β2-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 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, b-arrestin (Arr) 2-dependent activation of the mitogenactivated 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 β2-adrenoceptor agonist biased toward βArr2, nor formoterol promoted ERK1/2 phosphorylation in BEAS-2B cells, although β2-adrenoceptor desensitization was compromised in ARRβ2-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.