Molecular Mechanisms Governing Tumor‐Necrosis‐Factor‐Mediated Regulation of Plasminogen‐Activator Inhibitor Type‐2 Gene Expression

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 -1 100, 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 sitec 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 wggest 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.

Plasminogen Activator Inhibitor Type 2 Contains mRNA Instability Elements within Exon 4 of the Coding Region

“…Notwithstanding the important contribution of transcriptional control of PAI-2 expression (13,14), the role of posttranscriptional regulation of the PAI-2 gene has recently been highlighted (1,15). These investigations stemmed from earlier results whereby treatment of HT-1080 fibrosarcoma cells with a combination of PMA and TNF produced a 50 -100-fold increase in PAI-2 gene transcription but a 1500-fold increase in PAI-2 mRNA over a 24-h period (9).…”

“…Competition experiments confirmed that the two fastest migrating complexes (referred to as complexes I and II) were specific as only the unlabeled 4A RNA sequence competed for binding (lanes 3-5). Binding was not competed by cold RNA sequences harboring the 4B and 4C region of exon 4 (lanes 6 -11) or by RNA of unrelated (97-nt prothrombin 3Ј-UTR) sequence (lanes [12][13][14]. No specific binding of cytoplasmic proteins was observed to either the 4B or 4C probes (data not shown).…”

“…sense oligo 4 also substantially inhibited complex formation (lanes [12][13][14] and was essentially as effective as oligo 3 at inhibiting complex formation. As a negative control, antisense oligomers of unrelated sequence had no effect on binding activity (lane 15) at a concentration of 10 pmol.…”

“…Incubation of cytoplasmic extracts with the 4A probe produced two specific complexes (I and II, indicated with arrows). The formation of these complexes was competed with increasing concentrations (ϳ50-, 250-, and 500-fold molar excess) of unlabeled 4A RNA (lanes 3-5, respectively), but not with similar concentrations of unlabeled 4B (lanes 6 -8), 4C (lanes 9 -11), or an unrelated RNA (lanes [12][13][14]. No specific complex formation was produced when using the 4B or 4C RNA sequences as probes (data not shown).…”

Production of Plasminogen Activator Inhibitor-1 by Human Mast Cells and Its Possible Role in Asthma

“…PAI-3 regulates enzymes involved in fertilization rather than lung tissue fibrosis, whereas altered expression of both PAI-1 and PAI-2 is of potential relevance to the process of lung fibrosis (24,25). Although PAI-2 exhibits inhibitory activity toward tPA and uPA (26,27), the efficiency of PAI-2 is 20-to 100-fold less than what is observed for PAI-1 (28). PAI-2 is not generally detectable in human plasma, except during pregnancy (29,30).…”