Netrin-1 can affect morphogenesis and differentiation of the mouse mammary gland

Netrin-1 has been shown to regulate the function of the EGF-like protein Cripto-1 (Cr-1) and affect mammary gland development. Since Cr-1 is a target gene of Nanog and Oct4, we investigated the relationship between Netrin-1 and Cr-1, Nanog and Oct4 during different stages of development in the mouse mammary gland. Results from histological analysis show that exogenous Netrin-1 was able to induce formation of alveolar-like structures within the mammary gland terminal end buds of virgin transgenic Cripto-1 mice and enhance mammary gland alveologenesis in early pregnant FVB/N mice. Results from immunostaining and Western blot analysis show that Netrin-1, Nanog and Oct4 are expressed in the mouse embryonic mammary anlage epithelium while Cripto-1 is predominantly expressed outside this structure in the surrounding mesenchyme. We find that in lactating mammary glands of postnatal FVB/N mice, Netrin-1 expression is highest while Cripto-1 and Nanog levels are lowest indicating that Netrin-1 may perform a role in the mammary gland during lactation. HC-11 mouse mammary epithelial cells stimulated with lactogenic hormones and exogenous soluble Netrin-1 showed increased beta-casein expression as compared to control thus supporting the potential role for Netrin-1 during functional differentiation of mouse mammary epithelial cells. Finally, mouse ES cells treated with exogenous soluble Netrin-1 showed reduced levels of Nanog and Cripto-1 and higher levels of beta-III tubulin during differentiation. These results suggest that Netrin-1 may facilitate functional differentiation of mammary epithelial cells and possibly affect the expression of Nanog and/or Cripto-1 in multipotent cells that may reside in the mammary gland.     

Expression and Functional Role of Sprouty-2 in Breast Morphogenesis

Branching morphogenesis is a mechanism used by many species for organogenesis and tissue maintenance. Receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and the sprouty protein family are believed to be critical regulators of branching morphogenesis. The aim of this study was to analyze the expression of Sprouty-2 (SPRY2) in the mammary gland and study its role in branching morphogenesis. Human breast epithelial cells, breast tissue and mouse mammary glands were used for expression studies using immunoblotting, real rime PCR and immunohistochemistry. Knockdown of SPRY2 in the breast epithelial stem cell line D492 was done by lentiviral transduction of shRNA constructs targeting SPRY2. Three dimensional culture of D492 with or without endothelial cells was done in reconstituted basement membrane matrix. We show that in the human breast, SPRY2 is predominantly expressed in the luminal epithelial cells of both ducts and lobuli. In the mouse mammary gland, SPRY2 expression is low or absent in the virgin state, while in the pregnant mammary gland SPRY2 is expressed at branching epithelial buds with increased expression during lactation. This expression pattern is closely associated with the activation of the EGFR pathway. Using D492 which generates branching structures in three-dimensional (3D) culture, we show that SPRY2 expression is low during initiation of branching with subsequent increase throughout the branching process. Immunostaining locates expression of phosphorylated SPRY2 and EGFR at the tip of lobular-like, branching ends. SPRY2 knockdown (KD) resulted in increased migration, increased pERK and larger and more complex branching structures indicating a loss of negative feedback control during branching morphogenesis. In D492 co-cultures with endothelial cells, D492 SPRY2 KD generates spindle-like colonies that bear hallmarks of epithelial to mesenchymal transition. These data indicate that SPRY2 is an important regulator of branching morphogenesis and epithelial to mesenchymal transition in the mammary gland.     

Cripto-1 induces apoptosis in HC-11 mouse mammary epithelial cells

Cripto-1 (CR-1) is an epidermal growth factor (EGF)-related protein. CR-1 can inhibit beta-casein and whey acidic protein expression in mouse mammary epithelial cells. The present study demonstrates that CR-1 can induce apoptosis in HC-11 mouse mammary epithelial cells, as measured by bis-benzimide stained nuclei, TUNEL assay and cell death ELISA. Apoptosis could be observed after 2 days of exposure of confluent HC-11 cells to CR-1 in the absence of the survival factors EGF and insulin, with maximum apoptosis occurring at 3 days. A reduction in poly(ADP-ribose) polymerase (PARP) expression and an increase in beta-catenin cleavage was found after 18 h of exposure to CR-1 suggesting that apoptosis was preceded by the induction of a caspase activity since the caspase inhibitor ZFAD.FMK could block the CR-1-induced reduction in PARP expression and CR-1-induced apoptosis. CR-1 was found to increase the expression of caspase-3-like protease. Although, the levels of p27kip1 and p21Bax did not change after exposure to CR-1 for 18 h, the levels of Bcl-xL became undetectable. These studies suggest that CR-1 promotes apoptosis by mediating the induction of caspase-3-like protease and downregulating the expression of Bcl-xL.     

Cripto-1 indirectly stimulates the tyrosine phosphorylation of erb B-4 through a novel receptor

Cripto-1 (CR-1) is a recently discovered protein of the epidermal growth factor family that fails to directly bind to any of the four known erb B type 1 receptor tyrosine kinases. The present study demonstrates that CR-1 indirectly induces tyrosine phosphorylation of erb B-4 but not of the epidermal growth factor-related receptors erb B-2 and erb B-3 in different mouse and human mammary epithelial cell lines. In addition, down-regulation of erb B-4 in NMuMG mouse mammary epithelial cells and in T47D human breast cancer cells, using an anti-erb B-4 blocking antibody or a hammerhead ribozyme vector targeted to erb B-4 mRNA, impairs the ability of CR-1 to fully activate mitogen-activated protein kinase. Finally, chemical cross-linking of 125I-CR-1 to mouse and human mammary epithelial cell membranes results in the labeling of two specific bands with a molecular weight of 130 and 60 kDa, suggesting that the CR-1 receptor represents a novel receptor structurally unrelated to any of the known type I receptor tyrosine kinases. In conclusion, these data demonstrate that CR-1, upon binding to an unknown receptor, can enhance the tyrosine kinase activity of erb B-4 and that a functional erb B-4 receptor is required for CR-1-induced MAPK activation.     

Amphiregulin mediates self-renewal in an immortal mammary epithelial cell line with stem cell characteristics

Amphiregulin (AREG), a ligand for epidermal growth factor receptor, is required for mammary gland ductal morphogenesis and mediates estrogen actions in vivo, emerging as an essential growth factor during mammary gland growth and differentiation. The COMMA-D β-geo (CDβgeo) mouse mammary cell line displays characteristics of normal mammary progenitor cells including the ability to regenerate a mammary gland when transplanted into the cleared fat pad of a juvenile mouse, nuclear label retention, and the capacity to form anchorage-independent mammospheres. We demonstrate that AREG is essential for formation of floating mammospheres by CDβgeo cells and that the mitogen activated protein kinase signaling pathway is involved in AREG-mediated mammosphere formation. Addition of exogenous AREG promotes mammosphere formation in cells where AREG expression is knocked down by siRNA and mammosphere formation by AREG^−/− mammary epithelial cells. AREG knockdown inhibits mammosphere formation by duct-limited mammary progenitor cells but not lobule-limited mammary progenitor cells. These data demonstrate AREG mediates the function of a subset of mammary progenitor cells in vitro.     

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.     

Cripto: roles in mammary cell growth, survival, differentiation and transformation

Cripto-1 (Cr-1) protein, encoded by the teratocarcinoma-derived growth factor gene (TDGF-1), is highly correlated with transformation in breast cancer. Eighty-two percent of breast carcinomas express Cr-1 whereas it is undetected in normal human breast tissue. We confirmed and extended findings that Cr-1 protein is expressed during the pregnancy and lactating stages of normal murine mammary glands but is barely detectable in glands from virgin animals and is undetectable in involuted glands. Cr-1 was found to be expressed in CID 9 cells, a line of mammary epithelial cells derived from 14.5 day pregnant mice and we have used these cells to investigate the roles of this gene. Exogenous mouse Cr-1 expression from a retroviral vector caused CID 9 cells to grow at an increased rate and to increased cell densities compared to parental and control cells. CID 9 cells overexpressing Cr-1 did not differentiate efficiently. Infection of CID 9 cells with a Cr-1 antisense vector caused these cells to change in morphology, to grow slowly, to undergo apoptosis at a higher rate and to achieve lower saturation densities but the cells were still capable of differentiating. We concluded that Cr-1 is an autocrine growth factor for normal breast cells, that when over-expressed stimulates excessive cell proliferation at the expense of differentiation. In transplantation studies, Cr-1 over-expression stimulated the growth and survival of mammary cells, but did not stimulate tumorigenesis in vivo.     

Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and Mammary Epithelial Cells

Expression of the heparin-binding growth factor, pleiotrophin (PTN) in the mammary gland has been reported but its function during mammary gland development is not known. We examined the expression of PTN and its receptor ALK (Anaplastic Lymphoma Kinase) at various stages of mouse mammary gland development and found that their expression in epithelial cells is regulated in parallel during pregnancy. A 30-fold downregulation of PTN mRNA expression was observed during mid-pregnancy when the mammary gland undergoes lobular-alveolar differentiation. After weaning of pups, PTN expression was restored although baseline expression of PTN was reduced significantly in mammary glands of mice that had undergone multiple pregnancies. We found PTN expressed in epithelial cells of the mammary gland and thus used a monoclonal anti-PTN blocking antibody to elucidate its function in cultured mammary epithelial cells (MECs) as well as during gland development. Real-time impedance monitoring of MECs growth, migration and invasion during anti-PTN blocking antibody treatment showed that MECs motility and invasion but not proliferation depend on the activity of endogenous PTN. Increased number of mammospheres with laminin deposition after anti-PTN blocking antibody treatment of MECs in 3D culture and expression of progenitor markers suggest that the endogenously expressed PTN inhibits the expansion and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In vivo, PTN activity was found to inhibit ductal outgrowth and branching via the inhibition of phospho ERK1/2 signaling in the mammary epithelial cells. We conclude that PTN delays the maturation of the mammary gland by maintaining mammary epithelial cells in a progenitor phenotype and by inhibiting their differentiation during mammary gland development.     

Matriptase/epithin participates in mammary epithelial cell growth and morphogenesis through HGF activation

The epithelial-derived, type II transmembrane serine protease matriptase, the mouse homologue of which is epithin, has been shown to be involved in epidermal differentiation, hair formation, and thymus function. We show in this study that epithin/matriptase (Epi/MTP) plays a significant role in mammary epithelial cell growth and morphogenesis. Epi/MTP is expressed at low level in the mouse mammary epithelium of young animals and it accumulates at the terminal end-bud of the growing ducts. The level of Epi/MTP is elevated in the mammary glands at stages when epithelial proliferation and modeling occur. It is primarily present in the luminal epithelial cells of mouse mammary ducts and lobules. Using an ex vivo three-dimensional culture system for mammary epithelial functional assays, we show that mammary epithelial growth and morphogenesis in the presence of the latent form hepatocyte growth factor (pro-HGF) are blocked either by an inhibitor of the Epi/MTP protease activity or by siRNA knockdown of the Epi/MTP expression. These studies demonstrate that Epi/MTP participates in mammary epithelial growth and modeling through activation of pro-HGF. Our findings reveal an important pathway in normal mammary epithelial morphogenesis which may participate in breast cancer progression.     

Mammary epithelial-restricted expression of activated c-src rescues the block to mammary gland morphogenesis due to the deletion of the C-terminus of Patched-1

Mesenchymal dysplasia (mes) mice expressing a C-terminally truncated version of the Hedgehog (Hh)-ligand receptor, Patched-1 (Ptch1), exhibit a limited spectrum of developmental defects including blocked ductal morphogenesis of the mammary gland during puberty. Given that the Hh-ligands can stimulate signalling cascades distinct from the canonical pathway involving Smo and the Gli-family proteins and that Ptch1 binds to factors harbouring SH3-domains, we determined whether the mes mammary gland defect could be rescued by activating non-canonical signalling pathways downstream of Ptch1. We demonstrate here that expression of constitutively active c-src (c-src(Act)) in mammary epithelial cells overcomes the block to mammary epithelial morphogenesis in mes mice. Specifically, MMTV-directed expression of c-src(Act) rescued blocked ductal morphogenesis in mes mice, albeit only after animals were more than 15 weeks of age. The overall morphology resembled wild type mice expressing c-src(Act) although 40% of mes/MMTV-c-src(Act) mice exhibited terminal end buds at 24 weeks of age. C-src(Act) restored the proliferative capacity of mes epithelial cells, self-renewal capacity of mammary progenitor cells and increased the expression of Esr1, Ptch1 and Gli1. These data reveal the cooperative interactions between signalling cascades involving c-src and Ptch1 and suggest that Hh-signalling may be permissive for c-src/Esr1-dependent mammary gland morphogenesis.     

Prion protein modifies TGF-beta induced signal transduction

Members of the transforming growth factor-beta (TGF-beta) superfamily regulate a multitude of cellular processes as well as the expression of various proteins such as, e.g., matrix metalloproteinases (MMPs). These endopeptidases selectively degrade components of the extracellular matrix as well as non-matrix substrates like growth factors and cell surface receptors. MMPs are activated during embryonic development, morphogenesis, and tissue resorption/remodeling as well as in pathological conditions such as deranged wound healing and cancer metastasis. In this report we demonstrate that over-expression of cellular prion protein in mouse mammary gland epithelial cells is able to modulate TGF-beta induced signal transduction leading to a synergistic increase of secreted MMP-2 activity. This correlates with elevated substrate detachment of cells grown as an epithelial monolayer as well as interfering with morphogenesis of cells cultured in a three-dimensional collagen type I matrix.     

Mammary gland stem cells: current status and future challenges

Distinct subsets of cells, including cells with stem cell-like properties, have been proposed to exist in normal human breast epithelium and breast carcinomas. The cellular origins of epithelial cells contributing to gland development, tissue homeostasis and cancer are, however, still poorly understood. The mouse is a widely used model of mammary gland development, both directly by studying the mouse mammary epithelial cells themselves and indirectly, by studying development, morphogenesis, differentiation and carcinogenesis of xenotransplanted human breast epithelium in vivo. While in early studies, human or mouse epithelium was implanted as fragments into the mouse gland, more recent technical progress has allowed the self-renewal capacity and differentiation potential of distinct cell populations or even individual cells to be interrogated. Here, we review and discuss similarities and differences between mouse and human gland development with particular emphasis on the identity and localization of stem cells, and the influence of the surrounding microenvironment. It is concluded that while recent advances in the field have contributed immense insight into how the normal mammary gland develops and is maintained, significant discrepancies exist between the mouse and human gland which should be taken into consideration in current and future models of mammary stem cell biology.     

Essential functions of p21-activated kinase 1 in morphogenesis and differentiation of mammary glands

Although growth factors have been shown to influence mammary gland development, the nature of downstream effectors remains elusive. In this study, we show that the expression of p21-activated kinase (Pak)1, a serine/threonine protein kinase, is activated in mammary glands during pregnancy and lactation. By targeting an ectopic expression of a kinase-dead Pak1 mutant under the control of ovine beta-lactoglobulin promoter, we found that the mammary glands of female mice expressing kinase-dead Pak1 transgene revealed incomplete lobuloalveolar development and impaired functional differentiation. The expression of whey acidic protein and beta-casein and the amount of activated Stat5 in the nuclei of epithelial cells in transgenic mice were drastically reduced. Further analysis of the underlying mechanisms revealed that Pak1 stimulated beta-casein promoter activity in normal mouse mammary epithelial cells and also cooperated with Stat5a. Pak1 directly interacted with and phosphorylated Stat5a at Ser 779, and both COOH-terminal deletion containing Ser 779 of Stat5a and the Ser 779 to Ala mutation completely prevented the ability of Pak1 to stimulate beta-casein promoter. Mammary glands expressing inactive Pak1 exhibited a reduction of Stat5a Ser 779 phosphorylation. These findings suggest that Pak1 is required for alveolar morphogenesis and lactation function, and thus, identify novel functions of Pak1 in the mammary gland development.     

In VivoEffects of Hepatocyte Growth Factor/Scatter Factor on Mouse Mammary Gland Development

We have recently demonstrated the regulated expression ofHGF/SFand its receptor (c-met) during mouse mammary gland development together with the mitogenic, motogenic and morphogenic effects of exogenous HGF/SF on primary mammary epithelial cells in culture. This study was undertaken to analyze the influence of HGF/SF on reconstituted mouse mammary gland developmentin vivo.Here we report that overexpression of HGF/SF induces a range of alterations in the architecture of virgin mouse mammary gland. These include an enhancement of ductal end bud (mammary gland morphoregulatory control point) size and numbers and hyperplastic branching morphogenesis. These data are the first demonstration of the effects of HGF/SF on mammary epitheliumin vivo.