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 2

Nrf2 and CFTR colocalize in NhBE cells, and this colocalization is diminished in CFhBE cells.

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Nrf2 and CFTR colocalize in NhBE cells, and this colocalization is dimin...
(A–C) Representative photographs of IF for Nrf2 (green), CFTR (red), or DAPI (blue) in the apical (A), subapical (B), and perinuclear (C) views of NhBE and CFhBE cells, at ×60 original magnification. Merged composite images are shown in the far right column, with colocalization of Nrf2 and CFTR in yellow. To avoid nuclear permeabilization, mild detergent was used. (D) Slices view of IF in NhBE and CFhBE cells stained as above. Arrows indicate apical (A) and basolateral (B) sides. Each experiment was performed at least 10 times. (E) Representative IF images of NhBE and CFhBE cells, XY side sectioned after paraffin embedding, with staining as above. (F) CFTR association with Nrf2 was also analyzed by immunoprecipitation. NhBE and CFhBE cells were treated with the indicated doses of DMSO control, VX809, or VX661 for 48 hours. Whole cell lysates were incubated with anti-CFTR antibody, immunoprecipitated, subjected to SDS-PAGE, transferred to PVDF membrane, and probed with anti-Nrf2 antibody. Data are representative of 3–4 CF and 3 non-CF donors, and 3–4 replicates per treatment per donor. Scale bars: 10 μm.