From the Laboratory of Pathology, NCI, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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Thrombospondin-1 (TSP1) is a matricellular
protein that displays both pro- and anti-adhesive activities. Binding
to sulfated glycoconjugates mediates most high affinity binding of
soluble TSP1 to MDA-MB-435 cells, but attachment and spreading of these cells on immobilized TSP1 is primarily Thrombospondin-1 (TSP1)1
is an extracellular matrix glycoprotein that has diverse effects on
cell behavior (reviewed in Refs. 1 and 2). The five known
thrombospondin genes display distinct patterns of expression during
development and in several disease states. Disruption of the
thbs1 gene in mice results in lordosis of the spine and
abnormal proliferation and inflammatory responses in the lung (3).
Suppression of THBS1 expression by loss of wild type p53, by
activated Ras, Myc, nickel, and in metastatic clones of several tumor
cell lines suggested that loss of TSP1 expression may contribute to
tumor progression (reviewed in Ref. 4). Consistent with this
hypothesis, overexpression of THBS1 in breast carcinoma
cells (5), a transformed endothelial cell line (6), fibroblasts from Li
Fraumini patients (7), and glioblastoma cells (8) decreases tumor
growth in animal models. This suppressive activity is due at least in
part to the anti-angiogenic activity of TSP1 (reviewed in Refs. 4, 9,
and 10). TSP1 antagonizes growth factor-stimulated proliferation and
migration of endothelial cells. Its anti-angiogenic activity is thought to be the major mechanism for suppression of tumor growth in
THBS1-transfected MDA-MB-435 breast carcinoma cells, because
thrombospondin overexpression strongly inhibited tumor growth in
vivo but did not significantly alter in vitro
proliferation, motility, or the ability of the tumor cells to form
colonies in soft agar (5). However, higher doses of exogenous TSP1 and
some TSP1 peptides can directly inhibit proliferation of these cells
in vitro (11).
Defining the receptors that recognize TSP1 on endothelial and tumor
cells may provide insights into the differential effects of this
protein on each cell type. Receptors that mediate cell interactions
with TSP1 include integrins, proteoglycans, CD36, CD47, the low density
lipoprotein receptor-related protein, and sulfated glycolipids. Binding
of TSP1 to each of these receptors may elicit different cellular
responses. Thus both the relative levels of expression of each receptor
and, potentially, the activation state of each receptor may determine
the nature of the adhesive, motility, and proliferative responses of
cells to TSP1.
We have examined the role of integrins in the pro-adhesive activity of
TSP1 for human breast carcinoma cells. Although the integrin
Proteins and Peptides--
Calcium-replete TSP1 was purified
from human platelets as described (13). Proteolytic fragments of TSP1
were prepared as described previously (14). Synthetic peptides
containing TSP1 sequences were prepared as described previously
(15-17). Bovine type I collagen was obtained from Collaborative
Research, and vitronectin was from Sigma. Fibronectin was purified from
human plasma (National Institutes of Health Blood Bank) as described (18). Murine laminin-1 purified from the Engelbreth-Holm-Swarm sarcoma
was provided by Dr. Sadie Aznavoorian. Recombinant human EGF and
TGF- Monoclonal Antibodies--
Hybridomas producing the
Cell Lines and Reagents--
MDA-MB-435, MDA-MB-231, and MCF-7
breast carcinoma cells (American Type Culture Collection) were grown in
RPMI 1640 medium containing 10% FCS. Okadaic acid, PMA, pertussis
toxin (PT), herbimycin A, heparin, and sodium vanadate were purchased
from Sigma. Pertussis toxin B oligomer, staurosporine, wortmannin,
KT5823, guanosine-3',5'-cyclic monophosphothioate,
8-(4-chlorophenylthio)-, Rp isomer, and bisindolylmaleimide were from Calbiochem. KT5720 was from Kamiya Biomedical (Thousand Oaks, CA).
Adhesion Assays--
Cells were detached by replacing the growth
medium with PBS containing 2.5 mM EDTA and incubating 5-10
min at 37 °C. The cells were collected by centrifugation, suspended
in RPMI containing 0.1% BSA, and assayed for adhesion to
bacteriological polystyrene substrates coated with proteins as
described previously (14). Adhesion assays were terminated after 50 min
by washing to remove nonadherent cells and fixation with 1%
glutaraldehyde in PBS.
Chemotaxis--
Chemotaxis was measured in 48-well chambers
using Nucleopore 8 µm, polyvinylpyrrolidone-free filters (Neuroprobe
Inc, Gaithersburg, MD). To provide an integrin-independent substrate
for motility, the filters were coated with 10 µg/ml polylysine for
16 h at 4 °C prior to use. Motility was measured after 6.5 h and scored microscopically by counting nuclei of migrated cells on
the lower surface of the membrane.
Fluorescence Microscopy--
To examine integrin localization
and cytoskeletal rearrangement, 8-well glass chamber slides (Nalge Nunc
International, Naperville, IL) were coated with type I collagen, TSP1,
or fibronectin overnight at 4 °C. The chambers were then blocked
with 1% BSA in PBS, and cells were added in RPMI containing 0.1% BSA.
In some cases, antibodies were included in the medium. Cells were
allowed to attach and spread for 90 min. The unbound cells were then
removed along with the medium, and the chambers were rinsed with PBS
and fixed with 3.7% formaldehyde. Cells were stained with BODIPY TR-X
phallacidin (Molecular Probes, Inc. Eugene, OR) to visualize F-actin or
using primary antibodies followed by BODIPY FL anti-mouse IgG to
localize integrins or CD98. All staining procedures were carried out
according to the manufacturer's directions. Stained cells were
observed and photographed under a Zeiss fluorescent microscope using
appropriate filters.
Unstimulated MDA-MB-435 cells were evaluated for expression of
integrins or their subunits 1 day after plating in RPMI medium containing 10% FCS (Biofluids) by indirect immunofluorescence and flow
cytometry. Cells were washed with PBS, 0.2% BSA and incubated at
37 °C for 6 min with Puck's saline A with 0.2% EDTA and 10% FCS.
All subsequent procedures were performed on ice, and all washes were
with PBS containing 0.2% BSA. Cells were dislodged with a scraper, and
the resultant cell suspension was washed. Cell pellets were exposed to
mouse IgG or primary antibodies to integrins or integrin subunits in
PBS, 0.2% BSA, washed, and incubated with fluorescein
isothiocyanate-conjugated goat anti-mouse IgG (Tago, Inc., Burlingame,
CA). Following a wash, the cells were fixed in 1% paraformaldehyde and
analyzed in a FACScan flow cytometer (Becton Dickinson, San Jose, CA).
Initial gating was done using forward and side scatter to identify a
population of intact cells without debris.
Ligand Binding--
TSP1 was iodinated using Iodogen (Pierce) as
described previously (20). For some experiments, cells were grown in
sulfate-deficient medium containing chlorate to inhibit proteoglycan
and glycolipid sulfation as described previously (21).
Integrin Expression on Breast Carcinoma Cells--
Flow cytometric
analysis (Table I) and
immunoprecipitation using subunit-specific integrin antibodies (data
not shown) demonstrated that MDA-MB-435 cells express several
Binding of Soluble TSP1--
Previous studies using MDA-MB-231
breast carcinoma cells (25) concluded that sulfated glycoconjugates
including heparan sulfate and chondroitin sulfate proteoglycans play a
dominant role in both binding of soluble TSP1 and adhesion on
immobilized TSP1. We observed a similar dependence for
125I-TSP1 binding to MDA-MB-435 cells (Fig.
1). Binding was strongly inhibited by
heparin or a recombinant 18-kDa amino-terminal heparin-binding fragment
of TSP1, but the peptide GRGDS and
Although MDA-MB-435 cells express some
The
Several human breast cancer cell lines showed similar involvement of
Integrin Localization and Effects on Actin
Cytoskeleton--
Activation of Conformation Requirements for
Regulation of
Several pharmacological agents stimulated
Modulation of TSP1 Adhesion by G-protein Signaling--
Although
TSP1 peptides promote PT-sensitive integrin activation through binding
to CD47 (31, 33), we showed above that this pathway does not function
in MDA-MB-435 cells to activate Physiological Activators of TSP1 Adhesion and Chemotaxis--
We
noted that freshly passaged breast carcinoma cells exhibited stronger
Several growth factors were examined to define the basis of the serum
response for TSP1 adhesion (Fig. 9B). Addition of EGF to
serum-depleted medium increases adhesion of breast carcinoma cells to
some substrates (34) but in several experiments showed only a slight
stimulatory activity for spreading of MDA-MB-435 cells on TSP1 (Fig.
9B). FGF2 and TGF-
Acute addition of insulin, but not EGF, during the adhesion assay
produced a similar enhancement in adhesion of both cell lines to TSP1
as the 24-h pretreatment of the cells in culture (Fig. 9C
and results not shown). The dose dependence for the insulin response
was consistent with that for signaling through the IGF1 receptor (Fig.
9C), which is expressed in these breast carcinoma cells
(35). Both insulin and IGF1 strongly stimulated MDA-MB-435 cell
spreading on TSP1, moderately stimulated adhesion on type I collagen,
but did not stimulate adhesion on laminin-1 (Fig. 9C). EGF
(2 nM) was inactive in this assay (results not shown). IGF1
(EC50 = 1 nM) was 100-fold more potent than
insulin, as expected for a response mediated by the IGF1 receptor (35).
A similar difference in the potencies of IGF1 and insulin was also
observed in stimulation of TSP1 attachment of MDA-MB-231 cells (results not shown). Thus, occupancy of the IGF1 receptor specifically stimulates activity of the TSP1-binding integrin in both cell lines.
IGF1 also enhanced the chemotactic response of breast carcinoma cells
to TSP1. Addition of IGF1 to MDA-MB-435 cells in the upper well of a
modified Boyden chamber did not alter motility of the cells, but it
stimulated (2- to 5-fold) the chemotactic response to TSP1 added to the
lower chamber (Fig. 9D). This IGF1-stimulated motility to
TSP1 was mediated by the Modulation of TSP1 Adhesion by CD98--
Expression of the
transmembrane protein CD98 is induced by serum, and this protein was
recently shown to activate function of some The Several Although several signaling pathways have been identified that regulate
integrin activity by "inside-out" signaling (39), the mechanisms
for regulating activation states of specific integrins remain poorly
understood. In contrast to We have identified the IGF1 receptor as a specific regulator of
CD98 was recently identified as an activator of Only a small fraction of the TSP1 has diverse effects on breast carcinoma cell behavior, altering
their adhesion, motility, proliferation, protease expression, and
invasion. These cellular responses result in alterations of their
in vivo tumorigenic, angiogenic, and metastatic potentials (reviewed in Ref. 4). We have defined specific roles for the 1
integrin-dependent. The integrin
3
1 is the major mediator of breast
carcinoma cell adhesion and chemotaxis to TSP1. This integrin is
partially active in MDA-MB-435 cells but is mostly inactive in
MDA-MB-231 and MCF-7 cells, which require
1 integrin
activation to induce spreading on TSP1. Integrin-mediated cell
spreading on TSP1 is accompanied by extension of filopodia containing
1 integrins. TSP1 binding activity of the
3
1 integrin is not stimulated by
CD47-binding peptides from TSP1 or by protein kinase C activation,
which activate
v
3 integrin function in
the same cells. In MDA-MB-231 but not MDA-MB-435 cells, this integrin
is activated by pertussis toxin, whereas serum, insulin, insulin-like
growth factor-1, and ligation of CD98 increase activity of this
integrin in both cell lines. Serum stimulation is accompanied by
increased surface expression of CD98, whereas insulin-like growth
factor-1 does not increase CD98 expression. Thus, the pro-adhesive
activity of TSP1 for breast carcinoma cells is controlled by several
signals that regulate activity of the
3
1 integrin.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
v
3 is important for adhesion of several
cell types to TSP1 (12), we found that adhesion of breast carcinoma
cells on TSP1 substrates is not mediated by this integrin. We report here that the
3
1 integrin rather than
3 integrins play a dominant role in adhesion of several
breast carcinoma cell lines on TSP1. The activation state of the
3
1 integrin varies among the human breast
carcinoma cell lines examined and can be modulated by inside-out signaling, suggesting that the ability to receive pro-adhesive and
motility signals from TSP1 is tightly regulated in these breast carcinoma cell lines.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 were obtained from R & D Systems. Insulin was from Biofluids,
and recombinant human insulin-like growth factor-1 (IGF1) was from Bachem.
1 integrin-activating antibody TS2/16 (19) and the CD98
antibody 4F2 were obtained from the American Type Culture Collection.
Antibodies secreted in PFHM-II medium (Life Technologies, Inc.) were
purified by protein G affinity chromatography (Pierce). Integrin
function blocking antibodies used include LM609
(
v
3, provided by Dr. David Cheresh),
05-246 (
1
1, Upstate Biotechnology), 6D7
(
2
1, Dr. Harvey Gralnick, NIH), P1B5
(
3
1, Life Technologies, Inc.), 407279 (
4
1, Calbiochem), P1D6
(
5
1, Life Technologies, Inc.), and mAb13
(
1, Dr. Kenneth Yamada, NIH). Non blocking antibodies
recognizing
3
1 (M-KID2),
4
1 (HP2/1), and
5
1 (SAM1) were obtained from AMAC, Inc.
(Westbrook, ME), and
v (LM142) was provided by Dr. David Cheresh.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 integrins and
v
3. Integrin expression on MDA-MB-231 and
MCF-7 cells has been reported previously (22-24). MDA-MB-231 cells
express
2,
3,
4,
5,
6,
v, and
1 subunits. The MDA-MB-231 and MCF-7 cell
lines express only low levels of
3 subunits (24).
Integrin expression in MDA-MB-435 breast carcinoma cells
1 or
3
integrin function blocking antibodies had no effect. Conversely,
binding of 125I-TSP1 to MDA-MB-435 cells was not enhanced
by incubation with the
1 integrin-activating antibody
TS2/16, either alone or in the presence of 10 µg/ml heparin to
inhibit TSP1 binding to sulfated ligands (data not shown). Therefore,
high affinity binding to soluble TSP1 to these cells is mediated by
sulfated glycoconjugates and is independent of integrin binding.
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Fig. 1.
Specificity of
125I-thrombospondin binding to MDA-MB-435 cells. Cells
were harvested using 2.5 mM EDTA in PBS, resuspended in
RPMI 1640 medium containing 0.1% BSA, and incubated with
125I-thrombospondin for 1 h at 25 °C with the
indicated inhibitors. Cells were centrifuged through oil to remove
unbound labeled protein. The mean ± S.D. for triplicate
determinations is presented for binding determined in the absence
(control) or presence of presence of 204 µM GRGDS
peptide, 4 µM 18-kDa recombinant TSP1 heparin-binding
domain (HBD), 100 µg/ml heparin, 10 µg/ml mAb13
(anti- 1) or P1B5
(anti-
3
1).
1 Integrin-mediated Adhesion and Chemotaxis to
TSP1--
Although heparin and recombinant heparin binding domain from
TSP1 partially inhibited attachment of MDA-MB-435 cells on immobilized TSP1, the fraction of spread cells was unaffected (Fig.
2A). In the presence of a
1 integrin function blocking antibody at 2 µg/ml,
however, only spreading was inhibited, and a combination of heparin and
the
1 blocking antibody abolished spreading and markedly
inhibited attachment. At 50 µg/ml, the
1 antibody
completely inhibited adhesion to TSP1 (Fig. 2A). Thus,
interaction with a
1 integrin is essential for
spreading, but sulfated ligands may also contribute to adhesion of
these cells on TSP1. This was confirmed by inhibition of sulfation
following growth in chlorate. Adhesion was inhibited by 60% for
MDA-MB-435 cells with a 90% reduction in 35SO4
incorporation (Fig. 2B). RGD peptides did not inhibit
adhesion of MDA-MB-435 cells on TSP1 (results not shown).
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Fig. 2.
Role of integrins and sulfated
glycoconjugates in breast carcinoma cell adhesion and chemotaxis to
TSP1. A, MDA-MB-435 cell attachment (solid
bars) and spreading (striped bars) were measured after
50 min on polystyrene coated with thrombospondin (50 µg/ml) and
blocked with 1% BSA to reduce nonspecific adhesion.
Heparin-dependent adhesion was assessed by inhibition using
4 µM 18-kDa recombinant TSP1 heparin-binding domain
(HBD) or 50 µg/ml heparin. 1
integrin-dependent adhesion was inhibited using 2 or 50 µg/ml mAb13 (anti-
1). Results are presented as
mean ± S.D., n = 3. B, effect of
inhibiting sulfation on attachment of MDA-MB-435 cells. MDA-MB-435
cells were grown in Ham's F-12 medium (low sulfate) containing 10%
dialyzed fetal calf serum for 48 h. The medium was replaced with
the same medium containing 1% dialyzed serum with or without sodium
chlorate at the indicated concentrations. The cells were cultured for
24 h, harvested, and resuspended in F-12 medium containing 1 mg/ml
BSA with or without chlorate at the indicated concentrations. Cell
adhesion was quantified to polystyrene coated with 50 µg/ml
thrombospondin (striped bars) or 10 µg/ml fibronectin
(gray bars). 35S incorporation in MDA-435 cell
macromolecules (
) was assessed in duplicate cultures supplemented
with 25 µCi/ml [35S]sulfate. The cells were fixed and
washed in acetic acid/methanol, and incorporation of radioactivity in
macromolecules was determined by scintillation counting after
solubilization in 1% sodium dodecyl sulfate. C, integrin
v
3 mediates breast carcinoma cell
adhesion to vitronectin but not to TSP1. Adhesion of MDA-MB-435 cells
to 30 µg/ml TSP1 (solid bars) or 10 µg/ml vitronectin
(striped bars) was measured in the presence of the
v
3 function blocking antibody LM609 or
the
1-activating antibody TS2/16. D,
chemotaxis to TSP1 is
1 integrin-dependent.
MDA-MB-435 chemotaxis to 50 µg/ml TSP1 was determined in modified
Boyden chambers. Cells were added in the upper chamber with the
indicated concentrations of
1 integrin blocking antibody
mAb13 (
) or heparin (
). Spontaneous motility (
) was determined
in the absence of TSP1. Migrated cells were counted microscopically,
and results from triplicate wells are presented as a percent of
migration to TSP1 without inhibitors, mean ± S.D.
v
3
integrin (Table I), a function blocking antibody or an
v
3-specific RGD mimetic blocked adhesion
of the cells on vitronectin but had no effect on adhesion on TSP1 (Fig.
2C and results not shown). Conversely, in the presence of
the
1-activating antibody TS2/16, adhesion of MDA-MB-435
cells was enhanced on TSP1 but not on vitronectin (Fig. 2C).
Therefore, the
v
3 integrin is functional
in MDA-MB-435 cells, but it is apparently unable to recognize the RGD
motif in TSP1.
1 blocking antibody mAb13 inhibited chemotaxis to
TSP1, but heparin did not (Fig. 2D). For these experiments,
the filters were coated with polylysine to provide an
integrin-independent substrate for adhesion of the cells. Therefore,
chemotaxis of MDA-MB-435 cells to TSP1 is also primarily dependent on
the
1 integrin receptor.
1 integrins in their adhesion to TSP1 (Fig.
3). MDA-MB-231 cells attached poorly and
did not spread on substrates coated with low concentrations of TSP1. In
the presence of the
1-activating antibody, however, the
cells attached avidly on TSP1 and exhibited spreading (Fig.
3A). A third breast carcinoma cell line, MCF-7, behaved
similarly to the MDA-MB-231 cells and showed spreading on TSP1 only in
the presence of the
1-activating antibody (Fig. 3A). The apparent low avidity state of the integrin that
recognizes TSP1 on MDA-MB-435 cells was not an artifact from using EDTA
to dissociate the cells, because cells suspended by scraping from the
dish in the presence of divalent cations showed the same degree of
enhancement by TS2/16 for adhesion to TSP1 or type I collagen as cells
harvested using EDTA (Fig. 3B).
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Fig. 3.
1 integrins
recognizing TSP1 and type I collagen are partially inactive in human
breast carcinoma cell lines. A, spreading of three
breast carcinoma cell lines on 50 µg/ml TSP1 (solid bars)
or on TSP1 in the presence of 5 µg/ml TS2/16 (striped
bars). B, comparison of
1 integrin
activity in MDA-MB-435 cells harvested by scraping in RPMI medium or by
a 5-min treatment with 2.5 mM EDTA in PBS. Cells were
resuspended in RPMI medium with 0.1% BSA (solid bars) or
with 20 µg/ml TS2/16 (striped bars), and cell spreading
was assessed after 50 min on substrates coated with 20 µg/ml TSP1 or
5 µg/ml type I collagen.
3
1 Is the Major TSP1-binding Integrin
on Breast Carcinoma Cells--
Of the
subunit antibodies tested
for inhibiting adhesion to TSP1, only an
3 subunit
blocking antibody, P1B5, significantly inhibited adhesion of MDA-MB-231
cells to TSP1 (Fig. 4A,
p = 0.0007, 2-tailed t test). An
4 integrin blocking antibody slightly inhibited adhesion, but mixing this antibody with the
3 blocking
antibody produced no further inhibition than the latter antibody alone (Fig. 4A). MDA-MB-435 cell spreading on TSP1 was also
inhibited by the
3 blocking antibody, and somewhat by
the
4 antibody (Fig. 4B). Function blocking
antibodies for
1
1,
2
1, and
5
1
integrins had no effect on TSP1 adhesion, although the
2
1 and
5
1
antibodies inhibited adhesion of the same cells to known ligands for
these integrins (Fig. 4 and results not shown).
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Fig. 4.
Integrin subunit
specificity of TSP1 adhesion. A, MDA-MB-231 cell
attachment was quantified using substrates coated with 40 µg/ml TSP1
(solid bars) or 5 µg/ml type I collagen (gray
bars) in the presence of 5 µg/ml TS2/16 to activate
1 integrins and 5 µg/ml of the indicated function
blocking antibodies. B, inhibition of MDA-MB-435 cell
spreading on TSP1 (solid bars) or type I collagen
(gray bars) in the presence of TS2/16 and the indicated
subunit blocking antibodies.
1 integrins using
TS2/16 altered the morphology of cells attaching on TSP1 (Fig.
5). MDA-MB-435 cells extended a few
processes but exhibited no F-actin organization when attached on TSP1
alone (Fig. 5a), but addition of antibody TS2/16 stimulated spreading with redistribution of F-actin to the cell periphery (Fig.
5b). F-actin was also present in short spikes protruding from the spread cells but did not organize into stress fibers. Staining
with the
1 integrin antibody revealed numerous filopodia extending from these points (Fig. 5c). In some cells, these
filopodia were terminated with punctate
1 integrin
staining, possibly at sites of contact with the TSP1 substrate.
Formation of filopodia was specific to the TSP1 substrate, as
TS2/16-induced spreading of these cells on type I collagen (Fig.
5d) or fibronectin (results not shown) only rarely evoked
filopodia. These cytoskeletal rearrangements were specific for
1-dependent adhesion to intact TSP1 and were not observed in cells attaching on heparin-binding peptides or recombinant fragments of TSP1 (results not shown). Similar induction of
filopodia or microspikes by TSP1 have been observed in other cell types
(26).
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Fig. 5.
Actin organization and filopodia formation on
TSP1 is stimulated by 1 integrin
activation. Actin was visualized using BODIPY-phallacidin in
MDA-MB-435 cells attached on TSP1 (a) or TSP1 in the
presence of 5 µg/ml TS2/16 (b).
1 integrin
localization of the TS2/16-treated cells was visualized using BODIPY
FL-anti-mouse IgG on TSP1 (c) or type I collagen substrates
(d). Bar in a = 20 µm.
3
1-Mediated Adhesive Activity of
TSP1--
Differences in the conformation or folding of TSP1 could
account for discrepancies in its reported adhesive activity. The conformation of TSP1 and formation of specific intra-chain disulfide bonds are sensitive to the levels of divalent cations present during
its purification. Disulfide bonding also influences interactions of
TSP1 with several proteases and regulates the accessibility of the RGD
sequence to the
v
3 integrin (27, 28). We
therefore examined the influence of conformation on
3
1-dependent adhesion by absorbing TSP1 with or without divalent cations, at low pH
(29), or by reducing disulfide bonds using dithiothreitol (Fig.
6). Coating TSP1 at pH 4 in acetate
buffer enhanced MDA-MB-435 cell adhesion relative to TSP1 adsorbed in
PBS with Ca2+ and Mg2+, but use of PBS with 2.5 mM EDTA did not significantly affect
1-mediated adhesion. Although heparin only partially
inhibited MDA-MB-435 cell adhesion to TSP1 (20-50%) when the TSP1 was
adsorbed in Dulbecco's PBS (e.g. Fig. 2A),
adhesion to TSP1 adsorbed in pH 4 acetate buffer was inhibited 98% by
10 µg/ml heparin. Conversely, TS2/16 did not reproducibly increase
adhesion of MDA-MB-435 cells to TSP1 adsorbed in acetate buffer (data
not shown). Therefore, the enhanced adhesion to thrombospondin coated
at pH 4 was due primarily to enhancement of
heparin-dependent adhesion, whereas
1-integrins contributed less to adhesion on TSP1 coated
at the lower pH. Adhesion of MDA-MB-435 cells (Fig. 6) and MDA-MB-231 cells (results not shown) was strongly inhibited following reduction of
TSP1 with dithiothreitol. This contrasts with
v
3-dependent adhesion to
TSP1, which was reported to be enhanced following disulfide reduction
using the same conditions as used in Fig. 6 (28). Thus,
3
1-dependent adhesion of
breast carcinoma cells does not require Ca2+-replete TSP,
but some intact disulfide bonds are essential.
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Fig. 6.
Tertiary structure dependence for MDA-MB-435
cell adhesion on TSP1. MDA-MB-435 cell adhesion to 20 µg/ml TSP1
coated on polystyrene in 10 mM sodium acetate, 150 mM NaCl, pH 4 (29), Dulbecco's PBS with calcium and
magnesium (PBS-Ca2+), PBS with 2.5 mM EDTA, or
PBS with 2.5 mM EDTA and 2 mM dithiothreitol
(DTT). Attachment (solid bars) and spreading (striped
bars) were assessed in the absence or presence of 5 µg/ml
TS2/16.
1 Integrin Activation in Breast
Carcinoma Cells--
Adhesion of T lymphocytes to TSP1, mediated by
4
1 and
5
1
integrins, is stimulated by phorbol esters (30). PMA activation of
protein kinase C in MDA-MB-435 cells increased
v
3-mediated adhesion to vitronectin but
had no effect on
1 integrin-mediated adhesion to TSP1
(Fig. 7). Integrin-associated protein
(CD47) also regulates integrin function in several cell types (31, 32).
The carboxyl-terminal domain of TSP1 contains two peptide motifs that
activate integrin function through binding to CD47 (31). The
CD47-binding TSP1 peptide 7N3 activated adhesion of MDA-MB-435 cells on
vitronectin (Fig. 7) and a recombinant TSP1 fragment containing the RGD
sequence (results not shown) but had no effect on adhesion to native
TSP1 (Fig. 7). Thus MDA-MB-435 cells express functional
v
3 that can be activated by PMA or the
TSP1 7N3 peptide. This
v
3 integrin can
recognize the TSP1 RGD sequence in the context of a bacterial fusion
protein, but it does not play a significant role in adhesion of resting
or stimulated breast carcinoma cells to native platelet TSP1.
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Fig. 7.
Differential regulation of
1 and
3 integrin activity in MDA-MB-435
cells. Attachment of MDA-MB-435 cells on 5 µg/ml vitronectin
(striped bars) or 40 µg/ml TSP1 (solid bars)
was measured using cells treated with 20 µg/ml TS2/16, 10 ng/ml PMA,
or 3 µM of the CD47-binding TSP1 peptide 7N3
(FIRVVMYEGKK). Results are presented as a percentage of cell attachment
without additions, mean ± S.D., n = 3.
1-dependent adhesion to TSP1 (Table
II). The broad spectrum Ser/Thr protein
kinase inhibitor staurosporine increased spreading of all three cell lines. However, this activation in MDA-MB-435 cells was only partially replicated by specific inhibitors of protein kinase C
(bisindolylmaleimide), protein kinase A (KT5720), or protein kinase G
(KT5823 and guanosine-3',5'-cyclic monophosphorothioate,
8-(4-chloro-phenylthio)-, Rp isomer). Inhibition of
phosphatidylinositol 3-kinase using wortmannin had no significant effect on MDA-MB-435 cell spreading and weakly enhanced MDA-MB-231 cell
spreading on TSP1. Two calcium ionophores, ionomycin and A23187,
strongly enhanced spreading of MDA-MB-435 cells but had no effect on
MDA-MB-231 cell spreading on TSP1.
Modulation of breast carcinoma spreading on TSP1
1-activating antibody TS2/16 to measure basal and total
1-dependent adhesion and in cells pretreated
with and maintained in the following inhibitors: 10 nM
staurosporine (Ser/Thr kinase inhibitor), 100 nM KT5720
(protein kinase A), 200 nM bisindolylmaleimide (protein
kinase C), 1 µM KT5823 or 2 µM
guanosine-3',5'-cyclic monophosphorothioate, 8-(4-chlorophenylthio)-,
Rp isomer (protein kinase G), 2 nM wortmannin
(phosphatidylinositol 3-kinase), 1 µg/ml ionomycin or A23187 (calcium
ionophores), 1 µM herbimycin (tyrosine kinase), or 20 µM vanadate. The net increase in cell spreading in the
presence of the indicated drugs is expressed as a percent of that
induced by the
1-activating antibody TS2/16, mean ± S.D., n = 3.
3
1.
However, PT did influence MDA-MB-231 and MDA-MB-435 cell adhesion and
spreading on TSP1 or collagen (Fig. 8).
PT increased adhesion of MDA-MB-231 cells to TSP1 (Fig. 8A)
but inhibited both basal and TS2/16-stimulated adhesion of MDA-MB-435
cells on the same substrate (Fig. 8B). The effects of PT in
both cell lines were specific, since PT B-oligomer at the same
concentration had no effect (Fig. 8). The enhancement of MDA-MB-231
cell adhesion by PT is mediated by the
1 integrin,
because the
1 blocking antibody mAb13 inhibited the
PT-induced adhesion of MDA-MB-231 cells but heparin did not (results
not shown). However, not all
1 integrins in these breast
carcinoma cells were activated by PT. Adhesion of MDA-MB-231 cells to
collagen mediated by
2
1 (verified by the
blocking antibody 6D7, results not shown) was not altered by PT,
although the same adhesive pathway could be further activated by TS2/16
(Fig. 8A). In MDA-MB-435 cells, PT partially inhibited
2
1-mediated spreading on collagen
stimulated by TS2/16 (Fig. 8B).
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Fig. 8.
Pertussis toxin differentially regulates
MDA-MB-435 and MDA-MB-231 cell adhesion on TSP1. A,
MDA-MB-231 cell attachment on 40 µg/ml TSP1 (solid bars)
or 5 µg/ml type I collagen (gray bars) was measured alone
or in the presence of 5 µg/ml TS2/16, 1 µg/ml PT, or 1 µg/ml PT
B-oligomer. Results are mean ± S.D. for triplicate
determinations. B, MDA-MB-435 cell spreading was determined
on TSP1 (solid bars) or type I collagen substrates
(gray bars) in the presence of PT or PT B-oligomer added
alone or combined with 5 µg/ml TS2/16.
1 integrin-mediated adhesion on TSP1. This suggested that proliferation regulates
3
1-mediated
TSP1 adhesion. Serum induced a dose-dependent increase in
1 integrin-mediated attachment (Fig.
9A) and spreading of
MDA-MB-435 cells to TSP1 or type I collagen. A similar serum response
was observed in MDA-MB-231 cells for adhesion on TSP1, although
adhesion of the latter cell line to type I collagen was maintained in
the absence of serum (data not shown).
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Fig. 9.
Regulation of
1 integrin-mediated TSP1 interactions
by serum and growth factors. A, serum induces
attachment of MDA-MB-435 cells to TSP1 (solid bars) and type
I collagen (striped bars). Cells were grown for 24 h in
RPMI medium containing the indicated concentration of FCS.
B, insulin specifically induces adhesion of breast carcinoma
cells to TSP1. MDA-MB-435 cell spreading on surfaces coated with 40 µg/ml TSP1 was determined using cells grown for 24 h in RPMI
medium containing 2% serum and supplemented with the indicated growth
factors (10 ng/ml EGF, 100 ng/ml FGF2, 5 ng/ml TGF-
, or 10 µg/ml
insulin) or RPMI medium containing 10% serum. Spreading of cells grown
in 2% serum was also tested in the presence of 5 µg/ml antibody
TS2/16 (2% + TS2/16) to assess maximal
1
integrin-mediated spreading activity. C, dose dependence for
induction of TSP1 adhesion by insulin and IGF1. Cell spreading after 50 min (expressed as a percentage of maximal spreading elicited on each
substrate in the presence of 5 µg/ml TS2/16 antibody) was determined
in RPMI medium containing 0.1% BSA and supplemented with the indicated
concentrations of insulin (closed symbols) or IGF1
(open symbols) using substrates coated with 40 µg/ml TSP1
(
and
), 20 µg/ml laminin (
and
), or 5 µg/ml type I
collagen (
and
). D, IGF1 synergizes with TSP1 to
promote chemotaxis of MDA-MB-435 cells. Chemotaxis to 50 µg/ml TSP1
was determined in the presence of the indicated inhibitors or
stimulators at the following concentrations: 10 nM IGF1 5 µg/ml mAb13 (anti-
1), 5 µg/ml P1B5
(anti-
3), and 1 µg/ml PT. Results are mean ± S.D., n = 3-6.
1 were also ineffective, but addition
of insulin stimulated MDA-MB-435 cell adhesion to a greater extent than
10% serum (Fig. 9B). Insulin was also the only growth
factor tested that stimulated adhesion of MDA-MB-231 cells to TSP1
(results not shown).
3
1 integrin,
because mAb13 (anti-
1) and P1B5 antibodies
(anti-
3) strongly inhibited direct TSP1 chemotaxis and
that stimulated by IGF1. IGF1-stimulated chemotaxis to TSP1 was also
sensitive to PT inhibition (Fig. 9D).
1 integrins
(36). Clustering of CD98 using the antibody 4F2 stimulates small cell
lung carcinoma adhesion on fibronectin and laminin (36) and similarly
activated
3
1-mediated spreading of breast
carcinoma cells on TSP1 and
2
1-mediated
adhesion on type I collagen (Fig.
10A and results not shown).
Induction of
3
1-mediated TSP1 adhesion in
serum-containing growth medium may be mediated by induction of CD98
expression, because a 24-h exposure to 10% serum increased surface
expression of CD98 in MDA-MB-435 cells (Fig. 10B). IGF1
treatment for the same time, however, decreased CD98 expression (Fig.
10B), indicating that increased CD98 expression does not
mediate the response to IGF1.
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Fig. 10.
CD98 ligation stimulates breast carcinoma
cell adhesion to TSP1. A, basal (solid bars)
or stimulated MDA-MB-231 or MDA-MB-435 cell spreading on 25 µg/ml
TSP1 was determined in the presence of 5 µg/ml TS2/16 (striped
bars) or 20 µg/ml 4F2 (gray bars). B,
serum induces but IGF1 inhibits CD98 expression. MDA-MB-435 cells grown
24 h in RPMI medium containing 1% FCS, 10% FCS, or 1% FCS and
10 nM IGF1 as described in A were biotinylated,
and equal amounts of cell protein were immunoprecipitated with antibody
4F2. The immunoprecipitates were analyzed by SDS-gel electrophoresis
and Western blotting using streptavidin-peroxidase and chemiluminescent
detection. Markers indicate the migration of the 80- and 45-kDa
subunits of CD98.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3
1 integrin, with some
cooperation of sulfated glycoconjugates and
4
1 integrin, mediates adhesion of
MDA-MB-435 and MDA-MB-231 breast carcinoma cells to TSP1. This
1 integrin is maintained in an inactive or partially
active state in these cell lines but can be activated by exogenous
stimuli including serum, insulin, IGF1, and ligation of CD98. In
MDA-MB-231 cells, the inactive state of the
3
1 integrin is maintained by a
G-protein-mediated signal, but this suppression can also be overcome by
IGF1 receptor signaling. Stimuli that increase
1-dependent adhesion to TSP1 do not
stimulate
3-dependent adhesion to TSP1, even
though the cells express the known TSP1 receptor
v
3, and this integrin is functional and
inducible for vitronectin adhesion. We do not know why the
v
3 integrin on MDA-MB-435 cells cannot
recognize the RGD sequence in the type III repeat of platelet TSP1.
Other cell types, however, can utilize the same TSP1 preparations used for these experiments to support
v
3-dependent
adhesion.2
1 integrins have been implicated as TSP1
receptors in other cell types, including
2
1 on activated platelets (37),
3
1 on neurons (38), and
4
1 and
5
1
on activated T lymphocytes (30).
3
1 is
the dominant integrin for mediating adhesive activity of breast
carcinoma cells for TSP1, whereas
2
1
mediates adhesion of these cells to type I collagen but not to TSP1.
The integrin
4
1 may play a role in
adhesion of some breast carcinoma cell lines to TSP1, as we previously
reported for T lymphocytes (30). The mechanism for the apparent
differential recognition of TSP1 by
1 integrins among
these cell types remains to be defined. However, it is notable that
even within the breast carcinoma cell lines, pharmacological and
physiological stimuli can differentially modulate activity of the
3
1 integrin for promoting adhesion or
chemotaxis to TSP1. This finding implies a complex signaling process
that regulates the recognition of pro-adhesive signals from TSP1 in the
extracellular matrix. Both the IGF1 receptor and CD98 are components of
this regulatory complex in breast carcinoma cells, but the mechanisms
of their actions also remain to be defined.
v
3 integrin,
the
3
1 integrin in breast carcinoma cells
is not activated by engagement of CD47 by the TSP1 "VVM" peptides
or by protein kinase C activation. Rather, inhibition of Ser/Thr kinase
activity, but not Tyr kinase activity, increases
1-mediated adhesive activity of MDA-MB-435 cells for
TSP1. Conversely, phorbol ester activation of protein kinase C
increased adhesion via
v
3 but not
3
1 integrin. Thus, activation of
individual integrins in MDA-MB-435 cells can be differentially regulated.
3
1-mediated interactions with TSP1. The
insulin and IGF1 receptors were reported to be physically associated
with the
v
3 integrin but not with
1 integrins in fibroblasts (40). The
v
3 integrin also co-immunoprecipitated
with insulin receptor substrate-1 (41). Engagement of
v
3 integrin by vitronectin but not
2
1 integrin by collagen increased
mitogenic signaling through the insulin receptor (40, 41). Thus, the
specific activation of
3
1-mediated
spreading and chemotaxis to TSP1 by insulin or IGF1 was unexpected. We
observed a stronger response for stimulating adhesion to TSP1 than to
collagen or laminin, suggesting that the regulation of avidity by the
IGF1 receptor is specific for the
3
1
integrin. Other growth factors that utilize tyrosine kinase receptors
including FGF2 and EGF did not activate this integrin. We therefore
predict that specific coupling of
3
1
activation to IGF1 receptor signaling, rather than a general phosphorylation signal, mediates rapid activation of the TSP1 binding
integrin in breast carcinoma cells. The mechanism for this
specific signaling remains to be determined.
1
integrins by its ability to overcome Tac-
1 suppression of
1 integrin function (36, 42). Our data demonstrate that
clustering of CD98 can also increase
3
1-mediated TSP1 interactions. This may simply result from clustering of the CD98-associated
3
1 integrin, which increases the avidity
for cell adhesion to a surface coated with TSP1, or it may require
specific signal transduction from CD98. Regulation of CD98 levels is
probably responsible for the serum-induced increase in adhesion to
TSP1, since serum increases CD98 surface expression in MDA-MB-435
cells. The insulin and IGF1-induced stimulation of TSP1 spreading and
chemotaxis cannot be explained by regulation of CD98 levels, however,
since IGF1 down-regulates CD98 in these cells.
3
1 integrin
on MDA-MB-231 and MCF-7 cells is constitutively active to mediate
adhesion to TSP1. The inactive integrin appears to be on the cell
surface, since it can be rapidly activated by the TS2/16 antibody or by
IGF1 receptor ligands. The low basal activity of this integrin could result from absence of an activator or expression of an inhibitor in
MDA-MB-231 and MCF-7 cells. Several factors that suppress integrin function have been identified, including Ha-Ras (43), integrin-linked kinase, and protein kinase C (39). Additional proteins are known to
associate with the
3
1 integrin, including
some members of the TM4SF family and EMMPRIN (44, 45), but their roles
in regulating function are unknown. In MDA-MB-231 cells, suppression of
3
1 appears to be an active process that
can be disrupted by PT. Thus, a heterotrimeric G-protein signaling
pathway appears to maintain MDA-MB-231 cells in an inactive state. This
inhibitory pathway may also be specific for the
3
1 integrin in MDA-MB-231 cells, because
unstimulated MDA-MB-231 cells can spread on type I collagen using
2
1 integrin. Unstimulated MDA-MB-435
cells show the opposite phenotype, with better
3
1-dependent adhesion to TSP1
than
2
1-dependent adhesion to
collagen. The differential modulation of TSP1 interactions with these
two cell lines by PT as well as the calcium ionophores demonstrates
that regulation of
3
1 activity for TSP1
may differ even between two cell lines derived from the same type of
human cancer.
3
1 integrin in spreading, induction of
filopodia, and chemotactic responses to TSP1. In other cell types, the
low density lipoprotein receptor-related protein has been assigned a
role in internalization of TSP1 (46), and CD36 has been shown to play
an essential role in angiogenesis inhibition (47). The receptors that
mediate many responses to TSP1 remain to be defined. These responses
may require coordinated signaling through two or more TSP1 receptors. Defining the role of IGF1 and CD98 in regulating
1
integrin interactions with TSP1 provides our first insight into a
breast carcinoma TSP1 receptor that can be turned on or off in response
to known environmental stimuli. The ability to regulate the activity of
this TSP1 receptor will facilitate analysis of the signals resulting
from this interaction.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Ken Yamada, David Cheresh, and Harvey Gralnick for providing antibodies and Henry Krutzsch for synthesis of peptides.
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FOOTNOTES |
---|
* This work was supported in part by Department of Defense Grant DAMD17-94-J-4499.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: CBER/OTRR/DCTDA, Food and Drug Administration,
Woodmont Office Complex 1, 1401 Rockville Pike, Rockville, MD 20852.
§ To whom correspondence should be addressed: Bldg. 10, Rm. 2A33, 10 Center Dr. MSC 1500, National Institutes of Health, Bethesda, MD 20892-1500. Tel: 301-496-6264; Fax: 301-402-0043; E-mail: droberts{at}helix.nih.gov.
2 J. M. Sipes, H. C. Krutzsch, J. Lawler, and D. D. Roberts, submitted for publication.
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ABBREVIATIONS |
---|
The abbreviations used are:
TSP1, human
thrombospondin-1;
BSA, bovine serum albumin;
IGF1, insulin-like growth
factor-1;
PMA, phorbol 12-myristate 13-acetate;
PT, pertussis toxin;
RGD, Arg-Gly-Asp;
EGF, epidermal growth factor;
TGF-, transforming
growth factor-
;
FGF, fibroblast growth factor;
FCS, fetal calf
serum;
PBS, phosphate-buffered saline;
mAb, monoclonal antibody.
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REFERENCES |
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