Cholesterol depletion and genistein as tools to promote F508delCFTR retention at the plasma membrane.

Abstract

F508delCFTR-, but not wtCFTR-, expressing fibroblasts resemble Niemann Pick type C cells in the massive intracellular accumulation of free cholesterol. The recruitment and activation of F508delCFTR by cholesterol depletion was studied.
Filipin staining, forskolin-stimulated anion efflux and FITC-dextran uptake were studied in control cells and fibroblasts treated with 2-hydroxypropyl beta-cyclodextrin phosphatidylcholine large unilamellar vesicles to deplete cellular free cholesterol.
Treatment of F508delCFTR-, but not wtCFTR-, expressing fibroblasts with 2-hydroxypropyl beta-cyclodextrin resulted in a reduction in cellular cholesterol and a potentiation of the forskolin-induced anion efflux. In addition, forskolin also promoted a massive increase in the rate of endocytosis in F508delCFTR fibroblasts, which was absent in genistein- or cyclodextrin-treated cultures.
The results not only suggest that reducing cellular cholesterol may serve as pharmacotherapeutic tool in the treatment of cystic fibrosis but also reveal a novel mechanism for genistein regulation of F508delCFTR, i.e. retention by inhibition of endocytosis.