Steroid hormones modulate H19 gene expression in both mammary gland and uterus.

H19 is an imprinted and developmentally regulated gene whose product remains apparently untranslated. In a previous study on breast adenocarcinomas, we reported that overexpression of the H19 gene was significantly correlated with the presence of steroid receptors, suggesting the putative role of hormones in H19 transcription. To determine the mode of steroid action, we have detected levels of H19 RNA synthesis during mammary gland development by in situ hybridization (ISH): two peaks of H19 transcription occur during puberty and pregnancy. Furthermore, we demonstrated by ISH that in the uterus H19 RNA synthesis is high during estrus and metestrus phases. To test steroid control of H19 transcription, ovariectomized and adrenalectomized mice were supplemented, 1 week after surgery, with 17-beta-estradiol (E2, 20 microg/kg/day), progesterone (P, 1 mg/kg/day) or corticosterone (B, 0.3 mg/ kg/day) for 2 weeks. According to ISH data, E2 and to a lesser extent B stimulated H19 transcription in the uterus, whereas P inhibited it. To confirm the in vivo results, in vitro experiments were performed using cultures of MCF-7 cells (a hormone-sensitive mammary cell line). E2 stimulated the endogenous H19 gene of this cell line and tamoxifen inhibited this effect. Furthermore, we performed transient cotransfections in MCF-7, in HBL-100 (another hormone-sensitive mammary cell line) and in BT-20 (a hormone-insensitive mammary cell line) with various constructs of ERalpha (WT or mutated) and PR-A, in presence or absence of steroid hormones. We demonstrated that ERalpha up-regulated the H19 promoter in MCF-7 and in HBL-100, whereas PR-A did not have any effect per se. Moreover, in MCF-7, PR-A antagonized clearly the ERalpha-mediated promoter enhancement, but in HBL-100 this counteracting effect on the ERalpha up-regulation was not found. Interestingly, the same experiments performed in BT-20 cell line provided very similar results as those obtained in MCF-7 cells, with a clear down-regulation mediated by PR-A on the H19 promoter. All these in vitro data are in agreement with in vivo results. In addition, data obtained with ERalpha mutants indicate that H19 promoter activation is both ligand-dependent and ligand-independent. We have thus demonstrated that H19 gene expression is controlled by steroid hormones; furthermore, this gene is highly expressed in hormone-sensitive organs when the hormonal stimulation is accompanied with a morphological repair.

The expression of an Ets1 transcription factor lacking its activation domain decreases uPA proteolytic activity and cell motility, and impairs normal tubulogenesis and cancerous scattering in mammary epithelial cells.

Cell migration and invasion play a crucial role during normal and pathological development. The expression of several members of the Ets family of transcription factors has been shown to correlate with the occurrence of these processes. In the present study, we investigated the effect of the expression of Ets1-DB, the DNA-binding domain of c-Ets1, on the functional properties of NMuMG and MMT epithelial cell lines, from normal and cancerous mouse mammary tissues, respectively. We found that stable expression of this Ets1-DB mutant inhibited, in both cell types, the gene expression and activity of urokinase type-plasminogen activator (uPA), a potential target of c-Ets1. uPA is a key serine proteinase in the proteolytic cascade leading to the degradation of the extracellular matrix. In two-dimensional cultures, expression of the Ets1-DB mutant resulted in a decrease in cell migration and invasion in both cell lines. In three-dimensional collagen gels, NMuMG cells underwent tubular morphogenesis, while MMT cells developed as scattered structures. The Ets1-DB mutant impaired the capacity of NMuMG cells to form tubules and reduced the ability of MMT cells to invade these gels. Similar inhibition of cell migration, invasion and morphogenesis were observed in non-infected NMuMG and MMT cell lines treated with aprotinin, a serine proteinase inhibitor, suggesting that the inhibition of the plasmin cascade mediates in part the biological effects induced by the Ets1-DB mutant. These results demonstrate that Ets family members are involved in the control of uPA activity, cell motility and invasion during normal tubular morphogenesis and cancerous scattering in mammary epithelial cells.

Characterization of morphogenetic and invasive abilities of human mammary epithelial cells: correlation with variations of urokinase-type plasminogen activator activity and type-1 plasminogen activator inhibitor level.

Urokinase-type plasminogen activator (uPA) and one of its inhibitors, the PAI-1, are involved in the proteolytic cascade of matrix degradation during in vivo morphogenesis or metastasis. In the present study, we have characterized the in vitro morphological behavior of human normal and malignant mammary epithelial cells and determined the levels of uPA activity and PAI-1 during these events. Two-dimensional cultures in the presence of inductive fibroblast-conditioned medium (CM) allowed migration of HBL-100 cells and MDA-MB-231 cells. Normal human mammary epithelial cells (HMEC) and MCF-7 cells failed to migrate under these conditions. The epithelial cell migration correlated with an increase in the uPA activity whereas their immobility correlated with both increases in uPA activity and PAI-1 level. In three-dimensional cultures in collagen gel, fibroblasts or fibroblast CM induced branching tubular morphogenesis to HMEC, cord-like extensions to HBL-100 cells and a greater invasiveness ability to MDA-MB-231 cells. These events correlated with an increased uPA activity. In contrast, no morphological rearrangement was observed in MCF-7 cells and this correlated with both increases in uPA activity and PAI-1 level. Altogether, these results show that the in vitro mammary epithelial behavior is under the influence of mesenchymal inductive signals and is in agreement with modifications of uPA activity and PAI-1 levels. Our culture system gives a suitable model to study the mechanisms of mammary development and metastasis and to highlight the involvement of proteases and their inhibitors in cell-cell positioning and cell-matrix reorganization.

Expression of c-ets-1 and uPA genes is associated with mammary epithelial cell tubulogenesis or neoplastic scattering.

Although the inductive interactions which trigger epithelial morphogenesis have been extensively described, little is known about the transcription factors involved in these processes. During mammary gland morphogenesis, we report the expression of the transcription factor c-ets-1 and one of its target genes uPA in mesenchymal cells during early stages of epithelial invasion, and later in epithelial cells themselves. In vitro studies show that both c-ets-1 and uPA mRNAs can be induced in cultured normal mammary epithelial cells in response to medium conditioned by MRC-5 fibroblasts. In contrast, invasive tumorigenic cell lines from the mammary epithelium express constitutively c-ets-1 and uPA while non-invasive tumorigenic cells do not. In three dimensional co-cultures in collagen gels, a preferential expression of these genes is detected in epithelial cells migrating through the gel either at the tips of normal ducts or in cancerous cells which are scattering. These genes are also expressed in the neighboring fibroblasts. In MRC-5 fibroblasts, conditioned media from tumorigenic epithelial cells induce more efficiently c-ets-1 and uPA mRNA accumulation than do conditioned medium from normal cells. These results suggest that epithelial-mesenchymal interactions trigger c-ets-1 and uPA expression in both compartments during mammary gland morphogenesis. The expression of the genes correlates with invasiveness of epithelial cells irrespective of their being normal or cancerous.