ZIP8 mRNA Research Articles

Overexpression of Zip‐2 mRNA in the leukocytes of asthmatic infants

The increase in prevalence of asthma is strongly dependent on environmental factors, including foods. Significant decreases in the intake of dietary zinc may be an important contributing factor to the increasing incidence of asthma. As zinc can not passively diffuse across cell membranes, specific zinc transporters are required for zinc influx and efflux. There are two identified gene families involved in zinc homeostasis, ZnT transporters and ZIP transporters. In this study, we aimed to investigate the expression of these zinc transporters mRNA in the leukocytes of asthmatic infants. Asthmatic infants (n = 9) and healthy infants (n = 9) were included in this study. Five zinc transporters were chosen: ZnT-1, ZnT-3, ZnT-5, ZIP-1, and ZIP-2. Levels of zinc were determined in serum and expression of zinc transporters mRNA were measured. Serum zinc levels were significantly lower in asthmatic infants (P < 0.01); Expression of ZnT-1, ZnT-3, ZnT-5, ZIP-1 mRNA were not significantly different between two groups, but expression of ZIP-2 mRNA was significantly higher (P < 0.01) in the asthmatic group than the control group. In conclusion, overexpression of ZIP-2 mRNA was found in the leukocytes of asthmatic infants.

Localization of zip1 and zip4 mRNA in the adult rat brain

The localization of two members of the Slc39a (zip1 and zip4) family of zinc transporters was examined in the brains of adult mice. Zip1 was highly enriched in brain regions with high densities of neuronal cell bodies, including the hippocampus, thalamus, and perifontal cortex. Zip1 was also expressed in commissural fiber tracts such as the corpus callosum and anterior commissure, but little was found in the internal and external capsules. Also, very low amounts of zip1 mRNA were detected in resting astrocytes and reactive astrocytes that were examined at 14 days after inflicting a stab wound. Zip1 mRNA was detected in ependymal cells lining the third and lateral ventricles and epithelium cells in the choroid plexus. Interestingly, zip4 mRNA was detected in the choroid plexus but not in the ependymal cells or other neural elements. Zip4 mRNA was also detected in brain capillaries, but zip1 mRNA was not. In zip4 knockout heterozygotes that express green fluorescent protein regulated by the zip4 promoter, green fluorescent protein was detected in brain capillaries. Because zip4 levels are regulated by dietary Zn, our studies suggest that the brain has the potential of adapting to changes in Zn status.

Tissue-Specific Alterations in Zinc Transporter Expression in Intestine and Liver Reflect a Threshold for Homeostatic Compensation during Dietary Zinc Deficiency in Weanling Rats

Intestinal zinc (Zn) absorption and liver Zn mobilization are presumed to regulate Zn homeostasis. Several Zn transporters have been identified; however, their contribution to Zn homeostasis is poorly understood. Moreover, their regulation during periods of growth is unknown. To characterize the mechanisms that maintain Zn status, weanling rats were fed control (25 mg/kg), marginally low (MLZ; 15 mg/kg), low (LZ; 7 mg/kg), or very low (VLZ; <1 mg/kg) Zn diets for 3 wk and effects on jejunum Zip4 and ZnT1 and hepatic Zip1 and ZnT1 were assessed. Another control group was pair-fed (PF) to VLZ. The MLZ rats had lower jejunum ZnT1 protein abundance than the control. In the LZ group, we detected increased jejunum Zip4 mRNA expression and hepatic ZnT1 protein abundance and reduced jejunum Zip4 and ZnT1 and hepatic Zip1 protein abundance. VLZ had lower jejunum ZnT1 mRNA and protein abundance and hepatic Zip1 and ZnT1 protein abundance compared with the PF group. Zip4 protein was present at the intestinal villus tip in controls but was detected on the apical membrane throughout the entire villus in LZ rats. ZnT5 protein in jejunum was always detected at the apical membrane and also at the basolateral membrane of VLZ rats. In contrast, ZnT7 was found intracellularly in jejunum. Our data suggest that effects of Zn deficiency on Zn homeostasis occurs biphasically during marginal Zn deficiency through increased intestinal Zn uptake capacity and reduced intestinal Zn efflux, then during more pronounced degrees of Zn deficiency through decreased liver Zn accretion and increased hepatic Zn efflux back into circulation. These results assist in our understanding of how mammals regulate Zn homeostasis.

Interleukin-1β contributes via nitric oxide to the upregulation and functional activity of the zinc transporter Zip14 (Slc39a14) in murine hepatocytes

Zinc metabolism during chronic disease is dysregulated by inflammatory cytokines. Experiments with IL-6 knockout mice show that LPS regulates expression of the zinc transporter, Zip14, by a mechanism that is partially independent of IL-6. The LPS-induced model of sepsis may occur by a mechanism signaled by nitric oxide (NO) as a secondary messenger. To address the hypothesis that NO can modulate Zip14 expression, we treated primary hepatocytes from wild-type mice with the NO donor S-nitroso N-acetyl penicillamine (SNAP). After treatment with SNAP, steady-state Zip14 mRNA levels displayed a maximal increase after 8 h and a concomitant increase in the transcriptional activity of the gene. Chromatin immunoprecipitation documented the kinetics of activator protein (AP)-1 and RNA polymerase II association with the Zip14 promoter after NO exposure, indicating a role of AP-1 in transcription of Zip14. We then stimulated the primary murine hepatocytes with IL-1beta, an LPS-induced proinflammatory cytokine and a potent activator of inducible NO synthase (iNOS) and NO production. In support of our hypothesis, IL-1beta treatment led to a threefold increase in Zip14 mRNA and enhanced zinc transport, as measured with a zinc fluorophore, in wild-type but not iNOS-/- hepatocytes. These data suggest that signaling pathways activated by NO are factors in the upregulation of Zip14, which in turn mediates hepatic zinc accumulation and hypozincemia during inflammation and sepsis.

Zinc Transporters ZnT1 (Slc30a1), Zip8 (Slc39a8), and Zip10 (Slc39a10) in Mouse Red Blood Cells Are Differentially Regulated during Erythroid Development and by Dietary Zinc Deficiency

Zinc is essential for normal erythroid cell functions and therefore intracellular zinc homeostasis during erythroid differentiation is tightly regulated. However, a characterization of zinc transporters in erythrocytes has not been conducted. The membrane fraction of mature mouse RBC was screened for zinc transporter expression using western analysis as a first step in the characterization process. ZnT1, Zip8, and Zip10 were detected among the 12 transporter proteins tested. We examined expression of these zinc transporters during erythropoietin (EPO)-induced differentiation of splenic erythroid progenitor cells into reticulocytes. Both Zip8 and Zip10 mRNA increased by 2–6 h after addition of EPO to the cells. In contrast, maximal RNA levels for the zinc transporter ZnT1 and erythroid δ-aminolevulinic acid synthase were only produced by 24 h after EPO. We confirmed these changes in transcript abundance by western analysis. Dietary zinc status influences zinc-dependent functions of RBC. To determine whether the identified zinc transporters respond to dietary zinc status, mice were fed a zinc-deficient or control diet. Incorporation of 65Zn into erythrocytes in vitro was significantly increased in cells from the zinc-deficient mice. Western analysis and densitometry revealed that erythrocyte Zip10 was upregulated and ZnT1 was downregulated in the zinc-depleted mice. Zip8 was not affected by restricted zinc intake. Collectively, these data suggest that the zinc transporters ZnT1, Zip8, and Zip10 are important for zinc homeostasis in erythrocytes and that ZnT1 and Zip10 respond to the dietary zinc supply.

Restraint stress up-regulates expression of zinc transporter Zip14 mRNA in mouse liver

The zinc concentration in the liver was significantly higher in mice at 12 h after the onset of restraint stress than that without stress. The IL-6 protein level in the serum was transiently elevated at 3 h after the onset of restraint stress, and the IL-6-responsive zinc transporter Zip14 mRNA in the liver was expressed markedly at 6 h. These results suggest that Zip14 plays a major role in the mechanism responsible for accumulation of zinc in the liver under restraint stress.

Modulation of transferrin‐bound iron uptake by Zip14

All cells acquire iron through internalization of iron‐loaded transferrin (Tf) via the Tf receptor. The aim of the present study was to examine the effect of Zip14, a transmembrane metal ion transporter, on the uptake of iron from Tf. HEK293T cells were transfected with mouse Zip14 cDNA or empty vector and incubated for 2, 6, 8, 10, 16 h with 59Fe‐Tf, and the radioactivity incorporated into the cells was measured. At all time points, Zip14‐transfected cells took up more 59Fe from Tf than did control cells. At 8 h, Zip14‐overexpressing HEK cells took up 37 ± 6% more 59Fe than did controls. The effect of Zip14 on Tf‐bound iron uptake was also studied in HepG2 cells, which endogenously express abundant Zip14. Treatment of HepG2 cells with Zip14 siRNA decreased Zip14 mRNA and protein levels and markedly reduced Tf‐mediated iron uptake. These data strongly implicate a role for Zip14 in the uptake of iron from Tf, suggesting that Zip14 plays a role in normal iron metabolism. Funded by NIH grants DK065064 (MDK) and DK054488 (CAE).

Aberrant expression of zinc transporter ZIP4 (SLC39A4) significantly contributes to human pancreatic cancer pathogenesis and progression

Zinc is an essential trace element and catalytic/structural component used by many metalloenzymes and transcription factors. Recent studies indicate a possible correlation of zinc levels with the cancer risk; however, the exact role of zinc and zinc transporters in cancer progression is unknown. We have observed that a zinc transporter, ZIP4 (SLC39A4), was substantially overexpressed in 16 of 17 (94%) clinical pancreatic adenocarcinoma specimens compared with the surrounding normal tissues, and ZIP4 mRNA expression was significantly higher in human pancreatic cancer cells than human pancreatic ductal epithelium (HPDE) cells. This indicates that aberrant ZIP4 up-regulation may contribute to the pancreatic cancer pathogenesis and progression. We studied the effects of ZIP4 overexpression in pancreatic cancer cell proliferation in vitro and pancreatic cancer progression in vivo . We found that forced expression of ZIP4 increased intracellular zinc levels, increased cell proliferation by 2-fold in vitro , and significantly increased tumor volume by 13-fold in the nude mice model with s.c. xenograft compared with the control cells. In the orthotopic nude mice model, overexpression of ZIP4 not only increased the primary tumor weight (7.2-fold), it also increased the peritoneal dissemination and ascites incidence. Moreover, increased cell proliferation and higher zinc content were also observed in the tumor tissues that overexpressed ZIP4. These data reveal an important outcome of aberrant ZIP4 expression in contributing to pancreatic cancer pathogenesis and progression. It may suggest a therapeutic strategy whereby ZIP4 is targeted to control pancreatic cancer growth.

Novel zinc-responsive post-transcriptional mechanisms reciprocally regulate expression of the mouse Slc39a4 and Slc39a5 zinc transporters (Zip4 and Zip5)

Dietary zinc deficiency in mice is accompanied by enhanced expression of the zinc uptake transporter Slc39a4 (Zip4) and repressed expression of Slc39a5 (Zip5) in tissues which regulate zinc homeostasis (intestine, pancreas and visceral yolk sac). Herein, mechanisms controlling this differential expression were investigated. The induction of Zip4 mRNA during zinc deficiency, and its repression in response to zinc repletion were found to reflect changes in Zip4 mRNA stability and not changes in the relative rate of transcription of this gene. During zinc deficiency, ZIP4 protein levels are increased and this protein is localized on the apical membranes. Administration of an oral gavage of zinc caused ZIP4 internalization and degradation in enterocytes and visceral endoderm cells. Similarly, ZIP4 is induced by zinc deficiency in cultured mouse Hepa cells and is rapidly degraded in response to added zinc. Zip5 mRNA abundance does not change in response to zinc, but the translation of this mRNA was found to be zinc-responsive. During zinc deficiency, Zip5 mRNA remains associated with polysomes, while the protein is internalized and degraded in enterocytes, acinar cells and endoderm cells. After zinc-gavage, ZIP5 is rapidly resynthesized and targeted to the basolateral membranes of these cell types.

Expression of zinc transporters during the differentiation of erythroblasts from phenylhydrazine (PHZ)‐treated mice

Zinc is required for the functions of several proteins related to erythroid differentiation. Excessive intracellular free Zn2+ interferes with incorporation of Fe2+ during heme biosynthesis. Therefore, intracellular zinc homeostasis during erythroid differentiation must be tightly regulated. To examine the suggested hypothesis, we prepared erythroblasts from spleens of PHZ-treated, anemic mice, of which further differentiation was induced by in vitro erythropoietin (EPO)-treatment. Based on QRT-PCR results, Zip8 and ZnT1 expression showed the highest responsiveness to EPO among the ten zinc transporters tested. Zip8 mRNA expression was highest prior to the time-point when erythroid δ-aminolevulinic acid synthase mRNA reached its maximal level (i.e., 24 h after EPO-treatment), while the ZnT1 mRNA expression level peaked afterwards. Furthermore, western blots of ZnT1 with differentiating PHZ-splenocytes and erythrocyte ghost cells revealed a similar trend in protein expression. These results suggest that Zip8 and ZnT1 could be the zinc transporters most directly involved in the regulation of zinc homeostasis during terminal erythroid differentiation. Specifically, Zip8 may facilitate the zinc uptake required for normal erythroid differentiation, while ZnT1 removes excessive free Zn2+ that may compete with Fe2+ during heme biosynthesis. Supported by NIH Grant DK 31127 and Boston Family Endowment funds.

Zinc and its transporter ZIP10 are involved in invasive behavior of breast cancer cells

Zinc is an essential element, necessary for sustaining all life. Zinc deficiency causes taste impairments, immune deficiency, skin problems, and growth and mental retardation. Recent reports suggest that zinc is associated with an increased risk of cancer, although it is still unclear whether zinc or its transporters are involved in cancer progression. Here we show that zinc and its transporter ZIP10 are involved in the invasive behavior of breast cancer cells. The screening of clinical samples for ZIP10 mRNA expression suggested that ZIP10 was significantly associated with the metastasis of breast cancer to the lymph node. In addition, the expression of ZIP10 mRNA was higher in the invasive and metastatic breast cancer cell lines MDA-MB-231 and MDA-MB-435S than in less metastatic breast cancer cell lines, such as MCF7, T47D, ZR75-1 and ZR75-30. In in vitro cell migration assays, the depletion of zinc transporter ZIP10 and intracellular zinc inhibited the migratory activity of MDA-MB-231 and MDA-MB-435S cells. These results showed that zinc and ZIP10 play an essential role in the migratory activity of highly metastatic breast cancer cells, and suggest ZIP10 as a possible marker for the metastatic phenotype of breast cancer and a promising target of novel treatment strategies.

Zip14 expression in hepatic iron overload

The liver can acquire iron from plasma transferrin bound to the transferrin receptor (TfR) or from non-transferrin-bound iron (NTBI). During iron overload, hepatic TfR levels decrease whereas plasma NTBI levels increase, resulting in the accretion of NTBI in the liver. The molecular mechanisms responsible for hepatic NTBI uptake are poorly defined, but may involve Zip14, which has been shown to transport NTBI in AML12 cells, a mouse hepatocyte cell line (Liuzzi et al., PNAS, 2006). To explore this possibility further, we examined hepatic Zip14 mRNA levels in hypotransferrinemic (Trfhpx/hpx) mice that lack circulating transferrin but accumulate massive amounts of iron in the liver. Quantitative RT-PCR revealed no differences in levels of Zip14 transcripts (both long and short isoforms) between Trfhpx/hpx mice and controls (n=10/group). Similarly, treatment of AML12 hepatocytes with 0, 50, 250 or 500 μM of ferric nitrilotriacetate for 24 h had no effect on Zip14 mRNA levels, although TfR mRNA levels decreased by 75%. Collectively, these results suggest that Zip14 continues to be expressed during iron overload, and may thus contribute to NTBI uptake into the liver. Funded by NIH grant DK065064.

Enhanced cadmium-induced testicular necrosis and renal proximal tubule damage caused by gene-dose increase in a Slc39a8-transgenic mouse line

Resistance to cadmium (Cd)-induced testicular necrosis is an autosomal recessive trait defined as the Cdm locus. Using positional cloning, we previously identified the Slc39a8 (encoding an apical-surface ZIP8 transporter protein) as the gene most likely responsible for the phenotype. In situ hybridization revealed that endothelial cells of the testis vasculature express high ZIP8 levels in two sensitive inbred mouse strains and negligible amounts in two resistant strains. In the present study, we isolated a 168.7-kb bacterial artificial chromosome (BAC), carrying only the Slc39a8 gene, from a Cd-sensitive 129/SvJ BAC library and generated BAC-transgenic mice. The BTZIP8-3 line, having three copies of the 129/SvJ Slc39a8 gene inserted into the Cd-resistant C57BL/6J genome (having its normal two copies of the Slc39a8 gene), showed tissue-specific ZIP8 mRNA expression similar to wild-type mice, mainly in lung, testis, and kidney. The approximately 2.5-fold greater expression paralleled the fact that the BTZIP8-3 line has five copies, whereas wild-type mice have two copies, of the Slc39a8 gene. The ZIP8 mRNA and protein localized especially to endothelial cells of the testis vasculature in BTZIP8-3 mice. Cd treatment reversed Cd resistance (seen in nontransgenic littermates) to Cd sensitivity in BTZIP8-3 mice; reversal of the testicular necrosis phenotype confirms that Slc39a8 is unequivocally the Cdm locus. ZIP8 also localized specifically to the apical surface of proximal tubule cells in the BTZIP8-3 kidney. Cd treatment caused acute renal failure and signs of proximal tubular damage in the BTZIP8-3 but not nontransgenic littermates. BTZIP8-3 mice should be a useful model for studying Cd-induced disease in kidney.

Regulation of murine Zip14 during hepatic inflammation

The anemia of chronic disease is driven by inflammatory cytokines. These cytokines also regulate genes that produce hypozincemia and hepatic zinc accumulation. In the sterile abscess model of inflammation, up-regulation of the Zip14 zinc transporter by IL-6 is the mechanism responsible for the hypozincemia. However, experiments with IL-6 knockout mice show that LPS regulates Zip14 expression by a mechanism that is partially independent of IL-6. The LPS-induced model of sepsis may occur by a mechanism signaled by nitric oxide as a secondary messenger. IL-1β, an LPS-induced proinflammatory cytokine, is a potent activator of inducible nitric oxide synthase (iNOS), and nitric oxide production. Therefore, we hypothesized that nitric oxide can regulate Zip14 expression during LPS-induced inflammation. To address this hypothesis, we treated primary hepatocytes from wild-type mice with the nitric oxide donor s-nitroso n-acetyl penicillamine (SNAP). After treatment with 50 μM SNAP for 8 hr, Zip14 mRNA increased two-fold. We then took a more physiological approach by treating primary murine hepatocytes with IL-1β. As expected, IL-1β treatment increased iNOS mRNA as well as nitric oxide, as measured by nitrite concentration. In support of our hypothesis, IL-1β treatment also led to a six-fold increase in Zip14 mRNA and enhanced zinc transport as measured by the Zn2+-specific fluorophore, FluoZin-3 AM. Experiments utilizing siRNA to knockdown Zip14 expression and those using hepatocytes from iNOS knockout mice also support our hypothesis. These data suggest that signaling pathways activated by nitric oxide are factors in the up-regulation of Zip14 that in turn mediates hepatic zinc accumulation and reduction of serum zinc concentrations during inflammation and sepsis. Supported by NIH Grant DK 31127.

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, a 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.

Headliners: Cadmium-Induced Disease: Discovery of Gene Responsible for Cadmium Transport in Mice

Dalton TP, He L, Wang B, Miller ML, Jin L, Stringer KF, Chang X, Baxter CS, Nebert DW. 2005. Identification of mouse SLC39A8 as the transporter responsible for cadmium-induced toxicity in the testis. Proc Natl Acad Sci USA 102:3401–3406. Cadmium is a toxic and carcinogenic nonessential metal released from mining, smelting, battery manufacturing, and coal burning. Among other routes, humans are exposed to cadmium by smoking cigarettes and eating contaminated seafood or plants grown in contaminated soil. Environmental levels of cadmium have risen along with advances in industrialization, and the role of cadmium in human disease is therefore of increasing concern. Now NIEHS grantee Daniel W. Nebert and colleagues have discovered a gene responsible for the transport of cadmium in mice, which hints at a target that could be employed to prevent the toxic effects of cadmium in humans. Cadmium is readily absorbed through the lungs, intestines, and skin, and accumulates in the kidneys. The toxic mechanisms of cadmium are not well understood, but it is known to act intracellularly, causing damage particularly to the lungs, kidneys, bone, central nervous system, and reproductive tissues. Cadmium is also known to cause abnormalities in the developing embryo. The testis is a sensitive marker of cadmium exposure; cadmium-induced testicular necrosis is common across all studied animal species having testes. Exposure to this metal is a significant problem in many developing countries, where undernourishment and iron deficiency are associated with increased cadmium toxicity. Nebert and colleagues administered low doses of cadmium to mice and demonstrated that the SLC39A8 (ZIP8) protein, coded by the Slc39a8 gene, transported cadmium to the testis, causing vascular endothelial cell injury and subsequent testicular tissue death. Using in vitro models, the research team found that vascular endothelial cells from cadmium-insensitive mice did not accumulate ZIP8 mRNA, whereas cells from cadmium-sensitive mice did. The team suggests, therefore, that loss of vascular endothelial ZIP8 mRNA expression in the insensitive strains protected the mice’s testes against cadmium toxicity. Nebert and colleagues suspect that ZIP8 is normally responsible for the transport of other metals such as manganese and zinc, and that cadmium “participates as an opportunistic hitchhiker.” Because humans carry the analogous SLC39A8 gene, these findings have identified a potential target for exploration in preventing the toxic effects of cadmium in exposed people.

Interleukin-6 regulates the zinc transporter Zip14 in liver and contributes to the hypozincemia of the acute-phase response.

Infection and inflammation produce systemic responses that include hypozincemia and hypoferremia. The latter involves regulation of the iron transporter ferroportin 1 by hepcidin. The mechanism of reduced plasma zinc is not known. Transcripts of the two zinc transporter gene families (ZnT and Zip) were screened for regulation in mouse liver after turpentine-induced inflammation and LPS administration. Zip14 mRNA was the transporter transcript most up-regulated by inflammation and LPS. IL-6 knockout (IL-6(-/-)) mice did not exhibit either hypozincemia or the induction of Zip14 with turpentine inflammation. However, in IL-6(-/-) mice, LPS produced a milder hypozincemic response but no Zip14 induction. Northern analysis showed Zip14 up-regulation was specific for the liver, with one major transcript. Immunohistochemistry, using an antibody to an extracellular Zip14 epitope, showed both LPS and turpentine increased abundance of Zip14 at the plasma membrane of hepatocytes. IL-6 produced increased expression of Zip14 in primary hepatocytes cultures and localization of the protein to the plasma membrane. Transfection of mZip14 cDNA into human embryonic kidney cells increased zinc uptake as measured by both a fluorescent probe for free Zn(2+) and (65)Zn accumulation, as well as by metallothionein mRNA induction, all indicating that Zip14 functions as a zinc importer. Zip14 was localized in plasma membrane of the transfected cells. These in vivo and in vitro experiments demonstrate that Zip14 expression is up-regulated through IL-6, and that this zinc transporter most likely plays a major role in the mechanism responsible for hypozincemia that accompanies the acute-phase response to inflammation and infection.

Identification of mouse SLC39A8 as the transporter responsible for cadmium-induced toxicity in the testis

Testicular necrosis is a sensitive endpoint for cadmium (Cd(2+), Cd) toxicity across all species tested. Resistance to Cd-induced testicular damage is a recessive trait assigned to the Cdm locus on mouse chromosome 3. We first narrowed the Cdm-gene-containing region to 880 kb. SNP analysis of this region from two sensitive and two resistant inbred strains demonstrated a 400-kb haplotype block consistent with the Cd-induced toxicity phenotype; in this region is the Slc39a8 gene encoding a member of the solute-carrier superfamily. Slc39a8 encodes SLC39A8 (ZIP8), whose homologs in plant and yeast are putative zinc transporters. We show here that ZRT-, IRT-like protein (ZIP)8 expression in cultured mouse fetal fibroblasts leads to a >10-fold increase in the rate of intracellular Cd influx and accumulation and 30-fold increase in sensitivity to Cd-induced cell death. The complete ZIP8 mRNA and intron-exon splice junctions have no nucleotide differences between two sensitive and two resistant strains of mice; by using situ hybridization, we found that ZIP8 mRNA is prominent in the vascular endothelial cells of the testis of the sensitive strains of mice but absent in these cells of resistant strains. Slc39a8 is therefore the Cdm gene, defining sensitivity to Cd toxicity specifically in vascular endothelial cells of the testis.

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.

Effects of intracellular zinc depletion on metallothionein and ZIP2 transporter expression and apoptosis

Zinc is critical for the functional and structural integrity of cells. We have used the monocytic cell line THP-1 as a model in which to study both the responsiveness of metallothionein and ZIP2 transporter expression to zinc depletion induced by the intracellular zinc chelator TPEN [N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine] and the extent of concomitant apoptosis. Metallothionein expression increased proportionately with the addition of zinc to the medium and decreased with TPEN treatment. When treated with TPEN, both THP-1 cells and human peripheral blood mononuclear cells exhibited marked decreases in cellular zinc concentrations and increases in ZIP2 mRNA expression. These results suggest that cells attempt to homeostatically adjust to zinc depletion. When THP-1 cells were treated with >5 microM TPEN, cell viability decreased, and cells entered the early stages of apoptosis. These data show that metallothionein and ZIP2 expression are inversely related during zinc depletion and that apoptosis is concurrent with these changes.