title: Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.
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.
authors.affiliation: Laboratoire Génétique des Maladies Rares, Université de Poitiers, Poitiers, France.
authors.forename: Romain
authors.initials: R
authors.lastname: Ferru-Clément
authors.affiliation: Laboratoire Génétique des Maladies Rares, Université de Poitiers, Poitiers, France; Service de Génétique, Centre Hospitalier Universitaire de Poitiers, Poitiers, France.
authors.forename: Fleur
authors.initials: F
authors.lastname: Fresquet
authors.affiliation: Laboratoire Signalisation et Transports Ioniques Membranaires, CNRS, Université de Poitiers, Poitiers, France.
authors.forename: Caroline
authors.initials: C
authors.lastname: Norez
authors.affiliation: Laboratoire de Biophysique Médicale, Centre Hospitalier Universitaire de Poitiers, Poitiers, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Métayé
authors.affiliation: Laboratoire Signalisation et Transports Ioniques Membranaires, CNRS, Université de Poitiers, Poitiers, France.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Becq
authors.affiliation: Laboratoire Génétique des Maladies Rares, Université de Poitiers, Poitiers, France; Service de Génétique, Centre Hospitalier Universitaire de Poitiers, Poitiers, France.
authors.forename: Alain
authors.initials: A
authors.lastname: Kitzis
authors.affiliation: Laboratoire Génétique des Maladies Rares, Université de Poitiers, Poitiers, France.
authors.forename: Vincent
authors.initials: V
authors.lastname: Thoreau
mesh_terms: D008841:Actin Cytoskeleton / Q000378:metabolism; D002227:Carbazoles / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002540:Cerebral Cortex / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000378:metabolism*; D034285:Dynamin II / Q000378:metabolism; D004705:Endocytosis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D011412:Propanolamines / Q000494:pharmacology; D055550:Protein Stability / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011720:Pyrazoles / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000378:metabolism; D012137:Respiratory System / Q000378:metabolism* / Q000648:ultrastructure; D015398:Signal Transduction / Q000187:drug effects*; D013449:Sulfonamides / Q000494:pharmacology; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000096926:Benzenesulfonamides
chemical_list: D002227:Carbazoles; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D034285:Dynamin II; D004791:Enzyme Inhibitors; D011412:Propanolamines; D011720:Pyrazoles; D034741:RNA, Small Interfering; D013449:Sulfonamides; D051317:Wiskott-Aldrich Syndrome Protein, Neuronal; D020764:cdc42 GTP-Binding Protein; C581055:CID2950007; D000096926:Benzenesulfonamides; C495771:WASL protein, human; C489942:wiskostatin