1 Department of Pathology, Division of Molecular and Cellular Pathology and The
Cell Adhesion and Matrix Research Center, University of Alabama at Birmingham,
Birmingham, AL 35294-0019, USA
2 Research Service, Birmingham VA Medical Center, Birmingham, AL 35233-1996,
USA
3 Department of Pathology, Division of Laboratory Medicine, University of
Alabama at Birmingham, Birmingham, AL 35294-0019, USA
* Author for correspondence (e-mail: murphy{at}path.uab.edu)
Accepted 4 April 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Thrombospondin-1, Focal adhesion, Cell migration, Calreticulin, LDL receptor-related protein
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell migration is an integral process in tissue formation and remodeling,
and is dependent upon adhesion dynamics
(Kaverina et al., 2002;
Webb et al., 2002
). Nascent
adhesions form in the leading lamellipodia, stabilizing the protrusion and
generating traction to pull the cell body forwards
(Beningo et al., 2001
).
However, adhesions in the rear of the cell must disassemble to allow
retraction of the trailing edge of the cell. While focal adhesion plaques may
provide some of the tractional forces driving cell migration, the relative
stability of focal adhesions, compared with less-organized adhesive
structures, may retard rear retraction
(Lauffenburger and Horwitz,
1996
; Webb et al.,
2002
). Increased expression of vinculin and
-actinin
enhances focal adhesion formation and reduces cell migration, whereas
decreased expression reduces focal adhesion formation and stimulates migration
(Fernandez et al., 1992
;
Gluck and Ben-Ze'ev, 1994
).
Several promigratory signaling pathways, such as phosphoinositide 3-kinase (PI
3-kinase), src and focal adhesion kinase (FAK), also regulate focal adhesion
dynamics (Greenwood and Murphy-Ullrich,
1998
; Anand-Apte and Zetter,
1997
). Thus, modulating focal adhesion structure, by altering
either the expression of certain focal adhesion components or the signaling
pathways that regulate their assembly, significantly affects cell
migration.
Thrombospondin-1 (TSP1) is a large, homotrimeric, matricellular
glycoprotein, expressed in a highly regulated manner by numerous cell types in
developing and remodeling tissues. TSP1 is involved in numerous biological
functions, probably attributable to its multiple domains and cell-surface
receptors as well as its ability to act as either a soluble or matrix-bound
factor. Multiple domains of TSP1 affect cell migration, although regulation of
domain dominance remains poorly understood and probably depends on the cell
type and environmental context. Matrix-bound TSP1 supports a limited degree of
cell attachment and spreading, characterized by the formation of fascin
microspikes in the cell periphery and the absence of focal adhesions or stress
fibers (Adams, 1995;
Murphy-Ullrich and Höök,
1989
). Haptotactic migration to matrix-bound TSP1 occurs through
the pro-adhesive C-terminal domain, although other domains might also be
involved (Taraboletti et al.,
1987
). The heparin-binding domain (HBD) elicits cell migration in
neutrophils, monocytes, melanoma cells and endothelial cells, and is suggested
to mediate TSP1-induced chemotaxis
(Mansfield et al., 1990
;
Mansfield and Suchard, 1994
;
Taraboletti et al., 1987
;
Taraboletti et al., 1990
).
However, the mechanisms by which the HBD stimulates cell migration are not
established.
TSP1 destabilizes cell-ECM adhesions by stimulating focal adhesion
disassembly in highly adherent cells and preventing focal adhesion formation
in adhering cells. TSP1-induced focal adhesion disassembly involves unbundling
of actin stress fibers and the selective depletion of vinculin and
-actinin from the focal adhesion plaque
(Greenwood et al., 1998
;
Greenwood and Murphy-Ullrich,
1998
). This transition does not affect integrin clustering or cell
spreading, representing a reversion from a mature focal adhesion to a
less-organized adhesive structure. This spread cell depleted of focal
adhesions is termed the intermediate adhesive state by our group, and is
postulated to prime the cell for dynamic cellular processes
(Greenwood and Murphy-Ullrich,
1998
; Murphy-Ullrich,
2001
). TSP1 stimulates the transition to intermediate adhesion
through a 19-amino acid sequence (hep I peptide) in the HBD, which signals
focal adhesion turnover through a receptor co-complex of calreticulin (CRT)
and low-density lipoprotein (LDL) receptor-related protein (LRP)
(Murphy-Ullrich et al., 1993
;
Goicoechea et al., 2000
;
Orr et al., 2002
;
Orr et al., 2003
). We now
report that TSP1/hep I stimulates chemotaxis and chemokinesis in endothelial
cells and fibroblasts through ligation of the CRT-LRP receptor complex, and
selectively modulates fibroblast growth factor (FGF)-induced migration.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Proteins
TSP1 was isolated from human platelets from the American Red Cross, and
purified as previously described using heparin affinity and gel filtration
chromatography (Murphy-Ullrich et al.,
1993). Peptides hep I (ELTGAARKGSGRRLVKGPDC) and modified hep I
(ELTGAARAGSGRRLVAGPDC) were synthesized, purified and analyzed by the
University of Alabama at Birmingham Comprehensive Cancer Center/Peptide
Synthesis and Analysis shared facility and by AnaSpec (San Jose, CA).
Receptor-associated protein (RAP) was a generous gift of Dudley Strickland
(Jerome Holland Labs, ARC, Bethesda, MD). Bovine bFGF and aFGF were from
Calbiochem (San Diego, CA).
Cell culture
Bovine aortic endothelial (BAE) cells were isolated and cultured in DMEM
containing 4.5 g/l glucose, 2 mM glutamine and 10% FBS as described previously
(Murphy-Ullrich et al., 1993).
Wild-type (K41) and CRT-null (K42) mouse embryonic fibroblasts (MEFs) were a
gift of Marek Michalak (University of Alberta, Edmonton, AB, Canada). LRP-null
[PEA 13 (ATCC-CRL-2216)] MEFs were from American Type Culture Collection
(Manasses, VA). Growth conditions for MEFs were the same as described for BAE
cells.
Transwell assay
A 96-well chemotaxis chamber (Neuro Prob, Gaitherburg, MD) was used for the
transwell assays. BAE cells were grown to near confluence and fluorescently
labeled by incubation with 1 µM calcein AM for 10 minutes. Cells were
washed twice in DMEM and briefly trypsinized for re-plating on the transwell
membrane. The polyvinyl membrane containing 8 µm pores was coated for 5
hours with 10 µg/ml vitronectin and 10 µg/ml fibronectin, washed three
times in phosphate buffered saline (PBS) and air-dried. Increasing
concentrations of hep I, TSP1, modified hep I peptide or bFGF were added to
the lower chamber, and the membrane was fastened to the lower chamber per
manufacturer's instructions. Labeled BAE cells were then plated on the top
side of the filter at 2x104 cells per well in either
serum-free media or in serum-free media containing increasing concentrations
of hep I, TSP1, modified hep I or bFGF. Cells were allowed to migrate towards
the bottom well for 5 hours. The top side of the membrane was scraped using
pre-wetted cotton swabs and gently rinsed with PBS. The chamber was loaded
into a plate reader (emission 460 nm/absorbance 530 nm) and the remaining
well-associated fluorescence was read. Results for each assay were carried out
in triplicate and normalized to migration levels seen with serum-free media
alone.
Dunn chamber
Dunn chambers were from Weber Scientific International (Teddington, UK).
The Dunn chamber allows for generation of a stable chemotactic gradient and
observation of cell migration in the context of the gradient
(Zicha et al., 1991). Glass
coverslips were coated with 10 µg/ml fibronectin and 10 µg/ml
vitronectin for 5 hours. Cells were then sparsely plated onto the pre-coated
coverslips and allowed to attach in serum-free media for 3 hours. Serum-free
DMEM containing 23.8 mM HEPES, with or without 100 nM hep I, 7.8 nM TSP1 or
100 nM modified hep I, was added to both Dunn chamber wells. Glass coverslips
were then loaded onto the Dunn chamber, cells down, and sealed to the Dunn
chamber by an equal mixture of vacuum grease and Vaseline (blotted to remove
excess oil), with the outer well of the Dunn chamber remaining uncovered.
Media was removed from the outer well, and serum-free DMEM containing 23.8 mM
HEPES, with or without 100 nM hep I, 7.8 nM TSP1, 100 nM modified hep I, 0.1%
FBS, 61 pM bFGF or 67 pM aFGF, was added to the outer well, establishing a
concentration gradient. A computer-controlled stage (Prior Scientific,
Rockland, MA) was used to enable viewing of multiple fields on each of two
Dunn chambers over the time course of the experiment using software developed
in the Dennis Kucik laboratory. Cells were imaged on an Axiovert 100
microscope (Zeiss, Thornwood, NY) equipped with a CCD camera (Model 300T-RC,
Dage-MTI, Michigan City, IN). Temperature on the stage was kept constant at
37°C, and images of each field were captured at 2 minute-intervals for a
total of 6 hours and 40 minutes (200 frames) using a WinTV video card
(Hauppauge Computer Works, Hauppauge, NY) and software written in the Kucik
laboratory. Resulting time-lapse video was analyzed by Metamorph software
(Universal Imaging Corporation, Downingtown, PA) to track individual cell
migration paths through successively calculating (x,y) coordinates of the
centroid of the cell. Slight movements in position resulting from the use of a
movable stage were removed by simultaneous tracking of a stationary point, and
normalization of the resulting (x,y) coordinates generated by Metamorph.
Tracks were then analyzed using programs written by Dennis Kucik for this
purpose. Total track distance is determined as the sum of the incremental
distances between successive (x,y) coordinates. Cell speed was determined as
the total distance migrated divided by the time of the assay (generally 400
minutes). The total cellular displacement is calculated as the distance
between the final (x,y) coordinate and the initial (x,y) coordinate. For each
cell, x-displacement and y-displacement is also calculated using this method,
and can be used as final cell position given an initial cell position at the
origin (0,0).
Statistical analysis
Statistical significance was determined using Student's unpaired
t-tests and analysis of variance (ANOVA). Results were considered to
be significant at P<0.05 or P<0.01.
Online supplemental material
BAE and MEF cell migration in the Dunn chamber was recorded at 2-minute
intervals over 6 hours and 40 minutes (200 frames) using the WinTV video card.
Time-lapse videos were then created using Metamorph software. Videos were
recorded at 30 frames per second and compressed using the Cinepak codec. Movie
1 depicts BAE cell migration under serum-free conditions, whereas Movie 2
illustrates chemokinetic migration in BAE cells treated with hep I (100 nM).
Movie 3 illustrates the migratory defect in LRP-knockout MEFs under serum-free
conditions. Movie 4 shows chemotactic migration towards an aFGF (67 pM)
gradient, with the gradient highest on the right side of the screen, whereas
Movie 6 depicts the same aFGF-induced chemotaxis in the presence of
chemokinetic concentrations of hep I (100 nM). Movie 5 represents chemotactic
migration towards a bFGF (61 pM) gradient on the left side of the screen,
whereas Movie 7 illustrates the effect of chemokinetic hep I (100 nM)
treatment on bFGF-induced chemotaxis.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Since focal adhesion disassembly potentially affects the ability of cells
to migrate, we assessed the effect of hep I on random endothelial cell
migration (chemokinesis) by incubating cells with increasing concentrations of
hep I on both sides of the transwell membrane. Increases in chemokinetic
migration will result in increased movement of endothelial cells to the lower
surface of the transwell membrane despite the lack of a chemical gradient
directing the response. Increasing concentrations of hep I in both the upper
and lower wells stimulate an increase (2.5-fold) in the random motility
of endothelial cells (Fig. 2A),
and this response, like hep I-induced chemotaxis, was also maximal at 100 nM
hep I peptide. This concentration of hep I is also sufficient to stimulate
maximal focal adhesion turnover, suggesting a correlation between the presence
of focal adhesions and the ability of endothelial cells to move
(Murphy-Ullrich et al., 1993
).
The modified hep I peptide did not affect endothelial cell migration over the
range of concentrations tested. As positive controls, both TSP1 and bFGF were
tested for the ability to stimulate endothelial cell chemokinesis. Like
TSP1-induced chemotaxis, TSP1 stimulates a dose-dependent increase in
endothelial chemokinesis (
2-fold) that is maximal at 7.8 nM monomer
concentration (Fig. 2B) and,
similar to hep I-induced chemokinesis, this concentration is maximal for
induction of focal adhesion disassembly by TSP1. Consistent with previous
reports, bFGF (100 pM) stimulated a strong increase in endothelial cell
chemokinesis (
3-fold).
|
Viewing TSP1/hep I-induced migration in the Dunn chamber
The Dunn chamber, named for and developed by Graham Dunn, is a modified
helicobacter counting chamber capable of establishing chemical gradients
across a bridge region between an inner circular well and an outer concentric
well (Zicha et al., 1991).
Cells are plated onto glass coverslips and mounted on the chamber in the
presence of varying stimuli. The chamber is sealed, and the cells over the
bridge region monitored by time-lapse videomicroscopy. These videos can then
be analyzed by cell-tracking software to quantify the orientation,
directionality, distance and speed of the cells migrating over this bridge
region. BAE cell migration in response to serum-free DMEM or serum-free DMEM
with a TSP1 (7.8 nM), hep I (100 nM) or modified hep I (100 nM) gradient was
recorded, and individual cell migration tracks were determined. At least 20
cells were analyzed per field, and 3-6 fields were analyzed per condition
(60-120 cells total). A representative scatter plot is shown for each
condition, with the starting point of the cell designated as the origin of the
graph and the top of the graph representing the outer well or the source of
the gradient. In addition, the average distance migrated towards the outer
well is also shown. Whereas endothelial cell migration is not directionally
oriented under serum-free conditions (Fig.
3A), the addition of either TSP1 or hep I (P<0.01 and
P<0.05, respectively) to the outer well induced a markedly
directional response (Fig.
3B,C). By contrast, the inactive modified hep I peptide did not
orient BAE cell migration (Fig.
3D). Chemotactic concentrations of TSP1/hep I resulted in a
slight, but insignificant, increase in BAE cell migration distance, speed and
displacement (data not shown). These data suggest that TSP1/hep I stimulates a
chemotactic response in endothelial cells and that migration in response to
TSP1/hep I gradients in the transwell probably reflects chemotactic
migration.
|
Increased chemokinesis following TSP1/hep I treatment
The Dunn chamber can also be used to assess chemokinesis by observing
general migratory parameters, such as migration distance, speed, displacement
and directionality, in cells stimulated in the absence of a chemical gradient.
BAE cells were incubated in serum-free DMEM with or without TSP1 (7.8 nM), hep
I (100 nM) or modified hep I (100 nM), in both the inner and outer wells of
the Dunn chamber, and migration parameters were determined as described in the
Materials and Methods section. TSP1 and hep I induce endothelial cell
chemokinesis, characterized by an increase in the average cell migration
speed, the total cellular displacement and the percent of migrating cells
(Table 1; Movies 1 and 2
available at
jcs.biologists.org/supplemental).
The modified hep I peptide did not affect general migration parameters as
compared with media alone. As expected, TSP1/hep I-induced chemokinetic
migration did not show a preference for migration orientation, unlike
chemotactic migration towards TSP1/hep I (data not shown). Migration
directionality is measured by dividing the total distance of cell migration by
the resultant vector or total displacement
(Noble and Levine, 2000).
Under this method, a purely directional migration track will show a ratio of
cellular displacement to total migration distance equal to 1, meaning every
step was taken in the exact same direction. Chemokinetic TSP1/hep I stimulated
a slight, but insignificant, increase in BAE cell migration directionality.
Thus, TSP1/hep I enhances the percent of cells migrating and speed of cell
migration, without appreciably changing migration directionality, resulting in
a net increase in cellular displacement.
|
The apparent increase in average cell migration speed could occur either through stimulation of migration in a subpopulation of nonmigratory cells or through more-subtle alterations in migration speed over the cell population. To determine how TSP1/hep I affects migration speed within the BAE cell population, histograms of cells migrating at certain speed intervals in response to basal or TSP1/hep I-stimulated conditions were produced. Cells migrating under serum-free conditions display a Gaussian distribution of average cell migration speeds, with most cells migrating in the 0.030.09 µm/minute range (Fig. 4A). Treatment with TSP1 and hep I induces an increase in the percent of cells migrating at high speeds, while decreasing the percent of cells migrating at low speeds (Fig. 4B,C). This effect was not seen with cells treated with the inactive modified hep I peptide (Fig. 4D). The population of endothelial cells, both treated and untreated, displayed a wide range of average cell speeds, suggesting varying degrees of locomotion without clear distinctions between migrating and non-migrating cells. Whereas activation of a subpopulation of cells would have resulted in a biphasic distribution of cell speeds, TSP1/hep I treatment increased migration speed across the entire population of endothelial cells.
|
CRT and LRP mediate TSP1/hep I-induced cell migration
TSP1 signals focal adhesion disassembly through interactions between the
hep I sequence of TSP1 and cell-surface CRT
(Goicoechea et al., 2000).
Following hep I binding, the interaction between cell-surface CRT and LRP is
altered, resulting in activation of a downstream signaling cascade culminating
in focal adhesion disassembly. TSP1/hep I does not stimulate focal adhesion
disassembly or activation of downstream signaling pathways in fibroblasts
derived from either CRT-knockout or LRP-knockout mice, making these cells
useful tools for studying the specificity of this response
(Goicoechea et al., 2002
;
Orr et al., 2002
)
(Orr et al., 2003
).
The ability of TSP1/hep I to induce chemokinesis in wild-type and CRT-knockout fibroblasts was determined through Dunn chamber analysis. Wild-type fibroblasts demonstrated increased cell displacement (R) following TSP1/hep I treatment, illustrated by scatter plots of the final cell position (Fig. 5A,C,E). However, fibroblasts derived from CRT-knockout mice did not show increased migration in response to TSP1/hep I treatment (Fig. 5B,D,F). Both cell lines migrated similarly in response to 0.1% FBS (Fig. 5G,H), indicating that CRT-knockout fibroblasts are not migration deficient, but specifically fail to respond to TSP1/hep I. A closer look at the TSP1/hep I-induced changes in cell migration revealed a disparity in TSP1/hep I-induced cell migration between endothelial cells and fibroblasts. TSP1 and hep I stimulate migration to a similar extent in endothelial cells (Table 1). However, fibroblasts show different responses depending on whether they are stimulated with TSP1 or the hep I peptide. Treatment with hep I stimulates increased migration speed, total displacement and the percentage of migrating cells, similar to that seen in endothelial cells treated with TSP1/hep I (Fig. 6A-C). However, in fibroblasts, TSP1 did not stimulate as significant an increase in migration speed or the percent of migrating cells, but had a profound effect on migration directionality (Fig. 6A,B,D). However, the total cell displacement induced by TSP1 and hep I in the fibroblasts was similar with either treatment (Fig. 6C). The ability of TSP1 to act differently to hep I alone indicates that TSP1 affects fibroblast migration through other domains and receptors as well. However, the CRT-knockout fibroblasts showed impaired TSP1-induced migration, suggesting that, whereas hep I is not the only pro-migratory signal elicited by TSP1, signaling through the hep I sequence is required for maximal TSP1-induced fibroblast migration.
|
|
MEFs deficient for LRP, the second component of the TSP1/hep I receptor
complex, demonstrated a similar inability to respond to TSP1/hep I signaling
(Fig. 7A-D). However, unlike
CRT-knockout MEFs, LRP-knockout MEFs do not show significant migration in
response to 0.1% FBS treatment, suggesting a general migratory defect in these
cells (Movie 3, available at
jcs.biologists.org/supplemental).
Consistent with this, LRP-knockout cells did not migrate in response to
treatment with bFGF or tenascin C (data not shown). To demonstrate that LRP is
actually involved in TSP1/hep I-induced migration, endothelial cells were
pre-incubated with receptor-associated protein (RAP) (50 nM), which is an
endogenous LRP inhibitor, and stimulated with TSP1/hep I. This concentration
of RAP is sufficient to inhibit focal adhesion disassembly and intracellular
signaling in response to TSP1/hep I treatment
(Orr et al., 2003). Resulting
migratory responses are shown by scatter plots of final cell position, which
is an indication of total cellular displacement (R). Whereas RAP alone did not
affect endothelial cell migration (Fig.
8A,B), RAP pre-treatment significantly inhibited the ability of
endothelial cells to respond to TSP1/hep I-induced chemokinesis
(Fig. 8C-F). RAP has no
significant effect on FBS-induced endothelial cell migration
(Fig. 8G,H). Thus, TSP1/hep I
stimulates endothelial cell and fibroblast chemokinesis through the previously
described CRT- and LRP-dependent focal adhesion-labilizing pathway.
|
|
Chemokinetic hep I treatment alters endothelial cell migration toward
aFGF and bFGF
Cells encounter numerous pro-migratory and anti-migratory stimuli, each
with their own unique attributes, which the cell integrates into a specific
migratory response. Cell-ECM adhesions, particularly focal adhesions, can act
as sites of integration between growth factor-derived and matrix-derived
signals, but the effect of altering these structures on the integration of
these signals has not been fully developed
(Hauck et al., 2002;
Eliceiri, 2001
). Thus, we
sought to determine the effect of TSP1/hep I-induced chemokinesis, which
involves modulation of focal adhesion structure, on chemotaxis towards
separate growth factors, such as aFGF and bFGF. Cells were loaded onto the
Dunn chamber using either serum-free media or media containing hep I peptide
(100 nM) or TSP1 (7.8 nM), with the outer well also containing either aFGF (67
pM) or bFGF (61 pM), forming a chemotactic gradient. This allowed us to
observe aFGF- and bFGF-induced chemotaxis in the presence or absence of focal
adhesion-labilizing concentrations of hep I peptide and TSP1. Treatment with
an aFGF gradient induced only a slight chemotactic response in BAE cells
(Fig. 9A,D; Movies 1 and 4,
available at
jcs.biologists.org/supplemental),
whereas bFGF stimulated a potent chemotactic response
(Fig. 9A,G; Movies 1 and 5,
available at
jcs.biologists.org/supplemental).
When added in the presence of hep I or TSP1, aFGF displayed enhanced
chemotactic properties (Fig.
9B,E and Fig. 9C,F;
Movies 2 and 6, available at
jcs.biologists.org/supplemental),
whereas bFGF showed a reduced ability to orient BAE cell migration
(Fig. 9B,H and
Fig. 9C,I; Movies 2 and 7,
available at
jcs.biologists.org/supplemental).
Hep I neither enhanced nor inhibited the ability of 0.1% FBS to stimulate
endothelial cell chemotaxis, probably reflecting differing effects on the
multiple growth factors that are present in serum (data not shown). These data
suggest that the ability of cells to respond to specific growth factors might
depend on the adhesive state of the cell.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
TSP1 differentially affects migration of certain cell types in response to
the changing context of the ECM. Several studies have been performed
describing both a pro-migratory and an anti-migratory effect of TSP1 on
endothelial cell migration. TSP1 was the first endogenous anti-angiogenic
molecule discovered, and the ability of TSP1 to inhibit angiogenesis results
from the pro-apoptotic and anti-migratory effect of the CSVTCG sequence in the
type 1 repeats of TSP1 signaling through CD36 on endothelial cells
(Good et al., 1990;
Dawson et al., 1997
). However,
endothelial cells show differential expression of CD36 based on their
location, with no CD36 expressed on large vessel endothelial cells
(Febbraio et al., 2001
). Since
angiogenesis occurs mostly through capillary endothelial cells, TSP1 is likely
to be primarily anti-angiogenic. However, TSP1 may stimulate large vessel
endothelial cell migration, enhancing their ability to respond to vascular
wounding. Furthermore, platelet-derived histidine-rich glycoprotein binds TSP1
and inhibits TSP1-induced CD36 activation
(Simantov et al., 2001
). Under
these conditions, cellular responses to other domains of TSP1, such as the
HBD, might be selectively enhanced. Consistent with this, the HBD of TSP1
stimulates angiogenesis and upregulates the proangiogenic proteins matrix
metalloproteinase (MMP)-9 and tissue inhibitor of metalloproteinase (TIMP)-1
(Ferrari do Outeiro-Bernstein et al.,
2002
; Qian et al.,
1997
; Taraboletti et al.,
2000
). HBD-induced angiogenesis might involve hep I signaling,
since both TSP1-induced focal adhesion disassembly and HBD-induced
angiogenesis are inhibited by heparin
(Murphy-Ullrich et al., 1993
;
Ferrari do Outeiro-Bernstein et al.,
2002
). Signaling through the HBD might be enhanced by the release
of this domain from full-length TSP1 through the actions of several endogenous
proteases, thus separating the pro- and anti-angiogenic signals of TSP1. In
addition to hep I signaling, TSP1 might also enhance endothelial cell
migration through interactions with the
3ß1
integrin and syndecans (Lakshmi et al.,
2000
; Ferrari do
Outeiro-Bernstein et al., 2002
). The ability of
3ß1 integrin, heparan sulfate proteoglycans
(HSPGs) and CRT/LRP to mediate TSP1-induced cell migration might reflect
synergism in TSP1-derived pro-migratory signals, multiple sites of regulation
in the migration process, or environment-specific pro-migratory stimuli.
TSP1/hep I stimulates focal adhesion disassembly and cell migration through
the CRT-LRP receptor co-complex. Whereas basal cell migration is unaffected by
CRT inhibition, LRP-knockout cells demonstrate a severe migration deficiency.
In addition to being non-responsive to TSP1/hep I-induced cell migration,
LRP-knockout cells do not migrate in response to treatment with FBS, tenascin
C or bFGF. Since these cells lose focal adhesions but do not migrate in
response to tenascin C treatment, the migration deficiency in LRP-knockout
MEFs is not due to a defect in focal adhesion turnover. This suggests that
additional signals might be necessary for migration. LRP-knockout cells
exhibit reduced cell spreading compared with wild-type cells, and time-lapse
video microscopy demonstrates that LRP-knockout MEFs do not extend large,
stable lamellipodia (Movie 3, available at
jcs.biologists.org/supplemental;
A. W. Orr, D. Kucik and J. E. Murphy-Ullrich, unpublished). In apparent
contradiction to this, recent work (Ma et
al., 2002) indicates that LRP-knockout fibroblasts display
increased activation of Rac, a small GTPase known to play a pivotal role in
spreading and lamellipodia formation. However, in these studies, experiments
were performed under high serum conditions: in the absence of LRP, the
stability of Rac-activating growth factors might be enhanced because of a lack
of LRP-mediated endocytosis and clearance of activating factors.
A role for LRP in determining basal cell migration rate has not previously
been described although, under high serum conditions, LRP might modulate
growth factor- and protease-dependent cell migration
(Ma et al., 2002).
LRP-dependent endocytosis inhibits migration in response to urokinase-type
plasminogen activator (uPA) binding to its receptor (uPAR)
(Weaver et al., 1997
).
However, LRP mediates migration in response to complexes of plasminogen
activator inhibitor-1 (PAI-1) with uPAR-uPA, as well as platelet-derived
growth factor (PDGF)-induced cell migration through LRP transphosphorylation
(Chazaud et al., 2000
;
Chazaud et al., 2002
;
Loukinova et al., 2002
). LRP
localizes to the leading edge of migrating breast cancer cells, and LRP
expression correlates with breast cancer cell invasiveness
(Chazaud et al., 2002
;
Li et al., 1998-1999
),
suggesting that localized LRP signaling within the lamellipodia might regulate
normal protrusive activity and cell migration. However, preincubation of
endothelial cells with RAP, an endogenous LRP inhibitor, does not reduce basal
cell migration, indicating that ligand binding may not be the primary function
of LRP in determining basal cell migration rate.
aFGF and bFGF have long been associated with endothelial cell migration,
vascular development and angiogenesis
(Joseph-Silverstein and Rifkin,
1987; Slavin,
1995
). These heparin-binding growth factors, although similar in
structure and function, display distinct differences. aFGF alone does not
significantly affect BAE cell migration and requires co-stimulation with
heparin to mediate maximal endothelial cell responses
(Joseph-Silverstein and Rifkin,
1987
; Slavin,
1995
), whereas, bFGF does not require complementary signals to
stimulate endothelial cell migration and proliferation maximally. Our studies
show that hep I treatment enhances aFGF-induced chemotaxis but inhibits
chemotaxis to bFGF. It has previously been reported that TSP1 inhibits
bFGF-induced endothelial cell migration and angiogenesis
(Vogel et al., 1993
), although
this occurs through heparin-binding sequences in the type 1 repeats of TSP1
and not the amino-terminal HBD. Since both TSP1 and bFGF bind HSPGs, the
ability of TSP1 to inhibit bFGF-induced responses is suggested to occur
through competitive inhibition for binding to HSPGs
(Vogel et al., 1993
). However,
whereas both TSP1 and bFGF bind the HSPG perlecan on endothelial cells, the
sites mediating these interactions are distinct
(Feitsma et al., 2000
). In
addition, hep I-induced focal adhesion disassembly is insensitive to
heparitinase treatment, indicating that hep I does not bind endothelial cell
HSPGs and thus does not inhibit bFGF signaling though competition for
endogenous HSPGs (Murphy-Ullrich et al.,
1993
).
Cells integrate environmental signals, such as growth factors and ECM
proteins, to determine context-specific responses
(Hauck et al., 2002;
Eliceiri, 2001
). Althought
integrin-mediated adhesion regulates the ability of growth factors to induce
certain cellular responses, little is known about the consequence of altering
adhesion complexes on growth factor signaling. Focal adhesion disassembly can
potentially alter adhesion-derived signaling and the resulting growth factor
responses. Stimulation with aFGF results in endothelial cell focal adhesion
disassembly, whereas bFGF does not alter focal adhesion dynamics, suggesting
that bFGF targets other migratory processes
(Ding et al., 2000
;
Lee and Gotlieb, 2002
) (A. W.
Orr, M. A. Pallero and J. E. Murphy-Ullrich, unpublished). bFGF stimulates
endothelial cell chemotaxis through FGF receptor-1 (FGFR-1), which localizes
to focal adhesions, and activation of ERK within focal adhesions is required
for bFGF-induced endothelial cell migration
(Shono et al., 2001
;
Tanghetti et al., 2002
).
Alterations in focal adhesion structure might alter receptor localization or
activation of focal adhesion-derived signaling pathways required for
bFGF-induced chemotaxis. Although both aFGF and bFGF stimulate chemotaxis
through FGFR-1, no requirement for receptor localization to focal adhesions
has been described for aFGF function
(Slavin, 1995
). Furthermore,
since aFGF induces a similar loss of focal adhesion structure, TSP1-induced
intermediate adhesive signals probably affect aFGF and bFGF signaling
differently.
The work presented herein demonstrates that TSP1 induces a phenotypic switch in endothelial cell migration, both enhancing migration on its own and specifically modulating endothelial cell responses to the FGF family of growth factors. In addition to endothelial cells, TSP1 stimulates focal adhesion disassembly in every adherent cell type tested thus far, including both fibroblasts and smooth muscle cells, suggesting TSP1-mediated focal adhesion disassembly might have a more universal effect on localized areas of active cell migration. Furthermore, this work suggests a role for LRP in the regulation of basal cell motility. Future work will further characterize the role of this migratory response in various pathological states and explore the potential changes in proliferation, apoptosis and gene expression induced in response to this TSP1-derived signal.
![]() |
Acknowledgments |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Adams, J. C. (1995). Formation of stable
microspikes containing actin and the 55kDa actin-bundling protein, fascin, is
a consequence of cell adhesion to thrombospondin-1: implications for the
anti-adhesive activities of thrombospondin-1. J. Cell
Sci. 108,1977
-1990.
Adams, J. C. (2001). Thrombospondins: multifunctional regulators of cell interactions. Annu. Rev. Cell Dev. Biol. 17,25 -51.[CrossRef][Medline]
Adams, J. C. (2002). Regulation of protrusive
and contractile cell-matrix contacts. J. Cell Sci.
115,257
-265.
Anand-Apte, B. and Zetter, B. (1997). Signaling
mechanisms in growth factor-stimulated cell motility. Stem
Cells 15,259
-267.
Beningo, K. A., Dembo, M., Kaverina, I., Small, J. V. and Wang,
Y. (2001). Nascent focal adhesions are responsible for the
generation of strong propulsive forces in migrating fibroblasts. J.
Cell Biol. 153,881
-887.
Bornstein, P. (2001). Thrombospondins as
matricellular modulators of cell function. J. Clin.
Invest. 107,929
-934.
Chazaud, B., Bonavaud, S., Plonquet, A., Pouchelet, M., Gherardi, R. K. and Barlovatz-Meimon, G. (2000). Involvement of the [uPAR:uPA:PAI-1:LRP] complex in human myogenic cell motility. Exp. Cell Res. 258,237 -244.[CrossRef][Medline]
Chazaud, B., Ricoux, R., Christov, C., Plonquet, A., Gherardi,
R. K. and Barlovatz-Meimon, G. (2002). Promigratory effect of
plasminogen activator inhibitor-1 on invasive breast cancer cell populations.
Am. J. Pathol. 160,237
-246.
Couchman, J. R. and Rees, D. A. (1979). The behavior of fibroblasts migrating from chick heart explants: changes in adhesion, locomotion and growth, and in the distribution of actomyosin and fibronectin. J. Cell Sci. 39,149 -165.[Abstract]
Dawson, D. W., Pearce, S. F., Zhong, R., Silverstein, R. L.,
Frazier, W. A. and Bouck, N. P. (1997). CD36 mediates the in
vitro inhibitory effects of thrombospondin-1 on endothelial cells.
J. Cell Biol. 138,707
-717.
Ding, Q., Gladson, C. L., Guidry, C. R., Santoro, S. A., Dickeson, S. K., Shin, J. T. and Thompson, J. A. (2000). Extracellular FGF-1 inhibits cytoskeletal organization and promotes fibroblast motility. Growth Factors 18, 93-107.[Medline]
Eliceiri, B. P. (2001). Integrin and growth
factor receptor crosstalk. Circ. Res.
89,1104
-1110.
Febbraio, M., Hajjar, D. P. and Silverstein, R. L.
(2001). CD36: a class B scavenger receptor involved in
angiogenesis, atherosclerosis, inflammation, and lipid metabolism.
J. Clin. Invest. 108,785
-791.
Feitsma, K., Hausser, H., Robenek, H., Kresse, H. and Vischer,
P. (2000). Interaction of thrombospondin-1 and heparan
sulfate from endothelial cells. Structural requirements of heparin sulfate.
J. Biol. Chem. 275,9396
-9402.
Fernandez, J. L. R., Geiger, B., Salomon, D. and Ben-Ze'ev, A. (1992). Overexpression of vinculin suppresses cell motility in BALB/c 3T3 cells. Cell Motil. Cytoskeleton 22,127 -134.[Medline]
Ferrari do Outeiro-Bernstein, M. A., Nunes, S. S., Andrade, A. C., Alves, T. R., Legrand, C. and Morandi, V. (2002). A recombinant NH(2)-terminal heparin-binding domain of the adhesive glycoprotein, thrombospondin-1, promotes endothelial tube formation and cell survival: a possible role for syndecan-4 proteoglycan. Matrix Biol. 21,311 -324.[CrossRef][Medline]
Gluck, U. and Ben-Ze'ev, A. (1994). Modulation
of alpha-actinin levels affects cell motility and confers tumorigenicity on
3T3 cells. J. Cell Sci.
107,1773
-1782.
Goicoechea, S., Orr, A. W., Pallero, M. A., Eggleton, P. and
Murphy-Ullrich, J. E. (2000). Thrombospondin mediates focal
adhesion disassembly through interactions with cell surface calreticulin.
J. Biol. Chem. 275,36358
-36368.
Goicoechea, S., Pallero, M. A., Eggleton, P., Michalak, M. and
Murphy-Ullrich, J. E. (2002). The anti-adhesive activity of
thrombospondin is mediated by the N-terminal domain of cell surface
calreticulin. J. Biol. Chem.
277,37219
-37228
Good, D. J., Polverini, P. J., Rastinejad, F., le Beau, M. M., Lemons, R. S., Frazier, W. A. and Bouck, N. P. (1990). A tumor suppressor-dependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin. Proc. Natl. Acad. Sci. USA 87,6624 -6628.[Abstract]
Greenwood, J. A. and Murphy-Ullrich, J. E. (1998). Signaling of De-adhesion in cellular regulation and motility. Microsc. Res. Tech. 43,420 -432.[CrossRef][Medline]
Greenwood, J. A., Pallero, M. A., Theibert, A. B. and Murphy-Ullrich, J. E. (1998). Thrombospondin signaling of focal adhesion disassembly requires activation of phosphoinositide 3-kinase. J. Biol. Chem. 272,1755 -1763.[CrossRef]
Hauck, C. R., Hsai, D. A. and Schlaepfer, D. D. (2002). The focal adhesion kinasea regulator of cell migration and invasion. IUBMB Life 53,115 -119.[Medline]
Ingber, D. E. (1997). Integrins, tensegrity, and mechanotransduction. Gravit. Space Biol. Bull. 10, 29-55.
Joseph-Silverstein, J. and Rifkin, D. B. (1987). Endothelial cell growth factors and the vessel wall. Semin. Thromb. Hemost. 13,504 -513.[Medline]
Kaverina, I., Krylyshkina, O. and Small, J. V. (2002). Regulation of substrate adhesion dynamics during cell motility. Int. J. Biochem. Cell Biol. 34,746 -761.[CrossRef][Medline]
Lakshmi, C., He, C.-Z., Al-Barazi, H., Krutzsch, H. C.,
Iruela-Arispe, M. L. and Roberts, D. D. (2000). Cell
contact-dependent activation of 3ß1 integrin
modulates endothelial responses to thrombospondin-1. Mol. Biol.
Cell 11,2885
-2900.
Lauffenburger, D. A. and Horwitz, A. R. (1996). Cell migration: a physically integrated molecular process. Cell 84,359 -369.[Medline]
Lee, T. Y. and Gotlieb, A. I. (2002). Rho and basic fibroblast growth factor involvement in centrosome redistribution and actin microfilament remodeling during early endothelial wound repair. J. Vasc. Surg. 35,1242 -1252.[CrossRef][Medline]
Li, Y., Wood, N., Grimsley, P., Yellowlees, D. and Donnelly, P. K. (1998-1999). In vitro invasiveness of human breast cancer cells is promoted by low density lipoprotein receptor-related protein. Invasion Metastasis 18,240 -251.[CrossRef][Medline]
Loukinova, E., Ranganathan, S., Kuznetsov, S., Gorlatova, N.,
Migliorini, M. M., Loukinov, D., Ulery, P. G., Mikhailenko, I., Lawrence, D.
A. and Strickland, D. K. (2002). Platelet-derived growth
factor (PDGF)-induced tyrosine phosphorylation of the low density lipoprotein
receptor-related protein (LRP). Evidence for integrated co-receptor function
between LRP and PDGF. J. Biol. Chem.
277,15499
-15506.
Ma, Z., Thomas, K. S., Webb, D. J., Moravec, R., Salicioni, A.
M., Mars, W. M. and Gonias, S. L. (2002). Regulation of Rac1
activation by the low density lipoprotein receptor-related protein.
J. Cell Biol. 159,1061
-1070.
Mansfield, P. J., Boxer, L. A. and Suchard, S. J. (1990). Thrombospondin stimulates motility of human neutrophils. J. Cell Biol. 111,3077 -3086.[Abstract]
Mansfield, P. J. and Suchard, S. J. (1994). Thrombospondin promotes chemotaxis and haptotaxis of human peripheral blood monocytes. J. Immunol. 153,2419 -2429.
Murphy-Ullrich, J. E. (2001). The de-adhesive
activity of matricellular proteins: is intermediate cell adhesion an adaptive
state. J. Clin. Invest.
107,785
-790.
Murphy-Ullrich, J. E. and Höök, M. (1989). Thrombospondin modulates focal adhesions in endothelial cells. J. Cell Biol. 109,1309 -1319.[Abstract]
Murphy-Ullrich, J. E., Gurusiddappa, S., Frazier, W. A. and
Höök, M. (1993). Heparin-binding peptides from
thrombospondin-1 and 2 contain focal adhesion-labilizing activity.
J. Biol. Chem. 268,26784
-26789.
Noble, P. B. and Levine, M. D. (2000). Computer Assisted Analysis of Cell Locomotion and Chemotaxis (ed. X. Editor and Y. Editor), pp.95 -97. Elkins Park, PA: Franklin Book.
Orr, A. W., Pallero, M. A. and Murphy-Ullrich, J. E.
(2002). Thrombospondin stimulates focal adhesion disassembly
through Gi- and phosphoinositide 3-kinase-dependent ERK activation.
J. Biol. Chem. 277,20453
-20460.
Orr, A. W., Pedraza, C. E., Pallero, M. A., Elzie, C. A., Goicoechea, S., Strickland, D. K. and Murphy-Ullrich, J. E. (2003). Low density lipoprotein receptor-related protein is a calreticulin co-receptor that signals focal adhesion disassembly. (in press).
Qian, X., Wang, T. N., Rothman, V. L., Nicosia, R. F. and Tuszynski, G. P. (1997). Thrombospondin-1 modulates angiogenesis in vitro by up-regulation of matrix metalloproteinase-9 in endothelial cells. Exp. Cell Res. 235,403 -412.[CrossRef][Medline]
Shono, T., Kanetake, H. and Kanda, S. (2001). The role of mitogen-activated protein kinase activation within focal adhesions in chemotaxis toward FGF-2 by murine brain capillary endothelial cells. Exp. Cell Res. 264,275 -283.[CrossRef][Medline]
Simantov, R., Febbraio, M., Crombie, R., Asch, A. S., Nachman,
R. L. and Silverstein, R. L. (2001). Histidine-rich
glycoprotein inhibits the antiangiogenic effect of thrombospondin-1.
J. Clin. Invest. 107,45
-52.
Slavin, J. (1995). Fibroblast growth factors: at the heart of angiogenesis. Cell Biol. Int. 19,431 -444.[CrossRef][Medline]
Tanghetti, E., Ria, R., Dell'Era, P., Urbinati, C., Rusnati, M., Ennas, M. G. and Presta, M. (2002). Biological activity of substrate-bound basic fibroblast growth factor (FGF2): recruitment of FGF receptor-1 in endothelial cell adhesion contacts. Oncogene. 21,3889 -3897.[CrossRef][Medline]
Taraboletti, G., Roberts, D. D. and Liota, L. A. (1987). Thrombospondin-induced tumor cell migration: haptotaxis and chemotaxis are mediated by different molecular domains. J. Cell Biol. 105,2409 -2415.[Abstract]
Taraboletti, G., Roberts, D., Liotta, L. A. and Giavazzi, R. (1990). Platelet thrombospondin modulates endothelial cell adhesion, motility, and growth: a potential angiogenesis regulatory factor. J. Cell Biol. 111,765 -772.[Abstract]
Taraboletti, G., Morbidelli, L., Donnini, S., Parenti, A.,
Granger, H. J., Giavazzi, R. and Ziche, M. (2000). The
heparin binding 25 kDa fragment of thrombospondin-1 promotes angiogenesis and
modulates gelatinase and TIMP-2 production in endothelial cells.
FASEB J. 14,1674
-1676.
Vogel, T., Guo, N. H., Krutzsch, H. C., Blake, D. A., Hartman, J., Mendelovitz, S., Panet, A. and Roberts, D. D. (1993). Modulation of endothelial cell proliferation, adhesion, and motility by recombinant heparin-binding domain and synthetic peptides from the type I repeats of thrombospondin. J. Cell Biochem. 53, 74-84.[Medline]
Webb, D. J., Parsons, J. T. and Horwitz, A. F. (2002). Adhesion assembly, disassembly, and turnover in migrating cellsover and over and over again. Nat. Cell Biol. 4,E97 -E100.[CrossRef][Medline]
Weaver, A. M., Hussaini, I. M., Mazar, A., Henkin, J. and
Gonias, S. L. (1997). Embryonic fibroblasts that are
genetically deficient in low density lipoprotein receptor-related protein
demonstrate increased activity of the urokinase receptor system and
accelerated migration on vitronectin. J. Biol. Chem.
272,14372
-14379.
Zamir, E. and Geiger, B. (2001). Molecular complexity and dynamics of cell-matrix adhesions. J. Cell Sci. 114,3583 -3590.[Medline]
Zicha, D., Dunn, G. and Brown, A. F. (1991). A new direct-viewing chemotaxis chamber. J. Cell Sci. 99,769 -775.[Abstract]