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Activation of GluR6-containing Kainate Receptors Induces Ubiquitin-dependent Bcl-2 Degradation via Denitrosylation in the Rat Hippocampus after Kainate Treatment*

  • Jia Zhang
    Footnotes
    Affiliations
    Research Center of Biochemistry and Molecular Biology, Jiangsu Province Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou, Jiangsu 221002, China
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  • Hui Yan
    Footnotes
    Affiliations
    Research Center of Biochemistry and Molecular Biology, Jiangsu Province Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou, Jiangsu 221002, China
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  • Yong-Ping Wu
    Affiliations
    Jiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou, Jiangsu 221002, China
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  • Chong Li
    Affiliations
    Research Center of Biochemistry and Molecular Biology, Jiangsu Province Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou, Jiangsu 221002, China
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  • Guang-Yi Zhang
    Correspondence
    To whom correspondence should be addressed: Research Center of Biochemistry and Molecular Biology, Jiangsu Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou 221002, China. Tel./Fax: 86-516-8574-8486;
    Affiliations
    Research Center of Biochemistry and Molecular Biology, Jiangsu Province Key Laboratory of Brain Disease Bioinformation, Xuzhou Medical College, Xuzhou, Jiangsu 221002, China
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  • Author Footnotes
    * This work was supported by Project Grant 30870543 from the National Natural Science Foundation of China and Education Departmental Nature Science Funds of Jiangsu Province of China (09KJB310015).
    1 Both authors contributed equally to this work.
Open AccessPublished:December 10, 2010DOI:https://doi.org/10.1074/jbc.M110.156299
      We previously showed that Bcl-2 (B-cell lymphoma 2) is down-regulated in a kainate (KA)-induced rat epileptic seizure model. The underlying mechanism had remained largely unknown, but we here report for the first time that denitrosylation and ubiquitination are involved. Our results show that the S-nitrosylation levels of Bcl-2 are down-regulated after KA injection and that the GluR6 (glutamate receptor 6) antagonist NS102 can inhibit the denitrosylation of Bcl-2. Moreover, the ubiquitin-dependent degradation of Bcl-2 was found to be promoted after KA treatment, which could be suppressed by the proteasome inhibitor MG132 and the NO donors, sodium nitroprusside and S-nitrosoglutathione. In addition, experiments based on siRNA transfections were performed in the human SH-SY5Y neuroblastoma cell line to verify that the stability of Bcl-2 is causal to neuronal survival. At the same time, it was found that the exogenous NO donor GSNO could protect neurons when Bcl-2 is targeted. Subsequently, these mechanisms were morphologically validated by immunohistochemistry, cresyl violet staining, and in situ TUNEL staining to analyze the expression of Bcl-2 as well as the survival of CA1 and CA3/DG pyramidal neurons. NS102, GSNO, sodium nitroprusside, and MG132 contribute to the survival of CA1 and CA3/DG pyramidal neurons by attenuating Bcl-2 denitrosylation. Taken together, our data reveal that Bcl-2 ubiquitin-dependent degradation is induced by Bcl-2 denitrosylation during neuronal apoptosis after KA treatment.

      Introduction

      The agitation of glutamate receptors is thought to be the primary cause of epileptic seizures. Glutamate receptors are classified into metabolic glutamate receptors (GluRs)
      The abbreviations used are: GluR, glutamate receptor; KA, kainate; SNP, sodium nitroprusside; GSNO, S-nitrosoglutathione; NAC, N-acetyl-l-cysteine; MMTS, methyl methylthiomethyl sulfoxide; FMK, fluoromethyl ketone; AS-ODN, antisense oligodeoxynucleotides; S-ODN, sense oligodeoxynucleotides; MOPS, 4-morpholinepropanesulfonic acid; Trx, thioredoxin.
      and ionic GluRs. Glutamate itself gates three types of ionotropic receptors: NMDA, AMPA, and kainate (KA) receptors. There are also five types of kainate receptor subunits: GluR5, GluR6, GluR7, KA1, and KA2 (
      • Hollmann M.
      • Heinemann S.
      ). These subunits can be arranged in different ways to form a tetramer. GluR5–7 can form homomers (for example, a receptor composed entirely of GluR5) and heteromers (for example, a receptor composed of both GluR5 and GluR6). However, KA1 and KA2 can only form functional receptors by combining with one of the GluR5–7 subunits (
      • Bloss E.B.
      • Hunter R.G.
      ). Kainic acid (KA) is a potent exogenous agonist of the KA receptors, and the systemic administration of KA produces epilepsy in rats or mice accompanied by neuronal damage, mainly in limbic structures such as the hippocampal pyramidal neurons in particular (
      • Liu X.M.
      • Pei D.S.
      • Guan Q.H.
      • Sun Y.F.
      • Wang X.T.
      • Zhang Q.X.
      • Zhang G.Y.
      ). KA-induced seizures in rodents have been widely used as a model of human temporal lobe epilepsy.
      We have reported previously that the Bcl-2 (B-cell lymphoma gene 2) levels are down-regulated in a KA-induced rat epileptic seizure model (
      • Liu X.M.
      • Pei D.S.
      • Guan Q.H.
      • Sun Y.F.
      • Wang X.T.
      • Zhang Q.X.
      • Zhang G.Y.
      ). The Bcl-2 family proteins are mainly located in the mitochondrial outer membrane and are divided into pro-apoptotic and the anti-apoptotic groups (
      • Baliga B.C.
      • Kumar S.
      ,
      • Suzuki M.
      • Youle R.J.
      • Tjandra N.
      ,
      • Hirotani M.
      • Zhang Y.
      • Fujita N.
      • Naito M.
      • Tsuruo T.
      ,
      • Kelekar A.
      • Chang B.S.
      • Harlan J.E.
      • Fesik S.W.
      • Thompson C.B.
      ). Several mechanisms have been reported to underlie the anti-apoptotic function of Bcl-2, including the regulation of Ca2+ homeostasis or action as an antioxidant. Furthermore, as a heterodimer, the pro-apoptotic protein Bax is attenuated by Bcl-2. Moreover, Bcl-2 prevents the release of cytochrome c from mitochondria and inhibits the activation of caspase-9 and caspase-3 (
      • Breitschopf K.
      • Haendeler J.
      • Malchow P.
      • Zeiher A.M.
      • Dimmeler S.
      ).
      It has been well established that the Bcl-2 protein levels are essential for its anti-apoptotic function. The regulation of these levels mainly occurs via post-translational modifications and degradation (
      • Lutz R.J.
      ,
      • Glasgow J.N.
      • Qiu J.
      • Rassin D.
      • Grafe M.
      • Wood T.
      • Perez-Pol J.R.
      ,
      • Bentires-Alj M.
      • Dejardin E.
      • Viatour P.
      • Van Lint C.
      • Froesch B.
      • Reed J.C.
      • Merville M.P.
      • Bours V.
      ,
      • Mitchell K.O.
      • Ricci M.S.
      • Miyashita T.
      • Dicker D.T.
      • Jin Z.
      • Reed J.C.
      • El-Deiry W.S.
      ,
      • Li B.
      • Dou Q.P.
      ,
      • Yang Y.
      • Yu X.
      ). More recently, protein S-nitrosylation has emerged as the principal post-translational modification by which nitric oxide exerts a myriad of biological effects. S-Nitrosylation, the covalent attachment of a NO group to a Cys thiol side chain, has been postulated to be a fundamental mechanism in cellular signal transduction. The stability of proteins, cleavage of zymogens, and modification of active proteins can be controlled by this post-translational modification process (
      • Tannenbaum S.R.
      • Kim J.E.
      ,
      • Hess D.T.
      • Matsumoto A.
      • Kim S.O.
      • Marshall H.E.
      • Stamler J.S.
      ). More recently, nitrosylation has been found to be reversible. Nitrosylated Cys thiol groups can be reduced to free thiols, depending on the conditions, which is defined as denitrosylation and plays an important role in signal regulation in a broad range of diseases (
      • Benhar M.
      • Forrester M.T.
      • Stamler J.S.
      ). Mutational analysis of Bcl-2 has shown that two cysteine residues in this protein (Cys-158 and Cys-229) are important for the S-nitrosylation process (
      • Azad N.
      • Vallyathan V.
      • Wang L.
      • Tantishaiyakul V.
      • Stehlik C.
      • Leonard S.S.
      • Rojanasakul Y.
      ). Bcl-2 degradation is mainly mediated through ubiquitin-proteasome pathway. All four lysine residues in Bcl-2 (K17R, K22R, K218R, and K239R) target this protein for ubiquitin-dependent degradation (
      • Dimmeler S.
      • Breitschopf K.
      • Haendeler J.
      • Zeiher A.M.
      ). A more recent report has indicated that the S-nitrosylation of Bcl-2 prevents its ubiquitin-proteasomal degradation during the apoptotic cell death induced by chromium (VI) in lung cancers (
      • Azad N.
      • Vallyathan V.
      • Wang L.
      • Tantishaiyakul V.
      • Stehlik C.
      • Leonard S.S.
      • Rojanasakul Y.
      ). Similarly, it has been shown that the mechanism of cisplatin resistance involves the up-regulation of Bcl-2 expression by NO, which occurs by preventing its ubiquitin-dependent degradation in human lung carcinoma H-460 cells (
      • Chanvorachote P.
      • Nimmannit U.
      • Stehlik C.
      • Wang L.
      • Jiang B.H.
      • Ongpipatanakul B.
      • Rojanasakul Y.
      ).
      In this study, we demonstrate that KA induces Bcl-2 denitrosylation through the GluR6-KA receptor pathway, which is proposed to facilitate Bcl-2 ubiquitination and finally down-regulate its protein level. The GluR6 antagonist NS102, NO donor S-nitrosoglutathione (GSNO), sodium nitroprusside (SNP), and the proteasome inhibitor MG132 prevent the denitrosylation and the degradation of Bcl-2, playing an important role in neuron protection in hippocampal CA1 and CA3/DG regions.

      DISCUSSION

      In our present report, we demonstrate that GluR6-KA receptor-mediated Bcl-2 denitrosylation facilitates Bcl-2 ubiquitination and proteasomal degradation during KA-induced neuronal apoptosis. Pretreatment with the GluR6 antagonist NS102, the NO donors GSNO and SNP, as well as the proteasome inhibitor MG132 exerted neuroprotective effects by attenuating Bcl-2 denitrosylation and ubiquitin-dependent degradation.
      S-Nitrosylation and denitrosylation are crucial protein post-translation modifications that involve the regulation of protein function, such as phosphorylation and dephosphorylation, and that play important roles in the activity regulation of protein kinases under both physiological and pathological conditions. S-Nitrosylation can positively or negatively regulate the function of proteins, and denitrosylation has shown this same capacity (
      • Hess D.T.
      • Matsumoto A.
      • Kim S.O.
      • Marshall H.E.
      • Stamler J.S.
      ). For example, the pro-apoptosis proteinase matrix metalloproteinase 9 is activated by S-nitrosylation and induces neuronal apoptosis (
      • Gu Z.
      • Kaul M.
      • Yan B.
      • Kridel S.J.
      • Cui J.
      • Strongin A.
      • Smith J.W.
      • Liddington R.C.
      • Lipton S.A.
      ). Our previous study showed that JNK3, a pro-apoptosis kinase, is activated via S-nitrosylation during cerebral ischemia and reperfusion in the rat hippocampus, whereas the anti-apoptosis kinase, Akt/PKB, can be inactivated by S-nitrosylation (
      • Pei D.S.
      • Song Y.J.
      • Yu H.M.
      • Hu W.W.
      • Du Y.
      • Zhang G.Y.
      ,
      • Yasukawa T.
      • Tokunaga E.
      • Ota H.
      • Sugita H.
      • Martyn J.A.
      • Kaneki M.
      ). As for denitrosylation, when this occurs for caspase-3, a pro-apoptosis proteinase, caspase activity is inhibited, whereas Bcl-2 is hydrolyzed by this modification (
      • Benhar M.
      • Forrester M.T.
      • Stamler J.S.
      ,
      • Azad N.
      • Vallyathan V.
      • Wang L.
      • Tantishaiyakul V.
      • Stehlik C.
      • Leonard S.S.
      • Rojanasakul Y.
      ,
      • Mannick J.B.
      • Hausladen A.
      • Liu L.
      • Hess D.T.
      • Zeng M.
      • Miao Q.X.
      • Kane L.S.
      • Gow A.J.
      • Stamler J.S.
      ,
      • Benhar M.
      • Forrester M.T.
      • Hess D.T.
      • Stamler J.S.
      ). In accordance with previous reports and our present analyses, it is clear that Bcl-2 is also regulated by S-nitrosylation and denitrosylation. Bcl-2 is S-nitrosylated to a certain extent in its basal state, maintaining its stability. On this basis, the regulation or dysregulation of Bcl-2 S-nitrosylation could be employed in diverse cellular responses. For example, on the one hand the effects of stress inducers, including Cr (VI), FasL, and BSO, up-regulate Bcl-2 S-nitrosylation (
      • Azad N.
      • Vallyathan V.
      • Wang L.
      • Tantishaiyakul V.
      • Stehlik C.
      • Leonard S.S.
      • Rojanasakul Y.
      ). On the other hand, the dysregulation of Bcl-2 S-nitrosylation impairs the anti-apoptotic function of cells and reduces their resistance to KA-induced neuronal apoptosis and cisplatin-induced cell death in the case of human lung carcinoma H-460 cells (
      • Chanvorachote P.
      • Nimmannit U.
      • Stehlik C.
      • Wang L.
      • Jiang B.H.
      • Ongpipatanakul B.
      • Rojanasakul Y.
      ).
      Degradation mechanisms also play important roles in the regulation of protein levels, cell apoptosis, and survival and signaling pathways. Among the different protein degradation systems, the ubiquitin-dependent proteasomal degradation pathway is the most well studied (
      • Hochstrasser M.
      ). The ubiquitination system functions in a wide variety of cellular processes, including antigen processing, apoptosis, the cell cycle, DNA repair, immune responses, the modulation of cell surface receptors, the response to stress, and extracellular modulators. Our findings here indicate that in response to KA stress, Bcl-2 denitrosylation initiates the ubiquitin-dependent degradation of Bcl-2, giving rise to the apoptosis of hippocampal neurons. When Bcl-2 expression was decreased by AS-ODNs or siRNA, the neuroprotection of exogenous NO donor GSNO was weakened (as shown in Fig. 6). As is well established, the ubiquitin-proteasome system recycles proteins with a short half-life. It would be interesting to measure whether the ubiquitin-proteasome system controls the basal Bcl-2 levels in homeostasis. However, based on our present results (Fig. 3E), the Bcl-2 levels should therefore be noticeably increased at the indicated time points in the presence of MG132. Although our present data do not exclude the possibility of this regulatory mechanism, they cannot confirm that the ubiquitin-proteasome system is a general regulator of Bcl-2 degradation or whether another pathway performs this function.
      Although numerous theories have sought to explain the mechanism of protein S-nitrosylation, it remains largely unknown. Recently, two specific enzymatic systems of protein denitrosylation have been identified: the thioredoxin (Trx) system, which comprises Trx proteins, Trx reductase (TrxR) proteins, and NADPH; and the GSNO reductase system, which comprises GSH and GSNO reductase. Theoretically, the denitrosylation of target proteins is mediated either by catalyzing the conversion of reduced Trx-(SH)2 to oxidized Trx-S2 or by oxidizing glutathione (GSH). Moreover, candidate denitrosylases have been proposed, such as protein disulfide isomerase, xanthine oxidase, superoxide dismutase, glutathione peroxidase, and carbonyl reductase (
      • Benhar M.
      • Forrester M.T.
      • Stamler J.S.
      ). Additionally, receptor-coupled denitrosylation mechanisms have been identified that are mediated by Fas receptors, TNFα receptors, VEGF receptors, insulin receptors, and β-adrenergic receptors. In addition, a rise in intracellular calcium has been proven to play a crucial role in protein denitrosylation (
      • Chvanov M.
      • Gerasimenko O.V.
      • Petersen O.H.
      • Tepikin A.V.
      ). However, more studies are required to elucidate the mechanisms underlying GluR6-KA signaling. It will also be of great interest to determine the denitrosylase involved in Bcl-2 degradation and any other regulators that function in this process, such as calcium.
      In summary, we propose from our findings that the intracerebroventricular infusion of KA results in ubiquitin-dependent Bcl-2 degradation, which is facilitated by GluR6-KA receptor coupled Bcl-2 denitrosylation and is independent of its phosphorylation. Moreover, NS102, GSNO, SNP, and MG132 exert neuroprotective effects by preventing Bcl-2 degradation through the suppression of denitrosylation or ubiquitination. Further studies are needed to elucidate the mechanisms underpinning GluR6-KA receptor coupled denitrosylation, which may lead to better therapeutic approaches to the future treatment of epileptic seizures.

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