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Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants.

ABSTRACT: Analysis of the functional consequences and treatment response of rare CFTR variants is challenging due to the limited availability of primary airways cells.A Flp recombination target (FRT) site for stable expression of CFTR was incorporated into an immortalized CF bronchial epithelial cell line (CFBE41o-). CFTR cDNA was integrated into the FRT site. Expression was evaluated by western blotting and confocal microscopy and function measured by short circuit current. RNA sequencing was used to compare the transcriptional profile of the resulting CF8Flp cell line to primary cells and tissues.Functional CFTR was expressed from integrated cDNA at the FRT site of the CF8Flp cell line at levels comparable to that seen in native airway cells. CF8Flp cells expressing WT-CFTR have a stable transcriptome comparable to that of primary cultured airway epithelial cells, including genes that play key roles in CFTR pathways.CF8Flp cells provide a viable substitute for primary CF airway cells for the analysis of CFTR variants in a native context.

SUBMITTER: Gottschalk LB

PROVIDER: S-EPMC4879073 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants.

Gottschalk Laura B LB Vecchio-Pagan Briana B Sharma Neeraj N Han Sangwoo T ST Franca Arianna A Wohler Elizabeth S ES Batista Denise A S DA Goff Loyal A LA Cutting Garry R GR

Journal of cystic fibrosis : official journal of the European Cystic Fibrosis Society 20151213 3

<h4>Background</h4>Analysis of the functional consequences and treatment response of rare CFTR variants is challenging due to the limited availability of primary airways cells.<h4>Methods</h4>A Flp recombination target (FRT) site for stable expression of CFTR was incorporated into an immortalized CF bronchial epithelial cell line (CFBE41o-). CFTR cDNA was integrated into the FRT site. Expression was evaluated by western blotting and confocal microscopy and function measured by short circuit curr ...[more]

PMID: 26694805

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Project description:BACKGROUND: In airway epithelial cells, calcium mobilization can be elicited by selective autocrine and/or paracrine activation of apical or basolateral membrane heterotrimeric G protein-coupled receptors linked to phospholipase C (PLC) stimulation, which generates inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG) and induces Ca2+ release from endoplasmic reticulum (ER) stores. METHODS: In the present study, we monitored the cytosolic Ca2+ transients using the UV light photolysis technique to uncage caged Ca2+ or caged IP3 into the cytosol of loaded airway epithelial cells of cystic fibrosis (CF) and non-CF origin. We compared in these cells the types of Ca2+ receptors present in the ER, and measured their Ca2+ dependent activity before and after correction of F508del-CFTR abnormal trafficking either by low temperature or by the pharmacological corrector miglustat (N-butyldeoxynojirimycin). RESULTS: We showed reduction of the inositol 1,4,5-trisphosphate receptors (IP3R) dependent-Ca2+ response following both correcting treatments compared to uncorrected cells in such a way that Ca2+ responses (CF+treatment vs wild-type cells) were normalized. This normalization of the Ca2+ rate does not affect the activity of Ca2+-dependent chloride channel in miglustat-treated CF cells. Using two inhibitors of IP3R1, we observed a decrease of the implication of IP3R1 in the Ca2+ response in CF corrected cells. We observed a similar Ca2+ mobilization between CF-KM4 cells and CFTR-cDNA transfected CF cells (CF-KM4-reverted). When we restored the F508del-CFTR trafficking in CFTR-reverted cells, the specific IP3R activity was also reduced to a similar level as in non CF cells. At the structural level, the ER morphology of CF cells was highly condensed around the nucleus while in non CF cells or corrected CF cells the ER was extended at the totality of cell. CONCLUSION: These results suggest reversal of the IP3R dysfunction in F508del-CFTR epithelial cells by correction of the abnormal trafficking of F508del-CFTR in cystic fibrosis cells. Moreover, using CFTR cDNA-transfected CF cells, we demonstrated that abnormal increase of IP3R Ca2+ release in CF human epithelial cells could be the consequence of F508del-CFTR retention in ER compartment.

Dataset Information

Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants.

Publications

Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants.

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