PAJ-2 is a serine protease inhibitor involved in the regulation of urokinase-dependent proteolysis and implicated in the inhibition of tumor necrosis factor (TNF)-mediated apoptosis. The PAI-2 gene is one of the most TNF-responsive genes known and is also highly induced by the phorbol ester 12-phorbol 13-myristate acetate (PMA) and the phosphatase inhibitor, okadaic acid in HT-1080 and U-937 cells. Using a series of PAI-2 promoter deletion mutants fused to the chloramphenicol acetyl transferase (CAT) reporter gene in a transient expression system in HT-1080 fibrosarcoma cells, we identified two represser regions: one between -1859 and -1100, and one between -259 and -216. Cells transfected with constructs harboring more than 259 bp of promoter sequence generated a 10- to 15-fold increase in CAT activity when treated with PMA or with okadaic acid, but demonstrated only a 2.5-fold increase in response to TNF. Removal of the proximal repressor by deletion to position -216, or by internal deletion from either the -341 or -1100 PAI-2 CAT constructs, results in a marked increase in TNF-responsiveness suggesting that induction of PAI-2 gene transcription by TNF is associated with de-repression. Band shift analyses identified a single protein binding site (site A) within the proximal repressor region which was adjacent to a single CAGG motif. Tandem CAGG motifs act as binding sites for a TNF-responsive repressor protein (1). Site directed of site A resulted in a marked weakening in the basal and inducible expression indicating that it functions as an enhancer. Interestingly, disruption of the CAGG motif resulted in a profound increase in TNF-inducibility indicating that this motif is functionally important in the repressive response. Our findings suggest that TNF-mediated induction of PAI-2 transcription involves de-repression requiring a single CAGG motif adjacent to an enhancer region.
|Number of pages||1|
|Issue number||SUPPL. 3|
|Publication status||Published - 1 Dec 1996|