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Reactive oxygen species modulate endothelin-I-induced c-fos gene expression in cardiomyocytes

T.H. Cheng, N.L. Shih, S.Y. Chen, D.L. Wang, J.J. Chen
DOI: http://dx.doi.org/10.1016/S0008-6363(98)00275-2 654-662 First published online: 1 March 1999


Objectives: Recent evidence indicates that reactive oxygen species (ROS) may act as second messengers in receptor-mediated signaling pathways. The possible role of ROS during Et-1 stimulation in cardiomyocytes was therefore investigated. Methods: Intracellular ROS levels were measured with fluorescence probe 2′,7′-dichlorofluorescin diacetate by confocal microscopy in cultured neonatal rat cardiomyocytes. The ROS-inducible c-fos expression was analyzed by Northern blotting and promoter activity. Results: Et-1 applied to cardiomyocytes dose-dependently increased intracellular ROS levels. The increase of ROS levels was attenuated by pretreating cardiomyocytes with Et-A receptor antagonist-BQ485, but not with Et-B receptor antagonist. Cardiomyocytes pretreated with catalase or an antioxidant N-acetylcysteine (NAC) reduced Et-1-induced ROS levels. Et-1 or H2O2 treatment of cardiomyocytes rapidly induced the expression of an immediate early gene c-fos. Et-1-treated cardiomyocytes enhanced the c-fos gene expression as revealed by functional analysis using a reporter gene construct containing c-fos promoter region (−2.25 kb) and reporter gene chloramphenicol acetyltransferase. The induction of mRNA levels and the promoter activities of c-fos gene by Et-1 or H2O2 were abolished by pretreating cardiomyocytes with catalase or NAC. Cells transiently transfected with the dominant positive mutant of p21ras (RasL61) led to a significant increase in intracellular ROS. Concomitantly, the mRNA levels and the promoter activities of c-fos were also induced. In contrast, cells transfected with the dominant negative mutant of Ras (RasN17) inhibited Et-1-induced ROS. Consistently, the increase of c-fos mRNA levels and promoter activities by Et-1 were also inhibited. Conclusions: These findings clearly indicate that Et-1 treatment to cardiomyocytes can induce ROS via Ras pathway and the increased ROS are involved in the increase of c-fos expression. Our studies thus emphasize the importance of ROS as second messengers in Et-1-induced responses in cardiomyocytes.

  • Endothelin-1
  • Reactive oxygen species
  • c-fos
  • Gene expression
  • Ras
  • Rat, Cardiomyocyte

Time for primary review 28 days.

1 Introduction

Endothelin-1 (Et-1) is a potent 21-amino acid long vasopressor secreted from endothelial cells [1]. Apart from its constrictive activity on vascular smooth muscle, Et-1 has been demonstrated to stimulate hypertrophic growth in cardiomyocytes [2, 3]. Et-1 can induce a transient increase in expression of immediate early genes including c-fos and early growth response-1 (egr-1) [4]. It has been reported that Et-1 activates a phosphorylation cascade of protein kinases including Raf-1 and the extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase (MAPK) signaling pathway in cardiac myocytes [3]. Et-1 also activates phosphorylation cascade of MAPK/Jun amino-terminal kinase (JNK) in cardiac myocytes [5, 6]. Both ERK and JNK can phosphorylate Elk-1, one of several ternary complex factors, and resulting in an increase of c-fos transcription [7–9]. c-fos and c-jun make the heterodimer complex of activating protein-1 (AP-1), which preferentially binds to many genes that have 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive element (TRE) in their promoter region [10].

Recent evidence indicates that reactive oxygen species (ROS) may function as intracellular messengers to modulate signaling pathways [11]. ROS encompass many oxygen species including singlet oxygen, superoxide, hydrogen peroxide (H2O2) and hydroxyl radicals that are produced by virtually every type of cell [12]. ROS, when present in excess, are toxic to cells due to the potential damage they can cause to cellular components. However, there is growing evidence that ROS at low concentration serve as signaling molecules, providing a chemical link between alterations in intracellular redox status and responsive changes in the structure and function of transcription factors [12]. The changes of intracellular ROS have been detected in a variety of cells stimulated with cytokines, growth factors, agonists of receptor [12]. Studies have also demonstrated that Et-1 treatment can induce ROS levels in various cells [13, 14]. However, the origin of ROS and their signaling roles contributing to cellular responses in cardiomyocytes have not been well characterized.

The small G protein p21ras (Ras) is a potent regulator of myogenic cell growth and differentiation. Ras has been demonstrated to play a role in Et-1-mediated c-fos activity in cardiomyocytes [15]. Ras has been implicated to play a major role in the genesis of cardiac hypertrophy by activating the Raf-1/ERK pathway [16, 17]. It has been reported that Ras contributes to the induction of the c-fos by Et-1 in mesangial cells [18]. Recent findings further indicate that Ras is involved in the ROS generation in fibroblasts [19, 20]. Furthermore, Et-1 has also been reported to increase the ROS levels in broncho-alveolar cells [13]. The c-fos gene has been shown to be induced by Et-1 [21]or oxidants [22]in cardiomyocytes. However, the role of Ras in the generation of ROS and their relation to c-fos expression in the Et-1-treated cardiomyocytes have not been determined. Since ROS can act as second messengers, the roles of ROS in cardiomyocytes upon Et-1 treatment have thus been studied. In the present study, we clearly demonstrated that ROS mediate c-fos gene induction via Ras pathway in Et-1 treated cardiomyocytes.

2 Methods

2.1 Reagents

Endothelin-1, BQ-485 and BQ-788 were purchased from Calbiochem (San Diego, CA, USA). N-Acetylcystein (NAC), catalase and other chemicals were purchased from Sigma (St. Louis, MO, USA).

2.2 Cardiomyocytes culture

Primary cultures of neonatal rat ventricular myocytes were prepared as previously described [2]. The rats were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985). Briefly, ventricles from 1- to 2-day-old neonatal Sprague–Dawley rats were cut into chunks of approximately 1 mm3 using scissors and subjected to trypsin (0.125%, Gibco) digestion in phosphate-buffered saline (PBS). Trypsin digested cells were collected by centrifugation at 1200 rpm for 5 min. The cell pellet was resuspended in a medium containing 80% F10 nutrient mixture, 20% fetal calf serum, penicillin (100 U/ml) and streptomycin (68.6 μmol/l) and plated into a petri dish for 2.5 h. The suspended nonattached myocytes in the medium were collected and plated on a 100-mm diameter culture dishes with cell density at 1·107 cells/dish. After 2 days in culture, cells were transferred to medium containing 90% DMEM nutrient mixture, 10% fetal calf serum, penicillin (100 U/ml) and streptomycin (68.6 μmol/l). Myocytes cultures thus obtained were >80% pure as revealed by their contractile characteristics under light microscopy. Serum-containing medium from these cultured myocytes was replaced with serum-free medium and exposed to agents as indicated. For measurement of intracellular ROS generation, cardiomyocytes were plated to 30-mm diameter culture dishes with low cell density (∼1·104 cells/dish) to monitor intracellular oxidation in single cardiomyocyte.

2.3 Transfections

For the transient transfections, cardiac cells were transfected with different expression vectors by the calcium phosphate method [23]. The DNA–CaPO4 precipitates in each dish (10 ml) contained the following if not specifically identified: 15 μg c-fosCAT reporter plasmid; 1 or 5 μg RasL61; 5 μg RasN17; 5 μg pSV-β-galactosidase plasmid; and variable amounts of pSRα plasmid vector to adjust for total DNA. Briefly, cardiomyocytes were maintained in culture for 48 h prior to transfection. The indicated expression vectors were mixed with calcium phosphate and immediately added to the cardiomyocyte cell culture. After incubation for 5 h, cells were then washed three times with PBS and incubated with 10% serum DMEM. After 24 h, cells were washed with serum-free medium and incubated in the same medium for an additional 24 h. Cells were then treated with different agents. To correct for transfection efficiency, 5 μg of pSV-β-galactosidase plasmid, which contains a β-galactosidase gene driven by the simian virus 40 promoter and enhancer, was cotransfected into cells. The c-fos CAT vector included 5′ flanking regions of a 2.25 kb EcoR I-Nae I fragment containing upstream sequences and the promoter of the c-fos gene [24]. The constitutively activated Ras (RasL61) and the dominant-negative Ras (RasN17) plasmids have been previously reported [25]and were kindly provided by J.J. Shyy, (University of California, San Diego, CA, USA). In some experiments, RasL61 or RasN17 were transfected in the absence or presence of c-fosCAT. The empty vector pSRα was transfected and used as a control. Except for routine β-galactosidase activity, cells were constantly collected for measuring intracellular ROS, RNA levels and CAT activities as described below.

2.4 Assay of intracellular ROS

Intracellular ROS production was measured by using a fluorescent dye, 2′,7′-dichlorofluorescin diacetate (DCF-DA) (Molecular Probes, Eugene, OR, USA) with the ACAS Interactive Laser Cytometer (Meridian Instruments, Okemos, MI, USA). DCF-DA is a membrane-permeable compound. After intracellular deacetylation, DCF-DA forms a nonfluorescent product (DCF), which upon oxidation is transformed into fluorescent DCF [26]. A 10 mmol/l stock solution of DCF-DA was prepared in ethanol on a daily basis and diluted to a final concentration of 10 μmol/l just before the experiments. Myocytes with or without transfection were preincubated with 10 μmol/l DCF-DA in DMEM for 30 min at 37°C prior to treatment. Cells after dye exposure were rinsed with normal Tyrode solution. The cells were maintained in normal Tyrode solution and examined with the Laser Cytometer at 37°C. Excitation of DCF was achieved using the 488-nm line of a 20-mW argon-ion laser. The emission above 515-nm is quantitated from two dimensional image scans generated by a 1 micron laser beam and an X–Y scanning stage. A fluorescence value from single cells can be obtained. To provide a valid comparison, the same acquisition parameters were used for all observations. Quantification of the levels of DCF fluorescence was assessed on a relative scale from 0–4000 units. Baseline values from unstimulated or pSRα-tranfected cells were used as control values to compare with Et-1-stimulated or Ras-transfected cells. Values represent means±S.E.M. of DCF fluorescence from at least 20 randomly selected cells. This experiment was repeated for at least six times to avoid the transfection variation.

2.5 RNA isolation and northern hybridization

Total RNA was obtained by using guanidine thiocyanate as described previously [2]. Total RNA was collected and examined by mini-gel agarose electrophoresis. Ten micrograms of RNA was mixed with loading buffer containing ethidium bromide. The sample mixture was loaded and separated on 1% agarose gels containing 3.7% formaldehyde. RNA was transferred onto Nytran membrane (Schleicher & Schuell, Germany) by a vacuum blotting system (VacuGene XL, Pharmacia, Sweden) and immobilized by ultraviolet irradiation. A 2137 bp EcoR I/EcoR I fragment containing rat c-fos DNA [27]was used as the c-fos probe and was labeled by random priming with [32P]-dCTP using the random primer DNA labeling kit (Amersham, Buckinghamshire, UK). Individual blot was subsequently washed and reprobed with 18S ribosomal RNA cDNA probe (obtained from American Type Culture Collection) to normalize differences in loading and/or transfer to the filter. Autoradiographic results were scanned with a Bio-Rad densitometer.

2.6 Chloramphenicol acetyltransferase assays and β-galactosidase assays

The chloramphenicol acetyltransferase (CAT) and β-galactosidase assays were performed as previously described [2]. 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 every assay [23].

2.7 Statistical analysis

Data were presented as mean±S.E.M. Statistical analysis was performed using one-way analysis of variance (ANOVA) or an unpaired Student’s t-test. The significance level α was set at 0.05.

3 Results

3.1 Et-1 induced intracellular ROS levels in cardiomyocytes

Intracellular generation of ROS in cardiomyocytes was analyzed by the fluorescent product (DCF), a peroxidative product of DCF-DA with laser-scanning confocal microscopy. Exposure of cardiomyocytes to Et-1 (10 nmol/l) rapidly induced DCF fluorescence with a maximal increase of 2-fold at 15 min after stimulation (Fig. 1). This ROS induction was shown to be an Et-1 concentration dependent since ROS level appeared to reach plateau after treating with 10 nmol/l of Et-1 (Fig. 2A). To specify which Et-1 receptor subtype that was responsible for the generation of ROS in cardiomyocytes, we pretreated cardiomyocytes with Et-1 receptor type A antagonist (BQ485) or receptor type B antagonist (BQ788) before the Et-1 exposure. As shown in Fig. 2B, BQ485 (100 nmol/l) significantly inhibited the Et-1-induced generation of ROS. In contrast, BQ788 (100 nmol/l) had no significant effect on Et-1-induced generation of ROS. These results suggest that an increase of intracellular ROS levels upon Et-1 treatment is mediated mainly through the binding of Et-1 to Et-1 receptor type A.

Fig. 2

Et-1-induced ROS levels were attenuated by pretreatment of cardiomyocytes with Et-1 antagonists or antioxidant. (A) Et-1 induced ROS generation in a dose-dependent manner. (B) Cardiomyocytes from either control (C; column 1) or treated with Et-1 (10 nmol/l) in the absence (column 2) or presence of 100 nmol/l BQ485 (column 3), 100 nmol/l BQ788 (column 4), 10 mmol/l NAC (column 5) or 350 U/ml catalase (column 6) for 15 min. H2O2 (25 μmol/l) treated cells (column 7) are shown as positive controls. Fluorescence intensities of cells are shown as relative intensity of experimental groups compared to untreated control cells. At least 20 randomly selected cells were used for determination of each experiment. The results show mean±S.E.M. *P<0.05 vs. control; #P<0.05 vs. Et-1 treated cells. The experiment was repeated six times with reproducible results.

Fig. 1

Et-1 induced ROS generation in cardiomyocytes. (A) Cardiomyocytes treated with Et-1 increased intracellular ROS levels as revealed by fluorescent intensities of DCF. Cardiomyocytes preincubated with DCF-DA for 30 min and then treated with Et-1 for the indicated times (min). Fluorescence intensity was analyzed with confocal laser cytometer. (B) Cardiomyocytes were photographied by differential interference contrast microscopy. (C) Time course of Et-1-induced ROS generation in cardiomyocytes as revealed by DCF fluorescence. About 20 randomly selected cells were used for determination of each point. The results show mean±S.E.M. *P<0.05 vs. control untreated cells. The experiment was repeated six times with reproducible results.

To examine whether the ROS were responsible for the oxidation of DCF in cardiomycytes, we examined whether catalase, an enzyme that specifically hydrolyses H2O2, was able to prevent the oxidation of DCF. Cardiomyocytes were treated with Et-1 in the presence of catalase (350 U/ml). The addition of catalase to cultured cardiomyocytes completely inhibited Et-1-induced ROS levels as measured at 15 min after Et-1 treatment (Fig. 2B). NAC, a glutathione precursor and a free radical scavenger, also inhibited the increase in Et-1-induced intracellular ROS levels (Fig. 2B). These findings clearly demonstrate that Et-1 treatment to cardiomyocytes increases intracellular ROS levels.

3.2 Inhibitory effect of antioxidants on the expression of c-fos gene by Et-1

We then investigated whether ROS generated by Et-1 play a role in gene expression. Previous studies demonstrated that ROS stimulated the expression of immediate early response genes such as c-fos, c-jun, c-myc and egr-1 [28]. Of these genes, c-fos was found to respond to both Et-1 [4, 21]and H2O2 [22]treatment in cardiomyocytes. The c-fos mRNA levels were shown by Northern blotting at various time intervals after treating cardiomyocytes with either Et-1 (10 nmol/l) or H2O2 (25 μmol/l) (Fig. 3). As shown, the serum-deprived cultured cardiomyocytes incubated with Et-1 (10 nmol/l) rapidly increased c-fos mRNA levels within 10 min of Et-1 exposure. This induction reached a maximal level at 30 min, i.e., an 11-fold induction vs. control cells (Fig. 3A). Consistently, when cardiomyocytes were exposed to H2O2 (Fig. 3A), a time-dependent increase of c-fos mRNA levels was noticed. The H2O2 treatment for 60 min induced c-fos mRNA levels in cardiomyocytes about 13-fold as compared with controls (Fig. 3A). All these results suggest that Et-1-induced c-fos mRNA in cardiomyocytes are modulated through ROS generated during Et-1 exposure.

Fig. 3

Antioxidant inhibited Et-1- or H2O2- induced c-fos mRNA in cardiomyocytes. (A) Time course study of c-fos mRNA induction by Et-1 and H2O2. Cardiomyocytes were from untreated controls (lane1 and 5) or from Et-1 (10 nmol/l, lane 2–4) or H2O2 (25 μmol/l, lane 6–8) treatment for various time intervals. (B) Inhibition of Et-1-induced c-fos mRNA by antioxidant. Cardiomyocytes were either untreated controls (lane 1), or treated with Et-1 (10 nmol/l) in the absence (lane 2) or presence of 10 mmol/l NAC (lane 3) or 350 U/ml catalase (lane 4) for 30 min. For H2O2 studies, cells were treated with H2O2 (25 μmol/l) in the absence (lane 5) or presence of NAC (lane 6) or catalase (lane 7) for 60 min. Cells treated with PMA (0.8 μmol/l; lane 8) for 30 min were used as positive controls. Total RNA was extracted and Northern hybridization was performed with 32P-labeled rat c-fos as probe. 18S ribosomal RNA was used to normalize the RNA applied in each lane. Data was presented as folds increase of experimantal groups compared to untreated controls. *P<0.05 vs. control; #P<0.05 vs. Et-1 alone; +P<0.05 vs. H2O2 alone. Results are shown as mean±S.E.M. from five experiments in time course study and three experiments in panel B with reproducible results.

We then examined the effects of antioxidant or free radical scavenger, i.e. catalase and NAC, on the induction of c-fos by Et-1. As shown in Fig. 3B both Et-1 (10 nmol/l) and H2O2 (25 μmol/l) increased c-fos mRNA levels in cardiomyocytes. However, the addition of catalase (350 U/ml) and NAC (10 mmol/l) 30 min prior to the Et-1 and H2O2 treatment abolished the c-fos gene induction (Fig. 3B). Cardiomyocytes treated with PMA (phorbol 12-myristate 13-acetate; 0.8 μmol/l) for 30 min as positive controls induced c-fos mRNA levels about 21-folds as compared with controls (Fig. 3B). However, the c-fos induction by PMA was only partially inhibited after these antioxidants treatment (data not shown) indicating that the inhibitory effect of antioxidants was specific for Et-1-induced c-fos gene expression. These findings are consistent with the earlier results of ROS induction in cardiomyocytes after Et-1 treatment (Fig. 2). Taken together, these results suggest that Et-1-induced ROS levels are involved in the c-fos gene expression since antioxidant pretreatment of cardiomyocytes inhibits this c-fos induction.

3.3 ROS modulated Et-1-increased c-fos transcriptional activity

To further determine whether the ROS modulating the Et-1-induced c-fos expression was a transcriptional event, a c-fos promoter activity was examined by using a reporter gene construct containing c-fos promoter region (−2.25 kb) and reporter gene chloramphenicol acetyltransferase (CAT). This chimera was transfected into cardiomyocytes followed by Et-1 or H2O2 stimulation. As demonstrated in Fig. 4A, either catalase or NAC alone had no effect on the basal c-fos promoter activity. However, cardiomyocytes treated with Et-1 (10 nmol/l) and H2O2 (25 μmol/l) for 24 h led to a 2.7- and 2.1-fold increase in CAT activity, respectively, as compared to control unstimulated cells (Fig. 4B). In the presence of catalase (or NAC), Et-1- or H2O2-induced c-fos promoter activities were significantly inhibited. These data clearly indicate that ROS modulate the increase of c-fos promoter activity in cardiomyocytes after Et-1 or H2O2 treatment.

Fig. 4

Antioxidant inhibited Et-1- or H2O2- induced c-fos promoter activity. (A) Cardiomyocytes were transfected with 15 μg of c-fosCAT reporter gene construct for 24 h. Some cells were pretreated with NAC or catalase for 30 min. Cardiomycytes were then treated with Et-1 (10 nmol/l) or H2O2 (25 μmol/l) for 24 h. PMA-treated cells are shown as positive controls. CAT2 (lane 11) and CAT3 (lane 12) are shown as positive and negative controls of a CAT assay system. Cells were harvested and CAT assays were performed as described in Section 2. (B) CAT activities are shown as percentage incorporation after normalizing to that of β-galactosidase activities. The results are shown as mean±S.E.M. *P<0.05 vs. control; #P<0.05 vs. Et-1 alone; +P<0.05 vs. H2O2 alone. The experiment was repeated three times with reproducible results.

3.4 Ras mediated Et-1-induced ROS generation and c-fos expression

Ras has been demonstrated to be involved in the intracellular ROS generation in fibroblasts [20]. In order to study the role of Ras in the Et-1-induced ROS generation, expression plasmid encoding a constitutively activated Ras protein (RasL61) was used to transfect into cardiomyocytes. RasL61 is an active form of p21ras in which the Gln-61 in the wild type has been replaced by Leu [25]. As shown in Fig. 5A, RasL61 (5 μg) transfection into cardiomyocytes led to a 4.5-fold increase in intracellular ROS levels as compared to control pSRα-transfected cells. pSRα-transfected cardiomyocytes after Et-1 treatment increased intracellular ROS levels. In contrast, cardiomyocytes transfected with dominant negative Ras mutant (RasN17; 5 μg), a dominant negative mutant of Ras in which Ser-17 in the wild type has been replaced by Asn so that the affinity to GTP is dramatically reduced [25], abolished the Et-1-induced ROS production (Fig. 5A). To understand whether these Ras-mediated ROS were involved in the Et-1-induced c-fos expression, Ras-transfected cells were analyzed for their c-fos mRNA levels. Transient expression of RasL61 (5 μg) for 48 h significantly increased the c-fos mRNA (Fig. 5B). The treatment of catalase 350 U/ml for 24 h only partially inhibited the RasL61-stimulated c-fos gene expression. Compared to control pSRα-transfected cells, c-fos induction by Et-1 was abolished in cells transfected with RasN17 (5 μg), but not in those that received H2O2 (25 μmol/l) treatment for 30 min (Fig. 5B). To further confirm that Ras mediated Et-1-induced c-fos promoter activity, we cotransfected c- fosCAT reporter gene with RasL61 (or RasN17). Cotransfections of the c-fosCAT reporter gene with RasL61 (5 μg) stimulated c-fosCAT activity by 3.8-fold as compared with control pSRα-transfected cells (Fig. 6). The treatment of catalase 350 U/ml for 24 h partially inhibited the RasL61 stimulated c-fos promoter activity. Cotransfections of the c-fosCAT reporter construct with RasN17 (5 μg) into cardiomyocytes inhibited c-fos promoter activity induced by Et-1 (Fig. 6). However, H2O2 (25 μmol/l) treatment for 24 h on cardiomyocytes cotransfected with c-fosCAT and RasN17 (5 μg) led to 2.2-fold increase in CAT activity as compared to that of RasN17-transfected cells (Fig. 6B). All these results suggest that Ras mediate ROS generation and c-fos gene induction in cardiomyocytes exposed to Et-1.

Fig. 6

Ras mediated Et-1-induced c-fos promoter activities. (A) Fifteen micrograms of c-fosCAT reporter gene construct was cotransfected with either 5 μg of pSRα-empty vector, RasN17, or RasL61 into cardiomyocytes for 24 h, followed by treating with 10 nmol/l Et-1 (lanes 2–4) or 25 μmol/l H2O2 (lanes 5 and 8). Some cells were pretreated with catalase (350 U/ml) (lane 3) before Et-1 treatment for 24 h. CAT2 (lane 9) and CAT3 (lane 10) are shown as positive and negative controls for CAT assay. Cells were harvested and CAT assays were performed as described in Section 2. (B) CAT activities were determined by normalizing percentage incorporation in each experimental group to that of β-galactosidase activities. Results are shown as mean±S.E.M. *P<0.05 vs. pSRα-transfected control; #P<0.05 vs. Et-1 alone; +P<0.05 vs. only RasN17-transfected cells; ‡P<0.05 vs. only RasL61-transfected cells. The experiment was repeated three times with reproducible results.

Fig. 5

Ras modulated intracellular ROS generation and c-fos mRNA induction. (A) Cardiomyocytes were transfected with either pSRα-empty vector (5 μg), RasL61 (1 or 5 μg) or RasN17 (5 μg). Cells after transfection for 48 h were loaded with DCF-DA followed with Et-1 treatment and intracellular ROS were imaged by laser confocal microscopy. (B) Ras mediated c-fos mRNA levels induced by Et-1. Five micrograms of pSRα-empty vector, RasN17, or RasL61 were transfected into cardiomyocytes for 48 h, followed by treating with 10 nmol/l Et-1 (lanes 2–4) or 25 μmol/l H2O2 (lanes 5 and 8) for 30 min. Some cells were treated with catalase (350 U/ml) (lanes 3 and 7) for 24 h. H2O2 treated cells were used as positive controls. c-fos mRNA levels were analyzed by using a densitometer and the results were normalized to those of 18S ribosomal RNA. Results are shown as mean±S.E.M. *P<0.05 vs. pSRα-transfected control; #P<0.05 vs. Et-1 alone; +P<0.05 vs. only RasN17-transfected cells. The experiment was repeated three times with reproducible results.

4 Discussion

In the present study, we demonstrated that neonatal rat cardiomyocytes exposed to Et-1 increased intracellular reactive oxygen species and resulted in an induction of c-fos gene expression via Ras pathway. Recent observation indicates that cells from various sources (i.e. endothelial cells, smooth muscle cells, etc.) exposed to various growth factors or stimuli generate ROS as second messengers [12]. Present study has clearly demonstrated that cardiomyocytes exposed to Et-1 increase intracellular ROS levels via Et A receptor-dependent mechanism. This finding is consistent with the previous report that the Et A is the main receptor type present in the rat heart [29]and cardiomyocytes [30]. Furthermore, the increase of ROS by Et-1 treatment was completely abolished by co-incubation of cardiomyocytes with the antioxidant enzyme catalase. This indicates that H2O2 is a major reactive oxygen species that contribute to the increase of c-fos induction in Et-1-treated cardiomyocytes. Catalase is a big molecule which is unlikely to diffuse into cells but appeared to rapidly clear the intracellularly Et-1-induced H2O2. Because H2O2 is relatively stable, the intracellular generated H2O2 that is permeable to membrane can be easily removed by catalase. Alternatively, the extracellular catalase may be uptake by the cardiomyocytes and results in a decrease of intracellular ROS levels. The uptake of catalase into cells and the decrease of intracellular ROS levels have been previously shown in platelet-derived growth factor (PDGF)-treated smooth muscle cells [31]. Catalase has also been effectively used in treating various cells to prevent from cytokine- or growth factor-induced gene expression [31–33]. This is also in agreement with our previous report demonstrating the inhibitory effect of catalase on hemodynamic forces-induced gene expression by reducing intracellular ROS levels [34–36]. The induction of ROS by Et-1 was also blocked by the glutathione (GSH) precursor NAC. NAC treatment raised the levels of intracellular GSH that might remove the intracellular H2O2 via GSH peroxidase [11]. Our ROS induction by Et-1 was a time- and dose-dependent phenomenon. The origin of this increased ROS is not clear. ROS, including H2O2, may be derived from the activated electron transfer reaction or other superoxide producing enzyme systems. How the cells respond to a stimulus and transduce early signals to ROS-producing machineries remain to be defined. Protein kinase C activation after Et-1 treatment may be involved in this process [37]. Nevertheless, our results clearly indicate that Et-1 treatment of cardiomyocytes induces ROS production via Et A receptor.

Changes in the redox status of cells are thought to be able to induce modification of cellular signaling molecules including various protein kinases, protein phosphatases and transcription factors [12]. In the present study, we used oxidant-inducible gene c-fos as a target gene to demonstrate that this Et-1-induced c-fos expression was an event involving intracellular redox changes. Et-1 treatment of cardiomyocytes, similar to H2O2 exposure, induced a transient increase of c-fos expression. This transient induction by Et-1 was inhibited by treatment of cardiomyocytes with catalase or NAC. As a positive control, cardiomyocytes treated with PMA stimulated their c-fos expression. PMA treatment of cardiomyocytes greatly increased intracellular ROS levels. However, PMA-induced c-fos expression was only partially inhibited by NAC or catalase pretreatment (data not shown). The immediate proto-oncogene c-fos is normally expressed at a minimal level in the mammalian heart. Increased proto-oncogene expression has been implicated in the development of cardiac hypertrophy [38]. Induction of c-fos, a transcription factor that interacts with cis-regulatory element AP-1 in many genes, has important consequences for downstream gene expression that may determine the phenotypic response to Et-1. The c-fos promoter region contains at least three cis-regulatory regions that are involved in the regulation of c-fos transcription; i.e., the SIE (sis-inducible element), AP-1 (Fos–Jun transcription factor complex) and the SRE (serum responsive element) [39]. The activation of c-fos transcription is thought to be mediated by the activation of MAPK [40]. It has also been demonstrated that H2O2 is capable of stimulating MAPK [41, 42]. In cardiomyocytes, Et-1 has been shown to activate two closely related MAPK isoforms, p42MAPK and p44MAPK [43]via a Ras-dependent pathway. Recent data even indicate that Ras is a common signaling target of ROS [44]. Thus, Et-1-induced ROS are likely to activate the signaling pathway that results in the induction of transcriptional activity of c-fos.

Our results further demonstrated that Ras was involved in Et-1-induced ROS generation because a proportional increase of ROS production was noticed as the transfection dose of RasL61 increased. Although we could not rule out the possibility that transfected cells might release some factor and then acting on other cells. However, the cardiomyocytes after RasL61-transfection did get brighter with fluorescence intensity. Accordingly, increased c-fos mRNA levels in RasL61-transfected cardiomyocytes were noticed. Furthermore, Et-1-induced c-fos mRNA level was attenuated by co-transfection of cardiomyocytes with RasN17. Consistently, c-fos promoter activities were increased by RasL61 and attenuated by RasN17 transfection. All these results strongly support that Ras is involved in the Et-1-induced ROS production that results in the increase of c-fos mRNA levels. These findings are consistent with those of previous studies indicating that Et-1 stimulates a Ras-based signaling cascade in mesangial cells [18]. Ras-dependent ROS production has also been shown in NIH 3T3 fibroblasts. Furthermore, expression of dominant negative Ras suppressed ROS production has been reported [20]. All these are consistent with our results and the premise that ROS are generated by Et-1 via a Ras dependent mechanism. Taken together, these results provide strong evidence for a Ras-based signaling cascade linking Et-1 receptors to ROS generation and the activation of the c-fos gene in cardiomyocytes.

We have previously demonstrated that ROS work as second messengers in vascular endothelial cells subjected to hemodynamic forces [34–36]. These increased ROS induced AP-1 binding to a cis-acting element (TRE) in the MCP-1 promoter region [34]and possible serum response element (SRE) site in the egr-1 promoter region [45]. Because c-fos promoter region contains AP-1 and SRE sites, it is possible that ROS modulate AP-1 and/or SRE binding via Ras-dependent signaling pathway. The detailed mechanisms as to how the ROS modulate the signaling pathway remain largely unknown and warrant further study. Nevertheless, this study demonstrates that ROS act as cellular signals in Et-1-treated cardiomyocytes. The important role of Et-1 for cardiac hypertrophy has been well documented [2, 3]. Because Et-1 induces ROS that result in c-fos induction which subsequently may regulate the expression of late genes, the suppression of intracellular redox changes by increasing antioxidant capability may provide some insight for preventing Et-1-induced gene alterations in cardiomyocytes during the development of cardiac hypertrophy. Improvement of myocardial redox status with vitamin E therapy that can attenuate the development of hypertrophy to heart failure has recently been reported [46]. Our results provide further support of the importance of ROS as second messenger for Et-1-induced responses in cardiovascular systems. This finding may offer clinical implications for the therapeutic potential of antioxidant treatment against the transition from compensatory hypertrophy to heart failure.


The present work was partly supported by grant NSC 87-2314-B-002-229 from National Science Council, Taipei, Taiwan, R.O.C.


  • 1 Present address: Faculty of Art and Science, University of Toronto, Toronto, Ontario, Canada.


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