Francesca Fazioli

Proteolytic cleavage of the urokinase receptor substitutes for the agonist-induced chemotactic effect.

Physiological concentrations of urokinase plasminogen activator (uPA) stimulated a chemotactic response in human monocytic THP‐1 through binding to the urokinase receptor (uPAR). The effect did not require the protease moiety of uPA, as stimulation was achieved also with the N‐terminal fragment (ATF), while the 33 kDa low molecular weight uPA was ineffective. Co‐immunoprecipitation experiments showed association of uPAR with intracellular kinase(s), as demonstrated by in vitro kinase assays. Use of specific antibodies identified p56/p59hck as a kinase associated with uPAR in THP‐1 cell extracts. Upon addition of ATF, p56/p59hck activity was stimulated within 2 min and returned to normal after 30 min. Since uPAR lacks an intracellular domain capable of interacting with intracellular kinase, activation of p56/p59hck must require a transmembrane adaptor. Evidence for this was strongly supported by the finding that a soluble form of uPAR (suPAR) was capable of inducing chemotaxis not only in THP‐1 cells but also in cells lacking endogenous uPAR (IC50, 5 pM). However, activity of suPAR require chymotrypsin cleavage between the N‐terminal domain D1 and D2 + D3. Chymotrypsin‐cleaved suPAR also induced activation of p56/p59hck in THP‐1 cells, with a time course comparable with ATF. Our data show that uPA‐induced signal transduction takes place via uPAR, involves activation of intracellular tyrosine kinase(s) and requires an as yet undefined adaptor capable of connecting the extracellular ligand binding uPAR to intracellular transducer(s).

A urokinase-sensitive region of the human urokinase receptor is responsible for its chemotactic activity.

The role of urokinase‐type plasminogen activator (uPA) and its receptor (uPAR/CD87) in cell migration and invasion is well substantiated. Recently, uPA has been shown to be essential in cell migration, since uPA−/− mice are greatly impaired in inflammatory cell recruitment. We have shown previously that the uPA‐induced chemotaxis requires interaction with and modification of uPAR/CD87, which is the true chemoattracting molecule acting through an unidentified cell surface component which mediates this cell surface chemokine activity. By expressing and testing several uPAR/CD87 variants, we have located and functionally characterized a potent uPAR/CD87 epitope that mimics the effects of the uPA–uPAR interaction. The chemotactic activity lies in the region linking domains 1 and 2, the only protease‐sensitive region of uPAR/CD87, efficiently cleaved by uPA at physiological concentrations. Synthetic peptides carrying this epitope promote chemotaxis and activate p56/p59hck tyrosine kinase. Both chemotaxis and kinase activation are pertussis toxin sensitive, involving a Gi/o protein in the pathway.

Urokinase-type plasminogen activator and its receptor: new targets for anti-metastatic therapy?

Urokinase-type plasminogen activator (uPA) and its receptor are instrumental in cell invasion and metastasis; their high levels of expression in human tumours correlates with a high risk of recurrence. uPA has a pleiotropic effect on cell migration and spreading in vivo and in vitro through the activation of plasminogen or other protein factors at the cell surface or in the extracellular matrix. Three specific inhibitors, with different tissue-specificities and regulatory properties, modulate cell-surface exposure of uPA activity. Overall, uPA is at the centre of a complex system affecting cell movement and invasiveness, and inhibition of uPA is now a goal of anti-metastatic therapy. The role of uPA and its inhibition are discussed in this review by Francesca Fazioli and Francesco Blasi.

Integrin-dependent induction of functional urokinase receptors in primary T lymphocytes.

In order to reach the sites of inflammation, lymphocytes leave the bloodstream and migrate into peripheral tissues, in a process involving integrin-mediated adhesion to the vascular endothelium, followed by transmigration across the endothelial barrier and through the underlying interstitial matrix. We have investigated the role of the plasminogen activator/plasmin system in normal T cell migration. Receptors for urokinase plasminogen activator (uPAR) were not expressed in resting T lymphocytes, but could be efficiently induced at the mRNA and protein level by coclustering of the antigen receptor complex and beta1 or beta2 integrins, through a signalling pathway involving both protein kinase C activation and an increase in intracellular cyclic AMP. Catalytic activation of plasminogen by uPAR-expressing T cells promoted their migration through an extracellular matrix in vitro. Plasmin-induced invasion was inhibited by plasmin-and urokinase inhibitors and by anti-uPAR antibodies. Finally, cytofluorimetric and immunohistochemical analysis of primary human tumor specimens showed the presence of uPAR positive infiltrating T cells in vivo. Collectively, these findings suggest that plasminogen activation may play a role in lymphocyte migration in vivo, and that integrin-dependent expression of membrane-associated endopeptidases could represent an additional step in the regulated process of leukocyte transmigration.

The urokinase receptor: Structure, regulation and inhibitor-mediated internalization

Abstract The receptor for urokinase plasminogen activator (uPAR) acts as an anchorage site for uPA on the cell surface where it stimulates pro-uPA activation, allows the internalization of uPA:inhibitor and other complexes and sends directly or indirectly signals into the cell that may promote migration, adhesion and growth. It is a GPI-anchored, three-domain protein that belongs to the Ly6 family and is present at the focal and cell-to-cell contacts, where it concentrates uPA activity. Its activity appears to be important to regulate the invasiveness of human cancer cells both in vitro and in vivo, and its inhibition is now a target for antimetastatic therapy.

Removal of domain D2 or D3 of the human urokinase receptor does not affect ligand affinity.

The main ligand‐binding determinant of the human urokinase receptor (uPAR) is located in the amino terminal domain D1, but when isolated this domain presents a 1500 fold lower affinity than the intact three‐domain uPAR (D1D2D3) [1]. uPAR mutants missing either domain 2 (D1HD3) or domain 3 (D1D2) were expressed in murine LB6 cells and showed to be properly GPI‐anchored to the cell surface. Binding assays with [125I]ATF demonstrated that these mutants possessed a normal (D1D2) or slightly reduced (D1HD3) affinity, indicating that a high ligand‐affinity may be achieved by a combination of D1 with domain D2 or D3.

Biosynthesis and apical localization of the urokinase receptor in polarized MDCK epithelial cells.

The biosynthesis and the surface localization of the urokinase plasminogen activator receptor (uPAR) were analysed in MDCK epithelial cells and in unpolarized fibroblasts. No differences were observed with respect to rate of synthesis, nature of precursors and time of surface appearance. uPAR was localized particularly at the focal and cell‐cell contacts when expressed in fibroblasts. On the contrary, in MDCK cells uPAR was found mostly on the apical surface; in agreement with its localization, down‐regulation of uPAR by the uPA‐PAI‐1 complex was observed only from the apical membrane.

Increased Urokinase-Type Plasminogen Activator Receptor and Epidermal Growth Factor Receptor in Serum of Patients With Prostate Cancer

PURPOSE Prostate cancer cell motility and invasion have been linked to the up-regulated signaling of epidermal growth factor receptor and urokinase-type plasminogen activator receptor. We analyzed the expression of serum urokinase-type plasminogen activator receptor and epidermal growth factor receptor in the serum of patients with clinical suspicion of prostate cancer to evaluate the possible role as prostate cancer markers. MATERIALS AND METHODS Serum was collected from 79 consecutive patients referred to our institution for transrectal ultrasound guided prostate biopsy. All blood samples were obtained before prostate biopsy. Total urokinase-type plasminogen activator receptor and epidermal growth factor receptor antigen in serum were measured by specific enzyme-linked immunosorbent assays. Gleason score, the number of positive cores, maximum percent of cancer and inflammation were considered on biopsy. Patients determined to have prostate adenocarcinoma underwent radical retropubic prostatectomy. Gleason score, pathological stage (extraprostatic extension), surgical margins, seminal vesicle involvement, perineural infiltration, lymphovascular invasion and cancer volume were evaluated in radical retropubic prostatectomy specimens. RESULTS The 30 patients with prostate cancer had significantly higher levels of serum urokinase-type plasminogen activator receptor and epidermal growth factor receptor in comparison to those without prostate cancer but not significantly higher levels of prostate specific antigen. Urokinase-type plasminogen activator receptor and epidermal growth factor receptor levels closely correlated in the serum of patients with prostate cancer. In a multivariate model high serum epidermal growth factor receptor increased the probability of positive biopsies by 1.9 times. ROC analysis revealed that serum epidermal growth factor receptor had 93.3% sensitivity and 98% specificity for detecting prostate cancer at a cutoff of 67.9 ng/ml. Urokinase-type plasminogen activator receptor and epidermal growth factor receptor were significantly higher in patients with extraprostatic extension, seminal vesicle involvement and perineural infiltration in the radical retropubic prostatectomy specimens. Serum urokinase-type plasminogen activator receptor was the only independent predictive serum marker of extraprostatic extension, seminal vesicle involvement and perineural infiltration. CONCLUSIONS The measurement of urokinase-type plasminogen activator receptor and epidermal growth factor receptor in the serum of patients with clinical suspicion of prostate cancer might provide clinically relevant information on the state of the prostate gland. Measuring serum epidermal growth factor receptor could help predict which patients have prostate cancer, while serum urokinase-type plasminogen activator receptor over expression seems to be related to tumor extraprostatic spread.