Analysis of Mammary Gland Phenotypes by Transplantation of the Genetically Marked Mammary Epithelium

SummaryDuring puberty, robust morphogenesis occurs in the mammary gland; stem- and progenitor-cells develop into mature basal- and luminal-cells to form the ductal tree. The receptor signals that govern this process in mammary epithelial cells (MECs) are incompletely understood. The EGFR has been implicated and here we focused on EGFR’s downstream pathway component Rasgrp1. We find that Rasgrp1 dampens EGF-triggered signals in MECs. Biochemically and in vitro, Rasgrp1 perturbation results in increased EGFR-Ras-PI3K-AKT and mTORC1-S6 kinase signals, increased EGF-induced proliferation, and aberrant branching-capacity in 3D cultures. However, in vivo, Rasgrp1 perturbation results in delayed ductal tree maturation with shortened branches and reduced cellularity. Rasgrp1-deficient MEC organoids revealed lower frequencies of basal cells, the compartment that incorporates stem cells. Molecularly, EGF effectively counteracts Wnt signal-driven stem cell gene signature in organoids. Collectively, these studies demonstrate the need for fine-tuning of EGFR signals to properly instruct mammary epithelium during puberty.

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Loss of vitamin D receptor signaling from the mammary epithelium or adipose tissue alters pubertal glandular development

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00200.2014 ◽

2014 ◽

Vol 307 (8) ◽

pp. E674-E685 ◽

Cited By ~ 13

Author(s):

Abby L. Johnson ◽

Glendon M. Zinser ◽

Susan E. Waltz

Keyword(s):

Vitamin D ◽

Adipose Tissue ◽

Mammary Gland ◽

Epithelial Cell ◽

Epithelial Cells ◽

Mammary Epithelium ◽

Mammary Development ◽

Mammary Epithelial ◽

Cell Type ◽

Pubertal Mammary Development

Vitamin D3 receptor (VDR) signaling within the mammary gland regulates various postnatal stages of glandular development, including puberty, pregnancy, involution, and tumorigenesis. Previous studies have shown that vitamin D3 treatment induces cell-autonomous growth inhibition and differentiation of mammary epithelial cells in culture. Furthermore, mammary adipose tissue serves as a depot for vitamin D3 storage, and both epithelial cells and adipocytes are capable of bioactivating vitamin D3. Despite the pervasiveness of VDR in mammary tissue, individual contributions of epithelial cells and adipocytes, as well as the VDR-regulated cross-talk between these two cell types during pubertal mammary development, have yet to be investigated. To assess the cell-type specific effect of VDR signaling during pubertal mammary development, novel mouse models with mammary epithelial- or adipocyte-specific loss of VDR were generated. Interestingly, loss of VDR in either cellular compartment accelerated ductal morphogenesis with increased epithelial cell proliferation and decreased apoptosis within terminal end buds. Conversely, VDR signaling specifically in the mammary epithelium modulated hormone-induced alveolar growth, as ablation of VDR in this cell type resulted in precocious alveolar development. In examining cellular cross-talk ex vivo, we show that ligand-dependent VDR signaling in adipocytes significantly inhibits mammary epithelial cell growth in part through the vitamin D3-dependent production of the cytokine IL-6. Collectively, these studies delineate independent roles for vitamin D3-dependent VDR signaling in mammary adipocytes and epithelial cells in controlling pubertal mammary gland development.

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Transforming growth factor beta3 induces cell death during the first stage of mammary gland involution

Development ◽

10.1242/dev.127.14.3107 ◽

2000 ◽

Vol 127 (14) ◽

pp. 3107-3118 ◽

Cited By ~ 2

Author(s):

A.V. Nguyen ◽

J.W. Pollard

Keyword(s):

Cell Death ◽

Mammary Gland ◽

Transforming Growth Factor ◽

Alveolar Epithelium ◽

Mammary Epithelium ◽

Significant Inhibition ◽

Mammary Tissue ◽

Lactogenic Hormone ◽

Null Mutant Mice ◽

Transforming Growth Factor Beta3

Involution of the mammary gland following weaning is divided into two distinct phases. Initially, milk stasis results in the induction of local factors that cause apoptosis in the alveolar epithelium. Secondly after a prolonged absence of suckling, the consequent decline in circulating lactogenic hormone concentrations initiates remodeling of the mammary gland to the virgin-like state. We have shown that immediately following weaning TGFbeta3 mRNA and protein is rapidly induced in the mammary epithelium and that this precedes the onset of apoptosis. Unilateral inhibition of suckling and hormonal reconstitution experiments showed that TGFbeta3 induction is regulated by milk stasis and not by the circulating hormonal concentration. Directed expression of TGFbeta3 in the alveolar epithelium of lactating mice using a beta-lactoglobulin promoter mobilized SMAD4 translocation to the nucleus and caused apoptosis of these cells, but not tissue remodeling. Transplantation of neonatal mammary tissue derived from TGFbeta3 null mutant mice into syngenic hosts resulted in a significant inhibition of cell death compared to wild-type mice upon milk stasis. These results provide direct evidence that TGFbeta3 is a local mammary factor induced by milk stasis that causes apoptosis in the mammary gland epithelium during involution.

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Selective Response to Insulin Versus Insulin-Like Growth Factor-I and -II and Up-Regulation of Insulin Receptor Splice Variant B in the Differentiated Mouse Mammary Epithelium

Endocrinology ◽

10.1210/en.2008-0668 ◽

2009 ◽

Vol 150 (6) ◽

pp. 2924-2933 ◽

Cited By ~ 29

Author(s):

Chiara Berlato ◽

Wolfgang Doppler

Keyword(s):

Mammary Gland ◽

Insulin Receptor ◽

Lactate Production ◽

Terminal Differentiation ◽

Mammary Epithelium ◽

Epithelial Cell Line ◽

Human Form ◽

Igf Binding Proteins ◽

Stage Of Development ◽

Hc11 Cells

The terminal differentiation of the mouse mammary gland epithelium during lactation has been shown to require IGFs and/or superphysiological levels of insulin. It has been suggested that IGF receptor I (IGF-IR), in addition to its well-established role in the mammary gland during puberty and pregnancy, serves as the principal mediator of IGFs at this stage of development. However, our analysis of the expression levels of IGF-IR and the two insulin receptor (IR) splice variants, IR-A and IR-B, has revealed a 3- to 4-fold up-regulation of IR-B transcripts and a 6-fold down-regulation of IGF-IR transcripts and protein during terminal differentiation in the developing mammary gland. IR-B expression was also more than 10-fold up-regulated in murine mammary epithelial cell line HC11 during differentiation in vitro. As already described for the human form, murine IR-B cloned from HC11 exhibited selectivity for insulin as compared with IGFs. When differentiated HC11 cells were stimulated by 10 nm insulin, a concentration that is unable to activate IGF-IR, induction of milk protein and lipid synthetic enzyme gene expression, lactate production, and phosphorylation of Akt were observed. In contrast, on differentiated HC11 cells 10 nm IGF-I or 10 nm IGF-II were able to exert growth-promoting effects only. The lack of response of differentiated cells to low levels of IGFs could not be explained by inactivation of IGFs by IGF binding proteins. Our results suggest a previously unrecognized predominant role for IR-B in the differentiated mammary epithelium.

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Expression of xanthine oxidoreductase in mouse mammary epithelium during pregnancy and lactation: regulation of gene expression by glucocorticoids and prolactin

Biochemical Journal ◽

10.1042/bj3190801 ◽

1996 ◽

Vol 319 (3) ◽

pp. 801-810 ◽

Cited By ~ 32

Author(s):

Mami KUROSAKI ◽

Stefania ZANOTTA ◽

Marco LI CALZI ◽

Enrico GARATTINI ◽

Mineko TERAO

Keyword(s):

Gene Expression ◽

Mammary Gland ◽

Regulation Of Gene Expression ◽

Mammary Epithelium ◽

Receptor Activation ◽

Normal Breast ◽

Enzymic Activity ◽

Alveolar Epithelial Cells ◽

Xanthine Oxidoreductase ◽

Normal Breast Epithelium

In the mammary gland of virgin mice, xanthine oxidoreductase (XOR) enzymic activity is barely measurable. A high increase in the levels of the enzyme is observed during the last days of pregnancy and during lactation, and this is parallelled by an elevation in the amounts of the respective protein and transcript. In situ hybridization experiments demonstrate that the XOR mRNA is specifically expressed in the alveolar epithelial cells of the mammary gland. In HC11 cells, a model culture system for normal breast epithelium, the levels of XOR enzymic activity are dose- and time-dependently induced by dexamethasone, and a further synergistic augmentation is observed in the presence of dexamethasone plus prolactin. Increased XOR gene expression is consequent on glucocorticoid receptor activation, as indicated by sensitivity to the specific receptor antagonist RU486. In addition, the phenomenon is likely to involve protein phosphorylation and dephosphorylation events, as suggested by modulation of XOR mRNA by tyrosine kinase and phosphatase inhibitors.

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Post-secretory aggregation of caseins

Journal of Dairy Research ◽

10.1017/s0022029900017039 ◽

1979 ◽

Vol 46 (2) ◽

pp. 193-195 ◽

Cited By ~ 6

Author(s):

Brian E. Brooker ◽

Carl Holt

Keyword(s):

Mammary Gland ◽

Limited Proteolysis ◽

Mammary Epithelium ◽

Casein Micelles ◽

Golgi Vesicles

SUMMARYParticles as large as several µm diam. have been observed occasionally in normal milk and commonly in prepartum and postpartum colostrum. These particles can be dissociated by EDTA and their appearance closely resembles that of normal casein micelles. However, they are often too large to have been completely formed within the Golgi vesicles of mammary epithelium and hence some degree of post-secretory aggregation of caseins is thought to occur. Two possible mechanisms of post-secretory aggregation of caseins are: (1) a continuation of the normal processes of micelle assembly in the alveolus and (2) aggregation as a result of limited proteolysis of the caseins during the time the milk is in the mammary gland. Incubation of milk with fibrinolysin, however, failed to produce aggregation of normal micelles.

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Tissue distribution of the cellular binding protein for the mouse mammary tumour virus

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0070169 ◽

1991 ◽

Vol 7 (3) ◽

pp. 169-174 ◽

Cited By ~ 7

Author(s):

F. F. Bolander ◽

M. E. Blackstone

Keyword(s):

Salivary Gland ◽

Mammary Gland ◽

Binding Protein ◽

Mammary Epithelium ◽

Late Pregnancy ◽

Cell Surface Receptor ◽

Mammary Tumour ◽

Isolated Cells ◽

Mouse Mammary Tumour Virus ◽

Mouse Tissues

ABSTRACT The envelope protein, gp52, of the mouse mammary tumour virus (MMTV) binds to a cell-surface receptor as a first step in infection. A protein with characteristics of this receptor was measured on freshly isolated cells using, as ligand, 125I-labelled gp52 purified from C3H/HeN mice. The gp52-binding protein was found in all mouse tissues examined, but was present at highest concentrations in the mammary gland and spleen where it reached 4.2±0.3 (s.e.m.) pmol/mg protein; the dissociation constant was 30±7 pm. Binding to mammary epithelium could be displaced by either the RIII or 34I-R strains of MMTV, and binding was blocked by antibodies to gp52. Levels in the liver and adrenal glands were only 25% of those in the mammary gland, while the concentrations in the ovary and salivary gland were intermediate. Scatchard analyses of the binding data suggested that there was only a single set of high-affinity binding sites. During late pregnancy and lactation, receptor levels in mammary epithelium rose threefold, while those in the liver and salivary gland were unchanged. This induction would result in the mammary gland having 12 times the gp52-binding protein than other tissues and may result in the preferential reinfection of this tissue during lactation, with subsequent tumour formation.

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Electrical potentials and cell-to-cell dye movement in mouse mammary gland during lactation

AJP Cell Physiology ◽

10.1152/ajpcell.1984.247.1.c20 ◽

1984 ◽

Vol 247 (1) ◽

pp. C20-C25 ◽

Cited By ~ 32

Author(s):

S. E. Berga

Keyword(s):

Mammary Gland ◽

Lucifer Yellow ◽

Mammary Epithelium ◽

Mouse Mammary Gland ◽

Dye Transfer ◽

Alveolar Cells ◽

Electrical Potentials ◽

Lucifer Yellow Ch ◽

Electrophysiological Studies

Stable potentials were recorded with microelectrodes in an in vivo preparation of the mammary gland from the anesthetized lactating mouse. Location of the microelectrode tip was determined by ionophoretic injection of the fluorescent dye Lucifer yellow CH. Fifteen dye injections were localized to mammary alveolar cells; the average recorded potential for these penetrations was -49 +/- 2 mV. Cell-to-cell dye transfer between alveolar cells was observed with all intracellular Lucifer yellow injections. Ten dye injections were localized to the alveolar lumina with an average recorded potential of -35 +/- 2 mV. With these penetrations Lucifer yellow spread rapidly to many alveolar lumina. These findings indicate that stable potentials can be obtained from both cells and lumina in the in vivo mammary gland, demonstrating the feasibility of electrophysiological studies of the mammary epithelium. The presence of a large transepithelial potential provides evidence for physiologically tight junctions between mammary alveolar cells. In addition, the distribution of Lucifer yellow shows that mammary alveolar cells are coupled and suggests that milk flows freely between alveolar lumina.

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