Regulation of Plasminogen Receptor Expression on Monocytoid Cells by β1-Integrin-dependent Cellular Adherence to Extracellular Matrix Proteins

Plasminogen binding sites function to arm cell surfaces with the proteolytic activity of plasmin, critical for degradation of extracellular matrices. We have assessed the effects of adhesion of the representative monocytoid cell lines, THP-1 and U937, to purified extracellular matrix proteins on their expression of plasminogen receptors. After adhesion to immobilized fibronectin, adherent and nonadherent subpopulations of cells were separated. Plasminogen binding to the nonadherent population of cells increased 3-fold, whereas binding to the adherent population decreased by 60%. These changes were due to differences in the plasminogen binding capacities of the cells, while the affinities of the cells for plasminogen were unchanged. The up-regulation of receptor expression in the nonadherent cell population was: 1) induced rapidly and reversibly, 2) independent of new protein synthesis, 3) required an interaction between adherent and nonadherent cell populations, and 4) associated with an enhanced ability of the cells to promote plasminogen activation and to degrade fibronectin. Other immobilized adhesive proteins, laminin and vitronectin, also supported up-regulation of plasminogen receptors in the nonadherent cells. Carboxypeptidase B treatment eliminated the increment in the plasminogen binding capacity of the nonadherent cells, suggesting that the increase in binding was due to exposure of new carboxyl-terminal lysyl residues on the cell surfaces. Furthermore, both the adherence of the cells and up-regulation of plasminogen binding sites was abolished by beta1-integrin monoclonal antibodies. These results suggest that proteins found in extracellular matrices have the capacity to modulate the expression of plasminogen binding sites, thus regulating local proteolysis and cell migration.

The expression of cell surface binding sites for plasminogen is widespread among cells of both prokaryotic (reviewed in Refs. 1 and 2) and eukaryotic organisms (reviewed in Ref. 3). Among eukaryotes, plasminogen receptors are expressed on cells within the vasculature including platelets (4), monocytes (5), lymphocytes (5), and endothelial cells (6 -8), as well as on a variety of cells found within solid tissues including hepatocytes (9), fibroblasts (10), epidermal cells (11), and keratinocytes (12). By enhancing plasminogen activation (4,6) and protecting cell-bound plasmin from inactivation by ␣ 2 -antiplasmin (10,13,14), plasminogen receptors function to arm cell surfaces with the broad spectrum proteolytic activity of plasmin. Such cell surface proteolytic activity facilitates processes involving cell migration through extracellular matrices.
The ability to modulate plasminogen binding capacity provides a mechanism for regulation of cell surface proteolytic activity. Expression of plasminogen receptors on U937 monocytoid cells is up-regulated by interferon-␥ and vitamin D 3 (15,16). Down-regulation of plasminogen receptors is elicited by glucocorticoid treatment of HT-1080 fibrosarcoma cells (17) or thrombin treatment of endothelial cells (7). In the presence of differentiation inducing agents (phorbol 12-myristate 13-acetate and vitamin D 3 ), monocytoid cells (THP-1 and U937) change their adhesive properties and up-regulate plasminogen binding site expression (18). The importance of cell adhesion in modulating the cell-surface association of components of the plasminogen system has been emphasized by studies showing that adhesion modulates the expression and distribution of urokinase and its receptor (19) and the identification of the matrix-associated protein, vitronectin, as a ligand for the urokinase receptor (20).
It has now been established in many reports that cells respond to the presence and composition of the extracellular matrix by adhering, spreading, migrating, differentiating, and altering gene transcription and cellular phenotype (reviewed in Ref. 21). These matrix-dependent effects are exemplified in studies demonstrating that extracellular matrix attachment influences mammary epithelial cell differentiation (reviewed in Ref. 22) and expression of milk proteins (23). Adhesion of cells to the extracellular matrix also induces metalloproteinase expression (24). Many of these responses require attachment to the extracellular matrix via ␤ 1 -integrins (reviewed in Ref. 25). In other cell types, integrin-mediated attachment to the extracellular matrix suppresses apoptosis in both two-dimensional (26) and three-dimensional cultures (27).
In view of the close interrelationship between cell adhesion and proteolysis, we have examined the effect of adherence of monocytoid cells to purified extracellular matrix proteins (in the absence of exogenously added agonists) on plasminogen receptor expression. Adhesion is shown to directly influence the plasminogen binding capacity of cells, and a role for ␤ 1 -integrins in receptor expression is demonstrated. Data are developed to indicate that a specific subset of plasminogen receptors is altered by cell adhesion, and this subset influences plasmin generation. The ability of adhesion to modulate plasminogen receptor expression has important implications in several diverse physiological and pathophysiological processes.
Ligand Binding Assay-Cells were prepared for the ligand binding assays by washing two times in Hanks' balanced salt solution (HBSS) containing 1.2 mM CaCl 2 , 1.6 mM MgSO 4 , and 50 mM HEPES, pH 7.35 (HBSS-HEPES), and resuspended in HBSS-HEPES containing 1% bovine serum albumin (BSA) (Calbiochem) (HBSS-BSA). The cells (at 10 6 /ml) were incubated with 125 I-plasminogen (100 nM), or cells (at 5 ϫ 10 6 /ml) were incubated with 125 I-scu-PA (1 nM) in a total volume of 200 l at 37°C for 1 h. Triplicate 50-l aliquots were layered over 300 l of 20% sucrose in HBSS-BSA and centrifuged for 2.5 min in a Beckman Microfuge (Beckman Instruments). The tube tips were cut off and counted in a gamma counter (Iso Data, Inc., Palatine, IL). Specific binding data are given for all experiments and were determined by subtracting the nonspecific binding. For plasminogen, nonspecific binding was defined as counts bound to the cells in the presence of 200 mM ⑀-aminocaproic acid. For scu-PA, nonspecific binding was determined as counts bound in the presence of 100 nM unlabeled scu-PA. The level of nonspecific binding determined for both ligands was ϳ10% at the ligand concentrations specified above. The number of molecules of ligand bound per cell was calculated based on the specific activities of the ligands.
Establishment of Cell Adhesion-Fn (100 g, unless otherwise indicated), Vn (100 g), or Lm (160 g) in volumes of 10 ml in PBS were coated onto 100-mm Petri dishes (Corning Glass Works, Corning, NY) for 18 h at 4°C. The dishes were postcoated with 1% BSA for 90 min at 22°C. THP-1 cells (1.5 ϫ 10 7 ) in 10-ml volumes of cell culture medium were added to the coated dishes and incubated for 1 h at 37°C in an atmosphere with 95% humidity and 5% CO 2 . Nonadherent cells were recovered by gentle decanting and collected, followed by addition of 5 ml of the cell culture medium and recovery of additional cells with a plastic Pasteur pipette (twice). Adherent cells were recovered by flushing with a plastic Pasteur pipette until no cells were detected microscopically. Control cells were incubated in uncoated Petri dishes that also had been postcoated with 1% BSA. Cell viability was assessed by trypan blue exclusion and cell recovery by counting in a hemocytometer.
SDS-Polyacrylamide Gel Electrophoresis-Samples were prepared with sample buffer (2% SDS, 63 mM Tris-HCl, pH 6.8, 20 mM dithiothreitol, 10% sucrose and 0.02% bromphenol blue) containing 10 mM EDTA, 10 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride, 10 g/ml soybean trypsin inhibitor and 1.2 units/ml Trasylol, boiled for 5 min, and electrophoresed under reducing condition on 5% polyacrylamide slab gels in the buffer system of Laemmli (36). Gels were stained with Coomassie Blue and dried, and autoradiograms were developed with XRP-1 film (Eastman Kodak Co.) using an intensifier screen. Autoradiograms were scanned by laser densitometry. Molecular weights were estimated relative to protein standards obtained from Bio-Rad.
Statistics-Data are given as mean Ϯ S.D.
Reagents-Carboxypeptidase B was from Sigma. The plasmin substrate, D-Val-Leu-Lys-pNA (S-2251) was from Kabi Vitrum, Malmo, Sweden. Goat anti-mouse IgG and normal mouse IgG were from Calbiochem. Transwell dishes were from Costar, Cambridge, MA. Ascites containing mAb W632 was kindly provided by Dr. Martin Schwartz, The Scripps Research Institute.

Role of Cell Adhesion in Modulation of Plasminogen Receptor
Expression-We tested whether adhesion of THP-1 monocytoid cells to isolated extracellular matrix proteins could modulate plasminogen receptor expression. THP-1 cells were allowed to adhere to Petri dishes coated with either Fn, Vn, or Lm or to uncoated control wells. The cells adhered to the three matrix proteins, but not to the uncoated dishes. By producing a limited surface, a nonadherent and an adherent cell population could be recovered separately from each substrate and 125 I-plasminogen binding was measured. Plasminogen binding to the nonadherent cell population was greatly enhanced compared to the control cells incubated in uncoated dishes. A typical experiment is shown in Fig. 1. With the different matrix proteins, the percent plasminogen binding (relative to control cells at 100%) was 310 Ϯ 55 (Fn) (n ϭ 22), 167 Ϯ 20 (Vn) (n ϭ 3), and 140 Ϯ 35 (Lm) (n ϭ 3). In contrast, plasminogen binding to the adherent population decreased with each matrix protein compared to the control cells: the percent 125 I-plasminogen binding was 61 Ϯ 16 (Fn, n ϭ 22), 45 Ϯ 9 (Vn, n ϭ 3), and 64 Ϯ 35 (Lm, n ϭ 3).
The relationship between extent of adhesion and receptor up-regulation was then explored. Since the greatest degree of adherence and receptor up-regulation was attained with Fn ( Fig. 1), this substrate was used in subsequent experiments. Petri dishes were coated with varying quantities of Fn to attain different degree(s) of cellular adhesion. Maximal adhesion of the cells (85% adherence) was attained when the wells were coated with 50 g of Fn (Fig. 2, panel A). Plasminogen receptor expression in the nonadherent cells increased with increasing adhesion (Fig. 2, panel B). In contrast, plasminogen binding to the adherent cells decreased to 60 Ϯ 16% (n ϭ 6) relative to control cells (data not shown) without any apparent relation to the extent of adhesion. As specific examples, with 85, 64, and 38% adhesion, the decrease in plasminogen binding of the adherent cells was 31, 18, and 40%. The role of adhesion was also examined by comparing up-regulation of plasminogen binding sites in an additional cell type. In parallel experiments, using dishes coated with 100 g of Fn, THP-1 cells were 94% adherent and U937 monocytoid cells were 76% adherent. Plasminogen binding to the nonadherent THP-1 cells increased 2-fold over controls, while plasminogen binding to the nonadherent U937 cells increased 1.6-fold. Moreover, the up-regulation of plasminogen receptors did not reflect a general effect on cell surface receptors. When 125 I-scu-PA binding to the cells was examined in parallel with a 1 nM input concentration of 125 I-scu-PA, 1.19 Ϯ 0.26 ϫ 10 4 (n ϭ 3) and 1.38 Ϯ 0.29 ϫ 10 4 (n ϭ 3) molecules were bound per cell in the nonadherent and adherent THP-1 cell populations, respectively.
To further explore the requirement for adherence to substratum, we tested whether soluble Fn could induce plasminogen receptor up-regulation in the nonadherent cell population. Cells (1.5 ϫ 10 7 ) were incubated in polypropylene tubes in the presence of 100 g of soluble Fn. Under this condition, no up-regulation of plasminogen binding sites was observed.
In parallel experiments in which the cells were incubated with immobilized Fn, 70% adhesion was achieved, resulting in a 4.5-fold up-regulation of plasminogen receptor expression in the nonadherent population.
Next, the time dependence of plasminogen receptor up-regulation in the nonadherent cell population was investigated (Fig. 3). THP-1 cells were incubated with Fn-coated dishes for varying times. The nonadherent population was recovered and plasminogen binding and percent adhesion were determined. Plasminogen receptor up-regulation was induced rapidly, and could be detected by 10 min, reaching a maximum between 30 and 120 min. The receptor up-regulation decayed rapidly after 120 min. Interestingly, the extent of adhesion paralleled the receptor up-regulation until 120 min, but decayed more slowly.
We tested also whether the up-regulation of plasminogen binding sites was reversible. Cells were incubated with Fncoated dishes, and 69% of the cells adhered. The nonadherent population was recovered and then allowed to adhere to a second Fn-coated dish. Sixty-eight percent adherence was obtained. Therefore, the distinction between adherent and nonadherent populations was not due to an unique subpopulation of cells, but, apparently, to the availability of surface for adherence. As discussed above, plasminogen receptors were upregulated ϳ2-fold in the nonadherent cell population during the first adhesion step (Table I). Following the second adhesion of the nonadherent population of cells, plasminogen receptor expression was further up-regulated by ϳ2-fold in the newly nonadherent population and was downregulated ϳ2.5-fold in the newly adherent population (Table I). Thus, both up-regulation and down-regulation of plasminogen binding site exposure was reversible.
Mechanisms of Plasminogen Receptor Up-regulation-To determine whether the increase in plasminogen binding was due to a change in affinity and/or capacity of the nonadherent cells for plasminogen, binding isotherms were constructed for the cells that did not adhere to Fn and for control cells incubated in uncoated dishes. Under equilibrium binding conditions, the isotherms gave evidence of saturation and, therefore, the data were plotted in Scatchard plots (Fig. 4). Straight lines were obtained and provided good fits for both sets of data points (r ϭ Ϫ0.98 for control cells and r ϭ Ϫ0.99 for the nonadherent cells), suggesting a single class of plasminogen binding sites with respect to affinity. The affinities of both cell populations for plasminogen were not statistically different: K d values of 447 Ϯ 141 nM and 393 Ϯ 25 nM were calculated for nonadherent and control cell populations, respectively (n ϭ 3). However, the number of binding sites was 4-fold higher in the nonadherent population: ␤ max ϭ 28 Ϯ 5 ϫ 10 6 molecules/nonadherent cell versus 7 Ϯ 1 ϫ 10 6 molecules/control cell. Thus, the increase in plasminogen binding to the nonadherent cells was due to a change in capacity but not in affinity for plasminogen.
In order to determine whether de novo protein synthesis was required for plasminogen receptor up-regulation, THP-1 cells were treated with cycloheximide prior to exposure to the immobilized Fn. To insure that cycloheximide was exerting its anticipated effects, we verified that [ 35 S]methionine incorporation into total cellular protein was inhibited by greater than 90% (Table II). Under this condition, plasminogen receptor up-regulation was not affected (Table II). In controls, cycloheximide treatment altered neither cell viability nor the ability of the cells to adhere to Fn.
The possibility that soluble factors released during the adhesion step could be responsible for the up-regulation was explored. Conditioned medium was collected after adhesion of the cells to Fn for 1 h and was then added to fresh THP-1 cells in uncoated dishes for 1 h. No plasminogen receptor up-regulation was observed in the cells exposed to the conditioned medium (106 Ϯ 3% (n ϭ 3) relative to control cells exposed to The role of ␤ 1 -integrins in up-regulation of plasminogen binding sites following adherence to fibronectin was explored. Cells were preincubated with two anti-␤ 1 -integrin monoclonal antibodies that block adhesion to fibronectin, SG19 (32) and 4B4 (33). Under these conditions, no adherent cells were recovered (Fig. 5B), and plasminogen binding sites were not increased in the nonadherent population (Fig. 5A). Using the activating mAb, 8A2 (34), adherence was not blocked and plasminogen binding sites were up-regulated 3.7-fold compared to control cells incubated in the absence of Fn. Cells incubated with Fn in the presence of an irrelevant mAb exhibited a 2.5-fold up-regulation in plasminogen binding ability compared to the control cells. We found no up-regulation of plasminogen binding sites when cells were incubated with the activating antibody, 8A2, in uncoated dishes (data not shown), excluding the possibility that 8A2 could stimulate the cells to increase plasminogen binding sites in the absence of a ␤ 1 -integrin ligand.
To examine whether the up-regulation observed in the nonadherent population might require attachment followed by release of cells, we tested whether up-regulation could occur when adherent cells were prevented from detaching. Petri dishes were incubated with goat-anti-mouse IgG to capture either mAb W632, directed against the major histocompatiblity complex, or normal mouse IgG as described (37). The THP-1 cells adhered to each type of coating, presumably via an interaction with Fc receptors and/or the major histocompatiblity complex (Fig. 6B). Nonadherent and adherent cell populations were recovered from these dishes and plasminogen binding compared with nonadherent and adherent cells recovered from fibronectin-coated dishes. Under all conditions that induced adherence, plasminogen binding was increased to a similar extent in the nonadherent cell populations and decreased in the adherent populations, compared to control cells incubated in uncoated dishes (Fig. 6A). Therefore, the up-regulation in receptor expression in the nonadherent population cannot be attributed to changes occurring when cells detach from a surface.
Nature of the Up-regulated Plasminogen Receptor Population-Proteins with carboxyl-terminal lysyl residues are candidate plasminogen receptors (38). We sought to determine the extent to which plasminogen receptor up-regulation depended upon exposure of carboxyl-terminal lysyl residues. Adherent and nonadherent populations were generated in the presence of immobilized Fn. Then these populations and control cells were separately treated with carboxypeptidase B (CPB) prior to measuring 125 I-plasminogen binding. CPB treatment did not decrease viability of either the nonadherent, adherent or control cells, as determined by trypan blue exclusion. With the control and adherent cells, CPB treatment decreased plasmin-ogen binding in a dose-dependent manner, reaching a plateau at approximately 60% inhibition (Fig. 7), consistent with previously published data (38 -40). With the nonadherent cell population, CPB treatment decreased plasminogen binding to the level of the CPB-treated control cells (Fig. 7). These results suggested that the up-regulated plasminogen binding sites are composed predominantly of proteins exposing carboxyl-terminal lysyl (or arginyl) residues on the cell surface.
Functional Consequences of Plasminogen Receptor Up-regulation-We sought to determine whether up-regulation of plas-  6. Effect of irreversible adherence on plasminogen binding site expression. Petri dishes were incubated with 4.8 ml of goatanti-mouse IgG at 25 g/ml at 4°C for 18 h. Then either 2.5 ml of a 1/500 dilution of an ascites containing mAb W632, normal mouse IgG (NM IgG) (10 g/ml), or buffer were added, and the dishes were further incubated at 22°C for 60 min. Fibronectin-coated wells were prepared as described under "Experimental Procedures," and all dishes were postcoated with 1% BSA. THP-1 cells (1.5 ϫ 10 7 ) in 10 ml were incubated with the coated dishes or control (uncoated) wells. The adherent (cross-hatched bars) and nonadherent (filled bars) were recovered separately and incubated at 1.25 ϫ 10 6 cells/ml with 125 I-plasminogen (100 nM). minogen receptors was associated with an increased ability of the cells to promote plasminogen activation. Adherent and nonadherent cell populations were produced after incubation with Fn-coated dishes. These cells and control cells were incubated with 15 M Glu-plasminogen. The unbound plasminogen was removed by centrifugation and u-PA and the plasmin substrate, S-2251, were added. Plasmin activity was measured as cleavage of the S-2251. After 30 min, the plasmin amidolytic activity of the nonadherent cells was 3.5 times higher than that of the adherent cells and 2.5 times higher than the control cells (Fig. 8). The extent of promotion of plasminogen activation correlated with the plasminogen binding capacities of the cells in this experiment. Plasminogen binding to the nonadherent cells was approximately 2.5-and 2-fold higher than to either the adherent or control cells, respectively.
The relationship between plasminogen receptor expression and the ability of the cells to degrade a representative extracellular matrix component, Fn, was explored. Nonadherent cell populations were generated by adherence of THP-1 cells to Fn-coated plates and control cells were incubated in uncoated plates. These cells were incubated with 10 M Glu-plasminogen and washed to remove unbound ligand. Then u-PA and soluble 125 I-Fn were added and incubated with the cells. After incubation for 2 h, 125 I-Fn was degraded to a major product with a M r(app) of 190,000 (Fig. 9, lanes 2 and 3), while the integrity of either Fn alone or Fn plus u-PA was not changed between 0 and 2 h (data not shown). The extent of degradation in the nonadherent cells was ϳ2-fold greater than that of the control cells, as determined by laser densitometric scanning of autoradiograms of the gels. After 20 h of incubation, intact 125 I-Fn was no longer detectable after incubation with the cells (Fig. 8,  lanes 4 and 5). At this time point, the 190,000 band was further degraded into smaller fragments. The decrease in intensity of the 190,000 band was more extensive (ϳ4-fold) in the nonadherent cell population compared to control cells. Greater than 95% of the 125 I-Fn remained intact following incubation with buffer or with u-PA for 20 h (data not shown). Thus, the ability of the cells to degrade Fn was enhanced in the nonadherent cell population coinciding with plasminogen receptor up-regulation. DISCUSSION In this study, we provide the first evidence that adhesion of monocytoid cells to extracellular matrix proteins, via ␤ 1 -inte-grins, induces increased expression of plasminogen binding sites. The up-regulation of these sites occurred in the subpopulation of cells that was nonadherent, although the presence of adherent cells was required. Three different adhesive proteins, fibronectin, vitronectin, and laminin, supported this up-regulation. The following conclusions can be drawn from our analyses. 1) The presence of a population of cells adhering to the extracellular matrix in an integrin-dependent manner is sufficient to induce up-regulation of plasminogen binding sites in the nonadherent population of cells. In contrast, plasminogen binding to the adherent cells is decreased compared to cells maintained in suspension. 2) The up-regulation of plasminogen binding sites is a reversible process; when nonadherent cells are allowed to adhere to substratum, exposure of plasminogen binding sites is down-regulated. This reversibility suggests that the nonadherent cells did not represent a distinct subpopulation within the cell line. Furthermore, this implies that proteolysis does not play a role in the up-regulation. 3) The up-regulation is rapid and independent of new protein synthesis. 4) The newly available plasminogen receptors are proteins exposing carboxyl-terminal lysines or arginines. These receptors exhibit the same affinity for plasminogen as the receptors on resting cells. 5) Up-regulation of plasminogen binding sites increases the ability of the cells to promote plasminogen activation and to degrade Fn.
The ability of cells to promote plasminogen activation is absolutely dependent upon binding of plasminogen to the cell surface (41,42). Both protein (38) and nonprotein (e.g. gangliosides) (43) components of the cell membrane bind plasminogen. In the present study, the increase in plasminogen binding capacity of the nonadherent cells was entirely sensitive to CPB. Thus, these new binding sites are predominantly proteins exposing carboxyl-terminal lysines. Furthermore, the nonadherent cells exhibiting enhanced plasminogen binding were 3.5fold more effective than control cells in promoting plasminogen activation. These observations indicate a role of newly exposed carboxyl-terminal lysines in enhancing plasminogen activation and support the dependence of cell surface plasminogen activation on plasminogen receptor occupancy.
The presence of a population of cells that adhered to extracellular matrix proteins was necessary and sufficient to induce up-regulation of plasminogen receptors in the nonadherent cell population. The up-regulation of plasminogen binding sites did not require the addition of exogenous agonists. The adherence of the THP-1 cells to Fn was dependent upon ␤ 1 -integrin(s).  1 -Integrins are known to transmit external signals from the extracellular matrix to stimulate intracellular signaling pathways (reviewed in Ref. 44). However, activation of ␤ 1 -integrins on the monocytoid cells using an activating antibody in the absence of ␤ 1 ligands, was not sufficient to up-regulate plasminogen binding site expression. The ability to induce plasminogen receptor up-regulation was not limited to extracellular matrix proteins: Adherence of cells to immobilized antibodies also induced plasminogen receptor up-regulation in the nonadherent cell population, suggesting that the predominant determinant of up-regulation is the presence of adherent cells and may not be dependent upon intracellular signaling.
In analyzing plasminogen binding site up-regulation, we were unable to detect the presence of a soluble factor which could induce receptor up-regulation in the nonadherent cell population. Furthermore, plasminogen binding site up-regulation was not diminished by pretreatment with cycloheximide, suggesting that new protein synthesis was not required to achieve up-regulation. Another potential mechanism which could explain up-regulation is proteolytic modification of cell surfaces to increase the number of plasminogen binding sites (39,45). However, plasminogen binding site expression was down-regulated when nonadherent cells were allowed to adhere to a new surface. This observation argues that proteolytic digestion of membrane proteins is not responsible for the upregulation induced by adherence.
With elimination of the foregoing potential mechanisms to explain up-regulation of plasminogen receptor expression in the nonadherent cells, the following model is most compatible with our results. Direct contact between the adherent cell population and nonadherent cells induces reversible reorganization of the cell membrane which increases surface exposure of proteins with carboxyl-terminal lysines. The adhesion step induced down-regulation of receptor expression in the adherent cells. Furthermore, when the nonadherent cells (that exhibited receptor up-regulation) were allowed to adhere, adhesion induced receptor down-regulation. Down-regulation, likewise, may be induced by reorganization of the cell membrane during adhesion.
The ability of cells to regulate their expression of plasminogen receptors is critical for local regulation of proteolysis. When cells encounter an adhesive substrate, receptor expression is up-regulated in the nonadherent cell population. When this increase is combined with clustering of urokinase receptors at the leading edge of the cell (46), focal proteolytic activity is generated on the cell surface to promote matrix degradation. After the cells adhere, plasminogen receptors and, consequently, cell surface proteolytic activity are down-regulated, to aid in the establishment of stable cell-substratum contacts. This regulation of proteolytic activity at local environments should have important consequences for biological processes in which cells must degrade extracellular matrices in order to migrate, such as in wound healing, inflammation, metastasis, and angiogenesis.