EDITORIAL FOCUS
Sodium 4-phenylbutyrate downregulates HSC70 expression by facilitating mRNA degradation

Ronald C. Rubenstein1,2 and Bridget M. Lyons1

1 Division of Pulmonary Medicine, Children's Hospital of Philadelphia, and 2 Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Intracellular trafficking of the Delta 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-Delta F508 CFTR complex that may be important in the intracellular degradation of Delta 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE MOST COMMON MUTATION of the cystic fibrosis (CF) transmembrane conductance regulator (Delta F508 CFTR) is a temperature-sensitive trafficking mutant (8). Delta 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). Delta 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 Delta 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 Delta 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 Delta F508 homozygous CF patients. Similar effects of 4PBA to improve Delta 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 Delta 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 Delta 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 Delta F508 CFTR might result from a prolonged association of Delta F508 CFTR and HSC70. This model is supported by deoxyspergualin improving Delta 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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.

The CHO promoter 5' deletion mutations were constructed by recircularization of the pGL3 reporter plasmid containing the forwardly oriented promoter after digestion with SpeI (-1.4 kb), StuI (-0.6 kb), or StuI-PvuII (-0.5 kb), by insertion of an ~0.4-kb SpeI-liberated promoter fragment into the NheI site of pGL3 basic (-1.7 right-arrow -1.4 kb), or by inserting an ~1-kb XhoI-BsshII fragment from the pSK(-) promoter plasmid into the XhoI and MluI sites of pGL3 basic (-1.0 kb). Orientation was again confirmed by restriction mapping.

A 443-bp fragment of the human HSC70 promoter (GenBank accession no. S72464) inserted into the HincII site of pUC19 (pg21RPS500; a generous gift from Dr. Tohru Marunouchi, Fujita Health University, Tokyo, Japan) was liberated by digestion with HindIII and BamHI and subcloned into pSK(-). The promoter fragment was then liberated by digestion with XhoI and SpeI and cloned in the forward direction into the XhoI and NheI sites of pGL3 basic. A reverse orientation construct was constructed by liberating the promoter fragment with KpnI and SpeI and ligating it into the KpnI and NheI sites of pGL3 basic. These orientations were confirmed by restriction mapping.

All plasmids were propagated in Escherichia coli strain DH5-alpha and prepared with QIAGEN Mini or Spin Maxi kits (QIAGEN, Valencia, CA). The plasmid concentration was determined by measuring the absorbance at 260 nm.

For assessment of promoter function, IB3-1 cells in 35-mm dishes were cotransfected with the LipofectAMINE reagent (GIBCO BRL) with 1 µg of reporter plasmid and 1 µg of a beta -galactosidase expression plasmid (pCMV-SPORT beta -gal, GIBCO BRL), the latter as a control for transfection efficiency. The pGL3 basic plasmid was cotransfected similarly to assess baseline luciferase expression from the "promoterless" parental plasmid. Transfections were performed according the manufacturer's protocol in unmodified LHC-8 medium. Mock control transfections for assessment of background activity were performed identically except that the plasmid was excluded. After transfection, cells were incubated in standard growth medium for 24 h. The medium was then changed to control medium or control medium with 1 mM 4PBA for an additional 48 h before cell lysis and assay of reporter gene expression. These incubation periods were chosen to allow optimal reporter gene expression (which typically occurs ~72 h after transfection) while allowing sufficient time for incubation with 4PBA to exert any potential influence. Rubenstein et al. (23) have previously demonstrated that the trafficking of Delta F508 CFTR is improved after incubation of IB3-1 cells with 1 mM 4PBA for 24-48 h.

Cells were lysed with dual-assay lysis buffer, and the lysates were assessed for luciferase and beta -galactosidase activities with commercial kits (all from Promega). Luciferase activity was monitored with a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA). For data analysis, the background luciferase and beta -galactosidase activities (as determined from the mock transfections) were subtracted before normalization of luciferase activity by beta -galactosidase activity and then again normalizing these data by the luciferase or beta -galactosidase activity from the promoterless pGL3 basic transfections.

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 [alpha -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.

Slot blots for hybridization of runoff RNA were prepared as described (10) with Immobilon Ny+ membranes (Millipore, Bedford, MA). Six micrograms per slot of pSK(-) plasmid containing either human beta -actin cDNA (a kind gift from Dr. L. Gonzalez, Dept. of Neonatology, Children's Hospital of Philadelphia) or human HSC70 cDNA were immobilized on the hybridization membrane by ultraviolet cross-linking (5,000 µJ/cm2; Stratalinker, Stratagene). The membranes were prehybridized with Perfect Hyb Plus (Sigma) overnight at 68°C and hybridized to the runoff RNAs for 60 h at 55°C. The blots were subsequently washed with 2× SSC-0.1% SDS at room temperature. Hybridization was detected by fluorography and quantitated by densitometry of the fluorographic images.

The human HSC70 cDNA used in these studies was cloned by PCR of oligo(dT)-primed human leukocyte cDNA (a gift from Dr. Paul Anziano, Department of Neonatology, Children's Hospital of Philadelphia). The primers used (with italics indicating BamHI linkers) were 5'-TTTCTAGATTTTTGTGGCTTCCTTCGTTATTG and 3'-TTTCTAGATTAATCAACCTCTTCAATGGTGGG. The resulting cDNA was ligated into the BamHI site of pSK(-) and sequenced in the Nucleic Acid and Protein Core Facility of the Children's Hospital of Philadelphia. The sequence was identical to that of the deduced HSC70 cDNA from NCBI Entrez (Y00371) with the exception of a single nucleotide change in a third base of a codon that does not result in a change in amino acid in the translated protein.

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 beta -actin (pTRI-beta -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. [alpha -32P]UTP-labeled probes [50-70 kilocounts/min (kcpm) for HSC70, 5-10 kcpm for the RNA 18S control, and ~100 kcpm for beta -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.


    RESULTS
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INTRODUCTION
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DISCUSSION
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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 Delta F508/W1282X) (33) that 4PBA results in an ~40-50% reduction in steady-state expression of HSC70 mRNA and protein (25) and improved Delta 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- right-arrow -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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Fig. 1.   Functional features and analysis of the Chinese hamster ovary (CHO) 70-kDa heat shock cognate protein (HSC70) promoter. C, CCAAT/enhancer binding element; H, heat shock transcription factor consensus binding element; T, TATA box; ^, Sp1 consensus binding element; ATG, initiation methionine. The indicated fragments of the CHO HSC70 promoter were cloned into the pGL3 basic luciferase reporter vector as described in METHODS. The resulting plasmid constructs were cotransfected with the control plasmid pCMV-SPORT-beta Gal into IB3-1 cells with LipofectAMINE. Transfected cells were incubated in the absence and presence of 1 mM sodium 4-phenylbutyrate (4PBA) before assay of luciferase and beta -galactosidase (beta -Gal) activities as described in METHODS. Luciferase activity for each construct was first normalized by beta -Gal activity (to control for transfection efficiency) and is expressed relative to that of the transfected parent (promoterless) pGL3 basic plasmid. Data are means ± SE of 6-7 independent transfections for each construct. Nos. in middle, kb.

To ensure that this lack of effect of 4PBA on the HSC70 promoter was not a result of species-related issues, we assessed the activity of a 443-bp fragment of the human HSC70 promoter in the luciferase reporter system. This fragment corresponds to and is homologous in structure to the high-activity -1.0- right-arrow -0.6-kb region of the CHO promoter (Fig. 2). As observed for the CHO promoter, this human HSC70 promoter fragment efficiently directed luciferase reporter gene expression; this activity was similarly insensitive to 4PBA treatment. This promoter fragment maintained the directionality of the CHO HSC70 promoter; reporter gene activity was ~10-fold lower when the promoter was present in the reverse orientation (data not shown). These data are consistent with the 4PBA not altering the activity of the HSC70 promoter in this reporter gene expression system.


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Fig. 2.   Activity of a human HSC70 promoter fragment. The 440-bp human HSC70 promoter fragment was inserted into the pGL3 basic luciferase reporter vector as described in METHODS. Luciferase reporter activity was assessed in transfected IB3-1 cells incubated without and with 1 mM 4PBA after transfection as described in METHODS and Fig. 1. Data are means ± SE from 6-7 individual transfections for each construct relative to that of the promoterless pGL3 basic parent plasmid as described in Fig. 1. Short bars over H and T elements, regions of high promoter homology.

Promoter activity and the rate of mRNA transcription may be affected by factors not amenable to study in a reporter gene expression system. For example, alterations in local chromatin structure may influence the rate of mRNA transcription from chromosomal DNA; this does not significantly influence transfected expression plasmids. Because one proposed mechanism of action of the butyrates, including 4PBA, is the modulation of histone deacetylase activity and subsequent alterations in chromatin structure, we directly assessed the influence of 4PBA on the nuclear transcription of HSC70 mRNA in nuclear runoff assays. Figure 3 demonstrates that relative to the rate of transcription of beta -actin mRNA, 4PBA pretreatment of IB3-1 cells does not alter the rate of HSC70 mRNA synthesis in the cell nuclei. These data suggest that 4PBA does not significantly influence the rate of HSC70 mRNA synthesis through alteration of the chromatin structure local to the HSC70 gene as the mechanism by which it decreases steady-state HSC70 mRNA expression.


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Fig. 3.   Nuclear transcription of HSC70 mRNA. IB3-1 cells were incubated under control conditions and in the presence of 1 mM 4PBA for 48 h before isolation of nuclei as described in METHODS. Approximately 1 × 107 nuclei were incubated in the presence of ATP, CTP, GTP, and [alpha -32P]UTP for 30 min at 30°C to allow nuclear transcription of RNA. RNA transcripts were isolated and hybridized to nylon membrane-immobilized cDNAs for human HSC70 and beta -actin as described in METHODS. Membranes were washed and imaged by fluorography, and the fluorographic images were quantitated by densitometry. A: representative fluorogram. B: densitometric analysis. Data are means ± SE of HSC70 hybridization density normalized by the beta -actin density for duplicate hybridizations of 13 independent assays for each condition. The P value was determined by the Mann-Whitney U-test.

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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Fig. 4.   HSC70 mRNA degradation. A: IB3-1 cells were preincubated under control conditions and in the presence of 1 mM 4PBA for 24 h. Actinomycin D (10 µg/ml) was then added to inhibit mRNA synthesis. HSC70 mRNA was detected at the indicated times by ribonuclease protection assay as described in METHODS. The hybridization of an 18S rRNA as an internal standard was constant under these conditions (data not shown). B: relative amount of HSC70 mRNA remaining compared with the 0 time point was determined by densitometry of 4 independent experiments performed in duplicate. These data were fit to an exponential decay to derive the apparent first-order rate constant (kapp) for HSC70 mRNA degradation. Values are means ± SE. C: kapp was determined as above with 1 mM 4PBA either included or excluded from the incubation with actinomycin D. P values were determined by one-way ANOVA.

This cellular adaptation to 4PBA treatment did not result in a general increase in RNA turnover. In parallel experiments, 4PBA treatment did not alter the abundance of 18S rRNA (which was used as an internal standard in Fig. 4) nor did it significantly alter the rate of degradation of beta -actin mRNA. The apparent first-order rate of disappearance of beta -actin mRNA, determined as described for HSC70 mRNA in Fig. 4, was -0.042 ± 0.003 (SE) h-1 under control conditions and -0.046 ± 0.003 h-1 after 24 h of treatment with 1 mM 4PBA (P = 0.394 by Mann-Whitney U-test; n = 3 independent experiments performed in duplicate).

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 min-1; 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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Fig. 5.   Initial rate of HSC70 protein synthesis. IB3-1 cells were preincubated under control conditions and in the presence of 1 mM 4PBA for 24 h. Cells were then pulse labeled with a mixture of [35S]methionine and [35S]cysteine for the indicated times before radioimmunoprecipitation assay (RIPA) lysis as described in METHODS. HSC70 was immunoprecipitated from equal amounts of cellular lysate protein, resolved by SDS-PAGE, and detected by fluorography. A: representative fluorogram. B: densitometric analysis of 5 independent experiments. Data are means ± SE expressed relative to the density of the 240-min control value.

Steady-state 35S-HSC70 expression is approached at ~1 h of labeling. This is consistent with the turnover of HSC70 protein being rapid relative to that of mRNA, which is necessary for protein expression to correlate with mRNA expression. The steady-state expression of 35S-HSC70 in 1 mM 4PBA-treated IB3-1 cells was ~60% of that in control cells. These data confirm, by an alternate method, published observations by Rubenstein and Zeitlin (25) of decreased HSC70 protein expression after 4PBA treatment.

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 min-1; 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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Fig. 6.   Rate of HSC70 protein degradation. IB3-1 cells were preincubated under control conditions and in the presence of 1 mM 4PBA for 24 h. Cells were then pulse labeled with a mixture of [35S]methionine and [35S]cysteine for 20 min and chased in complete medium with the addition of 50 mM methionine and 10 mM cysteine for the indicated times before RIPA lysis. HSC70 was immunoprecipitated from equal amounts of cellular lysate protein, resolved by SDS-PAGE, and detected by fluorography as described in METHODS. A: representative fluorogram. B: relative amount of HSC70 protein remaining compared with the control 0 time point was determined by densitometry of 9 independent experiments. These data were fit to an exponential decay to derive kapp. , No 4PBA; open circle , 1 mM 4PBA. Values are means ± SE.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 Delta 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-alpha ) 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 Delta 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 Delta 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.


    ACKNOWLEDGEMENTS

We thank Drs. Paul Anziano and Thomas R. Kleyman for helpful discussions and Dr. Kleyman for a critical review of the manuscript.


    FOOTNOTES

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.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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