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(Circulation Research. 2001;89:661.)
© 2001 American Heart Association, Inc.

Molecular Medicine

ERK and p38 MAPK, but not NF-B, Are Critically Involved in Reactive Oxygen Species–Mediated Induction of IL-6 by Angiotensin II in Cardiac Fibroblasts

Motoaki Sano, Keiichi Fukuda, Toshihiko Sato, Haruko Kawaguchi, Makoto Suematsu, Satoshi Matsuda, Shigeo Koyasu, Hideo Matsui, Keiko Yamauchi-Takihara, Masaki Harada, Yoshihiko Saito, Satoshi Ogawa

From the Cardiopulmonary Division, Department of Internal Medicine (M. Sano, T.S., S.O.); the Institute for Advanced Cardiac Therapeutics (K.F., H.K.); the Department of Biochemistry (M. Suematsu); the Department of Microbiology and Immunology (S.M., S.K.), Keio University School of Medicine, Shinjuku, Tokyo, Japan; the Department of Molecular Medicine (H.M., K.Y.-T.), Osaka University Graduate School of Medicine, Suita, Osaka, Japan; and the Department of Medicine and Clinical Science (M.H., Y.S.), Kyoto University Graduate School of Medicine, Sakyo-ku, Kyoto, Japan.

Correspondence to Keiichi Fukuda, MD, Ph.D, Institute for Advanced Cardiac Therapeutics, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. E-mail kfukuda{at}sc.itc.keio.ac.jp

	Abstract

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Abstract—— We recently reported that angiotensin II (Ang II) induced IL-6 mRNA expression in cardiac fibroblasts, which played an important role in Ang II–induced cardiac hypertrophy in paracrine fashion. The present study investigated the regulatory mechanism of Ang II–induced IL-6 gene expression, focusing especially on reactive oxygen species (ROS)-mediated signaling in cardiac fibroblasts. Ang II increased intracellular ROS in cardiac fibroblasts, and the increase was completely inhibited by the AT-1 blocker candesartan and the NADH/NADPH oxidase inhibitor diphenyleneiodonium (DPI). We first confirmed that antioxidant N-acetylcysteine, superoxide scavenger Tiron, and DPI suppressed Ang II–induced IL-6 expression. Because we observed that exogenous H₂O₂ also increased IL-6 mRNA, the signaling pathways downstream of Ang II and exogenous H₂O₂ were compared. Ang II, as well as exogenous H₂O₂, activated ERK, p38 MAPK, and JNK, which were significantly inhibited by N-acetylcysteine and DPI. In contrast with exogenous H₂O₂, however, Ang II did not influence phosphorylation and degradation of IB-/ß or nuclear translocation of p65, nor did it increase NF-B promoter activity. PD98059 and SB203580 inhibited Ang II–induced IL-6 expression. Truncation and mutational analysis of the IL-6 gene promoter showed that CRE was an important cis-element in Ang II–induced IL-6 gene expression. NF-B–binding site was important for the basal expression of IL-6, but was not activated by Ang II. Ang II phosphorylated CREB through the ERK and p38 MAPK pathway in a ROS-sensitive manner. Collectively, these data indicated that Ang II stimulated ROS production via the AT1 receptor and NADH/NADPH oxidase, and that these ROS mediated activation of MAPKs, which culminated in IL-6 gene expression through a CRE-dependent, but not NF-B–dependent, pathway in cardiac fibroblasts.

Key Words: angiotensin II • interleukin-6 • reactive oxygen species • mitogen-activated protein kinase • cardiac fibroblast

	Introduction

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Clinical, experimental, and genetic data have demonstrated that the renin angiotensin system is linked to the pathogenesis of a variety of cardiac diseases. Expression of angiotensin II (Ang II), the key effector molecule of the renin angiotensin system, is increased under various pathophysiological conditions and stimulates cardiomyocyte hypertrophy and interstitial fibrosis coinciding with accumulation of extracellular matrix. Recent reports had shown that Ang II stimulates membrane-bound NAD(P)H oxidase, which generates reactive oxygen species (ROS) in a variety of nonphagocytic cells.¹ ROS may act as a second messenger that regulates various intracellular signal transduction cascades and the activity of various transcription factors. NF-B and AP-1 are the best characterized transcription factors to be influenced by the cellular oxidation-reduction (redox) state.^2,3 The primary target of activation of NF-B by ROS appears to be the phosphorylation and subsequent degradation of IB.⁴ Another well-characterized redox sensitive signaling pathway is that of MAPKs.^5,6 The increase in ROS production results in activation of MAPK pathways, and ultimately activates AP-1 through phosphorylation and induction of the c-fos and c-jun family of protooncogenes. Ang II may contribute to atherosclerosis through induction of ROS, which leads to expression of redox-sensitive vascular inflammatory genes, such as monocyte chemoattractant protein-1 (MCP-1) and vascular cell adhesion molecule-1 (VCAM-1), and to cell growth.^7–9 The potential role of ROS in the regulation of signal transduction and gene expression in the heart has been recently elucidated. Administration of antioxidants inhibited Ang II– and tumor necrosis factor (TNF)-–induced cardiac hypertrophy.¹⁰

We recently reported that Ang II induces production of interleukin (IL)-6, leukemia inhibitory factor, and cardiotrophin-1 in cardiac fibroblasts, which strongly mediated Ang II–induced cardiomyocyte hypertrophy in a paracrine manner.¹¹ These findings can explain the discrepancy that Ang II converting enzyme inhibitor or AT-1 blocker were very effective in the prevention of cardiac hypertrophy, although cardiomyocytes only expresses less than 10% of the number of AT-1 receptors expressed by cardiac fibroblasts. Among these cytokines, IL-6 is a pleotrophic cytokine, which may exert primary effects on myocardial function, such as reduction of cardiac contractility,¹² hypertrophy,¹³ and cytoprotection against apoptosis.¹⁴ Various kinds of stimuli such as TNF-, IL-1ß, Ang II, endothelin-1, mechanical stretch, ischemia, and reperfusion elicit IL-6 synthesis in the heart.^11,15–18 These stimuli appear to be able to increase intracellular ROS. The 5'-flanking sequence upstream of the IL-6 gene contains several response elements to the transcription factors AP-1, CREB, C/EBP, and NF-B.¹⁹ Although the significance of IL-6 production from cardiac fibroblasts is well understood, the precise mechanism of IL-6 production by Ang II remains unclear. In the present study, we investigated the role of redox-sensitive mechanisms in the modulation of Ang II–induced augmentation of IL-6 gene expression in cardiac fibroblasts. We further determined which redox-sensitive signal transduction pathways and transcription factors were involved in this process.

	Materials and Methods

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Cell Culture
Primary cardiac fibroblasts were cultured from the ventricles of 1-day-old neonatal Wistar rats (Japan Clea Inc, Tokyo, Japan), as described previously.^11,20 Human cardiac fibroblasts were obtained from the right atrial appendage at the time of cardiac surgery according to the guidelines of the Japanese Ministry of Health and Welfare.

Measurement of Intracellular Oxidant Generation
Cardiac fibroblasts were plated at density of 10⁴/cm². Cells were loaded with the oxidant-sensitive fluorogenic probe dichlorodihydrofluorescein diacetate (DCF-DA) for 30 minutes. This reagent is known to enter the cells, hydrolyze into dichlorodihydrofluorescein, and oxidize irreversibly into the fluorophore dichlorofluorescein (DCF).²¹ Cells were then washed with PBS, replaced with the fresh medium, and stimulated with Ang II. Intracellular DCF fluorescence was visualized by a laser-confocal video microscopy as described.²² Under observation through a 40x objective lens, 20 to 30 cells in several different fields were chosen at random for DCF measurements. The fluorescence intensities were measured by determining 8-bit gray levels (1 to 256) as described.²²

Extraction of Nuclear Fraction
Washed cells were suspended in a cold hypotonic buffer (10 mmol/L HEPES[pH 7.9], 10 mmol/L KCl, 0.1 mmol/L EDTA, 0.1 mmol/L EGTA, 1 mmol/L dithiothreitol [DTT], and 0.5 mmol/L phenylmethylsulfonyl fluoride [PMSF]). The cells were allowed to swell on ice for 15 minutes and then Dounce homogenized. The homogenates were centrifuged at 5000 rpm for 5 minutes. The pellets containing the nuclear fraction were resuspended in cold hypotonic buffer added with 0.1% Triton X, gently pipetted up and down, and centrifuged at 15 000 rpm for 5 minutes. The pellets were resuspended with a high-salt buffer (20 mmol/L HEPES [pH7.9], 0.4 mol/L NaCl, 1% triton X, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L DTT, and 1 mmol/L PMSF], and pipetted up and down. After centrifugation for 15 000 rpm for 15 minutes, the supernatants were recovered and used as nuclear extracts.

Western Blot Analysis
Rabbit polyclonal anti–phospho-specific p38 MAPK, anti–phospho-specific ERK1/2, anti–phospho-specific JNK, anti–phospho-specific I-B, and anti–phospho-specific CREB antibodies were purchased from New England Biolabs Inc (Beverly, MA). Anti-ERK1, anti–p38 MAPK, anti-JNK, anti-IB, anti-IBß and anti-p65 antibodies were purchased from Santa Cruz Biotechnology. Western blot analysis was performed as described.¹¹

RT-PCR and Northern Blot Analysis
Total RNA was extracted by TRIzol Reagent (Gibco-BRL). RT-PCR was performed as described previously.¹⁵ GAPDH or ß-actin were used as internal controls for each sample. Poly(A)⁺ RNA was isolated, and Northern blotting was performed as described previously.¹¹

Transfection and Luciferase Assay
A luciferase reporter plasmid carrying the NF-B binding site (pNFB-luc) was purchased from Stratagene. pIRES-EGFP-IB was provided by Dr S. Matsuda. The various lengths of IL-6 promoter–containing luciferase plasmids (p840, p417, p140, or p60) were provided by Dr K. Yamauchi-Takihara.²³ Luciferase plasmids containing a point-mutated IL-6 promoter (p1168huIL-6p-luc+, AP-1–mutant, CRE-mutant, C/EBP-mutant, and NF-B–mutant) were provided by Dr G. Haegeman (University of Gent, Belgium).²⁴ Transient transfection was performed using Effectene Transfection Reagent (Qiagen) according to the manufacturer’s instruction. Within 24 hours, cells were incubated with a transfection mixture with 0.32 µg of reporter plasmids and 0.08 µg of pRL-SV40 (Promega) as an internal control plasmid. Total cell lysates were collected at 6 hours and luciferase activity was measured by Dual Luciferase Reporter Assay System (Promega).

Statistical Analysis
All values are mean±SD. The significance of differences among mean values was determined by ANOVA. Statistical comparison of the control group with treated group was performed using Fisher’s multiple comparison tests. The accepted level of significance was P<0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ang II Increased Intracellular H₂O₂ in Cardiac Fibroblasts
Recent studies have demonstrated that growth factors stimulate production of ROS in various cell types. To determine whether Ang II induces intracellular ROS in cardiac fibroblasts, we measured intracellular oxidant levels using the hydroperoxide-sensitive fluorophore DCF-DA by laser confocal microscopy. Cells treated with Ang II had significantly higher fluorescence than cells treated with vehicle (Figure 1). The rise in DCF fluorescence was completely blocked by candesartan. Pretreatment with a flavoprotein containing the NADH/NADPH oxidase inhibitor diphenyleneiodonium (DPI) or catalase-reduced Ang II stimulated fluorescence. We also confirmed that preincubation with antisense oligonucleotide to p22phox inhibited the Ang II–induced increase in DCF fluorescence. These findings suggested that Ang II increases H₂O₂ in cardiac fibroblasts, and that the rise in ROS by Ang II is mediated by NADH/NADPH oxidase.

View larger version (42K): [in this window] [in a new window]   Figure 1. Angiotensin II increased intracellular H2O2 in cardiac fibroblasts. Cardiac fibroblasts were loaded with DCF-DA for 30 minutes and stimulated with Ang II. Intracellular H2O2 levels were measured by laser-confocal microscopy at 60 minutes. Ang II increased H2O2 levels in cardiac fibroblasts, and these increases were completely prevented by candesartan (CS: 10-6 mol/L), the NAD(P)H oxidase inhibitor DPI (10-6 mol/L), catalase, or antisense oligonucleotide to p22phox. The lower panel showed the results of the densitometry of 4 separate experiments. In each experiment, the densitometric analysis was performed on at least 50 cells. Data were mean±SD. Insert panel was a time course of H2O2 levels. *P<0.01 vs control, #P<0.01 vs angiotensin II alone.

Ang II–Induced IL-6 Gene Expression Is Regulated Through a Redox-Sensitive Mechanism
To confirm that Ang II induces IL-6 gene expression in cardiac fibroblasts, we performed poly(A)⁺RNA Northern blot analysis (Figure 2A). IL-6 mRNA was induced by Ang II as early as 30 minutes, gradually decreased, but was still elevated at 120 minutes.

View larger version (54K): [in this window] [in a new window]   Figure 2. Angiotensin II–induced IL-6 gene expression was mediated through a redox-sensitive pathway. A, Cardiac fibroblasts were stimulated with Ang II, and IL-6 mRNA expression was determined by Northern blot. Poly(A)+ RNA (4 µg) was loaded in each lane. Similar results were obtained in 2 separate experiments. B, Cells were preincubated with either NAC (10 mmol/L), Tiron (5 mmol/L), or DPI (10 µmol/L), and stimulated with Ang II for 30 minutes. IL-6 gene expression was examined by RT-PCR. ß-actin was used as an internal control. The results were reproducible in 4 separate experiments. M indicates XHaeIII/HindIII marker.

To examine the involvement of ROS in the Ang II–induced IL-6 gene expression, cardiac fibroblasts were pretreated with cell-permeable thiol antioxidants N-acetylcysteine (NAC), O^2- scavenger Tiron, or NADH/NADPH oxidase inhibitor DPI for 1 hour, and stimulated with Ang II. RT-PCR analysis showed that both NAC, Tiron, and DPI inhibited IL-6 mRNA expression, but did not affect ß-actin mRNA (Figure 2B). These findings suggested that intracellular ROS generation plays an important role in Ang II–induced IL-6 gene expression.

Effect of Exogenous H₂O₂ on IL-6 Gene Expression
To determine whether ROS-mediated signaling pathways induce IL-6 gene expression, we stimulated cells with various concentrations of exogenous H₂O₂ for 30 minutes, and measured IL-6 expression by using both RT-PCR and poly(A)⁺ RNA Northern blot analysis. Exogenous H₂O₂ augmented IL-6 gene expression in a dose-dependent manner, with a 50 to 100 µmol/L threshold and a maximal effect occurring at 200 µmol/L (Figures 3A and 3B). These concentrations are similar to those previously reported for H₂O₂-stimulated p38 MAPK activation in vascular smooth muscle cells (VSMCs).^6,25

View larger version (28K): [in this window] [in a new window]   Figure 3. Effect of exogenous H2O2 on IL-6 gene expression and signaling pathways in cardiac fibroblasts. A, Exogenous H2O2 induced IL-6 gene expression in cardiac fibroblasts. Cardiac fibroblasts were stimulated with various concentration of H2O2, and RNA was isolated at 30 minutes. RT-PCR was performed as described in Materials and Methods. Similar results were obtained in 3 separate experiments. M indicates XHaeIII/HindIII marker. B, Poly(A)+ RNA Northern blot analysis of IL-6 in cells stimulated with H2O2. Poly(A)+ RNA (4 µg) was loaded in each lane. C, Effect of exogenous H2O2 on phosphorylation of ERK, JNK, and p38 MAPK in cardiac fibroblasts. Cardiac fibroblasts stimulated by exogenous H2O2 were lysed, and Western blot analysis was performed. Both ERK, JNK, and p38 MAPK were activated by exogenous H2O2. Note that the activation of ERKs was also indicated by their band shift. Similar results were obtained in 2 separate experiments.

Exogenous H₂O₂ Activated Three MAPK Cascades
Recent studies have shown that exogenous H₂O₂ influences the MAPK cascade in various cell types. Thus, we next assessed the effect of exogenous H₂O₂ on MAPK activation in cardiac fibroblasts. We stimulated cells with 200 µmol/L H₂O₂ and detected the phosphorylation of individual MAPKs. ERK1/2 and JNK1 were activated by H₂O_2, which peaked at 15 minutes and declined thereafter. H₂O₂ induced rapid and sustained activation of p38 MAPK, which peaked at 5 minutes and was still detectable at 60 minutes (Figure 3C).

Ang II Activates p38 MAPK, ERK, and JNK in a Redox-Sensitive Manner
Previous reports revealed that Ang II activates ERK, JNK, and p38 MAPK in cardiac fibroblasts. However, it remains unclear whether the activation of these pathways is redox-dependent or not. To determine whether these pathways were redox-sensitive, we examined the effect of DPI or NAC on individual MAPK phosphorylation. We first confirmed that Ang II elicits a rapid and robust phosphorylation of p38 MAPK, ERK1/2, and JNK-1 with a peak at 5 minutes in cardiac fibroblasts (data not shown). Both DPI (10 µmol/L) and NAC (10 mmol/L) significantly inhibited Ang II–induced phosphorylation of p38 MAPK, ERK1/2 and JNK-1 (Figure 4A) in a dose-dependent manner. To exclude the possibility of these findings owing to nonspecific or toxic effects of these inhibitors, we preincubated the cells with sense and antisense oligonucleotides to p22phox, stimulated with Ang II, and detected phosphorylations of ERK, JNK, and p38 MAPK. Antisense oligonucleotides significantly inhibited the phosphorylation of these kinases, whereas sense oligonucleotides did not (Figures 4B and 4D). We also observed that NAC, DPI, and antisense oligonucleotide failed to attenuate Ang II–induced activation of JAK2 (Figure 4E). We also confirmed that antisense oligonucleotides to p22phox markedly decreased its mRNA expression, whereas the sense oligonucleotides did not (Figure 4F). These findings suggest that p38 MAPK, ERK1/2, and JNK-1 might be critical components of the redox-sensitive signaling pathways activated by Ang-II in cardiac fibroblasts.

View larger version (25K): [in this window] [in a new window]   Figure 4. Angiotensin II–induced activation of ERK, JNK, and p38 MAPK was partially mediated by ROS-sensitive pathway. A, Cells were pretreated with NADH/NADPH oxidase inhibitor DPI (10 µmol/L) or thiol antioxidant NAC (10 mmol/L) for 60 minutes and simulated with Ang II. Phosphorylation of ERK, JNK, or p38 MAPK was detected by Western blotting using anti–phospho-ERK, phospho-JNK, and phospho–p38 MAPK antibodies. Both NAC and DPI inhibited Ang II–induced activation of ERK, JNK, and p38 MAPK in a dose-dependent manner. Similar results were obtained in 3 separate experiments. B through E, Densitometric analyses of the phosphorylaions of ERK, JNK, p38 MAPK, and JAK2 in Ang II–stimulated cardiac fibroblasts. Cardiac fibroblasts were preincubated with p22phox: 5'-GGTCCTCACCATGGGGCAGATC-3' and antisense p22phox: 5'-GATCTGCCCCATGGTGAGGACC-3' oligonucleotides to NADPH oxidase, DPI, and NAC, and stimulated with Ang II. Phosphorylations of ERK, JNK, and p38 MAPK were detected, and densitometric analyses were performed. Results were obtained from 6 separate experiments. *P<0.01 vs control, #P<0.01 vs Ang II alone. NS indicates not significant vs Ang II alone. F, Antisense oligonucleotides decreased mRNA expression of p22phox in cardiac fibroblasts. Cardiac fibroblasts were treated with sense and antisense oligonucleotides for p22phox for 6 hours, and total RNA was isolated. PCR primers were prepared for p22phox (sense: 5'-TCCTTCAGACCTATCGGGCCATCGC-3'; antisense: 5'-TGTCGCTGGGCCTGGG-TTATCTCC-3'), and RT-PCR was performed as described12 for 30 cycles. Antisense oligonucleotides suppressed the mRNA expression of p22phox, whereas sense oligonucleotides did not.

Effect of ERK and/or p38 MAPK Pathways on Ang II—Induced Expression of IL-6 Gene
We next determined the role of redox-sensitive activation of MAPKs in Ang II—induced IL-6 gene expression. PD98059, a specific inhibitor of MKK-1 (MEK), inhibited augmentation of IL-6 gene expression after Ang II stimulation. SB203580, a specific inhibitor of p38 MAPK, also inhibited this expression (Figure 5). These findings suggest that activation of both ERK and p38 MAPK is a necessary step for IL-6 gene expression by Ang II.

View larger version (86K): [in this window] [in a new window]   Figure 5. Angiotensin II–induced IL-6 gene expression was attenuated by PD98059 and SB203580. Cardiac fibroblasts were stimulated with Ang II in the presence of PD98059 (20 µmol/L) or SB203580 (30 µmol/L), and total RNA was isolated at 30 minutes. RT-PCR was performed. Both PD98059 and SB203580 attenuated Ang II–induced IL-6 gene expression. The results were reproducible in 4 separate experiments.

Ang II Does Not Activate NF-B in Cardiac Fibroblasts
Another potential target of the ROS might be the NF-B pathways. The previous study reported that NF-B plays an important role in the transcriptional regulation of IL-6 expression, and that the activation of NF-B might be redox dependent. To investigate the molecular mechanism of Ang II–induced IL-6 expression, we determined whether Ang II could activate NF-B in cardiac fibroblasts. Interleukin-1ß (IL-1ß) was used as a positive control. Agents that activate NF-B induce phosphorylation of IB-/ß, which leads to ubiquitination/proteosomal degradation of IB-/ß. Degradation of IB unmasks the nuclear localization sequence of the NF-B complex, which is composed of p65 and p50 subunits, and allows NF-B to enter the nucleus.

IL-1ß induced rapid phosphorylation of IB- in cardiac fibroblasts (Figure 6A), but Ang II did not. Next, we investigated whether Ang II degrades IB- and IB-ß proteins in cardiac fibroblasts. IL-1ß degraded IB- from 10 minutes and IB-ß from 30 minutes in cardiac fibroblasts. On the other hand, Ang II did not induce degradation of these proteins (Figures 6B and 6C). We also performed Western blot analysis of the NF-B p65 subunit using the nuclear fraction. It should be noted that a small but significant amount of p65 exists in the nucleus under basal condition. However, Ang II had a marginal effect on the amount of p65 in the nucleus, whereas IL-1ß caused nuclear accumulation of p65 (Figure 6D). We also determined whether these findings were reproducible in human cardiac fibroblasts. IL-1ß induced rapid phosphorylation of IB-, although Ang II did not (Figure 6E).

View larger version (17K): [in this window] [in a new window]   Figure 6. NF-B–mediated pathway is not involved in angiotensin II–induced IL-6 gene expression. A, Effect of Ang II and IL-1ß on phosphorylation of IB- in cardiac fibroblasts. Cells were stimulated with Ang II or IL-1ß (10 ng/mL). Phosphorylation of IkB- was detected by anti–phospho-IB- antibody. IL-1ß strongly induced phosphorylation of IB- but not Ang II. Similar results were obtained in 3 separate experiments. B, Effect of Ang II and IL-1ß on degradation of IB- in cardiac fibroblasts. IL-1ß induced degradation of IB-, whereas Ang II had no effect on that of IB-. We obtained similar results in 2 additional experiments. C, Effect of Ang II and IL-1ß on degradation of IB-ß in cardiac fibroblasts. Similar results were obtained in 3 separate experiments, and degradation of IB-ß was detected. D, Effect of Ang II and IL-1ß on nuclear translocation of NF-B p65 in cardiac fibroblasts. After cells were stimulated with Ang II or IL-1ß, nuclear extracts were isolated. Western blot analysis was performed to detect nuclear translocation of NF-B p65. IL-1ß induced translocation of p65, but Ang II did not. E, Effects of Ang II and IL-1ß on phosphorylation of IB- in human cardiac fibroblasts. Human cardiac fibroblasts were stimulated as described above, and phosphorylation of IB- was observed. F, Cardiac fibroblasts were transfected with a luciferase reporter plasmid driven by NF-B promoter activity with pIRES-EGFP-IB or empty plasmid and stimulated with Ang II or IL-1ß. Note that IL-1ß enhanced NF-B promotor activity 2.8-fold, whereas Ang II did not. Data were expressed as mean±SD from the 4 separate experiments. *P<0.01 vs control.

To confirm that the NF-B pathway is not activated by Ang II in cardiac fibroblasts, cells were transfected with a luciferase plasmid driven by NF-B (pNFB-luc), and stimulated with Ang II or IL-1ß. IL-1ß enhanced NF-B promoter activity by 2.8-fold compared with the control, whereas Ang II had no effect (Figure 6F). These findings indicate that the NF-B pathway might play a marginal role in the Ang II–mediated signaling pathway in cardiac fibroblasts.

Identification of Ang II–Responsive Elements in the IL-6 Promoter
The IL-6 promoter contains a complex control region that includes AP-1, CRE, C/EBP, and NF-B sites that can be triggered by multiple activation pathways. We isolated the Ang II–responsive elements of the IL-6 promoter in cardiac fibroblasts. The various IL-6 promoter–luciferase plasmids used previously were transfected into cardiac fibroblasts and luciferase activity was measured with Ang II stimulation. Ang II stimulation for 2 hours significantly increased luciferase activity in p417 (multiple transcription factor binding sites) by 3.3-fold. After truncation of the IL-6 promoter from the 5'-end, the Ang II response disappeared in p140 (Figure 7A). These results clearly show that the NF-B binding element is not responsible for the induction of IL-6 mRNA by Ang II.

View larger version (17K): [in this window] [in a new window]   Figure 7. Identification of Ang II—responsive elements in IL-6 promotor. A, A series of deletion mutants of IL-6 promotor gene plasmids were cotransfected into cardiac fibroblasts with control plasmid pRL-SV40. Transfected cells were stimulated with Ang II for 2 hours, and luciferase activity was measured using a dual luciferase assay kit. NF-B was not responsible for the induction of IL-6 gene expression by Ang II. GRE indicates glucocorticoid receptor response element. B, Wild type, CRE-mutant, or C/EBP-mutant of IL-6 promoter–luciferase plasmids was cotransfected into cardiac fibroblasts with pRL-SV40. Cells were stimulated with Ang II for 6 hours. The mutation of CRE strongly abolished the responsiveness to Ang II. C, pIRES-EGFP-IB plasmid was cotransfected into cardiac fibroblasts with pNF-B–luciferase plasmid. IB is a truncated form of IB- that lacks phosphorylation sites essential for the activation of NF-B. IB reduced NF-B promotor activity to 14% of the control cell level under in unstimulated conditions, indicating that NF-B was activated to some extent in basal conditions in cardiac fibroblasts.

To determine which element is responsible for Ang II–induced transcriptional activation, point-mutated luciferase reporter plasmids for CRE, C/EBP, and NF-B were transfected into cardiac fibroblasts. Mutation of the C/EBP faintly decreased the baseline and slightly decreased the Ang II response. On the other hand, 5'mutation of the CRE-binding site strongly abolished the Ang II response (Figure 7B). These results indicated that the CRE binding element was mainly responsible for the induction of IL-6 gene expression by Ang II in cardiac fibroblasts.

Interestingly, we found that IL-6 promoter activity in unstimulated conditions was markedly reduced when the NF-B site was mutated in this assay (data not shown). We suspect that NF-B is activated in basal conditions in cardiac fibroblasts (see Figure 6D). To specifically inhibit NF-B activation, we cotransfected pIRES-EGFP-IB plasmid with pNF-B–luciferase plasmid and compared the luciferase activity with the control cells (Figure 7C). IB is a truncated form of IB- that lacks phosphorylation sites essential for the degradation of IB. The luciferase activity of the IB-transfected cells was suppressed to 14% of the control cell level, indicating that NF-B was always activated to some extent in cardiac fibroblasts.

Ang II Phosphorylated CREB Via ERK and p38 MAPK by a Redox-Sensitive Manner
One well-characterized CRE-binding transcription factor is CREB. CREB is phosphorylated by MAPK-activated protein kinase, which is downstream of ERK and/or p38 MAPK. Finally, we investigated whether CREB is activated by Ang II via a redox-dependent pathway (Figures 8A and 8B). We found CREB to be phosphorylated by Ang II, and that this phosphorylation was suppressed by both PD98059 and SB203580. Moreover, phosphorylation of CREB was dose-dependently inhibited by NAC and DPI. These findings indicated that CREB was phosphorylated via the ERK and p38 MAPK pathways in a redox-sensitive manner.

View larger version (24K): [in this window] [in a new window]   Figure 8. Ang II phosphorylated CREB via ERK and p38 MAPK by a redox-sensitive manner. Cells were preincubated with either NAC, DPI, PD98059, or SB203580, and stimulated with Ang II. Phosphorylation of CREB was observed by Western blot analysis.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Reactive oxygen species have recently been recognized to act as an intracellular second messenger, which can regulate various intracellular signal transduction cascades and transcription factors. In this study, we demonstrated that Ang II activated NADH/NADPH oxidase to generate intracellular ROS via an AT-1 receptor in cardiac fibroblasts, and that ROS mediated Ang II–induced activation of MAPK pathways, which culminated in IL-6 gene expression. We also found that CRE was a crucial cis-element for Ang II–mediated induction of IL-6 in cardiac fibroblasts. The present study demonstrated that Ang II activates ERK1/2, p38 MAPK, and JNK partly through ROS in cardiac fibroblasts. In VSMC, however, Ang II–generated ROS stimulated p38 MAPK, but not ERKs.⁶ Thus, the sensitivity of ERKs to oxidative signals appears to vary from cell type to cell type.^5,26,27 It remains unknown how ROS mediate ERK activation and why the role of ROS on activation of this pathway cannot be generalized. There are multiple pathways leading to ERK activation by Ang II, which also differ between cell types. These distinctive pathways might result in different sensitivity of ROS. Previous reports revealed that Ang II activates ERK via either Src- or EGF receptor–mediated pathway in cardiac fibroblasts.²⁸ In this tyrosine kinase-mediated upstream pathway, the candidate-direct targets of ROS were Src or protein tyrosine phosphatase.^29,30 Pu et al²⁹ demonstrated that ROS activated Src through selective phosphorylation at tyrosine 416 in NIH3T3 cells. Wang et al³¹ showed that NAC inhibited Src phosphorylation at tyrosine 416 by Ang II in cardiac fibroblasts. Mukhin et al³² mapped the major point of intersection to ROS to upstream of or at the level of Src in the GißSrcERK pathway using 5-HT1A receptor-transfected CHO cells. They noted that Src inhibitor PP1 did not block Ang II–induced ROS generation, and suggested that NAD(P)H oxidase is located upstream of Src. Because all tyrosine phosphatases have a conserved cysteine residue in their catalytic domain, it has been proposed that inhibition of tyrosine phosphatase (PTPase) activity by ROS may account for another mechanism of stimulation of tyrosine phosphorylation by ROS. Furthermore, the specific activities of PTPase in vitro are 10 to 1000 times higher than those of protein tyrosine kinases. Therefore, concurrent inhibition of PTPase by ROS might be necessary for any increase in the level of tyrosine phosphorylation.³³

Previous studies revealed that NF-B participates in Ang II–mediated signal transduction in hepatocytes, monocytes,³⁴ VSMCs, and mesangial cells, and regulates induction of MCP-1, IL-6, and angiotensinogen genes. Although Ang II increases NF-B transcriptional activity in several cell lines, the molecular mechanisms have not been determined, nor has it been established whether this phenomenon occurs ubiquitously. Several evidences suggest that ROS serve as a messenger in NF-B activation.⁴ PDTC has been shown to block Ang II–induced NF-B activation in VSMCs and mesangial cells.³⁵ Because Ang II induced intracellular ROS production, we investigated whether Ang II–induced ROS activated NF-B in cardiac fibroblasts. As reported previously, IL-1ß and H₂O₂ induced a sequential degradation of IB- and IB-ß, followed by the translocation of p65 to the nucleus (Figure 6 and data not shown). However, Ang II did not influence phosphorylation or degradation of IB or nuclear translocation of p65. We also found that IL-1ß increased NF-B–luciferase activity, but Ang II did not. Promoter analysis revealed that NF-B did not contribute to IL-6 induction by Ang II in cardiac fibroblasts. These findings indicated that, although the precise mechanism remains unknown, the activation of NF-B pathway in response to Ang II differs between cell types.

The deletion and mutation analysis of the IL-6 gene promoter showed that NF-B plays an important role in IL-6 gene expression in basal conditions. We found that a significant amount of p65 was located in the nucleus in unstimulated cardiac fibroblasts, and that transfection of IB plasmid suppressed the basal NF-B–luciferase activity to 14%, indicating that NF-B was activated to some extent. We think that this finding explains why NF-B was important for basal expression of IL-6, and that IL-6 is constitutively expressed in cardiac fibroblasts.¹¹ Kranzhofer et al³⁴ reported that human peripheral lymphocytes have constitutive NF-B binding activity which was not influenced by Ang II. Their observation was consistent with our results in cardiac fibroblasts.

Finally, we determined the Ang II–responsive elements in the IL-6 gene promoter. Truncation and mutational analysis of the IL-6 gene promoter showed that CRE was an important cis-element for Ang II–induced IL-6 gene expression. We demonstrated that CREB, which was one of the best-characterized CRE-binding transcription factors, was activated by ERK and p38 MAPK in a redox-sensitive manner. Funakoshi et al³⁶ showed that Ang II increased binding of CREB to the CRE site of the IL-6 gene promoter using gel mobility shift assays in VSMCs. Moreover, we showed that p38 MAPK and ERK were critical in Ang II–stimulated IL-6 gene expression. Collectively, the CREB might be involved in redox-sensitive CRE-dependent IL-6 gene induction by Ang II.

In conclusion, Ang II activated NADH/NADPH oxidase to generate ROS via the AT-1 receptor, and these ROS were at least partly involved in the activation of ERK, JNK, and p38 MAPK pathways, but not the NF-B pathway, in cardiac fibroblasts. The ROS-MAPKs (ERK and p38 MAPK)-meditated CRE-dependent transcription plays an important role in Ang II–induced IL-6 gene expression in cardiac fibroblasts. The mechanism by which ROS activated various signaling pathways remains undetermined and should be clarified in the near future.

	Acknowledgments

 
This study was supported in part by a Research Grant from the Research for the Future Program from the Japan Society for the Promotion of Science (JSPS-RFTF97I00201), a Research Grant from the Ministry of Education, Science, and Culture, Japan, and a Health Science Research Grant for Advanced Medical Technology from the Ministry of Welfare, Japan.

Received September 11, 2000; revision received May 23, 2001; accepted September 6, 2001.

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