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Abstract
It has been reported recently that the cystic fibrosis transmembrane conductance regulator (CFTR) besides transcellular chloride transport, also controls the paracellular permeability of bronchial epithelium. The aim of this study was to test whether overexpressing wtCFTR solely regulates paracellular permeability of cell monolayers. To answer this question we used a CFBE41o– cell line transfected with wtCFTR or mutant F508del-CFTR and compered them with parental line and healthy 16HBE14o– cells. Transepithelial electrical resistance (TER) and paracellular fluorescein flux were measured under control and CFTR-stimulating conditions. CFTR stimulation significant decreased TER in 16HBE14o– and also in CFBE41o– cells transfected with wtCFTR. In contrast, TER increased upon stimulation in CFBE41o– cells and CFBE41o– cells transfected with F508del-CFTR. Under non-stimulated conditions, all four cell lines had similar paracellular fluorescein flux. Stimulation increased only the paracellular permeability of the 16HBE14o– cell monolayers. We observed that 16HBE14o– cells were significantly smaller and showed a different structure of cell-cell contacts than CFBE41o– and its overexpressing clones. Consequently, 16HBE14o– cells have about 80% more cell-cell contacts through which electrical current and solutes can leak. Also tight junction protein composition is different in ‘healthy’ 16HBE14o– cells compared to ‘cystic fibrosis’ CFBE41o– cells. We found that claudin-3 expression was considerably stronger in 16HBE14o– cells than in the three CFBE41o– cell clones and thus independent of the presence of functional CFTR. Together, CFBE41o– cell line transfection with wtCFTR modifies transcellular conductance, but not the paracellular permeability. We conclude that CFTR overexpression is not sufficient to fully reconstitute transport in CF bronchial epithelium. Hence, it is not recommended to use those cell lines to study CFTR-dependent epithelial transport.
Highlights
In the apical and basolateral membrane, embedded ion channels and transporters together provide for epithelial transport
Continous Transepithelial Electrical Resistance Epithelial cell monolayers can be characterised by Continues Transepithelial Electric Resistance (cTER), which is determined primarily by the electrical resistance of the apical and basolateral cell membrane and the paracellular resistant, generated by the TJ complex [25]
We could not completely exclude that the differences between those two cell lines originated from the fact that they came from different donors instead of different CFTR expression in cell membrane
Summary
In the apical and basolateral membrane, embedded ion channels and transporters together provide for epithelial (transcellular) transport. The CF phenotype is the consequence of CFTR insufficiency in terms of its chloride conductance and concerning its regulatory function on other ion channels and intracellular interaction partners [6,7,8]. In this line, CFTR is assumed to be involved in the regulation of paracellular permeability [9,10,11,12]. Paracellular transport of solutes and water is driven by the transepithelial electrochemical gradient [13] and modulated by tight junctions (TJ), a multi-protein complex, which acts as a permeability barrier [14,15]. Through the leak pathway only a small amount of larger molecules can pass [17]
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