The transcription of the intercellular adhesion molecule-1 is regulated by Ets transcription factors Yvan de Launoit 1 , Marie Audette 2 , HeÂleÁne Pelczar 1 , Serge Plaza 3 and Jean-Luc Baert 1 1 UMR 319 CNRS - Institut Pasteur de Lille, Institut de Biologie, 1 rue Calmette, BP 447, 59021 Lille, Cedex - France; 2 Laboratory of Molecular Endocrinology, CHUL Research Center, 2705 Laurier Boulevard, G1V 4G2 Ste Foy, PQ - Canada; 3 EP 560 CNRS - Institut Pasteur de Lille, Institut de Biologie, 1 rue Calmette, BP 447, 59021 Lille, Cedex - France The Ets family of transcription factors comprises several members which are involved to regulate gene transcrip- tion. Although several consensus binding sites for Ets proteins can be found in a wide series of promoter, only a limited number of them are indeed activated by these transcription factors. The human intercellular adhesion molecule-1 (ICAM-1) plays a crucial role in immune responses by enabling the binding of eector cells to various target cell types. ICAM-1 is constitutively expressed at dierent levels in the absence of stimuli in dierent cell types, and its expression is upregulated by several proin¯ammatory cytokines. We have here examined the transcriptional regulation of human ICAM-1 expression by Ets proteins, and more particu- larly by ERM, a member of this family of transcription factors. Transient transfection assays revealed that Ets-2 and ERM signi®cantly activate the transcription of ICAM-1 promoter, whereas the less-related Ets family member, Spi-1/Pu.1, failed to do so. Transfection of a series of ICAM-1 promoter deletion mutants together with ERM expression plasmids have shown that an Ets responsive element is located within the ®rst 176 bp upstream from the translational start site. Electrophore- tic mobility shift assays and DNase I footprinting analysis have enabled us to identify two Ets binding sites at positions 7158 and 7138 from the ATG, respectively. Site directed mutagenesis of these elements has shown that the distal site is the major element required for the ERM-mediated activation of the ICAM- 1 promoter. We can thus conclude that expression of ICAM-1 can be regulated by Ets transcription factors. Keywords: ICAM-1; Ets; ERM; transcription factor Introduction The Ets family of transcription factors is an ubiquitously expressed group of proteins involved to regulate gene transcription in normal and cancerous tissues as well as during embryonic development. The hallmark of these transcription factors is a DNA- binding domain ± the ETS domain ± of &85 amino acids (Karim et al., 1990), which recognizes the core sequence GGAA/T, present in the regulatory region of a wide array of responsive genes, including membrane receptors, growth factors, transcription factors, and extracellular metalloproteinases (Wasylyk et al., 1993; Janknecht and Nordheim, 1993; CreÂpieux et al., 1994). The Ets family comprises several members classi®ed into dierent groups with respect to their sequence homology within the ETS domain. The PEA3 group (for review, see de Launoit et al., 1997) is composed of three members: PEA3 (Xin et al., 1992; Higashino et al., 1993), ER81 (Brown and McKnight, 1992; Jeon et al, 1995; Monte et al., 1995) and ERM (Monte et al., 1994). These three factors share more than 95% identity within the ETS domain (Monte et al., 1994). The human intercellular adhesion molecule-1 (ICAM-1, CD54), a member of the immunoglobulin supergene family, is an adhesion receptor of the immune system which mediates antigen-independent cell-cell contact between eector cells expressing the b2-integrin molecules LFA-1 and Mac-1, and target cells of various origins (Rothlein et al., 1986; Marlin and Springer, 1987; Diamond et al., 1990). Interaction between LFA-1 or Mac-1 and ICAM-1 is essential for immune responses. For instance, it increases the avidity of the interaction between the T-cell receptor and antigen presented in the context of class II MHC molecules (Altman et al., 1989). ICAM-1-b2-integrin interaction is also required for T-cell cytotoxicity, and leukocyte tracking and migration into in¯amed tissues (for review, see Dustin et al., 1988; Albelda et al., 1994). ICAM-1 expression is regulated by in¯ammatory mediators such as tumor necrosis factor-a (TNFa), interleukin-1b (IL-1b), interferon-g (IFNg), as well as by bacterial liposaccharide, phorbol esters and retinoic acid (RA) (for review, see Stratowa and Audette, 1995 and references therein). The stimulation of the ICAM-1 protein expression corre- lates with increased levels of ICAM-1 mRNA, thus suggesting that ICAM-1 expression could occur at the transcriptional level (Simmons et al., 1988). The 5'-regulatory region of the human ICAM-1 gene has been cloned and characterized (Stade et al., 1990; Voraberger et al., 1991; Wawryk et al., 1991), and two transcriptional start sites have been mapped, respec- tively 41 bp and 319 bp upstream from the transla- tional start site (Voraberger et al., 1991). The analysis of the 6 kb-region upstream from the ATG indicated the presence of one silencing and four enhancing elements contributing to the constitutive promoter activity (Jahnke et al., 1995). Deletion mutant analyses of the 5'-regulatory region of the ICAM-1 gene demonstrated the crucial role of the TATA-box located at position 770, 30 bp upstream from the proximal start site. Several responsive elements for binding transcription factors have been identi®ed in the Correspondence: Y de Launoit Y de Launoit and M Audette should both be considered as ®rst authors Received 23 July 1997; revised 12 November 1997; accepted 12 November 1997 Oncogene (1998) 16, 2065 ± 2073  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc