Role of NAD(P)H Oxidase in Angiotensin II–Induced JAK/STAT Signaling and Cytokine Induction
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, STAT1α/β, STAT3, and IL-6-synthesis can be abolished by DPI, as determined by immunoprecipitations and Northern blot analysis. Electroporation of neutralizing antisera targeted against p47phox, 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.
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 p47phox, p67phox, p40phox, and Rac GTP.10 Activation of the NAD(P)H oxidase is initiated by the cytosolic assembly of p47phox and p67phox and targeted to the plasma membrane by Rac GTP.9 10 p47phox itself seems to play a central role in the scenario of NAD(P)H oxidase activation, because p67phox fails to assemble with the NAD(P)H oxidase in the absence of p47phox, and p47phox 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-612 13 and NF-κB.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
Protein A/G agarose, cell culture medium (DMEM), and supplements were from Life Technologies, Inc. Antiphosphotyrosine (PY20) and monoclonal p47phox (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.
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 (EP) was performed as reported recently5 15 using a plate electrode (model P/N 747, BTX Inc) in Ca2+- and Mg2+-free HBS solution containing p47phox antibody (100 μg/mL). Unspecific IgG was used as controls.
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 4×104 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
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 p47phox
The expression of p47phox in RASM cultures was determined by RT-PCR (94°C 5 hours; 38× 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).
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 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.
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.
p47phox Detection and Activation
RASM cell p47phox 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 p47phox 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 p47phox. This increase can be abolished by DPI (Figure 2A⇓). Regulation of p47phox protein synthesis was tested by EP of p47phox antibodies into RASM cells. EP alone had no influence on the p47phox synthesis, whereas p47phox antibody prevented p47phox protein synthesis (Figure 2B⇓). Activation of the NAD(P)H oxidase system was determined by membrane translocation of p47phox (Figure 2C⇓) and O2− anion generation (Figure 2D⇓). Western blots of membrane fractions revealed that Ang II induces p47phox membrane translocation at 1 minute. Both DPI and p47phox antibodies abolished p47phox membrane translocation. Controls using unspecific IgG and EP alone had no influence on p47phox membrane translocation.
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 p47phox 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 p47phox antibody EP abolished JAK2 tyrosine phosphorylation (Figure 3D⇓). Similarly, JAK2 kinase activity was blocked by p47phox antibodies. Both EP and unspecific IgG did not affected Ang II–induced JAK2 kinase activity (Figure 3C⇓).
Subsequently, STAT-factor tyrosine phosphorylation and nuclear translocation were investigated. First, Ang II–induced increase in STAT1α/β and STAT3 tyrosine phosphorylation was determined. DPI abolished Ang II–induced STAT1α/β and STAT3 tyrosine phosphorylation (Figures 4A⇓ and 4B⇓). p47phox antibody EP blocked STAT1α/β and STAT3 tyrosine phosphorylation, whereas unspecific IgG had no significant influence on STAT1α/β (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 p47phox antibody EP, whereas unspecific IgG had no significant influence on STAT-factor nuclear translocation (P<0.003 p47phox EP versus IgG EP) (Figure 4D⇓).
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 p47phox 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 STAT1α/β 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 p47phox antisera EP significantly. Blockade of JAK2 by AG490 also abolished IL-6 release significantly (P<0.01 versus Ang II for all).
The present study demonstrates that Ang II–induced O2− anion formation depends on the p47phox 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 STAT1α/β antisera significantly abolished Ang II–induced IL-6 synthesis, indicating that JAK2 or STAT1α/β, 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 p47phox and demonstrated that p47phox protein expression is induced by Ang II. Moreover, blockade of p47phox either by DPI or p47phox 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 p67phox.9 25
We demonstrate that p47phox antisera abolishes Ang II–induced O2− anion formation. This may involve complex mechanisms beyond the blockade of p47phox by its antisera. Recent evidence suggested that p47phox, p67phox, and Rac exhibit cooperative binding, and protein-protein interaction may regulate the activation of the NAD(P)H oxidase. p67phox binds to p47phox 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 p47phox binds to the proline-rich region of p67phox, and the N-terminal half of p67phox contains a binding region for Rac. Because the p47phox antibody used in this study is targeted against the SH3 domain of p47phox, it may also block p67phox SH3 domains and thereby abolish binding of Rac to this complex. Although it was not investigated in this study, the inhibition of the p47phox-p67phox-Rac complex formation may be responsible for the complete blockade of NAD(P)H oxidase–induced O2− anion formation. Moreover, in the absence of p47phox, p67phox fails to assemble with the NAD(P)H oxidase, supporting an adaptor function for p47phox in the binding of p67phox.9 10 Finally, p47phox 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 p47phox.10 Similarly, binding of p47phox antisera may protect p47phox 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 p47phox and thereby blockade of the NAD(P)H oxidase system prevents the de novo protein synthesis of p47phox (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 STAT1α/β 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-κB, 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-κB,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-κB) 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-κB, 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.
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.
- © 2000 American Heart Association, Inc.
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