Contribution of cyclic AMP and β‐arrestin‐2‐dependent mechanisms to β2‐adrenoceptor‐mediated gene expression changes in human airway epithelial cells

Abstract

Long‐acting β2‐adrenoceptor agonists (LABAs) are mainstay drugs used routinely in asthma management. Our recent work revealed that β2‐adrenoceptor (β2‐AR) agonists promote a significant number of gene expression changes in BEAS‐2B airway epithelial cells that could contribute to their therapeutic activity and adverse effects (AEs) in asthma. In this study, we investigated the extent to which canonical (G‐protein/cAMP/protein kinase A [PKA]) and non‐canonical (β‐arrestin/extracellular signal‐regulated kinase [ERK]) signalling regulate gene expression in human primary airway epithelial cells (HpAECs) using formoterol and salmeterol as an example of a high and low efficacy LABA, respectively.Previous studies have shown that β‐arrestin‐2 is responsible for β2‐AR internalization and ERK1/2 phosphorylation. To investigate these functions in airway epithelial cells, we transfected BEAS‐2B cells with HA‐tagged β2‐AR and monitored internalization by confocal microscopy. When stimulated with formoterol or salmeterol, HA‐tagged β2‐AR redistributed from the plasma membrane to endosomes by a mechanism that was inhibited in cells deficient in β‐arrestin‐2. In BEAS‐2B cells and HpAECs, salmeterol (100nM) and formoterol (1nM) rapidly dephosphorylated basal ERK1/2 expression in a time‐dependent manner. This dephosphorylation was abolished in cells pre‐treated with a selective, β2‐AR antagonist, ICI‐118551 (1μM), and by overexpressing a PKA inhibitor transgene via adenoviral transduction with no evidence of ERK phosphorylation. Moreover, salmeterol‐ and formoterol‐induced ERK dephosphorylation was unaffected in BEAS‐2B cells lacking in β‐arrestin‐2.To evaluate the genomic effect of β2‐AR agonists, HpAECs from five normal subjects were treated with vehicle, formoterol (1nM), forskolin (FSK,10μM), tumor necrosis factor‐α (TNFα, 10ng/ml) and formoterol/TNFα or FSK/TNFα in combination for 1, 2, 6 and 18h. Total RNA was extracted, quantified, and sequenced on a NextSeq 500 instrument (Illumina). The sequenced reads were quantified by Kallisto and differential gene expression analysis performed using the R package, Sleuth. At a FDR of 5%, formoterol promoted a significant number of gene expression changes at both early and late time points; functional annotation clustering determined that many of these genes have both beneficial and AE potential. Formoterol also significantly enhanced or repressed many gene expression changes induced by TNFα at early and late time points. The gene expression profile induced by formoterol in HpAECs was replicated by FSK in the absence and presence of TNFα highlighting the cAMP dependency of this genomic effect.These data indicate that cAMP/PKA signalling plays a dominant role in regulating LABA‐induced gene expression changes in human airway epithelial cells. We submit that changes in gene expression through canonical signalling represents a previously unappreciated mechanism that contributes to the therapeutic activity and AEs of β2‐adrenoceptor agonists in asthma.Support or Funding InformationCanadian Institutes for Health Research (CIHR) ‐ The Lung Association of Alberta & NWTThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.