The major objective in generating a cell culture system for CF research is to provide in vitro
models that resemble as closely as possible the properties of the native tissue from which they were derived. A number of immortalized airway epithelial cell lines generated in the past have been critical for enhancing understanding of the pathways responsible for CF pathology (reviewed in [14
]). Currently, all available cell models lack one or more of the following characteristics critical for a CF-relevant airway epithelial cell model: 1) Epithelial polarization and tight junction formation, 2) isogenic cell lines expressing wt and ΔF508CFTR, 3) high levels of ΔF508CFTR expression in CF cell lines, and 4) stable expression of CFTR constructs. Thus, a prudent approach is to select a clonal cell line from the pool of available lines and select and optimize according to these criteria. Currently, a cell line that meets the above criteria is not available. The CFBE41o− cell line and the complemented CFBE41o− subclones introduced in this study do meet the above criteria. However, one notable limitation is the lack of an airway-typical ENaC-mediated Na absorption in both non-complemented and complemented CFBE41o− cells (data not shown). This characteristic is difficult to maintain under simple culture conditions and is generally lost in most human cell culture systems, whether primary or transformed.
Stable CF airway epithelial cell lines have been critical for both academic and commercial CF research. Basic mechanistic studies as well as screening drugs for their therapeutic potential have benefited from the availability of these human cell lines. Although a number of matched CF and nonCF cell lines have been developed over the years, CFTR expression is often variable, airway epithelial-specific phenotypic characteristics are lacking, or they have been derived from different individuals and thereby have different genetic backgrounds. Correction of the ΔF508CFTR trafficking defect in human airway epithelial cell lines turned out to be difficult. As a result many drug studies testing small molecules that correct this defect have used heterologous and/or non-epithelial cell systems, such as Fisher rat thyroid cells [63
], MDCK canine kidney epithelial cells [64
], LLC-PK1 porcine kidney epithelial cells [66
], HEK293 human embryonic kidney cells [67
], HT500 kidney cells [68
], CHO Chinese hamster ovary cells [71
], C127i murine mammary carcinoma cells [72
], and 3T3 fibroblasts [67
]. To overcome potential limitations of these heterologous cell systems that can lead to a misinterpretation of results, this study strived to generate stable and effectively isogenic CF airway cell lines that have electrophysiological characteristics that reflect both the wt and ΔF508CFTR and account for the affect of overexpressing CFTR.
Both the stability and the level of CFTR expression determine the value of a complemented cell line for CF research. Currently it is not clear what level of CFTR expression is required for normal function. This also relates to the question of how much CFTR function needs to be recovered for CF treatment to normalize defective Cl secretion. This study quantifies the relative CFTR mRNA levels and the resulting CFTR-mediated currents and indicates that there are 3.8 µA/cm2
of CFTR current per unit increase in CFTR mRNA levels (), where one unit is defined as the amount of endogenous wtCFTR mRNA in 16HBE14o− cells. It is estimated that there are ~43 active apical wtCFTR channels per cell per fold increase in the amount of CFTR mRNA generated by the 6.2 kb wtCFTR constructs in the CFBE41o− clones assuming ~106
cells per cm2
, an apical driving force for Cl of −22 mV [74
], and a single channel conductance of CFTR of 8 pS with an open probability of 0.5 [75
]. By comparison, the efficiency of generating a functional CFTR must be considerably higher in 16HBE14o− cells given that CFTR mRNA levels in these cells were significantly lower than those detected in the complemented cell clones. Chloride currents were about 1/3 at 5-fold higher mRNA expression levels (c7-6.2wt) and 1.6 times more at 14-fold higher mRNA expression levels (c10-6.2wt) (). Using a similar calculation as above, there are ~330 active CFTR channels per cell in 16HBE14o−, i.e., the natively expressed mRNA in 16HBE14o− was more efficient for the overall chloride secretory response and might be due to a substantially more efficient expression and/or processing of CFTR protein.
Since both the life-time of ΔF508CFTR is reduced and normal trafficking to the membrane of ΔF508CFTR is largely inhibited compared to wtCFTR, increasing the levels of ΔF508CFTR expression appears as a prudent strategy for testing whether overexpressed ΔF508CFTR has a functional role in CF airway epithelial cells. Although the parental CFBE41o− is homozygous for ΔF508CFTR, native expression levels are low [51
] and no significant CFTR-mediated currents can be detected. Clone c4-4.7ΔF showed a small, but consistent, forskolin-stimulated and GlyH101-blocked current at high levels of recombinant ΔF508CFTR mRNA suggesting that there is some CFTR-dependent function in these cells. Using the same values for driving forces and channel conductance as above, but with an open probability of 0.1 for ΔF508 CFTR [72
], there would be ~9 active apical ΔF508CFTR channels per cell per unit increase in the amount of ΔF508CFTR mRNA. This estimation suggests that enhanced expression of ΔF508CFTR increases the presence of ΔF508CFTR in the apical membrane of CF bronchial epithelial cells. However, the number of active channels associated with the ΔF508CFTR is lower than the number of active channels in the clonal isolates expressing recombinant wtCFTR. Since a 4.7kb ΔF508CFTR cDNA construct was used for the expression of recombinant ΔF508CFTR mRNA and a full-length 6.2kB wtCFTR cDNA construct was used for the wtCFTR complemented cell clones, it is possible that the 4.7kB construct was not ideal for optimal expression of functional ΔF508CFTR.
The correlation between CFTR mRNA levels and Cl transport represents an important consideration for designing CF therapies that rely on modulating the levels of CFTR mRNA whether through genetic means or through pharmacological enhancement. While others have carried out studies in heterologous systems, the paucity of data in cells that are polarized and normally express CFTR is noteworthy. The studies described here suggest a direct relationship between the amount of CFTR mRNA and the number of active CFTR channels in the apical membrane of polarized airway epithelial cells. The efficacy of mRNA generated from recombinant transgene appears to be significantly diminished when compared to CFTR mRNA expressed from the endogenous gene in terms of the ability to generate CFTR-associated cAMP-dependent Cl conductances. Furthermore, while the increased expression and ΔF508CFTR-associated function in this episomal vector complementation system indicates that the c4-4.7ΔF clone has a potentially useful role in the development of pharmacological agents that augment ΔF508CFTR expression, additional studies will be needed to evaluate the potential advantages of using a full-length 6.2kB vs a 4.7kB ΔF508CFTR construct to optimize the efficacy of ΔF508CFTR in CF bronchial epithelial cells.