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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 86-94, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.4881

Downregulation of the Epidermal Growth Factor Receptor by Human Cytomegalovirus Infection in Human Fetal Lung Fibroblasts

Thomas Beutler, Conny Höflich, Paul A. Stevens, Detlev H. Krüger and Susanna Prösch

Institutes of Virology and Medical Immunology, and Department of Neonatology, University Hospital Charité, Humboldt University, Berlin, Germany

Address correspondence to: Dr. Susanna Prösch, Ph.D., Institute of Virology, Univ. Hospital Charité, Humboldt University, Schumannstraße 20/21, D-10117 Berlin, Germany. E-mail: susanna.proesch{at}charite.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Epidermal growth factor plays a key role in late fetal lung development and differentiation as well as in regulating surfactant protein A synthesis, which is involved in innate immunity of the lung. Here we show that human cytomegalovirus (HCMV), a known lung pathogen in connatal and postnatal infection of neonates as well as transplant recipients, completely down-regulates EGF receptor (EGF-R) on the surface of human fetal lung fibroblasts. Inhibition of EGF-R synthesis occurs on the transcriptional rather than on the posttranscriptional level. The effect essentially depends on expression of viral immediate early and/or early genes, as binding of ultraviolet light-inactivated virus to the cells had no effect on EGF-R expression. Furthermore, the anti-HCMV drug ganciclovir, which blocks HCMV DNA replication and late gene expression, cannot overcome HCMV-mediated inhibition of EGF-R, suggesting that immediate early or early gene products may be responsible for down-regulation of EGF-R. Interestingly, the glucocorticoid dexamethasone, which is used for its antiinflammatory action to prevent chronic lung disease in preterm infants, promotes HCMV-associated downregulation of the EGF-R by stimulation of viral gene expression. From these data it can be hypothesized that the pathogenesis of HCMV lung infection involves down-regulation of EGF-R and that congenital HCMV infection may cause retardation in lung maturation and surfactant protein synthesis.

Abbreviations: bronchopulmonary dysplasia, BPD • chemiluminescence detection system, CCD • cycloheximide, CHX • dexamethasone, Dexa • epidermal growth factor, EGF • fluorescence-activated cell sorter, FACS • fetal calf serum, FCS • ganciclovir, GCV • human cytomegalovirus, HCMV • human fetal lung fibroblasts, HFLF • horseradish peroxidase, HRP • immediate early, IE • immunoglobulin, Ig • multiplicity of infection, MOI • messenger RNA, mRNA • phosphate buffered saline, PBS • phycoerythrin, PE • phenyl-methyl-sulfonyl fluoride, PMSF • sodium dodecyl sulfate, SDS • surfactant protein A, SP-A • TBST • tumor growth factor, TGF • ultraviolet light, UV


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Late fetal alveolar epithelial cell maturation is mediated through mesenchymal–epithelial cell communication. As a key mediator, epidermal growth factor (EGF) has been shown to accelerate fetal lung maturation both in vivo and in vitro, as it increases alveolarization and differentiation of type II cells (1, 2). EGF also plays an essential role in the development of the surfactant system, as it stimulates the production of surfactant-associated phospholipids and of surfactant proteins (SP) A and C (3–7). In rhesus monkey infants, EGF treatment decreased the severity of respiratory distress syndrome and improved pulmonary compliance (8, 9). Also, treatment of mouse embryo lungs or fetal mice with EGF-specific antisense oligonucleotides and anti-EGF serum, respectively, significantly reduced branching morphogenesis and alveolar type II cell differentiation (10, 11).

EGF functions by binding to and activing its receptor, EGF-R. Ligand–receptor interaction results in receptor dimerization, which in turn stimulates intrinsic tyrosine kinase activity leading to autophosphorylation of the receptor (12). The activated receptor then induces an intracellular mitogenic signal transduction cascade resulting in activation of mitogen-activated protein kinases and signal-regulated kinase, which lead to diverse cellular responses like stimulation of gene expression (13, 14). In late fetal development, the level of EGF-R activation regulates alveolar epithelial cell maturation. In EGF-R–deficient mice, the alveolar epithelium remained undifferentiated and the lungs were inadequately inflated (15–18). In human fetal lung explants, antisense inhibition of EGF-R decreased transcription of surfactant protein A (SP-A) (7), the main component of lung innate immunity (19).

Mesenchymal–epithelial communication occurs between lung fibroblasts in the mesenchym and type II cells in the alveolar epithelium. EGF-R in fetal lung fibroblasts rather than in type II cells controls the timing of mesenchymal-epithelial interaction leading to surfactant synthesis. Fetal lung fibroblasts in rats expressed much larger amounts of EGF-R than type II cells (20) indicating that fibroblasts signal differentiation of type II cells and surfactant synthesis. The highest levels of EGF-R on lung fibroblasts were detectable at the time of the onset of surfactant synthesis by type II cells (21, 22).

Human cytomegalovirus (HCMV) is a ubiquitous herpes virus in man. It is rarely pathogenic in healthy adults but is associated with severe infection and diseases in immunosuppressed patients (e.g., patients with human immunodeficiency viral infection and transplant recipients). Clinically, interstitial pneumonia is the most fatal infection. Furthermore, HCMV is the most common agent of congenital and perinatal infection in humans (23). In addition, about 20% of preterm infants become postnatally infected with HCMV by breastfeeding (24, 25). In a very recent clinical study among preterm infants, 18% of infants were found to be connatally or postnatally infected with HCMV (26). In all infected infants, viral DNA was detectable in tracheal or pharyngeal aspirates, indicating that the respiratory tract or even the lung was involved in infection. Notably, we (26), like Sawyer and coworkers (27), observed an increased incidence of HCMV infections in preterm newborns developing bronchopulmonary dysplasia during the first month of life. Others reported on HCMV-associated pneumonia and Wilson-Mikity syndrome in an infant after intrauterine HCMV infection, as well as chronic lung injury after postnatal HCMV infection in preterm infants (28, 29).

Pathogenesis of cytomegalovirus lung infection is not fully understood. Aside from a direct virus-induced pathogenesis leading to irreversible damage to infected lung cells, an immunopathogenic component caused by changes in local cytokine expression has been discussed (30–32). In the lung, HCMV may infect fibroblasts and type II cells as well as alveolar tissue macrophages (33–35). As shown very recently, SP-A may support infection of type II cells and alveolar macrophages by cytomegalovirus rather than elimination of the virus (36).

Our present work was intended to make a contribution to the understanding of HCMV-induced lung pathology. We could show that HCMV down-regulates EGF-R expression in fetal lung fibroblasts, and this effect essentially depends on viral gene expression. Dexamethasone, a drug currently used in the treatment of infants with chronic lung disease, supports the effect of HCMV on EGF-R expression. Furthermore, the antiviral drug ganciclovir (GCV) cannot block HCMV-mediated downregulation of EGF-R.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells and Viruses
Human fetal lung fibroblasts (HFLF) Fi 301 were maintained in Minimal Essential Eagle Medium (MEM; Biochrom AG, Berlin, Germany) supplemented with 2% fetal calf serum (FCS; Biochrom AG) certified as mycoplasma-free and low endotoxin. Virus stocks of the HCMV strain AD169 were obtained from supernatant of infected cell cultures 10 d p.i. by ultracentrifugation (90 min at 40,000 x g and 4°C) and stored in aliquots in liquid nitrogen. Ultraviolet (UV)-inactivation of HCMV AD169 was carried out by exposure of virus to UV light (220 V, 12 W) for 15 min (kindly done by J. Cinatl and J. U. Vogel, Frankfurt, Germany). UV-irradiated virus stocks were free of infectious virus as confirmed by plaque titration.

Generally, HFLF were infected with HCMV at a multiplicity of infection (MOI) of 1. Adsorption was allowed for 1 h at 37°C. Infected cells were propagated at 37°C.

Fluorescence-activated Cell Sorting Analysis
A total of 1 x 106 infected or mock-infected cells were washed two times with phosphate-buffered saline (PBS) and collected with cell dissociation solution (Sigma, Taufkirchen, Germany), washed again with PBS and with PBS/1% FCS. Cells were stained with phycoerythrin-conjugated anti-EGF-R antibody or phycoerythrin-conjugated isotype control immunoglobulin (both from BD PharMingen, Heidelberg, Germany) in adequate concentrations in PBS/10% FCS for 1 h, washed with PBS/1% FCS and again with PBS. Surface EGF-R amounts were analyzed using a FACScalibur analyser and Cellquest software (Becton Dickinson, Heidelberg, Germany).

Western Blot Analysis
For preparation of protein extracts, virus-infected and mock-infected HFLF (1 x 106 cells) were washed twice with PBS, harvested by cell scraping, washed again with PBS, and solubilized in 200 µl lysis buffer containing 20 mM Tris-hydrochloric acid, pH 7.5, 150 mM sodium chloride, 1% Nonidet P40, 0.02% sodium nitrate, aprotinin (1 µg/ml), antipain (1 µg/ml), leupeptin (2 µg/ml), phenyl-methyl-sulfonyl fluoride (PMSF) (2 mM) (all inhibitors from Roche, Mannheim, Germany) and 2.5 mM ethylenediaminetetraacetic acid, shaken for 1 h at 350 rpm and centrifuged for 50 min at 18,000 x g and 4°C to remove lipids and cell debris. The protein-containing supernatant was stored at -70°C. Protein quantification was performed using Advanced Protein Assay Reagent (TEBU, Frankfurt, Germany).

Four Western blot analysis probes (50–80 µg per lane) were electrophoresed on a 7.5% sodium dodecyl sulfate (SDS)-polyacrylamid gel and transferred (Mini Tank Electroblotter, OWL Scientific) to a cellulosenitrate membrane Protran BA 85 (Schleicher & Schuell, Dassel, Germany). The membrane was blocked overnight in TBST (10 mM Tris hydrochloride, pH 7.4, 150 mM sodium chloride, 0.05% Tween 20) supplemented with 3% bovine serum albumin (Boehringer Ingelheim, Heidelberg, Germany), followed by blocking for 1 h in TBST containing 1% gelatin. Membranes were then incubated for 2 h at room temperature with either sheep polyclonal anti-human EGF-R (1:1,500; Upstate Biotechnology, NY), mouse anti-HCMV IE antigen, clone E13 (1:500; Harlan Sera-Lab, Loughborough, UK), mouse monoclonal anti-HCMV p68 late protein (1:5,000; Advanced Biotechnology, Columbia, MD), or goat anti-actin antibody (1:1,000; SantaCruz Biotechnology Inc., Heidelberg, Germany). After washing five times with TBST, the blots were incubated for 1 h with the second, anti–species-specific antibody: rabbit anti-sheep horseradish peroxidase (HRP)-conjugated Ig (immunoglobulin) G (1:4,000; Upstate Biotechnology, New York, NY); goat anti-mouse HRP-conjugated IgG (1:4,000; Boehringer Mannheim, Indianapolis, IN); or donkey anti-goat HRP-conjugated IgG (1:4,000; SantaCruz Biotechnology, Heidelberg, Germany). The immunoreactive bands were visualized and quantified using SuperSignal substrate (Pierce, Rockford, IL) and a chemiluminescence detection system (CCD) Camera (Raytest Isotopenmeßgeräte GmbH, Straubenhardt, Germany).

RNA Isolation and Northern Blot Analysis
For total RNA isolation, infected and mock-infected HFLF (1 x 106 cells) were washed twice with PBS and lysed with the Trizol Reagent (GIBCO BRL, New York, NY). For Northern blot analysis a 10 µg sample of total RNA was electrophoresed on a 1% denaturing formaldehyde-agarose gel and transferred to nylon membrane (Boehringer Mannheim, Mannheim, Germany). Membranes were hybridized with 32P-labeled complementary DNA (Ready TO GO DNA Labelling Beads [-dCTP]; Amersham Pharmacia Biotech, Freiburg, Germany) coding for human EGF-R–like protein (clone 206, kindly provided by H. P. Saluz, Jena, Germany). After prehybridization for 4 h at 42°C in 3 x SSC, 5 x Denhardts, 1.0% SDS, 50% formamide, 1 mM NapyroPO4 in 1 M NaPO4, pH 7.0 and 1 µg/ml salmon sperm DNA, hybridization occurred at 42°C for 18 h. The blots were washed to a stringency of 1 x SSC/0.2% SDS at room temperature and exposed to a phosphor screen, and visualized and quantified by phosphorimaging (PhosphorImager, type SI) and ImageQuant software (both from Molecular Dynamics, Amersham Pharmacia Biotech, Freiburg, Germany).

EGF-R messenger RNA Turnover Studies
Mock-infected and HCMV-infected (MOI = 1) HFLF were treated with the transcription inhibitor actinomycin D (5 µg/ml, Sigma, Taufkirchen, Germany) 48 h after infection and harvested at 0, 4, 8, and 12 h thereafter. Total RNA was isolated from the cells and subjected to Northern blot analysis as described above.

Immunocytochemical Staining of HCMV-infected Cells for Immediate Early Antigens IE1 and IE2
For immuncytochemical staining, monolayers of HCMV-infected and mock-infected HFLF were washed twice with PBS, fixed with ethanol/acetone 95:5 for at least 30 min at –20°C, and blocked with 10% FCS, 0.2% Tween in PBS for 30 min at 37°C. Staining was carried out with mouse anti-HCMV IE antibody clone E13, 1:100 (Oxford Biotechnology, Oxford, UK) in blocking solution for 1 h at 37°C. After five washes with PBS goat anti-mouse HRP-conjugated IgG (1:4,000; Boehringer Mannheim, Indianapolis, IN) was added for an additional 1 h at 37°C. After five washes, the cells were incubated for 30 min with 3-amine-9-ethylcarbazole (AEC) substrate (Sigma, Taufkirchen, Germany) and visualized with a standard inverted light microscope.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Downregulation of EGF-R on the Surface of HFLF after Infection by HCMV
HFLF were infected with HCMV strain AD169 at MOI = 1. Infection was controlled by immunostaining of infected and mock-infected cells for IE1 and IE2 proteins 24 h after infection. Nuclei of all infected cells showed red staining, indicating that, indeed, all cells were productively infected (Figure 1A, top) while mock-infected cell culture showed no staining (Figure 1A, bottom). With fluorescence-activated cell sorting (FACS) analysis we observed a rapid loss of EGF-R on the surface of HCMV-infected cells that continued over time (Figures 1B and 1C). The influence of HCMV infection on EGF-R expression on the surface of HFLFs was determined: at Day 2 after infection, the EGF-R levels were reduced by about 50% when compared with mock-infected cells; at Day 4 after infection more than 90% of EGF-R was lost from the surface of infected cells (Figure 1C). In contrast, EGF-R on mock-infected HFLFs decreased only marginally over 4 d. Binding of the isotype-matched antibody was not significantly decreased after 4 d, indicating that the obtained decrease in EGF-R antibody binding as visible in Figure 1B is highly specific.





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  		Figure 1. Influence of HCMV on surface EGF-R expression on HFLF. (A) Immunocytochemical staining of HCMV-infected (top; MOI = 1) and mock-infected (bottom) HFLF at Day 1 after infection with antibody clone E13, specific for HCMV IE1 and IE2 antigens. (double arrow = 20 µm). (B) EGF-R expression on HCMV-infected and mock-infected HFLF at Days 1 (d1) and 4 (d4) after infection. Cells were stained with phycoerythrin (PE)-conjugated anti-EGF-R or PE-conjugated isotype control immunoglobulin, or were left unlabeled. (C) Kinetics of EGF-R expression on HFLFs following HCMV infection. Cells were stained with PE-labeled EGF-R antibody on the indicated days after infection and measured by flow cytometry, or were left unlabeled. Results are shown as fluorescence intensities (geo mean) for one representative experiment that was repeated twice. Data presented are for cells that were: uninfected, unlabeled (diamonds, dotted-dashed line); uninfected, anti-EGF-R (squares, thin solid line); and virus-infected, anti-EGF-R (triangles, thick solid line).

 
Decrease of EGF-R Protein Levels in HFLF after HCMV Infection
To determine whether the total level of EGF-R decreases after HCMV infection, protein extracts from infected and mock-infected HFLF were analyzed by Western blot analysis with an EGF-R–specific antibody. Infected (MOI = 1) and mock-infected cells were harvested at 1-d intervals. Similarly to the data obtained by FACS analysis, significant reduction of total EGF-R protein became obvious. At Days 3 and 4 after infection EGF-R was reduced by 20% and 60%, respectively, while EGF-R amounts in mock-infected cells remained unchanged (Figure 2, lanes 1 and 6). Immunoblotting of the same probes with antibodies against HCMV IE1 and IE2 as well as p68 late antigen showed the expected expression beginning at 24 (IE1 and IE2) and 48 h (p68) after infection, and an increase until Days 3 and 4 (Figure 2, middle panels). By comparison, expression of actin was not influenced by HCMV infection (Figure 2, bottom panel).



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  		Figure 2. Time-dependent effect of HCMV infection on EGF-R protein in HFLFs. Representative Western blot of total protein from HFLF mock-infected (lanes 1 and 6) and HCMV-infected (lanes 2–5) harvested at Days 1, 2, 3, and 4 after infection (d1–d4) are shown. The specimens were immunoblotted with sheep anti-human EGF-R (EGF-R) (top panel); anti-HCMV IE1 and IE2 clone E13 (IEACMV, p86 IE2 and p72 IE1) and anti-HCMV late protein p68 (p68CMV) (middle panels); anti-actin antibody (Actin) (Bottom panel).

 
HCMV Inhibits EGF-R Expression at the Transcriptional Level
To elucidate the mechanism of HCMV-associated down-regulation of EGF-R its transcription was studied by Northern blot analysis. When hybridizing total RNA from HCMV-infected and mock-infected cells a time-dependent reduction of EGF-R-specific messenger RNA (mRNA) in virus-infected cells was observed (Figures 3A and 3B). At Day 3 after infection parallel to the observed reduction of EGF-R on the protein level (Figure 2, lane 4), EGF-R mRNA was reduced by about 60% (Figure 3A, lane 4 versus lane 1; Figure 3B). On Day 4 after infection no significant amount of EGF-R mRNA could be detected in virus-infected cells (Figure 3A, lane 5) and inhibition reached more than 90% (Figure 3B). For control, EGF-R mRNA levels in mock-infected cells harvested at Days 1 and 4 after infection remained unchanged (Figure 3A, lanes 1 and 6).




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  		Figure 3. Effect of HCMV infection on EGF-R gene transcription in HFLFs (A) Representative Northern blot of total RNA from mock-infected HFLF (lanes 1 and 6, controls) and HCMV-infected HFLF (lanes 2–5) hybridized with 32P-radiolabeled plasmid p206 containing human EGF-R complementary DNA (EGF-R) and with 32P-radiolabeled complementary DNA specific for human actin (Actin). d1 to d4: cells harvested at Days 1, 2, 3 and 4 after infection. (B) Quantitative analysis of Northern blots of total RNA from mock-infected and HCMV-infected HFLF. The bars represents mean values (± SEM) of relative mRNA levels measured by phosphorimaging in three independent experiments. Vd1 to Vd4: virus-infected cells harvested on Days 1, 2, 3 or 4 after infection; Cod4: mock-infected control cells harvested at Day 4 after infection.

 
To test the possibility that HCMV infection influences stability of the EGF-R mRNA, total RNA was isolated from mock-infected and HCMV-infected cells after treatment with the transcription blocking agent actonomycin D and hybridized to the EGF-R–specific probe. After quantification by phosphorimaging the data of two independent experiments (calculated as a percentage of control levels at the time of adding actinomycin D) from all mock-infected and HCMV-infected cells were plotted against time (Figure 4). The resulting curves show that downregulation of EGF-R transcription following HCMV infection is not associated with destabilization of the EGF-R mRNA. Therefore, it seems unlikely that posttranscriptional mechanisms contribute to the observed virus-induced inhibition of EGF-R synthesis.



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  		Figure 4. Effect of HCMV infection on EGF-R mRNA stability HFLF were mock-infected (closed circles) or infected with HCMV (MOI = 1) (open circles) and after 48 h transcription was blocked by addition of actinomycin D (5 µg/ml). Total RNA was extracted from the cells at 0, 4, 8 and 12 h after addition of actinomycin D and analyzed by Northern blot using a radiolabeled EGF-R-specific complementary DNA probe. Data were obtained by Northern blots of total RNA from mock-infected and HCMV-infected HFLF after actinomycin D treatment as measured by phosphorimaging. Results of two independent experiments are shown.

 
Downregulation of EGF-R by HCMV Depends Essentially on Viral IE/E Gene Expression
Next, we investigated whether binding of the virus to the cells or viral gene expression is responsible for inhibition of EGF-R expression. Cells were incubated with either UV-inactivated virus or replication-competent virus and probed for EGF-R expression 4 d after infection. Both UV-inactivated and wild-type virus did bind to the cell; however, as shown in Figure 5A, no expression of viral proteins occurred. As expected, incubation of HFLF with the UV-inactivated virus had no effect on EGF-R expression (Figure 5A), indicating that binding of the virus to the cell by itself has no effect on EGF-R expression. For a positive control, productive infection of cells with replication-competent HCMV AD169 led to the previously described downregulation of EGF-R within 4 days after infection (Figure 5B).



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  		Figure 5. Influence of UV-irradiated HCMV on EGF-R expression in HFLF. (A) Total protein from mock-infected (co) and UV-irradiated HCMV-infected HFLF (HCMVUV) immunoblotted with EGF-R antibody (EGF-R) (top panel) and anti-HCMV IE1 and IE2 antibody clone E13 (IEACMV) (bottom panel). (B) Same procedure as in A, but nonirradiated virus (HCMV) was used instead of HCMVUV. Representative blots, which have been repeated twice, are shown.

 
In a second approach, the influence of GCV on HCMV-mediated inhibition of EGF-R was studied. GCV is the most frequently used anti-human HCMV drug (e.g., in transplant recipients with pneumonia) known to inhibit viral DNA replication and expression of late viral proteins (for review, see Ref. 37 and Figure 6, bottom panel, lane 4). To limit HCMV-associated disease, GCV therapy is now also common for treatment of congenital HCMV infection (38–40). HCMV- and mock-infected HFLF were treated with 10 µM GCV and studied for EGF-R expression by Western blot analysis at Day 4 after infection. GCV was added to the culture medium immediately after adsorption of the virus. As demonstrated in Figure 6 (top panel, lane 4), GCV seems to be unable to prevent HCMV-induced inhibition of EGF-R expression, although late gene expression was fully inhibited (Figure 6, bottom panel, lane 4). This indicates that HCMV late proteins are not involved in EGF-R suppression. The remaining but significantly reduced quantity of EGF-R in GCV-treated cells (as compared with untreated mock-infected and HCMV-infected cells; Figure 6, top panel, lane 4 versus lane 1 versus lane2) reflects the decreased overall expression of IE1/2 protein in the GCV-treated HCMV-infected culture (for comparison, see the untreated HCMV-infected culture, Figure 6, lane 4 versus lane 2). Thus, the decreased expression of IE1/2 (and E) proteins in the GCV-treated culture resulted in incomplete suppression of EGF-R synthesis.



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  		Figure 6. Effect of ganciclovir (GCV) on HCMV-associated down-regulation of EGF-R and viral gene expression in HFLF. Representative blots of total protein from mock-infected (co), HCMV-infected (HCMV), mock-infected cells treated with 10 µM GCV (GCV), or HCMV-infected HFLF treated with 10 µM GCV (HCMV+GCV) were immunoblotted with antibodies specific for human EGF-R (EGF-R) (top panel), anti-HCMV IE1 and IE2 antibody clone E13 (IEACMV) (middle panel), and anti-HCMV late protein p68 (p68CMV) (bottom panel).

 
Dexamethasone Triggers HCMV-induced Reduction of EGF-R Expression
Glucocorticoids, such as dexamethasone, are clinically used for immunosuppressive and antiinflammatory therapy (41). In preterm infants, dexamethasone has been shown to prevent respiratory distress syndrome (42, 43). However, dexamethasone treatment has also been reported to stimulate HCMV replication in HFLF by increasing HCMV protein expression and viral DNA synthesis (44–46). Analogously, in patients under dexamethasone therapy, enhancement of viral load can be observed frequently (unpublished observations). Therefore, we examined the effect of dexamethasone treatment on HCMV-induced downregulation of EGF-R. In preliminary investigations, a 3.3-fold maximal stimulation of HCMV IE1/2 gene expression was measured in the presence of 10-5 M dexamethasone added to virus-infected cultures after adsorption (data not shown). At this concentration, it can be inferred that dexamethasone strengthened the negative effect of HCMV on EGF-R expression because complete downregulation of EGF-R was already observed on Day 3 after infection, compared with Day 4 in the absence of dexamethasone (Figure 7A, top panel versus middle panel; Figure 7B). In the presence of dexamethasone, EGF-R protein level at Day 2 after infection was considerably reduced when compared with the untreated infected cell culture (40% versus 80%, Figure 7B). As a control, actin expression was not changed by dexamethasone (Figure 7A, bottom panel).




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  		Figure 7. Influence of dexamethasone on HCMV-dependent suppression of EGF-R in HFLF. (A) HFLF were mock-infected (co) or infected with HCMV AD169 (HCMV) and grown in the absence (top panel) or presence (middle panel) of 10-5 M dexamethasone. Cells were harvested on Days 1, 2, 3 or 4 after infection (d1 to d4) for immunoblot analysis of EGF-R total protein (EGF-R, top and middle panels). For control, the protein extracts were immunoblotted with actin-specific antibody (Actin). Representative immunoblots, which have been repeated twice, are shown. (B) Quantitative analysis of steady-state EGF-R protein levels in HCMV-infected HFLF grown in the absence (no dexa; black bars) or in the presence (plus dexa; gray bars) of 10-5 M dexamethasone. Mean values (± SEM) of relative EGF-R expression as estimated by phosphorimaging of three independent experiments are presented. Cod1, mock-infected control cells (control) harvested at Day 1 after infection; Vd1 to Vd4, virus-infected cells harvested on Days 1, 2, 3 and 4 after infection.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The EGF-R belongs to the family of receptor tyrosine kinases that are expressed in many different cell types. Activation of tyrosine kinase leads to autophosphorylation of the EGF-R itself, as well as initiating intracellular mitogenic signal transduction through a cascade of second messengers (12–14). In the lung, EGF-R plays a major role in the fibroblast–type II cell communication, which is essential for both lung maturation and type II cell function. Fetal lung fibroblasts in vivo as well as in vitro (as shown here) constitutively express detectable levels of EGF-R on their surface (20). In vivo EGF-R expression in lung fibroblasts increases during late gestation in a sex-specific manner (22, 47). EGF-R synthesis is positively regulated by its natural ligands EGF and tumor growth factor (TGF) {alpha} as well as by phorbol ester at the transcriptional and posttranscriptional level, while thyroid hormones and retinoic acid down-regulate EGF-R synthesis (48–50). Our study demonstrates that HCMV infection of fetal lung fibroblasts completely down-regulates synthesis of EGF-R within 3 to 4 d after infection. As a consequence, EGF-R irreversibly disappears from the surface of infected cells. Similarly, Sinclair and colleagues (personal communication) observed EGF-R down-regulation on HFLF after HCMV infection when measuring EGF-R–associated tyrosine kinase activity and surface EGF-R.

Our data show that the loss of EGF-R protein is strongly correlated with inhibition of EGF-R transcription, while the stability of the EGF-R mRNA remained unchanged. Thus, inhibition of EGF-R by HCMV occurs at the transcriptional rather than at the posttranscriptional level (mRNA stability). Degradation of the existing receptor by the virus could not be completely ruled out; however, this mechanism, if it exists, should play a much less important role compared to the transcriptional mechanism. In all our experiments, we observed a delayed decrease of EGF-R protein levels as compared with the decrease of EGF-R mRNA.

We have shown that downregulation of EGF-R in HCMV-infected cells strongly depends on de novo synthesis of viral proteins. Binding and internalization of irradiated, nonreplicating virus to the cells had no influence on EGF-R expression.

HCMV may code for up to 200 different proteins as predicted from the viral genome sequence (51). Expression of these proteins is strongly regulated as a cascade. After entry into the cell, first, immediate early (IE) proteins are synthesized, which peak at 24 h after infection. Early (E) gene expression starts at 24 h after infection and reaches its maximum at 48 h after infection. Late gene transcription depends on DNA replication and starts at about 48 h after infection. A complete replication cycle in permissive cells in vitro needs about 72 h (52). GCV, the main anti-HCMV drug, blocks HCMV DNA replication and late gene expression, and consequently, the spreading of the virus. However, GCV does not inhibit IE and E gene expression in infected cells (37). As shown in this article, GCV was not able to block HCMV-induced downregulation of EGF-R in infected cells. These data strongly suggest that IE and/or E proteins of the virus, but not late (structural) proteins, are responsible for inhibition of EGF-R transcription. The involvement of IE and/or E proteins in EGF-R downregulation is also supported by the time course of both surface EGF-R protein and EGF-R mRNA down-regulation. The half-life of EGF-R was determined to be 9 to 40 h (53). Thus, EGF-R detected in cells on Days 1 through 3 after infection may reflect the amount of receptor existing before infection. Downregulation of EGF-R mRNA was observed as early as 2 d after infection, when HCMV IE and E protein are synthesized. The major IE proteins IE1 and IE2 are involved in modulation of cellular processes like positive or negative regulation of cellular gene expression. They are also essential for expression of HCMV E genes (54, 55). Because of their ability to regulate gene expression, they are possible candidates for the observed effect on EGF-R transcription. However, as both IE1 and IE2 are essential for viral replication (56, 57), their role in EGF-R down-regulation is difficult to prove. Another possible candidate may be the glycoprotein B, which is synthesized with early kinetics and has also very recently been shown to modulate expression of several cellular genes (58).

Glucocorticoids, such as dexamethasone, are widely used for the prevention of respiratory distress syndrome in preterm infants because of their antiinflammatory effect (41–43). However, glucocorticoids did not influence EGF-R activation and expression, indicating that their effect targets processes other than the EGF/EGF-R system (47). On the other hand, dexamethasone was found to stimulate HCMV replication in vitro in fetal lung fibroblasts by stimulation of viral gene expression and viral DNA replication (44–46). Here we demonstrate that dexamethasone accelerates HCMV-mediated inhibition of EGF-R synthesis on HFLFs. This effect correlated with increased IE1/2 protein expression. Treatment of infected cells with 10-5 M dexamethasone stimulates a 3.3-fold increase in IE1/2 protein expression over that of the infected but untreated control cells (data not shown). Consequently, EGF-R downregulation in dexamethasone-treated cells occurs 24 h earlier than in untreated cells. This may be of special interest for the therapeutic management of preterm infants congenitally or postnatally infected with HCMV. In these infants, treatment with dexamethasone may increase viral load, promoting HCMV-associated pathogenesis (59). As a consequence, downregulation of EGF-R may cause a further delay in lung maturation and surfactant synthesis. These dexamethasone-treated infants may benefit from additional GCV administration to reduce or even block viral spread.

HCMV is not the only virus that was found to interfere with EGF-R surface expression and signaling. Several years ago, adenovirus, another lung pathogen, was found to down-regulate EGF-R (60, 61). However, in contrast to HCMV, adenovirus does not interfere with synthesis of the receptor, but stimulates endosome-mediated internalization and degradation of surface EGF-R. These effects are caused by the interaction of the adenoviral proteins E3 with the cytosolic tail of the EGF-R, mimicking oligomerization of the receptor, followed by its internalization and degradation. Normally these processes are induced by interaction of EGF-R with its ligands EGF or TGF-{alpha} (60, 61). Another virus known to interfere with EGF-R signaling is poxvirus, which codes for an EGF/TGF-{alpha}–like protein, vaccinia growth factor, acting in an autocrine fashion as a ligand of EGF-R (62, 63).

In summary, we could demonstrate that HCMV, an important congenital lung pathogen, negatively interferes with the EGF/EGF-R signal system which is involved in the regulation of lung maturation, surfactant lipid and surfactant protein A synthesis. Therefore, HCMV may interfere with lung maturation and functions, such as innate immunity, in which SP-A is involved. As HCMV was suggested to act as a co-factor for development of BPD and chronic lung injury in preterm infants (26, 27), the observed interference of the virus with the EGF/EGF-R signal system may be one component contributing to the development of chronic lung failure.


   Acknowledgments
 
The authors thank C. Priemer for providing fetal lung fibroblast cultures, J. Sinatl and J.-U. Vogel for providing UV-irradiated AD169, and M. Raftery for help in preparing the manuscript. The work was supported by Grant 01ZZ9511 from the German Federal Ministry of Education and Research, German Research Foundation, Deutsche Forschungsgemeinschaft SFB 421, and Humboldt University Berlin, Charité.

Received in original form April 18, 2002

Received in final form August 19, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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