THE HUMAN ICAM-2 PROMOTER IS ENDOTHELIAL CELL-SPECIFIC IN-VITRO AND IN-VIVO AND CONTAINS CRITICAL SP1 AND GATA BINDING-SITES

The expression of intercellular adhesion molecule 2 (ICAM-2) in adult tissues is restricted to vascular endothelial cells and megakaryocytes . We have previously shown that the endothelial-specific in vivo activ ity of the human ICAM-2 promoter is contained within a small (0.33-kil obase (kbp)) 5'-flanking region of the gene. Here we describe the in v itro characterization of this region. The ICAM-2 promoter is TATA-less , and transcription in endothelial cells initiates at four sites. Repo rter gene expression directed by the promoter was 125-fold greater tha n vector alone in bovine aortic endothelial cells but less than a-fold vector alone in non-endothelial (COS) cells, confirming that specific ity in vivo was paralleled in vitro. The addition of 2.7 kbp of 5'-fla nking region to the 0.33-kbp fragment had no effect on promoter activi ty or specificity. The mutation of an Spl motif centered at base pair -194 or an eight-base pair palindrome at -268 each reduced promoter ac tivity by 70%. Mutation of GATA motifs at -145 and -53 reduced promote r activity by 78 and 61%, respectively. Specific binding of bovine aor tic endothelial cells nuclear proteins to the Spl and GATA sites was d emonstrated by gel shift analysis. Promoter activity in COS cells was transactivated 3-4-fold by overexpression of GATA-8. The results prese nted here suggest that transcription from the ICAM-2 promoter in endot helial cells is regulated by the interplay of several positive-acting factors and provide the basis for further analysis of endothelial-spec ific gene expression.