© 1998 American Heart Association, Inc.
Original Contributions |
Urokinase Receptor-Dependent Upregulation of Smooth Muscle Cell Adhesion to Vitronectin by Urokinase
From the University of Pennsylvania School of Medicine, Philadelphia (A.W.C., A.K., E.S.B.); Centocor, Inc (E.S.B.), Malvern, Pa; and the Division of Cardiology, Department of Medicine, Georgetown University, Washington, DC (S.S.O.).
Correspondence to S. Steve Okada, Division of Cardiology, Building D, Room 396B, Georgetown University Medical Center, 4000 Reservoir Rd NW, Washington, DC 20007-2197. E-mail okadas{at}medlib.georgetown.edu
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Abstract |
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Abstract—The plasminogen activator system has been implicated in the modulation of the response to vascular injury. Although urokinase-type plasminogen activator (uPA) and its receptor (uPAR) may enhance matrix degradation as well as migration and invasion by smooth muscle cells (SMCs), their roles in cell adhesion are uncertain. Therefore, we examined the ability of uPA and uPAR to modulate adhesion of cultured human vascular SMCs to various matrices. We demonstrated a dose-dependent stimulation of adhesion by single-chain uPA (scuPA) to vitronectin (maximum 1.55-fold [±0.04-fold] increase, 10 nmol/L, P<0.002) but not to laminin, collagen I, or collagen IV. Baseline adhesion to vitronectin was completely inhibited by both EDTA and RGD peptide but was restored to >40% of control in the presence of scuPA (P=0.001 and 0.046, respectively). Adhesion to vitronectin was also significantly enhanced by the amino-terminal fragment of uPA (P=0.007) and two-chain, high-molecular-weight uPA (P<0.01) but not by the low-molecular-weight fragment of uPA, which lacks the receptor-binding domain. Aprotinin, a plasmin inhibitor, had no effect on baseline or scuPA-stimulated adhesion, suggesting a plasmin-independent process. Preincubation of scuPA with soluble uPAR inhibited scuPA stimulation of adhesion by 88±14% (P=0.01), as did pretreatment of SMCs with phosphatidylinositol-specific phospholipase C, which removes glycophosphatidylinositol-anchored proteins, including uPAR. Antibodies to both
vß3 and
vß5 integrin inhibited baseline adhesion
but not scuPA stimulation. Finally, coating plates with scuPA alone
enabled cell adhesion, which could be inhibited by both soluble uPAR
and anti-uPAR antibodies. These data suggest that uPA stimulates
adhesion of SMCs specifically to vitronectin and that it is
mediated by an interaction with uPAR. Upregulation of both proteins
after vascular injury may facilitate migration through stimulation of
both matrix degradation and cell adhesion.
Key Words: smooth muscle cell adhesion • upregulation • urokinase • vitronectin • urokinase receptor
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Introduction |
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After vascular injury, smooth muscle cells (SMCs) proliferate within the media of the vessel and migrate to the intima, where they continue to proliferate and contribute to atheroma formation and restenosis, common problems after vascular procedures.1 Adhesive functions of vascular SMCs play a central role in this inflammatory process. Cells use receptors expressed on the cell surface, growth factors produced by cells, and matrices secreted into the extracellular milieu.2 3 4 5
Traditionally, integrins have been thought of as the major
cellular receptors mediating adhesion.6 SMCs bind
to fibronectin, laminin, collagen I, and collagen IV primarily by means
of ß1 integrins.7 8 9
After vessel injury, however, SMCs use a different set of integrins and
matrices for adhesion and migration.10
Vitronectin, a serum protein that inhibits complement
activation and binds heparin, localizes to atherosclerotic
plaques.11 Vitronectin has been shown
to mediate migration of cells in vitro, suggesting that cells may use
vitronectin to adhere and migrate after vascular injury.
The major integrin receptors for vitronectin are
vß5,
vß3, and
vß1. In carcinoma
cells, vß5 promotes
cell attachment to vitronectin but not migration. When
transfected with ß3, these cells are capable of
migrating on vitronectin,12 13
demonstrating that cells use different cellular receptors in response
to various stimuli. Glioblastoma cells are thought to use
vitronectin and
vß3 receptor to invade
normal brain tissue.14 Abciximab, a monoclonal
antibody that binds with equal affinity to both
IIbß3, and
vß3, is used in humans
to reduce ischemic complications and possibly to reduce
restenosis after angioplasty.15 RGD
peptide, which blocks ligand binding of various integrin receptors
(including IIbß3 and
vß3 receptors),
inhibited neointima formation in damaged hamster carotid
arteries, presumably by interfering with these receptors on
platelets and SMCs.16 Thus, SMCs appear to
respond to vascular injury by using
vß5 and
vß3 integrin receptors
to adhere to and migrate on vitronectin, which localizes to
areas of vessel damage.11 Upregulation of these
factors by SMCs represents a potential target for therapies to
limit the vascular injury response.
Recently, the plasminogen activator system has
been suggested to play a role in atheroma formation and
regulation of cellular adhesion. Tissue-type plasminogen
activator (tPA), urokinase-type plasminogen
activator (uPA), and uPA receptor (uPAR) are upregulated in
atherosclerotic lesions and are localized to macrophages and
neointimal SMCs.17 18 19 tPA and
uPA are serine proteases secreted by migrating cells. They convert
plasminogen to plasmin, which mediates cellular migration
and matrix degradation, and aid in a diverse number of cellular
processes, such as angiogenesis and tumor
invasion.20 21 22 23 uPAR has also been implicated as
a nonintegrin cellular adhesion receptor.24 uPAR,
which is expressed on a wide variety of cells (including human SMCs),
is a glycoprotein with a molecular mass of 
45 to 60 kDa
and a glycophosphatidylinositol (GPI)anchor.25 26
Recombinant uPAR binds vitronectin, and this binding is
augmented by the addition of uPA. Transfected epithelial cells
expressing membrane-anchored uPAR become significantly more adhesive
than cells expressing soluble receptor.24
Cytokine-stimulated myelomonocytic cells adhere to
vitronectin in an RGD-independent manner, and this adhesion
is tightly linked to uPAR occupancy.27
We sought to define the factors, related to both integrin and plasminogen activator, involved in SMC adhesion to various matrices given the central role of SMCs in atheroma formation and recent evidence linking the plasminogen activator system to cellular adhesion. We demonstrate that uPA stimulates SMC adhesion selectively to vitronectin in a plasmin-independent but uPAR-dependent manner. We also demonstrate that uPA can act as an adhesive substrate for SMCs.
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Methods |
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Reagents
Human collagen I, laminin, collagen IV, and anti-human integrin
vß5 antibody P1F6
(unpurified, from mouse ascites fluid) were obtained from Gibco BRL.
Murine chimeric antibodies against human integrin
vß3 and
IIbß3 (abciximab and
7E3, respectively) and anti-human
IIbß3 antibody (10E5)
were supplied by Centocor (gift of M. Nakada). Human
vitronectin was obtained from Promega. Murine monoclonal
antibody 3936 against human uPAR was obtained from American
Diagnostica (gift of J. Bognacki). Single-chain uPA
(scuPA), two-chain, high-molecular-weight uPA (tcuPA), the
low-molecular-weight (LMW) fragment of uPA, the amino-terminal fragment
of uPA (ATF), and recombinant soluble uPAR (suPAR) were obtained from
Abbott Laboratories (gifts of A. Mazar and J. Henkin). Higazi et
al28 showed that the suPAR preparation does not
cleave scuPA. Phosphatidylinositol-specific phospholipase C (PIPLC) was
provided by M. Low (Columbia University). RGD peptide was purchased
from Sigma. The sequence of the RGD peptide was
Arg-Gly-Asp-Ser.
Cell Culture
SMCs were grown using the explant technique from human umbilical
veins or human adult aorta as previously
described.29 All use of human tissue was approved
by our institutional review board. In brief, an umbilical vein or
aortic media was isolated surgically and minced on culture plates.
Unless otherwise noted, experiments were performed with human umbilical
vein SMCs (HUVSMCs). SMCs were grown in a mixture of Dulbecco's
modified Eagle's medium (DMEM) and F12 Ham's solution supplemented
with 10% FBS, penicillin, streptomycin, and Fungizone. Cells were
passed at confluence with trypsin-EDTA. Passages 3 to 12 were used for
experiments.
Assay for Cell Adhesion
Nunc microwell 96-well plates were coated with matrix for 1 hour
at 37°C and then blocked with 1% BSA-PBS for 30 minutes at 37°C.
SMCs were removed with 2 mmol/L EDTA in calcium- and
magnesium-free PBS after they were washed with calcium- and
magnesium-free PBS. Cells were resuspended in Hanks' balanced salt
solution with 0.1% BSA. Unless otherwise specified, reagents and SMCs
(100 µL/well, at a concentration of 2x105
cells/mL) were added directly to wells and allowed to incubate for 2
hours at 37°C. Cells were then washed with PBS 3 times and fixed with
10% formaldehyde and 0.1 M acetate buffer (pH 5.2) for 15 minutes at
room temperature. Cells were washed twice with acetate buffer, twice
with deionized water, and allowed to air-dry. Fixed cells were stained
with 0.05% naphthol blue-black acetate buffer for 30 minutes at room
temperature. Cells were washed 3 times with deionized water and allowed
to air-dry. Stain was then solubilized with 0.1N NaOH and shaken for 30
minutes at room temperature. Absorbance was measured at 630 nm. All
experiments were performed in triplicate with data
representing average optical density minus background.
Student's t test was used to compare treatment groups with
control cells with P<0.05 considered significant.
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Results |
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Effect of scuPA on HUVSMC Adhesion
Given the recent evidence suggesting a role of the plasminogen activator system in cellular adhesion, we investigated the effect of scuPA on SMC adhesion to different matrices. Plates were coated with 5 µg/mL (67 nmol/L) vitronectin, 5 µg/mL (40 nmol/L) collagen I, 5 µg/mL (5 nmol/L) laminin, or 5 µg/mL (28 nmol/L) collagen IV. Simultaneous addition of 10 nmol/L scuPA with the cells to the well increased adhesion to vitronectin by 45±3% above the control level (P=0.02) (Figure 1
). No significant increase in adhesion
was seen with the other matrices, despite the various levels of
absolute adhesion with different matrices. Stimulation of adhesion was
dose-dependent (Figure 2, squares) and
was seen with both HUVSMCs and adult aortic SMCs (not shown). At 10
nmol/L scuPA, adhesion was increased by 55±4% (P=0.002),
with half-maximal stimulation between 0.1 and 0.2 nmol/L.
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) and with (
) 10 nmol/L scuPA and SMCs. Values are reported as
percentage of control adhesion to vitronectin and
represent the mean±SEM of 3 experiments. All experiments were
performed in triplicate. *P<0.05 versus control.
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) and with P1F6 (
), an
anti-
Integrin-mediated cellular adhesion to vitronectin is
RGD-dependent and inhibited by EDTA. Baseline adhesion of SMCs to
vitronectin was inhibited by 98±1.2% (P=0.01)
and 98±0.4% (P=0.01) by 100 µmol/L RGD and 10
mmol/L EDTA, respectively. However, when scuPA was added to cells in
addition to RGD or EDTA, adhesion was restored to 50±4%
(P=0.046) and 56±2% (P=0.001) of the control
level for RGD peptide and EDTA, respectively (Figure 3). scuPA-mediated stimulation of SMC
adhesion to vitronectin in the presence of RGD suggests
that a nonintegrin cellular receptor may mediate this adhesion.
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) was added at the
beginning of incubation for each condition. Values represent
adhesion as the percentage of control adhesion (no
inhibitor, no scuPA). Each point was tested in triplicate.
Data represent the mean±SEM of 5 experiments.
*P<0.05 versus control.
Inhibition of SMC Adhesion With Anti-integrin Antibodies
To better characterize the integrin receptors used by SMCs, we
repeated the experiments in the presence of an antibody to
vß5 (P1F6),
vß3 (abciximab or
7E3), or IIbß3
(control, 10E5) (Figure 4). The level of
inhibition varied from 13±5% (P=0.09) for abciximab to
70±2% (P=0.001) for P1F6. There was no inhibition of SMC
adhesion to collagen I, laminin, or collagen IV induced by any of these
antibodies (not shown). Thus, to adhere to vitronectin,
SMCs use primarily vß5
integrin, although vß3
integrin also contributes to adhesion. Nevertheless, in the presence of
P1F6, the level of adhesion increased in a dose-dependent manner from
51% to 110% of the control level (P=0.04) with the
addition of scuPA (Figure 2
, circles).
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SMC Adhesion to Vitronectin: Effect of Various Domains
of uPA
To further define the role of scuPA in augmenting SMC adhesion to
vitronectin, ATF and LMW uPA were incubated with cells
under the same conditions as scuPA. Equimolar concentrations of scuPA,
ATF, LMW uPA, tcuPA, or a combination of ATF and LMW uPA were added to
vitronectin-coated wells along with SMCs and incubated at
37°C for 2 hours. scuPA increased SMC adhesion by 41±5% above the
control level (P=0.009), and active tcuPA increased adhesion
by 35±6% (P=0.009). ATF, which possesses the
receptor-binding domain of scuPA without catalytic activity, increased
adhesion by 29±5% (P=0.007), whereas LMW uPA, which
contains the catalytic site of scuPA but not the receptor-binding
domain, had no effect on adhesion (Figure 5) and did not enhance the effect of ATF
when the 2 were combined. Finally, aprotinin, a plasmin
inhibitor, was unable to significantly inhibit either
baseline or scuPA-augmented adhesion (data not shown). These results
suggest that the effect of scuPA on SMC adhesion to
vitronectin is related to its binding to uPAR and does not
require plasmin activity.
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Inhibition of SMC Adhesion to Vitronectin by
scuPA/suPAR Complex and PIPLC
On the basis of the data presented above, uPAR
appears to function as a cellular adhesion molecule, mediating adhesion
to vitronectin when scuPA is present. We sought to
directly determine whether uPAR was necessary for the effect of scuPA
on SMC adhesion by inhibition with suPAR. scuPA (10 nmol/L) was
preincubated with increasing concentrations of suPAR (0, 0.5, 1, and
2 µmol/L) at 37°C for 1.5 hours. The scuPA/suPAR solution was
then added to vitronectin-coated wells along with the SMCs
and incubated for an additional 2 hours. Preincubation with suPAR
significantly inhibited the stimulatory effect of scuPA (Figure 6) by 88±14% (P=0.01) at a
50-fold molar excess of suPAR. In the absence of scuPA, suPAR inhibited
baseline adhesion by only 2±2% (P=0.42), indicating that
suPAR inhibits only adhesion stimulated by scuPA. Prior studies by
Higazi et al28 demonstrated that the suPAR
preparation does not cleave scuPA and thus excludes degradation of
scuPA as a mechanism for inhibition by
suPAR.28
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Similar results were obtained with PIPLC treatment of cells. PIPLC
cleaves the GPI anchor of uPAR. Preincubation with 1 U/mL PIPLC for 1
hour at 37°C eliminated the increase in adhesion associated with
scuPA (Figure 7). In PIPLC-treated SMCs
in the presence of P1F6 antibody, scuPA stimulation was blunted but not
eliminated.
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SMC Adhesion to scuPA-coated Wells
Finally, to further investigate the ability of uPAR to act as a
cellular receptor and to test whether scuPA could act as a ligand,
wells were coated only with 20 µg/mL scuPA and blocked with 1%
BSA-PBS. SMCs were then added to wells in the presence of various
inhibitors. Cells were able to adhere and spread to
scuPA-coated wells without difficulty and appeared normal. suPAR
inhibited this adhesion by 95±1% (P=0.01). In addition, a
monoclonal antibody to uPAR that blocks binding of uPA to uPAR (3936)
inhibited adhesion by 21±3% (P=0.008). This adhesion to
scuPA-coated wells was not inhibited by RGD peptide (5±3%,
P=0.23). Control wells not precoated with scuPA did not
support adhesion of SMCs at all (3±1% of control level) (Figure 8).
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Discussion |
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These data demonstrate that uPA is capable of promoting SMC adhesion to vitronectin but not to other extracellular matrices. This stimulation is RGD- and EDTA-independent. In the presence of anti-
vß3
and anti-vß5
antibodies, scuPA was still able to increase SMC adhesion to
vitronectin. Similarly, Waltz et
al,27 using transforming growth
factor-ß1/D1–stimulated
U937 cells, demonstrated a scuPA-mediated increase in adhesion to
vitronectin independent of both EDTA and RGD peptide.
Integrin-dependent adhesion to vitronectin is mediated by
v receptors, primarily
vß3,
vß5, and
vß1. The
v integrins recognize the RGD sequence in
vitronectin,6 10 and the fact that
RGD peptide does not inhibit the scuPA-mediated increase in SMC
adhesion to vitronectin most likely points to a nonintegrin
cellular receptor capable of promoting adhesion to
vitronectin when properly stimulated by scuPA. Although
upregulation of v integrin receptors may be an
important component of the SMC response to vascular injury, the
stimulatory effect of uPA on cellular adhesion appears to act
independently of this system. The effect of ATF and LMW uPA on SMC adhesion suggests that scuPA increases adhesion by means of receptor binding rather than protease activity. ATF, which contains the receptor-binding domain of scuPA, was able to significantly increase SMC adhesion to vitronectin, whereas the catalytic fragment, LMW uPA, had no significant effect on SMC adhesion. However, when added to ATF, LMW uPA seemed to inhibit the increase in adhesion seen with ATF alone. Low-affinity binding of LMW uPA to uPAR may cause conformational changes in uPAR and thus inhibit the binding of ATF to uPAR.
Stimulation of SMC adhesion to vitronectin by scuPA was
inhibited by pretreatment of cells with PIPLC, suggesting that the
cellular receptor involved is a GPI-linked protein, such as uPAR.
However, there was still a significant increase in SMC adhesion with
scuPA after PIPLC treatment in the presence of P1F6. This may have been
due to incomplete cleavage of uPAR from the cell surface, which was
confirmed by binding experiments. When125I-labeled scuPA binding to SMC was measured immediately
after PIPLC treatment, there was an 80% reduction in binding but
not a 100% reduction, suggesting that PIPLC treatment does not
completely cleave all the receptors (S.S. Okada and E.S. Barnathan,
unpublished data, 1991). In addition, SMCs may be able to regenerate
uPAR after PIPLC treatment during the 2-hour incubation period.
However, we cannot exclude the possibility of another
scuPA-activated receptor that is not GPI-anchored mediating
adhesion to vitronectin.
scuPA stimulation was also eliminated by preincubation of scuPA with suPAR, further implicating uPAR as a mediator of scuPA-induced adhesion. Whether preincubation with suPAR inhibits adhesion by directly blocking uPA binding to sites on SMCs, by blocking vitronectin-binding sites, or by another mechanism remains to be determined. The fact that suPAR inhibited only adhesion stimulated by scuPA and not baseline adhesion suggests the first mechanism. Adhesion in the absence of scuPA may occur by means of a "lower-affinity" interaction with uPAR that is not significant enough to be inhibited by suPAR. In fact, studies have shown that suPAR binds to immobilized vitronectin in the absence of scuPA but that concurrent receptor binding of uPA markedly promotes binding to vitronectin.24
The precise mechanism by which scuPA interacts with uPAR to mediate adhesion in general is not well understood. Wei et al24 originally postulated that uPA stabilized an active conformation of uPAR. Kanse et al30 demonstrated in endothelial cells that uPA increased vitronectin binding by increasing the affinity of vitronectin for uPAR. Moser et al,31 however, hypothesized that uPA mediated its effects by direct binding of uPA to vitronectin. Cytokines have been shown to augment the effect of uPA on cellular adhesion in monocytes,32 and monocyte adhesion is inhibited by cycloheximide and actinomycin D,33 which implies a receptor-mediated signal transduction pathway for the effect of uPA on cellular adhesion.
Recent studies have demonstrated that the uPAR may associate with
various integrins.34 35 It has been suggested
that the uPAR may interact directly with transmembrane integrins, which
are in turn bound to the cytoskeleton, thus enabling interaction with
the cytoskeleton. This interaction may occur independently of any RGD
sites on the integrin receptors. Wei et al34
found that uPAR complexes with ß1 integrin and
caveolin to inhibit native adhesion to fibronectin and to promote
adhesion to vitronectin in human embryonic kidney cells
transfected with uPAR cDNA. Other data have suggested a role of
integrins in association with plasminogen
activator inhibitor-1 (PAI-1), the major
physiological inhibitor of
plasminogen activator activity. PAI-1 forms
irreversible complexes with uPA and tPA and binds
vitronectin.36 37 Stefansson et
al35 demonstrated that PAI-1 shares a
vitronectin-binding site with
vß3 integrin,
suggesting that uPA increases cellular adhesion by binding to PAI-1,
thereby exposing binding sites on vitronectin for
vß3 integrin. Finally,
data demonstrating that uPAR competes directly with PAI-1 for binding
to vitronectin have been reported. Deng et
al38 mapped the uPAR-binding sequence in
vitronectin to the somatomedin-binding domain, which
contains the PAI-1–binding motif. Kjøller et
al39 recently demonstrated that PAI-1 inhibits
cell migration by interfering with the binding of uPAR to
vitronectin, independently of its function as a
plasminogen activator
inhibitor.35 38 Thus, uPA may
increase adhesion by binding PAI-1 and freeing up binding sites within
the vitronectin molecule for uPAR rather than for
vß3.
Our data support a model of uPA stimulation of SMC adhesion (Figure 9) in which uPA binds to uPAR and
promotes adhesion to vitronectin by means of a
conformational change in uPAR or exposure of a latent
vitronectin-binding site on uPAR, the end result being
increased affinity of uPAR for vitronectin. uPAR binding
occurs at a site independent from the RGD sequence recognized by the
v integrin receptors, explaining the
upregulation of adhesion by urokinase in the presence of RGD peptide.
Interaction between uPAR and integrin receptors, however, may still
occur.
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An unexpected finding was that scuPA supports adhesion of SMCs. This adhesion most likely uses uPAR as the adhesion receptor because the adhesion was inhibited by both suPAR and an anti-uPAR antibody but not an RGD peptide. The inhibition of adhesion by anti-uPAR antibody (3936) was only 21%, probably because 3936 is a monoclonal antibody that inhibits binding of uPA to suPAR but may not recognize the site (or sites) on uPAR that mediates adhesion to the scuPA matrix. This result potentially defines a ligand role of urokinase with uPAR acting as its complementary cellular receptor. As such, uPA secreted by SMCs could promote adhesion and migration by acting as a matrix on which cells migrate, in addition to its well-known ability to activate plasminogen and generate plasmin, facilitating matrix breakdown. Interestingly, mice deficient in plasminogen, as well as those deficient in uPA, have reduced neointima formation after vascular injury.40 This novel role of scuPA-uPAR interaction could augment upregulation of cellular adhesion to vitronectin after vessel injury and contribute to the process of neointimal thickening. More importantly, it demonstrates the possibility that nonintegrin membrane receptor proteins may at times mediate cellular adhesion at discrete sites where ligand is present and facilitate cell-matrix interactions.
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Acknowledgments |
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This work was supported by the National Institutes of Health (grants HL47839 to E.S.B. and HL02870 to S.S.O.). A.W.C. is a recipient of an American Heart Association Medical Student Research Fellowship. S.S.O. is a recipient of a National Institutes of Health Clinician Investigator Development Award.
Received October 29, 1997; accepted May 18, 1998.
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