Louis A. Lichten

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.

STAT5-glucocorticoid receptor interaction and MTF-1 regulate the expression of ZnT2 (Slc30a2) in pancreatic acinar cells

The exocrine pancreas plays an important role in endogenous zinc loss by regulating excretion into the intestinal tract and hence influences the dietary zinc requirement. The present experiments show that the zinc transporter ZnT2 (Slc30a2) is localized to the zymogen granules and that dietary zinc restriction in mice decreased the zinc concentration of zymogen granules and ZnT2 expression. Excess zinc given orally increased ZnT2 expression and was associated with increased pancreatic zinc accumulation. Rat AR42J acinar cells when induced into a secretory phenotype, using the glucocorticoid analog dexamethasone (DEX), exhibited increased ZnT2 expression and labile zinc as measured with a fluorophore. DEX administrated to mice also induced ZnT2 expression that accompanied a reduction of the pancreatic zinc content. ZnT2 promoter analyses identified elements required for responsiveness to zinc and DEX. Zinc regulation was traced to a MRE located downstream from the ZnT2 transcription start site. Responsiveness to DEX is produced by two upstream STAT5 binding sites that require the glucocorticoid receptor for activation. ZnT2 knockdown in the AR42J cells using siRNA resulted in increased cytoplasmic zinc and decreased zymogen granule zinc that further demonstrated that ZnT2 may mediate the sequestration of zinc into zymogen granules. We conclude, based upon experiments with intact mice and pancreatic acinar cells in culture, that ZnT2 participates in zinc transport into pancreatic zymogen granules through a glucocorticoid pathway requiring glucocorticoid receptor and STAT5, and zinc-regulated signaling pathways requiring MTF-1. The ZnT2 transporter appears to function in a physiologically responsive manner involving entero-pancreatic zinc trafficking.

MTF-1-mediated repression of the zinc transporter Zip10 is alleviated by zinc restriction.

The regulation of cellular zinc uptake is a key process in the overall mechanism governing mammalian zinc homeostasis and how zinc participates in cellular functions. We analyzed the zinc transporters of the Zip family in both the brain and liver of zinc-deficient animals and found a large, significant increase in Zip10 expression. Additionally, Zip10 expression decreased in response to zinc repletion. Moreover, isolated mouse hepatocytes, AML12 hepatocytes, and Neuro 2A cells also respond differentially to zinc availability in vitro. Measurement of Zip10 hnRNA and actinomycin D inhibition studies indicate that Zip10 was transcriptionally regulated by zinc deficiency. Through luciferase promoter constructs and ChIP analysis, binding of MTF-1 to a metal response element located 17 bp downstream of the transcription start site was shown to be necessary for zinc-induced repression of Zip10. Furthermore, zinc-activated MTF-1 causes down-regulation of Zip10 transcription by physically blocking Pol II movement through the gene. Lastly, ZIP10 is localized to the plasma membrane of hepatocytes and neuro 2A cells. Collectively, these results reveal a novel repressive role for MTF-1 in the regulation of the Zip10 zinc transporter expression by pausing Pol II transcription. ZIP10 may have roles in control of zinc homeostasis in specific sites particularly those of the brain and liver. Within that context ZIP10 may act as an important survival mechanism during periods of zinc inadequacy.

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 delta-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 (65)Zn 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.

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.

Plenary Lecture 2 Transcription factors, regulatory elements and nutrient―gene communication

Dramatic advances have been made in the understanding of the differing molecular mechanisms used by nutrients to regulate genes that are essential for their biological roles to carry out normal metabolism. Classical studies have focused on nutrients as ligands to activate specific transcription factors. New interest has focused on histone acetylation as a process for either global or limited gene activation and is the first mechanism to be discussed. Nuclear ATP-citrate lyase generates acetyl-CoA, which has been shown to have a role in the activation of specific genes via selective histone acetylation. Transcription factor acetylation may provide a second mode of control of nutrient-responsive gene transcription. The third mechanism relates to the availability of response elements within chromatin, which as well as the location of the elements in the gene may allow or prevent transcription. A fourth mechanism involves intracellular transport of Zn ions, which can orchestrate localized enzyme inhibition-activation. This process in turn influences signalling molecules that regulate gene expression. The examples provided in the present review point to a new level of complexity in understanding nutrient-gene communication.