NHE-RF1 protein rescues DeltaF508-CFTR function

In cystic fibrosis (CF), the DeltaF508-CFTR anterograde trafficking from the endoplasmic reticulum to the plasma membrane is inefficient. New strategies for increasing the delivery of DeltaF508-CFTR to the apical membranes are thus pathophysiologically relevant targets to study for CF treatment. Recent studies have demonstrated that PDZ-containing proteins play an essential role in determining polarized plasma membrane expression of ionic transporters. In the present study we have hypothesized that the PDZ-containing protein NHE-RF1, which binds to the carboxy terminus of CFTR, rescues DeltaF508-CFTR expression in the apical membrane of epithelial cells. The plasmids encoding DeltaF508-CFTR and NHE-RF1 were intranuclearly injected in A549 or Madin-Darby canine kidney (MDCK) cells, and DeltaF508-CFTR channel activity was functionally assayed using SPQ fluorescent probe. Cells injected with DeltaF508-CFTR alone presented a low chloride channel activity, whereas its coexpression with NHE-RF1 significantly increased both the basal and forskolin-activated chloride conductances. This last effect was lost with DeltaF508-CFTR deleted of its 13 last amino acids or by injection of a specific NHE-RF1 antisense oligonucleotide, but not by NHE-RF1 sense oligonucleotide. Immunocytochemical analysis performed in MDCK cells transiently transfected with DeltaF508-CFTR further revealed that NHE-RF1 specifically determined the apical plasma membrane expression of DeltaF508-CFTR but not that of a trafficking defective mutant potassium channel (KCNQ1). These data demonstrate that the modulation of the expression level of CFTR protein partners, like NHE-RF1, can rescue DeltaF508-CFTR activity.     

Myosin Vb is required for trafficking of the cystic fibrosis transmembrane conductance regulator in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl(-) secretion across fluid-transporting epithelia is regulated, in part, by modulating the number of CFTR Cl(-) channels in the plasma membrane by adjusting CFTR endocytosis and recycling. However, the mechanisms that regulate CFTR recycling in airway epithelial cells remain unknown, at least in part, because the recycling itineraries of CFTR in these cells are incompletely understood. In a previous study, we demonstrated that CFTR undergoes trafficking in Rab11a-specific apical recycling endosomes in human airway epithelial cells. Myosin Vb is a plus-end-directed, actin-based mechanoenzyme that facilitates protein trafficking in Rab11a-specific recycling vesicles in several cell model systems. There are no published studies examining the role of myosin Vb in airway epithelial cells. Thus, the goal of this study was to determine whether myosin Vb facilitates CFTR recycling in polarized human airway epithelial cells. Endogenous CFTR formed a complex with endogenous myosin Vb and Rab11a. Silencing myosin Vb by RNA-mediated interference decreased the expression of wild-type CFTR and DeltaF508-CFTR in the apical membrane and decreased CFTR-mediated Cl(-) secretion across polarized human airway epithelial cells. A recombinant tail domain fragment of myosin Vb attenuated the plasma membrane expression of CFTR by arresting CFTR recycling. The dominant-negative effect was dependent on the ability of the myosin Vb tail fragment to interact with Rab11a. Taken together, these data indicate that myosin Vb is required for CFTR recycling in Rab11a-specific apical recycling endosomes in polarized human airway epithelial cells.     

Nanomolar affinity small molecule correctors of defective Delta F508-CFTR chloride channel gating

Deletion of Phe-508 (Delta F508) is the most common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) causing cystic fibrosis. Delta F508-CFTR has defects in both channel gating and endoplasmic reticulum-to-plasma membrane processing. We identified six novel classes of high affinity potentiators of defective Delta F508-CFTR Cl- channel gating by screening 100,000 diverse small molecules. Compounds were added 15 min prior to assay of iodide uptake in epithelial cells co-expressing Delta F508-CFTR and a high sensitivity halide indicator (YFP-H148Q/I152L) in which Delta F508-CFTR was targeted to the plasma membrane by culture at 27 degrees C for 24 h. Thirty-two compounds with submicromolar activating potency were identified; most had tetrahydrobenzothiophene, benzofuran, pyramidinetrione, dihydropyridine, and anthraquinone core structures (360-480 daltons). Further screening of >1000 structural analogs revealed tetrahydrobenzothiophenes that activated DeltaF508-CFTR Cl- conductance reversibly with Kd < 100 nm. Single-cell voltage clamp analysis showed characteristic CFTR currents after Delta F508-CFTR activation. Activation required low concentrations of a cAMP agonist, thus mimicking the normal physiological response. A Bayesian computational model was developed using tetrahydrobenzothiophene structure-activity data, yielding insight into the physical character and structural features of active and inactive potentiators and successfully predicting the activity of structural analogs. Efficient potentiation of defective Delta F508-CFTR gating was also demonstrated in human bronchial epithelial cells from a Delta F508 cystic fibrosis subject after 27 degrees C temperature rescue. In conjunction with correctors of defective Delta F508-CFTR processing, small molecule potentiators of defective Delta F508-CFTR gating may be useful for therapy of cystic fibrosis caused by the Delta F508 mutation.     

Genistein restores functional interactions between Delta F508-CFTR and ENaC in Xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its Cl(-) channel properties, has regulatory interactions with other epithelial ion channels including the epithelial Na(+) channel (ENaC). Both the open probability and surface expression of wild type CFTR Cl(-) channels are increased significantly when CFTR is co-expressed in Xenopus oocytes with alphabetagamma-ENaC, and conversely, the activity of ENaC is inhibited following wild type CFTR activation. Using the Xenopus oocyte expression system, a lack of functional regulatory interactions between DeltaF508-CFTR and ENaC was observed following activation of DeltaF508-CFTR by forskolin and isobutylmethylxanthine (IBMX). Whole cell currents in oocytes expressing ENaC alone decreased in response to genistein but increased in response to a combination of forskolin and IBMX followed by genistein. In contrast, ENaC currents in oocytes co-expressing ENaC and DeltaF508-CFTR remained stable following stimulation with forskolin/IBMX/genistein. Furthermore, co-expression of DeltaF508-CFTR with ENaC enhanced the forskolin/IBMX/genistein-mediated activation of DeltaF508-CFTR. Our data suggest that genistein restores regulatory interactions between DeltaF508-CFTR and ENaC and that combinations of protein repair agents, such as 4-phenylbutyrate and genistein, may be necessary to restore DeltaF508-CFTR function in vivo.     

Diffusional mobility of the cystic fibrosis transmembrane conductance regulator mutant, delta F508-CFTR, in the endoplasmic reticulum measured by photobleaching of GFP-CFTR chimeras

Mutations in the cystic fibrosis transmembrane conductance regulator protein (CFTR) cause cystic fibrosis. The most common disease-causing mutation, DeltaF508, is retained in the endoplasmic reticulum (ER) and is unable to function as a plasma membrane chloride channel. To investigate whether the ER retention of DeltaF508-CFTR is caused by immobilization and/or aggregation, we have measured the diffusional mobility of green fluorescent protein (GFP) chimeras of wild type (wt)-CFTR and DeltaF508-CFTR by fluorescence recovery after photobleaching. GFP-labeled DeltaF508-CFTR was localized in the ER and wt-CFTR in the plasma membrane and intracellular membranes in transfected COS7 and Chinese hamster ovary K1 cells. Both chimeras localized to the ER after brefeldin A treatment. Spot photobleaching showed that CFTR diffusion (diffusion coefficient approximately 10(-9) cm(2)/s) was not significantly slowed by the DeltaF508 mutation and that nearly all wt-CFTR and DeltaF508-CFTR diffused throughout the ER without restriction. Stabilization of molecular chaperone interactions by ATP depletion produced remarkable DeltaF508-CFTR immobilization ( approximately 50%) and slowed diffusion (6.5 x 10(-10) cm(2)/s) but had little effect on wt-CFTR. Fluorescence depletion experiments revealed that the immobilized DeltaF508-CFTR in ATP-depleted cells remained in an ER pattern. The mobility of wt-CFTR and DeltaF508-CFTR was reduced by maneuvers that alter CFTR processing or interactions with molecular chaperones, including tunicamycin, geldanamycin, and lactacystin. Photobleaching of the fluorescent ER lipid diOC(4)(3) showed that neither ER restructuring nor fragmentation during these maneuvers was responsible for the slowing and immobilization of CFTR. These results suggest that (a) the ER retention of DeltaF508-CFTR is not due to restricted ER mobility, (b) the majority of DeltaF508-CFTR is not aggregated or bound to slowly moving membrane proteins, and (c) DeltaF508-CFTR may interact to a greater extent with molecular chaperones than does wt-CFTR.     

Calcium-pump inhibitors induce functional surface expression of Delta F508-CFTR protein in cystic fibrosis epithelial cells

The most common mutation in cystic fibrosis, Delta F508, results in a cystic fibrosis transmembrane conductance regulator (CFTR) protein that is retained in the endoplasmic reticulum (ER). Retention is dependent upon chaperone proteins, many of which require Ca(++) for optimal activity. Interfering with chaperone activity by depleting ER Ca(++) stores might allow functional Delta F508-CFTR to reach the cell surface. We exposed several cystic fibrosis cell lines to the ER Ca(++) pump inhibitor thapsigargin and evaluated surface expression of Delta F508-CFTR. Treatment released ER-retained Delta F508-CFTR to the plasma membrane, where it functioned effectively as a Cl(-) channel. Treatment with aerosolized calcium-pump inhibitors reversed the nasal epithelial potential defect observed in a mouse model of Delta F508-CFTR expression. Thus, ER calcium-pump inhibitors represent a potential target for correcting the cystic fibrosis defect.     

The DeltaF508-CFTR mutation results in increased biofilm formation by Pseudomonas aeruginosa by increasing iron availability

Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing DeltaF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and DeltaF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on DeltaF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing DeltaF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The DeltaF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.     

Annexin A5 increases the cell surface expression and the chloride channel function of the DeltaF508-cystic fibrosis transmembrane regulator

Cystic fibrosis (CF) is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. In CF, the most common mutant DeltaF508-CFTR is misfolded, is retained in the ER and is rapidly degraded. If conditions could allow DeltaF508-CFTR to reach and to stabilize in the plasma membrane, it could partially correct the CF defect. We have previously shown that annexin V (anxA5) binds to both the normal CFTR and the DeltaF508-CFTR in a Ca(2+)-dependent manner and that it regulates the chloride channel function of Wt-CFTR through its membrane integration. Our aim was to extend this finding to the DeltaF508-CFTR. Because some studies show that thapsigargin (Tg) increases the DeltaF508-CFTR apical expression and induces an increased [Ca(2+)](i) and because anxA5 relocates and binds to the plasma membrane in the presence of Ca(2+), we hypothesized that the Tg effect upon DeltaF508-CFTR function could involve anxA5. Our results show that raised anxA5 expression induces an augmented function of DeltaF508-CFTR due to its increased membrane localization. Furthermore, we show that the Tg effect involves anxA5. Therefore, we suggest that anxA5 is a potential therapeutic target in CF.     

Novel short chain fatty acids restore chloride secretion in cystic fibrosis

Phenylalanine deletion at position 508 of the cystic fibrosis transmembrane conductance regulator (DeltaF508-CFTR), the most common mutation in cystic fibrosis (CF), causes a misfolded protein exhibiting partial chloride conductance and impaired trafficking to the plasma membrane. 4-Phenylbutyrate corrects defective DeltaF508-CFTR trafficking in vitro, but is not clinically efficacious. From a panel of short chain fatty acid derivatives, we showed that 2,2-dimethyl-butyrate (ST20) and alpha-methylhydrocinnamic acid (ST7), exhibiting high oral bioavailability and sustained plasma levels, correct the DeltaF508-CFTR defect. Pre-incubation (>or=6h) of CF IB3-1 airway cells with >or=1mM ST7 or ST20 restored the ability of 100microM forskolin to stimulate an (125)I(-) efflux. This efflux was fully inhibited by NPPB, DPC, or glibenclamide, suggesting mediation through CFTR. Partial inhibition by DIDS suggests possible contribution from an additional Cl(-) channel regulated by CFTR. Thus, ST7 and ST20 offer treatment potential for CF caused by the DeltaF508 mutation.     

Protein kinase CK2, cystic fibrosis transmembrane conductance regulator, and the deltaF508 mutation: F508 deletion disrupts a kinase-binding site

Deletion of phenylalanine 508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common mutation in cystic fibrosis. The F508 region lies within a surface-exposed loop that has not been assigned any interaction with associated proteins. Here we demonstrate that the pleiotropic protein kinase CK2 that controls protein trafficking, cell proliferation, and development binds wild-type CFTR near F508 and phosphorylates NBD1 at Ser-511 in vivo and that mutation of Ser-511 disrupts CFTR channel gating. Importantly, the interaction of CK2 with NBD1 is selectively abrogated by the DeltaF508 mutation without disrupting four established CFTR-associated kinases and two phosphatases. Loss of CK2 association is functionally corroborated by the insensitivity of DeltaF508-CFTR to CK2 inhibition, the absence of CK2 activity in DeltaF508 CFTR-expressing cell membranes, and inhibition of CFTR channel activity by a peptide that mimics the F508 region of CFTR (but not the equivalent DeltaF508 peptide). Disruption of this CK2-CFTR association is the first described DeltaF508-dependent protein-protein interaction that provides a new molecular paradigm in the most frequent form of cystic fibrosis.     

PDZ domain interaction controls the endocytic recycling of the cystic fibrosis transmembrane conductance regulator

The C terminus of CFTR contains a PDZ interacting domain that is required for the polarized expression of cystic fibrosis transmembrane conductance regulator (CFTR) in the apical plasma membrane of polarized epithelial cells. To elucidate the mechanism whereby the PDZ interacting domain mediates the polarized expression of CFTR, Madin-Darby canine kidney cells were stably transfected with wild type (wt-CFTR) or C-terminally truncated human CFTR (CFTR-DeltaTRL). We tested the hypothesis that the PDZ interacting domain regulates sorting of CFTR from the Golgi to the apical plasma membrane. Pulse-chase studies in combination with domain-selective cell surface biotinylation revealed that newly synthesized wt-CFTR and CFTR-DeltaTRL were targeted equally to the apical and basolateral membranes in a nonpolarized fashion. Thus, the PDZ interacting domain is not an apical sorting motif. Deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane from approximately 24 to approximately 13 h but had no effect on the half-life of CFTR in the basolateral membrane. Thus, the PDZ interacting domain is an apical membrane retention motif. Next, we examined the hypothesis that the PDZ interacting domain affects the apical membrane half-life of CFTR by altering its endocytosis and/or endocytic recycling. Endocytosis of wt-CFTR and CFTR-DeltaTRL did not differ. However, endocytic recycling of CFTR-DeltaTRL was decreased when compared with wt-CFTR. Thus, deletion of the PDZ interacting domain reduced the half-life of CFTR in the apical membrane by decreasing CFTR endocytic recycling. Our results identify a new role for PDZ proteins in regulating the endocytic recycling of CFTR in polarized epithelial cells.     

Rescue of DeltaF508-CFTR trafficking and gating in human cystic fibrosis airway primary cultures by small molecules

Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in cftr, a gene encoding a PKA-regulated Cl(-) channel. The most common mutation results in a deletion of phenylalanine at position 508 (DeltaF508-CFTR) that impairs protein folding, trafficking, and channel gating in epithelial cells. In the airway, these defects alter salt and fluid transport, leading to chronic infection, inflammation, and loss of lung function. There are no drugs that specifically target mutant CFTR, and optimal treatment of CF may require repair of both the folding and gating defects. Here, we describe two classes of novel, potent small molecules identified from screening compound libraries that restore the function of DeltaF508-CFTR in both recombinant cells and cultures of human bronchial epithelia isolated from CF patients. The first class partially corrects the trafficking defect by facilitating exit from the endoplasmic reticulum and restores DeltaF508-CFTR-mediated Cl(-) transport to more than 10% of that observed in non-CF human bronchial epithelial cultures, a level expected to result in a clinical benefit in CF patients. The second class of compounds potentiates cAMP-mediated gating of DeltaF508-CFTR and achieves single-channel activity similar to wild-type CFTR. The CFTR-activating effects of the two mechanisms are additive and support the rationale of a drug discovery strategy based on rescue of the basic genetic defect responsible for CF.     

Role for phospholipid interactions in the trafficking defect of Delta F508-CFTR

Cystic fibrosis commonly occurs as a consequence of the DeltaF508 mutation in the first nucleotide binding fold domain (NBF-1) of CFTR. The mutation causes retention of the mutant CFTR molecule in the endoplasmic reticulum, and this aberrant trafficking event is believed to be due to defective interactions between the mutant NBF-1 domain and other cellular factors in the endoplasmic reticulum. Since the NBF-1 domain has been shown to interact with membranes, we wanted to investigate whether NBF-1 and CFTR interactions with specific phospholipid chaperones might play a role in trafficking. We have found that the recombinant wild-type NBF-1 interacts selectively with phosphatidylserine (PS) rather than phosphatidylcholine (PC). By contrast, NBF-1 carrying the DeltaF508 mutation loses the ability to discriminate between these two phospholipids. In cells expressing DeltaF508-CFTR, replacement of PC by noncharged analogues results in an absolute increase in CFTR expression. In addition, we detected progressive expression of higher molecular weight CFTR forms. Thus, phospholipid chaperones may be important for CFTR trafficking, and contribute to the pathology of cystic fibrosis.     

Comparative processing and function of human and ferret cystic fibrosis transmembrane conductance regulator

The most common cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation is ΔF508, and this causes cystic fibrosis (CF). New CF models in the pig and ferret have been generated that develop lung, pancreatic, liver, and intestinal pathologies that reflect disease in CF patients. Species-specific biology in the processing of CFTR has demonstrated that pig and mouse ΔF508-CFTR proteins are more effectively processed to the apical membrane of airway epithelia than human ΔF508-CFTR. The processing behavior of ferret WT- and ΔF508-CFTR proteins remains unknown, and such information is important to predicting the utility of a ΔF508-CFTR ferret. To this end, we sought to compare processing, membrane stability, and function of human and ferret WT- and ΔF508-CFTR proteins in a heterologous expression system using HT1080, HEK293T, BHK21, and Cos7 cells as well as human and ferret CF polarized airway epithelia. Analysis of the protein processing and stability by metabolic pulse-chase and surface On-Cell Western blots revealed that WT-fCFTR half-life and membrane stability were increased relative to WT-hCFTR. Furthermore, in BHK21, Cos7, and CuFi cells, human and ferret ΔF508-CFTR processing was negligible, whereas low levels of processing of ΔF508-fCFTR could be seen in HT1080 and HEK293T cells. Only the WT-fCFTR, but not ΔF508-fCFTR, produced functional cAMP-inducible chloride currents in both CF human and ferret airway epithelia. Further elucidation of the mechanism responsible for elevated fCFTR protein stability may lead to new therapeutic approaches to augment CFTR function. These findings also suggest that generation of a ferret CFTR(ΔF508/ΔF508) animal model may be useful.     

Targeting CAL as a negative regulator of DeltaF508-CFTR cell-surface expression: an RNA interference and structure-based mutagenetic approach

PDZ domains are ubiquitous peptide-binding modules that mediate protein-protein interactions in a wide variety of intracellular trafficking and localization processes. These include the pathways that regulate the membrane trafficking and endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel mutated in patients with cystic fibrosis. Correspondingly, a number of PDZ proteins have now been identified that directly or indirectly interact with the C terminus of CFTR. One of these is CAL, whose overexpression in heterologous cells directs the lysosomal degradation of WT-CFTR in a dose-dependent fashion and reduces the amount of CFTR found at the cell surface. Here, we show that RNA interference targeting endogenous CAL specifically increases cell-surface expression of the disease-associated DeltaF508-CFTR mutant and thus enhances transepithelial chloride currents in a polarized human patient bronchial epithelial cell line. We have reconstituted the CAL-CFTR interaction in vitro from purified components, demonstrating for the first time that the binding is direct and allowing us to characterize its components biochemically and biophysically. To test the hypothesis that inhibition of the binding site could also reverse CAL-mediated suppression of CFTR, a three-dimensional homology model of the CAL.CFTR complex was constructed and used to generate a CAL mutant whose binding pocket is correctly folded but has lost its ability to bind CFTR. Although produced at the same levels as wild-type protein, the mutant does not affect CFTR expression levels. Taken together, our data establish CAL as a candidate therapeutic target for correction of post-maturational trafficking defects in cystic fibrosis.     

Increased functional cell surface expression of CFTR and DeltaF508-CFTR by the anthracycline doxorubicin

Cystic fibrosis (CF) is a disease that is caused by mutations within the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The most common mutation, DeltaF508, accounts for 70% of all CF alleles and results in a protein that is defective in folding and trafficking to the cell surface. However, DeltaF508-CFTR is functional when properly localized. We report that a single, noncytotoxic dose of the anthracycline doxorubicin (Dox, 0.25 microM) significantly increased total cellular CFTR protein expression, cell surface CFTR protein expression, and CFTR-associated chloride secretion in cultured T84 epithelial cells. Dox treatment also increased DeltaF508-CFTR cell surface expression and DeltaF508-CFTR-associated chloride secretion in stably transfected Madin-Darby canine kidney cells. These results suggest that anthracycline analogs may be useful for the clinical treatment of CF.     

Pseudomonas aeruginosa inhibits endocytic recycling of CFTR in polarized human airway epithelial cells

The most common mutation in the CFTR gene in individuals with cystic fibrosis (CF), F508, leads to the absence of CFTR Cl^– channels in the apical plasma membrane, which in turn results in impairment of mucociliary clearance, the first line of defense against inhaled bacteria. Pseudomonas aeruginosa is particularly successful at colonizing and chronically infecting the lungs and is responsible for the majority of morbidity and mortality in patients with CF. Rescue of F508-CFTR by reduced temperature or chemical means reveals that the protein is at least partially functional as a Cl^– channel. Thus current research efforts have focused on identification of drugs that restore the presence of CFTR in the apical membrane to alleviate the symptoms of CF. Because little is known about the effects of P. aeruginosa on CFTR in the apical membrane, whether P. aeruginosa will affect the efficacy of new drugs designed to restore the plasma membrane expression of CFTR is unknown. Accordingly, the objective of the present study was to determine whether P. aeruginosa affects CFTR-mediated Cl^– secretion in polarized human airway epithelial cells. We report herein that a cell-free filtrate of P. aeruginosa reduced CFTR-mediated transepithelial Cl^– secretion by inhibiting the endocytic recycling of CFTR and thus the number of WT-CFTR and F508-CFTR Cl^– channels in the apical membrane in polarized human airway epithelial cells. These data suggest that chronic infection with P. aeruginosa may interfere with therapeutic strategies aimed at increasing the apical membrane expression of F508-CFTR. cystic fibrosis     

Use of Kinase Inhibitors to Correct {Delta}F508-CFTR Function

The most common mutation in cystic fibrosis (CF) is a deletion of Phe at position 508 (ΔF508-CFTR). ΔF508-CFTR is a trafficking mutant that is retained in the ER, unable to reach the plasma membrane. To identify compounds and drugs that rescue this trafficking defect, we screened a kinase inhibitor library enriched for small molecules already in the clinic or in clinical trials for the treatment of cancer and inflammation, using our recently developed high-content screen technology (Trzcinska-Daneluti et al. Mol. Cell. Proteomics 8:780, 2009). The top hits of the screen were further validated by (1) biochemical analysis to demonstrate the presence of mature (Band C) ΔF508-CFTR, (2) flow cytometry to reveal the presence of ΔF508-CFTR at the cell surface, (3) short-circuit current (Isc) analysis in Ussing chambers to show restoration of function of the rescued ΔF508-CFTR in epithelial MDCK cells stably expressing this mutant (including EC(50) determinations), and importantly (4) Isc analysis of Human Bronchial Epithelial (HBE) cells harvested from homozygote ΔF508-CFTR transplant patients. Interestingly, several inhibitors of receptor Tyr kinases (RTKs), such as SU5402 and SU6668 (which target FGFRs, VEGFR, and PDGFR) exhibited strong rescue of ΔF508-CFTR, as did several inhibitors of the Ras/Raf/MEK/ERK or p38 pathways (e.g. (5Z)-7-oxozeaenol). Prominent rescue was also observed by inhibitors of GSK-3β (e.g. GSK-3β Inhibitor II and Kenpaullone). These results identify several kinase inhibitors that can rescue ΔF508-CFTR to various degrees, and suggest that use of compounds or drugs already in the clinic or in clinical trials for other diseases can expedite delivery of treatment for CF patients.     

S-Nitrosoglutathione induces functional DeltaF508-CFTR in airway epithelial cells

S-Nitrosoglutathione (GSNO) is an endogenous bronchodilator levels of which are reduced in the airways of cystic fibrosis (CF) patients. GSNO has recently been shown to increase maturation of CFTR in CF cell lines at physiological concentrations. The ability of S-nitrosoglutathione to direct the DeltaF508-CFTR to the plasma membrane and restore the function of the cAMP-dependent chloride transport in cultured human airway epithelial cells has been studied. Immunocytochemistry showed a time- and dose-dependent increase of apically located CFTR after GSNO treatment. Chloride transport studies with the fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) showed that GSNO was able to induce a fourfold increase of cAMP-dependent chloride transport. Our data and the fact that endogenous GSNO levels are lower in the airways of CF patients make GSNO an interesting candidate for pharmacological treatment of cystic fibrosis.     

Beta-oestradiol rescues DeltaF508CFTR functional expression in human cystic fibrosis airway CFBE41o- cells through the up-regulation of NHERF1.

BACKGROUND INFORMATION: CF (cystic fibrosis) is a disease caused by mutations within the CFTR (CF transmembrane conductance regulator) gene. The most common mutation, DeltaF508 (deletion of Phe-508), results in a protein that is defective in folding and trafficking to the cell surface but is functional if properly localized in the plasma membrane. We have recently demonstrated that overexpression of the PDZ protein NHERF1 (Na(+)/H(+)-exchanger regulatory factor 1) in CF airway cells induced both a redistribution of DeltaF508CFTR from the cytoplasm to the apical membrane and the PKA (protein kinase A)-dependent activation of DeltaF508CFTR-dependent chloride secretion. In view of the potential importance of the targeted up-regulation of NHERF1 in a therapeutic context, and since it has been demonstrated that oestrogen treatment increases endogenous NHERF1 expression, we tested the hypothesis that oestrogen treatment can increase NHERF1 expression in a human bronchiolar epithelial CF cell line, CFBE41o(-), with subsequent rescue of apical DeltaF508CFTR chloride transport activity. RESULTS: We found that CFBE41o(-) cells do express ERs (oestrogen receptors) in the nuclear fraction and that beta-oestradiol treatment was able to significantly rescue DeltaF508CFTR-dependent chloride secretion in CFBE41o(-) cell monolayers with a peak between 6 and 12 h of treatment, demonstrating that the DeltaF508CFTR translocated to the apical membrane can function as a cAMP-responsive channel, with a significant increase in chloride secretion noted at 1 nM beta-oestradiol and a maximal effect observed at 10 nM. Importantly, knock-down of NHERF1 expression by transfection with siRNA (small interfering RNA) for NHERF1 inhibited the beta-oestradiol-dependent increase in DeltaF508CFTR protein expression levels and completely prevented the beta-oestradiol-dependent rescue of DeltaF508CFTR transport activity. CONCLUSIONS: These results demonstrate that beta-oestradiol-dependent up-regulation of NHERF1 significantly increases DeltaF508CFTR functional expression in CFBE41o(-) cells.