Loss of vitamin D receptor signaling from the mammary epithelium or adipose tissue alters pubertal glandular 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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Developmental role of the SNF1-related kinase Hunk in pregnancy-induced changes in the mammary gland

Development ◽

10.1242/dev.127.20.4493 ◽

2000 ◽

Vol 127 (20) ◽

pp. 4493-4509

Author(s):

H.P. Gardner ◽

G.K. Belka ◽

G.B. Wertheim ◽

J.L. Hartman ◽

S.I. Ha ◽

...

Keyword(s):

Mammary Gland ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Mammary Epithelium ◽

Ovarian Hormones ◽

Mammary Development ◽

Mammary Epithelial ◽

Serine Threonine Kinase ◽

Proliferation And Differentiation ◽

Induced Changes

The steroid hormones 17 beta-estradiol and progesterone play a central role in the pathogenesis of breast cancer and regulate key phases of mammary gland development. This suggests that developmental regulatory molecules whose activity is influenced by ovarian hormones may also contribute to mammary carcinogenesis. In a screen designed to identify protein kinases expressed in the mammary gland, we previously identified a novel SNF1-related serine/threonine kinase, Hunk (hormonally upregulated Neu-associated kinase). During postnatal mammary development, Hunk mRNA expression is restricted to a subset of mammary epithelial cells and is temporally regulated with highest levels of expression occurring during early pregnancy. In addition, treatment of mice with 17 beta-estradiol and progesterone results in the rapid and synergistic upregulation of Hunk expression in a subset of mammary epithelial cells, suggesting that the expression of this kinase may be regulated by ovarian hormones. Consistent with the tightly regulated pattern of Hunk expression during pregnancy, mammary glands from transgenic mice engineered to misexpress Hunk in the mammary epithelium manifest temporally distinct defects in epithelial proliferation and differentiation during pregnancy, and fail to undergo normal lobuloalveolar development. Together, these observations suggest that Hunk may contribute to changes in the mammary gland that occur during pregnancy in response to ovarian hormones.

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Chemerin Regulates Epithelial Barrier Function of Mammary Glands in Dairy Cows

Animals ◽

10.3390/ani11113194 ◽

2021 ◽

Vol 11 (11) ◽

pp. 3194

Author(s):

Yutaka Suzuki ◽

Sachi Chiba ◽

Koki Nishihara ◽

Keiichi Nakajima ◽

Akihiko Hagino ◽

...

Keyword(s):

Mammary Gland ◽

Epithelial Cell ◽

Epithelial Cells ◽

Dairy Cows ◽

Barrier Function ◽

Mammary Epithelial Cells ◽

Mammary Epithelial ◽

Epithelial Barrier ◽

Epithelial Tissue ◽

Epithelial Barrier Function

Epithelial barrier function in the mammary gland acts as a forefront of the defense mechanism against mastitis, which is widespread and a major disorder in dairy production. Chemerin is a chemoattractant protein with potent antimicrobial ability, but its role in the mammary gland remains unelucidated. The aim of this study was to determine the function of chemerin in mammary epithelial tissue of dairy cows in lactation or dry-off periods. Mammary epithelial cells produced chemerin protein, and secreted chemerin was detected in milk samples. Chemerin treatment promoted the proliferation of cultured bovine mammary epithelial cells and protected the integrity of the epithelial cell layer from hydrogen peroxide (H2O2)-induced damage. Meanwhile, chemerin levels were higher in mammary tissue with mastitis. Tumor necrosis factor alpha (TNF-α) strongly upregulated the expression of the chemerin-coding gene (RARRES2) in mammary epithelial cells. Therefore, chemerin was suggested to support mammary epithelial cell growth and epithelial barrier function and to be regulated by inflammatory stimuli. Our results may indicate chemerin as a novel therapeutic target for diseases in the bovine mammary gland.

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Proteinases of the mammary gland: developmental regulation in vivo and vectorial secretion in culture

Development ◽

10.1242/dev.112.2.439 ◽

1991 ◽

Vol 112 (2) ◽

pp. 439-449 ◽

Cited By ~ 2

Author(s):

R.S. Talhouk ◽

J.R. Chin ◽

E.N. Unemori ◽

Z. Werb ◽

M.J. Bissell

Keyword(s):

Mammary Gland ◽

Epithelial Cells ◽

Cell Function ◽

Mammary Development ◽

Mammary Epithelial ◽

The Other ◽

Mammary Tissue ◽

Tissue Inhibitor Of Metalloproteinases ◽

Tissue Extracts

The extracellular matrix (ECM) is an important regulator of mammary epithelial cell function both in vivo and in culture. Substantial remodeling of ECM accompanies the structural changes in the mammary gland during gestation, lactation and involution. However, little is known about the nature of the enzymes and the processes involved. We have characterized and studied the regulation of cell-associated and secreted mammary gland proteinases active at neutral pH that may be involved in degradation of the ECM during the different stages of mammary development. Mammary tissue extracts from virgin and pregnant CD-1 mice resolved by zymography contained three major proteinases of 60K (K = 10(3) Mr), 68K and 70K that degraded denatured collagen. These three gelatinases were completely inhibited by the tissue inhibitor of metalloproteinases. Proteolytic activity was lowest during lactation especially for the 60K gelatinase which was shown to be the activated form of the 68K gelatinase. The activated 60K form decreased prior to parturition but increased markedly after the first two days of involution. An additional gelatin-degrading proteinase of 130K was expressed during the first three days of involution and differed from the other gelatinases by its lack of inhibition by the tissue inhibitor of metalloproteinases. The activity of the casein-degrading proteinases was lowest during lactation. Three caseinolytic activities were detected in mammary tissue extracts. A novel 26K cell-associated caseinase—a serine arginine-esterase—was modulated at different stages of mammary development. The other caseinases, at 92K and a larger than 100K, were not developmentally regulated. To find out which cell type produced the proteinases in the mammary gland, we isolated and cultured mouse mammary epithelial cells. Cells cultured on different substrata produced the full spectrum of gelatinases and caseinases seen in the whole gland thus implicating the epithelial cells as a major source of these enzymes. Analysis of proteinases secreted by cells grown on a reconstituted basement membrane showed that gelatinases were secreted preferentially in the direction of the basement membrane. The temporal pattern of expression of these proteinases and the basal secretion of gelatinases by epithelial cells suggest their involvement in the remodelling of the extracellular matrix during the different stages of mammary development and thus modulation of mammary cell function.

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Fibronectin fragments induce MMP activity in mouse mammary epithelial cells: evidence for a role in mammary tissue remodeling

Journal of Cell Science ◽

10.1242/jcs.113.5.795 ◽

2000 ◽

Vol 113 (5) ◽

pp. 795-806 ◽

Cited By ~ 2

Author(s):

P. Schedin ◽

R. Strange ◽

T. Mitrenga ◽

P. Wolfe ◽

M. Kaeck

Keyword(s):

Extracellular Matrix ◽

Mammary Gland ◽

Epithelial Cell ◽

Epithelial Cells ◽

Protease Activity ◽

Mammary Epithelial Cells ◽

Cell Loss ◽

Mammary Epithelial ◽

Epithelial Cell Line ◽

Fibronectin Fragments

Mammary gland form and function are regulated by interactions between epithelium and extracellular matrix. Major glycoprotein components of extracellular matrix have been identified that give survival, proliferation and differentiation signals to mammary epithelial cells. We provide evidence that proteolytic fragments of the extracellular matrix glycoprotein, fibronectin, suppress growth and can promote apoptosis of mouse mammary epithelial cells. During mammary gland involution, total fibronectin and fibronectin fragment levels are increased. The peak levels of fibronectin protein and fragments are observed 4–6 days post-weaning, coincident with the peak in epithelial cell death. Using a model for hormone withdrawal-induced death of mammary epithelium, elevated levels of fibronectin proteolytic fragments were associated with apoptosis in TM-6 cells, a tumorigenic mouse mammary epithelial cell line. Treatment of TM-6 cells with exogenous fibronectin fragments (FN120) reduced cell number, and induced apoptosis and matrix degrading protease activity. Inhibition of matrix protease activity rescued TM-6 cell viability, indicating that FN120-induced cell loss is mediated through matrix protease activity. In a three-dimensional model for mammary gland development, FN120 reduced alveolar-like and promoted ductal-like development by a matrix protease-dependent mechanism. These data suggest that during post-lactational involution, fibronectin fragments may contribute to epithelial cell loss and dissolution of mammary alveoli by inducing matrix degrading proteinases.

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The Type 7 Serotonin Receptor, 5-HT7, Is Essential in the Mammary Gland for Regulation of Mammary Epithelial Structure and Function

BioMed Research International ◽

10.1155/2015/364746 ◽

2015 ◽

Vol 2015 ◽

pp. 1-8 ◽

Cited By ~ 6

Author(s):

Vaibhav P. Pai ◽

Laura L. Hernandez ◽

Malinda A. Stull ◽

Nelson D. Horseman

Keyword(s):

Mammary Gland ◽

Epithelial Cells ◽

Serotonin Receptor ◽

Mammary Epithelial Cells ◽

Mammary Epithelium ◽

Mammary Epithelial ◽

Lactation Performance ◽

Epithelial Structure ◽

And Function ◽

First Time

Autocrine-paracrine activity of serotonin (5-hydroxytryptamine, 5-HT) is a crucial homeostatic parameter in mammary gland development during lactation and involution. Published studies suggested that the 5-HT7receptor type was important for mediating several effects of 5-HT in the mammary epithelium. Here, using 5-HT7receptor-null (HT7KO) mice we attempt to understand the role of this receptor in mediating 5-HT actions within the mammary gland. We demonstrate for the first time that HT7KO dams are inefficient at sustaining their pups. Histologically, the HT7KO mammary epithelium shows a significant deviation from the normal secretory epithelium in morphological architecture, reduced secretory vesicles, and numerous multinucleated epithelial cells with atypically displaced nuclei, during lactation. Mammary epithelial cells in HT7KO dams also display an inability to transition from lactation to involution as normally seen by transition from a columnar to a squamous cell configuration, along with alveolar cell apoptosis and cell shedding. Our results show that 5-HT7is required for multiple actions of 5-HT in the mammary glands including core functions that contribute to changes in cell shape and cell turnover, as well as specialized secretory functions. Understanding these actions may provide new interventions to improve lactation performance and treat diseases such as mastitis and breast cancer.

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Zip3 plays a major role in zinc uptake into mammary epithelial cells and is regulated by prolactin

AJP Cell Physiology ◽

10.1152/ajpcell.00471.2004 ◽

2005 ◽

Vol 288 (5) ◽

pp. C1042-C1047 ◽

Cited By ~ 57

Author(s):

Shannon L. Kelleher ◽

Bo Lönnerdal

Keyword(s):

Mammary Gland ◽

Epithelial Cell ◽

Epithelial Cells ◽

Mammary Epithelial Cell ◽

Mammary Epithelial Cells ◽

Cell Model ◽

Mammary Epithelial ◽

Maternal Circulation ◽

Physiological Explanation ◽

Secretory Phenotype

During lactation, a substantial amount of Zn2+ is transferred by the mammary gland from the maternal circulation into milk; thus secretory mammary epithelial cells must tightly regulate Zn2+ transport to ensure optimal Zn2+ transfer to the suckling neonate. To date, six Zn2+ import proteins (Zip1–6) have been identified; however, Zip3 expression is restricted to tissues with unique requirements for Zn2+, such as the mammary gland, which suggests that it may play a specialized role in this tissue. In the present study, we have used a unique mammary epithelial cell model (HC11) to characterize the role of Zip3 in mammary epithelial cell Zn2+ transport. Confocal microscopy demonstrated that Zip3 is localized to the cell surface in mammary epithelial cells and transiently relocalized to an intracellular compartment in cells with a secretory phenotype. Total 65Zn transport was higher in secreting cells, while gene silencing of Zip3 decreased 65Zn uptake into mammary epithelial cells, particularly in those with a secretory phenotype. Finally, reduced expression of Zip3 ultimately resulted in cell death, indicating that mammary epithelial cells have a unique requirement for Zip3-mediated Zn2+ import, which may reflect the unique requirement for Zn2+ of this highly specialized cell type and thus provides a physiological explanation for the restricted tissue distribution of this Zn2+ importer.

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ScRNA-seq Reveals a Role of Mammary Luminal Epithelium in Adipocyte Adaptations

Journal of the Endocrine Society ◽

10.1210/jendso/bvab048.111 ◽

2021 ◽

Vol 5 (Supplement_1) ◽

pp. A55-A55

Author(s):

Luis Santos ◽

Douglas Arneson ◽

Karthickeyan Chella Krishnan ◽

In Sook Ahn ◽

Graciel Diamante ◽

...

Keyword(s):

Mammary Gland ◽

Epithelial Cells ◽

Cold Exposure ◽

Milk Fat ◽

Ex Vivo ◽

Mammary Epithelium ◽

Mammary Epithelial ◽

Lipocalin 2 ◽

Luminal Cells

Abstract Almost four decades of research suggest a dynamic role of ductal epithelial cells in adipocyte adaptation in mammary gland white adipose tissue (mgWAT), but factors that mediate such communication are not known. Here, we identify a complex intercellular crosstalk in mgWAT revealed by single-cell RNA-seq (scRNA-seq) and comprehensive data analysis suggest that epithelial luminal cells during cold exposure undergo major transcriptomic changes that lead to the expression of an array of genes that encode for secreted factors involved in adipose metabolism such as Adropin (Enho), neuregulin 4 (Nrg4), angiopoietin-like 4 (Angptl4), lipocalin 2 (Lcn2), milk fat globule-EGF factor 8 (Mfge8), Insulin-like growth factor-binding protein 1 (Igfbp1), and haptoglobin (Hp). To define the mammary epithelial secretome, we coin the phrase “mammokines”. We validated our cluster annotations and cluster-specific transcriptomics using eight different adipose scRNA-seq datasets including Tabula Muris and Tabula Muris Senis. In situ mRNA hybridization and ex vivo isolated mgWAT luminal cells show highly localized expression of mammokines in mammary ducts. Trajectory inference demonstrates that cold-exposed luminal cells have similar transcriptional profiles to lactation post-involution (PI), a phase defined by reappearance and maintenance of adipocytes in the mammary gland. Concomitantly, we found that under cold exposure female mgWAT maintains more adipogenic and less thermogenic potential than male scWAT and ex vivo removal of luminal epithelial cells from mgWAT markedly potentiates beige adipocyte differentiation. Conditioned media from isolated mammary epithelial cells treated with isoproterenol suppressed thermogenesis in differentiated beige/brown adipocytes and treatment of beige/brown differentiated adipocyte with mammokine LCN2 suppresses thermogenesis and increases adipogenesis. Finally, we find that mice lacking LCN2 show markedly higher cold-dependent thermogenesis in mgWAT than controls, and reconstitution of LCN2 in the mgWAT of LCN2 knockout mice promotes inhibition of thermogenesis. These results show a previously unknown role of mammary epithelium in adipocyte metabolism and suggest a potentially redundant evolutionary role of mammokines in maintaining mgWAT adiposity during cold exposure. Our data highlight mammary gland epithelium as a highly active metabolic cell type and mammokines could have broader implications in mammary gland physiology and lipid metabolism.

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Photoperiod effects on milk production in goats: Are they mediated by the molecular clock in the mammary gland?

10.32747/2014.7598164.bard ◽

2014 ◽

Author(s):

Therese Casey ◽

Sameer J. Mabjeesh ◽

Avi Shamay ◽

Karen Plaut

Keyword(s):

Growth Rate ◽

Mammary Gland ◽

Epithelial Cell ◽

Milk Production ◽

Mammary Epithelial Cell ◽

Mammary Epithelial Cells ◽

Physiological State ◽

Mammary Development ◽

Mammary Epithelial ◽

Protein Levels

US scientists, Dr. Theresa Casey and Dr. Karen Plaut, collaborated with Israeli scientists, Dr. SameerMabjeesh and Dr. AviShamay to conduct studies proposed in the BARD Project No. US-4715-14 Photoperiod effects on milk production in goats: Are they mediated by the molecular clock in the mammary gland over the last 3 years. CLOCK and BMAL1 are core components of the circadian clock and as heterodimers function as a transcription factor to drive circadian-rhythms of gene expression. Studies of CLOCK-mutant mice found impaired mammary development in late pregnancy was related to poor lactation performance post-partum. To gain a better understanding of role of clock in regulation of mammary development studies were conducted with the mammary epithelial cell line HC11. Decreasing CLOCK protein levels using shRNA resulted in increased mammary epithelial cell growth rate and impaired differentiation, with lower expression of differentiation markers including ad herens junction protein and fatty acid synthesis genes. When BMAL1 was knocked out using CRISPR-CAS mammary epithelial cells had greater growth rate, but reached stationary phase at a lower density, with FACS indicating cells were growing and dying at a faster rate. Beta-casein milk protein levels were significantly decreased in BMAL1 knockout cells. ChIP-seq analysis was conducted to identify BMAL1 target genes in mammary epithelial cells. Studies conducted in goats found that photoperiod duration and physiological state affected the dynamics of the mammary clock. Effects were likely independent of the photoperiod effects on prolactin levels. Interestingly, circadian rhythms of core body temperature, which functions as a key synchronizing cue sent out by the central clock in the hypothalamus, were profoundly affected by photoperiod and physiological state. Data support that the clock in the mammary gland regulates genes important to development of the gland and milk synthesis. We also found the clock in the mammary is responsive to changes in physiological state and photoperiod, and thus may serve as a mechanism to establish milk production levels in response to environmental cues.

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Epimorphin Functions as a Key Morphoregulator for Mammary Epithelial Cells

The Journal of Cell Biology ◽

10.1083/jcb.140.1.159 ◽

1998 ◽

Vol 140 (1) ◽

pp. 159-169 ◽

Cited By ~ 97

Author(s):

Yohei Hirai ◽

André Lochter ◽

Sybille Galosy ◽

Shogo Koshida ◽

Shinichiro Niwa ◽

...

Keyword(s):

Mammary Gland ◽

Growth Factors ◽

Growth Factor ◽

Epithelial Cell ◽

Epithelial Cells ◽

Mammary Epithelial Cells ◽

Branching Morphogenesis ◽

Mammary Epithelial ◽

Collagen Gels ◽

Stromal Compartment

Hepatocyte growth factor (HGF) and EGF have been reported to promote branching morphogenesis of mammary epithelial cells. We now show that it is epimorphin that is primarily responsible for this phenomenon. In vivo, epimorphin was detected in the stromal compartment but not in lumenal epithelial cells of the mammary gland; in culture, however, a subpopulation of mammary epithelial cells produced significant amounts of epimorphin. When epimorphin-expressing epithelial cell clones were cultured in collagen gels they displayed branching morphogenesis in the presence of HGF, EGF, keratinocyte growth factor, or fibroblast growth factor, a process that was inhibited by anti-epimorphin but not anti-HGF antibodies. The branch length, however, was roughly proportional to the ability of the factors to induce growth. Accordingly, epimorphin-negative epithelial cells simply grew in a cluster in response to the growth factors and failed to branch. When recombinant epimorphin was added to these collagen gels, epimorphin-negative cells underwent branching morphogenesis. The mode of action of epimorphin on morphogenesis of the gland, however, was dependent on how it was presented to the mammary cells. If epimorphin was overexpressed in epimorphin-negative epithelial cells under regulation of an inducible promoter or was allowed to coat the surface of each epithelial cell in a nonpolar fashion, the cells formed globular, alveoli-like structures with a large central lumen instead of branching ducts. This process was enhanced also by addition of HGF, EGF, or other growth factors and was inhibited by epimorphin antibodies. These results suggest that epimorphin is the primary morphogen in the mammary gland but that growth factors are necessary to achieve the appropriate cell numbers for the resulting morphogenesis to be visualized.

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Differential regulation of GLUT1 and GLUT8 expression by hypoxia in mammary epithelial cells

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00093.2014 ◽

2014 ◽

Vol 307 (3) ◽

pp. R237-R247 ◽

Cited By ~ 14

Author(s):

Yong Shao ◽

Theresa L. Wellman ◽

Karen M. Lounsbury ◽

Feng-Qi Zhao

Keyword(s):

Mammary Gland ◽

Epithelial Cells ◽

Glucose Uptake ◽

Mammary Epithelial Cells ◽

Physiological Mechanism ◽

Glucose Transporters ◽

Mammary Glands ◽

Mammary Development ◽

Mammary Epithelial ◽

Early Lactation

Glucose is a major substrate for milk synthesis and is taken up from the blood by mammary epithelial cells (MECs) through facilitative glucose transporters (GLUTs). The expression levels of GLUT1 and GLUT8 are upregulated dramatically in the mammary gland from late pregnancy through early lactation stages. This study aimed to test the hypothesis that this increase in GLUT1 and GLUT8 expression involves hypoxia signaling through hypoxia inducible factor-1α (HIF-1α) in MECs. Mouse mammary glands showed significantly more hypoxia in midpregnancy through early lactation stages compared with in the virgin stage, as stained by the hypoxia marker pimonidazole HCl. Treatment with hypoxia (2% O2) significantly stimulated glucose uptake and GLUT1 mRNA and protein expression, but decreased GLUT8 mRNA expression in bovine MECs. In MECs, hypoxia also increased the levels of HIF-1α protein in the nuclei, and siRNA against HIF-1α completely abolished the hypoxia-induced upregulation of GLUT1, while having no effect on GLUT8 expression. A 5′-RCGTG-3′ core HIF-1α binding sequence was identified 3.7 kb upstream of the bovine GLUT1 gene, and HIF-1α binding to this site was increased during hypoxia. In conclusion, the mammary glands in pregnant and lactating animals are hypoxic, and MECs respond to this hypoxia by increasing GLUT1 expression and glucose uptake through a HIF-1α-dependent mechanism. GLUT8 expression, however, is negatively regulated by hypoxia through a HIF-1α-independent pathway. The regulation of glucose transporters through hypoxia-mediated gene transcription in the mammary gland may provide an important physiological mechanism for MECs to meet the metabolic demands of mammary development and lactation.

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