From the Departments of Pathology and
¶ Biochemistry and Molecular Genetics, University of Virginia
Health Sciences Center, Charlottesville, Virginia 22908
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ABSTRACT |
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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 t1/2 was
8.2 h. uPA·PAI-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 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)1 is a member of the
LDL receptor family, which includes the LDL receptor-related protein
(LRP) and gp330/megalin (1, 2). These receptors have equivalent
structural motifs and bind many of the same ligands, including
apolipoprotein E-enriched chylomicron remnants, lipoprotein lipase,
thrombospondin I, urokinase-type plasminogen activator (uPA),
uPA-plasminogen activator inhibitor-1 (PAI-1) complex, and
receptor-associated protein (RAP) (3-10). RAP is a 39-kDa protein
chaperone which normally remains entirely intracellular (11, 12);
however, when incubated with cells in culture, RAP blocks the binding
of all known ligands to the VLDLr, LRP, and gp330/megalin (1, 13-15).
Some ligands do not bind interchangeably to different members of the
LDL receptor family. For example, activated
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 wild-type 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. uPA·PAI-1 complex,
which is bound to uPAR, still binds to LRP (29). This interaction not
only results in the internalization of uPA·PAI-1 complex, but
promotes uPAR internalization as well (30). Thus, it has been proposed
that uPA·PAI-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.
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 µM 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.
Na125I and [35S]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).
Cell Culture--
MDA-MB-435, which were from the ATCC, were
cultured in L-15 medium (Life Technologies, Inc.) supplemented with
10% FBS, 10 µg/ml insulin, and penicillin/streptomycin. Low-passage
(25-35) MCF-7 cells were kindly provided by Dr. Richard Santen
(University of Virginia). These cells were cultured in RPMI (Life
Technologies, Inc.) supplemented with 10% fetal bovine serum (FBS)
(Hyclone Laboratories, Logan, UT), 100 units/ml penicillin, and 100 µg/ml streptomycin. MDA-MB-435 and MCF-7 cells were passaged at
subconfluence with Cell Dissociation Buffer (Enzyme Free, Hank's
based, Life Technologies, Inc.).
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 µg/ml E-64, 10 µg/ml aprotinin,
and 10 µg/ml leupeptin. Equal amounts of cellular protein (50 µg)
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 CaCl2, 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 125I-GST-RAP--
GST-RAP
was radioiodinated, using Iodo-Beads (Pierce), to a specific activity
of 1-2 µCi/µg. MCF-7 cells were washed with EBSS, 25 mM HEPES, pH 7.4, and 5 mg/ml bovine serum albumin (EHB medium). 125I-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 125I-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 µCi/µg. The cultures were washed three
times and then incubated with 125I-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 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 µM 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 µM [Glu1]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 µM) or
vehicle for 15 min at 37 °C. uPA·PAI-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 uPA·PAI-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 125I-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 [35S]methionine (10 µCi/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 uPA·PAI-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
CaCl2, 1 mM MgCl2, 100 mM N-octyl glucoside, 10 µg/ml E-64, 10 µg/ml aprotinin, and 10 µg/ml leupeptin, pH 7.4. [35S]Methionine-labeled uPAR was recovered from equal
amounts of each cell extract by immunoprecipitation with uPAR-specific
antibody (42 µg/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.
[35S]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 µg/ml leupeptin, 2 µg/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 µm pore
membranes (Costar). The underside surface of each membrane was coated
with 20% FBS or with 5 µg/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 106 cells/ml (100 µl). 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 125I-GST-RAP to MCF-7 cells are shown in Fig.
2. Binding was specific and saturable;
the Scatchard transformation (not shown) was linear
(r2 = 0.94), suggesting that a single class of
binding sites was detected. The KD was 6 nM and the Bmax was 110 fmol/mg of
cell protein (n = 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 Bmax
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 125I-GST-RAP (10 nM). RAP degradation is
mediated only by receptors in the LDL receptor family (1, 13). Fig. 2,
panel B, shows that after an anticipated lag phase, specific
RAP degradation occurred at a nearly constant rate (1.4 ± 0.2 × 105 molecules/cell·h) for at least 6 h.
Non-radiolabeled RAP (200 nM) inhibited
125I-GST-RAP degradation by 95 ± 3% (not shown).
Based on this result, we chose to utilize 200 nM RAP to
neutralize the VLDLr in the uPAR catabolism and cellular migration
experiments (presented below). Assuming that all of the RAP-binding
sites, detected in our equilibrium binding experiments, represent
VLDLr, then approximately two molecules of RAP are internalized per
copy of cell surface VLDLr per hour.
The linear RAP degradation curve shown in Fig. 2 indicates that cell
surface VLDLr expression is not rapidly down-regulated by ligand. To
determine whether prolonged culturing in RAP alters cell surface VLDLr
expression, MCF-7 cells were cultured in the presence of 200 nM GST-RAP for 5 days. No change in the total level of
VLDLr antigen was detected by immunoblot analysis (results not shown).
Furthermore, the rate of GST-RAP degradation was unchanged; cells that
were cultured in RAP degraded 549 ± 52 fmol of
125I-GST-RAP/mg of cell protein/h. Cells that were cultured
for 5 days in vehicle degraded 532 ± 30 fmol of
125I-GST-RAP/mg of cell protein/h.
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 125I-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-uPA-binding 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 KD was 1.5 ± 0.3 nM and the
Bmax was 5.9 ± 0.4 fmol/mg of cell protein
(3,400 receptors/cell) (n = 4); these values are consistent with previously reported results (32). For RAP-treated cells, the KD was unchanged (1.4 ± 0.4 nM), as would be expected if the increase in uPA binding
was due to an increase in cell surface uPAR. The
Bmax was 19 ± 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 uPA·PAI-1 complex for 20 min at 37 °C. After acid
washing the cells to remove uPAR-associated ligands, the level of cell
surface uPAR was determined by measuring the binding of
125I-DIP-uPA (10 nM). Without prior ligand
challenge, MDA-MB-435 cells bound 115 ± 5 fmol of DIP-uPA per mg
of cell protein (Fig. 4). Cells that were
treated with RAP for 20 min demonstrated unchanged specific
125I-DIP-uPA binding, as expected. 125I-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 uPA·PAI-1 complex, demonstrated a 90%
decrease in 125I-DIP-uPA binding. Thus, uPA·PAI-1 complex
promoted uPAR endocytosis in MDA-MB-435 cells. When the cells were
pretreated with RAP and then exposed to uPA·PAI-1 complex, uPAR
endocytosis was blocked by 80%. These results suggest that the VLDLr
is required for uPA·PAI-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 [35S]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, panel
B. With both RAP-treated and control cells, linear graphs were obtained when the amount of labeled uPAR was plotted against time, according to the equation: log
[at=0]/[at=x] = kt/2.3 (Fig. 5, panel B). This result suggests
that uPAR catabolism follows first-order kinetics. RAP significantly
decreased the rate of uPAR catabolism. In the absence of RAP, the rate
constant for uPAR catabolism was 8.5 × 10
As a control, we studied the catabolism of
[35S]methionine-labeled CD44. CD44 is a glycoprotein
receptor which is expressed by MDA-MB-435 cells (50). When cells were
cultured for 24 h in the absence of RAP, 70 ± 5% of the
[35S]methionine-labeled CD44 remained (n = 4). In the presence of RAP, CD44 survival was unchanged; after
24 h, 68 ± 4% of the labeled CD44 remained. Thus, the
effects of RAP on uPAR survival in MDA-MB-435 cells are specific.
Our results demonstrated that uPA·PAI-1 complex promotes uPAR
internalization. To determine whether uPA·PAI-1 complex accelerates uPAR catabolism, we cultured metabolically labeled MDA-MB-435 cells in
the presence of 0.5 nM uPA·PAI-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 × 10 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 ± 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 ± 0.1-fold, n = 8, p < 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 ± 0.3-fold, n = 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 that
autocrine activation of uPAR by endogenously produced uPA may be
responsible for the increased motility of LRP-deficient 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 uPA-specific antibody that blocks uPA binding to uPAR
(25 µg/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, non-immune mouse IgG (25 µg/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 ± 8 pM (n = 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 KD 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 < 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 125I-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 immunoblot
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 ± 0.5- and 2.6 ± 0.6-fold, respectively
(n = 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.
Previous studies have shown that LRP and the VLDLr express similar
activities as receptors for free uPA and uPAR-associated uPA·PAI-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 uPA·PAI-1 complex. When MDA-MB-435 cells
were treated with uPA·PAI-1 complex at 37 °C, uPAR was rapidly
internalized by a RAP-inhibited pathway. The most likely explanation
for this result is that uPA·PAI-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
uPA·uPAR 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 uPA·PAI 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 uPA·PAI-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 exogenously added uPA·PAI-1 complex. At this time, we do
not know whether this process depended on the formation of uPA·PAI-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 vitronectin-coated 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 104 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 KD 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.
INTRODUCTION
Top
Abstract
Introduction
References
2-macroglobulin and Pseudomonas exotoxin A
bind only to LRP (16, 17).
MATERIALS AND METHODS
-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 ± 0.07 ng/cell
(n = 5).
RESULTS
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Fig. 1.
VLDLr expression in breast cancer cell
lines. Cell extracts prepared from MCF-7 and MDA-MB-435 human
breast cancer cells and human embryonic fibroblasts were subjected to
SDS-PAGE on 5% slabs and transferred to nitrocellulose. In RAP ligand
blots, membranes were incubated with GST-RAP, followed by GST-specific
monoclonal antibody and goat anti-mouse IgG-peroxidase conjugate. For
immunoblots, the VLDLr was detected using polyclonal anti-human VLDLr
antibody and LRP heavy chain was detected with monoclonal antibody
8G1.
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Fig. 2.
Binding and cellular degradation of GST-RAP
by MCF-7 cells. In panel A, MCF-7 cells were incubated
with increasing concentrations of 125I-GST-RAP for 4 h
at 4 °C. The specific binding isotherm is shown. In panel
B, degradation of GST-RAP by MCF-7 cells was determined by
measuring trichloroacetic acid soluble radioactivity in the medium.
Specific GST-RAP degradation is plotted as a function of time. Each
point represents the mean of results from four separate
experiments, each with duplicate determinations.
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Fig. 3.
Binding of DIP-uPA to MCF-7 cells cultured in
RAP. Panel A, MCF-7 cells were cultured in the presence
or absence of GST-RAP for 1-5 days. Specific binding of
125I-DIP-uPA (10 nM) to RAP-treated MCF-7 cells
( ) and vehicle-treated MCF-7 cells (
) was determined. Panel
B, MCF-7 cells were cultured in GST-RAP or in vehicle for 3 days.
Specific binding of 125I-DIP-uPA was then studied. Specific
binding isotherms are shown for RAP-treated MCF-7 cells (
) and
vehicle-treated MCF-7 cells (
). Each point represents the mean of
results from four separate experiments, each with duplicate
determinations.
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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,
uPA·PAI-1 complex or vehicle for 20 min and immediately chilled to
4 °C. After acid washing, specific binding of
125I-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
uPA·PAI-1 complex. RAP + uPA-PAI-1 represents cultures
that were pretreated with RAP and then pulse-exposed to uPA·PAI-1
complex.
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Fig. 5.
Catabolism of uPAR in breast cancer
cells. MDA-MB-435 cells were metabolically labeled with
[35S]methionine and then incubated for various periods of
time, in the presence or absence of RAP. 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 "+
RAP" or vehicle " RAP" for the indicated times.
In panel B, the amount of labeled uPAR recovered in
immunoprecipitates from RAP-treated cells (
) and vehicle-treated
cells (
) was plotted against time, as described in the text.
Panel C shows equivalent graphs analyzing labeled uPAR
recovery from cells treated with uPA·PAI-1 complex (
) or
uPA·PAI-1 complex plus RAP (
).
2
h
1, corresponding to a uPAR survival
t1/2 of 8.2 h. In the presence of RAP, the uPAR
catabolism rate constant was 4.1 × 10
2
h
1, corresponding to a t1/2 of
16.9 h. Thus, RAP treatment caused an approximate doubling of the
uPAR survival t1/2.
1 h
1, which corresponds to a
t1/2 of 1.8 h. When RAP was added to the
culture medium with uPA·PAI-1 complex, uPAR catabolism was inhibited;
the rate constant was 8.9 × 10
2 h
1
and the t1/2 was 7.8 h. Thus, recycling of
internalized uPAR is not 100% efficient. Instead, a significant fraction of the uPAR, which is internalized via a pathway that requires
both uPA·PAI-1 complex and the VLDLr, is targeted for degradation.
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Fig. 6.
Migration of RAP-treated MCF-7 cells
and the role of the uPA/uPAR system. Panel A, MCF-7
cells that 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 serum-free 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.
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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 µg/ml uPA-specific antibody (uPA-Ab) or 25 µg/ml
uPAR-specific antibody (uPAR-Ab).
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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 +. 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 +.
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.
DISCUSSION
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant CA-53462 and Department of the Army Breast Cancer Research Program Grant 94-J-4447.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Fellow of the American Heart Association, Virginia Affiliate.
To whom correspondence should be addressed: University of
Virginia Health Sciences Center, Depts. of Pathology and Biochemistry and Molecular Genetics, Box 214, Charlottesville, VA 22908. Tel.: 804-924-9192; Fax: 804-982-0283; E-mail: SLG2T{at}VIRGINIA.EDU.
2 D. H. D. Nguyen et al., unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: VLDLr, very low density lipoprotein receptor; LDL, low density lipoprotein receptor; LRP, low-density lipoprotein receptor-related protein; RAP, receptor-associated protein; uPA, urokinase-type plasminogen activator; uPAR, urokinase receptor; MEF, murine embryonic fibroblasts; PAI-1, plasminogen activator inhibitor-1; ERK, extracellular signal-regulated kinase; GST, glutathione S-transferase; FBS, fetal bovine serum; CM, conditioned medium; VSMC, vascular smooth muscle cells; scuPA, single-chain uPA; tcuPA, two-chain uPA; DIP-µPA, diisopropylphospho-uPA; MAP, mitogen-activated protein kinase; PAGE, polyacrylamide gel electrophoresis; VLK-AMC, Val-Leu-Lys-7-amido-4-methylcoumarin.
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