MAPK activation is involved in posttranscriptional regulation of RSV-induced RANTES gene expression

Konrad Pazdrak1, Barbara Olszewska-Pazdrak1, Tianshuang Liu1, Ryuta Takizawa1, Allan R. Brasier2,3, Roberto P. Garofalo1,4, and Antonella Casola1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa B (NF-kappa 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

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 [gamma -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-beta -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 beta -galactosidase reporter activity, as previously described (8). Luciferase was normalized to the internal control beta -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-kappa B1 binding site and ISRE, the sequences of which are as follows: 5'-GATCCATTTT GGAAACTCCCCTTAT-3' and 3'-TAAAACCTTTGAGGGGAATATCTAG-5' for NF-kappa 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Tyrosine phosphorylation and activation of extracellular signal-regulated kinase (ERK) in respiratory syncytial virus (RSV)-infected A549 cells. Total cell lysates prepared from control (C) and infected (V) A549 cells were resolved on 10% SDS-polyacrylamide gels or immunoprecipitated with anti-ERK antibody for in vitro kinase assay. A: Western blot using an antibody detecting tyrosine-phosphorylated ERK1 and ERK2 (pERK1 and pERK2, respectively). The same blot was stripped and reprobed with an antibody recognizing total ERK1 and ERK2 proteins. B: in vitro kinase assay. ERK catalytic activity in RSV-infected cells is expressed as percentage of increase over activity detected in control cells. Values are means ± SD (n = 3).

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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Fig. 2.   RSV infection results in activation of Raf-1. Control and RSV-infected A549 cells were harvested at different times. Total cell lysates were subjected to Western blotting with anti-Raf-1 antibody recognizing total expression of Raf-1 or immunoprecipitated with Raf-1 and then subjected to in vitro kinase assay. A: Western blot of 7% SDS-polyacrylamide gel-resolved Raf-1 protein. Arrow, slower electrophoretic mobility band present in infected cells. B: in vitro kinase assay. Raf-1 catalytic activity in RSV-infected cells is expressed as percentage of increase over activity detected in control cells. Values are means ± SD (n = 3).

We next evaluated the activation of MEK1 in RSV-infected A549 cells. MEK1 lies directly downstream of Raf-1 in a signal transduction pathway that is activated by growth factors. Raf-1 activates MEK1 by phosphorylation at Ser217 and Ser221. To determine whether MEK1 was phosphorylated as a result of RSV infection, total cell lysates from the experiments described above were subjected to Western blot with an antibody recognizing the phosphorylated form of MEK1. We found sustained phosphorylation of MEK1 protein in all samples obtained from RSV-infected epithelial cells, but not in those from control cells. Equivalent loading in all lanes was established by restaining the membrane with an anti-MEK1 antibody. A kinase assay using immunoprecipitated MEK1 protein and a synthetic substrate specific for MEK1 demonstrated a parallel increase in MEK1 catalytic activity in infected cells compared with control cells (Fig. 3B).


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Fig. 3.   RSV activates mitogen-activated protein (MAP) ERK kinase (MEK1) in A549 cells. A549 cells were treated with RSV for 3, 6, 12, and 24 h. Total cell lysates of control and RSV-infected A549 cells were resolved on 10% SDS-polyacrylamide gels or immunoprecipitated with anti-MEK1 antibody for in vitro kinase assay. A: Western blot detecting MEK1 kinase phosphorylated on serine residues (pMEK1). The same Western blot was stripped and reprobed with antibody recognizing total MEK1 protein. B: in vitro kinase assay. MEK1 catalytic activity in RSV-infected cells is expressed as percentage of increase over activity detected in control cells. Values are means ± SD (n = 3).

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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Fig. 4.   RSV activates Raf-1, MEK1, and ERK in small airway epithelial (SAE) cells. SAE cells were treated with RSV for 3, 6, 12, and 24 h. Total cell lysates were immunoprecipitated with appropriate antibodies, and in vitro kinase assay was performed using specific substrates for Raf-1 (A), MEK1 (B), and ERK (C). Percent increase in catalytic activity of kinases in RSV-infected cells is compared with activity in control noninfected cells. Values are means ± SD (n = 2).

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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Fig. 5.   Effect of intact vs. ultraviolet (UV)-inactivated RSV on Raf-1 and ERK activation. A549 cells were treated with replicating or UV-inactivated RSV for 6 h and then lysed and subjected to Western blotting with anti-Raf-1 and antiphospho-ERK antibodies. C, uninfected cells; V, RSV; UV, UV-inactivated RSV. Results are representative of 2 independent experiments.

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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Fig. 6.   Activation of p38 in RSV-infected A549 cells. Total cell lysates prepared from control and infected A549 cells were immunoprecipitated with anti-p38 antibody for in vitro kinase assay. Phosphorylated activating transcription factor-2 (pATF-2) was detected by Western blot. Results are representative of 2 independent experiments.

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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Fig. 7.   Effect of MEK1 inhibition on RSV-induced ERK activation. A549 cells were pretreated with 1, 10, and 50 µM PD-98056 (PD) before infection with RSV. Total cell lysates were obtained after 12 h of infection and tested for ERK activity. Results are representative of 2 independent experiments.



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Fig. 8.   Effect of PD-98056 on RSV-induced p38 activation. A549 cells were pretreated with 50 µM PD-98056 before infection with RSV. Total cell lysates were obtained after 12 h of infection and tested for p38 kinase activity. Results are representative of 2 independent experiments.



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Fig. 9.   Effect of mitogen-activated protein kinase (MAPK) inhibition on RSV-induced regulated on activation, normal T cell expressed, and presumably secreted (RANTES) secretion. A549 cells were infected with RSV in the absence or presence of 50 µM PD-98059 (PD) or 10 µM SB-203580 (SB) alone or in combination. Culture supernatants, from control and infected cells, were assayed 24 h later for RANTES production by ELISA. Values are means ± SD of 2 independent experiments performed in triplicate.

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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Fig. 10.   Northern blot of RANTES mRNA in RSV-infected A549 cells. A549 cells were infected with RSV for 18 h in the absence or presence of 50 µM PD-98059 (A) or 10 µM SB-203580 (B). Total RNA was extracted from control and infected cells, and 20 µg of RNA were fractionated on a 1.2% agarose-formaldehyde gel, transferred to a nylon membrane, and hybridized to a radiolabeled RANTES cDNA probe. Membrane was stripped and hybridized with a radiolabeled 18S cDNA probe to show equal loading of samples. Results are representative of 2 independent experiments.

To determine whether MAPK inhibition affected RANTES gene expression at the transcriptional level, the same experiment was conducted in A549 cells transiently transfected with a construct containing the first 974 nucleotides of the human RANTES promoter linked to the luciferase reporter gene, pGL2-974 (7). Treatment with PD-98056 or SB-203580 did not affect RSV-induced luciferase activity (Fig. 11), indicating that inhibition of RANTES gene expression does not occur by interference with RANTES gene transcription. The ISRE as well as the NF-kappa B site of the RANTES promoter are the two major cis-regulatory elements involved in regulation of RANTES gene transcription after RSV infection (7). Using electrophoretic mobility shift assays, we previously showed that three DNA-protein binding complexes formed on the NF-kappa B oligonucleotide: two are RSV inducible and one is not. These complexes are formed mainly by p65 homo- and heterodimers (7). Inhibition of MAPK did not affect the abundance of the two RSV-induced complexes binding to the RANTES NF-kappa B site (Fig. 12A). For the ISRE site, two complexes formed on the oligonucleotide: one is RSV inducible and is formed by homo- and heterodimers of interferon regulatory factor-1 (IRF-1), -3 and -7, and one is not. Inhibition of MAPK did not affect the abundance of the RSV-induced complex binding to the RANTES ISRE (Fig. 12B). Together, these data indicate a role for ERK and p38 MAPK in posttranscriptional control of RANTES expression after RSV infection.


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Fig. 11.   Effect of MAPK inhibition on RANTES promoter activation after RSV infection. A549 cells were transiently transfected with pGL2-974 plasmid and infected with RSV in the absence or presence of 50 µM PD-98059 or 10 µM SB-203580. At 24 h after infection, cells were harvested to measure luciferase activity. Uninfected plates served as controls. For each plate, luciferase was normalized to beta -galactosidase reporter activity. Values (means ± SD of 2 independent experiments performed in triplicate) are expressed as normalized luciferase activity.



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Fig. 12.   Electrophoretic mobility shift assay of nuclear factor-kappa B (A) and RANTES interferon-stimulated responsive element (B) binding complexes in response to MAPK inhibitor treatment. Nuclear extracts were prepared from control A549 cells and A549 cells infected with RSV for 12 h in the absence or presence of 50 µM PD-98059 or 10 µM SB-203580 and used for binding to the RANTES interferon-stimulated responsive element and nuclear factor-kappa B sites. Arrows, RSV-inducible complexes formed on oligonucleotides. Results are representative of 1 of 3 independent experiments.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa 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-kappa 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-kappa 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.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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