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Molecular Basis of Vitamin E Action

TOCOTRIENOL POTENTLY INHIBITS GLUTAMATE-INDUCED pp60c-Src KINASE ACTIVATION AND DEATH OF HT4 NEURONAL CELLS*
Open AccessPublished:April 28, 2000DOI:https://doi.org/10.1074/jbc.275.17.13049
      HT4 hippocampal neuronal cells were studied to compare the efficacy of tocopherols and tocotrienol to protect against glutamate-induced death. Tocotrienols were more effective than α-tocopherol in preventing glutamate-induced death. Uptake of tocotrienols from the culture medium was more efficient compared with that of α-tocopherol. Vitamin E molecules have potent antioxidant properties. Results show that at low concentrations, tocotrienols may have protected cells by an antioxidant-independent mechanism. Examination of signal transduction pathways revealed that protein tyrosine phosphorylation processes played a central role in the execution of death. Activation of pp60c-Src kinase and phosphorylation of ERK were observed in response to glutamate treatment. Nanomolar amounts of α-tocotrienol, but not α-tocopherol, blocked glutamate-induced death by suppressing glutamate-induced early activation of c-Src kinase. Overexpression of kinase-active c-Src sensitized cells to glutamate-induced death. Tocotrienol treatment prevented death of Src-overexpressing cells treated with glutamate. α-Tocotrienol did not influence activity of recombinant c-Src kinase suggesting that its mechanism of action may include regulation of SH domains. This study provides first evidence describing the molecular basis of tocotrienol action. At a concentration 4–10-fold lower than levels detected in plasma of supplemented humans, tocotrienol regulated unique signal transduction processes that were not sensitive to comparable concentrations of tocopherol.
      ROS
      reactive oxygen species
      DCF
      dichlorofluorescein
      DCFH-DA
      dichlorodihydrofluorescein diacetate
      PBS
      phosphate-buffered saline
      HPLC
      high performance liquid chromatography
      AAPH
      2,2′-azobis[2-amidinopropane]hydrochloride
      ERK
      extracellular signal-regulated kinase
      Vitamin E is a generic term for tocopherols and tocotrienols that qualitatively exhibit the biological activity of α-tocopherol (
      • Brigelius-Flohe R.
      • Traber M.G.
      ). Compared with tocopherols, tocotrienols have been poorly studied (
      • Traber M.G.
      • Packer L.
      ,
      • Traber M.G.
      • Sies H.
      ). Tocotrienols are minor plant constituents especially abundant in palm oil, cereal grains, and rice bran that can provide a significant source of vitamin E activity. Tocotrienols differ from tocopherols by possessing a farnesyl (isoprenoid) rather than a saturated phytyl side chain. Dietary tocotrienols become incorporated into circulating human lipoproteins where they react with peroxyl radicals as efficiently as the corresponding tocopherol isomers (
      • Suarna C.
      • Hood R.L.
      • Dean R.T.
      • Stocker R.
      ,
      • Serbinova E.
      • Kagan V.
      • Han D.
      • Packer L.
      ). Consistently, tocotrienol supplementation has been reported to influence beneficially the course of carotid atherosclerosis in humans (
      • Tomeo A.C.
      • Geller M.
      • Watkins T.R.
      • Gapor A.
      • Bierenbaum M.L.
      ). Following supplementation to humans, the level of α-tocotrienol in the plasma has been estimated to be 0.98 ± 0.8 μm (
      • O'Byrne D.
      • Traber M.G.
      • Packer L.
      • Grundy S.
      • Jialal I.
      ). A possible neuroprotective property of tocotrienols was indicated in a study testing the efficacy of the tocotrienol-rich fraction from palm oil to protect against oxidative damage of rat brain mitochondria. The tocotrienol-rich fraction from palm oil was significantly more effective than α-tocopherol in protecting the brain against damage caused by exposure to ascorbate-Fe2+, the free radical initiator azobis(2-amidopropane)dihydrochloride, or photosensitization. (
      • Kamat J.P.
      • Devasagayam T.P.
      ). At concentrations 25–50 μm, α-tocopherol is known to regulate signal transduction pathways by mechanisms that are independent of its antioxidant properties. α-Tocopherol, but not β-tocopherol having comparable antioxidant properties, inhibited inducible protein kinase C activity in smooth muscle cells (
      • Azzi A.
      • Boscoboinik D.
      • Marilley D.
      • Ozer N.K.
      • Stauble B.
      • Tasinato A.
      ,
      • Boscoboinik D.O.
      • Chatelain E.
      • Bartoli G.M.
      • Stauble B.
      • Azzi A.
      ). The signal transduction regulatory properties of tocotrienols, however, are yet unknown.
      ROS1 represent a major contributor to brain damage in disorders such as epilepsy (
      • Kabuto H.
      • Yokoi I.
      • Ogawa N.
      ,
      • Mahle C.
      • Dasgupta A.
      ), head trauma (
      • Beit-Yannai E.
      • Zhang R.
      • Trembovler V.
      • Samuni A.
      • Shohami E.
      ), and ischemia-reperfusion (
      • Keller J.N.
      • Kindy M.S.
      • Holtsberg F.W.
      • St Clair D.K.
      • Yen H.C.
      • Germeyer A.
      • Steiner S.M.
      • Bruce-Keller A.J.
      • Hutchins J.B.
      • Mattson M.P.
      ,
      • Kristian T.
      • Siesjo B.K.
      ,
      • Murakami K.
      • Kondo T.
      • Kawase M.
      • Li Y.
      • Sato S.
      • Chen S.F.
      • Chan P.H.
      ). Oxidative damage is also implicated in neurodegenerative diseases such as Huntington's (
      • Borlongan C.V.
      • Kanning K.
      • Poulos S.G.
      • Freeman T.B.
      • Cahill D.W.
      • Sanberg P.R.
      ), Alzheimer's (
      • Smith M.A.
      • Sayre L.M.
      • Anderson V.E.
      • Harris P.L.
      • Beal M.F.
      • Kowall N.
      • Perry G.
      ), and Parkinson's. In the pathogenesis of these diseases, oxidative damage may accumulate over a period of years leading to massive neuronal loss. Glutamate toxicity is a major contributor to pathological cell death within the nervous system and appears to be mediated by ROS (
      • Coyle J.T.
      • Puttfarcken P.
      ). There are two forms of glutamate toxicity as follows: receptor-initiated excitotoxicity (
      • Choi D.W.
      ) and non-receptor-mediated glutamate-induced toxicity (
      • Tan S.
      • Sagara Y.
      • Liu Y.
      • Maher P.
      • Schubert D.
      ). One model used to study oxidative stress-related neuronal death is to inhibit cystine uptake by exposing cells to high levels of glutamate (
      • Murphy T.H.
      • Schnaar R.L.
      • Coyle J.T.
      ). High glutamate levels block cystine uptake via the amino acid transporter Xc and impairs reduced glutathione (GSH) cell homeostasis. The induction of oxidative stress by glutamate in this model has been demonstrated to be a primary cytotoxic mechanism in C6 glial cells (
      • Kato S.
      • Negishi K.
      • Mawatari K.
      • Kuo C.H.
      ,
      • Han D.
      • Sen C.K.
      • Roy S.
      • Kobayashi M.S.
      • Tritschler H.J.
      • Packer L.
      ), PC-12 neuronal cells (
      • Froissard P.
      • Monrocq H.
      • Duval D.
      ,
      • Pereira C.M.
      • Oliveira C.R.
      ), immature cortical neurons cells (
      • Murphy T.H.
      • Schnaar R.L.
      • Coyle J.T.
      ), and oligodendroglia cells (
      • Oka A.
      • Belliveau M.J.
      • Rosenberg P.A.
      • Volpe J.J.
      ). Recently, the mitochondrial electron transport chain has been shown to be a source of ROS production during glutamate-induced apoptosis in HT22 neuronal cells, a sub-clone of HT4 cells used in the current study (
      • Tan S.
      • Sagara Y.
      • Liu Y.
      • Maher P.
      • Schubert D.
      ). At micromolar concentrations, antioxidants such as α-tocopherol, probucol, and α-lipoic acid have been shown to protect these cells against glutamate-induced cytotoxicity (
      • Murphy T.H.
      • Schnaar R.L.
      • Coyle J.T.
      ,
      • Kato S.
      • Negishi K.
      • Mawatari K.
      • Kuo C.H.
      ,
      • Han D.
      • Sen C.K.
      • Roy S.
      • Kobayashi M.S.
      • Tritschler H.J.
      • Packer L.
      ,
      • Miyamoto M.
      • Murphy T.H.
      • Schnaar R.L.
      • Coyle J.T.
      ,
      • Murphy T.H.
      • Miyamoto M.
      • Sastre A.
      • Schnaar R.L.
      • Coyle J.T.
      ).
      In the current study, rat hippocampal neuronal HT4 cells (
      • Morimoto B.H.
      • Koshland Jr., D.E.
      ) were exposed to elevated levels of extracellular glutamate, and the ability of tocotrienols and tocopherol to protect the neuronal cells was examined. This study presents first evidence showing that at amounts 4–10-fold lower than levels of tocotrienol detected in plasma of human supplemented with the vitamin E molecule (
      • O'Byrne D.
      • Traber M.G.
      • Packer L.
      • Grundy S.
      • Jialal I.
      ), α-tocotrienol has potent signal transduction regulatory properties that account for its neuroprotective function.

      DISCUSSION

      Previously we have observed in C6 glial cells as well as in HT4 cells that glutamate-induced death may be prevented by antioxidant treatment (
      • Han D.
      • Sen C.K.
      • Roy S.
      • Kobayashi M.S.
      • Tritschler H.J.
      • Packer L.
      ,
      • Tirosh O.
      • Sen C.K.
      • Roy S.
      • Kobayashi M.S.
      • Packer L.
      ). Compared with α-tocopherol, α-tocotrienol is more uniformly distributed in the membrane bilayer, more efficiently recycled from its corresponding chromanoxyl radical form, and more strongly disorders membrane lipid allowing for a better interaction of chromanols with lipid radicals (
      • Serbinova E.
      • Kagan V.
      • Han D.
      • Packer L.
      ). Because of these advantages, α-tocotrienol has better antioxidant activity than α-tocopherol (
      • Serbinova E.
      • Kagan V.
      • Han D.
      • Packer L.
      ,
      • Kamat J.P.
      • Devasagayam T.P.
      ,
      • Suzuki Y.J.
      • Tsuchiya M.
      • Wassall S.R.
      • Choo Y.M.
      • Govil G.
      • Kagan V.E.
      • Packer L.
      ). Although it is tempting to assume that the increased antioxidant activity of α-tocotrienol is responsible for its enhanced cytoprotective effect, results from experiments where cells were treated with 100 nm tocotrienol at various time points after glutamate challenge do not support the contention.
      The ability of 100 nm α-tocotrienol to protect against glutamate-induced loss of cell viability was retained only if the cells were treated up to 1 h after glutamate challenge. However, at high concentrations α-tocotrienol could completely protect the cells even when treated 6 h after glutamate addition. These results indicate that the mechanism of α-tocotrienol action is dependent on the concentration of tocotrienol used. In the cascade of events leading to cell death, low concentrations of tocotrienol influenced an early event, whereas at higher concentrations tocotrienol protected cells apparently by regulating a late event. Because 1 h of glutamate treatment did not cause elevation of intracellular ROS, it seems unlikely that 100 nm of α-tocotrienol protected cells via an antioxidant mechanism. A compelling line of evidence supporting this contention is that although α-tocotrienol completely prevented intracellular peroxide accumulation even when treated several hours after glutamate exposure, it did not completely protect cell viability when added 90 min after glutamate treatment. At nanomolar concentrations α-tocotrienol does not have potent antioxidant property. Micromolar amounts of this compound was necessary to protect cells against peroxyl radical-induced loss of viability. Furthermore, trolox (the water-soluble analog of tocopherol) as well as geldanamycin completely prevented glutamate-induced cell death without decreasing glutamate-induced accumulation of intracellular peroxides (not shown). Taken together, this evidence indicates that intracellular oxidants may not play a key role in the death pathway.
      Previously it has been suggested that compared with α-tocotrienol, γ-tocotrienol has more potent antioxidant properties (
      • Kamat J.P.
      • Devasagayam T.P.
      ). Although the differences were marginal, γ-tocotrienol tended to be less effective than α-tocotrienol in protecting the cells. These results lend further support to the hypothesis that at low concentrations the protective effect of α-tocotrienol against glutamate-induced cytotoxicity may not be related to antioxidant activity. The uptake of both α- and γ-tocotrienols by HT4 cells was clearly much better than that of α-tocopherol. It is generally believed that the chromanol nucleus of α-tocopherol is localized at the polar-hydrocarbon membrane interface whereas its phytyl chain interacts with the acyl chains of membrane phospholipids. Compared with α-tocopherol, α-tocotrienol is significantly less associated in clusters and is more uniformly distributed in the bilayer of dimyristoyl-phosaphatidylcholine liposomes (
      • Serbinova E.
      • Kagan V.
      • Han D.
      • Packer L.
      ). It is unlikely that the difference between the ability of tocotrienol and tocopherol to protect cells against glutamate challenge may be explained by differences in the uptake of these two forms of vitamin E by the cell. Although treatment of cells with 100 nm of α-tocotrienol for 5 min resulted in negligible loading of cells, these cells were completely resistant to glutamate-induced death, whereas cells loaded with the vitamin E molecule (1 μm, 2 h) were not. Furthermore, although γ-tocotrienol was more efficiently taken up by cells than α-tocotrienol, it was less efficient than the latter in protecting the cells against glutamate-induced death. Treatment of cells with the free form of α-tocotrienol resulted in a higher concentration of free tocotrienol in the cell compared with cells treated with esterified α-tocotrienol. It is possible that such differences are because of limited esterase activity available in the membrane environment where tocotrienol acetate is likely to partition. At low concentration, the observed neuroprotective property of tocotrienol is unlikely to be mediated via iron chelation because deferoxamine mesylate appeared to protect by influencing a late event in the death pathway.
      Oxidant challenge has been shown to be associated with increased [Ca2+]i (
      • Sen C.K.
      • Packer L.
      ,
      • Sen C.K.
      • Roy S.
      • Packer L.
      ) resulting from mobilization of the Ca2+ pool of sarcoendoplasmic reticulum (
      • Hu Q.
      • Corda S.
      • Zweier J.L.
      • Capogrossi M.C.
      • Ziegelstein R.C.
      ). In glutamate-treated HT4 cells, accumulation of intracellular ROS was followed by elevated levels of [Ca2+]i. Such an oxidant-induced increase in [Ca2+]i has been shown to contribute to cell death (
      • Karczewski J.M.
      • Peters J.G.
      • Noordhoek J.
      ,
      • Liu H.
      • Miller E.
      • van de Water B.
      • Stevens J.L.
      ). Decreased intracellular GSH resulted in calcium-mediated cell death in PC12 neuronal cells (
      • Jurma O.P.
      • Hom D.G.
      • Andersen J.K.
      ). Tocotrienol treatment prevented elevation of [Ca2+]i despite marked depletion of intracellular GSH.
      The involvement of signal transduction pathways in glutamate-induced cell death was evident. Inhibitors of the protein- tyrosine kinase activity completely prevented glutamate-induced cell death. Herbimycin and geldanamycin potently inhibited pp60c-Src tyrosine kinase activity (
      • Yoneda T.
      • Lowe C.
      • Lee C.H.
      • Gutierrez G.
      • Niewolna M.
      • Williams P.J.
      • Izbicka E.
      • Uehara Y.
      • Mundy G.R.
      ,
      • Hall T.J.
      • Schaeublin M.
      • Missbach M.
      ), whereas lavendustin A is an inhibitor of extracellular growth factor receptor protein-tyrosine kinase activity (
      • Hsu C.Y.
      • Persons P.E.
      • Spada A.P.
      • Bednar R.A.
      • Levitzki A.
      • Zilberstein A.
      ). The observation that herbimycin and geldanamycin, but not lavendustin A, prevent glutamate-induced death of HT4 neuronal cells suggested the involvement of c-Src kinase activity in the death pathway. Immunoprecipitation of tyrosine-phosphorylated protein from cellular extracts confirmed that protein tyrosine phosphorylation reactions were indeed triggered by exposure of cells to elevated levels of glutamate and that such reactions were inhibited by nanomolar concentrations of α-tocotrienol.
      The involvement of pp60c-Src kinase activity in the death pathway was verified by experiments involving the overexpression of catalytically active or inactive Src kinase. Tocotrienol treatment completely prevented glutamate-induced death even in active c-Src kinase overexpressing cells indicating that it either inhibited c-Src kinase activity or regulated one or more events upstream of c-Src kinase activation. Further evidence supporting this contention was provided by results obtained from the determination of c-Src kinase activity in HT4 cells. SH2 and SH3 domains are known to play a central role in regulating the catalytic activity of Src protein-tyrosine kinase. High resolution crystal structures of human SRC, in their repressed state, have provided a structural explanation for how intramolecular interactions of the SH3 and SH2 domains stabilize the inactive conformation of Src (
      • Thomas S.M.
      • Brugge J.S.
      ). The observation that α-tocotrienol inhibited glutamate-induced Src activation in HT4 cells but did not influence the catalytic activity of recombinant Src suggests that α-tocotrienol inhibited events leading to glutamate-induced reorganization of the SH domains and activation of Src kinase. Many intracellular pathways can be stimulated upon Src activation, and a variety of cellular consequences can result, including morphological changes and cell proliferation. For example, the activity of c-Src kinase is implicated in the progression of breast cancer (
      • Campbell D.H.
      • deFazio A.
      • Sutherland R.L.
      • Daly R.J.
      ,
      • Muthuswamy S.K.
      • Muller W.J.
      ). Mammary tumors expressing the neu proto-oncogene possess elevated c-Src tyrosine kinase activity (
      • Muthuswamy S.K.
      • Siegel P.M.
      • Dankort D.L.
      • Webster M.A.
      • Muller W.J.
      ). Markedly elevated levels of c-Src kinase activity have been detected in human skin tumors (
      • Barnekow A.
      • Paul E.
      • Schartl M.
      ). Because of the key involvement of Src kinases in driving receptor-mediated oncogenesis (
      • Maa M.C.
      • Leu T.H.
      • McCarley D.J.
      • Schatzman R.C.
      • Parsons S.J.
      ), inhibitors of these kinases are being studied as candidates for anti-cancer drugs (
      • Levitzki A.
      ).
      Further evidence suggesting that signal transduction processes related to the cell death pathway are involved in glutamate-induced cytotoxicity was obtained from the study of ERK1 and ERK2 activation. When activated, p44 and p42 mitogen-activated protein kinases (ERK1 and ERK2) are phosphorylated at specific threonine and tyrosine residues. ERK has been implicated in mediating the signaling events that precede apoptosis. ERK2 plays an active role in mediating anti-IgM-induced apoptosis of WEHI 231 B cells (
      • Lee J.R.
      • Koretzky G.A.
      ). H2O2induces the activation of multiple mitogen-activated protein kinases in oligodendrocyte progenitors, and the activation of ERK is associated with oxidant-mediated cytotoxicity (
      • Bhat N.R.
      • Zhang P.
      ). Rapid activation of ERK, particularly ERK2, in response to glutamate treatment was completely inhibited in cells treated with α-tocotrienol suggesting that α-tocotrienol influences an early event in the glutamate-induced death pathway. In some cases Src kinase activity is required for the activation of ERK (
      • Aikawa R.
      • Komuro I.
      • Yamazaki T.
      • Zou Y.
      • Kudoh S.
      • Tanaka M.
      • Shiojima I.
      • Hiroi Y.
      • Yazaki Y.
      ). Thus, it is likely tocotrienol treatment may have inhibited inducible ERK activation by down-regulating Src kinase activity.
      At 25–50 μm, α-tocopherol is known to regulate signal transduction pathways by mechanisms that are independent of its antioxidant properties (
      • Azzi A.
      • Boscoboinik D.
      • Marilley D.
      • Ozer N.K.
      • Stauble B.
      • Tasinato A.
      ,
      • Boscoboinik D.O.
      • Chatelain E.
      • Bartoli G.M.
      • Stauble B.
      • Azzi A.
      ). This study provides the first evidence describing a signal transduction regulatory role of tocotrienol. Protein tyrosine phosphorylation-related signal transduction pathways were observed to be involved in mediating glutamate-induced cytotoxicity. At amounts 4–10-fold lower than levels of tocotrienol detected in plasma of human supplemented with the vitamin E molecule (
      • O'Byrne D.
      • Traber M.G.
      • Packer L.
      • Grundy S.
      • Jialal I.
      ), tocotrienol inhibited glutamate-induced Src kinase activation, an early event in the pathway to death. At nanomolar levels tocotrienol regulated unique signal transduction processes that were not sensitive to comparable concentrations of tocopherol.

      Acknowledgments

      We thank Doran Kim for expert technical assistance.

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