Epithelial induction of stromal tenascin in the mouse mammary gland: from embryogenesis to carcinogenesis

The distribution of the extracellular matrix glycoprotein tenascin was studied by immunofluorescence in the developmental history of the mouse mammary gland from embryogenesis to carcinogenesis. Tenascin appeared only in the mesenchyme immediately surrounding the epithelia just starting morphogenesis, that is, in embryonic mammary glands from 13th to 16th day of gestation, in mammary endbuds which are a characteristic structure starting development during maturation of the mammary gland, and in the stroma of malignant mammary tumors. However, tenascin was absent in the elongating ducts of embryonic, adult, proliferating, and involuting mammary glands and preneoplastic hyperplastic alveolar nodules. The transplantation of embryonic submandibular mesenchyme into adult mammary glands induces the development of duct-alveolus nodules, which morphologically resemble developing endbuds. Tenascin reappeared around those nodules during the initial stages of their development. Tenascin expression could be induced experimentally in several ways. First, tenascin was detected at the site where the first mammary tumor cells GMT-L metastasized. Second, tenascin was detected in the connective tissue in the tumors derived from the injected C3H mammary tumor cell line CMT315 into Balb/c nude mouse. Cross-strain marker anti-CSA antiserum clearly showed that the tenascin-positive fibroblasts were of Balb/c origin. Third, when embryonic mammary epithelium was explanted on to embryonic mammary fat pad cultures, the mesenchymal cells condensed immediately surrounding the epithelium. Tenascin was detected in these condensed cells. From these three observations we conclude that both embryonic and neoplastic epithelium induced tenascin synthesis in their surrounding mesenchyme.     

Murine progesterone receptor expression in proliferating mammary epithelial cells during normal pubertal development and adult estrous cycle. Association with eralpha and erbeta status.

The ovarian steroids estrogen and progesterone are important in directing the normal growth and development of the mouse mammary gland. Previously, we have demonstrated that the majority of proliferating mammary epithelial cells do not express estrogen receptor-alpha (ERalpha). In this study we examined the relationship between progesterone receptor (PR) expression and proliferation in mammary epithelial cells using simultaneous immunohistochemistry for progesterone receptor (PR) and tritiated thymidine [(3)H]-Tdr) autoradiography. Results showed that the majority (>80%) of mammary epithelial cells labeled with [(3)H]-Tdr were PR-positive in the terminal end buds (TEBs) of pubertal mice and the ducts of pubertal and adult mice. Whereas the majority of mammary epithelial cells were also PR-positive, the basal cell population, which comprises the minority of mammary epithelial cells in the mammary ducts, was predominantly PR-negative. Nevertheless, the PR-positive phenotype remained the major proliferating cell type in the basal population. These findings suggest that the progesterone signaling pathway is involved in the proliferation of basal cell populations, potentially directing formation of tertiary side branching during pubertal development and alveolar bud formation in adult glands. A proportion of the basal cells exhibited weak expression of ERbeta, suggesting that the role of ERbeta in mediating normal estrogen-induced responses should be further studied. (J Histochem Cytochem: 47:1323-1330, 1999)     

Sustained mammary gland-directed, ponasterone A-inducible expression in transgenic mice.

The ability to regulate temporal- and spatial-specific expression of target genes in transgenic mice will facilitate analysis of gene function and enable the generation of murine models of human diseases. The genetic analysis of mammary gland tumorigenesis requires the development of mammary gland-specific transgenics, which are tightly regulated throughout the adult mammary epithelium. Analysis of genes implicated in mammary gland tumorigenesis has been hampered by mosaic transgene expression and the findings that homozygous deletion of several candidate genes (cyclin D1, Stat5A, prolactin receptor) abrogates normal mammary gland development. We describe the development of transgenic mouse lines in which sustained transgene expression was inducibly regulated, both specifically and homogeneously, in the adult mammary gland epithelium. Transgenes encoding RXRalpha and a chimeric ecdysone receptor under control of a modified MMTV-LTR, which targets mammary gland expression, were used. These transgenic 'receptor' lines were crossed with transgenic 'enhancer' lines in which the ecdysone/RXR binding site induced ligand-dependent expression of transgenic beta-galactosidase. Pharmacokinetic analysis of a highly bioactive ligand (ponasterone A), identified through screening ecdysteroids from local plants, demonstrated sustained release and transgene expression in vivo. This transgenic model with both tightly regulated and homogeneous transgene expression, which was sustained in vivo using ligands readily extracted from local flora, has broad practical applicability for genetic analysis of mammary gland disease.     

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.     

Circular RNA expression profiles and features in human tissues: a study using RNA-seq data

Background

Circular RNA (circRNA) is one type of noncoding RNA that forms a covalently closed continuous loop. Similar to long noncoding RNA (lncRNA), circRNA can act as microRNA (miRNA) ‘sponges’ to regulate gene expression, and its abnormal expression is related to diseases such as atherosclerosis, nervous system disorders and cancer. So far, there have been no systematic studies on circRNA abundance and expression profiles in human adult and fetal tissues.

Results

We explored circRNA expression profiles using RNA-seq data for six adult and fetal normal tissues (colon, heart, kidney, liver, lung, and stomach) and four gland normal tissues (adrenal gland, mammary gland, pancreas, and thyroid gland). A total of 8120, 25,933 and 14,433 circRNAs were detected by at least two supporting junction reads in adult, fetal and gland tissues, respectively. Among them, 3092, 14,241 and 6879 circRNAs were novel when compared to the published results. In each adult tissue type, we found at least 1000 circRNAs, among which 36.97–50.04% were tissue-specific. We reported 33 circRNAs that were ubiquitously expressed in all the adult tissues we examined. To further explore the potential “housekeeping” function of these circRNAs, we constructed a circRNA-miRNA-mRNA regulatory network containing 17 circRNAs, 22 miRNAs and 90 mRNAs. Furthermore, we found that both the abundance and the relative expression level of circRNAs were higher in fetal tissue than adult tissue. The number of circRNAs in gland tissues, especially in mammary gland (9665 circRNA candidates), was higher than that of other adult tissues (1160–3777).

Conclusions

We systematically investigated circRNA expression in a variety of human adult and fetal tissues. Our observation of different expression level of circRNAs in adult and fetal tissues suggested that circRNAs might play their role in a tissue-specific and development-specific fashion. Analysis of circRNA-miRNA-mRNA network provided potential targets of circRNAs. High expression level of circRNAs in mammary gland might be attributed to the rich innervation.

     

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.     

c-Myc affects mRNA translation, cell proliferation and progenitor cell function in the mammary gland

Background  

The oncoprotein c-Myc has been intensely studied in breast cancer and mouse mammary tumor models, but relatively little is known about the normal physiological role of c-Myc in the mammary gland. Here we investigated functions of c-Myc during mouse mammary gland development using a conditional knockout approach.     

Comparative expression of endogenous retrotransposons in adult mouse mammary gland during normal development and differentiation

Amplified expression of the endogenous retrotransposons, intracisternal A particles (IAPs) and murine leukemia virus-related elements (MLVEs), along with decreased expression of VL30 elements frequently occurs during mouse mammary tumorigenesis. We have now analyzed the expression of these retroelements during the normal developmental and differentiation cycle of the mammary gland as found in virgin, pregnant, lactating, and post-lactation adult female BALB/c mice. Retrotransposon expression was either unchanged or decreased during the progressive stages of the cycle compared to virgin tissue. Likewise, growth of mammary epithelial cells in primary culture had little or no effect on expression of IAPs, MLVEs and VL30 sequences. Thus, the dramatic changes involving these retrotransposons in many mouse mammary tumors appear unrelated to any normal state.     

Defects in mouse mammary gland development caused by conditional haploinsufficiency of Patched-1.

In vertebrates, the hedgehog family of cell signaling proteins and associated downstream network components play an essential role in mediating tissue interactions during development and organogenesis. Loss-of-function or misexpression mutation of hedgehog network components can cause birth defects, skin cancer and other tumors. The mammary gland is a specialized skin derivative requiring epithelial-epithelial and epithelial-stromal tissue interactions similar to those required for development of other organs, where these interactions are often controlled by hedgehog signaling. We have investigated the role of the Patched-1 (Ptc1) hedgehog receptor gene in mammary development and neoplasia. Haploinsufficiency at the Ptc1 locus results in severe histological defects in ductal structure, and minor morphological changes in terminal end buds in heterozygous postpubescent virgin animals. Defects are mainly ductal hyperplasias and dysplasias characterized by multilayered ductal walls and dissociated cells impacting ductal lumens. This phenotype is 100% penetrant. Remarkably, defects are reverted during late pregnancy and lactation but return upon involution and gland remodeling. Whole mammary gland transplants into athymic mice demonstrates that the observed dysplasias reflect an intrisic developmental defect within the gland. However, Ptc1-induced epithelial dysplasias are not stable upon transplantation into a wild-type epithelium-free fat pad, suggesting stromal (or epithelial and stromal) function of Ptc1. Mammary expression of Ptc1 mRNA is both epithelial and stromal and is developmentally regulated. Phenotypic reversion correlates with developmentally regulated and enhanced expression of Indian hedgehog (Ihh) during pregnancy and lactation. Data demonstrate a critical mammary role for at least one component of the hedgehog signaling network and suggest that Ihh is the primary hedgehog gene active in the gland.     

Intracellular cAMP levels in normal rat mammary gland and adenocarcinoma. In vivo vs in vitro.

Intracellular cAMP levels determined by radioimmunoassay technique were compared in normal rat mammary gland and DMBA-induced mammary adenocarcinoma as well as in epithelial cells derived from these tissues and grown in monolayer cultures. It was found that cAMP levels were higher in mammary tumors (0.643 p mole/mg wet weight) than in normal gland (0.158 p mole/mg). In contrast, cAMP levels in cultured adenocarcinoma cells were lower than those in normal mammary epithelial cells. The apparent contradiction may be a consequence of the fact that $\text{in vivo}$ cAMP values represent the average value of the composite cell types and not the epithelial components in question.     

Cadherins in the mammary gland and the melanocyte lineage

Cadherins are transmembrane glycoproteins involved in cell-cell adhesion, signalling, proliferation and differentiation. In this review, we have focused upon in vivo cadherin expression and function in two different biological systems, the mammary gland epithelium and the melanocyte lineage. Development of the mammary gland represents a paradigm of in situ epithelial differentiation and the melanocyte lineage of a model of non-epithelial (or mesenchymal) cell differentiation where cells migrate extensively from their site of origin towards the skin compartment. In the mammary epithelium, the predominantly expressed cadherin is E-cadherin, a cell surface molecule that directs morphogenesis and maintenance of the epithelial structure. In the melanocyte lineage, the expression of a number of cadherins is strictly spatiotemporally regulated during development and adult life. The specific functions mediated by this very dynamic cadherin expression are not yet known and their characterisation represents a challenge for the future.     

Presence of sub-epithelial lymphoid tissues in the teat of ewe-lambs and adult ewes

In the first part of the study, 24 clinically healthy teats from non-lactating ewe-lambs were examined bacteriologically and histologically. No bacteria were isolated from any of these teats; lymphocytes were observed in teat cisterns of six teats (25%) from three ewes. In the second part, 87 teats from adult ewes were examined; their origin was from lactating mammary glands with no bacteria isolated (n = 23), from glands after lactation with no bacteria isolated (n = 25), from lactating glands with bacteria isolated (n = 22) or from glands after lactation with bacteria isolated (n = 17). The salient histological feature was sub-epithelial leucocytic infiltration. In teat cisterns, lymphocytes were the predominant cell type and in teat ducts, lymphocytes and neutrophils were seen in equal proportions. Sub-epithelial lymphoid nodules, some with germinal centers, were detected in 43 (49%) teats; their majority was observed at the border between teat duct and teat cistern. Presence of bacteria was significantly associated with presence of leucocytic activity (P < 0.001) and with presence of lymphoid nodules (P = 0.032). We conclude that the presence of induced sub-epithelial lymphoid tissue at the border between teat duct and teat cistern appears to be important in protecting the mammary gland during the early stages of bacterial invasion.     

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

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

Enzymic changes in rabbit and rat mammary gland during the lactation cycle

1. Activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), isocitrate dehydrogenase (EC 1.1.1.42), malate dehydrogenase (EC 1.1.1.37), malate dehydrogenase (decarboxylating) (EC 1.1.1.40), and pyruvate carboxylase (EC 6.4.1.1) were determined in subcellular fractions of mammary gland from rabbits during pregnancy, at different stages of lactation and during weaning. The results were compared with those obtained in similar experiments with rat mammary gland. 2. Three bases of expression of the activity of enzymes in the particle-free supernatant fraction of mammary gland were compared. During lactation, activity expressed per mg. of particle-free supernatant protein (uncorrected for milk protein) correlated well with that expressed per mug. of DNA phosphorus. The disadvantages of expressing activities per g. wet wt. are discussed. 3. The major differences between the two tissues were: (a) neither malate dehydrogenase (decarboxylating) nor a soluble form of pyruvate carboxylase could be detected in rabbit mammary gland at any stage of the lactation cycle; (b) isocitrate dehydrogenase increased in activity during lactation in rabbit mammary gland, but not in that of the rat. 4. Pyruvate carboxylase in the mitochondrial fraction of rabbit mammary gland, and in both the mitochondrial and the soluble fractions of rat mammary gland, did not change in activity during lactation. 5. For each tissue, the NADP-dependent dehydrogenases studied had a high activity at all stages of the lactation cycle compared with the rate of fatty acid synthesis at mid-lactation. The significance of these results is discussed with respect to the supply of NADPH via NADH.