The urokinase receptor. A cell surface, regulated chemokine

The urokinase receptor. A cell surface, redated chemokine Review article

FRANCESCO BLASI

Dipartimento di Ricerca Biologica e Tecnologica (DIBIT), H.S. Raffaele, and Dipartimento di Genetica e Biologia dei Microrganismi, Universita di Milano, Milano, Italy

Blasi F. The urokinase receptor. A cell surface, regulated chemokine. APMIS 1999;107:96 101.

Mice deficient for the urokinase plasminogen activator (uPA) gene are deficient in the recruitment of T cells and macrophages and succumb to bacterial infections. High levels of uPA or of its receptor (uPAR, CD87) are produced in human cancers and are strong prognostic indicators of relapse. Thus uPA and uPAR have a profound influence on cell migration. This set of molecules is known to regulate surface proteolysis, cell adhesion and chemotaxis. We have investigated the mechanism involved in uPAR-dependent chemotaxis. Chemotaxis is induced through an uPA-dependent conformational change in uPAR which uncovers a very potent chemotactic epitope acting through a pertussis-toxin sensitive step and activating intracellular tyrosine kinases. The epitope is located in the linker region between domain D1 and D2 of uPAR. Binding of uPA transforms uPAR from a receptor for uPA into a pleiotropic ligand (“activated uPAR”) for other still unidentified surface molecules. Through these “adaptors”, uPAR causes cytoskeletal changes, activation of kinases and directional cell migration. The conformational change can be substituted by cleavage between domain DI and D2. in an area that can be cleaved by uPA itself at high efficiency.

Key words: Urokinase receptor; chemotaxis; cytoskeleton; tyrosine kinases; G proteins.

Francesco Blasi, Molecular Genetics Unit, DIBIT, H.S. Raffaele. via Olgettina 58, 201 32 Milan. Italy.

THE uPA SYSTEM

Cell recruitment and invasion are multi-step processes induced by multiple molecular signaling path- ways and requiring coordination between different types of cells ( 1 4 ) . Cytokines, growth factors and chemokines are involved as signaling mediators in these processes. Novel information has recently underlined the importance of another signaling system mediating and regulating cell recruitment and metastasis, the urokinase plasminogen activator (uPA) system. Two major functional domains make up the uPA molecule: the growth factor domain at the N-terminus, and the protease domain at the C- terminus. The protease moiety activates plasminogen and hence generates plasmin, a serine protease capable of digesting basement membrane and extracellular matrix proteins, an important mechanism by which uPA affects cell migration, since plasminogen- deficient mice are also deficient in wound healing ( 5 ) . However, plasmin formation is not the only import-

ant mechanism in uPA-dependent cell migration, since plasmin can also be formed via the other plasminogen activator, tPA. The growth factor domain has no protease activity but can bind a specific. high- affinity cell surface receptor, uPARKD87, a 270 residues GPI-membrane anchored molecule. This receptor is made up of three 80-residues domains. homologous to the Ly6ineurotoxins family. and two short linker regions. Chymotrypsin cleaves uPAR a t resi- due 87 between domains D1 and D2; the amino terminal domain DI is endowed with uPA binding activity but all three domains are necessary to achieve high affinity (6).

The inhibitors of uPA, PAI-I and PAI-2 (plasminogen activator inhibitor-type 1 and type-2) belong to the serpin family (7) and can specifically bind to and inhibit not only free, but also receptor-bound uPA. Active uPA is stably associated to its receptor uPAR at the plasma membrane but the binding of PAI-I changes uPAR properties, since the uPA-PAI- I complex also displays a binding site for the trans-meni-

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THE UROKINASE RECEPTOR

brane a2-macroglobulin receptoriLDL-receptor related protein (LRP). Through the combined actions of uPAR and LRF! the uPA-PAI-I complex is internalized and degraded while uPAR is recycled back to the cell surface (8-1 I ) . Through these properties, PAI-I controls not only cell surface proteolytic activity but also the physical location of uPAR onto the plasma membrane.

The importance of the uPA system in cell migration in vivo is underlined by a series of recent findings. Homologous recombinant mice lacking uPA are extremely deficient in T-cells and macrophage recruitment and succumb to bacterial (C. neofbrmans) infection (12). In addition, they are also deficient in supporting the growth and malignant development of chemically induced melanomas ( 13). Further, neutrophils from patients suffering of paroxysmal nocturnal hemoglobinuria (pnh), a disease in which GPI-anchored proteins (such as uPAR). are impaired in their trans-endothelial migration in vitro (14). Finally. high expression of uPA. uPAR and PAI-1 in human tumors is an important negative prognostic marker (1416).

Functionally, the uPA system displays regulated extracellular proteolysis, activates chemotaxis, regulates cell adhesion and causes profound changes in the cytoskeletal structure. In this respect, it has the properties of a cell-surface, regulated chemokine (17).

PROTEOLYTIC STIMULATION O F CELL MIGRATION

Cancer cells require a well regulated pericellular proteolysis to migrate: they must cleave linkages to the extracellular matrix and to other cells and degrade barriers like the basement membrane, the destruction of which is a common observation in invasive cancer. Regulated proteolysis can occur through different proteases. but it has been long recognized an important role for the plasminogen activation system ( I ) . Among plasminogen activators. uPA is clearly promi- nent in cancer mostly because the presence of a specific receptor. uPAR. provides a mechanism to localize the enzymatic activity on the cell surface.

Another important mechanism in uPA-stimulation of cell migration, is its ability to directly or indirectly activate motogenic factors. Indeed, uPA can activate pro-HGE while plasmin can d o the same for basic F G F and TGFP (18-20). A direct correlation between proteolytic activity and migration has been established for TGFP and bFGF (19-20). However, uPA derivatives deprived of proteolytic activity can also stimulate bovine endothelial cell migration, sug- gesting that uPAR occupancy on its own may have an independent stimulating activity (21-23) and indeed

migrating endothelial cells up-regulate the expression of uPAR (24).

uPAR A N D CELL ADHESION

Adhesion of differentiating monocytes or lympho- kine-treated HL60 and U937 monocyte-like cells can be inhibited by an anti-uPA antibody which essenti- ally prevents autocrine binding of biosynthetic uPA to uPAR (25). Lack of adhesion can be reversed by addition of exogenous uPA or of receptor-binding derivatives of it (like the ATE amino terminal fragment) and hence does not require proteolytic activity. ATF influences not only cell adhesion, but also changes the expression of genes like cathepsin B and the 92 K metalloprotease (26, 27).

It was first noticed that adhesion of myeloid cells to vitronectin (VN) could be linked to uPAR occupancy, and subsequently that uPAR could itself bind VN and mediate an RGD-independent cell adhesion process (28-32). Binding of VN to uPAR induces specific uPAR-dependent cell adhesion and spreading on VN (29). Binding of vitronectin to uPAR also causes inhibition of adhesion to different substrates like fibronectin or fibrinogen, and inhibits fibrinogen internalization and degradation via CR3iMac I integrins (the complement receptor type 3, i.e. CDl lb/ CD18) (31, 33). Again, this suggests that uPAR inter- venes in the competition for adhesion substrates. and in the choice between adhesion to and internalization-degradation of fibrinogen. In addition to being an adhesion receptor. Mac1 also mediates binding, internalization and degradation of fibrinogen, a major non-plasmin pathway for fibrin clearance by monocytoid cells (34). These effects appear to be mediated by a direct interaction of uPAR with the integrin receptors: co-immunoprecipitation of uPAR and Pl integrins has been observed upon solubilization of cytoskeleton-engaged integrins (30). Co-immunoprecipitation of uPAR and integrins had already been observed in lymphocytes (35). The direct interaction between uPAR and integrins is also shown by the iso- lation of a uPAR-binding peptide that does not inter- fere with uPA nor with VN binding, but interferes with cell adhesion and uPAR-integrins co-immunoprecipitation (30).

Interaction between uPAR and integrins has been observed also in neutrophils: the leukocyte integrin CDI lbiCD18, aMP2, (CR3iMacl) has been shown to physically associate with uPAR (36). This interaction is reversible and correlates with cell shape. In resting cells, uPAR and Mac1 co-localize, but follow- ing spontaneous cell polarization and migration. the two receptors dissociate with Mac1 concentrating in the uropodia and uPAR in the lamellipodia of polar- ized cells (36). Since Mac1 regulates cell adhesion,

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chemotaxis and cell migration at inflammatory sites (37), the interaction with uPAR may possibly mediate the migration-promoting activity of uPA/uPAR.

ACTIVATED uPAR AND CHEMOTAXIS

A chemotactic and chemokinetic property of uPA has been reported on a variety of cell types (19-24, 3842). This activity is exerted through its specific high affinity cell surface receptor (uPAR) (41,42) present in activated blood leukocytes, endothelial cells, macrophages, fibroblasts and in cancer cells. The receptor anchors uPA at the leading edge of cell migration and localizes it at the focal contacts and at cell to cell contact sites (4345). These sites also con- tain adhesion molecules, integrins, cadherins, and signal transducing substrates. Clustering and polarization of uPAR is regulated by receptor occupancy (42, 45, 46).

UPMUPAR appear to be involved also in chemotaxis induced by other attractant molecules, like FMLP. Indeed, exposure of human neutrophils to a chemotactic gradient of FMLP localizes uPAR to the leading edge of the migrating cell; anti-uPAR antibodies, or the expression of uPAR antisense, ablate the chemotactic activity of the peptide (41).

A direct chemotactic effect of uPA has been observed in several types of cells, bovine adrenal capillary endothelial cells, keratinocytes (2 l), monocyte- like cells, fibroblasts (42), and neutrophils (38, 39). Chemotaxis does not require the proteolytic moiety of uPA, as it can also be obtained with enzymatically inactive derivatives. The requirement for uPAR was thoroughly ascertained in the case of monocyte-like THP-1 and other cells since specific uPAR mono- clonal antibodies blocked the effect, and uPA was inactive on cells lacking uPAR; the effect was recovered upon uPAR cDNA transfection (42).

In THP-1 cells, ATF-induced chemotaxis is ac- companied by a time-dependent, transient, activation of a tyrosine kinase of the src family, p56/p58 hck, and is inhibited by tyrosine-kinase inhibitors. In addition, uPAR itself associates with ~ 5 6 1 ~ 5 8 hck, through an unidentified transmembrane molecule. The existence of a transmembrane adaptor was sug- gested to explain how could extracellular uPAR contact intracellular p56/p58 hck tyrosine kinase. Indeed, in cells lacking uPAR, and therefore not responding to ATF, a potent chemotactic signal is obtained with a soluble form of uPAR cleaved in two fragments between domain 1 and 2 (42). This effect also occurs in fibroblasts derived from uPAR-/- mice but not from Src-/- mice (Degryse, Blasi and Fazioli, unpub- lished). The current interpretation is that uPA binding causes a conformational change in uPAR similar to that obtained by chymotrypsin cleavage, trans-

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forming it from a receptor for uPA into a ligand for an unidentified transmembrane molecule which in turn mediates signal transduction (42). An important step in determining the nature of the mediator(s) of uPAR-dependent chemotaxis is the identification of the minimal amino acids sequence responsible for the chemotactic activity of uPAR. It has recently been shown that this sequence resides in the region con- necting domains D1 and D2. Indeed, a fragment of uPAR (“activated uPAR”) comprising the D2 plus D3 domains, and having an amino terminal SRSRY sequence, as well as linear peptides covering this region, have a very potent chemotactic activity, in the 0.1 pM range and display all other properties observed with chymotrypsin-cleaved uPAR (2).

Similar results are obtained with rat smooth muscle cells, where a gradient of pro-uPA induces uPAR-dependent chemotaxis. In the process, reor- ganization of the actin filaments, loss of stress fibers and change in the position of the focal contacts can be observed. All these effects are inhibited by pertussis toxin (Degryse, Fazioli, Rabbani and Blasi, un- published data). This result suggests the involvement of G-protein-coupled receptors, like the chemokine receptors, in uPAR-dependent chemotaxis. Also in this system, activated uPAR can bypass uPA/uPAR interaction and cause pertussis-toxin sensitive chemotaxis. The same is true for synthetic, short chemotactic uPAR peptides. Whether the unidentified adaptor is represented by a chemokine receptor, or whether the involvement of the chemokine receptor represents a downstream step, remains to be deter- mined.

The chemotactic epitope of uPAR is located in the linker region between domain D1 and D2 (2). Cell surface uPAR can be efficiently cleaved by physio- logical concentrations of uPA (47); the cleavage pro- duces the release of a soluble D1 domain leaving a cell-surface attached D2D3 fragment, potentially chemotactically-active. A secondary cleavage site for uPA also exists in the linker region: the occurrence of both cleavages would produce a chemotactically active uPAR peptide. The question of whether uPA-dependent cleavage(s) occurs physiologically has not yet been addressed: however, other proteases can cleave the linker region of uPAR and produce similar prod- ucts.

CONNECTION BETWEEN uPAR, ADHESION AND CHEMOTAXIS

The dual effect of uPAR on adhesion and chemotaxis may seem intriguing. However, also chemokines display a dual effect by stimulating migration at low concentrations, and adhesion onto specific extracellular matrix substrates at higher concentrations.


At the latter concentrations, migration is inhibited. Thus the same molecule is capable of activating migration through a specific receptor (in this case a seven trans-membrane chemokine receptor) and inhibiting through an interaction with integrins(48). When analysing the concentration-dependence of uPAR chemotactic action, a similar result has been observed: indeed, activated uPAR stimulates migration below 100 pM, while inhibiting it at higher concentrations (42). Since uPAR indeed interacts with integrins (30, 33 , this is the mechanism that may possibly be involved in the adhesion step. Whether or not the chemotactic activity of uPAR also involves a seven trans-membrane receptor, has not yet been established.

THE uPAR uPA PAI-1 CROSSROAD

The direct adhesive and chemotactic effects of the uPA system require a fine regulation in order to stimulate cell migration. The uPA inhibitor PAI-1 can regulate not only proteolysis, but also adhesion and chemotaxis. In addition to direct effects on adhesion and migration (49, 50), PAI-1 can internalize uPAR-bound uPA. In the process, uPAR is first internalized and subsequently recycled back to the cell surface (8, 10, 1 I ) . Through this recycling, contiguity (and hence interaction) of uPAR with different molecules (plasminogen, integrins, transmembrane chemotactic adaptors) can be changed, thus also changing the type of signal that it can send.

uPA/uPAR AND CANCER

That uPA/uPAR play a very important role in determining the malignancy of most human tumors is no longer controversial and is based on a large number of experimental studies in both model and human cancer. First, high levels of uPA, uPAR and PAI-1 are important negative prognostic criteria in many cancers. Second, in human or murine model systems, expression or administration of uPAR antagonists has a drastic effect on the metastatic ability of cancer cells (51), as well as on their growth and vasculariz- ation (52) . Third, inhibition of uPA activity or of uPAR expression by antisense RNA also drastically reduces tumor invasiveness (53-55).

Analysis of human tumors to identify the cells which are producing uPA, uPAR and PAI-1, produced the surprising results that uPA and uPAR are not necessarily produced by tumor cells and that the picture may differ in different tumors (reviewed in 56). In all cases, however, UPMUPAR expression is highest in, or even confined to, the leading edge of the tumor.

The fact that UPMUPAR may be produced by different types of cells in different types of cancer while being of severe prognostic significance in all cases, indicates that cancer may exploit the UPMUPAR system through different mechanisms, all inducing migration and invasiveness of cancer cells. Indeed, expression of uPAR in cancer cells suggests a mechanism whereby uPA stimulates migration by concentrating the proteolytic activity to the surface of the invading cell, or by inducing cancer cells chemotaxis. On the other hand, production of uPAR by stromal cells suggests a mechanism whereby uPAR can act at a distance, through either cell-cell contacts or by releasing a soluble chemotactic molecule. The chemoattractant activity of the linker region between domain D1 and D2, released by uPA or other proteases, might stimulate migration of neighbouring cancer cells.

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The urokinase receptor. A cell surface, regulated chemokine

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