Mechanism of Endothelial Cell NADPH Oxidase Activation by Angiotensin II

ROLE OF THE p47phox SUBUNIT*

Jian-Mei Li and Ajay M. ShahDagger

From the Department of Cardiology, Guy's, King's, and St. Thomas's School of Medicine, King's College London, Bessemer Road, London SE5 9PJ, United Kingdom

Received for publication, September 24, 2002, and in revised form, December 18, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelial cells express a constitutively active phagocyte-type NADPH oxidase whose activity is augmented by agonists such as angiotensin II. We recently reported (Li, J.-M., and Shah, A. M. (2002) J. Biol. Chem. 277, 19952-19960) that in contrast to neutrophils a substantial proportion of the NADPH oxidase in unstimulated endothelial cells exists as preassembled intracellular complexes. Here, we investigate the mechanism of angiotensin II-induced endothelial NADPH oxidase activation. Angiotensin II (100 nmol/liter)-induced reactive oxygen species production (as measured by dichlorohydrofluorescein fluorescence or lucigenin chemiluminescence) was completely absent in coronary microvascular endothelial cells isolated from p47phox knockout mice. Transfection of p47phox cDNA into p47phox-/- cells restored the angiotensin II response, whereas transfection of antisense p47phox cDNA into wild-type cells depleted p47phox and inhibited the angiotensin II response. In unstimulated human microvascular endothelial cells, there was significant p47phox-p22phox complex formation but minimal detectable p47phox phosphorylation. Angiotensin II induced rapid serine phosphorylation of p47phox (within 1 min, peaking at ~15 min), a 1.9 ± 0.1-fold increase in p47phox-p22phox complex formation and a 1.6 ± 0.2-fold increase in NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production (p < 0.05). p47phox was redistributed to "nuclear" and membrane-enriched cell fractions. These data indicate that angiotensin II-stimulated endothelial NADPH oxidase activity is regulated through serine phosphorylation of p47phox and its enhanced binding to p22phox.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Angiotensin II (AngII)1 has pleiotropic acute and chronic effects on many cell types and plays an important role in the pathophysiology of cardiovascular diseases, including hypertension, atherosclerosis, and heart failure (1). A significant body of evidence supports a role for the intracellular production of reactive oxygen species (ROS) in the signal transduction of AngII-dependent cellular responses via activation of redox-sensitive signaling cascades (2-4). Recent studies (5) suggest that a significant source of intracellular ROS in cardiovascular cells is a phagocyte-type NADPH oxidase. AngII activates NADPH oxidases in vascular smooth muscle (5, 6), fibroblasts (7), endothelial cells (EC) (8, 9), and cardiomyocytes (10). Furthermore, NADPH oxidase activation and increased ROS production are implicated in AngII-stimulated vascular smooth muscle hypertrophy (3, 5) and in AngII-dependent hypertension and the associated endothelial dysfunction (11-13). The mechanisms through which AngII activates NADPH oxidase are therefore of interest.

To date, NADPH oxidase has been best characterized in neutrophils where it is integral to nonspecific host defense. The neutrophil oxidase comprises a membrane-bound cytochrome b558 composed of a p22phox-gp91phox heterodimer and several cytosolic subunits (p47phox, p40phox, p67phox, and Rac) (14). The enzyme is normally dormant but upon neutrophil stimulation, the cytosolic subunits translocate to the membrane and associate with cytochrome b558, resulting in rapid activation of the oxidase. Phosphorylation of the regulatory subunit p47phox plays a key role in this process. The kinetics of NADPH oxidase activation parallel the kinetics of p47phox phosphorylation (15). Phosphorylation of p47phox is thought to induce conformational changes that allow subsequent binding of phospho-p47phox to cytochrome b558 and the other cytosolic subunits (14-16).

NADPH oxidase in non-phagocytic cells such as EC and vascular smooth muscle exhibits significant differences from the neutrophil enzyme. In particular, in contrast to the neutrophil enzyme, the oxidase in non-phagocytic cells is constitutively active at a low level even in unstimulated cells, yet it can be further stimulated acutely by agonists such as AngII and cytokines (5). Recent studies (17, 18) have indicated the presence of a number of isoforms of gp91phox, termed Noxs (for NADPH oxidase), and it has been suggested that the substitution of gp91phox (also known as Nox2) by Nox1 or Nox4 may account for the different behavior of non-phagocytic enzymes. For example, it has been shown in rat aortic smooth muscle cells that the predominant Nox isoforms are Nox4 and Nox1 with very low to undetectable levels of gp91phox expressed (18). In EC, however, all the classic NADPH oxidase subunits, including gp91phox, are expressed (19, 20). Recently, we have reported that, in contrast to neutrophils, in unstimulated quiescent cultured EC a substantial proportion of the NADPH oxidase is present as already fully preassembled complexes in a predominantly perinuclear location associated with the intracellular cytoskeleton (20). Thus, the "cytosolic" subunits p47phox, p40phox, p67phox, and Rac1 could be co-immunoprecipitated down with p22phox and gp91phox in unstimulated EC. The presence of these preassembled complexes is likely to account for the constitutive NADPH oxidase activity of unstimulated EC. However, if the oxidase is already preassembled in unstimulated EC, what is the precise nature of the mechanism(s) underlying the increase in oxidase activity following addition of agonists such as AngII? Recently, we reported that the stimulation of EC NADPH oxidase activity by TNFalpha or phorbol ester (PMA) required the presence of p47phox, although the mechanisms involved were not defined in that study (21). Likewise, in vascular smooth muscle cells, the p47phox subunit has been shown to be essential for ROS production in response to PMA, AngII, thrombin, and platelet-derived growth factor (22, 23), and compelling evidence has been provided for its involvement in atherosclerosis (23). Potential mechanisms that could underlie the increase in NADPH oxidase activity induced by AngII may include (a) an increase in the number of fully assembled oxidase complexes, (b) the translocation of p47phox to partially assembled complexes, and/or (c) the phosphorylation of p47phox (or other subunits) that are already part of the assembled oxidase complex in EC.

In the present study, we have therefore investigated the mechanisms of AngII-induced EC NADPH oxidase activation, focusing on the role of p47phox, its phosphorylation, and its possible translocation and association with cytochrome b558. Our results provide an insight into the fundamental mechanisms of AngII-induced EC NADPH oxidase activation and ROS production.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents-- 5-(and 6)-chloromethyl-2',7'-dichlorohydrofluorescein diacetate (DCF) was purchased from Molecular Probes. Goat polyclonal antibodies to p22phox and p47phox and the corresponding blocking peptides were from Santa Cruz Biotechnology. Affinity-purified rabbit polyclonal antibodies to p47phox and p22phox were a kind gift from Dr. F. Wientjes (University College London, UK). The anti-phosphoserine-specific monoclonal antibody was from Sigma. All other reagents were from Sigma except where specified.

Cell Culture-- p47phox null mice (p47phox-/-) on a 129 sv background were generated as described (24) and were kindly provided by Dr. Jurgen Roes (University College London, UK). All studies conformed with the Guidance on the Operation of Animals (Scientific Procedures) Act, 1986 (Her Majesty's Stationery Office, London, UK). Coronary microvascular EC (CMEC) were isolated from the hearts of 8-10-week-old p47phox-/- and matched wild-type mice exactly as described previously (25). Cultured CMEC were used at passage 2. Human microvascular EC (HMEC-1) (26) were obtained from the Centers for Disease Control and Prevention (Atlanta, GA). HL-60 cells were from the American Type Culture Collection. They were differentiated to neutrophils by incubation with 1.3% Me2SO for seven days and stimulated with PMA (100 ng/ml) for 30 min to activate NADPH oxidase (21).

Detection of ROS Production-- A number of complementary methods and assays were used for the detection of ROS (either O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> or H2O2). For visualization of ROS production, EC were cultured onto chamber slides, washed in Hanks' buffer, and then exposed to AngII (100 nmol/liter) or buffer alone for 10 min. Cells were incubated with 10 µmol/liter DCF in Hanks' buffer for 30 min at room temperature, and DCF fluorescence (which detects mainly H2O2) was then acquired as described (21). Fluorescence intensity was quantified microscopically from at least three random fields (1024 × 1022 pixels; 269.7 × 269.2 µm) per slide (50 cells assessed per slide), three slides per experimental condition.

In some experiments, ROS generation in DCF-loaded cells was also measured by flow cytometry on a FACScalibur instrument (BD PharMingen). For these studies, EC just reaching confluence were detached by trypsin and stimulated with AngII (100 nM). Cellular fluorescence was quantitated by the geometric means of data distributions (GMean).

Specific NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production was assessed by lucigenin (5 µmol/liter) chemiluminescence as described previously (19, 21). We measured this both in living cells and in total EC homogenate. For the latter, cells stimulated with AngII (100 nmol/liter) or buffer in serum-free medium were scraped into ice-cold Hank's balanced salt solution supplemented with MgCl2 0.8 mmol/liter and CaCl2 1.8 mmol/liter. Cells were disrupted by rapid freezing in liquid nitrogen followed by sonication. O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production was measured in the presence of NADPH (100 µmol/liter) for 20 min and was expressed as mean arbitrary light units per min (19, 21). In experiments with an AT1 or AT2 receptor antagonist (losartan and PD123319, respectively), these were added 30 min prior to AngII.

Immunoprecipitation and Immunoblotting-- Immunoprecipitation was performed as described previously using protein samples (250 µg) diluted in Tris-HCl 0.05 mol/liter buffer (pH 7.4) with protease inhibitors (20). p22phox or p47phox was immunoprecipitated down with affinity-purified goat polyclonal antibodies coupled to protein G-agarose beads. Immunoblotting of immunoprecipitates was undertaken with affinity-purified rabbit polyclonal antibodies to p47phox and p22phox or an anti-phosphoserine-specific monoclonal antibody. Protein isolated from differentiated, PMA-stimulated HL-60 cells was used as a positive control.

Subcellular Protein Fractionation-- Differential centrifugation was undertaken exactly as described previously (20). The following fractions were separated: (a) nuclei-enriched (N fraction) pelleted at 1,475 × g; (b) primary mitochondria and other large organelles (C fraction) pelleted at 10,800 × g; (c) submitochrondrial particles, smaller organelles, and some microsomes (D fraction) pelleted at 29,000 × g; (d) microsomes, microperoxisomes, and membrane fractions (E fraction) pelleted at 100,000 × g; and (e) soluble cytoplasmic protein (S fraction). The "purity" of fractions was confirmed by marker enzyme activities as described previously (20).

Sense and Antisense p47phox cDNAs Transfection and Confocal Microscopy-- Full-length neutrophil sense and antisense p47phox cDNAs were generated and CMEC were transfected as described previously (21). After 72 h of transfection, cells were examined by confocal microscopy for the expression of p47phox protein using a BioRad 1024 microscope. Optical sections were taken at 0.5-µm intervals, and images were captured and stored digitally for analysis.

Statistics-- Data are presented as mean ± S.D. of at least three different experiments for each condition. Six mice per group were used for each CMEC isolation, and at least three independent isolates were studied. Comparisons were made by one-way analysis of variance or unpaired Student's t test as appropriate. p < 0.05 was considered statistically significant.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AngII-induced ROS Production Is Inhibited in EC Lacking p47phox-- To investigate whether p47phox is necessary for AngII-stimulated increases in NADPH oxidase activity, wild-type and p47phox-/- CMEC were studied in parallel (Fig. 1A). Using flow cytometry in DCF-loaded cell suspensions, basal ROS production was detected in unstimulated EC of both types. Unstimulated p47phox-/- CMEC had significantly higher DCF fluorescence than wild-type cells (GMean 207.6 ± 11.26 versus 162.9 ± 13.44; p < 0.01). Wild-type EC incubated with AngII for 30 min showed a significant increase in fluorescence signal (GMean 241.1 ± 12.40 versus 162.9 ± 13.44; p < 0.01). In marked contrast, AngII treatment induced no increase in fluorescence in p47phox-/- cells; on the contrary, DCF fluorescence was reduced after AngII treatment (GMean 168.8 ± 13.45 versus 207.6 ± 11.26; p < 0.01).


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Fig. 1.   ROS production by wild-type and p47phox-/- CMEC detected by DCF fluorescence. A, ROS generation in EC suspensions detected by flow cytometry. The experiment was repeated five times and quantitative analyses performed on geometric means of the data distributions. B, representative microscopic images of DCF fluorescence in adherent wild-type and p47phox-/- EC with and without AngII. C, mean data for DCF fluorescence intensity in adherent EC, quantified microscopically (n > 300 cells from three independent experiments). *, p < 0.05 compared with respective wild-type or p47phox-/- control.

Similar results were obtained in experiments on adherent CMEC cultured onto chamber slides and examined by fluorescence microscopy (Fig. 1, B and C). A small amount of DCF fluorescence was detected in a mainly perinuclear distribution in unstimulated EC from both groups, with a higher level in p47phox-/- cells. AngII significantly increased fluorescence (by ~50%) in wild-type EC. However, no increase was observed after AngII in p47phox-/- cells. The addition of the cell-permeable O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> scavenger, Tiron, abolished DCF fluorescence in both groups, indicating that the primary source of H2O2 detected by DCF was likely to be O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>.

As an alternative approach, NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production with and without AngII was examined by lucigenin chemiluminescence both in living cells and cell homogenates (Fig. 2). Unstimulated p47phox-/- cells had slightly but significantly higher NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production than wild-type cells. AngII significantly increased NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production in intact wild-type cells (by 38.5 ± 9.8%), whereas this response was completely blocked in p47phox-/- cells. NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production was fully inhibited by DPI (50 µmol/liter) in both groups (Fig. 2) but was unaltered by rotenone, oxypurinol, or L-NAME (data not shown). Similar results were obtained with cell homogenate (Fig. 2, middle panel). Again basal O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production was higher in p47phox-/- cells compared with wild-type. AngII caused an ~2-fold increase in basal O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production in wild-type cells, whereas no increase was observed in p47phox-/- cells. AngII-induced NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production by wild-type cell homogenate was inhibited by DPI, superoxide dismutase, or Tiron, but was unaffected by rotenone, oxypurinol, or L-NAME (Fig. 2, middle and lower panels), consistent with NADPH oxidase as the source. Collectively, these results suggest that the p47phox subunit is essential for the AngII-induced increase in NADPH oxidase activity in EC.


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Fig. 2.   NADPH-dependent O<UP><SUB><B>2</B></SUB><SUP><B>&cjs1138;</B></SUP></UP> production by intact wild-type and p47phox-/- CMEC or cell homogenate measured by lucigenin chemiluminescence. The top panel shows results for intact cells, the middle and lower panels for cell homogenate. MLU, mean light units. *, p < 0.05 compared with the respective wild-type or p47phox-/- control. In the bottom panel, the effect of a number of inhibitors or antagonists on the maximal AngII-induced NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production by wild-type EC homogenate is shown. *, p < 0.05 compared with oxidase activity in the absence of inhibitors.

Effects of p47phoxSense or Antisense cDNA Transfection on the Oxidase Response to AngII-- To confirm that the loss of ROS response to AngII stimulation was directly attributable to the absence of p47phox, we undertook experiments to acutely deplete or restore p47phox levels in EC by transfection of antisense or sense p47phox cDNA, respectively. Fig. 3A shows a representative example of the effect of antisense p47phox cDNA transfection on p47phox expression in wild-type CMEC, assessed by confocal microscopy. There was an ~60.4 ± 7.3% reduction in p47phox fluorescence (mean data from 300 cells from three independent transfections). In wild-type EC transfected with vector control, AngII significantly increased NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production; this effect was totally lost in antisense cDNA transfected cells (Fig. 3B, top panel).


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Fig. 3.   Effect of sense and antisense p47phox cDNA transfection of EC. A, representative confocal micrographs showing wild-type or knockout cells stained for p47phox after transfection with empty vector or cDNA as labeled. B, AngII-induced changes in NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production (lucigenin chemiluminescence) in wild-type and p47phox-/- cells. *, p < 0.05 comparing AngII versus respective control.

On the other hand, transfection of p47phox-/- cells with sense p47phox cDNA resulted in significant expression of p47phox (Fig. 3A, lower panel). In parallel experiments, NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production in response to AngII was restored after sense p47phox cDNA transfection (Fig. 3B, bottom panel). These experiments clearly indicate an essential role of p47phox in AngII-induced EC NADPH oxidase activation and ROS production.

AngII Induces p47phox Phosphorylation and Increases Binding to p22phox-- To examine the mechanism through which p47phox is involved in AngII-induced NADPH oxidase activation, we studied p47phox phosphorylation and the binding of p47phox to p22phox. These studies were undertaken in HMEC-1, which were available in greater numbers than the primary murine EC isolates. Adherent cells were stimulated with AngII for 1-60 min, p47phox was immunoprecipitated from cell homogenate, and serine phosphorylation of p47phox was detected with a phosphoserine-specific antibody. In unstimulated cells, no significant serine phosphorylation of p47phox was detected (Fig. 4A, upper panel). AngII induced rapid p47phox phosphorylation within 1 min, peaking at ~15 min and thereafter declining gradually. The same membrane was reprobed with an anti-p22phox polyclonal antibody to assess the relationship between p47phox phosphorylation and p47phox-p22phox complex formation (Fig. 4A, lower panel). p22phox protein was readily detectable even in the p47phox immunoprecipitate of unstimulated EC, indicating complex formation in these cells in the absence of significant p47phox phosphorylation. However, the amount of p22phox co-immunoprecipitated with p47phox rapidly increased in AngII-stimulated EC, peaking at ~15 min.


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Fig. 4.   AngII-induced phosphorylation of p47phox and p22phox-p47phox complex formation in HMEC-1. A, upper panel, p47phox was immunoprecipitated (IP) and then immunoblotted (IB) for serine phosphorylation; the lower band, which is also present in the IgG control lane, is a nonspecific band. Lower panel, the same membrane was re-probed for p22phox co-immunoprecipitated down with p47phox. B, upper and middle panels, mean data for changes in p47phox phosphorylation and complex formation after AngII treatment. Immunoblots were scanned densitometrically, and results were expressed as arbitrary absorbance units. Lower panel, NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production by cell homogenates was measured by chemiluminescence and expressed as mean light units (MLU) per mg protein. *, p < 0.05 compared with unstimulated cells (i.e. time 0).

Fig. 4B shows the mean data from three different experiments and also shows data for NADPH oxidase activity measured in parallel in EC homogenates. It is evident that AngII-induced increases in p47phox phosphorylation and p22phox-p47phox complex formation were paralleled by significant increases in oxidase activity.

Effect of AT Receptor Antagonists on p47phox Phosphorylation and Binding to p22phox-- Because both AT1 and AT2 receptors are expressed on EC and could mediate different biological actions (8, 9, 27), we examined their role in AngII-induced p47phox phosphorylation. HMEC-1 were preincubated for 30 min with either the AT1 antagonist losartan (1 µmol/liter) or the AT2 antagonist PD123319 (0.1 µmol/liter) before treatment with AngII. p47phox was immunoprecipitated and examined for serine phosphorylation (Fig. 5A, upper panel). Both losartan or PD123319 inhibited AngII-induced p47phox phosphorylation, although the AT1 antagonist was more effective (Fig. 5B, upper panel).


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Fig. 5.   Effect of AT1 and AT2 antagonists on p47phox phosphorylation and p22phox-p47phox complex formation. A, upper panel, p47phox was immunoprecipitated (IP) and immunoblotted (IB) for serine phosphorylation. Lower panel, p22phox was immunoprecipitated and detected for the co-existence of p47phox. B, upper and middle panels, mean data for changes in p47phox phosphorylation and complex formation. Immunoblots were scanned densitometrically and expressed as arbitrary absorbance units. Lower panel, NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production by cell homogenates measured by chemiluminescence and expressed as mean light units (MLU) per min per mg protein. *, p < 0.05 compared with unstimulated cells (control).

To examine complex formation, we immunoprecipitated p22phox and then probed for the co-existence of p47phox (Fig. 5A, lower panel). After 30 min of AngII treatment, the amount of p47phox co-immunoprecipitated with p22phox had increased significantly (p < 0.05; Fig. 5B, top panel). Interestingly, p47phox was detected as a doublet (Fig. 5A, lower panel); the additional band may represent phospho-p47phox because it was not detected in unstimulated EC and was decreased in cells preincubated with losartan or PD123319. Both losartan and PD123319 significantly inhibited AngII-induced p47phox-p22phox complex formation, with losartan again being more effective (Fig. 5B, middle panel). This was accompanied by inhibition of AngII-induced NADPH oxidase activity, which occurred to a greater extent with losartan than with PD123319 (Fig. 5B, lower panel).

Subcellular Redistribution of p47phox after AngII Treatment-- Finally, we examined the effect of AngII treatment on the subcellular distribution of p47phox in HMEC-1. In unstimulated cells, after fractionation by differential centrifugation, the majority of p47phox (44 ± 3%) was detected in the 100,000 × g (microsomal and membrane) E fraction with a significant proportion (30 ± 4%) also found in the 1457 × g (nuclei-enriched) N fraction (Fig. 6A, upper panel). After AngII stimulation, 59 ± 4% of p47phox was detected in the N fraction and was clearly detected as a doublet band (Fig. 6A, lower panel).


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Fig. 6.   Distribution of p47phox studied by subcellular fractionation. A, p47phox expression in each fraction before and after AngII stimulation, assessed by immunoblotting. B, NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production by each fraction assessed by chemiluminescence. *, p < 0.05 compared with results without AngII.

In line with these results, NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production was significantly increased in the N fraction in AngII-stimulated EC. A significant increase was also found in the E fraction (Fig. 6B).

To assess whether a change in p47phox subcellular distribution could also be detected by a complementary method, we undertook immunocytochemical staining and confocal microscopy in HMEC-1 with and without AngII stimulation (Fig. 7). In unstimulated cells, p47phox had a mainly perinuclear and rather punctate distribution. After AngII stimulation, the perinuclear p47phox labeling appeared more organized in a reticular pattern, and there was also clear labeling of the cell surface membranes (Fig. 7, lower panels, white arrows), consistent with a redistribution of p47phox.


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Fig. 7.   Confocal micrographs showing immunocytochemical staining of HMEC-1 for p47phox with and without AngII stimulation. The right panels show higher magnification images. The white arrows indicate areas of cell surface membrane labeling in AngII-stimulated cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

AngII is well known to increase EC ROS production, and recent studies have implicated the stimulation of NADPH oxidase as an important component of this response (4, 5, 8, 9). In neutrophils, the activation of NADPH oxidase involves the translocation and binding of cytosolic subunits (p47phox, p67phox, p40phox, and rac) to cytochrome b558; the phosphorylation of p47phox is considered to be a key step in this process (14). In EC, however, we have recently shown that a significant proportion of the oxidase is present as fully preassembled intracellular complexes, even in unstimulated ("resting") cells (20). The main new findings of the present study are that (a) AngII-stimulated increases in EC NADPH oxidase activity and ROS production are absolutely dependent on the p47phox subunit; (b) the phosphorylation of p47phox and its binding to the cytochrome b558 to form new complexes is required for the AngII response; and (c) in unstimulated cells, the p47phox that is bound to cytochrome b558 appears to be in an unphosphorylated or minimally phosphorylated state.

The requirement for p47phox in the AngII response was clearly demonstrated in experiments on EC isolated from p47phox-/- and wild-type mice. Furthermore, in vitro transfection of p47phox cDNA into p47phox-/- EC successfully restored the ROS response to AngII, whereas in vitro depletion of p47phox from wild-type EC by antisense cDNA transfection resulted in loss of the ROS response. In the present study, we used lucigenin chemiluminescence or DCF fluorescence for ROS detection either in adherent cells, cell suspensions, and/or cell homogenates. Lucigenin chemiluminescence detects both intracellular and extracellular O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production, whereas DCF fluorescence detects mainly intracellular H2O2 (28). Evidence that the primary species leading to the H2O2 detected by DCF fluorescence in this study was O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> was provided by the finding that the cell-permeable O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> scavenger Tiron substantially inhibited the DCF signal (Fig. 1C). The data from these different assays (i.e. Figs. 1-3) were broadly comparable in indicating a requirement for p47phox in the AngII response; the small quantitative differences between assays are likely to reflect the differing experimental conditions (i.e. adherent versus trypsinized cells and intact cells versus cell homogenate).

The oxidase subunit p47phox contains several serine residues clustered within the carboxyl terminus that form potential protein kinase C phosphorylation sites (29). Native p47phox is a highly basic protein that undergoes stepwise charge shifts with successive phosphorylation events (29, 30). In neutrophils, p47phox phosphorylation is thought to be a prerequisite for its interaction with p22phox and the formation of a functional oxidase complex (15, 16). In EC, however, a significant proportion of the NADPH oxidase is present as fully preassembled intracellular complexes even in unstimulated ("resting") cells (20). Nevertheless, the oxidase can be further stimulated by agonists such as AngII. In the present study, we confirmed that p47phox could be co-immunoprecipitated with p22phox in the absence of AngII stimulation. An interesting and new finding was that the p47phox associated with p22phox in unstimulated EC appeared to be largely unphosphorylated (or minimally phosphorylated). Although it is unclear how unphosphorylated p47phox may bind to p22phox in EC, it has previously been reported that unphosphorylated p47phox can bind to p22phox in in vitro assays in the presence of arachidonic acid but that this binding only supports low O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production (31). The binding of unphosphorylated rather than phosphorylated p47phox to cytochrome b558 may therefore account for the fact that oxidase activity in unstimulated EC is relatively low despite the presence of fully assembled complexes.

The mechanism of further increases in oxidase activity in a setting where there are already existing preassembled complexes could either involve the phosphorylation of already bound p47phox or the formation of additional (new) complexes through the association of p47phox with other oxidase components. In neutrophils, initial p47phox phosphorylation is thought to occur in the cytosol; additional p47phox phosphorylation occurs upon interaction with membrane components, which then leads to immediate and irreversible binding of p47phox to cytochrome b558 (15). In the present study, we found that AngII very rapidly induced p47phox phosphorylation (<1 min), which was accompanied by an increase in p47phox-p22phox complex formation and O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production. These data are consistent with a mechanism where p47phox phosphorylation promotes stable binding of p47phox to p22phox (and thus oxidase activation), perhaps by inducing appropriate conformational changes in p47phox (16, 31), i.e. that p47phox phosphorylation promotes the formation of new oxidase complexes. Consistent with this, we found that AngII stimulation was associated with an ~20% translocation of p47phox from other fractions to the N (nuclei-enriched) fraction, where we have previously shown that the majority of oxidase components are found in EC (20). Interestingly, AngII also induced a small but significant translocation of p47phox to the membrane-enriched E fraction, consistent with some of the oxidase being present also on the plasma membrane. Indeed, immunocytochemical staining and confocal microscopy demonstrated p47phox labeling of the plasma membrane in AngII-treated cells as well as a suggestion of a more organized reticular labeling in the perinuclear region. The temporal relationship between p47phox phosphorylation, binding of phospho-p47phox to p22phox, and the increase in NADPH oxidase activity suggests that, as in neutrophils (14-16), the serine phosphorylation of p47phox is the key step for oxidase activation by AngII.

AngII exerts its biological effects through the activation of two distinct classes of receptors, AT1 and AT2. Both AT1 and AT2 receptors are known to be expressed in endothelial cells (9, 27, 32). Most of the stimulatory effects of AngII on cardiovascular cells are mediated by AT1 receptors, whereas there is less data on the AT2 receptor subtype, which in some settings can be antagonistic. The results of the present study suggested that both AT1 and AT2 receptors were involved in AngII-induced p47phox phosphorylation and NADPH oxidase activation because the latter could be inhibited either by losartan or PD123319, which are known to be specific inhibitors of the AT1 and AT2 receptor, respectively. However, the effects of losartan appeared to be more potent than those of PD123319, in accordance with a previous study in EC (9).

An interesting finding in the present study was that unstimulated p47phox-/- cells maintained a significant level of NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production and, in fact, had slightly but significantly higher ROS production than wild-type cells, as we have reported previously (21). The underlying basis for this "background" ROS production remains to be established. One interpretation of these results would be that the p47phox subunit is not essential for "basal" NADPH oxidase activity in EC. Consistent with this possibility, it has been reported that in cell-free systems NADPH oxidase can be activated in the absence of p47phox if high concentrations of p67phox and Rac1 are present (33, 34). However, an alternative interpretation would be that the background ROS production emanates from a completely different source. The findings of the present and previous (21) studies that the background NADPH-dependent O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> production is abolished by DPI but is unaffected by rotenone, oxypurinol, or L-NAME indicates that any such source would have to be an NADPH-dependent flavoprotein enzyme. Because DPI is not a specific inhibitor just of NADPH oxidase, definitive analysis of the underlying source of this background ROS production in EC may require studies in which multiple NADPH oxidase subunits are knocked out.

In summary, this study demonstrates that AngII-induced ROS production by EC is critically dependent on the NADPH oxidase subunit p47phox. Serine phosphorylation of p47phox, followed by p47phox translocation and the stable binding of phospho-p47phox to p22phox are the key steps that initiate AngII-induced NADPH oxidase activation. This process appears to involve the formation of new oxidase complexes. ROS generated intracellularly in the perinuclear region may serve as signaling molecules for the activation of redox-sensitive enzymes and transcription factors (1, 5), whereas extracellular ROS may contribute to endothelial dysfunction (11-13).

    FOOTNOTES

* This work was supported by British Heart Foundation (BHF) Program Grant RG/98008.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Holds the BHF Chair of Cardiology in King's College London. To whom correspondence should be addressed. Tel.: 44-207-346-3865; Fax: 44-207-346-4771; E-mail: ajay.shah@kcl.ac.uk.

Published, JBC Papers in Press, January 30, 2003, DOI 10.1074/jbc.M209793200

    ABBREVIATIONS

The abbreviations used are: AngII, angiotensin II; ROS, reactive oxygen species; NOX, NADPH oxidase; PMA, phorbol 12-myristate 13-acetate; DCF, 5-(and 6)-chloromethyl-2',7'-dichlorohydrofluorescein diacetate; EC, endothelial cell; CMEC, coronary microvascular EC; HMEC, human microvascular EC; DPI, diphenyleneiodonium; L-name, NG-nitro-L-arginine methyl ester.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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