scholarly journals Requirement of basement membrane for the suppression of programmed cell death in mammary epithelium

1996 ◽  
Vol 109 (3) ◽  
pp. 631-642 ◽  
Author(s):  
S. Pullan ◽  
J. Wilson ◽  
A. Metcalfe ◽  
G.M. Edwards ◽  
N. Goberdhan ◽  
...  
Keyword(s):  
Cell Death ◽  
Mammary Gland ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Single Cells ◽  
Control Point ◽  
Collagen I ◽  
Cell Phenotype ◽  
Cell Contact ◽  
Isolated Cells

Apoptosis is an active mechanism of cell death required for normal tissue homeostasis. Cells require survival signals to avoid the engagement of apoptosis. In the mammary gland, secretory epithelial cells are removed by apoptosis during involution. This cell loss coincides with matrix metalloproteinase activation and basement membrane degradation. In this paper we describe studies that confer a new role for basement membrane in the regulation of cell phenotype. We demonstrate that the first passage epithelial cells isolated from pregnant mouse mammary gland die by apoptosis in culture, but that cell death is suppressed by basement membrane. The correct type of extracellular matrix was required, since only a basement membrane, not plastic or a collagen I matrix, lowered the rate of apoptosis. Attachment to a matrix per se was not sufficient for survival, since apoptotic cells were observed when still attached to a collagen I substratum. Experiments with individually isolated cells confirmed the requirement of basement membrane for survival, and demonstrated that survival is enhanced by cell-cell contact. A function-blocking anti-beta1 integrin antibody doubled the rate of apoptosis in single cells cultured with basement membrane, indicating that integrin-mediated signals contributed to survival. We examined the cell death-associated genes bcl-2 and bax in mammary epithelia, and found that although the expression of Bcl-2 did not correlate with cell survival, increased levels of Bax were associated with apoptosis. We propose that basement membrane provides a survival stimulus for epithelial cells in vivo, and that loss of interaction with this type of matrix acts as a control point for cell deletions that occur at specific times during development, such as in mammary gland involution.

scholarly journals Development of mouse mammary gland: identification of stages in differentiation of luminal and myoepithelial cells using monoclonal antibodies and polyvalent antiserum against keratin.

1986 ◽  
Vol 34 (8) ◽  
pp. 1037-1046 ◽  
Author(s):  
A Sonnenberg ◽  
H Daams ◽  
M A Van der Valk ◽  
J Hilkens ◽  
J Hilgers
Keyword(s):  
Monoclonal Antibodies ◽  
Mammary Gland ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Cell Types ◽  
Mouse Mammary Gland ◽  
Type I ◽  
Basal Cells ◽  
Alveolar Cells ◽  
Luminal Type

The development of the mouse mammary gland was studied immunohistochemically using monoclonal antibodies against cell surface and basement membrane proteins and a polyclonal antibody against keratin. We have identified three basic cell types: basal, myoepithelial, and epithelial cells. The epithelial cells can be subdivided into three immunologically related cell types: luminal type I, luminal type II, and alveolar cells. These five cell types appear at different stages of mammary gland development and have either acquired or lost one of the antibody-defined antigens. The cytoplasmic distribution of several of these antigens varied according to the location of the cells within the mammary gland. Epithelial cells which did not line the lumen expressed antigens throughout the cytoplasm. These antigens were demonstrated on the apical site in situations where the cells lined the lumen. One antigen became increasingly basolateral as the cells became attached to the basement membrane. The basal cells synthesize laminin and deposit it at the cell base. They are present in endbuds and ducts and are probably the stem cells of the mammary gland. Transitional forms have been demonstrated which developmentally link these cells with both myoepithelial and (luminal) epithelial cells.

scholarly journals Prototheca zopfiiGenotype II induces mitochondrial apoptosis in models of bovine mastitis

2019 ◽  
Author(s):  
Muhammad Shahid ◽  
Eduardo R. Cobo ◽  
Liben Chen ◽  
Paloma A. Cavalcante ◽  
Herman W. Barkema ◽  
...  
Keyword(s):  
Cell Death ◽  
Mammary Gland ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Bovine Mastitis ◽  
Mammary Epithelial ◽  
Mitochondrial Apoptosis ◽  
Prototheca Zopfii ◽  
Severe Inflammation ◽  
Mammary Tissues

AbstractPrototheca zopfiiis an alga increasingly isolated from bovine mastitis. Of the two genotypes ofP. zopfii(genotype I and II (GT-I and II)),P. zopfiiGT-II is the genotype associated with acute mastitis and decreased milk production by unknown mechanisms. The objective was to determine inflammatory and apoptotic roles ofP. zopfiiGT-II in cultured mammary epithelial cells (from cattle and mice) and murine macrophages and using a murine model of mastitis.Prototheca zopfiiGT-II (but not GT-I) invaded bovine and murine mammary epithelial cells (MECs) and induced apoptosis, as determined by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay. ThisP. zopfiiGT-II driven apoptosis corresponded to mitochondrial pathways; mitochondrial transmembrane resistance (ΔΨm) was altered and modulation of mitochondrion-mediated apoptosis regulating genes changed (increased transcriptionalBax, cytochrome-c andApaf-1and downregulatedBcl-2), whereas caspase-9 and -3 expression increased. Apoptotic effects byP. zopfiiGT-II were more pronounced in macrophages compared to MECs. In a murine mammary infection model,P. zopfiiGT-II replicated in the mammary gland and caused severe inflammation with infiltration of macrophages and neutrophils and upregulation of pro-inflammatory genes (TNF-α,IL-1βandCxcl-1) and also apoptosis of epithelial cells. Thus, we concludedP. zopfiiGT-II is a mastitis-causing pathogen that triggers severe inflammation and also mitochondrial apoptosis.Author summaryBovine mastitis (inflammation of the udder) reduces milk production and quality, causing huge economic losses in the dairy industry worldwide. Although the algaPrototheca zopfiiis a major cause of mastitis in dairy cows, mechanisms by which it damages mammary tissues are not well known. Here, we used cell cultures and a mouse model of mastitis to determine howProtothecacaused inflammation and cell death in mammary tissues.Protothecainvaded mammary gland cells, from cattle and mice, as well as macrophages (white cells that take up and kill pathogens) and caused cell death by interfering with mitochondria. Furthermore,Protothecacauses severe inflammation and tissue damage when injected into the mammary glands of mice. Although there are two genotypes ofP. zopfii, only genotype II causes tissue damage, whereas gentotype I, common in farm environments, does not damage mammary tissues. SinceP. zopfiiis an alga and not a bacterium, antibiotic treatments, frequently used to treat mastitis in cattle, are not effective against this organism. Understanding howP. zopfiidamages mammary tissue and causes mastitis is important new knowledge to promote future development of evidence-based approaches to prevent and treat mammary gland infections with this organism.

scholarly journals Developmental regulation of Bcl-2 family protein expression in the involuting mammary gland

1999 ◽  
Vol 112 (11) ◽  
pp. 1771-1783 ◽  
Author(s):  
A.D. Metcalfe ◽  
A. Gilmore ◽  
T. Klinowska ◽  
J. Oliver ◽  
A.J. Valentijn ◽  
...  
Keyword(s):  
Cell Death ◽  
Mammary Gland ◽  
Epithelial Cells ◽  
De Novo ◽  
Mammary Epithelial Cells ◽  
Expression Profiles ◽  
Mammary Epithelial ◽  
Mammary Tissue ◽  
Pregnancy And Lactation

Epithelial cells within the mammary gland undergo developmental programmes of proliferation and apoptosis during the pregnancy cycle. After weaning, secretory epithelial cells are removed by apoptosis. To determine whether members of the Bcl-2 gene family could be involved in regulating this process, we have examined whether changes in their expression occur during this developmental apoptotic program in vivo. Bax and Bcl-x were evenly expressed throughout development. However, expression of Bak and Bad was increased during late pregnancy and lactation, and the proteins were present during the time of maximal apoptotic involution. Thereafter, their levels declined. In contrast, Bcl-w was expressed in pregnancy and lactation but was downregulated at the onset of apoptosis. Bcl-2 was not detected in lactating or early involuting mammary gland. Thus, the pro-apoptotic proteins Bax, Bak and Bad, as well as the death-suppressors Bcl-x, Bcl-2 and Bcl-w, are synthesised in mouse mammary gland, and dynamic changes in the expression profiles of these proteins occurs during development. To determine if changes in Bak and Bcl-w expression could regulate mammary apoptosis, their effect on cultured mouse mammary epithelial cells was examined in transient transfection assays. Enforced expression of Bak induced rapid mammary apoptosis, which could be suppressed by coexpression of Bcl-w. In extracts of mammary tissue in vivo, Bak heterodimerized with Bcl-x whereas Bax associated with Bcl-w, but Bak/Bcl-w heterodimers were not detected. Thus, Bak and Bcl-w may regulate cell death through independent pathways. These results support a model in which mammary epithelial cells are primed for apoptosis during the transition from pregnancy to lactation by de novo expression of the death effectors Bak and Bad. It is suggested that these proteins are prevented from triggering apoptosis by anti-apoptotic Bcl-2 family proteins until involution, when the levels of Bcl-w decline. Our study provides evidence that regulated changes in the expression of cell death genes may contribute to the developmental control of mammary apoptosis.

Response of single, pairs, and clusters of epithelial cells to substratum topography

1995 ◽  
Vol 73 (7-8) ◽  
pp. 473-489 ◽  
Author(s):  
C. Oakley ◽  
D. M. Brunette
Keyword(s):  
Epithelial Cells ◽  
Actin Filaments ◽  
Cell Shape ◽  
Single Cells ◽  
Cell Spreading ◽  
Contact Guidance ◽  
Adjacent Cell ◽  
Cell Contact ◽  
Topographic Effects ◽  
Control Surfaces

Cells cultured on grooved substrata change their shape, orientation, and direction of locomotion in response to substratum topography, a phenomenon called contact or topographic guidance. Porcine epithelial cells (E-cells) spread on micromachined grooved or smooth control surfaces were examined by epifluorescence and confocal microscopy to determine area, cell shape, and orientation in conjunction with distributions and orientations of actin filaments and microtubules. Single cells, cells within a pair or cluster, and pairs or clusters considered as a unit were compared. As expected, cell contact increased cell spreading, but surprisingly, increased cell contact influenced cell shape on smooth and grooved surfaces and increased alignment of cells spread on grooves. Both actin filaments and microtubules aligned initially and most consistently along the walls and ridge–groove edges. Single E-cells displayed the least variability of aligned cytoskeletal patterns. E-cells within clusters displayed the most variability as local topographic effects on the cytoskeleton could be overridden by adjacent cell contact. Overall, contact guidance of E-cells was neither synonymous with nor contingent upon an elliptical morphology oriented to the topography. E-cells also differed from fibroblasts in their response to cell contact and in their lack of a relationship between cell polarity and locomotion.Key words: microtubules, actin, topographic guidance, micromachined substrata.

The Human Mammary Gland Basement Membrane Is Integral to the Polarity of Luminal Epithelial Cells

1999 ◽  
Vol 247 (1) ◽  
pp. 267-278 ◽  
Author(s):  
Martin J. Slade ◽  
Rebecca C. Coope ◽  
Jennifer J. Gomm ◽  
R.Charles Coombes
Keyword(s):  
Mammary Gland ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Human Mammary Gland ◽  
Luminal Epithelial Cells

scholarly journals A strategy to address dissociation-induced compositional and transcriptional bias for single-cell analysis of the human mammary gland

2021 ◽  
Author(s):  
Lisa K. Engelbrecht ◽  
Alecia-Jane Twigger ◽  
Hilary M. Ganz ◽  
Christian J. Gabka ◽  
Andreas R. Bausch ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Single Cell ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Cellular Heterogeneity ◽  
Normal Mammary Gland ◽  
Isolated Cells ◽  
Tissue Dissociation ◽  
New Strategy ◽  
Gland Function

SummarySingle-cell transcriptomics provide insights into cellular heterogeneity and lineage dynamics that are key to better understanding normal mammary gland function as well as breast cancer initiation and progression. In contrast to murine tissue, human mammary glands require laborious dissociation protocols to isolate single cells. This leads to unavoidable procedure-induced compositional and transcriptional bias. Here, we present a new strategy on how to identify and minimize systematic error by combining different tissue dissociation strategies and then directly comparing composition and transcriptome of isolated cells using single-cell RNA sequencing and flow cytometry. Depending on the tissue isolation strategy, we found dramatic differences in abundance and heterogeneity of certain stromal cells types. Moreover, we identified lineage-specific dissociation-induced gene expression changes that, if left unchecked, could lead to misinterpretation of cellular heterogeneity and, since the basal epithelial population is particularly affected by this, wrongful assignment of putative stem cell populations.

Role of laminin polymerization at the epithelial mesenchymal interface in bronchial myogenesis

Development ◽  
1998 ◽  
Vol 125 (14) ◽  
pp. 2621-2629 ◽  
Author(s):  
Y. Yang ◽  
K.C. Palmer ◽  
N. Relan ◽  
C. Diglio ◽  
L. Schuger
Keyword(s):  
Smooth Muscle ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Mesenchymal Cell ◽  
Cell Types ◽  
Mesenchymal Cells ◽  
Cell Contact ◽  
Alpha Actin ◽  
Muscle Myosin

Undifferentiated mesenchymal cells were isolated from mouse embryonic lungs and plated at subconfluent and confluent densities. During the first 5 hours in culture, all the cells were negative for smooth muscle markers. After 24 hours in culture, the mesenchymal cells that spread synthesized smooth muscle alpha-actin, muscle myosin, desmin and SM22 in levels comparable to those of mature smooth muscle. The cells that did not spread remained negative for smooth muscle markers. SM differentiation was independent of cell-cell contact or proliferation. In additional studies, undifferentiated lung mesenchymal cells were cocultured with lung embryonic epithelial cells at high density. The epithelial cells aggregated into cysts surrounded by mesenchymal cells and a basement membrane was formed between the two cell types. In these cocultures, the mesenchymal cells in contact with the basement membrane spread and differentiated into smooth muscle. The rest of the mesenchymal cells remained round and negative for smooth muscle markers. Inhibition of laminin polymerization by an antibody to the globular regions of laminin beta1/gamma1 chains blocked basement membrane assembly, mesenchymal cell spreading and smooth muscle differentiation. These studies indicated that lung embryonic mesenchymal cells have the potential to differentiate into smooth muscle and the process is triggered by their spreading along the airway basement membrane.

A new monoclonal antibody to a cell-surface antigen that distinguishes luminal epithelial and myoepithelial cells in the rat mammary gland

1989 ◽  
Vol 94 (3) ◽  
pp. 545-552
Author(s):  
R.S. Mahendran ◽  
M.J. O'Hare ◽  
M.G. Ormerod ◽  
P.A. Edwards ◽  
R.A. McIlhinney ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Mammary Gland ◽  
Epithelial Cells ◽  
Milk Fat ◽  
Single Cells ◽  
Myoepithelial Cells ◽  
Stages Of Development ◽  
Leukaemia Cell Line ◽  
Flow Cytometric ◽  
Rat Mammary Gland

A monoclonal antibody (25.5) has been produced that recognises luminal epithelial cells of the rat mammary gland. This antibody together with monoclonal anti-CALLA antibodies, which react with mammary myoepithelial cells, has been used in biochemical, immunocytochemical and flow cytometric studies. Antibody 25.5 bound to proteins of molecular weight 70K and 25K (K = 10(3) Mr) in both the rat milk fat globule membrane and in single cell suspensions prepared from the virgin adult rat mammary gland. Anti-CALLA antibody (J5), recognised a 93–100K protein in the gland extracts, which co-electrophoresed with the CALLA/CD-10 antigen from NALM-6 acute lymphoblastic leukaemia cell line. Antibody 25.5 bound to the luminal surface of rat mammary epithelial cells at all stages of development from neonatal through to pregnancy, lactation and involution. CALLA immunoreactive staining has previously been shown on basally located presumptive myoepithelial cells at all stages of development. Flow cytometric analyses demonstrated that 25.5 and anti-CALLA antibodies stained independent cell populations in suspensions of single cells prepared from purified epithelial elements from the mammary gland of adult virgin rat.

scholarly journals Control of mammary epithelial differentiation: basement membrane induces tissue-specific gene expression in the absence of cell-cell interaction and morphological polarity.

1991 ◽  
Vol 115 (5) ◽  
pp. 1383-1395 ◽  
Author(s):  
C H Streuli ◽  
N Bailey ◽  
M J Bissell
Keyword(s):  
Gene Expression ◽  
Basement Membrane ◽  
Single Cells ◽  
Mammary Epithelial ◽  
Cell Contact ◽  
Specific Gene ◽  
Tissue Specific Gene ◽  
Morphological Polarity ◽  
Beta Casein ◽  
Cell Cell

Functional differentiation in mammary epithelia requires specific hormones and local environmental signals. The latter are provided both by extracellular matrix and by communication with adjacent cells, their action being intricately connected in what appears to be a cascade of events leading to milk production. To distinguish between the influence of basement membrane and that of cell-cell contact in this process, we developed a novel suspension culture assay in which mammary epithelial cells were embedded inside physiological substrata. Single cells, separated from each other, were able to assimilate information from a laminin-rich basement membrane substratum and were induced to express beta-casein. In contrast, a stromal environment of collagen I was not sufficient to induce milk synthesis unless accompanied by cell-cell contact. The expression of milk proteins did not depend on morphological polarity since E-cadherin and alpha 6 integrin were distributed evenly around the surface of single cells. In medium containing 5 microM Ca2+, cell-cell interactions were impaired in small clusters and E-cadherin was not detected at the cell surface, yet many cells were still able to produce beta-casein. Within the basement membrane substratum, signal transfer appeared to be mediated through integrins since a function-blocking anti-integrin antibody severely diminished the ability of suspension-cultured cells to synthesize beta-casein. These results provide evidence for a central role of basement membrane in the induction of tissue-specific gene expression.

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