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Cellular Localization and Activity of Ad-Delivered GFP-CFTR in Airway Epithelial and Tracheal Cells

¹ Laboratoire de Virologie et Pathologie Humaine, Université de Lyon 1 and CNRS FRE 3011, Faculté de Médecine Laennec and IFR Lyon-Est, Lyon, France; ² Institut de Physiologie et Biologie Cellulaires, CNRS UMR 6187, Université de Poitiers, Poitiers, France; ³ Department of Internal Medicine and ⁴ Howard Hughes Medical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa; ⁵ Transgene SA, Strasbourg, France; and ⁶ Laboratoire de Virologie Médicale, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, Bron, France

Correspondence and requests for reprints should be addressed to Saw-See Hong, Ph.D., Laboratoire de Virologie et Pathologie Humaine, CNRS FRE 3011, Faculté de Médecine RTH Laennec, 7 rue Guillaume Paradin, 69372 Lyon, France. E-mail: sawsee.hong{at}sante.univ-lyon1.fr

Abstract

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and the cellular trafficking of the CFTR protein is an essential factor that determines its function in cells. The aim of our study was to develop an Ad vector expressing a biologically active green fluorescent protein (GFP)-CFTR chimera that can be tracked by both its localization and chloride channel function. No study thus far has demonstrated a GFP-CFTR construct that displayed both of these functions in the airway epithelia. Tracheal glandular cells, MM39 (CFTRwt) and CF-KM4 (CFTRF508), as well as human airway epithelial cells from a patient with cystic fibrosis (CF-HAE) and from a healthy donor (HAE) were used for the functional analysis of our Ad vectors, Ad5/GFP-CFTRwt and Ad5/GFP-CFTRF508. The GFP-CFTRwt protein expressed was efficiently addressed to the plasma membrane of tracheal cells and to the apical surface of polarized CF-HAE cells, while GFP-CFTRF508 mutant was sequestered intracellularly. The functionality of the GFP-CFTRwt protein was demonstrated by its capacity to correct the chloride channel activity both in CF-KM4 and CF-HAE cells after Ad transduction. A correlation between the proportion of Ad5-transduced CF-KM4 cells and correction of CFTR function showed that 55 to 70% transduction resulted in 70% correction of the Cl^– channel function. In reconstituted CF-HAE, GFP-CFTRwt appeared as active as the nontagged CFTRwt protein in correcting the transepithelial Cl^– transport. We show for the first time a GFP-CFTR chimera that localized to the apical surface of human airway epithelia and restored epithelial chloride transport to similar levels as nontagged CFTR.

Key Words: cystic fibrosis • GFP-CFTR • Cl^– channel • adenovirus • CFTR-deficient airway epithelia

CLINICAL RELEVANCE We show a green fluorescent protein–cystic fibrosis transmembrane conductance regulator chimera that localizes to the apical surface of the airway epithelia and restored the Cl– transport. The cystic fibrosis transmembrane conductance regulator protein can now be tracked in human epithelia which will allow rapid translation of therapeutic interventions for cystic fibrosis.

 
Cystic fibrosis (CF) is an autosomal recessive genetic disease caused by mutations in a single gene, the CF transmembrane conductance regulator (CFTR) gene, and therefore represents an ideal candidate for gene therapy (1). At present, more than 1,500 different mutations in the CFTR gene have been identified which are associated with CF (2) (Cystic Fibrosis Mutation database; www.genet.sickkids.on.ca/cftr/). The CFTR gene consists of 27 exons, and the resulting spliced transcript is translated into a 1,480–amino acid protein (168 kD) with twelve putative transmembrane domains, two nucleotide-binding domains, and a regulatory domain (3). CFTR functions as a cAMP-regulated chloride (Cl^–) channel located mainly in the apical membrane of epithelial cells. CF primarily involves epithelial cells in the intestine, respiratory system, pancreas, gall bladder, and sweat glands. Two critical consequences of impaired CFTR function are a reduction in the volume of airway surface liquid and an increased adherence of bacterial pathogens such as Pseudomonas aeruginosa to airway epithelial cells (4–6). The main cause of CF mortality and morbidity is due to pulmonary complications associated with impaired clearance and obstruction by viscous secretions, which makes the airway epithelial cells the principle target for CF treatment.

The cellular trafficking of the CFTR protein is an essential factor that determines its function in cells. For example, the most common mutation found in patients with CF is the F508 mutation, which results in the abnormal processing and cell trafficking of the CFTR protein. The direct visualization of the CFTR protein or its mutants within cells have been made possible by fusing the protein to the green fluorescent protein (GFP) at its N-terminus (7). However, the genetic fusion of GFP to the CFTR could have some negative influence on the function of the CFTR protein. Vais and coworkers described a GFP-CFTR clone that was functional in patch-clamp studies but failed to localize to the plasma membrane of transduced cells (8). No study thus far has demonstrated both apical localization and functionality of a GFP-CFTR in human airway epithelia.

Gene transfer of CFTR in the airway epithelial represents a promising treatment for CF lung disease. However, the understanding of the exact mechanism of pulmonary disease and the consequences of correction is incomplete. The Ad vector has several advantages, such as its capacity to incorporate large transgenes; is relatively easy to manipulate genetically; and is useful in the functional study of a transgene in vitro using cell lines, ex vivo in tissues as well as in vivo animal systems. The aim of our study was to develop an Ad vector expressing a biologically active GFP-CFTR chimera that could be tracked in terms of localization and function in human airway epithelia. The GFP-CFTRwt construct in the present study differed from other constructs by its short flexible and basic linker, which allowed for the proper conformation of both the GFP and CFTR domains. When expressed in CFTR-deficient tracheal and polarized human epithelia cells, the CFTR chimera localized efficiently to the cell plasma membrane and the apical surface of the epithelium, respectively. Moreover, in reconstituted epithelia, the GFP-CFTRwt was as active as the non-tagged CFTRwt protein in correcting the transepithelial Cl^– transport. We show for the first time that wild-type CFTR linked to GFP is addressed to the apical surface of human airway epithelia, where it restored epithelial chloride transport to similar levels as wild-type CFTR.

MATERIALS AND METHODS

Cells
HEK293 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA), and maintained as monolayers in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Cergy Pontoise, France) supplemented with 10% fetal bovine serum (FBS; Invitrogen), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 37°C, 5% CO₂. Human tracheal gland serous cell lines MM-39 and CF-KM4 were isolated from the tracheal mucosa of a young healthy adult (9) and from a patient with CF carrying a F508-F508 mutation, respectively (10). Both cell lines were SV40-transformed and maintained as monolayers in a 1:1 mixture of DMEM and Ham's F12 supplemented with 1% Ultroser G (BioSepra, Villeneuve-la-Garenne, France), sodium pyruvate (1 mM), glutamine (5 mM), penicillin (200 U/ml), streptomycin (200 µg/ml), and epinephrine (3 µM), on collagen I–coated flasks (BioCoat; BD Biosciences, Le Pont-de-Claix, France).

Primary Human Airway Epithelial Cell Culture Model
Human airway epithelial cells (HAE) were isolated from tracheae and bronchi of donor lungs. Cells were seeded onto collagen-coated, semi-permeable membranes (0.6 cm² Millicell-HA; Millipore Corp., Bedford, MA), and grown at an air–liquid interface as previously described (11, 12). At 36 to 48 hours after seeding, the cells form a confluent culture with electrically tight junctions, and between Days 3 and 14 the epithelial cells differentiate into a predominantly ciliated phenotype (12). HAE and CFTR-deficient CF-HAE cells were cultured in a 1:1 mixture of DMEM and Ham's medium supplemented with 2% Ultroser G, penicillin (100 U/ml), streptomycin (100 µg/ml), gentamycin (10 µg/ml), colimycin (25 µg/ml), ceftazidime (75 µg/ml), imipenem (25 µg/ml), cilastin (25 µg/ml), and fluconazole (2 µg/ml). Basolateral culture medium was changed every 2 to 4 days. Samples were collected with approval from the University of Iowa Institutional Review Board.

Plasmids
The CFTRwt and CFTRF508 mutant genes were cloned into the pEGFP-C1 expression plasmid (Clontech Europe, St. Germain-en-Laye, France) placing the CFTR in frame with the C-terminus of the GFP protein, with a seven–amino acid linker of sequence SGLRSRA. Both CFTRwt and CFTRF508 DNA fragments were generated by PCR from the plasmids pcDNA3-CFTRwt and pcDNA3-CFTRF508, using the following pairs of primers: the first pair consisted of primers 5'-T CGA GCT ATG CAG AGG TCG CCT CTG-3' and 5'-GC CTA AAG CCT TGT ATC TTG CAC-3', and the second pair were 5'-GCT ATG CAG AGG TCG CCT CTG-3' and 5'-CCG GGC CTA AAG CCT TGT ATC TTG CAC-3'. Two PCR reactions were performed for each clone using the above pairs of primers. The PCR products from both reactions were then mixed, denatured, and hybridized to obtain DNA fragments with Xho I and Xma I cohesive ends competent for ligation to pEGFP-C1 linearized with Xho I and Xma I. The resulting pEGFP-CFTRwt and pEGFP-CFTRDF508 clones were then verified by DNA sequencing.

Ad Transfer Vectors
The Ad5/GFP-CFTRwt and Ad5/GFP-CFTRF508 recombinant vectors were generated by homologous recombination using the pEGFP-CFTRwt and pEGFP-CFTRF508 plasmids, respectively, with the adenovirus transfer plasmid pTG14682, following a method previously described (13). The backbone of the Ad5-based vector was deleted of the E1 and E3 regions and the recombinant vectors were propagated in HEK-293 cells. Virus stocks were purified by CsCl gradient ultracentrifugation according to conventional protocols (14). The infectivity index defined as the ratio of infectious particles to total physical particles (plaque-forming units [PFU]:PP) was 1:25, 1:20, and 1:10 for Ad5/GFP-CFTRwt, Ad5/GFP-CFTRF508, and Ad5/GFP, respectively. First-generation Ad5 vector Ad5/CFTR expressing nontagged CFTR used as control has been previously described (15–17).

Immunocytochemistry
Cell monolayers were infected with recombinant Ad at a multiplicity of infection (MOI) of 60 PFU/cell. Primary human epithelia were infected at MOI 200. After virus adsorption for 1 hour at 37°C, the cells were rinsed and further incubated with complete medium at 37°C, and Ad infection was followed up directly by GFP expression. At 48 hours after infection, cell monolayers and epithelia were processed for analysis by confocal microscopy. Cell monolayers were fixed with 3% paraformaldehyde in PBS, incubated with DAPI, and mounted on slides. Epithelia were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 and 2% bovine serum albumin (BSA) in SuperBlock (Pierce, Perbio Science France, Brebières, France). After blocking with 2% BSA in SuperBlock for 1 hour at room temperature, cells were incubated with primary antibody (mouse anti-human ZO-1 antibody, 1:250; BD Biosciences) for 3 hours at 37°C, rinsed in PBS, then incubated with secondary antibody conjugated to fluorophore (1:1,000; Alexa Fluor 568–conjugated goat anti-mouse IgG, Invitrogen) for 90 minutes at 37°C. After several rinses, cells were mounted on glass slides, a coverslip was placed with Vectashield + DAPI mounting solution (Vector Labs, Burlingame, CA), and samples were studied by confocal fluorescence microscopy. Samples were observed using a Leica TCS SP2 (Leica Microsystems, Wetzlar, Germany) or an Olympus FluoView V1000 (Olympus Corp., Center Valley, PA) confocal microscope using a x60 oil immersion lens.

GFP-Antibody Pull-Down
Aliquots of 10⁶ CF-KM4 cells were mock-infected or infected with Ad5GFP or Ad5GFP-CFTRwt (60 PFU/cell) and labeled with ³⁵S-amino acids (Redivue Pro-mix L-(³⁵S), 1,000 Ci/mmol; Amersham Biosciences Europe, Otelfingen, Switzerland), added at 100 µCi/ml in methionine-free medium from 18 to 72 h post-infection. Cells were lysed in 0.2 ml RIPA buffer (1% NP-40, 1% deoxycholate, 0.1% SDS, 150 mM NaCl, 1 mM Tris-HCl, pH 7.4) containing anti-protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), and freeze-thawed five times. Cell lysates were incubated with anti-GFP polyclonal antibody (5 µg; Invitrogen) for 1 h at 4°C, and post-incubated with protein G-Sepharose (1 mg) overnight at 4°C. Affinity gel was rinsed, resuspended in SDS-PAGE loading buffer, and eluted material analyzed by SDS-PAGE in 8% polyacrylamide gels. Gels were dried under vacuum and autoradiographed.

Immunoelectron Microscopy of GFP-CFTR–Transduced Cells
For immunoelectron microscopy (IEM), mouse monoclonal anti-CFTR antibody MM13–4 (Chemicon, Upstate Biotechnology, Lake Placid, NY), rabbit polyclonal anti-GFP (Invitrogen), and secondary antibodies of 20-nm colloidal gold grain–conjugated anti-rabbit IgG (GAR-20; British Biocell International, Cardiff, UK) and 10-nm gold-conjugated anti-mouse IgG (GAM-10; British Biocell International) were used. Ad-infected cells were fixed with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), pelleted, and post-fixed with osmium tetroxide (1% in 0.1 M cacodylate buffer, pH 7.4). The cell specimens were dehydrated and embedded in metacrylate resin (LR white; London Resin Co., Reading, UK), sectioned, and processed as previously described (18). Sections on grids were reacted with one single antibody (rabbit polyclonal anti-GFP or mouse monoclonal anti-CFTR antibody) or a mixture of both. The samples were examined under a Jeol 1200-EX electron microscope, equipped with a MegaView II high resolution TEM camera and a Soft Imaging System of analysis (Eloïse, Roissy, France).

The notion of co-localization for molecules or epitopes observed under the EM was defined by the following criterion. The antibody molecule size under the EM is approximately 15 nm, and in our IEM analyses, primary (15-nm) and secondary (15-nm) gold-labeled antibody molecules were used. Thus, if one antibody is labeled with a 10-nm gold grain and the other one with a 20-nm grain, localization of two gold grains that are 90 nm apart (15 + 15 + 10 + 15 + 15 + 20 nm) would theoretically mark two adjacent epitopes carried by the same protein molecule (e.g., the N-terminal GFP tag and a C-terminal epitope in CFTR), or alternatively two epitopes belonging to two neighboring molecules. Therefore, immunogold grains which are seen within a distance range of approximately 100 nm would indicate molecules/epitopes that colocalize in the same cell compartment.

Pictures of double-labeled sections of mock- or Ad-transduced cells were divided into separate fields, and gold grains were counted in each field to determine the grain density, that is, the number of gold grains of each species, 10 or 20 nm, per 10 µm² surface area. The plasma membrane compartment was delineated by the limit of the external leaflet and a cytoplasmic area of 50 nm underneath, for the same reasons as the ones evoked above.

Radiotracer Flux Analysis of CFTR Channel Activity
Cells were cultured in 24-multiwell plates and analyzed for CFTR channel activity 48 and 96 hours after infection with Ad vectors. The CFTR Cl^– channel activity was assayed by measuring the rate of iodide (¹²⁵I) effux from cells as previously described (19–21). All experiments were performed with a MultiPROBEIIex robotic liquid handling system (Perkin Elmer Life Sciences, Courtaboeuf, France). At the start of each experiment, cells were washed twice with efflux buffer (136.9 mM NaCl, 5.4 mM KCl, 0.3 mM KH₂PO₄, 0.3 mM NaH₂PO₄, 4.2 mM NaHCO₃, 0.5 mM MgCl₂, 1.3 mM CaCl₂, 0.4 mM MgSO₄, 5.6 mM glucose, and 10 mM HEPES, pH 7.4). The cells were then incubated in efflux buffer containing Na¹²⁵I (1 mCi Na¹²⁵I/ml; NEN, Boston, MA) for 1 hour, to allow the ¹²⁵I to reach equilibrium. At the end of the incubation period, the medium was removed and cells briefly washed with efflux buffer to remove extracellular ¹²⁵I. The progressive loss of intracellular ¹²⁵I was then determined by removing the medium and replacing it with fresh efflux buffer every minute for 10 minutes. The first three aliquots were used to establish a stable baseline of efflux buffer alone. Medium containing the appropriate drug was used for the remaining aliquots. The residual radioactivity was extracted with 0.1% SDS/0.1 N NaOH, and determined using a Packard Cobra II counter (Perkin Elmer Life Sciences). The fraction of initial intracellular ¹²⁵I lost during each time point was measured and the time-dependent rates of ¹²⁵I efflux were calculated using the formula ln (¹²⁵I_t1/¹²⁵I_t2)/(t₁ – t₂), where ¹²⁵I_t is the intracellular ¹²⁵I at time t, and t₁ and t₂ successive time points (22). Curves were constructed by plotting the rate of ¹²⁵I versus time. All comparisons were based on maximal values for the time-dependent rates (k = peak rates, min^–1) excluding the points used to establish the baseline (k peak-k basal, min^–1) (19–21). All experiments were done in triplicate.

Statistics
Results were expressed as mean ± SEM of n observations. Sets of data were compared with an ANOVA or a Student's t test. Differences were considered statistically significant when P < 0.05. All statistical tests were performed using GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA).

Transepithelial Ion Transport
The short-circuit current (I_sc) was measured in modified Ussing chambers (Jim's Instruments, Iowa City, IA) as previously described (23). Primary human airway epithelia were treated with forskolin (10^–5 M) and 3-isobutyl-2-methylxanthine (IBMX, 10^–4 M) for 18 to 24 hours before transfer to Ussing chamber to minimize basal CFTR current. For conditions with symmetrical Cl^– concentrations, solutions on both surfaces of the epithelia contained (in mM): 135 NaCl, 5 HEPES, 1.2 MgCl₂, 1.2 CaCl₂, 2.4 K₂HPO₄, 0.6 KH₂PO₄, and 5 dextrose and gassed with 100% O₂. To create a Cl^– concentration gradient, NaCl was replaced with Na-gluconate on the apical side to give a Cl^– concentration of 4.8 mM. Transepithelial voltage was clamped to zero. After measuring baseline current, the following reagents were added sequentially: (1) apical amiloride (10^–4 M), which inhibits apical Na⁺ channels and hyperpolarizes the apical membrane, thereby generating a driving force for a Cl^– secretory I_sc; (2) apical 4,4'-diisothiocyanotostilbene-2,2'-disulfonic acid (DIDS, 10^–4 M), which inhibits non CFTR-Cl^– channels; (3) apical forskolin (10^–5 M) and IBMX (10^–4 M), which increase cellular levels of cAMP leading to phosphorylation of CFTR and subsequent activation; and (4) apical bumetamide, which inhibits all Cl^– transport. The experiments were performed in quadruplicate.

RESULTS

Rationale for the Construction of GFP-CFTR Chimera
The main aim of this study was to construct GFP-tagged CFTR chimeras, the wild-type and F508 mutant, which would be biologically tracked in human airway epithelia by both its function and cellular localization. The first rationale of our construction was to fuse the GFP sequence to the N-terminus of CFTR to avoid disruption of the PDZ motif at the C-terminus. Second, a short flexible linker was placed between the GFP and CFTR polypeptides to ensure the proper three-dimensional conformation of both GFP and CFTR domains.

The cDNA fragments corresponding to the CFTRwt and CFTRF508 mutant genes were cloned into the expression plasmid pEGFP-C1 such that the CFTR gene sequence was fused in frame to the C-terminal codon of the GFP protein, with a seven–amino acid peptide linker carrying the sequence SGLRSRA, between the GFP and CFTR. The resulting plasmids pEGFP-CFTRwt and pEGFP-CFTRF508 were sequenced and 293 cells were transfected to verify that the GFP-CFTRwt fusion protein had plasma membrane localization and the GFP-CFTRF508 mutant was cytoplasmic (data not shown), before the two corresponding Ad5 recombinants Ad5/GFP-CFTRwt and Ad5/GFP-CFTRF508 were constructed.

Cellular Localization of GFP-CFTRwt and GFP-CFTRF508 in Human Tracheal Gland Cell Lines
The cellular localization of the GFP-CFTR proteins was analyzed in MM-39 cells (tracheal glandular cells from a normal individual) and CF-KM4 cells (tracheal glandular cells from a patient with CF). Cells were transduced by Ad vectors at a MOI of 50 and the GFP-CFTR expression was observed at 24, 48, and 72 hours after infection. The fluorescence signal of the GFP-fused CFTR proteins was optimal for observation at 48 hours after infection, and this was therefore the time choice for fluorescence analyses and IEM.

Both the GFP and CFTR fluorescent signals superimposed at the plasma membrane and the intracellular reticulum network in CF-KM4 cells (Figures 1a–1c). The same pattern was observed in MM-39 cells (not shown). The localization of the GFP-CFTRwt was compatible with a protein that functions as a Cl^– channel and recycles between the plasma membrane and different vesicular compartments within the cytoplasm. In contrast, the GFP-CFTRF508 protein was not observed at the plasma membrane, but appeared mainly as a reticulated fluorescence in the cytoplasm (Figures 1d–1f), consistent with an impaired trafficking to the cell surface. In cells infected with the control Ad5/GFP vector, the GFP fluorescence was diffused and localized in both cytoplasmic and nuclear compartments (not shown).

View larger version (80K): [in this window] [in a new window]   Figure 1. Confocal fluorescence microscopy of CF-KM4 cells after transduction by (a–c) Ad5/GFP-CFTRwt, and (d–f) Ad5/GFP-CFTRF508. GFP-CFTRwt and GFP-CFTRF508 fusion proteins were detected by the green fluorescent protein (GFP) signal (a, d), and by anti–cystic fibrosis transmembrane conductance regulator (CFTR) monoclonal antibody (clone MM13–4; b, e). The nuclei of the cells were counterstained by DAPI (blue signal in c and f), and the merged images (c, f) show the co-localization of GFP and CFTR signals.

View larger version (64K): [in this window] [in a new window]   Figure 2. Analysis of the GFP-CFTR fusion protein. (A) Immunoelectron microscopy of CF-KM4 cells infected with Ad5/GFP-CFTRwt (a–c), Ad5/GFP-CFTRF508 (d, e), and Ad5/GFP (f). The cell sections were double immunolabeled with anti-CFTR monoclonal antibody (clone MM13–4; revealed by 10-nm gold grain–tagged complementary antibody) and anti-GFP polyclonal antibody (revealed by 20-nm gold grain–tagged complementary antibody). The co-localization of the 10-nm and 20-nm gold grains are shown within circles in a, b, and e. Cy, cytoplasm; Nu, nucleus; PM, plasma membrane. Scale is given by the 20-nm gold grains. (B) GFP pull-down assays. Autoradiogram of 35S-amino acid–labeled cell lysates after immunoprecipitation with anti-GFP antibody. Lane 1, Ad5/GFP; lane 2, Ad5/GFP-CFTRwt; lane 3, mock-infected cells. Inset: Western blot analysis of anti-GFP immunoprecipitated material, using anti-CFTR monoclonal antibody (clone 24.1; R&D Systems, Minneapolis, MN). Note that the diffuse material visible at the top of the gel (lane 2) could be separated upon prolonged electrophoresis in SDS-PAGE into several discrete bands of anti-CFTR antibody–reacting bands, ranging from 180 to 230 kD in apparent molecular mass and characteristic of partially and fully glycosylated CFTR species. mm, molecular mass markers.

The CFTR-Cl^– channel activity was undetectable in mock-infected cells and cells infected with Ad5/GFP-CFTRF508 (Figure 3). However, a progressive increase in the level of ¹²⁵I efflux was observed in cells transduced with Ad5/CFTR and Ad5/GFP-CFTRwt (Figure 3), suggesting a correction of the Cl^– channel function upon expression of the nontagged CFTR and GFP-CFTRwt proteins. In both cases, the recovery of the Cl^– channel activity was detectable from MOI 40, and not at lower MOI, implying that probably a threshold of expression of functional GFP-CFTRwt proteins had to be attained before the correction of CFTR activity could be detected. It is noteworthy that the correction level of the Cl^– channel between cells transduced by Ad5/CFTR and Ad5/GFP-CFTR at the same MOIs differed by about 10 to 15%, suggesting that the activity of the GFP-tagged CFTR protein was minimally hindered by the GFP protein.

View larger version (22K): [in this window] [in a new window]   Figure 3. CFTR Cl– channel activity in CF-KM4 cells after mock- or Ad5-transduction at various MOI. Histogram representations of the iodide efflux in cells at 30 hours after infection with Ad5/CFTR, Ad5/GFP-CFTRwt, and Ad5/GFP-CFTRF508. The measurements were made in cell monolayers which were mock-infected (NI/NT) or infected by Ad at multiplicity of infections (MOI) of 20, 40, 60, and 80 plaque-forming units per cell.

View larger version (43K): [in this window] [in a new window]   Figure 4. CFTR Cl– channel activity in MM-39 (a, b) and CF-KM4 (c, d) in mock-transduced cells (NI/NT) or cells transduced by Ad5/GFP-CFTRwt at various MOI. The curve plots of the 125I efflux presented were obtained at 48 hours (shaded bars) and 96 hours (solid bars) after infection. NI/MPB91 refers to a set of control samples treated with MPB91 at 250 mM for 2 hours. * or °, P < 0.05; ** or °°P < 0.01; *** or °°°P < 0.001. fsk, forskolin; gst, genistein; ns, no significant difference. Flow cytometry analysis of Ad5/GFP-CFTRwt transduced CF-KM4 cells (e, f). Cells infected with Ad5/GFP-CFTRwt at various MOI, as indicated on the x-axis, were harvested at 48 hours (shaded bars) or 96 hours after infection (solid bars). Results are expressed as percentage of GFP-positive cells (e), and as mean fluorescence intensity (MFI; f), expressed as arbitrary units (AU).

Functionality of GFP-CFTRwt and GFP-CFTRF508 in Primary Human Airway Epithelial Cells
Normal human airway epithelial cells (HAE) and CFTR-deficient epithelial cells (CF-HAE) were grown as reconstituted epithelia on an air–liquid interface and infected with Ad5/GFP-CFTRwt or Ad5/GFP-CFTRF508 at MOI 200. The GFP-CFTR localization was analyzed at 48 hours after infection by fluorescence confocal microscopy and immunofluorescence staining of the cell tight junctions (ZO-1) at the apical pole. In CF-HAE cells, the GFP-CFTRwt protein was localized to the apical pole with the ZO-1 protein (red) marker (Figures 5a and 5b). There were some GFP-CFTR observed at the basal region together with the nuclei of the cells that were stained blue with DAPI (Figure 5c). In contrast, the GFP-CFTRF508 fluorescence was found more in the central and basal areas of the cytoplasm (Figures 5d–5f). The same fluorescence profile was observed for Ad5/GFP-CFTRwt– and Ad5/GFP-CFTRF508–transduced HAE cells (not shown).

View larger version (67K): [in this window] [in a new window]   Figure 5. Confocal microscopy of the cellular localization of GFP-CFTRwt (a–c) and GFP-CFTRF508 mutant (d–f) in polarized CFTR-deficient airway epithelia after Ad5 transduction. (a, d) X-Z images of CF-HAE cells with apical membrane at the top and filter support at the bottom. (b, e) X-Y images taken at the level of the apical membrane. (c, f) X-Y images taken through the basolateral membrane. GFP-tagged CFTR proteins were detectable by their green fluorescent signal, the nuclei were stained in blue with DAPI, and the apical tight junctions (red staining) were immunostained by mouse monoclonal anti–ZO-1 antibody and Alexa 568–conjugated secondary antibody.

View larger version (14K): [in this window] [in a new window]   Figure 6. Effect of expression of GFP-tagged and non-tagged CFTR on the transepithelial Cl– current in CFTR-deficient airway epithelia. Well-differentiated epithelia were basolaterally infected with Ad5/GFP-CFTRwt, Ad5/GFP-CFTRF508, or Ad5/CFTR at MOI 200 (2,000 particles/cell), or mock-infected (control). Three days after Ad-transduction, the cells were analyzed in Ussing chambers for transepithelial Cl– current, measured under short-circuit conditions. Data shown are bumetanide-sensitive current (Isc); n = 4 epithelia.

In this study, we investigated different biological and cellular aspects of GFP-tagged CFTR proteins delivered by adenovirus vectors to cells from the human respiratory tract, two tracheal glandular cell lines, MM-39 (CFTRwt) and CF-KM4 (CFTRF508), and human primary airway epithelial cells from a patient with CF (CF-HAE) and from a healthy donor (HAE). Of note, in healthy lungs, the most predominant location of CFTR expression are the tracheal and bronchial submucosal gland serous cells (24).

The cellular localization of the two GFP-CFTR fusion proteins, CFTRwt and CFTRF508 mutant, was analyzed by confocal fluorescence microscopy and IEM. The GFP-CFTRwt protein expressed in CFTR-deficient CF-KM4 cells by Ad5/GFP-CFTRwt clearly showed localization at the plasma membrane with some localization in the cytoplasm (Figure 1), and quantitative immunoelectron microscopic analysis confirmed the localization of the GFP-CFTRwt protein at the plasma membrane and in association with cytoplasmic membranes and vesicular structures (Figure 2A). In contrast, the mutant GFP-CFTRF508 protein was found to accumulate in the perinuclear area of the cytoplasmic compartment (Figures 1 and 2A).

When primary CF-HAE cells grown as polarized epithelia were transduced by Ad5/GFP-CFTRwt and Ad5/GFP-CFTRF508, the GFP-CFTRwt protein was observed at the apical surface, whereas the GFP-CFTRF508 mutant remained sequestered in the central and basal regions of the epithelium (Figure 5). Taken together, our results showed that our Ad5/GFP-CFTRwt vector expressed a GFP-fused CFTR protein, which was correctly addressed to the plasma membrane of CF-tracheal cells and the apical pole of reconstituted human airway epithelia. This is the first report showing the apical surface localization of an exogenous GFP-tagged CFTR protein on polarized human epithelial cells after Ad-mediated gene transfer.

A previous report showed that a GFP-fused CFTR protein with a 23-residue long linker was competent for apical localization in polarized canine MDCK cells (7); however, the same could not be demonstrated in human epithelial cells (personal communication, M. J. Welsh and L. S. Ostedgaard). Likewise, a GFP-CFTRwt fusion protein construct with an eight–amino acid spacer with sequence REPSARET, expressed by an Ad vector, failed to localize to the plasma membrane of A549 cells (8). The kink provoked by the proline residue in the linker could have consequences in its flexibility and therefore in the mobility between the two domains. Moreover, the REPSARET sequence has homology with PEST motifs, which could be responsible for undesired proteolytic cleavage between the GFP and CFTR domains (25, 26). Our GFP-CFTRwt construct carried a seven–amino acid flexible linker with the sequence SGLRSRA, which contained no bulky or peptide chain–bending amino acid residues. Although some spontaneous cleavage (10–15%) of our GFP-CFTRwt protein was observed, a majority of the GFP remained fused to the CFTRwt protein. It is likely that the difference in protein stability, functionality, and trafficking between the different GFP-CFTR chimeras is due to the nature of the linker sequence.

The GFP-CFTRwt was found to be functional in both CFTR-deficient tracheal cells and reconstituted epithelia. The GFP-fused CFTRwt protein was almost equivalent to the nonfused native protein in terms of Cl^– channel function, implying that, in contrast to previous reports, the GFP-tag had minimum influence on the CFTR activity. The GFP-CFTRwt correcting gene delivered by an Ad vector to CFTR-deficient CF-KM4 cells restored the Cl^– channel activity to subnormal levels. We were able to correlate the gain in CFTR function in Ad-transduced CF-KM4 cells with the vector input and the number of transduced cells. This gain in function occurred in an Ad dose–dependent manner, but within a relatively narrow MOI range (20–60 PFU/cell). Within this range, 55 to 70% of transduced CF-KM4 cells corresponded to a correction level of 70% of the Cl^– channel activity found in control MM-39 cells, which were of the same cell type. It was previously shown that the addition of 5 to 10% of CFTR-positive cells to CFTR-deficient epithelial cells was sufficient to correct the Cl^– channel defect of the entire culture (15, 27), and that overexpression could have an adverse effect on cellular localization and functionality of CFTR (15). Likewise, results obtained from a mouse model suggested that expression of 5% of the normal CFTR level was sufficient to correct 50% of the chloride ion transport defect and obtain a complete rescue of CFTR-associated intestinal disease (28). It was thus predicted that modest levels of CFTR gene expression and partial correction of the CFTR channel activity would have a significant therapeutic clinical impact. If this is the case, our 70% restoration of the Cl^– channel function would be more than sufficient to provide a net therapeutic benefit, and this correction was obtained at reasonably moderate MOI. Higher vector input (MOI of 80–100 PFU/cell) had a negative effect on the Cl^– channel activity (Figures 4b and 4d). The same negative effect was observed in CFTR-deficient cells (CF-KM4) and in cells with functional endogenous CFTR activity (MM-39) after infection with the two control vectors Ad5/GFP and Ad5/GFP-CFTRF508 used at high MOI (data not shown). Our data with control Ad5/GFP vector lacking CFTR in normal MM-39 cells excluded a specific downregulation of endogenous CFTR function by interference with exogenous CFTR at the gene expression level, and suggested that the negative effect on endogenous CFTR function was due to the Ad vector toxicity at high doses. The MM-39 and CF-KM4 cells do not to express CAR (the Coxsackie-Adenovirus receptor [29]), and a relatively high vector input was needed to obtain satisfactory Ad transduction. Using Ad vectors modified for efficient gene delivery into these cells (30) would effectively allow satisfactory correction at low vector doses and thus avoid vector toxicity.

The two vectors Ad5/GFP-CFTRwt and Ad5/GFP-CFTRF508 described in this work represent biological tools that can be further used to study the functional properties of normal and mutant CFTR in other systems, and to further dissect their molecular interactions with cell partners in situ in a variety of living tissues and experimental conditions. The expression of exogenous CFTR proteins can now be biologically tracked by both its function and cellular localization, which will allow rapid translation of therapeutic interventions for cystic fibrosis.

Acknowledgments

The authors are grateful for the technical assistance of L. Franqueville (Boulanger Lab), S. Peyrol, and C. Cassin (CeCIL, Laennec) in electron microscopy, and of B. Smatti and D. Ressnikoff (CCQ, Domaine Rockefeller) in confocal fluorescence microscopy.

Footnotes

^* These authors contributed equally to this work.

This work was supported by the French Cystic Fibrosis Foundation (Vaincre la Mucoviscidose, VLM) (to O.G. and C.N.), the University of Claude Bernard Lyon, the University of Poitiers, and the Centre National de la Recherche Scientifique (CNRS). S.-S.H. is an INSERM Chargé de Recherche.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0026TE on July 19, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form January 29, 2007

Accepted in final form June 17, 2007

References

1. Davies JC, Geddes DM, Alton EWF. Gene therapy for cystic fibrosis. J Gene Med 2001;3:409–417.[CrossRef][Medline]
2. Rowntree RK, Harris A. The phenotypic consequences of CFTR mutations. Ann Hum Genet 2003;67:471–485.[CrossRef][Medline]
3. Riordan JR, Rommens JM, Kerem B-S. Identification of the cystic fibrosis gene: cloning and characterization of complementary DNA. Science 1989;245:1066–1073.[Abstract/Free Full Text]
4. Imundo L, Barasch J, Prince A, Al-Awqati Q. Cystic fibrosis epithelial cells have a receptor for pathogenic bacteria on their apical surface. Proc Natl Acad Sci USA 1995;92:3019–3023.[Abstract/Free Full Text]
5. Lee TWR, Matthews DA, Blair GE. Novel molecular approaches to cystic fibrosis gene therapy. Biochem J 2005;387:1–15.[CrossRef][Medline]
6. Matsui H, Grubb BR, Tarran R, Randell SH, Gatzy JT, Davis W, Boucher RC. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airway disease. Cell 1998;95:1005–1015.[CrossRef][Medline]
7. Moyer BD, Loffing J, Schwiebert EM, Loffing-Cueni D, Halpin PA, Karlson KH, Ismailov II, Guggino WB, Langford GM, Stanton BA. Membrane trafficking of the cystic fibrosis gene product, cystic fibrosis transmembrane conducance regulator, tagged with green fluorescent protein in Madin-Darby Canine Kidney cells. J Biol Chem 1998;273:21759–21768.[Abstract/Free Full Text]
8. Vais H, Gao G-P, Yang MPT, Louboutin J-P, Somananthan S, Wilson JM, Reenstra W. Novel adenoviral vectors coding for GFP-tagged wtCFTR and delF508-CFTR: characterization of expression and electrophysiological properties in A549 cells. Pflugers Arch Eur. J Physiol 2004;449:278–287.[Medline]
9. Merten MD, Kammouni W, Renaud W, Birg F, Mattei MG, Figarella C. A transformed human tracheal gland cell line, MM-39, that retains serous secretory functions. Am J Respir Cell Mol Biol 1996;15:520–528.[Abstract]
10. Kammouni W, Moreau B, Becq F, Saleh A, Pavirani A, Figarella C, Merten MD. A cystic fibrosis tracheal gland cell line, CF-KM4. Correction by adenovirus-mediated CFTR gene transfer. Am J Respir Cell Mol Biol 1999;20:684–691.[Abstract/Free Full Text]
11. Zabner J, Zeiher BG, Friedman E, Welsh MJ. Adenovirus-mediated gene transfer to ciliated airway epithelia requires prolonged incubation time. J Virol 1996;70:6994–7003.[Abstract/Free Full Text]
12. Zabner J, Scheetz TE, Almabrazi HG, Casavant TL, Huang J, Keshavjee S, McCray PBJ. CFTR DF508 mutation has minimal effect on the gene expression profile of differentiated human airway epithelia. Am J Physiol Lung Cell Mol Physiol 2005;289:L545–L553.[Abstract/Free Full Text]
13. Chartier C, Degryse E, Gantzer M, Dieterlé A, Pavirani A, Mehtali M. Efficient generation of recombinant adenovirus vectors by homologous recombination in Escherichia coli. J Virol 1996;70:4805–4810.[Abstract]
14. Defer C, Belin MT, Caillet-Boudin ML, Boulanger P. Human adenovirus-host cell interactions: comparative study with members of subgroups B and C. J Virol 1990;64:3661–3673.[Abstract/Free Full Text]
15. Farmen SL, Karp PH, Ng P, Palmer DJ, Koehler DR, Hu J, Beaudet AL, Zabner J, Welsh MJ. Gene transfer of CFTR to airway epithelia: low levels of expression are sufficient to correct Cl- transport and overexpression can generate basolateral CFTR. Am J Physiol Lung Cell Mol Physiol 2005;289:L1123–L1130.[Abstract/Free Full Text]
16. Zabner J, Couture LA, Gregory RJ, Graham SM, Smith AE, Welsh MJ. Adenovirus-mediated gene transfer transiently corrects the chloride transport defect in nasal epithelial of patients with cystic fibrosis. Cell 1993;75:207–216.[CrossRef][Medline]
17. Zabner J, Karp P, Seiler M, Phillips SL, Mitchell CJ, Saavedra M, Welsh MJ, Klingelhutz AJ. Development of cystic fibrosis and noncystic fibrosis airway cell lines. Am J Physiol Lung Cell Mol Physiol 2003;284:L844–L854.[Abstract/Free Full Text]
18. Violot S, Hong SS, Rakotobe D, Petit C, Gay B, Moreau K, Billaud G, Priet S, Sire J, Schwartz O, et al. The human polycomb group EED protein interacts with the integrase of human immunodeficiency virus type 1. J Virol 2003;77:12507–12522.[Abstract/Free Full Text]
19. Derand R, Bulteau-Pignoux L, Mettey Y, Zegarra-Moran O, Howell LD, Randak C, Galietta LJ, Cohn JA, Norez C, Romio L, et al. Activation of G551D CFTR channel with MPB-91: regulation by ATPase activity and phosphorylation. Am J Physiol Cell Physiol 2001;281:C1657–C1666.[Abstract/Free Full Text]
20. Dormer RL, Derand R, McNeilly CM, Mettey Y, Bulteau-Pignoux L, Metaye T, Vierfond J, Gray MA, Galietta LJ, Morris MR, et al. Correction of delF508-CFTR activity with benzo(c)quinolizinium compounds through facilitation of its processing in cystic fibrosis airway cells. J Cell Sci 2001;114:4073–4081.[Medline]
21. Melin P, Thoreau V, Norez C, Bilan F, Kitzis A, Becq F. The cystic fibrosis mutation G1349D within the signature motif LSHGH of NBD2 abolishes the activation of CFTR chloride channels by genistein. Biochem Pharmacol 2004;67:2187–2196.[CrossRef][Medline]
22. Venglarik CJ, Bridges RJ, Frizzell RA. A simple assay for agonist-regulated Cl and K conductances in salt-secreting epithelial cells. Am J Physiol 1990;259:C358–C364.[Medline]
23. Ostedgaard LS, Randak C, Rokhlina T, Karp P, Vermeer D, Ashbourne Excoffon KJ, Welsh MJ. Effects of C-terminal deletions on cystic fibrosis transmembrane conductance regulator function in cystic fibrosis airway epithelia. Proc Natl Acad Sci USA 2003;100:1937–1942.[Abstract/Free Full Text]
24. Koehler DR, Hitt MM, Hu J. Challenges and strategies for cystic fibrosis lung gene therapy. Mol Ther 2001;4:84–91.[CrossRef][Medline]
25. Rogers S, Wells R, Rechsteiner M. Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science 1986;234:364–368.[Abstract/Free Full Text]
26. Rechsteiner M, Rogers SW. PEST sequences and regulation by proteolysis. Trends Biochem Sci 1996;21:267–271.[CrossRef][Medline]
27. Johnson LG, Olsen JC, Sarkadi B, Moore KL, Swanstrom R, Boucher RC. Efficiency of gene transfer for restoration of normal airway epithelial function in cystic fibrosis. Nat Genet 1992;2:21–25.[CrossRef][Medline]
28. Dorin JR, Farley R, Webb S, Smith SN, Farini E, Delaney SJ, Wainwright BJ, Alton EW, Porteous DJ. A demonstration using mouse models that successful gene therapy for cystic fibrosis requires only partial gene correction. Gene Ther 1996;3:797–801.[Medline]
29. Gaden F, Franqueville L, Hong SS, Legrand V, Figarella C, Boulanger P. Mechanism of restriction of normal and cystic fibrosis transmembrane conductance regulator-deficient human tracheal gland cells to adenovirus (Ad) infection and Ad-mediated gene transfer. Am J Respir Cell Mol Biol 2002;27:628–640.[Abstract/Free Full Text]
30. Gaden F, Franqueville L, Magnusson MK, Hong SS, Merten MD, Lindholm L, Boulanger P. Gene transduction and cell entry pathway of fiber-modified Adenovirus type 5 vectors carrying novel endocytic peptide ligands selected on human tracheal glandular cells. J Virol 2004;78:7227–7247.[Abstract/Free Full Text]

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