Articles in PresS. Am J Physiol Lung Cell Mol Physiol (November 21, 2003). 10.1152/ajplung.00280.2003
Glucocorticoid Inhibition of Surfactant Protein A (SP-A) Gene Expression in Lung Type II Cells is Mediated via the TTF-1 Binding Element
Joseph L. Alcorn1*, Kazi N. Islam2, Pampee P. Young2,4 and Carole R. Mendelson2,3*
1
Department of Pediatrics, University of Texas Medical School at Houston, 6431 Fannin, Suite 3.222, Houston, TX 77030
2
Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390
3
Department of Obstetrics & Gynecology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390
4
Current address: Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN 37232
Running Title: Glucocorticoid regulation of SP-A gene expression
*Address correspondence to: Carole R. Mendelson, Ph.D. Department of Biochemistry University of Texas Southwestern Medical Center 5323 Harry Hines Boulevard Dallas, Texas 75390-9038 Phone: 214-648-2944 Fax: 214-648-3214 E-mail:
[email protected]
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Copyright (c) 2003 by the American Physiological Society.
Glucocorticoid regulation of SP-A gene expression
ABSTRACT Cyclic AMP and interleukin-1 (IL-1) induction of surfactant protein-A (SP-A) expression in fetal lung type II cells is mediated by increased binding of thyroid transcription factor-1 (TTF-1) and NF-κB proteins p50 and p65 to the TBE (TTF-1binding element) at -183 bp. In type II cell transfections, dexamethasone (Dex) markedly inhibits cAMP induced expression of rabbit SP-A:hGH fusion genes containing as little as ~300 bp of SP-A 5’-flanking sequence. Dex inhibition is blocked by RU486, suggesting a role of the glucocorticoid receptor (GR). The present study was undertaken to define the mechanisms for GR inhibition of SP-A expression. Cotransfection of primary cultures of type II cells with a GR expression vector abrogated cAMP induction of SP-A promoter activity, while at the same time causing 60-fold induction of co-transfected mouse mammary tumor virus (MMTV) promoter. In lung cells transfected with a fusion gene containing 3-TBEs fused to the basal SP-A promoter, Dex prevented the stimulatory effect of IL-1 on TTF-1 induction of SP-A promoter activity, suggesting that GR inhibits SP-A promoter activity through the TBE. In gel shift assays using nuclear extracts from human fetal type II cells cultured in the absence or presence of cAMP, Dex markedly reduced binding of nuclear proteins to the TBE and blocked the stimulatory effect of cAMP on TBE binding activity. Our finding that Dex increased expression of the NF-κB inhibitory partner, IκBα, suggests that the decrease in TBE binding activity may be caused, in part, by GR inhibition of NF-κB interaction with this site. Keywords: Surfactant protein-A (SP-A), fetal lung, glucocorticoid receptor, cyclic AMP, thyroid transcription factor I (TTF-I)
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Glucocorticoid regulation of SP-A gene expression
INTRODUCTION Pulmonary surfactant, a developmentally regulated phospholipid-rich lipoprotein produced by type II pneumonocytes, reduces surface tension at the alveolar air-liquid interface and prevents alveolar collapse. In fetal lung, type II cells containing stored surfactant are first evident only after ~70% of gestation is completed. The fundamental discoveries that administration of synthetic glucocorticoids to fetal lambs resulted in accelerated lung maturation (37) and that glucocorticoid treatment of fetal rabbits caused enhanced surfactant production and accelerated appearance of type II cells (32, 59), led to numerous studies that supported the concept that glucocorticoids enhance surfactant glycerophospholipid synthesis (6, 44). Based on such findings, the antenatal administration of glucocorticoids has been used clinically to enhance lung maturation and prevent respiratory distress syndrome (RDS) in infants of mothers likely to deliver preterm (38). A number of factors, including gestational age, sex, and treatment regimen influence the effectiveness of antenatal glucocorticoid regulation of the lipid and protein components of surfactant (7). Surfactant glycerophospholipid synthesis is, in fact, under multifactorial control; in addition to glucocorticoids, a number of other steroid and polypeptide hormones, as well as cAMP, are important in its regulation (44). Surfactant is primarily comprised of phosphatidylcholine, with lesser amounts of other glycerophospholipids and cholesterol. Surfactant also contains four associated proteins, surfactant protein A (SP-A)1, SP-B, SP-C and SP-D, which are expressed in a 1
Abbreviations: Bt2cAMP, dibutyryl cyclic AMP; CRE, cAMP-response element; CREB, CRE-binding protein; CBP, CREB-binding protein; CMV, cytomegalovirus; Dex, dexamethasone; EMSA, electrophoretic mobility shift assay; FBS, fetal bovine serum; β-gal, β-galactosidase; GR, glucocorticoid receptor; GRE, glucocorticoid response element; hGH, human growth hormone; IL-1, interleukin-1; IκB, inhibitor of κB; MMTV, mouse mammary tumor virus; NF-κB, nuclear factor kappa B; PKA, protein kinase A; SP, surfactant protein; SRC-1, steroid receptor coactivator-1; TK, thymidine kinase; TTF-1, thyroid transcription factor-1; TBE, TTF-1-binding element; USF, upstream stimulatory factor.
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Glucocorticoid regulation of SP-A gene expression
lung-specific manner and developmentally regulated in fetal lung tissue (22, 44). Synthesis of the major surfactant protein, SP-A, a C-type lectin that plays an important role in immune defense within the lung alveolus, is initiated in fetal lung during the third trimester of gestation in concert with augmented surfactant glycerophospholipid synthesis (45, 57). Cyclic AMP increases expression of the SP-A gene in rabbit (9, 45), baboon (52) and human (49) fetal lung tissue in culture. The stimulatory effects of cAMP appear primarily to be mediated at the level of SP-A gene transcription (9, 11). In studies using transgenic mice and transfected type II cells, we have found that a ~400 bp region flanking the 5'-end of the SP-A gene mediates lung type II cell-specific and appropriate developmental regulation of expression (2). Within this region, five response elements have been identified that are each critical for basal and cAMP induction of SP-A promoter activity (63). One of these, termed TBE (TTF-1-binding element), binds the homeodomain transcription factor, thyroid transcription factor-1 (TTF-1/Nkx2.1) (34, 35). Cyclic AMP acting though protein kinase A (PK-A) causes increased TTF-1 phosphorylation, TBE-binding and transcriptional activity (34, 62). We recently found that treatment of human fetal type II cells with the cytokine interleukin-1 (IL-1), a known activator of NF-κB, increases SP-A expression and has additive stimulatory effects with cAMP. Interestingly, the TBE at ~-180 bp within the human and baboon (b) SP-A2 5’-flanking regions contains a ‘reverse-oriented’ NF-κB binding site; IL-1 and cAMP treatment of human fetal type II cells enhance binding of NF-κB proteins p50 and p65, together with TTF-1 to the TBE (27). Although we were unable to ascertain whether p65 and p50 bind to the TBE directly, in co-immunoprecipitation assays, it was found that p65 interacts with TTF-1 in type II cells (27). Furthermore, p65
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Glucocorticoid regulation of SP-A gene expression
and p50 act cooperatively with TTF-1 to stimulate SP-A promoter activity (27). Glucocorticoids have complex effects on SP-A expression that may be speciesspecific and dependent upon the stage of development at which treatment is initiated. In studies
using
midgestation
human
fetal
lung
explants,
we
observed
that
dexamethasone (Dex) caused a dose-dependent induction of hSP-A gene transcription and acted synergistically with Bt2cAMP (11, 12); however, in the same tissues, Dex caused a dose-dependent decrease in the levels of SP-A mRNA and protein and antagonized the stimulatory effect of Bt2cAMP on SP-A mRNA and protein levels (48). This apparent inhibitory effect of glucocorticoids is due to a dominant action to decrease SP-A mRNA stability (11, 12, 24). The inhibitory effects of Dex on SP-A mRNA stability were found to be dose-dependent, completely reversible and blocked by the glucocorticoid receptor (GR) antagonist RU486 (12). Unlike the rabbit, which has a single copy SP-A gene (10, 14), the genomes of humans (30, 39) and baboons (18) contain two highly similar SP-A genes, SP-A1 and SP-A2. We previously observed that the hSP-A2 gene is more responsive to the inductive effects of cAMP and the inhibitory effects of glucocorticoids than that encoding hSP-A1 (40). In studies using lung explants from 21-day fetal rabbits (term = day 31), Dex (10-7 M) caused an acute (6-24 h) inhibition of SP-A gene transcription and reduced the magnitude of the stimulatory effect of Bt2cAMP (9). However, after 48-72 h of incubation, a stimulatory effect of glucocorticoid was observed and an additive effect with Bt2cAMP on SP-A gene transcription was found (9). In type II cell transfection studies, we previously observed that Dex markedly inhibited cAMP-induced expression of rabbit SP-A:hGH fusion genes containing as little as 380 bp of SP-A 5'-flanking DNA
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Glucocorticoid regulation of SP-A gene expression
(1). The finding that this effect of Dex was half-maximal at 10-9 M and blocked by the glucocorticoid receptor (GR) antagonist RU486 suggests a role of GR in Dex inhibition of SP-A promoter activity. While Dex alone had little effect to alter basal expression of the SP-A:hGH fusion gene constructs, it had a marked effect to antagonize the stimulatory action of cAMP (1). It was our objective in this study to define the mechanisms for the effects of glucocorticoids acting through the GR on SP-A promoter activity. We have focused on the TBE in light of its importance in cAMP and IL-1 induction of SP-A gene expression and because it was found that the TBE interacts with TTF-1 and NF-κB proteins p65 and p50 (27). Glucocorticoids acting through the GR have been reported to antagonize NF-κB transcriptional activity via several mechanisms that appear to be cell-typespecific (17). Our findings suggest that in the absence of a functional glucocorticoidresponse element (GRE) within ~1000 bp upstream of the SP-A transcription initiation site, the inhibitory effects of Dex acting through GR are mediated via GR inhibition of the binding of type II cell nuclear proteins to the TBE. Furthermore, the inhibitory actions of glucocorticoids may be mediated, in part, through GR-induced expression of IκBα, an inhibitory partner of NF-κB, thereby reducing the levels of p65 and p50 available to bind to the TBE.
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Glucocorticoid regulation of SP-A gene expression
MATERIALS AND METHODS Fusion genes and recombinant adenoviruses. Fusion genes and expression vectors incorporated into the genomes of recombinant adenoviruses and used in the present study are shown in Figure 1. DNA containing the promoterless human growth hormone (hGH) gene, as reporter (53), was fused downstream of rabbit (r)SP-A sequences comprised of 20 bp of exon I and 990 bp of exon I 5’-flanking DNA (rSP-A-990:hGH) as described previously (1) and to the mouse mammary tumor virus long terminal repeat (MMTV) (MMTV-770:hGH), a sequence known to contain functional glucocorticoid receptor responsive elements (GRE) (19). Rabbit SP-A 5’-flanking sequences from -990 to -47 bp (excluding the TATA box) were subcloned upstream of 250 bp of thymidine kinase (TK) promoter sequences controlling hGH in pTKGH (53) to create rSP-A-990:TK:hGH for analysis of effects of GR on SP-A 5’-flanking sequences acting on a heterologous promoter. Several fusion genes were also constructed that contained various amounts of 5’-flanking DNA from the hSP-A2 gene and +20 bp of the first exon fused upstream of the promoterless hGH gene (64) to evaluate the effect of Dex on human SP-A2 promoter activity. These fusion genes include hSP-A2-1500:hGH, hSP-A2-296:hGH and hSP-A2-47:hGH. The cDNA encoding the human glucocorticoid receptor (hGR) (23) was placed under the control of the strong, ubiquitously expressed cytomegalovirus E1 (CMV) promoter (CMV:hGR) (60). These fusion genes were incorporated independently into the genomes of recombinant replication-defective adenoviruses using methods described by Graham and Prevec (21). An SP-A promoter construct was also generated that consisted of a concatamer of three repeats of baboon (b)SP-A2 gene 5’-flanking sequence between -185 and –165 bp containing TBE1
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Glucocorticoid regulation of SP-A gene expression
(underlined) (5’-GTGCTCCCCTCAAGGGTCCTA-3’) subcloned upstream of 50 bp of bSP-A2 5’-flanking sequence and +40 bp of bSP-A2 exon I, fused to hGH (TBE3:hGH) (34). This was introduced into A549 cells using standard transfection methodology (see below). Organ culture of fetal lung tissues, type II pneumonocyte isolation and primary culture. The procedure used to isolate and maintain human, rat and rabbit fetal type II pneumonocytes in primary culture has been described previously (4). Mid-gestation human fetal lung tissues were obtained from Advanced Bioscience Resources, Inc. (Alameda, CA) in accordance with the Donors Anatomical Gift Act of the State of Texas; protocols were approved by the Human Research Review Committee of the University of Texas Southwestern Medical Center at Dallas. Lung tissues from 18-day gestational age Sprague-Dawley fetal rats were obtained in accordance with the guidelines established by the Animal Welfare Information Center, and approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center at Dallas. The tissues were minced and cultured in serum-free Waymouth's MB756/1 medium (Gibco\BRL Inc., Gaithersburg, MD) in the presence of dibutyryl cAMP (1 mM, Bt2cAMP; Boehringer Mannheim Corp., Indianapolis, IN). After 3-5 days of organ culture with daily medium changes, lung explants were dissociated by digestion with collagenase type I (0.5 mg/ml; Sigma Chemical Co., St. Louis, MO) and collagenase type IA (0.5 mg/ml; Sigma Chemical Co.). Following collagenase digestion, the cell suspension was depleted of fibroblasts (4) and plated onto 60 mm tissue culture dishes or on Thermanox coverslips (Nunc, Inc., Naperville, IL) that were coated with extracellular matrix (ECM) prepared from MDCK cells (ATCC CRL 6253) (2-5 X 106
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Glucocorticoid regulation of SP-A gene expression
cells per 60 mm dish) (4).
Plated epithelial cell-enriched cultures were incubated
overnight in Waymouth's medium with 10% fetal bovine serum (FBS; Gibco\BRL Inc., Gaithersburg, MD). Dishes were washed twice with medium to eliminate dead and nonadherent cells, and then incubated in Waymouth's MB756/1 medium without FBS. The plating density of the cells after overnight incubation was approximately 50-60%. Infection of human type II pneumonocytes with recombinant adenoviruses. Type II pneumonocytes were infected with recombinant replication-defective human adenoviruses containing the appropriate fusion genes as described previously (1, 4). Briefly, type II pneumonocytes that were plated onto 60 mm ECM-coated dishes and maintained overnight in Waymouth's MB752/1 medium containing FBS (10% v/v) were then washed several times with medium to remove non-adherent cells and debris, and incubated for 1 h with recombinant adenovirus. The adenovirus-containing medium was then removed and replaced with fresh medium without serum. Cell culture and transfection.
The lung adenocarcinoma cell line A549 (36) was
maintained in Waymouth's MB756/1 medium (Gibco\BRL Inc., Gaithersburg, MD) containing FBS (10% vol/vol). The recombinant DNA was introduced into the cell line by infection using recombinant adenoviruses as described for type II cells in primary culture or by standard transfection techniques. In transfection studies to analyze the effects of Dex and GR on TTF-1 and IL-1 induction SP-A promoter activity using the (TBE)3SP-A:hGH fusion gene as reporter, A549 cells were grown to logarithmic phase (70% confluence) in Waymouth’s MB752/1 medium containing 10% FBS in 35-mm culture dishes. The cells were transfected with 2 µg of the (TBE)3SP-A:hGH fusion gene, together with either 1 µg of pCMV5/TTF-1 or the empty expression vector
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Glucocorticoid regulation of SP-A gene expression
(pCMV5), or 1 µg of RSV/p50, RSV/p65 alone or in combination and compensatory amounts of RSV empty vector. All cells were cotransfected with 0.25 µg RSV/β-gal as an internal control for transfection efficiency. The plasmids were combined with 8 µl of Fugene 6 (Roche Molecular Biochemicals, Indianapolis, IN) in Waymouth's MB752/1 medium and incubated at room temperature for 30 min. The cells were washed once with medium and incubated with reporter and expression plasmid/Fugene mixtures in 1 ml of serum-free Waymouth's MB752/1 medium for 15 h at 37oC. The medium was then removed, fresh Waymouth's medium (1 ml) was added to each well with or without IL-1β (10 ng/ml), and the cells were incubated at 37oC for 24 h. Media were then collected and assayed for hGH content by radioimmunoassay (Nichols Institute, San Juan Capistrano, CA). Variations in transfection efficiency were corrected by normalizing hGH to β-galactosidase activity, which was assayed using a Galato-lightTM kit (TROPIX, Inc, Bedford, MA). Immunoblot analysis.
Cellular proteins were isolated as described previously (3).
Briefly, cells were scraped from the plates with a rubber policeman and homogenized in ice-cold homogenization buffer (0.25 M sucrose, 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride [PMSF]) using 10 strokes in a teflon-glass homogenizer.
The homogenized samples were centrifuged at 600 xg for 5 min to
sediment nuclei and debris, and the resulting supernatant was assayed for protein content. Cellular proteins (50 µg) were subjected to one-dimensional sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the separated proteins transferred to nitrocellulose by electrophoretic transfer. The proteins on the resulting blots were analyzed for SP-A and IκB-α content as described previously (45),
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Glucocorticoid regulation of SP-A gene expression
using antibodies to human SP-A (49) and to IκB-α (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The resulting immune complexes were visualized using the ECL western blotting system (Amersham Pharmacia Biotech Inc., Piscataway, NJ).
The
blots were subjected to autoradiography using Kodak X-OMAT radiographic film for visualization of the resulting protein-antibody complexes. Quantitative analysis of hGH production in transfected human type II pneumonocytes and cell lines. Media from transfected cells or from cells infected with the recombinant adenoviruses containing promoter fusion constructs with hGH reporter genes were collected at 24 h intervals. The concentration of hGH in the medium was quantitated by radioimmunoassay using an Allegro hGH kit (Nichols Institute Diagnostics, San Juan Capistrano, CA). Electrophoretic mobility shift assays. Nuclear extracts were prepared from lung type II cells as described previously (33). Protein concentrations were determined by a modified Bradford assay (Bio-Rad, Richmond, CA). Double-stranded oligonucleotides corresponding
to
the
TBE
(TBE,
GTGCTCCCCTCAAGGGTCCT-3')
and
underlined) to
an
and
flanking
NF-κB
sequences
consensus
binding
(5'site
(underlined) (5'-AGTTGAGGGGACTTTCCCAGGC-3') (Integrated DNA Technologies, Inc., Coralville, IA) were end-labeled using [γ32P]ATP (ICN, Costa Mesa, CA) and used as probes. Nuclear proteins (7 µg) were incubated with
32
P-labeled TBE and NF-κB
oligonucleotides for 30 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 of poly(dI-dC)-poly(dI-dC) (Pharmacia, Piscataway, NJ) as nonspecific competitor. Protein-DNA complexes were
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Glucocorticoid regulation of SP-A gene expression
separated from free probe on a 4% non-denaturing polyacrylamide gel and visualized by autoradiography.
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Glucocorticoid regulation of SP-A gene expression
RESULTS Dexamethasone acting through GR inhibits SP-A promoter activity and stimulates MMTV promoter activity in type II pneumonocytes in primary culture. Previously, we observed that dexamethasone (Dex) antagonized the stimulatory effect of Bt2cAMP on expression of rabbit SP-A:hGH fusion genes containing 1766, 990 and 378 bp of SP-A 5’-flanking DNA; the effect of Dex was half-maximal at 10-9 M and blocked by the GR antagonist RU486 (1). To determine whether Dex had the capacity to stimulate gene expression in lung type II cells when acting through classical GREs, we compared effects of Dex ± GR overexpression on SP-A promoter activity with effects on activation of the mouse mammary tumor virus (MMTV) promoter, which is known to be positively regulated by glucocorticoids. MMTV promoter activity is regulated by direct binding of GR to cognate GREs within the LTR (19). Consequently, recombinant adenoviruses were created containing the human (h)GR coding sequence under control of the ubiquitously-expressed CMV E1 promoter, CMV:hGR, and a fusion gene comprised of 770 bp of the glucocorticoid-responsive mouse mammary tumor virus LTR linked to the hGH structural gene, as reporter (MMTV-770:hGH) (Fig. 1). Rat type II cells in primary culture were infected with recombinant adenoviruses containing either rSP-A-990:hGH (containing 990 bp of 5’-flanking DNA from the rabbit SP-A gene linked to hGH) or MMTV-770:hGH reporter constructs with or without co-transfected CMV:hGR. The transfected cells were incubated in the absence or presence of Bt2cAMP, Dex, or both agents in combination for up to 5 days in culture, and the amount of hGH secreted into the medium during each 24 h period was determined by radioimmunoassay. As can be seen in Figure 2A, in type II cells transfected with SP-A-990:hGH, Dex had little effect to
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Glucocorticoid regulation of SP-A gene expression
alter fusion gene expression as compared to cells incubated in control medium. Similar findings were obtained in type II cells co-transfected with SP-A-990:hGH and CMV:hGR (Fig. 2B). As we observed previously, Bt2cAMP increased fusion gene expression >25fold (Fig. 2A,B), while Dex antagonized the stimulatory effect of Bt2cAMP by ~70% (Fig. 2A). In the absence of Dex treatment, co-expression of CMV:hGR had little effect on SP-A-990:hGH expression in cells treated with Bt2cAMP; however, hGR overexpression enhanced the suppressive effect of Dex on cAMP stimulation of SP-A promoter activity, reducing hGH production by 99%, as compared to Bt2cAMP alone (Fig. 2B). These findings suggest that the antagonistic effect of Dex is mediated through the GR. In type II cells transfected with MMTV-770:hGH, Dex caused a modest increase in hGH production, as compared to untreated cells (Fig. 2C). Interestingly, Bt2cAMP increased MMTV-770:hGH expression in a pattern similar to that observed in cells transfected with SP-A-990:hGH. In cells co-transfected with CMV:hGR, Dex caused a rapid and marked increase in MMTV-770:hGH expression; no further stimulation was observed in cells co-incubated with Bt2cAMP + Dex (Fig. 2D). These findings suggest that cultured rat type II cells contain the cofactors required to mediate GR stimulation of MMTV expression; however, it is evident that these cells contain low levels of endogenous GR. On the other hand, it appears that the low endogenous levels of GR in rat type II cells are sufficient to mediate pronounced glucocorticoid inhibition of cAMP induction of SP-A promoter activity. To ascertain whether the inhibitory effect of Dex was mediated by interaction of GR with SP-A 5’-flanking sequences upstream of the TATA box or with sequences within the hGH structural gene, fetal rat type II cells were infected with recombinant
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Glucocorticoid regulation of SP-A gene expression
adenoviruses containing an hGH fusion gene comprised of SP-A 5’-flanking sequences from -990 to -47 bp (excluding the TATA box and +20 bp of exon I), fused upstream of the 250 bp herpes simplex virus thymidine kinase (TK) promoter (TK:SP-A-990:hGH), or with the 250 bp TK promoter fused to hGH (TK:hGH) (42) (Fig. 1). Whereas, no effect of Dex or cAMP were observed on expression of TK:hGH, Dex caused a 50% inhibition of cAMP induced SP-A-990:TK:hGH expression (data not shown), suggesting that Dex inhibition is mediated through SP-A 5’-flanking sequences between –47 and –990 bp. These findings differ from those of Floros and colleagues (25) who reported that a region between -32 and +63 bp of the hSP-A1 gene mediates Dex repression of transcription. . Effects of Dex on SP-A and MMTV promoter activity in rat type II cells and A549 lung adenocarcinoma cells are likely dependent upon endogenous levels of GR. In light of the apparently low levels of endogenous GR in primary cultures of lung type II cells, we performed similar experiments in the human lung adenocarcinoma cell line, A549, which is of presumed type II cell origin. The A549 cells were cultured and infected in a manner identical to that used for the rat type II cells. Cells were infected with recombinant adenoviruses containing either SP-A-990:hGH or MMTV-770:hGH fusion genes with or without co-transfection with CMV:hGR. The cells were cultured for 5 days in the absence or presence of Bt2cAMP, Dex, or both factors in combination, and the amounts of hGH secreted into the medium over a 24 h period between days 4 and 5 of culture were determined by radioimmunoassay. The data shown in Figure 3A for rat type II cells correspond to the results obtained after 5 days of culture that are shown in Figure 2.
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Glucocorticoid regulation of SP-A gene expression
In contrast to findings in primary cultures of rat type II cells, and as reported previously (1), Bt2cAMP had only a modest effect to increase SP-A promoter activity in A549 cells (Fig. 3). On the other hand, the effect of Dex to stimulate MMTV-770:hGH expression and to inhibit SP-A-990:hGH expression in the absence of cotransfected GR was much more pronounced in the A549 cells than in the primary cultures of rat type II cells, suggesting the presence of elevated levels of endogenous functional GR in the former (Fig. 3B). Accordingly, in the A549 cells, cotransfection of CMV:GR had a relatively modest effect to further increase Dex-inhibition of SP-A promoter activity and Dex-stimulation of MMTV promoter activity (Fig. 3B). Glucocorticoid inhibition of human SP-A2 promoter activity is mediated by sequences within 296 bp upstream of the transcription initiation site and is associated with decreased binding of type II cell nuclear proteins to the TTF-1response element (TBE). Since the inhibitory effect of Dex on SP-A promoter activity in rat type II cells is manifest only in the presence of Bt2cAMP, it is likely that the effect of Dex is mediated by GR inhibition of DNA-binding and/or transcriptional activity of a cAMP activated transcription factor. In previous studies, we observed that Dex antagonized cAMP stimulation of SP-A:hGH fusion genes containing as little as 378 bp of rSP-A 5’-flanking sequence (1), suggesting that the transcription factor(s) with which GR interacts lies within the proximal 5’-flanking region. In recent studies, we have found that the TBEs of the human and baboon SP-A2 genes, which contain a ‘reverseoriented’ NF-κB binding site and bind NF-κB proteins p65 and p50 together with TTF-1 (27), play an important role in cAMP and interleukin-1 (IL-1) stimulation of SP-A promoter activity (27). Since GR has been reported to interact with p65 (61) and to
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Glucocorticoid regulation of SP-A gene expression
block NF-κB transcriptional activity (17), it was of interest to determine whether the inhibitory effect of Dex is mediated via the TBE. Although the TBE is highly conserved among the SP-A genes of a variety of species (43), we elected to focus our studies on the TBE of human SP-A2 gene because of our prior studies on its characterization. It was first important to evaluate the effect of Dex on hSP-A2 promoter activity and to delineate the genomic region that mediates glucocorticoid inhibition. As we observed using the rabbit SP-A promoter constructs (Figs. 2 and 3), Dex antagonized cAMP induction of hSP-A2:hGH expression in transfected human fetal type II cells (Fig. 4). The inhibitory effect of Dex on cAMP-induced hSP-A2 promoter activity appears to be mediated by sequences between –47 and –296 bp of hSP-A2 5’-flanking DNA. It should be noted that in contrast to findings using rat type II cells (Fig. 2, 3), Dex inhibited basal levels of hSP-A2:hGH expression in the human fetal type II cells (Fig. 4). This may be due, in part, to the relatively high basal levels of fusion gene expression in the human, as compared to the rat type II cells. As reported previously, the hSP-A2 5’flanking region between –296 bp and -1500 bp appears to contain ‘silencer’ element(s) (64). Despite the presence of these putative ‘silencers,’ an inhibitory effect of Dex on basal and cAMP-induced expression was still evident (Fig. 4). A ‘silencer’ region also has been reported upstream of the rat SP-A gene (56). Since the –296 bp genomic region contains the TBE, it was of interest to analyze the effect of Dex on binding of type II cell nuclear proteins to this element using electrophoretic mobility shift assays (EMSA); nuclear protein binding to a consensus NF-κB response element was also studied for comparison. Cytoplasmic fractions of these cells were analyzed for SP-A content by immunoblotting. As reported previously,
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Glucocorticoid regulation of SP-A gene expression
cAMP treatment of human fetal type II cells caused a pronounced increase in SP-A protein levels as compared to those of untreated cells; Dex, which had little effect on SP-A levels in the absence of cAMP treatment, markedly inhibited cAMP induction of SP-A expression (48, 49) (Fig. 5A). It should be noted, however, that the mechanisms for this inhibitory effect of Dex on SP-A protein levels are complex and mediated, at least in part, by its action to decrease SP-A mRNA stability (11). As observed previously, cAMP increased nuclear protein binding to the TBE (27, 34) (Fig. 5B), as well as to the NF-κB consensus sequence (27) (Fig. 5C). Interestingly, Dex inhibited the binding of type II cell nuclear proteins to the TBE and NF-κB response elements and markedly antagonized the stimulatory effect of Bt2cAMP on nuclear protein binding to these sites (Fig. 5B,C). These findings suggest that Dex inhibition of SP-A promoter activity is mediated, in part, by reduced binding of TTF-1 and/or NF-κB to the TBE. Dexamethasone inhibits TTF-1 and IL-1 stimulation of SP-A promoter activity acting through the TBE. To directly assess whether GR and Dex inhibit SP-A promoter activity through the TBE, A549 cells, which do not express endogenous TTF-1 (34) but contain NF-κB (Islam, K.N. and Mendelson, C.R., unpublished observations), were transfected with a fusion gene comprised of three TBEs fused upstream of -50/+40 bp of promoter/first exon DNA from the bSP-A2 gene, linked upstream of the hGH structural gene, as reporter (TBE3:hGH). A549 cells were co-transfected with empty CMV expression vector, or CMV expression vector containing TTF-1 or hGR, alone or in combination; β-gal was co-transfected as a control for transfection efficiency. After 15 h, cells were incubated for 24 h with IL-1β (10 ng/ml) or Dex (10-7 M), alone or in combination. IL-1β has previously been reported to increase NF-κB binding and
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Glucocorticoid regulation of SP-A gene expression
transcriptional activity in A549 cells (29). On the other hand, effects of cAMP were not analyzed in the present study because cAMP has little or no effect on SP-A expression these cells (Fig. 3), likely due to a deficiency in protein kinase A activity (34). Reporter gene expression was evaluated by measuring the hGH that accumulated in the culture medium during this period. As we have observed previously (27), TTF-1 caused a twofold induction of SP-A promoter activity; a similar fold-stimulation was observed in cells incubated with IL-1β (Fig. 6). In cells transfected with CMV:TTF-1 and treated with IL-1β, SP-A promoter activity was increased >4-fold. The stimulatory effects of TTF-1 and IL-1β alone or in combination were markedly inhibited by overexpression of hGR in the presence of Dex (Fig. 6). GR had no effect in the absence of Dex (data not shown). These findings suggest that the inhibitory effect of Dex/GR on SP-A promoter activity is mediated through the TBE. Furthermore, the finding that Dex/GR inhibited IL-1βinduced fusion gene expression in the absence of TTF-1 co-transfection, suggests that glucocorticoid inhibition of hSP-A2 promoter activity is likely mediated by GR inhibition of endogenous NF-κB DNA-binding activity. Dexamethasone stimulates IκB-α expression in human fetal lung type II cells. Glucocorticoids have been reported to increase IκB-α expression in certain systems (5, 51); however, this effect appears to be cell type and promoter-specific (17). Glucocorticoid induction of IκB is suggested to cause increased retention of NF-κB p50 and p65 in inactive complexes within the cytoplasm. To further define the mechanism(s) by which Dex inhibits SP-A gene expression, human fetal lung type II cells were cultured for 3 days in control medium, in medium containing Bt2cAMP, Dex or IL-1α, alone and in various combinations. Cytoplasmic fractions from these cells were isolated
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and analyzed for IκB-α by immunoblotting. Levels of IκB-α protein were relatively unaffected by either IL-1α or Bt2cAMP, added alone or in combination (Fig. 7). By contrast, Dex markedly stimulated IκB-α levels in the absence or presence of Bt2cAMP (Fig. 7). Interestingly, Dex failed to stimulate IκB-α levels in type II cells treated with IL-1α or IL-1α + Bt2cAMP. These findings suggest that Dex antagonism of basal and cAMP-induced SP-A expression may be mediated, in part, by increased IκB expression, resulting in inhibition of NF-κB transcriptional activity. On the other hand, Dex antagonism of IL-1- or IL-1 + Bt2cAMP-induced SP-A expression cannot be explained by this mechanism.
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DISCUSSION The regulation of SP-A gene expression by glucocorticoids is complex and multifaceted, involves transcriptional and posttranscriptional effects (11, 12, 24, 26), and may be mediated by direct and indirect actions of the steroid on type II cells. In type II cell transfection studies using either rabbit (1) or human SP-A:hGH fusion gene constructs, we consistently observed an inhibitory effect of Dex on cAMP stimulation of SP-A promoter activity. The inhibitory effect of Dex appears to be mediated by sequences within ~380 (1) and ~300 bp upstream of the rabbit and human SP-A gene transcription initiation sites, respectively. This genomic region contains a number of highly conserved response elements that each are essential for cAMP induction of SP-A promoter activity (46). In the present study, we have begun to investigate the mechanisms for the inhibitory effects of glucocorticoids on SP-A gene transcription in lung type II cells. In type II cell transfection studies, we compared the effect of Dex in the absence or presence of overexpressed hGR on SP-A promoter activity and on activation of the MMTV promoter, which is known to be glucocorticoid responsive (19). Whereas, Dex in the presence of overexpressed GR had a pronounced effect to inhibit cAMP induction of SP-A promoter activity, a marked stimulatory effect on MMTV promoter activity also was observed. This indicates that type II cells have the necessary factors to support GR induction of a glucocorticoid-responsive promoter and suggests the absence of a stimulatory GRE within the SP-A 5’-flanking sequence. The lung cell transfection studies reported herein also revealed that functional levels of endogenous GR in primary cultures of fetal rat type II cells are quite low.
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Although a pronounced inhibitory effect of Dex on cAMP-induced SP-A promoter activity was observed in rat type II cells in the absence of co-transfected CMV:hGR, a robust induction by Dex of MMTV promoter activity required hGR overexpression. These findings suggest that the inhibitory action of glucocorticoids on cAMP induction of SP-A expression requires lower concentrations of GR than does Dex stimulation of MMTV promoter activity. On the other hand, the finding that Dex markedly inhibited SP-A promoter activity and stimulated MMTV promoter activity in A549 cells in the absence of cotransfected GR indicates that the adenocarcinoma cell line contains relatively high levels of functional GR. The results of a number of morphological studies indicate that GR is localized both to mesenchyme and epithelium in fetal lung (16). An autoradiographic study of [3H]Dex binding in sections of fetal mouse lung at various stages of development and in human fetal lung at 8 weeks of gestation revealed that mesenchyme directly adjacent to the more proximal portions of the bronchiolar network was heavily labeled, while the epithelium destined to later differentiate into bronchi and bronchioles was relatively unlabeled. Nuclear localization of Dex binding to distal portions of the growing epithelium, destined to become alveolar ducts and alveoli, was also evident (8). By in situ hybridization, GR mRNA levels were found to increase in embryonic mouse lung from E13 to E15/17; expression was mainly localized to the mesenchyme, while ductular epithelium was less intensely labeled (31). In another study of fetal mouse lung during the latter stages of gestation, GR was found to be localized to mesenchyme adjacent to the bronchial and air sac epithelia (28). These findings suggest that glucocorticoid actions on fetal lung development may be mediated by indirect effects on
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lung mesenchyme to reduce expression of inhibitory factors and/or enhance expression of stimulatory factors. It has been suggested that glucocorticoids act on the fetal lung fibroblast to promote synthesis of a ‘differentiation factor’ termed fibroblast pneumonocyte factor (FPF), which acts on lung epithelium to enhance alveolar type II cell differentiation and surfactant synthesis (55). To date, FPF has not been isolated and characterized. The finding that mice homozygous for targeted deletion of the GR gene (GR-/-) die within several hours of birth as a result of respiratory failure caused by atelectatic underdeveloped lungs (15) emphasizes the importance of GR in lung development. It was suggested that the lack of GR impaired development of the terminal bronchioles and alveoli beyond E15.5. Of importance, however, was the finding of comparable numbers of alveolar type II cells and apparently normal levels of mRNA encoding SP-A, SP-B and SP-C in lung tissues of newborn GR-/- mice, as compared to heterozygous or wild-type animals (15). On the other hand, mice homozygous for a point mutation in the GR that prevents its dimerization and DNA-binding are viable, despite lack of inducibility of a number of GR-regulated genes. This suggests that the actions of glucocorticoids to enhance lung development occur through DNA-binding-independent mechanisms (50). In the present study, we found that Dex/GR inhibition of SP-A promoter activity was most pronounced in lung cells that manifested elevated basal and/or cAMP induction of SP-A promoter activity. GR repression of AP-1 and NF-κB-induced gene expression has been reported to be mediated by the interaction of GR with these stimulatory transcription factors (50). In this regard, it is of interest that Dex treatment of human fetal lung type II cells inhibited nuclear protein binding to the TBE and prevented
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the stimulatory effect cAMP on TBE-binding activity. In addition to the homeodomain transcription factor TTF-1, the TBE also interacts with NF-κB p50 and p65 (27). Although we have as yet to obtain evidence that NF-κB binds directly to the TBE, by antibody-mediated supershift EMSA, we have found that p50 and p65 interact with TTF-1 at this site. Furthermore, in co-immunoprecipitation studies using type II cell nuclear extracts, we have observed that TTF-1 and p65 interact in vivo (27). TTF-1 and NF-κB also were found to cooperatively interact through the TBE to increase SP-A promoter activity; this was further enhanced by IL-1 (27). Our finding, in the present study, that Dex/GR acted through the TBE to inhibit the stimulatory effects of TTF-1 and IL-1 on SP-A promoter activity in transfected lung A549 adenocarcinoma cells suggests that glucocorticoids acting through GR may inhibit SP-A expression by blocking binding and transcriptional activity of TTF-1 and NF-κB at the TBE. As mentioned earlier, A549 cells don’t express endogenous TTF-1 (34) although they do contain NF-κB. Since the inhibitory effect of Dex/GR on SP-A promoter activity was manifest in IL-1 treated cells in the absence of TTF-1 co-transfection, our findings suggest that glucocorticoids exert their inhibitory actions in A549 cells through interaction with NF-κB. We have not as yet determined whether GR interacts directly with TTF-1 in type II cells to block its DNA binding and transcriptional activity. However, GR has been found to inhibit prolactin gene expression in non-pituitary cells through a response element that binds the POU-homeodomain transcription factor, Oct-1, and the homeodomain transcription factor Pbx-1 (58). Interestingly, addition of in vitro translated GR DNA-binding domain to nuclear extracts prevented the binding of Oct-1 and Pbx-1 to this response element (58). A similar mechanism, involving interaction of GR with
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Oct-1 bound to its response element has been proposed for glucocorticoid inhibition of the mouse gonadotropin-releasing hormone promoter (13). As mentioned above, there is abundant evidence to suggest that glucocorticoids exert their anti-inflammatory and immunosuppressive actions on cells by GR antagonism of NF-κB-mediated transactivation, and that NF-κB antagonizes the actions of glucocorticoids by inhibition of GR function (17). Several mechanisms have been proposed to explain these phenomena; however, they appear to be cell type- and promoter-specific (17). These include: [1] direct (61) or indirect (through CREB-binding protein [CBP]) (41) interaction between the N-terminus of p65 and DNA-binding domain of GR, which prevents functional association with the preinitiation complex and blocks transactivation; [2] competition between GR and NF-κB for limiting amounts of coactivators, CBP/p300 and steroid receptor coactivator (SRC)-1 (54) and/or; [3] glucocorticoid/GR induction of IκBα gene expression (5, 51).(5) In the present study, we observed that Dex increased IκB-α expression in human fetal type II cells cultured in the absence or presence of Bt2cAMP. It is suggested that the resulting increase in IκBα protein causes trapping of p65 and p50 in inactive complexes within the cytoplasm, blocking their transcriptional activity. Accordingly, we recently found that overexpression of dominant negative, phosphorylation-defective mutant forms of IκB in lung type II cells reduced binding of nuclear proteins to the TBE and inhibited cAMP and IL-1 induction of SP-A expression (27). On the other hand, an effect of Dex to increase IκB expression was not manifest in type II cells treated with cAMP ± IL-1. This is most likely due to a dominant effect of IL-1 to increase degradation of IκB. Since Dex inhibition of nuclear protein binding to the TBE and of SP-A promoter
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activity were also manifest in cells treated with IL-1, it is improbable that Dex antagonism of IL-1 ± Bt2cAMP stimulation of SP-A expression can be explained by IκB induction. We recently reported that the coactivators CBP/p300 and SRC-1, which contain intrinsic histone acetyltransferase activity, interact directly with TTF-1 and act synergistically to increase SP-A promoter activity (63). Cyclic AMP treatment of cultured type II cells was found to increase TTF-1 acetylation. This suggests that cAMPmediated TTF-1 phosphorylation facilitates interaction with CBP and SRC-1, resulting in TTF-1 hyperacetylation, which further enhances its DNA-binding and transcriptional activity (63). In light of the importance of these coactivators in TTF-1, as well as NF-κB (20, 47) transcriptional activity, it is possible that GR antagonism of cAMP and IL-1 induction of SP-A gene expression may be mediated, in part, by competition for essential coactivators and formation of a repressed chromatin structure. In summary, our present findings suggest that glucocorticoid inhibition of SP-A gene transcription is complex and occurs at several levels. The inhibitory actions of glucocorticoids do not appear to be mediated by direct binding of GR to DNA sequences surrounding the SP-A promoter. Rather, negative effects of glucocorticoids are likely caused by GR inhibition of binding and transcriptional activity of NF-κB and/or TTF-1 at the TBE, a response element that is crucial for cAMP and IL-1 induction of SP-A expression. Glucocorticoid-induced expression of the NF-κB inhibitor, IκB-α, may also play a role in Dex/GR inhibition of cAMP stimulated SP-A promoter activity. In consideration of previous findings that the coactivators CBP/p300 and SRC-1 interact with NF-κB (20, 47) and with TTF-1, and play an important role in the induction of SP-A
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promoter activity (63), it also is likely that negative effects of Dex/GR are mediated, in part, by competition for limiting amounts of these coactivators.
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ACKNOWLEDGMENTS The authors are grateful to Ms. Margaret Smith for her expert assistance in the preparation of primary cultures of type II cells. This research was supported by NIH Grants R37-HL-50022 and U01-HL52647 (C.R.M.) and by Grant 92G-083 from the American Heart Associations, Texas Affiliate, Inc (J.L.A).
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FIGURE LEGENDS Figure 1. Schematic Diagram of the Fusion Genes Incorporated into Recombinant Adenoviruses. Structures of the various fusion genes used in the study are shown in schematic fashion. CMV:hGR: A full-length cDNA encoding the human glucocorticoid receptor (hGR) was placed under control of 600 bp of the ubiquitously expressed cytomegalovirus (CMV) promoter. MMTV-770:hGH: The human growth hormone (hGH) structural gene (used as a reporter of promoter activity) was placed under control of 770 bp of the mouse mammary tumor virus (MMTV) long terminal repeat. rSP-A-990:hGH: The hGH structural gene was placed under control of 990 bp of DNA flanking the 5’-end of the rabbit SP-A gene plus 20 bp of SP-A exon I. TK:hGH: The 250 bp herpes simplex virus thymidine kinase (TK) promoter, which is unaffected by glucocorticoids or cAMP, was subcloned upstream of the hGH structural gene. rSP-A-990:TK:hGH: -990 to -47 bp of the rabbit SP-A 5’-flanking sequence was fused upstream of the TK promoter. Several fusion genes were utilized to analyze the effects of Dex on the hSP-A2 gene promoter. These included hSP-A2-1500:hGH, hSP-A2-296:hGH and hSP-A2-47:hGH, which contained 1500, 296 and 47 bp of 5’-flanking sequence and 20 bp of exon I from the hSP-A2 gene fused to the hGH structural gene. These fusion genes were incorporated into the genome of a recombinant human adenovirus (Ad5), as previously described (1). TBE3:hGH: This fusion gene is comprised of three repeats of baboon (b)SP-A2 gene 5’-flanking sequence between -185 and –165 bp containing TBE1 (underlined) (5’-GTGCTCCCCTCAAGGGTCCTA-3’) subcloned upstream of 50 bp of bSP-A2 5’-flanking sequence and +40 bp of bSP-A2 exon I, fused to hGH.
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Glucocorticoid regulation of SP-A gene expression
Figure 2.
Effects of dexamethasone ± cAMP in the absence or presence of
overexpressed
human
glucocorticoid
receptor
(hGR)
on
expression
of
SP-A-990:hGH and MMTV-770:hGH fusion genes in fetal rat type II pneumonocytes in primary culture.
Rat type II pneumonocytes were infected with recombinant
adenoviruses containing either the SP-A-990:hGH (Panels A and B) or MMTV-770:hGH (Panels C and D) fusion genes in the absence (Panels A and C) or presence (Panels B and D) of adenovirus expressing CMV:hGR. The cells were cultured in the absence or presence of Bt2cAMP (1 mM), dexamethasone (Dex, 10-7 M), or both agents in combination. Shown are the levels of hGH that accumulated in the culture medium during each 24-h period over 5 days. Data are the mean ± S.E.M. of values from two independent experiments each conducted in triplicate. Figure 3. Effects of dexamethasone ± cAMP in the absence or presence of overexpressed
human
glucocorticoid
receptor
(hGR)
on
expression
of
SP-A-990:hGH and MMTV-770:hGH fusion genes in primary cultures of fetal rat type II cells and in the A549 lung adenocarcinoma cell line. Rat type II pneumonocytes in primary culture (Panel A) and A549 lung adenocarcinoma cells (Panel B) were infected with recombinant adenoviruses containing either the SP-A-990:hGH or MMTV770:hGH
fusion genes alone or in combination with or without CMV:hGR. The cells were
cultured in the absence or presence of Bt2cAMP (1 mM), dexamethasone (Dex, 10-7 M), or both agents in combination. Shown are the levels of hGH secreted by the cells during a 24 h period between days 4 and 5 of culture. Data are the mean ± S.E.M. values from 2 independent experiments each conducted in triplicate.
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Glucocorticoid regulation of SP-A gene expression
Figure 4. Effects of Dex ± Bt2cAMP on human SP-A2 promoter activity. Primary cultures of human fetal type II cells were infected with recombinant adenoviruses containing hSP-A2:hGH fusion genes comprised of –47, -296 and –1500 bp of hSP-A2 5’-flanking DNA and +20 bp of exon I. The cells were cultured for 5 days in the absence or presence of Dex (10-7 M), Bt2cAMP (1 mM), or both agents in combination. Shown are the levels hGH secreted by cells over a 24-h period between days 4 and 5 of culture. Data are the mean ± S.E.M. of values from 2 independent experiments each conducted in triplicate. Figure 5. Effects of Dex ± Bt2cAMP on binding of type II cell nuclear proteins to radiolabeled TBE and NF-κB probes using EMSA. Nuclear and cytoplasmic extracts were prepared from human fetal type II cells after 3 days of culture in control medium or in medium containing Bt2cAMP (1 mM), Dex (10-7 M), or Bt2cAMP and Dex in combination. Cytoplasmic fractions were analyzed for immunoreactive SP-A by immunoblotting (Panel A). Type II cell nuclear proteins were incubated with
32
P-labeled
oligonucleotides containing TBE (underlined) (5’-GTGCTCCCCTCAAAGGGTCCT-3’) (Panel B) and NF-κB consensus (underlined) (5’-AGTTGAGGGGACTTTCCCAGGC-3’) (Panel C) binding elements and analyzed by EMSA. Figure 6. Effects of Dex/GR on TTF-1 and IL-1 activation of the SP-A promoter via the TBE. A549 cells lung adenocarcinoma cells were transfected with a fusion gene comprised of three TTF-1 binding elements fused upstream of –50/+40 bp of promoter/first exon DNA from the bSP-A2 gene, linked upstream of the hGH structural gene, as reporter (TBE3:hGH). The cells were co-transfected with empty CMV expression vector, or CMV expression vector containing TTF1 or hGR cDNAs, alone or
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Glucocorticoid regulation of SP-A gene expression
in combination; β-gal was co-transfected as a control for transfection efficiency. After 15 h, cells were incubated with IL-1β (10 ng/ml) or Dex (10-7 M), alone or in combination. SP-A promoter activity was evaluated by measuring the hGH that accumulated in the culture medium over a 24-h period. Data are the mean ± SEM of corrected values from three independent experiments, each conducted in triplicate. Figure 7. Effect of Dex on IκB-α expression in human fetal lung type II cells cultured in the absence or presence of Bt2cAMP and IL-1α alone and in combination. Human fetal lung type II cells were cultured for 3 days in control medium, in medium containing Bt2cAMP (1 mM), Dex (10-7 M) or IL-1α (10 ng/ml) alone and in various combinations. Cytoplasmic fractions from these cells were isolated and analyzed for IκB-α by immunoblotting.
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