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Thrombospondin-1 Peptides Signaling by Thrombospondin-1

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Thrombospondin-1 Peptides Signaling by Thrombospondin-1 b 1 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. Wilson,1 Zhuqing Li, Murat Kara, Kevin L. Gardner, and David D. Roberts2 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 b of Jurkat T cells on substrates coated with TSP1 or TSP1-derived peptides is mediated by 1 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 b on a 1 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. The Journal of Immunology, 1999, 163: 3621–3628. hrombospondins are a family of matricellular proteins expression may modulate local immune responses. Although the that have diverse effects on cell adhesion, motility, pro- tumor-suppressive activity of TSP1 has been primarily associated liferation, and survival (1–6). Thrombospondin-1 with its anti-angiogenic activity, immune modulation may also T 3 (TSP1), the first identified member of this family, is highly ex- contribute to the effects of TSP1 expression on tumor growth in pressed during wound repair and inflammatory responses (re- several animal models (reviewed in Ref. 6). viewed in Refs. 7 and 8). Expression of the THBS1 gene encoding TSP1 is a large multifunctional protein composed of three iden- TSP1 is induced by several growth factors, including platelet-de- tical subunits (7, 16, 17). The diverse effects of TSP1 on cell be- b rived growth factor and TGF- 1. TSP1 is also a major component havior have been associated with several functional sequences (re- a of platelet -granules and is released following platelet activation viewed in Refs. 6 and 7). The amino-terminal domain contains a at sites of injury. Elevated TSP1 levels at these sites can alter high affinity heparin-binding site and a binding site for the low functions of several cell types including endothelial cells, mono- density lipoprotein receptor-related protein that mediates internal- cytes (9, 10), macrophages (11), and NK cells (12). In addition to ization of TSP1 in some cells. The central stalk region of TSP1 its direct actions through binding to TSP1 receptors on target cells, contains anti-angiogenic sequences, sequences that bind to heparin TSP1 can alter cell behavior through activating latent TGF-b (13, (18), CD36 (19, 20), and fibrinogen (21), and an RGD sequence 14). These in vitro activities of TSP1 combined with the observed recognized by b integrins (22). Peptide sequences within the C- inflammatory disease in thbs1-null mice (15) suggested that TSP1 3 terminal domain bind to the integrin-associated glycoprotein CD47 b (23), which has been shown to modulate aV 3 integrin function (3) and T cell activation (24, 25). Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 T lymphocytes can interact with TSP1 via both integrin-depen- Received for publication May 7, 1999. Accepted for publication July 16, 1999. dent and integrin-independent pathways. CD47, which is a recep- The costs of publication of this article were defrayed in part by the payment of page tor for the C-terminal cell-binding domain of TSP1 (23), provides charges. This article must therefore be hereby marked advertisement in accordance costimulatory signals to T lymphocytes, probably via an integrin- with 18 U.S.C. Section 1734 solely to indicate this fact. independent pathway (24, 25). However, it has not been estab- 1 Current address: Dr. Wilson, Molecular Medicine Unit, St. James’s University Hos- lished that TSP1 binding to CD47 can elicit such a costimulatory pital Leeds, U.K. b 2 signal. 1 integrins mediate adhesion of peripheral T lymphocytes Address correspondence and reprint requests to Dr. David D. Roberts, Building 10, 1 Room 2A33, 10 Center Drive, MSC 1500, National Institutes of Health, Bethesda, on TSP1 (26). Activation-dependent adhesion of peripheral CD4 MD 20892-1500. E-mail address: [email protected] T cells to TSP1 was inhibited by function-blocking Abs to the 3 Abbreviations used in this paper: TSP1, thrombospondin-1; CAT, chloramphenicol a b a b b 4 1 and 5 1 integrins. 1 integrin interactions with other extra- acetyltransferase; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; LAT, linker for activation of T cells; MAP, mitogen-activated protein; MEK, cellular matrix ligands can modulate the recruitment and activation MAP/ERK kinase; RBD, Ras-binding domain. of T lymphocytes (27–30; reviewed in Ref. 31), suggesting that Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 3622 T CELLS USE MULTIPLE THROMBOSPONDIN RECEPTORS Table I. TSP1 peptides and analogs Peptide Amino Acid Sequence Activities 246 KRFKQDGGWSHWSPWSS Binds heparin, promotes cell adhesion, inhibits cell proliferation & angiogenesis (18, 45) 388 KRFKQDGGASHASPASS Inactive control for 246 7N3 FIRVVMYEGKK CD47 ligand; activates integrins (23) 604 FIRGGMYEGKK Inactive control for 7N3 Hep-I ELTGAARKGSRRLVKGPD Disrupts focal adhesions (2) Mal II SPWSSCSVTCGDGVITRIR CD36 ligand; inhibits angiogenesis (43, 56) 500 NGVQYRNC-am CD36 ligand; inhibits angiogenesis (43, 56) b TSP1 binding to 1 integrins on T cells may also have functional To investigate the role of sulfated glycoconjugates in adhesion, cells consequences. were grown in the presence of chlorate to inhibit sulfation (38). Cells for Although we have previously demonstrated that TSP1 can dif- these experiments were grown in Hams-F12 medium containing 10% di- alyzed FCS, 20 mM HEPES, and 2 mM glutamine for 48 h before being ferentially promote adhesion of resting and activated T lympho- transferred to the same media containing 2% dialyzed FCS with or without cytes and of naive and memory T cells (26), the effects of TSP1 on sodium chlorate for 24 h. To perform the adhesion assay, the cells were function of T cells have not been examined. To begin to define the resuspended in Hams-F12 medium containing 0.1% BSA and the relevant molecular responses of T cells to TSP1, we have examined the concentration of chlorate, and adhesion assays were performed as above. adhesive and signaling responses of T cells to intact TSP1 and Western blot analysis several defined functional sequences from TSP1. We demonstrate b Bacteriological polystyrene 35-mm petri dishes (Falcon 1008) were coated in this study that several cell surface receptors, including 1 inte- with the described combinations of Abs and TSP1/peptides overnight at grins, CD47, and heparan sulfate proteoglycans, can transduce sig- 4°C. Cells were added to the plates (5 3 105 cells in 0.1% BSA in RPMI) nals following interaction with TSP1 or specific TSP1 peptides. with soluble peptides or TSP1, where applicable. Cells were incubated at Binding to these receptors modulates the activation of p21 Ras and 37°C for 5 min to 4 h, the suspended cells were centrifuged for 5 min at 3 m phosphorylation of several MAP kinase pathways, and induces 600 g, and 50 l 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) so- AP-1-dependent transcription activity. dium deoxycholate, 1 mM EGTA, 1 mM Na3VO4, and protease inhibitors at 10 mg/ml antipain, pepstatin A, chymostatin, leupeptin, soybean trypsin Materials and Methods inhibitor, aprotinin, and 1 mM phenylmethane sulfonyl fluoride) was added Proteins and peptides 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 3 Human TSP1 was isolated from the supernatant of thrombin-stimulated g for 30 min at 4°C. The proteins were separated by SDS-PAGE on a human platelets by gelatin and heparin affinity, followed by gel filtration 4–15% gradient polyacrylamide gel and transferred to nitrocellulose mem- chromatography (32). Purified TSP1 was stored in aliquots at 270°C. Syn- brane by semidry electroblotting. thetic peptides derived from several functional domains of TSP1 were pre- Tyrosine-phosphorylated proteins were detected using the HRP-conju- pared as previously described (2, 18, 23) and are summarized in Table I. gated anti-phosphotyrosine Ab RC20 (Transduction Laboratories, Lexing- ton, KY). The phosphorylated forms of the MAP kinases ERK, stress- Antibodies activated protein/JNK, and p38 were identified using phospho-specific mAbs (New England Biolabs, Beverly, MA), followed by HRP-conjugated Anti-human CD3 mAb (PharMingen, San Diego, CA; clone HIT3a) was anti-rabbit Ab. Proteins were visualized by chemoluminescent detection.
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