Pro-adhesive and Chemotactic Activities of Thrombospondin-1 for Breast Carcinoma Cells Are Mediated by alpha 3beta 1 Integrin and Regulated by Insulin-like Growth Factor-1 and CD98*

Subramaniam Chandrasekaran, Neng-hua Guo, Rui G. Rodrigues, James KaiserDagger , and David D. Roberts§

From the Laboratory of Pathology, NCI, National Institutes of Health, Bethesda, Maryland 20892

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta 1 integrin-dependent. The integrin alpha 3beta 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 beta 1 integrin activation to induce spreading on TSP1. Integrin-mediated cell spreading on TSP1 is accompanied by extension of filopodia containing beta 1 integrins. TSP1 binding activity of the alpha 3beta 1 integrin is not stimulated by CD47-binding peptides from TSP1 or by protein kinase C activation, which activate alpha vbeta 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 alpha 3beta 1 integrin.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 alpha vbeta 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 alpha 3beta 1 integrin rather than beta 3 integrins play a dominant role in adhesion of several breast carcinoma cell lines on TSP1. The activation state of the alpha 3beta 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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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-beta 1 were obtained from R & D Systems. Insulin was from Biofluids, and recombinant human insulin-like growth factor-1 (IGF1) was from Bachem.

Monoclonal Antibodies-- Hybridomas producing the beta 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 (alpha vbeta 3, provided by Dr. David Cheresh), 05-246 (alpha 1beta 1, Upstate Biotechnology), 6D7 (alpha 2beta 1, Dr. Harvey Gralnick, NIH), P1B5 (alpha 3beta 1, Life Technologies, Inc.), 407279 (alpha 4beta 1, Calbiochem), P1D6 (alpha 5beta 1, Life Technologies, Inc.), and mAb13 (beta 1, Dr. Kenneth Yamada, NIH). Non blocking antibodies recognizing alpha 3beta 1 (M-KID2), alpha 4beta 1 (HP2/1), and alpha 5beta 1 (SAM1) were obtained from AMAC, Inc. (Westbrook, ME), and alpha v (LM142) was provided by Dr. David Cheresh.

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).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta 1 integrins and alpha vbeta 3. Integrin expression on MDA-MB-231 and MCF-7 cells has been reported previously (22-24). MDA-MB-231 cells express alpha 2, alpha 3, alpha 4, alpha 5, alpha 6, alpha v, and beta 1 subunits. The MDA-MB-231 and MCF-7 cell lines express only low levels of beta 3 subunits (24).

                              
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Table I
Integrin expression in MDA-MB-435 breast carcinoma cells

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 beta 1 or alpha 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 beta 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-beta 1) or P1B5 (anti-alpha 3beta 1).

beta 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 beta 1 integrin function blocking antibody at 2 µg/ml, however, only spreading was inhibited, and a combination of heparin and the beta 1 blocking antibody abolished spreading and markedly inhibited attachment. At 50 µg/ml, the beta 1 antibody completely inhibited adhesion to TSP1 (Fig. 2A). Thus, interaction with a beta 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. beta 1 integrin-dependent adhesion was inhibited using 2 or 50 µg/ml mAb13 (anti-beta 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 (open circle ) 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 alpha vbeta 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 alpha vbeta 3 function blocking antibody LM609 or the beta 1-activating antibody TS2/16. D, chemotaxis to TSP1 is beta 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 beta 1 integrin blocking antibody mAb13 () or heparin (open circle ). Spontaneous motility (black-triangle) 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.

Although MDA-MB-435 cells express some alpha vbeta 3 integrin (Table I), a function blocking antibody or an alpha vbeta 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 beta 1-activating antibody TS2/16, adhesion of MDA-MB-435 cells was enhanced on TSP1 but not on vitronectin (Fig. 2C). Therefore, the alpha vbeta 3 integrin is functional in MDA-MB-435 cells, but it is apparently unable to recognize the RGD motif in TSP1.

The beta 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 beta 1 integrin receptor.

Several human breast cancer cell lines showed similar involvement of beta 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 beta 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 beta 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.   beta 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 beta 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.

alpha 3beta 1 Is the Major TSP1-binding Integrin on Breast Carcinoma Cells-- Of the alpha  subunit antibodies tested for inhibiting adhesion to TSP1, only an alpha 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 alpha 4 integrin blocking antibody slightly inhibited adhesion, but mixing this antibody with the alpha 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 alpha 3 blocking antibody, and somewhat by the alpha 4 antibody (Fig. 4B). Function blocking antibodies for alpha 1beta 1, alpha 2beta 1, and alpha 5beta 1 integrins had no effect on TSP1 adhesion, although the alpha 2beta 1 and alpha 5beta 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 alpha  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 beta 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 alpha  subunit blocking antibodies.

Integrin Localization and Effects on Actin Cytoskeleton-- Activation of beta 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 beta 1 integrin antibody revealed numerous filopodia extending from these points (Fig. 5c). In some cells, these filopodia were terminated with punctate beta 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 beta 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 beta 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). beta 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.

Conformation Requirements for alpha 3beta 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 alpha vbeta 3 integrin (27, 28). We therefore examined the influence of conformation on alpha 3beta 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 beta 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 beta 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 alpha vbeta 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, alpha 3beta 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.

Regulation of beta 1 Integrin Activation in Breast Carcinoma Cells-- Adhesion of T lymphocytes to TSP1, mediated by alpha 4beta 1 and alpha 5beta 1 integrins, is stimulated by phorbol esters (30). PMA activation of protein kinase C in MDA-MB-435 cells increased alpha vbeta 3-mediated adhesion to vitronectin but had no effect on beta 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 alpha vbeta 3 that can be activated by PMA or the TSP1 7N3 peptide. This alpha vbeta 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 beta 1 and beta 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.

Several pharmacological agents stimulated beta 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.

                              
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Table II
Modulation of breast carcinoma spreading on TSP1
MDA-MB-435 or MDA-MB-231 cell spreading on TSP1 was measured in untreated cells in the presence or absence of the beta 1-activating antibody TS2/16 to measure basal and total beta 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 beta 1-activating antibody TS2/16, mean ± S.D., n = 3. 

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 alpha 3beta 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 beta 1 integrin, because the beta 1 blocking antibody mAb13 inhibited the PT-induced adhesion of MDA-MB-231 cells but heparin did not (results not shown). However, not all beta 1 integrins in these breast carcinoma cells were activated by PT. Adhesion of MDA-MB-231 cells to collagen mediated by alpha 2beta 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 alpha 2beta 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.

Physiological Activators of TSP1 Adhesion and Chemotaxis-- We noted that freshly passaged breast carcinoma cells exhibited stronger beta 1 integrin-mediated adhesion on TSP1. This suggested that proliferation regulates alpha 3beta 1-mediated TSP1 adhesion. Serum induced a dose-dependent increase in beta 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 beta 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-beta , 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 beta 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 (open circle  and ), 20 µg/ml laminin (black-triangle and triangle ), or 5 µg/ml type I collagen (black-square 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-beta 1), 5 µg/ml P1B5 (anti-alpha 3), and 1 µg/ml PT. Results are mean ± S.D., n = 3-6.

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-beta 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).

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 alpha 3beta 1 integrin, because mAb13 (anti-beta 1) and P1B5 antibodies (anti-alpha 3) strongly inhibited direct TSP1 chemotaxis and that stimulated by IGF1. IGF1-stimulated chemotaxis to TSP1 was also sensitive to PT inhibition (Fig. 9D).

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 beta 1 integrins (36). Clustering of CD98 using the antibody 4F2 stimulates small cell lung carcinoma adhesion on fibronectin and laminin (36) and similarly activated alpha 3beta 1-mediated spreading of breast carcinoma cells on TSP1 and alpha 2beta 1-mediated adhesion on type I collagen (Fig. 10A and results not shown). Induction of alpha 3beta 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

The alpha 3beta 1 integrin, with some cooperation of sulfated glycoconjugates and alpha 4beta 1 integrin, mediates adhesion of MDA-MB-435 and MDA-MB-231 breast carcinoma cells to TSP1. This beta 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 alpha 3beta 1 integrin is maintained by a G-protein-mediated signal, but this suppression can also be overcome by IGF1 receptor signaling. Stimuli that increase beta 1-dependent adhesion to TSP1 do not stimulate beta 3-dependent adhesion to TSP1, even though the cells express the known TSP1 receptor alpha vbeta 3, and this integrin is functional and inducible for vitronectin adhesion. We do not know why the alpha vbeta 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 alpha vbeta 3-dependent adhesion.2

Several beta 1 integrins have been implicated as TSP1 receptors in other cell types, including alpha 2beta 1 on activated platelets (37), alpha 3beta 1 on neurons (38), and alpha 4beta 1 and alpha 5beta 1 on activated T lymphocytes (30). alpha 3beta 1 is the dominant integrin for mediating adhesive activity of breast carcinoma cells for TSP1, whereas alpha 2beta 1 mediates adhesion of these cells to type I collagen but not to TSP1. The integrin alpha 4beta 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 beta 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 alpha 3beta 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.

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 alpha vbeta 3 integrin, the alpha 3beta 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 beta 1-mediated adhesive activity of MDA-MB-435 cells for TSP1. Conversely, phorbol ester activation of protein kinase C increased adhesion via alpha vbeta 3 but not alpha 3beta 1 integrin. Thus, activation of individual integrins in MDA-MB-435 cells can be differentially regulated.

We have identified the IGF1 receptor as a specific regulator of alpha 3beta 1-mediated interactions with TSP1. The insulin and IGF1 receptors were reported to be physically associated with the alpha vbeta 3 integrin but not with beta 1 integrins in fibroblasts (40). The alpha vbeta 3 integrin also co-immunoprecipitated with insulin receptor substrate-1 (41). Engagement of alpha vbeta 3 integrin by vitronectin but not alpha 2beta 1 integrin by collagen increased mitogenic signaling through the insulin receptor (40, 41). Thus, the specific activation of alpha 3beta 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 alpha 3beta 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 alpha 3beta 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.

CD98 was recently identified as an activator of beta 1 integrins by its ability to overcome Tac-beta 1 suppression of beta 1 integrin function (36, 42). Our data demonstrate that clustering of CD98 can also increase alpha 3beta 1-mediated TSP1 interactions. This may simply result from clustering of the CD98-associated alpha 3beta 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.

Only a small fraction of the alpha 3beta 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 alpha 3beta 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 alpha 3beta 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 alpha 3beta 1 integrin in MDA-MB-231 cells, because unstimulated MDA-MB-231 cells can spread on type I collagen using alpha 2beta 1 integrin. Unstimulated MDA-MB-435 cells show the opposite phenotype, with better alpha 3beta 1-dependent adhesion to TSP1 than alpha 2beta 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 alpha 3beta 1 activity for TSP1 may differ even between two cell lines derived from the same type of human cancer.

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 alpha 3beta 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 beta 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.

    ACKNOWLEDGEMENTS

We thank Drs. Ken Yamada, David Cheresh, and Harvey Gralnick for providing antibodies and Henry Krutzsch for synthesis of peptides.

    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.

Dagger 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.

    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-beta , transforming growth factor-beta ; FGF, fibroblast growth factor; FCS, fetal calf serum; PBS, phosphate-buffered saline; mAb, monoclonal antibody.

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