Stephen R. Hennigar

Essential Role for Zinc Transporter 2 (ZnT2)-mediated Zinc Transport in Mammary Gland Development and Function during Lactation

Background: ZnT2 is expressed in non-secreting and secreting mammary epithelium; however, the physiological role is not understood. Results: ZnT2-null mice have impaired mammary expansion and compromised mammary differentiation and milk secretion during lactation. Conclusion: ZnT2-mediated zinc transport is critical for mammary development and function during lactation. Significance: This study identifies novel consequences of ZnT2 function in the mammary gland. The zinc transporter ZnT2 (SLC30A2) imports zinc into vesicles in secreting mammary epithelial cells (MECs) and is critical for zinc efflux into milk during lactation. Recent studies show that ZnT2 also imports zinc into mitochondria and is expressed in the non-lactating mammary gland and non-secreting MECs, highlighting the importance of ZnT2 in general mammary gland biology. In this study we used nulliparous and lactating ZnT2-null mice and characterized the consequences on mammary gland development, function during lactation, and milk composition. We found that ZnT2 was primarily expressed in MECs and to a limited extent in macrophages in the nulliparous mammary gland and loss of ZnT2 impaired mammary expansion during development. Secondly, we found that lactating ZnT2-null mice had substantial defects in mammary gland architecture and MEC function during secretion, including fewer, condensed and disorganized alveoli, impaired Stat5 activation, and unpolarized MECs. Loss of ZnT2 led to reduced milk volume and milk containing less protein, fat, and lactose compared with wild-type littermates, implicating ZnT2 in the regulation of mammary differentiation and optimal milk production during lactation. Together, these results demonstrate that ZnT2-mediated zinc transport is critical for mammary gland function, suggesting that defects in ZnT2 not only reduce milk zinc concentration but may compromise breast health and increase the risk for lactation insufficiency in lactating women.

Zinc networks: the cell-specific compartmentalization of zinc for specialized functions.

Abstract Zinc (Zn2+) is the most abundant trace element in cells and is essential for a vast number of catalytic, structural, and regulatory processes. Mounting evidence indicates that like calcium (Ca2+), intracellular Zn2+ pools are redistributed for specific cellular functions. This occurs through the regulation of 24 Zn2+ transporters whose localization and expression is tissue and cell specific. We propose that the complement and regulation of Zn2+ transporters expressed within a given cell type reflects the function of the cell itself and comprises a ‘Zn2+ network.’ Importantly, increasing information implicates perturbations in the Zn2+ network with metabolic consequences and disease. Herein, we discuss our current understanding of Zn2+ transporters from the perspective of a Zn2+ network in four specific tissues with unique Zn2+ requirements (mammary gland, prostate, pancreas, and brain). Delineating the entire Zn2+ transporting network within the context of unique cellular Zn2+ needs is important in identifying critical gaps in our knowledge and improving our understanding of the consequences of Zn2+ dysregulation in human health and disease.

ZnT2 is a critical mediator of lysosomal-mediated cell death during early mammary gland involution

Mammary gland involution is the most dramatic example of physiological cell death. It occurs through an initial phase of lysosomal-mediated cell death (LCD) followed by mitochondrial-mediated apoptosis. Zinc (Zn) activates both LCD and apoptosis in vitro. The Zn transporter ZnT2 imports Zn into vesicles and mitochondria and ZnT2-overexpression activates cell death in mammary epithelial cells (MECs). We tested the hypothesis that ZnT2-mediated Zn transport is critical for mammary gland involution in mice. Following weaning, ZnT2 abundance increased in lysosomes and mitochondria, which paralleled Zn accumulation in each of these organelles. Adenoviral expression of ZnT2 in lactating mouse mammary glands in vivo increased Zn in lysosomes and mitochondria and activated LCD and apoptosis, promoting a profound reduction in MECs and alveoli. Injection of TNFα, a potent activator of early involution, into the mammary gland fat pads of lactating mice increased ZnT2 and Zn in lysosomes and activated premature involution. Exposure of cultured MECs to TNFα redistributed ZnT2 to lysosomes and increased lysosomal Zn, which activated lysosomal swelling, cathepsin B release, and LCD. Our data implicate ZnT2 as a critical mediator of cell death during involution and importantly, that as an initial involution signal, TNFα redistributes ZnT2 to lysosomes to activate LCD.

The biology of zinc transport in mammary epithelial cells: implications for mammary gland development, lactation, and involution.

Zinc plays a critical role in a vast array of cellular functions including gene transcription, protein translation, cell proliferation, differentiation, bioenergetics, and programmed cell death. The mammary gland depends upon tight coordination of these processes during development and reproduction for optimal expansion, differentiation, and involution. For example, zinc is required for activation of matrix metalloproteinases, intracellular signaling cascades such as MAPK and PKC, and the activation of both mitochondrial-mediated apoptosis and lysosomal-mediated cell death. In addition to functional needs, during lactation the mammary gland must balance providing optimal zinc for cellular requirements with the need to secrete a substantial amount of zinc into milk to meet the requirements of the developing neonate. Finally, the mammary gland exhibits the most profound example of programmed cell death, which is driven by both apoptotic and lysosomal-mediated cell death. Two families of zinc-specific transporters regulate zinc delivery for these diverse functions. Members of the ZIP family of zinc transporters (ZIP1–14) import zinc into the cytoplasm from outside the cell or from subcellular organelles, while members of the ZnT family (ZnT1–10) export zinc from the cytoplasm. Recently, the ion channel transient receptor potential mucolipin 1 (TRPML1) has also been implicated in zinc transport. Herein, we review our current understanding of the molecular mechanisms through which mammary epithelial cells utilize zinc with a focus on the transport of zinc into discrete subcellular organelles for specific cellular functions during mammary gland development, lactation, and involution.

Metallothionein and Zinc Transporter Expression in Circulating Human Blood Cells as Biomarkers of Zinc Status: a Systematic Review

Zinc is an essential nutrient for humans; however, a sensitive biomarker to assess zinc status has not been identified. The objective of this systematic review was to compile and assess studies that determined zinc transporter and/or metallothionein expression in various blood cell types and to determine their reliability and sensitivity to changes in dietary zinc. Sixteen studies were identified that determined the expression of zrt-, irt-like protein (ZIP) 1 [solute carrier family (SLC) 39A1], ZIP3 (SLC39A3), ZIP5 (SLC39A5), ZIP6 (SLC39A6), ZIP7 (SLC39A7), ZIP8 (SLC39A8), ZIP10 (SLC39A10), ZIP14 (SLC39A14), zinc transporter (ZnT)1 (SLC30A1), ZnT2 (SLC30A2), ZnT4 (SLC30A4), ZnT5 (SLC30A5), ZnT6 (SLC30A6), ZnT7 (SLC30A7), ZnT9 (SLC30A9), and/or metallothionein in various blood cells isolated from healthy adult men and women in response to zinc supplementation or depletion. Cell types included leukocytes, peripheral blood mononuclear cells, T lymphocytes, monocytes, and erythrocytes. ZIP1, ZnT1, and metallothionein were the most commonly measured proteins. Changes in ZIP1 and ZnT1 in response to zinc supplementation or depletion were not consistent across studies. Leukocyte metallothionein decreased with zinc depletion (-39% change from baseline, <5 mg Zn/d, n = 2 studies) and increased with zinc supplementation in a dose-dependent manner (35%, 15-22 mg Zn/d, n = 7 studies; 267%, 50 mg Zn/d, n = 2 studies) and at the earliest time points measured; however, no change or delayed response was observed in metallothionein in erythrocytes. A greater percentage of studies demonstrated that metallothionein in leukocyte subtypes was a more reliable (100%, n = 12; 69%, n = 16) and responsive (92%, n = 12; 82%, n = 11) indicator of zinc exposure than was plasma zinc, respectively. In conclusion, current evidence indicates that metallothionein in leukocyte subtypes may be a component in determining zinc status.

TNFα Post‐Translationally Targets ZnT2 to Accumulate Zinc in Lysosomes

Mammary epithelial cells undergo widespread lysosomal‐mediated cell death (LCD) during early mammary gland involution. Recently, we demonstrated that tumor necrosis factor‐α (TNFα), a cytokine released during early involution, redistributes the zinc (Zn) transporter ZnT2 to accumulate Zn in lysosomes and activate LCD and involution. The objective of this study is to determine how TNFα retargets ZnT2 to lysosomes. We tested the hypothesis that TNFα signaling dephosphorylates ZnT2 to uncover a highly conserved dileucine motif (L294L) in the C‐terminus of ZnT2, allowing adaptor protein complex‐3 (AP‐3) to bind and traffic ZnT2 to lysosomes. Confocal micrographs showed that TNFα redistributed wild‐type (WT) ZnT2 from late endosomes (Pearsons coefficient = 0.202 ± 0.05 and 0.097 ± 0.03; P < 0.05) to lysosomes (0.292 ± 0.03 and 0.649 ± 0.03; P < 0.0001), which increased lysosomal Zn (P < 0.0001) and activated LCD (P < 0.0001) compared to untreated cells. Mutation of the dileucine motif (L294V) eliminated the ability of TNFα to redistribute ZnT2 from late endosomes to lysosomes, increase lysosomal Zn, or activate LCD. Moreover, TNFα increased (P < 0.05) AP‐3 binding to wt ZnT2 but not to L294V immunoprecipitates. Finally, using phospho‐ and dephospho‐mimetics of predicted phosphorylation sites (T281, T288, and S296), we found that dephosphorylated S296 was required to target ZnT2 to accumulate Zn in lysosomes and activate LCD. Our findings suggest that women with variation in the C‐terminus of ZnT2 may be at risk for inadequate involution and breast disease due the inability to traffic ZnT2 to lysosomes. J. Cell. Physiol. 230: 2345–2350, 2015.

Prolactin (PRL)-stimulated Ubiquitination of ZnT2 Mediates a Transient Increase in Zinc Secretion Followed by ZnT2 Degradation in Mammary Epithelial Cells

Background: ZnT2 regulates zinc export from mammary cells. Results: Prolactin stimulates ZnT2 ubiquitination, targeting ZnT2 to vesicles and activating zinc accumulation to transiently enhance zinc secretion dependent upon Lys4/Lys6, after which ZnT2 is degraded. Conclusion: Prolactin is a critical regulator of transient ZnT2-mediated zinc secretion. Significance: This work provides insight into post-translational hormonal mechanisms that regulate zinc transport. The zinc transporter ZnT2 imports zinc into secretory vesicles and regulates zinc export from the mammary epithelial cell. Mutations in ZnT2 substantially impair zinc secretion into milk. The lactogenic hormone prolactin (PRL) transcriptionally increases ZnT2 expression through the Jak2/STAT5 signaling pathway, increasing zinc accumulation in secretory vesicles and zinc secretion. Herein, we report that PRL post-translationally stimulated ZnT2 ubiquitination, which altered ZnT2 trafficking and augmented vesicular zinc accumulation and secretion from mammary epithelial cells in a transient manner. Ubiquitination then down-regulated zinc secretion by stimulating degradation of ZnT2. Mutagenesis of two N-terminal lysine residues (K4R and K6R) inhibited ZnT2 ubiquitination, vesicular zinc accumulation and secretion, and protein degradation. These findings establish that PRL post-translationally regulates ZnT2-mediated zinc secretion in a multifactorial manner, first by enhancing zinc accumulation in vesicles to transiently enhance zinc secretion and then by activating ubiquitin-dependent ZnT2 degradation. This provides insight into novel mechanisms through which ZnT2 and zinc transport is tightly regulated in mammary epithelial cells.

Obesity-Induced Inflammation Is Associated with Alterations in Subcellular Zinc Pools and Premature Mammary Gland Involution in Lactating Mice

BACKGROUND Lactation failure is common in overweight and obese women; however, the precise mechanism remains unknown. OBJECTIVE We tested the hypothesis that obesity-induced inflammation in the mammary gland (MG) redistributes subcellular zinc pools to promote cell death of mammary epithelial cells (MECs) and premature involution. METHODS Female DBA/2J mice were fed a high-fat (obese; 45% kcal from fat, n = 60) or control diet (lean; 10% kcal from fat, n = 50) for 5 wk and bred. MG cytokines and macrophage infiltration were determined by reverse transcriptase-polymerase chain reaction and F4/80 staining, respectively. Zinc concentration was analyzed by atomic absorption spectroscopy, and zinc transporters and markers of endoplasmic reticulum (ER) stress, autophagy, and involution were measured by immunoblot. To confirm effects of inflammation, tumor necrosis factor-α (TNF) or vehicle was injected into adjacent MGs of lean lactating C57BL/6 mice (n = 5) and cultured MECs (HC11 cells) were treated with TNF in vitro. RESULTS Seventy-seven percent of obese mice failed to lactate (lean: 39%; P < 0.001). Obese mice capable of lactating had greater macrophage infiltration (obese: 135 ± 40.4 macrophages/mm(2); lean: 63.8 ± 8.9 macrophages/mm(2); P < 0.001) and elevated TNF expression (P < 0.05), concurrent with lower zrt- irt-like protein 7 abundance (P < 0.05) and higher ER zinc concentration (obese: 0.36 ± 0.004 μg Zn/mg protein; lean: 0.30 ± 0.02 μg Zn/mg protein; P < 0.05) compared with lean mice. Heat shock protein 5 (HSPA5) expression (P < 0.05) was suppressed in the MG of obese mice, which was consistent with HSPA5 suppression in TNF-injected MGs (P < 0.01) and MECs treated with TNF in vitro (P < 0.01). Moreover, obesity increased lysosomal activity (P < 0.05) and autophagy in the MG, which corresponded to increased zinc transporter 2 abundance and lysosomal zinc concentration compared with lean mice (obese: 0.20 ± 0.02 μg Zn/mg protein; lean: 0.14 ± 0.01 μg Zn/mg protein; P < 0.05). Importantly, MGs of obese mice exhibited markers of apoptosis (P = 0.05) and involution (P < 0.01), which were not observed in lean mice. CONCLUSIONS Diet-induced obesity created a proinflammatory MG microenvironment in mice, which was associated with zinc-mediated ER stress and autophagy and the activation of premature involution.

Homeostatic regulation of trace mineral transport by ubiquitination of membrane transporters

Post-translational modification is a critical mechanism by which trace mineral transporters rapidly adapt to their environment to homeostatically regulate ion transport. Recently, a novel pathway was described whereby iron stimulates the ubiquitination and proteasomal degradation of the trace mineral transporter ZIP14. Discovery of this pathway suggests the proteasome as a potential therapeutic target for regulation of iron storage. Moreover, these findings contribute to a theoretical framework that can be applied to other ubiquitinated trace mineral transporters. This review will detail the current state of knowledge regarding the ubiquitination of trace mineral transporters, focusing on iron and zinc transporters, and the potential utility of post-translational modification of trace mineral transporters in the treatment of disease.

Nutritional Immunity Starving Pathogens of Trace Minerals

Nutritional immunity is a process by which a host organism sequesters trace minerals in an effort to limit pathogenicity during infection. Circulating concentrations of minerals, such as iron and zinc, decline rapidly and dramatically with the inflammation associated with infection. The decline in iron and zinc is thought to starve invading pathogens of these essential elements, limiting disease progression and severity. The mechanisms contributing to the hypoferremia and hypozincemia of inflammation and potential interventions that exploit this process for the management of infection will be discussed.