Cellular localization and activity of Ad-delivered GFP-CFTR in airway epithelial and tracheal cells.
Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the cellular trafficking of the CFTR protein is an essential factor that determines its function in cells. The aim of our study was to develop an Ad vector expressing a biologically active green fluorescent protein (GFP)-CFTR chimera that can be tracked by both its localization and chloride channel function. No study thus far has demonstrated a GFP-CFTR construct that displayed both of these functions in the airway epithelia. Tracheal glandular cells, MM39 (CFTRwt) and CF-KM4 (CFTRDeltaF508), as well as human airway epithelial cells from a patient with cystic fibrosis (CF-HAE) and from a healthy donor (HAE) were used for the functional analysis of our Ad vectors, Ad5/GFP-CFTRwt and Ad5/GFP-CFTRDeltaF508. The GFP-CFTRwt protein expressed was efficiently addressed to the plasma membrane of tracheal cells and to the apical surface of polarized CF-HAE cells, while GFP-CFTRDeltaF508 mutant was sequestered intracellularly. The functionality of the GFP-CFTRwt protein was demonstrated by its capacity to correct the chloride channel activity both in CF-KM4 and CF-HAE cells after Ad transduction. A correlation between the proportion of Ad5-transduced CF-KM4 cells and correction of CFTR function showed that 55 to 70% transduction resulted in 70% correction of the Cl- channel function. In reconstituted CF-HAE, GFP-CFTRwt appeared as active as the nontagged CFTRwt protein in correcting the transepithelial Cl- transport. We show for the first time a GFP-CFTR chimera that localized to the apical surface of human airway epithelia and restored epithelial chloride transport to similar levels as nontagged CFTR.
Granio
Ophélia
O
Laboratoire de Virologie et Pathologie Humaine, Université de Lyon 1 and CNRS FRE 3011, Faculté de Médecine Laennec and IFR Lyon-Est, Lyon, France.
Norez
Caroline
C
Ashbourne Excoffon
Katherine J D
KJ
Karp
Philip H
PH
Lusky
Monika
M
Becq
Frédéric
F
Boulanger
Pierre
P
Zabner
Joseph
J
Hong
Saw-See
SS
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D000328:Adult; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating; D018613:Microscopy, Confocal; D050505:Mutant Proteins / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020545:Respiratory Mucosa / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D014132:Trachea / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D014161:Transduction, Genetic
D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins