Mitochondrial calcium-mediated reactive oxygen species are essential for the rapid induction of the grp78 gene in 9L rat brain tumour cells

Cell Signal. 2003 Jan;15(1):57-64. doi: 10.1016/s0898-6568(02)00055-4.


The glucose-regulated protein grp78 gene is rapidly transactivated in 9L rat brain tumour (RBT) cells treated with okadaic acid (OA) followed by heat shock (HS) (termed OA-->HS treatment). By Northern blotting analyses and transient transfection assays, we herein show that transactivation of grp78 by OA-->HS is abolished by an intracellular calcium chelator, bis(aminophenoxy)ethane N,N'-tetraacetic acid (BAPTA), and an inhibitor of mitochondrial Ca(2+) uniporter, ruthenium red (RR), while unaffected by cyclosporin A (CsA), an inhibitor of mitochondrial permeability transition pore (MTP). The inhibitory effects of BAPTA and RR also present in OA-->HS induction of transient elevation of intracellular hydrogen peroxide. The requirement of reactive oxygen intermediates (ROIs) is confirmed by substitutional addition of antioxidants, N-acetyl cysteine (NAC) and pyrrolidinedithiocarbamate (PDTC) during OA-->HS treatment, mimicking these inhibitory effects of BAPTA and RR. Western blotting analyses show that phosphorylation of transcription factor CREB is diminished only by BAPTA but not by RR, while phosphorylation of ATF-2 is unaffected by either agent. Conclusively, we present that both the disturbances of mitochondrial calcium homeostasis and reactive oxygen intermediates are essential for rapid transactivation of grp78, and this pathway is separate from protein kinase A (PKA)-dependent CREB activation or p38 mitogen-activated protein kinase (p38(MAPK))-dependent ATF-2 activation and signalling.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Activating Transcription Factor 2
  • Animals
  • Antioxidants / pharmacology
  • Brain Neoplasms
  • Calcium / physiology*
  • Calcium Channels
  • Calcium-Binding Proteins / antagonists & inhibitors
  • Carrier Proteins / biosynthesis
  • Carrier Proteins / genetics*
  • Chelating Agents / pharmacology
  • Cyclic AMP Response Element-Binding Protein / metabolism
  • Egtazic Acid / analogs & derivatives*
  • Egtazic Acid / pharmacology
  • Endoplasmic Reticulum Chaperone BiP
  • Heat-Shock Proteins*
  • Heat-Shock Response
  • Hydrogen Peroxide / metabolism
  • Kinetics
  • Mitochondria / metabolism*
  • Molecular Chaperones / biosynthesis
  • Molecular Chaperones / genetics*
  • Okadaic Acid / pharmacology
  • Promoter Regions, Genetic
  • RNA, Messenger / biosynthesis
  • Rats
  • Reactive Oxygen Species / metabolism*
  • Ruthenium Red / pharmacology
  • Signal Transduction
  • Transcription Factors / metabolism
  • Transcriptional Activation*
  • Tumor Cells, Cultured


  • Activating Transcription Factor 2
  • Antioxidants
  • Atf2 protein, rat
  • Calcium Channels
  • Calcium-Binding Proteins
  • Carrier Proteins
  • Chelating Agents
  • Cyclic AMP Response Element-Binding Protein
  • Endoplasmic Reticulum Chaperone BiP
  • Heat-Shock Proteins
  • Molecular Chaperones
  • RNA, Messenger
  • Reactive Oxygen Species
  • Transcription Factors
  • mitochondrial calcium uniporter
  • Ruthenium Red
  • Okadaic Acid
  • Egtazic Acid
  • Hydrogen Peroxide
  • 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
  • Calcium