Roles of Gas and ß-arrestin-2 in the regulation of ß2-adrenoceptor-mediated ERK1/2 signaling and gene expression changes in human airway epithelial cells
Creators: name: Hamed, Omar
Publisher: name: Cumming School of Medicine
Year: 2021
Subjects: subject: beta-2-adrenoceptor
subject: ERK1/2
subject: beta-arrestin-2
subject: cAMP
subject: epithelial cells
subject: respiratory pharmacology
subject: airways
subject: Health Sciences--Pharmacology
subject: Health Sciences--Pharmacy

description: Chronic use of ?2-adrenoceptor agonists as a monotherapy in asthma is associated with a loss of disease control and an increased risk of mortality. In murine models of asthma, ?2-adrenoceptor agonists were proposed to stimulate G?s in airway smooth muscle to provide beneficial bronchodilation while exacerbate an asthma-like phenotype in airway epithelium via non-canonical, ?-arrestin 2 (?Arr2)-dependent ERK1/2 signaling. However, to date, no direct evidence supports this proposal. Therefore, in this thesis, we tested the hypothesis that ?2-adrenoceptor agonists, including formoterol, promote biased, ?Arr2-dependent activation of ERK1/2 signaling in human airway epithelial cells and, thereby, effect changes in gene expression that could contribute to their adverse clinical outcomes. Three airway epithelial cell models were used: the BEAS-2B cell line, human primary bronchial epithelial cells (HBEC) grown in submersion culture and HBEC that were highly differentiated at an air-liquid interface. Unexpectedly, treatment of all epithelial cell models with formoterol decreased basal ERK1/2 phosphorylation. This was mediated by cAMP-dependent protein kinase A (PKA) and involved the inactivation of C-rapidly-activated fibrosarcoma. Neither carvedilol, a ?2-adrenoceptor agonist biased towards ?Arr2, nor formoterol promoted ERK1/2 phosphorylation in BEAS-2B cells although ?2-adrenoceptor desensitization was compromised in ?Arr2-deficient cells. Analyses of RNA-sequencing data derived from formoterol- and forskolin-treated HBEC showed that gene expression changes (including adverse effect genes) were highly rank-order correlated indicating that they were dependent on cAMP. This was confirmed in BEAS-2B cells in which formoterol-induced inflammatory gene expression changes were inhibited in cells lacking PKA catalytic subunits but not ?Arr2. These findings are the antithesis of results obtained in murine models of asthma and in HEK293 cells and highlight the context and cell-type-dependence of ?2-adrenoceptor-mediated signaling. A context-related issue of particular interest was the divergent signaling that may occur after acute and chronic agonist exposure (i.e., ?2-adrenoceptor-mediated ERK1/2 inactivation and activation, respectively). This was addressed using HEK293 cells in which G?s knockdown and knockout revealed the necessity of G?s for ?2-adrenoceptor-mediated ERK1/2 activation. Collectively, these results show that G?s-dependent signaling was responsible for changes in ERK1/2 phosphorylation status (in airway epithelia and HEK293 cells) and inflammatory gene expression produced by ?2-adrenoceptor agonists and suggest a potential role of G?s in mediating their adverse effects in asthma.