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α-Tocopherol Inhibits the Respiratory Burst in Human Monocytes

ATTENUATION OF p47phox MEMBRANE TRANSLOCATION AND PHOSPHORYLATION*
Open AccessPublished:December 04, 1998DOI:https://doi.org/10.1074/jbc.273.49.32801
      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.
      LDL
      low density lipoprotein
      ROS
      radical oxygen species
      phox
      phagocyte oxidase
      PKC
      protein kinase C
      PMA
      phorbol 12-myristate 13-acetate
      PS
      phosphatidylserine
      DG
      diacylglycerol
      PMSF
      phenylmethylsulfonyl fluoride
      PAGE
      polyacrylamide gel electrophoresis
      PIPES
      1,4-piperazinediethanesulfonic acid.
      Oxidative modification of low density lipoprotein (LDL)1 appears to be a key event in the early stages of atherogenesis (
      • Steinberg D.
      ). Monocyte release of superoxide anion (O⨪2), a reactive oxygen species (ROS), induces LDL oxidation (
      • Li Q.
      • Cathcart M.K.
      ). Vitamin E (α-tocopherol), a lipophilic molecule present in plasma lipoproteins, is one of the most important natural antioxidants (
      • Liebler D.C.
      • Burr J.A.
      ). It is able to scavenge acyl-peroxyl radicals in membrane structures and may prevent or delay cardiovascular diseases (
      • Janero D.R.
      ,
      • Stephens N.G.
      • Parsons A.
      • Schofield P.M.
      • Kelly F.
      • Cheeseman K.
      • Mitchinson M.J.
      • Brown M.J.
      ), possibly through its capacity to increase LDL resistance to oxidative modification (
      • Princen H.M.G.
      • van-Duyvenvoorde W.
      • Buytenhek R.
      • van-der-Laarse A.
      • van-Poppel G.
      • Gevers-Leuven J.A.
      • van-Hinsbergh V.W.M.
      ). The possibility that α-tocopherol has additional antioxidant effects by inhibiting ROS generation has received little attention. We (
      • Cachia O.
      • Léger C.L.
      • Descomps B.
      ) and others (
      • Devaraj S.
      • Li D.
      • Jialal I.
      ,
      • Sakamoto W.
      • Fujie K.
      • Handa H.
      • Ogihara T.
      • Mino M.
      ,
      • Kanno T.
      • Utsumi T.
      • Kobuchi H.
      • Takehara Y.
      • Akiyama J.
      • Yoshioka T.
      • Horton A.A.
      • Utsumi K.
      ) have shown that α-tocopherol inhibits O⨪2 production by human monocytes (
      • Cachia O.
      • Léger C.L.
      • Descomps B.
      ), rat macrophages, and neutrophils (
      • Devaraj S.
      • Li D.
      • Jialal I.
      ,
      • Sakamoto W.
      • Fujie K.
      • Handa H.
      • Ogihara T.
      • Mino M.
      ,
      • Kanno T.
      • Utsumi T.
      • Kobuchi H.
      • Takehara Y.
      • Akiyama J.
      • Yoshioka T.
      • Horton A.A.
      • Utsumi K.
      ), 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 b 558 and cytosolic proteins (p47phox, p67phox, Rac1/2, and p40phox) (
      • Chanock S.J.
      • el Benna J.
      • Smith R.M.
      • Babior B.M.
      ) that translocate to the membrane during stimulation to form a catalytically active oxidase (
      • DeLeo F.R.
      • Quinn M.T.
      ). During NADPH-oxidase activation, p47phox is phosphorylated on several serine residues (
      • el Benna J.
      • Faust L.P.
      • Babior B.M.
      ). Protein kinase C (PKC) is involved in NADPH-oxidase activation and can phosphorylate p47phox (
      • Babior B.M.
      ,
      • el Benna J.
      • Faust L.R.P.
      • Johnson J.L.
      • Babior B.M.
      ). PKC is a family of Ca2+- and phospholipid-dependent serine/threonine kinases that play a pivotal role in agonist-stimulated cell functions (
      • Nishizuka Y.
      ). Several PKC isoforms have been described (
      • Newton A.C.
      ). The conventional PKCs αPKC and βPKC are present in monocytes and are activated by phorbol esters such as phorbol 12-myristate 13-acetate (PMA) (
      • Chang Z.L.
      • Beezhold D.H.
      ). In human monocytes and neutrophils, PMA induces the production of O⨪2 and the phosphorylation of several proteins, one of which is p47phox(
      • Babior B.M.
      ).
      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.

      DISCUSSION

      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 (
      • el Benna J.
      • Faust L.R.P.
      • Johnson J.L.
      • Babior B.M.
      ,
      • Nauseef W.M.
      • Volpp B.D.
      • McCormick S.
      • Leidal K.G.
      • Clark R.A.
      ). 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. 3 A), a potent inhibitor of PKC (
      • Toullec D.
      • Pianetti P.
      • Coste H.
      • Bellevergue P.
      • Grand-Perret T.
      • Ajakane M.
      • Baudet V.
      • Boissin P.
      • Boursier E.
      • Loriolle F.
      • Duhamel L.
      • Charon D.
      • Kirilovsky J.
      ). 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 (
      • el Benna J.
      • Faust L.R.P.
      • Johnson J.L.
      • Babior B.M.
      ). 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 (
      • Urano S.
      • Yano K.
      • Matsuo M.
      ,
      • Srivastava S.
      • Phadke R.S.
      • Govil G.
      ) and would thus be potentially able to modify PKC activity. Several lipids known to incorporate into membrane structures are able to modulate PKC activity (
      • Epand R.M.
      • Lester D.S.
      ) and O⨪2 production in human adherent monocytes stimulated by PMA (
      • Kadri-Hassani N.
      • Léger C.L.
      • Descomps B.
      ). The present results support a membrane location for the interaction between PKC and α-tocopherol, as α-tocopherol (
      • Sakamoto W.
      • Fujie K.
      • Handa H.
      • Ogihara T.
      • Mino M.
      ,
      • Machlin I.J.
      ) 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 (
      • Esterbauer H.
      ). Epidemiological studies and intervention trials have suggested a potential effect of vitamin E in the prevention of atherosclerosis (
      • Stephens N.G.
      • Parsons A.
      • Schofield P.M.
      • Kelly F.
      • Cheeseman K.
      • Mitchinson M.J.
      • Brown M.J.
      ,
      • Gey K.F.
      ). This beneficial role has been defined as the ability of vitamin E to increase LDL resistance to oxidation (
      • Princen H.M.G.
      • van-Duyvenvoorde W.
      • Buytenhek R.
      • van-der-Laarse A.
      • van-Poppel G.
      • Gevers-Leuven J.A.
      • van-Hinsbergh V.W.M.
      ), LDL oxidation being a key event in atherogenesis (
      • Berliner J.A.
      • Heinecke J.W.
      ). Vitamin E may also protect LDL by attenuating the respiratory burst, as NADPH-oxidase activity and O⨪2 production by monocytes and macrophages are able to induce LDL oxidation (
      • Li Q.
      • Cathcart M.K.
      ,
      • Aviram M.
      • Rosenblat M.
      • Etzioni A.
      • Levy R.
      ). Monocytes from volunteers taking oral vitamin E showed lower O⨪2 production together with an impaired capacity to oxidize LDL (
      • Devaraj S.
      • Li D.
      • Jialal I.
      ). Similarly, J774 macrophages supplemented in vitrowith vitamin E lost their power to oxidize LDL (
      • Suzukawa M.
      • Abbey M.
      • Clifton P.
      • Nestel P.J.
      ).
      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.

      ACKNOWLEDGEMENTS

      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.

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