1 Division of Pulmonary Medicine, Children's Hospital of Philadelphia and Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104; and 2 Eudowood Division of Pediatric Respiratory Sciences, Johns Hopkins Medical Institutes, Baltimore, Maryland 21287
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ABSTRACT |
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The most common mutation of
the cystic fibrosis transmembrane conductance regulator
(CFTR), F508, is a trafficking mutant that has prolonged
associations with molecular chaperones and is rapidly degraded, at
least in part by the ubiquitin-proteasome system. Sodium
4-phenylbutyrate (4PBA) improves
F508-CFTR trafficking and function
in vitro in cystic fibrosis epithelial cells and in vivo. To further
understand the mechanism of action of 4PBA, we tested the hypothesis
that 4PBA modulates the targeting of
F508-CFTR for ubiquitination
and degradation by reducing the expression of Hsc70 in cystic fibrosis
epithelial cells. IB3-1 cells (genotype
F508/W1282X) that were
treated with 0.05-5 mM 4PBA for 2 days in culture demonstrated a
dose-dependent reduction in Hsc70 protein immunoreactivity and mRNA
levels. Immunoprecipitation with Hsc70-specific antiserum demonstrated
that Hsc70 and CFTR associated under control conditions and that
treatment with 4PBA reduced these complexes. Levels of immunoreactive
Hsp40, Hdj2, Hsp70, Hsp90, and calnexin were unaffected by 4PBA
treatment. These data suggest that 4PBA may improve
F508-CFTR
trafficking by allowing a greater proportion of mutant CFTR to escape
association with Hsc70.
cystic fibrosis; cystic fibrosis transmembrane conductance regulator; chaperones; Hsc70; phenylbutyrate
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INTRODUCTION |
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THE MOST COMMON MUTATION OF the cystic fibrosis
transmembrane conductance regulator (CFTR), F508-CFTR (deletion of
phenylalanine at position 508 of CFTR), is a temperature-sensitive
trafficking mutant (15).
F508-CFTR is retained in the endoplasmic
reticulum (ER), where it has prolonged associations with calnexin (28) and the 70-kDa heat shock protein family (37).
F508-CFTR is targeted
for rapid intracellular degradation (35), at least in part by the
ubiquitin-proteasome system (20, 36), and does not reach its
appropriate subcellular location at the apical plasma membrane (10,
22).
Treatment with protein stabilizing agents, chemical chaperones (4, 33),
or the transcriptional regulator butyrate (9) results in correction
of the F508-CFTR trafficking defect and cell surface
CFTR function in vitro. Recently, we demonstrated that sodium
4-phenylbutyrate (4PBA), a butyrate analog that is approved for
pharmaceutical use, similarly corrects the
F508-CFTR trafficking
defect and restores CFTR function at the plasma membrane of cultured
cystic fibrosis epithelial cells (30). We also recently demonstrated
that 4PBA caused a small but significant improvement in nasal
epithelial chloride transport in
F508-homozygous cystic fibrosis
patients (31). However, the mechanism of 4PBA action remains elusive.
4PBA, like butryate, regulates gene transcription. We (30) and others
(19) were unable to demonstrate a significant stimulation of
transcription of F508-CFTR after 4PBA treatment. We therefore hypothesized that 4PBA might regulate the transcription of a protein in
the intracellular protein trafficking pathway.
F508-CFTR has a
prolonged association with the 70-kDa heat shock protein family relative to that of wild-type CFTR (37). Hsc70 (Hsp73), the constitutively expressed member of this family, has a role in the
lysosomal degradation of intracellular proteins (11, 16) and was
recently shown to be required for the ubiquitin-dependent degradation
of a number of cellular proteins (3). Hsc70 forms a stable association
with, and prevents the aggregation of, a peptide derived from
F508-CFTR in an in vitro folding system (34). The rapid
intracellular degradation of
F508-CFTR occurs, at least in part, via
the ubiquitin-proteasome system (20, 36) and therefore might result
from a prolonged association of
F508-CFTR and Hsc70. We therefore
specifically tested the hypothesis that 4PBA would regulate Hsc70
expression and, subsequently, the interaction of
F508-CFTR and Hsc70.
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METHODS |
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Cell culture. IB3-1 cells (38) were grown on uncoated tissue culture plasticware in a 5% CO2 incubator at 37°C, or at 25°C as noted. Standard growth medium was LHC-8 (Biofluids, Rockville, MD) supplemented with 5% fetal bovine serum (Sigma Chemical, St. Louis, MO, or Biofluids), 100 U/ml penicillin-streptomycin (GIBCO BRL, Gaithersburg, MD), 0.2 mg/ml Primaxin (Imipenim, Merck, West Point, PA), 80 µg/ml tobramycin (Eli Lilly, Indianapolis, IN), and 2.5 µg/ml Fungizone (Biofluids). Cells for control experiments were cultured under these routine conditions. Growth medium for the treated cells was composed of the indicated agent at the indicated concentration added to the routine growth medium and incubated at 37°C in a 5% CO2 incubator. We previously determined that 4PBA maintains a constant concentration under these culture conditions for at least 2 days (30).
Antibodies. Rabbit anti-CFTR antiserum 181 (directed against CFTR amino acids 415-427 prior to the first nucleotide binding fold) was described previously (25). A rabbit polyclonal antiserum specific for Hsc70 (5) was a generous gift of Drs. C. R. Brown and W. J. Welch (University of California at San Francisco). A rat monoclonal antibody specific for Hsc70, clone 1B5, was a generous gift of Dr. A. Laszlo (Washington University, St. Louis, MO). This antibody is also commercially available (Stressgen Biotechnologies, Victoria, BC, Canada). A mouse monoclonal antibody directed against Hsp90 (clone AC88) and rabbit polyclonal antisera specific for Hsp40 and Hsp70 were purchased from Stressgen Biotechnologies. A mouse monoclonal antibody directed against calnexin (clone AF8) (18) was a generous gift of Dr. Michael Brenner (Harvard University). A mouse monoclonal antibody to Hdj2 (clone KA2A5.6) was from NeoMarkers (Union City, CA). Donkey anti-rabbit IgG-horseradish peroxidase conjugate and sheep anti-mouse IgG-horseradish peroxidase conjugates were purchased from Amersham (Arlington Heights, IL). Goat anti-rat IgG-horseradish peroxidase conjugate was purchased from Boehringer-Mannheim (Indianapolis, IN) or Amersham.
Immunoblot analysis.
Whole cell lysates were prepared by solubilization with 2% SDS at
95°C. Protein concentration in the lysates was determined using the
Bio-Rad DC assay reagents with bovine plasma -globulin as a standard
(Bio-Rad Laboratories, Hercules, CA). Equal amounts of protein were
resolved on 5, 7, 8, or 9% SDS-polyacrylamide gels. Proteins were
transferred to nitrocellulose, and immunodetection was performed as
previously described (25). Nonspecific binding was blocked by
incubation of the nitrocellulose with 2% gelatin or 10% nonfat dry
milk. Primary antisera and secondary antibodies were applied in buffer
containing 0.4% BSA overnight at 4°C and for 1 h at room
temperature, respectively. Detection of immunoreactivity was performed
with the enhanced chemiluminescence reagent (ECL, Amersham) and fluorography.
RNase protection.
An Hsc70-specific probe for RNase protection was constructed by
isolating a 500-bp EcoR I fragment
from American Type Culture Collection (ATCC) plasmid 77659 (ATCC,
Manassas, VA) and ligating this fragment into the
EcoR I site of pSK()
(Bluescript, Stratagene, La Jolla, CA). The resulting plasmid was
sequenced in the Genetics Core Facility at the Johns Hopkins Hospital
and found to be identical to sequences in exons 8 and 9 of the human
Hsc70 sequence, with sequencing using the T3 primer and T7 primer
leading to sense and antisense sequence, respectively.
Immunoprecipitation. Cultured cells were solubilized by incubation for 1 h at 4°C in RIPA [50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1% Triton X-100 (Bio-Rad or Fisher Scientific), 1% sodium deoxycholate (Sigma), and protease inhibitor cocktail (Sigma; used at 1:1,000 final dilution)]. Solubilized cells were then homogenized by passage 10 times through a 20-gauge needle and cleared by centrifugation at 15,000 g for 20 min at 4°C. Protein concentration was determined using the Bio-Rad DC reagents as above. Polyclonal Hsc70 antiserum was added to the cell lysates (2 µl/250 µg total protein, with equal amounts of protein at equal final concentrations for each condition within an experiment) and incubated at 4°C overnight with gentle agitation. Immune complexes were captured with protein A-Sepharose 4B (Pharmacia Biotechnologies, Piscataway, NJ) that had been preabsorbed with BSA for 45 min at 4°C. Precipitated complexes were collected by centrifugation and washed twice with cold RIPA and twice with cold TBS (50 mM Tris-Cl, pH 7.6, and 150 mM NaCl). Immunoprecipitated protein was released from the beads by incubation in SDS-PAGE sample buffer for 1 h at 70°C and resolved on 5 or 7% SDS-polyacrylamide gels. Immunodetection of immunoprecipitated Hsc70 or CFTR was performed as described above.
Densitometric analysis. Fluorographic images were digitized using an AlphaImager 2000 digital analysis system (AlphaInnotech, San Leandro, CA). Densitometric analysis of these images was performed using AlphaImager image analysis software (version 4.0, AlphaInnotech) with two-dimensional integration of the selected band. Density of the lane surrounding the band was similarly determined by two-dimensional integration and used as a baseline density for background subtraction. For comparisons within an experiment, the density of the control lane, the 100-ng lane for bovine Hsc70 standard curve experiments and the 10-µg lane for CFTR standard curve experiments, was arbitrarily set to 1.0. A one-way ANOVA was used to determine statistical significance of changes in density of fluorographic bands (SPSS software, version 7.0).
Reagents. Pharmaceutical grade 4PBA, manufactured by Triple Crown America (Perkasie, PA), was a gift of Dr. Saul Brusilow (Johns Hopkins School of Medicine). The sources for other reagents were as follows: reagent grade butyric acid and phenylacetic acid, Sigma; ACS reagent grade glycerol, J. T. Baker (Phillipsburg, NJ) or Fisher; Geneticin (G418), GIBCO BRL; nitrocellulose, Schleicher & Schuell (Keene, NH) or Amersham. Electrophoresis grade chemicals were obtained 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 decreases expression of Hsc70 but not
calnexin, Hsp90, Hsp70, Hdj2, or Hsp40.
We studied the immortalized cystic fibrosis bronchiolar epithelial cell
line IB3-1 (38). IB3-1 has the CFTR genotype F508/W1282X and is a
model system for study of the intracellular trafficking of
F508-CFTR
because the W1282X allele gives rise to an unstable and therefore
untranslated mRNA. This results in IB3-1 cells containing only
F508-CFTR (17). We previously demonstrated that treatment of IB3-1
cells with 4PBA results in restoration of appropriate intracellular
trafficking of
F508-CFTR (30). IB3-1 cells were treated with
increasing concentrations of 4PBA in culture for 2 days. As shown in
Fig. 1, total Hsc70
immunoreactivity in whole cell lysates declined in a dose-dependent
fashion with increasing concentrations of 4PBA as detected by a
Hsc70-specific rabbit polyclonal antiserum (Fig.
1A, representative immunoblot; Fig. 1B, compiled densitometric analysis of
Fig. 1A and 7 other immunoblots). Similar data were obtained when a rat monoclonal antibody to Hsc70 was
used to probe the immunoblots. These data are consistent with 4PBA
inducing a dose-dependent reduction of cellular Hsc70 protein.
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4PBA treatment results in decreased Hsc70 mRNA expression.
Because 4PBA is known to regulate transcription, we next assessed
whether the concentration-dependent decrease in Hsc70 protein expression was reflective of a decrease in Hsc70 mRNA expression. We
measured Hsc70 mRNA in lysates of IB3-1 cells by RNase protection. We
observed, in comparison to levels of 18S rRNA as an internal standard
for total RNA assayed and recovered, a concentration-dependent decrease
in steady-state Hsc70 mRNA levels after 4PBA treatment (Fig.
3) that correlated with the decrease in
Hsc70 immunoreactivity observed in Fig. 1. There was a maximum decrease
of ~50% of control expression with continuous exposure to 5 mM 4PBA.
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F508-CFTR forms a complex with Hsc70 that is
decreased by 4PBA, low temperature, butyrate, and glycerol.
Immunoprecipitation with an antiserum that recognizes both Hsp70 and
Hsc70 results in the coimmunoprecipitation of
F508-CFTR (37). We
confirmed a direct interaction between CFTR and Hsc70 by testing
whether specific immunoprecipitation of Hsc70 would result in
coimmunoprecipitation of CFTR immunoreactivity (Fig. 4A). The
relative mobility of CFTR in this experiment was ~170 kDa, which is
consistent with the ER glycosylated "band B" form. Our recovery
of Hsc70 by immunoprecipitation was ~10% of input, which is typical
in this kind of experiment when recovery has been measured (26). We
assume that this is representative of the total cellular pool of Hsc70.
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DISCUSSION |
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The data presented in these experiments suggest that 4PBA, which was
previously shown to facilitate trafficking of F508-CFTR to the
plasma membrane (30), downregulates Hsc70 at the protein and mRNA
levels. Consistent with these findings was the reduction in
F508-CFTR/Hsc70 complexes. Similar effects on Hsc70 protein and mRNA
expression and
F508-CFTR/Hsc70 complex formation were observed for
butyrate and glycerol, both of which restore
F508-CFTR trafficking.
Interaction with Hsc70 is suggested to be a key step in targeting a
number of cellular proteins for ubiquitination and degradation by the
proteasome (3). The usual intracellular fate of
F508-CFTR is
degradation, at least in part by the ubiquitin-proteasome system (20,
36). Therefore, 4PBA may promote
F508-CFTR trafficking by inhibiting
its recognition by the intracellular degradation pathway.
The decrease in Hsc70 protein expression induced by 4PBA, butyrate, and glycerol is ~40-60%. Although this may seem a relatively small effect, similar decreases in Hsc70 expression in rat glial precursor cells lead to their differentiation into oligodendrocytes, as demonstrated by myelin basic protein expression and process formation (1). Hsc70 expression in ventromedial hypothalamus, pituitary, and uterus varies by ~40% during the estrus cycles in rats and appears to be regulated by estrogen but not by progesterone (23). In embryonic chickens, Hsc70 expression is increased ~25% by exogenous insulin and decreased by treatment with antisense oligonucleotides to (pro)insulin; such small changes can influence apoptosis in the developing chick (13). These data are consistent with our observations that small perturbations in Hsc70 expression can result in alterations in cellular function.
Butyrate is typically thought to act as a transcriptional activator, which apparently is in contrast with these data. However, decreased expression of surfactant proteins A and B mRNA in fetal rat lung has been reported in response to butyrate treatment (27). This is consistent with our data suggesting a decrease in Hsc70 expression at the protein and mRNA levels after treatment with butyrate and 4PBA.
F508-CFTR degradation by the ubiquitin-proteasome
pathway.
F508-CFTR is typically degraded by the ubiquitin-proteasome system
(20, 36). However, inhibition of the proteolytic component of this
system with lactacystin or
N-acetyl-L-leucinyl-L-leucinyl-L-leucinal does not promote
F508-CFTR trafficking to the cell surface (20, 36).
These observations suggest that the committed step for intracellular
degradation occurs earlier in the pathway than the actual proteolysis.
F508-CFTR trafficking vs. degradation: a
hypothetical relative rate model.
Under physiological conditions, <1% of
F508-CFTR is trafficked
via the normal pathway; >99% is targeted for and subsequently degraded (35). In contrast, only ~75% of wild-type CFTR is targeted for and subsequently degraded, whereas 25% of wild-type CFTR is trafficked to the cell surface (35). Based on these proportions, the
"trafficking" pathway for
F508-CFTR is disfavored by at least 2-3 kcal/mol compared with that of wild-type CFTR (7). This may
result from either an intrinsic instability of the
F508-CFTR protein, as is suggested by the temperature sensitivity of the process
(15), or a stronger interaction of
F508-CFTR with the recognition
protein for the degradative pathway. In the latter case, a higher
proportion of
F508-CFTR would enter the degradative pathway compared
with wild-type CFTR.
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ACKNOWLEDGEMENTS |
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We thank Matthew J. Harley and Bridget Lyons for expert technical assistance.
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FOOTNOTES |
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This work was supported by Cystic Fibrosis Foundation Leroy Matthews Individual Physician Scientist Award RUBENS96LO (to R. C. Rubenstein), National Heart, Lung, and Blood Institute Grant P01-HL-51811 (to P. L. Zeitlin), and Cystic Fibrosis Foundation Grants R881 (to R. C. Rubenstein) and ZEITLI98PO (to P. L. Zeitlin).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. C. Rubenstein, Pulmonary Medicine, Abramson 410C, Children's Hospital of Philadelphia, 34th St. and Civic Center Blvd., Philadelphia, PA 19104 (E-mail: rrubenst{at}mail.med.upenn.edu).
Received 30 June 1999; accepted in final form 20 September 1999.
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