Urokinase-type plasminogen activator (uPA) is suggested to exert its proliferatory, migratory and invasive action through binding with its membrane receptor, promoting pericellular proteolysis and mediating cell signal transduction. One of the possible actions of urokinase can be related to the regulation of activity and/or the expression of proteolytic enzymes participating in extracellular matrix degradation. In the present study, the role of uPA in regulating matrix metalloproteinase (MMP) expression and release by the monocyte cell line THP-1 was investigated. Recombinant uPA induced the release of MMP9/gelatinase B, as detected by zymography and Western blotting, and this release was abolished by actinomycin D and cycloheximide (inhibitors of DNA transcription and protein synthesis) and partially suppressed by monensin (an inhibitor of secretion). Proteolytically inactive urokinase with substitution of His(204) for Gln was able to reproduce about 70% of the effect induced by the wild-type recombinant uPA. The reverse transcription-PCR and Northern blot data indicated that the action of r-uPA on THP-1 cells resulted in formation of MMP9 mRNA, which depended on time, within 6-48 h, of the cell incubation with r-uPA. These results suggest that urokinase upregulates MMP9 expression in monocytes via MMP9 gene transcription and protein biosynthesis.
Objective: Urokinase-type plasminogen activator (uPA) regulates cell migration and invasion by pericellular proteolysis and signal transduction events. We characterized the mechanisms by which uPA regulates matrix metalloproteinase-9 (MMP9) function in THP-1 monocytes. Methods and Results: In THP-1 monocytes, MMP9 production induced by urokinase was completely inhibited by the ERK1/2 inhibitor, PD98059, but not by the p38 mitogen-activated protein kinase inhibitor, SB202190. A dominant negative MEK1 adenovirus also blocked MMP9 expression. The effect of urokinase was completely suppressed by genistein and by herbimycin A indicating that tyrosine kinase(s) are required for MMP9 production. Bisindolylmaleimide, a protein kinase C (PKC) inhibitor, did not decrease MMP9 expression suggesting that PKC activation is not required. Key roles for cytosolic phospholipase A2 (PLA2) and eicosanoid production were shown by complete inhibition with methyl arachidonyl fluorophosphonate (an inhibitor of cytosolic PLA2), and indomethacin (a cyclooxygenase inhibitor), with no effect of monoalide, a secretory PLA2 inhibitor. uPA stimulated phosphorylation of cytosolic PLA2. Conclusions: Induction of MMP9 by uPA in THP-1 monocytes is via a pathway involving MEK1-ERK1/2-mediated activation of cytosolic PLA2 and eicosanoid generation. These data suggest important roles for eicosanoids in monocyte migration induced by uPA and MMP9.
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