Matrix Mo scutes AND THEIR LIGANDS Bjorn Reino Olsen INTRODUCTION ‘lular growth and differentiation, in two-dimensional cll culture a well s inthe three-di- /ncasional space ofthe developing organism, requir the presence of structured environment vwith which th cells can interact. This extracellular matiix (ECM) is composed of polymeric net- ‘works of several types of macromolecules in which smaller molecules, ions, and water are bound. “The major types of macromolecules are the fibrous proteins, such as collagens, elastin, fibrillins, fibronectin, and laminins, and the hydrophilic heteropolysaccharides, such as glycosaminoglycan chains in hyaluronan and proteoglycans. Is the combination of fibrous protein polymers and hy- ‘rated proteoglycans that gives extracellular matrices their resistance to tensile and compressive ‘mechanical forces. The macromolecular components of the polymeric assemblies of the ECM are in many ‘cases secreted by cells as precursor molecules that are significantly modified (proteolytically processed, sulfated, oxidized, and crosslinked) before they assemble with other components into functional polymers (Fig. 5.1). The formation of mattix assemblies in wivo is therefore in ‘most instances a unidirectional, ireversible process and the disissembly of the matrix is not simple reversal of assembly, but involves multiple, highly regulated processes. One consequence ‘of this is that polymers reconstituted in the laboratory with components extracted fiom extra- cellular matrices do not have all the properties they have when assembled by cells in vive. The ECM in vitois also modified by cells as they proliferate, differentiate, and migrate, and cells in ‘tuzn continuously interact with the matrix and communicate with each other through it (Hay, 1991). ‘The ECM is therefore not an inert product of secretory activites, but influences cellular shape, fate, and metabolism in ways that are as important to tissue and organ structure and function as che effects of many cytoplasmic processes. This realization has ed toa reassessment of the need fora detailed molecular understanding of the ECM. In the past the ECM was pri- marly appreciated for its challenge to biochemists interested in protein and complex catbo- hydrate structure; a deailed characterization of ECM constituents is now considered essential for understanding cell behavior in the context of tissue and organ development and function. Some of these constituents are obviously most important for their steuctual properties (colla- gens, elastin, and fibrillin), whereas others (matrix-bound fibroblast growth factors, trans- forming growth factor B, bone morphogenetic proteins are signaling molecules. In a third cat- gory are multidomain molecules (fibronectin, laminin, chrombospondin, tenascin, syndecans, and other proteoglycans) that are both structural constituents and regulators of cell behavior ig. 5.2), Proce of re ning Capra 02000 tennis Src on gas oapedicion yo ed CHAPTER 5 7jor Reino Olsen s | Fig. 5.1. Extracellular matrices are synthesized and assembled 4 i through many steps. Soluble matrix molecules are secreted by i cell, modified, and assembled into polymeric complexes. These | ‘complexes serve asa scaffold forces and asa reservoir for mall ‘molecules such as growth and difierentiation factors. FIBRILLAR COLLAGENS: MAJOR SCAFFOLD PROTEINS IN THE ECM CCollagens constitute a large family of proteins that represent the major proteins (about 25%) {in mammalian tissues. A subfamily ofthese proteins, the fibrillar collagens, contains rigid, rodlike | ‘molecules with three subunits, c chains, folded into a right-handed collagen triple helix (Linsen- | ‘mayer, 1991). Within fibrillar collagen tripl-elical domain, each a chain consists of about 1000 amino acid residues and is coiled into an extended, left-handed polyprolin I helix; three «chains I are in turn twisted into a right-handed superhelx (Fig. 5.2). The extended conformation of each «chain does not allow the formation of intrachain hydrogen bonds, andthe stability ofthe tiple i helix is instead due to interchain hydrogen bonds. Such interchain bonds can form only if every ‘third residue of each chain does not have a side chain and is packed close to the tipl-helical axis, Only glycine residues can therefore be accommodated in this postion. This explains why the amino acid sequence of each a chain in fibrillar collagens consists of about 300 Gly-X-Y tripep- tide repeats, where X and Y can be any residue, but ¥ is frequently proline or hydroxyproline. Ie also provides an explanation for why mutations in collagens chat lead to a replacement oftiple- | helical glycine residues with more bulky residues can cause severe abnormalities (Olsen, 1995). | Fibrilla collagen molecules are the major components of collagen fibrils. Ther chains are synthesized as precursors, proa. chains, with large propeptide regions flanking the central triple- helical domain. The carboxyl propeptide (C-propepride) is important forthe assembly of trimer- ie molecules in the rough endoplasmic reticulum. Formation of C-propeptide times, stabilized by intra- and interchain disulfide bonds, isthe first step inthe intracellular assembly and folding of trimeric procollagen molecules (Olsen, 1991). The folding ofthe tiple helical domain proceeds ina zipperike fashion from the carboxyl roward the amino end of procollagen molecules, with a rate thats limited by cis-trans isomerization of prolyl peptide bonds. Mutations in fibrillar pro collagens tha affect the structure and folding of the C-propeptide domain ate therefore likely to | affect the participation of the mutated chains in tiple-helical assemblies. In contrast, mutations upstream ofthe C-propeptide, such as in-frame deletions or gicine subsicaions inthe tiple-he- lial domains, exe dominant negative effect, in thatthe mutated chains wil participate in trimer assembly, but will inteefere with subsequent folding of che triple-helical domain. Lo5 Matrix Molecules and Their Ligands 59. Fig. 5.2. Diagram showing a segment ofa triple-helical collagen molecule. The tiple he- li i composed of three left-handed helices (x chains) that ae twisted into aright-hand- ed supethelix. The sequence ofeach a chain isa repeat ofthe tripeptide Gly XV. The Gly residues are packed close tothe trple-helical axis (indicated by a triangle). Only lycine (without a side chain) can be accommodated in this poston. Although any residue can fit into the X and ¥ positions, Pro is frequenty found in the ¥ postion, Fibillar procollagen chains are the products of nine genes (Vaorio and de Crombrugghe, 1990). The similarities ofthese genes suggest that they arose by multiple duplications from a sin- tle ancestral gene. Despite ther similarities and the high degree of sequence identity of their pro- tein products, chey exhibit specificity in the interactions of thet C-propeptides dusing trimeric a- semblics. Thus within triple-helical procollagens a relatively small number of chain combinations is found; these combinations represent fibrillar collagen types (Jacenko et al, 1991). Cottacens V/XI—Recutators oF Fisrit. ASSEMBLY, ‘SPATIAL ORGANIZATION, AND CELL. DIFFERENTIATION Som collagen types are heterotimers (types I, Vand XI), whereas others are homotrimers (pes Il and Il). Some chains participate in more than one type: for example, the (1) chain forms the homotrimeric collagen Il, bt is aso one of cree different chains in collagen XI mole- cules, Between collagens V and XI there is extensive sharing of polypeptide subunits, and fibrillar collagen molecules previously described as belonging o either collagen V or XI are now referred to as belonging to the V/XI type. Thus, fibrillar procollagen molecules secreted by cells are mem- bers ofa large group of homologous proteins. They all contain a C-propepride cat is completely removed by an endoproteinase after secretion, and thei triple helical rodlike domains polymerize ina staggered fashion into fibrillar arays (Fig, 5.3) They differ, however, inthe structure ofeheir amino propeptide (N-propeptide) domains and in the extent co which this domain is protelyt- cally removed. For some collagen types, such as collagen I and I, the N-propeptide processing is complete in molecules within mavure fibril. For other types, such as collagens V/XI, this is not the cas, in tha a large portion of the N-propepties in these molecules remains attached to the twiple-helical domain (Fig. 5.3). Itis believed that che incomplete processing of type V/XI mole- calles allows them to serve as regulators of fibril assembly (Linseamayer etal, 1993). Collagen fi- brs are heterotypic, ic, contain more than one collagen type, sich that collagen I fibrils contain 10% collagen V and collagen Il ibrils contain 5~-10% collagen XI. The presence of N-propep- ‘de domains on V/XI molecules represents a steric hindrance ro addition of molecules at fibril surfaces, This heterotypic/ steric hindrance model predicts that collagen fibril diamerers ina tssue are determined by the ratio ofthe “minor” component (V/XI) to the “major” component (Tor I). Ahigh ratio results in thin fibrils a low ratio results in thick fibrils. Direct support for this comes from studies of mutant and transgenic mice. For example, mice that are homozygous for a func- ‘tional null mutation in a1 (XD) collagen and transgenic mice overexpressing collagen II have car~ tilage collagen fibrils chat are abnormally chick (Garofalo eal, 1993; Li eral, 1995). ‘A characteristic feature of collagen fibrillar scaffolds is their precise three-dimensional pat- tems. These patterns follow mechanical stres lines and ensure a maximum of tensile strength with minimum of material. Examples are the criss-cossing lamellae of collagen fibers in lamellar bone or in comea, the arcades of collagen fibrils under the surface of articular cartlge, and the paral- lel fiber bundles in tendons and ligaments. Ultimately, cells are responsible for establishing these patterns, bu the mechanisms they use probably involve specific molecule and interactions. The Ee| 60 Bjom Reino Olsen Fig, 5.3. Diagram of cartilage collagen vil. Col- lagen it molecule are the major components Mol ecules of collagen XI and IX are located on the sur- face. Collagen 1 molecules, eterovime of tee | diferent a chains have amino-terminal coma i that are tought to block sterically the addition of | collagen Itmolecules atthe hb sue / caitical patrern-forming interactions are not known; however, the information for spatial organi | zation is not contained within the diferent fibrillar collagen types, because the same kind of het- fi crorypic fibril canbe parc of scaffolds with very different spatial organization. Transgenic mice with | an alteration in the N-propeptide region of collagen V molecules show a disruption of dhe lamel- | | lar arrangement of fibrils in the cornea, suggesting a role fr fibril surface domains in generating the spatial patern (Andrikopoulos et a, 1995), Finally, members ofa unique subfamily of fibril | associated collagens with interrupted triple helices (FACIT) (Olsen etal, 1995) are good candi- i dates for molecules that allow tissue-specific fibril patterns to be generated by modulating the sar- I face properties of fibril ‘The phenotypic consequences of mutations in fibrillar collagen genes indicate that the major fanction ofthese proteins isto provide elements oF high tensile strength atthe tissue evel. Muta- tions in collagen V/XI genes also suggest chat fibrillar collagen scaffolds are essential for normal ‘cllular growth and differentiation. For example, a functional null mutation in a (XI) collagen in ‘cartilage causes a severe disproportionate dwarfism in mice and perinatal death of homezygotes Gi erat, 1995). Histology of the mutant growth plaes reveals a disorganized spatial distribution of ells and a defect in chondrocyte differentiation to hypertrophy. The explanation for this is ike- | 1y tobe relate to the fact that in growth plates the proliferation and differentiation of chondro- i ‘nts is regulated by locally produced growth factors and cytokines. Calls that produce these fac- tors must therefore be localized close to calls that express the appropriate receptors. The aC) ‘mutation may dsrupe this relationship, because ic rsuls ina dramatic decrease in cohesive prop- ‘mtes ofthe matrix and a loss of cellular organization, Tiansgenic mice with a mutation in @2(V) collagen have a large number of har fllcles of unusual localization in the hypodermis; this may also be related to a defect in the mechanical properties of the fibrillar collagen scaffold (An. diikopoulos eal, 1995). FACIT Coitacens—Moputarors or CottacEn Frann, Sunrace Prorerrres Molecules tha are associated with collagen fibrils, contain two or more rple-hlical domains, and share characteristic protein domains (modules) are clasified as FACIT collagens (Olsen eal, 1995; Shaw and Olsen, 1991). OF the five currently known members in the group (collagens IX, XL, XIV, XVI, and XD collagen IX is che best characterized both structurally and functionally, Collagen IX molecules ate heterottimers of three diferent gene products (van der Rest eral, 1985). Each ofthe three a chains contains three triple helical domains separated and flanked by non. triple-helical sequence regions (Fig. 5.4). Between the amino-terminal and central triple helical domains a flexible hinge gives the molecule a kinked structure with two arms. Type IX molecules ate located on the surface of type H/XL-containing fibrils with the long arm parallel to the fibril surface and the short arm projecting into the perfibillar space (Vaughan e al, 1988) (Fg, 5.3).5 Matrix Molecules and Their Ligands 61 Fig, 5.4. Diagrams of collagen IX and XII (long form) molecules. Collagen 1X molecules contain th three chainsa1(X), a2(X), and ‘@3{1X), Each chain contains three triple-helical domains (COLI, COL2, COL3), interrupted and flanked by non-tiple-helical se- quences. In cartilage, the a(IX) chain contains a large globular amino-terminal domain. The a2(X) chain serves as a proteogly ‘ean core protein in that it contains a chondroitin sulfate (CS) side ‘chain attached tothe non-triple-helical region between the COLZ and COL3 domains. Collagen Xi! molecules are homotrimers of ‘a1(X chains. The three chains form two short triple-helical do- ‘mains separated by a flexible hinge region. A central globule is ‘composed of three globular domains that are homologous to the amino-terminal globular domain of a1(X) collagen chains. The ‘amino-terminal regions of the tee a1 (XI) chains contain rulti- ple fibronectin type I repeats and von Willebrand factor A-like domains. These regions form three “fingers” that extend from the central globule. Through alternative splicing a portion of the fi agers (white region in diagram) is spliced out in the short form of can be extracted from tissues, It is beicved that collagen IX may function as abridging molecule between fibrils or between fib- rils and other matrix constituents. Support from this comes from studies of transgenic mice and ‘mutations in humans. Transgenic mice with a dominant negative mutation in the a(IX) chain (Nakata eal, 1993), as well as mice that are homozygous for 1 (IX) null alleles (Passer etal, 1994), exhibit osteoarthritis in knee joints and mild chondrodysplasi. In humans, a splice site ‘mutation leading to exon skipping and an in-frame deletion inthe «2(0X) collagen chain has been shown to cause a form of multiple epiphyseal dysplasia, an autosomal dominant disorder charac- terized by early-onset osteoarthritis in lage joints associated with shore stature and stubby fingers (Muragaki eral, 1996). ‘Molecules of collagens XII and XIV are homorrimers of chains that are made up of several kinds of modules. Some modules are homologous to modules found in collagen IX, whereas oth- «ts show homology to von Willebrand factor A-domains and fibronectin type III repeats. Both. types of molecules contain a central globule with three fingerlike extensions and a thin triple- helical tail attached (Fig, 5.4) For collagen XU, two forms tha differ greatly inthe lengths of the fingerlike extensions are generated by alternative splicing of RNA transcripts. Variations in the car- boayl regions aso occur (Olsen ef al, 1995). Both collagens are found in connective tissues con- taining type I collagen fibrils, except mineralized bone matrix, and immunolabeling seudies show 4 fibrl-associated distribution. Type XIV collagen has been reported to bind to heparin slfare and the small fbrl-associated proteoglycan decorin (Brown etal, 1993; Font eral, 1993). This would suggest an indirect fibril association. direct association cannot be ruled out, however, because collagen XII molecules form copolymers with collagen I even in the absence of proteoglycans. A functional interaction beeween fibrils and collagens XII and XIV is implied by studies showing shat addition of the two collagens to type I collagen gels promote gel contraction mediated by fi- broblasts (Nishiyama etal, 1994). The effeceis dose dependent and can be prevented by denatu- zation or addition of specific antisera. The association with fibrils of collagens XII and XIV may therefore modulate the ftictional properties of fibril surfaces. The synthesis of diferent isoforms could be importantin this contest, because they could bind to fibrils with different afte. Also, ‘because the long form of collagen XIL is a proteoglycan, whereas the short form is not, variations inthe relative proportion of the two splice variants may serve to modulate the hydrophilic prop- cextcs of interfibrillar matrix compartments. Finally, the discovery thatthe collagen I N-propep- ‘tide processing enzyme binds o collagen XIV and can be purified as part of a complex with anti- bodies against collagen XIV raises the posibility thatthe FACIT collagens provide binding sites for fibrl-modifying extracellular matrix enzymes (Colige eta, 1995). collagen Xil. Hybrid molecules with both long and short fingers62 Bjorn Reino Olsen BaszMenr MemBranr CoLtAGENS AND ASSOCIATED CouLaczn Motecutes Atepithlil (and endothelial) stromal boundaries, basement membranes serve as specialized seas of ECM for cll attachment. By end-to-end and lateral interactions collagen IV molecules form a networklike scaffold for basement membranes (Yurchenco and O’Reat, 1994). Six differ- cnt collagen TV genes exist in mammals, and their products interact to form atleast thece difer- ent types of heterotimeric collagen IV molecules. These different isoforms show characteristic ts- sue specific expression patterns. ‘Within basement membranes the collagen TV networks are associated with a large number of oncollagenous molecules, such as vaious isoforms of laminin, nidogen, and the heparin sulfate proteoglycan perlecan. Additional collagens are also associated with basement membranes. These include the transmembrane collagen XVII in hemidesmosomes and collagen VII in anchoring f- bis. Collagens XVII and VII are important in regions of significant mechanical stress, such as skin, in that they anchor epithelial cells to the basement membrane (collagen XVII) and strap the basement membrane to the underlying stroma (collagen VID. In bullous pemphigoid, autoant- bodies agninse collagen VII cause blisters that separate epidermis from the basement membrane (iu eral, 1994); dominant and recessive forms ofepidermolyss bullosa can be caused by muta- ‘ions in collagens VII and XVII (McGrath et al, 1995; Uito etal, 1994). The physiological im- portance ofollagen IV isoformsis highlighted by Alpore syndrome (Tryggvason et al, 1993). This Alsease, characterized by progressive hereditary nephritis associated with sensorineural hearing loss and ocular lesions, can be caused by mutations within «3(TV) and ee4(IV) collagen genes (auto- somal Alport syndrome) (Mochizuki etal, 1994) or mutations in a5(IV) collagen (X-linked Al- port syndrome) (Batker eal, 1990). In cases of large deletions, including both the a5(IV) and the neighboring a(IV) collagen gene, renal disease i associated with inherited smooth muscle ‘mots (Zhou etal, 1993). ‘Two additional basement membrane-associated collagens, collagen VII and collagen XVI, are of interest because oftheir fanction in vascular physiology and pathology. Collagen VIII is 2 short-chain, nonfibilla collagen with significant homology to collagen X, a product of hyper ‘wophie chondrocytes (Muragaki cal, 1991; Yamaguchi eral, 1989, 1991). Is expression appears 1 be up-regulated during cardiac morphogenesis (Iruela-Arispe and Sage, 1991), in human ath- crosclerotc lesions (MacBeath etal, 1996), and following experimental lesions to the endotheli- um in large arceries (Bendeck etal, 1996; Sibinga eal, 1997). Ichas been suggested that colla- gen VIII may be important in medi Jayer into the intima during neointimal thickening following endothelial ell injury. Collagen XVII, together with collagen XV, belongs toa distinct subfamily of collagens called ‘multiples (60 named because of their multiple trple-helx domains and interruptions) (Oh et al, 1994; Rehn and Pihlajaniemi, 1994), Because ofthe alternative utilization of two promoters and alternative splicing, the COLI8A/ gene gives rise to three different transcript that are trans- lated into three protein variants These are localized in various basement membranes, including ‘those thac separate vascular endothelial cells from the undedying intima in blood vessels (Hallter et ab, 1998; Muagaki er al, 1995; Rehn and Pihlajaniemi, 1995; Saarela etal, 1998). When col- Jagen XVIT was intially cloned and sequenced it was noted thatthe sequence contained multiple consensus sequences for attachment of heparan sulfate side chain (Oh eal, 1994), Subsequent studies have, in fact, confirmed that collagen XVIII forms the core protein of a basement mem: brane proteoglycan Halfier er aL, 1998). Proteolytic processing of the carboxy! non-itiple-helical domain of collagen XVII in tissues leads to the release of heparin-binding molecules with anti~ angiogenic activity (Sasaki etal, 1998). One ofthese fragments, named endostatin, represents the carboxyl-terminal 20-kDa portion ofcollagen XVII chains (O'Reilly etal, 1997). Recombinane preparations ofendostain have been shown to inhibit the proliferation and migration of vascular endothelial cells, inhibit che groweh ‘of rumors in mice and rats, and cause regression of rumors in mice (Boehm et al, 1997; Dhana- bal al, 1999; O'Reilly e¢ al, 1997; Yamaguchi etal, 1999). The limited data available suggest that these effets are mediated by inhibition of tumor-induced angiogenesis. The Xcray crystallo- ‘sraphic structure ofendostatin has been determined for both the mouse and human proteins (Ding ral, 1998; Hohenester etal, 1998).'The compact structure consists of two ct helices and a large numberof B strands, stabilized by two intramolecular disulfide bonds. A coordinated zinc atom the migration of smooth muscle cells from the medial5 Matrix Molecules and Their Ligands is part of the structure (Ding er al, 1998) and on the surface a patch of arginyl residues forms a potential binding ste for heparin (Hohenester etal, 1998). Scudies of mutant endostatins have shown that aginines within this patch are required for heparin binding (Yamaguchi eal, 1999). Incerestingly, heparin or zinc binding is not required for biological activity (Yamaguchi er al, 1999). ELASTIC FIBERS AND MICROFIBRILS Collagen molecules and their fibers evolved as struccues of high tensile strength, equivalent to that of steel when compared on the bass of the sume cross-sectional area, but thre times bet- tecon a per unit weight basis In contas, elastic fibers, composed of molecules of elastin, provide tissues with elasticity so that they can recoil after transient sterch (Rosenbloom etal, 1993). In ‘organs such as the large arteries, skin, and lungs, elasticity is obviously crucial for normal func- tioning, Elastin fibers derive their impressive clastic properties, an extensibility that is about five ‘times that ofa rubber band with the same cros-sectional area, from the structure of elastin mol- several laminin isoforms are now known (Fig. 5.6). Several forms have a cross-shaped structure 25 visualize by rotary shadowing electron microscopy; some forms contain Y-shaped molecules. The laminins are important components of basement membranes, where they provide interaction sites5__Matrix Molecules and Their Ligands Fig. 5.6. Diagrams of wo types of laminins, Laminin 1 has a cross- shaped structure, whereas laminin 5 is Y-shaped, due to a shorter on a Bt a Bs 2 Lawns Lawns aechain, for many other constituents, including cell surface recxpeors (Timpl, 1996; Timpl and Brown, 1994). The funcrional and structural mapping ofthese sites and che complete sequencing of many laminin chains have provided detailed insights into the organization of laminin molecules. With- inthe cros-shaped laminin 1 molecule, chee similar shore arms are formed by the N-terminal r- sions ofthe al, BI, and +1 chains, whereas along arm is composed of the carboxy regions ofall ‘three chains, flded into a coiled-coil strucruresThe three chains are connected at the center ofthe «ross by three interchain disulfide bridges. The shore arms contain multiple EGFlike repeats of about 60 amino acid residues, terminated and interrupted by globular domains. The long arm con- sists of eprad repeats covering about 600 residues in all hree chains. The all chain is about 1000 ‘amino acid residues longer than the Bandy chains and forms five homologous globular repeats at the “base” of the cros; these globular repeats are similar to repeats found in the proteoglycan ‘molecule perlecan (lozzo, 1994). Calcium-dependent polymerization of laminin is based on in- teractions berween the globular domains atthe N termini and is thought to be important for the ‘assembly and organization of basement membranes. Also of significance forthe assembly of base- ‘ment membranes isthe high-affinity interaction with nidogen (Timpl and Brown, 1994). The binding site in laminin for nidogen ison the 71 chain, close to the center of the cross. On nido- sen, 2rodlike molecule with three globular domains, the binding st for laminin isin the carboxyl slobular domains; another globular domain binds o collagen IV. Thus, nidogen is abridging mol- coule that connects the laminin and collagen IV networks, and is probably crucial for the assem- bly of normal basement membranes. Laminin does not bind directly to collagen IV, but has binding sites for several other mole- cules besides nidogen. These are heparin, perlecan, and fbulin-1, which bind ro the end of the long arm of the laminin cross. The biologically most significant interactions of laminin, however, involve a varity of cell surface receptor, both integrins and nonintegrin receptors. Several inte. 4grins are laminin receptors (Kramer 1994). They show distinct preferences for different laminins, and recognize binding sites on either the short or long arms of laminin molecules In striated mus. cle cells laminin binds to the dystroglycan—dystrophin complex (Matsumura and Campbell, 1994). A disulfide-linked complex of laminins 5 and 6 is crucial forthe firm attachment of ker. atinocytes to the basement membrane in skin by is interaction with a6 integrins in hemides- ‘mosomes (Niessen etal, 1994; Pulkkinen etal, 1994; Sonnenberg et al, 1991). Laminins with 2 chains appear to be important for guiding axons to neuromuscular junctions (Noakes etal, 1995). Through interactions with the integrin av3, laminin is important for angiogen: (Brooks eral, 1994) Finally, laminin has important roles in induction and maintenance of fetentiated cellular phenotypes and in control of cell proliferation (Grantee al, 1989),66 Bjorn Reino Olsen EGF -tiee domains i \ / Oe 7 \ and the position of two cysteines that are involved in interchain disulfide (S— s 5) bonds within the trimeric molecules. Car- binding domains MODULATORS OF CELL-MATRIX INTERACTIONS “Whereas proteins such s fibronectin and laminin are important for adhesion of cells to ex- tracellular matrices, other ECM molecules appear to function as both positive and negative mod- ulators of such adhesive interactions. Examples of such modulators ate thrombospondin (Born- sein and Sage, 1994) and tenascin (Erickson, 1993). “Thrombospondins are a group of four homologous trimeric matrix proteins composed of sev- cral Ca2*-binding domains, EGF-like repeats, as well as other modules (Bornstein, 1992) (Fig. 5.7). Different members ofthe group show differences in cellular expression and functional prop-;« cries. Thrombospondin-1, a component of c. granules in platelets, is released when platelets are ted and forms multimers that bind fibrin and fibrinogen in the hemostatic plug, The pro- tein is also produced by many other cell types (fibroblasts, endothelial cells, and smooth muscle cells) and binds to a variety of matrix molecules, including fibrillar collagen, fibronectin, laminin, and heparan sulfate proteoglycan. Ichas a growth-promoting effect on fibroblasts and may desta- bilize cell-matrix interactions during wound healing and angiogenesis (Chiquet-Ehrismann, 1995). Ie therefore promotes cell proliferation and angiogenesis. The cellular receptor for throm- bospondin appears to be a chondroitin sulfate proteoglycan; cellular contact sites with throm- bospondin have a cytoskeletal organization chat is differen than the integrin-mediated focal ad- hhesions seen with molecules such as fibronectin (Adams, 1995). Cartilage oligomeric matrix protein (COMP) is a pentameric protein thats structurally re- lated to thrombospondin and is thought to have evolved from the thrombespondin-3 and -& branch ofthe family (Newton, 1994). COMP isa secretory product of chondrocytes and is local ied in their territorial matrix in cartilage. Mutations in COMP have been shown to be the cause of pscudoachondroplaia and a form of multiple epiphyscal dysplasia (Briggs et al, 1995). "The three members ofthe tenascin fumily (C, R, and X) are large hexameric (tenascin-C and or trimeric (tenascin-X) proteins with subunits composed of multiple protein modules (Er- ickson, 1993). The modules include fibronectin type HI repeats, EGF-like domains, and a car- boxyl domain with homology to the carboxyl-rerminal domains of B- and +-fibrinogen chains. “These modules form rodlike structures that interact with their amino-terminal domains within the ‘oligomers. Although mice that are homozygous for tenascin-C mull alleles have no obvious phe- notype (Saga et al, 1992), the multiple interactions and tissue expression patterns defined for ‘enascins strongly sugges tha they have important Functions. Tenascin-R is expressed in the czt- tral nervous system during development, tenascin is expressed particularly in muscle (smooth smuscle as well as cardiac and skeletal), and tenascin-C is found in healing wounds, ina wide vari- ‘ty of tumors and in the bran. In certain experimental conditions tenascin-C can bean adhesive ‘molecule fr cells it an also, however, ave antiadhesve effects (Chiquet-Ehrismann, 1995). The adhesive activity can be mediated by ether cell surface proteoglyean or integrins, depending om call type. Tenascin-C can bind heparin and this may be responsible for interactions with cell sur- face proteoglycans such as glypican (Erickson, 1993; Vaughan ef al, 1994). Tenascin-C can also block adhesion by covering up adhesive sites in other matrix molecules, such as fibronectin, ster- ically Blocking their interactions with cells, Interactions with specific “antiadhesive" cell surface re- ceptors are also conceivable. cell surface molecule on neurons, contactn, binds to both tenascin- ‘Cand-Rand may mediate thie effects on neurons; tenascin can both stimulate and inhibie neurite ourgrowth (Vaughan et, 1994).5_Matrix Molecules and Their Ligands o PROTEOGLYCANS: MULTIFUNCTIONAL ECM AND CELL SURFACE MOLECULES ‘Avatiery of proteoglycans play important roles in cellular growth and differentiation and in matrix structure. They range fiom the large hydrophilic space ling complexes of aggrecan and : vetsican with hyaluronan, tothe cell surface syndecan receptors. In basement membranes the ma- jor heparan sulfite proteoglycan is perlecan(lozzo, 1994; Timpl, 1994). With thee heparan sul fae side chains arached to the amino-terminal region, its core protein is mulkimodular in stuc. ture, having borrowed structural motif from a variety of other genes. These include a low-density lipoprotein (LDL) receptor-like module, regions with extensive homology to laminin chains, Jong stretch of N-CAMlike IgG repeats, and a carboxyl-terminal region with three globular and four EGF like repeats similar to a region of laminin. Alternative splicing can generate molecules of different lengths. Perlecan is present ina number of basement membranes, but is also found in the pericellular matrix of fibroblasts, Tn fac, fibroblasts, rather than epithelial eel, appear to be :major producers of petlecan (for example, in skin) In liver, perlecan is expressed by sinusoidal en- dothelial cells and is localized in the perisinusoidal space. The precise functions of perlecan have not been defined, but mutations in the wnc-52 gene in Caenorhabditis elegans, encoding a short version of perlocan, cause disruptions of skeletal muscle (Rogalski et al, 1993). This suggests that the molecule, asa component of skeletal muscle basement membranes, is important for assembly ‘of myofilaments and their attachment to cell membranes. Binding of growth factors and cytokines to the heparan sulfate side chains also enables perlecan to serve asa storage vehicle for biological. ly active molecules such as basic fibroblast growth factor (bFGF). 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