A molecular technique for detecting the liberation of intracellular zinc in cultured neurons.
We have previously reported that oxidative stimuli liberate Zn(2+) from metalloproteins, a phenomenon that can trigger neuronal cell death. Excessive intracellular Zn(2+) in many cell types triggers the expression of genes that encode metal binding proteins, such as metallothionein, via the activation and nuclear translocation of metal response element (MRE)-binding transcription factor-1 (MTF-1). Cd(2+) strongly induces nuclear translocation of MTF-1 in non-neuronal cells, but it does so by displacing Zn(2+) from its metal binding sites within the cell and increasing the intracellular concentration of this ion. Here, we describe the use of MRE-driven expression of a luciferase reporter gene as a sensitive molecular assay for detecting increases in intracellular zinc concentrations. MRE transactivation was induced in primary cortical neurons upon brief exposure to Zn(2+) or Cd(2+). Enhanced MRE transactivation was observed upon co-exposure of neurons to Cd(2+) together with NMDA, as this metal can permeate through the receptor channel. Luciferase expression was observed regardless of whether or not neurons had been co-transfected with an MTF-1-containing plasmid, suggesting the presence of an endogenous MTF-1-like protein. Indeed, RT-PCR revealed that MTF-1 mRNA is present in neurons. In contrast, MTF-1 deficient dko7 cells were only observed to have MRE transactivation when co-transfected with MTF-1. Our results indicate that Cd(2+) can effectively induce transactivation of MRE in neurons by liberating Zn(2+) from its intracellular binding sites.
Hara
Hirokazu
H
Department of Neurobiology, University of Pittsburgh School of Medicine, E1456-BST, Pittsburgh, PA 15261, USA.
Aizenman
Elias
E
D000818:Animals; D002104:Cadmium / Q000494:pharmacology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology*; D004268:DNA-Binding Proteins; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004622:Embryo, Mammalian; D004789:Enzyme Activation / Q000187:drug effects; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017930:Genes, Reporter / Q000502:physiology; D042541:Intracellular Space / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D008959:Models, Neurological; D015336:Molecular Probe Techniques; D016202:N-Methylaspartate / Q000494:pharmacology; D009474:Neurons / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014157:Transcription Factors / Q000378:metabolism; D014633:Valine / Q000031:analogs & derivatives* / Q000494:pharmacology; D015032:Zinc / Q000032:analysis*; D000097809:Transcription Factor MTF-1
D002104:Cadmium; D004268:DNA-Binding Proteins; D018690:Excitatory Amino Acid Agonists; D008156:Luciferases; D016202:N-Methylaspartate; D012333:RNA, Messenger; D014157:Transcription Factors; D014633:Valine; D015032:Zinc; D000097809:Transcription Factor MTF-1; C056639:2-amino-5-phosphopentanoic acid