Up-Regulation of Urokinase-Type Plasminogen Activator in Rabbit Corneal Epithelial Cells by Human Annexin A5

Abstract

Abstract: : Purpose:Urokinase-type plasminogen activator (uPA) is essential for the migration of corneal epithelial cells. We previously showed that the extent of epithelial migration after wounding was correlated with the activity of uPA in the culture medium of the rabbit corneal organ culture system and that the plasmin inhibitor blocked rabbit corneal epithelial cell migration. Human annexin A5, a calcium-dependent phospholipid binding protein, promotes the migration of corneal epithelial cells. To elucidate the mechanism of action of annexin A5, we determined the effect of annexin A5 on the production of uPA. Methods:The cultured medium of rabbit corneal epithelial (RCE) cells in the presence of recombinant human annexin A5 for 48 hours was collected. To determine the type of PA, the medium was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin-agarose zymography. Confluent monolayers of RCE cells were wounded with a scraper and subsequently cultured with recombinant human annexin A5. Twenty-four hours after wounding, the cultured medium was collected. The activity of PA in the medium was measured by a spectrofluorometric method using a plasmin-specific fluorogenic peptide substrate, t-butyloxycarbonyl-L-valyl-L-leucyl-L-lysine-4-methyl-coumaryl-7-amide (Boc-Val-Leu-Lys-MCA). Furthermore, the concentration of uPA in the medium was measured by enzyme-linked immunosorbent assay (ELISA). Results: Fibrinolytic zymography revealed that the PA released from RCE cells was uPA and the intensity of fibrinolytic band was increased by annexin A5. By the spectrofluorometric assay and ELISA, the amount of uPA released from the wounded monolayers of RCE cells was up-regulated by annexin A5 in a dose dependent manner. Conclusions:Human annexin A5 promotes the production of uPA in RCE cells. Human annexin A5 may have the promotive effect in corneal epithelial cell migration, in part, through the up-regulation of uPA production.

Keywords: wound healing • cornea: epithelium • cornea: basic science