Signaling through the stromal epidermal growth factor receptor is necessary for mammary ductal development

Stromal-epithelial interactions are critical in determining patterns of growth, development and ductal morphogenesis in the mammary gland, and their perturbations are significant components of tumorigenesis. Growth factors such as epidermal growth factor (EGF) contribute to these reciprocal stromal-epithelial interactions. To determine the role of signaling through the EGF receptor (EGFR) in mammary ductal growth and branching, we used mice with a targeted null mutation in the Egfr. Because Egfr-/- mice die perinatally, transplantation methods were used to study these processes. When we transplanted neonatal mammary glands under the renal capsule of immuno-compromised female mice, we found that EGFR is essential for mammary ductal growth and branching morphogenesis, but not for mammary lobulo-alveolar development. Ductal growth and development was normal in transplants of mammary epithelium from Egfr-/- mice into wild-type (WT) gland-free fat pads and in tissue recombinants prepared with WT stroma, irrespective of the source of epithelium (StromaWT/Epi-/-, StromaWT/EpiWT). However, ductal growth and branching was impaired in tissue recombinants prepared with Egfr-/- stroma (Stroma-/-/EpiWT, Stroma-/-/Epi-/-). Thus, for ductal morphogenesis, signaling through the EGFR is required only in the stromal component, the mammary fat pad. These data indicate that the EGFR pathway plays a key role in the stromal-epithelial interactions required for mammary ductal growth and branching morphogenesis. In contrast, signaling through the EGFR is not essential for lobulo-alveolar development. Stimulation of lobulo-alveolar development in the mammary gland grafts by inclusion of a pituitary isograft under the renal capsule as a source of prolactin resulted in normal alveolar development in both Egfr-/- and wild-type transplants. Through the use of tissue recombinants and transplantation, we have gained new insights into the nature of stromal-epithelial interactions in the mammary gland, and how they regulate ductal growth and branching morphogenesis.     

Interchangeability of Caenorhabditis elegans DSL proteins and intrinsic signalling activity of their extracellular domains in vivo

Ligands of the Delta/Serrate/lag-2 (DSL) family and their receptors, members of the lin-12/Notch family, mediate cell-cell interactions that specify cell fate in invertebrates and vertebrates. In C. elegans, two DSL genes, lag-2 and apx-1, influence different cell fate decisions during development. Here we show that APX-1 can fully substitute for LAG-2 when expressed under the control of lag-2 regulatory sequences. In addition, we demonstrate that truncated forms lacking the transmembrane and intracellular domains of both LAG-2 and APX-1 can also substitute for endogenous lag-2 activity. Moreover, we provide evidence that these truncated forms are secreted and able to activate LIN-12 and GLP-1 ectopically. Finally, we show that expression of a secreted DSL domain alone may enhance endogenous LAG-2 signalling. Our data suggest ways that activated forms of DSL ligands in other systems may be created.     

The insulin-like growth factors (IGF) and IGF type I receptor during postnatal growth of the murine mammary gland: sites of messenger ribonucleic acid expression and potential functions

The goals of this study were to determine the cellular sites of insulin-like growth factor (IGF) and IGF type-I receptor (IGF-IR) expression and to begin to elucidate functional roles for the IGFs during postnatal development of the murine mammary gland. Using in situ hybridization analyses, we determined that IGF-I, IGF-II, and IGF-IR messenger RNAs were expressed in the highly proliferative terminal end buds during pubertal ductal growth. Consistent with these data, IGF-I (in combination with mammogenic hormones) promoted ductal growth in pubertal stage mammary glands cultured in vitro. During postpubertal and pregnancy stages, IGF-II and IGF-IR continued to be expressed in ductal epithelium. Expression of IGF-II in ductal and alveolar epithelium correlated with the pattern of rapidly proliferating cells, as determined by incorporation of 5-Bromo-2'-deoxyuridine, suggesting a potential autocrine or paracrine role for IGF-II as a mitogen for ductal epithelial cells. IGF-I expression was reinitiated in mammary epithelium in the differentiated alveoli at the end of pregnancy, suggesting an additional role for this factor in maintenance of the alveoli during lactation. Taken together, these data support an in vivo role for locally-produced IGFs in promoting ductal growth during puberty and suggest that IGF-I and IGF-II may have distinct functions during pregnancy-induced alveolar development.     

Paralogous mouse Hox genes, Hoxa9, Hoxb9, and Hoxd9, function together to control development of the mammary gland in response to pregnancy

Although the role of Hox genes in patterning the mammalian body plan has been studied extensively during embryonic and fetal development, relatively little is known concerning Hox gene function in adult animals. Analysis of mice with mutant Hoxa9, Hoxb9, and Hoxd9 genes shows that these paralogous genes are required for mediating the expansion and/or differentiation of the mammary epithelium ductal system in response to pregnancy. Mothers with these three mutant genes cannot raise their own pups, but the pups can be rescued by fostering by wild-type mothers. Histologically, the mammary glands of the mutant mothers seem normal before pregnancy but do not develop properly in response to pregnancy and parturition. Hoxa9, Hoxb9, and Hoxd9 are expressed normally in adult mammary glands, suggesting a direct role for these genes in the development of mammary tissue after pregnancy. Because loss-of-function mutations in these Hox genes cause hypoplasia of the mammary gland after pregnancy, it may be productive to look for misexpression of these genes in mammary carcinomas.     

MMTV-Fgf8 transgenic mice develop mammary and salivary gland neoplasia and ovarian stromal hyperplasia

Prior studies have identified Fibroblast Growth Factor-8 (Fgf8) as a possible proto-oncogene in mouse mammary tumorigenesis. We now report on the generation of two types of Fgf8 transgenic mice that each utilize the mouse mammary tumor virus (MMTV) promoter. The first transgene (MMTV-Fgf8b) results in the overexpression of the FGF8b isoform exclusively. Male and female MMTV-Fgf8b transgenic mice are viable and fertile. RNA for FGF8b is detected in mammary gland and salivary gland tissues of transgenic mice by Northern blot analysis. Nearly 85% of breeding transgenic female mice developed mammary lobular adenocarcinomas by 12 months of age, while no tumors developed in non-transgenic littermates. Salivary gland tumors occurred in some animals, always in association with mammary tumors. Several MMTV-Fgf8b transgenic mice had lung metastases at necropsy. The second transgene (MMTV-Fgf8) uses the entire Fgf8 gene and potentially encodes all FGF8 isoforms. Fgf8 is expressed by this transgene in several tissues in addition to those described above, notably the ovaries. The two MMTV-Fgf8 founders developed mammary ductal adenocarcinomas at five and eight months of age, and both displayed ovarian stromal hyperplasia. The founders expressing either transgene did not successfully nurse their pups. These results demonstrate that production of FGF8b, and possibly other FGF8 isoforms, in the mammary and salivary glands contributes to oncogenesis, and that ovarian expression results in stromal hyperplasia.     

Analysis of phenotypic abnormalities and cell fate changes caused by dominant activated and dominant negative forms of the Notch receptor in Drosophila development

The Notch gene of Drosophila plays an important role in cell fate specification throughout development. The Notch protein contains a large extracellular domain of 36 EGF-like repeats as well as 3 Notch/lin-12 repeats and an intracellular domain with 6 cdc10/ankyrin repeats, motifs which are highly conserved in several vertebrate Notch homologues [1-7]. In this review we summarize the results of two recent studies which attempt to establish structure-function relationships of the various domains of the Notch gene product [8, 9]. The functions of various structural domains of the Notch protein in vivo were investigated using a series of deletion mutants which have been ectopically expressed either under the hsp70 heat-shock promoter or under the sevenless eye-specific promoter. Truncation of the extracellular domain of Drosophila Notch produces an activated receptor as judged by its ability to cause phenotypes matching those of gain-of-function alleles or duplications of the Notch locus [8]. Equivalent truncations of vertebrate Notch-related proteins have been associated with malignant neoplasms and other developmental abnormalities [3, 6, 10, 11]. In contrast, dominant negative phenotypes result from overexpression of a protein lacking most intracellular sequences. These results were extended by an analysis of activated Notch function at single-cell resolution in the Drosophila compound eye [9]. It was shown that while overexpression of full-length Notch in defined cell types has no apparent effects, overexpression of activated Notch in the same cells transiently blocks their proper cell-fate commitment, causing them to either adopt incorrect cell fates or to differentiate incompletely. Moreover, an activated Notch protein lacking the transmembrane domain is translocated to the nucleus, raising the possibility that Notch may participate directly in nuclear events.     

Activation and function of the epidermal growth factor receptor and erbB-2 during mammary gland morphogenesis

The hormonal stimulation of mammary gland morphogenesis is believed to occur through growth factor receptor signaling pathways. To determine the importance of the epidermal growth factor receptor (EGFR) pathway, we examined extracts of inguinal mammary glands from prepubertal and pubertal mice for tyrosine-phosphorylated EGFR and other erbB receptors. Tyrosine phosphorylation of both EGFR and erbB-2 was detected in normal female BALB/c mice at 5-6 weeks of age, but not during the prepubertal stage, e.g., 24 days of age. Treatment of mice with estradiol or epidermal growth factor also stimulated the formation of mammary EGFR/erbB-2 phosphotyrosine. Waved-2 mice, which have impaired EGFR kinase activity, exhibited less mammary development than did wild-type (wt) mice when both were evaluated at 36 days of age. Because EGFR knockout (KO) mice die shortly after birth, glands from the newborns were implanted under the renal capsules of female nude mice. Under these conditions, extensive ductal growth was observed in mammary glands from wt animals; in contrast, glands from EGFR KO mice failed to grow beyond rudimentary structures. Tissue recombinants revealed that the wt fat pad supported the morphogenesis of EGFR KO epithelium, whereas the EGFR KO fat pad did not. Taken together, these data suggest that EGFR is essential for morphogenesis of the mammary ducts and functions during this period of mammary development as a heterodimer with erbB-2 in the mammary stroma.     

Characterization of the INT6 mammary tumor gene product

INT6 is a unique gene, highly conserved throughout evolution and associated with mammary tumorigenesis in the mouse. Although it is expressed in all adult tissues of the mouse and early in embryonic development, its function is unknown. To study the normal distribution and the potential function of the Int6 gene products, we produced antibodies against synthetic peptides specific for the Int6 protein. Western blot and immunoprecipitation analysis demonstrated a 43-kD major gene product that is localized in the cytosolic fraction of mammary cell homogenates. This latter observation is supported by immunoperoxidase analysis, which shows a strong staining anti-Int6 peptide in the perinuclear region of the HC11 mammary epithelial cell line, suggesting a possible localization in the Golgi apparatus. Further immunocytochemical studies in the mouse embryo show that Int6 expression is prevalent in migrating neural crest cells, in the notochord, and in condensing cartilage between 9.5 and 14.5 days of development. In these embryonic tissues, Int6 staining co-localizes with the staining of ricinus lectin, and giantin, proteins that are specifically associated with the Golgi apparatus. The restricted expression of the protein within the Golgi apparatus and its strong conservation throughout evolution suggest that Int6 may perform an essential cellular function.     

The Hytrin Community Assessment Trial study: a one-year study of terazosin versus placebo in the treatment of men with symptomatic benign prostatic hyperplasia. HYCAT Investigator Group

Functional differentiation of mammary tissue progresses in distinct phases spanning puberty and pregnancy. Here we have analyzed and compared the effects of transforming growth factor beta 1 (TGF beta 1), TGF alpha, and whey acidic protein (WAP), the Notch-related cell fate protein Int3, and p53 and pRb on mammary development. We chose transgene expression from the WAP gene promoter which is only active in mammary alveolar cells. The imbalanced expression of these molecules specifically altered development and differentiation of the gland. While TGF alpha did not disturb alveolar outgrowth, little or no alveolar structures developed in the presence of Int3. TGF beta 1, WAP, and the expression of SV40 T-antigen-which inactivates p53 and PRb-reduced overall alveolar development. The expression of individual milk protein genes was affected differentially by the transgenes. A WAP-lacZ transgene served as an additional indicator of terminal differentiation of alveolar cells, Homogeneous expression of lacZ was seen in mice transgenic for lacZ, or for TGF alpha and lacZ. In contrast, only a few differentiated cells were observed in the presence of TGF beta 1 and Tag. Thus, the expression of growth regulators in the same defined subset of mammary cells results in distinct developmental changes and a specific pattern of alveolar differentiation.     

B and T cells are required for mouse mammary tumor virus spread within the mammary gland

Mouse mammary tumor virus (MMTV) is an infectious retrovirus transmitted through milk from mother to newborns. MMTV encodes a superantigen (SAg) whose activity is indispensable for the virus life cycle, since a genetically engineered virus with a mutation in the sag gene neither amplified in cells of the immune system of suckling pups nor infected their mammary glands. When wild-type MMTV was injected directly into the mammary glands of uninfected pubescent mice, their lymphoid as well as mammary gland cells became virus infected. To test whether this infection of lymphoid cells was dependent on SAg activity and required for virus spread within the mammary gland, we performed mammary gland injections of wild-type MMTV(C3H) into two strains of transgenic mice that lacked SAg-cognate, V beta 14+ T cells. Neither the MTV-ORF or LEL strains showed infection of their mammary glands. Moreover, no MMTV infection of their peripheral lymphocytes was detected. Similar experiments with mice lacking B cells (mu-chain knockouts) showed no detectable virus spread in the mammary glands or lymphoid tissues. These data suggest that SAg activity and MMTV-infected lymphocytes are required, not only for initial steps of viral infection, but also for virus spread within the mammary gland. Virus spread at late times in infection determines whether MMTV induces mammary tumors.     

Involution of the lactating mammary gland is inhibited by the IGF system in a transgenic mouse model

Development of the mammary gland during puberty, pregnancy, and lactation is controlled by steroid and peptide hormones and growth factors. To determine the role of the insulin-like growth factors (IGFs) in this process we developed a transgenic model using the whey acidic protein (WAP) gene to direct expression of rat IGF-I and human IGF binding protein-3 (IGFBP-3) to mammary tissue during late pregnancy and throughout lactation. High levels of expression of transgenic IGF-I and IGFBP-3 were seen in lobular-alveolar cells by in situ hybridization. There was no obvious effect on mammary development during pregnancy and lactation; indeed, mothers were capable of nursing their pups normally and the only structural difference seen in the mammary glands at peak lactation was an overall smaller size of the alveoli. We also evaluated the role of IGF-I and IGFBP-3 in the remodeling of mammary tissue during involution. Compared with control animals, the process of involution was modified in both transgenic lines. The degree of apoptotic cells was lower in the WAP-IGF-I and WAP-BP-3 expressing mice. In addition, there was a more quiescent pattern of involution with residual lobular secretary ability and a muted host inflammatory reaction with fewer lumenal microcalcifications. These results demonstrate that IGF-I and IGFBP-3 may modulate the involutionary process of the lactating mammary gland.     

An entire functional mammary gland may comprise the progeny from a single cell

Any epithelial portion of a normal mouse mammary gland can reproduce an entire functional gland when transplanted into an epithelium-free mammary fat pad. Mouse mammary hyperplasias and tumors are clonal dominant populations and probably represent the progeny of a single transformed cell. Our study provides evidence that single multipotent stem cells positioned throughout the mature fully developed mammary gland have the capacity to produce sufficient differentiated progeny to recapitulate an entire functional gland. Our evidence also demonstrates that these stem cells are self-renewing and are found with undiminished capacities in the newly regenerated gland. We have taken advantage of an experimental model where mouse mammary tumor virus infects mammary epithelial cells and inserts a deoxyribonucleic acid copy(ies) of its genome during replication. The insertions occur randomly within the somatic genome. CzechII mice have no endogenous nucleic acid sequence homology with mouse mammary tumor virus; therefore all viral insertions may be detected by Southern analysis provided a sufficient number of cells contain a specific insertional event. Transplantation of random fragments of infected CzechII mammary gland produced clonal-dominant epithelial populations in epithelium-free mammary fat pads. Serial transplantation of pieces of the clonally derived outgrowths produced second generation glands possessing the same viral insertion sites providing evidence for self-renewal of the original stem cell. Limiting dilution studies with cell cultures derived from third generation clonal outgrowths demonstrated that three multipotent but distinct mammary epithelial progenitors were present in clonally derived mammary epithelial populations. Estimation of the potential number of multipotent epithelial cells that may be evolved from an individual mammary-specific stem cell by self-renewal is in the order of 10(12)-10(13). Therefore, one stem cell might easily account for the renewal of mammary epithelium over several transplant generations.     

Interactions of EGF, Wnt and HOM-C genes specify the P12 neuroectoblast fate in C. elegans

We investigate how temporal and spatial interactions between multiple intercellular and intracellular factors specify the fate of a single cell in Caenorhabditis elegans. P12, which is a ventral cord neuroectoblast, divides postembryonically to generate neurons and a unique epidermal cell. Three classes of proteins are involved in the specification of P12 fate: the LIN-3/LET-23 epidermal growth factor signaling pathway, a Wnt protein LIN-44 and its candidate receptor LIN-17, and a homeotic gene product EGL-5. We show that LIN-3 is an inductive signal sufficient to promote the P12 fate, and the conserved EGF signaling pathway is utilized for P12 fate specification; egl-5 is a downstream target of the lin-3/let-23 pathway in specifying P12 fate; and LIN-44 and LIN-17 act synergistically with lin-3 in the specification of the P12 fate. The Wnt pathway may function early in development to regulate the competence of the cells to respond to the LIN-3 inductive signal.     

Relationship between the number of mast cells and neoplastic prolideration of the mammary gland epithelium

The results of the experiments make it possible to regard the number of mast in mammary glands as an index correlating with the intensity of the proliferative reaction of the mammary gland epithelium reflecting the course of the pathological process in the tissue. This index is higher to the moment of benign tumor formation. The second more intensive rise of this index characterizes malignant transformation of the epithelium. A decrease of the mast cells level since the moment of malignant transformation and their rather low content in malignant tumors manifest the decompensation of the organism antiblastic system.     

Localization of nitric oxide synthases and nitric oxide production in the rat mammary gland

We investigated nitric oxide (NO) production and the presence of nitric oxide synthase (NOS) in the mammary gland by use of an organ culture system of rat mammary glands. Mammary glands were excised from the inguinal parts of female Wistar-MS rats primed by implantation with pellets of 17beta-estradiol and progesterone and were diced into approximately 3-mm cubes. Three of these cubes were cultured with 2 ml of 10% FCS/DMEM plus carboxy-PTIO (an NO scavenger, 100 microM) in the presence or absence of LPS (0.5 microgram/ml) for 2 days. The amount of NO produced spontaneously by the cultured mammary glands was relatively minute at the end of the 2-day culture period, and the NO production was significantly enhanced by the presence of LPS. This enhancement of NO production was completely eliminated by addition of hydrocortisone (3 microM), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immunoblot analyses with specific antisera against NOS isoforms such as iNOS, endothelial NOS (eNOS), and brain NOS (bNOS) showed immunoreactive bands of iNOS (122 +/- 2 kD) and eNOS (152 +/- 3 kD) in extracts prepared from the mammary glands in the culture without LPS. The immunoreactive band of iNOS was highly intense after the treatment of mammary glands with LPS, whereas the corresponding eNOS immunoreactive band was faded. The immunohistochemical study of anti-iNOS antiserum on frozen sections of the cultured mammary glands showed that an immunoreactive substance with the antiserum was localized to the basal layer (composed of myoepithelial cells of alveoli and lactiferous ducts) of the mammary epithelia and to the endothelium of blood vessels that penetrated into the interstitium of the mammary glands. Histochemical staining for NADPH-diaphorase activity, which is identical to NOS, showed localization similar to that of iNOS in the mammary glands. Similar observations were noted in the immunohistochemistry of eNOS. In contrast, the immunoreactive signal with the bNOS antiserum was barely detected in the epithelial parts of alveoli and lactiferous ducts of the mammary glands. These observations demonstrate that three isoforms of NOS are present not only in the endothelium of blood vessels but also in the parenchymal cells (the glandular epithelium) of the rat mammary gland, such as epithelial cells and myoepithelial cells, and suggest that NO may have functional roles in the physiology of the mammary glands.     

Identification of specific relaxin-binding cells in the human female

Relaxin is secreted during pregnancy, but it has no verified effects in humans. The objective of the present study was to identify the cells containing specific relaxin-binding sites in the uterine cervix, vagina, uterus, mammary glands, mammary nipples, and term placenta in the human. The uterine cervix, vagina, and uterus were obtained from hysterectomy specimens. Mammary glands and nipples were obtained after modified radical mastectomy. Placenta was obtained after normal delivery. Tissue samples were cut into slices (0.5-3 cm3), frozen in liquid nitrogen, and cryosectioned (8 microm). Cells that bind relaxin were identified by sequential application of biotinylated porcine relaxin probe, antibiotin immunoglobulin G conjugated to 1 nm colloidal gold, and silver enhancement for signal amplification. Relaxin bound with specificity to epithelial cells, smooth muscle cells, and blood vessels in the cervix, vagina, uterus, and mammary nipples; to epithelial cells and blood vessels in the mammary glands; and to skin of the mammary nipples. In addition, relaxin bound to individual cell types within the term placenta (amnion epithelium, syncytiotrophoblasts, blood vessels), and to sebaceous glands within the nipples. We conclude that the specific relaxin-binding cells probably contain relaxin receptors. Identification of putative relaxin receptors may provide insight into physiological and/or therapeutic roles of relaxin in the human.