Loss of ZnT2‐mediated Zinc Transport Leads to Cytoplasmic Zinc Accumulation and Impairs Mammary Gland Involution

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The zinc (Zn) transporter ZnT2 is expressed in highly specialized exocrine tissues and imports Zn into mitochondria and vesicles. ZnT2‐mediated Zn transport is particularly critical in the secreting mammary gland (MG). In addition to importing Zn into secretory vesicles for secretion into milk, we recently reported ZnT2‐null mice (ZnT2ko) have impaired MG differentiation and secretion, which leads to reduced milk volume and sub‐optimal milk composition. Moreover, re‐localization of ZnT2 from secretory vesicles to lysosomes by TNF‐α stimulates lysosomal Zn import and initiates lysosomal mediated cell death, thus activating MG remodeling and involution. We hypothesized that loss of ZnT2 function would lead to impaired execution of mechanisms that drive MG remodeling. This may be particularly relevant to breast health, as non‐synonymous genetic variation in ZnT2 that impairs ZnT2 function is common and insufficient MG remodeling may predispose women to breast cancer. To determine the effects of the loss of ZnT2 function on involution we used ZnT2ko mice and their wild‐type (WT) littermates. Whole mount analysis revealed the MG of ZnT2ko mice, forced to undergo involution by removal of their offspring, more closely resembled a lactating MG than that of involuting MGs from WT mice. The MG area repopulated by adipocytes increased by 12% in ZnT2ko mice compared to a 37% increase in WT mice (P<0.001). To determine if the loss of ZnT2 impaired Zn sequestration, Zinpyr‐1, a cell permeable fluorescent probe was used to image labile Zn. In WT mice, labile Zn accumulation was only observed in numerous extruded apoptotic cells. In contrast, in ZnT2ko mice fewer apoptotic cells were noted, and cytoplasmic Zn accumulation was observed in the non‐apoptotic mammary epithelial cells, which was associated with reduced Stat3 activation (P<0.05). Furthermore, macrophage infiltration, a late event that occurs during MG involution, was reduced by 83% in ZnT2ko mice (P<0.05) and macrophages in ZnT2ko mice displayed a circular morphology, compared to the irregular morphology observed in WT mice, suggesting impaired macrophage activation. Our results indicate that the loss of ZnT2 leads to cytoplasmic Zn accumulation, which inhibits activation of key signals initiating or potentiating the cellular events characterizing involution.Support or Funding InformationIntramural funds to SLK