Mechanism of dexamethasone-mediated interleukin-8 gene suppression in cultured airway epithelial cells

Mary Mann-Jong Chang1,2,3, Maya Juarez, Dallas M. Hyde1,3, and Reen Wu1,2,3

1 Center for Comparative Respiratory Biology and Medicine, 2 Department of Internal Medicine, School of Medicine, and 3 Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, California 95616


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The effects of dexamethasone, a glucocorticoid analog, on interleukin 8 (IL-8) gene expression were studied in cultures of primary human tracheobronchial epithelial cells and an immortalized human bronchial epithelial cell line, HBE1 cells. Dexamethasone inhibited IL-8 mRNA and protein expression in a concentration- and time-dependent manner. The inhibition did not occur at the transcriptional level since both nuclear run-on activity and IL-8 promoter-reporter gene expression assay revealed no significant effect. Instead, there was a change in IL-8 mRNA stability in dexamethasone-treated cultures. Under actinomycin D treatment, IL-8 mRNA was quite stable in dexamethasone-depleted cultures, while in dexamethasone-pretreated cultures, IL-8 message was rapidly degraded within the first hour, then leveled off. When dexamethasone and actinomycin D were added simultaneously to dexamethasone-depleted cultures, IL-8 mRNA remained rather stable. When cycloheximide was used to inhibit new protein synthesis, dexamethasone-dependent inhibition was not observed. These results suggest that a posttranscriptional mechanism, which requires dexamethasone-dependent new protein synthesis, is involved in the regulation of IL-8 mRNA by dexamethasone in airway epithelial cells.

mRNA stability; transcription; posttranscriptional regulation


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

INTERLEUKIN-8 (IL-8), a chemotactic and inflammatory cytokine, was originally referred to as the monocyte-derived neutrophil chemotactic factor (40) and characterized as the major chemoattractant for neutrophil migration both in vivo and in vitro (21). However, a wide variety of biological effects of IL-8 have been demonstrated since then. Those effects include neutrophil activation (11), angiogenesis promotion (18), modulation of histamine release (10), regulation of cell adhesion properties (16), production of superoxide and hydrogen peroxide reactive oxygen metabolites (2), and induction of the release of azurophil granules (7). Pathologically, IL-8 has been implicated as a key factor in abnormalities such as sepsis (33) and adult respiratory distress syndrome (24), as well as in many other airway diseases.

A variety of cells can be induced to produce IL-8, including lymphocytes, keratinocytes, monocytes, endothelial cells, fibroblasts, and epithelial cells (3, 21, 27, 36, 37). Airway epithelial cells, being the front line of pulmonary defense, not only protect the airway by providing a physical barrier and clearing the airway surface (9) but can also modulate the movement of inflammatory cells by expressing cytokines and lipid mediators (4, 25, 30, 31). Airway epithelial cells in culture not only can be induced to produce IL-8 by proinflammatory stimuli, such as tumor necrosis factor-alpha (TNF-alpha ), IL-1beta , IL-6, and lipopolysaccharide (LPS) (19, 29), they can also produce IL-8 constitutively in both cell line and primary cultures (20).

Dexamethasone, an analog of glucocorticoids, is widely used clinically as an anti-inflammatory and immunosuppressive agent, especially in the treatment of asthma, and it remains the most potent agent for treatment. The effects of glucocorticoids mainly result from their ability to inhibit cellular release of various inflammatory mediators and cytokines such as IL-1 (22), IL-2 (1), TNF-alpha (5), interferon-beta (34), and interferon-gamma (34). It has also been reported in various cell culture systems that glucocorticoids can inhibit the production of IL-8, including epithelial and fibroblast cultures derived from human airway and lung tissues (19, 38). The inhibition can occur at either the transcriptional or posttranscriptional level. Mukaida et al. (26, 28) demonstrated a transcriptional mechanism of dexamethasone in the inhibition of IL-1-induced IL-8 gene expression in human glioblastoma cell lines and suggested that this inhibition is due to an impairment of nuclear factor-kappa B (NF-kappa B) activation (28). A similar observation was demonstrated in a rat kidney cell line, NRK-52E, in which the IL-1beta -induced expression of the neutrophil chemoattractant CINC/gro, a member of IL-8 family, was inhibited by dexamethasone at the transcriptional level (32). However, Tobler et al. (38) demonstrated a posttranscriptional effect of dexamethasone on IL-8 mRNA stability in normal human embryonic lung fibroblasts. All of the above studies were carried out in the presence of proinflammatory cytokines such as TNF-alpha and IL-1beta , which are known to be involved in activating the transcriptional system of the IL-8 gene.

In contrast to the enhanced IL-8 gene expression under activated conditions, the nature of dexamethasone inhibition on the basal level of IL-8 gene expression is not clear. Kwon and his colleagues (19, 20) have suggested that dexamethasone inhibited both the basal and TNF-alpha -induced IL-8 gene expression in the A549 cell line and the primary human airway epithelial cultures at the transcription level; however, in their studies, the exact mechanism of regulation was not demonstrated.

Our investigation focused on the effects of dexamethasone on basal level IL-8 gene expression in airway epithelial cells. We observed that dexamethasone caused a rapid decrease of IL-8 mRNA, and this acceleration of mRNA degradation might be due to an indirect mechanism that involved the synthesis of new protein. The regulation of dexamethasone on IL-8 gene expression at the transcriptional level in cultures of human airway epithelial cells appeared to be insignificant.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Sources of airway epithelial cells and culture conditions. Primary cultures of tracheobronchial epithelial (TBE) cells were derived from airway tissues of hospital donors and cultured in a serum-free F-12 medium supplemented with five different growth factors/hormones including insulin (5 µg/ml; Sigma, St. Louis, MO), epidermal growth factor (10 ng/ml; Upstate Biotechnologies, Lake Placid, NY), cholera toxin (20 ng/ml; List, Campbell, CA), transferrin (5 µg/ml, Sigma), and bovine hypothymus extract (30 µg/ml) (39). Dexamethasone (0.1 µM; Sigma) or hydrocortisone (1 nM; Sigma) was added as conditions required. Human tissues were obtained from organ donor patients or from autopsy through the University of California, Davis Medical Center. A human bronchial epithelial cell line, HBE1, was also used in this study, and it was cultured in the same serum-free hormone-supplemented medium. The HBE1 cells, a papilloma virus-immortalized human bronchial epithelial cell line, was generously provided by Dr. J. Yankaskas (University of North Carolina, Chapel Hill, NC). Cells were plated on plastic tissue culture dishes at a density of 50,000 cells/ml in the serum-free hormone-supplemented medium as described above. Because glucocorticoid hormone is an important supplement for airway epithelial cell growth, 1 nM hydrocortisone was added as supplement (1 nM is 1% of the dexamethasone concentration under dexamethasone-supplemented conditions) during plating. After 4-5 days, hydrocortisone was removed and cultures were maintained in a glucocorticoid hormone-free medium for at least 4 days. When cells were confluent, 0.1 µM dexamethasone was added to one-half of the cultures, while the other half remained untreated. Cells were harvested 24 h later. Cycloheximide (Sigma) was used for translation blocking study at 10 µg/ml, and actinomycin D (Sigma) was used for mRNA stability study at 10 µg/ml.

Quantitation of IL-8 production by ELISA. The levels of IL-8 in the secreted media collected from cultures were quantified by an ELISA kit obtained from Biosource International (Camarillo, CA) according to the manufacturer's suggested protocol. The minimum detectable dose of IL-8 is <10 pg/ml. The expression value was normalized with the cell number in the dish.

RNA preparation and Northern blot hybridization. Total RNA was isolated by the single-step acid guanidinium thiocyanate-phenol-chloroform extraction method described previously (8). A random primer labeling procedure was used to prepare 32P-labeled cDNA probes for hybridization. PG486, a pGEM-4Z-based clone (Promega, Madison, WI) containing a 486-base pair cDNA insert of human IL-8 sequence from nucleotides +1049 to +1525, was used as template for cDNA probe preparation. 18S ribosomal RNA as described before (6) was used as internal control for standardizing the Northern blot hybridization and the nuclear run-on assay. Northern blot hybridization was carried out as previously described (15).

Nuclear run-on assay. Nuclear run-on assay was performed according to the method of Greenberg and Ziff (14) with some modifications. Briefly, nuclei were isolated from dexamethasone-treated and untreated TBE cultures by Dounce homogenization in a hypotonic Tris-Cl buffer (pH 7.8) containing 0.4% Nonidet P-40 followed by resuspension in a 50% glycerol solution. Nascent transcripts were labeled by incubation of nuclei in a reaction buffer containing 500 µM each ATP, GTP, and CTP and 200 µCi of [alpha -32P]UTP (3,000 mCi/mmol; ICN). Run-on nuclear RNA was isolated by DNase I and proteinase K treatment, followed by ethanol precipitation. The precipitated RNA was then hybridized with a Nytran membrane that contained 5 µg of IL-8 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA inserts (6). Equal amounts of radioactive run-on [32P]RNA from these cultures were used for the hybridization.

Preparation of promoter-reporter gene chimeric constructs. Using human genomic DNA as a template, six different sizes of DNA sequence at the IL-8 gene 5'-flanking region that spans nucleotides +19 to -1472 were produced by the PCR method with appropriate primers. These DNA fragments, corresponding to nucleotides +19 to -1481, -1325, -481, -343, or -165 relative to the transcription start site, were then subcloned into pCAT3 (Promega) vector at the restriction sites of Bgl II and Hind III. The resulting clones that have the IL-8 promoter region upstream of the chloramphenicol acetyltransferase (CAT) reporter gene are pCAT 165 (from +19 to -165), pCAT 343 (+19 to -343), pCAT 481 (+19 to -481), pCAT 1325 (+19 to -1325), and pCAT 1481 (+19 to -1481). DNA sequencing was carried out on these chimeric constructs to confirm their corresponding positions in the human IL-8 promoter region. pSV-beta -gal plasmid DNA containing the beta -galactosidase reporter gene was obtained from Promega.

DNA transfection and reporter gene assay. The chimeric construct DNA was purified by QIAGEN column (QIAGEN, Valencia, CA) according to the manufacturer's suggested protocol. The DNA transient transfection study was carried out in a HBE1 cell line by the LIPOFECTIN-mediated method (12). Briefly, HBE1 cells were cultured in dexamethasone-free, serum-free, hormone-supplemented medium as described above. At 70% confluence, cultures were transfected with a DNA-LIPOFECTIN mixture. After 24 h of incubation, cultures were split in two: the medium of one set of culture dishes was changed to medium containing dexamethasone (0.1 µM) and the medium in the second set of culture dishes was changed to dexamethasone-free medium. After an additional 48 h, cultures were harvested for both CAT and beta -galactosidase assays. CAT activity was determined by a CAT ELISA kit from Boehringer Mannheim (Indianapolis, IN). The beta -galactosidase activity of pSV-beta -Gal (Promega)-transfected cells was used as an internal control for the normalization of transfection efficiency.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Dose-dependent inhibition. Constitutive secretions of IL-8 in airway cultures were inhibited by dexamethasone, and the inhibition was dose dependent. In primary human TBE cells with dexamethasone-free medium, the IL-8 secretion was 17.5 ± 0.9 ng/ml, while in HBE1 cells with the same condition, the IL-8 secretion amount was 6.8 ± 0.26 ng/ml (Fig. 1). Dexamethasone significantly inhibited the IL-8 production as well as the mRNA level. In both primary human TBE and HBE1 cells, the decrease in IL-8 by 0.1 µM dexamethasone treatment was ~60%. A similar dose-dependent inhibition by dexamethasone on the IL-8 mRNA level was also observed in these cultures (Fig. 2): the inhibition of IL-8 mRNA at 0.1 µM dexamethasone treatment was ~50% in both human TBE and HBE1 cells.


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Fig. 1.   Dose-dependent effects of dexamethasone (Dex) on interleukin-8 (IL-8) protein secretion in airway epithelial cultures. Primary human tracheobronchial epithelial (TBE) cells (solid bars) and human bronchial epithelial (HBE1) cells (open bars) were cultured as described in text. Various amounts of Dex were added to these cultures 24 h before harvest. Two hours before harvest, media were replaced by a fresh medium for further incubation. At the end of incubation, conditioned media of 2-h incubation were collected. An ELISA kit was used to measure the IL-8 secretion in the conditioned media. Cells were trypsinized and cell numbers were determined. The amount of IL-8 produced in the cultures is based on 3 independent experiments. Values are means ± SE of n = 3 experiments per group. * Significantly decreased (P <=  0.05) compared with no Dex treatment.



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Fig. 2.   Dose-dependent effects of Dex on IL-8 mRNA level in airway epithelial cultures. Experiments were carried out as described in Fig. 1, and various concentrations of Dex were added to these Dex-depleted cultures; 24 h later, total RNA was isolated and analyzed by Northern blot hybridization. A: primary human TBE cultures. B: HBE1 cell line. The autoradiographs shown are representative of at least 2 individual experiments; 18S rRNA-normalized densitometric values of those autoradiographs were plotted for easy visualization. * Significantly decreased (P <=  0.05) compared with control.

Time-course effect. The effect of dexamethasone on IL-8 mRNA was time dependent. As shown in Fig. 3A, the IL-8 message level rapidly decreased in primary human TBE cells after dexamethasone treatment. The rate of decrease reached a plateau after 5 h of dexamethasone treatment. An equally fast IL-8 mRNA- decreasing rate in the dexamethasone-treated culture was also observed in the HBE1 cell line (Fig. 3B). These data suggest that the downregulation of the IL-8 message levels by dexamethasone is an early event.


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Fig. 3.   Time-course effects of Dex treatment on IL-8 mRNA in airway epithelial cultures. Experiments were carried out as described in Fig. 1. Dex at 0.1 µM was added to the Dex-depleted cultures, and cultures at various times after the treatment were harvested for RNA isolation. IL-8 mRNA densitometric values were normalized with the 18S rRNA densitometric value. A: primary human TBE culture. B: HBE1 cell line culture. The autoradiographs shown are representative of at least 2 individual experiments, and 18S-normalized densitometric values of those autoradiographs were plotted for easy visualization.

Effects of dexamethasone on transcriptional regulation. Both nuclear run-on assays and IL-8 promoter-reporter gene transfection assays were performed to determine whether dexamethasone regulated IL-8 gene expression at the transcriptional level. Nuclei were isolated from dexamethasone-treated and untreated human primary TBE cultures. These nuclei were used for the run-on assay as described in METHODS. As shown in Fig. 4, dexamethasone treatment had no effect on the run-on activity of the IL-8 gene compared with GAPDH. To further verify this observation, DNA transient transfection assay was carried out on dexamethasone-treated and untreated HBE1 cultures (normalized with beta -galactosidase activity) with five different IL-8 promoter-reporter gene chimeric constructs (Fig. 5). The difference of activity between dexamethasone-treated and untreated cells was not significant, and this further suggested that transcriptional regulation is not involved in dexamethasone-mediated IL-8 gene suppression.


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Fig. 4.   Nuclear run-on assay of IL-8 transcription in Dex-treated and untreated cultures. Primary human TBE cells were cultured as described in Fig. 1. Dex (0.1 µM) was added to these Dex-depleted cultures as described in text. Nuclei were isolated from these cultures and used for nuclear run-on assay as described in text. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcription as a control, Dex treatment had no effect on IL-8 transcription. The autoradiographs shown are representative of at least 2 individual experiments.



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Fig. 5.   Effects of Dex on IL-8 promoter-chloramphenicol acetyltransferase (CAT) reporter gene activity. HBE1 cells at 80% confluency were transfected with various IL-8 promoter-CAT (pc) chimeric constructs as shown by the LIPOFECTIN-mediated gene transfer as described in text. Dex (0.1 µM) was added 1 day after transfection, and cells were maintained in that media for at least 24 h. Lysate of the cells was used for ELISA. Relative CAT activity, normalized to beta -galactosidase (beta -gal) activity, was plotted. Values are means ± SE of n = 3 experiments per group.

Effects of dexamethasone on RNA stability. Using actinomycin D to block new transcription, the half-life of IL-8 mRNA in both dexamethasone-pretreated (24 h) and untreated cultures was examined. As shown in Fig. 6, IL-8 mRNA was quite stable in both primary and HBE1 cultures under a dexamethasone-depleted condition, with a half-life of >8 and 4 h, respectively. In contrast, there was a rapid drop of IL-8 mRNA within the first 2 h after actinomycin D treatment in dexamethasone-supplemented cultures. Both the human primary TBE cells and HBE1 cells had a half-life of ~78 min, which is close to the 1 h reported by Kwon et al. (19). These results suggest that dexamethasone can affect IL-8 mRNA stability.


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Fig. 6.   Effects of actinomycin D treatment on IL-8 mRNA stability in airway epithelial cultures. Airway epithelial cultures were prepared as described in Fig. 1. Briefly, primary human TBE cells and HBE1 cells were maintained either under Dex-depleted conditions (-Dex) or under Dex (0.1 µM)-supplemented conditions (+Dex) for at least 24 h. Actinomycin D (10 µg/ml) was then added to these cultures, and RNA was isolated at various times after the treatment. Northern blot hybridization was carried out, and the IL-8 message level was normalized to the 18S rRNA level. The levels of IL-8 mRNA at various times after actinomycin D treatment were further normalized to the level at zero hour of actinomycin D treatment in each corresponding culture. A: primary culture of human TBE cells. B: HBE1 cell line. open circle , Dex-depleted culture condition. , Dex-treated cultures. Note that for primary cultures data have been duplicated in a separate primary epithelial culture derived from a different human. The autoradiographs shown are representative of at least 2 individual experiments, and 18S rRNA-normalized densitometric values of those autoradiographs were plotted for easy visualization.

To further elucidate the nature of the initial drop of IL-8 message in dexamethasone-pretreated cultures after actinomycin D treatment, we then determined whether this phenomenon could also occur if dexamethasone treatment happened at the same time as the arrest of DNA transcription. Treatment of dexamethasone and actinomycin D simultaneously in dexamethasone-depleted primary TBE cultures did not cause an initial drop of IL-8 mRNA (Fig. 7A). In contrast, dexamethasone treatment of dexamethasone-depleted cultures in the absence of actinomycin D caused a rapid decline of IL-8 mRNA with a half-life close to 1 h. A similar result was observed in the HBE1 cell line (Fig. 7B). These results suggest that the initial drop of IL-8 mRNA level by dexamethasone treatment is actinomycin D sensitive.


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Fig. 7.   Time-course study of IL-8 mRNA downregulation by Dex and Dex-actinomycin D combined treatment. Both primary cultures of human TBE cells and the HBE1 cell line were maintained in Dex-depleted culture conditions as described in text. Dex (0.1 µM) or Dex combined with actinomycin D (10 µg/ml) was added to the cells, and RNA was isolated from these cultures at various times after the treatment. Northern blotting analysis and quantitation were the same as in Fig. 6, and data are represented as percentage of IL-8 mRNA by using the mRNA amount of zero-hour treatment as 100%. A: primary culture of human TBE cells. B: HBE1 cell line. , Cultures treated with Dex. open circle , Cultures treated with Dex and actinomycin D (act D) simultaneously. Note that data have been repeated in a separate primary culture of a different human airway tissue. The autoradiographs shown are representative of at least 2 individual experiments, and 18S rRNA-normalized densitometric values of those autoradiographs were plotted for easy visualization.

Effects of cycloheximide treatment. To further understand the downregulation of the IL-8 message, we looked into whether the downregulation of the IL-8 message by dexamethasone requires new protein synthesis. Using cycloheximide to inhibit new protein synthesis, we added cycloheximide to dexamethasone-pretreated and untreated cultures. Unfortunately, cycloheximide treatment caused a superinduction phenomenon by increasing the IL-8 message (Fig. 8). This superinduction phenomenon was observed in both dexamethasone-treated and untreated cultures. The extent of superinduction between dexamethasone-treated and untreated cultures was apparently similar (Fig. 8). These data suggested that dexamethasone had no effect on the superinduction phenomenon. We then sought to see if pretreatment of cultures with cycloheximide by adding it before dexamethasone treatment could prevent dexamethasone-mediated IL-8 message downregulation. As shown in Fig. 9, such treatment could prevent dexamethasone-dependent downregulation in both TBE and HBE1 cells. This result supports the notion that new protein synthesis is needed to account for dexamethasone-dependent downregulation of the IL-8 message.


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Fig. 8.   Effects of protein synthesis inhibition on IL-8 mRNA level. Primary cultures of human TBE cells and HBE1 cells were grown in a Dex-depleted culture condition (open circle ) or pretreated with Dex (0.1 µM; ) for 24 h as described in text. Cultures were treated with cycloheximide, and RNA was isolated thereafter at various times. The level of IL-8 message was normalized to 18S rRNA. Note that data have been repeated in a separate primary culture that derived from a different human airway tissue. Autoradiographs shown are representative of at least 2 individual experiments, and 18S-normalized densitometric values of those autoradiographs were plotted for easy visualization.



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Fig. 9.   Effects of protein synthesis inhibition on Dex-dependent downregulation of IL-8 mRNA level. Primary human TBE culture and HBE1 cells were maintained under Dex-depleted culture conditions. Cycloheximide (10 µg/ml) was added to cultures 15 min before the addition of Dex (0.1 µM), and at various times after Dex treatment, RNA was isolated from these cultures. open circle , Dex-depleted culture. , Dex-treated culture. The autoradiographs shown are representative of at least 2 individual experiments, and 18S-normalized densitometric values of those autoradiographs were plotted for easy visualization.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Glucocorticoids are potent immunosuppressive agents. In airway epithelial cultures, dexamethasone, an analog of glucocorticoids, is an important growth supplement. In this investigation, we observed that dexamethasone inhibited production of IL-8 in various airway epithelial cultures, including primary TBE cells from human tissues and a human bronchial epithelial cell line, HBE1 cells. The inhibition was demonstrated at both the protein and mRNA levels. This inhibition was dose and time dependent on dexamethasone treatment. These results are consistent with the previous experiments reported by Kwon et al. (19, 20). However, our study has attained a conclusion that is quite different from the previous one. Previously, it was observed that dexamethasone preincubation did not change the stability of IL-8 mRNA in TNF-alpha -stimulated cells, suggesting that destabilization of mRNA was not involved in the action of dexamethasone on the downregulation of IL-8 mRNA expression (19, 20). However, there were no nuclear run-on data or other transcriptional experiments in the studies to further support this notion. Here we demonstrated a change in IL-8 mRNA stability with dexamethasone treatment. In dexamethasone-depleted cultures, the IL-8 message was quite stable. In dexamethasone-pretreated cells, the message was rapidly degraded within the first hour or two. These results support posttranscriptional regulation. Furthermore, we demonstrated that dexamethasone treatment had minimal effect on nuclear run-on activity and IL-8 promoter-reporter CAT activity. The reason for the difference in conclusions between our study and those of Kwon et al. (19, 20) may be due to the culture conditions and the extensiveness of dexamethasone depletion. In both studies, hydrocortisone was added at the initial stage and removed after the cells were established. In our study, initial plating media had only 1 nM hydrocortisone instead of 1 µM. When the cultures had been established, which usually occurred ~4-5 days after plating, hydrocortisone was removed through extensive washing (3-5 times rinsing at each medium change). Our starvation period was at least 4 days, and there were at least two medium changes before using the cells for experiments. Cell growth under such conditions was healthy and morphologically similar in comparison to the cells grown under dexamethasone-supplemented conditions. In the studies of Kwon et al. (19, 20), they used 1 µM hydrocortisone, which is 1,000 times the concentration of the hydrocortisone used in our media, and their starvation period was only 2 days. Another difference is that their cells were grown with the supplement of 0.1 µM retinoic acid, whereas we did not use retinoic acid in our culture media.

To exert a transcriptional inhibition, the dexamethasone-glucocorticoid receptor (GR) complex has either to bind to the GR-responsive site (GRE) of the IL-8 promoter to negatively regulate the transcriptional activity (26) or to interfere with the transactivation potential of the NF-kappa B system (28, 32). The IL-8 promoter has a putative GRE motif; however, the involvement of this GRE site in dexamethasone-mediated transcription is questionable (26). Using various IL-8 promoter-CAT reporter constructs that include the NF-kappa B binding site of the IL-8 promoter for transfection study, we observed a small decrease in CAT reporter gene activity in transfected cells after dexamethasone treatment, and the difference was statistically insignificant. Therefore, if there is any transcriptional regulation, the effect should be quite small.

Our conclusion of posttranscriptional regulation of IL-8 mRNA by dexamethasone is consistent with the finding that dexamethasone decreases IL-8 gene expression in normal human embryonic lung fibroblasts by reducing the stability of its mRNA (38). The IL-8 message contains an AUUUA sequence in the 3'-untranslated region. This AUUUA sequence is involved in the rapid degradation of mRNAs of inflammatory cytokines and protooncogenes (17, 35). Moreover, several reports have shown that dexamethasone decreases the stability of mRNAs containing the AUUUA sequence in the 3'-untranslated region (34). A specific protein that binds to RNAs containing AUUUA has been identified (13, 23), and it is thought that the formation of this complex may target susceptible mRNAs for rapid cytoplasmic degradation. A similar mechanism may involve airway epithelial cells, such that dexamethasone treatment enhances the complex formation and results in a rapid degradation of the IL-8 message.

The use of a protein synthesis inhibitor revealed several interesting findings that suggest the involvement of dexamethasone in the formation of the AUUUA binding complex for mRNA degradation. Using cycloheximide, we observed a superinduction phenomenon of the IL-8 message. The induction is rapid and reached two- to threefold within 2 h. Our data showed that this phenomenon was apparently not affected by dexamethasone, suggesting a dexamethasone-independent mechanism. However, pretreatment with cycloheximide could prevent the dexamethasone-dependent suppression phenomenon, suggesting that new protein synthesis is required for dexamethasone-dependent mRNA instability.

Actinomycin D experiments also revealed several interesting findings. First, the treatment caused a rapid decrease of IL-8 mRNA within the first couple of hours, and then the IL-8 mRNA leveled off in dexamethasone-pretreated cultures. In contrast, there was no rapid decrease of IL-8 mRNA in dexamethasone-depleted cultures. The rapid decrease of the IL-8 message after actinomycin D treatment suggested that there is a RNA destabilizing mechanism involved in the downregulation of IL-8 gene expression by dexamethasone. However, the leveling off phenomenon late in dexamethasone-treated cultures is difficult to understand. One possible explanation is that the protein(s) responsible for IL-8 mRNA degradation is labile and a continuous expression of this protein(s) is required for mRNA degradation. This notion is further supported by the experiment described in Fig. 7 in which simultaneous treatment of dexamethasone-depleted cultures with dexamethasone and actinomycin D did not demonstrate a sudden drop in the IL-8 mRNA level similar to that seen in dexamethasone-pretreated cultures. This result also suggests that the synthesis of this protein factor(s) (that accelerates the degradation of IL-8 mRNA) is transcriptionally dependent.

In summary, we have demonstrated that a dexamethasone-dependent new protein is involved in the degradation of the IL-8 message. This putative protein(s) is probably labile and dependent on a dexamethasone-GR-mediated transcriptional mechanism. This putative protein(s) may be distinguished from the mechanism that regulates the superinduction of IL-8 mRNA. How this new protein synthesis contributes to mRNA degradation is currently unknown. One possibility is that the dexamethasone-dependent new protein is a new RNase that is quite specific for IL-8 mRNA degradation. Another possibility is that this dexamethasone-dependent new protein(s) is a part of a RNA-protein complex that is responsible for the regulation of IL-8 mRNA stability. How this protein(s) interacts with the translation-dependent mRNA degradation is currently unknown. Nevertheless, we have demonstrated a posttranscriptional mechanism involved in the regulation of IL-8 mRNA stability in dexamethasone-treated airway epithelial cells. Further studies on the identification of the dexamethasone-dependent protein and its function on mRNA degradation are essential for the understanding of the posttranscriptional downregulation of IL-8 mRNA.


    ACKNOWLEDGEMENTS

We would like to express our thanks for the technical assistance of Yu Hua Zhao.


    FOOTNOTES

This work was supported by National Institutes of Health Grants ES-00628, ES-06230, HL-35635, HL-35635, and ES-09701 and by the California Tobacco-Related Disease Research Program (7RT-0149).

Address for reprint requests and other correspondence: M. M. J. Chang, Center for Comparative Respiratory Biology and Medicine, Surge 1 Bldg., Rm. 1121, Univ. of California, Davis, One Shields Ave., Davis, CA 95616 (E-mail: mjchang{at}ucdavis.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 30 September 1999; accepted in final form 17 July 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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