Abstract
Metal response element-binding transcription factor-1 (MTF-1) is a six-zinc finger protein that plays an essential role in activating metallothionein expression in response to the heavy metals zinc and cadmium. Low affinity interactions between zinc and specific zinc fingers in MTF-1 reversibly regulate its binding to the metal response elements in the mouse metallothionein-I promoter. This study examined the subcellular distribution and DNA binding activity of MTF-1 in cells treated with zinc or cadmium. Immunoblot analysis of cytosolic and nuclear extracts demonstrated that in untreated cells, about 83% of MTF-1 is found in the cytosolic extracts and is not activated to bind to DNA. In sharp contrast, within 30 min of zinc treatment (100 microM), MTF-1 is detected only in nuclear extracts and is activated to bind to DNA. The activation to bind to DNA and nuclear translocation of MTF-1 occurs in the absence of increased MTF-1 content in the cell. Furthermore, immunocytochemical localization and immunoblotting assays demonstrated that zinc induces the nuclear translocation of MTF-1-FLAG, expressed from the cytomegalovirus promoter in transiently transfected dko7 (MTF-1 double knockout) cells. Immunoblot analysis of cytosolic and nuclear extracts from cadmium-treated cells demonstrated that concentrations of cadmium (10 microM) that actively induce metallothionein gene expression cause only a small increase in the amount of nuclear MTF-1. In contrast, an overtly toxic concentration of cadmium (50 microM) rapidly induced the complete nuclear translocation and activation of DNA binding activity of MTF-1. These studies are consistent with the hypothesis that MTF-1 serves as a zinc sensor that responds to changes in cytosolic free zinc concentrations. In addition, these data suggest that cadmium activation of metallothionein gene expression may be accompanied by only small changes in nuclear MTF-1.
Highlights
Metallothioneins (MT)1 are small cysteine-rich proteins, which play a role in zinc homeostasis, cadmium detoxication, and protection from reactive free radicals [1,2,3,4,5,6]
Recombinant mouse Metal response element-binding transcription factor-1 (MTF-1) synthesized in vitro in a TnT lysate system was used as a positive control (Fig. 1, lane 1), and an extract from dko7 (MTF-1 double knockout) cells was used as a negative control (Fig. 1, lane 2)
The MTF-1 band was absent in extracts from dko7 cells (Fig. 1, lane 2) but was detected in whole cell extracts from mouse Hepa cells (Fig. 1, lane 3) and from dko7 cells transiently transfected with an MTF-1 expression vector
Summary
Metallothioneins (MT)1 are small cysteine-rich proteins, which play a role in zinc homeostasis, cadmium detoxication, and protection from reactive free radicals [1,2,3,4,5,6]. In experiments in which preparation of nuclear and cytosolic extracts was performed, cells were treated with 4 l/well LipofectAMINE, 100 ng/well CMV-MTF-1 expression vector, and 1 g/well SV--gal in 1.2 ml of DMEM. The amount of immunoreactive MTF-1 detected in nuclear extracts increased about 4-fold after zinc treatment of the cells.
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