how matrix metalloproteinases regulate cell behavior

8 Sep 2001 14:3 AR AR139-16.tex AR139-16.SGM ARv2(2001/05/10)P1: GSR

Annu. Rev. Cell Dev. Biol. 2001. 17:463–516Copyright c© 2001 by Annual Reviews. All rights reserved

HOW MATRIX METALLOPROTEINASES

REGULATE CELL BEHAVIOR

Mark D. Sternlicht and Zena WerbDepartment of Anatomy, University of California, San Francisco, California 94143-0452;e-mail: [email protected]; [email protected]

Key Words proteinases, tissue inhibitors of metalloproteinases, extracellularmatrix, proteolytic signaling

■ Abstract The matrix metalloproteinases (MMPs) constitute a multigene familyof over 25 secreted and cell surface enzymes that process or degrade numerous pericel-lular substrates. Their targets include other proteinases, proteinase inhibitors, clottingfactors, chemotactic molecules, latent growth factors, growth factor–binding proteins,cell surface receptors, cell-cell adhesion molecules, and virtually all structural extra-cellular matrix proteins. Thus MMPs are able to regulate many biologic processes andare closely regulated themselves. We review recent advances that help to explain howMMPs work, how they are controlled, and how they influence biologic behavior. Theseadvances shed light on how the structure and function of the MMPs are related andon how their transcription, secretion, activation, inhibition, localization, and clearanceare controlled. MMPs participate in numerous normal and abnormal processes, andthere are new insights into the key substrates and mechanisms responsible for regu-lating some of these processes in vivo. Our knowledge in the field of MMP biologyis rapidly expanding, yet we still do not fully understand how these enzymes regulatemost processes of development, homeostasis, and disease.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464OVERVIEW OF THE METALLOPROTEINASES. . . . . . . . . . . . . . . . . . . . . . . . . . . 465

The Metzincin Superfamily. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 465MMP Structure and Function. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467

REGULATION OF MMP ACTIVITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 470Transcriptional Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 470Post-Transcriptional Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473Regulation of MMP Secretion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474Activation of Latent Metalloproteinases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 474Endogenous Metalloproteinase Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 477Pericellular Localization of Proteolytic Activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . 479MMP Catabolism and Clearance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481MMP Substrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 482

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464 STERNLICHT ¥ WERB

REGULATION OF CELLULAR SIGNALS BY MMPs . . . . . . . . . . . . . . . . . . . . . . . 484Extracellular Matrix Remodeling and Cell Behavior. . . . . . . . . . . . . . . . . . . . . . . . 484Cell Surface Proteolysis and Cellular Signals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486Regulation of Paracrine Signals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487Generation and Inactivation of Bioactive Molecules. . . . . . . . . . . . . . . . . . . . . . . . 487

MMPs IN DEVELOPMENT AND DISEASE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488Genetic Dissection of Biologic Rolesand Critical In Vivo Substrates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 488

Determination of Biologic Significance UsingSubstrate Cleavage Site Mutations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 491

MMPs in Bone Development. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494MMPs in Vascular Remodeling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496MMPs in Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498

PERSPECTIVES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503

INTRODUCTION

Extracellular proteinases are required for numerous developmental and disease-related processes. The ability to degrade extracellular proteins is essential forany individual cell to interact properly with its immediate surroundings and formulticellular organisms to develop and function normally. This was obvious longbefore it was first shown that diffusible enzymes produced by fragments of in-voluting tadpole tail could degrade gels made of native fibrillar collagen (Gross& Lapiere 1962). Since then, a family of related enzymes has been identified inspecies from hydra to humans and collectively called matrix metalloproteinases(MMPs) because of their dependence on metal ions for catalytic activity, theirpotent ability to degrade structural proteins of the extracellular matrix (ECM), andspecific evolutionary sequence considerations that distinguish them from otherclosely related metalloproteinases (St¨ocker et al. 1995). In addition to their ECMsubstrates, MMPs also cleave cell surface molecules and other pericellular non-matrix proteins, thereby regulating cell behavior in several ways (Sternlicht et al.2000). Thus like the many proteins they modify, the MMPs influence diverse phys-iologic and pathologic processes, including aspects of embryonic development,tissue morphogenesis, wound repair, inflammatory diseases, and cancer (Nelsonet al. 2000, Sternlicht et al. 2001).

With the ability to alter cell fates and developmental outcomes comes the neces-sity for higher levels of control. Nevertheless, it took nearly a decade from the timecollagenolytic activities were first demonstrated to realize that MMPs are synthe-sized as inactive zymogens that require activation (Harper et al. 1971), and evenlonger to demonstrate the existence of the first of at least four endogenous metallo-proteinase inhibitors, now called tissue inhibitors of metalloproteinases, or TIMPs(Bauer et al. 1975). Since then, other levels of MMP regulation have been eluci-dated, although several layers of complexity are still left to unravel. In addition tobeing differentially regulated at the level of transcription, MMPs can be controlledat the protein level by their endogenous activators and inhibitors and by factors that


MATRIX METALLOPROTEINASES 465

influence their secretion, their cell surface localization, and their own degradationand clearance. Moreover, higher organisms express multiple MMPs, each with itsown profile of expression, localization, activation, inhibition, and clearance, as wellas its own, sometimes broad, range of preferred substrates. Thus multiple modi-fiers, each with its own regulatory inputs, control different MMP functions in vivo.

The multiplicity of MMPs with distinct but somewhat overlapping functionsprobably acts as a safeguard against any losses of regulatory control. Althoughsuch redundant and compensatory mechanisms are advantageous to the organism,they often confound efforts to fully understand how MMPs function in vivo. Asthe field has grown, the unforeseen complexity of MMPs has emerged, oftenrevealing surprising mechanisms of action. Frequently, the belief that an MMPis involved in a given biologic process is based on the strength of its associationwith that process and the existence of plausible mechanisms and then tested usingvarious experimental approaches. For example, MMPs are invariably upregulatedin rheumatoid arthritis and malignant disease, with more severe increases oftenindicating a worse prognosis. Moreover, a major hallmark of these diseases isthe capacity of cells to cross tissue boundaries and, in the case of cancer, spreadto distant sites of the body. Thus ECM-degrading enzymes must be present tobreak down the structural barriers to invasion. Indeed, extensive experimentalevidence supports this straightforward and conceptually appealing supposition,but the mechanisms may be more complex than originally thought. Furthermore,in vivo genetic approaches that test the consequences of selective gains or lossesof MMP function have led to the surprising finding that MMPs promote the initialstages of cancer development itself but may decrease the severity of the ultimatemalignancy (Coussens et al. 2000, Pozzi et al. 2000). In arthritis, the loss ofcertain MMPs surprisingly exacerbates rather than alleviates the disease (Mudgettet al. 1998). Considerable evidence implicates MMPs as important players inseveral biologic processes, yet the actual mechanisms underlying their influenceare mostly unsolved. It is hoped that understanding these processes will result ina more rational approach toward reducing or entirely alleviating the ill effects ofMMPs in disease while maintaining their necessary and beneficial functions.

OVERVIEW OF THE METALLOPROTEINASES

The Metzincin Superfamily

Proteolytic enzymes are classified as either exopeptidases or endopeptidases basedon whether they cleave terminal or internal peptide bonds, respectively. Most endo-peptidases are classified as serine, cysteine, aspartic or metalloproteinases basedon their catalytic mechanism and inhibitor sensitivities, and the metalloproteinasesare further separated into five superfamilies based on sequence considerations. Ofthese, the metzincin superfamily is distinguished by a highly conserved motifcontaining three histidines that bind zinc at the catalytic site and a conservedmethionine turn that sits beneath the active site zinc (St¨ocker et al. 1995). Their



signature zinc-binding motif reads HEBXHXBGBXHZ, where histidine (H), glu-tamic acid (E) and glycine (G) residues are invariant, B is a bulky hydrophobicresidue, X is a variable residue, and Z is a family-specific amino acid. The metz-incins are further subdivided into four multigene families, the serralysins, astacins,ADAMs/adamalysins, and MMPs, based primarily on the identity of the Z residue,which is serine in all but a few MMPs (St¨ocker et al. 1995).

The serralysins, which have a proline in the Z position, are large bacterialenzymes that often play an important role in bacterial virulence and pathogenicity(Sternlicht & Werb 1999). Astacins, on the other hand, contain glutamic acid inthe Z position. They include bone morphogenetic protein-1 (BMP-1), which isthe procollagen C-proteinase that removes the C-terminal propeptides of fibrillarprocollagens,Drosophilaand mammalian tolloid and tolloid-like proteins, whichactivate certain growth factors, and secreted and transmembrane meprins A and Bthat can process peptide hormones (reviewed in Sternlicht & Werb 1999).

The adamalysins, ADAMs and ADAMTSs have an aspartic acid in the Z po-sition. The adamalysins are soluble snake venom enzymes with potent ECM-degrading activity. The ADAMs are transmembrane cell surface proteins that havea disintegrin and metalloproteinase domain (Primakoff & Myles 2000). Each ofthe ADAMs has an N-terminal signal sequence followed by a propeptide domain,a functional or nonfunctional metalloproteinase domain, a disintegrin-like domainthat is similar to snake venom disintegrins but often lacks an Arg-Gly-Asp (RGD)sequence, a cysteine-rich domain, EGF-like repeats, a transmembrane domain, anda C-terminal cytoplasmic tail. Individual ADAMs may participate in proteolysisvia their metalloproteinase domain, adhesion via their disintegrin domain, cell-cellfusion via a putative hydrophobic fusion peptide in their cysteine-rich domain, andcell signaling via SH3-recognition sequences that are sometimes present in theirintracellular domain. Seventeen of the 30 known ADAMs have a functional zinc-binding motif, including ADAM17 (TNF-α converting enzyme, TACE), whichcleaves membrane-bound TNF-α to generate active soluble TNF-α. TACE alsoprobably contributes to the shedding of several other cell surface molecules andappears to be an essential activator of TGF-α in vivo (Peschon et al. 1998). Con-sidering their localization, other ADAMs are also likely to regulate the sheddingof several important cell surface molecules (Werb & Yan 1998).

The secreted ADAMTS proteins also have signal, propeptide, metallopro-teinase, and disintegrin-like domains. However, unlike the ADAMs, their disinte-grin domain is followed by a thrombospondin (TS) type I repeat, a cysteine-richdomain, one or more additional TS domains (except for ADAMTS-4, which lacks asecond TS repeat), and, in some cases, a C-terminal domain of variable length (Tang& Hong 1999). They include ADAMTS-1 and ADAMTS-8, which potently inhibitangiogenesis via their TS repeats (Iruela-Arispe et al. 1999); ADAMTS-2, whichis a procollagen amino-propeptidase that is required for the proper assembly offibrillar collagens I and II (Colige et al. 1997); and ADAMTS-4 and ADAMTS-5/11(aggrecanases 1 and 2, respectively), which can degrade the cartilage proteoglycanaggrecan (Abbaszade et al. 1999).



MMP Structure and Function

At present, 25 vertebrate MMPs and 22 human homologues have been identified(Nagase & Woessner 1999, Sternlicht & Bergers 2000, Lohi et al. 2001). In addi-tion, several nonvertebrate MMPs have been identified, including the embryonicsea urchin hatching enzyme envelysin (Lepage & Gache 1990);CaenorhabditiselegansMMPs C31, H19, and Y19 (Wada et al. 1998); aDrosophilaMMP (Llanoet al. 2000); an MMP in hydra that regulates cell differentiation and foot pro-cess development (Leontovich et al. 2000); soybean leaf metalloendopeptidase-1(McGeehan et al. 1992); an MMP in the flowering mustard plantArabidopsisthaliana (Maidment et al. 1999); and gamete lytic enzyme from green alga(Kinoshita et al. 1992). Each of the vertebrate MMPs has distinct but often over-lapping substrate specificities, and together they can cleave numerous extracellularsubstrates, including virtually all ECM proteins (reviewed in Sternlicht et al. 2001).In addition to their conserved zinc-binding motif (usuallyHEF/LGHS/ALGLXHS,where bold-noted amino acids are always present) and “Met turn” (usuallyALMYP), the MMPs share added stretches of sequence homology, giving them afairly conserved overall structure (St¨ocker et al. 1995).

Individual MMPs are referred to by their common names or according to asequential numeric nomenclature reserved for the vertebrate MMPs (Table 1).In addition, they are often grouped according to their modular domain structure(Figure 1). In this regard, all MMPs have an N-terminal signal sequence (or“pre” domain) that is removed after it directs their synthesis to the endoplasmicreticulum. Thus most MMPs are secreted; however, six display transmembranedomains and are expressed as cell surface enzymes. The pre domain is followedby a propeptide “pro” domain that maintains enzyme latency until it is removed ordisrupted, and a catalytic domain that contains the conserved zinc-binding region(reviewed in Nagase & Woessner 1999). The catalytic domain dictates cleavage-site specificity through its active site cleft, through specificity sub-site pockets thatbind amino acid residues immediately adjacent to the scissile peptide bond, andthrough secondary substrate-binding exosites located outside the active site itself(Overall 2001).

With the exception of MMP7 (matrilysin), MMP26 (endometase/matrilysin-2),and MMP23, all MMPs have a hemopexin/vitronectin-like domain that is con-nected to the catalytic domain by a hinge or linker region. MMP7 and MMP26merely lack these extra domains, whereas MMP23 has unique cysteine-rich,proline-rich, and IL-1 type II receptor-like domains instead of a hemopexin do-main (Gururajan et al. 1998, Park et al. 2000). When present, the hemopexindomain influences TIMP binding, the binding of certain substrates, membraneactivation, and some proteolytic activities. For example, chimeric enzyme studiesindicate that both ends of MMP1 (collagenase-1) are required for it to cleavenative fibrillar collagens (Murphy et al. 1992, Sanchez-Lopez et al. 1993). Thiscollagenolytic activity requires the initial binding and orientation of the collagenfibril, local unwinding of its triple-helical structure, and sequential cleavage of eachα-chain individually because the catalytic cleft is too narrow to accommodate the



TABLE 1 The vertebrate MMPs

MMP Common name(s) Domain organizationa

MMP1 Collagenase-1 B

MMP2 Gelatinase A C

MMP3 Stromelysin-1 B

MMP7 Matrilysin A


MMP9 Gelatinase B C

MMP10 Stromelysin-2 B

MMP11 Stromelysin-3 D

MMP12 Macrophage metalloelastase B


MMP14 MT1-MMP E

MMP15 MT2-MMP E

MMP16 MT3-MMP E

MMP17 MT4-MMP F

MMP18 Collagenase-4 (Xenopus) B

MMP19 RASI-1 B

MMP20 Enamelysin B

MMP21 XMMP (Xenopus) G

MMP22 CMMP (chicken) B

MMP23 H

MMP24 MT5-MMP E

MMP25 MT6-MMP F

MMP26 Endometase, Matrilysin-2 A

MMP27 B

MMP28 Epilysin D

aSee Figure 1 for domain arrangements.

entire triple helix (Overall 2001). Apparently, the hemopexin domain participatesin all but the last of these steps. The hinge region, in turn, varies in length andcomposition among the various MMPs and also influences substrate specificity(Knauper et al. 1997). Gelatinases A and B (MMP2 and MMP9, respectively)are further distinguished by the insertion of three head-to-tail cysteine-rich repeatswithin their catalytic domain. These inserts resemble the collagen-binding typeII repeats of fibronectin and are required to bind and cleave collagen and elastin(Murphy et al. 1994, Shipley et al. 1996). In addition, MMP9 has a unique type Vcollagen-like insert of unknown importance at the end of its hinge region. Finally,the membrane-type (MT) MMPs have a single-pass transmembrane domain anda short cytoplasmic C-terminal tail (MMPs 14, 15, 16, and 24) or a C-terminal



Figure 1 Domain structure of the MMPs. Pre, signal sequence; Pro, propeptide witha free zinc-ligating thiol (SH) group; F, furin-susceptible site; Zn, zinc-binding site;II, collagen-binding fibronectin type II inserts; H, hinge region; TM, transmembranedomain; C, cytoplasmic tail; GPI, glycophosphatidyl inositol-anchoring domain; C/P,cysteine/proline; IL-1R, interleukin-1 receptor. The hemopexin/vitronectin-like do-main contains four repeats with the first and last linked by a disulfide bond.



hydrophobic region that acts as a glycophosphatidyl inositol (GPI) membrane-anchoring signal (MMP17 and MMP25) (Itoh et al. 1999, Kojima et al. 2000).These domains play an essential role in the localization of several important pro-teolytic events to specific regions of the cell surface.

REGULATION OF MMP ACTIVITY

To accomplish their normal (or pathologic) functions, MMPs must be present inthe right cell type and pericellular location, at the right time, and in the rightamount, and they must be activated or inhibited appropriately. Thus MMPs aretightly regulated at the transcriptional and post-transcriptional levels and are alsocontrolled at the protein level via their activators, their inhibitors, and their cellsurface localization (Figure 2).

Transcriptional Regulation

Because MMP substrate specificities tend to overlap, the biologic function ofindividual MMPs is largely dictated by their differential patterns of expression.Indeed, differences in the temporal, spatial, and inducible expression of the MMPsare often indicative of their unique roles. Accordingly, most MMPs are closelyregulated at the level of transcription, with the notable exception of MMP2, whichis often constitutively expressed and controlled through a unique mechanism ofenzyme activation (Strongin et al. 1995) and some degree of post-transcriptionalmRNA stabilization (Overall et al. 1991). Nevertheless, data indicate that thebasal expression of MMP2, MMP14 (MT1-MMP), and TIMP2 is co-regulated,which is consistent with their cooperation during MMP2 activation and with spe-cific similarities in their gene promoters (Lohi et al. 2000). Otherwise, MMPgene expression is regulated by numerous stimulatory and suppressive factors thatinfluence multiple signaling pathways (Fini et al. 1998). For example, the expres-sion of various MMPs can be up- or downregulated by phorbol esters, integrin-derived signals, extracellular matrix proteins, cell stress, and changes in cell shape(Kheradmand et al. 1998; reviewed in Sternlicht & Werb 1999). Type I collagenacts as a ligand for discoidin domain-containing receptor-like tyrosine kinases thatinduce MMP1 expression when they are activated by intact collagen and become in-active when they bind MMP1-cleaved collagen (Vogel et al. 1997, Shrivastava et al.1997). Thus MMP1 expression can be induced by its own substrate and specifi-cally repressed once it cleaves that substrate and is no longer needed. In addition,

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→Figure 2 Regulation of the MMPs. MMP regulatory mechanisms include inductiveand suppressive signaling (1), intracellular signal transduction (2), transcriptional acti-vation and repression (3), post-transcriptional mRNA processing (4), mRNA degrada-tion (5), intracellular activation of furin-susceptible MMPs (6), constitutive secretion(7), regulated secretion (8), cell surface expression (9), proteolytic activation (10),proteolytic processing and inactivation (11), protein inhibition (12), ECM localization(13), cell surface localization (14), and endocytosis and intracellular degradation (15).



MMP expression is regulated by several cytokines and growth factors, includinginterleukins, interferons, EGF, KGF, NGF, basic FGF, VEGF, PDGF, TNF-α, TGF-β, and the extracellular matrix metalloproteinase inducer EMMPRIN (Fini et al.1998). Many of these stimuli induce the expression and/or activation of c-fosandc-jun proto-oncogene products, which heterodimerize and bind activator protein-1(AP-1) sites within several MMP gene promoters.

Although AP-1 complexes play a critical role in the regulation of several MMPgenes, other factors are also involved. In some cases, one signal may coordinatelyregulate some MMP genes and differentially regulate others. For example, TGF-β

suppresses the transcription of theMMP1 andMMP3 (stromelysin-1) genes, butinduces the expression of MMP13 (collagenase-3) (Uria et al. 1998). In addition,some MMPs are expressed in only a small repertoire of cell types, e.g., MMP20 ex-pression appears to be confined to the enamel organ of developing teeth (Sternlichtet al. 2000), and normal MMP9 expression is largely limited to osteoclasts, macro-phages, trophoblast cells, hippocampal neurons, and migrating keratinocytes at themargins of healing wounds (Mohan et al. 1998, Munaut et al. 1999). The use ofmice carryingβ-galactosidase reporter transgenes under the control of various por-tions of the MMP9 gene promoter have helped to identify the 5′ regions responsiblefor most of this cell-specific expression in vivo (Mohan et al. 1998). Cell-specificinduction of MMP expression has also been observed in culture. For example,phorbol esters induce the expression of MMP3 rather than MMP10 in fibroblasts,yet the opposite occurs in keratinocytes (Windsor et al. 1993). Thus how an MMPgene responds to a given input depends on the organization of its transcriptionalpromoter and the presence or absence of other signals, i.e., on cellular context.

Severalcis-regulatory elements influence MMP gene expression depending ontheir proximity to one another in the gene promoter. AP-1 sites give several MMPgenes the ability to be induced by phorbol esters and act synergistically with ad-jacent Ets-binding sites in genes such asMMP1, but not in others such asMMP13(Pendas et al. 1997). This difference is presumably because the Ets and AP-1 sitesare 9 nucleotides apart in theMMP1 gene promoter and 20 nucleotides apart intheMMP13promoter and because the distance between these two sites is critical(Gutman & Wasylyk 1990). As an indication of the importance of these sites,targeted disruption of the murine Ets2 transcription factor results in early embry-onic lethality and deficient MMP9 expression, as well as the deficient induction ofMMP3 and MMP13 in Ets2-deficient fibroblasts (Yamamoto et al. 1998). More-over, their expression in the Ets2-deficient fibroblasts is restored by the artificialexpression of Ets2. Several other putative regulatory elements have been identifiedwithin various MMP gene promoters, and many have been shown by functionalanalysis to regulate cell- and circumstance-specific gene expression. These in-clude an osteoblast-specific element in the MMP13 gene promoter that respondsto core-binding factor 1 (CBFA1) (Jimenez et al. 1999); aβ-catenin-regulatedLEF/TCF recognition site near the MMP7 transcription start site (Crawford et al.1999); TGF-β inhibitory elements in several MMP genes; and AP-2, Sp1, Sp3,NF-κB, CCAAT/enhancer-binding protein-β, or retinoic acid response elementsthat are also found in several MMP genes (Fini et al. 1998, Lohi et al. 2000, Ludwig



et al. 2000). In addition, a functional p53-binding site has been identified in theMMP2 gene promoter (Bian & Sun 1997), and wild-type p53 downregulates basaland inducible MMP1 gene expression in human fibroblasts and osteogenic sar-coma cells, whereas some mutant forms do not (Sun et al. 1999). On the otherhand, the downregulation of p53 using SV40 T-antigen suppresses the expressionof MMP2, MMP3, and MMP9 in human placental trophoblast-like cells (Loganet al. 1996). Despite numerous advances in our understanding of MMP gene reg-ulation, the cross-talk between the many signaling pathways, nuclear factors, andgene regulatory elements that regulate MMP expression are barely understood.

Basal and inducible levels of MMP gene expression can also be influenced bygenetic variations that may, in turn, influence the development or progression ofseveral diseases. Common bi-allelic single-nucleotide polymorphisms (SNPs) thatinfluence the rate of transcription have been identified within several MMP genepromoters (Ye 2000). For example, anMMP1SNP contains one or two guanidines1607 basepairs (bp) upstream of the transcription start site (Rutter et al. 1998).Here, the insertion of an extra G creates a functional Ets-binding site immediatelyadjacent to an AP-1 site and, as a result, transcription is enhanced up to 37-fold.Interestingly, the high-expressing 2G allele has been associated with higher levelsof MMP1 expression in vivo and is present more often in tumor cell lines andovarian cancer patients than in the general population (Rutter et al. 1998, Kanamoriet al. 1999). Moreover, an unusually large proportion of metastatic melanomaswith loss of heterozygosity at this site retains the high-expressing 2G allele (Nollet al. 2001). Thus enhancedMMP1 transcription may contribute to human cancersusceptibility and progression as it does in mice (Di Colandrea et al. 2000). AnotherSNP located 1306 bp upstream of theMMP2 transcription start site contains eithera cytidine or thymidine, such that the less common T allele disrupts an otherwisefunctional Sp1-binding site and diminishes promoter activity by about 50% (Priceet al. 2001). AnMMP3SNP is located 1171 bp upstream of the transcription startsite and contains a run of five or six adenosines (Ye et al. 2000). In this case, the 6Aallele binds an 89-kDa nuclear factor more readily than the 5A allele and has 50%less transcriptional activity. Another SNP contains either a cytidine or thymidine1562 bp upstream of theMMP9 transcription start site (Zhang et al. 1999). Here,nuclear protein complexes bind the C allele most readily, and the less common Tallele is about 1.5-fold more potent than the C allele. Finally, an A-to-G transitionexists 82 bp upstream of theMMP12transcription start site, such that the A allelehas higher AP-1 binding affinity and about 1.2-fold higher promoter activity thanthe less common G allele (Jormsjo et al. 2000). Most notably, however, theMMP3,MMP9, andMMP12SNPs have each been associated with coronary artery diseaseprogression despite their modest influence on gene transcription (Ye 2000).

Post-Transcriptional Regulation

Post-transcriptional mechanisms can also influence MMP expression. For exam-ple, mRNA transcripts that encode MMP1 and MMP3 are stabilized by phor-bol esters and EGF, whereas MMP13 transcripts are stabilized by PDGF and



glucocorticoids and destabilized by TGF-β (Delany et al. 1995, Vincenti 2001).The turnover of MMP1 mRNA is apparently regulated by AU-rich sequences inthe 3′ untranslated region, and similar sequences may also regulate the stability ofother MMP transcripts (Vincenti 2001). In addition, a soluble and proteolyticallyactive form of MT3-MMP is generated by alternative mRNA splicing rather thanmembrane shedding (Matsumoto et al. 1997), whereas the multiple transcripts ofMMP13, MMP17, and MMP20 probably result from alternative polyadenylation(reviewed in Sternlicht & Werb 1999).

Regulation of MMP Secretion

Although most MMPs are constitutively secreted once they become translated, con-spicuous instances of secretory control do exist. MMP8 (collagenase-2, neutro-phil collagenase) and MMP9 are synthesized by differentiating granulocytes inthe bone marrow, stored in the specific and gelatinase (teriary) granules of cir-culating neutrophils, respectively, and released following neutrophil activationby inflammatory mediators (Hasty et al. 1990). In macrophages, plasmin andthrombin induce the secretion of MMP12, but do not alter its rate of transcrip-tion (Raza et al. 2000). Instead, post-translational release of pre-formed MMP12from macrophages occurs in response to protein kinase C activation downstream ofthe G protein–coupled thrombin receptor PAR-1 (proteinase-activated receptor-1),which becomes activated after binding a ligand that is generated from its ownN-terminal end by thrombin-dependent cleavage.

Activation of Latent Metalloproteinases

Like other proteolytic enzymes, MMPs are first synthesized as inactive proenzymesor zymogens. Their latency is maintained by an unpaired cysteine sulfhydryl groupnear the C-terminal end of the propeptide domain. This sulfhydryl acts as a fourthligand for the active site zinc ion, and activation requires that this cysteine-to-zincswitch be opened by normal proteolytic removal of the propeptide domain or byectopic perturbation of the cysteine-zinc interaction (Van Wart & Birkedal-Hansen1990). Once displaced, the thiol group is replaced by a water molecule that canthen attack the peptide bonds of MMP targets.

Although most MMPs are secreted as latent zymogens, MMP11 (stromelysin-3), MMP27 (epilysin), and the MT-MMPs contain an RXK/RR furin-like enzymerecognition motif between their propeptide and catalytic domains. This allowsthem to be activated by intracellular subtilisin-type serine proteinases before theyreach the cell surface or are secreted (Pei & Weiss 1995). MMP23 also has afurin-susceptible cleavage site and is a likely target of intracellular proprotein con-vertases, but unlike all other MMPs, it lacks the conserved cysteine that is requiredfor enzyme latency in the first place (Gururajan et al. 1998). All other MMPs lacka furin-susceptible insert and are thus activated outside the cell following theirsecretion.

The extracellular activation of most MMPs can be initiated by other alreadyactivated MMPs or by several serine proteinases that can cleave peptide bonds



within MMP prodomains (Woessner & Nagase 2000). However, MMP2 is refrac-tory to activation by serine proteinases and is instead activated at the cell surfacethrough a unique multistep pathway involving MT-MMPs and TIMP2 (Figure 3)(Strongin et al. 1995). Indeed, MT1-MMP is a particularly efficient MMP2 acti-vator, whereas MT4-MMP and human (but not mouse) MT2-MMP are the onlyMT-MMPs that are unable to activate MMP2 (Zucker et al. 1998, Miyamori et al.2000, English et al. 2000). First, a cell surface MT-MMP binds and is inhibited bythe N-terminal domain of TIMP2, and the C-terminal domain of the bound TIMP2acts as a receptor for the hemopexin domain of ProMMP2. Then, an adjacent,uninhibited MT-MMP cleaves and activates the tethered ProMMP2. Followingthe initial cleavage of ProMMP2 by MT1-MMP, a residual portion of the MMP2propeptide is removed by another MMP2 molecule to yield a fully active, matureform of MMP2 (Deryugina et al. 2001).

It has been assumed that proteolytic removal of the MT-MMP prodomain byfurin-like enzymes in thetrans-Golgi network or by plasmin at the cell surface(Okumura et al. 1997) is required for MT-MMPs to activate MMP2. Such process-ing does appear to be necessary in some cell types (Strongin et al. 1995), but thesecells display both processed and full-length MT-MMPs on their surface so that thefunction of either individual form can not be distinguished. However, removal ofthe prodomain is not required for MT1-MMP to bind TIMP2 or activate MMP2in transfected COS-1 cells that lack endogenous MT1-MMP and are unable toprocess latent MT-MMPs (Cao et al. 1998). Even more surprisingly, COS-1 cellsfail to bind TIMP2 or activate MMP2 if the MT1-MMP prodomain is absent orreplaced by the MMP13 prodomain. However, COS-1 cells can activate MMP2 ifthey co-express a cDNA for prodomain-deleted MT1-MMP together with an inde-pendent cDNA for the MT1-MMP prodomain, but not if either cDNA is expressedalone (Cao et al. 2000). Therefore, the MT1-MMP prodomain is actually requiredfor the cell surface activation of MMP2 to proceed, but it does not have to remaincovalently attached. Furthermore, co-expression of the MMP1 prodomain insteadof the MT1-MMP prodomain does not rescue the ability of prodomain-deletedMT1-MMP to activate MMP2, thus indicating that the MT1-MMP prodomainhas specific attributes that enable it to do so. Direct exogenous addition of theMT1-MMP prodomain also fails to restore the function of processed MT1-MMP,and whereas full-length MT1-MMP is prominently expressed at the cell surface,much of the prodomain-deleted MT1-MMP is retained in the secretory pathway.Therefore, the MT1-MMP propeptide may act as an intramolecular chaperone thatis necessary for the efficient trafficking of MT1-MMP to the cell surface. Althoughprocessed MT1-MMP is eventually expressed at the cell surface, it lacks the abilityon its own to bind TIMP2 or activate MMP2. MT1-MMP may also be conforma-tionally activated through interactions with the cell membrane, and its retainedpropeptide domain may facilitate the binding of TIMP2.

The role of TIMP2 in MMP2 activation is its dominant in vivo function, asshown by targeted mutagenesis in mice (Z. Wang et al. 2000). Nevertheless, whilethe C-terminal domain of TIMP2 participates in the cell surface docking andactivation of MMP2, its N-terminal domain is an MMP inhibitor. Not surprisingly,



Figure 3 Cell surface activation of MMP2. A ProMT-MMP is activated during trans-port to the cell surface by an intracellular furin-like serine proteinase, at the cell surfaceby plasmin, or by non-proteolytic conformational changes. The activated MT-MMPis then inhibited by TIMP2 and the hemopexin domain of ProMMP2 binds to the C-terminal portion of TIMP2 to form a trimolecular complex. An uninhibited MT-MMPthen partially activates the ProMMP2 by removing most of the MMP2 propeptide.The remaining portion of the propeptide is removed by a separate MMP2 moleculeat the cell surface to yield fully active mature MMP2. Mature MMP2 can then be re-leased from the cell surface or bound by another cell surface MMP2-docking protein.It can also be inhibited by another TIMP molecule or left in an uninhibited active statedepending on local MMP:TIMP molar ratios.



low-to-moderate levels of TIMP2 promote the activation of MMP2, whereas higherlevels inhibit its activation by saturating free MT-MMPs that are needed to removethe MMP2 prodomain (Strongin et al. 1995). TIMP2 protein levels are reducedand MMP2 activation is enhanced in the presence of the MMP2 substrate, type IVcollagen (Maquoi et al. 2000). Furthermore, the ability of collagen to induce MMP2activation on demand probably results from TIMP2 degradation because there areno accompanying changes in MMP2, MT1-MMP, or TIMP2 mRNA expression orin the synthesis or activation of MT1-MMP. Therefore, local accumulation of typeIV collagen may trigger its own degradation by somehow lowering local TIMP2concentrations to levels that favor MMP2 activation.

Endogenous Metalloproteinase Inhibitors

The TIMPs represent a family of at least four 20–29-kDa secreted proteins (TIMPs1–4) that reversibly inhibit the MMPs in a 1:1 stoichiometric fashion (reviewed inEdwards 2001, Sternlicht & Werb 1999, Gomez et al. 1997). They share 37–51%overall sequence identity, a conserved gene structure, and 12 similarly separatedcysteine residues. These invariant cysteines form six intrachain disulfide bridgesto yield a conserved six-loop, two-domain structure. Truncated “tiny” TIMPs 1and 2 retain their inhibitory activity despite containing only the first three loops,thus indicating that portions of the N-terminal domain interact with the MMPcatalytic site (Murphy & Willenbrock 1995). Mutational analyses (O’Shea et al.1992, Willenbrock & Murphy 1994, Huang et al. 1997) and peptide- and antibody-blocking experiments (Bodden et al. 1994) have helped to further specify whichregions of the N-terminal domain influence inhibitory function. In addition, NMR(Williamson et al. 1997) and X-ray crystallographic studies (Gomis-R¨uth et al.1997) have revealed which TIMP residues interact directly with the MMP3 cat-alytic domain and how they inhibit MMP activity. Although these studies indicatethat the inhibitory activity of the TIMPs resides almost entirely in the N-terminaldomain alone, both domains influence enzyme-inhibitor binding (Willenbrock &Murphy 1994). For example, the C-terminal domain (loops 4–6) of TIMP1 bindsthe hemopexin domain of MMP9 more readily than it does the hemopexin do-main of MMP2, whereas the C-terminal domain of TIMP2 preferentially binds thehemopexin domain of MMP2 (Murphy & Willenbrock 1995).

Individual TIMPs differ in their ability to inhibit various MMPs (reviewedin Woessner & Nagase 2000). For example, TIMP2 and TIMP3 inhibit MT1-MMP, whereas TIMP1 does not. Likewise, TIMP1 is a relatively poor inhibitorof MT3-MMP, and TIMP3 appears to be a more potent inhibitor of MMP9 thanother TIMPs. TIMP3 is also unique in its ability to inhibit ADAMs-10 and -17,ADAMTS-4, and ADAMTS-5 (Kashiwagi et al. 2001), whereas TIMP1 can inhibitADAMTS-1 (Tortorella et al. 1999). In addition, the TIMPs differ in terms of theirgene regulation and tissue-specific patterns of gene expression (Edwards 2001).TIMP3 also has the unique ability to bind via its C-terminal domain to heparansulfates proteoglycans within the ECM, thereby concentrating it to specific regionswithin tissues and basement membranes (Langton et al. 1998).



The particular importance of TIMP3 in the eye is indicated by its increased ex-pression in various degenerative retinal diseases, its immunolocalization to Bruch’smembrane and drusen (deposits of extracellular matrix associated with maculardegeneration), and the presence of TIMP3 mutations in patients with Sorsby’s fun-dus dystrophy (SFD), an autosomal-dominant form of early retinal degeneration(Langton et al. 1998). Several missense mutations that introduce an extra cysteineinto the TIMP3 C-terminal domain, a nonsense mutation that truncates most ofthe C-terminal domain but leaves an unpaired cysteine, and a splice-site mutationthat may also yield an unpaired cysteine have been found in the affected membersof various SFD families (Langton et al. 2000). Although the presence of an un-paired cysteine could result in aberrant, function-perturbing disulfide bonds, SFDmutations apparently give rise to TIMP3 molecules that dimerize and show dimin-ished turnover but still retain their MMP-inhibitory and ECM-binding properties(Langton et al. 2000).

There is a long history indicating that TIMPs exert growth-promoting activ-ity independent of their metalloproteinase inhibitory activity. Indeed, TIMP1 wasfirst cloned as EPA for its erythroid-potentiating activity, and TIMPs 1, 2, and 3have since been shown to act as mitogens in several other cell types (Gomez et al.1997). Moreover, their mitogenic activity persists despite the presence of addedmutations that abolish their MMP inhibitory activity, thus indicating that theseactivities occur independently, perhaps as a distinct function of the C-terminaldomain (Chesler et al. 1995, Wingfield et al. 1999). Although it is still unclearhow TIMPs promote cell growth, this activity may explain several unexpectedassociations between TIMPs and cancer progression. However, TIMPs may alsopromote cell death or suppress mitogenic signals. For example, apoptosis is in-duced in various cell types by TIMP3, but not by TIMP1, TIMP2 or syntheticMMP inhibitors, thus suggesting that the mechanism may not rely on the inhi-bition of a metalloproteinase (Bond et al. 2000). However, the introduction of amutation that abolishes the metalloproteinase-inhibitory activity of TIMP3 alsoabolishes its ability to promote apoptosis, indicating that its inhibitory activity isnecessary and suggesting that its target may be an ADAM or ADAMTS rather thanan MMP. TIMP2, on the other hand, can suppress growth factor-responsivenessby interfering with the activation of tyrosine kinase-type growth factor receptors,and its ability to block mitogenic signaling is independent of its MMP-inhibitoryactivity (Hoegy et al. 2001). Nevertheless, no TIMP receptors have yet been iden-tified, suggesting that TIMPs may act as decoys for various signaling molecules.Moreover, these growth-promoting and growth-suppressive activities may not beentirely MMP-independent because the mutant TIMPs retain their ability to inter-act with MMPs via secondary non-inhibitory sites, such as those of their C-terminaldomains (Howard & Banda 1991). Therefore, these growth-altering activities maystill reflect the ability of TIMPs to indirectly modify MMP activity.

TIMPs are not the only endogenous MMP inhibitors. Indeed,α2-macroglobulinis a major endogenous inhibitor of the MMPs (Sottrup-Jensen & Birkedal-Hansen1989), and its importance may have been overlooked due to the recent emphasisplaced on the TIMPs. Becauseα2-macroglobulin is an abundant plasma protein,



it represents the major inhibitor of MMPs in tissue fluids, whereas TIMPs may actlocally. Moreover, becauseα2-macroglobulin/MMP complexes are removed byscavenger receptor-mediated endocytosis,α2-macroglobulin plays an importantrole in the irreversible clearance of MMPs, whereas TIMPs inhibit MMPs in areversible manner.

Another, recently recognized class of MMP inhibitors, protein subdomains,have structural similarity to the TIMPs. For example, proteolytic processing of theprocollagen C-terminal proteinase enhancer protein PCPE releases a C-terminalfragment with MMP inhibitory activity and structural similarity to the N-terminaldomain of the TIMPs (Mott et al. 2000). A primary sequence alignment search alsouncovered similarities between the TIMPs and the noncollagenous NC1 domain oftype IV collagen (Netzer et al. 1998). Moreover, functional analyses indicate thatthe NC1 domain has MMP inhibitory activity (Netzer et al. 1998) and can inhibitangiogenesis and tumor growth (Petitclerc et al. 2000). Nevertheless, the physiolo-gic targets of these inhibitory fragments remain uncertain. Although their activityagainst MMP2 is substantially lower than that of the TIMPs (Mott et al. 2000,Netzer et al. 1998), other MMPs or metzincins may be their true physiologic targets.

Pericellular Localization of Proteolytic Activity

Many of the extracellular signaling events that regulate cell behavior occur at ornear the cell membrane, and many cellular signals are created or canceled via peri-cellular proteolysis (Werb 1997, Werb & Yan 1998). Thus because it is irreversible,the processing of pericellular proteins by proteolysis is an ideal means of regulatingextracellular signal transduction. Although pericellular proteolysis may sometimesreflect the exclusive expression of a critical substrate at or near the cell surface,there are specific mechanisms that confine or concentrate proteinases in the imme-diate pericellular microenvironment. These mechanisms for localizing MMPs tothe cell surface and to specific cell surface subdomains include the expression ofmembrane-bound MT-MMPs; the binding of MMPs to cell surface receptors; thepresence of cell surface receptors for MMP-activating enzymes such as uPA, plas-min(ogen), thrombin, and elastase; and the concentration of MMPs on pericellularECM molecules. These localization mechanisms often enhance MMP activation,limit the access of MMP inhibitors, concentrate MMPs within the vicinity of theirtargets, and limit the extent of proteolysis to discrete pericellular regions.

Transmembrane and GPI-linked MT-MMPs are the most obvious mediators ofproteolytic activity at the cell surface. Removal of the transmembrane domains ofMT1, MT2, and MT3-MMP abolishes their ability to promote cellular invasion(Hotary et al. 2000). Moreover, MT-MMPs can concentrate within specific cellsurface domains such as cellular protrusions known as invadopodia (or invasivepseudopodia), where active ECM degradation takes place. Localization studies us-ing chimeric constructs indicate that the cytoplasmic and transmembrane domainsof MT1-MMP are required for it to cluster on invadopodia, and functional studiesindicate that invadopodial recruitment of MT1-MMP is necessary for cellular in-vasion to take place (Nakahara et al. 1997). Because GPI-anchored proteins tend to



partition into lipid rafts and caveolae where considerable signaling activity takesplace (Brown & London 1998), the GPI-linked MT-MMPs should also be enrichedin such regions.

Another means of localizing MMPs to the cell surface is via cell surface dockingreceptors. For example, activated MMP2 can bind to integrinαvβ3 on the surface ofangiogenic endothelial cells and invasive cancer cells (Brooks et al. 1996). Becausethe C-terminal domain of MMP2 is required for the formation ofαvβ3/MMP2complexes in vitro, the catalytic domain probably remains exposed so that it canstill carry out proteolysis. Interestingly, MT1-MMP generates only an MMP2activation intermediate, and another already activated MMP2 is required to removethe residual portion of the MMP2 propeptide and achieve full MMP2 activation(Deryugina et al. 2001). Thus cell surface MMP2 receptors may cooperate withMT1-MMP to facilitate MMP2 maturation, and data suggest that integrinαvβ3promotes such maturation by providing a platform for autocatalytic interactionsbetween fully and partially activated MMP2 (Deryugina et al. 2001). Moreover,colocalization data suggest that integrinαvβ3 may also cooperate with MT1-MMPin the clustering of active MMP2 on invadopodia.

MMP1 can also interact with a cell surface integrin. In this case, active andinactive MMP1 binds to the I domain ofα2 integrin, whereas MMPs 3 and 13do not (Stricker et al. 2000). Optimal binding of chimeric constructs requires theMMP1 hinge and hemopexin domains, but not its propeptide or catalytic domains,so that the latter domain may remain available to interact with its targets. Bindingof α2β1 integrin to type I collagen induces MMP1 expression in keratinocytes,and MMP1 is necessary forα2β1-dependent migration of keratinocytes over type Icollagen (Pilcher et al. 1999). Thusα2β1 integrin can act both as an MMP1 inducerand as an MMP1 receptor, and the binding of MMP1 toα2β1 integrin may play animportant role in the migration of keratinocytes that contact interstitial collagensduring epidermal wound repair.

Another molecule that can both induce MMP1 expression and then localize itto the cell surface is CD147/EMMPRIN (Guo et al. 2000). EMMPRIN is enrichedon the surface of cancer cells and induces adjacent fibroblasts to produce MMP1,which then apparently binds to the same cell surface EMMPRIN molecules thatelicited its expression in the first place. Therefore, tumor-derived EMMPRIN maypromote tumor cell invasion by both inducing fibroblasts to produce MMP1 andthen concentrating it on the surface of the invasive cells. MMPs can also be bound tothe cell surface by their own substrates. Latent ProMMP9, for example, binds withhigh affinity to type IV collagenα2 chains on the surface of several cell types, thusconcentrating it at the cell-ECM interface in anticipation of any future need and indirect contact with its target (Olson et al. 1998, Toth et al. 1999). This interface iswhere MMP9 is found in nascent skin cancers (Coussens et al. 2000). ActivatedMMP9 binds to the cell surface hyaluronan receptor CD44 (Bourguignon et al.1998). Moreover, the localization of MMP9 to the cell surface by CD44 appears topromote tumor cell invasion and angiogenesis and may mediate the activation oflatent TGF-β by MMP9 (Yu & Stamenkovic 2000). Recent data also indicate thatMMP7 binds to cell surface and ECM heparan sulfate moeities that may enhance



the stability, activation, and activity of MMP7 (Yu & Woessner 2000). Moreover,cell surface heparan sulfates may act as docking molecules for other MMPs, aswell as TIMP3.

Although many of the above docking mechanisms have not been definitivelyproven, they suggest the existence of localized pericellular feed-back networks thatcoordinate the need for a given MMP with its appropriate expression, activation,and physical placement. The specific cell surface molecules that mediate theseinteractions have not been definitively elucidated, in part, because such moleculesare frequently enriched in specialized signaling domains such as caveolae and rafts.Furthermore, by combining many of the functions of a given feed-back loop withina few components (for instance by binding an MMP to its own inducer, its ownsubstrate or a molecule that normally ligates its substrate), these signaling networksmay be particularly compact and efficient. By the same token, the multifunctionalnature of these interactions will undoubtedly make them all the more difficult tosort out in functional experiments.

MMP Catabolism and Clearance

An obvious means of regulating MMPs is via their own proteolytic inactivationand physical clearance. Although considerable progress has been made in under-standing the progressive proteolytic processing of MMP propeptides, relativelylittle is known about the further autoproteolysis of active MMPs. Nevertheless,it is clear that some cleavages inactivate MMPs, whereas others, such as thosethat specifically remove the hemopexin domain, can generate truncated enzymesthat lose their ability to cleave some substrates but retain their ability to cleaveothers (reviewed in Woessner & Nagase 2000). Such processing can also diminishtheir affinity for and ability to be inhibited by TIMPs, as occurs with C-terminallytruncated MMP2 (Y. Itoh et al. 1998). Removal of the hemopexin-like domain alsocancels the ability of certain MMPs to localize to the cell surface. In addition, MT-MMPs can be secreted if they are cleaved at a juxtamembrane site before or afterthey reach the cell surface (Imai et al. 1996). Thus factors that influence MMPdegradation can alter the steady-state concentrations of MMPs, their substratespecificities, their localization, and their ability to be activated or inhibited.

Another means of regulating extracellular MMP levels is by the direct clear-ance of intact enzymes. Most MMPs cleave the bait region ofα2-macroglobulin,thereby initiating a conformational change in the large tetrameric macroglobu-lin that irreversibly traps the enzyme (Sottrup-Jensen & Birkedal-Hansen 1989).Although the catalytic activity of the MMP is not inhibited per se, its physicalentrapment keeps the enzyme from interacting with natural substrates, and theα2-macroglobulin/MMP complex is eventually endocytosed and permanently cleared.

Thrombospondin 2 (TS2) has also been implicated in the clearance of MMPs.Interestingly, TS2-deficient mice exhibit a number of connective tissue abnormal-ities, and their fibroblasts have an adhesion defect that is the result of increasedMMP2 levels (Yang et al. 2000). The increased MMP2 levels occur becauseTS2 normally binds both latent and active MMP2 and because TS2 is normally



endocytosed by the low-density lipoprotein receptor-related protein LRP and prob-ably carries any bound MMP2 with it (Yang et al. 2001). The cellular internalizationof TS2/MMP2 complexes by the LRP scavenger receptor may therefore play an im-portant role in regulating MMP2 levels outside fibroblasts and other cells. Evidencealso indicates that MMP13 is rapidly cleared after it binds to an MMP13-specific170-kDa high-affinity receptor present on various cell types (Barmina et al. 1999).The binding requires calcium, and the subsequent internalization and degradationof MMP13 requires LRP because LRP-null cells bind MMP13 but fail to internal-ize it. Moreover, the internalization of both MMP13 and TS2/MMP2 complexesis inhibited by the 39-kDa receptor-associated protein RAP, which binds and in-hibits LRP. Thus MMPs are tightly regulated by several variously characterizedmechanisms during virtually every aspect of their life-span, from their inductionto their ultimate destruction.

MMP Substrates

Although very few bona fide MMP substrates have been definitively identifiedin vivo, numerous candidates have been tested and identified in vitro (Table 2).

TABLE 2 Common matrix metalloproteinase substratesa

MMP 1 2 3 7 8 9 10 11 12 13 14 16 18 19 26

ECM Proteins:Aggrecan + + + + + + + + + +Collagen I + + − + + − + + + + + −Collagen II + + − + + −Collagen III + + + − + − + + + +Collagen IV − + + + − + + − + − + +Collagen V − + + − − + + −Collagen VI − − − − +Collagen VII + + +Collagen VIII +Collagen IX − − + +Collagen X + + + − +Collagen XI + + + + −Collagen XIV − − − + +Decorin − + + + +Elastin − + + + + + + −Entactin/Nidogen + + + + + +Fibrillin + + + + + +Fibronectin + + + + − − + + + + + + +Fibulins + +Gelatin I + + + + + + + + + + +IGFBPs + + + +Laminin + + + + + − + + −



TABLE 2 (Continued)

MMP 1 2 3 7 8 9 10 11 12 13 14 16 18 19 26

Link Protein + + + + + +Myelin Basic + + + + + +Osteonectin + + + + +Tenascin + + + + − +Vitronectin + + + + + + +

Other Proteins:α1-AC + + + −α2-M + − + + + + + + +α1-PI + + + + + + + + + +Casein + − + + + + − + + −C1q + + + + + +E-cadherin + +Factor XII − + + +Fibrin + + + + +Fibrinogen + + + + + + + + + + +IL1α − − − −IL1β + + + +ProMMP2 + +ProTGFβ + +ProTNFα + + + + + + +Plasminogen + + + + + −Substance P + + + +T kininogen − + −

aThe symbols indicate whether the indicated protein is (+) or is not (−) digested by the indicated MMP.

Abbreviations:α1-AC, α1-antichymotrypsin;α2-M, α2-macroglobulin;α1-PI, α1-proteinase inhibitor. Adapted fromWoessner & Nagase 2000 with additional data from Ashworth et al. 1999, Hiller et al. 2000, Hiraoka et al. 1998, Maneset al. 1997, Noe et al. 2001, Park et al. 2000, Stracke et al. 2000, Sternlicht et al. 2001, and Uria & Lopez-Otin 2000.

For interstitial collagens, aggrecan and link protein, the cleavage sites identifiedin breakdown products isolated from tissue extracts match those that have beenestablished in vitro; however, multiple MMPs can generate these same cleavages.In a limited number of cases, the MMP substrates and responsible MMPs havebeen identified genetically. The restricted expression pattern of an enzyme mayalso help to predict its proteolytic targets. For example, MMP20/enamelysin isprincipally expressed in the enamel organ of developing teeth, where ameloblastcells secrete amelogenin, the major protein that forms the organic matrix of toothenamel. Amelogenin is continuously degraded and replenished as enamel forma-tion proceeds, and it is hydrolyzed by recombinant MMP20 in vitro (Li et al. 2001).Moreover, an inherited missense mutation in the amelogenin gene is associatedwith X-linked amelogenesis imperfecta and alters an amelogenin cleavage site sothat it is poorly hydrolyzed by MMP20 (Li et al. 2001). Therefore, amelogeninis almost certainly a natural substrate of MMP20, and inherited mutations that



render it resistant to MMP20 cleavage may lead to defective tooth enamel forma-tion. Nevertheless, such a concordance between the isolated temporal and spatialexpression of an MMP and its substrate, together with the existence of disease-linked mutations that alter the proteolytic susceptibility of the substrate, is theexception. In virtually all other cases, the identification of key in vivo substratesand the responsible enzymes will require rigorous experimental approaches.

Screening methods are now emerging as another means of identifying potentialphysiologic MMP substrates. For example, monocyte chemoattractant protein-3(MCP-3) was identified as an MMP2 substrate after using the MMP2 hemopexindomain as bait to search for potential binding partners in yeast two-hybrid screens(McQuibban et al. 2000). Thus the hemopexin domain of MMP2 contains at leastone exosite that is required for the binding of MCP-3. A phage-displayed hexapep-tide library has also been used to map the preferred substrate specificity of humanMMP13 (Deng et al. 2000). Subsequent database searches for proteins with match-ing peptide sequences confirmed the ability of MMP13 to degrade aggrecan andcollagens II and IV, but also identified a sequence within the prodomain of TGF-β3as a potential target. This is particularly intriguing because cell surface-associatedMMP2 and MMP9 can activate TGF-β and because TGF-β activation is blockedby metalloproteinase inhibitors (Yu & Stamenkovic 2000). Moreover, MMP13 canbe activated at the cell surface by MT1-MMP, thus potentially placing it in the rightlocation to activate TGF-β. As is so often the case, however, a determination ofwhether such processing actually occurs in vivo awaits further testing and will un-doubtedly be difficult to prove. Therefore, although the list of potential substratesis long, the list of known in vivo substrates remains relatively short.

REGULATION OF CELLULAR SIGNALS BY MMPs

Although MMPs can cleave virtually all structural ECM molecules, they can alsocleave several circulating, cell surface and pericellular proteins, which enablesthem to regulate cell behavior in numerous ways (Figure 4). These mechanismsinclude the alteration of cell-matrix and cell-cell interactions; the release, activa-tion, or inactivation of autocrine or paracrine signaling molecules; and the potentialactivation or inactivation of cell surface receptors.

Extracellular Matrix Remodeling and Cell Behavior

ECM-degradation can be viewed, on the one hand, as merely disrupting and re-modeling structural barriers and, indeed, such degradative processes permit cellularinvasion to take place. However, extracellular matrices are not just passive cellularscaffolds; they influence cell behavior by sequestering signaling molecules, suchas growth factors and growth factor-binding proteins, and by acting as contextualligands for cellular adhesion receptors, such as integrins, that transduce signalsto the cell interior (Streuli 1999). Indeed, extracellular matrices regulate suchbasic processes as cell shape, movement, growth, differentiation, and survival bycontrolling cell adhesion and the cytoskeletal machinery (reviewed in Lukashev



Figure 4 Potential mechanisms of MMP-mediated cellular signaling.

& Werb 1998). By extension, MMPs also influence these same processes by al-tering the composition and structural organization of the ECM, thereby alteringmatrix-derived signals. Moreover, proteolytic ECM remodeling results in the re-lease of modular breakdown products with biologic activity. For example, theMMP-dependent cleavage of native fibrillar collagen exposes otherwise crypticRGD sites that can then be ligated byαvβ3 integrin, and this interaction promotesthe survival and growth of melanoma cells (Petitclerc et al. 1999). In addition, thecleavage of intact laminin-5 by MMP2 generates aγ2-chain fragment (γ2x) thatinduces epithelial cell motility, presumably by exposing an otherwise inaccessi-ble site (Giannelli et al. 1997). ECM molecules also act as binding reservoirs forvarious growth factors and cytokines that are released once the ECM moleculesare degraded. For example, the small collagen-associated proteoglycan decorinacts as a depot for TGF-β, and its degradation by various MMPs makes the other-wise sequestered TGF-β available to carry out its biologic functions (K Imai et al.1997). One such function is to inhibit the expression of several MMP genes. ThusMMP-mediated activation and release of TGF-β may act as a negative feed-backmechanism to limit MMP expression and further TGF-β release. Likewise, MMP-cleaved collagen can feed back through discoidin domain receptor-2 to suppresscollagenase expression and further collagen degradation, whereas intact collagenhas the opposite effect (Vogel et al. 1997).

Most cells must adhere to a natural or provisional matrix to survive. Thus MMP-mediated disruption of subcellular matrices can induce apoptosis in anchorage-dependent cells and plays an important role in normal physiologic cell deathin tissues such as the involuting mammary gland (Alexander et al. 1996). In



addition, pericellular matrix destruction could defy cancer by raising the cell deathrate, or promote it by selecting for anchorage-independent and apoptosis-resistantsubclones.

Cell Surface Proteolysis and Cellular Signals

MMPs can potentially influence cell behavior by cleaving cell-cell adhesion pro-teins, by releasing bioactive cell surface molecules, or by cleaving cell surfacemolecules that transduce signals from the extracellular environment. For example,MMP3 and MMP7 both cleave the adherens junction protein E-cadherin, and thesoluble extracellular fragment that is released disrupts cell aggregation and pro-motes cell invasion in a paracrine manner independent of the cleavage event itself(Lochter et al. 1997, Noe et al. 2001). It is tempting to speculate that the MMP-mediated cleavage of E-cadherin may abrogate its tumor suppressive activity andinfluence other aspects of cancer associated with E-cadherin and MMP alterations,such as epithelial-to-mesenchymal phenotypic conversions, invasion, and genomicinstability (Sternlicht et al. 2000). Transmembrane MT-MMPs can also degradecell surface tissue transglutaminase, an integrin-binding co-receptor that promotesthe adhesion and spreading of cells on fibronectin and can thus regulate cancer cellmigration either positively or negatively, depending on the type of ECM the cellsencounter (Belkin et al. 2001).

MMPs can also release cell surface molecules. For example, MMP3 can releasesoluble L-selectin from leukocytes (Preece et al. 1996) and can release activeheparin-binding EGF-like growth factor (HB-EGF) from cell surfaces by cleavingit at a site just outside the cell membrane (Suzuki et al. 1997). MMP7 can induce Fasreceptor-mediated apoptosis by releasing active soluble Fas ligand from the surfaceof the same target cells, and this shedding appears to influence involution of theprostate following castration, since prostatic involution is significantly diminishedin MMP7-deficient mice (Powell et al. 1999). The shedding of soluble Fas ligandcould prevent cancer progression, or it could promote progression either by exertingpressure for the selection of subpopulations that are refractory to Fas-mediated celldeath or by merely removing a critical component of the Fas-mediated apoptoticpathway. Indeed, the constitutive expression of MMP7 in cells that express both Fasligand and its receptor results in the emergence of clones that are resistant to Fas-mediated and chemotherapy-mediated apoptosis (Fingleton et al. 1999, Mitsiadeset al. 2001). Cell surface localized MMP2 and MMP9 can activate latent TGF-β

(Yu & Stamenkovic 2000). At least seven MMPs can activate recombinant solubleproTNF-α (Woessner & Nagase 2000) and, like ADAM17, GPI-linked MT4-MMPcan cleave membrane-bound proTNF-α to generate active soluble TNF-α (Englishet al. 2000). Under the right circumstances, several MMPs may process TGF-β

and TNF-α in vivo.In addition to causing the activation and release of cytokines and growth factors,

MMPs can also cleave their cell surface receptors. MMP2, for example, cleaves cellsurface fibroblast growth factor (FGF) receptor 1 at a specific juxtamembrane site,thereby releasing a soluble receptor fragment that retains its ability to bind FGF



(Levi et al. 1996). Soluble FGF receptor type 1 has been found in the circulationand in vascular basement membranes and may indirectly influence FGF availabil-ity. It is not known whether the removal of cell surface FGF receptors directlyblocks FGF responsiveness in the receptor-depleted cells as occurs for other re-ceptors. For example, MMP9 cleaves IL2 receptorα (IL2Rα) on T cells and signi-ficantly downregulates their proliferative response to IL2 in culture (Sheu et al.2001). Furthermore, the in vivo responsiveness of tumor-infiltrating lymphocytes issuppressed by the metalloproteinase-dependent downregulation of IL2Rα protein(but not mRNA) levels (Sheu et al. 2001).

Regulation of Paracrine Signals

In addition to releasing several cell surface and matrix-bound growth factors andcleaving cell surface receptors to release soluble growth factor-binding proteins,MMPs may regulate the availability of paracrine signals either by inactivating themdirectly or by inactivating their soluble binding proteins. For example, MMPs caninactivate angiotensins I and II, bradykinin, and substance P, thereby limiting theirrespective physiologic effects directly (Woessner & Nagase 2000). On the otherhand, the degradation of insulin-like growth factor (IGF) binding proteins (IGF-BPs) 3 and 5 by MMPs 1, 2 and 3, and of IGFBP-1 by MMP11, indirectly increasesthe availability of IGF survival and growth factors (Fowlkes et al. 1999, Manes et al.1997). For example, IGFBP-1 inhibits the growth of human breast cancer cells,whereas the degradation of IGFBP-1 by MMP11 restores their IGF-1-dependentgrowth in culture and in vivo (Manes et al. 1997). Conversely, TIMP1 blocks hep-atocyte growth and hepatocellular carcinogenesis in a bitransgenic carcinogenesismodel by protecting IGFBP-3 from proteolysis, thereby limiting the availability ofIGF-2 (Martin et al. 1999). Likewise, an MMP-resistant form of IGFBP-5 inhibitsthe responsiveness of smooth muscle cells to IGF-1 (Y Imai et al. 1997).

As inflammatory cell products, MMPs participate in and promote inflamma-tory processes, but they may also blunt such processes. For example, MMP2 cancleave monocyte chemoattractant protein-3, thereby inactivating it and generat-ing a chemokine receptor-binding antagonist that further impedes inflammation(McQuibban et al. 2000) and, perhaps, immune surveillance against cancer. In aco-culture model of herniated disk resorption, MMP3 from chondrocytes is re-quired to generate a macrophage chemotactic factor (Haro et al. 2000a), whereasmacrophage MMP7 is required to release soluble TNF-α from the macrophages,thereby enabling them to infiltrate the cultured intervertebral discs (Haro et al.2000b). In addition, MMPs can degrade the inflammatory cytokine IL1β, whichis also a potent inducer of MMP expression, but not IL1α (Ito et al. 1996).

Generation and Inactivation of Bioactive Molecules

In addition to altering the activity and availability of various cytokines, growthfactors, and hormones, MMPs can cleave a number of other nonmatrix proteins,sometimes abolishing their normal biologic function while generating breakdownproducts with entirely new biologic activities. MMPs can cleave serine proteinases,



such as plasminogen and urokinase-type plasminogen activator, as well as seve-ral serpin-type serine proteinase inhibitors, includingα1-proteinase inhibitor (α1-PI),α1-antichymotrypsin, antithrombin III, and plasminogen activator inhibitor-2(Sternlicht & Werb 1999). These cleavages often inactivate the proteinase or pro-teinase inhibitor and simultaneously create bioactive breakdown products. Forexample, MMPs 2, 7, 9, and 12 can cleave plasminogen to generate the angiogen-esis inhibitor angiostatin (Patterson & Sang 1997, O’Reilly et al. 1999). Likewise,the inactivation ofα1-PI by MMPs creates a cleavage product that acts as a potentchemoattractant for neutrophils (Banda et al. 1988) and assists tumor growth andinvasion, possibly by modulating NK cell cytotoxicity (Kataoka et al. 1999). Vir-tually all MMPs can degrade fibrinogen, and MMPs 12, 13, and 14 can inactivateFactor XII (Hageman factor), thereby suppressing normal clotting mechanisms(Hiller et al. 2000). In addition, MMPs can degrade insoluble fibrin in a plasmin-independent manner, and this fibrinolytic capacity plays an important role in newblood vessel formation, dermal wound repair, glomerular function, and other bio-logic processes (Hiraoka et al. 1998, Lund et al. 1999, Lelongt et al. 2001). SeveralMMPs can also cleave the collagen-like region of C1q, a subunit of the C1 compo-nent of the classic complement cascade; however, the cleaved collagen-like regionstill retains its ability to induce an oxidative burst in neutrophils (Ruiz et al. 1999).Undoubtedly, some of the MMP-mediated mechanisms outlined above contributeto a variety of biologic processes, but it is still largely unclear which mechanismsare crucial.

MMPs IN DEVELOPMENT AND DISEASE

Genetic Dissection of Biologic Rolesand Critical In Vivo Substrates

One of the major challenges left in understanding the biology of the MMPs is theidentification of their essential in vivo substrates. Even when a candidate proteincan be cleaved by a given MMP in vitro and is, in fact, degraded during a particularbiologic process; and even though its cleavage may make perfect mechanisticsense, an entirely different substrate may actually regulate the process. This isparticularly well illustrated by the recent finding thatα1-PI is the critical substratefor MMP9 in bullous pemphigoid, an autoimmune skin-blistering disease (Liu et al.2000). Prior to this finding, evidence indicated that the key MMP9 substrate wasthe hemidesmosomal protein BP180 (collagen XVII), which helps anchor basalepidermal cells to the underlying basement membrane. BP180 is one of two majorautoantigens recognized by the autoantibodies that initiate bullous pemphigoid, itis cleaved during blister formation, and it can be cleaved by MMP9 in solution.Moreover, MMP9 is upregulated in bullous pemphigoid, and knockout mice thatlack MMP9 are resistant to the blistering that is elicited by BP180 antibodies inwild-type mice. The MMP9-deficient mice do, however, develop blisters if theyare reconstituted with MMP9-expressing neutrophils. Thus it appears that MMP9



is responsible for BP180 cleavage and subsequent blister formation. However,neutrophil elastase (NE) can also cleave BP180; it too is upregulated in bullouspemphigoid, and mice lacking this enzyme are also refractory to antibody-inducedblistering unless they are reconstituted with NE-competent neutrophils. Moreover,BP180 is cleaved and blisters do form if MMP9-null mice are reconstituted withneutrophils from NE-null mice, or if NE-null mice receive MMP9-null neutrophils,thus indicating that both enzymes participate and act along the same pathway.However, even though both enzymes can cleave BP180 in solution, only purifiedNE causes the blister-like separation of isolated skin sections. In addition, MMP9-null mice do form blisters if they are reconstituted with a large excess of MMP9-null (but NE-competent) neutrophils. Thus MMP9 acts upstream of NE and onlyindirectly participates in BP180 cleavage, whereas NE is absolutely required forBP180 cleavage and dermal-epidermal separation.

The principal endogenous inhibitor of NE,α1-PI, is also elevated in the skinof antibody-treated MMP9- and NE-deficient mice. This suggests that the roleof MMP9 in blistering is to inactivateα1-PI, thereby removing the major con-straint against NE and allowing it to cleave BP180. Indeed,α1-PI degradation wasonly absent in antibody-treated MMP9-deficient mice, even when they were givenexcess MMP9-null neutrophils. Nevertheless, BP180 degradation and blister for-mation did take place in MMP9-deficient mice that received excess MMP9-nullneutrophils. Apparently, the additional MMP9-null neutrophils enable blister for-mation by providing enough NE to overcome the elevated levels of intactα1-PI.Moreover, the administration of excessα1-PI blocks antibody-induced blisteringin wild-type mice, but not if theα1-PI is pre-treated with MMP9. Thus MMP9indirectly contributes to antibody-induced skin blistering by inactivatingα1-PIand allowing NE to cleave BP180 in an unrestricted manner (Figure 5), whereasdirect cleavage of BP180 by MMP9, if it occurs at all, is probably minor.

Similar interactions between MMPs, serine proteinases, and their inhibitorsmay also participate in other diseases. In pulmonary emphysema, for example, theirreversible enlargement of peripheral air spaces is thought to result from prote-olytic degradation of interstitial elastic fibers, which otherwise have a cross-linkedelastin core and a fibrillin-rich microfibrillar coat (Ashworth et al. 1999). Thelong-standing yet controversial elastase-antielastase theory states that the progres-sive erosion of alveolar walls results from inflammatory stimuli, such as cigarettesmoke, that alter the balance between NE and its natural inhibitor,α1-PI. Thistheory derives primarily from the observations that (a) intrapulmonary instillationof NE causes experimental emphysema, (b) individuals withα1-PI deficiency arepredisposed to develop emphysema, and (c) cigarette smoke can inactivateα1-PI (Dhami et al. 2000). Moreover, both exogenousα1-PI and an anti-neutrophilantibody protect against acute matrix degradation in mice exposed to cigarettesmoke (Dhami et al. 2000). However, over 90% of the inflammatory cells insmokers’ lungs are macrophages rather than neutrophils, and macrophages cansecrete MMPs 2, 7, 9, and 12, all of which can degrade elastin and inactivateα1-PI (Dhami et al. 2000). Moreover, MMPs 2, 9, and 12 (as well as NE and



Figure 5 BP180 cleavage in bullous pemphigoid. Infiltrating neutrophils (PMNs)in and around bullous pemphigoid lesions release substantial amounts of neutrophilelastase (NE) and gelatinase B (MMP9) from large azurophilic primary granules andsmaller tertiary granules, respectively. In addition,α1-proteinase inhibitor (α1-PI) iselevated, but because it is inactivated by MMP9, its ability to inhibit NE is abolished,thus enabling NE to cleave hemidesmosomal type XVII collagen (BP180) in an un-restricted manner. Furthermore, cleavedα1-PI fragments may exacerbate this processby enhancing PMN recruitment. Although MMP9 can also cleave BP180 in vitro, itsdirect contribution to BP180 cleavage in vivo is minimal or non-existent.

other enzymes) can degrade fibrillin within elastic fibers (Ashworth et al. 1999).Thus macrophage-derived proteinases that are increased in the lungs of smokersmay be responsible for the matrix degradation of emphysema. Indeed, the chronicexposure of wild-type mice to cigarette smoke elicits significant accumulation oflung macrophages (but not neutrophils), induction and activation of MMP12 (butnot NE), and emphysematous enlargement of pulmonary air spaces (Hautamakiet al. 1997). By comparison, smoke-exposed MMP12-deficient mice show no suchpulmonary changes even after the forced recruitment of macrophages in responseto monthly intratracheal administration of monocyte chemoattractant protein-1(MCP-1), indicating that the lack of MMP12, rather than the lack of macrophages,protects them from smoke-induced pulmonary damage (Hautamaki et al. 1997). Inaddition, the relatively mild accumulation of neutrophils and the absence of NE insmoke-exposed wild-type and MMP12-deficient mice suggests the direct involve-ment of MMP12, rather than NE. Nevertheless, it remains unclear whether otherenzymes participate or if elastin, fibrillin,α1-PI, or other substrates are critical tothe natural development of emphysema.

The breakdown of elastic fibers has also been implicated in the formation andrupture of intracranial and aortic aneurysms. Elastolytic MMPs 2, 9, and 12 areprominent within human aortic aneurysms, and MMP12 immunolocalizes to resid-ual elastic fiber fragments (Curci et al. 1998). In mice, the development of experi-mental aortic aneurysms is suppressed by the administration of a nonselective



MMP inhibitor and by targeted disruption of the MMP9 gene, but not byselective disruption of the MMP12 gene (Pyo et al. 2000). Furthermore, resistanceto aneurysms can be conferred by bone marrow transplantation from MMP9-deficient donors to wild-type hosts, and it can be abolished in MMP9-deficientmice by transplanting wild-type bone marrow cells (Pyo et al. 2000). Thus MMP9appears to play an essential role in aneurysm formation, probably (though notconclusively) by virtue of its ability to cleave elastin and/or fibrillin.

One in vivo role for an MMP that has been definitively identified is the activa-tion of intestinal cryptα-defensins by MMP7 (Wilson et al. 1999). The defensins,which play an important role in innate immunity, are antimicrobial (membrane-disrupting) peptides that are initially produced as inactive precursors by granu-locytes and specific epithelial cells, including Paneth cells of the small intestine.In mice, crypt defensins (cryptdins) colocalize with MMP7 in apical Paneth cellgranules, and the absence of MMP7 in gene-targeted mice has no effect on crypt-din production. MMP7 can, however, activate latent pro-cryptdins in vitro byremoving their prodomain, and unlike wild-type mice, MMP7-deficient mice failto process and instead accumulate inactive pro-cryptdins in their small intestines.As a result, intestinal extracts from MMP7-deficient mice have less antimicrobialactivity than those of their wild-type counterparts, and orally administered bacteriashow greater intestinal survival and overall virulence in MMP7-deficient mice thanin wild-type controls. Thus MMP7 contributes to innate immunity by regulatingcryptdin activation in the small intestine and may do so in other tissues as well.

These and other examples (Table 3) illustrate how genetically modified micecan reveal the proteinases and, if circumstances permit, the substrates that regulatephysiologic and pathologic processes. Often, however, the conclusiveness of thisapproach is limited by the expression of redundant or compensatory enzymeswith overlapping activity, the modest yet nonessential influence of an enzyme onthe process in question, or the complete absence of any influence. For example,MMP3 is thought to be an important mediator of cartilage loss in rheumatoidand osteoarthritis, yet MMP3-deficient and wild-type mice are equally susceptibleto collagen-induced arthritis, and both show cleavage of the aggrecan Asn341-Phe342 peptide bond (Mudgett et al. 1998). Thus even if MMP3 cleaves aggrecan,other redundant or compensatory enzymes are likely to participate and mightmask the influence of MMP3, or MMP3 may not participate at all. Unmaskingthe responsible substrates and mechanisms may therefore require the generationof animals that are deficient in multiple enzymes. Functional overlap betweendistinct enzymes also probably explains the viability of most MMP-deficient miceand the subtle nature of their developmental phenotypes, with the notable exceptionof MT1-MMP null animals, which exhibit severe skeletal and connective tissueabnormalities and early postnatal lethality (Holmbeck et al. 1999, Zhou et al. 2000).

Determination of Biologic Significance UsingSubstrate Cleavage Site Mutations

Mutations in amelogenin that decrease its susceptibility to MMP20 are associ-ated with inherited defects in tooth enamal formation (Li et al. 2001). These data



TABLE 3 Influence of MMPs in genetically modified mice: non-malignant phenotypes

Genotype Phenotype Reference

Haptoglobin-MMP1 Emphysematous enlargement of (D’Armiento et al. 1992)pulmonary air spaces

WAP-MMP3 Increased mammary apoptosis (Alexander et al. 1996)in pregnancy

Precocious mammary differentiation (Sympson et al. 1994)

MMTV-MMP7 Precocious mammary differentiation, (Rudolph-Owen et al.male infertility 1998a)

β-Actin-TIMP1 Accelerated mammary adipogenesis (Alexander et al. 2001)

MMP2−/− Delayed mammary gland development a

MMP3−/− Hypomorphic mammary gland a

developmentAccelerated mammary adipogenesis (Alexander et al. 2001)Delayed incisional wound closure (Bullard et al. 1999)Resistance to contact dermatitis (Wang et al. 1999)Normal cardiac rupture following (Heymans et al. 1999)infarction

Normal collagen-induced arthritis (Mudgett et al. 1998)development

Impairedex vivoherniated disk (Haro et al. 2000a)resorption

MMP7−/− Deficient innate intestinal immunity (Wilson et al. 1999)Impaired tracheal wound (Dunsmore et al. 1998)reepithelialization

Diminished prostate involution (Powell et al. 1999)after castration

Impaired ex vivo herniated disk (Haro et al. 2000b)resorption

MMP9−/− Delayed growth plate vascularization (Vu et al. 1998)Delayed endochondral ossification (Vu et al. 1998)Defective osteoclast recruitment (Engsig et al. 2000)Resistance to experimental bullous (Liu et al. 2000)pemphigoid

Resistance to experimental aortic (Pyo et al. 2000)aneurysms

Prolonged contact dermatitis (Wang et al. 1999)Protection from ventricular enlargement (Ducharme et al. 2000)after infarction

Protection from cardiac rupture following (Heymans et al. 1999)infarction

Diminished motor defects following (Wang et al. 2000a)brain trauma



TABLE 3 (Continued)

Genotype Phenotype Reference

Diminished PMN infiltration in early b

glomerulonephritisEnhanced susceptibility to late-stage (Lelongt et al. 2001)glomerulonephritis

Abnormal decidual implantation sites c

Normal renal Alport syndrome (Andrews et al. 2000)progression

Normal LPS-induced lung injury (Betsuyaku et al. 1999a)Normal neutrophil emigration (Betsuyaku et al. 1999b)

MMP11−/− No overt developmental phenotype (Masson et al. 1998)Accelerated neointima formation after (Lijnen et al. 1999)vessel injury

MMP12−/− Resistance to cigarette smoke-induced (Hautamaki et al. 1997)emphysema

Normal susceptibility to experimental (Pyo et al. 2000)aortic aneurysms

Normal cardiac rupture following (Heymans et al. 1999)infarction

MMP14−/− Severe bone and connective tissue (Holmbeck et al. 1999)abnormalities

Inadequate collagen turnover, early (Holmbeck et al. 1999)postnatal death

Impaired endochondral ossification (Zhou et al. 2000)and angiogenesis

TIMP1−/− Resistance to cornealPseudomonas (Osiewicz et al. 1999)infections

Normal renal interstitial fibrosis after (Eddy et al. 2000)proteinuria

TIMP2−/− Deficient MMP2 activation; otherwise (Wang et al. 2000b)normal

TIMP3−/− Accelerated mammary gland involution d

Emphysematous enlargement of pulmonaryd

air spaces

aMD Sternlicht, BS Wiseman & Z Werbunpublished observations.bJ-P Rougier & Z Werb unpublished observations.cN Rougier, JL Rinkenberger & Z Werb unpublished observations.dJ Fata, K Leco & R Khokha personal communication.

Abbreviations: LPS, lipopolysaccharide; MMTV, mouse mammary tumor virus promoter; PMN, neutrophil; WAP,Whey acidic protein gene promoter.



complement the enamel organ-specific expression of both MMP20 and amelogeninto strongly implicate amelogenin as a physiologic target of MMP20. A similar ex-perimental approach is to generate animals that express mutant substrates thatresist enzyme cleavage. For example, several collagenolytic MMPs cleave theα

chains of native fibrillar collagens at a single peptide bond, such that type I colla-gen fibrils containing mutantα1(1) chains with an amino acid substitution at thissite are resistant to cleavage. The introduction of this mutation into the endoge-nousCOL1A1gene of mice leads to marked dermal fibrosis resembling humanscleroderma and impaired postpartum uterine involution (Liu et al. 1995). Thesestudies suggest that collagen resorption can also be accomplished through colla-genase cleavage at a novel site in the nonhelical N-telopeptide domain. Never-theless, these studies do not identify which MMP cleaves type I collagen withinits triple-helical domain during any specific processes. Moreover, considerabledifferences between collagenase expression patterns in humans and mice (Balbinet al. 2001) and differences between the catalytic activities of human and mouseMMP orthologues (Noel et al. 1995) may limit our ability to extrapolate murinedata to humans and other species. Nevertheless, despite some inherent limitations,gene-targeting technologies provide the most powerful and potentially definitivemeans of determining how MMPs regulate biologic processes in vivo.

MMPs in Bone Development

Bone development can proceed in two distinct ways. Skeletal long bones and ver-tebrae form by the multistep process of endochondral ossification, whereas the flatbones of the skull develop by intramembranous ossification. In endochondral ossi-fication, chondrocytes proliferate and differentiate to form a zone of hypertrophiccartilage that is then resorbed and replaced by mineralized bone matrix. It appearsthat resorption of the hypertrophic cartilage ECM is accomplished by chondro-clasts that make way for invading capillary endothelial cells. Osteoblasts are thenrecruited to form trabecular bone that is subsequently remodeled by osteoclasts andmaintained by the opposing but coordinated activities of bone-forming osteoblastsand bone-resorbing osteoclasts. With intramembranous ossification, osteoblastsdeposit bone matrix onto a cartilage template that is later removed, and vascu-larization appears to be accomplished via osteoclast-mediated matrix remodeling.Bone development thus requires active remodeling of cartilage and bone matricesand the concerted differentiation and activity of several cell types. It is thereforenot surprising that at least three MMPs, MMP2, MMP9, and MT1-MMP, influencebone development and homeostasis.

The only MMP gene mutations that are known to cause an inherited humandisease were recently found in theMMP2gene (Martignetti et al. 2001). These mu-tations abolish the expression of functional MMP2 and cause nodulosis, arthropa-thy, and osteolysis (NAO) syndrome, a rare autosomal recessive osteolysis (or“disappearing-bone”) disorder. Affected individuals present between one and tenyears of age with subcutaneous fibrous nodules, arthritis, minor facial anoma-lies, sclerotic cranial sutures, generalized osteopenia, and multicentric osteolytic



changes primarily involving the carpal and tarsal bones of the hands and feet. Theseemingly counterintuitive finding that the absence of a matrix-degrading enzymeleads to progressive bone loss instead of an osteopetrotic increase in bone masssuggests that MMP2 is not a critical bone matrix-dissolving enzyme. Rather, it sug-gests that MMP2 promotes osteoblastic bone formation and/or inhibits osteoclas-tic bone resorption. Because TGF-β promotes osteoblastic activity (Filvaroff et al.1999), and because mutations that favor unopposed TGF-β activity cause a humanosteopetrotic disorder (Janssens et al. 2000), it is tempting to speculate that the ab-sence of MMP2 might hinder bone deposition (and thus favor resorption) if MMP2were a critical activator of TGF-β during bone remodeling. Alternatively, the lackof MMP2 might favor bone resorption if MMP2 is required to remove the collagenscaffold onto which osteoblasts lay new bone matrix and if such collagen removalwere a prerequisite to the latter step of bone deposition. Although identifying theactual mechanisms clearly requires further experimentation, MMP2-deficient miceappear to develop normally and lack an overt osteolytic or arthritic phenotype. Thismay be due to compensatory mechanisms such as a lack of contributing factorsthat might reveal a phenotype that is otherwise too subtle to detect in mice, or theactual lack of a role for MMP2 in murine as opposed to human bone remodeling.Nevertheless, other MMP-deficient mice do exhibit skeletal phenotypes.

Mice lacking MMP9 accumulate hypertrophic cartilage within the growing re-gion of their long bones and ultimately exhibit subtle bone shortening (Vu et al.1998). This expansion of the hypertrophic cartilage zone results from deficientendochondral ossification rather than any changes in chondrocyte proliferationor differentiation. The apoptotic loss of hypertrophic chondrocytes, which onlyoccurs near invading capillary endothelial cells, is delayed. This delay appearsto be secondary to diminished vascular invasion into the hypertrophic cartilage.Indeed, MMP9-deficient hypertrophic cartilage fails to release normal levels ofpro-angiogenic activity in culture. Thus the absence of MMP9 may diminish the re-lease of an angiogenic factor from the hypertrophic cartilage ECM, thereby slowingvascular invasion and the subsequent death of hypertrophic chondrocytes. Severalangiogenic factors are expressed in the growth plate of developing long bones, in-cluding vascular endothelial cell growth factor (VEGF), which is highly expressedin terminal hypertrophic chondrocytes (Gerber et al. 1999). Moreover, the inhibi-tion of VEGF signaling with a soluble VEGF receptor chimeric protein (mFlt-IgG)inhibits capillary invasion into hypertrophic cartilage and leads to a bone pheno-type that is similar to that seen in MMP9-null mice. In addition, mFlt-IgG inhibitsthe recruitment of chondroclasts, osteoblasts and osteoclasts (Gerber et al. 1999,Engsig et al. 2000). Thus MMP9 appears to play an essential role in releasingECM-bound VEGF from hypertrophic cartilage stores and in the VEGF-mediatedrecruitment of several cell types that are necessary for proper bone development.

Whereas MMP9-null mice show a specific delay in endochondral ossification,targeted inactivation of the MT1-MMP gene in mice causes severe defects in bothendochondral and intramembranous bone formation (Holmbeck et al. 1999, Zhouet al. 2000). Because MT1-MMP is a critical activator of MMP2, it is importantto note that many of the features of the MT1-MMP-deficient mice (craniofacial



dysmorphism, arthritis, osteopenia, and soft tissue fibrosis) resemble those de-scribed in MMP2-deficient humans with NAO syndrome (Martignetti et al. 2001).Similar defects have also been seen in mice harboring a mutation that renderstheir type I collagen fibrils resistant to collagenase cleavage (Liu et al. 1995), andcells isolated from MT1-MMP-null mice exhibit deficient collagenolytic activityin vitro (Holmbek et al. 1999). Thus impaired remodeling of collagen-containingskeletal and periskeletal matrices may lead to the defects seen in MT1-MMP-deficient mice. Bone marrow stromal cells from MT1-MMP-deficient mice showdecreased bone formation, suggesting that MT1-MMP may influence the differen-tiation and activity of osteoblasts, and MT1-MMP-deficient mice have increasednumbers of osteoclasts, suggesting that MT1-MMP may influence the differen-tiation and recruitment of the latter cell type as well. A better understanding ofthe mechanisms whereby MMPs influence skeletal development and homeostasismay have important implications in terms of avoiding any unwanted side-effectsof clinical interventions aimed at inhibiting MMP activity.

MMPs in Vascular Remodeling

The formation of new blood vessels from existing vessels (angiogenesis) is re-quired for tissue development and repair and for the growth of solid tumors. MMPscan contribute to angiogenesis in at least three ways: They can enable endothe-lial cell migration through surrounding tissues by disrupting ECM barriers, theycan promote it by liberating sequestered angiogenic factors, or they can defy itby generating anti-angiogenic breakdown products. In addition, the balance be-tween MMPs and their inhibitors must be restored during the maturation of newlyformed blood vessels to favor basement membrane assembly and endothelial celldifferentiation and quiescence (Kraling et al. 1999).

Insoluble fibrin is one of the barriers that angiogenic endothelial cells mustpenetrate during new blood vessel formation. Endothelial cell MMPs are requiredfor neovascularization of fibrin to occur in culture and in vivo, whereas the fib-rinolytic plasminogen/plasminogen activator system is surprisingly not required(Hiraoka et al. 1998). Moreover, the potent pericellular fibrinolysin MT1-MMPenables otherwise non-invasive cells to penetrate fibrin gels and form tubularstructures. Invasion and tubulogenesis also proceed in the absence of MMP2 butare blocked by natural and synthetic MMP inhibitors and fail to take place if atransmembrane-deleted form of MT1-MMP is expressed instead of the full-lengthmembrane-associated form. Thus membrane-associated MMPs play a critical rolein angiogenesis by virtue of their pericellular fibrinoytic activity. Undoubtedly, theyplay a similar role in enabling endothelial cells to traverse other ECM barriers aswell.

During the early development of solid cancers, an “angiogenic switch” initi-ates the formation of new blood vessels prior to the emergence of invasive tumors(Hanahan & Folkman 1996). One possibility is that various stimuli induce the ex-pression of pro-angiogenic genes thereby activating this switch. This appears not tobe the case, however, in a transgenic model of pancreatic islet cell carcinogenesis



(Bergers et al. 2000). In this model, VEGF, its receptors, and acidic FGF are allexpressed at stable constitutive levels before and after angiogenesis begins, yet asynthetic inhibitor of VEGF signaling inhibits angiogenesis and tumor growth. Onthe other hand, MMPs 2 and 9 are upregulated in angiogenic islets, and synthetic in-hibitors of MMP activity delay the onset of angiogenesis and inhibit tumor growth.The genetic ablation of MMP9 also suppresses angiogenesis and tumor growth,whereas the lack of MMP2 inhibits tumor formation, but does not alter the develop-ment of angiogenic islets. Moreover, exogenous MMP9 (but not MMP2) releasesVEGF from cultured islets, thereby eliciting an angiogenic response in co-culturedendothelial cells. Therefore, MMP9 contributes to the induction of angiogenesisby releasing sequestered VEGF, although other mechanisms may also exist.

Although MMP2 does not appear to influence angiogenesis in the above model,its downregulation by targeted gene disruption and antisense methods does sup-press tumor-induced angiogenesis in other models (T Itoh et al. 1998, Fang et al.2000). Integrinαvβ3, which recognizes RGD sequences on vitronectin and otherECM molecules, is highly expressed on angiogenic endothelial cells, and anti-bodies and peptides that block its function inhibit angiogenesis in several modelsystems (Eliceiri & Cheresh 1999). Moreover, the fewαv-deficient mice that sur-vive to term die shortly thereafter with extensive vascular abnormalities in sometissues. The RGD-independent binding of MMP2 to integrinαvβ3 may influ-ence angiogenesis by facilitating vascular invasion (Brooks et al. 1996). However,MMP2-deficient mice have no overt angiogenic phenotype but do display lesstumor-induced angiogenesis than wild-type controls (T Itoh et al. 1998). Becauseinaccessible RGD sites on native collagen are unmasked by the MMP-dependentcleavage of collagen, focal MMP-mediated collagen degradation may not onlyremove a physical barrier to endothelial cell invasion, but may also fosterαvβ3-mediated endothelial cell migration through the proteolyzed ECM. In favor of thishypothesis, the recombinant hemopexin domain of MMP2 prevents active MMP2from binding toαvβ3 and inhibits angiogenesis in culture (Brooks et al. 1998)and in vivo (Pfeifer et al. 2000). An organic compound that blocks the ability ofαvβ3 integrin to bind MMP2 (but not vitronectin) is also a potent inhibitor ofangiogenesis and tumor growth in vivo (Silletti et al. 2001). Because this com-pound does not appear to alter the activation or activity of MMP2 directly,αvβ3integrin may influence angiogenesis in part by concentrating MMP2 at the cellsurface. However, other data indicate thatαvβ3 integrin promotes the maturationof MMP2 to a fully active form following the MT1-MMP-mediated formation ofan MMP2 activation intermediate (Deryugina et al. 2001).

Part of the anti-angiogenic activity of TS1, TS2, and other TS domain-containingproteins may derive from their ability to downregulate MMP2 and MMP9 undercertain conditions. However, even though TS1 generally acts as an angiogenesis in-hibitor, it may also promote angiogenesis at low concentrations (Qian et al. 1997).Under these conditions, TS1 upregulates endothelial cell MMP9 activity in a dose-dependent manner, and endothelial cell tube formation is suppressed in culture byantibodies against either TS1 or MMP9 (Qian et al. 1997). By yeast two-hybridand immunoprecipitaion analysis, MMP2 and MMP9 bind the pro-perdin-like type



1 repeats of TS1 and TS2 via their fibronectin type II gelatin-binding domains,but neither TS is degraded (Bein & Simons 2000). Purified TS type 1 repeats andintact TS1 also inhibit the degradation of type IV collagen and appear to suppressthe activation of proMMP2 and proMMP9 in culture. However, the diminishedMMP activity could also reflect reduced steady-state MMP levels. Indeed, TS2binds both latent and active MMP2, but instead of directly inhibiting the activa-tion or activity of MMP2, it apparently promotes MMP2 clearance through theLRP-dependent internalization of TS2/MMP2 complexes (Yang et al. 2001).

MMPs can also negatively regulate angiogenesis by generating anti-angiogenicpeptides. Angiostatin is an N-terminal breakdown product of plasminogen thatcan be generated by MMPs 2, 3, 7, 9, and 12 (Patterson & Sang 1997, O’Reillyet al. 1999); endostatin and tumstatin are anti-angiogenic protein fragments fromthe C-terminal portions of collagens XVIII and IV, respectively (Maeshima et al.2000); and the free hemopexin domain of MMP9 itself may act as an angiogenesisinhibitor (Brooks et al. 1998). In addition, by cleaving cell surface FGF receptor1, MMP2 generates a circulating and matrix-associated binding protein that canmodulate the potent angiogenic effects of FGF by altering its availability (Levi et al.1996). FGF receptor shedding may also downregulate the FGF responsiveness ofthose cells that have lost their receptor. To date, however, the importance of MMPsin down-modulating angiogenesis is unclear, whereas they are clearly importantpositive regulators of angiogenesis.

MMPs in Cancer

MMPs are generally present in greater amounts and activated more often in andaround malignant cancers than in normal, benign, or premalignant tissues, withthe highest expression taking place in areas of active invasion at the tumor-stromainterface (reviewed in Sternlicht & Bergers 2000). Indeed, several MMPs were firstcloned and have been repeatedly re-cloned as cancer-associated genes (reviewedin Sternlicht & Werb 1999). Significant positive correlations have been foundbetween MMP expression and various indicators of a poor prognosis in virtuallyall types of cancer, and in some instances, increased MMP levels represent anindependent predictor of shortened disease-free and overall survival (reviewed inSternlicht & Bergers 2000). In epithelial cancers, however, most of the upregulatedMMPs are expressed by the supporting stromal cells rather than by the carcinomacells themselves (McKerrow et al. 2000). Thus MMPs from adjacent stromal cellsare often induced and commandeered by the malignant epithelial cells.

In addition to the extensive correlative data linking MMP overexpression withmore aggressive malignant behavior and poor clinical outcome, compelling exper-imental data show that MMPs actively contribute to cancer progression. Relativelybenign cells acquire malignant properties when MMP activity is increased or TIMPactivity diminished, and highly malignant cells are made less aggressive by reduc-ing their MMP levels or suppressing their MMP activity. Several MMPs have beenimplicated as key agonists in tumor invasion, metastasis, and angiogenesis, includ-ing MMPs 1, 2, 3, 9, and 14 (reviewed in Sternlicht & Bergers 2000). Without



the aid of ECM-degrading MMPs, endothelial cells would probably be unable topenetrate the ECM, and cancer cells would be unable to cross the matrix barriersthat otherwise contain their spread. However, recent data indicate that MMPs dofar more to influence cancer than merely remove the physical barriers to inva-sion and metastasis. A number of MMPs can promote early cancer development,and MMP3 can also promote late epithelial-to-mesenchymal phenotypic changesthat are associated with more aggressive malignant behavior (Lochter et al. 1997,Sternlicht et al. 1999). These data support the notion that MMPs can contributeto virtually all stages of cancer evolution, both early and late, thus expanding thepotential clinical utility of MMP inhibitors. Conversely, some MMPs may defycancer progression, and others may not participate in cancer, but undoubtedly playnormal physiologic roles. These possibilities must be considered and the mecha-nisms underlying the influence of MMPs in cancer must be understood in the designof therapeutic agents in order to optimize their efficacy and minimize their toxicity.

EARLY NEOPLASIA A number of recent studies indicate that stromal influences pro-mote cancer development (Barcellos-Hoff & Ravani 2000, Moinfar et al. 2000).Fibrotic and inflammatory conditions increase cancer risk, and some familial can-cer syndromes are the result of gene defects that produce stromal changes beforeepithelial changes ever occur (Kinzler & Vogelstein 1998, Willenbucher et al.1999). These are also conditions in which stromal MMPs are upregulated, and theattendant rise in MMP activity may contribute to cancer susceptibility (Sternlichtet al. 1999). Indeed, fibroblasts foster the growth of human cancer cells in nudemice, yet fibroblasts lacking MMP11 do not (Masson et al. 1998).

Several studies in genetically modified mice also support the notion that MMPsparticipate in early cancer development (Table 4). In general, MMP-overproducingtransgenic mice develop spontaneous hyperproliferative lesions. Moreover, whenMMP transgenic and knockout mice receive various oncogenic stimuli, the MMP-overexpressing mice develop more cancers than usual, whereas those that lackvarious MMPs or overproduce TIMP1 tend to get fewer cancers than normal.For instance, MMP1 transgenic mice show increased sensitivity to chemical car-cinogens (D’Armiento et al. 1995, Di Colandrea et al. 2000), whereas MMP11-deficient mice and transgenic mice that have elevated levels of circulating TIMP1show a diminished sensitivity to carcinogens (Masson et al. 1998, Buck et al.1999). The overexpression of MMP7 accelerates mammary cancer developmentin mice carrying a mammary-targetedHer2/neutransgene (Rudolph-Owen et al.1998b), whereas its absence in mice carrying theApcmin mutation slows the for-mation of intestinal adenomas (Wilson et al. 1997). The absence of MMP9 defiesthe development but accelerates the progression of human papilloma virus-16-induced squamous cell carcinomas in transgenic mice, whereas normal carcino-genesis is restored by transplanting MMP9-expressing bone marrow cells to theMMP9-deficient transgenic mice, thus indicating the importance of inflammatorycell-derived MMP9 in skin carcinogenesis (Coussens et al. 2000). Most notably,however, independent lines of mammary-targeted MMP3 transgenic mice developspontaneous premalignant lesions and malignant mammary cancers despite an



TABLE 4 MMPs promote carcinogenesis in genetically modified mice

MMP/TIMP Oncogenicgenotype stimulus Phenotype Reference

Haptoglobin-MMP1 none hyperkeratosis, acanthosis (D’Armientoet al. 1995)

DMBA + ↑ skin carcinogenesis (D’ArmientoPMA et al. 1995)

DMBA + ↑ skin papilloma formation (DiColandreachrysarobin et al. 2000)

WAP-MMP3 none mammary hyperplasias (Sternlichtand cancers et al. 1999)

MMTV-MMP3 none mammary hyperplasias (Matrisian 1999)and cancers

MMTV-MMP7 none mammary hyperplasias (Rudolph-Owenet al. 1998b)

MMTV- neu ↑ mammary carcinogenesis (Rudolph-Owenet al. 1998b)

MMTV-MMP14 none mammary hyperplasias (Ha et al. 2001)and cancers

MMP2−/− RIP-TAg ↓ pancreatic tumor growth (Bergers et al. 2000)Injected cells ↓ angiogenesis and tumor (Itoh et al. 1998a)

growth

MMP7−/− Apcmin ↓ intestinal adenoma (Wilson et al. 1997)formation

MMP9−/− K14-HPV16 ↓ skin carcinogenesis (Coussens et al. 2000)RIP-TAg ↓ pancreatic carcinogenesis (Bergers et al. 2000)

MMP11−/− DMBA ↓ mammary carcinogenesis (Masson et al. 1998)

H2-TIMP1 Crp-TAg ↓ liver carcinogenesis (Martin et al. 1996)

H2-TIMP1 (as) Crp-TAg ↑ liver carcinogenesis (Martin et al. 1996)

WAP-TIMP1 WAP-MMP3 ↓ mammary neoplasia (Sternlicht et al. 1999)

Albumin-TIMP1 DMBA + PMA ↓ mammary carcinogenesis (Buck et al. 1999)

LFABP-TIMP1 Apcmin ↑ intestinal adenoma (Heppner-Gossformationa et al. 1998)

aIntestinal adenoma formation was, however, inhibited by a broad-range synthetic MMP inhibitor.

Abbreviations:Apcmin, adenomatous polyposis coli gene with multiple intestinal neoplasia mutation; as, antisense; Crp,C-reactive protein gene promoter; DMBA, dimethylbenz-anthracine; H2, major histocompatability complex class I promoter;HPV16, human papilloma virus 16 early region; K14, keratin-14 promoter; LFABP, liver fatty acid binding protein promoter;MMTV, mouse mammary tumor virus promoter; PMA, phorbol myristate acetate; RIP, rat insulin promoter; TAg, SV40 Tantigen; WAP, whey acidic protein promoter.



absence of carcinogens or pre-existing gene mutations (Sternlicht et al. 1999,Matrisian 1999). In addition, these changes are blocked by the co-expression ofa TIMP1 transgene (Sternlicht et al. 1999). Moreover, mammary-targeted MT1-MMP transgenic mice also develop spontaneous mammary cancers (Ha et al.2001). Although each of these examples suggest that MMPs participate in earlycancer development, the spontaneous development of premalignant and malignantlesions in the latter studies lends considerable support to the likelihood that MMPsact as natural tumor promoters and influence cancer susceptibility.

If, in fact, MMPs promote cancer development, then their inhibitors should defyit. However, this is not always the case (Sternlicht & Bergers 2000). Data favoringthe likelihood that MMPs promote cancer include those indicating that murine3T3 cells become tumorigenic after antisense depletion of TIMP1 (Khokha et al.1989) and that TIMPs inhibit the transforming activity of phorbol esters (Shojiet al. 1997) and the growth of some cancer cells in vivo (Khokha 1994, Kr¨ugeret al. 1997). Moreover, TIMP1 overproduction slows chemical skin carcinogen-esis (Buck et al. 1999) and SV40 large T antigen-induced liver carcinogenesisin transgenic mice (Martin et al. 1996). In the latter model, TIMP1 appears toprotect IGFBP-3 thereby inhibiting IGF signaling and hepatocyte growth (Martinet al. 1999). Surprisingly, inApcmin mice, TIMP1 overexpression had no effecton tumor development in one line and augmented intestinal adenoma forma-tion in another, whereas a synthetic MMP inhibitor slowed adenoma formation(Heppner-Goss et al. 1998). This discrepancy may reflect the MMP-independentgrowth-promoting activity of TIMP1, which persists in mutant TIMPs that lackMMP-inhibitory activity (Chesler et al. 1995). TIMP1 may also confer a growthadvantage by upregulating VEGF expression in vivo. Indeed, TIMP1-transfectedhuman and rat mammary cancer cells show a direct relationship between TIMP1levels and VEGF production, tumor growth, and tumor vascularization and prolif-eration in vivo, but are unaffected by TIMP1 in terms of their growth in cul-ture (Yoshiji et al. 1998). TIMP1 may also act as an anti-apoptotic survivalfactor. For example, cultured B cells show increased survival in the face of variousapoptotic stimuli if they are grown in the presence of TIMP1, but not if TIMP2or a synthetic MMP inhibitor is added instead (Guedez et al. 1998). In anotherset of experiments, co-isogenic pairs of transformed fibroblasts were establishedfrom wild-type and TIMP1-deficient littermates and injected into the tail veinsof wild-type and TIMP1-null recipients (Soloway et al. 1996). Interestingly, lungcolonization depended on the TIMP1 status of the tumor cells rather than thatof the host. As predicted, two TIMP1-deficient lines formed substantially morelung colonies than their respective wild-type partners, yet the opposite was truein two other independently isolated pairs, with the TIMP1-expressing cells in-stead showing enhanced colony formation. Thus TIMP1 may promote or suppresstumor growth depending on the context of the individual tumor cells and the po-tential for cross-talk with other signaling pathways. These studies may explainwhy TIMPs are often upregulated rather than downregulated in human cancersand why their upregulation is often associated with a poor prognosis (Sternlicht &



Bergers 2000). In addition, TIMPs may inhibit the MMP-mediated infiltration oflymphocytes and immune surveillance. Alternatively, increased TIMP1 levels maymerely reflect the increased matrix remodeling that occurs during invasion, and thecritical parameter may be the balance between MMPs and their inhibitors ratherthan the expression of one component alone. For TIMP2, conflicting data fromclinical samples may also reflect the fact that it participates both in the activationand inhibition of MMP2 in a dose-dependent manner. Thus increasing levelsof TIMP2 should increase MMP2 activation to a point, after which the acti-vation and proteolytic activity of MMP2 would be blocked by the inhibitoryactivity of TIMP2. Ultimately, the TIMPs may block some aspects of cancerby inhibiting certain metalloproteinases, yet promote other aspects of cancer byinhibiting different metalloproteinases and by influencing cells in a metalloprotei-nase-independent manner. Although it is clearly difficult to distinguish whetherMMP-independent and -dependent TIMP activities are involved in a given process,it will be necessary to do so in order to gain a better understanding of their role incancer.

MALIGNANT PROGRESSION Malignant cancers are distinguished from in situ can-cers and benign neoplasms by their ability to cross tissue boundaries and spread toother organs of the body. To do so, cancer cells must cross multiple ECM barriersas they traverse the epithelial basement membrane and interstitial stroma, enterblood vessels or lymphatics, and extravasate to form metastatic deposits at a distantsite. Because of this requirement and because invasive behavior is associated withthe upregulation of MMPs in virtually all human cancers, MMPs are thought tofacilitate invasion and metastasis by degrading structural ECM components. How-ever, although in vitro and in vivo cell invasion assays have generally confirmedthat the levels of MMP activity influence the ability of cells to penetrate native andreconstituted basement membranes, the data supporting a positive role for MMPsin metastasis are limited. The MMP9 promoter is induced during progression fromin situ to invasive disease in transgenic mice that carry an MMP9 promoter-drivenβ-galactosidase transgene (Kupferman et al. 2000), and mice that lack MMP2show diminished lung colonization after intravenous administration of cancer cells(T Itoh et al. 1998). Moreover, PCR-based detection methods suggest that MMP9and perhaps other MMPs are required for intravasation and hematogenous spreadin vivo, since MMP inhibitors block intravasation, and only MMP9-expressingcells are able to enter the blood stream in this assay (Kim et al. 1998). Surprisinglyhowever, intravital videomicroscopy indicates that TIMP1-overexpressing cancercells exit the vasculature just as well as their parental counterparts, but yield fewerand smaller metastases owing to diminished tumor growth after they leave thebloodstream (Koop et al. 1994). Therefore, extravasation may not require MMPactivity, and although it is generally accepted that MMPs contribute to invasionand metastasis, the underlying mechanisms appear to be more complex than origi-nally thought. In other words, the MMP-dependent invasive capacity of malignantcells may rely on more than just the disruption of ECM barriers. For example,



downregulation of E-cadherin is causally involved in the malignant progressionof some cancers, and MMP-generated E-cadherin breakdown products promotetumor cell invasion (Noe et al. 2001). In addition, MMPs can trigger epithelial-to-mesenchymal phenotypic conversions, a process that is associated with moreaggressive malignant behavior and one that occurs naturally during developmentand wound repair when stationary and otherwise adherent epithelial cells becomemigratory and invasive (Lochter et al. 1997, Sternlicht et al. 1999). Thus MMPscan potentially contribute, both negatively and positively, to all stages of cancerevolution by numerous distinct pathways.

PERSPECTIVES

Not long ago, MMPs and other ECM-degrading enzymes were thought to act bysimply excavating the structural groundwork that supported cells and separateddistinct tissue compartments. Their capacity to remodel the ECM and promotecellular invasion was thought to come only from their ability to destroy underly-ing cellular foundations and disrupt the barriers that otherwise keep cells withintheir own specialized compartments. This viewpoint changed with the realizationthat extracellular matrices convey their own inherent information and that thesestructural data act as critical modifiers of cell behavior and misbehavior. More-over, the ECM harbors several other signaling molecules such as bound growthfactors and growth factor-binding proteins. Thus MMP-mediated remodeling ofthe ECM alters the informational content seen by nearby cells because remodelingcan destroy current structural inputs, unearth hidden structural information, andrelease otherwise sequestered signaling molecules. In addition, it is now clear thatalthough MMPs were initially named for their potent matrix-degrading capacity,they do more than just hydrolyze ECM molecules. They can irreversibly cleavemany cell surface and pericellular proteins. Thus they can activate or inactivatevarious signaling pathways and can increase or decrease the availability of signal-ing molecules by degrading their binding partners or generating new ones. Severalmechanisms that dock MMPs at the cell surface have evolved that not only focusMMP activity to specific regions of the pericellular microenvironment, but alsobalance MMP supply and demand. The existence of multi-functional MMP dock-ing proteins on the cell surface provides a versatile mechanism for establishingpowerful local extracellular signaling networks.

Proteolytic mechanisms regulate many normal developmental and homeostaticprocesses, whereas inappropriate proteolysis causes or exacerbates a number ofdiseases. The challenge now is to determine exactly which of the many potentialmechanisms and substrates worked out in in vitro systems actually regulate thesephysiologic and pathologic processes. Considerable headway has been made inelucidating the critical proteolytic pathways that regulate some of these processes,often with surprising results. However, far more mechanisms remain unidentifiedor unproven, thus leaving the field ripe for further progress and important newinsights.



ACKNOWLEDGMENTS

This work was supported by grants from the National Cancer Institute (CA72006,CA58207 and CA37395).

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