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(Circulation Research. 2000;87:1195.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Role of NAD(P)H Oxidase in Angiotensin II–Induced JAK/STAT Signaling and Cytokine Induction

Bernhard Schieffer, Maren Luchtefeld, Sabine Braun, Andres Hilfiker, Denise Hilfiker-Kleiner, Helmut Drexler

From the Department of Cardiology and Angiology, Medizinische Hochschule Hannover, Germany.

Correspondence to Bernhard Schieffer, MD, Department of Cardiology and Angiology, Medizinische Hochschule Hannover, Carl Neuberg Strasse 1, 30625 Hannover, Germany. E-mail Schieffer.Bernhard{at}MH-Hannover.de

	Abstract

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Abstract—Inflammatory processes involve both synthesis of inflammatory cytokines, such as interleukin-6 (IL-6), and the activation of their distinct signaling pathways, eg, the janus kinases (JAKs) and signal transducers and activators of transcription (STAT). Superoxide (O₂^-) anions activate this signaling cascade, and the vasoconstrictor angiotensin II (Ang II) enhances the formation of O₂^- anions via the NAD(P)H oxidase system in rat aortic smooth muscle cells. Ang II activates the JAK/STAT cascade via its type 1 (AT₁) receptor and induces synthesis and release of IL-6. Therefore, we investigated the role of O₂^- anions generated by the NAD(P)H oxidase system on the Ang II activation of the JAK/STAT cascade and its impact on IL-6 synthesis. Ang II stimulation of rat aortic smooth muscle cells induced a rapid increase in O₂^- anions determined by laser fluoroscopy, which can be abolished by DPI, a flavoprotein inhibitor. Ang II–induced phosphorylation of JAK2, STAT1/ß, STAT3, and IL-6-synthesis can be abolished by DPI, as determined by immunoprecipitations and Northern blot analysis. Electroporation of neutralizing antisera targeted against p47^phox, a NAD(P)H oxidase subunit, abolished Ang II–induced JAK/STAT activation and IL-6 synthesis. Inhibition of JAK2 by its inhibitor AG490 (10 µmol/L) blocked not only JAK2 activation but also IL-6 synthesis. These results suggest that stimulation of the JAK/STAT cascade by Ang II requires O₂^- anions generated by the NAD(P)H oxidase system, and O₂^- anion–dependent activation of the JAK/STAT cascade seems to be additionally involved in Ang II–induced IL-6 synthesis. Thus, Ang II–induced inflammatory effects seem to require O₂^- anions generated by the NAD(P)H oxidase system.

Key Words: angiotensin receptors • oxidant stress • cell signaling • atherosclerosis pathophysiology • gene regulation

	Introduction

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Inflammatory processes represent a hallmark of atherosclerosis and involve the release of inflammatory cytokines, the activation of their distinct signaling cascades, and, presumably, increased oxidative stress, ie, locally generated superoxide (O₂^-) anions.¹ Signaling cascades involved in the transmission of inflammatory processes leading directly from the activated cytokine receptor to gene transcription include the janus kinases (JAKs) and signal transducers and activators of transcription (STAT) factors.^{2 3} Recent evidence suggests that the JAK/STAT cascade also conveys angiotensin II (Ang II) signals from the plasma membrane to the nucleus via the stimulation of the Ang II type 1 (AT₁) receptor.⁴ In addition, the JAK/STAT signaling cascade was shown to be an important link between the activation of the AT₁ receptor and nuclear transcriptional changes leading to cell growth.⁵ However, the mechanism by which the G protein–coupled AT₁ receptor activates a tyrosine kinase like JAK2 remains unclear. Although JAK2 seems to physically associate with the AT₁ receptor,⁴ its binding does not directly lead to JAK2 activation.⁶ In this regard, Simon et al⁷ reported that JAK2 activation in vitro depends on the presence of reactive oxygen species (ROS). Because Ang II activates the generation of O₂^- anions,⁸ we hypothesized that a ROS-generating system is involved in Ang II–induced activation of the JAK/STAT cascade and, subsequently, in interleukin-6 (IL-6) synthesis.

In cellular systems, a major source of ROS derives from the membrane-bound NAD(P)H oxidase system.⁹ The primary products of this system are superoxide anions (O₂^-), which are rapidly dismutated, for example, to hydrogen peroxide.⁹ Activation of NAD(P)H oxidase system requires the participation of its cytosolic factors p47^phox, p67^phox, p40^phox, and Rac GTP.¹⁰ Activation of the NAD(P)H oxidase is initiated by the cytosolic assembly of p47^phox and p67^phox and targeted to the plasma membrane by Rac GTP.^{9 10} p47^phox itself seems to play a central role in the scenario of NAD(P)H oxidase activation, because p67^phox fails to assemble with the NAD(P)H oxidase in the absence of p47^phox, and p47^phox regulates the electron transfer from FAD to the heme center of cytochrome b₅₅₈ leading to O₂^- anion generation.⁹

Previous observations from our group demonstrated that Ang II induces IL-6 synthesis and release in smooth muscle cells in vitro, and both factors are colocalized in stable and unstable atherosclerotic lesions.¹¹ In addition, promoter studies of STAT-regulated genes revealed that STAT-binding sites are in close proximity to binding sites for other transcription factors known to be involved in IL-6 gene transcription, such as nuclear factor (NF)–IL-6^{12 13} and NF-B.¹⁴ Therefore, the present study investigated whether IL-6 induction by Ang II is regulated, in part, by the JAK/STAT cascade. Moreover, because Ang II causes the generation of O₂^- anions via the NAD(P)H oxidase⁸ and O₂^- anions are involved in the activation of the JAK/STAT cascade,⁷ we investigated whether Ang II activates the JAK/STAT cascade via O₂^- anions and whether this ROS-dependent mechanism may be involved in Ang II–induced IL-6 synthesis.

	Materials and Methods

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Reagents
Protein A/G agarose, cell culture medium (DMEM), and supplements were from Life Technologies, Inc. Antiphosphotyrosine (PY20) and monoclonal p47^phox (clone1) were from Transduction Laboratories, and polyclonal JAK2, STAT1, and STAT3 were from Santa Cruz Biotech. All chemicals, DPI, Ang II, 2',7'-dichlorofluorescein (DCF) and 2',7'-DCF-diacetate (DCF-DA) were from Sigma.

Cell Culture
Rat aortic smooth muscle (RASM) cells were maintained in DMEM supplemented with 10% (vol/vol) FBS, 10 µg/mL streptomycin, and 100 U/mL penicillin. Cells were growth-arrested in serum-free medium for 24 hours. Cell number used was 10⁷ cells per 100 mm².^{4 5}

Immunoprecipitation and Western Blot Analysis
RASM cells were stimulated with Ang II (10^-7 mol/L) for the indicated times. Immunoprecipitations with antiphosphotyrosine antibodies (PY20) were performed as described.⁴ The proteins were separated by SDS-PAGE, transferred to membranes, and probed with anti-JAK2, anti-STAT1, and anti-STAT3 antibody (1:1000). Proteins were visualized by chemiluminescence.^{4 5 15}

In Vitro Kinase Assay
JAK2 tyrosine kinase activity was measured by autophosphorylation.⁴ After stimulation, JAK2 was immunoprecipitated. The pellet was resuspended in kinase buffer and allowed to autophosphorylate in the presence of 15 µmol/L ATP. Proteins were separated by SDS-PAGE, transferred to membranes, and probed with antiphosphotyrosine antibody.⁴

Electroporation Procedure
Electroporation (EP) was performed as reported recently^{5 15} using a plate electrode (model P/N 747, BTX Inc) in Ca²⁺- and Mg²⁺-free HBS solution containing p47^phox antibody (100 µg/mL). Unspecific IgG was used as controls.

Carboxy-Dichlorofluorescein Fluoroscopy
Generation of O₂^- anions was monitored by oxidation of carboxy-dichlorofluorescein (DCFH-DA) to DCF¹⁶ by laser fluoroscopy at 485/538 nm (Fluoroscan, Labsystems ), and 4x10⁴ RASM cells per well were plated under serum-free conditions and incubated with the inhibitors. DCFH-DA (5 µmol/L) was added before Ang II stimulation, and developing fluorescence was determined. Each experiment was performed in triplicate.

Northern Blot Analysis
Total RNA was isolated, separated, and transferred to membranes (Amersham). Rat IL-6 cDNA probes were generated by polymerase chain reaction (PCR) (533 bp, 5'-TGTTGTTGACAGCCACTGC-3' and 5'-TTTCAAGATGAGTTGGATGGTC-3') and labeled.¹¹ The blots were visualized by autoradiography.¹¹

Reverse Transcriptase–PCR
Total RNA was isolated, and first-strand synthesis was carried out with total cDNA using reverse transcriptase (RT) (Superscript II, Life Technologies Inc) and oligo d(T)primers. Semiquantitative PCR was carried out by normalizing all cDNAs to GAPDH. IL-6 primer sequences are given above. PCR fragments were densitometrically analyzed (GelDoc 2000, BioRad).

Sequencing of p47^phox
The expression of p47^phox in RASM cultures was determined by RT-PCR (94°C 5 hours; 38x 94°C 10 minutes, 57°C 30 minutes, 72°C 40 minutes; 72°C 7 hours, 4°C ; forward 5'-CCAG-CCAGCACTATGTGTACA-3', reverse 5'-ACGCTGTTGCGGCG-ATA-3'), which revealed a 931-bp fragment that was sequenced. This sequence was submitted to GenBank (accession number AF260779).

Membrane Preparation
RASM cells were lysed in hypotonic buffer (20 mmol/L Tris-HCL, pH 7.6) supplemented with 1 mmol/L Na₃VO₄, 1 mmol/L phenylmethylsulfonyl fluoride, and 10 µg/mL leupeptin. The cells were dounced and centrifuged, and the pellet was resuspended in buffer (20 mmol/L Tris-HCL, pH 7.4; 25 mmol/L EDTA, 1% Triton X-100; 10% glycerol; 0.1% SDS; 50 mmol/L NaF; 10 mmol/L Na₂P₂O₇; and 1% desoxycholate). Proteins were processed for Western blot analysis.¹⁷

IL-6 Concentration
IL-6 protein in the supernatant media was determined by enzyme-linked immune absorbance assay (Quantikine rat IL-6, R&D Systems) at 420 nm.¹¹ Each experiment was performed in triplicate.

Statistical Analysis
All data are given as mean±SEM of at least 4 separate experiments. Results were processed using SigmaPlot 5.0 (SPSS Inc). Differences were evaluated by t test analysis. Statistical significance was defined as P<0.05.

	Results

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p47^phox Detection and Activation
RASM cell p47^phox was amplified by RT-PCR, and a 931-bp fragment was sequenced. A total of 863 of 931 bp were identical (92% identity) to the homologue mouse p47^phox sequence (Figure 1). BLAST analysis revealed 94% identity and 95% similarity to the mouse sequence at protein levels, 86% identity and 93% similarity to the bovine fragment, and 80% identity and 89% similarity to the human fragment, respectively. Western blot analysis revealed that Ang II induces the protein synthesis of p47^phox. This increase can be abolished by DPI (Figure 2A). Regulation of p47^phox protein synthesis was tested by EP of p47^phox antibodies into RASM cells. EP alone had no influence on the p47^phox synthesis, whereas p47^phox antibody prevented p47^phox protein synthesis (Figure 2B). Activation of the NAD(P)H oxidase system was determined by membrane translocation of p47^phox (Figure 2C) and O₂^- anion generation (Figure 2D). Western blots of membrane fractions revealed that Ang II induces p47^phox membrane translocation at 1 minute. Both DPI and p47^phox antibodies abolished p47^phox membrane translocation. Controls using unspecific IgG and EP alone had no influence on p47^phox membrane translocation.

View larger version (21K): [in this window] [in a new window]   Figure 1. Figure 1. Expression of p47phox in various tissues by semiquantitative RT-PCR. Expression of p47phox was detectable in cultured RASM cells and rat myocardium. Human macrophage cell lines (U937) and human umbilical vein endothelial cells (HUVECs) were used as controls.

View larger version (29K): [in this window] [in a new window]   Figure 2. Figure 2. A, Ang II stimulates the protein synthesis of p47phox in RASM cells (right). Inhibition of NAD(P)H oxidase by DPI abolished Ang II–induced p47phox synthesis (left). B, Ang II–induced p47phox protein synthesis. EP of neutralizing p47phox antibodies abolished Ang II–induced p47phox protein synthesis. EP alone or unspecific IgG had no influence on the p47phox synthesis. C, Activation of NAD(P)H oxidase by Ang II. Ang II induces the rapid and transient membrane translocation of p47phox, as shown by Western blot analysis of membrane fractions. EP of p47phox antisera and DPI abolished p47phox membrane translocation, whereas unspecific IgG and the EP had no influence on p47phox membrane translocation. D, Ang II–induced O2- anion generation was determined by DFC-DA to DFC conversion, and results are given as percent increase. Ang II–induced O2- anion generation was abolished by DPI. p47phox antibody EP abolished O2- anion generation by Ang II. EP of unspecific IgG had no influence on Ang II–dependent O2- anion formation. Serum-free controls showed no increase in fluoroscopic signal. Blockade of the AT1 receptor by LOS abolished Ang II–induced O2- anion formation.

Membrane translocation of components of the NAD(P)H oxidase reflects the activation of the NAD(P)H oxidase system and the initiation of O₂^- anion generation. Laser fluoroscopy experiments revealed an Ang II–dependent increase in O₂^- anions peaking at 5 minutes. DPI abolished the Ang II–induced O₂^- anion generation (P<0.0001 versus Ang II). DPI dose-response curves revealed a maximum inhibition of O₂^- anion formation at 100 µmol/L (data not shown). The AT₁-receptor antagonist losartan (LOS) (10^-5 mol/L) abolished Ang II–induced O₂^- anion generation. EP of p47^phox abolished Ang II–induced O₂^- anion generation (P<0.0001), whereas controls using unspecific IgG did not influenced O₂^- anion formation. Serum-free controls showed no increase in O₂^- anion generation (Figure 2D).

O₂^- Anion–Dependent Activation of the JAK/STAT Cascade
Ang II induces JAK2 and STAT factor tyrosine phosphorylation,⁴ which can be abolished by DPI (Figure 3A). Tyrosine phosphorylation of JAK2 is associated with an increase in tyrosine kinase activity.⁴ Ang II–induced JAK2 kinase activity can be abolished by DPI (Figure 3B). EP alone did not alter JAK2 tyrosine phosphorylation (Figure 3C), whereas p47^phox antibody EP abolished JAK2 tyrosine phosphorylation (Figure 3D). Similarly, JAK2 kinase activity was blocked by p47^phox antibodies. Both EP and unspecific IgG did not affected Ang II–induced JAK2 kinase activity (Figure 3C).

View larger version (27K): [in this window] [in a new window]   Figure 3. Figure 3. A, Ang II–induced JAK2 phosphorylation depends on NAD(P)H oxidase. RASM cells were stimulated with Ang II, and immunoprecipitations with phosphotyrosine antisera were performed followed by Western blot analysis using anti-JAK2 antisera. Preincubation with the flavoprotein inhibitor DPI abolished Ang II–induced JAK2 tyrosine phosphorylation.4 B, Ang II–induced JAK2 kinase activity depends on NAD(P)H oxidase. RASM cells were stimulated, and lysates were subjected to JAK2 kinase assay.4 The Ang II–induced increase in JAK2 kinase activity4 was abolished by DPI. C, p47phox and JAK2 kinase activity. EP of p47phox antibodies abolished Ang II–induced JAK2 kinase activity, whereas EP alone or unspecific IgG had no influence on JAK2 kinase activity. Data were scanned (n=4) and blotted as mean±SEM. D, p47phox and JAK2 tyrosine phosphorylation. RASM cells were electroporated with p47phox antibodies and stimulated. Lysates were subjected to immunoprecipitations with phosphotyrosine antisera followed by Western blot analysis with anti-JAK2 antibodies. p47phox antibodies abolished JAK2-tyrosine phosphorylation induced by Ang II.

Subsequently, STAT-factor tyrosine phosphorylation and nuclear translocation were investigated. First, Ang II–induced increase in STAT1/ß and STAT3 tyrosine phosphorylation was determined. DPI abolished Ang II–induced STAT1/ß and STAT3 tyrosine phosphorylation (Figures 4A and 4B). p47^phox antibody EP blocked STAT1/ß and STAT3 tyrosine phosphorylation, whereas unspecific IgG had no significant influence on STAT1/ß (P>0.07) and STAT3 (P>0.05) phosphorylation (Figure 4C). Second, Ang II–induced nuclear translocation of STAT factors was abolished by DPI. Similar results were obtained with p47^phox antibody EP, whereas unspecific IgG had no significant influence on STAT-factor nuclear translocation (P<0.003 p47^phox EP versus IgG EP) (Figure 4D).

View larger version (70K): [in this window] [in a new window]   Figure 4. Figure 4. A, NAD(P)H oxidase–dependent STAT activation. RASM cells were stimulated with Ang II, and lysates were subjected to immunoprecipitations with phosphotyrosine antisera followed by Western blot analysis with anti-STAT1/ß or anti-STAT3 antibodies. Results revealed an increase in STAT1/ß (A) or STAT3 (B) tyrosine phosphorylation at 10 minutes, which was abolished by DPI. C, p47phox-dependent STAT activation. RASM cells were electroporated with p47phox antibodies and stimulated with Ang II. Lysates were subjected to immunoprecipitations with phosphotyrosine antisera followed by Western blot analysis with anti-STAT1/ß (top) or anti-STAT3 (bottom) antibodies. Ang II–induced STAT1/ß or STAT3 phosphorylation peaked at 10 minutes4 and was abolished by p47phox antibodies. Controls using unspecific IgG had no influence on STAT factor phosphorylation. D, NAD(P)H oxidase–dependent STAT1/STAT3 nuclear translocation. RASM cells were electroporated with p47phox antibodies and stimulated with Ang II, and nuclear extracts were analyzed by Western blot. Ang II–induced STAT1/ß (top) or STAT3 (bottom) nuclear translocation was abolished by either DPI or p47phox antibody EP. Unspecific IgG had no influence on STAT factor nuclear translocation.

O₂^- Anions and IL-6 Production
To determine whether Ang II–induced IL-6 synthesis and release is O₂^- anion–dependent, IL-6 transcription was analyzed.¹⁵ Both DPI and EP of p47^phox antibodies abolished Ang II–induced IL-6 transcription (Figures 5A and 5B). EP or unspecific IgG reduced IL-6 transcription significantly (P<0.05); however, comparing electroporated cells with or without IgG antisera revealed no significant difference in IL-6 transcription. AT₁-receptor blockade by LOS abolished IL-6 transcription (Figure 5C). To test the involvement of the JAK/STAT cascade, JAK2 blockade by AG490 (10 µmol/L) was used.^{5 18} AG490 blocked Ang II–induced IL-6 transcription and release (Figures 5A through 5C). Semiquantitative RT-PCR analysis of IL-6 transcription (5 separate experiments) revealed additionally that EP of STAT3 and JAK2 antisera reduced IL-6 transcription, but differences were not significant. In contrast, EP of STAT1/ß antisera reduced IL-6 transcription significantly (P<0.03) (Figure 5C). Similarly, IL-6 protein release was determined and peaked 6 hours after Ang II stimulation.¹¹ Ang II–induced IL-6 release was inhibited by both DPI and p47^phox antisera EP significantly. Blockade of JAK2 by AG490 also abolished IL-6 release significantly (P<0.01 versus Ang II for all).

View larger version (27K): [in this window] [in a new window]   Figure 5. Figure 5. A, NAD(P)H oxidase–dependent IL-6 transcription. RASM cells were stimulated with Ang II. Northern blot analysis showed that Ang II induced a rapid increase of 2 transcripts that peaked at 30 minutes (left).11 DPI abolished Ang II–induced IL-6 transcription (middle). Blockade of JAK2 by its inhibitor AG490 abolished IL-6 transcription (right). B, EP with p47phox antibodies abolished Ang II–induced IL-6 transcription, whereas controls using unspecific IgG or EP procedure alone did not block IL-6 transcription. C, RT-PCR analysis of STAT3 and JAK2 antibody EP had no significant influence on IL-6 transcription, whereas STAT1 /ß antibodies reduced Ang II–induced IL-6 transcripts significantly. D, NAD(P)H oxidase–dependent IL-6 release. Ang II–induced increase of IL-6 protein release peaked at 6 hours in RASM cells.11 DPI abolished Ang II–induced IL-6 release. EP of p47phox antibodies and blockade of JAK2 by AG490 abolished IL-6 release. Unspecific IgG had no influence on IL-6 release. n=4 (each in triplicate).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study demonstrates that Ang II–induced O₂^- anion formation depends on the p47^phox subunit of the NAD(P)H oxidase. O₂^- anions are additionally required for Ang II–induced activation of the JAK/STAT cascade. Blockade of the tyrosine kinase JAK2 by its inhibitor and EP of STAT1/ß antisera significantly abolished Ang II–induced IL-6 synthesis, indicating that JAK2 or STAT1/ß, when activated by O₂^- anions, participates in the Ang II–induced IL-6 synthesis.

Traditionally, G protein–coupled receptors control the activation of signaling events via heterotrimeric G proteins.¹⁸ Recent evidence suggests that reversible phosphorylation involving kinases and phosphatases plays a critical role in G protein–coupled receptor signaling leading to cell growth and proliferation.^{5 15} Nevertheless, receptors like the AT₁ receptor lack intrinsic tyrosine kinase activity.¹⁹ This observation suggests that additional second messenger systems may be recruited into the AT₁-receptor signaling complex. The fact that the AT₁ receptor lacks classic binding motifs, for example, SH2, SH3, or PH domains, emphasizes that these additional signaling systems may be activated independent of traditional protein-binding motifs.¹⁸

In this regard, we hypothesized that ROS-generating systems are potential candidates as independent signal-transduction mediators that overcome traditional protein-binding motifs, because their products do not rely on physical association of signaling proteins yet stimulate signaling events transiently and rapidly.⁹ In mammalian cells, a major source of ROS derives from the membrane-bound NAD(P)H oxidase system.⁹ Superoxide anions, generated by the NAD(P)H oxidase system, are easily produced (no protein de novo synthesis is required) and effectively controlled, especially by superoxide dismutases and glutathione peroxidases.⁹

The present study suggests that ROS are critically involved in early signaling events by connecting apparently unconnected signaling cascades, that is, the G protein–coupled AT₁ receptor with soluble tyrosine kinases, such as JAK2. However, it remained to be determined whether ROS-induced activation of target proteins relies more on the protein redox sensitivity than on specific protein-binding motifs. In this regard, Simon et al⁷ demonstrated that JAK2 activation in vitro is regulated by ROS. The present study provides evidence that Ang II–induced JAK2 activation requires O₂^- anions generated by the NAD(P)H oxidase system. These observations suggest that redox-sensitive signaling molecules, such as JAK2, in close proximity to ROS-generating systems, eg, the NADP(H) oxidase, may represent a novel mechanism by which the G protein–coupled AT₁ receptors activate apparently unconnected signaling pathways irrespective of available classic protein-binding motifs.

Several groups demonstrated that the NAD(P)H oxidase system exists in nonphagocytic cells of the vascular wall, for example, fibroblasts, vascular smooth muscle cells, and endothelial cells.^{10 20 21 22 23 24} However, the role of the various NAD(P)H oxidase subunits remained unclear. The present study isolated and sequenced rat smooth muscle cell p47^phox and demonstrated that p47^phox protein expression is induced by Ang II. Moreover, blockade of p47^phox either by DPI or p47^phox antisera abolished its protein synthesis, suggesting a redox-sensitive autoregulatory mechanism by which Ang II may induce NAD(P)H oxidase expression. However, we cannot exclude the competing influence of other members of the NAD(P)H oxidase family, such as mox-1, or additional NAD(P)H oxidase family subunits, such as p67^phox.^{9 25}

We demonstrate that p47^phox antisera abolishes Ang II–induced O₂^- anion formation. This may involve complex mechanisms beyond the blockade of p47^phox by its antisera. Recent evidence suggested that p47^phox, p67^phox, and Rac exhibit cooperative binding, and protein-protein interaction may regulate the activation of the NAD(P)H oxidase. p67^phox binds to p47^phox via a tail-to-tail interaction, using the SH3 domain in the C-terminus of the former to bind to the proline-rich sequence in the C-terminus of the latter.^{9 10} The second SH3 domain of p47^phox binds to the proline-rich region of p67^phox, and the N-terminal half of p67^phox contains a binding region for Rac. Because the p47^phox antibody used in this study is targeted against the SH3 domain of p47^phox, it may also block p67^phox SH3 domains and thereby abolish binding of Rac to this complex. Although it was not investigated in this study, the inhibition of the p47^phox-p67^phox-Rac complex formation may be responsible for the complete blockade of NAD(P)H oxidase–induced O₂^- anion formation. Moreover, in the absence of p47^phox, p67^phox fails to assemble with the NAD(P)H oxidase, supporting an adaptor function for p47^phox in the binding of p67^phox.^{9 10} Finally, p47^phox is phosphorylated on serine during the oxidase complex formation and translocates to plasma membranes. Phosphorylation is one of the key events in NAD(P)H oxidase activation. Protein kinase C, protein kinase A, and mitogen-activated protein kinase have been shown to regulate phosphorylation of p47^phox.¹⁰ Similarly, binding of p47^phox antisera may protect p47^phox from phosphorylation by various kinases.

Exposure of vascular smooth muscle cells to Ang II resulted in an increase in O₂^- anion generation, as first determined by Griendling et al⁸ and subjected to the activation of the NAD(P)H oxidase system.⁸ The authors reported that the Ang II–induced NAD(P)H oxidase activation occurs in a rather slow fashion (within hours), whereas results from other studies,⁷ including the present one, demonstrated that O₂^- anion generation occurred within seconds. In addition, the present study demonstrated that blockade of p47^phox and thereby blockade of the NAD(P)H oxidase system prevents the de novo protein synthesis of p47^phox (Figure 2). Thus, it is tempting to speculate that the delayed activation of the NAD(P)H oxidase system that was observed by Griendling et al⁸ is a more likely a result of the protein de novo synthesis rather then a delayed activation of the preexisting NAD(P)H oxidase by Ang II.⁸

Recent clinical observations indicated that proinflammatory factors, such as IL-6, seemed to be a trigger for an acute coronary syndrome,²⁶ and chronic blockade of Ang II formation showed beneficial effects on the development of an acute coronary syndrome. The latter is potentially attributable to a reduction of inflammatory processes, because Ang II induces synthesis and release of IL-6 in smooth muscle cells and macrophages and both Ang II and IL-6 were colocalized at the shoulder region of human atherosclerotic coronary plaques.^{26 11} These observations suggest a close interaction between Ang II and IL-6, with potential relevance for the development of an unstable atherosclerotic lesion.

What is the role of common signaling intermediates, such as the JAK/STAT cascade and O₂^- anions? The present study demonstrates that O₂^- anions are required for Ang II–dependent JAK/STAT activation and that blockade of JAK2 by its inhibitor AG490 or EP of STAT1/ß antisera reduced IL-6 synthesis and release significantly.

In addition, although the tryphostin-class kinase inhibitor AG490 is known to abolish JAK2 kinase activity selectively,²⁷ it is also an accepted inhibitor of epidermal growth factor signaling.²⁸ Transactivation of epidermal growth factor receptors is an established mechanism of G protein–coupled receptors, such as the AT₁ receptor, as reported by Eguchi et al.²⁹ Thus, it is not surprising that blockade of AG490 reduces IL-6 release to a greater extend compared with JAK2 or STAT antibody EP (see Figures 5C and 5D). Because cytokines such as IL-6 regulate their synthesis and release via the activation of the JAK/STAT cascade,² these findings are consistent with the notion that the activation of the JAK/STAT pathway may be involved in Ang II–induced IL-6 release and that this effect is triggered by O₂^- anions generated via the NAD(P)H oxidase system.

Previous observations demonstrated that other transcription factors, such as NF-B, are involved in Ang II–induced IL-6 transcription in vitro.³⁰ In this context, promoter studies of STAT-regulated genes revealed that STAT-binding sites are in close proximity to binding sites for other transcription factors. For example, STAT3 interaction was reported with C/EBP/NF–IL-6,^{12 13} NF-B,¹⁴ and activator protein-1,³¹ whereas STAT1 interacts with IRF-1³² and SP1.³³ Thus, it is possible that not one transcription factor alone (eg, STAT-factors or NF-B) but several transcription factors in symphony are involved in Ang II–induced IL-6 synthesis and thereby control IL-6 gene transcription. The potential interaction of the G protein–coupled AT₁ receptors [via the NAD(P)H oxidase system] with other redox-sensitive transcription factors, such as NF-B, and their synergistic impact on IL-6 transcription need to be specifically addressed in future studies. However, this scenario may represent a novel and integrative concept by which multiple redox-sensitive transcription factors together modulate the transcription rate of a single gene, such as IL-6.

In summary, we demonstrate that Ang II–induced JAK/STAT activation requires O₂^- anions generated by the NAD(P)H oxidase. This observation suggests that redox-sensitive signaling cascades, such as the JAK/STAT cascade, in close proximity to ROS-generating systems, eg, the NADP(H) oxidase, may represent a novel mechanism by which the AT₁ receptor activates signaling pathways irrespective of available classic protein-binding motifs.

	Acknowledgments

 
This work was supported by the Deutsche Forschungsgemeinschaft (DFG) grant Schie 386/3-1 and DFG-Sonderforschungsbereich 244: C14. The authors are indebted to Elisabeth Schieffer, MD, for critical discussions.

Received May 31, 2000; revision received October 12, 2000; accepted October 12, 2000.

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