Retrovirally expressed metal response element-binding transcription factor-1 normalizes metallothionein-1 gene expression and protects cells against zinc, but not cadmium, toxicity.
Metal response element (MRE) transcription factor-1 (MTF1), a member of the Cys2-His2 class of zinc-finger transcription factors, is best known for its robust transcriptional regulation of mammalian metallothionein (MT) genes. MTF1 is also believed to play a generalized role in regulating genes involved in protection against heavy metals and oxidative stress. MTF1 binding to MRE motifs is regulated by changes in intracellular zinc (Zn(2+)) concentration. Molecular dissection of MTF1 has been hindered by its high constitutive trans-activity following transient transfection and the failure of these systems to examine genes packaged in native chromatin. In developing a system to avoid these problems, we employed a high-efficiency retroviral transduction system to reintroduce MTF1 into mouse Mtf1(-/-) knockout cells (dko7). Electrophoretic mobility shift assays demonstrated that MTF1 retrovirally transduced dko7 cells (MTF1dko7) possess levels of inducible MTF1-MRE binding activity similar to that seen in mouse hepatoma Hepa-1 cells, and MTF1 binding could be modulated over a 20-fold range by varying the concentration of Zn(2+) present in the culture medium. The dko7 cells exhibited no change in Mt1 gene expression upon Zn(2+) or cadmium (Cd(2+)) treatment; in contrast, in MTF1dko7 cells, Zn(2+) or Cd(2+) induced MT1 mRNA accumulation in a dose-dependent manner. Interestingly, MTF1dko7 cells showed resistance to Zn(2+) toxicity, but negligible resistance to Cd(2+). Concomitantly, MT1 protein levels in MTF1dko7 cells were inducible to the same degree as that in Hepa-1 cells when treated with Zn(2+), but not with Cd(2+). Together, our studies suggest that MTF1-mediated regulation of gene expression is sufficient to protect cells against Zn(2+) toxicity and may be necessary but not sufficient to protect cells against Cd(2+) toxicity.
Solis
Willy A
WA
Center for Environmental Genetics, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0056, USA.
Childs
Nicole L
NL
Weedon
Michael N
MN
He
Lei
L
Nebert
Daniel W
DW
Dalton
Timothy P
TP
D000818:Animals; D002104:Cadmium / Q000037:antagonists & inhibitors* / Q000633:toxicity*; D002470:Cell Survival / Q000187:drug effects; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins; D004586:Electrophoresis; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D017403:In Situ Hybridization; D008114:Liver Neoplasms, Experimental / Q000235:genetics / Q000378:metabolism; D008668:Metallothionein / Q000096:biosynthesis* / Q000235:genetics*; D051379:Mice; D018345:Mice, Knockout; D010957:Plasmids / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D012190:Retroviridae; D014157:Transcription Factors / Q000096:biosynthesis / Q000235:genetics*; D014161:Transduction, Genetic; D014407:Tumor Cells, Cultured; D015032:Zinc / Q000037:antagonists & inhibitors* / Q000633:toxicity*; D000097809:Transcription Factor MTF-1
D002104:Cadmium; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D008668:Metallothionein; D012333:RNA, Messenger; D014157:Transcription Factors; D015032:Zinc; D000097809:Transcription Factor MTF-1