Correspondence to: Steven L. Gonias, Department of Pathology, Department of Biochemistry and Molecular Genetics, University of Virginia Health Sciences Center, Box 214, Charlottesville, VA 22908., SLG2T{at}VIRGINIA.EDU (E-mail), (804) 924-9192 (phone), (804) 982-0283 (fax)
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Abstract |
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Urokinase-type plasminogen activator (uPA) activates the mitogen activated protein (MAP) kinases, extracellular signal-regulated kinase (ERK) 1 and 2, in diverse cell types. In this study, we demonstrate that uPA stimulates migration of MCF-7 breast cancer cells, HT 1080 fibrosarcoma cells, and uPAR-overexpressing MCF-7 cells by a mechanism that depends on uPA receptor (uPAR)-ligation and ERK activation. Ras and MAP kinase kinase (MEK) were necessary and sufficient for uPA-induced ERK activation and stimulation of cellular migration, as demonstrated in experiments with dominant-negative and constitutively active mutants of these signaling proteins. Myosin light chain kinase (MLCK) was also required for uPA-stimulated cellular migration, as determined in experiments with three separate MLCK inhibitors. When MCF-7 cells were treated with uPA, MLCK was phosphorylated by a MEK-dependent pathway and apparently activated, since serine-phosphorylation of myosin II regulatory light chain (RLC) was also increased. Despite the transient nature of ERK phosphorylation, MLCK remained phosphorylated for at least 6 h. The uPA-induced increase in MCF-7 cell migration was observed selectively on vitronectin-coated surfaces and was mediated by a ß1-integrin (probably Vß1) and
Vß5. When MCF-7 cells were transfected to express
Vß3 and treated with uPA, ERK was still phosphorylated; however, the cells did not demonstrate increased migration. Neutralizing the function of
Vß3, with blocking antibody, restored the ability of uPA to promote cellular migration. Thus, we have demonstrated that uPA promotes cellular migration, in an integrin-selective manner, by initiating a uPAR-dependent signaling cascade in which Ras, MEK, ERK, and MLCK serve as essential downstream effectors.
Key Words: urokinase-type plasminogen activator, myosin light chain kinase, integrins, vitronectin, cellular migration
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Introduction |
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UROKINASE-type plasminogen activator (uPA)1 is a serine proteinase which is synthesized as a 54-kD single chain (sc) zymogen and converted into the active two chain form (tcuPA) by various proteinases, including plasmin (
uPAR is linked to the cell surface by a glycosyl-phosphatidylinositol (GPI) anchor (V-containing integrins (
The signaling cascades that are activated when uPA binds to uPAR are only partially characterized. Known protein tyrosine kinases in the Src-family, including p60fyn, p53/p56lyn, p56/p59hck, and p59fgr, have been coimmunoprecipitated with uPAR ( (
uPA promotes the migration of diverse cell types (
The goal of this investigation was to elucidate the mechanism by which uPA promotes cellular migration. Our results demonstrate that the uPA response requires uPAR-ligation and activation of the Ras/ERK-signaling pathway. MLCK functions downstream of Ras/ERK and is also an essential mediator of uPA-promoted cellular migration, suggesting that the uPA/uPAR system may regulate cytoskeletal contractility. MCF-7 cell migration on vitronectin was promoted by uPA only when migration was under the control of Vß5 and a ß1 subunitcontaining integrin (probably
Vß1), but not
Vß3, thus defining yet another novel functional relationship between integrins and the uPA/uPAR system. The activity of MLCK in uPA-promoted cellular migration and the integrin-selectivity of the response suggest a model in which uPAR-initiated signal transduction orchestrates integrin function and cytoskeletal reorganization during cellular migration.
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Materials and Methods |
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Antibodies and Reagents
scuPA and tcuPA were provided by Drs. Jack Henkin and Andrew Mazar of Abbott Laboratories. tcuPA was inactivated with 20 mM diisopropylfluorophosphate (Sigma Chemical Co.), as described previously (Vß5 (P1F6),
Vß3 (LM609), and ß1-containing integrins (6S6) were from Chemicon International. The MEK inhibitor, PD098059, the MLCK inhibitors, ML-7, ML-9, and W-7, and actinomycin D were from Calbiochem. Calcein-AM was from Molecular Probes. Leupeptin was from Boehringer Mannheim. Cycloheximide, PMSF, aprotinin, benzamidine, NaF, sodium orthovanadate, and G418 were from Sigma Chemical Co.
Cell Culture and Transfection Methods
Low-passage MCF-7 cells were kindly provided by Dr. Richard Santen (University of Virginia, Charlottesville, VA) and cultured in RPMI (Life Technologies, Inc.) supplemented with 10% FBS (Hyclone), penicillin (100 U/ml), and streptomycin (100 µg/ml) (Life Technologies, Inc.). HT 1080 human fibrosarcoma cells were from the ATCC. These cells were cultured in MEM supplemented with FBS, penicillin, and streptomycin. Cells were passaged with enzyme-free cell dissociation buffer and maintained in culture at 37°C for 48 h before conducting experiments.
The full-length human uPAR cDNA was obtained from the ATCC and subcloned into pBK-CMV (Stratagene). To generate stable MCF-7 cell lines which overexpress uPAR, 5 x 105 cells were transfected with 2 µg of the uPAR expression construct, using 10 µl of Superfect (Qiagen) for 2.5 h at 37°C. The cells were then washed with serum-free RPMI, cultured in serum-supplemented medium for 48 h, and selected in G418 (1 mg/ml) for 14 d. Single-cell clones were prepared by serial dilution and screened for uPAR overexpression by flow cytometry. Cell-surface uPAR was quantitated by measuring specific binding of 125I-DIP-uPA, as described previously (
Expression constructs which encode constitutively active rat MEK1 (S218D/S222
D) and dominant-negative rabbit MEK1 (S217
A), in pCHA and pBABE, respectively, were described previously (
V) and dominant-negative H-Ras (S17
N), in pDCR, were described by
The full-length cDNA for the ß3-integrin subunit was kindly provided by Dr. David Cheresh (Scripps Research Institute, La Jolla, CA). The cDNA was subcloned into pBK-CMV and transfected into MCF-7 cells (2 µg cDNA/5 x 105 cells) using 10 µl Superfect. The cells were cultured for 3648 h and selected in G418 (1 mg/ml) for 14 d. The transfectants were then subjected to flow cytometry, using antibody LM609 and fluorescein-conjugated antimouse IgG, to detect cell-surface Vß3 expression. After flow cytometry, the cells were cultured in RPMI supplemented with G418 (25 µg/ml), FBS, penicillin, and streptomycin. ERK activation experiments and migration assays were performed within 2 wk of obtaining flow cytometry results.
ERK Activation in Transfected Cells
To confirm that ERK activity was regulated in cells transfected to express mutant MEK1 or H-Ras, 5 x 105 MCF-7 cells were transfected with 5 µg of each cDNA construct and with 1.25 µg of a construct encoding HA-tagged ERK1, as described by
ERK phosphorylation, in ß3-integrin subunit-expressing MCF-7 cells, was also detected by immunoblot analysis. In brief, MCF-7 cells, that had been transfected and selected, were transferred to serum-free medium for 6 h and then treated with 10 nM DIP-uPA, 25 ng/ml EGF, or vehicle. Cell extracts were subjected to SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies for phosphorylated ERK1/2 and total ERK1/2, as described previously (
MLCK Phosphorylation Experiments
MCF-7 cells were cultured in 100-mm dishes until 7080% confluent, washed with phosphate-free RPMI, and metabolically labeled for 2 h at 37°C with [32P]orthophosphate (250 µCi) in serum-free RPMI supplemented with 1 mg/ml BSA and 1 mg/ml sodium orthovanadate. The labeled cells were treated with 10 nM DIP-uPA for up to 6 h. Control cells were treated with vehicle instead of DIP-uPA. In some cultures, 50 µM PD098059 was added to inhibit MEK. The PD098059 was added 15 min before adding the DIP-uPA and remained present throughout the assay. After washing the cultures, the cells were extracted with 1% NP-40, 10 mM Tris-HCl, 140 mM NaCl, 2 mM EDTA, 100 KIU/ml aprotinin, 0.1 mg/ml leupeptin, 2 mM PMSF, 50 mM NaF, 1 mM sodium vanadate, 20 mM sodium pyrophosphate, pH 8.1, for 30 min on ice and centrifuged at 800 g for 10 min. The supernatants were precleared with protein Aagarose for 1 h at 22°C. MLCK in the supernatants was then immunoprecipitated by incubation with MLCK-specific monoclonal antibody (6 µg) for 12 h at 4°C, rabbit antimouse IgG (7.5 µg) for 4 h at 4°C, and finally with protein Aagarose for 1 h at 22°C. The immunoprecipitates were subjected to SDS-PAGE on 8% acrylamide slabs and transferred to nitrocellulose. Phosphorylated MLCK was detected by autoradiography.
Serine-phosphorylation of RLC
Suspended MCF-7 cells (105 in 100 µl) were treated with 10 nM DIP-uPA or with vehicle for the indicated times at 37°C. Reactions were terminated by adding SDS sample buffer at 95°C. The whole-cell lysates were then subjected to SDS-PAGE on 15% acrylamide slabs and transferred to nitrocellulose. Immunoblot analysis was performed to detect serine-phosphorylated RLC (primary antibody at 0.5 µg/ml). The same blots were also probed to detect total RLC. In some experiments, the cells were pretreated for 15 min with drugs that inhibit MEK or MLCK, before adding uPA or vehicle.
Migration Assays
We demonstrated previously that uPA promotes MCF-7 cell migration across serum-coated Transwell membranes irrespective of whether both sides of the membrane are coated with serum or just the underside (
Transwell membranes (6.5 mm, 8.0-µm pores) (Costar) were coated with 20% FBS, purified vitronectin (5 µg/ml), or type I collagen (25 µg/ml) for 2 h at 37°C. Both membrane surfaces were blocked with 10 mg/ml BSA. MCF-7 cells, uPAR-overexpressing MCF-7 cells, and ß3-integrin subunit-expressing MCF-7 cells (105 cells in 100 µl) were pretreated with 10 nM DIP-uPA or with vehicle for 15 min, in suspension, and then added to the top chamber. Before DIP-uPA exposure, some cells were treated for 15 min with actinomycin D (10 µg/ml), cycloheximide (3 µg/ml), ML-7 (3 µM), ML-9 (30 µM), W-7 (51 µM), or with the following antibodies: uPA-specific antibody, uPAR-specific antibody, LM609, P1F6, or 6S6 (at concentrations up to 32 µg/ml). When cells were pretreated with DIP-uPA, 10 nM DIP-uPA was added to both Transwell chambers. Drugs or antibodies were added to the top chamber. The bottom chamber always contained 10% FBS. After terminating a study, cells were removed from the top surface of each membrane using a cotton swab. Cells which penetrated to the underside surfaces of the membranes were stained with Diff-Quik (Dade Diagnostics) and counted. In some experiments, migration of uPAR-overexpressing MCF-7 cells was quantitated by fixing the membranes in methanol and staining the migratory cells with 0.1% crystal violet. The dye was eluted with 10% acetic acid and the absorbance of the eluate was determined at 600 nm. In control experiments, we confirmed that crystal violet absorbance is linearly related to cell number.
HT 1080 cell migration was studied in Transwell chambers containing membranes that were coated on both surfaces with 20% FBS. 5 x 105 cells were added to the top chamber in serum-free medium and allowed to migrate for 6 h in the presence or absence of 10 nM DIP-uPA. FBS was not added to the bottom chamber. Thus, there was no chemotactic or haptotactic stimulus, suggesting that chemokinesis was detected. Nonmigrating cells were removed with a cotton swab. Cellular migration was then determined by the crystal violetstaining method.
To study the migration of GFP-expressing cells, translucent Biocoat Cell Culture Inserts (Becton Dickinson) were used instead of Transwell chambers. The insert membranes had 8-µm pores and were coated with serum or purified vitronectin. The response to uPA was not affected in this alternative system as determined by counting Diff-Quikstained cells. Cells (5 x 105) that were cotransfected with signaling effector mutants and pEGFP, or with pEGFP alone, were added to the top chamber and allowed to migrate for 6 h. In some experiments, cells were treated with inhibitors or antibodies and allowed to migrate in the presence or absence of DIP-uPA. Migrating cells were fixed in 4% paraformaldehyde and counted by fluorescence microscopy. To standardize results obtained with transfected and untransfected cells, the pEGFP-transfection efficiency was determined for each experiment. The number of GFP-positive cells which migrated across the membrane was then divided by the transfection efficiency (typically 0.20.3).
Mannosamine Treatment Protocol
Mannosamine inhibits a critical enzyme involved in the attachment of GPI-linked proteins to their anchors (
Cell Adhesion Assays
Vitronectin-coated cell culture wells were prepared by incubating purified vitronectin (5 µg/ml) in 96-well cell culture plates (Costar) for 2 h at 37°C. The wells were then blocked with BSA. MCF-7 cells that were cotransfected to express GFP and dominant-negative or constitutively active MEK1 or H-Ras (105 cells in 100 µl) were allowed to adhere for the indicated times at 37°C. To assess cellular adhesion, the wells were washed with 10 mM Hepes, 150 mM NaCl, pH 7.4, and adherent cells were quantitated by fluorescence emission. The excitation and emission wavelengths were 488 nm and 507 nm, respectively. Fluorescence emission was also determined for the total number of cells added to each well. Cellular adhesion was quantitated as a percentage of the total number of GFP-expressing cells added. In control experiments, we determined that cell adhesion and spreading do not affect fluorescence emission.
MCF-7 cells that did not express GFP were allowed to adhere to vitronectin-coated cell culture wells in the presence of integrin-specific antibodies (P1F6, LM609, 6S6) or drugs that inhibit MEK or MLCK. After washing, adherent cells were stained with Calcein-AM (
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Results |
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uPA-stimulated Cellular Migration Is Antagonized by uPAR-specific Antibody and by PD098059 in Parent and uPAR-overexpressing MCF-7 Cells
We demonstrated previously that uPA activates ERK1/2, rapidly but transiently, in MCF-7 cells and stimulates MCF-7 cell migration (
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MCF-7 cells express 3,300 copies of cell-surface uPAR, as determined by 125I-DIP-uPA binding and the experimentally determined mean cellular mass (0.94 ng) (
uPA-stimulated HT 1080 Cell Migration Requires ERK Activation
HT 1080 fibrosarcoma cells express uPAR (
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The lack of an effect of uPA- and uPAR-specific antibodies on HT 1080 cell migration, in the absence of exogenously added DIP-uPA, suggested that endogenously produced uPA is insufficient to activate autocrine uPAR-signaling in these cells. However, others have demonstrated that HT 1080 cells express substantial levels of uPA (
MEK Is Essential in the uPAR-initiated Signaling Pathway which Increases MCF-7 Cell Migration
To further explore the relationship between the uPA/uPAR system and ERK activation in promoting cellular migration, we transfected MCF-7 cells to express dominant-negative or constitutively active MEK1. To demonstrate that the MEK1 mutants were functional as upstream-modulators of ERK activation, MCF-7 cells were cotransfected to express HA-tagged ERK1. The transfectants were treated with DIP-uPA (10 nM) or vehicle for 1 min; cell extracts were immunoprecipitated using an antibody directed against the HA-epitope and phosphorylated ERK1 was detected by immunoblot analysis. As shown in Figure 3 A, MCF-7 cells that were transfected to express dominant-negative MEK1 did not contain detectable levels of phosphorylated HA-ERK1, irrespective of whether these cells were treated with DIP-uPA or not. By contrast, substantial levels of phosphorylated HA-ERK1 were detected in cells that were transfected to express constitutively active MEK1. However, when these cells were treated with DIP-uPA, no further increase in phosphorylated HA-ERK1 was observed. As a control, we cotransfected MCF-7 cells with the HA-tagged ERK1 construct and with the empty vector (pCHA) which had been used to prepare the constitutively active MEK1 mutant. In the absence of uPA, only trace levels of phosphorylated HA-ERK1 were observed; however, DIP-uPA substantially increased phosphorylated HA-ERK1 in these cells. These results demonstrate that the MEK1 mutants were functional as regulators of ERK activation in the presence and absence of uPA.
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To study the migration of MCF-7 cells, which were transiently transfected to express mutant forms of MEK1, we cotransfected the cells with pEGFP. Cotransfection efficiencies were always >90%, allowing us to selectively detect the migration of transfected cells by GFP fluorescence. As shown in Figure 3 B, cells that were transfected only with pEGFP demonstrated increased migration when treated with 10 nM DIP-uPA. When MCF-7 cells were transfected to express dominant-negative MEK1, basal migration was not significantly altered (24% decrease, P > 0.1); however, the ability of uPA to stimulate cellular migration was entirely blocked. MCF-7 cells that were transfected to express constitutively active MEK1 demonstrated increased migration in the absence of uPA; however, these cells demonstrated no change in migration when treated with DIP-uPA. Since uPAR synthesis may be regulated by a MAP kinasedependent pathway (
In Transwell migration assays, increased cellular migration may reflect increased cellular penetration of the Transwell membranes or a change in the kinetics of cellular adhesion to the membranes. To rule out the latter possibility, GFP-expressing MCF-7 cells were allowed to adhere to vitronectin-coated cell culture wells for 10, 20, 30, 40, or 60 min. Adhesion was detected by measuring fluorescence emission in a Cytofluor 2350. The kinetics of MCF-7 cell adhesion were not affected by DIP-uPA or by either MEK mutant (data not shown). PD098059 also did not affect MCF-7 cell adhesion, in the presence or absence of uPA. Thus, the differences observed in the Transwell assays reflect changes in the migration of cells which have already adhered to the Transwell membranes.
Ras Is Essential in the uPAR-initiated Signaling Pathway which Increases MCF-7 Cell Migration
Ras activation is frequently but not always necessary as an upstream activator of ERK in growth factorstimulated cells (
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To determine whether the H-Ras mutants affect the ability of uPA to stimulate cellular migration, MCF-7 cells were cotransfected to express GFP. As shown in Figure 4 B, MCF-7 cells that expressed dominant-negative H-Ras demonstrated only a slight decrease in migration in the absence of uPA, which was not statistically significant. However, these cells failed to demonstrate increased migration when treated with DIP-uPA. MCF-7 cells that expressed constitutively active H-Ras demonstrated 2.3 ± 0.3-fold increased migration in the absence of uPA, but no further increase in motility when DIP-uPA was added. Antibodies against uPA and uPAR had no effect on the migration of constitutively active H-Rasexpressing MCF-7 cells (data not shown). Thus, constitutively active H-Ras did not increase migration by regulating uPA or uPAR expression. Instead, Ras functioned as an essential mediator of the uPAR-initiated signal transduction response which stimulates MCF-7 cell migration.
uPA-stimulated MCF-7 Cell Migration Does Not Require New Gene Transcription or Protein Synthesis
When activated, ERK may translocate to the nucleus and regulate gene expression by modifying transcription factors, such as Elk-1 (
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MLCK Is Phosphorylated and Activated by a MEK-dependent Pathway in uPA-treated Cells
MLCK is a Ca2+/calmodulin-dependent kinase that phosphorylates RLC, promoting contraction of the actin-based cytoskeleton (
To determine whether uPAR-initiated signal transduction results in MLCK phosphorylation, MLCK was immunoprecipitated from MCF-7 cells that had been metabolically labeled with [32P]orthophosphate and treated with DIP-uPA. Figure 6 A shows that MLCK was phosphorylated within 1 h of exposure to uPA; however, unlike ERK1/2, MLCK phosphorylation was sustained for at least 6 h. PhosphorImager analysis was used to quantitate phosphorylated MLCK. When the results were standardized for total MLCK recovery, we did not detect significant variation in the level of phosphorylated MLCK between 1 and 6 h (Figure 6 B). Pretreatment of MCF-7 cells with PD098059, before DIP-uPA exposure, blocked MLCK phosphorylation. Thus, uPA-induced MLCK phosphorylation is MEK-dependent. We have demonstrated that purified ERK1 directly phosphorylates MLCK in vitro (data not shown), confirming the work of others (
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To determine whether MLCK, which is phosphorylated in uPA-treated cells, is also activated, we examined RLC phosphorylation in whole-cell extracts using an antibody which is specific for phosphoserine. As shown in Figure 6 C, the major serine-phosphorylated species demonstrated an apparent mass of 23 kD. This species was identical in mobility to RLC, as determined by probing the same blots with RLC-specific antibody. DIP-uPA significantly increased RLC phosphorylation. In five separate experiments, the increase in RLC phosphorylation was 2.4 ± 0.2-fold and 3.0 ± 0.4-fold after treatment with 10 nM DIP-uPA for 45 and 60 min, respectively. A number of enzymes may phosphorylate RLC on serine, in addition to MLCK (
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MLCK Activity Is Necessary for uPA-promoted Cellular Migration
To determine whether MLCK activity is necessary for uPA-promoted MCF-7 cell migration, Transwell assays were performed in the presence of the MLCK inhibitors. In the absence of uPA, ML-7 and ML-9, at concentrations up to 10-fold the reported Ki values for MLCK inhibition, had little or no effect on MCF-7 cell migration (Figure 7 A). W-7 was also inactive at concentrations up to 51 µM, which is 10-fold the reported Ki for MLCK inhibition. These results suggest that MLCK activity is not essential for basal MCF-7 cell migration. W-7, at concentrations exceeding 0.1 mM, blocked MCF-7 cell migration, probably reflecting the ability of W-7 to inhibit Ca2+/calmodulin-dependent kinases other than MLCK (
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When MCF-7 cells were treated with DIP-uPA, in the presence of the MLCK inhibitors, the uPA-induced increase in cellular migration was blocked (Figure 7 B). Equivalent results were obtained in experiments with uPAR-overexpressing MCF-7 cells and HT 1080 cells. None of three drugs affected the basal level of migration of these two cell lines; however, uPA-stimulated cellular migration was inhibited. In control experiments, ML-7, ML-9, and W-7 had no effect on MCF-7 cell adhesion to vitronectin (data not shown). These studies demonstrate a critical role for MLCK activity in uPA-stimulated cellular migration in three distinct model systems.
uPA-promoted MCF-7 Cell Migration Is Matrix Proteinselective
When surfaces are coated with serum, vitronectin serves as the major attachment and spreading factor (
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Mannosamine inhibits the membrane-anchoring of all GPI-linked proteins, including uPAR (
Vß5 Mediates MCF-7 Cell Adhesion to Vitronectin
Vß5 and
Vß1, but not
Vß3. Our flow cytometry experiments, with antibodies directed against
Vß5 and
Vß3, confirmed their results (data not shown). We also demonstrated substantial levels of cell-surface ß1 subunit; however, our antibody was not specific for
Vß1. To compare the function of various integrins in MCF-7 cell adhesion to vitronectin, in the presence and absence of DIP-uPA (10 nM), integrin-neutralizing antibodies were used. In the absence of uPA, antibody P1F6, which blocks
Vß5, substantially inhibited MCF-7 cell adhesion whereas LM609, which blocks
Vß3, was ineffective as anticipated (Figure 9). Interestingly, when the ß1-integrinblocking antibody, 6S6, was added in combination with P1F6, the extent of inhibition was significantly increased (P < 0.05). These results suggest that a ß1 subunitcontaining integrin (probably
Vß1) plays a significant supporting role in MCF-7 cell adhesion to vitronectin. DIP-uPA neither promoted nor inhibited MCF-7 cell adhesion in the presence or absence of any of the antibodies, suggesting that uPA does not affect the function of the major vitronectin-binding integrins as mediators of MCF-7 cell adhesion.
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A ß1-Integrin and Vß5 Function in uPA-promoted MCF-7 Cell Migration
In the absence of uPA, Vß3-blocking antibody had no effect on MCF-7 cell migration on vitronectin, as anticipated (Figure 10 A). However,
Vß5-blocking antibody was also inactive and ß1 subunitblocking antibody inhibited migration by <25%. When added in combination,
Vß5-blocking antibody and the ß1-blocking antibody inhibited migration by up to 79 ± 5%. These results suggest that
Vß5 and a ß1-containing integrin (probably
Vß1) function interchangeably in MCF-7 cell migration on vitronectin, in the absence of uPA.
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Figure 10 B compares the activities of the vitronectin-binding integrins in mediating uPA-stimulated MCF-7 cell migration. The Vß3-blocking antibody, LM609, had no effect on the uPA response. Antibody P1F6, which neutralizes
Vß5, reduced the magnitude of the uPA response; however, a statistically significant increase in cellular migration was still observed (P < 0.005). The uPA response was reduced still further when migration was allowed to proceed in the presence of antibody 6S6. Importantly, when antibodies P1F6 and 6S6 were added in combination, the response of MCF-7 cells to uPA was entirely abrogated. These results demonstrate that uPA-stimulated MCF-7 cell migration on vitronectin is mediated by the same integrins which function in the absence of uPA. A ß1-containing integrin (probably
Vß1) and
Vß5 are both involved.
uPA Does Not Promote the Migration of MCF-7 Cells that Express Vß3
Vß5 but not when they express
Vß3. To determine whether the pattern of integrin expression influences the ability of MCF-7 cells to respond to exogenously added uPA, MCF-7 cells were transfected to express the ß3-integrin subunit.
Vß3 expression was demonstrated in cells that had been selected in G418 for 14 d, by flow cytometry (Figure 11 A). DIP-uPA stimulated ERK1/2 phosphorylation in the ß3-expressing cells (Figure 11 B). Interestingly, ERK phosphorylation was sustained for an increased period of time (at least 40 min), compared with the parent cell line (<5 min) (
Vß3 may affect the duration of ERK activation in other cell systems as well (
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Vß3-expressing MCF-7 cells demonstrated increased migration on serum-coated membranes, in the absence of uPA, compared with the parent cell line (control) and MCF-7 cells which had been transfected with empty vector (Figure 11 C). However, DIP-uPA (10 nM) did not stimulate migration of the
Vß3-expressing cells. When LM609 was added to block the activity of
Vß3, cellular migration returned to the pretransfection level; however, the cells also regained responsiveness to DIP-uPA. Identical results were obtained in transient transfection experiments; cells that were cotransfected to express GFP and ß3-integrin subunit demonstrated 2.6-fold increased migration compared with cells that were transfected with pEGFP alone. However, these cells did not respond to DIP-uPA (data not shown). Antibody LM609 decreased the migration of the transiently transfected cells but restored responsiveness to uPA. These results suggest that
Vß3 serves as the dominant integrin responsible for the migration of ß3-transfected cells and that uPA does not promote
Vß3-mediated MCF-7 cell migration. When
Vß3 is blocked, the naturally occurring vitronectin-binding integrins remain available and the function of these integrins is enhanced by uPA.
Vß3-Mediated MCF-7 Cell Migration Is MLCK-independent
Since the ability of uPA to promote MCF-7 cell migration depends on MLCK activity, we performed experiments to determine whether Vß3-expressing cells, which were refractory to uPA stimulation, are also refractory to MLCK inhibitors. Migration of ß3-expressing cells was studied in the presence of ML-7, ML-9, and W-7, at concentrations that abolished the uPA response in untransfected cells. As shown in Table 2, none of the inhibitors significantly affected the migration of ß3-expressing MCF-7 cells on vitronectin. Thus, MLCK does not play an essential role in
Vß3-mediated MCF-7 cell migration.
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Discussion |
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Cellular migration is an integrated, multistep process which is regulated by growth factors and extracellular matrix proteins that bind integrins (
We demonstrated previously that uPA promotes MCF-7 cell migration, in vitro, in serum-coated Transwell chambers (
Although recent studies have elucidated diverse signaling pathways that may be activated by uPA (
In MCF-7 cells, uPA-induced ERK activation is highly transient; however, the effects of uPA on cellular migration are sustained (
In uPA-treated MCF-7 cells, MLCK was phosphorylated by a MEK-dependent pathway and remained phosphorylated for at least 6 h, which was the duration of our standard Transwell migration assay. The phosphorylated MLCK was also apparently activated since levels of serine-phosphorylated RLC increased and this increase was prevented by ML-7 and ML-9. The same MLCK inhibitors blocked the motility-stimulating activity of uPA in three separate model systems. Thus, MLCK provides a link between uPAR ligation and increased cytoskeletal contractility which drives cellular migration. Although the increase in RLC phosphorylation was only about two- to threefold, our analysis was not sensitive to possible compartmentalization of the enzyme. Thus, it is possible that RLC phosphorylation was increased to a much greater level within specific regions of the cell. Since MLCK phosphorylation and activation are sustained in uPA-treated cells, continuous ERK activity may not be necessary for motility stimulation. We propose that the uPAR-activated signaling pathway, which promotes cellular motility, includes RasRaf
MEK
ERK
MLCK.
In the absence of uPA, the level of phosphorylated MLCK in MCF-7 cells was near or below the detection limit of our assay. Furthermore, MLCK inhibitors, which entirely blocked uPA-stimulated MCF-7 cell migration, did not affect the level of phosphorylated RLC or cellular migration, in the absence of uPA. Similar results were obtained in migration assays with uPAR-overexpressing MCF-7 cells and HT 1080 cells. These results may be explained if RLC phosphorylation is stable in the time course of our experiments, so that only new RLC phosphorylation is blocked by the inhibitors. However, it is also possible that MLCK does not play a major role in regulating the basal level of RLC phosphorylation, in the absence of stimulants such as uPA, in MCF-7 and HT 1080 cells. Evidence supporting the latter hypothesis has been reported in other systems (
promoted FG cell migration. Although the mechanism of uPA action was not fully characterized, it is tempting to speculate that in the FG cells, endogenously synthesized uPA functioned comparably to exogenously added uPA, as defined in the present study. Plasminogen activator inhibitor (PAI)-1 and PAI-2 inhibited FG cell migration, suggesting a role for uPA proteinase activity in this process (
Vß3 mediates cellular migration in the absence of exogenous stimulants whereas
Vß5 may mediate cellular migration only in cells that are treated with growth factors such as EGF or insulin-like growth factor-1 (
Vß5 and a ß1 subunit containing integrin (probably
Vß1) mediated cellular migration. When these cells were transfected to express
Vß3, the cells still responded to uPA, as determined in ERK phosphorylation experiments; however, uPA no longer stimulated cellular migration. Furthermore, MLCK antagonists did not inhibit the migration of
Vß3-expressing cells. Thus, the Ras/ERK-dependent uPAR-signaling pathway may not promote motility in uPA-treated cells that utilize
Vß3 to migrate on vitronectin. Interestingly, despite expression of
Vß3 in the transfected cells, alternative vitronectin-binding integrins were still available and competent to mediate cellular migration when
Vß3 was blocked with antibody. Under these conditions, the ability of uPA to promote cellular migration was restored.
HT 1080 cells express substantial levels of Vß5 and utilize this integrin, as opposed to
Vß3, to adhere and migrate on vitronectin (
The reason why uPA-promoted activation of ERK and MLCK stimulates cellular migration in an integrin-selective manner remains to be determined. Various integrin properties, including their subcellular localization, avidity for ligand, and strength of focal adhesions may be involved. The ability of uPA to regulate RLC phosphorylation, as well as integrin function (results presented here and by
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Acknowledgements |
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We would like to thank J. Slack for advice on transfection strategies and K. Boardman for excellent technical assistance.
This work was supported by grant CA-53462 from the National Institutes of Health and grant 94-J-4447 from the Department of the Army Breast Cancer Research Program. D. Nguyen is supported by an American Cancer Society Training Grant.
Submitted: September 30, 1998; Revised: June 3, 1999; Accepted: June 8, 1999.
1.used in this paper: ERK, extracellular signal-regulated kinase; GFP, green fluorescent protein; GPI, glycosyl-phosphatidylinositol; HA, hemagglutinin; MAP kinase, mitogen activated protein kinase; MEK, MAP kinase kinase; MLCK, myosin light chain kinase; PAI, plasminogen activator inhibitor; RLC, myosin II regulatory light chain; sc, single chain; tc, two chain; uPA, urokinase-type plasminogen activator; uPAR, urokinase-type plasminogen activator receptor
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