Regulation of zinc transporters by dietary flaxseed lignan in human breast cancer xenografts

Zinc is essential for cell growth. Previous studies have shown that zinc concentration in breast cancer tissues is higher than that in normal breast tissues. Zinc cannot passively diffuse across cell membranes and specific zinc transporter proteins are required. Two gene families have been identified involved in zinc homeostasis. ZnT transporters reduce intracellular zinc while ZIP transporters increase intracellular zinc. In this study, three human zinc transporter members: ZnT-1, ZIP2 and LIV-1 were chosen. We aimed to determine the effect of flaxseed lignan on the growth of ER-negative breast cancer cells in a nude mice model and observe the effect of flaxseed lignan on the regulation of the three zinc transporter in mRNA level. Nude mice were xenografted with human breast cancer cell line MDA-MB-231 and 6 weeks later were fed either the basal diet (BD) or BD supplemented with 10% FS and SDG for 5 weeks. The SDG levels were equivalent to the amounts in the 10% FS. RT-PCR was performed. Compared with the BD group, the tumor growth rate was significantly lower (P < 0. 05) in the FS and SDG group. ZnT-1 mRNA level in mammary tumor was increased in SDG group and decreased in FS group, but no significant difference was found. Extremely low amplification of ZIP2 from mRNA was detected, with no difference between the treatment groups. LIV-1 mRNA expression of SDG group increases compared with BD group. In FS group, it significantly increases nearly 9 times than that in BD group (P < 0. 005).     

Over-expression of Zip-13 mRNA in kidney and lung during dietary zinc deficiency in Wistar rats

Zinc is an essential nutrient for all organisms, which is involved in the function of numerous key enzymes in metabolism. Two gene families have been identified involved in zinc homeostasis. ZnT transporters reduce intracellular zinc while Zip transporters increase intracellular zinc. Previous studies in our laboratory have shown that Zip-1, ZnT-1, Zip-2 and LIV-1 mRNA are associated with zinc level in established human breast cancer in nude mice model. In this study, six zinc transporters: ZnT-1, ZnT-2, ZnT-4, Zip-1, Zip-8 and Zip-13 were chosen. We aim to determine the relation between zinc transporters and zinc level in kidney and lung of Wistar rats. Eighteen Wistar rats were randomly divided into three groups: normal group, zinc-deficiency group and pair-fed group. After 22 days, the rats were killed and organs samples were taken, then zinc transporters mRNA were detected by RT-PCR. Compared with the normal group, Zip-13 shows an up-regulation (P < 0.05) in zinc-deficiency group both in kidney and lung, and Zip-8 was significantly lower (P < 0.05) in zinc-deficiency group in kidney.     

Influence of DNA-methylation on zinc homeostasis in myeloid cells: Regulation of zinc transporters and zinc binding proteins

The distribution of intracellular zinc, predominantly regulated through zinc transporters and zinc binding proteins, is required to support an efficient immune response. Epigenetic mechanisms such as DNA methylation are involved in the expression of these genes. In demethylation experiments using 5-Aza-2′-deoxycytidine (AZA) increased intracellular (after 24 and 48 h) and total cellular zinc levels (after 48 h) were observed in the myeloid cell line HL-60. To uncover the mechanisms that cause the disturbed zinc homeostasis after DNA demethylation, the expression of human zinc transporters and zinc binding proteins were investigated. Real time PCR analyses of 14 ZIP (solute-linked carrier (SLC) SLC39A; Zrt/IRT-like protein), and 9 ZnT (SLC30A) zinc transporters revealed significantly enhanced mRNA expression of the zinc importer ZIP1 after AZA treatment. Because ZIP1 protein was also enhanced after AZA treatment, ZIP1 up-regulation might be the mediator of enhanced intracellular zinc levels. The mRNA expression of ZIP14 was decreased, whereas zinc exporter ZnT3 mRNA was also significantly increased; which might be a cellular reaction to compensate elevated zinc levels. An enhanced but not significant chromatin accessibility of ZIP1 promoter region I was detected by chromatin accessibility by real-time PCR (CHART) assays after demethylation. Additionally, DNA demethylation resulted in increased mRNA accumulation of zinc binding proteins metallothionein (MT) and S100A8/S100A9 after 48 h. MT mRNA was significantly enhanced after 24 h of AZA treatment also suggesting a reaction of the cell to restore zinc homeostasis. These data indicate that DNA methylation is an important epigenetic mechanism affecting zinc binding proteins and transporters, and, therefore, regulating zinc homeostasis in myeloid cells.     

Increased level of exogenous zinc induces cytotoxicity and up-regulates the expression of the ZnT-1 zinc transporter gene in pancreatic cancer cells

A balance between zinc uptake by ZIP (SLC39) and efflux of zinc from the cytoplasm into subcellular organelles and out of the cell by ZnT (SLC30) transporters is crucial for zinc homeostasis. It is not clear whether normal and cancerous pancreatic cells respond differently to increased extracellular zinc concentrations. Use of flow cytometry-based methods revealed that treatment with as little as 0.01 mM zinc induced significant cytotoxicity in two human ductal adenocarcinoma cell lines. In contrast, normal human pancreatic islet cells tolerated as high as 0.5 mM zinc. Insulinoma cell lines of mouse and rat origin also succumbed to high concentrations of zinc. Exposure to elevated zinc concentrations enhanced the numbers of carcinoma but not primary islet cells staining with the cell-permeable zinc-specific fluorescent dye, FluoZin-3, indicating increased zinc influx in transformed cells. Mitochondrial membrane depolarization, superoxide generation, decreased antioxidant thiols, intracellular acidosis and activation of intracellular caspases characterized zinc-induced carcinoma cell death. Only the antioxidant glutathione but not inhibitors of enzymes implicated in apoptosis or necrosis prevented zinc-induced cytotoxicity in insulinoma cells. Immunoblotting revealed that zinc treatment increased the ubiquitination of proteins in cancer cells. Importantly, zinc treatment up-regulated the expression of ZnT-1 gene in a rat insulinoma cell line and in two human ductal adenocarcinoma cell lines. These results indicate that the exposure of pancreatic cancer cells to elevated extracellular zinc concentrations can lead to cytotoxic cell death characterized by increased protein ubiquitination and up-regulation of the zinc transporter ZnT-1 gene expression.     

Zinc transporter mRNA expression in the RWPE-1 human prostate epithelial cell line

The human prostate gland undergoes a prominent alteration in Zn+2 homeostasis during the development of prostate cancer. The goal of the present study was to determine if the immortalized human prostate cell line (RWPE-1) could serve as a model system to study the role of zinc in prostate cancer. The study examined the expression of mRNA for 19 members of the zinc transporter gene family in normal prostate tissue, the prostate RWPE-1 cell line, and the LNCaP, DU-145 and PC-3 prostate cancer cell lines. The study demonstrated that the expression of the 19 zinc transporters was similar between the RWPE-1 cell line and the in situ prostate gland. Of the 19 zinc transporters, only 5 had levels that were different between the RWPE-1 cells and the tissue samples; all five being increased (ZnT-6, Zip-1, Zip-3A, Zip-10, and Zip-14). The response of the 19 transporters was also determined when the cell lines were exposed to 75 microM Zn+2 for 24 h. It was shown for the RWPE-1 cells that only 5 transporters responded to Zn+2 with mRNA for ZnT-1 and ZnT-2 being increased while mRNA for ZnT-7, Zip-7 and Zip-10 transporters were decreased. It was shown for the LNCaP, DU-145 and PC-3 cells that Zn+2 had no effect on the mRNA levels of all 19 transporters except for an induction of ZnT-1 in PC-3 cells. Overall, the study suggests that the RWPE-1 cells could be a valuable model for the study of the zinc transporter gene family in the prostate.     

Differential expression of zinc transporters in functionally contrasting tissues involved in zinc homeostasis

Abstract

Zinc homeostasis is maintained by 24 tissue-specific zinc transporters which include ZnTs (ZnT1-10), ZIPs (ZIP1-14), in addition to metallothionein (MT). Current study aimed the role of zinc transporters in maintaining the basal levels of zinc in functionally contrasting tissue specific THP-1 (monocyte), RD (muscle), and Saos-2 (bone) cells. Zinc transporters expression was assessed by qRT-PCR. The mRNA levels of ZnTs (ZnT5-7 & ZnT9), ZIPs (ZIP6-10, ZIP13-14), and MT were significantly (p?<?0.05) higher in Saos-2 compared to THP-1 and RD. The present study suggests that distinct expression pattern of zinc transporters and metallothionein might be responsible for the differential zinc assimilation.     

The ZIP7 gene (Slc39a7) encodes a zinc transporter involved in zinc homeostasis of the Golgi apparatus

It has been suggested that ZIP7 (Ke4, Slc39a7) belongs to the ZIP family of zinc transporters. Transient expression of the V5-tagged human ZIP7 fusion protein in CHO cells led to elevation of the cytoplasmic zinc level. However, the precise function of ZIP7 in cellular zinc homeostasis is not clear. Here we report that the ZIP7 gene is ubiquitously expressed in human and mouse tissues. The endogenous ZIP7 was associated with the Golgi apparatus and was capable of transporting zinc from the Golgi apparatus into the cytoplasm of the cell. Moreover, by using the yeast mutant strain Deltazrt3 that was defective in release of stored zinc from vacuoles, we found that ZIP7 was able to decrease the level of accumulated zinc and in the meantime to increase the nuclear/cytoplasmic labile zinc level in the ZIP7-expressing zrt3 mutant. We showed that the protein expression of ZIP7 was repressed under zinc-rich condition, whereas there were no effects of zinc on ZIP7 gene expression and intracellular localization. Neither did zinc deficiency affect the intracellular distribution of ZIP7 in mammalian cells. Our study demonstrates that ZIP7 is a functional zinc transporter that acts by transporting zinc from the Golgi apparatus to the cytoplasm of the cell.     

The LZT proteins; the LIV-1 subfamily of zinc transporters

Zinc is an essential ion for cells with a vital role to play in controlling the cellular processes of the cell, such as growth, development and differentiation. Specialist proteins called zinc transporters control the level of intracellular zinc in cells. In mammals, the ZIP family of zinc transporters has a pivotal role in maintaining the correct level of intracellular zinc by their ability to transport zinc into cells from outside, although they may also transport metal ions other than zinc. There are now recognised to be four subfamilies of the ZIP transporters, including the recently discovered LIV-1 subfamily which has similarity to the oestrogen-regulated gene LIV-1, previously implicated in metastatic breast cancer. We call this new subfamily LZT, for LIV-1 subfamily of ZIP zinc Transporters. Here we document current knowledge of this previously uncharacterised group of proteins, which includes the KE4 proteins. LZT proteins are similar to ZIP transporters in secondary structure and ability to transport metal ions across the plasma membrane or intracellular membranes. However, LZT proteins have a unique motif (HEXPHEXGD) with conserved proline and glutamic acid residues, unprecedented in other zinc transporters. The localisation of LZT proteins to lamellipodiae mirrors cellular location of the membrane-type matrix metalloproteases. These differences to other zinc transporters may be consistent with an alternative role for LZT proteins in cells, particularly in diseases such as cancer.     

Cloning and characterization of a novel mammalian zinc transporter, zinc transporter 5, abundantly expressed in pancreatic beta cells

Intracellular homeostasis for zinc is achieved through the coordinate regulation of specific transporters engaged in zinc influx, efflux, and intracellular compartmentalization. We have identified a novel mammalian zinc transporter, zinc transporter 5 (ZnT-5), by virtue of its similarity to ZRC1, a zinc transporter of Saccharomyces cerevisiae, a member of the cation diffusion facilitator family. Human ZnT-5 (hZnT-5) cDNA encodes a 765-amino acid protein with 15 predicted membrane-spanning domains. hZnT-5 was ubiquitously expressed in all tested human tissues and abundantly expressed in the pancreas. In the human pancreas, hZnT-5 was expressed abundantly in insulin-containing beta cells that contain zinc at the highest level in the body. The hZnT-5 immunoreactivity was found to be associated with secretory granules by electron microscopy. The hZnT-5-derived zinc transport activity was detected using the Golgi-enriched vesicles prepared from hZnT-5-induced HeLa/hZnT-5 cells in which exogenous hZnT-5 expression is inducible by the Tet-on gene regulation system. This activity was dependent on time, temperature, and concentration and was saturable. Moreover, zinc at a high concentration (10 mm) inhibited the growth of yeast expressing hZnT-5. These results suggest that ZnT-5 plays an important role for transporting zinc into secretory granules in pancreatic beta cells.     

Imbalance in zinc homeostasis enhances lung Tissue Loss following cigarette smoke exposure

Cigarette smoke exposure is a major cause of chronic obstructive pulmonary disease. Cadmium is a leading toxic component of cigarette smoke. Cadmium and zinc are highly related metals. Whereas, zinc is an essential metal required for normal health, cadmium is highly toxic. Zrt- and Irt-like protein 8 (ZIP8) is an avid transporter of both zinc and cadmium into cells and is abundantly expressed in the lung of smokers compared to nonsmokers. Our objective was to determine whether disturbed zinc homeostasis through diet or the zinc transporter ZIP8 increase susceptibility to lung damage following prolonged cigarette smoke exposure.MethodsCigarette smoke exposure was evaluated in the lungs of mice subject to insufficient and sufficient zinc intakes, in transgenic ZIP8 overexpressing mice, and a novel myeloid-specific ZIP8 knockout strain.ResultsModerate depletion of zinc intakes in adult mice resulted in a significant increase in lung cadmium burden and permanent lung tissue loss following prolonged smoke exposure. Overexpression of ZIP8 resulted in increased lung cadmium burden and more extensive lung damage, whereas cigarette smoke exposure in ZIP8 knockout mice resulted in increased lung tissue loss without a change in lung cadmium content, but a decrease in zinc.ConclusionsOverall, findings were consistent with past human studies. Imbalance in Zn homeostasis increases susceptibility to permanent lung injury following prolonged cigarette smoke exposure. Based on animal studies, both increased and decreased ZIP8 expression enhanced irreversible tissue damage in response to prolonged tobacco smoke exposure. We believe these findings represent an important advancement in our understanding of how imbalance in zinc homeostasis and cadmium exposure via tobacco smoke may increase susceptibility to smoking-induced lung disease.     

Structure, function, and regulation of a subfamily of mouse zinc transporter genes

Zinc is an essential metal for all eukaryotes, and cells have evolved a complex system of proteins to maintain the precise balance of zinc uptake, intracellular storage, and efflux. In mammals, zinc uptake appears to be mediated by members of the Zrt/Irt-like protein (ZIP) superfamily of metal ion transporters. Herein, we have studied a subfamily of zip genes (zip1, zip2, and zip3) that is conserved in mice and humans. These eight-transmembrane domain proteins contain a conserved 12-amino acid signature sequence within the fourth transmembrane domain. All three of these mouse ZIP proteins function to specifically increase the uptake of zinc in transfected cultured cells, similar to the previously demonstrated functions of human ZIP1 and ZIP2 (Gaither, L. A., and Eide, D. J. (2000) J. Biol. Chem. 275, 5560-5564; Gaither, L. A., and Eide, D. J. (2001) J. Biol. Chem. 276, 22258-22264). No ZIP3 orthologs have been previously studied. Furthermore, this first systematic comparative study of the in vivo expression and dietary zinc regulation of this subfamily of zip genes revealed that 1) zip1 mRNA is abundant in many mouse tissues, whereas zip2 and zip3 mRNAs are very rare or moderately rare, respectively, and tissue-restricted in their accumulation; and 2) unlike mouse metallothionein I and zip4 mRNAs (Dufner-Beattie, J., Wang, F., Kuo, Y.-M., Gitschier, J., Eide, D., and Andrews, G. K. (2003) J. Biol. Chem. 278, 33474-33481), the abundance of zip1, zip2, and zip3 mRNAs is not regulated by dietary zinc in the intestine and visceral endoderm, tissues involved in nutrient absorption. These studies suggest that all three of these ZIP proteins may play cell-specific roles in zinc homeostasis rather than primary roles in the acquisition of dietary zinc.     

Zinc accumulation in N-methyl-N-nitrosourea-induced rat mammary tumors is accompanied by an altered expression of ZnT-1 and metallothionein

Zinc is essential for cell proliferation. Several human studies have shown that in breast cancer tissues, zinc concentration expressed on a per tissue weight basis is higher than that in normal breast tissues. However, the mechanisms involved are unknown. N-methyl-N-nitrosourea (MNU)-induced rat mammary tumorigenesis is one of the most widely used rodent mammary tumorigenesis models for studying human breast cancer due to their similarities in hormone dependency, pathogenesis, histological classification, and immunocytochemical markers. This study was to establish if there was an accumulation of zinc in MNU-induced rat mammary tumors and, if there was, to explore the possible mechanisms involved. Sprague-Dawley rats were sham-treated or MNU-treated (50 mg/kg; n = 12) for 100 days. In MNU-induced mammary tumors (mammary tumors), zinc concentration expressed on a per dry weight basis was 12 times of that in normal mammary glands. Moreover, the mRNA level of ZnT-1 (a transporter involved in zinc efflux) in mammary tumors was reduced by 55% as compared with that in normal mammary glands. The mRNA level of Nramp2 (a divalent cation importer) and ZnT-4 (another transporter involved in zinc efflux) was unaffected by MNU-induced mammary tumorigenesis. The mRNA and protein levels of metallothionein (a putative zinc storage protein) in mammary tumors were 1.3 and 3.5 times of that in normal mammary glands, respectively. Collectively, our observations showed that zinc is accumulated in MNU-induced rat mammary tumors and this accumulation is accompanied by an altered expression of ZnT-1 and metallothionein, suggesting that zinc homeostasis might be altered in MNU-induced rat mammary tumorigenesis. Because zinc is essential to cell proliferation and cell proliferation is increased in mammary tumors, zinc accumulation is likely a part of an integrated effort to ensure sufficient zinc supply to sustain tumor growth.     

Zinc nutritional status influences ZnT1 and ZIP4 gene expression in children with a high risk of zinc deficiency

IntroductionSubclinical deficiency of zinc is associated with impairment of immune system function, growth, and cognitive development in children. Although plasma zinc is the best available biomarker of the risk of zinc deficiency in populations, its sensitivity for early detection of deficiency is limited. Therefore, we aimed to investigate zinc deficiency among preschool children and its relationship with whole blood gene expression of zinc transporters ZIP4 and ZnT1.Material and methodsThis cross-sectional study included 139 children aged 32–76 months enrolled in philanthropic day-care centers. We performed an anthropometric evaluation, weighed food record and dietary record for dietary assessment, blood sample collection for zinc, and whole blood gene expression analyses of ZnT1 (SLC30A1) and ZIP4 (SLC39A4).ResultsZinc deficiency was observed in 26.6 % of the children despite adequate zinc intake and a phytate:zinc molar ratio < 18. Usual zinc intake did not affect whole blood gene expression of zinc transporters, but zinc status influenced ZnT1 and ZIP4 whole blood mRNA. Children with zinc deficiency exhibited 37.1 % higher ZnT1 expression and 45.3 % lower ZIP4 expression than children with adequate zinc (p < 0.05).ConclusionChildren with plasma zinc deficiency exhibited higher expression of ZnT1 and lower expression of ZIP4 in whole blood mRNA, reinforcing the existence of strong regulation of mineral homeostasis according to the nutritional status, indicating that this analysis may be useful in the evaluation of dietary interventions.     

Increased expression of zinc transporter ZIP4, ZIP11, ZnT1, and ZnT6 predicts poor prognosis in pancreatic cancer

IntroductionZinc homeostasis is regulated by SLC39A/ZIP, SLC30A/ZnT, and metallothionein (MT) families in human cells. Zinc dyshomeostasis may affect or be affected by the abnormal behavior of cancer cells. Although decreased serum zinc levels are observed in patients with pancreatic adenocarcinoma (PAAD), limited information is available regarding the expression pattern and prognostic roles of zinc homeostasis-related genes in PAAD.ObjectivesThe primary objective of this study was to explore the expression pattern and prognostic roles of zinc homeostasis-related genes in PAAD.MethodsThe expression pattern of 35 known zinc homeostasis-related genes in PAAD was systemically explored based on RNA-sequencing data from the Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) projects. The association between the expression levels of zinc homeostasis-related genes and survival of PAAD patients was evaluated using the Kaplan-Meier method and the log-rank test. Expressional correlation between zinc homeostasis-related genes with potential prognostic value in PAAD and normal pancreatic controls was evaluated using Pearson’s correlation analysis. Functional enrichment analyses were performed to elucidate possible mechanisms for the potential prognostic and therapeutic roles of these zinc homeostasis-related genes in PAAD. Effects of ZIP11, ZnT1, or ZnT6 knockdown on the proliferation and the migration of Capan-1 pancreatic cancer cells were assessed by the CCK-8 assay and the wound healing assay respectively.ResultsWe demonstrated that the expression levels of ZIP1, ZIP3, ZIP4, ZIP6, ZIP7, ZIP9, ZIP10, ZIP11, ZIP13, ZnT1, ZnT5, ZnT6, ZnT7, and ZnT9 were increased, whereas the expression levels of ZIP5, ZIP14, ZnT2, MT1 G, MT1H, and MT1X were decreased in PAAD tumors compared with normal pancreatic controls. Among these differentially-expressed genes related to zinc homeostasis, higher expression of ZIP4, ZIP11, ZnT1 or ZnT6 predicted poorer prognosis with the possible involvement of several cancer-related processes and pathways in PAAD patients. We further demonstrated that knockdown of ZIP11 attenuated Capan-1 cell proliferation with decreased activation of ERK1/2 pathway; knockdown of ZnT1 attenuated Capan-1 cell proliferation with decreased activation of ERK1/2, p38 MAPK, NF-kB, and mTOR pathways; knockdown of ZnT6 attenuated Capan-1 cell proliferation with decreased activation of ERK1/2, p38 MAPK, and NF-kB pathways.ConclusionsHigher expression of the zinc transporter ZIP4, ZIP11, ZnT1 or ZnT6 predicted poorer prognosis in patients with PAAD. These findings provide new clues for understanding the complex relationship between zinc homeostasis and pancreatic cancer.     

Biochemical characterization of human ZIP13 protein: a homo-dimerized zinc transporter involved in the spondylocheiro dysplastic Ehlers-Danlos syndrome

The human SLC39A13 gene encodes ZIP13, a member of the LZT (LIV-1 subfamily of ZIP zinc transporters) family. The ZIP13 protein is important for connective tissue development, and its loss of function is causative for the spondylocheiro dysplastic form of Ehlers-Danlos syndrome. However, this protein has not been characterized in detail. Here we report the first detailed biochemical characterization of the human ZIP13 protein using its ectopic expressed and the purified recombinant protein. Protease accessibility, microscopic, and computational analyses demonstrated that ZIP13 contains eight putative transmembrane domains and a unique hydrophilic region and that it resides with both its N and C termini facing the luminal side on the Golgi. Analyses including cross-linking, immunoprecipitation, Blue Native-PAGE, and size-exclusion chromatography experiments indicated that the ZIP13 protein may form a homo-dimer. We also demonstrated that ZIP13 mediates zinc influx, as assessed by monitoring the expression of the metallothionein gene and by detecting the intracellular zinc level with a zinc indicator, FluoZin-3. Our data indicate that ZIP13 is a homo-dimerized zinc transporter that possesses some domains that are not found in other LZT family members. This is the first biochemical characterization of the physiologically important protein ZIP13 and the demonstration of homo-dimerization for a mammalian ZIP zinc transporter family member. This biochemical characterization of the human ZIP13 protein provides important information for further investigations of its structural characteristics and function.     

Longitudinal changes in zinc transport kinetics, metallothionein and zinc transporter expression in a blood-brain barrier model in response to a moderately excessive zinc environment

A blood-brain barrier (BBB) model composed of porcine brain capillary endothelial cells (BCEC) was exposed to a moderately excessive zinc environment (50 micromol/L Zn) in cell culture, and longitudinal measurements were made of zinc transport kinetics, ZnT-1 (SLC30A1) expression and changes in the protein concentration of metallothionein (MT), ZnT-1, ZnT-2 (SLC30A2) and Zip1 (SLC39A1). Zinc release by cells of the BBB model significantly increased after 12-24 h of exposure, but decreased back to control levels after 48-96 h, as indicated by transport across the BBB from both the ablumenal (brain) and the lumenal (blood) directions. Expression of ZnT-1, the zinc export protein, increased by 169% within 12 h, but was no longer different from controls after 24 h. Likewise, ZnT-1 protein content increased transiently after 12 h of exposure, but returned to control levels by 24 h. Capacity for zinc uptake and retention increased from both the lumenal and the ablumenal directions within 12-24 h of exposure and remained elevated. MT and ZnT-2 were elevated within 12 h and remained elevated throughout the study. Zip1 was unchanged by the treatment. The BBB's response to a moderately high zinc environment was dynamic and involved multiple mechanisms. The initial response was to increase the cells' capacity to sequester zinc with additional MT and to increase zinc export with the ZnT-1 protein. But the longer-term strategy involved increasing ZnT-2 transporters, presumably to sequester zinc into intracellular vesicles as a mechanism to protect the brain and to maintain brain zinc homeostasis.     

Zinc and Zinc Transporters in Macrophages and Their Roles in Efferocytosis in COPD

Our previous studies have shown that nutritional zinc restriction exacerbates airway inflammation accompanied by an increase in caspase-3 activation and an accumulation of apoptotic epithelial cells in the bronchioles of the mice. Normally, apoptotic cells are rapidly cleared by macrophage efferocytosis, limiting any secondary necrosis and inflammation. We therefore hypothesized that zinc deficiency is not only pro-apoptotic but also impairs macrophage efferocytosis. Impaired efferocytic clearance of apoptotic epithelial cells by alveolar macrophages occurs in chronic obstructive pulmonary disease (COPD), cigarette-smoking and other lung inflammatory diseases. We now show that zinc is a factor in impaired macrophage efferocytosis in COPD. Concentrations of zinc were significantly reduced in the supernatant of bronchoalveolar lavage fluid of patients with COPD who were current smokers, compared to healthy controls, smokers or COPD patients not actively smoking. Lavage zinc was positively correlated with AM efferocytosis and there was decreased efferocytosis in macrophages depleted of Zn in vitro by treatment with the membrane-permeable zinc chelator TPEN. Organ and cell Zn homeostasis are mediated by two families of membrane ZIP and ZnT proteins. Macrophages of mice null for ZIP1 had significantly lower intracellular zinc and efferocytosis capability, suggesting ZIP1 may play an important role. We investigated further using the human THP-1 derived macrophage cell line, with and without zinc chelation by TPEN to mimic zinc deficiency. There was no change in ZIP1 mRNA levels by TPEN but a significant 3-fold increase in expression of another influx transporter ZIP2, consistent with a role for ZIP2 in maintaining macrophage Zn levels. Both ZIP1 and ZIP2 proteins were localized to the plasma membrane and cytoplasm in normal human lung alveolar macrophages. We propose that zinc homeostasis in macrophages involves the coordinated action of ZIP1 and ZIP2 transporters responding differently to zinc deficiency signals and that these play important roles in macrophage efferocytosis.     

Correlation between ZIP2 messenger RNA expression and zinc level in rat lateral prostate

Zinc content in rat lateral prostate (LP) is higher compared with the other tissues, but the zinc retention system in the prostate remains unclear. In the present study, we examined the expression of ZRT, and IRT-like protein (ZIP) family transporter in rat prostate. The zinc level in rat LP was higher compared with the ventral (VP) and dorsal prostate (DP). The predicted ZIP2 mRNA was really expressed in LP at a high level. The expression was decreased in LP from castrated rats, associated with a decrease in zinc level, and these changes were prevented by testosterone replacement. Moreover, ZIP2 expression levels in LP positively correlated with the zinc levels. These findings strongly suggest that ZIP2 is involved in zinc homeostasis of rat prostate.     

A role for ZnT-1 in regulating cellular cation influx

The ZnTs are a growing family of proteins involved in lowering or sequestration of cellular zinc. Using fluorescent measurements of zinc transport we have addressed the mechanism of action of the most ubiquitously expressed member of this family, ZnT-1. This protein has been shown to lower levels of intracellular zinc though the mechanism has remained elusive. The rate of zinc efflux in HEK293 cells expressing ZnT-1 was not accelerated in comparison to control cells, suggesting that ZnT-1 may be involved in regulating influx rather than efflux of zinc. Co-expression of the L-type calcium channel, a major route for zinc influx, and ZnT-1 resulted in a 3-fold reduction in the rate of zinc influx in HEK293 and PC-12 cells, indicating that ZnT-1 modulates zinc permeation through this channel. Immunoblot analysis indicates that ZnT-1 expression does not modulate LTCC expression. Our findings therefore indicate that ZnT-1 modulates the permeation of cations through LTCC, thereby, regulating cation homeostasis through this pathway. Furthermore, ZnT-1 may play a role in cellular ion homeostasis and thereby confer protection against pathophysiological events linked to cellular Ca(2+) or Zn(2+) permeation and cell death.