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Molecular mechanisms governing tumor-necrosis-factor-mediated regulation of plasminogen-activator inhibitor type-2
1Monash University Department of Medicine, Boxhill Hospital, Victoria, Australia.
Abstract:
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.
Insights
This study identifies regulatory elements controlling Plasminogen-activator inhibitor type 2 (PAI-2) gene expression. Tumor necrosis factor (TNF) induction of PAI-2 involves derepression and specific protein binding site interactions.
Area of Science:
- Molecular Biology
- Gene Regulation
- Cancer Biology
Background:
- Plasminogen-activator inhibitor type 2 (PAI-2) regulates proteolysis and inhibits tumor necrosis factor (TNF)-mediated apoptosis.
- The PAI-2 gene is highly responsive to TNF, phorbol 12-myristate 13-acetate (PMA), and okadaic acid in fibrosarcoma and histiocytic cells.
Purpose of the Study:
- To identify and characterize regulatory DNA elements and protein factors controlling basal and inducible PAI-2 gene transcription.
- To elucidate the mechanisms underlying TNF-induced PAI-2 gene expression.
Main Methods:
- Transfection of chloramphenicol acetyl transferase (CAT) reporter gene constructs with PAI-2 promoter deletions into HT-1080 cells.
- Electrophoretic mobility shift assay (EMSA) to analyze protein-DNA interactions within the PAI-2 promoter.
Main Results:
- Two repressor regions in the PAI-2 promoter were identified: distal (-1859 to -1100) and proximal (-259 to -219).
- Deletion of the proximal repressor region selectively enhanced TNF responsiveness, indicating derepression is key to TNF induction.
- EMSA identified two protein-binding sites (A and B) in the proximal repressor region; TNF treatment significantly reduced binding activity at site B.
Conclusions:
- TNF-mediated induction of PAI-2 gene expression is associated with derepression.
- Specific cis-acting and trans-acting factors, particularly those interacting with binding site B near the proximal repressor, are crucial for TNF-regulated PAI-2 expression.