Thrombospondin-1

Vol. 29, No. 6, pp. 861-865, 1997 {3 1997 Elsevier ScienceLtd. All rights reserved Printed in Great Britain

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IN FOCUS

Thrombospondin-1 JOSEPHINE

C. ADAMS

MRC-Laboratory fbr Molecular Cell Biology and Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WClE 6BT, U.K. Thromhospondin-1 is a glycoprotein that is released from platelet a-granules in response to thromhin stimulation and that is also a transient component of extracelhdar matrix in developing and repairing tissues. It is a 420 kDa homotrimer, each subunit of which consists of multiple structural domains. A variety of factors regulate thrombospondin-1 expression and the protein is degraded by both extracellular and intracellular routes. Thrombospondin-1 functions as a cell adhesion molecule and also modulates cell movement, cell proliferation, neurite outgrowth and angiogenesis. The molecular mechanisms underlying these activities are beginning to be examined. Medical interest in thrombospondin-1 centres on its roles in haemostasis and its effects on angiogenesis. 0 1997 Elsevier Science Ltd Int. J. Biochem. Cell Biol. (1997) 29, 861-865

gene family. The original thrombospondin is now termed thrombospondin-1 (TSP-I), to Thrombospondin-1 (TSP-1) is a glycoprotein signify that it was the first to be discovered, and first discovered in the early 1970s as a protein the other family members are termed TSP-2, associated with the surfaces of thombin- TSP-3, TSP-4 and COMP/TSP-5. TSP-1 and stimulated platelets (Baenziger et al., 1971). TSP-2 are trimeric, whereas TSP-3, TSP-4 and This protein was isolated in the presence of COMP are pentameric. These proteins are reducing agent as a 190 kDa monomer and was highly conserved across vertebrates, but to date designated ‘thrombin-sensitive protein’. Sub- have not been identified in invertebrates. sequently, it became apparent that the protein was stored intracellularly in platelet a-granules STRUCTURE OF THROMBOSPONDIN-1 and was releasedupon platelet activation. In the late 1970s a method by which the protein could The TSP-1 molecule is a homotrimer. Each be isolated under non-reducing conditions was subunit comprises a 1152 amino acid residue devised by Lawler and co-workers: this revealed polypeptide, post-translationally modified by that the intact protein was a 420 kDa trimer. N-linked glycosylation and fi-hydroxylation of The name thrombospondin was proposed, to asparagine residues. Under the transmission indicate that the protein was released in electron m icroscope (TEM), each subunit of the responseto thrombin (Lawler et al., 1978). The trimer is seen to form amino- and carboxylcDNA encoding this protein was cloned in 1986 terminal globular domains connected by long, (Lawler and Hynes, 1986). In the period thin, flexible arms. In primary sequence, the between 1991 and 1994,three proteins identified amino- and carboxyl-domains consist of unique by molecular cloning and a protein purified sequences,which are unrelated to other proteins from articular cartilage, cartilage oligomeric in the databases,and the rod-like arms contain matrix protein (COMP), were found to have a short linear region containing the two cysteine overall structural and sequencehomology with residues, which are involved in trimer forthrombospondin, indicating the existence of a mation; a region with homology to procollagen, and three types of repeated domains termed the Received 15 April 1996; accepted 2 December 1996. type 1, type 2 and type 3 repeats. INTRODUCTION

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Josephtne

The three type 1 repeats are each framed around six cysteine residues and have homology to repeated domains present in complement components such as properdin, the proteins UNC-5 and F-spondin, which are involved in axon guidance, and the coat protein of malarial circumsporozoites. This category of repeat is referred to either as a ‘thrombospondin repeat’ or a ‘properdin repeat’. The three type 2 repeats are similar to epidermal growth factor, and thus have homology to many cell-surface and secreted proteins of vertebrates and invertebrates. The seven type 3 repeats are rich in aspartic acid residues and each repeat contains one or two sequences that are similar to the E/F hand, calcium-binding motifs of calmodulin. The calcium-binding properties of TSP- 1 were appreciated from the time that it was first purified, since (a) the length of the arms appeared shorter in TSP-1 molecules prepared for TEM in the absence of calcium; and (b) limited proteolysis of calcium-replete or EGTAtreated TSP-1 gave rise to different sets of proteolytic fragments. Recent direct binding measurements have shown that each TSP-1 molecule binds on average 35 calcium ions (reviewed in Adams et al., 1995). As described below, the binding of calcium plays an important role in the functional activities of TSP- 1. The thrombospondin-1 gene is located on human chromosome 15ql5 and mouse chromosome 2 band F. The human and mouse TSP-1 genes are both about 20 kb in size and in both species the protein is encoded in 22 exons. The promoter regions contains binding sites for many transcription factors including serum response factor. Sites for endonuclease cleavage are present in the 3’ untranslated region of the mRNA, which may contribute to rapid turnover. A single mRNA transcript of about 6 kb is detected in human and mouse tissues and cell lines: putative alternatively spliced transcripts encoding the amino-terminal portion of the protein have been isolated by PCR from some transformed cell lines (Adams et al., 1995). SYNTHESIS

AND DEGRADATION

The abundance and distribution of the TSP-1 transcript has been examined in mouse embryos, where it displays a temporally and spatially restricted expression pattern in many tissues, presumably as a consequence of the

C’. Adams

regulated expression and activity of the necessary transcription factors (Iruela-Arispe cl/ 111.. 1993). TSP-1 is synthesised by many cell types i/z u’tro, probably because of the presence of a serum-response element in the promoter. However, many factors alter the level of TSP-I mRNA expression (Adams ct (I/.. 1995; Bornstein, 1992, 1995). In serum-starved cell cultures, TSP-1 mRNA levels are low but are upregulated rapidly in response to serum or PDGF treatment. TSP-1 thus falls into the category of ‘immediate early’ response genes. TSP-1 expression decreases as cell density increases and is upregulated by stress conditions such as heat shock or hypoxia. TSP-1 mRNA and protein levels are upregulated by polypeptide growth factors including PDGF, TGF-P, and bFGF and are also regulated by the level of expression of the ~53 tumour supressor gene product (Dameron eI ill., 1994). TSP-1 protein levels are down-regulated by IL-lb or TNFcr: this does not involve an alteration in mRNA levels, suggesting that post-translational processing is in some way altered. Finally, transcription of the TSP-1 gene is downregulated in cells transformed by cyf&s,c,jun or LJ-.SK(Mettouchi et al., 1994). Taken together, these observations suggest various mechanisms by which the production of TSP-1 protein could be acutely regulated in rice so that protein synthesis is concentrated at appropriate sites in the context of normal developmental or tissue cell rearrangements, wound healing or angiogenesis. For example, TSP-1 is upregulated by progesterone in the human endometrium during a phase associated with cessation of capillary growth (Iruela-Arispe, 1996). Inappropriate regulation of TSP-1 expression may contribute to the high levels of protein observed in association with malignant carcinomas of the breast (Tuszynski and Nicosia. 1996). TSP-I can be degraded both extracellularly or intracellularly. In the cardiovascular system. TSP-1 released from platelets and incorporated into the fibrin clot is a substrate for thrombin and Factor XIIIa. In tissues and in cell culture systems, TSP-1 is detected as small, granular patches within the matrix or in association with cell surfaces. The appearance of these patches is quite distinct from the network of fibrils formed by fibronectin, collagens or elastin microfibrils (Vischer et al., 1988). If iodinated TSP-I is added to growing cultures of fibroblasts. endothelial cells or smooth muscle cells, a portion is retained in the matrix but most of the

Thrombospondin-1

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LDL receptorrelated wotein

Fig. 1. The interactions of thrombospondin- 1 with cell surfaces.The structural features of a single subunit of the TSP-I trimer are indicated. Peptide sequencesinvolved in cell adhesion are indicated below the appropriate domain. The adhesive motifs in the heparin-binding domain are also responsible for focal contact disassembly and the type 1 repeat peptides also have anti-angiogenic activity. For each adhesive domain, several types of cell-surface binding proteins have been identified on various cell types and these are shown below the appropriate domain. LDL receptor-related protein mediates endocytosis of TSP-I in smooth muscle cells. The proteoglycan syndecan-1 binds to the heparin-binding domain; the proteoglycans which are thought to bind to the other domains have not yet been identified at the molecular level. CD36 binds to a CSVTCG-peptide affinity matrix, as does the 50 kDa protein. CD47/IAP binds to both carboxy-terminal domain peptides. Integrin a,jI, binds to the RGD site in a conformation-dependent manner. Little is known about the intracellular events triggered by these interactions, but individual adhesive domains are known to function in a co-operative manner to bring about the many activities of TSP-1.

protein is endocytosed and released within 30 m in after degradation in lysosomes. This process is mediated by binding of the aminoterminal domain of TSP-1 to proteoglycans and the low density lipoprotein receptor-related protein (Adams et al., 1995; see Fig. 1).

binds and activates latent TGFfl, it may have additional indirect effects on the activities of immune cells. TGFj binding involves the peptide motif RFK located betweenthe first and second type 1 repeats (Schultz-Cherry et al., 1995). The function of TSP-1 in tissue matrix is less well understood, but in vitro assays using cell BIOLOGICAL Ii-UNCTION types derived from the cardiovascular system, TSP-1 released by activated platelets partici- solid tissues or various types of tumours have pates in the formation and resolution of the indicated that TSP-1 may have a generic role in fibrin clot, by binding to fibrin, plasminogen, the regulation of cell adhesive, motile and urokinase and histidine-rich glycoprotein. proliferative behaviour (reviewed in Adams TSP-1 also participates in the formation of et al., 1995; Bornstein, 1992).These assayshave molecular bridges between platelets and leuco- examined the responses of cells to substrata cytes recruited as part of an inflammatory coated with TSP-1 or the effects of adding response. These bridges may involve inter- soluble TSP-1 to pre-adherent cells. The actions between TSP- 1 and cell-surface proteins mechanismsinvolved are currently under active such as CD36 and the avp3 integrin or with investigation and appear to differ in various fibrinogen bound to the platelet-specific aIIb/fi3 ways from integrin-mediated adhesion and integrin. Another bridging role of TSP-1 is in signalling as exemplified by cellular interactions the recognition of apoptotic neutrophils by with fibronectin. The current picture is summarmacrophages (Savill et al., 1993). Since TSP-1 ised in Fig. 1.

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Jo,ephme C Adam\

Cell adhesion to TSP-I requires concurrent interactions with multiple domains of the molecule and the presence of calcium ions in the type 3 repeats is critical for adhesive activity. The different TSP-1 adhesive domains are recognised by a diversity of cell-surface binding molecules including proteoglycans, CD36 glycolipids and in some cases the a,p; integrin. Neurite outgrowth on TSP-1 is mediated by integrin a,P, (DeFreitas et ul., 1995). Cellular interactions with the FY VVMWK and IRVVM peptide motifs in the carboxyl-terminal domain involve CD47, the integrin-associated protein (1AP; Gao et al., 1996). It is attractive to speculate that through these multiple interactions, TSP-1 activates a series of intracellular signalling events, which can ‘cross-talk’ with integrin or growth factor signalling pathways and so subtly modify cell behaviour. However. little is known about the nature of these intracellular events. Interactions with the type 3 repeats and carboxyl-terminal domain may affect intracellular calcium levels (Tsao and Mousa, 1995). Other responses involve the actin cytoskeleton. Thus, soluble TSP- 1 causes disassembly of focal contacts and perinuclear microfilament bundles. Cells adherent on platelet TSP-1 respond by organising substratum adhesion contacts composed of large arrays of cortical microspikes, which contain the actin-bundling protein, fascin (Adams, 1995: also reviewed in Adams et ul., 1995). POSSIBLE

MEDICAL

APPLICATIONS

Interest in exploiting the biology of TSP- 1 for medical purposes has focused principally on the role of TSP-1 in the cardiovascular system, where its multiple activities may have potential for therapeutic intervention. These include the role of TSP-1 in haemostasis, specifically its interactions with platelets and its roles in fibrinolysis. There is growing evidence that TSP-1 may function as a natural modulator of angiogenesis, its activities being context- and concentration-dependent, so leading to positive or negative regulation of angiogenesis. This has raised interest in the possibility that TSP-I or appropriate mimetics could be used to regulate angiogenesis in acute wound healing situations or to counteract tumour growth and metastasis (Tuszynski and Nicosia, 1996). Further studies on the expression of TSP-1 by tumours will show if its overexpression has any diagnostic or prognostic value. The recent appreciation that

TSP-I and other thrombospondin family members are present in cartilage and bone and that the COMP gene is mutated in human pseudoachondroplasia (reviewed in Adams ~‘1al., 1995) is likely to stimulate much research into the roles of thrombospondins in these tissues, again with the aim of uncovering diagnostic or therapeutic applications. Ackno~k[,~/~c,rnt,/~~,s I thank Jack Lawler for many stimulating conversations. 1 gratefully acknowledge the financial support of the Wellcome Trust (grants number 038284 and 046105).

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Thrombospondin-1 Savill J., Fadok V. and Henson P. (1993) Phagocytic recognition of cells undergoing apoptosis. Immunol. Today 14, 131-135. Schultz-Cherry S., Chen H., Mosher D., Misenheimer T. M., Krutzsch H. C., Roberts D. D. and Murphy-Ullrich J. E. (1995) Regulation of transforming growth factor-b by discrete sequences of thrombospondin-1. J. Biol. Chem. 270, 73047310. Tsao P. W. and Mousa S. A. (1995) Thrombospondin mediates calcium mobilisation in fibroblasts via its

ArggGly-Asp

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Tuszynski G. P. and Nicosia R. F. (1996) The role of thrombospondin-I in tumor progression and angiogenesis. Bioessuys 18, 71-76. Vischer P., Volker W., Schmidt A. and Sinclair N. (1988) Association of thrombospondin of endothelial cells with other matrix proteins and cell attachment sites and migration tracks. Eur. J. Cell Biol. 47, 36 46.