RT Journal Article SR Electronic T1 β2-Adrenoceptor Agonists Promote ERK1/2 Dephosphorylation in Human Airway Epithelial Cells by Canonical, cAMP-driven Signaling Independently of β-Arrestin 2 JF Molecular Pharmacology JO Mol Pharmacol FD American Society for Pharmacology and Experimental Therapeutics SP MOLPHARM-AR-2021-000294 DO 10.1124/molpharm.121.000294 A1 Omar Hamed A1 Radhika Joshi A1 Aubrey N. Michi A1 Cora Kooi A1 Mark A. Giembycz YR 2021 UL http://molpharm.aspetjournals.org/content/early/2021/08/01/molpharm.121.000294.abstract AB Chronic use of β2-adrenoceptor agonists as a monotherapy in asthma is associated with a loss of disease control and an increased risk of mortality. Herein, we tested the hypothesis that β2-adrenoceptor agonists, including formoterol, promote biased, β-arrestin 2 (βArr2)-dependent activation of the mitogen-activated protein (MAP) kinases, ERK1/2, 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 and involved the inactivation of C-rapidly-activated fibrosarcoma, which attenuated down-stream ERK1/2 activity, and the induction of dual-specificity phosphatase-1. Formoterol also inhibited the basal expression of early growth response-1, an ERK1/2-regulated gene that controls cell growth and repair in the airways. Neither carvedilol, a β2-adrenoceptor agonist biased towards βArr2, nor formoterol promoted ERK1/2 phosphorylation in BEAS-2B cells although β2-adrenoceptor desensitization was compromised in ARRB2-deficient cells. Collectively, these results contest the hypothesis that formoterol activates ERK1/2 in airway epithelia by nucleating a βArr2 signaling complex; instead, they indicate that β2-adrenoceptor agonists inhibit constitutive ERK1/2 activity in a cAMP-dependent manner. These findings are the antithesis of results obtained using acutely challenged native and engineered HEK293 cells, which have been used extensively to study mechanisms of ERK1/2 activation, and highlight the cell-type-dependence of β2-adrenoceptor-mediated signaling. Significance Statement It has been proposed that the adverse-effects of β2-adrenoceptor agonist monotherapy in asthma are mediated by genomic mechanisms that occur principally in airway epithelial cells and are the result of β-arrestin 2-dependent activation of ERK1/2. This study shows that β2-adrenoceptor agonists, paradoxically, reduced ERK1/2 phosphorylation in airway epithelia by disrupting upstream Ras-C-Raf complex formation and inducing DUSP1. Moreover, these effects were PKA-dependent suggesting that β2-adrenoceptor agonists were not biased toward β-arrestin 2 and acted via canonical, cAMP-dependent signaling.