T HROMBOSPONDINS : Multifunctional Regulators of Cell Interactions

24 Sep 2001 11:3 AR AR139-02.tex AR139-02.SGM ARv2(2001/05/10)P1: GPQ

Annu. Rev. Cell Dev. Biol. 2001. 17:25–51Copyright c© 2001 by Annual Reviews. All rights reserved

THROMBOSPONDINS: MultifunctionalRegulators of Cell Interactions

Josephine C. AdamsMRC Laboratory for Molecular Cell Biology and Department of Biochemistry andMolecular Biology, University College London, Gower Street, London WC1E 6BT,United Kingdom; e-mail: [email protected]

Key Words extracellular matrix, adhesion, cytoskeleton, signaling, angiogenesis

■ Abstract Thrombospondins are secreted, multidomain macromolecules that actas regulators of cell interactions in vertebrates. Gene knockout mice constructed fortwo members of this family demonstrate roles in the organization and homeostasisof multiple tissues, with particularly significant activities in the regulation of angio-genesis. This review discusses the functions of thrombospondins with regard to theircellular mechanisms of action and highlights recent advances in understanding howmultifactorial molecular interactions, at the cell surface and within extracellular ma-trix, produce cell-type-specific effects on cell behavior and the organization of matrixand tissues.

CONTENTS

AN INTRODUCTION TO THROMBOSPONDINS. . . . . . . . . . . . . . . . . . . . . . . . . . 26Structural Organization of Thrombospondins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Major Sites of Expression. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27Molecular Connections. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29Special Properties of Pentameric Thrombospondins. . . . . . . . . . . . . . . . . . . . . . . . . 32

CONTEXT-SPECIFIC ROLES OF THROMBOSPONDINSIN VIVO: THE EVIDENCE FROM GENE KNOCKOUTMICE AND HUMAN GENETIC DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Activation of TGF-β1 by TSP-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33TSP-2 and Connective Tissue Organization. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34TSP-1, TSP-2 and Angiogenesis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35The Role of COMP in Cartilage Biology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

CELLULAR MECHANISMS OF ACTION OFTHROMBOSPONDINS: THE EVIDENCE FROMTISSUE CULTURE ASSAYS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Cell Adhesion and Motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Regulation of Cytoskeleton and Cell Shape. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Regulation of Proliferation and Apoptosis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

1081-0706/01/1115-0025$14.00 25

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26 ADAMS

PERSPECTIVE AND CONCLUSIONS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41Emergent Paradigm: Thrombospondinsas Multifunctional Regulators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Paradoxes and Open Questions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

AN INTRODUCTION TO THROMBOSPONDINS

The thrombospondins are a gene family of five members in vertebrates. All throm-bospondins are multimeric, multidomain glycoproteins that function at cellsurfaces and in the extracellular matrix milieu. They have been termed adhesion-modulating or matricellular components of the extracellular matrix. Previousreviews have focused on the properties and interactions of thrombospondin-1(TSP-1) and, more recently, on the highly related TSP-2 (Adams et al. 1995,Bornstein 1995, Bornstein et al. 2000, Lawler 2000). Here, I present an integratedappraisal and perspective of the biological roles and molecular mechanisms ofaction of the whole thrombospondin family.

Structural Organization of Thrombospondins

The thrombospondin gene family is divided into two subfamilies, A and B, accord-ing to their overall molecular organization (Figure 1). The subgroup A proteins,TSP-1 and -2 , are assembled as trimers (Lawler & Hynes 1986, Bornstein et al.1991, LaBell et al. 1992, Laherty et al. 1992). The subgroup B thrombospondins,designated TSP-3, -4, and COMP (cartilage oligomeric matrix protein, also desig-nated TSP-5), are distinct in that they contain unique N-terminal regions, lack theprocollagen homology domain and type 1 repeats, contain four copies of the type2 repeat, and are assembled as pentamers (Oldberg et al. 1992, Vos et al. 1992,Bornstein et al. 1993, Lawler et al. 1993, Qabar et al. 1995) (Figure 1).

The molecular architecture of type 2 and type 3 repeats and of the C-terminaldomain is thus the hallmark of a thrombospondin (Figure 1). Indeed, the primarysequences of thrombospondins are most closely related in the type 3 repeats and Cterminus: TSP-1 and TSP-2 have 82 % identity in this region and subgroups A andB thrombospondins show a mean sequence identity of 60%. The type 1 repeats arepresent in a wide range of evolutionarily and structurally diverse proteins, termedthe thrombospondin type 1 repeat, or TSR, superfamily (reviewed by Adams &Tucker 2000), and thus appear a late addition by exon shuffling in subgroup A. In-deed, a single prototypic thrombospondin is present in theDrosophilagenome thatmost closely resembles TSP-4 (predicted product CG11326). Protein analysis isnow needed to determine whether this ancestral molecule is pentameric or trimeric.

Transmission electron microscope (TEM) views of thrombospondins show thatthe globular N- and C-terminal domains of each subunit are connected by a thin,highly flexible stalk region (see for example, Lawler et al. 1985, 1995; Morgelinet al. 1992; Qabar et al. 1995; H Chen et al. 2000). In addition, the physical struc-ture is altered in the absence of calcium ions: The stalk region elongates and theC-terminal globule decreases in diameter (Lawler et al. 1985). Information on the

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THROMBOSPONDINS 27

three-dimensional structures of the domains and sequence repeat units is patchy.Circular dichroism studies of monomeric N-terminal globular domain indicateβ-sheet content (Misenheimer et al. 2000). Residues 271–293 in the TSP-1 oligomer-ization sequence are predicted to form a coiled-coil, as does the pentamerizationsequence of COMP (Misenheimer et al. 2000; see below). The procollagen regionof TSP-1, which is also involved in stable trimer assembly, folds into a compact,stable, disulfide-bonded, monomeric structure (Misenheimer et al. 2000). X-raysolution scattering studies of the type 1 repeats of properdin, a component of thecomplement cascade consisting of six thrombospondin type 1 repeats, indicate a4-nm, elongated, ellipsoid shape for each repeat (Smith et al. 1991). NMR stud-ies of HB-GAM (heparin-binding growth-associated molecule), a small secretedprotein that contains two distantly related type 1 repeats, show each repeat to bemade up of three antiparallelβ-sheets (Kilpelainen et al. 2000). The type 2 repeatsare EGF-like and, based on structural measurements of other EGF domains, arethought to each form a small globular domain of about 3 nm diameter (for examplesee Baron et al. 1992). Expression of the type 3 repeats of COMP as a single unitshowed that the polypepetide forms rods of mean length 14.2 nm (Maddox et al.2000). Direct physical characterizations of the isolated C-terminal domain havenot been published, but from the dimensions of TSP-1 molecule under TEM, itappears that the type 3 repeats likely fold together with the C-terminal domainwhen calcium-replete to form the C-terminal globule (Lawler et al. 1985). Thusthe type 3 repeats and C terminus should be viewed as a structural cassette ratherthan as discrete entities.

The subunits of thrombospondins are assembled post-translationally into oli-gomers by interchain disulfide bonds formed between two closely positionedcysteines within the oligomerization sequence and by interactions between theoligomerization sequences that lead to the formation of three or five-strandedα-helical coiled-coil bundles (Sottile et al. 1991, Efimov et al. 1994, Meisenheimeret al. 2000). Within each subunit, the type 3 repeats are highly disulfide-bonded(Lawler et al. 1985, Speziale & Detwiler 1990). The subunits are also modified byaddition of N- and O-linked sugars (Furokawa et al. 1989, Hofsteenge et al. 2000).The motif WXXW in each type 1 repeat is a recognition site for C-mannosylation ofthe first tryptophan, and these modifications are made with different stoichiometriesin different cell types. Serine or threonine residues in adjacent CXXS/TCG motifscarry a novel O-linked disaccharide, glucose-fucose-O (Hofsteenge et al. 2000).The functional significance of these unusual modifications is not known; how-ever, O-linked fucose tetrasaccharides have been characterized on the EGF repeatsof certain proteins. In the case ofDrosophila Notch, synthesis of the tetrasac-charide through the action of Fringe modulates the interaction of Notch withDelta and Serrate and thereby alters intercellular signaling pathways (Moloneyet al. 2000).

Major Sites of Expression

Each thrombospondin has a distinct pattern of expression in developing tis-sues and thoroughout life (Table 1). Many developing tissues express multiple

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28 ADAMST

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THROMBOSPONDINS 29

thrombospondins; however, within a given tissue, individual family members areoften expressed by non-overlapping cell populations (for example see Tucker et al.1995). Under pathophysiological conditions, major changes occur in the expres-sion of thrombospondins in adult organisms (Table 1). In particular, TSP-1 is re-leased from plateletα-granules; is upregulated in healing wounds in skin, muscle,and the central nervous system through expression by macrophages or microglia,fibroblasts, and endothelial cells; and is present in atherosclerotic lesions, tumor-associated stroma, and rheumatoid synovium. TSP-2 expression is increased infibroblasts within skin wound granulation tissue (Kyriakides et al. 1999b). Tran-script array analysis has documented 15- and 23-fold upregulations of TSP-4in adult human muscle from Duchenne muscular dystrophy andα-sarcoglycan-deficiency patients, respectively (Y-W Chen et al. 2000). Increased levels of COMPand COMP fragments in serum and synovial fluid correlate with osteoarthritis,rheumatoid arthritis, joint injury, and cartilage degradation (Neidhart et al. 1997,Petersson et al. 1998, Clark et al. 1999). In culture, TSP-1 is most highly secretedby proliferative sparse cells and is deposited amorphously around migratory cells(Raugi et al. 1982, Adams 1997b). In denser, static cultures TSP-1 localizes withheparan sulfate proteoglycans in small cell-surface patches (Vischer et al. 1988,Murphy-Ullrich et al. 1988).

Molecular Connections

As secreted components of extracellular matrix, thrombospondins bind to otherextracellular molecules and also connect to the intracellular compartment throughinteractions with transmembrane glycoproteins (Figure 1).

EXTRACELLULAR CONNECTIONS A second hallmark of the thrombospondins isthe ability to bind large numbers of calcium ions (Figure 1). These are boundcooperatively through the type 3 repeats, which are similar to calmodulin EFhand repeats in terms of the positioning of oxygenated residues (Lawler & Hynes1986). Each molecule of TSP-1 binds an average of 35 calcium ions, with aKd of52µM at 4◦C (Misenheimer & Mosher 1995). Thus subgroup B thrombospondinsare predicted to bind 60 calcium ions per molecule. Calcium ion-binding inducesmajor conformational changes in the type 3 repeats, which affect the physical prop-erties, sensitivity to proteolysis, and cell-attachment activity of the whole molecule.All thrombospondins are also heparin-binding proteins (Qabar et al. 1994, 1995;Lawler et al. 1995; Chen & Mosher 1996a; H Chen et al. 2000). In subgroup A,the N-terminal domains contain consensus high-affinity BBXB heparin-bindingmotifs and the type 1 repeats probably contribute multiple lower-affinity sites, theactivity of which may differ between glycoforms (Lawler et al. 1992, Hofsteengeet al. 2000). The heparin-binding sites of subgroup B thrombospondins have notbeen mapped.

Interactions of thrombospondins with other extracellular molecules have beenprincipally studied with regard to TSP-1. Through its multiple domains and

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30 ADAMS

TABLE 2 Active peptide motifs in thrombospondin-1

Domain Peptide motif Binding molecule Reference

NTD RKGSGRR/KKTR heparin/HSPG Lawler et al. 1992MKKTRG decorin Merle et al. 1997FQGVLQNVRFVF activatedα3β1 Krutzsch

integrin et al. 1999

Type 1 rep. CSVTCG CD36 Asch et al. 1992GGWSHW fibronectin Sipes et al. 1993KRFK/WSHWSPW TGFβ Schultz-Cherry

et al. 1995WSHWSPWS heparin/HSPG Guo et al. 1992EWSPCSVTCGNGIQVRIK heparin/HSPG Gantt et al. 1997

Type 3 rep. RGD αvβ3,αIIbβ3 Lawler andintegrins Hynes, 1989

NCPFHYNP cathepsin G, elastase Hogg et al. 1994NCQYVYNV elastase Hogg et al. 1994

CTD RFYVVMWK CD47 Gao et al. 1996

repeats, TSP-1 binds to a large number of matrix components, proteases, cy-tokines, and growth factors (Figure 1). A number of these interactions are sharedby TSP-2. Active peptide motifs within TSP-1 have been mapped for some of theseinteractions (Table 2). In many cases, the binding of TSP-1 modulates the activityof the binding partner. The catalytic activities of thrombin, plasmin, neutrophilcathepsin G, and elastase are all decreased by bound TSP-1, and the activationof plasminogen is regulated in trimolecular complexes with urokinase plasmino-gen activator or plasminogen activator inhibitor (reviewed by Hogg 1994). Theseproperties are thought to be significant for the formation and resolution of fibrinclots, although more recent analyses of knockout mice have not revealed uniqueessential roles for TSP-1 or TSP-2 in this process. TSP-1 and TSP-2 also bindand inhibit matrix metalloproteinase 2 (MMP-2) (Bein & Simon 2000, Yang et al.2000a). For TSP-2, this is an indirect effect by which the extracellular active poolof MMP-2 is decreased because of cellular uptake of a TSP-2/MMP-2 complexby LDL receptor–related protein (LRP) (Yang et al. 2000b). This process mayhave functional consequences in collagen fibrillogenesis and the regulation ofangiogenesis.

With regard to cytokine binding by TSP-1, the interaction with TGF-β1 is ofparticular interest. The WSHWSPW motif in the second type 1 repeat of TSP-1binds to the small latent TGF-β1 complex [TGF-β1-LAP (latency-associated pep-tide)]. Mature active TGF-β1 is then released by an intermolecular activation effectof the KRFK motif in the first type 1 repeat of TSP-1, which involves binding ofKRFK to a LSKL motif at the N terminus of LAP and presumably leads to re-lease of LAP from TGF-β1 (Schultz-Cherry et al. 1995, Ribeiro et al. 1999). This

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THROMBOSPONDINS 31

interaction is of significance in the homeostatic function of TSP-1. TSP-2 containsno KRFK motif; thus although it binds to TGF-β1-LAP, it cannot activate latentTGF-β1 (Schultz-Cherry et al. 1995).

The extracellular interactions of subgroup B thrombospondins have been lessintensively studied. COMP and TSP-4 bind to other matrix components, inparticular fibrillar collagens I and II (Rosenburg et al. 1998). COMP also bindscollagen IX (Holden et al. 2000), and TSP-4 also binds to laminin, fibronectin,and matrilin-2 and does not bind to collagen IV or nidogen (Narouz-Ott et al.2000). COMP is a substrate for matrix metalloproteinases, in particular MMP-19and MMP-20 (Stracke et al. 2000), but whether COMP has regulatory effects onprotease activity has not been reported.

CELL-SURFACE INTERACTIONS Thrombospondins also interact with cell surfacesand in general support cell attachment. The molecular basis for this property hasbeen most intensively studied for TSP-1. Cell attachment is calcium dependent,and four regions of each subunit contribute to the activity: the NTD, type 1 repeats,type 3 repeats, and CTD (Figure 1). Each region interacts with different types ofcell-adhesion receptors, and active peptide motifs have been identified for anumber of these interactions (Figure 1 and Table 2). The interactions work co-operatively and combinatorially to support stable cell attachment, spreading, andlonger-term cell activities such as cytoskeletal organization, regulation of prolif-eration, and cell migration. The mechanisms of cell attachment to TSP-1 showconsiderable cell-type specificity, and this has been ascribed to the expressionof different repertoires of adhesion receptors (reviewed by Adams et al. 1995,Adams 1997a).

Although the activities of most of the receptor-binding peptide motifs have notbeen examined in the context of the intact molecule, these motifs are thought to beconstitutively active. In contrast, many cell types do not undergo RGD-dependentattachment to TSP-1 (for example, Adams & Lawler 1993, 1994). The availabilityof the RGD motif is highly regulated according to the exact pattern of disulfidepairing in the type 3 repeats. Intramolecular thiol-disulfide exchange occurs withinthe type 3 repeats such that TSP-1 exists in multiple conformations, and the rate ofthiol exchange is increased in calcium-depleted TSP-1 (Speziale & Detwiler 1990,Sankarapandi et al. 1995). This process probably restricts access to the RGD sitebecause RGD-dependent attachment activity is increased in dithiothreitol-reducedTSP-1 or TSP-2 (Sun et al. 1992, Chen et al. 1994). In vivo, extracellular proteindisulfide isomerase (PDI) may catalyze disulfide exchange on TSP-1. In vitro,PDI alters the protease inhibitory properties of TSP-1 and increases the RGD-dependent attachment of endothelial cells to TSP-1 (Hotchkiss et al. 1998). Attach-ment to theα3β1 integrin depends on intracellular activation ofα3β1 by signalingcrosstalk downstream of CD98 or insulin-like growth factor-1 (Chandrasekaranet al. 1999).

The interaction with LRP is critical for endocytosis of TSP-1 and TSP-2. Forcell types that express LRP, the binding of soluble TSP-1 to LRP in conjunction

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with heparan sulfate proteoglycans results in rapid uptake and lysosomal degra-dation in a timeframe of minutes to hours (Mikhailenko et al. 1995, Chen et al.1996b). The identity of the proteoglycan is not clear, but syndecan-1 is a likelycandidate because of its known interaction with the NTD (Sun et al. 1989). Itis not known whether matrix-bound TSP-1 could also be internalized by thismechanism.

The mechanism of cell attachment to TSP-2 has not been rigorously exam-ined but also appears to depend on multiple, cell-type-dependent interactions withthe various domains of TSP-2 (Chen et al. 1994). Cell attachment activity hasbeen demonstrated for TSP-4 and COMP, but the binding sites and receptors areunknown (Adams & Lawler 1994, DiCesare et al. 1994b). TSP-4 also supportsneurite outgrowth and may participate in synaptic organization in vivo (Arber &Caroni 1995).

Special Properties of Pentameric Thrombospondins

In contrast to the matrix interactions of TSP-1 and TSP-2, which are mediatedby the type 1 repeats and central stalk region (Table 2), TSP-4 and COMP bindcollagens I, II, and IX through their C-terminal domains (Rosenburg et al. 1998,Narouz-Ott et al. 2000) (Figure 1). The binding interactions are of nanomolaraffinity, are zinc-ion dependent, and occur at zinc ion concentrations that wouldbe in the physiological range for cartilage. Both thrombospondins bind to a site ineach of the collagen N- and C-propeptides and to two sites within the triple-helicaldomain (Holden et al. 2000, Narouz-Ott et al. 2000). In combination with thepentameric nature of these thrombospondins, these multipoint interactions mostlikely provide strong multivalent linkage to group and organize collagen monomerpolypeptides and polymeric fibrils. In the case of COMP, a candidate-binding motiffor collagens II and IX has been mapped by peptide inhibition to residues 579 to595 within the C-terminal domain (Holden et al. 2000).

Another unique feature of the subgroup B thrombospondins derives from thenature of the pentamerization sequence. The region critical for pentamerizationof COMP is within residues 20–83. Pentamerization involves formation of inter-chain disulfide bridges between cysteine residues 68 and 71 and the assemblyof a five-stranded alpha-helical bundle (Efimov et al. 1994). Crystallographicstudies of the recombinant pentamer module revealed that the bundle contains a7.3-nm-long hydrophobic pore of diameter 0.2–0.6 nm, with a potential ion trapnear the C-terminal end. This structure has striking similarities to ion channelmodels (Malashkeviah et al. 1996). Furthermore, the pore of the COMP pen-tamer also has the capacity to bind hydrophobic compounds, as demonstratedfor vitamin D and all-trans retinoic acid, with micromolar affinity (Guo et al.1998). The physiological significance of this binding remains to be established butmight be a general feature of subgroup B thrombospondins that enables them tostore and concentrate hydrophobic signaling molecules and facilitate their deliveryto cells.

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CONTEXT-SPECIFIC ROLES OF THROMBOSPONDINSIN VIVO: THE EVIDENCE FROM GENE KNOCKOUTMICE AND HUMAN GENETIC DISEASE

Mice null for TSP-1 or TSP-2 are capable of normal development and are fertile(Lawler et al. 1998, Kyriakides et al. 1998). The two types of mice show dis-tinctive abnormalities that affect multiple tissues and, in the case of TSP-1, alsoaffect embryo viability (Table 3). Thus despite their great structural relatednessand partially overlapping sites of expression during development, the essentialfunctions of TSP-1 and TSP-2 are distinct and nonredundant. Furthermore, theloss of expression from either gene does not involve a compensatory upregu-lation of the other thrombospondin (Lawler et al. 1998; Kyriakides et al. 1998a,1999b). The molecular processes underlying these alterations to phenotype, whichaffect cell differentiation, tissue organization, and immune response, are not fullyunderstood, but the available information indicates mechanisms of considerablecomplexity that involve multiple interactions of TSP-1 and TSP-2 with other ex-tracellular components and with cell surfaces.

Activation of TGF-ββ1 by TSP-1

The pneumonia and abnormal inflammation in the lungs and pancreas of TSP-1-null mice arise as a consequence of a specific lack of TGF-β1 activation inthese tissues. By histology, the lungs and pancreas of TGF-β1-null mice havemany phenotypic similarities with those of TSP-1-null mice (Shull et al. 1992).Intraperitoneal injection of TSP-1-null mice with the KRFK peptide from TSP-1that activates TGF-β1 increased the level of active TGF-β1 and restored a wild-type phenotype in both tissues. Blockade of normal TGF-β1 activation in wild-typemice with the LSKL peptide from LAP caused hyperplasia of the lung epithelium,as seen in TSP-1-null mice (Crawford et al. 1998). Thus in these two tissues, TSP-1has a dominant role in TGF-β1 activation that is a required part of normal epithelialhomeostasis and control of immune response. In the normal lung, latent TGF-β1 isproduced by the airway epithelial cells or by macrophages recruited in response toinjury (Yehualaeshet et al. 1999). Clearly, TSP-1 is not the sole activator of TGF-β1

TABLE 3 Major phenotypes of TSP-1 and TSP-2 gene knockout mice

TSP-1 TSP-2

Decreased embryonic viability Fragile skin

Pneumonia from 1month of age Lax tendons

Spinal lordosis Two-fold increase in bone density

Two-fold increase in circulating monocytes Increased vascular densityProlonged bleeding timeAccelerated skin wound healing

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34 ADAMS

because TGF-β1-null mice show a much more severe and general phenotype, andother tissues of the TSP-1-null mice, such as bronchial smooth muscle, containactive TGF-β1 (Crawford et al. 1998). Moreover, the quantity of active TGF-β1released from activated platelets is equal in the presence or absence of TSP-1(Abdelouahed et al. 2000).

TSP-2 and Connective Tissue Organization

The increased bone density of TSP-2-null mice has been related to an increasedproliferation of osteoblast precursors in the absence of any change to osteoclastfunction and thus presumably relates to increased matrix deposition (Hankensonet al. 2000). The defects of skin and tendon relate to unsuspected roles of TSP-2 incollagen fibrillogenesis. In TSP-2-null mice, dermal collagen fibrils are enlarged,with an average fibril diameter of 145 nm, compared with 122 nm in wild-typemice, and are disordered in packing, with increased spacing between the fibrils anda randomized orientation to the epidermis. These alterations manifest as a reducedtensile strength and increased ductility of skin tissue (Kyriakides et al. 1998a).These alterations in collagen fibril assembly are also apparent in the fibrotic re-sponse during foreign body reaction or the healing of full-thickness epidermalexcision wounds (Kyriakides et al. 1999a,b). Skin wounds showed increased for-mation of rete ridges during the regeneration of the epithelium and healed withmarkedly less scarring (Kyriakides et al. 1999b).

Collagen fibril assembly in vivo is thought to be initiated by association ofmature single collagen molecules within narrow channels that are formed betweenextended projections from fibroblast cell surfaces (Birk & Trelstad 1986; reviewedby Adams 2001). Early collagen fibers associate end-to-end by their C-terminaltips. Interactions with decorin and other proteoglycans regulate and size limitthe lateral association of fibrils by direct binding at the fibril surface (reviewedby Kadler et al. 2000). These molecules are abundant components of connectivetissue matrix. In contrast, TSP-2 is only detectable at very low levels in the dermisof wild-type mice, and thus it is not clear whether its interaction with collagen isdirect or if it modulates interactions of collagens with proteoglycans, or indeedwhether its site of action is at the cell surface or within the ECM.

Interestingly, primary TSP-2-null dermal fibroblasts showed a 30% reductionin the ability to attach to matrix-coated surfaces (Kyriakides et al. 1998a). Thisphenotype relates to the ability of TSP-2 to bind and inactivate MMP-2 by in-creasing its endocytosis by an LRP-dependent mechanism (Yang et al. 2000a,b).Thus TSP-2-null fibroblasts produce increased amounts of active MMP-2 that in-hibit cell attachment and spreading, perhaps by a general proteolysis of adhesivereceptors or the pericellular matrix. Loss of normal spatial order could also beimportant. Active MMPs are reported to be concentrated at defined points on thecell surface associated with lamellipodia, and changes in three-dimensional spatialassembly owing to increased protease activity could be envisioned to affect theorganization of other cell surface projections and the fibril-forming channels. Itwould be interesting to know whether over-expression of specific MMP inhibitors

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such as tissue inhibitor of metalloproteinases (TIMP) would normalize collagenfibril organization or abrogate the lax skin phenotype of TSP-2-null mice.

TSP-1, TSP-2 and Angiogenesis

TSP-1 was first identified as natural protein inhibitor of angiogenesis in an in vitroendothelial cord formation assay (Good et al. 1990), and this antiangiogenic ac-tivity is shared by TSP-2 (Volpert et al. 1995). Because of the high interest in thepotential of angiogenesis regulators as inhibitors of tumor growth, the developmen-tal and adult regulation of blood vessel growth in the TSP-1 and TSP-2-null micehas formed a major focus of interest. Blood vessel density, platelet function, andbleeding times are quantitatively normal in TSP-1-null mice, whereas TSP-2-nullmice show an approximately twofold increase in blood vessel density in varioustissues and increased tail wound bleeding times (Lawler et al. 1998, Kyriakideset al. 1998a). Thus neither protein appears to have a major impact on developmen-tal angiogenesis. In contrast, both proteins have significant effects in wound-repairangiogenesis. Both types of mice showed increased vascularity in skin wounds, andin TSP-2-null mice, vascularization of the granulation tissue was prolonged and thewounds healed more rapidly (Lawler et al. 1998, Kyriakides et al. 1999b). Trans-genic over-expression of TSP-1 in basal keratinocytes did not affect the archi-tecture or developmental vascularization of the epidermis or dermis but resultedin delayed healing and impaired granulation tissue formation of full-thicknessexcision wounds (Streit et al. 2000). This effect involved decreased proliferationof endothelial cells, reduced fibroblast migration into granulation tissue, a 30%reduction in the density of blood vessels in the granulation tissue, and loss of thenormal fourfold increase in vessel size during wound healing.

Evidence that over-expression of TSP-1 or TSP-2 inhibits tumor growth andangiogenesis has been reproduced convincingly in a number of tumor cell linesand experimental models (Weinstat-Saslow et al. 1994; Volpert et al. 1998; Bleuelet al. 1999; Streit et al. 1999a,b). In most cases, the anti-angiogenic effect involveddecreases in both vessel size and number. In a squamous carcinoma model, in-creased matrix assembly around the tumor also suggested a possible barrier effecton tumor growth and vascularization (Bleuel et al. 1999). Reports on the relativeefficacies of TSP-1 or TSP-2 are conflicting. TSP-1 was found more effective inblockade of corneal angiogenesis (Volpert et al. 1995), but in a nude mouse tumormodel, tumor growth was inhibited 50% by TSP-1 and 90% by TSP-2. The com-bination of TSP-1 and TSP-2 completely inhibited tumor growth over a 12-weekperiod (Streit et al. 1999a). These results imply some difference in the mecha-nisms of action of TSP-1 and TSP-2. Peptides corresponding to two regions ofthe type 1 repeats of TSP-1 independently inhibit neovascularization of retina orchick chorioallentoic membrane (Iruela-Arispe et al. 1999, Shafiee et al. 2000).

The Role of COMP in Cartilage Biology

The significance of COMP in human cartilage was clearly established by the dis-covery that mutations in COMP lead to pseudoachondroplastic dysplasia (PSACH)

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and the Fairbanks and Ribbings forms of multiple epiphyseal dysplasia (MED)(Hecht et al. 1995, Briggs et al. 1995). These autosomal-dominant bone dysplasiasare characterized by disproportionate short stature as a result of premature arrestof bone growth and joint laxity (PSACH; OMIM entry 117170), or by mild shortstature and hip pain (MED; OMIM entry 132400). Both conditions also involveearly onset arthritis (reviewed by Francomano et al. 1996). Mutations of COMPinvolving amino acid deletions, non-conservative substitutions, insertions, or veryshort triplet expansions have been documented in affected individuals. The muta-tions map within the type 3 repeats and the C-terminal domain, with one third ofpatients showing heterozygous deletion of single aspartic acid residues within thesixth type 3 repeat (OMIM entry 600310).

Because COMP is assembled as a pentamer, even if the wild-type and mu-tant alleles are equivalently transcribed and translated, only 1 out of 32 COMPmolecules will be assembled as entirely wild-type. This is hypothesized to accountfor the dominant-negative effects of the mutations. Most of the mutations affectthe calcium-binding repeats and are proposed to affect protein conformation byeffects on calcium ion-binding or by alterations in the distribution of disulfidebonds or free sulfhydryl groups within the type 3 repeats and C-terminal domain(Hecht et al. 1995, Briggs et al. 1995). PSACH cartilage shows reduced or absentdeposition of COMP and collagen IX into ECM and the chondrocytes containenlarged rough endoplasmic reticulum vesicles filled with lamellar material thatincludes COMP, collagen IX, and aggrecan (Maddox et al. 1997, Delot et al. 1998,Hecht et al. 1998b).

These defects in secretion depend on the terminal differentiation status of chon-docytes. Monolayer dedifferentiated chondrocytes from PSACH and MED pa-tients secreted normal levels of pentameric COMP, and the deposition of COMPinto PSACH tendon was normal (Maddox et al. 1997, Delot et al. 1998, Hechtet al. 1998a). Thus the disease is modeled to have a multifactorial basis: Quanti-tative reductions in matrix COMP are caused by impaired processing of structu-rally abnormal COMP molecules and, overall quantitative and qualitativealterations in cartilage ECM arise through the intracellular aggregation and al-tered extracellular interactions of other matrix molecules with mutant COMPmolecules (Maddox et al. 1997, Delot et al. 1998, Hecht et al. 1998b) (Figure2). These abnormalities indicate a normal role for COMP in the assembly andstability of cartilage matrix and possibly also in signaling effects of ECM onchondrocytes.

Aspects of this model have been explicitly tested by comparing the properties ofrecombinant mutant COMPs in vitro and in cell culture. Deletion of residue D469in the type 3 repeats did not affect pentamerization but resulted in a 50% decreasein calcium ion binding and loss of the normal calcium ion concentration-dependentalteration in physical properties (Hou et al. 2000, Thur et al. 2000). By electronmicroscopy, pentamers containing an equivalent mutation, termed MUT3, did notshow major calcium-dependent alterations in conformation. MUT3 protein wasalso altered in protease sensitivity and had reduced calcium ion-binding capacity

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(H Chen et al. 2000). Molecules containing the MED mutation D361Y showedequivalent physical changes (Thur et al. 2000). A D446N mutation also decreasedcalcium ion binding and altered the physical structure of monomeric type 3 repeatmodules (Maddox et al. 2000). These data demonstrate that single residue changeswithin the type 3 repeats can affect both calcium ion binding and the physicalstructure of the whole molecule. The1469 and D361Y mutants also showedalterations in zinc binding and proteolytic sensitivities in the presence of zinc(Thur et al. 2000). This is of interest because the binding of COMP to collagens I,II, and XI is zinc ion dependent, and mutant COMPs show reduced binding to thesecollagens (Rosenburg et al. 1998, Holden et al. 2000, Thur et al. 2000). These dataindicate a mechanism by which mutant COMP molecules that are successfullysecreted could destabilize organization of residual cartilage matrix.

CELLULAR MECHANISMS OF ACTION OFTHROMBOSPONDINS: THE EVIDENCE FROMTISSUE CULTURE ASSAYS

The mechanisms of action of thrombospondins have chiefly been studied withregard to TSP-1, initially using protein purified from platelets and more recentlywith recombinant protein. TSP-1 has many effects on cell function, including adhe-sion, cytoskeletal organization, migration, neurite outgrowth, cell-cell aggregation,proliferation, apoptosis, differentiation, and transcriptional regulation (Figure 1).Attempts to probe the molecular basis of these effects initially yielded a bewil-dering complexity in the cell-type specificity of functional effects and also inthe cell surface interactions involved for any single cellular response in a givencell type. The literature for this area up to 1995 has been reviewed (Lahav 1993,Adams et al. 1995). It is now recognised that this complexity derives from theconformational flexibility of TSP-1 and also from the distinct expression profilesand activity status of TSP-1 receptors in different cell types. Thus, mechanisms ofaction of TSP-1 need to be defined in individual cell types, and caution is neededin making extrapolations between different cell types. Here, I focus on the mech-anisms of effects on cell function that are relevant to the established in vivo rolesof thrombospondins.

Cell Adhesion and Motility

TSP-1 supports the attachment of many cell types, and depending on the cell type,the cells remain round and static or spread and become motile. Differences inattachment or spreading behavior do not correlate with cell binding to particularthrombospondin domains (Adams & Lawler 1993) and are therefore thought torelate to the intracellular coupling of thrombospondin receptors. Cell types thatspread and are motile on TSP-1 include glomerular mesangial cells, monocytes,neutrophils, smooth muscle, skeletal myoblasts, and carcinoma cells. In most cases,

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interactions with the N- and/or C-terminal domains are necessary for motility. Thetype 1 repeats and CTD are also implicated in the stimulation of endothelial cellmotility (reviewed by Adams et al. 1995, Bornstein 1995, Adams 1997a).

Effects of TSP-1 on smooth muscle motility have been a focus of attention,because of the localization of TSP-1 in atherosclerotic lesions and at sites of vas-cular injury where normally contractile smooth muscle cells undergo a phenotypicswitch to an inappropriate migratory state (for example, Raugi et al. 1990, Mianoet al. 1993, Roth et al. 1998). Vascular smooth muscle cell chemotaxis to TSP-1depends on the C terminus of TSP-1 and anαv-type integrin (Yabkowitz et al.1993b) and correlates with activation of FAK and the extracellular regulated ki-nases ERK1/2 (Gahtan et al. 1999a,b; Sajid et al. 2000). The activities of proteinkinase C and phosphoinositide 3-kinase (PI 3-K) are also involved (Willis et al.2000). CD47 is needed in the migratory response to TSP-1 and also functionssynergistically in cross-talk between TSP-1 and collagen-mediated chemotaxisthrough a physical association withα2β1 integrin (Wang & Frazier 1998). Thiseffect onα2β1 function is dependent on the activity of a Gi-type heterotrimericG protein that results in decreased cyclic AMP levels and inhibition of ERK(Wang et al. 1999). How activation of CD47 results in appropriate organization ofthe cytoskeleton for cell motility has not been studied. Although these cell culturedata support a positive role of TSP-1 on smooth muscle cell migration, and treat-ment of injured arteries with antibody to the CD47-binding site of TSP-1 reducesneointimal thickening (Chen et al. 1999a), no particular effects on smooth musclehave been described in TSP-1-null mice, transgenic TSP-1 over-expressor mice,or CD47-null-mice. However, the effects of TSP-1 and TSP-2 on blood vesselsize and number must, at some level, involve effects on the supporting smoothmuscle cells and pericytes as well as endothelial cells.

In certain contexts, TSP-1 acts as a molecular bridge that promotes cell-celladhesion or aggregation. In addition to its involvement in platelet aggregation andfibrin clot formation, TSP-1 participates in the attachment of malarial-parasitized(sickle) red blood cells to vascular endothelium (Roberts et al. 1985). This pro-cess results in vascular occlusions and appears to depend on the binding of TSP-1by CD47 (Brittain et al. 2001). The binding of CD47 on T cells to TSP-1 andSIRP1alpha on endothelial surfaces contributes to T cell arrest on activated en-dothelium (Ticchioni et al. 2001). TSP-1 bound to the surfaces of macrophages byCD36 andαvβ3 integrin promotes the recognition of apoptotic neutrophils, T cells,and eosinophils (Savill et al. 1992, Stern et al. 1996). Further studies in TSP-1-nullmice are needed to evaluate the biological significance of these effects in malarialinfection and the resolution of inflammatory lesions.

Regulation of Cytoskeleton and Cell Shape

TSP-1 has both positive and regulatory effects on the actin cytoskeleton. Cellspreading and migration on TSP-1 substrata are clearly associated with a dis-tinctive organization of the cortical cytoskeleton that involves the formation of

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large lamellipodia with ribs and spikes containing the actin-binding protein fascin.These elements form a matrix-contact structure and are also involved in cell mi-gration on TSP-1 (Adams 1995, 1997b). The mechanism of fascin spike formationby TSP-1 has been studied mainly in skeletal muscle cells, which spread ex-tensively on TSP-1. Syndecan-1 was necessary in transducing spike formationat the plasma membrane (Adams et al. 2001). The spatial assembly of spikesand lamellipodia requires the activities of the small GTPases Cdc42 and Racbut not the activity of Rho (Adams & Schwartz 2000). In contrast, the activ-ity of protein kinase C has an inhibitory effect on spike formation because itphosphorylates fascin and thereby downregulates its actin-binding activity, caus-ing a loss of spikes (Adams et al. 1999). Because protein kinase C is generallyactivated byβ1 integrins, these results suggest mechanisms by which the com-position of ECM and its content of TSP-1 could alter the relative activity andnet-working of cell signaling pathways through differential ligation of cell ad-hesion receptors (Figure 3). Other pathways activated by TSP-1 modulate cellspreading. Muskelin is an intracellular kelch-repeat protein that participates in theprocess of skeletal myoblast cell spreading on TSP-1 and downmodulates fascinspike formation when over-expressed (Adams et al. 1998). Kelch repeats formβ-propeller structures that mediate protein-protein interactions (reviewed byAdams et al. 2000). Such interactions might be important in the functional activitiesof muskelin.

Treatment of fibronectin-adherent cells with soluble TSP-1 results in a reorga-nization of matrix-contact structures that may also be relevant to the regulationof cell migration. Fibroblasts and endothelial cells adhere to fibronectin throughfocal adhesions, which are assembled as a result of integrin ligation and clustering(reviewed by Adams 2001). Soluble TSP-1 causes disassembly of focal adhesionsand microfilament bundles located in the central regions of cells, without affectingcell spreading (Murphy-Ullrich & Hook 1989). This property localizes to the motifELTGAARKGSGRRLVKGPD in the NTD and is shared by the equivalent pep-tides of TSP-2 (Murphy-Ullrich et al. 1993). The process depends on the activitiesof PI 3-K and cyclic GMP-dependent kinase (Greenwood et al. 1998). Furtherstudies showed that the production of phosphatidylinositol (3, 4, 5)-trisphosphateby PI 3-K activity resulted in a release ofα-actinin and vinculin from the ad-hesion plaque. Other structural components, including talin andα5β1 integrin,were retained. TSP-1 thus probably moderates the adhesive strength of the con-tact without altering matrix attachment. This reduction in cell contractility couldhave a pro-migratory effect (Greenwood et al. 2000). For endothelial cells to mi-grate, cadherin-based cell-cell adhesions also need to be loosened. Soluble TSP-1also induces the formation of intercellular gaps between adherent endothelial cellsin association with reorganization of F-actin and clustering of phosphotyrosine-containing proteins at cell-cell margins (Figure 3). This process depends on pro-tein tyrosine kinase activity and correlates with tyrosine phosphorylation of focaladhesion kinase, paxillin,γ -catenin, and p120cas, that are known componentsof cell matrix and cell-cell contacts (Goldblum et al. 1999). Thus through its

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multiple cell-binding sites, TSP-1 exerts diverse and cell-type-specific effects onactin cytoskeleton.

Regulation of Proliferation and Apoptosis

The effects of TSP-1 on cell proliferation are also cell-type dependent. For smoothmuscle cells, TSP-1 synergizes with growth factors such as EGF or PDGF topromote proliferation (Majack et al. 1988). A number of signaling events have beenimplicated downstream of CD47 andαvβ3 integrin that have general roles in theregulation of proliferation. Use of anti-TSP-1 antibodies to arrest cell proliferationdemonstrated effects on cdk2 kinase activity, expression of cyclins A and E, andinduction of p21Cip1/WAF1, a negative regulator of cdk2 and general growthsuppressor. Upregulation of p21 is essential for inhibition of proliferation, raisingthe hypothesis that regulation of p21 by TSP-1 is needed for its stimulatory effecton smooth muscle proliferation (Chen et al. 1999b).

TSP-1 also stimulates the activation and clonal expansion of T cells. Attachmentof T cells to intact TSP-1 was mediated primarily byβ1 integrins, characterizedasα4β1 andα5β1 (Yabkowitz et al. 1993a), but interactions with CD47 and hepa-ran sulfate proteoglycans were detected on peptide substrata (Wilson et al. 1999).TSP-1 or the peptides provided costimulatory signals that amplified T cell signal-ing responses to T cell receptor/CD3 ligation and resulted in enhanced transcrip-tional activity, and also directly activated mitogen-activated protein kinase (MAPK)signaling (Wilson et al. 1999). TSP-1 also stimulates T cell proliferation; this ef-fect is transduced by CD47 and CD36 (Vallejo et al. 2000). However, transcriptprofiling by cDNA microarray analysis showed intact TSP-1 to have both positiveand negative effects on the changes in gene expression induced by ligation of CD3.At the cellular level, inhibition of T cell activation by intact TSP-1 is dependenton CD47 and heparan sulfate proteoglycans (Li et al. 2001). Thus TSP-1 that isreleased by multiple cell types within inflammatory lesions may provide a regu-latory signal for clonal expansion of T cells, as well as a matrix appropriate for Tcell adhesion, migration, and clustering (Vallejo et al. 2000, Li et al. 2000).

For endothelial cells, TSP-1 acts as a negative regulator of proliferation thatactively induces apoptosis. The anti-angiogenic activity of TSP-1 maps to theCSVTCG CD36-binding motif and also adjacent peptide motifs that have beenassociated with glycosaminoglycan-binding activity (Tolsma et al. 1993, Iruela-Arispe et al. 1999) (Table 2). CD36 is a critical receptor because TSP-1 doesnot show anti-angiogenic activity in a CD36-null mouse model (Jimenez et al.2000). The intracellular effector mechanisms depend on the activity of fyn kinase,which associates with CD36, and on induction of apoptosis by a MAPK andcaspase-3-dependent pathway (Jimenez et al. 2000, Nor et al. 2000) (Figure 3).However, in macrophages, CD36-ligation by TSP1 is not sufficient to induce apop-tosis (Wintergerst et al. 2000). The other peptide motifs that inhibit angiogenesismight enhance the binding of TSP-1 to CD36, for example through ligation of pro-teoglycan coreceptors, or could have indirect effects such as sequestration of pro-angiogenic morphoregulatory factors (Guo et al. 1997, Iruela-Arispe et al. 1999).

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PERSPECTIVE AND CONCLUSIONS

Emergent Paradigm: Thrombospondinsas Multifunctional Regulators

Thrombospondins have complex and cell-type-specific effects on cell functionthat relate to their multidomain and multivalent interactions with cell surfaces andin matrix organization. The biological effects of deleting the genes for TSP-1 orTSP-2 demonstrate that these activities do not operate with equal importance inall tissues. Particular functions appear to dominate in certain tissues or cell types,and only a small subset of the many functions that have been characterized in tis-sue culture are evidently uniquely essential. However, the molecular mechanismsthat underlie the biological defects apparent in the knockout mice or humans withmutant COMP indicate the complexity of molecular connections made by throm-bospondins and demonstrate how the regulatory interactions of thrombospondinswith matrix, cytokines, proteases, and cell surfaces are integrated at the tissue levelinto the circuitries of delicate, cell-type-specific, homeostatic mechanisms.

Paradoxes and Open Questions

These evolving views of the biological roles and mechanisms of action of throm-bospondins still contain significant uncertainties. The use of peptides to map func-tional sites within the type 1 repeats implicates heparan sulfate proteoglycans, inaddition to CD36, in inhibition of angiogenesis activity and raises questions as tothe identity of the core proteins that might signal cooperatively with CD36 in en-dothelial cell apoptosis. The peptides map two independent active sites within thetype 1 repeats, however, although the short peptides have heparin-binding activities(Guo et al. 1992, 1997), and full-length recombinant TSP-1 molecules mutatedin the N-terminal heparin-binding site retain an affinity for heparin (Lawler et al.1992); recombinant type 1 repeats from TSP-1 do not bind heparin (Panetti et al.1999). A resolution to these complexities may be found in further studies of type 1repeat glycoforms. Although the functional significance of C-mannosylation isunknown, it is of note that synthetic peptides would not contain this modificationand the recombinant proteins would contain it variably. These conflicting data alsopoint to the need to evaluate the peptides as functional motifs in the context ofintact recombinant domains or full-length TSP-1 and to obtain higher-resolutioninformation on the type 1 repeats of TSP-1 and TSP-2 from crystal structure.

In consideration of the whole thrombospondin family, the type 1 repeats areunique to TSP-1 and TSP-2, and many of the specific effects of TSP-1 and TSP-2 oncells and in matrix derive from the type 1 repeats. This leaves open the question,are there core functions of thrombospondins and, if so, what are they? In thisregard, the major functions of the type 2, type 3 repeats and C-terminal domainshared by all thrombospondins relate to cation binding, cell attachment, and matrixinteractions and assembly (Figure 1). The CD47-binding motif is present in allthrombospondins, but whether CD47 is a generic receptor is unknown. In binding

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42 ADAMS

to TSP-1, CD47 forms a receptor complex with RGD-boundαvβ3 integrin, yet thesubgroup B thrombospondins either lack an RGD site or have a site positioned inthe third, rather than the seventh, type 3 repeats. This can be envisioned to posespatial constraints on assembly of the receptor complex and raises the possibilitythat CD47 could also act in cooperation with a non-RGD-dependent adhesionreceptor. It is also an open question whether CD47 is the sole receptor for theC-terminal domain. Non-integrin proteins of 105 and 80 kDa have been identifiedby affinity chromatography (Yabkowitz & Dixit 1991).

In the matricellular hypothesis, TSP-1 and TSP-2 are viewed as contextualadaptor proteins that do not participate in the structure of matrix (Bornstein 1995,Bornstein et al. 2000). Although their effects on cells clearly have a cell-type-specific molecular basis, the effects of TSP-1 on the structure of fibrin filamentsin blood clots (Bale & Mosher 1986), the interactions of TSP-4 and COMP withfibrillar collagens, the role of COMP in cartilage matrix, and the newly discoveredeffects of TSP-2 deficiency on collagen fibril assembly in mice, all challenge theview that thrombospondins do not participate in matrix organization and assembly.Further studies of all thrombospondins should rapidly bring new answers to thesequestions.

ACKNOWLEDGMENTS

I thank Deane Mosher, Juergen Engel, and members of my lab for discussions.I apologize to those whose work is not directly cited here. I thank the WellcomeTrust for financial support (grant 038284).

Visit the Annual Reviews home page at www.AnnualReviews.org

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28 Sep 2001 14:39 AR AR139-02-COLOR.tex AR139-02-COLOR.SGM ARv2(2001/05/10)P1: GDL

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Figure 2 Model for the processing, secretion, and matrix organizational role ofCOMP in cartilage and the hypothetical molecular basis for the cartilage defects inPSACH and MED. Not to scale.

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Figure 3 Context-specific activation of signaling molecules and cytoskeletal organizationby TSP-1. PanelsA–C show three examples of how TSP-1 engages alternate cell surfacereceptors in different cell types to activate specific sets of signaling pathways that producecomplex phenotypic responses. Effects on the actin cytoskeleton, exemplified in skeletalmyoblasts and endothelial cells, also depend on distinct sets of signaling molecules.

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P1: FRK

September 11, 2001 11:18 Annual Reviews AR139-FM

Annual Review of Cell and Developmental BiologyVolume 17, 2001

CONTENTS

CHEMICAL AND BIOLOGICAL STRATEGIES FOR ENGINEERING CELLSURFACE GLYCOSYLATION, Eliana Saxon and Carolyn Bertozzi 1

THROMBOSPONDINS: MULTIFUNCTIONAL REGULATORS OF CELLINTERACTIONS, Josephine C. Adams 25

SALMONELLA INTERACTIONS WITH HOST CELLS: TYPE III SECRETIONAT WORK, Jorge E. Galan 53

PATTERNING MECHANISMS CONTROLLING VERTEBRATE LIMBDEVELOPMENT, Javier Capdevila and Juan Carlos Izpisua Belmonte 87

IDENTIFICATION OF SELF THROUGH TWO-DIMENSIONAL CHEMISTRYAND SYNAPSES, Michael L. Dustin, Shannon K Bromley, Mark M. Davis,and Cheng Zhu 133

POLARIZED CELL GROWTH IN HIGHER PLANTS, Peter Hepler, L. Vidali,and A. Cheung 159

BOUNDARIES IN DEVELOPMENT: FORMATION AND FUNCTION,Kenneth D. Irvine and Cordelia Rauskolb 189

MOLECULAR BASES OF CIRCADIAN RHYTHMS, Stacey L. Harmer,Satchidananda Panda, and Steve A. Kay 215

EARLY EYE DEVELOPMENT IN VERTEBRATES, Robert L. Chow andRichard A. Lang 255

RNP LOCALIZATION AND TRANSPORT IN YEAST, Pascal Chartrand,Robert H. Singer, and Roy M. Long 297

VERTEBRATE SOMITOGENESIS, Olivier Pourquie 311

ANIMAL CELL CYTOKINESIS, Michael Glotzer 351

STEM AND PROGENITOR CELLS: ORIGINS, PHENOTYPES, LINEAGECOMMITMENTS, AND TRANSDIFFERENTIATIONS, Irving L. Weissman,David J. Anderson, and Fred Gage 387

RECENT ADVANCES IN CHEMICAL APPROACHES TO THE STUDY OFBIOLOGICAL SYSTEMS, Michael A. Shogren-Knaak, Peter J. Alaimo,and Kevan M. Shokat 405

EMBRYO-DERIVED STEM CELLS: OF MICE AND MEN, Austin G. Smith 435

vii

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P1: FRK

September 11, 2001 11:18 Annual Reviews AR139-FM

viii CONTENTS

HOW MATRIX METALLOPROTEINASES REGULATE CELL BEHAVIOR,Mark D. Sternlicht and Zena Werb 463

BIOLOGICAL BASKET WEAVING: FORMATION AND FUNCTION OFCLATHRIN-COATED VESICLES, Frances M. Brodsky, Chih-Ying Chen,Christine Knuehl, Mhairi C. Towler, and Diane E. Wakeham 517

GETTING THE MESSAGE ACROSS: THE INTRACELLULARLOCALIZATION OF mRNAS IN HIGHER EUKARYOTES,Isabel M. Palacios and Daniel St. Johnston 569

CELLULAR FUNCTION OF PHOSPHOINOSITIDE 3-KINASES:IMPLICATIONS FOR DEVELOPMENT, IMMUNITY, HOMEOSTASIS, ANDCANCER, Roy Katso, Klaus Okkenhaug, Khatereh Ahmadi, Sarah White,John Timms, and Michael D. Waterfield 615

CONTROL OF EARLY SEED DEVELOPMENT, Adbul M. Chaudhury, AnnaKoltunow, Thomas Payne, Ming Luo, Mathew R. Tucker, E. S. Dennis,and W. J. Peacock 677

PEROXISOME BIOGENESIS, P. Edward Purdue and Paul B. Lazarow 701

THE MOLECULAR BASIS OF SISTER-CHROMATID COHESION,Janice Y. Lee and Terry L. Orr-Weaver 753

LEFT-RIGHT ASYMMETRY DETERMINATION IN VERTEBRATES,Mark Mercola and Michael Levin 779

INDEXESSubject Index 807Cumulative Index of Contributing Authors, Volumes 13–17 841Cumulative Index of Chapter Titles, Volumes 13–17 844

ERRATAAn online log of corrections to Annual Review of Cell and DevelopmentalBiology chapters may be found http://cellbio.AnnualReviews.org/errata.shtml

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Documents

T HROMBOSPONDINS : Multifunctional Regulators of Cell Interactions