Robert J. Cousins

Mammalian Zinc Transport, Trafficking, and Signals * □

Structural, catalytic, and regulatory functions of zinc in biology continue to be defined. The number of genes coding for proteins with zinc-binding domains is conservatively estimated at 3% of the human genome but possibly is asmuch as 10% (1, 2). Zinc utilization in abundant, yet diverse, applications illustrateswhy organisms have evolved specific pathways to homeostatically regulate availability of this essential micronutrient at specific cellular sites through an array of transporters. Animals regulate zinc gain and loss efficiently. In humans, about 1% of the total body zinc content is replenished daily by the diet (3). This is accomplished principally by tight control of two systems, absorption from the intestine and endogenous loss via pancreatic and other intestinal secretions. Loss-of-function studies have not provided a delineation of how zinc participates in physiologic activities. Nevertheless, immune function and resistance to infection and control of nitrosative and/or oxidative stress of inflammation are two areas of particular contemporary interest. Unfolding knowledge of cell type-specific expression of zinc transporter genes that respond differentially to hormonal and cytokine stimulation should aid in understanding the role of zinc in these physiologic systems. Coordination of intracellular zinc trafficking has focused on the cysteine-rich protein metallothionein (MT).2 Zinc-dependent expression of Mt and its well documented response to multiple mediators, including oxidants, cytokines, hormones, and nitric oxide, have been extensively investigated. Here we review recent findings of how zinc transporters andMT influencemammalian cellular zincmetabolism and signaling pathways.

Mammalian Zinc Transporters: Nutritional and Physiologic Regulation

Research advances defining how zinc is transported into and out of cells and organelles have increased exponentially within the past five years. Research has progressed through application of molecular techniques including genomic analysis, cell transfection, RNA interference, kinetic analysis of ion transport, and application of cell and animal models including knockout mice. The knowledge base has increased for most of 10 members of the ZnT family and 14 members of the Zrt-, Irt-like protein (ZIP) family. Relative to the handling of dietary zinc is the involvement of ZnT1, ZIP4, and ZIP5 in intestinal zinc transport, involvement of ZIP10 and ZnT1 in renal zinc reabsorption, and the roles of ZIP5, ZnT2, and ZnT1 in pancreatic release of endogenous zinc. These events are major factors in regulation of zinc homeostasis. Other salient findings are the involvement of ZnT2 in lactation, ZIP14 in the hypozincemia of inflammation, ZIP6, ZIP7, and ZIP10 in metastatic breast cancer, and ZnT8 in insulin processing and as an autoantigen in diabetes.

Zip14 (Slc39a14) mediates non-transferrin-bound iron uptake into cells.

Zip14 is a member of the SLC39A zinc transporter family, which is involved in zinc uptake by cells. Up-regulation of Zip14 by IL-6 appears to contribute to the hepatic zinc accumulation and hypozincemia of inflammation. At least three members of the SLC39A family transport other trace elements, such as iron and manganese, in addition to zinc. We analyzed the capability of Zip14 to mediate non-transferrin-bound iron (NTBI) uptake by overexpressing mouse Zip14 in HEK 293H cells and Sf9 insect cells. Zip14 was found to localize to the plasma membrane, and its overexpression increased the uptake of both 65Zn and 59Fe. Addition of bathophenanthroline sulfonate, a cell-impermeant ferrous iron chelator, inhibited Zip14-mediated iron uptake from ferric citrate, suggesting that iron is taken up by HEK cells as Fe2+. Iron uptake by HEK and Sf9 cells expressing Zip14 was inhibited by zinc. Suppression of endogenous Zip14 expression by using Zip14 siRNA reduced the uptake of both iron and zinc by AML12 mouse hepatocytes. Zip14 siRNA treatment also decreased metallothionein mRNA levels, suggesting that compensatory mechanisms were not sufficient to restore intracellular zinc. Collectively, these results indicate that Zip14 can mediate the uptake of zinc and NTBI into cells and that it may play a role in zinc and iron metabolism in hepatocytes, where this transporter is abundantly expressed. Because NTBI is commonly found in plasma of patients with hemochromatosis and transfusional iron overload, Zip14-mediated NTBI uptake may contribute to the hepatic iron loading that characterizes these diseases.

Tissue-specific regulation of zinc metabolism and metallothionein genes by interleukin 1.

Interleukin 1 (IL 1) production is stimulated by infection, cellular injury, and inflammation. This cytokine directs a wide spectrum of host responses. Human interleukin 1α (IL 1α) was used to examine the time course of effects on zinc metabolism as part of the acute phase response. IL 1 produced a transient depression in the serum zinc concentration and increased serum ceruloplasmin. Metallothionein levels were increased in liver 14‐fold after IL 1. Increased expression of metallothionein‐1 and ‐2 genes following IL 1 were observed in liver, bone marrow, and thymus. Pulse‐labeling experiments with i.v.‐administered 65Zn showed that IL 1 drastically altered zinc distribution kinetics among tissues. More 65Zn was taken up (and/or retained) by the liver, bone marrow, and thymus 6 h after IL 1, whereas correspondingly less 65Zn was found in bone, skin, and intestine. Uptake by other tissues was not affected by IL 1. Chromatography of cytosol from tissues with increased 65Zn uptake suggests the IL 1‐induced redistribution may be driven by enhanced metallothionein synthesis. Collectively, the results show that IL 1 regulates zinc metabolism and may direct its preferential, tissue‐specific distribution via elevated metallothionein‐1 and ‐2 gene expression.—Cousins, R. J.; Leinart, A. S. Tissue‐specific regulation of zinc metabolism and metallothionein genes by interleukin 1. FASEB J. 2: 2884‐2890; 1988.

Integrative Aspects of Zinc Transporters

Cells maintain zinc concentrations with relatively narrow limits. Nevertheless, physiologically relevant changes in free Zn(II) pools or changes in Zn bound to specific ligands or within vesicles may occur without a major change in total cellular zinc concentrations. The task of maintaining such levels rests in part with zinc transporter proteins. The genes for some putative zinc transporters have recently been cloned. As of this time, most have not been directly shown to transport zinc in functional studies, albeit evidence is strong that they have such a function. Zinc transporter (ZnT)-1 was identified as a rescue agent for cells maintained in very high extracellular zinc conditions; therefore, ZnT-1 has been suggested to function as an exporter. ZnT-1 is expressed in a variety of tissues, including intestine, kidney and liver. Intestinal expression is regional, being much greater in duodenum and jejunum and in villus versus crypt cells. Immunolocalization places ZnT-1 at the basolateral membrane of intestinal enterocytes and epithelial cells of the distal renal tubules. Regulation of ZnT-1 mRNA and ZnT-1 protein does not change markedly with changes in dietary zinc level except when a large single oral zinc supplement is provided. ZnT-1 is induced by transient ischemia of the forebrain. ZnT-2 and ZnT-3 may function in tissue-specific vesicular zinc transport. ZnT-4 is believed to be abundant in mammary gland and may be associated with zinc secretion into milk. A mutation of the ZnT-4 gene may account for the lethal milk (lm) syndrome. The putative zinc transporters identified thus far appear to have characteristics commensurate with functions in integrative zinc acquisition and homeostasis.

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.

A global view of the selectivity of zinc deprivation and excess on genes expressed in human THP-1 mononuclear cells

Among the micronutrients required by humans, zinc has particularly divergent modes of action. cDNA microarray and quantitative PCR technologies were used to investigate the zinc responsiveness of known genes that influence zinc homeostasis and to identify, through global screening, genes that may relate to phenotypic outcomes of altered dietary zinc intake. Human monocytic/macrophage THP-1 cells were either acutely zinc depleted, using a cell-permeable zinc-specific chelator, or were supplemented with zinc to alter intracellular zinc concentrations. Initially, genes associated with zinc homeostasis were evaluated by quantitative PCR to establish ranges for fold changes in transcript abundance that might be expected with global screening. Zinc transporter-1 and zinc transporter-7 expression increased when cellular zinc increased, whereas Zip-2 expression, the most zinc-responsive gene examined, was markedly increased by zinc depletion. Microarrays composed of ≈22,000 elements were used to identify those genes responsive to either zinc depletion, zinc supplementation, or both conditions. Hierarchal clustering and ANOVA revealed that ≈5% or 1,045 genes were zinc responsive. Further sorting based on this pattern of the zinc responsiveness of these genes into seven groups revealed that 104 genes were linearly zinc responsive in a positive mode (i.e., increased expression as cellular zinc increases) and 86 genes that were linearly zinc responsive in a negative mode (i.e., decreased expression as cellular zinc increases). Expression of some genes was responsive to only zinc depletion or supplementation. Categorization by function revealed numerous genes needed for host defense were among those identified as zinc responsive, including cytokine receptors and genes associated with amplification of the Th1 immune response.

Zinc transporter ZIP8 (SLC39A8) and zinc influence IFN-γ expression in activated human T cells

The zinc transporter ZIP8 is highly expressed in T cells derived from human subjects. T cell ZIP8 expression was markedly up‐regulated upon in vitro activation. T cells collected from human subjects who had received oral zinc supplementation (15 mg/day) had higher expression of the activation marker IFN‐γ upon in vitro activation, indicating a potentiating effect of zinc on T cell activation. Similarly, in vitro zinc treatment of T cells along with activation resulted in increased IFN‐γ expression with a maximum effect at 3.1 μM. Knockdown of ZIP8 in T cells by siRNA decreased ZIP8 levels in nonactivated and activated cells and concomitantly reduced secretion of IFN‐γ and perforin, both signatures of activation. Overexpression of ZIP8 by transient transfection caused T cells to exhibit enhanced activation. Confocal microscopy established that ZIP8 is localized to the lysosome where ZIP8 abundance is increased upon activation. Loss of lysosomal labile zinc in response to activation was measured by flow cytometry using a zinc fluorophore. Zinc between 0.8 and 3.1 μM reduced CN phosphatase activity. CN was also inhibited by the CN inhibitor FK506 and ZIP8 overexpression. The results suggest that zinc at low concentrations, through inhibition of CN, sustains phosphorylation of the transcription factor CREB, yielding greater IFN‐γ expression in T cells. ZIP8, through control of zinc transport from the lysosome, may provide a secondary level of IFN‐γ regulation in T cells.

A role of zinc in the regulation of gene expression

Zn, without question, has important functions related to gene expression. Newer technologies applied to address these functions are providing answers relating to the importance of this micronutrient in human and animal health.

Zip14 is a complex broad-scope metal-ion transporter whose functional properties support roles in the cellular uptake of zinc and nontransferrin-bound iron

Recent studies have shown that overexpression of the transmembrane protein Zrt- and Irt-like protein 14 (Zip14) stimulates the cellular uptake of zinc and nontransferrin-bound iron (NTBI). Here, we directly tested the hypothesis that Zip14 transports free zinc, iron, and other metal ions by using the Xenopus laevis oocyte heterologous expression system, and use of this approach also allowed us to characterize the functional properties of Zip14. Expression of mouse Zip14 in RNA-injected oocytes stimulated the uptake of (55)Fe in the presence of l-ascorbate but not nitrilotriacetic acid, indicating that Zip14 is an iron transporter specific for ferrous ion (Fe(2+)) over ferric ion (Fe(3+)). Zip14-mediated (55)Fe(2+) uptake was saturable (K(0.5) ≈ 2 μM), temperature-dependent (apparent activation energy, E(a) = 15 kcal/mol), pH-sensitive, Ca(2+)-dependent, and inhibited by Co(2+), Mn(2+), and Zn(2+). HCO(3)(-) stimulated (55)Fe(2+) transport. These properties are in close agreement with those of NTBI uptake in the perfused rat liver and in isolated hepatocytes reported in the literature. Zip14 also mediated the uptake of (109)Cd(2+), (54)Mn(2+), and (65)Zn(2+) but not (64)Cu (I or II). (65)Zn(2+) uptake also was saturable (K(0.5) ≈ 2 μM) but, notably, the metal-ion inhibition profile and Ca(2+) dependence of Zn(2+) transport differed from those of Fe(2+) transport, and we propose a model to account for these observations. Our data reveal that Zip14 is a complex, broad-scope metal-ion transporter. Whereas zinc appears to be a preferred substrate under normal conditions, we found that Zip14 is capable of mediating cellular uptake of NTBI characteristic of iron-overload conditions.