Clinically approved CFTR modulators rescue Nrf2 dysfunction in cystic fibrosis airway epithelia
Dana C. Borcherding, … , Scott M. Plafker, Assem G. Ziady
Dana C. Borcherding, … , Scott M. Plafker, Assem G. Ziady
Published May 30, 2019
Citation Information: J Clin Invest. 2019;129(8):3448-3463. https://doi.org/10.1172/JCI96273.
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Clinically approved CFTR modulators rescue Nrf2 dysfunction in cystic fibrosis airway epithelia

Abstract

Cystic fibrosis (CF) is a multiorgan progressive genetic disease caused by loss of functional cystic fibrosis transmembrane conductance regulator (CFTR) channel. Previously, we identified a significant dysfunction in CF cells and model mice of the transcription factor nuclear factor E2–related factor-2 (Nrf2), a major regulator of redox balance and inflammatory signaling. Here we report that the approved F508del CFTR correctors VX809 and VX661 recover diminished Nrf2 function and colocalization with CFTR in CF human primary bronchial epithelia by proximity ligation assay, immunoprecipitation, and immunofluorescence, concordant with CFTR correction. F508del CFTR correctors induced Nrf2 nuclear translocation, Nrf2-dependent luciferase activity, and transcriptional activation of target genes. Rescue of Nrf2 function by VX809/VX661 was dependent on significant correction of F508del and was blocked by inhibition of corrected channel function, or high-level shRNA knockdown of CFTR or F508del CFTR. Mechanistically, F508del CFTR modulation restored Nrf2 phosphorylation and its interaction with the coactivator CREB-binding protein (CBP). Our findings demonstrate that sufficient modulation of F508del CFTR function corrects Nrf2 dysfunction in CF.

Authors

Dana C. Borcherding, Matthew E. Siefert, Songbai Lin, John Brewington, Hesham Sadek, John P. Clancy, Scott M. Plafker, Assem G. Ziady

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Figure 3

Nrf2 dysfunction and diminished colocalization with F508del occur physiologically and can be rescued in primary CFhBE cells by CFTR modulation.

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Nrf2 dysfunction and diminished colocalization with F508del occur physio...
(A) Representative 3D micrographs of a proximity ligation assay (PLA) showing that Nrf2 interacts closely with CFTR in NhBE, but not CFhBE, cells (red dots: PLA signals), and that 10 μM VX809 treatment rescues Nrf2-CFTR interaction, visualized by increased red PLA signal. (B) PLA for NhBE cells incubated with antibodies for Nrf2 and either CFTR (top panel) or mouse IgG (bottom panel) as negative control. (C) CFhBE cells incubated with DMSO control or 1–10 μM VX809, and/or 20 μM CFTRinh-172 (Inh172), a CFTR inhibitor, for 48 hours. CFTR-Nrf2 interaction determined by PLA and fluorescent imaging. Arrows indicate apical and basolateral sides. Each experiment repeated in 3–4 CF and 3 non-CF donors with 3–5 replicates per treatment per donor. (D) Nrf2-activated gene expression is decreased in whole lungs from CF mouse models compared with WT mice, as determined by qPCR. Data for n = 5–10 mice per group are expressed as box-and-whisker plots. Horizontal bars indicate the median, box borders indicate 25th and 75th percentiles, and whiskers indicate 5th and 95th percentiles. Outliers are indicated by points outside the box plots. *P < 0.05, **P < 0.01, ***P < 0.001 vs. WT mice by 1-way ANOVA and Dunnett’s multiple-comparisons test. (E) Nrf2-CFTR colocalization is decreased in the lung airway epithelial cells of DF508 CF mice versus WT mice. Representative micrographs of IF for Nrf2 (green), CFTR (red), and DAPI (blue), with Nrf2-CFTR colocalization in yellow, at ×60 original magnification, for at least n = 4 mice per group with 3–5 replicates per donor. Scale bars: 10 μm.