TY - JOUR
T1 - CFTR gene transfer to human cystic fibrosis pancreatic duct cells using a sendai virus vector
AU - Rakonczay, Zoltan
AU - Hegyi, Peter
AU - Hasegawa, Mamoru
AU - Inoue, Makoto
AU - You, Jun
AU - Iida, Akihiro
AU - Ignath, Imre
AU - Alton, Eric W. F. W.
AU - Griesenbach, Uta
AU - Ovari, Gabriella
AU - Vag, Janos
AU - Da Paula, Ana C.
AU - Crawford, Russell M.
AU - Varga, Gabor
AU - Amaral, Margarida D.
AU - Mehta, Anil
AU - Lonovics, Janos
AU - Argent, Barry E.
AU - Gray, Michael A.
PY - 2008/2
Y1 - 2008/2
N2 - Cystic fibrosis (CF) is a fatal inherited disease caused by the absence or dysfunction of the CF transmembrane conductance regulator (CFTR) Cl- channel. About 70% of CF patients are exocrine pancreatic insufficient due to failure of the pancreatic ducts to secrete a HCO3--rich fluid. Our aim in this study was to investigate the potential of a recombinant Sendai virus (SeV) vector to introduce normal CFTR into human CF pancreatic duct (CFPAC-1) cells, and to assess the effect of CFTR gene transfer on the key transporters involved in HCO3- transport. Using polarized cultures of homozygous F508del CFPAC-1 cells as a model for the human CF pancreatic ductal epithelium we showed that SeV was an efficient gene transfer agent when applied to the apical membrane. The presence of functional CFTR was confirmed using iodide efflux assay. CFTR expression had no effect on cell growth, monolayer integrity, and mRNA levels for key transporters in the duct cell (pNBC, AE2, NHE2, NHE3, DRA, and PAT-1), but did upregulate the activity of apical Cl-/HCO3- and Na+/H+ exchangers (NHEs). In CFTR-corrected cells, apical Cl-/HCO3- exchange activity was further enhanced by cAMP, a key feature exhibited by normal pancreatic duct cells. The cAMP stimulated Cl-/HCO3- exchange was inhibited by dihydro-4,4'diisothiocyanostilbene-2,2'-disulfonic acid (H-2-DIDS), but not by a specific CFTR inhibitor, CFTRinh-172. Our data show that SeV vector is a potential CFTR gene transfer agent for human pancreatic duct cells and that expression of CFTR in CF cells is associated with a restoration of Cl- and HCO3- transport at the apical membrane.
AB - Cystic fibrosis (CF) is a fatal inherited disease caused by the absence or dysfunction of the CF transmembrane conductance regulator (CFTR) Cl- channel. About 70% of CF patients are exocrine pancreatic insufficient due to failure of the pancreatic ducts to secrete a HCO3--rich fluid. Our aim in this study was to investigate the potential of a recombinant Sendai virus (SeV) vector to introduce normal CFTR into human CF pancreatic duct (CFPAC-1) cells, and to assess the effect of CFTR gene transfer on the key transporters involved in HCO3- transport. Using polarized cultures of homozygous F508del CFPAC-1 cells as a model for the human CF pancreatic ductal epithelium we showed that SeV was an efficient gene transfer agent when applied to the apical membrane. The presence of functional CFTR was confirmed using iodide efflux assay. CFTR expression had no effect on cell growth, monolayer integrity, and mRNA levels for key transporters in the duct cell (pNBC, AE2, NHE2, NHE3, DRA, and PAT-1), but did upregulate the activity of apical Cl-/HCO3- and Na+/H+ exchangers (NHEs). In CFTR-corrected cells, apical Cl-/HCO3- exchange activity was further enhanced by cAMP, a key feature exhibited by normal pancreatic duct cells. The cAMP stimulated Cl-/HCO3- exchange was inhibited by dihydro-4,4'diisothiocyanostilbene-2,2'-disulfonic acid (H-2-DIDS), but not by a specific CFTR inhibitor, CFTRinh-172. Our data show that SeV vector is a potential CFTR gene transfer agent for human pancreatic duct cells and that expression of CFTR in CF cells is associated with a restoration of Cl- and HCO3- transport at the apical membrane.
KW - TRANSMEMBRANE CONDUCTANCE REGULATOR
KW - APICAL CL-/HCO3-EXCHANGER
KW - CONGENITAL CHLORIDE DIARRHEA
KW - DEPENDENT HCO3-TRANSPORT
KW - ADENOMA DRA
KW - BICARBONATE SECRETION
KW - HETEROLOGOUS CELLS
KW - NA+/H+ EXCHANGER
KW - INTRACELLULAR PH
KW - PROTEIN
U2 - 10.1002/jcp.21220
DO - 10.1002/jcp.21220
M3 - Article
SN - 1040-0605
VL - 214
SP - 442
EP - 455
JO - American Journal of Physiology: Lung Cellular and Molecular Physiology
JF - American Journal of Physiology: Lung Cellular and Molecular Physiology
IS - 2
ER -