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

J Mammary Gland Biol Neoplasia

Gallagher

et al. 2015

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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.

Section: Subcellular Localization Of Znt2 Variantsmentioning

confidence: 99%

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

Alam

J Mammary Gland Biol Neoplasia

Gallagher

et al. 2015

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“…Cells were maintained in growth medium (RPMI 1640 supplemented with 10% fetal bovine serum, 5 g/ml insulin, 10 ng/ml epidermal growth factor, and gentamycin). Cells were transfected with ZnT2 siRNA (5Ј-CCAUCUGCCUGGUGU UCAU-3; Sigma) using Lipofectamine 2000 (Life Technologies) as previously described (14) to attenuate ZnT2 (ZnT2KD). Transfected cells were stimulated into a secreting cell with prolactin (1 g/ml) and cortisol (2 M) for 24 h. Cell lysates and conditioned medium (CM) were collected and immunoblotted for p-Stat5 or ␤-casein as described above.…”

Section: Methodsmentioning

confidence: 99%

“…lactin, leading to N-terminal (Lys-4/Lys-6) ubiquitination, which retargets ZnT2 to secretory vesicles to facilitate zinc accumulation, traffics ZnT2-containing vesicles to the cell membrane for zinc efflux into milk, and then activates proteasomal degradation to abrogate zinc secretion (10,14). ZnT2 function in the mammary gland is not limited to lactation as it is also detected in the non-lactating mammary gland and nonsecreting MECs (13,15).…”

mentioning

confidence: 99%

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

Lee

Journal of Biological Chemistry

Alam

et al. 2015

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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.

“…Thus, an attempt to protect against Zn toxicity may underlie the sparse mammary epithelium in ZnT4-null mice. Alternatively, increased ZnT2 expression might augment Zn import into mitochondria (41), reducing ATP production (22) and/or increasing apoptosis (41), either of which would have important implications for MEC function. In addition, these defects may also be a consequence of the adipocyte-rich stroma that is retained in ZnT4-null mammary glands, which may secrete estrogen (3), proinflammatory cytokines (50), such as TNF-␣, and other antilactogenic factors.…”

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

American Journal of Physiology-Regulatory, Integrative and Comp

et al. 2016

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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...