Polarized Expression of Cystic Fibrosis Transmembrane Conductance Regulator and Associated Epithelial Proteins during the Regeneration of Human Airway Surface Epithelium in Three-Dimensional Culture

Abstract

SUMMARY:We have previously shown that, in normal human airway tissue, localization of the cystic fibrosis transmembrane conductance regulator (CFTR) can be affected by epithelial maturation, polarity, and differentiation and that CFTR trafficking and apical localization depend on the integrity of the airway epithelium. In this study, we addressed the question of whether the three-dimensional (3-D) organization of adult human airway epithelial cells in suspension culture under rotation, leading to spheroid-like structures, could mimic the in vivo phenomenon of differentiation and polarization. The kinetics of the differentiation, polarity, and formation of the CFTR-ZO-1-ezrin complex was analyzed by transmission, scanning, and immunofluorescence microscopy. Functional activity of the airway surface epithelium was assessed by monitoring the degree of cAMP-stimulated chloride efflux from cultured cells. Our results show that after the initial step of dedifferentiation, characterized by a loss of ciliated cells and disappearance of epithelial subapical CFTR-ezrin-ZO-1 complex, the isolated cells formed 3-D spheroid structures within 24 hours. After 15 days, progressive ciliogenesis was observed and secretory cells could be identified. After 35 days of 3-D culture, ZO-1, CFTR, ezrin, and CD59 were apically or subapically located, and well-differentiated secretory and ciliated cells were identified. CFTR functionality was assessed by analyzing the Cl Ϫ secretion after amiloride and forskolin perfusion. After 35 days of culture of spheroids in suspension, a significant increase in Cl Ϫ efflux was observed in well-differentiated ciliated cells. (Lab Invest 2002, 82:989 -998).

Foxj1 is required for apical localization of ezrin in airway epithelial cells

“…Localization of apical ezrin in ciliated airway epithelial cells is, in agreement with prior reports of ezrin expression in the apical membrane of epithelial cells, lining the airways and ciliated nasal epithelial cells (Berryman et al, 1993;Brezillon et al, 1997;Mohler et al, 1999;Laoukili et al, 2001;Castillon et al, 2002). Our use of multiple airway cell markers in vivo and in vitro (primary cell culture), and high-resolution imaging now confirms specific expression in the ciliated cell of the airway.…”

The Short Apical Membrane Half-life of Rescued ΔF508-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Results from Accelerated Endocytosis of ΔF508-CFTR in Polarized Human Airway Epithelial Cells

“…Furthermore, both WT-CFTR and ⌬F508-CFTR undergo trafficking in the Rab11a-specific apical recycling compartment. Substantial evidence indicates that membrane trafficking events are cell type-specific (42)(43)(44)(92)(93)(94). Thus, it is not surprising that the endocytic trafficking of rescued ⌬F508-CFTR differs in polarized human airway epithelial cells such as CFBE41oϪ cells and in non-polarized fibroblasts such as BHK-21 cells.…”

Stimulation of औ2-Adrenergic Receptor Increases Cystic Fibrosis Transmembrane Conductance Regulator Expression in Human Airway Epithelial Cells through a cAMP/Protein Kinase A-independent Pathway

“…Moreover, HAEC can be cultured under different conditions in vitro with variable degrees of differentiation (23,24). Under our culture conditions, primary HAEC showed tight junctions, elevated transepithelial resistance, and a polarized distribution of CFTR.…”