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
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ABSTRACT |
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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
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INTRODUCTION |
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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- (TNF-
), IL-1
, 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- (5), interferon-
(34), and
interferon-
(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-
B (NF-
B) activation
(28). A similar observation was demonstrated in a rat
kidney cell line, NRK-52E, in which the IL-1
-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-
and IL-1
, 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--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.
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METHODS |
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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 [-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-
-gal plasmid DNA containing the
-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 -galactosidase assays. CAT
activity was determined by a CAT ELISA kit from Boehringer Mannheim
(Indianapolis, IN). The
-galactosidase activity of pSV-
-Gal (Promega)-transfected cells was used as an internal control for the
normalization of transfection efficiency.
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RESULTS |
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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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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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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 -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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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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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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DISCUSSION |
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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--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-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-
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.
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ACKNOWLEDGEMENTS |
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We would like to express our thanks for the technical assistance of Yu Hua Zhao.
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FOOTNOTES |
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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.
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