ETS1 lowers capillary endothelial cell density at confluence and induces the expression of VE-cadherin.

Ets1 is a transcription factor expressed in endothelial cells during angiogenesis but its target genes and function in blood vessel formation are still unknown. We have over-expressed Ets1 as a tagged protein in brain capillary endothelial cells and in 3T3 fibroblasts using a retroviral vector. Over-expression of Ets1 reduced by nearly half cell density at confluence of endothelials but not of fibroblasts. As density at confluence is controlled in part by cadherins, this growth arrest could be due to the up-regulation of these cell contact molecules. Indeed, Ets1 increased the expression of the endothelial-specific VE-cadherin without affecting N-cadherin expression levels. In parallel, both a dominant negative mutant of Ets members and an Ets1 anti-sense oligonucleotide inhibited VE-cadherin expression in endothelial cells. Ets1 bound to two Ets-binding sites located in the proximal region of the VE-cadherin promoter. Mutation of these sites abolished Ets1-induced transactivation of the promoter. The present work is the first demonstration of a function of Ets1 in the regulation of a specific endothelial marker based on its endogenous gene and protein expression.

Constitutive expression of the DNA-binding domain of Ets1 increases endothelial cell adhesion and stimulates their organization into capillary-like structures.

We previously reported that the Ets1 transcription factor is expressed in endothelial cells during angiogenesis both in normal and pathological development. We analyse here the effects of the stable expression of an Ets transdominant negative mutant (Ets1-DB), consisting in an Ets1 protein lacking its transactivation domain. A retrovirus containing the Ets1-DB sequence fused to an IRES-Neo sequence was designed and used to infect brain capillary (IBE) and aorta (MAE) mouse endothelial cell lines. Cells expressing this Ets1 mutant were examined for proliferation, migration and adhesion. Consistent changes were observed on cell morphology, with increased spreading and modifications in the organization of the cytoskeleton, and increased cell adhesion. We investigated the ability of endothelial cells to organise into capillary-like structures using three-dimensional gels. On Matrigel, all endothelial cell lines formed a cord-like network within 24 h, with an increased ability of Ets1-DB cells to spread on this substrate. In long term cultures, IBE cells expressing Ets1-DB showed a higher capacity to form branched structures; this effect was potentiated by FGF2. These results demonstrate a role of the Ets transcription factors in the regulation of the adhesive and morphogenetic properties of endothelial cells.

The avian transcription factor c-Rel is induced and translocates into the nucleus of thymocytes undergoing apoptosis.

This study investigates the involvement of the avian transcription factor c-Rel in thymocyte apoptosis occurring either in vivo or in organotypic culture. In vivo, only a few cortical thymocytes express the c-Rel protein. Their number, localization and morphology resemble that of apoptotic cells evidenced by TUNEL staining. In organotypic culture, the expression of c-Rel is induced in medullary thymocytes as apoptosis is triggered. This induction would be post-transcriptional since no increase in the c-rel gene expression is detected. Moreover, c-Rel translocates into the nucleus of medullary thymocytes during the time course of apoptosis. This translocation is preceded by a decrease in ikba expression, the gene which encodes the avian homologue of IkappaBalpha. Altogether these results suggest that the proto-oncogene c-rel could take an active part in apoptosis of cortical thymocytes occurring in vivo during T-cell selection as well as in experimentally-induced apoptosis of medullary thymocytes.

[New inhibitors of tumor angiogenesis]. / De nouveaux outils pour bloquer l'angiogenèse tumorale.

During the past few years, our understanding of the molecular mechanisms of angiogenesis has steadily increased. These informations allowed the design of new strategies aimed to kill cancer cells by shutting off the blood vessels through which they get the oxygen and nutrients they need to grow. Therapies that target tumor blood vessels cause the regression of solid tumors in mice. The possibility to obtain synergistic effects by combining antiangiogenic and cytotoxic therapy, and the therapeutic applications of these studies will be discussed, following a meeting on angiogenesis held in Boston on February 10th and 11th 1997.

[Experimental models of angiogenesis for the study of the Ets1 transcription factor]. / Modèles expérimentaux d'angiogenèse pour l'étude du facteur de transcription Ets1.

The Ets1 transcription factor gene is expressed in endothelial cells during blood vessel formation under normal or pathological conditions. The proposed hypothesis aims to involve Ets1 in the regulation of angiogenesis processes by activating the transcription of genes encoding matrix-degrading proteases. In vivo experiments allowing specific manipulation of ets1 gene expression or activity within endothelial cells are necessary to investigate the functional role of Ets1. Two experimental models using retroviruses expressing ets1 mutants have been chosen: Chicken embryo development and tumour-induced angiogenesis in mice. Another approach targeted on the vascular endothelium has been developed in order to obtain transgenic mice expressing specifically in endothelial cells an ets1 transdominant negative mutant under the control of the tek and tie promoters. These tools should allow interference with Ets1 activity at defined stages during normal or pathological development.

[Experimental angiogenesis : strategy for the functional study of the transcription factors of the Ets family during morphogenesis of the vascular tree]. / Angiogenèse expérimentale : stratégies pour l'étude fonctionnelle des facteurs de transcription de la famille Ets dans la morphogenèse du réseau vasculaire.

During morphogenesis of the vascular tree, the massive outgrowth of primitive capillaries is followed by the development and the maturation of some capillary branches whereas others regress. The direct observation and the manipulation of in vivo models, including a series of recent knock-out experiments, allow to delineate the mechanisms controlling this process, and to identify factors involved in the formation of a mature capillary, surrounded with a basal lamina and pericytes. The expression of several members of the Ets family of transcription factors, Ets1, Erg and Fli, correlates with the occurrence of invasive processes, such as angiogenesis during normal and pathological development. The description of the phenotype of cultured endothelial cells expressing the DNA binding domain of Ets1 suggests that members of the Ets family take part in the morphogenesis of the -vascular tree. Although transient transfection experiments allowed the identification of putative targets genes for Ets1 during angiogenesis, deciphering the Ets1 regulation networks remains a major goal for the future.

HIF-2alpha specifically activates the VE-cadherin promoter independently of hypoxia and in synergy with Ets-1 through two essential ETS-binding sites.

The mechanisms that are responsible for the restricted pattern of expression of the VE-cadherin gene in endothelial cells are not clearly understood. Regulation of expression is under the control of an approximately 140 bp proximal promoter that provides basal, non-endothelial specific expression. A larger region contained within the 2.5 kb genomic DNA sequence located ahead of the transcription start is involved in the specific expression of the gene in endothelial cells. We show here that the VE-cadherin promoter contains several putative hypoxia response elements (HRE) which are able to bind endothelial nuclear factors under normoxia. The VE-cadherin gene is not responsive to hypoxia but hypoxia-inducible factor (HIF)-2alpha specifically activates the promoter while HIF-1alpha does not. The HRE, that are involved in this activity have been identified. Further, we show that HIF-2alpha cooperates with the Ets-1 transcription factor for activation of the VE-cadherin promoter and that this synergy is dependent on the binding of Ets-1 to DNA. This cooperative action of HIF-2alpha with Ets-1 most probably participates to the transcriptional regulation of expression of the gene in endothelial cells. This mechanism may also be involved in the expression of the VE-cadherin gene by tumor cells in the process of vascular mimicry.

Invasive tumors induce c-ets1 transcription factor expression in adjacent stroma.

The stroma reaction plays a central role in tumor growth, invasion and metastasis. Tumor growth is dependent on angiogenesis and requires the vascular supply provided by new capillary blood vessels of the stroma. The expression of the gene encoding the transcription factor c-ets1 is localized within fibroblasts and endothelial cells of the stromal compartment. This expression correlates with the accumulation of transcripts for potential target genes such as collagenase I and stromelysin I in stromal fibroblasts surrounding malignant cells in invasive tumors. We suggest that c-Ets1 protein might regulate the transcription of the genes coding for matrix-degrading proteases necessary for both angiogenesis and tumor invasion.

[Transcription factors of the Ets family and morphogenesis of the vascular tree]. / Les facteurs de transcription de la famille Ets et la morphogenèse du réseau vasculaire.

The expression of several members of the Ets family of transcription factors, Ets1, Erg and Fli, correlates with the occurrence of invasive processes such as angiogenesis during normal and pathological development. The description of the phenotype of cultured endothelial cells expressing the DNA binding domain of Ets1 suggests that members of the Ets family take part in the morphogenesis of the vascular tree. Although transient transfection experiments allowed the identification of putative targets genes for Ets1 during angiogenesis, deciphering the Ets1 regulation networks remains a major goal for the future.

The ETS1 transcription factor is expressed during epithelial-mesenchymal transitions in the chick embryo and is activated in scatter factor-stimulated MDCK epithelial cells.

In embryos and in human tumors, the expression of the ETS1 transcription factor correlates with the occurrence of invasive processes. Although this was demonstrated in cells of mesodermal origin, the expression of ETS1 was not detected in epithelial cells. In the present study, we show that during early organogenesis in the chick embryo, ETS1 mRNA expression was transiently induced in epithelial structures, during emigration of neural crest cells and dispersion of somites into the mesenchymal sclerotome. In contrast, the expression of ETS1 was not detected in situations where epithelial layers stayed cohesive while forming a new structure, such as the dermomyotome forming the myotome. The involvement of ETS1 in epithelial cell dissociation was examined in MDCK epithelial cells stimulated by scatter factor/hepatocyte growth factor (SF/HGF), a potent inducer of cell dissociation and motility. SF/HGF was found to stimulate ETS1 mRNA and protein expressions, and these increases coincided with the dispersion of cells and the expression of protease mRNAs, such as urokinase-type plasminogen activator and collagenase, but not with the protease inhibitor, plasminogen activator inhibitor type 1. Furthermore, we showed that SF/HGF was able to induce a transcriptional response involving ETS1 by using artificial as well as cellular promoters, such as the urokinase-type plasminogen activator and collagenase 1 promoters, containing RAS-responsive elements with essential ETS-binding sites. These data demonstrate expression of ETS1 during epithelial-mesenchymal transitions in the developing embryo and show that ETS1 can act as a downstream effector of SF/HGF in MDCK epithelial cells. Taken together, these data identify ETS1 as a molecular actor of epithelia cell dissociation.

Expression of interstitial collagenase is restricted to skeletal tissue during mouse embryogenesis.

Collagenases are thought to be involved in physiological and pathological processes that require extracellular matrix remodeling. Using the in situ hybridization technique, we describe the expression of interstitial collagenase gene during mouse embryogenesis between E6.5 and E17. We demonstrate that interstitial collagenase expression is exclusively detected in one event, namely the onset of bone formation. Transcripts accumulate in hypertrophied chondrocytes, found in the mature cartilaginous matrix of long-bone growth plates or ribs, and in osteoblasts and/or in endothelial cells that have migrated into the shafts of developing long bones. The expression of the tissue inhibitor of metalloproteinases (TIMP-2) gene precedes the expression of interstitial collagenase in developing bones. These data suggest that interstitial collagenase plays a specific role in bone development and that the tight regulation of its activity during development is achieved not only by post-translational mechanisms with TIMPs, as previously suggested, but also at the transcriptional level.

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.

Targeted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs VEGF-mediated endothelial survival and angiogenesis.

Vascular endothelial cadherin, VE-cadherin, mediates adhesion between endothelial cells and may affect vascular morphogenesis via intracellular signaling, but the nature of these signals remains unknown. Here, targeted inactivation (VEC-/-) or truncation of the beta-catenin-binding cytosolic domain (VECdeltaC/deltaC) of the VE-cadherin gene was found not to affect assembly of endothelial cells in vascular plexi, but to impair their subsequent remodeling and maturation, causing lethality at 9.5 days of gestation. Deficiency or truncation of VE-cadherin induced endothelial apoptosis and abolished transmission of the endothelial survival signal by VEGF-A to Akt kinase and Bcl2 via reduced complex formation with VEGF receptor-2, beta-catenin, and phosphoinositide 3 (PI3)-kinase. Thus, VE-cadherin/ beta-catenin signaling controls endothelial survival.