Regulation of cAMP-Dependent Gene Expression in Airway Epithelial Cells: A Transcriptional and Pharmacodynamic Analysis
Date
2019-08-02
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Abstract
Asthma and chronic obstructive pulmonary disease (COPD) are leading causes of death worldwide and their prevalence is predicted to increase over the next 20 years. Current treatments focus on providing symptomatic relief, with bronchodilators, and gaining disease control by suppressing inflammation. Long-acting β2-adrenoceptor agonists (LABAs) are effective bronchodilators that work by elevating cAMP in airway smooth muscle (ASM). Less appreciated and understood is the ability of cAMP to regulate gene expression. For example, previous studies have shown LABAs to protect ASM against pro-contractile stimuli by up-regulating RGS2, which attenuates signalling mediated through Gq-linked receptors. Based on these historical data, the first objective of the work reported here was to “mine” the LABA-regulated transcriptome for additional genes with disease-relevant functions. In the BEAS-2B airway epithelial cell line and in human primary bronchial epithelial cells (HBEC), the LABAs, indacaterol and salmeterol, promoted robust transcriptional responses. Functional annotation and gene ontology analyses identified genes with both anti-inflammatory and adverse effect potential. Moreover, there was significant overlap in gene expression changes between BEAS-2B and HBEC, and between the two LABAs. These observations suggested that pro- inflammatory gene expression changes could explain the known toxicity of chronic, β2- adrenoceptor agonist monotherapy initially reported in patients with asthma in the 1960s. Conversely, the expression of anti-inflammatory genes could help prevent exacerbations. Roflumilast is a phosphodiesterase (PDE) 4 inhibitor approved by the United States Food and Drug Administration as an ‘add-on’ therapy for patients with severe, bronchitic COPD with a history of frequent exacerbations. The second objective was to determine if inhibiting cAMP degradation could augment the expression of the LABA-regulated transcriptome and assess whether this could help explain the clinical activity of roflumilast. In BEAS-2B cells, roflumilast N-oxide (the active metabolite of roflumilast) augmented gene expression changes induced by a submaximal concentration of salmeterol, although the magnitude of the effect was dependent on the gene of interest. Further analysis established that this was because the sensitivity of the salmeterol-regulated transcriptome varied by a factor of 7.5-fold, which could be consistent with gene- dependent differences in regulation downstream of cAMP-dependent protein kinase. The final objective was to examine the pharmacology of GS-5759. This is a novel, bifunctional ligand composed a b2-adrenoceptor agonist linked to an analogue of the PDE4 inhibitor, GSK 256066, by a pent-1-yn-1ylbenzene spacer. Pharmacodynamic studies established that the PDE4 inhibitor pharmacophore produced a 35.5-fold increase in affinity for the b2-adrenoceptor relative to the monofunctional parent compound in the absence of any change in efficacy. However, this was not related to inhibition of PDE4 but to non-allosteric, ‘forced proximity’ binding, which predicts an increased in retention time of a compound near the orthosteric site of the receptor. In the context of obstructive lung diseases LABAs are established bronchodilators. The data presented in this thesis demonstrate that these drugs alone and in combination with a PDE4 inhibitor also have significant genomic actions. The possibility that gene expression changes occur in patients taking inhaled b2-adrenoceptor agonists and have functional consequences should be considered.
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Keywords
Transcription; cAMP; LABA; Pharmacology; COPD; Asthma; Forced Proximity
Citation
Yan, D. (2019). Regulation of cAMP-Dependent Gene Expression in Airway Epithelial Cells: A Transcriptional and Pharmacodynamic Analysis (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.