Activation of estrogen receptor-alpha by the heavy metal cadmium.
Previous studies from this laboratory have shown that the heavy metal cadmium (Cd) mimics the effects of estradiol in estrogen-responsive breast cancer cell lines. To understand the mechanism by which cadmium activates estrogen receptor-alpha (ER-alpha), the ability of cadmium to bind to and activate wild-type and various mutants of ER-alpha was examined. When tested in transient cotransfection assays in COS-1 cells, cadmium concentrations as low as 10(-11) M activated ER-alpha. Scatchard analysis employing either purified human recombinant ER-alpha or extracts from ER-containing MCF-7 cells demonstrated that l09Cd binds to the ER with an equilibrium dissociation constant of approximately 4 to 5 x 10(-10) M. Cadmium also blocks the binding of estradiol to ER-alpha in a noncompetitive manner (K(i) = 2.96 x 10(-10) M), suggesting that the heavy metal interacts with the hormone-binding domain of the receptor. To study the role of the hormone-binding domain in cadmium activation, COS-1 cells were transiently cotransfected with GAL-ER, a chimeric receptor containing the DNA-binding domain of the transcription factor GAL4 and the hormone-binding domain of ER-alpha, and a GAL4-responsive reporter gene. Treatment of the transfected cells with either 10(-6) M cadmium or 10(-9) M estradiol resulted in a 4-fold increase in reporter gene activity. The effect of cadmium on the chimeric receptor was blocked by the antiestrogen, ICI-164,384, suggesting that cadmium activates ER-alpha through an interaction with the hormone-binding domain of the receptor. Transfection and binding assays with ER-alpha mutants identified C381, C447, E523, H524, and D538 as possible interaction sites of cadmium with the hormone-binding domain of ER-alpha.
Stoica
A
A
Department of Biochemistry and Molecular Biology, Vincent T. Lombardi Cancer Center, Georgetown University, Washington, DC 20007, USA.
Katzenellenbogen
B S
BS
Martin
M B
MB
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001943:Breast Neoplasms; D019556:COS Cells; D002104:Cadmium / Q000378:metabolism / Q000494:pharmacology*; D004268:DNA-Binding Proteins; D004958:Estradiol / Q000378:metabolism; D047628:Estrogen Receptor alpha; D005260:Female; D005656:Fungal Proteins / Q000235:genetics; D017930:Genes, Reporter; D006801:Humans; D009154:Mutation; D011960:Receptors, Estrogen / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D047628:Estrogen Receptor alpha; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D002104:Cadmium; D004958:Estradiol