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An important feature of the mammary gland is its ability to undergo profound morphological, physiological, and intracellular changes to establish and maintain secretory function. During this process, key polarity proteins and receptors are recruited to the surface of mammary epithelial cells (MECs), and the vesicle transport system develops and matures. However, the intracellular mechanisms responsible for the development of secretory function in these cells are unclear. The vesicular zinc (Zn2+) transporter ZnT2 is critical for appropriate mammary gland architecture, and ZnT2 deletion is associated with cytoplasmic Zn2+ accumulation, loss of secretory function and lactation failure. The underlying mechanisms are important to understand as numerous mutations and non-synonymous genetic variation in ZnT2 have been detected in women that result in severe Zn2+ deficiency in exclusively breastfed infants. Here we found that ZnT2 deletion in lactating mice and cultured MECs resulted in Zn2+-mediated degradation of phosphatase and tensin homolog (PTEN), which impaired intercellular junction formation, prolactin receptor trafficking, and alveolar lumen development. Moreover, ZnT2 directly interacted with vacuolar H+-ATPase (V-ATPase) and ZnT2 deletion impaired vesicle biogenesis, acidification, trafficking and secretion. In summary, our findings indicate that ZnT2 and V-ATPase interact and that this interaction critically mediates polarity establishment, alveolar development and secretory function in the lactating mammary gland. Our observations implicate disruption in ZnT2 function as a modifier of secretory capacity and lactation performance. [ABSTRACT FROM AUTHOR]

Suboptimal lactation is a common, yet underappreciated cause for early cessation of breastfeeding. Molecular regulation of mammary gland function is critical to the process lactation; however, physiological factors underlying insufficient milk production are poorly understood. The zinc (Zn) transporter ZnT2 is critical for regulation of mammary gland development and maturation during puberty, lactation, and postlactation gland remodeling. Numerous genetic variants in the gene encoding ZnT2 ( SLC30A2 ) are associated with low milk Zn concentration and result in severe Zn deficiency in exclusively breastfed infants. However, the functional impacts of genetic variation in ZnT2 on key mammary epithelial cell functions have not yet been systematically explored at the cellular level. Here we determined a common mutation in SLC30A2 /ZnT2 substituting serine for threonine at amino acid 288 (Thr288Ser) was found in 20% of women producing low milk volume ( n = 2/10) but was not identified in women producing normal volume. Exploration of cellular consequences in vitro using phosphomimetics showed the serine substitution promoted preferential phosphorylation of ZnT2, driving localization to the lysosome and increasing lysosome biogenesis and acidification. While the substitution did not initiate lysosome-mediated cell death, cellular ATP levels were significantly reduced. Our findings demonstrate the Thr288Ser mutation in SLC30A2 /ZnT2 impairs critical functions of mammary epithelial cells and suggest a role for genetic variation in the regulation of milk production and lactation performance.

Mammary gland involution, a tightly regulated process of tissue remodeling by which a lactating mammary gland reverts to the prepregnant state, is characterized by the most profound example of regulated epithelial cell death in normal tissue. Defects in the execution of involution are associated with lactation failure and breast cancer. Initiation of mammary gland involution requires upregulation of lysosome biogenesis and acidification to activate lysosome-mediated cell death; however, specific mediators of this initial phase of involution are not well described. Zinc transporter 2 [ZnT2 ( SLC30A2)] has been implicated in lysosome biogenesis and lysosome-mediated cell death during involution; however, the direct role of ZnT2 in this process has not been elucidated. Here we showed that ZnT2-null mice had impaired alveolar regression and reduced activation of the involution marker phosphorylated Stat3, indicating insufficient initiation of mammary gland remodeling during involution. Moreover, we found that the loss of ZnT2 inhibited assembly of the proton transporter vacuolar ATPase on lysosomes, thereby decreasing lysosome abundance and size. Studies in cultured mammary epithelial cells revealed that while the involution signal TNFα promoted lysosome biogenesis and acidification, attenuation of ZnT2 impaired the lysosome response to this involution signal, which was not a consequence of cytoplasmic Zn accumulation. Our findings establish ZnT2 as a novel regulator of vacuolar ATPase assembly, driving lysosome biogenesis, acidification, and tissue remodeling during the initiation of mammary gland involution.