Ets-1 Is a Critical Transcriptional Regulator of Reactive Oxygen Species and p47phox Gene Expression in Response to Angiotensin II
Angiotensin (Ang) II is a potent mediator of vascular inflammation. A central mechanism by which Ang II promotes inflammation is through the generation of reactive oxygen species (ROS). In the current study, we investigated the role of the transcription factor Ets-1 in regulating Ang II–induced ROS generation. ROS generation was measured in the thoracic aorta of Ets-1−/− mice compared with littermate controls after continuous infusion of Ang II. H2O2 and superoxide anion (O2−) production were significantly blunted in the Ets-1−/− mice. Inhibition of Ets-1 expression by small interfering RNA in primary human aortic smooth muscle cells also potently inhibited ROS production and the induction of the NAD(P)H oxidase subunit p47phox in response to Ang II. To evaluate the therapeutic potential of inhibiting Ets-1 in wild-type mice, dominant negative Ets-1 membrane-permeable peptides were administered systemically. Ang II–induced ROS production and medial hypertrophy in the thoracic aorta were markedly diminished as a result of blocking Ets-1. In summary, Ets-1 functions as a critical downstream transcriptional mediator of Ang II ROS generation by regulating the expression of NAD(P)H oxidase subunits such as p47phox.
Angiotensin (Ang) II is widely recognized as a critical regulator of the development of hypertension and atherosclerosis.1 Several studies support a central role for reactive oxygen species (ROS) in Ang II–mediated cellular events.2 ROS promote lipoprotein oxidation, foam cell formation, smooth muscle cell hypertrophy, and endothelial damage.3 Production of ROS in response to Ang II within the vessel wall promotes vascular inflammation by inducing the expression of inflammatory genes such as the chemokine monocyte chemoattractant protein-1 and the adhesion molecule vascular cell adhesion molecule-1.4,5 Other Ang II–mediated cellular effects include medial hypertrophy and endothelial dysfunction. It is generally recognized that a major source of ROS is NAD(P)H oxidase.6
Activation of NAD(P)H oxidase involves the assembly of phosphorylated NAD(P)H subunits, including p40phox, p47phox, and p67phox, into a macromolecular complex together with gp91phox and p22phox.7 Although several of the NAD(P)H subunits are constitutively expressed in vascular cells, further induction of the individual subunits also occurs via increased gene transcription. For example, expression of the gp91phox homolog Nox1 is highly induced in response to Ang II or thrombin.8 Ang II stimulation of human vascular (V)SMCs is associated with 2- to 3-fold increases in the level of expression of p40phox, p47phox, p67phox, and p22phox within 4 to 24 hours.9
The regulation of NAD(P)H oxidase gene expression at the transcriptional level in the setting of inflammation remains incompletely understood. We recently identified a novel role for the ETS transcription factor Ets-1 as a transcriptional mediator of vascular inflammation and remodeling.10 Ets-1 is induced in VSMCs in response to a variety of inflammatory mediators.11–13 The induction of the chemokine monocyte chemoattractant protein-1, the adhesion molecule vascular cell adhesion molecule-1, and plasminogen activator inhibitor-1 by Ang II is largely dependent on Ets-1.10 In the current study, we identified a novel role for Ets-1 as a transcriptional mediator of Ang II–mediated ROS generation.
Materials and Methods
Tissue Culture and Reagents
Primary human aortic (HA)SMCs (Cambrex Bio Science Inc, Walkersville, Md) and rat aortic (RA)SMCs were grown in DMEM with 10% FBS as previously described.14 Cells were made quiescent by culture in 0.1% FBS-containing media for 24 to 48 hours before Ang II stimulation.
Expression Vector and Luciferase Reporter Gene Constructs
A 3067-bp fragment corresponding to nucleotides −3024 to +53 of the human p47phox promoter was cloned from human genomic DNA (BD Biosciences, Palo Alto, Calif) by PCR as previously described.10 Additional details are in the expanded Materials and Methods section of the online data supplement at http://circres.ahajournals.org.
Site-directed mutagenesis on the p47phox promoter was performed using the quickChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, Calif) according to the protocols of the manufacturer.
DNA Transfection and Reporter Assay
Transfection of RASMCs was performed as described previously.10 Briefly, cotransfection of 0.3 μg of reporter gene construct and 0.15 μg of expression vector was performed with 4 μL of Lipofectamine (Invitrogen, Carlsbad, Calif) using 2×105 cells per well.
Electrophoretic Mobility-Shift Analysis and Chromatin Immunoprecipitation Analysis
To determine the DNA–protein interactions, electrophoretic mobility-shift analysis was performed as previously described.10,15 The sequences for the oligonucleotide probes and detailed methods are provided in the expanded Materials and Methods section in the online data supplement.
All peptides were synthesized at the Tufts University Core Facility (Boston, Mass) as previously described.16 The HIV-1 TAT peptide (TryGlyArgLysLysArgArgGlnArgArgArgGly) was added to the carboxyl terminus.
Small Interfering RNA Preparation and Transfection
Ets-1 sense (ACUUGCUACCAUCCCGUAC-dTT) and antisense (GUACGGGAUGGUAGCAAGU-dTT) small interfering (si)RNAs, and control scrambled siRNA (sense, AGGAGAUAUUUCGAGCUU-dTT; antisense, AAGCUCGAAAUAUCUCCU-dTT) were synthesized, high-performance liquid chromatography purified, and annealed (Invitrogen) as previously reported.17 Additional details are provided in the expanded Materials and Methods section in the online data supplement.
Western Blot Analysis
Western blot analysis was performed as previously described.15,16 Additional details are provided in the expanded Materials and Methods section in the online data supplement.
Mouse Ang II and Peptide Infusion
The peptide infusion experiments were performed using C57BL/6 mice (The Jackson Laboratory, Bar Harbor, Me). The ALZET minipump (DURECT Corporation, Cupertino, Calif) filled with 1.4 mg/kg per day Ang II (Sigma, St Louis, Mo) or saline alone (sham treatment) were implanted subcutaneously. For the peptide infusion, mice were divided into an untreated group (n=8) and 3 treatment groups (n=8), in which mice were infused with Ang II alone or Ang II with a second pump filled with either a dominant negative Ets-1 peptide (DN-Ets-1) (10 mg/kg per day) or a mutant control peptide (10 mg/kg per day). The presence of the peptide within the thoracic aorta was verified by immunofluorescence (Figure IV in the online data supplement).
Measurement of medial thickness was performed as previously described.10 Five sections in each animal were used for analysis. Additional details are provided in the expanded Materials and Methods section in the online data supplement.
Sections (5 μm) from paraffin-embedded aorta or tissues were used. Briefly, sections were deparaffinized, rehydrated, and microwave retrieved in 10 mmol/L Tris–EDTA (pH 7.5) at 93°C for 5 minutes. Sections were incubated with rabbit polyclonal p47phox antibody (sc14015; Santa Cruz Biotechnology, Inc, Santa Cruz, Calif) at 4°C overnight. Additional details are provided in the expanded Materials and Methods section in the online data supplement.
In Situ Detection and Quantitation of O2− in Aorta Sections
To measure the superoxide anion production in vessels, frozen cross sections of aortas were stained with dihydroethidium (DHE) (Invitrogen) as described previously.18 Additional details are provided in the expanded Materials and Methods section in the online data supplement.
Quantitative Measurement of Intracellular Superoxide Production in Aorta Segments Using
Three 2-mm thoracic aorta rings from each animal were incubated with 50 μmol/L DHE in fresh Krebs/Hepes buffer at 37°C and homogenized in 300 μL of methanol on ice (n=6 to 7 animals per group). Intracellular superoxide production was measured by the formation of 2-hydroxyethidium from DHE and superoxide. Additional details are provided in the expanded Materials and Methods section in the online data supplement.
The results are expressed as means±SEM. The data were analyzed by ANOVA followed by Dunnett or Bonferroni post hoc test for multiple comparisons. A probability value of less than 0.05 was considered to be statistically significant.
To evaluate the potential role of Ets-1 in Ang II–mediated ROS generation, H2O2 and superoxide (O2−) production were measured in the thoracic aortic segments obtained from wild-type and Ets-1−/− mice before and after stimulation with Ang II. The generation of H2O2 was significantly blunted in the Ets-1−/− mice compared with the littermate controls (Figure 1A). H2O2 production was similarly blunted in HASMCs stimulated with Ang II, when transfected with siRNA directed against Ets-1 compared with the scrambled control (Figure 1B). DHE staining was performed to evaluate intracellular O2− production in response to Ang II (Figure 1C). Significant reductions in O2− production were observed in aortic segments stimulated with Ang II obtained from Ets-1−/− mice compared with littermate controls (Figure 1D).
Ets-1 is rapidly induced in HASMCs in response to Ang II (Figure 1E). One of the major mechanisms by which Ang II induces oxidative stress in VSMCs is by activating the membrane-bound NAD(P)H oxidase.6 We observed a similar induction of the NAD(P)H subunit p47phox by Western blot analysis in response to Ang II in HASMCs at a slightly later time point than Ets-1 (Figure 1F).
To determine whether increased p47phox expression is dependent on Ets-1, HASMCs were treated with increasing doses of siRNA directed against Ets-1. This led to a marked reduction in the induction of p47phox by Ang II, whereas no decrease in p47phox expression was observed in cells treated with a scrambled control siRNA (Figure 2A).
To further define the potential role of Ets-1 in the regulation of the p47phox gene, a 3-kb fragment encoding the p47phox gene promoter was isolated and subcloned into the PGL3 luciferase reporter. A series of 6 deletion constructs were also made (Figure 2B). Transient transfections in RASMCs with a mammalian expression plasmid encoding Ets-1 resulted in an ≈6-fold increase in the activity of the reporter (Figure 2C). Similarly, we observed a 4- to 5-fold increase in the activity of the p47phox luciferase reporter in RASMCs stimulated with Ang II. Deletion analysis demonstrated that the majority of the increase in transactivation by Ets-1 and Ang II is attributable to a region of the p47phox promoter located between nucleotide −86 and −36, containing a putative Ets-1–binding site (−45).
A gel mobility-shift assay was used to evaluate the ability of in vitro translated Ets-1 to bind to the oligonucleotide probe encoding the −45 Ets site compared with a consensus Ets-1–binding site. A similar size complex was formed with oligonucleotide probes encoding both Ets sites (Figure 2D, lanes 2 and 5). Competition with a cold wild-type oligonucleotide effectively inhibited binding of Ets-1 (lane 6), whereas an Ets-1 mutant oligonucleotide did not (lane 7). An antibody to Ets-1 also inhibited binding (lane 8), whereas an isotype-matched control antibody (lane 9) did not, thereby demonstrating the specificity of the protein binding to this oligonucleotide probe as being Ets-1. Gel mobility-shift assays were similarly conducted using nuclear extracts derived from HASMCs stimulated with Ang II for different amounts of time (Figure 2E). Whereas minimal Ets-1 binding was observed in nuclear extracts obtained from unstimulated HASMCs, there was a marked increase in binding of proteins to this Ets-1–binding site within 1 hour after stimulation with Ang II. The specificity of this bound complex was demonstrated using an antibody directed against Ets-1 (Figure 2E, lanes 10 and 12) that completely inhibited protein–DNA complex formation, whereas an isotype control antibody had no effect (lanes 11 and 13).
Site-directed mutagenesis was used to further define the functional significance of the identified −45 Ets-1–binding site in the p47phox promoter, within either the entire 3 kb or the 224- or 86-bp p47phox luciferase reporter constructs. Mutation of this site significantly diminished transactivation by Ets-1 or inducibility by Ang II (Figure 3A), further supporting the importance of this site in the regulation of the p47phox gene in VSMCs.
A chromatin immunoprecipitation (ChIP) assay was used to confirm binding of Ets-1 to specific binding sites within the p47phox promoter using cellular extracts obtained from HASMCs stimulated with Ang II (Figure 3B). PCR primers were made for 4 different regions of the p47phox promoter, denoted ChIP1 through ChIP4 in Figure 3B. HASMCs were stimulated for different amounts of time (0, 2, 4 hours). Immunoprecipitation of the protein–DNA cross-linked fragments using an Ets-1–specific antibody confirmed binding of Ets-1 to the region (ChIP1) containing the −45 Ets-1 site, whereas no evidence of binding was observed to three other regions containing putative Ets binding sites (Figure 3B).
Immunohistochemistry was used to evaluate the expression of p47phox. The expression of p47phox in wild-type and Ets-1−/− mice before and after Ang II stimulation. p47phox expression was significantly blunted in thoracic aorta of Ets-1−/− mice compared with littermate controls after stimulation with Ang II. (Figure 3C). The main induction of p47phox appears to be within the VSMCs of the media, suggesting that the major cellular source of ROS in response to Ang II is VSMCs.
As an alternative approach toward inhibiting the function of Ets-1, we developed a DN-Ets-1 encoding the terminal portion of the Ets-1 DNA-binding domain in tandem with a 14-aa peptide encoding an HIV-TAT peptide that has previously been shown to promote cellular uptake and nuclear localization of other peptides (Figure 4A).16,19 The DN-Ets-1 peptide inhibited the induction of p47phox in response to Ang II in HASMCs compared with no effect of the Mut-Ets-1 peptide (Figure 4B). The DN-Ets-1 peptide inhibits transactivation of the p47phox promoter by Ets-1 in a dose-dependent manner (Figure 4C). Basal levels of p47phox as well as induction by Ang II were partially inhibited in VSMCs by administration of the DN-Ets-1 peptide but not the Mut-Ets-1 peptide (supplemental Figure I). To evaluate the ability of the DN-Ets-1 to inhibit Ang II–mediated ROS generation, HASMCs were cultured in the presence of the DN-Ets-1, Mut-Ets-1 peptide, or saline control. Measurement of H2O2 was performed using the 2′,7′-dichlorofluorescein diacetate assay (Figure 4D). A significant reduction in Ang II–mediated ROS generation was observed in the cells treated with the DN-Ets-1 peptide compared with those treated with saline or the mutant Ets-1 (Mut-Ets-1) peptide.
To further define the therapeutic potential of the dominant-negative peptides, we evaluated the ability of these peptides to inhibit Ang II–mediated ROS generation in vivo. Systemic administration of the DN-Ets-1, the Mut-Ets-1 peptide, or saline (control) was performed in the presence or absence of Ang II. The infusion of the DN-Ets-1 and Mut-Ets-1 peptide did not significantly alter the increase in systolic blood pressure associated with Ang II infusion over 2 weeks compared with control (saline) mice (Figure 5A). This is similar to what we have observed in the Ets-1−/− mice.10 No reduction in blood pressure was observed in Ets-1−/− mice in response to Ang II administration. Nevertheless, significant changes in vascular remodeling were observed in Ets-1−/− mice treated with Ang II compared with littermate controls. In particular, we observed a reduction in Ang II–induced medial hypertrophy. Administration of the DN-Ets-1 peptide was similarly associated with a significant reduction in Ang II–mediated increases in aortic medial thickness and media to lumen ratio (Figure 5B through 5D).
The thoracic aorta was harvested 2 weeks after infusion of Ang II, and ROS generation was assessed using the Amplex Red assay. H2O2 production was significantly blunted in the mice treated with the DN-Ets-1 peptide compared with the mutant peptide (Figure 6A). DHE was used to estimate intracellular superoxide production (see Materials and Methods). The DN-Ets-1 peptide significantly blunted the production of 2-hydroxyethidium from DHE, a measure of superoxide production, in animals treated with Ang II, whereas the Mut-Ets-1 peptide did not have this effect (Figure 6B). In summary, Ets-1 functions to amplify ROS generation in response to Ang II by increasing the expression of NAD(P)H oxidase subunits such as p47phox in addition to its broader role as a mediator of Ang II–mediated vascular inflammation.
Oxidative stress has been shown to play an important role in the initiation and propagation of vascular inflammation and remodeling in a number of vascular diseases.20,21 Ang II has been increasingly recognized as a major proinflammatory mediator in the vasculature. The cellular effects of Ang II are initiated through the activation of several redox-sensitive signaling pathways.22 In addition to signal transduction pathways, several transcription factors have been implicated in mediating the effects of Ang II effects in VSMCs and endothelial cells. Stimulation of VSMCs with Ang II leads to a dose-dependent and rapid induction in the immediate early genes c-fos, c-jun, and early growth response gene (Egr)-1.23–25 Targeted disruption of the Kruppel-like zinc finger transcription factor KLF5 leads to marked reductions in Ang II–mediated effects on vascular remodeling and cardiac hypertrophy.26 The signal transducers and activators of transcription STAT1 and STAT2 and nuclear factor κB are rapidly activated in response to Ang II.27,28
The role of Ets-1 as a downstream transcriptional effector of ROS has recently been demonstrated in several different cell types.The expression of Ets-1 is induced in a variety of ovarian cancer cell lines by H2O2.29 The induction of Ets-1 in these cells is linked to increased binding of the transcription factor Nrf2 to an antioxidant response element within the Ets-1 gene promoter. H2O2 is a tumor-derived factor that can stimulate cellular growth at low concentrations. Ets-1 has also been shown to cooperate with the transcription factor activator protein-1 to regulate gene expression of the matrix metalloproteinase-1 in response to H2O2 in the HT-1080 fibrosarcoma cell line.30 These studies support a role of Ets-1 as a downstream mediator of ROS in addition to regulating the expression genes that are involved in the generation of ROS.
Stimulation of human monocytes with interferon-γ or tumor necrosis factor-α is associated with the increased expression of several NAD(P)H subunits. The induced expression of gp91phox and p22phox requires activation and binding of the ETS factor PU.1 to regulatory regions of these genes.18 Interferon-γ induction of p67phox and gp91phox requires the recruitment of CREB-binding protein by PU.1 and additional factors including interferon-γ regulatory factor 1 and interferon-γ consensus binding protein (ICSBP).31 The transcription factor activator protein-1 is also required for regulation of the p67phox gene in human monocytes.32 In the current study we have identified the p47phox gene as a downstream target of Ets-1. p47phox has previously been shown to be critical for Ang II–mediated ROS generation.33 Because ROS generation was blunted at time points before the induction of p47phox by Ang II, this suggests that Ets-1 plays an important role in early phases of Ang II–mediated ROS generation, possibly by affecting the basal expression of genes such as the NAD(P)H subunits involved in early phases of ROS generation. Superoxide production is markedly reduced in p47phox−/− mice after administration of Ang II compared with littermate controls. Proliferation of VSMCs in response to mitogenic stimuli such as serum or thrombin is dependent on p47phox.34 Our results also demonstrate that Ang II can stimulate p47phox gene expression. Protein levels of p47phox are similarly increased in the balloon-injured rat aorta or in atherosclerotic lesions of ApoE deficient mice.34
The reduction in Ang II–mediated medial hypertrophy and ROS generation we observed after systemic administration of DN-Ets-1 peptides was independent of changes in blood pressure. Reductions in vascular remodeling in response to Ang II stimulation in Ets-1–deficient mice compared with littermate controls that we have previously reported were also independent of changes in blood pressure.10 Other studies evaluating the effects of inhibiting Ang II–mediated ROS generation have been associated with variable effects on blood pressure. For example, when a cell-permeant inhibitor of gp91phox, gp91phox docking sequence(gp91 ds)-tat peptide, which inhibits the association of gp91phox with p47phox, is administered systemically, this leads to a marked reduction in ROS generation and the recruitment of inflammatory cells and medial hypertrophy. These changes occurred despite similar increases in blood pressure in response to Ang II in the presence or absence of the gp91 ds tat peptide.35 Furthermore, when gp91phox knockout mice were crossed with transgenic mice expressing the active human renin gene (TTRhRen) compared with the transgenic mice alone (TTRhRen), the blood pressure levels were the same.36 The TTRHRen mice have chronically elevated levels of Ang II that are associated with hypertension. Despite the absence of a reduction in blood pressure, superoxide production is markedly diminished in the aortas of the gp91phox−/− and gp91phox−/− /TTRhRen mice. In another study, transgenic mice that express increased amounts of the enzyme catalase in VSMCs were generated. Catalase expression promotes the breakdown of hydrogen peroxide to water. Ang II–mediated vascular hypertrophy was markedly attenuated in the catalase-expressing transgenic mice compared with control mice, despite similar increases in blood pressure.37 The results of these studies suggest that ROS production is a critical determinant of vascular remodeling mediated by Ang II and that these effects may occur independent of changes in blood pressure.
Membrane permeable peptides have been used to block the function of other transcription factors. A 14-aa peptide encoding part of the HIV-1 TAT protein (48 to 60) is sufficient not only to promote transport of a peptide across the cell membrane but also promotes accumulation of the peptide in the cell nucleus.19 We recently demonstrated that a membrane-permeable peptide encoding the terminal portion of the DNA-binding domain of the ETS transcription factor ELF-1 and the 14-aa HIV-1 TAT peptide was able to specifically block DNA binding and transactivation of the Tie2 promoter by ELF-1 and did not interfere with the binding or function of other ETS factors.16 The results of our current study similarly support the therapeutic potential of membrane permeable DN-Ets-1 peptides to inhibit the function of Ets-1 in wild-type mice expressing the endogenous Ets-1 protein and thereby reduce the expression of target genes such as p47phox and the generation of ROS in response to Ang II. The use of membrane permeable peptides has previously been used to inhibit the function of NAD(P)H oxidase. The gp91ds-tat peptide interferes with protein–protein interactions of the docking sequence of gp91phox.35
In summary, the studies reported here demonstrate that Ets-1 is a critical transcriptional mediator of ROS generation by Ang II by regulating the expression of NAD(P)H oxidase subunits such as p47phox. The results of our previous studies support a role for Ets-1 as a transcriptional mediator of several downstream targets of Ang II, including monocyte chemoattractant protein-1, vascular cell adhesion molecule-1, and plasminogen activator inhibitor-1.10 Finally the studies support the therapeutic potential of inhibiting Ets-1 via systemic administration of DN-Ets-1 peptides in the setting of vascular inflammation.
Sources of Funding
This work was supported by NIH grants PO1 HL76540 and HL082717.
Original received March 23, 2007; revision received August 27, 2007; accepted September 5, 2007.
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