© 2002 American Thoracic Society DOI: 10.1165/rcmb.2002-0011OC Constitutive and Cytokine-Induced Expression of the ETS Transcription Factor ESE-3 in the LungDivision of Pulmonary and Critical Care Medicine and Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Boston; New England Baptist Bone and Joint Institute, Beth Israel Deaconess Medical Center, Boston; Harvard Medical School, Boston; Department of Environmental Health, Harvard School of Public Health, Boston, Massachusetts; Department of Epidemiology and Biostatistics, Case Western Reserve University, Cleveland, Ohio; and Parc Cientific Barcelona, Barcelona, Spain Address correspondence to: Eric S. Silverman, M.D., Department of Environmental Health, Harvard School of Public Health, 667 Huntington Ave., Boston, MA 02115. E-mail: esilverm{at}hsph.harvard.edu
Family studies of asthma suggest that the genes ESE-2 and ESE-3 contain polymorphisms that contribute to disease susceptibility. Each gene codes for an ETS transcription factor that is characterized by epithelium-restricted constitutive expression and may function as a context-dependent activator or repressor of transcription; however, nothing is known about the role of these genes in lung homeostasis or the pathogenesis of airway disease. In this study, we show that ESE-3 mRNA and protein are constitutively expressed in bronchial and mucous gland epithelial cells. Consistent with these findings, ESE-3 mRNA is constitutively expressed in human bronchial epithelial cells grown in tissue culture. In contrast, ESE-2 mRNA could not be detected in the lung or cultured human bronchial epithelial cells. Human bronchial smooth muscle cells and fibroblasts do not constitutively express ESE-3; however, after stimulation with interleukin-1ß or tumor necrosis factor-  , levels of ESE-3 mRNA and protein increase dramatically by 24 h. This cytokine induction is dose-dependent and abrogated by specific inhibitors of the MEK1/2 (U0126) and p38 (SB03580) signal transduction pathways. Overexpression of ESE-3 protein in 3T3 cells and human bronchial smooth muscle cells inhibits MMP-1 promoter activity, suggesting that ESE-3 may function as a transcriptional repressor.
Abbreviations: Ets binding site, EBS • epithelium-specific expression, ESE • interleukin, IL • human bronchial epithelial cells, HBEC • human bronchial fibroblasts, HBFB • human bronchial smooth muscle cells, HBSMC • mitogen-activated protein kinase, MAPK • matrix metalloproteinase, MMP • phosphate-buffered saline, PBS • tumor necrosis factor, TNF
The genes ESE-21 and ESE-32 code for proteins that belong to an ETS transcription factor subfamily characterized by epithelium-specific expression (ESE) (1–3). Both proteins contain a conserved DNA-binding motif, called the ETS domain, and a conserved Pointed domain that is involved in protein–protein interactions (2). The ETS domain has high affinity for the consensus cis element -GGA(A/T)-, which is found in promoters and other regulatory sequences of many genes (4, 5). Although ESE-2 and ESE-3 are thought to be involved in the context-dependent regulation of specific genes in epithelial cells, their functions are largely unknown. It has been hypothesized that they are involved in tubulogenesis and branching morphogenesis in glandular organs such as the lung, induction or repression of epithelium-specific genes in the context of an inflammatory microenvironment, and oncogenesis of epithelial-derived tumors such as bronchogenic carcinoma (1–3). ESE-2 and ESE-3 are of particular interest to pulmonologists because they have been identified in family studies as "asthma candidate genes." Both genes are adjacently located on chromosome 11p12–15, a genomic region linked to asthma and atopy in some, but not all genome scans, and have been identified as strong positional candidates in a fine-mapping study involving the Tristan da Cunha population (6–8). Follow-up studies involving a number of different populations support an association between polymorphisms near these genes and the diagnosis of asthma (7, 9); however, no DNA sequence variants in ESE-2 or ESE-3 have been associated with the diagnosis of asthma, and the involvement of these genes in the pathogenesis of airway disease remains unknown.
The purpose of this study was to examine the constitutive and cytokine-inducible expression of ESE-2 and ESE-3 in the airways. We show that ESE-3, but not ESE-2, is expressed constitutively in human bronchial epithelial cells (HBEC) and mucous gland epithelial cells. Other cells of the airways do not express ESE-3 under nonstimulated conditions; however, ESE-3 expression is induced in human bronchial smooth muscle cells (HBSMC) and fibroblasts (HBFB) after treatment with interleukin (IL)-1ß and tumor necrosis factor (TNF)-
Tissue Culture, Recombinant IL-1ß, and TNF- MAPK InhibitorsHBEC, HBSMC, and HBFB were obtained from Clonetics/BioWhittaker (Walkersville, MD) and grown in supplied SmGM medium (MCDB-131, supplemented with human epidermal growth factor, human basic fibroblast growth factor, dexamethasone and 5% fetal bovine serum) as instructed. HBEC for transfection studies were a generous gift of R. Panettieri (University of Pennsylvania, Philadelphia, PA). 3T3-Swiss albino (mouse embryo fibroblasts) were obtained from the American Type Culture Collection (Rockville, MD) and grown in DMEM medium (Life Technologies, Gaithersburg, MD) containing streptomycin 50 µg/ml, penicillin 50 IU/ml, and 10% fetal calf serum. All cells were grown in 60-mm petri dishes at 37°C, 5% CO2, and passaged at confluence every 4–5 d.
Recombinant human IL-1ß and TNF- U0126 and SB203580 were obtained from Calbiochem (LaJolla, CA), diluted in dimethylsulfoxide, and stored at -80°C.
RNA Extraction, Reverse Transcription–Polymerase Chain Reaction, and Northern Blot Analysis Reverse transcription (RT)-polymerase chain reaction (PCR) was performed with 2 µg total RNA, 1 µg random hexamers (Life Technologies), 50 nmoles dNTP, 25 U rRNasin RNase inhibitor (Promega, Madison, WI), 200 U M-MLV RT (Promega) in a 25 µl total volume consisting of 50 mM Tris-HCL (pH 8.3), 75 mM KCl, 3 mM MgCl2, and 10 mM DTT at 37°C for 60 min. RT reactions were diluted 1:5 in water and stored at –20°C. PCR reactions were performed with 4 µl of RT diluted reactions, 40 nmoles dNTP, 10 pmoles primers, 2 U Taq polymerase (Promega) in a 50 µl total volume consisting of 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, and 1.5 mM MgCl2. ESE-2 primers were (2a sense) 5'-GCCTCTGATTTGTGTGACACTGA, (2b sense) 5'-TGGACCTAGCCACCGCTGCC, and (antisense) 5'-ATTGAAAGTACAGGTACTCGCCGC. ESE-3 primers were (sense) 5'-CCTGGACACCAACCAGCTGGATGC and (antisense) 5'-CCTGAAGACGCCCTCAGATCGGTC. MMP-1 primers were (sense) 5'-GATGGGAGGCAAGTTGAAAA and (antisense) 5'-ACCGGACTTCATCTCTGTCG. ETS-1 primers were (sense) 5'-AGCCGACTCTCACCATCATC and (antisense) 5'-GGATGGAGCGTCTGATAGGA. Annealing temperature was 58 to 60°C with 25–30 PCR cycles. PCR products were separated on a 1.5–2% agarose gel and photographed. Northern blot analysis was performed by standard technique (10). MultiTissue blots were obtained from Clontech (Palo Alto, CA) and processed according to the manufacturer's instructions. ESE-2 and ESE-3 cDNA probes, which have been described in detail (1, 2), were labeled by random oligonucleotide extension (10).
Lung Tissue and Immunohistochemistry
Protein Radiolabeling and Immunoprecipitation
Plasmids, Transient Transfection Analysis, and Luciferase Assays Cells were transiently transfected with SuperFect Transfection Reagent (Qiagen) or Fugene 6 (Roche, Indianapolis, IN) according to the manufacturer's instructions, and incubated for 24 to 48 h at 37°C in growth medium. Luciferase assays were performed with Reporter Lysis Buffer and Assay Reagent (Promega) according to the manufacturer's instructions. Luciferase data were normalized such that unstimulated promoter activity was arbitrarily set at 100. Transfection efficiency was monitored by cotransfection with pSV-ß-galactosidase (Promega). Transfection efficiencies were 40% for 3T3 cells and 10% for HBSMCs as determined by visualization of ß-galactosidase activity or green fluorescent protein (10).
Statistical Analysis
ESE-3 mRNA Is Constitutively Expressed in the Lung Tissue and leukocyte expression profiles of ESE-2 and ESE-3 mRNA were determined by Northern blot analysis (Figure 1). The blot was sequentially probed with radiolabeled ESE-2 and ESE-3 cDNAs. ESE-2 mRNA could not be detected in normal lung, but could be found in the kidney. In contrast, the 5.9-kb transcript of ESE-3 mRNA was detected in normal lung tissue. ESE-3 mRNA signal also was detected in colon, with weaker levels found in the kidney and small intestine. Multiple mRNA transcripts, presumably arising from alternative utilization of polyadenylation signals, were detected at 5.6, 4.6, and 1.3 kb (not shown) in some of the more intensely labeled tissues, as has been previously described (3). Neither ESE-2 nor ESE-3 mRNA was detected in peripheral leukocytes.
ESE-3 Protein Is Constitutively Expressed in Mucous Gland and Bronchial Epithelial Cells Cells in the lung expressing ESE-3 protein were identified by subjecting human lungs obtained from transplant donors to immunohistochemical analysis with monoclonal antibody 5A5.5 directed against ESE-3 (Figures 2A–2C). ESE-3 protein was found to be restricted to the nuclei of mucous gland (Figure 2A) and bronchial epithelial cells (Figures 2B and 2C). Staining was most intense in the mucous gland epithelial cells, with the majority of cells showing nuclear immunoreactivity. In contrast, only a minority of bronchial epithelial cells had ESE-3 immunoreactivity in the nucleus. Immunoreactivity was not detected in airway smooth muscle cells, fibroblasts, alveolar epithelial cells, or leukocytes (data not shown).
ESE-3 mRNA Is Constitutively Expressed in Human Bronchial Epithelial Cells in Tissue Culture RT-PCR and Northern blot analysis of HBECs grown in tissue culture confirmed the tissue-specific expression of ESE-3. ESE-3 was easily detected by RT-PCR (Figure 3A) and Northern blot analysis (Figure 3B). Stimulation of cells with the inflammatory cytokines TNF- (5 ng/ml) and IL-1ß (5 ng/ml) for up to 24 h did not change the steady-state levels of ESE-3 (Figures 3A and 3B). In contrast, the two alternatively spliced forms of ESE-2, namely ESE-2a and ESE-2b, could not be detected in HBEC even after stimulation with TNF- (5 ng/ml) and IL-1ß (5 ng/ml) for up to 24 h (Figure 3A). These findings are consistent with the multiple-tissue Northern blot.
ESE-3 mRNA and Protein Are Inducibly Expressed in Human Bronchial Smooth Muscle Cells after Treatment with TNF- or IL-1ßTo test the hypothesis that ESE-3 may be induced in other cells of the airways in response to inflammatory cytokines of relevance to asthma, we exposed HBSMC to TNF- and IL-1ß. ESE-3 mRNA was induced 6 to 24 h after treatment with both cytokines, each at a concentration of 5 ng/ml, and detected by RT-PCR (Figure 4A). Without stimulation (time 0 h), message was undetectable in both types of cells. ESE-3 mRNA peaked at  24 to 48 h, and the combination of TNF- and IL-1ß accentuated the induction, resulting in an earlier and more intense signal. The presence or absence of 5% FBS in medium had no effect on induction. Northern blot analysis quantitatively confirmed these findings and suggests that the combination of both cytokines has a synergistic effect on ESE-3 induction (Figure 4B). Consistent with these findings, ESE-3 protein could be detected by immunopreciptitation of radiolabeled nuclear extracts with anti–ESE-3 antibodies after 48 h of treatment with TNF- and IL-1ß (Figure 5). Similar induction of ESE-3 mRNA was detected when HBFBs were treated with TNF- and IL-1ß (data not shown). ESE-2 mRNA could not be detected in HBSMC, even after stimulation with TNF- (5 ng/ml) and IL-1ß (5 ng/ml) for up to 24 h (data not shown).
ESE-3 mRNA Induction in Bronchial Smooth Muscle Cells Is Dose-Dependent and MAPK-Dependent The threshold of ESE-3 induction by TNF- and IL-1ß was examined by exposing HBSMCs to increasing amounts of each cytokine for 48 h and measuring mRNA by RT-PCR. ESE-3 mRNA was low, but detectable, with as little as 0.05 ng/ml of TNF- or IL-1ß and increased in a dose–response fashion up to 5.0 ng/ml (Figure 6A). This induction was in part dependent on the MEK1/2 and p38 components of the MAPK signal transduction pathway. Addition of either U0126, a specific and potent inhibitor of MEK1/2, or SB203580, a specific and potent inhibitor of p38, nearly abolished induction of ESE-3 mRNA (Figure 6B). Addition of both reagents inhibited induction at lower doses.
Overexpression of ESE-3 Represses MMP-1 Promoter Activity in Transiently Transfected 3T3 Cells and HBSMCs ESE-3 may function as a transcriptional activator or repressor in epithelial cells, depending on the promoter with which it interacts. In this regard its function in HBSMC and HBFB after cytokine induction is also likely to be context-dependent. To study the role of ESE-3 in nonepithelial cells, we transiently transfected 3T3 cells and HBSMCs with an ESE-3 expression construct to determine its effect on MMP-1 promoter activity. MMP-1 promoter was chosen as the target because it is induced by inflammatory cytokines in tissue culture, and levels are increased in the airway smooth muscle of individuals with asthma (12). Moreover, the MMP-1 promoter contains multiple functional ETS consensus-binding sites that interact with ETS-1 and other ETS factors (13, 14).
The time course of MMP-1 upregulation in HBSMC after cytokine stimulation is shown in Figure 7 by RT-PCR (Figure 7A) and Northern blot analysis (Figure 7B). MMP-1 mRNA is induced early after cytokine stimulation with maximal levels reached at
To determine which hypothesis is correct, we generated an MMP-1 promoter–reporter construct (MMP-1pGL3 basic), cotransfected it into 3T3 cells and HBSMCs with an ESE-3 expression construct, and measured luciferase activity 24 h later. Basal levels were arbitrarily normalized to a value of assigned 100 (Figure 8A). As anticipated, cotransfection with 1 µg pCI-ETS1 increased MMP-1 promoter activity 3-fold (n = 5, P < 0.001) (Figure 8A). In contrast, transfection with 1 µg of pCI-ESE3 decreased basal levels by 70% in a dose-dependent fashion (P = 0.001, R2=0.552) (Figures 8A and 8B). These data suggest that ESE-3 may function as a transcriptional repressor of MMP-1. As a control, cotransfection studies were performed using the MatA12-Luc promoter–reporter construct (matrilysin promoter) (3). ESE-3 significantly increased luciferase activity (n = 3 each, P < 0.001), suggesting that it may also function as an activator of transcription in 3T3 cells depending upon the promoter (Figure 8C).
ETS transcription factors are characterized by a conserved winged helix-turn-helix DNA binding domain (Ets domain) that confers the ability of these proteins to bind specific cis-regulatory elements containing a core -GGA(A/T)- DNA sequence (Ets binding site [EBS]). More than 30 ETS genes have been identified by sequence homology, making the ETS family one of the largest and most diverse groups of transcription factors (for reviews see Refs. 4, 15). ETS factors may function as transcriptional activators or repressors of target genes, depending on the precise sequence geometry of the EBS in relation to other cis-regulatory elements and their ability to form complexes with other transcriptional regulators (16). Most ETS transcription factors are ubiquitously expressed in the majority of tissues, and more than 200 target genes have already been discovered (5). By regulating the expression of a diverse group of target genes in a temporal- and cell-specific fashion, ETS transcription factors are thought to play a variety of roles in normal and abnormal cellular events, including cell proliferation, differentiation, development, inflammation, hematopoiesis, apoptosis, metastasis, tissue remodeling, angiogenesis, and transformation. The ESE subfamily of ETS transcription factors consists of three known members, ESE-1/ELF3/ESX, ESE-2/ELF-5/ASTH1-I, and ESE-3/EHF/ASTH1-J, and are unique among ETS proteins in that their expression is thought to be limited to epithelial cells (1–3, 17, 18). Although these transcription factors are expressed exclusively in epithelial cells, they are not expressed in all epithelial cells. In adults, the highest levels of ESE-1 mRNA are found in the intestine, with much lower levels expressed in the lung (17). The highest levels of ESE-2 mRNA are found in the salivary gland, mammary glands, and trachea, with much lower levels expressed in the lung (1). ESE-3 mRNA is strongly expressed in a wider variety of tissues than ESE-2, with the highest levels of ESE-3 mRNA in the trachea, lung, colon, pancreas, and prostate (2, 3). It has been hypothesized that ESE members, through their ability to regulate epithelial growth and differentiation, play a role in tubulogenesis and branching morphogenesis in organs containing glandular epithelium, such as the lung and trachea, and in oncogenesis of epithelium-derived tumors, such as bronchogenic carcinoma (2, 3). ESE-2 and ESE-3 are of particular interest to geneticists because they are highly polymorphic and thought to be asthma candidate genes. ESE-2 and ESE-3 are located on chromosome 11p12–15, a genomic region linked to asthma susceptibility in several whole-genome scans (19). Moreover, ESE-2 and ESE-3 were positionally cloned in a fine-mapping study performed on the Tristan da Cunha population (7, 8). Tristan da Cunha is a small volcanic island in the South Atlantic Ocean whose population is suited for such a study because it has a stable, homogeneous population with a 30% prevalence of asthma (6). Subsequent studies involving a number of different populations support an association between polymorphisms in ESE-2 and ESE-3 and the diagnosis of asthma; however, no variant forms of these genes have been associated with the diagnosis of asthma, and even the role of these proteins in airway biology and the pathogenesis of asthma has not been established (7, 9). To build on these data and further explore the role of ESE-2 and ESE-3 in the context of airway biology and asthma, we examined the expression profile of these genes in cells of the lung. The first unexpected finding was the inability to detect ESE-2 mRNA in whole lung or airway epithelial cells by Northern blot analysis and RT-PCR (Figures 1 and 3A) for both ESE-2a and ESE-2b mRNA, the two alternative-splice variants of ESE-2 (1). In the original description of ESE-2, Oettgen and coworkers detected low levels of ESE-2 mRNA in the lung on a dot-blot analysis of 64 different tissues (1). It is possible that ESE-2 was induced in response to temporal, cytokine, mechanical, or inflammatory stimulation in the tissue studied by Oettgen and coworkers. Another possibility is that the lung tissue studied by Oettgen and coworkers may have contained trachea.
ESE-3 mRNA and protein were easily detected in the lung and located in the nuclei of bronchial and mucous gland epithelial cells (Figures 1–3). ESE-3 was not detected in other cells of the normal lung or in unstimulated peripheral leukocytes, results consistent with the epithelial cell–restricted expression of ESE-3 as noted by others (2, 3). However, we hypothesized that ESE-3 may be induced in other airway cells in response to inflammatory cytokines or growth factors associated with asthma. This hypothesis was correct. We found that IL-1ß and TNF- ESE-3 expressed in bronchial smooth muscle cells and fibroblasts may activate or repress a completely different set of target genes than those regulated in bronchial epithelial cells. To test this hypothesis, we chose induction of the MMP-1 promoter as a model of ESE-3–regulated expression for four reasons: (i) MMP-1 is expressed in bronchial smooth muscle cells and is transcriptionally induced in response to inflammatory cytokines (Figure 7) (12); (ii) MMP-1 may enhance airway smooth muscle hyperplasia by degrading insulin-like growth factor (IGF)-binding proteins and increasing IGF levels (12); (iii) MMP-1 has multiple ETS consensus binding sites that are required for ETS-1–, ETS-2–, or ERG-mediated induction (14); and (iv) the promoter has been shown to be repressed by ESE-3 overexpression in HepG2 hepatocarcinoma and HCT-15 colon adenocarcinoma cells (3, 18).
The time course of ESE-3 induction in relation to ETS-1 and MMP-1 induction is consistent with the role of ESE-3 as a repressor of MMP-1 activation. In our study, MMP-1 levels peaked In summary, our study demonstrates the expression of the asthma candidate gene ESE-3 in cells of the airway. Constitutive expression was found in bronchial and mucous gland epithelial cells and, contrary to expectation, cytokine-inducible expression was found in bronchial smooth muscle cells and fibroblasts. Data consistent with a role for ESE-3 as a transcriptional repressor downstream of MAPK signaling cascades are presented. We further speculate that variant forms of ESE-3 increase susceptibility to the development of asthma by altering the role ESE-3 as a transcriptional regulator in multiple tissues of the airway.
This project was supported by NIH RO1 HL70573 (E.S.S.), NIH RO1 CA076323 (T.A.L.), and UO1 HL65899 (S.T.W.).
1 GenBank accession numbers AF115402, AF115403, AF170583. 
2 GenBank accession numbers AF124438, AF124439, AF212848. Received in original form January 29, 2002 Received in final form July 8, 2002
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Abstract
Introduction
B did not attenuate ESE-3 induction (data not shown). It is likely that other cytokines implicated in the pathogenesis of asthma induce ESE-3 in bronchial smooth muscle cells and fibroblasts. 
