Partial restoration of cAMP-stimulated CFTR chloride channel
activity in
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
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ABSTRACT |
Deletion of the codon encoding phenylalanine 508 (
F508) is
the most common mutation in cystic fibrosis (CF) and results in a
trafficking defect. Mutant
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
F508-CFTR to the cell membrane. We show that
DSG treatment of immortalized human CF epithelial cells (
F508) and
cells expressing recombinant
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
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 |
THE MOST COMMON CAUSE of cystic fibrosis (CF) is
deletion of the phenylalanine residue at position 508 (
F508) of the
cystic fibrosis transmembrane conductance regulator (CFTR). Studies
have shown that this mutation results in the synthesis of a variant CFTR (
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
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
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
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
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
F508-CFTR is unable to dissociate
from either calnexin or HSP70 and does not exit the ER to the Golgi. In
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
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
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
F508-CFTR, cells expressing the mutant
protein were exposed to DSG. We report here that addition of DSG to
cells expressing recombinant
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 |
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
F508 variant (8, 12, 29).
C127-
F508-low (mouse mammary tumor) and
LLC-PK1-
F508 (pig kidney
epithelial) are two recombinant cell lines stably expressing low levels
of the mutant
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-
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
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
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 M
. 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
[
-32P]ATP,
and polyacrylamide gel electrophoresis have all been described previously (3, 15).
 |
RESULTS |
Detection of functional CFTR Cl
channel activity in C127 cells expressing recombinant
F508-CFTR following treatment with DSG.
C127-
F508-low is a recombinant cell line stably transfected with the
cDNA encoding the mutant
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
F508-CFTR with its
associated cellular chaperones and thereby alter its subcellular
location, C127-
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
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
F508-CFTR had
exited the ER to the Golgi (Fig. 1).

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Fig. 1.
Immunoprecipitation analysis of C127- F508-low cells. Lysates were
prepared from C127 cells stably expressing 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
[ -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- 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 F508-CFTR (n = 9, where
n = no. of cells), for cells
expressing 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.
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To ascertain whether a small amount of
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
F508-CFTR-low cells with sodium butyrate (3) to augment the
expression of
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-
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-
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-
F508-low cells were most likely due to the presence
of mutant
F508-CFTR at the cell surface.
Because the structure of DSG resembles that of the polyamine
spermidine, C127-
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-
F508-low cells were also observed with
LLC-PK1-
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
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-
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-
B
(NF-
B) (23). This block may have reduced the transcriptional activity of the cytomegalovirus promoter (which contains several consensus NF-
B binding sites) used to express
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.
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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
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
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
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
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
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.
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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 ( ) and after addition of 200 µM
CPT-cAMP ( ) 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.
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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
F508-CFTR suggests that DSG
treatment can result in the relocation of at least some of the mutant
protein to the plasma membrane.
 |
DISCUSSION |
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
F508, are the most common among CF patients. The variant
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
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
F508-CFTR in recombinant and immortalized human CF epithelial
cell lines.
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
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
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-
B, thereby reducing the expression of
F508-CFTR in the
recombinant cells (Fig. 1). It should also be noted that the stability
of the mutant
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
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
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
F508-CFTR besides affecting its interaction with
the chaperones. For example, because it has been suggested that a small
amount of
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-
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
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
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
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
F508-CFTR trafficking defect.
If the mechanism by which DSG affected the presence of
F508-CFTR at
the plasma membrane was indeed mediated through its interaction with
the chaperones that normally associate with
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
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.
 |
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