Division of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Physiology Program, Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts 02115; and Respiratory Research Group, University of Calgary, Calgary, Alberta, Canada T2N 4N1
Submitted 13 June 2003 ; accepted in final form 21 November 2003
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ABSTRACT |
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hyperresponsiveness; remodeling; gene transcription; serum response factor
Several lines of evidence suggest that EGR-1 may be involved in the pathogenesis of asthma. 1) EGR-1 is expressed in several airway cell types grown in cell culture and is induced by platelet-derived growth factors (PDGF) and other growth factors, cytokines, and prophlogistic stimuli found in the airways of asthmatic patients (15, 18, 27). 2) EGR-1 is thought to control the expression of several genes involved in the regulation of airway inflammation, smooth muscle growth, and airway tone (11, 22, 23). 3) Cytokine expression, IgE regulation, and hyporesponsive airways are altered in egr-1-deficient mice (23). 4) The EGR-1 gene is located in the "cytokine-gene cluster" (q3133) of chromosome 5, a genomic region linked to atopy, airway hyperresponsiveness, and asthma (17). However, it is unclear whether EGR-1 plays a role in the pathogenesis of asthma.
Induction of EGR-1 occurs primarily at the transcriptional level and is mediated, in part, through different subgroups of mitogen-activated protein kinases (MAPKs), including the extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 pathways (22). Induction through these pathways involves phosphorylation of ternary complex factors, such as E26 transcription factor (ETS)-like protein 1 (ELK-1), and the stabilization of serum response factors (SRF) at multiple serum response elements (SRE) in the EGR-1 promoter; however, the specifics of EGR-1 transcriptional activation depend on cell type and specific stimulus (8, 19).
On the basis of these data, we formed two hypotheses: 1) EGR-1 is induced by PDGF in human airway smooth muscle cells (HASMC) in cell culture, and 2) EGR-1 expression is increased in vivo in airway smooth muscle of asthmatic patients compared with nonasthmatic controls. We found that PDGF induces EGR-1 in HASMC in cell culture by a signal transduction pathway involving ERK1/2; however, no significant differences in EGR-1 expression were found in the lungs of asthmatic patients compared with nonasthmatic controls or in the lungs of mice with ovalbumin (OVA)-induced airway inflammation compared with lungs of normal mice.
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MATERIALS AND METHODS |
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Recombinant human PDGF-B homodimer (Calbiochem, La Jolla, CA) was diluted in PBS containing 0.1% bovine serum albumin to a concentration of 10 µg/ml and stored at -80°C.
The MAPK kinase inhibitor U-0126 and the control compound U-0124 were obtained from Calbiochem, diluted in dimethyl sulfoxide to a concentration of 1 µg/µl, and stored at -80°C. Cells were pretreated with inhibitor or control at a final concentration of 1, 10, or 30 µM for 30 min before PDGF treatment.
Northern blot analysis and RT-PCR. Total RNA was extracted with TRIzol reagent (Life Technologies) in accordance with the manufacturer's instructions. RNA samples (8 µg) were separated on a 1.25% formaldehyde-agarose gel by standard techniques, transferred to a nylon membrane (Schleicher & Schuell, Dassel, Germany) with the Turboblotter (Schleicher & Schuell), and cross-linked to the membrane with ultraviolet light (Stratalinker UV cross-linker, Stratagene, La Jolla, CA). EGR-1 and GAPDH cDNA probes were radiolabeled with [-32P]dCTP (NEN Life Science Products, Boston, MA) with a random-labeling kit (Roche Diagnostics, Mannheim, Germany). Hybridization was carried out at 68°C in an ExpressHyb hybridization solution (Clontech Laboratories, Palo Alto, CA), and the blots were washed in accordance with the manufacturer's instructions. The blots were exposed to autoradiography film (X-Omat-AR film, Kodak, Rochester, NY) at -80°C for
12 h.
Quantitative real-time RT-PCR was performed on RNA from mouse lungs with the use of an iCycler iQ real-time detection system and iQ SYBR green supermix in accordance with the manufacturer's instructions (Bio-Rad, Hercules, CA). Primers for EGR-1 are shown in Table 1. Data were normalized to GAPDH mRNA.
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Construction of promoter-reporter plasmids, cell transfections, and luciferase assays. EGR-1 promoter fragments were amplified from human genomic DNA with PCR primers (Table 1) and the advantage-GC 2 PCR protocol (Clontech) and ligated into the KpnI and HindIII sites of pGL3-basic vector (Promega, Madison, WI). Megaprep kit (Qiagen) was used to purify plasmids, and the correct orientation was confirmed by sequencing.
3T3 cells were transiently transfected using Superfect transfection reagent (Qiagen), and HASMC were transfected using FuGENE 6 (Roche) in accordance with the manufacturer's instructions. After 3 h, the transfection medium was replaced by growth-arrest medium, stimulated with PDGF (100 ng/ml), and harvested 2448 h later for luciferase activity. Luciferase activity was measured with a luminometer (model TD-20/20, Turner) and luciferase-reporter assay reagent in accordance with the manufacturer's instructions (Promega). Transfection efficiency was monitored by cotransfection with pSV--galactosidase (Promega) and determination of total protein content in cell extracts; normalization did not significantly alter results. Transfection efficiencies were
40% for 3T3 cells and 10% for HASMC as determined by visualization of
-galactosidase activity or green fluorescent protein (2).
Preparation of protein extracts, Western blots, immunoprecipitation, and EMSAs. Nuclear and cytosolic proteins were prepared as described by Dignam et al. (5) and stored at -80°C in small aliquots.
Western blots were performed as previously described in detail (24). Each lane contained 10 µg of protein extract. The membranes were probed sequentially with polyclonal antibodies to EGR-1, serum protein 3 (SP3), ERK1/2, phosphorylated ERK1/2 (Thr202/Tyr204), stress-activated protein (SAP) kinase (SAPK/JNK), phosphorylated SAP/JNK (Thr183/Tyr185), p38, and phosphorylated p38 (Thr180/Tyr182; Santa Cruz Biotechnology, Santa Cruz, CA) at dilutions of 1:10,000 and subjected to enhanced chemiluminescence detection (Amersham, Arlington Heights, IL) with 1:10,000 horseradish peroxidase-linked secondary antiserum. Blots were exposed to photographic film for 115 min and stained with Coomassie blue to confirm even loading.
Nuclear extracts from untreated or PDGF-pretreated HASMC were precleared with protein A-agarose and normal rabbit serum and subjected to immunoprecipitation with rabbit polyclonal IgG ELK-1 antibody (Santa Cruz Biotechnology). Phosphorylated ELK-1 (pELK-1) was identified by the use of a mouse monoclonal IgG pELK-1 antibody (Santa Cruz Biotechnology) at a dilution of 1:500 as described above.
In vitro binding reactions between oligonucleotides and nuclear extract were done in a total volume of 20 µl containing 2 µl of 10x binding buffer (0.1 M Tris·HCl, pH 7.5, 50% glycerol, 10 mM EDTA, and 10 mM dithiothreitol), 1 µl of 1 µg/µl poly(dI-dC) poly(dI-dC) (Sigma, St. Louis, MO), 1 µl of 1 µg/µl salmon sperm DNA (Sigma), 24 µl of nuclear extract (normalized to 8 µg/µl of total protein), and 1 µl of radiolabeled oligonucleotide (specific activity 50,000 cpm/µl). The reaction was allowed to proceed for 30 min at 22°C before the addition of 2 µl of nondenaturing loading buffer (0.2% bromphenol blue, 0.2% xylene cyanol, and 20% glycerol). Samples were electrophoresed on 1.5-mm-thick 48% polyacrylamide gels with Tris-boric acid-EDTA running buffer at 200 V for
12.5 h. Gels were dried under vacuum and autoradiographed overnight with Kodak X-Omat-AR film.
Oligonucleotides for EMSAs (Table 1) were synthesized by GeneLink (Hawthorne, NY), annealed, and end-labeled using Klenow enzyme as previously described (24).
Supershift and competition studies were performed as described above, except 1 µl (2 µg) of antibody (Santa Cruz Biotechnology) was added 10 min before the addition of radiolabeled oligonucleotide.
Immunohistochemistry and immunocytochemistry. The protocol for tissue collection was approved by the Brigham and Women's Hospital Institutional Review Board. Immunostaining was performed on paraffin-embedded tissue samples of human lung obtained from patients who died from status asthmaticus (n = 8), patients with asthma who were dying from other reasons (n = 6), and unused lung donor tissue (n = 11). Asthma was defined according to standard American Thoracic Society criteria (1).
Briefly, 5-µm-thick sections were cut, dewaxed in graded xylenes, and rehydrated in graded alcohols. The slides were treated with 0.3% Triton X-100 (Sigma) and blocked with 10% normal goat serum diluted in phosphate-buffered saline (PBS) with 2% bovine serum albumin. Rabbit polyclonal antiserum to EGR-1 (EGR-1 588 and C-19, Santa Cruz Biotechnology) at a dilution of 1:1,500 was used as the primary antibody. Negative controls consisted of absorbing the primary antiserum with a specific EGR-1 peptide (Santa Cruz Biotechnology) and substituting nonspecific rabbit IgG (Sigma) for the primary antiserum. The primary antibody was applied, and the slides were incubated overnight at 4°C. The slides were washed, and biotinylated goat anti-rabbit antibody (Vector Laboratories, Burlingame, CA), diluted 1:200 in PBS, was applied to the slides and incubated for 2 h at 4°C. Endogenous peroxidases were then blocked by incubation in 3% hydrogen peroxide in methanol for 10 min at room temperature, and streptavidin-horseradish peroxidase (Jackson Immunoresearch Laboratories, West Grove, PA), diluted 1:1,000, was applied to the slides for 30 min at room temperature. Immunopositivity was visualized with diaminobenzidine (0.025%) in PBS (Sigma) with 0.1% hydrogen peroxide. The slides were then counterstained with 2% methyl green (Sigma). Staining intensity was semiquantitatively analyzed separately for large airways (defined as containing cartilage) and small airways (noncartilaginous) as follows: no immunostaining (0), minimal staining (0.5), light staining (
1), moderate staining (
2), and intense staining (
3). All asthmatic patients were prospectively grouped for analysis and compared with nonasthmatic controls.
HASMC were seeded (50,000 cells/chamber) on Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL), growth arrested overnight, and treated with PDGF (100 ng/ml) for 1 h before fixation. After two washes with PBS, cells were fixed for 10 min in 1:1 acetone-methanol, washed with PBS again, and left to air dry. The slides were then hydrated in PBS and immunostained for EGR-1 as described above, except the counterstain was 4% methyl green.
Mice and OVA-induced airway inflammation. Male BALB/c mice were housed in isolation cages in a specific pathogen-free, 12:12-h light-dark cycle environment; sterilized rodent chow and water were provided ad libitum. At 10 wk of age, mice were subjected to an OVA-induced airway inflammation protocol as follows: on day 0, all mice were immunized via intraperitoneal injection with 10 µg of chicken OVA (grade III, Sigma) mixed with 1 µg of Al(OH)3 (J. T. Baker Chemical, Phillipsburg, NJ) in 0.2 ml of PBS. An identical booster injection was given on day 7. On days 1420, mice were exposed to aerosolized 4% OVA dissolved in PBS or PBS alone for 25 min/day. Lungs were harvested for RNA and frozen sections on day 21 of the protocol, 24 h after the last OVA exposure. All animal protocols were approved by the Harvard Medical School Standing Committee on Animals.
Statistical analysis. Relative luciferase activities of the constructs and immunohistochemical scoring results were compared by t-test or one-way ANOVA (Tukey's post hoc analysis) using Statistica for Windows 5.0 (Statsoft, Tulsa, OK). Tests were conducted at the 5% significance level, and results are expressed as means ± SE.
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RESULTS |
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A Western blot (Fig. 1B) for EGR-1 protein after addition of PDGF (100 ng/ml) is consistent with the findings from the Northern blot. EGR-1 protein peaks at 1 h after addition of PDGF and then decreases over time, approaching baseline levels by 21 h. Levels of SP3 protein remain unchanged and serve as a control.
Immunocytochemical analysis of HASMC grown in cell culture have no detectable EGR-1 staining in the absence of PDGF (Fig. 2A). The addition of PDGF (100 ng/ml for 1 h) shows a dramatic induction of EGR-1 protein in the cytoplasm and nucleus of these cells (Fig. 2B).
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EGR-1 protein induced in these smooth muscle cells after PDGF induction is capable of binding DNA containing the consensus sequence GCGGGGCG, as demonstrated by EMSA and supershift analysis. Nuclear protein extracts were prepared from HASMC under growth-arrest conditions and after treatment with PDGF (100 ng/ml) for 116 h and incubated with a radiolabeled oligonucleotide containing a consensus EGR-1-binding site. A predominant gel shift appeared transiently at 1 h (Fig. 3A), a finding consistent with the Western blots. The gel shift supershifts with EGR-1 antibody and not with control antibodies to transcription factors SP3 or globin transcription factor 1 (GATA-1; Fig. 3B).
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PDGF activates EGR-1 promoter-reporter constructs in HASMC and 3T3 cells. The proximal 1,351 bp1 of the human EGR-1 promoter contain multiple SRE, ETS consensus sites, and GC boxes implicated in EGR-1 transcriptional control. This core promoter region was placed upstream of a luciferase cDNA to form the promoter-reporter construct 1351pEGRPro-Luc and transfected into untreated and PDGF (100 ng/ml)-treated HASMC. Relative luciferase levels at 48 h were 1,000 ± 436 for untreated cells compared with 5,539 ± 1,714 for PDGF-treated cells (Fig. 4, inset), representing a 5.5-fold increase (n = 12, P < 0.03) in promoter activity.
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Similar results were obtained in 3T3 cells, a mouse embryo fibroblast cell line with a transfection efficiency fourfold higher than that of HASMC. Because of the greater transfection efficiency, these cells were used to map functional promoter elements. The deletion series, consisting of sequentially smaller regions of the EGR-1 promoter, suggest that SRE-3 (Fig. 4), between 663 and 654 bp from the ATG start site, is the most important SRE mediating PDGF-induced EGR-1 promoter activity. Figure 5 shows relative luciferase levels at 24 h for all deletion constructs with and without PDGF (100 ng/ml) treatment. 1351pEGRPro-Luc and 1110pEGRPro-Luc contain SRE-3 and respond to PDGF with a 4.4- and 4.5-fold induction of luciferase activity relative to basal conditions: 4,384 ± 810 vs. 1,000 ± 93 (n = 6, P < 0.001) and 8,379 ± 1,617 vs. 1,859 ± 145 (n = 6, P < 0.001), respectively. In contrast, constructs 645-, 607-, 466-, 364-, and 0pEGRPro-Luc are missing SRE-3 and do not respond significantly to PDGF. Construct 645pEGRPro-Luc has similar baseline activity but is not PDGF responsive; the baseline activity of constructs pEGRPro-Luc466 and pEGRPro-Luc364 are dramatically reduced.
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SRF/ELK/SP1 complexes bind to the EGR-1 promoter. To identify the nuclear proteins interacting with the EGR-1 promoter in the 465-bp region implicated in PDGF-induced transcription, EMSAs were performed with HASMC nuclear extract and oligonucleotides spanning this region. These oligonucleotides contained one or more of the ETS (CCGGAA), SRE (CCTTATTTGG), and SP1 (CCGCCC) consensus elements implicated in EGR-1 regulation by the deletion series. Antibodies to SRF and the ETS factors ELK-1, pELK-1, SAP-1, and SP1 were used for supershift analysis. An antibody to GATA-1 was used as a negative control.
Many nuclear protein complexes interact with this promoter region. Some of the DNA-binding proteins were identifiable by supershift analysis (Fig. 5). The highest mobility shift identified was SP1, which bound to oligonucleotides containing the GC box (GGCCCGGCCGC) located downstream of SRE-2 between bp 624 and 613. The next band of slightly lower mobility was SRF, which bound to SRE-3 and SRE-2. Very faint bands of lowest mobility were produced by ELK-1 and pELK-1 and were detected only with oligonucleotides containing SRE-3 and SRE-2. Antibodies to SAP-1 and GATA-1 failed to supershift any band. These data, interpreted in the context of the deletion series, suggest that SP1/GC boxes may contribute to constitutive EGR-1 promoter activity and that SRE-3 may be necessary for PDGF-induced EGR-1 transcription.
PDGF induction of EGR-1 in HASMC is associated with ELK-1 phosphorylation and requires the MAPK pathway. Depending on cell type and stimulus, transcriptional activation of EGR-1 may involve phosphorylation of ternary complex factors such as ELK-1. Supershift analysis, such as shown in Fig. 5, detected subtle band shifts with antibodies directed to ELK-1 or pELK-1 but was inadequate to assess changes in ELK-1 phosphorylation in HASMC after PDGF treatment. To test the hypothesis that EGR-1 induction in HASMC after treatment with PDGF is associated with ELK-1 phosphorylation, we performed immunoprecipitation using antibodies specific for pELK-1. A Western blot of immunoprecipitated pELK-1 after PDGF (100 ng/ml) treatment shows that ELK-1 phosphorylation follows a time course similar to EGR-1 induction, with increased levels 0.5 h after PDGF treatment (Fig. 6A). By 21 h after treatment, levels have returned to near-baseline values.
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ELK-1 is known to be phosphorylated through pathways involving ERK1/2 phosphorylation and activation. To test the hypothesis that treatment of HASMC with PDGF results in phosphorylation of ERK1/2 at time points coinciding with ELK-1 phosphorylation, we performed Western blot analysis to determine the phosphorylation status of ERK1/2. Cytoplasmic extracts were harvested from HASMC under basal conditions and after treatment with PDGF (100 ng/ml). Phosphorylated ERK1/2 increased 0.5 h after PDGF treatment relative to undetectable basal levels (Fig. 6B). Levels remained elevated for up to 21 h. In contrast, total ERK1/2 levels remained relatively constant. No changes in JNK or p38 phosphorylation were detected (data not shown).
EGR-1 induction by PDGF was dependent on the MEK1/2 components of the MAPK signal transduction pathway. The addition of U-0126, a specific and potent inhibitor of MEK1/2, abolished induction of EGR-1 mRNA (Fig. 6C) and protein (Fig. 6D) after PDGF treatment (1 h). Dimethyl sulfoxide and the negative control U-0124 did not affect EGR-1 induction or inhibition.
EGR-1 is expressed in airway smooth muscle of asthmatic patients and nonasthmatic controls and mice with and without OVA-induced airway inflammation. EGR-1 immunohistochemistry was performed on human lung tissue from 8 patients (3 women) who died from status asthmaticus, 6 patients (3 women) with diagnosed asthma who died for reasons other than their asthma, and 11 transplant donor patients (5 women) without asthma. Mean ages of patients at the time lung tissue was obtained were 35.8 ± 5.4, 35.0 ± 4.2, and 36.7 ± 2.6 yr, respectively (P = 0.9). IgE levels were 315 ± 113, 214 ± 166, and 56 ± 20 U/ml, respectively (P = 0.3).
In the tissue obtained from asthmatic patients, 12 of 14 specimens showed EGR-1 staining, whereas 8 of 11 of the control tissues showed EGR-1 staining. Staining was most evident in cytoplasm and nuclei of airway smooth muscle cells and is consistent with the immunocytochemistry studies discussed above (Fig. 2, A and B). Some staining was evident in airway epithelial cells, but this was less consistent and intense. Representative images of bronchioles demonstrate the presence of EGR-1 staining in smooth muscle airway and epithelial cells of asthmatic patients (Fig. 7, A and B). The intensity of staining varied significantly between subjects; however, there were no significant differences in airway smooth muscle (Table 2) or epithelial cells (data not shown) between nonasthmatic subjects and asthmatic patients in proximal or distal airways. Retrospective comparisons of fatal and nonfatal asthma showed no significant difference. Controls demonstrate the absence of staining in specimens from asthmatic patients with serum IgG as the primary antibody (Fig. 7C) and after peptide absorbance (Fig. 7D).
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EGR-1 immunohistochemistry was performed on mouse lung tissue from eight mice challenged with OVA and eight mice challenged with PBS. OVA-challenged mice had a moderate-to-severe eosinophilic inflammatory process characterized by focal cellular infiltrates around airways and vessels (Fig. 7F). PBS-challenged mice had no inflammation and normal-appearing lungs (Fig. 7E). EGR-1 staining was evident in airway epithelium, airway smooth muscle, and vascular smooth muscle without any difference in intensity between OVA- and PBS-challenged mice (Fig. 7, E and F; data not shown). Quantitative RT-PCR on total RNA from whole mouse lungs confirmed that there was no difference between OVA- and PBS-challenged mice in EGR-1 mRNA (Fig. 8, Table 2).
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DISCUSSION |
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In this study, transient transfection analysis with EGR-1 promoter-reporter constructs suggests that at least part of the induction occurs at the level of transcription. Deletion analysis of the EGR-1 promoter implicates SRE-3 as the most important for PDGF-induced EGR-1 transcription (Fig. 4). SRE, ELK-1, and SP1 are present in nuclear protein extracts from HASMC and bind to SRE-3 on the EGR-1 promoter. Moreover, in HASMC, ELK-1 is phosphorylated by PDGF at a time when EGR-1 is maximally induced by PDGF (Figs. 5 and 6A). Finally, we show that ERK1/2 is phosphorylated by PDGF and that its activity is essential for EGR-1 induction (Fig. 6, B and C). Our findings for HASMC are consistent with the observations of Clarkson et al. (4), who studied the mechanisms by which growth hormone upregulates EGR-1 in preadipocytes. However, we were unable to detect the phosphorylation of p38 or JNK, which has been reported for other cell types (data not shown) (19). PDGF has been shown to phosphorylate ERK1/2 in bovine (16), canine (3), and human airway smooth muscle (10). In addition to the SRE, ETS, and SP1 consensus-binding sites implicated by our study, several other promoter regulatory elements may be involved in the modulation of EGR-1 transcription in airway smooth muscle.
Although we have used PDGF as a model agonist, EGR-1 mRNA and protein may be induced in vitro by numerous stimuli found in the airways of asthmatic patients. For example, angiotensin II treatment induces EGR-1 in airway smooth muscle cells grown in cell culture, and this may be responsible for the increased cell growth that occurs after treatment. McKay et al. (12) proposed that the observed smooth muscle cell hypertrophy is caused by the induction of transforming growth factor-1 (TGF-
1) by EGR-1 and other immediate-early transcription factors. Other stimuli of relevance to airway inflammation and remodeling that may induce EGR-1 include mechanical forces, hypoxia, reactive oxygen species, endothelin, granulocyte/macrophage colony-stimulating factor, TNF-
, TGF-
1, epidermal growth factor, and fibroblast growth factor (11, 22).
On the basis of these in vitro observations, we hypothesized that EGR-1 levels are increased in asthmatic patients compared with nonasthmatic controls. However appealing this hypothesis is, we have been unable to detect an increase in EGR-1 in the airways of asthmatic patients compared with nonasthmatic controls (Fig. 7, Table 2). We found EGR-1 protein in the nucleus and cytoplasm of airway and vascular smooth muscle cells and, to a lesser extent, in bronchial epithelial cells in asthmatic patients and nonasthmatic controls. In a study of such a heterogeneous disease as asthma, it is not surprising to find large interindividual variance in several parameters. One could speculate that different degrees of airway remodeling or airway inflammation could affect the expression of EGR-1 and that certain asthma (or other) medications could influence the EGR-1 expression. Unfortunately, it was not possible to investigate such relations with the limited numbers of available specimens. It is also conceivable that factors such as certain medications, tissue hypoxia, and mechanical forces during handling of the tissue postmortem could lead to a rapid induction of EGR-1 also in the nonasthmatic tissue.
Studies involving animal models also suggest that EGR-1 may be expressed in the lung and that it plays a role in allergen-induced airway disease (23). We previously showed that EGR-1 can modulate airway hyperresponsiveness in the context of allergen-induced airway inflammation (23). egr-1 knockout mice had decreased airway responsiveness to methacholine in an OVA-induced model of airway inflammation. Although there were no obvious differences in airway inflammation, TNF- levels were decreased in the knockout mice. Decreased TNF-
may partially account for the observed airway hyporesponsiveness but is unlikely to be the only factor contributing to this phenomenon. These data suggest that EGR-1 regulates the transcription of several genes involved in the various pathways controlling airway contractility and remodeling. Genes known to be regulated by EGR-1 that may contribute to asthma development or severity include PDGF-A, PDGF-B, TGF-
1, fibroblast growth factor-2, macrophage colony-stimulating factor, interleukin-2, intracellular adhesion molecule-1, copper-zinc superoxide dismutase gene, CD44,
1-adrenergic receptor, fibronectin, and 5-lipoxygenase (7, 11, 22). Each of these human genes contains at least one EGR-1 consensus-binding site within its promoter region, and the modulated expression of these genes may be attributable to a direct effect of EGR-1 on the promoters. Nevertheless, we did not find induction of EGR-1 protein or mRNA in the lungs of mice with asthmalike inflammation, findings consistent with the immunohistochemical assessment of EGR-1 protein in the lungs of asthmatic patients.
In conclusion, we have shown that the transcription factor EGR-1 may be induced by PDGF in HASMC grown in cell culture. The transcriptional pathway mediating this expression requires ERK1/2 activation and is associated with phosphorylation of ELK-1 and the binding of SRF on SRE-3. We could find no evidence to support the hypothesis that EGR-1 is increased in the lungs of asthmatic patients. It remains to be established whether the EGR-1 pathway is a critical component of the airway remodeling process in asthma.
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ACKNOWLEDGMENTS |
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GRANTS
This study was funded by the Swedish Heart-Lung Foundation, the Swedish Asthma and Allergy Association's Research Foundation, and National Heart, Lung, and Blood Institute Grants HL-70573 and HL-03827.
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FOOTNOTES |
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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.
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REFERENCES |
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