Activation of ΔF508 CFTR in an epithelial monolayer

Abstract

The ΔF508 mutation leads to retention of cystic fibrosis transmembrane conductance regulator (CFTR) in the endoplasmic reticulum and rapid degradation by the proteasome and other proteolytic systems. In stably transfected LLC-PK1(porcine kidney) epithelial cells, ΔF508 CFTR conforms to this paradigm and is not present at the plasma membrane. When LLC-PK1 cells or human nasal polyp cells derived from a ΔF508 homozygous patient are grown on plastic dishes and treated with an epithelial differentiating agent (DMSO, 2% for 4 days) or when LLC-PK1 cells are grown as polarized monolayers on permeable supports, plasma membrane ΔF508 CFTR is significantly increased. Moreover, when confluent LLC-PK1 cells expressing ΔF508 CFTR were treated with DMSO and mounted in an Ussing chamber, a further increase in cAMP-activated short-circuit current (i.e., ∼7 μA/cm2; P < 0.00025 compared with untreated controls) was observed. No plasma membrane CFTR was detected after DMSO treatment in nonepithelial cells (mouse L cells) expressing ΔF508 CFTR. The experiments describe a way to augment ΔF508 CFTR maturation in epithelial cells that appears to act through a novel mechanism and allows insertion of functional ΔF508 CFTR in the plasma membranes of transporting cell monolayers. The results raise the possibility that increased epithelial differentiation might increase the delivery of ΔF508 CFTR from the endoplasmic reticulum to the Golgi, where the ΔF508 protein is shielded from degradative pathways such as the proteasome and allowed to mature.

ΔF508 CFTR protein expression in tissues from patients with cystic fibrosis

“…The dependence of ∆F508 CFTR maturation and targeting upon epithelial polarization has recently been demonstrated in stably transfected cells, as well as in endogenous CFTR-expressing epithelial cells (51,52).…”

Conformational and Temperature-sensitive Stability Defects of the ΔF508 Cystic Fibrosis Transmembrane Conductance Regulator in Post-endoplasmic Reticulum Compartments

“…Development of novel strategies to stabilize the native ⌬F508 CFTR would complement the present therapeutic approaches aiming to correct the folding defect at the ER, in tissue culture models (10,(22)(23)(24)(25)45), in CF mice (21), and in human trials (46,47).…”

“…Reduced temperature (10,21,22), chemical chaperones (23)(24)(25), and down-regulation of Hsp70 (26,27) activity are thought to partially revert the folding defect of ⌬F508 CFTR and promote the accumulation of the functional channel at the cell surface. Importantly, using more sensitive electrophysiological techniques, constitutive accumulation of ⌬F508 CFTR was documented in the plasma membrane of primary epithelia from ⌬F508 homozygous mice (21,28) and in the intestinal and gallbladder epithelia of homozygous ⌬F508 patients (29,30).…”

Chemical Chaperones: A Pharmacological Strategy for Disorders of Protein Folding and Trafficking

“…Brown et al showed that glycerol and two other low molecular weight organic compounds, deuterated water and TMAO, mediated increased posttranslational maturation of CFTR⌬F508 in a cell culture model system and this was associated with increased chloride transport activity (14). Other studies have shown that chloride transport is increased significantly by the effect of DMSO (DMSO) on polarized epithelial cell lines expressing CFTR⌬F508 (15). Specific amino acids also appear to affect maturation of CFTR⌬F508 by a chaperone-like mechanism as evidenced by the effect of glutamine in a cell culture model system of CFTR⌬F508 (16).…”