LOCALIZATION AND CHARACTERIZATION OF A RETINOIC ACID RESPONSE-LIKE ELEMENT IN THE PLASMINOGEN-ACTIVATOR INHIBITOR-2 GENE PROMOTER

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 line s.(1) To identify the promoter elements required for RA induced potent iation, deletion mutants of the PAI-2 promoter fused to the chloramphe nicol acetyl transferase (CAT) reporter gene were transiently expresse d in U937 cells. These studies demonstrated that promoter sequences lo cated between -1839 and -1063 were functionally relevant in generating this response. Exonuclease III protection analysis revealed that nucl ear 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 myelomonocy tic leukemic cell factors to this region. Extracts prepared from RA st imulated and unstimulated cells resulted in the same pattern of comple x formation, but the mobilities of the complexes were slightly differe nt, which suggests RA binding to the complexes. Our results suggest th e role of PAI-2 promoter elements in generating the response to RA. Th e binding characteristics of nuclear factors prepared from myelomonocy tic cells to these sequences are indicative of RA receptor-DNA interac tion.