Modulation of endocytic trafficking and apical stability of CFTR in primary human airway epithelial cultures

Abstract

CFTR is a highly regulated apical chloride channel of epithelial cells that is mutated in cystic fibrosis (CF). In this study, we characterized the apical stability and intracellular trafficking of wild-type and mutant CFTR in its native environment, i.e., highly differentiated primary human airway epithelial (HAE) cultures. We labeled the apical pool of CFTR and subsequently visualized the protein in intracellular compartments. CFTR moved from the apical surface to endosomes and then efficiently recycled back to the surface. CFTR endocytosis occurred more slowly in polarized than in nonpolarized HAE cells or in a polarized epithelial cell line. The most common mutation in CF, DeltaF508 CFTR, was rescued from endoplasmic reticulum retention by low-temperature incubation but transited from the apical membrane to endocytic compartments more rapidly and recycled less efficiently than wild-type CFTR. Incubation with small-molecule correctors resulted in DeltaF508 CFTR at the apical membrane but did not restore apical stability. To stabilize the mutant protein at the apical membrane, we found that the dynamin inhibitor Dynasore and the cholesterol-extracting agent cyclodextrin dramatically reduced internalization of DeltaF508, whereas the proteasomal inhibitor MG-132 completely blocked endocytosis of DeltaF508. On examination of intrinsic properties of CFTR that may affect its apical stability, we found that N-linked oligosaccharides were not necessary for transport to the apical membrane but were required for efficient apical recycling and, therefore, influenced the turnover of surface CFTR. Thus apical stability of CFTR in its native environment is affected by properties of the protein and modulation of endocytic trafficking.

Pharmacological Rescue of the Mutant Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Detected by Use of a Novel Fluorescence Platform

“…To date, C4 is one of the most well-studied correctors for F508del-CFTR rescue. However, the efficiency of rescue for C4 is quite limited across multiple cell types that have been tested (15,18,32). This result is in agreement with data obtained from both F508del-CFTR FAP constructs in HEK293 cells as well as the currents associated with F508del-CFTR endogenously expressed from HBE cells, where functional C4 correction efficacy was poor (Table 1).…”

“…Even after corrector treatment, F508del-CFTR has <15% the activity of CFTR wild-type (WT), and the corrector efficacy may vary depending on the cell type examined (13)(14)(15)(16). More robust rescue can be achieved by using a combination of correctors, which enhances rescue to levels greater than individual compound actions (17)(18)(19).…”

Correctors of mutant CFTR enhance subcortical cAMP–PKA signaling through modulating ezrin phosphorylation and cytoskeleton organization

“…Finally, as it is known that the low-temperature-rescued F508del CFTR (rF508del CFTR) is unstable on the cell surface and is rapidly internalized and degraded at 37°C (Cholon et al, 2010;Sharma et al, 2004), we monitored the effect of T567D ezrin expression on the cell surface stability of rF508del CFTR by following the surface pool of rF508del CFTR using the surface biotinylation assay previously described (Favia et al, 2014;Jurkuvenaite et al, 2010). Fig.…”

Disease-relevant proteostasis regulation of cystic fibrosis transmembrane conductance regulator

“…The abundance of CFTR at the PM is mainly determined by its recycling through the Rab11 þ endocytic pathway. 52,53 It has been reported that ubiquitylated CFTR becomes unstable at the PM and is diverted to lysosomal degradation. 54 Moreover, SQSTM1 can also function as a critical regulator of internalization, trafficking and sorting of ubiquitinylated surface proteins.…”