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¹ Gregory Fleming James Cystic Fibrosis Research Center
² Department of Cell Biology
⁵ Department of Pediatrics
⁶ Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, USA
³ Tranzyme, Inc., Birmingham, AL, USA
⁴ Southern Research Institute, Birmingham, AL, USA

	Abstract

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The cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-regulated chloride channel. Mutations in the CFTR gene result in cystic fibrosis (CF). The most common mutation, F508, results in endoplasmic reticulum-associated degradation (ERAD) of CFTR. F508 CFTR has been described as a temperature-sensitive mutation that can be rescued following growth at 27°C. In order to study the processing and function of wild-type and rescued F508 CFTR at the cell surface under non-polarized and polarized conditions, we developed stable cell lines expressing F508 or wild-type CFTR. CFBE41o^– is a human airway epithelial cell line capable of forming high resistance, polarized monolayers when cultured on permeable supports, while HeLa cells are normally grown under non-polarizing conditions. Immunoprecipitation, cell surface biotinylation, immunofluorescence, and functional assays confirmed the presence of F508 CFTR at the cell surface in both cell lines after incubating the cells for 48 h at 27°C. However, stimulators of wild-type CFTR such as forskolin, ß₂-adrenergic or A_2B-adenosine receptor agonists failed to activate rescued F508 CFTR in CFBE41o^– monolayers. Rescued F508 CFTR could be stimulated with genistein independent of pretreatment with cAMP signalling agonists. Interestingly, rescued F508 CFTR in HeLa cells could be efficiently stimulated with either forskolin or genistein to promote Cl^– transport. These results indicate that F508 CFTR, when rescued in CFBE41o^– human airway epithelial cells, is poorly responsive to signalling pathways known to regulate wild-type CFTR. Furthermore, the differences in rescue and activation of F508 CFTR in the two cell lines suggest that cell-type specific differences in F508 CFTR processing are likely to complicate efforts to identify potentiators and/or correctors of the F508 defect.

(Received 17 August 2005; accepted after revision 4 October 2005; first published online 6 October 2005)
Corresponding author J. P. Clancy: Department of Pediatrics, University of Alabama at Birmingham, 620 ACC, 1600 7th Ave South, Birmingham, AL 35233, USA. Email: jpclancy{at}peds.uab.edu

	Introduction

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Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that encodes the CFTR protein. Deletion of phenylalanine at position 508 (F508) is the most common disease-associated mutation, and this mutation is found in approximately 70% of mutant chromosomes and in nearly 90% of CF affected individuals (Cheng et al. 1990; Pilewski & Frizzell 1999; Davis 2001). Patients who are homozygous for the F508 defect frequently have a ‘severe’ phenotype, at least with respect to exocrine pancreatic function (Kerem & Kerem 1996). The F508 mutation causes misfolding and ER-associated degradation (ERAD) of CFTR (Ward & Kopito 1994; Ward et al. 1995; Gelman et al. 2002). This results in severely reduced or absent CFTR at the cell surface and defective cAMP-dependent Cl^– conductance in affected tissues.

It has been shown that F508 CFTR is a temperature-sensitive mutant and may reach the cell surface in cells cultured at low (27°C) temperature such as Xenopus oocytes or Sf9 insect cells where it retains cAMP- and PKA-dependent activity (Denning et al. 1992; Bear et al. 1992). Furthermore, small molecules called ‘chemical chaperones’ can also be used to release F508 CFTR from ERAD (Brown et al. 1996; Zeitlin 2000). Based on these observations, large scale efforts have been undertaken to identify rescue agents that ‘correct’F508 CFTR processing and that could be used in vivo (Zeitlin 1998; Welch & Howard 2000; Galietta et al. 2001; Welch 2004).

More recent studies on isolated membrane patches from cells treated to promote F508 CFTR plasma membrane localization suggest that the rescued surface protein may not retain all functions of the wild-type CFTR, and that it may be resistant to activation by PKA and ATP (Hwang et al. 1997; Wang et al. 1998; Al-Nakkash & Hwang 1999; Yang et al. 2003). The magnitude and cell-type specificity of F508 CFTR responsiveness to activating stimuli, however, remains controversial. Furthermore, because activation of wild-type CFTR in vivo is likely to be accomplished through tightly regulated receptor-based mechanisms, it is important to test whether rescued F508 CFTR retains this function (Hentchel-Franks et al. 2004). Therefore, functional studies in polarized airway cell model systems with wild-type or F508 CFTR expression have significance and are needed in order to compare their function in the same cellular background.

Two G protein coupled receptor (GPCR) signalling cascades (ß₂-adrenergic receptors (ß₂-ARs) and A_2B-adenosine receptors (A_2B-ARs)) efficiently activate wild-type CFTR in vitro and in vivo by stimulating adenyl cyclase, and raising cellular cAMP (Clancy et al. 1999; Huang et al. 2001; Cobb et al. 2002; Naren et al. 2003). Although mechanistic distinctions have been drawn between these pathways, they both activate CFTR in a spatially restricted manner, probably through protein–protein interactions between CFTR and CFTR-binding partners at the epithelial plasma membrane (Hall et al. 1998; Welch & Howard 2000; Naren 2000; Sun et al. 2000).

In order to study the functional activity of rescued F508 compared to wild-type CFTR on different cellular backgrounds, we developed and characterized stable wild-type and F508 CFTR expressing human bronchial epithelial (CFBE41o^– WT and CFBE41o^–F) and HeLa (HeLa WT and HeLa F) cell lines. Here we show that while forskolin or GPCR pathways are functional in CFBE41o^– cells, activation of rescued F508 CFTR through these pathways is defective in CFBE41o^–F monolayers. In contrast, raising cAMP levels activates F508 CFTR in HeLa F cells. Failure of these physiological GPCR pathways to activate F508 CFTR in human airway cell monolayers suggests that pharmacological correctors of F508 processing defects need to be evaluated in relevant cell models before clinical trials begin. Our findings support the existence of the previously suggested, but unappreciated, defects in F508 CFTR signal reception and transduction that are attributable to the F508 CFTR mutation.

	Methods

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Development of stable cell lines expressing WT and F508 CFTR

Wild-type and F508 CFTR cDNA was stably introduced into HeLa and CFBE41o^– cells using TranzVector (Tranzyme, Inc., Birmingham, AL, USA). The TranzVector system represents an HIV-based lentiviral vector with unique safety features as described by Wu et al. (2000). To generate vector stock, CFTR cDNA was first cloned into the gene transfer component under the control of the human cytomegalovirus (hCMV) promoter. Expression of CFTR was also coupled to the puromycin-N-acetyltransferase gene (puro) gene via the internal ribosomal entry site (IRES) of encephalomyocarditis virus, allowing for rapid selection of cells expressing CFTR in media containing puromycin. HeLa and CFBE41o^– cells were transduced at multiplicity of infection of one followed by puromycin (4 µg ml^–1) selection. Puromycin-resistant cells were expanded to form a pool of stable CFTR expressors.

Ussing chamber studies in CFBE41o^– cell monolayers

Inserts were mounted in Ussing chambers, and I_sc was measured under voltage clamp conditions as previously described (Hentchel-Franks et al. 2004; Cobb et al. 2002). Briefly, cells grown at an air–liquid interface were mounted in modified Ussing chambers (Jim's Instruments, Iowa City, IA, USA), and initially bathed on both sides with identical Ringer solutions containing (mM): 115 NaCl, 25 NaHCO₃, 2.4 KH₂PO₄, 1.24 K₂HPO₄, 1.2 CaCl₂, 1.2 MgCl₂, 10 D-glucose (pH 7.4). Bath solutions were vigorously stirred and gassed with 95% O₂–5% CO₂. Solutions and chambers were maintained at 37°C. Short circuit current (I_sc) measurements were obtained by using an epithelial voltage clamp (University of Iowa Bioengineering, Iowa City, IA, USA). A 3-mV pulse of 1 s duration was imposed every 100 s to monitor resistance, which was calculated using Ohm's law. To measure stimulated I_sc, the mucosal bathing solution was changed to a low Cl^– solution containing 1.2 mM NaCl, 115 mM sodium gluconate and all other components as above plus 100 µM amiloride. Increasing agonist concentrations were added to the bathing solutions as indicated (minimum 5 min of observation at each concentration). Unless otherwise noted, time points were taken at 500 s intervals. Glybenclamide (200 µM) was added to the mucosal bathing solution at the end of experiments to block CFTR-dependent I_sc.

The CFBE41o^– WT and CFBE41o^–F cells were handled in exactly the same fashion during our studies, except for the period of growth at low temperature for 48 h prior to placement in Ussing chambers (where indicated for the CFBE41o^–F monolayers). Specifically, chambers were maintained at 37°C, and agonist stimulation was initiated within 1 h of placement in the chambers. Control experiments performed in our laboratory have not found differences in agonist responses of CFBE41o^–F monolayers (following temperature correction) that are subsequently studied in chambers maintained at 27°C versus 37°C. The data are shown for 37°C chambers to allow direct comparison with the WT-CFTR condition.

SPQ studies of halide efflux in HeLa cells

HeLa cells stably expressing WT or F508 CFTR were studied with the halide quenched dye 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ, Molecular Probes Inc., Eugene, OR, USA) as previously described (Clancy et al. 1998; Cobb et al. 2002). Briefly, cells were seeded at 5 x 10⁵ cells/coverslip and grown in Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) at 37°C for 48 h. The medium was changed, and cells were subsequently grown for 48 h at either 37°C or 27°C. On the day of study, cells were loaded with hypotonic SPQ (10 mM) for 10 min, and then placed in a NaI buffer to quench cellular fluorescence. The cells were then placed in a specially designed perfusion chamber and studied at room temperature. Fluorescence of individual cells was measured using a Zeiss inverted microscope (excitation at 340 nm, emission at > 410 nm), a PTI imaging system, and a Hamamatsu camera. Baseline fluorescence was measured in isotonic NaI buffer, followed by perfusion with isotonic dequench buffer (NaNO₃ replaced NaI) to measure unregulated efflux, and then NaNO₃ buffer with 20 µM forskolin, 50 µM genistein, or forskolin and genistein as indicated. At the end of each experiment, cells were returned to the NaI buffer for requench (1100 s). Increase in fluorescence above the basal (NaI quenched) level is shown (% increase F > basal). The data are cumulative from three coverslips in each condition studied in a paired fashion on three separate days (n= 50 cells/curve). The bottom 10% of cells in all conditions (attributable to inadequate SPQ loading, cell detachment, etc.) were discarded and the data obtained from the top 90% of cells in each condition were analysed as previously described (Clancy et al. 1998, 1999).

Real time RT-PCR to quantify CFTR expression

TaqMan One Step RT-PCR protocol (Applied Biosystems, Foster City, CA, USA) was used to quantify CFTR mRNA transcripts using ‘Assays on Demand’ Gene Expression Products, coupled with the ABI Prism 7500 sequence detection system (Applied Biosystems). Briefly, total RNA was isolated using the Qiagen RNeasy mini kit according to the manufacturer's instructions (Qiagen, Valencia, CA, USA). To prevent possible DNA contamination, the samples were pretreated with RNase-free DNase (Qiagen). Sequence specific primers and probes for human CFTR and 18S rRNA were purchased from Assays on Demand (ABI); Assay ID for CFTR: Hs00357011_m1; the probe extends across the exon 21/22 boundary of the CFTR sequence. TaqMan One Step PCR Master Mix Reagents Kit (ABI) was used for reverse transcriptase and PCR. The reaction volume was 25 µl including 12.5 µl of 2x Master Mix without UNG (AmpErase uracil-N-glycosylase), 0.625 µl of 40x MultiScribe and RNase Inhibitor Mix, 1.25 µl of 20x target primer and probe, 5.625 µl of nuclease-free water (Ambion), and 5 µl of RNA sample. The reaction plates were covered with an optical cap and centrifuged briefly to remove bubbles. The thermocycler conditions were as follows: Stage 1: 48°C for 30 min; Stage 2: 95°C for 10 min; Stage 3: 95°C for 15 s, repeat 40 cycles, 60°C for 1 min. All experiments were run in triplicate on two separate days. The absolute value of the slope of log input amount versusC_t was > 0.1, implying that the efficiencies of CFTR and 18S rRNA amplification were not equal. Therefore, the relative quantification of transcript levels (CFTR compared with endogenous 18S rRNA) was performed using the standard curve method.

Immunocytochemistry

CFBE41o^– cells were grown on 12 mm diameter polycarbonate filters (Corning-Costar) for 8–10 days, and HeLa cells were grown on glass coverslips. To rescue F508 CFTR, cells were grown at 27°C for 48 h. After fixation in 4% buffered formaldehyde for 20 min, cells were permeabilized in 0.01% Triton X-100 in PBS for 5 min and non-specific protein binding sites were blocked using goat serum diluted 1: 20 in PBS for 30 min. Anti-CFTR C terminal (24–1, ATCC no. HB-11947) monoclonal antibody (5 µg ml^–1 for 2 h at room temperature) was used to detect CFTR and anti-ZO1 polyclonal, rabbit antibody (1 µg ml^–1 for 2 h at room temperature, Zymed Laboratories) was used to stain tight junctions. Nuclei were stained with Vectashield with 4',6-diamidino-2-phenylindole (DAPI) (Vector Laboratories).

Microscopy

Images were captured on an Olympus IX170 inverted epifluorescence microscope equipped with step motor, filter wheel assembly (Ludl Electronics Products, Hawthorne, NY, USA), and 83000 filter set (Chroma Technology, Brattleboro, VT, USA) and SenSys-cooled charge-coupled high-resolution camera (Photometrics, Tucson, AZ, USA). Partial deconvolution of images was performed using IPLab software (Scanalytics, Fairfax, VA, USA).

CFTR immunoprecipitation

CFTR was immunoprecipitated from 500 µg of total cellular proteins using 1 µg 24–1, anti-C-terminal antibody (ATCC no. HB-11947) coupled to 20 µl Protein A agarose beads (Roche Biochemicals) and in vitro phosphorylated with [-³²P]ATP (NEN) and PKA catalytic subunit (Promega, Madison, WI, USA). Labelled CFTR was analysed by SDS-PAGE and autoradiography as previously described (Bebok et al. 1998).

Cell surface biotinylation

Cell surface glycoproteins were biotinylated as previously described (Peter et al. 2002). Total CFTR and biotinylated CFTR was immunoprecipitated, separated by SDS-PAGE and Western blotted. CFTR was detected with anti-CFTR NBD2 polyclonal antibody and biotinylated CFTR was detected with avidin–HRP followed by ECL (Pierce Biotechnology, Inc., Rockford, IL, USA).

cAMP measurements

Cellular cAMP was measured using an ELISA-based detection kit as previously described (Cayman Chemicals, Ann Arbor, MI, USA) (Cobb et al. 2003). Briefly, cells grown on 60 mm dishes (7 x 10⁶ cells/dish) were stimulated with agonist for 10 min (at 37°C), and the cellular cAMP was extracted with ice-cold ethanol. The supernatants were dried, re-suspended in phosphate buffer, and the cAMP levels were quantified according to the manufacturer's directions.

ATP (nucleotide) measurements

The amount of ATP in acid-soluble extracts of CFBE41o^– monolayers were measured using Partisil strong anion exchange (SAX, Keystone Scientific Inc., Bellefonte, PA, USA) HPLC as described (Parker et al. 1999). ATP levels were determined by measuring its UV absorbance at 260 nm. Nucleotide concentrations were derived from HPLC standard curves.

Statistical analysis

For I_sc, phosphor images, cAMP, ATP levels, and RT- PCR measurements, descriptive statistics (mean and S.E.M.) and paired and unpaired t tests were performed using SigmaStat software (Systat Software Inc., Point Richmond, CA, USA). All statistical tests were two-sided and were performed at a 5% significance level (i.e. = 0.05).

	Results

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Characterization of the model cell lines and rescue of F508 CFTR at 27°C

RT-PCR.  To characterize the newly developed model cell lines used in our studies, we first measured CFTR mRNA levels in HeLa WT, HeLa F, CFBE41o^– WT, and CFBE41o^–F cells using real time RT-PCR. Parental HeLa and CFBE41o^– cells were included as controls. Figure 1A summarizes CFTR mRNA levels as measured by real time RT-PCR and plotted as a ratio of 18S RNA. Recombinant wild-type and F508 CFTR transcripts were detected in transduced HeLa and CFBE41o^– cell lines in similar ranges, whereas expression of endogenous CFTR in parental cells (HeLa and CFBE41o^–) was below the level of detection. These cell models therefore serve as useful tools to study recombinant wild-type and F508 CFTR in the absence of detectable endogenous CFTR expression.

View larger version (46K): [in this window] [in a new window]   Figure 1.  Characterization of HeLa WT, HeLa F, CFBE41o– WT and CFBE41o–F cell lines A, real time RT-PCR to measure CFTR mRNA levels. CFTR transcript levels were measured using TaqMan quantitative PCR using 18S rRNA as control. Samples were internally normalized to 18S rRNA, and plotted as CFTR mRNA levels relative to 18S rRNA. Parental HeLa and CFBE41o– cells were used as controls. Mean and S.D. of n= 6 samples amplified under the same conditions. B, CFTR protein expression. CFTR was immunoprecipitated from 500 µg total cellular proteins using 24–1, anti-C terminal antibody, in vitro phosphorylated using [-32P]ATP, PKA (protein kinase A), separated on 6% PAGE and detected using Phosphorimager analysis. Calu-3 cells were used as controls for wild-type CFTR expression. B and B represents core glycosylated, ER form of CFTR. B and C represent fully glycosylated CFTR. Comparisons shown are from one day of study, and representative of > three studies in each of the conditions. C, low temperature (27°C) rescue of F508 CFTR in HeLa F and CFBE41o– F cells. Representative gels show the presence of B and B only (lane 1) in HeLa F and CFBE41o–F cells when the cells were grown at 37°C and the appearance of B and C after a 48 h incubation at 27°C (lanes 2–3). D, summary 27°C rescue experiments. Results are plotted as C/B band ratios after growing the cells at 27°C for 48 h, mean ±S.E.M., n= 12 per condition (P < 0.01 for HeLa C/B ratios compared with CFBE41o– C/B ratios).

CFTR detection at the plasma membrane.  WT and F508 CFTR localization to the plasma membrane was assessed by immunofluorescence and surface biotinylation (Fig. 2). In Fig. 2A, HeLa F cells grown at 37°C (upper left panel) and 27°C (upper middle panel) are compared, demonstrating redistribution of F508 CFTR to the plasma membrane at low temperatures. HeLa WT cells are shown as controls (upper right panel). The presence of F508 CFTR in the plasma membrane was confirmed by cell surface biotinylation (Fig. 2B, lower left panel). Only the rescued B and C was biotinylated, but there was a significant increase in both B and B and B and C levels at 27°C (total). Cell surface biotinylation experiments from HeLa WT cells grown at 37 or 27°C are shown as controls (lower right panel) and exhibit an increase in total CFTR levels at low temperature. Although rescue of F508 CFTR at 27°C in HeLa F cells was efficient, the biotinylated, surface pool of F508 CFTR (27°C) was considerably less than the surface WT-CFTR pool (37°C) (Fig. 2B).

View larger version (54K): [in this window] [in a new window]   Figure 2.  CFTR detection in HeLa F, HeLa WT, CFBE41o–F, and CFBE41o– WT cells by immunofluorescence and cell surface biotinylation A, cell immunofluorescence studies in HeLa cells. HeLa F508 CFTR cells grown at 37°C (Left) and 27°C for 48 hr (Middle). F508 CFTR staining (in green) moves from perinuclear (nucleus in blue) to cytoplasmic/surface at low temperature. HeLa Wt CFTR cells grown at 37°C are shown to the right. Colour figures can be seen in the online versions, while the accompanying journal figures are provided in greyscale. B, cell biotinylation experiments in HeLa cells. Total protein input was matched (25 µg lane–1). Left, HeLa F cells. Representative gels are shown with total (T) and biotinylated (B) CFTR. Biotinylated B and C is detected following growth at low temperature. Right, HeLa WT cells. Representative gels are shown with total (T) and biotinylated (B) CFTR. Biotinylated B and C is detected following growth at 27°C and 37°C. C, immunofluorescence studies in CFBE41o– monolayers. Left, CFBE41o–F cells grown on permeable supports at 37°C. En face images at the nuclear level (left) and at the level of tight junctions (right). ZO-1 was stained to demonstrate well-developed tight junctions (in red). F508 CFTR staining (in green) is only perinuclear at 37°C. Images below show side views of monolayers, and demonstrate F508 CFTR localization to the perinuclear region. Right, CFBE41o–F cells grown on permeable supports at 27°C. En face images at the nuclear level (left) and at the level of tight junctions (right). ZO-1 was stained to demonstrate well-developed tight junctions (red). After 48 h incubation at 27°C, F508 CFTR staining increased and could be seen at the level of tight junctions at the apical cell surface. Images in the middle show side views of monolayers and demonstrate F508 CFTR localization to the apical membrane after growth at 27°C. Colour figures can be seen in the online versions, while the accompanying journal figures are provided in greyscale. D, cell biotinylation experiments in CFBE41o– monoalyers. Total protein input was matched (25 µg lane–1). Left, CFBE41o–F cells. Representative gels are shown with total (T) and biotinylated (B) CFTR. Biotinylated B and C is detected following growth at low temperature. Right, CFBE41o– WT cells. Representative gels are shown with total (T) and biotinylated (B) CFTR. Biotinylated B and C is detected following growth at 27°C > 37°C.

Activation of CFTR in HeLa and CFBE41o^– cells

The results shown in Figs 1 and 2 demonstrate vastly different F508 CFTR rescue efficiencies in HeLa F and CFBE41o^–F cells at low temperature (27°C). In HeLa cells surface localized (biotinylated) WT-CFTR levels were higher at 37°C than rescued F508 CFTR at 27°C. In contrast, in the CFBE41o^– cells the rescued, cell surface localized F508 CFTR pool at 27°C was significantly higher than the surface wild-type CFTR pool at 37°C. Based on these biochemical observations, we compared activation of WT-CFTR (in cells grown at 37°C) to F508 CFTR (in cells grown at 27°C) within the two cell lines.

Wild-type and F508 CFTR activation in HeLa WT (37°C) and HeLa F (27°C) by forskolin, genistein and the combination of both agonists were compared using SPQ assay. The results shown in Fig. 3 indicate that halide efflux in both cells was activated by forskolin and genistein in a qualitatively similar and additive fashion. HeLa F cells grown at 37°C (no detectable cell surface CFTR) failed to respond to forskolin and genistein, confirming the CFTR dependence of stimulated halide efflux.

View larger version (40K): [in this window] [in a new window]   Figure 3.  Halide efflux in HeLa cells A, HeLa WT cells. Cells were grown at 37°C prior to study by SPQ as described in Methods. Each curve is the mean ±S.E.M. of 50 cells studied in the indicated condition. Halide efflux was activated by the addition of forskolin (10 µM), genistein (50 µM) or forkolin and genistein together at the time indicated by the arrow (agonist). Additive effects for the two agonists are demonstrated. Fluorescence was requenched in NaI buffer at the end of the experiments. B, HeLa F cells grown at 27°C and 37°C. Cells were stimulated with forskolin (10 µM), genistein (50 µM), or forskolin + genistein indicated by the arrow (agonist). Additive effects for the two agonists are demonstrated. Cells grown at 37°C failed to respond to the combination of forskolin + genistein.

View larger version (22K): [in this window] [in a new window]   Figure 4.  Cl– conductance in CFBE41o– WT monolayers Cells were grown as monolayers at 37°C and studied in Ussing chambers as described in Methods. LoCl–, switch to apical LoCl– buffer; Am, addition of amiloride (100 µM) to the apical compartment; Glyb, addition of glybenclamide (200 µM) to the apical compartment. Axes are noted within each panel (µA –Y axis, time in minutes –X axis). A–C, examples of monolayers stimulated with forskolin (10 µM, A), NECA (10 µM, B), or albuterol (10 µM, C). Brisk currents are produced by all three agonists, with blockade following addition of glybenclamide. D, summary of dose–response experiments performed in CFBE41o– WT monolayers. Cells were stimulated with increasing concentrations of agonists (0.1, 1.0, 10 µM) as shown. Forskolin, NECA, and albuterol produced maximal currents at 0.1–1.0 µM, and Ado produced maximal currents at 1.0–10 µM (consistent with stimulation of the low affinity A2B AR). n= 6–8 filters studied in each condition. *NECA, ALB and Forskolin currents at 2000 s > 1000 s (P < 0.001). Ado currents at 3000 s > 2000 s (P < 0.001).

View larger version (26K): [in this window] [in a new window]   Figure 5.  Cl– conductance in parental CFBE41o– monolayers (no CFTR transduction) Parental cells were grown as monolayers at 27°C and studied in Ussing chambers as described in Methods. LoCl–, switch to apical LoCl– buffer; Am, addition of amiloride (100 µM) to the apical compartment; Glyb, addition of glybenclamide (200 µM) to the apical compartment. A, cells were stimulated with 10 µM agonists as indicated (forskolin, NECA, albuterol) followed by genistein (50 µM). No currents were stimulated by any of the agonists (n= 6 filters/condition). B, cells were stimulated with the calcium mobilizing agents ionomycin (2 µM) or A23187 (5 µM) as indicated. Both agonists produced rapid spikes in current followed by sustained conductance (*P < 0.01 compared with 1000 s currents, n= 6 filters/condition).

View larger version (25K): [in this window] [in a new window]   Figure 6.  Cl– conductance in CFBE41o–F monolayers grown at 27°C (48 h) Cells were grown as monolayers at 27°C and studied in Ussing chambers as described in Methods. LoCl–, switch to apical LoCl– buffer; Am, addition of amiloride (100 µM) to the apical compartment; Glyb, addition of glybenclamide (200 µM) to the apical compartment. Axes are noted within each panel (µA –Y axis, time in minutes –X axis). A–C, examples of monolayers stimulated with albuterol (10 µM, A), NECA (10 µM, B), or forskolin (10 µM, C). Minimal currents are produced by all three agonists. Subsequent stimulation with genistein (50 µM, apical and basolateral) produces large, sustained currents that are sensitive to glybenclamide blockade. D, summary of dose–response experiments performed in CFBE41o–F508 monolayers. Cells were stimulated with increasing concentrations of agonists (0.1, 1.0, 10 µM) as shown. Forskolin, NECA and albuterol produced minimal currents at all concentrations. In contrast, genistein (50 µM, apical and basolateral) stimulated robust Cl– conductance that was sensitive to glybenclamide blockade (*P < 0.001 compared with at 3000 s currents for each condition). n= 8–12 filters studied in each condition.

View larger version (18K): [in this window] [in a new window]   Figure 7.  cAMP and ATP levels in CFBE41o–F cells For cAMP measurements, cells were grown in 60 mm dishes at 37°C or 27°C (48 h), stimulated with agonists (10 min, 37°C), and then cell cAMP was measured as described in Methods. For ATP measurements, monolayers were grown on 24 mm diameter inserts at an air–liquid interface until confluent, and placed in incubators set at 37°C or 27°C (48 h). On the day of study, monolayers were placed in 37°C incubators for 30 min (to simulate Ussing chamber conditions), and then cell ATP was measured as described in Methods. A, cAMP levels produced by 10 µM forskolin, albuterol, or NECA. cAMP levels produced by forskolin, albuterol and NECA (10 µM agonists) were > Controls (*P < 0.001); cAMP levels produced by forskolin and albuterol were > NECA (P < 0.001); cAMP produced by forskolin > albuterol (P < 0.001). n= 4 dishes/condition. B, low temperature growth does not inhibit cAMP production. cAMP levels produced by NECA and albuterol in CFBE41o– cells grown at 37°C and 27°C (48 h). cAMP levels are normalized (%) to the mean value obtained at 37°C for the two agonists. Growth of cells at 27°C had no effect on cAMP production by either agonist (n= 4 dishes/condition). C, low temperature growth does not reduce cell ATP levels. Monolayers grown at 27°C (CFBE41o–F) had similar cell ATP levels compared with CFBE41o– WT cells (n= 3 inserts/condition).

Genistein activates WT and F508 CFTR in CFBE41o^– monolayers

CFTR-mediated Cl^– currents were activated in temperature-corrected CFBE41o^–F monolayers by genistein (Fig. 6), following prestimulation with cAMP signalling agents. To assess the relative responsiveness of F508 CFTR to genistein alone or genistein + cAMP in CFBE41o^–F cells, temperature-corrected CFBE41o^–F monolayers were tested in dose–response experiments. Increasing concentrations of genistein with and without cAMP agonists were tested. The cells were stimulated with increasing concentrations of genistein (1, 10, 50 µM) in the presence or absence of NECA or albuterol prestimulation (10 µM) (Fig. 8A). CFBE41o^– WT monolayers (grown at 37°C) stimulated with increasing concentrations of genistein are included for comparison (no NECA or albuterol treatment). In contrast to the forskolin + genistein studies shown in HeLa F cells (Fig. 3), no synergism between albuterol, NECA and genistein could be demonstrated in CFBE41o^–F monolayers. Genistein-stimulated currents were sensitive to inhibition of PKA by H89 (40 µM, 30 min treatment prior to dose–response experiments (Fig. 8A)). Maximal genistein-stimulated currents were similar in WT-CFTR and temperature corrected F508 CFTR expressing monolayers (Fig. 8A).

View larger version (32K): [in this window] [in a new window]   Figure 8.  Genistein-stimulated Cl– conductance in CFBE41o– monolayers Cells were grown as monolayers at 27°C (48 h) or 37°C and studied in Ussing chambers as described in Methods. LoCl–, switch to apical LoCl– buffer; Am, addition of amiloride (100 µM) to the apical compartment; Glyb, addition of glybenclamide (200 µM) to the apical compartment. A, no additive effects of cAMP agonists and genistein on Cl– conductance are seen in CFBE41o–F monolayers. CFBE41o–F cells (open symbols) were grown at 27°C (48 h) prior to study. CFBE41o– WT (filled symbols) cells were grown at 37°C. Increasing concentrations of genistein (1, 10, 50 µM) were applied to activate Isc in NECA or albuterol pretreated (10 µM) or Control (no prestimulation) CFBE41o–F monolayers. H89 treated monolayers were exposed to H89-Cl (40 µM) for 30 min prior to stimulation. Neither receptor agonist had additive/synergistic effects on Isc when combined with genistein compared with genistein alone (‘Control’), and genistein currents were sensitive to PKA blockade with H89. CFBE41o– WT cells stimulated with increasing concentrations of genistein (alone) displayed similar maximal Cl– conductance compared with CFBE41o–F cells. *P < 0.001 compared with F508 condition. n= 8–12 filters/condition. B, genistein stimulated Isc measurements in CBE41o–F monolayer grown at 37°C. Parental CFBE41o– cells (no CFTR transduction) and CFBE41o–F cells grown at 27°C were used as controls testing the presence of minimal levels of F508 CFTR in the apical membrane of CFBE41o–F cells grown at 37°C. Increasing concentrations of forskolin (0.1, 1.0, 10 µM) failed to stimulate Isc. Small genistein stimulated currents were seen in CFBE41o–F grown at 37°C (2.18 ± 0.28 µA cm–2). No currents in parental CFBE41o– cells grown at 27°C were recorded. Robust Isc was measured in monolayers stimulated with genistein after low temperature correction. *P < 0.001 for CFBE41o–F monolayers grown at 27°C versus 37°C and parental controls; P < 0.001 for CFBE41o–F grown at 37°C compared with parental CFBE41o– cells at 27°C. n= 8–12 filters/condition.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We developed and characterized F508 CFTR expressing cell lines that are in high demand for studies of epithelial ion transport. Using these cell lines, we showed that low temperature-rescued F508 CFTR in CFBE41o^–F cells (as determined by biochemical, immunofluorescence, and functional measures) was a poor target for activation by potent cAMP signalling stimuli. This includes two receptor signalling pathways that are known to regulate CFTR in vivo (A_2B ARs and ß₂ ARs), as well as a direct activator of adenyl cyclase (forskolin). In contrast, forskolin activated anion transport in HeLa F cells after low temperature correction. The failure of these pathways to activate F508 CFTR in a polarized human airway epithelial cell line is significant, since the findings establish that surface localized F508 CFTR after low temperature rescue is profoundly unresponsive to the normal signalling pathways used by airway epithelial cells to activate endogenous, wild-type CFTR in vivo. The results also bear upon human studies designed to test new pharmacological agents (‘correctors’) for achieving F508 CFTR surface localization, since conventional methods for activating CFTR (e.g. with A₂ AR or ß₂ AR agonists during the nasal potential difference measurement) may not reveal the presence of F508 CFTR in the surface membrane in vivo, even if some mutant protein has reached its correct location at the airway mucosal surface.

In our experiments, activation of F508 CFTR-dependent currents in a human airway epithelial cell line monolayer (CFBE41o^–F) required stimulation with genistein, a soy-derived product that has been shown to inhibit tyrosine kinase (Illek et al. 1995). Genistein has been shown to activate CFTR by a cyclic AMP-independent process and can increase cAMP-dependent activation of wild-type CFTR (Reenstra et al. 1996). Genistein has also been shown to increase the open channel probability (P_o) of F508 CFTR in excised membrane patches (Hwang et al. 1997). Our results are in agreement with those of Hwang and colleagues, who showed that F508 CFTR displays a markedly reduced rate of activation to classic cAMP-mediated pathways compared to wild-type CFTR (Wang et al. 1998; Wang et al. 2000). Near normal levels of CFTR activity, however, could eventually be obtained in the previously published studies. Here we show that similar deficiencies in cAMP responsiveness apply to F508 CFTR expressed in airway epithelial monolayers. In contrast to the findings of Wang and colleagues completed in heterologous expression systems, only minimal activation of F508 CFTR was seen in our studies despite prolonged exposure to cAMP-dependent agonists (up to 30 min). These findings suggest that in polarized monolayers, the refractoriness of rescued F508 CFTR to cAMP/PKA activation may be even more severe than it was previously observed in single channel studies of non-polarized cells. Endogenous (unstimulated) PKA activity was necessary, however, to permit genistein activation of F508 CFTR in airway cell monolayers (Fig. 8A). Our results also extend those recently published by Pedemonte & Lukacs (2005) in which new ‘correctors’ of F508 CFTR processing were identified and tested in primary human CF bronchial epithelia (derived from F508/F508 subjects). They showed that activation of maximal F508 CFTR-mediated currents required costimulation of the monolayers with forskolin and genistein and represented only 8% of WT-CFTR-mediated currents. Interestingly, some of the newly identified correctors were shown to increase the responsiveness of F508 CFTR to forskolin (compared with temperature corrected protein), with a corresponding elongated F508 CFTR residence time in the cell membrane. This intriguing observation suggests that pharmacologically corrected F508 CFTR may differ from temperature-corrected F508 CFTR in terms of its function. Future studies assessing the responsiveness of pharmacologically corrected F508 CFTR to activation by physiological pathways will be necessary to clarify the functional defects of the mutant protein and may reveal mechanistic differences between pharmacological rescue agents and low temperature

A recent report from Andersson et al. (2003) provided evidence that F508 CFTR expressing airway cell lines (CFBE41o^– and CFSMEo^–) demonstrated small but statistically significant genistein and forskolin-stimulated Cl^– efflux when grown under non-polarizing conditions at 37°C. Furthermore, studies by Kelley (1996) found that ß₂ AR agonists, when coupled with phosphodiesterase inhibitors also stimulated F508 CFTR-specific Cl^– efflux from CF nasal cells grown under non-polarizing conditions at 37°C. Neither report, however, quantified total or surface F508 CFTR levels. The discrepancies between these previously published findings and our results presented here highlight the potential impact that cell type, cell polarization status, and assay methodology may have on detection of F508 CFTR processing and function.

The two main pathways (A_2B ARs and ß₂ ARs) known to regulate WT-CFTR in vivo have been postulated to spatially compartmentalize with CFTR through binding partner interactions, providing a means for local regulation of CFTR activity (Huang et al. 2000; Naren et al. 2003). Our findings in CFBE41o^– monolayers are consistent with compartmentalized signalling, as both A_2B and ß₂ AR stimulated cAMP production that was far less than forskolin (Fig. 7A), despite similar maximal currents in CFBE41o^– WT cells (Fig. 4). The studies presented here suggest that the F508 mutation may cause significant conformational changes (even after rescue) that could alter these interactions and abolish normal regulation. This assumption is supported by previous finding that the F508 CFTR protein is only partially functional when present at the cell membrane (Wang et al. 1998; Wang et al. 2000). Careful comparative studies of surface wild-type and F508 CFTR interactions with membrane binding partners may identify differences that impart the refractory phenotype to F508 CFTR.

Because F508 CFTR rescue efficiency was higher in HeLa F than in CFBE41o^–F cells as shown by immunoprecipitation (Fig. 1C), it could be suggested that deficient activation of F508 CFTR by cAMP-mediated pathways is only dependent on cell surface expression levels. However, our results rather support the interpretation that WT and F508 CFTR exhibit differential regulation at the cell membrane that is dependent on the cell type. First, experiments performed in our laboratory were unable demonstrate discordant regulation of WT-CFTR by cAMP versus genistein at different expression levels (CFBE41o^– cells, WT-CFTR expression titrated with Ad-WT-CFTR – data not shown). Second, even minimal, biochemically undetectable levels of F508 could be activated in CFBE41o^–F monolayers by genistein but not by forskolin (Fig. 8B). Third, biotinylated F508 CFTR B and C levels were 2.5-fold greater at 27°C than biotinylated wild-type CFTR B and C levels at 37°C (Fig. 2). In spite of the significantly lower cell surface CFTR levels in CFBE41o^– WT cells, the wild-type protein could be efficiently activated through cAMP-mediated pathways. Actvation of rescued F508 CFTR, however, required subsequent treatment by genistein (Figs 6 and 8). Genistein activation served as an internal positive control, and confirmed that significant levels of F508 CFTR were at the cell membrane. The absence of these genistein-stimulated currents in the temperature corrected parental cells (Fig. 5A) points towards F508 CFTR as the responsible transporter in the transduced cells. In addition, similar maximal currents following genistein stimulation were seen for surface WT-CFTR and F508 CFTR (Fig. 8A). Finally, despite lower levels of surface F508 CFTR (27°C) relative to WT-CFTR (37°C, Fig. 2B) in transduced HeLa cells, forskolin was an effective activator of F508 CFTR-dependent halide efflux (Fig. 3A). While our results cannot exclude that these observations are a unique feature of CFBE41o^– airway cells, the findings suggest that the defect in rescued F508 CFTR regulation was specific for F508 CFTR and was not due to low level surface localization.

We speculate that failed activation of F508 CFTR through A_2B ARs, ß₂ ARs, and cAMP could result in part from altered interactions of the rescued protein with binding partners known to regulate CFTR Cl^– channel activity such as EBP50 (Hall et al. 1998), E3KARP (Sun et al. 2000), CAP70 (Wang et al. 2000), or syntaxin 1A (Naren 2000; Naren et al. 1997). Previously we have shown that membrane localized CFTR in airway cells exists in a macromolecular complex involving CFTR–EBP50–ß₂ AR, and that the assembly of this complex is regulated by PKA-dependent phosphorylation of the R domain (Naren et al. 2003). More recently, it has been reported that the F508 mutation alters the interdomain interactions of CFTR (Du et al. 2005). If the rescued F508 protein is still in a misfolded state as suggested by Sharma et al. (2001, 2004), it may alter assembly of this macromolecular complex, resulting in deficient regulation. In either case, our findings indicate that the choice of expression system utilized to test new correctors of F508 CFTR can dramatically influence outcome. Permissive cell lines may not predict the responsiveness of F508 CFTR to endogenous or exogenous activating stimuli seen in more stringent (polarized) cellular expression systems.

Our results compare and contrast the behaviour of F508 CFTR in a cell line that does not express CFTR endogenously (HeLa cells) with a CF airway epithelial cell line capable of forming high resistance monolayers (CFBE41o^– cells). Although raising cAMP (by forskolin) was a reasonably effective stimulus for both wild-type and rescued F508 CFTR in HeLa cells compared with genistein, the effect was dramatically different compared to the complete lack of currents in CFBE41o^–F cells after forskolin stimulation (Figs 6C and D and 8B). Furthermore, F508 CFTR-dependent halide efflux in HeLa cells stimulated by genistein and forskolin was synergistic, while we were unable to demonstrate additive effects of forskolin or receptor agonists and genistein over a wide range of genistein doses in CFBE41o^– monolayers (Fig. 8). These fundamental differences in F508 CFTR behaviour in the two cell types probably involve a variety of factors, such as levels of B and C, assay sensitivity (I_scversus SPQ), and/or responsiveness of surface F508 CFTR to activating stimuli. More importantly, these differences should be carefully considered when attempting to generalize findings reported in certain CFTR expression systems.

CFBE41o^–F and CFBE41o^– WT cells demonstrated several features that suggest that they are a useful model system to study CFTR biogenesis, trafficking and regulation. In addition to ease of growth and propagation, they exhibit a polarizing phenotype, retain A_2B AR and ß₂ AR expression (Clancy et al. 2003; Hentchel-Franks et al. 2004), and produce a robust and highly specific signal (wild-type or F508 CFTR) to monitor CFTR activity. They also demonstrate several other epithelial features that make them a useful model system to study ion transport, including apical localization of CFTR, basolateral localization of Na⁺–K⁺–2Cl^– cotransporter activity, and preservation of calcium-activated Cl^– conductance (Fig. 5, and Clancy et al. 2003). The CFBE41o^– cells that we have characterized here differ from those originally developed by Gruenert and colleagues (Kunzelmann et al. 1993). The parental CFBE41o^– cells which were used to develop the cell lines characterized here have no detectable endogenous CFTR mRNA and protein. However, the cells maintained many features desirable for studies of CFTR function in polarizing human airway cells after transduction with wild-type or F508 CFTR.

In summary, our results indicate that despite cell surface localization of F508 CFTR in a polarized airway epithelial cell line (with or without temperature rescue), its activation by receptor-based, cAMP-dependent pathways was profoundly defective. CFTR is a tightly regulated protein with multiple levels of regulation including ATP binding and hydrolysis, phosphorylation, and interactions with binding partners. Regulation of CFTR by in vivo signalling pathways is likely to be crucial to restore normal pulmonary physiology in cystic fibrosis. Our results support the hypothesis that CFTR is regulated in a cell-type specific manner, and that activation of surface F508 CFTR (for purposes of detection or therapy) will require combinational therapies that include agents geared towards correcting F508 CFTR processing, coupled with strategies to restore normal regulation at the plasma membrane.

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