News and Publications

Regulation of the Plasminogen Activation System

* Institute de Recerca Oncologica, Barcelona, Spain
** Department of Immunology, TSRI
*** University of California, San Diego, California

Interactions of proteins of the plasminogen activation system with cell surfaces modulate cellular functions, including cell migration and prohormone processing. In a reciprocal fashion, cell-surface receptors for plasminogen promote plasminogen activation, for further modulation of cellular functions. We are investigating the regulation of plasmin activity at the levels of cellular receptors and gene expression.

Previously, we showed that localization of Glu-plasminogen on the surface of endothelial cells enhances its conversion to Lys-plasminogen by exogenous plasmin. This conversion leads to stimulation of plasminogen activation because Lys-plasminogen is the preferred substrate for plasminogen activators on cell surfaces. Recently, we found that Glu-plasminogen was converted to Lys-plasminogen on monocytoid cells in the absence of exogenous plasmin. Culture of cells under serum-free conditions did not affect this conversion, suggesting that the enzymatic activity was cell derived. In further tests, we found that endogenous monocytoid plasminogen could provide a source of plasmin to convert cell-associated Glu-plasminogen to Lys-plasminogen. Our results suggest that plasmin converts cell-bound Glu-plasminogen to Lys-plasminogen and that plasmin is produced by activation of monocytoid plasminogen by endogenous monocytoid plasminogen activators to enhance plasminogen activation on the monocytoid cell surface.

Identification and characterization of cell-surface plasminogen receptors are confounded because nonviable cells bind approximately 10- to 50-fold more plasminogen than viable cells do. Using a proteomic approach, we are developing a method to specifically isolate cell-surface plasminogen receptors from viable cells. We found that the capacity of viable peripheral blood monocytes to bind and activate plasminogen increased 28-fold as the cells differentiated into tissue macrophages in response to colony-stimulating factor 1, thereby arming the cells with plasmin, an important protease involved in cell migration.

Plasminogen is expressed broadly in neuroendocrine tissues, where synthesis of proteins is regulated by neurotrophins such as nerve growth factor (NGF). Our recent results suggest that plasmin activity is regulated at the level of expression of the plasminogen gene. Thus, regulation of plasminogen gene expression by neurotrophins could be a mechanism for regulating local proteolysis in these tissues. We found that NGF treatment of cultured NGF-responsive rat PC12 pheochromocytoma cells increased the plasminogen content of the culture medium 3-fold and that steady-state levels of plasminogen mRNA were increased approximately 6-fold. When PC12 cells were transfected with a construct consisting of a 2400-bp fragment of the murine plasminogen promoter cloned upstream of the pGL2 firefly luciferase reporter plasmid, luciferase activity was increased 5-fold after treatment of the cells with NGF. Treatment of the transfected cells with PD98059, an inhibitor of MAP kinase kinase, before stimulation with NGF decreased luciferase expression in response to NGF to the level present in untreated cells. Furthermore, cotransfection with a dominant-negative mutant Ha-Ras completely blocked NGF-induced luciferase activity, suggesting that the Ras-extracellular signal-regulated kinase cascade is required for stimulation of plasminogen gene expression in response to NGF.

In deletional experiments, the region between -255 and -106 of the plasminogen promoter contained cis-acting elements that were responsible for the response to NGF. In mutational studies, 2 binding sites for the transcription factor Sp1 within a cluster of 3 Sp1 sites in this region were both necessary and sufficient for the response of the plasminogen promoter to NGF. These results suggest that plasminogen gene expression is upregulated by neurotrophins, which may provide a previously unrecognized mechanism for regulation of neuronal function via the proteolytic activity of plasmin.

Publications

Albrecht, S., Magdolen, V., Herzog, U., Miles, L.A., Kirschenhofer, A., Baretton, G., Luther, T. Soluble tissue factor interferes with angiostatin-mediated inhibition of endothelial cell proliferation by lysine-specific interaction with plasminogen kringle domains. Thromb. Haemost. 88:1054, 2002.

Garcia-Bannach, F., Gutierrez, A., Fowler, B.J., Bugge, T.H., Degen, J.L., Parmer, R.J., Miles, L.A. Localization of regulatory elements mediating constitutive and cytokine-stimulated plasminogen gene expression. J. Biol. Chem. 277:38579, 2002.

Gutierrez, A., Garcia-Bannach, F., Zhang, L., Jenkins, G.R., Parmer, R.J., Miles, L.A. Regulation of plasminogen gene expression. In: Plasminogen: Structure, Activation, and Regulation. Waisman, D.M., Rescigno, A.M. (Eds.). Plenum, New York, in press.

Jiang, Q., Yasothornsrikul, S., Taupenot, L., Miles, L.A., Parmer, R.J. The local chromaffin cell plasminogen/plasmin system and the regulation of catecholamine secretion. Ann. N. Y. Acad. Sci. 971:445, 2002.

López-Alemany, R., Longstaff, C., Hawley, S., Mirshahi, M., Fábregas, P., Jardi, M., Merton, E., Miles, L.A., Félez, J. Inhibition of cell surface mediated plasminogen activation by a monoclonal antibody against α-enolase. Am. J. Hematol. 72:324, 2003.

Miles, L.A., Andronicos, N.M., Félez, J., Grella, D.K., Castellino, F.J., Gong, Y. The role of lys-pg in cell-mediated plasmin production. In: Plasminogen: Structure, Activation, and Regulation. Waisman, D.M., Rescigno, A.M. (Eds.). Plenum, New York, in press.

Miles, L.A., Castellino, F.J., Gong, Y. Critical role for conversion of Glu-plasminogen to Lys-plasminogen for optimal stimulation of plasminogen activation on cell surfaces. Trends Cardiovasc. Med. 13:21, 2003.

Miles, L.A., Hawley, S.B., Parmer, R.J. Chromaffin cell plasminogen receptors, Ann. N. Y. Acad. Sci. 971:454, 2002.

Tauri, T., Manjumdar, M., Miles, L.A., Ruf, W., Takada, Y. Plasmin-induced migration of endothelial cells: a potential target for the anti-angiogenic action of angiostatin. J. Biol. Chem. 277:33564, 2002.

Zhang, L., Gong, Y., Grella, D.K., Castellino. F.J., Miles, L.A. Endogenous plasminogen converts Glu-plasminogen to Lys-plasminogen on the monocytoid cell surface. J. Thromb. Haemost. 1:1264, 2003.