Paracellular Transport through Healthy and Cystic Fibrosis Bronchial Epithelial Cell Lines – Do We Have a Proper Model?

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.

E‐cadherin is downregulated in benign prostatic hyperplasia and required for tight junction formation and permeability barrier in the prostatic epithelial cell monolayer

“…10 Disruption of the mucosal epithelial barrier has been associated with the development and progression of many diseases, including pancreatitis, asthma, cystic fibrosis, Crohn's disease, and cancer. 11,[13][14][15][16][17] The human prostate is composed of a series of glandular ducts lined by a mucosal layer of luminal secretory cells and an underlying basal cell layer. 18 Loss of epithelial barrier integrity in the prostate could result in the leakage of PSA and other secreted proteins into the stromal compartment, and thus promote BPH pathogenesis and/or progression.…”

“…Adherens junctions are composed of cadherins, and tight junctions are composed of a complex of transmembrane proteins, scaffolding proteins, and regulatory molecules . Disruption of the mucosal epithelial barrier has been associated with the development and progression of many diseases, including pancreatitis, asthma, cystic fibrosis, Crohn's disease, and cancer . The human prostate is composed of a series of glandular ducts lined by a mucosal layer of luminal secretory cells and an underlying basal cell layer .…”

Claudins: Gatekeepers of lung epithelial function

“…However, measures of tight junction permeability such as transepithelial resistance are sensitive to whether primary cells are being examined or a cell line model is being used. The method of immortalization can also influence barrier function, so results obtained in vitro do need to be interpreted with caution [93]. Biochemical data support a role for CFTR directly interacting with ZO-1 via a PDZ motif [82] as well as indirectly through another scaffold protein, Na + /H + exchanger regulatory factor 1 (NHERF1) [83].…”

The role of mucus on drug transport and its potential to affect therapeutic outcomes

“…This cell line was generated by transformation of CF airway cells with the SV40 virus and is homozygous for the most common CF mutation, the F508-CFTR mutation. Of particular interest to pharmaceutical research is the ability of this cell line to express tight-junction proteins such as claudin-1, ZO-1, and occludin (15), which confer on CFBE41o-monolayers significant epithelial barrier properties evidenced by high transepithelial electrical resistance (TEER) values (15)(16)(17)(18). This cell line also expresses a number of proteins relevant for pulmonary drug transport, including P-glycoprotein (P-gp), lung resistance-related P protein (LRP), and caveolin-1 (15).…”