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Endocrinology Vol. 147, No. 11 5187-5195
Copyright © 2006 by The Endocrine Society

Estrogen Related Receptor-{alpha} Enhances Surfactant Protein-A Gene Expression in Fetal Lung Type II Cells

Dongyuan Liu, Margaret M. Hinshelwood, Vincent Giguère and Carole R. Mendelson

Departments of Biochemistry (D.L., C.R.M.) and Obstetrics and Gynecology (M.M.H., C.R.M.), The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9038; and Molecular Oncology Group (V.G.), McGill University Health Center, Montreal, Quebec, Canada H3A 1A1

Address all correspondence to: Carole R. Mendelson, Ph.D., Department of Biochemistry, The University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9038. E-mail: carole.mendelson{at}utsouthwestern.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Surfactant protein-A (SP-A) gene expression is developmentally regulated in fetal lung type II cells in concert with surfactant glycerophospholipid synthesis. In studies using transfected type II cells, we characterized a nuclear receptor element (NRESP-A, 5'-TGACCTTA-3') at –242 bp in the 5'-flanking sequence of human SP-A2 (hSP-A) gene that is essential for basal and cAMP-induced expression. NRESP-A has high sequence similarity to the consensus binding site for estrogen-related receptor (ERR). In the present study, we observed that ERR{alpha} and ERR{gamma}, but not ERRß, were expressed in human fetal lung type II cells. In vitro transcribed/translated ERR{alpha} and ERR{gamma} bound to the NRESP-A; DNase I footprinting using bacterially expressed ERR{alpha} revealed a single DNase I protected region that included NRESP-A. In transient transfection assays of COS-7 and primary cultures of lung type II cells, ERR{alpha} acting through NRESP-A increased hSP-A promoter activity, whereas ERR{gamma} had no effect. ERR{alpha} overexpression in lung type II cells enhanced cAMP induction of endogenous hSP-A expression, whereas cotransfection of protein kinase A catalytic subunit enhanced ERR{alpha} stimulation of hSP-A promoter activity in lung adenocarcinoma cells. Mice homozygous null for the ERR{alpha} gene manifested decreased SP-A expression relative to wild-type and heterozygous littermates. The ERR{alpha}-specific inverse agonist XCT790 inhibited cAMP induced hSP-A expression in human fetal lung type II cells in a concentration-dependent manner, suggesting a role of peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha}. These findings suggest that ERR{alpha} acting through NRESP-A is an important mediator of hSP-A gene expression and its induction by cAMP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PULMONARY SURFACTANT, a phospholipid-rich, developmentally regulated lipoprotein secreted by type II cells of the lung alveolus acts to reduce alveolar surface tension, thereby preventing alveolar collapse on exhalation of air. Four lung-specific surfactant-associated proteins (SPs) have been characterized, SP-A, SP-B, SP-C, and SP-D, which are expressed in a lung-specific and developmentally timed manner (1, 2). Synthesis of the major surfactant protein, SP-A, a C-type lectin that plays an important role in immune defense within the lung alveolus (3), is initiated after approximately 70% of gestation is completed and reaches maximal levels just before birth (4). Because SP-A is expressed in concert with surfactant glycerophospholipid synthesis (5), it serves as an excellent marker of fetal lung maturity.

The SP-A gene is expressed primarily in lung type II cells and to a lesser extent in bronchioalveolar epithelial (Clara) cells (6, 7). SP-A expression is subject to multifactorial regulation; agents that increase cAMP (5, 8) and cytokines, such as IL-1 (9, 10, 11), appear to play the most important roles in its regulation. In studies using transgenic mice and transfected type II cells, we identified an approximately 300-bp region just upstream of the SP-A gene that mediates appropriate developmental, lung type II cell-specific, and cAMP-regulated expression (12, 13). Within this region there are a number of highly conserved response elements, including a thyroid transcription factor (TTF-1/Nkx2.1)-binding element, which binds TTF-1 (14) and nuclear factor-{kappa}B (NF-{kappa}B) (15), an E-box element, which binds upstream stimulatory factors-1 and -2 (16, 17), a GT-box, which binds Sp1 and other related transcription factors (18) and a putative nuclear receptor element half-site (NRESP-A) (19, 20). Mutation of any one of these elements markedly reduces basal and blocks cAMP induction of SP-A promoter activity. Thus, these conserved response elements act in a cooperative manner to mediate lung-specific, developmental, and hormonal stimulation of SP-A gene expression.

In humans, SP-A is encoded by two highly similar genes, hSP-A1 and hSP-A2 (21, 22). Although both genes contain these critical response elements within their 5'-flanking regions (23), the hSP-A2 is more highly regulated during fetal development and by cAMP than is hSP-A1 (24). Thus, we focused our studies on human (h)SP-A2 (hereafter referred to as hSP-A). NRESP-A (5'-TGACCT(C/T)A-3'), which is located approximately 240 bp upstream of the hSP-A gene, differs by one nucleotide from the palindromic consensus cAMP response element (CRE, 5'-TGACGTCA-3'), which is known to bind CRE binding protein (CREB) as a homodimer. However, we demonstrated that CREB and related family members, CREM and ATF-1, cannot bind to this element (20). Based on EMSAs using mutated NRESP-A oligonucleotides as nonradiolabeled competitors, we found that the critical protein-binding nucleotides in NRESP-A constituted the hexameric element, 5'-TGACCTCA-3', which corresponded to a half-site for binding of members of the nuclear receptor superfamily (20). In fact, NRESP-A was found to be highly similar to the consensus binding sequence for the estrogen-related receptors (ERRs/NR3B) [ERR element (ERRE), 5'-TNAAGGTCA-3', reverse 5'-TGACCTTNA-3'].

The ERRs are orphan members of the nuclear receptor superfamily comprised of three closely related members: ERR{alpha}/NR3B1, ERRß/NR3B2, and ERR{gamma}/NR3B3. Due to the high degree of sequence conservation in their DNA binding domains, the three ERR isoforms are capable of binding to the same response element (25). The genes encoding ERR{alpha} and ERR{gamma} are expressed broadly with some overlap of expression in tissues such as brain and kidney (26, 27, 28). During early embryonic development, ERRß is expressed exclusively in extraembryonic ectoderm and chorion by 7.5 d post coitum (dpc) (29) Mice homozygous null for ERRß die at 10.5 dpc because of severe defects in chorion formation and trophoblast differentiation (29). On the other hand, mice with a targeted deletion for ERR{alpha} manifest a reduced fat mass, resistance to high-fat diet-induced obesity, and decreased lipogenesis in adipose tissue (30). Gene expression profiling experiments in adipose tissues from ERR{alpha} knockout mice revealed alterations in the expression of genes implicated in the regulation of adipogenesis and energy metabolism (30). Thus, ERR{alpha} may play an important role in lipid homeostasis.

Despite numerous studies of the actions of ERRs in gene regulation in a variety of cell types, there are no reports regarding the roles of ERRs in lung type II cells, which effect high levels of lipogenesis for synthesis of pulmonary surfactant (31). The initiation of SP-A gene expression in fetal lung type II cells is closely coupled to the developmental increase in synthesis of surfactant glycerophospholipids and their storage in the cytoplasm as lamellar bodies (32). This is thought to be analogous to the synthesis and accumulation of triacylglycerols in lipid droplets of adipocytes (33). Thus, we postulated that ERRs may play an important role in activating SP-A gene expression in fetal lung. In this report, we demonstrate that ERR{alpha}, acting through NRESP-A, enhances hSP-A promoter activity. This action of ERR{alpha} was increased by cotransfection of the catalytic subunit of cAMP-dependent protein kinase A (PKA). Moreover, cAMP treatment of lung type II cells increased ERR{alpha} stimulation of SP-A expression. The finding that mice homozygous null for the ERR{alpha} gene manifested decreased SP-A expression relative to wild-type and heterozygous littermates suggests a role of endogenous ERR{alpha} in SP-A gene expression. Furthermore, an ERR{alpha}-specific inverse agonist XCT790, which blocks ERR{alpha} interaction with the coactivator peroxisome proliferator-activated receptor coactivator (PGC)-1{alpha} (34), inhibited hSP-A expression in cAMP-treated human fetal lung type II cells in a concentration-dependent manner. Collectively, these findings suggest that ERR{alpha} plays an important role in the regulation of SP-A gene expression and its induction by cAMP.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Fusion genes and recombinant adenoviruses
Fusion genes were constructed containing 291 and 62 bp of 5'-flanking DNA from the hSP-A gene and +27 bp of the first exon (GenBank accession no. AF061969) fused upstream of the firefly luciferase gene (pGL3; Promega Co., Madison, WI) (hSP-A-291:LUC and hSP-A-62:LUC) to evaluate the effects of ERR on hSP-A promoter activity. To evaluate the importance of NRESP-A in hSP-A promoter activity, an hSP-A:LUC fusion gene was constructed containing a scramble mutation (underlined) in NRESP-A (5'-GTGGGttctagaaGCCAG-3'). This was subcloned into the pGL3 basic plasmid (hSP-A-291NRESP-Amut:LUC). NRESP-Ax1:LUC and NRESP-Ax3:LUC constructs were made by inserting one and three copies of NRESP-A (underlined) and flanking sequence (5'-GTGGGTGACCTTAGCCAG-3'), respectively, upstream of –62 bp of the minimal hSP-A promoter and +27 bp of the first exon of hSP-A, fused to luciferase.

Human ERR{alpha} and ERR{gamma} expression plasmids (CMV/ERR{alpha} and CMV/ERR{gamma}) were kindly provided by Dr. Tim Willson (GlaxoSmithKline, Research Triangle Park, NC). Recombinant adenoviruses containing these expression vectors were generated as described previously (35). Briefly, CMV/ERR{alpha} and CMV/ERR{gamma} were subcloned into the pShuttle vector (Stratagene, La Jolla, CA) to generate pShuttle-CMV/ERR{alpha} and pShuttle-CMV/ERR{gamma}, respectively. Recombinant adenoviral particles containing the pShuttle-CMV/ERR{alpha} and -CMV/ERR{gamma} fusion genes were then generated by cotransformation of electrocompetent BJ5183 bacteria with these fusion genes and pAdEasy-1. PacI-digested recombinant adenoviral fusion genes were then transfected into 293 cells for recombinant adenoviral packaging and propagation. Viral DNA was analyzed to confirm the presence of the fusion genes by restriction endonuclease digestion, PCR, and DNA sequencing. The recombinant adenoviruses were then titered in 293 cells at least three times to determine the number of infectious viral particles (plaque-forming units). CMV-ß-gal adenovirus was kindly provided by Dr. Joseph Alcorn (University of Texas Medical School, Houston, TX). Recombinant adenoviruses containing hSP-A-296:hGH and hSP-A-296NRESP-Amut:hGH fusion genes were constructed as described above.

Culture of human fetal lung explants and isolation and culture of lung type II cells
Human fetal type II pneumonocytes were isolated and cultured as described in detail previously (36). Briefly, midgestation human fetal lung tissues were obtained from Advanced Bioscience Resources (Alameda, CA) in accordance with the Donors Anatomical Gift Act of the State of Texas. The protocols were approved by the Human Research Review Committee of the University of Texas Southwestern Medical Center at Dallas. Tissues were minced and rinsed in serum-free Waymouth’s MB752/1 medium (Gibco Invitrogen Corp., Carlsbad, CA). Lung explants were placed on lens paper supported by stainless steel grids in 35-mm sterile dishes containing 0.5 ml serum-free Waymouth’s media. To isolate type II cells, the explants were cultured in the presence of 1 mM dibutyryl cAMP (Bt2cAMP) (Roche Molecular Biochemicals, Indianapolis, IN) for 5 d to enrich the population of differentiated cells. Cells were dispersed from the explants by digestion with collagenases type I (0.5 mg/ml; Sigma Chemical Co., St. Louis, MO) and type IA (0.5 mg/ml; Sigma) for approximately 15 min. The resulting cell suspension was depleted of fibroblasts by incubation with diethylaminoethyl-dextran (250 µg/ml) for 30 min at 37 C, followed by centrifugation at 400 x g for 5 min (36). The cell pellet was resuspended in Waymouth’s MB752/1 medium containing 10% (vol/vol) fetal bovine serum (FBS; Gemini Bio-Products, Woodland, CA), plated onto 60-mm tissue culture dishes or Thermanox coverslips (Nunc, Naperville, IL) coated with extracellular matrix prepared from Madin-Darby canine kidney cells (CRL 6253; American Type Culture Collection, Manassas, VA; 2–5 x 106 cells/60-mm dish) (36) and incubated overnight. Cells were then washed twice with medium to eliminate dead and nonadherent cells and incubated in Waymouth’s medium without FBS. The plating density of the cells after overnight incubation was approximately 50–60%. COS-7 cells were cultured in DMEM containing 10% FBS.

Transient transfections
Before transfection, COS-7 or lung A549 cells were plated onto 35-mm dishes and grown to logarithmic phase at 50–80% confluence. After washing with PBS, the cells were transfected with 1.5 µg of hSP-A-291:LUC, hSP-A-291NRESP-Amut:LUC, NRESP-Ax3:LUC, or NRESP-Ax1:LUC reporter plasmids, with or without expression vectors for ERR{alpha} or ERR{gamma} (0.3 µg) as well as the corresponding amounts of empty vectors. To evaluate the effects of PKA on ERR{alpha} stimulation of hSP-A promoter activity, A549 lung adenocarcinoma cells were transfected with hSP-A-291:LUC (1 µg), with or without cotransfection of expression vectors for ERR{alpha}, PKA ß-catalytic subunit (PKAcat) (RSV/PKAcat-ß) or a catalytically inactive mutant form of PKAcat-ß (RSV/PKAcat-ßm) (both kindly provided by Dr. Richard Maurer, Oregon Health and Science University, Portland, OR). In all experiments, 0.2 µg of phRL-TK (Renilla luciferase; Promega) was cotransfected as a control for transfection efficiency, and each experimental condition was assayed in triplicate. Two micrograms of plasmid DNA for each transfection were incubated with Superfect (QIAGEN, Inc., Valencia, CA) in Waymouth’s MB752/1 medium without serum, as suggested by the manufacturer, before adding to cells. The cells were incubated with the Superfect/DNA mixture for 4 h at 37 C before washing in culture medium. The cells were then incubated for 48 h, and the cell lysates were collected and assayed for luciferase activity by use of the Dual-Glo luciferase assay kit (Promega) and a 7715 Microplate luminometer (Cambridge Technology, Cambridge, MA).

Expression of SP-A fusion genes in transfected type II cells
Type II cells plated at a density of 5–9 x 106 cells per 60-mm dish were maintained overnight in Waymouth’s MB 752/1. Cells were then washed twice with medium and incubated with recombinant adenoviruses containing ERR{alpha} or ERR{gamma} expression vectors or CMV:ß-gal at a multiplicity of infection of 10. After 10 h, cells were washed and medium was added containing 1 x 106 recombinant adenoviruses containing hSP-A:hGH fusion genes, resulting in a multiplicity of infection of 0.1–0.2. In this manner, approximately the same number of cells (1 x 106) were transfected with the reporter genes in each experiment. After 1 h, medium containing the recombinant adenoviruses was aspirated and replaced with fresh medium. Media from transfected cells were collected every 24 h and assayed for hGH by RIA (Nichols Institute, San Juan Capistrano, CA).

Semiquantitative RT-PCR
Total RNA was extracted from cells by the use of the TRIzol method (Invitrogen). After DNase I treatment of RNA, first-strand cDNA synthesis was catalyzed by SuperScript II RNase H-reverse transcriptase (Invitrogen). Amplification of target cDNAs was implemented by PCR. The oligonucleotides used were as follows: ERR{alpha} (NM_004451.3), forward (5'-AGTGCTGGCCCATTTCTATG-3') (1359–1378 bp) and reverse (5'-TCTCCAAGTCCCACTCTGCT-3') (1861–1842 bp); ERRß (NM_004452.2), forward (5'-CACTGCAGGACTACGAGCTG-3') (1453–1472 bp) and reverse (5'-GGTGAGCCAGAGATGCTTTC-3') (2043–2024 bp); and ERR{gamma} (NM_001438.2), forward (5'-CAGTGACATCAAAGCCCTCA-3') (943–962 bp) and reverse (5'-AGCTTCTGAACGGCTTCAAC-3') (1338–1319 bp). The PCR conditions were as follows: 94 C for 30 sec, 60 C for 30 sec, 72 C for 30 sec for 28 cycles.

EMSA
Nuclear extracts were prepared from lung type II cells as described previously (18). Double-stranded oligonucleotides containing NRESP-A (underlined) and flanking sequences (5'-GTGGGTGACCTTAGCCA-3') as well as the ERRE consensus and flanking sequences (5'-CCGGGGCTTTCAAGGTCATATGCA-3') and ERREmut and flanking sequences (5'-CCGGGGCTTTCAAccTCATATGCA-3') were end labeled using polynucleotide kinase and [{gamma}-32P]ATP (PerkinElmer Life and Analytical Sciences, Inc., Shelton, CT) and used as probes. In vitro-transcribed/translated proteins were synthesized using the TNT coupled transcription/translation system (Promega). The ERR{alpha} and -{gamma} proteins were radiolabeled by carrying out in vitro transcription/translation in the presence of [35S]methionine. Ten microliters of in vitro-transcribed/translated ERR{alpha} and ERR{gamma} proteins were incubated with the radiolabeled DNA probes for 20 min at room temperature in reaction buffer [20 mM HEPES (pH 7.6), 75 mM KCl, 0.2 mM EDTA, 20% glycerol] and 1 µg poly(dI-dC)-poly(dI-dC) (Amersham Biosciences, Piscataway, NJ) as nonspecific competitor. Protein-DNA complexes were resolved on 5% nondenaturing polyacrylamide gels and visualized by autoradiography.

DNase I footprinting
Human ERR{alpha} cDNA was subcloned into the bacterial expression vector pGEX4T1 (Amersham Biosciences) in-frame with glutathione S-transferase (GST). The GST-ERR{alpha} polypeptide was prepared from Escherichia coli according to procedures provided by the manufacturer. DNA probes for footprinting analyses were prepared by digesting the hSP-A-291:LUC plasmid with HindIII or MluI to linearize. These fragments, which contain 291 bp of 5'-flanking DNA and 27 bp of the first exon of the hSP-A gene, were then end labeled using DNA Polymerase I Large (Klenow) fragment and [32P]dCTP. The DNase I footprinting assays were performed in a 200-µl reaction volume. DNA-binding reactions were carried out in a mixture containing 10 mM Tris-HCl (pH 8.0), 5 mM MgCl2, 1 mM CaCl2, 2 mM dithiothreitol, 50 µg/ml BSA, 2 µg/ml calf thymus DNA, and 100 mM KCl. Bacterially expressed proteins (either GST or GST-ERR{alpha}) were incubated with 20,000 cpm of radiolabeled DNA fragment. After 30 min of incubation at room temperature, the reaction mixtures were digested with DNase I (1 U/µl) for 2 min and stopped by the addition of 700 µl DNase I stop solution containing 645 µl of 100% ethanol, 5 µg tRNA, and 50 µl of saturated ammonium acetate. The reaction products were fractionated on 6% polyacrylamide-7 M urea sequencing gels; double-stranded dideoxy sequencing of the probe was performed using the vector primers both forward (5'-TTC GGC TTG TCT GAA CTA AAC CAG GC-3') and reverse (5'-CAG TGT GCT GGA ATT CGG CTT GCT GCT T-3') (U.S. Biochemical Corp., Cleveland, OH), and a sample of the sequencing reaction was loaded adjacent to the samples analyzed by DNase I footprinting.

Immunoblot and shift Western blot analyses
For immunoblotting, nuclear extracts prepared as described above, were fractionated in gradient polyacrylamide gels (Invitrogen) and transferred onto Hybond-P (Amersham Biosciences). Blots were probed first by using rabbit antibodies for ERR{alpha} (kindly provided by Dr. P. J. Willy, Exelixis, Inc., San Francisco, CA) or SP-A (8) and then with horseradish peroxidase-conjugated goat antirabbit IgG (1:10,000) (Amersham Biosciences) as the secondary antibody. The ERR{alpha} antibody specifically interacted with a protein of approximately 51 kDa in immunoblots of lung nuclear extracts from wild-type fetal mice, whereas no immunoreactive band of this size was detected in lung nuclear extracts from ERR{alpha}–/– fetuses (data not shown). Immunoreactive bands were visualized by using an enhanced chemiluminescence system according to the manufacturer’s recommendations (Amersham Biosciences). Shift Western blot analysis was performed as described previously (37). Briefly, nuclear extracts from type II cells were incubated with double-stranded radiolabeled probes for NRESP-A, the consensus ERRE, or a NF-{kappa}B consensus binding site (5'-AGTTGAGGGGACTTTCCCAGGC-3'), as a control (as described under EMSA). Protein-DNA complexes were separated in a native PAGE gel and then transferred onto a nitrocellulose membrane. Binding proteins were detected by immunoblot analysis using an ERR{alpha} antibody, and immunoreactive bands were visualized by enhanced chemiluminescence, as described above.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ERR{alpha} and ERR{gamma} are expressed in human type II cells and bind to NRESP-A
In our initial studies of the regulation of hSP-A gene expression, we characterized a number of response elements essential for mediating basal and cAMP-induced hSP-A promoter activity in transfected type II cells and transgenic mice. Among these is NRESP-A located 240 bp upstream of the hSP-A gene, which corresponds to a half-site for binding members of the nuclear receptor superfamily. This element is highly similar to the consensus binding site (ERRE) for the ERR family of transcription factors. Because there are three closely related ERR isoforms; ERR{alpha}, ERRß, and ERR{gamma}, we first analyzed the expression pattern of these isoforms in human fetal lung type II cells. RT-PCR analysis using human lung type II cell RNA demonstrated that transcripts for ERR{alpha} and ERR{gamma} were present, whereas ERRß expression was not detected (Fig. 1Go).


Figure 1
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  		FIG. 1. ERR{alpha} and ERR{gamma} are expressed in lung type II cells. Total RNA (0.16 µg) isolated from human fetal lung type II cells was analyzed for ERR{alpha}, ERRß, and ERR{gamma} mRNA transcripts using RT-PCR. mRNA transcripts for ERR{alpha} and ERR{gamma} were found to be present in type II cells, whereas ERRß expression was not detectable.

 
To determine whether ERR{alpha} and ERR{gamma} can bind to the NRESP-A, EMSA was performed using a radiolabeled NRESP-A oligonucleotide and in vitro-transcribed-translated ERR{alpha} and ERR{gamma} proteins. Expressed ERR{alpha} and ERR{gamma} bound to the radiolabeled NRESP-A as a single complex (Fig. 2AGo). Binding of ERR{alpha} and ERR{gamma} to radiolabeled NRESP-A was effectively competed by excess nonradiolabeled NRESP-A probe or an ERRE palindromic consensus binding sequence but not by a mutated ERRE sequence. Thus, both ERR{alpha} and ERR{gamma} can specifically bind to the NRESP-A in vitro.


Figure 2
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  		FIG. 2. ERR{alpha} binds to NRESP-A. A, In vitro-transcribed/translated ERR{alpha} and ERR{gamma} bind specifically to the NRESP-A. EMSAs were performed using equivalent amounts of in vitro-transcribed/translated ERR{alpha} or ERR{gamma} proteins and a radiolabeled NRESP-A probe (5'-GTGGGTGACCTTAGCCA-3'). For competition assays, ERR{alpha} or ERR{gamma} was incubated with radiolabeled NRESP-A in the absence (–) or presence of 200-fold molar excess of a nonradiolabeled consensus ERRE (5'-CCGGGGCTTTCAAGGTCATATGCA-3'), mutated ERRE (ERREmut; mutated nucleotides in lowercase, 5'-CCGGGGCTTTCAAccTCATATGCA-3'), or 200- or 400-fold excess of NRESP-A. B, DNase I footprinting of the hSP-A promoter. DNase I footprinting assays were carried out using a radiolabeled probe encompassing –291 to +27 bp surrounding the hSP-A transcription start site incubated in the absence of expressed protein (none), in the presence of expressed GST (30 µg), or with increasing amounts of GST-ERR{alpha} fusion protein (GST-ERR{alpha} fusion protein; 10, 20, and 30 µg). Products were fractionated on a 6% polyacrylamide-urea gel. Lanes 1–4 represent the nucleotide sequence of this genomic region. The protected region is indicated. C, Endogenous ERR{alpha} in human fetal lung type II cells binds to NRESP-A. Shift Western blot analysis was used to assess ERR{alpha} binding to NRESP-A. Nuclear proteins (10 and 20 µg) from human fetal lung type II cells were incubated with radiolabeled NRESP-A (lanes 1 and 2), ERRE consensus (lanes 3 and 4), or NF{kappa}B response element (lanes 7 and 8). Binding reactions were resolved by electrophoresis on a native polyacrylamide gel, and DNA-protein complexes were analyzed for ERR{alpha} after transfer and immunoblotting with anti-ERR{alpha} antibody.

 
To further assess the presence of ERR binding sites within a 291-bp region upstream of the hSP-A gene, DNase I footprinting assays were carried out using a bacterially expressed GST-ERR{alpha} peptide and a radiolabeled probe encompassing –291 to +27 bp surrounding the hSP-A transcription start site. As shown in Fig. 2BGo, the only region of the SP-A promoter protected by expressed ERR{alpha} was between –231 and –250 bp, a region that included NRESP-A. By contrast, no protection occurred when expressed GST peptide alone was used in the DNAase I footprinting assay.

Finally, to determine whether ERR{alpha} in lung type II cell nuclear extracts can bind to NRESP-A, a shift Western blot assay was performed using nuclear extracts from human fetal lung type II cells and an ERR{alpha} antibody. This method was used in lieu of antibody-mediated supershift EMSA because the ERR{alpha} antibody used has greater apparent specificity for the denatured than the native ERR{alpha} protein. Using the shift Western blotting method, ERR{alpha} in type II cells was found to bind to both the NRESP-A and ERRE palindromic consensus sequences; binding was increased with increasing amounts of nuclear extracts (Fig. 2CGo). Conversely, no ERR{alpha} binding was found using an NF-{kappa}B response element probe as a control.

ERR{alpha} but not ERR{gamma} can activate the hSP-A promoter through the NRESP-A
The above findings suggest that both ERR{alpha} and ERR{gamma} can bind to the NRESP-A in vitro and that ERR{alpha} in type II cell nuclear extracts has the ability to bind to NRESP-A. To determine whether ERR{alpha} and/or ERR{gamma} can activate the hSP-A gene promoter, cotransfection studies were carried out in human fetal lung type II cells in primary culture. Because these cells are resistant to standard transfection protocols (13, 36), hSP-A-296:hGH fusion genes containing wild-type or mutated NRESP-A were incorporated into recombinant adenoviruses and introduced into primary cultures of human fetal type II cells by infection. The cells were coinfected with recombinant adenoviruses containing expression vectors for ERR{alpha}, ERR{gamma}, or ß-gal as control. ERR{alpha} caused a greater than 2-fold induction of reporter gene expression over the control vector (Fig. 3Go). Mutation of the NRESP-A greatly reduced basal expression and completely abolished ERR{alpha} induction of SP-A promoter activity. In contrast to ERR{alpha}, ERR{gamma} failed to stimulate SP-A promoter activity (Fig. 3Go). Furthermore, ERR{gamma} modestly inhibited expression, compared with levels observed with the control vector. In type II cells coinfected with expression vectors for ERR{alpha} + ERR{gamma}, promoter activity was modestly reduced, compared with the effect of ERR{alpha} alone (Fig. 3Go). The finding that basal expression was markedly reduced on mutagenesis of NRESP-A suggests that endogenous ERR{alpha} and/or other transcription factors expressed in type II cells up-regulate SP-A promoter activity through binding to this site.


Figure 3
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  		FIG. 3. ERR{alpha} acting via NRESP-A enhances hSP-A promoter activity in human fetal type II cells. Human fetal lung type II cells in monolayer culture were infected with recombinant adenoviruses containing hSP-A:hGH fusion genes comprised of 296 bp of hSP-A 5'-flanking DNA fused to the hGH structural gene, as reporter, with or without mutations in NRESP-A and expression vectors for ß-gal [control (Con)], ERR{alpha}, ERR{gamma}, or ERR{alpha} and ERR{gamma} in combination. After infection, the cells were incubated for up to 3 d in serum-free medium. Culture media were harvested every 24 h and analyzed for hGH by RIA. Values are means ± SEM of data from two independent experiments, each conducted in triplicate.

 
Transfection studies also were carried out using COS-7 cells because they express very low levels of nuclear receptors. Human ERR{alpha} and ERR{gamma} expression plasmids were cotransfected into COS-7 cells with the hSP-A-291:LUC reporter construct. Cotransfection with ERR{alpha} conferred a greater than 6-fold increase in hSP-A promoter activity, compared with the empty expression plasmid (Fig. 4AGo). As we observed in lung type II cells, in contrast to ERR{alpha}, ERR{gamma} had no apparent effect on hSP-A promoter activity. Again, when both ERR{alpha} and ERR{gamma} were transfected together, there was a modest repression of SP-A reporter gene activity, compared with the effect of ERR{alpha} alone (Fig. 4AGo).


Figure 4
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  		FIG. 4. ERR{alpha} stimulation of hSP-A promoter activity is mediated by NRESP-A. A, Overexpression of ERR{alpha} but not ERR{gamma} enhances hSP-A promoter activity in transfected COS-7 cells. Human ERR{alpha} and/or ERR{gamma} expression plasmids (0.3 µg) were cotransfected into COS-7 cells with a reporter construct containing 291 bp of 5'-flanking sequence and 27 bp of exon I from the hSP-A gene (1.5 µg). A Renilla luciferase expression plasmid (phRL-TK) was used to correct for transfection efficiency. Cells were harvested after 48 h and cell extracts were assayed for firefly luciferase and Renilla luciferase activities. Data are presented as the mean ± SEM of duplicate samples from three independent experiments. B, NRESP-A mediates ERR{alpha} induction of hSP-A promoter activity in a copy-dependent manner. One or three copies of the NRESP-A sequence were inserted upstream of a basal hSP-A promoter sequence (–62/+27), which includes the GT- and TATA boxes, fused to luciferase. The recombinant constructs NRESP-Ax3:LUC and NRESP-Ax1:LUC were transfected into COS-7 cells together with ERR{alpha} or ERR{gamma} expression plasmids transfected either alone or in combination or an empty vector as control (Con). The phRL-TK expression plasmid was cotransfected to correct for transfection efficiency. Cells were harvested after 48 h, and cell extracts were assayed for firefly luciferase and Renilla luciferase activities. Data are presented as the mean ± SEM of duplicate samples from three independent experiments. RLU, Relative light units.

 
To determine whether ERR{alpha} acts solely through NRESP-A to increase hSP-A promoter activity, luciferase reporter constructs containing one and three copies of NRESP-A and flanking sequence fused upstream of the hSP-A minimal promoter (–62 bp/+27 bp) were transfected into COS-7 cells with or without cotransfected ERR{alpha}. Parallel experiments were conducted using these reporter constructs and ERR{gamma} in the absence or presence of cotransfected ERR{alpha}. As can be seen, cotransfection of ERR{alpha} caused an approximately 3.5- and 11-fold induction of NRESP-Ax1:LUC expression and NRESP-Ax3:LUC, respectively, compared with the empty expression vector (Fig. 4BGo). Whereas ERR{gamma} had no effect to stimulate expression of the NRESP-Ax1:LUC, ERR{gamma} caused a 4-fold stimulation of NRESP-Ax3:LUC promoter construct. When both ERR{alpha} and ERR{gamma} were cotransfected, the stimulatory effect on SP-A promoter activity was comparable with that observed with ERR{alpha} alone. The decreased transcriptional efficacy of ERR{gamma} is, therefore, partially overcome by increasing the number of tandem ERR response elements. Collectively, these findings demonstrate that ERR{alpha} has a greater potential to activate expression of the hSP-A promoter through NRESP-A than does ERR{gamma}.

Lack of ERR{alpha} results in decreased SP-A expression in fetal mouse lung during late gestation.
To define the role of endogenous ERR{alpha} in the regulation of SP-A gene expression during fetal lung development, SP-A protein was measured in the lungs of ERR{alpha} gene targeted mice. Male and female ERR{alpha}+/– mice were bred and at 18.5 dpc, the fetuses in two litters were genotyped as previously described (30) and fetal lungs were harvested and analyzed for SP-A by immunoblotting. As can be seen in the immunoblot in Fig. 5Go, SP-A protein levels in lung tissues of ERR{alpha}–/– fetuses were considerably reduced, compared with those of heterozygote and wild-type fetuses in the same litters.


Figure 5
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  		FIG. 5. SP-A gene expression is decreased in the lungs of ERR{alpha}–/– fetal mice at 18.5 dpc. Mice heterozygous for targeted deletion of the ERR{alpha} gene were bred. At 18.5 dpc, cytoplasmic proteins (15 µg) were isolated from lung tissues of the fetuses that were genotyped and SP-A was analyzed by immunoblotting. Lanes 1–3, ERR{alpha}–/– pups; lanes 4–6, ERR{alpha}+/– pups; lanes 7–9, wild-type pups.

 
ERR{alpha} up-regulates cAMP induction of endogenous hSP-A expression in human fetal lung type II cells
From the data presented thus far, we conclude that ERR{alpha} binds to NRESP-A and enhances hSP-A promoter activity. As mentioned, we previously observed that NRESP-A is critical for cAMP induction of hSP-A promoter activity in fetal lung type II cells (12, 19, 20). To investigate whether overexpression of ERR{alpha} can enhance endogenous SP-A expression and the effect of cAMP, human fetal lung type II cells cultured in serum-free medium in the absence or presence of Bt2cAMP were infected with recombinant adenoviruses expressing ERR{alpha} or ERR{gamma}, and hSP-A protein expression was analyzed by immunoblotting. As can be seen in Fig. 6Go, neither overexpressed ERR{alpha} nor ERR{gamma} altered SP-A protein levels in type II cells cultured in the absence of Bt2cAMP. By contrast, when type II cells were cultured in the presence of Bt2cAMP, endogenous SP-A protein was up-regulated by overexpressed ERR{alpha}, compared with the ß-gal control (Fig. 6Go). On the other hand, overexpression of ERR{gamma} had a modest inhibitory effect on endogenous SP-A protein levels (Fig. 6Go). These findings again suggest that ERR{gamma} may potentially inhibit basal levels of SP-A promoter activity, possibly by competing with endogenous ERR{alpha} for binding to NRESP-A.


Figure 6
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  		FIG. 6. ERR{alpha} up-regulates cAMP induction of endogenous hSP-A expression in human fetal lung type II cells. Human fetal lung type II cells in monolayer culture were infected with recombinant adenoviruses expressing ERR{alpha} or ERR{gamma} (multiplicity of infection = 10). After infection, the cells were incubated for 48 h in the absence or presence of 1 mM Bt2cAMP. Cytoplasmic proteins (10 µg) isolated from human fetal type II cells were analyzed for SP-A protein by immunoblotting.

 
PKAcat enhances ERR{alpha} stimulation of SP-A promoter activity in transfected lung cells
The above findings suggest that cAMP may stimulate hSP-A expression by enhancing ERR{alpha} transcriptional activity. Because cAMP exerts its effects through activation of PKA, we analyzed the effects of overexpression of the PKAcat on ERR{alpha} stimulation of a hSP-A-291:LUC fusion gene in transfected A549 cells, a lung adenocarcinoma cell line that contains very low levels of PKA activity and does not express endogenous SP-A (14). As can be seen in Fig. 7Go, cotransfection of ERR{alpha} caused an approximately 3-fold induction of hSP-A promoter activity. Although PKAcat alone had no stimulatory effect on hSP-A promoter activity, it enhanced the stimulatory effect of ERR{alpha} to levels more than 6-fold greater than control. By contrast, a mutated inactive form of PKAcat-ß had no effect to alter the stimulatory effect of ERR{alpha} on reporter gene expression.


Figure 7
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  		FIG. 7. Overexpression of PKA in lung A549 cells enhances ERR{alpha} stimulation of hSP-A promoter activity. Lung A549 adenocarcinoma cells transfected with an hSP-A-291:LUC fusion gene were cotransfected with expression vectors for ERR{alpha}, PKAcat, or a mutated inactive form of PKAcat-ß (PKAcatmut) alone or in various combinations or with an empty expression vector as control (Con). The phRL-TK expression plasmid was cotransfected to correct for transfection efficiency. Cells were harvested after 48 h, and cell extracts were assayed for firefly luciferase and Renilla luciferase activities. Data are presented as the mean ± SEM of triplicate samples from a representative experiment.

 
The synthetic ERR{alpha} inverse agonist XCT790 inhibits hSP-A expression in human fetal lung type II cells in culture
A selective ERR{alpha} inverse agonist, XCT790 (kindly provided by Dr. Trish Willy, Exelixis) was used to treat type II cells cultured in medium containing Bt2cAMP. XCT790 acts to prevent ERR{alpha} from interacting with the coactivator, PGC-1{alpha}, thereby inhibiting ERR{alpha} transcriptional activity (34). Cells were collected 48 h after treatment and cytoplasmic extracts were made. SP-A expression was detected by immunoblotting. As can be seen in Fig. 8Go, SP-A expression was inhibited by XCT790 in a dose-dependent manner. These findings suggest that ERR{alpha} interaction with PGC-1{alpha} may mediate increased SP-A expression in fetal lung type II cells in culture.


Figure 8
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  		FIG. 8. The synthetic ERR{alpha}-inverse agonist XCT790 inhibits SP-A expression in human fetal lung type II cells in culture. Human fetal lung type II cells were cultured for 48 h in medium containing Bt2cAMP (1 mM) and/or XCT790 (5 µM, 10 µM). Cytoplasmic fractions from these cells (10 µg) were isolated and analyzed for SP-A by immunoblotting.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In previous studies using transgenic mice (12) and transfected type II cells (13, 14, 18, 19, 20, 38), we found that lung cell-specific, developmental and cAMP stimulation of SP-A expression is mediated by the cooperative interaction of transcription factors bound to response elements within an approximately 300-bp region upstream of the SP-A gene. One of the response elements found to be critical for cAMP induction of SP-A promoter activity has sequence homology to a half-site for binding of members of the nuclear receptor family (NRESP-A). In UV cross-linking assays using type II cell nuclear proteins, we found that this site bound a protein of approximately 50 kDa (19). The ERRs, which are proteins of approximately 50 kDa, bind as monomers or homodimers to an extended half-site in DNA, 5'-TNAAGGTCA-3' (39), referred to as ERRE (26). NRESP-A (5'-CTAAGGTCA-3') of the hSP-A gene is highly similar to the ERRE consensus binding site.

In the present study, we observed using RT-PCR that transcripts for ERR{alpha} and ERR{gamma}, but not ERRß, were expressed in human fetal lung type II cells. Furthermore, in vitro-transcribed/translated ERR{alpha} and -{gamma} specifically bound to the NRESP-A response element of the hSP-A gene. DNase I footprinting analysis of an approximately 300-bp region upstream of the human SP-A gene using bacterially expressed ERR{alpha} revealed the presence of a single DNase I protected region between –250 and –231 bp (5'-AGAGTGGGTGACCTTAGCC-3'), which included the putative ERRE (underlined). Immunoblot analysis after EMSA, using an antibody to ERR{alpha}, indicated that endogenously expressed ERR{alpha} in human fetal type II cells specifically binds to this site. Collectively, these results suggest that ERR{alpha} and -{gamma} are expressed in human fetal lung type II cells and have the capacity to bind to NRESP-A. It was observed that the presence of a T at the N' (2 position) of the extended ERRE consensus (5'-TNAAGGTCA-3') predicts that ERR{alpha} will bind as a dimer rather than a monomer (39). Interestingly, the NRESP-A sequence has a T in the N' position, suggesting that ERR{alpha} may bind as a homodimer to this site.

To determine whether ERR{alpha}, ERR{gamma}, or both regulate hSP-A promoter activity, cotransfection studies were carried out in human fetal lung type II cells and COS-7 cells. We observed that ERR{alpha} induced expression of hSP-A-291 and hSP-A-296 reporter genes in both cell types that was dependent on NRESP-A. Furthermore, ERR{alpha} activated expression of a minimal SP-A promoter construct fused downstream of one or three copies of NRESP-A. By contrast, ERR{gamma} had no effect to increase hSP-A-291:LUC fusion gene expression and modestly inhibited the stimulatory effect of ERR{alpha}. Whereas ERR{gamma} also had no effect to increase expression of the minimal SP-A promoter construct fused to one copy of NRESP-A, ERR{gamma} caused a 4-fold stimulation of the hSP-A promoter construct containing three tandem copies of NRESP-A. The lack of transcriptional efficacy of ERR{gamma} is therefore partially overcome by increasing the number of tandem response elements. These findings suggest that the sequence of the response element and flanking DNA may play an important role in the selective recognition of ERR family members. In this regard, it has been observed that heterodimerization of ERR{alpha} and ERR{gamma} can inhibit the transcriptional activity of both nuclear receptors (40). Furthermore, in studies of transfected type II cells, overexpression of ERR{gamma} was found to inhibit hSP-A promoter activity and expression. On the other hand, we consider an inhibitory role of endogenous ERR{gamma} in type II cells to be unlikely because ERR{gamma} mRNA appears to be expressed at levels approximately 60-fold lower than ERR{alpha} in this cell type, as analyzed by quantitative real-time RT-PCR (data not shown).

To investigate the role of ERR{alpha} on SP-A expression in fetal lung during development, we analyzed the lungs of fetal mice with a targeted deletion in the ERR{alpha} gene (30). At 18.5 dpc, SP-A protein levels in lung tissues of ERR{alpha}–/– fetuses were considerably reduced, compared with those of ERR{alpha}+/– and wild-type fetuses in the same litter. These findings suggest an important role of ERR{alpha} in the developmental regulation of SP-A expression. In fetal lung, developmental regulation of SP-A expression is known to be closely associated with the synthesis of surfactant glycerophospholipids (5), which are essential for air breathing. The fact that ERR{alpha} null mice do not manifest an apparent surfactant deficiency suggests that ERR{alpha} does not play a significant role in surfactant glycerophospholipid metabolism in lung type II cells, in contrast to its known function in lipid homeostasis in adipose tissue, liver, and intestine (26, 30, 41). Furthermore, it should be noted that SP-A predominantly plays a role in the modulation of the innate immune defense within the lung alveolus (3) and does not appear to influence the surface-active properties of lung surfactant. Interestingly, macrophages from ERR{alpha}–/– mice exhibit decreased clearance of the intracellular bacterial pathogen Listeria monocytogenes, and these mice are more susceptible to Listeria infection than wild-type (Sonoda J., I. Mehl, J. Laganière, L.-W. Chong, G. Barish, V. Giguère, and R. M. Evans, unpublished observations). Thus, a lung phenotype in the ERR{alpha} null mouse may manifest itself only in the presence of an environmental challenge that impacts energy metabolism and/or pulmonary infection.

In preliminary studies, we observed that ERR{alpha} as well as ERR{gamma} expression remained relatively unchanged in fetal mouse lung from 15 dpc to term and during differentiation of human fetal lung in culture in concert with the developmental induction of SP-A expression (Liu, D., and C. R. Mendelson, unpublished observations). In previous studies, it was similarly found that expression levels of the essential transcription factor TTF-1/Nkx2.1 remained relatively constant during fetal lung development (42) and with cAMP and IL-1 treatment of human fetal type II cells in culture (14). However, cAMP and IL-1 treatment of the cultured type II cells enhanced TTF-1 phosphorylation (14) and binding to its DNA response element with recruitment of essential coactivators and increased local acetylation of histone H3 (Lys 9) to mediate increased SP-A gene expression (43). We therefore suggest that an inductive effect of ERR{alpha} on SP-A gene expression during development may be dependent on changes in ERR{alpha} posttranslational modification, DNA-binding, and/or interaction with essential coactivators.

In this regard, we observed in the present study that cAMP stimulation of SP-A expression in human fetal lung type II cells was enhanced by overexpression of ERR{alpha}. Interestingly, overexpressed ERR{alpha} had no effect to increase SP-A protein levels in type II cells cultured in the absence of cAMP. Furthermore, overexpression of PKA catalytic subunit enhanced ERR{alpha} stimulation of hSP-A promoter activity in transfected A549 cells, a lung adenocarcinoma cell line that contains very low levels of PKA activity. Based on these findings, we postulate that cAMP/PKA may enhance ERR{alpha} transcriptional activity through effecting changes in its posttranslational modification. Despite its homology to the estrogen receptor, no natural ligands for ERRs have been found (44). Structural studies of ERR{alpha} and ERR{gamma} revealed a transcriptionally active conformation in the absence of a ligand (45). ERR{alpha} transcriptional activity may require posttranslational modification, such as phosphorylation (46), followed by dimerization and interaction with coactivators, including steroid receptor coactivators-1, -2, and -3 (47, 48) and PGC-1{alpha}. PGC-1{alpha} is known to be critical for lipid, glucose and energy homeostasis in a number of tissues (49, 50) and has been identified as a strong coactivator of ERR{alpha} (34, 51, 52, 53). Interestingly, PGC-1{alpha} also up-regulates ERR{alpha} gene expression through its interaction with ERR{alpha} on the ERR{alpha} promoter (53).

To investigate the role of ERR{alpha} coactivator interaction in the regulation of hSP-A gene expression, human fetal type II cells were treated with the ERR{alpha} inverse agonist XCT790 (34, 54), which selectively blocks ERR{alpha}-PGC-1{alpha} interaction. Treatment with XCT790 caused a dose-dependent inhibition of SP-A expression in cAMP-treated human fetal lung type II cells, suggesting a possible role of PGC-1{alpha} and related coactivators in transcriptional activation of SP-A expression by endogenous ERR{alpha}.

In summary, although ERR{alpha} has been well studied in a variety of tissues, including heart, adipose, skeletal muscle, adrenal, liver, bone, and intestine and human breast cancer, this is the first report of the potential role of ERR{alpha} in gene regulation in the lung. The results of our studies suggest that ERR{alpha} is expressed in lung type II cells in which it acts to increase expression of the SP-A gene through a response element in its 5'-flanking region. This action of ERR{alpha} appears to require interaction with PGC-1{alpha}. Our findings of reduced SP-A expression in ERR{alpha} gene targeted mice further suggest a role of ERR{alpha} in the regulation of SP-A gene expression during fetal lung development.


   Acknowledgments
 
The authors thank Ms. Margaret Smith for the expert isolation of human fetal type II cells and Céline Champigny for skilled technical support with the ERR{alpha} gene targeted mice.


   Footnotes
 
This work was supported by National Institutes of Health Grant 5-R37-HL050022 (to C.R.M.) and a grant from the Canadian Institutes of Health Research (to V.G.).

The authors D.L., M.M.H., V.G., and C.R.M. have nothing to declare.

First Published Online August 17, 2006

Abbreviations: Bt2cAMP, Dibutyryl cAMP; CRE, cAMP response element; CREB, CRE binding protein; dpc, days post coitum; ERR, estrogen-related receptor; ERRE, ERR element; FBS, fetal bovine serum; GST, glutathione S-transferase; h, human; NF{kappa}B, nuclear factor-{kappa}B; NRE, nuclear receptor element; PGC, peroxisome proliferator-activated receptor coactivator; PKA, protein kinase A; PKAcat, PKA ß-catalytic subunit; SP, surfactant-associated protein; TTF, thyroid transcription factor.

Received May 18, 2006.

Accepted for publication August 10, 2006.


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