β 2-Adrenoceptor Agonists Promote Extracellular Signal-Regulated Kinase 1/2 Dephosphorylation in Human Airway Epithelial Cells by Canonical, cAMP-Driven Signaling Independently of β-Arrestin 2.

Abstract

Chronic use of β 2-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 β 2-adrenoceptor agonists, including formoterol, promote biased, β-arrestin (Arr) 2-dependent activation of the mitogen-activated protein 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 downstream 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 β 2-adrenoceptor agonist biased toward βArr2, nor formoterol promoted ERK1/2 phosphorylation in BEAS-2B cells, although β 2-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 β 2-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 β 2-adrenoceptor-mediated signaling. SIGNIFICANCE STATEMENT: It has been proposed that the adverse effects of β 2-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 β 2-adrenoceptor agonists, paradoxically, reduced ERK1/2 phosphorylation in airway epithelia by disrupting upstream rat sarcoma-C-rapidly accelerated fibrosarcoma complex formation and inducing dual-specificity phosphatase 1. Moreover, these effects were cAMP-dependent protein kinase-dependent, suggesting that β 2-adrenoceptor agonists were not biased toward β-arrestin 2 and acted via canonical, cAMP-dependent signaling.