Differential metal response and regulation of human heavy metal-inducible genes

Abstract

A number of heavy metal-inducible genes have been reported, but their ranges of response to various metal species are not well known. It is also unclear if these genes are regulated through common mechanisms. To answer these questions, we compared induction kinetics of human metal-inducible genes including the MT-IIA (coding for a metallothionein isoform), hsp70 (coding for the 70-kDa heat-shock protein), and c-fos genes in HeLa cells exposed to Zn, Cd, Ag, Hg, Cu(II), Co, or Ni ions. Transcripts from these three genes were increased after exposure to wide ranges of metals, but each gene was unique in its induction kinetics. Generally, induction was observed at lower metal concentrations in the order of MT-IIA, hsp70, and c-fos. These genes also showed differential responses in time course: more rapid induction was observed in the order of c-fos, hsp70, and MT-IIA after exposure to Zn or Cd. Since the metal-responsive element (MRE) and heat shock element (HSE) of the MT-IIA and hsp70 genes, respectively, are thought to be the cis-acting DNA elements that mediate metal response, we compared the properties of proteins that specifically bind to these elements. MRE-binding activity was detected only in the extract from cells exposed to Zn. By contrast, HSE-binding activity was detected in extracts from cells treated with Zn, Cd, Ag, and Cu. The former was also activated by Zn in vitro, while the latter was not. Each of these DNA-binding activities showed no affinity to the recognition sequence of the other. These results demonstrate that the human metal-inducible genes have broad ranges of response to a variety of heavy metals, but suggest that they are probably regulated through independent mechanisms.

Overexpression of Heat Shock Factor 1 Masks the Heavy Metal Response of the Heat Shock Protein 70 (hsp70) Gene Promoter

“…7) It has been confirmed by a supershift analysis that these complexes contain HSF1 (S. Koizumi, unpublished results). To examine the dose-dependent effect of Zn, WCEs were prepared from HeLa cells treated with different concentrations of Zn, and the HSF1/ HSE complexes were detected by EMSA (Fig.…”

“…The Zn dependency of the HSF1/HSE complexes observed in this experiment is closely related to that of hsp70 mRNA expression. 7) However, activation did not occur by the ad- dition of Zn in vitro. The HSF1 complexes were not induced by the addition of 200 µM Zn, which is sufficient for induction in vivo, to the protein-DNA binding reaction (Fig.…”

“…WCEs were prepared as previously described. 7) Western Blot Analysis ---Western blot analysis was performed as previously described. 8) To detect HSF1, a rabbit anti-human HSF1-IgG and a goat anti-rabbit IgG-IgG/peroxidase conjugate (Santa Cruz Biotechnology, Inc., CA, U.S.A.) were used as the primary and secondary antibodies, respectively.…”

“…EMSA and Supershift Assay ---EMSA was performed as previously described. 7) Synthetic 20-bp double-stranded oligonucleotides containing the MREa of the human MT-llA gene and the HSE of the human hsp70 gene 7) were end-labeled with [γ-32 P] ATP (ICN Biomedicals, Inc., CA, U.S.A.) and T4 polynucleotide kinase (Takara Bio, Inc., Shiga, Japan), and used as probes. For supershift assay, a rabbit IgG directed to human HSF1 (Santa Cruz Biotechnology, Inc. or Affinity BioReagents, Inc., CO, U.S.A.) or HSF2 (Santa Cruz Biotechnology, Inc.) was added to the EMSA reaction mixture after binding reaction, and incubated at 25°C for 20 min further before electrophoresis.…”

“…5) With regard to this gene, the heat shock element (HSE) and heat shock transcription factor 1 (HSF1) binding to it were assumed to be involved in the metal regulation. 6,7) No cross-recognition of the target sequences was observed between MTF-1 and HSF1, 7) suggesting that HSF1 mediates metal response through a pathway distinct from the MTF-1/MRE system. In the present study, we approached the mechanism of the heavy metal-induced activation of hsp70 by means of electrophoretic mobility shift assay (EMSA) and transient transcription assay.…”