Cell, Vol. 41, 9-10, May 1985, Copyright 0 1985 by MIT

Metallothioneins: Proteins in Search of Function

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Michael Karin Department of Microbiology University of Southern California School of Medicine 2011 Zonal Avenue Los Angeles, California 90033

Metallothioneins (MTs) are ubiquitous low molecular weight proteins that are characterized by an unusually high cysteine content (30 mole O/O) and a selective capac- ity to bind heavy metal ions, such as zinc (Zn), cadmium (Cd), and copper (Cu). There are two major subgroups of metallothioneins, which have been referred to as MT-I and MT-II, based on their electrophoretic properties; other methods further resolve the MT-I group into several dis- tinct molecular forms known as MT-In, MT-la, etc. The various isoforms exhibit small differences in their binding affinity for metal ion ligands, but otherwise, appear to have similar biochemical properties.

The multiplicity of MTs has been confirmed by gene cloning experiments. In most vertebrate species exam- ined, the MTs are encoded by a multigene family. In- dividual members of the MT gene family exhibit different responses to inducers because they possess distinct pro- moter elements (Richards et al., Cell 37, 263-272, 1984).

Much of the recent interest in MTs has focused on the regulation of transcription of the genes that encode them, with relatively little effort directed towards understanding the physiology of the proteins themselves. However, several new findings discussed below may shed some light on the biological roles of these unusual proteins. (For a review of earlier work, see Metallothionein, eds. J. H. R. Kagi and M. Nordberg, Birkhauser-Verlag, 1979.) Induction by Heavy Metal ions and Protection Against Toxicity Injection of animals with Cd or Zn salts causes MTs to ac- cumulate in the liver and kidney. Durnam and Palmiter have shown that this is due to transcriptional activation of MT genes by heavy metal ions that bind to the proteins (JBC 256, 5712-5716, 1981). These and earlier findings led to the hypothesis that MTs function as a protective system against heavy metal toxicity. Indeed, MTs do seem to play such a role in cultured mammalian cells. Cells that have been selected for Cd resistance produce higher than nor- mal levels of MTs, as a result of gene amplification (Beach and Palmiter, PNAS 78, 2110-2114, 1981). Similarly, cells become resistant to Cd after they have been genetically engineered to produce high levels of MTs (Karin et al., PNAS 80, 4040-4044, 1983).

It seems unlikely that protection against heavy metals would be the primary function of MTs. First, these ions are not present at high levels in most biotopes and probably do not exert a selection pressure significant enough to justify the existence of a special detoxification system. Second, if the role of MTs were purely protective, one would expect to find these proteins only after exposure to

toxic heavy metals; in fact, the basal level of expression of MT is relatively high.

The MTs that are naturally present in an animal’s liver and kidney serve as the major storage form for the essen- tial trace elements, Zn and Cu. The level of these ele- ments is highly influenced by the dietary status of the ani- mal. These observations, together with the finding that MTs are expressed in an inducible manner in essentially every tissue (Searle et al., MCB 4, 1221-1230, 1984), pro- vide evidence that MTs play a role in both the extracellular (homeostatic) and intracellular control of Zn and Cu me- tabolism. Once either ion reaches a certain threshold level, the transcription rate of MT genes increases, lead- ing to synthesis of more protein to bind the excess metal. In addition, the level of intracellular Zn regulates the turn- over rate of MTs; when Zn is in short supply, MTs are rap- idly degraded (Karin et al., J. Cell Physiol. 706, 63-74, 1981). Regulation by Hormones and Expression During Development A variety of acute stresses, including the injection of bac- terial lipopolysaccharide (LPS), increase hepatic synthe- sis of MTs. This may be explained in part by the resultant increase in plasma levels of glucocorticoid hormones, which have been shown to induce biosynthesis of MTs (Karin et al., Nature 308, 513-519, 1984). However, the in- duction of MTs is not solely mediated by these hormones; Durnam et al. (PNAS 87, 1053-1056, 1984) have shown that a MT-thymidine kinase fusion gene in transgenic mice can be induced by the injection of LPS but not by ad- ministration of a synthetic glucocorticoid.

LPS injection and other stressful stimuli are known to trigger the acute phase response, a pleiotropic response to tissue injury and inflammation. During the acute phase response, the expression of a number of liver-specific genes is induced, probably by the action of interleukin 1 (IL-l), a polypeptide hormone secreted by macrophages (Bornstein, Ann. NY Acad. Sci. 389, 323-337, 1982). Pre- liminary experiments reported by Durnam et al. (op. cit.) indicate that injection of IL-l into mice leads to increased expression of MTs. Interferon (IFN), another lymphokine, has been shown to increase the transcription rate of MT genes (Friedman et al., Cell 38, 745-755, 1984).

The transcription of MT genes is modulated during em- bryogenesis. In the early mouse embryo, high levels of MT mRNA are found in the primitive endoderm and later (i.e., days 9-12 of gestation), in the parietal and visceral endoderm cells of the yolk sac and in the newly formed fetal liver. After day 15 of gestation, however, the level of these transcripts decreases abruptly (Andrews et al., Dev. Biol. 703, 294-303, 1984). It is unclear whether these changes result from extracellular signals, such as metal ions or hormones, or alternatively, from developmentally controlled changes in the level of transcription factors responsible for the basal level of MT expression. MTs and the Regulation of Cellular Metabolism Proteins whose synthesis is subject to such complex and

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fine regulation as MTs are likely to occupy a central role in cellular metabolism. As the major Zn-binding proteins in the cell, MTs can potentially modulate many important biological processes that involve Zn-requiring enzymes;

e.g., replication, transcription, protein synthesis and degradation, energy metabolism. Their involvement can

either be direct, via interaction with inactive apoenzymes, or indirect, by regulation of available Zn in the cell. Indeed, it has been shown that several Zn-requiring apoenzymes can be reactivated by the transfer of Zn from MTs to the apoenzymes (Udom and Brady, Biochem J. 787329-335, 1980).

MTs may thus constitute a regulatory system whose function is analogous to that of calmodulin in calcium me- tabolism. Conceivably, different variants of MT could carry Zn to different intracellular compartments or else in-

teract with different classes of enzymes. Hormones that control MT gene expression could effectively modulate a cell’s metabolic and proliferative status by altering the in- tracellular distribution of Zn. MTs and the Control of Differentiation and Proliferation Recent cytological observations have raised the possibil- ity that MTs are involved in the control of cellular growth. LeBeau et al. (Nature, 373, 709-711, 1985) have found that the functional human MT gene cluster is split in leukemic cells of patients suffering from acute myelomonocytic leukemia. Approximately one half of the gene cluster, nor- mally present on the long arm of chromosome 16 at band q22, is translocated to the short arm of the same chromo- some. This rearrangement is found in cells that contain either an inversion of chromosome 16 or a 16:16 transloca- tion; in each case, the breakpoint occurs in the middle of the MT gene cluster.

One possible explanation for these data is that a resi- dent cellular oncogene on the short arm of chromosome 16 is activated by enhancer elements associated with MT genes (Karin et al., Cell 36, 371-379, 1984; Haslinger and Karin, Nature, in press). Alternatively, the perturbation of MT gene expression may itself be the primary factor in the development of neoplasia. This would implicate MTs in the myeloid-monocyte differentiation pathway or in the general control of cellular proliferation, possibly through the regulation of intracellular Zn. This idea is supported by the finding that growth arrested human fibroblasts con- tain significantly lower levels of MT mRNA than do actively proliferating cells (Angel et al., Cancer Cells 3, in press). The dramatic changes in MT gene expression throughout the early development of the mouse embryo may also re- flect the involvement of MTs in growth control processes, MTs and Free Radicals Since MTs are directly induced by hormones whose levels are elevated in response to stress (glucocorticoids and IL-

l), and by IFNs, which are released in response to viral in- fection, it seems likely that they are in some way involved in the first line of defense against exogenous assaults.

Both IL-l and Y-IFN are responsible for the activation of neutrophils and macrophages, leading to the release of massive amounts of active oxygen species. While these free radicals facilitate the destruction of bacteria, para- sites, and virus-infected cells during the inflammatory re- sponse, they are also extremely cytotoxic and could cause severe tissue damage to the host in the absence of protec- tive measures. Special free radical scavengers, such as superoxide dismutase, constitute one form of protection for the cell.

MTs have been shown to be extraordinarily efficient scavengers of free hydroxyl radicals in vitro (Thornalley and Vagak, BBA 827, 36-44, 1985), but unlike superoxide dismutase, they are not active toward superoxide radicals. Hydroxyl radical damage to MT itself appears to occur at the metal-thiolate clusters, which may be repaired in the cell by reduced glutathione. While there is still no direct proof that MTs function as free radical scavengers in vivo, the high intracellular concentrations of MTs achieved after induction (up to2 mM) and their regulation by IL-l and IFN would tend to suggest that these proteins are part of such a protective system. MTs and the UV Response Free radicals are responsible for many of the adverse bio- logical effects of ionizing radiation. Eukaryotic cells pos- sess a specialized system, the UV response, which is similar to the bacterial SOS response in providing protec- tion against radiation damage (Schorpp et al., Cell 37, 861-868, 1984). After exposure to UV light, cultured hu- man fibroblasts secrete a factor, EPIF (for extracellular protein synthesis inducing factor), which can elicit the UV response in nonirradiated cells, thereby protecting them against subsequent challenge. EPIF has been shown to be an efficient inducer of MT mRNA and may be respon- sible for its induction by direct exposure to UV light (Angel et al., EMBO J., in press). This finding, coupled with the earlier finding that mammalian cells expressing high lev- els of MTs are resistant to X-ray damage (Bakka and Webb, Biochem. Pharmacol. 30, 721-725, 1981), suggests that MTs may be important in the mammalian UV response, perhaps serving as free radical scavengers or as sources of Zn for DNA repair enzymes that are activated after irradiation.

In summary, the evidence provided by investigations of MT gene expression indicates that the metallothioneins are involved in the control of normal cellular metabolism and in cellular adaptation to various types of stress. A bet- ter understanding of their biochemical roles in these re- sponses will require additional studies both at the protein level and at the DNA level.