urokinase and urokinase receptor: a paracrine/autocrine system regulating cell migration and...

Summary Urokinase and its receptor are essential components of the cell migration machinery, providing an inducible, transient and localized cell surface proteolytic activity. This activity has been shown to be required in normal and pathological forms of cellular invasiveness (i.e. in several embryonic developmental processes, during inflammatory responses and cancer metastasis and spreading). It represents one of the best known of the proteolytic systems which are currently under investigation in this field. The urokinase receptor allows a continuous regulatinn of the proteolytic activity at cell contacts, utilizing the different localization of urokinase and its inhibitors. The receptor, in fact, in addition to focusing the enzymatic activity at focal and cell-cell contacts, also regulates it by internalizing and degrading only the inhibited form of urokinase. Internalized receptor releases the ligands to the lysosomes and recycles back to surface. In this way, the proteolytically active areas of the cell surface can be continuously monitored for their activity and their location modified. The cell can thus coordinate its migration efforts with a step-wise modifi- cation of the proteolytic activity-map of the cell surface. The urokinase cycle can be supported by one individual cell (autocrine) or by two or more cells. In the latter case, complementation and synergism of urokinase and its receptor are found.

Introduction Cell-cell and cell-environment (extracellular matrix and basement mcmbrdnesj interfaces represent physical barriers to migration and need to be continuously disrupted and re- established by migrating cells. Migration and invasion are properties of many cells; however, these processes occur only at defined limes andor in response to particular stimuli. Invasion is thus an inducible property subjected to a strict cellular and molecular control. In order to invade, the cells must thereforc be equipped with an inducible system that allows them to overcome intercellular barriers.

The important role of adhesion in contact reconstitution, the basis of organ selectivity and homing, has recently becn

discussed(') and will not be dealt with here. The emphasis will be on localized proteolysis, which is an important mechanism regulating cellular interactions, given that proteins constitulc the major element of specificity in all cell contacts. A requirerncnt for proteolysis unifies phenomena as different as oocyte maturation, cmbryo implantation, embryonic cell migration, angiogenesis, myogcnic differentiation, cancer metastasis, and more.

Plasma contains proteins that participate in controlled proteolysis both intra- and extra-vascularly (fibrinogen, fibronectin, vitronectin, von Willebrandt factor, alpha-2- antiplasmin, plasminogen activators, plasminogen activator inhibitors etc. j. Interestingly, these proteins have been found to be components of the extracellular matrix or of the basement membrane, and to have a functional role in cell adhesion and migration(2-5). These matrix componcnts are them- selves regulators or substrates of the extracellular proteolytic processes.

Plasminogen activation is onc significant inducible extracellular proteol ytic system involved in the regulation of cellular interactions. Other proteolytic systems are probably as important, like the metalloproteinases, but, for these, the reader will have to consult other reviews(@. Plasminogen activators convert the inactive plasminogen into the trypsin-like serine protease plasmin. This broad spectrum protease can degrade circulating and tissue proteins as well as activate zymogens or growth-factor precursors. Two plasminogen activators exist, urokindse-(uPA)t and tissue-type (tPA)(').

uPA has an essential role in cell migration and invasiveness, as demonstrated by a multitude of studies in several different systems. Tumor invasiveness and metastasis can be blocked or stimulated in several model systems by a directed increase or decrease of uPA activity (7-13). The invasion of the basement membranes by tumor cells and human monocytes is inhibited by the uPA inhibitor PAL-I or by blocking surface binding of uPA(I4-l9). The degradation of extracellular matrix proteins by tumor cells is blocked by inhibitors of uPA and plasmin(20.21). The in vitro migration of keratinocytes, endothelial and myoblastic cells is also inhibited by uPA inhibitor^(^^-^^). Finally, the fusion of myoblasts into myotubes is modulated by such inhibitors and by uPA antibodies(27). In this review, I will concentrate on the structural and biochemical features of the uPA system, focussing on those aspects with potential regulatory properties.

The Cell-Surface Plasmin-Forming Pathway The activation of plasminogen by uPA mostly occurs at the

?-Abbreviations: uPA: urokinase-type plasminogen activator; tPA: tissue-type plasminogen activator; pro-uPA: the proenzyme pre- cursor of uPA (needs cleavage at residue 158 to become activated into uPA); uPAR: receptor Tor uPA; PAI-1: plasminogen activator inhibitor type 1; PAI-2: plasminogen activator inhibitor type 2; uPA:PAI: a covalent complex between uPA and PAl-1 or PAL2 which inactivates uPA; EGF: epidermal growth factor; TGFp: transforming growth factor beta; PI-PLC: phosphatidylinositol specific phospholipase C.

cell surface. uPA is synthesizcd as an inactive proenzyme (pro-uPA) and after secretion is bound to specific cellular receptors by both autocrine and paracrine mechanisms (sce below). The initiating cvent in the conversion of pro-uPA to active uPA in vivo is still not known and may not always involve proteolysis. In vitro, plasmin can activate pro-uPA both in solution(2x,29) and on the cell s ~ r f a c e ( ~ ~ j . An arginine- specific protease distinct froni plasmin and resistant to trasy- 101, detected in the membrane fraction of RSV-transformed chicken embryo fibroblasts. is able to convert receptor- bound pro-uPA to uPA(~') . The membrane attachment of pro-uPA has major regulatory importance since the rate of activation of receptor-bound pro-uPA by plasmin is at least twenty-fold higher than in solution(32). In in vitru cultured cells, production of active uPA has been shown to occur almost exclusively at the cell s u r f a ~ e ( ~ ~ , ~ ~ ) . Moreover, receptor binding has been shown to stimulate the activity of a mutant pro-uPA that cannot be cleaved at lys158, suggesting that receptor binding can cause a change in conformation of pro-uPA which confers enzymatic activity(34). Possibly, receptor bindmg of pro-uPA provides the initial step in plasminogen activation in the absence of proteases. Subse- quently, proteolytic activation of pro-uPA by plasmin or other proteases would sustain plasminogen activation.

Plasminogen binds to spccific cell surface rcccptor~(~~) . This interaction requires the same lysine binding sites recog- nized by plasmatic plasmin inhibitors Binding to the cell surface, therefore, renders plasmin res stant to a2-antiplasmin(33,35). And in fact plasminogen activation occurs at the cell surface in a reaction in which uPA and plasminogen are

surface bound(33). The ccll surface provides therefore a suit- able local environment for activating plasminogen, by both stimulating plasminogen activator activity and preventing plasmin inhibition. In in vitm cultured cells. in fact, active plasmin generation does not occur in the fluid phase because of the presence of the inhibitors. In cells that produce either pro-uPA or uPA receptor, co-cultivation of the two cell types is required in order to obtain activation of pro-uPA and effi- cient plasmin activityQ1).

Cells possess specific receptors for uPA (uPAR) that recognise pro-uPA and uPA at the EGF-likc amino tcrminal domain (amino acid residues 20-32)(36-3si. The interaction of uPA with its receptor is characterizcd by high affinity, with a Kd of about 0.2 TIM(".^'). The uPA receptor is a heavily gly- cosylated protein of about 55-65 kIS3') composed of 3 13 amino acid residues(40). Thc carboxy terminal hydrophobic domain of uPAR is processed during biosynthesis and substi- tuted by a glycolipid anchor (GPI) which allows its attachment to the membrane(41). The protein is organized in three domains of ca. 90 amino acids each, and the binding determi- nants are contained in the aniino terminal This property provides a simple way to analyse for surface expression of uPAR since phosphatidyl-inositol specific phospholipase C (PI-PLC) can solubilizc uPAR.

The uPA receptor is expressed (10.000 to 250,000 per cell) by circulating whitc blood cclls (monocyles, B lymphocytcs and granulocytes) and by many cultured cell lines (Fig. 1) but its distribution in the tissues is not yet The intcrac- tion of uPA with its receptor appears to be tightly regulated. Phorbol esters, TGFp and EGF increase the synthesis of the

Fig. 1. Immunofluoreicence analysis of the uPA receptor on in v i m cultured human myohlasts for 10 days. using anti-uPAR moiioclonal antibody R4. (Courtesy of J. Pel- lainen and G. Barlovatz Mei- inon).

receptor by activating gene transcription(44). Increase in receptor number is accompanied by a decrease in its affinity but the structural basis for this effect is not k n o ~ n ( ~ ~ 9 ~ 6 ) .

The other components of the uPA system are the fast-act- ing high affinity plasminogen activator inhibitors, PAL 1, PAI-2 and protease nexin 1, all members of the serpins (inhibitors of serine proteases) protein Binding of these inhibitors to active uPA results in the formation of an SDS-stable, inactive covalent complex. PAI-1 is an extracellular matrix protein found in a complex with vitronectin, a molecule involved in cell adhesion(3. The other inhibitor, PAI-2, does not appear to be an extracellular matrix pro- t ~ i n ( ~ ~ ) .

The uPA system is thus composed of one enzyme (uPA), one substrate (plasminogen), two receptors (uPAR and the less characterized plasminogen receptor), and three high affinity inhibitors. Plasminogen receptors are characterized by low affinity and high capacily, while uPAR has high affinity and low capacityi4”). However, surface plasmin is resistant to a 2 - ‘ ~ t i p I a ~ m i n ( ~ ~ , ~ ~ ) while receptor bound uPA is sensitive to PAT-1 and PAI-2(48749). These properties suggest that plasminogen needs to be readily available on the cell surface for activation, which however occurs only at specific positions, namely sites occupied by uPAR. The different lig- and affinities of the uPA and plasminogen receptors, in fact, suggests that surface plasmin may be easily dissociated, while active uPA is known to have a longer half-life on the

Dissociation of plasmin from the cell surface results in inhibition of its activity; uPA activity, on the other hand, can be inhibited while still on the surface (see below). The plasmin activity-map of the cell surface, therefore, might be essentially controlled by the location of the uPA receptors.

uPA has been previously localized at the cell-to-cell and cell-to-substratum (focal) contact sites(50). It has now been shown that uPAR is also present at these same sites (Vaheri, personal communication; Pollanen and Blasi: see Figure 1). Additionally, plasminogen has been shown to bind to components of the extracellular matrix‘51). The focal contact sites, i.e. the very sites where plasmin formation is required for cell migration, are therefore equipped with all the neces- sary components allowing a very precise localization of Lhc plasmin enzymatic activity. Thus uPA activity may regulate cellular adhesion at the focal contacts. This hypothesis is supported by the stabilization of vitroiiectin-dependent adhesion of human HT 1080 cells by the inhibitor of uPA, PAI- 1(52),

The uPA Cycle Cells are capable of supporting a full functional cycle of uPA (Fig. 2). The cycle can be completed by a single in vitro cultured cell synthesizing all components of the uPA system (except plasminogen provided with the serum). However, complementation between different cells may be the rule under in vit~o conditions (see below). The product of the LIPA gene is the secreted 5inglc chain pro-uPA that can bind to the uPA receptor(i0). When secreted in large amounts, pro-uPA can totally saturate the receptor in an autocrine fashion(s3). As discussed above, receptor binding increaser cellular

I 1 pro-uPA 1 Surface

\ 4 ~ t

Fig. 2. A scheme of the cellular cycle of uPA. The suffixes i and s indicate the intracellular or surface localization. respectively. R indicates UPAR.

activity of uPA and allows a strong, focalized and transient surface proteolytic activity. The inhibition of receptor-bound uPA by PAls and the dissociation of plasmin, which in soluble form is sensitive to the inhibitor, regulate this activity. In fact, covalent uPA:PAI-I complexes are formed with cell surface-hound uPA, resulting in inhibition of surface uPA activity(49) with kinetic constants comparable to those obtained in solution(54).

Data accumulated in several laboratories showed that receptor bound active uPA is not internalized nor degraded.

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Time in hours Fig. 3. Synergistic action of uPA and uPAR in the degradation of extracellular matrix. Mouse LB6 cells have been engineered to product human pro-uPA or human uPAR. A total of 10’ cells has been plated onto tritiated extracellular matrix. 5 x lo3 pro-uPA producing cells have been co-cultivated with 9.5 x lo4 uPAR-expressing cells in thc presence (full circles) or absence (full triangles) of the uPA antagonist ATF. The diamonds represent the degrading activity of 5 x lo3 pro-uPA producing cells co-cultivated with 9.5 x lo4 un- transfected control LBh cells. Reproduced from ref. 22. Cells were plated on tritium-labeled extracellular matrices from rat muscle cells, and matrix degradation measured by the assay of soluble radioactivity at various times.

Fig. 4. In ~ i t u hybridization of adjacent sections of a colon cancer speciinen with labeled antisense RNA probcs for uPA (a. a l ) or uPAR (b, bl ). Both signals are located at the contour$ of malignant epithelium but uPA mRNA signal is in fibroblast-like stromal cells (a, arrows) and uPAR mRNA in malignant cells (b, arrows). Control sense-RNA gave no signal (not shown). a I and b I are the dark field images of a and b. Reproduced from ref. 6 I .

However, uPA complexed with PAI- 1 is efficiently internalized and degraded(s5) and this process is mediated by the uPA receptor(56). uPAR, therefore, can exist in two conformations which are distinguished by their ability to internalize the lig- and. Interconversion is dependent on PAI- lw Once internalized, the uPA:PAI complex is degraded in the lyso- some(55,s6) but the internalized receptor is recycled to the cell surface (Conese, Olson, Pedersen and Blasi, submitted for publication; see Figure 6). Recently, the possibility has been raised that a trans-membrane protein, the a2-macroglobulin receptorLDL-receptor related protein (LRP), mediates the internalization of the UPAR:LIPA:PAI complexes(57).

The different fate of uPA:PAI-1 and uPAR after internalization can provide cells with a mechanism for continously changing the proteolytically active surface areas by re-posi- tioning their uPA receptors. This would be dependent on the local concentration of, for example, PAI-1 which is present in the extracellular matrix. Thu\ cells are able to carry out a complete cycle of uPA, from synthesis, secretion and autocrine receptor binding, through surface activation and inhibition to internalization and lysosomal degradation. However, the same cycle can also occur by a paracrine mechanism requiring two different cells, one producing uPA, the other the receptor (see below).

Internalization and degradation of the uPA:PAI- 1 complex may be regulated by phosphorylation. It has recently been found that pro-uPA secreted by several cells is phosphorylated in serine. Surprisingly, while the kinetic parame- ters and the specific activity of the phosphorylated enzyme were found to be similar to that of un-phosphorylated uPA, its PAI-1 sensitivity was found to be decreased several fold(58). The presence of phosphorylated, PAI- 1 -resistant uPA may result in a local increase in extracellular proteoly-tic activity and in a decrease of uPA internalization. The biochemistry and the physiological significance of pro-uPA phosphorylation await elucidation.

Complementation of uPA and Receptor in vitro and in vivo: Invasion as a Multicellular Phenomenon An essential role of the uPA system in invasiveness, suggested long ago by Ossowslu and R e i ~ h @ , ~ ~ , , has been demonstrated by a variety of approaches. In order to under- stand the role of uPAR in invasion, it would be desirable to engineer cells to produce single components of the uPA pathway to allow an analysis of their role by genetic complementation. Mouse LU6 cells produce no plasminogen activators

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surface were produced by genetic engineering methods. These cells are found to complement each other with respect to receptor saturation and surface enzymatic activity; most interestingly, they are also observed to cooperate in two assays that reproduce some of the steps in tumor invasiveness. Co-cultivation of LB6 cells producing either human uPA or uPAR leads to a synergistic amplification of the degradation of the extracellular matrix by the tumor cells(*l); an example is reproduced in Figure 3. Tn a second system measuring invasion into the chorion allantoic membrane of the chicken embryo, co-cultivation of uPA-producing cells strongly enhanced the invasive ability of uPAR-producing cells(60). This synergism correlates with the enhanced activation of single-chain pro-uPA in the presence of uPAR- expressing cells(21). This type of analysis can now be extended to other components of the uPA pathway (PAI-1 and PAI-2) and is now being tested in other more representa- tive systems of invasion and metastasis.

This complementation study in v i m gives relevance to recent findings by in situ hybridization that identify those cells that synthesize uPA and uPAR in human tumors. In human colon adenocarcinomas, uPAR is produced largely by the invading tumor cells, while uPA is made by some adjacent fibroblast-like stromal cells(61); an example is shown in Figure 4. A gradient of uPA and uPAR mRNA is observed in the tumor with the highest levels localized at the leading edge of the tumor. In addition to uPA, stromal cells of human tumors may contribute to the invasive process through other proteases, like collagenase (Pyke, Ralfkjaer, Tryggvason and Dan& submitted for publication). These and the uPA/uPAR data suggest a cross-feeding between the tumor and stromal cells, with tumor cells (uPAK-positive) inducing uPA and collagenase synthesis from neighbouring cells and thus actively utilizing stromal products for invasion. The invasive process in tumors, therefore, appears to be, at least in some cases, the result of multicellular cooperation orches-

Fig. 5. Schematic drawing of how saturation of the urokinase receptors of a tumor cell (C) can be obtained by a paracrine (A, B) or autocrine (Dj mcchanism.

and their receptor cannot bind human uPA because of species-specificity of binding(38). Cell clones that either secrete human uPA or express human uPA receptor on their

Cleavage Detachment lnhi bition Recycling

Fig. 6. Schematic drawing of the PAI-1 induced continuous change of surface proteolytic activity and of its connection to the contact sites and cell movement. uPAR, the large grey ball, can bind uPA (the curvcd arrow) leading to the degradation of some adhesive junction. This cleavage allows, for example, a rotation of thc cell (follow the change of the black dot position marker) with formation of new junctions. PAL1 (dark grey, C-shaped) can bind and inactivate receptor-bound uPA leading to internalization and degradation of uPA. In the process uPAR recycles and relocates close to a different adhesive junction. The binding of uPA to this uPAR starts the process again.

trated by factors (inducers) produced by tumor cells (Pyke et al., op. cit.). Such a paracrine mechanism, in principle, resembles that which occurs during embryogenesis where gradients of certain molecules can cause vectorial cell migra-

However, the results of colon carcinomas may not be generalized to all types of cancers. And in fact many carcinoma cell lines produce both uPA and Thus the UPNUPAR system in human tumors may operate by both autocrine and paracrine mechanisms (Fig. 5).

If the role of the uPA/uPAR system is often critical in tumor invasion, one might expect invasive tumors to have selected mutations in the uPA or uPAR gene. If such mutations are sought and not found, however, their absence might suggest that alternative mechanisms are available to the cells for invasion. Nevertheless, cell surface extracellular proteolysis by the uPAIuPAR system needs such a finely tuned regulation involving multiple proteins (i.e. PAI-1, focal contact proteins, etc), to make it unlikely that a mutation in a single gene would make a cell more invasive. Additional mutations in other genes may be required to allow proper regulation of extracellular proteolysis.

The Cell Surface as a Dynamic Proteolytic Structure Little doubt exists as to the role of the uPA system in cell migration, cancer spreading and metastasis. The information obtained on the biochemical mechanisms may have practical significance and is being investigated in many types of cancer for diagnostic and therapeutic goals. One major advance has been the understanding of how cells can regulate the location of cell surface proteolytic activity, and therefore of cell migration. The data suggest that after cleavage of one or more proteins at a specific contact site, PAI-1-induced inhibition of uPA, followed by internalization and degradation of the uPA:PAI-1 complex, favors formation of new contacts at that very site which meanwhile may have migrated. Recy- cling of uPAR to the cell surface, on the other hand, exposes a new area to which uPA can bind and activate proteolysis, and so on (Fig. 6). This suggests that PAI-1 may cause a con- stant change of uPAR position, and therefore of the proteolytic activity-map, on the cell surface. The recycling receptor can reappear at a different contact site, bind uPA and therefore activate proteolysis at a site different from the pre- vious. The cell surface can thus be regarded as a composite of proteolytically active and adhesion promoting areas of con- tinuosly changing map. The coupling of the change of surface proteolytic map to the physical movement of the cell, is at the moment totally obscure.

In this view, both the proteolytic enzyme and its inhibitor are required for cell migration, adhesion and invasion. This probably explains why high levels of both uPA and PAI-I are prognostic indicators of high probability of tumor spreading and metastasis as assessed through the retrospective study of hundreds of patients with mammary carcinoinasi6’); Gr@n- dahl-Hansen et al. (submitted). The temporal changes of surface plasmin activity and location allowing both the cleavage and the subsequent reformation of cellular contacts during cell movement, require not only uPA but also PAI- 1. The

final outcome and the rate with which it is attained will depend on the relative concentration of the components, and on their spatial and temporal localization.

Acknowledgements I am very grateful to all my collaborators for their enthusi- asm, their criticisms and the accuracy and thoroughness of their work. In particular thanks are due to Jari Polliinen, Mas- simo Conese, Roberta Benfante and Anna Brandazza for the many fruitful discussions. The long-standing collaboration and the many stimulating discussions with Keld Dan@ are gratefully acknowledged. The work from my laboratory has been supported by the Danish Cancer Society, the Danish Biotechnology Program and the European Community “Sci- ence” Program.

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Francesco Blasi is at the Dipartimento di Genetica e Biologia dei Microrgaiiismi, University of Milano, via Celoria 26,20133 Milano, Italy.

urokinase and urokinase receptor: a paracrine/autocrine system regulating cell migration and...

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