Abstract
Abstract Metallothionein (MT) is a low molecular weight, cystein-rich and metal-binding protein, which plays a role in zinc homeostasis, cadmium detoxification and protection from reactive free radicals. The rapid induction of MT-I and MT-II gene transcription by heavy metals is mediated by metal response elements (MREs) and a protein that binds specifically to MREs and that transactivates MT gene expression has been cloned from mouse and human, and is termed MTF-1 (MRE-binding transcription factor-1). In this study, we characterized the MTF-1 binding activity of CHO CdR cell by EMSA (electrophoretic mobility shift assay). Incubation of CHO CdR cells in medium containing cadmium did not lead to significant increase in the amount of MTF-1 DNA binding activity, whereas zinc led to rapid increase within 15 min. This change was not due to the fluctuation of MTF-1 level as demonstrated by Western blot analysis. The MTF-1 binding activity of untreated cells was increased after the in vitro addition of zinc, but was inhibited by addition of glutathion or cadmium simultaneously. The increase of MTF-1 activity by zinc can be diminished when cells were pretreated with intracellular zinc chelator TPEN. These results imply that zinc play a important role in MTF-1 binding activity. Previous studies have revealed that protein kinase C (PKC) inhibitor, rottlerin or H7 could inhibit MT gene expression induced by heavy metals. In this work we found the MTF-1 binding activity increased. It seems that metallothionein gene expression was uncoupled with the MRE binding activity of MTF-1 in certain situations. We also used MRE×2, which contains two MRE core sequences as a probe in the EMSA. We found that two MTF-1 molecules were able to bind to the MRE×2 after the cells were treated with zinc. To investigate interaction between MT-II promoter and MTF-1 after the cells were treated with heavy metals, we tried to establish a system for in vivo footprinting. However, we were not able to abtain an optimal condition to detect the binding between MTF-1 and MREs.