MINIREVIEW Proteoglycans in health and disease: the multiple roles of syndecan shedding Tina Manon-Jensen 1 , Yoshifumi Itoh 2 and John R. Couchman 1 1 Deparment of Biomedical Sciences, University of Copenhagen, Denmark 2 Kennedy Institute of Rheumatology, Imperial College London, UK Introduction Syndecans are type 1 transmembrane heparan sulfate proteoglycans (HSPGs) that have important roles dur- ing development, wound healing and tumour progres- sion by controlling cell proliferation, differentiation, adhesion and migration. The heparan sulfate (HS) chains substituted on the extracellular domains interact with a wide range of ligands such as extracellular matrix glycoproteins, collagens, cytokines, chemokines, growth factors and enzymes, including metzincin pro- teinases. The ectodomain of each syndecan is constitu- tively shed in some cultured cells, but is accelerated in response to wound healing, and some pathophysio- logical events. Ectodomain shedding is an important regulatory mechanism, because it can rapidly generate soluble ectodomains that can function as paracrine or autocrine effectors or competitors. Mammals have four syndecan family members, syndecan-1 to -4 (Fig. 1), whereas invertebrates and primitive chordates possess only one syndecan, which is essential for neuronal development and axon guidance [1,2]. All cells express at least one member of the syndecan family [3], with the exception of erythrocytes. Syndecan-4 can be found in most tissues, but seems to be less abundant and is frequently coexpressed with other syndecans. Syndec- an-1 is highly expressed in epithelia, syndecan-2 in endothelia and fibroblasts, whereas high expression of Keywords cell adhesion; cell migration; glycosaminoglycan; growth factor; heparan sulfate; metzincin; proteinase; proteoglycan; receptors; signaling Correspondence J. R. Couchman, Department of Biomedical Sciences, University of Copenhagen Biocenter, Ole Maaløes Vej 5, 2200 Copenhagen N, Denmark Fax: +45 353 25669 Tel: +45 353 25670 E-mail: john.couchman@bric.ku.dk (Received 5 May 2010, revised 26 July 2010, accepted 28 July 2010) doi:10.1111/j.1742-4658.2010.07798.x Proteolytic processes in the extracellular matrix are a major influence on cell adhesion, migration, survival, differentiation and proliferation. The syndecan cell-surface proteoglycans are important mediators of cell spread- ing on extracellular matrix and respond to growth factors and other bio- logically active polypeptides. The ectodomain of each syndecan is constitutively shed from many cultured cells, but is accelerated in response to wound healing and diverse pathophysiological events. Ectodomain shed- ding is an important regulatory mechanism, because it rapidly changes sur- face receptor dynamics and generates soluble ectodomains that can function as paracrine or autocrine effectors, or competitive inhibitors. It is known that the family of syndecans can be shed by a variety of matrix pro- teinase, including many metzincins. Shedding is particularly active in prolif- erating and invasive cells, such as cancer cells, where cell-surface components are continually released. Here, recent research into the shed- ding of syndecans and its physiological relevance are assessed. Abbreviations ADAM, a disintegrin and metalloproteinase; GAG, glycosaminoglycan; GlcA, glucuronic acid; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine, HS, heparan sulfate; HSPG, heparan sulfate proteoglycan; MMP, matrix metalloproteinase; PKC, protein kinase C; PMA, phorbol myristate acetate; TIMP, tissue inhibitor of metalloproteinases. 3876 FEBS Journal 277 (2010) 3876–3889 ª 2010 The Authors Journal compilation ª 2010 FEBS syndecan-3 can mostly be found in neuronal tissues and some musculoskeletal tissue. Here, our under- standing of syndecan shedding and its function in wound healing and tumour progression is reviewed. Other reviews on syndecan structure and function have been recently published [4–6]. Structural organization of syndecans The syndecan core proteins range from 20 to 40 kDa and have cytoplasmic domains that are highly con- served across species, but have diversity in their ectod- omains. All comprise an ectodomain, a single transmembrane domain and a short cytoplasmic domain (Fig. 1). The cytoplasmic domain consists of membrane-proximal C1 and distal C2 conserved region flanking a variable region (V) that is unique to each syndecan, but highly conserved across species within each individual syndecan gene. The C2 region inter- acts with a number of PSD-95 ⁄ Discs-large ⁄ Zonula occludens-domain-containing proteins such as syntenin, Ga-interacting protein (GAIP)-interacting C-terminus ⁄ synectin and calc ium ⁄ calmodulin-associated serine kinase, since the C2 region contains a class II PSD-95 ⁄ Discs-large ⁄ Zonula occludens protein-binding motif FXF, where F represent a hydrophobic residue and X any amino acid residue. Although information is sparse, current evidence suggests that the C1 region can interact with ezrin, at least for syndecan-2, which provides a link to the actin cytoskeleton [7]. The cen- tral V-region probably contains sites for syndecan-spe- cific interaction partners, although this is only well understood for syndecan-4 [4,8]. A ternary signalling complex with phosphatidylinositol 4,5-bisphosphate and protein kinase Ca has been described [9], whereas others partners are the actin-associated protein a-acti- nin as well as syndesmos, about whose function rather little is known [10]. The transmembrane domain of all syndecans contains a GXXXG motif that promotes formation of SDS-resistant dimers [11,12]. The N-ter- minal ectodomain has glycosaminoglycan (GAG) chain substitution sites. These are predominantly HS cova- lently linked to serine residues in a serine–glycine motif surrounded by acidic residues. In addition to HS, syndecan-1 and -3 can be substituted with chondroitin or dermatan sulfate at sites closer to the transmem- brane domain. The synthesis of GAG chains in the Golgi apparatus is a highly complex process, but both HS and chon- droitin sulfate chains are linked to serine residues on Syndecan-1 Syndecan-2 Syndecan-4Syndecan-3 Chondroitin sulphate Heparan sulphate 33 kDa 23 kDa 43 kDa 22 kDa C1 C2 V Ser Xyl Gal GlcA GlcNAc IdoA GalNAc Ser 6-O 6-O 2-0 2-0 2-0 6-0 NNN 6-0 NN Fig. 1. Schematic of the four vertebrate syndecans. Syndecans-1 and -3 core proteins are larger than those of syndecan-2 and -4, and can bear both heparan and chondroitin sulfate chains. The GAG chains are substituted on core protein serine residues and have a common stem tetrasaccharide of xylose (xyl), two galactose units (gal) and a glucuronic acid residue (GlcA). The repeating disaccharide of HS is N-acetylglu- cosamine and uronic acid, followed by several modifications in terms of sulfate and uronic acid epimerization to iduronic acid. The glucosa- mine can be N-, 6-O or (rarely) 3-O sulfated, whereas the iduronic acid can be 2-O sulfated. In most cases, there are regions of low sulfation, for example, adjacent to the core protein, with regions of intermediate or high sulfation. This yields a polysaccharide of immense variability and complexity. Chondroitin sulfate contains N-acetylgalactosamine, which may be 6-O or 4-O sulfated. The cytoplasmic domains have two highly conserved regions (C1 and C2) with an intervening syndecan-specific variable (V) region. T. Manon-Jensen et al. Syndecan shedding at the cell surface FEBS Journal 277 (2010) 3876–3889 ª 2010 The Authors Journal compilation ª 2010 FEBS 3877 the core protein through a tetrasaccharide linker con- sisting of xylose–galactose–galactose–uronic acid resi- dues, followed by the repeating disaccharide units. The repeating unit of HS and chondroitin sulfate back- bones are glucuronic acid (GlcA)–N-acetylglucosamine (GlcNAc) or GlcA–N-acetylgalactosamine (GalNAc), respectively. These chains range from 50 to 200 disac- charides in length and undergo extensive modification in which some uronic acid residues are epimerized and a number of sulfation events occur (Fig. 1). In the case of HS, chain modifications are not uniform but local- ized along the chain. Subdomains of low sulfation are interspersed among regions that are highly sulfated, and small regions of intermediate sulfate lie at the boundaries of these subdomains [13,14]. How the syn- thesis of such complex polysaccharides is controlled remains unknown. Syndecan shedding Syndecans undergo regulated proteolytic cleavage, usu- ally near the plasma membrane, in a process known as shedding. The release of syndecan extracellular domains may not only downregulate signal transduc- tion, but also convert the membrane-bound receptors into soluble effectors ⁄ or antagonists. Soluble syndecan ectodomain can compete with intact syndecans for extracellular ligands in the pericellular environment [15] (Fig. 2). The remaining portion of the membrane- bound receptor loses its ability to bind ligands, and can be further processed by the presenilin ⁄ c-secretase complex. Like many other type I transmembrane proteins [16], syndecan-3 has been shown to undergo restricted intramembrane proteolysis by the membrane presenilin ⁄ c-secretase complex within the hydrophobic environment (mainly between Leu403 and Val404) of the phospholipid bilayer of the membrane [17]. In turn, there is decreased plasma membrane targeting of the transcriptional cofactor calcium ⁄ calmodulin-associ- ated serine kinase. Signaling is not restricted to the syndecan proteoglycans but can be evoked by extracel- lular proteoglycans binding to cell-surface receptors. The leucine-rich proteoglycans are discussed in this context by Iozzo & Schaefer [18] in this minireview series. Matrix metalloproteinases Ectodomain shedding itself is a highly regulated pro- cess that requires the direct action of enzymes gener- ally referred to as sheddases. All mammalian syndecan family members can be cleaved by extracellular prote- ase [3]. The matrix metalloproteinases (MMPs) are known sheddases of syndecans, and are endopeptidases belonging to the family of metzincins (zinc endopeptid- ases) which contain three major multigene families: seralysins, astacins and a disintegrin and metallo- proteinase (ADAM) ⁄ adamlysins. Substrate specificity for MMPs is broad, therefore they function in many physiological processes and are key to normal matrix turnover, but also have essential roles in development and reproduction, and in pathological tissue remodel- ling during inflammatory disease, cancer invasion and metastasis. Normally, MMPs cleave substrates before HS MMP9 Heparanase ERK Syndecan CS Soluble ectodomain Intramembrane proteolysis by the membrane presenilin/γ-secretase complex Intracellular Extracellular Fig. 2. Shedding of syndecans by metzincin proteinases. Several metzincin enzymes can cleave the syndecan core proteins, for example MMP9, the site(s) being mem- brane-proximal. Shedding is reported to be enhanced if the HS chains are first cleaved by heparanase. The shed syndecan may be deposited in the pericellular matrix, whereas the remnant core protein at the cell surface may be further processed by intramembrane cleavage by the presenilin ⁄ c-secretase com- plex. There may also be signalling through MAP kinases. Syndecan shedding at the cell surface T. Manon-Jensen et al. 3878 FEBS Journal 277 (2010) 3876–3889 ª 2010 The Authors Journal compilation ª 2010 FEBS a hydrophobic residue like Leu, Ile, Met, Phe or Tyr, whereas cleavage before a charged residue is rarely seen [19]. Twenty-three human MMPs have been identified which can be divided into eight distinct structural groups, five of which are secreted and three are mem- brane-bound (MT-MMPs) (Fig. 3). The general form of MMPs include an N-terminal signal sequence that directs them to the endoplasmic reticulum, a propep- tide (Pro) containing a cysteine switch motif PRCGXPD (except for MMP23 which lacks the cysteine switch motif) that maintains them as inactive zymogens, and a catalytic domain with a z inc-binding site (Zn, HEXXHXXGXXH) and a conserved methionine (Met-turn) supporting the catalytic zinc. Interaction between cysteine–zinc maintains proMMPs in an inac- tive state by preventing a water molecule from binding to the zinc atom. All MMPs, with the exception of MMP-7, MMP-23 and MMP-26, also contain a hemopexin-like domain that is connected to the catalytic domain by a hinge region and mediates inter- actions with tissue inhibitors of metalloproteinases, cell-surface molecules and proteolytic substrates. The first and last of the four repeats in the hemopexin-like domains are linked by a disulfide bond (S–S) [19]. Two gelatinase MMPs (MMP-2 and MMP-9) con- tain additional inserts that resemble collagen-binding type II repeats of fibronectin. MMP-11 and MMP-28 contain a basic amino acid motif [KX(R ⁄ K)R] recog- nized by furin-like serine proteinases between their propeptide and catalytic domains that results in their intracellular activation. This motif is also found in MMP-21 with the vitronectin-like insert (Vn), MMP- 23 and the membrane-type MMPs (MT-MMPs) [19]. All soluble MMPs that do not harbour the basic motif at the end of propeptide are secreted as zymogens and activated extracellularly through proteolytic removal of propeptide. Active MMPs, plasmin, cathepsin G and neutrophil elastase have all been associated with this function. MT-MMPs can be subdivided into transmembrane (TM) forms and those that are glycosylphosphatidylinositol anchored. The TM-type MT-MMPs (MMP-14, MMP-15 and MMP-24) have a single-span transmembrane domain and a very short cytoplasmic domain. Alternately MMP-17 and MMP- 25 are glycosylphosphatidylinositol-anchored MMPs. The type II membrane-linked MMP, MMP-23, has an N-terminal signal anchor targeting it to the cell mem- brane. Also, it is characterized by unique cysteine array and immunoglobulin-like domains. In healthy adults, activity of MMPs is difficult to detect, except under conditions of tissue remodelling, for example, in wound healing and menstrual endo- metrium. Under physiological conditions, the activity of MMPs is regulated by transcription, activation of the precursor zymogen and by interactions with spe- cific extracellular matrix components. In addition, endogenous tissue inhibitors of metalloproteinases provide a balance to prevent excessive degradation of extracellular matrix. This physiological balance may be disrupted in cancer. In many cancers, MMP expression is upregulated and correlates with poor prognosis [20,21]. Nevertheless, under some circum- stances specific MMPs have a dual antitumour effect [22]. Tissue inhibitor of metalloproteinases The catalytic activity of MMPs can be inhibited by the family of tissue inhibitor of metalloproteinases (TIMP), of which there are four members (TIMP1-4). TIMP-1, -2 and -4 are diffusible secreted proteins, Type I transmembrane GPI-anchored Gelatin-binding Minimal Simple hemopexin-containing Furin-activated secreted MMP17 (MT4-MMP) MMP25 (MT6-MMP) MMP7 MMP26 MMP1 MMP12 MMP10 MMP8 MMP3 MMP28 MMP11 MMP9 MMP2 MMP27 MMP20 MMP19 MMP18 MMP13 MMP15 (MT2-MMP) MMP14 (MT1-MMP) Type II transmembrane MMP23 MMP24 (MT5-MMP) MMP16 (MT3-MMP) Vitronectin-like MMP21 pros cat Hpx Hpx Hpx Hpx FNII Fu V TM Cyt TM IgCysR GPI Fig. 3. Schematic of mammalian matrix me- talloproteinases. The domain structures of the various groups are shown, with a list of some members. S, signal peptide; Cat, catalytic domain; Pro, pro domain; TM, transmembrane domain; Cyt, cytoplasmic domain; Fu, furin cleavage site; Hpx, hemopexin domain; Fn, fibronectin type II repeats; V, vitronectin-like domain; CysR, cysteine array; Ig, immunoglobin-like domain, GPI, glycosylphosphatidylinositol linker. T. Manon-Jensen et al. Syndecan shedding at the cell surface FEBS Journal 277 (2010) 3876–3889 ª 2010 The Authors Journal compilation ª 2010 FEBS 3879 whereas TIMP-3 is matrix associated because of its heparin-binding characteristics which promote its asso- ciation with matrix proteoglycans [23,24]. TIMP-3 binds to sulfated glycosaminoglycans such as heparin, HS, chondroitin 4- and 6-sulfates, dermatan sulfate, and sulfated compounds such as suramin and pento- san, enabling interaction with GAG chains of synde- cans [25]. Only TIMP-3 of the TIMP family has been shown to effectively block shedding of syndecan-1 and -4 in mouse mammary epithelial cells [26]. Each TIMP can inhibit most MMPs, except TIMP-1 that, in particular, fails to inhibit several of the mem- brane-type MMPs, MMP-14, -15, -16 and -24. The inhibitory effect of TIMP-3 is different from the oth- ers, as it also inhibits other metzincin subgroups, for example the ADAM ⁄ adamlysins, including ADAM-17 (TACE) [27], ADAM-10 [28] and ADAM-12 [29], and the ADAMs with thrombospondin motifs (ADAMTS) including the aggrecanases ADAMTS4 and ADAM- TS5 [30]. Kinetic studies have shown that TIMP-3 is effective inhibitor of ADAM-17 (TACE) and aggre- canases [27,30]. All mammalian TIMPs consist of two distinct domains, N-terminal ( 125 amino acids) and C-terminal ( 65 amino acids), where the N-terminal domain usually is responsible for inhibition of protein- ase activity. However, recently it has been shown that the isolated N-terminal domains of TIMP-1 and TIMP-3 are insufficient for ADAM10 inhibition, whereas full-length TIMP-1 and TIMP-3 are [31]. The C-terminal domain of TIMPs can stabilize proMMP by binding to its hemopexin domain, leaving the N-ter- minal fully capable of interacting with other MMPs. Most cell types secrete proMMP-9 in complex with TIMP-1, which complex can be found in the Golgi apparatus [32]. TIMPs -2, -3 or -4 can bind proMMP2, whereas TIMP-1 and -3 can interact with proMMP9. TIMPs also facilitate activation of MMPs, by for example, functioning as an adaptor between MT1- MMP and Pro-MMP-2. MT1-MMP alone cannot bind proMMP2, but the N-terminal region of TIMP-2 binds the catalytic domain of MT1-MMP inhibiting its activity, whereas its C-terminal domain binds to the hemopexin-like domain of Pro-MMP-2 forming a ternary complex. The complexed MT1-MMP cannot cleave Pro-MMP-2, but requires a second MT1-MMP molecule (without TIMP-2). Thus cleavage and activa- tion of proMMP-2 require both active and inactive MT1-MMP [33,34]. This process is facilitated by ho- modimerization of two MT1-MMP molecules through its hemopexin and transmembrane domains [35]. Syndecan sheddases The glycosaminoglycan-bearing ectodomains of mam- malian and Drosophila syndecans can be constitutively shed from the cell surface as part of the normal turn- over [3,26,36–39]. This constitutive shedding involves metalloproteinases, but may be distinct from the metal- loproteinase activity that mediates accelerated shedding in response to wound healing, for example [26]. Evidence indicates the involvement of several MMPs in syndecan cleavage in vitro and in vivo (Fig. 4). Matrilysin (MMP-7) cleaves syndecan-1 [40], gelatinas- es MMP-2 and MMP-9 can cleave syndecans-1, -2 and Syndecan-1 Syndecan-4 CS HS ADAMT-S1 and -S4 Plasmin Lys114-Arg115 and Lys 129-Val130 Thrombin Lys114-Arg115 MMP2 MMP9 MMP7 MT1-MMP Near the 1st GAG chain MMP2 MMP9 Intracellular Extracellular Human: Gly245-Leu246 Mouse: Ala243-Ser244 MT3-MMP ADAM17 ADAM17 Fig. 4. Documented examples of metzincin proteinases that shed syndecans-1 and -4. Only in a few cases are the precise cleavage sites known. Most sites are believed to be membrane-proximal, although ADAMTS-1 and -4 may cleave syndecan-4 close to the N-terminus. CS, chondroitin sulphate; HS, heparan sulphate. Syndecan shedding at the cell surface T. Manon-Jensen et al. 3880 FEBS Journal 277 (2010) 3876–3889 ª 2010 The Authors Journal compilation ª 2010 FEBS -4 [41,42], whereas the membrane-associated metallo- proteinases MT1-MMP and MT3-MMP are known to cleave syndecan-1 [43]. However, current knowledge of precise cleavage-specific sites on syndecan core proteins is sparse. Human syndecan-4 is cleaved by the serine proteases, plasmin and thrombin, at Lys114– Arg115 ⁄ Lys192–Val130 and Lys114–Arg115, respec- tively [44]. Despite high sequence homology between human and mouse syndecan-1, they have distinct MT1-MMP cleavage sites: human syndecan-1 is cleaved at Gly245–Leu246, whereas cleavage of mouse syndecan-1 occurs at Ala243–Ser244 [43,45]. The ADAM family of disintegrin and metallopro- teinase membrane-anchored proteinases [46] also par- ticipate in syndecan shedding. ADAM17 (TACE) has recently been reported to shed syndecan-1 and syndec- an-4 [47]. The cysteine-rich domain of human ADAM12 was shown to associate with the ectodomain of syndecan-4 and is regulated by HS; however, direct ectodomain interactions with other members of the ADAM family are not known [48,49]. The ADAMTS family (disintegrin and metallopro- teinase with thrombospondin motifs) [50] also associ- ates with syndecans. It has been reported that the p53 form ADAMTS4 binds HS and chondroitin sulfate chains of syndecan-1 and aggrecan [51,52]. A recent study also reported that syndecan-4 may regulate acti- vation of ADAMTS-5 via engagement of HS chains and regulation of MAPK-dependent synthesis of MMP3 during cartilage damage in osteoarthritis [53]. Therefore, lack of syndecan-4 may be chondroprotec- tive in some models of osteoarthritis. Both ADAMTS-1 and ADAMTS-4 have been demonstrated to cleave syndecan-4 near the first GAG-attachment site, rather than close to the membrane. This was shown to decrease cell adhesion and promote cell migration [54]. Regulation of syndecan shedding Syndecan shedding occurs through the direct action of sheddases, although a variety of extracellular stimuli including growth factors [55], chemokines [40,41,56], bacterial virulence factors [57,58], trypsin [36], insulin [59], heparanase [60] and cell stress [26] are known to induce syndecan shedding. It is not yet clear how extracellular stimuli influence sheddases to mediate syndecan cleavage, but different agonists appear to activate distinct intracellular signalling pathways to activate shedding. Chemical inhibitor studies suggest involvement of various signal transduction cascades, such as protein kinase C (PKC), protein tyrosine kinase, nuclear factor jB and mitogen-activated pro- tein kinase pathways. For example, epidermal growth factor- and thrombin receptor-mediated shedding cor- relates with activation of the ERK ⁄ MAPK pathway, and does not appear to involve PKC activation. Inhibition of PKC activity prevents phorbol myristate acetate (PMA)- and cellular stress-induced shedding of syndecans, but does not affect thrombin or epidermal growth factor receptor-activated shedding [26,55]. Interestingly, some pathogens usurp the host cell shedding machinery to neutralize the host innate sys- tem to promote their own pathogenesis by elevation of syndecan shedding in response to bacterial virulence factors [61–63]. For example, Staphylococcus aureus, a common Gram-positive bacterium implicated in life- threatening diseases like endocarditis and osteomyeli- tis, enhances shedding of syndecan-1 through a-toxin and b-toxin [58]. Beta-toxin, but not a-toxin, also mediates shedding of syndecan-4. Alpha- and b-toxins do not directly trigger syndecan-1 shedding, but acti- vate protein tyrosine kinase-dependent intracellular sig- nalling pathways that stimulate syndecan-1 shedding [58]. Bacterial proteases can also enhance syndecan shedding by mimicking the direct shedding effect of syndecan sheddases [64]. For example, Streptococ- cus pneumoniae sheds syndecan-1 directly through ZmpC, a metalloproteinase virulence factor, where the size of the shed soluble ectodomain is smaller than that derived from a-orb-toxin mediated shedding [57]. Other pathogens may utilize HSPGs as attachment receptors to facilitate either their entry into the host cells or their survival in the host environment. For example, the capsid ORF2 protein of hepatitis E virus interacts mainly with 6-O-sulfate of syndecan-1 in Huh-7 liver cells for productive infection [65]. Intracellular regulatory mechanisms play important roles in agonist-induced shedding. Syndecans possess highly conserved transmembrane and cytoplasmic domains, the latter having three conserved tyrosine res- idues and a variable number of serine ⁄ threonine resi- dues that can serve as phosphorylation sites [66]. Phosphorylation of tyrosine residues has been sug- gested to positively regulate syndecan-1 shedding [26,55,67]. The phosphatase inhibitor pervanadate and activation of intracellular kinases leads to tyrosine phosphorylation and shedding of syndecan-1 [68]. Hayashida et al. [69] confirmed the pervanadate effect on syndecan-1 shedding, but showed that S. aureus b-toxin and PMA-mediated shedding was not accom- panied by tyrosine phosphorylation. However, tyrosine to phenylalanine mutation reduced the syndecan shedding, suggesting mechanisms other than phosphor- ylation, such as binding to other cytoplasmic compo- nents is critical in agonist-mediated shedding. For example, syndecan-1 cytoplasmic domain interacts with T. Manon-Jensen et al. Syndecan shedding at the cell surface FEBS Journal 277 (2010) 3876–3889 ª 2010 The Authors Journal compilation ª 2010 FEBS 3881 the inactive, GDP-bound form of Rab5, a small GTPase that regulates intracellular trafficking and triggers its conversion to an active GTP-bound state in response to shedding promoters. A dominant negative form of Rab5, unable to switch between active and inactive states, significantly inhibited syndecan-1 shed- ding, suggesting that trafficking is a key regulator of syndecan-1 shedding [69]. Wound healing Wound healing is a regulated process that can be divided into three sequential, yet overlapping, phases; inflammation, proliferation and remodelling [70]. Synd- ecan-4 is upregulated in a range of inflammatory con- ditions like ischaemic myocardial injury [71], and dermal wound repair [72]. For example, atherosclerosis is a chronic inflammatory disease marked by aberra- tions in cell migration, proliferation and low-density lipoprotein internalization [73]. Oxidized linoleic acid, the major oxidized fatty acid in low-density lipopro- tein, upregulates expression of syndecan-4, and as a consequence, accelerated shedding of syndecans-4 involving the MEK pathway [74]. Increased levels of syndecan-1 ectodomain are present in dermal wound fluid, and in serum from patients with acute graft-ver- sus-host disease [75]. A key inflammatory response is chemokine-mediated recruitment of leukocytes into sites of inflammation [76]. Many chemokines bind HS chains of syndecans and evoke MMP-mediated shedding of syndecans with potential loss from the site of injury [40,41,56]. MMP- 7 is upregulated in injured mucosal epithelium of the lung, and promotes inflammation by shedding a synd- ecan-1 ⁄ KC (CXCL8) complex that directs neutrophil influx to the sites of injury [40]. Soluble syndecan-1 may maintain the proteolytic balance of acute wound fluids, because it can bind the inflammation-related neutrophil proteases cathepsin G and elastase, conse- quently decreasing their affinity for their physiological targets [37]. The function of syndecan-1 shedding in wound heal- ing is not restricted to inflammation, but serves also to promote re-epithelialization; however, this is not fully clarified. Proliferating keratinocytes at the wound edge and endothelial cells in the wound bed transiently express syndecan-1 [77], whereas keratinocytes migrat- ing into the wound lose their cell-surface syndecan-1 expression [37]. Syndecan-1 and syndecan-4 are shed and may accumulate in dermal wound fluids [55]. Using a noncancerous simple epithelium cell line (BEAS-2b) and organotypic cultures derived from pri- mary epithelial cells, it has been demonstrated that syndecan-1 is shed primarily by MMP-7 from epithe- lial cells after injury [78], which enhances cell migra- tion and facilitates wound closure. Therefore, syndecan-1 shedding appears to be an important response in wound healing. MMP-7 null mice demon- strate a severely diminished re-epithelialization in response to lung injury. Suppression of syndecan-1 expression in simple epithelial cells induces a promigra- tory phenotype [79,80], consistent with decreased synd- ecan levels in injured stratified epithelium (cornea and skin) during repair [81,82]. Furthermore, knockdown of syndecan-1 expression resulted in slowed cell migra- tion in an A549 (a carcinoma-derived alveolar type II) cell line [83]. Interestingly, soluble syndecan-1 ectodo- main inhibited wound repair in mice overexpressing syndecan-1, by exhibiting delay in wound closure, re-epithelialization, granulation tissue formation and remodelling [84]. Overall, the studies reveal that MMP-7 cleavage of syndecan-1 is essential for effective re-epithelialization; however, a balance is critical because soluble syndecan-1 overexpression or complete absence of syndecan-1 in the knockout lead to impair- ment. The function of syndecan-1 may be tissue spe- cific, because syndecan-1 null primary dermal fibroblasts migrated faster than wild-type cells [85]. E-cadherin, a known mediator of cell–cell contact, is also shed in vivo from injured lung epithelium by MMP-7 [86], and has been shown to be coordinately regulated with syndecan-1 [79]. It is not known if shedding of E-cadherin and syndecan-1 happen contig- uously, but could synergistically promote a migratory epithelial phenotype. It is well known that syndecans are functionally cou- pled to integrins [4], which represent the major group of cell-surface receptors for extracellular matrix macro- molecules. There are 24 heterodimeric integrins in mammals, each composed of an a and a b subunit derived from combinations of 8 b and 18 a subunits. Interaction between syndecan and integrins may be direct [87] or indirect through an intermediate ‘recep- tor’ [88]. This adhesion mechanism can be HS indepen- dent, because the cell adhesion properties of syndecans are not only limited to the HS chains, but can also be mediated through the ectodomain core protein. The evolutionarily conserved NXIP motif of syndecan-4 has been shown to promote b1-integrin-dependent cell adhesion [89]. Syndecan-1 ectodomain regulates avb3 and avb5 integrin-mediated attachment and spreading in human mammary carcinoma cells and B82L fibro- blasts, respectively. The activity has been mapped to residues 88–252 within the syndecan-1 ectodomain [90,91]. This association can be blocked by synstatin, a peptide inhibitor corresponding to the active site of the Syndecan shedding at the cell surface T. Manon-Jensen et al. 3882 FEBS Journal 277 (2010) 3876–3889 ª 2010 The Authors Journal compilation ª 2010 FEBS syndecan-1 core protein, and which can suppress angiogenesis in vitro and in vivo, perhaps signifying syndecan-1 as a critical mediator of tumour progres- sion [87]. Another motif, the AVAAV (amino acids 222-226), only present within the syndecan-1 ectodomain, has been suggested to be an invasion regulatory domain, because mutation within this region abolishes syndec- an-1-mediated inhibition of cell invasion [92]. How- ever, the mechanism remains unknown. Integrins and syndecans together may influence the outcome of cell adhesion and migration because their different activation states and clustering on the cell surface result in varying degrees of mechanical force exerted on the extracellular matrix [5]. Syndecan-1 shedding by MMP-7 from repairing simple epithelial (BEAS-2b) cells after injury [77] enhances cell migra- tion and facilitates wound closure by causing the a2b1 integrin to assume a less-active conformation, compati- ble with migration. It has previously been shown that syndecan-1 facilitates integrin a2b1-mediated adhesion to collagen [93]. Tumour progression In addition to genetic and epigenetic changes, tumour progression links a series of steps involving adhesion, motility and growth, resulting in metastatic spread, a major cause of death among cancer patients. These steps are influenced by the activity of tumour-derived MMPs. MMPs facilitate metastasis by degrading extra- cellular matrix components, such as collagens, laminins and proteoglycans, and they modulate cell adhesion, enabling turnover of matrix contacts or adhesions. Novel roles for proteoglycans in malignancy are also discussed elsewhere in this volume [94]. As part of the regulation of MMPs, rate-limiting effects, such as zymogen activation and the availability of TIMPs are important. Another control element may be contributed by HS chains of proteoglycans, which interact with many extracellular protease, with exam- ples from all four classes of proteases (aspartyl-, seryl-, cysteyl-protease and metalloproteases). Heparan sulfate also interacts with protease inhibitors, for example TIMP-3 [95] and antithrombin III (ATIII). These interactions may control extracellular matrix degrada- tion, by either modifying enzymatic activity through activation or inhibition, or providing a reservoir of latent enzyme that is positioned for directed proteolytic attack on extracellular matrix proteins. For example, highly sulfated HS has been shown to inhibit the proteolytic degradation of aggrecan, in part through direct inhibition of aggrecanase activity [96]. Further- more, HS chains of syndecans bind tumour-associated MMPs, MMP-2, -7, -9 and -13 [97], in which MMP-2 catalytic activity is inhibited by its interaction with HS chains of syndecan-2 [98], whereas MMP-1, -7 and -13 catalytic activity increases in the presence of heparin [97]. MMP-7 has been shown to promote syndecan-1 shedding upon growth factor activation (FGF-2), achieving its own release although still being attached to HS chains [97]. Other attributes of HS chains include the ability of TIMP-3 to interact with cell-sur- face HS. This may lead to inhibition or internalization of cell-surface MMPs or ADAMs, because it has been discovered that TIMP-3 is internalized in HEK293 and HTB94 chondrosarcoma cells [99], a process that is mediated by cell-surface glycosaminoglycans [99,100]. Overall, HS chains of syndecans may support inva- sion of tumour cells by protecting and anchoring matrix-degrading proteases, while also harbouring sig- nalling molecules that promote growth and directional migration. However, the MMP-13 C-terminal domain has been shown using yeast two-hybrid analysis to associate with syndecan-4 without HS chains, suggest- ing alternative MMP interaction sites than GAG chains [101]. The role of syndecans in tumour progression may vary with tumour stage and type, because syndecan-1 is reported to be downregulated in several types of breast cancer [102], but upregulated in several tumours, such as pancreatic cancer. Soluble syndecan- 1 ectodomain can be found in the serum of lung can- cer patients [103] and Hodgkin’s lymphoma patients [104], in the extracellular matrix of myeloma biopsies, as well in the serum of myeloma patients [105,106], to a much greater degree than in healthy individuals [107]. A recent study has distinguished the roles between membrane-bound and shed form of syndecan-1 in breast cancer epithelial cells (MCF-7) in vitro. The membrane-bound form of syndecan-1 increased prolif- eration and inhibited invasiveness, whereas the soluble form had the opposite effect, by promoting invasive- ness and inhibiting proliferation [108]. Perhaps the best evidence for the importance of shedding in cancer is shown for syndecan-1 in mye- loma. Multiple myeloma is a malignant proliferation of the bone marrow plasma cells increasing angiogene- sis and development of osteolytic bone disease. Soluble syndecan-1 promotes the growth of myeloma tumours in vivo [109]. High levels of shed syndecan-1 in the sera of myeloma patients are a marker of poor prognosis [105,107,110]. Heparanase seems to play a distinct role in shedding syndecans in myeloma. Mammalian heparanase T. Manon-Jensen et al. Syndecan shedding at the cell surface FEBS Journal 277 (2010) 3876–3889 ª 2010 The Authors Journal compilation ª 2010 FEBS 3883 (endo-b-d-glucuronidase) is known to modulate synde- cans by cleaving the less-sulfated regions along the HS chain releasing fragments of 10–20 sugar residues [111] (Fig. 2). It may function in tumour progression by promoting tumour growth, angiogenesis and metastasis [112] by both enzymatic and nonenzymatic mecha- nisms. A recently described nonenzymatic mechanism of heparanase is its ability to facilitate cell adhesion and spreading by clustering of syndecan-1 and syndec- an-4 through interaction with their HS chains [113]. Knockdown of heparanase in myeloma cell lines decreases soluble syndecan-1 [114]. In support, active heparanase was shown to accelerate myeloma cell growth and promote bone metastasis by increasing the number and size of blood vessels within the tumour [115,116]. Heparanase function in tumour progression is discussed by Barash et al. [117] in this minireview series. Elevated active heparanase has been demonstrated to enhance syndecan-1 shedding through ERK signal- ling, which in turn upregulates expression of two pro- teases, MMP-9 and urokinase-type plasminogen activator [118]. Recently, it has been shown that hepa- ranase-enhanced shedding of syndecan-1 by myeloma cells promoted endothelial invasion and angiogenesis [118]. Heparanase also increased urokinase-type plas- minogen activator receptor expression levels [119], and can even initiate syndecan-1 expression in the ARH-77 (human plasma cell leukemia) cell line that is normally negative for syndecan-1 [60]. The expression of uroki- nase-type plasminogen activator and its receptor may also be a predictor of poor prognosis, just as with shed syndecan-1 and heparanase [120]. The gelatinase MMP-9 sheds syndecan-1 directly [41], and has been suggested as a useful prognostic index of bone disease [121]. In addition, myeloma cell invasiveness can be promoted by MMP-9 in vitro [122], consistent with data suggesting that MMP-9 inhibition has antimyeloma effects [123]. Urokinase, by contrast, has a more indirect effect on syndecan shedding. Its activity in generating plasmin from plasminogen has been suggested to be a major activator of MMPs in vivo, where it can process proMMP into active MMP. In turn, these shed syndecans directly and⁄ or activate other MMP sheddases. For example, plasmin directly activates proMMP-1, proMMP-3, proMMP-9, proMMP-10 and proMMP-13 in vitro [124]. Conclusions and perspectives Syndecan shedding is subject to highly complex regula- tion. 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