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

Lee, Sooyeon; Hennigar, Stephen R.; Alam, Samina; Nishida, Keigo; Kelleher, Shannon L.

doi:10.1074/jbc.m115.637439

Cited by 58 publications

(70 citation statements)

References 79 publications

(84 reference statements)

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“…Interestingly, many women in our study carried compound substitutions in ZnT2, similar to what Itsumura and colleagues [10] recently reported in a woman with reduced milk [Zn]. Because we recently reported that ZnT2-null mice had profound defects in mammary gland function and milk secretion in general [14], our study implicates genetic variation in SLC30A2 as a modifier of mammary gland biology, which may have consequences on both maternal and infant health.…”

Section: Discussionsupporting

confidence: 85%

“…However, we recently showed that the redistribution of ZnT2 from secretory vesicles to lysosomes activates lysosomal-mediated cell death and mammary gland remodeling during involution [13]. Moreover, ZnT2-null mice have profound defects in mammary gland function, secretion and milk composition [14], implicating ZnT2-mediated Zn transport in processes that extend well-beyond Zn secretion into milk. Because appropriate Zn management is critical for modulating a vast array of biological processes including endoplasmic reticulum (ER) stress [15], intracellular signaling [16], vesicular trafficking [4] and autophagy [17], inappropriate sub-cellular Zn transport may have important implications for cell function.…”

Section: Introductionmentioning

confidence: 98%

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Exome Sequencing of SLC30A2 Identifies Novel Loss- and Gain-of-Function Variants Associated with Breast Cell Dysfunction

Alam

Hennigar

Gallagher

et al. 2015

J Mammary Gland Biol Neoplasia

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The zinc (Zn) transporter ZnT2 (SLC30A2) is expressed in specialized secretory cells including breast, pancreas and prostate, and imports Zn into mitochondria and vesicles. Mutations in SLC30A2 substantially reduce milk Zn concentration ([Zn]) and cause severe Zn deficiency in exclusively breastfed infants. Recent studies show that ZnT2-null mice have low milk [Zn], in addition to profound defects in mammary gland function during lactation. Here, we used breast milk [Zn] to identify novel non-synonymous ZnT2 variants in a population of lactating women. We also asked whether specific variants induce disturbances in intracellular Zn management or cause cellular dysfunction in mammary epithelial cells. Healthy, breastfeeding women were stratified into quartiles by milk [Zn] and exonic sequencing of SLC30A2 was performed. We found that 36% of women tested carried non-synonymous ZnT2 variants, all of whom had milk Zn levels that were distinctly above or below those in women without variants. We identified 12 novel heterozygous variants. Two variants (D(103)E and T(288)S) were identified with high frequency (9 and 16%, respectively) and expression of T(288)S was associated with a known hallmark of breast dysfunction (elevated milk sodium/potassium ratio). Select variants (A(28)D, K(66)N, Q(71)H, D(103)E, A(105)P, Q(137)H, T(288)S and T(312)K) were characterized in vitro. Compared with wild-type ZnT2, these variants were inappropriately localized, and most resulted in either 'loss-of-function' or 'gain-of-function', and altered sub-cellular Zn pools, Zn secretion, and cell cycle check-points. Our study indicates that SLC30A2 variants are common in this population, dysregulate Zn management and can lead to breast cell dysfunction. This suggests that genetic variation in ZnT2 could be an important modifier of infant growth/development and reproductive health/disease. Importantly, milk [Zn] level may serve as a bio-reporter of breast function during lactation.

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Section: Discussionsupporting

confidence: 85%

Section: Introductionmentioning

confidence: 98%

Exome Sequencing of SLC30A2 Identifies Novel Loss- and Gain-of-Function Variants Associated with Breast Cell Dysfunction

Alam

Hennigar

Gallagher

et al. 2015

J Mammary Gland Biol Neoplasia

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“…ZnT2 is a key mediator of zinc transport into vesicles in mammary gland epithelial cells (2,26). It is the sole transporter, the loss of function of which was found to cause zinc-deficient breast milk and TNZD (6 -11, 27).…”

Section: Discussionmentioning

confidence: 99%

Molecular Basis of Transient Neonatal Zinc Deficiency

Golan¹,

Itsumura

Glaser³

et al. 2016

Journal of Biological Chemistry

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A gradually increasing number of transient neonatal zinc deficiency (TNZD) cases was recently reported, all of which were associated with inactivating ZnT2 mutations. Here we characterized the impact of three novel heterozygous ZnT2 mutations G280R, T312M, and E355Q, which cause TNZD in exclusively breastfed infants of Japanese mothers. We used the bimolecular fluorescence complementation (BiFC) assay to provide direct visual evidence for the in situ dimerization of these ZnT2 mutants, and to explore their subcellular localization. Moreover, using three complementary functional assays, zinc accumulation using BiFC-Zinquin and Zinpyr-1 fluorescence as well as zinc toxicity assay, we determined the impact of these ZnT2 mutations on vesicular zinc accumulation. Although all three mutants formed homodimers with the wild type (WT) ZnT2 and retained substantial vesicular localization, as well as vesicular zinc accumulation, they had no dominant-negative effect over the WT ZnT2. Furthermore, using advanced bioinformatics, structural modeling, and site-directed mutagenesis we found that these mutations localized at key residues, which play an important physiological role in zinc coordination (G280R and E355Q) and zinc permeation (T312M). Collectively, our findings establish that some heterozygous loss of function ZnT2 mutations disrupt zinc binding and zinc permeation, thereby suggesting a haploinsufficiency state for the unaffected WT ZnT2 allele in TNZD pathogenesis. These results highlight the burning need for the development of a suitable genetic screen for the early diagnosis of TNZD to prevent morbidity.In the past 50 years, the key role of zinc in human health was established (1). Zinc is crucial for central physiological processes including DNA and protein synthesis, enzyme activity, intracellular signaling, immune function, fertility, as well as growth and development (1-3). Zinc homeostasis is primarily regulated by two zinc transporter families as well as by zincbinding proteins. The SLC39A gene family encodes for ZIP1-14 transporters, which are responsible for zinc uptake into the cytosol from the extracellular fluid or from intracellular vesicles (4). In contrast, the SLC30A gene family encodes for the transporters ZnT1-10, which are responsible for zinc secretion from the cytosol to intracellular organelles or through the plasma membrane outside the cells (2, 4).The recent position of the Academy of Nutrition and Dietetics (5) is that breastfeeding in the first 6 months of life can provide adequate micronutrients and macronutrients for optimal growth and development. This statement is correct for the vast majority of infants around the world. However, recent reports indicate that some exclusively breastfed infants suffer from severe zinc deficiency due to lack of zinc in the breast milk they consume (6 -11). These cases were referred to as transient neonatal zinc deficiency (TNZD), 4 as the symptoms can be treated with zinc supplementation to the diet of the infant (12, 13). In contrast to TNZD, which appe...

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“…However, changes in ZnT4 or ZnT2 expression are not responsible; thus, the molecular underpinnings remain unknown. While ZnT2-null mice also have a substantial reduction in secretory epithelium, this results from cytoplasmic Zn accumulation, the loss of phospho-STAT5 signaling, and profound defects in mammary gland differentiation and secretion, as opposed to precious involution (21). In contrast, while ZnT4-null mice have a substantial reduction in secretory epithelium, these defects are associated with increased ZnT2 and TNF-␣ expression and precocious activation of phospho-STAT3, which is indicative of mammary gland remodeling (9,10).…”

Section: Ck8mentioning

confidence: 99%

“…Recent studies have shown that cell signaling can be affected by alterations in subcellular Zn pools (reviewed in Ref. 24), which has critical implications for mammary gland function (5,10,21,42). For example, LMO4, a member of the LIM-only class of Zn finger transcription factors, associates with gp130 (30), coactivates JAK1, the tyrosine phosphatase SHP2, and suppressor of cytokine signaling 3 (SOCS3) (30), is a positive regulator of MEC proliferation, and is critical for mammary expansion during pregnancy (46).…”

Section: Ck8mentioning

confidence: 99%

ZnT4 (SLC30A4)-null (“lethal milk”) mice have defects in mammary gland secretion and hallmarks of precocious involution during lactation

McCormick

Lee

Hennigar

et al. 2016

American Journal of Physiology-Regulatory, Integrative and Comp

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McCormick NH, Lee S, Hennigar SR, Kelleher SL. ZnT4 (SLC30A4)-null ("lethal milk") mice have defects in mammary gland secretion and hallmarks of precocious involution during lactation. Am J Physiol Regul Integr Comp Physiol 310: R33-R40, 2016. First published November 4, 2015 doi:10.1152/ajpregu.00315.2014.-During lactation, highly specialized secretory mammary epithelial cells (MECs) produce and secrete huge quantities of nutrients and nonnutritive factors into breast milk. The zinc (Zn) transporter ZnT4 (SLC30A4) transports Zn into the trans-Golgi apparatus for lactose synthesis, and across the apical cell membrane for efflux from MECs into milk. This is consistent with observations in "lethal milk" (lm/lm) mice, which have a truncation mutation in SLC30A4, and present with not only low milk Zn concentration, but also smaller mammary glands, decreased milk volume, and lactation failure by lactation day 2. However, the molecular underpinnings of these defects are not understood. Here, we used lactating C57BL/6J lm/lm (ZnT4-null) mice to explore the consequences of a ZnT4-null phenotype on mammary gland function during early lactation. Lactating C57BL/6J lm/lm mice had significantly fewer, smaller, and collapsed alveoli comprising swollen, lipid-filled MECs during early lactation. These defects were associated with decreased Akt expression and STAT5 activation, indicative of defects in MEC secretion. In addition, increased expression of ZnT2, TNF-␣, and cleaved e-cadherin concomitant with increased activation of STAT3 implicated the loss of ZnT4 in precocious activation of involution. Collectively, our study indicates that the loss of ZnT4 has profound consequences on MEC secretion and may promote tissue remodeling in the mammary gland during early lactation.zinc; lactation; mammary gland; SLC30A4; ZnT4 ZNT4 (SLC30A4) IS A ZINC (Zn) transporter that is expressed in numerous tissues, including the liver (13), lung (13), small intestine (28), and mammary gland (13). ZnT4 is particularly important for mammary gland function, as ZnT4 expression increases dramatically during lactation (23). Previous studies in vitro established that ZnT4 transports Zn into the transGolgi apparatus of the mammary epithelial cell (MEC) and delivers Zn to critical Zn-dependent proteins, including galactosyltransferase (a resident Golgi protein that is vital for lactose production) and carbonic anhydrase VI (a secreted milk enzyme that is vital for infant alimentary tract pH balance) (15). The "lethal milk" mouse (C57BL/6J lm/lm ) carries a spontaneous truncation mutation in SLC30A4, resulting in non-sense-mediated decay of ZnT4 mRNA (29). During lactation, ZnT4-null mice have ϳ35% lower milk Zn concentration than their wild-type littermates (37). Although this implicates ZnT4 in Zn secretion into milk, studies in breastfeeding women have yet to identify defects in ZnT4 in women with low milk Zn levels or find an association with transient neonatal Zn deficiency in exclusively breastfed infants (4,14,20). Moreover, ZnT4-null mice have s...

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

Cited by 58 publications

References 79 publications

Exome Sequencing of SLC30A2 Identifies Novel Loss- and Gain-of-Function Variants Associated with Breast Cell Dysfunction

Exome Sequencing of SLC30A2 Identifies Novel Loss- and Gain-of-Function Variants Associated with Breast Cell Dysfunction

Molecular Basis of Transient Neonatal Zinc Deficiency

ZnT4 (SLC30A4)-null (“lethal milk”) mice have defects in mammary gland secretion and hallmarks of precocious involution during lactation

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