Failure of cAMP agonists to activate rescued ΔF508 CFTR in CFBE41o- airway epithelial monolayers

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated chloride channel. Mutations in the CFTR gene result in cystic fibrosis (CF). The most common mutation, Delta F508, results in endoplasmic reticulum-associated degradation (ERAD) of CFTR. Delta F508 CFTR has been described as a temperature-sensitive mutation that can be rescued following growth at 27 degrees C. In order to study the processing and function of wild-type and rescued Delta F508 CFTR at the cell surface under non-polarized and polarized conditions, we developed stable cell lines expressing Delta F508 or wild-type CFTR. CFBE41o(-) is a human airway epithelial cell line capable of forming high resistance, polarized monolayers when cultured on permeable supports, while HeLa cells are normally grown under non-polarizing conditions. Immunoprecipitation, cell surface biotinylation, immunofluorescence, and functional assays confirmed the presence of Delta F508 CFTR at the cell surface in both cell lines after incubating the cells for 48 h at 27 degrees C. However, stimulators of wild-type CFTR such as forskolin, beta(2)-adrenergic or A(2B)-adenosine receptor agonists failed to activate rescued Delta F508 CFTR in CFBE41o(-) monolayers. Rescued Delta F508 CFTR could be stimulated with genistein independent of pretreatment with cAMP signalling agonists. Interestingly, rescued Delta F508 CFTR in HeLa cells could be efficiently stimulated with either forskolin or genistein to promote Cl- transport. These results indicate that Delta F508 CFTR, when rescued in CFBE41o(-) human airway epithelial cells, is poorly responsive to signalling pathways known to regulate wild-type CFTR. Furthermore, the differences in rescue and activation of Delta F508 CFTR in the two cell lines suggest that cell-type specific differences in Delta F508 CFTR processing are likely to complicate efforts to identify potentiators and/or correctors of the Delta F508 defect.