Abstract
Among all tissues and organs, the mammary gland is unique because most of its development occurs in adulthood. Notch signaling has a major role in mammary gland development and has been implicated in breast cancer. The vacuolar-ATPase (V-ATPase) is a proton pump responsible for the regulation and control of pH in intracellular vesicles and the extracellular milieu. We have previously reported that a2V-ATPase (a2V), an isoform of ‘a' subunit of V-ATPase, regulates processing of Notch receptor and alters Notch signaling in breast cancer. To study the role of a2V in mammary gland development, we generated an a2V-KO model (conditional mammary knockout a2V mouse strain). During normal mammary gland development, the basal level expression of a2V increased from puberty, virginity, and pregnancy through the lactation stage and then decreased during involution. Litters of a2V-KO mice weighed significantly less when compared with litters from wild-type mice and showed reduced expression of the lactation marker β-casein. Whole-mount analysis of mammary glands demonstrated impaired ductal elongation and bifurcation in a2V-KO mice. Consequently, we found disintegrated mammary epithelium as seen by basal and luminal epithelial staining, although the rate of proliferation remained unchanged. Delayed mammary morphogenesis in a2V-KO mice was associated with aberrant activation of Notch and TGF-β (transforming growth factor-β) pathways. Notably, Hey1 (hairy/enhancer-of-split related with YRPW motif) and Smad2, the key downstream mediators of Notch and TGF-β pathways, respectively, were upregulated in a2V-KO mice and also in human mammary epithelial cells treated with a2V siRNA. Taken together, our results show that a2V deficiency disrupts the endolysosomal route in Notch and TGF signaling, thereby impairing mammary gland development. Our findings have broader implications in developmental and oncogenic cellular environments where V-ATPase, Notch and TGF-β are crucial for cell survival.
Highlights
The mammary gland is one of the few organs whose development occurs postnatally
The mammary epithelium forms the main ductal network of the bilayered gland that consists of an outer layer of myoepithelial cells and inner layer of luminal epithelial cells
Confocal microscopy confirmed the translocation of phosphorylated Smad[2] in the nucleus of mammary epithelial cells in a2V-KO mice (Figure 7c). These results demonstrate the involvement of Notch and Transforming growth factor-β (TGF-β) pathways in a2V-mediated pubertal mammary gland development
Summary
The mammary gland is one of the few organs whose development occurs postnatally. The gland is transformed from a nonfunctional organ into a mature, milk-producing gland evolved to nurture the newborn.[1]. A2V is a key factor in pregnancy and cancer through its role in autophagy and immunomodulation.[19,22] Notably, genetic mutations in human a2V gene result in Cutis Laxa syndrome where patients present with wrinkly skin because of impaired protein glycosylation and elevated TGF-β signaling.[23] Recent reports suggest the involvement of V-ATPase in Notch pathway regulation in Drosophila as well as mammalian cells.[24,25] We have previously reported that a2V regulates lysosomal degradation of Notch receptor Notch 1 and alters Notch signaling in breast cancer.[26] a2V regulates Notch signaling in inflammation-induced preterm labor in mice.[27] the involvement of a2V in context of Notch and TGF-β pathways in mammary gland development has not been explored. In vitro knockdown (KD) of a2V in human mammary epithelial cells (HMEpCs) resulted in alterations in Notch and TGF-β activity Taken together, these results suggest that a2V has a key role in the mammary gland and identify a novel role for a2V in regulating Notch signaling during development
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