Heat and Heavy Metal Stress Synergize to Mediate Transcriptional

Synergistic Activation of Metallothionein Promoter by Heat and Heavy Metal Stress

1

Heat and Heavy Metal Stress Synergize to Mediate

Transcriptional Hyperactivation by Metal-Responsive

Transcription Factor MTF-1

Nurten Saydam, Florian Steiner, Oleg Georgiev and Walter Schaffner*

Institute of Molecular Biology, University of Zurich, Winterthurerstr. 190,

CH-8057 Zurich, Switzerland

*Corresponding author:

Dr. Walter Schaffner

Institute of Molecular Biology, University of Zurich Winterthurerstr. 190

CH-8057 Zurich, Switzerland

phone : + 41 1 6353150/51

Fax : + 41 1 6356811

E-Mail: [email protected]

Key Words: MTF-1, metallothionein, heat shock, heavy metal stress

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on June 12, 2003 as Manuscript M302138200 by guest on A

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Summary

Mammalian cells react to heavy metal stress by transcribing a number

of genes that contain metal-response elements (MREs) in their

promoter/enhancer region; this activation is mediated by metal-responsive

transcription factor-1 (MTF-1). Well-known target genes of MTF-1 are

those encoding metallothioneins, small, cysteine-rich proteins with a high

affinity for heavy metals. The response to heat shock, another cell stress, is

mediated by heat shock transcription factor 1 (HSF1) which activates a

battery of heat shock genes. Little is known about the crosstalk between the

different antistress systems of the cell. Here we report a synergistic

activation of metal-responsive promoters by heavy metal load (zinc or

cadmium) and heat shock. An obvious explanation, cooperativity between

MTF-1 and heat shock transcription factor 1 (HSF1), seems unlikely:

transfected HSF1 boosts the activity of an Hsp70 promoter but hardly affects

an MRE-containing promoter upon exposure to metal and heat shock. A clue

to the mechanism is given by our finding that heat shock leads to

intracellular accumulation of heavy metals. We propose that the known anti-

apoptotic effect of heat shock proteins allows for cell survival in spite of

heavy metal accumulation, and consequently results in a hyperactivation of

the metal response pathway.

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Introduction

The mammalian metal-responsive transcription factor-1 (MTF-1) is a

zinc finger transcription factor that regulates the transcription of target genes

in response to heavy metals (1-3). The best characterized target genes of

MTF-1 are those encoding metallothioneins (MTs), a family of small,

cysteine-rich metal-binding proteins with roles in heavy metal detoxification

and homeostasis of heavy metals, radical scavenging, and maintenance of

cellular redox state (4-7). The expression of metallothioneins can be induced

by a variety of physiological and environmental stresses such as heavy

metals, oxidizing agents, hypoxia, phorbol esters, ultraviolet and ionizing

radiation, glucocorticoid hormones and infectious agents.

MTF-1 activates metallothionein gene expression through MRE

(metal response element) sequences of core consensus TGCRCNC; multiple

MREs are present in the promoter regions of MT-I and MT-II genes (1,8).

In resting cells, MTF-1 resides in the cytoplasm, and translocates to the

nucleus in response to heavy metal exposure (9-11). Recently, we have

shown that this translocation occurs not only after heavy metal stress but

also after heat shock (11).

Heat is another important stress condition which elevates, through

HSF1, transcription of the genes for heat shock proteins (Hsps) which can


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maintain/restore cellular protein functions via chaperone activity (12-15).

The expression of heat shock proteins is also associated with pathological

states, including inflammation, fever, infection, ischemia, and cancer (16-

18). Moreover, some Hsps, notably Hsp70, have been shown to protect cells

against apoptosis (19-22).

In yeast cells, the CUP1 copper metallothionein gene is activated not

only by excess copper but also by heat shock through the HSE sequences in

the promoter (23,24). Conversely, in several species, cadmium induces heat

shock genes as well as metallothionein genes (25-27). In mammalian cells,

heat shock-induced nuclear translocation of MTF-1 has been shown to be

insufficient to activate transcription from a metallothionein gene promoter

(11). Consistent with this finding, we could not find any heat shock element

(HSE) consensus sequence in the mouse metallothionein gene promoter.

In this study, we report that a metallothionein promoter is

hyperactivated in an MTF-1 dependent manner by a combined exposure to

heat and heavy metals. This induction is associated with an accumulation of

heavy metals in the cell during heat exposure. Unlike a similar

hyperactivation of the heat shock 70 gene promoter by heat and cadmium

where heat shock transcription factor HSF-1 plays a crucial role, HSF-1 is

apparently not required for metallothionein promoter activation.


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Materials and Methods

Transient Transfections and Luciferase Assays

HEK293 (Adenovirus-transformed human embryonic kidney) cells and Hela

(cervix carcinoma) cells were used for transient transfections. Reporter

genes consisted of the firefly luciferase coding sequence driven either by a

mouse metallothionein I promoter, a synthetic 4xMREd/TATA box

promoter, or by the mouse Hsp70 promoter (kindly provided by Olivier

Bensaude). References were ß-galactosidase (CMV-LacZ) or renilla

luciferase (pRL-CMV) genes under the control of the ubiquitously active

CMV promoter. Reporter and reference genes were transfected into cells by

the calcium phosphate method (28). 36 h post-transfection, cells were

exposed to heat shock at 43 oC during the indicated time periods. Heavy

metals, H2O2, serum, low pH (6.0) and cycloheximide were administered to

the culture either during heat shock at 43 oC or at 37 oC as indicated. After

heat treatments, cells were transferred to 37 oC and incubated for another 3 h

to allow for the recovery of the reporter protein, luciferase (29). Cells were

harvested and analysed by measuring luciferase activities according to

Promega’s instruction. Firefly luciferase units were normalised to either ß-

galactosidase values or renilla luciferase values (30).


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Transcript Analysis by S1 Mapping

S1 nuclease mapping of transcripts was performed according to the standard

procedure (31,32). OVEC-4xMREd promoter or OVEC-mMT-I promoter

(10 µg/100mm plate) and OVEC-reference driven by the CMV promoter (1

µg/100mm plate) were transfected into 293 cells. Cells were treated with

zinc, cadmium, heat or heavy metal/heat shock combinations for the

indicated times. Cells were then harvested and analysed for the transcripts.

Western Blot

HEK293 cells were transfected with a VSV tagged MTF-1 expression vector

(11), and 36 h posttransfection cells were treated with either zinc (100 µM),

cadmium (60 µM) and/or heat (42°C, 1.5 h). After a 3 h recovery period of

cells at 37°C, nuclear extracts were prepared as described (33). 30 µg of

nuclear extracts were loaded onto an SDS-polyacrylamide gel (7.5%) and

transferred to polyvinylidene difluoride membranes (Amersham Pharmacia

Biotech) using Transblot-SD Semi-dry Transfer Cell (Bio-Rad). Before

blocking, the membrane was stained with Ponceau to verify the amount of

proteins loaded into each well. Before incubation with the primary antibody,

the membrane was incubated in blocking buffer (5% non-fat milk, 0.5%

Tween 20 in 1 x PBS) for 1 h at room temperature. The membrane was then

incubated with mouse anti-VSV antibody (1:10,000, Sigma) for 1 h at room


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temperature. After washing three times for 10 min each with washing buffer

(0.5% Tween 20 in 1 x PBS), the membrane was incubated for 1 h at room

temperature with the secondary antibody, anti-mouse horseradish

peroxidase-labeled (1:10,000, Vector Laboratories) in a buffer containing

2.5% non-fat milk, 0.5% Tween 20 in 1 x PBS. After three washes of 15 min

each, the horseradish peroxidase signal was detected using ECL Plus

(Amersham Pharmacia Biotech) following the manufacturer's instructions.

Heavy Metal Determination in cell extracts

HEK293 cells were treated with 100µM zinc or 60µM cadmium (final

concentration in DMEM culture medium containing 5% fetal calf serum),

and left either at 37°C or 42°C for one hour. The cells were scraped off with

5 ml PBS suprapur (137mM NaCl, 2.7mM KCl, 10mM Na2HPO4, 2mM

KH2PO4). A total of 1ml cell suspension was diluted with 1ml 65% HNO3.

All samples were microwave digested (MLS Ethos 900, MLS, Leutkirch,

Germany) at 210°C and subsequently diluted with Milli Q water up to 10ml

before inductively coupled plasma mass spectrometry (ICP-MS) analysis.

For analysis of metal concentrations ICP-MS was performed using a

HP4500 Series 300 ShieldTorch System instrument (Agilent, Waldbronn,

Germany) in peak-hopping mode with a spacing at 0.05 amu, 3 points/peak,

3 scans/sample, and an integration time of 300 ms/point. The rate of plasma


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flow was 15.5 L/min with an auxiliary flow of 1.0 L/min. The RF power was

1.18 kW. The samples were introduced using a cross-flow nebulizer at a

flow rate of 1.02 L/min. The apparatus was calibrated using a 6.5% HNO3

multielement solution containing Cu (1.95, 3.9, 19.5, 39, 97.5 and 195 ppb),

Zn (2, 4, 20, 40, 100 and 200 ppb) and Cd (1.82, 3.64, 18.2, 36.4, 91 and

182 ppb) with 103Rh, the internal standard for all isotopes of Cu, Zn, and Cd.


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Results

The transport of MTF-1 to the nucleus is induced not only by

treatment with zinc and cadmium but also by a number of other stress

conditions (11), which led us to test whether any of these could synergize

with heavy metal induction. A luciferase reporter gene driven by a natural

mouse metallothionein-I promoter or a synthetic metal responsive promoter

with four copies of the MREd sequence (“4xMREd”) was tested in HEK293

cells. While heat shock alone did not induce reporter gene transcription,

strong synergistic activation of the 4xMREd promoter was observed with

zinc (34 fold versus 3 fold with metal alone) and even more pronounced

with cadmium (85 fold versus 1,6 fold) in the synthetic MTF-1-dependent

promoter (Figure 1A). A less pronounced but still impressive synergy was

observed at the natural metallothionein promoter with zinc (8.7x versus

1.9x) and with cadmium (9.1x versus 2.5x) (Figure 1B). By contrast, none of

the other stress conditions, including hydrogen peroxide, low extracellular

pH and cycloheximide, or a high serum concentration, acted in synergy with

heavy metal treatment; in some cases metal inducibility was even reduced

(Figure 1A). The synergistic activation of the MTF-1-dependent 4xMREd

promoter by heat and heavy metal load was also observed in another human

cell line (Hela) (data not shown). To test whether the strong activation


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observed was perhaps a peculiarity of the luciferase reporter system, we

repeated the experiment in HEK293 cells with the established OVEC

reporter that measures RNA levels directly (34). Again there was a clear

synergy of heavy metal and heat shock treatment, particularly evident with

cadmium (8 fold instead of 1.8 fold with zinc, and 52 fold instead of 1.4 fold

with cadmium; Figure 2A). One possible explanation for this boost of

activity might have been an increased accumulation of MTF-1 in the nucleus

upon a combined heat/metal treatment. This was however not the case; the

amount of MTF-1 that became associated with nuclear structures was the

same with both metals irrespective of heat shock (Figure 2B).

Next we considered the possibility that the heat shock factor 1

(HSF1), the major transcription factor involved in the heat shock response,

was directly or indirectly participating in this synergy effect. HSF1 is

conserved from yeast to humans and binds to so called heat shock elements

(HSEs) of consensus sequence nGAAnnTTCn (35-38). Unlike the situation

in yeast, where heat shock activates metallothionein (CUP1) expression via a

heat shock response element, there are no obvious binding sites for HSF-1 in

the mouse metallothionein-1 promoter, let alone the synthetic 4xMREd

promoter; however, an indirect effect seemed also possible, e.g., with a

cofactor-like binding of HSF-1 to MTF-1, rather than to DNA. To test this


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possibility, we cotransfected an expression plasmid for HSF1 together with

the 4xMREd promoter. The effect of HSF1 on this promoter was at most

marginal (Figure 3). By contrast, activity of an Hsp70 promoter was strongly

boosted by the combination of HSF1 and heavy metal (Figure 4). (The poor

inducibility by heat without transfected HSF1 can be explained by the

relatively high basal activity of the Hsp70 promoter in HEK293 cells) (39).

From this data it still seems possible that if not HSF-1 itself, at least

some heat shock proteins might be involved in the hyperactivation of the

4xMREd promoter. Chaperones are known to facilitate formation of some

transcriptional regulatory complexes, which in turn activate or repress

transcription from certain promoters (40-45). Firstly, to test if Hsp90 is

involved in the transcriptional activation of MTF-1, we treated HepG2 cells

with geldanamycin, which specifically blocks the ATP binding cassette of

Hsp90 and thereby abolishes its chaperone activity (46). In our case,

however, neither the transcriptional activity nor the nuclear translocation of

MTF-1 was affected by geldanamycin (data not shown).

In contrast a role in assisting the formation of transcription

complexes, recent studies by Freeman and Yamamoto indicate that some

chaperones, notably p23 and Hsp90, can interfere with transcription by

disassambling regulatory complexes; this was revealed by tethering


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chaperone-Gal4 fusion proteins to a reporter promoter (47). Along this vein

of thought, it could be argued that upon heat shock, such interfering hsps are

titrated by unfolded proteins which would boost the transcriptional response

of some promoters. To test whether the molecular chaperones p23, Hsp90

and Hsp70 mediate disassembly of the MTF-1 containing transcriptional

complexes, each of these proteins was fused to the DNA binding domain of

Gal4 and tested on a luciferase gene driven by three Gal4 binding sites

followed by four metal response elements (MREd). However, neither Gal4-

p23, Gal4-Hsp90 nor Gal4-Hsp70 significantly affected basal or heat/heavy

metal stimulated levels of transcription from this reporter gene (data not

shown).

Since we could not observe any involvement of the key proteins of the

heat shock response in the synergistic activation of MTF-1, we considered

the possibility that MTF-1 was the principal effector of transcriptional

activation, while heat shock somehow altered the cellular handling of heavy

metals. This indeed appears to be the case, as determination of zinc and

cadmium concentrations in HEK293 cells shows that heat stimulates the

intracellular accumulation of these metals (Figure 5).


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Discussion

Recent work from our laboratory pointed to a relationship between

MTF-1 and heat shock stress: not only heavy metals, but also heat causes the

translocation of MTF-1 from the cytoplasm to the nucleus. Other conditions

tested such as low pH (6.0), H2O2 and high serum concentration, were also

found to induce, at least to some extent, this translocation. However, none of

these other conditions was able per se to activate MTF-1-dependent

transcription under our assay conditions (11). Here we show that heat shock

is ineffective by itself, but heat and heavy metals can synergize to

hyperactivate metal-inducible promoters.

A straightforward explanation for this effect, cooperative action of

MTF-1 and heat shock transcription factor (HSF1), was ruled out. Unlike the

mouse Hsp70 promoter, which is also synergistically activated by heat and

cadmium and where transfected HSF1 potentiates the effect, the presence or

absence of HSF1 had no influence on the 4xMREd promoter.

Since zinc is the main physiological inducer of MTF-1 in mammalian

cells, one could simply postulate that heat shock triggers a zinc release from

cellular zinc stores, which in turn binds to MTF-1 allowing it to activate its

target genes. However, zinc liberated by heat shock alone would be expected

to activate MTF-1, which is not the case: transcription is only induced when


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exogenous zinc or cadmium is provided during heat shock. Interestingly,

relatively low concentrations of these heavy metals, which by themselves

hardly induce transcription, are sufficient for synergistic activation.

We find that the amount of MTF-1 associated with nuclear structures

is not increased by heat/metal treatment vs. metal alone (Figure 2B).

Alterations in the phosphorylation state of MTF-1 have been invoked in the

process of transcriptional induction (48,49). It is unknown at present

whether heat treatment results in additional modifications of MTF-1 and/or

of a putative co-factor that remains to be identified.

Taken together, the hyperactivation of the metallothionein promoter

by heat and metal is best explained by the propensity of cells to accumulate

heavy metal under heat shock. Indeed we find that at a given heavy metal

concentration in the culture medium, heat shock resulted in 2.5 fold and 2.3

fold higher intracellular concentrations of zinc and cadmium, respectively.

However, our attempts to achieve the same high level of activation by

merely increasing extracellular heavy metal concentration at 37oC were

unsuccessful, invariably resulting in massive cell death (not shown). This

finding of course raises the question why the cells did not die in the

combination of metal and heat shock. It is well-documented that heat shock

proteins, notably Hsp70, exert a strong anti-apoptotic effect (13). Therefore,


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we propose that heat treatment not only results in heavy metal accumulation

but also allows for survival at intracellular metal concentrations that could

not be tolerated otherwise. While cadmium is not a physiological trace

element, zinc is essential for proper functioning of the immune system (50),

and it is tempting to speculate that fever, a natural heat shock condition (51),

contributes to the defense against infectious agents by promoting zinc

uptake, among other effects. Furthermore, upon infection stress, an increased

production of MT (52-57) might also protect cells against the reactive

oxygen species produced by the innate immune system.


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Acknowledgements

We are indebted to Dr. Gerd Multhaup and Andreas Simons (Heidelberg) for

metal determinations. We also thank Drs. Olivier Bensaude (Paris) for the

Hsp70 promoter construct, Didier Picard (Geneva) for Hsp90 expression

vector and anti-Hsp90 antibody, Vincenzo Zimarino (Milano) for HSF1

expression vector, Ivor J. Benjamin (Dallas) for HSF1 knockout cells, Brian

C. Freeman and Keith R. Yamamoto (San Francisco) for the Gal4-p23,

Gal4-Hsp90 and Gal4-Hsp70 expression vectors, Fritz Ochsenbein for

preparing the figures, and Jason Kinchen for critical reading of the

manuscript. This work was supported by the Swiss National Science

Foundation and the Kanton Zürich.


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Figure Legends

Figure 1. Heat/zinc and heat/cadmium combinations activate the

4xMREd promoter synergistically.

HEK293 cells were transfected with either 4xMREd-Luc and CMV-LacZ

(A) or mMTI-Luc and pRL-CMV (B) as reporter and reference genes,

respectively. 16 h after transfection, cells were washed and maintained in

5% FCS-DMEM. 24 h later, media were changed to 0.5% BSA-DMEM and

cells were incubated for another 24 h. As indicated below the graph, cells

were treated with heat (43 oC, 1.5 h), H2O2 (500 µM), serum (10% dialysed

FCS), low pH (6.0) or cycloheximide (CHX) (10 µg/ml) in the presence or

absence of either 100 µM zinc chloride, 60 µM cadmium chloride, or 500

µM copper sulphate (for further details see Materials and Methods). The

cells exposed to heat were transferred to 37 oC for another 3 h to allow for

their recovery. Cells were then collected and processed for luciferase

activity. The basal level was taken as 1 in order to calculate the fold

activation.


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Figure 2. Synthetic metal responsive promoter (4xMREd) is activated

by heat shock/heavy metal treatment at the transcriptional level.

4xMREd-OVEC and CMV-OVEC-ref constructs were transfected into 293

cells. 36 h after transfection, cells were simultaneously treated with heavy

metals and heat (43 oC) as indicated in the concentrations and time course.

Cells were then collected and transcripts analysed by S1 nuclease mapping.

ct, correct transcripts of reporter gene; ref, reference gene transcripts. The

basal level was taken as 1 in order to calculate the fold activation (A). 293

cells transfected with a VSV tagged MTF-1 expression vector were treated

with heat (42°C, 1.5 h) and heavy metals as above. After 3 h recovery at

37°C, nuclear extacts were prepared and Western blot was performed. VSV

tagged MTF-1 was detected by anti-VSV antibody (B). by guest on April 1, 2018

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Figure 3. HSF1 has no effect on the activation of 4xMREd promoter in

Hela cells.

Hela cells were transfected with the reporter gene 4xMREd-Luc, the

reference gene CMV-LacZ and the mouse HSF1 (heat shock transcription

factor-1) expression vector. 36 h after transfection, cells were exposed to

heat shock at 43 oC for 3h in the presence or absence of heavy metals, 100

µM zinc chloride or 60 µM cadmium chloride. 3 h after recovery at 37 oC,

cells were harvested and luciferase activities were measured. The basal level

was taken as 1 in order to calculate the fold activation.


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Figure 4. Expression of the Hsp70 promoter by cadmium and heat in

presence or absence of HSF1.

HEK293 cells were transfected with the Hsp70-Luc promoter-reporter

construct, the CMV-LacZ reference construct and the mouse HSF1

expression vector. 36 h after transfection, cells were treated with 100 µM

zinc chloride or 60 µM cadmium chloride with or without heat shock at 43

oC for 1 h. Cells were collected and reporter gene activities determined by

luciferase assay. The basal level was taken as 1 in order to calculate the fold

activation.


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Figure 5. Cellular accumulation of zinc and cadmium is boosted by heat

shock.

After addition of zinc and cadmium to a final concentration of 100µM and

60µM, respectively, with or without heat shock (42 °C for one hour),

HEK293 cells were harvested and analysed by ICN-MS. The data from the

three independent determinations are shown.


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Fig.1


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Fig.2


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Fig.3


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01 2 3 4 5 6 7 8 9 10 11

1

Fol

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Zinc (100µM)

Cadmium (60µM)

mHSF-1 (2µg)

Heat Shock (43°C, 1h)

2

3

4

5

6

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8

Fig.4


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Fig.5


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Nurten Saydam, Florian Steiner, Oleg Georgiev and Walter Schaffnerby metal-responsive transcription factor MTF-1›

Heat and heavy metal stress synergize to mediate transcriptional hyperactivation

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Heat and Heavy Metal Stress Synergize to Mediate Transcriptional