1 Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, Cardiff, CF14 4XN, UK
2 Laboratoire physiologie des régulations cellulaires, UMR6558, Université de Poitiers, 40 avenue du recteur Pineau, 86022 Poitiers, France
3 Laboratoire de chimie organique, Faculté de médecine et de pharmacie de Poitiers, 34 rue du jardin des plantes, 86005 Poitiers, France
4 Department of Physiological Sciences, University Medical School, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK
5 Laboratorio di genetica molecolare, Istituto Giannina Gaslini, 16148 Genova, Italy
6 Department of Child Health, University Hospital of Wales, Heath Park, Cardiff, CF14 4XN, UK
*Authors for correspondence (e-mail: frederic.becq{at}univ-poitiers.fr; dormer{at}cardiff.uk)
Accepted July 31, 2001
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SUMMARY |
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Key words: CFTR, delF508, Immunolocalisation, Human airway cells, Pharmacology, Trafficking
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INTRODUCTION |
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To gain insight into the pharmacology of CFTR chloride channel having disease-causing mutations at NBD1 such as delF508 it is necessary to develop compounds that can restore the defective processing of mutant CFTR. A limited number of molecules have been shown to restore some function in delF508 cells (Schultz et al., 1999), such as the xanthine derivatives IBMX (Becq et al., 1994) and CPX (Srivastava et al., 1999), the benzimidazolone NS004 (Gribkoff et al., 1994) and the isoflavone derivative genistein (Illek et al., 1995). CPX is believed to bind to delF508 protein and to affect the trafficking process of delF508 (Srivastava et al., 1999).
The degree of mislocalisation of delF508-CFTR in native epithelial cells, which also show reduced Cl channel activity (Kelley et al., 1996), is unclear because reports vary from different tissues and type of preparation (Kartner et al., 1992; Engelhardt et al., 1992; Denning et al., 1992b; Puchelle et al., 1992; Kälin et al., 1999). We have developed a system to study the location of CFTR in freshly isolated native airway epithelial cells from cystic fibrosis patients, which allows quantification of the percentage of cells with a defined CFTR location. We have determined the degree of mislocalisation of delF508-CFTR and whether the new CFTR-activating benzo(c)quinolizinium compounds (Becq et al., 1999) affect delF508-CFTR activity and trafficking. Moving delF508-CFTR from within the cell to the apical membrane would be a major step forward in the development of a rational cystic fibrosis therapy and would be applicable to other disorders of protein trafficking (Aridor and Balch, 1999).
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MATERIALS AND METHODS |
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CFTR antibody
An antibody against a peptide consisting of the 23 C-terminal amino acids of CFTR (MPCT-1) was raised as previously described (Lloyd Mills et al., 1992; Pereira et al., 1998) and affinity-purified using peptide coupled to CH-Sepharose 4B (Pharmacia), with elution of antibody fractions in 0.1 M glycine-HCl and immediate neutralisation. The antibody has been previously shown to crossreact with CFTR in transfected but not mock transfected CHO cells (Pereira et al., 1998) and to recognise CFTR in native submandibular and pancreatic tissues (Lloyd Mills et al., 1992).
Incubation and fixation of nasal epithelial cells
Airway epithelial cells obtained by nasal brushing were from five non-CF individuals, four delF508/delF508 CF individuals and one delF508/G551D CF individual undergoing flexible bronchoscopy under sedation. Two brushing samples were obtained from the non-anaesthetised nostril by gently brushing a standard 0.5 cm cytology brush along the inferior turbinate. The study was approved by the local ethics committee of Bro Taf Health Authority. The brushes were placed immediately into DMEM/F12 medium and incubated in the presence or absence of MPB-07 or MPB-91 at 37°C, as described in the text. After incubation, the brushes were removed from the medium and cells smeared gently onto Snowcoat x-tra microslides (Surgipath). The samples were left to air-dry, fixed for 5 minutes at 20°C in 5% acetic acid in ethanol, washed in PBS and stored at 20°C. The samples were treated with appropriate block (normal goat or rabbit serum; 1:20) for 30 minutes at room temperature.
Culture and fixation of nasal epithelial cells
Nasal polyps were obtained immediately following polypectomy from non-CF or delF508/delF508 CF individuals. Epithelial cells were digested from the surface with protease XIV (Sigma) for 1-2 hours at 37°C and cultured on glass coverslips in DMEM/F12 medium (GIBCOBRL) for 6-8 days before incubation in the presence or absence of MPB-07 at 37°C, as described in the text. Following incubation cells were washed twice with phosphate buffered saline (PBS) and fixed for 30 minutes in 4% paraformaldehyde in PBS. Cells were permeabilised with 0.2% Triton X-100 for 20 minutes and blocked by incubation first with 50 mM glycine for 30 minutes and then with 10% normal goat serum for 1 hour.
Immunofluorescence localisation
Anti-CFTR antibody (MPCT-1; 1:100), anti-cytokeratin (clone AE1/AE3; 1:50, from Sigma), and anti-CD59 (BRIC229; 1:100, from National Blood Service, Bristol, UK) were used as primary antibodies. The fixed cells were incubated with primary antibody overnight at 4°C, followed by three washes and then incubation with the appropriate secondary antibody (FITC- or Cy3-conjugated; 1:100) for 30 minutes at room temperature. For ER localisation, fixed cells were incubated for 2 hours at room temperature with 2 µM BODIPY-thapsigargin (Molecular Probes). Some cells from nasal brushings were stained with the red nuclear stain propidium iodide (Sigma) for 5 minutes at room temperature. Slides were then mounted in Fluorosave Reagent (Calbiochem, La Jolla, CA). Fluorescence was detected using confocal laser scanning microscopy on a Leitz DMRBE microscope (Leica, Germany) fitted with a TCS4D scanner (Leica, Germany). Confocal images were collected at a magnification of x100 under oil immersion and displayed as Kalman averages on a screen 512x512 pixels; 72 dpi. Images were processed using Scanware software (Leica, Germany) and image manipulation was performed using Adobe PhotoShop.
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RESULTS |
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To investigate the specificity of MPB compounds on chloride transport activity, we studied their actions on calcium-dependent and volume-sensitive chloride transport in IB3-1 cells (Fig. 3A,B). MPB-91 treatment (250 µM for 2 hours at 37°C) did not alter the calcium-dependent chloride transport stimulated with 1 µM of the calcium ionophore A23187 (relative rates to control: A23187 no MPB: 2.10±0.16, n=4 vs after treatment: 2.07±0.15, n=4) (Fig. 3A,C). By contrast, a volume-sensitive chloride transport that was stimulated following a hypo-osmotic challenge (relative rates to control 8.56±0.89, n=4) was significantly inhibited in treated cells (relative rates to control 5.83±0.36, n=4) (Fig. 3B,C). These data support an interaction between CFTR and the volume-sensitive chloride transport as suggested by Vennekens et al. (Vennekens et al., 1999).
Immunoprecipitation and in vitro phosphorylation of CFTR after two hours treatment with MPB-91 is shown Fig. 4. The low molecular weight (band B) is detected (Fig. 4, lanes 4,5). When IB3-1 cells were pretreated with 250 µM MPB-91 a mature, fully processed form of CFTR was detected (Fig. 4, lane 5) at a molecular weight similar to that seen in Calu-3 cells expressing wt-CFTR (Fig. 4, lane 2). Densitometric analysis of CFTR after in vitro phosphorylation is shown Fig. 4B. The percentages of band C and B were compared in the presence or absence of MPB-91. The band C form increased from 2.9% to 22% after MPB-91 treatment, whereas the band B form decreased from 97% in non-treated cells to 78% in MPB-91 treated cells.
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CFTR immunolocalisation in cells obtained by nasal brushing of non-cystic fibrosis and cystic fibrosis individuals
We investigated the location of wild-type and delF508-CFTR in freshly isolated nasal cells obtained from non-CF and delF508/delF508 CF individuals. Cells were predominantly ciliated and elongated, having a polarised morphology. Cilia, visible at the apical end of the cell (Fig. 5A), continued to beat for the 2 hour incubation prior to fixation. The nasal cells expressed cytokeratins (Fig. 5B, green), typical of epithelial cells, throughout the cell. Fig. 4B also shows that, in cells from non-cystic fibrosis individuals, wild-type CFTR (red) was located at the apical end of the cell. Wild-type CFTR (Fig. 5C, red) was shown to colocalise with the GPI-anchored apical membrane protein, CD59 (Davies et al., 1989) (Fig. 5D, green), also in dual-labelled cells. Fig. 6 shows a direct comparison between the cellular location of wild-type and delF508-CFTR. In non-cystic fibrosis cells (Fig. 6A), the apical location of wild-type CFTR (green) was readily observed in cells counterstained for nuclei with propidium iodide (red). By contrast, in cystic fibrosis nasal cells, delF508-CFTR was restricted to a region surrounding the nucleus (perinuclear) (Fig. 6C,E,G), or to a discrete region adjacent to the nucleus (juxtanuclear). In a small number (less than 10%) of untreated cystic fibrosis cells, delF508-CFTR was located throughout the cell (Fig. 6I).
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By counting cells with a defined pattern of delF508-CFTR location, we quantified the effect of MPB-07 in changing delF508-CFTR from a perinuclear to an apical location. Fig. 7A shows that in most wild-type cells CFTR had a distinct apical location (Fig. 5, Fig. 6A,B) with no cells showing a perinuclear CFTR location. In the rest of the wild-type cells CFTR (and also CD59) was present within the cell as well as apically located. The latter category of cells are not included in the histogram (Fig. 7) for clarity. The reason for the widespread distribution of both CFTR and CD59 in a minority of cells is not clear: this may reflect methodology in collecting and analysing native human cells but may equally reflect variation in CFTR distribution in the epithelial cell population. Nevertheless, MPB-07 did not change the pattern of wild-type CFTR location (Fig. 7B), which was very different from that of delF508-CFTR. Thus, in contrast to wild-type cells, the majority of untreated cystic fibrosis cells had a characteristically perinuclear location of delF508-CFTR (Fig. 6C,E,G) with less than 10% showing an apical location (Fig. 7C) and the remainder showing more widespread distribution throughout the cell. In a minority of delF508/delF508 cells, some delF508-CFTR may therefore escape to the apical region; however, the lack of complete uniformity of distribution in all delF508/delF508 cells may also reflect methodology in examining native human cells. Nevertheless, MPB-07 treatment resulted in a significant increase in the number of cells showing a marked focussing of delF508-CFTR towards the apical membrane (Fig. 7D), with a corresponding decrease in the number of cells showing the perinuclear location characteristic of delF508/delF508 cystic fibrosis cells (Fig. 7D). MPB-07 did not change the percentage of cells showing juxtanuclear location of delF508-CFTR (20-25% with or without MPB-07 treatment) but did increase the number of cells in which delF508-CFTR was distributed throughout the cell (Fig. 6I) from 9.5±8.8% to 28.1±5.4% (P<0.05 for n=4 individuals). Thus, MPB-07 changed delF508-CFTR location such that, before treatment, 80% of cells showed a restricted intracellular location that could be distinguished from wild-type, whereas, after treatment, the majority of cystic fibrosis cells (60-70%) showed a more apical CFTR location that could not be distinguished from wild-type.
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DISCUSSION |
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Other non-selective treatments such as low temperature (25-27°C), glycerol, 4-phenylbutyrate or DMSO have required long-term exposure to show modest effects on delF508-CFTR trafficking in transfected cells or cultured cell lines (Cheng et al., 1990; Denning et al., 1992a; Rubenstein et al., 1997; Sato et al., 1996; Bebök et al., 1998). Here we have shown for the first time a dramatic effect of a pharmacological agent in native cells in which the naturally occurring delF508 CFTR protein is abnormally retained in the endoplasmic reticulum. The action of MPB compounds on delF508-CFTR trafficking and appearance of CFTR Cl channel activity was selective and readily observed after 2 hours, indicative of an acute effect on release from the endoplasmic reticulum by a direct action on the malfolded protein. Interestingly, correction of defective protein kinesis of human P-glycoprotein mutants in the presence of substrates and modulators such as capsaicin, cyclosporin, vinblastine or verapamil has been demonstrated (Loo and Clarke, 1996). These effects occurred within a few hours (2-4 hours) after the addition of drugs (Loo and Clarke, 1996), a time scale remarkably similar to our study. Both the Loo and Clarke study and the present report may indicate a common mechanism of action for compounds that can modulate the activity of P-glycoprotein and CFTR. Upregulation of CFTR expression was shown using butyrate and its analog sodium 4-phenylbutyrate (Rubenstein et al., 1997). Unexpectedly, a direct inhibitory action of both compounds on wild-type CFTR was recently demonstrated at a single-channel level (Lindsell, 2001) further indicating that some drugs that act on the biosynthesis of CFTR may also be able to directly interfere with the channel transport function. One hypothesis is that within the CFTR structure, occupation of a drug-binding site by MPB (and/or other compounds) may stabilize the conformation of delF508-CFTR protein such that correction of malfolding occurs. This would allow the complex formed by MPB and the protein to escape the quality control system and reach the apical compartment of cells, where activation of chloride transport by cAMP agonists occurs.
The data indicate that, in the majority of untreated delF508/delF508 cystic fibrosis cells, delF508-CFTR is restricted to the endoplasmic reticulum. The juxtanuclear and more widespread cellular distribution, present in a small percentage of cells (Fig. 6I) and increased by MPB-07 treatment, are likely to represent more distal compartments on the pathway to the apical membrane. This data broadly agrees with other recent studies on nasal epithelial cells from polyps or brushings (Kälin et al., 1999; Penque et al., 2000), which also suggest that at least some delF508-CFTR escapes from the endoplasmic reticulum in native cystic fibrosis cells. However, the demonstration that the majority of delF508-CFTR is mislocalised emphasizes the importance of the present findings that MPB compounds correct delF508-CFTR location. Their application to the majority of CF patients is highlighted by our results in the present report that nasal cells obtained from a compound heterozygote individual (delF508/G551D) have an altered CFTR location similar to delF508/delF508 cells. The data suggest that delF508-CFTR is more strongly expressed than G551D or that its restriction to the endoplasmic reticulum also retains G551D. Incubation of delF508/G551D cells with MPB-91 markedly altered the location of mutant CFTR towards a wild-type distribution, although we cannot distinguish whether the apically located CFTR is delF508 or G551D. Thus, MPB-91 provides a new class of compounds that not only activate G551D-CFTR (R.D., L.B.-P. and F.B., unpublished) but also traffic mutant CFTR to the apical membrane in CF compound heterozygotes with a delF508/G551D mutation.
In conclusion, the present study demonstrates that benzoquinolizinium compounds, which directly activate wild-type (Becq et al., 1999) and G551D-CFTR Cl channels (R.D., L.B.-P. and F.B., unpublished), have a dramatic effect in restoring defective chloride transport in CF cells by increasing delF508-CFTR at the apical membrane. This is a major step forward in the goal of devising a rational drug treatment aimed at repair or rescue of the activity of the mutant cystic fibrosis gene protein and has implications for other disorders of protein trafficking (Aridor and Balch, 1999).
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ACKNOWLEDGMENTS |
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