Response Of Mammary Gland Research Articles

Uteroplacental insufficiency alters the mammary gland response to lactogenic hormones in vitro

Adequate mammary development and coordinated actions of lactogenic hormones are essential for the initiation of lactation. Pregnancies compromised by uteroplacental insufficiency impair mammary development and lactation, further slowing postnatal growth. It is not known whether the initiation of lactation or galactopoesis is compromised. Uteroplacental insufficiency induced in rats by bilateral uterine vessel ligation (Restricted) or sham surgery (Control) on Day 18 of gestation preceded collection of mammary tissue on Day 20 of pregnancy. Mammary explants were cultured with combinations of insulin, cortisol and prolactin and analysed for alpha-lactalbumin and beta-casein gene expression. Mammary tissue from late pregnant Restricted rats had elevated alpha-lactalbumin, but not beta-casein, mRNA, which is consistent with premature lactogenesis resulting from an early decline in peripheral maternal progesterone. Explants from Restricted rats were more responsive to hormone stimulation after 3 days in culture, indicating that compromised galactopoesis, not lactogenesis, most likely leads to the reduced growth of suckled pups.

Effect of chronic oxytocin-treatment on the bovine mammary gland immune system

The aim of this study was to evaluate the effect of chronic oxytocin (OT) treatment on the mammary gland immune system. In <I>Experiment I</I> fourteen healthy cows were used to study the effect of chronic intramuscular (im) OT administration on concentration of milk somatic cells and white blood cells (WBC). Cows in the OT-group (6) were im injected with 50 IU OT (5 ml) whereas animals of the C-group (6) were im injected with 5 ml of saline (9 g/l) for eight days (Day 1–8) before each milking. Milk samples were taken during normal milking time on Day 0–3, 5, 7, 9–11 and 18. Blood samples were taken immediately after each milking and analysed for WBC count, polymorphonuclear neutrophils, potassium (K), sodium (Na) and chloride (Cl) ions, and blood lactose. All milk samples were analysed for somatic cell counts (SCC), lactose, Na, Cl and electrical conductivity (EC). Furthermore mRNA expression of tumor necrosis factor-α (TNFα), interleukin (IL)-1β, IL-6, IL-8 and cyclooxygenase-2 (COX2) in milk cells were measured via real-time RT-PCR. None of the investigated milk and blood parameters changed significantly in response to the OT treatment. The mRNA-expression of TNFα decreased (<I>P</I> < 0.05) to a minimum on Day 3 in response to OT administration. IL-1β and IL-6-mRNA expression decreased (<I>P</I> < 0.05) to a minimum within three day. IL-8 and COX2 expression did not change in response to OT treatment. In <I>Experiment II</I> twelve cows, randomly divided into two groups of six, were used to investigate the effect of chronic im OT administration on mammary tissue. Cows were im administered 50 IU OT (OT-group) or 5 ml saline (9 g/l; C-group) before each milking during eight days. Biopsy samples were taken after every morning milking. The mRNA expression of various inflammatory factors and the tight junction (TJ) proteins occludin (OCLN) and zonula occludens (ZO)-1, ZO-2 and ZO-3 were measured via real-time RT-PCR. TNFα-mRNA expression decreased (Day 2 with <I>P</I> < 0.05) within the first four days of OT administration and increased (<I>P</I> < 0.05) in the C-group on Day 2. IL-1β expression levels of the OT-group increased transiently and decreased on Day 3 and in the C-group values increased significantly on Day 3 as compared to Day 0. IL-6 expression in the OT-group decreased (<I>P</I> < 0.05) to a minimum on Day 1 and increased (<I>P</I> < 0.05) as compared to Day 0 on Day 7 and increased significantly on Day 1 and Day 5 compared to Day 0 in group C. IL-8 and COX2 expression did not change in response to OT administration. The mRNA-expression of OCLN and ZO-3 decreased (<I>P</I> < 0.05) as compared to Day 0 with a minimum on Day 7. ZO-1 and ZO-2 expression did not change due to OT administration. ZO-2-mRNA expression in C-group decreased significantly on Day 2 compared to Day 0. In conclusion, chronic OT administration induced increasing SCC and EC levels in milk as well as K and lactose in blood while nearly all investigated cytokines in milk cells and mammary tissue were down regulated. The mRNA expressions of the TJ proteins OCLN and ZO-3 were down-regulated in response to the OT treatment what indicates an increasing TJ permeability. Besides the effect on TJ proteins there was no obvious change of the immunological competence of the mammary gland in response to OT. However, a more complete milk ejection should help to remove pathogens during milking.

Regulation of Gene Expression in the Bovine Mammary Gland by Ovarian Steroids

It is well established that estrogen is required for mammary epithelial cell proliferation and ductal development in the growing animal, and that lobuloalveolar development during gestation is dependent on progesterone. The effects of these steroid hormones on gene expression in the mammary gland are mediated primarily by their respective nuclear hormone receptors, which function as hormone-bound transcription factors. To gain insight into how estrogen and progesterone regulate mammary gland growth and function in cattle, we and others have characterized the expression patterns of their cognate nuclear hormone receptors in the bovine mammary gland throughout development, pregnancy, and lactation. This work has identified a lack of expression of estrogen receptor β and a greater abundance of progesterone receptor during lactation in the bovine mammary gland, compared with the rodent gland. We speculate that interactions among the estrogen receptor isoforms that regulate progesterone receptor expression may contribute to these species differences. Further, demonstrated expression of substantial quantities of estrogen receptor within the prepubertal bovine mammary fat pad, along with coordinated insulin-like growth factor-I expression, suggests that this tissue may stimulate parenchymal growth via an estrogen-responsive paracrine mechanism. In addition, the recent availability of bovine genomic sequence information and microarray technologies has permitted the study of global gene expression in the mammary gland in response to the steroid environment. We have identified more than 100 estrogen-responsive genes, of which the majority are novel estrogen gene targets. Estrogen-induced changes in gene expression were consistent with increased mammary epithelial cell proliferation, increased extracellular matrix turnover in parenchyma, and increased extracellular matrix deposition in the fat pad. A comparison of estrogen-responsive genes in the mammary glands of humans, mice, and cattle suggests considerable variation among species, as well as potential differences in regulatory elements in common estrogen receptor gene targets. Continuing studies using advanced molecular techniques should assist in elucidating the complex regulation of mammary function at the transcript level.

Differential cytokine and chemokine responses of bovine mammary epithelial cells to Staphylococcus aureus and Escherichia coli

We studied the inflammatory and immune responses of bovine mammary epithelial cells (bMEC) infected by mastitis isolates of Staphylococcus aureus. Primary cultures of bMEC were co-incubated separately with three strains of S. aureus and one strain of Escherichia coli. Transcriptional levels and/or protein release of interleukin-8 (IL-8), growth related oncogene alpha (GRO-α), growth related oncogene beta (GRO-β), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), transforming growth factor β1 (TGF-β1) and interleukin-10 (IL-10) were measured at 3, 10 and 24 h post-infection (PI). The results indicated that at earlier hours of co-culture, bMEC infected with S. aureus or E. coli expressed more IL-1β, TNF-α, IL-8 and GRO-α mRNA than uninfected bMEC. Furthermore, infected bMEC released more TNF-α, IL-8, GRO-α and GRO-β proteins than uninfected bMEC. However, differential transcription and release of some cytokines/chemokines from bMEC was observed according to the strain of S. aureus and bacteria Gram type. In conclusion, bMEC did not show an anti-inflammatory potential through IL-10 or TGF-β1 release. Nevertheless, bMEC were able to release neutrophil-mobilizing chemokines and pro-inflammatory cytokines upon bacterial stimulation, strongly suggesting that bMEC are active contributors to immune and inflammatory responses of mammary gland. In addition, the clinical characteristics and resolution of mastitis may be partly determined by the responses of bMEC according to S. aureus strains and bacteria Gram type.

Differential Cell-Specific Modulation of HOXA10 by Estrogen and Specificity Protein 1 Response Elements

HOX genes are highly evolutionarily conserved regulators of embryonic development. HOXA10 also regulates differentiation of the adult reproductive tract and mammary gland in response to sex steroids. We recently identified two HOXA10 estrogen response elements (EREs). Here we demonstrate that estrogen-responsive HOXA10 expression is cell type specific. We conducted an in vitro study at an academic medical center. Reporter assay, gel shift assays (electrophoretic mobility shift assay), and immunohistochemistry were done. The HOXA10 EREs and a specificity protein 1 (Sp1) binding site differentially drive the cell-type-specific E2 response. In electrophoretic mobility shift assays, both estrogen receptor-alpha and -beta bound both EREs but not the Sp1 site. In reporter assays, both EREs and the Sp1 site demonstrated estrogen responsiveness and tissue specificity; transiently transfected uterine Ishikawa cells or breast MCF-7 cells showed differential responses to E2 treatment. Each response element (Sp1, ERE1, and ERE2) drove distinct differential expression in each cell type. Sp1 protein was expressed in a menstrual-cycle stage-specific expression pattern in endometrium, first expressed in perivascular cells. Tissue specificity inherent to a regulatory element as well as differential cellular expression of transcription factors imparts differential tissue-specific estrogen responsiveness.

The Rat Mammary Gland: Morphologic Changes as an Indicator of Systemic Hormonal Perturbations Induced by Xenobiotics

The development and morphology of the rat mammary gland are dependent upon several hormones including estrogens, androgens, progesterone, growth hormone and prolactin. In toxicology studies, treatment with xenobiotics may alter these hormones resulting in changes in the morphology of reproductive tissues such as the mammary gland. In the rat, male and female mammary glands exhibit striking morphologic differences that can be altered secondary to hormonal perturbations. Recognizing these morphologic changes can help the pathologist predict potential xenobiotic-induced perturbations in the systemic hormonal milieu. This review examines the development of the rat mammary gland and the influence of sex hormones on the morphology of the adult male and female rat mammary gland. Specific case examples from the literature and data from our laboratory highlight the dynamic nature of the rat mammary gland in response to hormonal changes.

Steroid Receptor Coactivator 2 is Required for Female Fertility and Mammary Morphogenesis: Insights from the Mouse, Relevance to the Human

Although the importance of the progesterone receptor (PR) to female reproductive and mammary gland biology is firmly established, the coregulators selectively co-opted by PR in these systems have not been clearly delineated. A selective gene-knockout approach applied to the mouse, which abrogates gene function only in cell types that express PR, recently disclosed steroid receptor coactivator 2 (SRC-2, also known as TIF-2 or GRIP-1) to be an indispensable coregulator for uterine and mammary gland responses that require progesterone. Uterine cells positive for PR (but devoid of SRC-2) were found to be incapable of facilitating embryo implantation, a necessary first step toward the establishment of the materno-fetal interface. Importantly, such an implantation defect is not exhibited by knockouts for SRC-1 or SRC-3, underscoring the unique coregulator importance of SRC-2 in peri-implantation biology. Moreover, despite normal levels of PR, SRC-1 and SRC-3, progesterone-dependent branching morphogenesis and alveologenesis fails to occur in the murine mammary gland in the absence of SRC-2, thereby establishing a critical coregulator role for SRC-2 in signaling cascades that mediate progesterone-induced mammary epithelial proliferation. Finally, the recent detection of SRC-2 in the human endometrium and breast suggests that this coregulator may represent a new clinical target for the future management of female reproductive health and/or breast cancer.

The mammary gland response to estradiol: Monotonic at the cellular level, non-monotonic at the tissue-level of organization?

The role of hormones in mammary gland development has been studied in detail using surgical and genetic models. These studies have indicated roles for estrogen in ductal elongation and terminal end bud formation. However, no comprehensive study has quantified how different doses of estrogen affect morphological parameters of mammary gland development. Additionally, comparisons between the estrogen-responsiveness of the mammary gland and uterus, the model organ for estrogen action are incomplete. In this study, immature mice were ovariectomized and implanted with osmotic pumps releasing one of eight doses of 17β-estradiol for 10 days. As expected from the classical uterotrophic assay, the uterus showed a monotonic dose–response curve for all measured endpoints. In contrast, the mammary gland showed a non-monotonic, inverted-U shaped response to estrogen with regard to morphometric parameters, and a monotonic response with regard to gene expression parameters. These results indicate that estrogen has opposing effects in mammary gland morphogenesis depending on estrogen dose, i.e. low to moderate doses induce terminal end bud formation and ductal elongation while higher doses inhibit these processes. This non-monotonic dose–response in the mammary gland may reflect complex interactions, where estrogen can act on multiple targets either as an agonist or antagonist.

P-511: Differential cell specific modulation of HOXA10 by estrogen and SP1 response elements

HOX genes are highly evolutionarily conserved regulators of embryonic development. HOXA10 continues to regulate differentiation of the adult reproductive tract and mammary gland in response to sex steroids. HOXA10 estrogen response elements ERE1 and ERE2 were recently characterized and are differentially expressed in response to 17B-estradiol (E2) or diethylstilbestrol (DES) in uterine cells. Here we demonstrate that estrogen responsive HOXA10 expression is not only ligand specific but cell type specific.

Innate immune response of bovine mammary gland to pathogenic bacteria responsible for mastitis

Mastitis (mammary gland inflammation) is one of the most important bovine diseases causing economic losses to dairy producers. Mammary gland inflammation is a consequence of the activity of a number of cell and soluble factors that function together to eliminate invading microorganisms. The factors involved in this inflammatory response differ depending on the infectious agent. This review analyzes the factors involved in the immunologic mechanisms against the main pathogenic bacteria causing mastitis, and emphasizes the innate immune response of the mammary gland. Knowledge, at the molecular level, of the mammary gland immune response during infection by pathogenic bacteria is fundamental to the design of effective therapies to control and eradicate bovine mastitis.

The pleiotropic roles of transforming growth factor beta in homeostasis and carcinogenesis of endocrine organs

Transforming growth factor beta (TGF-beta) is a ubiquitous cytokine that plays a critical role in numerous pathways regulating cellular and tissue homeostasis. TGF-beta is regulated by hormones and is a primary mediator of hormone response in uterus, prostate and mammary glands. This review will address the role of TGF-beta in regulating hormone-dependent proliferation and morphogenesis. The subversion of TGF-beta regulation during the processes of carcinogenesis, with particular emphasis on its effects on genetic stability and epithelial to mesenchymal transition, will also be examined. An understanding of the multiple and complex mechanisms of TGF-beta regulation of epithelial function, and the ultimate loss of TGF-beta function during carcinogenesis, will be critical in the design of novel therapeutic interventions for endocrine-related cancers.

Study of cytokines involved in the prevention of a murine experimental breast cancer by kefir

Previous studies have shown that compounds released during milk fermentation by Lactobacillus helveticus are implicated in the antitumour effect of this product. Here the effects of the consumption, during 2 or 7 days, of kefir or kefir cell-free fraction (KF) on the systemic and local immune responses in mammary glands and tumours using a murine hormone-dependent breast cancer model were studied. In the tumour control group, mice did not receive these products. At the end of the feeding period, mice were injected subcutaneously with tumour cells in the mammary gland. Four days post-injection, they received kefir or KF on a cyclical basis. Rate of tumour development, cytokines in serum; mammary gland tissue, and tumour isolated cells were monitored. Two-day cyclical administration of both products delayed tumour growth. Both kefir and KF increased IL-10 in serum and decreased IL-6(+) cells (cytokine involved in oestrogen synthesis) in mammary glands. Two-day cyclical administration of KF increased IL-10(+) cells in mammary glands and in tumours and decreased IL-6(+) cells in tumour. This study demonstrated the modulatory capacity of KF on the immune response in mammary glands and tumours and the importance of the administration period to obtain this effect.

Parous mammary glands exhibit distinct alterations in gene expression and proliferation responsiveness to carcinogenic stimuli in Lewis rats

Early full-term pregnancy affords lifetime protection against the development of breast cancer. Parity-induced protection can be reproduced in a carcinogen-induced rat mammary carcinoma model, but the molecular mechanisms of this protection against carcinogenic stimuli in rat mammary glands have not been fully characterized. To gain a better understanding of these molecular mechanisms, we used an oligonucleotide microarray to examine gene expression in parous and age-matched virgin (AMV) mammary glands of Lewis rats before and after carcinogen (N-methyl-N-nitrosourea; MNU) treatment. Parous mammary glands before MNU treatment showed up-regulation of multiple differentiation-related genes, such as whey acidic protein (Wap), casein beta (Csn2), casein gamma (Csng), lipopolysaccharide binding protein (Lbp), secreted phosphoprotein 1 (Spp1) and glycosylation-dependent cell adhesion molecule 1 (Glycam1). Also, parous mammary glands before MNU treatment exhibited down-regulation of growth-related genes such as regenerating islet-derived 3 alpha (Reg3a), mesothelin (Msln), insulin-like growth factor 2 (Igf2) and insulin-like growth factor binding protein 4 (Igfbp4). After MNU treatment, AMV mammary glands exhibited up-regulation of growth-related genes, such as Msln, cell division cycle 2 homolog A (Cdc2a), Igf2, Igfbp4, stathmin 1 (Stmn1) and homeobox, msh-like 1 (Msx1), whereas expression of these genes remained low in parous mammary glands. AMV mammary glands also exhibited marked up-regulation of Cdc2a and Stmn1 in response to MNU. After MNU treatment, the PCNA labeling index increased significantly in AMV mammary epithelial cells (13.7+/-1.1%), but remained low in parous mammary glands (3.6+/-0.4%). The response of AMV mammary glands to carcinogenic stimuli includes up-regulation of growth-related genes and increased cell proliferation. The lack of a similar response in parous mammary glands may explain parity-induced protection against mammary tumor development.

Role of several Staphylococcus aureus virulence factors on the inflammatory response in bovine mammary gland

Staphylococcus aureus causes many serious diseases in humans and animals, and it is the most common aetiologic agent of contagious bovine mastitis. The bacteria produce several virulence factors and the importance of evaluating the combination of these virulence factors has been recently emphasized. In study, the combination of several virulence factors: coagulase gene ( coa), protein A gene ( spa), collagen-binding protein gene ( cna), fibrinogen-binding protein gene ( efb), Panton-Valentin leukocydin gene ( pvl) and enterotoxins ( sea, seb, sec, sed, see, seg, seh, sei, sej) was considered. The analysis of the relationship between presence/absence of the different genes and the udder inflammatory response measured by milk somatic cell counts was performed by general linear models and logistic regression. The classification of isolates in clusters by virulence genes combinations showed that at least one cluster induced a higher inflammatory response. Moreover, the analysis of the association between virulence genes and the presence of a subclinical mastitis showed the role of spa and sej gene as risk factors. These results support that the development of subclinical mastitis could be related to strains characteristics and to the expression of specific combinations of the virulence factors.

Effects of milk fermented by Lactobacillus helveticus R389 on immune cells associated to mammary glands in normal and a breast cancer model

Antitumour activity is an effect attributed to probiotics and fermented foods. Here, the immune cells in mammary glands and cytokine concentration in serum were analyzed using mice fed with milk fermented by Lactobacillus helveticus R389 or L89 (proteolytic-deficient variant), injected or not with breast tumour cells. Mice were fed 7 days with fermented milk, injected with breast tumour cells and 4 days post-injection, they received fermented milk. IgA, CD4, CD8, cytokines and Bcl-2 positive cells in mammary glands and cytokine in serum were determined. Mice fed with L. helveticus R389 fermented milk and injected with tumour cells increased IgA and CD4 positive cells in mammary glands (tumour control increased CD8+ cells). Mice from fermented milk control groups (without tumour cell injection) did not show changes in immune cell or cytokine positive cell numbers. IL-10 increases and IL-6 decreases were more pronounced in mice fed with milk fermented by L. helveticus R389 than in the other groups. This study demonstrated the immunoregulatory capacity of milk fermented by L. helveticus R389 on the immune response in mammary glands in presence of a local pathology (breast tumour). Orally administered fermented products could be used to modify the immune cell activation in distant mucosal sites and maintain these cells alert, but local stimulus was necessary to produce the activation of a local immune response in mammary glands, which could modulate the immune-endocrine relationship in these glands.

Characterization of the infection-responsive bovine lactoferrin promoter

The concentration of lactoferrin in bovine milk is dramatically increased in response to infection. The high levels of lactoferrin may have a role in the prevention of microbial infection of the mammary gland. However, molecular mechanisms of how the lactoferrin gene is regulated in the mammary gland in response to infection remain unknown. In this study, we isolated and characterized the 5′ flanking region of the bovine lactoferrin gene. An 8.2 kilobase (kb) fragment of the bovine lactoferrin gene, containing 4.4 kb of 5′ flanking region, exon 1, intron 1, and exon 2, was isolated from a bovine genomic library on two overlapping bacterial artificial chromosome (BAC) clones. Sequence analysis of the isolated lactoferrin gene revealed that the promoter region contains a high GC content, a noncanonical TATA box, multiple stimulating protein 1 (SP1)/GC elements, and other putative binding sites for transcription factors including nuclear factor-κB (NF-κB), activator protein 1 (AP1), signal transducer and activator of transcriptions 3 and 5 (STAT3 and STAT5), and steroid hormone receptors. To demonstrate that the isolated promoter is functional, 4.4 kb of 5′ flanking region was inserted upstream from the firefly luciferase gene and the chimeric construct was transiently transfected into murine mammary epithelial cells. Transfection studies showed that the basal promoter activity is quite potent, being similar in strength to that of the simian virus 40 (SV40) promoter/enhancer. In addition, a 24-h treatment with Escherichia coli lipopolysaccharide (LPS) significantly stimulated its activity up to 2.3-fold in a dose-dependent manner. Furthermore, promoter deletion analysis indicated that the sequence up to − 543 was sufficient for basal activity, whereas the sequence up to − 1029 was required for maximal basal activity. The basal activity of the promoter is affected by both positive regulatory regions (− 2462/− 1879 and − 1029/− 75) and a negative regulatory region (− 1407/− 1029). LPS-responsive regions of the promoter were localized to the region from − 1029 to − 543 containing one STAT3 site and two NF-κB sites, and the region from − 4355 to − 2462 containing three AP1 sites and six NF-κB sites. Taken together, our findings suggested that the lactoferrin promoter responds to infection via the NF-κB pathway.

Effects of milk fermented by Lactobacillus helveticusR389 on a murine breast cancer model

IntroductionAntitumour activity is one of the health-promoting effects attributed to the lactic acid bacteria and their products of fermentation. Previous studies in mice demonstrated that bioactive compounds released in milk fermented by Lactobacillus helveticus R389 contribute to its immunoenhancing and antitumour properties. The aim of the present work was to study the effects of the consumption of milk fermented by L. helveticus R389 or its proteolytic-deficient variant, L. helveticus L89, on a murine hormone-dependent breast cancer model.MethodsMice were fed with milk fermented by L. helveticus R389 or L. helveticus L89, during 2 or 7 days. The tumour control group received no special feeding. At the end of the feeding period, the mice were challenged by a subcutaneous injection of tumour cells in the mammary gland. Four days post-injection, the mice received fermented milk on a cyclical basis. The rate of tumour development and the cytokines in serum, mammary gland tissue and tumour-isolated cells were monitored. Bcl-2-positive cells in mammary glands and cellular apoptosis in tumour tissue were also studied.ResultsSeven days of cyclical administration of milk fermented by either bacterial strain delayed or stopped the tumour development. Cytokines demonstrated that L. helveticus R389 modulated the immune response challenged by the tumour. IL-10 and IL-4 were increased in all the samples from this group. In comparison with the tumour control, all test groups showed a decrease of IL-6, a cytokine involved in oestrogen synthesis. Seven days of cyclical feeding with milk fermented by L. helveticus R389 produced an increase in the number of apoptotic cells, compared with all other groups.ConclusionThis study demonstrated that 7 days of cyclical administration of milk fermented by both strains of L. helveticus diminishes tumour growth, stimulating an antitumour immune response. Compounds released during milk fermentation with L. helveticus R389 would be implicated in its immunoregulatory capacity on the immune response in mammary glands and tumour, which were correlated with the cytokines found at the systemic level. The milk fermented by L. helveticus R389 was able to modulate the relationship between immune and endocrine systems (by IL-6 diminution), which is very important in oestrogen-dependent tumour and induced cellular apoptosis.

Epithelial-stromal interactions in the mouse and human mammary gland in vivo.

This review deals with the development and hormonal responses of mouse and human mammary glands. A major focus of the review is the role of mesenchymal-epithelial interactions in embryonic mammary development and the role of stromal-epithelial interactions in mammary gland biology. Finally, we present a new model for studying growth, differentiation and hormonal response in human breast epithelium grown in vivo in nude mouse hosts. This new model involves the construction of tissue recombinants composed of human or mouse mammary fibroblasts plus human breast epithelium in polymerized collagen gels. In the model, mouse mammary fibroblasts and human breast fibroblasts appear to support the normal differentiation and growth of human breast epithelium equally. This observation raises the possibility of using mouse mammary fibroblasts from various mutant mice to assess the role of specific paracrine-acting gene products in human mammary gland biology and carcinogenesis.

Null Mutation of Peroxisome Proliferator-activated Receptor-interacting Protein in Mammary Glands Causes Defective Mammopoiesis

To investigate the role of nuclear receptor coactivator peroxisome proliferator-activated receptor-interacting protein (PRIP) in mammary gland development, we generated a conditional null mutation of PRIP in mammary glands. In PRIP-deficient mammary glands, the elongation of ducts during puberty was not affected, but the numbers of ductal branches were decreased, a condition that persisted long after puberty, indicating that the potential of ductal branching was impaired. During pregnancy, PRIP-deficient mammary glands exhibited decreased alveolar density. The lactating PRIP-deficient glands contained scant lobuloalveoli with many adipocytes, whereas the wild type glands were composed of virtually no adipocytes but mostly lobuloalveoli. As a result, PRIP mammary-deficient glands could not produce enough milk to nurse all the pups during lactation. The ductal branching of mammary glands in response to estrogen treatment was attenuated in PRIP mutant glands. Whereas the proliferation index was similar between wild type and PRIP-deficient glands, increased apoptosis was observed in PRIP-deficient glands. PRIP-deficient glands expressed increased amphiregulin, transforming growth factor-alpha, and betacellulin mRNA as compared with wild type glands. The differentiated function of PRIP-deficient mammary epithelial cells was largely intact, as evidenced by the expression of abundant beta-casein, whey acidic protein (WAP), and WDNM1 mRNA. We conclude that PRIP is important for normal mammary gland development.

The expression of oestrogen receptor (ER)-beta and its variants, but not ERalpha, in adult human mammary fibroblasts.

Whilst oestrogen receptor (ER)-alpha and ERbeta have been shown to be important in the development of the mammary gland, the cell-specific expression pattern of these two receptors within the human breast is not clear. Although it is well established that in the developing rodent mammary gland stromal ERalpha mediates the secretion of growth factors which stimulate the proliferation of the ductal epithelium, the expression of ERalpha in human adult breast stromal fibroblasts is controversial, and the expression of ERbeta has not been properly defined. In the present study, we have evaluated the expression of ERalpha and ERbeta by immunohistochemistry in normal tissue samples, and in purified human breast fibroblasts by Western blotting, RT-PCR analysis and ligand-binding sucrose gradient assay. Our data clearly demonstrated that ERbeta variants, including ERbeta1, ERbeta2, ERbeta5, ERbetadelta and ERbetains, but not ERalpha, are expressed in human adult mammary fibroblasts. These results are supported by the findings that an ERbeta-selective ligand, BAG, but not the ERalpha high-affinity ligand oestradiol, can induce fibroblast growth factor-7 release and activate transcription from an oestrogen-responsive element promoter in these adult human mammary fibroblasts. Together, these observations revealed that, in the adult breast and in breast cancer, the proliferative signals derived from the stroma of adult mammary glands in response to oestrogen are not mediated by ERalpha and provide new insights into the nature of stromal-epithelial interactions in the adult mammary gland. In addition, the expression of these ERbeta variants in cells where there is no ERalpha suggested that these ERbeta splice forms may have functions other than that of modulating ERalpha activity.