Evidence against the acidification hypothesis in cystic
fibrosis
Gregory A.
Gibson1,
Warren G.
Hill2, and
Ora A.
Weisz1,2
1 Laboratory of Epithelial Cell Biology, Renal-Electrolyte
Division, and 2 Department of Cell Biology and Physiology,
University of Pittsburgh, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
The pleiotropic effects of cystic fibrosis (CF) result from the
mislocalization or inactivity of an apical membrane chloride channel,
the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR
may also modulate intracellular chloride conductances and thus affect
organelle pH. To test the role of CFTR in organelle pH regulation, we
developed a model system to selectively perturb the pH of a subset of
acidified compartments in polarized cells and determined the effects on
various protein trafficking steps. We then tested whether these effects
were observed in cells lacking wild-type CFTR and whether
reintroduction of CFTR affected trafficking in these cells. Our model
system involves adenovirus-mediated expression of the influenza virus
M2 protein, an acid-activated ion channel. M2 expression selectively
slows traffic through the trans-Golgi network (TGN) and
apical endocytic compartments in polarized Madin-Darby canine kidney
(MDCK) cells. Expression of M2 or treatment with other pH perturbants
also slowed protein traffic in the CF cell line CFPAC, suggesting that
the TGN in this cell line is normally acidified. Expression of
functional CFTR had no effect on traffic and failed to rescue the
effect of M2. Our results argue against a role for CFTR in the
regulation of organelle pH and protein trafficking in epithelial cells.
endosome; Golgi; Madin-Darby canine kidney
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INTRODUCTION |
CYSTIC FIBROSIS
(CF) results from the mislocalization or inactivity of the apical
membrane cystic fibrosis transmembrane conductance regulator chloride
channel (CFTR). In addition to its activity at the apical membrane of
polarized cells, CFTR has also been postulated to modulate chloride
conductances in acidified intracellular compartments and thereby
regulate the pH of organelles along the secretory and endocytic
pathways (2, 4). Comparison of the pH of the
trans-Golgi network (TGN) and endosomal compartments in CF
versus normal cells suggested that CF cells fail to adequately acidify
these compartments (4). The consequences of disrupted Golgi and endosome pH in CF cells could include altered glycoconjugate composition at the cell surface [which could explain enhanced bacterial binding to CF cells (17, 39, 59, 60)] as well as altered membrane trafficking (4, 6, 10). However, the role of CFTR in modulating organelle pH and membrane trafficking has
remained controversial for various reasons. One problem has been the
lack of closely matched polarized CF and control cell lines in which to
perform these studies. As a result, previous studies that examined the
effect of CFTR on endosomal pH or membrane trafficking events were
performed by using heterologous expression systems or nonpolarized CF
and control cells (6, 10, 20, 21, 58, 62, 67). Although
most of these studies found no effect of CFTR on either organelle pH or
membrane trafficking, the presence of alternative chloride conductances
in these systems could mask a functional requirement for CFTR. Another
major complication in identifying the role of CFTR in regulating
organelle pH is that the importance of acidification in protein traffic
is not well understood. Previous studies employing global pH
perturbants [such as weak bases or vacuolar H+-ATPase
(V-ATPase) inhibitors] to disrupt acidification have yielded conflicting results regarding the consequences of disrupting organelle acidification on biosynthetic and postendocytic traffic (16, 47,
49, 51, 73, 77, 78). Thus it has not been possible to correlate
any differences in membrane traffic between CF and normal cells with a
defect in acidification of CF cells.
We have generated a model system to selectively perturb the pH of a
subset of acidified compartments in polarized epithelial cells. This
system involves expression of the influenza virus M2 protein, an
acid-activated proton-selective channel that increases the pH of a
subset of acidified compartments in cells (14, 15, 26, 27, 30,
33, 42, 50, 54, 61, 68, 71, 74). We have previously demonstrated
that expression of M2 disrupts acidification of the TGN and apical
recycling endosomes, but not that of basolateral endosomes or lysosomes
in polarized Madin-Darby canine kidney (MDCK) cells (31).
Importantly, the TGN and apical endosomes are the same compartments
whose pH is predicted to be disrupted in CF cells. Thus we asked
whether M2 expression in polarized epithelial cells mimics the CF
phenotype and whether expression of epitope-tagged, fully functional
CFTR in cells lacking functional chloride channel has any effect on
biosynthetic or postendocytic traffic. Our previous data suggested that
even closely matched CF and control cells are inappropriate for
comparing protein processing/glycosylation to determine the function of
CF (40). Therefore, we used recombinant adenoviruses to
generate matched CFTR-expressing and -nonexpressing cell lines that
differ only by overnight culture. We used two cell lines for our
studies: MDCK II, which form sealed monolayers on permeable filter
supports, and CFPAC, a pancreatic adenocarcinoma cell line that forms
polarized islands of cells; neither of these cell lines express
endogenous wild-type CFTR (46, 64).
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MATERIALS AND METHODS |
Cell lines.
Low-passage MDCK cells (type II) were maintained in minimal essential
medium (Cellgro; Fisher Scientific, Pittsburgh, PA) supplemented with
10% fetal bovine serum (Atlanta Biologicals, Norcross, GA),
streptomycin (100 µg/ml), and penicillin (100 U/ml). Generation and
characterization of the MDCK T23 cell line, which stably expresses the
tetracycline-repressible transactivator tTA (25), was
described previously (5). These cells also express the
polymeric immunoglobulin receptor (pIgR) under control of the
butyrate-inducible cytomegalovirus promoter. By indirect
immunofluorescence, >90% of the cells express detectable levels of
pIgR after overnight induction with 2 mM butyrate. For all experiments,
cells were seeded at high density (~2 × 105
cells/well) in 12-mm Transwells (0.4-µm pore; Costar, Cambridge, MA)
for 2-3 days before infection with recombinant adenovirus. Experiments were performed the following day. CFPAC cells were obtained
from American Type Culture Collection (Rockville, MD) and maintained in
DMEM/F-12 supplemented with 10% fetal bovine serum. Cells were plated
in 35-mm dishes (3 × 105 cells/dish) or 12-well
dishes (1 × 105 cells/well) 2-3 days before infection.
Recombinant adenoviruses.
The generation of E1-substituted recombinant adenoviruses (AV) encoding
M2 in the correct and reverse orientations (AV-M2 and AV-M2rev,
respectively) and influenza hemagglutinin (AV-HA) was described
previously (31). Generation of an adenovirus encoding the
rabbit pIgR was also described previously (32).
Construction of AV-M2901, which encodes fully functional,
epitope-tagged CFTR (M2-901/CFTR; Refs. 36 and 65) was described
previously (37). Viruses were purified as described
previously (31).
Adenoviral infection.
Viral infection of filter-grown MDCK T23 cells was performed as
described previously (31). CFPAC cells grown on plastic dishes were washed with a large volume of calcium-free
phosphate-buffered saline (PBS) containing 1 mM MgCl2
(PBS-M). After 5 min at room temperature, the PBS-M was replaced with a
small volume (0.4 ml for 35-mm dishes, 0.3 ml for 12-well plates) of
PBS-M containing recombinant adenovirus(es). The dishes were rocked
briefly by hand, and the cells were returned to an incubator for
1-2 h. Mock-infected cells were treated identically except that
virus was omitted during the incubation period. Dishes were then rinsed
with PBS-M, and cells were incubated overnight in growth medium
containing 2 mM butyrate.
Indirect immunofluorescence.
Indirect immunofluorescence of fixed, virally infected cells was
performed as described previously (34). Briefly, cells were rinsed once with PBS, fixed for 20 min at ambient temperature in
3% paraformaldehyde, and then incubated briefly with PBS containing 10 mM glycine and 0.02% sodium azide (PBS-G). Cells were permeabilized for 3 min in 0.5% Triton X-100 in PBS-G. Nonspecific binding was blocked with 0.25% ovalbumin in PBS-G before incubation with
antibodies. M2 was detected using the monoclonal antibody 5C4 (1:250
dilution). Samples were then incubated with Cy3-conjugated
affinity-purified goat anti-mouse antibody (2 mg/ml, 1:1,000 dilution;
Jackson ImmunoResearch Laboratories, Avondale, PA). ZO-1 was detected
using a polyclonal anti-ZO-1 antibody (Zymed Laboratories, South San
Francisco, CA), followed by FITC-conjugated goat anti-rabbit IgG (2 mg/ml, 1:100 dilution; Jackson ImmunoResearch Laboratories). For
live-cell staining of epitope-tagged CFTR, cells were rapidly chilled
to 0°C by being washed with ice-cold PBS and were maintained on ice throughout the staining. Nonspecific binding sites were blocked by
incubation for 20 min with 5% normal goat serum in PBS, and the cells
were sequentially incubated with monoclonal anti-FLAG antibody
(Stratagene, La Jolla, CA) and Cy3-conjugated goat anti-mouse secondary
antibody for 30 min each with several washes in between. After cells
were washed further, they were fixed for 10 min at 0°C and then 20 min at ambient temperature with 3% paraformaldehyde. Cells were viewed
with a Nikon Optiphot microscope (Fryer, Carpentersville, IL), and
images were acquired with a Hamamatsu C5985 chilled coupled charge-device camera (8 bit, 756 × 483 pixels; Hamamatsu
Photonics Systems, Bridgewater, NJ) and printed with a Kodak 8650PS
dye-sublimation printer (Rochester, NY).
Transcytosis and recycling assays.
Recycling and transcytosis assays in filter-grown MDCK T23 cells were
performed essentially as described previously (31). To
measure recycling of IgA in CFPAC cells, virally infected cells in
12-well dishes were rinsed with MEM/BSA (minimum essential medium,
Hanks' balanced salt solution, 0.6% BSA, and 20 mM HEPES, pH 7.4) and
then incubated with 0.3 ml of MEM/BSA containing
125I-labeled IgA for 30 min at 37°C. Cells were rinsed
once with MEM/BSA and washed three times for 5 min each with MEM/BSA on ice, the medium was replaced with prewarmed MEM/BSA, and the cells were
returned to 37°C. At the designated time points, the media were
collected and replaced. After the final time point, cells were
solubilized and the amount of 125I-IgA in all samples was
determined using a gamma counter (Packard Instrument, Downers Grove,
IL). An equal number of mock-infected CFPAC cells (not expressing the
pIgR) were treated identically to determine nonspecific IgA uptake and
recycling, and these values were subtracted from the experimental
samples. Where indicated, 10 µM forskolin (FSK; Calbiochem, San
Diego, CA) was added during the last 10 min of radioligand uptake and
included in subsequent steps. Recycling of 125I-labeled
iron-loaded human transferrin (Tf) was performed as described above
except that cells were preincubated in MEM/BSA for 45 min before
125I-Tf uptake for 45 min at 37°C. After cells were
washed, they were incubated at 37°C for 3 min, the medium was
replaced, and the time course was initiated.
TGN-to-cell surface delivery of influenza HA.
Surface delivery of newly synthesized HA was performed essentially as
described previously (31). Filter-grown T23 cells were
coinfected with AV-HA [multiplicity of infection (MOI) 25] and
AV-M2rev, AV-M2, or AV-M2901 (MOI 250). Plastic-grown CFPAC cells were
coinfected with AV-HA (MOI 250), AV-TA (MOI 200), and AV-M2rev, AV-M2,
or AV-M2901 (MOI 500) as described in Adenoviral infection. The following day, cells were rinsed once with
PBS and then starved for 30 min in medium A (cysteine-free,
methionine-free MEM containing 0.35g/l NaHCO3, 10 mM HEPES,
and 10 mM MES, pH 7.0). Where indicated, the M2 ion channel blockers
amantadine (AMT; 5 µM; Sigma Chemical, St. Louis, MO), BL-1743 (10 µM), or the V-ATPase inhibitor bafilomycin A1
(BafA1, 1 µM; Sigma Chemical) were added at the
beginning of the starvation and included during the pulse and chase
periods. Cells were metabolically labeled with 50-100 µCi/ml
[35S]Express (NEN, Boston, MA) in the same medium and
then chased for 2 h at 19°C in the same medium supplemented with
four times the normal amount of cysteine and methionine
(medium B). At various times, individual filters or
dishes were removed, rapidly chilled to 0°C by being rinsed with
ice-cold PBS, and incubated on ice for 30 min in 1 ml of medium
B containing 100 µg/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin (Sigma Chemical) for 30 min. Trypsin cleaves HA into two subunits (HA1 and HA2) that remain associated via disulfide bonds during immunoprecipitation. Only the
apical chamber of filter-grown cells was treated with trypsin. Trypsinization was stopped by incubating the cells twice for 10 min
with ice-cold medium B containing 200 µg/ml soybean
trypsin inhibitor (Sigma Chemical). Cells were rinsed with PBS and
lysed in 0.5 ml of detergent solution (50 mM Tris · HCl, 2%
NP-40, 0.4% deoxycholate, and 62.5 mM EDTA, pH 8.0) containing 1 µg/ml aprotinin and 20 µg/ml soybean trypsin inhibitor. Lysates
were centrifuged briefly to remove nuclei, and SDS was added to the
supernatant to a final concentration of 0.2%. HA was
immunoprecipitated with the use of a monoclonal antibody, and
antibody-antigen complexes were collected with the use of fixed
Staphylococcus aureus (Pansorbin; Calbiochem) and washed
three times with radioimmunoprecipitation assay (RIPA) buffer (10 mM
Tris · HCl, 0.15 M NaCl, 1% Triton X-100, 1% NP-40, and 0.1%
SDS, pH 7.4). After electrophoresis was carried out on 10%
SDS-polyacrylamide gels, the percentage of cleaved HA was quantitated
using a phosphorimager (GS-363 Molecular Imager System; Bio-Rad,
Hercules, CA).
Immunoprecipitation of virally expressed M2.
CFPAC cells were mock infected or infected with AV-M2 as described in
Adenoviral infection. The following day, cells were rinsed once with PBS, starved for 30 min in medium A, and
then radiolabeled in a humidified chamber for 2 h by placing the
filters on a 25-µl drop of medium A containing 1.5 mCi/ml
[35S]Express. After labeling was completed, filters were
rinsed once with PBS and then cut out of the plastic insert, and the
cells were solubilized with 0.5 ml of 60 mM octylglucoside and 0.1% SDS in HEPES-buffered saline (10 mM HEPES and 0.15 M NaCl, pH 7.4)
containing 1 µg/ml aprotinin. Lysates were centrifuged for 7 min at
16,000 g at room temperature, and the supernatants were immunoprecipitated with 5C4. Antibody-antigen complexes were collected with the use of fixed S. aureus (Pansorbin) and washed
three times with RIPA buffer. After samples were eluted in Laemmli
sample buffer, they were electrophoresed on 12% SDS-polyacrylamide
gels and analyzed using a phosphorimager.
Detection of functional epitope-tagged CFTR using SPQ.
cAMP-dependent anion efflux was monitored by
6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ; Molecular
Probes, Eugene, OR) fluorescence changes in living cells as described
previously (40). Subconfluent cells grown on 25-mm glass
coverslips were loaded with 10 mM SPQ in hypotonic NaI buffer. Iodide
binds to SPQ and quenches its fluorescence. Cells were mounted in a
perfusion chamber placed in a heating stage set to 37°C and perfused
with buffers throughout the experiment. Imaging was performed on a
Nikon Diaphot 300 inverted microscope equipped with a ×40
oil-immersion objective, an image intensifier, and a video camera.
Excitation was at 330 nm, and image acquisition and analysis were
performed by using Metafluor software (Universal Imaging, West Chester,
PA). The average fluorescence intensity of individual cells in a field
was monitored every 15 s throughout the assay. Cells were perfused
for 2 min with isotonic NaI buffer, for 4 min with nitrate buffer to
assess the rate of iodide leakage/exchange from nonstimulated cells,
for 4 min with nitrate buffer supplemented with 10 µM FSK and 200 µM 3-isobutyl-1-methylxanthine (IBMX; Calbiochem), and then for 4 min
with iodide buffer to requench intracellular SPQ. Functional
CFTR was detected as an increase in the rate of dequenching of SPQ upon
addition of FSK/IBMX. Assays on mock-infected and AV-M2901-infected
cells were performed blindly.
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RESULTS |
Adenovirus-mediated expression of CFTR and M2 in polarized MDCK
cells.
We used indirect immunofluorescence to confirm that polarized MDCK T23
cells were efficiently infected with recombinant adenoviruses encoding
M2 or epitope-tagged CFTR. Filter-grown cells were infected with AV-M2
or AV-M2901 at an MOI of 250 and processed for indirect immunofluorescence the following day (Fig.
1). Approximately one-half of the cells
expressed detectable levels of M2 or CFTR under these conditions.
Previous experiments in which M2-expressing cells were infected under
identical conditions gave maximal effects on all transport steps
measured, although M2 could be detected in only 10-70% of the
cells (31). In addition, infection of these cells with
10-fold lower levels of recombinant adenovirus that encodes influenza
HA resulted in >90% expressing cells (31). Thus we
suspect that essentially all of the cells on the filter insert are
infected under our conditions but that immunofluorescence detection of
M2 and CFTR is not as sensitive as detection of HA.

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Fig. 1.
Expression of M2 and epitope-tagged cystic fibrosis transmembrane
conductance regulator (CFTR) in Madin-Darby canine kidney (MDCK) T23
cells. Filter-grown MDCK T23 cells were infected with adenoviruses
encoding M2 (AV-M2) or M2901 (AV-M2901) at a multiplicity of infection
(MOI) of 250. On the following day, cells expressing M2 were fixed and
processed for indirect immunofluorescence, whereas cells expressing
M2901 were processed for live-cell staining using anti-FLAG antibody as
described in MATERIALS AND METHODS. Scale bar: 10 µm.
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M2 but not CFTR slows protein traffic in polarized MDCK cells.
Although CFTR has been postulated to regulate TGN pH, the role of
counterion conductance in regulating organelle acidification is
controversial (3, 18, 44). We and others have previously determined that expression of M2, which increases TGN pH, inhibits surface delivery of newly synthesized HA in HeLa cells (33, 34,
61) as well as in polarized MDCK cells (32). The
effect of M2 occurs at the level of HA exit from the TGN and is
inhibited by inclusion of the ion channel blocker AMT (33,
34). The role of counterion conductance in regulating organelle
acidification is controversial (3, 18, 44). By contrast,
expression of CFTR might be expected to enhance acidification of the
TGN, with unpredictable consequences on protein traffic through this
compartment. Therefore, we compared the effects of M2 and CFTR on
apical biosynthetic traffic in MDCK T23 cells. Polarized MDCK cells
expressing HA and either M2 or CFTR were radiolabeled and chased for
2 h at 19°C to accumulate newly synthesized HA in the TGN
(45). Control cells were infected with an adenovirus
encoding M2 in the reverse orientation (M2rev) in addition to HA to
maintain a constant MOI in all samples. Cells were then rapidly warmed,
and the rate of HA delivery to the apical cell surface was quantitated
(Fig. 2). As expected, M2 slowed the rate
of HA delivery to the apical surface. The effect of M2 on this step in
HA transport was similar to that of the V-ATPase inhibitor
BafA1 (Fig. 2, compare upright and inverted triangle
symbols) and was blocked by inclusion of AMT during the experiment (not
shown). However, expression of epitope-tagged CFTR (Fig. 2, square
symbols) had no effect on the rate of HA delivery to the cell surface.

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Fig. 2.
Expression of M2 but not CFTR slows apical delivery of
hemagglutinin (HA) in polarized MDCK cells. MDCK T23 cells were
infected with AV-M2rev, AV-M2, or AV-M2901 as described in
MATERIALS AND METHODS. On the following day, cells were
starved for 30 min in cysteine-free, methionine-free medium,
radiolabeled for 15 min, and then chased for 2 h at 19°C.
Bafilomycin A1 (BafA1; 1 µM) was included in
1 set of AV-M2rev-infected cells at the beginning of the starvation
period and in subsequent steps. The medium was replaced with prewarmed
chase medium, and the cells were incubated at 37°C for the indicated
times before rapid chilling and cell surface trypsinization. Cells were
solubilized, HA was immunoprecipitated, and the percentage of cleaved
HA was quantitated from SDS-PAGE gels with the use of a phosphorimager.
Similar results were obtained in 3 experiments.
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Next, we investigated the effect of epitope-tagged CFTR on the rate of
postendocytic trafficking of preinternalized IgA in these cells. In
polarized MDCK cells, newly synthesized pIgR is delivered to the
basolateral surface, where it can bind IgA. The receptor is then
transcytosed across the cell and cleaved to release IgA bound to a
portion of the receptor into the apical medium. The remaining uncleaved
receptor can recycle at the apical surface. Sensitive assays are
available to measure the rate of IgA transcytosis and apical recycling,
and we previously demonstrated that M2 expression slows the rate of
both of these pathways in an AMT-inhibited manner (31).
This suggests that both transcytosis and apical recycling are sensitive
to altered organelle pH. We therefore compared the effects of M2 and
CFTR expression on IgA transcytosis (Fig.
3). Unlike M2 expression, which inhibited
the rate of transcytosis of IgA (Fig. 3A), CFTR expression
had no effect on the rate of basolateral-to-apical transcytosis of IgA
(Fig. 3B). In addition, because elevation of intracellular
cAMP levels has previously been reported to stimulate the rate of IgA
transcytosis across MDCK monolayers (29) and also
activates CFTR channel activity, we tested whether stimulation of cAMP
production by FSK would unmask an effect of CFTR in this assay (Fig.
3B). Although transcytosis was stimulated in all samples
treated with FSK, we did not detect any difference between control and
CFTR-expressing samples.

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Fig. 3.
M2 but not CFTR expression slows immunoglobulin A
(IgA) transcytosis across MDCK monolayers. Filter-grown MDCK T23 cells
were mock infected or infected with AV-M2 (A) or AV-M2901
(B), and cells were incubated overnight with 2 mM butyrate
to induce expression of polymeric immunoglobulin receptor. Cells were
incubated with basolaterally added 125I-labeled IgA for 10 min and then washed extensively. The rate of basolateral-to-apical
transcytosis of 125I-IgA was quantitated as described in
MATERIALS AND METHODS. Forskolin (FSK) was added to the
indicated samples at the beginning of the IgA uptake period and was
included during subsequent steps. Values are means ± SD for
triplicate samples. Similar results were obtained in at least 3 independent experiments for each condition.
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As an additional test to determine whether CFTR expression affects
apical postendocytic traffic in polarized MDCK T23 cells, we measured
the effects of CFTR and M2 expression on the apical recycling of
preinternalized IgA. As previously demonstrated, expression of M2 had a
modest but reproducible effect on the rate of apical recycling of IgA
(Fig. 4A). By contrast,
expression of CFTR in the presence (Fig. 4B) or absence (not
shown) of FSK stimulation had no effect on the rate of IgA recycling.

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Fig. 4.
M2 but not CFTR expression slows apical recycling of IgA
in polarized MDCK cells. Filter-grown MDCK T23 cells were mock infected
or infected with AV-M2 (A) or AV-M2901 (B), and
cells were induced with 2 mM butyrate. On the following day, cells were
incubated with apically added 125I-IgA for 10 min and then
washed extensively. The rate of 125I-IgA apical recycling
was quantitated as described in MATERIALS AND METHODS. FSK
was added to the samples shown in B at the beginning of the
IgA uptake period and was included during subsequent steps. Values are
means ± SD for triplicate samples. Similar results were obtained
in at least 3 independent experiments for each condition.
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Expression of functional CFTR in CFPAC cells.
MDCK II cells do not express endogenous CFTR, yet they are able to
acidify both apical and basolateral endocytic compartments to a similar
extent (Dunn K, personal communication). Together with the experiments
described above, these data suggest that exogenous expression of CFTR
does not affect pH regulation in these cells. However, it could be
argued that MDCK cells use an alternative mechanism to regulate
organelle pH but that CFTR fulfills this function in cells that
normally express the protein. Therefore, we tested the effect of CFTR
expression on protein traffic in the CF cell line CFPAC, derived from a
pancreatic adenocarcinoma from a CF patient (64). If the
acidification hypothesis is correct, organelles in CFPAC cells should
be relatively alkaline, and expression of functional CFTR in CFPAC
cells should stimulate both biosynthetic and postendocytic
transport. By contrast, expression of M2 or incubation with
global pH perturbants might be expected to have little or no effect on
traffic in these cells. In our hands, these cells do not form confluent
monolayers when grown on permeable filter supports; however, cells
grown on plastic form large islands of relatively flat cells connected
by tight junctions, suggesting that they are somewhat polarized (Fig.
5C). Infection of these cells
with AV-M2901 resulted in high levels of CFTR expression (Fig.
5B) and restored FSK-stimulated halide-transport in these cells as measured using the SPQ assay (Fig.
6). Infection of these cells with AV-M2
resulted in nearly undetectable fluorescence labeling (not shown),
perhaps because of the large size of these cells, but M2 could be
readily immunoprecipitated from AV-M2-infected CFPAC cells (Fig.
5D).

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Fig. 5.
Expression of CFTR in CFPAC cells (a cystic fibrosis pancreatic
adenocarcinoma cell line). CFPAC cells plated on glass coverslips were
mock infected (A) or infected with AV-M2901 (MOI 250;
B) and induced with 2 mM butyrate. On the following day,
cells were rapidly chilled and processed for live-cell staining with
the use of anti-FLAG antibody as described in MATERIALS AND
METHODS. C: AV-infected CFPAC cells were fixed and
processed for indirect immunofluorescence to localize the tight
junction marker ZO-1. A-C: scale bar, 10 µM.
D: CFPAC cells were mock infected or infected with AV-M2 at
an MOI of 250 or 2500. AV-transactivator (TA) was included at an MOI of
200. On the following day, cells were starved, radiolabeled for 3 h with 100 µCi/ml [35S]cysteine and
[35S]methionine, and then solubilized and M2
immunoprecipitated.
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Fig. 6.
CFTR expressed in CFPAC cells is functional. CFPAC cells
were mock infected (control) or infected with AV-M2901 (+CFTR) at an
MOI of 50. Cells were induced overnight with 2 mM butyrate and tested
for expression of functional CFTR using the
6-methoxy-N-(3-sulfopropyl)quinolinium assay as described in
MATERIALS AND METHODS. The arrows at 3, 7, and 11 min
denote the switch to nitrate buffer, the addition of FSK and
3-isobutyl-1-methylxanthine (IBMX), and the return to iodide buffer,
respectively.
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CFTR expression does not affect protein traffic in CFPAC cells.
To determine whether expression of M2 or CFTR affects biosynthetic
traffic in CFPAC cells, we quantitated the kinetics of HA delivery from
the TGN to the plasma membrane in AV-infected cells (Fig.
7A). Expression of active M2
decreased the rate of HA cell surface delivery, and the effect of M2
was completely reversed by AMT, suggesting that the effect of M2 was
due to altered TGN pH. Treatment with BafA1 also decreased
TGN-to-cell surface delivery kinetics of HA. By contrast, expression of
epitope-tagged CFTR had no effect on the rate of HA delivery from the
TGN to the cell surface. Interestingly, addition of FSK stimulated HA delivery kinetics in these cells, but the effect was independent of
CFTR expression (Fig. 7B).

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Fig. 7.
M2 but not CFTR alters HA delivery in CFPAC cells.
A: CFPAC cells were infected with AV-HA, AV-TA, and
AV-M2rev, AV-M2901, or AV-M2 and induced with 2 mM butyrate. On the
following day, cells were starved, radiolabeled for 15 min, and then
chased for 2 h at 19°C. The medium was replaced with prewarmed
chase medium, and the cells were incubated at 37°C for the indicated
times. Cells were rapidly chilled, treated with trypsin, and quenched
with soybean trypsin inhibitor. After solubilization and
immunoprecipitation of HA, the kinetics of trans-Golgi
network-to-cell surface delivery were analyzed by SDS-PAGE and
quantitated using a phosphorimager. AMT, amantadine. B: HA
delivery in CFPAC cells is stimulated by FSK. Cells were infected with
AV-M2rev or AV-M2901 and induced as described in A. On the
following day, cells were starved, radiolabeled for 15 min, and then
chased for 2 h at 19°C. FSK (10 µM) was added to the indicated
samples starting 10 min before the end of the 19°C incubation. The
medium was replaced with prewarmed chase medium (±FSK), the cells were
incubated at 37°C, and HA delivery to the cell surface was
quantitated as described in A. Similar results were obtained
in 3 experiments.
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Because M2 alkalinizes compartments by essentially increasing the
proton leak rate, it is possible that V-ATPase activity is actually
increased in organelles that express M2. If V-ATPase activity were
limited by lack of a counterion conductance in CFPAC cells, then CFTR
expression might help drop the pH of these compartments in cells
expressing M2. To test this possibility, we examined whether
coexpression of CFTR would reverse the effects of M2 on HA cell surface
delivery (Fig. 8). As we previously
observed, M2 expression inhibited the amount of HA delivered to the
plasma membrane during a 60-min chase period. Inhibition of M2 ion
channel activity using AMT or another selective inhibitor (BL-1743;
Ref. 72) restored normal delivery. However, expression of CFTR did not
rescue the effect of M2 on HA delivery. Together, these results suggest
that M2 expression and BafA1 treatment are able to
alkalinize the TGN and that CFTR expression does not substantially
alter TGN pH. Furthermore, cAMP-mediated stimulation of apical
biosynthetic delivery is CFTR independent.

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Fig. 8.
CFTR expression does not reverse the effect of M2 on HA
cell surface delivery in CFPAC cells. CFPAC cells were infected with
the indicated adenoviruses in addition to AV-TA. On the following day,
the HA was starved, radiolabeled for 15 min, chased for 2 h at
19°C, and then warmed to 37°C for 1 h, and the amount of HA at
the cell surface was quantitated as described in MATERIALS AND
METHODS. M2 ion channel inhibitors AMT and BL-1743 were included
where indicated. CFTR expression had no effect on the M2-mediated delay
in HA cell surface delivery, whereas inclusion of either AMT or BL-1743
blocked the effect of M2. Similar results were obtained in 4 experiments.
|
|
We then determined the effects of M2 and CFTR expression on
postendocytic traffic in CFPAC cells. Initially, we measured the effect
of M2 and CFTR expression on the exocytosis of preinternalized 125I-IgA. Because the CFPAC cells grow in polarized
islands, we have access to primarily the apical surface of these cells,
although some basolateral surface is also accessible. Therefore, the
IgA recycling assay actually reports a combination of recycling and transcytosis, which cannot be distinguished. Surprisingly, we found
that neither M2 nor epitope-tagged CFTR expression affected the rate of
exocytosis of preinternalized IgA in this assay (Fig. 9A). However, treatment with
the global pH perturbant chloroquine resulted in significant inhibition
of IgA exocytosis, suggesting a requirement for organelle acidification
in either transcytosis or recycling in these cells. Increasing the
level of M2 or CFTR expression by infection with 10-fold more virus
(MOI 2,500) did not affect the results (not shown). In addition,
inclusion of FSK did not stimulate exocytosis of preinternalized IgA in
CFPAC cells under any conditions (Fig. 9B). Finally, we
examined the effect of M2 or CFTR expression on the recycling of
preinternalized 125I-Tf in polarized CFPAC cells. Using
this assay, which is a very sensitive reporter for changes in endosomal
pH, we found no effect of M2 or CFTR, even when they were expressed at
a very high MOI (Fig. 10). Thus our
data suggest that CFTR does not regulate organelle pH or membrane
trafficking in polarized epithelial cells.

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Fig. 9.
CFTR expression does not affect apical recycling in
CFPAC. A: CFPAC cells were infected with AV-TA (MOI 100),
AV-pIgR (MOI 250), and AV-M2rev, AV-M2901, or AV-M2 (MOI 250 each) and
then induced with 2 mM butyrate. Indicated samples were incubated with
200 µg/ml chloroquine starting 2 h before the experiment.
Recycling of preinternalized 125I-IgA was quantitated the
following day as described in MATERIALS AND METHODS.
B: FSK does not affect IgA exocytosis in CFPAC cells. Cells
were infected as described in A. Where indicated, FSK (10 µM) was added during the last 10 min of radioligand uptake and in
subsequent steps. The rate of 125I-IgA recycling in
FSK-stimulated or nonstimulated cells was unaffected by either M2 or
M2901 expression. Values are means ± SD for triplicate samples.
Similar results were obtained in at least 4 experiments for each
condition.
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Fig. 10.
CFTR expression does not affect recycling of transferrin
(Tf) in CFPAC. CFPAC cells were infected with AV-TA (MOI 200)
and AV-M2rev, AV-M2, or AV-M2901 (MOI 2,500). FSK was added to the
indicated samples during the 125I-Tf uptake and in
subsequent steps. The rate of 125I-Tf recycling was
determined as described in MATERIALS AND METHODS. Values
are means ± SD for triplicate samples. Similar results were
obtained in 2 experiments.
|
|
 |
DISCUSSION |
Many organelles in the secretory pathway are acidified by
electrogenic proton-translocating ATPases. As these ATPases pump protons into the lumen of an organelle, they generate a membrane potential gradient that inhibits further acidification. In normal cells, this membrane potential is collapsed by a parallel chloride conductance, allowing maximal acidification (1, 3, 24). The acidification hypothesis suggests that CFTR may play this role in
the late Golgi and endosomal compartments of epithelial cells. The
original studies by Barasch et al. (2, 4) used the
membrane permeant weak base DAMP to estimate that the pH of the TGN and
endosomes in several CF cell lines (including immortalized respiratory
epithelia and the pancreatic adenocarcinoma line CFPAC-1) and primary
cultures (from nasal polyps) was elevated by ~0.2 pH units compared
with controls. Furthermore, they showed that a light vesicle fraction
isolated from CF cells was slow to acidify compared with that from
normal cells and that degradation of internalized endocytosed
2-macroglobulin was delayed significantly in CF compared with normal cells, although lysosomal pH was unaltered
(4). Together, these data suggested that CFTR plays a role
in regulating and maintaining the pH of the TGN and endosomes.
CFTR has been localized to endocytic compartments and found to recycle
from the plasma membrane in several cell types (6, 7, 44, 55,
76). Moreover, although CFTR has not been localized directly to
the Golgi complex, it is not unreasonable to suppose its presence there
at steady state. Many recycling proteins and mucins transiently pass
through the trans-Golgi or TGN as monitored by their
repeated exposure to glycosyltransferases (43, 69). In
addition, the half-life of mature CFTR is ~7-8 h
(75), which is relatively short for an apical membrane
protein (66). Therefore, there is likely to be a small
amount of newly synthesized CFTR traversing the secretory pathway at
any given time. Because CFTR is functional in the endoplasmic reticulum (53), the newly synthesized protein may be active as it
transits the Golgi. By contrast, the most common mutant of CFTR (CFTR
F508) is retained in the endoplasmic reticulum and does not reach
the plasma membrane (13, 19). Although a recent report
suggests that CFTR
F508 can be detected at the plasma membrane in
some tissues (41), we did not detect any cAMP-stimulated
halide efflux in mock-infected CFPAC cells by using a highly sensitive
assay. Moreover, these cells are thought to express little or no CFTR
F508 protein. Thus it is very unlikely that these cells express functional CFTR in endocytic or Golgi compartments.
The goal of our studies was to determine whether CFTR regulates
organelle pH in polarized epithelial cells. The acidification hypothesis predicts that the pH of the TGN and apical endosomes, two
compartments that should contain CFTR at steady state, is disrupted in
CF cells. To test this hypothesis, we developed a model system to
selectively disrupt the pH of these two compartments by expression of
influenza M2 protein and compared the effects of M2 and CFTR expression
in polarized cells. Although M2 activity disrupted protein export from
the TGN as well as the trafficking of transcytosing and recycling
proteins at the apical surface of polarized MDCK cells, expression of
CFTR had no effect on any of these pathways. Furthermore, expression of
functional CFTR in a polarized CF cell line did not affect these
trafficking steps. Finally, coexpression of CFTR was unable to reverse
the effects of M2 on trafficking. Together, our data suggest that
1) perturbation of TGN and endocytic pH disrupts protein
traffic, 2) TGN pH in CFPAC cells is normally acidic, and
3) CFTR does not regulate TGN or endosome pH in MDCK or
CFPAC cells.
Several studies by other groups have also failed to observe any effects
of CFTR on organelle pH or protein trafficking. For example, no
significant elevation in the pH of endosomes was observed when CFTR was
transfected into Chinese hamster ovary, 3T3, or L cells (7, 20,
44, 57, 58, 62). However, these cells do not normally express
functional CFTR and likely have other functional chloride conductances
that regulate organelle pH. Therefore, heterologous expression of CFTR
in these cells is unlikely to disrupt normal pH regulation.
Interestingly, one study found that CFTR expression in 3T3 cells
stimulated endosome fusion (6); this is an intriguing
observation because regulation of endosome fusion requires
acidification and appears to be regulated by membrane potential
(28). Thus it is possible that CFTR expression could alter
membrane potential in endosomes without affecting pH. Another study
found no effect of CFTR expression on the kinetics of transferrin recycling or on endosomal pH in isolated CFPAC cells (21).
However, because transferrin recycles almost exclusively from the
basolateral surface in most polarized epithelial cells, an effect of
CFTR on trafficking through an apical compartment might have been
missed in these studies. Similarly, because the CFPAC cells used in our studies also were not fully polarized, it is possible that they also
expressed a redundant counterion conductance that would be absent from
apical endocytic compartments in fully polarized cells. Therefore,
definitive resolution of the role of CFTR in regulating apical endosome
pH awaits the development of well-matched, -characterized, and
-polarized CF and rescued epithelial cells.
Recently, the regulation of membrane insertion into and retrieval from
the cell surface has been linked to the activation of CFTR (10,
38, 70). Bradbury et al. (10) compared endocytosis and exocytosis in CFPAC cells transfected with CFTR or with vector alone. Under basal conditions the extent of endocytosis of rhodamine dextran was identical in both cell types; however, FSK treatment inhibited endocytosis and stimulated exocytosis only in cells expressing CFTR. Similarly, recycling of preinternalized wheat germ
agglutinin was stimulated by FSK in CFTR-expressing cells. This
suggests that CFTR activation can modulate membrane recycling in some
cells. By contrast, in our experiments, we observed FSK-stimulated exocytosis of newly synthesized HA in CFPAC cells independently of CFTR
expression and saw no effect of FSK on the rate of exocytosis of IgA.
Although we do not understand the reason for this discrepancy, we can
envision several possibilities to explain this difference. First, the
corrected CFPACs used by Bradbury et al. (10) were a
subclone of the original CFPAC line and might have diverged from the
original clone; we have previously (40) demonstrated that
even clonally related cell lines can be inappropriate for studying the
role of CFTR in protein processing. Thus it is possible that the
rescued CFPAC subclone has distinct membrane trafficking properties
that are unrelated to expression of wild-type CFTR. Second, our assay
measured the effect of CFTR expression on the endocytosis of a specific
protein (IgA) that is known to be internalized via clathrin-mediated
endocytosis at the apical surface of polarized cells. CFTR has also
been demonstrated to be internalized via clathrin-coated vesicles
(8, 9). By contrast, the previous study followed the
internalization of the rhodamine-dextran and wheat germ agglutinin,
which are internalized via both clathrin-dependent and
clathrin-independent pathways. Clathrin-independent mediated endocytosis from the apical surface of other polarized epithelial cells
is stimulated by FSK (22); thus it is possible that CFTR expression indirectly stimulates the clathrin-independent pathway.
We were surprised to find that expression of M2 had no effect on IgA
traffic in CFPAC cells, even when it was expressed at 10-fold higher
levels than our usual condition. IgA recycling was slowed by cell
treatment with the global pH perturbant chloroquine, suggesting that
organelle acidification is important for apical postendocytic traffic
in these cells. One possibility is that M2 is not efficiently localized
to apical endosomes in these cells. M2 does not contain a functional
endocytosis signal (Henkel JR and Weisz OA, unpublished observations);
thus its endosomal localization depends on either bulk flow membrane
trafficking or transient passage of newly synthesized protein through
endosomes en route to the plasma membrane. Although the effects of M2
on apical recycling are maximal at the relatively low expression levels
used in this and previous studies (31), it is possible
that much higher expression levels are necessary to accumulate M2 in
endosomes in CFPAC cells. Alternatively, because the CFPAC cells do not
grow as fully polarized monolayers, our "recycling" assay reports a
combination of apical recycling, basolateral recycling, and
transcytosis. The total uptake and the amount of preinternalized IgA
released via these pathways will be different depending on whether a
cell is fully polarized or is at the edge of an island of cells. Thus,
if basolateral recycling contributes to a majority of the release, an
effect of M2 on apical recycling or transcytosis would be masked.
Although there is accumulating evidence against a role for CFTR
in the regulation of organelle pH and membrane trafficking, the
contribution of CFTR activity to protein processing and glycosylation in the TGN remains unclear (see Ref. 63 for review). Whereas we and
others have found no effect of CFTR expression on glycosylation (40, 48, 56), several studies have linked the CF phenotype to increased glycoconjugate sulfation (11, 12, 23, 35, 80). The data presented here suggest that this increase in
sulfation is due not to altered TGN pH but, rather, to other defects in CF cells. For example, CFTR has been reported to transport the sulfate
donor adenosine-3'-phosphate 5'-phosphosulfate (52), and
this could account for the observed defect in sulfation in CF cells.
Alternatively, other modifying genes may be responsible for the
sulfation defect (79). Thus future studies are needed to
address the mechanism by which CFTR may modulate glycoconjugate processing.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Xiaosui Jiang for performing preliminary experiments,
Dr. Robert Lamb, Northwestern University, Evanston, IL, for the gift of
monoclonal antibody 5C4, and Dr. Mark Krystal, Bristol-Myers Squibb
Pharmaceutical Research Institute, Wallingford, CT, for the gift of M2
ion channel inhibitor BL-1743.
 |
FOOTNOTES |
This work was supported by grants from the Cystic Fibrosis Foundation,
by National Institute of Diabetes and Digestive and Kidney Diseases
(NIDDK) Grant DK-54407 (to O. A. Weisz), and by Dialysis Clinic,
Inc. W. G. Hill was supported by NIDDK Grant DK-50829 (to R. A. Frizzell).
Address for reprint requests and other correspondence: O. A. Weisz, Renal-Electrolyte Division, Univ. of Pittsburgh, 3550 Terrace
St., Pittsburgh, PA 15261 (E-mail:
weisz{at}msx.dept-med.pitt.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 22 February 2000; accepted in final form 24 April 2000.
 |
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