Clinically approved CFTR modulators rescue Nrf2 dysfunction in cystic fibrosis airway epithelia
Dana C. Borcherding, Matthew E. Siefert, Songbai Lin, John Brewington, Hesham Sadek, John P. Clancy, Scott M. Plafker, Assem G. Ziady
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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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 9

CFTR modulation increases Nrf2 phosphorylation and interaction with CBP.

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CFTR modulation increases Nrf2 phosphorylation and interaction with CBP....
NhBE and CFhBE cells were treated with the indicated doses of VX809 or VX661 for 48 hours. (A and B) Cell lysates were immunoprecipitated at 4°C with anti-Nrf2 antibody, then subjected to Western blot analysis with anti-phosphoserine antibody (A); or with anti-CBP antibody, then subjected to Western blot analysis with anti-Nrf2 antibody (B). (C and D) Average levels of Nrf2 immunoprecipitated with CBP from cell lysates from 4 CF (C) and 3 non-CF (D) donors following treatment with DMSO, VX809, VX661, or treatments plus CFTRinh-172, demonstrate that CFTR modulation increases Nrf2-CBP interaction in a CFTR function–dependent manner. Aliquots of the input lysates were analyzed by Western blot with antibody against total Nrf2 to confirm the presence of equal levels of Nrf2 in lysates subjected to immunoprecipitation (Supplemental Figure 14). Representative blots are shown for 4 independent experiments in 4 CF and 3 non-CF donors. 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. *P < 0.05, ***P < 0.001 vs. vehicle control; or #P < 0.05 vs. cells treated with the same dose of VX809/VX661 by mixed-effects ANOVA with Dunnett’s multiple-corrections test.