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Chronic β2AR stimulation limits CFTR activation in human airway epithelia
John J. Brewington, … , L. Jason Lu, John P. Clancy
John J. Brewington, … , L. Jason Lu, John P. Clancy
Published February 22, 2018
Citation Information: JCI Insight. 2018;3(4):e93029. https://doi.org/10.1172/jci.insight.93029.
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Research Article Cell biology Pulmonology

Chronic β2AR stimulation limits CFTR activation in human airway epithelia

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Abstract

Traditional pulmonary therapies for cystic fibrosis (CF) target the downstream effects of CF transmembrane conductance regulator (CFTR) dysfunction (the cause of CF). Use of one such therapy, β-adrenergic bronchodilators (such as albuterol), is nearly universal for airway clearance. Conversely, novel modulator therapies restore function to select mutant CFTR proteins, offering a disease-modifying treatment. Recent trials of modulators targeting F508del-CFTR, the most common CFTR mutation, suggest that chronic β-agonist use may undermine clinical modulator benefits. We therefore sought to understand the impact of chronic or excess β-agonist exposure on CFTR activation in human airway epithelium. The present studies demonstrate a greater than 60% reduction in both wild-type and modulator-corrected F508del-CFTR activation following chronic exposure to short- and long-acting β-agonists. This reduction was due to reduced cellular generation of cAMP downstream of the β-2 adrenergic receptor–G protein complex. Our results point towards a posttranscriptional reduction in adenylyl cyclase function as the mechanism of impaired CFTR activation produced by prolonged β-agonist exposure. β-Agonist–induced CFTR dysfunction was sufficient to abrogate VX809/VX770 modulation of F508del-CFTR in vitro. Understanding the clinical relevance of our observations is critical for CF patients using these drugs, and for investigators to inform future CFTR modulator drug trials.

Authors

John J. Brewington, Jessica Backstrom, Amanda Feldman, Elizabeth L. Kramer, Jessica D. Moncivaiz, Alicia J. Ostmann, Xiaoting Zhu, L. Jason Lu, John P. Clancy

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Figure 6

Chronic albuterol exposure reduces cell surface β2AR, but not CFTR, localization in wtCFTR+ and VX809-corrected F508del-CFTR+ primary human airway epithelial cells (HAECs).

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Chronic albuterol exposure reduces cell surface β2AR, but not CFTR, loca...
wtCFTR+ and VX809-corrected F508del-CFTR+ HAECs were exposed to albuterol (10 μM) for 72 hours. Cells were then fixed and immunofluorescence for CFTR (green), F-actin (red), and β-2 adrenergic receptor (β2AR) (purple) was performed. Colocalization analyses were performed to determine the colocalization of CFTR and β2AR at the cell surface (manually delineated) and the entire captured images. Colocalization analysis included both single images in the x-y and x-z planes (shown) and 3-dimensional reconstruction images in the x-y-z plane. wtCFTR+ HAECs are shown under control conditions (A) and with albuterol pretreatment (B), demonstrating reduced apical β2AR staining with albuterol pretreatment. Colocalization analysis of a manually delineated apical surface segment or the native images (C and D, respectively; n = 3), confirms reduced colocalization of wtCFTR and β2AR following albuterol exposure. Similarly, VX809-corrected F508del-CFTR+ HAECs are shown in the absence (E) or presence of albuterol (F). Equivalent colocalization analysis again demonstrates reduced CFTR/β2AR colocalization in both the apical surface (G) and the entire images (H); n = 3 for both analyses. Data presented represent the mean ± SEM. Scale bars: 25 μm. *P < 0.05; **P < 0.01 by 2-tailed t test.

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