The Δ F508 Mutation Causes CFTR Misprocessing and Cystic Fibrosis–Like Disease in Pigs

Abstract

Mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel cause the autosomal recessive disease, cystic fibrosis (CF). The most common mutation is ΔF508, which deletes phenylalanine508. In vitro studies indicate that CFTR-ΔF508 is misprocessed, though in vivo consequences of the mutation are uncertain. To better understand effects of the ΔF508 mutation, we produced CFTRΔF508/ΔF508 pigs. Our biochemical, immunocytochemical and electrophysiological data on CFTR-ΔF508 in newborn pigs paralleled in vitro results. They also indicated that CFTRΔF508/ΔF508 airway epithelia retain a small residual CFTR conductance; maximal stimulation produced ~6% of wild-type function. Interestingly, cAMP agonists were less potent at stimulating current in CFTRΔF508/ΔF508 epithelia, suggesting that quantitative tests of maximal anion current may overestimate transport under physiological conditions. Despite residual CFTR function, four older CFTRΔF508/ΔF508 pigs developed lung disease strikingly similar to human CF. These results suggest that this limited CFTR activity is insufficient to prevent lung or gastrointestinal disease in CF pigs. These data also suggest that studies of recombinant CFTR-ΔF508 misprocessing predict in vivo behavior, which validates its use in biochemical and drug discovery experiments. These findings help elucidate the molecular pathogenesis of the common CF mutation and will guide strategies for developing new therapeutics.

A microRNA network regulates expression and biosynthesis of wild-type and ΔF508 mutant cystic fibrosis transmembrane conductance regulator

“…epithelial cells in which endogenous CFTR is studied have less band B protein than that detected when recombinant CFTR is expressed (31,32). Treatment with the miR-138 mimic and the SIN3A DsiRNA increased cAMP-stimulated G t (Fig.…”

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

“…CFTR ΔF508/ΔF508 (CF), CFTR +/ΔF508 (heterozygote), and CFTR +/+ pigs were the product of CFTR +/ΔF508 matings (29). These studies were approved by the University of Iowa Animal Care and Use Committee.…”

“…An advantage is that we used a porcine model of CF that develops the characteristic features of CF lung disease (29,30). To avoid the effects of longterm infection and inflammation, including potential epigenetic changes in epithelial cells, we generated airway epithelia from newborn animals, which we previously showed exhibit airway host defense defects (30,31,33).…”

“…To determine the relationship between CFTR expression and anion secretion, we studied epithelia with varying ratios of CFTR −/− (CF) and CFTR +/+ (WT) cells. Because the most common CFTR mutation in humans is deletion of phenylalanine at position 508 (ΔF508) (3), we also examined airway epithelia produced by mixing cells from CFTR ΔF508/ΔF508 pigs with cells from CFTR +/+ littermates (29). After epithelia were studied, quantitative PCR of genomic DNA indicated that the percentages of CF and WT cells in epithelia were as predicted by the seeding ratio (Fig.…”

“…We recently developed CFTR −/− and CFTR ΔF508/ΔF508 pigs (28,29). At birth, their airways lack infection and inflammation, but, over the ensuing weeks and months, they develop the hallmark features of CF airway disease (30).…”