Functional analysis of phospholipid-metabolizing enzyme signal transduction during early phase of cell death after ischemia

2012 Fiscal Year Final Research Report

• Project/Area Number: 22791745
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Single-year Grants
• Research Field: Emergency medicine
• Research Institution: Yamagata University
• Principal Investigator: MASASHI Okada 山形大学, 医学部, 助教 (70512614)
• Project Period (FY): 2010 – 2012
• Keywords: DGK / 虚血 / イノシトールリン脂質 / ユビキチン / 細胞周期

Research Abstract

In previous study, we demonstrated that diacylglycerol kinase DGKzeta which is distributed at nucleus in normal hippocampal neuron in vivo, is translocated to cytoplasm from nucleus by middle cerebral artery infarction in rat. But it is unclear that the mechanisms of ischemia induced translocation of DGKzeta and the physiological functions after localization in cytoplasm of DGKzeta. In this study, we demonstrated that transient exposure to excitotoxic concentration of glutamate led to cytoplasmic accumulation of DGKζ followed by its down-regulation. Results showed that DGKζ down-regulation was caused by proteolytic degradation through the ubiquitin.proteasome system UPSrather than transcriptional inhibition. DGKζ-deficient hippocampus exhibited a significant increase in Ser807811 phosphorylated retinoblastoma protein levels together with up-regulation of the expression of type D and E cyclins, indicative of cell cycle reentry. From a functional perspective, in vitro gene silencing of D GKzeta via specific siRNA enhanced DNA fragmentation in cultured neurons after glutamate exposure. At the organismal level, hippocam-pal neurons of DGKzeta-deficient mice showed vulnerability to kainate-induced seizures. In addition, nucleosome assembly pro-tein NAP1-like 1 NAP1L1and NAP1-like 4 NAP1L4are identified as novel DGKzeta binding partners. The molecular interaction of DGKzeta and NAP1Ls prohibits nuclear import of DGKzeta because binding of NAP1Ls to DGKzeta blocks import carrier proteins, Qip1 and NPI1, to interact with DGKzeta, leading to cytoplasmic tethering of DGKzeta. In addition, overexpression of NAP1Ls ex-erts a protective effect against doxorubicin-induced cytotoxicity. Additionally, we identified a small GTPase effector protein, IQGAP1, as a novel DGKzeta-associated complex protein. A bacterial endotoxin, lipopolysaccharide LPSfacilitated the complex formation in macrophages. RNA interference-mediated knockdown of DGKzeta or IQGAP1 impaired LPS-induced Rac1 activation. Primary macrophages derived from DGKzeta-mice attenuated LPS-induced phagocytosis of bacteria. DGKzeta is involved in IQGAP1Rac1-mediated phagocytosis upon LPS stimulation in macrophages.

Research Products

• [Journal Article] DGKζ is degraded through the cytoplasmic ubiquitin-proteasome system under excitotoxic conditions, which causes neuronal apoptosis because of aberrant cell cycle reentry (2012)
  • Author(s): Okada M, Hozumi Y, Tanaka T, Suzuki Y, Yanagida M, Araki Y, Evangelisti C, Yagisawa H, Topham MK, Martelli AM, Goto K
  • Journal Title: Cellular Signalling Volume: 24 Pages: 1573-1582
  • DOI: DOI:10.1016/j.cellsig.2012.03.021.
  • Peer Reviewed
• [Journal Article] DGKζ through IQGAP1/Rac1 pathway (2012)
  • Author(s): Okada M^*, Hozumi Y, Iwazaki K, Misaki K, Yanagida M, Araki Y, Watanabe T, Yagisawa H, Topham MK, Kaibuchi K, Goto K^* (^*Double corresponding)
  • Journal Title: Biochem Biophys Res Commun Volume: 420 Pages: 479-484
  • DOI: DOI:10.1016/j.bbrc.2012.03.057.
  • Peer Reviewed
• [Journal Article] NMDA receptor-mediated Ca^<2+> influx triggers nucleocytoplasmic translocation of diacylglycerol kinase ζ under oxygen?glucose deprivation conditions, an in vitro model of ischemia, in rat hippocampal slices (2012)
  • Author(s): Yusuke Suzuki, Yoshihiko Yamazaki, Yasukazu Hozumi, Masashi Okada Toshiaki Tanaka, Ken Iseki, Nobuo Ohta, Masaru Aoyagi, Satoshi Fujii, Kaoru Goto
  • Journal Title: Histochem Cell Biol. Volume: 137 Pages: 499-511
  • DOI: DOI:10.1007/s00418-011-0907-y
  • Peer Reviewed
• [Journal Article] Interaction of nucleosome ssembly proteins abolishes nuclear localization of DGKζ by attenuating its association with importins (2011)
  • Author(s): Okada M, Hozumi Y, Ichimura T, Tanaka T, Hasegawa H, Yamamoto M, Takahashi N, Iseki K, Yagisawa H, Shinkawa T, Isobe T, Goto K.
  • Journal Title: Exp Cell Res Volume: 317 Pages: 2853-63
  • DOI: DOI:10.1016/j.yexcr.2011.09.014
  • Peer Reviewed
• [Presentation] DGK ζ is a suppressor of excitotoxic neuronal cell death (2011)
  • Author(s): Masashi Okada, Kaoru Goto
  • Organizer: 分子生物学会年会
  • Place of Presentation: 横浜、パシフィコ横浜
  • Year and Date: 2011-12-16
• [Presentation] DGKζis a suppressor of excitotoxic neuronal cell death (2011)
  • Author(s): Masashi Okada, Kaoru Goto
  • Organizer: JBS バイオフロンティア国際シンポジウム九州大学創立百周年記念
  • Place of Presentation: 福岡、Hotel Luigans-Spa and Resort
  • Year and Date: 2011-11-15
• [Presentation] Nucleaosome assembly protein はDGKz の核-細胞質間輸送を制御する (2010)
  • Author(s): 岡田雅司、後藤薫
  • Organizer: 日本分子生物学会年会、日本生化学大会合同大会
  • Place of Presentation: 神戸、神戸ポートアイランド
  • Year and Date: 2010-12-09
• [Remarks]
  • URL: http://www.id.yamagata-u.ac.jp/AnatomyII/anaII.htm