Phosphorylation is involved in the activation of metal-regulatory transcription factor 1 in response to metal ions.

We have studied the role of phosphorylation in the activation of metal-regulatory transcription factor-1 (MTF-1) and metallothionein (MT) gene expression. We showed that MTF-1 is phosphorylated in vivo and that zinc stimulates MTF-1 phosphorylation 2-4-fold. Several kinase inhibitors were used to examine the possible involvement of kinase cascades in the activation of MTF-1. Metal-induced MT gene expression was abrogated by protein kinase C (PKC), c-Jun N-terminal kinase (JNK), phosphoinositide 3-kinase, and tyrosine-specific protein kinases inhibitors, as assayed by Northern analysis and by cotransfection experiments using a metal regulatory element-luciferase reporter plasmid. The extracellular signal-activated protein kinase and the p38 kinase cascades did not appear to be essential for the activation of MT gene transcription by metals. By using dominant-negative mutants of PKC, JNK, mitogen-activated kinase kinase 4 (MKK4), and MKK7, we provide further evidence supporting a role for PKC and JNK in the activation of MTF-1 in response to metals. Notably, increased MTF-1 DNA binding in response to zinc and MTF-1 nuclear localization was not inhibited in cells preincubated with the different kinase inhibitors despite strong inhibition of MTF-1-mediated gene expression. This suggests that phosphorylation is essential for MTF-1 transactivation function. We hypothesize that metal-induced phosphorylation of MTF-1 is one of the primary events leading to increased MTF-1 activity. Thus, metal ions such as cadmium could activate MTF-1 and induce MT gene expression by stimulating one or several kinases in the MTF-1 signal transduction pathway.

Characterization of the mouse metal-regulatory-element-binding proteins, metal element protein-1 and metal regulatory transcription factor-1.

Metal activation of metallothionein gene transcription depends mainly on the presence of regulatory DNA sequences termed metal-regulatory elements (MREs) and involves MRE-binding transcription factor-1 (MTF-1) interacting with the MREs in a Zn(2+)-dependent manner. We previously identified and characterized a nuclear protein, termed metal element protein-1 (MEP-1), specifically binding with high affinity to MRE elements. The precise relationship between MTF-1 and MEP-1 was unclear, and to determine whether MEP-1 and MTF-1 were distinct protein species, we performed DNA binding analyses to characterize the binding properties of both proteins. Electrophoretic mobility-shift assays showed that MTF-1, produced in COS cells, produces a slower-migrating band compared with that obtained with purified MEP-1. Using an anti-MTF-1 antibody, we showed that both the MTF-1-MRE and the MEP-1-MRE complexes are supershifted by an anti-MTF-1 antibody, thus demonstrating that MEP-1 is antigenically related to MTF-1. RNase protection analyses carried out with RNA prepared from different tissues and cell lines failed to reveal the presence of MTF-1 splicing variants. This indicates that MEP-1 may be a proteolytic fragment of MTF-1. MTF-1 DNA-binding activity was rapidly activated in vivo by Zn(2+) ions but not by Cd(2+), UV irradiation or PMA, and occurred on ice as well as at 21 degrees C. In control and Zn(2+)-treated cell extracts, DNA-binding activity was not enhanced in vitro following the addition of exogenous Zn(2+) or a preincubation at 37 degrees C. However, recombinant MTF-1 produced in vitro required Zn(2+) activation for DNA binding. Interestingly, treatment of nuclear extracts with calf intestine phosphatase completely abrogated MTF-1 DNA-binding activity, thus suggesting that phosphorylation is involved in the regulation of MTF-1 activity.

Characterization of cis-acting elements in the promoter of the mouse metallothionein-3 gene. Activation of gene expression during neuronal differentiation of P19 embryonal carcinoma cells.

The metallothionein (MT)3 gene is expressed predominantly in the brain and the organs of the reproductive system, and fails to respond to metal ions in vivo. A CTG repeat was proposed to function as a potential repressor element in nonpermissive cells, and a sequence similar to the JC virus silencer element was found to function as a negative element in permissive primary astrocytes. The objective of this study was to characterize further the mechanisms governing cell-type specific MT-3 gene transcription. We searched for a suitable cell line expressing the MT-3 gene to be used for determination of MT-3 promoter tissue specificity, and showed that MT-3 expression is activated during neuroectodermal differentiation of P19 cells induced by retinoic acid to levels similar to those found in whole brain. Deletion of the CTG repeat or of the JC virus silencer did not promote MT-3 promoter activity in nonpermissive cells, or enhance expression in permissive cells. We identified MT-3 promoter sequences interacting with liver and brain nuclear proteins, as assayed by DNase I footprinting analyses and electrophoretic mobility shift assay, and assessed the role of these sequences in the regulation of MT-3 expression by cotransfection experiments. We generated stable transfectants in permissive C6 and nonpermissive NIH-3T3 cells, and analysed the methylation status of the MT-3 gene. These studies show that regulation of tissue-specific MT-3 gene expression does not appear to involve a repressor, and suggest that other mechanisms such as chromatin organization and epigenetic modifications could account for the absence of MT-3 gene transcription in nonpermissive cells.