Biochemical and Biophysical Research Communications 278, 584 –589 (2000) doi:10.1006/bbrc.2000.3848, available online at http://www.idealibrary.com on
Functional Expression of NAD(P)H Oxidase p47 in Lung Microvascular Endothelial Cells Hedwig S. Murphy,* ,† ,1 Chuliang Yu,† and Jawaid Quddus† *Pathology and Laboratory Medicine, Veterans Administration Medical Center; and †Department of Pathology, University of Michigan, Ann Arbor, Michigan 48109
Received October 23, 2000
Vascular endothelial cell superoxide (O 2•) has an important role in intracellular signaling, in interaction with other reactive species such as nitric oxide, and in vascular dysfunction. Little is known regarding the source and function of O 2• from microvascular endothelial cells from specific tissues. Mouse lung microvascular endothelial cells stimulated with phorbol ester (PMA) or NADPH generated significant O 2•, which was inhibited by diphenyleneiodonium (DPI) but not by allopurinol, rotenone, indomethacin, or quinacrine. Optimal O 2• generation required cytosolic as well as particulate cell fractions of cells. In parallel studies, PMA induced increased expression of the p47 component of the NAD(P)H oxidase in the particulate fraction, which was inhibited by staurosporine and calphostin. These data demonstrate that NAD(P)H oxidase is an important source of O 2• generation in lung microvascular endothelial cells. © 2000 Academic Press Key Words: endothelial cells; superoxide; NAD(P)H oxidase; p47; reactive oxygen species (ROS); lung.
Reactive oxygen species (ROS) play an important role in inflammation, atherosclerosis, ischemia/reperfusion injury and hypertension (1– 4). Endothelial cell dysfunction, the hallmark of acute lung injury occurring in inflammation and ischemia, has been directly linked to the generation of reactive oxygen species including superoxide (O 2•). While evidence suggests that an NAD(P)H oxidase is responsible for O 2• generation in macrovascular endothelial cells, little is known regarding the source of O 2• in microvascular endothelial cells from specific tissues. Inflammatory cells such as macrophages and neutrophils are well known to genSupported by a grant from the American Heart Association, Michigan Affiliate and by Research Enhancement Award Program (REAP) funds from the Department of Veterans Affairs. 1 To whom correspondence and reprint requests should be addressed at Department of Pathology, University of Michigan, 5240 Medical Science I, 1301 Catherine, Ann Arbor, Michigan 48109. Fax: 734-761-5037. E-mail:
[email protected]. 0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
erate substantial quantities of ROS but vascular endothelial cells derived from microvessels generate significant O 2• in response to inflammatory mediators as well (5–9). Furthermore, microvascular endothelial cells from different tissues differ in their generation of O 2•, suggesting that this reactive species may have an important role in tissue-specific responses (9). An increasing number of functions are ascribed to endothelial cell derived O 2•. This radical appears to be important in endothelium-dependent responses and development of hypertension by modulation of endothelium-derived relaxing factor (EDRF/NO) (10 –14). Toxic oxygen radicals are well-known to play a central role in neutrophil-mediated endothelial cell injury in acute lung inflammation. In tissue microvasculature, O 2• may interact with nitric oxide ( •NO) to scavenge this radical thereby decreasing •NO mediated signal transduction events. This interaction may also result in generation of the toxic radical peroxynitrite or may decrease availability of O 2• which, along with iron, is required for the iron-catalyzed reduction of H 2O 2 to hydroxyl radical ( •OH) (9, 15–18). A number of enzymes may be involved in generation of O 2• including xanthine oxidase, mitochondrial enzymes and cytochrome P450 enzymes, but evidence suggests that a neutrophil-like NAD(P)H oxidase is responsible, at least in part, for generation of substantial O 2• by endothelial cells (10, 19 –26). In human neutrophils, activation of NAD(P)H oxidase results in a surge of oxygen radical production (the respiratory burst). During activation of the oxidase, cytosolic p47phox and p67-phox associate with the membraneassociated flavocytochrome b 558 (p22-phox, gp91 phox subcomponents). Activation of the enzyme requires sequential phosphorylation of two serines of p47, after which membrane association of p47 and p67 takes place (27, 28). Other O 2•⫺ producing cells types including osteoclasts (29), B lymphocytes (30), mesangial cells (31) and fibroblasts (32), possess oxidase components similar to those found in neutrophils suggesting homology among O 2• producing cells. Although accumu-
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lating evidence suggests that a neutrophil-like NAD(P)H oxidase is responsible for O 2• generation in vascular cells as well, this evidence is largely derived from studies of human umbilical vein endothelial cells and other large vessel endothelial cells and may not reflect tissue specific microvascular responses. Endothelial cells from a number of tissues express mRNA and protein for the oxidase components gp91, p22phox, p67-phox and p47-phox suggesting the presence of an NAD(P)H oxidase in the microvasculature (10, 22, 15, 33, 34). Studies of vascular NAD(P)H oxidase in vivo and in whole vessel explants have been complicated not only by the presence of neutrophils but by the existence of NAD(P)H oxidases in smooth muscle and fibroblasts (26, 34). The purpose of this study was to determine if activation of a neutrophil-like NAD(P)Hoxidase was responsible for O 2• generation in mouse lung microvascular endothelial cells. METHODS Reagents. RPMI 1640, fetal calf serum, penicillin–streptomycin, and L-glutamine were purchased from GIBCO Laboratories (Grand Island, NY). Endothelial cell growth supplement was from Collaborative Biomedical (Bedford, MA). Phorbol 12-myristate 13-acetate (PMA), NADPH, diphenyleneiodonium (DPI), rotenone, allopurinol, indomethacin, and quinicrine were obtained from Sigma Chemical (St. Louis, MO). Antibodies were the generous gift of John Curnutte (Genentech, Inc., Palo Alto, CA). Anti-peptide antibodies were raised in rabbits against the protein-G-purified whole recombinant p47phox component of human neutrophil NAD(P)H oxidase. Endothelial cell isolation and culture. Microvascular endothelial cells were isolated from peripheral lungs of 21-day-old AKR mice as previously described (35). Briefly, strips of peripheral lung were removed, minced and incubated in gelatin-coated tissue culture flasks in growth media (RPMI 1640, 20% fetal calf serum, endothelial cell growth supplement and penicillin–streptomycin). After 65 h, tissues were removed and cultures consisted uniformly of endothelial cells, Cultured cells were characterized cells by their cobblestone morphology, uptake of DiI-Ac-LDL, angiotensin converting enzyme (ACE) activity and adhesion molecule expression as we have previously described (35). Cells were maintained in culture and were used at 80 –90% confluence at passage 2 for all experimental studies. Each experiment described was repeated on 3– 4 separate isolates of endothelial cells. HL-60 cells. HL-60 cells were grown in media (RPMI 1640, 20% fetal calf serum, 2 mM L-glutamine, Penicilin–streptomycin) plus 1.25% DMSO for 9 days as previously described. Differentiated HL-60 cells generate O 2• by activation of NAD(P)H oxidase (36). O 2• generation. O 2• generation in endothelial cells was determined by the superoxide dismutase (SOD)-inhibitable reduction of ferricytochrome c as we have previously described (5). Cells were grown to 80 –90% confluence in 6-well tissue culture dishes. To inhibit endogenous SOD, cells were incubated for 1 h at 37°C in 1 mM diethyldithiocarbamate (DETC) in HBSS plus 0.2% BSA, as we have previously described (4). In studies evaluating the effect of inhibitors, cells were pretreated with allopurinol for 1 h, DPI, rotenone, indomethacin or quinicrine for 10 min. After cells were washed, they were incubated in a reaction mixture containing 80 mM ferricytochrome c ⫾ SOD (23.2 mg/ml) in HBSS plus 0.2% BSA. Cells were stimulated in the presence and absence of the inhibitors. Ferricytochrome c was measured by absorbance at 550 nm and the change in OD determined by comparison of stimulated cells in the presence or absence
of SOD. Concentration of O 2• was calculated using the extinction coefficient of 18.5 cm ⫺1 mM ⫺1 for ferricytochrome c. Cell viability at the conclusion of the assay was determined by exclusion of trypan blue. For HL-60 cells, 1 ⫻ 10 6 cells were suspended in 1 ml of reaction mixture containing 80 mM ferricytochrome c ⫾ SOD (23.2 mg/ml) in HBSS. Preparation of subcellular fractions. Cells in 100 mm plates were incubated with appropriate agonists at 37°C, washed three times with ice cold PBS and were harvested by scraping. Cells were centrifuged and resuspended in 80 l of extraction buffer (50 mM Tris– HCl, pH 7.4, 50 mM 2-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mg/ml leupeptin, 10 mg/ml pepstatin, 2 mM EGTA, and 2 mM EDTA). Cells were disrupted by sonication on ice with three 15-s bursts (Ultrasonic processor W-384, Heat SystemsUltrasonic Inc.). Subsequent procedures were performed at 4°C. Sonicates were centrifuged at 500g for 10 min, the nuclei-rich pellet discarded and the supernatant fluid centrifuged at 100,000g for 1 h at 4°C. The supernatant fluid was removed as the cytosolic fraction and the pellet containing the particulate fraction was resuspended in 80 l extraction buffer and sonicated with three 15-s bursts. Samples were stored at ⫺20°C overnight and heated at 100°C for 5 min in standard protein loading buffer. Immunoblot analysis of expression of oxidase components. Cytosolic and particulate fractions from stimulated and unstimulated endothelial cells were subjected to SDS–PAGE on 10% acrylamide gels and compared to fractions from human neutrophils. After electrophoretic transfer to PVDF membrane, blots were probed with 1:5000 dilution of anti-p47 Phox antibody. The membrane was washed with PBS-Tween and incubated in a 1:5000 dilution of horseradish-peroxide-labeled anti-rabbit IgG. The blots were developed using ECL Western blotting reagents (Amersham, UK). Scanning densitometry was performed and analyzed using an MCID imaging system from Imaging Research. Statistical analysis. Where appropriate, data were expressed as means ⫾ SEM. A paired t test was used to compare response between two treatments. Statistical significance was defined as P ⬍ 0.05.
RESULTS Endothelial Cells Use an NAD(P)H Oxidase to Generate O 2• Generation of O 2• was evaluated in murine lung microvascular endothelial cells. Cells were isolated and grown as monolayers to 80 –90% confluence. Parallel studies were performed on differentiated HL-60 cells (Table 1). Endothelial cells generated significant O 2• when exposed to NADPH (100 M) or PMA (1 M) for 90 min. Cells were then incubated for 10 min with the NAD(P)H oxidase inhibitor DPI (diphenyliodonium, 1 M) (37) prior to and simultaneous with stimulation with either NADPH or PMA. In endothelial cells as well as HL-60 cells, O 2• release in response to both stimuli was suppressed by pretreatment with DPI. To rule out alternate enzyme sources of O 2•, the effects of additional inhibitors were evaluated. Addition of allopurinol (1 M) a xanthine oxidase inhibitor, rotenone (10 M) an inhibitor of the mitochondrial respiratory chain, indomethacin (10 M), a non-steroidal antiinflammatory agent, and cyclooxygenase-2 inhibitor, or quinicrine (10 M) an anti-malarial drug and phospholipase A2 inhibitor (which inhibits oxygen radicals re-
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Superoxide Generation Lung endothelial cells
HL-60 cells
Stimulus
Inhibitor
O 2• nmoles/5 ⫻ 10 5 cells
Stimulus
Inhibitor
O 2• nmoles/1 ⫻ 10 6 cells
None NADPH
None None DPI None DPI Allopurinol Rotenone Indomethacin Quinicrine
0.70 ⫾ 0.12 4.75 ⫾ 0.33* 1.14 ⫾ 0.55** 3.49 ⫾ 0.28* 0.73 ⫾ 0.51** 3.89 ⫾ 0.07 3.01 ⫾ 0.70 3.32 ⫾ 0.18 3.33 ⫾ 0.39
None PMA
None None DPI Rotenone Indomethacin Quinicrine
0.00 ⫾ 0.00 78.9 ⫾ 2.30* 0.00 ⫾ 0.00** 78.0 ⫾ 2.00 71.0 ⫾ 2.00 80.0 ⫾ 4.50
PMA
Note. Cells were stimulated for 90 min with PMA or NADPH in the presence and absence of inhibitors: NADPH, 100 M, PMA, 1 M; DPI, 100 M; Allopurinol, 1 M; Indomethacin, 10 M; Rotenone, 10 M; and Quinicrine, 10 M. Values represent the mean ⫾ SEM of separate experiments on three separate isolates of cells, with each data point in duplicate. * P ⬍ 0.05 compared to values for unstimulated cells. ** P ⬍ 0.05 compared to values for stimulated cells.
lease from human alveolar macrophages) had no effect on the release of O 2• from either HL-60 or endothelial cells (38, 39). Only DPI significantly inhibited release of O 2• from either HL-60 or endothelial cells. Cytosolic and particulate fractions of lung endothelial cells were assayed for O 2• release in response to PMA (Fig. 1). Cells were fractionated as described in the Methods and cytosolic fraction, particulate fraction and mixed cytosolic and particulate fractions were incubated for 30 min with PMA (1 M). While particulate fractions alone were capable of generating O 2•, optimal release required the presence of both cytosolic and particulate cell fractions. A Functional NAD(P)H Oxidase Is Expressed in Endothelial Cells Expression of p47-phox protein in stimulated and unstimulated cells was determined by Western blot analysis (Fig. 2). Cytosolic as well as particulate frac-
FIG. 1. Generation of O 2• by cell fractions. Cytosolic (C) and particulate (P) fractions and cytosolic plus particulate fraction (C ⫹ P) of endothelial cells incubated for 30 min with PMA (1 M) and assayed for O 2• generation. Data represent the mean ⫾ SEM of two data points per sample. Two separate experiments on separate cell isolates yielded similar results.
tions of unstimulated endothelial cells have low level expression of a 47 kDa protein reactive with anti-p47phox. The p47 antibody reacted with similar proteins in extracts of HL-60 cells as well as human neutrophils (data not shown). Exposure of cells to PMA (1 M) for 10 min resulted in a 6-fold increase in p47 expression in the particulate fraction with a slight decrease in cytosolic expression. When cells were pretreated with the protein kinase C inhibitors staurosporine (200 nM ⫻ 10 min) or Calphostin (300 nM ⫻ 18 h) followed by exposure to PMA (1 M) for 10 min, the increase in particulate fraction p47 was inhibited (Figs. 3A and 3B). In some experiments, unstimulated endothelial cells released low levels of O 2•, suggesting that even without exposure to exogenous agents, the cells were basally activated. Likewise, in some experiments, pretreatment with protein kinase C inhibitors resulted in
FIG. 2. PMA-induced increase in p47 expression. Cytosolic (C) and particulate (P) fractions of endothelial cells incubated for 10 min in the presence and absence of PMA (1 M) with equal protein loading in all lanes. Scanning densitometry data of the same immunoblot expressed as percent of control. Data are representative of three separate experiments performed on separate cell isolates.
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FIG. 3. Effect of PKC inhibitors on PMA induced increase in p47 expression. (A) Particulate fraction of endothelial cells incubated with PMA (1 M) for 5 min or PMA (1 M) for 5 min after exposure to staurosporine (200 nM) for 10 min. (B) Particulate fraction of endothelial cells incubated with PMA (1 M) for 5 min or PMA (1 M) for 5 min after exposure to calphostin (300 nM) for 18 h. Immunoblots with anti-p47 antibody, with equal protein loading in all lanes. Scanning densitometry data of the same immunoblot expressed as percent of control. Data are representative of two separate experiments performed on separate cell isolates.
a decrease in p47 expression to a level below the controls (untreated cells) (Fig. 3A). Western blot analysis of p47 protein expression in lung endothelial cells exposed to PMA (1 M) for 0 –10 min demonstrated time dependent increase in p47 expression in the particulate fractions (Fig. 4). This increase was evident within the first minute of exposure to PMA. These findings suggest that activation of endothelial cells is associated with the appearance of this oxidase component in the particulate fraction of endothelial cells, in a manner similar to the neutrophil NAD(P)H oxidase. DISCUSSION Generation of O 2• by vascular endothelial cells in response to a variety of agonists has been reported, but
FIG. 4. Time course of PMA induced increase in p47 expression. Particulate fraction of endothelial cells incubated with PMA (1 M) for the duration of time indicated. Immunoblots with anti-p47 antibody, with equal protein loading in all lanes. Scanning densitometry data of the same immunoblot expressed as percentage of control. Data are representative of three separate experiments performed on separate cell isolates.
the enzyme source for microvascular endothelial cell O 2• has not previously been determined. This study provides evidence that an NAD(P)H oxidase is responsible for generation of O 2• by lung microvascular endothelial cells. Indirect evidence has implicated a neutrophil-like NAD(P)H oxidase in the generation of O 2• in vascular endothelial cells. In a variety of intact vessels, vessel rings and isolated endothelial cells, protein kinase C inhibitors as well NAD(P)H oxidase inhibitors reduce O 2• generation while NADH and NADPH stimulate generation of O 2• suggesting the existence of this endothelial NAD(P)H oxidase. Protein as well as mRNA for p22, gp91, p47 and p67 components have been detected in endothelial cells from human and animal tissues (19 –25). Evidence suggests that a number of enzymes may be involved in generation of endothelial cell O 2• including xanthine oxidase, mitochondrial enzymes, cytochrome P450 enzymes and nitric oxide synthase. PMA has not been shown to activate nitric oxide synthase and in the present study, it is unlikely that nitric oxide synthase contributes to the O 2• generation which occurs rapidly. Endothelial cells are known to have a xanthine dehydrogenase/xanthine oxidase which catalyzes the oxidation of hypoxanthine to xanthine and xanthine to uric acid, reducing O 2 to O 2• (40) but our studies have consistently demonstrated that allopurinol fails to inhibit lung endothelial cell O 2• release (5). Inhibitors of mitochondrial oxidase, cyclooxygenase and phospholipase A2 failed to inhibit O 2• release as well. The suppression of O 2• release by DPI is therefore most likely attributable to inhibition of the NAD(P)H oxidase. DPI is an inhibitor of flavin-dependent enzymes including xanthine oxidase, nitric oxide synthase and NAD(P)H oxidase. DPI inhibits O 2• generation in neutrophils and macrophages by binding to the same
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NAD(P)H oxidase protein, p47, which is phosphorylated during activation (37). In a similar manner, DPI inhibited O 2• release from endothelial cells. In neutrophils, p47-phox is phosphorylated and then has the capability of translocating to the membrane, transporting with it the p67-phox component. This function, while perhaps not essential to oxidase activity, appears to enhance oxidase activity (Rev. in 27). PMA directly activates protein kinase C, which has been implicated in phosphorylation of the p47 component of the neutrophil NAD(P)H oxidase(41– 42). In our study, translocation of p47 in response to PMA stimulation was inhibited by the protein kinase C inhibitors staurosporine and calphostin suggesting a similar phosphorylation of the endothelial cell oxidase. While neutrophil-derived O 2• has an important bactericidal role, this is likely limited in the case of the endothelial cell-derived O 2• which is generated in 10- to 100-fold lower concentrations. The importance of ECderived O 2• cannot, however, be underestimated. Accumulating evidence indicates that in large vessels the interaction of O 2• with •NO occurs rapidly and may account for impaired endothelium-dependent relaxation. In tissue microvasculature, O 2• interaction with • NO also serves important biological functions resulting in alteration of •NO mediated signal transduction events or generation of the toxic radical peroxynitrite. The cytoprotective effect of exogenous as well as endogenous nitric oxide in neutrophil-mediated endothelial cell injury may be related to O 2• scavenging and subsequent reduction of O 2• available for generation of OH (1, 9, 11–18). Our study demonstrates that the p47 component of the neutrophil-like endothelial NAD(P)H oxidase has an important role in generation of O 2• in lung microvascular endothelial cells and this component may prove to be a useful target for modulation of the enzyme.
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