Fibrinolysis (1994) $, 113-119 01994 Longman Group UK Ltd Localization and Characterization of a Retinoic Acid Response-like Element in the Plasminogen Activator Inhibitor-2 Gene Promoter W. A. Schuster, R. L. Medcalf, E. K. O. Kruithof SUMMARY. We have previously shown that all-trans retinoic acid (RA) potentiates phorbol ester-mediated induction of plasminogen activator inhibitor-2 (PAI-2) gene transcription in human myelomonocytic leukemic cell lines. 1 To identify the promoter elements required for RA induced potentiation, deletion mutants of the PAI-2 promoter fused to the chloramphenicol acetyl transferase (CAT) reporter gene were transiently expressed in U937 cells. These studies demonstrated that promoter sequences located between -1839 and -1063 were functionally relevant in generating this response. Exonuclease III protection analysis revealed that nuclear factors prepared from HL-60 cells bind to a region (-1659 to -1620), which contains a retinoic acid receptor element half-site separated by seven nucleotides from a glucocorticoid response element half-site. Gel retardation analysis using an oligonucleotide of PAI-2 promoter sequences -1660 to -1620 confirmed the specific binding of myelomonocytic leukemic cell factors to this region. Extracts prepared from RA stimulated and unstimulated cells resulted in the same pattern of complex formation, but the mobilities of the complexes were slightly different, which suggests RA binding to the complexes. Our results suggest the role of PAI-2 promoter elements in generating the response to RA. The binding characteristics of nuclear factors prepared from myelomonocytic cells to these sequences are indicative of RA receptor-DNA interaction. The vitamin A derivative, retinoic acid (RA), exerts profound effects on development and differentiation (for review see 2). These effects are mediated via a growing family of nuclear receptors which are members of the steroid hormone receptor superfa- mily. Like other members of this family, RA recep- tors mediate ligand-dependent regulation of gene transcription as a consequence of binding to specific DNA sequences termed retinoic acid response elements (RARE). Despite the broad spectrum of RA-mediated actions, naturally occurring RARE's have only been shown for a small number of genes. These include the human oxytocin gene, 3 the laminin B1 gene, 4 the osteocalcin gene, 5 the gene for comple- ment H factor 6 and the RAR-ot and -0 genes them- selves. 7-~~ Comparison of these RARE's points to a palindromic feature consisting of inverted repeats. However, Umesono and co-workers ~l identified a W. A. Schuster, E. K. O. Kruithof, Division of Haematology, Department of Medicine, University Hospital, CHUV, 1011 Lausanne, Switzerland, R. L. Medcalf, Monash University Department of Medicine, Nelson Road, Box Hill 3128, Victoria, Australia. This work was supported by the Swiss National Foundation for ScientificResearch Grants 32.29034.90and 31.26399.89and by the Foundation for Research on Atherosclerosisand Thrombosis. 113 thyroid hormone response element (TRE) consisting of a direct repeat, not a palindrome, of the two half-sites. Furthermore, by increasing the spacing between the half sites, this region could be converted into a RARE. In conclusion, no unequivocal RARE consensus sequence has thus far been described. Our initial observation that RA potentiates the induction of plasminogen activator inhibitor-2 (PAI-2) mRNA and antigen by phorbol myristate acetate (PMA) in myelomonocytic leukemic cell lines, and that this effect was due to increases in PAI-2 gene transcription rates, suggested a possible interaction of the RA receptor with cis-acting DNA elements in the promoter region of the PAI-2 gene. Here we describe the identification and characteri- zation of a novel cis-acting RARE-like PAI-2 gene promoter element composed of two heterologous half sites which may be responsible for conferring retinoic acid potentiation of the PMA effect on this gene. MATERIALS AND METHODS Cell Culture The promyelocytic HL-60 cell line was originally derived from a patient with acute promyelocytic