Normal Mammary Gland Function Research Articles

Loss of Panx1 Impairs Mammary Gland Development at Lactation: Implications for Breast Tumorigenesis.

Pannexin1 (Panx1) subunits oligomerize to form large-pore channels between the intracellular and extracellular milieu that have been shown to regulate proliferation, differentiation and cell death mechanisms. These key cellular responses are ultimately necessary for normal tissue development and function but the role of Panx1 in development, differentiation and function in many tissues remains unexplored, including that of the breast. Panx1 was identified to be expressed in the mammary gland through western blot and immunofluorescent analysis and is dynamically upregulated during pregnancy and lactation. In order to evaluate the role of Panx1 in the context of mammary gland development and function, Panx1-/- mice were evaluated in comparison to wild-type mice in the mammary glands of virgin, lactating and involuting mice. Our results revealed that Panx1 ablation did not affect virgin or involuting mammary glands following histological and whole mount analysis. Panx1 was necessary for timely alveolar development during early lactation based on a decreased number of alveolar lumen following histological analysis and reduced proliferation following Ki67 immunofluorescent labelling. Importantly, the loss of Panx1 in lactating mammary glands did not overtly affect epithelial or secretory differentiation of the mammary gland suggesting that Panx1 is not critical in normal mammary gland function. In addition, PANX1 mRNA expression was correlated with negative clinical outcomes in patients with breast cancer using in silico arrays. Together, our results suggest that Panx1 is necessary for timely alveolar development following the transition from pregnancy to lactation, which may have implications extending to patients with breast cancer.

Effect of glycogen synthase kinase-3 inactivation on mouse mammary gland development and oncogenesis.

Many components of the Wnt/β-catenin signaling pathway have critical functions in mammary gland development and tumor formation, yet the contribution of glycogen synthase kinase-3 (GSK-3α and GSK-3β) to mammopoiesis and oncogenesis is unclear. Here, we report that WAP-Cre-mediated deletion of GSK-3 in the mammary epithelium results in activation of Wnt/β-catenin signaling and induces mammary intraepithelial neoplasia that progresses to squamous transdifferentiation and development of adenosquamous carcinomas at 6 months. To uncover possible β-catenin-independent activities of GSK-3, we generated mammary-specific knockouts of GSK-3 and β-catenin. Squamous transdifferentiation of the mammary epithelium was largely attenuated, however, mammary epithelial cells lost the ability to form mammospheres suggesting perturbation of stem cell properties unrelated to loss of β-catenin alone. At 10 months, adenocarcinomas that developed in glands lacking GSK-3 and β-catenin displayed elevated levels of γ-catenin/plakoglobin as well as activation of the Hedgehog and Notch pathways. Collectively, these results establish the two isoforms of GSK-3 as essential integrators of multiple developmental signals that act to maintain normal mammary gland function and suppress tumorigenesis.

Parity-Induced Decrease in Systemic Growth Hormone Alters Mammary Gland Signaling: A Potential Role in Pregnancy Protection from Breast Cancer

Early full-term pregnancy is an effective natural protection against breast cancer in both humans and experimental rodents. The protective effect of an early pregnancy is, in part, linked to changes in circulating hormones that are involved in both normal breast development and breast cancer. For example, a reduction in circulating growth hormone (GH) has been shown to protect rats from carcinogen-induced mammary tumors. We examined the ability of a full-term pregnancy to alter the endocrine GH/insulin-like growth factor-I (IGF-I) axis and how this change affected normal mammary gland function in two commonly used rat models (Sprague-Dawley and Wistar Furth). Circulating GH and IGF-I were measured in blood drawn every 30 minutes from parous and age-matched virgin female rats. Mean serum GH levels were significantly decreased (P < 0.01) in parous compared with age-matched virgin rats for both strains. Changes in GH levels were independent of estrous cycle, indicated by a significant (P < 0.05) reduction in circulating levels of GH during estrus and diestrus in both parous strains. Despite the decrease in circulating GH, pituitary GH mRNA levels were unaltered in parous rats. Circulating IGF-I and hepatic IGF-I mRNA were also unaltered by parity in either rat strain. Immunoblot analysis of mammary glands showed decreases in phosphorylation of signal transducer and activator of transcription 5A and Janus-activated kinase 2, suggesting reduced action of GH in the mammary gland. Therefore, although the parity reduction in circulating GH does not affect circulating IGF-I levels, it is possible that reduced GH acts directly at the mammary gland and may play a role in pregnancy protection from breast cancer.

Bovine glucose transporter GLUT8: cloning, expression, and developmental regulation in mammary gland

GLUT8 is a newly identified member of the facilitative glucose transporter family, which characteristically exhibits high-affinity glucose transport activity. The expression of GLUT8 has been shown to depend on gonadotropin secretion in human testes and to be regulated by insulin in the blastocyst. To characterize GLUT8 and investigate its role in normal mammary gland function, we cloned and sequenced the full-length cDNA of bovine GLUT8. The 2073-base-pair cDNA sequence is predicted to encode a protein of 478 amino acids, with a molecular weight of approximately 51 kDa. The deduced amino acid sequence of bovine GLUT8 is 90%, 84%, 84% and 58% identical to human, mouse, rat and chicken GLUT8, and is 26%, 27% and 24% identical to bovine GLUT1, GLUT3 and GLUT4, respectively. Bovine GLUT8 retains the characteristic structural features of GLUT8 proteins previously identified from other species including membrane spanning helices, glucose transporter motifs, an N-linked glycosylation site on loop 9 and a putative dileucine internalization motif. The major in vitro transcription and translation product of bovine GLUT8 cDNA migrated at an apparent molecular weight of 38 kDa similar to the sizes reported for GLUT8 from other mammalian species. In the presence of canine microsomal membranes, the translation product increased to 40 kDa suggesting glycosylation. Transient transfection studies using a FLAG epitope tagged construct in COS-7 cells revealed that bovine GLUT8 is localized to the cytoplasm in non-stimulated conditions. A 2.1-kb GLUT8 mRNA transcript was detected at high levels in bovine testes, at moderate levels in lactating bovine mammary gland, lung, kidney, spleen, intestine and skeletal muscle, and at low levels in bovine liver. GLUT8 mRNA expression in bovine mammary gland increased about 10-fold ( P<0.001) during late pregnancy and early lactation, similar to the pattern of change in GLUT1 mRNA and more dramatic than the increase seen in mouse mammary gland. These results suggest that GLUT8 expression may be regulated by lactogenic hormones and that GLUT8 may play a role in glucose uptake in the lactating mammary gland.

Inappropriate P-cadherin expression in the mouse mammary epithelium is compatible with normal mammary gland function

Cadherins comprise a family of cell-cell adhesion proteins critical to the architecture and function of tissues. Expression of family members E-, N-, and P-cadherin is regulated in a spatial and temporal fashion in the developing and adult organism. Using in vivo and in vitro experimental systems, perturbation of cadherin expression by genetic deletion, overexpression, mutant dominant-negative constructs, and, to a lesser degree, expression of an inappropriate cadherin have all been shown to alter embryogenesis, tissue architecture, and cell behavior. Here we studied how expression of an inappropriate cadherin affects the adult mouse mammary gland. Human P-cadherin was expressed in mammary epithelial cells under control of the mouse mammary tumor virus (MMTV) promoter, and the effect on mammary gland behavior was studied. Typically, E-cadherin is expressed by mammary epithelial cells, whereas P-cadherin is found in myoepithelial cells and cap cells of the ductal terminal end bud. However, breast cancers frequently express P-cadherin, even though they are thought to arise from epithelial cells, and it is a marker of poor prognosis. We developed two independent transgenic mouse lines that exhibited high levels of P-cadherin protein expression in the mammary epithelium. P-cadherin was detected in most, but not all, luminal epithelial cells, and was appropriately localized to cell-cell borders. It was detected in the mammary glands of virgin, pregnant, lactating, post-lactation, and aged parous female mice. Despite the robust and widespread expression of an inappropriate cadherin, no effect was observed on mammary gland morphogenesis, architecture, lactation, or involution in transgenic mice compared to wild-type mice. No mammary tumors formed spontaneously in either wild-type or transgenic mice. Moreover, mammary tumors induced by the neu oncogene, which was introduced by a breeding strategy, showed no differences between mice with or without hP-cadherin. Surprisingly, however, none of the tumors expressed hP-cadherin protein. Together, our studies show no apparent effect on adult mammary gland or tumor behavior by inappropriate expression of P-cadherin in normal mammary epithelial cells.

Human CREB-binding Protein/p300-interacting Transactivator with ED-rich Tail (CITED) 4, a New Member of the CITED Family, Functions as a Co-activator for Transcription Factor AP-2

Members of the CREB-binding protein/p300-interacting transactivator with ED-rich tail (CITED) family bind CREB-binding protein and p300 with high affinity and regulate gene transcription. Gene knockout studies indicate that CITED2 is required for neural crest and neural tube development and that it functions as a co-activator for transcription factor AP-2 (TFAP2). Here we describe human CITED4, a new member of this family, which is encoded by a single exon mapping to chromosome 1p34--1p35. CITED4 and p300/CREB-binding protein are present in endogenous naturally occurring complexes, indicating that they interact physiologically. The interaction occurs between the cysteine-histidine-rich domain 1 of p300 and the carboxyl terminus of CITED4. In keeping with this, CITED4 functions as a transactivator when artificially targeted to a promoter element. CITED4 physically interacts with all TFAP2 isoforms in vitro and strongly co-activates all TFAP2 isoforms in Hep3B cells. Co-activation of TFAP2 requires amino-terminal and carboxyl-terminal residues of CITED4. In HepG2 cells, CITED4 is significantly weaker than CITED2 for TFAP2C co-activation. These results suggest that CITED4 may function as a co-activator for TFAP2. They also suggest the existence of cell type- and TFAP2 isoform-specific co-activation by CITED2 and CITED4, which may result in differential modulation of TFAP2 function.

Pathological Changes in Bovine Mammary Glands Following Intramammary Infusion of Recombinant Interleukin-2

Eighteen lactating dairy cows were used to evaluate the physiological response of mammary glands to increasing doses of recombinant bovine interleukin-2. Right front and rear quarters were intramammarily infused with five different doses (.1 to 100μg per quarter) of interleukin-2 as either a single or multiple treatment. Left front and rear quarters were intramammarily infused with a saline placebo and served as within-animal controls. Milk secretion samples for compositional analysis were collected from each quarter prior to infusion and at 12, 24, 36, and 48h following infusion. Animals were slaughtered by exsanguination immediately following the 48-h sampling period, and mammary gland tissue was obtained for morphometric analysis. No changes in milk composition were observed between control quarters and those infused with up to 10 pg of interleukin-2 per quarter, administered as either a single or multiple treatment. Quarters infused with a single 100-μg dose of interleukin-2 or three consecutive doses of 25 and 100μg of interleukin-2 had significantly lower lactose concentrations; there was a concomitant increase in bovine serum albumin, pH, and SCC compared with preinfusion concentrations or with control quarters. Morphometric analysis of tissue demonstrated an increase in stroma, a decrease in lumenal area, and a marked increase in the number of infiltrating leukocytes in those quarters infused with the higher doses of interleukin-2. Results suggest that interleukin-2 can be intramammarily infused at doses as high as 10μg per quarter without adversely affecting milk quality or normal mammary gland function.

Cell to Cell Interactions and Normal Mammary Gland Function

The nonepithelial components of the mammary gland are reviewed and their potential for regulatory cell-cell interactions with the epithelial cells are discussed. Studies undertaken to examine the regulatory potential of mammary stromal fibroblasts using an in vitro cell culture system are presented. The influence was examined of epithelial-fibroblast interactions on estrogenic regulation of progesterone receptor concentration in epithelial cells and on epithelial and fibroblast DNA synthesis. Mammary fibroblasts affect estrogen responsiveness in epithelial cells by two different mechanisms. In the case of progesterone receptor regulation, fibroblasts promote estrogen-dependent increases in the receptor via a substratum effect possibly by the production of collagen type I. By contrast, the fibroblast effect promoting estrogen-dependent cell proliferation requires fibroblasts to be metabolically active and in close contact with the epithelium. Additionally, under coculture conditions, estrogen-dependent stimulation of fibroblast DNA synthesis is also observed, indicating a bidirectional, interactive phenomenon between the two types of cells. It is possible that the modulations in epithelial responsiveness to estrogen that are associated with the presence of mammary fibroblasts in vitro reflect regulatory mechanisms that operate in vivo.

Suppression of lactation by tumor promoters in mice.

AbstractAs a possible step to evaluate the role of tumor promoters in normal cells, the effects of phorbol and its ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), on normal mammary gland function and pituitary secretion of growth hormone (GH) and prolactin, were examined in lactating C3H/He mice. Daily subcutaneous injections of 400 μg phorbol or 4 μg TPA during Days 12 and 14 of lactation resulted in a significant decline in litter growth rate and the ratio of RNA synthesis: DNA synthesis of mammary glands associated with an increase in mammary gland DNA synthesis. On the other hand, pituitary synthesis and release of GH and prolactin, the pattern of estrous cycles and ovarian structures were affected little by these treatments. All findings strongly suggest that these tumor promoters can directly inhibit lactation by shifting mammary gland cells from a functional state to a mitotic state.