OXIDATION OF THE β2-ADRENERGIC RECEPTOR AND SIGNALING IN AIRWAY EPITHELIAL CELLS

Abstract

Asthma is a chronic airway inflammatory disease characterized by airway obstruction and acute bronchospasm, leading to symptoms such as difficulty breathing, coughing, and tightness in the chest. Although the etiology remains elusive, asthma is a heterogeneous disease with multifactorial pathogenesis, including the generation of reactive oxygen species (ROS) within the airway epithelium due to the upregulation of ROS generating enzyme NADPH oxidase (NOX), which exacerbates airway inflammation and contributes greatly to pathophysiology. While there is no panacea for asthma treatment, symptomatic relief is achieved by various therapeutic interventions, particularly β2-adrenergic receptor (β2AR) agonists, which produce airway relaxation, reduce bronchospasms and improve pulmonary function. Although β2-agonists remain the gold standard for the treatment of asthma, chronic administration can lead to loss of its bronchodilatory efficacy, a process termed β2-agonist tachyphylaxis, for which, no mechanistic explanation exists. Our laboratory has recently demonstrated that agonism of β2AR leads to robust formation of ROS, and that ROS are required for β2AR function, as inhibition of ROS abrogates both β2AR-mediated G-protein and β-arrestin signaling. Additionally, we have shown that β2AR is oxidized by ROS, forming reversible cysteine-S-sulfenic acids, which are required for proper receptor function. Given this, the first objective of the study was to determine the mechanisms of β2-agonist-dependent ROS generation, in particular H2O2 in primary airway epithelial cells derived from healthy and asthma-diseased patients, which endogenously express β2AR. Using H2O2-selective fluorescent chemical probes in conjugation with appropriate receptor and/or enzyme inhibitors, our results demonstrate that β2 agonists induce robust H2O2 production in a manner dependent on both β2AR and NOX. Further, given the highly oxidative nature of the asthmatic disease state, as well as β2-driven ROS generation, we aimed to investigate the role of β2AR-overoxidation from Cys-S-sulfenic acids to β2AR-Cys-S-sulfinic acids on β2AR signaling, and to test the hypothesis whether over-oxidation of β2AR to S-sulfinic acids contributes to clinical tachyphylaxis. Our results demonstrate that β2AR agonism induces overoxidation of β2AR leading to the formation of Cys-S-sulfinic acid oxomodifications. Overall, our findings address ROS-mediated functional alterations on β2AR by comparing the differences in β2AR signaling in airway epithelial cells derived from non-diseased and asthmatic patients and our results implicate that, higher-order irreversible oxidation states inhibit β2AR-mediated cAMP formation, which is critical for bronchodilation highlighting its role in clinically observed β2-agonist tolerance.