Partial restoration of cAMP-stimulated CFTR chloride channel activity in Delta F508 cells by deoxyspergualin

Canwen Jiang1, Shaona L. Fang1, Yong-Fu Xiao2, Sean P. O'Connor1, Steven G. Nadler3, Des W. Lee4, Douglas M. Jefferson4, Johanne M. Kaplan1, Alan E. Smith1, and Seng H. Cheng1

1 Genzyme Corporation, Framingham 01701-9322; 2 Department of Medicine, Harvard Medical School, Boston 02215; 4 Tufts University School of Medicine, Department of Physiology, and New England Medical Center, Department of Pediatrics and Medicine, Boston, Massachusetts 02111; and 3 Bristol-Myers Squibb, Princeton, New Jersey 08540

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Deletion of the codon encoding phenylalanine 508 (Delta F508) is the most common mutation in cystic fibrosis (CF) and results in a trafficking defect. Mutant Delta F508-CF transmembrane conductance regulator (CFTR) protein retains functional activity, but the nascent protein is recognized as abnormal and, in consequence, is retained in the endoplasmic reticulum (ER) and degraded. It has been proposed that this retention in the ER is mediated, at least in part, by the cellular chaperones heat shock protein (HSP) 70 and calnexin. We have investigated the ability of deoxyspergualin (DSG), a compound known to compete effectively for binding with HSP70 and HSP90, to promote trafficking of Delta F508-CFTR to the cell membrane. We show that DSG treatment of immortalized human CF epithelial cells (Delta F508) and cells expressing recombinant Delta F508-CFTR partially restored cAMP-stimulated CFTR Cl- channel activity at the plasma membrane. Although there are several possible explanations for these results, one simple interpretation is that DSG may have altered the interaction between Delta F508-CFTR and its associated chaperones. If this is correct, agents capable of altering the normal functioning of cellular chaperones may provide yet another means of restoring CFTR Cl- channel activity to CF subjects harboring this class of mutations.

cystic fibrosis; cellular chaperones; 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence; whole cell patch clamp

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE MOST COMMON CAUSE of cystic fibrosis (CF) is deletion of the phenylalanine residue at position 508 (Delta F508) of the cystic fibrosis transmembrane conductance regulator (CFTR). Studies have shown that this mutation results in the synthesis of a variant CFTR (Delta F508-CFTR) that is defective in its ability to traffic normally to the apical membrane surface where it functions as a Cl- channel (4). Rather, most of the nascent Delta F508-CFTR is retained in the endoplasmic reticulum (ER) where it is degraded by a process that involves ubiquitination (13, 25). However, functional cAMP-stimulated CFTR Cl- channel activity can be detected at the plasma membrane when Delta F508-CFTR is synthesized at a reduced temperature (6) or in the presence of chemical chaperones (2, 21) and when overexpressed (3), indicating that the deletion of phenylalanine 508 does not completely abolish CFTR function. Therefore, strategies that facilitate the relocation of mutant Delta F508-CFTR at the plasma membrane may be therapeutically beneficial for the treatment of CF.

The proper folding and assembly of many newly synthesized proteins in the ER are facilitated by cellular chaperones (10). These chaperones are thought to promote productive folding in part by preventing aggregation of folding intermediates. Both wild-type and mutant Delta F508-CFTR interact with the ER-resident chaperone calnexin and the cytosolic chaperone heat shock protein (HSP) 70 (19, 28). However, in contrast to wild-type CFTR, mutant Delta F508-CFTR is unable to dissociate from either calnexin or HSP70 and does not exit the ER to the Golgi. In Delta F508-CFTR-producing cells, only the partially glycosylated band B form but none of the fully glycosylated band C form of CFTR is generated. Presumably, the mutant Delta F508-CFTR is recognized as abnormal, perhaps by the chaperones themselves, and is retained in the ER where it is subsequently degraded. The finding that HSP70 and calnexin may be responsible for the ER retention of Delta F508-CFTR raises the possibility of therapeutic intervention in CF by agents capable of interfering with the normal functioning of these chaperones.

One potential candidate that we considered was deoxyspergualin (DSG), a stable synthetic analog of the natural product spergualin (24). DSG has demonstrated potent immunosuppressive activity in a number of T cell-dependent assays and animal models. It has been suggested that this immunosuppressive activity is mediated, at least in part, through its ability to interact with heat shock cognate (HSC) 70 and HSP90 (16, 17). The dissociation constant values of the DSG-HSP complexes are 4-5 µM and, as such, are predicted to compete effectively with protein or peptide binding to HSC70 and HSP90 and thereby affect protein trafficking (16). To test whether this binding to the chaperones is sufficient to alter the trafficking and hence the subcellular location of Delta F508-CFTR, cells expressing the mutant protein were exposed to DSG. We report here that addition of DSG to cells expressing recombinant Delta F508-CFTR resulted in the appearance of functional cAMP-stimulated CFTR Cl- channel activity at the cell surface. More importantly, DSG also restored cAMP-mediated CFTR Cl- channel activity in immortalized human CF airway and biliary epithelial cells.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Chemicals. DSG was obtained from Bristol-Myers Squibb; forskolin was from Calbiochem; 6-methoxy-N-(3-sulfopropyl)-quinolinium (SPQ) was from Molecular Probes; diphenylamine carboxylic acid (DPC) was from Fluka; and sodium butyrate, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP), ionomycin, UTP, and IBMX were from Sigma.

Cells. CFT1 and JME/CF15 are two immortalized human CF airway epithelial cell lines, and IBE-1 is an immortalized human CF intrahepatic biliary epithelial cell line; all contain the Delta F508 variant (8, 12, 29). C127-Delta F508-low (mouse mammary tumor) and LLC-PK1-Delta F508 (pig kidney epithelial) are two recombinant cell lines stably expressing low levels of the mutant Delta F508-CFTR protein (3, 15). C127-mock is a cell line that had been stably transfected with the backbone of the expression vector used to generate C127-Delta F508-low (3). The details of the generation, characterization, and routine propagation of all these cell lines have been described (3, 8, 12, 15, 29).

The cells were treated with between 5 and 100 µg/ml of DSG for up to 72 h. Concentrations of DSG >50 µg/ml were toxic to most of the cell types tested. Although some responses were observed at shorter incubation times, most of the experiments were performed with cells that had been treated with DSG for >24 h as this generated more consistent results. Because DSG is modified by polyamine oxidase present in fetal bovine serum (23), cells were routinely replenished with fresh medium containing DSG and aminoguanidine every 24 h. As a control in some experiments, C127 cells were also treated with 5 mM sodium butyrate for 24 h to enhance expression of Delta F508-CFTR (3). In addition, as another control, cells were cultured at 23°C for 24-48 h (6) to facilitate folding of the mutant Delta F508-CFTR at the ER.

Assessment of CFTR functional activity using fluorescence digital imaging microscopy. The cAMP-dependent CFTR Cl- channel activity was assessed using the halide-sensitive fluorophore SPQ essentially as described previously (3, 15). Briefly, the cells were treated with different amounts of DSG for the times specified. At the end of the treatment period, the cells were loaded with SPQ by hypotonic shock for 4 min at room temperature. SPQ fluorescence initially was quenched by incubating the cells for up to 30 min in a NaI buffer (composition in mM: 135 NaI, 2.4 K2HPO4, 0.6 KH2PO4, 1 MgSO4, 1 CaSO4, 10 dextrose, and 10 HEPES, pH 7.4). After the baseline fluorescence (Fo) was measured for 2 min, the NaI solution was replaced with one containing 135 mM NaNO3, and fluorescence was measured for another 16 min. Forskolin (20 µM) and IBMX (100 µM) were added 5 min after the anion substitution to increase intracellular levels of cAMP. An increase in halide permeability is reflected by a more rapid increase in SPQ fluorescence. It is the rate of change rather than the absolute change in signal that is the important variable in evaluating anion permeability. Differences in absolute levels reflect quantitative differences between groups in SPQ loading, size of cells, or number of cells studied. The data are presented as means ± SE of fluorescence at time t (Ft) minus the Fo (the average fluorescence measured in the presence of I- for 2 min before ion substitution) and are representative of results obtained under each condition. For each experiment, between 50 and 100 cells were examined on a given day and studies under each condition were repeated on at least 2 days. For each experiment, the responses were compared with those obtained with control or untreated cells. Cells were scored as positive if they exhibited a rate of change in fluorescence that was greater than the signal observed with the control cells. Under the conditions specified above, control cells were unresponsive to added cAMP agonists. There was a broad spectrum in the rate of change in SPQ fluorescence observed with responsive cells. Normally, we scored cells as responsive if the slope of the response curve, which is indicative of the rate of increase in SPQ fluorescence, was >= 0.364 following stimulation with cAMP agonists. Because the response was heterogeneous, the data shown are for the 10% of cells in each experiment showing the greatest response. All the cells in the field were evaluated, but, for clarity of presentation, only the top 10% of responders are illustrated in Figs. 2-4.

Whole cell patch-clamp recording. Whole cell patch-clamp recordings were performed essentially as described previously (1, 7, 9). Briefly, cells on coverslips were placed in a chamber mounted on a Nikon Diaphot inverted microscope. Patch pipettes had resistances of 2-4 MOmega . Whole cell configuration was achieved with an additional pulse suction to rupture the gigaseal. The pipette (intracellular) solution contained (in mM) 130 CsCl, 20 tetraethylammonium (TEA) chloride, 10 HEPES, 10 EGTA, 10 MgATP, and 0.1 LiGTP, pH 7.4. The bath (extracellular) solution contained (in mM) 140 N-methyl-D-glucamine, 2 CaCl2, 1 MgCl2, 0.1 CdCl2, 10 HEPES, 4 CsCl, and 10 glucose, pH 7.4. These solutions were designed so only currents flowing through Cl- channels were studied, since Cl- was the only significant permeant ion in the solutions. Furthermore, Ca2+ and Ca2+-activated Cl- currents were minimized by inclusion of 10 mM EGTA in the intracellular solution and 100 µM of Cd2+ in the extracellular bath. K+ currents were minimized by including 20 mM TEA in the intracellular solution. Aspartate was used as the replacement anion in experiments in which extracellular Cl- concentration was changed. Forskolin (10 µM), IBMX (100 µM), CPT-cAMP (200 µM), DPC (200 µM), UTP (100 µM), and ionomycin (1 µM) were added to the bath solutions as indicated. In some experiments, forskolin and IBMX were used to raise intracellular levels of cAMP and, in others, CPT-cAMP was used. Similar results were obtained with both approaches. Current recordings were made from the same cells before, during, and after exposure to the solutions containing the different agonists or inhibitors. All experiments were performed at room temperature (22°C). Currents were filtered at 2 kHz. Data acquisition and analysis were performed using the pCLAMP 5.5.1 software (Axon Instruments, Foster City, CA).

Biochemical analysis of CFTR. Our procedures for cell lysate preparation, immunoprecipitation, phosphorylation of CFTR using protein kinase A and [gamma -32P]ATP, and polyacrylamide gel electrophoresis have all been described previously (3, 15).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Detection of functional CFTR Cl- channel activity in C127 cells expressing recombinant Delta F508-CFTR following treatment with DSG. C127-Delta F508-low is a recombinant cell line stably transfected with the cDNA encoding the mutant Delta F508-CFTR (3, 15). These cells produce solely the immature, partially glycosylated band B form of CFTR (characteristic of processing only in the ER) and do not exhibit detectable CFTR Cl- channel activity at the cell surface (3) (Figs. 1 and 2). To examine if DSG was able to affect the stable interaction of Delta F508-CFTR with its associated cellular chaperones and thereby alter its subcellular location, C127-Delta F508-low cells were treated with between 10 and 50 µg/ml DSG for 48-72 h. We first analyzed for evidence of the mature band C form of CFTR (4), which would be indicative of Delta F508-CFTR processing in the Golgi. Biochemical analysis of lysates from these cells showed no discernible evidence of the mature band C form of CFTR, indicating that very little if any Delta F508-CFTR had exited the ER to the Golgi (Fig. 1).


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Fig. 1.   Immunoprecipitation analysis of C127-Delta F508-low cells. Lysates were prepared from C127 cells stably expressing Delta F508-cystic fibrosis transmembrane conductance regulator (CFTR) (lanes 1-3) or wild-type CFTR (WT, lane 4). Cells were treated with either 10 µg/ml deoxyspergualin (DSG) (lane 2) or 50 µg/ml DSG (lane 3) or were left untreated (lanes 1 and 4) for 72 h before lysis. Immunoprecipitates obtained using the anti-CFTR monoclonal antibody 24-1 (15) were phosphorylated in vitro by the addition of the catalytic subunit of protein kinase A and [gamma -32P]ATP. Positions of band B (core-glycosylated CFTR) and band C (mature form of CFTR) are indicated on right. Amount of total protein loaded into each lane was normalized; exposures shown were the same for each lane.


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Fig. 2.   6-Methoxy-N-(3-sulfopropyl)quinolinium (SPQ) analysis of C127-Delta F508-low cells following treatment with DSG. NO-3 was substituted for I- in the bathing medium at 0 min. Cells were stimulated 4 min later (arrow) with 20 µM forskolin and 100 µM IBMX. Changes in fluorescence are shown for C127 cells expressing Delta F508-CFTR (n = 9, where n = no. of cells), for cells expressing Delta F508-CFTR that had been treated with 5 mM sodium butyrate for 24 h (n = 11) or with 10 µg/ml DSG for 72 h (n = 10) or incubated at 23°C for 24 h (n = 12), and for mock-transfected C127 cells that had been similarly pretreated with DSG (n = 7). Data are presented as the fluorescence at time t (Ft) minus the baseline fluorescence (Fo, average fluorescence measured in the presence of I- for 2 min before ion substitution). Data are means ± SE and are representative of responses obtained from several experiments for each condition.

To ascertain whether a small amount of Delta F508-CFTR, below the sensitivity of detection with the biochemical assay, may have traversed the Golgi to the plasma membrane, we employed the more sensitive single cell membrane halide permeability assay using the Cl- indicator SPQ (3, 15). In this assay, a rapid change in SPQ fluorescence on stimulation with cAMP agonists is indicative of the presence of active CFTR at the plasma membrane. As we had previously reported, either treating the Delta F508-CFTR-low cells with sodium butyrate (3) to augment the expression of Delta F508-CFTR or culturing them at a reduced temperature (23°C) to enhance folding (6) generated cAMP-stimulated halide efflux (Fig. 2). Cells that were grown in the presence of DSG for 3 days also restored cAMP-stimulated anion efflux albeit to a lesser extent than was observed with sodium butyrate treatment or following a temperature shift (Fig. 2). Approximately 17% of the DSG-treated C127-Delta F508-low cells generated a measurable response compared with 90% with sodium butyrate treatment or following growth at low temperature (average of 5 experiments). This disparity in response was not unexpected because treatment of these cells with sodium butyrate or growth at reduced temperature, unlike treatment with DSG, results in synthesis of detectable amounts of band C form of CFTR (3, 6). Exposure to higher concentrations of DSG (>50 µg/ml) was toxic to the cells and did not improve either the intensity or frequency of the signal. No response was observed in C127-Delta F508-low cells that were left untreated or in C127-mock cells (parental C127 cells mock-transfected with expression vector alone) that had been treated with DSG (Fig. 2). These results suggest that the Cl- channels observed in the DSG-treated C127-Delta F508-low cells were most likely due to the presence of mutant Delta F508-CFTR at the cell surface.

Because the structure of DSG resembles that of the polyamine spermidine, C127-Delta F508-low cells were also treated with 5 µg/ml spermidine for 72 h as a negative control. No measurable cAMP-stimulated Cl- channel activity was detected following treatment with spermidine, arguing against a nonspecific effect (data not shown). Data similar to those described for C127-Delta F508-low cells were also observed with LLC-PK1-Delta F508 cells, a recombinant pig kidney epithelial cell line stably expressing the variant CFTR (data not shown).

Effect of DSG on immortalized CF airway epithelial cells. To test whether DSG had a similar effect on human CF cells, an immortalized airway epithelial cell line (JME/CF15) obtained from a CF patient homozygous for the Delta F508 mutation (12) was treated with DSG. Attempts to detect changes in the glycosylation state of CFTR following treatment with DSG or sodium butyrate or growth at reduced temperature by immunoprecipitation assays were unsuccessful due to the low amounts of CFTR in these cells. This was not surprising, since many similar labeling experiments in the past using primary normal human airway epithelial cells also failed to detect CFTR, due to its low abundance. Examination of the untreated JME/CF15 cells using the SPQ assay showed, as expected, a lack of detectable cAMP-stimulated Cl- channel activity (Fig. 3A). In addition, consistent with expectations, when these cells were grown at 23°C for 24 h, measurable cAMP-regulated Cl- channel activity could be detected in a proportion of the cells (Fig. 3). Cells pretreated with between 10 and 100 µg/ml DSG for 72 h also displayed cAMP-responsive Cl- channel activity (Fig. 3A). The effect was specific for DSG and was not replicated with the structurally related analog spermidine (data not shown). The response observed with DSG appeared more robust than that attained when cells were cultured at low temperature. For example, the cAMP-stimulated rate of change in SPQ fluorescence observed with DSG was consistently greater (Fig. 3A) and the total number of responsive cells (~10-15%) was slightly higher (Fig. 3B) than that observed when the cells were cultured at low temperature. This is contrary to what was observed with the recombinant C127-Delta F508-low cells. However, it should be noted that DSG also has an ascribed role in blocking the nuclear translocation of the nuclear transcription factor-kappa B (NF-kappa B) (23). This block may have reduced the transcriptional activity of the cytomegalovirus promoter (which contains several consensus NF-kappa B binding sites) used to express Delta F508-CFTR in the C127 cells and therefore reduced the levels of mutant protein produced in these cells. Although the percentage of responsive cells observed with DSG was only ~12% (Fig. 3B), it should be noted that this determination was limited by the sensitivity of the SPQ assay and that the number of cells affected may be greater.


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Fig. 3.   SPQ analysis of the human cystic fibrosis airway epithelial cell line JME/CF15 following treatment with DSG. A: shifts in fluorescence are shown for JME/CF15 cells that had been treated for 72 h with 10 µg/ml DSG (n = 8, where n = no. of responding cells) or with 100 µg/ml DSG (n = 11), incubated at 23°C for 24 h (n = 7), or had no added treatment (n = 7). Cells were stimulated with 20 µM forskolin and 100 µM IBMX 4 min after ion substitution (arrow). Data are presented as Ft minus Fo (average fluorescence measured in the presence of I- for 2 min before ion substitution). Data are means ± SE and are representative of responses obtained from several experiments for each condition. B: percentage of responsive JME/CF15 cells following treatment with different concentrations of DSG or following growth at 23°C for 24 h. Data are expressed as means ± SE. Statistical analysis was performed using ANOVA followed by unpaired Student's t-test. ** P < 0.01 and * P < 0.05, signifies significance from untreated controls.

Although a greater number of responsive cells was observed when 10 µg/ml of DSG was used instead of 5 µg/ml, no further significant increment in response was noted at concentrations higher than 10 µg/ml (Fig. 3B). As with the C127 cells, some toxicity was evident at DSG concentrations above 50 µg/ml. We conclude that DSG would appear to be capable of generating functional cAMP-stimulated Cl- channel activity in at least a proportion of the immortalized Delta F508 human airway epithelial cells. Because many studies have indicated that these cells lack cAMP-dependent Cl- channel activity other than CFTR (12), the observed response after DSG treatment was most likely due to Delta F508-CFTR at the cell surface. We have repeated the above experiments using another immortalized human CF airway epithelial cell line (CFT1) (29) with very similar results (data not shown).

Effect of DSG on immortalized CF biliary epithelial cells. The ability of DSG to influence the presence of endogenous mutant Delta F508-CFTR at the plasma membrane was also assessed in IBE-1 cells, an immortalized human CF intrahepatic biliary epithelial cell line that harbors the Delta F508 and G542X (premature stop mutation at residue 542) mutations (8). Consistent with previous reports (8), IBE-1 cells did not exhibit any measurable cAMP-stimulated CFTR Cl- channel activity (Fig. 4A). However, on exposure to between 5 and 50 µg/ml DSG for 72 h, up to 20% of the cells exhibited measurable cAMP-stimulated Cl- channel activity (Fig. 4). The effect of DSG on IBE-1 cells was concentration dependent, with higher concentrations of DSG giving rise to greater transport rates and higher numbers of positively responding cells (Fig. 4). Moreover, the response observed at the higher concentrations was similar to that attained when these cells were cultured at reduced temperature. Together, these data demonstrate that exposure of Delta F508-expressing cells to DSG partially corrects the trafficking defect of the mutant protein, as evidenced by the presence of cAMP-stimulated Cl- channel activity at the cell surface.


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Fig. 4.   SPQ halide efflux assay of IBE-1 cells following treatment with DSG. A: changes in fluorescence are shown for IBE-1 cells that were treated for 72 h with 10 µg/ml DSG (n = 9, where n = no. of cells) or with 50 µg/ml DSG (n = 14), incubated at 23°C for 24 h (n = 10), or had no added treatment (n = 6). Cells were stimulated with 20 µM forskolin and 100 µM IBMX 4 min after ion substitution (arrow). Data are presented as Ft minus Fo (average fluorescence measured in the presence of I- for 2 min before ion substitution). Data are means ± SE and are representative of responses obtained from several experiments for each condition. B: percentage of responsive IBE-1 cells following treatment with different concentrations of DSG or following growth at 23°C for 24 h. Data are expressed as means ± SE. Statistical analysis was performed using ANOVA followed by unpaired Student's t-test. ** P < 0.01 and * P < 0.05, signifies significance from untreated controls.

Whole cell patch-clamp analysis of IBE-1 cells treated with DSG. To confirm that the signals observed using the SPQ fluorescence assay were truly CFTR mediated, whole cell patch-clamp experiments were also performed on the IBE-1 cells. Figure 5 shows representative current tracings from one such experiment. In these studies, the holding potential was 0 mV (which inactivates the voltage-gated Na+ and Ca2+ channels) and the voltage was stepped from -100 to +80 mV in 20-mV increments to activate whole cell currents. Intracellular and extracellular solutions were designed to study only current flowing through Cl- channels, since Cl- was the only significant permeant ion in the solutions. Currents from Ca2+ and K+ channels were minimized by omitting K+ from both intra- and extracellular solutions and by inclusion of 100 µM Cd2+ in the extracellular solution and 20 mM TEA and 10 mM EGTA in the intracellular solution. Under these conditions, 100 µM UTP and 1 µM ionomycin failed to activate whole cell currents (data not shown). Finally, any contribution from the outwardly rectifying Cl- channel was minimized by performing the experiments at room temperature.


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Fig. 5.   Whole cell patch-clamp analysis of IBE-1 cells. Currents shown are in response to voltage steps from a holding potential of 0 mV to between -100 and +80 mV in steps of 20-mV increments. Representative whole cell currents under basal (unstimulated) conditions from IBE-1 cells (A) and from IBE-1 cells that had been treated with DSG (10 µg/ml) for 48 h (C) are shown. B: recording from the same IBE-1 cells following stimulation with 200 µM 8-(4-chlorophenylthio) (CPT)-cAMP. D: recording from the IBE-1 cells treated with DSG following stimulation with 200 µM CPT-cAMP. Similar results were observed with the cAMP agonists forskolin and IBMX. E: current-voltage relationships obtained under basal conditions (black-square) and after addition of 200 µM CPT-cAMP (bullet ) of 7 responder cells from 7 different coverslips treated with DSG (10 µg/ml) for 48-72 h are summarized. Currents showed linear current-voltage behavior and no time dependence. Data are presented as means ± SE.

Consistent with previous studies, no currents were activated when untreated IBE-1 cells (n = 6) were stimulated with cAMP agonists or CPT-cAMP (Fig. 5, A and B). However, when the same cells were treated with 10 µg/ml DSG for 48 h, significant activation of whole cell currents in response to CPT-cAMP was observed in 7 of 19 successfully patched cells (Fig. 5, C and D). The currents were ascertained to be Cl- currents by the change in reversal potential when the extracellular Cl- concentration was reduced from 150 to 20 mM. The whole cell properties were qualitatively similar to those observed with wild-type CFTR (5, 26) and those attained following infection of the IBE-1 cells with adenovirus vectors encoding wild-type CFTR (data not shown). Whole cell currents were reversibly activated by CPT-cAMP, were time independent, were markedly reduced by DPC (92% ± 4, n = 4), an agent shown to inhibit CFTR activity, and displayed a linear current-voltage relationship (Fig. 5E). Together, these data strongly indicate that functional CFTR Cl- channel activity was present on the surface of IBE-1 cells following treatment with DSG. That these cells only contained Delta F508-CFTR suggests that DSG treatment can result in the relocation of at least some of the mutant protein to the plasma membrane.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Several therapeutic approaches are being developed concurrently for the treatment of CF. These include 1) use of agents that improve the antibacterial activity and viscosity of the mucous fluids lining the airways, 2) use of agents that bypass the CFTR Cl- channel defect, 3) protein and gene augmentation therapy, and 4) use of agents that reverse the mutant phenotype. Examples of the last group include aminoglycosides to suppress disease-associated stop mutations (11) and phenylbutyrate (3, 20) and chemical chaperones (2, 21) to reverse trafficking mutants.

The trafficking mutations, or class II-type mutations, as exemplified by Delta F508, are the most common among CF patients. The variant Delta F508-CFTR is recognized as abnormal and is purportedly retained by the cellular chaperones HSP70 and calnexin in the ER where it is subsequently degraded (19, 28). We rationalized therefore that agents capable of disrupting the interaction of Delta F508-CFTR with its cellular chaperones might facilitate escape of the variant protein from the quality control apparatus in the ER and thereby allow transit to the plasma membrane. DSG, an immunosuppressant presently under clinical investigation, binds HSC70 and HSP90 with affinities that are predicted to compete effectively for the binding of these chaperones to nascent polypeptides (16). We report here that DSG was indeed capable of partially reversing the trafficking defect associated with Delta F508-CFTR in recombinant and immortalized human CF epithelial cell lines. Delta F508-CFTR cells exposed to DSG exhibited cAMP-stimulated Cl- channel activity, a function that was otherwise lacking in these cells. We interpret these results to mean that DSG was able to salvage a fraction of the mutant CFTR normally targeted for degradation by HSP70 and calnexin and thereby allowed for the translocation of at least some Delta F508-CFTR to the plasma membrane. However, although functional cAMP-stimulated Cl- channels were detected in a proportion of the DSG-treated cells, we were unable to demonstrate the presence of any mature band C form of CFTR by immunoprecipitation analysis. This result would argue either that a very small amount of Delta F508-CFTR that was below the level of detection using the biochemical assays escaped the ER to the Golgi and thence to the plasma membrane or that the form that trafficked to the plasma membrane was indistinguishable from the core-glycosylated band B form. If any conversion to band C had occurred, this was likely to be <1% of total CFTR synthesized. Our ability to detect mature band C CFTR following DSG treatment may also have been hampered by the fact that DSG also decreased nuclear translocation of the transcription factor NF-kappa B, thereby reducing the expression of Delta F508-CFTR in the recombinant cells (Fig. 1). It should also be noted that the stability of the mutant Delta F508-CFTR at the plasma membrane is reportedly much shorter than that for wild-type CFTR (14).

Although it is possible that DSG affected the subcellular location of Delta F508-CFTR by altering its relationship with the cellular chaperones, because we were unable to detect band C form of CFTR we cannot exclude other mechanisms. Our proposed basis for the effect of DSG assumes that DSG disrupted the interaction between Delta F508-CFTR and HSP70 and calnexin and, in consequence, the retention of the mutant protein within the ER. However, it has been shown that HSP70 also has an ascribed role in promoting the folding of nascent CFTR by inhibiting off-pathway associations that lead to the formation of high-molecular-weight aggregates (22). As such, DSG may have a more complex effect on Delta F508-CFTR besides affecting its interaction with the chaperones. For example, because it has been suggested that a small amount of Delta F508-CFTR can escape the quality control apparatus in the ER, DSG may also act to influence the rate of degradation or stability of band C-Delta F508-CFTR. Furthermore, although the ability of DSG to bind HSP70 is well characterized, there is no indication that it has a similar effect on calnexin. Therefore, it remains to be determined whether DSG affected the trafficking of Delta F508-CFTR by altering the relationship between the mutant protein and its associated cellular chaperones.

We also compared the response observed with DSG with other interventions shown previously to result in the presence of Delta F508-CFTR at the plasma membrane. In both the CF airway and biliary epithelial cell lines, the response attained with DSG was comparable to that observed when these cells were cultured at a reduced temperature. In recombinant cells, the effect of incubation at low temperature has been shown to be as effective as treatment with the chemical chaperone glycerol in eliciting the presence of Delta F508-CFTR at the cell surface (2, 21). In this regard, DSG would appear to be as effective as any other treatment shown previously to be capable of rescuing the Delta F508-CFTR trafficking defect.

If the mechanism by which DSG affected the presence of Delta F508-CFTR at the plasma membrane was indeed mediated through its interaction with the chaperones that normally associate with Delta F508-CFTR, then other interventions aimed at eliciting a similar release of the chaperones from the newly synthesized mutant CFTR might induce a portion of the protein to undergo maturation and transit to the cell surface. For example, heat shock treatment, which results in a rapid redistribution of HSP73 from the cytoplasm to the nucleus, might also result in the release of a small proportion of the mutant CFTR. Immunosuppressive allotrap peptides derived from highly conserved regions of human major histocompatibility complex class I molecules are capable of binding HSP70 and may also be similarly efficacious (18). However, all these interventions are nonspecific and as such are likely to result in a general disruption of the quality control apparatus that normally regulates proper folding and trafficking of proteins in the cell. It is unclear whether such a general disruption would adversely affect long-term cell viability. In addition, because DSG and the allotrap peptides are immunosuppressants, they may not be useful for the long-term treatment of CF. Nevertheless, our results suggest that identification of agents like DSG, which perhaps are more specific for Delta F508-CFTR or which act only transiently, may be efficacious for the treatment of CF. Furthermore, one may also consider inclusion of compounds like genistein and calyculin, shown recently to enhance the activity of CFTR Cl- channels at the cell surface (27).

    ACKNOWLEDGEMENTS

We thank members of the CF Research Group for their comments and formative discussions throughout this project and P. Rafter and J. Marshall in particular for their technical assistance. We also thank S. Eastman, J. Marshall, R. Scheule, and N. Yew for their constructive comments on the manuscript.

    FOOTNOTES

Address for reprint requests: S. H. Cheng, Genzyme Corporation, One Mountain Road, Framingham, MA 01701-9322.

Received 13 November 1997; accepted in final form 14 April 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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