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 inhibitor involved in the regulation of urokinase-dependent proteolysis, is also 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 phorbol 12-myristate 13-acetate (PMA) and the phosphatase inhibitor, okadaic acid, in 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 transcription. A series of promoter deletion mutants (nucleotides -1859 to -91) fused to the chloramphenicol acetyl transferase (CAT) reporter gene were transfected into HT-1080 cells. Two repressor regions were identified; one distally between positions -1859 and -1100, and one proximally between positions -259 and -219. Cells transfected with constructs harboring more than 259 bp promoter sequence produced a 10-15-fold increase in CAT activity when treated with PMA or okadaic acid, but produced only a minimal (2.5-fold) increase in response to TNF. Removal of the proximal repressor by deletion to position -219, or by internal deletion from the -1100 PAI-2 CAT construct, resulted in a selective increase in TNF responsiveness, suggesting that induction of PAI-2 gene transcription 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 while site B is situated immediately adjacent to the 3' boundary. Treatment of cells with PMA or okadaic acid produced no change in the binding activity of proteins recognising sites A or B. However, treatment of cells with TNF results in a profound selective reduction in site-B-binding activity, suggesting that this site plays a significant role in TNF-mediated regulation of PAI-2 gene expression. Our findings suggest that TNF-mediated induction of PAI-2 gene expression involves derepression and is associated with cis-acting and trans-acting factors located within and adjacent to the proximal repressor region.
Dear
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Monash University Department of Medicine, Boxhill Hospital, Victoria, Australia.
Shen
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Rüegg
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Medcalf
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RL
D001483:Base Sequence; D001665:Binding Sites; D015342:DNA Probes / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009687:Nuclear Proteins / Q000737:chemistry / Q000378:metabolism; D017396:Plasminogen Activator Inhibitor 2 / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics / Q000494:pharmacology; D017421:Sequence Analysis; D017384:Sequence Deletion / Q000235:genetics; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D015342:DNA Probes; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D017396:Plasminogen Activator Inhibitor 2; D012097:Repressor Proteins; D014409:Tumor Necrosis Factor-alpha