 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. Roberts 2 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  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 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. T hrombospondins are a family of matricellular proteins that have diverse effects on cell adhesion, motility, pro- liferation, and survival (1– 6). Thrombospondin-1 (TSP1), 3 the first identified member of this family, is highly ex- pressed during wound repair and inflammatory responses (re- viewed in Refs. 7 and 8). Expression of the THBS1 gene encoding TSP1 is induced by several growth factors, including platelet-de- rived growth factor and TGF-1. TSP1 is also a major component of platelet -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, mono- cytes (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- (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 iden- tical subunits (7, 16, 17). The diverse effects of TSP1 on cell be- havior have been associated with several functional sequences (re- viewed in Refs. 6 and 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 internal- ization 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  3 integrins (22). Peptide sequences within the C- terminal domain bind to the integrin-associated glycoprotein CD47 (23), which has been shown to modulate a V  3 integrin function (3) and T cell activation (24, 25). T lymphocytes can interact with TSP1 via both integrin-depen- dent and integrin-independent pathways. CD47, which is a recep- tor 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 estab- lished that TSP1 binding to CD47 can elicit such a costimulatory signal.  1 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  4  1 and  5  1 integrins.  1 integrin interactions with other extra- cellular matrix ligands can modulate the recruitment and activation of T lymphocytes (27–30; reviewed in Ref. 31), suggesting that Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Received for publication May 7, 1999. Accepted for publication July 16, 1999. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Current address: Dr. Wilson, Molecular Medicine Unit, St. James’s University Hos- pital Leeds, U.K. 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: droberts@helix.nih.gov 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. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00