The Low Density Lipoprotein Receptor-related Protein/α2-Macroglobulin Receptor Regulates Cell Surface Plasminogen Activator Activity on Human Trophoblast Cells*

The low density lipoprotein receptor-related protein/α2-macroglobulin receptor (LRP/α2MR) mediates the internalization of numerous ligands, including prourokinase (pro-UK) and complexes between two-chain urokinase (tc-u-PA) and plasminogen activator inhibitor type-1 (PAI-1). It has been suggested that through its ability to internalize these ligands, LRP/α2MR may regulate the expression of plasminogen activator activity on cell surfaces; this hypothesis, however, has not been experimentally confirmed. To address this issue, we assessed the ability of LRP/α2MR to regulate plasminogen activator activity on human trophoblast cells, which express both LRP/α2MR and the urokinase receptor (uPAR). Trophoblasts internalized and degraded exogenous125I-pro-UK (primarily following its conversion to tc-u-PA and incorporation into tc-u-PA·PAI complexes) in an LRP/α2MR-dependent manner, which was inhibited by the LRP/α2MR receptor-associated protein. Receptor-associated protein also caused a ∼50% reduction in cell surface plasminogen activator activity and delayed the regeneration of unoccupied uPAR by cells on which uPAR were initially saturated with pro-UK. Identical effects were caused by anti-LRP/α2MR antibodies. These results demonstrate that LRP/α2MR promotes the expression of cell surface plasminogen activator activity on trophoblasts by facilitating the clearance of tc-u-PA·PAI complexes and regeneration of unoccupied cell surface uPAR.

Homozygous disruption of the LRP/␣ 2 MR gene leads to embryonic death, demonstrating an essential role for LRP/␣ 2 MR during development (10,11). However, the specific function(s) of LRP/␣ 2 MR necessary for successful development has not been defined. Furthermore, despite characterization of the processes by which pro-UK and tc-u-PA⅐PAI-1 complexes bind to and are internalized by LRP/␣ 2 MR, an effect of LRP/␣ 2 MR on cellular plasminogen activator activity has not been demonstrated. To address this, we assessed the effect of LRP/␣ 2 MR inhibition on the cell surface plasminogen activator activity of human trophoblast cells, which express both uPAR and LRP/ ␣ 2 MR (30,37). Despite a lack of impaired fertility in mice with homozygous disruption of the uPAR gene (38,39), a potentially more important role for uPAR in human reproduction is suggested by the polarized expression of this receptor on invading trophoblasts (40). Our studies demonstrate that LRP/␣ 2 MR promotes cell surface plasminogen activator activity on these cells.
Cells-Three trophoblast cell lines were used in these studies. ED 27 and ED 77 cells were derived from chorionic villous biopsies, performed for medically indicated cytogenetic analyses. These cells stain immunohistochemically for cytokeratin and the ␣and ␤-chains of human chorionic gonadotropin, but not vimentin (46 -48). HTR 8 cells were derived from outgrowths of first-trimester chorionic villi (49), express cytokeratin and HLA framework antigens (50), and produce ␤-chain of human chorionic gonadotropin (49,51). Human aortic smooth muscle cells, which express both LRP/␣ 2 MR and the VLDL receptor, were isolated and cultured as described previously (52).
Characterization of uPAR and LRP/␣ 2 MR expression by trophoblast cell lines-The expression of uPAR by trophoblast cells was quantitated by measuring the binding of 125 I-pro-UK to cell monolayers, at 4°C, after elution of endogenous u-PA (37,53). Specific binding was defined as described previously (37), and linear (54) and nonlinear curve fitting performed by the least squares method, using the Kaleidagraph software program (Abelbeck Software, Reading PA). The expression of LRP/␣ 2 MR and VLDL receptors was assessed by immunoblotting and ligand blotting, using RAP as the ligand (42).
Measurement of 125 I-pro-UK internalization and degradation-The internalization and degradation of 125 I-pro-UK was measured as described previously (12). Briefly, cells were incubated at 37°C in the presence of 2 nM 125 I-pro-UK. At various intervals, degraded radioligand was determined by measuring the amount of radioactivity in conditioned medium that remained soluble in 10% ice-cold trichloroacetic acid. The amount of 125 I-pro-UK bound to the cell surface was determined by measuring the radioactivity within eluates prepared by incubating cells for 3 min in 50 mM glycine-HCl, 0.1 M NaCl, pH 3.0 (53), and the amount of 125 I-pro-UK within cells was determined by lysing cells in 0.1 M NaOH following elution of surface-bound radioactivity. Specific degradation was defined as the difference between the amount of 125 I-pro-UK degraded in the absence and presence of a 100-fold molar excess of unlabeled pro-UK. The effect of RAP or anti-LRP/␣ 2 MR antibodies on 125 I-pro-UK degradation was assessed using the formula, where A ϭ specific 125 I-pro-UK degradation (cpm), and B ϭ 125 I-pro-UK degradation (cpm) in the presence of RAP. The significance of differences between the amount of 125 I-pro-UK internalized, degraded, or bound to control and RAP-exposed cells was determined using the Student's two-tailed t test for paired samples. The Effect of RAP and Anti-LRP/␣ 2 MR Antibodies on Cell Surface Plasminogen Activator Activity-Cell surface plasminogen activator activity was measured as described by Ellis et al. (1,55). Briefly, cells were cultured in 96-well plates and incubated in the absence (HTR 8 cells) or presence (ED 27 and ED 77 cells) of 2 nM pro-UK, for varying intervals. Cells were then washed and further incubated in fresh medium containing 0.2 M plasminogen and 0.6 mM H-D-Val-Leu-Lys-AMC for 75 min. Plasmin generation was determined by measuring substrate hydrolysis (excitation wavelength, 360 nm; emission wavelength, 460 nM) using a Cytofluor 2300 fluorescence plate reader (Millipore, Bedford MA).
To confirm the dependence of this assay on the assembly of pro-UK and plasminogen on the cell surface, we compared the u-PA-dependent activation of plasminogen in the absence and presence of ED 27 cells. Briefly, cells were incubated with 2.0 nM pro-UK and washed, and plasminogen activator activity was measured as described above. In parallel, 0.1 nM pro-UK, a concentration chosen to reflect the amount of pro-UK expected to bind to cellular uPAR under these conditions (based on the affinity of pro-UK binding; Table I), was incubated in empty wells. Concentrated stock solutions of plasminogen and H-D-Val-Leu-Lys-AMC were then directly added to the pro-UK-containing wells to achieve final concentrations equivalent to those in wells containing cells.
To determine the effect of RAP on the expression of cell surface plasminogen activator activity by HTR 8 cells (which produce endogenous prourokinase), 4 l of either phosphate-buffered saline or a 500 g/ml solution of RAP were added to quadruplicate wells of cells cultured in 96-well plates. Sixteen and 18 h later, a similar amount of RAP was added to each of two additional sets of wells. Medium was then removed, and cell surface plasminogen activator activity was measured. Similar assays were utilized to assess the effect of RAP on the plasminogen activator activity of ED 27 and ED 77 cells. However, because these cells did not produce pro-UK, they were incubated for 6 h with 0, 0.5, or 2.0 nM pro-UK, in the absence or presence of 20 g/ml RAP, prior to measurement of plasminogen activator activity.
In selected experiments, the effects of RAP and anti-LRP/␣ 2 MR antibodies on cell surface plasminogen activator activity were measured in parallel.
Assessment of the Species of 125 I-u-PA That Accumulated on the Surface of Cells Incubated with 125 I-Pro-UK in the Absence and Presence of RAP-To assess the species of 125 I-u-PA that accumulated on the surface of cells incubated with 125 I-pro-UK and RAP, ED 27 cells were incubated with 4 nM 125 I-pro-UK for 30 min and then washed and further incubated, at 37°C, in the absence or presence of 20 g/ml RAP. Immediately after incubation with 125 I-pro-UK, as well as after 4 and 6 h of incubation in the absence or presence of RAP, equal volumes (15 l) of cell eluates were analyzed in parallel using 7.5% SDS-polyacrylamide gel electrophoresis (56) and autoradiography. Autoradiograms were prepared by exposing dried gels to Reflection™ autoradiography film (New England Nuclear) for 24 h. Bands were quantitated by scanning the autoradiogram into Adobe Photoshop (Adobe Systems, Mountain View, CA) and determining the relative amount of radioactivity within each band by measuring the number of pixels contained within it, using NIH Image, version 1.59.
Effect of RAP on the Regeneration of Unoccupied uPAR-Because RAP-mediated inhibition of ligand uptake by LRP/␣ 2 MR might impair the regeneration of uPAR, we assessed the effect of RAP on the expression of unoccupied uPAR by cells on which uPARs were initially saturated with pro-UK. ED 27 cells were incubated with 4 nM unlabeled prourokinase, to saturate cellular uPAR, in the absence or presence of 20 g/ml of RAP. Cells were then washed, and those initially incubated with pro-UK and RAP or with pro-UK alone were further incubated in fresh medium containing either 20 g/ml RAP or no additions, respectively. At selected time points (0, 0.5, 2.0, 4.0, and 6.0 h), cells were chilled to 4°C and washed, and their expression of unoccupied uPAR was assessed by measuring the binding of 125 I-pro-UK.
To ensure that RAP had no direct effect on uPAR expression, the expression of unoccupied uPAR by these cells was assessed at selected time points following incubation with RAP alone.

Expression of the Urokinase Receptor, u-PA and PAI-1 by
Trophoblast Cells-In initial studies, the expression of uPAR, u-PA and PAI-1 by the trophoblast cells used in these studies was measured (Table I). All three cell lines expressed uPAR, as demonstrated by their high affinity binding of 125 I-pro-UK. All cells also produced PAI-1, measured using an enzyme-linked immunosorbent assay that detects both free and complexed inhibitor. However, u-PA was produced only by HTR 8 cells, and as a consequence, these were the only cells to express cell surface plasminogen activator activity in the absence of added pro-UK.
Internalization and Degradation of 125 I-Pro-UK by Trophoblast Cells-We next determined whether the cells internalized and degraded 125 I-pro-UK in an LRP/␣ 2 MR-dependent manner. Each of the cell lines internalized and degraded 125 I-pro-UK, as demonstrated by the accumulation of trichloroacetic acid-soluble radioactivity in conditioned medium (Fig. 1). The amount of cell surface-associated radioactivity diminished with time, whereas the amount of u-PA within the cells remained relatively constant. Qualitatively similar patterns of 125 I-pro-UK degradation were observed for all three cell lines, although HTR 8 cells demonstrated less cell surface-associated and internalized 125 I-pro-UK at all time points. This observation is explained by the expression of fewer uPAR by these cells and the fact that HTR 8 cells produced endogenous pro-UK, which would be expected to compete with 125 I-pro-UK for binding to uPAR (Table I). Serial analyses of the 125 I-pro-UK in conditioned medium over the course of these studies demonstrate that it remained predominantly (Ͼ95%) in the single chain form.
To determine the role of LRP/␣ 2 MR in the internalization and degradation of u-PA by trophoblast cells, the ability of 20 g/ml (512 nM) RAP to inhibit the degradation of exogenous 125 I-pro-UK by ED 27 and ED 77 cells was determined. RAP affected the disposition of the radioligand in two ways. First, degradation was inhibited by approximately 75% (Table II), consistent with the results of studies employing other cell types (12). Second, RAP caused the accumulation of significantly (p Ͻ 0.001) more radioactivity (ϳ25%) on the cell surface, an observation not previously reported (Table II). Similar results were obtained using HTR 8 cells (not shown).
To confirm that these effects were mediated through LRP/ ␣ 2 MR, the effect of RAP on the degradation of 125 I-pro-UK by ED 77 cells was compared with that of an affinity-purified, anti-LRP/␣ 2 MR antibody. Both RAP and the antibody significantly inhibited degradation ( Fig. 2A) and caused similar increases in cell surface-associated radioactivity (Fig. 2B). No changes in either of these parameters was caused by nonimmune rabbit IgG.
These findings confirmed that LRP/␣ 2 MR mediated the internalization and degradation of exogenous 125 I-pro-UK by trophoblast cells. We hypothesized that by preventing the binding of 125 I-pro-UK (or 125 I-tc-u-PA⅐PAI complexes formed on the cell surface) to LRP/␣ 2 MR, RAP inhibited ligand internalization, thereby causing an accumulation of cell-surface radioactivity. Although such an increase might also result from increased uPAR expression, 125 I-pro-UK binding assays revealed no increase in receptor expression after a 6 h incubation of cells with RAP.
Previous studies have demonstrated that in addition to LRP/ ␣ 2 MR, subpopulations of trophoblasts express the VLDL receptor (57), which also mediates internalization and degradation of tc-u-PA⅐PAI-1 complexes (58,59). However, immunoblots using anti-LRP/␣ 2 MR and anti-VLDL receptor antibodies demonstrated that only the former was expressed by the cells used in these studies (Fig. 3).
Effect of RAP on Cell Surface PA Activity-Prior to assessing the effects of RAP on cell surface plasminogen activator activity, we confirmed that the activation of plasminogen observed in our assays reflected events occurring on the cell surface. To address this issue, the abilities of similar (estimated) concentrations of cell-bound and fluid-phase u-PA to activate plasminogen were compared. Essentially no plasmin generation (59 arbitrary fluorescence units) occurred in the absence of cells, whereas a markedly greater amount (5047 arbitrary fluorescence units) occurred in their presence (Fig. 4). Even a concentration of pro-UK in solution (1.0 nM) that was 10-fold greater than that estimated to bind to the cells activated less than 10% as much plasminogen as the cell-associated ligand. These studies established that the activation of plasminogen in this assay was dependent upon assembly of pro-UK and plasminogen on the cell surface (2,55).
We next determined the effect of inhibition of LRP/␣ 2 MR ligand uptake by RAP on cell surface plasminogen activator activity. Incubation of HTR 8 cells (which produce u-PA and express cell surface plasminogen activator activity) with RAP caused a time-dependent inhibition of cell surface plasminogen  activator activity (Fig. 5). Inhibition was apparent within 2 h after addition of RAP and maintained for the 20 h experimental period. In contrast, RAP did not inhibit cell surface plasminogen activator activity when included only in the medium used to wash cells incubated in the absence of RAP, nor did it inhibit the activation of plasminogen by tc-u-PA in solution (not shown). These experiments suggest that the inhibitory effect of RAP on cell surface plasminogen activator activity resulted from its interaction with LRP/␣ 2 MR. We next measured the effects of RAP on the expression of cell surface plasminogen activator activity by ED 27 and ED 77 cells. Activity was measured after incubating cells with 0, 0.5, or 2.0 nM pro-UK for 6 h, at 37°C, in the absence or presence of RAP. RAP caused a significant decrease (ϳ50%; p Ͻ 0.005) in cell surface plasminogen activator activity by both cell lines (Fig. 6, A and B). The specificity of this effect was demonstrated by the observation that anti-LRP/␣ 2 MR antibodies, used at a concentration of 20 g/ml (ϳ130 nM), caused similar, and  highly significant (p Ͻ 0.001) inhibition (Fig. 6C). Inhibition of plasminogen activator activity by RAP occurred in a concentration-dependent manner; approximately 25% inhibition was caused by 128 nM RAP, whereas 768 nM RAP, the highest concentration tested, inhibited activity by 65%. No significant inhibition was caused by nonimmune rabbit IgG (Fig. 6C), ␤ 2 -glycoprotein I, or bovine serum albumin, used as control proteins (not shown).
RAP Induces the Selective Accumulation of u-PA⅐PAI Complexes on the Cell Surface-Our studies to this point demonstrated that RAP and/or anti-LRP/␣ 2 MR antibodies 1) inhibited the internalization and degradation of exogenous 125 I-pro-UK, 2) caused an accumulation of 125 I-pro-UK (or another u-PA species) on the cell surface, and 3) inhibited cell surface plasminogen activator activity. To reconcile these observations, we hypothesized that inhibition of LRP/␣ 2 MR by RAP might inhibit cell surface plasminogen activator activity through two mechanisms. First, rather than impairing the internalization of pro-UK, RAP might selectively inhibit the clearance of proteolytically inactive tc-u-PA⅐PAI complexes generated on the cell surface. These complexes bind more avidly than pro-UK to LRP/␣ 2 MR (12,18), but with equal affinity to uPAR (60). Hence, accumulated complexes might compete with pro-UK and/or tc-u-PA for binding to a fixed number of uPARs, thereby inhibiting plasmin generation. RAP-mediated inhibition of tcu-PA⅐PAI complex uptake by LRP/␣ 2 MR might also impair the regeneration of unoccupied uPAR on the cell surface, further contributing to its inhibitory effect on plasminogen activator activity.
To determine the species of u-PA that accumulated on the surface of cells in the presence of RAP, cells were preincubated with 125 I-pro-UK and then washed and further incubated in the absence or presence of RAP. Eluates were prepared immediately after preincubation of cells with 125 I-pro-UK and after 4 and 6 h of incubation in the absence or presence of RAP. Analysis of these eluates by SDS-polyacrylamide gel electrophoresis and autoradiography revealed that pro-UK, which migrated with an M r of ϳ55,000 on both nonreduced (Fig. 7) and reduced gels, was the major species of u-PA associated with the cell surface at all time points. In addition, two high molecular weight tc-u-PA⅐PAI complexes were detected. These migrated with M r values of ϳ92,000 and ϳ100,000, consistent with those of complexes between tc-u-PA and PAI-1 (61), and tc-u-PA and the higher molecular mass (67 kDa) form of plasminogen activator inhibitor type-2 (PAI-2) (62, 63), a second plasminogen activator inhibitor produced by trophoblasts (37,64) (Fig. 7). Complexes were detectable within 1-2 h after incubation at 37°C (not shown) and present in greater amounts after 4 and 6 h. Furthermore, more of each complex accumulated in the presence of RAP. For example, after 4 h, approximately 3.7-fold more tc-u-PA⅐PAI-1 complex was present on cells incubated with RAP, and whereas the ϳ100-kDa complex was easily detectable in the eluate of RAP-treated cells, a corresponding complex was present only at levels insufficient for quantitation in eluates of control cells. Furthermore, although equal amounts of 125 I-pro-UK were present in the eluates of control and RAP-treated cells, 125 I-pro-UK comprised a smaller percentage of the total radioactivity on the surface of the latter. For example, after 4 h of incubation, pro-UK comprised 85%, whereas tc-u-PA⅐PAI-1 and tc-u-PA⅐PAI-2 complexes comprised 10.6 and 4.4%, respectively, of the total radioactivity within eluates of RAP-treated cells. In contrast, pro-UK comprised 96%, whereas tc-u-PA⅐PAI-1 complexes comprised only 4% of the radioactivity within eluates of control cells. These results demonstrate that RAP selectively impairs the clearance of tc-u-PA⅐PAI complexes, leading to the occupancy of a greater percentage of uPAR by these proteolytically inactive ligands. The reason that lesser amounts of 125 I-pro-UK were not clearly demonstrated in eluates of RAP-treated cells reflects the relatively high concentrations of 125 I-pro-UK used in these studies and the fact that equal volumes, rather than equal cpm of eluates were compared. However, we cannot exclude the possibility that the marked reduction in cell surface plasminogen activator activity induced by RAP and/or anti-LRP/␣ 2 MR antibodies may reflect, in part, additional effects of tc-u-PA⅐PAI complexes independent of those attributable to direct competition with pro-UK or tc-u-PA for binding to uPAR.
RAP Impairs the Regeneration of Unoccupied Cell Surface uPAR-To assess the effects of RAP on the regeneration of unoccupied uPAR, ED 27 cell uPARs were saturated by incubation with unlabeled pro-UK, in the absence or presence of RAP. Cells preincubated with either pro-UK and RAP, or pro-UK alone, were then further incubated in medium containing either 20 g/ml RAP or no additives, respectively. As depicted in Fig. 8A, immediately after preincubation with unlabeled pro-UK, or pro-UK and RAP, cells bound equivalent amounts of 125 I-pro-UK. These were small in comparison to control cells, as cellular uPARs were occupied by unlabeled ligand. After incubation at 37°C, however, progressively more 125 I-pro-UK bound to cells incubated in the absence of RAP, whereas comparatively little change was noted in the amount of 125 I-pro-UK that bound to cells incubated in the presence of RAP. These differences achieved statistical significance within 30 min of initiating the 37°C incubation (p Ͻ 0.05), and they demonstrate that by inhibiting the uptake of uPAR ligands, RAP also inhibited the regeneration of unoccupied uPAR. In contrast, RAP did not alter the binding of 125 I-pro-UK to cells not preincubated with pro-UK (Fig. 8B), confirming that it had no direct effect on uPAR expression. These results demonstrate that through its ability to mediate the clearance of tc-u-PA⅐PAI complexes, LRP/␣ 2 MR promotes the regeneration of unoccupied uPAR on the cell surface. DISCUSSION These studies demonstrate that LRP/␣ 2 MR is expressed by trophoblast cells, on which it mediates the uptake and degradation of pro-UK and/or tc-u-PA⅐PAI complexes. The latter selectively accumulate on the cell surface in the presence of RAP, an observation that extends the studies of Nykjaer et al. (18), who demonstrated that RAP caused an accumulation of 125 I-tc-u-PA⅐PAI-1 complexes in lysates of cells incubated with 125 I-pro-UK. Most importantly, our results demonstrate that inhibition of LRP/␣ 2 MR by RAP or anti-LRP/␣ 2 MR antibodies diminishes the expression of cell surface plasminogen activator activity.
The cascade of events involving activation of uPAR-bound pro-UK, inhibition of receptor-bound tc-u-PA by plasminogen activator inhibitors, and binding and internalization of tc-u-PA⅐PAI complexes by LRP/␣ 2 MR is dependent upon multiple Cells were then washed, and incubated for 4 or 6 h in the absence or presence of 20 g/ml RAP. At the end of these periods (as well as immediately after the incubation with 125 I-pro-UK), cells were washed, and cell surface radioactivity was eluted. Equal volumes of eluate were separated using 7.5% SDS-polyacrylamide gel electrophoresis, and dried gels were exposed to autoradiography film for 24 h. This figure depicts the analysis of eluates prepared from cells immediately after the 30-min incubation with 125 I-pro-UK, in the presence (ϩ) or absence (Ϫ) of RAP (time ϭ 0), or after 4 or 6 h of additional incubation in the presence (ϩ) or absence (Ϫ) of RAP. The diffuse bands with a molecular mass of ϳ110 kDa in 0 disappeared following mild reduction, and likely represent pro-UK dimers. Similar results were obtained using ED 77 cells.
FIG. 8. Effect of RAP-mediated inhibition of ligand uptake by LRP/␣ 2 MR on the regeneration of cell surface uPAR. A, ED 27 cells were preincubated for 30 min with 4 nM unlabeled pro-UK, to saturate uPAR, in the absence or presence of 20 g/ml RAP. Medium was then removed, and cells preincubated with pro-UK and RAP were subsequently incubated at 37°C in fresh medium containing RAP (white bars). Cells that had been preincubated with only pro-UK were further incubated in fresh medium alone (black bars). After 0.5, 2.0, 4.0, and 6.0 h of incubation at 37°C, cells were washed, and the binding of 125 I-pro-UK was measured. B, control cells were treated in the same manner as the cells described in A, but preincubated with either fresh medium containing RAP (white bars) or medium alone (black bars), in the absence of pro-UK. Data represent the mean Ϯ S.D. of triplicate points from an experiment representative of three so performed. Similar results were obtained using ED 77 cells.
interactions that are difficult to assess kinetically in an integrated manner. In our studies, the role of LRP/␣ 2 MR in the regulation of cell surface plasminogen activator activity was investigated by inhibiting the function of the endocytic receptor using RAP (9,14,41,42). This strategy avoids the difficulty of identifying agonists that selectively influence LRP/␣ 2 MR expression and/or activity without altering that of other secreted and/or cell-associated proteins involved in plasminogen activation (65). Our premise that the effects of RAP on the uptake and clearance of 125 I-pro-UK (or 125 I-tc-u-PA⅐PAI complexes) and cell surface plasminogen activator activity were mediated through interactions with LRP/␣ 2 MR is supported by the observation that these effects were mimicked by anti-LRP/␣ 2 MR antibodies.
Our studies are consistent with a model in which inhibition of LRP/␣ 2 MR-mediated ligand uptake by RAP and/or anti-LRP/ ␣ 2 MR antibodies selectively impairs the clearance of proteolytically inactive tc-u-PA⅐PAI complexes formed on the cell surface after the binding and activation of pro-UK. These complexes compete with pro-UK and tc-u-PA for binding to uPAR and hence impair the ability of uPAR to promote cell surface plasmin generation. This model is supported by several prior observations. First, although pro-UK may be directly internalized by LRP/␣ 2 MR (12,66), this process most likely does not involve prior binding to uPAR and occurs slowly in cells that do not overexpress the endocytic receptor (61); this was confirmed in our studies by the observation that RAP did not grossly affect the disposition of cell-associated 125 I-pro-UK (Fig. 7). In contrast, tc-u-PA⅐PAI-1 complexes bind to LRP/␣ 2 MR with 10 -20fold greater affinity than pro-UK (12,18) and are internalized preferentially; hence, the clearance of these complexes is selectively impaired in the presence of RAP. Indeed, the fact that relatively minor amounts of tc-u-PA⅐PAI-1 complexes were detectable on the surface of cells incubated with 125 I-pro-UK in the absence of RAP confirms reports demonstrating that these complexes are internalized rapidly (23,24,37,61,67). Furthermore, because tc-u-PA⅐PAI-1 complexes bind to uPAR with an affinity similar to that of pro-UK or tc-u-PA (60), the ongoing generation of complexes, in conjunction with selective inhibition of their clearance, results in an increased concentration of proteolytically inactive complexes that compete for binding to a fixed number of uPARs. Occupancy by tc-u-PA⅐PAI complexes impairs the ability of uPAR to promote plasminogen activator activity and is likely to be primarily accountable for inhibition of such activity by RAP and/or anti-LRP/␣ 2 MR antibodies. However, the fact that relatively minor (ϳ12%) reductions in the percentage of uPAR occupied by pro-UK led to a marked reduction (ϳ50%) in cell surface plasminogen activator activity suggests that tc-u-PA⅐PAI complexes might also inhibit cellular plasminogen activation through additional pathways. Alternatively, the assay used for measurement of cell surface plasminogen activator activity, which reflects amplification of pro-UK activation by plasmin, as well as reciprocal activation of plasminogen by uPAR-bound tc-u-PA (1) or pro-UK (68), may be more sensitive to minor reductions in cell surface uPAR occupancy by u-PA.
In addition to complexes between tc-u-PA and PAI-1, a complex with molecular mass consistent with that of tc-u-PA and PAI-2 also accumulated on cells in the presence of RAP. Should additional studies confirm that this complex indeed consists of tc-u-PA and PAI-2, these studies would extend previous reports (60,69,70) by suggesting that the clearance of tc-u-PA⅐PAI-2 complexes might be mediated by LRP/␣ 2 MR. Nevertheless, because these complexes bind to u-PAR with high affinity (60), they would be expected to compete with pro-UK or tc-u-PA for binding to the receptor in a manner similar to that of tc-u-PA⅐PAI-1 complexes.
By inhibiting the clearance of tc-u-PA⅐PAI complexes, RAP also impaired the regeneration of unoccupied uPAR. Urokinase receptors are internalized concurrently with tc-u-PA⅐PAI complexes (22), and although the complexes undergo lysosomal degradation, uPARs are recycled (25). This observation is supported by our studies, which demonstrate that increased binding of 125 I-pro-UK to cells on which uPAR were saturated with unlabeled pro-UK was detectable after only 30 min of incubation at 37°C (Fig. 8). In the presence of RAP, however, this process was significantly delayed. Thus, inhibition of LRP/ ␣ 2 MR-mediated clearance of tc-u-PA⅐PAI complexes by RAP also impaired the regeneration of cell surface uPAR, suggesting a second mechanism by which RAP and/or anti-LRP/␣ 2 MR antibodies inhibited the expression of cell surface plasminogen activator activity.
Taken together, these studies demonstrate that on trophoblasts, LRP/␣ 2 MR promotes cell surface plasminogen activator activity. However, the effects of LRP/␣ 2 MR on cellular plasmin generation is likely to be cell-specific and dependent upon the levels of uPAR, u-PA, and plasminogen activator inhibitors produced by specific cell types. However, previous studies suggest the potential importance of cellular plasmin generation in processes such as cell migration. For example, a plasmin-dependent pathway of migration, which is inhibited by anti-LRP/ ␣ 2 MR antibodies, has been demonstrated on smooth muscle cells (31). In contrast, the migration of murine embryonic fibroblasts lacking LRP/␣ 2 MR (MEF-2) is enhanced (71). Increased migration of the latter cells might be explained, in part, by their increased expression of uPAR, and the higher levels of u-PA present in their conditioned medium (71). Nevertheless, reconciliation of these contrasting observations will require more detailed evaluation of the effect of LRP/␣ 2 MR on specific cellular processes, including uPAR-dependent adhesion (72,73), activation of u-PA/uPAR-initiated signaling pathways (33), and, as suggested by the current study, cell surface plasmin generation.