Activation of mouse metallothionein I promoter by cadmium in human neuroblastoma cells

Abstract

SummaryMetallothioneins (MTs) are cysteine-rich proteins that bind metal ions with high affinity. In all cell types that have been examined, mouse MT-I (mMT-I) and MT-II are up-regulated by zinc (Zn), cadmium (Cd), as well as pro-oxidants (i.e. tert-butyl-hydroquione, tBHQ). The 5’-flanking region of the mMT-I gene contains multiple cis-acting elements that bind the trans-acting factors Sp1, MRE-binding transcription factor-1 (MTF-1) and upstream stimulatory factor (USF). The promoter also includes an antioxidant responsive element (ARE) which overlaps the USF binding site. Transcriptional activation of mMT-I has been extensively studied, however there is little data on regulation of the mMT-I promoter in neuronal cells. Truncations of the mMT-I promoter were inserted into a luciferase (luc) reporter construct. IMR-32 cells were cotransfected with the mMT-I luciferase constructs and a RSV-galactosidase constitutive reporter, and treated with tBHQ (10 μM), Cd (0.5 μM), and Zn (100 μM). The —250 mMT-I-luc, —150 mMT-I-luc, —150 mMT-IΔUSF/ARE-luc (USF/ARE deleted) and MREd5-luc were activated by Cd. Remarkably, these same constructs were not activated by Zn or tBHQ. Electrophoretic mobility shift assays suggest that a functional MTF-1 is not present in the IMR-32 cells. Thus, the absence of MTF-1 could account for the lack of a Zn response. To test this hypothesis, IMR-32 cells were cotransfected with increasing concentrations of a mammalian expression vector for mouse MTF-1 (CMV-mMTF-1) and the -250 mMT-I-luc reporter construct. These conditions regenerated a Zn-mediated induction of the mMT-I reporter gene. Notably, activation this construct by Cd was unaffected by overexpression of mMTF-1. These data suggest that Cd activates the MRE by a MTF-1-independent mechanism in human IMR-32 neuroblastoma cells.