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Mammary glands of adult human females are secretory organs comprised of interdependent epithelial and mesenchymal cells. These cells constitute an assemblage of collecting ducts that end in terminal duct lobular units with hollow alveolar ductules that can differentiate to produce and expel milk. Systemic and maternal hormones, autocrine and paracrine growth factors, and cytokines regulate virtually all phases of mammary gland development. During organogenesis, epithelial and mesenchymal cells interact to form precursors of the parenchyma and stroma in the mature gland. Organogenesis precedes five stages of postnatal development: puberty, pregnancy, lactation, involution, and menopause. Each stage requires a specific set of morphogenetic changes in glandular structure and function. Cycles of cell proliferation, differentiation, and involution may recur until menopause. In addition, physiological responses such as inflammation and pathological events such as tumorigenesis are remarkable for their similarities to embryonic morphogenesis. Here we take a succinct look at the ever-improving understanding of stroma–epithelial interactions and mesenchyme function in mammary gland biology.

Mice lacking the RelA (p65) subunit of NF-κ B die between days 14 and 15 of embryogenesis because of massive liver destruction. Fibroblasts and macrophages isolated from relA-/- embryos were found to be highly sensitive to tumor necrosis factor (TNF) cytotoxicity, raising the possibility that endogenous TNF is the cause of liver cell apoptosis. To test this idea, we generated mice lacking both TNF and RelA. Embryogenesis proceeds normally in such mice, and TNF/RelA double-deficient mice are viable and have normal livers. Thus, the RelA-mediated antiapoptotic signal that protects normal cells from TNF injury in vitro can be shown to be operative in vivo.

Tenascin-C (TNC), an extracellular matrix glycoprotein, plays important roles in tissue remodeling. TNC is not normally expressed in adults but reappears under pathologic conditions. The present study was designed to clarify the contribution of TNC to ventricular remodeling after myocardial infarction. We examined the expression of TNC after experimental myocardial infarction in the rat by immunohistochemistry and in situ hybridization. Within 24 hours of permanent coronary ligation, interstitial fibroblasts in the border zone started to express TNC mRNA. The expression of TNC was down-regulated on Day 7 and was no longer apparent by Day 14 after infarction. During the healing process, TNC protein and TNC-producing cells were found at the edges of the residual myocardium. Some of the TNC-producing cells were immunoreactive for α-smooth muscle actin. In culture, TNC increased the number of cardiomyocytes attached to laminin but inhibited the formation of focal contacts at costameres. The results indicate that during the acute phase after myocardial infarction, interstitial cells in the border zone synthesize TNC, which may loosen the strong adhesion of surviving cardiomyocytes to connective tissue and thereby facilitate tissue reorganization.

The mammary gland develops from the placode at ectodermal invagination. The rudimentary parenchyma (mammary bud) develops mammary trees and alveolar structures, suggesting that the mammary bud consists of stem/progenitor cells. Here, we established a clonal stem cell line from a mammary bud of a p53 null female embryo at day 14.5. FP5-3-1 line was a homogeneous cell population with polygonal epithelial morphology and spontaneously became heterogeneous during passages. Recloning gave rise to four sublines; three sublines have basal epithelial property and one subline has luminal epithelial property. The former sublines generate functional mammary glands when injected into cleared fat pads and the latter subline does not. The cell lines also express many stemness-related genes. The clonal cell lines established in the present study are shown to be mammary stem cells and not tumorigenic. They provide useful models for normal and tumor biology of the mammary gland in vivo and in vitro.

It has become clear that deposition of extracellular matrix(ECM) proteins is a major cause of human restenosis after percutaneous coronary angioplasty (PTCA). To define the composition and organization of the involved ECM in human restenotic tissue, we morphologically and semiquantitatively analyzed specimens obtained by means of directional coronary atherectomy at various stages after PTCA with anti-fibronectin, tenascin-C, collagens I and III, and PG-M/versican antibodies. Tenascin-C deposition transiently increased within 1 month after PTCA, when smooth muscle cell migration and proliferation was active. Following the disappearance of tenascin-C, PG-M/versican accumulation increased and peaked between 1 month and 3 months when clinical restenosis was most actively progressing. At later stages, the PG-M/versican was replaced by a more mature ECM consisting of collagens I and III. The volume ratio of elastin remained at a low level throughout. Our results demonstrate that the matrix proteins of human restenotic lesions sequentially change after angioplasty and that tenascin-C could be a key molecule in the early stages.

The expression of fibronectin (FN) isoforms including extra domain A (EDA) and extra domain B (EDB) segments, was investigated in 36 invasive ductal carcinomas and 13 benign tumors of human breast tissues by in situ hybridization using probes specific to alternative splicing sites. Signals for the constant region of FN mRNA in cancer cells were found in 53% of the invasive ductal carcinomas. The EDA+ and EDB+ mRNA signals were found in 47% and 33%, respectively. Stromal cells expressing FN, EDA+ and EDB+ mRNA signals were present in 100%, 69% and 14% of cases, respectively. Expression of FN mRNAs by cancer cells was most frequent in intraductal lesions or large cancer nests, and that by stromal cells was associated with desmoplastic areas. In representative cases, proportions of FN mRNA‐positive cancer cells expressing EDA and EDB segments were 45% and 39%, respectively, signals for both being frequently found in the same cells. EDA+ and EDB+ mRNA were labeled in 25% and 6% of the FN mRNA‐positive stromal cells, a large proportion thus being EDA‐/EDB‐ FN. In conclusion, the splicing pattern of FN pre‐mRNA is dependent on the cell type and histology of breast cancer tissues. The observed lack of expression in fibroadenomas and other benign conditions suggests a link with tumor progression.

Tenascins (TNs) are a family of extracellular matrix glycoproteins. The first member of this family to be recognized, tenascin-C (TN-C), is known to be expressed in various tumors including human astrocytomas. Tenascin-X (TN-X) is the latest member of the TN family to be reported, and its expression in tumor tissues has not yet been examined. In this study, we found expression of TN-X in glioma cell lines and human astrocytomas by immunoblot analysis using anti-mouse TN-X antibodies. We also examined the expression of TN-C and TN-X immunohistochemically in a series of 32 human astrocytomas and tissue from 5 normal brains. Expression of TN-X was up-regulated to a higher degree in low-grade astrocytomas than in high-grade astrocytomas. TN-X was mainly localized in the perivascular stroma around tumor vessels, and weakly expressed in the intercellular spaces among tumor cells. In contrast, TN-C was more strongly expressed in the intercellular spaces and in tumor vessels in high-grade astrocytomas (anaplastic astrocytomas and glioblastomas) than in low-grade astrocytomas. In the tissues expressing both TNs, the distribution of TN-X was often reciprocal to that of TN-C. These findings indicate that the expression of TN-C and TN-X in astrocytomas is different, and that these glycoproteins could be involved in neovascularization in different manners.

Nasal mucosa covered by pseudostratified ciliated epithelia can be injured by microbial infection and physical and chemical agents. To elucidate mechanisms of regeneration, erosion of rat nasal mucosa was produced by intranasal instillation of trichloroacetic acid, and tissue specimens were then sequentially obtained after 1–14 days. Since tenascin-C (TN-C) and its receptor, α9β1 integrin, are assumed to play important roles in regeneration of stratified squamous epithelia, their expression was evaluated by immunohistochemistry and in situ hybridization. Three to five days after the injury, TN-C mRNA was found in epithelial cells of migrating fronts and in epithelial sheets recovering ulcerated surfaces between the fronts and normal regions. TN-C deposition was increased under such sheets. Enhanced α9 staining was also evident in the involved epithelium. 5-Bromo-2’-deoxyuridine incorporation assays revealed significant increase in proliferating cells in cell sheets over TN-C deposits at 3–7 days. Therefore, we conclude that regenerating epithelial cells produce and secrete TN-C, associated with an increase in α9 expression, and that interactions between these molecules could regulate migration and proliferation of the epithelial cells in an autocrine manner.

A rat strain carrying the human c-Ha-ras protooncogene, established by our laboratory, is highly susceptible to mammary chemical carcinogens. The transgenic rats exhibit increased number of terminal endbuds (TEBs) at the tips of developing ducts in the mammary gland compared to non-transgenic littermates. Confocal microscopy revealed the level of active mitogen-activated protein kinase to be elevated in these TEBs, and a close correlation between their numbers and tumorigenic response initiated by 7,12-dimethylbenz[a]anthracene was confirmed. Single injections of N-methyl-N-nitrosourea into the transgenic rats caused mutations in codon 12 of human c-Ha-ras transgene in TEBs before tumor development, supporting the conclusion that these structures are the major targets of chemical carcinogens. In contrast, with spontaneous development of lesions, alveolar hyperplasia with elevated expression levels of rat and human c-Ha-ras protooncogenes is the first morphological alteration which becomes apparent. Some but not all hyperplastic alveolar nodules were found to harbor mutations in the transgene. The results indicate that elevated expression of c-Ha-ras protooncogene is sufficient in itself to cause a highly proliferative phenotype of mammary alveoli. Our data suggest that TEBs and acini are the major targets for chemical and sporadic carcinogenesis, respectively, in the mammary glands of human c-Ha-ras protooncogene transgenic rats.