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Current Drug Targets, 2006, 7, 607-627

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Zinc and its Specific Transporters as Potential Targets in Airway Disease C. Murgia3, C. J. Lang1, A.Q. Truong-Tran2, D. Grosser1, L. Jayaram1, R.E. Ruffin1, G. Perozzi3 and P.D. Zalewski 1,* 1

Department of Medicine, University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia 5011; Northwestern University, Division of Allergy-Immunology, Feinberg School of Medicine, 240 E. Huron St, Chicago IL 60611, USA; 3Istituto Nazionale di Ricerca per gli Alimenti e la Nutrizione, 00178, Rome, Italy 2

Abstract: The dietary group IIb metal zinc (Zn) plays essential housekeeping roles in cellular metabolism and gene expression. It regulates a number of cellular processes including mitosis, apoptosis, secretion and signal transduction as well as critical events in physiological processes as diverse as insulin release, T cell cytokine production, wound healing, vision and neurotransmission. Critical to these processes are the mechanisms that regulate Zn homeostasis in cells and tissues. The proteins that control Zn uptake and compartmentalization are rapidly being identified and characterized. Recently, the first images of sub-cellular pools of Zn in airway epithelium have been obtained. This review discusses what we currently know about Zn in the airways, both in the normal and inflamed states, and then considers how we might target Zn metabolism by developing strategies to monitor and manipulate airway Zn levels in airway disease.

Key Words: Zinc, Zinc transporters, Airway epithelium, Airway inflammation, Asthma, Zinc fluorophores, Zinc deficiency, Zinc supplementation. INTRODUCTION Much of the pioneer work on Zn metabolism occurred at least four decades ago, commencing with the initial studies of experimental Zn deficiency in rodents, the finding of stoichiometric quantities of Zn in carbonic anhydrase and other metalloproteins and, later, the identification of human Zn deficiency syndromes [1-4]. This was followed in the 1980s by discovery of the unique role of Zn in the function of Zn finger transcription factors [5]. Recently, two developments have led to a dramatic upsurge of interest in the cellular biology of Zn ions. We, and others, have developed a panel of Zn fluorophores with different Zn affinities, excitation/emission wavelengths and fluorescence intensities, that are enabling the imaging of more dynamic pools of Zn in a range of cell types and tissues [6-9]. Fluorescence imaging of Zn, along with the elegant and powerful technique of autometallography developed by Danscher and colleagues [10] for visualization of Zn by electron microscopy, are beginning to define the role of Zn in a number of physiological and pathological events. The second development has been the discovery of two large and growing families of specific Zn transporter proteins [11, 12]. Understanding how these transporters cooperate to regulate cellular Zn levels, and to deliver it to metalloproteins, is central to understanding the normal physiology of Zn and abnormalities that might arise in disease. Mutations in two Zn transporters have now been linked to the Zn deficiency diseases acrodermatitis enteropathica in humans [13] and lethal milk syndrome in mice [14]. Future research is likely to identify other diseases in *Address correspondence to this author at the Department of Medicine, University of Adelaide, The Queen Elizabeth Hospital, Woodville, South Australia 5011; Tel: +61 8 82227344; Fax: +61 8 82226042; E-mail: [email protected]

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which changes in Zn transporter levels, distribution or activities are involved. The above studies have also shown the profound influence of Zn ions in the functional integrity of a number of epithelial tissues (e.g. epidermis and gastrointestinal epithelium) [15-17]. Our studies over the last few years have pointed to an important cytoprotective role for Zn in airway epithelium (AE), a role for the Zn transporter ZnT4 in AE Zn homeostasis and a loss of AE Zn during airway inflammation in mice [18-23]. These studies add to other investigations that have reported low dietary Zn intake and low circulating and hair Zn concentrations in human subjects with asthma [reviewed in 21]. This review aims to bring together these and other findings pertinent to this area, with the hope of stimulating new research and designing new strategies to monitor and regulate airway Zn and Zn transporter pools. The review begins with a discussion of general aspects of whole body and cellular Zn homeostasis and then proceeds to what is specifically known about Zn in AE. Next, we describe alterations in Zn homeostasis in airway inflammation in mice and humans and address the question of whether subjects with asthma are Zn deficient. Finally, we discuss current strategies to monitor and supplement levels of airway Zn and suggest some future research priorities in this area. ZINC HOMEOSTASIS AND DEFICIENCY The human body comprises 2-3 g of Zn, which is distributed non-uniformly throughout all organs, secretions, fluids and tissues. This metal ion is particularly abundant in the retina, prostate, pancreatic islets and sperm, reaching concentrations as high as 3000 µg/g [3]. Zn is an essential dietary component required for the functional integrity of many organ systems, as well as for growth, development, and tissue repair [24]. The overall levels of Zn in the body reflect a © 2006 Bentham Science Publishers Ltd.

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balance between absorption of dietary Zn and loss of Zn from the body (Fig. 1). Zn homeostasis in an adult human involves the absorption of 3-4 mg Zn per day and the excretion or loss of a similar amount [25]. Homeostatic mechanisms in the intestine can maintain this balance during short periods of Zn deprivation by increasing absorption and decreasing excretion. However, if Zn losses are prolonged, a state of Zn deficiency will ensue. This is accompanied by a number of clinical manifestations, which may prove fatal if intracellular Zn levels fall below threshold concentrations. Conversely, excesses of the metal interfere with absorption of copper and iron and may also be toxic for cells [24]. Dietary Zn Intake and Absorption The current recommended daily allowances for Zn are 5 mg for infants, 10 mg for children, 15 mg for teenagers, adults and pregnant women and 16-19 mg for lactating women [26]. Zn is normally obtained in foods such as red meat, seafood, dairy food, grains and nuts [27]. In an Australian national random survey in 1991, the major individual food sources for Zn in men were steak (11% of total Zn intake), bread (9%), milk (8%), cheese (6%), minced meat (4%) and breakfast cereals (3%) [28]. Zn-fortified cereals have recently become a major source of Zn for young children [29]. There are no stores of Zn in the body (unlike iron), although bone and liver Zn may act as reservoirs. Zn homeostasis is regulated largely at the level of the gastrointestinal tract, by absorption and excretion [25]. The efficiency by which Zn is absorbed across the intestine depends on several factors, but most importantly it is the formation of chelates and other complexes that either promote or impede absorption (Fig. 1A), depending upon the nature of the bound ligand. Thus, while green vegetables, corn and rice contain adequate levels of Zn, this Zn is in a form that is less bioavailable, due to its binding to negatively-charged phytates (inositol hexa- and pentaphosphates) which are not absorbed [30]. Both stable isotopes and radioisotopes have been utilized in tracer studies to provide information on efficiency of Zn uptake across the intestine [31]. As part of normal Zn homeostasis, fractional absorption of Zn decreases as the intralumenal concentration of Zn is increased, while in Zn deficiency or situations of high Zn demand (e.g. in pregnancy, lactation and infancy), fractional absorption of Zn is increased [32]. A substantial proportion (> 60%) of ingested Zn is excreted in feces, originating from either unabsorbed Zn or absorbed Zn returned via the pancreatic and biliary secretions (Fig. 1B) [24]. Loss of Zn by these processes may be important in homeostatic regulation of body Zn status since it has been suggested that amounts of Zn excreted increase with Zn intake [32]. The mechanisms underlying absorption of Zn across the intestinal mucosa are still being defined, but involve the actions of Zn transporter proteins residing in both the lumenal and serosal membranes of enterocytes [reviewed in 33]. Some of these molecules are members of a large family of Zn transporters that regulate Zn homeostasis in cells and tissues (discussed later). Others include multi-specific metal ion transporters such as Divalent Cation Transporter-1 (DCT-1). DCT-1 is thought to mediate Zn, copper and iron absorption at the lumenal surface of enterocytes, thereby

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providing an explanation for why prolonged Zn supplementation can lead to deficiencies of these other metals [33]. After absorption across the brush border, Zn is coupled to cysteine-rich intestinal protein (CRIP), a 77 amino acid cytosolic protein that shuttles Zn from apical to basolateral surfaces. During periods of high intake, Zn is bound to another cysteine-rich cytosolic protein, metallothionein to prevent Zn overload. At the basolateral surface, the Zn transporter, ZnT1, mediates Zn export. Zn is then carried by plasma albumin to the liver and other tissues [3] (Fig. 1C). A small proportion of plasma Zn, which is bound to amino acids, enters the glomerular filtrate but is reabsorbed along kidney proximal tubules (Fig. 1D), preventing significant urinary loss of Zn, at least in non-pathological conditions [34]. In many potentially harmful situations or altered physiological states, such as stress, trauma, acute infection, acute inflammation, severe burns, malignancy and rheumatoid arthritis, there is a redistribution of Zn from plasma to liver (Fig. 1E). This process, which is mediated by glucocorticoids and interleukins (IL-1, IL-6), can lead to a doubling of the liver Zn content within 24 h of onset of acute inflammation [35, 36]. Mobilization of Zn to the liver is transient, with Zn returning to the plasma following cessation of the inflammation or stress. It is not clear whether the released Zn re-enters tissue pools or is destined for excretion. In addition to excretion of Zn via the feces, significant losses of Zn from the body can result from secretion of Zn across epithelial surfaces into breast milk, airway lining fluid and other body fluids (Fig. 1F), sloughing of epithelial cells and exudation of inflammatory cells into gut and airway lumens or from skin (Fig. 1G) and during excessive cell turnover (Fig. 1H; e.g. in patients with severe burns) [24]. Zinc Deficiency Any prolonged condition in which Zn losses from the body exceed its absorption will result in a state of Zn deficiency. The classic features of Zn deficiency include retardation of growth in children, immunodeficiency, hypogeusia, loss of appetite, diarrhea, skin changes and delayed sexual maturation [4, 24]. Zn is limiting in the diet of some indigenous peoples, including Australian aborigines, but severe primary Zn deficiency is relatively rare. A major cause of Zn deficiency is the consumption of diets that are low in animal protein and rich in phytate such as in many developing countries [1]. Amongst people in developed countries, the greatest risk of Zn deficiency due to inadequate Zn intake occurs in infants (1-3 y), adolescent females and the elderly (> 70 y) [37]. Zn deficiency due to inadequate intake may also follow parenteral nutrition lacking extra Zn supplementation and in anorexia nervosa and protein-energy malnutrition [24]. Other causes of Zn deficiency are Zn malabsorption (e.g. in alcoholism and acrodermatitis enteropathica) and excessive Zn losses from the body due to diarrheal diseases, hyperzincuria (e.g. in diabetes mellitus) and widespread losses of epithelial cells (e.g. due to scaling of skin in psoriasis) [reviewed in 23]. Sub-Cellular Pools of Zinc Zn is largely an intracellular ion and, as a group IIb metal, it occurs as the divalent ion Zn(II), coordinating with ligands in the +2 oxidation state in a preferred tetrahedral

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Fig. (1). Mechanisms of Zn homeostasis. Whole body Zn homeostasis involves a balance between dietary Zn intake and losses of Zn from the body. (A) Zn, bound to various ligands, is absorbed across the intestinal mucosa by both Zn-specific and multi-cation transporters in the brush border. (B) Unabsorbed Zn, which may constitute ~ 60% of dietary Zn, and Zn returned to the intestinal lumen via bile and pancreatic ducts, is excreted in feces. (C) Following, absorption, Zn is carried by plasma albumin to liver and other tissues. (D) Some Zn is lost in urine, and this is increased markedly in diabetes mellitus. Most of the Zn, however, is reabsorbed in the proximal tubules. (E) In infection, inflammation and other stresses, a significant portion of plasma Zn is mobilized to the liver, in a process mediated by glucocorticoids and cytokines. The fate of this Zn (e.g. whether it is eventually excreted) and the reasons for the mobilization are not known. Three other mechanisms by which Zn can be lost from the body, and which may be particularly relevant for epithelial tissues include (F) secretion of Zn (e.g. into breast milk), (G) shedding of epithelial cells and exudation of inflammatory cells (e.g. eosinophils) into the lumen and (H) excessive cell turnover. If there are prolonged periods in which Zn losses exceed Zn absorption (~ 3-4 mg Zn per day in a typical adult), a partial Zn deficiency may arise secondarily.

configuration; because of a very high affinity for electrons, it is able to bind to sulphur and nitrogen in cysteines and histidines of metalloproteins [38]. Cellular Zn occurs either tightly bound to metalloproteins (a largely fixed pool of cellular Zn that only turns over slowly and comprises 80-90% of cell Zn) or in more dynamic, free or loosely bound (labile) pools [39]. As a general rule, the fixed pools of Zn correspond to Zn in metalloenzymes (which number in the several hundred) and Zn finger proteins and are mainly responsible for housekeeping functions in cellular metabolism and gene expression [40], although some of this Zn may be more labile and exchangeable than previously thought [41]. The Zn, which is most susceptible to depletion in Zn deficiency, resides in the minor, readily exchangeable labile pools that are loosely associated with proteins, lipids, cytoskeletal processes or sequestered in vesicles [6, 39]. Labile Zn is concentrated in certain tissues and within specific regions of these tissues, where it performs special functions beyond those of general metabolism and gene expression, including secre-

tion, cytoprotection and signal transduction [23]. Thus, abundant levels of labile Zn are predominantly seen in those cells and tissues involved in secretion, including the prostatic epithelium (secretion of seminal Zn), pancreatic islets (secretion of insulin and glucagon), certain presynaptic neurons (secretion of glutamate) and mast cells (secretion of histamine) [7, 39, 42]. Zinc Transporters Cells need a constant supply of Zn. However, free Zn ions are potent inhibitors of some cytoplasmic enzymes and therefore, are potentially toxic to cells [43]. In order to maintain controlled levels of this metal ion in the appropriate subcellular compartments, tightly regulated homeostatic mechanisms need to be in place. This is achieved by the concerted actions of a number of specialized proteins. Metallothioneins (MTs) are cytosolic proteins with a high content of cysteine (25-30%) that bind Zn, as well as other metal

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ions, with high affinity. MTs are thought to be responsible for intracellular metal ion detoxification [44] and Zn and other stress factors induce their synthesis. Early studies of the active transport mechanisms by which fibroblasts take up Zn from the medium [45], predicted the existence of specific transporters to aid Zn movement across biological membranes. In the past decade, several mammalian Zn transporters have been identified and the corresponding genes cloned. Based on their sequence similarities and on structural properties, they have been assigned to two distinct families. Solute Carrier family 39 (SLC39) includes mammalian ZRT/ IRT-related proteins (ZIPs), while Solute Carrier Family 30 (SLC30), comprises the mammalian zinc transporters (ZnTs).

porters (LZTs), respectively [47]. Most of the ZIP proteins share a similar molecular structure (as shown for ZIP4 in Fig. 2A). Although experimental evidence has not been produced for all of them, ZIP proteins are most likely involved in the homeostatic control of Zn uptake. Specific Zn influx activity has been shown so far for 6 family members (ZIPs 1-5 and ZIP14) in transfected cells [48-54]. The tissue distribution of ZIP proteins differs widely and is relevant for their function in Zn homeostatic processes. ZIPs 1, 3 and 8 are widely expressed in several tissues [53, 55], while Zip2 expression is more restricted (skin, liver, ovary and visceral yolk sac) [53]. Zip4 and Zip5 are most abundant in the organs involved in body Zn homeostasis (intestine, pancreas, liver and kidney) [56]. Neither mouse ZIP4 nor mouse ZIP5 are expressed in lungs [56].

ZIP Subfamily of SLC39

Mutations in the human ZIP4 gene have been found in patients affected with Acrodermatitis enterophatica, a human autosomal recessive disease characterized by markedly impaired intestinal absorption of Zn [57] and consequent severe Zn deficiency [13]. Zn deficiency is thought to induce ZIP4mediated Zn uptake from the intestinal lumen via transcriptional up-regulation [12, 53] and membrane localization [58] as well as reduce ZIP5-mediated Zn uptake from the blood via a signaling cascade leading to endocytosis of ZIP5 protein [56]. ZIP4 is inactivated through the same endocytosis mechanism when Zn supply is adequate. Similarly, in the pancreas endocrine β cells exclusively express ZIP4, while ZIP5 is present in the acinar cells of the exocrine portion only in Zn-adequate animals [56]. Taken together, these data

The SLC39 transporter family includes several members identified in prokaryotic and eukaryotic organisms, including archea, bacteria, fungi, plants and mammals (Table 1). Amongst mammals, the best-characterized members of the ZIP subfamily of SLC39 are the ZIP proteins. 14 ZIPencoding genes have been identified in the human genome [46], and although molecular characterization of their corresponding proteins is still in progress, much work has been done on ZIPs 1-5, and more recently ZIPs 6-8 and 14. According to their molecular features ZIP proteins have been grouped into 4 sub-families. Of particular interest are subfamilies II and IV, which consists of mammalian, nematode and insect genes, and the LIV-1 subfamily of ZIP Zn transTable 1.

Properties of the ZIP Transporters of the SLC39 Family

Human gene name

Protein name

Lung expression

Subcellular localization

Human gene locus

Human sequence accession number

SLC39A1a

ZIP1

Yes

Plasma membrane/ intracellular vesicles

1q21

NM_014437

b

ZIP2

No

plasma membrane

14q11.1

NM_014579

SLC39A3 c

ZIP3

Unk

Unk

19p13.3

NM_144564

ZIP4

No

Apical membrane

8q24.3

NM_017767

ZIP5

No

Basolateral membrane

12q13.13

NM_173596

ZIP6/LIV-1

Yes

Plasma-membrane

18q12.1

NM_012319

ZIP7/KE4

Yes

Golgi/ER

6p21.3

NM_006979

ZIP8/BIGM103

Yes

Plasma-membrane

4q22-q24

NM_022154

SLC39A9

ZIP9

Unk

Unk

14q24.1

NM_018375

SLC39A10

ZIP10

Unk

Unk

2q33.1

NM_020342

SLC39A11

ZIP11

Unk

Unk

17q25.1

NM_139177

SLC39A12

ZIP12

Unk

Unk

10p12.33

NM_152725

ZIP13

Unk

Unk

11p11.12

NM_152264

ZIP14

Yes

Plasma-membrane

8p21.2

NM_015359

SLC39A2

SLC39A4

d,e

SLC39A5 f SLC39A6

g

SLC39A7 h SLC39A8

i

SLC39A13 SLC39A14

j

SLC, solute carrier family; ZIP, ZRT/IRT-related transporters; ER, endoplasmic reticulum; Golgi, Golgi apparatus; Y, yes; N, no; Unk, unknown; a [51]; b [52]; d [13] e [224] ; f [225]; g [49]; h [226]; i [55]; j [62].



demonstrate that the two proteins likely cooperate in maintaining Zn homeostasis in the organism [56]. Zn-dependent intracellular trafficking between plasma membrane and intracellular organelles was also recently demonstrated for mouse ZIP1 and mouse ZIP3 in transfected cells [59]. As for the less characterized members, human ZIP6 (hZIP6) is a Zn-influx transporter [48] that is up-regulated in breast cancer and regulated by estrogen [49]. Mouse and human ZIP7 is capable of transporting Zn from the Golgi vesicles into the cytoplasm [60]. ZIP8, initially described as a transcript induced by immune activation in monocytes [55], was later identified as the transporter responsible for cadmium-induced toxicity in the testis [61]. ZIP8 is present in a variety of tissues, including lung, an important target for cadmium toxicity. Overall, these findings point to the Zn transporters as a major intracellular route for toxic heavy metal contaminants. The most recently characterized member of the ZIP family, ZIP14, is widely distributed in all tissues examined, including lung, and contains the metalloprotease motif characteristic of the LZT subfamily. ZIP14 was shown to reside on the plasma membrane and to be able to take up Zn from the extracellular medium [62]. Further insights into the functions of ZIP proteins have been recently gained by looking at their function in development and cellsignaling events in model organisms such as Zebrafish and Drosophila [63, 64] (see later). ZnT Subfamily of SLC30 The other group of Zn transporters is the SLC30 or Cation Diffusion Facilitator (CDF) family, comprising mammalian ZnTs (Table 2) (Fig. 2B). In the human genome, to date, 10 members have been identified and their corresponding proteins characterized [11, 12, 65]. With the exception of ZnT5 [66], ZnTs are mainly involved in Zn efflux, Table 2.

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as well as compartmentalization of the metal in intracellular organelles (Fig. 2C). Increasing evidence supports the hypothesis that by mobilizing Zn in intracellular organelles, Zn transporters not only contribute to Zn storage and detoxification but also supply the metal to Zn-dependent proteins [67, 68]. ZnT requirement for specific enzymatic activities could be a more general mechanism. For example, ZnT5 and ZnT7 are required for the proper function of membraneanchored alkaline phosphatase [67]. Other Zn-dependent enzymes such as Cu/Zn SOD are also likely to depend on ZnT proteins for their Zn supply. The distinct tissue distribution and sub-cellular localization of these transporters suggests that they play distinct roles in the intracellular movements of Zn ions. ZnT1 is widely expressed and thought to be predominantly involved in membrane efflux of Zn and detoxification [69]; it may also regulate export of Zn into plasma [70] and is essential in early development [71]. ZnT3 is largely brain-specific and is required to load Zn into presynaptic glutamate-containing secretory granules [72, 73]. ZnT2 and ZnT4 are localized in cytoplasmic vesicles and may sequester Zn in this compartment [74-76]. A point mutation in the ZnT4 gene causes a major defect in the secretion of Zn in milk and is the molecular basis of the lethal milk mouse syndrome [14]. According with its role in milk Zn secretion, ZnT4 is expressed in breast epithelial cells [77]. ZnT4 is also widely expressed in most tissues and cell lines, where it localizes in the membrane of intracellular vesicles [76], suggesting a broader role in Zn metabolism. For example, altered Zn levels in prostate benign prostatic hyperplasia and carcinoma are well documented and human ZnT4 was decreased in benign and tumor samples compared to normal tissue [78]. ZnT5 is localized in the membranes of Zn-rich insulin secretory granules of pancreatic islet β cells [79], but is also expressed in other

Properties of the SLC30 (ZnT) Family of Transporters

Human gene name

Protein name

Lung expression

Subcellular localization

KO phenotype

Human gene locus

Human sequence accession number

SLC30A1a,b

ZnT1

Yes

Plasma-membrane and vescicles

Embryonic lethal

1q32.3

NM_021194

SLC30A2c

ZnT2

Yes

Intracellular vesicles

1p35.3

NM_032513

SLC30A3d, e

ZnT3

No

Synaptic vesicles

Seizures, Alzheimer's

2p23.3

NM_003459

SLC30A4,f,g,h

ZnT4/Dri27

Yes

Endosomes

Lethal milk*

15q21.1

NM_013309

SLC30A5,I, j k

ZnT5/ZTL1

Yes

Secretory granules/ intracellular vesicles

Bone abnormalities/ heart failure

5q13.1

NM_022902

SLC30A6g

ZnT6

No

Golgi/intracellular vesicles

2p22.3

NM_017964

SLC30A7k,l

ZnT7

Yes

Intracellular vesicles

1p21.2

NM_133496

SLC30A8m

ZnT8

No

Secretory granules of pancreatic endocrine cells

1q41

NM_173851

SLC30A9o

ZnT9/C4orf1

Yes

cytoplasmic

4p13

NM_006345

SLC30A10k

ZnT10

Unk

Unk

1q41

NM_018713

SLC, solute carrier family; ZnT, zinc transporter family, Unk, unknown; KO, knock-out phenotype; a [69]; b [71]; c [74] ; d [75] ; e [73] ; f [227]; g [76]; h [81]; I [66]; j [80]; k [79]; l [81]; m [65]; n [82]; o [228]; * naturally occurring point mutation resulting in a truncated protein.

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Fig. (2). Two families of zinc (Zn) transporters. A:. Structure of ZIP4, typical of many ZIP family Zn transporters. This transporter resides in the apical plasma membrane of intestinal enterocytes. Most ZIP protein share a similar structure with 8 predicted trans-membrane domains, two histidine-rich domains (green oblongs), one in the extracellular N-terminal region and the other in the cytoplasmic loop between transmembrane domains IV and V, extracellular N and C termini and an amphipathic region from domains III to V that may form the wall of a Zn ion channel [11]. Members of the LZT subfamily also contain the conserved Zn-metalloprotease signature HEXPHEXGD in the transmembrane segment V [48]. B: Structure of ZnT4, typical of many SLC30 Zn transporters. This transporter normally resides in vesicle (or granule) membranes. ZnT proteins consist of 6 trans-membrane domains, with N and C termini on the cytoplasmic side of the membrane. They also have a conserved histidine-rich domain between trans-membrane segments IV and V, which binds metal ions including Zn [76] and an amphipathic trans-membrane segment which may line the Zn ion channel [11]. C: Schema of the sub-cellular distribution of Zn transporters. Zn is taken up across the plasma membrane via the family of human ZIP transporters (e.g. hZIP1, hZIP2 and hZIP4). Zn transporters of the SLC30 family mediate the sub-cellular localization of Zn into the Golgi apparatus (ZnT6 and ZnT7), vesicles (ZnT2 and ZnT4) and secretory granules (ZnT3 and ZnT5). The transporters responsible for localization of Zn to nucleus, mitochondria and other organelles have not yet been identified. Zn may exit cells via dedicated efflux transporters (eg ZnT1) or by secretion of granular contents.


tissues [66]. ZnT5–/– mice show several intriguing features including poor growth, lean phenotype, decreased bone density and weak muscle. Moreover, more than 60% of ZnT5–/– male mice die suddenly of bradyarhythmias [80]. ZnT6 and ZnT7 are localized to the Golgi apparatus but little else is known about their tissue distribution and function [81, 82]. ZnT8 is specific for the endocrine pancreas and localizes in the insulin secretory granules of β cells [65, unpublished observations]. Little information is available on the last two members of this subfamily: ZnT9 was isolated from human embryonic lung cells and contains a cation efflux motif. It is widely expressed in adult tissues and it moves from a cytoplasmic localization, presumably associated with the cytoskeleton, to the nucleus during S-phase. However, it shares little sequence homology with the other ZnTs [83]. ZnT10 was identified through computer analysis of the genomic databases, and its expression appears to be restricted to fetal liver and brain [65]. Following molecular characterization of ZnT proteins, several studies have addressed the question of how they are regulated. The emerging scenario points to both transcriptional and post-transcriptional levels of regulation. Transcription of ZnT1 and ZnT2 mRNAs is up-regulated in rat small intestine, kidney and liver by dietary Zn supplementation, as well as in response to a short-term, acute increase in Zn intake [84]. In the pancreas, ZnT1 and ZnT2 transcript levels decrease dramatically during Zn deficiency [85]. ZnT4 and ZnT6 expression in the small intestine parallels Zn nutritional status, while ZnT2 and ZnT5 expression is reduced in Zn deficiency, but almost unaffected by excess Zn in the diet [85]. In addition, several ZnT proteins appear to reside in the membrane of intracellular vesicles, whose cytoplasmic localization changes in response to Zn supplementation. In mouse mammary epithelial cells, ZnT1 and ZnT4 localize primarily to the perinuclear region and Golgi apparatus, respectively, in the absence of added Zn, while more peripheral vesicular staining was observed following Zn addition. ZnT2, on the contrary, showed disperse cytoplasmic vesicular staining in normal culture conditions that became perinuclear following Zn supplementation [86]. Zn-dependent re-localization of ZnT4 and ZnT6 vesicles from the perinuclear region towards the cell periphery was observed also in NRK cells [81] and in CHO cells (unpublished observations). Overall, these studies point to a role for ZnTs in the efflux of excess Zn from the cell. However, unlike the picture emerging on ZIP protein function, investigations on ZnTs have been greatly impaired by the lack of functional assays. A genetic approach [87] recently demonstrated a role of ZnT1 and metallothionein in Zn detoxification, providing further evidence that chelation of zinc ions by MTs and the Zn efflux activity of ZnT1 act in concert to provide an efficient mechanism that lowers intracellular zinc toxicity. For excellent reviews on Zn transporters see [11, 12, 88] and for further reading on general aspects of Zn biology, we recommend the series of papers that constitute an issue of Biometals 14, Issues 3-4, 2001. ZINC IN NORMAL AIRWAY EPITHELIUM Airway Epithelium The airway epithelium (AE) is a pseudo-stratified columnar epithelium that lines the length of the conducting airways


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with the exception of the terminal respiratory bronchioles. Its major functions are as a physical barrier separating the airway connective tissue and smooth muscle from the airway lumen as well as removal of foreign particles. The AE comprises three cell types. The predominant airway epithelial cell (AEC) is a columnar polarized cell, with a basolateral side adjacent to the basement membrane and an apical side that contains several hundred cilia, which protrude into the lumen of the airway. To provide energy for ciliary beating, the apical cytoplasm contains abundant mitochondria [89]. Interspersed amongst the ciliated cells are mucin-producing and other secretory cells (goblet cells, serous cells, clara cells, small mucous granule cells, brush cells and neuroendocrine cells) [90, 91]. Mucin-producing AEC secrete a layer of high molecular weight mucopolysaccharides (mucin) into the epithelial lining fluid (ELF) which trap foreign particles. The coordinating beating of the cilia propels the mucus and debris back up the airways in a process known as mucociliary clearance [92]. Secretory AEC also release into the ELF a variety of protective substances that are involved in innate immunity. These include anti-bacterial agents such as lysozyme, collectins and β-defensins [93, 94] and anti-oxidants such as glutathione and ascorbic acid [95]. Thus, an intact and functioning AE is critical for maintaining sterile, undamaged airway tracts. In addition, the AE is an important regulator of airway function, producing smooth muscle relaxant factors such as prostaglandin E2 and nitric oxide and enzymes that catabolize smooth muscle contractile agonists [96]. The function of basal AEC remains unclear but they may serve to anchor columnar cells to the basement membrane, as well as being precursors of the other two types of AEC [97]. Localization of Zinc in Ciliated Airway Epithelial Cells Five years ago, our group, using the Zn fluorophore, Zinquin, initiated the first studies of the levels and distribution of labile Zn pools in AE. Cryosections of pig and sheep trachea and lung showed intense Zinquin fluorescence in the apical cytoplasm; this fluorescence was completely quenched in the presence of the Zn chelator TPEN (N,N,N′,N′tetrakis-[98]-ethylenediamine) [20]. This pattern was confirmed later in the AE of mouse trachea and lung [19]. Although these studies did not indicate whether the labeling was in ciliated, secretory or both types of AE, similar intense apical fluorescence was observed in isolated ciliated sheep AEC (brushed from the trachea) and in isolated ciliated human AEC (obtained during bronchoscopy and by nasal brushing) [22]. In more than 95% of ciliated AEC (sheep or human), Zinquin fluorescence was most intense in the apical cytoplasm [22]. In addition to a distinct perinuclear fluorescence that often lined the entire nuclear membrane, there was most often an intense, diffuse apical fluorescence, which we have proposed to represent Zn in apical cytoplasmic vesicles, as well as other vesicles en-route between perinuclear and apical compartments [23]. Perinuclear Zinquin staining in these cells likely represents a pool of Zn in the golgi body. In some ciliated human and sheep AEC, fluorescence was largely or exclusively restricted to apical structures close to, but not coincident with, the ciliary basal bodies. Some Zn may be disposed around the apical mitochondria or interact with cytoplasmic metalloproteins in this region. Other Zn may be destined for secretion into ELF. Currently, there has

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been no measurement of Zn in this fluid although we have begun to measure Zn in induced sputum and bronchoalveolar lavage fluid (BALF) of humans and mice, respectively (discussed later). Zinc Transporters in Lung and AE We have shown that the AE is rich in Zn particularly in the apical cytoplasm beneath the cilia [19, 21]. While most airway epithelial Zn likely derives from sub-epithelial capillaries, we cannot exclude that some Zn enters at the mucosal surface of the lumen by re-absorption of secreted Zn. Therefore, there is likely to be a considerable flux of Zn into and across the AE. Firstly, there is Zn uptake at the basolateral surface from Zn-containing plasma albumin in sub-epithelial capillaries. From studies in other tissues [21], ZIP proteins are probably involved in the plasma membrane uptake. Once absorbed Zn is packaged in cytoplasmic vesicles that migrate to the perinuclear region and apical cytoplasm; this function is likely to be carried out by ZnT transporters. Different ZnTs are also probably involved in supplying the metal to Zn-dependent proteins. Any given cell type expresses a set of zinc transporter genes, that can provide the metal required for the specific needs of the cell. Northern blot and RT-PCR analysis have shown that some of the described zinc transporters are expressed in lung [87, 99]. In most cases, these analyses were carried out on RNA extracted from whole lung, and therefore do not take into account the complexity of this organ which is composed of several different tissues. Therefore, there is still very little information about the specific set of zinc transporters expressed in AE and their relative quantities. Our studies have shown ZnT4 to be highly expressed in AE, suggesting that this transporter is a key player in airway Zn homeostasis. Using RT-PCR we have shown abundant expression of ZnT4 mRNA in human bronchial and nasal AEC and in mouse AE; this was matched by strong immunostaining for ZnT4 in the apical cytoplasm of these cells (unpublished observations). This transporter has not previously been found to mediate cellular Zn uptake in other cell types. Rather, ZnT4 may play a primary role in vesicular uptake of Zn and/or subcellular compartmentalization because it is largely localized to the membranes of cytoplasmic vesicles [76]. The ZnT4 immunofluorescence pattern overlapped, but did not entirely coincide, with that of Zinquin (i.e. Zn) fluorescence, confirming other findings in cultured MDCK cells, mammary epithelium and rat kidney, which concluded that there may be distinct vesicular pools of ZnT4 and Zn [34, 77]. In addition, immunofluorescence studies with human ciliated AEC, revealed lower levels of ZnT4 protein on the basal side of the cells. This distribution of ZnT4 suggests a role in picking up Zn from sites at the basolateral membrane and delivery to the apical cytoplasm. Zinc Metalloproteins Relevant to the Airways While Zn metalloenzymes and transcription factors are ubiquitous throughout the cells and tissues of the body, there are a number of Zn metalloproteins that are especially relevant to airway function and disease (Fig. 3). Zinc Metalloproteinases Zn metalloproteinases (Fig. 3A) are important for extracellular matrix changes during remodeling of the airways.

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These comprise several families. One of these, the metzincin family, includes the family of metalloproteases known by the acronym of ADAM (a disintegrin- and metalloproteinasecontaining protein). ADAM33 has attracted special interest because of its potential role as a susceptibility gene in asthma [100], although this conclusion has recently been challenged [101, 102]. Nevertheless, an association between this gene and bronchial hyper-responsiveness has been confirmed [101]. ADAM33 is an 812 amino acid membrane protein with a metalloproteinase domain whose catalytic activity is inhibited by Zn chelators [103]. It is not expressed in AE but is expressed in lung fibroblasts and bronchiolar smooth muscle [101, 104]. Suggested roles for ADAM33 include proteolysis, adhesion, fusion, cell-cell communication and signaling [104]. Crystal structures of the catalytic domain of ADAM33 have confirmed that, like other matrix metalloproteinases, the active site contains Zn and water atoms that are necessary for proteolysis, the catalytic Zn ion is coordinated by His345, His349, and His355 which comprise a conserved Zn-binding motif HEXXHXXGXXH (Fig. 3A) and the 4th ligand-binding site of Zn is to a thiol group located in the prodomain [105]. The latter maintains the enzyme in an inactive state and activation is a consequence of disruption of this bond either by proteolysis or movement of the prodomain away from the catalytic domain. In addition to ADAM33, other Zn-containing matrix metalloproteinases (e.g. MMP-2, MMP-9) have been implicated in extracellular matrix cleavage and remodeling events that occur during airway inflammation [106]. Zn in these MMPs may be exchangeable [107] since the addition of the Zn-binding molecule, calprotectin inhibits, in a Zn-reversible manner, the activity of all MMPs tested (MMP-1, 2, 3, 7, 9, 13) [107]. However, different concentrations of calprotectin were necessary to produce a 50% inhibition of activity, depending on the MMP tested, suggesting that these enzymes do have different affinities for Zn [107]. Alkaline Phosphatase and Ecto-5'-Nucleotidase ATP and other extracellular nucleotides play important roles in several airway epithelial functions including mucociliary clearance. However, an excess of these nucleotides, as occurs during lung trauma and airway inflammation, can cause damage to the AE [108]. Alkaline phosphatase (Fig. 3B), along with another Zn metalloenzyme, ecto-5'nucleotidase, play an important role in the dephosphorylation of ATP into adenosine on the airway mucosal epithelial surface and are major regulators of airway nucleotide concentrations [108]. Alkaline phosphatase has also been implicated in bacterial endotoxin neutralization and signaling via the sphingosine 1-phosphate receptor (see [108] for discussion). Binding of Zn is required for both catalysis and structural stabilization of alkaline phosphatase [109] and levels of this enzyme (e.g. in plasma) have been used as an indicator of Zn status in humans and experimental animals [110]. Interestingly, very recent studies implicate ZnT5 and ZnT7 in the activation of membrane-anchored alkaline phosphatase [67]. Cu/Zn Superoxide Dismutase Superoxide dismutases (SOD) are major anti-oxidant enzymes in AE, which catalyze the dismutation of superoxide anion (O2-) to oxygen and hydrogen peroxide, and are the first line of defense against reactive oxygen species (ROS) in


cells. Hydrogen peroxide is, in turn, removed by catalase or glutathione peroxidase. The AE contains two types of SOD, Mn SOD that is found within mitochondria and Cu/Zn SOD (Fig. 3C), which is restricted to the cytosol [reviewed in 111]. Larsen and colleagues [112] demonstrated that transgenic mice with elevated levels of Cu/Zn SOD in the lungs were more resistant to allergen-induced hyper-responsiveness than wild-type mice. Moreover, activity of Cu/Zn SOD may be decreased in the AE of human asthmatics [113, 114]. Using immunofluorescence in human bronchial and sheep tracheal AEC, we have shown that Cu/Zn SOD is localized to the same apical cytoplasmic region as labile Zn [22]. This observation promotes speculation as to whether Zn levels in this enzyme are influenced by cytoplasmic Zn concentrations and/or whether Zn in Cu/Zn SOD comprises a significant portion of the Zinquin-stainable Zn in these cells. The Zn in this metalloenzyme is bound to three histidines and an aspartate, but is still readily exchangeable [115, 116]. The role of Zn in Cu/Zn SOD is controversial. Severe depletion of intracellular Zn in keratinocytes by treatment with TPEN in vitro resulted in apoptosis but did not affect the Cu/Zn SOD activity of cells; however, this study did not determine whether Zn was depleted from the enzyme [117]. Another group reported that lung Cu/Zn SOD was paradoxically increased in Zn deficiency [118]. In contrast, mutations in Cu/Zn SOD that decreased the enzyme’s affinity for Zn and led to loss of Zn, were found to promote catalysis of peroxynitrite-mediated tyrosine nitration, resulting in protein oxidation and consequent cellular damage [116]. Thus, Zn may be required in Cu/Zn SOD to maintain normal antioxidant function. STAT3/Snail Signal transducers and activators of transcription (STATs) mediate biological actions such as cell proliferation, differentiation and survival in response to cytokines and growth factors affecting organogenesis, wound healing and cancer progression [119]. It was recently shown that the zebrafish homolog of ZIP6/LIV1 is a downstream target of STAT3 and it is essential for its role in epithelial-mesenchymal transition during zebrafish gastrulation [64]. ZIP6/LIV1 regulates nuclear translocation of the transcriptional repressor snail (Fig. 3D). It is interesting to note that the snail protein requires its Zn-finger domain to accumulate in the nucleus. In the same report it was shown that the expression of ZIP6/LIV1, in the human prostate cancer cell line DU145, is inhibited by siRNA for STAT3 [64]. These observations therefore implicate ZIP6/LIV1 in breast cancer and embryonic development [120]. Along the same lines, another recent report describes the role of the Drosophila fear of intimacy (FOI) gene, that encodes a protein belonging to the ZIP family and related more closely to the LIV subgroup, in regulation of signaling events. The FOI protein interacts with Hedgehog signaling to promote cell migration. Regulated activation of Hedgehog signaling is necessary for embryonic cell growth and differentiation in both invertebrates and vertebrates. Deregulation of this pathway was shown to promote growth of a significant number of endodermderived tumors, including small-cell lung cancer [121, 122]. NADPH Oxidase and Ion Channel Proteins In addition to promoting the removal of superoxide anion via Cu/Zn SOD, Zn may also inhibit the production of su-


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peroxide by eosinophils, neutrophils and other cells. Eosinophils, activated by IL-5 and other stimuli, generate extracellular superoxide anions through a plasma membrane NADPH oxidase, which is expressed at high levels in these cells [123]. The inhibitory effect of Zn is not due to a direct action on the oxidase but rather on an associated plasma membrane proton channel (Fig. 3E). The generation of superoxide anion requires the transfer of electrons across the plasma membrane with the concomitant release of protons into the cytoplasm. Since NADPH oxidase is inhibited by intracellular acidification, eosinophils have an efficient mechanism for removing protons from the cytosol to maintain oxidase activity. At least two studies have shown that this proton channel in eosinophils is very sensitive to inhibition by Zn [124, 125], providing an explanation for Znmediated suppression of superoxide production in these cells. This was nicely confirmed by experiments showing that COS-7 cells transfected with four NADPH oxidase components, but lacking H+ channels, produce superoxide anion in the presence of Zn concentrations that normally inhibit superoxide production in neutrophils and eosinophils [126]. The mechanism by which Zn blocks the channels is not clear but one study in rat alveolar epithelial cells showed that Zn binds to external sites on the H(+) channel and modulates gating [127]. At least one airway ion channel may be stimulated by Zn ions. In the presence of ATP, Zn induced a sustained increase in cytosolic Ca2+ in AEC that was apparently mediated by activation of P2X purinergic receptor channels and resulted in a significant Cl- secretory response, in vitro and in vivo [128]. An issue that needs to be addressed here is how concentrations of Zn ions, known to modulate these channels, are achieved in ELF and whether ELF Zn concentrations change during inflammation or disease. In view of the evidence that another chloride channel CIC-3 co-localizes with ZnT3 in synaptic vesicles and regulates Zn uptake by the vesicles [72], it will be interesting to see whether ZnT4 is also in association with other ion channels and whether pools of apical Zn in AEC are determined by similar anion shunts. Raf-1 and Other Signal Transduction Proteins The concept that Zn ions may participate, like calcium ions, as second messengers in cell signaling was first proposed two decades ago [38]; however evidence for this is only now beginning to emerge. In one recent report, ZnT1 was shown to promote Raf-1 protein kinase activation by binding to its amino-terminal regulatory portion in a Zndependent manner (Fig. 3F) [68]. As previously demonstrated genetically in C. elegans [129], these results confirm in mammalian cells a link between Zn, ZnT1 and Rasmediated signaling pathways that regulate growth, survival and differentiation. Zn ions have also been shown to dynamically regulate two other important signaling pathways, stimulating the phosphatidylinositol 3-kinase/Akt pathways and suppressing protein kinase C [130-132]. For example, Wu and colleagues [132] showed that Zn supplementation of primary human AEC in vitro and rat AE in vivo resulted in proteasomemediated degradation and loss of function of PTEN, a tumor suppressor protein which dephosphorylates PIP3 and negatively regulates the phosphatidylinositol 3-kinase/Akt path-

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way, thereby stimulating cell growth and suppressing apoptosis in AE. β2 Adrenoreceptor Two further studies are of particular relevance to airway smooth muscle contraction. Firstly, Zn (at concentrations as low as 1µM) caused a positive allosteric modulation of agonist binding to the β2 adrenoreceptor (Fig. 3G), apparently by bridging two trans-membrane domains of the receptor [133]. Agonists of β2 adrenoreceptor relax airway smooth muscle and are widely used bronchodilators in the treatment of asthma. It will be interesting to determine whether a lowering of Zn concentrations in the airways, below a certain threshold, can lead to an enhancement of bronchoconstriction and whether Zn supplements given with bronchodilators can enhance their effect.

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Procaspase-3 Finally, Zn interacts with procaspase-3 (Fig. 3H), the zymogen form of a major effector of apoptosis [reviewed in 18]. Our studies, and those of other laboratories, have demonstrated increases in apoptotic cells in a variety of tissues of Zn deficient animals, including the intestinal epithelium, skin, thymus, testis, retina and pancreas [reviewed in 20]. We have shown increased apoptosis in the inflamed AE of mice on low Zn diets compared with inflamed AE of mice on normal Zn diets [19]. We have also shown that increasing intracellular Zn in AEC, using the Zn ionophore sodium pyrithione and exogenous ZnSO4, resulted in a significant inhibition of H 2O2-induced caspase-3 activation, while depletion of labile Zn by TPEN caused a marked increase in caspase-3dependent apoptosis, as well as facilitating caspase-3 activation by low concentrations of ROS [22, 134]. Furthermore,

Fig. (3). Zn-containing metalloproteins that are relevant to airway function. A: ADAM33 is a large trans-membrane protein with an extracellular region that includes an N terminus prodomain, an active site that contains Zn, a transmembrane region and a cytoplasmic tail. The Zn is thought to be exchangeable and therefore, ADAM33 activity may be influenced by Zn concentrations in the microenvironment of the enzyme and inhibited in Zn deficiency. B: Alkaline phosphatase is a Zn-dependent enzyme in the apical region of the AE that is important in regulating airway nucleotide concentrations and inhibited in Zn deficiency. Alkaline phosphatase may also be dependent on ZnT5 and ZnT7. C: Cu/Zn SOD, which is the first line of cellular defense against reactive oxygen species (ROS), is dependent on Zn (as well as Cu) for structural and functional activity. It is co-localized with ZnT4 and Zn in the apical cytoplasm of AEC but whether it requires ZnT4 or other Zn transporter for insertion of Zn is not known. D: Zn binds to and inhibits apical proton channels in AE, resulting in decreased acidity of epithelial lining fluid (ELF) and inhibition of NADPH oxidase-dependent superoxide anion production. E: Zn effects the Ras signaling pathway and ZnT-1 is an activator of Raf-1, thereby influencing downstream kinase cascades. F: Zn binds to the trans-membrane domains of the β2 adrenoreceptor, thereby promoting agonist binding. By this mechanism Zn may promote bronchodilation, while Zn deficiency may be a factor in bronchoconstriction. G: Zn is a potent inhibitor of procaspase-3 activation, thereby suppressing apoptosis. As with Cu/Zn SOD, the co-localization of procaspase-3 with ZnT4 raises the question of whether this transporter has a special relationship in regulating this enzyme.


procaspase-3, the zymogen precursor of the active enzyme, is almost exclusively localized to the Zn-rich apical cytoplasm of AE [19, 22, 135]. We propose that the localization of procaspase-3 to the apical cytoplasm could provide an early response mechanism to lumenal toxins, allowing the AEC to die in a controlled manner by apoptosis. A critical step in the activation of this enzyme may be removal of inhibitory Zn. In addition, Zn depletion may activate procaspase-3 by indirect effects on oxidative stress (see below). Other Functions of Zn in Airway Epithelium Zn has a number of other properties and actions, which are likely to be beneficial for the AE. The AE is particularly vulnerable to a range of oxidants derived internally (e.g. as a by-product of aerobic respiration) or externally from atmospheric oxidants (e.g. ozone and cigarette smoke) and from inflammatory cells during acute or chronic inflammation of the airways [reviewed in 136]. Therefore, the AE and its secretions contain a variety of anti-oxidants including catalase, vitamins C and E, glutathione, and two forms of superoxide dismutase [136]. Zn has a significant impact on the antioxidant capacity of cells and tissues. Cell cultures grown in Zn-deficient media have increased oxidant production [137] and Zn-depleted primary sheep and human AEC showed extensive lipid peroxidation in the apical membranes [22]. In vivo, Zn deficiency increases susceptibility to oxidative injury. For example, Zn deficient rats exposed to hyperoxia have higher levels of oxidative stress in their lungs than Zn normal rats exposed to the same stress; this is prevented by dietary Zn repletion [138-141]. Conversely, Zn supplementation protects against oxidative damage and has been shown to be beneficial in ROS-induced disease progression [142, 143]. The precise mechanism by which Zn acts as an antioxidant is unclear. Part of its action is due to its role in Cu/Zn SOD, as described earlier. Other mechanisms include the stabilization of protein thiol and sulfhydryl groups against oxidation [reviewed in 144], the stabilization of membrane lipids against peroxidation [22] and the suppression of nitric oxide production [16]. In addition, Zn can antagonise redox-active transition metals, such as iron or copper, thus, preventing hydroxyl radical formation [145, 146]. Finally, Zn can induce the expression of metallothionein, a metalloprotein with potent antioxidant activity [41]. Zn is essential for some aspects of the immune response and depletion of Zn may predispose to Th2-driven proinflammatory cytokine production. For reviews on the role of Zn in immune responses, see [8, 147]. Immune cells are not only responsible for mucosal immunity in the airways but also trigger allergic reactions via production of IgE. The role of Zn in each of these processes needs to be clarified. However, there is evidence that Zn can influence the composition of T lymphocyte subsets, their proliferation, production of interleukins and cytokines such as IL-1, IL-4 and IFN-γ and their death by apoptosis [reviewed in 147, 148, 149]). Of particular relevance to asthma, Zn regulates the balances between CD4+ and CD8+ T cells and between Th1 and Th2 subsets [147, 150]. Even 4 weeks of mild nutritional Zn deprivation in experimental human volunteers resulted in a decline in the ratio of CD4+ (helper)/CD8 + (suppressor) T cells and diminished Th1-dependent cytokine production; Th2-dependent cytokine production was unimpaired, creating


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a relative Th 1 deficiency [150]. It has been proposed that the shift towards a Th2–dependent response in Zn deficient individuals could facilitate mucosal IgE production in atopic individuals and the production of inflammatory cytokines in diseases of airway inflammation [151]. That Zn is anti-inflammatory has largely emerged from the well-known effects of topically-administered Zncontaining creams and lotions, dating as far back as 1500 BC when Zn oxide as calomine was applied to heal eye lesions [152]. There is also now good evidence that Zn has antiinflammatory effects in the intestine and that Zn metabolism is perturbed in a number of acute and chronic inflammatory disorders, both in experimental animals and in humans [reviewed in 153]. For example, Zn levels in neutrophils (an index of systemic Zn levels) decrease significantly in the inflammatory disorders psoriasis and seborrhoeic dermatitis [154]. It has yet to be determined whether a fall in Zn levels promotes inflammation in these diseases or whether the fall in Zn levels is a secondary manifestation of the disease. Both may be correct and this will be discussed later in the context of asthma. A number of hypotheses have been advanced as to why Zn is anti-inflammatory. One proposed target is NFκβ, a cytoplasmic transcription factor, which in activated cells translocates to the nucleus and is critical for the expression of many pro-inflammatory cytokines [155]. We have shown that depletion of Zn in mast cells stimulated nuclear translocation of NF-κβ, while increasing intracellular Zn using the Zn ionophore, pyrithione, blocked translocation [42]. It has been proposed that Zn blocks translocation of this factor by suppression of phosphorylation and degradation of the inhibitory proteins that normally sequester NF-κβ in the cytoplasm [156]. Another major anti-inflammatory effect of Zn is by reducing infiltration of inflammatory cells, including neutrophils [157]. This is discussed further in a later section, where we describe effects of Zn supplementation and depletion on airway eosinophilia in allergic mice. Other actions of Zn relate to its effects on cell growth and wound healing. Zn is a growth co-factor for cells, being important for RNA and DNA synthesis, while at the same time suppressing endonucleases and caspases involved in cell death [reviewed in 20]. Zn is particularly important for epithelial tissues which exhibit rapid cell turnover and this can be seen from the diverse epidermal and gastrointestinal epithelial mucosal lesions that occur in Zn deficiency [16, 17, 158]. Effects on airway epithelium have yet to be properly studied, although our group has begun to address this in a murine model of allergic airway inflammation (see later). The possible role of Zn in fibroblast proliferation and collagen synthesis is interesting. Zn is likely to be beneficial for normal epithelial repair, promoting fibroblast growth and extracellular matrix production. In Zn deficiency, fibroblast proliferation and collagen synthesis are impaired and wound healing is delayed [159]. Zn is required for events during early-mid G1 [160] and acts synergistically with epidermal growth factor in promoting re-epithelialization and enhancing deposition of collagen [161]. However, these same processes contribute to airway remodeling in inflamed airways subjected to repeated episodes of damage and repair and, in these circumstances, Zn may be detrimental. The role of Zn in AE repair and airway remodeling needs further investigation.

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Finally, in view of evidence that most secretory tissues use Zn to package secretory products in granules destined for exocytosis, we might predict a role for Zn in mucin secretion by AE and secretory glands. While mucins are known to bind Zn in vitro [162], there is as yet no evidence that mucins contain Zn. Since extracellular Zn negatively regulates the secretory process in a number of secretory cell types [9, 39], it will be important to determine whether AE secretions contain Zn and whether levels of Zn in ELF can influence mucin secretion. ZINC AND AIRWAY INFLAMMATION Asthma Asthma is a disease characterized by recurrent episodes of wheezing, breathlessness and chest tightness due to narrowing and obstruction of the airways [reviewed in 163]. It affects an increasing proportion of our society, including 15% of children and 12% of adults in Australia and accounts for 3% of the disease burden in this country [164, 165]. The binding of complexes of IgE with allergen to the surface of lung mast cells causing degranulation and release of histamine and other mediators initially trigger asthmatic episodes. These mediators trigger an immediate bronchoconstriction [166]. Later, there is a chronic infiltration of airways by inflammatory cells, especially eosinophils, but sometimes neutrophils [167] and consequent damage to the epithelium, associated with worsening of clinical symptoms [96, 168]. Tissue injury in inflamed airways leads to epithelial desquamation and shedding [169]. It is also accompanied by mucous hyperplasia with consequent over-production and release of mucins into the lumen [92]. Eventually, the airways become remodeled with smooth muscle hyperplasia and extensive collagen deposition that further limit airflow [170]. Although the usual treatment for persistent asthma involves a combination of bronchodilator and inhaled corticosteroid, there is a need to develop strategies to better control other aspects of the disease, including minimizing the ongoing damage to AE and subsequent repair and remodeling. Asthma and Zn deficiency Loss of Apical Zn in a Murine Model of Allergic Airway Inflammation Our interest in the question of whether asthmatics are Zn deficient arose out of our studies using Zinquin to determine the distribution and levels of Zn in AE, before and after induction of allergic airway inflammation in mice [19]. We used a well-established murine airway inflammation model in which Balb/c mice were sensitized to OVA by intraperitoneal injections and then exposed to an aerosol of 1% OVA [171]. In this model, there is airway hyper-responsiveness, eosinophilia (as manifest by high eosinophil counts in blood, BALF and around bronchioles), mucosal cell hyperplasia and increased levels of IL-4, -5 and -12. Evidence of remodeling in some OVA-treated animals includes thickening of the epithelial basement membrane region and smooth muscle alterations [171]. Zinquin staining of lung cryosections from control mice (receiving saline instead of OVA) resulted in a normal pattern of intense, TPEN-sensitive Zinquin fluorescence in the apical cytoplasm of AE. By contrast, AE from OVA-treated

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mice was largely depleted of stainable Zn [19]. The reduction in available Zn amounted to 2-3 fold (p