Departments of 3 Pediatrics, 1 Biochemistry and Molecular Biology, and 2 Neuroscience, University of Florida, Gainesville, Florida 32610
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ABSTRACT |
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Endothelin (ET)-1
is a potent vasoconstrictor elicited from endothelial cells in response
to a variety of stimuli and an important mediator for a variety of
vascular diseases including pulmonary hypertension. In this paper, we
describe the molecular regulation of the ET-1 gene in response to a
vasoactive mediator, thrombin, in human pulmonary endothelial cells.
Thrombin induces preproET-1 mRNA through a transcriptionally dependent
mechanism, with a peak induction after 1 h of exposure. Analysis of
chromatin structure identified several DNase I-hypersensitive regions
under both basal and thrombin-stimulated conditions that reside in the
5'-promoter region, indicating that the ET-1 promoter is a
constitutive promoter. Deletion analysis was employed as a functional
assay to identify regions of the ET-1 promoter that are important in
transcriptional regulation. We found that sites between 141 and
378 bp are essential for basal activity and that those between
378 and
484 bp are essential for thrombin-stimulated
activity. However, full expression under both conditions required an
element(s) within
952 bp.
chromatin studies; deletion analysis; pulmonary vascular disease
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INTRODUCTION |
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IT HAS BEEN well established that the endothelium is vital in the regulation of reactivity in vascular tissues via release of endothelium-derived factors that act on adjacent smooth muscle cells. One such factor produced by vascular endothelial cells is the potent vasoconstrictive peptide endothelin (ET)-1, which is regulated by a number of inflammatory and vasoactive mediators (11, 15, 20, 36). Regulation of ET-1 secretion by these mediators is most likely critical in maintenance of vascular tone in certain pathophysiological situations.
Our laboratory is particularly interested in the mechanisms present in pulmonary disease states, especially pulmonary hypertension and adult respiratory distress syndrome. Mounting evidence implicates ET-1 as a mediator of these pathologies (8, 10, 16, 30, 31). Therefore, studies aimed at understanding the molecular mechanisms underlying ET-1 gene expression in human pulmonary endothelial cells may lead to data relevant to treatment of these human pathologies.
The mature 21-amino acid ET-1 peptide is derived by proteolytic processing from preproET-1, a 212-amino acid precursor. Understanding ET-1 gene expression under constitutive as well as stimulated conditions is imperative for our understanding of the role of ET-1 in pulmonary and vascular disease. Functional assays have been used by two different groups to provide a fairly detailed picture of the basal regulation of ET-1 in bovine endothelial cells (6, 14, 17, 18, 34) and in COS cells (1). In addition, a very limited analysis of stimulus-induced ET-1 expression, using functional studies, has been conducted (19, 23). However, there are limitations to these previous investigations, as these studies have been conducted in a heterologous system whereby regulation of the human ET-1 gene was assessed in bovine or porcine aortic endothelial cells, COS cells, HeLa cells, or NIH/3T3 cells, in some cases with variable results. On the basis of reports of species- and tissue-dependent differences in ET-1 gene expression (26) and reports that endothelial cells derived from different species and tissues can express dramatic variations in metabolic and enzymatic properties (3, 7, 25, 29), we believe that investigation of human gene regulation should be conducted in human tissue-derived cells relevant to disease. Additionally, we need a better understanding of the regulation of this gene, not only under basal conditions but also under stimulated conditions that mimic the vascular environment to which endothelial cells are exposed during disease states.
In this work, we provide a detailed analysis of the expression and regulation of the human ET-1 gene in human pulmonary endothelial cells. The aim of these studies was to investigate the effect of thrombin, a physiological mediator potentially involved in the pathogenesis of pulmonary vascular disease, on the molecular regulation of ET-1.
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METHODS |
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Cell culture. Isolation of human pulmonary arterial (HPA) endothelial cells was performed essentially as described (32), with some modifications. Cells were obtained from HPA segments from heart transplant donors by collagenase (Sigma, St. Louis, MO) treatment and scraping of the endothelium. The scraped cells were placed in endothelial basal medium (EBM; Clonetics, San Diego, CA), pelleted, resuspended in EBM plus 10% fetal bovine serum (FBS), and plated in cell culture flasks. The cells were incubated at 37°C in room air containing 5% CO2, and the medium was changed within 24 h and then every two days until confluence. Once the cells reached confluence, they were trypsinized using 0.025% trypsin and 0.02 mM EDTA and passed at a ratio of 1:3 or 1:4 in EBM plus 10% FBS. Human pulmonary microvascular (HMV) endothelial cells were obtained commercially from Clonetics. These cells were passed under the same conditions as the HPA cells. All cells used for the following investigations were at low passage, with no studies performed above passage 11.
Northern analysis. HPA and HMV endothelial cells were grown to confluence on 100-mm cell culture dishes. ET-1 mRNA levels were assessed under both basal and thrombin-stimulated conditions. Cells were exposed to bovine thrombin (10 U/ml; Sigma) or to purified bovine thrombin (ICN) for a period of 15 min to 4 h. Similar results were obtained with both products; therefore, the majority of the experiments were performed with the Sigma preparation. Each treated plate had a corresponding control plate from the same cell line and passage number. Inhibitor studies were performed to better characterize the regulatory mechanisms involved in ET-1 gene expression at the transcriptional level. Cells were cotreated with thrombin (10 U/ml) and either actinomycin D (4 µM; Sigma), an RNA synthesis inhibitor, or cycloheximide (20 µM; Sigma), a protein synthesis inhibitor.
Total cellular RNA was isolated according to the procedures presented by Chomczynski and Sacchi (4), and Northern analysis was performed as described previously (11), with human ET-1 or cathepsin B cDNAs used as probes. The human ET-1 cDNA was isolated by our laboratory as previously described (32). The cathepsin B cDNA was a gift from S. J. Chan (Univ. of Chicago) and was used as an internal control to correct for any variations in RNA loading. Northern blots were exposed to autoradiography with an intensifying screen atPreparation of permeabilized cells, DNase I
digestion, and DNA isolation.
Six 150-mm cell culture dishes of HPA endothelial cells grown to
confluence were placed in EBM containing 1% FBS. The next day, three
plates were kept as controls, receiving no treatment, and three plates
were treated with 10 U/ml of thrombin for 20-30 min. These were
washed and trypsinized, and 5 ml of cold medium were added to each
plate. The cells from each set were scraped, collected, and centrifuged
at 600 g, 4°C, for 5 min and
resuspended in 4.5 ml of solution A
(150 mM sucrose, 80 mM KCl, 35 mM HEPES, pH 7.4, 5 mM potassium
phosphate, and 5 mM MgCl2). Then
1.5 ml of 0.2%
L--lysolecithin
in 60 mM KCl, 15 mM NaCl, 60 mM Tris, pH 7.8, and 0.25 M sucrose were
added; this mixture was incubated for 2 min on ice; and 30 ml of
solution A were added to stop the reaction. The cell suspension was spun again and resuspended in 1,200-1,500 µl of solution A,
yielding 2 × 107
cells/suspension. Cells from each suspension were then split into
4-5 aliquots of 300 µl each. The cell suspensions were treated with increasing concentrations of DNase I (2.5 mg/ml of stock; Worthington Biochemical, Freehold, NJ) at 37°C for 4 min. Lysis buffer (100 mM EDTA, 1% SDS, and 5 µg/ml of proteinase K) was added
to each sample and rocked for 3 h at 55°C. Genomic DNA was extracted by standard phenol-chloroform purification procedures. The
DNA was treated with RNase A (50 µg/ml; Sigma) followed by ethanol
precipitation and resuspended in water.
Southern analysis. Twenty micrograms of DNA were digested and size fractionated on an agarose gel, and Southern analysis was performed (27). The DNA was electrotransferred to an uncharged nylon membrane (Zetabind, Cuno, Meriden, CT) and cross-linked by ultraviolet irradiation. The method of indirect end labeling was used to map the positions of hypersensitive sites (35). Both high- and low-resolution chromatin structure was evaluated. For low-resolution studies, DNA was size fractionated on a standard 1% agarose gel, and high-resolution analysis was performed with the use of MetaPhor (FMC BioProducts, Rockland, ME) agarose gel system, which more accurately resolves smaller fragments than standard agarose.
A full-length ET-1 genomic clone was obtained commercially (Genome Systems, St. Louis, MO) by screening a human P1 bacteriophage library for clones positive for the 5'- and 3'-ends of the ET-1 gene. Subclones of the ET-1 gene in pUC19 were constructed for use in making probes and promoter deletion fragments for chromatin structure and promoter deletion analysis. Probes for DNase I analysis were created either by isolation of restricted fragments from ET-1 gene subclones using gratuitous restriction sites or by amplification of the desired fragment using PCR primers. Labeling of probes by [32P]dATP was accomplished by random-primer extension (GIBCO BRL).Plasmid constructions.
Portions of the ET-1 promoter were cloned into a promoterless human
growth hormone (hGH) reporter vector, pØGH (Nichols Institute, San
Juan Capistrano, CA), for use in promoter deletion analysis. The
largest ET-1-pØGH reporter plasmids were created by cloning portions of the ET-1 gene using gratuitous restriction enzyme sites and
with ligation into the polylinker of pØGH. These plasmids contain
8, 5.2, and 2.4 kb of ET-1 sequence 5' to the transcriptional start site. Each of these plasmids has a common 3' site located 166 bp 3' to the transcriptional start site, created
by a Bgl II restriction enzyme site at
this position. The 8- and 5.2-kb ET-1-promoter fragments were created
by cleavage of the ET-1 gene with Bgl
II and Xba I, respectively. The
desired fragments were cloned into the
BamH I site or the
Xba I and
BamH I sites, respectively, of
pØGH. Similarly, the 2.4-kb ET-1 fragment, which is flanked by an
Xho I site at 2.4 kb, was
created by liberating the desired fragment from a second subclone of
the ET-1 gene by digestion of this clone with
Xba I (in the polylinker of pUC19) and
Bgl II (at +166 bp of ET-1). This
product was directionally cloned into the
Xba I and
BamH I sites of pØGH. All
positive clones were confirmed by restriction mapping and sequence
analysis.
Transient transfection methods. HPA or HMV endothelial cells were grown to ~70% confluence in 150-mm cell culture dishes. Plasmid DNA (8.1 µg) was brought to 80-µl volume in Tris-buffered saline and combined with 224 µl of a 10 mg/ml stock solution of DEAE-dextran (Sigma). The DNA-DEAE-dextran solution was added to cells in 10.8 ml of EBM supplemented with 10% NuSerum (Collaborative Biomedical Products, Bedford, MA) for a final DEAE-dextran concentration of 200 µg/ml. To inactivate the lysosomes of the cell and thereby prevent degradation of the transfected DNA, chloroquine diphosphate (Sigma) was added to the media at a final concentration of 75 µM. Cells were incubated for 4 h at 37°C in room air with 5% CO2. Media were poured off, and 13.5 ml of 10% DMSO (Sigma) in PBS were added to each plate for 1.5 min. The cells were washed in PBS, EBM plus 10% FBS was added, and cells were incubated at 37°C. The next day, cells were passed to 4 × 100-mm culture dishes (27). On day 3 after transfection, the medium was changed to EBM containing 1% FBS, and several hours later, two of the four plates were kept as control plates and two were treated with thrombin (10 U/ml). Media were collected 72 h after treatment, and hGH levels were measured using a commercially available radioimmunoassay (Nichols Institute).
To evaluate plate-to-plate variability of transfection efficiency, a Southern analysis of DNA isolated from cells transfected with the ET-1 promoter-hGH plasmid was performed (22). At the termination of the experiment, the cells from control and stimulus-treated plates were lysed, and DNA was isolated and digested to generate a unique fragment. The DNA fragments were separated by size on an agarose gel and transferred to a nylon membrane that was hybridized to a probe within the promoter deletion fragment. Autoradiography was performed, and the relative amounts of plasmid were evaluated by densitometry.Data analysis. Statistical comparisons were made for each vector individually to detect differences under stimulus versus control conditions. Values were calculated using a paired two-tailed t-test. All data are expressed as means ± SE, and significance was set at P < 0.05.
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RESULTS |
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Detailed analysis of thrombin-stimulated ET-1 mRNA levels. Figure 1, A and B, illustrates ET-1 mRNA levels in HMV and HPA cells in response to thrombin stimulation over the course of 4 h. Figure 1A is a representative Northern blot from HMV cells, with nearly identical results for HPA cells. The graph in Fig. 1B illustrates a comparison of the multiples of induction of ET-1 mRNA for both cell lines. As illustrated, ET-1 mRNA levels are elevated over control values as early as 30 min after thrombin stimulation. After a peak induction at 1 h, ET-1 mRNA levels decline out to 4 h. Shown is a summary of three separate experiments for both cell lines.
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Promoter activity analysis. Our data, using actinomycin D to block transcription, indicated that ET-1 mRNA is induced in response to stimuli as a result of de novo transcriptional events. However, this does not prove that ET-1 itself has undergone de novo transcription. We have chosen to employ transient transfection of a promoter fragment of ET-1 fused to the hGH reporter gene to assay for de novo transcription of ET-1. An 8.0-kb fragment of the ET-1 promoter fused to the hGH reporter vector (pØGH) was introduced into both HPA and HMV endothelial cells and assayed for reporter protein expression. Results of this analysis are shown in Fig. 2, A and B. The 5' region of the ET-1 gene possesses de novo transcriptional activity under basal conditions in both cell lines as evidenced by expression of the hGH reporter protein. No secreted hGH was detectable with the vector (pØGH) alone. Additionally, the ET-1 promoter confers increased transcriptional activity in response to thrombin stimulation, since hGH protein levels are induced over control values after stimulation. This response is seen for both HPA (Fig. 2A) and HMV (Fig. 2B) endothelial cells. A metallothionein promoter-driven hGH vector was also transfected as a control for thrombin-dependent changes of growth hormone, and thrombin was shown to have no differential effects compared with control conditions (data not shown).
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Chromatin structure analysis.
To provide evidence of potential binding sites for
trans-acting factors on
cis-acting DNA sequences, ET-1 gene
chromatin structure was evaluated by mapping DNase I-hypersensitive
sites surrounding the gene in HPA endothelial cells. The strategy for
locating these sites involved first scanning the entire gene at low
resolution from both the 5'- and the 3'-end. As illustrated
in Fig.
3A,
genomic DNA of basal and thrombin-stimulated HPA cells was cleaved with Xho I, which liberated a 10-kb
fragment that encompassed the ET-1 gene. Probe
I, which is 378 bp in size and abuts the
Xho I site at the 3'-end of the
gene, was employed to indirectly label this fragment and to scan the
gene from the 3' to 5' perspective. When no DNase I was
added (lane 0), the expected 10-kb
fragment was detected, whereas with increasing concentrations of DNase
I, a very strong hypersensitive site is evident in the promoter region just upstream of transcriptional initiation, with two additional sites
near the 5'-end of exon V. At this level of resolution, there are
no detectable differences in DNase I sensitivity between control and
thrombin-stimulated conditions. Similar analysis of this region from
the 5'-end toward the 3'-end using a probe that abuts the
Xho I site at 2,459 bp showed
identical results.
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Deletion analysis of the ET-1 promoter.
To test the functionality of the putative
cis-regulatory regions identified by
chromatin structure analysis, plasmids containing various
5'-flanking sequences of the ET-1-promoter region were cloned
into the hGH gene reporter vector pØGH. A linear map of each of
the ET-1-promoter deletion fragments relative to the ET-1 gene is shown
in Fig.
4A. As
indicated, these fragments range in size from 8.0 kb to
88 bp of the 5'-flanking region, and all have a common
Bgl II site at the 3'-end that
maps to position +166 bp within exon 1. These constructs were
transiently transfected into HPA and microvascular endothelial cells
utilizing a combination of techniques that include DEAE-dextran with
NuSerum, chloroquine diphosphate, and DMSO to improve transfection
efficiency.
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DISCUSSION |
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This study is the first detailed functional analysis of the human ET-1
promoter under both basal and thrombin-stimulated conditions. Previous
studies of ET-1 gene regulation involving an endothelial cell model
system relied on animal-derived or human umbilical vein endothelial
cells (6, 11, 14, 15, 17-20, 23, 34, 36). However, it is possible
that animal and umbilical vein endothelial cell-based models do not
accurately reflect ET-1 regulation in human systemic or pulmonary
vascular endothelial cells. Numerous examples provide evidence of
differential gene regulation in endothelial cells derived from
different tissues and species (2, 3, 7, 29). In addition, there are a
limited number of reports detailing stimulus-dependent ET-1 gene
regulation. These reports indicate an increase in ET-1-promoter
activity after stimulation with insulin (23), thrombin, interleukin-1,
or transforming growth factor- (19); however, these
analyses were cursory in that single constructs of 4.4 kb (23) and 2.9 kb (19) of the ET-1-promoter region were the only promoter fragments
tested. Furthermore, these studies on the human promoter were performed in bovine aortic endothelial cells.
Thrombin plays an important role in a variety of vascular pathophysiologies, including pulmonary hypertension and acute lung injury (9, 13). Thrombin also has been shown to induce ET-1 expression (11, 19, 36), thus potentially elevating the levels of this potent vasoconstrictor in a variety of vascular diseases. Therefore, we evaluated thrombin-dependent regulation of ET-1 in both HPA and HMV endothelial cells. On the basis of the potency of ET-1, small alterations in ET-1 levels can cause dramatic changes in vascular tone. We therefore feel that understanding the increases in ET-1 expression will provide important physiological information.
In conjunction with our functional studies on the promoter, we have examined the chromatin structure of the ET-1 locus via DNase I treatment of lysolecithin-permeabilized cells. This in situ (24) method of analysis allows the cells to remain intact during DNase I treatment and has been shown to display very efficient DNA replication and transcription activities (5, 21). We observed numerous DNase I-hypersensitive regions in the ET-1 locus, with the majority of these regions concentrated in the 5'-promoter region of the gene. We found no detectable difference in sensitivity to DNase I in basal versus thrombin-stimulated cells, suggesting that the ET-1 gene promoter is a preset promoter in which the promoter is poised for transcriptional activation (12, 33) rather than having an inducible hypersensitive site that would be associated with remodeling promoters.
The functional significance of these hypersensitive sites was
established for both basal and thrombin stimulation of human pulmonary
endothelial cells. These analyses demonstrate that an important element
for basal regulation of ET-1 lies between 378 and
141 bp.
Furthermore, thrombin-stimulated induction minimally required an
additional element between
484 and
378 bp. However, neither of these putative elements was sufficient for full expression of the reporter gene. There was a gradual increase in reporter expression, with increasing sizes of promoter fragment transfected, under both basal and thrombin-stimulated conditions. Full expression was apparent for both conditions with the
952-bp fragment, and larger fragments showed no additional increases in reporter expression.
A composite of the estimated regions of the DNase I-hypersensitive
sites in the ET-1 gene and 5'-flanking region deletion analysis
are shown in Fig. 5. Only those constructs
that contain regions of DNase I hypersensitivity with an increase in
construct size are shown. The putative
cis-regulatory regions closely defined by DNase I correlate with those regions shown to be important for basal and thrombin-stimulated expression of ET-1 in transient transfection assays. It should be noted that no hypersensitive sites
are detected when basal expression is lost (both the 88- and
141-bp constructs). The detection of the first three
hypersensitive regions directly correlates with basal expression found
in the
378-bp construct. Thrombin-inducible expression is first
detected with the
484-bp construct, which does not correlate
with any additional region of hypersensitivity. However, it might be
hypothesized that the 5' most hypersensitive site contained
within the
378-bp construct may in fact be responsible for
thrombin-inducible expression of ET-1. This site may lie too close to
the boundary of the
378-bp construct, thereby interfering with
its function and not allowing the
378-bp construct to confer
thrombin-stimulated expression. Also, the hypersensitive sites are
estimated based on the size of the fragments and size markers on the
gel, with some margin of error. Full basal and thrombin-stimulated
reporter gene expression is only detected with the
952-bp
construct, which contains the next hypersensitive site at approximately
700 bp, suggesting that this site may be responsible and
necessary for conferring full basal and thrombin-stimulated expression
of the ET-1 promoter. Although additional hypersensitive sites become
apparent further upstream, these do not seem to have an effect on
reporter gene expression. These sites may be constitutive sites that
are important for other stimuli.
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The number of hypersensitive regions and the increasing level of
expression with both basal and thrombin stimulation demonstrated in our
deletion analysis of the ET-1-promoter region suggest a complex
regulatory mechanism. We believe that several interacting regions with
a number of possible cis-acting
elements cooperate to allow complete basal and stimulated
transcription. With the requirement of a number of interacting
regulatory regions for full basal and regulated expression and thus the
lack of a clearly defined unique regulatory site, further deletion
analyses will not provide additional information. Generation of
constructs using partial fragments of the ET-1-promoter region will
result in the loss of integrity of the ET-1 promoter and may interrupt
the interactions of these regulatory sites. In fact, we have inserted a
region of the ET-1 promoter from 2.4 kb to
500 bp in a
minimal thymidine kinase promoter-hGH plasmid that resulted in no
reporter expression (Golden, unpublished data). To further
identify the specific regions involved, higher resolution studies that
still maintain the integrity of the gene are necessary. This can be
accomplished by in vivo footprinting of the ET-1 gene promoter region
under basal and thrombin stimulation to identify important protein-DNA
contacts from
952 to
141 bp.
Our results of ET-1 regulation differ from those described in
heterologous systems, in which the human ET-1-promoter regions were
transfected into bovine aortic endothelial cells (17, 34). We obtained
88- and
141-bp ET-1-hGH constructs that were previously used in evaluating basal expression in bovine cells (34). The
88-bp construct resulted in no basal expression in both human and bovine endothelial cells, and the
144-bp construct resulted in basal expression in bovine aortic endothelial cells but not in human
pulmonary arterial endothelial cells. We have performed similar studies
in human aortic endothelial cells and have found them to be similar to
the human pulmonary endothelial cells (data not shown); therefore, the
differences found between our study and the bovine aortic endothelial
cell studies are not due to the difference between aortic and pulmonary
endothelial cells. Also, our studies in human cells and the studies in
bovine cells are remarkably different than what has been observed for
basal human ET-1-promoter activity in COS cells (monkey kidney
epithelial cells) (1). Variable results for basal human ET-1-promoter activity have also been observed in HeLa cells, with the
143- and
141-bp constructs of the promoter region
having no activity and some basal activity, respectively (17, 34), and
the
129-bp region generating no activity (1). In bovine aortic
endothelial cells, the region between
141 and
127 bp of
the ET-1 promoter is required for full basal transcriptional activity,
and a protein-binding motif, essential for basal regulation, lies
within this region between
136 and
131 bp (17, 34). Our
results using a homologous system with the human promoter in human
pulmonary endothelial cells demonstrate that additional regions aside
from the
141-bp promoter region are required for ET-1-promoter
activity. We observed no detectable level of basal expression by ET-1
fusion constructs when the size of the ET-1-promoter fragment was
141 bp or smaller, and this is corroborated by the fact that
there are no resident hypersensitive sites within this region of the
gene. On the basis of our results, we believe that the initial basal
expression requires a region between
378 and
141 bp,
whereas full expression is not apparent until a much larger fragment,
952 bp, is transfected.
Computer analysis of the ET-1-promoter region has identified a number
of elements that may have a regulatory role. For example, several
activator protein-1-like consensus sequences and a
nuclear factor-B sequence have been identified. However, not all of
these sites are located near the hypersensitive sites identified or in
the region demonstrating functional activity. In addition, Scarpati and
DiCorleto (28) previously identified a thrombin response element
(CCACCCACC); however, we were unable to detect this element in the
sequenced region of the ET-1 promoter that includes the
thrombin-dependent regulation, indicating that we have detected a novel
thrombin cis-acting element.
Our studies have provided an analysis of the transcriptional activity of the ET-1 promoter. These data are the first such analyses of the function of the human ET-1 promoter in a homologous system using a tissue type relevant to human disease, specifically, pulmonary disease. It is also the first detailed analysis of stimulus-induced ET-1-promoter function. At present, the transcriptional regulation of the ET-1 gene appears to be a complex system, with the possibility of a number of interacting regulatory sites involved in full basal and thrombin-dependent expression. This may not be surprising, since ET-1 is an extremely potent vasoconstrictor and its expression must be highly regulated to ensure the maintenance of appropriate vascular tone.
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ACKNOWLEDGEMENTS |
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We thank J. Philip Kohler and Sandra Fogg for technical support on this project and Dr. David Wilson for kindly providing a number of the ET-1-hGH constructs.
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FOOTNOTES |
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This study was supported by an American Heart Association of Florida Grant-in-Aid and a National Aeronautics and Space Administration Space Grant Fellow Award to C. L. Golden.
Address for reprint requests: G. A. Visner, 1600 SW Archer Rd., Rm. D2-15, PO Box 100296, Dept. of Pediatrics, Univ. of Florida, Gainesville, FL 32610.
Received 3 April 1997; accepted in final form 12 February 1998.
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