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Am J Physiol Lung Cell Mol Physiol 292: L1085-L1094, 2007. First published January 19, 2007; doi:10.1152/ajplung.00445.2005
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NHE-RF1 protein rescues {Delta}F508-CFTR function

Florian Bossard ,1,* Amal Robay,1,* Gilles Toumaniantz,1,3 Shehrazade Dahimene,1 Frédéric Becq,2 Jean Merot,1 and Chantal Gauthier1,3

1Institut National de la Santé et de la Recherche Médicale Unité 533, l' Institut du Thorax, Faculté de Médecine, Nantes; 2Centre National de la Recherche Scientifique Unité Mixte de Recherches 6187, Institut de Physiologie et Biologie Cellulaires, Université de Poitiers, Poitiers; and 3Faculté des Sciences et Techniques, Université de Nantes, Nantes, France

Submitted 19 October 2005 ; accepted in final form 6 December 2006


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In cystic fibrosis (CF), the {Delta}F508-CFTR anterograde trafficking from the endoplasmic reticulum to the plasma membrane is inefficient. New strategies for increasing the delivery of {Delta}F508-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 {Delta}F508-CFTR expression in the apical membrane of epithelial cells. The plasmids encoding {Delta}F508-CFTR and NHE-RF1 were intranuclearly injected in A549 or Madin-Darby canine kidney (MDCK) cells, and {Delta}F508-CFTR channel activity was functionally assayed using SPQ fluorescent probe. Cells injected with {Delta}F508-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 {Delta}F508-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 {Delta}F508-CFTR further revealed that NHE-RF1 specifically determined the apical plasma membrane expression of {Delta}F508-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 {Delta}F508-CFTR activity.

cystic fibrosis; {Delta}F508 cystic fibrosis transmembrane conductance regulator; Na+/H+ exchanger regulatory factor isoform 1; polarized expression; traffic

THE CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR), a cAMP-activated chloride (Cl–) channel, is expressed in a wide variety of epithelial cells, including airway and kidney (10, 42). Mutations in the CFTR gene lead to the genetic disease cystic fibrosis (CF), a lethal autosomal recessive disorder. The major disease-causing mutation of CF is a deletion of phenylalanine at position 508 ({Delta}F508) in the cytoplasmic domain known as the first nucleotide binding fold (NBF1). This mutation accounts for nearly 70% of the CF alleles (46), and ~90% of CF patients carry at least one {Delta}F508-CFTR allele (6). Nascent wild-type CFTR (wtCFTR) is translated and core-glycosylated in the endoplasmic reticulum. A fraction (25–40%) of the core-glycosylated CFTR protein (25, 49) is transported to the Golgi apparatus, where glycosylation is further processed in mature CFTR targeted to the plasma membrane (5). In contrast to wtCFTR, nearly all {Delta}F508-CFTR is retained in the endoplasmic reticulum as a core-glycosylated form, which is sorted to the ubiquitin-proteasome degradation pathway instead of the Golgi apparatus (5, 19). However, when {Delta}F508-CFTR is located at the plasma membrane, it is functional and presents a partial cAMP-activated Cl– channel activity (9). Thus identification of strategies aimed at increasing {Delta}F508-CFTR anterograde trafficking and channel activity to the apical plasma membrane of airway epithelial cells would have important therapeutic implications for treating CF.

PDZ domains are modular protein interaction domains that play a role in protein targeting and protein complex assembly. These domains of ~90 amino acids are known by the acronym of the first three PDZ-containing proteins identified: the postsynaptic protein PSD-95/SAP90, the Drosophila septate junction protein Discs-large, and the tight junction protein ZO-1 (18). PDZ-containing proteins are typically involved in the assembly of supramolecular complexes that perform localized signaling functions at particular subcellular locations. Also, it was established that PDZ domain-based interactions are essential for polarized distribution of numerous membrane proteins in neurons and epithelial cells (21, 40). Organization around a PDZ-based scaffold allows the stable localization of interacting proteins and enhances the rate and fidelity of signal transduction within the complex. Some PDZ-containing proteins are more dynamically regulated in distribution and also may be involved in the trafficking of interacting proteins within the cell (40).

The last four amino acids of CFTR (Asp-Thr-Arg-Leu) constitute a consensus sequence known to bind to PDZ domain proteins. The Na+/H+ exchanger regulatory factor isoform 1, NHE-RF1 [also called EBP50, ERM-binding phosphoprotein (50)], which contains two PDZ domains, is able to bind to the COOH terminus of CFTR through its PDZ1 domain (41, 47), whereas the PDZ2 domain remains available to interact with other proteins (30), including another CFTR channel when NHE-RF1 is bound to ezrin (23). Because NHE-RF1 associates with ezrin, which binds to the regulatory subunit of protein kinase A (PKA) (12), it has been hypothesized that NHE-RF1 anchors CFTR to the cytoskeleton at a subapical compartment, targeting PKA near CFTR (41). NHE-RF1 plays a key role in the polarization of CFTR to the apical plasma membrane in epithelial cells (32). NHE-RF1 binding to CFTR also increases the open probability of CFTR channel (38). In addition, deletion of the PDZ-interacting domain of CFTR reduced its half-life at the apical membrane of polarized epithelial cells by decreasing CFTR recycling from endosomes. This suggests that NHE-RF1 presents an apical membrane retention motif (3).

Based on these data, the aim of the present study was to determine the consequences of NHE-RF1 overexpression on the trafficking and Cl– channel activity of the {Delta}F508-CFTR protein in a recombinant system. We demonstrated that NHE-RF1 overexpression was able to restore the Cl– channel activity of {Delta}F508-CFTR in nonpolarized (A549) as well as in polarized Madin-Darby canine kidney (MDCK) epithelial cells. This effect was dependent on direct NHE-RF1 and CFTR interactions, because {Delta}F508-CFTR missing its 13 COOH-terminal amino acids was not activated by NHE-RF1 overexpression. This result was associated with an increased apical membrane expression of {Delta}F508-CFTR in MDCK cells.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture. The human lung epithelium-derived cell line (A549) was provided by the American Type Culture Collection (Rockville, MD). A549 cells were cultured as previously reported (2). MDCK type II epithelial cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). All cells were cultured at 37°C in a 5% CO2 humidified atmosphere.

Plasmids. Transgene cDNAs were subcloned into pcDNA3 or pcb6 mammalian expression vectors under the control of a cytomegalovirus promoter. The pcDNA3-CFTR and pcDNA3-{Delta}F508-CFTR plasmids (a gift from J. Ricardo, Lisbon, Portugal) encode the human wtCFTR and the mutated {Delta}F508-CFTR proteins, respectively. To construct the {Delta}F508-K1468X mutant plasmid, the KpnI/HpaI restriction fragment of the pcDNA3-{Delta}F508-CFTR plasmid, containing the {Delta}F508 mutation, was cloned in frame in pcDNA3-CFTR-K1468X. The double mutation was verified by sequencing.

The pcDNA3-NHE-RF1 plasmid (a gift from V. Ramesh, Boston, MA) encodes the human NHE-RF1 protein. The pcb6-E1Q1 plasmid encodes a human fusion protein KCNE1-KCNQ1 exposing an extracellular VSV tag. KCNQ1 and KCNE1 are an apical potassium channel and its transmembrane regulatory subunit, respectively; they are responsible for the IKs potassium current. The pcb6-P117L plasmid encodes a mutant of KCNE1-KCNQ1 presenting a trafficking defect (7).

PCR experiments. MDCK cells were grown to confluence, and total RNAs were extracted following the modified guanidinium-thiocyanate-phenol-chloroform method. Total cDNAs were synthetized using the murine Moloney leukemia virus reverse transcriptase (GIBCO BRL, Villiers-Le-Bel, France). PCR products were generated using specific NHE-RF1 (5'-GAGACCAAGCTGCTGGTG-3' sense and 5'-GGCCAGGGAGATGTTGAAG-3' antisense) and CFTR primers (5'-AATGTAACAGCCTTCTGGGAG-3' sense and 5'-GTTGGCATGCTTTGATGAC-3' antisense) targeting homologous regions of canine and human sequences. NHE-RF1 or CFTR encoding plasmids were used as positive controls, and H2O as a negative control.

Sense and antisense oligonucleotides for NHE-RF1. The sense and antisense oligonucleotides purchased from Genosis were 5'-ATGAGCGCGGACGCAGCGGC-3' and 5'-GCCGCTGCGTCCGCGCTCAT-3', respectively. The antisense oligoprobe was designed to match the NHE-RF1 initiation ATG position (underlined sequence).

Intranuclear injection of plasmids. For functional assays, cells were microinjected with plasmids (30 µg/ml for human wtCFTR and {Delta}F508-CFTR and 50 µg/ml for human NHE-RF1 and sense and antisense oligonucleotides of NHE-RF1) at 1 day after plating on glass coverslips (Nunclon; InterMed Nunc, Roskilde, Denmark). Our protocol to intranuclearly microinject individual cells has been reported in detail elsewhere (29). Plasmids were diluted in an injection buffer made of 50 mM HEPES, 50 mM NaOH, and 40 mM NaCl, pH 7.4. Fluorescein isothiocyanate (FITC)-labeled dextran (0.5%) was added to the injection medium to visualize successfully microinjected cells.

Plasmid transfection. For immunocytochemical assays, cells were transiently transfected with indicated plasmids following JetPEI manufacturer's protocol (Polyplus Transfection, Illkirch, France).

Fluorescence measurements of Cl– efflux. The Cl– channel activity of CFTR was assessed using the halide-sensitive fluorescent probe 6-methoxy-N-(3-sulfopropyl) quinolinium (SPQ; Molecular Probes, Leiden, Netherlands). Twenty-four hours postinjection, cells were loaded with the intracellular SPQ dye by incubation in Ca2+-free hypotonic (50% dilution) medium containing 10 mM SPQ at 37°C for 15 min. The coverslips were mounted on the stage of an inverted microscope (Nikon Diaphot) equipped for fluorescence and illuminated at 360 nm. The emitted light was collected at 456 nm by a high-resolution image intensifier coupled to a video camera (extended ISIS camera system; Photonic Science, Roberts-bridge, UK) connected to a digital image processing board controlled by FLUO software (Imstar, Paris, France). Cells were maintained at 37°C and continuously superfused with an extracellular solution containing 145 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, and 5 mM glucose, pH 7.4. A microperfusion system allowed local application and rapid change of the different experimental mediums. Iodide (I–)- and nitrate (NO3–)-containing mediums were identical to the extracellular solution except that I– and NO3– replaced Cl– as the dominant extracellular anions. All extracellular mediums also contained 10 µM bumetanide to inhibit the Na+/K+/2Cl– cotransporter. Single-cell fluorescence intensity was measured from digital image processing and displayed against time. Fluorescence intensity was standardized according to the equation F = (F – Fo)/Fo x 100, where F is the relative fluorescence and Fo is the fluorescence intensity measured in the presence of I–. The membrane permeability to halides was determined as the rate of SPQ dequenching upon perfusion with nitrates. At least three successive data points were collected immediately after the NO3–-containing medium application and then fitted using a linear regression analysis. The slope of the straight line reflecting the membrane permeability to halides (p, in min–1) was used as an index of CFTR activity.

Immunocytochemical assays. To evaluate the cell polarity of A549 and MDCK cells, they were grown to confluence on glass coverslips. They were then washed with PBS and fixed in paraformaldehyde 4% for 20 min at room temperature. After washing with PBS, the fixed cells were permeabilized with 0.2% Triton X-100 (Sigma, Saint Quentin Fallavier, France) in PBS and incubated with anti-ZO-1 monoclonal antibody (1:20 dilution; Biogenesis, Poole, UK) for 1 h at room temperature. This antiserum was revealed with Alexa Fluor 488-conjugated secondary antibody (1:1,000 dilution; Molecular Probes) for 1 h at room temperature.

To determine the cellular localization of NHE-RF1 and CFTR, MDCK cells were transiently transfected with pcDNA3-CFTR alone, pcDNA3-{Delta}F508-CFTR plasmid alone, or in association with the human pcDNA3-NHE-RF1 plasmid following standard protocol for JetPEI (Polyplus Transfection, Illkirch, France). After 24 h, cells were washed with PBS and fixed with 4% paraformaldehyde. Cells were immunolabeled with primary monoclonal antibody raised against the first extracellular loop of human CFTR (MATG 1031, 1:20 dilution; Transgene, Stasbourg, France) and Alexa Fluor 488-conjugated secondary antibody (1:1,000 dilution). Cells were then also permeabilized and incubated with anti-human NHE-RF1 monoclonal antibody (1:1,000 dilution; BD Transduction Laboratories, Le-Pont-de-Claix, France) and Alexa Fluor 594-conjugated secondary antibody as described above. Cells were mounted with 50% glycerol in PBS and analyzed by confocal microscopy.

Control experiments were performed using the wild-type, trafficking-competent VSV-KCNE1-KCNQ1 (E1Q1) potassium channel and the trafficking-defective VSV-KCNE1-KCNQ1-P117L mutant (P117L) that contain an extracellular VSV tag (7). MDCK cells were transiently transfected with pcb6-E1Q1 alone, pcb6-P117L alone, or in association with pcDNA3-NHE-RF1 following standard protocol for JetPEI. After 24 h, to detect membrane expression of the KCNE1-KCNQ1 or P117L proteins, nonpermeabilized cells were immunolabeled with anti-VSV monoclonal antibody (1:500 dilution; Sigma) as previously described. Cells were then fixed with 4% paraformaldehyde, permeabilized, and incubated with anti-NHE-RF1 antibody and Alexa Fluor 594-conjugated secondary antibody. Alternatively, to detect intracellular expression of the trafficking mutant P117L, the cells were fixed with 4% paraformaldehyde, permeabilized, and immunolabeled using an antibody raised against the intracellular COOH-terminal tail of KCNQ1 (1:500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) and anti-NHE-RF1 antibody as described above. Cells were mounted with 50% glycerol in PBS and analyzed by confocal microscopy.

Fluorescence intensity quantitation. Imaging conditions and acquisition were identical for all experimental conditions. The fluorescence intensity of apical CFTR staining was assessed using Metamorph software (Universal Imaging, Media, PA).

Drugs. Intracellular cAMP was increased with a mixture containing 10 µM forskolin plus 100 µM 3-isobutyl-1-methylxanthine (IBMX) (both from Sigma). For SPQ experiments, drugs were dissolved in dimethyl sulfoxide (Sigma) so that final concentration of the solvent was <0.1%.

Statistical analysis. Data are expressed as means ± SE of n number of experiments. The statistical significance of a drug effect versus baseline was assessed using a paired Student's t-test. The significant difference between two experimental conditions was assessed using an unpaired Student's t-test.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The polarity of A549 and MDCK cells grown on glass coverslips was assessed by immunocytochemistry using the tight junction-associated protein ZO-1 (zonula occludens-1) as a marker. Confocal microscopy analysis showed a discontinuous staining localized at cell contacts in A549 cells when using a focal plane parallel to the cellular monolayer (Fig. 1A). On the other hand, a typical polyedric staining surrounding MDCK cells was observed (Fig. 1B). Furthermore, as expected for polarized epithelial cells, transversal (xz) sections revealed that ZO-1 was specifically localized in the apex of MDCK lateral membranes (Fig. 1D), whereas no polarized distribution was observed in A549 membranes (Fig. 1C). Together, these data indicate that MDCK but not A549 cells undergo consistent morphological polarization when grown to confluence on glass coverslips.


Figure 1
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  		Fig. 1. Zonula occludens-1 (ZO-1) immunolocalization in confluent A549 and Madin-Darby canine kidney (MDCK) cells. A549 and MDCK cells were grown to confluence on glass coverslips. Cells were fixed and immunostained with Alexa Fluor 488-conjugated secondary antibody after incubation with monoclonal anti-ZO-1 antibody. Images in A and B are longitudinal confocal micrographs of immunostained A549 and MDCK cells, respectively. Images in C and D are transversal confocal micrographs of immunostained A549 and MDCK cells, respectively. Scale bars, 10 µm.

 
PCR experiments performed on total mRNA extracts revealed that both A549 and MDCK cells endogenously expressed NHE-RF1 but not CFTR transcripts (Fig. 2).


Figure 2
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  		Fig. 2. Endogenous expression of Na+/H+ exchanger regulatory factor isoform 1 (NHE-RF1) and cystic fibrosis transmembrane conductance regulator (CFTR) mRNAs in A549 and MDCK cells. PCR products were generated using primers specific for NHE-RF1 and CFTR and were observed under UV radiation after 1% agarose gel electrophoresis. –, negative control (H2O); +, positive control (pcDNA3-NHE-RF1 or pcDNA3-CFTR); NRT, non-reverse-transcribed cDNAs; RT, reverse-transcribed cDNAs.

 
In A549 cells microinjected with the plasmid encoding wtCFTR, the basal CFTR permeability to halides (p) was 0.15 ± 0.01 min–1 (Fig. 3). The application of 10 µM forskolin, a direct activator of adenylyl cyclase, plus 100 µM IBMX, a phosphodiesterase inhibitor, increased the p value sevenfold (P < 0.001 vs. p baseline; Fig. 3). The coexpression of NHE-RF1 with wtCFTR significantly increased the basal CFTR channel activity as well as the forskolin-induced activation of CFTR (P < 0.001 vs. basal activity obtained in cells expressing only wtCFTR; Fig. 3). In MDCK cells, the NHE-RF1 overexpression did not modify the basal wtCFTR channel activity but significantly increased the forskolin-induced activation of CFTR (P < 0.001 vs. activity obtained in cells expressing only wtCFTR; Fig. 3).


Figure 3
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  		Fig. 3. Activation of CFTR protein by NHE-RF1 overexpression in A549 and MDCK cells. A549 and MDCK cells were microinjected with wild-type CFTR (wtCFTR; 30 µg/ml) and NHE-RF1 (50 µg/ml) cDNAs. Membrane permeability to halides (p, in min–1) was measured under baseline conditions (open bars) and after application of 10 µM forskolin (Fsk; filled bars). Data are means ± SE of n cells. The significant effects of forskolin were analyzed using a paired Student's t-test: P < 0.001 vs. baseline. The significant effects of NHE-RF1 were analyzed using an unpaired Student's t-test: ***P < 0.001.

 
On the other hand, A549 and MDCK cells expressing {Delta}F508-CFTR alone presented a very low permeability to halides and a low but significant sensitivity to forskolin (Fig. 4). However, when coexpressed with NHE-RF1, {Delta}F508-CFTR presented a significant basal channel activity. More interestingly, {Delta}F508-CFTR channel activity was then increased more than threefold by forskolin (P < 0.001 vs. activity obtained in cells expressing only {Delta}F508-CFTR; Fig. 4) in both cell lines.


Figure 4
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  		Fig. 4. Activation of {Delta}F508-CFTR by NHE-RF1 overexpression in A549 and MDCK cells. Cells were injected with {Delta}F508-CFTR (30 µg/ml) and NHE-RF1 (50 µg/ml) encoding plasmids in the absence or the presence of sense or antisense oligonucleotides for NHE-RF1 (50 µg/ml). Membrane permeability to halides (p, in min–1) was measured under baseline conditions (open bars) and after application of 10 µM Fsk (filled bars). Data are means ± SE of n cells. The significant effects of forskolin were analyzed using a paired Student's t-test: *P < 0.05; **P < 0.01; ***P < 0.001 vs. baseline. The significant influence of the NHE-RF1 overexpression was determined using 2-way ANOVA: ##P < 0.01; ###P < 0.001 vs. cells only injected with {Delta}F508-CFTR. The significant influence of the antisense and sense oligonucleotides was determined using 2-way ANOVA: °°P < 0.01; °°°P < 0.001 vs. cells injected with NHE-RF1.

 
Antisense oligonucleotide strategy was used to ascertain the role of NHE-RF1 on CFTR channel activity. An antisense oligonucleotide was designed to target the NHE-RF1 mRNA and to block its translation. As illustrated in Fig. 4, NHE-RF1 antisense oligonucleotide blocked the effects of NHE-RF1 on the basal and forskolin-induced activations of {Delta}F508-CFTR in both A549 and MDCK cells. On the other hand, the sense oligonucleotide, used as a control, did not affect {Delta}F508-CFTR activity responses to NHE-RF1 (Fig. 4).

To establish whether increased wild-type or {Delta}F508 mutant CFTR activity resulted from improved membrane expression of the protein, we performed immunocytochemistry studies using a monoclonal antibody raised against the first extracellular loop of the human CFTR protein. MDCK cells grown on glass coverslips that exhibit a consistent morphological polarization (Fig. 1) were used in these experiments. As illustrated in Fig. 5, A and B, the extracellular antibody specifically detected wtCFTR localized in the apical membrane of nonpermeabilized MDCK cells, regardless of the presence or the absence of NHE-RF1. Moreover, wtCFTR and NHE-RF1 were at least partially colocalized in MDCK apical membranes (Fig. 5B, e and f). As expected, {Delta}F508-CFTR transfected alone was not detected at the cell surface (Fig. 6Aa) but was readily detected in the apical membranes when cotransfected with NHE-RF1 (Fig. 6A, b and c). Furthermore, both exogenous proteins were apically localized (Fig. 6A, c and f). Fluorescence quantification showed that apical immunostainings of wtCFTR were not significantly different in the presence and in the absence of NHE-RF1 (Fig. 6B), whereas {Delta}F508-CFTR apical immunostainings were null but reached the wild-type CFTR levels when coexpressed with NHE-RF1 (Fig. 6B).


Figure 5
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  		Fig. 5. Confocal imaging of MDCK cells transiently transfected with pcDNA3-CFTR alone or in the presence of pcDNA3-NHE-RF1. A: 24 h after transfection, cells were fixed and immunostained with a monoclonal antibody raised against the first extracellular loop of human CFTR and then incubated with a Alexa Fluor 488-conjugated secondary antibody. B: MDCK cells underwent the same treatment as in A, but in addition they were permeabilized and immunostained with monoclonal anti-human NHE-RF1 antibody that was visualized with Alexa Fluor 594-labeled secondary antibody. Longitudinal and transversal confocal micrographs are annotated xy and xz, respectively. AP, apical membrane; BM, basal membrane. See text for more detailed mention of individual panels.

 

Figure 6
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  		Fig. 6. Effects of the overexpression of NHE-RF1 on apical localization of {Delta}F508-CFTR. A: confocal imaging of MDCK cells transiently transfected with pcDNA3-{Delta}F508-CFTR plasmid alone or in the presence of pcDNA3-NHE-RF1. Twenty-four hours after transfection, cells were fixed and immunostained with a monoclonal antibody raised against the first extracellular loop of human CFTR and then incubated with a Alexa Fluor 488-conjugated secondary antibody. In addition, MDCK were permeabilized and immunostained with monoclonal anti-human NHE-RF1 antibody, which was visualized with Alexa Fluor 594-labeled secondary antibody. Longitudinal and transversal confocal micrographs are annotated xy and xz, respectively. B: quantitation of fluorescence intensity. The fluorescence intensity of apical wtCFTR and {Delta}F508-CFTR staining was assessed using Metamorph software (Universal Imaging, Media, PA). Data are means ± SE of n cells. The significant effects of NHE-RF1 were analyzed using a Student's t-test: ***P < 0.001.

 
To establish the role of PDZ interactions in {Delta}F508-CFTR apical and functional rescue, a {Delta}F508 mutant missing its PDZ interaction domain with NHE-RF1 was used. As illustrated in Fig. 7, coexpression of {Delta}F508-K1468X-CFTR with NHE-RF1 in MDCK cells did not result in any significant changes in the baseline or the forskolin-stimulated Cl– channel activity. To ascertain the effects of NHE-RF1 on CFTR trafficking did not result from a global nonspecific rescue of endoplasmic reticulum-retained proteins or a nonspecific increase of apical proteins expression, we analyzed its effect on the trafficking of an unrelated potassium channel, KCNQ1. As illustrated in Fig. 8, the extracellularly tagged fusion protein KCNE1-KCNQ1 but not its trafficking defective mutant (P117L) was readily detected in the apical membranes of nonpermeabilized MDCK cells using extracellular anti-VSV antibody (Fig. 8A) (7). In these conditions, the P117L mutant was never detected at the plasma membrane irrespective of the presence (Fig. 8B, b, e, and h) or the absence of NHE-RF1 (Fig. 8B, a, d, and g). The P117L mutant detected using an anti-KCNQ1 antibody raised against the cytoplasmic COOH-terminal tail of the protein (Fig. 8Bc) remained trapped in intracellular compartments that did not colocalize with those of NHE-RF1 (Fig. 8Bi).


Figure 7
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  		Fig. 7. Effect of NHE-RF1 overexpression on {Delta}F508-K1468X in MDCK cells. MDCK cells were microinjected with {Delta}F508-K1468X (30 µg/ml) and NHE-RF1 (50 µg/ml) cDNAs. Membrane permeability to halides (p, in min–1) was measured under baseline conditions (open bars) and after application of 10 µM Fsk (filled bars). Data are means ± SE of n cells. The significant effects of forskolin were analyzed using a paired Student's t-test.

 

Figure 8
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  		Fig. 8. Confocal imaging of MDCK cells transiently transfected with pcb6-E1Q1 alone, pcb6-P117L plasmid alone, or in the presence of pcDNA3-NHE-RF1. A: 24 h after transfection, cells were immunostained with a monoclonal antibody raised against the extracellular VSV tag of the KCNE1-KCNQ1 fusion protein and then incubated with a Alexa Fluor 488-conjugated secondary antibody. Longitudinal and transversal confocal micrographs are annotated xy and xz, respectively. B: MDCK cells underwent the same treatment as in A, but in addition they were permeabilized and immunostained with monoclonal anti-human NHE-RF1 antibody, which was visualized with Alexa Fluor 594-labeled secondary antibody (a, b, d, e, g, and h). MDCK cells were fixed, permeabilized, and immunolabeled with a monoclonal antibody raised against the cytoplasmic COOH-terminal tail of the KCNQ1 protein and then incubated with a FITC-conjugated secondary antibody (c, f, and i). They were also immunostained with monoclonal anti-human NHE-RF1 antibody, which was visualized with Alexa Fluor 594-labeled secondary antibody. All panels are transversal confocal micrographs.

 

   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study demonstrates that the overexpression of the CFTR protein partner, NHE-RF1, rescues {Delta}F508-CFTR activity. This conclusion is based on several lines of evidence. First, cells injected with {Delta}F508-CFTR alone presented a low Cl– channel activity, whereas the coexpression with NHE-RF1 significantly increased both the basal and the forskolin-activated {Delta}F508-CFTR conductances. Second, these effects were abolished by a specific NHE-RF1 antisense oligonucleotide. Third, immunocytochemical analysis demonstrated an increased {Delta}F508-CFTR expression in the apical plasma membrane of the cells cotransfected with NHE-RF1. Finally, the involvement of PDZ domains in such effect is strongly suggested by the use of {Delta}F508-K1468X-CFTR, a {Delta}F508 mutant lacking the 13 last amino acids involved in PDZ interaction with NHE-RF1. In these last experimental conditions, the overexpression of NHE-RF1 did not rescue the {Delta}F508-CFTR activity.

Our study was performed in heterologous systems (A549 and MDCK cells) overexpressing {Delta}F508-CFTR and NHE-RF1. Because CFTR channel is targeted to the apical membrane in a number of epithelial cells, including those of the airways, intestine, and kidney (2, 10, 31, 42), we first ascertained that MDCK cells were morphologically polarized in our experimental conditions. Indeed, we showed that the tight junction marker ZO-1 was localized in the upper third of the lateral membranes of confluent MDCK cells grown on glass coverslips. This observation is consistent with previous studies performed on cells growing on permeable supports and indicates that MDCK cells grown in our experimental conditions exhibit a significant morphological polarization to separate apical and basolateral membrane domains. However, it is worth noting that in these latter conditions, the height of the cells remained less than when cells are grown on permeable support (43). Also, we showed that both cell lines did not express endogenous CFTR albeit they expressed endogenous NHE-RF1.

We demonstrated that NHE-RF1 overexpression produced an increase in wtCFTR basal activity in A549 but not in polarized MDCK cells. The increase in wtCFTR basal activity obtained in A549 cells is in agreement with Raghuram et al. (38), who showed that NHE-RF1 overexpression increases the Cl– channel activity of wtCFTR. Also, the absence of NHE-RF1 effect on wtCFTR basal activity observed in MDCK cells is in accordance with three other studies reporting that the apical and functional expression of wtCFTR in MDCK cells does not necessitate NHE-RF1 binding (1, 17, 36). This corroborates our observation that NHE-RF1 did not increase wtCFTR apical staining in MDCK cells. On the other hand, it has been demonstrated that in MDCK cells, NHE-RF1 is essential for polarized and functional expression of wtCFTR (32, 33) even if it is not sufficient (27, 28). To make the scheme even more complicated, Raghuram et al. (38) further suggested that the regulation of CFTR channel activity by NHE-RF1 would be biphasic: activated at low concentrations and attenuated at high concentrations of NHE-RF1. Nevertheless, we have shown that in both cell lines, NHE-RF1 overexpression increased the forskolin-induced activation of wtCFTR. It is well admitted that NHE-RF1 is bound to ezrin, which is a PKA-anchoring protein (12), leading to the formation of macromolecular complexes in which may be gathered CFTR, ezrin, and PKA. Thus the NHE-RF1 overexpression may facilitate CFTR phosphorylation by anchoring the regulatory subunits of the PKA in the vicinity of the Cl– channel (24, 34, 38, 44).

Very interestingly, we showed that in A549 and MDCK cells, NHE-RF1 increased both the basal and the forskolin-activated {Delta}F508-CFTR conductances. These effects were abolished by a specific NHE-RF1 antisense oligonucleotide but not by a sense oligonucleotide. Furthermore, immunocytochemistry experiments showed that NHE-RF1 overexpression modified {Delta}F508-CFTR localization in MDCK cells. When {Delta}F508-CFTR protein was expressed alone, no plasma membrane staining was observed, whereas {Delta}F508-CFTR colocalized with NHE-RF1 in the apical cell membranes when coexpressed with the latter.

We showed that {Delta}F508 rescue was critically dependent on its COOH-terminal PDZ ligand domain, suggesting that physical interaction with NHE-RF1 was a prerequisite to its effect. Indeed, {Delta}F508-K1468X-CFTR exhibited a low basal Cl– channel activity that was insensitive to forskolin in the presence of NHE-RF1. Our results corroborate the results recently published by Guerra et al. (16). In the human cell line CFBE14o- endogenously expressing {Delta}F508-CFTR, transfection with vectors encoding wild-type mouse NHE-RF1 increased both apical CFTR expression and apical PKA-dependent CFTR-mediated Cl– efflux, whereas transfection with mouse NHE-RF1 mutated in the binding groove of the PDZ domains or truncated for the ERM domain inhibited both the apical CFTR expression and the CFTR-dependent Cl– efflux (16). Furthermore, the effect of NHE-RF1 on {Delta}F508-CFTR trafficking in MDCK cells was specific, because NHE-RF1 did not rescue the plasma membrane expression of another trafficking-defective mutant potassium channel, KCNQ1 (Fig. 5B, b and c) (7). Also, the apical expression of NHE-RF1 in MDCK cells is consistent with previous reports in human airway epithelial cells (41). On the basis of these results, one can speculate on the mechanisms involved in the improvement of {Delta}F508-CFTR trafficking by NHE-RF1. For example, NHE-RF1 overexpression may allow {Delta}F508-CFTR protein to leave the endoplasmic reticulum and thus enable its maturation process to finally facilitate its trafficking to the apical membrane. In addition, NHE-RF1 may also modulate the recycling of {Delta}F508-CFTR between the apical plasma membrane and the recycling compartments.

The molecular mechanisms involved in the functional rescue of {Delta}F508-CFTR by NHE-RF1 remain unclear. Several studies suggest some putative roles of NHE-RF1. NHE-RF1 may compete with another PDZ domain-containing protein known to bind and to inhibit CFTR, CFTR-activated-ligand (CAL; Refs. 3, 4). NHE-RF1 may interact with chaperone proteins involved in {Delta}F508-CFTR processing, like Hsc70/Hsp70, Hsp90, or calnexin (14, 26, 35, 37, 50), or with any of the cochaperones involved in their regulation. NHE-RF1 also may modulate the stoichiometry of CFTR Cl– channel and/or its association with specific signal transduction pathways (23, 51). In addition, it has been established that NHE-RF1 can self-associate through PDZ-PDZ interactions (15) and consequently acts as a scaffolding protein in macromolecular complexes (38, 48). Finally, NHE-RF1 may participate to the differential association of CFTR with beta-adrenergic transduction pathways. Indeed, Naren et al. (34) have shown that CFTR is associated with a beta2-adrenoceptor macromolecular signaling complex. This observation has been strengthened by the study showing that the treatment of primary human airway surface epithelial cells with a beta2-adrenoceptor agonist, salmeterol, increases the mature CFTR protein expression in a time-dependent fashion. This effect does not result from the activation of the cAMP/PKA pathway but involves the NHE-RF1 protein (45). In addition, we have demonstrated that beta3-adrenergic stimulation also increases CFTR Cl– channel activity through a cAMP/PKA-independent but mitogen-activated protein kinase-dependent transduction pathway (22, 39). Whether NHE-RF1 also is involved in the latter transduction pathway remains to be elucidated.

Together, the present results demonstrate that NHE-RF1 overexpression is able to restore a significant Cl– permeability in cells expressing {Delta}F508-CFTR. Considering that only a small amount (10–15%) of {Delta}F508-CFTR activity is required to restore epithelial function and moderate CF disease (11, 20), the improvement of CFTR function by NHE-RF1 may have therapeutic implications.


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This work was supported by grants from the Association Vaincre la Mucoviscidose and the Fondation Langlois.


   ACKNOWLEDGMENTS
 
We thank Karine Laurent and Mortéza Erfanian for expert technical assistance with cell cultures.

Present address of A. Robay: Children's Hospital of Philadelphia, Abramson Research Center, Philadelphia, PA 19104.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Gauthier, l'Institut du Thorax, INSERM U533, Faculté de Médecine, F-44035 Nantes, France (e-mail: chantal.gauthier{at}nantes.inserm.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* F. Bossard and A. Robay contributed equally to this work. Back


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