NHERF1/EBP50 controls lactation by establishing basal membrane polarity complexes with prolactin receptor

The development of the lactating mammary gland is a complex multifactorial process occurring in mammals during pregnancy. We show here that this process requires NHERF1/EBP50 (Na/H exchanger regulatory factor 1/ERM-binding phosphoprotein 50) expression and that successful lactation depends on NHERF1 allele copy number, with rates of 50 and 20% in NHERF1(+/−) and (−/−) mice, respectively. The prolactin receptor (PRLR)-STAT5 signaling provides the central axis triggering the differentiation of secretory mammary alveolar cells. In successfully lactating glands, NHERF1 is massively upregulated and forms complexes with PRLR, but also with β-catenin, E-cadherin and ezrin at the alveolar basal membrane, establishing basal polarity. In NHERF1-deficient glands, the basal polarity is disrupted, the PRLR levels and basal membrane localization are abolished, and the downstream STAT5 activation collapses with consequent reduction of milk protein synthesis. NHERF1/EBP50, a protein deregulated in breast cancer, thus emerges as an important physiological mediator of milk secretion, by engagement of PRLR in multimeric complexes at the alveolar basal membrane with subsequent network activation leading to cell differentiation.     

Effects of neonatal exposure to diethylstilbestrol, tamoxifen, and toremifene on the BALB/c mouse mammary gland

In this study, we compared the long-term effects of neonatal exposure to diethylstilbestrol (DES, 0.0125-50 microg), tamoxifen (TAM, 0.0125-50 microg), and toremifene (TOR, 53 microg) on mammary gland development and differentiation. Allometric growth of the mammary ducts was stimulated by neonatal DES exposure (12.5 microg) and impaired by exposure to TAM (25 microg). Neonatal treatment with high doses of DES resulted in mammary ducts that displayed extensive dilatation and precocious lactogenesis in postpubertal, nulliparous females. Initiation of this precocious differentiation coincided with the absence of corpora lutea, increased levels of serum prolactin (PRL), and the induction of Prl mRNA expression within the mammary glands. Neonatal exposure to 1.25 microg TAM increased alveolar development in postpubertal, nulliparous females similar to that recorded in females treated with low doses of DES. Lower doses of TAM did not affect alveolar development, whereas branching morphogenesis and alveolar development were impaired by higher doses. Increased alveolar development in females exposed to 1.25 microg TAM was associated with elevated serum progesterone (P) and increased alveolar development in response to exogenous P. Taken together, our findings demonstrate that neonatal exposure to both DES and TAM exerts long-lasting effects on the proliferation and differentiation of the mammary glands in female BALB/c, primarily as the result of endocrine disruption.     

A novel LacZ reporter mouse reveals complex regulation of the progesterone receptor promoter during mammary gland development

To further our understanding of progesterone (P) as an endocrine mammogen, a PR(lacz) knockin mouse was generated in which the endogenous progesterone receptor (PR) promoter directly regulated lacZ reporter expression. The PR(lacz) mouse revealed PR promoter activity was restricted to the epithelial compartment during the prenatal and postnatal stages of mammary gland development. At puberty, PR promoter activity was unexpectedly robust and restricted to the body cells within the terminal end buds and to the luminal epithelial cells in the subtending ducts. In the adult, the preferential localization of PR(lacz) positive cells to the distal regions of ductal side branches provided a cellular context to the recognized mandatory role of P in ductal side-branching, and segregation of these cells from cells that undergo proliferation supported an intraepithelial paracrine mode of action for P in branching morphogenesis. Toward the end of pregnancy, the PR(lacz) mouse disclosed a progressive attenuation in PR promoter activity, supporting the postulate that the preparturient removal of the proliferative signal of P is a prerequisite for the emergence of a functional lactating mammary gland. The data suggest that PR expression before pregnancy is to ensure the specification and spatial organization of ductal and alveolar progenitor cell lineages, whereas abrogation of PR expression before lactation is required to enable terminal differentiation of the mammary gland.     

Oestrogen receptor-alpha regulates non-canonical Hedgehog-signalling in the mammary gland

Mesenchymal dysplasia (mes) mice harbour a truncation in the C-terminal region of the Hh-ligand receptor, Patched-1 (mPtch1). While the mes variant of mPtch1 binds to Hh-ligands with an affinity similar to that of wild type mPtch1 and appears to normally regulate canonical Hh-signalling via smoothened, the mes mutation causes, among other non-lethal defects, a block to mammary ductal elongation at puberty. We demonstrated previously Hh-signalling induces the activation of Erk1/2 and c-src independently of its control of smo activity. Furthermore, mammary epithelial cell-directed expression of an activated allele of c-src rescued the block to ductal elongation in mes mice, albeit with delayed kinetics. Given that this rescue was accompanied by an induction in estrogen receptor-alpha (ERα) expression and that complex regulatory interactions between ERα and c-src are required for normal mammary gland development, it was hypothesized that expression of ERα would also overcome the block to mammary ductal elongation at puberty in the mes mouse. We demonstrate here that conditional expression of ERα in luminal mammary epithelial cells on the mes background facilitates ductal morphogenesis with kinetics similar to that of the MMTV-c-src^Act mice. We demonstrate further that Erk1/2 is activated in primary mammary epithelial cells by Shh-ligand and that this activation is blocked by the inhibitor of c-src, PP2, is partially blocked by the ERα inhibitor, ICI 182780 but is not blocked by the smo-inhibitor, SANT-1. These data reveal an apparent Hh-signalling cascade operating through c-src and ERα that is required for mammary gland morphogenesis at puberty.     

Pseudophosphorylated prolactin (S179D PRL) inhibits growth and promotes beta-casein gene expression in the rat mammary gland

We have investigated the individual roles of unmodified prolactin (U-PRL) and a mimic of phosphorylated PRL (S179D PRL) in mammary development. Recombinant versions of the PRLs were delivered to rats throughout pregnancy at a rate of 6 microg/24 h per rat and to non-pregnant females at a rate of 24 microg/24 h per rat. Measurement of progesterone, corticosterone, and estradiol showed no effect of the administered PRLs on the levels of these other mammotropic hormones. Histological and morphometric analysis showed U-PRL to cause mammary growth, whereas S179D PRL inhibited growth. Molecular analysis demonstrated decreased beta-casein expression in the mammary glands of the U-PRL-treated animals at term and increased beta-casein expression in the mammary glands of the S179D PRL-treated animals. Superior beta-casein gene expression in response to S179D PRL versus U-PRL was confirmed in HC11 cells. We conclude that U-PRL is important for growth, whereas S179D PRL promotes at least one measure of differentiated function in the mammary gland.     

Matrix metalloproteinases are expressed during ductal and alveolar mammary morphogenesis, and misregulation of stromelysin-1 in transgenic mice induces unscheduled alveolar development.

The matrix-degrading metalloproteinases stromelysin-1, stromelysin-3, and gelatinase A are expressed during ductal branching morphogenesis of the murine mammary gland. Stromelysin-1 expression in particular correlates with ductal elongation, and in situ hybridization and three-dimensional reconstruction studies revealed that stromelysin-1 mRNA was concentrated in stromal fibroblasts along the length of advancing ducts. Transgenic mice expressing an activated form of stromelysin-1 under the control of the MMTV promoter/enhancer exhibited inappropriate alveolar development in virgin females. Ultrastructural analysis demonstrated that the basement membrane underlying epithelial and myoepithelial cells was amorphous and discontinuous compared with the highly ordered basal lamina in control mammary glands. Transgenic mammary glands had at least a twofold increase in the number of cells/unit area and a 1.4-fold increase in the percent of cycling cells by 13 wk of age compared with nontransgenic littermates. In addition, transgenic glands expressed beta-casein mRNA, but not protein, and resembled the proliferative and differentiated state of an animal between 8 and 10 days pregnant. An analysis of metalloproteinase expression in the glands of normal pregnant females demonstrated that the same matrix metalloproteinase family members, including stromelysin-1, were expressed in connective tissue cells surrounding epithelial clusters during the time of lobuloalveolar development. These results suggest that metalloproteinases may assist in remodeling ECM during normal ductal and alveolar branching morphogenesis, and that disruption of the basement membrane by an activated metalloproteinase can affect basic cellular processes of proliferation and differentiation.     

Synergism between prolactin and ovarian hormones of DNA synthesis in rats mammary gland.

Autoradiography with [30H] thymedine has been used to measure the rate of DNA synthesis in the mammary epithelium of hypophysectomized-ovariectomized rats under the influence of estradiol, progesterone, and prolactin. Controls and animals treated with estradiol did not increase [3-H] thymidine incorporation, while progesterone alone had a slight stimulatory effect. Prolactin alone stimulated some [3-H] thymidine uptake in ductal and alveolar epithelium, but when combined with either estradiol or progesterone synergistic effects were observed. Estradiol with prolactin stimulated incorporation primarily in the ductal epithelium, whereas progesterone with prolactin stimulated both ductal and alveolar epithelium.     

The pattern of beta-catenin responsiveness within the mammary gland is regulated by progesterone receptor

Experiments involving beta-catenin loss- and gain-of-function in the mammary gland have decisively demonstrated the role of this protein in normal alveologenesis. However, the relationship between hormonal and beta-catenin signaling has not been investigated. In this study, we demonstrate that activated beta-catenin rescues alveologenesis in progesterone receptor (PR; Pgr)-null mice during pregnancy. Two distinct subsets of mammary cells respond to expression of DeltaN89beta-catenin. Cells at ductal tips are inherently beta-catenin-responsive and form alveoli in the absence of PR. However, PR activity confers beta-catenin responsiveness to progenitor cells along the lateral ductal borders in the virgin gland. Once activated by beta-catenin, responding cells switch on an alveolar differentiation program that is indistinguishable from that observed in pregnancy and is curtailed by PR signaling.     

Vitamin D(3) receptor ablation alters mammary gland morphogenesis

Postnatal mammary gland morphogenesis is achieved through coordination of signaling networks in both the epithelial and stromal cells of the developing gland. While the major proliferative hormones driving pubertal mammary gland development are estrogen and progesterone, studies in transgenic and knockout mice have successfully identified other steroid and peptide hormones that impact on mammary gland development. The vitamin D(3) receptor (VDR), whose ligand 1,25-dihydroxyvitamin D(3) is the biologically active form of vitamin D(3), has been implicated in control of differentiation, cell cycle and apoptosis of mammary cells in culture, but little is known about the physiological relevance of the vitamin D(3) endocrine system in the developing gland. In these studies, we report the expression of the VDR in epithelial cells of the terminal end bud and subtending ducts, in stromal cells and in a subset of lymphocytes within the lymph node. In the terminal end bud, a distinct gradient of VDR expression is observed, with weak VDR staining in proliferative populations and strong VDR staining in differentiated populations. The role of the VDR in ductal morphogenesis was examined in Vdr knockout mice fed high dietary Ca(2+) which normalizes fertility, serum estrogen and neonatal growth. Our results indicate that mammary glands from virgin Vdr knockout mice are heavier and exhibit enhanced growth, as evidenced by higher numbers of terminal end buds, greater ductal outgrowth and enhanced secondary branch points, compared with glands from age- and weight-matched wild-type mice. In addition, glands from Vdr knockout mice exhibit enhanced growth in response to exogenous estrogen and progesterone, both in vivo and in organ culture, compared with glands from wild-type mice. Our data provide the first in vivo evidence that 1,25-dihydroxyvitamin D(3) and the VDR impact on ductal elongation and branching morphogenesis during pubertal development of the mammary gland. Collectively, these results suggest that the vitamin D(3) signaling pathway participates in negative growth regulation of the mammary gland.     

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.     

Cortactin-binding protein 90 (CBP90) expression in the mouse mammary glands during prolactin-induced lobuloalveolar development

We have previously performed suppression subtractive hybridization to identify genes that were induced during prolactin (PRL)-driven lobuloalveolar development of the mammary gland. This suggested that cortactin-binding protein 90 (CBP90), which is known to be a brain-specific protein that binds to cortactin, was expressed under the regulation of PRL in the mammary glands (preliminary observation). In this study, the expression of CBP90 was examined in the mammary glands of mice under manipulated hormonal circumstances. PRL treatment by 9 days of pituitary grafting induced CBP90 expression in the normal mammary glands but not in the cleared fat pads, while cortactin was expressed constitutively in both the normal mammary glands and the cleared fat pads. Unlike milk proteins, longer treatment with PRL (36 days of pituitary grafting) did not increase the expression level of CBP90 mRNA, while it slightly increased the cortactin mRNA level. Mammary CBP90 mRNA expression was induced by pituitary grafting but not by progesterone treatment in PRL-deficient mice, while pituitary grafting induced mammary CBP90 expression in ovariectomized PRL-deficient mice only when estrogen and progesterone were appropriately supplemented to permit the formation of alveolar buds. The CBP90 protein was detected by immunohistochemistry in the luminal epithelium of the alveolar buds and more faintly in the ductal epithelium. Thus, from the unique expression pattern, CBP90 may be useful as a molecular marker for the hormone-stimulated development of mammary alveolar buds.     

PPARγ antagonist GW9662 induces functional estrogen receptor in mouse mammary organ culture: potential translational significance

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ) plays a central role in regulating metabolism, including interaction with the estrogen receptor-α (ERα). Significantly, PPARγ activity can be modulated by small molecules to control cancer both in vitro and in vivo (Yin et al., Cancer Res 69:687–694, 2009). Here, we evaluated the effects of the PPARγ agonist GW7845 and the PPARγ antagonist GW9662 on DMBA-induced mammary alveolar lesions (MAL) in a mouse mammary organ culture. The results were as follows: (a) the incidence of MAL development was significantly inhibited by GW 7845 and GW 9662; (b) GW9662 but not GW7845, in the presence of estradiol, induced ER and PR expression in mammary glands and functional ERα in MAL; (c) while GW9662 inhibited expression of adipsin and ap2, GW 7845 enhanced expression of these PPARγ-response genes; and (d) Tamoxifen caused significant inhibition of GW9662 treated MAL, suggesting that GW9662 sensitizes MAL to antiestrogen treatment, presumably through rendering functional ERα and induction of PR. The induction of ERα by GW9662, including newer analogs, may permit use of anti-ER strategies to inhibit breast cancer in ER? patients.     

Sequential requirement of hepatocyte growth factor and neuregulin in the morphogenesis and differentiation of the mammary gland

We have examined the role of two mesenchymal ligands of epithelial tyrosine kinase receptors in mouse mammary gland morphogenesis. In organ cultures of mammary glands, hepatocyte growth factor (HGF, scatter factor) promoted branching of the ductal trees but inhibited the production of secretory proteins. Neuregulin (NRG, neu differentiation factor) stimulated lobulo-alveolar budding and the production of milk proteins. These functional effects are paralleled by the expression of the two factors in vivo: HGF is produced in mesenchymal cells during ductal branching in the virgin animal; NRG is expressed in the mesenchyme during lobulo-alveolar development at pregnancy. The receptors of HGF and NRG (c-met, c-erbB3, and c-erbB4), which are expressed in the epithelial cells, are not regulated. In organ culture, branching morphogenesis and lobulo-alveolar differentiation of the mammary gland could be abolished by blocking expression of endogenous HGF and NRG by the respective antisense oligonucleotides; in antisense oligonucleotide-treated glands, morphogenesis could again be induced by the addition of recombinant HGF and NRG. We thus show that two major postnatal morphogenic periods of mammary gland development are dependent on sequential mesenchymal- epithelial interactions mediated by HGF and NRG.     

A short form of the prolactin (PRL) receptor is able to rescue mammopoiesis in heterozygous PRL receptor mice

The heterozygous prolactin (PRL) receptor (PRLR +/-) mouse fails to develop a fully functional mammary gland at the end of the first pregnancy and shows markedly impaired lobuloalveolar development and milk secretion in young females. The PRLR is expressed ubiquitously, with various proportions of long and short isoforms in different tissues. Conflicting data have appeared on the putative role of the receptor short forms, with both agonist and antagonistic actions proposed. To assess whether the mouse PR-1 short isoform of the PRLR is potentially able to transduce a signal, we overexpressed it in heterozygous mice and investigated its effect on the rescue of mammary development. PRLR+/- mice were not able to develop a functional mammary gland, but restoration of mammary alveolar development and an increase in the expressions of casein and whey acidic protein genes were observed in transgenic PRLR+/- mice expressing the short form of the PRLR, leading to a complete rescue of mammary gland development and function in young females. These results demonstrate that PR-1, the short form of the PRLR, can improve mammary development in PRLR+/- mice, which compensates for the haploinsufficiency of the receptor long form; this effect is probably caused by accelerated proliferation and an activation of the PRLR signaling cascade, resulting in activation of target genes involved in mammary development and milk synthesis.     

Metastasis-associated protein 1 deregulation causes inappropriate mammary gland development and tumorigenesis

Emerging data suggest that metastasis-associated protein 1 (MTA1) represses ligand-dependent transactivation functions of estrogen receptor-alpha in cultured breast cancer cells and that MTA1 is upregulated in human breast tumors. However, the role of MTA1 in tumorigenesis in a physiologically relevant animal system remains unknown. To reveal the role of MTA1 in mammary gland development, transgenic mice expressing MTA1 under the control of the mouse mammary tumor virus promoter long terminal repeat were generated. Unexpectedly, we found that mammary glands of these virgin transgenic mice exhibited extensive side branching and precocious differentiation because of increased proliferation of ductal and alveolar epithelial cells. Mammary glands of virgin transgenic mice resemble those from wild-type mice in mid-pregnancy and inappropriately express beta-casein, cyclin D1 and beta-catenin protein. Increased ductal growth was also observed in the glands of ovariectomized female mice, as well as of transgenic male mice. MTA1 dysregulation in mammary epithelium and cancer cells triggered downregulation of the progesterone receptor-B isoform and upregulation of the progesterone receptor-A isoform, resulting in an imbalance in the native ratio of progesterone receptor A and B isoforms. MTA1 transgene also increased the expression of progesterone receptor-A target genes Bcl-XL (Bcl2l1) and cyclin D1 in mammary gland of virgin mice, and, subsequently, produced a delayed involution. Remarkably, 30% of MTA1 transgenic females developed focal hyperplastic nodules, and about 7% exhibited mammary tumors within 18 months. These studies establish, for the first time, a potential role of MTA1 in mammary gland development and tumorigenesis. The underlying mechanism involves the upregulation of progesterone receptor A and its targets, Bcl-XL and cyclin D1.     

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.