Báo cáo khoa học: Proteoglycans in health and disease: the multiple roles of syndecan shedding ppt

Couchman1 1 Deparment of Biomedical Sciences, University of Copenhagen, Denmark 2 Kennedy Institute of Rheumatology, Imperial College London, UK Introduction Syndecans are type 1 transme

Proteoglycans in health and disease: the multiple roles of syndecan shedding

Tina Manon-Jensen1, Yoshifumi Itoh2and John R Couchman1

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 healdur-ing 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.

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 calcium⁄ 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

Chondroitin sulphate

Heparan sulphate

C1 C2 V

Ser

Xyl Gal GlcA GlcNAc IdoA GalNAc

Ser

6-O 6-O

2-0 2-0 2-0 6-0

N N N 6-0 N N

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.

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 metalloproteinprote-ases (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.

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 zinc-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

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 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

MMP27 MMP20 MMP19 MMP18 MMP13

MMP15 (MT2-MMP) MMP14 (MT1-MMP)

MMP24 (MT5-MMP) MMP16 (MT3-MMP)

pro

s cat

Hpx Hpx Hpx Hpx

FNII

Fu

V

TM Cyt

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.

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-MT1-MMP-2 MT1-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

CS

HS

ADAMT-S1 and -S4

Plasmin Lys114-Arg115 and Lys 129-Val130 Thrombin Lys114-Arg115

MMP2

MMP7

MT1-MMP

Near the 1st GAG chain

MMP2 MMP9

Intracellular

Extracellular

Human: Gly245-Leu246 Mouse: Ala243-Ser244 MT3-MMP

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.

-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 prometallopro-teinases [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- or b-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

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-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

synde1 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

(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 In tissue culture, there may be constitutive

shed-ding, and in vivo enhanced shedding in cases of injury

and disease Because syndecans are important co-receptors for adhesion and growth factor co-receptors, their loss from the cell surface may have multiple effects There is certainly a need for a deeper under-standing of these processes, because they may relate to diagnosis, prognosis or even treatment options for some diseases Better reagents for detecting syndecan cleavage will be a valuable aid in these analyses, both

in vitro and in vivo This may be difficult, not least because so many different proteases can cleave the syndecan core proteins There is much to learn about when and where these events take place

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