Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

Thierry Métayé,Julie G Donaldson,Alain Kitzis,Caroline Norez,Romain Ferru-Clément,Vincent Thoreau,Frédéric Becq,Fleur Fresquet

doi:10.1371/journal.pone.0118943

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

Highlights

Cystic fibrosis (CF) is the most common recessive inherited disorder in Caucasian populations
In CFTR-transfected BHK cells, pharmacological inhibition of N-WASP has been shown to result in a decrease of CFTR cell surface expression, via accelerated endocytosis and seemingly impaired recycling [18]
Two distinct approaches were chosen to investigate the consequences upon CFTR homeostasis of Cdc42 pathway impairments: (i) we exposed cells to the pharmacological inhibitor ML141, a new compound selectively inhibiting Cdc42 [20], or to wiskostatin; (ii) we performed depletions by RNA-interference of either Cdc42 itself or else its effector N-WASP

Summary

Introduction

Cystic fibrosis (CF) is the most common recessive inherited disorder in Caucasian populations It is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR) [1,2]. As folding of the newly synthesized protein in the endoplasmic reticulum (ER) is unsuccessful, it is fully degraded by the ubiquitin-proteasome pathway before reaching the Golgi complex [7,8]. It has been shown in CFTR-transfected BHK cells that endocytosis rates are identical for WT and rescued (r)F508del-CFTR, whereas recycling to PM is less efficient for the mutant [9]. Because of these dysfunctions of the most common mutant, the trafficking mechanisms of CFTR to and from PM (initial targeting, internalization, recycling) have been the subject over the years of considerable attention

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Results

Conclusion