The activity of plasmin, the major enzyme responsible for dissolving fibrin clots, is regulated by plasminogen activators, plasminogen activator inhibitors, ␣ 2 -antiplasmin, and inflammatory mediators. Recent studies suggest that plasmin activity can be regulated also at the level of plasminogen gene expression. In this study, we characterized the murine plasminogen promoter and 5-flanking region. The major transcription start site was identified at ؊83 bp relative to the ATG translational initiation codon. A series of 5-flanking sequences up to 2400 bp upstream of the transcription initiation site were fused to the luciferase reporter gene and transfected into hepatocytic cells. A 106-bp 5-flanking region of the murine plasminogen gene demonstrated sufficient functional promoter activity in plasminogenexpressing cells. IL-6 treatment stimulated luciferase activity driven by the 5-flanking region and an intact consensus IL-6-responsive element at ؊791, was required for maximal stimulation by this cytokine. These results indicate the presence of regulatory elements in the 5-flanking region of the murine plasminogen promoter that may regulate murine plasminogen gene expression and, hence, plasmin activity.
Summary. An emerging area of research has demonstrated that plasminogen functions in the acute-phase response to tissue injury, neoplastic growth or infection. We have previously shown that the acute-phase mediator, interleukin (IL)-6, increases circulating plasminogen levels via upregulation of plasminogen promoter activity. We also identified a putative IL-6 responsive element (nt )791 to )783; IL6-RE) in the plasminogen gene that is required for maximal stimulation of promoter activity by IL-6. For the present study, we investigated the transcription factors and signaling pathway mediating the response of the plasminogen gene to IL-6. In electrophoretic mobility shift assays (EMSAs), a radiolabeled oligonucleotide IL6-RE probe formed specific complexes with nuclear proteins from untreated hepatocytic cells. The extent of complex formation was markedly increased using nuclear proteins from IL-6-treated cells. Complex formation was abolished by an oligonucleotide with the consensus CCAAT/enhancer binding protein (C/EBP) sequence. Furthermore, complexes were supershifted by antibodies to C/EBPb. Treatment of Hepa 1-6 cells with the mitogen-activated protein kinase (MAPK) inhibitor, PD-98059, inhibited IL-6-stimulated plasminogen promoter activity. These results suggest that transcription factor C/EBPb and the MAPK pathway play key roles in the response of the plasminogen gene to IL-6, thus elucidating a major mechanism by which the plasminogen system is upregulated to perform its crucial functions in the acutephase response.
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