Caroline Norez

Rescue of functional delF508-CFTR channels in cystic fibrosis epithelial cells by the α-glucosidase inhibitor miglustat

In the disease cystic fibrosis (CF), the most common mutation delF508 results in endoplasmic reticulum retention of misfolded CF gene proteins (CFTR). We show that the α‐1,2‐glucosidase inhibitor miglustat (N‐butyldeoxynojirimycin, NB‐DNJ) prevents delF508‐CFTR/calnexin interaction and restores cAMP‐activated chloride current in epithelial CF cells. Moreover, miglustat rescues a mature and functional delF508‐CFTR in the intestinal crypts of ileal mucosa from delF508 mice. Since miglustat is an orally active orphan drug (Zavesca®) prescribed for the treatment of Gaucher disease, our findings provide the basis for future clinical evaluation of miglustat in CF patients.

9‐Phenanthrol inhibits human TRPM4 but not TRPM5 cationic channels

Background and purpose: TRPM4 and TRPM5 are calcium‐activated non‐selective cation channels with almost identical characteristics. TRPM4 is detected in several tissues including heart, kidney, brainstem, cerebral artery and immune system whereas TRPM5 expression is more restricted. Determination of their roles in physiological processes requires specific pharmacological tools. TRPM4 is inhibited by glibenclamide, a modulator of ATP binding cassette proteins (ABC transporters), such as the cystic fibrosis transmembrane conductance regulator (CFTR). We took advantage of this similarity to investigate the effect of hydroxytricyclic compounds shown to modulate ABC transporters, on TRPM4 and TRPM5.

Maintaining Low Ca2+ Level in the Endoplasmic Reticulum Restores Abnormal Endogenous F508del-CFTR Trafficking in Airway Epithelial Cells

The most common mutation in cystic fibrosis, F508del, results in cystic fibrosis transmembrane conductance regulator protein (CFTR) that is retained in the endoplasmic reticulum (ER). Retention is dependent on chaperone proteins, many of which, like calnexin, require calcium for optimal activity. Here, we show that a limited and a maintained ER calcium level is sufficient to inhibit the F508del–CFTR/calnexin interaction and to restore the cAMP‐dependent CFTR chloride transport, thus showing the correction of abnormal trafficking. We used Western blot analysis, iodide efflux and calcium measurement techniques applied to the human airway epithelial cystic fibrosis cell line CF15 (F508del/F508del). The inhibition of ER calcium pump, with thapsigargin, curcumin, 2,5‐di(t‐butyl)hydroquinone or cyclopiazonic acid, maintains a threshold levels of calcium that is correlated to the recovery of endogenous F508del‐CFTR transport activity. In particular, cyclopiazonic acid restores a 2‐aminoethyoxydiphenyl borate‐sensitive F508del‐CFTR trafficking with an EC50 of 915 nm. By contrast, the 1,4,5‐trisphosphate or IP3 receptor activators, i.e., ATP and histamine, while transiently emptying the ER intracellular calcium store, did not affect the trafficking of F508del‐CFTR. Our data suggest that decreasing the ER calcium level is not sufficient to restore the defective trafficking of F508del‐CFTR, whereas decreasing and also maintaining low ER calcium level allow correction of defective biosynthetic pathway of endogenous F508del‐CFTR in human airway epithelial cells.

Rescue of ΔF508-CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) by Curcumin: Involvement of the Keratin 18 Network

The most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, ΔF508, causes retention of ΔF508-CFTR in the endoplasmic reticulum and leads to the absence of CFTR Cl– channels in the plasma membrane. ΔF508-CFTR retains some Cl– channel activity so increased expression of ΔF508-CFTR in the plasma membrane can restore Cl– secretion deficiency. Recently, curcumin was shown to rescue ΔF508-CFTR localization and function. In our previous work, the keratin 18 (K18) network was implicated in ΔF508-CFTR trafficking. Here, we hypothesized that curcumin could restore a functional ΔF508-CFTR to the plasma membrane acting via the K18 network. First, we analyzed the effects of curcumin on the localization of ΔF508-CFTR in different cell lines (HeLa cells stably transfected with wild-type CFTR or ΔF508-CFTR, CALU-3 cells, or cystic fibrosis pancreatic epithelial cells CFPAC-1) and found that it was significantly delocalized toward the plasma membrane in ΔF508-CFTR-expressing cells. We also performed a functional assay for the CFTR chloride channel in CFPAC-1 cells treated or not with curcumin and detected an increase in a cAMP-dependent chloride efflux in treated ΔF508-CFTR-expressing cells. The K18 network then was analyzed by immunocytochemistry and immunoblot exclusively in curcumin-treated or untreated CFPAC-1 cells because of their endogenic ΔF508-CFTR expression. After curcumin treatment, we observed a remodeling of the K18 network and a significant increase in K18 Ser52 phosphorylation, a site directly implicated in the reorganization of intermediate filaments. With these results, we propose that K18 as a new therapeutic target and curcumin, and/or its analogs, might be considered as potential therapeutic agents for cystic fibrosis.

Disruption of CFTR chloride channel alters mechanical properties and cAMP‐dependent Cl− transport of mouse aortic smooth muscle cells

Chloride (Cl−) channels expressed in vascular smooth muscle cells (VSMC) are important to control membrane potential equilibrium, intracellular pH, cell volume maintenance, contraction, relaxation and proliferation. The present study was designed to compare the expression, regulation and function of CFTR Cl− channels in aortic VSMC from Cftr+/+ and Cftr−/− mice. Using an iodide efflux assay we demonstrated stimulation of CFTR by VIP, isoproterenol, cAMP agonists and other pharmacological activators in cultured VSMC from Cftr+/+. On the contrary, in cultured VSMC from Cftr−/− mice these agonists have no effect, showing that CFTR is the dominant Cl− channel involved in the response to cAMP mediators. Angiotensin II and the calcium ionophore A23187 stimulated Ca2+‐dependent Cl− channels in VSMCs from both genotypes. CFTR was activated in myocytes maintained in medium containing either high potassium or 5‐hydroxytryptamine (5‐HT) and was inhibited by CFTRinh‐172, glibenclamide and diphenylamine‐2,2′‐dicarboxylic acid (DPC). We also examined the mechanical properties of aortas. Arteries with or without endothelium from Cftr−/− mice became significantly more constricted (∼2‐fold) than that of Cftr+/+ mice in response to vasoactive agents. Moreover, in precontracted arteries of Cftr+/+ mice, VIP and CFTR activators induced vasorelaxation that was altered in Cftr−/− mice. Our findings suggest a novel mechanism for regulation of the vascular tone by cAMP‐dependent CFTR chloride channels in VSMC. To our knowledge this study is the first to report the phenotypic consequences of the loss of a Cl− channel on vascular reactivity.

Evidence that CFTR is expressed in rat tracheal smooth muscle cells and contributes to bronchodilation

BackgroundThe airway functions are profoundly affected in many diseases including asthma, chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF). CF the most common lethal autosomal recessive genetic disease is caused by mutations of the CFTR gene, which normally encodes a multifunctional and integral membrane protein, the CF transmembrane conductance regulator (CFTR) expressed in airway epithelial cells.MethodsTo demonstrate that CFTR is also expressed in tracheal smooth muscle cells (TSMC), we used iodide efflux assay to analyse the chloride transports in organ culture of rat TSMC, immunofluorescence study to localize CFTR proteins and isometric contraction measurement on isolated tracheal rings to observe the implication of CFTR in the bronchodilation.ResultsWe characterized three different pathways stimulated by the cAMP agonist forskolin and the isoflavone agent genistein, by the calcium ionophore A23187 and by hypo-osmotic challenge. The pharmacology of the cAMP-dependent iodide efflux was investigated in detail. We demonstrated in rat TSMC that it is remarkably similar to that of the epithelial CFTR, both for activation (using three benzo [c]quinolizinium derivatives) and for inhibition (glibenclamide, DPC and CFTRinh-172). Using rat tracheal rings, we observed that the activation of CFTR by benzoquinolizinium derivatives in TSMC leads to CFTRinh-172-sensitive bronchodilation after constriction with carbachol. An immunolocalisation study confirmed expression of CFTR in tracheal myocytes.ConclusionAltogether, these observations revealed that CFTR in the airways of rat is expressed not only in the epithelial cells but also in tracheal smooth muscle cells leading to the hypothesis that this ionic channel could contribute to bronchodilation.

Syntaxin 8 impairs trafficking of cystic fibrosis transmembrane conductance regulator (CFTR) and inhibits its channel activity

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-dependent chloride channel that mediates electrolyte transport across the luminal surface of epithelial cells. In this paper, we describe the CFTR regulation by syntaxin 8, a t-SNARE protein (target soluble N-ethylmaleimide-sensitive factor attachment protein receptor) involved in the SNARE endosomal complex. Syntaxin family members are key molecules implicated in diverse vesicle docking and membrane fusion events. We found that syntaxin 8 physically interacts with CFTR: recombinant syntaxin 8 binds CFTR in vitro and both proteins co-immunoprecipitate in HT29 cells. Syntaxin 8 regulates CFTR-mediated currents in chinese hamster ovary (CHO) cells stably expressing CFTR and syntaxin 8. Iodide efflux and whole-cell patch-clamp experiments on these cells indicate a strong inhibition of CFTR chloride current by syntaxin 8 overexpression. At the cellular level, we observed that syntaxin 8 overexpression disturbs CFTR trafficking. Confocal microscopy shows a dramatic decrease in green fluorescent protein-tagged CFTR plasma membrane staining, when syntaxin 8 is coexpressed in COS-7 cells. Using antibodies against Lamp-1, TfR or Rab11 we determined by immunofluorescence assays that both proteins are mainly accumulated in recycling endosomes. Our results evidence that syntaxin 8 contributes to the regulation of CFTR trafficking and chloride channel activity by the SNARE machinery.

A Cystic Fibrosis Respiratory Epithelial Cell Chronically Treated by Miglustat Acquires a Non–Cystic Fibrosis–Like Phenotype

Cystic fibrosis (CF) is a fatal, autosomal and recessive genetic disease that is mainly due to inactivating mutations in the chloride channel CF transmembrane conductance regulator (CFTR). Sodium hyperabsorption by the airways, profound lung inflammation, and dysregulation of calcium homeostasis, are presumably causally related to loss of CFTR-dependent chloride function in patients with CF. Miglustat (N-butyldeoxynojirimycin, Zavesca), an inhibitor of the alpha-1,2 glucosidase, has been proposed for clinical use in CF because of its effect as a corrector of the defective trafficking of F508del-CFTR. In the present study, we show that daily treatment for 2 months with low concentrations of miglustat on the human CF nasal epithelial cell line, JME/CF15 (F508del/F508del-CFTR), results in progressive, stable, reversible, and sustained correction of F508del-CFTR trafficking, down-regulation of sodium hyperabsorption, and regulation of the calcium homeostasis. In conclusion, we provide here the first evidence that a respiratory CF cell can acquire a non-CF-like phenotype when chronically treated with low concentrations of a pharmacological drug.

Discovery of novel potent ΔF508-CFTR correctors that target the nucleotide binding domain

The deletion of Phe508 (ΔF508) in the first nucleotide binding domain (NBD1) of CFTR is the most common mutation associated with cystic fibrosis. The ΔF508‐CFTR mutant is recognized as improperly folded and targeted for proteasomal degradation. Based on molecular dynamics simulation results, we hypothesized that interaction between ΔF508‐NBD1 and housekeeping proteins prevents ΔF508‐CFTR delivery to the plasma membrane. Based on this assumption we applied structure‐based virtual screening to identify new low‐molecular‐weight compounds that should bind to ΔF508‐NBD1 and act as protein–protein interaction inhibitors. Using different functional assays for CFTR activity, we demonstrated that in silico‐selected compounds induced functional expression of ΔF508‐CFTR in transfected HeLa cells, human bronchial CF cells in primary culture, and in the nasal epithelium of homozygous ΔF508‐CFTR mice. The proposed compounds disrupt keratin8‐ΔF508‐CFTR interaction in ΔF508‐CFTR HeLa cells. Structural analysis of ΔF508‐NBD1 in the presence of these compounds suggests their binding to NBD1. We conclude that our strategy leads to the discovery of new compounds that are among the most potent correctors of ΔF508‐CFTR trafficking defect known to date.

Discovery of pyrrolo[2,3-b]pyrazines derivatives as submicromolar affinity activators of wild type, G551D, and F508del cystic fibrosis transmembrane conductance regulator chloride channels

The cystic fibrosis transmembrane conductance regulator (CFTR) represents the main Cl- channel in the apical membrane of epithelial cells for cAMP-dependent Cl- secretion. Here we report on the synthesis and screening of a small library of 6-phenylpyrrolo[2,3-b]pyrazines (named RP derivatives) evaluated as activators of wild-type CFTR, G551D-CFTR, and F508del-CFTR Cl- channels. Iodide efflux and whole-cell patch-clamp recordings analysis identified RP107 [7-n-butyl-6-(4-hydroxyphenyl)[5H]-pyrrolo[2,3-b]pyrazine] as a submicromolar activator of wild-type (WT)-CFTR [human airway epithelial Calu-3 and WT-CFTR-Chinese hamster ovary (CHO) cells], G551D-CFTR (G551D-CFTR-CHO cells), and F508del-CFTR (in temperature-corrected human airway epithelial F508del/F508del CF15 cells). The structural analog RP108 [7-n-butyl-6-(4-chlorophenyl)[5H]pyrrolo[2,3-b]pyrazine], contrary to RP107, was a less potent activator only at micromolar concentrations. RP107 and RP108 did not have any effect on the cellular cAMP level. Activation was potentiated by low concentration of forskolin and inhibited by glibenclamide and CFTRinh-172 [3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl-)methylene]-2-thioxo-4-thiazolidinone]but not by calixarene or DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid). Finally, we found significant stimulation of short circuit current (Isc) by RP107 (EC50 = 89 nM) and RP108 (EC50 = 103 μM) on colon of Cftr+/+ but not of Cftr-/- mice mounted in Ussing chamber. Stimulation of Isc was inhibited by glibenclamide but not affected by DIDS. These results show that RP107 stimulates wild-type CFTR and mutated CFTR, with submicromolar affinity by a cAMP-independent mechanism. Our preliminary structure-activity relationship study identified 4-hydroxyphenyl and 7-n-butyl as determinants required for activation of CFTR. The potency of these agents indicates that compounds in this class may be of therapeutic benefit in CFTR-related diseases, including cystic fibrosis.