The JI Acurri Cytometers
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wilson, K. E.
Right arrow Articles by Roberts, D. D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wilson, K. E.
Right arrow Articles by Roberts, D. D.
The Journal of Immunology, 1999, 163: 3621-3628.
Copyright © 1999 by The American Association of Immunologists

ß1 Integrin- and Proteoglycan-Mediated Stimulation of T Lymphoma Cell Adhesion and Mitogen-Activated Protein Kinase Signaling by Thrombospondin-1 and Thrombospondin-1 Peptides

Katherine E. Wilson1, Zhuqing Li, Murat Kara, Kevin L. Gardner and David D. Roberts2

Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cell-cell and cell-matrix interactions play important regulatory roles in lymphocyte homeostasis. Thrombospondin-1 (TSP1) is a matricellular protein that differentially promotes the adhesion of resting and activated T cells. In this work, we show that adhesion of Jurkat T cells on substrates coated with TSP1 or TSP1-derived peptides is mediated by ß1 integrins, CD47, and heparan sulfate proteoglycans. Interactions with TSP1 or TSP1 peptides stimulated CD3-induced Ras activation and tyrosine phosphorylation of several T cell proteins. The signals from TSP1 and its derived peptides differentially synergized with activation of the TCR to induce phosphorylation of linker for activation of T cells (LAT) and extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase, and p38 kinases. The phosphorylation of ERK in the presence of full-length TSP1 was transient and dependent on a ß1 integrin receptor. Interestingly, peptides derived from the type 1 repeats of TSP1 and a CD47-binding peptide from the carboxyl-terminal domain of TSP1 also stimulated mitogen-activated protein (MAP) kinase phosphorylation. Moreover, the TSP1 heparin-binding peptide synergized with Ab-ligated TCR to transduce signals to the nucleus, detected by activation of AP-1- and Elk-dependent transcription. This TSP1 peptide-dependent activation of AP-1 was inhibited by both heparin and the MAP/ERK kinase inhibitor PD98059, providing a functional link between adhesion molecule interaction and nuclear transactivation events via the MAP kinase pathways. These findings have implications for the role of extracellular TSP1 and TSP1 fragments in the regulation of T cell function during hemostasis, wound repair, and other inflammatory responses.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Thrombospondins are a family of matricellular proteins that have diverse effects on cell adhesion, motility, proliferation, and survival (1, 2, 3, 4, 5, 6). Thrombospondin-1 (TSP1),3 the first identified member of this family, is highly expressed during wound repair and inflammatory responses (reviewed in Refs. 7, 8). Expression of the THBS1 gene encoding TSP1 is induced by several growth factors, including platelet-derived growth factor and TGF-ß1. TSP1 is also a major component of platelet {alpha}-granules and is released following platelet activation at sites of injury. Elevated TSP1 levels at these sites can alter functions of several cell types including endothelial cells, monocytes (9, 10), macrophages (11), and NK cells (12). In addition to its direct actions through binding to TSP1 receptors on target cells, TSP1 can alter cell behavior through activating latent TGF-ß (13, 14). These in vitro activities of TSP1 combined with the observed inflammatory disease in thbs1-null mice (15) suggested that TSP1 expression may modulate local immune responses. Although the tumor-suppressive activity of TSP1 has been primarily associated with its anti-angiogenic activity, immune modulation may also contribute to the effects of TSP1 expression on tumor growth in several animal models (reviewed in Ref. 6).

TSP1 is a large multifunctional protein composed of three identical subunits (7, 16, 17). The diverse effects of TSP1 on cell behavior have been associated with several functional sequences (reviewed in Refs. 6, 7). The amino-terminal domain contains a high affinity heparin-binding site and a binding site for the low density lipoprotein receptor-related protein that mediates internalization of TSP1 in some cells. The central stalk region of TSP1 contains anti-angiogenic sequences, sequences that bind to heparin (18), CD36 (19, 20), and fibrinogen (21), and an RGD sequence recognized by ß3 integrins (22). Peptide sequences within the C-terminal domain bind to the integrin-associated glycoprotein CD47 (23), which has been shown to modulate aVß3 integrin function (3) and T cell activation (24, 25).

T lymphocytes can interact with TSP1 via both integrin-dependent and integrin-independent pathways. CD47, which is a receptor for the C-terminal cell-binding domain of TSP1 (23), provides costimulatory signals to T lymphocytes, probably via an integrin-independent pathway (24, 25). However, it has not been established that TSP1 binding to CD47 can elicit such a costimulatory signal. ß1 integrins mediate adhesion of peripheral T lymphocytes on TSP1 (26). Activation-dependent adhesion of peripheral CD4+ T cells to TSP1 was inhibited by function-blocking Abs to the {alpha}4ß1 and {alpha}5ß1 integrins. ß1 integrin interactions with other extracellular matrix ligands can modulate the recruitment and activation of T lymphocytes (27, 28, 29, 30 ; reviewed in Ref. 31), suggesting that TSP1 binding to ß1 integrins on T cells may also have functional consequences.

Although we have previously demonstrated that TSP1 can differentially promote adhesion of resting and activated T lymphocytes and of naive and memory T cells (26), the effects of TSP1 on function of T cells have not been examined. To begin to define the molecular responses of T cells to TSP1, we have examined the adhesive and signaling responses of T cells to intact TSP1 and several defined functional sequences from TSP1. We demonstrate in this study that several cell surface receptors, including ß1 integrins, CD47, and heparan sulfate proteoglycans, can transduce signals following interaction with TSP1 or specific TSP1 peptides. Binding to these receptors modulates the activation of p21 Ras and phosphorylation of several MAP kinase pathways, and induces AP-1-dependent transcription activity.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Proteins and peptides

Human TSP1 was isolated from the supernatant of thrombin-stimulated human platelets by gelatin and heparin affinity, followed by gel filtration chromatography (32). Purified TSP1 was stored in aliquots at -70°C. Synthetic peptides derived from several functional domains of TSP1 were prepared as previously described (2, 18, 23) and are summarized in Table IGo.


View this table:
[in this window]
[in a new window]
  		Table I. TSP1 peptides and analogs

 
Antibodies

Anti-human CD3 mAb (PharMingen, San Diego, CA; clone HIT3a) was immobilized on plastic at 0.5 µg/ml. The function-blocking ß1 integrin Ab, mAb13 (provided by Dr. Ken Yamada National Institute of Dental and Craniofacial Research, Bethesda, MD) (33), was used in solution at 10 µg/ml, and the ß1 integrin-activating Ab TS2/16 (34) was purified from the hybridoma (American Type Culture Collection, Manassas, VA) and used in solution at 20 µg/ml. Anti-CD47 mAb (clone CIKm1; ICN Pharmaceuticals, Costa Mesa, CA) was immobilized on plastic at 0.5 µg/ml. Phospho-specific Abs to MAP kinases were obtained from New England Biolabs. Rabbit anti-LAT (35) was provided by Dr. Larry Samelson, National Cancer Institute (Bethesda, MD).

Cell culture

The human Jurkat T-lymphoma cell line (36) was routinely cultured in RPMI 1640 supplemented with 10% FCS, 2 mM L-glutamine, 100 IU/ml penicillin, and 100 µg/ml streptomycin at 37°C in an atmosphere of 5% CO2. All media and culture components were from Biofluids (Rockville, MD).

Adhesion assays

Purified TSP1 and peptides, diluted in Dulbecco’s PBS, were coated onto 96-well flat-bottom plates (Immulon 2) overnight at 4°C. After aspiration of the buffer, nonspecific adherence to plastic was blocked by incubation with Dulbecco’s PBS containing 1% BSA for 30 min at 37°C. Cells were washed in serum-free media and resuspended at 5 x 105 cells/ml in RPMI containing 0.1% BSA. Aliquots of cells (100 µl) were added to each well alone or with Abs at the indicated concentrations. The plate was incubated at 37°C for 15 min, before nonadherent cells were removed by washing. The number of adherent cells was quantified using the previously described colorimetric hexosaminidase assay (37).

To investigate the role of sulfated glycoconjugates in adhesion, cells were grown in the presence of chlorate to inhibit sulfation (38). Cells for these experiments were grown in Hams-F12 medium containing 10% dialyzed FCS, 20 mM HEPES, and 2 mM glutamine for 48 h before being transferred to the same media containing 2% dialyzed FCS with or without sodium chlorate for 24 h. To perform the adhesion assay, the cells were resuspended in Hams-F12 medium containing 0.1% BSA and the relevant concentration of chlorate, and adhesion assays were performed as above.

Western blot analysis

Bacteriological polystyrene 35-mm petri dishes (Falcon 1008) were coated with the described combinations of Abs and TSP1/peptides overnight at 4°C. Cells were added to the plates (5 x 105 cells in 0.1% BSA in RPMI) with soluble peptides or TSP1, where applicable. Cells were incubated at 37°C for 5 min to 4 h, the suspended cells were centrifuged for 5 min at 600 x g, and 50 µl of RIPA buffer containing protease inhibitors (50 mM Tris-HCl, pH 7.4, 0.15 M NaCl, 1% (w/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 1 mM EGTA, 1 mM Na3VO4, and protease inhibitors at 10 µg/ml antipain, pepstatin A, chymostatin, leupeptin, soybean trypsin inhibitor, aprotinin, and 1 mM phenylmethane sulfonyl fluoride) was added to the plate and cell pellet. These two fractions were then combined and incubated on ice for 30 min. The cell lysates were centrifuged at 14,000 x g for 30 min at 4°C. The proteins were separated by SDS-PAGE on a 4–15% gradient polyacrylamide gel and transferred to nitrocellulose membrane by semidry electroblotting.

Tyrosine-phosphorylated proteins were detected using the HRP-conjugated anti-phosphotyrosine Ab RC20 (Transduction Laboratories, Lexington, KY). The phosphorylated forms of the MAP kinases ERK, stress-activated protein/JNK, and p38 were identified using phospho-specific mAbs (New England Biolabs, Beverly, MA), followed by HRP-conjugated anti-rabbit Ab. Proteins were visualized by chemoluminescent detection.

Activated Ras interaction assay

An activated Ras interaction assay was used to measure Ras activation by TSP1 (39). In brief, Escherichia coli transformed with the pGEX-RBD plasmid was induced to produce the rGST-RBD fusion protein, which contains a Ras-binding domain comprising amino acids 1–149 of c-Raf-1 (39). The recombinant fusion protein was affinity purified using glutathione-Sepharose 4B beads. Mouse anti-human CD3 mAb (0.5 µg/ml, clone HIT3a; PharMingen) was coated on six-well plates (Nalge Nune, Naperville, IL) overnight in PBS at 4°C. Approximately 1.5 x 106 Jurkat cells suspended in RPMI 1640 with 0.1% BSA were added into each well. TSP1 and its peptides were added at the indicated concentrations, and the cells were incubated in 5% CO2 at 37oC for 15 min. The cells were collected by centrifugation and washed with PBS three times.

The cells were lysed by addition of magnesium lysis buffer (39). Cell lysates were normalized based on protein level determined by BCA assay (Pierce, Rockford, IL). Equal amounts of cell protein were incubated with purified GST-RBD immobilized on glutathione beads prepared according to the manufacturer’s instruction. After extensive washing, bound proteins were eluted by boiling in SDS-PAGE sample buffer for 5 min and separated on 4–12% SDS-PAGE gradient gel. Western blotting analysis was performed as described above, and Ras activity was detected by probing with an anti-Ras mAb (clone18; Transduction Laboratories) and chemoluminescent detection (Pierce).

Reporter assays

Jurkat cells were cultured in RPMI 1640 medium with 10% FCS at 37°C for 16–18 h. Cells were then transiently transfected with 5 µg of indicated CAT reporter constructs DNA by electroporation (40). For the Elk reporter assay, equal amounts of a Gal4-Elk construct (a generous gift from Dr. Silvio Gutkind, NIDCR, National Institute of Health, Bethesda, MD) and an E1B-CAT construct containing five copies of the UAS (Gal4 binding site) upstream of the E1B minimal promoter (41) were cotransfected. Transfected cells were diluted in RPMI 1640 containing 0.1% BSA to a final concentration of 2 x 106 cells/well and stimulated with the indicated TSP1 peptides in the presence of immobilized mouse anti-human CD3 Ab (1 µg/well; clone HIT3a). For the inhibition experiments using heparin and the MEK inhibitor PD98059 (Calbiochem, La Jolla, CA), transfected cells were stimulated with peptide 246 with or without heparin (100 µg/ml) or after pretreatment for 30 min with the MEK inhibitor PD98059 (10 µM). CAT activity in cell extracts was analyzed as previously described (42).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ß1 integrins mediate adhesion of Jurkat cells to TSP1

As previously reported for CD4+ peripheral T cells (26), Jurkat T lymphoma cells attached on substrates coated with TSP1 within 5 min, but adhesion was transient and the cells began to detach from TSP1 after 30 min. Adhesion to TSP1 was inhibited by the ß1 integrin-blocking Ab mAb13, which reduced the level of cells attached to 28% of control (Fig. 1Go). Conversely, addition of the ß1 integrin-activating Ab TS2/16 increased the level of adhesion to 163% of control levels on 50 µg/ml TSP1 (Fig. 1Go) and 251% of the control level on 5 µg/ml. More than 90% of the input cells attached on 50 µg/ml TSP1 in the presence of the ß1 integrin-activating Ab. Adhesion to TSP1 was not significantly inhibited by heparin or an anti-CD47 Ab (Fig. 1Go), indicating that proteoglycans and CD47 do not play a major role in adhesion of these cells to immobilized intact TSP1.



View larger version (17K):
[in this window]
[in a new window]
  		FIGURE 1. Adhesion of Jurkat T cells to intact TSP1 is mediated through a ß1 integrin. TSP1 (20 µg/ml) was coated onto all wells of a 96-well plate, except control wells that were coated only with BSA. After removing the unbound protein, the plate was blocked with 1% BSA. Jurkat cells were washed and suspended in RPMI containing 0.1% BSA. Before adding the cells to the plate (1 x 105 cells/well), the following reagents were added at the indicated concentrations: ß1 integrin-activating Ab TS2/16 (20 µg/ml), ß1 integrin function-blocking Ab mAb13 (10 µg/ml), heparin (100 µg/ml), and anti-CD47 (10 µg/ml). Adhesion was quantified after 15 min in quadruplicate wells by a colorimetric hexosaminidase assay, and is presented as a percentage of the adhesion to TSP1 measured in the absence of inhibitors (mean ± SD).

 
Adhesion was also examined using synthetic peptides from several regions of TSP1 coated onto plastic (Fig. 2GoA). Both peptide 246, a heparin-binding peptide from the type 1 repeats of TSP1 (18), and the CD47-binding peptide 7N3 from the C-terminal domain of TSP1 (23) promoted adhesion of Jurkat cells. Typically, 60–70% of input cells attached on these TSP1 peptides. No adhesion was observed on the CD36-binding peptides Mal II, from the type 1 repeats, and peptide 500, from the procollagen domain of TSP1 (43). The cells also failed to attach on the peptide Hep-I from the heparin-binding domain (2).



View larger version (21K):
[in this window]
[in a new window]
  		FIGURE 2. Jurkat cells adhere to peptides from the type 1 repeats and C-terminal domain of TSP1. A, Adhesion was examined on immobilized synthetic peptides from different regions of the TSP1 molecule. All peptides in this assay were coated at 100 µM, and adhesion was assayed as described in Fig. 1Go. B, Peptides 246 (•) and 7N3 ({blacktriangleup}) and their respective control peptides 388 ({circ}) and 605 ({triangleup}) were coated onto plastic at the indicated concentrations. Adhesion was assayed after 15 min in quadruplicate wells and presented as mean ± SD.

 
The specificities of the two active TSP1 peptides were verified using peptides with amino acid substitutions in essential residues (Fig. 2GoB and Table IGo). Cell adhesion on both peptides was specific, in that neither control peptide significantly promoted adhesion. Based on time-lapse video, cells on TSP1 and peptide 246 extended and retracted pseudopodia, but were less motile compared with those on fibronectin, where the cell bodies also moved (data not shown). The ß1 integrin-blocking mAb13 had no effect on adhesion to either peptide 246 or 7N3 (data not shown), confirming that adhesion to these peptides is not ß1 integrin dependent.

Sulfated glycosaminoglycans mediate adhesion to the type 1 peptide 246

To examine the role of sulfated glycosaminoglycans in Jurkat cell adhesion to TSP1 peptide 246, cells were cultured in media containing differing concentrations of the 3'-phosphoadenosine 5'-phosphosulfate synthase inhibitor chlorate. Chlorate treatment inhibited attachment of cells to immobilized peptide 246 (Fig. 3Go), approaching background adhesion at 40 mM chlorate. Growth with chlorate had no significant effect on cell adhesion to intact TSP1. Treatment of the Jurkat cells with heparatinase, but not with chondroitinase ABC, also significantly inhibited adhesion to peptide 246 (data not shown). Therefore, both heparan sulfate proteoglycans and CD47 can mediate adhesion of Jurkat cells to synthetic peptides from TSP1, but adhesion to the intact protein primarily depends on a ß1 integrin.



View larger version (15K):
[in this window]
[in a new window]
  		FIGURE 3. Adhesion of Jurkat T cells to TSP1 peptide 246 is mediated by a sulfated receptor. Jurkat cells, grown in the presence of the indicated concentrations of sodium chlorate to inhibit sulfation as described in Materials and Methods, were assayed for adhesion on plates coated with 5 µM peptide 246 (•), 50 µg/ml TSP1 ({blacktriangleup}), or BSA control ({circ}). Adhesion is presented as a percentage of that for untreated cells on TSP1 or peptide 246 (mean ± SD, n = 4).

 
TSP1 and TSP1 peptides induce rapid changes in tyrosine phosphorylation

Lysates were prepared from cells incubated for 15 min in the presence of TSP1, the heparin-binding TSP1 peptide 246, or the CD47-binding TSP1 peptide 7N3. TSP1 and the peptides had similar activities in solution or immobilized on plastic, so protein or peptides in solution were routinely used to ensure interaction with all of the cells. The cell lysates were separated by SDS-PAGE and transferred to nitrocellulose, and phosphorylated proteins were detected using the anti-phosphotyrosine Ab RC20. Phosphorylation of several proteins was enhanced in the presence of TSP1 or the TSP1 peptides, including 70- and 120-kDa species (Fig. 4GoA). Anti-CD3 Ab also stimulated phosphorylation of these proteins. Treatment with the peptides (Fig. 4GoA) or intact TSP1 (Fig. 4GoB) in the presence of anti-CD3 enhanced the CD3-dependent phosphorylation of a 36-kDa protein with similar mobility to the recently identified ZAP-70 tyrosine kinase substrate LAT (35). Although the TSP1 peptide 246 directly stimulated phosphorylation of this protein, intact TSP1 did not reproducibly enhance its phosphorylation, except in the presence of anti-CD3 (Fig. 4GoB).



View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 4. TSP1 and peptide 246 stimulate CD3-induced tyrosine phosphorylation of LAT. A, Lysates were prepared with 100 µl of RIPA buffer containing protease inhibitors, from 5 x 10 5 cells incubated for 15 min with TSP1 (50 µg/ml) peptide 7N3 (20 µM), or peptide 246 (20 µM) added in solution. Anti-CD3 Ab was immobilized onto the plate at 0.5 µg/ml, where indicated. Lysates were separated by SDS-PAGE and transferred to nitrocellulose. The separated proteins were incubated with the HRP-conjugated anti-phosphotyrosine Ab RC20 and visualized by chemoluminescence. Migration of m.w. standards is indicated in the right margin. B, Cells were treated with TSP1, anti-CD3, or both. Lysates were blotted with anti-phosphotyrosine (top panel) or immunoprecipitated with anti-LAT and blotted with anti-phosphotyrosine (middle panel) or anti-LAT (lower panel).

 
The 36-kDa phosphoprotein was identified as LAT by immunoprecipitation with a LAT Ab and blotting with anti-phosphotyrosine (Fig. 4GoB). TSP1 treatment alone did not stimulate LAT phosphorylation, but TSP1 markedly enhanced the phosphorylation of LAT in cells treated with anti-CD3. The increased phosphorylation signal was not due to alteration in LAT protein levels, as verified by blotting of the LAT immunoprecipitates with anti-LAT (Fig. 4GoB).

TSP1/peptides increase expression of activated p21 Ras

Ras activation is an important early signaling event in T cell responses to several external signals. To determine whether Ras participates in the responses of T cells to TSP1, we measured the level of activated p21 Ras in Jurkat cells stimulated by TSP1 using a Raf interaction assay (39). As shown in Fig. 5Go, the level of activated Ras in Jurkat cells detected by binding to Raf1 was increased significantly and in a dose-dependent manner following stimulation with TSP1 in the presence of anti-CD3. As was observed for induction of LAT phosphorylation, TSP1 did not activate Ras in the absence of anti-CD3 (results not shown). The two TSP1 peptides 246 and 7N3 also increased activation of Ras beyond that induced by treatment of Jurkat cells with suboptimal doses of CD3 Ab alone.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 5. Intact TSP1 and TSP1 peptides synergize with TCR signaling to activate Ras. Jurkat cells were cultured in RPMI 1640 medium with 10% FCS at 37°C for 16–18 h. Cells were then pelleted, resuspended in RPMI 1640 with 0.1% BSA to a final concentration of 2 x 106 cells/well, and stimulated with 5–45 µg/ml TSP1 or 20 µM of the TSP1 peptides 7N3 and 246 in the presence of immobilized anti-CD3. Treated and untreated control cells were lysed and incubated with GST-Raf1-RBD immobilized on glutathione-Sepharose 4B beads, as described in Materials and Methods. Bound proteins were separated by 4–15% gradient SDS-PAGE gel and analyzed by Western blotting using an anti-p21Ras mAb.

 
TSP1 and peptides induce ERK phosphorylation

The ERK signaling pathway is a target of Ras/Raf and is important in mediating T cell activation. To determine whether TSP1 utilizes this signaling pathway, cell lysates were analyzed by Western blot for the presence of Y204-phosphorylated ERK1/2. ERK phosphorylation was significantly stimulated in the presence of TSP1, peptide 246, or peptide 7N3 in the absence of TCR stimulation (Fig. 6GoA). At the concentration used, anti-CD3 alone significantly increased ERK phosphorylation in some experiments, but the CD3 Ab strongly and reproducibly enhanced the phosphorylation induced by TSP1 or the TSP1 peptides 246 and 7N3. The enhancement of CD3-stimulated ERK phosphorylation was dose dependent and specific in that a control peptide lacking the essential Trp residues (peptide 388) was inactive (Fig. 6GoB). Stimulation of ERK phosphorylation by the CD47-binding peptide 7N3 was also confirmed to be specific using the control peptide FIRGGMYEGKK (results not shown).



View larger version (48K):
[in this window]
[in a new window]
  		FIGURE 6. Intact TSP1 and TSP1 peptides induce ERK phosphorylation. Lysates were prepared as described previously and Western blotted using a phospho-specific ERK Ab. A, Cells were incubated for 15 min with immobilized TSP1 (50 µg/ml), peptides 246 and 7N3 (100 µM) alone, or CD3 Ab (0.5 µg/ml). B, The indicated concentrations (µM) of peptides 246 or the control peptide 388 were added in solution to Jurkat cells on plates with immobilized CD3 Ab. ERK phosphorylation was detected in lysates prepared after 15 min. C, Peptide 246 was used to study the time course of ERK phosphorylation. Parallel cultures were incubated on dishes coated with anti-CD3 (+) or uncoated dishes (-). Peptide 246 was added to the cells in solution (20 µM), and lysates were prepared after 15 min, 1 h, 4 h, and 24 h. ERK phosphorylation was quantified as above.

 
The phosphorylation of ERK induced by peptide 246 was rapid, consistent with the kinetics of adhesion (Fig. 6GoC). In some experiments, increased phosphorylation could be detected within 5 min. However, the phosphorylation response was not sustained and decreased over time, both in the presence (results not shown) and absence of anti-CD3 Ab (Fig. 6GoC).

We examined the role of specific TSP1 receptors in transducing signals into the ERK pathway. Addition of the ß1 integrin-blocking Ab mAb13 (Fig. 7GoA) inhibited phosphorylation stimulated by TSP1 alone by 44% and the synergy observed between TSP1 and anti-CD3 by 15%, but mildly stimulated both control phosphorylation and that stimulated by anti-CD3 alone. Similarly, when cells were prepared with 40 mM chlorate, the synergism between peptide 246 and CD3-stimulated phosphorylation was inhibited (Fig. 7GoB). Thus, both integrin signaling initiated by TSP1 binding to a ß1 integrin and interaction of peptide 246 with proteoglycans on Jurkat T cells can activate the ERK pathway.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 7. ß1 integrin and sulfated proteoglycans are required for transducing signals from TSP1 and peptides into the ERK pathway. A, Control cells and Jurkat cells treated with a ß1 integrin function-blocking Ab (mAb13, 10 µg/ml) were incubated for 15 min with the indicated combinations of soluble TSP1 (50 µg/ml) and immobilized anti-CD3 Ab (0.5 µg/ml). Lysates were prepared and analyzed by Western blotting, and the level of ERK phosphorylation was quantified by densitometry. B, Jurkat cells were cultured in sulfate-free medium in the presence or absence of 40 mM chlorate. Lysates were produced from the cells after incubation for 15 min with the indicated combinations of peptide 246 in solution (20 µM) and immobilized anti-CD3 Ab (0.5 µg/ml). The lysates were analyzed for the level of phosphorylated ERK.

 
Differential role of G protein signaling in peptide 246 and 7N3 responses

Although responses of melanoma cells to the CD47-binding TSP1 peptide 7N3 are mediated by Gi{alpha} and inhibited by pertussis toxin (3, 44), adhesive and motility responses of the same cells to TSP1 peptide 246 are mostly pertussis toxin resistant (45, 46). The ERK phosphorylation responses induced by these TSP1 peptides in Jurkat cells likewise were differentially sensitive to pertussis toxin (Fig. 8Go). The response to peptide 7N3 was completely reversed by pertussis toxin, whereas the response to peptide 246 was only partially reduced.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 8. Differential sensitivity of the ERK responses stimulated by TSP1 heparin-binding and CD47-binding peptides to pertussis toxin. Jurkat T cells were incubated with 0.5 µg/ml pertussis toxin (striped bars) or control medium (solid bars) and then treated with 20 µM of the indicated peptides or buffer control. The lysates were analyzed for the level of phosphorylated ERK. Results show densitometric analysis of a representative experiment.

 
TSP1 and peptides induce phosphorylation of JNK and p38 kinase

To determine whether MAP kinase signaling stimulated by TSP1 was specific to the ERK pathway, cell lysates were analyzed by Western blotting for the presence of phosphorylated JNK (Fig. 9GoA) and p38 kinase (Fig. 9GoB) after incubation with TSP1 or peptide 246. Phosphorylation of both kinases was observed when TSP1 or peptide 246 was present along with anti-CD3 Ab. No signal was produced with anti-CD3 alone or TSP1 alone. The 246 peptide, however, produced a weak signal in the absence of anti-CD3 Ab. In the Jurkat cell lysates, the predominant form of phosphorylated JNK was the 46-kDa isoform; the lower m.w. band corresponds to p38 kinase. Blotting with Abs to detect the total JNK and p38 kinase in the cell lysates confirmed that the differences observed with the phospho-specific Abs reflect changes in levels of p38 phosphorylation.



View larger version (49K):
[in this window]
[in a new window]
  		FIGURE 9. TSP1 and TSP1 peptide 246 synergize with anti-CD3 to induce phosphorylation of JNK and p38 kinase. Jurkat T cells were incubated for 15 min with the indicated combinations of immobilized TSP1 (50 µg/ml), peptide 246 (100 µM), and CD3 Ab (0.5 µg/ml), and lysates were prepared in RIPA buffer containing protease inhibitors. A, The lysates were analyzed by Western blotting using anti-JNK (top lanes) or a phospho-specific JNK Ab (lower lanes). B, The lysates were analyzed by Western blotting using a phospho-specific p38 kinase Ab (top lanes) or anti-p38 (lower lanes). The positive control (+) sample on this blot was a lysate from anisomycin-treated C-6 glioma cells.

 
A heparin-binding peptide from TSP1 transduces signals to the nucleus via Elk-1- and AP-1-dependent transcriptional activation

Because the TSP1 peptides 246 and 7N3 induced phosphorylation of MAP kinases, we used Elk-1 and AP-1 reporter assays to determine whether these responses to TSP1 altered downstream signaling from the MAP kinase pathways. Treatment with the TSP1 peptide 246 increased both AP-1- and Elk-1-dependent transcription of CAT in Jurkat cells plated on anti-CD3 (Fig. 10Go, A and B). The CD47-binding peptide 7N3 induced a slight stimulation of both AP-1 and Elk-1 activity, whereas ligation of CD47 by an Ab induced only AP-1 activity (Fig. 10Go, A and B). The AP-1 response stimulated by TSP1 peptide 246 was mediated by a sulfated surface receptor on the Jurkat cells, because the stimulation of AP-1 activity by this peptide was reversed in the presence of heparin (Fig. 10GoC). Heparin treatment alone did not alter AP-1 activity, verifying the specificity of the inhibitor. The AP-1 response to the TSP1 peptide also required the Ras/MEK/ERK pathway, because treatment of the Jurkat cells with the MEK inhibitor PD98059 inhibited AP-1-dependent CAT expression (Fig. 10GoD). Therefore, induction of AP-1 in response to the TSP1 peptide 246 is mediated by binding to cell surface proteoglycans and requires signal transduction through the Ras/MEK/ERK MAP kinase pathway.



View larger version (20K):
[in this window]
[in a new window]
  		FIGURE 10. Induction of Elk1 and AP-1 activity in Jurkat cells by TSP1 peptides. A, Jurkat cells were cotransfected with 5 µg of a Gal4-Elk expression plasmid and an E1B-CAT construct by electroporation, as described in Materials and Methods. Transiently transfected cells were stimulated with either peptide 246 (20 µM) or 7N3 (20 µM) in the presence of precoated anti-human CD3 mAb (1 µg/well). Anti-CD47 Ab was also tested by coimmobilizing with the anti-CD3. CAT activity was measured 4 h after stimulation, as described in Materials and Methods. The relative CAT activity is presented as mean ± SD of duplicate samples. B, Jurkat cells were transfected with 5 µg of an AP1-CAT construct. Transfected cells were then stimulated with peptides 246 (20 µM) or 7N3 (20 µM) in the presence of precoated anti-human CD3 mAb (1 µg/well). CAT activity was measured after 4 h. C, AP-1 activity induced by TSP1 peptide 246 is inhibited by heparin. Jurkat cells were transiently transfected with AP1-CAT construct. Cells were then stimulated with peptide 246 (20 µM) in the presence of precoated anti-human CD3 mAb (1 µg/well) either with or without 100 µg/ml of heparin. The relative CAT activity is presented as mean ± SD for duplicates. D, Stimulation of AP-1-dependent CAT activity was measured in untreated Jurkat cells or cells pretreated with 10 µM of the MEK inhibitor PD98059.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have demonstrated that TSP1 and specific TSP1 peptides can interact with Jurkat T-lymphoma cells and modulate their behavior through binding to at least three receptors. Jurkat T cells rapidly but transiently adhere to immobilized TSP1 and to immobilized peptides from the type 1 repeats and C-terminal globular domain of TSP1. Adhesion to the intact protein appears to be mediated primarily by ß1 integrins, as the addition of a ß1 integrin-enhancing Ab increases the level of adhesion, and adhesion could be inhibited by a ß1 integrin-blocking Ab. The cell surface receptor CD47 mediates interactions with a TSP1 peptide from the C-terminal domain, and sulfated proteoglycans are receptors for a heparin-binding peptide from the type 1 repeats of TSP1. Binding soluble TSP1 or TSP1 peptides to each of these three receptors induces several signaling responses directly or by costimulating signaling from the TCR and results in altered transcriptional activity. Synergism of intact TSP1 with TCR signaling occurs at a point upstream of Ras and LAT, since TSP1 only activated these in the presence of anti-CD3. However, direct activation of MAP kinase phosphorylation by TSP1 and its peptides suggests that a Ras- and LAT-independent pathway may allow TSP1 and its peptides to directly stimulate MAP kinase signaling.

CD47 is present at a high density on peripheral T lymphocytes and can serve as a costimulator of T cell activation (24, 25). Ligation of CD47 by some Abs enhances the proliferation of T cells by costimulation of the CD3/TCR pathway (24). Because TSP1 is a known CD47 ligand, it was proposed to mediate costimulation through binding to this receptor (24). Although our data demonstrating that a CD47-binding peptide from TSP1 activates the MAP kinase pathway support this hypothesis, we also identified two additional TSP1 receptors on T cells through which TSP1 or TSP1 fragments may activate or costimulate the same signaling pathways. ß1 integrins play a major role in adhesion of peripheral T lymphocytes (26) and Jurkat T lymphoma cells on immobilized TSP1 and mediate activation of the ERK pathway by intact soluble TSP1. A heparin-binding peptide from TSP1 induces phosphorylation of three MAP kinase pathways and increases Elk1- and AP-1-dependent transcriptional activities. Binding to a sulfated cell surface receptor is required for the activation of ERK signaling, and the MEK-ERK pathway is required for induction of AP-1-dependent transcription by this peptide. Binding of TSP1 peptides to CD47 also directly activates the ERK pathway through a pertussis toxin-sensitive pathway, but this signal is less efficient than that induced by the heparin-binding peptide 246 for costimulating AP-1 activity. Thus, ß1 integrins and proteoglycans are also important signaling receptors for TSP1 in T cells. Further studies will be required to understand how T cells integrate these three signaling pathways activated by TSP1 and the physiological significance of these signals to lymphocyte functions.

We have identified cell surface proteoglycans as a potential receptor that modulates TCR/CD3-dependent AP-1 activity through a signal transduction pathway that requires the MEK/ERK1/2 cascade, which may be stimulated through Ras activation of Raf. TSP1 is a high affinity heparin-binding protein and contains several sites implicated in heparin binding (reviewed in Ref. 6). Most studies of signal transduction through cell surface proteoglycans have focused on the transmembrane syndecans (47), which are known to bind to TSP1 (48, 49). Clustering of syndecans following ligand binding activates protein kinase C pathways (50). Interactions between proteoglycans and Ras or MAP kinase signaling have not been observed previously, nor has their ligation been shown to alter Elk- or AP-1-dependent transcription. Based on the present data, signals arising from binding to T cell proteoglycans are a new pathway for modulating TCR signaling. It will be important to identify the T cell proteoglycans that mediate these signals and the pathways through which the TSP1 peptides induce phosphorylation of ERK, JNK, and p38 kinase.

TCR signaling may not be the only pathway that is modulated by TSP1 binding to T cells. Interaction of TSP1 peptides with heparan sulfate proteoglycans or CD47 can also stimulate signaling through integrin receptors in melanoma cells, endothelial cells, and platelets (3, 46, 51). Because TSP1 and the TSP1 peptides also activated MAP kinase signaling in the absence of CD3 stimulation, signals arising from TSP1 binding in the absence of TCR signaling may significantly modulate T cell responses to other stimuli. Signaling through the MAP kinase pathway in T cells can induce IL-2 expression, anergy, activation, proliferation, or apoptosis (52, 53). TSP1 is known to modulate proliferation of other cell types (4, 5, 45, 54) and to induce apoptosis of endothelial cells (55).

Physiologically, T cells could encounter TSP1 or fragments of TSP1 either in solution or immobilized in the extracellular matrix. If TSP1 was bound to the surface of an APC, its engagement of ß1 integrins, CD47, or proteoglycans on T cells could modulate TCR responses, as modeled in this study, using immobilized anti-CD3. At present, no data exist to demonstrate TSP1 on APCs, so a more plausible hypothesis for TSP1 function is as a soluble modulator released at sites of inflammation or bound to the extracellular matrix at these sites. Intact TSP1 and fragments of TSP1 produced by local proteolysis may transduce distinct signals to T cells by interacting with one or more of the TSP1 receptors identified in this work. Based on our evidence that TSP1 induces signals that can alter gene expression in Jurkat T cells, T lymphocytes must be considered as one of the targets of TSP1 when it is expressed or released during hemostasis, inflammatory disease, wound repair, and in cancer.


   Acknowledgments
 
We thank Drs. Ken Yamada, Henry Krutzsch, Silvio Gutkind, Larry Samelson, and Stephen Taylor for providing reagents.


   Footnotes
 
1 Current address: Dr. Wilson, Molecular Medicine Unit, St. James’s University Hospital Leeds, U.K. Back

2 Address correspondence and reprint requests to Dr. David D. Roberts, Building 10, Room 2A33, 10 Center Drive, MSC 1500, National Institutes of Health, Bethesda, MD 20892-1500. E-mail address: Back

3 Abbreviations used in this paper: TSP1, thrombospondin-1; CAT, chloramphenicol acetyltransferase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; LAT, linker for activation of T cells; MAP, mitogen-activated protein; MEK, MAP/ERK kinase; RBD, Ras-binding domain. Back

Received for publication May 7, 1999. Accepted for publication July 16, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Roberts, D. D., J. A. Sherwood, V. Ginsburg. 1987. Platelet thrombospondin mediates attachment and spreading of human melanoma cells. J. Cell Biol. 104:131.[Abstract/Free Full Text]
  2. Murphy-Ullrich, J. E., S. Gurusiddappa, W. A. Frazier, M. Höök. 1993. Heparin-binding peptides from thrombospondins 1 and 2 contain focal adhesion-labilizing activity. J. Biol. Chem. 268:26784.[Abstract/Free Full Text]
  3. Gao, A. G., F. P. Lindberg, J. M. Dimitry, E. J. Brown, W. A. Frazier. 1996. Thrombospondin modulates {alpha}vß3 function through integrin-associated protein. J. Cell Biol. 135:533.[Abstract/Free Full Text]
  4. Phan, S. H., R. G. Dillon, B. M. McGarry, V. M. Dixit. 1989. Stimulation of fibroblast proliferation by thrombospondin. Biochem. Biophys. Res. Commun. 163:56.[Medline]
  5. Majack, R. A., L. V. Goodman, V. M. Dixit. 1988. Cell surface thrombospondin is functionally essential for vascular smooth muscle cell proliferation. J. Cell Biol. 106:415.[Abstract/Free Full Text]
  6. Roberts, D. D.. 1996. Regulation of tumor growth and metastasis by thrombospondin-1. FASEB J. 10:1183.[Abstract]
  7. Bornstein, P.. 1992. Thrombospondins: structure and regulation of expression. FASEB J. 6:3290.[Abstract]
  8. Mosher, D. F.. 1990. Physiology of thrombospondin. Annu. Rev. Med. 41:85.[Medline]
  9. Schuepp, B. J., T. W. Jungi. 1991. Thrombospondin-exposed human monocytes display augmented luminol-enhanced chemoluminescence upon receptor triggering. Biochem. Biophys. Res. Commun. 177:1087.[Medline]
  10. Mansfield, P. J., S. J. Suchard. 1994. Thrombospondin promotes chemotaxis and haptotaxis of human peripheral blood monocytes. J. Immunol. 153:4219.[Abstract]
  11. Savill, J., N. Hogg, Y. Ren, C. Haslett. 1992. Thrombospondin cooperates with CD36 and the vitronectin receptor in macrophage recognition of neutrophils undergoing apoptosis. J. Clin. Invest. 90:1513.
  12. Pierson, B. A., K. Gupta, W.-S. Hu, J. S. Miller. 1996. Human natural killer cell expansion is regulated by thrombospondin-mediated activation of transforming growth factor ß-1 and independent accessory cell-derived contact and soluble factors. Blood 87:180.[Abstract/Free Full Text]
  13. Schultz-Cherry, S., J. E. Murphy-Ullrich. 1993. Thrombospondin causes activation of latent transforming growth factor-ß secreted by endothelial cells by a novel mechanism. J. Cell Biol. 122:923.[Abstract/Free Full Text]
  14. Crawford, S. E., V. Stellmach, J. E. Murphy-Ullrich, S. M. F. Ribeiro, J. Lawler, R. O. Hynes, G. P. Boivin, N. Bouck. 1998. Thrombospondin-1 is a major activator of TGF-ß1 in vivo. Cell 93:1159.[Medline]
  15. Lawler, J., M. Sunday, V. Thibert, M. Duquette, E. L. George, H. Rayburn, R. O. Hynes. 1998. Thrombospondin-1 is required for normal murine pulmonary homeostasis and its absence causes pneumonia. J. Clin. Invest. 101:982.[Medline]
  16. Bornstein, P.. 1995. Diversity of function is inherent in matricellular proteins: an appraisal of thrombospondin 1. J. Cell Biol. 130:503.[Free Full Text]
  17. Lahav, J. 1993. Thrombospondin. CRC Press, Boca Raton.
  18. Guo, N. H., H. C. Krutzsch, E. Nègre, T. Vogel, D. A. Blake, D. D. Roberts. 1992. Heparin- and sulfatide-binding peptides from the type I repeats of human thrombospondin promote melanoma cell adhesion. Proc. Natl. Acad. Sci. USA 89:3040.[Abstract/Free Full Text]
  19. Asch, A. S., S. Silbiger, E. Heimer, R. L. Nachman. 1992. Thrombospondin sequence motif (CSVTCG) is responsible for CD36 binding. Biochem. Biophys. Res. Commun. 182:1208.[Medline]
  20. Li, W. X., R. J. Howard, L. L. Leung. 1993. Identification of SVTCG in thrombospondin as the conformation-dependent, high affinity binding site for its receptor, CD36. J. Biol. Chem. 268:16179.[Abstract/Free Full Text]
  21. Panetti, T. S., B. J. Kudryk, D. F. Mosher. 1999. Interaction of recombinant procollagen and properdin modules of thrombospondin-1 with heparin and fibrinogen/fibrin. J. Biol. Chem. 274:430.[Abstract/Free Full Text]
  22. Lawler, J., R. Weinstein, R. O. Hynes. 1988. Cell attachment to thrombospondin: the role of ARG-GLY-ASP, calcium, and integrin receptors. J. Cell Biol. 107:2351.[Abstract/Free Full Text]
  23. Gao, A.-G., F. P. Lindberg, M. B. Finn, S. D. Blystone, E. J. Brown, W. A. Frazier. 1996. Integrin-associated protein is a receptor for the C-terminal domain of thrombospondin. J. Biol. Chem. 271:21.[Abstract/Free Full Text]
  24. Reinhold, M. I., F. P. Lindberg, G. J. Kersh, P. M. Allen, E. J. Brown. 1997. Costimulation of T cell activation by integrin-associated protein (CD47) is an adhesion-dependent, CD28-independent signaling pathway. J. Exp. Med. 185:1.[Abstract/Free Full Text]
  25. Ticchioni, M., M. Deckert, F. Mary, G. Bernard, E. J. Brown, A. Bernard. 1997. Integrin-associated protein (CD47) is a comitogenic molecule on CD3-activated human T cells. J. Immunol. 158:677.[Abstract]
  26. Yabkowitz, R., V. M. Dixit, N. Guo, D. D. Roberts, Y. Shimizu. 1993. Activated T-cell adhesion to thrombospondin is mediated by the {alpha}4ß1 (VLA-4) and {alpha}5ß1 (VLA-5) integrins. J. Immunol. 151:149.[Abstract]
  27. Ybarrondo, B., A. M. O’Rourke, J. B. McCarthy, M. F. Mescher. 1997. Cytotoxic T-lymphocyte interaction with fibronectin and vitronectin: activated adhesion and cosignaling. Immunology 91:186.[Medline]
  28. Sturmhofel, K., C. Brando, F. Martinon, E. M. Shevach, J. E. Coligan. 1995. Antigen-independent, integrin-mediated T cell activation. J. Immunol. 154:2104.[Abstract]
  29. Woodside, D. G., T. K. Teague, B. W. McIntyre. 1996. Specific inhibition of T lymphocyte coactivation by triggering integrin ß1 reveals convergence of ß1, ß2, and ß7 signaling pathways. J. Immunol. 157:700.[Abstract]
  30. Udagawa, T., D. G. Woodside, B. W. McIntyre. 1996. {alpha}4ß1 (CD49d/CD29) integrin costimulation of human T cells enhances transcription factor and cytokine induction in the absence of altered sensitivity to anti-CD3 stimulation. J. Immunol. 157:1965.[Abstract]
  31. Rosales, C., R. L. Juliano. 1995. Signal transduction by cell adhesion receptors in leukocytes. J. Leukocyte Biol. 57:189.[Abstract]
  32. Roberts, D. D., J. Cashel, N. Guo. 1994. Purification of thrombospondin from human platelets. J. Tissue Culture Methods 16:217.
  33. Akiyama, S. K., S. S. Yamada, W. T. Chen, K. M. Yamada. 1989. Analysis of fibronectin receptor function with monoclonal antibodies: roles in cell adhesion, migration, matrix assembly, and cytoskeletal organization. J. Cell Biol. 109:863.[Abstract/Free Full Text]
  34. Hemler, M. E., F. Sanchez-Madrid, T. J. Flotte, A. M. Krensky, S. J. Burakoff, A. K. Bhan, T. A. Springer, J. L. Strominger. 1984. Glycoproteins of 210,000 and 130,000 m.w. on activated T cells: cell distribution and antigenic relation to components on resting cells and T cell lines. J. Immunol. 132:3011.[Abstract]
  35. Zhang, W., J. Sloan-Lancaster, J. Kitchen, R. P. Trible, L. E. Samelson. 1998. LAT: the ZAP-70 tyrosine kinase substrate that links T cell receptor to cellular activation. Cell 92:83.[Medline]
  36. Gillis, S., J. Watson. 1980. Biochemical and biological characterization of lymphocyte regulatory molecules. V. Identification of an interleukin 2-producing human leukemia T cell line. J. Exp. Med. 152:1709.[Abstract/Free Full Text]
  37. Landgren, U.. 1984. Measurement of cell numbers by means of the endogenous enzyme hexosaminidase: applications to detection of lymphokines and cell surface antigens. J. Immunol. Methods 67:379.[Medline]
  38. Guo, N. H., H. C. Krutzsch, T. Vogel, D. D. Roberts. 1992. Interactions of a laminin-binding peptide from a 33-kDa protein related to the 67-kDa laminin receptor with laminin and melanoma cells are heparin-dependent. J. Biol. Chem. 267:17743.[Abstract/Free Full Text]
  39. Taylor, S. J., D. Shalloway. 1996. Cell cycle-dependent activation of Ras. Curr. Biol. 6:1621.[Medline]
  40. Butscher, W. G., C. Powers, M. Olive, C. Vinson, K. Gardner. 1998. Coordinate transactivation of the interleukin-2 CD28 response element by c-Rel and ATF-1/CREB2. J. Biol. Chem. 273:552.[Abstract/Free Full Text]
  41. Duncan, R., I. Collins, T. Tomonaga, T. Zhang, D. Levens. 1996. A unique transactivation sequence motif is found in the carboxyl-terminal domain of the single-strand-binding protein FBP. Mol. Cell. Biol. 16:2274.[Abstract]
  42. Gorman, C. 1985. DNA Cloning. D. M. Glover, ed. IRL Press, Oxford, p. 143.
  43. Dawson, D. W., S. F. Pearce, R. Zhong, R. L. Silverstein, W. A. Frazier, N. P. Bouck. 1997. CD36 mediates the in vitro inhibitory effects of thrombospondin-1 on endothelial cells. J. Cell Biol. 138:707.[Abstract/Free Full Text]
  44. Frazier, W. A., A.-G. Gao, J. Dimitry, J. Chung, E. J. Brown, F. P. Lindberg, M. E. Linder. 1999. The thrombospondin receptor integrin-associated protein (CD47) functionally couples to heterotrimeric Gi. J. Biol. Chem. 274:8554.[Abstract/Free Full Text]
  45. Guo, N., V. S. Zabrenetzky, L. Chandrasekaran, J. M. Sipes, J. Lawler, H. C. Krutzsch, D. D. Roberts. 1998. Differential roles of protein kinase C and pertussis toxin-sensitive G-binding proteins in modulation of melanoma cell proliferation and motility by thrombospondin-1. Cancer Res. 58:3154.[Abstract/Free Full Text]
  46. Sipes, J. M., H. C. Krutzsch, J. Lawler, and D. D. Roberts. 1999. Cooperation between thrombospondin-1 type 1 repeat peptides and integrin {alpha}vß3 ligands to promote melanoma cell spreading and focal adhesion formation. J. Biol. Chem. In press.
  47. Couchman, J. R., A. Woods. 1996. Syndecans, signaling, and cell adhesion. J. Cell. Biochem. 61:578.[Medline]
  48. Rapraeger, A.. 1989. Transforming growth factor (type ß) promotes the addition of chondroitin sulfate chains to the cell surface proteoglycan (syndecan) of mouse mammary epithelia. J. Cell Biol. 109:2509.[Abstract/Free Full Text]
  49. Lebakken, C. S., A. C. Rapraeger. 1996. Syndecan-1 mediates cell spreading in transfected human lymphoblastoid (Raji) cells. J. Cell Biol. 132:1209.[Abstract/Free Full Text]
  50. Oh, E. S., A. Woods, J. R. Couchman. 1997. Syndecan-4 proteoglycan regulates the distribution and activity of protein kinase C. J. Biol. Chem. 272:8133.[Abstract/Free Full Text]
  51. Chung, J., A. G. Gao, W. A. Frazier. 1997. Thrombospondin acts via integrin-associated protein to activate the platelet integrin {alpha}IIbß3. J. Biol. Chem. 272:14740.[Abstract/Free Full Text]
  52. Cantrell, D.. 1996. T cell antigen receptor signal transduction pathways. Annu. Rev. Immunol. 14:259.[Medline]
  53. Van den Brink, M. R., R. Kapeller, J. C. Pratt, J. H. Chang, S. J. Burakoff. 1999. The extracellular signal-regulated kinase pathway is required for activation-induced cell death of T cells. J. Biol. Chem. 274:11178.[Abstract/Free Full Text]
  54. Taraboletti, G., D. Roberts, L. A. Liotta, R. Giavazzi. 1990. Platelet thrombospondin modulates endothelial cell adhesion, motility, and growth: a potential angiogenesis regulatory factor. J. Cell Biol. 111:765.[Abstract/Free Full Text]
  55. Guo, N., H. C. Krutzsch, J. K. Inman, D. D. Roberts. 1997. Thrombospondin 1 and type I repeat peptides of thrombospondin 1 specifically induce apoptosis of endothelial cells. Cancer Res. 57:1735.[Abstract/Free Full Text]
  56. Tolsma, S. S., O. V. Volpert, D. J. Good, W. A. Frazier, P. J. Polverini, N. Bouck. 1993. Peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity. J. Cell Biol. 122:497.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 GlycobiologyHome page
E. Muroi, S. Manabe, M. Ikezaki, Y. Urata, S. Sato, T. Kondo, Y. Ito, and Y. Ihara
C-Mannosylated peptides derived from the thrombospondin type 1 repeat enhance lipopolysaccharide-induced signaling in macrophage-like RAW264.7 cells
Glycobiology, September 1, 2007; 17(9): 1015 - 1028.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Futagami, S. Sugita, J. Vega, K. Ishida, H. Takase, K. Maruyama, H. Aburatani, and M. Mochizuki
Role of Thrombospondin-1 in T Cell Response to Ocular Pigment Epithelial Cells
J. Immunol., June 1, 2007; 178(11): 6994 - 7005.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
J. S. Isenberg, L. A. Ridnour, J. Dimitry, W. A. Frazier, D. A. Wink, and D. D. Roberts
CD47 Is Necessary for Inhibition of Nitric Oxide-stimulated Vascular Cell Responses by Thrombospondin-1
J. Biol. Chem., September 8, 2006; 281(36): 26069 - 26080.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
T. Numakawa, T. Ishimoto, S. Suzuki, Y. Numakawa, N. Adachi, T. Matsumoto, D. Yokomaku, H. Koshimizu, K. E. Fujimori, R. Hashimoto, et al.
Neuronal Roles of the Integrin-associated Protein (IAP/CD47) in Developing Cortical Neurons
J. Biol. Chem., October 8, 2004; 279(41): 43245 - 43253.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
P. P. Manna and W. A. Frazier
The Mechanism of CD47-Dependent Killing of T Cells: Heterotrimeric Gi-Dependent Inhibition of Protein Kinase A
J. Immunol., April 1, 2003; 170(7): 3544 - 3553.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Sci.Home page
J. S. Lymn, M. K. Patel, G. F. Clunn, S. J. Rao, K. L. Gallagher, and A. D. Hughes
Thrombospondin-1 differentially induces chemotaxis and DNA synthesis of human venous smooth muscle cells at the receptor-binding level
J. Cell Sci., November 15, 2002; 115(22): 4353 - 4360.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. O. Barazi, Z. Li, J. A. Cashel, H. C. Krutzsch, D. S. Annis, D. F. Mosher, and D. D. Roberts
Regulation of Integrin Function by CD47 Ligands. DIFFERENTIAL EFFECTS ON alpha vbeta 3 AND alpha 4beta 1 INTEGRIN-MEDIATED ADHESION
J. Biol. Chem., November 1, 2002; 277(45): 42859 - 42866.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Biol.Home page
Z. Li, M. J. Calzada, J. M. Sipes, J. A. Cashel, H. C. Krutzsch, D. S. Annis, D. F. Mosher, and D. D. Roberts
Interactions of thrombospondins with {alpha}4{beta}1 integrin and CD47 differentially modulate T cell behavior
J. Cell Biol., April 29, 2002; 157(3): 509 - 519.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
Y. Zaffran, O. Destaing, A. Roux, S. Ory, T. Nheu, P. Jurdic, C. Rabourdin-Combe, and A. L. Astier
CD46/CD3 Costimulation Induces Morphological Changes of Human T Cells and Activation of Vav, Rac, and Extracellular Signal-Regulated Kinase Mitogen-Activated Protein Kinase
J. Immunol., December 15, 2001; 167(12): 6780 - 6785.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
D. Merlin, S. Sitaraman, X. Liu, K. Eastburn, J. Sun, T. Kucharzik, B. Lewis, and J. L. Madara
CD98-mediated Links between Amino Acid Transport and beta 1 Integrin Distribution in Polarized Columnar Epithelia
J. Biol. Chem.,