Because MMP substrate specificities tend to overlap, the biologic function of individual MMPs is largely dictated by their differential patterns of expression. Indeed, differences in the temporal, spatial, and inducible expression of the MMPs are often indicative of their unique roles. Accordingly, most MMPs are closely regulated at the level of transcription, with the notable exception of MMP2, which is often constitutively expressed and controlled through a unique mechanism of enzyme activation (Strongin et al. 1995
) and some degree of post-transcriptional mRNA stabilization (Overall et al. 1991
). Nevertheless, data indicate that the basal expression of MMP2, MMP14 (MT1-MMP), and TIMP2 is co-regulated, which is consistent with their cooperation during MMP2 activation and with specific similarities in their gene promoters (Lohi et al. 2000
). Otherwise, MMP gene expression is regulated by numerous stimulatory and suppressive factors that influence multiple signaling pathways (Fini et al. 1998
). For example, the expression 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 collagen acts as a ligand for discoidin domain-containing receptor-like tyrosine kinases that induce MMP1 expression when they are activated by intact collagen and become inactive 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 specifically repressed once it cleaves that substrate and is no longer needed. In addition, MMP expression is regulated by several cytokines and growth factors, including interleukins, interferons, EGF, KGF, NGF, basic FGF, VEGF, PDGF, TNF-α, TGF-β, and the e
ducer EMMPRIN (Fini et al. 1998
). Many of these stimuli induce the expression and/or activation of c-fos
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 MMP genes, other factors are also involved. In some cases, one signal may coordinately regulate some MMP genes and differentially regulate others. For example, TGF-β suppresses the transcription of the MMP1
(stromelysin-1) genes, but induces 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 expression appears to be confined to the enamel organ of developing teeth (Sternlicht et al. 2000
), and normal MMP9 expression is largely limited to osteoclasts, macrophages, trophoblast cells, hippocampal neurons, and migrating keratinocytes at the margins of healing wounds (Mohan et al. 1998
, Munaut et al. 1999
). The use of mice carrying β-galactosidase reporter transgenes under the control of various portions of the MMP9 gene promoter have helped to identify the 5′ regions responsible for most of this cell-specific expression in vivo (Mohan et al. 1998
). Cell-specific induction 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 MMP gene responds to a given input depends on the organization of its transcriptional promoter and the presence or absence of other signals, i.e., on cellular context.
-regulatory elements influence MMP gene expression depending on their proximity to one another in the gene promoter. AP-1 sites give several MMP genes the ability to be induced by phorbol esters and act synergistically with adjacent Ets-binding sites in genes such as MMP1
, but not in others such as MMP13
(Pendas et al. 1997
). This difference is presumably because the Ets and AP-1 sites are 9 nucleotides apart in the MMP1
gene promoter and 20 nucleotides apart in the MMP13
promoter 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 embryonic lethality and deficient MMP9 expression, as well as the deficient induction of MMP3 and MMP13 in Ets2-deficient fibroblasts (Yamamoto et al. 1998
). Moreover, their expression in the Ets2-deficient fibroblasts is restored by the artificial expression of Ets2. Several other putative regulatory elements have been identified within various MMP gene promoters, and many have been shown by functional analysis to regulate cell- and circumstance-specific gene expression. These include an osteoblast-specific element in the MMP13 gene promoter that responds to core-binding factor 1 (CBFA1) (Jimenez et al. 1999
); a β-catenin-regulated LEF/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 elements that 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 the MMP2 gene promoter (Bian & Sun 1997
), and wild-type p53 downregulates basal and inducible MMP1 gene expression in human fibroblasts and osteogenic sarcoma cells, whereas some mutant forms do not (Sun et al. 1999
). On the other hand, the downregulation of p53 using SV40 T-antigen suppresses the expression of MMP2, MMP3, and MMP9 in human placental trophoblast-like cells (Logan et al. 1996
). Despite numerous advances in our understanding of MMP gene regulation, the cross-talk between the many signaling pathways, nuclear factors, and gene regulatory elements that regulate MMP expression are barely understood.
Basal and inducible levels of MMP gene expression can also be influenced by genetic variations that may, in turn, influence the development or progression of several diseases. Common bi-allelic single-nucleotide polymorphisms (SNPs) that influence the rate of transcription have been identified within several MMP gene promoters (Ye 2000
). For example, an MMP1
SNP contains one or two guanidines 1607 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 immediately adjacent 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 levels of MMP1
expression in vivo and is present more often in tumor cell lines and ovarian cancer patients than in the general population (Rutter et al. 1998
, Kanamori et al. 1999
). Moreover, an unusually large proportion of metastatic melanomas with loss of heterozygosity at this site retains the high-expressing 2G allele (Noll et al. 2001
). Thus enhanced MMP1
transcription may contribute to human cancer susceptibility and progression as it does in mice (Di Colandrea et al. 2000
). Another SNP located 1306 bp upstream of the MMP2
transcription start site contains either a cytidine or thymidine, such that the less common T allele disrupts an otherwise functional Sp1-binding site and diminishes promoter activity by about 50% (Price et al. 2001
). An MMP3
SNP is located 1171 bp upstream of the transcription start site and contains a run of five or six adenosines (Ye et al. 2000
). In this case, the 6A allele 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 thymidine 1562 bp upstream of the MMP9
transcription start site (Zhang et al. 1999
). Here, nuclear protein complexes bind the C allele most readily, and the less common T allele is about 1.5-fold more potent than the C allele. Finally, an A-to-G transition exists 82 bp upstream of the MMP12
transcription start site, such that the A allele has higher AP-1 binding affinity and about 1.2-fold higher promoter activity than the less common G allele (Jormsjo et al. 2000
). Most notably, however, the MMP3
, and MMP12
SNPs have each been associated with coronary artery disease progression despite their modest influence on gene transcription (Ye 2000
Activation of Latent Metalloproteinases
Like other proteolytic enzymes, MMPs are first synthesized as inactive proenzymes or zymogens. Their latency is maintained by an unpaired cysteine sulfhydryl group near the C-terminal end of the propeptide domain. This sulfhydryl acts as a fourth ligand for the active site zinc ion, and activation requires that this cysteine-to-zinc switch be opened by normal proteolytic removal of the propeptide domain or by ectopic perturbation of the cysteine-zinc interaction (Van Wart&Birkedal-Hansen 1990
). Once displaced, the thiol group is replaced by a water molecule that can then 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 enzyme recognition motif between their propeptide and catalytic domains. This allows them to be activated by intracellular subtilisin-type serine proteinases before they reach the cell surface or are secreted (Pei & Weiss 1995
). MMP23 also has a furin-susceptible cleavage site and is a likely target of intracellular proprotein convertases, but unlike all other MMPs, it lacks the conserved cysteine that is required for enzyme latency in the first place (Gururajan et al. 1998
). All other MMPs lack a furin-susceptible insert and are thus activated outside the cell following their secretion.
The extracellular activation of most MMPs can be initiated by other already activated MMPs or by several serine proteinases that can cleave peptide bonds within MMP prodomains (Woessner & Nagase 2000
). However, MMP2 is refractory to activation by serine proteinases and is instead activated at the cell surface through a unique multistep pathway involving MT-MMPs and TIMP2 () (Strongin et al. 1995
). Indeed, MT1-MMP is a particularly efficient MMP2 activator, whereas MT4-MMP and human (but not mouse) MT2-MMP are the only MT-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 by the N-terminal domain of TIMP2, and the C-terminal domain of the bound TIMP2 acts as a receptor for the hemopexin domain of ProMMP2. Then, an adjacent, uninhibited MT-MMP cleaves and activates the tethered ProMMP2. Following the initial cleavage of ProMMP2 by MT1-MMP, a residual portion of the MMP2 propeptide is removed by another MMP2 molecule to yield a fully active, mature form of MMP2 (Deryugina et al. 2001
Figure 3 Cell surface activation of MMP2. A ProMT-MMP is activated during transport to the cell surface by an intracellular furin-like serine proteinase, at the cell surface by plasmin, or by non-proteolytic conformational changes. The activated MT-MMP is then (more ...)
It has been assumed that proteolytic removal of the MT-MMP prodomain by furin-like enzymes in the trans
-Golgi network or by plasmin at the cell surface (Okumura et al. 1997
) is required for MT-MMPs to activate MMP2. Such processing does appear to be necessary in some cell types (Strongin et al. 1995
), but these cells display both processed and full-length MT-MMPs on their surface so that the function of either individual form can not be distinguished. However, removal of the prodomain is not required for MT1-MMP to bind TIMP2 or activate MMP2 in transfected COS-1 cells that lack endogenous MT1-MMP and are unable to process latent MT-MMPs (Cao et al. 1998
). Even more surprisingly, COS-1 cells fail to bind TIMP2 or activate MMP2 if the MT1-MMP prodomain is absent or replaced by the MMP13 prodomain. However, COS-1 cells can activate MMP2 if they co-express a cDNA for prodomain-deleted MT1-MMP together with an independent cDNA for the MT1-MMP prodomain, but not if either cDNA is expressed alone (Cao et al. 2000
). Therefore, the MT1-MMP prodomain is actually required for the cell surface activation of MMP2 to proceed, but it does not have to remain covalently attached. Furthermore, co-expression of the MMP1 prodomain instead of the MT1-MMP prodomain does not rescue the ability of prodomain-deleted MT1-MMP to activate MMP2, thus indicating that the MT1-MMP prodomain has specific attributes that enable it to do so. Direct exogenous addition of the MT1-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 that is necessary for the efficient trafficking of MT1-MMP to the cell surface. Although processed MT1-MMP is eventually expressed at the cell surface, it lacks the ability on its own to bind TIMP2 or activate MMP2. MT1-MMP may also be conformationally activated through interactions with the cell membrane, and its retained propeptide domain may facilitate the binding of TIMP2.
The role of TIMP2 in MMP2 activation is its dominant in vivo function, as shown by targeted mutagenesis in mice (Z. Wang et al. 2000
). Nevertheless, while the C-terminal domain of TIMP2 participates in the cell surface docking and activation of MMP2, its N-terminal domain is an MMP inhibitor. Not surprisingly, low-to-moderate levels of TIMP2 promote the activation of MMP2, whereas higher levels inhibit its activation by saturating free MT-MMPs that are needed to remove the MMP2 prodomain (Strongin et al. 1995
). TIMP2 protein levels are reduced and MMP2 activation is enhanced in the presence of the MMP2 substrate, type IV collagen (Maquoi et al. 2000
). Furthermore, the ability of collagen to induce MMP2 activation on demand probably results from TIMP2 degradation because there are no accompanying changes in MMP2, MT1-MMP, or TIMP2 mRNA expression or in the synthesis or activation of MT1-MMP. Therefore, local accumulation of type IV collagen may trigger its own degradation by somehow lowering local TIMP2 concentrations to levels that favor MMP2 activation.
Endogenous Metalloproteinase Inhibitors
The TIMPs represent a family of at least four 20–29-kDa secreted proteins (TIMPs 1–4) that reversibly inhibit the MMPs in a 1:1 stoichiometric fashion (reviewed in Edwards 2001
, Sternlicht & Werb 1999
, Gomez et al. 1997
). They share 37–51% overall sequence identity, a conserved gene structure, and 12 similarly separated cysteine residues. These invariant cysteines form six intrachain disulfide bridges to yield a conserved six-loop, two-domain structure. Truncated “tiny” TIMPs 1 and 2 retain their inhibitory activity despite containing only the first three loops, thus indicating that portions of the N-terminal domain interact with the MMP catalytic 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 which regions of the N-terminal domain influence inhibitory function. In addition, NMR (Williamson et al. 1997
) and X-ray crystallographic studies (Gomis-Rüth et al. 1997
) have revealed which TIMP residues interact directly with the MMP3 catalytic domain and how they inhibit MMP activity. Although these studies indicate that the inhibitory activity of the TIMPs resides almost entirely in the N-terminal domain alone, both domains influence enzyme-inhibitor binding (Willenbrock & Murphy 1994
). For example, the C-terminal domain (loops 4–6) of TIMP1 binds the hemopexin domain of MMP9 more readily than it does the hemopexin domain of MMP2, whereas the C-terminal domain of TIMP2 preferentially binds the hemopexin domain of MMP2 (Murphy & Willenbrock 1995
Individual TIMPs differ in their ability to inhibit various MMPs (reviewed in Woessner & Nagase 2000
). For example, TIMP2 and TIMP3 inhibit MT1-MMP, whereas TIMP1 does not. Likewise, TIMP1 is a relatively poor inhibitor of MT3-MMP, and TIMP3 appears to be a more potent inhibitor of MMP9 than other 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 inhibit ADAMTS-1 (Tortorella et al. 1999
). In addition, the TIMPs differ in terms of their gene regulation and tissue-specific patterns of gene expression (Edwards 2001
). TIMP3 also has the unique ability to bind via its C-terminal domain to heparan sulfates proteoglycans within the ECM, thereby concentrating it to specific regions within tissues and basement membranes (Langton et al. 1998
The particular importance of TIMP3 in the eye is indicated by its increased expression in various degenerative retinal diseases, its immunolocalization to Bruch’s membrane and drusen (deposits of extracellular matrix associated with macular degeneration), and the presence of TIMP3 mutations in patients with Sorsby’s fundus dystrophy (SFD), an autosomal-dominant form of early retinal degeneration (Langton et al. 1998
). Several missense mutations that introduce an extra cysteine into the TIMP3 C-terminal domain, a nonsense mutation that truncates most of the C-terminal domain but leaves an unpaired cysteine, and a splice-site mutation that may also yield an unpaired cysteine have been found in the affected members of various SFD families (Langton et al. 2000
). Although the presence of an unpaired cysteine could result in aberrant, function-perturbing disulfide bonds, SFD mutations apparently give rise to TIMP3 molecules that dimerize and show diminished 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 activity independent of their metalloproteinase inhibitory activity. Indeed, TIMP1 was first cloned as EPA for its e
ctivity, and TIMPs 1, 2, and 3 have 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 added mutations that abolish their MMP inhibitory activity, thus indicating that these activities occur independently, perhaps as a distinct function of the C-terminal domain (Chesler et al. 1995
, Wingfield et al. 1999
). Although it is still unclear how TIMPs promote cell growth, this activity may explain several unexpected associations between TIMPs and cancer progression. However, TIMPs may also promote cell death or suppress mitogenic signals. For example, apoptosis is induced in various cell types by TIMP3, but not by TIMP1, TIMP2 or synthetic MMP inhibitors, thus suggesting that the mechanism may not rely on the inhibition of a metalloproteinase (Bond et al. 2000
). However, the introduction of a mutation that abolishes the metalloproteinase-inhibitory activity of TIMP3 also abolishes its ability to promote apoptosis, indicating that its inhibitory activity is necessary and suggesting that its target may be an ADAM or ADAMTS rather than an MMP. TIMP2, on the other hand, can suppress growth factor-responsiveness by interfering with the activation of tyrosine kinase-type growth factor receptors, and its ability to block mitogenic signaling is independent of its MMP-inhibitory activity (Hoegy et al. 2001
). Nevertheless, no TIMP receptors have yet been identified, suggesting that TIMPs may act as decoys for various signaling molecules. Moreover, these growth-promoting and growth-suppressive activities may not be entirely MMP-independent because the mutant TIMPs retain their ability to interact with MMPs via secondary non-inhibitory sites, such as those of their C-terminal domains (Howard & Banda 1991
). Therefore, these growth-altering activities may still reflect the ability of TIMPs to indirectly modify MMP activity.
TIMPs are not the only endogenous MMP inhibitors. Indeed, α2-macroglobulin is a major endogenous inhibitor of the MMPs (Sottrup-Jensen & Birkedal-Hansen 1989
), and its importance may have been overlooked due to the recent emphasis placed on the TIMPs. Because α2-macroglobulin is an abundant plasma protein, it represents the major inhibitor of MMPs in tissue fluids, whereas TIMPs may act locally. Moreover, because α2-macroglobulin/MMP complexes are removed by scavenger receptor-mediated endocytosis, α2-macroglobulin plays an important role in the irreversible clearance of MMPs, whereas TIMPs inhibit MMPs in a reversible manner.
Another, recently recognized class of MMP inhibitors, protein subdomains, have structural similarity to the TIMPs. For example, proteolytic processing of the procollagen C-terminal proteinase enhancer protein PCPE releases a C-terminal fragment with MMP inhibitory activity and structural similarity to the N-terminal domain of the TIMPs (Mott et al. 2000
). A primary sequence alignment search also uncovered similarities between the TIMPs and the noncollagenous NC1 domain of type IV collagen (Netzer et al. 1998
). Moreover, functional analyses indicate that the NC1 domain has MMP inhibitory activity (Netzer et al. 1998
) and can inhibit angiogenesis and tumor growth (Petitclerc et al. 2000
). Nevertheless, the physiologic targets of these inhibitory fragments remain uncertain. Although their activity against 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 or near the cell membrane, and many cellular signals are created or canceled via pericellular proteolysis (Werb 1997
). Thus because it is irreversible, the processing of pericellular proteins by proteolysis is an ideal means of regulating extracellular signal transduction. Although pericellular proteolysis may sometimes reflect the exclusive expression of a critical substrate at or near the cell surface, there are specific mechanisms that confine or concentrate proteinases in the immediate pericellular microenvironment. These mechanisms for localizing MMPs to the cell surface and to specific cell surface subdomains include the expression of membrane-bound MT-MMPs; the binding of MMPs to cell surface receptors; the presence of cell surface receptors for MMP-activating enzymes such as uPA, plasmin(ogen), thrombin, and elastase; and the concentration of MMPs on pericellular ECM molecules. These localization mechanisms often enhance MMP activation, limit the access of MMP inhibitors, concentrate MMPs within the vicinity of their targets, and limit the extent of proteolysis to discrete pericellular regions.
Transmembrane and GPI-linked MT-MMPs are the most obvious mediators of proteolytic activity at the cell surface. Removal of the transmembrane domains of MT1, MT2, and MT3-MMP abolishes their ability to promote cellular invasion (Hotary et al. 2000
). Moreover, MT-MMPs can concentrate within specific cell surface domains such as cellular protrusions known as invadopodia (or invasive pseudopodia), where active ECM degradation takes place. Localization studies using chimeric constructs indicate that the cytoplasmic and transmembrane domains of MT1-MMP are required for it to cluster on invadopodia, and functional studies indicate that invadopodial recruitment of MT1-MMP is necessary for cellular invasion to take place (Nakahara et al. 1997
). Because GPI-anchored proteins tend to partition into lipid rafts and caveolae where considerable signaling activity takes place (Brown & London 1998
), the GPI-linked MT-MMPs should also be enriched in such regions.
Another means of localizing MMPs to the cell surface is via cell surface docking receptors. For example, activated MMP2 can bind to integrin αvβ3 on the surface of angiogenic endothelial cells and invasive cancer cells (Brooks et al. 1996
). Because the C-terminal domain of MMP2 is required for the formation of αvβ3/MMP2 complexes in vitro, the catalytic domain probably remains exposed so that it can still carry out proteolysis. Interestingly, MT1-MMP generates only an MMP2 activation intermediate, and another already activated MMP2 is required to remove the residual portion of the MMP2 propeptide and achieve full MMP2 activation (Deryugina et al. 2001
). Thus cell surface MMP2 receptors may cooperate with MT1-MMP to facilitate MMP2 maturation, and data suggest that integrin αvβ3 promotes such maturation by providing a platform for autocatalytic interactions between fully and partially activated MMP2 (Deryugina et al. 2001
). Moreover, colocalization data suggest that integrin αvβ3 may also cooperate with MT1-MMP in the clustering of active MMP2 on invadopodia.
MMP1 can also interact with a cell surface integrin. In this case, active and inactive MMP1 binds to the I domain of α2 integrin, whereas MMPs 3 and 13 do not (Stricker et al. 2000
). Optimal binding of chimeric constructs requires the MMP1 hinge and hemopexin domains, but not its propeptide or catalytic domains, so that the latter domain may remain available to interact with its targets. Binding of α2β1 integrin to type I collagen induces MMP1 expression in keratinocytes, and MMP1 is necessary for α2β1-dependent migration of keratinocytes over type I collagen (Pilcher et al. 1999
). Thus α2β1 integrin can act both as an MMP1 inducer and as an MMP1 receptor, and the binding of MMP1 to α2β1 integrin may play an important role in the migration of keratinocytes that contact interstitial collagens during epidermal wound repair.
Another molecule that can both induce MMP1 expression and then localize it to the cell surface is CD147/EMMPRIN (Guo et al. 2000
). EMMPRIN is enriched on the surface of cancer cells and induces adjacent fibroblasts to produce MMP1, which then apparently binds to the same cell surface EMMPRIN molecules that elicited its expression in the first place. Therefore, tumor-derived EMMPRIN may promote tumor cell invasion by both inducing fibroblasts to produce MMP1 and then concentrating it on the surface of the invasive cells. MMPs can also be bound to the cell surface by their own substrates. Latent ProMMP9, for example, binds with high affinity to type IV collagen α2 chains on the surface of several cell types, thus concentrating it at the cell-ECM interface in anticipation of any future need and in direct contact with its target (Olson et al. 1998
, Toth et al. 1999
). This interface is where MMP9 is found in nascent skin cancers (Coussens et al. 2000
). Activated MMP9 binds to the cell surface hyaluronan receptor CD44 (Bourguignon et al. 1998
). Moreover, the localization of MMP9 to the cell surface by CD44 appears to promote tumor cell invasion and angiogenesis and may mediate the activation of latent TGF-β by MMP9 (Yu & Stamenkovic 2000
). Recent data also indicate that MMP7 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, as well as TIMP3.
Although many of the above docking mechanisms have not been definitively proven, they suggest the existence of localized pericellular feed-back networks that coordinate the need for a given MMP with its appropriate expression, activation, and physical placement. The specific cell surface molecules that mediate these interactions have not been definitively elucidated, in part, because such molecules are frequently enriched in specialized signaling domains such as caveolae and rafts. Furthermore, by combining many of the functions of a given feed-back loop within a few components (for instance by binding an MMP to its own inducer, its own substrate or a molecule that normally ligates its substrate), these signaling networks may be particularly compact and efficient. By the same token, the multifunctional nature of these interactions will undoubtedly make them all the more difficult to sort out in functional experiments.
MMP Catabolism and Clearance
An obvious means of regulating MMPs is via their own proteolytic inactivation and physical clearance. Although considerable progress has been made in understanding the progressive proteolytic processing of MMP propeptides, relatively little is known about the further autoproteolysis of active MMPs. Nevertheless, it is clear that some cleavages inactivate MMPs, whereas others, such as those that specifically remove the hemopexin domain, can generate truncated enzymes that lose their ability to cleave some substrates but retain their ability to cleave others (reviewed in Woessner & Nagase 2000
). Such processing can also diminish their affinity for and ability to be inhibited by TIMPs, as occurs with C-terminally truncated MMP2 (Y. Itoh et al. 1998
). Removal of the hemopexin-like domain also cancels 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 after they reach the cell surface (Imai et al. 1996
). Thus factors that influence MMP degradation can alter the steady-state concentrations of MMPs, their substrate specificities, their localization, and their ability to be activated or inhibited.
Another means of regulating extracellular MMP levels is by the direct clearance of intact enzymes. Most MMPs cleave the bait region of α2-macroglobulin, thereby initiating a conformational change in the large tetrameric macroglobulin that irreversibly traps the enzyme (Sottrup-Jensen & Birkedal-Hansen 1989
). Although the catalytic activity of the MMP is not inhibited per se, its physical entrapment keeps the enzyme from interacting with natural substrates, and the α2-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 abnormalities, and their fibroblasts have an adhesion defect that is the result of increased MMP2 levels (Yang et al. 2000
). The increased MMP2 levels occur because TS2 normally binds both latent and active MMP2 and because TS2 is normally endocytosed by the low-density lipoprotein receptor-related protein LRP and probably carries any bound MMP2 with it (Yang et al. 2001
). The cellular internalization of TS2/MMP2 complexes by the LRP scavenger receptor may therefore play an important role in regulating MMP2 levels outside fibroblasts and other cells. Evidence also indicates that MMP13 is rapidly cleared after it binds to an MMP13-specific 170-kDa high-affinity receptor present on various cell types (Barmina et al. 1999
). The binding requires calcium, and the subsequent internalization and degradation of MMP13 requires LRP because LRP-null cells bind MMP13 but fail to internalize it. Moreover, the internalization of both MMP13 and TS2/MMP2 complexes is inhibited by the 39-kDa receptor-associated protein RAP, which binds and inhibits LRP. Thus MMPs are tightly regulated by several variously characterized mechanisms during virtually every aspect of their life-span, from their induction to their ultimate destruction.
Although very few bona fide MMP substrates have been definitively identified in vivo, numerous candidates have been tested and identified in vitro (). For interstitial collagens, aggrecan and link protein, the cleavage sites identified in breakdown products isolated from tissue extracts match those that have been established in vitro; however, multiple MMPs can generate these same cleavages. In a limited number of cases, the MMP substrates and responsible MMPs have been identified genetically. The restricted expression pattern of an enzyme may also help to predict its proteolytic targets. For example, MMP20/enamelysin is principally expressed in the enamel organ of developing teeth, where ameloblast cells secrete amelogenin, the major protein that forms the organic matrix of tooth enamel. Amelogenin is continuously degraded and replenished as enamel formation proceeds, and it is hydrolyzed by recombinant MMP20 in vitro (Li et al. 2001
). Moreover, an inherited missense mutation in the amelogenin gene is associated with X-linked amelogenesis imperfecta and alters an amelogenin cleavage site so that it is poorly hydrolyzed by MMP20 (Li et al. 2001
). Therefore, amelogenin is almost certainly a natural substrate of MMP20, and inherited mutations that render it resistant to MMP20 cleavage may lead to defective tooth enamel formation. Nevertheless, such a concordance between the isolated temporal and spatial expression of an MMP and its substrate, together with the existence of disease-linked mutations that alter the proteolytic susceptibility of the substrate, is the exception. In virtually all other cases, the identification of key in vivo substrates and the responsible enzymes will require rigorous experimental approaches.
Common matrix metalloproteinase substratesa
Screening methods are now emerging as another means of identifying potential physiologic MMP substrates. For example, monocyte chemoattractant protein-3 (MCP-3) was identified as an MMP2 substrate after using the MMP2 hemopexin domain 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 least one exosite that is required for the binding of MCP-3. A phage-displayed hexapeptide library has also been used to map the preferred substrate specificity of human MMP13 (Deng et al. 2000
). Subsequent database searches for proteins with matching peptide sequences confirmed the ability of MMP13 to degrade aggrecan and collagens II and IV, but also identified a sequence within the prodomain of TGF-β3 as a potential target. This is particularly intriguing because cell surface-associated MMP2 and MMP9 can activate TGF-β and because TGF-β activation is blocked by metalloproteinase inhibitors (Yu & Stamenkovic 2000
). Moreover, MMP13 can be activated at the cell surface by MT1-MMP, thus potentially placing it in the right location to activate TGF-β. As is so often the case, however, a determination of whether such processing actually occurs in vivo awaits further testing and will undoubtedly be difficult to prove. Therefore, although the list of potential substrates is long, the list of known in vivo substrates remains relatively short.