MOLECULAR MECHANISMS GOVERNING TUMOR-NECROSIS-FACTOR-MEDIATED REGULATION OF PLASMINOGEN-ACTIVATOR INHIBITOR TYPE-2 GENE-EXPRESSION

Plasminogen-activator inhibitor type 2 (PAI-2), a serine protease inhi bitor involved in the regulation of urokinase-dependent proteolysis, i s also implicated in the inhibition of tumor-necrosis-factor-(TNF)-med iated apoptosis. The PAI-2 gene is one of the most TNF-responsive gene s known and is also highly induced by the phorbol ester phorbol 12-myr istate 13-acetate (PMA) and the phosphatase inhibitor, okadaic acid, i n both HT-1080 fibrosarcoma and U-937 histiocytic cells. We sought to identify and characterize regulatory cis-acting DNA elements and trans -acting factors which mediate basal and inducible PAI-2 gene transcrip tion. A series of promoter deletion mutants (nucleotides -1859 to -91) fused to the chloramphenicol acetyl transferase (CAT) reporter gene w ere transfected into HT-1080 cells. Two repressor regions were identif ied; one distally between positions -1859 and -1100, and one proximall y between positions -259 and -219. Cells transfected with constructs h arboring more than 259 bp promoter sequence produced a 10-15-fold incr ease in CAT activity when treated with PMA or okadaic acid, but produc ed only a minimal (2.5-fold) increase in response to TNF. Removal of t he proximal repressor by deletion to position -219, or by internal del etion from the -1100 PAI-2 CAT construct, resulted in a selective incr ease in TNF responsiveness, suggesting that induction of PAI-2 gene tr anscription by TNF is associated with derepression. Detailed analysis of the proximal repressor utilizing the electrophoretic mobility shift assay (EMSA), identified two novel and distinct protein-binding sites (A and B). Site A is located within the 40-bp proximal repressor whil e site B is situated immediately adjacent to the 3' boundary. Treatmen t of cells with PMA or okadaic acid produced no change in the binding activity of proteins recognising sites A or B. However, treatment of c ells with TNF results in a profound selective reduction in site-B-bind ing activity, suggesting that this site plays a significant role in TN F-mediated regulation of PAI-2 gene expression. Our findings suggest t hat TNF-mediated induction of PAI-2 gene expression involves derepress ion and is associated with cis-acting and trans-acting factors located within and adjacent to the proximal repressor region.