The acrodermatitis enteropathica gene ZIP4 encodes a tissue-specific,zinc-regulated zinc transporter in mice

The human ZIP4 gene (SLC39A4) is a candidate for the genetic disorder of zinc metabolism acrodermatitis enteropathica. To understand its role in zinc homeostasis, we examined the function and expression of mouse ZIP4. This gene encodes a well conserved eight-transmembrane protein that can specifically increase the influx of zinc into transfected cells. Expression of this gene is robust in tissues involved in nutrient uptake, such as the intestines and embryonic visceral yolk sac, and is dynamically regulated by zinc. Dietary zinc deficiency causes a marked increase in the accumulation of ZIP4 mRNA in these tissues, whereas injection of zinc or increasing zinc content of the diet rapidly reduces its abundance. Zinc can also regulate the accumulation of ZIP4 protein at the apical surface of enterocytes and visceral endoderm cells. These results provide compelling evidence that ZIP4 is a zinc transporter that plays an important role in zinc homeostasis, a process that is defective in acrodermatitis enteropathica in humans.     

The Zinc Transporter Zip5 (Slc39a5) Regulates Intestinal Zinc Excretion and Protects the Pancreas against Zinc Toxicity

Background

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.

Conclusions

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.     

Expression and regulation of SLC39A family zinc transporters in the developing mouse intestine

Several ZIP genes (SLC39A family of metal transporters) play roles in zinc homeostasis. Herein, the temporal and spatial patterns of expression of the mouse ZIP1, 3, 4, and 5 genes in the developing intestine and the effects of maternal dietary zinc deficiency on these patterns of expression were examined. ZIP1 and ZIP3 genes, conserved members of the ZIP subfamily II, were found to be coexpressed during development. Expression of these genes was detected on day 14 of gestation in smooth muscle and the pseudostratified endoderm. By 5 days post-partum, prominent expression became restricted to muscle and connective stroma. In contrast, expression of ZIP4 and ZIP5 genes, members of the ZIP subfamily called LIV-1, coincided with epithelial morphogenesis. ZIP5 expression was detected on d16 of gestation and localized to the basolateral membranes of the single-layered epithelium. ZIP4 expression was detected on d18 of gestation and localized to the apical membrane of villus epithelial cells. When dams were fed a zinc-deficient diet beginning at parturition, ZIP4 expression in the nursing neonate was greatly induced. In contrast, neonatal ZIP5 expression remained unchanged, but this protein was removed from the basolateral membrane of the enterocyte. These responses to dietary zinc deficiency mimic those found in the adult intestine. These studies reveal cell-type-specific expression of ZIP genes during development of the intestine, and suggest that the mouse intestine can elicit an adaptive response to dietary zinc availability at birth.     

The mammalian Zip5 protein is a zinc transporter that localizes to the basolateral surface of polarized cells

The mouse and human Zip5 proteins are members of the ZIP family of metal ion transporters. In this study, we present evidence that mouse Zip5 is a zinc uptake transporter that is specific for Zn(II) over other potential metal ion substrates. We also show that, unlike many other mammalian ZIP proteins, the endocytic removal of mZip5 from the plasma membrane is not triggered by zinc treatment. Thus, the activity of mZip5 does not appear to be down-regulated by zinc repletion. Zip5 expression is restricted to many tissues important for zinc homeostasis, including the intestine, pancreas, liver, and kidney. Zip5 is similar in sequence to the Zip4 protein, which is involved in the uptake of dietary zinc. Co-expression of Zip4 and Zip5 in the intestine led to the hypothesis that these proteins play overlapping roles in the uptake of dietary zinc across the apical membrane of intestinal enterocytes. Surprisingly, however, we found that mZip5 localizes specifically to the basolateral membrane of polarized Madin-Darby canine kidney cells. These observations suggest that Zip5 plays a novel role in polarized cells by carrying out serosal-to-mucosal zinc transport. Furthermore, given its expression in tissues important to zinc homeostasis, we propose that Zip5 plays a central role in controlling organismal zinc status.     

Mouse ZIP1 and ZIP3 genes together are essential for adaptation to dietary zinc deficiency during pregnancy

Subfamily II of the solute-linked carrier 39A superfamily contains three well-conserved zinc transporters (ZIPs1, 2, 3) whose physiological functions are unknown. We generated mice homozygous for knockout alleles of ZIP1 and both ZIP1 and ZIP 3 (double-knockout). These mice were apparently normal when dietary zinc was replete, but when dietary zinc was limited during pregnancy embryos from ZIP1 or ZIP3 knockout mice were two to three times more likely to develop abnormally than those in wildtype mice, and 91% (71/78) of embryos developed abnormally in ZIP1, ZIP3 double-knockout mice. Analysis of the patterns of expression of these genes in mice revealed predominate expression in intestinal stromal cells, nephric-tubular epithelial cells, pancreatic ductal epithelial cells, and hepatocytes surrounding the central vein. This suggests that these zinc transporters function, at least in part, in the redistribution and/or retention of zinc rather than its acquisition from the diet. In conclusion, mutations in the ZIP1 and ZIP3 zinc transporter genes are silent when dietary intake of zinc is normal, but can dramatically compromise the success of pregnancy when dietary intake of zinc is limiting.     

A mouse model of acrodermatitis enteropathica: loss of intestine zinc transporter ZIP4 (Slc39a4) disrupts the stem cell niche and intestine integrity

Mutations in the human Zip4 gene cause acrodermatitis enteropathica, a rare, pseudo-dominant, lethal genetic disorder. We created a tamoxifen-inducible, enterocyte-specific knockout of this gene in mice which mimics this human disorder. We found that the enterocyte Zip4 gene in mice is essential throughout life, and loss-of-function of this gene rapidly leads to wasting and death unless mice are nursed or provided excess dietary zinc. An initial effect of the knockout was the reprogramming of Paneth cells, which contribute to the intestinal stem cell niche in the crypts. Labile zinc in Paneth cells was lost, followed by diminished Sox9 (sex determining region Y-box 9) and lysozyme expression, and accumulation of mucin, which is normally found in goblet cells. This was accompanied by dysplasia of the intestinal crypts and significantly diminished small intestine cell division, and attenuated mTOR1 activity in villus enterocytes, indicative of increased catabolic metabolism, and diminished protein synthesis. This was followed by disorganization of the absorptive epithelium. Elemental analyses of small intestine, liver, and pancreas from Zip4-intestine knockout mice revealed that total zinc was dramatically and rapidly decreased in these organs whereas iron, manganese, and copper slowly accumulated to high levels in the liver as the disease progressed. These studies strongly suggest that wasting and lethality in acrodermatitis enteropathica patients reflects the loss-of-function of the intestine zinc transporter ZIP4, which leads to abnormal Paneth cell gene expression, disruption of the intestinal stem cell niche, and diminished function of the intestinal mucosa. These changes, in turn, cause a switch from anabolic to catabolic metabolism and altered homeostasis of several essential metals, which, if untreated by excess dietary zinc, leads to dramatic weight loss and death.     

Zn2+-stimulated endocytosis of the mZIP4 zinc transporter regulates its location at the plasma membrane

Zinc is an essential nutrient for all organisms. Its requirement in humans is illustrated dramatically by the genetic disorder acrodermatitis enteropathica (AE). AE is caused by the reduced uptake of dietary zinc by enterocytes, and the ensuing systemic zinc deficiency leads to dermatological lesions and immune and reproductive dysfunction. The gene responsible for AE, SLC39A4, encodes a member of the ZIP family of metal transporters, hZIP4. The mouse ZIP4 protein, mZIP4, stimulates zinc uptake in cultured cells, and studies in mice have demonstrated that zinc treatment decreases mZIP4 mRNA levels in the gut. In this study, we demonstrated using transfected cultured cells that the mZIP4 protein is also regulated at a post-translational level in response to zinc availability. Zinc deficiency increased mZIP4 protein levels at the plasma membrane, and this was associated with increased zinc uptake. Significantly, treating cells with low micromolar zinc concentrations stimulated the rapid endocytosis of the transporter. Zinc-regulated localization of the human ZIP4 protein was also demonstrated in cultured cells. These findings suggest that zinc-regulated trafficking of human and mouse ZIP4 is a key mechanism controlling dietary zinc absorption and cellular zinc homeostasis.     

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.     

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.     

A histidine-rich cluster mediates the ubiquitination and degradation of the human zinc transporter, hZIP4, and protects against zinc cytotoxicity

Zinc is an essential nutrient. Genetic evidence for this nutritional requirement in humans is the zinc deficiency disease, acrodermatitis enteropathica. This disorder is caused by mutations in hZIP4 (SLC39A4), a zinc importer required for zinc uptake in enterocytes and other cell types. Studies in mice have demonstrated that levels of the mZIP4 mRNA are reduced by elevated dietary zinc, resulting in a decreased abundance of the ZIP4 protein at the plasma membrane. Moreover, studies in cultured cells have demonstrated that low micromolar concentrations of zinc stimulate the endocytosis of the mZIP4 protein resulting in a reduction in cellular zinc uptake. In this study, we demonstrate an additional level of hZIP4 regulation involving ubiquitination and degradation of this transporter in elevated zinc concentrations. Mutational analysis identified a cytoplasmic histidine-rich domain that was essential for ubiquitin-dependent degradation of ZIP4 and protection against zinc toxicity. However, this motif was dispensable for zinc-induced endocytosis. These findings indicate that ubiquitin-mediated degradation of the ZIP4 protein is critical for regulating zinc homeostasis in response to the upper tier of physiological zinc concentrations, via a process that is distinct from zinc-stimulated endocytosis.     

Generation and characterization of mice lacking the zinc uptake transporter ZIP3

The mouse ZIP3 (SLC39A3) gene encodes an eight-transmembrane-domain protein that has been conserved in mammals and can function to transport zinc. To analyze the expression of ZIP3 in the early embryo and neonate and to determine its in vivo function, we generated ZIP3 null mice in which the ZIP3 open reading frame was replaced with that of the enhanced green fluorescent protein (EGFP) reporter. EGFP fluorescence revealed that ZIP3 was expressed in the inner cell mass of the blastocyst and later during embryonic development in many tissues. Elevated expression was apparent in the embryonic brain and neurotube and neonatal gonads. Homozygous knockout mice were viable and fertile and under normal growth conditions exhibited no obvious phenotypic abnormalities. Deletion of ZIP3 did not alter zinc homeostasis at the molecular level as assessed by essential metal levels and the expression of zinc-responsive genes. In knockout mice stressed with a zinc-deficient diet during pregnancy or at weaning, a subtle increase in the sensitivity to abnormal morphogenesis of the embryo and to depletion of thymic pre-T cells, respectively, was noted. These results suggest that this protein plays an ancillary role in zinc homeostasis in mice.     

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.     

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.     

ZIP4 confers resistance to zinc deficiency-induced apoptosis in pancreatic cancer

Emerging evidence implicates the zinc importer ZIP4 as a critical factor that enhances pancreatic cancer proliferation; however, the role of ZIP4 in promoting pancreatic cancer progression by regulating apoptosis requires elucidation. To determine the effect of ZIP4 on apoptosis, we used cell lines where ZIP4 levels were upregulated or silenced in combination with Chelex 100 treatment to deplete intracellular zinc. Pancreatic cancer xenografts derived from those cells were also included. TUNEL and flow cytometry analysis were used to measure apoptosis and western blotting was used to analyze protein expression for PARP and multiple caspases. Cell cycle profiles were examined by flow cytometry. Zinc depletion by Chelex induced more apoptosis of pancreatic cancer cells in comparison to normal medium, where almost no apoptosis was observed. ZIP4 stably overexpressed MIA PaCa-2 (MIA-ZIP4) cells were more resistant to zinc depletion-induced apoptosis compared with vector control. Conversely, AsPC-1 (AsPC-shZIP4) cells with stable knockdown of ZIP4 were more sensitive to zinc deficiency than control. Resistance to apoptosis mediated by ZIP4 was accomplished by the caspase pathway. In vivo data also confirmed that ZIP4 overexpressed xenografts showed less apoptosis than controls. Cell cycle profiles indicate that silencing of ZIP4 leads to decreased cell population in S phase and G₀/G₁ arrest. These results described a previously uncharacterized role of ZIP4 in apoptosis resistance and elucidated a novel pathway through which ZIP4 regulates pancreatic cancer growth. This research provides additional evidence for ZIP4 and related signaling cascade as a molecular target for therapeutic intervention in pancreatic cancer.     

Zinc-stimulated endocytosis controls activity of the mouse ZIP1 and ZIP3 zinc uptake transporters

The mouse mZip1 and mZip3 zinc transporters have been implicated in zinc acquisition by the cells of many tissues. This hypothesis raised the question of whether activity of these proteins is regulated to maintain zinc homeostasis. Neither mZIP1 nor mZIP3 mRNA levels are highly regulated by zinc status. Therefore, we investigated whether zinc controls the activity of these proteins post-translationally by altering their subcellular distribution. When expressed in transfected cells grown in zinc-replete medium, both mZip1 and mZip3 were largely present in intracellular organelles. However, these proteins were found to rapidly transit between the plasma membrane and intracellular compartments in zinc-replete cells. Zinc deficiency increased plasma membrane levels of mZip1 and mZip3 by decreasing their rates of endocytosis. Greater zinc deficiency was required to alter mZip3 distribution than was needed to affect mZip1. Increased surface levels correlated with increased zinc uptake activity. Taken together, these results suggest that post-translational control of mZip1 and mZip3 localization plays a role in zinc homeostasis. Moreover, our results indicate that zinc-responsive endocytosis is a conserved mechanism controlling activity of many mammalian zinc uptake transporters.     

Effect of dietary iron deficiency and overload on the expression of ZIP metal-ion transporters in rat liver

The mammalian ZIP (Zrt-, Irt-like Protein) family of transmembrane transport proteins consists of 14 members that share considerable homology. ZIP proteins have been shown to mediate the cellular uptake of the essential trace elements zinc, iron, and manganese. The aim of the present study was to determine the effect of dietary iron deficiency and overload on the expression of all 14 ZIP transporters in the liver, the main site of iron storage. Weanling male rats (n = 6/group) were fed iron-deficient (FeD), iron-adequate (FeA), or iron-overloaded (FeO) diets in two independent feeding studies. In study 1, diets were based on the TestDiet 5755 formulation and contained iron at 9 ppm (FeD), 215 ppm (FeA), and 27,974 ppm (3% FeO). In study 2, diets were based on the AIN-93G formulation and contained iron at 9 ppm Fe (FeD), 50 ppm Fe (FeA), or 18916 ppm (2% FeO). After 3 weeks, the FeD diets depleted liver non-heme iron stores and induced anemia, whereas FeO diets resulted in hepatic iron overload. Quantitative RT-PCR revealed that ZIP5 mRNA levels were 3- and 8-fold higher in 2% FeO and 3% FeO livers, respectively, compared with FeA controls. In both studies, a consistent downregulation of ZIP6, ZIP7, and ZIP10 was also observed in FeO liver relative to FeA controls. Studies in H4IIE hepatoma cells further documented that iron loading affects the expression of these ZIP transporters. Overall, our data suggest that ZIP5, ZIP6, ZIP7, and ZIP10 are regulated by iron, indicating that they may play a role in hepatic iron/metal homeostasis during iron deficiency and overload.