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1.  Age-Dependent Signature of Metallothionein Expression in Primary CD4 T Cell Responses Is Due to Sustained Zinc Signaling 
Rejuvenation Research  2008;11(6):1001-1011.
The ability to mount adaptive immune responses to vaccinations and viral infections declines with increasing age. To identify mechanisms leading to immunosenescence, primary CD4 T cell responses were examined in 60- to 75-year-old individuals lacking overt functional defects. Transcriptome analysis indicated a selective defect in zinc homeostasis. CD4 T cell activation was associated with zinc influx via the zinc transporter Zip6, leading to increased free cytoplasmic zinc and activation of negative feedback loops, including the induction of zinc-binding metallothioneins. In young adults, activation-induced cytoplasmic zinc concentrations declined after 2 days to below prestimulation levels. In contrast, activated naïve CD4 T cells from older individuals failed to downregulate cytoplasmic zinc, resulting in excessive induction of metallothioneins. Activation-induced metallothioneins regulated the redox state in activated T cells and accounted for an increased proliferation of old CD4 T cells, suggesting that regulation of T cell zinc homeostasis functions as a compensatory mechanism to preserve the replicative potential of naïve CD4 T cells with age.
PMCID: PMC2848531  PMID: 19072254
2.  Uptake epithelia behave in a cell-centric and not systems homeostatic manner in response to zinc depletion and supplementation† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c3mt00212h Click here for additional data file.  
Metallomics  2013;6(1):154-165.
Global transcriptomic analysis, non-invasive real-time flux, nutritional profiling and metallomics reveal cell-centric response to zinc supplementation/depletion in zebrafish uptake epithelia.
Much remains to be understood about systemic regulation of zinc uptake in vertebrates, and adequate zinc status is far from always achieved in animals or human. In addition to absorbing zinc from the diet, fish are able to take up zinc directly from the water with the gills. This provides an elegant system to study zinc uptake, how it relates to zinc status, and the expression of genes for proteins involved in zinc acquisition. A 21-day experiment was conducted in which zebrafish were acclimated to deficient, control or excess zinc concentrations in the water and feed. Deficient provision of zinc reduced whole body zinc, potassium, sodium and calcium levels whilst zinc concentrations in the uptake epithelia (gills and gut) remained unchanged. Excess levels of zinc caused accumulation of zinc in the gills, intestine and carcass, but impaired whole body iron, sodium and calcium concentrations. Fish subjected to zinc deficiency had, surprisingly, a reduced zinc influx across the gill epithelium, even when tested at a high concentration of zinc in the water. Zinc influx in the excess group was indistinct from the control. Expression of genes for metallothionein-2 (Mt2) and zinc transporters-1, -2, and -8 (Znt1, Znt2, Znt8) in uptake epithelia showed in general a direct relationship with zinc supply, while mRNA for Zip4 was inversely related to zinc supply. Transcripts for the epithelial calcium channel (Ecac/Trpv6) showed time-dependent increased expression in the gills of the deficiency group, and a transient decrease of expression during zinc excess. Transcriptome profiling by microarrays showed that in both gills and intestine, the most markedly affected biological functions were those related to cell growth, proliferation and cancer, closely followed by processes of gene transcription and protein synthesis in general. Whilst changes in zinc supply had profound effects in the intestine on genes associated with uptake and metabolism of macronutrients, many of the unique categories of genes preferentially regulated in the gill could be mapped onto signalling pathways. This included pathways for PPAR/RXR, LXR/RXR, ATM, chemokine, and BMP signalling. Overall, the responses of epithelial tissue to zinc deficiency and excess are best explained by local epithelial homeostasis with no evidence of systemic control.
PMCID: PMC4157650  PMID: 24301558
3.  Metallothionein regulates intracellular zinc signaling during CD4+ T cell activation 
BMC Immunology  2016;17:13.
The ultra-low redox potential and zinc binding properties of the intracellular pool of mammalian metallothioneins (MT) suggest a role for MT in the transduction of redox signals into intracellular zinc signals. Increased expression of MT after exposure to heavy metals, oxidative stress, or inflammatory cytokines leads to an increased intracellular redox-mobilizable zinc pool that can affect downstream zinc-sensitive signaling pathways. CD4+ T helper cells are poised to be influenced by MT transduced zinc signaling because they produce intracellular reactive oxygen species following activation through the T cell receptor and are sensitive to small changes in intracellular [Zn2+].
MT expression and intracellular [Zn2+] are both increased during primary activation and expansion of naïve CD4+ T cells into the Tr1 phenotype in vitro. When Tr1 cells from wildtype mice are compared with congenic mice lacking functional Mt1 and Mt2 genes, the expression of intracellular MT is associated with a greater increase in intracellular [Zn2+] immediately following exposure to reactive oxygen species or upon restimulation through the T cell receptor. The release of Zn2+ from MT is associated with a greater increase in p38 MAPK activation following restimulation and decreased p38 MAPK activation in MT knockout Tr1 cells can be rescued by increasing intracellular [Zn2+]. Additionally, IL-10 secretion is increased in MT knockout Tr1 cells compared with wildtype controls and this increase is prevented when the intracellular [Zn2+] is increased experimentally.
Differences in zinc signaling associated with MT expression appear to be a result of preferential oxidation of MT and concomitant release of Zn2+. Although zinc is released from many proteins following oxidation, release is greater when the cell contains an intracellular pool of MT. By expressing MT in response to certain environmental conditions, CD4+ T cells are able to more efficiently release intracellular zinc and regulate signaling pathways following stimulation. The link between MT expression and increased zinc signaling following activation represents an important immunomodulatory mechanism of MT and illuminates the complex role MT plays in shaping immune responses.
Electronic supplementary material
The online version of this article (doi:10.1186/s12865-016-0151-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4890327  PMID: 27251638
Metallothionein; Zinc signal; CD4+ T helper cell; p38 MAPK; Tr1; Redox; T cell receptor
4.  Activity of Metal-Responsive Transcription Factor 1 by Toxic Heavy Metals and H2O2 In Vitro Is Modulated by Metallothionein 
Molecular and Cellular Biology  2003;23(23):8471-8485.
Metallothioneins are small, cysteine-rich proteins that avidly bind heavy metals such as zinc, copper, and cadmium to reduce their concentration to a physiological or nontoxic level. Metallothionein gene transcription is induced by several stimuli, notably heavy metal load and oxidative stress. Transcriptional induction of metallothionein genes is mediated by the metal-responsive transcription factor 1 (MTF-1), an essential zinc finger protein that binds to specific DNA motifs termed metal-response elements. In cell-free DNA binding reactions with nuclear extracts, MTF-1 requires elevated zinc concentrations for efficient DNA binding but paradoxically is inactivated by other in vivo inducers such as cadmium, copper, and hydrogen peroxide. Here we have developed a cell-free, MTF-1-dependent transcription system which accurately reproduces the activation of metallothionein gene promoters not only by zinc but also by these other inducers. We found that while transcriptional induction by zinc can be achieved by elevated zinc concentration alone, induction by cadmium, copper, or H2O2 additionally requires the presence of zinc-saturated metallothionein. This is explained by the preferential binding of cadmium or copper to metallothionein or its oxidation by H2O2; the concomitant release of zinc in turn leads to the activation of transcription factor MTF-1. Conversely, thionein, the metal-free form of metallothionein, inhibits activation of MTF-1. The release of zinc from cellular components, including metallothioneins, and the sequestration of zinc by newly produced apometallothionein might be a basic mechanism to regulate MTF-1 activity upon cellular stress.
PMCID: PMC262672  PMID: 14612393
5.  ttm-1 Encodes CDF Transporters That Excrete Zinc from Intestinal Cells of C. elegans and Act in a Parallel Negative Feedback Circuit That Promotes Homeostasis 
PLoS Genetics  2013;9(5):e1003522.
Zinc is an essential metal involved in a wide range of biological processes, and aberrant zinc metabolism is implicated in human diseases. The gastrointestinal tract of animals is a critical site of zinc metabolism that is responsible for dietary zinc uptake and distribution to the body. However, the role of the gastrointestinal tract in zinc excretion remains unclear. Zinc transporters are key regulators of zinc metabolism that mediate the movement of zinc ions across membranes. Here, we identified a comprehensive list of 14 predicted Cation Diffusion Facilitator (CDF) family zinc transporters in Caenorhabditis elegans and demonstrated that zinc is excreted from intestinal cells by one of these CDF proteins, TTM-1B. The ttm-1 locus encodes two transcripts, ttm-1a and ttm-1b, that use different transcription start sites. ttm-1b expression was induced by high levels of zinc specifically in intestinal cells, whereas ttm-1a was not induced by zinc. TTM-1B was localized to the apical plasma membrane of intestinal cells, and analyses of loss-of-function mutant animals indicated that TTM-1B promotes zinc excretion into the intestinal lumen. Zinc excretion mediated by TTM-1B contributes to zinc detoxification. These observations indicate that ttm-1 is a component of a negative feedback circuit, since high levels of cytoplasmic zinc increase ttm-1b transcript levels and TTM-1B protein functions to reduce the level of cytoplasmic zinc. We showed that TTM-1 isoforms function in tandem with CDF-2, which is also induced by high levels of cytoplasmic zinc and reduces cytoplasmic zinc levels by sequestering zinc in lysosome-related organelles. These findings define a parallel negative feedback circuit that promotes zinc homeostasis and advance the understanding of the physiological roles of the gastrointestinal tract in zinc metabolism in animals.
Author Summary
Zinc is an essential mineral nutrient involved in many physiological processes, and it plays a critical role in human health. Insufficient dietary zinc causes a wide range of health problems, and excess dietary zinc causes toxicity. Furthermore, genetic mutations affecting zinc metabolism have been implicated in a variety of human diseases. Therefore, animals require homeostatic mechanisms that effectively regulate zinc metabolism in response to dietary fluctuations. The gastrointestinal tract is a major tissue that orchestrates zinc metabolism in animals, and zinc transporters are key molecular regulators involved in this process. To understand these regulatory mechanisms, we used bioinformatic techniques to identify 14 genes that encode predicted Cation Diffusion Facilitator (CDF) family zinc transporters in the C. elegans genome. We demonstrated that one of these, ttm-1, functions in intestinal cells to promote zinc excretion, and this activity protects animals from zinc toxicity. Genetic analysis revealed that zinc excretion mediated by TTM-1B is coordinated with zinc storage mediated by CDF-2, and these transporters function in a parallel negative feedback circuit to maintain zinc homeostasis in intestinal cells. These findings provide molecular and physiological insight into the regulatory mechanisms of zinc metabolism in animals.
PMCID: PMC3662639  PMID: 23717214
6.  Roles of zinc and metallothionein-3 in oxidative stress-induced lysosomal dysfunction, cell death, and autophagy in neurons and astrocytes 
Molecular Brain  2010;3:30.
Zinc dyshomeostasis has been recognized as an important mechanism for cell death in acute brain injury. An increase in the level of free or histochemically reactive zinc in astrocytes and neurons is considered one of the major causes of death of these cells in ischemia and trauma. Although zinc dyshomeostasis can lead to cell death via diverse routes, the major pathway appears to involve oxidative stress.
Recently, we found that a rise of zinc in autophagic vacuoles, including autolysosomes, is a prerequisite for lysosomal membrane permeabilization and cell death in cultured brain cells exposed to oxidative stress conditions. The source of zinc in this process is likely redox-sensitive zinc-binding proteins such as metallothioneins, which release zinc under oxidative conditions. Of the metallothioneins, metallothionein-3 is especially enriched in the central nervous system, but its physiologic role in this tissue is not well established. Like other metallothioneins, metallothionein-3 may function as metal detoxicant, but is also known to inhibit neurite outgrowth and, sometimes, promote neuronal death, likely by serving as a source of toxic zinc release. In addition, metallothionein-3 regulates lysosomal functions. In the absence of metallothionein-3, there are changes in lysosome-associated membrane protein-1 and -2, and reductions in certain lysosomal enzymes that result in decreased autophagic flux. This may have dual effects on cell survival. In acute oxidative injury, zinc dyshomeostasis and lysosomal membrane permeabilization are diminished in metallothionein-3 null cells, resulting in less cell death. But over the longer term, diminished lysosomal function may lead to the accumulation of abnormal proteins and cause cytotoxicity.
The roles of zinc and metallothionein-3 in autophagy and/or lysosomal function have just begun to be investigated. In light of evidence that autophagy and lysosomes may play significant roles in the pathogenesis of various neurological diseases, further insight into the contribution of zinc dynamics and metallothionein-3 function may help provide ways to effectively regulate these processes in brain cells.
PMCID: PMC2988061  PMID: 20974010
7.  Zinc homeostasis and neurodegenerative disorders 
Zinc is an essential trace element, whose importance to the function of the central nervous system (CNS) is increasingly being appreciated. Alterations in zinc dyshomeostasis has been suggested as a key factor in the development of several neuropsychiatric disorders. In the CNS, zinc occurs in two forms: the first being tightly bound to proteins and, secondly, the free, cytoplasmic, or extracellular form found in presynaptic vesicles. Under normal conditions, zinc released from the synaptic vesicles modulates both ionotropic and metabotropic post-synaptic receptors. While under clinical conditions such as traumatic brain injury, stroke or epilepsy, the excess influx of zinc into neurons has been found to result in neurotoxicity and damage to postsynaptic neurons. On the other hand, a growing body of evidence suggests that a deficiency, rather than an excess, of zinc leads to an increased risk for the development of neurological disorders. Indeed, zinc deficiency has been shown to affect neurogenesis and increase neuronal apoptosis, which can lead to learning and memory deficits. Altered zinc homeostasis is also suggested as a risk factor for depression, Alzheimer's disease (AD), aging, and other neurodegenerative disorders. Under normal CNS physiology, homeostatic controls are put in place to avoid the accumulation of excess zinc or its deficiency. This cellular zinc homeostasis results from the actions of a coordinated regulation effected by different proteins involved in the uptake, excretion and intracellular storage/trafficking of zinc. These proteins include membranous transporters (ZnT and Zip) and metallothioneins (MT) which control intracellular zinc levels. Interestingly, alterations in ZnT and MT have been recently reported in both aging and AD. This paper provides an overview of both clinical and experimental evidence that implicates a dysfunction in zinc homeostasis in the pathophysiology of depression, AD, and aging.
PMCID: PMC3715721  PMID: 23882214
zinc; zinc transporters; metallothioneins; depression; aging; Alzheimer's disease; neurodegeneration
8.  Zinc-bound metallothioneins and immune plasticity: lessons from very old mice and humans 
The capacity of the remodelling immune responses during stress (named immune plasticity) is fundamental to reach successful ageing. We herein report two pivotal experimental models in order to demonstrate the relevance of the immune plasticity in ageing and successful ageing. These two experimental models will be compared with the capacity in remodelling the immune response in human centenarians. With regard to experimental models, one model is represented by the circadian rhythms of immune responses, the other one is the immune responses during partial hepatectomy/liver regeneration (pHx). The latter is suggestive because it mimics the immunosenescence and chronic inflammation 48 h after partial hepatectomy in the young through the continuous production of IL-6, which is the main cause of immune plasticity lack in ageing. The constant production of IL-6 leads to abnormal increments of zinc-bound Metallothionein (MT), which is in turn unable in zinc release in ageing. As a consequence, low zinc ion bioavailability appears for thymic and extrathymic immune efficiency, in particular of liver NKT cells bearing TCR γδ. The remodelling during the circadian cycle and during pHx of zinc-bound MT confers the immune plasticity of liver NKT γδ cells and NK cells in young and very old mice, not in old mice. With regard to human centenarians and their capacity in remodelling the immune response with respect to elderly, these exceptional individuals display low zinc-bound MT associated with: a) satisfactory intracellular zinc ion availability, b) more capacity in zinc release by MT, c) less inflammation due to low gene expression of IL-6 receptor (gp130), d) increased levels of IFN-gamma and number of NKT cell bearing TCR γδ. Moreover, some polymorphisms for MT tested in PBMCs from human donors are related to successful ageing. In conclusion, zinc-bound MT homeostasis is fundamental to confer the immune plasticity that is a condition "sine qua non" to achieve healthy ageing and longevity.
PMCID: PMC2082024  PMID: 17903270
9.  Effects of zinc supplementation and zinc chelation on in vitro β-cell function in INS-1E cells 
BMC Research Notes  2014;7:84.
Zinc is essential for the activities of pancreatic β-cells, especially insulin storage and secretion. Insulin secretion leads to co-release of zinc which contributes to the paracrine communication in the pancreatic islets. Zinc-transporting proteins (zinc-regulated transporter, iron-regulated transporter-like proteins [ZIPs] and zinc transporters [ZnTs]) and metal-buffering proteins (metallothioneins, MTs) tightly regulate intracellular zinc homeostasis. The present study investigated how modulation of cellular zinc availability affects β-cell function using INS-1E cells.
Using INS-1E cells, we found that zinc supplementation and zinc chelation had significant effects on insulin content and insulin secretion. Supplemental zinc within the physiological concentration range induced insulin secretion. Insulin content was reduced by zinc chelation with N,N,N’,N-tektrakis(2-pyridylmethyl)-ethylenediamine. The changes in intracellular insulin content following exposure to various concentrations of zinc were reflected by changes in the expression patterns of MT-1A, ZnT-8, ZnT-5, and ZnT-3. Furthermore, high zinc concentrations induced cell necrosis while zinc chelation induced apoptosis. Finally, cell proliferation was sensitive to changes in zinc the concentration.
These results indicate that the β-cell-like function and survival of INS-1E cells are dependent on the surrounding zinc concentrations. Our results suggest that regulation of zinc homeostasis could represent a pharmacological target.
PMCID: PMC3923740  PMID: 24502363
Zinc; Insulin; Zinc transporter; Metallothionein; Chelation; TPEN; INS-1E cells; β –cell; Diabetes
10.  A Family Knockout of All Four Drosophila Metallothioneins Reveals a Central Role in Copper Homeostasis and Detoxification†  
Molecular and Cellular Biology  2006;26(6):2286-2296.
Metallothioneins are ubiquitous, small, cysteine-rich proteins with the ability to bind heavy metals. In spite of their biochemical characterization, their in vivo function remains elusive. Here, we report the generation of a metallothionein gene family knockout in Drosophila melanogaster by targeted disruption of all four genes (MtnA to -D). These flies are viable if raised in standard laboratory food. During development, however, they are highly sensitive to copper, cadmium, and (to a lesser extent) zinc load. Metallothionein expression is particularly important for male viability; while copper load during development affects males and females equally, adult males lacking metallothioneins display a severely reduced life span, possibly due to copper-mediated oxidative stress. Using various reporter gene constructs, we find that different metallothioneins are expressed with virtually the same tissue specificity in larvae, notably in the intestinal tract at sites of metal accumulation, including the midgut's “copper cells.” The same expression pattern is observed with a synthetic minipromoter consisting only of four tandem metal response elements. From these and other experiments, we conclude that tissue specificity of metallothionein expression is a consequence, rather than a cause, of metal distribution in the organism. The bright orange luminescence of copper accumulated in copper cells of the midgut is severely reduced in the metallothionein gene family knockout, as well as in mutants of metal-responsive transcription factor 1 (MTF-1), the main regulator of metallothionein expression. This indicates that an in vivo metallothionein-copper complex forms the basis of this luminescence. Strikingly, metallothionein mutants show an increased, MTF-1-dependent induction of metallothionein promoters in response to copper, cadmium, silver, zinc, and mercury. We conclude that free metal, but not metallothionein-bound metal, triggers the activation of MTF-1 and that metallothioneins regulate their own expression by a negative feedback loop.
PMCID: PMC1430275  PMID: 16508004
11.  The Znt4 mutation inlethal milk mice affects intestinal zinc homeostasis through the expression of other Zn transporters 
Genes & Nutrition  2006;1(1):61-70.
The lethal milk mouse syndrome is caused by a point mutation in the zinc transporter gene ZnT4 resulting in defective zinc secretion in the milk of homozygous mutant dams. Pups of any genotype fed solely on lm milk die within the first two weeks of neonatal life, displaying zinc deficiency symptoms. Homozygous mutant pups survive when foster nursed by wild type dams and show signs of mild zinc deficiency in adulthood. To further investigate the role of ZnT4 in zinc secretion in the intestinal epithelium, we have studied the expression by real time quantitative PCR of mutant ZnT4 and of other zinc transporters of the Zip and ZnT families, in the jejunum of homozygous lm mice and of the isogenic wild type strain C57BL/ 6J. We report in this paper that expression of the mutant ZnT4 mRNA, carrying a premature translational termination codon (ZnT4/lm), is almost absent in tissues from lm mice, probably as a result of degradation by the Nonsense Mediated mRNA Decay (NMD) Pathway. In the jejunum of mutant mice, we also observed decreased expression of the uptake zinc transporter Zip4, paralleled by increased levels of both metallothionein genes MTI and MTII. Zinc supplementation of lm mice in the drinking water did not result in further decrease of Zip4 expression, but led to full induction of MT mRNAs. These results lead us to conclude that, although in the enterocytes of lm mice the absence of the zinc secretion activity mediated by ZnT4 results in increased intracellular zinc concentration, other zinc efflux activities are able to maintain the level of zinc ions below the threshold necessary for full induction of metallothioneins.
PMCID: PMC3454819  PMID: 18850221
Copper; Copper transporter; lm syndrome; Metallothionein; zinc deficiency; ZnT4; Zinc Transporter
12.  Zinc-induced upregulation of metallothionein (MT)-2A is predicted by gene expression of zinc transporters in healthy adults 
Genes & Nutrition  2015;10(6):44.
The usefulness of zinc transporter and metallothionein (MT) gene expressions to detect changes in zinc intake remains unclear. This pilot study aimed to determine the effects of zinc supplementation on zinc transporter and MT gene expressions in humans. Healthy adults (n = 39) were randomised to zinc treatment (ZT), receiving 22 mg Zn/day (n = 19), or no treatment (NT) (n = 20). Blood samples were collected on Days 0, 2, 7, 14, and 21. Plasma zinc and serum C-reactive protein concentrations were analysed. Gene expression of zinc transporters and MT in peripheral blood mononuclear cells was analysed using real-time PCR. Using repeated-measures ANOVA, MT-2A gene expression and fold change were found to be higher in the ZT group (P = 0.025 and P = 0.016, respectively) compared to the NT group, specifically at Day 2 (40 ± 18 % increase from baseline, P = 0.011), despite no significant increase in plasma zinc concentration. In a multiple regression model exploring the changes in gene expressions between Days 0 and 21, the change in MT-2A gene expression was correlated with changes in all zinc transporter expressions (r2 = 0.54, P = 0.029); the change in ZIP1 expression emerged as a univariate predictor (P = 0.003). Dietary zinc intake was predictive of zinc transporter and MT expressions (P = 0.030). Physical activity level was positively correlated with baseline ZIP7 expression (r = 0.36, P = 0.029). The present study shows that MT-2A expression is related to changing expression of zinc transporter genes, specifically ZIP1, in response to zinc supplementation. The current report adds to our understanding of MT in the coordinated nature of cellular zinc homeostasis.
Electronic supplementary material
The online version of this article (doi:10.1007/s12263-015-0494-y) contains supplementary material, which is available to authorized users.
PMCID: PMC4596130  PMID: 26446034
Zinc; Transporter; Metallothionein; Gene expression; Physical activity
13.  Profiling of zinc altered gene expression in human prostate normal versus cancer cells: a time course study 
We have demonstrated that zinc exposure induces apoptosis in human prostate cancer cells (PC-3) and benign hyperplasia cells (BPH), but not in normal prostate cells (HPR-1). However, the mechanisms underlying the effects of zinc on prostate cancer cell growth and zinc homeostasis remain unclear.
To explore the zinc effect on gene expression profiles in normal (HPR-1) and malignant prostate cells (PC-3), we conducted a time course study of Zn treatment with microarray analysis. Microarray data were evaluated and profiled using computational approach for the primary and secondary data analyses. Final analyses were focused on the genes: 1. highly sensitive to zinc, 2. associated with zinc homeostasis, i.e. metallothioneins (MTs), solute zinc carriers (ZIPs) and zinc exporters (ZnTs), 3. relevant to several oncogenic pathways. Zinc-mediated mRNA levels of MT isotypes were further validated by semi-quantitative RT-PCR.
Results showed that zinc effect on genome-wide expression patterns was cell type specific, and zinc appeared to have mainly down-regulatory effects on thousands of genes (1,953 in HPR-1; 3,534 in PC-3) with a threshold of ±2.5-fold, while fewer genes were up-regulated (872 in HPR-1; 571 in PC-3). The patterns of zinc effect on functional MT genes’ expression provided evidence for the cell-type dependent zinc accumulation and zinc-induced apoptosis in prostate cells. In PC-3 cells, zinc significantly up-regulated the expression of MT-1 isotypes -J and -M, denoted previously as “non-functional” MT genes, and now a depictive molecular structure of MT-1J was proposed. Examination of genes involved in oncogenic pathways indicated that certain genes, e.g. Fos, Akt1, Jak3 and PI3K were highly regulated by zinc with cell type specificity.
This work provided an extensive database on zinc related prostate cancer research. The strategy of data analysis was devoted to find genes highly sensitive to Zn, and the genes associated with zinc accumulation and zinc-induced apoptosis. The results indicate that zinc regulation of gene expression is cell-type specific, and MT genes play important roles in prostate malignancy.
PMCID: PMC2821158  PMID: 19071009
microarray; zinc; prostate malignant cells; gene isoform; metallothionein; zinc transporter
14.  Increased inflammatory response in aged mice is associated with age-related zinc deficiency and zinc transporter dysregulation☆ 
Aging is a complex process associated with physiological changes in numerous organ systems. In particular, aging of the immune system is characterized by progressive dysregulation of immune responses, resulting in increased susceptibility to infectious diseases, impaired vaccination efficacy and systemic low-grade chronic inflammation. Increasing evidence suggest that intracellular zinc homeostasis, regulated by zinc transporter expression, is critically involved in the signaling and activation of immune cells. We hypothesize that epigenetic alterations and nutritional deficits associated with aging may lead to zinc transporter dysregulation, resulting in decreases in cellular zinc levels and enhanced inflammation with age. The goal of this study was to examine the contribution of age-related zinc deficiency and zinc transporter dysregulation on the inflammatory response in immune cells. The effects of zinc deficiency and age on the induction of inflammatory responses were determined using an in vitro cell culture system and an aged mouse model. We showed that zinc deficiency, particularly the reduction in intracellular zinc in immune cells, was associated with increased inflammation with age. Furthermore, reduced Zip 6 expression enhanced proinflammatory response, and age-specific Zip 6 dysregulation correlated with an increase in Zip 6 promoter methylation. Furthermore, restoring zinc status via dietary supplementation reduced aged-associated inflammation. Our data suggested that age-related epigenetic dysregulation in zinc transporter expression may influence cellular zinc levels and contribute to increased susceptibility to inflammation with age.
PMCID: PMC3586240  PMID: 22981370
Aging; Epigenetics; Immunity; Inflammation; Zinc
15.  Accumulation of cholesterol and increased demand for zinc in serum-deprived RPE cells 
Molecular Vision  2016;22:1387-1404.
Having observed that confluent ARPE-19 cells (derived from human RPE) survive well in high-glucose serum-free medium (SFM) without further feeding for several days, we investigated the expression profile of RPE cells under the same conditions.
Expression profiles were examined with microarray and quantitative PCR (qPCR) analyses, followed by western blot analysis of key regulated proteins. The effects of low-density lipoprotein (LDL) and zinc supplementation were examined with qPCR. Immunofluorescence was used to localize the LDL receptor and to examine LDL uptake. Cellular cholesterol levels were measured with filipin binding. Expression patterns in primary fetal RPE cells were compared using qPCR.
Microarray analyses of gene expression in ARPE-19, confirmed with qPCR, showed upregulation of lipid and cholesterol biosynthesis pathways in SFM. At the protein level, the cholesterol synthesis control factor SRBEF2 was activated, and other key lipid synthesis proteins increased. Supplementation of SFM with LDL reversed the upregulation of lipid and cholesterol synthesis genes, but not of cholesterol transport genes. The LDL receptor relocated to the plasma membrane, and LDL uptake was activated by day 5–7 in SFM, suggesting increased demand for cholesterol. Confluent ARPE-19 cells in SFM accumulated intracellular cholesterol, compared with cells supplemented with serum, over 7 days. Over the same time course in SFM, the expression of metallothioneins decreased while the major zinc transporter was upregulated, consistent with a parallel increase in demand for zinc. Supplementation with zinc reversed expression changes for metallothionein genes, but not for other zinc-related genes. Similar patterns of regulation were also seen in primary fetal human RPE cells in SFM.
ARPE-19 cells respond to serum deprivation and starvation with upregulation of the lipid and cholesterol pathways, accumulation of intracellular cholesterol, and increased demand for zinc. Similar trends are seen in primary fetal RPE cells. Cholesterol accumulation basal to RPE is a prominent feature of age-related macular degeneration (AMD), while dietary zinc is protective. It is conceivable that accumulating defects in Bruch’s membrane and dysfunction of the choriocapillaris could impede transport between RPE and vasculature in AMD. Thus, this pattern of response to serum deprivation in RPE-derived cells may have relevance for some aspects of the progression of AMD.
PMCID: PMC5166821  PMID: 28003730
16.  Activation of Pattern Recognition Receptors Upregulates Metallothioneins, Thereby Increasing Intracellular Accumulation of Zinc, Autophagy, and Bacterial Clearance by Macrophages 
Gastroenterology  2014;147(4):835-846.
Background & Aims
Continuous stimulation of pattern recognition receptors (PRRs), including nucleotide-binding oligomerization domain-2 (NOD2) (variants in NOD2 have been associated with Crohn's disease), alters the phenotype of myeloid-derived cells, reducing production of inflammatory cytokines and increasing clearance of microbes. We investigated the mechanisms by which microbial clearance increases in macrophages under these conditions.
Monocytes were purified from human peripheral blood mononuclear cells and differentiated to monocyte-derived macrophages (MDMs). We also isolated human intestinal macrophages. Bacterial clearance by MDMs was assessed in gentamicin protection assays. Effects of intracellular zinc and autophagy were measured by flow cytometry, immunoblot, reverse transcription PCR, and microscopy experiments. Small interfering RNAs were used to knock down specific proteins in MDMs. NOD2–/– and C57BL/6J mice, maintained in a specific pathogen-free facility, were given antibiotics, muramyl dipeptide (to stimulate NOD2), or dextran sodium sulfate; intestinal lamina propria cells were collected and analyzed.
Chronic stimulation of human MDMs through NOD2 upregulated the expression of multiple genes encoding metallothioneins, which bind and regulate levels of intracellular zinc. Intestinal myeloid-derived cells are continually stimulated through PRRs; metallothionein expression was upregulated in human and mouse intestinal myeloid-derived cells. Continuous stimulation of NOD2 increased levels of intracellular zinc, thereby increasing autophagy and bacterial clearance. The metal-regulatory transcription factor-1 (MTF-1) was required for regulation of metallothionein genes in human MDMs. Knockdown of MTF-1 did not affect baseline clearance of bacteria by MDMs. However, the increase in intracellular zinc, autophagy, and bacterial clearance observed with continuous NOD2 stimulation was impaired in MDMs upon MTF-1 knockdown. Addition of zinc or induction of autophagy restored bacterial clearance to MDMs following metallothionein knockdown. NOD2 synergized with the PRRs TLR5 and TLR9 to increase the effects of metallothioneins in MDMs. In mice, the intestinal microbiota contributed to the regulation in expression of metallothioneins, levels of zinc, autophagy, and bacterial clearance by intestinal macrophages.
In studies of human MDMs and in mice, continuous stimulation of PRRs induces expression of metallothioneins. This leads to increased levels of intracellular zinc and enhanced clearance of bacteria via autophagy in macrophages.
PMCID: PMC4170054  PMID: 24960189
innate immunity; inflammatory bowel disease; signal transduction; intestinal inflammation
17.  Zinc: dietary intake and impact of supplementation on immune function in elderly 
Age  2012;35(3):839-860.
The diet in the elderly does not provide a sufficient level of nutrients needed to maintain an adequate healthy status leading to micronutrient deficiencies and impaired immune response with subsequent development of degenerative diseases. Nutrient “zinc” is a relevant micronutrient involved in maintaining a good integrity of many body homeostatic mechanisms, including immune efficiency, owing to its requirement for the biological activity of many enzymes, proteins and for cellular proliferation and genomic stability. Old people aged 60–65 years and older have zinc intakes below 50% of the recommended daily allowance on a given day. Many causes can be involved: among them, altered intestinal absorption, inadequate mastication, psychosocial factors, drugs interactions, altered subcellular processes (zinc transporters (Zip and ZnT family), metallothioneins, divalent metal transporter-1). Zinc supplementation may remodel the immune alterations in elderly leading to healthy ageing. Several zinc trials have been carried out with contradictory data, perhaps due to incorrect choice of an effective zinc supplementation in old subjects showing subsequent zinc toxic effects on immunity. Old subjects with specific IL-6 polymorphism (GG allele carriers; named C−) are more prone for zinc supplementation than the entire old population, in whom correct dietary habits with foods containing zinc (Mediterranean diet) may be sufficient in restoring zinc deficiency and impaired immune response. We summarise the main causes of low zinc dietary intake in elderly reporting an update on the impact of zinc supplementation upon the immune response also on the basis of individual IL-6 polymorphism.
PMCID: PMC3636409  PMID: 22222917
Dietary zinc intake; Zinc intestinal absorption; Zinc supplementation; IL-6 polymorphism; Ageing; Immunosenescence
18.  Zinc and the modulation of redox homeostasis 
Free radical biology & medicine  2012;53(9):1748-1759.
Zinc, a redox inactive metal, has been long viewed as a component of the antioxidant network, and growing evidence points to its involvement in redox-regulated signaling. These actions are exerted through several mechanisms based on the unique chemical and functional properties of zinc. Overall, zinc contributes to maintain the cell redox balance through different mechanisms including: i) the regulation of oxidant production and metal-induced oxidative damage; ii) the dynamic association of zinc with sulfur in protein cysteine clusters, from which the metal can be released by nitric oxide, peroxides, oxidized glutathione and other thiol oxidant species; iii) zinc-mediated induction of the zinc-binding protein metallothionein, which releases the metal under oxidative conditions and act per se scavenging oxidants; iv) the involvement of zinc in the regulation of glutathione metabolism and of the overall protein thiol redox status; and v) a direct or indirect regulation of redox signaling. Findings of oxidative stress, altered redox signaling, and associated cell/tissue disfunction in cell and animal models of zinc deficiency, stress the relevant role of zinc in the preservation of cell redox homeostasis. However, while the participation of zinc in antioxidant protection, redox sensing, and redox-regulated signaling is accepted, the involved molecules, targets and mechanisms are still partially known and the subject of active research.
PMCID: PMC3506432  PMID: 22960578
19.  The Zinc Transporter Zip5 (Slc39a5) Regulates Intestinal Zinc Excretion and Protects the Pancreas against Zinc Toxicity 
PLoS ONE  2013;8(11):e82149.
ZIP5 localizes to the baso-lateral membranes of intestinal enterocytes and pancreatic acinar cells and is internalized and degraded coordinately in these cell-types during periods of dietary zinc deficiency. These cell-types are thought to control zinc excretion from the body. The baso-lateral localization and zinc-regulation of ZIP5 in these cells are unique among the 14 members of the Slc39a family and suggest that ZIP5 plays a role in zinc excretion.
Methods/Principal Findings
We created mice with floxed Zip5 genes and deleted this gene in the entire mouse or specifically in enterocytes or acinar cells and then examined the effects on zinc homeostasis. We found that ZIP5 is not essential for growth and viability but total knockout of ZIP5 led to increased zinc in the liver in mice fed a zinc-adequate (ZnA) diet but impaired accumulation of pancreatic zinc in mice fed a zinc-excess (ZnE) diet. Loss-of-function of enterocyte ZIP5, in contrast, led to increased pancreatic zinc in mice fed a ZnA diet and increased abundance of intestinal Zip4 mRNA. Finally, loss-of-function of acinar cell ZIP5 modestly reduced pancreatic zinc in mice fed a ZnA diet but did not impair zinc uptake as measured by the rapid accumulation of 67zinc. Retention of pancreatic 67zinc was impaired in these mice but the absence of pancreatic ZIP5 sensitized them to zinc-induced pancreatitis and exacerbated the formation of large cytoplasmic vacuoles containing secretory protein in acinar cells.
These studies demonstrate that ZIP5 participates in the control of zinc excretion in mice. Specifically, they reveal a paramount function of intestinal ZIP5 in zinc excretion but suggest a role for pancreatic ZIP5 in zinc accumulation/retention in acinar cells. ZIP5 functions in acinar cells to protect against zinc-induced acute pancreatitis and attenuate the process of zymophagy. This suggests that it may play a role in autophagy.
PMCID: PMC3841122  PMID: 24303081
20.  LiZIP3 is a cellular zinc transporter that mediates the tightly regulated import of zinc in Leishmania infantum parasites 
Molecular microbiology  2015;96(3):581-595.
Cellular zinc homeostasis ensures that the intracellular concentration of this element is kept within limits that enable its participation in critical physiological processes without exerting toxic effects. We report here the identification and characterization of the first mediator of zinc homeostasis in Leishmania infantum, LiZIP3, a member of the ZIP family of divalent metal-ion transporters. The zinc transporter activity of LiZIP3 was first disclosed by its capacity to rescue the growth of Saccharomyces cerevisiae strains deficient in zinc acquisition. Subsequent expression of LiZIP3 in Xenopus laevis oocytes was shown to stimulate the uptake of a broad range of metal ions, among which Zn2+ was the preferred LiZIP3 substrate (K0.5 ≈ 0.1 μM). Evidence that LiZIP3 functions as a zinc importer in L. infantum came from the observations that the protein locates to the cell membrane and that its overexpression leads to augmented zinc internalization. Importantly, expression and cell-surface location of LiZIP3 are lost when parasites face high zinc bioavailability. LiZIP3 decline in response to zinc is regulated at the mRNA level in a process involving (a) short-lived protein(s). Collectively, our data reveal that LiZIP3 enables L. infantum to acquire zinc in a highly regulated manner, hence contributing to zinc homeostasis.
PMCID: PMC4964273  PMID: 25644708
Leishmania; trypanosomatid; ZIP; zinc-transport; zinc-homeostasis
21.  Zinc pyrithione impairs zinc homeostasis and upregulates stress response gene expression in reconstructed human epidermis 
Zinc ion homeostasis plays an important role in human cutaneous biology where it is involved in epidermal differentiation and barrier function, inflammatory and antimicrobial regulation, and wound healing. Zinc-based compounds designed for topical delivery therefore represent an important class of cutaneous therapeutics. Zinc pyrithione (ZnPT) is an FDA-approved microbicidal agent used worldwide in over-the-counter topical antimicrobials, and has also been examined as an investigational therapeutic targeting psoriasis and UVB-induced epidermal hyperplasia. Recently, we have demonstrated that cultured primary human skin keratinocytes display an exquisite sensitivity to nanomolar ZnPT concentrations causing induction of heat shock response gene expression and poly(ADP-ribose) polymerase (PARP)-dependent cell death (Cell Stress Chaperones 15:309–322, 2010). Here we demonstrate that ZnPT causes rapid accumulation of intracellular zinc in primary keratinocytes as observed by quantitative fluorescence microscopy and inductively coupled plasma mass spectrometry (ICP-MS), and that PARP activation, energy crisis, and genomic impairment are all antagonized by zinc chelation. In epidermal reconstructs (EpiDerm™) exposed to topical ZnPT (0.1–2% in Vanicream™), ICP-MS demonstrated rapid zinc accumulation, and expression array analysis demonstrated upregulation of stress response genes encoding metallothionein-2A (MT2A), heat shock proteins (HSPA6, HSPA1A, HSPB5, HSPA1L, DNAJA1, HSPH1, HSPD1, HSPE1), antioxidants (SOD2, GSTM3, HMOX1), and the cell cycle inhibitor p21 (CDKN1A). IHC analysis of ZnPT-treated EpiDerm™ confirmed upregulation of Hsp70 and TUNEL-positivity. Taken together our data demonstrate that ZnPT impairs zinc ion homeostasis and upregulates stress response gene expression in primary keratinocytes and reconstructed human epidermis, activities that may underlie therapeutic and toxicological effects of this topical drug.
PMCID: PMC4169675  PMID: 21424779
Zinc pyrithione; Keratinocyte; Reconstructed epidermis; Heat shock response; HSPA1A; ICP-MS
22.  The Zinc Transporter Zip14 Influences c-Met Phosphorylation and Hepatocyte Proliferation During Liver Regeneration in Mice 
Gastroenterology  2012;142(7):1536-46.e5.
Zinc homeostasis in cells is maintained through tight regulation of zinc influx, efflux, and distribution to intracellular organelles by zinc transporters. The Zrt-Irt-like protein (ZIP) transporters facilitate zinc influx to the cytosol. Expression of the ZIP family member Zip14 can be induced by inflammatory cytokines, which also initiate liver regeneration. Hepatocyte proliferation is required for liver regeneration. Zinc regulates cell proliferation, tissue growth, and many mitogenic signaling pathways; we investigated its role in hepatocytes.
Wild-type and Zip14−/− mice that underwent partial hepatectomy (70% of liver removed) were used as models of liver regeneration. We also analyzed AML12 hepatocytes that overexpressed Zip14. Proliferation was assessed with proliferating cell nuclear antigen, CD1, and Ki67 markers and along with assays of zinc content was related to protein tyrosine phosphatase 1B (PTP1B) and extracellular signal–regulated kinase 1/2 signaling.
Zip14 was up-regulated and hepatic zinc content increased during liver regeneration. Increased hepatic zinc inhibited activity of the phosphatase PTP1B and increased phosphorylation of c-Met, which promoted hepatocyte proliferation. AML12 cells that overexpressed Zip14 increased in zinc content and proliferation; PTP1B was inhibited and phosphorylation of c-Met increased. The increases in hepatic levels of zinc and hepatocyte proliferation that occurred following partial hepatectomy were not observed in Zip14−/− mice.
The transporter Zip14 mediates hepatic uptake of zinc during liver regeneration and for hepatocyte proliferation. These findings indicate that zinc transporter activity regulates liver tissue growth by sequestering zinc. Reagents that regulate ZIP14 activity might be developed as therapeutics to promote liver regeneration in patients with chronic liver disease.
PMCID: PMC3635537  PMID: 22374166
Micronutrient; Signal Transduction; Tissue Regeneration; Interleukin-6
23.  Zinc Transporter ZIP14 Functions in Hepatic Zinc, Iron and Glucose Homeostasis during the Innate Immune Response (Endotoxemia) 
PLoS ONE  2012;7(10):e48679.
ZIP14 (slc39A14) is a zinc transporter induced in response to pro-inflammatory stimuli. ZIP14 induction accompanies the reduction in serum zinc (hypozincemia) of acute inflammation. ZIP14 can transport Zn2+ and non-transferrin-bound Fe2+ in vitro. Using a Zip14−/− mouse model we demonstrated that ZIP14 was essential for control of phosphatase PTP1B activity and phosphorylation of c-Met during liver regeneration. In the current studies, a global screening of ZIP transporter gene expression in response to LPS-induced endotoxemia was conducted. Following LPS, Zip14 was the most highly up-regulated Zip transcript in liver, but also in white adipose tissue and muscle. Using ZIP14−/− mice we show that ZIP14 contributes to zinc absorption from the gastrointestinal tract directly or indirectly as zinc absorption was decreased in the KOs. In contrast, Zip14−/− mice absorbed more iron. The Zip14 KO mice did not exhibit hypozincemia following LPS, but do have hypoferremia. Livers of Zip14−/− mice had increased transcript abundance for hepcidin, divalent metal transporter-1, ferritin and transferrin receptor-1 and greater accumulation of iron. The Zip14−/− phenotype included greater body fat, hypoglycemia and higher insulin levels, as well as increased liver glucose and greater phosphorylation of the insulin receptor and increased GLUT2, SREBP-1c and FASN expression. The Zip14 KO mice exhibited decreased circulating IL-6 with increased hepatic SOCS-3 following LPS, suggesting SOCS-3 inhibited insulin signaling which produced the hypoglycemia in this genotype. The results are consistent with ZIP14 ablation yielding abnormal labile zinc pools which lead to increased SOCS-3 production through G-coupled receptor activation and increased cAMP production as well as signaled by increased pSTAT3 via the IL-6 receptor, which inhibits IRS 1/2 phosphorylation. Our data show the role of ZIP14 in the hepatocyte is multi-functional since zinc and iron trafficking are altered in the Zip14−/− mice and their phenotype shows defects in glucose homeostasis.
PMCID: PMC3480510  PMID: 23110240
24.  Influence of +1245 A/G MT1A polymorphism on advanced glycation end-products (AGEs) in elderly: effect of zinc supplementation 
Genes & Nutrition  2014;9(5):426.
Advanced glycation end-products (AGEs) stimulate reactive oxygen species (ROS) generation and represent a risk factor for atherosclerosis, while their formation seems to be prevented by zinc. Metallothioneins (MT), zinc-binding proteins exert an antioxidant function by regulating intracellular zinc availability and protecting cells from ROS damages. +1245 A/G MT1A polymorphism was implicated in type 2 diabetes and in cardiovascular disease development as well as in the modulation of antioxidant response. The purpose of this study was to investigate the influence of +1245 A/G MT1A polymorphism on AGEs and ROS production and to verify the effect of zinc supplementation on plasma AGEs, zinc status parameters and antioxidant enzyme activity in relation to this SNP. One hundred and ten healthy subjects (72 ± 6 years) from the ZincAge study were supplied with zinc aspartate (10 mg/day for 7 weeks) and screened for +1245 MT1A polymorphism. +1245 MT1A G+ (Arginine) genotype showed higher plasma AGEs and ROS production in peripheral blood mononuclear cells (PBMCs) than G− (Lysine) one at the baseline. No significant changes after zinc supplementation were observed for AGEs, ROS and MT levels as well as for enzyme antioxidant activity in relation to the genotype. Among zinc status parameters, major increases were observed for the intracellular labile zinc (iZnL) and the NO-induced release of zinc in PBMCs, in G+ genotype as compared to G− one. In summary, +1245 G+ carriers showed increased plasma AGEs and ROS production in PBMCs at baseline and a higher improvement in iZnL after zinc intervention with respect to G− individuals.
Electronic supplementary material
The online version of this article (doi:10.1007/s12263-014-0426-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4172651  PMID: 25149676
MT1A polymorphism; Advanced glycation end-products (AGEs); Zinc supplementation; Aging; Intracellular free zinc
25.  Characterization of Zinc Influx Transporters (ZIPs) in Pancreatic β Cells 
The Journal of Biological Chemistry  2015;290(30):18757-18769.
Background: Zinc influx transporters (ZIPs), together with zinc efflux transporters (ZnTs), regulate cellular zinc homeostasis.
Results: Down-regulation of ZIP6 and ZIP7 expression impairs glucose-stimulated insulin secretion via reduced first-phase insulin exocytosis.
Conclusion: ZIP6 and ZIP7 are functionally important for maintaining proper insulin secretion in pancreatic β cells.
Significance: ZIP6 and ZIP7 represent novel proteins that contribute to the insulin secretory pathway.
Zinc plays an essential role in the regulation of pancreatic β cell function, affecting important processes including insulin biosynthesis, glucose-stimulated insulin secretion, and cell viability. Mutations in the zinc efflux transport protein ZnT8 have been linked with both type 1 and type 2 diabetes, further supporting an important role for zinc in glucose homeostasis. However, very little is known about how cytosolic zinc is controlled by zinc influx transporters (ZIPs). In this study, we examined the β cell and islet ZIP transcriptome and show consistent high expression of ZIP6 (Slc39a6) and ZIP7 (Slc39a7) genes across human and mouse islets and MIN6 β cells. Modulation of ZIP6 and ZIP7 expression significantly altered cytosolic zinc influx in pancreatic β cells, indicating an important role for ZIP6 and ZIP7 in regulating cellular zinc homeostasis. Functionally, this dysregulated cytosolic zinc homeostasis led to impaired insulin secretion. In parallel studies, we identified both ZIP6 and ZIP7 as potential interacting proteins with GLP-1R by a membrane yeast two-hybrid assay. Knock-down of ZIP6 but not ZIP7 in MIN6 β cells impaired the protective effects of GLP-1 on fatty acid-induced cell apoptosis, possibly via reduced activation of the p-ERK pathway. Therefore, our data suggest that ZIP6 and ZIP7 function as two important zinc influx transporters to regulate cytosolic zinc concentrations and insulin secretion in β cells. In particular, ZIP6 is also capable of directly interacting with GLP-1R to facilitate the protective effect of GLP-1 on β cell survival.
PMCID: PMC4513131  PMID: 25969539
exocytosis; glucose; insulin secretion; pancreatic islet; zinc; GLP1; insulin granule; zinc influx transporter

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