Circulation Research, Vol 72, 330-340, Copyright © 1993 by American Heart Association

ARTICLES

Regulation of the endothelial cell urokinase-type plasminogen activator receptor. Evidence for cyclic AMP-dependent and protein kinase C- dependent pathways

DJ Langer, A Kuo, K Kariko, M Ahuja, BD Klugherz, KM Ivanics, JA Hoxie, WV Williams, BT Liang and DB Cines
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.

Binding of urokinase-type plasminogen activator (u-PA) to specific receptors (u-PAR) on the surface of endothelial cells contributes to the regulation of plasmin-dependent processes such as fibrinolysis and angiogenesis. We studied the effect of raising intracellular levels of cyclic AMP (cAMP) and/or activating protein kinase C on the expression of u-PAR in cultured human umbilical vein endothelial cells (HUVEC). Incubation of HUVEC with forskolin stimulated a time- and concentration- dependent increase in the expression of u-PAR, measured both by an increase in the specific binding of radiolabeled single-chain u-PA (scu- PA) and by increased binding of anti-u-PAR antibodies. Maximal increase in u-PAR expression (81 +/- 11% above control, n = 11) was not associated with a change in receptor affinity for scu-PA when HUVEC were incubated for 20 hours at 37 degrees C with 50 microM forskolin. Receptor induction by forskolin was inhibited when HUVEC were preincubated with deoxyadenosine monophosphate (DAM), an inhibitor of adenylyl cyclase. A similar increase in receptor expression (128 +/- 27% above control, n = 3) was induced by the cAMP analogue 8- bromoadenosine 3':5'-cyclic monophosphate (50 mM). Forskolin induced an approximately twofold increase in the expression of a single approximately 1.4-kb u-PAR messenger RNA (mRNA) transcript within 2 hours. Phorbol myristate acetate (PMA) also stimulated a time- and concentration-dependent increase in specific scu-PA binding. The maximal increase in u-PAR expression (254 +/- 27% above control, n = 11) was observed when HUVEC were preincubated with 10 nM PMA for 20 hours. Induction of u-PAR by PMA was inhibited when HUVEC were preincubated with either cycloheximide or H7 but was unaffected by DAM. u-PAR induced by PMA showed a reduced affinity for scu-PA (Kd, 14 +/- 2 nM versus 3.6 +/- 0.6 nM, p < 0.001; n = 8). PMA stimulation for 20 hours resulted in a sixfold increase in a single approximately 1.4-kb u- PAR mRNA transcript, with increased levels detectable within 30 minutes. Coincubation of HUVEC with optimal concentrations of forskolin and PMA for 20 hours produced a fully additive increase in u-PAR expression at both the mRNA and protein levels. These data suggest that both cAMP-dependent and protein kinase C-dependent protein kinase pathways may independently regulate u-PAR expression in human endothelial cells.

This article has been cited by other articles:

				A J Wilson and P R Gibson Role of urokinase and its receptor in basal and stimulated colonic epithelial cell migration in vitro Gut, July 1, 2000; 47(1): 105 - 111. [Abstract] [Full Text] [PDF]

				P. H. A. Quax, J. M. Grimbergen, M. Lansink, A. H. F. Bakker, M.-C. Blatter, D. Belin, V. W. M. van Hinsbergh, and J. H. Verheijen Binding of Human Urokinase-Type Plasminogen Activator to Its Receptor : Residues Involved in Species Specificity and Binding Arterioscler Thromb Vasc Biol, May 1, 1998; 18(5): 693 - 701. [Abstract] [Full Text] [PDF]

				E. O. Harrington, J. Loffler, P. R. Nelson, K. C. Kent, M. Simons, and J. A. Ware Enhancement of Migration by Protein Kinase Calpha and Inhibition of Proliferation and Cell Cycle Progression by Protein Kinase Cdelta in Capillary Endothelial Cells J. Biol. Chem., March 14, 1997; 272(11): 7390 - 7397. [Abstract] [Full Text] [PDF]

				X.-N. Li, V. K. Varma, J. M. Parks, R. L. Benza, J. C. Koons, J. R. Grammer, H. Grenett, E. M. Tabengwa, and F. M. Booyse Thrombin Decreases the Urokinase Receptor and Surface-Localized Fibrinolysis in Cultured Endothelial Cells Arterioscler Thromb Vasc Biol, March 1, 1995; 15(3): 410 - 419. [Abstract] [Full Text]