Biochem. J. (2009) 423, 343–351 (Printed in Great Britain)
343
doi:10.1042/BJ20090447
Urokinase-receptor-mediated phenotypic changes in vascular smooth
muscle cells require the involvement of membrane rafts
Julia KIYAN*1 , Graham SMITH†, Hermann HALLER* and Inna DUMLER*
*Hannover Medical School, Carl-Neuberg Straße 1, D-30625 Hannover, Germany, and †AstraZeneca, Alderley Park, Macclesfield SK10 4TF, U.K.
The cholesterol-enriched membrane microdomains lipid rafts
play a key role in cell activation by recruiting and excluding
specific signalling components of cell-surface receptors upon
receptor engagement. Our previous studies have demonstrated
that the GPI (glycosylphosphatidylinositol)-linked uPAR [uPA
(urokinase-type plasminogen activator) receptor], which can be
found in lipid rafts and in non-raft fractions, can mediate the
differentiation of VSMCs (vascular smooth muscle cells) towards
a pathophysiological de-differentiated phenotype. However, the
mechanism by which uPAR and its ligand uPA regulate VSMC
phenotypic changes is not known. In the present study, we
provide evidence that the molecular machinery of uPAR-mediated
VSMC differentiation employs lipid rafts. We show that the
disruption of rafts in VSMCs by membrane cholesterol depletion
using MCD (methyl-β-cyclodextrin) or filipin leads to the upregulation of uPAR and cell de-differentiation. uPAR silencing
by means of interfering RNA resulted in an increased expression
of contractile proteins. Consequently, disruption of lipid rafts
impaired the expression of these proteins and transcriptional
activity of related genes. We provide evidence that this effect
was mediated by uPAR. Similar effects were observed in VSMCs
isolated from Cav1−/− (caveolin-1-deficient) mice. Despite the
level of uPAR being significantly higher after the disruption
of the rafts, uPA/uPAR-dependent cell migration was impaired.
However, caveolin-1 deficiency impaired only uPAR-dependent
cell proliferation, whereas cell migration was strongly upregulated in these cells. Our results provide evidence that rafts are
required in the regulation of uPAR-mediated VSMC phenotypic
modulations. These findings suggest further that, in the context
of uPA/uPAR-dependent processes, caveolae-associated and nonassociated rafts represent different signalling membrane domains.
INTRODUCTION
VSMCs {vascular SMCs [SM (smooth muscle) cells]} play
an important role in the pathology of vascular remodellingassociated diseases. During cellular remodelling following
vascular injury and the development of atherosclerotic plaques,
VSMCs change from their physiological contractile phenotype to
the pathophysiological synthetic phenotype and migrate into the
intima, where they proliferate and produce extracellular matrix
[5,6]. uPA (urokinase-type plasminogen activator) and its specific
receptor (uPAR) have been implicated in a broad spectrum of
pathophysiological processes involved in cardiovascular diseases
and vascular remodelling. The fibrinolytic uPA/uPAR system is
induced after vascular injury and up-regulated in atherosclerotic
lesions, contributing to neointima formation and early lesion development [7–9]. Beyond the regulation of cell-surface-associated
proteolysis, uPAR orchestrates the signalling pathways underlying the functional changes in vascular cells, such as adhesion, migration and proliferation [10]. uPAR targeting in human organ cultures and in animal models leads to a strong inhibition of negative
vascular remodelling [11,12]. As uPAR is linked to the outer membrane leaflet by a GPI anchor and is devoid of any known catalytic
activity, all of its diverse biological functions are strictly dependent on its interactions with other proteins. Indeed, a large variety of
proteins capable of interacting with uPAR in a cell-specific fashion
have been identified over the last two decades. Our previous
findings have demonstrated that, in human VSMCs, uPAR is
Cholesterol is an essential component of the membranes of mammalian cells and an intricate system has been developed to regulate
cellular levels of this lipid. In part, cholesterol reaches cells
via the circulation, packaged in LDL (low-density lipoprotein)
particles. However, it is synthesized further by most cells using
enzymes residing in the endoplasmic reticulum [1]. Cholesterol
is a major component of eukaryotic cell membranes and is highly
enriched in plasma membranes, but not in other cell organelles.
It is believed that, in membranes, cholesterol plays a role in
forming specific segregated microdomains, called lipid rafts,
thought to regulate membrane protein sorting and construction of
signalling complexes [2]. According to lipid raft models of plasma
membrane organization, lipids of specific chemistry, namely
cholesterol and sphingolipids, spontaneously associate with each
other to form rigid platforms for the segregation of proteins such as
GPI (glycosylphosphatidylinositol)-anchored proteins. Performing biochemical studies to investigate rafts is technically
challenging. The main approaches used are cholesterol depletion
using cholesterol-extracting drugs, such as MCD (methyl-βcyclodextrin) [3], or cholesterol binding using filipin [4], and
inhibition of intracellular cholesterol biosynthesis, using, for
example, inhibitors of HMG-CoA (3-hydroxy-3-methylglutarylCoA) synthetase, namely statins.
Key words: caveolin, cell differentiation, lipid raft, plateletderived growth factor-receptor (PDGFR), urokinase-type plasminogen activator receptor (uPAR), vascular smooth muscle cell.
Abbreviations used: CTB, cholera toxin B; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPI, glycosylphosphatidylinositol; MCD, methyl-βcyclodextrin; PDGFRβ, platelet-derived growth factor-receptor β; RT–PCR, reverse transcription–PCR; siRNA, small interfering RNA; SM, smooth muscle;
SMA, SM α-actin; SMC, SM cell; uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; uPARsi, uPAR siRNA; VSMC, vascular SMC; WT,
wild-type.
1
To whom correspondence should be addressed (email [email protected]).
c The Authors Journal compilation c 2009 Biochemical Society
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J. Kiyan and others
associated with PDGFRβ (platelet-derived growth factor-receptor
β) to mediate intracellular signalling and cell functions [13]. uPA
binding to uPAR induced rapid PDGFRβ activation, phosphorylation and dimerization. As a GPI-linked protein, uPAR has been
found to localize to different membrane domains including lipid
rafts [14,15]. Our recent studies have revealed an important role
of lipid rafts in uPA/uPAR-directed signalling in human VSMCs
[16]. However, whether this might have functional consequences
for VSMC phenotypic modulations has remained unexplored.
Modulation of cholesterol content in the cell membrane leads
to changes in cell functions and behaviour. In VSMCs, cholesterol
might regulate cell phenotypic states. It has been shown previously
that loading VSMCs with cholesterol rapidly induces cell transdifferentiation [18]. On the contrary, decreasing the cholesterol
content represses VSMC de-differentiation in culture [19].
Together with the observation that increased cholesterol levels
represent a risk factor for the development of cardiovascular
diseases, these findings suggest that cholesterol is a powerful
regulator of VSMC functions. We have demonstrated recently
that VSMC inhibition of cholesterol biosynthesis by rosuvastatin
leads to inhibition of cell proliferation in both ex vivo and
in vitro models, and consequently exerts a positive effect on
injury-induced vascular remodelling [20]. We found that this
beneficial effect of rosuvastatin was mediated by uPAR-mediated
promotion of the differentiated VSMC phenotype. However, the
role of rosuvastatin, as well as other statins, in VSMC transdifferentiation has not yet been elucidated. One possibility is that
deregulation of uPAR-directed cell signalling via membrane rafts
leads to changes in cell behaviour and phenotype. Our present
study was designed to verify this hypothesis.
Cells were cultivated in DMEM (Dulbecco’s modified Eagle’s
medium) containing 10 % (v/v) fetal bovine serum.
For VSMC nucleofection, a Basic Smooth Muscle Cell
Nucleofector kit from Amaxa was used in accordance with the
manufacturer’s instructions. Overexpression or down-regulation
of proteins was proved by Western blot analysis 24–72 h after cell
nucleofection and was stable for at least 4–5 days.
Chemiluminescent images were captured using VersaDoc3000 and quantified using Quantity One software (Bio-Rad
Laboratories).
Quantitative RT (reverse transcription)–PCR analysis of uPAR in
human VSMCs
Total RNA was isolated from VSMCs using an RNeasy miniprep
kit (Quiagen), and real-time quantitative RT–PCR for uPAR
mRNA was performed on a TaqMan ABI 7700 sequence detection
system (Applied Biosystems). GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as a reference gene, as described
previously [22].
FACS analysis
VSMCs treated with MCD or filipin were de-attached using 5 mM
EDTA/PBS (pH 7.4) and labelled with anti-uPAR monoclonal
antibodies (R&D Systems), according to the manufacturer’s
recommendation, and Alexa Fluor® 488-conjugated secondary
antibodies (Invitrogen). Analysis was performed using FACS
CANTO system (BD Biosciences) using acquisition software
FACS DIVA (BD Biosciences). Data were analysed using Summit
software (Dako).
MATERIALS AND METHODS
Immunofluorescent confocal microscopy
Reagents and antibodies
For immunocytochemical staining, cells were cultured on glass
coverslips. Cells were fixed with 2 % paraformaldehyde in PBS
for 15 min at 4 ◦C, permeabilized with 0.1 % Triton X-100 in
PBS for 3 min at 4 ◦C, and blocked overnight at 4 ◦C in 1 % (w/v)
BSA in PBS. Cells were labelled with primary antibody (2 h at
18–22 ◦C) and fluorescently labelled secondary antibodies (1 h at
room temperature). Lipid rafts were labelled using Alexa Fluor®
594-conjugated CTB (cholera toxin B) subunit (raft labelling
kit; Molecular Probes), according to manufacturer’s instructions.
After staining, cells were embedded in Aqua-Poly-Mount
mounting medium (Polysciences). The fluorescence cell images
were captured using a Leica TCS-SP2 AOBS confocal microscope. All of the images were taken with oil-immersed × 63
objective (numerical aperture = 1.4). Images were recorded with
detection wavelengths range for Alexa Fluor® 488- and Alexa
Fluor® 594-double staining of 505 to 550 nm and 605 to 675 nm
respectively. All the images were acquired with a resolution of
1024 × 1024 pixels. The fluorograms were obtained by plotting
each pixel of the overlay Alexa Fluor® 488/Alexa Fluor® 594
colour fluorescence cell image [13]. Pixel values of Alexa
Fluor® 488 and Alexa Fluor® 594 fluorescence images represent
horizontal and vertical axis values for each point respectively. The
points of the fluorograms are coloured in accordance with pixel
counts with specific Alexa Fluor® 488 and Alexa Fluor® 594
fluorescence intensities. The pixel count is shown in logarithmic
scale.
High-quality commercial grade chemicals were purchased from
Sigma, Amersham Pharmacia Biotech and Merck. Monoclonal
anti-h-caldesmon, anti-calponin and anti-SMA (SM α-actin) antibodies were from Sigma. Monoclonal anti-PDGFRβ and
anti-uPAR antibodies were from R&D Systems. Monoclonal anticaveolin-1 antibodies were from Transduction Laboratories (BD
Biosciences). Polyclonal anti-caveolin-1 antibodies were from
Cell Signaling. Fluorescent Alexa Fluor® 488- and Alexa
Fluor® 594-conjugated secondary antibodies, and raft labelling
kits were from Molecular Probes (Invitrogen). uPARsi [uPAR
siRNA (small interfering RNA)] duplexes, caveolin-1 siRNA
duplexes and control non-specific siRNA duplexes were from
Santa Cruz Biotechnology. MCD and filipin were from Sigma.
Rosuvastatin was provided by AstraZeneca. The construct to
express luciferase under the SMA promoter was generously given
by Professor Gary Owens, Department of Molecular Physiology
and Biological Physics University of Virginia School of Medicine,
Charlottesville, VA, U.S.A.
Cell culture and cell nucleofection
Primary human coronary artery SMCs were purchased from
PromoCell and Lonza BioScience Walkersville, cultivated in
the medium recommended by the suppliers and used between
passages three and six. VSMCs derived from aortas of Cav1−/−
(calveolin-1 deficient) and corresponding WT (wild-type) mice
were isolated as described previously [21]. Experiments were
performed in accordance with the legal requirements of the relevant local and national guidelines regarding animal experiments.
c The Authors Journal compilation c 2009 Biochemical Society
Isolation of lipid raft fractions
A detergent-free raft isolation method employing centrifugation
in OptiPrep gradient [23] was used with some modifications,
Membrane rafts and uPAR in VSMC phenotypic modulations
namely a discontinuous OptiPrep gradient was used. After
centrifugation [20 500 rev./min for 90 min at 4 ◦C using an SW-41
rotor (Beckman)], gradients were fractionated into 1 ml aliquots,
and the distribution of proteins was assessed by Western blot
analysis using the same amount of protein from each fraction.
Fractions collected between 0 and 5 % OptiPrep (fraction 2),
5 and 10 % OptiPrep (fraction 5), and 10 and 20 % OptiPrep
(fraction 7) are shown. Fraction 2 contained most of the raft
marker GM1 as determined using dot-blot analysis of fractions
using HRP (horseradish peroxidase)-conjugated CTB.
Chemotaxis assay, cell proliferation assay and luciferase assay
The chemotaxis assay was performed using a wounding model
on Cell Locate grid coverslips (Eppendorf). Briefly, cells
were seeded on to grid coverslips reaching approx. 80 %
confluence after attachment. After cell starvation, wounding
was performed under a microscope and the wound position
documented. Cells were allowed to migrate in the presence
of various stimuli for 12–24 h. Additionally, cell migration
was assessed using a Boyden chamber, as described previously
[13].
VSMC proliferation was quantified using the colorimetric cell
proliferation ELISA kit (Roche Applied Bioscience) in accordance with the manufacturer’s instructions. The mean +
− S.D. absorbance at 450 nm was calculated for three independent experiments
is indicated.
Luciferase activity was measured using the Promega ONEGloTM luciferase assay system, according to manufacturer’s
instructions.
Statistical analysis
All the experiments were performed in triplicate. Values are
means +
− S.E.M. Statistical significance analysis (P < 0.05) was
performed using a Student’s t test.
RESULTS
uPA induces raft enrichment in uPAR and PDGFRβ
In human VSMCs, PDGFRβ serves as a transmembrane interactor
of uPAR, mediating its cellular and functional effects [13]. The
observation that both uPAR and PDGFRβ could be found in
lipid rafts [14,24] persuaded us to test whether this localization
is uPA-dependent. We applied an immunocytochemical approach
and used Alexa Fluor® 594-conjugated CTB to indicate lipid
rafts. As shown in Figures 1(A) and 1(B), co-localization of
uPAR and PDGFRβ with fluorescently labelled CTB was upregulated in response to uPA, thus pointing to the role of rafts in
uPAR/PDGFRβ-dependent cellular responses.
These visual observations were confirmed by their quantification in fluorograms. Distribution of the points in the fluorograms confirmed increased co-localization of both receptors with
lipid rafts in VSMCs stimulated with uPA for 20 min. These
results are in agreement with our earlier observation showing
increased association and co-localization of uPAR and PDGFRβ
in response to uPA [13]. Furthermore, co-localization of uPAR
with caveolin-1 was also increased after uPA stimulation of
VSMCs (Figure 1C).
Rafts regulate uPAR-mediated VSMC phenotypic changes
Recently, we have demonstrated that uPAR contributes to vascular
remodelling by promoting VSMC phenotypic modulations [20].
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To elucidate the role of rafts in these processes, we first
examined whether raft disruption might affect the abundance
of uPAR, as this is critical for the propagation of trans-differentiation. We targeted membrane rafts by cholesterol depletion
using the extracting drug MCD and filipin. As shown in
Figure 2(A), both MCD and filipin treatment led to uPAR upregulation. Immunocytochemical staining of the cells under nonpermeabilizing conditions showed that uPAR was accumulated
on the cell membrane (Figure 2B). In order to clarify whether
uPAR up-regulation was transcriptional, we performed TaqMan
analysis. Figure 2(C) shows that both MCD and filipin induced
significant up-regulation of uPAR mRNA. Further FACS analysis
of VSMCs treated with both substances confirmed that MCD
treatment did indeed increase uPAR on the cell surface
(Figure 2D). Filipin treatment, although increasing the total
amount of uPAR (Figure 2A) and uPAR transcription (Figure 2C),
led to only a small increase in uPAR on the cell surface
(Figure 2D).
To investigate the consequences of this effect on uPARmediated VSMC phenotypic modulation, we examined the
expression of SM marker proteins after uPAR silencing and raft
disruption. Cells were nucleofected with uPARsi duplexes or
control duplexes, and then the expression of SMA, calponin and
SM high-molecular-mass caldesmon was analysed by Western
blotting. Down-regulation of uPAR promoted the expression of
contractile markers (Figure 3A). Conversely, cell treatment with
MCD or filipin decreased the expression of contractile marker
proteins (Figures 3B and 3C). We found that the down-regulation
of uPAR using siRNA duplexes prevented the MCD-dependent
decrease in contractile marker expression (Figure 3D), confirming
further the role of uPAR in this process. To elucidate the functional
consequences of this effect, we examined the possibility of a direct
transcriptional activation of SM-specific genes in these cells.
For this purpose, control and uPARsi VSMCs were transfected
with luciferase reporter constructs driven by an SMC-specific
promoter from the SMA gene. As shown in Figure 3(E), SMA
promoter was activated in uPARsi VSMCs and the inhibitory
effect of MCD was abolished in these but not in control
cells.
Together, these findings indicate that the level of uPAR is an
important regulator of VSMC phenotype. Thus a decrease in
uPAR expression leads to cell differentiation, whereas accumulation of uPAR by cell treatment with raft-disrupting drugs leads
to VSMC de-differentiation.
uPAR-related cellular effects are differently regulated by
caveolae-associated and non-associated membrane rafts
Caveolae, a major specialized subclass of lipid rafts, are membrane invaginations enriched in particular lipids (e.g. cholesterol
and glycosphingolipids) and are characterized by an abundance
of palmitoylated scaffolding proteins (e.g. caveolins) that cause
rafts to polymerize and interact with a wide variety of proteins.
The number and distribution of caveolae are different in synthetic
and contractile VSMC phenotypes [19]. By regulating cholesterol
trafficking and multiple receptor-mediated signalling pathways,
caveolae influence vascular function and cardiovascular disease
development [25].
We observed uPA-dependent redistribution of caveolin-1,
together with uPAR and PDGFRβ, to raft fractions in response
to uPA (Figures 4A–4D). Therefore we investigated whether uPA
stimulation might also regulate the uPAR/caveolin-1 association.
As shown in Figure 4(E), these proteins had an increased
association in response to uPA in co-immunoprecipitation
experiments.
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Figure 1
J. Kiyan and others
uPA induces the co-localization of uPAR and PDGFRβ with lipid rafts
VSMC were stimulated with uPA for 20 min, the stimulation medium was removed, cells were placed on ice, labelled for lipid rafts using an Alexa Fluor® 594 vibrant raft labelling kit, then fixed,
blocked and labelled with anti-uPAR (A) or anti-PDGFRβ (B) antibodies and the corresponding Alexa Fluor® 488-conjugated secondary antibodies. Fluorograms were obtained as described in the
Materials and methods section. (C) VSMCs were stimulated with uPA for 20 min, then fixed and stained for uPAR and caveolin-1.
Caveolae-associated and non-associated rafts have been shown
to differ, at least partially, in specific protein clustering and,
consequently, to mediate different cellular responses [26]. To
distinguish between the roles of caveolar and non-caveolar rafts
in uPAR-dependent VSMC cellular functions, we investigated
c The Authors Journal compilation c 2009 Biochemical Society
VSMCs after down-regulation of caveolin-1 using siRNA. We
found that expression of SM markers was activated in these cells;
consequently uPAR was down-regulated (Figure 4F).
Finally, we analysed the uPA-dependent functional responses
of Cav1−/− murine VSMCs generated previously [27] and human
Membrane rafts and uPAR in VSMC phenotypic modulations
Figure 2
Raft disruption increases uPAR expression in VSMCs
(A) Expression of uPAR in VSMCs treated with 0.1 % MCD or 5 μM filipin for 24 h was assessed
by Western blotting. The positions of molecular-mass markers (in kDa) are indicated on the
right. Lower panels, Western blotting with tubulin as a loading control. (B) VSMCs treated
with 0.1 % MCD (right-hand panel) were stained under non-permeabilizing conditions with
anti-uPAR primary antibodies, then fixed, blocked overnight and stained with Alexa Fluor®
488-conjugated secondary antibodies. (C) Effect of VSMC treatment with 0.1 % MCD or 5 μM
filipin on uPAR expression as determined by TaqMan analysis. (D) Amount of surface uPAR after
VSMC treatment with 0.1 % MCD or 5 μM filipin assessed using FACS analysis, as described
in the Material and methods section. N. control, negative (isotype IgG) control.
VSMCs treated with raft-disrupting drugs. As shown in Figure 5,
caveolin-1 deficiency elicited effects on cell behaviour that were
different from those of raft disruption. Thus raft targeting by
rosuvastatin resulted in the down-regulation of uPA-induced cell
migration, whereas cell proliferation was not significantly affected
(Figure 5A). By contrast, only uPA-initiated cell proliferation was
impaired in Cav1−/− VSMCs, whereas the migratory response
of these cells was strongly up-regulated by uPA (Figure 5B).
We can conclude, therefore, that, at least in the context of
uPA-dependent processes, rafts and caveolae represent different
signalling membrane domains.
DISCUSSION
Signalling cascades activated by cell-surface receptors are
tremendously complicated processes involving both acute- and
longer-term signalling events. Although early studies did not
generally address the membrane environment of receptors, a
body of work has now demonstrated that heterogeneity exists in
the composition of the plasma membrane and that distinct lipid
environments play a role in receptor function. Cholesterol-rich
microdomains, often referred to as lipid rafts and more recently
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as membrane rafts, have become a central facet of signalling
research, as the function of many receptors and their downstream
effectors is dependent on rafts [2,28]. Numerous proteins may
cluster in membrane rafts, including GPI-anchored proteins,
G-protein-coupled receptors, receptor tyrosine kinases and
phosphatases, thereby accounting for the potentially important
role of rafts in cell signalling [29,30]. Lipid rafts may or may not
be enriched in caveolin-1, the major structural protein found in
the anatomic membrane structures referred to as caveolae. Raftassociated caveolin-1 may function in maintaining high cholesterol levels. Previous studies have implicated lipid rafts in various
disease processes including vascular disease [31,32]. These
studies have demonstrated that alteration in lipid raft expression
and occupancy is decisive for cell–cell contact and cell signalling, and that these are abnormal in patients with vascular disease
[33–35]. Moreover, translocation of some receptors to lipid
rafts and associated signalling can differ between prognostically
important groups of patients and serve as a prognostic marker
[36].
uPAR, a multi-functional cell-surface receptor, is an important
participant in the molecular machinery underlying vascular
remodelling and cardiovascular diseases [9,37–39]. Our recent
findings revealed a novel aspect of uPAR in these processes,
which is linked to the regulation of VSMC trans-differentiation
[20]. Although uPAR, as a GPI-anchored protein, has been
identified previously in lipid rafts [14], little is known regarding
the dependence on rafts for eliciting downstream signalling of
uPAR and consequent biological outcomes in VSMCs.
In the present study, we have identified a novel role for membrane lipid rafts in uPA/uPAR-mediated promotion of VSMC
functional responses, in particular in directing VSMC phenotypic
modulations. The evidence we present in support of a role for lipid
rafts in VSMC phenotypic changes is as follows: (i) raft disruption
by depletion of cholesterol in the plasma membrane resulted in
uPAR up-regulation; (ii) raft disruption led to decreased expression of contractile proteins and transcriptional activity of SM
genes, an effect abolished in uPARsi VSMCs; (iii) uPAR and
its transmembane adaptor PDGFRβ were redistributed to lipid
rafts in response to uPA; and (iv) raft disruption influenced
some uPA-initiated cellular responses. Our findings provide the
first evidence that VSMCs employ a cholesterol-rich membrane
raft compartment for transmission of the uPAR-mediated
differentiation signal. Since uPAR has been identified in rafts
and non-raft fractions [14], our present findings suggest further
that raft-associated uPAR represents a distinct and separately
functioning pool of receptors. Our present study demonstrates that
three-dimensional cell morphology and uPAR-directed signal
transduction become integrated to regulate cell phenotype.
Different classes of rafts exist in mammalian cells. Some lipid
rafts lack structural protein components, but when enriched with
a specific protein raft morphology function can change. The
first structural protein component to be identified was caveolin-1
[40]. Although incompletely understood, caveolin-1 appears to
function as a scaffolding molecule that regulates the activities of
signalling molecules clustered in caveolar domains. However, a
growing body of evidence points to a clear distinction between
signalling mechanisms and related functional responses in lipid
rafts compared with caveolae [26]. Results of our experiments on
VSMCs isolated from Cav1−/− mice support this concept. Thus
the functional responses were different in VSMCs with disrupted
rafts and in Cav1−/− VSMCs. We favour the hypothesis that
certain components of the uPAR interactome may be regulated by
caveolin-1 in the context of plasma-membrane-attached caveolae
or perhaps by the recruitment of other regulatory molecules in the
caveolae, but not to a raft. We can conclude that, at least in terms of
c The Authors Journal compilation c 2009 Biochemical Society
348
Figure 3
J. Kiyan and others
uPAR regulates the expression of contractile marker proteins
(A) VSMC were nucleofected with control (Co) or uPARsi duplexes using an Amaxa Basic Smooth Muscle Cell Nucleofector kit. At 48 h after nucleofection, Western blotting was performed to
demonstrate the decreased expression of uPAR and increased expression of SMA (α-SM Actin), calponin and h-caldesmon. Tubulin served as a loading control. After 24 h VSMC treatment with
0.1 % MCD (B) or 5 μM filipin (C), expression of contractile proteins was down-regulated. (D) Effects of MCD on contractile protein expression are mediated by uPAR. VSMCs were nucleofected to
down-regulate uPAR expression as in (A). At 48 h after nucleofection, cells were stimulated with 0.1 % MCD for 24 h and the expression of SMA was measured. (E) VSMCs were nucleofected with
the construct expressing luciferase under the control of the SMA promoter simultaneously with control (si Control) or uPARsi duplexes. At 24 h after nucleofection, cells were stimulated with 0.1 %
MCD for 3 h and luciferase activity was measured, as described in Materials and methods section.
uPA-dependent processes, rafts and caveolae represent different
signalling membrane domains.
One intriguing observation of our present study was the strong
dependence of uPAR levels in VSMCs on the integrity of rafts.
Raft disruption resulted in a strong uPAR up-regulation. This,
in turn, initiated VSMC phenotypic transition to the de-differentiated pathophysiological phenotype. uPA stimulation induced
translocation of uPAR and its transmembrane adaptor PDGFRβ
to lipid rafts. We have reported recently that association between
components of the uPAR interactome was increased after raft
disruption, as well as basal phosphorylation levels of ERK
(extracellular-signal-regulated kinase), SHP-2 (Src homology 2
domain-containing protein tyrosine phosphatase-2) and RhoA
activity [16]. However, uPA-initiated functional responses, such
as cell migration and proliferation, were abrogated in these cells.
This raises the idea that the control of uPAR-related events
in VSMCs provided by rafts may be of at least two different
functional compartments. The first one is the strict regulation of
uPAR cellular levels that determines cell phenotype. The second
implies that rafts in VSMCs may function as the platform for a
network of signalling molecules that start to assemble in response
c The Authors Journal compilation c 2009 Biochemical Society
to external stimuli such as uPA. These molecules may work in
concert with downstream targets known to regulate cytoskeletal
organization, such as PI3K (phosphoinositide 3-kinase), Rho,
Rac and Cdc42 [13,41]. Additionally, lipid rafts may also help
to preserve the membrane uPAR capable of uPA binding and
signalling, and have an impact on regulators for efficient signal
transduction. What the molecular mechanism is underlying uPARdirected VSMC phenotypic transition is an intriguing question
and the answer is largely obscure and requires specific study.
Our preliminary results suggest the involvement of myocardin
(J. Kiyan and I. Dumler, unpublished work), a serum-response
factor cofactor that is expressed specifically in SMC and cardiac
muscle cell lineages and which plays an important role in VSMC
differentiation in vivo.
Finally, our results suggest that cellular cholesterol levels and,
in particular membrane lipids, regulate uPAR-directed VSMC
phenotypic changes. They suggest further that uPAR signalling
and cellular functions can be modified in VSMCs by statin treatment; in particular to shift cells to a physiologically differentiated
phenotype. It is tempting to speculate that some of the pleiotropic
effects of statins on the cardiovascular system could result from
Membrane rafts and uPAR in VSMC phenotypic modulations
Figure 4
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uPA induces the redistribution of uPAR-associated signalling molecules to the lipid raft membrane fraction
(A) Typical GM1 distributions in fractions collected from the OptiPrep gradient. (B) uPA-induced re-distribution of uPAR (B), PDGFRβ (C) and caveolin-1 (D) in the OptiPrep gradient fractions.
Lower panels, quantification of proteins in the fractions. (E) Lysates of uPA-stimulated VSMCs were used to immunoprecipitate (IP) uPAR. The presence of caveolin-1 in the immunoprecipitates
was assessed by immunoblotting (WB). Lower panel, immunoblotting with anti-uPAR antibodies to demonstrate the equal amount of immunoprecipitated uPAR. (F) VSMCs were nucleofected with
control or caveolin-1 siRNA (Cav si) duplexes. At 48 h after nucleofection, silencing was verified. Expression of uPAR and contractile proteins was assessed by Western blotting. a.u., arbitrary units;
Co, control.
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J. Kiyan and others
Figure 5 Effects of cholesterol depletion and caveolin-1 knockout on uPAinduced migration and proliferation
(A) uPA-induced migration and proliferation of human coronary artery VSMCs were measured
after 24 h of treatment with rosuvastatin (RS). (B) uPA-induced migration and proliferation of
VSMCs isolated from Cav1−/− mice and their corresponding WT genetic control (Cav1+/+ ).
changes in membrane lipids, at least in the context of uPARrelated vascular remodelling. These findings may have applications in the understanding and potential treatment of these
diseases.
AUTHOR CONTRIBUTION
Julia Kiyan performed the experiments and prepared the manuscript, Graham Smith
designed and optimized the experiments on the cell treatment with rosuvastatin, Hermann
Haller approved the paper, and Inna Dumler, the scientific surpervisor, prepared and edited
the manuscript prior to submission.
ACKNOWLEDGEMENTS
We are grateful to Iris Kilian and Petra Wubbolt for excellent technical assistance, Professor
Gary Owens for giving us the construct expressing luciferase under the SMA promoter,
and Maren White for editing the manuscript prior to submission.
FUNDING
This work was supported by the Deutsche Forschungsgemeinschaft [grant numbers DU
344/1-4, DU 344/6-1] and an ERA-AGE FLARE grant financed by Bundesministerium für
Bildung und Forschung [grant number 01 ET 0802].
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Received 18 March 2009/16 July 2009; accepted 20 August 2009
Published as BJ Immediate Publication 20 August 2009, doi:10.1042/BJ20090447
c The Authors Journal compilation c 2009 Biochemical Society