Proteolysis and cell migration: creating a path?

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Both serine and metalloproteinases have been implicated inthe complex integrated events underlying cell migration but nodefinitive single mechanism has emerged. Work over the pasttwo years linking both membrane and soluble proteinases withintegrins and other adhesion proteins and with intracellularsignalling systems could herald the beginnings of a potentialexpansion of our understanding of the role and regulation ofproteolysis in cell migration.

AddressesSchool of Biological Sciences, University of East Anglia, Norwich NR47TJ, UK*e-mail: [email protected]†e-mail: [email protected]

Current Opinion in Cell Biology 1999, 11:614–621

0955-0674/99/$ — see front matter © 1999 Elsevier Science Ltd. All rights reserved.

AbbreviationsEAE experimental autoimmune encephalomyelitisECM extracellular matrixMMP matrix metalloproteinaseMT1 membrane-type 1PAI plasminogen activator PAI-1 PA inhibitorTGFαα transforming growth factor αTIMP tissue inhibitor of MMPstPA tissue type plasminogen activatoruPA urokinase-type plasminogen activatoruPAR uPA receptorVSMC vascular smooth muscle cell

IntroductionCell migration plays a key role in a plethora of biologicalevents including morphogenesis, wound healing andtumour metastasis. The molecular mechanisms behind thevarious cellular strategies employed are being analysed atall levels, from interactions between the cell and its extra-cellular matrix (ECM), cytoskeletal changes, cell signallingand gene regulation. The efficient integration of theseprocesses is a key determinant of different cell migrationpatterns and a major challenge to a rational understandingof events [1,2]. The migration of different cell types isdetermined by variables such as their origin, adhesionreceptor function and the environment, including thenature of the ECM. In vivo the movement of cells withinor through tissue barriers is clearly a complex process, andcan only provide us with some of the clues to the precisemechanisms involved, but two-dimensional or three-dimensional migration/invasion models set up in vitro canhelp to establish some of the basic principles. The majorquestion is ‘Do cells always use proteolytic mechanisms tomodify the ECM in their path and is this simply a pathclearing mechanism, or a way of reorganising the matrix tofacilitate cellular interactions?’. The further implications ofmatrix degradation also cannot be ignored, including the

release of growth factors, and the generation of modulato-ry neo-epitopes.

Early studies placed most emphasis on the role of theurokinase-type plasminogen activator, (uPA), system andplasmin generation in the facilitation of cell migration [3],but the contribution of matrix metalloproteinases (MMPs)has become evident more recently [4]. Evidence that thetwo systems interact has also emerged, although no singlemechanism can be defined (Table 1). Some cell typesexpress proteinases when they simply bind to an ECM orduring reorganisation and contraction of a three-dimen-sional matrix and this is likely to be of relevance to cellmigration processes [5,6,7•,8,9•,10–12].

The uPA systemIt has long been hypothesized that cells might focus pro-teinases at their leading edge, where proteolysis can directmigration, but this has been technically challenging todemonstrate. The plasmin cascade system driven by uPAoperates by this type of mechanism, as the uPA receptor(uPAR) was shown to be spatially and temporally associatedwith cellular structures that regulate cell adhesion, migra-tion and invasion, colocalising with integrins in focalcontacts and at the leading edge of migrating cells [3](Figure 1). Its substrate, plasminogen is also cell associated.In fact, this is not just a way to focus plasmin activity as theuPAR also functions as an adhesion receptor for vitronectin(and is associated with caveolin) [13] (Figures 2,3). Thismay explain the interesting data showing that the plas-minogen activator inhibitor (PAI-1) gene knockout micecannot support the local invasion of a transplanted malig-nant keratinocyte tumour model or neovascularisation ofthe developing ‘tumour’ [14••]. PAI-1 may act by blockingthe binding between vitronectin and uPAR–uPA orbetween vitronectin and integrins [3]. The role ofplasmin — either directly, or in the activation of MMPs intumour cell invasion models [15] — has recently been con-firmed in several studies indicating that uPA may beessential, but not sufficient, for tumour cell invasion ofmatrigel [16–18].

Following injury to the vascular wall, for example afterballoon angioplasty, vascular smooth muscle cells (VSMCs)migrate from the media to create a neointima or new innerlining. The uPA/plasminogen system appears to play a rolein VSMC migration during this process. Gene targetingapproaches have revealed that neointimal formation isreduced in plg–/– or uPA–/– mice, whereas it is normal intissue type plasminogen activator (tPA)–/– mice (reviewed byCarmeliet and Collen [19••]). Studies with the uPAR–/–

mice, however, indicate that uPAR is unnecessary for themigration of VSMCs. In this case uPA may be concentrated

Proteolysis and cell migration: creating a path?Gillian Murphy* and Jelena Gavrilovic†

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Proteolysis and cell migration Murphy and Gavrilovic 615

in the pericellular ECM in sufficient quantities to initiateplasmin based proteolysis [20•].

The role of matrix metalloproteinasesMMPs have been implicated in the remodelling of theECM and the penetration of both normal and tumour cellsthrough tissue barriers, although no definitive single mech-anism can be ascribed to all situations. A role for MMPs inVSMC migration has been convincingly demonstrated bythe overexpression of TIMPs (tissue inhibitors of MMPs) in

the vascular wall in vivo [21,22] and in vitro [23,24].Although these studies provide evidence that MMPs areinvolved in VSMC migration through the ECM, as yet it isuncertain which matrix components are degraded andwhether pro-migratory neo-epitopes are generated. Fibringel population by endothelial cells (emerging from murinemuscle explants) and the formation of neovessel tubuleswas not blocked by serine proteinase inhibitors includingthose for the plasminogen activator (PA) system, but wasprevented by the MMP inhibitors [25••]. Muscle explants

Table 1

Proteinases implicated in cell migration.

Approach Cell type References

Localisation of proteinases at cell surface Melanoma (MT1 MMP) [39]Osteoclasts (MT1 MMP) [46]Tumour (MMP9) [52•]

[55,56•]

Use of inhibitors to block cell migration/invasion In vitro: keratinocytes, osteoclasts, [23,24,45,61]vascular smooth muscle cellsIn vivo: vascular smooth muscle cells [21,22]

Blockade of specific proteinases Mammary tumour cells (MMP3) [26]Tumour (MMP9) [56•]Ovarian cancer cells (MMP9) [16]Melanoma (MMP2) [47,48]

Gene ablation Vascular smooth muscle cells (Plasminogen, uPA) [19••]

Macrophages (MMP12) [29]Endothelial cells (MMP2) [50•]Chondroclasts, endothelial cells (MMP9) [51••]

Overexpression of proteinases 3T3, MDCK, melanoma (MT1 MMP) [38,25••]Mammary tumour cells (MMP2) [47]

Exogenous degradation Neurons (MMP2) [60]Mammary epithelial cells (MMP2) [58]

Figure 1

Depiction of immunofluorescence ofproteinases at the surface of migrating cells.(a) Immunofluorescence staining of Met-1mammary tumour cells for MMP9 (arrowhead).Staining for the splice variant, CD44v3,8-10shows that they co-localise. (i) negative control[56•]. (b) A 'walking' osteoclast stained withphalloidin reveals actin both at the leadingedge (arrowhead) and retraction fibres at rear.MT1 MMP is found in the same cell at theleading edge [46]. (c) In a 'sitting' osteoclastMT1 MMP (shown) and actin are found in thepodosomes at the cell periphery [46]. (d) uPAlocalised at cell–substratum contact sites ofHT1080 fibrosarcoma cells [68].Co-localisation with both uPA receptor and theαvβ3 integrin have also been demonstrated atthe focal adhesion sites [3].

(a) (b) (c)

(d)

(i)

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from mice in which both the tPA and the uPA genes hadbeen ablated showed no diminution in the growth of theneo vessels into the fibrin gel. Endothelial cells expressedmembrane-type 1 (MT1) MMP (MMP14), and MMPs 1, 2and 3, but tissue from mice that also had the MMP3 geneablated did not show impaired tubulogenesis. As MT1MMP had the strongest fibrinolytic activity it was felt thatthis enzyme might mediate the endothelial cell migration infibrin gels. In contrast, mouse mammary tumour cell linesinvaded a reconstituted basement membrane by astromeslysin (MMP3)-dependent mechanism, as shown by

the inhibitory effect of antisense oligonucleotides to MMP3[26]. Using blocking antibodies the α1 and α2 integrinswere shown to be involved in this invasion and appeared tofunction through the regulation of MMP3 expression. Anti-integrin antibodies had no effect on two-dimensionalmigration of the cells, however [27]. The mechanism bywhich MMP3 promotes cellular invasion has not yet beenelucidated; however, overexpression of MMP3 in mammaryepithelial cells leads to activation and upregulation of otherMMPs and the loss of cell–cell contacts, due in part to thedegradation of E-cadherin [28]. Targeted disruption ofMMP12 (mouse macrophage metalloelastase) results in thereduction of macrophage invasion through matrigel both invitro and in vivo (in sponges containing matrigel implantedin MMP12 knockout mice [29]). In skin diseases with pro-nounced numbers of infiltrating macrophages, cellsexpressing metalloelastase were localised in regions of base-ment membrane disruption, implying that this enzyme mayaid macrophage invasion [30].

Using the uPA receptor–uPA–plasmin cascade as a para-digm, MMP activity focused at the cell surface wouldappear to be the most logical mechanism to efficientlyeffect and regulate invasive processes. Hence the MT1MMP mediated activation of MMP2 would seem to fulfilthese criteria [31], where the generation of an MTIMMP–TIMP2 complex at the cell surface can act as areceptor for MMP2 [32,33] (Figure 3). MT1 MMP andMMP2 have collagenolytic properties and their concentra-tion at the cell surface would represent a powerful buthighly localised means of regulating cell–collagen interac-tions by cleavage of the collagen. Studies on themodulation of this system during cell–matrix interactionshave implicated the aggregation of β1 integrins as being akey event in MMP2 activation via MT1 MMP [12,34].Interestingly, the MT1 MMP becomes degraded to aninactive 43 kDa form during MMP2 activation [35].Hence, both the activation and subsequent proteolyticprocessing of proteinases to inactive forms can be con-trolled via integrin–matrix interactions. MT1 MMP canalso degrade a number of other ECM components, includ-ing cross-linked fibrin gels [25••], laminin and fibronectin[36,37] and may be functional in isolation from otherMMPs. Glioma cells appear to utilise MT1 MMP tomigrate on a non-permissive myelin substrate and 3T3cells overexpressing MT1 MMP acquire this ability and caninfiltrate into rat optic nerve explants [38].

When MTI MMP was overexpressed in human melanomacells, the cells activated MMP2 and degraded and invaded anECM substratum. The proteinases localised predominantlyto ‘invadopodia’, specialised membrane extensions that arethe sites of ECM degradation [39]. A similar localisation ofMT1 MMP has also been described in the lamellipodia oftransfected 3T3 cells [38]. Invadopodial localisation of MTIMMP was essential for ECM degradation and melanoma cellinvasion. Neither a truncated MTI MMP mutant, lacking thetransmembrane and cytoplasmic domains, nor a chimeric

616 Cell-to-cell contact and extracellular matrix

Figure 2

Potential interactions of proteinases with the cell surface.Accumulation of proteinases at the cell surface, as depicted inFigure 1, occurs by different mechanisms, which are slowly beingdissected. UPA–uPAR localisation at focal adhesion sites may becaused by binding to vitronectin (VN) and/or to integrins, notably αvβ3the vitronectin receptor, but also β1 and β2 integrins [13]. The adhesiveand signalling functions of uPAR may be mediated by furtherinteractions with caveolin and associated molecules. The uPA inhibitorPAI-1 interferes with vitronectin binding to both integrins and the uPAreceptor. MMP1 is thought to bind to collagen through the hemopexindomain but may also interact with the A domain of the collagen bindingintegrin β2 [64]. MT1 MMP may also interact with integrins orcomponents of focal adhesion complexes via its cytoplasmic domain.As an MT1 MMP–TIMP2 complex this can also act as a receptor tosequester MMP2 [31]. MMP9 may associate with α2 (IV) collagen atthe cell surface (not shown; [53•] or with isoforms of CD44 that haveheparan sulphate chains [55,56•], in association with CD44 clusteringand ERM (ezrin/radixin/moesin) family proteins–cytoskeletalinteractions during hyaluronan binding.

α2

β1 MT1-MMP

?

?

??

Focal adhesioncomplex

Actin filaments

αv

β3

Actin filaments

α2

β1

Focal adhesioncomplex

Hyaluronan

Actin filaments

MMP-9

MMP-1

ERMs

CD44

uPAR

uPAVN VN CollagenCollagen


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MTI MMP, containing the interleukin 2 receptor α chaintransmembrane and cytoplasmic domains, were able tolocalise to the invadopodia, or effect an increase in cell inva-sion of matrix relative to the wild-type [39]. The role of thecytoplasmic domain of MTI MMP in the trafficking of thisenzyme is now under intensive investigation. Comparison ofthis domain within all the forms of MT MMP that activateproMMP2, (MT1, 2, 3 and 5-MMPs) indicates a commonpotential tyrosine phosphorylation site and a carboxy-termi-nal motif terminating in a valine, similar to transforminggrowth factor α, (TGFα) [40••]. This may provide clues forthe elucidation of signalling mechanisms and intracellularbinding partners, perhaps linked to the integrin activationevents (Figures 2,3). TGFα has been shown to bind to pro-teins containing PDZ domains including syntenin, whichcolocalises with immature intracellular proTGFα and is nec-essary for its targeting to the cell surface [41]. Syntenin playsa similar role in the trafficking of syndecan-2 [42], a mem-brane-associated heparan sulphate proteoglycan thatco-distributes with F-actin and regulates the formation offilopodia, which protrude from the leading edges of migrato-ry cells [43]. It should be noted, however, that Hiraoka et al.[25••] have demonstrated that MDCK epithelial cells areinduced to invade a fibrin gel when overexpressing eitherMT1 MMP encoding the full-length protein or a mutant MT1MMP encoding a protein which lacks the cytoplasmic tail.

Osteoclasts, the cells responsible for the resorption of bonematrix, need to migrate to their sites of activity [44] e.g. themovement of preosteoclasts to the developing marrow cav-ity of long bones. Migration is inhibited by syntheticinhibitors of MMPs and by TIMP2 both in vivo through theosteoid of developing bone and in vitro through a collagen

gel [45]. Immunolocalisation studies have shown that MTIMMP was associated with lamellipodia and invadopodia ofisolated rabbit osteoclasts with a ‘walking’ phenotype (Fig-ure 1) [46]. Cells with a ‘sitting’ phenotype often had MTIMMP localised to small dots of the cell periphery and thiscolocalised with actin staining indicative of arrest andattachment of the osteoclasts (Figure 1c). Previous studieshad identified MMP9 and MMP12 in osteoclasts but thepresence of MMP2 seems to be variable. It is difficult toconclude whether MTI MMP has a direct role in osteoclastmigration until further studies have been performed.

Other cell types are thought to utilise MMP2 for cellmigration although the involvement of MTI MMP in itsactivation has not been completely addressed. Transfec-tion of MMP1, 2 or 3 into a mammary epithelioid cell lineshowed that only MMP2 could promote invasion in thesecells using the reconstituted basement membrane assay,although both isolated MMP2 and 3 had the ability todegrade the matrix. In contrast to the wild-type MMP2,expression of a catalytically active but hemopexin-domain-truncated form of MMP2 did not result in increasedinvasiveness or lung colonisation of nude mice by the cells,indicating that this cell-binding domain of the enzyme wasnecessary for functionality in vivo. Furthermore, adminis-tration of a full-length, catalytically inactive form of MMP2with the MMP2 transfected cells inhibited their colonisa-tion of the lung and nodular growth, suggesting that itcould compete for the wild-type enzyme binding site [47].In a comparable study, direct interactions of MMP2 withthe αVβ3 integrin, through the hemopexin domain, havebeen reported as significant in angiogenesis but the rela-tionship with the MT MMP system was not considered


Figure 3

Intracellular and extracellular interactions allowplasmin and MT1 MMP to modulate pericellularproteinase activation cascades. The potentialinteractions between the plasminogen,plg/plasmin and the MMP activation cascadesat the cell surface has been well documented(p=latent, a=active forms) [3,15,31]. Suchpericellular proteolytic processing may bepartially protected from the action of the naturalinhibitors of the component enzymes. Theregulation of the key cell surface activities uPAand MT1 MMP, by intracellular signallingcascades linked to integrins and thecytoskeleton is still largely conjectural. The uPAreceptor may be linked to integrins, caveolinand associated kinases [13]. MT1 MMP isknown to be activated intracellularly by theserine proteinase furin. Interaction with PDZproteins may also be involved in the traffickingof MT1 MMP to the cell surface. Further workis required to establish the nature of thecurrently highly speculative MT1 MMPinteractions with components of thecytoskeleton and adhesion complexes, such askinases, integrins and syndecans.

Intracellularinteractions

Extracellular interactions

SrcShcCaveolinIntegrins

Actin

Actin Pericellularspace

Integrin

PlasminuPA

plg

pMMP-13

aMMP-2

aMMP-1

aMMP-3

aMMP-9

EnhancedECM

proteolysis

Inhibitorsα2APPAITIMPS

aMMP-13

TIMP-2

Furinactivation

PDZ proteins?Syndecan-2?Actin?Integrin?

??

?P

α

aMT-MMP

pMMP-2β

β

uPAR


α

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[48]. Transmigration of T lymphocytes into perivasculartissues involves movement across the endothelial cell layerand is a dynamic process in which both T cells andendothelial cells actively modulate their cell–cell andcell–ECM interactions. The binding of autoreactive T cellclones expressing α4 integrin to V-CAM 1 (vascular celladhesion molecule 1) on endothelial cells in experimentalautoimmune encephalomyelitis (EAE) caused the induc-tion and activation of MMP2 in the T cells, apparentlyfacilitating migration as two synthetic inhibitors of MMPsprevented this process as well as EAE induction [49]. Totallevels of MTI MMP protein did not appear to change onα4 ligation but the activation, clustering and localisationwere not studied. Studies with the MMP2 and MMP9knockout mice have revealed that host MMP2 and MMP9synthesis have important roles in the angiogenic response[50•,51••]. Although it is unknown which ECM compo-nents were degraded in these systems, it is highly likelythat endothelial cell migration and invasion through thevascular basal lamina was impeded through the lack ofthese MMPs.

MMP9 (gelatinase B) has been implicated in cell invasionin a number of model systems but little is known of itsmechanism of action in this regard including its ability tointeract with cell surfaces. The very recent observationthat MMP9 is specifically located in lung epithelial cellswith an active migratory phenotype at the front edge of awound is of particular interest. MMP9 appears to be asso-ciated with type IV collagen production at primordial cellsubstratum contacts where vinculin and actin also accumu-late [52•]. Confinement of proMMP9 in areas ofcell–matrix contact have been described in breast epithe-lial cells [53•] and in endothelial cells [54] and activationby a plasmin-mediated mechanism proposed [55]. ActiveMMP9 has also been found in association with theCD44V3,8–10 splice variant on the invadapodia of a breastcancer cell line. This form of CD44 is known to be prefer-entially expressed on the surface of metastatic tumour cellsand to promote cell migration [56•,57•] (Figure 1). Murinecarcinoma cells overexpressing another isoformCD44V3,7–10 showed an enhanced ability to degrade colla-gen IV which could be blocked by a functional blockingantibody to MMP9. The transfected cells were also able toinvade myoblast monolayers in an augmented fashion,with a requirement for MMP9 activity. Hyaluronan-medi-ated clustering of CD44 appeared to be a feature of thecoclustering of CD44 and MMP9 (Figure 2). A membrane-anchored serine-protease-inhibitor-like glycoprotein,RECK has been shown to suppress invasive activity of var-ious malignant cells by the downregulation of MMP9secretion [58]. RECK appears to bind to MMP9 but itsmechanism of action is otherwise unclear.

MMP modification of matrix: effects on cellmigrationThe question of what variety of functions cell surfacelocalised MMP2 may have, besides ‘creating a path’, is also

of importance. Giannelli et al. [59] reported that exogenousMMP2 induces the migration of breast epithelial cells onlaminin 5. MMP2 specifically cleaved the α2 subunit oflaminin 5 exposing a putative cryptic pro-migratory sitethat triggered cell motility.

Recent studies suggest that modification of the basal lam-ina of the endoneurium is essential to peripheral nerveregeneration following injury. Dorsal root ganglion neuronscan only extend neurites when cultured on frozen sectionsof predegenerated adult nerve but are unable to do so onnormal nerve [60]. Treatment of peripheral nerve sectionswith MMP2 results in the removal of a putative chon-droitin sulphate proteoglycan and exposure of epitopespermissive for neurite extension [61]. The precise natureof this new substrate is unclear but antibody blocking stud-ies reveal that neurite extension following MMP2 cleavageis mediated by one or more laminin isoforms.

Keratinocyte migration across a dermal matrix in woundhealing has been shown to depend on MMP1 (collagenase 1)activity [62]. The expression of MMP1 was shown to bedependent on interaction of α2β1 integrin with type I col-lagen and migration of keratinocytes over a collagenousmatrix was prevented by functionally blocking antibodiesto α2 integrin. It is possible that keratinocytes that havecleaved type I collagen do not adhere well to the cleavedfragments and move forward searching for intact collagento which they can attach. Perhaps a more likely scenario isthat keratinocyte expression of αvβ5 integrin could beinvolved in migration over collagenous fragments. It isknown from several studies that collagenase cleavage oftype I collagen results in the exposure of Arg-Gly-Asp(RGD)-dependent integrin binding sites [63]. Recent dataindicate that there is a close association of MMP1 with theα2 integrin subunit (Figure 2), such that these entities canbe co-immunoprecipitated from keratinocyte extracts [64].MMP1 was shown to bind to the A domain of α2 integrinin a cation-dependent manner. Even more interesting isthe fact that MMP1 and type I collagen may bind at twoindependent sites on the α2 integrin, as mutated A domainwhich no longer binds type I collagen, can still bind toMMP1. These results raise the intriguing possibility thatMMP1 expression may be tightly focussed to the leadingedge of migrating keratinocyte where α2β1 integrin hasbeen localised in other cell types (Figure 2).

Conclusions and future directionsThe concept that cells focus proteolytic activities at theircell surface to help remove ECM barriers to migration, orto promote detachment, is upheld by the recent dataemerging in this area. The next year should see the devel-opment of studies relating cell-surface proteinases with thecytoskeleton and with cellular signalling activities.A promising key area of research in this regard is the uPAreceptor–caveolin–integrin interactions and their conse-quences. The role of growth factors with distinctive effectson cell–cell and cell–matrix binding, such as hepatocyte


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growth factor, will also be prominent. The potential associ-ations of the MT MMPs with the cytoskeleton and PDZproteins or other scaffolds for signalling complexes is alsolikely to be a burgeoning area. Other proteinase systemsmay emerge as being of more importance for cell migra-tion, including the membrane associated serine proteinase,seprase [65,66] and the mammalian astacins such asmeprin [67] and the reprolysins (ADAMs [a disintegrin andmetalloproteinase]).

AcknowledgementsWe are indebted to Vera Knäuper for drawing the figures and to Jill Gortonfor manuscript preparation. Our research is funded by the Medical ResearchCouncil, the Wellcome Trust, the Arthritis Research Campaign and theBritish Heart Foundation. Due to the limited number of citationspermitted, we apologise to all those whose work is not featured.

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest••of outstanding interest

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Proteolysis and cell migration: creating a path?

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