Eudowood Division of Pediatric Respiratory Sciences, Department of Pediatrics, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287-2533
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ABSTRACT |
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The
F508 cystic fibrosis transmembrane conductance regulator (CFTR) is a
temperature-sensitive trafficking mutant that is detected as an
immature 160-kDa form (band B) in gel electrophoresis. The goal of this
study was to test the hypothesis that HSP70, a member of the 70-kDa
heat shock protein family, promotes
F508 CFTR processing to the
mature 180-kDa form (band C). Both pharmacological and genetic
techniques were used to induce HSP70. IB3-1 cells were treated with
sodium 4-phenylbutyrate (4PBA) to promote maturation of
F508 CFTR to
band C. A dose-dependent increase in band C and total cellular HSP70
was observed. Under these conditions, HSP70-CFTR complexes were
increased and 70-kDa heat shock cognate protein-CFTR complexes were
decreased. Increased
F508 CFTR maturation was also seen after
transfection with an HSP70 expression plasmid and exposure to
glutamine, an inducer of HSP70. With immunofluorescence techniques, the
increased appearance of CFTR band C correlated with CFTR distribution
beyond the perinuclear regions. These data suggest that induction of
HSP70 promotes
F508 CFTR maturation and trafficking.
cystic fibrosis; cystic fibrosis transmembrane conductance regulator; 70-kilodalton heat shock protein chaperones; phenylbutyrate; plasmid transfection; glutamine
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INTRODUCTION |
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CYSTIC
FIBROSIS (CF) is a systemic autosomal recessive disease that
results from the functional absence of a single membrane glycoprotein,
the CF transmembrane conductance regulator (CFTR) (48).
CFTR is a cAMP-dependent Cl channel present in both
surface and submucosal gland epithelia. Mutations in the CF gene result
in a polyexocrinopathy affecting several exocrine organs including the
airways, pancreas, sweat ducts, gastrointestinal tract, and
reproductive tract. With the exception of the sweat glands, the
recurring theme in all organs is the obstruction of passages by mucus,
which, in turn, leads to chronic airway infection, pancreatic
insufficiency, intestinal obstruction, and infertility.
The most common mutation of the CF gene (F508 CFTR), which leads to
the deletion of a single phenylalanine residue at position 508 on the
protein molecule (32), occurs in ~70% of CF chromosomes (61). The
F508 mutant protein is still functional when
induced in the oocyte expression system (16), a planar
lipid bilayer (33), or high-level expression systems that
allow a small fraction to reach the cell surface (10). The
major consequence of the
F508 mutation is failure of the mutant
protein to be correctly processed and delivered to the cell membrane
(11, 62). Treatment with chemical chaperones (5, 7,
54) or reduction in growth temperature to 27°C (15,
29) has been shown to overcome this trafficking defect and
result in processed and functional
F508 CFTR on the cell surface.
Small-chain fatty acid derivatives of butyric acid, including sodium
4-phenylbutyrate (4PBA), promote
F508 CFTR trafficking by mechanisms
distinct from those attributed to the chemical chaperones (40,
50, 51). We hypothesize that these compounds regulate the
expression of molecular chaperones that interact with newly synthesized
CFTR in the endoplasmic reticulum.
Molecular chaperones participate in the biogenesis of proteins,
including their synthesis, folding assembly, disassembly, and
translocation (30). Several cellular proteins, including members of the heat shock protein (HSP) family have been identified as
CFTR chaperones (14, 21, 35, 38, 44, 57). Although the
constitutively expressed 73-kDa HSP [70-kDa heat shock cognate protein
(HSC70)] is one of the more extensively studied CFTR chaperones, its
exact role has not been clearly defined. HSC70 promotes folding of the
NH2-terminal nucleotide-binding domain (NBD1) of both
F508 and wild-type CFTR (57) and in conjunction with
human DnaJ 2 (Hdj-2) transiently interacts with CFTR translation
intermediates to facilitate a putative complex formation between NBD1
and the regulatory domain of the CFTR channel (38). On the
other hand, HSC70 is required for the ubiquitin-dependent degradation
of a number of intracellular proteins (6). Because
F508
CFTR forms a prolonged association with HSC70, unlike wild-type CFTR,
this chaperone may also be a signal for mutant CFTR
ubiquitination and subsequent degradation by the proteasomal
system (31, 52, 65).
Whether or not HSC70 labels F508 CFTR for degradation, its reduction
by 4PBA may not be sufficient to promote further processing without
additional alternative chaperones to interact and keep CFTR on the
trafficking pathway. A previously described cellular response to HSC70
inhibition is the induction of the stress-inducible 72-kDa heat shock
protein, commonly known as HSP70 (2). HSC70 and HSP70 are
often considered indistinguishable in terms of their function and role
in cellular metabolism, with the exception that HSP70 is more efficient
than HSC70 and replaces its constitutive counterpart during times of
stress (1, 24, 58). We therefore specifically tested the
hypothesis that 4PBA induces HSP70 expression and promotes
HSP70-
F508 CFTR interaction. We also overexpressed HSP70 by plasmid
transfection and with an alternative pharmacological agent, leaving
HSC70 levels unchanged. Elevation of HSP70 and subsequent HSP70-CFTR
association promotes
F508 CFTR maturation.
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METHODS |
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Cell culture. IB3-1 cells (66) were grown on uncoated tissue culture plasticware or on uncoated glass coverslips for immunocytochemistry. The standard growth medium was LHC-8 (Biofluids, Rockville, MD) supplemented with 5% fetal bovine serum (Biofluids), 100 U/ml of penicillin-streptomycin (GIBCO BRL, Gaithersburg, MD), 0.2 mg/ml of Primaxin (imipenem, Merck, West Point, PA), 80 µg/ml of tobramycin (Eli Lilly, Indianapolis, IN), and 2.5 µg/ml of Fungizone (Biofluids). Cells for control experiments were cultured in 5% CO2 incubators in standard growth medium at 37 or 27°C as noted. The growth medium for the treated cells was composed of the indicated agent at the indicated concentration added to the routine growth medium when cells had reached 80% confluence.
Antibodies. Antisera 169 and 181 directed against human CFTR peptides in the regulatory domain and before NBD1, respectively, were generated in rabbits as previously described by our laboratory (13). Antiserum 169 was used for immunoprecipitation, and antiserum 181 was used at a 1:1,000 dilution for immunoblot detection. A rat monoclonal antibody specific for HSC70 (clone 1B5) and a mouse monoclonal antibody specific for HSP70 were purchased from StressGen Biotechnologies (Victoria, BC) (8). Donkey anti-rabbit IgG-horseradish peroxidase conjugate and sheep anti-mouse IgG-horseradish peroxidase conjugate were purchased from Amersham (Arlington Heights, IL). Goat anti-rat IgG-horseradish peroxidase conjugate was obtained from Boehringer Mannheim (Indianapolis, IN). For immunocytochemistry, sheep anti-rabbit Cy3 conjugate and goat anti-rat FITC conjugate were purchased from Sigma (St. Louis, MO). Goat anti-mouse Cy5 conjugate was obtained from Jackson Immunoresearch Laboratories (West Grove, PA).
Protein lysates.
The cells were washed twice in PBS and lysed with
radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris-Cl (pH 8.0),
150 mM NaCl, 1% Triton X-100 (Bio-Rad, Hercules, CA), and 1% sodium deoxycholate (Sigma)] containing freshly added inhibitors (2 mM phenylmethylsulfonyl fluoride, 0.2 trypsin inhibitor units/ml of aprotinin, and 10 µM sodium orthovanadate) at 4°C. The lysates were passed through a 26-gauge needle 10 times to shear DNA, and additional 2 mM phenylmethylsulfonyl fluoride was added to each sample.
The samples were incubated on ice for 30 min and then centrifuged at
15,000 g for 20 min to obtain the supernatant. The protein
concentration of the supernatant was determined with Bio-Rad DC assay
reagents with bovine plasma -globulin as a standard (Bio-Rad).
Immunoblot analysis. Equal amounts of protein were resolved on 9% SDS-polyacrylamide gels. The proteins were transferred to nitrocellulose, and immunodetection was performed as previously described (37). Nonspecific binding was blocked by incubation of the nitrocellulose with 5% nonfat dry milk in 1% BSA (HSC70 and CFTR) or 5% nonfat dry milk in PBS-0.05% Tween 20 (HSP70). Primary antisera and secondary antibodies were applied in blocking buffer overnight at 4°C and for 1 h at room temperature, respectively. Detection of immunoreactivity was performed with enhanced chemiluminescence reagent (Amersham) and fluorography.
Recombinant human HSP70 (~90% purity) was purchased from StressGen Biotechnologies for use in constructing a standard curve of HSP70 immunoreactivity. Immunoblots containing the recombinant HSP70 were probed with the mouse monoclonal HSP70-specific antibody.Immunoprecipitation. CFTR antiserum 169 (10 µl) or mouse monoclonal anti-HSP70 antibody (10 µg) was added to the cell lysates (800 µg of total protein, with equal amounts of protein at equal final concentrations for each condition within an experiment) and incubated for 1 h at 4°C. Protein A Sepharose 4B (Pharmacia Biotechnologies, Piscataway, NJ) preabsorbed with 3% BSA for 1 h at 4°C was then added, and each sample was incubated at 4°C overnight with gentle agitation to capture the immune complexes. Precipitated complexes were collected by centrifugation at 2,500 g for 5 min and washed four times with PBS at 4°C to remove nonspecifically adsorbed proteins. Bound antigen was eluted from the beads by incubation in SDS sample buffer for 15 min at 70°C and resolved on 9% SDS-polyacrylamide gels. Immunodetection of immunoprecipitated HSC70, HSP70, or CFTR was performed as described in Immunoblot analysis.
Immunocytochemistry.
Cells grown on uncoated glass coverslips under the indicated conditions
were washed twice in cold PBS and permeabilized by immersion in 100%
methanol at 70°C. Nonantigenic sites were blocked by immersion in
cold 0.2% BSA-PBS for 10 min. The cells were then incubated with
anti-CFTR antibody 181 (1:100), anti-HSC70 antibody (1:100), and
anti-HSP70 antibody (1:100) for 1 h at room temperature. After a
wash with cold PBS to remove excess primary antibody, fluorochrome-conjugated antisera (1:500) were added for 30 min. The
cells were washed again and overlaid with glycerol-PBS Slowfade antifade reagent (Molecular Probes, Eugene, OR) before microscopic examination. Fluorescent images were visualized with a Zeiss Axiovert microscope (Thornwood, NY) and captured with a high-performance digital
charge-coupled device camera. Images were then analyzed with IP Lab
Spectrum software (Scanalytics, Fairfax, VA).
Transfection and overexpression of HSP70. An expression plasmid (pcDNA3-HSP70) containing human HSP70 cDNA (64) under control of the cytomegalovirus promoter was a generous gift from Dr. Hector R. Wong (Children's Hospital Medical Center, Cincinnati, OH). IB3-1 cells were grown to 80% confluence in a T75 tissue culture flask. For each transfection, 5 µg DNA/250 µl OptiMem reduced-serum medium (GIBCO BRL) and 40 µl LipofectAMINE reagent (GIBCO BRL)/250 µl OptiMem were combined and incubated for 40 min at room temperature to allow DNA-liposome complexes to form. The standard growth medium was removed, and the cells were washed with OptiMem before the addition of complex solution. An additional 500 µl of OptiMem were added to each flask to ensure coverage of the entire surface. The cells were incubated with diluted complexes for 4 h at 37°C in a 5% CO2 incubator. The transfection mixture was removed and replaced with standard medium, which was changed every 24 h until the cells were lysed. A control population of IB3-1 cells was transfected with the pcDNA3 plasmid (Invitrogen, San Diego, CA) lacking HSP70 cDNA under similar conditions.
Densitometric analysis. Fluorographic images were digitalized into eight-bit tagged image format files with a Scan Jet Plus (Hewlett-Packard). Baseline density of the area surrounding the bands was determined by two-dimensional integration with Quantity One image analysis software (Bio-Rad) and subtracted as background. Densitometric volume analysis of selected bands was similarly performed. For comparison within an experiment, the density of the 37°C control lane, the 10-ng lane for bovine HSP70 standard curve experiments, and the 25-µg lane for CFTR standard curve experiments was arbitrarily set to 1.0. Differences between the mean densities of the fluorographic bands were compared by one-way ANOVA, followed by least significant difference multiple comparisons test as appropriate (SPSS Software version 7.0).
Reagents. Pharmaceutical grade 4PBA was a generous gift from Medicis Pharmaceutical (Scottsdale, AZ). Glutamine was purchased from GIBCO BRL, and nitrocellulose was from Schleicher and Schuell (Keene, NH). Electrophoresis-grade chemicals were purchased from Fisher, Bio-Rad, or GIBCO BRL. All other reagents were of reagent grade or better.
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RESULTS |
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4PBA treatment of IB3-1 cells increases expression of HSP70 and
promotes HSP70-F508 CFTR interaction at the expense of HSC70-
F508
CFTR complex formation.
We studied the immortalized CF bronchial epithelial cell line IB3-1
(66). IB3-1 has the CFTR genotype
F508/W1282X but
expresses only
F508 CFTR because the W1282X mutation produces an
unstable, and therefore untranslated, mRNA (27, 28). Our
laboratory (50) previously demonstrated that treatment of
IB3-1 cells with 4PBA resulted in restoration of appropriate
intracellular trafficking of
F508 CFTR and CFTR-like
Cl
channels on the cell surface. In Fig.
1, IB3-1 cells were exposed to
concentrations of 4PBA from 10 µM to 5 mM at 37°C for 48 h and
lysed, and the proteins were resolved by SDS-PAGE. Immunoblots with
anti-CFTR antibody 181 were analyzed by densitometry. The relative
amounts of CFTR band C and band B are shown for each concentration
(Fig. 1, A and B).
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Increased HSP70 expression leads to higher molecular mass forms of
F508 CFTR.
Because 4PBA may have multiple effects on cellular metabolism, we
sought to determine the effect of isolated HSP70 overexpression on
F508 CFTR production. IB3-1 cells were transfected with either pcDNA3-HSP70 (64) or mock transfected with empty pcDNA
plasmid. The cells were grown in standard medium for up to 6 days after transfection at 37°C. Immunoblotting of protein lysates taken at
different time intervals demonstrated maximal expression of HSP70 (~3
times) at 2 days, with a decline at 6 days, which was still greater
than that in mock-transfected and wild-type IB3-1 cells (Fig.
3). With a HSC70-specific rat monoclonal
antibody, we detected no significant change in total HSC70
immunoreactivity in whole cell lysates after transfection as expected
(Fig. 3A). In terms of
F508-CFTR processing, there was a
gradual increase in the amount of the higher molecular mass form, band
C, from posttransfection day 2 to day 6, in the
cells transfected with the HSP70 expression plasmid (Fig. 3).
Mock-transfected cells failed to demonstrate a significant change in
their CFTR profile up to 6 days after transfection. These data
demonstrate that increased expression of HSP70 alone can maintain
F508 CFTR on its biogenic pathway, allowing the mutant protein to
exit the endoplasmic reticulum and traverse the Golgi apparatus where
it acquires its matured glycosylation pattern. Using our previously
constructed standard curve for CFTR band C immunoreactivity, we
estimated that pcDNA3-HSP70 transfection increases
F508 CFTR
maturation over twofold, a degree similar to the growth of cells at
27°C.
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Glutamine induces production of HSP70 and promotes trafficking of
the F508 CFTR mutant.
Glutamine (2-20 mM) has previously been shown to induce HSP70 in
an intestinal epithelial cell line (IEC-18), and to confer protection
against lethal heat (49°C) and oxidant injury (41, 53,
63). We therefore tested whether glutamine would be capable of
inducing HSP70 in cells of respiratory origin as well as the effect
glutamine may have on
F508 CFTR trafficking. As before, IB3-1 cells
were grown to 70-80% confluence in standard medium (2 mM
glutamine) before the addition of extra glutamine from 1 to 100 mM. The
cells were incubated in additional glutamine for 48-72 h in a 5%
CO2 incubator at 37°C. As represented in Fig. 4, glutamine supplementation resulted in
a dose-dependent induction of HSP70. Furthermore, although we
occasionally observed a small amount of CFTR band C even in untreated
IB3-1 cells grown at 37°C (Fig. 4A), there was still a
significant increase in the relative amount of band C after glutamine
supplementation. The greatest increase in CFTR band C occurred with 50 mM glutamine (~2.5 times), although significant changes were seen
with as low as 5 mM supplementation (~1.5 times; Fig. 4). No
appreciable difference was seen in HSC70 expression after glutamine
treatment (Fig. 4A).
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Effect of HSP70 induction on F508 CFTR localization.
To provide additional support for the observation that 4PBA and
glutamine alter the expression and localization of
F508 CFTR, we
performed immunofluorescence microscopy studies. The vast majority of
untreated IB3-1 cells expressed low levels of
F508 CFTR located predominantly in the perinuclear region (Fig.
5A) (25, 65). Growth at lowered temperature, treatment with 4PBA, or glutamine increased the intensity of the CFTR staining and also produced an
expanded distribution beyond the endoplasmic reticulum region throughout the cytoplasm and periphery of the cells (Fig. 5,
B-D). Consistent with our Western blotting
analyses, immunofluorescence microscopy demonstrated an increase in
HSP70 signal after treatment with 4PBA or glutamine. Under these
experimental conditions, we detected no qualitatively apparent change
in HSC70. We also performed immunofluorescence studies on IB3-1 cells
after plasmid transfer. There was no significant change in CFTR or
HSC70 after mock transfection (Fig. 5E); however, there was
an increased HSP70 signal primarily from the nucleus, suggesting a
stress response (8, 42). Immunocytochemistry confirmed an
increase in HSP70 after pcDNA3-HSP70 transfection. There was also a
progressive increase in CFTR signal intensity, with a greater
peripheral distribution from day 2 to day 6 posttransfection that was consistent with our immunoblot results (Fig.
5, F and G). These data establish that after 4PBA
treatment, pcDNA3-HSP70 transfection, or glutamine supplementation, one
common denominator in restoring
F508 CFTR intracellular trafficking
is increased expression of HSP70.
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DISCUSSION |
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The present studies demonstrate, for the first time, that the
specific induction of HSP70 production by plasmid transfection or
pharmacological techniques is capable of correcting the F508 CFTR
trafficking defect in respiratory epithelial cells. This conclusion is
supported by the finding of increased HSP70-
F508 CFTR complex
formation after 4PBA treatment as well as at lowered growth
temperature, conditions that facilitate
F508 CFTR trafficking (15, 50, 51). Although we have shown that 4PBA can
downregulate HSC70 under certain conditions and reduce HSC70-
F508
CFTR association (52), in this study, we were unable to
detect any significant change in the overall levels of HSC70 protein
after glutamine supplementation or after HSP70 gene transfer. Thus even
if prolonged interaction with HSC70 eventually labels misfolded mutant
CFTR for degradation, the induction of HSP70 without specific
inhibition of HSC70 appears to promote
F508 CFTR trafficking.
The most compelling evidence that HSP70 induction is sufficient to
restore F508 CFTR processing comes from our pcDNA3-HSP70 transfection data. This approach isolated the effect of HSP70 from the
myriad of other potential effects of 4PBA and glutamine. Specifically,
we did not observe a significant change in HSC70 expression under these
conditions. After mock transfection, we did observe a smaller increase
in HSP70, which likely reflects a cellular stress response to gene
transfer (8). This increase in HSP70 was not accompanied
by an increase in mature
F508 CFTR, suggesting that a critical
amount of HSP70 is required. An alternative explanation is that the
HSP70 induced by transfection stress is located predominantly in the
nucleus (8, 42) and not around the endoplasmic reticulum
where it is in close proximity to CFTR translation intermediates.
The HSP70 class of molecular chaperones is thought to interact with
early-stage translational intermediates of CFTR in the endoplasmic
reticulum (57). Although others may exist, at least one
binding site for HSC70 has been identified within NBD1. This region,
which corresponds to the residues Gly545 to
Ala561, includes several CF-associated mutations that
affect both folding (46) and maturation (26,
60). HSC70 has been shown to interact with both immature
wild-type and F508 CFTR in vivo; however, the mutant protein has an
extended interaction with HSC70 and fails to acquire a mature
glycosylation pattern (65). Under the model proposed by Qu
and Thomas (47),
F508 is a kinetic folding
mutation that results in a significant energy barrier between the
native state and the step affected by the mutation, precluding the use
of the native state chaperones, including HSC70, to promote folding.
Additional evidence for this model comes from experiments that
demonstrate that wild-type CFTR, but not
F508 CFTR, is capable of
attaining a protease-resistant configuration while in the endoplasmic
reticulum (36). Metabolic energy is required for the
conformational transition but not to maintain the stability of the
protease-resistant form that is now capable of exiting the endoplasmic reticulum.
Although encoded by different genes, both HSC70 and HSP70 exhibit an extremely high degree of sequence relatedness. At the nucleotide sequence level, the Hsc70 and Hsp70 genes display a homology of 74%, whereas the homology increases to 81% at the predicted amino acid sequence level, with higher divergence at the carboxy-terminal regions of the proteins (17). A number of studies, however, support the idea that they are truly distinct proteins with respect to cell physiology. One major difference and potential advantage is that the HSP70 gene lacks introns. By overcoming the requirement for mRNA processing, high levels of HSP70 can be more rapidly achieved in times of cellular stress. Other unique attributes of HSP70 include the ability to confer "thermotolerance" or protection to a subsequent stress after initial exposure to mild or sublethal stress (34, 39) and the ability to reduce cell growth rate (19). In terms of chaperone function, HSC70 and HSP70 have been thought of more interchangeably (8, 20, 58). The majority of previous articles pertaining to CFTR has not distinguished between these two cellular chaperones (31, 65). However, our observation of increased HSP70 expression after a reduction in HSC70 is not unique. Aquino et al. (2) noted a sharp induction of HSP70 in oligodendrocyte precursor cells after transfection with an antisense oligonucleotide specific for HSC70. The induced HSP70 provided replacement chaperone function and restored synthesis of myelin basic protein.
In terms of CFTR synthesis, our laboratory (52) previously failed to demonstrate a significant change in HSP70 expression after 4PBA treatment. Since that time, we have modified our immunoblotting technique, replacing the standard nonfat dry milk blocking solution with a PBS-0.05% Tween-based solution recommended by the manufacturer. This change, we believe, has led to a dramatic decrease in nonspecific background staining and increased sensitivity to detect changes in HSP70 protein expression.
Strickland et al. (57) demonstrated that in an in vitro
folding system, HSC70 interacts with the NBD1 of both wild-type and
F508 CFTR to increase the folding yield of this domain. However, the
addition of ATP to this system, which mimics the intracellular environment, negated the folding properties of HSC70. Because ATP
alters the rate of substrate binding and release, a critical determinant of the folding efficiency of a chaperone appears to be its
kinetic properties. Therefore, one possible explanation for our current
observations is that although HSC70 and HSP70 may both promote CFTR
folding, HSP70 is kinetically more favorable and is subsequently able
to promote folding of even the nascent
F508 CFTR polypeptide. In
this scenario, the increased HSP70-
F508 CFTR interaction seen after
HSP70 induction would represent a transient but determinant step in
further posttranslational processing and not merely HSC70 substitution
and continued endoplasmic reticulum retention. If, in reality,
prolonged interaction with HSC70 targets
F508 CFTR for
ubiquitin-dependent degradation, an alternative explanation for our
observations might be that HSP70 replaces HSC70 in complexes with CFTR.
Along similar lines, because HSC70 has been shown to form stable
heterocomplexes with newly translated HSP70 (8) after the
induction or overexpression of HSP70, there may be competition for
HSC70 interaction between
F508 CFTR and HSP70, resulting in fewer
HSC70-
F508 CFTR complexes susceptible to ubiquitin-dependent
degradation. Support for this mechanism comes from our observation of
an HSC70-HSP70 association free of CFTR after 4PBA treatment. Although
the HSC70-HSP70 heterocomplex is necessary for nuclear translocation of
HSP70 (8), its significance in CFTR trafficking is unclear.
Additional evidence for HSP70 being involved in restoring F508 CFTR
biosynthesis comes from observations of other pharmaceutical agents and
from another disease caused by a trafficking defect. The
original member of the butyrate class of drugs shown to increase surface expression of
F508 CFTR was sodium butyrate. This drug stimulates CFTR gene transcription (29) but is also likely
to produce its effect on
F508 CFTR processing by altering the
intracellular folding environment. Sodium butyrate has been studied
extensively in the cancer field and has been shown to induce HSP70 in a
variety of leukocyte cell lines (22, 23).
8-Cyclopentyl-1,3-dipropylxanthine (CPX), an adenosine receptor
antagonist that binds directly to NBD1 and activates CFTR Cl transport (4), has also been observed to
promote trafficking of the
F508 CFTR protein (45).
Using cDNA microarrays to study global gene expression, Srivastava et
al. (56) demonstrated differences in non-CFTR gene
expression patterns between cells expressing wild-type and
F508
CFTR. Treatment of
F508 CFTR cells with CPX induced substantial
changes in gene expression, some of which reversed the mutant gene
expression profile toward that of wild-type cells. Interestingly, of
the genes previously implicated in CFTR trafficking, the most profound
effect of CPX treatment was upregulation of HSP70.
Finally, the most common disease causing mutation of
1-antitrypsin, PiZ, produces a trafficking mutant that
shares some common features with
F508 CFTR. Recently, Novoradaskaya
et al. (43) demonstrated a dose-dependent increase in the
Z-mutant
1-antitrypsin secretion after treatment with
4PBA in alveolar macrophages from PiZ individuals and in a Chinese
hamster ovary cell line model expressing Z
1-antitrypsin. Concurrent with the restoration of Z
1-antitrypsin trafficking, there were increases in the
chaperones HSP90 and HSP70. HSP90 may also be required for CFTR
trafficking because its inhibition by geldanamycin leads to more rapid
degradation of
F508 CFTR (35). However, 4PBA treatment
has been previously shown not to alter steady-state levels of HSP90
expression in IB3-1 cells (52). In addition to
demonstrating an increased secretion of
1-antitrypsin in
different cell types after 4PBA treatment, Burrows et al.
(9) observed an increase in blood levels of
1-antitrypsin in PiZ mice gavage fed 4PBA. Although there has yet to be established a causal effect, these observations are
consistent with the hypothesis that induction of HSP70 and its
interaction with translational intermediates are capable of overcoming
the barriers imposed by kinetic folding mutations.
Although, to our knowledge, this is the first study to describe a
potential therapeutic role for glutamine in both CF and respiratory
epithelia, the role of this nonessential amino acid has been studied
extensively in the gastrointestinal system. Glutamine is the primary
metabolic fuel for enterocytes under normal circumstances (49,
55), but after a variety of injuries, glutamine supplementation has been shown to improve gut morphology and survival (12, 18, 41, 59). The protective effect of glutamine in the gut has been
attributed in part to its induction of HSP70 because treatment with the
nonmetabolizable glutamine analog
6-diazo-5-oxo-L-norleucine also confers protection from
heat and oxidant stress (63). Maximal induction of HSP70
was seen at 10 mM glutamine, but an increase could be seen with
concentrations as low as 2 mM. Using a cDNA probe, Wischmeyer et al.
(63) demonstrated a dose-dependent induction of HSP70 at
the mRNA level. In our experiments, the higher concentrations of
glutamine (50-100 mM) may also exert an osmotic effect on the cell
and promote
F508 CFTR folding through a mechanism similar to the
endogenous intracellular solutes myo-inositol, betaine, and
taurine (7). However, this potential mechanism is not
likely to account for the increase in mature
F508 CFTR observed at
lower glutamine concentrations.
The observations in this report suggest that HSP70 induction overcomes
the F508 CFTR folding defect and results in the surface expression
of mature glycosylated CFTR. Although we did not test whether there was
a corresponding increase in cAMP-stimulated Cl
secretion,
Rubenstein et al. (50) have previously shown that 4PBA increases detection of CFTR band C on an immunoblot and increases CFTR channels on the plasma membrane. We also limited our observations of the effect of HSP70 induction to the
F508 CFTR mutant. Whether this approach would also overcome the defects caused by other trafficking mutants such as N1303K, P574H, or G480C remains to be tested.
In conclusion, we demonstrate increased mature F508 CFTR expression
after induction of HSP70 by gene transfer and pharmaceutical manipulation. These data suggest that HSP70 provides a chaperone function that is not sufficient in the native cellular state but if
augmented, is capable of overcoming the folding block imposed by the
F508 mutation. Screening for compounds that upregulate HSP70 may
accelerate the identification of molecules that might be beneficial in
the therapy for CF.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge Dr. William G. Kearns (Children's Hospital of the King's Daughters, Norfolk, VA) for assistance with immunofluorescence microscopy. We thank Dr. Hector R. Wong (Children's Hospital Medical Center, Cincinnati, OH) for the pcDNA-70-kDa heat shock protein plasmid and Medicis Pharmaceutical for providing sodium 4-phenylbutyrate.
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FOOTNOTES |
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This work was supported by a Cystic Fibrosis Foundation Clinical Fellowship Training Grant (to L. R. Choo-Kang) and National Heart, Lung, and Blood Institute Grant P01-HL-51811 (to P. L. Zeitlin). Microscopy was performed at the Imaging Core Facility (supported by a Cystic Fibrosis Research Development Grant) in Dr. William B. Guggino's laboratory (Johns Hopkins School of Medicine, Baltimore, MD).
A licensing agreement exists between the Johns Hopkins University, Ucyclyd Pharmaceuticals (Phoenix, AZ), and P. L. Zeitlin. The terms of this arrangement are being managed by the University in accordance with its conflict of interest policies.
Original submission in response to a special call for papers on "CFTR Trafficking and Signaling in Respiratory Epithelium."
Address for reprint requests and other correspondence: P. L. Zeitlin, Eudowood Division of Pediatric Respiratory Sciences, Johns Hopkins Medical Institutions, Park 316, 600 North Wolfe St., Baltimore, MD 21287-2533 (E-mail: pzeitli{at}jhmi.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 2 October 2000; accepted in final form 13 February 2001.
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