Regulation of the Endothelial Cell Urokinase-Type Plasminogen Activator Receptor Evidence for Cyclic AMP-Dependent and Protein Kinase C-Dependent Pathways

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°C with 50 μM 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 ≊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