Browsing by Author "Newton, Robert"
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Item Open Access A bronchoprotective role for Rgs2 in a murine model of lipopolysaccharide-induced airways inflammation(2018-10-01) George, Tresa; Chakraborty, Mainak; Giembycz, Mark A; Newton, RobertAbstract Background Asthma exacerbations are associated with the recruitment of neutrophils to the lungs. These cells release proteases and mediators, many of which act at G protein-coupled receptors (GPCRs) that couple via Gq to promote bronchoconstriction and inflammation. Common asthma therapeutics up-regulate expression of the regulator of G protein signalling (RGS), RGS2. As RGS2 reduces signaling from Gq-coupled GPCRs, we have defined role(s) for this GTPase-activating protein in an acute neutrophilic model of lung inflammation. Methods Wild type and Rgs2−/− C57Bl6 mice were exposed to nebulized lipopolysaccharide (LPS). Lung function (respiratory system resistance and compliance) was measured using a SCIREQ flexivent small animal ventilator. Lung inflammation was assessed by histochemistry, cell counting and by cytokine and chemokine expression in bronchoalveolar lavage (BAL) fluid. Results Lipopolysaccharide inhalation induced transient airways hyperreactivity (AHR) and neutrophilic lung inflammation. While AHR and inflammation was greatest 3 h post-LPS exposure, BAL neutrophils persisted for 24 h. At 3 h post-LPS inhalation, multiple inflammatory cytokines (CSF2, CSF3, IL6, TNF) and chemokines (CCL3, CCL4, CXCL1, CXCL2) were highly expressed in the BAL fluid, prior to declining by 24 h. Compared to wild type counterparts, Rgs2−/− mice developed significantly greater airflow resistance in response to inhaled methacholine (MCh) at 3 h post-LPS exposure. At 24 h post-LPS exposure, when lung function was recovering in the wild type animals, MCh-induced resistance was increased, and compliance decreased, in Rgs2−/− mice. Thus, Rgs2−/− mice show AHR and stiffer lungs 24 h post-LPS exposure. Histological markers of inflammation, total and differential cell counts, and major cytokine and chemokine expression in BAL fluid were similar between wild type and Rgs2−/− mice. However, 3 and 24 h post-LPS exposure, IL12B expression was significantly elevated in BAL fluid from Rgs2−/− mice compared to wild type animals. Conclusions While Rgs2 is bronchoprotective in acute neutrophilic inflammation, no clear anti-inflammatory effect was apparent. Nevertheless, elevated IL12B expression in Rgs2−/− animals raises the possibility that RGS2 could dampen Th1 responses. These findings indicate that up-regulation of RGS2, as occurs in response to inhaled corticosteroids and long-acting β2-adrenoceptor agonists, may be beneficial in acute neutrophilic exacerbations of airway disease, including asthma.Item Open Access A Pharmacodynamics Analysis of Glucocorticoid Receptor-Mediated Gene Expression in BEAS-2B Human Airway Epithelial Cells(2016) Joshi, Taruna; Giembycz, Mark; Newton, Robert; McKay, Derek; von der Weid, Pierre; Hirota, Simon; Cameron, LisaInternational treatment guidelines recommend that inhaled glucocorticoids be used as a monotherapy to control mild-to-moderate asthma. However, a combination of an inhaled glucocorticoid with a long-acting β2-adrenoceptor agonist is recommended for managing moderate-to-severe asthma, which cannot be controlled by glucocorticoids alone. Increasing evidence supports the idea that glucocorticoids acting through the glucocorticoid receptor (GR) can attenuate inflammatory responses either by inducing anti-inflammatory genes (a process called transactivation) or by suppressing pro-inflammatory genes (a process called transrepression). Gene transactivation by glucocorticoids was initially thought to be responsible for causing the metabolic side-effects and, hence, is relatively understudied when compared to transrepression. However, it has become increasingly clear that transactivation plays an important role in the anti-inflammatory actions of glucocorticoids. Moreover, the extent to which clinically-relevant glucocorticoids are equivalent in their ability to promote gene expression is unclear. This thesis describes the first pharmacodynamic approach to evaluate the transactivation potential of a panel of glucocorticoids alone and in combination with indacaterol (Ind), a long-acting β2-adrenoceptor agonist. Pharmacodynamic analyses showed that magnitude of luciferase gene induction was agonist dependent (i.e. seven different glucocorticoids tested displayed varying degrees of agonism). In addition, there were significant differences in agonist potency and, more importantly, the relationship between GR occupancy and response. To complement the reporter studies, similar analyses were performed on four glucocorticoid-inducible candidate genes. Three of these (GILZ, p57kip2 and CRISPLD2) are genes with potential anti-inflammatory activity and a fourth gene, PDK4 is predicted to promote metabolic side-effects. Similar to the luciferase reporter system, the expression of these genes was agonist-dependent and displayed markedly different GR occupancy-response relationships. Furthermore, Ind, when combined with the seven GR agonists tested, synergistically enhanced transactivation, the magnitude of which was agonist and gene dependent. These studies demonstrate that when gene transactivation is used as a functional output, glucocorticoids used to treat asthma are not biologically-equivalent. It is proposed that these differences may be exploited to therapeutic advantage. Thus, the generation of gene expression ‘fingerprints’ in target and off-target human tissues may allow new GR agonists to be rationally designed for asthma with an improved therapeutic index.Item Open Access Characterizing the role of NR4A1 in the regulation of intestinal smooth muscle cell phenotype and function(2021-04-06) Szczepanski, Holly Elizabeth; Hirota, Simon Andrew; Newton, Robert; von der Weid, Pierre-YvesIntestinal fibrosis and stricture formation are common complications of Crohn’s disease (CD). Recently, smooth muscle hypertrophy and hyperplasia have gained greater recognition as a driver of stricture formation, rather than an increase in fibrosis alone. Despite advances in treatment of CD, current therapies do little to prevent or reverse strictures. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an orphan nuclear receptor (NR) that is anti-fibrotic in non-intestinal systems and exhibits anti-proliferative effects in smooth muscle cells (SMCs). NR4A1 gene variants have been associated with increased risk of IBD, however, mechanisms regulating NR4A1 expression and its role in intestinal SMC function have not been investigated. We therefore hypothesized that presence and activation of NR4A1 regulates cell proliferation and metabolism by modulating intestinal SMC phenotype. Primary intestinal SMCs isolated from Nr4a1+/+ and Nr4a1-/- mice and a commercially sourced human primary intestinal SMC line were used as in vitro models. We employed the chronic model of dextran sulfate sodium (DSS) and the SAMP1/YitFc model of spontaneous ileitis as in vivo models. In alignment with our hypothesis, NR4A1 presence and exposure to identified agonists, cytosporone-B (Csn-B) and 6-mercaptopurine (6-MP), regulated key cellular mechanisms involved in excessive smooth muscle hypertrophy and hyperplasia. NR4A1 expression was significantly induced in human colonic SMCs by platelet-derived growth factor (PDGF)-BB suggesting a potential negative feedback mechanism to control mitogen-induced SMC proliferation. Taken together, in this study, NR4A1 is shown to be a vital brake in intestinal SMC phenotypic modulation by limiting excessive proliferation and other associated characteristics that could contribute to pathogenic tissue remodelling observed in fibrostenotic CD.Item Open Access Evidence for anti-inflammatory properties of glucocorticoid-inducible genes in the airways(2011) King, Elizabeth Mary; Newton, RobertItem Open Access Expanding Allergen-specific Tr-1 CD4+ T cells to treat Allergic Asthma(2017) Chakraborty, Mainak; Santamaria, Pere; Mody, Christopher; Newton, Robert; Giembycz, MarkAsthma is a chronic inflammatory disease of the lungs, which is one of the most frequent chronic disease in industrialized nations. The associated morbidity and occasional mortality rates are secondary to an underlying dysfunction of the airway driven by immune-mediated inflammation. Our lab has been developing a nanoparticle based therapy to deliver antigen-specific peptide-major histocompatibility complex to treat autoimmunity, which triggers the induction and expansion of cognate autoregulatory CD4+ Tr1-like cells that suppresses the autoimmune response via secretion of anti-inflammatory cytokines such as IL-10 and TGF-β. We tested this therapeutic avenue in a chronic model of asthma in BALB/c mice, and found that the pMHC-NP therapy triggers allergen-specific Tr-1 cell formation and expansion that can suppress inflammation, promoting the resolution of airways hyper-responsiveness and airway remodeling. Asthma-relevant pMHC class II-coated NPs may therefore represent a viable alternative to current approaches to restore immune homeostasis in allergic individuals.Item Open Access Exploring the combinatorial effects of inflammatory stimuli and glucocorticoids on BIRC3 and BIRC2 expression: Differential regulation and functional investigations(2024-01-16) Thorne, Andrew James; Newton, Robert; Hirota, Simon; Yipp, BryanInflammation serves as a crucial innate mechanism in response to injury, infection, or harmful stimuli that activates the immune system and aims to restore homeostasis. Chronic inflammatory disorders like asthma present with ongoing inflammation whereby pulmonary epithelial cells release inflammatory mediators, intensifying airway inflammation, and worsening disease. Synthetic glucocorticoids represent the primary treatment for asthma by utilizing the anti-inflammatory properties of their endogenous counterparts to reduce inflammatory mediators. Their mechanism of action involves binding to the glucocorticoid receptor (GR) in cells and interacting with DNA elements to enhance anti-inflammatory gene expression. However, certain inflammatory genes play protective roles by promoting tissue repair or self-regulatory feedback mechanisms to limit inflammatory signaling. Some of these genes are spared the repressive effects of glucocorticoids and are even augmented, presumably for protection. The BIRC3 gene, while being associated with the inflammatory NF-κB pathway, is a gene upregulated in models of the pulmonary epithelium and in vivo by glucocorticoids. BIRC3, along with its family member BIRC2, may contribute to regulating NF-κB pathways, but also inhibit apoptosis, although their specific roles remain unclear. Consequently, the rationale of glucocorticoid-enhanced BIRC3 remain ambiguous. Given their similarities, BIRC3 and BIRC2 were both investigated, focusing on their expression, regulation, and the interactions between the GR and NF-κB at the gene promoter. These analyses detailed robust expression patterns differentiating BIRC3 and BIRC2 by their response to inflammatory cytokines and glucocorticoids over time. Promoter analysis revealed insights into possible GR and NF-κB interactions to explain the regulation of BIRC3 expression, thus providing an example of GR-NF-κB positive interactions leading to inflammatory gene expression. Further, investigations into the NF-κB pathway as a platform for BIRC2/3 function revealed the established canonical NF-κB pathway may challenge established dogma. This exploratory research highlighted the significance of TAK1 as central kinase within the NF-κB pathway. Moreover, possible redundancy between IKK1 and IKK2, and the presence of additional regulators within canonical NF-κB signaling may exist. While this study provided no greater insights for redundant, or individual roles for BIRC2/3 in NF-κB signaling or apoptosis, it facilitated the development of tools for future investigations.Item Open Access Glucocorticoid-driven transcriptomes in human airway epithelial cells: commonalities, differences and functional insight from cell lines and primary cells(2019-01-31) Mostafa, Mahmoud M; Rider, Christopher F; Shah, Suharsh; Traves, Suzanne L; Gordon, Paul M K; Miller-Larsson, Anna; Leigh, Richard; Newton, RobertAbstract Background Glucocorticoids act on the glucocorticoid receptor (GR; NR3C1) to resolve inflammation and, as inhaled corticosteroids (ICS), are the cornerstone of treatment for asthma. However, reduced efficacy in severe disease or exacerbations indicates a need to improve ICS actions. Methods Glucocorticoid-driven transcriptomes were compared using PrimeView microarrays between primary human bronchial epithelial (HBE) cells and the model cell lines, pulmonary type II A549 and bronchial epithelial BEAS-2B cells. Results In BEAS-2B cells, budesonide induced (≥2-fold, P ≤ 0.05) or, in a more delayed fashion, repressed (≤0.5-fold, P ≤ 0.05) the expression of 63, 133, 240, and 257 or 15, 56, 236, and 344 mRNAs at 1, 2, 6, and 18 h, respectively. Within the early-induced mRNAs were multiple transcriptional activators and repressors, thereby providing mechanisms for the subsequent modulation of gene expression. Using the above criteria, 17 (BCL6, BIRC3, CEBPD, ERRFI1, FBXL16, FKBP5, GADD45B, IRS2, KLF9, PDK4, PER1, RGCC, RGS2, SEC14L2, SLC16A12, TFCP2L1, TSC22D3) induced and 8 (ARL4C, FLRT2, IER3, IL11, PLAUR, SEMA3A, SLC4A7, SOX9) repressed mRNAs were common between A549, BEAS-2B and HBE cells at 6 h. As absolute gene expression change showed greater commonality, lowering the cut-off (≥1.25 or ≤ 0.8-fold) within these groups produced 93 induced and 82 repressed genes in common. Since large changes in few mRNAs and/or small changes in many mRNAs may drive function, gene ontology (GO)/pathway analyses were performed using both stringency criteria. Budesonide-induced genes showed GO term enrichment for positive and negative regulation of transcription, signaling, proliferation, apoptosis, and movement, as well as FOXO and PI3K-Akt signaling pathways. Repressed genes were enriched for inflammatory signaling pathways (TNF, NF-κB) and GO terms for cytokine activity, chemotaxis and cell signaling. Reduced growth factor expression and effects on proliferation and apoptosis were highlighted. Conclusions While glucocorticoids repress mRNAs associated with inflammation, prior induction of transcriptional activators and repressors may explain longer-term responses to these agents. Furthermore, positive and negative effects on signaling, proliferation, migration and apoptosis were revealed. Since many such gene expression changes occurred in human airways post-ICS inhalation, the effects observed in cell lines and primary HBE cells in vitro may be relevant to ICS in vivo.Item Open Access Investigation into the Bronchoprotective and Anti-inflammatory Properties of RGS2(2016) George, Tresa; Newton, Robert; Giembycz, Mark; Kelly, MargaretThe clinical management of asthma involves treatment with inhaled corticosteroids (ICS) and long-acting β2-adrenoceptor agonists (LABAs). Regulator of G-protein signalling (RGS) 2 inhibits signalling from Gq protein-coupled receptors. In humans, combinations of a glucocorticoid and a LABA synergistically enhanced the expression of RGS2. In mice, 3 weeks of HDM challenge or 30 min of LPS challenge reduced lung function and induced airways inflammation. Compared to wild-type, Rgs2-/- mice showed significantly increased airways resistance and reduced compliance, in both HDM- and LPS-challenges. There was no difference between wild-type and Rgs2-/- mice in HDM-induced and LPS-induced inflammation. There was a trend towards increased inflammatory cell counts in the BALF of Rgs2-/- compared to wild-type. There was a trend towards increased expression for many HDM-induced cytokines/chemokines in Rgs2-/-. There was no difference between wild-type and Rgs2-/- mice in LPS-induced expression of cytokines/chemokines. These data show Rgs2 is bronchoprotective in HDM-induced inflammation and suggest a modest anti-inflammatory role. Rgs2 is bronchoprotective in LPS-induced inflammation, but more studies are required to address a possible anti-inflammatory role. If applicable to humans, these data suggest that therapeutics, for example ICS/LABA combination therapies, which are designed to maximize RGS2 expression, will be beneficial for asthma control and management.Item Open Access Modulation of glucocorticoid-inducible gene expression: Effects of inflammatory stimuli and long-acting β2-adrenoceptor agonists(2014-09-15) Rider, Christopher Francis; Newton, Robert; Giembycz, Mark AnthonyGlucocorticoids, acting on the glucocorticoid receptor (GR; NR3C1), are the most effective medication for controlling inflammation in the majority of asthmatics. However, some asthmatics, particularly those with severe disease, have reduced responses to glucocorticoids, a condition known as glucocorticoid resistance. Likewise, glucocorticoids have reduced effectiveness in asthmatics who smoke and during exacerbations, which are frequently induced by human rhinovirus (HRV) infection. Glucocorticoids reduce inflammatory protein production by directly inhibiting inflammatory transcription factors, including nuclear factor-kappa B (NF-κB) and by enhancing the expression of potentially anti-inflammatory genes (transactivation). The importance of transactivation in glucocorticoid activity is increasingly apparent, but the impact of inflammatory mediators on anti-inflammatory gene expression is understudied. Human bronchial epithelial, BEAS-2B, cells stably transfected with a 2×glucocorticoid response element (GRE) reporter system, which models glucocorticoid-inducible gene expression, demonstrate concentration-dependent activation by glucocorticoids. However, dexamethasone-induced 2×GRE activation was time-dependently reduced by pre-treatment with inflammatory mediators, including tumor necrosis factor (TNF), interleukin-1β (IL1B) and cigarette smoke extract (CSE). Furthermore, TNF pre-treatment decreased dexamethasone-induced mRNA expression of genes with potentially anti-inflammatory activity, including cyclin dependent kinase inhibitor 1C (CDKN1C) and TSC22 domain family protein 3 (TSC22D3/GILZ), in bronchial epithelial and airway smooth muscle cells. Likewise, pre-incubations with HRV or the synthetic double-stranded viral RNA mimetic polyinosinic:polycytidylic acid (poly(I:C)) reduced dexamethasone-induced 2×GRE activation. Poly(I:C) also reduced dexamethasone-induced CDKN1C expression. Approaches to reverse TNF-induced glucocorticoid hyporesponsiveness were evaluated, including addition of long-acting β2-adrenoceptor agonists (LABAs), use of novel GR agonists and inflammatory signalling pathway inhibition. LABAs, such as formoterol, potentiated 2×GRE reporter activation and CDKN1C expression through a time- and PKA-dependent mechanism that did not enhance GR expression, agonist affinity or translocation, but instead allows for gene specific control. Therefore, LABA addition functionally reversed glucocorticoid hyporesponsiveness induced by TNF or poly(I:C). However, novel GR agonist-induced 2×GRE activation was repressed by TNF or poly(I:C) treatment, with the degree of repression correlating with agonist efficacy. Finally, inhibition of the NF-κB and c-Jun N-terminal kinase (JNK) mitogen activated protein kinase pathways partially reversed TNF-induced glucocorticoid hyporesponsiveness. These results may contribute to the development of improved treatments for combating glucocorticoid hyporesponsiveness during exacerbations and in severe asthma.Item Open Access Molecular mechanisms of anti-inflammatory glucocorticoid action: Evidence for a role of glucocorticoid-inducible genes and implications for glucocorticoid insensitivity(2016) Shah, Suharsh Vinaykumar; Newton, Robert; Proud, David; MacNaughton, WallaceThe anti-inflammatory activity of glucocorticoids, in diseases such as asthma, is attributed to their ability to reduce the expression of multiple inflammatory mediators. While glucocorticoid receptor (NR3C1)-mediated transcriptional activation, or transactivation, was believed to primarily mediate side-effects, accumulating evidence indicates that transactivation is also important for the inhibition of inflammatory gene expression. This illustrates the need to investigate possible functional roles for specific glucocorticoid-inducible genes. In this thesis, a repressive role for DUSP1, a phosphatase that inhibits MAPKs, was investigated. DUSP1 was induced by IL1B and dexamethasone in both human pulmonary A549 and primary HBE cells. IL1B-induced DUSP1 negatively regulated MAPK activity and in the further presence of dexamethasone, DUSP1 played a transient, typically partial, role in repressing expression of inflammatory genes, including CXCL1, CXCL2 and PTGS2. Thus, additional glucocorticoid-induced gene products are necessary for repression. Regulation of the mRNA destabilizing protein, ZFP36, by DUSP1 was examined. Following the loss of DUSP1, ZFP36 expression was enhanced and this attenuated IL1B-induced TNF expression. Despite a modest ability of dexamethasone to induce ZFP36, thereby off-setting loss of ZFP36 due to reduced MAPK activity, neither silencing of dexamethasone-induced ZFP36, DUSP1, nor both together, prevented repression of TNF by dexamethasone. Finally, while DUSP1 over-expression attenuated IL1B-induced expression of many inflammatory genes (e.g. IL8, CSF2 etc.), others, including, the inflammatory transcription factor, IRF1, and downstream target genes, such as CXCL10, were profoundly enhanced. While MAPK inhibition prolonged IRF1 expression, silencing of IL1B plus dexamethasone-induced DUSP1 reduced IRF1 and CXCL10 expression. Since, CXCL10 expression was largely unaffected by dexamethasone, these data suggest a mechanism whereby dexamethasone-induced DUSP1 expression maintains CXCL10 expression. In conclusion, this study demonstrates interlinked counterregulatory networks, in which IL1B-induced DUSP1 and ZFP36 regulate MAPK activation and inflammatory gene expression respectively. The transient and partial repressive effect of dexamethasone-induced DUSP1 on only few IL1B-induced inflammatory genes provides a rational for functional screening of glucocorticoid-inducible genes that may show repressive functions. Furthermore, by switching off MAPKs, DUSP1 maintains IRF1 expression and may contribute to glucocorticoid insensitivity.Item Open Access New perspectives on G-protein coupled receptor signaling in the pregnant myometrium: Exploring the role of regulator of G-protein signaling 2(2024-01) Urrego, Daniela; Slater, Donna; Newton, Robert; Wood, Stephen; Cole, WilliamPrematurity at birth results in neonatal death, or lifelong disability. Early deliveries due to preterm labour may be preventable but remain a significant problem as there are no treatments to reliably stop preterm labour. The ineffectiveness of existing treatments for preterm labour are possibly owing to targeting a limited variety of pro-labour signals that are believed to participate in uterine contractions, such as oxytocin and prostaglandins. Interestingly, prostaglandins (e.g. PGE2) also have functions that may counteract labour activation and maintain uterine quiescence during pregnancy, and these mechanisms may be important to consider in the search for new therapies for preterm labour. This thesis aimed to explore whether the regulator of G protein signaling 2 (RGS2) is involved in the mechanism by which certain prostaglandins suppress uterine contractility. RGS2 functions by inhibiting signals from some pro-contractile G protein-coupled receptors, and it is clinically relevant in the function of other smooth muscle organs. We hypothesized that prostaglandins and glucocorticoids (relevant in the treatment of preterm labour) increase RGS2 expression, and consequently reduce the myometrial pro-contractile response to GPCR agonists like oxytocin. Conversely, pro-inflammatory cytokines implicated in labour were expected to reduce RGS2 expression. We anticipated conditions under which RGS2 expression was reduced would have an opposite effect, thereby increasing uterine contractility. First, we investigated whether the expression of RGS2 in human myometrial smooth muscle (MSM) cells is regulated by mediators relevant to labour including prostaglandins, glucocorticoids, and pro-inflammatory cytokines. Prostaglandin E2 enhanced RGS2 mRNA expression, and the addition of glucocorticoids prolonged this effect. Additionally, PGE2 rescued RGS2 expression that was suppressed by pro-inflammatory cytokines. To understand the function of RGS2 in the uterus, sought to understand the diversity of pro-contractile receptors present in the human myometrium towards the end of pregnancy since these may be acted upon by RGS2. In addition to the well-known highly expressed oxytocin receptor, there were high levels of expression of histamine receptor 1 which is known to have a pro-contractile effect dampened by RGS2 in other smooth muscle. Based on this we selected histamine and oxytocin to study the effects of RGS2 on pro-contractile signaling. In MSM cells prostaglandin- and glucocorticoid-mediated increases in RGS2 failed to significantly reduce either histamine or oxytocin-induced calcium signals which were taken as a measure of contractile activity. The histamine, but not the oxytocin calcium response was reduced by adenoviral overexpression of RGS2 in MSM cells. We next investigated whether a reduction in RGS2 expression increased uterine contractility. We utilized a total Rgs2-knockout mouse model and compared uterine contractility of tissues and cells to that of wildtype mice. The absence of RGS2 increased overall contractility in knockout tissues and in freshly dispersed uterine cells the exaggerated response to oxytocin. Furthermore, the relaxant effects of PGE2 on uterine tissue contractility were in part RGS2-dependent, as they were diminished in knockout tissues. Despite the effects of Rgs2-knockout on isolated myometrial tissues and cells, knockout mice did not undergo labour earlier overall. Studying the effect of RGS2 on the timing of labour in humans is difficult since samples are obtained only at the time of delivery and/or labour. Nevertheless, we found that RGS2 was decreased with preterm but not term labour, implicating different mechanisms that give rise to labour in each setting. Whether the reduction of RGS2 seen in preterm labour gives rise to increased uterine contractility, as we observed in the Rgs2-knockout mouse uterus, remains to be determined. In conclusion we demonstrate that RGS2 expression is regulated by PGE2 and that its function appears to involve the suppression of uterine contractility, as demonstrated through both loss and gain of function experiments in complementary models of study. The effects of RGS2 on the receptors that elicit uterine contractility in labour should be more carefully studied, considering a wider variety of receptors acting in concert. As well, the effects of RGS2 on mouse pregnancies can later be examined to determine whether perturbations in RGS2 expression leave the uterus vulnerable to preterm labour.Item Open Access The Effect of Phosphodiesterase Inhibitors on the Induction of Gene Expression by Long-Acting beta2-Adrenoceptor Agonists and Glucocorticoids(2013-09-13) BinMahfouz, Hawazen; Newton, Robert; Giembycz, MarkRecently, phosphodiesterases (PDE)4 inhibitors have received approval as a complementary anti-inflammatory treatment for chronic obstructive pulmonary disease (COPD) patients who are already taking long-acting β2-agonists (LABA)/inhaled corticosteroid (ICS) combination therapy. However, this benefit is seen only in patients of the severe, bronchitic, frequent exacerbator phenotype. Several strategies have been proposed to enhance the clinical efficacy of PDE4 inhibitors. One of these is the use of dual PDE3/4 inhibitors, which in addition to providing superior anti-inflammatory activity when compared to a PDE4 inhibitor alone, will also promote bronchodilatation. The present study demonstrates that a PDE3 plus a PDE4 inhibitor successfully sensitized BEAS-2B airway epithelial cells transfected with a cyclic adenosine 3',5'-monophosphate (cAMP)-response element luciferase reporter to the LABA, formoterol. Furthermore, PDE3 plus PDE4 inhibitors in combination prolonged and/or sensitized the ability of formoterol to induce several genes in BEAS-2B cells that have anti-inflammatory potential in the absence and presence of the glucocorticoid, dexamethasone. Collectively, these data suggest that LABA/ICS combination therapy in conjunction with an inhibitors of PDE3 and PDE4 may together improve clinical outcomes in larger a population of severe COPD patients.Item Open Access Transcriptional regulation by glucocorticoids: a comparative transcriptome analysis preceding mechanistic and functional assessments of KLF9 induction(2020-09-15) Mostafa, Mahmoud; Newton, Robert; Giembycz, Mark A.; Cobb, Jennifer A.Regulation of gene expression by glucocorticoid receptor (GR) is not only central to numerous endocrine processes, but also critical for the control of inflammation by glucocorticoids administered for the treatment of inflammatory diseases, including asthma. Advancing our knowledge of glucocorticoid biology as well as developing GR ligands with improved therapeutic profile are hindered by an inadequate understanding of the mechanisms and impacts of gene regulation by glucocorticoids. Furthermore, owing to their pleiotropic effects on many different target cells, transcriptomic responses to glucocorticoids vary widely between cell types. This confounds the identification of key glucocorticoid-regulated genes. In this thesis, transcriptomic responses to glucocorticoid were compared between A549, BEAS-2B, and primary HBE cells. These are models for airway epithelial cells, which represent a key player in asthma pathogenesis and response to inhaled glucocorticoid therapies. While the variability in glucocorticoid-modulated gene expression between epithelial cell variants was surprisingly high, the genes regulated in common may represent key players in eliciting glucocorticoid effects. Among the genes that were induced in common, transcriptional regulators, such as KLF9, were highly enriched and hypothesized to contribute to later gene expression changes, including repression of inflammatory genes. Mechanistically, ligand-activated GR binds and activates multiple conserved enhancer regions upstream of the KLF9 gene. These include a promoter proximal region that was appeared to be essential for the constitutive expression and glucocorticoid-mediated induction of KLF9. In fact, GR recruitment and transcriptional activation at KLF9 upstream enhancers were highly conserved among cell lines and primary cells, demonstrating reliability of cell lines in mechanistic interrogations of conserved genes. Assessment of KLF9 functions was attempted via transcriptome analysis of CRISPR-edited KLF9 KO lines. While procedural artifacts hindered the identification of transcriptomic impacts of KLF9 induction, candidate gene investigations following overexpression or knockdown of KLF9 suggested a role for the constitutively expressed KLF9 in limiting the expression of AKAP12 and RGS2. Yet, the functional impacts of glucocorticoid-induced KLF9 in pulmonary epithelial cells remain undetermined. Collectively, these results establish a platform for identifying key glucocorticoid-regulated genes in the airways, and guiding the selection of cell line models for in vitro investigations of such genes.