title: Establishment and characterization of a novel polarized MDCK epithelial cellular model for CFTR studies.
abstract: F508del is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that is responsible for the genetic disease Cystic Fibrosis (CF). It results in a major failure of CFTR to traffic to the apical membrane of epithelial cells, where it should function as a chloride (Cl-) channel. Most studies on localization, processing and cellular trafficking of wild-type (wt) and F508del-CFTR have been performed in non-epithelial cells. Notwithstanding, polarized epithelial cells possess distinctly organized and regulated membrane trafficking pathways. We have used Madin-Darby canine kidney (MDCK) type II cells (proximal tubular cells which do not express endogenous CFTR) to generate novel epithelial, polarized cellular models stably expressing wt- or F508del-CFTR through transduction with recombinant lentiviral vectors. Characterization of these cell lines shows that wt-CFTR is correctly processed and apically localized, producing a cAMP-activated Cl- conductance. In contrast, F508del-CFTR is mostly detected in itsimmature form, localized intracellularly and producing only residual Cl- conductance. These novel cell lines constitute bona fide models and significantly improved resources to investigate the molecular mechanisms of polarized membrane traffic of wt- and F508del-CFTR in the same cellular background. They are also useful to identify/validate novel therapeutic compounds for CF.
authors.affiliation: Department of Chemistry and Biochemistry, University of Lisboa, Portugal. filipamendes@hotmail.com
authors.forename: Filipa
authors.initials: F
authors.lastname: Mendes
authors.forename: John
authors.initials: J
authors.lastname: Wakefield
authors.forename: Tanja
authors.initials: T
authors.lastname: Bachhuber
authors.forename: Margarida
authors.initials: M
authors.lastname: Barroso
authors.forename: Zsuzsa
authors.initials: Z
authors.lastname: Bebok
authors.forename: Deborah
authors.initials: D
authors.lastname: Penque
authors.forename: Karl
authors.initials: K
authors.lastname: Kunzelmann
authors.forename: Margarida D
authors.initials: MD
authors.lastname: Amaral
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D016764:Cell Polarity; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004285:Dogs; D004847:Epithelial Cells / Q000737:chemistry / Q000166:cytology* / Q000378:metabolism; D005260:Female; D007668:Kidney / Q000166:cytology; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D014176:Protein Biosynthesis; D021381:Protein Transport; D014161:Transduction, Genetic; D019076:Transgenes
chemical_list: C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator