Constitutive expression of the DNA-binding domain of Ets1 increases endothelial cell adhesion and stimulates their organization into capillary- like structures Virginie Mattot 1,2 , Chantal Vercamer 1 , Fabrice Soncin 1 , Thierry Calmels 1,3 , Christelle Huguet 1,4 , Ve ronique Fafeur 1 and Bernard Vandenbunder* ,1 1 CNRS EP560 ± Institut Pasteur de Lille, Institut de Biologie de Lille, 1 rue Calmette, 59021 Lille, France 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 eects of the stable expression of an Ets transdomi- nant 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 modi®cations in the organization of the cytoskeleton, and increased cell adhesion. We investi- gated 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 eect 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. Oncogene (2000) 19, 762 ± 772. Keywords: Ets; transcription factor; endothelial cells; adhesion; angiogenesis Introduction Ets1 is the cellular progenitor of the v-ets oncogene found in the genome of the avian leukaemia retrovirus E26. Ets1 encodes a transcription factor which is the founder of a family containing more than 30 related proteins, identi®ed in species ranging from Caenorhab- ditis elegans to human (reviewed in CreÂpieux et al., 1994; Ghysdael and Boureux, 1997). The signature of this family is a 85 amino acids sequence, which corresponds to the DNA-binding domain. Indeed this domain confers the ability to recognise a sequence containing a purine rich core motif GGAA/T, also named the Ets binding site. Functional Ets binding sites have been identi®ed in various cellular promoters (reviewed in Ghysdael and Boureux, 1997), including the promoters of genes encoding transcription factors such as JunB, growth factor receptors such as VEGFR-1 (Wakiya et al., 1996), Tie-1 and Tie-2 (Dube et al., 1999; Iljin et al., 1999), matrix degrading proteases and their inhibitors (Gum et al., 1996; Logan et al., 1996) and adhesion molecules such as b2 integrin, ICAM and VE-cadherin (Bottinger et al., 1994; de Launoit et al., 1998; Gory et al., 1998). The Ets1 gene is not ubiquitously expressed in embryonic and adult tissues. Rather, we and others previously evidenced the speci®c expression of Ets1 during invasive processes, including the formation of new blood vessels during normal and pathological development (reviewed in Vandenbunder et al., 1995). In embryos, Ets1 is expressed in the blood islands of the yolk sac where are located the hemangioblasts, the precursors of the hematopoietic and vascular lineages, and in endothelial cells (Pardanaud and Dieterlen- LieÁ vre, 1993; Vandenbunder et al., 1989). This expression is detected in endothelial cells during both vasculogenesis and angiogenesis. In adults, Ets1 is expressed in endothelial cells during wound healing, tumour angiogenesis, and during re-endothelialization after denuding injury (Bolon et al., 1995; Wernert et al., 1992, 1994). In agreement with its expression during new blood vessel formation but not in mature vessels, ets1 mRNA expression was found to be higher in proliferating but not in con¯uent human endothelial cells (Wernert et al., 1992). Ets1 mRNA levels have been shown to be increased upon treatment with angiogenic factors, such as TNFa, PMA, FGF, and VEGF (Chen et al., 1997; Iwasaka et al., 1996; Wernert et al., 1992, and our unpublished data). These results suggest that Ets1 can be involved in angiogenesis associated with normal development and tumour growth. Recently, Ets1 de®cient mice have been obtained by homologous recombination (Barton et al., 1998). Although no detailed description of their vasculature has been published, the existence of viable ets17/7 mice suggests that Ets1 is not essential for blood vessel formation in the embryo. Redundancy between Ets family members may account for this result, since the expression pattern of Ets1 overlaps the patterns of Erg and Fli, but not of Ets2, in endothelial cells during early embryonic development (Dhordain et *Correspondence: B Vandenbunder Current addresses: 2 Virginie Mattot, Center for Transgene Technology and Gene Therapy, Flanders InterUniversity Institute for Biotechnology, Campus Gasthuisberg, O&N, B3000 Leuven, Belgium; 3 Thierry Calmels, SmithKline Beecham, Laboratoires Pharmaceutiques, 4 rue du Chesnay Beauregard, BP 58, 35762, Saint GreÂgoire, France; 4 Christelle Huguet, P®zer Limited, Discovery Biology Department, Sandwich, Kent CT13 9NJ, UK Received 28 April 1999; revised 20 September 1999; accepted 28 September 1999 Oncogene (2000) 19, 762 ± 772 ã 2000 Macmillan Publishers Ltd All rights reserved 0950 ± 9232/00 $15.00 www.nature.com/onc