Increased gene expression of endothelin-1 and vasoactive intestinal contractor/endothelin-2 in the mammary gland of lactating mice

In an attempt to understand the roles of endothelin-1 (ET-1) and vasoactive intestinal contractor/endothelin-2 (VIC/ET-2), we have studied the genes for both peptides to be expressed in the mammary gland of lactating mice. We observed through real-time PCR analysis that ET-1 and VIC/ET-2 gene expression gradually increase after parturition and that ET-1 gene expression is significantly higher than that of VIC/ET-2. The distribution of ET-1 peptide was found to be localized mainly in the epithelial cells of the mammary gland at 14th day of lactation. ET-1 gene expression increases significantly, parallel to the increase in beta-casein gene expression, in epithelial cell lines (HC11) of mouse mammary gland after hormonal stimulation by addition of dexamethazone and prolactin. The observed increase in ET-1 expression in differentiated epithelial cells suggests physiological roles for ET-1, including milk production and secretion in the mammary gland of lactating mice.     

Parity-induced mammary epithelial cells are multipotent and express cell surface markers associated with stem cells

Parity-induced mammary epithelial cells (PI-MECs) are defined as a pregnancy hormone-responsive cell population that activates the promoter of late milk protein genes during the second half of pregnancy and lactation. However, unlike their terminally differentiated counterparts, these cells do not undergo programmed cell death during post-lactational remodeling of the gland. We previously demonstrated that upon transplantation into an epithelial-free mammary fat pad, PI-MECs exhibited two important features of multipotent mammary epithelial progenitors: a) self-renewal, and b) contribution to ductal and alveolar morphogenesis. In this new report, we introduce a new method to viably label PI-MECs. Using this methodology, we analyzed the requirement of ovarian hormones for the maintenance of this epithelial subtype in the involuted mammary gland. Furthermore, we examined the expression of putative stem cell markers and found that a portion of GFP-labeled PI-MECs were part of the CD24(+)/CD49f(high) mammary epithelial subtype, which has recently been suggested to contain multipotent stem cells. Subsequently, we demonstrated that isolated PI-MECs were able to form mammospheres in culture, and upon transplantation, these purified epithelial cells were capable of establishing a fully functional mammary gland. These observations suggest that PI-MECs contain multipotent progenitors that are able to self renew and generate diverse epithelial lineages present in the murine mammary gland.     

Mouse mammary epithelial cells express the Na-K-Cl cotransporter,NKCC1: characterization,localization, and involvement in ductal development and morphogenesis

Despite the fact that physiological evidence points to the existence of a functional Na-K-Cl cotransporter in the mammary gland, the molecular identity of this transport process remains unknown. We now show that the Na-K-Cl cotransporter isoform, NKCC1, is expressed in mammary tissue. Developmental profiling revealed that the level of NKCC1 protein was significantly influenced by the stage of mammary gland development, and immunolocalization studies demonstrated that NKCC1 was present on the basolateral membrane of mammary epithelial cells. To examine whether functional NKCC1 is required for mammary epithelial cell development, we used NKCC1 -/- mice. We demonstrate that NKCC1 -/- mammary epithelium exhibited a significant delay in ductal outgrowth and an increase in branching morphogenesis during virgin development. These effects were autonomous to the epithelium as assessed by mammary gland transplantation. Although the absence of NKCC1 had no apparent effect on gross mammary epithelial cell morphology during lactation, pups born to NKCC1 -/- mice failed to thrive. Finally, analysis of NKCC1 protein in mouse models that exhibit defects in mammary gland development demonstrate that high levels of NKCC1 protein are indicative of ductal epithelial cells, and the presence of NKCC1 protein is characteristic of mammary epithelial cell identity.     

An adjunct mammary epithelial cell population in parous females: its role in functional adaptation and tissue renewal

Mammary gland biologists have long assumed that differentiated secretory epithelial cells undergo programmed cell death at the end of lactation and that the alveolar compartment is reconstituted from undifferentiated precursor cells in subsequent pregnancies. It is generally agreed that the remodeled gland in a parous animal resembles that of a mature virgin at the morphological level. However, several physiological differences have been noted in comparing the responses of mammary epithelia from nulliparous versus parous females to hormonal stimulation and carcinogenic agents. We present genetic evidence that an involuted mammary gland is fundamentally different from a virgin gland, despite its close morphological resemblance. This difference results from the formation of a new mammary epithelial cell population that originates from differentiating cells during pregnancy. In contrast to the majority of fully committed alveolar cells, this epithelial population does not undergo cell death during involution or remodeling after lactation. We show that these cells can function as alveolar progenitors in subsequent pregnancies and that they can play an important role in functional adaptation in genetically engineered mice, which exhibit a reversion of a lactation-deficient phenotype in multiparous animals. In transplantation studies, this parity-induced epithelial population shows the capacity for self-renewal and contributes significantly to the reconstitution of the resulting mammary outgrowth (i.e. ductal morphogenesis and lobulogenesis). We propose that this parity-induced population contributes importantly to the biological differences between the mammary glands of parous and nulliparous females.     

The estrogen-responsive Agr2 gene regulates mammary epithelial proliferation and facilitates lobuloalveolar development

Agr2 is a putative protein disulfide isomerase (PDI) initially identified as an estrogen-responsive gene in breast cancer cell lines. While Agr2 expression in breast cancer is positively correlated with estrogen receptor (ER) expression, it is upregulated in both hormone dependent and independent carcinomas. Several in vitro and xenograft studies have implicated Agr2 in different oncogenic features of breast cancer; however, the physiological role of Agr2 in normal mammary gland development remains to be defined. Agr2 expression is developmentally regulated in the mammary gland, with maximum expression during late pregnancy and lactation. Using a mammary gland specific knockout mouse model, we show that Agr2 facilitates normal lobuloalveolar development by regulating mammary epithelial cell proliferation; we found no effects on apoptosis in Agr2(-/-) mammary epithelial cells. Consequently, mammary glands of Agr2(-/-) females exhibit reduced expression of milk proteins, and by two weeks post-partum their pups are smaller in size. Utilizing a conditional mouse model, we show that Agr2 constitutive expression drives precocious lobuloalveolar development and increased milk protein expression in the virgin mammary gland. In vitro studies using knock down and overexpression strategies in estrogen receptor positive and negative mammary epithelial cell lines demonstrate a role for Agr2 in estradiol-induced cell proliferation. In conclusion, the estrogen-responsive Agr2, a candidate breast cancer oncogene, regulates epithelial cell proliferation and lobuloalveolar development in the mammary gland. The pro-proliferative effects of Agr2 may explain its actions in early tumorigenesis.     

Insulin-like growth factor binding protein (IGFBP)-5 is upregulated during both differentiation and apoptosis in primary cultures of mouse mammary epithelial cells

We have previously demonstrated that insulin-like growth factor binding protein-5 (IGFBP-5) is upregulated following treatment of the mouse mammary epithelial cell line HC11 with lactogenic hormones (dexamethasone, insulin, and prolactin-DIP). In addition, we have also shown that IGFBP-5 is upregulated in mammary epithelial cells in vivo during involution of the rodent mammary gland. We have, therefore, postulated that there may be a dual regulation of IGFBP-5 expression during the temporally separated processes of differentiation and apoptosis of mammary epithelial cells. To test this hypothesis further, we have used a phenotypically differentiated model, which comprises primary cultures of mouse mammary epithelial cells grown on a layer of EHS (Engelbreth-Holm-Swarm) extracellular matrix. We show that lactogenic hormone treatment (hydrocortisone, insulin, and prolactin-HIP) of these cultures induces the upregulation of IGFBP-5 thus replicating the results obtained with the HC11 cell line. In addition, following the induction of apoptosis in primary cultures of mammary epithelial cells by treatment with TGFbeta-3, IGFBP-5 expression is also upregulated. In parallel with this upregulation of IGFBP-5, there is also an increase in the levels of cleaved caspase-3, a well-characterized marker of cellular apoptosis. These findings confirm previous in vivo work demonstrating an increase in IGFBP-5 expression during involution of the mouse mammary gland. When HC11 cells are cultured under serum-free conditions (a well-characterized apoptotic insult in cell culture), there is also an increase in cleaved caspase-3 levels. Unexpectedly, in the presence of TGFbeta-3, caspase-3 levels are attenuated. In the presence of DIP, caspase-3 levels are also decreased in HC11 cells. As described previously, TGFbeta-3 inhibits beta-casein synthesis in HC11 cells. In the HC11 cell line (in contrast to primary cultures of mammary epithelial cells), there is no evidence for TGFbeta-3 induction of IGFBP-5 under either serum-free or DIP-supplemented conditions. We believe our data with primary cultures of mammary epithelial cells support the hypothesis of dual regulation of IGFBP-5 expression during both differentiation and apoptosis in the mammary gland and emphasizes the importance of using appropriate cell culture models to investigate such phenomena in this tissue. We discuss the possible implications of our observations in relation to the physiological processes of pregnancy, lactation, and involution in the mammary gland and the associated changes in mammary epithelial cell function.     

Receptor activator of NF-kappaB ligand regulates the proliferation of mammary epithelial cells via Id2

Receptor activator of NF-kappaB ligand (RANKL) is a key regulator for mammary gland development during pregnancy. RANKL-deficient mice display impaired development of lobulo-alveolar mammary structures. Similar mammary gland defects have been reported in mice lacking Id2. Here we report that RANKL induces the proliferation of mammary epithelial cells via Id2. RANKL triggers marked nuclear translocation of Id2 in mammary epithelial cells. In vivo studies further demonstrated the defective nuclear translocation of Id2, but the normal expression of cyclin D1, in the mammary epithelial cells of rankl-/- mice. In vitro studies with nuclear localization sequence-tagged Id2 revealed that the nuclear localization of Id2 itself is critical for the downregulation of p21 promoter activity. Moreover, RANKL stimulation failed to induce cell growth and to downregulate p21 expression in Id2-/- mammary epithelial cells. Our results indicate that the inhibitor of helix-loop-helix protein, Id2, is critical to control the proliferation of mammary epithelial cells in response to RANKL stimulation.     

Mouse mammary epithelial histamine system.

Histamine is suggested to play a role in mammary gland growth regulation, differentiation and functioning during pregnancy and lactation. Two pools of histamine are thought to be involved in these processes: mastocyte- and epithelial cell related histamine. In the present study we focused on epithelial cells. Immunohistochemistry has shown that the epithelial cells positive for histamine and L-histidine decarboxylase (HDC), the primary enzyme regulating histamine biosynthesis, were mainly found in cells forming alveolar structures in the mammary gland. Cultured primary mouse mammary epithelial cells (MMEC) expressed strong HDC immunoreactivity, especially dividing cells and non-differentiated ones. Histidine decarboxylase activity undergoes significant changes during pregnancy and lactation. Pregnancy associated intensive growth of the mammary gland coincided with an increase and the first days of lactation with a decrease of HDC protein expression. Binding studies with mammary tissue membranes and epithelial cell membranes revealed the presence of H1 and H3 but not H2 receptors. Summarizing, our data have shown that mammary epithelial cells are capable of synthesizing and excreting histamine and they bear histamine receptors. These findings further substantiate the role of histamine in mammary gland physiology.     

Cellular prion protein in the bovine mammary gland is selectively expressed in active lactocytes.

The cellular prion protein (PrP(c)) is a highly conserved glycoprotein with a still enigmatic physiological function. It is mainly expressed in the central nervous system but accumulating data suggest that PrP(c) is also found in a broad spectrum of non-neuronal tissue. Here we investigated the cell-type-related PrP(c) expression in the bovine mammary gland by using immunohistochemistry (IHC), ELISA, Western blot, and real-time RT-PCR. Specific immunostaining of serial sections revealed that PrP(c) is selectively localized in mammary gland epithelial cells. Particularly strong expression was found at the basolateral surface of those cells showing active secretion. Results obtained by RT-PCR and ELISA complemented IHC findings. No correlation was found between the level of PrP(c) expression and other parameters such as age of the animals under study or stage of lactation.     

Biphasic regulation of mammary epithelial resistance by serotonin through activation of multiple pathways

Mammary gland homeostasis and the lactation-to-involution switch are regulated by serotonin (5-hydroxytryptamine (5-HT)). Mammary epithelial tight junctions are physiological targets of 5-HT, and their disruption marks an early stage of mammary gland involution. In these studies, we have identified signal transduction mechanism employed by 5-HT during regulation of mammary gland transepithelial resistance. Transepithelial electrical resistance and tight junction protein architecture were studied in cultures of MCF10A human mammary epithelial cells. Serotonin had biphasic effects on mammary epithelial resistance. At lower concentrations and earlier time points, 5-HT potentiated epithelial transmembrane resistance, whereas at higher concentrations and later time points, 5-HT decreased transepithelial electrical resistance and disrupted tight junctions. Both the early and delayed actions of 5-HT were mediated by the 5-HT7 receptor through activation of G(s)/cAMP. 5-HT induced the activities of both protein kinase A and p38 mitogen-activated protein kinase. Inhibition of p38 mitogen-activated protein kinase abrogated 5-HT-induced disruption of mammary epithelial tight junctions (the delayed effect). In contrast, inhibition of protein kinase A prevented the increased epithelial resistance in response to 5-HT (the transient effect). These studies imply an integrated set of mechanisms whereby transient, modest activation of 5-HT7 promotes tight junction integrity, and sustained 5-HT7 activation drives involution by disrupting tight junctions.