Involvement of reactive oxygen species in angiotensin II-induced endothelin-1 gene expression in rat cardiac fibroblasts
Tzu-Hurng Cheng, PhD* ,
Pao-Yun Cheng, MS ,
Neng-Lang Shih, PhD,
Iuan-Bor Chen, BS||,
Danny Ling Wang, PhD and
Jin-Jer Chen, MD ,*
* Department of Medicine, Taipei Medical University-Wan Fang Hospital, Taipei, Taiwan
Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan, R.O.C.
|| Present address: Faculty of Art and Science, University of Toronto, Toronto, Ontario, Canada
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Figure 1 Characteristics of the activation of DNA synthesis by angiotensin II (Ang II) in cardiac fibroblasts. All experiments were performed with the incorporation of [3H]thymidine into DNA. (A) Effect of Ang II concentration on the DNA synthesis. Cells were incubated with the indicated doses of Ang II for 24 h and then assayed for [3H]thymidine incorporation. (B) Effect of Ang II or endothelin-1 receptor antagonists on [3H]thymidine incorporation. Cells were preincubated with either losartan (Losa) (1 M) or BQ485 (1 M) for 1 h followed by an incubation with 100 nM Ang II for 24 h. Experimental details are given in the Experimental Procedures section. [3H]thymidine incorporation is expressed as the percentage of increase relative to the [3H] content (100%) in the control (C). All data are shown as the means ± SEM of 9 to 12 determinations in three to four different cell preparations. *p < 0.05 versus control (Student t test); #p < 0.05 versus Ang II alone (analysis of variance).
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Figure 2 Effect of angiotensin II (Ang II) on endothelin-1 (ET-1) gene expression in neonatal rat cardiac fibroblasts. (A) Time course of Ang II on ET-1 messenger RNA (mRNA) expression. Cells were incubated with Ang II (100 nM) for the indicated times. (B) Dose-dependent effect of Ang II on ET-1 mRNA expression. Cells were incubated with various doses of Ang II for 30 min. (C) Time course of Ang II-increased ET-1 promoter activity. Cardiac fibroblasts were transfected with chimeric ET-1 promoter-chloramphenicol acetyltransferase (CAT) fusion genes followed by treatment with Ang II (100 nM) for the time indicated or pretreated with losartan (Losa) (1 M) for 1 h followed by Ang II stimulation. (D) Induction of ET-1 promoter activity by different concentration of Ang II. Cells were harvested, and CAT activities were measured as described in the Experimental Procedures section. Control (C), no drugs; CAT2 and CAT3 are shown as positive and negative control; CAT activities are shown as the percentage of incorporation after normalizing to that of ß-galactosidase activities. Data are represented as difference relative to control groups. The results are shown as mean ± SEM (n = 3 per group). *p < 0.05 vs. control (Student t test); #p < 0.05 vs. Ang II alone (analysis of variance). The experiment was repeated three times with reproducible results.
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Figure 3 Angiotensin II (Ang II)-induced proliferation and endothelin-1 (ET-1) gene expression were mediated by reactive oxygen species (ROS) in cardiac fibroblasts. (A) Angiotensin II increased intracellular ROS in cardiac fibroblasts. Cardiac fibroblasts were loaded with dichlorofluorescin diacetate for 30 min and stimulated with Ang II for 30 min. Intracellular ROS levels were measured by laser-confocal microscopy. Angiotensin II (100 nM) increased ROS levels in cardiac fibroblasts, and these increases were abolished by losartan (Losa; 1 µM), the NAD(P)H oxidase inhibitor diphenyleneiodonium (DPI) (1 µM), catalase (350 U/ml), or N-acetylcysteine (NAC) (10 mM). Cells treated with H2O2 are used as positive control. In each experiment, the densitometric analysis was performed on at least 20 cells. (B) Effect of antioxidants on Ang II-induced DNA synthesis in cardiac fibroblasts. Cells were preincubated with NAD(P)H oxidase inhibitor DPI (1 µM), catalase (350 U/ml), or NAC (10 mM) for 30 min followed by incubation with 100 nM Ang II for 24 h. Increases in [3H]thymidine incorporation are each expressed relative to the [3H] content (100%) in the respective control (C). (C) Effect of antioxidants on Ang II-induced ET-1 messenger RNA in cardiac fibroblasts. Cells were preincubated with either the catalase (350 U/ml) or NAC (10 mM) for 30 min followed by an incubation with 100 nM Ang II for 30 min. (D) Effect of antioxidants on Ang II-increased ET-1 promoter activity in cardiac fibroblasts. Cells were preincubated with either the catalase (350 U/ml) or NAC (10 mM) for 30 min followed by an incubation with 100 nM Ang II for 24 h. The results are shown as mean ± SEM (n = 3 per group). *p < 0.05 vs. control (Student t test); #p < 0.05 vs. Ang II alone (analysis of variance).
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Figure 4 Angiotensin II (Ang II) increased ET-1 gene expression via extracellular signal-regulated kinase (ERK) in a redox-sensitive manner. (A to C) Angiotensin II-induced activation of ERK, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38MAPK) was mediated by reactive oxygen species-sensitive pathway. Cells were preincubated with either the catalase (350 U/ml) or N-acetylcysteine (NAC) (10 mM) for 30 min and stimulated with Ang II (100 nM) for 30 min. Phosphorylation of ERK, JNK, or p38MAPK was detected by Western blotting using anti-phospho-ERK, phospho-JNK, and phospho-p38MAPK antibodies. Both catalase and NAC inhibited Ang II-induced activation of ERK, JNK, or p38MAPK. Phosphorylations of ERK, JNK, or p38MAPK were detected, and densitometric analyses were performed. The results are shown as mean ± SEM (n = 4 per group). (D) Angiotensin II-induced ET-1 messenger RNA was attenuated by PD98059 in cardiac fibroblasts. Cardiac fibroblasts were stimulated with Ang II (100 nM) in the presence of PD98059 (PD; 20 M) or SB203580 (SB; 20 M), and total RNA was isolated at 30 min. (E) Angiotensin II-increased ET-1 promoter activity was inhibited by PD98059 in cardiac fibroblasts. Cardiac fibroblasts were stimulated with Ang II (100 nM) in the presence of PD98059 (PD; 20 M) or SB203580 (SB; 20 M), and chloramphenicol acetyltransferase (CAT) activity was assayed after 24 h. (F) Angiotensin II-increased ET-1 promoter activity via Ras/Raf/ERK pathway in cardiac fibroblasts. Cells, transfected with either pSR -empty vector (5 µg), or an expression plasmid encoding the dominant negative mutant mERK, Raf301, or RasN17 (5 µg), were co-transfected with 15 µg of ET-1 promoter-CAT plasmid. Cells co-transfected with ET-1 promoter-CAT plasmid and an expression plasmid encoding MEK1 (5 µg) or RasL61 (5 µg) were used as positive controls. The results are shown as mean ± SEM (n = 3 per group). *p < 0.05 vs. control (Student t test); #p < 0.05 versus Ang II alone (analysis of variance).
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Figure 5 Identification of angiotensin II (Ang II)-responsive cis-elements in endothelin 1 (ET-1) promoter. (A) A series of deletion mutants containing various lengths of ET-1 promoter region were transfected into cardiac fibroblasts. Transfected cells were stimulated with Ang II (100 nM) for 24 h, and chloramphenicol acetyltransferase (CAT) activities were measured. Stepwise 5'-deletion constructs were depicted (top). Bars represent mean (±SEM, n = 3 per group). CAT activity of each construct in the presence or absence of Ang II were assayed. CAT 2 and CAT 3 were used as positive or negative controls for CAT assay respectively (bottom). (B) Wild type (204 bp) or AP-1 mutant of ET-1 promoter-CAT plasmid was transfected into cardiac fibroblasts. Cells were stimulated with Ang II (100 nM) for 24 h. The mutation of AP-1 strongly abolished the responsiveness to Ang II stimulation. The results are shown as mean ± SEM (n = 3 per group). *p < 0.05 versus control (Student t test); #p < 0.05 versus Ang II alone (analysis of variance).
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