Effects of epidermal growth factor,estrogen, and progestin on DNA synthesis in mammary cells in vivo are determined by the developmental state of the gland

Estrogen (E), progesterone (P), and epidermal growth factor (EGF) are involved in the growth and development of the normal mammary gland. While studies have been carried out to investigate the in vivo effects of EGF in the immature mammary gland, nothing is known about the growth effects of EGF or its potential interactions with E and/or P in the adult mammary gland. The present studies were undertaken to investigate the effects of EGF, E, and P on mammary cell proliferation in immature, peripubertal vs. adult, sexually mature mice. We have found that EGF promotes epithelial and stromal cell proliferation in both the immature and adult mammary glands. In the immature gland, the end bud epithelium is most responsive to the proliferative effects of EGF and there is no apparent interaction between EGF, E, and/or P. In contrast, in the mature gland EGF adds to the proliferative effects of E+P in the ductal epithelium resulting in more extensive ductal sidebranching. Thus these results demonstrate that the developmental state of the mammary gland determines the nature and extent of the interactions between EGF, E, and P in growth and development. © 1993 Wiley-Liss, Inc.     

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.     

Transforming growth factor-alpha messenger RNA localization in the developing adult rat and human mammary gland by in situ hybridization

Transforming growth factor-alpha (TGF alpha) has been implicated in the autocrine growth control of a number of different rodent and human tumor cells, including breast cancer cells. Although TGF alpha has been detected in a limited number of normal tissues, its distribution and physiological function in the mammary gland are relatively unknown. TGF alpha mRNA expression was detected by in situ hybridization with a labeled TGF alpha antisense RNA probe and quantitated by application of computer-assisted digital image processing in both the ductal and alveolar epithelial cells in the virgin rat and nulliparous and parous human mammary glands. During pregnancy and lactation, the level of TGF alpha mRNA expression in the ductal and alveolar epithelial cells increased two- to threefold, while a heterogeneous yet strong expression of TGF alpha mRNA could also be detected in approximately 10-15% of the surrounding stromal cells in the pregnant mammary gland.     

Receptor-mediated effects on ligand availability influence relative mitogenic potencies of epidermal growth factor and transforming growth factor α

Epidermal growth factor (EGF) and transforming growth factor α (TGFα) elicit quantitatively different cell proliferation responses even though they act via a common receptor, the epidermal growth factor receptor (EGFR). We hypothesized that differential cellular trafficking of available ligand is responsible for the different mitogenic responses elicited by EGF and TGFα. Mitogenesis and ligand depletion were determined simultaneously in NR6 mouse fibroblasts expressing either wild-type (WT) or internalization-deficient cytoplasmic domain-truncated (c'973) EGFR. Thus we could determine the effects of both ligand-induced and low level constitutive ligand/receptor processing. For a given initial amount of growth factor, TGFα is a weaker stimulus than EGF in cells expressing either form of the EGFR. This difference in the mitogenic potencies correlates with increased depletion of TGFα observed during the growth assays. When this difference in ligand depletion is accounted for, or minimized, EGF and TGFα elicit quantitatively similar growth responses. Therefore, the relative mitogenic potencies of EGF and TGFα depend on ligand availability, as determined by the cellular trafficking of these ligands in conjunction with environmental circumstances. Interestingly, our data demonstrate that TGFα can be a less potent mitogenic stimulus than EGF under conditions where ligand availability is limited. Further, in our assays, differences in ligand processing are sufficient to explain the different mitogenic potencies of these growth factors in either of the receptor trafficking scenarios. Our results suggest a model of regulation of hormone responsiveness which favors dissociative ligands (such as TGFα) in receptor-limited situations and non-dissociative ligands (such as EGF) in the face of high receptor levels. © 1996 Wiley-Liss, Inc.     

Prolactin and epidermal growth factor regulation of the proliferation,morphogenesis, and functional differentiation of normal rat mammary epithelial cells in three dimensional primary culture

The epithelial cell-specific effects of prolactin and epidermal growth factor (EGF) on the development of normal rat mammary epithelial cells (MEC) were evaluated using a three dimensional primary culture model developed in our laboratory. Non-milk-producing MEC were isolated as spherical end bud-like mammary epithelial organoids (MEO) from pubescent virgin female rats. The cultured MEO developed into elaborate multilobular and lobuloductal alveolar organoids composed of cytologically and functionally differentiated MEC. Prolactin (0.01–10 μg/ml) and EGF (1–100 ng/ml) were each required for induction of cell growth, extensive alveolar, as well as multilobular branching morphogenesis, and casein accumulation. MEO cultured without prolactin for 14 days remained sensitive to the mitogenic, morphogenic, and lactogenic effects of prolactin upon subsequent exposure. Similarly, cells cultured in the absence of EGF remained sensitive to the mitogenic and lactogenic effects of EGF, but were less responsive to its morphogenic effects when it was added on day 14 of a 21-day culture period. If exposure to prolactin was terminated after the first week, the magnitude of the mitogenic and lactogenic effects, but not the morphogenic response was decreased. Removal of EGF on day 7 also reduced the mitogenic response, but did not have any effect on the magnitude of the lactogenic or morphogenic responses. These studies demonstrate that physiologically relevant development of normal MEC can be induced in culture and that this model system can be used to study the mechanisms by which prolactin and EGF regulate the complex developmental pathways operative in the mammary gland. © 1995 Wiley-Liss, Inc.     

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.     

Members of the fatty acid binding protein family are differentiation factors for the mammary gland

Mammary gland development is controlled by systemic hormones and by growth factors that might complement or mediate hormonal action. Peptides that locally signal growth cessation and stimulate differentiation of the developing epithelium have not been described. Here, we report that recombinant and wild-type forms of mammary-derived growth inhibitor (MDGI) and heart-fatty acid binding protein (FABP), which belong to the FABP family, specifically inhibit growth of normal mouse mammary epithelial cells (MEC), while growth of stromal cells is not suppressed. In mammary gland organ culture, inhibition of ductal growth is associated with the appearance of bulbous alveolar end buds and formation of fully developed lobuloalveolar structures. In parallel, MDGI stimulates its own expression and promotes milk protein synthesis. Selective inhibition of endogenous MDGI expression in MEC by antisense phosphorothioate oligonucleotides suppresses appearance of alveolar end buds and lowers the beta-casein level in organ cultures. Furthermore, MDGI suppresses the mitogenic effects of epidermal growth factor, and epidermal growth factor antagonizes the activities of MDGI. Finally, the regulatory properties of MDGI can be fully mimicked by an 11-amino acid sequence, represented in the COOH terminus of MDGI and a subfamily of structurally related FABPs. This peptide does not bind fatty acids. To our knowledge, this is the first report about a growth inhibitor promoting mammary gland differentiation.     

Cyclin-dependent kinase (cdk) inhibitors/cdk4/cdk2 complexes in early stages of mouse mammary preneoplasia.

The level of circulating ovarian hormones (estrogen and progesterone) alone or in combination with pituitary hormones have a potent mitogenic impact in the normal mammary gland, and they also play a pivotal role in the development and progression of mammary carcinoma. The differential effects of hormones on the molecular components of cyclin-dependent kinase (cdk) complexes in mammary epithelium of the hormone-dependent ductal outgrowth line, EL11, and the hormone-independent alveolar outgrowth line, TM2L, were the focus of this study. The two outgrowth lines, which represent early stages in mammary hyperplasia, were compared with normal mammary gland at different hormonal conditions: control, hormone stimulated by pituitary isograft, and hormone depleted by ovariectomy. Hormonal stimulation by a pituitary isograft resulted in DNA synthesis and lobuloalveolar development of normal mammary ducts, DNA synthesis but no lobuloalveolar development in the EL11 ductal outgrowth, and no changes either in DNA synthesis or in lobuloalveolar morphology in the TM2L outgrowth. The levels of cdk4- and cyclin D1-associated kinase activities were correlated with cell proliferation in only the alveolar phenotypes (i.e., in only hormonally stimulated normal virgin gland and in alveolar mammary outgrowth), whereas cyclin D2-dependent kinase activity was correlated with cell proliferation in only the alveolar preneoplasia. p16(INK4a) and p21(Cip1) protein levels were decreased at the earliest stages of preneoplasia, i.e., at immortalization, and were independent from changes in cyclin D1, which occurred later in preneoplasia. Although all cdk inhibitors changed in concordance with hormonal status reflected by proliferation levels, p27(Kip1) was the only cdk inhibitor that was up-regulated at the earliest stages of preneoplasia and may have a unique role in blocking alveolar differentiation in response to the loss of one or more of the cell cycle-negative regulators. We hypothesize that up-regulation of p27(Kip1) prevents immortalized ductal outgrowths (EL11) from progressing to the neoplastic state, even under hormonal stimulation.     

Genetic manipulation of individual somatic mammary cells in vivo reveals a master role of STAT5a in inducing alveolar fate commitment and lactogenesis even in the absence of ovarian hormones

Assessing the molecular control of development and cell fate in individual cells in the intact mammary epithelium has not been possible to date. By exploiting an intraductal retrovirus (RCAS)-mediated gene delivery method to introduce a marker gene, we found that ductal epithelial cells are turned over with a half time of approximately 1 month in adult virgin mice. However, following RCAS-mediated introduction of a constitutively activated STAT5a (caSTAT5a), caSTAT5a-activated ductal epithelial cells expand and replace other cells in the epithelium, eventually forming a mammary gland resembling that in a late pregnant mouse, suggesting that STAT5a activation alone is sufficient to mediate pregnancy-induced mammary cell expansion, alveolar cell fate commitment, and lactogenesis. Furthermore, such caSTAT5a-induced alveolar differentiation does not require ovarian functions, although caSTAT5a-induced cell proliferation is partly reduced in ovariectomized mice. In conclusion, in this first report of studying the developmental role of a gene in a few cells in a normally developed virgin mammary ductal tree, STAT5a activation causes alveolar fate commitment and lactogenesis, and with the help of ovarian hormones, drives alveolar expansion.     

The regulation of expression of mouse mammary tumor virus DNA by steroid hormones and growth factors

Mouse mammary tumor virus (MMTV) expression is associated with hyperplastic alveolar growth and subsequent development of mammary cancers in the mouse. The expression of this virus is also controlled by factors involved in the normal proliferation and differentiation of the mammary epithelium. During pregnancy when the mammary gland undergoes massive proliferation, MMTV expression is increased. Steroid hormones and growth factors that play an important role in the proliferation of mammary gland cells are responsible for the increased MMTV expression. In sarcomatous transformation of mouse mammary epithelial cells, MMTV expression is repressed. This repression is due to negative control of MMTV expression by transforming growth factor-beta (TGF beta). This growth factor is produced in high amounts when mammary epithelial cells progress into the transformed state. The expression of MMTV is therefore under multiple control by steroid hormones and growth factors.     

The metastasis-promoting protein S100A4 regulates mammary branching morphogenesis

High levels of the S100 calcium binding protein S100A4 also called fibroblast specific protein 1 (FSP1) have been established as an inducer of metastasis and indicator of poor prognosis in breast cancer. The mechanism by which S100A4 leads to increased cancer aggressiveness has yet to be established; moreover, the function of this protein in normal mammary gland biology has not been investigated. To address the role of S100A4 in normal mammary gland, its spatial and temporal expression patterns and possible function in branching morphogenesis were investigated. We show that the protein is expressed mainly in cells of the stromal compartment of adult humans, and during active ductal development, in pregnancy and in involution of mouse mammary gland. In 3D culture models, topical addition of S100A4 induced a significant increase in the TGFα mediated branching phenotype and a concomitant increase in expression of a previously identified branching morphogen, metalloproteinase-3 (MMP-3). These events were found to be dependent on MEK activation. Downregulation of S100A4 using shRNA significantly reduced TGFα induced branching and altered E-cadherin localization. These findings provide evidence that S100A4 is developmentally regulated and that it plays a functional role in mammary gland development, in concert with TGFα by activating MMP-3, and increasing invasion into the fat pad during branching. We suggest that S100A4-mediated effects during branching morphogenesis provide a plausible mechanism for how it may function in breast cancer progression.     

Dual roles for macrophages in ovarian cycle-associated development and remodelling of the mammary gland epithelium

Each ovarian cycle, the mammary gland epithelium rotates through a sequence of hormonally regulated cell proliferation, differentiation and apoptosis. These studies investigate the role of macrophages in this cellular turnover. Macrophage populations and their spatial distribution were found to fluctuate across the cycle. The number of macrophages was highest at diestrus, and the greatest number of macrophages in direct contact with epithelial cells occurred at proestrus. The physiological necessity of macrophages in mammary gland morphogenesis during the estrous cycle was demonstrated in Cd11b-Dtr transgenic mice. Ovariectomised mice were treated with estradiol and progesterone to stimulate alveolar development, and with the progesterone receptor antagonist mifepristone to induce regression of the newly formed alveolar buds. Macrophage depletion during alveolar development resulted in a reduction in both ductal epithelial cell proliferation and the number of alveolar buds. Macrophage depletion during alveolar regression resulted in an increased number of branch points and an accumulation of TUNEL-positive cells. These studies show that macrophages have two roles in the cellular turnover of epithelial cells in the cycling mammary gland; following ovulation, they promote the development of alveolar buds in preparation for possible pregnancy, and they remodel the tissue back to its basic architecture in preparation for a new estrous cycle.     

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.     

Cellular turnover in the mammary gland is correlated with systemic levels of progesterone and not 17beta-estradiol during the estrous cycle

Adult mammary tissue has been considered "resting" with minimal morphological change. Here, we reveal the dynamic nature of the nulliparous murine mammary gland. We demonstrate specific changes at the morphological and cellular levels, and uncover their relationship with the murine estrous cycle and physiological levels of steroid hormones. Differences in the numbers of higher-order epithelial branches and alveolar development led to extensive mouse-to-mouse mammary variations. Morphology (assigned grades 0-3) ranged from a complete lack of alveoli to the presence of numerous alveoli emanating from branches. Morphological changes were driven by epithelial proliferation and apoptosis, which differed between ductal versus alveolar structures. Proliferation within alveolar epithelium increased as morphological grade increased. Extensive alveolar apoptosis was restricted to tissue exhibiting grade 3 morphology, and was approximately 14-fold higher than at all other grades. Epithelial proliferation and apoptosis exhibited a positive relationship with serum levels of progesterone, but not with 17beta-estradiol. Compared with other estrous stages, diestrus was unique in that the morphological grade, epithelial proliferation, apoptosis, and progesterone levels all peaked at this stage. The regulated tissue remodeling of the mammary gland was orchestrated with mRNA changes in specific matrix metalloproteinases (MMP-9 and MMP-13) and specific tissue inhibitors of metalloproteinases (TIMP-3 and TIMP-4). We propose that the cyclical turnover of epithelial cells within the adult mammary tissue is a sum of spatial and functional coordination of hormonal and matrix regulatory factors.     

Signaling pathways in mammary gland development.

Unlike most other organs, development of the mammary gland occurs predominantly after birth, under the control of steroid and peptide hormones. Once the gland is established, cycles of proliferation, functional differentiation, and death of alveolar epithelium occur repeatedly with each pregnancy. Although it is unique in this respect, the signaling pathways utilized by the gland are shared with other cell types, and have been tailored to meet the needs of this secretory tissue. Here we discuss the signaling pathways that have been adopted by the mammary gland for its own purposes, and the functions they perform.     

Role of growth factors, steroid and peptide hormones in the regulation of human prostatic tumor growth

Previous work carried out in the authors' laboratory has shown that LHRH agonists directly inhibit the proliferation of hormone-responsive and hormone-independent human prostatic cancer cell lines (respectively LNCaP and DU145). In addition, the hormone-dependent LNCaP cells respond to a challenge with testosterone with an increase in growth rate. The following experiments have been performed to investigate whether the LHRH agonists might act by interfering with the stimulatory actions of either the EGF/TGF system or androgens. The results obtained in LNCaP and DU145 cells show that LHRH agonists counteract the mitogenic action of the EGF/TGF system. This effect is mediated by a decrease in the concentration of EGF receptors. In addition, in the hormone-dependent LNCaP cells, the treatment with LHRH agonists antagonizes the proliferation promoting effect of testosterone, which in turn appears to be mediated by the activation of the locally expressed EGF/TGF system. Finally, the results suggest the presence in LNCaP cells of a soluble peptidase able to degrade LHRH. In conclusion, the present data suggest an intimate interplay among the actions of LHRH agonists, of androgens and of growth factors, thus, supporting the hypothesis that LHRH agonists may interfere with the EGF/TGF stimulatory loop and with androgens in the control of the proliferation of human prostatic tumors.     

Proteolytic processing of TGFα redirects its mitogenic activity: the membrane-anchored form is autocrine,the secreted form is paracrine

Wild-type transforming growth factor α (TGFα) expression in lactotrope cells in the pituitary gland led to lactotrope-specific pituitary hyperplasia and adenomata. To indicate whether the EGF receptor is involved in this TGFα-mediated phenotype, we bred TGFα mice with mice expressing the cytoplasmic truncated-EGF receptor (EGFR-tr), which is dominant-negative in other models. These bitransgenic mice developed pituitary pathology despite expression of the dominant-negative receptor. To further characterize this observation, we generated two lineages of transgenic mice that overexpress mutant forms of TGFα: a processed soluble form (s TGFα) and a cytoplasmic-deleted form (TGFαΔC). While sTGFα expression in lactotrope cells failed to induce autocrine lactotrope hyperplasia, the pituitary became very enlarged due to proliferation of neighboring interstitial cells. In contrast, the TGFαΔC mice did not develop a phenotype, although the mRNA and protein were present in the pituitary and this form of TGFα was confirmed to be biologically active and targeted properly to the plasma membrane of cultured CHO cells. The results suggest that the cytoplasmic domain of TGFα is required for autocrine parenchymal tumor formation in the pituitary gland. This signal cannot be inhibited by the EGFR-tr. Conversely, the released form of TGFα appears to have primarily paracrine activity.