EDITORIAL FOCUS
Induction of HSP70 promotes Delta F508 CFTR trafficking

Lee R. Choo-Kang and Pamela L. Zeitlin

Eudowood Division of Pediatric Respiratory Sciences, Department of Pediatrics, Johns Hopkins Medical Institutions, Baltimore, Maryland 21287-2533


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The Delta 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 Delta 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 Delta 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 Delta 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 Delta F508 CFTR maturation and trafficking.

cystic fibrosis; cystic fibrosis transmembrane conductance regulator; 70-kilodalton heat shock protein chaperones; phenylbutyrate; plasmid transfection; glutamine


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Delta 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 Delta 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 Delta 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 Delta F508 CFTR on the cell surface. Small-chain fatty acid derivatives of butyric acid, including sodium 4-phenylbutyrate (4PBA), promote Delta 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 Delta 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 Delta 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 Delta 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-Delta 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 Delta F508 CFTR maturation.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 gamma -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.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

4PBA treatment of IB3-1 cells increases expression of HSP70 and promotes HSP70-Delta F508 CFTR interaction at the expense of HSC70-Delta F508 CFTR complex formation. We studied the immortalized CF bronchial epithelial cell line IB3-1 (66). IB3-1 has the CFTR genotype Delta F508/W1282X but expresses only Delta 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 Delta 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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Fig. 1.   Dose-dependent increase in cystic fibrosis transmembrane conductance regulator (CFTR) band C is mediated by sodium 4-phenylbutyrate (4PBA). A: IB3-1 cells were incubated with the indicated concentrations of 4PBA for 48 h. Protein lysates were prepared with radioimmunoprecipitation assay (RIPA) buffer containing freshly added inhibitors as described in METHODS. Total protein (15 µg) was resolved on 9% SDS-PAGE. Proteins were electrophoretically transferred to nitrocellulose, and immunodetection of CFTR was performed as described in METHODS. Primary antiserum was rabbit anti-CFTR antiserum 181. Band B, position of immature CFTR. Nos. on left, molecular mass in kDa. B: densitometry was performed as described in METHODS. Density of 0-4PBA (control) lane was set to 100, and values are means ± SE expressed relative to control value from a total of 6 immunoblots from 6 individual experiments. * P < 0.05 vs. control by 1-way ANOVA followed by the least significant difference (LSD) multiple comparison test. C: CFTR densitometry standard curve construction. Immunodetection of CFTR band C in indicated amounts of IB3-1 lysate protein was performed as described in METHODS. D: densitometric analysis was performed on experiment in C and 3 similar experiments as described in METHODS. Density of 25-µg lane was set to 1, and values are means ± SE expressed relative to 1.

To better quantify the change produced by 4PBA as well as by subsequent experimental conditions, we constructed a densitometric standard curve of CFTR band C immunoreactivity using protein lysate from IB3-1 cells grown at 27°C (Fig. 1, C and D). Although the rate of change of the densitometric signal is less than the absolute change in IB3-1 protein, our standard curve suggests that CFTR band C immunoreactivity as detected by densitometry is linearly related to the amount of CFTR protein between 5 and 25 µg of IB3-1 lysate. CFTR immunoreactivity was not consistently detected in samples containing <= 2.5 µg of IB3-1 protein. Thus even the smallest (approximately twofold) increase in the CFTR band C densitometric signal seen with lower concentrations of 4PBA reflects a greater increase in actual protein expression.

Because 4PBA treatment has been previously shown to reduce cellular HSC70 protein levels and to decrease HSC70-Delta F508 CFTR interaction (52) and because HSC70 inhibition induces HSP70 in other models (2, 3), we chose to examine the effects of 4PBA (10 µM to 5 mM) on HSP70 levels in IB3-1 cells. As shown in Fig. 2, 4PBA treatment increased total cellular HSP70 immunoreactivity as detected by a HSP70-specific mouse monoclonal antibody (Fig. 2, A and B). In comparison, HSP70 protein levels did not demonstrate a consistent or significant change when the cells were incubated at 27°C.


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Fig. 2.   4PBA treatment induces total cellular 70-kDa heat shock protein (HSP70) expression and increases the amount of HSP70 coimmunoprecipitated with Delta F508 CFTR. A: IB3-1 cells were grown in indicated concentrations of 4PBA for 48 h. Cells were solubilized with RIPA buffer, and 15 µg of total protein were analyzed on 9% SDS-PAGE. Immunodetection for HSP70 was performed with a mouse HSP70-specific monoclonal antibody as described in METHODS. B: densitometric analysis of immunoblot in A and similar immunoblots (7 independent experiments) was performed as in Fig. 1. Values are means ± SE relative to control value for both total HSP70 and HSP70 complexed with CFTR. For comparison with total cellular HSP70 expression, densitometric analysis of immunoblot in E and 6 similar immunoblots (7 independent experiments) demonstrates a corresponding increase in amount of HSP70 coimmunoprecipitated with Delta F508 CFTR after 4PBA treatment. Significantly different compared with control (P < 0.05): * total HSP70; + HSP70 complexed for CFTR (both by 1-way ANOVA followed by the LSD multiple comparison test). C: standard curve construction. IB3-1 lysate protein (15 µg) or indicated amount of purified recombinant human HSP70 was resolved on 9% SDS-PAGE. Proteins were electrophoretically transferred to nitrocellulose, and immunodetection of HSP70 was performed as described in METHODS. Primary antiserum was mouse monoclonal antiserum specific for HSP70. D: densitometry was performed as described in METHODS. black-lozenge , Mean relative density; diamond , relative density of 15 µg of IB3-1 lysate, corresponding to ~8.7 ng of human HSP70 immunoreactivity. Density of 10-ng lane was set to 1, and values are means ± SE expressed relative to 1 from 4 identical experiments; error bars not seen are within symbols. E: analysis of protein complex isolated with anti-CFTR antibody 169. After treatment with 4PBA, IB3-1 cells were solubilized, and 800 µg of total protein were incubated with anti-CFTR antibody 169 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 with mouse monoclonal anti-HSP70-specific antibody, rat monoclonal anti-70-kDa heat shock cognate protein (HSC70)-specific antibody, and anti-CFTR antiserum 181 as described in METHODS. Nos. on left, molecular mass in kDa.

To estimate the increase in HSP70 protein represented by the two- to threefold increase in total HSP70 immunoreactivity after 4PBA treatment, a densitometric standard curve of immunoreactivity for recombinant human HSP70 was constructed (Fig. 2, C and D). We performed both log and linear regressions for the data in Fig. 2D, and both were acceptable fits (r2 for log and linear were 0.9443 and 0.9682, respectively). Using the linear model (y = 0.1074x + 0.0139), we predicted that IB3-1 cells contain ~8.7 ng HSP70/15 µg total cellular protein. These data are consistent with 4PBA treatment inducing a two- to threefold increase in cellular HSP70 protein.

To test whether 4PBA would also alter the potential interaction between HSP70 and Delta F508 CFTR, we immunoprecipitated IB3-1 cell lysates with anti-CFTR antiserum 169 and probed with HSP70-specific mouse monoclonal antibody. 4PBA treatment increased the amount of HSP70 coimmunoprecipitated with Delta F508 CFTR in a dose-dependent manner (Fig. 2, B and E). We also confirmed that 4PBA reduces the amount of HSC70 associated with Delta F508 CFTR (52) by immunoblotting the resolved anti-CFTR antibody 169-protein complexes with anti-HSC70 monoclonal antiserum (Fig. 2E). Untreated IB3-1 cells incubated in a 5% CO2 atmosphere at 27°C for 48 h served as a positive trafficking control (15). Although incubation at 27°C did not significantly increase cellular HSP70 content, there was a sixfold increase in the amount of this chaperone associated with Delta F508 CFTR. To determine whether these observations were secondary to increased HSP70-substrate interaction or reflected the presence of more CFTR, we performed immunoblotting on the anti-CFTR antibody 169-protein complexes with anti-CFTR antibody 181. Although immature band B was the predominant form of CFTR immunoprecipitated by the anti-CFTR antibody 169, trace amounts of band C were also detected after treatment with 4PBA or incubation at 27°C (Fig. 2E). We detected no significant change in the amount of total CFTR immunoprecipitated with anti-CFTR antibody 169 after 4PBA or with growth at 27°C. Therefore, the increased HSP70-Delta F508 CFTR interaction observed after 4PBA treatment is likely secondary to the higher cellular content of HSP70. However, we cannot discount the possibility that the lowered temperature slows the rate of HSP70-Delta F508 CFTR complex dissociation and contributes to its accumulation.

We attempted the reciprocal experiment using anti-HSP70 monoclonal antiserum to immunoprecipitate HSP70-substrate complexes. Immunoblotting the resolved anti-HSP70 antibody-protein complexes with anti-CFTR antibody 181 failed to detect any CFTR. A likely explanation for this observation is the nuclear presence of the majority of HSP70 such that a very small fraction of HSP70 is available to associate with CFTR. On the other hand, there is a relatively larger fraction of CFTR available to associate with HSP70. Nevertheless, immunodetection with a monoclonal anti-HSC70-specific antibody revealed an association between the two HSP70 chaperones distinct from their interactions with CFTR (data not shown).

Increased HSP70 expression leads to higher molecular mass forms of Delta F508 CFTR. Because 4PBA may have multiple effects on cellular metabolism, we sought to determine the effect of isolated HSP70 overexpression on Delta 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 Delta 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 Delta 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 Delta F508 CFTR maturation over twofold, a degree similar to the growth of cells at 27°C.


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Fig. 3.   HSP70 overexpression by plasmid transfection promotes Delta F508 CFTR maturation. A: IB3-1 cells were grown to 80% confluence before transfection with an expression plasmid containing human HSP70 cDNA or mock transfected with an empty pcDNA plasmid. Cells were lysed at 2-day intervals for up to 6 days after transfection, and 15 µg of total protein were analyzed on 9% SDS-PAGE. Blots are mock-transfected cells lysed at 6 days posttransfection. Immunodetection for CFTR, HSP70, and HSC70 were performed as described in METHODS. Nos. on left, molecular mass in kDa. B: densitometric analysis of immunoblot in A and 5 other immunoblots (3 independent experiments done in duplicate) was performed as in Fig. 1. Values are means ± SE relative to values for untransfected control IB3-1 cells for total HSP70 and CFTR. Significantly different from control (P < 0.05): * HSP70; + CFTR band C (both by 1-way ANOVA followed by the LSD multiple comparison test).

Glutamine induces production of HSP70 and promotes trafficking of the Delta 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 Delta 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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Fig. 4.   Glutamine induces HSP70 expression and promotes Delta F508 CFTR maturation. A: IB3-1 cells were incubated with the indicated concentrations of glutamine for 48 h. Protein lysates were prepared in RIPA buffer, and total protein (15 µg) was resolved on 9% SDS-PAGE. Proteins were electrophoretically transferred to nitrocellulose, and immunodetection of CFTR, HSP70, and HSC70 was performed as described in METHODS. Nos. on left, molecular mass in kDa. B: densitometry was performed as described in METHODS for HSP70 and CFTR band C on a total of 6 immunoblots from 6 individual experiments. Density of 0 supplemental glutamine (control) lane was set to 100, and values are means ± SE expressed relative to the control value. Significantly different compared from control (P < 0.05): * HSP70; + CFTR band C (both by 1-way ANOVA followed by LSD multiple comparisons test).

Effect of HSP70 induction on Delta F508 CFTR localization. To provide additional support for the observation that 4PBA and glutamine alter the expression and localization of Delta F508 CFTR, we performed immunofluorescence microscopy studies. The vast majority of untreated IB3-1 cells expressed low levels of Delta 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 Delta F508 CFTR intracellular trafficking is increased expression of HSP70.


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Fig. 5.   Immunolocalization of Delta F508 CFTR (red) in IB3-1 cells after HSP70 (blue) induction by pharmacotherapy and plasmid transfection. HSC70 is indirectly visualized by the green signal. Untreated IB3-1 cells grown at 37°C (A) expressed mutant CFTR predominantly in a perinuclear location (arrowheads) similar to HSC70 expression. After incubation at 27°C (B) and treatment with 4PBA (C) or glutamine (D), there was an increase in CFTR signal intensity along with a more peripheral distribution (arrows). Mock-transfected cells (E) demonstrated an increase in HSP70 signal predominantly in the nucleus and no significant change in the perinuclear localization (arrowheads) of Delta F508 CFTR. Two days (F) after pcDNA-HSP70 transfection, there was still significant nuclear staining for HSP70 but an increased CFTR signal throughout the cytoplasm (arrows) that was more pronounced at 6 days (G) despite a decline in HSP70 signal intensity.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta F508 CFTR trafficking defect in respiratory epithelial cells. This conclusion is supported by the finding of increased HSP70-Delta F508 CFTR complex formation after 4PBA treatment as well as at lowered growth temperature, conditions that facilitate Delta F508 CFTR trafficking (15, 50, 51). Although we have shown that 4PBA can downregulate HSC70 under certain conditions and reduce HSC70-Delta 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 Delta F508 CFTR trafficking.

The most compelling evidence that HSP70 induction is sufficient to restore Delta 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 Delta 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 Delta 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), Delta 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 Delta 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 Delta 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 Delta F508 CFTR polypeptide. In this scenario, the increased HSP70-Delta 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 Delta 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 Delta F508 CFTR and HSP70, resulting in fewer HSC70-Delta 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 Delta 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 Delta F508 CFTR was sodium butyrate. This drug stimulates CFTR gene transcription (29) but is also likely to produce its effect on Delta 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 Delta 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 Delta F508 CFTR. Treatment of Delta 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 alpha 1-antitrypsin, PiZ, produces a trafficking mutant that shares some common features with Delta F508 CFTR. Recently, Novoradaskaya et al. (43) demonstrated a dose-dependent increase in the Z-mutant alpha 1-antitrypsin secretion after treatment with 4PBA in alveolar macrophages from PiZ individuals and in a Chinese hamster ovary cell line model expressing Z alpha 1-antitrypsin. Concurrent with the restoration of Z alpha 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 Delta 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 alpha 1-antitrypsin in different cell types after 4PBA treatment, Burrows et al. (9) observed an increase in blood levels of alpha 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 Delta 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 Delta F508 CFTR observed at lower glutamine concentrations.

The observations in this report suggest that HSP70 induction overcomes the Delta 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 Delta 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 Delta 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 Delta F508 mutation. Screening for compounds that upregulate HSP70 may accelerate the identification of molecules that might be beneficial in the therapy for CF.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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