Abstract
After weaning, during mammary gland involution, milk-producing mammary epithelial cells undergo apoptosis. Effective clearance of these dying cells is essential, as persistent apoptotic cells have a negative impact on gland homeostasis, future lactation and cancer susceptibility. In mice, apoptotic cells are cleared by the neighboring epithelium, yet little is known about how mammary epithelial cells become phagocytic or whether this function is conserved between species. Here we use a rat model of weaning-induced involution and involuting breast tissue from women, to demonstrate apoptotic cells within luminal epithelial cells and epithelial expression of the scavenger mannose receptor, suggesting conservation of phagocytosis by epithelial cells. In the rat, epithelial transforming growth factor-β (TGF-β) signaling is increased during involution, a pathway known to promote phagocytic capability. To test whether TGF-β enhances the phagocytic ability of mammary epithelial cells, non-transformed murine mammary epithelial EpH4 cells were cultured to achieve tight junction impermeability, such as occurs during lactation. TGF-β3 treatment promoted loss of tight junction impermeability, reorganization and cleavage of the adherens junction protein E-cadherin (E-cad), and phagocytosis. Phagocytosis correlated with junction disruption, suggesting junction reorganization is necessary for phagocytosis by epithelial cells. Supporting this hypothesis, epithelial cell E-cad reorganization and cleavage were observed in rat and human involuting mammary glands. Further, in the rat, E-cad cleavage correlated with increased γ-secretase activity and β-catenin nuclear localization. In vitro, pharmacologic inhibitors of γ-secretase or β-catenin reduced the effect of TGF-β3 on phagocytosis to near baseline levels. However, β-catenin signaling through LiCl treatment did not enhance phagocytic capacity, suggesting a model in which both reorganization of cell junctions and β-catenin signaling contribute to phagocytosis downstream of TGF-β3. Our data provide insight into how mammary epithelial cells contribute to apoptotic cell clearance, and in light of the negative consequences of impaired apoptotic cell clearance during involution, may shed light on involution-associated breast pathologies.