Zinc homeostasis and signaling in health and diseases
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The essential trace element zinc (Zn) is widely required in cellular functions, and abnormal Zn homeostasis causes a variety of health problems that include growth retardation, immunodeficiency, hypogonadism, and neuronal and sensory dysfunctions. Zn homeostasis is regulated through Zn transporters, permeable channels, and metallothioneins. Recent studies highlight Zn’s dynamic activity and its role as a signaling mediator. Zn acts as an intracellular signaling molecule, capable of communicating between cells, converting extracellular stimuli to intracellular signals, and controlling intracellular events. We have proposed that intracellular Zn signaling falls into two classes, early and late Zn signaling. This review addresses recent findings regarding Zn signaling and its role in physiological processes and pathogenesis.
KeywordsZinc Signaling Zinc transporter Metallothionein Disease
Bone morphogenetic protein
3′,5′-Cyclic adenosine monophosphate
3′-5′-Cyclic guanosine monophosphate
Experimental autoimmune encephalomyelitis
Extracellular signal-regulated kinase 1 and 2
Early zinc signaling
Fc epsilon receptor I
Fibroblast growth factor receptor
G protein-coupled receptor
G protein-coupled receptor 39
Late zinc signaling
MT1 and MT2 double knockout
Nuclear factor kappa B
Protein kinase C
Reactive oxygen species
Signal transducers and activators of transcription 3
Transforming growth factor beta
Tumor necrosis factor alpha
T helper 17
The presence of zinc (Zn) was discovered in Aspergillus niger, the common bread mold, in the nineteenth century . Zn was not recognized as indispensable for human life for almost another 100 years until the important discovery by Prasad et al. . Although Zn salts and Zn-related compounds are normally colorless, unlike those of metals such as copper and iron, making a biological study of Zn more difficult, recent advances in life science research have contributed to unfolding its basic requirement for mammalian life , including the fact that Zn is pivotal for mammalian oocytogenesis, even before conception [4, 5].
Here, we review new findings on the role of Zn signaling in physiological processes and disease status, and discuss the impact of EZS and LZS on biological events.
Zn transporters and MTs in Zn homeostasis, health, and disease
Zn has wide-ranging effects on cellular functions [25, 26], and imbalances in its homeostasis cause various types of abnormalities in humans and in animal models [2, 15, 27]. The intracellular and extracellular Zn concentration and distribution is controlled by MTs , and by Zn transporters of the Slc39/ZIP and Slc30/ZnT families; these transporters increase and decrease, respectively, the cytosolic Zn level [29, 30] (Fig. 1).
Molecular and genetic advances have uncovered the biological significance of Zn transporters. Mice with a targeted disruption of ZIP1, ZIP2, or ZIP3 are sensitive to dietary Zn deficiency during pregnancy [31, 32]. Mutations in the human ZIP4 gene cause the inherited disorder acrodermatitis enteropathica [33, 34], in which the intestine’s ability to absorb dietary Zn is impaired. ZIP13-knockout (KO) mice suffer from disorganization in hard and connective tissues, including bone, teeth, skin, and eyes [23, 24]. ZIP13 is involved in bone morphogenetic protein (BMP)/transforming growth factor beta (TGF-β)-mediated Smad localization to the nucleus  (Fig. 2b). In humans, the loss of ZIP13 function causes the spondylocheiro dysplastic form of Ehlers–Danlos syndrome [23, 35]. ZIP14-KO mice exhibit retarded growth and impaired gluconeogenesis, and ZIP14 has been shown to regulate G protein-coupled receptor (GPCR) signaling, including that through the parathyroid hormone receptor 1, growth hormone releasing hormone receptor, and glucagon receptor, by inhibiting phosphodiesterase activity (Fig. 2c) . The lethal milk mutant mice, which have a ZnT4 loss-of-function mutation, suffer from an inherited Zn deficiency in the milk . A similar abnormality, in which a ZnT2 loss-of-function mutation reduces the concentration of Zn in the milk, has been found in a human case . ZnT8 is an islet β-cell-specific Zn transporter that provides Zn to form insulin complexes ; its single-nucleotide polymorphism is associated with type 2 diabetes , and its deletion is accompanied by impaired insulin secretion . ZnT5-KO mice suffer from growth retardation and osteogenic problems , and exhibit impaired cytokine production in mast cells  (Fig. 2d).
Molecular and genetic approaches using fruit flies have contributed to our understanding of Zn transporter roles. The Drosophila gene fear of intimacy, which shares similarities with mammalian ZIP6/Liv1 and ZIP10 , is essential for proper gonad formation, E-cadherin expression, and glial cell migration [45, 46, 47]. Zebrafish Liv1 controls the epithelial–mesenchymal transition under STAT3 activation  (Fig. 2a), suggesting that ZIP6 and/or ZIP10 may have critical roles in cell migration; this was also supported by studies using mammalian cells . The Drosophila counterpart of ZIP7, Catsup, controls melanin synthesis . Investigation of the Drosophila breathless mutant, which lacks functional fibroblast growth factor receptors (FGFRs), suggested that Catsup protein facilitates FGFR signaling by inhibiting FGFR protein downregulation . A functional connection between ZIP7 and signaling was proposed by a study using cancer cells; ZIP7 positively regulates tyrosine kinases through Zn inhibition of protein tyrosine phosphatases . These reports indicate that ZIP7’s importance in various biological events may be evolutionarily conserved.
MT molecules are also important in Zn homeostasis ; they will be only briefly mentioned here, since they are well described in other articles in this volume. MT was first discovered as a cadmium-binding protein in horse kidneys . Their unique cysteine-rich structure, Zn-binding ability, and expression regulation have drawn many researchers to study MTs, and genetic advances using mouse models have enlarged our understanding of MT activity both in physiological and pathogenic processes such as inflammation. MTs protect against fibrinolytic disturbance and organ damage induced by the bacterial endotoxin lipopolysaccharide (LPS) and ozone (O3) . MTs may protect against allergic inflammation through their antioxidant potential, and by suppressing inflammatory cytokines, including interleukin-1 beta . MT defects may cause severe inflammatory responses in multiple sclerosis, Helicobacter-induced gastritis, and LPS-mediated lung injury [55, 56, 57], suggesting that MTs and Zn may coordinate to protect against various inflammatory conditions in mice . Collectively, these reports strongly suggest that MTs and Zn transporters are linked with a wide variety of biological events, disorders, and regulatory functions.
Zn in allergy and immunology
Zn deficiency in humans accompanies many chronic diseases and is frequently a dietary problem. Zn homeostasis experiments using mouse models appear to be ideal for determining the impact of a single element nutritional deficiency on immune function, at both the cellular level and the molecular level . Numerous results from animal models have confirmed that Zn deficiency induces thymic atrophy and lymphopenia, compromises cell- and antibody-mediated responses, and results in increased infection rates and durations [60, 61, 62, 63, 64].
Intracellular Zn levels and distribution have been investigated using Zn probes, which enable the imaging of distinct pools of Zn in allergy-related cells. For instance, mast cell granules fluoresce intensely with zinquin . Airway epithelial cells are also rich in Zn . In addition, Zn is found in the intracellular and extracellular matrix and accumulates in skin tissues following injury . Furthermore, Zn accelerates wound healing and the reepithelialization process [68, 69]. It has been reported that Zn deficiency increases allergic eosinophilic inflammation, whereas dietary Zn supplementation attenuates its intensity . Zn deficiency is a risk factor for developing asthma [71, 72]. These reports suggest that Zn is involved in the development of allergic disease. In addition, high levels of ZIP2 messenger RNA (mRNA) are found in the leukocytes of asthmatic infants . However, the precise roles of Zn and Zn transporters in allergy-related cells are not well understood.
In addition to its role in allergy-related cells, Zn is involved in the control of both dendritic and T cells. The intracellular free Zn concentration decreases in mouse dendritic cells exposed to the endotoxin LPS, which is a Toll-like receptor (TLR) stimulant. Furthermore, artificially depleting intracellular Zn with a Zn chelator triggers dendritic cell maturation. On the other hand, artificially elevating the intracellular Zn level suppresses the dendritic cells’ ability to respond to LPS. LPS stimulation affects Zn transporter expression, effectively increasing (ZIP family) or decreasing (ZnT family) the level of intracellular Zn. Importantly, the ectopic expression of ZIP6, which is reduced by LPS stimulation, suppresses dendritic cell maturation and inhibits CD4+ T cell-stimulatory activity . A similar Zn effect is observed in live animals; LPS injection reduces the intracellular free Zn and ZIP6 mRNA levels in dendritic cells, and treatment with Zn-depleting agents increases dendritic cell maturation . These results show that extracellular stimuli, by affecting Zn transporter expression and thus changing the intracellular Zn levels, play a role in the process of dendritic cell maturation, an important step in T cell activation and immune response (Fig. 2e).
It has also been reported that MTs are involved in asthma and ultraviolet B-induced contact hypersensitivity. Ovalbumin-induced airway inflammation is worse in the MT1 and MT2 double-KO (MT1/2-KO) mice than in wild-type mice. The cellular profile of the bronchoalveolar fluid, lung histology findings, and expression of proinflammatory molecules in the lungs were consistent with increased airway inflammation in MT1/2-KO mice . In another allergy model, moderate daily ultraviolet B doses caused significantly more immunosuppression in MT1/2-KO mice than in wild-type mice . These findings indicate that Zn transporters and Zn-related molecules are indispensably involved in allergic and immune responses and regulate a variety of signaling processes (Figs. 4, 5).
Zn as a neurotransmitter, or the first messenger in cell–cell communication
An exchangeable Zn ion in the brain was first documented by Maske  in 1955. Zn is highly concentrated in synaptic vesicles and is released with glutamate in an activity-dependent manner . Recently, Zn-imaging techniques using fluorescence sensor molecules, which allow the Zn concentration and distribution to be analyzed in real time, have been widely applied . Microfluorescence imaging of Zn secretion showed that Zn is released from hippocampal mossy fiber terminal vesicles into the surrounding milieu upon exocytotic presynaptic stimulation [93, 94, 95]. It is then taken up into the cytoplasm of neighboring cells through gated Zn channels. The rapid influx of Zn through Ca-ion-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate/kainate channels triggers the generation of reactive oxygen species (ROS) and is potentially neurotoxic .
In the above scenario, Zn’s activity is similar to that of neurotransmitters, which are stored in membrane-enclosed synaptic vesicles, are released by exocytosis, and activate postsynaptic cells through transmitter-gated ion channels [97, 98, 99]. Since Zn modulates both the current response (mediated by excitatory and inhibitory neurotransmitter receptors) and the efficacy of transporter-driven neurotransmitter reuptake , synaptically released Zn has been proposed to function as an important regulator of synaptic transmission and plasticity [101, 102], or as an “atypical neurotransmitter” . ZnT3 is highly expressed on synaptic vesicle membranes and is essential for loading Zn into synaptic vesicles.
Although ZnT3-KO mice show a loss of stainable Zn in synapses , they are behaviorally normal except for an enhanced susceptibility to kainite-induced seizures . Recently, however, it was revealed that the ZnT3-KO mice exhibit age-dependent deficits in learning and memory that are not apparent in young animals. This age-dependent cognitive phenotype might be owing to its decreased level in the hippocampus . In addition, extracellular-signal-regulated kinase 1 and 2 (Erk1/2) activation is reduced in the hippocampal mossy fibers in ZnT3-KO mice, suggesting that ZnT3 is involved in the presynaptic Erk1/2 signaling required for hippocampus-dependent memory . Takeda et al.  reported that Zn chelation with clioquinol inhibits hippocampal long-term potentiation and cognitive behavior. This evidence suggests that synaptic Zn acts as a neurotransmitter for maintaining the synaptic environment.
Zn has been identified as an endogenous GPCR 39 (GPR39) agonist, and the concept that GPR39 is the Zn-sensing receptor in the brain is consistent with Zn’s role as a neurotransmitter or first messenger [108, 109]. Intercellular Zn communication mediated through the Zn-sensing GPR39 is also involved in regulating epithelial cell repair  and endocrine pancreatic function . Also supporting Zn’s role in neurotransmission, Hosie et al.  identified two Zn-binding sites and characterized a third in the γ-aminobutyric acid receptor, using site-directed mutagenesis. In addition, Hirzel et al.  produced knock-in mice carrying the mutation D80A (a constructed Zn-binding site) in the glycine receptor alpha1 subunit gene to show that Zn modulates neurotransmission. It is also known that Zn inhibits N-methyl-d-aspartate receptor activity through a dual mechanism, a voltage-dependent channel block and a voltage-independent reduction in the probability of the channel opening [114, 115, 116]. Thus, it is likely that Zn acts as a neurotransmitter in addition to its other roles in the neural system.
Zn as an intracellular signaling molecule, or the second messenger
Several intracellular second messengers have been identified, including 3′,5′-cyclic adenosine monophosphate (cAMP), Ca2+, 3′,5′-cyclic guanosine monophosphate (cGMP), nitric oxide (NO), ROS, and lipid mediators . Zn has also appeared as a potential intracellular signaling molecule in mitosis in starfish oocytes . Recently, an essential role for Zn in arresting second metaphase exit has been reported. This suggests that Zn has the potential to act as an intracellular signaling molecule to regulate the developmental program in vertebrate fertilization [4, 5]. It has also been reported that Zn itself affects a variety of signaling molecules, including PKC, Ca/calmodulin-dependent protein kinase II, Erk1/2, cAMP-dependent protein kinase, protein tyrosine phosphatase, and caspase-3 [119, 120, 121, 122, 123, 124, 125]. In addition, Zn activates ion channels such as the transient receptor potential ankyrin 1 [126, 127], ATP-sensitive K+ , and large-conductance Ca-activated K+  channels. Together, these findings suggest that Zn may function as an intracellular signaling molecule or second messenger, if extracellular stimuli such as cytokines and growth factors cause the intracellular Zn status to change, either independently, or in a manner dependent on the transcriptional changes of Zn transporters or MTs.
We have proposed that Zn signaling can be divided into EZS, which is directly and rapidly induced by an extracellular stimulus, and LZS, which depends on transcriptional changes to MTs, Zn transporters, and other Zn-related molecules  (Fig. 3). ZIP6/Liv1 is required for the epithelial–mesenchymal transition in the zebrafish gastrula organizer, because it is essential for the nuclear retention of the E-cadherin repressor Snail (Fig. 2a) . Since ZIP6/Liv1 expression in the zebrafish organizer is dependent on STAT3 activation, any extracellular stimulus regulating STAT3 activation could change the intracellular Zn level by inducing changes in Zn transporters such as ZIP6/Liv1. TLR4 also alters the intracellular free Zn level, which it decreases by inducing changes in Zn transporter expression in dendritic cells (Fig. 2e)  and in pulmonary endothelial cells . These observations support the idea that Zn acts as an intracellular second messenger, that is, as a molecule whose intracellular status is altered in response to an extracellular stimulus and that is capable of transducing the extracellular stimulus into an intracellular signaling event.
The role of Zn as a second messenger is further supported by our findings that ZIP13 is required for the BMP/TGF-β-induced nuclear localization of Smad proteins (Fig. 2b) , that ZIP14 is involved in GPCR-mediated signal transduction through cAMP basal level regulation (Fig. 2c) , and that FcεRI-stimulation-induced PKC activation is dependent on ZnT5 in mast cells (Fig. 2d) . Another example showing that Zn transporters are involved in signaling pathways is that cation diffusion facilitator 1, a structural homologue of ZnT1 in Caenorhabditis elegans, might mediate Raf-1 activation by directly interacting with Raf-1 and removing Zn from the Raf-1 cysteine-rich domain . Thus, extracellular stimuli can affect intracellular signaling pathways by changing the intracellular Zn status through changes in the expression of Zn transporters and other Zn-related molecules; this exemplifies the LZS type (Fig. 3) .
Outside intracellular compartments, Zn-buffering proteins such as MTs are believed to be a major source of free Zn released into the cytosol space in response to an extracellular stimulus [135, 136, 137]. Zn is rapidly released from MTs by nitrosylation or by thiol ligand oxidation. Ultraviolet A irradiation can induce the generation of ROS, which might oxidize thiols in MTs and induce transient increases in intracellular free Zn level . In pathological situations, N-methyl-d-aspartate receptor activation might induce endogenous NO synthesis and increase intracellular free Zn level by liberating Zn from MTs . The liberation of intracellular Zn and the overactivation of potassium channels were proposed to be important components of nitrosative stress-induced neuronal death . The liberated Zn induces its toxicity to neurons via activation of 12-lipoxygenase and mitogen-activated protein kinase . The NO donor S-nitrosocysteine (SNOC) both increases free Zn level and inhibits LPS-induced tumor necrosis factor alpha and interleukin-1 beta release. This SNOC-induced increase in intracellular free Zn level may therefore mediate SNOC’s inhibitory effect via increased cGMP level . In addition, cytokine stimulation sometimes induces NO production, thereby enhancing the intracellular free Zn level . These reports indicate that Zn released from oxidative-reaction-stimulated MTs contributes to the EZS-type regulation of intracellular phenomena (Fig. 7b).
Conclusion and future prospects
As discussed in this review, Zn is clearly involved in regulating a wide variety of physiological responses. Although most Zn studies have focused on the general maintenance of its homeostasis, recent studies have shown that Zn’s functions are far more extensive than might be implied by its simple definition as an essential nutrient. The intracellular Zn status is dynamically altered in response to a variety of extracellular stimuli, and this change in Zn status is capable of transducing extracellular stimuli into intracellular signaling events. On the basis of the evidence accumulated, we have proposed that Zn acts as a signaling molecule in at least two modes: transcription-independent EZS and transcription-dependent LZS (Fig. 3) . Several important questions remain to be resolved: What extracellular stimulus, in which cells or tissues, regulates the expression of the Zn transporters and channels involved in Zn signaling? How are these Zn transporters or channels activated in response to the extracellular stimulus? How does each Zn transporter specifically regulate the intracellular signaling pathway? How do Zn transporters transfer Zn into their target molecules? Is there any chaperon which transfers Zn from a given transporter to its specific target molecule? What are the target molecules and biological roles of Zn signaling? Another question is how to specifically monitor and visualize intracellular free Zn in vivo with high resolution; this will require the use of chemical compounds , multitracer detection devices , and two-photon and X-ray fluorescence microscopy [145, 146]. The development of drugs for Zn-related diseases is another emerging avenue of study. Modified Zn-containing pharmaceuticals may speed the discovery of Zn-signaling action points . A deeper understanding of the early and late Zn signals and signaling dysfunctions will provide novel insights into Zn’s roles as a signaling molecule in mammalian health and diseases.
We thank Masami Kawamura, Ayumi Ito, and Mayumi Hara for their indispensable support. We also thank Mizuki Shimura and Ryoko Masuda for their secretarial assistance. This work was supported by KAKENHI and the JST-CREST program.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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