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 β2adrenoceptor 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 β2adrenoceptor 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 STATEMENTIt 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, cAMPdependent signaling.

α and β catalytic subunits of cAMP‐dependent protein kinase regulate formoterol‐induced inflammatory gene expression changes in human bronchial epithelial cells

“…Previously, we determined by western blotting that βArr2 is the predominant, non‐visual arrestin in BEAS‐2B cells with relatively little βArr1 (Hamed et al, 2021). In that study, we also reported using CRISPR/Cas9 genome editing with different sgRNAs the generation of three BEAS‐2B cell clones deficient in βArr2 (Hamed et al, 2021).…”

“…This resulted in a frameshift mutation and the generation of a premature stop codon. BEAS-2B cells deficient in βArr2 were generated as described previously (Hamed et al, 2021).…”

“…One example is the Elk-1-regulated gene, EGR1, which was repressed by formoterol in HBEC and BEAS-2B cells. Mechanistically, this seemingly occurs by the PKA-dependent phosphorylation and subsequent uncoupling of c-Raf from rat sarcoma such that constitutive, down-stream MEK1/2-ERK1/2-Elk-1 signalling is impaired (Hamed et al, 2021).…”

“…We have reported previously that neither carvedilol, a β 2 ‐adrenoceptor agonist biased toward βArr2 recruitment, nor the LABA, formoterol, promoted ERK1 or ERK2 phosphorylation in human primary bronchial epithelial cells (HBEC) (Hamed et al, 2021), which are known to be regulated by numerous upstream effectors including βArr2 (Luttrell & Gesty‐Palmer, 2010). Instead, LABAs suppressed constitutive ERK1/2 signalling via PKA‐dependent mechanisms that involved the inactivation of the MAPK kinase, C‐rapidly accelerated fibrosarcoma (c‐Raf) (Hamed et al, 2021). In the present study, we hypothesized that cAMP and not βArr2 mediates the genomic effects of β 2 ‐adrenoceptor agonists.…”

Long-Acting β2 Adrenergic Receptor Agonist Ameliorates Imiquimod-Induced Psoriasis-Like Skin Lesion by Regulating Keratinocyte Proliferation and Apoptosis

“…The inhibition of ERK relieved skin symptoms in a mouse imiquimod-induced model ( Wu et al, 2018 ; Huang X et al, 2019 ). A latest study reported that β 2 -AR agonists promote ERK dephosphorylation in human airway epithelial cells by cAMP-PKA signaling ( Hamed et al, 2021 ), although some earlier studies showed that PKA mediates the switch of coupling of β 2 -AR from Gs to Gi and initiate the activation of ERK pathway ( Daaka et al, 1997 ; Zamah et al, 2002 ; Smith et al, 2021 ). Thus, we examined ERK phosphorylation in psoriasis-like skin lesions.…”