The ΔF508-CFTR mutation inhibits wild-type CFTR processing and function when co-expressed in human airway epithelia and in mouse nasal mucosa

Abstract

BackgroundRescue or correction of CFTR function in native epithelia is the ultimate goal of CF therapeutics development. Wild-type (WT) CFTR introduction and replacement is also of particular interest. Such therapies may be complicated by possible CFTR self-assembly into an oligomer or multimer.ResultsSurprisingly, functional CFTR assays in native airway epithelia showed that the most common CFTR mutant, ΔF508-CFTR (ΔF-CFTR), inhibits WT-CFTR when both forms are co-expressed. To examine more mechanistically, both forms of CFTR were transfected transiently in varying amounts into IB3-1 CF human airway epithelial cells and HEK-293 human embryonic kidney cells null for endogenous CFTR protein expression. Increasing amounts of ΔF-CFTR inhibited WT-CFTR protein processing and function in CF human airway epithelial cells but not in heterologous HEK-293 cells. Stably expressed ΔF-CFTR in clones of the non-CF human airway epithelial cell line, CALU-3, also showed reduction in cAMP-stimulated anion secretion and in WT-CFTR processing. An ultimate test of this dominant negative-like effect of ΔF-CFTR on WT-CFTR was the parallel study of two different CF mouse models: the ΔF-CFTR mouse and the bitransgenic CFTR mouse corrected in the gut but null in the lung and airways. WT/ΔF heterozygotes had an intermediate phenotype with regard to CFTR agonist responses in in vivo nasal potential difference (NPD) recordings and in Ussing chamber recordings of short-circuit current (ISC) in vitro on primary tracheal epithelial cells isolated from the same mice. In contrast, CFTR bitransgenic +/− heterozygotes had no difference in their responses versus +/+ wild-type mice.ConclusionsTaken altogether, these data suggest that ΔF-CFTR and WT-CFTR co-assemble into an oligomeric macromolecular complex in native epithelia and share protein processing machinery and regulation at the level of the endoplasmic reticulum (ER). As a consequence, ΔF-CFTR slows WT-CFTR protein processing and limits its expression and function in the apical membrane of native airway epithelia. Implications of these data for the relative health of CF heterozygous carriers, for CFTR protein processing in native airway epithelia, and for the relative efficacy of different CF therapeutic approaches is significant and is discussed.

Stimulation of β2-adrenergic receptor increases CFTR function and decreases ATP levels in murine hematopoietic stem/progenitor cells

“…Calu-3, an epithelial cell line derived from submucosal gland, was used as positive control since it has been already shown that β 2 -AR and CFTR interact with each other in this cell line [7]. Western blotting for CFTR showed a predominant mature form of CFTR in Calu-3 cells as compared with immature form of CFTR in agreement with previous studies [11,12] (not shown). HSPCs displayed both immature and mature forms, as previously demonstrated [5].…”

Highly Efficient Gene Editing of Cystic Fibrosis Patient-Derived Airway Basal Cells Results in Functional CFTR Correction

“…Although it might be assumed that carrier state cells would have CFTR activity similar to that of non-CF cells, it has been suggested that presence of the DF508 protein can adversely affect the processing and chloride channel function of the wildtype CFTR protein. 39 While the mutation-specific editing approach achieved efficient correction and restored CFTR activity, there are at least two potential drawbacks to this strategy. First is the potential requirement for a new set of editing reagents (e.g., sequence specific nuclease and donor) for each mutation or small region spanning several mutations.…”

Relationships among CFTR expression, HCO 3 − secretion, and host defense may inform gene- and cell-based cystic fibrosis therapies

“…One approach to answering this question is to study airway epithelia from people who are heterozygous for a CF-causing mutation (CF carriers). A few studies in humans and mice measured transepithelial voltage (Vt) across nasal epithelia before and during perfusion of a solution that is Cl − -free and contains an agent to increase cellular levels of cAMP to phosphorylate and activate CFTR (15)(16)(17)(18). Two reports also studied cultured mouse and human cells (17,18).…”