Báo cáo khoa học: Localizing matrix metalloproteinase activities in the pericellular environment doc

Impor-tantly, MT1-MMP activates proMMP2 via a mecha-nism in which the tissue inhibitor of MMPs, tissue inhibitor of metalloproteinase TIMP-2, bound to the MT1-MMP catalytic domain acts a

Localizing matrix metalloproteinase activities in the

pericellular environment

Gillian Murphy1 and Hideaki Nagase2

1 Department of Oncology, University of Cambridge, Cancer Research UK Cambridge Institute, Li Ka Shing Centre, Cambridge, UK

2 Department of Matrix Biology, The Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, UK

Introduction

Timely alteration of extracellular matrix (ECM)

com-position and the pericellular environment is essential in

many biological processes such as embryonic

develop-ment, morphogenesis, cell migration, differentiation,

apoptosis and tissue remodeling In diseases such as

cancer [1], atheroma, arthritis, neurodegenerative

dis-eases and various connective tissue disdis-eases, these

pro-cesses become dysregulated Matrix metalloproteinases

(MMPs) are pivotal effectors of the cellular

microenvi-ronment and modulate cellular activities and tissue

structure throughout development and physiological

and pathological remodeling [2] All 23 human MMPs

harbor signals that direct them to the endoplasmic

reticulum and hence to the cell surface or to secretion

The extracellular activities of the MMPs are multiple, cleaving not only the components of the ECM, but also many of the bioactive molecules at or around the cell surface Because there is considerable overlap in substrate specificity amongst the MMPs, mechanisms

to preclude redundancy exist A number of post-secre-tory regulations of MMP activities have been described [3] Specific MMPs do have greater affinity for specific substrates and the concentrations of the active enzyme and the preferred substrate relative to other substrates may be determinants of efficacy [4] The mechanisms

by which cells can regulate their function in a spatial fashion pose intriguing questions and are clearly of critical importance in relation to remodeling events,

Keywords

CD44; collagen; extracellular matrix; integrin;

proteoglycan; receptor; tetraspanin

Correspondence

H Nagase, Department of Matrix Biology,

The Kennedy Institute of Rheumatology

Division, Faculty of Medicine, Imperial

College London, London W6 8LH UK

Fax: +44 02083834488

Tel: 02083834994

E-mail: h.nagase@imperial.ac.uk;

h.nagase@ic.ac.uk

(Received 26 June 2010, revised 15

September 2010, accepted 22 September

2010)

doi:10.1111/j.1742-4658.2010.07918.x

Matrix metalloproteinases (MMPs) are a group of structurally related pro-teolytic enzymes containing a zinc ion in the active site They are secreted from cells or bound to the plasma membrane and hydrolyze extracellular matrix (ECM) and cell surface-bound molecules They therefore play key roles in morphogenesis, wound healing, tissue repair and remodeling in dis-eases such as cancer and arthritis Although the cell anchored membrane-type MMPs (MT-MMPs) function pericellularly, the secreted MMPs have been considered to act within the ECM, away from the cells from which they are synthesized However, recent studies have shown that secreted MMPs bind to specific cell surface receptors, membrane-anchored proteins

or cell-associated ECM molecules and function pericellularly at focussed locations This minireview describes examples of cell surface and pericellu-lar partners of MMPs, as well as how they alter enzyme function and cellular behaviour

Abbreviations

ECM, extracellular matrix; GPI, glycosylphosphatidylinositol; HB-EGF, heparin-binding epidermal growth factor; LRP, low-density lipoprotein receptor-related protein; MMP, matrix metalloproteinase, MT-MMP, membrane-type matrix metalloproteinase; TGF, transforming growth factor; TIMP, tissue inhibitor of metalloproteinase.

directed cell migration and the directed interactions

with other cells Among all the MMPs, six are

mem-brane-type (MT)-MMPs that have specific domains

sequestering them at the cell membrane The majority

of MMPs are secreted into the extracellular space,

although there is accumulating evidence that they may

be recruited back to the local cell environment by

interactions with cell surface proteins and the

pericellu-lar matrix (Table 1) This minireview describes some of

the ingenious devices that have been constructed to

focus and regulate the proteolytic activity of the

MMPs in the pericellular environment

MMP domain structure

Besides the archetypal secretory signal sequence,

regu-latory propeptide and catalytic domain, the MMPs

have a C-terminal hemopexin-like domain, with the

exception of MMP-7, MMP-23 and MMP-26 MMP-2

and MMP-9 also have three repeats of the fibronectin

type II motif inserted into the catalytic domain There

are six membrane-anchored MMPs, termed ‘mem-brane-type’ (MT); four are transmembrane proteins with short cytoplasmic domains (MT1-, MT2-, MT3-and MT5-MMP) MT3-and two with glycosylphosphatidyli-nositol (GPI) anchors (MT4- and MT6-MMP) [5] Although many studies initially focused on the cata-lytic domain, particularly in relation to the develop-ment of active site inhibitors, it is now appreciated that the extracatalytic domains of the MMPs also play important roles in their function The N-terminal pro-peptide acts to maintain the latency of the MMPs by the presence of a cysteine residue in the active site coordinated to the catalytic zinc ion [6–9] The activa-tion mechanism (known as the ‘cysteine switch’) involves proteolytic cleavages of the propeptide caus-ing a destabilization of the cysteine–zinc interaction [10] The extra catalytic domains of the MMPs can contribute to macromolecular substrate specificity; the fibronectin type II domains of MMP-2 and MMP-9 are important for the cleavage of denatured collagens, type IV collagen and elastin [11,12] The hemopexin

Table 1 Cell surface-binding partners of MMPs Some representative examples of the associations of MMPs with cell surface molecules and the domain involved in interactions are shown It is possible that such interactions confer site specificity for MMP action and could form the basis for therapeutic targeting outside the catalytic cleft.

domain of MMP-2 was found to bind chemokines

such as monocyte chemoattractant protein-3 and hence

facilitated its cleavage [13] The hemopexin domains of

the collagenolytic MMPs (MMP-1, MMP-2, MMP-8,

MMP-13 and MT1-MMP) are essential for cleaving

native triple helical collagen monomers It has been

shown that collagenases locally unwind the triple helix

to allow access of the individual a chains to the active

site center [14] Hence, the collagen-binding site may

be composed of elements from both the catalytic

and the hemopexin domains It is also clear that

inter-domain flexibility is key for the specificity of the

colla-genases and possibly other MMPs [15]

Cell membrane-anchored MMPs

Unlike most of the soluble MMPs,

membrane-anchored MT-MMPs are already active at the cell

sur-face because they are cleaved intracellularly by

furin-like proprotein convertases at their specific recognition

sequence, RX[R⁄ K]R, located at the C-terminus of the

propeptide [16–18] MT1-MMP is the best studied of

this subfamily and is known to promote cell invasion

and motility by degrading pericellular ECM molecules

and eliciting the ‘shedding’ of CD44 and syndecan1

ectodomains It also degrades a plethora of other

extracellular and cell surface proteins [18,19]

Impor-tantly, MT1-MMP activates proMMP2 via a

mecha-nism in which the tissue inhibitor of MMPs, tissue

inhibitor of metalloproteinase (TIMP)-2, bound to the

MT1-MMP catalytic domain acts as a ‘receptor’ for

proMMP-2 at the cell surface In this system, modules

within blades III and IV of the hemopexin domain of

proMMP-2 bind to the C-terminal domain of TIMP-2

and this ternary complex formation allows proteolytic

activation of proMMP-2 by an adjacent molecule of

MT1-MMP that is free of TIMP-2 MT1-MMP can

also activate proMMP-13 [20] It is therefore regarded

as one of the key factors influencing the input of the

cellular microenvironment into cell signaling pathways

[18] Dimerization of MT1-MMP via the hemopexin

domain is essential for both collagenolysis and

effec-tive proMMP-2 activation and it may be the basis

of its function as an oligomer at the cell surface [21]

Miyamori et al [22], on the other hand, reported that

claudin-5 at endothelial cell tight junctions recruits

MT1-MMP and proMMP-2 on the cell surface to

achieve elevated focal concentrations, leading to

enhanced proMMP-2 activation independent of

TIMP-2 Similar enhancements of proMMP-2

activa-tion were reported for other MT-MMPs, suggesting

that clustering is an important factor for proMMP-2

activation [22]

To achieve focal degradation, cells localize MT1-MMP at lamellipodia, the migration front of the cells [23–25] This localization may be achieved by interac-tion with CD44 and integrins (see below) MMP-2 is also enriched in invadopodia and it has been suggested that it could be bound to MT1-MMP through TIMP-2,

as described above

MT1-MMP is largely sequestered intracellularly in membrane vesicles and may be derived from newly synthesized material, or from surface enzyme recycled through endocytosis Studies to determine MT1-MMP localization in invading cells are ongoing, although they have identified pathways necessary for the forma-tion of invasive structures and the movement of secre-tory vesicles involving microtubules and Rab proteins [26–29] The short 20 amino acid cytoplasmic domain

of MT1-MMP appears to be of importance in linking the enzyme proteolytic activity to efficient cell migra-tion and invasion; several studies have shown that deletion of this domain markedly reduces cell migra-tion triggered by the enzyme without affecting the cell surface proteolytic activity of the enzyme [30–33] However, there is no universal agreement on this point because, in some over-expression studies, MT1-MMP with the cytoplasmic domain deleted can efficiently drive cell migration through collagen [34] This may be

a result of overloading of the secretory pathway with

an excess of enzyme, over-riding the normal regulatory mechanisms In the same study, the transmembrane domain was found to be essential because secreted col-lagenases such as MMP-1 or MT1-MMP without the transmembrane domain did not drive cell invasion through a 3D collagen matrix Membrane-anchoring

of either MMP-1, or even the catalytic domain of MT1-MMP alone, could drive collagenolytic migra-tion, albeit with considerably less efficiency than MT1-MMP itself [34] Cell invasion through a fibrin gel also required intact membrane tethered MT1-MMP, although the tethered catalytic domain alone was not effective in this case [34] How the hemopexin domain interacts with a fibrin substrate has not been studied

Of the other MT-MMPs, only MT2-MMP has been shown to play a similar role to MT1-MMP in cell invasion of collagen I gels [35] Studies looking at cell invasion through intact basement membranes have shown that MT2-MMP and MT3-MMP, as well as MT1-MMP, may have important roles [36] These enzymes can degrade laminin and type IV collagen and the importance of membrane tethering and the lack

of requirement for the hemopexin domain for cell invasion were demonstrated The two GPI-anchored members of the MT-MMP family, MT4-MMP and MT6-MMP, have a broad substrate repertoire that is

still being defined, including ECM proteins, and have

been shown to activate proMMP-2 and proMMP-9

MT4-MMP also activates proADAMTS-4 [37] It is

considered that MT4 and MT6-MMP may have

unique roles, related to their localization in specific

cel-lular microenvironments via the GPI anchor within

lipid rafts of cell membranes [16] GPI-MT-MMP

activity at the cell surface is also regulated by

endocy-tosis and recycling, as reported for MT1-MMP [16]

There is also some preliminary evidence for

homodi-merization of the GPI-MT-MMPs, although this has

not been studied in depth [16]

Integrins

Studies of wound healing in keratinocyte monolayers

using blocking-antibodies indicated that the proteolytic

activity of MMP-1 is required for migration of human

keratinocytes on native collagen I Upon wounding,

keratinocytes contact dermal collagen I fibrils and the

interaction with a2b1 integrin stimulates the cell to

produce proMMP-1 [38] Biochemical and cell-based

studies have further shown that both proMMP-1 and

MMP-1 bind the a2b1 integrin via the I domain of the

a2 integrin subunit and that the linker peptide and the

hemopexin domain of MMP-1 are required for optimal

binding [39] Basal keratinocytes constitutively express

the a2b1 integrin on their basolateral surfaces and, in

wounds, this receptor accumulates at the forward-basal

tip of migrating keratinocyte in contact with dermal

type I collagen [38,39] Because the a2b1 integrin binds

native collagen I with high affinity, clustering this

inte-grin at contact points would tightly tether resting

keratinocytes to the dermis, although MMP-1 bound

to a2b1 integrin focally cleaves the collagen matrix

This results in denaturation of collagen fragments,

which weakens the adhesion to the matrix and allows

keratinocyte migration [38,39] Hence, a2b1 integrin,

MMP-1 and collagen substrate coordinate together to

drive and regulate migrating keratinocyte during

re-epithelialization This phenomenon, however, has

not been further studied in vivo, probably because

MMP-13, the predominant collagenase in mice, does

not appear to interact with integrins It is also not

known whether a similar system operates in other cell

types, although other epithelial cells that move in two

dimensions may utilize MMP-1 in a similar way

MT1-MMP can be colocalized with b1 integrin in

some cell types [40,41], and this appears to regulate its

function It is not clear whether this the result of a

direct interaction, although these reports suggest that

MT1-MMP and b1 integrin are functioning in the

same area on the cell surface In ovarian cancer cells,

antibody-induced clustering of a3b1 integrin stimulates polarized trafficking and cell surface expression of MT1-MMP, colocalization to aggregated integrin com-plexes and activation of pro-MMP-2 In the case of endothelial cells, MT1-MMP is up-regulated and colo-calizes with b1 integrin at the intercellular contacts of confluent cells on b1 integrin-interacting matrices such

as collagen I, fibronectin or fibrinogen This up-regula-tion was also shown to be the result of an impairment

of internalization of the cell surface MT1-MMP [42]

On migrating endothelial cells, MT1-MMP was found

to be associated with avb3 integrin at motility-associ-ated structures and the two proteins could be co-im-munoprecipitated [42] It has also been reported that the integrin avb3 binds MMP-2 via its hemopexin domain [43,44] and it is colocalized with degraded col-lagen type I [45] Physical interaction of MT1-MMP with avb3 can process the av subunit and increases outside-in signaling via avb3 [46] Furthermore, it is possible that MMP-2 and MT1-MMP activities could

be colocalized through their avb3-binding to modify integrin-ligand interactions rapidly in situ [42,47] The isolated hemopexin domain of MMP-2 was reported to block angiogenesis in model systems and to inhibit the interaction of MMP-2 with integrin avb3 [43,44] How-ever, there remains some controversy on the direct binding of avb3 and the MMP-2 hemopexin domain [48,49] Cells expressing avb3 could not use MMP-2 coated surfaces either to attach or spread It is possible that specific forms of the integrin and MMP-2 are involved in binding interactions and that this cannot always be recapitulated in vitro

MT1-MMP has been described as interacting with avb8 to activate transforming growth factor (TGF)-b1 It has been proposed that, upon ligation of avb8 with latent TGFb (latency associated peptide-b1⁄ latency-associated peptide-b1), avb8 and MT1-MMP become closely associated and form a complex on the cell surface [50] The mechanism for this is unknown, although it is postulated that the interaction may be indirect because the cell surface appears to be required for productive interactions and the secreted forms of avb8 and MT1-MMP did not activate

TGF-b [50] Because the localization of MT1-MMP in latency-associated peptide-b1 substrate contacts is dependent on the presence of b8, it is likely that avb8–latent TGF b interaction initiates the recruit-ment of MT1-MMP [50]

Tetraspanins Tetraspanins are evolutionarily conserved membrane proteins that tend to associate laterally with one

another and to cluster dynamically with numerous

partner proteins in membrane microdomains

Conse-quently, members of this family are involved in the

coordination of intracellular and intercellular

pro-cesses, including signal transduction, cell proliferation,

adhesion and migration Recent characterization of

tet-raspanin-enriched microdomains suggests that they

might be specially suited for the regulation of avidity

of adhesion receptors and the compartmentalization of

enzymatic activities Shiomi et al [51] reported that

CD151 binds to the propeptide of proMMP-7 and

actives the enzyme on the cell surface It is considered

to be the result of conformational changes in

proM-MP-7 induced by interacting with CD151, thus

facilitating a spontaneous activation of proMMP-7

pericellularly The tetraspanin CD151 may also be a

key regulator of MT1-MMP function at the surface of

endothelial cells MT1-MMP colocalizes with

tetra-spanin CD151 and its associated partner a3b1 integrin

at lateral endothelial cell junctions Biochemical and

fluorescence resonance energy transfer analyses by

Yan˜ez-Mo´ et al [52] showed that the MT1-MMP

hemopexin domain associates with CD151 and forms

an a3b1 integrin⁄ CD151 ⁄ MT1-MMP ternary complex

Ablation of CD151 expression enhanced

MT1-MMP-mediated activation of MMP-2, although collagen

degradation was reduced around the cell periphery

MT1-MMP subcellular localization and its inclusion

into detergent-resistant membrane domains, as well as

association with the a3b1 integrin, were affected [52]

Thus, CD151 can finely regulate not only proteolytic

activities of MT1-MMP, but also the sites of action

through complex formation with a3b1

Another tetraspanin, CD63, a component of late

endosomal and lysosomal membranes, interacts with

MT1-MMP directly through the N-terminus of CD63

and the hemopexin domain [53] and accelerates

inter-nalization and lysosomal degradation of MT1-MMP

[53], thus acting as a further regulator of MT1-MMP

trafficking

CD44

The hyaluronan receptor CD44, of which some forms

are heavily glycosylated and sulfated, is an important

mediator of cell migration and tissue remodeling

events CD44v(3,8-10) was reported to be associated

with active MMP-9 within the invadopodia of

meta-static breast cancer cells [54] The complex links to

ankyrin and the membrane-associated actomyosin

con-tractile system required for ‘invadopodia’ formation,

thus coupling matrix degradation and tumor cell

migration during breast cancer progression [54] Yu

and Stamenkovic [55] also showed that active MMP-9 can associate with CD44 on mammary carcinoma cells and activate latent TGFb TIMP-1 binding to a proM-MP-9–CD44 complex is also a prerequisite for anti-apoptotic signaling in erythroid cells [56] Redondo-Munoz et al [57] found that CD44v and a4b1integrin colocalize with MMP-9 in invading lymphoid cells, and MMP-9 produced by chronic lymphocytic leuke-mia B cells is considered to contribute to their tissue infiltration by degrading extracellular and membrane-anchored substrates This interaction is mediated by the hemopexin domain Binding of soluble or immobi-lized MMP-9, or the MMP-9 hemopexin domain, to a4b1 integrin and CD44v prevents B-cell leukemia lymphocyte apoptosis by inducing Lyn activation, STAT3 phosphorylation and Mcl-1 up-regulation [58], although the target(s) of MMP-9 activity in this con-text are not yet known

As discussed above, CD44 also interacts with MT1-MMP, and directs the presence of MT1-MMP in lamellipodia of the invasive front of migrating cells [25] This localization may be achieved by interaction with CD44 through the hemopexin domain of the enzyme and stem region of CD44, and CD44 associ-ates with F-actin through its cytoplasmic domain by interacting with Ezrin⁄ Radixin ⁄ Moesin proteins MT1-MMP is also localized to F-actin-rich invasive struc-tures found in some cells, termed invadopodia, and detailed time-lapse studies have demonstrated that cortactin and actin aggregates at membrane regions adherent to matrix where MT1-MMP accumulates [26] Matrix degradation leads to cortactin dissociation from the area, although MT1-MMP remains associated with foci of degraded matrix [26] Hence, proteolytic shedding of CD44 from the cell surface by MT1-MMP promotes cell migration on a hyaluronan based 2D matrix [59] MT1-MMP may therefore act to regulate the adhesion properties of cellular lamellipo-dia Again, the hemopexin domain of MT1-MMP is required for CD44 shedding and cell migration to occur in this model Marrero-Diaz et al [60] carried out time-lapse confocal microscopy and fluorescence resonance energy transfer imaging of carcinoma cells embedded in a 3D collagen I matrix containing hyal-uronan and showed that MT1-MMP interacted with CD44 preferentially at the trailing edge of the invading tumor cells and that the proteolytic processing of the CD44 extracellular domain was enriched in the retracting rear ends It was concluded that the role of MT1-MMP in CD44-mediated tumor-cell invasion is cell retraction, although CD44 is not essential for MT1-MMP-mediated invasion into the 3D matrix of hyaluro-nan-collagen

Membrane proteoglycans and

glycosaminoglycans

Proteoglycans, which contain either heparan sulfate or

chondroitin sulfate glycosaminoglycan chains attached

to a protein core, are an important class of cell surface

and ECM molecules regulating activation and activity

of MMPs Many MMPs are bound to tissues through

interaction with glycosaminoglycans, and MMP-7 is

one of the most tightly bound MMP-7 is often found

to be bound to heparan sulfate proteoglycans on or

around epithelial cells and in the underlying basement

membrane, and it may be released by heparinase

diges-tion [61] When it is bound to heparin, the activity of

MMP-7 is greatly enhanced Two putative

heparin-binding peptides were identified near the C- and

N-ter-minal regions of proMMP-7, although molecular

mod-eling suggested an extensive binding track crossing

multiple peptide strands Evidence was also found for

the binding of MMP-2, -9, -13 and -16 [48,61] As

sug-gested by Ra and Parks [4], binding of those MMPs to

heparan sulfate in the extracellular space could prevent

the loss of secreted enzyme, provide a reservoir of

latent enzyme, and facilitate cellular sensing and

regu-lation of enzyme levels Furthermore, binding to the

cell surface could position the enzyme for directed

pro-teolytic attack for activation of (or by) other MMPs

and for regulation of other cell surface proteins

Forms of the hyaluronan receptor CD44 bearing

sulfated glycosaminoglycans bind MMP-9, as discussed

above Yu et al [62] reported that such forms of

CD44 recruit proteolytically active MMP-7 and the

substrate heparin-binding epidermal growth factor

pre-cursor (proHB-EGF) via the sulfated sugars, forming

a complex on the surface of tumor cell line The

pro-HB-EGF within this complex is processed by MMP-7,

and the resulting mature HB-EGF engages and

acti-vates its receptor, ErbB4, leading to cell survival In

CD44() ⁄ )) mice, postpartum uterine involution is

accelerated and maintenance of lactation is impaired

as a result of altered MMP-7 localization and

decreased ErbB4 activation in both uterine and

mam-mary epithelia [62] Because MMP-7 is known as an

important regulator of many proteolytic events at the

cell surface, it is possible that CD44 interaction could

be a means of localizing the enzyme to key sites It is

likely that charge interactions with sulfated

glycosami-noglycans of CD44 are important, although the

mole-cular nature of this is not fully understood

Syndecans and glypicans are other examples of

membrane associated proteoglycans with highly

sul-fated glycosaminoglycan chains that regulate cell

sur-face events by interactions with effectors, including

growth factors, integrins and proteinase inhibitors Endometrial epithelial cells and carcinoma cells from various tissues bind to active MMP-7 at the cell sur-face MMP-7-binding could be decreased by interfering with heparan sulfate proteoglycans, and by interacting with TIMP-2 or a synthetic MMP inhibitor The bound MMP-7 remains fully active towards a macro-molecular substrate but is resistant to inhibition by TIMP-2 [63] MMP-9 associated with the heparan sul-fate chains of the GPI-anchored cell surface proteogly-can glypiproteogly-can of murine colon adenocarcinoma cells [64] This allows the accumulation of MMP-9 on the tips of invasive protrusions of the cells and promotes their motility Treatment of the cells with heparitinase-I

or heparin released MMP-9 from the cell surface, which resulted in the suppression of their motility to a level similar to that exhibited by an MMP inhibitor However, the heparan sulfate-interacting domain of MMP-9 is not known Iida et al [48] reported that melonoma cell specific cell surface chondrotin sulfate proteoglycan enhances the activation of proMMP-2 by MT3-MMP and cell invasion in vitro The complex is formed through glycosaminoglycan components inter-acting with the catalytic domain of MT3-MMP and the hemopexin domain of proMMP-2 This effect was also observed with isolated chondroitin 4-sulfate, but not chondroitin 6-sulfate, hyaluronan or heparin, sug-gesting that a specific sulfation pattern is important in those reactions

Low-density lipoprotein receptor-related protein (LRP) and Ku LRP is a member of the low-density lipoprotein recep-tor superfamily and a heterodimeric endocytic receprecep-tor for a large number of proteins, and also has signaling properties LRP internalizes many diverse ligands, including a2-macroglobulin-proteinase complexes, sev-eral serine proteinases, proteinase inhibitors and pro-teinase–inhibitor complexes [65] MMP-2, MMP-9 and MMP-13 have been reported to be endocytosed by LRP, introducing another level of regulation of peri-cellular proteolysis by MMPs ProMMP-2 by itself has

a relatively low affinity to LRP but forms complexes with either thrombospondin [66] or TIMP-2 [67] that are readily endocytosed by LRP The study of proMMP-2–TIMP-2 complex internalization indicated that both the fibronectin II domain of MMP-2 and TIMP-2 have independent binding sites in LRP, which enhances the uptake of the complex [67] ProMMP-9 is internalized as the proMMP-9–TIMP-1 complex [68] Internalization of MMP-13 is initiated by binding to

an unidentified 170 kDa receptor and the enyzme is

transferred to LRP for internalization and intracellular

degradation [69] Troeberg et al [70] reported that

TIMP-3, is also internalized by LRP, which also

requires a sulfated proteoglycan as a co-receptor

The hemopexin domain of MMP-9 contains a

bind-ing site for LRP-1 and LRP-2 and these receptors have

been implicated in regulating MMP-9 activity [71] The

64 amino acid linker region of MMP-9 between the

catalytic and hemopexin domains is heavily

O-glycosy-lated The linker region is required to correctly orient

the hemopexin domain for inhibition by TIMP-1 and

internalization by LRP-1 and LRP-2, hence regulating

active enzyme bioavailability [71,72]

Another interesting molecule interacting with MMP-9

on the cell surface is the heterodimeric DNA repair

molecule Ku (Ku70⁄ Ku80) Ku not only is present in

the cytosol and nucleus, but also is found on cell

surface of certain cell types Monferran et al [73]

reported that MMP-9 is bound to Ku on the leading

and tailing edge of the leukemic cell surface and helps

the cell to migrate into type IV collagen matrices,

indi-cating the importance of the membrane bound Ku in

ECM turnover The interaction of MMP-9 and Ku is

mediated by the hemopexin domain of MMP-9 and

Ku80

Emmprin

Emmprin, CD147 (Basigin) is an important cell surface

bound MMP regulator It is a transmembrane

glyco-protein with two Ig-like domains [74] It was identified

as an MMP-inducer expressed on epithelial cells and is

known to enhance cell proliferation and multidrug

resistance by promoting hyaluronan synthesis and

angiogenesis via the augmentation of vascular

endothe-lial growth factor production [75] It interacts with

sev-eral molecules including caveolin, cyclophilin 60 and

monocarboxylate transporters [75] Although epithelial

Emmprin stimulates surrounding stromal cells to

pro-duce a number of proMMPs, proMMP-1 was found

to interact with Emmprin on human lung carcinoma

cells [76], and both proMMP-1 and active MMP-1

bind to Emmprin on glandular epithelium in the

human endometrium [77] Although the activity of

MMP-1 on the cell surface has not been examined, it

may be another way to specifically regulate pericellular

collagenolytic activity

Endo180

Endo180 was originally identified as constitutively

recycling cell surface receptor [78] It was also found

as a macrophage mannose receptor type C lectin [79]

and as urokinase-type plasminogen activator receptor associated protein [80] It has also been characterized

as a collagen-binding and collagen-internalization receptor [81] MT1-MMP was shown to have a critical role in collagen phagocytosis [82] and recent studies by Messaritou et al [83] have demonstrated that Endo180

is a negative regulator of MT1-MMP activity and thus down-regulates MT1-MMP-dependent MMP-2 activa-tion in HT1080 cells Depleactiva-tion of Endo180 by siRNA led to the accumulation of collagen in the medium as a result of reduced collagen endocytosis, and resulted in the collagen-dependent increase of MT1-MMP activity

on the cell surface However, Messaritou et al [83] could not show direct binding of Endo180 and MT1-MMP, suggesting that the effect of Endo180 on the regulation of MT1-MMP activity involves another molecule Their study indicates an intricate coordina-tion of collagen clearance in the pericellular environ-ment mediated both collagen internalization and regulated MT1-MMP activity

Cholesterol sulfate MMP-7 cleaves many ECM molecules and other pro-teins in the cellular microenvironment and is consid-ered to have ‘sheddase’ activities comparable with those of the disintegrin MMPs It is known to induce adhesion of colon cancer cells by the cleavage of cell surface proteins, although binding of MMP-7 to cell surface cholesterol sulfate is essential for this activity and the induction of cell aggregation [84] The choles-terol sulfate-binding site has been identified on the opposite side of the catalytic cleft of MMP-7 [85], and

it has been proposed that this makes it possible for the enzyme to cleave both cell surface protein substrates and those in the pericellular ECM

Pericellular ECM MMP-binding to the extracellular matrix has been noted in many immunohistochemical and biochemical extraction studies and examples of the potential effects

of matrix macromolecule association with MMPs on the activity of the latter have been postulated Because many ECM proteins are associated with the cell sur-face, it is likely that these act as important binding sites and modulators of pericellular proteolysis The gelatinases, MMP-2 and MMP-9, were the first MMPs

to be recognized as binding to fibrillar collagens such

as type I and their denatured forms within the ECM

It was shown that this was effected by a ‘gelatin-bind-ing domain’, consist‘gelatin-bind-ing of three repeats of the fibro-nectin type II motif inserted into the catalytic domain

[11,86] Subsequently, proMMP-9 and its TIMP-1

complex were shown to bind with high affinity to a

number of cell lines via cell surface a2 chain of type

IV collagen [87] Binding of pro-MMP-9 to cells does

not result in zymogen activation and is not followed

by ligand internalization, even after complex formation

with TIMP-1 Interestingly, the proenzyme does not

bind to secreted triple-helical collagen IV It was

pro-posed that this unique interaction between pro-MMP-9

and a2(IV) may play a role in targeting the zymogen

to cell matrix contacts and in the degradation of the

collagen IV network Preliminary studies have

impli-cated the gelatin-binding domain of MMP-9 in a2(IV)

collagen-binding [88] However, the nature of a2(IV)

collagen association to cells is itself not clear Owen

et al [89] showed that MMP-9 secreted by activated

PMN leucocytes binds to the cell surface by an

unknown mechanism Significantly, the bound enzyme

is fully functional proteolytically, although is no longer

regulated by TIMP-1 or TIMP-2 Bannikov et al [90]

found that the gelatinolytic activity of MMP-9 could

be detected in situ in tissue sections of term placenta,

However, all the enzyme extracted from this tissue was

in a proform They found that purified proMMP-9

acquired activity against gelatin substrates, although

its propeptide remained intact These results suggest

that, although activation of all known MMPs in vitro

is accomplished by proteolytic processing of the

pro-peptide, other mechanisms, such as binding to a ligand

or to a substrate, may lead to a disengagement of the

propeptide from the active center of the enzyme,

caus-ing its activation This observation could have

implica-tions for the association of other MMPs to cell surface

molecules Bone sialoprotein is a member of a family

of glycoprotein ligands for integrins and can therefore

be cell surface associated Bone sialoprotein has

been shown to induce limited gelatinase activity in

proMMP-2 without removal of the propeptide and to

restore enzymatic activity to MMP-2 previously

inhib-ited by TIMP-2 [91]

More recently Freise et al [87] found that proforms

of MMPs were closely associated with collagenous

sep-tae in fibrotic liver tissue and that the triple helical

domain of a2 chain of collagen VI bound with

nanom-olar affinity to procollagenases (proMMP-1, -8 and

-13) and proMMP-3, as well as proMMP-2 and -9

The binding of collagen VI to those zymogens or

acti-vated MMPs reduced the levels of auto-activation and

enzymatic activities, respectively, with the exception of

an observed increase in proMMP-2 activation and

MMP-2 activity It was suggested that the a2(VI) chain

modulates MMP availability by sequestering proMMPs

in the ECM and blocking proteolytic activity Using

tandem affinity expression tagged MMP-13 hinge-hemopexin domains as a bait, Zhang et al [92] found that they bound TIMP-1 and a2-macroglobulin, fibro-nectin, type VI collagen, xylosyltransferase 1, decorin, syndecan 4 and serglycin in the medium of human chondrocytes in culture Although the consequences of these interactions remain to be demonstrated, studies suggest that MMP-13 activity may be either targeted

at a more specific site in the connective tissue matrix,

or that matrix proteins may regulate its activity during cartilage degradation

The mechanisms of interaction of the collagenases with fibrillar collagen substrates is key to directed cleavage As exemplified with MT1-MMP, cell surface collagenolysis is implicated in directional cell move-ment along the collagen fibrils In the tissue, interstitial collagen forms insoluble fibrils that are highly resistant

to digestion, even by collagenases such as MMP-1, MMP-13 and MT1-MMP This may be particularly a result of collagenase cleavage sites in collagen fibrils being covered mainly by the C-terminal telopeptide of the neighboring collagen molecule, as shown in the 3D structure of rat tendon collagen fibrils recently solved using X-ray diffraction analysis [93,94] This suggests that the removal of the C-telopeptide or its structural alteration needs to take place before collagenase can act on collagen fibrils This would also remove impor-tant cross linking sites from the fibrils On the other hand, Saffarian et al [95] reported that active MMP-1 moves to one direction on collagen fibrils analgous to a molecular ratchet, which is driven by proteolysis This directional movement may be explained by the Orgel model of collagen fibrils [93]: upon destabilization or cleavage of the C-telopeptide, collagenase can remove the C-terminal ¼ fragment including the C-telopep-tide, which then exposes the C-terminally adjacent col-lagenase-cleavage site Subsequent cleavage of this site will expose another site on the C-terminal side Such directional movement of the collagenase may promote cells to move in one direction when the enzyme is attached to the cell surface This may also occur with MT-MMPs and MMP-1 attached to Emmprin or a2b1 integrin Cells such as inflammatory cells may use such

a mechanism to move along one direction depending

on the orientation of collagen fibrils In Orgel’s model, the a2b1 integrin-binding site in collagen I is also blocked by the adjacent collagen molecule [96] To make these sites available to the integrins, collagenoly-sis and denaturation of the ¾ fragment is probably necessary Further investigations are necessary to determine how pericellular collagenolysis and integrin engagement are coordinated during the cell movement

on collagen fibrils Those questions may apply to the

migration of many cell types, including cancer cells,

inflammatory cells, smooth muscle cells and

endothe-lial cells, as well as the directionality of collagen fibres

in the tissue

Conclusions and future prospects

Subsequent to the discovery of vertebrate collagenase

in the tadpole tail undergoing metamorphosis [97],

MMPs have largely been characterized in relation to

their respective abilities to degrade ECM molecules

However, for the last two decades, researchers have

recognized that non-ECM proteins, such as serpins,

cytokines, chemokines, growth factors and growth

fac-tor-binding proteins, are also MMP substrates [2]

Along with the cryptic function of ECM molecules

exposed by MMP cleavage, these observations

empha-size that MMPs play diverse roles in many cellular

activities, such as proliferation, differentiation,

migra-tion and apoptosis The proteolytic acmigra-tions associated

with these events most effectively occur at or close to

the cell surface Thus, the MT-MMPs have been

regarded as prime candidates in such activities [19]

However, numerous studies have demonstrated that

secreted MMPs are also recruited to the cell surface,

interacting with cell surface receptors or pericellular

macromolecules, including those of the ECM (Table 1)

Such interactions introduce intricate regulatory

mecha-nisms that create gradients or directionality of

prote-olytic activity Of particular note is the role of

interacting proteins in endocytic mechanisms to

down-regulate MMP function Although only a few MMPs

have been so far studied, the diversity of mechanisms

is proving to be enormous These examples have led us

to consider that many, if not all, of the MMPs may

frequently function pericellularly, rather than at sites

distal to the cells, where the communication between

the cells and the surrounding ECM and other bioactive

molecules takes place For example, MMP-19 is

another member found to be associated with myeloid

cell surface, although the binding partner is yet to be

identified [98] Identification of the partner molecule

would help us understand the role of MMP-19 in

blood-derived cell migration Careful

immunocyto-chemical studies of more recently discovered MMPs

certainly deserve further attention, which may help to

elucidate the molecular mechanism of the proposed

function or introduce new biological functions The

use of modern techniques for protein identification will

also further the characterization of proteins that

inter-act with MMPs [99,100] and further define cell surface

focussing and regulation of their function in relation

to specific cellular activities

Acknowledgements

We would like to thank all the individuals who have contributed to MMP research and apologize that we have been unable to cite all the relevant studies in this minireview Our work is supported by grants from Cancer Research UK, Medical Research Council, European Union Framework 6 Program to G.M., the Wellcome Trust, Arthritis Research UK and National Institutes of Health (AR40994) to H.N

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