Endothelin-1 (ET-1), a peptide consisting of 21 amino acids, is one of the most powerful vasoconstrictors known.1 It is also a chemoattractant and a mitogen for endothelial cells,2,3 smooth muscle cells, and tumor cells.4,5 Therefore, ET-1 plays a major role in many pathophysiologic conditions, including pulmonary hypertension,6 acute renal failure,7 and cancer.5 Thus, a very tight regulation is needed for ET-1 levels.
ET-1 induces its action by binding to 2 receptors —ETA and ETB, located primarily in smooth muscle and endothelial cells, respectively.8,9 ET-1 binding to ETA receptor leads to vasoconstrictive and proliferative responses. ETB is known to regulate ET-1 plasma levels by the internalization of ET-1 after its binding to the receptor.10,11 Hence, blockade of ETB elevates ET-1 extracellular levels.11
ET-1 is initially synthesized as preproendothelin-1 (PPE-1).12 We hypothesized that because ET-1 receptor blockade modulates ET-1 levels in the extracellular compartment, it may also modulate its intracellular levels, and therefore may also affect PPE-1 transcription, as a feedback regulatory mechanism.
Previous studies demonstrated that ET-1 receptor antagonists elevate the plasma immunoreactive ET-1 (irET-1).11,13 However, there is conflicting data regarding PPE-1 mRNA levels in cultured endothelial cells; whereas Sanchez et al14 found an increase in PPE-1 mRNA levels in response to ETB blockade, others demonstrated the opposite.15,16
In the present study, we investigated the effect of ET-1 receptor blockade on PPE-1 transcription in bovine aortic endothelial cells (BAEC) and in transgenic mice expressing the luciferase reporter gene under the control of PPE-1 promoter.17 PPE-1 transcription was examined in vitro in endothelial cells after treatment with BQ-123 (ETA receptor selective antagonist), BQ-788 (ETB receptor selective antagonist), and Bosentan (a dual ET-1A/B receptor antagonist). PPE-1 transcription was also examined in vivo in transgenic mice that were treated with Bosentan; organs from these mice were tested for luciferase activity, PPE-1 mRNA levels, and plasma irET-1 levels.
BAEC (kindly provided by Professor Nafthali Savion, Goldshlager Institute, Sheba Medical Center, Tel-Hashomer, Israel) were grown in Dulbecco's modified eagle medium with 10% fetal calf serum and incubated at 37°C in 5% CO2-containing humidified atmosphere.
BAEC were transiently transfected with PPE-1 luciferase plasmid17 or with SV40-luciferase reporter plasmid (Promega Gmbh, Manheim, Germany) as a nonselective promoter control. Lipofectamine and Plus reagents (Invitrogen Life Technologies, Carlsbad, CA) were used for transfection. Forty-eight hours after transfection, cells were treated for 1 hour with different concentrations of ET-1 antagonists: Bosentan (Actelion Pharmaceuticals, Allschwil, Switzerland), a dual endothelin receptor antagonist, BQ123 (Sigma-Aldrich, St Louis, MO), endothelin receptor subtype A antagonist, and BQ788 (Sigma-Aldrich, St Louis, MO), endothelin receptor subtype B antagonist.
Luciferase Activity in Cells and Tissues
For assays of β-gal or luciferase activities in cell extracts, cells were lysed in Reporter Lysis Buffer (Promega Gmbh, Manheim, Germany). Luciferase activity in vitro and in vivo was determined as previously described.17 Protein determination of extracts was performed using the BioRad Bradford protein assay (Hercules, CA). Relative luciferase activity was calculated as the ratio of light units to β-galactosidase units, with β-galactosidase activity resulting from cotransfection of the constitutively active lacZ construct in plasmid cytomegalovirus-galactosidase (Promega Gmbh, Manheim, Germany), used as an internal standard for the transfection efficiency.
β-gal was assayed photometrically. The standard assay was performed by adding a diluted extract sample to an assay buffer (final concentrations: 0.1 M sodium phosphate buffer, pH 7.5, 1 mM MgCl2, 45 mM β-mercaptoethanol) containing o-nitrophenyl β-D-galactopyranoside (0.8 mg/mL). The reactions were incubated at 37 °C for 30 minutes, and the absorbance at 405 nm was detected. The readings were corrected using blank values obtained from extracts from mock transfections.
We used transgenic mice expressing firefly luciferase reporter gene under the control of the murine PPE-1 promoter.17 These mice were prepared using cDNA of the luciferase enzyme and crossbred for 8 generations with BALB/c mice (LUC/BALB/c). The transgenic mice expressing luciferase in endothelial cells were housed in the animal care facility. Transgenic mice were fed normal powder chow diet or powder chow diet supplemented with grinded Bosentan tablets (Actelion Ltd, Allschwil, Switzerland). Assuming that 1 animal consumed 4 g chow per day, each mouse was administered with 100 mg Bosentan/kg body weight for 30 days. Animals were killed and tested for luciferase activity in several organs, lung ET-1 mRNA levels, and plasma irET-1 levels.
All animal procedures were in accordance with the guidelines of the Animal Care and Use Committee at the Sheba Medical Center.
Semiquantitative PCR Analysis of PPE-1 mRNA
Lungs were frozen in liquid nitrogen. Total RNA from lungs was isolated with the RNeasy Mini Kit (QIAGEN GmbH, Hilden, Germany). One microgram of total RNA from each tissue sample was reverse transcribed into cDNA. Using the Titan One Tube RT-PCR Kit (Roche Diagnostics GmbH, Mannheim, Germany), this RNA was then amplified with specific primers for PPE-1 or β-actin as a control. The reaction mixture was prepared according to the kit protocol and contained either the forward primer 5′-CCAAGGCCAACCGCGAGAAGATGAC-3′ and the reverse primer 5′-AGGGTACATGGTGGTGCCGCCAGAC-3′ for β-actin, or the forward primer 5′-CTTCCCAATAAGGCCACAGAC-3′ and the reverse primer 5′-AGCCACACAGATGGTCTTGCTAAG-3′ for PPE-1. The reverse transcriptase-polymerase chain reaction conditions were as instructed by the manufacturer's protocol. The resulting DNA fragments were analyzed by gel electrophoresis; the expected product was 470 or 587 bp for PPE-1 or â-actin, respectively.
The irEndothelin-1 (1 to 21) levels in plasma were measured by an ELISA kit from BIOMEDICA (Graz, Austria).
Results are expressed as mean±SEM. Statistical differences between means were determined by Student t test.
Bosentan, a Dual ET-1A/B Receptor Antagonist, and BQ788, ETB Selective Antagonist, Increased Preproedothelin-1 Promoter Activity in Endothelial Cells
To test the effect of ET-1 receptor blockade on PPE-1 transcription, BAEC were transfected with luciferase expression plasmids under the control of PPE-1 promoter or SV40 promoter, followed by 1-hour treatment with ET-1 receptor antagonists.
A significant increase in luciferase activity under the control of PPE-1 promoter was measured with Bosentan and BQ788 treatment but not with BQ123 (ET-1 receptor subtype A antagonist). Bosentan and BQ788 treatment (at 1 μM) resulted in a 1.6-fold and 1.3-fold increase, respectively, in luciferase activity compared with untreated control (Fig. 1). No significant increase was observed in the SV40-luciferase transfected cells (data not shown).
Bosentan, a Dual ET-1A/B Receptor Antagonist, Increased ET-1 Expression in Transgenic Mice
Luciferase activity, lung PPE-1 mRNA levels, and plasma irET-1 levels were tested in transgenic mice expressing firefly luciferase reporter gene under the control of the murine PPE-1 promoter to further study the regulatory mechanism of PPE-1 transcription by ET-1 receptor antagonists in vivo. Bosentan treatment for 30 days resulted in a 1.6-fold to 2-fold increase in luciferase levels (Fig. 2A) in several tissues (heart, lungs, kidneys, and brain) and in ET-1 lung mRNA levels (Fig. 2B), compared with the control group. Lung mRNA levels were tested, because ET-1 is known to be involved in the lung's circulation and blood pressure homeostasis.18
Plasma irET-1 levels were increased by 2.7-fold in mice treated with Bosentan (Fig. 2C), in agreement with previous studies.11,13
In the present study, we demonstrated that the ET-1 receptor blockade in endothelial cells induces an increase in PPE-1 promoter activity. This increase can be attributed to the ETB receptor blockade, as both Bosentan and BQ-788 increased transcription by the PPE-1 promoter, and BQ-123 had no effect. We also demonstrated an increase in PPE-1 promoter activity, PPE-1 mRNA levels, and irET-1 plasma levels in transgenic mice expressing the luciferase reporter gene under the control of PPE-1 promoter, when these mice were treated with Bosentan for 30 days.
Although previous studies demonstrated an increase in ET-1 extracellular levels after treatment with ET-1 receptor antagonists,11,13 the effect of ET-1 and its receptor antagonist administration on PPE-1 transcription is controversial. Previous studies demonstrated an increase in PPE-1 mRNA levels16 or ET-1 protein synthesis15 in endothelial cells treated with ET-1, and the prevention of this increase with a dual ET-1A/B antagonist and not with BQ123.16 In contrast, Sanchez et al.14 demonstrated that ET-1 treatment resulted in a reduction in PPE-1 mRNA synthesis and that ETB blockade by BQ788 increases preproedothelin-1 mRNA levels, with BQ123 having no effect. The difference between these studies may be explained either by the different endothelial cell-lines used, rat endothelial cells16 versus porcine aortic endothelial cells,14 or by the different incubation times with ET-1, 4 hours16 versus 24 hours,14 respectively. However, both studies used northern blot analysis to test the effect of ET-1 and its receptor antagonists on PPE-1 transcription.14,16 In our work, semiquantitative PCR analysis was used to demonstrate the difference in PPE-1 mRNA. To test whether the different mRNA levels are due to enhanced transcription, luciferase assay was performed. This assay also provides a quantitative validation for the PCR results.
Our results demonstrate, in accordance with Sanchez et al,14 that the increased PPE-1 mRNA levels in response to ETB blockade are in association with increased PPE-1 promoter activity, implying for enhanced transcription in response to ET-1 receptor blockade. Moreover, we have demonstrated for the first time that this increase can be detected both in vitro and in vivo, for short-term and long-term period treatments, respectively.
These results may suggest a feedback regulation mechanism – ET-1 reduces its own transcription after its internalization by binding to ETB receptors on endothelial cells. Intracellular levels of ET-1 may also be important for cell signaling, as it was recently found that the nuclear membrane of several cell-lines contains functional ET-1 receptors.19,20
As demonstrated in the present study, PPE-1 mRNA levels correlated with luciferase activity in transgenic mice expressing the luciferase reporter gene under the control of PPE-1 promoter. Thus, this model could be used as a basis for evaluation of ET-1 expression in different pathophysiologic states, including hypertension, cancer, and acute renal failure.
In the present study, we demonstrated that ETB receptor blockade in endothelial cells induces an increase in PPE-1 promoter activity, in vitro and in vivo, and that ETA receptor blockade has no effect on the promoter activity. This difference might have consequences on the treatment of cardiovascular diseases. For example, it was previously demonstrated that rebound pulmonary hypertension after withdrawal of nitric oxide therapy is induced by elevated levels of plasma ET-1.21 Therefore, usage of an ETA antagonist, in which there is no increase in PPE-1 transcription, might prevent rebound pulmonary hypertension. This should further be evaluated in the clinical setting.
In summary, we suggest that treatment with selective ETA antagonist, instead of a nonselective ETA/B antagonist, may avoid the increase in plasma ET-1 levels, thus preventing the vasoconstrictive effect and other undesired effects that might be induced by the increased ET-1.
1. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 1988;332:411–415.
2. Morbidelli L, Orlando C, Maggi CA, et al. Proliferation and migration of endothelial cells is promoted by endothelins via activation of ETB receptors. Am J Physiol. 1995;269:H686–H695.
3. Noiri E, Hu Y, Bahou WF, et al. Permissive role of nitric oxide in endothelin-induced migration of endothelial cells. J Biol Chem. 1997;272:1747–1752.
4. Alberts GF, Peifley KA, Johns A, et al. Constitutive endothelin-1 overexpression promotes smooth muscle cell proliferation via an external autocrine loop. J Biol Chem. 1994;269:10112–10118.
5. Bagnato A, Tecce R, Moretti C, et al. Autocrine actions of endothelin-1 as a growth factor in human ovarian carcinoma cells. Clin Cancer Res. 1995;1:1059–1066.
6. Schiffrin EL. Role of endothelin-1 in hypertension and vascular disease. Am J Hypertens. 2001;14:S83–S89.
7. Yanagisawa H, Nodera M, Umemori Y, et al. Role of angiotensin II, endothelin-1, and nitric oxide in HgCl2
-induced acute renal failure. Toxicol Appl Pharmacol. 1998;152:315–326.
8. Arai H, Hori S, Aramori I, et al. Cloning and expression of a cDNA encoding an endothelin receptor. Nature. 1990;348:730–732.
9. Sakurai T, Yanagisawa M, Takuwa Y, et al. Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor. Nature. 1990;348:732–735.
10. Dupuis J, Stewart DJ, Cernacek P, et al. Human pulmonary circulation is an important site for both clearance and production of endothelin-1. Circulation. 1996;94:1578–1584.
11. Ozaki S, Ohwaki K, Ihara M, et al. ETB-mediated regulation of extracellular levels of endothelin-1 in cultured human endothelial cells. Biochem Biophys Res Commun. 1995;209:483–489.
12. Barnes K, Turner AJ. The endothelin system and endothelin-converting enzyme in the brain: molecular and cellular studies. Neurochem Res. 1997;22:1033–1040.
13. Opgenorth TJ, Wessale JL, Dixon DB, et al. Effects of endothelin receptor antagonists on the plasma immunoreactive endothelin-1 level. J Cardiovasc Pharmacol. 2000;36:S292–S296.
14. Sanchez R, MacKenzie A, Farhat N, et al. Endothelin B receptor-mediated regulation of endothelin-1 content and release in cultured porcine aorta endothelial cell. J Cardiovasc Pharmacol. 2002;39:652–659.
15. Saijonmaa O, Nyman T, Fyhrquist F. Endothelin-1 stimulates its own synthesis in human endothelial cells. Biochem Biophys Res Commun. 1992;188:286–291.
16. Saito S, Hirata Y, Imai T, et al. Autocrine regulation of the endothelin-1 gene in rat endothelial cells. J Cardiovasc Pharmacol. 1995;26(Suppl 3):S84–S87.
17. Harats D, Kurihara H, Belloni P, et al. Targeting gene expression to the vascular wall in transgenic mice using the murine preproendothelin-1 promoter. J Clin Invest. 1995;95:1335–1344.
18. Rubin LJ, Badesch DB, Barst RJ, et al. Bosentan therapy for pulmonary arterial hypertension. N Engl J Med. 2002;346:896–903.
19. Bkaily G, Choufani S, Hassan G, et al. Presence of functional endothelin-1 receptors in nuclear membranes of human aortic vascular smooth muscle cells. J Cardiovasc Pharmacol. 2000;36:S414–S417.
20. Boivin B, Chevalier D, Villeneuve LR, et al. Functional endothelin receptors are present on nuclei in cardiac ventricular myocytes. J Biol Chem. 2003;278:29153–29163.
21. McMullan DM, Janine MB, Johengen MJ, et al. Inhaled nitric oxide-induced rebound pulmonary hypertension: role for endothelin-1. Am J Physiol Heart Circ Physiol. 2001;280:H777–H785.