Analysis of cysteine and histidine residues required for zinc response of the transcription factor human MTF-1.

Metal responsive element (MRE)-binding transcription factor-1 (MTF-1) is a zinc finger (ZF) transcription factor that plays a key role in heavy metal homeostasis by regulating relevant genes in response to metals. MTF-1 is known to be activated by heavy metals such as Zn and Cd, but the mechanism of activation remains unclear. In the present study, Cys and His residues of human MTF-1 (hMTF-1), some of which may be involved in interaction with metals or with each other, were screened for their contribution to Zn-dependent transcription. To avoid poor induction ratios of previous transfection assays, we re-examined experimental conditions to establish an assay able to correctly detect Zn-responsive transcription. Using this assay, a series of Cys and/or His substitution mutants were analyzed over the entire hMTF-1 molecule. In five out of the six ZFs (ZF1 to ZF5), Cys mutations that disrupt the ZF structure abolished response to Zn. Of these, ZF5 was shown for the first time to be essential for Zn-responsive transcription, despite it being unnecessary for Zn-induced DNA binding. These results indicate that Zn activation of hMTF-1 involves an additional process besides induction of DNA binding activity. Our assay also confirmed the importance of Cys in the acidic activation domain, as well as those in the C-terminal Cys cluster, implicated in transcription in other studies. The identified Cys residues might contribute to metal response of hMTF-1 through direct metal binding and/or intramolecular interactions, analysis of which will be helpful in understanding the mechanism of metal response.

Heavy metal response of the heat shock protein 70 gene is mediated by duplicated heat shock elements and heat shock factor 1.

Heavy metals induce transcription of a number of mammalian genes, but in most cases the mechanism of induction has not been well characterized. The human heat shock protein 70 gene (hsp70) is activated by several heavy metals such as Cd and Zn, and the heat shock element (HSE) has been proposed to mediate metal response by previous studies. However, it was observed that the lack of further upstream sequences rendered the hsp70 promoter unresponsive to metals. A detailed deletion analysis of the promoter revealed that the distal HSE-like sequence (dHSE) is required for heavy metal response. A supershift assay showed that the heat shock factor 1 (HSF1) recognized dHSE, as well as the proximal HSE (pHSE). An hsp70 promoter variant with a dHSE mutation that blocks HSF1 binding completely lost metal response, demonstrating that the HSF1/dHSE interaction is crucial. Another promoter variant with a similar pHSE mutation partially lost metal response, indicating that both HSEs are required for full activity. Knockdown of HSF1 dramatically reduced the metal response of hsp70, demonstrating its essential role. Furthermore, a reporter gene regulated by the human hsp70 promoter, which lacked metal response in HSF1-null mouse cells, acquired the response upon over-expression of a recombinant human HSF1. These results demonstrate that the duplicated HSEs and HSF1 constitute the mechanism for the heavy metal response of hsp70, which is distinct from the known metal regulatory system for the metallothionein genes.

A versatile transfection assay system to evaluate the biological effects of diverse industrial chemicals.

Gene expression processes are now recognized as important targets of the toxic effects exerted by industrial chemicals. The transient transfection assay is a powerful tool to evaluate such effects. Thus, we developed a versatile assay system by constructing a basic reporter plasmid in which the regulatory DNA sequence to be studied can easily be substituted. To verify the performance of this system, reporter plasmids carrying any of the three distinct regulatory sequences, estrogen responsive element (ERE), glucocorticoid responsive element (GRE) and xenobiotic responsive element (XRE) were constructed. After transfection of human cells, these plasmids successfully expressed the relevant reporter genes in response to specific inducers, ß-estradiol, dexamethasone and 3-methylcholanthrene, respectively. Several industrial chemicals were assayed using these reporter plasmids, and the ability of p-dimethylaminoazobenzene to elevate GRE- and XRE-mediated transcription was detected. α-Naphthylamine and o-tolidine were also observed to increase the XRE-mediated response. The transfection assay system established here will be useful to evaluate the effects of a wide variety of industrial chemicals.

An improved transfection assay for evaluating the effects of heavy metals.

The transfection assay is an important tool for evaluating the health effects of industrial chemicals, with the reporter gene expression as an indicator. However, heavy metals often influence the expression of the reference plasmids used to correct variations in transfection efficiency between assay plates, reducing the reliability of this assay. We found that the target of heavy metals is the reporter, rather than the promoter used in the reference plasmid. Of the reporters we tested, luciferase (Luc) enzyme activity was affected by heavy metals, whereas gene product levels of the chloramphenicol acetyltransferase (CAT) or beta-galactosidase (betaGal) gene were not. Neither heavy metals nor extracts from cells exposed to heavy metals showed an effect when directly added to the Luc enzyme, suggesting that heavy metals act through an indirect mechanism. These data indicate that the use of CAT or betaGal as a reporter is appropriate for a reliable assay of heavy metal effects.

Cadmium-induced alterations of gene expression in human cells.

We have reported the changes in gene expression in human HeLa cells exposed to a low concentration (5µM) of Cd. In the present study, cells exposed to a higher concentration of Cd were analyzed using a DNA microarray with 9182 human cDNA probes, in an attempt to obtain a comprehensive view on the biological effects of Cd. After a 6h exposure to 50µM Cd, 48 genes were up-regulated 2.5-fold or greater and 14 genes were down-regulated to 40% or less. Marked up-regulation of genes coding for metallothioneins, anti-oxidant proteins, and heat shock proteins was observed. Cd appeared to repress cell proliferation by modulating genes involved in multiple pathways. Cd also affected a number of genes related to apoptosis. Interestingly, it appeared that a series of genes were regulated to accelerate the intrinsic pathway of apoptosis, while others were directed to suppress the extrinsic pathway. Of these, rapid and transient induction of the TR3 gene was noted as a possible key process in Cd-induced apoptosis. Effects on several genes that may reflect mechanistic backgrounds of Cd toxicity were also observed. The present study disclosed a complex pleiotypic response of human cells to Cd, which was composed of a variety of changes in gene expression directed to defense, growth arrest, recovery from damage, apoptosis and so on.

Gene expression profile in human cells exposed to zinc.

Although Zn is an essential trace metal for humans, a comprehensive view of its effects on cellular functions has not been obtained. We used a DNA microarray to assess transcriptional alterations in human HeLa cells after exposure to a moderate concentration of Zn (100 muM ZnSO(4)). Out of 9,182 human genes, expression was increased in 7 genes and decreased in 4 genes twofold or greater. Four of the 7 upregulated genes were those coding for metallothionein isoforms or related proteins. An unexpectedly small extent of changes in gene expression might reflect rapid sequestration of Zn ions by metallothioneins, and the absence of most of the other protective responses indicated the non-toxic nature of Zn at this concentration. Comparison with our previous DNA microarray results for 5 muM CdSO(4)-exposed HeLa cells revealed several genes that are regulated by both metals in parallel, and a gene reciprocally regulated by them.

[Heavy metal responses of the human metallothionein isoform genes].

Metallothioneins (MTs) are proteins known to be involved in defense mechanisms against heavy metals and reactive oxygen species. In human, more than ten MT isoform genes have been identified, in contrast to much fewer isoforms in other mammalian species. The increased number of isoforms in human may have some biological significance; for example, isoforms may have been functionally differentiated to deal with various environmental factors in the evolutional process. However, we know little about the functions of the individual MT isoforms. To clarify functional differences between human MT isoforms, we developed a method to determine individual isoform mRNA levels using real-time polymerase chain reaction (PCR), and studied responses of the isoform genes against heavy metals (Zn, Cd, Cu) and As in HeLa cells. These metals induced all MT isoforms except for MT-1A by Cu, though their induced levels were different. Furthermore, these metals preferentially induced isoforms MT-2A and MT-1X suggesting that these isoforms may be important in protecting from their cytotoxicity.

[Mechanism of metallothionein gene activation mediated by heavy-metal dependent transcription factor MTF-1].

Transcriptional activation of metallothionein (MT) genes by heavy metals is a valuable system for understanding the functions of MT as well as the cellular response against heavy metals. Although it is now known that heavy metal signals culminating in MT induction converge upon a transcription factor MTF-1, the mechanism underlying the MTF-1 response to heavy metals has not been elucidated. To address this issue, we investigated various aspects of the in vivo response of MTF-1 against heavy metals. Chromatin immunoprecipitation assay showed that heavy metal-dependent DNA binding of MTF-1 is the critical step in vivo. MTF-1 is primarily localized in the nucleus so that heavy metal-dependent nuclear translocation demonstrated by other groups does not seem to be universal and hence may not be critical for activation of MTF-1. In the six Zn finger motifs, the hallmark of MTF-1, the third and the fourth fingers are essential for the nuclear localization of MTF-1. Furthermore, all fingers except the last are important for transcriptional activation function of MTF-1, suggesting their key role for MTF-1 function. Also, a cysteine cluster structure located in the C-terminal region of MTF-1 is critical for transactivating function of MTF-1. These results suggest a central role of the Zn-finger domain and intramolecular cooperation through a structural change of MTF-1 for its response to heavy metal challenge.

Cadmium-responsive element of the human heme oxygenase-1 gene mediates heat shock factor 1-dependent transcriptional activation.

Transcription of a number of mammalian genes is activated by heavy metals, but mechanisms of signaling and transcriptional regulation are not well understood. From a comparison of heavy metal responses of several human genes, it was noted that the heme oxygenase-1 (HO-1) gene is quite similar in the spectrum of metal response and induction kinetics to the heat shock protein 70 (HSP70) gene, suggesting a common regulatory mechanism shared by these genes. The cadmium-responsive element (CdRE) known to be responsible for the metal regulation of ho-1 formed complexes with proteins from heavy metal-treated HeLa cells in an electrophoretic mobility shift assay (EMSA). These complexes were indistinguishable in mobility from those formed by the heat shock factor 1 (HSF1) and the heat shock element involved in hsp70 regulation, suggesting the involvement of HSF1 also in the CdRE complexes. Competitive EMSA and supershift analysis with an anti-HSF1 antibody revealed that HSF1 was in fact a component of the CdRE complexes. A fine analysis on the affinity of HSF1 to a series of mutant CdRE sequences showed that HSF1 recognizes a sequence motif TnCTAGA. Transient transfection analysis with overexpressed recombinant HSF1 demonstrated that CdRE has HSF1-dependent enhancer-like activity that requires direct binding of HSF1. In the absence of overexpressed HSF1, however, CdRE by itself was insufficient to mediate heavy metal-induced transcription, suggesting requirement of additional regulatory sequences. The finding that HSF1 is directly involved in the regulation of ho-1 with an anti-oxidative role revealed a new aspect of the biological defense mechanism.

Analysis of human proteins that have an affinity to heavy metals by metal-chelating column chromatography.

To clarify the molecular basis of toxicities of industrial chemicals, it is demanded to develop appropriate methods whereby their cellular target molecules can be directly identified. In the present study, we focused on target proteins of heavy metals and established the method to detect them using a combination of metal-chelating column chromatography and a subsequent analysis by electrophoresis. Protein samples prepared from HeLa cells were applied to the Zn- or Cd-chelating column, and the bound proteins were analyzed by SDS-polyacrylamide gel electrophoresis followed by either silver staining, or fluorography when using radiolabel protein samples. Among several protein species trapped in the columns, a 36-kDa protein apparently has an affinity to both Zn and Cd, indicating the possibility that Cd can exchange essential Zn on this protein. These results suggest that the established method is useful for the target protein screening and further analyses of separated proteins.

Cross talk of heat shock and heavy metal regulatory pathways.

Heavy metals induce transcription of human genes including those coding for metallothionein and heat shock protein 70 (HSP70). It has been suggested that these processes are mediated by metal-activated transcription factors, MTF-1 and HSF1, respectively, and are independent of each other. We raised an antibody against human MTF-1 which efficiently supershifts the protein-DNA complex formed by MTF-1 and its cognate binding sequence, MRE. We discovered that this antibody could also supershift complexes formed by HSF1 and its recognition sequence HSE, which suggested the involvement of MTF-1 in these complexes. This supershift was observed for HSF1/HSE complexes induced by Zn, Cd, Ag, and heat shock. Furthermore, overexpression of MTF-1 in HeLa cells markedly reduced metal-induced transcription from the hsp70-1 gene promoter which depends on HSF1. These data indicate that MTF-1 represses HSF1-mediated transcription probably through a direct protein-protein interaction, suggesting a cross talk of two lines of stress-responsive regulatory pathways.

Gene expression profiles in the liver and kidney of metallothionein-null mice.

It has been reported that the expression of certain genes was altered in rodent cells lacking metallothioneins (MTs). To further explore the effects of MT deficiency, we screened genes differentially expressed in the liver and kidney of MT-null mice by cDNA microarray analysis. In the liver, 29 of 8737 genes analyzed were altered in their expression levels: 19 and 10 genes were up-regulated and down-regulated, respectively. Particularly, 14 of the 29 genes were related to energy metabolism, and some of these suggested that loss of MTs might lead to obesity and irregular ATP synthesis. In the kidney, 41 differentially expressed genes were observed: 27 and 14 genes were up-regulated and down-regulated, respectively. Eleven of the 41 genes were also related to energy metabolism. Microarray results were confirmed by Northern blot analysis for five of the energy metabolism-related genes.

Ultraviolet irradiation increases the sensitivity of cultured human skin cells to cadmium probably through the inhibition of metallothionein gene expression.

We previously developed an apparatus that can irradiate cultured cells with monochromatic ultraviolet (UV) rays to exactly assess the biological effects of UV components on mammalian cells. Using this device, we studied the effects of UV in and near the UVB region on the general as well as specific protein synthesis of the human skin-derived NB1RGB cells. We found that Cd-induced synthesis of metallothioneins (MTs), which are the proteins involved in the protection against heavy metals and oxidative stress, is inhibited by UV at 280 nm more extensively than total protein synthesis. Such an inhibition was observed when MTs were induced by different inducers such as Cd, Zn, and dexamethasone in three human cell lines, indicating that it is not an event specific to a certain inducer or a certain cell type. By contrast, UV at 300 or 320 nm showed only a marginal effect. UV at 280 nm was likely to block MT gene transcription because Cd-induced increase of MT mRNA was strongly inhibited by irradiation. Cd induction of 70-kDa heat shock protein mRNA was also inhibited by UV irradiation, suggesting that the expression of inducible genes are commonly sensitive to UV. Furthermore, we observed that the irradiation of UV at 280 nm renders NB1RGB cells extremely susceptible to Cd, probably due to the reduced MT synthesis. These observations strongly suggest that UV at 280 nm severely damages cellular inducible protective functions, warning us of a new risk of UV exposure.

Application of DNA microarrays in occupational health research.

The profiling of gene expression patterns with DNA microarrays is recently being widely used not only in basic molecular biological studies but also in the practical fields. In clinical application, for example, this technique is expected to be quite useful in making a correct diagnosis. In the pharmacological area, the microarray analysis can be applied to drug discovery and individualized drug treatment. Although not so popular as these examples, DNA microarrays could also be a powerful tool in studies relevant to occupational health. This review will describe the outline of gene expression profiling with DNA microarrays and prospects in occupational health research.

DNA microarray analysis of altered gene expression in cadmium-exposed human cells.

Cadmium (Cd) is a heavy metal known to be toxic and carcinogenic, but its mechanism of action remains to be elucidated. Development of the DNA microarray technology has recently made the comprehensive analysis of gene expression possible, and it could be a powerful tool also in toxicological studies. With microarray slides containing 7,000-9,000 genes, we have been studying the gene expression profiles of a human cell line exposed to Cd. By exposure to a non-lethal concentration of Cd, 46 upregulated and 10 downregulated genes whose expression levels changed twofold or greater were observed. The expression of genes related to cellular protection and damage control mechanisms such as those encoding metallothioneins, anti-oxidant proteins and heat shock proteins was simultaneously induced. In addition, altered expression of many genes involved in signaling, metabolism and so on was newly observed. As a whole, a number of genes appear to be coordinately regulated toward survival from Cd toxicity. When cells were exposed to a higher concentration of Cd, more remarkable effects were observed both in the number of affected genes and in the extent of altered expression. These findings will contribute to the understanding of the complicated biological effects of Cd.

Effect of ultraviolet irradiation on the protein synthesis of human skin cells: a study with a monochromatic ultraviolet irradiation apparatus.

Human beings always suffer from serious risk of exposure to ultraviolet (UV) rays in general as well as in work environments but the health effects of UV are not yet fully understood. To enable fine analysis of the biological effects of UV at any given wavelength, we developed an apparatus that can experimentally irradiate cultured mammalian cells with monochromatic UV rays at any given wavelength. With this device, the effects of UV at 260, 280, 300 and 320 nm on the total protein synthesis of human skin-derived NB1RGB cells were examined. An inhibitory effect of UV at 260 and 280 nm was evident, whereas UV at 300 and 320 nm had only a marginal effect. UV at 280 nm also decreased amino acid uptake, which appears, at least partly, to be responsible for reduced protein synthesis. Metallothioneins (MTs), known as proteins defending against heavy metal toxicity, have been reported to be induced after UV irradiation, but UV at 280 nm did not induce MTs in NB1RGB cells. Cadmium pretreatment of cells, that had been assumed to protect cells from UV damage via MT induction, did not increase cell viability after exposure to UV at 280 nm. The present study unambiguously revealed the biological effects of monochromatic UV rays, also showing that the UV-irradiating apparatus can be a powerful tool for studying the health effects of UV.

DNA microarray analysis of human gene expression induced by a non-lethal dose of cadmium.

Cadmium (Cd) is a hazardous heavy metal present in working and living environments. Cd affects many cellular functions, but little is known about the mechanisms of its toxicity and cellular defense against it. Recently, advanced gene expression analysis employing DNA microarrays provided us the means to profile the expression of thousands of genes simultaneously. We describe here a study of Cd-induced gene expression profile. Messenger RNA was prepared from HeLa cells exposed to a non-lethal dose of CdSO4, and analyzed by the use of an array consisting of 7,075 human cDNAs. Many stress response genes including those coding for metallothioneins and heat shock proteins were observed to be induced by Cd. The cellular metabolism inclined toward the synthesis of cysteine and glutathione after Cd exposure. Anti-oxidant genes also appeared to be induced to protect cell components and to quench reactive oxygen species. Ubiquitin pathway was activated as well probably to degrade proteins which might not be renatured. These data suggest that human cells mobilize every genomic resource (induction of some genes and repression of others) to overcome cytotoxicity caused by Cd.