Metallothionein-I1II in neuroprotection

Mie . Pedersen,1 Rikke Jensen,1 Dan S. Pedersen,1,2 Anders D. Skjolding,1 Casper Hempel,1,3,4 Lasse Maretty,1,3,4 and Milena Penkowa1*
1

Section of Neuroprotection, Institute of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark Department of Neurobiology, Institute of Anatomy, Faculty of Health Sciences, University of Aarhus, Aarhus, Denmark Centre for Medical Parasitology at Department of Clinical Microbiology and Department of Infectious Diseases, Copenhagen University Hospital (Rigshospitalet), Copenhagen, Denmark

2 3

Centre for Medical Parasitology at Department of International Health, Immunology, and Microbiology, University of Copenhagen, Copenhagen, Denmark

Abstract.
Metallothionein (MT)-III synthesis is induced in the central nervous system (CNS) in response to practically any pathogen or disorder, where it is increased mainly in reactive glia. MT-III are involved in host defence reactions and neuroprotection during neuropathological conditions, in which MT-III decrease inflammation and secondary tissue damage (oxidative stress, neurodegeneration, and apoptosis) and promote post-injury repair and regeneration (angiogenesis, neurogenesis, neuronal sprouting and tissue remodelling). Intracellularly the molecular MT-III actions involve metal ion control and scavenging of reactive oxygen
C V 2009 International Union of Biochemistry and Molecular Biology, Inc. Volume 35, Number 4, July/August 2009, Pages 315325  E-mail: M.PENKOWA@sund.ku.dk

species (ROS) leading to cellular redox control. By regulating metal ions, MT-III can control metal-containing transcription factors, zinc-finger proteins and p53. However, the neuroprotective functions of MT-III also involve an extracellular component. MT-III protects the neurons by signal transduction through the low-density lipoprotein family of receptors on the cell surface involving lipoprotein receptor-1 (LRP1) and megalin (LRP2). In this review we discuss the newest data on cerebral MT-III functions following brain injury and experimental autoimmune encephalomyelitis.
Keywords: metallothionein, neuroprotection, neurodegenerative disease, neuroinflammation

1. Introduction
Mammalian metallothioneins (MTs) constitute a family of ubiquitous, low molecular weight proteins (67 kDa), characterized by a high content of cystein residues (30%) organized in unique sequences, which enable the formation of distinct metal thiolate clusters within each of the MT domains

Abbreviations: BBB, blood brain barrier; CNS, central nervous system; CREB, cAMP response element binding protein; EAE, experimental autoimmune encephalomyelitis; EPO, erythroprotein; ERK, extracellular signal-regulated kinase; IL, interleukin; LRP, lipoprotein receptor; MT, metallothionein; MTKO, metallothionein-I/II knockout; ROS, reactive oxygen species; STAT, signal transducer and activator of transcription; TBI, traumatic brain injury; TgMT, transgenic MT-I overexpressing; TNFa, tumor necrosis factor alpha. *Address for correspondence: Milena Penkowa, M.D., Ph.D., D.MSc., Section of Neuroprotection, Institute of Neuroscience and Pharmacology, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, building 18.1.44, DK-2200 Copenhagen, Denmark. Tel.: 45 35 32 72 22; Fax: 45 35 32 72 20; E-mail: M.PENKOWA@sund.ku.dk. Received 27 February 2009; revised 1 April 2009; accepted 1 April 2009 DOI: 10.1002/biof.44 Published online 27 May 2009 in Wiley InterScience (www.interscience.wiley.com)

[1,2]. Under physiological conditions, Zn(II)and to a lesser extent Cu(I)comprise the main MT associated metals in vivo; however a range of other transition metals (Cd(II), Hg(II), Fe(II), Pb(II) etc.) also engage in MT thiolate clusters in vitro and/or in vivo [3]. Metal/thiolate stochiometries depend mainly on metal coordination properties; for divalent metals, the a- and b-domains coordinate four and three ions in a tetrahedral manner, respectively [1]. The lack of any apparent secondary structure in apothionein indicates the template functions of metal ions on MT protein folding [4]. Furthermore, the impact of cellular oxidative and nitrosative status on metal saturation and MT poly-/dimerization (through the formation of intra- and intermolecular disulfide bonds) place redox status as yet another variable affecting MT structure [57]. In mammals, MTs are divided into four subfamilies (MT IIV). All known human MT genes are located in the q13 locus on chromosome 16 and encode seven functional MT-I genes (MT-IA, B, E, F, G, H, X) and a single functional gene for each of the three other MT isoforms; in mice, all functional MT isoforms are encoded by single genes located on chromosome 8 315

Fig. 1. This figure depicts how the main inducers of MT-I1II expression regulate cerebral MT gene transcription by signaling through intracellular pathways and response elements present in the gene regulatory region. Some of the inducers comprise: hormones (glucocorticoids, catecholamines) signaling through binding to glucocorticoid response factors (GRF) that subsequently bind to glucocorticoid response elements (GRE); proinflammatory cytokines (e.g., interleukins (IL) and tumor necrosis factor alpha (TNFa)) signalling through signal transducer and activator of transcription (STAT); reactive oxygen species (e.g., hydrogen peroxide (H2O2), nitrogen oxide (NO), hydroxyl radical (OH)) signaling through antioxidant response elements (ARE); and metal ions (e.g., zinc (Zn), copper (Cu), cadmium (Cd)), which bind to metal transcription factors (MTF), whereupon these bind to metal response elements (MRE) in the gene promotor region. [14,16,17]. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] [813]. MT-III differ by only a single amino acid insertion and are the best studied and most widely expressed isoforms; MTIII and MT-IV exhibit a more tissue specific expression and will not be addressed further in this review [3,14]. response elements, antioxidant response elements, glucocorticoid response elements (Fig. 1) [14,16,17]. MT-III are involved in host defence reactions to damage as MT-III mediate neuroprotection and tissue recovery in vivo as shown during a range of neuropathological conditions including brain injury and experimental autoimmune encephalomyelitis (EAE) [15] (Fig. 2), which will be the focus of this review.

2. Cerebral MT-I1II
MT-III in the central nervous system (CNS) are induced by practically any pathogen or disorder, which results in increased synthesis of MT-III mRNA and proteins [15]. Increased MT-III proteins are mainly observed in reactive astroglia, but also in microglia, macrophages, and endothelial cells. In the brain, a wide range of MT-III inducers exist and they comprise proinflammatory cytokines (for example, interleukin (IL)-6, IL-3, interferon, tumor necrosis factor alpha (TNFa)), reactive oxygen species (ROS), metal ions (cations, eg. Zn, Cu, Cd, Hg, Pb) and hormones (glucocorticoids, catecholamines) [3,15]. The inducers increase MT-III by signaling through intracellular pathways and response elements present in the gene regulatory region that in brief include signal transducer and activator of transcription (STAT)-3, metal 316

3. MT-I1II roles in neuropathology

3.1. Brain injury
By studies of experimental brain injury, the in vivo inducibility, regulation and expression of MT-III were characterized [18]. Such studies revealed that cerebral MT-III proteins and their temporospatial responses to injury are quite consistent in different animal species and strains, which were studied during rodent lifespan, that is, from the embryonic (prenatal) development to neonatal, young and old age [18 22]. In general after injury, MT-III are rapidly induced and are de novo synthesized in reactive glia and phagocytic cells. The MT-III increase is transient and after having
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Fig. 2. This figure provides an overview of the neuroprotective and host defense reactions of MT-I1II in response to tissue damage during neuropathological conditions such as brain injury and experimental autoimmune encephalomyelitis. See text for further details. Abbreviations: BDNF, brain-derived neurotrophic factor; EPO, Erythropoietin; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; GDNF, glial cell line-derived neurotrophic factor; IL, interleukin; NGF, nerve growth factor; NT, neurotrophin; TGF, transforming growth factor-beta; TGFR, transforming growth factor-beta receptor; VEGF, vascular endothelial growth factor. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

peaked within the first week post-injury, it slowly returns to baseline (premorbid) levels. The neurobiological effects exerted by MT-III during tissue damage were initially identified by means of genetically modified mice such as the MT-III deficient mice (MT-III knockout (MTKO) mice) and the transgenic MT-I overexpressing (TgMT) mice. The first study of in vivo neuropathology in MTKO mice pointed towards MT-III as neuroprotectants, as brain injured MTKO mice display increased inflammation, oxidative stress and apoptosis, while the astroglial scar formation is impaired, when compared to those of wild-type mice [23]. Further studies showed chronically impaired brain tissue recovery including incomplete angiogenesis, deficient expression of growth/trophic factors, and increased levels of proinflammatory and/or neurotoxic mediators in the MTKO mice relative to controls [24]. Accordingly, only wild-type controls show regeneration and recovery of the cortical parenchyma at 2 months post-injury, while MTKO mice fail to restore the injury, which remain as a necrotic lesion cavity at 3 months post-injury [24]. More recent studies of brain injured MTKO mice reveal that MT-III are crucial for activation and mobilization of endogenous neural stem/precursor cells and for
Metallothionein-III in neuroprotection

the transcriptional activation of factors controlling cellular growth and neuronal growth cones [20]. In case of TgMT mice overexpressing (intracellular) MTI form, the responses to brain injury are quite opposite or complementary to those seen in the MTKO mice. Brain injured TgMT mice display minimized secondary tissue damage with decreased inflammation, oxidative stress, neurodegeneration and apoptosis [25]. Also, MT-I overexpression enhances expression levels of various growth/trophic factors (including basic fibroblast growth factor, basic fibroblast growth factor-receptor, transforming growth factor-beta, transforming growth factor-beta receptor, neurotrophins-3, -4/5, nerve growth factor, vascular endothelial growth factor, and platelet-derived growth factor receptor), increases neuritogenic growth cones, and stimulates vascular remodeling and angiogenesis during the first weeks post-injury and when compared to those of wild-type controls [25]. The data obtained in the genetically modified mice were all relating to intracellular expression of MT-III, however, data have shown that exogenous (extracellular) MT-II causes similar neuroprotective effects as those observed in the TgMT mice [25]. Accordingly, ip administration of MT-II 317

protein in both wildtype and MTKO mice with brain injury results in the same neurobiological effect as those caused by transgenic (intracellular) MT-I-overexpression [25]. Mounting data from neuronal cell studies and from brain injured animals support such extracellular and/or paracrine mechanisms of MT-III actions [2628]. Recently, it became clear that MT-III are in fact secreted actively from glial cells into the extracellular fluid, from which MT-III are rapidly internalized by neurons, which express their surface receptor (megalin) [29,30]. This highlights that exogenously injected proteins exert analogous neuroprotective functions as those of the endogenous MT-III, and it points towards the potential of MT-III in terms of future development and pharmaceutical strategies to be used against brain injury. MT-III administration is effective in experimental traumatic brain injury (TBI) due to breakdown of the blood-brain barrier (BBB) at the site of the injury, allowing MT to enter the brain [25]. As described previously [20,25,31,32], i.p. injection of MT-I or MT-II after a brain injury and/or after EAE onset of attack results in increased MT-III in the brain, as peripherally injected MT-III proteins emerge after 1545 min post-injection in the extracellular tissue of the brain, and by 68 h, the exogenous and extracellular MT-III have disappeared. Supporting this, an extracellular pool of MT-III proteins were detected in a matrix from the injured brain [30]. As shown in independent experiments, the extracellular MT-III proteins are taken up by neurons through surface receptors megalin and LRP, for which MT-III are ligands [29,30]. This poses great challenge on the future development of exogenous MT-III treatment for brain diseases. In a study by Chung et al. (2003), filling a brain injury (lesion-cavity) with MT-II gel foam promoted wound healing, decreased cellular degradation, and increased progression of reactive axonal proceses [33]. Also, injection of gel foam containing genetically modified MT-III overexpressing cells into the brain parenchyma with the aid of 3D stereotactical surgical equipment including neuronavigation, could be another possibility. MTIII could hereby have a place in future pharmacological treatment of patients with traumatic brain injury, stroke and possible also after surgical treatment of brain tumors.

3.2 Multiple sclerosis

EAE is an animal model of human multiple sclerosis, and it is the first in vivo model of a human brain disease in which exogenous MT pharmaceuticals were applied and characterized [34]. This investigation showed how MT-II treatment ameliorates clinical severity, neurological deficits, and mortality in EAE sensitized rats when compared to the course seen in controls. MT-II injected on the first day of clinical disease reduced the severity of EAE symptoms, the time duration of the EAE attack, as well as MT-II minimized the mortality rate from 1015% in rats receiving placebo to 23% in the MT-II treated group. Also, the EAE lesions consisting of demyelination and autoimmune/inflammatory infiltrates (due to invading T lymphocytes and macrophages) were decreased in MT-II treated animals relative to controls [34]. 318

As a result, the delayed damage such as axonal transection, neuronal degeneration and cell death associated with EAE were reduced by the MT-II treatment [34]. During the remission phase (around 1 month after EAE immunization) increased neuroregenerative responses and remyelination due to recruitment of oligodendroglial stem/precursor cells were seen in animals receiving MT-II treatment relative to placebo [34]. The mechanisms of MT-III actions in EAE were further investigated by means of MTKO mice, in which the clinical severity and the histopathology are more severe than in wildtypes [35]. In this study, the clinical evaluation of the mice with EAE included animal susceptibility to disease induction (incidence), mortality, day of onset, mean and maximum scores of neurological symptoms. The MTKO mice are more susceptible to EAE induction as 70% showed fullscale EAE, while only 43.75% of wildtypes developed clinical symptoms of EAE. The mortality rates, mean daily EAE score and the maximum disease score also pointed towards a worsened course in MTKO mice relative to controls [35]. In line with this, the MTKO mice with EAE display more demyelination throughout the CNS along with increased inflammation consisting mainly of infiltrating macrophages and T lymphocytes, which causes higher levels of proinflammatory cytokines and oxidative stress [34]. Axonal degeneration and transection were increased in MTKO mice, which also showed increased apoptotic cell loss in neurons, oligodendrocytes, and astroglia relatively to wildtype controls [34]. As a likely consequence, remyelination by oligodendroglial precursors, expression of growth/ repair factors, growth cone formation and neurological recovery from EAE are diminished by MT-III deficiency relatively to controls [34]. A dual role of apoptosis is proposed in EAE/multiple sclerosis, where apoptosis of neurons and oligodendrocytes is harmful, while apoptosis of autoreactive T-cells may lead to protection [3638]. The fact that neuronal cells seem to benefit the most from MT-III treatment at the cellular level [34] is therefore highly important when discussing the pharmaceutical potential of MT-III in multiple sclerosis treatment. Seeing that MT-III treatment inhibits both cellular, physiological, and clinical effects during EAE development and progression MT-III may present promising pharmaceutical agents. In the following sections, the molecular mechanisms of action and signaling pathways will be discussed.

4. MT-I1II receptors and signal transduction

MT-III play an important role in intracellular processes such as metal ion homeostasis, regulation of redox states, scavenging of ROS, and regulation of zinc-containing transcription factors, zinc-finger proteins and p53 [15,17,39,40]. Until now little has been understood about the molecular mechanisms that regulate these actions. As MT-III lack signaling peptides, their well-characterized free radical scavenging and heavy metal binding properties have traditionally
BioFactors

Fig. 3. This figure shows the receptor megalin, which is a 600-kDa, transmembrane glycoprotein with a large NH2terminal extracellular domain consisting of 4,400 amino acids, a single transmembrane domain of 22 amino acids, and a short cytoplasmic tail of 213 amino acids. The extracellular domain contains four cysteine-rich clusters comprising lowdensity lipoprotein-receptor type A repeats, which constitute the ligand-binding regions, and are separated and followed by 17 epidermal growth factor (EGF)-type repeats and eight spacer regions [42,43]. Abbreviations: APO, apolipoprotein; MT, metallothionein; Rbp, Retinol-binding protein; Trt, transthyretin; Shh, Sonic hedgehog. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

been applied to explain their functions, but there is increasing realization that the cellular and physiological neuroprotective functions of MT-III may also involve an extracellular component. Recent studies report that exogenous MT-III interacts directly with neurons to promote neuronal survival, neurite outgrowth and axonal regeneration, both in animal and tissue culture models of neuronal trauma [27,29]. In vitro, secreted MT-III are internalized through the low-density lipoprotein family of receptors, including LRP1 and megalin/LRP2 (Fig. 3) [30,41]. Megalin is a 600-kDa, transmembrane glycoprotein with a large NH2-terminal extracellular domain, a single transmembrane domain, and a short cytoMetallothionein-III in neuroprotection

plasmic tail [42,43]. Within the CNS, megalin is expressed in the ependyma of the choroid plexus [42,43] and neurons [29,30], while LRP1 is detected in astrocytes and neurons [44,45]. The role of megalin in neuronal MT-III uptake is shown by blocking the receptor with specific antibodies. This reduces MT-III uptake into neurons and abolishes the stimulation on neurite outgrowth in vitro [30,41]. By binding to megalin, MTIII can promote neuronal survival and this may in part be due to activation of signaling molecules and transcription factors extracellular signal-regulated kinase (ERK), Phophoinositide 3-kinase/Akt and cAMP response element binding protein 319

(CREB) in neurons in vitro [27,29]. LRP1 also promotes MT-III uptake into neurons, and promotes survival through the same intracellular signaling molecules [29,46]. How MT-III binding to megalin directly activates signal transduction pathways is not yet fully understood, but the work of Ambjrn et al. (2008) suggests that the receptors may act in a tyrosine kinase receptor type fashion. In addition to a direct activation of signaling pathways, megalin and LRP1 are endocytosis receptors, which bind their extracellular ligands before an endocytotic uptake [27]. Upon ligand binding megalin is internalized via clathrincoated pits, delivered to early endosomes where the ligand dissociates, and finally travel to late endosomes [47,48]. Dissociation of the complex occurs in late endosomes (most likely triggered by the low ph (5.5) of this compartment), upon which megalin is finally recycled to the cell surface [47,48]. While megalin is recycled to the membrane, its ligands are transferred to vesicles such as lysosomes [42]. Wolff et al. (2006) demonstrated megalin-mediated uptake of cadmium-MT by renal cells, which results in cadmiuminduced nephrotoxicity, suggesting that following internalization cadmium is released from MT. If a similar situation occurs in neurons, it is possible that internalized MT-III is redirected to other cellular compartments within the neuron, such as the nucleus. It is well established that MT-III can be translocated from the cytoplasm to the nucleus in a regulated fashion [14,17,39]. Within the nucleus, MT could protect the DNA from oxidative damage, or act as a regulator of gene expression by controlling the availability of zinc to zinc-dependent transcription factors and zing fingers. Taken together the results from ligand-receptor studies have begun to shed light upon MT-III signal transduction through megalin and LRP1, although other and yet uncharacterized receptors for MT-III are most likely to exist.

5. Mechanisms of MT-I1II neuroprotective actions

In vivo MT-III are efficient ROS scavengers and protect against ROS-induced cellular damage [49,50], as well as acute and chronic heavy metal toxicity in several organs and cell types [5154]. For example, MTKO mice display higher mortality than wildtype mice in murine models of Menckes disease [51], and MTKO mice have been shown to be more sensitive to renal malformations during zinc-deficiency, as well as displaying increased pancreatic damage in response to zinc-overload, when compared with wildtype mice [52]. In addition, zinc-MT has shown to have a significantly higher protective effect against radiation-induced DNA damage, when compared to glutathione and albumin in vitro [55]. MT bound to copper and cadmium, also mediated protection of radiation-induced DNA damage, but with a lower capacity than zinc-MT. This study indicates that MT are highcapacity antioxidants. Previous studies show that MT-III scavenge superoxide anions, hydroxyl radicals, phenoxyl radicals and nitric oxide [3,56]. MT-III act as thiol donors that protect against 320

hydroxyl radical-mediated DNA degradation with higher molar efficiency (almost 800-fold) relatively to glutathione in vitro [57]. Also, MT-III functionally substitute for Cu/Znsuper oxide dismutase in the defense against oxidation [58,59], and MT-III compensate for the effects of glutathion and/or catalase depletion [60]. The data support that cells with a high content of MT-III are more resistant to free radical-induced damage in vitro and in vivo, than cells having a low or absent MTIII expression [15,34,6163]. The same functions of MT-III in the CNS are supported by studies of MTKO mice and TgMT mice, indicating that MTKO mice suffer more extensive ROS-induced cellular damage, than TgMT mice during brain pathology [35,64]. MT-III are intracellular redox active proteins that are oxidized and reduced when MT-III release and bind zinc, respectively [65,66]. When MT-III are oxidized, zinc is released and becomes available for metalloproteins such as carboxypeptidase A, carbonic anhydrase, alkaline phosphatase, and sorbitol dehydrogenase [65,67]. Mitochondrial aconitase (m-aconitase) accepts zinc from MTIII through direct protein-protein interactions [6769]. Once m-aconitase is separated from MT-III by a membrane that is only permeable to zinc ions, no zinc exchange is observed. Without the membranous separation, m-aconitase and MT-III proteins mix leading to swift exchange of zinc [69]. This observation supports that zinc is primarily a ligand-coupled metal that is less likely free floating as unbound metal ions inside cells [69], indicating that intracellular transfer of zinc is tightly controlled and that MT-III act as specific chaperones with regard to zinc transport. After MT-III have released zinc, the proteins may form intramolecular disulfide bonds or in case all zinc atoms are released apothionin. During zinc overload, the metal binds to the thiolate cluster and contributes to the synthesis of MT-III [65,70]. When availability or accessibility of zinc is low, the metal is donated by MT-III to zinc acceptors [66,71]. Zinc is important for cells both in terms of survival and death, and oxidative stress can disturb this redox capacity by homocysteinylating MT-III, which makes MTIII unable to scavenge ROS [66,71]. Zinc is also recognized to be imperative for gene transcription and translation of the mRNA, which is due to its regulation of DNA binding metalloproteins [65,69]. The oxidoreductive mechanisms whereby MT-III exchange zinc are dependent on glutathione disulfide and its reduced form glutathione [65,70]. The rate limiting step is glutathione disulfide; which oxidizes MT-III resulting in zinc release [65]. Glutathione on the other hand mediates the transfer of zinc to apothionein. However, high glutathione levels concomitant with a depletion of intracellular glutathione disulfide lead to an inactivation of MT-IIIs zinc release [65]. Consequently, the redox state and the metal ion concentration modulate MT-III proteins and vice versa, which indicates that MT-III have significant impact upon multiple signaling pathways, metabolism and cytoprotection. Also, it is important to note that the glutathione disulfide-mediated release of metal from MT-III under conditions of oxidative stress may cause a disturbance of metal metabolism with important consequences for the
BioFactors

progression of diseases such as Alzheimers and Parkinsons disease where metal-induced oxidative stress occurs in affected brain tissue [72]. The impact of this increase in free metal must be carefully investigated before initiating any MT based pharmacological treatment for CNS disorders. ROS are critical regulators of the intracellular apoptotic cascade. As described, MT-III reduce ROS formation and apoptotic cell death, which is in part due to an inhibition of caspase-1 and -3, reduction of mitochondrial leakage of cytochrome c and p53 [34]. Excess ROS or deficiency in antioxidants cause oxidative stress, which per se stimulate cytochrome c leakage from the mitochondria, and this can in turn activate the caspase cascade (i.e., caspase-3, caspase-9) leading to DNA fragmentation and apoptosis. Studies have shown that MT-III impedes the release of cytochrome c from the mitochondria and thereby inhibits this cascade in apoptosis [34,68,73]. The molecular mechanisms behind the antiapoptotic effects could also be explained by the metal chelating properties of MT-III. Following CNS injury zinc is released from damaged neurons [74], and has been shown to mediate neuronal apoptosis in vitro by increasing the expression of the proapoptotic protein Bim, as well as activating the mitochondrial apoptotic pathway, which includes cytochrome c leakage and activation of caspase-9 and -3 [75]. By chelating free zinc atoms, MT-III could contribute to neuronal protection. In addition, MT-III has been shown to increase the expression of B-cell lymphoma-2 protein [50,76], which is an antiapoptotic protein that antagonizes Bim [75]. The metal-binding properties of MT-III also affect other zinc-dependent proteins, such as p53, which is a transcription factor and tumor suppressor arresting cell cycle progression and activate proapoptotic signals when DNA damage is severe [7779]. p53 is highly dependent on zinc for folding and DNA-binding [7779]. Several in vitro studies have shown that apo-MT-III, acts as a metal chelator, sequestering zinc from p53 and thereby mediating a p53null state [7780]. It has therefore been suggested by some studies that an excess of apo-MT inhibits p53 activation due to its metal chelating abilities, hereby preventing apoptosis [77,79,80]. On the other hand, when MT-III and p53 are expressed in the same ratios, MT-III can have a positive effect on p53 transcriptional activities by acting as zinc donor [7780], suggesting that it is primarily the overexpression of apothionein that might contribute to p53 inhibition.

6. Mechanisms of MT-I1II neuroregenerative actions

Inflammation plays an important role in the cellular, physiological and clinical pathology of both TBI and EAE, and has dual roles in providing both detrimental and protective mechanism [8183]. Neuronal damage is rapidly sensed by astrocytes, which respond by triggering a stereotypical pattern of molecular and morphological alterations termed reactive astrogliosis [84]. Reactive astrocytes have several roles in downregulation of inflammation as they may isolate the injured area, prevent ectopic neuritogenesis and they
Metallothionein-III in neuroprotection

are the main source of growth/trophic factors, antioxidants, hematopoietins and survival signals [84,85]. Within the injured brain, MT-III are consistently upregulated in reactive astrocytes. At the intracellular level MT-III may influence the expression of other astroglial growth/ trophic factors in both an autocrine and paracrine fashion by influencing intracellular signaling molecules and transcription factors, such as ERK, protein kinase C, Phophoinositide 3-kinase, protein kinase B/Akt and CREB [27,29]. ERK has been shown to be involved in regulating the expression of brainderived neurotrophic factor [86], glial cell line-derived neurotrophic factor [87], neurotrophins [88], nerve growth factor [89], and transforming growth factor-beta [90] in vitro, while cAMP and CREB are involved in regulation of vascular endothelial growth factor [91,92], and glial cell line-derived neurotrophic factor [87,93]. Together with MT-III these growth/ trophic factors may reinforce neuroprotective effects in a synergistic manner [9497]. These effects, which are mediated through the activation of different extracellular receptors, may mutually augment the intracellular signaling crucial in repair and regeneration. Also, these factors may activate common intracellular pathways or signals (e.g., ERK, Janus-activated kinases, Src family kinases), and transcription factors (e.g., STAT-3, and CREB). In addition, the growth/trophic factors induced by MT-III may themselves increase the expression of MT-III [98], hereby working as a positive feedback loop. The pro-angiogenic actions of MT-III may also relate to such positive feedback interactions with vascular growth factors and angiopoietins. However, MT-III may also directly influence the endothelial cell cycle. A downregulation of MT-I expression in endothelial cells inhibits proliferation, migration, and network formation in vitro, as well as angiogenesis in vivo [99]. Inhibition of MT-I or II by siRNA treatment also increased G0/G1-phase cell population, decreased S-phase population, inhibited cell migration and network formation in endothelial cells [99], indicating direct actions of MT-III in endothelial cell cycle progression and angiogenesis. MT-III localization varies with cell cyle progression [14,17,100]. During G0 and G1 phase MT-III are localized in the cytoplasm, during the S and G2 phase they are also seen in the nucleus [14,17,39,100]. At the cellular level MT-III are essential for the recruitment, proliferation and migration of neuroglial precursor cells and their directional mobilization towards sites of injury [19,20]. In vitro studies have recently characterized the intraneuronal signaling cascades that mediate neurite outgrowth in response to MT-III and these include activation of protein kinase C, protein kinase B/Akt, mitogen activated protein kinase, and CREB (Personal communication from collegue Johanne W Asmussen).

7. MT interactions with erythropoietin (EPO)

Erythropoietin (EPO) is a hematopoietic growth factor, which stimulates proliferation and differentiation of erythroid cells. During the last decade, EPOs neuroprotective effects have been characterized in various brain disorders and injuries 321

(e.g., cryogenic injury, EAE, stroke, concussion, Parkinsons disease) [101103]. EPO causes multifaceted neuroprotective effects via EPO receptors, and EPO and MT-III exert several common pharmaceutical actions with regard to neuroprotection and regeneration following pathology. In vitro studies show that EPO induces MT-III transcripts in erythrocyte cell lines, whilst high MT-III levels were followed by decreased expression of EPO receptor [104]. Also, data indicates that MT might be involved in EPO signal transduction as well as in its feedback regulation and thus has a role during EPOinduced erythrocyte proliferation [104], whereby MT-III might be involved in EPO signal transduction as well as in its feedback regulation. This notion of MT-III being a molecular mechanism of EPO action was also supported by in vivo studies. Hence, EPO administration induces MT-III mRNA and proteins in astrocytes of the cerebral cortex of mice, and during cerebral ischemia, EPO potentiated the infarct-mediated expression of MT-III proteins [105]. To this end, it has been shown that EPO is neuroprotective in wildtype animals suffering an ischemic insult, as EPO significantly reduces infarct area and volume [101103,106]. However, in MT-III deficient mice, EPO had no neuroprotective effects and failed to alter the infarct volume relative to controls receiving vehicle (placebo) treatment [105]. In fact, this interaction is in agreement with the in vitro results obtained by Abdel-Mageed et al. (2003). According to this, EPO signal transduction and functions are likely dependent on MT-III pathways or the intracellular levels of MT-III expression, even if MT-III proteins might exert negative feedback regulation upon the EPO receptor [104]. In fact, the data point towards MT-III as a possible intracellular regulator of the tissue response and/or sensitivity to EPO, and this interaction is likely relevant both inside and outside the CNS. Also in other experimental neuropathologies (including, but not limited to, TBI and EAE), EPO has been demonstrated as a neuroprotective agent providing new therapeutic strategies [101103]. However, the same molecular mechanism may apply to these conditions, in which MT-III also act as neuroprotective agents [27,107]. The factors linking EPO and MT-III functions are likely manifold. Yet, data support that both EPO and MT-III utilize several common pathways and affects a multitude of intracellular signals including, but not limited to various branches of mitogen activated protein kinase pathways, Rho-like GTPase signaling pathways, ERK, phosphatidylinositol-3-kinase/Akt pathways, and Janus-activated kinase 2/STAT-3 pathways. Many of these are common to both EPO and MT-III, which may contribute to the common actions of EPO and MT-III administration. However, we cannot rule out that MT-III may signal through other receptors than megalin and LRP1, and in fact, a receptor with specificity for MT-III has yet to be identified.

and EAE, as well as presenting data on their molecular mechanisms of actions. MT-III shows obvious pharmaceutical potential. The MT-mediated activation of stem/progenitor cell populations derived from the stem cell niches in the brain, opens exciting perspectives regarding the putative therapeutically use of these proteins in treating neurodegenerative diseases, such as TBI and multiple sclerosis. The question remaining to be answered is how the experimental studies will translate into clinical trials. Also, we remain to determine more precisely the possible side effects of pharmacological MT-III administration inside and outside the CNS.

References
[1] Braun, W., Vasak, M., Robbins, A. H., Stout, C. D., Wagner, G., Kagi, J. H., and Wuthrich, K. (1992) Comparison of the NMR solution structure and the x-ray crystal structure of rat metallothionein-2. Proc. Natl. Acad. Sci. USA 89, 1012410128. [2] Kagi, J. H., Himmelhoch, S. R., Whanger, P. D., Bethune, J. L., and Vallee, B. L. (1974) Equine hepatic and renal metallothioneins. Purification, molecular weight, amino acid composition, and metal content. J. Biol. Chem. 249, 35373542. [3] Hidalgo, J., Aschner, M., Zatta, P., and Vasak, M. (2001) Roles of the metallothionein family of proteins in the central nervous system. Brain. Res. Bull. 55, 133145. [4] Duncan, K. E., Ngu, T. T., Chan, J., Salgado, M. T., Merrifield, M. E., and Stillman, M. J. (2006) Peptide folding, metal-binding mechanisms, and binding site structures in metallothioneins. Exp. Biol. Med. (Maywood) 231, 14881499. [5] Feng, W., Benz, F. W., Cai, J., Pierce, W. M., and Kang, Y. J. (2006) Metallothionein disulfides are present in metallothionein-overexpressing transgenic mouse heart and increase under conditions of oxidative stress. J. Biol. Chem. 281, 681687. [6] Pearce, L. L., Gandley, R. E., Han, W., Wasserloos, K., Stitt, M., Kanai, A. J., McLaughlin, M. K., Pitt, B. R., and Levitan, E. S. (2000) Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein. Proc. Natl. Acad. Sci. USA 97, 477482. [7] Zangger, K., Shen, G., Oz, G., Otvos, J. D., and Armitage, I. M. (2001) Oxidative dimerization in metallothionein is a result of intermolecular disulphide bonds between cysteines in the alpha-domain. Biochem. J. 359, 353360. [8] Cox, D. R. and Palmiter, R. D. (1983) The metallothionein-I gene maps to mouse chromosome 8: implications for human Menkes disease, Hum. Genet. 64, 6164. [9] Karin, M., Eddy, R. L., Henry, W. M., Haley, L. L., Byers, M. G., and Shows, T. B. (1984) Human metallothionein genes are clustered on chromosome 16. Proc. Natl. Acad. Sci. USA 81, 54945498. [10] Palmiter, R. D., Findley, S. D., Whitmore, T. E., and Durnam, D. M. (1992) MT-III, a brain-specific member of the metallothionein gene family. Proc. Natl. Acad. Sci. USA 89, 63336337. [11] Quaife, C. J., Findley, S. D., Erickson, J. C., Froelick, G. J., Kelly, E. J., Zambrowicz, B. P., and Palmiter, R. D. (1994) Induction of a new metallothionein isoform (MT-IV) occurs during differentiation of stratified squamous epithelia. Biochemistry 33, 72507259. [12] Stennard, F. A., Holloway, A. F., Hamilton, J., and West, A. K. (1994) Characterisation of six additional human metallothionein genes. Biochim. Biophys. Acta. 1218, 357365. [13] West, A. K., Stallings, R., Hildebrand, C. E., Chiu, R., Karin, M., and Richards, R. I. (1990) Human metallothionein genes: structure of the functional locus at 16q13. Genomics 8, 513518. [14] Coyle, P., Philcox, J. C., Carey, L. C., and Rofe, A. M. (2002) Metallothionein: the multipurpose protein. Cell Mol. Life. Sci. 59, 627647. [15] Penkowa, M. (2006) Metallothioneins are multipurpose neuroprotectants during brain pathology. FEBS J. 273, 18571870.
BioFactors

8. Concluding remarks
In this review we present the latest knowledge on the role of MT-III in neuroprotection and regeneration following TBI 322

[16] Haq, F., Mahoney, M., and Koropatnick, J. (2003) Signaling events for metallothionein induction. Mutat. Res. 533, 211226. [17] Miles, A. T., Hawksworth, G. M., Beattie, J. H., and Rodilla, V. (2000) Induction, regulation, degradation, and biological significance of mammalian metallothioneins. Crit. Rev. Biochem. Mol. Biol. 35, 3570. [18] Penkowa, M. and Moos, T. (1995) Disruption of the blood-brain interface in neonatal rat neocortex induces a transient expression of metallothionein in reactive astrocytes. Glia 13, 217227. [19] Penkowa, M. (2006) Metallothionein III expression and roles during neuropathology in the CNS. Dan. Med. Bull. 53, 105121. [20] Penkowa, M., Caceres, M., Borup, R., Nielsen, F. C., Poulsen, C. B., Quintana, A., Molinero, A., Carrasco, J., Florit, S., Giralt, M., and Hidalgo, J. (2006) Novel roles for metallothionein-III (MT-III) in defense responses, neurogenesis, and tissue restoration after traumatic brain injury: insights from global gene expression profiling in wild-type and MT-III knockout mice. J. Neurosci. Res. 84, 14521474. [21] Penkowa, M., Giralt, M., Thomsen, P. S., Carrasco, J., and Hidalgo, J. (2001) Zinc or copper deficiency-induced impaired inflammatory response to brain trauma may be caused by the concomitant metallothionein changes, J. Neurotrauma 18, 447463. [22] Penkowa, M., Tio, L., Giralt, M., Quintana, A., Molinero, A., Atrian, S., Vasak, M., and Hidalgo, J. (2006) Specificity and divergence in the neurobiologic effects of different metallothioneins after brain injury. J. Neurosci. Res. 83, 974984. [23] Penkowa, M., Carrasco, J., Giralt, M., Moos, T., and Hidalgo, J. (1999) CNS wound healing is severely depressed in metallothionein I- and II-deficient mice. J. Neurosci. 19, 25352545. [24] Penkowa, M., Carrasco, J., Giralt, M., Molinero, A., Hernandez, J., Campbell, I. L., and Hidalgo, J. (2000) Altered central nervous system cytokine-growth factor expression profiles and angiogenesis in metallothionein-III deficient mice. J. Cereb. Blood Flow. Metab. 20, 11741189. [25] Giralt, M., Penkowa, M., Lago, N., Molinero, A., and Hidalgo, J. (2002) Metallothionein-12 protect the CNS after a focal brain injury. Exp. Neurol. 173, 114128. [26] Chung, R. S., Adlard, P. A., Dittmann, J., Vickers, J. C., Chuah, M. I., and West, A. K. (2004) Neuron-glia communication: metallothionein expression is specifically up-regulated by astrocytes in response to neuronal injury. J. Neurochem. 88, 454461. [27] Chung, R. S., Hidalgo, J., and West, A. K. (2008) New insight into the molecular pathways of metallothionein-mediated neuroprotection and regeneration. J. Neurochem. 104, 1420. [28] Kohler, L. B., Berezin, V., Bock, E., and Penkowa, M. (2003) The role of metallothionein II in neuronal differentiation and survival. Brain Res. 992, 128136. [29] Ambjorn, M., Asmussen, J. W., Lindstam, M., Gotfryd, K., Jacobsen, C., Kiselyov, V. V., Moestrup, S. K., Penkowa, M., Bock, E., and Berezin, V. (2008) Metallothionein and a peptide modeled after metallothionein, EmtinB, induce neuronal differentiation and survival through binding to receptors of the low-density lipoprotein receptor family. J. Neurochem. 104, 2137. [30] Chung, R. S., Penkowa, M., Dittmann, J., King, C. E., Bartlett, C., Asmussen, J. W., Hidalgo, J., Carrasco, J., Leung, Y. K., Walker, A. K., Fung, S. J., Dunlop, S. A., Fitzgerald, M., Beazley, L. D., Chuah, M. I., Vickers, J. C., and West, A. K. (2008) Redefining the role of metallothionein within the injured brain: extracellular metallothioneins play an important role in the astrocyte-neuron response to injury. J. Biol. Chem. 283, 1534915358. [31] Penkowa, M., Giralt, M., Camats, J., and Hidalgo, J. (2002) Metallothionein 12 protect the CNS during neuroglial degeneration induced by 6-aminonicotinamide. J. Comp. Neurol. 444, 174189. [32] Penkowa, M. and Hidalgo, J. (2000) Metallothionein III expression and their role in experimental autoimmune encephalomyelitis. Glia. 32, 247263. [33] Chung, R. S., Vickers, J. C., Chuah, M. I., and West, A. K. (2003) Metallothionein-IIA promotes initial neurite elongation and postinjury reactive neurite growth and facilitates healing after focal cortical brain injury. J. Neurosci. 23, 33363342.

[34] Penkowa, M. and Hidalgo, J. (2001) Metallothionein treatment reduces proinflammatory cytokines IL-6 and TNF-alpha and apoptotic cell death during experimental autoimmune encephalomyelitis (EAE). Exp. Neurol. 170, 114. [35] Penkowa, M., Espejo, C., Martinez-Caceres, E. M., Poulsen, C. B., Montalban, X., and Hidalgo, J. (2001) Altered inflammatory response and increased neurodegeneration in metallothionein III deficient mice during experimental autoimmune encephalomyelitis. J. Neuroimmunol. 119, 248260. [36] Comi, C., Leone, M., Bonissoni, S., DeFranco, S., Bottarel, F., Mezzatesta, C., Chiocchetti, A., Perla, F., Monaco, F., and Dianzani, U. (2000) Defective T cell fas function in patients with multiple sclerosis. Neurology 55, 921927. [37] Huang, W. X., Huang, M. P., Gomes, M. A., and Hillert, J. (2000) Apoptosis mediators fasL and TRAIL are upregulated in peripheral blood mononuclear cells in MS. Neurology 55, 928934. [38] Segal, B. M. and Cross, A. H. (2000) Fas(t) track to apoptosis in MS: TNF receptors may suppress or potentiate CNS demyelination. Neurology 55, 906907. [39] Cherian, M. G. and Kang, Y. J. (2006) Metallothionein and liver cell regeneration. Exp. Biol. Med. (Maywood.) 231, 138144. [40] Masters, B. A., Kelly, E. J., Quaife, C. J., Brinster, R. L., and Palmiter, R. D. (1994) Targeted disruption of metallothionein I and II genes increases sensitivity to cadmium. Proc. Natl. Acad. Sci. USA 91, 584588. [41] Fitzgerald, M., Nairn, P., Bartlett, C. A., Chung, R. S., West, A. K., and Beazley, L. D. (2007) Metallothionein-IIA promotes neurite growth via the megalin receptor. Exp. Brain Res. 183, 171180. [42] Christensen, E. I. and Birn, H. (2001) Megalin and cubilin: synergistic endocytic receptors in renal proximal tubule. Am. J. Physiol. Renal. Physiol. 280, F562F573. [43] Christensen, E. I. and Birn, H. (2002) Megalin and cubilin: multifunctional endocytic receptors. Nat. Rev. Mol. Cell Biol. 3, 256266. [44] Moestrup, S. K., Gliemann, J., and Pallesen, G. (1992) Distribution of the alpha 2-macroglobulin receptor/low density lipoprotein receptorrelated protein in human tissues. Cell. Tissue Res. 269, 375382. [45] Zheng, G., Bachinsky, D. R., Stamenkovic, I., Strickland, D. K., Brown, D., Andres, G., and McCluskey, R. T. (1994) Organ distribution in rats of two members of the low-density lipoprotein receptor gene family, gp330 and LRP/alpha 2MR, and the receptor-associated protein (RAP). J. Histochem. Cytochem. 42, 531542. [46] Campana, W. M., Li, X., Dragojlovic, N., Janes, J., Gaultier, A., and Gonias, S. L. (2006) The low-density lipoprotein receptor-related protein is a pro-survival receptor in Schwann cells: possible implications in peripheral nerve injury. J. Neurosci. 26, 1119711207. [47] Czekay, R. P., Orlando, R. A., Woodward, L., Lundstrom, M., and Farquhar, M. G. (1997) Endocytic trafficking of megalin/RAP complexes: dissociation of the complexes in late endosomes. Mol. Biol. Cell 8, 517532. [48] Fisher, C. E. and Howie, S. E. (2006) The role of megalin (LRP-2/ Gp330) during development. Dev. Biol. 296, 279297. [49] Futakawa, N., Kondoh, M., Ueda, S., Higashimoto, M., Takiguchi, M., Suzuki, S., and Sato, M. (2006) Involvement of oxidative stress in the synthesis of metallothionein induced by mitochondrial inhibitors. Biol. Pharm. Bull. 29, 20162020. [50] Theocharis, S. E., Margeli, A. P., Klijanienko, J. T., and Kouraklis, G. P. (2004) Metallothionein expression in human neoplasia. Histopathology 45, 103118. [51] Kelly, E. J. and Palmiter, R. D. (1996) A murine model of Menkes disease reveals a physiological function of metallothionein. Nat. Genet. 13, 219222. [52] Kelly, E. J., Quaife, C. J., Froelick, G. J., and Palmiter, R. D. (1996) Metallothionein I and II protect against zinc deficiency and zinc toxicity in mice. J. Nutr. 126, 17821790. [53] Kondo, Y., Rusnak, J. M., Hoyt, D. G., Settineri, C. E., Pitt, B. R., and Lazo, J. S. (1997) Enhanced apoptosis in metallothionein null cells. Mol. Pharmacol. 52, 195201. [54] Michalska, A. E. and Choo, K. H. (1993) Targeting and germ-line transmission of a null mutation at the metallothionein I and II loci in mouse. Proc. Natl. Acad. Sci. USA 90, 80888092.

Metallothionein-III in neuroprotection

323

[55] Cai, L. and Cherian, M. G. (2003) Zinc-metallothionein protects from DNA damage induced by radiation better than glutathione and copperor cadmium-metallothioneins. Toxicol. Lett. 136, 193198. [56] Formigari, A., Irato, P., and Santon, A. (2007) Zinc, antioxidant systems and metallothionein in metal mediated-apoptosis: biochemical and cytochemical aspects. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 146, 443459. [57] Abel, J. and de Ruiter, N. (1989) Inhibition of hydroxyl-radical-generated DNA degradation by metallothionein. Toxicol. Lett. 47, 191196. [58] Ghoshal, K., Majumder, S., Li, Z., Bray, T. M., and Jacob, S. T. (1999) Transcriptional induction of metallothionein-I and -II genes in the livers of Cu,Zn-superoxide dismutase knockout mice. Biochem. Biophys. Res. Commun. 264, 735742. [59] Tamai, K. T., Gralla, E. B., Ellerby, L. M., Valentine, J. S., and Thiele, D. J. (1993) Yeast and mammalian metallothioneins functionally substitute for yeast copper-zinc superoxide dismutase. Proc. Natl. Acad. Sci. USA 90, 80138017. [60] Haidara, K., Moffatt, P., and Denizeau, F. (1999) Metallothionein induction attenuates the effects of glutathione depletors in rat hepatocytes. Toxicol. Sci. 49, 297305. [61] Andrews, G. K. (2000) Regulation of metallothionein gene expression by oxidative stress and metal ions. Biochem. Pharmacol. 59, 95104. [62] Kondoh, M., Inoue, Y., Atagi, S., Futakawa, N., Higashimoto, M., and Sato, M. (2001) Specific induction of metallothionein synthesis by mitochondrial oxidative stress. Life Sci. 69, 21372146. [63] Stankovic, R. K., Chung, R. S., and Penkowa, M. (2007) Metallothioneins I and II: neuroprotective significance during CNS pathology. Int. J. Biochem. Cell Biol. 39, 484489. [64] Penkowa, M., Florit, S., Giralt, M., Quintana, A., Molinero, A., Carrasco, J., and Hidalgo, J. (2005) Metallothionein reduces central nervous system inflammation, neurodegeneration, and cell death following kainic acid-induced epileptic seizures. J. Neurosci. Res. 79, 522534. [65] Bell, S. G. and Vallee, B. L. (2009) The metallothionein/thionein system: an oxidoreductive metabolic zinc link. Chembiochem. 10, 5562. [66] Maret, W. (2008) Metallothionein redox biology in the cytoprotective and cytotoxic functions of zinc. Exp. Gerontol. 43, 363369. [67] Feng, W., Cai, J., Pierce, W. M., Franklin, R. B., Maret, W., Benz, F. W., and Kang, Y. J. (2005) Metallothionein transfers zinc to mitochondrial aconitase through a direct interaction in mouse hearts. Biochem. Biophys. Res. Commun. 332, 853858. [68] Singh, K. K., Desouki, M. M., Franklin, R. B., and Costello, L. C. (2006) Mitochondrial aconitase and citrate metabolism in malignant and nonmalignant human prostate tissues. Mol. Cancer 5, 14. [69] Maret, W. (2004) Zinc and sulfur: a critical biological partnership. Biochemistry 43, 33013309. [70] Kang, Y. J. (2006) Metallothionein redox cycle and function. Exp. Biol. Med. (Maywood) 231, 14591467. [71] Maret, W. (2000) The function of zinc metallothionein: a link between cellular zinc and redox state. J Nutr., 130, 1455S1458S. [72] Maret, W. (1995) Metallothionein/disulfide interactions, oxidative stress, and the mobilization of cellular zinc. Neurochem. Int. 27, 111117. [73] Cai, L., Wang, Y., Zhou, G., Chen, T., Song, Y., Li, X., and Kang, Y. J. (2006) Attenuation by metallothionein of early cardiac cell death via suppression of mitochondrial oxidative stress results in a prevention of diabetic cardiomyopathy. J. Am. Coll. Cardiol. 48, 16881697. [74] Doering, P., Danscher, G., Larsen, A., Bruhn, M., Sondergaard, C., and Stoltenberg, M. (2007) Changes in the vesicular zinc pattern following traumatic brain injury. Neuroscience 150, 93103. [75] Mann, J. J. and Fraker, P. J. (2005) Zinc pyrithione induces apoptosis and increases expression of Bim. Apoptosis 10, 369379. [76] Tekur, S. and Ho, S. M. (2002) Ribozyme-mediated downregulation of human metallothionein II(a) induces apoptosis in human prostate and ovarian cancer cell lines. Mol. Carcinog. 33, 4455. [77] Hainaut, P. and Mann, K. (2001) Zinc binding and redox control of p53 structure and function. Antioxid. Redox. Signal. 3, 611623. [78] Meplan, C., Richard, M. J., and Hainaut, P. (2000) Metalloregulation of the tumor suppressor protein p53: zinc mediates the renaturation of

[79]

[80]

[81]

[82]

[83]

[84] [85]

[86]

[87]

[88]

[89]

[90]

[91]

[92]

[93] [94]

[95]

[96]

[97]

[98]

p53 after exposure to metal chelators in vitro and in intact cells. Oncogene 19, 52275236. Ostrakhovitch, E. A., Olsson, P. E., Jiang, S., and Cherian, M. G. (2006) Interaction of metallothionein with tumor suppressor p53 protein. FEBS Lett. 580, 12351238. Ostrakhovitch, E. A., Olsson, P. E., Hofsten von, J., and Cherian, M. G. (2007) P53 mediated regulation of metallothionein transcription in breast cancer cells. J. Cell Biochem. 102, 15711583. Lenzlinger, P. M., Morganti-Kossmann, M. C., Laurer, H. L., and McIntosh, T. K. (2001) The duality of the inflammatory response to traumatic brain injury. Mol. Neurobiol. 24, 169181. Lucas, S. M., Rothwell, N. J., and Gibson, R. M. (2006) The role of inflammation in CNS injury and disease. Br. J. Pharmacol. 147 (Suppl 1), S232S240. Nair, A., Frederick, T. J., and Miller, S. D. (2008) Astrocytes in multiple sclerosis: a product of their environment. Cell. Mol. Life Sci. 65, 27022720. Sofroniew, M. V. (2005) Reactive astrocytes in neural repair and protection. Neuroscientist 11, 400407. Correa-Cerro, L. S. and Mandell, J. W. (2007) Molecular mechanisms of astrogliosis: new approaches with mouse genetics. J. Neuropathol. Exp. Neurol. 66, 169176. Soto, I., Rosenthal, J. J., Blagburn, J. M., and Blanco, R. E. (2006) Fibroblast growth factor 2 applied to the optic nerve after axotomy up-regulates BDNF and TrkB in ganglion cells by activating the ERK and PKA signaling pathways. J. Neurochem. 96, 8296. Hisaoka, K., Maeda, N., Tsuchioka, M., and Takebayashi, M. (2008) Antidepressants induce acute CREB phosphorylation and CRE-mediated gene expression in glial cells: a possible contribution to GDNF production. Brain Res. 1196, 5358. Kanda, N., Koike, S., and Watanabe, S. (2005) Prostaglandin E2 enhances neurotrophin-4 production via EP3 receptor in human keratinocytes. J. Pharmacol. Exp. Ther. 315, 796804. Peng, C. H., Huang, C. N., Hsu, S. P. and Wang, C. J. (2007) Penta-acetyl geniposide-induced apoptosis involving transcription of NGF/p75 via MAPK-mediated AP-1 activation in C6 glioma cells. Toxicology 238, 130139. Dhandapani, K. M., Khan, M. M., Wade, F. M., Wakade, C., Mahesh, V. B., and Brann, D. W. (2007) Induction of transforming growth factorbeta1 by basic fibroblast growth factor in rat C6 glioma cells and astrocytes is mediated by MEK/ERK signaling and AP-1 activation, J. Neurosci. Res., 85, 10331045. Akiyama, H., Tanaka, T., Maeno, T., Kanai, H., Kimura, Y., Kishi, S., and Kurabayashi, M. (2002) Induction of VEGF gene expression by retinoic acid through Sp1-binding sites in retinoblastoma Y79 cells. Invest. Ophthalmol. Vis. Sci. 43, 13671374. Tanaka, T., Kanai, H., Sekiguchi, K., Aihara, Y., Yokoyama, T., Arai, M., Kanda, T., Nagai, R., and Kurabayashi, M. (2000) Induction of VEGF gene transcription by IL-1 beta is mediated through stress-activated MAP kinases and Sp1 sites in cardiac myocytes. J. Mol. Cell Cardiol. 32, 19551967. Metsis, M. (2001) Genes for neurotrophic factors and their receptors: structure and regulation. Cell Mol. Life Sci. 58, 10141020. Marzella, P. L., Gillespie, L. N., Clark, G. M., Bartlett, P. F., and Kilpatrick, T. J. (1999) The neurotrophins act synergistically with LIF and members of the TGF-beta superfamily to promote the survival of spiral ganglia neurons in vitro. Hear Res. 138, 7380. Xiao, Z., Kong, Y., Yang, S., Li, M., Wen, J., and Li, L. (2007) Upregulation of Flk-1 by bFGF via the ERK pathway is essential for VEGF-mediated promotion of neural stem cell proliferation. Cell Res. 17, 7379. Marzella, P. L., Clark, G. M., Shepherd, R. K., Bartlett, P. F., and Kilpatrick, T. J. (1998) Synergy between TGF-beta 3 and NT-3 to promote the survival of spiral ganglia neurones in vitro. Neurosci. Lett. 240, 7780. Krieglstein, K., Farkas, L., and Unsicker, K. (1998) TGF-beta regulates the survival of ciliary ganglionic neurons synergistically with ciliary neurotrophic factor and neurotrophins. J. Neurobiol. 37, 563572. Joshi, B., Ordonez-Ercan, D., Dasgupta, P., and Chellappan, S. (2005) Induction of human metallothionein 1G promoter by VEGF and heavy

324

BioFactors

metals: differential involvement of E2F and metal transcription factors. Oncogene 24, 22042217. [99] Miyashita, H., and Sato, Y. (2005) Metallothionein 1 is a downstream target of vascular endothelial zinc finger 1 (VEZF1) in endothelial cells and participates in the regulation of angiogenesis. Endothelium 12, 163170. [100] Sato, M. and Kondoh, M. (2002) Recent studies on metallothionein: protection against toxicity of heavy metals and oxygen free radicals. Tohoku J. Exp. Med. 196, 922. [101] Brines, M. L., Ghezzi, P., Keenan, S., Agnello, D., de Lanerolle, N. C., Cerami, C., Itri, L. M., and Cerami, A. (2000) Erythropoietin crosses the blood-brain barrier to protect against experimental brain injury. Proc. Natl. Acad. Sci. USA 97, 1052610531. [102] Maiese, K., Li, F., and Chong, Z. Z. (2005) New avenues of exploration for erythropoietin. JAMA 293, 9095.

[103] Rabie, T. and Marti, H. H. (2008) Brain protection by erythropoietin: a manifold task. Physiology (Bethesda) 23, 263274. [104] Abdel-Mageed, A. B., Zhao, F., Rider, B. J., and Agrawal, K. C. (2003) Erythropoietin-induced metallothionein gene expression: role in proliferation of K562 cells. Exp. Biol. Med. (Maywood.) 228, 10331039. [105] Wakida, K., Shimazawa, M., Hozumi, I., Satoh, M., Nagase, H., Inuzuka, T., and Hara, H. (2007) Neuroprotective effect of erythropoietin, and role of metallothionein-1 and -2, in permanent focal cerebral ischemia. Neuroscience 148, 105114. [106] Liu, X. B., Wang, J. A., Yu, S. P., Keogh, C. L., and Wei, L. (2008) Therapeutic strategy of erythropoietin in neurological disorders. CNS Neurol. Disord. Drug. Targets 7, 227234. [107] West, A. K., Hidalgo, J., Eddins, D., Levin, E. D., and Aschner, M. (2008) Metallothionein in the central nervous system: Roles in protection, regeneration and cognition. Neurotoxicology 29, 489503.

Metallothionein-III in neuroprotection

325