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Opposite Roles of Selenium-dependent Glutathione Peroxidase-1 in Superoxide Generator Diquat- and Peroxynitrite-induced Apoptosis and Signaling*

Open AccessPublished:November 16, 2001DOI:https://doi.org/10.1074/jbc.M106946200
      Oxidative injuries including apoptosis can be induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) in aerobic metabolism. We determined impacts of a selenium-dependent glutathione peroxidase-1 (GPX1) on apoptosis induced by diquat (DQ), a ROS (superoxide) generator, and peroxynitrite (PN), a potent RNS. Hepatocytes were isolated from GPX1 knockout (GPX1−/−) or wild-type (WT) mice, and treated with 0.5 mm DQ or 0.1–0.8 mm PN for up to 12 h. Loss of cell viability, high levels of apoptotic cells, and severe DNA fragmentation were produced by DQ in only GPX1−/− cells and by PN in only WT cells. These two groups of cells shared similar cytochromec release, caspase-3 activation, and p21 WAF1/CIP1 cleavage. Higher levels of protein nitration were induced by PN in WT than GPX1−/− cells. Much less and/or slower cellular GSH depletion was caused by DQ or PN in GPX1−/− than in WT cells, and corresponding GSSG accumulation occurred only in the latter. In conclusion, it is most striking that, although GPX1 protects against apoptosis induced by superoxide-generator DQ, the enzyme actually promotes apoptosis induced by PN in murine hepatocytes. Indeed, GSH is a physiological substrate for GPX1 in coping with ROS in these cells.
      ROS
      reactive oxygen species
      RNS
      reactive nitrogen species
      DQ
      diquat
      PN
      peroxynitrite
      GPX
      glutathione peroxidase
      GPX1
      cellular glutathione peroxidase
      GPX1−/−
      GPX1 knockout
      WT
      wild-type
      MAPK
      mitogen-activated protein kinase
      JNK
      c-Jun NH2-terminal kinase
      TUNEL
      terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling
      Reactive oxygen species (ROS)1 and reactive nitrogen species (RNS) are constantly generated in aerobic metabolism and involved in pathogenesis of many diseases (
      • Evans M.D.
      • Griffiths H.R.
      • Lunec J.
      ,
      • Sies H.
      ). Pro-oxidants such as diquat (DQ) also induce cellular production of ROS including superoxide anion (O⨪2), hydrogen peroxide (H2O2), and hydroxyl radical (OH) (
      • Farrington J.A.
      • Ebert M.
      • Land E.J.
      • Fletcher K.
      ). Peroxynitrite (PN), a potent RNS, may be formed by O⨪2 and nitric oxide (NO) at a diffusion-limited rate (
      • Koppenol W.H.
      ). As PN nitrates a variety of biomolecules (
      • Beckman J.S.
      • Koppenol W.H.
      ), formation of nitrotyrosine in proteins is often used to assess its cellular activity (
      • Haddad I.Y.
      • Pataki G.
      • Hu P.
      • Galliani C.
      • Beckman J.S.
      • Matalon S.
      ). Selenium is an essential antioxidant nutrient (
      • Stadtman T.C.
      ) that has potential in preventing cancer (
      • Clark L.C.
      • Combs Jr., G.F.
      • Turnbull B.W.
      • Slate E.H.
      • Chalker D.K.
      • Chow J.
      • Davis L.S.
      • Glover R.A.
      • Graham G.F.
      • Gross E.G.
      • Krongrad A.
      • Lesher Jr., J.L.
      • Park H.K.
      • Sanders Jr., B.B.
      • Smith C.L.
      • Taylor J.R.
      ), viral infection (
      • Beck M.A.
      • Shi Q.
      • Morris V.C.
      • Levander O.A.
      ), and chronic disease (
      • Gu B.Q.
      ). Among the 18 identified mammalian selenoproteins (
      • Flohé L.
      • Andreesen J.R.
      • Brigelius-Flohé R.
      • Maiorino M.
      • Ursini F.
      ,
      • Gladyshev V.N.
      • Hatfield D.L.
      ), glutathione peroxidase-1 (EC 1.11.19, GPX1) was the first discovered (
      • Rotruck J.T.
      • Pope A.L.
      • Ganther H.E.
      • Swanson A.B.
      • Hafeman D.G.
      • Hoekstra W.G.
      ,
      • Flohé L.
      • Gunzler W.A.
      • Schock H.H.
      ) and the most abundant (
      • Cheng W.H.
      • Ho Y.-S.
      • Ross D.A.
      • Valentine B.A.
      • Combs Jr., G.F.
      • Lei X.G.
      ). Using GPX1 knockout mice (GPX1−/−) (
      • Ho Y.S.
      • Magnenat J.L.
      • Bronson R.T.
      • Cao J.
      • Gargano M.
      • Sugawara M.
      • Funk C.D.
      ), we have demonstrated that GPX1 is the metabolic mediator of body selenium to protect mice against pro-oxidant-induced death and oxidative injuries (
      • Cheng W.H.
      • Fu Y.X.
      • Porres J.M.
      • Ross D.A.
      • Lei X.G.
      ,
      • Fu Y.X.
      • Cheng W.H.
      • Porres J.M.
      • Ross D.A.
      • Lei X.G.
      ). In contrast to such strong evidence for the long-assumed role of GPX1 in coping with ROS in vivo (
      • Hoekstra W.G.
      ), the impact of GPX1 on RNS-mediated oxidative stress in various organisms is virtually unknown.
      Earlier, Sies et al. (
      • Sies H.
      • Sharov V.S.
      • Klotz L.-O.
      • Briviba K.
      ) showed that adding GPX1 in human fibroblast lysates was able to reduce PN to nitrite and thus attenuated the PN-mediated protein nitration in the presence of adequate glutathione (GSH). Because nitration of protein tyrosine residues may impair the tyrosine phosphorylation-related signaling and function (
      • Brito C.
      • Naviliat M.
      • Tiscornia A.C.
      • Vuillier F.
      • Gualco G.
      • Dighiero G.
      • Radi R.
      • Cayota A.M.
      ), their finding has physiological relevance. However, the metabolic role of GPX1 in intact cells in coping with PN might be different from that in cell lysates, because of a strong reactivity between PN and CO2 to form more active intermediates such asNO2 or CO⨪3 (
      • Squadrito G.L.
      • Pryor W.A.
      ), a possible inactivation of GPX1 by PN in oxidative state (
      • Padmaja S.
      • Squadrito G.L.
      • Pryor W.A.
      ), and modulations of cellular ROS on PN cytotoxicity (
      • Day B.J.
      • Patel M.
      • Calavetta L.
      • Chang L.Y.
      • Stamler J.S.
      ).
      Because apoptosis is induced by moderate levels of ROS in many types of cells (
      • Gardner A.M.
      • Xu F.H.
      • Fady C.
      • Jacoby F.J.
      • Duffey D.C.
      • Tu Y.
      • Lichtenstein A.
      ), and by PN in HL-60 (
      • Lin K.T.
      • Xue J.Y.
      • Nomen M.
      • Spur B.
      • Wong P.Y.
      ), PC12 (
      • Estevez A.G.
      • Radi R.
      • Barbeito L.
      • Shih J.T.
      • Thompson J.A.
      • Beckman J.S.
      ), and human endothelial cells (
      • Szabo C.
      • Zingarelli B.
      • O'Connor M.
      • Salzman A.L.
      ), it can be used to assess oxidative injury. Two key events in the induced apoptosis include cytochrome c release from mitochondria and activation of caspase-3 (
      • Green D.R.
      • Reed J.C.
      ). During the early stage of apoptosis, the activated caspase-3 cleaves p21 WAF1/CIP1 , a cyclin-dependent kinase inhibitor that protects cells from apoptosis (
      • Gorospe M.
      • Wang X.
      • Guyton K.Z.
      • Holbrook N.J.
      ), at a specific aspartate residue (Asp-112) and causes the loss of its localization and function in nuclei (
      • Levkau B.
      • Koyama H.
      • Raines E.W.
      • Clurman B.E.
      • Herren B.
      • Orth K.
      • Roberts J.M.
      • Ross R.
      ). c-Jun NH2-terminal protein kinase (JNK) and p38 kinase, two mitogen-activated protein kinases (MAPK), are also activated in apoptosis induced by diverse stimuli (
      • Tibbles L.A.
      • Woodgett J.R.
      ). It is unknown how GPX1 affects the DQ- and PN-induced apoptosis and related signaling.
      Intracellular GSH may play three roles in metabolism: as an independent antioxidant, as a presumed physiological substrate of GPX1 to be oxidized to GSSG and regenerated by NADPH-dependent glutathione reductase (EC 1.6.4.2) reaction (
      • Sies H.
      • Gerstenecker C.
      • Menzel H.
      • Flohé L.
      ,
      • Meister A.
      • Anderson M.E.
      ), and as a regulator of apoptosis (
      • Coppola S.
      • Ghibelli L.
      ). It is fascinating to find out how GPX1 knockout affects the responses of cellular GSH/GSSG to ROS and RNS. Therefore, our objective was to dissect the metabolic role of GPX1 in cell death, apoptotic signaling, protein nitration, and GSH/GSSG responses induced by the ROS generator DQ and RNS donor PN in primary hepatocytes isolated from the GPX1−/− and the WT mice. Most strikingly, we found that GPX1 knockout did not attenuate, but enhanced hepatocyte resistance to the PN-mediated apoptosis, which was completely opposite to its impact on the DQ-mediated apoptosis or our expectation.

      DISCUSSION

      It is remarkable that GPX1 knockout exerted completely opposite impacts on susceptibility of mouse hepatocyte to DQ and PN-induced apoptotic death. Because high levels of H2O2could be produced by DQ (
      • Farrington J.A.
      • Ebert M.
      • Land E.J.
      • Fletcher K.
      ), the substantial loss of cellular defense against DQ-induced apoptosis in GPX1−/− over WT cells is consistent with the whole body responses of the GPX1−/− mice challenged with ROS generators (
      • Fu Y.X.
      • Cheng W.H.
      • Porres J.M.
      • Ross D.A.
      • Lei X.G.
      ,
      • Cheng W.H.
      • Ho Y.-S.
      • Valentine B.A.
      • Ross D.A.
      • Combs Jr., G.F.
      • Lei X.G.
      ,
      • de Haan J.B.
      • Bladier C.
      • Griffiths P.
      • Kelner M.
      • O'Shea R.D.
      • Cheung N.S.
      • Bronson R.T.
      • Silvestro M.J.
      • Wild S.
      • Zheng S.S.
      • Beart P.M.
      • Hertzog P.J.
      • Kola I.
      ). However, the positive impact of GPX1 knockout on hepatocyte resistance to PN cytotoxicity is rather striking and does not agree with the notion that selenoproteins such as GPX1 (
      • Sies H.
      • Sharov V.S.
      • Klotz L.-O.
      • Briviba K.
      ) and selenoprotein P (
      • Arteel G.E.
      • Briviba K.
      • Sies H.
      ) may protect against PN-induced oxidative stressin vivo. Although PN is highly reactive with a short half-life, our results are reproducible and physiologically relevant. This is because we demonstrated a PN-dose dependent response of cell viability and nitrotyrosine formation, a reliable indicator of PN activity in the cell (
      • Haddad I.Y.
      • Pataki G.
      • Hu P.
      • Galliani C.
      • Beckman J.S.
      • Matalon S.
      ). Our selected PN dose (0.4 mm), similar to that used by others (
      • Sies H.
      • Sharov V.S.
      • Klotz L.-O.
      • Briviba K.
      ,
      • Go Y.M.
      • Patel R.P.
      • Maland M.C.
      • Park H.
      • Beckman J.S.
      • Darley-Usmar V.M.
      • Jo H.
      ), was the minimal level that distinguished GPX1−/− from WT cells. Comparable results were obtained by using different sources or manipulations of PN treatment (data not shown). The enhanced nitrotyrosine formation in WT cells over that in GPX1−/− cells treated with 0.2 or 0.4 mm PN reflects a promoting role of GPX1, similar to that of other peroxidases (
      • Sampson J.B.
      • Ye Y.
      • Rosen H.
      • Beckman J.S.
      ), in the PN-mediated protein nitration.
      A fundamental question is how GPX1 affects the PN-mediated apoptosis and protein nitration. In cell lysate, extrinsic GPX1 was able to reduce PN to nitrite using GSH in a two-electron catalysis (
      • Sies H.
      • Sharov V.S.
      • Klotz L.-O.
      • Briviba K.
      ). However, we did not see a difference in PN reduction or medium nitrite level between GPX1−/− and WT cells treated with 0–0.8 mmPN for 12 h (data not shown). Because of the strong reactivity between PN and CO2 (
      • Squadrito G.L.
      • Pryor W.A.
      ), GPX1 in the cultured cells, unlike in the cell lysates (
      • Sies H.
      • Sharov V.S.
      • Klotz L.-O.
      • Briviba K.
      ), might be encountered with not only authentic PN, but also more reactive PN intermediates such asNO2 or CO⨪3. As PN inactivated a good portion of GPX1 (
      • Padmaja S.
      • Squadrito G.L.
      • Pryor W.A.
      ) in WT cells, the projected enzymatic reduction of PN by GPX1 (
      • Sies H.
      • Sharov V.S.
      • Klotz L.-O.
      • Briviba K.
      ) might not be as effective in competing against thiols or CO2 as for direct reactions with PN (
      • Arteel G.E.
      • Briviba K.
      • Sies H.
      ) in these cells. However, this PN-mediated inactivation of GPX1 could not explain the enhanced sensitivity of WT cells to PN toxicity, because those cells still had much higher GPX activity than the GPX1−/−hepatocytes (207–316 versus 4.2 milliunits/mg of protein). More likely, GPX1 exerted its role by affecting H2O2 removal and thus cellular balances of ROS and RNS that could modulate PN toxicity (
      • Day B.J.
      • Patel M.
      • Calavetta L.
      • Chang L.Y.
      • Stamler J.S.
      ,
      • Brune B.
      • Gotz C.
      • Messmer U.K.
      • Sandau K.
      • Hirvonen M.R.
      • Lapetina E.G.
      ,
      • Eu J.P.
      • Liu L.
      • Zeng M.
      • Stamler J.S.
      ).
      The DQ-treated GPX1−/− and the PN-treated WT cells clearly underwent apoptosis and exhibited similar apoptotic signaling, because cytochromec release and pro-caspase-3 cleavages preceded the appearance of apoptotic cells and severe DNA fragmentation in these cells. As a critical step in stress-induced apoptosis, cytochromec release from mitochondria enables it to bind to Apaf-1 and caspase-9, leading to the activation of caspase-9 that in turn activates caspase-3 (
      • Green D.R.
      • Reed J.C.
      ). Caspase-3 is an executioner of apoptosis with many target proteins, including p21 WAF1/CIP1 (
      • Levkau B.
      • Koyama H.
      • Raines E.W.
      • Clurman B.E.
      • Herren B.
      • Orth K.
      • Roberts J.M.
      • Ross R.
      ) that protects against apoptosis (
      • Gorospe M.
      • Wang X.
      • Guyton K.Z.
      • Holbrook N.J.
      ). Cleavage of p21 WAF1/CIP1 mediated by caspase-3 and the consequent activation of cyclin A/Cdk2 have been shown as prerequisite for the execution of apoptosis in human hepatoma cells SK-HEP-1 induced by ginsenoside Rh2 (
      • Jin Y.H.
      • Yoo K.J.
      • Lee Y.H.
      • Lee S.K.
      ). In the present study, the initial up-regulation of p21 WAF1/CIP1 protein expression at 3 h over the base line was maintained later only in the DQ-treated WT cells and the PN-treated GPX1−/− cells that showed no induced apoptosis. In contrast, the DQ-treated GPX1−/− and the PN-treated WT cells exhibited significant decreases of p21 WAF1/CIP1 at 6 and 9 h over the levels at 0 or 3 h. Both p38 MAPK and JNK, two kinases involved in stress-induced apoptosis (
      • Tibbles L.A.
      • Woodgett J.R.
      ,
      • Park H.-S.
      • Park E.
      • Kim M.-S.
      • Ahn K.
      • Kim I.Y.
      • Choi E.-J.
      ), were activated by DQ or PN at 30 min, but their responses were not consistent with the changes of the three assayed apoptotic signal molecules. Seemingly, GPX1 exerted its role in the PN- or DQ-induced apoptotic events downstream or independent of activation of these two kinases. Inhibition of the PN-induced activation of p38 MAPK and JNK by selenite in the cultured rat liver epithelial cells has been suggested to be through selenium-containing proteins, including GPX (
      • Schieke S.M.
      • Briviba K.
      • Klotz L.-O.
      • Sies H.
      ). In our study, activation of p38 MAPK was slightly stronger by DQ and much so by PN in WT than GPX1−/− cells. Thus, GPX1 promoted its activation mediated by ROS or RNS in mouse hepatocytes, indicating a possible cell-specific or GPX1-independent effect of selenite on these kinases.
      Distinct differences in the DQ-induced cellular GSH/GSSG changes between the GPX1−/− and WT cells in the present study support the idea that GSH is a physiological substrate of GPX1 in metabolism (
      • Sies H.
      • Gerstenecker C.
      • Menzel H.
      • Flohé L.
      ,
      • Meister A.
      • Anderson M.E.
      ). In the presence of GPX1, WT cells displayed a sharp decrease in GSH, along with an abrupt rise of GSSG, within 60 min after the DQ treatment. Although this GSH depletion attenuated after 60 min, probably because of the decrease in ROS production and(or) an accelerated regeneration of GSH from GSSG by glutathione reductase, GSH was indeed oxidized to GSSG by GPX1 to reduce the DQ-generated H2O2 and other hydroperoxides at a very high rate initially. In contrast, GPX1−/− cells responded to DQ or PN with much less and slower depletion of GSH than WT cells, without any GSSG accumulation at all. Clearly, lack of GPX1 spared the oxidation of GSH to GSSG and left it for direct and/or GPX1-independent protections (
      • Sies H.
      • Gerstenecker C.
      • Menzel H.
      • Flohé L.
      ,
      • Meister A.
      • Anderson M.E.
      ,
      • Koppenol W.H.
      • Moreno J.J.
      • Pryor W.A.
      • Ischiropoulos H.
      • Beckman J.S.
      ). In the PN-treated WT cells, GSH seemed to act as a substrate of GPX1 initially and then became more like a GPX1-indpendent protector because a sharp rise in GSSG along with the GSH depletion was not seen after 60 min. In comparison with these distinct roles of GSH in functioning as a GPX1 substrate and a major antioxidant, the suggested necessity of certain amount of cellular GSH for cells to undergo apoptosis instead of necrosis (
      • Coppola S.
      • Ghibelli L.
      ) was not fully shown in our study. Although apoptotic events occurred in the DQ-treated GPX1−/− cells in which cellular GSH was indeed greater than in WT cells, these events were also exhibited in the PN-treated WT cells in which cellular GSH was depleted to a very low level initially. Thus, cellular GSH alteration alone may not be sufficient to regulate apoptosis.
      Elucidating the opposite role of GPX1 in DQ- and PN-induced oxidative injury has broad implications. It teaches us that antioxidant protection for a given enzyme or protein such as GPX1 may not be a general property, but depends on the specific nature of oxidants. Although pro-oxidant properties of high levels of vitamin E or C (
      • Maiorino M.
      • Zamburlini A.
      • Roveri A.
      • Ursini F.
      ,
      • Podmore I.D.
      • Griffiths H.R.
      • Herbert K.E.
      • Mistry N.
      • Mistry P.
      • Lunec J.
      ) and overexpression of Cu,Zn-superoxide dismutase (
      • Midorikawa K.
      • Kawanishi S.
      ) have been reported previously, our study provides the first evidence to show the “double-edged sword” function of an “antioxidant” enzyme at its physiological expression level in metabolically normal primary cells. The potent role of GPX1 in turning off the DQ-induced and in switching on the PN-induced apoptosis will help us in elucidating mechanisms of ROS/RNS in regulating cell death and related signaling (
      • Hensley K.
      • Robinson K.A.
      • Gabbita S.P.
      • Salsman S.
      • Floyd R.A.
      ), and in developing novel therapeutic strategies for the ROS and RNS involved diseases (
      • Stadtman E.R.
      • Levine R.L.
      ). Our findings also caution the public that blind antioxidant supplementation in clinic or nutrition may not always be desirable. In line of our view, knockout of GPX1 enhanced mouse brain resistance to the kainic acid-induced epileptic seizure (
      • Jiang D.
      • Akopian G.
      • Ho Y.-S.
      • Walsh J.P.
      • Andersen J.K.
      ), whereas overexpressing GPX1 promoted acetaminophen toxicity to mice (
      • Mirochnitchenko O.I.
      • Weisbrot-Lefkowitz M.
      • Reuhl K.
      • Chen L.
      • Yang C.
      • Inouye M.
      ) and tumorigenesis (
      • Lu Y.P.
      • Lou Y.R.
      • Yen P.
      • Newmark H.L.
      • Mirochnitchenko O.I.
      • Inouye M.
      • Huang M.T.
      ). Likewise, vitamin C was able to induce decomposition of lipid hydroperoxides to endogenous geneotoxins (
      • Lee S.H.
      • Oe T.
      • Blair I.A.
      ).

      Acknowledgments

      We thank Dr. Hiroki Ueda for technical help in TUNEL assay and Professor Leon Heppel for critical review of the manuscript.

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