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Cardiovascular Research 2004 61(1):159-168; doi:10.1016/j.cardiores.2003.10.019
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Angiotensin II induces endothelin-1 gene expression via extracellular signal-regulated kinase pathway in rat aortic smooth muscle cells

Hong-Jye Honga, Paul Chanb, Ju-Chi Liub, Shu-Hui Juanb, Meng-Ting Huangb, Jaung-Geng Linc and Tzu-Hurng Cheng*,b,d

aSchool of Chinese Medicine, China Medical University, Taichung, Taiwan, ROC
bDepartment of Medicine and Clinical Research Center, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan, ROC
cInstitute of Chinese Medical Science, China Medical University, Taichung, Taiwan, ROC
dDepartment of Pharmacology, National Defense Medical Center, Taipei, Taiwan, ROC

* Corresponding author. Department of Medicine, Taipei Medical University-Wan Fang Hospital, Taipei 115, Taiwan, ROC. Tel.: +886-2-27899135; fax: +886-2-29383273. thcheng{at}gate.sinica.edu.tw

Received 9 June 2003; revised 12 October 2003; accepted 23 October 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Angiotensin II (Ang II) increases vascular endothelin-1 (ET-1) tissue levels, which in turn mediate a major part of Ang II-stimulated vascular growth and hypertension in vivo. Ang II also stimulates reactive oxygen species (ROS) generation in vascular smooth muscle cells (SMCs). However, whether ROS are involved in Ang II-induced ET-1 gene expression and the related intracellular mechanisms in vascular SMCs remains to be determined. Methods: Cultured rat aortic SMCs were stimulated with Ang II, [3H]thymidine incorporation and the ET-1 gene expression was examined. Antioxidants pretreatment on Ang II-induced extracellular signal-regulated kinase (ERK) phosphorylation were performed to elucidate the redox-sensitive pathway in proliferation and ET-1 gene expression. Results: Ang II-increased DNA synthesis was inhibited by AT1 receptor antagonist (olmesartan) and ETA receptor antagonist (BQ485). ET-1 gene was induced with Ang II as revealed by Northern blotting and promoter activity assay. Ang II-increased intracellular ROS levels were inhibited by olmesartan and antioxidants. Antioxidants suppressed Ang II-induced ET-1 gene expression and ERK phosphorylation. An ERK inhibitor U0126 fully inhibited Ang II-induced ET-1 expression. Co-transfection of dominant negative mutant of Ras, Raf and MEK1 attenuated the Ang II-increased ET-1 promoter activity, suggesting that the Ras-Raf-ERK pathway is required for Ang II-induced ET-1 gene. Truncation and mutational analysis of the ET-1 gene promoter showed that activator protein-1 (AP-1) binding site was an important cis-element in Ang II-induced ET-1 gene expression. Moreover, Ang II- or H2O2-induced AP-1 reporter activities were also inhibited by antioxidants. Conclusions: Our data suggest that ROS are involved in Ang II-induced proliferation and the redox-sensitive ERK pathway plays a role in ET-1 gene expression in rat aortic SMCs.

KEYWORDS Angiotensin II; Endothelin-1; Reactive oxygen species; Extracellular signal-regulated kinase; Smooth muscle cells

Abbreviations: Ang II, angiotensin II • ET-1, endothelin-1 • AT1, Ang II type 1 receptor • ETA, ET-1 type A receptor • CAT, chloramphenicol acetyltransferase • MAPKs, mitogen-activated protein kinases • ERK, extracellular signal-regulated kinase • JNK, c-Jun N-terminal kinase • MEK, MAPK/ERK kinase • SMCs, smooth muscle cells


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Vascular smooth muscle cell (SMC) proliferation is a key event in the pathogenesis of arteriosclerosis and many other vascular diseases [1,2]. There is increasing evidence that the renin–angiotensin system, which, may contribute to the pathogenesis of chronic vascular disease. Angiotensin II (Ang II), an important component of the renin–angiotensin system and a vasoactive peptide [3], is also a known growth factor for vascular SMCs both in vivo and in vitro [4,5]. Literatures indicate that Ang II can turn on the synthesis of the very potent vasoconstrictor peptide endothelin-1 (ET-1) in different vascular cell types, including cultured vascular SMCs [5–7]. ET-1 was shown to mediate the growth-promoting effect of Ang II and thus play an important role in cardiovascular disease and vascular remodeling [5–7] (for review, see Ref. [8]). Although the significance of Ang II-induced ET-1 gene expression in vascular tissues is determined [5,6], the regulatory mechanism of ET-1 gene induction by Ang II in vascular SMCs remains unclear.

Recent reports have shown that Ang II stimulates membrane-bound NAD(P)H oxidase, which generates reactive oxygen species (ROS) in vascular SMCs [9]. Excess ROS generation is considered to be a likely initiator of atherosclerotic events, causing the increased synthesis of numerous mitogenic factors that contribute to the hyperproliferation of SMCs and vascular plaque formation [10]. The potential role of ROS in the regulation of signal transduction and gene expression in the cardiovascular system has recently been elucidated [11,12]. Reports have indicated that ROS are capable of stimulating vascular SMC proliferation [13]. ROS may also act as second messengers that regulate various intracellular signal transduction cascades and the activity of various transcription factors such as activator protein-1 (AP-1) [14,15]. We recently reported that ROS mediates ET-1 gene induction in cardiac fibroblasts and vascular endothelial cells [15,16]. However, the role of ROS in the Ang II-induced proliferation and the ET-1 gene induction in vascular SMCs still remains to be clarified.

The aim of the present study was to clarify the regulation of ET-1 by Ang II in vascular SMCs and their role in SMC proliferation. We further determined the intracellular signal transduction pathways focusing especially on ROS-mediated signaling and transcription factors involved in this process. This study shows that ROS are essential for Ang II-induced proliferation and ET-1 gene expression in vascular SMCs. Our results further indicate that redox-sensitive transcription factor AP-1 play a role in Ang II-induced ET-1 gene expression and Ang II up-regulates the ET-1 gene at least in part via Ras/Raf/extracellular signal-regulated kinase (ERK) signaling pathway in vascular SMCs.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Materials
Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, and tissue culture reagents were from Life Technologies. A rat ET-1 cDNA probe (Accession No. M64711 [GenBank] ) was obtained as previously described [17]. A series of deletion mutants containing various lengths of the ET-1 promoter region fused to chloramphenicol acetyltransferase (CAT) reporter gene and the catalytically inactive mutant of ERK2 (mERK2), RasN17, RasL61 and Raf301 were previously described [16]. The ECL detection system was from Amersham Pharmacia Biotech. U0126 was obtained from Tocris Cookson (Bristol, UK). Olmesartan was provided by Sankyo (Tokyo, Japan). The plasmid AP-1-Luc containing the firefly luciferase reporter gene driven by a basic promoter element (TATA box) joined to tandem repeats of AP-1 binding element were obtained from Stratagene (La Jolla, CA, USA). Ang II, NAC and all other chemicals were purchased from Sigma (St. Louis, MO, USA).

2.2 Culture of rat aortic smooth muscle cells
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and approved by the Institutional Animal Care and Use Committee of Taipei Medical University. Thoracic aortae of male Sprague–Dawley rats were excised rapidly and immersed in DMEM containing penicillin (100 U/ml) and streptomycin (100 µg/ml). Connective tissue and adherent fat were cleaned. Isolated arteries were cut open, and the endothelium was removed by gently rubbing off the intimal surface with sharp scissors. Denuded aortae were cut into ~3-mm pieces and placed with the intimal face down into three 35-mm culture dishes (Iwaki, Osaka, Japan). DMEM containing 10% fetal calf serum and penicillin/streptomycin was gently added to the dishes to cover the tissues without disturbing the orientation of the explants. Vascular SMCs were allowed to grow from the tissue (7–10 days), and the tissues were removed using sterilized fine forceps and washed with culture medium. After reaching confluence in three 35-mm dishes, cells were harvested by brief trypsinization and grown in T-75 flasks (Iwaki) (passage 1). Subcultured cells from passages 3 to 12 were used in the experiments and showed 99% of purity as estimated by cell morphology and positive immunostaining with smooth muscle {alpha}-actin antibody (Sigma).

2.3 DNA synthesis
To measure synthesis of new DNA, cells (1 x 105/well) were plated in six-well (35-mm) dishes 24 h before experiments as previously described [15]. Cells were incubated with [3H]thymidine (5 µCi/ml). After addition of agent indicated, cells were harvested by incubation at 4 °C with trichloroacetic acid (5%) followed by solubilization in 0.1N NaOH, and radioactivity was determined by scintillation counting. Data are presented as the mean±SEM of 9–12 determinations in three to four different cell preparations and normalized to the untreated sample x 100 (i.e. percentage of control).

2.4 Assay of intracellular ROS
ROS were measured using a previously described method [16]. Prior to the chemical or Ang II treatment, smooth muscle cells were incubated in culture medium containing a fluorescent dye, 2'7'-dichlorofluorescin diacetate (DCF-DA) (Molecular Probes, Eugene, OR, USA) of 30 µM for 1 h to establish a stable intracellular level of the probe. The same concentration of DCF-DA was maintained during the chemical or Ang II treatment. Subsequently, the cells were washed with PBS, removed from Petri dishes by brief trypsinization, and measured for 2',7'-dichlorofluorescein (DCF) fluorescence intensity. The DCF fluorescence intensity of the cells is an index of intracellular levels of ROS; and it can be determined by fluorescence spectrophotometry with excitation and emission wavelengths at 475 and 525 nm, respectively. The cell number in each sample was counted in an automatic cell counter (S.ST.II/ZM, Coulter Electronics, Miami, FL, USA) and utilized to normalize the fluorescence intensity of DCF.

2.5 Assay of ET-1 peptide secretion
ET-1 levels were measured in culture medium using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Amersham-Pharmacia, Amersham, UK). Results were normalized to cellular protein content in all experiments and expressed as a percentage relative to the cells incubated with the vehicle.

2.6 RNA isolation and northern blot analysis
RNA isolation and Northern blotting were performed as previously described [16]. Expression of ET-1 mRNA was quantitated and was normalized to the 18S signal.

2.7 Transfection and chloramphenicol acetyltransferase assays
For the transient transfections, cells were transfected with different expression vectors by the calcium phosphate method [11]. DNA concentration for all samples was adjusted to equal amount with empty vector pSR{alpha} in each experiment. To correct for variability in transfection efficiency, 5 µg of pSV-β'-galactosidase plasmid DNA was cotransfected in all the experiments. Total RNA was isolated from SMCs without transfection treatment, but for CAT assay, cell extracts were prepared 36 h after transfection. The CAT and β'-galactosidase assays were performed as previously described [14]. The relative CAT activity was corrected by normalizing the respective CAT value to that of β-galactosidase activity. Cotransfected β-galactosidase activity varied by <10% within a given experiment and was not affected by any of the experimental manipulations described. As positive and negative controls, pBLCAT2 (with thymidine kinase promoter) and pBLCAT3 (without promoter) were included in each assay.

2.8 Western blot analysis
Rabbit polyclonal anti-phospho-specific ERK antibodies were purchased from New England Biolabs (Beverly, MA, USA). Anti-ERK antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Western blot analysis was performed as previously described [16].

2.9 Luciferase assay
SMCs plated on six-well (35-mm) dishes were transfected with the luciferase reporter construct possessing consensus AP-1 binding sites (AP-1-Luc) (Stratagene). After incubation for 24 h in serum-free DMEM, smooth muscle cells were cultured under different treatments as indicated for 48 h. SMCs were assayed for luciferase activity with a luciferase reporter assay kit (Stratagene). The firefly luciferase activities as AP-1 transcriptional activity were normalized for transfection efficiency to its respective β-galactosidase activity and expressed as relative activity to control.

2.10 Statistical analysis
Results are expressed as mean±SEM of at least three experiments unless designated otherwise. Statistical analysis was performed using Student's t-test and analysis of variance (ANOVA) followed by a Dunnett multiple comparison test using GraphPad Prism (GraphPad Software, San Diego, CA, USA). A value of p<0.05 was considered to be statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Role of endogenous ET-1 in Ang II-induced proliferation of smooth muscle cells
Ang II-stimulated rat aortic SMC proliferation was assessed by analyzing DNA synthesis with [3H]thymidine incorporation. Ang II increased DNA synthesis in SMCs in a dose-dependent manner (Fig. 1A). Ang II (100 nM)-stimulated DNA synthesis in SMCs was inhibited by either AT1 receptor antagonist olmesartan (1 µM) or ETA receptor antagonist BQ485 (1 µM) treatment (Fig. 1B). Both olmesartan and BQ485 had no effect on basal [3H]thymidine uptake. These data suggest the possible role of endogenous ET-1 as an autocrine growth factor for the proliferation of SMCs under Ang II stimulation.


Figure 1
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  		Fig. 1 Characteristics of the activation of DNA synthesis by Ang II in SMCs. Increases in [3H]thymidine incorporation are each expressed relative to the [3H] content (100%) in their respective controls (C). All data are shown as the mean±SEM for 9–12 determinations in three to four cell preparations. *p<0.05 versus control. #p<0.05 versus Ang II alone. (A) Effect of Ang II concentration on DNA synthesis. Cells were incubated with the indicated doses of Ang II for 24 h, and [3H]thymidine incorporation was then assayed. (B) Effect of Ang II or ET-1 receptor antagonists on [3H]thymidine incorporation. Cells were preincubated with either olmesartan (Olmesa; 1 µM) or BQ485 (1 µM) for 1 h after their incubation with 100 nM Ang II for 24 h.

 
3.2 Ang II-induced ET-1 gene expression in smooth muscle cells
To examine whether Ang II increases ET-1 mRNA levels in SMCs, we performed Northern blot analysis (Fig. 2A,B). ET-1 mRNA was induced by Ang II (100 nM) as early as 1 h (Fig. 2A). When SMCs were treated with Ang II for 6 h, the Ang II-induced ET-1 mRNA expression was dose-dependent with the maximum induction at 100 nM (Fig. 2B). To determine whether the Ang II-induced ET-1 expression is regulated at the transcriptional level, an ET-1 promoter construct containing the ET-1 promoter region (–4.4 kb) and the reporter gene CAT was constructed and transiently transfected into smooth muscle cells. SMCs exposed to 24 h of Ang II (100 nM) significantly increased ET-1 promoter activity (Fig. 2C). Ang II dose-dependently increased ET-1 promoter activity. 100 nM of Ang II also gave maximum induction (Fig. 2D). The effect of AT1 receptor blocker olmesartan on the Ang II-increased ET-1 promoter activity was also investigated (Fig. 2D). SMCs were pretreated with olmesartan (1 µM) for 1 h and subsequently stimulated with Ang II for 24 h. The Ang II-induced ET-1 promoter activity was inhibited by olmesartan. Time course and dose-dependency of ET-1 expression by Ang II had also been performed at the protein level (Fig. 2E,F). SMCs exposed to 24 h of Ang II (100 nM) significantly increased ET-1 peptide secretion (Fig. 2E). Ang II dose-dependently increased ET-1 peptide secretion (Fig. 2F). These data show that Ang II directly induces ET-1 gene expression in SMCs.


Figure 2
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  		Fig. 2 Effect of Ang II on ET-1 gene expression in SMCs. Data are represented as the difference relative to the data in the control groups. The results are shown as the mean±SEM (n = 3). *p<0.05 versus control. #p<0.05 versus Ang II alone. (A) Time course of Ang II on ET-1 mRNA expression. Cells were incubated with Ang II (100 nM) for the indicated times. (B) Dose–response effect of Ang II on ET-1 mRNA expression. Cells were incubated with various doses of Ang II for 6 h. (C) Time course of Ang II on ET-1 promoter activity. Cells were transfected with chimeric CAT fusion genes and then treated with Ang II (100 nM) for the time indicated. Cells were harvested, and CAT activities were measured. CAT activities are shown as the percentage incorporation after the data were normalized to β-galactosidase activities. C indicates control (no drugs). CAT2 and CAT3 are positive and negative controls, respectively. (D) Induction of ET-1 promoter activity by different concentrations of Ang II. Some cells were preincubated with olmesartan (Olmesa; 1 µM) for 1 h after their incubation with 100 nM Ang II for 24 h or not. (E) Time course of Ang II on ET-1 peptide secretion. Confluent cells were exposed to Ang II (100 nM) for times indicated. ET-1 peptide in overlying medium was determined by use of ELISA. White or black columns denote the cells incubated with the vehicle or Ang II, respectively. (F) Induction of ET-1 peptide secretion by different concentrations of Ang II. Cells were incubated with various doses of Ang II for 24 h.

 
3.3 ROS mediate Ang II-induced proliferation and ET-1 gene expression in smooth muscle cells
We and others have demonstrated that Ang II stimulates ROS production in various cell types, including cardiomyocytes, cardiac fibroblasts and SMCs [9,18,19]. To confirm previous observations that Ang II induces intracellular ROS in SMCs, we measured intracellular ROS level by analyzing the fluorescent product DCF, a peroxidative product of DCF-DA. SMCs treated with Ang II (100 nM) had significantly higher ROS levels than those cells treated with vehicle only (Fig. 3A). The increase of ROS was completely blocked by pretreatment of cells with either olmesartan or antioxidants such as N-acetylcysteine (NAC) or diphenyleneiodonium (DPI), a flavoprotein containing the NADH/NADPH oxidase inhibitor. Olmesartan (1 µM), NAC (10 mM) and DPI (10 µM) all showed a significant reduction in ROS production (Fig. 3A). To elucidate the involvement of ROS in the Ang II-induced proliferation, SMCs were pretreated with NAC or DPI for 30 min followed by Ang II treatment. SMCs pretreated with NAC (10 mM) or DPI (10 µM) significantly suppressed Ang II-induced [3H]thymidine uptake (Fig. 3B). To further examine the ROS involvement in the Ang II-induced ET-1 gene expression, SMCs were preincubated with an antioxidant NAC or DPI for 30 min and then treated with Ang II. As shown in Fig. 4A, SMCs pretreated with NAC (10 mM) or DPI (10 µM) significantly suppressed Ang II-induced ET-1 mRNA level. Similarly, cells pretreated with NAC or DPI also suppressed Ang II-increased ET-1 promoter activity (Fig. 4B). These findings suggest that intracellular ROS generation apparently mediate Ang II-induced proliferation and ET-1 gene expression in rat aortic SMCs.


Figure 3
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  		Fig. 3 ROS involvement in Ang II-induced SMC proliferation. All data are shown as the mean±SEM for 9–12 determinations in three to four cell preparations. *p<0.05 versus control. #p<0.05 versus Ang II alone. (A) Effect of olmesartan and antioxidants on Ang II-related increases in ROS. Cells were loaded with DCF-DA for 30 min and stimulated with Ang II. Ang II (100 nM) increased ROS levels, which were abolished by olmesartan (Olmesa; 1 µM), NAC (10 mM), and DPI (10 µM). Cells treated with H2O2 (100 µM) are shown as positive controls. (B) Effect of antioxidants on Ang II-induced DNA synthesis in SMCs. Cells were preincubated with NAC (10 mM), or DPI (10 µM) for 30 min after incubation with 100 nM Ang II for 24 h. Increases in [3H]thymidine incorporation are expressed relative to the [3H] content (100%) in their respective controls (C).

 

Figure 4
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  		Fig. 4 Ang II-induced ET-1 gene expression mediated by ROS in SMCs. The results are shown as the mean±SEM (n = 3). *p<0.05 versus control. #p<0.05 versus Ang II alone. (A) Effect of antioxidants on Ang II-induced ET-1 mRNA in SMCs. Cells were preincubated with DPI (10 µM) or NAC (10 mM) for 30 min after incubation with 100 nM Ang II for 6 h. (B) Effect of antioxidants on Ang II-induced increases in ET-1 promoter activity in SMCs. Cells were preincubated with DPI (10 µM), or NAC (10 mM) for 30 min after incubation with 100 nM Ang II for 24 h.

 
3.4 Effect of redox-sensitive ERK pathway in Ang II-induced ET-1 gene expression
Ang II has been shown to activate ERK and the activation of this pathway is redox-sensitive [20,21]. We recently reported that ROS were involved in the activation of ERK pathway, which culminated in ET-1 gene expression [15,16]. To study whether ERK pathway was involved in Ang II-induced ET-1 gene expression in SMCs, we examined the effect of antioxidants on ERK pathway and determined the effect of ERK inhibitor on Ang II-induced ET-1 gene expression. We first confirmed the role of ROS in Ang II-increased phosphorylation of ERK in SMCs (Fig. 5A). Both NAC (10 mM) and DPI (10 µM) significantly inhibited Ang II-induced phosphorylation of ERK (Fig. 5A). These data suggest that ERK is a redox-sensitive signaling pathway activated by Ang-II in SMCs. We next determined the role of redox-sensitive activation of ERK in Ang II-induced ET-1 gene expression. Both NAC and U0126, a specific inhibitor of MKK-1 (MEK), inhibited augmentation of ET-1 mRNA expression stimulated with Ang II (Fig. 5B). Similarly, coincubation with either NAC or U0126 also completely abolished Ang II-increased ET-1 promoter activity (Fig. 5C). These findings suggest that activation of ERK is a necessary step for ET-1 gene expression induced with Ang II.


Figure 5
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  		Fig. 5 Ang II-induced increases in ET-1 gene expression by means of ERK in a redox-sensitive manner. The results are shown as the mean±SEM (n = 3). *p<0.05 versus control. #p<0.05 versus Ang II alone. (A) Ang II-induced activation of ERK was mediated by a ROS-sensitive pathway. Cells were preincubated with DPI (10 µM), or NAC (10 mM) for 30 min and stimulated with Ang II (100 nM) for 30 min. Both DPI and NAC inhibited Ang II-induced activation of ERK. (B) Ang II-induced ET-1 mRNA was attenuated by U0126, a MEK1 inhibitor, in SMCs. SMCs were stimulated with Ang II (100 nM) in the presence of U0126 (10 µM), or NAC (10 mM). Total RNA was isolated at 6 h. (C) Ang II-induced increases in ET-1 promoter activity were inhibited by U0126 in SMCs. SMCs were stimulated with Ang II (100 nM) in the presence of U0126 (10 µM), or NAC (10 mM). CAT activity was assayed after 24 h. (D) Ang II-induced increases in ET-1 promoter activity via the Ras/Raf/ERK pathway in SMCs. Cells were transfected with pSR{alpha} empty vector (5 µg) or an expression plasmid encoding the dominant negative mutant mERK, Raf301, or RasN17 (5 µg) was cotransfected with 15 µg of a plasmid for ET-1 promoter and CAT. Cells cotransfected with an expression plasmid encoding MEK1 (5 µg) or RasL61 (5 µg) were used as positive controls.

 
To identify the signaling pathway involved in the Ang II-induced ET-1 expression, we also cotransfected SMCs with various dominant negative mutants Ras (RasN17), Raf-1 (Raf301) or a catalytically inactive mutant of ERK2 (mERK), all of which are associated with the Ras/Raf/ERK pathway. SMCs cotransfected with RasN17, Raf301 or mERK resulted in a significant inhibition in Ang II-induced ET-1 promoter activity (Fig. 5D). In contrast, cells cotransfected with a dominant positive mutant of Ras (RasL61) or MEK1 greatly increased their ET-1 promoter activities. These results further suggest that the Ras/Raf/ERK signaling pathway plays an important role in Ang II-induced ET-1 gene expression in SMCs.

3.5 Identification of Ang II-responsive regulatory elements in the ET-1 promoter in smooth muscle cells
The ET-1 promoter contains a number of AP-1 and GATA sites, which can be regulated by multiple activation pathways [22]. We dissected the Ang II-responsive elements of the ET-1 promoter in SMCs. As previously described [16], a series of deletion mutants containing various lengths of the ET-1 promoter region fused to CAT reporter gene were transfected into SMCs and CAT activity was measured in response to Ang II stimulation. Ang II stimulation for 24 h significantly increased CAT activity in –4400 CAT, –700 CAT and –204 CAT, all of which contain multiple transcription factor binding sites including GATA (bp –136 to –131) and AP-1 (bp –108 to –102) sites (Fig. 6). However, after further truncation of the GATA and subsequent AP-1 site from the 5'-end, the increase of the Ang II-induced ET-1 promoter activity were almost completely abolished in both –129 CAT and –98 CAT. It is interesting that deletion of these two sites also resulted in a significant decrease in basal promoter activity (Fig. 6). These findings suggest that the GATA site as well as AP-1 site is necessary for Ang II-stimulated ET-1 gene induction. Using the electrophoretic mobility shift assay, the role of GATA as well as AP-1 element was assayed in cells treated with Ang II for 6 h. The formation of GATA complexes following Ang II treatment was only slightly affected in SMC nuclear extracts. In contrast with the GATA binding, the formation of AP-1 complexes following Ang II treatment was enhanced in SMCs (data not shown). We further transfected cells with the luciferase reporter construct possessing consensus AP-1 binding sites to quantify the role of AP-1 in the induction of ET-1 gene by Ang II. Ang II (1–100 nM) dose-dependently increased AP-1 reporter activity in SMCs (Fig. 7A). Pretreating cells with antioxidants, NAC or DPI attenuated the Ang II-stimulated AP-1 reporter activity (Fig. 7B). Moreover, cells transfected with reporter construct-204 CAT containing both GATA and AP-1 sites with two-bp mutation in the AP-1 site, the Ang II- or H2O2-induced ET-1 promoter activity was completely abolished. In addition, the basal promoter activity also decreased as compared with control (Fig. 7C). These findings suggest that the AP-1 binding element is essential for the induction of ET-1 gene by Ang II. These results clearly indicate that ROS mediate the transcriptional activity of AP-1 induced by Ang II and the AP-1 binding element is responsible for the induction of ET-1 gene expression by Ang II in SMCs.


Figure 6
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  		Fig. 6 A series of deletion mutants of ET-1 promoter gene plasmids were cotransfected into SMCs. Transfected cells were stimulated with Ang II (100 nM) for 24 h, and CAT activities were measured. Stepwise 5'-deletion constructs were depicted (top). The results are shown as the mean±SEM (n = 3). *p<0.05 versus control. #p<0.05 versus Ang II alone.

 

Figure 7
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  		Fig. 7 Crucial role of AP-1 motif in Ang II-induced increases in ET-1 promoter activity. The results are shown as the mean±SEM (n = 3). *p<0.05 versus control. #p<0.05 versus Ang II alone. (A) SMCs, transfected with vector or AP-1-Luc, were treated for 24 h with indicated concentrations of Ang II. Luciferase activity was expressed as relative activity to untreated control (C). (B) SMCs, transfected with AP-1-Luc, were incubated for 24 h with either no drug (C), 10 µM DPI, or 10 mM NAC in the absence or presence of Ang II (100 nM). Cells treated with H2O2 (100 µM) are shown as positive controls. (C) Wild-type (204 bp) or AP-1 mutants of the plasmids for the ET-1 promoter and CAT were cotransfected into SMCs. Cells were stimulated with Ang II (100 nM) or H2O2 (100 µM) for 24 h. The mutation of AP-1 strongly abolished the responsiveness to Ang II or H2O2.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
It is clear that the proliferation of vascular SMCs contributes to the pathophysiology of hypertension, atherosclerosis, coronary artery restenosis after angioplasty, and stent placement [23]. In addition, the production of ROS in the vessel wall is increased in models associated with vascular remodeling such as hypercholesterolemia, hypertension, diabetes, and balloon injury to the coronary arteries [24–26]. Thus a better understanding of the mechanisms involved may lead to additional treatments for such diseases. In the present study, we demonstrated that ROS mediate Ang II-induced proliferation and ET-1 gene expression in SMCs, and antioxidants significantly inhibit these effects. Ang II has been shown to stimulate both hyperplasia and hypertrophy in vascular SMCs via the AT1 receptor [27]. We characterized the proliferative response to Ang II using agents that inhibited the AT1 receptor (olmesartan) and the ETA receptor (BQ485). The Ang II-induced DNA synthesis in vascular SMCs was inhibited by both olmesartan and BQ485. The effect of Ang II on ET-1 gene expression is also mediated by the activation of the AT1 receptor. These data are compatible with Moreau et al. that Ang II infusion in intact animals increases blood pressure and induces vascular hypertrophy together with increased vascular ET-1 levels [5]. In addition, Chen et al. [28] reported that Ang II-induced vasoconstriction in some blood vessel preparations in vitro, can be blocked by an ET receptor antagonist. Similarly, ET receptor antagonism prevents Ang II-induced increase of total peripheral and renal vascular resistance as well as urinary protein excretion in vivo [29]. In keeping with these observations, our findings strongly suggest that the pathological effects attributed to Ang II in the vascular system are mediated, at least in part, by the modulation of vascular ET-1 production.

Recent studies have shown that a major source of ROS in vascular tissues is NAD(P)H oxidase, and the activity of NAD(P)H oxidase is increased by Ang II [9]. Thus, the source of ROS in the present study can be Ang II-stimulated NAD(P)H oxidase. It is also known that antioxidants are capable of antagonizing the actions of Ang II in vivo [30]. To better understand the role that ROS might play in influencing Ang II action in SMCs, we have characterized the induction of ET-1 gene and the phosphorylation of ERK in these cells by Ang II and examined the effects of antioxidants NAC and DPI on this pathway. In this study, we demonstrated that ROS are involved in Ang II-induced activation of ERK pathway, which leads to ET-1 gene expression. We also found that AP-1 site in the promoter region is a crucial cis-element for Ang II-mediated induction of ET-1 gene in SMCs. Co-transfection experiments with dominant negative Ras, Raf, and ERK suggested that the Ras-Raf-ERK pathway is involved in the transcriptional activation of the ET-1 by Ang II. Wang et al. [31] previously reported that MEK1 was activated by Ang II and inhibited by NAC, suggesting that it could be a potential site of action for these drugs, although any site upstream of MEK1 also could be implicated. Contrary to our present findings and others [20,21], Ushio-Fukai et al. [32] showed that DPI inhibited p38 MAPK, but not ERK stimulated by Ang II in vascular SMCs. Although no data were available to explain these discrepancies, they may be due to the different experimental methods and/or phenotypes of SMCs. Further studies are needed that will enable us to understand the role of ROS in the overall growth promotion by Ang II in SMCs. We characterized the Ang II-induced ET-1 gene expression using U0126 that implicated the requirement for MEK1 activation. However, the molecular mechanisms how ROS regulate upstream signaling which lead to Ang II-induced ERK phosphorylation in SMCs remains further determined. Several evidences suggest that ROS serve as messengers in AP-1 activation [14,15]. Truncation and mutational analysis of the ET-1 gene promoter showed that AP-1 binding sequence is an important cis-element for Ang II-induced ET-1 gene expression and the GATA site is also necessary for ET-1 gene induction by Ang II. These results are consistent with the previous report by Kawana et al. [22], showing that both the GATA and AP-1 sites are essential for ET-1 promoter function and cooperative interaction of GATA and AP-1 regulates transcription of the ET-1 gene in endothelial cells.

In conclusion, Ang II via AT1 receptor increased intracellular ROS, which were at least partly involved in Ang II-increased activation of ERK pathway in SMCs. Moreover, we showed that ERK activation plays a role in Ang II-stimulated ET-1 gene expression in SMCs. Recently, pharmacological intervention with angiotensin converting enzyme inhibitors and AT1 receptor antagonists are used clinically for cardiovascular disease, diabetes complications such as hypertension and other vascular disorders [33,34]. In vivo study revealed that Ang II-induced vascular hypertrophy and the increase in blood pressure is mediated at least in part by an increased production of endogenous ET, which then activates ETA receptors to produce the observed changes on the cardiovascular system [5]. In the present study, we demonstrated that ROS mediate Ang II-induced proliferation and ET-1 gene expression in vascular SMCs, and antioxidants significantly inhibit these effects. Ang II inhibition blocks ET-1 which presumably blocks vasoconstriction which is associated with hypertension and end-organ damage (e.g. diabetic nephropathy and diabetic retinopathy). Our findings provide substantial evidence for new therapeutic options with AT1 and ETA receptor antagonists combined with antioxidants in the treatment of cardiovascular diseases associated with an increased activity of the renin–angiotensin system.


   Acknowledgements
 
This study was in supported with grants from Shin Kong Wu Ho-Su Memorial Hospital (SKH-TMU-92-15 to T.H. Cheng) and National Science Council (NSC92-2320-B039-044 to H.J. Hong), Taiwan, R.O.C. We are thankful to Sankyo (Tokyo, Japan) for generously providing olmesartan used in this study.


   Notes
 
Time for primary review 20 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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