the biology of zinc transport in mammary epithelial cells: implications for mammary gland...

The Biology of Zinc Transport in Mammary Epithelial Cells:Implications for Mammary Gland Development,Lactation, and Involution

Nicholas H. McCormick & Stephen R. Hennigar &

Kirill Kiselyov & Shannon L. Kelleher

Received: 19 August 2013 /Accepted: 4 December 2013# Springer Science+Business Media New York 2013

Abstract Zinc plays a critical role in a vast array of cellularfunctions including gene transcription, protein translation, cellproliferation, differentiation, bioenergetics, and programmedcell death. The mammary gland depends upon tight coordina-tion of these processes during development and reproductionfor optimal expansion, differentiation, and involution. Forexample, zinc is required for activation of matrix metallopro-teinases, intracellular signaling cascades such as MAPK andPKC, and the activation of both mitochondrial-mediated apo-ptosis and lysosomal-mediated cell death. In addition to func-tional needs, during lactation the mammary gland must balanceproviding optimal zinc for cellular requirements with the needto secrete a substantial amount of zinc into milk to meet therequirements of the developing neonate. Finally, the mammarygland exhibits the most profound example of programmed celldeath, which is driven by both apoptotic and lysosomal-mediated cell death. Two families of zinc-specific transportersregulate zinc delivery for these diverse functions. Members ofthe ZIP family of zinc transporters (ZIP1–14) import zinc into

the cytoplasm from outside the cell or from subcellular organ-elles, while members of the ZnT family (ZnT1–10) export zincfrom the cytoplasm. Recently, the ion channel transient recep-tor potential mucolipin 1 (TRPML1) has also been implicatedin zinc transport. Herein, we review our current understandingof the molecular mechanisms through which mammary epithe-lial cells utilize zinc with a focus on the transport of zinc intodiscrete subcellular organelles for specific cellular functionsduringmammary gland development, lactation, and involution.

Keywords Zinc . Zinc transporter . TRPML1 .Mammarydevelopment . Lactation . Involution

AbbreviationsTRPML1 Transient receptor potential mucolipin 1

Introduction

To transition into a secreting organ capable of producing milkof optimal quality and quantity, the mammary gland mustundergo several critical stages of growth and developmentduring discrete periods of time (reviewed in [1]). Duringpubertal development, the mammary gland experiences ex-tensive ductal growth and elongation into the mammary fatpad, which is driven primarily by growth hormone, estrogen,epidermal growth factor (EGF), and insulin like growth factor-1 (IGF-1). In addition, a modest amount of secondary andtertiary branching occurs with the onset of each estrous cycle,which is driven largely by progesterone. The terminal endbuds (TEB) located at the distal end of the ducts, migratethrough the mammary fat pad until encountering the edge,serving as the mechanical impedance required to terminateductal elongation. During pregnancy, secondary and tertiarybranching is augmented and leads to the formation of

N. H. McCormick : S. R. Hennigar : S. L. KelleherDepartment of Nutritional Sciences, The Pennsylvania StateUniversity, University Park, PA, USA

K. KiselyovDepartment of Biological Sciences, University of Pittsburgh,Pittsburgh, PA, USA

S. L. KelleherDepartment of Surgery, Penn State Hershey College of Medicine,Hershey, PA, USA

S. L. KelleherDepartment of Cell and Molecular Physiology, Penn State HersheyCollege of Medicine, Hershey, PA, USA

S. L. Kelleher (*)222 Chandlee Laboratory, University Park, PA 16802, USAe-mail: [email protected]

J Mammary Gland Biol NeoplasiaDOI 10.1007/s10911-013-9314-4

supernumerary ductal branching. Additionally, the formationof lobular-alveolar units, driven primarily by progesterone andthe lactogenic hormone prolactin, create a tree-like ductalstructure capable of milk synthesis and secretion. Duringlactation, the withdrawal of progesterone at parturition com-bined with high levels of circulating and episodically secretedprolactin stimulates the secretory activation of the mammarygland. This process involves a vast expansion of the luminalspace as well as the synthesis and secretion of copiousamounts of milk containing numerous proteins and othernutritive and non-nutritive factors. Finally, following the ces-sation of lactation the mammary gland regresses in a highlyregulated and step-wise fashion. First, the milk-producingepithelial cells are removed by programmed cell death mech-anisms, resulting in the collapse of functional alveolar struc-tures. Second, the extracellular matrix is broken down andvasculature is remodeled, cell debris and milk components arephagocytosed, and the stroma is repopulated with differenti-ated adipocytes. As noted above, it has long been recognizedthat numerous hormones and cytokines are critical for thephenotypic transition of the mammary gland, primarilythrough regulating biological processes such as gene tran-scription and key signaling pathways. What is much lessunderstood are factors that modulate these biological process-es and are responsible for mammary gland expansion, differ-entiation, secretion and regression.

Zinc is the second most abundant trace element in thehuman body [2] and the most abundant trace element in cells.As a micronutrient, zinc plays a catalytic, structural or regula-tory role in a vast array of biological processes. At the cellularlevel, zinc is required by over 10 % of the eukaryotic proteomeand plays critical roles in numerous processes including DNAsynthesis and repair, transcription, intracellular signaling, pro-tein translation, cell proliferation, differentiation, apoptosis,autophagy, and motility [3–8]. These processes are tightlyregulated by zinc delivery to critical regulatory factors facili-tated by an extensive network of zincmanagement proteins thatinclude zinc transporting proteins, ion channels, and zinc bind-ing proteins (Fig. 1). Similar to calcium, zinc is taken up by thecell and transferred between subcellular compartments in re-sponse to regulatory cues, including hormones and cytokines.Zinc is transported into the cell and between these subcellularcompartments by members of two different families of zinctransporters (ZIP and ZnT proteins) and ion channels. The ZIPfamily of zinc transporters (members of the SLC39A genefamily; ZIP1–14) import zinc into the cytoplasm, either fromthe extracellular milieu or fromwithin an intracellular compart-ment. In contrast, the ZnT family of zinc transporters (membersof the SLC30A gene family; ZnT1–10) export zinc from thecytoplasm. Little is known about the driving force responsiblefor regulating zinc movement through zinc transporters, how-ever zinc binds directly to histidine clusters in multiple zinctransporters, suggesting a possible mechanism for transfer ([9]

and reviewed in [10]). ZIP1 and ZIP2 have been shown tofunction in an energy independent manner [11, 12] and ZIP2transport is stimulated by the presence of bicarbonate ions,suggesting a Zn2+/[HCO3

−]2 symport mechanism [11]. ZnT5,which imports zinc into the Golgi apparatus, is catalyzed by aZn2+/H+ exchange [13], implicating a proton gradient as adriving force behind ZnT-mediated zinc import into vesicles.More recently, ion channels (members of the transient receptorpotential (TRP) superfamily), were shown to transport zinc(reviewed in [14]). Although many of these ion channelsimport zinc across the cell membrane, some are localized tointracellular membranes in the endosomal pathway, includingsecretory vesicles (e.g. TRPM7), recycling endosomes (e.g.TRPMV6), and late endosomes and lysosomes (TRPML1)(reviewed in [15]) and may function to import zinc from theseorganelles into the cytoplasm [14]. The impact of TRP chan-nels on zinc transport, as well as mechanisms of zinc perme-ation through TRP channels in the presence of zinc-bindingproteins are not well understood [14]. Finally, metallothioneins(MT) are zinc binding proteins that have a high affinity for zincand buffer cytoplasmic zinc pools to protect against cytotoxic-ity ([16] and reviewed in [17, 18]). Endogenous expression andlocalization of zinc management proteins under normal phys-iological conditions varies greatly by tissue, cell type, andcell/tissue phenotype. Importantly, dysregulation in zinc man-agement has been associated with numerous diseases and con-ditions including impaired breast function [19] and breastcancer [20, 21].

Research over the past decade has uncovered several ex-citing roles for the transport of zinc mediated through specificzinc transporters in mammary gland biology, mostly centeredon understanding the regulated transfer of copious amounts ofzinc into milk [22–24]. However, the role of zinc in such adiverse array of biological processes that are central to theunique features of mammary gland biology suggests that thetransport of zinc is critical to mammary gland developmentand function in general. The regulation of mammary glandsecretion has been extensively reviewed elsewhere [25–30].This review focuses on the potential roles for zinc in mam-mary gland development, lactation, and involution, with spe-cific emphasis on the molecular mechanisms that regulate thetransport of zinc to modulate these processes.

Zinc and Mammary Gland Expansion and Development

Central to mammary gland development during puberty is thegrowth and elongation of the primary ducts through the mam-mary fat pad. The TEB, located at the distal end of the primaryducts, serves as the principal enzymatic and mechanical driv-ing force behind ductal elongation. A delicate balance be-tween proliferation, apoptosis, and differentiation of TEBcells is critical for ductal elongation.While zinc plays a critical

J Mammary Gland Biol Neoplasia

role in numerous mechanisms required for TEB function, themolecular events that regulate the transport of zinc for deliv-ery to these functions are much less understood.

Zinc Acquisition by the Proliferating Mammary EpithelialCell

Much has been learned about the role and regulation ofmammary gland zinc transport through the use of mousemodels and the mouse mammary epithelial cell line HC11[31–35]. Zinc must first enter the cell. Robust expression ofmost ZIP proteins (with the exception of ZIP2, ZIP3, ZIP6,ZIP7, ZIP9 and ZIP11) has been documented in the nullipa-rous, adult mouse mammary gland [22]. Studies in HC11 cellsindicate that while ZIP3, ZIP7, ZIP11, ZIP12, ZIP13 andZIP14 are expressed, they are actually localized to intracellu-lar compartments ([22] and Fig. 2) and thus may not play arole in basal zinc acquisition. We have detected ZIP5 at the

cell surface (Fig. 3), implicating ZIP5 in zinc uptake in pro-liferating mammary epithelial cells. Localization of the re-maining ZIP proteins (ZIP1, ZIP4, ZIP6, ZIP8 and ZIP10)in normal mammary epithelial cells has not been ascertainedand thus their contribution to zinc import into the cell remainsto be understood.

Once in the cell, very little “free” zinc exists in the cyto-plasm. Zinc is usually found bound to MT, or associated withcytoplasmic anions, proteins and amino acids [36]. Preciselyhow much zinc is “free” is widely debated, thus, quantifica-tion of cytoplasmic and organelle zinc pools should beregarded with caution. However, promising new technologyutilizes genetically encoded sensors to monitor and quantifycytoplasmic and organelle zinc pools [37, 38]. For example,Palmer and colleagues have shown that cytoplasmic [38] andorganelle zinc concentrations are cell-specific [39], reflectingthe cell-specific need for zinc-mediated functions. In addition,they found that cytoplasmic zinc concentration in HeLa cells

Fig. 1 A model for the transportof zinc for specialized functionsin mammary epithelial cells. zinctransporters and TRP ionchannels that transport zinc andtheir known (bold font) orproposed (regular font)localization in mammaryepithelial cells.M7 TRPM7;ML1TRPML1; MV6 TRPMV6; MTmetallothionein; MMP matrixmetalloproteinase; SV secretoryvesicle; EE early endosome; RErecycling endosome; LE lateendosome; LY lysosome; APautophagosome; Mitomitochondrion; GA Golgiapparatus; RER roughendoplasmic reticulum; Nnucleus; APM apical membrane;BSM basolateral membrane

Fig. 2 ZIP12, ZIP13 and ZIP14 reside in intracellular vesicles innormal mouse mammary epithelial (HC11) cells. Representativeimages of ZIP12 (green ) and the trans-Golgi apparatus markerp58 (red ); ZIP13 (green ) and the late endosome marker man-nose-6-phosphate receptor (M6PR); and ZIP14 (red ) and the

trans-Golgi apparatus marker p58 (green ). ZIP protein antibodieswere obtained from Sigma-Aldrich and used at 1 μg/mL. Anti-bodies against M6PR and p58 were obtained from Abcam andused at 1 μg/mL. Proteins were detected with Alexa Fluor®488or 568. Magnification, 40× under oil


is approximately 100 times higher than the zinc concentrationin the endoplasmic reticulum (ER) and Golgi apparatus [40].The use of zinc-specific fluorophors such as FluoZin-3 [34]and Zinpyr [41] suggest that organelle zinc pools are at leastpartially labile. Intriguingly, they have shown that HC11 cellstreated with prolactin decrease mitochondrial zinc pools [39],consistent with a role for zinc in modulating bioenergetics andor apoptosis [42] in mammary epithelial cells. Thus, zinc mustbe efficiently transported into subcellular compartments toregulate zinc-dependent functions, such as those describedbelow, and to protect against cytotoxicity.

Cell Proliferation

Mammary gland development has a multi-faceted require-ment for cell growth and proliferation, and zinc transport playsa central role in several aspects including modulating enzymeactivity involved in nucleic acid synthesis [3–5] and providingstructure to over 1,000 zinc-finger transcription factors(reviewed in [43]).

To our knowledge, no zinc transporters have been impli-cated in the delivery of zinc specifically for DNA synthesis;however, inhibition of metal transcription factor-1 (MTF-1), azinc-responsive transcription factor that regulates expressionof several zinc management proteins that protect against zinctoxicity including ZnT1 [44], ZnT2 [45], and MT [46], de-creases EGF-dependent extracellular signal-related kinase(ERK) phosphorylation, an essential reaction for DNA syn-thesis [47]. Chesters et al. demonstrated that the zinc chelatorDTPA decreases thymidine incorporation by ~90 % in 3T3mouse fibroblasts, and subsequent zinc supplementation re-versed this effect, thereby demonstrating the need for zinc inDNA synthesis for cell growth and proliferation [3]. More-over, zinc deficiency decreases the activity of two key en-zymes in this process, thymidylate synthetase and thymidylatekinase [5]. Collectively, this suggests that the ability to buffercytoplasmic zinc through MT binding and ZnT-mediated ef-flux or vesicular sequestration at critical junctures may regu-late DNA synthesis and cell proliferation.

Cell Signaling

Cytoplasmic zinc availability plays a critical role in the exe-cution of cell signaling cascades by modulating kinase andphosphatase activities [7]. For example, mitogen-activatedprotein kinases (MAPKs) are a diverse family of enzymesresponsible for mediating the cellular response to a multitudeof extracellular stimuli, most importantly, those that stimulategrowth, proliferation, and cell survival. Lefebvre et al. showedthat zinc exposure in rat fibroblasts increased MAPK activity,while conversely, zinc chelation partially decreased MAPKstimulation [6]. In accordance with this principle, zinc expo-sure stimulates MAPK signaling in murine fibroblasts [48],providing evidence that zinc is a critical modulator of MAPKsignaling. Additionally, protein kinase C (PKC) phosphory-lates (thereby activating or deactivating) various proteins in-volved in a multitude of signal transduction pathways includ-ing those involved in regulating cell growth and transcription.PKC contains two identical zinc binding motifs in the N-terminal, regulatory region of the enzyme [49]. Nanomolarconcentrations of zinc stimulate PKC translocation to the cellmembrane [50], implicating ZnT/ZIP-mediated cytoplasmiczinc influx and efflux in modulating cell signaling.

To activate/deactivate proliferative cellular signaling path-ways such as MAPK and PKC, the cytoplasmic zinc poolmust be modulated. Hirano et al. have implicated zinc as apotential signaling ion due to three specific factors: 1) thecreation of a “zinc wave”, or the increase in intracellular zincconcentration, which occurs in response to an external stimuli;2) the source of the “zinc wave” is an intracellular compart-ment; and 3) intracellular zinc at concentrations similar to thatcreated by the “zinc wave” affects intracellular signalingmolecules, and therefore may induce a response originallystimulated by an extracellular prompt [7]. The ER and Golgiapparatus are likely two key zinc storage pools that aremobilizable in nature [40]. We have previously shown thatthe Golgi apparatus serves as a mobilizable zinc pool in themammary epithelial cell [51]. This suggests that subcellularzinc influx/efflux, and therefore zinc transporters as molecularmediators, are important components of cell signaling regula-tion. Recently, modulation of zinc pools in the ER and/orGolgi apparatus has been shown to play critical roles insignaling pathways specifically vital for cell proliferation[52, 53]. Several zinc transporters are localized to the ER/Golgi apparatus in mammary epithelial cells including ZIP6,ZIP7 and ZnT4, ZnT5, ZnT6, and ZnT7. A recent report byLue and colleagues reported that ZIP6, which exports zincfrom the ER, is required to drive EGF signaling in prostatecells [54]. Furthermore, MAPK signaling activated by EGFinduces expression of the zinc -dependent matrix metallopro-teinases (MMP) MMP-1, −2 and −9 in human mammary cells[55]. Collectively, this links the efflux of zinc from the ER,cell signaling and a cellular process critical for ductal

Fig. 3 ZIP5 resides at the cell surface in normal mouse mammaryepithelial cells. HC11 cells were treated with biotin (+) and biotinylatedcell surface proteins were captured with avidin-labeled agarose beads,precipitated and separated on an SDS-PAGE gel. Cells not treated withbiotin (−) were used as a negative control; a sample of a total membranefraction (TM) was used as a positive control. Proteins were transferred tonitrocellulose membranes and immunoblotted for ZIP5. ZIP5 antibodywas obtained from Aviva and used at 1 μg/mL. Immunoblots weredetected with SuperSignal West Femto Chemiluminescent Substrateand exposed to autoradiography film


elongation (discussed below). These functional connectionsremain to be directly interrogated in the mammary gland butprovide important precedence. Additionally, ZIP7 mediateszinc release from the Golgi apparatus and regulates both cellgrowth and differentiation pathways involving EGFR, HER2,IGF1R and Src signaling, in which cytosolic zinc is proposedto inhibit the activity of phosphatases, promoting tyrosinekinase activity [53, 56, 57]. Specifically, recent work byTaylor et al. found that phosphorylation of ZIP7, which islocalized to the trans -Golgi apparatus in mammary epithelialcells [52], is associated with the gated release of zinc fromintracellular stores. Interestingly, elimination of cytoplasmiczinc through ZnT1 over-expression [58] or ZIP9 silencing[59] also mediates the activation of ERK signaling in othercell types, suggesting that zinc may control cell signalingevents in a biphasic manner. In contrast, PKC activation isassociated with the transport of zinc into the Golgi apparatus.ZnT5-null mice have defects in the ability of PKC to translo-cate to the cell membrane in mast cells. Because ZnT5 islocalized to the Golgi apparatus this suggests that the transportof zinc into the Golgi apparatus via ZnT5 is critical for PKCactivation [60]. But again, these functional connections re-main to be interrogated in the mammary gland. Clearly, tightregulation of zinc transport within the mammary epithelial cellis likely critical to maintaining the delicate balance of zinc-dependent intracellular signaling pathways that promote thegrowth and proliferation of ductal structures.

Apoptosis

The regulation of apoptosis in mammary epithelial cells dur-ing the process of ductal elongation is critical for optimaldevelopment because proper lumen formation requires apo-ptosis for clearance of cells in the acini center [61]. Mitochon-dria play a fundamental role in the intrinsic (mitochondrial)-mediated apoptotic pathway through the release of apoptosis-inducing factors such as cytochrome c . Regulation of mito-chondrial zinc pools are critical for several cellular processesincluding bioenergetics and apoptosis (reviewed in [62]).Thus far, two zinc transporters have been associated with thetransport of zinc in mitochondria. To our knowledge, ZnT2 isthe only zinc transporter that has been directly implicated inmitochondrial zinc import. Moreover, Seo et al. demonstratedthat ZnT2-overexpression in the mitochondria of mammaryepithelial cells increases apoptosis and decreases the produc-tion of ATP [42], suggesting mitochondrial zinc managementis critical for mammary gland expansion and regression. En-dogenous ZnT2 expression inmammary glands of nulliparousfemale mice [22] and proliferating mammary epithelial cells[63] also reinforces the notion that ZnT2 serves a criticalpurpose in mammary gland during times of growth anddevelopment.

ZIP8 translocates to mitochondria in response to the cyto-kine tumor necrosis factor-alpha (TNFα) in lung epithelialcells [64]. TNFα-induced ZIP8 expression and translocationincreases mitochondrial zinc content. Because ZIP8 attenua-tion reduces mitochondrial zinc content and impairs mito-chondrial function it has been suggested that ZIP8 plays a rolein mitochondrial zinc transport. However, a direct effect ofZIP8 on mitochondrial zinc import has not been delineated. Infact, based on predicted topological orientation, ZIP8 likelyimports zinc out of mitochondria. Because ZIP8 is also foundon the cell membrane [64], perhaps increased cytoplasmiczinc levels result in indirect zinc accumulation in mitochon-dria. Due to the network-driven design of zinc transport ob-served in other organelles, additional zinc transporters local-ized to the mitochondria (and their role in the mediation ofapoptosis) likely remain to be identified. Overall, the transportof zinc into mitochondria provides an important, if not drivingcontribution to apoptosis, which is critical for optimal ductalelongation and mammary gland development.

Invasion

MMPs are secreted zinc-dependent proteins whose cleavagesubstrates include many mammary gland extracellular matrix(ECM) proteins such as collagen, fibronectin, and E-cadherin,and whose activity renders the release of numerous bioactivecomponents including IGF and TGF-ß [65]. Specific MMPsare metallated in the Golgi apparatus, while others aremetallated extracellularly. Therefore, zinc transport into theGolgi apparatus and across the cell membrane is required forMMP activation and function. Consistent with a key role ofMMPs in mammary gland development, MMP-2-null andMMP-3-null mice have substantial defects in mammary glanddevelopment including decreased ductal invasion (MMP-2)and lateral branching (MMP-3) [66]. Prior to the zinc-dependent activation of MMPs, many pro-MMPs must becleaved intracellularly in order to expose the zinc-specificactive site. Furin, a proprotein convertase, is responsible forthe cleavage of many pro-MMP proteins in the trans -Golgiapparatus including pro-MT-MMP-3 [67] and pro-MMP-2[68]. Furin activity is greatly inhibited by the presence of zinc[69], therefore ZIP and ZnT proteins localized to the trans -Golgi apparatus must play a role in modulating the activity offurin and its ability to cleave pro-MMP proteins. Candidatesinclude ZnT4, transporting zinc into the trans -Golgi appara-tus [19], and ZIP7 [70], transporting zinc out of the trans -Golgi apparatus into the cytoplasm. MMP activation is a step-wise process that concludes with extracellular binding of zincto a newly exposed MMP active site. Given the localizedenvironment from which zinc is acquired for MMP binding,it is reasonable to believe that this zinc may be provided byzinc transporters on the cell surface of the mammary epithelialcells from which it was secreted. Candidates include ZnT1,


ZnT2, and ZnT4, all of which have been localized to the cellsurface of mammary epithelial cells [22, 34]. In contrast,inactivation of MMPs is critical to balancing an appropriateamount of ductal expansion without creating an environmentof excess proliferation. Tissue inhibitors of metalloproteinases(TIMPs) are zinc-dependent enzymes that serve to inhibit theactivity of the aforementioned MMPs, and furthermore, arecarefully regulated during mammary gland development. Toour knowledge there is no direct evidence that the transport ofzinc directly affects TIMP activity; however, it is reasonable toassume that future research may reveal a role for zinc trans-porters in mediating TIMP activity.

Zinc and Mammary Gland Function During Lactation

To produce milk during lactation, the mammary gland un-dergoes several critical physiological changes induced first byprogesterone withdrawal followed by increased prolactin andcortisol secretion at the time of parturition. Continued activa-tion of prolactin secretion is required to maintain the secretoryepithelium. Zinc serves a critical role in several facets regu-lating the development and maintenance of a secretory organincluding cell differentiation, maintenance of a secretory phe-notype, protein synthesis, and possibly in the process ofsecretion. Studies have shown that the mammary gland accu-mulates and transports a tremendous amount of zinc, particu-larly into the Golgi apparatus and secretory system of themammary epithelial cell during lactation [51] for two signif-icant reasons: 1) to provide zinc for the numerous zinc-dependent proteins critical to lactation and mammary glandfunction; and 2) for possible secretion into milk to meet thenutritional demands of the nursing infant.

Zinc Acquisition by the Secreting Mammary Epithelial Cell

Multiple zinc transporters play central roles in zinc transportinto the secreting mammary epithelial cell, into/from variousorganelles for specific cell functions, and for secretion intobreast milk. In circulation, zinc is found associated withmultiple proteins including albumin and α2-macroglobulin[2] and serum zinc levels return to pre-pregnant levels duringlactation. Zinc uptake from maternal circulation into the se-creting mammary epithelial cell is not understood; however,based on localization and expression data in lactating mice,several zinc transporters serve as likely candidates for this role[22, 72]. Studies indicate that ZIP5, ZIP8 and ZIP10 arelocalized to the basolateral membrane of the mammary epi-thelial cell and expression is significantly higher in lactatingthan in non-lactating mice. This suggests that they may playimportant roles in zinc transport frommaternal circulation intomammary epithelial cells [22].

Differentiation

Mammary epithelial cell differentiation is primarily stimulatedby prolactin, which in turn initiates intracellular signalingcascades that promote the synthesis of proteins necessary fordifferentiation. Prolactin-stimulated Jak2 (Janus-kinase2)/STAT5 (Signal Transducer and Activator of Transcription-5)signaling is the primary driver of secretory differentiation,lactogenesis, and galactopoiesis in mammary epithelial cells.As discussed above, the regulation of zinc transport into/fromthe cytoplasm is critical for proper intracellular signaling,including Jak2/STAT5, and zinc is likely released from intra-cellular organelles such as the Golgi apparatus or ER in aregulated manner. Based on expression and subcellular local-ization data generated in mouse models, numerous zinc trans-porters could play a role in modulating zinc supply for(de)phosphatase activity. For example, expression of ZIP7increases dramatically during lactation, suggesting that themobilization of zinc pools from the Golgi apparatus is impor-tant to cellular function. Similarly, uptake of zinc into theGolgi apparatus occurs through ZnT4 and ZnT5. Previously,we have shown that ZnT4 transports zinc out of the cytoplasmand into the trans -Golgi apparatus [34] and overexpression ofzinc induces the phosphorylation of Jak2 (NHM, unpublishedobservations). Alternatively, modulation of vesicular zincpools through ZnT2, ZIP3, and ZIP11 or zinc uptake throughZIP5, ZIP8, and ZIP10 may also play important roles inregulating Jak2/STAT5 signaling as well, due to their cellsurface localization in the mammary epithelial cell [22]. Fur-ther studies are required to understand the role zinc transportplays in regulating cell signaling in differentiated, secretorycells.

Protein Synthesis/Modification

We and others have shown that zinc is sequestered in the ERand Golgi apparatus [34, 40, 51]. Recent reports indicate thatprecise control of zinc transport into/from the ER and Golgiapparatus is key to both the synthesis of secreted proteins andthe enzymatic machinery required for their production. ZnT4and ZnT5 are localized to the Golgi apparatus of mammaryepithelial cells and are expressed in greater abundance duringlactation. It is plausible that increased abundance of these zinctransporters is important in mediating zinc availability in theGolgi apparatus for general protein synthesis and modifica-tion. One example is galactosyltransferase, which is a zinc-dependent protein that resides in the Golgi apparatus. As partof the lactose synthase enzyme complex, galactosyltransferaseplays a critical role in lactose synthesis. The production oflactose drives water movement into milk and thusgalactosyltransferase activity indirectly contributes to milkvolume. Moreover, secreted proteins are produced in theGolgi apparatus and many of them are zinc-dependent


proteins. We have previously shown that zinc transport intothe trans -Golgi apparatus of mammary epithelial cellsthrough ZnT4 is necessary for the stability of carbonicanhydrase VI (CA VI) protein, a zinc-dependent secretedenzyme [34]. CAVI binds zinc at its active site during syn-thesis in the Golgi apparatus, which is critical for the structuralintegrity of the protein itself [73]. CAVI is a component ofbreast milk and implicated in the support of infant alimentarytract development [74–76], indirectly implicating zinc avail-ability in the Golgi apparatus of the mammary epithelial cell ingastrointestinal development in the newborn.

Additionally, the export of zinc out of the ER and Golgiapparatus is critical to mammary cell function. Expression ofZIP7, which exports zinc from the Golgi apparatus in mamma-ry epithelial cells, is also more highly expressed during lacta-tion. A recent report in Drosophila noted that the loss of Catsup(ZIP7) causes ER stress [77], indicating that the inability toremove zinc from the ER increases the level of mis-foldedproteins within the secretory compartment, which could havemajor implications in the lactating mammary gland.

Zinc Secretion into Milk

In milk, zinc is found loosely bound to citrate [78], serumalbumin [79], and associated with caseins [80]. Zinc transportinto breast milk is not well understood but is likely accom-plished by multiple zinc transporters localized to the Golgiapparatus, secretory vesicles and directly on the mammaryepithelial cell apical membrane. To date, both ZnT2 and ZnT4have been implicated in zinc secretion into milk [23, 24].However, the relative contributions of ZnT2- and ZnT4-mediated zinc transport remain to be defined. Two distincthuman ZnT2 isoforms have been reported to reside in twodifferent intracellular locations within the mammary epithelialcell [63]. A 42 kDa ZnT2 isoform localizes to the endosomal/secretory compartment, while a slightly smaller 35 kDa iso-form, resulting from the splicing of exon 2 [63] localizes to thecell membrane. Both of these isoforms have proven functionalin the context of zinc secretion. Overexpression of the 42 kDaisoform in cultured mammary epithelial cells increases zincvesicularization, and overexpression of both isoformsincreases zinc export out of the cell [63]. Endogenous expres-sion of the vesicularized isoform is higher in secreting mam-mary epithelial cells compared with non-secreting cells,further strengthening the hypothesis that ZnT2-medaited ve-sicular zinc secretion serves a significant role in the mammaryepithelium during lactation. Four different mutations in thegene encoding human ZnT2 (SLC30A2 ) have been identifiedin breastfeeding women, all of which reduce milk zinc con-centration by ~75 % [24, 35, 81] and results in a disorderreferred to as “transient neonatal zinc deficiency” [24]. Thefirst mutation identified substitutes arginine with histidine atamino acid 54, which results in the mislocalization and

degradation of ZnT2 [24]. Subsequently, a mutation that sub-stitutes an arginine with histidine at amino acid 87 was iden-tified that results in the retention of ZnT2 in the ER/Golgiapparatus and decreases zinc secretion [35]. Most recently awoman with compound heterozygous mutations substitutingtryptophan with arginine at amino acid 152 and serine withleucine at amino acid 296 was identified [81]. W152R abol-ishes the ability of ZnT2 to dimerize, while S296L reducesprotein stability. This provides compelling evidence that ZnT2is vital to the process of vesicular zinc secretion into milk.However, it is possible that the defect is not confined toreduced milk zinc levels and that ZnT2 may play a larger rolein mammary gland biology as previously discussed.

ZnT4 is localized to the trans -Golgi apparatus [34], sug-gesting that ZnT4 plays a vital role in zinc-dependent process-es in the Golgi apparatus. As described previously, the trans-port of zinc into the ER/Golgi apparatus may have importantimplications with respect to the regulation of cell signaling,the maturation and function of enzymes that reside in theGolgi apparatus, and perhaps the control of endoplasmicreticulum/Golgi apparatus stress. In addition, we found thatZnT4 accumulates in lysosome-like vesicles and at the cellmembrane in response to zinc, suggesting that Golgi-derivedvesicles and perhaps ZnT4 directly, exports zinc into milk[34]. Mice expressing a spontaneous mutation in the gene thatencodes ZnT4 renders the ZnT4 protein non-functional. Con-sistent with a role for ZnT4 in milk zinc secretion, milk fromZnT4-null mice contain ~35 % less zinc than their wild-typelittermates [82]. It should be noted that defects in ZnT4 havenot yet been associated with low milk zinc levels in humans[83]. While a specific role for ZnT4 in zinc secretion into milkhas been postulated [23], ZnT4 likely serves other criticalroles within the mammary gland due to other mammary glanddefects in ZnT4-null mice including decreased mammarygland weight and milk secretion. However, the precise mech-anism(s) through which ZnT4 participates in the functionalintegrity of the mammary gland remains to be determined.

Interestingly, numerous ZIP proteins are localized to theapical cell membrane of the mammary epithelial cell, suggest-ing that zinc is also taken up from the secreted milk pool. Forexample, ZIP3 is localized to the apical membrane of thelactating mouse mammary gland and milk zinc concentrationfrom ZIP3-null mice is significantly higher than in wild-typemice, indicating that ZIP3 plays a fundamental role in the re-uptake of zinc from the previously secreted milk pool [72].While the biological relevance of zinc re-uptake is not under-stood, ZIP3-null mice have increased apoptosis suggesting aspecific role for the transport of zinc via ZIP3. Intriguingly,prolactin stimulates the re-localization of ZIP3 to the cellmembrane of mammary epithelial cells, providing furtherevidence that ZIP3-mediated zinc transport plays an importantrole during lactation and may assist in constraining apoptoticcell death [32]. However, the mechanisms and regulatory


factors responsible for zinc acquisition by the mammary epi-thelium remains largely unknown.

Zinc and Post-Lactational Involution

Mammary gland involution is a highly complex process thatoccurs in two phases. The first phase is characterized bymassive programmed cell death, with ~80 % of the milk-producing mammary epithelial cells [84] removed in a matterof days. This phase can be further divided into an early-waveof lysosomal-mediated cell death (which occurs <24 h post-weaning) [85, 86], followed by multiple waves ofmitochondrial-mediated apoptosis (which occurs >48 h post-weaning) [87–89]. Prior to 48 h, involution is reversible andlactation can resume in response to suckling [88, 89]. Asecond wave of apoptosis follows and initiates irreversibletissue remodeling with protease-mediated degradation of thebasement membrane and redifferentiation of the adipocytes[88]. Many reports show that zinc is a potent regulator of bothlysosomal- and mitochondrial-mediated cell death (reviewedby [90, 91]) and therefore zinc transport into these subcellularcompartments is critical to understanding the biology of mam-mary gland involution.

Lysosomal-mediated Cell Death

In contrast to apoptotic cell death, where pro-apoptotic factorsare released in response to mitochondrial outer membranepermeabilization [92], lysosomal-mediated cell death occursin response to a stimulus, which induces lysosomal membranepermeabilization (LMP) and lysosomal contents are releasedinto the cytosol to act as executioner proteases [93]. Recently,zinc accumulation in lysosomes was shown to cause LMP andinduce cell death in neurons [91, 94, 95], retinal pigmentepithelial and photoreceptor cells [96], and breast cancer cells[97]. Interestingly, unpublished data from our laboratory showsthat zinc accumulates in lysosomes isolated from involutingmouse mammary gland, suggesting an important role for zinctransport into lysosomes in post-lactational regression.

Information on lysosomal zinc transport is scarce. ZnT2localizes to lysosomes in M1 fibroblasts [98] and possibly inprostate [99] and kidney [100]. Although ZnT2 is not found inlysosomes in the mammary glands of nulliparous or lactatingmice or mammary epithelial cells, when we fractionate sub-cellular organelles from involuting mammary glands by den-sity gradient fractionation we find increased activity of thezinc-dependent lysosomal enzyme acid phosphatase in frac-tions that also contain ZnT2 (Fig. 4). In addition to ZnT2,ZnT4 may play a key role in lysosomal zinc accumulationunder certain circumstances. ZnT4 is detected in lysosomes ofHeLa cells [102]. Although ZnT4 is not detected in lysosomesof mammary epithelial cells under normal conditions [51],

zinc exposure stimulates ZnT4 accumulation in lysosome-like vesicles and at the cell membrane [34], suggesting thatZnT4 may participate in lysosome-mediated zinc accumulation.The mechanisms through which ZnT4 is redistributed to lyso-somes are not understood. However, similar to ZnT2 anddiscussed below, ZnT4 contains a dileucine motifs in the N-and C-termini that may play a role in trafficking ZnT4 tolysosomes under certain conditions.

The role of zinc in LMP and lysosomal-mediated cell deathis unclear. One possibility is that zinc is required to activatezinc-dependent lysosomal enzymes involved in LMP andlysosomal-mediated cell death. Lysosomes contain dozens ofhydrolytic enzymes such as proteases, lipases, nucleases, gly-cosidases, phosphatases, and sulfatases that reach maximalactivity at a low pH (pH<5). Many of these enzymes arezinc-dependent and are highly upregulated during involution,including cathepsins [103, 104] and acid phosphatase [105,106]. In addition, acid sphingomyelinase is a zinc-dependentphosphodiesterase that hydrolyzes sphingomyelin, a majormembrane phospholipid, to produce phosphocholine and cer-amide. Ceramide is further processed into sphingosine, whichis proposed to accumulate in lysosomes and permeabilizemembranes. This suggests that ZnT2 may import zinc intolysosomes to activate zinc-dependent lysosomal hydrolases(e.g. acid phosphatase, acid sphingomyelinase) and thereforemodulate LMP and lysosomal-mediated cell death duringinvolution. Another explanation for lysosomal zinc accumu-lation is that it may play a role as a biologically active zincsink that is released back into the cytoplasm to activate thesecond wave of apoptotic cell death [102]. This would requirethe presence of ZIP proteins or ion channels on the lysosomalmembrane to export zinc back into the cytoplasm in responseto intra- or extracellular cues. For example, ZIP8 relocalizes to

3 4 51 20.000

0.001

0.005

0.010

0.015

Density gradient fraction

Aci

d ph

osph

atas

e ac

tivity

(Uni

ts/m

L)/fr

actio

n

IB: ZnT2

Fig. 4 ZnT2 is redistributed to lysosomes during involution. Mammaryglands from lactating and 24 h involuting mice were homogenized on icein homogenization buffer, centrifuged at 5,000×g for 15min, and densitygradient fractionation was performed as previously described [101].Fractions were collected, pooled, and either assayed for acid phosphataseactivity or ZnT2 by immunoblotting. Acid phosphatase activity wasmeasured using a commercially available kit from Sigma-Aldrich(CS0740). Acid phosphatase activity was only detected in involutingmammary glands. Data represent mean activity (U/mL)/fraction ± SD.For detection of ZnT2, pooled fractions were pelleted by ultracentrifuga-tion at 150,000×g for 30 min prior to immunoblotting for ZnT2


lysosomes in response to T-cell receptor activation [107].Although ZIP8 is not localized to lysosomes in mammaryepithelial cells, extra- or intracellular signals may relocalizeZIP8 to lysosomes to modulate zinc export. In addition,lyso/endosomal ion channel TRPML1 conducts zinc [108]and has been shown to regulate the efflux of zinc fromlysosomes [102, 109]. Interestingly, TRPML1 co-localizeswith ZnT2 and ZnT4 in HeLa cells [102], suggesting ZnT2and/or ZnT4 may work in a coordinated manner withTRPML1 to modulate lysosomal zinc pools in mammaryepithelial cells. Intriguingly, TRPML1 levels are below detec-tion levels in virgin and lactating mammary glands and HC11cells, but is highly expressed in the involuting mammarygland (Fig. 5), implicating TRPML1-mediated lysosomal zincefflux in the process of mammary gland remodeling.

Another key question that remains is, what factors regulatethe re-localization of zinc transporters to lysosomes, thusplaying key roles in modulating cell function? As noted pre-viously, ZnT2 [63], ZnT4 [51], and ZIP8 [22] are not found inlysosomes in mammary epithelial cells [63] nor does zincaccumulate in lysosomes in mammary epithelial cells underbasal conditions or in response to lactogenic stimuli [51].Lysosomal proteins contain specific sorting signals such asdileucine-based motifs in the cytoplasmic domains, whichinteract with components of clathrin coats or adaptor proteincomplexes. Intriguingly, both ZnT2 and ZnT4 containdileucine motifs in their N- and C-termini.We have previouslyshown that the lactogenic hormone prolactin regulates thesubcellular localization and function of ZIP3 [32] and ZnT2[63]. Therefore, it is reasonable that factors upregulated duringweaning may post-translationally modify ZnT2 (and perhapsZnT4) to uncover a lysosomal targeting motif, thereby medi-ating the localization and function of ZnT2 and/or ZnT4during involution. Milk stasis and the accumulation of localstimuli produced by the mammary gland are responsible forthe first phase of involution [89]. Therefore, we speculate thatin the absence of suckling, these local stimuli such as LIF,TGF-β, or TNFα may modulate signaling pathways thatuncover lysosomal targeting motifs in ZnT2 (and perhapsZnT4) to target them to lysosomes. In this model, ZnT2-mediated lysosomal zinc accumulation would lead to LMPand initiate the early wave of lysosomal-mediated cell death ofthe mammary epithelial cells. Understanding how zinc

transporters are post-translationally regulated is an area cur-rently receiving attention in the field.

Apoptosis

Within 24–48 h post-weaning, mammary epithelial cells un-dergo a second wave of programmed cell death driven byapoptosis. In contrast to cell death during early involutionwhich is independent of executioner caspases [85], the intrin-sic mitochondrial apoptotic pathway is activated by initiatorcaspases that cleave the pro-apoptotic Bcl-2 family member,Bid, into its truncated form (tBid). tBid is translocated to themitochondria and initiates mitochondrial-mediated apoptosisthrough the release of cytochrome c and other factors thatactivate effector caspases. Although zinc is recognized as apotent regulator of mitochondrial-mediated cell death [90],studies to date have not examinedmitochondrial zinc transportduring involution. As discussed above, ZnT2-mediated ex-pansion ofmitochondrial zinc pools decreases ATP biogenesisand induces apoptosis in mammary epithelial cells [42].Therefore a key question in understanding programmed celldeath mechanisms during mammary gland involution iswhether involuting mammary glands accumulate zinc in mi-tochondria? If this is the case, we predict ZnT2 (and likelyother zinc transporters) is upregulated to expand mitochondri-al zinc pools and activate apoptosis as discussed previously.

The specific actions of zinc in apoptosis are less clear dueto the complex, diverse, and cell-specific nature of its effects.In general, it seems that intracellular zinc concentrationsabove and below a likely predetermined and cell-specificthreshold induce apoptosis. Apoptosis is inversely correlatedto the level of labile intracellular zinc [110]. Endogenous zincinhibits the activity of caspases that initiate (caspase-8) andexecute (caspase-3, −6, and −7) apoptosis [111]. Zalewski andcolleagues put forth the postulate that zinc binds to the sulf-hydryl in Cys163 of the pro-caspase molecule, preventing itsoxidation and reversibly inhibiting its activity [112]. Recentlyand perhaps a more physiologically relevant contributingfactor to the anti-apoptotic effect of zinc, is its role in main-taining the functional configuration of inhibitor of apoptosisproteins (IAPs) [113]. Zinc is especially important for the BIRand RING domains which contain zinc-finger-like domainsthat incorporate zinc in order to interact with the catalytic siteof caspases and prevent their activation and recruit ubiquitinconjugating enzymes to ubiquitinate IAP antagonists forproteasomal degradation, respectively. Indeed, IAP expres-sion decreases with zinc deficiency (notably the potent cas-pase inhibitor, X-linked IAP (XIAP)) which is suggested to“release an XIAP-mediated brake”, increasing vulnerability toapoptosis [113]. Interestingly, zinc depletion in Caco-2/TC7cells rapidly reduces XIAP expression and inhibits TNFα-induced NF-κB translocation to the nucleus, skewing the fateof the cells from survival to cell death [114]. Microarray data

Fig. 5 TRPML1 protein increases during involution. Immunoblot ofTRPML1 in total membrane protein from virgin, lactating, and 48 hinvoluting mouse mammary gland and HC11 mammary epithelial cells(MEC). β-actin was used as a loading control


from the same study found a marked increase in ZnT2 whenTNFα and zinc were added to the culture medium followingTPEN treatment, supporting a role for zinc in cytoplasmic zincdepletion during programmed cell death. This suggests thatthe second wave of apoptotic programmed cell death duringinvolution may depend on the depletion of cytoplasmic zinc,possibly through ZnT2-mediated sequestration of zinc in 1)mitochondria, or 2) lysosomes during the first wave of pro-grammed cell death. Although these events are most likely notmutually exclusive, the latter could implicate zinc in potenti-ating the lysosomal-mitochondrial axis of programmed celldeath [115]. Cross-talk between lysosomes and mitochondriaduring cell death involves lysosomal hydrolases released fol-lowing LMP which directly or indirectly (e.g. Bid or Baxactivation) activate mitochondrial-mediated apoptosis throughMOMP and the release of cytochrome c and other pro-apoptotic proteins that engage apoptosis. Therefore, in thismodel the second wave of mitochondrial-mediated pro-grammed cell death would be contingent on factors regulatingthe transport of zinc into lysosomes and the activation of LMPas discussed above. Alternatively, the second wave of pro-grammed cell death may be independent of lysosomes andrely solely on mitochondrial zinc accumulation. Interestingly,we observed an expansion of mitochondrial zinc pools con-comitant with apoptosis of mammary epithelial cells thatresulted in decreased milk production in lactating mice fed amarginally zinc deficient diet [19]. Not only does this suggestthat the regulation of mitochondrial zinc pools may be impor-tant for lactation success, but also for sufficient involution.

Concluding Remarks

In order to better understand mammary gland development andfunction, it is imperative that the role and regulation of zinctransport be explored. The vast array of biological processesthat require zinc are critical to mammary gland developmentand function, which raises several important questions: How iszinc delivery to various biological processes regulated? Howdo dietary, environmental and genetic factors affect zinc deliv-ery during critical periods of mammary gland development andfunction? Clearly, the inability to import adequate zinc to fulfillthese vital requirements may compromise mammary glanddevelopment and the ability to produce an adequate volumeof high quality milk [19]. Moreover, defects in zinc metabolismmay prematurely activate involution and perhaps put women atrisk for breast disease and cancer. Given that ~80 % of womendo not consume adequate zinc and that chronic diseases ofinflammation such as obesity and diabetes, which are increas-ingly prevalent in young children, are associated with lowsystemic zinc levels, many women may have sub-optimalmammary gland development and function. While most ofour knowledge comes from studies in humans and mouse

models, these studies likely inform our understanding of mam-mary gland biology in production animals as well. A moreinsightful understanding of the transport of zinc and its role inregulating such a diverse array of processes including transcrip-tion, intracellular signaling, cell proliferation, differentiation,apoptosis, autophagy and motility is needed to optimize mam-mary gland development and lactation.

Acknowledgments The authors thank Dr. Veronica Lopez and VanessaVelasquez for technical assistance. This work was supported byR01HD058614, R01HD058614-S1, W82XWH-07-1-0692 andW81XWH-09-1-356 to SLK.

References

1. Sternlicht MD, Kouros-Mehr H, Lu P, et al. Hormonal and localcontrol of mammary branching morphogenesis. Differentiation.2006;74(7):365–81.

2. Vallee BL, Falchuk KH. The biochemical basis of zinc physiology.Physiol Rev. 1993;73(1):79–118.

3. Chesters JK, Petrie L, Vint H. Specificity and timing of the Zn2+requirement for DNA synthesis by 3 T3 cells. Exp Cell Res.1989;184(2):499–508.

4. Chesters JK, Boyne R. Nature of the Zn2+ requirement for DNAsynthesis by 3 T3 cells. Exp Cell Res. 1991;192(2):631–4.

5. Ishikawa Y, KudoH, Suzuki S, et al. Down regulation by a low-zincdiet in gene expression of rat prostatic thymidylate synthase andthymidine kinase. Nutr Metab (Lond). 2008;5:12.

6. Lefebvre D, BoneyCM,Ketelslegers JM, et al. Inhibition of insulin-like growth factor-I mitogenic action by zinc chelation is associatedwith a decreased mitogen-activated protein kinase activation inRAT-1 fibroblasts. FEBS Lett. 1999;449(2–3):284–8.

7. Hirano T, Murakami M, Fukada T, et al. Roles of zinc and zincsignaling in immunity: zinc as an intracellular signaling molecule.Adv Immunol. 2008;97:149–76.

8. Guo B, Yang M, Liang D, et al. Cell apoptosis induced by zincdeficiency in osteoblastic MC3T3-E1 cells via a mitochondrial-mediated pathway. Mol Cell Biochem. 2012;361(1–2):209–16.

9. Murgia C, Vespignani I, Cerase J, et al. Cloning, expression, andvesicular localization of zinc transporter Dri 27/ZnT4 in intestinaltissue and cells. Am J Physiol. 1999;277(6 Pt 1):G1231–9.

10. Jeong J, Eide DJ. The SLC39 family of zinc transporters. MolAspects Med. 2013;34(2–3):612–9.

11. Gaither LA, Eide DJ. Functional expression of the human hZIP2zinc transporter. J Biol Chem. 2000;275(8):5560–4.

12. Gaither LA, Eide DJ. The human ZIP1 transporter mediates zincuptake in human K562 erythroleukemia cells. J Biol Chem.2001;276(25):22258–64.

13. Ohana E, Hoch E, Keasar C, et al. Identification of the Zn2+ bindingsite and mode of operation of a mammalian Zn2+ transporter. J BiolChem. 2009;284(26):17677–86.

14. Bouron A, Oberwinkler J. Contribution of calcium-conductingchannels to the transport of zinc ions. Pflugers Arch 2013.

15. Abe K, Puertollano R. Role of TRP channels in the regulation of theendosomal pathway. Physiology (Bethesda). 2011;26(1):14–22.

16. Krezel A, Maret W. Zinc-buffering capacity of a eukaryotic cell atphysiological pZn. J Biol Inorg Chem. 2006;11(8):1049–62.

17. Colvin RA, Holmes WR, Fontaine CP, et al. Cytosolic zinc buffer-ing and muffling: their role in intracellular zinc homeostasis.Metallomics. 2010;2(5):306–17.


18. Maret W. Metallothionein redox biology in the cytoprotective andcytotoxic functions of zinc. Exp Gerontol. 2008;43(5):363–9.

19. Dempsey C,McCormick NH, Croxford TP, et al. Marginal maternalzinc deficiency in lactating mice reduces secretory capacity andalters milk composition. J Nutr. 2012;142(4):655–60.

20. Lopez V, Kelleher SL. Zip6-attenuation promotes epithelial-to-mesenchymal transition in ductal breast tumor (T47D) cells. ExpCell Res. 2010;316(3):366–75.

21. Hogstrand C, Kille P, Ackland ML, et al. A Mechanism forEpithelial-Mesenchymal Transition and Anoikis Resistance inBreast Cancer Triggered by Zinc Channel ZIP6 and SignalTransducer and Activator of Transcription 3 (STAT3). Biochem J2013.

22. Kelleher SL, Velasquez V, Croxford TP, et al. Mapping the zinc-transporting system in mammary cells: Molecular analysis reveals aphenotype-dependent zinc-transporting network during lactation. JCell Physiol. 2012;227(4):1761–70.

23. Huang L, Gitschier J. A novel gene involved in zinc transport isdeficient in the lethal milk mouse. Nat Genet. 1997;17(3):292–7.

24. Chowanadisai W, Lönnerdal B, Kelleher SL. Identification of amutation in SLC30A2 (ZnT-2) in women with low milk zincconcentration that results in transient neonatal zinc deficiency. JBiol Chem. 2006;281(51):39699–707.

25. McManaman JL, Reyland ME, Thrower EC. Secretion and fluidtransport mechanisms in the mammary gland: comparisons with theexocrine pancreas and the salivary gland. J Mammary Gland BiolNeoplasia. 2006;11(3–4):249–68.

26. Clermont Y, Xia L, Rambourg A, et al. Transport of caseinsubmicelles and formation of secretion granules in the Golgi appa-ratus of epithelial cells of the lactating mammary gland of the rat.Anat Rec. 1993;235(3):363–73.

27. Smith JJ, Nickerson SC, Keenan TW. Metabolic energy and cyto-skeletal requirements for synthesis and secretion by acini from ratmammary gland-II. Intracellular transport and secretion of proteinand lactose. Int J Biochem. 1982;14(2):99–109.

28. Chat S, Layani S, Mahaut C, et al. Characterisation of the potentialSNARE proteins relevant to milk product release by mouse mam-mary epithelial cells. Eur J Cell Biol. 2011;90(5):401–13.

29. Truchet S, Ollivier-Bousquet M. Mammary gland secretion: hor-monal coordination of endocytosis and exocytosis. Animal.2009;3(12):1733–42.

30. ShennanDB. Calcium transport bymammary secretory cells: mech-anisms underlying transepithelial movement. Cell Mol Biol Lett.2008;13(4):514–25.

31. Kelleher SL, Lonnerdal B. Zn transporter levels and localizationchange throughout lactation in rat mammary gland and are regulatedby Zn in mammary cells. J Nutr. 2003;133(11):3378–85.

32. Kelleher SL, Lonnerdal B. Zip3 plays a major role in zinc uptakeinto mammary epithelial cells and is regulated by prolactin. Am JPhysiol Cell Physiol. 2005;288(5):C1042–7.

33. Qian L, Lopez V, Seo YA, et al. Prolactin regulates ZNT2 expres-sion through the JAK2/STAT5 signaling pathway in mammarycells. Am J Physiol Cell Physiol. 2009;297(2):C369–77.

34. McCormick NH, Kelleher SL. ZnT4 provides zinc to zinc-dependent proteins in the trans-Golgi network critical for cell func-tion and Zn export in mammary epithelial cells. Am J Physiol CellPhysiol. 2012;303(3):C291–7.

35. Lasry I, Seo YA, Ityel H, et al. A dominant negative heterozygousG87Rmutation in the zinc transporter, ZnT-2 (SLC30A2), results intransient neonatal zinc deficiency. J Biol Chem. 2012;287(35):29348–61.

36. Suzuki T, Ishihara K, Migaki H, et al. Zinc transporters, ZnT5 andZnT7, are required for the activation of alkaline phosphatases,zinc-requiring enzymes that are glycosylphosphatidylinositol-anchored to the cytoplasmic membrane. J Biol Chem.2005;280(1):637–43.

37. Miranda JG, Weaver AL, Qin Y, et al. New alternately coloredFRET sensors for simultaneous monitoring of Zn2+ in multiplecellular locations. PLoS ONE. 2012;7(11):e49371.

38. Qin Y, Miranda JG, Stoddard CI, et al. Direct Comparison of aGenetically Encoded Sensor and Small Molecule Indicator:Implications for Quantification of Cytosolic Zn(2+). ACS ChemBiol 2013.

39. Park JG, Qin Y, Galati DF, et al. New sensors for quantitativemeasurement of mitochondrial Zn(2+). ACS Chem Biol.2012;7(10):1636–40.

40. Qin Y, Dittmer PJ, Park JG, et al. Measuring steady-state anddynamic endoplasmic reticulum and Golgi Zn2+ with geneti-cally encoded sensors. Proc Natl Acad Sci U S A. 2011;108(18):7351–6.

41. Tomat E, Nolan EM, Jaworski J, et al. Organelle-specific zincdetection using zinpyr-labeled fusion proteins in live cells. J AmChem Soc. 2008;130(47):15776–7.

42. Seo YA, Lopez V, Kelleher SL. A histidine-rich motif mediatesmitochondrial localization of ZnT2 tomodulate mitochondrial func-tion. Am J Physiol Cell Physiol. 2011;300(6):C1479–89.

43. Berg JM, Shi Y. The galvanization of biology: a growing appreci-ation for the roles of zinc. Science. 1996;271(5252):1081–5.

44. Langmade SJ, Ravindra R, Daniels PJ, et al. The transcription factorMTF-1 mediates metal regulation of the mouse ZnT1 gene. J BiolChem. 2000;275(44):34803–9.

45. Guo L, Lichten LA, Ryu MS, et al. STAT5-glucocorticoid receptorinteraction andMTF-1 regulate the expression of ZnT2 (Slc30a2) inpancreatic acinar cells. Proc Natl Acad Sci U S A. 2010;107(7):2818–23.

46. Westin G, Schaffner W. A zinc-responsive factor interacts with ametal-regulated enhancer element (MRE) of the mousemetallothionein-I gene. EMBO J. 1988;7(12):3763–70.

47. Kimura T, Itoh N, Sone T, et al. Role of metal-responsive transcrip-tion factor-1 (MTF-1) in EGF-dependent DNA synthesis in primaryhepatocytes. J Cell Biochem. 2006;99(2):485–94.

48. Hansson A. Extracellular zinc ions induces mitogen-activated pro-tein kinase activity and protein tyrosine phosphorylation inbombesin-sensitive Swiss 3T3 fibroblasts. Arch BiochemBiophys. 1996;328(2):233–8.

49. Parker PJ, Coussens L, Totty N, et al. The complete primarystructure of protein kinase C–the major phorbol ester receptor.Science. 1986;233(4766):853–9.

50. Csermely P, Szamel M, Resch K, et al. Zinc can increase the activityof protein kinase C and contributes to its binding to plasma mem-branes in T lymphocytes. J Biol Chem. 1988;263(14):6487–90.

51. McCormick N, Velasquez V, Finney L, et al. X-ray fluorescencemicroscopy reveals accumulation and secretion of discrete intracel-lular zinc pools in the lactating mouse mammary gland. PLoSONE.2010;5(6):e11078.

52. Taylor KM, Hiscox S, Nicholson RI, et al. Protein kinase CK2triggers cytosolic zinc signaling pathways by phosphorylation ofzinc channel ZIP7. Sci Signal. 2012;5(210):ra11.

53. Hogstrand C, Kille P, Nicholson RI, et al. Zinc transporters andcancer: a potential role for ZIP7 as a hub for tyrosine kinaseactivation. Trends Mol Med. 2009;15(3):101–11.

54. Lue HW, Yang X, Wang R, et al. LIV-1 promotes prostate cancerepithelial-to-mesenchymal transition and metastasis through HB-EGF shedding and EGFR-mediated ERK signaling. PLoS ONE.2011;6(11):e27720.

55. Zhang Y, Gonzalez RM, Zangar RC. Protein secretion in humanmammary epithelial cells following HER1 receptor activation: in-fluence of HER2 and HER3 expression. BMC Cancer. 2011;11:69.

56. Murakami M, Hirano T. Intracellular zinc homeostasis and zincsignaling. Cancer Sci. 2008;99(8):1515–22.

57. Taylor KM. A distinct role in breast cancer for two LIV-1 familyzinc transporters. Biochem Soc Trans. 2008;36(Pt 6):1247–51.


58. Mor M, Beharier O, Levy S, et al. ZnT-1 enhances the activity andsurface expression of T-type calcium channels through activation ofRas-ERK signaling. Am J Physiol Cell Physiol. 2012;303(2):C192–203.

59. Taniguchi M, Fukunaka A, Hagihara M, et al. Essential role of thezinc transporter ZIP9/SLC39A9 in regulating the activations of Aktand Erk in B-cell receptor signaling pathway in DT40 cells. PLoSONE. 2013;8(3):e58022.

60. Nishida K, Hasegawa A, Nakae S, et al. Zinc transporterZnt5/Slc30a5 is required for the mast cell-mediated delayed-typeallergic reaction but not the immediate-type reaction. J Exp Med.2009;206(6):1351–64.

61. Mailleux AA, Overholtzer M, Brugge JS. Lumen formation duringmammary epithelial morphogenesis: insights from in vitro andin vivo models. Cell Cycle. 2008;7(1):57–62.

62. Alam S, Kelleher SL. Cellular mechanisms of zinc dysregulation: aperspective on zinc homeostasis as an etiological factor in thedevelopment and progression of breast cancer. Nutrients.2012;4(8):875–903.

63. Lopez V, Kelleher SL. Zinc transporter-2 (ZnT2) variants are local-ized to distinct subcellular compartments and functionally transportzinc. Biochem J. 2009;422(1):43–52.

64. Besecker B, Bao S, Bohacova B, et al. The human zinc transporterSLC39A8 (Zip8) is critical in zinc-mediated cytoprotection in lungepithelia. Am J Physiol Lung Cell Mol Physiol. 2008;294(6):L1127–36.

65. McCawley LJ, Matrisian LM. Matrix metalloproteinases: they’renot just for matrix anymore! Curr Opin Cell Biol. 2001;13(5):534–40.

66. Wiseman BS, Sternlicht MD, Lund LR, et al. Site-specific inductiveand inhibitory activities of MMP-2 and MMP-3 orchestrate mam-mary gland branching morphogenesis. J Cell Biol. 2003;162(6):1123–33.

67. Kang T, Nagase H, Pei D. Activation of membrane-type matrixmetalloproteinase 3 zymogen by the proprotein convertase furin inthe trans-Golgi network. Cancer Res. 2002;62(3):675–81.

68. Cao J, Rehemtulla A, Pavlaki M, et al. Furin directly cleavesproMMP-2 in the trans-Golgi network resulting in a nonfunctioningproteinase. J Biol Chem. 2005;280(12):10974–80.

69. Podsiadlo P, Komiyama T, Fuller RS, et al. Furin inhibition bycompounds of copper and zinc. J Biol Chem. 2004;279(35):36219–27.

70. Huang L, Kirschke CP, Zhang Y, et al. The ZIP7 gene (Slc39a7)encodes a zinc transporter involved in zinc homeostasis of the Golgiapparatus. J Biol Chem. 2005;280(15):15456–63.

71. Cragg RA, Christie GR, Phillips SR, et al. A novel zinc-regulatedhuman zinc transporter, hZTL1, is localized to the enterocyte apicalmembrane. J Biol Chem. 2002;277(25):22789–97.

72. Kelleher SL, Lopez V, Lönnerdal B, et al. Zip3 (Slc39a3) functionsin zinc reuptake from the alveolar lumen in lactating mammarygland. Am J Physiol Regul Integr Comp Physiol. 2009;297(1):R194–201.

73. Závodszky P, Johansen JT, Hvidt A. Hydrogen-exchange study ofthe conformational stability of human carbonic-anhydrase B and itsmetallocomplexes. Eur J Biochem. 1975;56(1):67–72.

74. Karhumaa P, Leinonen J, Parkkila S, et al. The identification ofsecreted carbonic anhydrase VI as a constitutive glycoprotein ofhuman and rat milk. ProcNatl Acad Sci U SA. 2001;98(20):11604–8.

75. Parkkila S, Parkkila AK, Lehtola J, et al. Salivary carbonicanhydrase protects gastroesophageal mucosa from acid injury. DigDis Sci. 1997;42(5):1013–9.

76. Thatcher BJ, Doherty AE, Orvisky E, et al. Gustin from humanparotid saliva is carbonic anhydrase VI. Biochem Biophys ResCommun. 1998;250(3):635–41.

77. Groth C, Sasamura T, Khanna MR, et al. Protein trafficking abnor-malities in Drosophila tissues with impaired activity of the ZIP7zinc transporter Catsup. Development. 2013;140(14):3018–27.

78. Lönnerdal B, Stanislowski AG, Hurley LS. Isolation of a lowmolecular weight zinc binding ligand from human milk. J InorgBiochem. 1980;12(1):71–8.

79. Lönnerdal B, Hoffman B, Hurley LS. Zinc and copper bindingproteins in human milk. Am J Clin Nutr. 1982;36(6):1170–6.

80. Harzer G, Kauer H. Binding of zinc to casein. Am J Clin Nutr.1982;35(5):981–7.

81. Itsumura N, Inamo Y, Okazaki F, et al. Compound HeterozygousMutations in SLC30A2/ZnT2 Results in Low Milk ZincConcentrations: A Novel Mechanism for Zinc Deficiency in aBreast-Fed Infant. PLoS ONE. 2013;8(5):e64045.

82. Piletz JE, Ganschow RE. Zinc deficiency in murine milk underliesexpression of the lethal milk (lm) mutation. Science.1978;199(4325):181–3.

83. Michalczyk A, Varigos G, Catto-Smith A, et al. Analysis of zinctransporter, hZnT4 (Slc30A4), gene expression in amammary glanddisorder leading to reduced zinc secretion into milk. Hum Genet.2003;113(3):202–10.

84. Marti A, Jehn B, Costello E, et al. Protein kinaseA andAP-1 (c-Fos/JunD) are induced during apoptosis of mouse mammary epithelialcells. Oncogene. 1994;9(4):1213–23.

85. Kreuzaler PA, Staniszewska AD, Li W, et al. Stat3 controlslysosomal-mediated cell death in vivo. Nat Cell Biol. 2011;13(3):303–9.

86. Arnandis T, Ferrer-Vicens I, García-Trevijano ER, et al. Calpainsmediate epithelial-cell death during mammary gland involution:mitochondria and lysosomal destabilization. Cell Death Differ.2012;19(9):1536–48.

87. Stein T, Salomonis N, Gusterson BA.Mammary gland involution asa multi-step process. J Mammary Gland Biol Neoplasia.2007;12(1):25–35.

88. Baxter FO, Neoh K, Tevendale MC. The beginning of the end:death signaling in early involution. J Mammary Gland BiolNeoplasia. 2007;12(1):3–13.

89. Watson CJ. Involution: apoptosis and tissue remodelling that con-vert the mammary gland from milk factory to a quiescent organ.Breast Cancer Res. 2006;8(2):203.

90. Sunderman FW. The influence of zinc on apoptosis. Ann Clin LabSci. 1995;25(2):134–42.

91. Lee SJ, Cho KS, Koh JY. Oxidative injury triggers autophagy inastrocytes: the role of endogenous zinc. Glia. 2009;57(12):1351–61.

92. Bender T, Martinou JC. Where killers meet–permeabilization of theouter mitochondrial membrane during apoptosis. Cold Spring HarbPerspect Biol. 2013;5(1):a011106.

93. Foghsgaard L, Wissing D, Mauch D, et al. Cathepsin B acts as adominant execution protease in tumor cell apoptosis induced bytumor necrosis factor. J Cell Biol. 2001;153(5):999–1010.

94. Park MH, Lee SJ, Byun HR, et al. Clioquinol induces autophagy incultured astrocytes and neurons by acting as a zinc ionophore.Neurobiol Dis. 2011;42(3):242–51.

95. Hwang JJ, Lee SJ, Kim TY, et al. Zinc and 4-hydroxy-2-nonenalmediate lysosomal membrane permeabilization induced by H2O2in cultured hippocampal neurons. J Neurosci. 2008;28(12):3114–22.

96. Cho KS, Yoon YH, Choi JA, et al. Induction of autophagy and celldeath by tamoxifen in cultured retinal pigment epithelial and pho-toreceptor cells. Investig Ophthalmol Vis Sci. 2012;53(9):5344–53.

97. Hwang JJ, Kim HN, Kim J, et al. Zinc(II) ion mediates tamoxifen-induced autophagy and cell death in MCF-7 breast cancer cell line.Biometals. 2010;23(6):997–1013.

98. Falcón-Pérez JM, Dell’Angelica EC. Zinc transporter 2 (SLC30A2)can suppress the vesicular zinc defect of adaptor protein 3-depleted


fibroblasts by promoting zinc accumulation in lysosomes. Exp CellRes. 2007;313(7):1473–83.

99. Iguchi K, Usui S, Inoue T, et al. High-level expression of zinctransporter-2 in the rat lateral and dorsal prostate. J Androl.2002;23(6):819–24.

100. Palmiter RD, Cole TB, Findley SD. ZnT-2, a mammalian proteinthat confers resistance to zinc by facilitating vesicular sequestration.EMBO J. 1996;15(8):1784–91.

101. Kelleher SL, Lönnerdal B. Mammary gland copper transport isstimulated by prolactin through alterations in Ctr1 and Atp7Alocalization. Am J Physiol Regul Integr Comp Physiol.2006;291(4):R1181–91.

102. Kukic I, Lee JK, Coblentz J, et al. Zinc-dependent lysosomalenlargement in TRPML1-deficient cells involves MTF-1 transcrip-tion factor and ZnT4 (Slc30a4) transporter. Biochem J.2013;451(2):155–63.

103. Burke MA, Hutter D, Reshamwala RP, et al. Cathepsin L plays anactive role in involution of the mouse mammary gland. Dev Dyn.2003;227(3):315–22.

104. Clarkson RW, Watson CJ. Microarray analysis of the involutionswitch. J Mammary Gland Biol Neoplasia. 2003;8(3):309–19.

105. Helminen HJ, Ericsson JL, Niemi M. Lysosomal changes duringcastration-induced prostatic involution in the rat. Acta PatholMicrobiol Scand A. 1970;78(4):493–4.

106. Helminen HJ, Ericsson JL. Quantitation of lysosomal enzymechanges during enforced mammary gland involution. Exp CellRes. 1970;60(3):419–26.

107. Aydemir TB, Liuzzi JP, McClellan S, et al. Zinc transporter ZIP8(SLC39A8) and zinc influence IFN-gamma expression in activatedhuman T cells. J Leukoc Biol. 2009;86(2):337–48.

108. Dong XP, Cheng X, Mills E, et al. The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release chan-nel. Nature. 2008;455(7215):992–6.

109. Eichelsdoerfer JL, Evans JA, Slaugenhaupt SA, et al. Zincdyshomeostasis is linked with the loss of mucolipidosis IV-associatedTRPML1 ion channel. J Biol Chem. 2010;285(45):34304–8.

110. Zalewski PD, Forbes IJ, Betts WH. Correlation of apoptosis withchange in intracellular labile Zn(II) using zinquin [(2-methyl-8-p-toluenesulphonamido-6-quinolyloxy)acetic acid], a new specificfluorescent probe for Zn(II). Biochem J. 1993;296(Pt 2):403–8.

111. Stennicke HR, Salvesen GS. Biochemical characteristics ofcaspases-3, -6, -7, and -8. J Biol Chem. 1997;272(41):25719–23.

112. Truong-Tran AQ, Carter J, Ruffin RE, et al. The role of zinc incaspase activation and apoptotic cell death. Biometals. 2001;14(3–4):315–30.

113. Roscioli E, HamonR, Lester S, et al. Zinc-rich inhibitor of apoptosisproteins (IAPs) as regulatory factors in the epithelium of normal andinflamed airways. Biometals. 2013;26(2):205–27.

114. Ranaldi G, Ferruzza S, Canali R, et al. Intracellular zinc is requiredfor intestinal cell survival signals triggered by the inflammatorycytokine TNFα. J Nutr Biochem. 2013;24(6):967–76.

115. Terman A, Gustafsson B, Brunk UT. The lysosomal-mitochondrialaxis theory of postmitotic aging and cell death. Chem Biol Interact.2006;163(1–2):29–37.


the biology of zinc transport in mammary epithelial cells: implications for mammary gland...

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