Induction of the stress response with prostaglandin A₁ increases I-B gene expression

^a Division of Critical Care Medicine, Children's Hospital Medical Center, Cincinnati, Ohio 45229, USA

	ABSTRACT

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I-B is an intracellular protein that functions as a primary inhibitor of the proinflammatory transcription factor NF-B. Induction of the stress response with heat shock was previously demonstrated to induce I-B gene expression. Because the stress response can also be induced by nonthermal stimuli, we determined whether induction of the stress response with prostaglandin A₁ (PGA₁) would induce I-B gene expression. Treatment of human bronchial epithelium (BEAS-2B cells) with PGA₁ induced nuclear translocation of heat shock factor 1, thus confirming that PGA₁ induces the stress response in BEAS-2B cells. Induction of the stress response with PGA₁ increased I-B mRNA expression in a time-dependent manner and increased I-B peptide expression. Transient transfection assays involving a human I-B promoter-luciferase reporter construct demonstrated that induction of the stress response with PGA₁ activated the I-B promoter. Induction of the stress response with PGA₁ and concomitant induction of I-B were associated with inhibition of TNF--mediated secretion of interleukin 8 and with inhibition of TNF--mediated nuclear translocation and activation of NF-B. These data demonstrate that induction of the stress response, by a nonthermal stimulus, increases I-B gene expression by a mechanism involving activation of the I-B promoter. Coupled with previous data demonstrating heat shock-mediated induction of I-B gene expression, these data suggest that I-B may be considered to be one of the stress proteins. The functional consequences of stress response-mediated I-B gene expression may involve attenuation of cellular proinflammatory responses.—Thomas, S. C., Ryan, M. A., Shanley, T. P., Wong, H. R. Induction of the stress response with prostaglandin A₁ increases I-B gene expression. FASEB J. 12, 1371–1378 (1998)

Key Words: PGA₁ • NF-B • interleukin 8 • inflammation

	INTRODUCTION

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THE FAMILY OF INHIBITORY proteins known as I-B sequester the proinflammatory transcription factor NF-B in the cytoplasm (reviewed in refs 1, 2). The I-B isoform is one of the most studied and best understood members of the I-B protein family. I-B binds to NF-B at a position that blocks NF-B nuclear localization sequences. Phosphorylation-dependent degradation of I-B and subsequent dissociation of I-B from NF-B free NF-B to translocate to the nucleus to direct transcription of various proinflammatory genes. Since increased expression of proinflammatory genes is thought to play an important role in the pathophysiology of diseases such as sepsis and acute respiratory distress syndrome, there is a great deal of interest in defining pathways to inhibit NF-B activation.

A potential strategy to inhibit NF-B activation is to increase intracellular expression of I-B. Evidence to support this strategy includes 1) the existence of a negative feedback loop whereby NF-B nuclear translocation increases I-B expression (3, 4); 2) the demonstration that increased expression of wild-type I-B (5) or a dominant negative I-B isoform inhibits in vitro NF-B activation (6); 3) the demonstration that in vivo somatic gene transfer of I-B inhibits NF-B activation (7); and 4) the demonstration that I-B knockout mice have increased and prolonged activation of NF-B (8, 9).

Heat shock causes intracellular expression of a specific group of proteins, called heat shock proteins, that have broad cytoprotective properties (reviewed in refs 10, 11). This characteristic response, called the heat shock response, is highly conserved throughout evolution. Nonthermal stimuli such as heavy metals and oxidants can induce the same cellular response. Because of the highly conserved nature, the variety of described inducers, and the broad cytoprotective properties of the heat shock response, the more general terms `stress response' and 'stress proteins' have been suggested as being more appropriate (11).

An important feature of the stress response is that it can inhibit cellular proinflammatory responses. For example, previous studies demonstrated that induction of the stress response inhibited 1) inducible nitric oxide synthase gene expression in a variety of cells treated with cytokines (12–16) and in the lungs of rats treated with endotoxin (17); 2) mononuclear cell expression of tumor necrosis factor (TNF-)² and interleukin 1ß (IL-1ß) (18, 19); and 3) cyytokine-mediated NF-B nuclear translocation (14–16, 20). We recently demonstrated a novel feature of the stress response. In a cultured human distal respiratory epithelial cell line, induction of stress response with thermal stress (heat shock) caused increased expression of I-B (20). This observation was subsequently confirmed in cultured porcine endothelium by DeMeester et al. (21). The demonstration that heat shock induces I-B gene expression has two important implications. First, it suggests a potential novel mechanism by which the stress response can modulate proinflammatory responses. Second, and more fundamental, it suggests that I-B may be considered one of the stress proteins.

To address the possibility that I-B is a stress protein, we determined whether a nonthermal inducer of the stress response increases I-B gene expression. We hypothesized that prostaglandin A₁ (PGA₁), a potent nonthermal inducer of the stress response (22, 23), would induce I-B gene expression in a human bronchial epithelial cell line. In the current study we demonstrate that PGA₁ induces I-B gene expression by a mechanism involving activation of the I-B promoter. Furthermore, PGA₁-mediated induction of the stress response and concomitant induction of I-B are associated with inhibition of interleukin 8 (IL-8) secretion and activation of NF-B.

	MATERIALS AND METHODS

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Cell culture
All experiments involved BEAS-2B cells (American Type Culture Collection, Rockville, Md.), a human bronchial epithelial cell line transformed by an adenovirus 12-SV40 hybrid virus. Cell cultures were maintained in a room air/5% CO₂ incubator at 37°C using DMEM (Gibco BRL, Gaithersburg, Md.) containing 8% fetal bovine serum and penicillin/streptomycin (Gibco BRL).

Stress response models
As a positive control for induction of the stress response, one group of cells was subjected to heat shock (43°C for 1 h) in a room air/5% CO₂ incubator, then returned to 37°C. Another group of cells was treated with PGA₁ (Cayman Chemical, Ann Arbor, Mich.) at a concentration of 30 µg/ml. In preliminary experiments, we determined that 30 µg/ml of PGA₁ induced the stress response in BEAS-2B cells without causing cellular injury. Stock concentrations of PGA₁ (5 mg/ml) were prepared in 100% ethanol. Control cells were treated with equivalent amounts of the ethanol diluent. To document induction of the stress response, treated cells were analyzed for nuclear translocation of heat shock factor 1 (HSF-1) using electromobility shift assays (EMSA).

Nuclear protein extraction
Nuclear proteins were isolated from treated cells grown to 90% confluence in 100 mm² dishes. All nuclear protein extraction procedures were performed on ice with ice-cold reagents. Cells were washed twice with phosphate-buffered saline (PBS), harvested by scraping into 1 ml of PBS, and pelleted at 6000 rpm for 5 min. Pellets were washed twice with PBS, resuspended in one packed cell volume of lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl₂, 0.2% v/v Nonidet P-40, 1 mM DTT, and 0.1 mM PMSF), and incubated for 5 min with occasional vortexing. After centrifugation at 6000 rpm, one cell pellet volume of extraction buffer (20 mM HEPES pH 7.9, 420 mM NaCl, 0.1 mM EDTA, 1.5 mM MgCl₂, 25% v/v glycerol, 1 mM DTT, and 0.1 mM PMSF) was added to the nuclear pellet and incubated on ice for 15 min with occasional vortexing. The nuclear proteins were isolated by centrifugation at 14,000 rpm for 15 min. Protein concentrations were determined by Bradford assay (BioRad, Hercules, Calif.) and stored at -70°C until used for EMSA.

EMSA
An oligonucleotide probe corresponding to a previously published heat shock element consensus sequence (24) was synthesized by the University of Cincinnati DNA Core Facility. An oligonucleotide probe corresponding to the NF-B consensus sequence was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.). Oligonucleotide probes were labeled with -[³²P]dATP using T4 polynucleotide kinase (Gibco BRL) and purified in Bio-Spin chromatography columns (BioRad). Ten micrograms of nuclear proteins were preincubated in EMSA buffer (12 mM HEPES pH 7.9, 4 mM Tris-HCl pH 7.9, 25 mM KCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM DTT, 50 ng/ml poly [d(I-C)], 12% glycerol v/v, and 0.1 mM PMSF) on ice for 10 min before addition of the respective radiolabeled oligonucleotide probe for an additional 10 min. Protein–nucleic acid complexes were resolved using a nondenaturing polyacrylamide gel consisting of 5% acrylamide (29:1 ratio of acrylamide:bisacrylamide) and run in 0.5X TBE (45 mM Tris-HCl, 45 mM boric acid, 1 mM EDTA) for 1 h at constant current (30 mA). Gels were transferred to Whatman 3M paper, dried under a vacuum at 80°C for 1 h, and exposed to photographic film at -70°C with an intensifying screen. Specificity of the binding reactions was determined by coincubating nuclear extracts with radiolabeled oligonucleotide plus 100-fold molar excess of unlabeled oligonucleotide. Supershift assays were performed by coincubating nuclear extracts with radiolabeled oligonucleotide plus an anti-HSF-1 antibody (Stressgen, Victoria, British Columbia) or an anti-NF-B (p65 subunit) antibody (Santa Cruz Biotechnology).

Northern blot analysis
Total cellular RNA was recovered using the Trizol reagent (Gibco, BRL). RNA was quantified by spectrophotometry (260 nM), and 15 µg of total RNA per condition underwent electrophoresis on a 1% agarose gel containing 3% formaldehyde. The integrity of the RNA after electrophoresis was confirmed by ethidium bromide staining and brief UV illumination. RNAs were transferred to nylon membranes (Micron Separations Inc., Westboro, Mass.) and UV auto-cross-linked (UV Stratalinker 1800; Stratagene, La Jolla, Calif.). After 4 h prehybridization at 42°C, membranes were hybridized overnight at 42°C with either a radiolabeled human I-B cDNA probe (a kind gift of Dr. Albert Baldwin, Jr., University of North Carolina, Chapel Hill) or a radiolabeled I-Bß cDNA probe (a kind gift of Dr. Sankar Ghosh, Yale University). The cDNAs were labeled with -[³²P]dCTP (specific activity 3000 Ci/mM, New England Nuclear Research Products, Boston, Mass.) by random priming (Pharmacia, Piscataway, N.J.). The hybridized filters were serially washed at 53°C using 2X sodium citrate/sodium chloride/0.1% sodium dodecyl sulfate (SDS) and 25 mM NaHPO₄/1 mM EDTA/0.1% SDS solutions. After washing, exposure was carried out overnight and analyzed using a Phosphor Imager screen and software (Molecular Dynamics, Sunnyvale, Calif.). To normalize results for loading differences, membranes were stripped with boiling 5 mM EDTA and rehybridized with a -[³²P]dATP end-labeled oligonucleotide probe for 18s rRNA.

Metabolic labeling and immunoprecipitation of I-B
Cells were incubated with methionine- and cysteine-deficient MEM for 45 min prior to treatment. Cells were treated with PGA₁ or vehicle and labeled for 4 h with 0.5 mCi/ml [³⁵S]methionine/cysteine. After 4 h, cells were lysed with solubilization buffer (60 mM Tris pH 7.4, 190 mM NaCl, 6 mM EDTA, and 4% SDS), followed by the addition of 1 volume of water and 8 volumes of dilution buffer (60 mM Tris pH 7.4, 190 mM NaCl, 6 mM EDTA, and 2.5% Triton X-100). Cell lysates were cleared overnight at 4°C with 5 µl of normal rabbit serum and 30 µl of a 1:1 suspension of protein G Sepharose and immunoprecipitation buffer (1 volume of solubilization buffer, 1 volume of water, 8 volumes of dilution buffer). Protein G was removed by centrifugation, then 5 µl of an anti-human I-B antibody (Santa Cruz Biotechnology, Inc.) and 30 µl of protein G Sepharose were added to the supernatants and incubated overnight at 4°C. Protein G–antibody–protein complexes were collected by centrifugation, washed four times with wash buffer I (50 mM Tris pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.1% Triton X100, and 0.02% SDS ), and twice with wash buffer II (50 mM Tris pH 7.5, 150 mM NaCl, and 5 mM EDTA). The pellet was resuspended in 15 µl of electrophoresis sample buffer (60 mM Tris pH 6.8, 0.2% SDS, 10% glycerol, 5% 2-mercaptoethanol, and 0.01% bromophenol blue), boiled for 3 min, centrifuged for 1 min, and separated electrophoretically on an 8–16% polyacrylamide gradient gel (Novex, San Diego, Calif.). Gels were fixed in 40% methanol, 10% acetic acid, and 3% glycerol for 30 min and dried. Gels were exposed and analyzed using a Phosphor Imager screen after drying.

Construction of the I-B promoter-luciferase reporter plasmid
Oligonucleotide primers were designed by the University of Cincinnati DNA Core Facility corresponding to the most 5' and 3' regions of the published human I-B promoter sequence (3). Using these primers, an I-B promoter fragment (1.3 kb from the transcription initiation site) was recovered from a human genomic DNA template using the polymerase chain reaction (PCR). The PCR product was ligated into the plasmid pCR2.1 (Invitrogen, San Diego, Calif.) and sequenced by the Sanger dideoxy method. Sequence analysis demonstrated >99% correlation between the PCR product and the published human I-B promoter sequence (3). A Kpn 1/Xho 1 fragment incorporating the 1.3 kb I-B promoter fragment was excised from pCR2.1 and subcloned into the plasmid pGL2 (Promega, Madison, Wis.) such that the I-B promoter regulated expression of the reporter gene firefly luciferase. This human I-B promoter-luciferase plasmid was previously demonstrated to be functional in that it was inducible by TNF-, a well-known inducer of the I-B promoter (H. Wong, unpublished data).

Transient transfections, functional promoter analyses, and luciferase assays
Functional analysis of the I-B promoter was performed by transiently transfecting cells with the I-B promoter-luciferase reporter plasmid. Cells were transfected in duplicate, in 6-well plates, at a density of 300,000 cells per well by incubation with cationic liposomes (lipofectin, Gibco BRL) for 5 h in Opti-MEM (Gibco BRL). The liposome:DNA ratio used was 20:3 µg. After transfection, cells were washed once with PBS and allowed to recover overnight. After exposure to experimental conditions, cellular proteins were extracted and analyzed for luciferase activity according to the manufacturer's instructions (Promega), using a Berthold AutoLumat LB953 liminometer. Luciferase activity is reported as light units corrected for total cellular protein.

Induction and measurement immunoreactive IL-8
IL-8 secretion was induced by treating cells for 4 h with recombinant human TNF- (Boehringer-Mannheim, Indianapolis, Ind.) at a concentration of 10 ng/ml. One group of cells was treated with PGA₁ (30 µg/ml) for 2 h before treatment with TNF-. Immunoreactive IL-8 concentrations were measured in the media of treated cells using a commercially available sandwich ELISA (R&D Systems Inc., Minneapolis, Minn.). All procedures were performed according to the manufacturer's instructions.

Measurement of NF-B activation
NF-B activation was measured by transiently transfecting cells with an NF-B-dependent luciferase reporter plasmid. Transfection and luciferase assays were performed as described above. The plasmid used in these studies contained three tandem B motifs upstream of a minimal interferon ß promoter (3xBLuc, a kind gift of Dr. Roland M. Schimd, University of Ulm, Ulm, Germany). 3xBLuc has been described in detail previously and demonstrated to be a sensitive tool to measure in vitro NF-B activation (25).

Statistical analysis
Differences in luciferase activity and immunoreactive IL-8 concentrations between the experimental groups were evaluated by one-way analysis of variance and Student-Newman-Keuls test. P < 0.05 was considered statistically significant.

	RESULTS

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PGA₁ induces nuclear translocation of HSF-1
HSF-1 is the primary transcription factor that regulates stress protein expression, and the presence of HSF-1 in the nucleus is one of the earliest detectable events in cells undergoing the stress response (reviewed in refs 10, 11). Amici et al. (22) previously demonstrated that PGA₁ induced nuclear translocation of HSF-1 in K562 cells. Treatment of BEAS-2B cells with PGA₁ (30 µg/ml for 2 h) caused nuclear translocation of HSF-1, as demonstrated by formation of an HSF–1/DNA complex ( Fig. 1, lane 2). Coincubation of nuclear extracts with 100-fold molar excess of cold oligonucleotide inhibited formation of the HSF–1/DNA complex ( Fig. 1, lane 3), confirming the specificity of binding. Coincubation of nuclear extracts with an anti-HSF-1 antibody caused a supershift of the HSF–1/DNA complex, further confirming the specificity of binding ( Fig. 1, lane 4). As a positive control for nuclear translocation of HSF-1, another group of cells was subjected to heat shock (43°C for 1 h). One hour after heat shock, there was formation of an HSF-1/DNA ( Fig. 1, lane 5) identical to that of cells treated with PGA₁. Coincubation of nuclear extracts from heat-shocked cells with an anti-HSF-1 antibody caused a supershift of the HSF–1/DNA complex ( Fig. 1, lane 6) identical to that of cells treated with PGA₁. There was minimal detection of HSF-1 nuclear translocation in control cells ( Fig. 1, lane 1). These data confirm that PGA₁ induces the stress response in BEAS-2B cells.

View larger version (53K): [in this window] [in a new window]   Figure 1. Representative EMSA demonstrating that PGA1 induces HSF-1 nuclear translocation. Lane 1: control cells; lane 2: 2 h after treatment with PGA1; lane 3: cold competitor assay 2 h after treatment with PGA1; lane 4: supershift assay 2 h after treatment with PGA1; lane 5: 1 h after heat shock; and lane 6: supershift assay 1 h after heat shock.

Induction of the stress response with PGA₁ increases I-B gene expression
Having demonstrated that PGA₁ induces the stress response in BEAS-2B cells, we next determined whether PGA₁ would increase I-B gene expression. Treatment with PGA₁ (30 µg/ml) increased I-B mRNA expression in a time-dependent manner ( Fig. 2). Of the times tested, peak expression of I-B mRNA occurred 2 h after treatment with PGA₁ and was similar to that of a group of cells treated with TNF- (10 ng/ml for 2h, positive control for I-B mRNA expression). I-B mRNA levels returned to control levels within 8 to 24 h after treatment with PGA₁.

View larger version (44K): [in this window] [in a new window]   Figure 2. Representative Northern blot analysis demonstrating that PGA1 increases I-B mRNA expression. Control cells (`C') were treated with equivalent amounts of ethanol diluent and tumor necrosis factor -treated cells (`TNF') were treated for 2 h (10 ng/ml). Other cells were treated with PGA1 (30 µg/ml) for the indicated times. 18s rRNA was used to control for loading differences. Data represent 1 of 4 similar experiments.

To determine whether induction of the stress response with PGA₁ also increased I-B peptide expression, cells were labeled with [³⁵S]methionine/cysteine and the cellular lysates were immunoprecipitated with an anti-human I-Bß antibody. Assuming that peak expression of I-B peptide would lag behind peak expression of I-B mRNA (2 h after PGA₁), labeling and treatment with PGA₁ were carried out for 4 h in these experiments. In cells treated with PGA₁, I-B peptide levels increased above control cells ( Fig. 3). Collectively, these data demonstrate that induction of the stress response with PGA₁ increases I-B gene expression in BEAS-2B cells.

View larger version (31K): [in this window] [in a new window]   Figure 3. Representative metabolic labeling and immunoprecipitation demonstrating increased I-B peptide expression after treatment with PGA1. Lane 1 represents control cells treated for 4 h with an equivalent amount of ethanol diluent. Lane 2 represents cells treated with PGA1 (30 µg/ml) for 4 h.

To determine whether induction of the stress response affects mRNA expression of the I-Bß isoform, another group of cells was subjected to the classic inducer of the stress response (heat shock) and analyzed for expression of I-Bß mRNA. Induction of the stress response with heat shock did not affect steady-state levels of I-Bß mRNA in a manner that was detectable ( Fig. 4).

View larger version (48K): [in this window] [in a new window]   Figure 4. Representative Northern blot analysis demonstrating the effects of the stress response on steady state levels of I-Bß mRNA. Control cells (`C') were incubated at 37°C in basal growth media. Other cells were subjected to heat shock and harvested for Northern blot analysis at the indicated times. 18s rRNA was used to control for loading differences.

Induction of the stress response with PGA₁ activates the I-B promoter
Having demonstrated that PGA₁ increases I-B gene expression, we next determined whether PGA₁ activates the I-B promoter. Cells were transiently transfected with a reporter plasmid in which the reporter gene firefly luciferase was placed under control of the human I-B promoter. Treatment of transfected cells with PGA₁ caused an increase in luciferase activity ~2.6-fold above control cells ( Fig. 5), thus demonstrating that induction of the stress response with PGA₁ activates the I-B promoter.

View larger version (8K): [in this window] [in a new window]   Figure 5. Luciferase activity in cells transiently transfected with a human I-B promoter-luciferase plasmid and treated with PGA1 (30 µg/ml) for 4 h. Control cells were treated with equivalent amounts of ethanol diluent for 4 h. Data are plotted as mean light units (±SEM), corrected for total cellular protein, and represent 5 separate experiments in duplicate. *P < 0.05 vs. control.

Induction of the stress response with PGA₁ is associated with inhibition of TNF--mediated secretion of immunoreactive IL-8
IL-8 secretion is an important proinflammatory response of respiratory epithelium (26). To determine whether PGA₁-mediated induction of I-B gene expression affects the proinflammatory response of BEAS-2B cells, we measured concentrations of immunoreactive IL-8 in the media of cells treated with TNF- and PGA₁. Treatment with TNF- alone for 4 h increased concentrations of immunoreactive IL-8 in the media ( Fig. 6). Treatment with PGA₁ for 2 h before treatment with TNF- inhibited secretion of immunoreactive IL-8. Immunoreactive IL-8 was not detectable in the media of control cells or in cells treated with PGA₁ alone. Viability was not altered in cells pretreated with PGA₁ compared to cells treated with TNF- alone (data not shown). These data demonstrate that induction of the stress response with PGA₁ is associated with attenuation of the proinflammatory response of BEAS-2B cells.

View larger version (8K): [in this window] [in a new window]   Figure 6. ELISA data demonstrating the effect of PGA1-mediated induction of the stress response on secretion of immunoreactive IL-8. TNF--treated cells were treated for 4 h. Another group of cells were treated with PGA1 for 2 h before treatment with TNF. Data represent 5 separate experiments and are plotted as means ±SEM. *P < 0.05 vs. cells treated with TNF- alone.

The effect of PGA₁-mediated induction of the stress response and concomitant induction of I-B on NF-B
Increased levels of intracellular I-B inhibit NF-B activity (5, 7). In these experiments, we determined whether stress response (PGA₁)-mediated induction of I-B affects nuclear translocation and activation of NF-B.

NF-B nuclear translocation was determined by EMSA. Treatment with TNF- (10 ng/ml, for 30 min) caused nuclear translocation of NF-B in BEAS-2B cells ( Fig. 7, lane 3). The specificity of the band seen in lane 3 was confirmed by competitor assay (lane 4) and supershift assay (lane 5). Induction of the stress response and I-B with PGA₁ attenuated TNF--mediated NF-B nuclear translocation ( Fig. 7, lane 6). There was minimal detection of NF-B in nuclear extracts from control cells (lane 1). Treatment with PGA₁ alone did not detectably increase nuclear translocation of NF-B compared to control cells (lane 2).

View larger version (90K): [in this window] [in a new window]   Figure 7. Representative EMSA demonstrating the effect of PGA1-mediated induction of I-B on TNF--mediated NF-B nuclear translocation. Lane 1: control cells; lane 2: cells treated with PGA1 alone; lane 3: cells treated with TNF-, 10 ng/ml, for 0.5 h; lane 4: cold competitor assay after TNF- treatment; lane 5: supershift assay (anti-p65) after TNF- treatment; and lane 6: cells treated with PGA1 for 2 h, then treated with TNF- for 0.5 h.

Activation of NF-B was measured by transiently transfecting cells with an NF-B-dependent luciferase reporter plasmid (3xBLuc, ref 25). Treatment with TNF- (10 ng/ml, for 4 h) significantly increased luciferase activity above control levels, indicating activation of NF-B ( Fig. 8). Previous induction of the stress response and I-B with PGA₁ inhibited TNF--mediated luciferase activity, indicating inhibition of NF-B activation.

View larger version (9K): [in this window] [in a new window]   Figure 8. Luciferase activity in cells transiently transfected with 3xBLuc. One group of cells was treated with TNF- (10 ng/ml, for 4 h). Another group of cells was treated with PGA1 for 2 h, then treated with TNF-. Data are plotted as mean light units (±SEM), corrected for total cellular protein, and represent 4 separate experiments in duplicate. *P < 0.05 vs. control cells and cells treated with PGA1 and TNF-.

Collectively, these data demonstrate that induction of the stress response with PGA₁ and concomitant induction of I-B are associated with inhibition of NF-B nuclear translocation and activation.

	DISCUSSION

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Heat shock is the best-known inducer of the stress response, but a variety of chemical agents are also known to induce the stress response (reviewed in ref 27). Treatment of BEAS-2B cells with PGA₁ caused nuclear translocation of HSF-1 in a manner similar to cells subjected to heat shock, thus confirming previous data demonstrating that PGA₁ is a potent nonthermal inducer of the stress response (22, 23). The main finding of the current study is that induction of the stress response with a nonthermal inducer, PGA₁, caused increased expression of the I-B gene. Induction of the stress response with PGA₁ caused a time-dependent increase of I-B mRNA. The amount of I-B mRNA expression was similar to that of cells treated with TNF-, a well-known inducer of I-B (3, 4). Stress response-mediated increases of I-B mRNA led to de novo translation of I-B peptide, as demonstrated by experiments involving metabolic labeling and immunoprecipitation. This effect appears to be specific for the I-B isoform because heat shock, the classic inducer of the stress response, did not detectably affect expression of I-Bß mRNA in BEAS-2B cells.

Our data also demonstrate that the stress response increases I-B gene expression by a mechanism involving activation of the I-B promoter. It is known that the I-B promoter can be regulated by NF-B, thus providing an important negative feedback loop whereby nuclear translocation of NF-B causes increased expression of I-B, which then becomes available to attenuate ongoing NF-B activation (3, 4). However, in our experiments, PGA₁ or heat shock (20) did not appear to cause nuclear translocation of NF-B. These data suggest that the stress response activates the I-B promoter by a novel, non-NF-B-dependent pathway. The proximal factors that regulate stress response-mediated activation of the I-B promoter are currently under investigation. Specifically, we previously identified a potential heat shock responsive element in the I-B promoter (20) and are currently investigating the role of this region in stress response-mediated induction of I-B.

Coupled with previous data demonstrating that heat shock increased I-B gene expression (20, 21), the current data support the concept that I-B may be regarded as one of the stress proteins. The stress protein family encompasses a broad group of proteins sharing the common feature of being inducible by severe cellular stresses such as heat shock, heavy metals, and oxidants (reviewed in ref 27). Several examples exist of proteins that were described to have certain functions and subsequently found to be stress-inducible, including heme oxygenase (28), ubiquitin (29), thrombomodulin (30), and calreticulin (31). Although the functions of stress proteins are not fully understood, they appear to serve broad cytoprotective functions. Many of the stress proteins are also known to function as molecular chaperones. As molecular chaperones, stress proteins directly interact with other intracellular proteins and assist in stabilizing, refolding, and transporting intracellular proteins under basal conditions and during times of cellular stress (reviewed in ref 32). The primary known function of I-B, cytoplasmic sequestration of NF-B, is generally consistent with this molecular chaperone concept.

The functional consequences of stress response-mediated induction of I-B remain to be fully elucidated. Recent studies demonstrated that the stress response has potent anti-inflammatory effects (12–20). A greater understanding of these effects seems to be important, because the stress response also has broad protective effects against inflammation-associated cellular and tissue injury (17, 33–35). Since increased expression of I-B inhibits nuclear translocation of the proinflammatory transcription factor NF-B (5, 7), one potential consequence of stress response-mediated induction of I-B is attenuation of cellular proinflammatory responses. Our current data support this assertion by demonstrating that induction of the stress response with PGA₁ is associated with inhibition of TNF--mediated IL-8 secretion, a response previously demonstrated to be NF-B dependent (36). In addition, we demonstrated that induction of the stress response with PGA₁ was associated with inhibition of NF-B nuclear translocation and activation. Rossi et al. (37) provided the first demonstration that induction of the stress response with PGA₁ inhibits NF-B activation. These studies involved Jurkat T cells, HeLa cells, and CEM-SS lymphoid cells. The study by Rossi et al. also demonstrated that induction of the stress response with PGA₁ inhibited degradation of I-B. However, the role of PGA₁ in de novo synthesis of I-B was not directly examined.

Together, our current data and the data cited above suggest a potential novel pathway by which the stress response inhibits cellular proinflammatory responses. We propose that stress response-mediated induction of I-B may be a central mechanism by which the stress response inhibits cellular proinflammatory responses. By increasing intracellular levels of I-B, the stress response can potentially attenuate activation of NF-B and subsequent proinflammatory gene expression.

	ACKNOWLEDGMENTS

 
Supported by a research grant from the American Lung Association and a Pediatric Center for Gene Expression and Development Pediatrician/Scientist Career Development Award (National Institutes of Health grant HD28827–05) to H.R.W.

	FOOTNOTES

 
¹ Correspondence: Division of Critical Care Medicine-OSB5, Children's Hospital Medical Center, 3333 Burnet Ave., Cincinnati, OH 45229, USA. E-mail: wonghr{at}chmcc.org

² Abbreviations: PGA₁, prostaglandin A₁; IL, interleukin; HSF, heat shock factor; EMSA, electromobility shift assays; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; SDS, sodium dodecyl sulfate; TNF, tumor necrosis factor.

Received for publication February 17, 1998.

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