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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Research Article Pulmonology

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 1

CFTR modulation dose-dependently increases Nrf2 nuclear localization and activity.

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CFTR modulation dose-dependently increases Nrf2 nuclear localization and...
(A and B) Gene expression of CFTR, NFE2L2, and Nrf2-regulated genes GCLC and NQO1 in primary NhBE (A) and CFhBE (B) cells, after incubation with DMSO control or the indicated doses of VX809 for 48 hours, determined by real-time quantitative PCR (qPCR). Data presented as fold changes versus untreated cells; calculated from cycle threshold measurements and normalized to 18S rRNA for n = 3 independent experiments and 3 donors per cell type. Data 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. (C) VX809 induces Nrf2-mediated luciferase expression in CFhBE cells. Cells were transiently transfected with a plasmid containing the Nrf2-binding promoter ARE driving firefly and a transfection control Renilla luciferase plasmid, then treated with VX809 (1–10 μM) for 48 hours. Expression in cell lysates was measured by luminometer. Relative activity is firefly/Renilla luciferase activity normalized to total protein, and is expressed as fold change versus DMSO control. Mean ± SEM for n = 7 independent experiments. (D) Representative micrographs of basolateral view of immunofluorescence for Nrf2 (green) or DAPI nuclear staining (blue). Cells were treated with VX809 (10–100 μM) for 48 hours. Scale bars: 10 μm. (E) Western blotting for Nrf2 protein in nuclear and cytosolic fractions of NhBE and CFhBE cells after VX809 treatment for 48 hours; β-actin and proliferating cell nuclear antigen (PCNA) are loading controls. (F) Aggregate short-circuit current data for cells pretreated with VX809 or VX661 (symbols color-coded by donor). Mean ± SEM for 3–4 replicates per condition per donor, from at least 3 unique patient donors, normalized as percentage of donor DMSO control. Dashed lines represent percentage of NhBE control average. For A–C, *P < 0.05, **P < 0.01, ***P < 0.001 vs. DMSO control cells by 1-way ANOVA and Dunnett’s multiple-comparisons test.