NOTCH2 expression. These findings may have implications for the understanding of B cell malignancies with dysregulated IRF4 and NOTCH2 activity.
PMCID: PMC3865479  PMID: 24323359
22.  Identification of Natural Human Glucocorticoid Receptor (hGR) Mutations or Polymorphisms and their Functional Consequences at the Hormone-Receptor Interaction Level 
Glucocorticoids regulate a broad spectrum of physiologic functions essential for life and play an important role in the maintenance of basal and stress-related homeostasis. At the cellular level, the actions of glucocorticoids are mediated by the human glucocorticoid receptor α (hGRα), a ligand-dependent transcription factor ubiquitously expressed in almost all tissues and cells. The molecular mechanisms of hGRα action involve (1) binding to glucocorticoids, (2) cytoplasmic to nuclear translocation, (3) binding/association to DNA/chromatin, (4) transcriptional activation or repression by interacting with cofactors and other transcription factors. Mutations or polymorphisms in the hGR gene may impair these molecular mechanisms of hGRα action, thereby altering tissue sensitivity to glucocorticoids. The latter may take the form of glucocorticoid resistance or glucocorticoid hypersensitivity and may be associated with significant morbidity. The identification of natural pathologic mutations in patients' hGR gene and the subsequent examination of the functional defects of the natural mutant hGRα receptors enhances our understanding of the molecular mechanisms of hGRα action and highlights the importance of integrated cellular and molecular signaling mechanisms for maintaining homeostasis and preserving normal physiology.
PMCID: PMC2788239  PMID: 19763496
cytoplasmic to nuclear translocation; dexamethasone binding assay; gene sequencing; fluorescent recovery after photobleaching; glucocorticoid receptor; glucocorticoid resistance; glucocorticoid hypersensitivity; green fluorescent protein; glutathione-S transferase pull-down assay; reporter assay; thymidine incorporation assay; transfection
23.  Increased Glucocorticoid Receptor β Alters Steroid Response in Glucocorticoid-insensitive Asthma 
Rationale: Glucocorticoids (GCs) are highly effective in the treatment of asthma. However, some individuals have GC-insensitive asthma.
Objectives: To evaluate the functional response to steroids of bronchoalveolar lavage (BAL) cells from sites of airway inflammation from patients with GC-insensitive versus GC-sensitive asthma. As well, to attempt to define the functional role of glucocorticoid receptor (GCR)β (a splicing variant, and dominant negative inhibitor of, the classic GCRα) in controlling GCRα nuclear translocation and transactivation at a molecular level.
Methods and Measurements: Fiberoptic bronchoscopy with collection of BAL fluid was performed on seven patients with GC-sensitive asthma and eight patients with GC-insensitive asthma. GCRα cellular shuttling in response to 10−6 M dexamethasone treatment and GCRβ expression were analyzed in BAL cells by immunofluorescence staining. The effects of overexpression and silencing of GCRβ mRNA on GCRα function were assessed.
Main Results: Significantly reduced nuclear translocation of GCRα in response to steroids was found in BAL cells from patients with GC-insensitive asthma. BAL macrophages from patients with GC-insensitive asthma had significantly increased levels of cytoplasmic and nuclear GCRβ. It was demonstrated that GCRα nuclear translocation and its transactivation properties were proportionately reduced by level of viral transduction of the GCRβ gene into the DO-11.10 cell line. RNA silencing of GCRβ mRNA in human BAL macrophages from patients with GC-insensitive asthma resulted in enhanced dexamethasone-induced GCRα transactivation.
Conclusions: GC insensitivity is associated with loss of GCRα nuclear translocation in BAL cells and elevated GCRβ, which may inhibit GCRα transactivation in response to steroids.
PMCID: PMC2662945  PMID: 16387802
asthma; bronchoalveolar lavage cells; glucocorticoid insensitivity; glucocorticoid receptor
24.  Regulation of CYP3A genes by glucocorticoids in human lung cells 
F1000Research  2013;2:173.
Inhaled glucocorticoids are the first-line treatment for patients with persistent asthma.  However, approximately thirty percent of patients exhibit glucocorticoid insensitivity, which may involve excess metabolic clearance of the glucocorticoids by CYP3A enzymes in the lung.  CYP3A4, 3A5, and 3A7 enzymes metabolize glucocorticoids, which in turn induce CYP3A genes.  However, the mechanism of CYP3A5 mRNA regulation by glucocorticoids in lung cells has not been determined.  In hepatocytes, glucocorticoids bind to the glucocorticoid receptor (GR), which induces the expression of the constitutive androstane receptor or pregnane X receptor; both of which bind to the retinoid X receptor alpha, leading to the induction of CYP3A4, 3A5, and 3A7.  There is also evidence to suggest a direct induction of CYP3A5 by GR activation in liver cells. In this study, these pathways were evaluated as the mechanism for CYP3A5 mRNA induction by glucocorticoids in freshly isolated primary tracheal epithelial, adenocarcinomic human alveolar basal epithelial (A549), immortalized bronchial epithelial (BEAS-2B), primary normal human bronchial/tracheal epithelial (NHBE), primary small airway epithelial (SAEC), and primary lobar epithelial lung cells. In A549 cells, beclomethasone 17-monopropionate ([M1]) induced CYP3A5 mRNA through the glucocorticoid receptor. CYP3A5 mRNA induction by five different glucocorticoids was attenuated by inhibiting the glucocorticoid receptor using ketoconazole, and for beclomethasone dipropionate, using siRNA-mediated knock-down of the glucocorticoid receptor. The constitutive androstane receptor was not expressed in lung cells. SAEC cells, a primary lung cell line, expressed CYP3A5, but CYP3A5 mRNA was not induced by glucocorticoid treatment despite evaluating a multitude of cell culture conditions. None of the other lung cells expressed CYP3A4, 3A5 or 3A7 mRNA. These studies demonstrate that CYP3A5 mRNA is induced by glucocorticoids in A549 cells via the glucocorticoid receptor, but that additional undefined regulatory processes exist in primary lung cells.
PMCID: PMC3869485  PMID: 24555085
25.  Regulation of CYP3A genes by glucocorticoids in human lung cells 
F1000Research  2013;2:173.
Inhaled glucocorticoids are the first-line treatment for patients with persistent asthma.  However, approximately thirty percent of patients exhibit glucocorticoid insensitivity, which may involve excess metabolic clearance of the glucocorticoids by CYP3A enzymes in the lung.  CYP3A4, 3A5, and 3A7 enzymes metabolize glucocorticoids, which in turn induce CYP3A genes.  However, the mechanism of CYP3A5 mRNA regulation by glucocorticoids in lung cells has not been determined.  In hepatocytes, glucocorticoids bind to the glucocorticoid receptor (GR), which induces the expression of the constitutive androstane receptor or pregnane X receptor; both of which bind to the retinoid X receptor alpha, leading to the induction of CYP3A4, 3A5, and 3A7.  There is also evidence to suggest a direct induction of CYP3A5 by GR activation in liver cells. In this study, these pathways were evaluated as the mechanism for CYP3A5 mRNA induction by glucocorticoids in freshly isolated primary tracheal epithelial, adenocarcinomic human alveolar basal epithelial (A549), immortalized bronchial epithelial (BEAS-2B), primary normal human bronchial/tracheal epithelial (NHBE), primary small airway epithelial (SAEC), and primary lobar epithelial lung cells. In A549 cells, beclomethasone 17-monopropionate ([M1]) induced CYP3A5 mRNA through the glucocorticoid receptor. CYP3A5 mRNA induction by five different glucocorticoids was attenuated by inhibiting the glucocorticoid receptor using ketoconazole, and for beclomethasone dipropionate, using siRNA-mediated knock-down of the glucocorticoid receptor. The constitutive androstane receptor was not expressed in lung cells. SAEC cells, a primary lung cell line, expressed CYP3A5, but CYP3A5 mRNA was not induced by glucocorticoid treatment despite evaluating a multitude of cell culture conditions. None of the other lung cells expressed CYP3A4, 3A5 or 3A7 mRNA. These studies demonstrate that CYP3A5 mRNA is induced by glucocorticoids in A549 cells via the glucocorticoid receptor, but that additional undefined regulatory processes exist in primary lung cells.
PMCID: PMC3869485  PMID: 24555085

Results 1-25 (755392)