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(Hypertension. 1995;25:683-687.)
© 1995 American Heart Association, Inc.

Articles

Characterization and Functional Analysis of the Rat Endothelin-1 Promoter

Martin Paul; Martin Zintz; Wolfgang Böcker; Mark Dyer

From the Max-Delbrück Center for Molecular Medicine and University Hospital Benjamin Franklin, Free University, Berlin, Germany; and Sandoz Ltd, Basel, Switzerland (M.D.).

Correspondence to Martin Paul, MD, Free University Berlin, University Hospital Benjamin Franklin, Department of Clinical Pharmacology, Hindenburgdamm 30, D-12200 Berlin, FRG. E-mail paul@ovid.uks.fu-berlin.de.

	Abstract

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Abstract To define the molecular mechanisms of endothelin-1 (ET-1) gene regulation, we cloned, sequenced, and characterized the rat ET-1 promoter. A sequence consisting of the first 1329 bp of the rat ET-1 promoter was investigated in greater detail. Sequence analysis identified putative binding sites for a number of transcriptional factors that may be involved in ET-1 gene regulation. Several of these factors have been proposed earlier to be involved in cell-specific gene regulation and may be responsible for directing ET-1 expression in vivo. For functional analysis of the ET-1 promoter, we generated a reporter gene construct using luciferase as reporter gene under control of the promoter fragment isolated. The construct was transfected transiently into bovine aortic endothelial cells, and luciferase expression was evaluated. The results indicated that the promoter segment used showed high expression in endothelial cells comparable to that induced by viral promoters. Since ET-1 is regulated by a number of vasoactive substances, we studied the effect of angiotensin II on endothelin transcription. We could demonstrate a dose-dependent transcriptional activation of ET-1 transcription by angiotensin.

Key Words: cloning, molecular • transcription factors • angiotensin II • gene expression regulation • luciferase • endothelium

	Introduction

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Endothelin-1 (ET-1) is one of the strongest in vivo vasoconstrictors known.^{1 2 3} Although the ultimate physiological and pathophysiological role of the peptide is unknown, it has been implicated in cardiovascular disorders such as hypertension, atherosclerosis, and ischemic conditions.^{4 5 6 7 8} Previous studies have suggested that the peptide acts predominantly as a paracrine-autocrine substance^{9 10 11} and can be tightly regulated at the tissue level by a number of factors such as shear stress,¹² thrombin,^{13 14} and vasopressin and angiotensin II (Ang II).^{9 15} Endothelin regulation by these substances has been demonstrated at the peptide and mRNA levels, but little is known regarding the transcriptional mechanisms involved. Previous studies have focused predominantly on the characterization of the basal mechanisms of endothelin transcription, for example, by defining the sequences responsible for the endothelial cell–specific expression of the human endothelin gene^{16 17} and by drawing attention to the role of the transcription factor GATA-2¹⁸ in endothelin gene expression. To address the transcriptional mechanisms of rat endothelin regulation by other vasoactive peptides, we cloned, sequenced, and characterized the rat ET-1 promoter and identified several consensus sequences that are putatively involved in endothelin gene regulation. In a second group of experiments, we functionally characterized the promoter and specifically studied the transcriptional mechanisms of endothelin regulation by Ang II in transfected endothelial cells. Our results indicate that Ang II has direct effects on ET-1 transcription, which may be relevant for the interaction between the two vasoactive systems at the cellular and molecular levels.

	Methods

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Cloning of the Rat ET-1 Promoter
We cloned the rat ET-1 promoter region from a rat genomic library (Clonetech RL1022) using a ³²P-labeled DNA probe consisting of a 574-bp HindIII fragment of the human ET-1 promoter region that had been previously cloned by M.D. (unpublished work, 1993). Positive plaques were further purified and their DNA isolated and subjected to Southern blot analysis. A 2.0-kb Sac I promoter fragment containing the 3' part of the promoter region was cloned into a pT7T3 18U vector (Pharmacia). The entire insert was sequenced and further analyzed. A HindIII–BglII fragment of the rat endothelin promoter, consisting of DNA spanning from position -1329 on the promoter sequence to position +166 on the coding region, was cloned into the Sma I site of the plasmid pdLux (kindly supplied by M. Bader, MDC, Berlin, FRG). The resulting vector, pdLux/pret-1, was used for transfection of bovine aortic endothelial cells (BAECs). Sequence analysis was performed by the didesoxy method using Sequenase II (USB).

Cell Culture
Bovine aortas were obtained at a local slaughterhouse. The aortic lumen was rinsed once in phosphate-buffered saline (PBS) to remove blood and immediately processed with the use of sterile procedures. The aortic arches were opened lengthwise with a scalpel, and the intimal surface was washed with Ca²⁺-free PBS and incubated with dispase (4 mg/mL) and glucose (2 mg/mL) for 45 minutes in 80 mL HBS solution (1.7 mol/L NaCl, 125 mmol/L HEPES, 50 mmol/L KCl, pH 7.4) The endothelial cell layer was removed with a sterile cotton swab. The endothelial cells were trapped in the cotton fibers by rolling the swab with gentle pressure along the endothelium. The swab was then swirled in culture medium to liberate the cells. Cells were centrifuged at 800 rpm for 10 minutes, resuspended, and seeded into Falcon dishes coated with collagen A (0.1% in PBS, Biochrom KG/Berlin) in Dulbecco's modified Eagle's medium (DMEM) with high glucose (4.5 g/L), 10% fetal calf serum (GIBCO), 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin and incubated at 37°C in a 95% air/5% CO₂ humidified atmosphere. These cells were identified by their typical "cobblestone" appearance on phase-contrast microscopy and by their immunofluorescence after staining for factor VIII antigen. Endothelial cells harvested between passages 3 and 8 were used in all experiments.

Transfection and Luciferase Assay
BAECs at 40% to 60% confluence were transfected with 5 µg pdLux/pret-1 or the expression vector pCMVluc (Clonetech) with the use of the CaPO₄ coprecipitation method.¹⁹ As an internal control for transfection efficiency, 3 µg of pCH110 containing lacZ, under the control of the SV40 early gene promoter, was added to 237.5 µL precipitation mixture containing (mmol/L) HEPES-NaOH 20, pH 7.1, NaCl 135, KCl 10, Na₂HPO₄ 1.5, and sucrose 6. Then, 12.5 µL of 2.5 mol/L CaCl₂ was added; the mixture was incubated at room temperature for 20 minutes and transferred directly into 3 mL of tissue culture medium that had been replaced 2 hours before. After 4 hours of incubation at 37°C in a 5% CO₂ atmosphere, the cells were subjected to a glycerol shock. The medium was removed, and the cells were washed twice with PBS and incubated at room temperature for 2 minutes in 15% glycerol in PBS. The cells were washed again with PBS and supplied with 3 mL fresh culture medium. Forty-eight hours later, the medium was removed, and the cells were washed twice with PBS. Cells were harvested after 5 minutes of incubation in 1 mL TEN (40 mmol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA, 150 mmol/L NaCl). The cells were pelleted at low speed, resuspended in 100 µL of 250 mmol/L Tris-HCl, pH 7.5, and 5 mmol/L EDTA and lysed by vortexing and three freeze/thaw steps. Cell debris was removed by centrifugation at 15 000 rpm for 10 minutes at 4°C. Luminescence assays were used to measure luciferase and lacZ activity in the supernatant. Luciferase was measured according to de Wet et al.²⁰ Fifty microliters of cell extract was mixed with 50 µL of 2x GG buffer (50 mmol/L glycylglycine-HCl, pH 7.8, 30 mmol/L MgSO₄) and 300 µL of 10 mmol/L ATP, pH 7.2. This mixture was placed into the injection chamber of a Berthold LB953 luminometer and injected with 300 µL of 0.3 mmol/L luciferin (Serva) in 2x GG buffer. Light emission was detected and integrated over a 10-second interval. LacZ activity was measured by addition of 300 µL of 20 µg/mL galacton (Tropix) in 100 mmol/L NaH₂PO₄, pH 7.8, to 50 µL cell extract and incubation of the mixture for 1 hour at room temperature in the dark. The mixture was then placed in the injection chamber of the luminometer and injected with 300 µL of 1 mol/L NaOH, and light emission was integrated over a 10-second interval. The values for luciferase activity were divided by the lacZ activity and statistically analyzed.

Stimulation Experiments With Ang II
Preconfluent cultures of BAECs in tissue culture dishes (30 mm) were transfected with the plasmid pdLux/pret-1 48 hours before use. The culture medium was removed, and the cell monolayers were washed twice with Ca²⁺-free PBS and supplied with 3 mL serum-free DMEM for 2 hours. Human Ang II (Bachem) was then added to the replacement medium in concentrations between 10^-6 and 10^-8 mol/L, and both the control and treated cultures were incubated for 3 hours. After incubation medium was aspirated, the attached cells were collected for luciferase assay and for extraction of total RNA. In separate experiments, endothelial cells of the same origin and passage were stimulated with Ang II (10^-7 mol/L) as above and collected for RNA extraction.

Measurement of Endothelin mRNA
The medium of a confluent BAEC monolayer grown in 100-mm culture flasks was replaced with 10 mL fresh serum-free DMEM and preincubated for 12 hours. Ang II was added and incubated with BAECs for 1 hour. Cells were harvested as described above, and total RNA was prepared based on the method described by Wilkinson.²¹ Twenty micrograms of total RNA was loaded on a 1% agarose gel, separated by gel electrophoresis, and blotted onto nylon membranes (Pall) with the use of standard methodology. Membranes were hybridized to a full-length probe of the rat ET-1 cDNA (kindly provided by Dr M. Yanagisawa, Dallas, Tex) as described.¹² Hybridization signals were analyzed with a computer-based scanning system (Fujix Bas 200, Fuji).

Statistical Analysis
Data were analyzed using Student's t test for unpaired samples. Statistical significance was accepted at a value of P<.05.

	Results

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The rat ET-1 promoter region was cloned by screening a rat genomic library. In total, 3x10⁶ phages were screened with the use of a human ET-1 promoter fragment. Two hybridizing phages were detected, purified, and further characterized. The two phages contained overlapping segments of the rat genome as was determined by comparative Southern blotting with rat genomic DNA (data not shown). Both phage sequences contained a Sac I fragment, which contained the promoter region directly adjacent to the transcriptional start site and which was also present in genomic rat DNA hybridized with the endothelin promoter probe. The fragments from both phages were gel-purified and subcloned into a vector as described above. The complete inserts were sequenced and found to be identical. The complete promoter sequence of the Sac I fragment is shown in Fig 1. The position (5) of the TATAA box and consensus sequences for the putative cis-acting regions are indicated. Comparison of the rat promoter with the human promoter sequence revealed an 85% similarity with the DNA sequences investigated.¹⁶ The promoter region contained a TATAA box at positions -32 to -28 of the coding sequence. Computer-based sequence analysis revealed the presence of a number of consensus sequences for cis-acting factors such as AP-1 (-109 to -102), two GATA-2 sequences (-135 to -131 and -910 to -905), a GHF-1 consensus sequence (-1289 to -1283), and a putative calcium-responsive element (CaRe) (-599 to -590). These sequences may be involved in directing cell-specific expression and regulating rat endothelin.

View larger version (51K): [in this window] [in a new window]   Figure 1. Sequence of the rat endothelin-1 promoter, including the sequence of the BglII–HindIII fragment used for functional analysis. Arrows indicate the locations of consensus sequences for putative binding sites of transcription factors GHF-1, LBP-1, GATA, calcium-responsive element (CaRE), TF-IIIA, and AP-1. The transcriptional start site is found at position +1. The TATAA box extends from position -32 to -28.

For functional analysis, a rat ET-1 promoter fragment, extending from the HindIII site (position -1329 on the sequence) to a BglII site (position +166) of the sequence, was cloned into an expression vector containing the luciferase cDNA that was used as a reporter sequence. The 3' end of the fragment was located between the transcriptional start site and the initiation codon of the ET-1 gene sequence. Primer extension analysis of the transcript originating from this chimeric construct after transfection into BAECs revealed transcript initiation at the predicted position (+1) of the cloned sequence (data not shown).

Functional studies were carried out after the rET-1/luciferase construct was transfected into BAECs with the use of the calcium phosphate method. Measurement of luciferase activity in transfected and nontransfected cells showed that the ET-1 promoter induced transcription levels of approximately 40% of the expression levels induced by a cytomegaly (CMV) promoter–driven luciferase construct transfected in parallel experiments (Fig 2).

View larger version (33K): [in this window] [in a new window]   Figure 2. Bar graph shows relative promoter activity of the rat endothelin-1 (ET-1) promoter after transfection of the chimeric construct into bovine aortic endothelial cells. For this experiment, we compared the expression of the rat ET-1 promoter to that of the cytomegaly (CMV) promoter (n=3 for each experiment). *P<.000001 between both constructs and untransfected controls; #P<.001 between the construct driven by the endothelin and CMV promoters.

To investigate the effects of Ang II on endothelin transcription, we first treated nontransfected BAECs with the peptide and determined steady-state endothelin mRNA concentrations by Northern blotting. Results showed that Ang II at a concentration of 10^-7 mol/L led to a visible increase in ET-1 mRNA levels (Fig 3). In separate experiments, BAECs transfected with the chimeric promoter construct were treated with Ang II concentrations between 10^-6 and 10^-8 mol/L (n=5 for each experiment), and luciferase activity was determined. Results indicated that Ang II has a dose-dependent stimulatory effect on endothelin transcription (35%) that was significant at all doses used (P<.05). The maximal increase was detected at a concentration of 10^-6 mol/L with a stimulation of approximately 30% (Fig 4).

View larger version (20K): [in this window] [in a new window]   Figure 3. Northern blot analysis shows stimulation of endothelin mRNA in bovine aortic endothelial cells by angiotensin II (10-7 mol/L). Equal loading of the electrophoresis gel before blotting was verified by assessment of ribosomal RNA bands. Blots were hybridized with a rat endothelin-1 cDNA fragment that cross-hybridizes with bovine endothelin-1. Note the difference between unstimulated (C) and stimulated cells.

View larger version (46K): [in this window] [in a new window]   Figure 4. Bar graph shows regulation of endothelin-1 promoter by angiotensin II (ANG II). Transfected bovine aortic endothelial cells were incubated with various concentrations of the peptide. Results indicate a direct transcriptional effect of angiotensin II on endothelin-1 transcription (n=5 for each experiment). Endothelin-1 transcription was significantly enhanced (*P<.05) over unstimulated cells at each of the doses used.

	Discussion

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We report on the cloning, analysis, and functional characterization of the rat ET-1 promoter. A 1329-bp segment of the promoter has been studied in greater detail. This fragment contained a single TATAA box, as well as the transcriptional initiation site of the endothelin sequence, and showed high similarity to the human ET-1 promoter sequence. Previous studies of the human gene promoter have shown that DNA sequences 143 bp upstream from the transcription start site are sufficient for endothelial cell–specific expression.¹⁶ The highly conserved rat promoter is likely to function in a similar fashion. Sequence analysis of the promoter revealed several consensus sequences for putative cis/trans interactions in the regulation of endothelin transcription.

Similar to various other eukaryotic genes, the endothelin gene can be induced by phorbol esters. This induction is mediated via a complex of proto-oncogene products named jun and fos, which bind to the corresponding cis-acting element called "TPA-responsive element" (TPA, 12-O-tetradecanoylphorbol-13-acetate) or "AP-1/jun–binding element." Indeed, it has been shown previously that endothelin can be regulated by fos and jun complexes.²² We identified an AP-1 binding site within the rat ET-1 promoter, located between -102 and -109, that contains the consensus sequence GTGACTAA. This binding site matches with the AP-1 octapeptide consensus sequence ^C/GTGACT^C/AA.

The rat ET-1 promoter also contains the cis-acting sequence TACA or GATA at positions -135 and -910, which have been found to bind the transcriptional factor GATA-2 (formerly NF-E1b). GATA-2 belongs to the zinc finger DNA-binding proteins, a family of transcriptional activators that recognize the GATA core sequence. Among the GATA binding proteins, the amino acid sequences binding to the corresponding DNA are highly conserved. Two other transcriptional factors have been identified to be highly similar to GATA-2. Both, GATA-1 and GATA-3, have been implicated in cell-specific transcriptional regulation. Exonuclease footprinting, transactivation studies, and treatment with retinoic acid clearly demonstrated that GATA-2 binds to GATA consensus sequence in the human ET-1 promoter and stimulates its transcription.¹⁸

GHF-1 expression is normally restricted to GH-, prolactin-, and thyroid stimulating hormone–expressing cells. In normal pituitary cells, the pituitary specific transcriptional factor Pit-1/GHF-1 is responsible for tissue-specific gene expression of prolactin and GH and therefore regulates differentiation and proliferation. Future studies will be directed toward clarifying the function of the identified cis-acting sequences and their importance for endothelin gene regulation.

It is interesting to note that the rat ET-1 promoter also contains a consensus sequence for a calcium-responsive element (CaRE) that may be involved in endothelin regulation as described.^{9 14 23}

Functional analysis of the rat endothelin promoter was carried out after transfection of the promoter/luciferase construct into BAECs. Results indicate that the sequence conferred strong transcriptional activity in this cell line. To study the regulation of the promoter, we chose Ang II as a stimulus. The observation that potent endogenous vasoconstrictors induce endothelin in endothelial cells is consistent with the idea that the vascular endothelium is a transducer within the vessel wall. Ang II, like vasopressin and epinephrine, significantly increases ET-1 mRNA production.^{3 15 24 25 26} Since previous studies²⁷ have demonstrated that BAECs express both subtype 1 and subtype 2 angiotensin receptors and since endothelins are considered to have low species specificity, we used these cells for studying the effects of Ang II on rat ET-1 transcription.

Ang II stimulated the expression of the native ET-1 mRNA as well as of the chimeric promoter/luciferase construct in BAECs. There was an obvious discrepancy in the degree of activation, which was considerably stronger in the Northern blot experiments compared with the reporter gene assays (transfected cells). This could be explained by the fact that the reporter gene studies measure the direct transcriptional effects of Ang II on the ET-1 promoter, whereas the steady-state mRNA measurements are a reflection of both synthesis and degradation of ET-1 mRNA. It is therefore conceivable that Ang II also affects endothelin mRNA stability. Although the responsible molecular mechanisms are unknown at present, it may be possible that the effects of Ang II on its receptor(s) induce the binding of fos and jun complexes via the described second-messenger pathways to the AP-1 site on the endothelin promoter sequence. Previous experiments have suggested such a mechanism for Ang II action via the AP-1 site in transfected hepatoma cells.²⁸ Alternatively, it could be possible that Ang II binds directly to the promoter sequence, as has been hypothesized previously.²⁸ We will address these issues in future experiments by studying the cis/trans interactions of endothelin regulation in greater detail. The use of specific Ang II receptor antagonists will be useful to characterize the specificity of the endothelin response.

Our findings suggest that the regulation of vasoactive factors may be closely linked at the molecular and cellular levels. This is apparently true not only for the stimulation of endothelin by Ang II^{24 25 26} but also vice versa. In this context, a previous study has suggested a reciprocal mechanism by demonstrating that endothelin can stimulate the activity of angiotensin-converting enzyme, resulting in an enhanced formation of Ang II.²⁹ Such interactions may contribute to the overall regulation of vascular tone and could be relevant for the pathophysiology of cardiovascular disease.

	Acknowledgments

 
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG PA 332-1). The technical help of Heike Marquardt and Heide Kistel is gratefully acknowledged. We would like to thank Dr Chris E. Talsness for critically reading the manuscript.

	References

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