Urokinase (u-PA)-mediated plasmin generation on the cell surface plays a central role in extracellular matrix turnover in both physiological and pathological states. u-PA is localised to the cell surface via a specific GPIlinked receptor. Receptor bound u-PA is subject to inhibitory regulation by the plasminogen activator inhibitors (PAI's). PAI-2 is the predominant inhibitor of u-PA in the extracellular matrix. In addition to localising cell surface plasminogen activation recent studies have implicated u-PAR in transmembrane signal transduction including modulation ofc-fos gene expression'. We postulated that u-PA, on binding its receptor, modulates PAI-2 gene expression via altered fos/jun (AP-1) activity. Electrophoretic mobility shift assays (EMSA) utilising nuclear extracts prepared from u-PA treated U-937 histiocytic lymphoma and HT-1080 fibrosarcoma cell lines demonstrated a significant increase in binding to oligonucleotides harbouring the AP-1 consensus sequence. Transient transfection of HT-1080 cells with PAI-2 promoter constructs, known to harbour two AP-1 sequences, fused to the chloramphenicol acetyl transferase (CAT) reporter gene demonstrated increased CAT activity with u-PA treatment alone and a synergistic increase in CAT activity in combination with phorbol ester (PMA). The synergistic effect of u-PA on PMA mediated induction of PAI-2 was confirmed at the mRNA level. Our observations provide the first description of u-PA mediated modulation of AP-1 activity correlating with an increase in PAI-2 gene expression. Together these findings indicate a novel auto regulatory loop in the regulation of plasminogen activation.
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|Published - 1 Dec 1996