Departments of 1 Pediatrics, 2 Internal Medicine, and 4 Microbiology and Immunology and 3 Sealy Center for Molecular Sciences, University of Texas Medical Branch, Galveston, Texas 77555
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ABSTRACT |
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Airway epithelial cells represent the primary cell target of respiratory syncytial virus (RSV) infection. They actively participate in the lung immune/inflammatory response that follows RSV infection by expressing chemokines, small chemotactic cytokines that recruit and activate leukocytes. Regulated on activation, normal T cell expressed, and presumably secreted (RANTES) is a member of the CC chemokine subfamily and is strongly chemotactic for T lymphocytes, monocytes, basophils, and eosinophils, cell types that are present or activated in the inflammatory infiltrate that follows RSV infection of the lung. RSV infection of airway epithelial cells induces RANTES expression by increasing gene transcription and stabilizing RNA transcripts. The signaling pathway regulating RANTES gene expression after RSV infection has not been determined. In this study, we examined the role of extracellular signal-regulated kinase (ERK) and p38, members of the mitogen-activated protein (MAP) kinase (MAPK) family, in RSV-induced RANTES production. RSV infection of alveolar epithelial cells induced increased phosphorylation and catalytic activity of ERK and the upstream kinases Raf-1 and MAP ERK kinase. Induction of the MAP signaling cascade required a replication-competent virus. RSV infection of alveolar epithelial cells also induced activation of p38 MAPK. Inhibition of ERK and p38 activation significantly reduced RSV-induced RANTES mRNA and protein secretion without affecting RANTES gene transcription or transcription factor activation. These results indicate that the MAPK signaling cascade regulates RANTES production in alveolar epithelial cells through a posttranscriptional mechanism.
respiratory syncytial virus; chemokine; signal transduction; gene regulation; mitogen-activated protein kinase; regulated on activation; normal T cell expressed, and presumably secreted
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INTRODUCTION |
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RESPIRATORY SYNCYTIAL VIRUS (RSV) is an enveloped, negative-sense single-stranded RNA virus (for review see Ref. 16). Since its isolation, RSV has been identified as a leading cause of epidemic respiratory tract illness in children in the United States and worldwide. RSV is so ubiquitous that it will infect 100% of children before the age of 3 yr. It is estimated that 40-50% of children hospitalized with bronchiolitis and 25% of children with pneumonia are infected with RSV, resulting in 100,000 hospital admissions annually in the United States alone (16). RSV has been shown to predispose those infected to the development of hyperreactive airway disease (38), and recurrent episodes of wheezing in asthmatic children are often precipitated by RSV infection. The mechanisms of RSV-induced airway disease and its long-term consequences are largely unknown. However, experimental evidence suggests that early inflammatory and immune events of the host in response to RSV infection may be crucial (11).
The epithelium of the respiratory mucosa, the main function of which is to provide a protective physical barrier against injurious inhaled stimuli, is the main target of RSV. After inhalation or self-inoculation of the virus into the nasal mucosa and infection of the local respiratory epithelium, RSV spreads along the respiratory tract mainly by cell-to-cell viral transfer through the intracytoplasmic bridges (15). A number of molecules that are produced by human epithelial cells as a consequence of RSV infection have been described. Among them are potent immunomodulatory and inflammatory mediators, including cytokines and chemokines, a family of small chemotactic cytokines that direct leukocyte transendothelial migration and movement through the extracellular matrix.
We and others have shown that CXC and CC chemokines are strongly expressed in vitro in RSV-infected respiratory epithelial cells (2, 4, 26, 27, 31). Recent in vivo studies have demonstrated elevated concentrations of the same chemokines in nasal washes and bronchoalveolar lavages of children infected with RSV (12, 34, 35) and in lungs of RSV-infected mice (14), suggesting that, indeed, chemokines produced by infected epithelial cells can play an important role in the pathogenesis of RSV-induced airway inflammation.
Regulated on activation, normally T cell expressed, and presumably
secreted (RANTES) is a member of the CC branch of the chemokine family
and is strongly chemotactic for T lymphocytes, monocytes, basophils,
and eosinophils (3), cell types that are present or
activated in the inflammatory infiltrate that follows RSV infection of
the lung. We recently investigated the mechanisms of RANTES induction
in alveolar epithelial cells after RSV infection. RSV induces RANTES
expression by increasing gene transcription through the activation of
multiple cis-regulatory elements, with nuclear factor-B
(NF-
B) and interferon-stimulated responsive element (ISRE) being the
most important (7). However, the intracellular signaling
pathway regulating RANTES gene expression after RSV infection has not
been determined.
Many stimuli have been shown to elicit specific biological responses through activation of the mitogen-activated protein (MAP) kinase (MAPK) signaling cascade. The MAPK family includes five different molecules, with extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 being the best described (for review see Ref. 39). Activation of MAPK has been involved in a variety of cellular functions, including proliferation, differentiation, and apoptosis (for review see Ref. 19). Recent studies have indicated that they can also play an important role in the induction of proinflammatory molecules such as chemokines (17, 18, 21). In this study, we examined the role of the MAPKs ERK and p38 in RSV-induced RANTES production. We show that RSV infection of alveolar epithelial cells results in activation of ERK, the upstream kinases Raf-1 and MAP ERK kinase (MEK), and p38. Induction of the signaling cascade requires a replicating virus. Inhibition of ERK and p38 activation significantly reduces RSV-induced RANTES mRNA and protein secretion without affecting RANTES gene transcription or transcription factor activation.
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MATERIALS AND METHODS |
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Reagents. Phosphospecific antibodies that recognize the activated forms of ERK1 and ERK2 (phospho-Tyr204 of ERK1 and ERK2; sc-7383) and MAPK kinase (MEK1, phospho-Ser217) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and New England Biolabs (Beverly, MA), respectively. Antibodies specific for Raf-1 (sc-133), MEK1 (sc-219), ERK1, and ERK2 (sc-154) were obtained from Santa Cruz Biotechnology. The MEK-specific inhibitor PD-98059 and the p38-specific inhibitor SB-20350 were purchased from Calbiochem (San Diego, CA). Stock solution of 50 mM was prepared in Me2SO.
RSV preparation.
The human Long strain of RSV (A2) was grown in Hep-2 cells and purified
by centrifugation on discontinuous sucrose gradients as described
elsewhere (37). The virus titer of the purified RSV pools
was 8-9 log10 plaque-forming units per milliliter as determined by a methylcellulose plaque assay. No contaminating cytokines were found in these sucrose-purified viral preparations (28). Lipopolysaccharide, assayed using the
Limulus hemocyanin agglutination assay, was not detected.
Virus pools were aliquoted, quick-frozen on dry ice-alcohol, and stored
at 70°C until use.
Cell culture and infection of epithelial cells with RSV. A549, human alveolar type II-like epithelial cells (American Type Culture Collection, Manassas, VA), were maintained in Ham's F-12K medium containing 10% (vol/vol) fetal bovine serum, 10 mM glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin. Small alveolar epithelial (SAE) cells were obtained from Clonetics (San Diego, CA) and grown according to the manufacturer's instructions. Cell monolayers were infected with RSV at multiplicity of infection of 1 (unless otherwise stated), as described elsewhere (13). An equivalent amount of a 20% sucrose solution was added to uninfected A549 cells as a control. When MAPK inhibitor was used, cells were pretreated with the inhibitor for 1 h and then infected in its presence. Because MAPK inhibitors were diluted in DMSO, equal amounts of DMSO were added to untreated infected cells as a control. Total number of cells, cell viability, and viral replication, after inhibitor treatment, were measured by trypan blue exclusion and plaque assay, respectively.
Western blotting. RSV-infected and control cells were lysed by addition of ice-cold lysis buffer (50 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1 mM EGTA, 0.25% sodium deoxycholate, 1 mM Na3VO4, 1 mM NaF, 1% Triton X-100, and aprotinin, leupeptin, and pepstatin at 1 µg/ml each). After incubation on ice for 10 min, the lysates were collected, and detergent-insoluble materials were removed by centrifugation at 4°C at 14,000 g. Cell lysates (20 µg/sample) were then boiled in 2× Laemmli buffer for 2 min and resolved on SDS-polyacrylamide gel. Proteins were transferred for 6 h onto Hybond-enhanced chemiluminescence (ECL) nitrocellulose membrane (Amersham), and nonspecific binding sites were blocked by immersing the membrane in blocking solution [Tris-buffered saline-Tween 20 (TBST): 10 mM Tris · HCl, pH 7.6, 150 mM NaCl, 0.05% Tween 20 (vol/vol)] containing 5% skim milk powder or 5% bovine serum albumin for 30 min. After a short wash in TBST, the membranes were incubated in a 1:2,000 dilution of primary antibody overnight at 4°C or for 2 h at room temperature (antiphospho-ERK). Bound antibody was coated with peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) diluted 1:10,000 in TBST for 30 min at room temperature. After the membranes were washed, the proteins were detected using ECL (Amersham Pharmacia Biotech) according to the manufacturer's protocol.
Kinase assay.
For in vitro ERK, MEK, and Raf-1 kinase assays, 200 µg of total cell
extracts were incubated with A/G Plus agarose (sc-2003, Santa Cruz
Biotechnology)-immobilized appropriate antibody overnight at 4°C in
500 µl of lysis buffer. Immunocomplexes were collected by
centrifugation and washed twice in 1 ml of the lysis buffer and then
twice in kinase buffer (in mM: 10 HEPES, pH 7.4, 50 NaCl, 5 MgCl2, and 0.1 Na3VO4). Kinase
assays were performed by resuspension of the samples in 50 µl of
kinase buffer containing 0.25 mCi/ml [-32P]ATP and
substrate for 45 min at room temperature. Substrates for Raf-1
(syntide-2, sc- 3015) and MEK1 (sc-3014) were purchased from Santa Cruz
Biotechnology; substrate for MAPK, myelin basic protein, was obtained
from Upstate Biotechnology (Waltham, MA). The phosphorylation reaction
was terminated by spotting 15-µl aliquots of the assay mixture on a
1 × 1-cm Whatman phosphocellulose paper. Then filters
were washed five times for 10 min in 0.5% orthophosphoric acid, and
the amount of [32P]phosphate transfer was determined by
liquid scintillation counting. The p38 kinase assay was performed
according to the instructions provided with the kinase assay kit (Cell
Signaling Technology, Beverly, MA). Briefly, 200 ml of total cell
lysates were incubated with an agarose-conjugated anti-p38 antibody and
immunoprecipitated overnight. Immunocomplexes were washed and
resupended in kinase buffer containing 2 µg of recombinant activating
transcription factor-2 (ATF-2) and 200 µM ATP as substrate. After 30 min, reactions were terminated by addition of SDS sample buffer, and
samples were loaded on a 12% SDS-polyacrylamide gel. Proteins were
transferred to a nitrocellulose membrane, and phosphorylated ATF-2 was
detected by Western blot.
RANTES enzyme-linked immunosorbent assay. Immunoreactive RANTES was quantitated by a double-antibody enzyme-linked immunosorbent assay kit (DuoSet, R & D Systems, Minneapolis, MN) following the manufacturer's protocol.
Northern blot.
Total RNA was extracted from control and infected A549 cells by the
acid guanidinium thiocyanate-phenol-chloroform method (32). RNA (20 µg) was fractionated on a 1.2%
agarose-formaldehyde gel, transferred to nylon membranes, and
hybridized to a radiolabeled RANTES cDNA (the RANTES cDNA plasmid was a
generous gift of Dr. A. Krensky, Stanford, CA), as previously described
(5). After they were washed, membranes were exposed for
autoradiography using Kodak XAR film at 70°C using intensifying
screens. After exposure, membranes were stripped and rehybridized with
an 18S cDNA probe.
Cell transfection.
In transient transfection assays, we used a fragment of the human
RANTES promoter spanning nucleotides 974 to +55, relative to the mRNA
start site designated +1, cloned into the luciferase reporter gene
vector pGL2 (Promega, Madison, WI) and designated pGL2-974, as
previously described (7). Logarithmically growing A549
cells were transfected in triplicate in 60-mm petri dishes by
DEAE-dextran, as previously described (8). Cells were
incubated in 2 ml of HEPES-buffered DMEM (10 mM HEPES, pH 7.4)
containing 20 µl of 60 mg/ml DEAE-dextran (Pharmacia) premixed with 6 µg of RANTES-pGL2 plasmids and 1 µg of
cytomegalovirus-
-galactosidase internal control plasmid. After
3 h, medium was removed and 0.5 ml of 10% (vol/vol) DMSO in PBS
was added to the cells for 2 min. Cells were washed with PBS and
cultured overnight in 10% fetal bovine serum-DMEM. On the next
morning, cells were infected with RSV, and at 24 h after
infection, cells were lysed to measure independently luciferase and
-galactosidase reporter activity, as previously described
(8). Luciferase was normalized to the internal control
-galactosidase activity. All experiments were performed in duplicate
or triplicate.
Electrophoretic mobility shift assay.
Nuclear extracts of uninfected and infected A549 cells were prepared
using hypotonic/nonionic detergent lysis, as previously described
(8). Proteins were normalized by protein assay (Protein Reagent, Bio-Rad, Hercules, CA) and used to bind to duplex
oligonucleotides corresponding to the RANTES
NF-B1 binding site and ISRE, the sequences of which are
as follows: 5'-GATCCATTTT GGAAACTCCCCTTAT-3' and
3'-TAAAACCTTTGAGGGGAATATCTAG-5' for NF-
B1 and
5'-GATCCATATTTCAG TTTT C TTTT CCGT-3' and
3'-TATAAAGTCAAAAGAAAAGGCATCTAG-5' for ISRE. DNA-binding
reactions contained 10-15 µg of nuclear proteins, 5% glycerol,
12 mM HEPES, 80 mM NaCl, 5 mM dithiothreitol, 5 mM Mg2Cl,
0.5 mM EDTA, 1 µg of poly(dI-dC), and 40,000 cpm of
32P-labeled double-stranded oligonucleotide in a total
volume of 20 µl. The nuclear proteins were incubated with the probe
for 15 min at room temperature and then fractionated by 6%
nondenaturing polyacrylamide gels in Tris-boric acid-EDTA buffer (22 mM
Tris · HCl, 22 mM boric acid, and 0.25 mM EDTA, pH 8). After
electrophoretic separation, gels were dried and exposed for
autoradiography using Kodak XAR film at
70°C using intensifying screens.
Statistical analysis. Data from experiments involving multiple samples subject to each treatment were analyzed by the Student-Newman-Keuls t-test for multiple pairwise comparisons. Results were considered significantly different at P < 0.05.
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RESULTS |
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RSV infection results in ERK1 and ERK2 activation.
To determine whether RSV infection of A549 cells induced ERK
activation, we first performed Western blot analysis of total cell
proteins extracted from A549 cells uninfected or infected for various
lengths of time using a monoclonal antibody that recognizes tyrosine-phosphorylated ERK1 and ERK2. Uninfected cells had a detectable basal level of ERK phosphorylation (Fig.
1A). On infection with RSV,
ERK1 and ERK2 phosphorylation had substantially increased within 3 h, reaching a maximum after 12 h. Membrane was stripped and
reprobed with an antibody recognizing total ERK to verify equal loading
of the samples. Detection of tyrosine-phosphorylated ERK was
accompanied by an increase in catalytic activity, investigated by in
vitro kinase assay. ERK showed an eightfold increase in kinase activity
at 3 h after infection, which peaked at 12 h, similar to
RSV-induced ERK phosphorylation (Fig. 1B).
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RSV activates Raf-1 and MEK kinase in airway epithelial cells.
Because ERK is most commonly phosphorylated through induction of
Raf-1-MEK kinases (39), we determined whether RSV
infection of airway epithelial cells was able to activate these two
upstream kinases. At 3, 6, 12, and 24 h after RSV infection, total
cell protein extracts were probed by Western blot using an antibody to
Raf-1. A slower electrophoretic mobility of a single band of ~72 kDa
was consistently observed in protein extracts from RSV-infected cells
compared with control cells, indicating the phosphorylation of Raf-1 in
infected cell extracts (Fig.
2A). This phenomenon could be
detected within the first 3 h of RSV infection and was sustained
for up to 24 h. These findings were confirmed using an in vitro
kinase assay in which immunoprecipitated Raf-1 catalyzed the
incorporation of 32P into Syntide-2, a synthetic Raf-1
substrate (29). Raf-1 from RSV-infected A549 cells
exhibited a four- to sixfold increase in catalytic activity at all time
points tested compared with control cells (Fig.
2B).
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RSV activates the Raf-MEK-MAPK pathway in SAE cells.
To confirm these results in a normal cell type, we assayed Raf-1, MEK,
and ERK kinase activity in SAE cells infected with RSV. SAE cells are
derived from the small bronchioli of the lung, and they show a pattern
of RANTES induction, after RSV infection, similar to A549 cells
(27). Starting at 3 h, a progressive increase in
Raf-1, MEK1, and ERK1/2 activity could be detected in RSV-infected SAE
cells. However, the Raf-MAPK pathway activation in SAE cells was
characterized by slower kinetics in SAE than in A549 cells (Fig.
4).
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Activation of Raf-MAPK pathways occurs in the presence of
replicating RSV.
To determine whether activation of the Raf-MAPK signaling pathway by
RSV requires viral replication, A549 cells were stimulated with
ultraviolet (UV)-inactivated RSV. We previously showed that UV-inactivated RSV binds to A549 cells but does not induce production of chemokines in A549 cells (27). Total cell proteins were
extracted from 6-h-infected A549 cells and used for Western blot. As
shown in Fig. 5, only replicating RSV was
able to induce ERK phosphorylation and Raf-1 increased mobility
shift, indicating that viral replication is necessary for
Raf-MAPK activation. UV-inactivated RSV failed to have an effect on
Raf-1 and MAPK also in earlier and later time points (data not shown).
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RSV infection results in p38 activation.
To determine whether RSV infection of A549 cells induced p38
activation, first, we used a monoclonal antibody that recognizes serine-phosphorylated p38 to perform Western blot analysis of total
cell proteins extracted from A549 cells uninfected or infected for
various lengths of time. We were not able to detect a significant difference in the level of p38 phoshorylation between control and
infected cells (data not shown). To determine whether RSV infection was
inducing p38 catalytic activity, we performed an in vitro kinase assay.
As shown in Fig. 6, p38 kinase activity was increased at 3 h after infection, peaking at 6 h, with
kinetics similar to kinetics observed for RSV-induced ERK
phosphorylation.
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MAPK inhibition interferes with RSV-induced RANTES secretion.
To determine whether the activation of ERK after RSV stimulation occurs
through activation of MEK1, we pretreated A549 cells with an MEK1
inhibitor, PD-98056, before stimulation with RSV. The RSV-induced
increase in ERK catalytic activity was inhibited by PD-98056 in a
dose-dependent manner (Fig. 7), reaching
maximum of inhibition (~75%) at 50 µM. PD-98056 had no effect on
Raf-1, as expected (data not shown). Because PD-98056 has been shown to
potentially affect p38 activation in certain cell types, we also
investigated p38 kinase activity in RSV-infected A549 cells treated
with the ERK inhibitor. PD-98056 did not affect RSV-induced p38
activation (Fig. 8). We recently
demonstrated that RSV is a potent stimulus for RANTES production in
cultured human nasal, bronchial, and airway epithelial cells (27,
31). In all epithelial cell types, synthesis of RANTES required
replicating virus and was dose and time dependent, with increased
steady-state levels of RANTES mRNA starting 6-12 h after infection
(27, 31). To determine the contribution of RSV-induced ERK
and p38 activation in RANTES secretion, A549 cells were infected with
RSV in the absence or presence of 50 µM PD-98056 or 10 µM
SB-203580, a specific p38 inhibitor. RANTES protein secretion was then
measured by enzyme-linked immunosorbent assay from supernatants of
control and 24-h-infected cells. Addition of PD-98056 or SB-203580,
alone or in combination, significantly blocked RSV-induced RANTES
production (Fig. 9) without affecting
cell viability or viral replication (data not shown), indicating that
both kinases play an important role in RSV-induced RANTES production. A
similar experiment was also performed in SAE cells infected with RSV in
the absence or presence of 50 µM PD-98056. As in A549 cells, PD-98056
significantly reduced RSV-induced RANTES production (50, 1,800, and
1,000 pg/ml for control, RSV, and RSV + PD-98056, respectively),
indicating that, indeed, inducible RANTES secretion is partially
regulated through MAPK activation.
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MAPK inhibition affects RSV-induced RANTES expression without
affecting gene transcription.
To determine whether the reduction in RSV-induced RANTES secretion by
PD-98056 and SB-203580 was paralleled by changes in steady-state level
of RANTES mRNA, A549 cells were infected with RSV for 18 h, in the
presence or absence of 50 µM PD-98056 or 10 µM SB-203580, and total
RNA was extracted from control and infected cells for Northern blot
analysis. Densitometric analysis showed that treatment with PD-98056 or
SB-203580, compared with infection alone, reduced RSV-induced RANTES
mRNA induction by 40-50% (Fig.
10). This change was not due to a
nonspecific effect, because steady-state levels of 18S RNA and total
cell number (not shown) were unchanged.
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DISCUSSION |
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RANTES is a CC chemokine that plays a major role in allergic inflammation (1, 24). It is highly produced by airway epithelial cells after RSV infection and is present in elevated concentration in nasal washing and bronchoalveolar lavage of RSV-infected children, suggesting an important role also in RSV-induced lung inflammation (12, 27, 31, 34, 35). Recent studies have shown that RANTES gene expression after RSV infection is controlled at the transcriptional and posttranscriptional levels (7, 20). However, the signaling pathways leading to RSV-induced RANTES secretion have not been previously investigated. In this study, we show that RSV infection of alveolar epithelial cells activates ERK and p38 MAPK and that both kinases are involved in RANTES gene expression.
We found that RSV infection of A549 cells induced tyrosine phosphorylation of ERK1 and ERK2 and an increase in their catalytic activity. The kinetics of induction paralleled the RSV replication cycle, starting at early time points of infection and peaking at ~12 h after infection (23). Importantly, ERK activation precedes significant accumulation of RANTES transcripts (7), indicating a temporal relationship between ERK activation and RANTES expression. The upstream kinases involved in ERK induction, Raf-1 and MEK, were also activated after RSV infection, with similar kinetics. Viral replication was necessary for Raf-MEK-MAPK induction, since UV-inactivated virus, which does not replicate in cells, was unable to activate the signaling cascade. These results are in agreement with our previous reports that RANTES gene expression in A549 cells is dependent on replicating RSV (7, 27). Our study confirms a previous finding that RSV infection can activate ERK (9) and shows for the first time that RSV can also activate p38, another member of the MAPK cascade, an event that occurs in the setting of other viral infections, such as human immunodeficiency virus, cytomegalovirus, Epstein-Barr virus, adenovirus, and influenza (6, 10, 21, 22, 30). Although most of these observations have been obtained using transformed cell lines, we were able to confirm some of our findings in normal human SAE cells. The slower kinetics of RSV-induced ERK activation in SAE cells could be ascribed to different kinetics of viral replication in this cell type vs. the A549 cells, as suggested by Becker et al. (4), who found differences in RSV production/release between epithelial cells of the upper and lower respiratory tract.
Inhibition of ERK and p38 activation significantly blocked RSV-induced RANTES gene expression and protein secretion. RSV-induced ERK activation has been previously shown to be involved in interleukin (IL)-8 protein production, since its inhibition significantly reduced IL-8 secretion, with no effect on mRNA levels (9). Influenza virus infection of respiratory cells results in activation of all three major branches of the MAPK family. Although inhibition of JNK and p38 significantly attenuated RANTES secretion, ERK inhibition had no effect (21). However, ERK has been shown to regulate RANTES production in human endothelial and smooth muscle cells (25, 33). Therefore, it is likely that the signal transduction pathway regulating RANTES induction is stimulus and, possibly, cell type specific.
Although these studies have identified a role of MAPKs in RANTES
production, they have not investigated the link between the activation
of these kinases and the mechanism of RANTES gene expression. We
recently studied the mechanism of RANTES gene expression in RSV-infected alveolar epithelial cells. RANTES gene transcription is
activated after RSV infection of A549 cells, and the presence of
multiple cis-regulatory elements is required for full
induction of the RANTES promoter, with the ISRE and NF-B sites
playing a major role in RANTES transcription (7).
Induction of MAPK can activate various transcription factors that are
involved in the inducible expression of cytokine and chemokine genes,
including members of the NF-
B, NF-IL-6, activator protein (AP)-1,
ATF, and IRF families of transcription factors (for review see Ref. 36). The results of this study show that inhibition of ERK
and p38 does not affect RSV-induced RANTES gene transcription and transcription factor binding to the RANTES NF-
B and ISRE sites. However, increased transcription is not the only mechanism by which
RANTES gene expression is regulated in epithelial cells after viral
infection. Koga et al. (20) recently reported that RSV
infection of bronchial epithelial cells markedly increased RANTES mRNA
half-life, which was identified as the major mechanism responsible for
viral-induced mRNA accumulation in that cell type (20),
and mRNA stabilization plays an important role in RANTES gene
expression in a variety of cell types, including SAE and intestinal
epithelial cells (7). Activation of p38 MAPK has been
shown to induce mRNA stabilization of IL-8 and IL-6 genes in HeLa
cells, while activation of the JNK pathway induced IL-8 gene
transcription (18, 40). Therefore, a similar mechanism could account for the reduction of RSV-induced RANTES gene expression after inhibition of ERK and p38 in A549 cells. We are currently investigating the role of JNK in RSV-induced chemokine production. A
better understanding of the signaling pathways involved in chemokine gene expression is critical for developing strategies to reduce airway
inflammation associated with RSV infection.
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institutes of Health Grants AI-01763-01, AI-15939, PO1 AI-46004, and P30 ES-6676. A. Casola is a Child Health Research Center Young Investigator (National Institute of Child Health and Human Development Grant HD-27841).
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FOOTNOTES |
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Address for reprint requests and other correspondence: A. Casola, Div. of Child Health Research Center, Dept. of Pediatrics, 301 University Blvd., Galveston, TX 77555-0366 (E-mail: ancasola{at}utmb.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.
February 15, 2002;10.1152/ajplung.00331.2001
Received 17 August 2001; accepted in final form 11 February 2002.
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