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* 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. § Recipient of a grant from Association pour la Recherche Contre le Cancer.
Vitamin E (α-tocopherol), one of the most important natural antioxidants, is assumed to be beneficial in the prevention of cardiovascular diseases. α-Tocopherol exhibits acyl-peroxyl-radical scavenger properties and exerts cell-mediated actions in the hemovascular compartment, such as inhibition of superoxide anion (O⨪2) production by leukocytes. The aim of this study was to examine the mechanism underlying the inhibitory effect of α-tocopherol on O⨪2 production by human monocytes. In activated monocytes O⨪2 is produced by the NADPH-oxidase enzyme complex. The oxidase activation elicited by phorbol myristate acetate (PMA) requires membrane translocation of several cytosolic factors. We found that in human PMA-stimulated adherent monocytes, α-tocopherol (but not β-tocopherol) inhibited O⨪2production in intact cells but had no effect on a membrane preparation containing activated NADPH-oxidase, suggesting that α-tocopherol impairs the assembly process of the enzyme complex. We showed that translocation and phosphorylation of the cytosolic factor p47phox were reduced in monocytes preincubated with α-tocopherol. We verified that the tryptic phosphopeptide map of monocyte p47phox was similar to that of neutrophil p47phox, indicating that several serine residues were phosphorylated. Peptides whose phosphorylation is dependent on protein kinase C (PKC) were phosphorylated to a lesser degree when p47phox was immunoprecipitated from α-tocopherol-treated monocytes. In vitro, the activity of PKC from monocytes was inhibited by α-tocopherol in a specific manner compared with that of β-tocopherol or Trolox®. Membrane translocation of PKC was not affected. These results show that α-tocopherol inhibits O⨪2production by human adherent monocytes by impairing the assembly of the NADPH-oxidase and suggest that the inhibition of phosphorylation and translocation of the cytosolic factor p47phox results from a decrease in PKC activity.
low density lipoprotein
radical oxygen species
protein kinase C
phorbol 12-myristate 13-acetate
polyacrylamide gel electrophoresis
Oxidative modification of low density lipoprotein (LDL)1 appears to be a key event in the early stages of atherogenesis (
), but the mechanism(s) underlying this inhibitory effect are unknown.
The system responsible for O⨪2 production in phagocytic cells is the multicomponent enzyme NADPH-oxidase. This complex includes membrane-bound cytochrome b558 and cytosolic proteins (p47phox, p67phox, Rac1/2, and p40phox) (
The purpose of this study was to explore the underlying mechanism of α-tocopherol-induced inhibition of O⨪2 production by monocytes. We found that α-tocopherol treatment of human adherent monocytes inhibited PMA-induced translocation and phosphorylation of the cytosolic oxidase component p47phox.
dl-α-Tocopherol,dl-β-tocopherol, and PMA were from Sigma; SDS-PAGE reagents were from Bio-Rad; and [32P]orthophosphate and [γ-32P]ATP were from NEN Life Science Products. Anti-p47phox antibody was a kind gift from Dr. Babior (Scripps Research Institute). Anti-PKC antibody was from Santa Cruz Biotechnology.
Mononuclear cells were obtained from healthy subjects by dextran sedimentation and Ficoll-Paque fractionation of freshly drawn blood. The cells were resuspended in RPMI medium + 10% fetal calf serum and then plated in Petri dishes for 2 h at 37 °C (20 × 106 cells/10 ml), in humidified air with 5% CO2. The adherent monocytes remaining after washing with RPMI (about 5 × 106cells/dish) were incubated with α-tocopherol or ethanol in basal conditions for 30 min and then stimulated with PMA (30 ng/ml) for 10 min. For the phosphorylation experiments, cells were resuspended in phosphate-free Dulbecco's modified Eagle's medium, treated with 2.7 mm diisopropylfluorophosphate on ice for 10 min, and then washed. After 2 h in Petri dishes, non-adherent cells were removed, and the remaining monocytes were incubated in phosphate-free Dulbecco's modified Eagle's medium containing 250 μCi of [32P]orthophosphate/107 cells/ml for 1.5 h at 37 °C. α-Tocopherol and PMA were then added as described above. The purity and viability of the monocyte preparations after the adherence step were higher than 95 and 90%, as assessed by trypan blue dye exclusion and neutral red staining, respectively.
Superoxide Production Measurement
The effect of α-tocopherol on O⨪2 production was measured in a lucigenin-enhanced chemiluminescence assay. Adherent monocytes purified in a 48-well plate were preincubated with various amounts of α-tocopherol or its ethanol vehicle for 30 min before stimulation with 60 ng/ml PMA in the presence of 100 μm lucigenin. Luminescence was measured by an ultrasensitive photon-counting imaging camera monitored by a computer-assisted image processor (Argus 100, Hamamatsu Photonics, Japan). Photon emission was recorded for 60 min after PMA stimulation and was proportional to O⨪2 production (
). The membrane fraction containing active NADPH-oxidase was obtained by sonication of stimulated human neutrophils or monocytes (1 μg/ml PMA) in 0.34m sucrose + 1 mm PMSF and ultracentrifugation (100,000 × g for 30 min at 4 °C in a Beckman TL 100). The pellet (membrane fraction) was solubilized in 0.34m sucrose. The production of O⨪2 was measured (
Resting and stimulated cells were scraped from the Petri dishes (5 × 106 cells/ml), sonicated on ice (2 × 10 s) in relaxation buffer (100 mm KCl, 3 mm NaCl, 3.5 mm MgCl2, 10 mm PIPES, 1 mm EGTA, 10 μg/ml pepstatin, 10 μg/ml leupeptin, 0.5 mm PMSF), and centrifuged (600 × g for 10 min at 4 °C) to remove nuclei and unbroken cells; the supernatant was then ultracentrifuged (100,000 × g for 30 min at 4 °C). Pellets were washed in relaxation buffer, dissolved in Laemmli sample buffer, and submitted to 10% SDS-PAGE (
). The upper part of the gel was revealed with anti-PKC antibody (1/1000 dilution), and the lower part with anti-p47phox (1/5000) and labeling by secondary antibodies (1/2000), followed by ECL detection.
p47phoxphosphorylation was analyzed as described previously (
). Resting and activated 32P-labeled monocytes were scraped (107 cells/ml) in ice-cold lysis buffer (20 mmTris-HCl, pH 7.4, 150 mm NaCl, 0.25 m sucrose, 5 mm EGTA, 5 mm EDTA, 15 μg/ml leupeptin, 10 μg/ml pepstatin, 10 μg/ml aprotinin, 1.5 mm PMSF, 1 mm diisopropylfluorophosphate, 0.5% Triton X-100, 25 mm NaF, 5 mm NaVO4, 5 mm β-glycerophosphate, 1 mmp-nitrophenyl phosphate, 1 mg/ml DNase I), sonicated, and ultracentrifuged. P47phox was immunoprecipitated and analyzed as described previously (
The nitrocellulose area containing 32P-labeled p47phoxwas incubated for 30 min at 37 °C with polyvinylpyrrolidone, washed five times with water and twice with 50 mm ammonium bicarbonate, and then incubated overnight with trypsin (
). The plate was analyzed in an Instant Imager apparatus equipped with Instant Imager software (Packard).
Assay of PKC Activity
Resting adherent monocytes were scraped in lysis buffer (20 mm Tris-HCl, pH 7.5, 2 mm EDTA, 10 mm EGTA, 0.3 m sucrose, 2 mm dithiothreitol, 2 mm PMSF, 10 μg/ml leupeptin, 10 μg/ml pepstatin) at 3 × 107 cells/ml. They were then sonicated and ultracentrifuged to obtain the cytosolic fraction as described above. PKC activity in aliquots of cytosolic fraction was measured by PS/DG-stimulated histone III-S phosphorylation. Activating liposomes containing α- or β-tocopherol were prepared as follows. PS and DG were dissolved in CHCl3and then mixed with tocopherol in ethanol; the mixture was dried under N2, resuspended in 20 mm Tris-HCl, pH 7.5, and sonicated for 2 min on ice under N2. Histone III-S (200 μg/ml) was incubated with the PKC- containing cell fraction in reaction buffer (20 mm Tris-HCl, pH 7.5, 10 mmMgCl2) with 1 mm CaCl2 and lipids (final concentrations 200 μg/ml PS, 10 μg/ml DG, 20 μg/ml tocopherol). Phosphorylation was performed by adding 10 μm ATP containing 0.1 μCi of [γ-32P]ATP/ml for 10 min at 30 °C and stopped on P81 filter paper. After extensive washing in 0.8% phosphoric acid, the amount of remaining 32P-histone was measured by scintillation counting. Specific activity was the difference between activity in the presence and absence of Ca2+, PS, and DG. When Trolox was used it was added to the reaction mixture at the same molarity as α-tocopherol (46 μm).
α-Tocopherol Inhibits O⨪2 Production in Intact Human Adherent Monocytes
PMA-stimulated production of O⨪2 by monocytes was depressed in the presence of α-tocopherol in a concentration-dependent manner (Fig. 1). The inhibition was not complete, reaching a maximum of 50% at 45 μg/ml α-tocopherol. It is noteworthy that the basal activity of unstimulated monocytes (representing 15 ± 5% (n = 4) of the activity of stimulated cells) was not inhibited by α-tocopherol. O⨪2production by xanthine/xanthine oxidase was not decreased by α-tocopherol (data not shown), suggesting that inhibition did not result from scavenging of O⨪2. This lack of O⨪2scavenging was previously found even in the presence of LDL (
), showing that it is not due to the absence of a lipid phase in the medium. α-Tocopherol also failed to decrease O⨪2 production in a membrane preparation containing NADPH-oxidase (Fig. 1). This showed that α-tocopherol did not directly interfere with activated NADPH-oxidase but may instead have inhibited an event preceding oxidase activation. In contrast to this depressive effect of α-tocopherol, β-tocopherol enhanced O⨪2 production by monocytes.
), we investigated the effect of α-tocopherol on this process by using Western blot analysis of the membrane fractions. We chose a concentration of α-tocopherol that clearly inhibited O⨪2 production (Fig. 1). When monocytes were stimulated with PMA, pretreatment with 20 μg/ml α-tocopherol inhibited membrane translocation of p47phox (Fig. 2). In resting monocytes, p47phoxwas barely detectable in the membrane fraction, and α-tocopherol alone did not induce detectable movement of p47phox between the cytosolic and membrane compartments (Fig. 2). Western blots were scanned, and the combined densitometry data indicated that in the presence of α-tocopherol the level of p47phox translocation was 70.9 ± 4.3% of control (n = 4)(p < 0.05).
α-Tocopherol Inhibits p47phox Phosphorylation
The phosphorylation of p47phox in human monocytes was analyzed after 32P labeling, p47phox immunoprecipitation, and SDS-PAGE. PMA stimulation triggered the phosphorylation of p47phox, which was abolished in the presence of GF109203X (Fig. 3A). α-Tocopherol inhibited PMA-induced p47phox phosphorylation (Fig. 3A) (30.6 ± 3.9% of the control (n = 3) (p < 0.05) as determined by densitometry analysis). Western blot results showed that the same amount of p47phox was loaded in each lane (Fig. 3B). In resting monocytes, p47phox was slightly phosphorylated (Fig. 3A), and α-tocopherol had no detectable effect on phosphorylation (data not shown).
PMA is a direct agonist of conventional and novel isoforms of PKC (
). The PKC pathway was therefore a possible target for the action of α-tocopherol. To determine whether α-tocopherol inhibited specific phosphorylation sites, we carried out two-dimensional phosphopeptide mapping of p47phox. The pattern of tryptic p47phox phosphopeptides obtained in human adherent monocytes under PMA stimulation (Fig. 4) was close to that of human neutrophils (
). α-Tocopherol inhibited the phosphorylation of the major PKC-dependent sites (shown by arrows), whereas two phosphopeptides that are phosphorylated in resting cells (broken arrows) were not affected by α-tocopherol. This provides further evidence that the inhibitory effect of α-tocopherol is mediated by PKC.
PKC Translocation Is Insensitive to α-Tocopherol
To assess whether α-tocopherol inhibits p47phox translocation and phosphorylation by modulating PKC activation, we used Western blot analysis to visualize the translocation of PKC from cytosol to membrane. The antibody used to reveal PKC cross-reacted with α and β isoforms of PKC present in monocytes (
). Fig. 5 shows the massive translocation of PKC from cytosol to membrane after PMA stimulation, with only a trace of PKC remaining in the cytosolic fraction. α-Tocopherol did not modify this process (100.5 ± 4.5% of the control (n = 3) as determined by densitometry analysis). It did, however, inhibit p47phox membrane translocation in the same cell extracts. Western blot analysis of p47phox in the cytosols confirmed that equivalent amounts of cells were used (Fig. 5). Taken together, these results suggest that α-tocopherol interferes with the activity of membrane-bound conventional PKC rather than with its membrane translocation but do not exclude an action of α-tocopherol on other PKC isoforms.
α-Tocopherol Inhibits PKC Activity in Vitro
We then tested the effect of α-tocopherol on PKC activity. A PKC preparation from the cytosolic fraction was incubated in the presence of PS/DG liposomes, with or without incorporated α-tocopherol. Fig. 6 shows that α-tocopherol strongly inhibited PKC activity, whereas β-tocopherol was less effective and Trolox had a weak effect; this is consistent with a specific action of α-tocopherol.
In this study α-tocopherol depressed the PMA-induced respiratory burst of human adherent monocytes without affecting the assembled oxidase activity of membrane preparations originating from PMA-activated monocytes or neutrophils. This strongly suggests that the α-tocopherol inhibition of O⨪2 production is at least in part related to a functional impairment of the NADPH-oxidase assembly. Indeed, we found that α-tocopherol inhibited p47phoxtranslocation to the membrane and impaired p47phoxphosphorylation.
In human neutrophils, oxidase activation by PMA is believed to be mediated by PKC (
). The results described here in human monocytes suggest that the PKC pathway is involved in O⨪2 production, as PMA-induced p47phox phosphorylation was inhibited by GF109203X (Fig. 3A), a potent inhibitor of PKC (
). We further showed the following: (i) α-tocopherol inhibited PMA-induced but not basal O⨪2 production; (ii) α-tocopherol inhibited PMA-induced p47phox translocation and phosphorylation; and (iii) the peptides whose phosphorylation was inhibited by α-tocopherol are known PKC substrates (
). These results suggest that the PKC pathway is a target of α-tocopherol in human monocytes. The lack of effect of α-tocopherol on basal activity of unstimulated cells suggests that PKC isoforms that can be activated by PMA are sensitive to α-tocopherol.
The mechanism by which α-tocopherol interacts with PKC is unclear. In our model α-tocopherol failed to inhibit PKC translocation to the membrane (Fig. 5), suggesting that it modulates only the activity of membrane-bound enzyme. In vitro, α-tocopherol inhibited monocyte PKC activity only when α-tocopherol was incorporated in PS/DG liposomes (Fig. 6). There was no action when α-tocopherol was supplied as a solution in ethanol (data not shown) or when it was replaced by Trolox, a hydrophilic analog of α-tocopherol that is unable to penetrate liposomes but has a preserved antioxidant moiety (Fig. 6). These results suggest that α-tocopherol incorporation into the lamellar (pseudo-membrane) structure of liposomes is a prerequisite for its inhibitory action on PKC. Tocopherol compounds have been shown to alter the structural organization of liposomes (
) and activated PKC (Fig. 5) are both located in the membrane fraction. Our results further suggest an additional specific effect of α-tocopherol on PKC, as α-tocopherol inhibited PKC activity much more strongly than did β-tocopherol (Fig. 6).
Taken together these results suggest that the inhibition of PKC activity (i) is not directly due to the antioxidant capacity of α-tocopherol, (ii) requires the integration of α-tocopherol in a pseudo-membrane structure, and (iii) is due to an interaction between the α-tocopherol molecular structure and PKC. In addition, as α-tocopherol inhibited both PKC activity and superoxide production whereas β-tocopherol and Trolox did not (this report and Ref, 10), these data support a link between the effect of α-tocopherol on PKC activity and O⨪2 production.
Vitamin E is a natural lipophilic antioxidant compound that protects lipid structures from peroxidation. It is the first antioxidant to be consumed during oxidative modification of LDL (
Our results provide a model for a monocyte-mediated effect of vitamin E in preventing LDL oxidation; following PKC inhibition, p47phoxtranslocation and phosphorylation are inhibited, thereby decreasing superoxide production. As well as elucidating the way in which vitamin E delays and/or decreases oxygen-radical injury of LDL, this cellular model provides new insights into the potential beneficial effect of vitamin E on atherogenesis.
We thank Dr. Bernard M. Babior from the Scripps Research Institute, La Jolla, CA, for p47phox antibody. We also thank Dr. Axel Perianin from CNRS, Hôpital Cochin, Paris, France, for helpful discussions. We are also grateful to Dr. Najib Kadri-Hassani for giving us the opportunity to report results on the effects of β-tocopherol on O⨪2 production in PMA-stimulated human monocytes.