This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 25/3/512    most recent 01.ATV.0000154141.66879.98v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Touyz, R.M. Articles by Schiffrin, E.L. Search for Related Content PubMed PubMed Citation Articles by Touyz, R.M. Articles by Schiffrin, E.L. Related Collections Oxidant stress ACE/Angiotension receptors Cell biology/structural biology Cell signalling/signal transduction

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2005;25:512.)
© 2005 American Heart Association, Inc.

Vascular Biology

p47phox Associates With the Cytoskeleton Through Cortactin in Human Vascular Smooth Muscle Cells

Role in NAD(P)H Oxidase Regulation by Angiotensin II

R.M. Touyz; G. Yao; M.T. Quinn; P.J. Pagano; E.L. Schiffrin

From Multidisciplinary Research Group on Hypertension (R.M.T., G.Y., E.L.S.), Clinical Research Institute of Montreal, University of Montreal, Canada; Department of Veterinary Molecular Biology (M.T.Q.), Montana State University, Bozeman; Hypertension and Vascular Research Division and Biostatistics Department (P.J.P.), Henry Ford Hospital, Detroit, Mich.

Correspondence to Rhian M Touyz, MD, PhD, Clinical Research Institute of Montreal, 110 Pine Ave West, Montreal, Quebec H2W 1R7, Canada. E-mail touyzr{at}ircm.qc.ca

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Objective— We tested the hypothesis that p47phox associates with the actin cytoskeleton, enabling site-directed activation of NAD(P)H oxidase, and assessed whether these actions influence reactive oxygen species (ROS) generation and signaling by angiotensin II (Ang II) in vascular smooth muscle cells (VSMCs) from human resistance and coronary arteries.

Methods and Results— Electroporation of anti-p47phox antibody into VSMCs abrogated Ang II-mediated O₂ generation, establishing the requirement for p47phox in this response. Immunfluorescence confocal microscopy demonstrated a cytosolic distribution of p47phox in basal conditions. After Ang II stimulation, p47phox rearranged in a linear fashion, colocalizing with F-actin. Co-immunoprecipitation studies confirmed an association between p47phox and actin and demonstrated an interaction with the actin-binding protein cortactin. Cytoskeletal disruption with cytochalasin prevented p47phox:actin interaction and attenuated ROS formation and p38MAP kinase and Akt phosphorylation by Ang II. Intracellular ROS generation in response to LY83583 (O₂ generator) or exogenous H₂O₂ and Ang II-induced ERK1/2 activation were unaltered by cytochalasin.

Conclusions— The p47phox:actin interaction, through cortactin, plays an important role in Ang II-mediated site-directed assembly of functionally active NAD(P)H oxidase, ROS generation, and activation of redox-sensitive p38MAP kinase and Akt, but not ERK1/2. These findings demonstrate the importance of an intact actin-cytoskeleton in NAD(P)H oxidase regulation and redox signaling by Ang II in human VSMCs.

We demonstrate that p47phox:actin interaction, through cortactin, is involved in Ang II-mediated site-directed assembly of NAD(P)H oxidase, ROS generation, and activation of redox-sensitive p38MAPK and Akt, but not ERK1/2. These findings demonstrate the importance of an intact actin-cytoskeleton in NAD(P)H oxidase regulation and redox-signaling by Ang II in human VSMCs.

Key Words: reactive oxygen species • cytochalasin B • cortactin • LY83583 • hydrogen peroxide

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Compelling evidence indicates that reactive oxygen species (ROS), particularly superoxide (O₂) and hydrogen peroxide (H₂O₂), are important in angiotensin II (Ang II)–mediated signal transduction in vascular smooth muscle cells (VSMCs).^1,2 Redox-sensitive signaling in VSMCs is involved in Ang II-induced phosphorylation of mitogen activated protein (MAP) kinases, particularly p38MAP kinase and JNK, PI3K/AKT, intracellular Ca²⁺ homeostasis, and activation of transcription factors, such as AP-1, NFB, and HIF-1.^3–7 These molecular events influence VSMC function, including contraction, growth, apoptosis, and extracellular matrix protein production. Among the many enzymatic sources of O₂, nonphagocytic nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] oxidase appears to be important in the vasculature.^2,8,9 The prototypical phagocytic NAD(P)H oxidase is a multicomponent enzyme comprising membrane-bound p22phox and gp91phox, 3 cytoplasmic subunits, p47phox, p67phox, and p40phox, and the small GTPase Rac 1/2.^10,11 Most vascular cell types, including endothelial cells, adventitial fibroblasts, and smooth muscle cells, express components of the phagocytic NAD(P)H oxidase, as well as gp91phox (nox2) homologues nox1, nox4, and nox5.^2,12–14 We recently demonstrated that all 5 of the major neutrophil subunits are present and regulated by Ang II in VSMC from human small arteries.^15,16

NAD(P)H oxidase, which is constitutively active at low levels in resting VSMCs, becomes fully activated on exposure to stretch, cytokines, growth factors, and vasoactive agents.^17–19 These factors are implicated in vascular pathology, probably through their oxidative stress-inducing capacity. Of the numerous vasoactive agents regulating vascular NAD(P)H oxidase, Ang II appears to be one of the most important.^8,9 Although exact mechanisms of VSMC NAD(P)H oxidase activation have not yet been fully elucidated, evidence indicates that cytosolic p47phox, p67phox, and p40phox translocate to membrane-associated gp91phox and p22phox, resulting in assembly of the active oxidase, which generates O₂.^15,16 In polymorphonuclear leukocytes, p47phox is essential in the assembly process. This is exemplified in cells of patients with chronic granulomatous disease, in which no translocation occurs because of p47phox deficiency.²⁰ The p47phox also plays a major role in functionally active NAD(P)H oxidase activity in cardiovascular cells, as evidenced by studies using p47phox^–/– mice.^21,22 Lavigne et al demonstrated that aortic VSMCs derived from aorta of p47phox^–/– mice failed to generate O₂ in response to phorbol esters, Ang II, or platelet-derived growth factor (PDGF).²¹ Li and Shah reported that Ang II regulates pre-assembled NAD(P)H oxidase in endothelial cells, which is inhibited in cells from p47phox^–/– mice.²² We recently demonstrated in VSMCs from human resistance arteries that c-Src-mediated phosphorylation of p47phox is a prerequisite for assembly and translocation of the p40-p47-p67 phox complex.¹⁶ What remains unclear is how the p47phox-regulated cytosolic complex migrates to the cell membrane to facilitate site-directed complex formation with p22phox and gp91phox/nox and consequent activation of NAD(P)H oxidase.

Studies in polymorphonuclear leukocytes and endothelial cells suggest that the cytoskeleton may play a role. In activated neutrophils, p47phox, p67phox, and p40phox associate with filamentous (F)-actin.^23–28 In endothelial cells, p47phox localizes with the cytoskeleton to induce tumor necrosis factor- signaling.²⁶ However, van Bruggen et al recently reported that p67phox and Rac2 translocation do not depend on a rearrangement of the actin cytoskeleton in phagocytic cells.²⁷ To our knowledge, nothing is known about the relationship between the cytoskeleton and NAD(P)H oxidase, specifically p47phox, in Ang II-mediated ROS regulation and signal transduction in human VSMCs.

We tested the hypothesis that Ang II-activated p47phox associates with cytoskeletal proteins, which facilitates site-directed targeting of functionally active NAD(P)H oxidase in VSMCs.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Cell Culture
VSMCs derived from human small arteries from gluteal biopsy samples were cultured as previously described.^15,16 Human coronary VSMCs (Clonetics San Diego, Calif) were also studied.

Immunofluorescence Confocal Microscopy
VSMCs were stimulated with Ang II (10^–7 mol/L, 5 to 30 minutes). In some experiments, cells were pre-exposed to cytochalasin B (10^–6 mol/L, 60 minutes), which arrests new F-actin assembly. Washed cells were fixed and incubated with primary antibody (anti-p47phox, anti-cortactin) and proteins detected with secondary antibody. Imaging was acquired with a Zeiss Axiovert S100TV LSM 510 laser scanning system.

Immunoprecipitation and Immunoblotting
p47phox Immunoprecipitation
Cells were lysed and p47phox was immunoprecipitated as previously detailed (online data).^15,16,29

Western Immunoblotting
After SDS-PAGE separation of proteins, samples were transferred to polyvinylidene fluoride membranes. Membranes were incubated with the following antibodies: anti-smooth muscle cell -actin, anti-cortactin, anti-p47phox, anti-phospho-ERK1/2, anti-phospho-p38MAP kinase, and anti-phospho-Akt. Washed membranes were incubated with horseradish peroxidase-conjugated second antibody. Immunoreactive proteins were detected by chemiluminescence.

Electroporation
VSMCs were electroporated with anti-p47phox antibody, anti-IgG, or in the absence of any antibodies as we previously described (online data).³⁰

Viability Test
Trypan blue dye exclusion, which was used to evaluate viability of electroporated VSMCs before plating them in DMEM.

Detection of Electroporated Antibody by Immunofluorescence
To determine efficiency of antibody incorporation, cells were also electroporated in the presence of secondary antibodies (Alexa fluor 488; Molecular Probes, Eugene, Ore). Fluorescence was assessed by epifluorescence microscopy just before experimentation. Cells that fluoresced were considered successfully electroporated.

Measurement of ROS in Intact Cells
Intracellular O₂ and H₂O₂ levels were measured with the fluoroprobes tempo-9-AC¹⁶ and CMH₂-DCFDA,^15,29 respectively, in unstimulated cells and in cells exposed to Ang II (10^{9 to} 10^–6 mol/L) in the absence and presence of 10^–6 mol/L cytochalasin B (10^–6 mol/L, 60-minute pre-incubation). In some experiments cells were stimulated with LY83583 (O₂ generator, 10^–6 to 10^–4, 10 minutes)³¹ or exogenous H₂O₂ (10^–7 to 10^–5 mol/L, 10 minutes), in the absence and presence of cytochalasin B (10^–6 mol/L, 60-minute pre-incubation).

Measurement of NAD(P)H Oxidase Activity
Quiescent VSMCs were stimulated with Ang II for 5 to 15 minutes in the absence and presence of apocynin (3x10^–5 mol/L, 20-minute pre-incubation), which blocks association of p47phox with membrane-associated subunits, p22phox and gp91phox,^15,16 or with gp91ds-tat, a novel competitive inhibitor of NAD(P)H oxidase assembly³² (5x10^–6 mol/L). The lucigenin-derived chemiluminescence assay was used to determine NAD(P)H oxidase activity in whole-cell homogenates as previously described.^15,16 Data are presented as relative light units (RLU)/min per milligram of protein.

Analysis
Data obtained from digital imaging studies, in which multiple cells were examined in each experimental field, were calculated as the mean per experiment and then as the mean of multiple experiments. Each experiment was performed at least 3 times. Values are presented as means±SEM. Data were analyzed by ANOVA or Student t test. Tukey-Kramer correction was used to compensate for multiple testing procedures. P<0.05 was significant.

An expanded Methods section is available online. Please see http://atvb.ahajournals.org.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Functionally Active p47phox Is Essential for Ang II-stimulated O₂ Production in VSMCs
To establish the functional importance of p47phox in the generation of intracellular ROS by Ang II, we disrupted p47phox function by electroporating cells with Alexa-labeled anti-p47phox antibody. When cells were exposed to a 1.3-ms pulse of 750 V/cm, >70% of cells survived and 60% were brightly fluorescent after overnight incubation. In comparison, cells that were incubated in the presence of labeled antibody without electroporation failed to fluoresce.

Electroporated cells were loaded with tempo-9-AC and analyzed for O₂ production. Electroporation did not significantly influence basal O₂ generation (Figure I, available online at http://atvb.ahajournals.org). In p47phox antibody-electroporated cells, Ang II-stimulated O₂ formation was significantly reduced (P<0.01) compared with cells electroporated with anti-IgG or no antibodies (Figure I).

Interruption of p47phox-gp91phox/p22phox Association Inhibits Ang II-Stimulated NAD(P)H Oxidase Activation
To verify the importance of p47phox interaction with cell membrane-associated subunits in the activation of NAD(P)H oxidase in our cell model, effects of Ang II in the presence of apocynin, a catechol-methyl derivative that blocks p47phox interaction with gp91phox/p22phox, and gp91ds-tat, a competitive inhibitor of p47phox-dependent NAD(P)H oxidase assembly,³¹ were assessed. Whereas Ang II significantly increased NAD(P)H oxidase activation (3886±142 versus basal 85±12 RLU/min per milligram (x10⁴), P<0.01) in control cells, Ang II-induced responses were significantly attenuated (P<0.01) in cells pre-exposed to apocynin (65±14 RLU/min per milligram [10⁴] and gp91ds-tat (213±39 RLU/min per milligram [x10⁴]).

p47phox Is a Cytoskeletal-Associated Protein
Immunofluorescent image analysis demonstrated that in the basal state, p47phox is localized in the cytoplasmic region (Figure II, available online at http://atvb.ahajournals.org). After Ang II stimulation, p47phox aligns in a reticular-type pattern and migrates to the cell periphery. Costaining with actin-microfilament-binding rhodamine phalloidin revealed a somewhat linear colocalization of p47phox and actin (Figure 1A to 1D), suggesting an association between p47phox and F-actin in stimulated cells. Confocal immunofluorescence images demonstrated that in addition to associating with F-actin, p47phox colocalizes with the actin-binding protein cortactin in Ang II-stimulated cells (Figure 2A).

View larger version (47K): [in this window] [in a new window]   Figure 1. Effect of chytochalasin B on p47phox and actin localization in VSMCs. VSMCs were stimulated with Ang II (10–7 mol/L, 5 minutes) and analyzed by confocal immunofluorescence microscopy using x63 (A to C) and x100 (D) oil immersion objectives. Cells were labeled with monoclonal anti-p47phox (blue fluorescence) and rhodamine-phalloidin for F-actin detection (red fluorescence). A and B, Individual staining patterns for actin and p47phox, respectively. C and D, Dual staining pattern. VSMCs were pretreated with cytochalasin B (10–6 mol/L, 1 hour) and analyzed as described (E to G). E, Cytochalasin B alone (p47phox, blue fluorescence; actin, red fluorescence), x63 oil immersion objective. F, Ang II plus cytochalasin B, x63 oil immersion objective. G, Ang II plus cytochalasin B, x100 oil immersion objective. H, No staining in the negative control.

View larger version (37K): [in this window] [in a new window]   Figure 2. p47phox associates with the actin-binding protein cortactin in Ang II-stimulated cells. A, VSMCs were stimulated with Ang II (10–7 mol/L, 10 minutes), followed by labeling with anti-cortactin polyclonal antibody (Alexa-594, anti-rabbit secondary antibody, red fluorescence) and monoclonal anti-p47phox (Alexa-488 anti-mouse secondary antibody, green fluorescence). Colocalization of cortactin and p47phox are revealed as yellow-orange fluorescence in the merged image. B to D, Cortactin co-immunoprecipitates with p47phox in Ang II-stimulated cells. Coronary VSMCs were stimulated with Ang II, immunoprecipitated (IP) with anti-p47phox antibody, and immunoblotted (IB) with anti-cortactin antibody (labeling at 80 to 85 kDa) (B). Membranes were reprobed with anti-p47phox antibody to demonstrate equal p47phox protein content (C). Bar graphs are means±SEM of 3 separate experiments (D). Results are expressed as percentage of control (H2O-treated cells) taken as 100%. *P<0.05 versus control. IgG was used as a negative control.

To further evaluate whether Ang II-stimulated p47phox organization is related to actin association, cells were pretreated with cytochalasin B. As shown in Figure 1E to 1G, p47phox remains mainly in the cytoplasmic area in cytochalasin B-treated cells and does not orientate in a linear alignment after Ang II stimulation, suggesting that cytochalasin B interferes with p47phox:actin interaction.

Co-immunoprecipitation of p47phox With Actin and Cortactin in Ang II-Stimulated VSMCs
To confirm these results and to evaluate whether the actin-binding protein cortactin plays a role in p47phox:actin association, p47phox was isolated by using affinity-purified anti-p47phox to immunoprecipitate p47phox from VSMC homogenate. The p47phox immunoprecipitates were analyzed by Western blotting for the presence of actin and cortactin using specific anti--actin and anti-cortactin antibodies, respectively. As shown in Figures 2B to 2D and 3 , cortactin and actin co-immunoprecipitated with p47phox, with enhanced effects in Ang II-stimulated cells compared with vehicle-treated cells. In the presence of cytochalasin B, Ang II-induced p47phox:actin co-immunoprecipitation was reduced compared with cells not exposed to cytochalasin B (Figure 3).

View larger version (26K): [in this window] [in a new window]   Figure 3. Association of p47phox with the actin-cytoskeleton in Ang II-stimulated VSMC. Coronary artery VSMCs were stimulated with vehicle (H2O) (control) or Ang II (5 minutes) in the absence and presence of cytochalasin B (Cytoch) (10–6 mol/L, 1-hour pretreatment), immunoprecipitated (IP) with anti-p47phox antibody, and immunoblotted (IB) with -actin antibody. VSMC lysate was used as a positive control (PC) for -actin immunoblotting. Upper panel, Representative immunoblot. Bar graphs are means±SEM of 3 separate experiments. Results are expressed as percentage of control (H2O-treated cells) taken as 100%. *P<0.05, **P<0.01.

Cytochalasin B Attenuates Ang II-Stimulated ROS Production Without Influencing ROS Formation Induced by LY83583 and H₂O₂
To investigate the functional significance of p47phox:actin interaction, we investigated whether Ang II-mediated production of ROS is altered in cells in which actin assembly is arrested. Pretreatment of VSMCs with cytochalasin B significantly attenuated (P<0.01) Ang II-stimulated increase in DCFDA fluorescence (P<0.01) (Figure 4). However, responses were not completely abolished, indicating that cytochalasin B-independent events also contribute to Ang II-mediated O₂ generation

View larger version (16K): [in this window] [in a new window]   Figure 4. Effects of cytochalasin B on ROS production in VSMC. Cytochalasin B inhibits Ang II-induced ROS formation in VSMCs. Control cells (white symbols) and cells exposed to cytochalasin B (10–6 mol/L) for 1 hour (black symbols) were stimulated with the indicated doses of Ang II, and DCFDA fluorescence was monitored as an indicator of ROS production. *P<0.05, **P<0.01 versus Ang II counterpart.

To establish whether cytoskeletal integrity is also required for NAD(P)H oxidase-independent mechanisms of ROS production, cytochalasin B actions were determined in cells stimulated with LY83583 (Figure III, available online at http://atvb.ahajournals.org) or exogenous H₂O₂ (Figure III). LY83583 and H₂O₂ dose-dependently increased VSMC O₂ and H₂O₂, respectively. These effects occur independently of NAD(P)H oxidase activation and are not inhibited by apocynin (data not shown). Cytochalasin B did not modify ROS formation in response to LY83583 or H₂O₂.

Cytochalasin B Does Not Alter ERK1/2 Signaling but Attenuates p38MAP Kinase and Akt Phosphorylation by Ang II
To evaluate whether cytochalasin B interferes with Ang II-mediated signaling, we assessed effects of Ang II on ERK1/2, p38MAP kinase, and Akt. Ang II stimulation induced rapid phosphorylation of ERK1/2, p38MAP kinase, and Akt (Figure 5). Whereas cytochalasin B did not alter ERK1/2 phosphorylation, it significantly attenuated Ang II-induced activation of p38MAP kinase and Akt (Figure 5).

View larger version (22K): [in this window] [in a new window]   Figure 5. Cytochalasin B attenuates Ang II-induced phosphorylation of p38MAP kinase and Akt, but not ERK1/2. VSMCs were stimulated with Ang II (10–7 mol/L) for 5 (Ang5) or 10 minutes (Ang10) in the absence and presence of cytochalasin B (10–6 mol/L, 1 hour). Phosphorylation of ERK1/2, p38MAP kinase, and Akt was assessed as described in the Methods. Immunoblots are representative of 3 different experiments. Data are percentage change relative to control taken as 100%. Results are means±SEM of 3 experiments. *P<0.05, **P<0.01 versus control (Cont) and cytochalasin B (cyt) groups. +P<0.05, ++P<0.01 versus Ang II counterpart.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Ang II-mediated ROS production in the vasculature derives, in part, from VSMC NAD(P)H oxidase.^8,9 The ep47phox plays a critical role in this process by initiating complex formation between the NAD(P)H oxidase cytosolic subunits and by promoting translocation to cell membrane-associated flavocytochrome b₅₅₈.^16,33 Intracellular ROS are involved in redox-sensitive signaling pathways by Ang II and have been implicated as important second messengers in VSMCs.^1–6 What remains unclear is how Ang II regulates p47phox migration and how it promotes site-direction of functionally active NAD(P)H oxidase. In the present study, we provide novel data indicating: (1) Ang II-activated p47phox associates with actin facilitating cytosolic complex translocation to the cell membrane to assemble active vascular NAD(P)H oxidase; (2) cortactin may be a putative adaptor protein linking p47phox and actin; (3) p47phox interaction with cytoskeletal proteins is important for NAD(P)H oxidase-mediated ROS generation in VSMCs; and (4) p38MAP kinase and Akt, but not ERK1/2, signaling by Ang II depends on an intact actin-cytoskeleton in VSMCs. The p47phox-cytoskeletal targeting may promote site-directed NAD(P)H-driven ROS production, which could contribute to efficient redox-dependent signaling by Ang II in VSMCs.

A relationship between NAD(P)H oxidase and the cytoskeleton has been demonstrated in neutrophils.^{23,24,34–37} Whether the cytoskeleton is also involved in NAD(P)H oxidase regulation and redox-sensitive signaling in non-phagocytic cells remains unclear. An association between p47phox and actin has been demonstrated in endothelial cells.^22,26,38,39 In VSMCs, Nox1 and Nox4 have been shown to colocalize with caveolae and vinculin, respectively.⁴⁰ Furthermore, Zuo et al recently showed that AT₁R trafficking and ROS signaling involved in VSMC growth requires the integrity of microtubules.⁴¹ Here we demonstrate, using a multidisciplinary approach, the novel findings that p47phox is closely associated with F-actin in Ang II-treated human VSMCs. On Ang II stimulation, p47phox migrates from the cytosol along actin fibers to the cell periphery to assemble functionally active NAD(P)H oxidase. This is evidenced by immunofluorescence studies, p47phox:actin co-immunoprecipitation, and by the findings that cytochalsin B disrupts p47phox:actin interaction.

Subcellular mechanisms whereby p47phox interacts with actin are unclear; however, p47phox contains numerous functional binding sites, including Src homology 3 (SH3) domains, a motif typically found in proteins known to associate with actin-binding proteins⁴² and Phox homology (PX) domains, which possess a PXXP motif, allowing binding to proteins containing SH3 domains.^43,44 In fact, p47phox has been shown previously to associate with actin-binding proteins coronin, cofilin, and moesin.^36,44 The interaction of p47phox with moesin, an ezrin-radixin-moesin family of F-actin-binding proteins, occurs through the PX domain.^45,46 In our study, cortactin, a Src-regulated actin-binding protein, which possesses SH3 domains,⁴⁷ colocalized and co-immunoprecipitated with p47phox, particularly in Ang II-stimulated VSMCs. This was significantly attenuated by cytochalasin B. These results suggest cortactin as a putative scaffolding protein linking actin to cytosolic phox proteins. Of significance, we previously showed that Ang II regulates p47phox activation through c-Src-dependent actions. Accordingly, we suggest that c-Src may regulate NAD(P)H oxidase through multiple mechanisms, including phosphorylation of p47phox, as well as through translocation of the cytosolic phox complex by facilitating p47-phox-cortactin-actin association. Cortactin is one of many actin-binding proteins and it is possible that other proteins are also involved. Exact molecular processes and identification of additional binding partners in this phenomenon await clarification.

Interactions between NAD(P)H oxidase and the cytoskeleton have important functional significance in VSMCs. This is evidenced by the results that Ang II-induced O₂ production was markedly attenuated in cytochalasin B-treated cells. Our findings that cytochalasin B did not alter ROS formation induced by LY83583 or by exogenous H₂O₂ suggests that a functionally intact cytoskeleton is important for NAD(P)H oxidase-dependent, but not necessarily for NAD(P)H oxidase-independent processes of ROS production, of which there are numerous enzymatic and nonenzymatic mechanisms.^2,3 Furthermore, Ang II-induced activation of ERK1/2 was unaltered by cytochalasin B, whereas phosphorylation of p38MAP kinase and Akt was significantly downregulated by cytochalasin B. These data have at least 3 implications. First, not all Ang II-mediated cellular actions are cytoskeleton-dependent. Second, Ang II signaling is intact and functional in cytochalasin B-treated cells. Finally, p38MAP kinase and Akt, which are redox-sensitive kinases,⁷ depend on an intact actin cytoskeleton for activation by Ang II.

In addition to the cytoskeleton acting as a scaffold for p47phox, it is possible that phox proteins and ROS are involved in cytoskeletal reorganization accompanying O₂ formation. In phorbol-activated polymorphonuclear leukocytes, cytosolic phox proteins associate with and regulate assembly of coronin, an actin-associated protein that accumulates at cortical sites of moving cells and contributes to the dynamics of the actin system.³⁷ In cells from patients lacking p47phox or p67phox, rearrangement of F-actin and coronin in activated cells is absent or markedly diminished.³⁷ Clements et al recently reported that peroxynitrite inhibits actin dynamics in phagocytic cells, with significant effect on actin-dependent cellular processes.⁴⁸ In our study, we cannot exclude the possibility that p47phox and ROS may also influence VSMC function by altering cytoskeletal arrangement.

In conclusion, we provide several lines of evidence that p47phox associates with actin in VSMCs, possibly through scaffolding processes involving the actin-binding, c-Src-regulated protein cortactin. Interactions between cytosolic phox proteins and the cytoskeleton play an important role in NAD(P)H-driven O₂ generation, which may contribute to site-directed activation of NAD(P)H oxidase and efficient redox-dependent Ang II signaling, such as Akt, in human VSMCs. These are not generalized phenomena because NAD(P)H oxidase-independent ROS formation and Ang II-mediated ERK1/2 signaling in VSMCs do not depend on an intact actin cytoskeleton

	Acknowledgments

 
This study was supported by grant 44018 and a Group grant to the Multidisciplinary Research Group on Hypertension, both from the Canadian Institutes for Health Research and grant AR42426 from the National Institutes of Health. R.M.T. received a scholarship from the Fonds de la Recherché en Sante du Quebec.

Received July 7, 2004; accepted December 3, 2004.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Touyz RM, Tabet F, Schiffrin EL. Redox-dependent signalling by angiotensin II and vascular remodelling in hypertension. Clin Exp Pharmacol Physiol. 2003; 30: 860–866.[CrossRef][Medline] [Order article via Infotrieve]
2. Lassegue B, Clempus RE. Vascular NAD(P)H oxidases: specific features, expression, and regulation. Am J Physiol Regul Integr Comp Physiol. 2003; 285: R277–R297.[Abstract/Free Full Text]
3. Torres M, Forman HJ. Redox signaling and the MAP kinase pathways. Biofactors. 2003; 17: 287–296.[Medline] [Order article via Infotrieve]
4. Viedt C, Fei J, Krieger-Brauer HI, Brandes RP, Teupser D, Kamimura M, Katus HA, Kreuzer J. Role of p22phox in angiotensin II and platelet-derived growth factor AA induced activator protein 1 activation in vascular smooth muscle cells. J Mol Med. 2004; 82: 31–38.[CrossRef][Medline] [Order article via Infotrieve]
5. El Bekay R, Alvarez M, Monteseirin J, Alba G, Chacon P, Vega A, Martin-Nieto J, Jimenez J, Pintado E, Bedoya FJ, Sobrino F. Oxidative stress is a critical mediator of the angiotensin II signal in human neutrophils: involvement of mitogen-activated protein kinase, calcineurin, and the transcription factor NF-kappaB. Blood. 2003; 102: 662–671.[Abstract/Free Full Text]
6. De Nigris F, Lerman LO, Condorelli M, Lerman A, Napoli C. Oxidation-sensitive transcription factors and molecular mechanisms in the arterial wall. Antioxid Redox Signal. 2001; 3: 1119–1130.[CrossRef][Medline] [Order article via Infotrieve]
7. Taniyama Y, Ushio-Fukai M, Hitomi H, Rocic P, Kingsley MJ, Pfahnl C, Weber DS, Alexander RW, Griendling KK. Role of p38 MAPK and MAPKAPK-2 in angiotensin II-induced Akt activation in vascular smooth muscle cells. Am J Physiol Cell Physiol. 2004; 287: C494–C499.[Abstract/Free Full Text]
8. Seshiah PN, Weber DS, Rocic P, Valppu L, Taniyama Y, Griendling KK. Angiotensin II stimulation of NAD(P)H oxidase activity: upstream mediators. Circ Res. 2002; 91: 406–413.[Abstract/Free Full Text]
9. Zafari AM, Ushio-Fukai M, Akers M, Yin Q, Shah A, Harrison DG, Taylor WR, Griendling KK. Role of NADH/NAD(P)H oxidase-derived H₂O₂ in angiotensin II-induced vascular hypertrophy. Hypertension. 1998; 32: 488–495.[Abstract/Free Full Text]
10. Babior BM. NAD(P)H oxidase. Curr Opin Immunol. 2004; 16: 42–47.[CrossRef][Medline] [Order article via Infotrieve]
11. Dang PM, Cross AR, Quinn MT, Babior BM. Assembly of the neutrophil respiratory burst oxidase: a direct interaction between p67phox and cytochrome b558 II. Proc Natl Acad Sci U S A. 2002; 99: 4262–4265.[Abstract/Free Full Text]
12. Rey FE, Pagano PJ. The reactive adventitia: fibroblast oxidase in vascular function. Arterioscler Thromb Vasc Biol. 2002; 22: 1962–1971.[Abstract/Free Full Text]
13. Sorescu D, Somers MJ, Lassegue B, Grant S, Harrison DG, Griendling KK. Electron spin resonance characterization of the NAD(P)H oxidase in vascular smooth muscle cells. Free Radic Biol Med. 2001; 30: 603–612.[CrossRef][Medline] [Order article via Infotrieve]
14. Ago T, Kitazono T, Ooboshi H, Iyama T, Han YH, Takada J, Wakisaka M, Ibayashi S, Utsumi H, Iida M. Nox4 as the Major Catalytic Component of an Endothelial NAD(P)H Oxidase. Circulation. 2004; 109: 227–233.[Abstract/Free Full Text]
15. Touyz RM, Chen X, Tabet F, Yao G, He G, Quinn MT, Pagano PJ, Schiffrin EL. Expression of a functionally active gp91phox-containing neutrophil-type NAD(P)H oxidase in smooth muscle cells from human resistance arteries: regulation by angiotensin II. Circ Res. 2002; 90: 1205–1213.[Abstract/Free Full Text]
16. Touyz RM, Yao G, Schiffrin EL. c-Src induces phosphorylation and translocation of p47phox: role in superoxide generation by angiotensin II in human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2003; 23: 981–987.[Abstract/Free Full Text]
17. Ungvari Z, Csiszar A, Huang A, Kaminski PM, Wolin MS, Koller A. High pressure induces superoxide production in isolated arteries via protein kinase C-dependent activation of NAD(P)H oxidase. Circulation. 2003; 108: 1253–1258.[Abstract/Free Full Text]
18. Grote K, Flach I, Luchtefeld M, Akin E, Holland SM, Drexler H, Schieffer B. Mechanical stretch enhances mRNA expression and proenzyme release of matrix metalloproteinase-2 (MMP-2) via NAD(P)H oxidase-derived reactive oxygen species. Circ Res. 2003; 92: 80–86.
19. Touyz RM, Schiffrin EL. Increased generation of superoxide by angiotensin II in smooth muscle cells from resistance arteries of hypertensive patients: role of phospholipase D-dependent NAD(P)H oxidase-sensitive pathways. J Hypertens. 2001; 19: 1245–1254.[CrossRef][Medline] [Order article via Infotrieve]
20. Allen LH, DeLeo FR, Gallois A, Toyoshima S, Suzuki K, Nauseef WM. Transient association of the nicotinamide adenine dinucleotide phosphate oxidase subunits p47phox and p67phox with phagosomes in neutrophils from patients with X-linked chronic granulomatous disease. Blood. 1999; 93: 3521–3530.[Abstract/Free Full Text]
21. Lavigne MC, Malech HL, Holland SM, Leto TL. Genetic demonstration of p47phox-dependent superoxide anion production in murine vascular smooth muscle cells. Circulation. 2001; 104: 79–84.[Abstract/Free Full Text]
22. Li JM, Shah AM. Intracellular localization and preassembly of the NAD(P)H oxidase complex in cultured endothelial cells. J Biol Chem. 2002; 277: 19952–19960.[Abstract/Free Full Text]
23. Woodman RC, Ruedi JM, Jesaitis AJ, Okamura N, Quinn MT, Smith RM, Curnutte JT, Babior BM. Respiratory burst oxidase and three of four oxidase-related polypeptides are associated with the cytoskeleton of human neutrophils. J Clin Invest. 1991; 87: 1345–1351.
24. Quinn MT, Parkos CA, Jesaitis AJ. The lateral organization of components of the membrane skeleton and superoxide generation in the plasma membrane of stimulated human neutrophils. Biochim Biophys Acta. 1989; 987: 83–94.[Medline] [Order article via Infotrieve]
25. El Benna J, Dang PM, Andrieu V, Vergnaud S, Dewas C, Cachia O, Fay M, Morel F, Chollet-Martin S, Hakim J, Gougerot-Pocidalo MA. p40phox associates with the neutrophil Triton X-100-insoluble cytoskeletal fraction and PMA-activated membrane skeleton: a comparative study with P67phox and P47phox. J Leukoc Biol. 1999; 66: 1014–10120.[Abstract]
26. Li JM, Mullen AM, Yun S, Wientjes F, Brouns GY, Thrasher AJ, Shah AM. Essential role of the NAD(P)H oxidase subunit p47(phox) in endothelial cell superoxide production in response to phorbol ester and tumor necrosis factor-alpha. Circ Res. 2002; 90: 143–150.[Abstract/Free Full Text]
27. van Bruggen R, Anthony E, Fernandez-Borja M, Roos D. Continuous translocation of Rac2 and the NAD(P)H oxidase component p67(phox) during phagocytosis. J Biol Chem. 2004; 5; 279: 9097–9102.
28. De Leo FR, Ulman KV, Davis AR, Jutila KL, Quinn MT. Assembly of the human neutrophil NAD(P)H oxidase involves binding of p67phox and flavocytochrome b to a common functional domain in p47phox. J Biol Chem. 1996; 271: 17013–17020.[Abstract/Free Full Text]
29. El Benna J, Ruedi JM, Babior BM. Cytosolic guanine nucleotide-binding protein Rac2 operates in vivo as a component of the neutrophil respiratory burst oxidase. Transfer of Rac2 and the cytosolic oxidase components p47phox and p67phox to the submembranous actin cytoskeleton during oxidase activation. J Biol Chem. 1994; 269: 6729–6734.[Abstract/Free Full Text]
30. Touyz RM, Wu XH, He G, Park JB, Chen X, Vacher J, Rajapurohitam V, Schiffrin EL. Role of c-Src in the regulation of vascular contraction and Ca²⁺ signaling by angiotensin II in human vascular smooth muscle cells. J Hypertens. 2001; 19: 441–449.[CrossRef][Medline] [Order article via Infotrieve]
31. Hasegawa T, Bando A, Tsuchiya K, Abe S, Okamoto M, Kirima K, Ueno S, Yoshizumi M, Houchi H, Tamaki T. Enzymatic and nonenzymatic formation of reactive oxygen species from 6-anilino-5,8-quinolinequinone. Biochim Biophys Acta. 2004; 1670: 19–27.[Medline] [Order article via Infotrieve]
32. Rey FE, Cifuentes ME, Kiarash A, Quinn MT, Pagano PJ. Novel competitive inhibitor of NAD(P)H oxidase assembly attenuates vascular O(2)(–) and systolic blood pressure in mice. Circ Res. 2001; 89: 408–414.[Abstract/Free Full Text]
33. Heyworth PG, Curnutte JT, Nauseef WM, Volpp BD, Pearson DW, Rosen H, Clark RA. Neutrophil nicotinamide adenine dinucleotide phosphate oxidase assembly. Translocation of p47phox and p67phox requires interaction between p47phox and cytochrome b558. J Clin Invest. 1991; 87: 352–356.
34. Umanskiy K, Robinson C, Cave C, Williams MA, Lentsch AB, Cuschieri J, Solomkin JS. NAD(P)H oxidase activation in fibronectin adherent human neutrophils: A potential role for beta1 integrin ligation. Surgery. 2003; 134: 378–383.[CrossRef][Medline] [Order article via Infotrieve]
35. Kobayashi T, Tsunawaki S, Seguchi H. Evaluation of the process for superoxide production by NAD(P)H oxidase in human neutrophils: evidence for cytoplasmic origin of superoxide. Redox Rep. 2001; 6: 27–36.[CrossRef][Medline] [Order article via Infotrieve]
36. Morimatsu T, Kawagoshi A, Yoshida K, Tamura M. Actin enhances the activation of human neutrophil NAD(P)H oxidase in a cell-free system. Biochem Biophys Res Commun. 1997; 230: 206–210.[CrossRef][Medline] [Order article via Infotrieve]
37. Grogan A, Reeves E, Keep N, Wientjes F, Totty NF, Burlingame AL, Hsuan JJ, Segal AW. Cytosolic phox proteins interact with and regulate the assembly of coronin in neutrophils. J Cell Sci. 1997; 110: 3071–3081.[Abstract]
38. Gu Y, Xu YC, Wu RF, Nwariaku FE, Souza RF, Flores SC, Terada LS. p47phox participates in activation of RelA in endothelial cells. J Biol Chem. 2003; 278: 17210–17217.[Abstract/Free Full Text]
39. Jian-Mei Li and Ajay M. Shah. Mechanism of Endothelial Cell NAD(P)H Oxidase Activation by Angiotensin II. Role of the p47^phox subunit. J Biol Chem. 2003; 4; 278: 12094–12100.
40. Zuo L, Ushio-Fukai M, Hilenski LL, Alexander RW. Microtubules regulate angiotensin II type 1 receptor and Rac1 localization in caveolae/lipid rafts - Role in redox signaling. Arterioscler Thromb Vasc Biol. 2004; 24: 1223–1228.[Abstract/Free Full Text]
41. Hilenski LL, Clempus RE, Quinn MT, Lambeth JD, Griendling KK. Distinct subcellular localization of Nox1 and Nox4 in vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 2004; 24: 1–8.[Free Full Text]
42. Mayer BJ. SH3 domains: complexity in moderation. J Cell Sci. 2001; 114: 1253–1263.[Abstract]
43. Wientjes FB, Segal AW. PX domain takes shape. Curr Opin Hematol. 2003; 10: 2–7.[CrossRef][Medline] [Order article via Infotrieve]
44. Ago T, Kuribayashi F, Hiroaki H, Takeya R, Ito T, Kohda D, Sumimoto H. Phosphorylation of p47phox directs phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NAD(P)H oxidase activation. Proc Natl Acad Sci U S A. 2003; 100: 4474–4479.[Abstract/Free Full Text]
45. Wientjes FB, Reeves EP, Soskic V, Furthmayr H, Segal AW. The NAD(P)H oxidase components p47phox and p40phox bind to Moesin through their PX domain. Biochem Biophys Res Commun. 2001; 289: 382–388.[CrossRef][Medline] [Order article via Infotrieve]
46. Wientjes FB, Segal AW, Hartwig JH. Immunoelectron microscopy shows a clustered distribution of NAD(P)H oxidase components in the human neutrophil plasma membrane. J Leukocyte Biol. 1997; 61: 303–312.[Abstract]
47. Weed SA, Parsons JT. Cortactin: coupling membrane dynamics to cortical actin assembly. Oncogene. 2001; 20: 6418–6434.[CrossRef][Medline] [Order article via Infotrieve]
48. Clements MK, Siemsen DW, Swain SD, Hanson AJ, Nelson-Overton LK, Rohn TT, Quinn MT. Inhibition of actin polymerization by peroxynitrite modulates neutrophil functional responses. J Leukoc Biol. 2003; 73: 344–355.[Abstract/Free Full Text]

This article has been cited by other articles:

				H. Kinoshita, N. Matsuda, H. Kaba, N. Hatakeyama, T. Azma, K. Nakahata, Y. Kuroda, K. Tange, H. Iranami, and Y. Hatano Roles of Phosphatidylinositol 3-Kinase-Akt and NADPH Oxidase in Adenosine 5'-Triphosphate-Sensitive K+ Channel Function Impaired by High Glucose in the Human Artery Hypertension, September 1, 2008; 52(3): 507 - 513. [Abstract] [Full Text] [PDF]

				T. M. Paravicini and R. M. Touyz NADPH Oxidases, Reactive Oxygen Species, and Hypertension: Clinical implications and therapeutic possibilities Diabetes Care, February 1, 2008; 31(Supplement_2): S170 - S180. [Abstract] [Full Text] [PDF]

				R. M. Wadsworth Oxidative stress and the endothelium Exp Physiol, January 1, 2008; 93(1): 155 - 157. [Full Text] [PDF]

				S. Li, X. Li, H. Zheng, B. Xie, K. R. Bidasee, and G. J. Rozanski Pro-oxidant effect of transforming growth factor-{beta}1 mediates contractile dysfunction in rat ventricular myocytes Cardiovasc Res, January 1, 2008; 77(1): 107 - 117. [Abstract] [Full Text] [PDF]

				A. Sachse and G. Wolf Angiotensin II Induced Reactive Oxygen Species and the Kidney J. Am. Soc. Nephrol., September 1, 2007; 18(9): 2439 - 2446. [Abstract] [Full Text] [PDF]

				P. V. Usatyuk, L. H. Romer, D. He, N. L. Parinandi, M. E. Kleinberg, S. Zhan, J. R. Jacobson, S. M. Dudek, S. Pendyala, J. G. N. Garcia, et al. Regulation of Hyperoxia-induced NADPH Oxidase Activation in Human Lung Endothelial Cells by the Actin Cytoskeleton and Cortactin J. Biol. Chem., August 10, 2007; 282(32): 23284 - 23295. [Abstract] [Full Text] [PDF]

				E. L. Schiffrin, M. L. Lipman, and J. F.E. Mann Chronic Kidney Disease: Effects on the Cardiovascular System Circulation, July 3, 2007; 116(1): 85 - 97. [Abstract] [Full Text] [PDF]

				A. M. Guo, A. S. Arbab, J. R. Falck, P. Chen, P. A. Edwards, R. J. Roman, and A. G. Scicli Activation of Vascular Endothelial Growth Factor through Reactive Oxygen Species Mediates 20-Hydroxyeicosatetraenoic Acid-Induced Endothelial Cell Proliferation J. Pharmacol. Exp. Ther., April 1, 2007; 321(1): 18 - 27. [Abstract] [Full Text] [PDF]

				J.-K. Park, R. Fischer, R. Dechend, E. Shagdarsuren, A. Gapeljuk, M. Wellner, S. Meiners, P. Gratze, N. Al-Saadi, S. Feldt, et al. p38 Mitogen-Activated Protein Kinase Inhibition Ameliorates Angiotensin II-Induced Target Organ Damage Hypertension, March 1, 2007; 49(3): 481 - 489. [Abstract] [Full Text] [PDF]

				Y. Wang, M. M. Zeigler, G. K. Lam, M. G. Hunter, T. D. Eubank, V. V. Khramtsov, S. Tridandapani, C. K. Sen, and C. B. Marsh The Role of the NADPH Oxidase Complex, p38 MAPK, and Akt in Regulating Human Monocyte/Macrophage Survival Am. J. Respir. Cell Mol. Biol., January 1, 2007; 36(1): 68 - 77. [Abstract] [Full Text] [PDF]

				P. K. Mehta and K. K. Griendling Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system Am J Physiol Cell Physiol, January 1, 2007; 292(1): C82 - C97. [Abstract] [Full Text] [PDF]

				Y. Wei, J. R. Sowers, R. Nistala, H. Gong, G. M.-E. Uptergrove, S. E. Clark, E. M. Morris, N. Szary, C. Manrique, and C. S. Stump Angiotensin II-induced NADPH Oxidase Activation Impairs Insulin Signaling in Skeletal Muscle Cells J. Biol. Chem., November 17, 2006; 281(46): 35137 - 35146. [Abstract] [Full Text] [PDF]

				L. S. Terada Specificity in reactive oxidant signaling: think globally, act locally J. Cell Biol., August 28, 2006; 174(5): 615 - 623. [Abstract] [Full Text] [PDF]

				R. E. Clempus and K. K. Griendling Reactive oxygen species signaling in vascular smooth muscle cells Cardiovasc Res, July 15, 2006; 71(2): 216 - 225. [Abstract] [Full Text] [PDF]

				T. M. Paravicini and R. M. Touyz Redox signaling in hypertension Cardiovasc Res, July 15, 2006; 71(2): 247 - 258. [Abstract] [Full Text] [PDF]