Plasminogen-Independent Initiation of the Pro-urokinase Activation Cascade in Vivo. Activation of Pro-urokinase by Glandular Kallikrein (mGK-6) in Plasminogen-Deficient Mice † Karin List,* ,‡ Ole N. Jensen, § Thomas H. Bugge, || Leif R. Lund, ‡ Michael Ploug, ‡ Keld Danø, ‡ and Niels Behrendt ‡ The Finsen Laboratory, Rigshospitalet, StrandbouleVarden 49, DK-2100 Copenhagen Ø, Denmark, Department of Molecular Biology, Odense UniVersity, DK-5230 Odense M, Denmark, and Oral and Pharyngeal Cancer Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892 ReceiVed July 23, 1999; ReVised Manuscript ReceiVed October 18, 1999 ABSTRACT: The plasminogen activation (PA) system is involved in the degradation of fibrin and various extracellular matrix proteins, taking part in a number of physiological and pathological tissue remodeling processes including cancer invasion. This system is organized as a classical proteolytic cascade, and as for other cascade systems, understanding the physiological initiation mechanism is of central importance. The attempts to identify initiation routes for activation of the proform of the key enzyme urokinase-type plasminogen activator (pro-uPA) in vivo have been hampered by the strong activator potency of the plasmin, that is generated during the progress of the cascade. Using gene-targeted mice deficient in plasminogen (Plg -/- mice) [Bugge, T. H., Flick, M. J., Daugherty, C. C., and Degen, J. L. (1995) Genes DeV.9, 794-807], we have now demonstrated and identified a component capable of initiating the cascade by activating pro-uPA. The urine from Plg -/- mice contained active two-chain uPA as well as a proteinase capable of activating exogenously added pro-uPA. The active component was purified and identified by mass spectrometry-based peptide mapping as mouse glandular kallikrein mGK-6 (true tissue kallikrein). The pro-uPA converting activity of the mGK-6 enzyme, as well as its ability to cleave a synthetic substrate for glandular kallikrein, was inhibited by the serine proteinase inhibitor leupeptin but not by other serine proteinase inhibitors such as aprotinin, antithrombin III, or R 1 -antitrypsin. We suggest that mouse glandular kallikrein mGK-6 is an activator of pro-uPA in the mouse urinary tract in vivo. Since this kallikrein is expressed in a number of tissues and also occurs in plasma, it can also be considered a candidate for a physiological pro-uPA activator in other locations. Proteolytic enzymes are involved in degradation of the extracellular matrix during the migration of cells through tissue barriers. Tissue degradation and cell migration occur in cancer invasion as well as in normal physiological processes such as trophoblast invasion, morphogenesis, inflammation, and wound healing. The plasminogen activa- tion (PA) 1 system plays an important role in many of these degradative events (for reviews, see refs 1 and 2). Further- more, it may undertake specific cleavage reactions in the activation of certain growth factors (3) and other proteolytic systems (4). The central target for the activation processes is the pro-enzyme plasminogen, which is present in high amounts in plasma and other extracellular fluids and is activated by either of two plasminogen activators, urokinase- type plasminogen activator (uPA) and tissue-type plasmi- nogen activator (tPA). The activation product, plasmin, is an efficient proteinase with a relatively broad, trypsin-like activity. Therefore, the stores of plasminogen represent a large, resting proteolytic potential which becomes activated locally under specific conditions. It follows that the mecha- nisms of this activation process play a trigger role in the whole proteolytic system. uPA is capable of cleaving plasminogen at the specific peptide bond critical for activa- tion and plays a central role in the pericellular degradation of extracellular matrix proteins, mentioned above, in addition to a role in fibrinolysis (1, 5, 6). uPA is secreted as a single polypeptide chain protein which undergoes specific cleavages during the progress of the cascade. This single-chain form, pro-uPA, is a real pro-enzyme with virtually no intrinsic activity (7), and the active two-chain form is obtained upon cleavage after Lys 158 . This activation can be efficiently mediated by plasmin, resulting in a strong feedback ampli- fication of the system in the presence of plasminogen as soon as the first active enzyme molecules are formed. This † This work was supported by H:S Copenhagen Hospital Corp., Denmark. * Corresponding author. Fax: +45 35 38 54 50; Phone: +45 35 58 76; E-mail: karin.list@finsenlab.dk. ‡ The Finsen Laboratory, Rigshospitalet. § Odense University. | Oral and Pharyngeal Cancer Branch, NIDCR. 1 Abbreviations: PA, plasminogen activation; uPA, urokinase-type plasminogen activator; tPA, tissue-type plasminogen activator; uPAR, urokinase-type plasminogen activator receptor; ATF, amino-terminal fragment of uPA; LMW-uPA, low molecular weight two-chain uPA; mGK-6, the protein product from the mouse glandular kallikrein-6 gene; NGF, nerve growth factor; EGF-BP, epidermal growth factor binding protein; hK1, human true tissue kallikrein; hK2, human prostatic kallikrein; PSA, prostate-specific antigen; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry; PAI-1, plasminogen activator inhibitor type 1. 508 Biochemistry 2000, 39, 508-515 10.1021/bi991701f CCC: $19.00 © 2000 American Chemical Society Published on Web 12/29/1999