EGF receptor regulation in normal mouse mammary gland.

Estrogen (E), progesterone (P), and epidermal growth factor (EGF) are known to regulate growth and development of the normal mammary gland, and it is possible that EGF may interact with E and/or P. Estrogen (ER), progesterone (PR), and EGF receptors (EGF-R) have been detected in both mammary epithelial and stromal cells, and the relative roles of the various cells types in hormone-dependent growth regulation are not known. The present studies were undertaken to determine if E and/or P influence EGF action by exerting a regulatory effect on EGF-R levels and which cell types are affected. The comparative effects of ovariectomy and hormone treatments on EGF-R levels were examined in immature, pubertal 5-week-old and sexually mature 10-week-old female mice. EGF-R were characterized as a single class of high affinity sites and EGF-R concentration was 2-fold higher in glands of 5-week-old mice. Ovariectomy had no significant effect on EGF-R concentration in either age group, and treatment with E and/or P had no effect on EGF-R levels in either epithelial or stromal cells in 5-week-old mice. In contrast, E+P treatment caused a 2-fold increase in receptor concentration in 10-week-old mice in the mammary epithelium. Thus it appears that the developmental state of the gland may determine the nature and extent of the interaction of of EGF, E, and P.     

Role of epidermal growth factor in the acquisition of ovarian steroid hormone responsiveness in the normal mouse mammary gland

The purpose of the present studies was to investigate the role of epidermal growth factor (EGF) in the acquisition of estrogen (E) and progestin (P) responsiveness in the mouse mammary gland in vivo. Using the Elvax 40P implant technique to introduce bioactive molecules directly into the mammary gland to produce a localized effect, we have made the novel observation that EGF implanted into glands of pubertal mice followed by E treatment resulted in the precocious acquisition of E-inducible progesterone receptors (PR). In sexually mature mice, EGF implants alone were able to increase PR. A neutralizing antibody specific for EGF blocked E-dependent stimulation of end-bud development and PR induction. Furthermore, the antiestrogen ICI 182,780 blocked the EGF-induced stimulation end-buds and PR induction, indicating that these EGF effects are mediated via estrogen receptors (ER). Immunohistochemical analysis showed that the endogenous EGF content of mammary glands of mature mice was higher than pubertal mice, that E implants caused a localized increase in mammary gland EGF content in both pubertal and mature mice, and that in mature mice E caused an increase in stromal cell EGF content. We have previously shown that the acquisition of E-inducible PR can be modulated by mammary stroma, and the present results indicate that mammary stroma could modulate hormonal responsiveness through control of local growth factor concentration. Taken together, these results provide evidence that E-dependent responses of mouse mammary gland in vivo, such as end-bud proliferation and PR regulation, may be mediated by EGF through an ER-dependent mechanism. J. Cell. Physiol. 174:251–260, 1998. © 1998 Wiley-Liss, Inc.     

Estrogen and estrogen plus progestin act directly on the mammary gland to increase proliferation in a postmenopausal mouse model

Hormone replacement therapy (HRT) with ovarian hormones is an important therapeutic modality for postmenopausal women. However, a negative side effect of HRT is an increased risk of breast cancer. Surgical induction of menopause by ovariectomy (OVX) in mice is an experimental model that may provide insights into the effects of hormone replacement therapy on the human breast. We have developed a mouse model of early and late postmenopausal states to investigate the effects of HRT on the normal mammary gland. The purpose of this study was to determine if HRT-induced proliferation was due to the direct action of the hormones on the mammary gland, or mediated systemically by hormones or growth factors produced elsewhere in the body. Estrogen (E) or E plus the synthetic progestin, R5020, were implanted directly into the mammary glands of early (1 week post OVX) and late (5 week post OVX) postmenopausal mice instead of administration by injection. We report that responses of early and late postmenopausal mice to implanted hormones were the same as those observed previously with systemically administered hormones. Implanted E conferred an enhanced proliferative response in the late postmenopausal gland characterized morphologically by enlarged duct ends. E+R5020 implants induced similar degrees of cell proliferation in both postmenopausal states but the morphological responses differed. Ductal sidebranching was observed in early postmenopausal mice, whereas duct end enlargement was observed in late postmenopausal mice. The differences in morphological response to E+R5020 in 5 week post OVX were associated with an inability of E to induce progesterone receptors (PR) in the late postmenopausal gland. The responses of the late postmenopausal glands to E and E+P were very similar to that observed previously in immature pubertal glands in ovary-intact mice. In pubertal mice, PR cannot be induced by E unless the mammary gland is pre-treated with EGF-containing implants. Similarly, herein pre-treatment of the late postmenopausal mammary gland with EGF-containing implants restored PR induction by E. Thus, EGF may determine the sensitivity of the mammary gland to E and E+P in late postmenopause and at puberty.     

EGF and TGFα modulate structural and functional differentiation of the mammary gland from pregnant mice in vitro: Possible role of the arachidonic acid pathway

Epidermal growth factor (EGF) has been suggested to be involved in mammary gland development by mitogenic stimulation of the ductal and alveolar epithelium in virgin mice. The present studies demonstrate that also in late-pregnant mice EGF leads to proliferation of the ductal, ductular, and alveolar epithelium. The mitogenic effect is associated with structural and functional dedifferentiation of alveolar cells as revealed by analysis of morphology, expression of cytosolic and secretory proteins, and fatty acid synthesis. Using a combination of metabolic inhibitors, the dedifferentiating effect of EGF could be blocked while the mitogenic action was not influenced. This finding demonstrates that the signal transduction pathway leading to dedifferentiation and mitosis can be separated, and that the dedifferentiating effect of EGF is independent of its mitogenic properties, but is probably mediated by activation of the arachidonic acid-dependent pathways (cyclo- and lipoxygenase pathways). Release of arachidonic acid from the endogenous phospholipid pool was found to be an early response of the explants to EGF. Accordingly, arachidonic acid itself proved to be capable of inducing epithelial dedifferentiation but failed to stimulate proliferation. TGFα showed qualitatively similar effects as EGF but was generally a stronger agonist. It is suggested that EGF and TGFα also play a role in mammary gland physiology during pregnancy by final developing and maintanance of the lobulo-alveolar structure in the mammary gland and prevention of premature onset of lactation, and that this is mediated through the PLA₂-arachidonic acid signalling cascade.     

Inhibition of mouse mammary ductal morphogenesis and down-regulation of the EGF receptor by epidermal growth factor

EGF, initially demonstrated to be a potent mitogen for a variety of cell types, has more recently been shown to inhibit proliferation of several cell lines. Few studies, however, have addressed the effects of EGF on growth and morphogenesis of tissues in vivo, particularly with regard to EGF as a possible inhibitor. We now demonstrate that EGF treatment of vigorously growing mammary ducts, administered directly to the glands by slow release plastic implants, inhibited normal ductal growth. Inhibition was restricted to the region around the implant and untreated glands in the same animal were normal, indicating direct effects of EGF. EGF-treated end buds were smaller and demonstrated reduced levels of DNA synthesis, although remnants of a stem (cap) cell layer persisted. Full inhibition of growth occurred within 3 days of implantation and required extended exposure to EGF, since treatment of 5 hr or less had no effect on ductal growth. At the lower inhibitory doses tested, growth resumed within 8 days, indicating reversibility of inhibition. No lobuloalveolar or hyperplastic response was seen. 125I-EGF autoradiography revealed that ductal growth inhibition was preceded by the disappearance of EGF receptors located in the cap cell layer of the end bud epithelium and in stromal cells adjacent to the buds. These results, in conjunction with our previous evidence demonstrating the growth-stimulatory effect by EGF on nonproliferating mammary ducts, suggest a growth regulatory role for EGF in mouse mammary ductal morphogenesis.     

Actions of oestrogens and antioestrogens on rat mammary gland development: relevance to breast cancer prevention

The proliferative actions of a series of antioestrogens on the development of the second thoracic mammary gland of ovariectomized immature Sprague-Dawley rats have been investigated. Evidence is presented that shows trans-tamoxifen, LY 117018 and LY 139481, like oestradiol-17 beta and cis-tamoxifen, promote full mammary gland ductal development and induce a high rate of cell proliferation in the undifferentiated epithelial cells of the terminal end buds, the main growth region for ductal growth. Conversely, ICI 164,384, a new antioestrogen, is without effect on ductal elongation. In vivo exposure of trans-tamoxifen and LY 117018 treated glands in medically castrated animals to the carcinogen DMBA, results in a high rate of mammary tumour development. Indeed, the actions of these so-called antioestrogens are equivalent to those observed in oestradiol-treated rats.     

Estradiol, progesterone and prolactin modulate mammary gland morphogenesis in adult female plains vizcacha (Lagostomus maximus)

We studied for the first time the mammary gland morphogenesis and its hormonal modulation by immunolocalizing estradiol, progesterone and prolactin receptors (ER, PR and PRLR) in adult females of Lagostomus maximus, a caviomorph rodent which shows a pseudo-ovulatory process at mid-gestation. Mammary ductal system of non-pregnant females lacks expression of both ERα and ERβ. Yet throughout pregnancy, ERα and ERβ levels increase as well as the expression of PR. These increments are concomitant with ductal branching and alveolar differentiation. Even though mammary gland morphology is quite similar to that described for other rodents, alveolar proliferation and differentiation are accelerated towards the second half of pregnancy, once pseudo-ovulation had occurred. Moreover, this exponential growth correlates with an increment of both progesterone and estradiol serum-induced pseudo-ovulation. As expected, PR and PRLR are strongly expressed in the alveolar epithelium during pregnancy and lactation. Strikingly, PRLR is also present in ductal epithelia of cycling glands suggesting that prolactin function may not be restricted to its trophic effect on mammary glands of pregnant and lactating females, but it also regulates other physiological processes in mammary glands of non-pregnant animals. In conclusion, this report suggests that pseudo-ovulation at mid-gestation may be associated to L. maximus mammary gland growth and differentiation. The rise in P and E2-induced pseudo-ovulation as well as the increased expression of their receptors, all events that correlate with the development of a more elaborated and differentiated ductal network, pinpoint a possible relation between this peculiar physiological event and mammary gland morphogenesis.     

Mammary gland development in transforming growth factor beta1 null mutant mice: systemic and epithelial effects

The cytokine-transforming growth factor beta1 (TGFB1) is implicated in development of the mammary gland through regulation of epithelial cell proliferation and differentiation during puberty and pregnancy. We compared mammary gland morphogenesis in virgin Tgfb1(+/+), Tgfb1(+/-), and Tgfb1(-/-) mice and transplanted Tgfb1(+/+) and Tgfb1(-/-) epithelium to determine the impact of TGFB1 deficiency on development. When mammary gland tissue was evaluated relative to the timing of puberty, invasion through the mammary fat pad of the ductal epithelium progressed similarly, irrespective of genotype, albeit fewer terminal end buds were observed in mammary glands from Tgfb1(-/-) mice. The terminal end buds appeared to be normal morphologically, and a comparable amount of epithelial proliferation was evident. When transplanted into wild-type recipients, however, Tgfb1(-/-) epithelium showed accelerated invasion compared with Tgfb1(+/+) epithelium. This suggests that the normal rate of ductal extension in Tgfb1(-/-) null mutant mice is the net result of impaired endocrine or paracrine support acting to limit the consequences of unrestrained epithelial growth. By adulthood, mammary glands in cycling virgin Tgfb1(-/-) mice were morphologically similar to those in Tgfb1(+/+) and Tgfb1(+/-) animals, with a normal branching pattern, and the tissue differentiated into early alveolar structures in the diestrous phase of the ovarian cycle. Transplanted mammary gland epithelium showed a similar extent of ductal branching and evidence of secretory differentiation of luminal cells in pregnancy. These results reveal two opposing actions of TGFB1 during pubertal mammary gland morphogenesis: autocrine inhibition of epithelial ductal growth, and endocrine or paracrine stimulation of epithelial ductal growth.     

Testosterone inhibits estrogen-induced mammary epithelial proliferation and suppresses estrogen receptor expression.

This study investigated the effect of sex steroids and tamoxifen on primate mammary epithelial proliferation and steroid receptor gene expression. Ovariectomized rhesus monkeys were treated with placebo, 17beta estradiol (E2) alone or in combination with progesterone (E2/P) or testosterone (E2/T), or tamoxifen for 3 days. E2 alone increased mammary epithelial proliferation by approximately sixfold (P:<0.0001) and increased mammary epithelial estrogen receptor (ERalpha) mRNA expression by approximately 50% (P:<0.0001; ERbeta mRNA was not detected in the primate mammary gland). Progesterone did not alter E2's proliferative effects, but testosterone reduced E2-induced proliferation by approximately 40% (P:<0.002) and entirely abolished E2-induced augmentation of ERalpha expression. Tamoxifen had a significant agonist effect in the ovariectomized monkey, producing a approximately threefold increase in mammary epithelial proliferation (P:<0.01), but tamoxifen also reduced ERalpha expression below placebo level. Androgen receptor (AR) mRNA was detected in mammary epithelium by in situ hybridization. AR mRNA levels were not altered by E2 alone but were significantly reduced by E2/T and tamoxifen treatment. Because combined E2/T and tamoxifen had similar effects on mammary epithelium, we investigated the regulation of known sex steroid-responsive mRNAs in the primate mammary epithelium. E2 alone had no effect on apolipoprotein D (ApoD) or IGF binding protein 5 (IGFBP5) expression, but E2/T and tamoxifen treatment groups both demonstrated identical alterations in these mRNAs (ApoD was decreased and IGFBP5 was increased). These observations showing androgen-induced down-regulation of mammary epithelial proliferation and ER expression suggest that combined estrogen/androgen hormone replacement therapy might reduce the risk of breast cancer associated with estrogen replacement. In addition, these novel findings on tamoxifen's androgen-like effects on primate mammary epithelial sex steroid receptor expression suggest that tamoxifen's protective action on mammary gland may involve androgenic effects.     

Development of Foreign Mammary Epithelial Morphology in the Stroma of Immunodeficient Mice

Systemic growth and branching stimuli, and appropriate interactions with the host stroma are essential for the development of foreign epithelia in the mammary gland of immunodeficient mice. These factors were manipulated to promote and investigate the generation of representative bovine epithelial morphology in the transplanted mouse mammary stroma. The bovine mammary epithelium is unique in its commitment to rapid proliferation and high rate of differentiation. Its morphological organization within a fibrotic stroma resembles that of the human breast, and differs significantly from the rudimentary ductal network that penetrates a fatty stroma in mice. Transplantation of bovine mammary epithelial cells into the cleared mammary fat pad of NOD-SCID mice led to continuous growth of epithelial structures. Multilayered hollow spheres developed within fibrotic areas, but in contrast to mice, no epithelial organization was formed between adipocytes. The multilayered spheres shared characteristics with the heifer gland’s epithelium, including lumen size, cell proliferation, cytokeratin orientation, estrogen/progesterone receptor expression and localization, and milk protein synthesis. However, they did not extend into the mouse fat pad via ductal morphology. Pre-transplantation of fibroblasts increased the number of spheres, but did not promote extension of bovine morphology. The bovine cells preserved their fate and rarely participated in chimeric mouse–bovine outgrowths. Nevertheless, a single case of terminal ductal lobuloalveolar unit (TDLU) development was recorded in mice treated with estrogen and progesterone, implying the feasibility of this representative bovine morphology’s development. In vitro extension of these studies revealed paracrine inhibition of bovine epithelial mammosphere development by adipocytes, which was also generalized to breast epithelial mammosphere formation. The rescue of mammosphere development by fibroblast growth factor administration evidences an active equilibrium between inhibitory and supportive effects exerted by the adipose and fibrotic regions of the stroma, respectively, which determines the development of foreign epithelium.     

Steroid receptors and proliferation in the human breast

Despite recent gains in our knowledge of the hormonal control of proliferation and differentiation in the rodent mammary gland, the factors regulating these processes in the human are poorly understood. We have developed a model in which intact normal human breast tissue is grafted subcutaneously into adult female athymic nude mice and treated with oestrogen (E) and/or progesterone (P) at human physiological serum levels. We have shown that (i) E and not P is the major epithelial cell mitogen in the adult non-pregnant, non-lactating breast, (ii) E induces progesterone receptor (PR) expression and (iii) PR expression is maximally induced at low E concentrations while a higher amount of E is required to stimulate proliferation. These data raised the question of whether one cell type demonstrated two different responses to the two different E concentrations or whether PR expression and proliferation occurred in separate cell populations. Using dual label immunofluorescence, we showed that steroid receptor expression and proliferation (Ki67 antigen) are detected in separate cell populations in normal human breast epithelium, and that cells expressing the oestrogen receptor-alpha (ERalpha) invariably contained the PR. We also reported that this separation between steroid receptor expression and proliferation observed in the normal human epithelium is disrupted at an early stage in breast tumourigenesis. One interpretation supported by our recent findings is that some ERalpha/PR-positive epithelial cells are quiescent breast stem cells that act as "steroid hormone sensors". Such hormone sensor cells might secrete positive or negative paracrine/juxtacrine factors dependent on the prevailing E or P concentration to influence the proliferative activity of adjacent ERalpha/PR-negative epithelial cells.     

Influence of hormone and growth factor interactions on the proliferative potential of normal rat mammary epithelial cells in vitro

These experiments were aimed at using a recently developed serum-free culture system for growth of normal rat mammary epithelial (RME) cells in vitro to examine the interactions of specific hormones and growth factors on the proliferative potential of these cells. RME cells were obtained by enzymatic dissociation of mammary tissues of Lewis rats. Primary cultures were started by plating 2 X 10(5) RME cells per 60-mm type I collagen-coated tissue culture dish. Cultures were maintained in a basal medium that consisted of Ham's F-12 medium supplemented with bovine serum albumin (BSA), ethanolamine (EA), and transferrin (Tf), which, by itself, did not support RME cell proliferation. Insulin (I), hydrocortisone (HC), and epidermal growth factor (EGF), when added to the basal medium interacted synergistically to stimulate RME cell proliferation, but this effect was dependent on the additional presence of cholera toxin (CT). Under these conditions a greater-than-tenfold increase in cell number over a 10-day culture period was obtained. Insulin could be replaced by physiological levels of insulin-like growth factor-I (IGF-I). CT could be replaced by other agents that elevate intracellular levels of cyclic adenosine 3':5' monophosphate (cAMP) such as dibutyryl-cAMP (db-cAMP), prostaglandin E1 (PGE-1), and/or isobutylmethylxanthine (IBMX). Prolactin (M) or progesterone (P) potentiated the effect of I, HC, EGF, and CT, resulting in an additional twofold increase in cell number over that found in their absence. However, addition of both hormones was no more effective than either one alone. Furthermore, addition of M or P in the absence of EGF had no effect on RME cell proliferation. Addition of 17-B-estradiol (E2) to the I-, HC-, EGF-, and CT-containing medium also resulted in enhanced RME cell proliferation. These results point to a number of hormone and growth factor interactions that influence the proliferation of normal RME cells in vitro.     

Subpopulations of cells in immature mouse mammary gland as detected by proliferative responses to hormones in organ culture

Organ culture of immature mouse mammary gland was used to demonstrate the presence of different epithelial cell types in this tissue. Whole glands were cultivated for 10 days in various hormone combinations, and the proliferative responses of the epithelium were evaluated by [3H]thymidine autoradiography. It was found that different regions of the gland responded to hormones dissimilarly. The large, primary duct required no added hormones for either maintenance (viability according to histological criteria) or proliferation. Secondary and tertiary ducts required insulin for maintenance and proliferation and exhibited hyperplasia when a mineralocorticoid plus either growth hormone, prolactin, or placental lactogen were also present. End buds required the most complex hormone environment for maintenance in culture, and did not exhibit proliferative activity as intense as that which occurs in vivo, even in the optimum hormone combinations used.     

An in vitro model of epithelial cell growth stimulation in the rodent mammary gland

Abstract. Mouse mammary epithelial cell cultures previously described bring about extensive proliferation and a cell population with the appropriate markers for luminal ductal epithelial cells, and also the ability to form normal tissue after implantation into mice. This success may result from a culture environment that resembles certain aspects of the environment in the mammary gland. Mouse mammary epithelial cells, whose proliferation is limited when plated alone, can be stimulated to multiply by contact with lethally irradiated cells of the LA7 rat mammary tumour line. Most of the proliferative stimulus is imparted by direct cell contact between LA7 and mouse mammary cells. Junctions, including adherens junctions, form among all cells in the culture, much as junctions form in the mammary gland. LA7 cells secrete TGFα and bFGF, factors found in the mammary gland, and factors to which mouse mammary cells respond in culture. Mouse mammary cells express keratins 8 and 18, markers for luminal cells of the mammary duct. LA7 cells express keratin 14 and vimentin, markers for myoepithelial cells. These facts, taken together, fit a model of cell replacement in an epithelial tissue and also imitate the relationship between luminal ductal cells and myoepithelial cells in the mammary gland. This method of culturing cells is useful, not only for in vitro – in vivo carcinogenesis studies, but also for the study of mechanisms by which growth signals are imparted from one cell to another.     

Nulliparous CCAAT/enhancer binding proteindelta (C/EBPdelta) knockout mice exhibit mammary gland ductal hyperlasia

CCAAT/Enhancer binding proteins (C/EBPs) are a family of nuclear proteins that function in the control of cell growth, death, and differentiation. We previously reported that C/EBPdelta plays a key role in mammary epithelial cell G(0) growth arrest. In this report, we investigated the role of C/EBPdelta in mammary gland development and function using female mice homozygous for a targeted deletion of C/EBPdelta (C/EBPdelta -/-). C/EBPdelta -/- females develop normally and exhibit normal reproductive and lactational performance. Adult nulliparous C/EBPdelta -/- females, however, exhibit mammary epithelial cell growth control defects. The mean number of mammary ductal branches is significantly higher in adult nulliparous C/EBPdelta -/- females compared with C/EBPdelta +/+ (wild-type control) females (66.8 +/- 5.2 vs 42.9 +/- 6.3 branch points/field, P < 0.01). In addition, the mean total mammary gland cellular volume occupied by epithelium is significantly higher in adult nulliparous C/EBPdelta -/- females compared with C/EBPdelta +/+ controls (29.0 +/- 1.4 vs 20.4 +/- 1.3, P < 0.001). Our results showed that the BrdU labeling index was significantly higher in mammary epithelial cells from nulliparous C/EBPdelta -/- females compared with C/EBPdelta +/+ controls during the proestrus/estrus (4.55 +/- 0.70 vs 2.14 +/- 0.43, P < 0.01) and metestrus/diestrus (6.92 +/- 0.75 vs 3.98 +/- 0.43 P < 0.01) phases of the estrus cycle. In contrast, the percentage of mammary epithelial cells undergoing apoptosis during both phases of the estrus cycle did not differ between C/EBPdelta -/- and C/EBPdelta +/+ females. The increased epithelial cell content and proliferative capacity was restricted to the nulliparous C/EBPdelta -/- females as no differences in mammary gland morphology, ductal branching or total epithelial content were observed between multiparous C/EBPdelta -/- and C/EBPdelta +/+ females. These results demonstrate that C/EBPdelta plays a novel role in mammary epithelial cell growth control that appears to be restricted to the nulliparous mammary gland.     

Regulation of epithelial cell turnover and macrophage phenotype by epithelial cell-derived transforming growth factor beta1 in the mammary gland

Transforming growth factor beta1 (TGFB1) is a multi-functional cytokine that regulates cell proliferation, apoptosis and immune system responses. In the breast, the mammary epithelium is the primary source of TGFB1 and increased expression is associated with increased breast cancer risk. This study was conducted to investigate the roles of epithelial cell-derived TGFB1 in regulation of epithelial cell activity and macrophage phenotype in the mammary gland. Tgfb1 null mutant and wildtype mammary epithelium was transplanted into contra-lateral sides of the cleared mammary gland of TGFB1 replete scid mice. Transplanted tissue was analysed for markers of proliferation and apoptosis to determine the effect of Tgfb1 null mutation on epithelial cell turnover, and was analysed by immunohistochemistry to investigate the location, abundance and phenotype of macrophages. The number of proliferating and dying ductal epithelial cells, determined by BrdU and TUNEL, was increased by 35% and 3.3-fold respectively in mammary gland transplanted with Tgfb1 null epithelium compared to wildtype epithelium (p < 0.05). Abundance of F4/80+ macrophages in between Tgfb1 null epithelial cells compared to wildtype epithelial cells was increased by 50%. The number of iNOS+ and CCR7+ cells in the stroma surrounding Tgfb1 null alveolar epithelium was increased by 78% and 2-fold respectively, and dendriform MHC class II+ cells within ductal epithelium were decreased by 30%. We conclude that epithelial cell-derived TGFB1 in the mammary gland has two functions: (1) regulation of cellular turnover of epithelial cells, and (2) regulation of local macrophage phenotype. These findings shed new light on the diversity of roles of TGFB1 in the mammary gland which are likely to impact on breast cancer risk.     

Smad3 in the mammary epithelium has a nonredundant role in the induction of apoptosis, but not in the regulation of proliferation or differentiation by transforming growth factor-beta.

Transforming growth factor-beta (TGF-beta) regulates proliferation, morphogenesis, and functional differentiation in the mammary gland and plays complex roles in mammary tumorigenesis. Here we show that the signaling mediators Smad1-Smad5 are expressed at all stages of mammary gland development. To begin to investigate which Smads mediate which TGF-beta responses, we have analyzed mammary gland development in Smad3 null mice. Smad3 null virgin females showed delayed mammary gland development. However, this phenotype was secondary to ovarian insufficiency because Smad3 null mammary epithelium developed normally in hormonally supplemented Smad3 null mice or when transplanted into wild-type hosts. Absence of Smad3 had no effect on the ability of TGF-beta to inhibit the growth of mammary epithelial cells in culture, and no compensatory changes in expression or activation of Smad2 were seen in the Smad3 null epithelium. A small but significant decrease in apoptotic cells was seen in involuting glands from Smad3 null transplants. The results suggest that epithelial Smad3 is dispensable for TGF-beta effects on proliferation and differentiation in the mammary gland, but that it contributes in a nonredundant manner to the induction of apoptosis.     

Mouse mammary epithelial cells express the Na-K-Cl cotransporter,NKCC1: characterization,localization, and involvement in ductal development and morphogenesis

Despite the fact that physiological evidence points to the existence of a functional Na-K-Cl cotransporter in the mammary gland, the molecular identity of this transport process remains unknown. We now show that the Na-K-Cl cotransporter isoform, NKCC1, is expressed in mammary tissue. Developmental profiling revealed that the level of NKCC1 protein was significantly influenced by the stage of mammary gland development, and immunolocalization studies demonstrated that NKCC1 was present on the basolateral membrane of mammary epithelial cells. To examine whether functional NKCC1 is required for mammary epithelial cell development, we used NKCC1 -/- mice. We demonstrate that NKCC1 -/- mammary epithelium exhibited a significant delay in ductal outgrowth and an increase in branching morphogenesis during virgin development. These effects were autonomous to the epithelium as assessed by mammary gland transplantation. Although the absence of NKCC1 had no apparent effect on gross mammary epithelial cell morphology during lactation, pups born to NKCC1 -/- mice failed to thrive. Finally, analysis of NKCC1 protein in mouse models that exhibit defects in mammary gland development demonstrate that high levels of NKCC1 protein are indicative of ductal epithelial cells, and the presence of NKCC1 protein is characteristic of mammary epithelial cell identity.     

Adipocyte derived paracrine mediators of mammary ductal morphogenesis controlled by retinoic acid receptors

We generated a transgenic (Tg)-mouse model expressing a dominant negative-(DN)-RARα, (RARαG303E) under adipocytes-specific promoter to explore the paracrine role of adipocyte retinoic acid receptors (RARs) in mammary morphogenesis. Transgenic adipocytes had reduced level of RARα, β and γ, which coincided with a severely underdeveloped pubertal and mature ductal tree with profoundly decreased epithelial cell proliferation. Transplantation experiments of mammary epithelium and of whole mammary glands implicated a fat-pad dependent paracrine mechanism in the stunted phenotype of the epithelial ductal tree. Co-cultures of primary adipocytes, or in vitro differentiated adipocyte cell line, with mammary epithelium showed that when activated, adipocyte-RARs contribute to generation of secreted proliferative and pro-migratory factors. Gene expression microarrays revealed a large number of genes regulated by adipocyte-RARs. Among them, pleiotrophin (PTN) was identified as the paracrine effectors of epithelial cell migration. Its expression was found to be strongly inhibited by DN-RARα, an inhibition relieved by pharmacological doses of all-trans retinoic acid (atRA) in culture and in vivo. Moreover, adipocyte-PTHR, another atRA responsive gene, was found to be an up-stream regulator of PTN. Overall, these results support the existence of a novel paracrine loop controlled by adipocyte-RAR that regulates the mammary ductal tree morphogenesis.     

Activation of retinoblastoma protein in mammary gland leads to ductal growth suppression, precocious differentiation, and adenocarcinoma.

The retinoblastoma (Rb) tumor suppressor controls cellular proliferation, survival, and differentiation and is functionally inactivated by mutations or hyperphosphorylation in most human cancers. Although activation of endogenous Rb is thought to provide an effective approach to suppress cell proliferation, long-term inhibition of apoptosis by active Rb may have detrimental consequences in vivo. To directly test these paradigms, we targeted phosphorylation-resistant constitutively active Rb alleles, Rb Delta Ks, to the mouse mammary gland. Pubescent transgenic females displayed reduced ductal elongation and cell proliferation at the endbuds. Post-puberty transgenic mice exhibited precocious cellular differentiation and beta-casein expression and extended survival of the mammary epithelium with a moderate but specific effect on the expression of E2F1, IGF1R alpha, and phospho-protein kinase B/AKT. Remarkably, approximately 30% Rb Delta K transgenic females developed focal hyperplastic nodules, and approximately 7% exhibited full-blown mammary adenocarcinomas within 15 mo. Expression of the Rb Delta K transgene in these mammary tumors was reduced greatly. Our results suggest that transient activation of Rb induces cancer by extending cell survival and that the dual effects of Rb on cell proliferation and apoptosis impose an inherent caveat to the use of the Rb pathway for long-term cancer therapy.