Expression Of ZnT-1 Research Articles

Calcium and zinc decrease intracellular iron by decreasing transport during iron repletion in an in vitro model.

Iron is an essential micronutrient that participates in a number of vital reactions and its absorption may be altered by various nutritional factors such as other micronutrients. Our hypothesis is that iron absorption is decreased because of the interactions with zinc and calcium. We evaluated the interaction between calcium and zinc on iron uptake and transport, intracellular Fe and Zn levels and mRNA expression of DMT1, ferroportin, Zip4 and ZnT1 in an in vitro model. Caco-2 cells were cultivated with 1mM Ca; 10 or 30µM Zn and/or 10, 20 or 30µM Fe for 24h. Intracellular Fe decreased in cells incubated with 30µM Zn or with the mix Ca/10µM Zn/Fe. Zn mostly increased under Ca, Zn and Fe treatment. DMT1 mRNA expression decreased when intracellular Fe increased. Ferroportin expression displayed no change in cells cultured with different Fe concentrations. The mix of Ca, Zn and Fe increased DMT1 and ferroportin expression mainly under high Zn concentration. Zip4 expression was mostly augmented by Ca and Fe; however, ZnT1 showed no change in all conditions studied. Fe uptake was higher in all the conditions studied compared to control cells; however, Fe transport increased only in cells incubated with Fe alone. In all the other conditions, Fe transport was lower than that in control cells. The present findings suggest that Ca and Zn interfere with iron metabolism. This interference is through an increase in ferroportin activity, which results in a diminished net iron absorption.

Effects of Hepcidin on Zinc Homeostasis in HepG2 cells

Hepcidin, a peptide hormone produced by theliver in response to iron loading and inflammation, regulates body iron storesby inhibiting iron transporter function. Recently, hepcidin was shown toinhibit zinc efflux in intestinal epithelial cells. The objective of this study was to determine whether hepcidin affects hepatic zinc homeostasis. Human HepG2cells were treated ± 1μM hepcidin for 24 h. Labile zinc pools were labeled with the zinc‐specific fluoroionophore FluoZin‐3‐AM (Kd 15 nM) and visualized and quantified using confocal microscopy and a fluorescent platereader. Expression of metallothionein, ZIP14, and ZnT1 was determined by Western blot. Intracellular zinc in control cells was localized to vesiclesdispersed throughout the cytoplasm; however, the number of vesicles and intensity of FluoZin‐3 fluorescence decreased by 38% in cells treated with hepcidin for 24 h (34.0 ± 11.3 and 21.0 ± 11.2 RFU/OD 562 nm; P<0.05). Metallothionein expression increased in cells treated with hepcidin as compared to control cells(P<0.05). ZIP14 expression remained unchanged (P>0.05), whereas hepcidin increased ZnT1 expression (P<0.05) as compared to control cells. These data suggest that hepcidin may affect zinc homeostasis in liver cells. Understanding the mechanism and role of hepcidin in zinc homeostasis may provide insight in to the hypozincemia associated with inflammation and infection.Support or Funding InformationThe views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government. Support provided by USAMRMC.

Expression Patterns and Correlations with Metabolic Markers of Zinc Transporters ZIP14 and ZNT1 in Obesity and Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) is associated with infertility, increased androgen levels, and insulin resistance. In adipose tissue, zinc facilitates insulin signaling. Circulating zinc levels are altered in obesity, diabetes, and PCOS; and zinc supplementation can ameliorate metabolic disturbances in PCOS. In adipose tissue, expression of zinc influx transporter ZIP14 varies with body mass index (BMI), clinical markers of metabolic syndrome, and peroxisome proliferator-activated receptor gamma (PPARG). In this study, we investigated expression levels of ZIP14 and PPARG in subcutaneous adipose tissue of 36 PCOS women (17 lean and 19 obese women) compared with 23 healthy controls (7 lean and 16 obese women). Further, expression levels of zinc transporter ZIP9, a recently identified androgen receptor, and zinc efflux transporter ZNT1 were investigated, alongside lipid profile and markers of glucose metabolism [insulin degrading enzyme, retinol-binding protein 4 (RBP4), and glucose transporter 4 (GLUT4)]. We find that ZIP14 expression is reduced in obesity and positively correlates with PPARG expression, which is downregulated with increasing BMI. ZNT1 is upregulated in obesity, and both ZIP14 and ZNT1 expression significantly correlates with clinical markers of altered glucose metabolism. In addition, RBP4 and GLUT4 associate with obesity, but an association with PCOS as such was present only for PPARG and RBP4. ZIP14 and ZNT1 does not relate to clinical androgen status and ZIP9 is unaffected by all parameters investigated. In conclusion, our findings support the existence of a zinc dyshomeostasis in adipose tissue in metabolic disturbances including PCOS-related obesity.

Zinc Dyshomeostasis in Cardiomyocytes after Acute Hypoxia/Reoxygenation.

Zinc dyshomeostasis may play a role in the pathogenesis of myocardial ischemia/reperfusion injury. The objective of this study was to investigate the expression profile of zinc regulated transporter like- and iron-regulated transporter-like proteins (ZIPs) and zinc transporter proteins (ZnTs) in cardiomyocytes and their modulation in response to hypoxia and reoxygenation. Adult rat ventricular myocytes (ARVMs) were subjected to 6h of hypoxia, followed by 18h of reoxygenation. Intracellular and extracellular zinc concentrations were determined using Fluozin-3 and Newport Green fluorescence, respectively. Expression of ZnTs 1, 2, 5, and 9 along with ZIPs 1, 2, 3, 6, 7, 9, 10, 11, 13, and 14 was detectable in the cardiomyocytes by real-time reverse transcriptase polymerase chain reaction. Hypoxia elicited accumulation of intracellular free zinc, but subsequent reoxygenation resulted in striking loss of intracellular free zinc and decreased the cardiomyocyte viability. Concomitantly, extracellular zinc levels dropped rapidly during hypoxia, but increased after reoxygenation. Immunoblotting analysis revealed that hypoxia increased the expression of ZnT1, but reoxygenation significantly increased the expression of ZnTs 2 and 5. Neither hypoxia nor reoxygenation altered the levels of ZnT9. Increased intracellular zinc at the end of hypoxia is related to enhanced expression of ZIPs, whereas decreased intracellular zinc during reoxygenation appears to be due to lowered expression of all ZIPs, in addition to elevated levels of ZnTs 2 and 5. These results thus suggest that there is impaired accumulation of intracellular zinc during reoxygenation, due to overexpression of specific ZnTs and downregulation of ZIP expression.

Down-regulation of zinc transporter-1 in astrocytes induces neuropathic pain via the brain-derived neurotrophic factor - K+-Cl− co-transporter-2 signaling pathway in the mouse spinal cord

We previously demonstrated, using a DNA microarray analysis, the down-regulated expression of the slc30a1 gene (zinc transporter 1, ZnT1) in a neuropathic pain model induced by partial sciatic nerve ligation (PSNL). Zinc is an essential trace mineral that plays important roles in physiological functions, and ZnT1 modulates intracellular zinc levels. In the present study, we examined the effects of the down-regulation of the ZnT1 gene in the spinal cord on tactile allodynia.The knockdown (KD) of ZnT1 by the intrathecal administration of siRNA against ZnT1 to mice induced allodynia, a characteristic syndrome of neuropathic pain, which persisted for at least one month. ZnT1 KD increased intracellular zinc concentrations in primary astrocyte cultures, and this was followed by enhanced PKCα membrane translocation and NFκB nuclear translocation as well as increases in the levels of IL-6 and BDNF expressed and the phosphorylation of CREB in vitro. Neuropathic pain induced by ZnT1 KD was inhibited by an IL-6, BDNF, and TrkB siRNA injection. The down-regulated expression of KCC2 in spinal cord was induced by ZnT1 KD and prevented by an intrathecal injection of IL-6, BDNF, and TrkB siRNA. These results indicate that PSNL via the down-regulated expression of ZnT1 increases intracellular zinc concentrations, enhances PKCα membrane translocation and NFκB nuclear translocation, up-regulates the expression of IL-6, increases the phosphorylation of CREB, and promotes the BDNF cascade reaction in astrocytes, thereby down-regulating the expression of KCC2 and inducing neuropathic pain in vivo. This mechanism is considered to be responsible for the activation of TrkB in neurons through the release of BDNF from astrocytes. The results of the present study also indicate that zinc signaling in astrocytes occurs upstream of the BDNF-TrkB-KCC2 cascade reaction.

Hepcidin attenuates zinc efflux in Caco‐2 cells

Hepcidin, a peptide hormone produced by theliver in response to iron loading and inflammation, regulates body iron storesby inhibiting iron transporter function. Zinc also regulates hepcidinexpression; however, the effects of hepcidin on zinc transport and zinctransporter function have not been documented. The objective of this study wasto determine whether hepcidin affects intestinal zinc transport. Differentiatedhuman intestinal Caco‐2 cells were treated ± 1μM hepcidin for 3–24 h. Zinctransport was assessed in cells seeded on Transwell inserts. Media was collected from the apical and basolateral chambers and 67Znconcentrations were determined by ICP‐MS. Expression of metallothionein (MT1A), ZIP4 (SLC39A4), and ZnT1 (SLC30A1)was determined using RT‐qPCR and Western blot. Hepcidin attenuated zinctransport, as cells treated with hepcidin exported 20 and 26% less 67Zninto the basolateral chamber at 3 h and 24 h (P<0.05), respectively, and retained more cellular 67Zn(P<0.05) as compared to control cells. Intracellular zinc in control cells was localized to vesicles dispersed throughout the cytoplasm; however, the number of vesicles and intensity of FluoZin‐3 fluorescence decreased (P<0.05)in cells treated with hepcidin for 24 h. The mRNA abundance of metallothionein increased 1.5‐fold (P<0.05). ZIP4 protein remained unchanged (P>0.05), whereas hepcidin reduced ZnT1 protein by 75% (P<0.05) as compared to control cells. These data suggest that hepcidin is a regulatory hormone that manages zinc transport in intestinal cells by downregulating ZnT1 to reduce zinc export and increasing metallothionein to buffer the rise in intracellular zinc. Understanding the mechanism and role of hepcidin in zinc homeostasis may provide insight into the hypozincemiaassociated with inflammation and infection.Support or Funding InformationSupport provided by USAMRMC.

A Feedback Loop between Inflammation and Zn Uptake.

ObjectiveZinc (Zn) has major effects on the immune system and inflammation is associated with systemic Zn deficiency. The aim of this work was to investigate how inflammation modifies Zn metabolism at the cellular level. Rheumatoid arthritis (RA) synoviocytes exposed to cytokines were used as a model of chronic inflammation. Osteoarthritis (OA) synoviocytes were used as control.MethodsZn levels were measured in medium and inside cells by Induced Coupled Plasma-Mass Spectrometry (ICP-MS), in the presence of minute quantities of stable spike 70Zn isotope and the addition or not of the pro-inflammatory cytokines interleukin-17 (IL-17) and tumor necrosis factor alpha (TNF-α). Gene expression of ZIP-8 importer, ZnT1 exporter and the homeostasis regulators metallothioneins (MTs) was evaluated after pre-exposure to cytokines, with or without exogenous Zn addition at increasing concentrations. IL-6 production was used as a marker of inflammation and measured by ELISA.ResultsExposure to IL-17 and TNF-α enhanced expression of the Zn-importer ZIP-8, regardless of the concentration of Zn in the culture medium. In contrast, the expression of the Zn-exporter ZnT1 and of the MTs was primarily dependent on Zn levels. Addition of Zn also increased the production of IL-6, thus further stimulating the inflammatory response.ConclusionIL-17/TNF-mediated inflammation enhanced the intracellular Zn uptake by synoviocytes, further increasing inflammation. These observations document the existence of a feedback loop between inflammation and Zn uptake. Based on these results, a mathematical model was developed to represent the cytokine-mediated Zn homeostasis alterations.

Accumulation of copper in the kidney of pigs fed high dietary zinc is due to metallothionein expression with minor effects on genes involved in copper metabolism

A study was conducted to determine the effect of high dietary zinc (Zn) oxide on trace element accumulation in various organs with special emphasis on the kidney. A total of 40 weaned piglets were allocated into two groups with 16 and 24 piglets each receiving a diet containing normal (NZn; 100mg Zn/kg) or high (HZn; 2,100mg Zn/kg) Zn concentration, respectively. After two weeks, eight piglets from each treatment were killed and organ samples were taken. Eight piglets from the remaining 16 pigs fed HZn diets were changed to NZn diets (CZn). All remaining piglets were killed after another two weeks for organ sampling. Trace element concentration was determined in the jejunum, liver, kidney, pancreas, bone (metacarpal IV), spleen, lung, thymus, tonsils and lymph nodes of jejunum, ileum and colon. Kidney mRNA expression of Zn transporter ZnT1 and ZIP4, genes involved in Cu metabolism (Ctr1, Atox1, SOD1, ATP7A, CCS, CP) and divalent metal ion transport (DMT1) and binding (MT-1a, MT-2b, MT-3) were determined. The Zn concentration in jejunum, liver, pancreas tissue and metacarpal IV was higher (P<0.05) in HZn group compared with NZn and CZn groups. Trace element concentration in organs of CZn pigs was similar to those fed NZn diets. Zn concentration in muscle, lung and lymphatic organs as thymus, tonsils, spleen and lymph nodes of jejunum, ileum and colon did not differ between the groups. Zn and Cu were positively correlated (R=0.67; P<0.05) in the kidney. No significant differences for Cu chaperones, Cu transporters and Cu-dependent factors were determined despite decreased expression of Atox1 after two weeks and increased Ctr1 expression over time in the HZn group. Expression of MT-1a, MT-2b and MT-3 were significantly higher in HZn fed pigs with most pronounced effects for MT-1a > MT-2b > MT-3. Gene expression of MTs in pigs fed CZn diets did not differ from pigs fed NZn diets. The data suggest that high dietary Zn feeding in pigs leads to Cu co-accumulation in the kidney of pigs with minor effect on genes relevant for Cu metabolism. In addition, the organ Zn and Cu accumulation is reversible after two weeks of withdrawal of high dietary Zn.

ZNT-1 Expression Reduction Enhances Free Zinc Accumulation in Astrocytes After Ischemic Stroke.

Excess intracellular zinc has been implicated in ischemic brain cell death. We previously reported that extracellular zinc increases intracellular free zinc level only in hypoxic astrocytes but not in normoxia astrocytes. However, the underlying mechanisms remain to be elucidated. Zinc transporters ZnTs and ZIPs mediate intracellular zinc efflux and extracellular zinc influx. In the present study, we determined the effect of hypoxia/reoxygenation on ZnT-1 and ZIP-1. Hypoxia/reoxygenation did not change the ZIP-1 level in astrocytes. Remarkably, hypoxia/reoxygenation dramatically decreased ZnT-1 expression, which can be difficult to reverse by the addition of extracellular zinc, although extracellular zinc treatment significantly increased ZnT-1 level at normoxia. These results suggest that hypoxia/reoxygenation blocked zinc efflux, whereas zinc influx may be at a similar level to that in normoxia, providing a novel mechanism for intracellular free zinc accumulation after ischemic stroke.

Zinc sensing by metal-responsive transcription factor 1 (MTF1) controls metallothionein and ZnT1 expression to buffer the sensitivity of the transcriptome response to zinc.

Only a small number of genes are known direct targets of the zinc-responsive transcription factor MTF1; therefore, the aim of this study was to gain a more complete understanding of the MTF-1 regulated zinc-responsive component of the transcriptome. A targeted siRNA was used to deplete MTF1 expression in the human intestinal cell line Caco-2. We predicted that the response to zinc of direct MTF1 target genes would be abrogated by MTF1 knockdown. Surprisingly, a greater number of genes were regulated by zinc following MFT1 knockdown, and most genes that responded to zinc under both control and MTF1-depleted conditions had an augmented response in the latter condition. Exceptions were the zinc effluxer ZnT1 and a suite of metallothionein genes, suggesting that responses of other genes to zinc are usually buffered by increases in these proteins. We propose that MTF1 heads a hierarchy of zinc sensors, and through controlling the expression of a raft of metallothioneins and other key proteins involved in controlling intracellular zinc levels (e.g. ZnT1) alters zinc buffering capacity and total cellular zinc content. We tested and validated this model by overexpressing metallothionein and observing the predicted curtailment in response of the zinc-repressed SLC30A5 (ZnT5) promoter. The model provides the framework for an integrated understanding of cellular zinc homeostasis. Because MTs can bind metals other than zinc, this framework links with overall cellular metal homeostasis.

Dosage Effect of Zinc Glycine Chelate on Zinc Metabolism and Gene Expression of Zinc Transporter in Intestinal Segments on Rat.

Zinc plays an essential role in various fundamental biological processes. The focus of this research was to investigate the dosage effect of zinc glycine chelate (Zn-Gly) on zinc metabolism and the gene expression of zinc transporters in intestinal segments. A total of 30 4-week-old SD rats were randomized into five treatment groups. The basal diets for each group were supplemented with gradient levels of Zn (0, 30, 60, 90, and 180mg/kg) from Zn-Gly. After 1-week experiment, the results showed that serum and hepatic zinc concentration were elevated linearly with supplemental Zn levels from 0 to 180mg Zn/kg. Serum Cu-Zn SOD activities resulted in a significant (P<0.01) quadratic response and reached the peak when fed 60mg Zn/kg. There were linear responses to the addition of Zn-Gly from 0 to 180mg Zn/kg on Cu-Zn SOD and AKP activities in the liver. In the duodenum, MT1 mRNA was upregulated with the increasing dietary Zn-Gly levels and reached the peak of 180mg Zn/kg (P<0.05). Zip4 mRNA expression was downregulated with the increasing zinc levels (P<0.05) in both duodenum and jejunum. In the jejunum, Zip5 mRNA expression in 60mg Zn/kg was higher compared with other groups (P<0.05). ZnT1 mRNA in duodenum was numerically increased with the rising levels of zinc content and was significantly higher (P<0.05) with 180mg Zn/kg. In the duodenum, adding 60 or 90mg Zn/kg increased PepT1 expression, but in the jejunum, 60mg Zn/kg did not differ from 0 added Zn. In summary, there is a dose-dependent effect of dietary Zn-Gly on serum and hepatic zinc levels and the activities of Cu-Zn SOD and AKP on rats. Dietary Zn-Gly has a certain effect on MT1, Zip4, Zip5, and ZnT1 expression, which expressed differently in intestinal segments with different levels of Zn-Gly load. Besides, Zn-Gly also could regulate PepT1 expression in intestinal segments.

Acute changes in cellular zinc alters zinc uptake rates prior to zinc transporter gene expression in Jurkat cells.

A coordinated network of zinc transporters and binding proteins tightly regulate cellular zinc levels. Canonical responses to zinc availability are thought to be mediated by changes in gene expression of key zinc transporters. We investigated the temporal relationships of actual zinc uptake with patterns of gene expression in membrane-bound zinc transporters in the human immortalized T lymphocyte Jurkat cell line. Cellular zinc levels were elevated or reduced with exogenous zinc sulfate or N,N,N',N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), respectively. Excess zinc resulted in a rapid 44 % decrease in the rate of zinc uptake within 10 min. After 120 min, the expression of metallothionein (positive control) increased, as well as the zinc exporter, ZnT1; however, the expression of zinc importers did not change during this time period. Zinc chelation with TPEN resulted in a rapid twofold increase in the rate of zinc uptake within 10 min. After 120 min, the expression of ZnT1 decreased, while again the expression of zinc importers did not change. Overall, zinc transporter gene expression kinetics did not match actual changes in cellular zinc uptake with exogenous zinc or TPEN treatments. This suggests zinc transporter regulation may be the initial response to changes in zinc within Jurkat cells.

Expression and Cellular Distribution of SLC30A Zinc Transporter Family Members in Human Normal and Diabetic Pancreases and in Human Insulin‐secreting b‐cells

Zinc has been shown to have important roles in insulin synthesis, maturation, and secretion in pancreatic ß‐cells. Human cellular zinc homeostasis is largely maintained by two zinc transporter families, SLC30A (ZNT1‐10) and SLC39A (ZIP1‐14). This study aimed to demonstrate the protein expression of 10 members of the ZNT family in human pancreases in normal and type 2 diabetic conditions by immunohistochemistry. The protein expression and cellular localization of ZNTs were also illustrated in a human‐derived pancreatic ß‐cell line (1.1B4‐KH5) by immunocytochemistry. Our results revealed that, nine and eight ZNTs were expressed at different levels in acini of normal and diabetic pancreases, respectively. Moreover, nine and ten ZNTs were detected in the islet of Langerhans of normal and diabetic pancreases, respectively. In ß‐cells, most ZNTs were expressed intracellularly except for ZNT1 and ZNT2, which were localized on the cell membrane. These intracellularly expressed ZNTs showed some levels of overlapping with insulin within the cell. Our study suggests that zinc homeostasis is vital for the pancreas function both in the exocrine and endocrine components evident by the extensive expression of ZNTs. Increase in expression of ZNT8 and ZNT10 or decrease in expression of ZNT1 and ZNT2 may complicate with type 2 diabetes.

Zinc transporter-1: a novel NMDA receptor-binding protein at the postsynaptic density.

Zinc (Zn(2+) ) is believed to play a relevant role in the physiology and pathophysiology of the brain. Hence, Zn(2+) homeostasis is critical and involves different classes of molecules, including Zn(2+) transporters. The ubiquitous Zn(2+) transporter-1 (ZNT-1) is a transmembrane protein that pumps cytosolic Zn(2+) to the extracellular space, but its function in the central nervous system is not fully understood. Here, we show that ZNT-1 interacts with GluN2A-containing NMDA receptors, suggesting a role for this transporter at the excitatory glutamatergic synapse. First, we found that ZNT-1 is highly expressed at the hippocampal postsynaptic density (PSD) where NMDA receptors are enriched. Two-hybrid screening, coimmunoprecipitation experiments and clustering assay in COS-7 cells demonstrated that ZNT-1 specifically binds the GluN2A subunit of the NMDA receptor. GluN2A deletion mutants and pull-down assays indicated GluN2A(1390-1464) domain as necessary for the binding to ZNT-1. Most importantly, ZNT-1/GluN2A complex was proved to be dynamic, since it was regulated by induction of synaptic plasticity. Finally, modulation of ZNT-1 expression in hippocampal neurons determined a significant change in dendritic spine morphology, PSD-95 clusters and GluN2A surface levels, supporting the involvement of ZNT-1 in the dynamics of excitatory PSD. Zn(2+) transporter-1 (ZNT-1) pumps cytosolic Zn(2+) to the extracellular space, but its function in the central nervous system is not fully understood. We show that ZNT-1 interacts with GluN2A-containing NMDA receptors at the glutamatergic synapse. Most importantly, ZNT-1/GluN2A complex is regulated by induction of synaptic plasticity. Modulation of ZNT-1 expression in hippocampal neurons determined a shrinkage of dendritic spines and a reduction of GluN2A surface levels supporting the involvement of ZNT-1 in the dynamics of the excitatory synapse.

Zinc transporters ZnT3 and ZnT6 are downregulated in the spinal cords of patients with sporadic amyotrophic lateral sclerosis.

The loss of homeostasis of essential metals is associated with various diseases, including neurodegenerative diseases. Previous studies have shown that the levels of zinc (Zn) are significantly higher in the cerebrospinal fluid of patients with amyotrophic lateral sclerosis (ALS). Zn transporters and metallothioneins tightly control intracellular and extracellular Zn levels. This study investigated the protein levels of ZnT, a Zn transporter family, in ALS patients and model mice. The mRNA expression of ZnT1, -3, -4, -5, -6, -7, and -10 was assessed in the spinal cords of human control subjects. ZnT3 and ZnT6 protein levels were significantly diminished in the spinal cords of sporadic ALS patients compared with controls. Furthermore, immunohistochemical staining demonstrated decreased ZnT3 and ZnT6 immunoreactivity in the ventral horn of the spinal cords in ALS patients. Moreover, immunohistochemical analysis revealed that all ZnTs expressed in the spinal cords were localized in a distinct subset of motor neurons. In addition, ZnT3 and ZnT6 protein levels were not altered in SOD1 (G93A) mutant transgenic mice before or after the onset of ALS symptoms compared with controls. These results suggest that ZnT3 and ZnT6 protein levels are decreased in the spinal cords of sporadic ALS patients; however, this did not occur merely via loss of motor neurons.

Stimulation of bone growth following zinc incorporation into biomaterials

Rapid development of zinc biology has broadened the applications of Zn-incorporated biomaterials to tissue engineering but also raised concerns about the long-term safety of released Zn2+ ions. Clinical success hinges on the amount of incorporated zinc and subsequent optimized release sufficient to stimulate osseointegration. In this study, zinc is incorporated into the sub-surface of TiO2 coatings by plasma immersion ion implantation and deposition (PIII&D). The Zn-implanted coatings show significant improvement compared to the “bulk-doped” coatings prepared by plasma electrolyte oxidation in terms of osteogenesis in vitro and in vivo. Molecular and cellular osteogenic activities demonstrate that rBMSCs cultured on the Zn-implanted coatings have higher ALP activity and up-regulated osteogenic-related genes (OCN, Col-I, ALP, Runx2) compared to the bulk-doped Zn coatings and controls. In vivo osseointegration studies conducted for 12 weeks on the rat model show early-stage new bone formation and the bone contact ratio (12 week) on the Zn-implanted coating is larger. The ZnT1 and ZIP1 gene expression studies demonstrate that the Zn-implanted coatings can better stimulate bone growth with reduced Zn release than those doped with zinc throughout the coatings.

Noccaea caerulescens populations adapted to grow in metalliferous and non-metalliferous soils: Ni tolerance, accumulation and expression analysis of genes involved in metal homeostasis

Three populations of the Brassicaceae Noccaea caerulescens: (i) the metallicolous Mt. Prinzera population (Italy), which have adapted to grow on serpentinite, a soil naturally rich in Ni, Co, Cr; (ii) the metallicolous La Calamine population (Belgium), which have adapted to growth on soil highly contaminated by Cd, Zn and Pb, and (iii) the non-metallicolous population growing near the town of Rožnov pod Radhoštěm (Czech Republic), were grown in hydroponics and treated with different Ni concentrations (0, 10 and 100μM NiSO4). Ni tolerance and accumulation were analysed along with the expression of genes belonging to different families involved in plant metal homeostasis: ZNT1, ZNT2, NRAMP3, NRAMP4 coding for non-ATP-hydrolysing plasma membrane and vacuolar metal transporters, HMA3, HMA4 coding for ATP-hydrolising metal transporters, NAS1, NAS3, NAS4 and MT1B involved in metal chelation. The three populations showed different levels of expression of some of the tested genes in condition of 0μM Ni. In addition, the Ni hyperaccumulator Mt. Prinzera showed the highest Ni translocation capacity at 10μM Ni and a specific up-regulation of ZNT1, ZNT2, NAS3, NRAMP3 and NRAMP4 genes. With the same Ni treatment, the La Calamine population induced HMA4 and MT1B genes, while the Rožnov pod Radhoštěm population only displayed an over-expression of all the genes at 100μM Ni, the condition in which this population suffers heavily from Ni stress. The expression of ZNT1, ZNT2, NRAMP3, NRAMP4 was also tested on N. caerulescens plants growing in the natural environment on Mt. Prinzera. Higher transcript levels of ZNT1 and ZNT2 were associated with higher levels of plant's total Ni content. Overall these results suggested that some of the genes considered can have a role in determining metal tolerance and accumulation in metallicolous N. caerulescens populations, while they were not involved in metal homeostasis in the non-metallicolous population.

Zinc transporter-1 concentrates at the postsynaptic density of hippocampal synapses

BackgroundZinc concentrates at excitatory synapses, both at the postsynaptic density and in a subset of glutamatergic boutons. Zinc can modulate synaptic plasticity, memory formation and nociception by regulating transmitter receptors and signal transduction pathways. Also, intracellular zinc accumulation is a hallmark of degenerating neurons in several neurological disorders. To date, no single zinc extrusion mechanism has been directly localized to synapses. Based on the presence of a canonical PDZ I motif in the Zinc Transporter-1 protein (ZnT1), we hypothesized that ZnT1 may be targeted to synaptic compartments for local control of cytosolic zinc. Using our previously developed protocol for the co-localization of reactive zinc and synaptic proteins, we further asked if ZnT1 expression correlates with presynaptic zinc content in individual synapses.FindingsHere we demonstrate that ZnT1 is a plasma membrane protein that is enriched in dendritic spines and in biochemically isolated synaptic membranes. Hippocampal CA1 synapses labelled by postembedding immunogold showed over a 5-fold increase in ZnT1 concentration at synaptic junctions compared with extrasynaptic membranes. Subsynaptic analysis revealed a peak ZnT1 density on the postsynaptic side of the synapse, < 10 nm away from the postsynaptic membrane. ZnT1 was found in the vast majority of excitatory synapses regardless of the presence of vesicular zinc in presynaptic boutons.ConclusionsOur study has identified ZnT1 as a novel postsynaptic density protein, and it may help elucidate the role of zinc homeostasis in synaptic function and disease.

Supplement zinc as an effective treatment for spinal cord ischemia/reperfusion injury in rats

ObjectiveBrain-derived neurotrophic factor (BDNF) plays a key role in the pathophysiology process and therapy of spinal cord injury (SCI). Accordingly, zinc regulates the expression of BDNF and its receptor in the central nervous system, the mechanism of which is still unknown. The present study investigates whether supplement zinc could reduce neurological damage in a rat model, with spinal cord ischemia–reperfusion (I/R) injury and how the effect of zinc transporter 1(ZnT-1) was involved. Methods: 100 Sprague–Dawley male rats were randomly and evenly divided into four groups. They were subjected to spinal cord ischemia by clamping the abdominal aorta for 45min. Rats in the zinc-deficient dietary model group (ZD), zinc-adequate dietary model group (ZA), and zinc-high dietary model group (ZH) were given free access to purified diet, containing 5, 30, or 180mg Zn/kg. Sham operation rats were subjected to laparotomy without clamping of the aorta and were fed by ZA diet (30mg Zn/kg). Neurological function was scored by Tarlov's score. The spinal cord segments (L5) were harvested for histological examination, auto-metallographic (AMG) analysis, myeloperoxidase (MPO) activity analysis, expression of ZnT-1 and BDNF. Results: The rats in the ZH group have shown the higher neurological scores, slighter histological changes and the attenuated MPO activity, compared with those in the ZD and ZA groups at the four observation time points (p<0.05). The AMG staining density in the ZH group was significantly higher than that of ZD group in 14 days later after the operation. Compared with other groups, ZH group's expression of Zn-T1 and BDNF were significantly increased, and was positively correlated with the same time points after surgery (Spearman rho=0.403, p=0.0152.) Conclusion: These findings suggest that zinc supplement can significantly reduce the spinal cord I/R injury in rats. The mechanism may be related with restraining the MPO activity and increasing of ZnT-1, which promoted the synthesis and release of BDNF.

Cobalt chloride speciation, mechanisms of cytotoxicity on human pulmonary cells, and synergistic toxicity with zinc

Cobalt is used in numerous industrial sectors, leading to occupational diseases, particularly by inhalation. Cobalt-associated mechanisms of toxicity are far from being understood and information that could improve knowledge in this area is required. We investigated the impact of a soluble cobalt compound, CoCl(2)*6H(2)O, on the BEAS-2B lung epithelial cell line, as well as its impact on metal homeostasis. Cobalt speciation in different culture media, in particular soluble and precipitated cobalt species, was investigated via theoretical and analytical approaches. The cytotoxic effects of cobalt on the cells were assessed. Upon exposure of BEAS-2B cells to cobalt, intracellular accumulation of cobalt and zinc was demonstrated using direct in situ microchemical analysis based on ion micro-beam techniques and analysis after cell lysis by inductively coupled plasma mass spectrometry (ICP-MS). Microchemical imaging revealed that cobalt was rather homogeneously distributed in the nucleus and in the cytoplasm whereas zinc was more abundant in the nucleus. The modulation of zinc homeostasis led to the evaluation of the effect of combined cobalt and zinc exposure. In this case, a clear synergistic increase in toxicity was observed as well as a substantial increase in zinc content within cells. Western blots performed under the same coexposure conditions revealed a decrease in ZnT1 expression, suggesting that cobalt could inhibit zinc release through the modulation of ZnT1. Overall, this study highlights the potential hazard to lung function, of combined exposure to cobalt and zinc.