Possible role for the FosB/JunD AP-1 transcription factor complex in glutamate-mediated excitotoxicity in cultured cerebellar granule cells.
The potent excitatory and neurotoxic actions of glutamate are known to influence the expression of a variety of genes, including those encoding the AP-1 transcription factor, which comprises proteins belonging to the Fos and Jun families. However, the precise role of Fos- and Jun-like transcription factors in these events remains elusive. Here we demonstrate, using primary cultures of mouse brain cerebellar granule cells as an in vitro model system, a possible involvement of the FosB/JunD heterodimer in excitotoxicity. Granule cells were grown for either 2 or 7 days in vitro (DIV) before exposure to varying concentrations (1-3000 microM) of the excitotoxin glutamate. In 7-DIV cells, glutamate induced a concentration-dependent neuronal death, whereas, in 2-DIV cells, no glutamate-induced neuronal damage was seen. We were particularly interested in comparing the protein composition of the AP-1 transcription factor complex in cells exposed to excitotoxic and to nontoxic conditions. AP-1 DNA binding activity was demonstrated by gel shift analysis in nuclear extracts derived from 7-DIV cells following exposure to either a nontoxic (10 microM) or an excitotoxic (250 microM) dose of glutamate and was similarly observed in extracts of 2-DIV cells exposed to the same levels of glutamate. Gel supershift analysis using antibodies against the different Fos and Jun family members allowed differentiation between AP-1 DNA binding in nuclear extracts as a function of both 1) viability status and 2) the stage of development. Of major significance was the finding that FosB could be detected as a component of AP-1 in 7-DIV cells only under excitotoxic conditions, whereas c-Fos, Fra-2, and JunD proteins were detectable under both excitotoxic and nontoxic conditions in cells of this age. In 2-DIV cells (in which glutamate is nontoxic), AP-1 comprised combinations of only Fra-1, Fra-2, c-Jun, and JunD. Because Fos family members are unable to form homodimers, this finding raises the possibility that the FosB/JunD heterodimer may have special significance in the mechanism of excitotoxic neuronal death.
Lidwell
K
K
School of Biology, University of St. Andrews, Fife, Scotland, United Kingdom.
Griffiths
R
R
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002455:Cell Division; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000378:metabolism* / Q000633:toxicity; D007424:Intracellular Fluid / Q000378:metabolism; D051379:Mice; D009474:Neurons / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism*
D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D018698:Glutamic Acid; D004247:DNA; D002118:Calcium