Am J Physiol Cell Physiol Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Cell Physiol 278: C259-C267, 2000;
0363-6143/00 $5.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (90)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rubenstein, R. C.
Right arrow Articles by Zeitlin, P. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rubenstein, R. C.
Right arrow Articles by Zeitlin, P. L.
Vol. 278, Issue 2, C259-C267, February 2000

EDITORIAL FOCUS
Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of Delta F508-CFTR

Ronald C. Rubenstein1 and Pamela L. Zeitlin2

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The most common mutation of the cystic fibrosis transmembrane conductance regulator (CFTR), Delta 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 Delta 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 Delta F508-CFTR for ubiquitination and degradation by reducing the expression of Hsc70 in cystic fibrosis epithelial cells. IB3-1 cells (genotype Delta 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 Delta 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE MOST COMMON MUTATION OF the cystic fibrosis transmembrane conductance regulator (CFTR), Delta F508-CFTR (deletion of phenylalanine at position 508 of CFTR), is a temperature-sensitive trafficking mutant (15). Delta 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). Delta 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 Delta 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 Delta 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 Delta 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 Delta F508-CFTR after 4PBA treatment. We therefore hypothesized that 4PBA might regulate the transcription of a protein in the intracellular protein trafficking pathway. Delta 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 Delta F508-CFTR in an in vitro folding system (34). The rapid intracellular degradation of Delta F508-CFTR occurs, at least in part, via the ubiquitin-proteasome system (20, 36) and therefore might result from a prolonged association of Delta F508-CFTR and Hsc70. We therefore specifically tested the hypothesis that 4PBA would regulate Hsc70 expression and, subsequently, the interaction of Delta F508-CFTR and Hsc70.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma -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.

Recombinant bovine Hsc70 (>95% purity) was purchased from Stressgen Biotechnologies for use in constructing a standard curve of Hsc70 immunoreactivity. Immunoblots containing the bovine Hsc70 were probed with the Hsc70-specific polyclonal antiserum.

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.

Hybridization probes were synthesized using a Maxiscript T7 kit (Ambion, Austin, TX) and [alpha -32P]UTP (Amersham or DuPont NEN, Boston, MA) according to the Maxiscript protocol. Templates for internal control hybridizations, pTRI-18S and pTRI-cyclophilin A, were purchased from Ambion, and probes were similarly synthesized using the Maxiscript T7 kit. Probes were isolated by acid phenol-chloroform (Ambion) extraction, separated from unincorporated nucleotide by gel filtration (Sephadex G25 RNA spin column, Boehringer-Mannheim), and ethanol-acetate precipitated before resuspension in hybridization buffer. The concentration of radioactivity in the synthesized probes was determined by liquid scintillation.

RNase protection experiments were performed using the Direct Protect RNase protection assay kit (Ambion) according to the manufacturer's protocol. IB3-1 cell lysates were prepared in Direct Protect lysis buffer according to the manufacturer's protocol after incubation under the appropriate condition for 48 h. Probe (50-70 and 5-10 thousands of counts/min for Hsc70 and control, respectively) and cellular RNA were hybridized overnight at 37°C and digested with RNase cocktail. Protected fragments were resolved by electophoresis on 5% acrylamide-8 M urea gels and detected by fluorography. Hsc70 mRNA concentration is expressed relative to control (18S or cyclophilin A) hybridization by densitometry (see Densitometric analysis). Results for hybridization of Hsc70 mRNA relative to the two control species were similar and were therefore grouped for data analysis.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta F508/W1282X and is a model system for study of the intracellular trafficking of Delta F508-CFTR because the W1282X allele gives rise to an unstable and therefore untranslated mRNA. This results in IB3-1 cells containing only Delta F508-CFTR (17). We previously demonstrated that treatment of IB3-1 cells with 4PBA results in restoration of appropriate intracellular trafficking of Delta 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.


View larger version (24K):
[in this window]
[in a new window]
  		Fig. 1.   Dose-dependent reduction in Hsc70 expression mediated by sodium 4-phenylbutyrate (4PBA). A: IB3-1 cells were incubated with indicated concentration of 4PBA for 48 h. Whole cell lysates were prepared with SDS as described in METHODS. Total protein (5 µg) was resolved on 8% SDS-polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose, and immunodetection of Hsc70 was performed as described in METHODS. Primary antiserum was rabbit polyclonal antiserum specific for Hsc70. B: densitometry was performed as described in METHODS on 8 total immunoblot experiments (4 experiments performed in duplicate). Density of 0 4PBA (control) lane was set to 1, and density (means ± SE) of other lanes is expressed relative to control. Statistical significance (P values indicated below error bars) was determined by a 1-way ANOVA in comparison with control. C: standard curve construction. IB3-1 lysate protein (5 µg) or indicated amount of purified recombinant bovine Hsc70 was resolved on 8% SDS-polyacrylamide gels. Proteins were electrophoretically transferred to nitrocellulose, and immunodetection of Hsc70 was performed as described in METHODS. Primary antiserum was rabbit polyclonal antiserum specific for Hsc70. D: densitometry was performed as described in METHODS on 3 identical experiments. Density of 100-ng lane was set to 1, and densities of other lanes are expressed relative to 100-ng lane. Mean relative density is shown by filled circles. Error bars (SE) are contained within symbols. Relative density of 5 µg of IB3-1 lysate is depicted by open circle and corresponds to ~35 ng of bovine Hsc70 immunoreactivity.

To estimate the decrease in Hsc70 protein represented by this ~50% decrease in immunoreactivity, we constructed a densitometric standard curve of immunoreactivity for recombinant bovine Hsc70 (Fig. 1C, representative immunoblot; Fig. 1D, standard curve derived from Fig. 1C and two other experiments). We performed both log and linear regressions for the data of Fig. 1D, and both were acceptable fits (r2 for log and linear were 0.973 and 0.930, respectively). The superiority of the log fit may be due to saturation of the X-ray film used for fluorography at high amounts of Hsc70, although the densitometer was still able to distinguish density variation. These data suggest that IB3-1 cells contain ~35 ng Hsc70/5 µg total cellular protein. Furthermore, these data demonstrate a good correlation of Hsc70 immunoreactivity and measured densitometry; a 50% decrease in measured density corresponds to an ~50-60% decrease in total Hsc70 protein immunoreactivity.

We next assessed whether 4PBA would regulate expression of a number of other molecular chaperones in IB3-1 cells (Fig. 2). Calnexin is a molecular chaperone present in the ER membrane that binds to glycoproteins in the ER via high-mannose core residues and has previously been shown to have a prolonged interaction with Delta F508-CFTR in heterologous cells expressing Delta F508-CFTR (28). Hsp90 is required for correct folding and function of a number of cellular proteins (16). Inhibition of Hsp90 function with geldanamycin leads to more rapid degradation of Delta F508-CFTR (24), suggesting that Hsp90 may be required for CFTR trafficking. Hsp70 (Hsp72) expression is induced by heat shock and the presence of denatured proteins within the eukaryotic cell (16). In Escherichia coli, the Hsp70 homologue DnaK and the Hsp40 homologue DnaJ act to promote protein folding (16). Hdj2 is the member of the Hsp40 family that specifically interacts with and regulates the ATPase activity of Hsc70. Hdj2 also interacts with CFTR during CFTR translation (26). Increasing concentrations of 4PBA did not affect the expression of calnexin, Hsp90, Hsp70, Hsp40, or Hdj-2 in IB3-1 cells. To substantiate these observations, densitometric analysis of the representative immunoblots of Fig. 2 and similar separate experiments (n = 3 independent experiments for each chaperone including intermediate concentrations of 4PBA) was performed (data not shown). These data and the data of Fig. 1 are consistent with selective regulation of only Hsc70, the constitutively expressed member of the 70-kDa heat shock protein family by 4PBA, and none of the other five molecular chaperones assessed.


View larger version (94K):
[in this window]
[in a new window]
  		Fig. 2.   Calnexin, Hsp90, Hsp70, Hsp40, and Hdj2 expression is unchanged by 4PBA treatment. IB3-1 cells were treated as described for Fig. 1. Immunoblotting with 5 µg of IB3-1 SDS lysate protein was performed as described in METHODS.

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.


View larger version (47K):
[in this window]
[in a new window]
  		Fig. 3.   Dose-dependent downregulation of Hsc70 mRNA expression by 4PBA. A: IB3-1 cells were treated as for Figs. 1 and 2. Hsc70 mRNA and 18S rRNA (as an internal reference) were measured by Direct Protect RNase protection as described in METHODS. B: densitometric analysis on 3 independent experiments was performed as described by first normalizing density of Hsc70 hybridization by internal reference RNA hybridization (either 18S rRNA or cyclophilin A mRNA hybridization; see METHODS) to control for total RNA in each hybridization and RNA recovery during assay. This ratio for each condition was subsequently made relative to Hsc70-to-reference RNA ratio for control lane. Means ± SE for 3 independent experiments are shown. P values (shown above respective error bars) were determined by 1-way ANOVA in comparison with control.

Delta 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 Delta 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.


View larger version (27K):
[in this window]
[in a new window]
  		Fig. 4.   4PBA-treatment of IB3-1 cells decreases amount of cystic fibrosis transmembrane conductance regulator (CFTR) immunoreactivity coprecipitated with Hsc70. IB3-1 cells were grown in indicated concentration of 4PBA for 2 days at 37°C. Cells were solubilized with RIPA, and 250 µg of total protein were incubated with Hsc70-specific rabbit polyclonal antiserum as described in METHODS. Immune complexes were recovered by centrifugation after incubation with protein A-Sepharose. Composition of precipitated immune complexes was analyzed by SDS-PAGE and protein immunoblot using anti-CFTR antiserum 181 and rabbit polyclonal anti-Hsc70 antibody as described in METHODS. Total protein (10 µg) was resolved in IB3-1 SDS lysate lane, and immunoprecipitate from equivalent of 80 µg of cellular protein was analyzed in immunoprecipitation lanes. Relative mobility of CFTR associated with Hsc70 was ~170 kDa. B: densitometric analysis of these and similar immunoblots (4 independent experiments) was performed as described for Fig. 1. Mean density (±SE) relative to control for 4 independent experiments is shown. P values vs. control were determined by 1-way ANOVA. C: CFTR densitometry standard curve construction. Immunodetection of CFTR in indicated amount of IB3-1 lysate protein was performed as described in METHODS. Densitometry was performed as described in METHODS on 4 independent concentration curves. For each independent experiment, density of CFTR immunoreactivity in 10-µg sample was set to 1, and densities of CFTR immunoreactivity in other samples are expressed relative to 10-µg lane. Mean relative density is shown by closed symbols. Error bars (SE) are contained within symbols where not visible.

As expected from the immunoblot data of Fig. 1, IB3-1 cells treated with increasing concentrations of 4PBA had decreased amounts of immunoprecipitable Hsc70 (Fig. 4A). With increasing concentrations of 4PBA, less immunoreactive CFTR was recovered in complex with Hsc70. At >1 mM 4PBA, CFTR was undetectable in the immunoprecipitates. Similarly, CFTR was not associated with Hsc70 when IB3-1 cells were incubated at 25°C. We previously showed that treatment with >0.1 mM 4PBA or incubation at 25°C leads to increased overall expression and the appearance of mature CFTR in IB3-1 cells (30). To better quantify this change, we performed densitometric analysis of this and similar experiments (Fig. 4B) and constructed a densitrometric standard curve of CFTR immunoreactivity using IB3-1 lysate protein (Fig. 4C). Our standard curve suggests that CFTR immunoreacitivity as detected by densitometry decreases linearly as a function of decreasing protein but that the decrease in densitometric signal exceeds the decrease in CFTR protein, i.e., a decrease in IB3-1 protein from 5 to 2.5 µg leads to an approximately two-thirds decrease in densitometric signal. CFTR immunoreactivity was not consistently detected in samples containing 2 µg of IB3-1 protein and was not detected in samples containing 1 µg of IB3-1 protein. Thus the slightly greater change in CFTR vs. Hsc70 densitometric signal in Fig. 4B actually reflects a similar decrease in immunoreactive protein of the two species.

The decrease in CFTR recovered by immunoprecipitation in proportion to the decrease in Hsc70 is consistent with 4PBA not directly influencing the binding affinity of CFTR and Hsc70. We further assessed this by immunoprecipitating Hsc70-CFTR complexes from untreated IB3-1 cells either with or without 1 mM 4PBA added to the RIPA lysis and wash buffers. The recovery of Hsc70 and associated CFTR was unaltered in the presence of 4PBA (data not shown), which is consistent with 4PBA not altering the in vitro affinity of Hsc70 for CFTR. Collectively, these data suggest that 4PBA treatment leads to an increased proportion of Delta F508-CFTR escaping association with Hsc70 due to a decrease in Hsc70 expression. If association with Hsc70 is necessary for CFTR ubiquitination, as it is for a number of other cellular proteins (3), then escape from this association may decrease the proportion of Delta F508-CFTR that is prematurely degraded.

We next examined the effects of two compounds that promote trafficking of Delta F508-CFTR to the plasma membrane, the transcriptional regulator butyrate (9) and the chemical chaperone glycerol (4, 30, 33). We also tested the major in vivo metabolite of 4PBA, phenylacetate, and the aminoglycoside antibiotic geneticin (G418). G418 promotes read through and stabilization of the otherwise unstable mRNA derived from the W1282X missense allele present in IB3-1 cells and results in the appearance of CFTR channel activity at the IB3-1 plasma membrane (2).

Representative data for these immunoblot experiments are shown in Fig. 5. The results again demonstrate a reduction in Hsc70 immunoreactivity with 1 mM butyrate and 1 M glycerol but no change in Hsp90, Hsp70, or Hsp40 immunoreactivity. These observations are consistent with the hypothesis that conditions that promote Delta F508-CFTR trafficking to the cell surface are associated with a reduction in Hsc70 expression, although we have yet to establish a causal relationship. Phenylacetate at 1 mM had little effect on Hsc70 or Hsp90 expression. In vivo, 4PBA is rapidly and completely converted by beta -oxidation to phenylacetate and then conjugated with glutamine to form phenacetylglutamine, which is excreted in the urine (6). Phenylacetate has a different potency profile from butyrate or 4PBA with respect to specific gene induction (8). These results suggest that 4PBA alone may regulate Hsc70 expression. G418, which acts on the W1282X allele and not Delta F508-CFTR, had little effect on Hsc70 or Hsp90 but increased Hsp40 and Hsp70 immunoreactivity. Although it seems logical that increased Hsc70 might lead to reduced trafficking of CFTR derived from the W1282X allele, the W1282X-derived CFTR would have wild-type structure in the region of F508, and the F508 region may be a critical determinant of CFTR affinity for Hsc70. These changes in Hsc70 protein expression again correlated with changes in steady-state Hsc70 mRNA expression, as determined by RNase protection (Fig. 6). In this experiment, 1 mM butyrate and 1 M glycerol were associated with ~50% reduction in steady-state Hsc70 mRNA levels. The glycerol effect was unexpected and may occur by a mechanism different from that of the butyrates.


View larger version (37K):
[in this window]
[in a new window]
  		Fig. 5.   Hsc70 expression in IB3-1 cells is decreased by butyrate (BA; 1 mM) and glycerol (Glyc, 1 M) but not phenylacetate (PAA; 1 mM) and G418 (0.2 mg/ml); Hsp90, Hsp70, and Hsp40 expression are not affected by these agents. A: IB3-1 cells were incubated at 37°C for 2 days under indicated conditions. Cellular homogenates were prepared in 2% SDS as described in METHODS, and 5 µg of total cellular protein were analyzed by immunoblot for Hsc70 using rabbit polyclonal antiserum, Hsp90, Hsp70, and Hsp40 as described in METHODS. Con, control. B and C: densitometric analysis of Hsc70 expression (B) and Hsp90, Hsp70, and Hsp40 expression (C) was performed as described for Fig. 1. B: means ± SE of density in 4 independnt experiments. C: means ± SE of relative density in 3 (Hsp70) or 4 (Hsp40 and Hsp90) independent experiments. P values vs. control (B and C) were determined by 1-way ANOVA.



View larger version (55K):
[in this window]
[in a new window]
  		Fig. 6.   Butyrate and glycerol also decrease Hsc70 mRNA expression. A: IB3-1 cells were incubated under indicated conditions for 2 days before preparation of cellular lysates for assay of Hsc70 mRNA and control RNA (18S rRNA) by Direct Protect RNase protection as described in METHODS. B: densitometric analysis was performed as described for 4 independent experiments by first normalizing density of Hsc70 hybridization to reference RNA hybridization (18S rRNA or cyclophilin A mRNA) to control for total RNA in each hybridization and RNA recovery during the assay. This ratio for each condition was subsequently made relative to Hsc70-to-reference RNA ratio for control lane. Plotted are means ± SE relative Hsc70 mRNA expression for 4 independent experiments. P values (shown above respective error bars) were determined by 1-way ANOVA in comparison with control.

Our results in Fig. 5 predict that conditions that do not affect Hsc70 expression would allow CFTR to associate with Hsc70. We tested this prediction by coimmunoprecipitation. Figure 7 demonstrates that treatment of IB3-1 cells with glycerol or butyrate decreases the amount of immunoreactive CFTR coprecipitated with Hsc70. Again, the densitometric analysis of this and similar experiments (Fig. 7B) suggests that the decrease in CFTR associated with Hsc70 resulted from decreased expression of Hsc70. These data are consistent with a model in which agents that improve Delta F508-CFTR intracellular trafficking decrease the total amount of Delta F508-CFTR/Hsc70 complex. There was little effect of phenylacetate or G418 treatment. The latter observation is consistent with G418 acting by a mechanism different from that of 4PBA, glycerol, or butyrate. This is also consistent with G418 acting on the W1282X-CFTR allele present in IB3-1 and not on the Delta F508 allele that is the target of 4PBA, glycerol, or butyrate.


View larger version (48K):
[in this window]
[in a new window]
  		Fig. 7.   Butyrate and glycerol treatment of IB3-1 cells decreases amount of CFTR immunoreactivity coprecipitated with Hsc70. A: IB3-1 cells were grown in indicated concentration of 4PBA for 2 days at 37°C. Cells were solubilized with RIPA, and 250 µg of total protein were incubated with Hsc70-specific rabbit polyclonal antiserum as described in METHODS. Immune complexes were recovered by centrifugation after incubation with protein A-Sepharose. Composition of precipitated immune complexes was analyzed by SDS-PAGE and protein immunoblot using anti-CFTR antiserum 181 and rabbit polyclonal anti-Hsc70 antibody as described in METHODS. Total protein (10 µg) was resolved in IB3-1 SDS lysate lane, and immunoprecipitate from equivalent of 80 µg of cellular protein was analyzed in immunoprecipitation lanes. Relative mobility of CFTR coimmunoprecipitated with Hsc70 was again ~170 kDa. B: densitometric analysis of these immunoblots (4 independent experiments) was performed as described for Fig. 1. Shown is mean density (±SE) relative to control for 4 independent experiments. P values vs. control were determined by 1-way ANOVA.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The data presented in these experiments suggest that 4PBA, which was previously shown to facilitate trafficking of Delta F508-CFTR to the plasma membrane (30), downregulates Hsc70 at the protein and mRNA levels. Consistent with these findings was the reduction in Delta F508-CFTR/Hsc70 complexes. Similar effects on Hsc70 protein and mRNA expression and Delta F508-CFTR/Hsc70 complex formation were observed for butyrate and glycerol, both of which restore Delta 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 Delta F508-CFTR is degradation, at least in part by the ubiquitin-proteasome system (20, 36). Therefore, 4PBA may promote Delta 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.

Delta F508-CFTR degradation by the ubiquitin-proteasome pathway. Delta 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 Delta 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.

The committed step for degradation may occur during recognition and/or subsequent ubiquitination of Delta F508-CFTR. Because Hsc70 is required for a number of intracellular proteins to undergo ubiquitination and degradation (3), decreasing Hsc70 levels in the cell would decrease the rate of intracellular degradation of these proteins. Movement along the folding pathway might therefore be promoted by decreasing the rate at which Delta F508-CFTR is recognized by Hsc70 and enters the degradative pathway.

This view may seem contradictory to the presumed pro-folding role of molecular chaperones; however, it is not inconsistent with previous studies. Hsc70 binds to a Delta F508 synthetic peptide derived from the first nucleotide binding fold of CFTR, prevents its aggregation in vitro, and improves its folding yield (34). However, inclusion of ATP in this experimental system, which allows peptide dissociation and may better mimic the intracellular environment, restored the propensity of the synthetic Delta F508 peptide to form aggregates and decreased its folding yield to that found when Hsc70 was not present. This suggests a more complex role of Hsc70 than simply promoting CFTR folding. Hsc70 associates with CFTR during CFTR translation, and the association with Delta F508-CFTR is greater and longer lived than with wild-type CFTR (26). CFTR also undergoes cotranslational ubiquitination (32), and the possibility of enhanced ubiquitin-dependent degradation of the Delta F508 peptide in the presence of Hsc70 is absent in the in vitro folding system (34). Collectively, these data are consistent with a model in which Hsc70 remains associated with species that are "partially structured" and likely to aggregate, thereby preventing aggregation. However, the prolonged association with Hsc70 would result in increased cotranslational and/or posttranslational ubiquitination and targeting for subsequent degradation.

Others have attempted pharmacologic manipulation of chaperone function to improve Delta F508-CFTR trafficking. Geldanamycin, which inhibits Hsp90 function, led to more rapid degradation of CFTR in heterologous cells expressing wild-type CFTR but did not alter the interaction of CFTR with Hsc70. In fact, Hsc70 expression, as detected by immunoreactivity with an Hsc70-specific monoclonal antibody (clone 1B5, also used in our experiments), increased with geldanamycin treatment and may have been important in the enhanced degradation of CFTR. These experiments did not specifically address the influence of geldanamycin on Delta F508-CFTR trafficking (24). Our data do not suggest a perturbation of Hsp90 expression or function by 4PBA and therefore are consistent with the observed effect of geldanamycin. Interestingly, deoxyspergualin, which competitively inhibits peptide binding to Hsc70 and Hsp90, leads to restoration of CFTR function in heterologous cells expressing Delta F508-CFTR (21). If the interaction with Hsp90 is counterproductive for Delta F508-CFTR trafficking, as the results with geldanamycin suggest, then the inhibition of peptide binding to Hsc70 caused by deoxyspergualin positively influences Delta F508-CFTR trafficking. These results are consistent with our data and model, in which decreased Hsc70 function due to decreasd Hsc70 expression after 4PBA treatment leads to improved Delta F508-CFTR trafficking.

Hsc70 is also important in other functions of the cell; its abundance may reflect this. It is found in the endocytic pathway and is required for ATP-dependent uncoating of clathrin-coated pits (14, 29). It also has a role in targeting intracellular proteins for lysosomal degradation (11). It is unclear what effect alterations in Hsc70 expression as reported in these studies would have on these intracellular processes and whether alterations in these processes would influence intracellular trafficking of Delta F508-CFTR. Hypothetically, complete knockout of Hsc70 would be undesirable. Our data are consistent with, at most, a 40-60% reduction in Hsc70 expression at clinically relevant 4PBA concentrations.

Delta F508-CFTR trafficking vs. degradation: a hypothetical relative rate model. Under physiological conditions, <1% of Delta 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 Delta 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 Delta F508-CFTR protein, as is suggested by the temperature sensitivity of the process (15), or a stronger interaction of Delta F508-CFTR with the recognition protein for the degradative pathway. In the latter case, a higher proportion of Delta F508-CFTR would enter the degradative pathway compared with wild-type CFTR.

In this model, decreasing the association of Delta F508-CFTR with the recognition protein would promote its trafficking to the cell surface. If this association is a typical bimolecular reaction, the rate of association is dependent on temperature (7). Delta F508-CFTR trafficking is improved by reduced temperature in vitro (15). A reduction of 10-12°C, as is accomplished by incubating cells at 25-27°C, is predicted to decrease the bimolecular rate of association of Delta F508-CFTR with a recognition protein by ~50-67% (7). The observed decrease in Hsc70 recovered by immunoprecipitation in Fig. 4 could also contribute to this effect. Decreasing the intracellular concentration of the recognition protein, such as Hsc70, by ~50% would similarly decrease its rate of association with Delta F508-CFTR and lead to a reduction in premature degradation of Delta F508-CFTR. More newly synthesized Delta F508-CFTR would thereby enter the trafficking pathway.

In conclusion, we demonstrate that 4PBA and butyrate decrease both the expression of Hsc70 mRNA and protein and also its association with Delta F508-CFTR. These data are consistent with a hypothetical model in which the association of Delta F508-CFTR with Hsc70 leads to ubiquitination and proteasomal degradation of Delta F508-CFTR. 4PBA- and butyrate-mediated reduction in Hsc70 may promote Delta F508-CFTR trafficking to the cell surface.


    ACKNOWLEDGEMENTS

We thank Matthew J. Harley and Bridget Lyons for expert technical assistance.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Aquino, D. A., D. Peng, C. Lopez, and M. Farooq. The constitutive heat shock protein-70 is required for optimal expression of myelin basic protein during differentiation of oligodendrocytes. Neurochem. Res. 23: 413-420, 1998[Web of Science][Medline].

2.   Bedwell, D. M., A. Kaenjak, D. J. Benos, Z. Bebok, K. Bubien, J. Hong, A. Tousson, J. P. Clancy, and E. J. Sorscher. Suppression of a CFTR premature stop mutation in a bronchial epithelial cell line. Nat. Med. 3: 1280-1284, 1997[Web of Science][Medline].

3.   Bercovich, B., I. Stancovski, A. Mayer, N. Blumenfeld, A. Laszlo, A. L. Schwartz, and A. Ciechanover. Ubiquitin-dependent degradation of certain protein substrates in vitro requires the molecular chaperone Hsc70. J. Biol. Chem. 272: 9002-9010, 1997[Abstract/Free Full Text].

4.   Brown, C. R., L. Q. Hong-Brown, J. Biwersi, A. S. Verkman, and W. J. Welch. Chemical chaperones correct the mutant phenotype of the Delta F508 cystic fibrosis transmembrane conductance regulator protein. Cell Stress Chaperones 1: 117-125, 1996[Web of Science][Medline].

5.   Brown, C. R., R. L. Martin, W. J. Hansen, R. P. Beckmann, and W. J. Welch. The constitutive and stress inducible forms of hsp 70 exhibit functional similarities and interact with one another in an ATP- dependent fashion. J. Cell Biol. 120: 1101-1112, 1993[Abstract/Free Full Text].

6.   Brusilow, S. W. Phenylacetylglutamine may replace urea as a vehicle for waste nitrogen excretion. Pediatr. Res. 29: 147-150, 1991.

7.   Castellan, G. W. Physical Chemistry. Reading, MA: Addison-Wesley, 1971.

8.   Chen, W. Y., E. C. Bailey, S. L. McCune, J. Y. Dong, and T. M. Townes. Reactivation of silenced, virally transduced genes by inhibitors of histone deacetylase. Proc. Natl. Acad. Sci. USA 94: 5798-5803, 1997[Abstract/Free Full Text].

9.   Cheng, S. H., S. L. Fang, J. Zabner, J. Marshall, S. Piraino, S. C. Schiavi, D. M. Jefferson, M. J. Welsh, and A. E. Smith. Functional activation of the cystic fibrosis trafficking mutant Delta F508-CFTR by overexpression. Am. J. Physiol. Lung Cell. Mol. Physiol. 268: L615-L624, 1995[Abstract/Free Full Text].

10.   Cheng, S. H., R. J. Gregory, J. Marshall, S. Paul, D. W. Souza, G. A. White, C. R. O'Riordan, and A. E. Smith. Defective intracellular transport and processing of CFTR is the molecular basis of most cystic fibrosis. Cell 63: 827-834, 1990[Web of Science][Medline].

11.   Chiang, H.-L., S. R. Terlecky, C. P. Plant, and J. F. Dice. A role for a 70-kilodalton heat shock protein in lysosomal degradation of intracellular proteins. Science 246: 382-385, 1989[Abstract/Free Full Text].

13.   De la Rosa, E. J., E. Vega-Nunez, A. V. Morales, J. Serna, E. Rubio, and F. de Pablo. Modulation of the chaperone heat shock cognate 70 by embryonic (pro)insulin correlates with prevention of apoptosis. Proc. Natl. Acad. Sci. USA 95: 9950-9955, 1998[Abstract/Free Full Text].

14.   DeLuca-Flaherty, C., D. B. McKay, P. Parham, and B. L. Hill. Uncoating protein (hsc70) binds a conformationally labile domain of clathrin light chain LCa to stimulate ATP hydrolysis. Cell 62: 875-887, 1990[Web of Science][Medline].

15.   Denning, G. M., M. A. Anderson, J. F. Amara, J. Marshall, A. E. Smith, and M. J. Welsh. Processing of mutant cystic fibrosis transmembrane regulator is temperature-sensitive. Nature 358: 761-764, 1992[Medline].

16.   Gething, M.-J., and J. Sambrook. Protein folding in the cell. Nature 355: 33-45, 1992[Medline].

17.   Hamosh, A., B. J. Rosenstein, and G. R. Cutting. CFTR nonsense mutations G542X and W1282X associated with severe reduction of CFTR mRNA in nasal epithelial cells. Hum. Mol. Genet. 1: 542-544, 1992[Free Full Text].

18.   Hochstenbach, F., V. David, S. Watkins, and M. B. Brenner. Endoplasmic reticulum resident protein of 90 kilodaltons associates with the T- and B-cell antigen receptors and major histocompatibility complex antigens during their assembly. Proc. Natl. Acad. Sci. USA 89: 4734-4738, 1992[Abstract/Free Full Text].

19.   Hyde, S. C., S. E. Smyth, D. C. Gruenert, and D. R. Gill. The effects of 4-phenylbutyric acid on CFTR mRNA levels (Abstract). Pediatr. Pulmonol. Suppl. 17: 211, 1998.

20.   Jensen, T. J., M. A. Loo, S. Pind, D. B. Williams, A. L. Goldberg, and J. R. Riordan. Multiple proteolytic systems, including the proteasome, contribute to CFTR processing. Cell 83: 129-135, 1995[Web of Science][Medline].

21.   Jiang, C., S. L. Fang, Y. F. Xiao, S. P. O'Connor, S. G. Nadler, D. W. Lee, D. M. Jefferson, J. M. Kaplan, A. E. Smith, and S. H. Cheng. Partial restoration of cAMP-stimulated CFTR chloride channel activity in Delta F508 cells by deoxyspergualin. Am. J. Physiol. Cell Physiol. 275: C171-C178, 1998[Abstract/Free Full Text].

22.   Kartner, N., O. Augustinas, T. J. Jensen, A. L. Naismith, and J. R. Riordan. Mislocalization of Delta F508 CFTR in cystic fibrosis sweat gland. Nat. Genet. 1: 321-327, 1992[Web of Science][Medline].

23.   Krebs, C. J., E. D. Jarvis, and D. W. Pfaff. The 70-kDa heat shock cognate protein (Hsc73) gene is enhanced by ovarian hormones in the ventromedial hypothalamus. Proc. Natl. Acad. Sci. USA 96: 1686-1691, 1999[Abstract/Free Full Text].

24.   Loo, M. A., T. J. Jensen, L. Cui, Y. Hou, X. B. Chang, and J. R. Riordan. Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome. EMBO J. 17: 6879-6887, 1998[Web of Science][Medline].

25.   McGrath, S. A., A. Basu, and P. L. Zeitlin. Cystic fibrosis gene and protein expression during fetal lung development. Am. J. Respir. Cell Mol. Biol. 8: 201-208, 1993.

26.   Meacham, G. C., Z. Lu, S. King, E. Sorscher, A. Tousson, and D. M. Cyr. The Hdj-2/Hsc70 chaperone pair facilitates early steps in CFTR biogenesis. EMBO J. 18: 1492-1505, 1999[Web of Science][Medline].

27.   Peterec, S. M., K. V. Nichols, D. W. Dynia, C. M. Wilson, and I. Gross. Butyrate modulates surfactant protein mRNA in fetal rat lung by altering mRNA transcription and stability. Am. J. Physiol. Lung Cell. Mol. Physiol. 267: L9-L15, 1994[Abstract/Free Full Text].

28.   Pind, S., J. R. Riordan, and D. B. Williams. Participation of the endoplasmic reticulum chaperone calnexin (p88, IP90) in the biogenesis of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 269: 12784-12788, 1994[Abstract/Free Full Text].

29.   Rothman, J. E., and S. L. Schmid. Enzymatic recycling of clathrin from coated vesicles. Cell 46: 5-9, 1986[Web of Science][Medline].

30.   Rubenstein, R. C., M. E. Egan, and P. L. Zeitlin. In vitro pharmacologic restoration of CFTR-mediated chloride transport with sodium 4-phenylbutyrate in cystic fibrosis epithelial cells containing Delta F508-CFTR. J. Clin. Invest. 100: 2457-2465, 1997[Web of Science][Medline].

31.   Rubenstein, R. C., and P. L. Zeitlin. A pilot clinical trial of oral sodium 4-phenylbutyrate (Buphenyl) in Delta F508-homozygous cystic fibrosis patients: partial restoration of nasal epithelial CFTR function. Am. J. Respir. Crit. Care Med. 157: 484-490, 1998[Abstract/Free Full Text].

32.   Sato, S., C. L. Ward, and R. R. Kopito. Cotranslational ubiquitination of cystic fibrosis transmembrane conductance regulator in vitro. J. Biol. Chem. 273: 7189-7192, 1998[Abstract/Free Full Text].

33.   Sato, S., C. L. Ward, M. E. Krouse, J. J. Wine, and R. R. Kopito. Glycerol reverses the misfolding phenotype of the most common cystic fibrosis mutation. J. Biol. Chem. 271: 635-638, 1996[Abstract/Free Full Text].

34.   Strickland, E. H., B.-H. Qu, L. Millen, and P. J. Thomas. The molecular chaperone Hsc70 assists in the in vitro folding of the N-terminal nucleotide-binding domain of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 272: 25421-25424, 1997[Abstract/Free Full Text].

35.   Ward, C. L., and R. R. Kopito. Intracellular turnover of cystic fibrosis transmembrane conductance regulator. Inefficient processing and rapid degradation of wild-type and mutant proteins. J. Biol. Chem. 269: 25710-25718, 1994[Abstract/Free Full Text].

36.   Ward, C. L., S. Omura, and R. R. Kopito. Degradation of CFTR by the ubiquitin-proteasome pathway. Cell 83: 121-127, 1995[Web of Science][Medline].

37.   Yang, Y., S. Janich, J. A. Cohn, and J. M. Wilson. The common variant of cystic fibrosis transmembrnae conductance regulator is recognized by hsp70 and degraded in a pre-Golgi nonlysosomal compartment. Proc. Natl. Acad. Sci. USA 90: 9480-9484, 1993[Abstract/Free Full Text].

38.   Zeitlin, P. L., L. Lu, J. Rhim, G. Cutting, G. Stetten, K. A. Kieffer, R. Craig, and W. B. Guggino. A cystic fibrosis bronchial epithelial cell line: immortalization by adeno-12-SV40 infection. Am. J. Respir. Cell Mol. Biol. 4: 313-319, 1991.

Am J Physiol Cell Physiol 278(2):C259-C267
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society


This article has been cited by other articles:


Home page
 J. Lipid Res.Home page
S. Basseri, S. Lhotak, A. M. Sharma, and R. C. Austin
The chemical chaperone 4-phenylbutyrate inhibits adipogenesis by modulating the unfolded protein response
J. Lipid Res., December 1, 2009; 50(12): 2486 - 2501.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
C. Norez, F. Antigny, S. Noel, C. Vandebrouck, and F. Becq
A Cystic Fibrosis Respiratory Epithelial Cell Chronically Treated by Miglustat Acquires a Non-Cystic Fibrosis-Like Phenotype
Am. J. Respir. Cell Mol. Biol., August 1, 2009; 41(2): 217 - 225.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
S. Das, T. D. Smith, J. D. Sarma, J. D. Ritzenthaler, J. Maza, B. E. Kaplan, L. A. Cunningham, L. Suaud, M. J. Hubbard, R. C. Rubenstein, et al.
ERp29 Restricts Connexin43 Oligomerization in the Endoplasmic Reticulum
Mol. Biol. Cell, May 15, 2009; 20(10): 2593 - 2604.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
A. Sobolewski, N. Rudarakanchana, P. D. Upton, J. Yang, T. K. Crilley, R. C. Trembath, and N. W. Morrell
Failure of bone morphogenetic protein receptor trafficking in pulmonary arterial hypertension: potential for rescue
Hum. Mol. Genet., October 15, 2008; 17(20): 3180 - 3190.
[Abstract] [Full Text] [PDF]

Home page
 Eur Respir JHome page
J. Li, Y-Y. Xiang, L. Ye, L-C. Tsui, J. F. MacDonald, J. Hu, and W-Y. Lu
Nonsteroidal anti-inflammatory drugs upregulate function of wild-type and mutant CFTR
Eur. Respir. J., August 1, 2008; 32(2): 334 - 343.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. C. Grifoni, S. E. McKey, and H. A. Drummond
Hsc70 regulates cell surface ASIC2 expression and vascular smooth muscle cell migration
Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2022 - H2030.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
C. M. Fuller and D. J. Benos
Putting the brakes on vascular smooth muscle cell migration
Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H1987 - H1988.
[Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
C. Norez, F. Bilan, A. Kitzis, Y. Mettey, and F. Becq
Proteasome-Dependent Pharmacological Rescue of Cystic Fibrosis Transmembrane Conductance Regulator Revealed by Mutation of Glycine 622
J. Pharmacol. Exp. Ther., April 1, 2008; 325(1): 89 - 99.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
N. Vij, M. O. Amoako, S. Mazur, and P. L. Zeitlin
CHOP Transcription Factor Mediates IL-8 Signaling in Cystic Fibrosis Bronchial Epithelial Cells
Am. J. Respir. Cell Mol. Biol., February 1, 2008; 38(2): 176 - 184.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
Z. Jin, I. Tietjen, L. Bu, L. Liu-Yesucevitz, S. K. Gaur, C. A. Walsh, and X. Piao
Disease-associated mutations affect GPR56 protein trafficking and cell surface expression
Hum. Mol. Genet., August 15, 2007; 16(16): 1972 - 1985.
[Abstract] [Full Text] [PDF]

Home page
 Neuro Oncol DukeHome page
M. W. Graner and D. D. Bigner
Chaperone proteins and brain tumors: Potential targets and possible therapeutics
Neuro-oncol, July 1, 2005; 7(3): 260 - 278.
[Abstract] [PDF]

Home page
 J. Biol. Chem.Home page
S. W. Fewell, C. M. Smith, M. A. Lyon, T. P. Dumitrescu, P. Wipf, B. W. Day, and J. L. Brodsky
Small Molecule Modulators of Endogenous and Co-chaperone-stimulated Hsp70 ATPase Activity
J. Biol. Chem., December 3, 2004; 279(49): 51131 - 51140.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
E. E. Deane and N. Y. S. Woo
Differential gene expression associated with euryhalinity in sea bream (Sparus sarba)
Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2004; 287(5): R1054 - R1063.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
M. Lim, K. McKenzie, A. D. Floyd, E. Kwon, and P. L. Zeitlin
Modulation of {Delta}F508 Cystic Fibrosis Transmembrane Regulator Trafficking and Function with 4-Phenylbutyrate and Flavonoids
Am. J. Respir. Cell Mol. Biol., September 1, 2004; 31(3): 351 - 357.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
E. Cormet-Boyaka, M. Jablonsky, A. P. Naren, P. L. Jackson, D. D. Muccio, and K. L. Kirk
Rescuing cystic fibrosis transmembrane conductance regulator (CFTR)-processing mutants by transcomplementation
PNAS, May 25, 2004; 101(21): 8221 - 8226.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
J. M. Wright, P. L. Zeitlin, L. Cebotaru, S. E. Guggino, and W. B. Guggino
Gene expression profile analysis of 4-phenylbutyrate treatment of IB3-1 bronchial epithelial cell line demonstrates a major influence on heat-shock proteins
Physiol Genomics, January 15, 2004; 16(2): 204 - 211.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
P. L. Zeitlin, D. B. Gail, and S. Banks-Schlegel
Protein Processing and Degradation in Pulmonary Health and Disease
Am. J. Respir. Cell Mol. Biol., November 1, 2003; 29(5): 642 - 645.
[Full Text]

Home page
 Am. J. Respir. Cell Mol. Bio.Home page
B. R. Cobb, L. Fan, T. E. Kovacs, E. J. Sorscher, and J. P. Clancy
Adenosine Receptors and Phosphodiesterase Inhibitors Stimulate Cl- Secretion in Calu-3 Cells
Am. J. Respir. Cell Mol. Biol., September 1, 2003; 29(3): 410 - 418.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
C. A. Bertrand and R. A. Frizzell
The role of regulated CFTR trafficking in epithelial secretion
Am J Physiol Cell Physiol, July 1, 2003; 285(1): C1 - C18.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
J. Zabner, P. Karp, M. Seiler, S. L. Phillips, C. J. Mitchell, M. Saavedra, M. Welsh, and A. J. Klingelhutz
Development of cystic fibrosis and noncystic fibrosis airway cell lines
Am J Physiol Lung Cell Mol Physiol, May 1, 2003; 284(5): L844 - L854.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
W.-J. Wang, S. Mulugeta, S. J. Russo, and M. F. Beers
Deletion of exon 4 from human surfactant protein C results in aggresome formation and generation of a dominant negative
J. Cell Sci., February 15, 2003; 116(4): 683 - 692.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. F. Poschet, J. Skidmore, J. C. Boucher, A. M. Firoved, R. W. Van Dyke, and V. Deretic
Hyperacidification of Cellubrevin Endocytic Compartments and Defective Endosomal Recycling in Cystic Fibrosis Respiratory Epithelial Cells
J. Biol. Chem., April 12, 2002; 277(16): 13959 - 13965.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Suaud, J. Li, Q. Jiang, R. C. Rubenstein, and T. R. Kleyman
Genistein Restores Functional Interactions between Delta F508-CFTR and ENaC in Xenopus Oocytes
J. Biol. Chem., March 8, 2002; 277(11): 8928 - 8933.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
J. L. Brodsky
Chaperoning the maturation of the cystic fibrosis transmembrane conductance regulator
Am J Physiol Lung Cell Mol Physiol, July 1, 2001; 281(1): L39 - L42.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
R. C. Rubenstein and B. M. Lyons
Sodium 4-phenylbutyrate downregulates HSC70 expression by facilitating mRNA degradation
Am J Physiol Lung Cell Mol Physiol, July 1, 2001; 281(1): L43 - L51.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
L. R. Choo-Kang and P. L. Zeitlin
Induction of HSP70 promotes {Delta}F508 CFTR trafficking
Am J Physiol Lung Cell Mol Physiol, July 1, 2001; 281(1): L58 - L68.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
G. Baroudi, V. Pouliot, I. Denjoy, P. Guicheney, A. Shrier, and M. Chahine
Novel Mechanism for Brugada Syndrome : Defective Surface Localization of an SCN5A Mutant (R1432G)
Circ. Res., June 22, 2001; 88 (12): e78 - e83.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Biol. CellHome page
Y. Zhang, G. Nijbroek, M. L. Sullivan, A. A. McCracken, S. C. Watkins, S. Michaelis, and J. L. Brodsky
Hsp70 Molecular Chaperone Facilitates Endoplasmic Reticulum-associated Protein Degradation of Cystic Fibrosis Transmembrane Conductance Regulator in Yeast
Mol. Biol. Cell, May 1, 2001; 12(5): 1303 - 1314.
[Abstract] [Full Text]

Home page
 Mol. Interv.Home page
H. B. Pollard, O. Eidelman, K. A. Jacobson, and M. Srivastava
Pharmacogenomics of Cystic Fibrosis
Mol. Interv., April 1, 2001; 1(1): 54 - 63.
[Abstract] [Full Text]

Home page
 Am. J. Physiol. Cell Physiol.Home page
N. A. Bradbury
Focus on "Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of Delta F508-CFTR"
Am J Physiol Cell Physiol, February 1, 2000; 278(2): C257 - C258.
[Full Text] [PDF]

Home page
 Circ. Res.Home page
G. Baroudi, S. Acharfi, C. Larouche, and M. Chahine
Expression and Intracellular Localization of an SCN5A Double Mutant R1232W/T1620M Implicated in Brugada Syndrome
Circ. Res., January 11, 2002; 90 (1): e11 - e16.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (90)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rubenstein, R. C.
Right arrow Articles by Zeitlin, P. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rubenstein, R. C.
Right arrow Articles by Zeitlin, P. L.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online