Establishment and characterization of a novel polarized MDCK epithelial cellular model for CFTR studies.
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.
Mendes
Filipa
F
Department of Chemistry and Biochemistry, University of Lisboa, Portugal. filipamendes@hotmail.com
Wakefield
John
J
Bachhuber
Tanja
T
Barroso
Margarida
M
Bebok
Zsuzsa
Z
Penque
Deborah
D
Kunzelmann
Karl
K
Amaral
Margarida D
MD
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
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator