1 Gregory Fleming James Cystic Fibrosis Research Center, Departments of 2 Physiology and Biophysics and 5 Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35294; 3 University Medical School of Pécs, Pécs H-7624, Hungary; and 4 Department of Pediatrics, Northwestern University Medical School, Evanston, Illinois 60201
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ABSTRACT |
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The F508 mutation leads to retention of cystic fibrosis
transmembrane conductance regulator (CFTR) in the endoplasmic reticulum and rapid degradation by the proteasome and other proteolytic systems.
In stably transfected LLC-PK1
(porcine kidney) epithelial cells,
F508 CFTR conforms to this
paradigm and is not present at the plasma membrane. When
LLC-PK1 cells or human nasal polyp cells derived from a
F508 homozygous patient are grown on plastic dishes and treated with an epithelial differentiating agent (DMSO, 2%
for 4 days) or when LLC-PK1 cells
are grown as polarized monolayers on permeable supports, plasma
membrane
F508 CFTR is significantly increased. Moreover, when
confluent LLC-PK1 cells expressing
F508 CFTR were treated with DMSO and mounted in an Ussing chamber, a
further increase in cAMP-activated short-circuit current (i.e., ~7
µA/cm2;
P < 0.00025 compared with untreated
controls) was observed. No plasma membrane CFTR was detected after DMSO
treatment in nonepithelial cells (mouse L cells) expressing
F508
CFTR. The experiments describe a way to augment
F508 CFTR maturation
in epithelial cells that appears to act through a novel mechanism and
allows insertion of functional
F508 CFTR in the plasma membranes of
transporting cell monolayers. The results raise the possibility that
increased epithelial differentiation might increase the delivery of
F508 CFTR from the endoplasmic reticulum to the Golgi, where the
F508 protein is shielded from degradative pathways such as the
proteasome and allowed to mature.
short-circuit current; dimethyl sulfoxide; cystic fibrosis transmembrane conductance regulator; cell differentiation
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INTRODUCTION |
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APPROXIMATELY 90% of cystic fibrosis (CF) patients
possess at least one cystic fibrosis transmembrane conductance
regulator (CFTR) allele that lacks a phenylalanine residue at CFTR
position 508 (F508). The findings of Cheng et al. (4) indicated that the
F508 mutation causes retention of CFTR in the endoplasmic reticulum (ER) and a failure to insert the protein in the plasma membrane. Butyrate, an epithelial differentiating agent, has been shown
to augment
F508 maturation in nonpolarized cells grown on plastic
(3). Another report suggested the possibility that butyrate increases
ion transport in CF nasal epithelia, although augmentation of
F508
maturation was not directly shown (36). Improvements in
F508 CFTR
due to butyrate have not been attributed to differentiating effects per
se but to increased CFTR mRNA and protein levels. The
F508 folding
defect is also temperature sensitive and influenced by high
concentrations of glycerol (7, 37). CFTR normally functions as a
Cl
channel in the context
of polarized epithelial monolayers. However, interventions that
activate maturation and Cl
secretion through
F508 CFTR in physiologically important systems such as polarized epithelial cell monolayers have been difficult to
establish.
An effort in many laboratories has been directed toward development of
channel openers for the F508 CFTR. This strategy depends on at least
some (e.g., small amounts of)
F508 protein in the plasma membrane
(19). Because studies of
F508 processing have focused on
undifferentiated cells, less information is available concerning CFTR
maturation in polarized epithelia. Previous observations in other
laboratories suggest that measurable amounts of
F508 protein might
be present in highly differentiated
F508 human nasal epithelial
cells (9), CF sweat ducts in situ (35), or in the nasal airways of
F508 CFTR mice (22). In the present experiments, we adapted
LLC-PK1 renal epithelial cells
transfected with CFTR cDNAs for measurement of transepithelial
Cl
transport. This cell
line readily forms polarized cell monolayers (30, 34, 39) and may have
advantages over primary CF airway epithelial cells (i.e., since lung or
nasal tissue from
F508 patients is limited or less frequently
available) for use in screening drugs for effects on CFTR. Although the
kidney-derived LLC-PK1 cell line
does not endogenously express wild-type or
F508 CFTR, the processing
of wild-type and mutant CFTR in this cell type has been carefully
studied and found to closely resemble the behavior of other epithelial
cells that do endogenously express CFTR (28). Moreover, kidney-derived
epithelia have been a useful model system for characterizing effects
due to epithelial differentiation. For example, Madin-Darby canine
kidney cells grown on filters have been used to study the distribution
of membrane proteins in polarized cells (1) and the mechanisms
underlying this distribution (12, 29).
LLC-PK1 cells have been used to
explore specific markers of differentiation (27), ion conductance and
membrane voltage (24), chemical induction of differentiation (26), and
the differentiation-dependent expression of the
Na+-glucose cotransporter (8). We
also studied the effect of DMSO on human nasal epithelial cells derived
from
F508 homozygous patients and on mouse L cells expressing
F508 or wild-type CFTR (46). In our studies, we found that growth of
epithelial cells under conditions designed to induce a more
differentiated phenotype (treatment with an epithelial differentiating
agent, DMSO, or growth of the cells as monolayers on permeable
supports) led to improvements in
F508 CFTR processing. Butyrate (5 mM for 24 h), glycerol (10% for 48 h), or growth at 27°C (for 48 h) had no detectable effect on
F508 maturation in these epithelial
cell monolayers.
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METHODS |
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Cell culture.
Parental LLC-PK1 cells were
cultured in M199 media supplemented with 5% fetal bovine serum (FBS).
LLC-PK1 cells with stable expression of the wild-type or F508 CFTR (generous gift of S. Cheng,
Genzyme; Ref. 28) were cultured in DMEM medium supplemented with 10%
FBS and 500 µg/ml G418 (Sigma). DMSO (Sigma) was diluted in medium.
Cells were treated with DMSO (2% vol/vol) for 4 days after confluency.
Cd2+ was added to induce CFTR
expression from the metallothionein promoter at 5 µM in experiments
and controls. Mouse fibroblast cells expressing
F508 or wild-type
CFTR (46) were cultured in DMEM medium supplemented with 10%
FBS.
Immunoprecipitation. LLC-PK1 cells grown on plastic were washed three times with PBS (pH 7.2, containing 1 mM MgCl2 and 0.1 mM CaCl2) on ice and then lysed in a buffer containing 150 mM NaCl, 20 mM HEPES, 1 mM EDTA, 1% NP-40, 100 µg/ml aprotinin, 100 µg/ml leupeptin, and 2 mM phenylmethylsulfonyl fluoride. Lysate supernatant (300 µg protein) was immunoprecipitated at 4°C for 2 h using an anti-CFTR COOH-terminal monoclonal antibody (Genzyme) and protein G agarose beads (Boehringer Mannheim). Immunoprecipitated proteins were phosphorylated in vitro as described previously (4, 33) and separated on a 7% polyacrylamide gel. Gels were placed on PhosphoScreen and analyzed with PhosphorImager (Molecular Dynamics). Images were further characterized with IPLab spectrum (Signal Analytics).
RT-PCR.
RT-PCR was carried out using Promega PolyATtract series 9600 mRNA
isolation and cDNA synthesis system, according to the manufacturer's protocol. Briefly, mRNA was extracted from control and DMSO-treated samples (1 × 105 cells) and
50% of each mRNA sample was used to synthesize cDNA. The remainder of
the sample was identically treated, but without RT, as a control to
exclude DNA contamination. Samples were homogenized and diluted in 40 µl of prewarmed (70°C) manufacturer's hybridization buffer, and
mRNA was purified on oligo(dT) matrix. After samples were washed,
10-µl samples were processed to cDNA by adding 5 µl of reverse
transcriptase. To ensure that PCR yield in these experiments was
roughly dependent on the amount of mRNA starting material, pBluescript
containing CFTR (gift of J. Rommens) was transcribed in vitro with T7
polymerase and mRNA added to cell lysates from parental
LLC-PK1 cells at known
concentrations. First-strand CFTR cDNA was then synthesized exactly as
above and studied in parallel with cDNA from cells expressing wild-type
or F508 CFTR. In all studies, cDNA synthesis was stopped after 30 min by incubating the samples at 95°C for 5 min. Control
experiments in which RT was omitted from the cDNA synthesis step led to
no detectable PCR product under any conditions. Quantitation was by
densitometry using IPLab spectrum (Signal Analytics).
CFTR detection by confocal immunofluorescence microscopy using
anti-first nucleotide binding domain (NBD1) (rabbit) polyclonal
antibody.
Coverslips were washed three times in PBS containing 1 mM
Ca2+ and 0.5 mM
Mg2+, fixed in 20°C
methanol for 20 min, and air dried at room temperature. The cells were
rehydrated in PBS for 5 min. Nonspecific protein binding sites were
blocked by incubating with 1% (wt /vol) porcine
-globulin in PBS
for 30 min at room temperature. Primary antibody [a polyclonal
(rabbit) antibody raised against the CFTR NBD1] (17, 33) was
applied and incubated at room temperature for 40 min. After subsequent
washing (5 × 3-min washes in PBS), the cells were
incubated with tetramethylrhodamine isothiocyanate (TRITC)-labeled
anti-rabbit IgG antibody for 40 min and then washed, mounted, and
visualized as described above. Controls in which the first antibody was
replaced by nonimmune rabbit serum were negative in all cases. Cells
were viewed with an Olympus IX70 inverted epifluorescence microscope at
623-nm light excitation using UPlanApo ×100 or UApo/340 ×40
objectives. Digital confocal images were captured using a Photometrics
SenSys digital camera and IPLab spectrum software with Power Microtome
(Signal Analytics).
Detection of tight junction formation by confocal
immunofluorescence microscopy using anti-zonula occludens-1 (rabbit)
polyclonal antibody.
Coverslips were washed three times in PBS supplemented with 1 mM
Ca2+ and 0.5 mM
Mg2+ and fixed in 4% formaldehyde
in PBS for 30 min at room temperature. Nonspecific protein binding
sites were blocked by incubating with 1% (wt /vol) porcine
-globulin (Sigma) in PBS for 30 min at room temperature. A rabbit
polyclonal antibody, anti-zonula occludens-1 (ZO-1) IgG (Zymed
Laboratories) (15), was applied and incubated at room temperature for
60 min. After subsequent washing (5 × 3 min washes in PBS), the
cells were incubated with TRITC-labeled anti-rabbit IgG antibody
(DAKOPATTS) for 40 min at room temperature, washed in PBS, and mounted
using Vectashield (Vector Labs) mounting medium. Controls in which the
first antibody was replaced by nonimmune rabbit serum were negative in
all cases. DMSO treatment (2%) was for 4 days.
Identification of F508 CFTR single-channel activity
in the plasma membranes of LLC-PK1 cells
following DMSO treatment.
DMSO-treated cells grown on plastic were studied in the cell-attached
configuration by the patch-clamp technique. The bath and pipette
solutions, electronics, pipette fabrication, and further details
regarding the patch-clamp technique have been described previously
(43). The pipette solution contained an impermeant cation (i.e.,
N-methyl-D-glutamine)
so that inward current could only be due to
Cl
flow into the pipette.
CFTR channels were activated after excision by 250 U/ml protein kinase
A (PKA) plus 200 µM ATP in the bath.
Transepithelial transport through F508 CFTR.
LLC-PK1 cell monolayers were grown
to confluency on permeable supports (Millipore, Anotec) for at least 5 days and then treated with 2% DMSO for 3-6 additional days.
Filters were mounted in an Ussing chamber, and short-circuit current
(Isc)
measurement was carried out as described in Ref. 42. Bumetanide was
added at 100 µM to both the mucosal and serosal surfaces. NaCl Ringer solution contained (in mM) 145 Na+, 5 K+, 124.8 Cl
, 1.2 Ca2+, 1.2 Mg2+, 25 HCO
3, 4.2 PO3
4, and 10 glucose (pH = 7.4). In 6 mM Cl
Ringer, 118.8 mM of
Cl
was replaced by the
impermeant anion gluconate.
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RESULTS |
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DMSO treatment of LLC-PK1 cells
expressing F508 CFTR.
DMSO, like butyrate, displays potent activity as an epithelial
differentiating agent (14, 23, 31, 40). Both drugs cause changes in the
display of plasma membrane glycoproteins in epithelial cells, although
the two compounds are believed to act by independent mechanisms (14,
23, 31, 40). Dose and time dependencies of DMSO treatment (0, 2, 5, and
10% DMSO for 1, 6, or 12 h and 1, 3, 4, and 5 days) were evaluated
systematically in LLC-PK1 cells
that express recombinant
F508 CFTR. We found that treatment for 4 days with 2% DMSO led to maximal effects on
F508 maturation and
transepithelial Cl
transport in these cells. This 2% DMSO schedule had no visible effect
on cell viability, and, in a quantitative assay of cell growth
(CellTiter 96, Promega), the well-known differentiating effects of DMSO
(which slow cell proliferation) decreased cell proliferation in the
LLC-PK1 cells by only ~15%. We
therefore settled on this schedule for future studies. We studied
LLC-PK1 cells expressing wild-type
or
F508 CFTR and the parental (non-CFTR expressing)
LLC-PK1 cell line (28).
DMSO treatment leads to a mature, band C form of
F508 CFTR.
Incomplete processing of CFTR can be identified by the absence of a
mature, fully glycosylated (band C) form of the protein (4).
Lower-molecular-weight forms of CFTR (e.g., band B) represent the
high-mannose, endoglycosidase H-sensitive forms of the
protein that are established cotranslationally in the ER. The
maturation of CFTR from core to complex glycosylation reflects the
transition of the protein from the ER to the Golgi, where attachment of
carbohydrates and trimming are accomplished as CFTR proceeds to the
trans-Golgi network. Immunoprecipitation of CFTR after a 4-day
pretreatment with DMSO is shown in Fig.
1. The result shows the
appearance of mature, fully glycosylated CFTR after DMSO treatment in
F508 cells. This result supports the notion that
F508 maturation
is augmented by DMSO. Figure 1 also indicates a small increase in total
CFTR protein in LLC-PK1 cells
after DMSO treatment. To determine whether this increase was due to
effects on steady-state levels of mRNA, we performed a semiquantitative
RT-PCR from total cellular mRNA. DMSO did not appear to cause increases
in CFTR mRNA levels as judged by this RT-PCR assay (Fig.
2). Unlike previous reports concerning
butyrate treatment (3), the experiment suggests that increased
F508
CFTR mRNA does not account for the modest increase in total
F508
protein after DMSO treatment or the increase in band C. These results
also suggest that DMSO promotes
F508 maturation through a mechanism
that is different from that observed with butyrate.
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F508 CFTR localization in the plasma membrane.
We used digital confocal fluorescent microscopy for CFTR localization
that was detected by a polyclonal (rabbit) antibody raised against CFTR
NBD1. The antibody used in these experiments has been validated
previously by its ability to immunoprecipitate either truncated CFTR or
full-length CFTR, immunolocalize CFTR to cells overexpressing the
protein, and correctly identify the presence or absence of CFTR in vivo
from salivary gland sections of CFTR wild-type or knockout mice of the
same strain (5, 16, 17, 33). In
LLC-PK1 cells expressing the
wild-type CFTR, the protein localized mainly to the perinuclear ER
compartment and to the region of the plasma membrane (Fig.
3A).
DMSO treatment only slightly increased the membrane staining of
wild-type CFTR. In contrast,
F508 CFTR was detected in the
perinuclear (ER) compartment in
LLC-PK1 cells with no significant
membrane staining. After DMSO treatment, staining in the region of the
cell membrane (similar to that observed in wild-type CFTR-expressing
cells) could be detected.
F508 CFTR localizes to the perinuclear ER
region of
F508 homozygous human nasal epithelial cells (Fig.
3B). Treatment of these cells with
DMSO (2% for 4 days) resulted in a more diffuse cytoplasmic staining
that included staining of cells in the plasma membranes. In contrast,
DMSO treatment had no significant effect on
F508 CFTR staining in
mouse fibroblast cells (mouse L cells, Fig.
3C). These studies suggest that the
specificity of effects of DMSO on
F508 CFTR processing occurs in
epithelial cells but not in fibroblast cells.
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Identification of F508 channels in the plasma
membranes of LLC-PK1 cells.
We also examined
F508 CFTR activity in the plasma membranes of
LLC-PK1 cells following DMSO
treatment (Fig. 4) using patch-clamp techniques. Cells grown on plastic were studied in the cell-attached and excised inside-out configurations. The pipette solution contained an impermeant cation (i.e.,
N-methyl-D-glucamine)
so that inward current could only be due to
Cl
flow. The holding
potential was approximately
60 mV. A current-voltage relationship and typical tracing are shown in Fig. 4, both consistent with the channel activity previously described for
F508 CFTR (6, 21,
32). The conductance of the channel is ~8-10 pS, and the channel
is activated after excision by 250 U/ml PKA plus 200 µM ATP in the
bath, as expected for a functional CFTR protein. We observed CFTR
channels in 2 of 27 seals in
F508
LLC-PK1 cells and in 12 of 48 seals in the same cells treated with DMSO. The wild-type CFTR was
observed in 8 of 25 seals. The open probability of the
F508 channels
was studied after stimulation by PKA and ATP. In the representative
tracing shown in Fig. 4, open probability before activation was 2%
(98% closed); open probability after activation was 97% (3% closed).
The wild-type CFTR also exhibited strong activation when cells attached
(open probability up to 98% following PKA stimulation).
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Transepithelial Cl transport
mediated by
F508 CFTR following DMSO treatment.
To extend our findings concerning
F508 CFTR in
LLC-PK1 cells grown in plastic
dishes, we also grew cells on permeable supports to form epithelial
monolayers. LLC-PK1 cells exhibit
a more differentiated phenotype under these conditions, as indicated by
tight junction formation, epithelial resistance measurements, and the
capability to perform vectoral ion transport (11, 30, 38, 39). Under these conditions,
F508 LLC-PK1
cells had low, but detectable, Cl
transport that was
absent in parental (non-CFTR expressing) cells (P < 0.05, Figs.
5 and 6).
Resistance in the cell monolayers was ~100
· cm2. A
3-day DMSO treatment of
F508 CFTR-expressing cells led to forskolin-activated currents in the direction of
Cl
secretion that were
greatly increased compared with untreated
F508
LLC-PK1 cells
(P < 0.00025). Transport was absent
in parental LLC-PK1 cells with or
without DMSO treatment, indicating specificity for
F508 CFTR
expression. When Cl
was
omitted from solutions bathing the monolayers,
Isc was not detectable either in wild-type or
F508 CFTR-expressing cells under
any treatment conditions, indicating that the
Isc observed in
the presence of Cl
was due
to vectoral Cl
transport.
Studies of wild-type CFTR-expressing cells are shown for comparison.
Modest increases in monolayer resistance (e.g., by 15-20%) were
noted in most paired experiments as a result of DMSO treatment, a
finding that suggests enhancement of tight junction formation in cells
grown on permeable supports in the presence of DMSO. A summary of these
studies is shown in Fig. 6.
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Isc studies of
alternative activators of F508 CFTR maturation in
LLC-PK1 cells.
We performed the same sorts of experiments shown above on
F508
LLC-PK1 cells following incubation
with 10% glycerol (for 48 h), 5 mM butyrate (for 24 h; longer
incubations led to substantial toxicity), or growth at 27°C (for 48 h). Each of these interventions has been shown to augment
F508
processing in certain cell types grown on plastic (3, 7, 37). We saw no
evidence with any of these approaches of either
Isc activation or
band C CFTR. These results are significant because they suggest that
DMSO acts to potentiate
F508 CFTR maturation through a mechanism
that 1) is different from that
observed with glycerol or butyrate,
2) is more potent than can be
obtained with glycerol or butyrate, at least in the
LLC-PK1 model, and
3) allows insertion of the
F508 CFTR in the plasma membrane of a transporting cellular monolayer in
vitro.
DMSO treatment leads to a more differentiated phenotype in recombinant LLC-PK1 cells. To determine whether DMSO altered the phenotype of LLC-PK1 cells, we probed cells grown on plastic with an antibody to ZO-1, a constituent of epithelial tight junctions (15, 41). Organization and assembly of tight junctions is an additional, useful measurement of polarity and differentiation in epithelial cell monolayers. Well-organized tight junctions occurring in an ordered distribution indicate a higher level of polarity and differentiation, whereas tight junction antigens that are absent, poorly developed, or distributed throughout the cytoplasm or plasma membrane may be indicative of a less differentiated phenotype. As shown in Fig. 7, a 4-day treatment with 2% DMSO led to qualitative increases in tight junction organization in LLC-PK1 cells, as detected by ZO-1 staining in the regions of cell-to-cell contact.
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DISCUSSION |
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In LLC-PK1 cells grown on plastic,
F508 CFTR is absent from the plasma membrane (Fig. 3) but can be
detected at the cell surface when cells are grown as a monolayer on a
permeable support (Fig. 5). The maturation of
F508 CFTR can be
augmented further by treatment with nontoxic concentrations of a
differentiation agent known to increase epithelial cell polarity, DMSO
(14, 23, 31, 40). The in vitro model described here for cell monolayers
(as opposed to previous studies of nonpolarized cells grown on plastic)
might be useful in the development of procedures to augment
F508
CFTR maturation or to study openers of
F508 CFTR
Cl
channels present in the
plasma membrane.
We tested the possibility that DMSO, like butyrate, increases F508
CFTR mRNA levels (Fig. 2). We saw no evidence of increased CFTR mRNA in
either wild-type or
F508
LLC-PK1 cells under the same DMSO
treatment conditions that partially corrected
F508 protein
processing. Moreover, butyrate treatment of
F508 cells at 5 mM
caused no improvement in Cl
transport in an Ussing chamber protocol. These results suggest that
DMSO augments the maturation of
F508 CFTR by a mechanism that is
different from the known effects of butyrate. DMSO has also recently
been suggested to act as a "chemical chaperone" and to correct
the folding and biogenesis of the pathogenic prion protein in vitro (W. J. Welch, personal communication). Although it is possible that DMSO
has effects on CFTR folding, there is no direct evidence in our
experiments that DMSO can reach cytoplasmic levels that alter the
kinetics, efficiency, or other aspects of CFTR folding in living cells
to augment
F508 CFTR processing. Therefore, although an effect on
CFTR folding remains an important consideration in our experiments,
further studies will be necessary to test a direct role for DMSO as a
chemical chaperone for CFTR.
In our experiments, interventions that increase epithelial cell
polarity and differentiation also conferred increases in F508 CFTR
maturation. Growth of epithelial cells on permeable supports is a
well-established method of augmenting epithelial cell differentiation and has been described previously as leading to dome formation and a
more differentiated phenotype in many cell types (13, 18, 25).
LLC-PK1 cells have previously been
reported to switch from an undifferentiated to a more differentiated
phenotype when grown on filters (48). This method was therefore
selected as one way to increase cell polarity in our experiments. We
showed that growth on permeable supports also led to functionally
detectable
F508 CFTR in the plasma membrane by
Isc measurements
(Fig. 6). DMSO treatment is another well-described method for
augmenting cell differentiation in epithelial cells derived from
different tissues (14, 23, 31, 40). Accordingly, we tested the
influence of DMSO on
F508 CFTR maturational processing. DMSO
treatment caused increased resistance in
LLC-PK1 monolayers (presumably on
the basis of greater organization of tight junctions), and the same
treatment led to further increases in
F508 CFTR activity at the cell
surface (Figs. 5 and 6). Moreover, we observed that organization of
tight junctions in LLC-PK1 cells
increased under the same conditions that led to
F508 maturation
(Figs. 1, 3, and 7). Finally, vectoral chloride transport (Fig. 5)
should only be detectable in a polarized epithelial monolayer and was
observed in our studies of anion transport. Together, these experiments indicate that a more differentiated phenotype also confers an increase
in
F508 CFTR maturation. The effect of DMSO on
F508 CFTR
processing was also observed in primary human nasal polyp cells but not
in nonepithelial cells (mouse fibroblast, L cells; Fig. 3). Experiments
in these other cell types suggest the usefulness of the approach for
activating
F508 processing in human epithelial cells.
Transmembrane proteins are often distributed throughout the plasma
membrane when cells are undifferentiated. With the onset of
differentiation and the development of adherens junctions, tight
junctions, and cell matrix interactions, structural polarity is
established. The process by which proteins target to the apical membrane appears to occur by a default mechanism (10). Our results may
indicate that cellular polarity helps establish part of this default
pathway. The possibility that F508 CFTR enters the apical targeting
pathway specifically after an increase in cellular differentiation is
suggested by results in Figs. 1, 3, and 5-7. Other contributors to
F508 maturation, including stabilization of the folded structure of
F508 protein, a change in
F508 CFTR interactions with cellular chaperones, or a decrease in the rate of degradation of
F508 CFTR in
the ER (so as to allow more time to reach the Golgi), are among the
alternative explanations for the findings shown here.
In summary, the ER in epithelial cells is a hostile environment,
insofar as the CFTR is concerned. Wild-type and F508 molecules, even
when folded into functional
Cl
channels, are
polyubiquitinated and degraded in short order (half time of ~30 min)
by the proteasome and possibly other proteolytic pathways (20, 44).
CFTR that escapes the ER and enters the Golgi may reach a "safe
haven," where half time increases to >12 h for the post-ER form.
Our data suggest, after DMSO treatment in
LLC-PK1 cells, an increased amount
of the post-ER complex-glycosylated and membrane-localized
F508 CFTR
is observed. Because neither block of ubiquitination nor
proteasome inhibition has been reported to enhance
F508 maturation,
it is less likely that the effects observed here are caused by blockade
of the proteasome or of ubiquitination. Our results raise the
possibility that induction of polarity in LLC-PK1 cells might increase a
transition of
F508 CFTR from the ER to the trans-Golgi
network (Figs. 1, 3 and 7). If the
F508 CFTR quality
control mechanism becomes "leaky" in differentiated cells
compared with the undifferentiated state, the findings could have
important implications concerning the in vivo processing of
F508
CFTR, which occurs in well-organized cells within tissues characterized
by tight junctions and polarity. Further experiments are needed to test
this possibility, as well as to test the effects of DMSO treatment in
F508 CF mice.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jeong Hong, V. K. Gadi, Jan Tidwell, Kynda Roberts and Bonnie Parrott for help with preparation of this manuscript, Eddie Walthall for technical assistance, and Drs. Vytas Bankaitis and Douglas Cyr for useful discussions.
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FOOTNOTES |
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This work was supported by the National Institutes of Health and the Cystic Fibrosis Foundation.
Address for reprint requests: E. J. Sorscher, Gregory Fleming James Cystic Fibrosis Research Center, Univ. of Alabama at Birmingham, 1918 Univ. Boulevard (BHSB 798), Birmingham, AL 35294-0005.
Received 25 April 1997; accepted in final form 27 April 1998.
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