ERK and p38 MAPK, but not NF-κB, Are Critically Involved in Reactive Oxygen Species–Mediated Induction of IL-6 by Angiotensin II in Cardiac Fibroblasts
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 IκB-α/β or nuclear translocation of p65, nor did it increase NF-κB 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-κB–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-κB–dependent, pathway in cardiac fibroblasts.
- angiotensin II
- reactive oxygen species
- mitogen-activated protein kinase
- cardiac fibroblast
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-κB 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-κB by ROS appears to be the phosphorylation and subsequent degradation of IκB.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)-α–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-α, IL-1β, 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-κB.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
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 40× 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 Iκ-Bα, and anti–phospho-specific CREB antibodies were purchased from New England Biolabs Inc (Beverly, MA). Anti-ERK1, anti–p38 MAPK, anti-JNK, anti-IκBα, anti-IκBβ 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 β-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-κB binding site (pNFκB-luc) was purchased from Stratagene. pIRES-EGFP-ΔIκB 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-κB–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).
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.
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.
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.
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), O2− 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 β-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
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 H2O2, 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.
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.
Ang II Does Not Activate NF-κB in Cardiac Fibroblasts
Another potential target of the ROS might be the NF-κB pathways. The previous study reported that NF-κB plays an important role in the transcriptional regulation of IL-6 expression, and that the activation of NF-κB might be redox dependent. To investigate the molecular mechanism of Ang II–induced IL-6 expression, we determined whether Ang II could activate NF-κB in cardiac fibroblasts. Interleukin-1β (IL-1β) was used as a positive control. Agents that activate NF-κB induce phosphorylation of IκB-α/β, which leads to ubiquitination/proteosomal degradation of IκB-α/β. Degradation of IκB unmasks the nuclear localization sequence of the NF-κB complex, which is composed of p65 and p50 subunits, and allows NF-κB to enter the nucleus.
IL-1β induced rapid phosphorylation of IκB-α in cardiac fibroblasts (Figure 6A), but Ang II did not. Next, we investigated whether Ang II degrades IκB-α and IκB-β proteins in cardiac fibroblasts. IL-1β degraded IκB-α from 10 minutes and IκB-β 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-κB 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-1β caused nuclear accumulation of p65 (Figure 6D). We also determined whether these findings were reproducible in human cardiac fibroblasts. IL-1β induced rapid phosphorylation of IκB-α, although Ang II did not (Figure 6E).
To confirm that the NF-κB pathway is not activated by Ang II in cardiac fibroblasts, cells were transfected with a luciferase plasmid driven by NF-κB (pNFκB-luc), and stimulated with Ang II or IL-1β. IL-1β enhanced NF-κB promoter activity by 2.8-fold compared with the control, whereas Ang II had no effect (Figure 6F). These findings indicate that the NF-κB 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-κB 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-κB binding element is not responsible for the induction of IL-6 mRNA by Ang II.
To determine which element is responsible for Ang II–induced transcriptional activation, point-mutated luciferase reporter plasmids for CRE, C/EBP, and NF-κB 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-κB site was mutated in this assay (data not shown). We suspect that NF-κB is activated in basal conditions in cardiac fibroblasts (see Figure 6D). To specifically inhibit NF-κB activation, we cotransfected pIRES-EGFP-ΔIκB plasmid with pNF-κB–luciferase plasmid and compared the luciferase activity with the control cells (Figure 7C). ΔIκB is a truncated form of IκB-α that lacks phosphorylation sites essential for the degradation of IκB. The luciferase activity of the ΔIκB-transfected cells was suppressed to 14% of the control cell level, indicating that NF-κB 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.
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 Giβγ→Src→→ERK 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-κB 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-κB 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-κB activation.4 PDTC has been shown to block Ang II–induced NF-κB activation in VSMCs and mesangial cells.35 Because Ang II induced intracellular ROS production, we investigated whether Ang II–induced ROS activated NF-κB in cardiac fibroblasts. As reported previously, IL-1β and H2O2 induced a sequential degradation of IκB-α and IκB-β, 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 IκB or nuclear translocation of p65. We also found that IL-1β increased NF-κB–luciferase activity, but Ang II did not. Promoter analysis revealed that NF-κB 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-κB pathway in response to Ang II differs between cell types.
The deletion and mutation analysis of the IL-6 gene promoter showed that NF-κB 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 ΔIκB plasmid suppressed the basal NF-κB–luciferase activity to 14%, indicating that NF-κB was activated to some extent. We think that this finding explains why NF-κB 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-κB 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-κB 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.
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.
Original received September 11, 2000; resubmission received May 23, 2001; revised resubmission received September 6, 2001. accepted September 6, 2001.
Griendling KK, Minieri CA, Ollerenshaw JD, Alexander RW. Angiotensin II stimulates NADH and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 1994; 74: 1141–1148.
Winyard PG, Blake DR. Antioxidants, redox-regulated transcription factors, and inflammation. Adv Pharmacol. 1997; 38: 403–421.
Sen CK, Packer L. Antioxidant and redox regulation of gene transcription. FASEB J. 1996; 10: 709–720.
Traenckner EB, Wilk S, Baeuerle PA. A proteasome inhibitor prevents activation of NF-κB and stabilizes a newly phosphorylated form of IκB-α that is still bound to NF-κB. EMBO J. 1994; 15: 5433–5441.
Baas AS, Berk BC. Differential activation of mitogen-activated protein kinases by H2O2 and O2− in vascular smooth muscle cells. Circ Res. 1995; 77: 29–36.
Ushio-Fukai M, Alexander RW, Akers M, Griendling KK. p38 Mitogen-activated protein kinase is a critical component of the redox-sensitive signaling pathways activated by angiotensin II: role in vascular smooth muscle cell hypertrophy. J Biol Chem. 1998; 273: 15022–15029.
Zafari AM, Ushio-Fukai M, Akers M, Yin Q, Shah A, Harrison DG, Taylor WR, Griendling KK. Role of NADH/NADPH oxidase-derived H2O2 in angiotensin II-induced vascular hypertrophy. Hypertension. 1998; 32: 488–495.
Ushio-Fukai M, Zafari A, Fukui T, Ishizaka N, Griendling KK. p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II–induced hypertrophy in vascular smooth muscle cells. J Biol Chem. 1996; 271: 23317–23321.
Tummala PE, Chen XL, Sundell CL, Laursen JB, Hammes CP, Alexander RW, Harrison DG, Medford RM. Angiotensin II induces vascular cell adhesion molecule-1 expression in rat vasculature: a potential link between the renin-angiotensin system and atherosclerosis. Circulation. 1999; 100: 1223–1229.
Nakamura K, Fushimi , Kouchi H, Mihara K, Miyazaki M, Ohe T, Namba M. Inhibitory effects of antioxidants on neonatal rat cardiac myocyte hypertrophy induced by tumor necrosis factor-α and angiotensin II. Circulation. 1998; 98: 794–799.
Sano M, Fukuda K, Kodama H, Pan J, Saito M, Matsuzaki J, Takahashi T, Makino S, Kato T, Ogawa S. Interleukin-6 family of cytokines mediate angiotensin II-induced cardiac hypertrophy in rodent cardiomyocytes. J Biol Chem. 2000; 275: 29717–29723.
Finkel MS, Oddis CV, Jacob TD, Watkins SC, Hattler BG, Simmons RL. Negative inotropic effects of cytokines on the heart mediated by nitric oxide. Science. 1992; 257: 387–389.
Hirota H, Yoshida K, Kishimoto T, Taga T. Continuous activation of gp130, a signal-transducing receptor component for interleukin 6-related cytokines, causes myocardial hypertrophy in mice. Proc Natl Acad Sci U S A. 1995; 92: 4862–4866.
Sheng Z, Knowlton K, Chen J, Hoshijima M, Brown JH, Chien KR. Cardiotrophin 1 inhibition of cardiac myocyte apoptosis via a mitogen-activated protein kinase-dependent pathway: divergence from downstream CT-1 signals for myocardial cell hypertrophy. J Biol Chem. 1997; 272: 5783–5791.
Sano M, Fukuda K, Kodama H, Takahashi T, Kato T, Hakuno D, Sato T, Manabe T, Tahara S, Ogawa S. Autocrine/Paracrine secretion of IL-6 family cytokines causes angiotensin II-induced delayed STAT3 activation. Biochem Biophys Res Commun. 2000; 269: 798–802.
Saito S, Aikawa R, Shiojima I, Nagai R, Yazaki Y, Komuro I. Endothelin-1 induces expression of fetal genes through the interleukin-6 family of cytokines in cardiac myocytes. FEBS Lett. 1999; 456: 103–107.
Pan J, Fukuda K, Kodama H, Sano M, Takahashi T, Makino S, Kato T, Manabe T, Hori S, Ogawa S. Involvement of gp130-mediated signaling in pressure overload-induced activation of the JAK/STAT pathway in rodent heart. Heart Vessels. 1998; 13: 199–208.
Pan J, Fukuda K, Saito M, Matsuzaki J, Kodama H, Sano M, Takahashi T, Kato T, Ogawa S. Mechanical stretch activates the JAK/STAT pathway in rat cardiomyocytes. Circ Res. 84, 1999; 1127–1136:.
Tanabe O, Akira S, Kamiya T, Wong GG, Hirano T, Kishimoto T. Genomic structure of the murine IL-6 gene: high degree conservation of potential regulatory sequences between mouse and human. J Immunol. 1988; 141: 3875–3881.
Kodama H, Fukuda K, Pan J, Makino S, Baba A, Hori S, Ogawa S. Leukemia inhibitory factor, a potent cardiac hypertrophic cytokine, activates the JAK/STAT pathway in rat cardiomyocytes. Circ Res. 1997; 81: 656–663.
Suematsu M, Schmid-Schoenbein GW, Chavez-Chavez RH, Yee TT, DeLano FA, Tamatani T, Miyasaka M, Zweifach BW. In vivo visualization of oxidative changes in microvessels during neutrophil activation. Am J Physiol. 1993; 264: H881–H891.
Hayashi S, Takamiya R, Yamaguchi T, Matsumoto K, Tojo SJ, Tamatani T, Kitajima M, Makino N, Ishimura Y, Suematsu M. Induction of heme oxygenase-1 suppresses venular leukocyte adhesion elicited by oxidative stress: role of bilirubin generated by the enzyme. Circ Res. 1999; 85: 663–671.
Matsui H, Ihara Y, Fujio Y, Kunisada K, Akira S, Kishimoto T, Yamauchi-Takihara K. Induction of interleukin-6 by hypoxia is mediated by nuclear factor-κB and NF-IL6 in cardiac myocytes. Cardiovasc Res. 1999; 42: 104–112.
Vanden Berghe W, De Bosscher K, Boone E, Plaisance S, Haegeman G. The nuclear factor-κB engages CBP/p300 and histone acetyltransferase activity for transcriptional activation of the interleukin-6 gene promoter. J Biol Chem. 1999; 274: 32091–32098.
Ushio-Fukai M, Alexander RW, Akers M, Yin Q, Fujio Y, Walsh K, Griendling KK. Reactive oxygen species mediate the activation of Akt/protein kinase B by angiotensin II in vascular smooth muscle cells. J Biol Chem. 1999; 274: 22699–22704.
Abe J, Kusuhara M, Ulevitch RJ, Berk BC, Lee JD. Big mitogen-activated protein kinase 1 is a redox-sensitive kinase. J Biol Chem. 1996; 271: 16586–16590.
Kyriakis JN, Avuruch J. Sounding the alarm: protein kinase cascades activated by stress and inflammation. J Biol Chem. 1996; 271: 24313–24316.
Murasawa S, Mori Y, Nozawa Y, Gotoh N, Shibuya M, Masaki H, Maruyama K, Tsutsumi Y, Moriguchi Y, Shibazaki Y, Tanaka Y, Iwasaka T, Inada M, Matsubara H. Angiotensin II type 1 receptor-induced extracellular signal-regulated protein kinase activation is mediated by Ca2+/calmodulin-dependent transactivation of epidermal growth factor receptor. Circ Res. 1998; 29: 1338–1378.
Pu M, Akhand AA, Kato M, Hamaguchi M, Koike T, Iwata H, Sabe H, Suzuki H, Nakashima I. Evidence of a novel redox-linked activation mechanism for the Src kinase which is independent of tyrosine 527-mediatedregulation. Oncogene. 1996; 13: 2615–2622.
Devary Y, Gottlieb RA, Smeal T, Karin M. The mammalian ultraviolet response is triggered by activation of Src tyrosine kinases. Cell. 1992; 71: 1081–1091.
Wang D, Yu X, Cohen RA, Brecher P. Distinct effects of N-acetylcysteine and nitric oxide on angiotensin II-induced epidermal growth factor receptor phosphorylation and intracellular Ca2+ levels. J Biol Chem. 2000; 275: 12223–12230.
Mukhin YV, Garnovskaya MN, Collinsworth G, Grewal JS, Pendergrass D, Nagai T, Pinckney S, Greene EL, Raymond JR. 5-Hydroxytryptamine1A receptor/Gibetagamma stimulates mitogen-activated protein kinase via NAD(P)H oxidase and reactive oxygen species upstream of src in chinese hamster ovary fibroblasts. Biochem J. 2000; 347: 61–67.
Fischer EH, Charbonneau H, Tonks NK. Protein tyrosine phosphatases: a diverse family of intracellular and transmembrane enzymes. Science. 1991; 253: 401–406.
Kranzhofer R, Browatzki M, Schmidt J, Kubler W. Angiotensin II activates the proinflammatory transcription factor nuclear factor-κB in human monocytes. Biochem Biophys Res Commun. 1999; 257: 826–828.
Rovin BH, Dickerson JA, Tan LC, Fassler J. Modulation of IL-1-induced chemokine expression in human mesangial cells through alterations in redox status. Cytokine. 1997; 3: 178–186.
Funakoshi Y, Ichiki T, Ito K, Takeshita A. Induction of interleukin-6 expression by angiotensin II in rat vascular smooth muscle cells. Hypertension. 1999; 34: 118–125.