The Very Low Density Lipoprotein Receptor Regulates Urokinase Receptor Catabolism and Breast Cancer Cell Motility in Vitro *

The very low density lipoprotein receptor (VLDLr) binds diverse ligands, including urokinase-type plasminogen activator (uPA) and uPA-plasminogen activator inhibitor-1 (PAI-1) complex. In this study, we characterized the effects of the VLDLr on the internalization, catabolism, and function of the uPA receptor (uPAR) in MCF-7 and MDA-MB-435 breast cancer cells. When challenged with uPA·PAI-1 complex, MDA-MB-435 cells internalized uPAR; this process was inhibited by 80% when the activity of the VLDLr was neutralized with receptor-associated protein (RAP). To determine whether internalized uPAR is degraded, we studied the catabolism of [35S]methionine-labeled uPAR. In the absence of exogenous agents, the uPAR catabolism t 1 2 was 8.2 h. uPA·PAI-1 complex accelerated uPAR catabolism (t 1 2 to 1.8 h), while RAP inhibited uPAR catabolism in the presence (t 1 2 of 7.8 h) and absence (t 1 2 of 16.9 h) of uPA·PAI-1 complex, demonstrating a critical role for the VLDLr. When MCF-7 cells were cultured in RAP, cell surface uPAR levels increased gradually, reaching a new steady-state in 3 days. The amount of uPA which accumulated in the medium also increased. Culturing in RAP for 3 days increased MCF-7 cell motility by 2.2 ± 0.1-fold and by 4.4 ± 0.3-fold when 1.0 nm uPA was added. The effects of RAP on MCF-7 cell motility were entirely abrogated by an antibody which binds uPA and prevents uPA binding to uPAR. MCF-7 cells that were cultured in RAP demonstrated increased levels of activated mitogen-activated protein kinases. Furthermore, the MEK inhibitor, PD098059, decreased the motility of RAP-treated cells without affecting control cultures. These studies suggest a model in which the VLDLr regulates autocrine uPAR-initiated signaling and thereby regulates cellular motility.

The very low density lipoprotein receptor (VLDLr) binds diverse ligands, including urokinase-type plasminogen activator (uPA) and uPA-plasminogen activator inhibitor-1 (PAI-1) complex. In this study, we characterized the effects of the VLDLr on the internalization, catabolism, and function of the uPA receptor (uPAR) in MCF-7 and MDA-MB-435 breast cancer cells. When challenged with uPA⅐PAI-1 complex, MDA-MB-435 cells internalized uPAR; this process was inhibited by 80% when the activity of the VLDLr was neutralized with receptor-associated protein (RAP). To determine whether internalized uPAR is degraded, we studied the catabolism of [ 35 S]methionine-labeled uPAR. In the absence of exogenous agents, the uPAR catabolism t1 ⁄2 was 8.2 h. uPA⅐PAI-1 complex accelerated uPAR catabolism (t1 ⁄2 to 1.8 h), while RAP inhibited uPAR catabolism in the presence (t1 ⁄2 of 7.8 h) and absence (t1 ⁄2 of 16.9 h) of uPA⅐PAI-1 complex, demonstrating a critical role for the VLDLr. When MCF-7 cells were cultured in RAP, cell surface uPAR levels increased gradually, reaching a new steady-state in 3 days. The amount of uPA which accumulated in the medium also increased. Culturing in RAP for 3 days increased MCF-7 cell motility by 2.2 ؎ 0.1-fold and by 4.4 ؎ 0.3-fold when 1.0 nM uPA was added. The effects of RAP on MCF-7 cell motility were entirely abrogated by an antibody which binds uPA and prevents uPA binding to uPAR. MCF-7 cells that were cultured in RAP demonstrated increased levels of activated mitogen-activated protein kinases. Furthermore, the MEK inhibitor, PD098059, decreased the motility of RAP-treated cells without affecting control cultures. These studies suggest a model in which the VLDLr regulates autocrine uPAR-initiated signaling and thereby regulates cellular motility.
In normal mouse development, LDL receptor homologues play distinct roles. Homozygous LRP deficiency is embryonic lethal (18). gp330/megalin-deficient mice survive gestation but die shortly thereafter due to abnormal lung development (19), whereas VLDLr-deficient mice survive but demonstrate decreased body weight, body mass index, and adipose tissue mass (20). These diverse phenotypes may reflect differences in the cells or tissues that express the various LDL receptor homologues. Alternatively, uncharacterized differences in receptor function may be involved.
Our laboratory recently identified a possible role for LRP as a regulator of cellular motility (21). We studied murine embryonic fibroblasts (MEFs) that are LRP-deficient and wild-type MEFs from the same mouse strain. These cells do not express the VLDLr or gp330/megalin (22). When allowed to migrate into denuded areas of vitronectin-coated cell culture wells, the LRP-deficient MEFs migrated almost twice as rapidly as wildtype cells (21). The increased motility of the LRP-deficient MEFs was at least partially explained by an increase in the level of cell surface uPAR and by an increase in the amount of uPA which accumulated in the conditioned medium of these cells (21). In diverse systems, uPA binding to uPAR promotes cellular migration by localizing cell surface proteinase activity, initiating signal transduction, and/or by regulating cellular adhesion (reviewed in Refs. 23 and 24).
Unlike LRP-deficient MEFs, vascular smooth muscle cells (VSMCs), which are treated with RAP to deactivate LDL receptor homologues, demonstrate decreased motility (25,26). Interestingly, when MEFs are treated with RAP, while the migration assay is underway, no change in motility is observed (21). Although it was suggested that the uPA/uPAR system may be responsible for the changes in VSMC motility which accompany RAP treatment (25,26), experiments were not performed to address this possibility. Other LRP ligands also may be involved. For example, thrombospondin 1 has been shown to inhibit the motility of VSMCs but not fibroblasts (27). It is also possible that RAP affects VSMCs differently than MEFs since VSMCs express VLDLr in addition to LRP (25,28).
The hypothesis that LRP regulates cellular motility by altering the activity of the uPA/uPAR system is supported by recent studies demonstrating a role for LRP in uPAR endocytosis. uPA⅐PAI-1 complex, which is bound to uPAR, still binds to LRP (29). This interaction not only results in the internalization of uPA⅐PAI-1 complex, but promotes uPAR internalization as well (30). Thus, it has been proposed that uPA⅐PAI-1 complex bridges uPAR to LRP by forming a tetramolecular complex that undergoes endocytosis as an intact unit (18,31). Internalized uPAR recycles back to the cell surface (31); however, the efficiency of recycling remains unclear. If the efficiency is less than 100%, then LRP may promote uPAR degradation in lysosomes, explaining why LRP-deficient MEFs have increased levels of cell surface uPAR (21).
The goal of the present investigation was to characterize the role of the VLDLr in the regulation of cell surface uPAR expression and cellular motility. Our studies were performed using breast cancer cell lines which express VLDLr but do not express LRP or gp330/megalin. When the VLDLr was neutralized, by culturing these cells in the presence of RAP, the level of cell surface uPAR increased gradually, reaching a new steady-state. The increase in cell surface uPAR was explained by a decrease in the rate of uPAR catabolism. Neutralizing the VLDLr also increased cellular motility. The increase in motility was entirely counteracted by an antibody which binds endogenously produced uPA and prevents uPAR ligation. We have previously shown that uPA promotes MCF-7 cell motility by activating the MAP kinases, extracellular signal-regulated kinase (ERK) 1 and ERK2 (32). In RAP-treated breast cancer cells, the levels of activated ERK1 and ERK2 were increased. Furthermore, the motility of RAP-treated cells was selectively inhibited by an antagonist of ERK-dependent signaling. These newly identified activities of the VLDLr indicate a potentially important role for this receptor as a regulator of cancer cell physiology.

MATERIALS AND METHODS
Proteins and Reagents-Single-chain uPA (scuPA), two-chain uPA (tcuPA), and a polyclonal antibody which specifically recognizes human uPAR were provided by Drs. Jack Henkin and Andrew Mazar (Abbott Laboratories). tcuPA was inactivated with diisopropyl fluorophosphate to form DIP-uPA, as described previously (32). A monoclonal antibody specific for the amino-terminal fragment of human uPA was from American Diagnostica. This antibody prevents the binding of uPA to cell surface uPAR (33). Polyclonal antibody 399R, which recognizes human uPAR, was also from American Diagnostica. PAI-1 was provided by Dr. Duane Day (Molecular Innovations). An expression construct encoding RAP as a glutathione S-transferase (GST) fusion protein was provided by Dr. Joachim Herz (University of Texas Southwestern Medical Center, Dallas, TX). GST-RAP was expressed and purified as described previously (34), and used without further modification. GST does not interfere with the function of RAP and does not independently affect any of the activities of LDL receptor homologues (34 -36). The GST-RAP preparations used in this study contained less than 5 ng/ml endotoxin in a 0.2 M solution, as determined by Pyrotell Limulus amebocyte clotting times. In control experiments, purified endotoxin, at 10 ng/ml, did not affect MCF-7 cell motility or uPAR expression. Na 125 I and [ 35 S]methionine were from Amersham. The specific MAP kinase kinase (MEK) inhibitor, PD098059, was from Calbiochem. A polyclonal antibody which recognizes only active, phosphorylated ERK1 and ERK2 (p44/42) was provided by Dr. Michael Weber (University of Virginia). The polyclonal antibody which recognizes total ERK1 and ERK2 was from Zymed Laboratories Inc. (San Francisco, CA).
Ligand Blot and Immunoblot Analysis-MDA-MB-435 and MCF-7 cells were solubilized in 50 mM HEPES, 0.5 M NaCl, 0.05% Tween 20, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml E-64, 10 g/ml aprotinin, and 10 g/ml leupeptin. Equal amounts of cellular protein (50 g) were subjected to SDS-PAGE on 5% slabs and electrotransferred to nitrocellulose membranes (Millipore). The membranes were blocked with 50 mM Tris, 150 mM NaCl, pH 7.4 (TBS), containing 5% milk for 12 h at 4°C. Ligand blot analysis was performed to detect membrane-associated RAP-binding proteins. The membranes were incubated with 200 nM GST-RAP in TBS with 5 mM CaCl 2 , 0.02% Tween 20, and 5% milk for 1 h at 25°C. The membranes were then probed with GST-specific monoclonal antibody (Sigma) for 1 h at 25°C, followed by goat anti-mouse IgG-peroxidase conjugate (Sigma). Secondary antibody was detected by enhanced chemiluminescence. VLDLr was detected by immunoblot analysis using similarly prepared nitrocellulose membranes and polyclonal anti-human VLDLr antibody (kindly provided by Drs. Keith McCrae and Mats Gåfvels). LRP heavy chain was detected with monoclonal antibody 8G1, provided by Dr. Dudley Strickland (American Red Cross, Rockville, MD).
Cellular Degradation of 125 I-GST-RAP-GST-RAP was radioiodinated, using Iodo-Beads (Pierce), to a specific activity of 1-2 Ci/g. MCF-7 cells were washed with EBSS, 25 mM HEPES, pH 7.4, and 5 mg/ml bovine serum albumin (EHB medium). 125 I-GST-RAP (10 nM) was then added to the cultures. A 100-fold molar excess of nonradiolabeled GST-RAP was added to some cultures so that specific RAP degradation could be determined. The cells were allowed to incubate for various periods of time at 37°C. Cellular degradation of 125 I-GST-RAP was detected by measuring the increase in trichloroacetic acid soluble radioactivity in the medium.
Binding of DIP-uPA to MCF-7 Cells-MCF-7 cells were cultured in the presence or absence of GST-RAP (200 nM) for up to 5 days. The medium and GST-RAP were replaced daily. Our analysis of RAP catabolism by MCF-7 cells demonstrated that the concentration of GST-RAP in the medium decreased by less than 1% in each 24-h culturing period. Specific binding of DIP-uPA to RAP-treated and control MCF-7 cells was compared. DIP-uPA was radioiodinated with Iodo-Beads to a specific activity of 2-4 Ci/g. The cultures were washed three times and then incubated with 125 I-DIP-uPA (0.15-10 nM) in EHB for 4 h at 4°C. In this uPA concentration range, high affinity binding to uPAR is selectively detected and low affinity interactions, such as those that might occur with the VLDLr, do not contribute significantly (3,32,37,38). To quantitate specific binding, a 50-fold molar excess of nonradiolabeled DIP-uPA was added to some cultures. At the end of each binding experiment, the cultures were washed four times at 4°C; cell associated radioactivity was recovered in 1 M NaOH and quantitated in a ␥-counter. Cellular protein was determined by the bicinchoninic acid assay (Sigma). To calculate the number of specific uPA-binding sites per cell, the average mass of the MCF-7 cell was determined. Suspended cells were counted using a hemocytometer or a Coulter counter (yielding equivalent results) and then extracted for protein determination. The mass was 0.94 Ϯ 0.07 ng/cell (n ϭ 5).
uPA Accumulation in Conditioned Medium-MCF-7 cells were incubated for 24 h in RPMI, without serum, in the presence or absence of 0.1 M GST-RAP. Conditioned medium (CM) was recovered and concentrated 30-fold using Centricon concentrators with 10-kDa exclusion filters (Amicon). To detect plasminogen activator, concentrated CM was diluted 1:10 into solutions that contained 1.0 M [Glu 1 ]plasminogen and 0.5 mM Val-Leu-Lys-7-amido-4-methylcoumarin (VLK-AMC). Fluorescence emission at 460 nm (excitation at 380 nm) was monitored for 1 h at 25°C. These tracings were converted using a first derivative function so that the resulting plots showed relative plasmin concentration against time. The absolute concentration of uPA in MCF-7 cell CM was determined by comparing the maximum velocity of plasminogen activation to a standard curve generated with different concentrations of purified tcuPA (0.1-10 nM). In control experiments, we determined that GST-RAP does not affect the kinetics of plasminogen activation or VLK-AMC hydrolysis. In additional control experiments, the selective uPA inhibitor, amiloride (1.0 mM) was used to demonstrate that uPA is the primary plasminogen activator in MCF-7 cell CM (39). Due to amiloride fluorescence, these experiments were performed using the plasmin-specific chromogenic substrate, VLK-p-nitroanilide.
VLDLr in uPAR Endocytosis-MDA-MB-435 cells were chosen for these experiments since these cells have high levels of cell surface uPAR, compared with MCF-7 cells (Ref. 40 and our results). The MDA-MB-435 cells were treated with GST-RAP (1 M) or vehicle for 15 min at 37°C. uPA⅐PAI-1 complex (10 nM), which was pre-formed by reacting tcuPA with PAI-1 at a 1:1 molar ratio, or DIP-uPA (10 nM) was added to the medium and incubation was allowed to proceed for 20 min at 37°C. The cultures were then placed on ice and washed 3 times with ice-cold EBSS, 10 mM HEPES, pH 7.4. A mild acid wash was then performed to dissociate uPA⅐PAI-1 complex or DIP-uPA (21,31). The acid wash sequence was: 50 mM glycine-HCl, 100 mM NaCl, pH 3.0, for 10 min; 0.5 M HEPES, 0.1 M NaCl, pH 7.5; and then three washes with ice-cold EBSS, 10 mM HEPES, pH 7.4. Cell surface uPAR was quantitated by measuring specific binding of 125 I-DIP-uPA (10 nM).
Kinetics of uPAR Catabolism and the Role of the VLDLr-MDA-MB-435 cells were cultured for 12 h in methionine-free Dulbecco's modified Eagle's medium and then for 24 h in methionine-free Dulbecco's modified Eagle's medium supplemented with [ 35 S]methionine (10 Ci/ml). The cultures were chased for 1 h with methionine-containing complete medium, washed, and incubated in fresh medium, in the presence or absence of uPA⅐PAI-1 complex (0.5 nM) and GST-RAP (200 nM). At various times, the cells were solubilized in 10 mM HEPES, 150 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 100 mM N-octyl glucoside, 10 g/ml E-64, 10 g/ml aprotinin, and 10 g/ml leupeptin, pH 7.4. [ 35 S]Methionine-labeled uPAR was recovered from equal amounts of each cell extract by immunoprecipitation with uPAR-specific antibody (42 g/ ml), in the presence of 0.1% (w/v) ovalbumin and 10 mM EDTA. Antibody-antigen complexes were isolated with Protein A-agarose (Sigma). In control experiments, glycoprotein CD44 was recovered from the same cell extracts by immunoprecipitation using an antibody from Endogen (41) and rabbit anti-mouse IgG (Jackson Immunoresearch Laboratories). Immunoprecipitated proteins were subjected to SDS-PAGE and electrotransferred to nitrocellulose. [ 35 S]Methionine-labeled uPAR or CD44 was quantitated by PhosphorImager analysis. Western blot analysis was performed to confirm the identity of the immunoprecipitated proteins.
Analysis of MAP Kinase Activation-MCF-7 cells were cultured for 3 days in the presence of 200 nM GST-RAP or vehicle. Activation of ERK1 and ERK2 was then determined as described previously (32). Briefly, the medium was aspirated and replaced with ice-cold phosphate-buffered saline containing 1 mg/ml sodium orthovanadate. The cells were extracted at 4°C with 1.0% Nonidet P-40, 50 mM HEPES, 100 mM NaCl, 2 mM EDTA, 1 g/ml leupeptin, 2 g/ml aprotinin, 0.4 mg/ml sodium orthovanadate, 0.4 mg/ml sodium fluoride, and 5 mg/ml dithiothreitol, pH 7.4. The extracts were subjected to SDS-PAGE on 12% slabs. Proteins were transferred to nitrocellulose membranes which were probed with antibodies that detect only phosphorylated (active) ERK1 and ERK2 or total ERK1 and ERK2.
Cellular Migration-Cellular migration was studied using tissue culture-treated 6.5-mm Transwell chambers with 8.0 m pore membranes (Costar). The underside surface of each membrane was coated with 20% FBS or with 5 g/ml purified vitronectin for 2 h at 37°C. The vitronectin was purified as described previously (42). When incubated with serum, the membranes become coated primarily with vitronectin, which serves as a major attachment and spreading factor (43). After coating, the membranes were blocked with 0.5% bovine serum albumin for 2 h at 37°C and then washed with serum-free RPMI. MCF-7 cells, which had been treated with GST-RAP (200 nM) for 3 days and untreated cells were dissociated from monolayer cultures with Cell Dissociation Buffer (Life Technologies), washed with serum-free medium, and transferred to the top chamber of each Transwell at a density of 10 6 cells/ml (100 l). The bottom chamber contained RPMI with 10% FBS. GST-RAP (200 nM) was added to both chambers of Transwells which contained cells that had been RAP treated. Cells were allowed to migrate for 6 h at 37°C. The Transwell membranes were then recovered. Non-migrating cells were removed from the top surfaces with a cotton swab. The membranes were then fixed and stained with Diff-Quik (Dade Diagnostics). Cells that had migrated to the lower surfaces of the membranes were counted.

VLDLr Expression in Breast Cancer Cell Lines-Previous
studies have demonstrated that MCF-7 cells express VLDLr, but not LRP or gp330/megalin (3,44). Our ligand blot analyses confirmed this result (Fig. 1). When GST-RAP was incubated with proteins that were extracted from MCF-7 cells and immobilized on nitrocellulose, a single band with an apparent mass of 105 kDa was detected. The mobility of this band was identical to that of the VLDLr, as determined by immunoblot analysis. MDA-MB-435 cells, which express increased levels of uPAR compared with MCF-7 cells (40), also expressed VLDLr but no other members of the LDL receptor family, as determined by RAP ligand blotting. As a control, we prepared extracts of human embryonic fibroblasts. GST-RAP bound to a single high molecular mass band in the human embryonic fibroblast extracts. The mobility of this band was identical to that of the LRP heavy chain, as determined by immunoblot analysis. VLDLr was not detected in the human embryonic fibroblast extracts.
Degradation of GST-RAP by MCF-7 Cells-Studies analyzing the binding of 125 I-GST-RAP to MCF-7 cells are shown in Fig. 2. Binding was specific and saturable; the Scatchard transformation (not shown) was linear (r 2 ϭ 0.94), suggesting that a single class of binding sites was detected. The K D was 6 nM and the B max was 110 fmol/mg of cell protein (n ϭ 4). Assuming that RAP binds exclusively to the VLDLr, in MCF-7 cells, and that there is one RAP-binding site per VLDLr, then the B max corresponds to 60,000 copies of cell surface VLDLr/cell. RAP binding studies should be interpreted with caution since RAP has been reported to bind to cell surface sites that are independent of the LDL receptor family (45).
To study MCF-7 cell VLDLr function, we examined the kinetics of RAP degradation, using a nearly saturating concentration of 125 I-GST-RAP (10 nM). RAP degradation is mediated only by receptors in the LDL receptor family (1,13). Regulation of Cell Surface uPAR Expression by the VLDLr-LRP-deficient MEFs express 3-5-fold increased levels of cell surface uPAR compared with wild-type MEFs (21). To determine whether the VLDLr regulates cell surface uPAR expression, MCF-7 cells were cultured in the presence of 200 nM GST-RAP for up to 5 days. Cell surface uPAR was detected by measuring the binding of 10 nM 125 I-DIP-uPA at 4°C. As shown in Fig. 3, panel A, DIP-uPA binding increased progressively with time, reaching a maximum in 3 days. To confirm that the increase in uPA binding was due to uPAR, complete DIP-uPAbinding isotherms were generated using MCF-7 cells that had been treated with RAP for 3 days and control cells that were not RAP-treated (Fig. 3, panel B). For the control cells, the K D was 1.5 Ϯ 0.3 nM and the B max was 5.9 Ϯ 0.4 fmol/mg of cell protein (3,400 receptors/cell) (n ϭ 4); these values are consist-ent with previously reported results (32). For RAP-treated cells, the K D was unchanged (1.4 Ϯ 0.4 nM), as would be expected if the increase in uPA binding was due to an increase in cell surface uPAR. The B max was 19 Ϯ 2 fmol/mg of cell protein (10,700 receptors/cell). Thus, prolonged culturing of MCF-7 cells in RAP increases the amount of available cell surface uPAR.
Evidence for VLDLr-mediated uPAR Endocytosis-To determine whether the VLDLr mediates uPAR endocytosis, MDA-MB-435 cells were pretreated with RAP or vehicle for 15 min and then challenged with DIP-uPA or uPA⅐PAI-1 complex for 20 min at 37°C. After acid washing the cells to remove uPARassociated ligands, the level of cell surface uPAR was determined by measuring the binding of 125 I-DIP-uPA (10 nM). Without prior ligand challenge, MDA-MB-435 cells bound 115 Ϯ 5 fmol of DIP-uPA per mg of cell protein (Fig. 4). Cells that were treated with RAP for 20 min demonstrated unchanged specific 125 I-DIP-uPA binding, as expected. 125 I-DIP-uPA binding was also unchanged when cells were pretreated with nonradiolabeled DIP-uPA. This result confirms that DIP-uPA does not promote rapid uPAR internalization (30). By contrast, MDA-MB-435 cells, which were pretreated with uPA⅐PAI-1 complex, demonstrated a 90% decrease in 125 I-DIP-uPA binding. Thus, uPA⅐PAI-1 complex promoted uPAR endocytosis in MDA-MB-435 cells. When the cells were pretreated with RAP and then exposed to uPA⅐PAI-1 complex, uPAR endocytosis was blocked by 80%. These results suggest that the VLDLr is required for uPA⅐PAI-1 complex-mediated uPAR endocytosis in MDA-MB-435 cells.
The VLDLr Promotes uPAR Catabolism-For VLDLr-mediated uPAR endocytosis to decrease the steady-state level of cell surface uPAR, either the distribution of uPAR between cell surface and intracellular pools must be shifted or a fraction of the internalized uPAR must be catabolized. To study uPAR catabolism, MDA-MB-435 cells were metabolically labeled with [ 35 S]methionine. The cells were then cultured in the presence or absence of RAP. At various times, uPAR was recovered by immunoprecipitation. Representative immunoprecipitates are shown in Fig. 5, panel A. We confirmed that the major band was uPAR by immunoblot analysis (results not shown). The minor bands are probably proteins which co-immunoprecipitate with uPAR, as previously demonstrated by others (46 -49).
The kinetics of uPAR catabolism are shown in Fig. 5,  Our results demonstrated that uPA⅐PAI-1 complex promotes uPAR internalization. To determine whether uPA⅐PAI-1 complex accelerates uPAR catabolism, we cultured metabolically labeled MDA-MB-435 cells in the presence of 0.5 nM uPA⅐PAI-1 complex. As shown in Fig. 5, panel C, the rate of uPAR catabolism was substantially increased; the first-order rate constant was 3.8 ϫ 10 Ϫ1 h Ϫ1 , which corresponds to a t1 ⁄2 of 1.8 h. When RAP was added to the culture medium with uPA⅐PAI-1 complex, uPAR catabolism was inhibited; the rate constant was 8.9 ϫ 10 Ϫ2 h Ϫ1 and the t1 ⁄2 was 7.8 h. Thus, recycling of inter-nalized uPAR is not 100% efficient. Instead, a significant fraction of the uPAR, which is internalized via a pathway that requires both uPA⅐PAI-1 complex and the VLDLr, is targeted for degradation.
Regulation of MCF-7 Cell Motility by the VLDLr and the Role of the uPA/uPAR System-Cellular migration was studied using serum-coated Transwell membranes (Fig. 6, panel A). When MCF-7 cells were not pre-cultured in RAP or treated with uPA, 80 Ϯ 18 cells penetrated the membranes within 6 h. ScuPA (1 nM) promoted MCF-7 cell migration, as previously reported (32). Pre-culturing in RAP for 3 days also increased MCF-7 cell motility (2.2 Ϯ 0.1-fold, n ϭ 8, p Ͻ 0.001); however, when RAP was incubated with the cells only while the migration assay was underway (no pre-culturing), cellular motility was unchanged (results not shown). ScuPA (1 nM) increased the motility of MCF-7 cells that had been pre-cultured in RAP still further (4.4 Ϯ 0.3-fold, n ϭ 8). When the Transwell membranes were coated with purified vitronectin instead of serum, identical results were obtained (results not shown). In control experiments, we demonstrated that GST-RAP does not affect MCF-7 cell proliferation.
In our previous study (21), results were presented to suggest FIG. 4. The effects of VLDLr on uPAR endocytosis. MDA-MB-435 cells were treated with RAP or vehicle for 15 min at 37°C. Cultures were then pulse-exposed to DIP-uPA, uPA⅐PAI-1 complex or vehicle for 20 min and immediately chilled to 4°C. After acid washing, specific binding of 125 I-DIP-uPA (10 nM) was determined. Control represents cultures that were pretreated and pulse-exposed to vehicle. RAP represents cultures that were pretreated with RAP and then pulse-exposed to vehicle. DIP-uPA represents cultures that were pretreated with vehicle and pulse-exposed to DIP-uPA. uPA-PAI-1 represents cultures that were pretreated with vehicle and then pulse-exposed to uPA⅐PAI-1 complex. RAP ϩ uPA-PAI-1 represents cultures that were pretreated with RAP and then pulse-exposed to uPA⅐PAI-1 complex. Cells extracts were incubated with uPAR-specific antibody followed by Protein A-agarose. The immunoprecipitates were subjected to SDS-PAGE on 8% slabs and transferred to nitrocellulose. Radioactivity was quantitated by PhosphorImager analysis. Panel A shows immunoprecipitates of metabolically labeled cultures that were treated with RAP "ϩ RAP" or vehicle "Ϫ RAP" for the indicated times. In panel B, the amount of labeled uPAR recovered in immunoprecipitates from RAPtreated cells (f) and vehicle-treated cells (OE) was plotted against time, as described in the text. Panel C shows equivalent graphs analyzing labeled uPAR recovery from cells treated with uPA⅐PAI-1 complex (OE) or uPA⅐PAI-1 complex plus RAP (f). that autocrine activation of uPAR by endogenously produced uPA may be responsible for the increased motility of LRPdeficient MEFs. To determine whether the uPA/uPAR system is responsible for the increased motility of RAP-treated MCF-7 cells, we performed migration assays in the presence of a uPAspecific antibody that blocks uPA binding to uPAR (25 g/ml). As shown in Fig. 6, the antibody had no effect on the motility of control cells, suggesting that autocrine activation of uPAR is not significant when the VLDLr is active. By contrast, the antibody completely neutralized the activity of exogenously added uPA, confirming the effectiveness of the antibody in this system. uPA-specific antibody also inhibited the motility of RAP-treated cells; these cells migrated comparably to cells that had not been precultured in RAP. In control experiments, nonimmune mouse IgG (25 g/ml) did not affect the migration of control MCF-7 cells or cells that had been cultured in RAP. Furthermore, non-immune IgG did not inhibit the response to exogenously added uPA. These results suggest that the increase in MCF-7 cell motility, which is induced by culturing in RAP, results from the activity of endogenously produced uPA.
Previous studies have either failed to detect uPA expression by MCF-7 cells or have detected very low levels (32,40). Thus, we re-examined the question of uPA synthesis and secretion by MCF-7 cells using a highly sensitive assay in which plasminogen activator is detected in CM based on its ability to activate plasminogen. In three separate experiments, low levels of plasminogen activator were detected in CM isolated from control MCF-7 cells (Fig. 7, panel B). RAP significantly increased the recovery of plasminogen activator in the CM. The plasminogen activator was uPA since amiloride inhibited 96% of the activity (results not shown). The concentration of uPA in the CM recovered from RAP-treated cells (before concentrating) was 53 Ϯ 8 pM (n ϭ 4), as determined by comparison to a standard curve generated with purified tcuPA. Our activity assay detects scuPA and tcuPA; however, if PAI-1 was present in the CM, then the uPA concentration may have been underestimated (21).
The concentration of uPA in MCF-7 cell CM was substantially lower than the K D for uPA binding to MCF-7 cell uPAR. Thus, new experiments were performed to determine whether exogenously added uPA, at very low concentrations, promotes MCF-7 cell motility. Fig. 7 shows that 50 pM uPA induced a statistically significant increase in MCF-7 cell motility (p Ͻ 0001). The activity of 50 pM scuPA was entirely neutralized by uPA-specific antibody and by uPAR-specific antibody 399R. In separate control experiments, we confirmed that antibody 399R completely inhibits the specific binding of 125 I-DIP-uPA to MCF-7 cells. We have also shown that antibody 399R inhibits MAP kinase activation in response to uPA. 2 Thus, these experiments demonstrate that uPA, at low concentrations, increases MCF-7 cell motility by a mechanism that requires binding to uPAR.
MAP Kinase Is Activated in RAP-treated MCF-7 Cells-uPA activates ERK1 and ERK2 in MCF-7 cells and this response is necessary for uPA-promoted migration (32). Since RAP-treated MCF-7 cells have increased levels of cell surface uPAR and accumulate increased amounts of uPA, we undertook experiments to determine whether the extent of activation of ERK1 and/or ERK2 is increased in these cells as well. MCF-7 cells were cultured in standard FBS-supplemented medium, in the presence or absence of 200 nM GST-RAP for 3 days, and isolated without adding exogenous stimulants 12 h after the last change in medium. Activated ERK1 and ERK2 were detected by im- were precultured in RAP for 3 days and control cells that were cultured in vehicle (C) were allowed to migrate in serum-coated Transwell chambers in the presence or absence of scuPA, as shown. uPA-specific antibody (uPA-Ab) or non-immune IgG were also added as indicated. The number of cells migrating across the membrane is expressed as a percentage of that observed with control cells (no RAP pretreatment or scuPA exposure). Each bar represents the results of four separate experiments with triplicate determinations. Panel B, to study uPA secretion by MCF-7 cells, cultures were incubated in serumfree medium for 24 h, in the presence or absence of RAP. CM was collected and concentrated. The concentrated CM was incubated with plasminogen and VLK-AMC. Substrate hydrolysis is shown. "Background" shows the results obtained when plasminogen was activated using un-conditioned medium. munoblot analysis. A single experiment, in which six separate cultures were analyzed, is shown in Fig. 8, panel A. Culturing in RAP increased the levels of activated ERK1 and ERK2 by 2.8 Ϯ 0.5-and 2.6 Ϯ 0.6-fold, respectively (n ϭ 6). Furthermore, PD098059, a selective inhibitor of MAP kinase kinase (MEK), inhibited the migration of RAP-treated cells without affecting the migration of control cells (Fig. 8, panel B). These studies suggest that a MAP kinase-dependent signaling pathway may be selectively activated in RAP-treated MCF-7 cells and that this pathway is required for enhanced motility on serum-coated surfaces.

DISCUSSION
Previous studies have shown that LRP and the VLDLr express similar activities as receptors for free uPA and uPARassociated uPA⅐PAI-1 complex (3,7,18,29). In this study, we demonstrated that the VLDLr, like LRP (30), mediates the endocytosis of uPAR through an indirect mechanism that depends on uPA⅐PAI-1 complex. When MDA-MB-435 cells were treated with uPA⅐PAI-1 complex at 37°C, uPAR was rapidly internalized by a RAP-inhibited pathway. The most likely explanation for this result is that uPA⅐PAI-1 complex bridges the VLDLr and uPAR, on the cell surface, so that uPAR is internalized in clathrin-coated pits with the VLDLr. uPA binding to uPAR causes conformational changes in the receptor which could also be involved in the VLDLr interaction (51). In our 20-min endocytosis assays, DIP-uPA did not promote uPAR internalization, consistent with previous studies demonstrating that uPA⅐uPAR complex is stable on the cell surface (52,53). However, we cannot rule out the possibility that uPA affects the rate of internalization of uPAR over a period of days.
In cells that express LRP, internalized uPAR is transferred to acidified endosomes where uPA⅐PAI complex is dissociated before the uPAR recycles back to the cell surface (18,31,54). If recycling is 100% efficient, the expected outcome of this pathway is to re-generate un-liganded uPAR on the cell surface, which is available to bind free uPA (1,18,31). In MCF-7 cells that were cultured for at least 3 days in RAP, the level of cell surface uPAR was increased. To explain these results, we studied the catabolism of metabolically labeled uPAR. When cultured in the presence of uPA⅐PAI-1 complex, MDA-MB-435 cells rapidly digested uPAR (t1 ⁄2 of 1.8 h versus 8.2 h) and this process was inhibited by RAP, indicating that at least a fraction of the uPAR, which is internalized in association with the VLDLr, is degraded. RAP also prolonged the survival of uPAR under standard cell culturing conditions, in the absence of FIG. 8. Activation of ERK1 and ERK2 in VLDLr-neutralized cells. Panel A, immunoblot analysis detecting phosphorylated ERK1 and ERK2 and total ERK1 and ERK2 in six separate MCF-7 cell cultures which were analyzed simultaneously. Three of the cultures, which were maintained in the presence of GST-RAP for 3 days are designated by ϩ. Levels of phosphorylated ERK1 and ERK2 were standardized by comparison to total ERK levels. The levels of phosphorylated ERK1 and ERK2 in RAP-treated cells were then compared with the levels detected in matched controls. Panel B, MCF-7 cells were allowed to migrate for 6 h through Transwells membranes that were precoated, on the underside surfaces, with FBS. The MEK inhibitor, PD098059, was added to the top chamber as indicated by ϩ. For bars labeled Control, the cells were precultured in vehicle for 3 days and then allowed to migrate. For bars labeled RAP treated, the cells were precultured in RAP for 3 days and then RAP was added to both Transwell chambers. Cellular migration was standardized to that observed in control cells which were not RAP pretreated or treated with drug. Each bar represents the results of three separate experiments with triplicate determinations .   FIG. 7. MCF-7 cell migration in response to low concentrations of uPA. MCF-7 cells were allowed to migrate for 6 h in serum-coated Transwell membranes. The indicated concentrations of scuPA were added to both Transwell chambers. The response of MCF-7 cells to 50 pM uPA was studied in the presence of 25 g/ml uPA-specific antibody (uPA-Ab) or 25 g/ml uPAR-specific antibody (uPAR-Ab). exogenously added uPA⅐PAI-1 complex. At this time, we do not know whether this process depended on the formation of uPA⅐PAI-1 complex from endogenously produced uPA and PAI-1.
uPAR was still catabolized, albeit at a slower rate, when MDA-MB-435 cells were cultured in the presence of RAP. The residual catabolism may reflect bulk plasma membrane turnover, enzymatic release of uPAR from the cell surface, or the function of receptors outside the LDL receptor family. Nykjaer et al. (49) recently demonstrated that the mannose 6-phosphate/insulin-like growth factor-II receptor interacts with cell surface uPAR and targets uPAR for catabolism in lysosomes. Thus, cell surface uPAR levels may be controlled by diverse plasma-membrane interactions.
MCF-7 cells that were cultured in the presence of RAP for 3 days demonstrated increased motility on serum-or vitronectincoated surfaces. When the cells were not precultured in RAP and allowed to migrate in Transwell chambers in the presence of RAP, motility was unchanged. Thus, the mechanism by which RAP promotes MCF-7 cell motility probably requires a change in the phenotype of the cell, which occurs slowly, as opposed to the more simple mechanism in which motility is influenced entirely by agents that accumulate at increased levels in solution when the VLDLr is blocked. Importantly, uPA-specific antibody inhibited the migration of MCF-7 cells that were exposed to exogenous uPA or precultured in RAP. The same antibody had no effect on the motility of control MCF-7 cells. These results provide evidence that autocrine activation of uPAR is responsible for the increase in the motility of RAP-treated cells. Apparently, in control cells, the level of cell surface uPAR and/or the amount of uPA which accumulates in the medium are too low to significantly affect motility.
Even though the level of cell surface uPAR in RAP-treated MCF-7 cells was increased to 10 4 sites per cell, the level of endogenously produced uPA was still low, compared with that observed in other cell lines, including LRP-deficient MEFs (21). Thus, we hypothesized that MCF-7 cells are highly sensitive to low concentrations of uPA and respond to low levels of uPAR ligation. When MCF-7 cells, which had not been precultured in RAP, were exposed to uPA, at concentrations from 50 pM to 1.0 nM, significant increases in motility were observed. Furthermore, an essential role for uPAR was demonstrated. Thus, MCF-7 cells are sensitive to concentrations of uPA that are substantially lower than the K D for uPAR binding. Although we did not measure the sensitivity to uPA of RAP pretreated MCF-7 cells, it is possible that these cells respond to even lower uPA concentrations, due to the increase in cell surface uPAR.
Further evidence that prolonged culturing in RAP affects the physiology of MCF-7 cells was obtained in studies of MAP kinase activation. Under standard cell-culturing conditions, the levels of activated ERK1 and ERK2 were significantly increased by RAP. While it is intriguing to speculate that the increase in activated ERK is caused by activation of the uPA/ uPAR system, we have not yet conclusively linked these two processes. When treated with a high concentration of uPA, levels of activated ERK1 and ERK2 increase rapidly but transiently in MCF-7 cells (32). This response is substantially different than the low-level sustained activation of ERK1 and ERK2 demonstrated with RAP-treated MCF-7 cells. When cells are treated with epidermal growth factor, the kinetics of ERK activation depend on the epidermal growth factor concentration (55). High epidermal growth factor concentrations induce transient activation of ERK1 and ERK2 while lower epidermal growth factor concentrations induce sustained responses (55). Whether activation of ERK1 and ERK2, in response to uPA, is transient or sustained may depend on whether the cells are pulse-exposed to a high concentration of uPA, as in our previous study (32), or continuously exposed to a low level of uPA, as is hypothesized for RAP-treated cells.
The ability of the VLDLr to regulate the uPA/uPAR system may be considered in relation to multicellular tissues such as intact breast cancers. If the VLDLr is expressed by malignant epithelial cells, as previously demonstrated (3,44), it should regulate pericellular uPA levels, irrespective of whether the uPA is synthesized by cancer cells or benign stromal cells. Alternatively, our results suggest that uPAR regulation depends on co-expression of uPAR and the VLDLr by the same cell type. LRP, which is expressed by macrophages and fibroblasts, may also regulate uPA levels in the microenvironment of the cancer but will not regulate cell surface uPAR levels in the malignant cells, if these cells are LRP negative. Thus, members of the LDL receptor family may regulate the activity of the uPA/uPAR system within cancers by both autocrine and paracrine mechanisms.