Regulation of the urokinase-type plasminogen activator receptor on vascular smooth muscle cells is under the control of thrombin and other mitogens.
The urokinase-type plasminogen activator receptor (u-PAR) was demonstrated on cultured smooth muscle cells (SMCs) of bovine aorta. Binding of 125I-urokinase-type plasminogen activator (u-PA) was concentration dependent and saturable within 45-60 minutes. A similar concentration and time dependence was found in functional plasminogen activation studies. Human two-chain high-molecular-weight u-PA and its proenzyme (pro-u-PA) bound specifically with identical affinity (Kd). Activation of pro-u-PA was strongly accelerated on binding to SMCs and occurred only in the presence of plasminogen on the cell surface. A 100-fold molar excess of unlabeled high-molecular-weight u-PA effectively blocked binding of the radiolabeled ligands; tissue-type plasminogen activator, plasminogen, low-molecular-weight u-PA, and unrelated proteins did not. 125I-u-PA binding was abolished by a monoclonal antibody against the specific u-PA sequence responsible for u-PAR binding. Binding of u-PA sharply decreased on SMC exposure to phosphatidylinositol-specific phospholipase C, confirming the glycan phospholipid cell anchorage of u-PAR. Bovine and human alpha-thrombin (240 nM) increased the binding of 125I-u-PA fivefold, translating into an increase in the number of sites per cell from about 10(5) to 5 x 10(5) without significant change in the Kd (1.29 +/- 0.39 nM). Active site blockade of thrombin by D-Phe-Pro-Arg-chloromethyl ketone resulted in the total loss of stimulatory activity, as did the use of the inactive active site thrombin mutant, S205A. Hirugen (100 microM), which blocks the anion-binding exosite of thrombin, blocked u-PAR stimulating activity. Thus, both the catalytic activity and integrity of the exosite are important for thrombin's stimulatory activity. Other SMC mitogens (epidermal growth factor, transforming growth factor-beta 1, basic fibroblast growth factor, platelet-derived growth factor, and phorbol 12-myristate 13-acetate) increased u-PAR expression on SMCs six- to 20-fold while concomitantly increasing Kd four- to 10-fold. In all cases the induction of u-PAR was dependent on de novo protein synthesis. These observations assign a possible role for thrombin and other mitogens in u-PAR regulation, thereby influencing the pericellular proteolysis that is important in SMC migration and atheromatous plaque development.
Reuning
U
U
Lilly Laboratories for Clinical Research, Eli Lilly & Company, Indianapolis, IN 46285.
Bang
N U
NU
D000818:Animals; D001011:Aorta / Q000378:metabolism; D001161:Arteriosclerosis / Q000378:metabolism; D001665:Binding Sites / Q000187:drug effects; D002417:Cattle; D002465:Cell Movement; D004815:Epidermal Growth Factor / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D017261:Glycosylphosphatidylinositols; D006629:Hirudins / Q000031:analogs & derivatives / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D010446:Peptide Fragments / Q000494:pharmacology; D010960:Plasminogen Activators / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D013917:Thrombin / Q000378:metabolism* / Q000494:pharmacology; D016212:Transforming Growth Factor beta / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D017261:Glycosylphosphatidylinositols; D006629:Hirudins; C524898:PLAUR protein, human; D010446:Peptide Fragments; D010982:Platelet-Derived Growth Factor; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D016212:Transforming Growth Factor beta; D016222:Fibroblast Growth Factor 2; C062510:hirugen; D004815:Epidermal Growth Factor; D010960:Plasminogen Activators; D013917:Thrombin; D014568:Urokinase-Type Plasminogen Activator