Surface Of Mammary Epithelial Cells Research Articles

Zinc transporter 2 interacts with vacuolar ATPase and is required for polarization, vesicle acidification, and secretion in mammary epithelial cells

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.

Inflammatory Mediators in Mastitis and Lactation Insufficiency

Mastitis is a common inflammatory disease during lactation that causes reduced milk supply. A growing body of evidence challenges the central role of pathogenic bacteria in mastitis, with disease severity associated with markers of inflammation rather than infection. Inflammation in the mammary gland may be triggered by microbe-associated molecular patterns (MAMPs) as well as danger-associated molecular patterns (DAMPs) binding to pattern recognition receptors such as the toll-like receptors (TLRs) on the surface of mammary epithelial cells and local immune cell populations. Activation of the TLR4 signalling pathway and downstream nuclear factor kappa B (NFkB) is critical to mediating local mammary gland inflammation and systemic immune responses in mouse models of mastitis. However, activation of NFkB also induces epithelial cell apoptosis and reduced milk protein synthesis, suggesting that inflammatory mediators activated during mastitis promote partial involution. Perturbed milk flow, maternal stress and genetic predisposition are significant risk factors for mastitis, and could lead to a heightened TLR4-mediated inflammatory response, resulting in increased susceptibility and severity of mastitis disease in the context of low MAMP abundance. Therefore, heightened host inflammatory signalling may act in concert with pathogenic or commensal bacterial species to cause both the inflammation associated with mastitis and lactation insufficiency. Here, we present an alternate paradigm to the widely held notion that breast inflammation is driven principally by infectious bacterial pathogens, and suggest there may be other therapeutic strategies, apart from the currently utilised antimicrobial agents, that could be employed to prevent and treat mastitis in women.

Abstract 5066: Matriptase mediated signaling in breast cancer.

Abstract Historically proteases have been associated with tumor progression and metastasis through degradation of the basement membrane. In recent years these enzymes have also been shown to play key roles in the activation of growth factors and cytokines, thereby activating pro-oncogenic signaling pathways. Matriptase is an epithelia-specific pericellular protease which has received considerable attention recently spurred by its consistent dysregulation in human epithelial tumors including breast cancer. Several institutions are currently developing small and large molecule inhibitors of matriptase, however thus far no studies have been published focusing on the role of matriptase in breast cancer growth and progression. Presently we do not know whether matriptase plays a causal or promotional role in breast carcinogenesis and therefore its potential as an anti-cancer drug target is still unexplored. In the following study we will perform both functional and mechanistic studies of matriptase in breast cancer using parallel, complementary in-vitro and in-vivo genetic “loss-of-function” strategies. Matriptase efficiently cleaves pro-hepatocyte growth factor (pro-HGF) into active HGF, the cognate ligand of the proto-oncogene tyrosine kinase receptor c-Met, and thereby elicits pro-tumorigenic downstream signaling events. Both c-Met and matriptase are expressed on the surface of mammary epithelial cells and breast carcinoma cells, whereas the c-Met ligand and matriptase proteolytic target pro-HGF is secreted by stromal fibroblasts thus defining a paracrine growth factor activation and signaling mechanism. Our goal with this project is to determine whether matriptase is a novel target which could provide an opportunity for developing improved breast cancer intervention drugs in the future. Citation Format: Gina L. Zoratti, Christopher Bergum, Julie L. Boerner, Karin List. Matriptase mediated signaling in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5066. doi:10.1158/1538-7445.AM2013-5066

G-helix of Maspin Mediates Effects on Cell Migration and Adhesion

Maspin is a member of the serine protease inhibitor (serpin) superfamily that lacks protease inhibitory ability, although displaying tumor metastasis-suppressing activity resulting from its influence on cell migration, invasion, proliferation, apoptosis, and adhesion. The molecular mechanisms of these actions of maspin are as yet undefined. Here, we sought to identify critical functional motifs by the expression of maspin with point mutations at sites potentially involved in protein-protein interactions: the G α-helix (G-helix), an internal salt bridge or the P1 position of the reactive center loop. Our findings indicate that only mutations in the G-helix attenuated inhibition of cell migration by maspin and that this structural element is also involved in the effect of maspin on cell adhesion. The action of maspin on cell migration could be mimicked by a 15-mer G-helix peptide, indicating that the G-helix is both essential and sufficient for this effect. In addition, we provide evidence that the effects of the G-helix of maspin are dependent on β1 integrins. These data reveal that the major extracellular functions associated with the tumor suppressive action of maspin likely involve interactions in which the G-helix plays a key role.

165. LOCATION OF ACTIVE TGFB1 IN THE MAMMARY GLAND DURING DIFFERENT STAGES OF DEVELOPMENT

Development of the mammary gland involves complex interactions between epithelial and stromal cells under the influence of hormones and cytokines. Transforming growth factor beta 1 (TGFB1) is a multi-functional cytokine that we have reported to be essential for normal mammary gland development. TGFB1 is produced and secreted as part of a latent complex, and requires activation at the site of action to have biological effects. In situ hybridisation studies have shown mRNA encoding Tgfb1 is mainly expressed by mammary epithelium; however, the expression pattern of active TGFB1 in the mammary gland during different developmental stages is still unclear. Mammary gland tissue was collected from mice at puberty (5 weeks old), adult diestrus and late pregnancy (day 18 pc). Frozen sections were stained with antibody specifically reactive with active TGFB1 protein (not latent TGFB1 or other TGFB isoforms) for immunofluorescent analysis. Qualitative assessment of the staining revealed different patterns of active TGFB1 localisation depending on developmental stage. The strongest expression of active TGFB1 was observed in the mammary gland at diestrus compared to puberty and pregnancy. At diestrus, active TGFB1 was located around the surface of mammary epithelial cells. The staining was heterogeneous, with distinct zones of active TGFB1 accumulated around some but not all epithelial cells. During puberty, active TGFB1 was observed only within the lumen of the ducts. During late pregnancy, TGFB1 was homogenously distributed within the alveolar epithelium. The different patterns of active TGFB1 observed during puberty, diestrus and pregnancy suggest that TGFB1 has different roles in the mammary gland dependent on developmental stage.

Bovine lactoferrin serves as a molecular bridge for internalization of Streptococcus uberis into bovine mammary epithelial cells

Streptococcus uberis, an environmental mastitis pathogen, is an important causative agent of mastitis in dairy cattle throughout the world. Research from our laboratory demonstrated that bovine lactoferrin (LF), a whey protein present in milk and nonlactating cow mammary secretions, significantly enhanced adherence of S. uberis to mammary epithelial cells in culture. Subsequent research from our laboratory identified S. uberis adhesion molecule (SUAM) showing an affinity for LF. The objective of the present investigation was to test the hypothesis that the interaction between SUAM, bovine LF, and a putative LF receptor on the bovine mammary epithelial cell surface could serve as a bridging molecule for internalization of S. uberis into mammary epithelial cells. When internalization assays were conducted using cell growth medium containing bovine LF, a significant increase in internalization of S. uberis into mammary epithelial cells was observed. However, this effect was reversed when assays were conducted in the presence of antibodies to bovine LF suggesting that internalization of S. uberis into mammary epithelial cells, at least in part, was mediated by LF ligands. When S. uberis was pretreated with antibodies to SUAM, internalization in the presence of LF was reduced in the same manner as observed with antibodies to LF. Transmission and scanning electron microscopy results demonstrated that streptavidin-coated gold particles specifically localized on biotinylated LF receptors on S. uberis and mammary epithelial cell surfaces supporting the availability of LF receptors. Collectively, these results suggest that LF serves as a bridging molecule between SUAM located on the surface of S. uberis and LF receptors located on the surface of mammary epithelial cells thus enhancing internalization of S. uberis into host cells. Exploitation of LF as a molecular bridge for internalization of S. uberis into mammary epithelial cells may confer a significant advantage allowing mammary gland infection.

Omega-3 Polyunsaturated Fatty Acids Regulate Syndecan-1 Expression in Human Breast Cancer Cells

Human epidemiologic studies and animal model studies support a role for n-3 polyunsaturated fatty acids (n-3 PUFA) in prevention or inhibition of breast cancer. However, mechanisms for this protection remain unclear. Syndecan-1 is a heparan sulfate proteoglycan, expressed on the surface of mammary epithelial cells and known to regulate many biological processes, including cytoskeletal organization, growth factor signaling, and cell-cell adhesion. We studied effects of n-3 PUFA on syndecan-1 expression in human mammary cell lines. PUFA were delivered to cells by low-density lipoproteins (LDL) isolated from the plasma of monkeys fed diets enriched in fish oil (n-3 PUFA) or linoleic acid (n-6 PUFA). Proteoglycan synthesis was measured by incorporation of [35S]-sodium sulfate. No effect of either LDL was observed in nontumorigenic MCF-10A cells, whereas in MCF-7 breast cancer cells, treatment with n-3-enriched LDL but not n-6-enriched LDL resulted in significantly greater synthesis of a proteoglycan identified by immunoprecipitation as syndecan-1. Using real-time reverse transcription-PCR (RT-PCR), it was shown that n-3-enriched LDL significantly increased the expression of syndecan-1 mRNA in a dose-dependent manner and maximal effective time at 8 hours of treatment. The effect was mimicked by an agonist for peroxisome proliferator-activated receptor gamma (PPARgamma) and eliminated by the presence of PPARgamma antagonist suggesting a role for PPARgamma in syndecan enhancement. Our studies show that n-3 LDL modifies the production of syndecan-1 in human breast cancer cells and suggest that biological processes regulated by syndecan-1 may be modified through LDL delivery of n-3 PUFA.

Expression and localisation of GLUT1 and GLUT12 glucose transporters in the pregnant and lactating rat mammary gland.

Glucose plays a major role in mammary gland function during lactation as it is used both as a fuel and as a precursor of milk components. In rats, previous studies have shown that the facilitative glucose transporter GLUT1 is expressed in mammary epithelial cells. We have used confocal immunofluorescence to localise GLUT1 and GLUT12, a recently identified member of the sugar transporter family, in pregnant and lactating rat mammary gland. GLUT12 staining was observed in the cytoplasm of mammary epithelial cells at day 20 of pregnancy, and at 1 and 6 days postpartum. Furthermore, GLUT12 staining was present at the apical plasma membrane of epithelial cells during lactation. In contrast, GLUT1 protein localised to the cytoplasm and basolateral surface of mammary epithelial cells. Forced weaning resulted in decreased cytoplasmic GLUT1 staining intensity, but no change in GLUT12 staining. The results suggest a possible role for GLUT12 in the metabolism of mammary epithelial cells during pregnancy and lactation.

Epimorphin mediates mammary luminal morphogenesis through control of C/EBPbeta.

We have shown previously that epimorphin (EPM), a protein expressed on the surface of myoepithelial and fibroblast cells of the mammary gland, acts as a multifunctional morphogen of mammary epithelial cells. Here, we present the molecular mechanism by which EPM mediates luminal morphogenesis. Treatment of cells with EPM to induce lumen formation greatly increases the overall expression of transcription factor CCAAT/enhancer binding protein (C/EBP)beta and alters the relative expression of its two principal isoforms, LIP and LAP. These alterations were shown to be essential for the morphogenetic activities, since constitutive expression of LIP was sufficient to produce lumen formation, whereas constitutive expression of LAP blocked EPM-mediated luminal morphogenesis. Furthermore, in a transgenic mouse model in which EPM expression was expressed in an apolar fashion on the surface of mammary epithelial cells, we found increased expression of C/EBPbeta, increased relative expression of LIP to LAP, and enlarged ductal lumina. Together, our studies demonstrate a role for EPM in luminal morphogenesis through control of C/EBPbeta expression.

Identification of the mammary tumor virus envelope glycoprotein (gp52) on mouse mammary epithelial cell surface

Lactoperoxidase radioiodination of mammary epithelial cells cultured in monolayers followed by SDS-PAGE analysis revealed only a few distinct peaks. One of these, identified as major envelope glycoptrotein (gp 52) of MTV, is present on the surface of mammary epithelial cells (both tumor and normal) from chronically infected BALB/cfC3H mice but not on the surface of normal mammary epithelial cells from virus-free sol BALB c mice. Its presence on the cell surface is influenced by both hormones and cell density, the same factors which greatly control the production and release of intact MTV virions into culture media. This suggests a correlation between abundance of radioiodinatable gp 52 on the cell surface and MTV found in culture media.