Mammalian Metallothionein-2A and Oxidative Stress

Mammalian metallothionein-2A (MT2A) has received considerable attention in recent years due to its crucial pathophysiological role in anti-oxidant, anti-apoptosis, detoxification and anti-inflammation. For many years, most studies evaluating the effects of MT2A have focused on reactive oxygen species (ROS), as second messengers that lead to oxidative stress injury of cells and tissues. Recent studies have highlighted that oxidative stress could activate mitogen-activated protein kinases (MAPKs), and MT2A, as a mediator of MAPKs, to regulate the pathogenesis of various diseases. However, the molecule mechanism of MT2A remains elusive. A deeper understanding of the functional, biochemical and molecular characteristics of MT2A would be identified, in order to bring new opportunities for oxidative stress therapy.


The Role of Zn
MT2A is a powerful scavenger of free radicals through its cysteine residues [15]. It is a Zn chelator when the amount of Zn is excessive and as a scavenger of ROS when oxidative stress is elevated [37]. The binding Zn of apoMT would maintain the stability of MT [42], and oxidative stress could also trigger Zn to integrate with apoMT [43]. Unfortunately, no further studies focusing on MT2A have been performed. Low dose of Zn (50 μM ZnSO4) could up-regulate MT2A expression in reducing cytotoxicity through inhibiting oxidative stress and DNA damage [44], whereas the high dose (100 μM ZnSO4) is responsible for neurotoxicity through ERK1/2 [45]. MT2A is capable of Figure 1. Structure of human metallothionein-2 (MT-2). Amino acid sequence and 3D structure were retrieved from UniProt (P02795). Class I MT2 contains 2 metal-binding domains: four divalent ions are chelated within cluster A of the α-domain and are coordinated via cysteinyl thiolate bridges to 11 cysteine ligands. Cluster B, the corresponding region within the β-domain, can ligate three divalent ions to 9 cysteines. The 3D structures show that cluster A could bind three metal ions (Cd 2+ ), and cluster B could bind four metal ions (Cd 2+ ), respectively.

The Role of Zn
MT2A is a powerful scavenger of free radicals through its cysteine residues [15]. It is a Zn chelator when the amount of Zn is excessive and as a scavenger of ROS when oxidative stress is elevated [37]. The binding Zn of apoMT would maintain the stability of MT [42], and oxidative stress could also trigger Zn to integrate with apoMT [43]. Unfortunately, no further studies focusing on MT2A have been performed. Low dose of Zn (50 µM ZnSO 4 ) could up-regulate MT2A expression in reducing cytotoxicity through inhibiting oxidative stress and DNA damage [44], whereas the high dose (100 µM ZnSO 4 ) is responsible for neurotoxicity through ERK1/2 [45]. MT2A is capable of binding Zn 2+ , known as Zn 7 MT2A, which affects ionic homeostasis and subsequent neurotoxicity of cultured cortical neurons [10]. The characterization of the metal-binding abilities of MT2A shows a clear preference towards Zn 2+ coordination, compared to Cd 2+ and Cu + [39].

Anti-Apoptosis
A wide range of adverse stimuli, such as oxidative stress could cause cell apoptosis [21]. MT2A reduces adriamycin-induced myocardial injury through inhibition of oxidative stress-mediated mitochondrial cytochrome-c release and activated caspase-3 [47], protects human umbilical vein endothelial cells from lipopolysaccharide (LPS)-associated apoptosis, and also influences cellular behaviors such as proliferation and chemotaxis by binding to membrane receptors [52]. MT2A could also protect endoplasmic reticulum (ER) stress-induced cardiac failure associated with attenuation of myocardial apoptosis [53]. Knockdown of MT2A could down-regulate Zn level and affect cell apoptosis [4]. Moreover, MT2A is a protective protein from apoptosis by down-regulating the expression of Bax, caspase-3, caspase-9, and caspase-12 [4,54].

Mitochondrial Stress
Oxidative stress-mediated damage to mitochondrial DNA could be observed in patients with diabetes mellitus and atherosclerosis [59]. MT2A exerts antioxidant effects against mitochondrial superoxide [60]. Over expression of MT2A can decrease oxygen consumption, down-regulate cellular ATP levels and decrease oxidative phosphorylation capacity, and interact with mitochondrial complexes indirectly, which might be involved in the inhibition of certain respiratory enzymes via metal binding [61]. MT2A could suppress ischemia/reperfusion (I/R)-induced myocardial apoptosis mediated by mitochondrial stress [62] (Figure 2). As for downstream signaling, intrinsic apoptotic signaling leads to mitochondrial membrane permeabilization and releases cytochrome-c into the cytosol through JNK signal [63].

Lysosomal Membrane Permeabilization (LMP) Stress (LMPS)
Oxidative stress could induce LMP through activation of lysosomal hydrolytic enzymes [70], which causes apoptosis [71]. MT-1/2A up-regulation has been reported to protect against LMP induced by various kinds of oxidative stress [72]. Lysosomal delivery of up-regulated MT2A is the key mechanism by which autophagy protects cells against LMPS [73] (Figure 2). LMPS is associated with activation of MAPKs, for instance, JNK has an important pro-apoptotic function, which mediates the upstream of LMP and phosphorylation [63].

Biological Membrane Lipid Peroxidation Injury (LPI)
MT2A could inhibit LPI and improve recovery after transient brain I/R in rats [74] and other researches have demonstrated that LPI increased by I/R-induced myocardial injury are dramatically decreased in MT-overexpressing mice and the oxidative damage in the lipid membranes is related to lipid peroxide (LPO)and MT levels [62] (Figure 2). ERS stimulates autophagy in a JNK-dependent manner and promotes cell survival during oxidative stress [64]. MT2A could suppress the expression of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP) during Ang II-induced ERS [65] (Figure 2). ERS augments left ventricular diameter, suppresses heart contractility, and induces liver injury, which are significantly attenuated or ablated by MTs [66,67]. Additionally, ERS leads to accumulation of unfolded proteins in ER, which could activate multiple signaling pathways including JNK, p38 and NF-κB [68,69].

Lysosomal Membrane Permeabilization (LMP) Stress (LMPS)
Oxidative stress could induce LMP through activation of lysosomal hydrolytic enzymes [70], which causes apoptosis [71]. MT-1/2A up-regulation has been reported to protect against LMP induced by various kinds of oxidative stress [72]. Lysosomal delivery of up-regulated MT2A is the key mechanism by which autophagy protects cells against LMPS [73] (Figure 2). LMPS is associated with activation of MAPKs, for instance, JNK has an important pro-apoptotic function, which mediates the upstream of LMP and phosphorylation [63].

Biological Membrane Lipid Peroxidation Injury (LPI)
MT2A could inhibit LPI and improve recovery after transient brain I/R in rats [74] and other researches have demonstrated that LPI increased by I/R-induced myocardial injury are dramatically decreased in MT-overexpressing mice and the oxidative damage in the lipid membranes is related to lipid peroxide (LPO)and MT levels [62] (Figure 2).  ERS stimulates autophagy in a JNK-dep oxidative stress [64]. MT2A could suppress (C/EBP) homologous protein (CHOP) during A ventricular diameter, suppresses heart contrac attenuated or ablated by MTs [66,67]. Addition in ER, which could activate multiple signaling  ERS stimulates autophagy in a JNK-dependent manner and promotes cell survival during ative stress [64]. MT2A could suppress the expression of CCAAT/enhancer-binding protein BP) homologous protein (CHOP) during Ang II-induced ERS [65] (Figure 2). ERS augments left ricular diameter, suppresses heart contractility, and induces liver injury, which are significantly uated or ablated by MTs [66,67]. Additionally, ERS leads to accumulation of unfolded proteins , which could activate multiple signaling pathways including JNK, p38 and NF-κB [68,69].
. Lysosomal Membrane Permeabilization (LMP) Stress (LMPS) Oxidative stress could induce LMP through activation of lysosomal hydrolytic enzymes [70], h causes apoptosis [71]. MT-1/2A up-regulation has been reported to protect against LMP ced by various kinds of oxidative stress [72]. Lysosomal delivery of up-regulated MT2A is the mechanism by which autophagy protects cells against LMPS [73] (Figure 2). LMPS is associated activation of MAPKs, for instance, JNK has an important pro-apoptotic function, which iates the upstream of LMP and phosphorylation [63].
. Biological Membrane Lipid Peroxidation Injury (LPI) MT2A could inhibit LPI and improve recovery after transient brain I/R in rats [74] and other rches have demonstrated that LPI increased by I/R-induced myocardial injury are dramatically eased in MT-overexpressing mice and the oxidative damage in the lipid membranes is related to peroxide (LPO)and MT levels [62] (Figure 2).

The Role of MT2A in MAPKs Signals
MAPKs comprise a family of serine/threonine phosphorylating proteins, which contain three main branches: ERK, JNK and p38 [75]. MTs play an important role in improving the LPS-induced cardiac dysfunction with activated MAPK [76] (Figure 3). However, MT plays a key role in preventing hypoxia-induced renal injury via Nrf2, owing to inactivation of AKT and ERK [58]. Arsenic trioxide could induce H9c2 cell death in a dose-and time-dependent manner with a significant activation of MAPKs, but not in MT-H9c2 cells [77]. Importantly, the protective effect of MT on arsenic trioxide-induced apoptotic cell is completely recaptured in heart with a significant prevention of MAPKs [77]. In Cd-induced apoptotic cells, MT is less expressed in Cd-sensitive cells but p-JNK is increased, and a strong activator of JNK, R0318220, could reverse the Cd-sensitive phenotype in Cd-resist cells, and this research also showed that p-JNK1/2 is markedly up-regulated in MT −/− cells compared with MT +/+ cells through Cd treatment, suggesting that MT might inhibit JNK1/2 activation [16]. Another study has indicated that the suppression of JNK is mediated by ROS [78]. Whether MT directly inhibits JNK phosphorylation or not remains elusive in present studies.

The Role of MT2A in MAPKs Signals
MAPKs comprise a family of serine/threonine phosphorylating proteins, which contain three main branches: ERK, JNK and p38 [75]. MTs play an important role in improving the LPS-induced cardiac dysfunction with activated MAPK [76] (Figure 3). However, MT plays a key role in preventing hypoxia-induced renal injury via Nrf2, owing to inactivation of AKT and ERK [58]. Arsenic trioxide could induce H9c2 cell death in a dose-and time-dependent manner with a significant activation of MAPKs, but not in MT-H9c2 cells [77]. Importantly, the protective effect of MT on arsenic trioxide-induced apoptotic cell is completely recaptured in heart with a significant prevention of MAPKs [77]. In Cd-induced apoptotic cells, MT is less expressed in Cd-sensitive cells but p-JNK is increased, and a strong activator of JNK, R0318220, could reverse the Cd-sensitive phenotype in Cd-resist cells, and this research also showed that p-JNK1/2 is markedly up-regulated in MT -/cells compared with MT +/+ cells through Cd treatment, suggesting that MT might inhibit JNK1/2 activation [16]. Another study has indicated that the suppression of JNK is mediated by ROS [78]. Whether MT directly inhibits JNK phosphorylation or not remains elusive in present studies.

Cardiovascular Disease
MT2A is a potent antioxidant in heart [37,53,79] (Table 1). More importantly, antioxidant is shown to exert beneficial effects in hypertension, atherosclerosis, ischemic heart disease, cardiomyopathy and congestive heart failure [17,80]. Although the mechanism underlying myocardial protection from I/R injury through MT has not been fully understood, a large pool of evidence has demonstrated that oxidative stress is a critical mediator for myocardial damage during I/R [27,62]. MT2A might play a role in cardiovascular protection through radical scavenging activities and suppression of lipid peroxidation [81]. MT2A polymorphism is associated with atherosclerosis on coronary artery [26], and carotid artery [82]. The cardioprotective property of MT

The Role of MT2A in MAPKs Signals
MAPKs comprise a family of serine/threonine phosphorylating proteins, which contain three main branches: ERK, JNK and p38 [75]. MTs play an important role in improving the LPS-induced cardiac dysfunction with activated MAPK [76] (Figure 3). However, MT plays a key role in preventing hypoxia-induced renal injury via Nrf2, owing to inactivation of AKT and ERK [58]. Arsenic trioxide could induce H9c2 cell death in a dose-and time-dependent manner with a significant activation of MAPKs, but not in MT-H9c2 cells [77]. Importantly, the protective effect of MT on arsenic trioxide-induced apoptotic cell is completely recaptured in heart with a significant prevention of MAPKs [77]. In Cd-induced apoptotic cells, MT is less expressed in Cd-sensitive cells but p-JNK is increased, and a strong activator of JNK, R0318220, could reverse the Cd-sensitive phenotype in Cd-resist cells, and this research also showed that p-JNK1/2 is markedly up-regulated in MT -/cells compared with MT +/+ cells through Cd treatment, suggesting that MT might inhibit JNK1/2 activation [16]. Another study has indicated that the suppression of JNK is mediated by ROS [78]. Whether MT directly inhibits JNK phosphorylation or not remains elusive in present studies.

Cardiovascular Disease
MT2A is a potent antioxidant in heart [37,53,79] (Table 1). More importantly, antioxidant is shown to exert beneficial effects in hypertension, atherosclerosis, ischemic heart disease, cardiomyopathy and congestive heart failure [17,80]. Although the mechanism underlying myocardial protection from I/R injury through MT has not been fully understood, a large pool of evidence has demonstrated that oxidative stress is a critical mediator for myocardial damage during I/R [27,62]. MT2A might play a role in cardiovascular protection through radical scavenging activities and suppression of lipid peroxidation [81]. MT2A polymorphism is associated with : caused definitely, brown dotted arrows (C/EBP) homologous protein (CHOP) during Ang II-induced ERS [65] (Figure 2). ERS augments left ventricular diameter, suppresses heart contractility, and induces liver injury, which are significantly attenuated or ablated by MTs [66,67]. Additionally, ERS leads to accumulation of unfolded proteins in ER, which could activate multiple signaling pathways including JNK, p38 and NF-κB [68,69].

Lysosomal Membrane Permeabilization (LMP) Stress (LMPS)
Oxidative stress could induce LMP through activation of lysosomal hydrolytic enzymes [70], which causes apoptosis [71]. MT-1/2A up-regulation has been reported to protect against LMP induced by various kinds of oxidative stress [72]. Lysosomal delivery of up-regulated MT2A is the key mechanism by which autophagy protects cells against LMPS [73] (Figure 2). LMPS is associated with activation of MAPKs, for instance, JNK has an important pro-apoptotic function, which mediates the upstream of LMP and phosphorylation [63].

Biological Membrane Lipid Peroxidation Injury (LPI)
MT2A could inhibit LPI and improve recovery after transient brain I/R in rats [74] and other researches have demonstrated that LPI increased by I/R-induced myocardial injury are dramatically decreased in MT-overexpressing mice and the oxidative damage in the lipid membranes is related to lipid peroxide (LPO)and MT levels [62] (Figure 2).

Cardiovascular Disease
MT2A is a potent antioxidant in heart [37,53,79] (Table 1). More importantly, antioxidant is shown to exert beneficial effects in hypertension, atherosclerosis, ischemic heart disease, cardiomyopathy and congestive heart failure [17,80]. Although the mechanism underlying myocardial protection from I/R injury through MT has not been fully understood, a large pool of evidence has demonstrated that oxidative stress is a critical mediator for myocardial damage during I/R [27,62]. MT2A might play a role in cardiovascular protection through radical scavenging activities and suppression of lipid peroxidation [81]. MT2A polymorphism is associated with atherosclerosis on coronary artery [26], and carotid artery [82]. The cardioprotective property of MT is involved in diabetes mellitus-, obesityand aging-induced cardiac damage [28,83]. ERS directly triggers cardiomyocyte dysfunction and MT could ablate the process through up-regulating the level of JNK phosphorylation [84]. ERS inhibitor tauroursodeoxycholic acid could reverse the process [85]. Moreover, MT is able to prevent myocardial anomalies through restoration of autophagy in hypertensive heart diseases [79].

Nervous System Disease
Multiple nervous system diseases are closely related to MT2A. MT2A is the most significantly up-regulated transcript in ischemic head [86], and it is a novel neuroprotective factor to prevent ischemic injury [74] (Table 1). Simultaneously, MT2A is a critical component in the maintenance of immune homeostasis, as it is demonstrated in autoimmune encephalomyelitis disease [87]. Parkinson's disease is one of the most common progressive neurodegenerative disorders with increased oxidative stress and MT2A released from astrocytes is a potent protector of dopaminergic neuron [88]. Alzheimer's disease is triggered by the deposition of insoluble extracellular b-amyloid (Ab) plaque, and MT2A is capable of protecting against Ab aggregation and toxicity for therapeutic approach to Alzheimer's disease [10]. Additionally, MT-1/2 knockout mice would result in embryonic lethality in a model of Menkes disease (a copper efflux disease) [89].

Cancers
MT-1/2 deficiency predisposes mice more sensitive to early life Pb exposure with regard to testes tumors, renal and urinary bladder preneoplastic lesions [33]. MT2A over-expression is associated with cell proliferation in cancerous breast tissue [90], with significantly modified breast cancer risk, and cell cycle is inhibited through silencing MT2A [91]. MT2A predicts high therapeutic value in hepatocellular carcinoma [92], whereas predicts poor survival in glioblastoma multiforme [93]. MT2A might be a chemosensitive indicator in gastric cancer (GC) [94], and another study found that MT2A might play a role in suppressing tumor activity through inhibiting NF-κB and might be a prognostic biomarker and potential target for individual therapy of GC [95] (Table 1). MT2A polymorphismis closely correlated with neoplasm, implicated in laryngeal cancer [96]. In addition, MT2A contributes to chemotherapy resistance in osteosarcoma [97]. Finally, it has to be mentioned that the MT2A has differential outcome in various types of cancer that may be tissue or cell type dependent, just like E2 factor, including cellular proliferation, apoptosis and tumor kinetics [98].

Aging
MT2A is crucial for the immune efficiency during aging and age-related diseases [99]. Up-regulated expression of MT2A in kidney with aging might play a protective role, which is closely related to aging [34]. Additionally, MT2A gene polymorphisms are associated with aging in Turkey [100] (Table 1).

Diabetes Mellitus
MT2A could prevent diabetes-induced cardiac ERS, which contributes to prevent dilated cardiomyopathy (DCM) [65] (Table 1). MT2A plays an important role in antioxidant defense in type2 diabetes mellitus through modulating glutathione, which promotes phosphorylation of insulin receptors through enhancing transportation of glucose into cells [101]. Moreover, MT2Ais correlated to diabetic atherosclerosis in patients [102].

Hepatal and Gastrointestinal Diseases
It was found that MT2A is slightly more expressed in both chronic hepatitis and Wilson's disease [103] (Table 1). Furthermore, MT2A could activate human hepatic stellate cells to up-regulate the expression of collagenase genes, which might exert the therapeutic effect upon liver fibrosis [104]. Additionally, MT2A could prevent colonic mucosal inflammation in murine experimental colitis [105]. Human: LX-2 cell from human hepatic stellate; pEGFP-N1-hMT-IIA and pEGFP-N1 were transfected into LX-2 cells; n = not disclosed Liver fibrosis might be treated by MT2A MT7: human MT-IIA over-expressing cardiac cell line; BBH: bovine brain homogenate rats; DA: Dark Agouti rats; IID: itai-itai disease; DCM: dilated cardiomyopathy.

Conclusions
MT2A is intimately associated with oxidative stress, mediated by subcellular pathways of mitochondria, ER, lysosomal, and lipidosome, as well as MAPKs (ERK, JNK and p38) signals.