Origin,concentration and structural features of human mammary gland cells cultured from breast secretions

Summary This study traced the origin of cells observed in human breast secretion samples obtained during lactation and describes the appearance of these cells following prolonged maintenance in vitro. Human milk contains a large number of single vacuolated foam cells and a small proportion of non-vacuolated epithelial cells in clusters. Foam cells are identified by their large size, the polarity of their cytoplasmic organelles, the variation in number and size of lipid vacuoles and the condensed chromatin of their eccentrically located nucleus. Both cell types originate by exfoliation from the mammary gland. This was established by comparing the structural characteristics of cells isolated from milk with those of the cuboidal cell linings of ducts and alveoli in lactating mammary tissue. Relatively pure populations of foam cells could be established from early lactation samples (3–7 days post/partum) while non-vacuolated epithelial cell clusters were more frequently cultured from late lactation specimens (1–10 days postweaning). Foam cells did not divide and lost cytoplasmic organization during prolonged culture. In contrast, non-vacuolated epithelium in clusters proliferated to form colonies of polygonal cells. These results, which imply that foam cells are an active form of the non-vacuolated mammary cells in clusters, call attention to one system for the study of the complex hormonal interactions necessary to induce and maintain lactation.Supported in part by NCI contract NO 1-CB-33898     

Estrogen receptor alpha is required for mammary development and the induction of mammary hyperplasia and epigenetic alterations in the aromatase transgenic mice

Aromatase transgenic mice exhibit hyperplastic and dysplastic changes, attesting to the importance of local estrogen in breast carcinogenesis. These mice also show increased levels of the estrogen receptor and β (ER, ERβ) suggesting that this receptor may play an important role in the initiation of estrogen-mediated mammary hyperplasia observed in these mice. To address the specific role of ER in the mammary development and in the induction of estrogen-mediated hyperplasia in aromatase transgenic mice, we have generated MMTV-aromatase × ER knockout cross (referred as aromatase/ERKO). Even though ERβ is expressed in aromatase/ERKO mice, lack of ER leads to impaired mammary growth in these mice. The data suggest that ER plays an important role in the mammary gland development as well as in the induction of mammary hyperplasia in aromatase transgenic mice. Lack of ER expression in the aromatase/ERKO mice resulted in a decrease in the expression of Cyclin D1, PCNA and TGFβ relative to the aromatase parental strain. The studies involving aromatase/ERKO mice show that lack of ER results in impaired mammary development even in the presence of continuous tissue estrogen, suggesting estrogen/ER-mediated actions are critical for mammary development and carcinogenesis.     

The RANKL signaling axis is sufficient to elicit ductal side-branching and alveologenesis in the mammary gland of the virgin mouse

Receptor of Activated NF-κB Ligand (RANKL) is implicated as one of a number of effector molecules that mediate progesterone and prolactin signaling in the murine mammary epithelium. Using a mouse transgenic approach, we demonstrate that installation of the RANKL signaling axis into the mammary epithelium results in precocious ductal side-branching and alveologenesis in the virgin animal. These morphological changes occur due to RANKL-induced mammary epithelial proliferation, which is accompanied by increases in expression of activated NF-kB and cyclin D1. With age, prolonged RANKL exposure elicits limited mammary epithelial hyperplasia. While these transgenics exhibit RANKL-induced salivary gland adenocarcinomas, palpable mammary tumors are not observed due to RANKL-suppression of its own signaling receptor (RANK) in the mammary epithelium. Together, these studies reveal not only that the RANKL signaling axis can program many of the normal epithelial changes attributed to progesterone and prolactin action in the normal mammary gland during early pregnancy, but underscore the necessity for tight control of this signaling molecule to avoid unwarranted developmental changes that could lead to mammary hyperplasia in later life.     

Structure and function of the human calcium-sensing receptor: insights from natural and engineered mutations and allosteric modulators

The human extracellular Ca(2+)-sensing receptor (CaR), a member of the G protein-coupled receptor family 3, plays a key role in the regulation of extracellular calcium homeostasis. It is one of just a few G protein-coupled receptors with a large number of naturally occurring mutations identified in patients. In contrast to the small sizes of its agonists, this large dimeric receptor consists of domains with topologically distinctive orthosteric and allosteric sites. Information derived from studies of naturally occurring mutations, engineered mutations, allosteric modulators and crystal structures of the agonist-binding domain of homologous type 1 metabotropic glutamate receptor and G protein-coupled rhodopsin offers new insights into the structure and function of the CaR.     

Annexin II (calpactin I) in the mouse mammary gland: immunolocalisation by light- and electron microscopy

Summary To elucidate the putative role of annexin II (calpactin I) in the secretory function of mammary tissue its immunolocalisation in the mammary gland of pregnant and lactating mice was investigated by light- and electron microscopy using the immunoperoxidase technique. A low level of fairly uniform annexin II staining was evident throughout the gland despite its mixed composition during pregnancy. In lactating tissue it was revealed that apparently mature alveoli contained a concentration of annexin II staining outlining their epithelium. The staining was localised by immuno-electron microscopy to the apical membrane of these alveolar epithelial cells and their microvillar extentions. There was also an apparent association of annexin II with vesicles of a range of sizes located near, or actually fused with, the apical membrane. Many of the small, stained vasicles could clearly be identified as casein-containing vesicle while the large vesicles were apparently associated with either casein granules or possibly lipid. The appearance of a selective concentration of annexin II in apparently actively secreting mammary epithelial cells, as revealed in this study, is consistent with a possible structural and/or functional role for this protein at the membranes participating in the secretion of protein and possibly lipid from these secretory cells.     

Localization of plasma membrane and secretory calcium pumps in the mammary gland

Until recently the mechanism for the enrichment of milk with calcium was thought to be almost entirely via the secretory pathway. However, recent studies suggest that a plasma membrane calcium ATPase, PMCA2, is the primary mechanism for calcium transport into milk, highlighting a major role for apical calcium transport. We compared the expression of the recently identified secretory calcium ATPase, SPCA2, and SPCA1, in the mouse mammary gland during development. SPCA2 levels increased over 35-fold during lactation with expression localized to luminal secretory cells, while SPCA1 increased only a modest 2-fold and was expressed throughout the cells of the mammary gland. We also observed major differences in the localization of PMCA2 and PMCA1. Our studies highlight the likely specific roles of PMCA2 and SPCA2 in lactation and indicate that calcium transport into milk is a complex interplay between apical and secretory pathways.     

Cyclin-dependent kinase inhibitor p27(Kip1) is required for mouse mammary gland morphogenesis and function

We have studied the role of the cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) in postnatal mammary gland morphogenesis. Based on its ability to negatively regulate cyclin/Cdk function, loss of p27 may result in unrestrained cellular proliferation. However, recent evidence about the stabilizing effect of p27 on cyclin D1-Cdk4 complexes suggests that p27 deficiency might recapitulate the hypoplastic mammary phenotype of cyclin D1-deficient animals. These hypotheses were investigated in postnatal p27-deficient (p27(-/-)), hemizygous (p27(+/)-), or wild-type (p27(+/+)) mammary glands. Mammary glands from p27(+/)- mice displayed increased ductal branching and proliferation with delayed postlactational involution. In contrast, p27(-/-) mammary glands or wild-type mammary fat pads reconstituted with p27(-/-) epithelium produced the opposite phenotype: hypoplasia, low proliferation, decreased ductal branching, impaired lobuloalveolar differentiation, and inability to lactate. The association of cyclin D1 with Cdk4, the kinase activity of Cdk4 against pRb in vitro, the nuclear localization of cyclin D1, and the stability of cyclin D1 were all severely impaired in p27(-/-) mammary epithelial cells compared with p27(+/+) and p27(+/-) mammary epithelial cells. Therefore, p27 is required for mammary gland development in a dose-dependent fashion and positively regulates cyclin D-Cdk4 function in the mammary gland.     

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.     

The human bitter taste receptor hTAS2R39 is the primary receptor for the bitterness of theaflavins

We purified several hundred mgs of four major theaflavins (theaflavin, theaflavin-3-O-gallate, theaflavin-3′-O-gallate, and theaflavin-3,3′-O-digallate). Among the 25 hTAS2Rs expressed in HEK293T cells, hTAS2R39 and hTAS2R14 were activated by theaflavins. Both hTAS2R39 and hTAS2R14 responded to theaflavin-3′-O-gallate. In addition, hTAS2R39 was activated by theaflavin and theaflavin-3,3′-O-gallate, but not by theaflavin-3-O-gallate. In contrast, hTAS2R14 responded to theaflavin-3-O-gallate.     

Unstructured loop is essential for the activation of mGluR2

Metabotropic Glutamate Receptors (mGluRs) are Class C G-protein coupled receptors (GPCRs) that are expressed throughout the central nervous system and are involved in several neurological and psychiatric disorders. Although, many studies focused on Glutamate induced activation of mGluR2, however, the role of unstructured loop (or “BC loop”) in activation of metabotropic Glutamate receptors is currently unknown. Here, using Förster Resonance Energy Transfer (FRET) based assay in live cells we show that unstructured loop is required for Glutamate induced conformation and hence the activation of the receptor.     

Vascular smooth muscle cells from small human omental arteries express P2X1 and P2X4 receptor subunits

Stimulation of P2X receptors by ATP in vascular smooth muscle cells (VSMCs) is proposed to mediate vascular tone. However, understanding of P2X receptor-mediated actions in human blood vessels is limited, and therefore, the current work investigates the role of P2X receptors in freshly isolated small human gastro-omental arteries (HGOAs). Expression of P2X1 and P2X4 receptor subunit messenger RNA (mRNA) and protein was identified in individual HGOA VSMCs using RT-PCR and immunofluorescent analysis and using Western blot in multi-cellular preparations. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l, a selective P2X receptor agonist, evoked robust increases in [Ca²⁺]_i in fluo-3-loaded HGOA VSMCs. Pre-incubation with 1 μmol/l NF279, a selective P2X receptor antagonist, reduced the amplitude of αβ-meATP-induced increase in [Ca²⁺]_i by about 70 %. ATP of 10 μmol/l and αβ-meATP of 10 μmol/l produced similar contractile responses in segments of HGOA, and these contractions were greatly reduced by 2 μmol/l NF449, a selective P2X receptor inhibitor. These data suggest that VSMCs from HGOA express P2X1 and P2X4 receptor subunits with homomeric P2X1 receptors likely serving as the predominant target for extracellular ATP.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-014-9415-6) contains supplementary material, which is available to authorized users.     

Fluvastatin suppresses native and recombinant human P2X4 receptor function

Statins have both cholesterol lowering and anti-inflammatory activities, whether mechanisms underlying their activities are independent remains unclear. The ATP-gated P2X(4) receptor is a pro-inflammatory mediator. Here, we investigate the action of fluvastatin and other cholesterol depleting agents on native and recombinant human P2X(4) receptor. Fluvastatin and mβCD suppressed P2X(4)-dependent calcium influx in THP-1 monocytes, without affecting P2Y receptor responses. mβCD or filipin III suppressed the current density of recombinant human P2X(4) receptors. Human P2X(2) was insensitive to cholesterol depletion. Cholesterol depletion had no effect on intrinsic P2X(4) receptor properties as judged by ATP concentration-response relationship, receptor rundown or current decay during agonist occupancy. These data suggest fluvastatin suppresses P2X(4) activity in monocytes through cholesterol depletion and not by modulating intrinsic channel properties.