1 Division of Pulmonary Medicine, Children's Hospital of Philadelphia, and 2 Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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Intracellular trafficking of the F508
cystic fibrosis transmembrane conductance regulator (CFTR) is repaired
by sodium 4-phenylbutyrate (4PBA) by an undetermined mechanism. 4PBA
downregulates protein and mRNA expression of the heat shock cognate
protein HSC70 (the constitutively expressed member of the 70-kDa heat
shock protein family) by ~40-50% and decreases formation of a
HSC70-
F508 CFTR complex that may be important in the intracellular
degradation of
F508 CFTR. We examined the potential mechanisms by
which 4PBA decreases HSC70 mRNA and protein expression. In IB3-1 cells,
1 mM 4PBA did not alter the activity of the Chinese hamster ovary HSC70
promoter or of a human HSC70 promoter fragment in luciferase reporter
assays nor did it alter HSC70 mRNA synthesis in nuclear runoff assays.
In contrast, preincubation with 4PBA increased the rate of HSC70 mRNA
degradation by ~40%. The initial rate of 35S-HSC70
protein synthesis in 4PBA-treated IB3-1 cells was reduced by ~40%,
consistent with the steady-state mRNA level, whereas its rate of
degradation was unaltered by 4PBA. 4PBA also reduced the steady-state
accumulation of 35S-HSC70 by ~40%. These data suggest
that 4PBA decreases the expression of HSC70 mRNA and protein by
inducing cellular adaptations that result in the decreased stability of
HSC70 mRNA.
cystic fibrosis; cystic fibrosis transmembrane conductance regulator chaperones; 70-kilodalton heat shock protein constitutive form; messenger ribonucleic acid
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INTRODUCTION |
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THE
MOST COMMON MUTATION of the cystic fibrosis (CF) transmembrane
conductance regulator (F508 CFTR) is a temperature-sensitive trafficking mutant (8).
F508 CFTR is retained in the
endoplasmic reticulum where it has prolonged associations with calnexin
(22) and the 70-kDa heat shock protein family
(32) and forms a functional protein (21).
F508 CFTR is targeted for rapid intracellular degradation
(27), at least in part, by the ubiquitin-proteasome system
(14, 28) and does not reach its appropriate subcellular location at the apical plasma membrane (6, 16). It is thus an attractive target for pharmaceutical therapies aimed at correcting its trafficking defect.
A number of in vitro manipulations, including protein stabilizing
agents or chemical chaperones (2, 26), the transcriptional regulator butyrate (5), and the immunomodulator
deoxyspergualin (15) also result in correction of the
trafficking defect of F508 CFTR and cell surface CFTR function in
vitro. Previous work by Rubenstein et al. (23) has
shown that sodium 4-phenylbutyrate (4PBA), a butyrate analog that is
approved for pharmaceutical use, similarly corrects the trafficking
defect of
F508 CFTR and restores CFTR function at the plasma
membrane of cultured CF epithelial cells. Rubenstein and Zeitlin
(24) also recently demonstrated that 4PBA caused a small
but significant improvement in nasal epithelial Cl
transport in
F508 homozygous CF patients. Similar effects of 4PBA to
improve
F508 CFTR trafficking and function in vitro have recently
been reported in other cell lines (12). However, the mechanism of 4PBA action remains elusive.
4PBA, like butyrate, regulates gene transcription, although apparently
not of CFTR or F508 CFTR (11, 23). While testing the
hypothesis that 4PBA might regulate the transcription of a protein in
the intracellular protein trafficking pathway, Rubenstein and Zeitlin
(25) observed that 4PBA as well as butyrate decreases the
expression of the heat shock cognate protein HSC70 (the constitutively expressed member of the 70-kDa heat shock protein family). They also
observed that HSC70 forms a complex with
F508 CFTR and that the
formation of this complex decreases with 4PBA or butyrate treatment. Because HSC70 has a role in the lysosomal
degradation of intracellular proteins (7, 9) and is
required for the ubiquitin-dependent degradation of a number of
cellular proteins (1), the rapid intracellular degradation
of
F508 CFTR might result from a prolonged association of
F508
CFTR and HSC70. This model is supported by deoxyspergualin improving
F508 CFTR trafficking because deoxyspergualin is a competitive
inhibitor of protein binding to HSC70 and the 90-kDa heat shock protein
(HSP90) (15). The observation that inhibition of HSP90
function with geldanamycin inhibits CFTR trafficking and promotes CFTR
degradation (19) suggests that modulation of HSC70
activity with deoxyspergualin is more likely its mechanism of
action in improving CFTR trafficking than its modulation of HSP90.
The aim of the present studies was to delineate the cellular mechanism by which 4PBA leads to a decrease in steady-state expression of HSC70 mRNA and protein. 4PBA and other butyrates are transcriptional regulators that can modify histone deacetylase activity and chromatin structure (4). We therefore anticipated that we would observe altered (decreased) transcription of HSC70 mRNA after 4PBA treatment. In contrast, we observed that 4PBA did little to HSC70 promoter activity, rate of mRNA synthesis, or protein turnover. Rather, the predominant effect of 4PBA was to induce a cellular adaptation that resulted in enhanced degradation of HSC70 mRNA.
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METHODS |
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Cell culture. IB3-1 cells (33) were grown on uncoated tissue culture plasticware in a 5% CO2 incubator at 37°C. 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, Life Technologies, 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 the control experiments were cultured under these routine conditions. The growth medium for the treated cells was composed of the indicated concentrations of 4PBA added to the routine growth medium and incubated at 37°C in a 5% CO2 incubator. Rubenstein et al. (23) have previously determined that 4PBA maintains a constant concentration under these culture conditions for at least 2 days.
Antibodies. A rabbit polyclonal antiserum specific for HSC70 (3) was a generous gift from Dr. W. J. Welch (University of California, San Francisco). A rat monoclonal antibody specific for HSC70, clone 1B5, was purchased from Stressgen Biotechnologies (Victoria, BC).
Reporter gene construction and assay.
A pSK() plasmid (Bluescript, Stratagene, La Jolla, CA) containing a
1.8-kb fragment of Cricetulus griseus [Chinese hamster ovary (CHO)] genomic DNA comprising the 5'-upstream or promoter region
of the HSC70 gene was a gift from Dr. Andrei Laszlo (Washington University, St. Louis, MO). Orientation and identity of the insert were
confirmed by sequencing in the Nucleic Acid and Protein Core Facility
of the Children's Hospital of Philadelphia. With standard techniques,
the 1.8-kb promoter fragment was excised from the pSK(
) vector with
HindIII and XbaI and inserted in the forward orientation into the HindIII and NheI sites of
the pGL3 basic luciferase reporter vector (Promega, Madison, WI). The
reverse orientation was similarly constructed by ligating a
KpnI-NheI fragment from the pSK(
) plasmid into
the KpnI and XbaI sites of pGL3 basic.
Orientation was confirmed by restriction mapping.
Nuclear runoff assays.
Nuclei from control or 4PBA-treated IB3-1 cells were prepared by
hypotonic lysis and Dounce homogenization essentially as described
(10) and stored at 80°C in 50 mM Tris-Cl (pH 8.3), 5 mM MgCl2, 0.1 mM EDTA, and 40% (vol/vol) glycerol before
use. Approximately 1 × 107 nuclei/reaction (as
determined by counting with a hemacytometer) were run off for 30 min at
30°C after the addition of an equal volume of 2× reaction buffer
containing 10 mM Tris-Cl (pH 8.0), 5 mM MgCl2, 0.3 M KCl, 5 mM dithiothreitol (Sigma), 1 mM ATP, 1 mM CTP, 1 mM GTP (Ultrapure
nucleotide triphosphates, Pharmacia Biotechnologies, Piscataway, NJ),
and 12 µCi of [
-32P]UTP (NEN, Boston, MA). Runoff
RNA was isolated with the RNAwiz reagent (Ambion, Austin, TX) according
to the manufacturer's protocol and resuspended in Perfect Hyb Plus
(Sigma) for hybridization.
RNase protection.
RNase protection experiments were performed essentially as previously
described (25) with the Direct Protect ribonuclease protection assay kit (Ambion). The template for the HSC70 probe was
derived from exons 8 and 9 of the human HSC70 gene and the template for
the control probe was pTRI RNA 18S (Ambion), both as previously
described (25). The template for human -actin (pTRI-
-actin) was similarly purchased from Ambion. IB3-1 cell lysates were prepared in Direct Protect lysis buffer according to the
manufacturer's protocol after incubation under the appropriate condition. [
-32P]UTP-labeled probes [50-70
kilocounts/min (kcpm) for HSC70, 5-10 kcpm for the RNA 18S
control, and ~100 kcpm for
-actin] were synthesized with
a MAXIscript T7 kit (Ambion), isolated by acid phenol-chloroform
(Ambion) extraction, separated from unincorporated nucleotide by gel
filtration (Sephadex G25 RNA spin column, Boehringer Mannheim), and
precipitated with ethanol-acetate before resuspension in hybridization
buffer. The radioactivity in the synthesized probes was determined by
liquid scintillation counting. The probes were then hybridized with
cellular RNA overnight at 37°C and digested with a RNase cocktail.
Protected fragments were resolved by electrophoresis on 5%
acrylamide-8 M urea gels and detected by fluorography. Fluorographic bands representing HSC70 mRNA and 18S rRNA hybridization were quantitated by densitometry (see Densitometric analysis).
35S protein labeling and immunoprecipitation. IB3-1 cells pretreated under control conditions or with 1 mM 4PBA were starved of methionine and cysteine by incubation in methionine-, cysteine-, and serum-free bronchial epithelial cell basal labeling medium (Clonetics, San Diego, CA) for 15 min. EasyTag Expre35S35S protein labeling kit (a mixture of [35S]methionine and [35S]cysteine, 100-300 µCi/35-mm dish; NEN) was then added in bronchial epithelial cell basal labeling medium. For pulse-chase experiments, the cells were pulse labeled with [35S]methionine-cysteine for 20 min, washed with buffered saline solution, and chased in standard growth medium containing 50 mM methionine and 10 mM cysteine.
The cell lysates for immunoprecipitation were prepared by solubilization for 1 h at 4°C in radioimmunoprecipitation assay (RIPA) buffer (50 mM Tris-Cl, pH 8.0, 150 mM NaCl, and 1% Triton X-100; Fisher Scientific, Pittsburgh, PA), 1% sodium deoxycholate (Sigma), 0.1% SDS (Fisher Scientific), and protease inhibitor cocktail (used at a 1:1,000 final dilution; Sigma). Solubilized cells were then homogenized by passage seven times through a 20-gauge needle and cleared by centrifugation at 15,000 g for 20 min at 4°C. The protein concentration was determined with Bio-Rad DC reagents (Bio-Rad, Hercules, CA). HSC70 polyclonal antiserum (1.5 µl) or rat monoclonal antibody (2.5 µg) was added to the cell lysates (100 µg of total protein with equal amounts of protein at equal final concentrations for each condition within an experiment) and incubated at room temperature for 2 h with gentle agitation. Immune complexes were captured with protein A Sepharose 4B (for the polyclonal antiserum) or GammaBind Plus (protein G Sepharose for the monoclonal antibody; both from Pharmacia) that had been preabsorbed with bovine serum albumin for 60 min at room temperature. Precipitated complexes were collected by centrifugation and washed twice with cold RIPA buffer and twice with cold Tris-buffered saline (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 7% SDS-polyacrylamide gels. Immunoprecipitated HSC70 was detected by fluorography, and the band intensity was quantitated by densitometry (see Densitometric analysis).Densitometric analysis. Fluorographic images were digitized with an AlphaImager 2000 digital analysis system (AlphaInnotech, San Leandro, CA). Densitometric analysis of these images was performed with 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 "0-h" lanes for control and 4PBA treatment (see Fig. 4), the "240-min" control (see Fig. 5), and the 0-h control (see Fig. 6) were individually and arbitrarily set to 1, with the remaining densities expressed relative to this reference density.Reagents. Pharmaceutical-grade 4PBA, manufactured by Triple Crown America (Perkasie, PA), was a gift from Dr. Saul Brusilow (The Johns Hopkins School of Medicine, Baltimore, MD). Electrophoresis-grade chemicals were obtained from Fisher, Bio-Rad, or GIBCO BRL. Restriction enzymes and other reagents for plasmid manipulation were from Promega. All other reagents were of reagent grade or better.
Statistical analysis. All statistical analysis and curve fitting were performed with SPSS software version 7.0. Apparent first-order rates of decay (see Figs. 4 and 6) were estimated for each individual time course based on the relative density remaining, and these rate constants were averaged and compared. Significance (P value) was determined by the nonparametric Mann-Whitney U-test or, where appropriate, one-way ANOVA.
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RESULTS |
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4PBA treatment of IB3-1 cells does not affect the synthesis of
HSC70 mRNA.
Our laboratory has previously demonstrated in the immortalized CF
bronchiolar epithelial cell line IB3-1 (genotype F508/W1282X) (33) that 4PBA results in an ~40-50% reduction in
steady-state expression of HSC70 mRNA and protein (25) and
improved
F508 CFTR intracellular trafficking (23). We
assessed whether this reduction in HSC70 mRNA was a result of
inhibition of HSC70 promoter activity by 4PBA in a luciferase reporter
system. A 1.8-kb fragment from the 5'-untranslated region of the
Cricetulus griseus (CHO) HSC70 gene inserted into the pGL3
basic luciferase reporter vector efficiently directed reporter gene
expression (Fig. 1). This
promoter fragment was ~1,000-fold less efficient when inserted into
the reporter plasmid in the reverse orientation (data not shown). Deletion mutagenesis revealed that a 5' deletion of up to ~800 bp of
this promoter did not significantly alter the activity of the promoter,
whereas deletion of the
1.0-
0.6-kb region, which contains
numerous transcription factor consensus binding sequences and the
untranslated exon 1, resulted in significantly decreased promoter
activity. Interestingly, none of these promoter constructs had
significantly altered activity in the presence of 4PBA, suggesting that
4PBA does not directly influence the activity of the HSC70 promoter.
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4PBA treatment enhances HSC70 mRNA degradation.
We next assessed the influence of 4PBA on the rate of HSC70 mRNA
degradation in IB3-1 cells. After pretreatment with 1 mM 4PBA for
24 h, the apparent first-order rate of disappearance of HSC70 mRNA
(with mRNA synthesis inhibited by actinomycin D) was increased
~1.4-fold (Fig. 4). This effect
required preincubation of the cells with 4PBA; it was not present if
4PBA was added only during the incubation with actinomycin D (Fig.
4C). Similarly, the presence of 4PBA during the incubation
with actinomycin D was not required to sustain the increased rate of
HSC70 mRNA degradation after 4PBA pretreatment. These data suggest that
4PBA decreases the steady-state expression of HSC70 mRNA primarily by
increasing the rate of HSC70 mRNA degradation. This effect requires
preincubation of IB3-1 cells with 4PBA, further suggesting that this
effect results from an overall cellular adaptation to 4PBA treatment rather than an acute effect.
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The initial rate of HSC70 protein synthesis reflects steady-state
mRNA expression.
To begin to assess the influence of 4PBA on HSC70 protein turnover, we
characterized the initial rate of HSC70 protein synthesis (Fig.
5). The initial rate of
35S-HSC70 synthesis for 20 min in 4PBA-treated IB3-1 cells
was ~60% of that in control cells [4PBA, 0.017 ± 0.002 (SE)
vs. control, 0.029 ± 0.004 min1; n = 5 independent experiments]. These data correlate well with previously reported measurements by Rubenstein and Zeitlin
(25) of steady-state HSC70 mRNA expression after 4PBA
treatment. These data suggest that that the rate of synthesis of
35S-HSC70 is proportional to and determined by the
steady-state concentration of HSC70 mRNA and that 4PBA treatment does
not influence the rate of translation of HSC70 mRNA by the ribosome.
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4PBA does not alter the kinetics of 35S-HSC70
degradation.
The kinetics of newly synthesized 35S-HSC70 degradation
were assessed in standard pulse-chase experiments (Fig.
6). IB3-1 cells pretreated under
control conditions or with 1 mM 4PBA had the same median
apparent first-order rate constant of 35S-HSC70 degradation
(control, 0.056 vs. 4PBA, 0.063 min1; P = 0.297 by Mann-Whitney U-test). Similar apparent first-order rate constants were observed when control cells were chased in the
presence of 1 mM 4PBA or when 4PBA-treated cells were chased in the
absence of 4PBA (data not shown). These rate constants were
significantly greater than those observed for HSC70 mRNA degradation
(Fig. 4), confirming that HSC70 protein turnover is more rapid than
HSC70 mRNA turnover.
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DISCUSSION |
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The present studies extend previous observations by Rubenstein and
Zeitlin (25) of the downregulation of HSC70 expression in
IB3-1 cells with 4PBA and butyrate by investigating the mechanism by
which 4PBA causes this effect. The butyrates, including 4PBA, are
generally thought to be transcriptional regulators that act by altering
chromatin structure via the modulation of histone deacetylase activity
(4). Surprisingly, our data do not support this mechanism
of action for 4PBA on HSC70 mRNA synthesis. In addition to HSC70
promoter activity not being altered by 4PBA, the synthesis of nascent
HSC70 mRNA transcripts was unaffected by 4PBA. Rather, our data suggest
that the F508 CFTR protein repair agent 4PBA acts primarily to
enhance the turnover of HSC70 mRNA by speeding its degradation.
These observations are still consistent with 4PBA acting as a transcriptional regulator. The HSC70 promoter has a number of putative binding sites for transcription factors. Although the present studies do not explicitly address this, the observed lack of effect of 4PBA on HSC70 promoter activity and mRNA synthesis may be a result of an increase in the activity of one transcription factor balanced by a concomitant decrease in the activity of another. Modulation of HSC70 promoter activity has been reported. For example, activity of the rat HSC70 promoter can be stimulated by treatment of transfected neuroblastoma cells with ethanol. An Sp1 consensus binding site is critical for this response to ethanol in the context of this promoter region, although ethanol treatment does not significantly alter Sp1 binding to the promoter. These observations suggest that another transcription factor(s) is regulated by ethanol and, in turn, modulates the activity of Sp1 through an interaction with Sp1 and/or that region of the HSC70 promoter (30).
Furthermore, the effect of 4PBA on HSC70 mRNA degradation required that the cells be pretreated with 4PBA. The effect was not present if 4PBA was added acutely at the same time as an inhibitor of mRNA synthesis, actinomycin D, and the effect did not quickly reverse if 4PBA was removed on inhibition of mRNA synthesis by actinomycin D. These data suggest that 4PBA causes a global cellular adaptation that requires mRNA synthesis, one of the results of this cellular adaptation being the enhancement of HSC70 mRNA degradation. Such alterations in cellular mRNA synthesis and protein expression could result from the effect of 4PBA on histone deacetylase activity and subsequent alterations in chromatin structure at genes other than HSC70.
AUUUA pentamers in the 3'-untranslated region of a mRNA have long been associated with destabilizing that mRNA species. The presence of these AUUUA pentamers serves to speed the removal of polyadenylation sequences and thus enhance mRNA turnover. Proteins such as AUF1 and HuR appear to bind to these AU-rich regions and regulate degradation (18) or stabilization (20), respectively, of these mRNAs.
The HSC70 gene has two nonoverlapping AUUUA motifs in its
3'-untranslated region. Significant destabilization of mRNA is better correlated with multiple and/or overlapping AUUUA motifs
(31). This may be reflected by the half-life that we
observed for HSC70 mRNA being much longer than that of the mRNA of
genes (e.g., cytokines such as granulocyte-macrophage
colony-stimulating factor or tumor necrosis factor-) where there are
multiple or overlapping AUUUA elements.
The physiological relevance of AUF1 and HuR in regulating the degradation of HSC70 mRNA is not known. It is also not known whether the action of 4PBA would lead to modulation of these proteins, although there are suggestions that interrelationships exist. AUF1 coimmunoprecipitates with HSC70 in HeLa cells and is degraded primarily by the ubiquitin- proteasome pathway. Proteasome inhibition leads to stabilization of AUUUA-containing mRNAs (17). Recent evidence suggests that the proteasome itself may contain endonuclease activity that will recognize and cleave mRNAs containing multiple or overlapping AUUUA elements (13). Because HSC70 is also found colocalized with the proteasome (29) and is a necessary cofactor for the ubiquitination and proteasome degradation of a number of cellular proteins (1), pharmacological modulations of the interaction of AUF1 with HSC70 could lead to alterations in the function of AUF1 and resulting alterations in cellular mRNA stability.
This hypothetical mechanism is similar to that which Rubenstein and
Zeitlin (25) recently proposed for 4PBA altering the trafficking of F508 CFTR, namely a decrease in HSC70, leading to the
target protein escaping proteasomal degradation. The target protein
would then retain its function and stimulate mRNA turnover (for AUF1)
or be allowed to traffic to the cell surface (for
F508 CFTR). The
major weakness of this hypothetical mechanism is that it does not
explain a priori the process by which the cell, in a response to 4PBA
that requires mRNA synthesis, initiates more rapid degradation of HSC70 mRNA.
In contrast to the effect of 4PBA on HSC70 mRNA, the cellular adaptation induced by 4PBA apparently does not affect the turnover kinetics of the protein. The initial rates of HSC70 synthesis in the presence and absence of 4PBA reflect the respective steady-state levels of HSC70 mRNA, suggesting that 4PBA treatment does not influence the rate of translation of HSC70 mRNA by the ribosome. Similarly, the apparent first-order rate of degradation of newly synthesized HSC70 protein is unaltered by 4PBA treatment, suggesting that the cellular adaptation to 4PBA does not alter the catabolism of HSC70 protein. These plots of the fraction of HSC70 remaining versus time are somewhat curvilinear, suggesting that the degradation kinetics are more complicated, perhaps reflective of "subpopulations" of HSC70 in the cell. Given the multitude of cellular functions attributed to HSC70, such a finding or supposition is not unexpected. Even with this curvilinearity, the plots of the fraction of HSC70 remaining versus time remain parallel, which is again consistent with 4PBA not altering the turnover or degradative pathway(s) of HSC70. The rates of HSC70 protein are significantly higher than the rates of HSC70 mRNA degradation. This is necessary for the expression level of a protein to correlate with and reflect steady-state mRNA expression.
In conclusion, we have demonstrated that 4PBA stimulates the degradation of HSC70 mRNA in IB3-1 cells without significantly altering HSC70 mRNA synthesis, promoter activity, or protein turnover. This response, which was sufficient to account for the previously observed decrease in steady-state HSC70 mRNA expression by Rubenstein and Zeitlin (25), required a preincubation with 4PBA, suggesting that it resulted from a cellular adaptation to 4PBA treatment. Further study, including experiments in other cell lines, will be required to delineate the mechanism by which 4PBA, a transcriptional regulator, induces this cellular adaptation, including altered HSC70 mRNA degradation, and whether this adaptation is a general response to 4PBA treatment.
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ACKNOWLEDGEMENTS |
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We thank Drs. Paul Anziano and Thomas R. Kleyman for helpful discussions and Dr. Kleyman for a critical review of the manuscript.
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FOOTNOTES |
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These studies were supported by Cystic Fibrosis Foundation Grant R881, Leroy Matthews Individual Physician Scientist Award RUBENS96LO, and National Institute of Diabetes and Digestive and Kidney Diseases Grant R01-DK-58046 (to R. C. Rubenstein).
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: 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).
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 11 September 2000; accepted in final form 30 November 2000.
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