Extracellular matrix remodelling: the role of matrix metalloproteinases

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1 Journal of Pathology J Pathol 2003; 200: Published online in Wiley InterScience ( DOI: /path.1400 Review Article Extracellular matrix remodelling: the role of matrix metalloproteinases Ivan Stamenkovic* Experimental Pathology Division, Institut Universitaire de Pathologie, Université de Lausanne, Lausanne, Switzerland *Correspondence to: Dr Ivan Stamenkovic, Experimental Pathology Division, Institut Universitaire de Pathologie, Université de Lausanne, 25 Rue du Bagnon, CH-1011 Lausanne, Switzerland. Abstract Matrix metalloproteinases (MMPs) are a growing family of metalloendopeptidases that cleave the protein components of the extracellular matrix and thereby play a central role in tissue remodelling. For many years following their discovery, MMPs were believed to function primarily as regulators of ECM composition and to facilitate cell migration simply by removing barriers such as collagen. It is becoming increasingly clear, however, that MMPs are implicated in the functional regulation of a host of non-ecm molecules that include growth factors and their receptors, cytokines and chemokines, adhesion receptors and cell surface proteoglycans, and a variety of enzymes. MMPs therefore play an important role in the control of cellular interactions with and response to their environment in conditions that promote tissue turnover, be they physiological, such as normal development, or pathological, such as inflammation and cancer. This review summarizes some of the recent discoveries that have shed new light on the role of MMPs in physiology and disease. Copyright 2003 John Wiley & Sons, Ltd. Keywords: matrix metalloproteinases; remodelling; cancer; inflammation; development Introduction The extracellular matrix (ECM) plays a key role in tissue architecture and homeostasis. In most organs, the principal proteinaceous components of the ECM are collagens that are produced and secreted by a variety of stromal cells, although predominantly fibroblasts, and which provide much of the scaffold necessary for the organization of cells that constitute the tissue. Numerous other proteins contribute to specialized components of the ECM structure, such as the basement membrane, including laminin, entactin, collagen IV, and various growth factors and proteases [1]. A second class of molecules that play an essential role in the composition of the ECM are secreted proteoglycans whose protein core is covalently bound to high-molecular-weight glycosaminoglycans, including chondroitin, heparan, and keratan sulphate. Proteoglycans are implicated in numerous processes, two of their major functions including the support of cell adhesion and the binding of a host of latent growth factors [2]. The ECM also contains one major glycosaminoglycan, hyaluronan (HA), which is typically not sulphated or bound to a protein core. Hyaluronan is a polymer built of repeating disaccharide units with the structure [D-glucuronic acid (1-β-3) N -acetyl-dglucosamine (1-β-4)] n, which is synthesized primarily by fibroblasts, although other stromal cells produce HA as well, and deposited in the ECM of virtually all organs. Hyaluronan participates in the regulation of numerous cellular functions, prominent among which are adhesion, trafficking, and signalling [3]. Together, the ECM components thus constitute a structure that in addition to helping maintain tissue integrity regulates cell migration and provides a reservoir of cytokines and growth factors. By its very nature, the ECM is constantly undergoing changes in response to a host of cellular stimuli. These range from dynamic homeostasis that typifies resting-state adult organs to full-blown tissue remodelling, as occurs during normal development, inflammation, wound healing, and cancer, characterized by major alterations in both ECM structure and composition. ECM remodelling is the result of multiple concurrent processes that vary according to the initiating stimulus. Thus, the structural and functional changes within the ECM of a given organ that occur during an acute inflammatory response differ from those that accompany limb development and tumour invasion. Nevertheless, several key mechanistic features of tissue remodelling are common to most aetiologies. Remodelling at the very minimum requires two events: synthesis and deposition of ECM components on the one hand and their proteolytic breakdown on the other. Numerous proteases have been implicated in the proteolytic degradation of the ECM, most prominent among which are members of the matrix metalloproteinase (MMP) family. Matrix metalloproteinases form a subfamily of the metzincin superfamily of proteases, which constitutes one of several metalloendopeptidase families [4 6]. The metzincins share a conserved structural topology, a consensus motif within the catalytic domain containing three histidines that provide a zinc binding site and Copyright 2003 John Wiley & Sons, Ltd.

2 Extracellular matrix remodelling 449 a conserved Met-turn motif that resides beneath the active site zinc ion [5]. Based on structural similarity, the metzincins are subdivided into four subfamilies: the serralysins, adamalysins, astracins, and matrixins (MMPs). The first insight into the function of MMPs was obtained as a result of the observation that their proteolytic activity was responsible for the dissolution of the tadpole tail [7]. It was subsequently recognized that the >20 human MMPs and homologues from other species could cleave practically all of the protein components of the ECM. Based on their perceived specificity for ECM proteins, MMPs have been divided into collagenases, gelatinases, stromelysins, and matrilysins. However, the high degree of overlap among MMP substrate specificities and the notion that MMPs can cleave a growing list of substrates that are not part of the ECM [8,9] render this nomenclature imprecise at best. MMPs are assigned a number that corresponds to the chronology of their identification, but a more rational approach as to their classification based on their structure is currently being adopted [9]. Thus several subclasses of MMPs have been identified, some of which are secreted whereas others are membrane bound (Figure 1). All MMPs are produced as zymogens containing a secretory signal sequence and a propeptide whose proteolytic cleavage is required for MMP activation. The propeptide is followed by the catalytic domain that contains the consensus zincbinding motif HEBXHXBGBXH, where X is a variable residue and B is a bulky hydrophobic residue. At least two MMPs (MMP-7 and MMP-26) are composed only of the signal peptide, propeptide, and catalytic domain, and are known as minimal domain MMPs (Figure 1). Most of the remaining MMPs contain a haemopexin-like domain that is thought to confer some degree of substrate specificity, and several have additional features, such as fibronectin-like repeats or serine protease recognition motifs (Figure 1). Finally, a subclass of MMPs contains a transmembrane and intracellular domain and are often referred to as MT- MMPs. Regulation of MMP activation and proteolytic activity MMP activity is controlled at least at three levels: transcription, proteolytic activation of the zymogen form, and inhibition of the active enzyme by a host of natural inhibitors. Most MMPs are expressed at low levels or not at all in resting-state adult tissues. However, numerous cytokines and growth factors as well as physical cellular interactions provide stimuli that can rapidly induce MMP expression (reviewed in [10] and references therein). The propeptide serves to maintain pro-mmps in an inactive state. A cysteine-sulphydryl group within the distinctive consensus PRCGXPDV motif in the propeptide domain binds to the active site zinc atom, thereby preventing activation. Disruption of this interaction by physical or chemical means constitutes the first step in MMP activation, which is followed by proteolytic cleavage of the COOH-terminal side of the PRCGXPDV site with irreversible loss of the cysteine residue [6,11,12]. Whereas activation of most MMPs occurs outside the cell, several MMPs, including MMP-11 and -28 and membrane-bound MMPs, contain furin protease cleavage sites and may be activated by intracellular furin-like serine proteases before reaching the cell surface [12]. Most MMPs can be activated by other MMPs (Table 1) and a variety of serine proteases in vitro [11]. However, with one notable exception, the mechanisms of physiological extracellular activation of MMPs remain to be elucidated. The exception is activation of MMP-2. It has been demonstrated that MMP-14 binds the tissue inhibitor of metalloproteinases 2 (TIMP-2) on the cell surface and recruits MMP-2, which binds to the immobilized TIMP-2 through its haemopexin-like domain [13,14]. A TIMP-free MMP-14 is then recruited to the complex and cleaves the MMP-2 propeptide, resulting in partial MMP-2 activation, which is completed by an adjacent active MMP-2 [15]. MMP activity is tightly controlled by several endogenous inhibitors. In tissue fluids, the principal MMP inhibitor is α 2 -macroglobulin, which binds to MMPs and creates a complex that is itself bound and irreversibly cleared by scavenger receptors [16,17]. The most thoroughly studied MMP inhibitors, however, are TIMPs. The four TIMPs that have been characterized thus far are small molecules of kda that bind to MMPs in a 1 : 1 stoichiometric ratio and reversibly block MMP activity (reviewed in [18]). TIMPs differ in their expression patterns and affinity for the various MMPs. Thus, TIMP-1 and TIMP- 2 inhibit the activity of a broad range of MMPs and, by analogy to the TIMP-2/pro-MMP-2 interaction, TIMP-1 may form a complex with pro-mmp-9 [18,19]. The TIMP-1/pro-MMP-9 complex is thought to recruit MMP-3, forming a stable ternary complex that causes MMP-3 inactivation [20]. However, the physiological relevance of this putative complex remains to be established. TIMP-3 preferentially inhibits the activity of MMP-1, -3, -7, and -13, and unlike other TIMPs blocks the activity of the more distantly related metalloproteinase ADAM-17 [21]. TIMP-4 has a more restricted inhibitory activity that primarily blocks MMP-2 and -7 and to a lesser extent MMP-1, -3, and -9 [22]. Recent evidence suggests that TIMPs may have additional functions to those of MMP inhibition [18]. A case in point is the observation that an important physiological function of TIMP-2 is the activation of MMP-2 [23]. Among other molecules capable of regulating MMP proteolytic activity are thrombospondin and RECK. Thrombospondin-2 binds MMP-2 in a complex that facilitates scavenger receptor-mediated endocytosis and clearance [17], whereas thrombospondin-1 binds to prommp-2 and -9 and directly blocks their

3 450 IStamenkovic MMP structure Minimal domain: MMP-7 MMP-26 S S Simple hemopexin domain: MMP-1 MMP-3 MMP-8 MMP-10 MMP-12 MMP-13 MMP-19 MMP-20 MMP-27 Type II fibronectin-like repeats: MMP-2 MMP-9 S S S S Simple hemopexin domain, furin-activated: MMP-11 MMP-28 S S Transmembrane MMPs: MMP-14 MMP-15 MMP-16 MMP-24 GPI-anchored MMPs: MMP-17 MMP-25 S S signal sequence pro-domain Zinc binding domain transmembrane domain furin recognition motif Type II fibronectin repeats catalytic domain S S Hinge region Hemopexin-like domain cytoplasmic domain GPI anchor Figure 1. Protein structure of MMPs. The principal structural subclasses of MMPs are shown and the different domains indicated. Individual MMPs that belong to each structural subclass are listed

4 Extracellular matrix remodelling 451 Table 1. Non-ECM substrates of matrix metalloproteinases MMP Growth factor precursors and binding proteins Cytokines chemokines Adhesion receptors Miscellany 1 IGFBP, perlecan CXCL12, protnf-α α 1 -Antichymotrypsin α 1 -Proteinase inhibitor prommp-1, 2 2 Decorin, protgf-β, IGF-BP, protnfα, MCP-3 (CCL7) prommp-1, -2, -13 FGFR1 3 IGFBP, perlecan, decorin, prohb-egf proil-1β protnf-α proil-1β CXCL12 7 Decorin, prohb-egf FasL, protnf-α E-cadherin E-cadherin β 4 -Integrin Syndecan-1 Plasminogen α 1 -Antichymotrypsin α 1 -Proteinase inhibitor prommp-1, -3, -7, -9, -13 Plasminogen Pro-α-defensin prommp-7 8 α 1 -Antichymotrypsin α 1 -Proteinase inhibitor prommp-8 9 ProTGF-β ProIL-1β IL-2Rα protnf-α CXCL7, CXCL8 CXCL1, CXCL12 KitL ICAM-1 Plasminogen α 1 -Proteinase inhibitor 10 prommp-1, IGFBP α 1 -Proteinase inhibitor 12 Plasminogen 13 CXCL12 prommp-9, CXCL12 CD44 αv integrin 15 ttg 16 prommp-2 17 prommp-2 Cell surface tissue Transglutaminase (ttg) pro-mmp-2 activation [24,25]. Interestingly, thrombospondin-1 has also been reported to increase MMP-2 and -9 activation [26]. A seemingly potent MMP inhibitor is the cell surface receptor known as reversion-inducing cysteine-rich protein with kazal motifs (RECK). It is the only cell surface MMP inhibitor characterized thus far [27]. A recently identified mechanism that controls MMP activity is cell surface localization. It has long been suggested that MMPs function at or in the vicinity of the cell surface [28]. Accumulating evidence now suggests that cell surface association may be critical for optimal MMP function. MT-MMPs have been shown to localize to invadopodia specialized plasma membrane protrusions believed to constitute the cellular structures that direct invasion [29,30] and to lose their proteolytic activity when expressed in secreted form [29,31]. Moreover, an increasing number of secreted MMPs are now recognized to at least transiently localize to the cell surface, most often in association with adhesion receptors or cell surface proteoglycans. Thus, MMP-1 associates with integrins [32] and EMMPRIN, an immunoglobulin superfamily member that induces MMP expression [33,34]. In addition to the MMP-14/TIMP-2 complex, MMP-2 interacts with αvβ 3 -integrin via its haemopexin-like domain [35]. MMP-7 binds cell surface heparan sulphate proteoglycans (HSPG), including HSPG isoforms of the hyaluronan receptor CD44 [36] (Figure 2), and MMP-9 binds several cell surface receptors including CD44 [37,38], ICAM-1 [39], and integrins (Lan and Stamenkovic, unpublished). MMP- 9 has also been suggested to use cell surface-bound collagen IV chains as a docking mechanism [40]. MMP-13 binds HSPG (Yu and Stamenkovic, unpublished) and MMP-19 localizes to the cell surface by a mechanism that is as yet undefined [41]. The potential functional importance of cell surface docking of secreted MMPs has recently been highlighted by several studies. Inhibition of cell surface localization of MMP-9 in a mouse mammary carcinoma cell line led to the loss of its invasive and metastatic properties that could be rescued by constitutive cell surface expression of an MMP-9 fusion protein [37,42]. Similarly, disruption of the interaction between CD44HSPG and MMP-7 was observed to result in MMP-7 relocation from the apical to the basal cell surface in postpartum uterine and lactating mammary epithelia that was associated with increased epithelial cell death and inappropriate tissue remodelling in vivo [36] (Figure 2). Cell surface docking may provide a mechanism to tether MMP activity to the regions of physical

5 452 IStamenkovic A model for CD44HSPG-dependent MMP-7-mediated HB-EGF processing extracellular Heparan sulfate MMP-7 prommp-7 Cell membrane CD44HSPG prohb-egf CD44HSPG prohb-egf intracellular A A model for CD44HSPG-dependent MMP-7-mediated HB-EGF processing HB-EGF HB-EGF Cell membrane CD44HSPG CD44HSPG ErbB4 perbb4 B Growth and survival signals Figure 2. A model for CD44HSPG-dependent, MMP-7-mediated HB-EGF processing. (A) The HSPG isoform of CD44 coordinates the formation of a cell surface complex containing prommp-7 and prohb-egf. MMP-7 becomes activated by a mechanism that remains to be determined. (B) Active MMP-7 cleaves prohb-egf and the mature HB-EGF moiety is presented by the heparan sulphate chains to ErbB4. Engagement of ErbB4 by HB-EGF results in phosphorylation of tyrosine residues in its intracellular domain and the triggering of survival and growth signals. ErbB family members interact with CD44 independent of heparan sulphate, and possibly via intracellular adaptor molecules. This mechanism is hypothetical, however, and the putative adaptors remain to be identified contact between the cell and ECM where ECM remodelling occurs. In support of this view, integrins and ECM-degrading proteases have been observed to cocluster in invadopodia [43,44]. Adhesion receptormediated cell surface docking of MMPs may not only concentrate MMP proteolytic activity at sites where it is most needed, but potentially provide the cell with a means to control ECM degradation, preserving a scaffold of partially degraded ECM proteins that facilitates migration and helps generate survival signals.

6 Extracellular matrix remodelling 453 Growth factor precursor processing on the cell surface, in proximity to the corresponding receptors, may provide MMPs with the ability to optimize the effectiveness of growth factor-mediated stimulation. It is also conceivable that cell surface localization may play a part in the control of MMP activation, as has been shown to be the case for MMP-2. Finally, the apparent persistent activity of cell membrane-anchored MMP-7 and MMP-9 [36,37,42] raises the possibility that the cell surface may provide a protective niche for MMPs from natural inhibitors. However, there is no indication at present as to the mechanisms that may underlie such protection. MMP substrates MMPs have long been thought to primarily degrade ECM proteins and their ECM substrate specificity has been extensively reviewed [8 10]. Facilitation of cell migration was believed to be the principal effect of the MMP-mediated breakdown of ECM barriers. However, the effects of ECM degradation on cell behaviour are likely to be considerably more complex, due, at the very least, to a combination of changes in the nature of physical interactions between the cell and the degraded ECM proteins on the one hand, and the release of a host of ECM-sequestered cytokines on the other. Several ECM degradation products have been shown to display unique biological properties at least in part by exposure of new recognition sites for cell surface ECM receptors that can trigger a variety of cellular signals. For example, cleavage of collagen IV and laminin-5 generates exposure of cryptic sites that promote migration [45,46]. Although ECM degradation remains an important physiological function of MMPs, recent observations provide convincing evidence that MMP substrates include a host of non-ecm molecules ranging from growth factor precursor and binding proteins to cell surface adhesion receptors (Table 1). These newly recognized properties suggest a more extensive involvement of MMPs in a variety of physiological and pathological processes than had previously been appreciated. Degradation of insulin-like growth factor binding protein (IGF-BP) by several MMPs releases IGFs [47,48], cleavage of perlecan releases fibroblast growth factors, [49] and proteolysis of latent TGFβ binding proteins, including decorin, augments the bioavailability of latent TGF-β. Latent TGF-β 1 and 2 can be proteolytically activated by MMP-2 and MMP-9 [42], and recent evidence suggests that active TGF-β can be released my MMP-14 from cell surface complexes involving αvβ 8 -integrin [50]. In addition, growth factor precursors can be activated and released by MMP-mediated proteolytic cleavage. Cell surface growth factor precursors that undergo MMP-mediated cleavage identified thus far include members of the EGF family, particularly heparin-binding EGF (HB- EGF) precursor, cleaved and activated by MMP-3 [51] and MMP-7 [36]. Indirect evidence suggests that several growth factor receptors are MMP substrates. Shedding of members of the EGF receptor family, including ErbB2 and ErbB4, is blocked by TIMP-1 [52,53], whereas the release of hepatocyte growth factor/scatter factor receptor c-met [54] is blocked by TIMP-3. Fibroblast growth factor receptor 1 is proteolytically cleaved by MMP-2 [55]. In all cases, cleavage occurs within the extracellular domain. Cell surface adhesion receptors and proteoglycans are also subject to MMP-mediated cleavage and shedding. E-cadherin is cleaved by MMP-3 and -7 [56] whereas the extracellular domain of the hyaluronan (HA) receptor CD44 is cleaved by MMP-14 [57]. Integrins have been observed to serve as MMP substrates, as exemplified by the cleavage of αv integrin chain by MMP-14 [58], and that of β4-integrin by MMP-7 [59]. Shedding of ICAM-1 has recently been shown to be MMP-9 mediated [39], and MMP-7 has been found to cleave the proteoglycan syndecan-1 [60]. Syndecan-1 associates with the CXC chemokine KC, and release of the syndecan/kc complex from the cell surface as a result of MMP-7-mediated proteolysis is reported to play an important role in regulating neutrophil influx to sites of injury [60]. Finally, a variety of cytokines, cytokine receptors, and chemokines have been observed to undergo MMPmediated cleavage. Tumour necrosis factor α (TNFα) is released from the cell surface by ADAM-17 and MMP-1, -3, and -7 [8,61], while Fas ligand (FasL) is shed as a result of MMP-7-mediated proteolysis [62,63]. Interleukin-2 receptor α has been proposed to undergo MMP-9-dependent downregulation [64]. MMP-2 cleaves and inactivates monocyte chemoattractant protein-3 (MCP-3, also known as CCL7 [65]). MMP-9 cleaves the neutrophil chemoattractant interleukin-8 (IL-8, also known as CXCL8), strongly increasing its activity [66]. By contrast, MMP-9 proteolytically inactivates connective tissue-activating peptide III (CTAP-III, also known as CXCL7), platelet factor 4 (PF4, also known as CXCL4), and growthrelated oncogene α (GROα, also known as CXCL1 [66]). Stromal cell-derived factor (SDF-1 also known as CXCL12) undergoes inactivating cleavage by several MMPs, including MMP-1, -3, -9, -13, and -14 [67]. Interestingly, induction of SDF-1 expression in the context of bone marrow ablation upregulates MMP-9 expression, resulting in the proteolytic cleavage of Kit ligand (KitL) and the recruitment of ckit+ stem cells [68]. MMPs and development The nature and variety of MMP substrates predict that a major physiological role of MMPs should lie in tissue morphogenesis and organization. Indeed,

7 454 IStamenkovic numerous experimental approaches have shown that MMPs help control processes that range from cell aggregation in vitro to branching morphogenesis of several organs and hormone-dependent tissue cycling. Adipocytes cultured on basement membrane organize into large multicellular clusters that secrete MMP- 2. Inhibition of MMP activity blocks migration of these cells and three-dimensional cluster organization [69]. Pancreatic islet cell morphogenesis also appears to depend on MMP activity. In vitro, embryonic pancreatic islet epithelial cells cultured on collagen gels differentiate and organize into aggregates that resemble the islets of Langerhans and secrete MMP-2. Inhibition of MMP activity abrogates cell organization into islet type structures without affecting endocrine cell differentiation [70]. MMPs are implicated in branching morphogenesis, although the mechanisms appear to vary according to the type of tissue. Culture of day 10 embryonic kidney explants results in ureteric bud development by branching morphogenesis that is inhibited by blocking anti-mmp-9 antibody [71]. This process may also implicate MMP-14, and can be enhanced by inhibition of TIMP-2 expression [72]. In contrast, branching of cultured salivary glands is enhanced by MMP inhibition [73]. This dichotomy may be explained by differences in the mechanisms that underlie branching of the two epithelia. Whereas kidney epithelium branches by budding and growing into the mesenchyme, a process that requires ECM degradation, salivary gland epithelium branches by the formation of clefts composed of collagen bundles in the epithelium in parallel to growth into the surrounding mesenchyme. It follows that in the kidney ECM degradation would be predicted to enhance branching by reducing the resistance of the ECM barrier, whereas in salivary glands MMPmediated degradation of collagen would be expected to destabilize the clefts and reduce branching. The ECM of the mammary gland is subject to major remodelling during development, lactation, and weaning, and several lines of evidence suggest that MMPs are implicated in each of these events. Mammary gland formation during embryonic development begins by the budding of an epithelial tubular structure into the mammary fat pad. Extensive growth and branching at the terminal end bud follow to form the epithelial ducts. These ducts end in structures that differentiate into alveoli, which constitute the milk-secreting units during lactation. Alveolar development during pregnancy and lactation dramatically alters mammary gland architecture, which regains its pre-pregnancy state during weaning by a process known as involution. Expression of an active form of MMP-3 in the mammary gland alters gland development [74,75]. Virgin female mice that express a low level of the transgene in mammary epithelia display abnormal gland morphology characterized by excessive branching of the primary ducts and early alveolar development with β-casein expression at levels comparable to those observed in early to mid-pregnancy glands. Lactating glands have high levels of transgene expression and show loss of basement membrane integrity with depletion of laminin and collagen IV. This is accompanied by a reduction in alveolar size and premature alveolar epithelial apoptosis during late pregnancy. The extracellular MMP activity correlates with increased cleavage of the basement membrane molecule entactin in the vicinity of cells undergoing apoptosis. Alveolar epithelial cells are rescued from apoptosis when mice overexpressing MMP-3 in the mammary epithelium are crossed with mice overexpressing TIMP-1 [76]. Interestingly, mice deficient in CD44, which provides a docking receptor for MMP-7 on the epithelial cell surface, display a similar mammary gland defect [36]. In these mice MMP-7 activity is mislocalized, such that instead of being confined to the apical epithelial surface, it diffuses to the basement membrane. Alveolar epithelial cells undergo premature apoptosis, presumably because of impaired MMP-7- mediated prohb-egf processing [36], and the glands undergo premature involution that may be due, at least in part, to ECM degradation resulting from inappropriate localization of MMP-7 proteolytic activity. Clearly, MMP activity can have a potent effect on mammary gland development and function, although the full extent of its involvement remains to be elucidated. MMP-7 has recently been found to play a role in postpartum uterine involution. It has been known that in the rat MMP-7 expression is induced in the uterus at the time of delivery and reaches a maximum at about 48 h postpartum, following which it decreases rapidly, becoming undetectable at 5 days [77]. In CD44-deficient mice, expression of MMP- 7 in postpartum uterus is comparable to that in wild-type tissue but the cellular distribution of the enzyme is altered. Instead of localizing to the apical cell surface, as in postpartum uteri of wild-type mice, MMP-7 diffuses to the basal compartment and is released into the basement membrane. This relocalization is accompanied by degradation of the ECM and accelerated involution compared to uteri of wild-type animals [36]. These observations support the notion that location plays a major role in the effect of MMP activity. MMPs in bone development There is increasing evidence that MMPs are implicated in bone development. Two types of ossification underlie skeletal development: intramembranous and endochondral [78]. Intramembranous ossification, restricted to the skull and clavicles, results from direct differentiation of the mesenchymal condensations that precede the future skeletal elements into osteoblasts. Endochondral ossification is the process whereby the rest of the future skeleton develops, characterized by the differentiation of the mesenchymal condensations into chondrocytes, which form the cartilage anlagen of

8 Extracellular matrix remodelling 455 the future bones. In the periphery of the anlage, cells from the perichondrium differentiate into osteoblasts, whereas the periphery of the anlage becomes hypertrophic as a result of chondrocyte proliferation and maturation. Hypertrophic cartilage is progressively resorbed by the action of chondroclasts that invade and penetrate the cartilage and replaced by bone matrix following the invasion of capillaries. The bone matrix is deposited by osteoblasts that follow capillary invasion, and is remodelled by the action of osteoclasts. Homeostasis in adult bone is maintained by the balance of osteoblast and osteoclast activity. Thus far, at least two MMPs have been implicated in the regulation of bone development: MMP-9 and -14. Mice in which the MMP-9 gene has been inactivated display a specific defect in endochondral bone formation, characterized by an accumulation of hypertrophic cartilage at the skeletal growth plates [79]. This inappropriate cartilage accumulation appears to be due to impaired vascular invasion into the hypertrophic cartilage zone, which leads to inhibition of endochondral ossification. In addition, apoptosis of terminal hypertrophic chondrocytes is delayed, suggesting that death of these cells is related to capillary invasion. This notion is supported by the observation that chondrocytes proximal to the capillaries are the ones that undergo cell death. Although it is not clear how MMP-9 activity regulates endochondral ossification, one possibility may be that MMP-9 helps release one or several angiogenic factors from the hypertrophic cartilage ECM [79]. This would explain the observed defect in angiogenesis in the absence of MMP-9, along with the inhibition of the processes that are dependent on it. In support of this notion, VEGF has been shown to be a major angiogenic factor at the growth plate, and its inhibition by recombinant soluble VEGF receptor (VEGFR) leads to abrogation of vascular ingrowth into hypertrophic cartilage, resulting in a phenotype that is similar to but more severe than that observed in MMP-9 null mice [80]. Expression of soluble VEGFR, however, has additional effects on bone development, including a decrease in chondroclast and osteoblast recruitment. It seems therefore that MMP- 9 may regulate bone formation by a partial effect on VEGF activity or on the activity of other angiogenic factors that cooperate with VEGF. It is noteworthy that impaired endochondral ossification is the only developmental defect in MMP-9 null mice, suggesting that the non-redundant functions of MMP-9 during development are highly restricted. In contrast to MMP-9 null mice, MMP-14 null animals display numerous skeletal defects [81]. In addition to craniofacial dysmorphisms caused by impaired intramembranous bone formation, these mice display dwarfism that may reflect defective endochondral ossification as well as osteopenia, arthritis, and fibrosis of soft tissues [81]. The cranial abnormalities are due in part to impaired removal of the calvarial cartilage primordia, which persist and transform into a fibrotic vestige. This may interfere with the normal formation of the calvarial bones and suture closure. In the long bones, there is defective ossification of the epiphysis. Under normal conditions, hypertrophic cartilage is formed in the centre of the epiphysis from the inward maturation of chondrocytes, and undergoes ossification by perichondral vessel invasion through vascular canals formed by the perichondrium. In MMP-14 null mice, no vascular canal is formed. Ossification is therefore delayed and occurs only when the hypertrophic cartilage has expanded into the perichondrium by direct invasion of perichondrial vessels. Delayed epiphyseal ossification in these mice is associated with growth plate disorganization and reduced chondrocyte proliferation, which may contribute to the observed dwarfism. Progressive fibrosis of the periskeletal soft tissues may also contribute to dwarfism and, interestingly, similar defects have been described in mice with a mutation in the collagenase cleavage site of type I collagen [82]. Bone marrow stromal cells and fibroblasts isolated from MMP-14 null mice display impaired collagenolytic activity in vitro, suggesting that the observed developmental defects may be due, at least in part, to inadequate remodelling of the skeletal matrix and periskeletal soft-tissue ECM. Decreased osteogenesis from bone marrow stromal cells in MMP- 14 null mice suggests that differentiation or function of osteoblasts may be impaired, while the observed increase in the osteoclast number suggests that MMP- 14 may play a regulatory role in the development of these cells as well. MMPs in physiological invasion and wound healing There is strong evidence that MMPs participate in normal cell invasion, migration, and process outgrowth in physiological settings. Physiologically invasive cells include, among others, trophoblasts and osteoclasts, whereas neurons require mechanisms that control neurite extension. At the implantation site, trophoblasts must penetrate into the maternal decidua. Trophoblasts express high levels of MMP-9, and blocking anti- MMP-9 antibody can abrogate their invasion of ECM in vitro [83,84]. Osteoclasts are recruited to bone surfaces during bone remodelling, and their migration through collagen gels is blocked by MMP inhibitors [85]. In a model of neuronal differentiation, the neuroblastoma cell line SKSNBE displays extensive neurite outgrowth in response to retinoic acid. MMP-9 is induced and expressed in the neurites, suggesting its participation in the outgrowth [86]. These observations are supported by studies showing that oligodendrocytes in culture express active MMPs at the tip of their processes and that blocking anti-mmp-9 antibody inhibits extension of the processes [87]. Oligodendrocytes from MMP-9-deficient mice fail to display process outgrowth in culture. Wound healing relies on processes that are similar to those required to promote physiological development and malignant tumour progression, including

9 456 IStamenkovic migration, ECM degradation, and invasion. In culture, keratinocytes migrate on collagen-1 in a manner that requires specific cleavage of collagen-1 by MMP-1. MMP inhibitors can block this migration and epithelial cells that do not express MMP-1 do not migrate on collagen type I. Consistent with this observation, keratinocytes do not migrate on collagen type I that bears a mutation in the MMP- 1 cleavage site [88]. Keratinocytes at the edge of the wound must migrate to cover, or re-epithelialize the wound surface. The fibrin-rich provisional matrix that is deposited following wounding must then be removed and the dermis contracts to facilitate wound closure. In wound-healing models, MMP inhibitors abrogate keratinocyte migration and delay wound healing in vivo [89]. Similar wound-healing impairment is observed in plasminogen-deficient mice, suggesting that both plasmin and MMPs play a significant role in keratinocyte migration during wound healing [90]. Keratinocyte migration is completely blocked in plasminogen-deficient mice treated with MMP inhibitors, indicating synergism between these two classes of enzymes [89]. Wound healing is delayed in MMP-3 null mice, although by a mechanism that does not appear to implicate impaired keratinocyte migration. MMP-3 activity appears to be important in fibroblast-mediated wound contraction as MMP-3 deficient fibroblasts have a reduced ability to contract collagen gels [91,92]. MMPs in cancer Most of the evidence that MMPs are implicated in cancer development and progression stems from experimental models in which expression of selected MMPs in tumour cells has been observed to promote tumour growth, invasion, and metastasis, whereas natural and synthetic MMP inhibitors have been shown to reduce and even abrogate tumour development (reviewed in [93]). In virtually all human cancers, MMP expression and activity has been found to be increased and to correlate with invasiveness and poor prognosis, supporting the experimental data and the notion that MMPs play a role in human cancer progression. Despite the conceptually straightforward relationship between MMP activity and tumour aggressiveness, there are instances where MMP expression appears to be associated with a more favourable prognosis. In colon cancer, expression of MMP-12 by the tumour cells and MMP-9 expression by infiltrating macrophages have been reported to correlate with reduced metastatic proclivity [94,95]. To what extent MMP activity is the driving force behind the biological behaviour of these tumours remains to be clarified. The notion that MMP activity correlates with tumour aggressiveness would imply an inverse relationship between MMP inhibitor expression and tumour progression. However, tumours with poor prognosis have been observed to express elevated levels of TIMP-1 and -2 (reviewed in [18]). One possible explanation may be that these tumours display high levels of MMP activity and that the seemingly elevated TIMPs reflect an ineffective attempt of the host tissue to control MMP-mediated proteolysis. However, several observations suggest that TIMPs may have functions other than the ability to block MMPs. First, as discussed above, TIMP-2 is a key component in MMP-2 activation. In addition, at least under some circumstances, TIMPs can induce VEGF expression and promote tumour angiogenesis [96], tumour cell survival [97], and tumour growth [98]. Thus the balance between MMP and TIMP expression may have effects that reach beyond the regulation of MMP activity alone. Although it was initially believed that tumourderived MMPs play the principal role in tumour progression, recent evidence from transgenic mouse models indicates that stromal cell-derived MMPs may play an equally important role. Moreover, whereas some MMPs such as MMP-7 are primarily expressed by tumour cells, others, including MMP-2, -3, and -9, are expressed by stromal cells, sometimes predominantly so. Stromal cell expression of MMPs may be induced by tumour cell infiltration, by direct cell cell contact, in paracrine manner via secretion of tumourderived growth factors, or as a result of stimulation by growth factors released from degraded ECM (Figure 3). Until recently, MMP activity has been associated primarily with tumour invasion and metastasis. It is now becoming clear, partly owing to transgenic mouse models (Table 2), that MMPs may play a critical role in early events in tumour development. Several lines of evidence indicate that MMPs regulate cell growth and survival. By activating cell surface growth factor precursors, releasing and activating latent growth factors sequestered in the ECM and altering the structure of essential ECM components, MMPs directly participate in the generation of signals that induce tumour cell proliferation. At least two classes of growth factor precursors that promote cell proliferation, TGF-α and IGFs, are known to be released and activated by MMPs [47,48]. Several mediators of tumour cell survival are also regulated by MMPs. It has been suggested that MMP-7 promotes carcinoma cell survival rather than invasiveness [99]. At least two mechanisms support this notion. First, MMP-7 cleaves Fas ligand (FasL) from the cell surface, yielding a soluble form whose effectiveness in triggering cell death by engaging its receptor Fas is significantly reduced in some settings [64], even though it may enhance cell death in others [63]. Second, MMP-7 cleaves the heparin-binding EGF precursor from the cell surface, releasing the mature active form that binds ErbB1 and ErbB4 receptors, generating signals that confer protection from apoptosis [36] (Figure 2). Another metalloproteinase that promotes tumour cell survival is MMP-11. Mouse fibroblasts expressing MMP-11 support carcinoma cell

10 Extracellular matrix remodelling 457 Endothelial cell fibroblast leukocyte GFs ECM PGs chemokines MMPs Growth factors cytokines Contactdependent signal exchange A Cancer cells Endothelial cell proliferation Fibroblast activation and migration GFs GFs ECM Cytokines/ chemokines Tissue remodeling Stimulation of migration and growth Leukocyte Recruitment and activation Tumor cells B Migration invasion survival proliferation Figure 3. MMPs in tumour host interactions. (A) Tumour cells invade quiescent stroma containing fibroblasts, endothelial cells, a few leucocytes, and an ECM composed of collagens (grid) and proteoglycans (PGs), and containing numerous growth factor precursors (GFs). Tumour cells express MMPs that are concentrated at the tips of invadopodia and secrete growth factors, some of which may behave as chemokines. (B) As a result of direct physical contact with stromal cells and the effect of their growth factors, tumour cells induce fibroblast activation, endothelial cell proliferation and leucocyte recruitment. Activated fibroblasts express MMPs and secrete growth factors and ECM components; endothelial cells also produce growth factors and express MMPs, and leucocytes display an activated phenotype, contributing their own MMPs and a variety of cytokines and chemokines. The combined effect of MMP activity derived from both the tumour and stromal cells accelerates ECM degradation and growth factor release, enhancing stromal cell activation the remodelling process. The net effect on the tumour cells is promotion of migration, invasion, survival, and proliferation growth, whereas MMP-11-deficient fibroblasts do not [100], and overexpression of MMP-11 reduces apoptosis in tumour xenografts [101]. The exact mechanisms whereby MMP-11 promotes tumour cell survival remain to be defined. MMP-9, which has primarily been associated with invasion and metastasis, also has the ability to protect tumour cells from apoptosis [102]. At least one mechanism whereby MMP-9 appears to promote survival in some tumour types is by proteolytic activation of latent TGF-β ([42] and unpublished observations), which may play a major role in promoting progression of tumours that are resistant to its growth-inhibitory effects [103]. Several lines of evidence indicate that MMPs play an active role in angiogenesis (reviewed in [104]). The sprouting of new blood vessels from existing

11 458 IStamenkovic Table 2. Tumour development in MMP transgenic and knockout mice MMP Promoter Additional stimulus Phenotype Reference MMP overexpression 1 Haptoglobin None Hyperkeratosis [136] DMBA + PMA Increased skin carcinogenesis [136] 3 Whey-acidic protein (WAP) None Mammary hyperplasia and cancer [137] Mouse mammary tumour virus (MMTV) None Mammary hyperplasia and cancer [138] 7 MMTV None Mammary hyperplasia [99] MMTV-HER2/neu Increased mammary carcinogenesis [99] 14 MMTV None Mammary hyperplasia and cancer [139] MMP deletion 2 RIP-Tag Reduced pancreatic carcinogenesis [102] 7 Apc Min Reduced Intestinal adenoma [140] formation 9 K14-HPV16 Reduced skin carcinogenesis [106] RIP-Tag Reduced pancreatic carcinogenesis [102] 11 DMBA Reduced mammary carcinogenesis [100] ones requires at the very least the creation of a path along which endothelial cells can migrate and form new tubular structures. Intuitively, ECM degradation should therefore provide one mechanism whereby MMPs facilitate angiogenesis. This notion is supported by the observation that degradation of collagen type I is necessary for endothelial cell invasion of the ECM and vascular development [105]. However, there appear to be multiple ways in which MMPs regulate angiogenesis. Convincing evidence that MMPs play a role in angiogenesis has been provided by the MMP-9 knockout mouse, which displays a delay in endochondral bone formation that has been attributed to delayed neovascularization [106]. Subsequently, pancreatic islet tumours that develop in the RIP-Tag model were shown to lack the angiogenic switch, characteristic of tumour progression, in the absence of MMP-9 [102]. Similar behaviour was observed in the K14-HPV16 skin cancer model in the absence of MMP-9 [106]. In addition to helping degrade collagen IV and other ECM components, MMP-9 has been proposed to augment VEGF bioavailability. In parallel studies, MMP-9 activation of latent TGF-β has been shown to contribute to capillary tube formation in vitro [42]. MMP-2 is also implicated in tumour angiogenesis. A reduction in MMP-2 expression in tumour cells decreases angiogenesis in the chicken chorioallantoic membrane model [107] and tumour angiogenesis and growth were reduced in MMP-2 deficient mice compared to normal counterparts (108). In contrast to MMP-9, MMP-2 does not appear to be required for the angiogenic switch in the RIP-Tag model [102]. MMP-14 also appears to play a role in angiogenesis as MMP-14-deficient mice display defective angiogenesis during development [109]. MMP-14 degrades the fibrin matrix that surrounds newly formed blood vessels [110], potentially facilitating endothelial cell penetration of tumour tissue. In addition, inhibition of MMP-14 function blocks endothelial cell migration and capillary tube formation in vitro [111]. Although there is strong support for MMP participation in the promotion of angiogenesis, there is also evidence that MMPs may regulate angiogenesis by generating angiogenic inhibitors from substrate cleavage products. Angiostatin, a cleavage product of plasminogen, is generated by MMP-2, -3, -9, and -12 [112,113], and endostatin, a cleavage product of basement membrane collagen type XVIII, can be produced by the proteolytic activity of MMP-3, -9, -12, and -13 [114]. Angiostatin and endostatin can inhibit endothelial cell proliferation and endostatin has been proposed to block MMP-14 activity [115]. However, the contribution of MMP-mediated generation of these cleavage products to the control of physiological and tumour angiogenesis remains to be defined. MMPs in invasion and metastasis Metastasis is the most devastating event associated with cancer because it heralds an irreversible stage of progression that responds poorly if at all to current therapeutic regimens. Cancer metastasis is a complex, multistage process, which includes cell detachment from the primary tumour mass, migration through the ECM, degradation of the vascular endothelial basement membrane and penetration into the vascular lumen a process known as intravasation survival within the circulation, which reflects resistance to both shear stress and immune surveillance, adhesion to and proliferation on distal vascular endothelia, and finally penetration into a new host tissue microenvironment and establishment of a relationship with the local stroma that is conducive to new tumour colony outgrowth. Several if not all of these steps depend at least in part on MMP activity. Detachment of cells from the primary tumour mass requires the downregulation of cell cell adhesion mechanisms. Loss of E-cadherin expression in various cancers has been associated with increased metastatic proclivity [116], and recent evidence suggests that one

12 Extracellular matrix remodelling 459 mechanism whereby cell surface E-cadherin is lost in tumour cells is proteolytic cleavage by MMP-3 and -7 [56]. Moreover, E-cadherin proteolytic cleavage fragments generated by MMPs appear to promote tumour cell migration [56], although the precise mechanism remains to be determined. E-cadherin shedding is associated with epithelial to mesenchymal transition, which often correlates with cancer aggressiveness [116]. Migration of tumour cells through the host tissue stroma requires partial degradation of the ECM and coordinated sequential attachment to and detachment from the ECM scaffold. Recent work using twophoton microscopy has provided spectacular realtime evidence that MMP proteolytic activity causes controlled degradation of collagen fibrils that are in contact with the invading tumour cell surface, leaving trail of released cell surface molecules in the cell s wake [117,118]. Interestingly, inhibition of MMPs does not result in abrogation of tumour cell migration through the collagen gel but rather transforms the crawling movement associated with collagen fibril cleavage into amoeboid movement that leaves the collagen lattice intact [117,118]. According to this model, MMP expression does not influence tumour cell migration rate through collagen but plays a defining role in the type of migratory activity that tumour cells display, and, by extension, in the types of signals they receive from the host tissue stroma. Moreover, this model strongly supports the notion that the MMP activity relevant to ECM degradation is associated with the tumour cell surface. As already discussed, cleavage of ECM components by MMPs generates proteolytic fragments that enhance tumour cell migration. Thus, cleavage of laminin-5 by MMP-2 and -14 results in laminin fragments that trigger migration signals in cells [45,119], and cleavage of collagen IV discloses cryptic sites that are recognized by integrins and contribute to migration stimuli [46]. MMPs also cleave adhesion receptors responsible for cell matrix interaction, thereby presumably participating in the detachment of cells from the ECM. The cell surface hyaluronan receptor and facultative proteoglycan CD44 is cleaved by MMP-14, and its cleavage promotes migration. Expression of CD44 containing a mutation of the proteolytic cleavage site abrogates cell migration on ECM [57]. Intravasation, the process whereby tumour cells penetrate the vascular endothelial wall, has been proposed to be a rate-limiting event in metastasis [120]. Although it is likely that a variety of MMPs may be involved in the degradation of the vascular endothelial basement membrane, MMP-9 has thus far been shown to play a potentially leading role [120,121]. Survival in the face of the immune response is key for the ability of tumour cells with metastatic potential to establish new colonies. Among the wide range of mechanisms that have been proposed to explain tumour cell evasion of immune surveillance, several are MMP-dependent. Tumour cells typically interact with neutrophils, macrophages, cytotoxic T cells (CTLs), and natural killer (NK) cells. T cell proliferation is controlled in large part by the engagement of the interleukin-2 receptor (IL-2R) by its natural ligand IL-2. MMPs, including MMP-9, have been shown to cleave the α-chain of IL-2R [65], resulting in the inhibition of T cell proliferation. MMP-9-mediated activation of latent TGF-β may also contribute to immune suppression, since TGF-β isa potentinhibitoroft cell function [122]. Cleavage by MMPs of FasL may confer partial protection from CTL-dependent Fasmediated cell death [64] and recent evidence indicates that MMP-9-mediated shedding of cell surface ICAM- 1 may block the ability of CTLs and NK cells to interact with target cells, thereby reducing the effectiveness of their cytotoxicity [39]. In MMP-11-deficient mice, tumours display a significant increase in the number of infiltrating neutrophils and macrophages, consistent with the possibility that MMP-11 may cleave and inactivate a chemokine [123]. Interestingly, an MMP-11 cleavage product of α1-proteinase inhibitor reduces the sensitivity of tumour cells to NK-mediated killing [124]. Recent evidence indicates that MMPs cleave a variety of chemokines in ways that can either enhance or block their function. MMP-2 cleaves and inactivates MCP-3 (CXCL7). Interestingly, the cleaved fragment becomes an antagonist to the corresponding receptors [65]. MMP-9 augments IL-8 activity, which should have a potent chemotactic effect on neutrophils, but proteolytically blocks the activity of several other chemokines that may have an important effect on leucocyte recruitment and survival [66]. Thus, tumour cell expression of MMP-9 or its induction in stromal cells may help contain the immune response by several mechanisms. SDF-1/CXCL12, which is inactivated by several MMPs [67] (Table 1) is a ligand for the CXC chemokine receptor 4 (CXCR4) on leucocytes and breast carcinoma cells [125]. Inhibition of CXCR4 engagement by its ligand using monoclonal antibodies reduces metastasis to lung and lymph nodes in vivo [125]. In an elegant recent study, MMP-9 has been shown to cleave cell surface KitL in bone marrow cells [68]. Shedding of KitL plays an important role in the transfer of endothelial and haematopoietic stem cells (HSC) from a quiescent to a proliferative niche. In MMP-9 null mice, KitL shedding is impaired, resulting in reduced HSC mobility and a correspondingly reduced haematopoietic recovery following bone marrow ablation. Thus, MMP-9-mediated KitL release allows bone marrow repopulating cells to home to an microenvironment that facilitates their differentiation and promotes the reconstitution of the progenitor/stem cell pool [68]. Extravasation was believed to be a key step in cancer metastasis. However, increasing evidence indicates that extravasation is not rate limiting (reviewed in [10,126]), and that it occurs following the proliferation of immobilized tumour cells on vascular endothelium.