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Published ahead of print on January 23, 2004, doi:10.1165/rcmb.2003-0424OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 844-852, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0424OC

Sphingosine Kinase Mediates Activation of Extracellular Signal–Related Kinase and Akt by Respiratory Syncytial Virus

Martha M. Monick, Kelli Cameron, Linda S. Powers, Noah S. Butler, Diann McCoy, Rama K. Mallampalli and Gary W. Hunninghake

University of Iowa Roy J. and Lucille A. Carver College of Medicine, and Veterans Administration Medical Center, Iowa City, Iowa

Address correspondence to: Martha M. Monick, Division of Pulmonary, Critical Care, and Occupational Medicine, Room 100, EMRB, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA 52242. E-mail: martha-monick{at}uiowa.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Respiratory syncytial virus (RSV) preferentially infects lung epithelial cells. Infected cells remain viable well into the infection. This prolonged survival results from RSV-induced activation of pro-survival pathways, including Akt and extracellular signal-related kinase (ERK). Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite with demonstrated links to cell survival. It is enzymatically generated by sequential activation of ceramidase (generation of sphingosine) and sphingosine kinase (generation of S1P). In these studies, we found that RSV stimulated neutral ceramidase and sphingosine kinase activities in lung epithelial cells. The combined effect of activation of these two enzymes would decrease proapoptotic ceramide and increase antiapoptotic S1P. S1P activated Akt and ERK within minutes, and inhibition of sphingosine kinase blocked RSV-induced ERK and Akt activation, leading to accelerated cell death after viral infection. RSV infection does eventually kill infected cells but activation of cell survival pathways significantly delays cell death. The studies are the first evidence linking sphingolipid metabolites to cell survival mechanisms in the context of a viral infection.

Abbreviations: extracellular signal–related kinase, ERK • human tracheobronchial epithelial cells, hTBEs • polymerase chain reaction, PCR • phosphatidylinositol 3, PI 3 • respiratory syncytial virus, RSV • reverse transcriptase, RT • sphingosine 1-phosphate, S1P


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Respiratory syncytial virus (RSV) is an important pathogen in young children and immunocompromised adults (1). Worldwide, it is the most common cause of bronchiolitis-associated hospitalization of children less than 2 yr of age (2). Early severe RSV infections (bronchiolitis with hospitalization) cause long-term morbidity and mortality by increasing the risk for recurrent wheezing and asthma symptoms throughout childhood (3). RSV is found ubiquitously in the environment. It is an enveloped, nonsegmented, negative-strand RNA virus, a member of the family Paramyxoviridae. Entry into the host cell (primarily the respiratory epithelium) is by cell-surface fusion. Eleven genes and their encoded proteins have been identified for RSV. These include the F or fusion protein, G attachment protein, nucleocapsid proteins, a polymerase subunit, two N or nonstructural proteins, and M2-2, an RNA regulatory protein (4). Infection of lung epithelial cells leads to viral replication and induction of an inflammatory response characterized by the production of chemokines and cytokines. In our previous work, we showed that the Akt and extracellular signal–related kinase (ERK) signaling pathways were involved in both inflammation and epithelial cell survival in RSV-infected cells (5–10). In this study, we evaluated the role of sphingolipid metabolites in RSV-induced lung epithelial cell signaling.

Sphingolipids, initially considered inert structural components of the plasma membrane, have been shown to have multiple and distinct roles in signal transduction (11). Ceramide, the common backbone of most sphingolipids, can be generated by de novo synthesis initiated with the condensation of serine and palmitoyl-CoA and completed by a series of enzyme modifications. Ceramide can also be generated more rapidly by the action of acid and neutral sphingomyelinases on sphingomyelin in the outer leaflet of the plasma membrane (12). Sphingomyelinases can be activated by proinflammatory cytokines, growth factors, and environmental stress (13). Once generated either by de novo synthesis or the actions of a sphingomyelinase, ceramide can be deacylated by ceramidases. This is the rate-limiting step in determining intracellular levels of sphingosine (14). In turn, sphingosine can be phosphorylated by sphingosine kinase yielding sphingosine 1 phosphate (S1P) (15). There are two isoforms of sphingosine kinase, SK1 and SK2. The most common form (and the one modulated by RSV in our system) is SK1. SK2, in contrast to the antiapoptotic effect of SK1, has been recently described as a BH3 only, proapoptotic protein (16). Ceramide has been shown to be antiproliferative and proapototic (17). In contrast, S1P has been implicated in cell proliferation and survival (18). S1P is released into the extracellular space, where it signals via Edg receptors. The S1P-specific Edg receptors include: S1P1/Edg1, S1P2/Edg5/AGR16/H218, S1P3/Edg3, S1P4/Edg6, and S1P5/Edg8 (19). Signaling via these receptors affects angiogenesis, vascular maturation, cell migration, cell survival, cell proliferation, and production of inflammatory mediators (19, 20).

ERK and Akt have been closely linked to cell survival. We have demonstrated that RSV activates both of these pathways in lung epithelial cells (6, 8) and that RSV-induced apoptosis in epithelial cells is delayed by activation of the phosphatidylinositol (PI) 3-kinase/Akt survival pathway. The PI 3-kinase/Akt pathway increases cell survival through a number of mechanisms, including inactivation of apoptosis relevant factors (GSK-3, Bad, caspase 9, and Forkhead transcription factors) and activation of nuclear factor-{kappa}B leading to transcription of the IAP family of antiapoptotic factors (21). Akt is activated downstream of PI 3-kinase after PH domain-dependent recruitment to PI3,4,5P at the plasma membrane, phosphorylation at the activation loop by PDK-1, followed by phosphorylation of serine 473 in the hydrophobic motif. Inhibition of Akt has been strongly linked to decreased cell survival (22, 23).

The mitogen-activated protein kinases (ERK, p38, and JNK) are an evolutionarily conserved family of enzymes that signal to regulatory targets both in the cytoplasm and nucleus (24). The ERK mitogen-activated protein kinase pathway has been linked to transcription of c-fos, activation of the transcription factors Elk-1, Sp-1, Egr1, and phosphorylation of the activator protein-1 subunits, fra 1 and 2 (25). Relevant to the present study, recent data has supported a cell-type specific role for ERK in cell survival (26–28). In osteoclasts, tumor necrosis factor–induced survival requires ERK activity (29). In fibroblasts, ERK activity prevents anchorage and serum removal–induced apoptosis (30). In squamous carcinoma cells, hepatocyte growth factor blocks suspension-induced apoptosis via activation of ERK (31). ERK effects on survival may be mediated by expression of survival-promoting genes, phosphorylation and activation of antiapoptotic protein, IEX-1, or phosphorylation and inhibition of caspase 9 (26, 28).

In these studies, we investigated the role of sphingolipid metabolism in the RSV-induced activation of survival pathways (ERK and Akt) and prolonged survival of infected cells. The data demonstrate that in RSV-infected lung epithelial cells, synergistic effects on both Akt and ERK activation contributed to prolonged survival of infected cells. We found that inhibition of sphingosine kinase (which would block S1P production) blocked RSV-induced activation of both Akt and ERK. RSV infection activated two enzymes, neutral ceramidase and sphingosine kinase. The combined effect of this activity would decrease proapoptotic ceramide and increase antiapoptotic S1P. Further, inhibition of sphingosine kinase activity shortened survival of RSV-infected lung epithelial cells. This is the first description of viral-induced sphingosine kinase activity contributing to prolonged survival of the infected cell.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) and Calbiochem (La Jolla, CA). Protease inhibitors were obtained from Boehringer Mannheim (St. Louis, MO). Antibodies to Akt and ERK were obtained from Santa Cruz (Santa Cruz, CA). Antibodies to phosphorylated ERK (Thr202/Tyr204) and Akt (Ser 473) were obtained from Cell Signaling Technology (Beverly, MA). Nitrocellulose and ECL Plus were obtained from Amersham (Arlington Heights, IL). Developing antibodies were from Santa Cruz. S1P, DL-threo-dihydrosphingosine, and D-erythro-N,N-Dimethylsphingosine were obtained from Biomol Research Laboratories (Plymouth Meeting, PA).

Epithelial Cell Culture and Viral Infection
A549 lung epithelial cells were obtained from American Type Culture Collection (Mannassas, VA). Cells were maintained in 100-mm tissue culture flasks (Corning, Corning, NY) in minimal essential medium (Invitrogen, Carlsbad, CA) with 10% fetal calf serum. For infection, cells at ~ 80% confluence were treated with human RSV, strain A-2 (multiplicity of infection [moi] of 5). Viral stocks were obtained from Advanced Biotechnologies Inc. (Columbia, MD). Because of a report of possible adenovirus contamination in some RSV stocks (32), we tested our stock for adenovirus by polymerase chain reaction (PCR) and found it to be completely free of adenoviral contamination. The initial stock (1 x 109 TCID50) was aliquoted and kept frozen at –135°C. A fresh aliquot was thawed for each experiment. The virus was never refrozen.

Primary Tracheobronchial Epithelial Cells
All protocols were approved by the University of Iowa Institutional Review Board. Human tracheobronchial epithelial cells (hTBEs) were obtained as previously described (33). Epithelial cells were isolated from tracheal and bronchial mucosa by enzymatic dissociation and cultured in laboratory of carcinogenesis (LHC)-8e medium on plates coated with collagen/albumin for study up to passage 10. For infection, cells at ~ 80% confluence were treated with human RSV, strain A-2 (moi of 5).

Cell Survival Analysis
For analysis of cell survival, A549 epithelial cells (seeded in 48-well tissue culture plates and infected with RSV [moi of 5]) were cultured alone or with pathway inhibitors (sphingosine kinase, DMS at 10 uM) (ERK, U0126 at 10 uM) (PI 3-kinase/Akt, LY294002 at 10 uM) for the described times. Triplicate cultures were performed on all experiments. After the incubation period, one sample was analyzed by the trypan blue exclusion method and the percentage of dead cells was calculated. At least 300 cells were counted for each sample from a minimum of six fields. Another sample was stained with ethidium homodimer (EthD-1; Molecular Probes, Eugene, OR) at 8 uM and images obtained of both brightfield and fluorescence using a Leica DMRB microscope equipped with a Qimaging RETICA 1,300 digital camera and imaging system. In some instances live cells were stained with calcein AM (1 µM; Molecular Probes); and the percentage of dead cells estimated by manually counting the EthD-1–positive cells in comparison to the calcein am–positive cells. After obtaining images, the percentage of EthD-1–positive cells was determined. Quantification was by direct cell count. Two hundred cells were counted from a minimum of four different fields. Average viability was determined and statistics performed using GraphPad software (GraphPad Software, Inc., San Diego, CA).

Isolation of Protein Extracts
Whole cell protein was obtained by lysing the cells on ice for 20 min, in 300 µl of lysis buffer (0.05 M Tris pH 7.4, 0.15 M NaCl, 1% NP-40, 1 protease minitab [Roche Biochemicals, Indianapolis, IN]/10 ml and 1x phosphatase inhibitor cocktail [#524625; Calbiochem]). The lysates were then sonicated for 20 s, incubated at 4° for 30 min, and the insoluble fraction removed by centrifugation at 15,000 x g for 10 min. Protein concentrations were determined using a protein measurement kit from Bio-Rad (Hercules, CA). Samples were stored at –70°C for future Western analysis.

Western Analysis
Western analysis for the presence of particular proteins or for phosphorylated forms of proteins was performed as previously described (6). 40 µg of protein was mixed 1:1 with 2x sample buffer (20% glycerol, 4% sodium dodecyl sulfate, 10% ß-mercaptoethanol, 0.05% bromophenol blue, and 1.25 M Tris pH 6.8) and loaded onto a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel and run at 100 V for 2 h. Cell proteins were transferred to nitrocellulose with a Bio-Rad (Hercules, CA) semidry transfer system, according to the manufacturer's instructions. Equal loading of the protein groups on the blots was evaluated using Ponceaus S (Sigma Chemical Co.), a staining solution designed for staining proteins on nitrocellulose membranes. The nitrocellulose was then blocked with 5% milk in TTBS (tris-buffered saline with 0.1% Tween 20) for 1 h, washed, and then incubated with the primary antibody at dilutions of 1:500 to 1:2,000 overnight. The blots were washed four times with TTBS and incubated for 1 h with horseradish peroxidase–conjugated anti-IgG antibody (1:5,000 to 1:20,000). Immunoreactive bands were developed using a chemiluminescent substrate, ECL Plus (Amersham Biosciences, Piscataway, NJ). An autoradiograph was obtained, with exposure times of 10 s to 2 min. Protein levels were quantifed using a FluorS scanner and Quantity one software for analysis (Bio-Rad). The data were analyzed and statistics performed using GraphPad software. Densitometry is expressed as fold increase (experimental value/control value).

Acidic and Neutral Ceramidase Assay
A549 cells were treated and homogenized in lysis buffer containing 50 mM sodium acetate, pH 4.5, 0.5% Triton X-100, 5 mM MgCl2, 1 mM EDTA, and 5 mM D-galactonic acid-{gamma}-lactone for the acidic ceramidase, and 50 mM Tris, pH 8.0, 0.5% Triton X-100, 5 mM MgCl2, 1 mM EDTA, 5 mM D-galactonic acid-{gamma}-lactone for the neutral ceramidase. Cell homogenates were centrifuged for 10 min at 14,000 x g, and the supernatant was taken for an in vitro assay. One hundred micrograms of protein in a total volume of 100 µl was incubated for 20 h at 37°C with 20 nCi of 14C ceramide. The reaction was stopped by the addition of 200 µl of water, and lipid extraction was performed by addition of 2 ml of chloroform/methanol (2:1; vol/vol). The lower phase was concentrated and lipids were resolved by thin layer chromatography (TLC) using chloroform/methanol/ammonia (90:20:0.5; vol/vol). Spots corresponding to ceramide and stearic acid were analyzed and quantitated using a phosphoimager (Bio-Rad).

Sphingosine Kinase Activity Assay
A549 cells were harvested into 500 µl sphingosine kinase buffer (0.2 M Tris-HCl pH 7.4, 1 mM EDTA, 0.5 mM deoxypyridoxine, 15 mM sodium fluoride, 1 mM 2-mercaptoethanol, 1 mM sodium vanadate, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml soybean trypsin inhibitor, 40 mM ß-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10% glycerol. Cell suspensions were lysed by 7 freeze/thaw cycles and centrifuged at 100,000 x g for 1 h. Cellular protein (2–10 µg) was mixed with 10 nmol of sphingosine substrate (stock is 1 mM sphingosine/EtOH with 5% Triton X-100; Biomol, Plymouth Meeting, PA), 180 nmol of cold ATP (magnesium salt), and 10 uCi 32-P-{gamma}ATP (NEN/Perkin-Elmer Life Sciences, Boston, MA). Final volumes were adjusted to 200 µl with sphingosine kinase buffer. Samples were incubated at 37° for 30 min to allow sphingosine kinase to convert sphingosine to S1P. Reactions were terminated by the addition of 800 µl of chloroform:methanol:HCl (100:200:1, vol/vol). Next, 240 µl each of chroroform and 2 M KCl were added and lipids extracted. The aqueous fractions were discarded, and 50 µl of the organic phase from each sample spotted in a lane of a TLC plate (K6 silica gel 60 A; Whatman, Clifton, NJ). Also spotted was 1–5 µg cold S1P standard and 1 nCi 32-P-ATP as size and intensity references, respectively. TLC was performed in butanol:ethanol:acetic acid:water (80:20:10:20, vol/vol) and data obtained from a phosphoimager (Bio-Rad).

Isolation of RNA
Total RNA was isolated using the Absolutely RNA RT-PCR Miniprep Kit (Stratagene, La Jolla, CA) following the manufacturer's instructions. RNA was quantitated using RiboGreen Kit (Molecular Probes). RNA samples were stored at –70°C.

Real-Time Reverse Transcriptase–PCR Detection of SK1 mRNA
One microgram of total RNA was reversed transcribed to cDNA using RETROscript reverse transcriptase (RT)-PCR Kit (Ambion, Austin, TX). The resulting cDNA was subjected to PCR in a Bio-Rad iCycler iQ system as follows: in a 0.2-ml PCR tube (Bio-Rad), 2 µl of cDNA (10% of synthesis reaction) was added to 48 µl of PCR reaction mixture containing 160 µM each dNTP (Invitrogen, Carlsbad, CA), 3.0 mM MgCl2 (Invitrogen), 1:15,000 SYBR Green I DNA Dye (Molecular Probes), 0.2 µM of each sense and antisense primers (IDT, Coralville, IA), and 2.5 U of Platimum Taq DNA (Invitrogen). Amplification and data collection was performed as previously described (34). Primers for human SK1 and HPRT genes are as follows (5' to 3'): SK1: forward, AAT-CTC-CTT-CAC-GCT-GAT-GC reverse, GAC-CTG-CTC-ATA-GCC-AGC-A HPRT: forward, TTG-GAA-AGG-GTG-TTT-ATT-CCT-C reverse, TCC-CCT-GTT-GAC-TGG-TCA-TT.

Quantitation of mRNA
Relative quantitative gene expression was calculated as follows. For each sample assayed, the Threshold Cycles (Ct) for reactions amplifying SK1 and HPRT were determined. The gene specific Ct for each sample was corrected by subtracting the Ct for HPRT ({Delta}Ct). Untreated controls were chosen as the reference samples, and the {Delta}Ct for all experimental samples were subtracted by the {Delta}Ct for the control samples ({Delta}{Delta}Ct). Finally, sample mRNA abundance, relative to control mRNA abundance was calculated by the formula 2-({Delta}{Delta}Ct). Validity of this approach was confirmed by using serial 10-fold dilutions of template containing experimental and HPRT genes. Using this set of template mixtures, the amplification efficiencies for experimental and HPRT amplimers were found to be identical.

siRNA for Sphingosine Kinase
Sphingosine kinase-1 si RNA and a non-silencing control siRNA were purchased from Qiagen (Catalog #1024921). A549 cells were plated in Opti-MEM (Gibco, Langley, OK) supplemented with 10% FBS (Hyclone, Logan, UT). The cells were cultured overnight and then the subconfluent cells were transfected. The transfection reaction was performed according to directions from Invitrogen (Lipofectamine 2000). Briefly, 20 pmol of siRNA in Opti-MEM was diluted with 2 µl of the transfection reagent for each reaction. The solution was incubated at room temperature for 20 min and then added to the cells. Twenty-four hours after transfection of siRNA, cells were infected with RSV for an additional 24 h. At this point, cellular lysates were assayed for sphingosine kinase activity and cell cultures were assayed for cell viability.

Statistical Analysis
Statistical analysis was performed on densitometry data, ELISA results and real-time PCR data. Significance was determined by Student's t test (GraphPad statistical analysis software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
RSV Infection of Lung Epithelial Cells Results in Significant Viral Replication before any Effect on Cell Survival
To evaluate the effect of infection on lung epithelial cell survival, A549 cells were infected with RSV. At various time points, whole cell lysates were obtained and viral replication analyzed by Western analysis for RSV-specific proteins. At the same time, duplicate cultures were analyzed for cell survival. Within 1 h there was increased detection of viral antigen that was maximal between 6–24 h of analysis (Figure 1A). In contrast, there was very little RSV-induced cell death until 48 h after infection (Figure 1B). By 72 h after infection the majority of the cells were dead. This is not novel data, but is included to point out that significant viral replication occurs before an effect on cell viability.



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  		Figure 1. RSV infection does not cause immediate cell death. A549 cells were infected with RSV (moi of 5) for various times. This moi has been determined previously to infect 90–100% of the cells. (A) Whole cell lysates were obtained and viral replication evaluated by Western analysis. Blots were stained with a polyclonal antibody to RSV proteins. (B) At various times after RSV infection, cell survival was analyzed by EthD-1 cell entry. The figure shows bright field photomicrographs of the cell monolayer and fluorescent photomicrographs of the EthD-1–positive cells. The graph depicts the percentage of dead cells, determined by counting 200 cells from a minimum of four different fields. Data are from three separate experiments.

 
RSV-Induced ERK and Akt Activation Have a Synergistic Effect on Cell Survival
In this experiment, we investigated the effect of blocking cell survival pathways in RSV-infected lung epithelial cells. Figure 2A demonstrates that at 24 h after infection, RSV has killed a small fraction of the cell population. If either ERK or Akt is inhibited, the amount of cell death is significantly increased. If both pathways are inhibited, close to 100% of the cells are dead. Figure 2B is a Western analysis, demonstrating that the inhibitors (10 µM for both U0126 and LY294002) specifically block RSV-induced Akt and ERK. The experiment also suggests that these two pathways are distinct and that inhibition of one does not substantially alter the activity of the other. The statistical analysis of densitometry of the Western analysis shows a nonsignificant increase in Akt activity with the ERK inhibitor. We hope to pursue this observation in the future. We have demonstrated before that RSV-induced Akt is involved in prolonged survival of infected cells. The link between ERK activity and survival of infected cells is a novel finding and suggests that these two pathways have complementary effects on prolonging survival of RSV-infected cells.



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  		Figure 2. Inhibition of ERK, Akt, or both pathways shortens cell survival after RSV infection. (A) A549 cells were infected with RSV (moi of 5) for 30 h. In some groups, the cells were treated with inhibitors (U0126 [10 µM] or LY294002 [10 µM] or both), beginning 30 min before RSV infection. Cell survival was analyzed by EthD-1 cell entry. The figure shows bright field photomicrographs of the cell monolayer and fluorescent photomicrographs of the EthD-1–positive cells. The images are representative of three separate experiments. The graph depicts the percentage of dead cells. Data are from three separate experiments. (B) A549 cells treated as describe above were incubated for 24 h. Whole cell lysates were obtained and analyzed for activated ERK and Akt. Equal loading was determined by stripping the blot and staining for ß actin. Densitometry was performed on three separate experiments and is shown in the graphs as fold increase. P values were determined for experimental samples versus control.

 
Inhibition of Sphingosine Kinase Blocks RSV-Induced ERK and Akt
The sphingolipid S1P has been implicated in cell survival in a number of systems (18). To investigate a possible role for S1P in RSV-induced survival pathways, we inhibited the enzyme responsible for S1P production, sphingosine kinase. Figure 3 demonstrates that inhibiting sphingosine kinase (with D-erythro-N,N-dimethyl-sphingosine [DMS]) blocked activation of both ERK and Akt, 24 h after infection. We found the same observation using another inhibitor of sphingosine kinase, DL-threo-dihydrosphingosine (DHS). The densitometry (expressed as fold increase) for the DHS experiments (three separate experiments) is as follows: (i) Active ERK, RSV 19 ± 3.78 compared with RSV and DMS 1 ± 0.50, P < 0.01; and (ii) Active Akt, RSV 5 ± 0.58 compared with RSV and DHS 1.8 ± 0.14, P < 0.01. While a useful model for studying pulmonary epithelial cells, A549 cells are not the same as primary human lung epithelial cells. They are a line that was initiated in 1972 by Giard and coworkers through explant culture of lung carcinomatous tissue from a 58-yr-old Caucasian male (35). To confirm our observations in a more relevant human cell, we evaluated the effect on sphingosine kinase inhibition on RSV-induced ERK and Akt activities in hTBE cells (see MATERIALS AND METHODS). Figure 3B demonstrates that in a primary tracheobronchial epithelial cell, inhibition of sphingosine kinase inhibits both ERK and Akt activation by RSV.



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  		Figure 3. RSV-induced ERK and Akt activity requires sphingosine kinase activity. (A) A549 cells were treated with the sphingosine kinase inhibitor, DMS (10 µM). RSV (moi of 5) was added to the cultures 30 min later and the cells cultured for an additional 24 h. Whole cell lysates were obtained and Western analysis performed for active ERK (phosphorylated Thr 202 and Tyr 204) and active Akt (phosphorylated Ser 473). Data are representative of three separate experiments. Densitometry was performed on three separate experiments and is shown in the graphs as fold increase. P values were determined for experimental samples versus control. (B) Identical experiments were performed using hTBE cells. Densitometry is shown as described above.

 
RSV Infection Activates Ceramidase and Sphingosine Kinase
To examine the enzymatic basis for RSV regulation of sphingolipid metabolism, we assayed two key steps involved in the generation of S1P. Sphingosine, the substrate for sphingosine kinase, is generated via deacylation of ceramide catalyzed by one of several ceramidases with varying pH optima. Two of these ceramidases, an acid ceramidase that is localized primarily in lysosomes (the site of lipid degradation) and a neutral/alkaline form that has been recently cloned in mammalian cells, were examined (36). These two ceramidases are the products of two different genes (37) with distinct pH requirements (acid: pH 4.5, neutral/alkaline: pH 7.2) (38). We analyzed the two different enzymes by altering the pH of the lysis buffer and the pH of the experimental buffer to optimize activity from either an acid or neutral ceramidase. Lung epithelial cells were infected with RSV for various times, and then whole cell lysates were obtained in either an acid or neutral buffer for analysis of these ceramidase activities. Figure 4A demonstrates that RSV infection rapidly stimulates significant amounts of neutral ceramidase activity. RSV modestly increased acid ceramidase activity, but the majority of viral-induced ceramidase activity occurred primarily in a neutral environment. The increased ceramidase activity generates increased amounts of sphingosine, a substrate for sphingosine kinase. We looked at the effect of RSV infection of SK1 mRNA. We found that RSV significantly increased expression of SK1 mRNA (Figure 4B). We also analyzed sphingosine kinase activity. Figure 4C demonstrates that lung epithelial cells constitutively express some sphingosine kinase activity and that activity of this enzyme is significantly increased after RSV infection (24 h). With the present available tools, we cannot definitively determine if the increased activity is due to increased protein, increased activity, or a combination of the two. However, we have shown that RSV infection significantly enhances sphingosine kinase activity and that this is accompanied by an increase in SK1 mRNA. We analyzed earlier time points, but found no activation of sphingosine kinase (data not shown). This is consistent with a role for sphingosine kinase in RSV replication-induced signaling. We have shown previously that there are two waves of inflammatory/survival signaling after RSV, one early activation linked to viral binding to the membrane and a latter activation due to viral replication (6, 8, 39). The time point of RSV-induced sphingosine kinase activity, after significant viral replication but before the appearance of cell death, is consistent with an effect of viral replication. To confirm the efficacy of our inhibitor (DMS), some groups were treated with DMS as well as RSV. Figure 4C demonstrates that exposure to DMS blocks RSV-induced sphingosine kinase activity. To confirm that this was not a finding unique to A549 cells, hTBEs were also analyzed for sphingosine kinase activity. Figure 4D demonstrates that in hTBEs, RSV induces sphingosine kinase activity that can be inhibited by DMS.




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  		Figure 4. RSV activates ceramidase and sphingosine kinase. A549 cells were infected with RSV (moi of 5) for various times. (A) Cells were lysed with either acid or neutral lysis buffers (see MATERIALS AND METHODS) and a ceramidase assay performed. Data was evaluated by phosphoimaging of the TLC plate. The graph represents three separate experiments (arbitrary units from the imaging software). (B) Cells were infected as describe above. At 24 h after infection, mRNA was isolated and SK1 levels determined by real-time RT-PCR. Data are a composite of three separate experiments. (C) Cells were lysed into sphingosine kinase buffer (see MATERIALS AND METHODS) and a sphingosine kinase assay performed. The panel contains TLC data (sphingosine kinase activity is reflected in the S1P production) and a graph with data combined from three separate experiments (Bio-Rad phosphoimaging with Personal FX). (D) hTBE cells were infected with RSV (moi of 5) for various times and with and without DMS for 24 h. Whole cell lysates were obtained and sphingosine kinase activity was determined as described in MATERIALS AND METHODS. In panel 1, a time course of RSV infection is shown. In panel 2, hTBEs were infected for 24 h with and without DMS and then sphingosine kinase activity assayed. Fold increase was determined by phosphoimaging of the TLC plate using the Bio-Rad Personal FX (experimental values/control values). The graph shows data from three separate experiments.

 
S1P Activates ERK and Akt in Lung Epithelial Cells
The prosurvival sphingolipid, S1P, has been shown to activate ERK and Akt in other systems (40, 41). Because sphingosine kinase inhibition blocked RSV-induced ERK and Akt (Figure 3), we examined the effect of exogenous S1P on lung epithelial cells. S1P induced activation of both ERK and Akt (Figure 5). The kinetics of ERK and Akt activation by S1P were slightly different. S1P increased ERK phosphorylation within 5 min, reaching maximal activation by 15 min, whereas Akt expression peaked 1 h after stimulation by the sphingolipid.



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  		Figure 5. S1P induces ERK and Akt activity in lung epithelial cells. A549 cells were treated with S1P (5 µM) for various times. Whole cell lysates were obtained and Western analysis performed for active ERK (phosphorylated Thr 202 and Tyr 204) and active Akt (phosphorylated Ser 473). Equal loading of the blot was determined by stripping the blots and reprobing with antibodies to total ERK and total Akt. Densitometry was performed and the data in the graph is expressed as fold increase (control value/experimental value). The blots are representative of three separate experiments.

 
Inhibition of Sphingosine Kinase Shortens Survival Times after RSV Infection
The previous data demonstrates that both Akt and ERK are important survival pathways for virally infected lung epithelial cells, that inhibiting ERK and AKT shortens survival time after RSV infection, that infection with RSV results in increased sphingosine kinase activity, and that blocking sphingosine kinase activity inhibits both ERK and Akt. To link sphingosine kinase–induced ERK and AKT to epithelial cell survival, lung epithelial cells were infected with RSV with and without an inhibitor of sphingosine kinase (DMS). Figure 6A demonstrates that inhibition of sphingosine kinase in the setting of RSV infection shortens survival time. The ~ 20% killing of RSV-infected cells at 30 h after infection is consistent with the survival data shown in Figure 1 (significant cell death from RSV alone does not occur until 48 h after infection). Sphingosine kinase also plays a role in normal lung epithelial cell survival, but the effects of inhibition are significantly greater with RSV infection. We obtained similar data with a trypan blue exclusion assay (data not shown). The same experimental design was repeated in hTBE cells. Viability was analyzed at 24 h after infection. As with A549 cells, inhibition of sphingosine kinase results in a significant increase in cell death in the RSV-infected cells (Figure 6B). This data replicated in a trypan blue exclusion assay (data not shown). To confirm the effect of sphingosine kinase inhibition on infected cell viability, we used siRNA techniques to block the RSV-induced sphingosine kinase activity. The sphingosine kinase siRNA alone had no effect on cell viability. However, as with the DMS data, inhibiting sphingosine kinase activity with siRNA shortened the survival of RSV infected cells (Figure 6C).




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  		Figure 6. Inhibition of sphingosine kinase shortens cell survival after RSV infection. (A) A549 cells were infected with RSV (moi of 5) for 30 h. In some groups, the cells were treated with an inhibitor of sphingosine kinase (DMS, 10 µM) beginning 30 min before RSV infection. Cell survival was analyzed by EthD-1 cell entry. The figure shows bright field photomicrographs of the cell monolayer and fluorescent photomicrographs of the EthD-1–positive cells. The graph depicts the percentage of dead cells. The data is representative of three separate experiments. (B) hTBE cells were infected with RSV (moi of 5) with and without the sphingosine kinase inhibitor, DMS (10 µM). Cell survival was analyzed by EthD-1 cell entry. The figure shows bright field photomicrographs of the cell monolayer and fluorescent photomicrographs of the EthD-1–positive cells. The graph depicts the percentage of dead cells. Data are representative of three separate experiments. (C) A549 cells were transfected with siRNA (specific for sphingosine kinase 1 or a nonspecific control). 24 h after transfection, cells were infected with RSV (moi of 5) and cultured for a further 24 h. At 24 h whole cell lysates were obtained and sphingosine kinase activity determined. Parallel samples were analyzed for cell viability with ethidium homodimer staining. Percentage of dead cells was determined by counting a total of 200 cells in three separate samples.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Epithelial cell death is an integral feature of airway inflammation. We have shown that RSV infection of lung epithelia results in cell death, but only after the cells have survived long enough to allow for the release of inflammatory mediators and support viral replication (5, 6, 8, 39). Herein, we show that RSV activates both ERK and Akt signaling and that both pathways are involved in maintaining cell survival in the setting of viral infection. The current studies demonstrate for the first time that sphingosine kinase activity is necessary to trigger these survival pathways. RSV sequentially activates a neutral ceramidase and then activates and induces sphingosine kinase. Consistent with the requirement for sphingosine kinase activity in ERK and Akt activation, sphingosine kinase activity was required for extended survival after RSV infection. RSV-induced ERK and Akt activities do not prevent viral-induced cell death, but they do delay cell death (50% cell death for RSV infected cells occurs at ~ 48 h, 50% cell death in the setting of sphingosine kinase inhibition and RSV infection occurs at ~ 24 h). Although most of the work described here was done in A549 cells, the data replicated in primary tracheobronchial epithelial cells. As a composite, these data suggest that RSV infection uses bioactive sphingolipids as an effector mechanism to prolong survival of infected epithelial cells (Figure 7).



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  		Figure 7. Model for prolonged cell survival after RSV infection. RSV infection activates ceramidase and then sphingosine kinase. This results in the activation of both ERK and Akt pathways. Combined activation of the two pathways significantly prolongs lung epithelial cell survival in the setting of RSV infection.

 
RSV's epidemiologic links to airway inflammatory disorders such as asthma are well recognized. S1P also has described links to asthma. For example, pulmonary S1P secretion triggered by allergen challenge induces mast cell degranulation via the S1P receptors, S1P1 and S1P2 (42, 43). These studies show that S1P can promote inflammation and airway remodeling by altering smooth muscle cell contraction, growth, and proinflammatory cytokine production (42). Our study does not directly address the inflammatory effects of increased S1P, but it is interesting to speculate that RSV induction of sphingosine kinase activity may contribute to airway inflammation and asthmatic symptoms by increasing extracellular levels of S1P. Thus, S1P, generated in response to RSV-activation of sphingosine kinase, might contribute to the inflammatory asthmatic phenotype.

S1P exposure has been previously linked to ERK activation. For example, VEGF stimulates endothelial cell growth via PKC-linked activation of the sphingosine kinase pathway. S1P generated in response to PKC activation in this study led to activation of Ras/Raf/MEK/ERK by binding to the S1P1 receptor; the PI 3-kinase and Akt survival pathway was also activated in this study (44). Likewise, we have shown that RSV activates multiple PKC isoforms leading to ERK activation (8). Indeed, initially during viral binding, RSV activates PKC {zeta}, but at later time points during viral replication (and the observed sphingosine kinase activity of this study), we found activation of PKC ß1, {delta}, and {epsilon}. This study does not address a specific link between PKC isoforms and RSV-induced sphingosine kinase activity, but the existing data suggest that PKC isoforms may be an additional metabolic target for viruses involved in airway inflammation.

S1P can signal both intracellularly and extracellularly via the S1P1–5 G protein–coupled receptors. Previous studies suggest that S1P signaling can be either intracellular or via a paracrine or autocrine pathway by release into the extracellular space and receptor binding (18). S1P has been shown to activate ERK by an intracellular signaling mechanism. In a study by Shu and colleagues, VEGF triggers sphingosine kinase activation and ERK activation that is independent of the S1P receptors (44). Akt activation, on the other hand, has been linked to extracellular S1P signaling via the S1P1 receptor. For example, S1P signaling via S1P1 receptors play a role in PDGF-induced cell motility (45). In the study by Hobson and coworkers, engagement of S1P to the S1P1 G protein–coupled receptor activates Rac, Fak, and PI3 kinase (and Akt), thereby regulating cell motility.

An interesting possibility in airway epithelia is that RSV-induced S1P signals to ERK via an intracellular pathway and to Akt via extracellular activation of the S1P1 receptor. This would have the added advantage of physically separating the two pathways. Akt, in some systems, has been shown to bind Raf-1 (upstream of ERK in RSV-infected cells) at serine residues (259), resulting in a reduced Raf kinase activity and decreased MEK and ERK1/2 activity (46). Our study demonstrates both ERK and Akt activation at 24 h after RSV infection. In addition, the inhibitor data suggest that in RSV-induced signaling, there is no significant crosstalk between these two pathways. One mechanism that would explain this observation is the spatial separation of the two pathways. If activation of Akt is downstream of S1P1 receptors and ERK is downstream of intracellular S1P signaling, physical separation of the activated Akt and Raf-1 is possible. Extracellular S1P can activate ERK and Akt in lung epithelial cells (this study), but we do not as yet know whether RSV-induced S1P is signaling intracellularly or via S1P receptors.

S1P, in contrast to ceramide, has been strongly linked to cell survival. In this study, we demonstrated that in lung epithelial cells infection with RSV led to sphingosine kinase–dependent activation of two survival pathways, ERK and Akt. Enhanced survival resulting from RSV-induced activation of these signaling pathways allows for increased viral replication and elaboration of inflammatory mediators by epithelial cells before viral-induced cell death. Thus, the generation of S1P might serve as a critical upstream signaling molecule in the pathogenesis of viral-associated airway disease. The results raise the possibility for interventions directed at modulation of sphingolipid metabolism after RSV infection to limit airway inflammation.


   Acknowledgments
 
This manuscript was supported by a VA Merit Review grant, NIH: HL-60316 and NIH: ES-09607, EPA: R826711 (G.W.H.), HL-55584 and HL 68135 (R.K.M.), and RR00059 from the General Clinical Research Centers Program, NCRR, NIH.

Received in original form November 24, 2003

Received in final form January 14, 2004


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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