Abstract
Metal response element-binding transcription factor-1 (MTF-1) binds specifically to metal response elements (MREs) and transactivates metallothionein (MT) gene expression in response to zinc and cadmium. This investigation contrasts the mechanism of mouse MT gene (mMT-I) promoter activation by cadmium and zinc in IMR-32 human neuroblastoma cells to determine whether MTF-1 binding to the MRE is necessary for activation by these metals. Cadmium activated a mMT-1 promoter (-150 base pairs) luciferase reporter 20-25-fold through a MRE-dependent mechanism. In contrast, zinc had little effect on the mMT-1 luciferase reporter. IMR-32 cells lacked MRE binding activity, and treatment with zinc in vitro or in vivo did not generate a MTF-1. MRE complex, suggesting that IMR-32 cells lack functional MTF-1. Overexpression of mMTF-1 regenerated a zinc-mediated induction of the MRE without affecting cadmium activation. Because no other transition metals tested activated the MRE, this effect appeared to be cadmium-specific. These data demonstrate that in IMR-32 human neuroblastoma cells, zinc and cadmium can use independent mechanisms for activation of the mMT-I promoter and cadmium-mediated MRE activation is independent of MTF-1 and zinc.
Highlights
Metallothioneins (MTs)1 constitute a conserved family of cysteine-rich heavy metal binding proteins [1]
The hypothesis that this human neuroblastoma cell line has no functional Metal response elements (MREs)-binding transcription factor-1 (MTF-1) was evaluated by EMSAs on whole cell extracts from IMR-32 cells compared with mouse hepatoma cells (Hepa) cells (Fig. 2)
The MRE Not upstream stimulatory factor (USF)/antioxidant responsive element (ARE) Mediates Induction of the mouse MT-I gene (mMT-I) Promoter by Cadmium—We have recently demonstrated that the USF/ARE from the mMT-I promoter participates in cadmium-mediated induction of mMT-I in Hepa cells and that this composite site does not contribute to induction of mMT-1 by zinc [13]
Summary
Materials—Cadmium chloride, zinc chloride, copper sulfate, sodium arsenite, sodium dichromate, cobalt chloride, nickel chloride, lead acetate, mercuric chloride, manganese chloride, bismuth nitrate, ferric chloride, and phorbol 12-myristate 13-acetate were obtained from Sigma. tert-Butylhydroquinone (tBHQ) was obtained from Acros Organics (Fairlawn, NJ). Materials—Cadmium chloride, zinc chloride, copper sulfate, sodium arsenite, sodium dichromate, cobalt chloride, nickel chloride, lead acetate, mercuric chloride, manganese chloride, bismuth nitrate, ferric chloride, and phorbol 12-myristate 13-acetate were obtained from Sigma. Tert-Butylhydroquinone (tBHQ) was obtained from Acros Organics (Fairlawn, NJ). D-Luciferin was obtained from Analytical Luminescence Laboratories, Inc.
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