REGULATION OF THE PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-2 GENE IN MONOCYTES - LOCALIZATION OF AN UPSTREAM TRANSCRIPTIONAL SILENCER

Transcriptional regulation of the plasminogen activator inhibitor type -2 (PAI-2) gene appears to be an important factor in the response of m ononuclear phagocytes to inflammation. We have investigated here the m olecular basis for PAI-2 synthesis in monocytic cells by reporter gene deletion analysis. A DNA fragment containing 5.1 kb of 5' flanking re gion through to the start of the second exon was fused to a chloramphe nicol acetyl transferase (CAT) reporter gene, transfected into macroph age and nonmacrophage cells and tested for PAI-2 promoter-directed CAT activity in the presence and absence of phorbol ester. Deletion analy sis showed the existence of three major transcription regulatory regio ns. (1) A positive regulatory region contained in the proximal promote r mediates basal transcription and 12-phorbol 13-myristate acetate ind ucibility. (2) A negative regulatory region, or silencer, present betw een -1977 and -1675, was found to repress PAI-2 promoter activity in a n orientation- and position-independent manner, but not in a cell-spec ific manner, (3) A second positive regulatory element, located upstrea m between approximately -5100 and -3300, appears to overcome inhibitio n mediated by the silencer in a cell-specific manner, suggesting a mec hanism for the regulation of this gene. We have localized the motif re sponsible for silencer activity to a 28-bp DNA sequence containing a u nique 12-bp palindrome centered at an Xba I restriction enzyme site, C TCTCTAGAGAG, which is designated the PAI-2-upstream silencer element-1 (PAUSE-1). This element binds a specific PAUSE-1 binding factor as de termined by mobility shift analysis, We conclude that PAI-2 gene trans cription is regulated by both positive and negative control mechanisms that may be important for the regulation of other genes as well. (C) 1996 by The American Society of Hematology.