Chapter 10 Metallothionein: Structure and regulation

Biochemistry and Molecular Biology of Fishes, vol. 6

T. P. Mommsen and T. W. Moon (Editors) 9 2005 Elsevier B.V. All rights reserved.

CHAPTER 10

Metallothionein" Structure and regulation PETER KLING* AND PER-ERIK OLSSON** *Department of Zoology, University of G6teborg SE-405 30, G6teborg, Sweden, and * *Department of Natural Sciences, Section of Biology, University of Orebro SE-701 82, Orebro, Sweden

I. II. III. IV. V. VI. VII. VIII.

Introduction MT structure MT synthesis and regulation Metal regulation of MT synthesis Free radical regulation of MT synthesis Involvement of MT in protection against oxidative stress Summary References

L Introduction Metallothionein (MT) is an ubiquitous cytosolic, cysteine-rich, metal- and free radical-binding protein. Since the discovery of MT four decades ago, extensive efforts have been directed to define the physiological role of MT. The first extensive chemical characterization of MT demonstrated that it contained 5.9% Cd 2+, 2.2% Zn 2+ and 8.5% S (w/w), and that 95% of the S was present as sulfhydryl groups of cysteine residues 4~ The sulfhydryl groups were all found to be involved in cation binding. Aromatic amino acids and histidine were absent and the protein had little or no absorbance at 280 nm. Initially, MT was proposed to protect cells from Cd toxicity 4~ The finding that large amounts of MT were formed in the liver of rabbits in response to repeated doses of Cd supported this suggestion 72. Thus, Piscator proposed that once thionein synthesis had been initiated, the animal develops resistance to Cd, and will consequently tolerate higher doses of this toxic metal 72. At about this time environmental cadmium pollution was identified as a causative factor in the Japanese Itai-itai disease 32. These observations promoted biochemical and chemical research into the toxicological importance of MT production as a defense mechanism against low levels of cadmium exposure. However, it soon became apparent that other metals, zinc and copper in

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particular, could also induce MT synthesis. Currently, the primary function of MT is believed to be the maintenance of homeostasis of these essential elements 35. While the primary physiological role of MT involves the homeostasis of zinc and copper, it remains that MT also has a role in the cellular defense against cadmium and mercury. In addition, being a thiol containing protein MT has the potential to be an effective free radical scavenger, therefore, important in regulating the cellular redox-state. During the processes of transcription, translation and replication, many Zn requiting enzymes are required. This suggests that MT could be involved in the control of development and differentiation 28. Recently, studies have revealed a role for MT in preventing oxidative stress. Disturbances in trace metal metabolism and generation of free radicals is often implicated in the development of pathological states and aging, and MT may be a link in these processes. The present review is focused on the regulation of teleost fish MT by heavy metals and free radicals. The conservation of regulatory mechanisms between fish and mammals is discussed and the unique characteristics of fish MT is highlighted.

II. M T structure The presence of MT in fish was first indicated in the goldfish, Carassius auratus 51. Other teleosts reported to contain MT included the copper rockfish, Sebastodes caurinus; eel, Anguilla anguilla; plaice, Pleuronectes platessa; and flounder, Parophrys v e t u l u s 14'15'23'59. The first characterization of fish MT demonstrated that two isoforms existed in eel liver and included the first preliminary amino acid data 58. It was established that the protein is inducible 59, had an apparent molecular weight close to 7000 D a 49, binds zinc 15, c a d m i u m 52, copper 23 and mercury 14 and had a relatively high cysteine c o n t e n t 58'66. Since then MT has been isolated and characterized from a number of teleosts. The first partial MT amino acid sequence was obtained from plaice 67 and revealed that fish and mammalian MT was closely related. In 1987 the first complete sequencing of a teleost MT was accomplished 13. This demonstrated the presence of two MT isoforms, related to mammalian MT1 and MT2, with a high sequence similarity. The sequences obtained from fish MT show that all fish species so far studied have the cysteines conserved in number and position. Two main differences from mammalian MTs are present (Fig. 1). One is the difference in amino-terminal sequence, the main antigenic epitope, which has been shown to result in low antibody reactivity between distantly related species. The second main difference is the positioning of the cysteine in position 55 in teleosts that is found in position 57 in mammals. The number of cysteines is conserved among fish except for the pupfish (Cyprinodon nevadensis) where an extra cysteine has replaced a serine at position 27 (EMBL accession number X97273). The pupfish inhabits desert streams with high daily fluctuations in water temperature. Studies are presently ongoing to determine the effect of this additional cysteine on MT function.

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III. M T synthesis a n d r e g u l a t i o n MT synthesis in fish and mammals is stimulated by a wide variety of agents, including heavy metals, glucocorticoids, inflammatory substances and oxidative stress 24'29'42'47. MT transcription does not depend on de novo protein synthesis and appears to be mainly regulated at the transcriptional level 4~. The use of MT as an indicator of heavymetal pollution has received a great deal of interest. The relationship between heavy metals and MT was early demonstrated in field s t u d i e s 6 ~ However, although elevated cadmium levels lead to increased MT levels in different tissues, there are also other compounds and circumstances that can regulate the MT levels in fish. MT induction by metals has been shown for fish and fish cell lines 38'91. Other studies have shown that hepatic MT levels are elevated by glucocorticoids, noradrenalin and progesterone 33'38. MT is endogenously regulated during different developmental life stages 62, during sexual maturation 61 and in response to water temperature 64. The use of MT as a biomonitoring tool should be performed with caution due to the multitude of circumstances where MT is endogenously regulated. It has been shown that binding of heavy metals such as Hg and Cd to MT decreases the toxicity of these metals (Fig. 2). Thus toxicity would first occur when the MT pool is saturated with the toxic metals, and the metals start to bind other proteins in the cell. This 'spillover' hypothesis states that toxicological changes coincide with the appearance of toxic metals bound to other proteins than MT 86. If high amounts of heavy metals accumulate, the basal levels of MT would not be sufficient for protection and de novo MT synthesis is required. Therefore, the rate of uptake of the metal and the rate of MT synthesis determine the protection 55. Several studies have shown that

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Fig. 2. MTs role in the subcellular distribution of heavy metals. Following uptake, metals activate MTF-1 to initiate transcription at MREs on MT promoters. Raised apo-thionein levels result in reduced toxicity of the heavy metal. Once the metals are bound to MT there is a decrease in free metals and a subsequent MTF-1 inactivation, and lowered MT gene transcription. When the rate of heavy metal accumulation exceeds that of MT synthesis, toxicity may occur. Binding of metals such as Cd and Hg may impair MTs role in regulating the levels of trace elements such as Zn and Cu, essential for maintaining enzyme activity. Other heavy metal binding proteins aid to protect from toxicity.

low MT expressing cell lines 22'68'69 are sensitive to Cd, whereas, excessive expression of MT confers resistance to Cd 9'43. Moreover, generation of MT-null mice by targeted disruption of MT genes in mice have revealed that those mice become hypersensitive to C d 54'56. These results argue in favor of the spillover hypothesis. However, if the main role of MT is to regulate the cellular levels of Zn and Cu, the binding of Hg and Cd would prevent MT from fulfilling its primary function, therefore even low levels would result in toxic effects. Although Cd and Hg detoxification is a property of MT, this is probably not its primary role. From an evolutionary point of view, it is unlikely that MT was evolved to protect from non-abundant elements. The toxicity of heavy metals such as Cd is more likely to depend on species-specific uptake and other low molecular weight (LMW) heavy metal binding proteins including G S H 7'78.

IV. Metal regulation of MT synthesis The regulatory regions of mammalian and yeast MTs have been studied in detail, but less is known about fish MT promoter regions. A hallmark of MTs is the rapid transactivation through multiple copies of metal responsive elements (MREs) within

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their promoters. Comparison of teleost and mammalian MT promoters reveals a high degree of conservation with respect to the MRE consensus sequence 5 ~ TGC(G/A)CNC 3~ and indicates that the heavy metal response is highly conserved. The rainbow trout MT-B (rtMT-B) gene was the first teleost MT gene to be isolated, and sequencing of that promoter consequently revealed conservation between mammalian and teleost MRE core sequences 9~ A unique feature among teleost MT promoters is the organization of proximal and distal MREs. This has been demonstrated in the rainbow trout 57'63'90, pike (Esox lucius) and stone loach (Noemachelius barbatulus) 45, Sockeye salmon (Oncorhynchus nerka) ~8, Antarctic icefish (Chionodraco hamatus) MTI177 and carp (Cyprinus carpio) (Genebank accession number AF001983). The MREs in the MTI promoter of Antarctic icefish is an exception among fish since the MREs are only localised within the proximal part of the promoter 77. Metal-induction of cells transfected with full length promoters correlated well with the number of MREs present in the MT genes, indicating that both proximal and distal MREs are needed to achieve maximum inducibility of MT transcription 63,65,9~ Isolation of the MRE binding transcription factor (MTF-1) from several mammalian species 16'73, Japanese puffer fish, Fugu rubripes 5 and zebrafish, Danio rerio 19 have revealed a high degree of evolutionary conservation of the heavy metal stress response. MTF-1 belongs to the Cys2-His2 family of transcription factors with separate transcriptional activation domains rich in acidic and proline residues 73. The binding of MTF-1 to MREs is strongly dependent on Zn or agents that mobilize cellular pools of Zn 25'26'69. The observation that cycloheximide treatment of BHK cells mediated MRE driven vector expression, as well as Zn, led to the speculation that MTF-1 may be under the control of an inhibitor (MTI) 69. However, more recent data suggest that MTF-1 is the zinc sensor in the cell with a very low binding affinity for Zn. Deletions within recombinant MTF-1 suggests that the Zn finger domain is involved in Zn-dependent activation, where MTF-1 and its Zn finger 1 acts as a sensor for available free Zn 12'25. The activation of MTF-1 in vitro appears to be exclusively dependent on Zn, although MTF-1 from puffer fish appears to be activated by Cd 5. Interestingly, when comparing the warm and cold-water species such as zebrafish and rainbow trout, it was observed that while the zebrafish contain one MTF-1 isoform, the rainbow trout appears to contain two. While Zn activated one isoform within a wide temperature range, the other isoform was only activated at high temperatures 27. These adapted MTF-1 isoforms of the rainbow trout allows temperature specific MT regulation in response to metals during the summer and winter season. In vivo studies of MTF-1 suggest an important role during development, since MTF-1 knockout mice die in utero from hepatic failure 34. Embryological studies have shown that MTF-1 is strongly and ubiquitously expressed already in the one-cell stage of the zebrafish, whereas MTF-1 is predominantly expressed in the neural parts after hatching, indicating that MTF-1 is important during early development in the zebrafish 19. The MTF-1 transcription factor is also activated in the zebrafish cell line, ZEM2S, in response to metal treatment 17. The activity of MTF-1 in the embryological teleost cell line CHSE-214 has been confirmed in three different generations (5, 34 and 59) of the CHSE-214

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TABLE 1 Comparison of MRE-dependent MTF- 1 activity in three different generations (5, 34 and 59) of the teleost CHSE-214 cell line in response to different concentrations of Zn Zn (/xM) 0 50 100 150 250

CHSE-214:5 3.1 3.6 11 12 1

CHSE-214:34 1.7 2.4 7.1 16 1.2

CHSE-214:59 1.4 1.6 5.8 15 1

CHSE-214: 5, 34 and 59 cells were transfected with a MRE-pEGFP reporter vector prior to the quantitative fluorescent activated cell sorting (FACS) determinations. Relative GFP activity is presented as the mean of six replicas in control or Zn (50, 100 and 150/xM) treated CHSE-214: 5, 34 and 59 cells for 24 h. The GFP activity of CHSE-214:59 Zn (250/xM) exposed cells, which exhibited the lowest activity, was arbitrarily set to 1.0. All other activities were adjusted accordingly.

cell line. In this experiment there was a correlation between high MTF-1 activity (CHSE-214: 59) and low MT expression following high Zn concentrations. The lower MTF-1 activity observed in CHSE-214:5 cells may thus be a consequence of the relatively high levels of MT that can sequester Zn (Table 1). MT was initially considered to be most important for sequestering Cd and thereby important to protect against Cd toxicity. However, more recent data suggest that Zn is the metal primarily sequestered by MT. These novel data were demonstrated in teleost cell lines, which indicate that high MT expression is correlated to Zn resistance but not to Cd resistance 4s. Similar results have also been obtained from studies on mouse, where MTF-1 expression, which is required for normal MT expression, protects against Zn, but not Cd induced toxicity 8~ Metal regulation of MT genes by activation of MTF-1, and its interaction with MRE cis-acting sequences, does not appear to be the only mechanism that induces MT transcription by metals. In mammalian species the composite USF/ARE element of the mouse MTI promoter has shown to mediate Cd induction 3. In addition Cd has also been demonstrated to activate MREs in cells lacking MTF-12o. Studies on fish species also suggest other mechanisms for metal-induced MT transcription. Ag has been proposed to act as a primary inducer of MT transcription in teleost cell lines, independently of MTF-1. Transcription was induced at 100-fold lower concentrations of Ag than of Zn in cells transfected with rtMT-A promoter containing six MREs. Thus a redistribution of Zn from cellular Zn pools in response to the low Ag concentrations is unlikely to induce MT transcription and no redistribution of Zn from different cellular compartments was observed in response to Ag. These data strongly support the idea that Ag is a primary inducer of MT transcription in teleosts 93.

V. Free radical regulation of MT synthesis During respiration, 02 is reduced to water. However, as a consequence of respiration, toxic by-products such as superoxide and H202 are produced. These ROS are potentially harmful to macromolecules such as DNA, protein and lipids. To protect

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from ROS, an organism must be able to regulate its antioxidants accordingly. There are several ways to protect the cellular compartments from oxidative stress, including repair of the oxidative damaged sites or enhancing gene expression of antioxidant genes. The expression can be enhanced either by activating or raising the cellular level of a certain transcription factor. In contrast to eukaryotes which through signal transduction upregulate antioxidant genes, prokaryotes, such as E. coli, is dependent on direct oxidative activation of the Oxy R and Sox R factors, leading to upregulation of antioxidant genes 92. Fish MT is induced in response to factors promoting oxidative stress. These factors include stress and inflammation 6'5~ and oxidant generating substances such as hydrogen peroxide 46'47'76. The genetic basis for MT induction by oxidative stress has recently been investigated in both fish and mammalian species. Mammalian MT genes contain an upstream stimulatory factor (formerly known as the major late transcription factor, MLTF)/antioxidant responsive element (USF/ARE) that directs H202 responsiveness 24. The transcription factors involved in regulating the ARE in response to oxidative stress is highly dependent of heterodimerization and subsequent binding of nrf2 and a small maf protein. Cytosolic nrf2 is released from its inhibitor, keapl, and translocated to the nucleus upon kinase phosphorylation 37. In mammals MTF-1 is activated in response to oxidative stress 26. Although MTF-1 activation studies have not been performed on fish, the presence of multiple AP-1 elements in the fish gene promoters support the observation that oxidative stress induces MT in fish via AP1 elements. AP1 binding occurs at cis-acting (5t-TGA G/CTA/C agc-3 ~) elements sharing homology to the ARE consensus sequence. AP1 was first identified as a transcriptional factor that binds and is required for optimal basal activity of the human MT-IIa promoter 4. The activity of AP 1 is modulated by a multitude of factors including oxidative stress 87. AP1 consists of dimers from the products of the protooncogene families jun and fos. AP1 exists in two forms, as homodimers of jun proteins or heterodimers of jun:fos proteins. The primary role of AP1 is to regulate gene expression in response to mitogenic signals on the cell surface 87. Therefore, in non-proliferating cells AP1 activity is usually low, consisting mainly of phosphorylated c-Jun homodimers. Like most transcription factors the DNA binding and transactivation domains of fos and jun proteins are physically separated. The activity of both proteins is controlled by phosphorylation. While Jun-N-terminal kinases (JNK) regulate the phosphorylation of c-jun, the kinases involved in c-fos regulation are yet to be identified 3~ Inducers of oxidative stress, such as UV-irradiation and H 2 0 2 lead to AP1 activation and has also been shown to induce the transcription of cjun and c-fos in several cell lines 7~ Under normal physiological conditions the cellular environment is strongly reducing. It has been observed that proliferating cells contain high levels of MT 82. The identification of AP1 elements on MT promoters and the observation that proliferating cells contain high levels of MT, raises the question whether MT controls cellular proliferation and differentiation. Several AP1 sites have been identified in the promoter of rainbow trout, pike, stone loach and Antarctic icel~sh 45'63'76'77. In response to oxidative stress, these AP1 elements are involved in the regulation of both rainbow trout MT-A and MT-B genes 63'76. The rtMT-A promoter contains four AP1 sites located in a proximal

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Fig. 3. In vivo inhibition of H202 induced rtMT-A gene activity by AP1 oligonucleotides. Luciferase activities of Hep G-2 cells transfected with a rtMT-A promoter plasmid containing the complete set of AP1 promoter elements alone ( - A P 1 ) or together with competing AP1 oligonucleotides (+ AP1). Cells were grown in media alone (control) or exposed to 200/zM H202 for 24 h. The luciferase activities are given in arbitrary units. All activities were normalized for/3-galactosidase activity. Significant reduction in H202 induced rtMT-A gene activity was observed following co-transfection with AP1 oligo as indicated by (*).

and a distal cluster. EMSA analysis and transfection experiments indicate that the AP1 sites are transactivated through the AP1 protein complex and mediate basal level expression of the rtMT-A gene 48. In addition, it has been demonstrated that rtMT-A induction by oxidative stress is dependent on endogenous AP1 protein. Cotransfections of rtMT-A AP1 oligonucleotides and AP1 containing plasmids demonstrated an inhibition of oxidative induced rtMT-A gene expression (Fig. 3). Unlike mammals, MTF-1 and MREs in fish do not seem to participate in MT regulation during oxidative stress, which indicate that the oxidative stress response is not conserved between fish and mammalian species.

VI. Involvement of MT in protection against oxidative stress The evolution of antioxidants in aerobic organisms started when photosynthetic bacteria began producing oxygen. Due to its toxicity, oxygen had posed a major threat to life 36. Teleosts, such as the rainbow trout, inhabit turbid cool rivers, with a high level of dissolved oxygen and this is a potential threat to the rainbow trout. In addition to ROS produced by the respiratory chain, they are also generated in response to UV-irradiation mediated photochemical processes in the w a t e r 39. Hence, fish may have evolved unique regulatory systems, such as MT, for protection from oxygen toxicity. MT is rich in sulfur, and therefore, represents a significant portion of total cellular protein thiols. Thiols are known to be highly reactive towards oxidants

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and thus MT could be an excellent free radical scavenger. In vitro studies have revealed MTs high reactivity towards hydroxyl and superoxide radicals 84. MT can also protect from DNA strand breaks in vitro 1 and in vivo 21. Several studies on aquatic organisms also suggest an important role for MT during oxidative stress. These studies suggest that fish MT are both induced and protected from oxidative stress, and hence suggest a physiological role for MT during free radical production. Inflammation is known to generate excess free radicals and injection of NIPll-LPH antigen in Atlantic salmon results in MT induction 5~ In addition, oxidative stress conditions such as capture stress 6 and paraquat treatment 71 result in MT induction in fish. In the teleost cell line, RTG-2, both MT mRNA and protein were upregulated in response to oxidative stress 47. Transcriptional induction of MT m R N A in response to oxidative stress was recently reported in the teleost RTH-149 cell line 76. Pretreatment with Zn or Cd of the rainbow trout cell line, RTG-2, indicate that elevated MT levels protect the cells against oxidative stress, without the interference from GSH mediated protection 46'47. In addition, the salmonid cell line (CHSE-214), which exhibit low MT expression, rendered resistant to H202 following over-expression of MT 47, which directly suggest MTs involvement in protecting against oxidative stress (Table 2). In the carp, it was suggested that both elevated GSH and MT afford H20 2 protection ss. Cd-pretreatment of the mussel (Mytilus galloprovincialis) result in decreased iron-induced oxyradical formation, which might be the result of Cd-induced MT expression 85. In addition, the role of MT in protecting from oxyradical-mediated inflammation has also been reported in oyster (Crassostrea virginica) hemocytes 2. Besides the direct interactions of MT with certain oxidants, indirect roles of MT in free radical scavenging have been suggested s3. Due to the potential toxicological role of heavy metals to alter the cellular and lysosomal membrane leading to lipid peroxidation, MT, as a major metal binding protein, may prevent the formation of heavy metal induced free radicals. Both Cd and Cu can induce lipid peroxidation in the sea bass, Dicentrarchus labrax 75. Hence, MT could inhibit radical formation by binding these metals. It has been suggested that Zn released from MT can stabilize TABLE 2 Dose-dependent H202 sensitivity in Zn pre-treated RTG-2 cells and MT over-expressed CHSE-214:59 cells RTG-2 CHSE-214:59 Untreated Zn-treated pBK-CMV pBK-CMV-MT 50 46 74 70 101 150 47 60 53 78 300 16 48 43 64 600 5 15 13 30 The relative H202 tolerance in untreated RTG-2 cells was calculated as the ratio of cells grown in media alone for 72 h, H202 exposed for 24 h divided by cells grown in media alone for 96 h. The relative H202 tolerance in Zn pre-treated RTG-2 cells was calculated as the ratio of Zn treated (150 ~M, 72 h), H202 (24 h) exposed cells divided by cells grown in Zn (150/xM) alone for 96 h. The effect of MT overexpression on H202 sensitivity of CHSE-214:59 cells was determined by comparing the survival of overexpressed (pBK-CMV-MT) and not over-expressed (pBK-CMV) cells by calculating the ratio of surviving, H202 exposed cells divided by untreated cells.

H202 (/xM)

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the cell membrane, preventing iron-mediated lipid peroxidation s3. As an important Cu binding protein, MT has a role in preventing Cu-mediated redox cycling. The ability to undergo redox cycling makes Cu a potential mediator of the highly reactive hydroxyl radical. During physiological, reducing conditions a role for MT in preventing Cu(I) mediated lipid peroxidation has been proposed. However, during oxidative stress, Cu is released by MT and enhances the formation of ROS 31. Thus, oxidant-induced release of Cu from MT may suggest the existence of physiologic redox mechanisms that control the transfer of Cu from MT to Cu-depending enzymes similar to what has been proposed for Zn transfer from MT during minute changes in the cellular redox potential 53.

VII. Summary Although ample data suggests that MT is involved in the regulation and interactions with metals and oxidants, its physiological role remains elusive. MT knockout models reveal little about the physiological significance of MT, since the animals develop and appear normal. However, it has been observed that MT knockout mice are obese 1~ This suggests a link between MT and energy balance. The animals are hyperphagic and deposit lipids at a high rate. Thus MT may prevent accumulation of those energy stores. Recently it was suggested that MT could inhibit the respiratory chain by donating Zn 89, resulting in lowered ATP and free radical production. This mechanism may explain how MT prevents obesity, while at the same time being able to regulate the levels of free radicals. Interestingly, defects in energy metabolism have been correlated to longevity in yeast, C. elegans and Drosophila 8~ and also to high MT expression in C. elegans 8. Since aging is correlated to an increased amount of free radicals ~, a possible role for MT is to function as a regulator of energy balance, thereby prolonging life. The correlation between age, energy expenditure, Zn status, free radical production and MT expression are fascinating future prospects.

VIII. References 1. Abel, J. and N. de Ruiter. Inhibition of hydroxyl-radical-generated DNA degradation by metallothionein. Toxicol. Lett. 47: 191-196, 1989. 2. Anderson, R.S., K.M. Patel and G. Roesijadi. Oyster metallothionein as an oxyradical scavenger: implications for hemocyte defence responses. Dev. Comput. Immunol. 23: 443-449, 1999. 3. Andrews, G.K. Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem. Pharmacol. 59: 95-104, 2000. 4. Angel, P. and M. Karin. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim. Biophys. Acta 1072: 129-157, 1991. 5. Auf der Maur, M.A., T. Belser, G. Elgar, O. Georgiev and S. Schaffner. Characterisation of the transcription factor MTF-1 from Japanese pufferfish (Fugu rubripes) reveals evolutionary conservation of heavy metal stress response. Biol. Chem. 380: 175-185, 1999. 6. Baer, K.N. and P. Thomas. Influence of capture stress, salinity and reproductive status on zinc, proteins in the liver of three marine teleost species. Mar. Environ. Res. 28: 277-287, 1990. 7. Baer, K.N. and P. Thomas. Isolation of novel metal-binding proteins distinct from metallothionein from spotted seatrout (Cynoscion nebulosus) and Atlantic croaker (Micropogonias undulatus) ovaries. Mar. Biol. 108: 31-37, 1991.

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