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. 2005 Oct 12;33(18):5715-27.
doi: 10.1093/nar/gki881. Print 2005.

Two major branches of anti-cadmium defense in the mouse: MTF-1/metallothioneins and glutathione

Ursula Wimmer  1 , Ying Wang, Oleg Georgiev, Walter Schaffner
Affiliations

Two major branches of anti-cadmium defense in the mouse: MTF-1/metallothioneins and glutathione

Ursula Wimmer et al. Nucleic Acids Res. .

Abstract

Metal-responsive transcription factor 1 (MTF-1) regulates expression of its target genes in response to various stress conditions, notably heavy metal load, via binding to metal response elements (MREs) in the respective enhancer/promoter regions. Furthermore, it serves a vital function in embryonic liver development. However, targeted deletion of Mtf1 in the liver after birth is no longer lethal. For this study, Mtf1 conditional knockout mice and control littermates were both mock- or cadmium-treated and liver-specific transcription was analyzed. Besides the well-characterized metallothionein genes, several new MTF-1 target genes with MRE motifs in the promoter region emerged. MTF-1 is required for the basal expression of selenoprotein W, muscle 1 gene (Sepw1) that encodes a glutathione-binding and putative antioxidant protein, supporting a role of MTF-1 in the oxidative stress response. Furthermore, MTF-1 mediates the cadmium-induced expression of N-myc downstream regulated gene 1 (Ndrg1), which is induced by several stress conditions and is overexpressed in many cancers. MTF-1 is also involved in the cadmium response of cysteine- and glycine-rich protein 1 gene (Csrp1), which is implicated in cytoskeletal organization. In contrast, MTF-1 represses the basal expression of Slc39a10, a putative zinc transporter. In a pathway independent of MTF-1, cadmium also induced the transcription of genes involved in the synthesis and regeneration of glutathione, a cadmium-binding antioxidant. These data provide strong evidence for two major branches of cellular anti-cadmium defense, one via MTF-1 and its target genes, notably metallothioneins, the other via glutathione, with an apparent overlap in selenoprotein W.

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Figures

Figure 1
Figure 1
Deletion of Mtf1 in adult mouse liver. (a) Generation of Mtf1 conditional knockout mice. The targeted allele was obtained by homologous recombination of wild-type (wt) allele and targeting vector in ES cells. Removal of the neomycin cassette (NEO) by Cre recombinase led to the conditional knockout allele Mtf1loxP. Conditional Cre-mediated deletion of exons 3 and 4 (Mtf1Δ) results in loss of function via loss of an essential part of the DNA-binding domain and the generation of a new stop codon right after exon 2. Exons 3 to 7 of Mtf1 are indicated by grey boxes, loxP sites by black triangles. TK, thymidine kinase cassette. Restriction enzymes: San, SanDI; B, BbvCI; S, SrfI; H, HpaI. The HpaI site indicated by the crossed H was lost during the cloning procedure for the targeting vector. (b) RT–PCRs with total liver RNA from pI–pC-induced male Mtf1Mx-cre or Mtf1loxP mice. The used primer pair results in products of 589 bp and 218 bp with full-length mRNA and mRNA without exons 3 and 4, respectively. (c) EMSA with liver protein extract of a pI–pC-induced male Mtf1Mx-cre or Mtf1loxP mouse. MTF-1 protein–DNA complex formation was tested with 32P-labeled MRE consensus oligonucleotide MRE-s. Specificity of binding was verified with excess of unlabeled competitor MREd or unrelated Gal4 oligonucleotide; Sp1 bandshifts with 32P-labeled Sp1 consensus oligonucleotide were included as a loading control.
Figure 2
Figure 2
Sepw1 basal expression depends on MTF-1. (a) Semiquantitative RT–PCRs for Sepw1 mRNA using total liver RNA from pI–pC-induced male Mtf1Mx-cre or Mtf1loxP mice. The animals had obtained either mock s.c. injections (−Cd) or s.c. injections with 20 µmol/kg body weight CdSO4 (+Cd) 6 h before sacrificing them. RT–PCRs for Hprt mRNA were used as internal control to adjust the amount of total RNA used. (b) S1 analysis for Sepw1 mRNA with RNA described in (a), using a 32P-labeled Sepw1 S1 probe. A 32P-labeled S1 probe for Hprt mRNA was used to adjust the amount of RNA used. (c) MRE core consensus sequences TGCRCNC (bold letters) and flanking sequences found in a region of 1000 bp upstream from Sepw1 transcription start; the position of each core sequence is indicated. (d) EMSA with liver protein extracts of a male Mtf1loxP or a pI–pC-induced, male Mtf1Mx-cre mouse, both mock-treated. MTF-1 protein–DNA complex formation was tested with 32P-labeled Sepw1 MRE1 or MRE2 oligonucleotide, respectively. Specificity of binding was verified with excess of unlabeled competitor MREd or unrelated Gal4 oligonucleotide. 32P-labeled MRE-s was included to indicate the position of an MTF-1-DNA complex; bandshifts for the common transcription factor Sp1 with 32P-labeled Sp1 consensus oligonucleotide were obtained as protein loading control.
Figure 3
Figure 3
Cadmium response of Ndrg1 depends on MTF-1. (a) Semiquantitative RT–PCRs for Ndrg1 mRNA using total liver RNA from pI–pC-induced male Mtf1Mx-cre or Mtf1loxP mice. The animals had obtained either mock s.c. injections (−Cd) or s.c. injections with 20 µmol/kg body weight CdSO4 (+Cd) 6 h before sacrificing them. RT–PCRs for Hprt mRNA were used as internal control to adjust the amount of total RNA used. (b) MRE core consensus sequences TGCRCNC (bold letters) and flanking sequences found in a region of 1000 bp upstream from Ndrg1 transcription start; the position of each core sequence is indicated. (c) EMSA with liver protein extracts of a male Mtf1loxP or a pI–pC-induced, male Mtf1Mx-cre mouse, both mock-treated. MTF-1 protein–DNA complex formation was tested with 32P-labeled Ndrg1 MRE1 or MRE2 oligonucleotide, or a 32P-labeled oligonucleotide including both MRE3 and MRE4 (MRE3,4). Specificity of binding was verified with excess of unlabeled competitor MREd or unrelated Gal4 oligonucleotide. 32P-labeled MRE-s was included to indicate the position of an MTF-1-DNA complex; Sp1 bandshifts with 32P-labeled Sp1 consensus oligonucleotide were obtained as protein loading control.
Figure 4
Figure 4
Cadmium response of Csrp1 depends on MTF-1. (a) Semiquantitative RT–PCRs for Csrp1 mRNA using total liver RNA from pI–pC-induced male Mtf1Mx-cre or Mtf1loxP mice. The animals had obtained either mock s.c. injections (−Cd) or s.c. injections with 20 µmol/kg body weight CdSO4 (+Cd) 6 h before sacrificing them. RT–PCRs for Hprt mRNA were used as internal control to adjust the amount of total RNA used. (b) MRE core consensus sequences TGCRCNC (bold letters) and flanking sequences found in a region of 1000 bp upstream from Csrp1 transcription start; the position of each core sequence is indicated. (c) EMSA with liver protein extracts of a male Mtf1loxP or a pI–pC-induced, male Mtf1Mx-cre mouse, both mock-treated. MTF-1 protein–DNA complex formation was tested with 32P-labeled Csrp1 MRE1, MRE2, MRE3 or MRE4 oligonucleotide, respectively. Specificity of binding was verified with excess of unlabeled competitor MREd or unrelated Gal4 oligonucleotide. 32P-labeled MRE-s was included to indicate the position of an MTF-1–DNA complex; Sp1 bandshifts with 32P-labeled Sp1 consensus oligonucleotide were obtained as protein loading control.
Figure 5
Figure 5
MTF-1 represses basal expression of Slc39a10. (a) Semiquantitative RT–PCRs for Slc39a10 mRNA using total liver RNA from pI–pC-induced male Mtf1Mx-cre or Mtf1loxP mice. The animals had obtained either mock s.c. injections (−Cd) or s.c. injections with 20 µmol/kg body weight CdSO4 (+Cd) 6 h before sacrificing them. RT–PCRs for Hprt mRNA were used as internal control to adjust the amount of total RNA used. (b) MRE core consensus sequences TGCRCNC (bold letters) and flanking sequences found in a region of 1000 bp upstream from Slc39a10 transcription start; the position of each core sequence is indicated. (c) EMSA with liver protein extracts of a male Mtf1loxP or a pI–pC-induced, male Mtf1Mx-cre mouse, both mock-treated. MTF-1 protein–DNA complex formation was tested with 32P-labeled Slc39a10 MRE1 or MRE2 oligonucleotide, respectively. Specificity of binding was verified with excess of unlabeled competitor MREd or unrelated Gal4 oligonucleotide. 32P-labeled MRE-s was included to indicate the position of an MTF-1–DNA complex; Sp1 bandshifts with 32P-labeled Sp1 consensus oligonucleotide were obtained as protein loading control.
Figure 6
Figure 6
Cells with reduced glutathione level that also lack MTF-1 are hypersensitive to cadmium. The viability of cells was assessed with the so-called MTT assay. Mouse embryonic fibroblasts with (ckoC) and without (delC19, delC21 and delC23) functional Mtf1 were compared. Cells were pre-incubated in medium containing 0, 5, 10, 25 or 50 µM BSO for 24 h and further exposed to 0, 5, 10 or 20 µM CdCl2 (Cd) in the specified pre-incubation medium for an additional 24 h. Results are expressed as mean values ± SD (n = 3) normalized to the respective value of untreated cells.
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