Progress in Histochemistry and Cytochemistry 47 (2012) 27–58
PROGRESS IN HISTOCHEMISTRY
AND CYTOCHEMISTRY
www.elsevier.de/proghi
Available online at www.sciencedirect.com
Review
Nuclear localization of Matrix metalloproteinases
Ferdinando Mannello ∗, Virginia MeddaDepartment of Biomolecular Sciences, Section of Clinical Biochemistry, Unit of Cell Biology,University “Carlo Bo” of Urbino, Via O. Ubaldini 7, 61029 Urbino (PU), Italy
Abstract
Matrix metalloproteinases (MMPs) were originally identified as matrixin proteases that act in theextracellular matrix. Recent works have uncovered nontraditional roles for MMPs in the extracellularspace as well as in the cytosol and nucleus. There is strong evidence that subspecialized and com-partmentalized matrixins participate in many physiological and pathological cellular processes, inwhich they can act as both degradative and regulatory proteases. In this review, we discuss the tran-scriptional and translational control of matrixin expression, their regulation of intracellular sorting,and the structural basis of activation and inhibition. In particular, we highlight the emerging roles ofvarious matrixin forms in diseases. The activity of matrix metalloproteinases is regulated at severallevels, including enzyme activation, inhibition, complex formation and compartmentalization. MostMMPs are secreted and have their function in the extracellular environment. MMPs are also foundinside cells, both in the nucleus, cytosol and organelles. The role of intracellular located MMPs is stillpoorly understood, although recent studies have unraveled some of their functions. The localization,activation and activity of MMPs are regulated by their interactions with other proteins, proteoglycancore proteins and / or their glycosaminoglycan chains, as well as other molecules. Complexes formedbetween MMPs and various molecules may also include interactions with noncatalytic sites. Suchexosites are regions involved in substrate processing, localized outside the active site, and are potentialbinding sites of specific MMP inhibitors. Knowledge about regulation of MMP activity is essential
∗ Corresponding author. Dipartimento di Scienze Biomolecolari, Sezione di Biochimica Clinica, Unità di Biolo-gia Cellulare, Università Studi “Carlo Bo” di Urbino, Via O. Ubaldini 7, 61029 Urbino (PU), Italia.Tel.: +39 0722 351479; fax: +39 0722 322370.
“Don’t be trapped by dogma, which is living with the results of other people’s thinking.Don’t let the noise of other’s opinions drown out your own inner and have the courageto follow your intuition”
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1. The Metzincin superfamily
Matrix metalloproteinases belong to a family of zinc and calcium dependentendopeptidases called Metzincin. The endopeptidases belong to the wide Metzincingroup, in turn constitutes one of several metalloendopeptidase families; according totheir structural characteristics all the metzincins are mainly subdivided into astacins,
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ADAMs/adamalysins/reprolysins, serralysins, matrix metalloproteinases/matrixins, sna-palysins, leishmanolysins, and pappalysins (Sterchi, 2008). All the metzincins are mostlymultidomain proteins with approximately 130-260-residue globular catalytic domainsshowing a common core architecture characterized by a long zinc-binding consensus motif,HEXXHXXGXX(H/D), and a methionine-containing Met-turn. Metzincins have been char-acterized to participate in unspecific protein degradation such as digestion of intake proteinsand tissue development, maintenance, and remodeling, but they are also involved in highlyspecific cleavage events to activate or inactivate themselves or other (pro)enzymes andbioactive peptides (Gomis-Ruth, 2009).
In particular, as our knowledge of the MMP family continues to grow, we are learningthat their activities are not limited to matrix degradation (McCawley and Matrisian, 2001;Morrison et al., 2009). MMPs seem to touch almost every aspect of mammalian biologyand, through more than 1,500 new publications each year, certain exciting discoveriessolidified the field, solving previous puzzles and pointing the research in new directions(starting from biochemical studies, discussing the flurry of activity that involved the generegulation through molecular biology approaches, and finally focusing the interest to MMPsas therapeutic targets in several diseases).
2. Definition and variety of Matrix metalloproteinases or Matrixins
The proteases play essential roles in a wide variety of biological processes and are alsoassociated with multiple physiological and pathological processes (Lopez-Otin and Bond,2008). The maintenance of a healthy organism largely relies upon controlled biosynthe-sis, maturation, function, and terminal breakdown of proteins, and proteolytic enzymescontribute to these processes by irreversibly cleaving peptide bonds. This can result indestruction of the substrate protein, its maturation, or modulation of the biological activitiesof the cleavage products (Mannello et al., 2005a; Mannello et al., 2005c).
The first “collagenolytic activity” was described about 50 years ago during the metamor-phosis in tadpole tails (Gross and Nagai, 1965), as an enzymatic activity able to degradeinterstitial collagen. The discovery of collagenase opened a new field of unexpected biomed-ical research and the way to study what has now become the matrix metalloproteinase family.Today, to accomplish the multitude of selective and well-controlled proteolytic events thatkeep us healthy, it has been identified the human genome encoding for more than 600 pro-teases and more than 200 endogenous protease inhibitors (Ugalde et al., 2010). A carefulanalysis of the human degradome classified proteinases mainly into five catalytic classes(Aspartyl, Cysteine, Serine, Threonine and Metallo- proteases) and about 70 families of pro-teinases with peculiar characteristics (for details, see the Mammalian Degradome Database;http://degradome.uniovi.es/dindex.html) (Quesada et al., 2009).
Among these proteinases, the matrix degrading metalloenzymes are the most commonenzymes, mainly named Matrixins or Matrix MetalloProteinases (MMP) (Mannello et al.,2005a). They form a multigenic family (encoded by 24 distinct genes in human)(Yan andBoyd, 2007) of proteolytic calcium/zinc-dependent enzymes (expressed as 26 distinct pro-teins), functioning at neutral pH, secreted in their latent form (proenzymes or inactive
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zymogens or pro-MMPs) and requiring proteolytic activation (Mannello et al., 2005b;Rosenblum et al., 2007a).
MMPs possess overlapping, but distinct substrate spectra (Quesada et al., 2009); theymay destroy almost the extracellular matrix (ECM) components to permit normal remod-eling and contribute to pathological tissue destruction and tumor cell invasion (McCawleyand Matrisian, 2001; Kessenbrock et al., 2010). The identification of specific matrix andnon-matrix substrates for MMPs and the elucidation of the biological consequence ofcleavage demonstrates that MMPs should be viewed more as pruning shears, playingsophisticated roles in modulating normal cellular behavior, cell-cell communication andtumor progression (Tonti et al., 2009;Roy et al., 2009; Hadler-Olsen et al., 2011). MMPsactivity is closely regulated by their main endogenous inhibitors, the tissue inhibitors ofMMPs (TIMPs) (Mannello and Gazzanelli, 2001; Troeberg and Nagase, 2007; Clark et al.,2008).
Most of MMPs occupy a central role in normal physiological conditions (such as stemcell differentiation, proliferation, cell motility, remodeling, wound healing, angiogenesis,apoptosis)(Mannello et al., 2005a; Malemud, 2006; Mannello et al., 2006;) controllingalso key reproductive events (such as ovulation, embryo implantation, uterine, breast, andprostate involution, menstruation, and endometrial proliferation) (Hadler-Olsen et al., 2011).On the other hand, imbalance between MMP and TIMP expression has been involved invarious medical conditions (such as tumor invasion, rheumatoid arthritis, atherosclerosis,aneurysms, nephritis, tissue ulcers, fibrosis and endometriosis), through different mecha-nisms (mainly linked to tissue destruction, fibrosis, and weakening of matrix) (Malemud,2006; Kessenbrock et al., 2010; Gialeli et al., 2011).
Although the biological roles of the MMPs have been traditionally associated with thedegradation and turnover of most of the components of the ECM, this functional misconcep-tion has been used for years to explain the involvement of the MMP family in developmentalprocesses, cell homeostasis and disease, and led to clinical trials of MMP inhibitors for thetreatment of cancer that failed to meet their endpoints and cast a shadow on MMPs as drug-gable targets. Actually, accumulated evidence from a great variety of MMP degradomicsstudies, is changing the dogma about MMP functions, recognizing the ability of process-ing of a large variety of processes (Rodriguez et al., 2010), with both beneficial/protective(Lopez-Otin et al., 2009) and deleterious effects (Roy et al., 2009).
2.1. Structural diversity
This superfamily shares a conserved structural topology (Fig. 1), which consists ofa catalytic domain containing three histidines that constitute the zinc binding site and a“methionin-turn” motif that lies beneath the active site zinc ion (Gomis-Ruth, 2009). Theion-binding motif reads HEBXHXHBGBXHZ, where histidine (H), glutamic acid (E) andglycine (G) are invariant, B is a bulky hydrophobic residue, X is a variable residue and Z isa family-specific amino acid (serine in MMPs). All MMPs have an N-terminal hydropho-bic signal sequence (predomain) which leads their synthesis to the endoplasmic reticulumand their secretion into the extracellular space; this predomain is followed by a 77-87amino acid-long prodomain that constitutes the N-terminus of the secreted enzyme and
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Fig. 1. Domain structure of secreted and membrane-bound Matrix metalloproteinases. MMPs arecomprised of different subdomains and have the “minimal domain” in common, which contains threeprincipal regions: an aminoterminal signal sequence (Pre) to be cleaved by the signal peptidase duringentry into the endoplasmic reticulum, a pro-domain (Pro) containing a thiol-group (-SH) and a furincleavage site, and the catalytic domain with a zinc-binding site (Zn2+). Interaction of the -SH groupof the pro-domain with the zinc ion of the catalytic domain keeps the enzyme as an inactive zymogen.Activation of the zymogen is often mediated by intracellular furin-like proteinases that target thefurin recognition motif (Fu) between the pro-domain and the catalytic domain. In addition to theminimal domain, most MMPs possess a hemopexin-like region, a domain composed of four repeatsthat resemble hemopexin and contain a disulfide bond (S-S) between the first and the last subdomain,which is linked to the catalytic domain via a flexible hinge region. Besides their differential domainstructure, MMPs can be principally divided into secreted (MMP-1, -2, -3, -7, -8, -9, -10, -11, -12, -13,-19, -20, -21, -22, -27, -28) and membrane anchored proteinases (MMP-14, -15, -16, -17, -23, -24,-25), the latter of which use either a transmembrane domain (TM) with a cytoplasmic domain (Cy)attached to it, a glycosylphosphatidylinositol (GPI) anchor, or an amino-terminal signal anchor (SA),which is only the case for MMP-23, as it is anchored in the plasma membrane. MMP-23 also containsthe unique cysteine array (CA) and an immunoglobulin (Ig)-like domain. The gelatinases MMP-2 and-9 show gelatin-binding repeats that resemble the collagen-binding type II motif of fibronectin (FN).
maintains it in its latent form until its removal or disruption. The prodomain keepsthe enzyme inactive through a mechanism identified as “cysteine switch” where theunpaired cysteine in the highly conserved “Pro-Arg-Cys-Gly-X-Pro-Asp” sequence formsa bridge with the catalytic zinc, thus preventing enzymatic activity. The enzyme acquires
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total proteolytic capacity when the prodomain becomes chemically removed by cleav-age (Rosenblum et al., 2007a). The active site is of great importance: it specificallybinds to selective substrates by means of its active site cleft, through specificity sub-site pockets that bind amino acids adjacent to the scissile peptide bond, and throughsecondary substrate binding exosites located outside the active site (Overall, 2001;Overall, 2002). These domains represent the minimal structure of MMPs found inMMP-7 (matrilysin) and MMP-26 (endometase/matrylisin-2) which lack any otherdomain. All the other MMPs have a hinge region varying in length and compositionwhich also influences substrate specificity, and a four-blade structure representing thehemopexin/vitronectin-like domain (Lauer-Fields et al., 2009). Two metalloproteinases,MMP-2 and MMP-9 (also named Gelatinase A and B), are further characterized by thepresence of three head-to-tail cystein-rich repeats within the catalytic domain (Xi et al.,2010). This structure resembles the collagen-binding type II repeats of fibronectin andis necessary for the binding and cleavaging activities of these MMPs. Not all MMPsare secreted enzymes; membrane-type (MT) MMPs have been identified to contain asingle-pass transmembrane domain and a short cytoplasmic C-terminal tail or to beanchored to the cell membrane by a glycosylphosphatidylinositol anchor (Minond et al.,2007).
2.2. Functional complexity
The MMPs can be divided into 5 main groups according to their domain composition andtheir ability to degrade individual component of ECM (Quesada et al., 2009): 1) Matrilysins(MMP-7 and MMP-26) are MMPs that lack the hemopexin C domain; 2) Collagenases(MMP-1, MMP-8 and MMP-13) are composed of a catalytic domain and hemopexin -like domain; 3) Stromelysins (MMP-3, MMP-10 and MMP-11) possess the same domainscomposition of collagenase class; 4) Gelatinases (MMP-2 and MMP-9) have within thecatalytic domain a compact collagen binding domain called fibronectin-like domain; 5)Membrane Type MMPs (MT-MMP) (MMP-14, MMP-15, MMP-16, MMP-17, MMP-24and MMP-25) are inserted in the plasma membrane by a transmembrane segment or aglycosylphosphatidylinositol (GPI). Many of the MMPs are specifically regulated at thelevel of gene expression, but their production as inactive proenzymes is another importantlevel of functional regulation (Clark et al., 2008).
All matrixins are synthesized as pre-pro-enzymes and secreted as inactive pro-MMPs inmost cases. The pro-peptide domain (about 80 amino acids) has a highly conserved uniquePRCG(V/N)PD sequence. The Cys within this sequence coordinates the catalytic zinc tomaintain the latency of pro-MMPs (Rosenblum et al., 2007a).
Structure of the pro-domain is known for MMP-2, MMP-3 and MMP-9, and it consistsof its three helices and connecting loops. The first loop between helix-1 and helix-2 isprotease-sensitive. An extended peptide region after helix lies in the substrate binding cleftof the catalytic domain.
The catalytic zinc is coordinated by the three histidine residues; in the absence of asubstrate or inhibitor, a water molecule forms an additional fourth ligand to zinc and isentrapped between the catalytic glutamic acid of the HEXXHXXGXXH motif and the metal.This sequence is missing in stromelysin (MMP-11) and MT1-MMP (MMP-14) which were
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shown to be activated intracellularly by furin, while MMP-23 has a pro-protein processingsequence RX(K/R)R at the C-terminal end of the propeptide (Murphy and Nagase, 2008).
In all MMPs the carbonyl group of scissile peptide bond points towards the catalyticzinc and therefore can be polarized. The peptide hydrolysis is assisted also by the carboxylgroup of the catalytic glutamate, in fact the catalytic water molecule intercalates betweenthis carbonyl group and the glutamate, thereby facilitating the nucleophilic attack of thewater molecule on the carboxyl carbon of the peptide scissile bond, and giving rise to apentacoordinate transition state (Rosenblum et al., 2007b).
The tetrahedral intermediate is presumably stabilized by both the zinc and a carbonylgroup of the first alanine residue of the edge strand. Simultaneously, one water proton couldbe transferred to the amino group via the glutamic carboxylate (acting as a proton shuttle);after the cleavage of the peptide bond and the transfer of a second proton this amino groupcould leave the enzyme-substrate complex (together with the N-terminal substrate fragment)In addition, proton transfer to the peptidic nitrogen possibly facilitated by the catalyticglutamate, completes the catalytic reaction, enabling the release of cleavage products andof the free enzyme (Bode, 2003).
2.3. Catalysis mechanism and substrate specificity
Although all MMP structures reported to date possess similar core domains, importantdifferences in the side-chains and surface loop alter the size, shape and chemical compositionof the specificity subsites (Pochetti et al., 2009). The active site is a cavity spanning theentire enzyme and it has been shown that a substrate containing at least six amino acids(three on each side of the scissile bond) is required for the proteolytic activity of MMPs:these six amino acids occupy the subsites S3-S3. Closer examination of the structures ofcatalytic domains revealed that a conserved aspartic acid is found in the vicinity of themethionine turn, the side chain of which is buried inside the core of the domain (Bertiniet al., 2005; Gomis-Ruth, 2009). The substrate binds into the catalytic site cleft from the leftto the right with respect to its N- and C-termini, and the carboxyl group of the peptide bondcoordinates with the active site zinc. However, in the X-ray structure of MMP-1 an argininedefines the bottom of the pocket, whereas in MMP-7 a tyrosine fulfills this purpose, leadingto a restriction of the pocket (Jackson et al., 2010).
The primed sites of active site have been described in detail, whereas the unprimed siteshave not been examined deeply. However, the importance of other parts of binding site,especially the unprimed site, for the design of more potent and selective inhibitors has beendemonstrated as well (Zhang et al., 2008).
The C-terminal domain of the MMPs, which is present in all members except thematrilysins, shows strong sequence similarity to members of the hemopexin family, includ-ing hemopexin, integrins and vitronectin (Elkins et al., 2002). The common functionalproperty possessed by these hemopexin-containing proteins seems to be the participationin quite specific protein-protein and protein-ligand interaction (which differ among variousMMPs). When present, the hemopexin-like domain also influences TIMP binding, the bind-ing of certain substrates, membrane activation and some proteolytic activities (Lauer-Fieldset al., 2009).
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The N- and C-domains of the MMPs seem to be packed as separate entities in the crystalwith connecting flexible linker peptide or hinge which is rather loosely arranged. The hingeis composed of 2-72 residues, being short (16 residues) in the collagenases where displaysa motif resembling those observed in each chain of triple helical collagen (Minond et al.,2004). The residues of the hinge region also influence substrate specificity; the capacityto cleave triple helical collagen obviously depends on the correct interplay between thecatalytic and the hemopexin-like domain (Baronas-Lowell et al., 2007).
The gelatinases have an additional domain consisting of three tandem copies of 58 aminoacid residues forming the fibronectin type II like module, which are inserted between thefifth strand and the catalytic helix (Bode, 2003). The structure of each fibronectin-likedomain consists of two antiparallel sheets, connected with a short helix and stabilized bytwo disulfide bonds. A portion of this domain is called collagen binding domain (CBD),since it appears to be important for binding gelatin and collagen (Lauer-Fields et al., 2002;Minond et al., 2004; Lauer-Fields et al., 2008).
2.4. Activation and inhibition mechanisms
Beyond their classical connective-tissue-remodeling functions, MMPs are known toprecisely regulate the function of bioactive molecules by proteolytic processing. Underphysiological conditions, MMP activity is controlled at least at three levels: 1) transcrip-tion, 2) proteolytic activation of zymogen form, and 3) inhibition of the active enzyme byendogenous inhibitors. Most MMPs are expressed in adult tissues at low levels or not at allin resting condition. However, several cytokines and growth factors as well as physical cel-lular interactions provide stimuli that can rapidly induce MMP expression (Vessillier et al.,2004; Bahar-Shany et al., 2010). Thus, cell use various strategies to regulate extracellularproteases; a greater understanding of regulatory mechanism that controls MMP activityprovides several new avenues for therapeutic intervention, since many drugs are designedto target these key regulatory points (Fingleton, 2007; Roy et al., 2009; Gialeli et al., 2011).
Most proMMPs are secreted from cells and activated extracellularly. MMPs can beactivated by proteinases or in vitro by chemical agents such as thiol-modifying agents (4-aminophenylmercuric acetate, HgCl2 and N-ethylmaleimide), oxidized glutathione, SDS,chaotropic agents and reactive oxygen species (Mannello et al., 2005b; Mannello, 2006).Low pH and slight heat treatment can also lead to activation. These agents most likelywork through the disturbance of cysteine-zinc interaction of the cysteine switch. Studiesof proMMP activation with a mercurial compound have indicated that the initial cleavageoccurs within the propeptide and this reaction is intramolecular rather than intermolecular(Rosenblum et al., 2007a). The subsequent removal of the rest of the propeptide is dueto intermolecular reaction of generated intermediates. In vivo, it has been shown that NO(nitric oxide) activates proMMP-9 by interacting with the thiol group of cysteine switchand forming an S-nitrosylated derivative (Okamoto et al., 2001; Pei et al., 2006).
Proteolytic activation of MMPs is a stepwise in many cases. The initial proteolytic attackoccurs at an exposed loop region between the first and the second helics of propeptide. Thecleavage specificity of the bait region is dictated by the sequence found in each MMP. Oncea part of the propeptide is removed, this probably destabilizes the rest of the propeptide,
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including the cysteine switch-zinc interaction, which allows the intermolecular processingby partially activated MMP intermediates or other active MMPs. Thus, the final step in theactivation is conducted by a MMP (Clark et al., 2008). The stepwise activation system mayhave evolved to accommodate finer regulatory mechanisms to control destructive enzymes,in as much as TIMPs may interfere with activation by interacting with the intermediateMMP before it is fully activated (Mannello and Gazzanelli, 2001; Troeberg and Nagase,2007).
The extracellular activation of most MMPs can be initiated by other already acti-vated MMPs or by several serine proteinases that can cleave peptide bonds within MMPprodomains. However, proMMP-2 is refractory to action of serine proteinases and it isinstead activated at the cell surface through a unique multistep pathway involving MT-MMPs (Iki et al., 1999; Itoh et al., 2008). According to the present knowledge, a cell surfaceMT1-MMP binds the N-terminal domain of TIMP-2 being inhibited; the C-terminal domainof the bound TIMP-2 acts as receptor for the hemopexin-like domain of proMMP-2. Then,an adjacent, MT1-MMP cleaves and activates tethered proMMP-2. Following the initialcleavage of proMMP-2 by MT1-MMP, a residual portion of MMP-2 propeptide is removedby another MMP-2 molecule to yield a fully active, mature form of MMP-2 (Worley et al.,2003; Itoh et al., 2008).
Activation by protease is mediated by cleavage of the bait region; this partly activates theMMP. Full activations achieved by completed removal of the propeptide by intermolecularprocessing. Chemical activation relies on modification of the cysteine switch sulphydryl,resulting in partial activation of the MMP and intramolecular cleavage of the propeptide.Full activity results from the removal of the remainder of the propeptide by intermolecularprocessing (Mannello et al., 2006).
MMP activity is tightly controlled by several endogenous inhibitors (Fig. 2). In tissuefluids, the principal MMP inhibitors is �2-macroglobulin, a large serum protein, whichbinds MMPs and creates a complex that is itself bound irreversibly (Arbelaez et al., 1997;Baker et al., 2002). However, the most thoroughly studied MMP inhibitors, are TIMPs:four human TIMPs have been characterized thus far; they are small molecules of 21-28kDa which bind MMPs in a 1:1 stoichiometric ratio and reversibly block MMP activ-ity. TIMPs, which are anchored to the extracellular matrix or secreted extracellularly,differ in their expression pattern (Mannello and Gazzanelli, 2001; Troeberg and Nagase,2007).
Among other molecules capable of regulating MMP proteolytic activity we underlinethrombospondin-2 and RECK (reversion-inducing cysteine-rich protein with Kazal domainmotifs), a GPI-anchored glycoprotein that suppresses angiogenic sprouting (Rhee andCoussens, 2002; Agah et al., 2005; van der Jagt et al., 2006; Chang et al., 2008). Otherkey regulators of extracellular matrix are TFPI2 (tissue-factor pathway-inhibitor-2), a ser-ine protease inhibitor that can act as a MMP inhibitor and PCPE (procollagen C-terminalproteinase enhancer), a molecule which possesses a significant inhibitory activity; otherlatent MMP inhibitors might be hidden in the NC1 domains of type IV collagen or in thelaminin-binding domain of agrin, which are structurally similar to TIMPs (Morrison et al.,2009; Rodriguez et al., 2010). However, it may be interesting to outline that endogenousinhibitors may also act as activators. This unexpected function has been quite well describedfor TIMP2 in the case of MMP2 activation (Overall and Butler, 2007).
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Fig. 2. Main characteristics of MMP inhibitors. MMP expression in the pericellular space is strictlycontrolled at the levels of transcription, secretion, activation, and inhibition of the activated enzyme,by nonspecific inhibitors and by stoichiometric binding of specific, locally produced tissue inhibitorsof metalloproteinases (TIMPs: TIMP–1, TIMP–2, TIMP–3, and TIMP–4). Several other proteins havebeen reported to inhibit MMPs (like tissue factor pathway inhibitor–2, C–terminal fragment of theprocollagen, C-terminal proteinases enhancer protein, membrane-bound �-amyloid precursor protein,RECK (reversion-inducing cysteine-rich protein with kazal motifs), chlorotoxin, etc.). Proteins suchas plasma �-macroglobulins are general endopeptidase inhibitors that inhibit most proteinases bytrapping them within the macroglobulin after proteolysis of the bait region of the inhibitor. Finally,several natural biocompounds (like squalamine, genistein, curcumin, catechin and resveratrol) havean inhibitory effect on MMP expression and activity, through mechanism chelating calcium and zincions, blocking the catalytic site or degrading the cysteine switch.
Several classes of synthetic compounds acting as MMP inhibitors have been discovered;the most common is containing a Zn-liganding hydroxamic acid, carboxylic acid or thiolgroup attached to a small peptide fragment capable of binding to specificity pockets of theMMP enzyme (Mannello et al., 2005b). Despite the high therapeutic potential of MMPinhibitors (MMPIs), all clinical trials have failed to date (Coussens et al., 2002), except
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for doxycycline for periodontal disease. This can be attributed to (i) poor selectivity of theMMPIs, (ii) poor target validation for the targeted therapy and (iii) poorly defined predictivepreclinical animal models for safety and efficacy (Mannello et al., 2005b). Lessons fromprevious failures, such as recent discoveries of oxidative/nitrosative activation and phos-phorylation of MMPs, as well as novel non-matrix related intra- and extracellular targets ofMMP, give new hope for MMPI development for both chronic and acute diseases (Ramnathand Creaven, 2004; Overall and Kleifeld, 2006; Dorman et al., 2010). On the other hand,among the biologically active components from natural products (Mannello, 2006; Zhangand Kim, 2009), green tea polyphenols caused the strong inhibition of MMPs. More par-ticularly, it has been reported that natural extracts components (such as epigallochatechingallate, resveratrol, curcumin) are able to inhibit the enzymatic activity of various MMPsas well as the activation of proMMP-2 (Aggarwal et al., 2004).
2.5. Involvement in physiological and pathological processes
MMPs roles in pathology may be grouped into the following main types: (1) tissuedestruction (e.g., cancer invasion and metastasis, arthritis, ulcers, periodontal diseases,brain degenerative diseases)(Gottschall and Deb, 1996; Yong et al., 2001; Malemud, 2006;Agrawal et al., 2008; Moor et al., 2009; Oyarzun et al., 2010; Gialeli et al., 2011; Hainardet al., 2011; Kessenbrock et al., 2011; Sexton et al., 2011; Skarmoutsou et al., 2011); (2)fibrosis (e.g., liver cirrhosis, fibrotic lung disease, otosclerosis, atherosclerosis, and multiplesclerosis)(Starckx et al., 2003; Fiotti et al., 2004; Chakrabarti and Patel, 2005; Roderfeldet al., 2007; Rybakowski, 2009; Lim et al., 2010; Ragino et al., 2010); (3) weakeningof matrix (e.g., dilated cardiomyopathy, aortic aneurysm and varicose veins)(Papalambroset al., 2003; Reddy et al., 2004; Mannello and Raffetto, 2011).
As for most biological processes, matrix degradation is a precise event, attributed toproteinases that are produced and released on demand from activated cells. By meansof specific cell surface receptors, the cell recognizes a particular matrix molecule and isinstructed to produce the appropriate metalloproteinase, which is then released into thepericellular space where it degrades its specific substrate (Murphy and Nagase, 2011). Thesites of matrix degradation may be isolated by reorganization of cell membrane; as thecell moves beyond the site of matrix degradation, excess proteinase would spill into theopen extracellular space. Thus, TIMPs may act in the tissue environment to neutralize usedproteinases thereby preventing excessive and unwanted degradation away from the sites ofmetalloproteinase production (Wagenaar-Miller et al., 2007).
The importance of proteolytic enzymes in facilitating invasive tumor growth had beenrecognized some considerable time before these enzymes were isolated and characterized,with a hypothetical secretion by fibroblasts (Tang et al., 2004), and subsequently iden-tified as hyaluronidases, serine proteinases, and matrix metalloproteinases. According tothe “three-step” hypothesis of tumor cell invasion (Liotta et al., 1983; Liotta et al., 1986;Saito et al., 2007), key events in the process of tumor invasion are tumor cell adhesionto ECM structures, ECM degradation by proteolysis, and then tumor cell migration intothe degraded area. Acting by tissue breakdown and remodeling during tumor invasion,intravasation into circulation, extravasation, and migration to metastatic sites and tumor
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angiogenesis (Gialeli et al., 2011), MMPs are overexpressed in a wide range of malignanttumors, with demonstrated correlation between their over-expression, tumor aggressive-ness, stage, and prognosis (Kessenbrock et al., 2010). The pattern of MMP expression isnow known to be more complex than simple secretion by tumor cells and in many humanmalignancies it is characterized by the induction of metalloproteinase expression in “host”stromal cells (Heppner et al., 1996; Mannello, 2011). It is also clear that the MMP activ-ity around a tumor is a feature of increased tissue remodeling as much as increased tissuedegradation. Although modifications or degradation of the ECM by tumor-derived proteaseswas originally thought to destroy physical barriers to cell migration, more recently profoundeffects on cell adhesion and migration were identified, related to MMP activation on varioustissue culture substrates including fibronectin, gelatin, and vitronectin (Egeblad et al., 2005;Kessenbrock et al., 2010; Peng et al., 2011). On the other hand, recent findings also suggestthat excessive proteolysis may inhibit this process by impairing tumor cell adhesion or dis-rupting and degrading the cell-matrix interactions or matrix signals required for migrationand invasion, demonstrating the existence of a certain critical range or a balance betweensome MMPs and TIMP expression in order for both tumor invasion and angiogenesis tooccur (Seo et al., 2003; Chernov et al., 2009).
The high expression of both proteinases and inhibitor associated with poor prognosisprobably reflects the need for some regulation of the increased metalloproteinase activity(Mannello, 2011). The tumor must “remodel” the local tissue, by generation of a modifiedvasculature and the generation of supportive stromal tissues, the stroma acting as a “collab-orator” with induction of proteinases and inhibitors by the adjacent tumor cells resulting inangiogenesis and invasive growth (Sun and Zhang, 2006).
The growth of the MMP family has inevitably contributed to a more complex role intumor growth and spread. Although gelatinase A, gelatinase B, stromelysin-3, matrilysin,MTI-MMP, collagenases, and stromelysin-1 seem to be associated most closely with theinvasive phenotype (Morrison et al., 2009; Kessenbrock et al., 2010), recent studies havehighlighted that some MMPs play many roles in cancer distinct from matrix destruction,influencing early steps of tumor evolution, and expanding their pro-tumorigenic properties(Lopez-Otin and Bond, 2008; Fanjul-Fernandez et al., 2010). However, these in vivo studieshave also shown that, unexpectedly, some MMP family members (like MMP8, ADAMTS15)may have paradoxical anti-tumor functions (Lopez-Otin et al., 2009). The identification andvalidation of some MMPs and related enzymes as anti-tumor proteases and speculates aboutthe molecular mechanisms underlying their protective roles in tumor development (Martinand Matrisian, 2007).
MMPs have been also associated with a variety of escaping mechanisms that cancercells develop to avoid host immune response (Van Kempen et al., 2006; Haiko et al., 2009),mainly through the suppression of T-lymphocytes proliferation by the disruption of theIL-2R� signaling (Sheu et al., 2001), or by decreasing the tumor cells sensitivity to naturalkiller cells via a bioactive fragment from �1-proteinase inhibitor (Kim et al., 2000; Coussenset al., 2002; Folgueras et al., 2004).
For what concerns the joint diseases, although a number of proteinases have been foundin the arthritic joints (Manicourt et al., 1995), matrixins are considered to be key enzymesfor the degradation of cartilage matrix (Seki et al., 1995; Alenius et al., 2001; Tardif et al.,2004; Malemud, 2006).
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The destructive enzymes largely originate from chondrocytes (Tardif et al., 2004),releasing/secreting active MMPs that cleave the core protein of aggrecan and link pro-tein, telopeptides of type II collagen, where intermolecular cross-linking occurs and typeIX collagen in vitro, finally acting as a depolymerizer of cartilage collagens (Tardif et al.,2004; Malemud, 2006).
In atherosclerotic plaques, MMP-3 (as well as MMP-1, MMP-2, and MMP-9) weredetected in both smooth muscle cells and macrophages, and (Papalambros et al., 2004;Newby, 2006) are thought to participate in weakening the connective tissue matrix in theintima, which leads to plaque rupture, acute thrombosis, and smooth muscle cell prolifera-tion and migration. The polymorphism analysis of some MMP genes identified a commonvariant in their promoters, significantly associated with greater progression to coronaryatherosclerosis than other genotypes (Papaspyridonos et al., 2006).
MMPs are also important in the etiopathogenesis of endometriosis, being involved in theECM invasion of the endometriotic cells in the ectopic situs. Consequently, increased levelsof MMP-1, MMP-2, MMP-3, MMP-7 and MMP-9 were mainly detected in peritoneal fluidof patients with endometriosis (Osteen et al., 1996; Osteen et al., 1999; Sharpe-Timms andCox, 2002; Braundmeier et al., 2006; Qiu et al., 2006; De et al., 2011). They are stimulated byTNF-� and IL-1 which decrease the expression of TIMP, increasing the imbalance betweenMMP and TIMP (Kang et al., 2008). Similarly to cancers, in endometriosis MMP-activityis increased, associated with cathepsin D and plasminogen (Gilabert-Estelles et al., 2003;Ramon et al., 2005), suggesting that selective MMP and uPA inhibitors may be useful inendometriosis management (Huang, 2008).
2.6. Intracellular compartmentalization
Through their motifs and modules, the secreted MMPs are directed to various compart-ments in the extracellular environment as well as to cell membranes (Baronas-Lowell et al.,2007; Cauwe and Opdenakker, 2010; Kessenbrock et al., 2010). The interaction with theirbinding partners (collagens, laminins, fibronectin, elastin, core proteins and GAG-chains ofPGs) (McCawley and Matrisian, 2001; Raeber et al., 2007; Rodriguez et al., 2010) variesin strength, which has implications for the ability to extract a given enzyme from a tissue.Examples are the binding of MMP-1, -2, -7, -8, -9 and -13 to heparin and heparan sulfate(Groth et al., 2009), where the interaction with heparin occurs through the HPX domain ofMMP-1, -2 and 9 (Wallon and Overall, 1997). MMP-7 lacks the HPX domain and interactsthrough the catalytic and the pro-domain; this MMP binds much stronger to the GAG-chainsthan the other MMPs (Yu and Woessner, Jr., 2000).
Binding of MMPs to cell membranes may 1) regulate their activity (that leads to theiractivation and promotion of cell migration and invasion through basement membranesand tissue) and 2) activate intracellular signaling cascades (an effect independent of theirproteolytic activities)(Koyama et al., 2008). Cell surface associated enzymes can also beinternalized and either directed to the lysosomes for destruction or be a source of intracellularactivity. An emerging concept in MMP regulation is their intra/extracellular location becauseboth secreted and membrane bound MMPs have been found localized to various intracellularsites.
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Unlike the other members of the MMP family, and despite the presence of the N-terminalsignal peptide, most of the MMP-26 (matrilysin-2/endometase) produced is reported to beretained inside the cell (Marchenko et al., 2004). The conserved PRCGXPD motif in theprodomain involved in the latency of other MMPs is replaced by the unique PH81CGVPDmotif in MMP-26. This motif, along with other atypical structures, is assumed to facilitateautocatalytic activation of the enzyme inside the cell. Normal intracellular calcium-levelsprobably maintain MMP-26 in an inactive state and the active enzyme may only be seenduring transient intracellular calcium influx.
Polymorphonuclear leukocytes and mast cells can store MMPs, as well as other pro-teinases and PGs, in exocytic vesicles and release them into the extracellular environmentupon activation of the cells (Bravo-Cordero et al., 2007). Recent studies have shown alsothat endothelial cells, chondrocytes and various cancer cells can store MMPs in intracel-lular vesicles. Endothelial cells could release MMP-2, MMP-9, MT1-MMP, TIMP-1 andTIMP-2 very rapidly, suggesting that they originate from intracellular storage compart-ments. The vesicle content of both pro- and active MMPs was increased by stimulationwith the angiogenic factors fibroblast growth factor-2 or vascular endothelial growth factor.The addition of isolated vesicles to endothelial cells increased their ability to invade andform capillary-like structures in vitro (Donnini et al., 2004). Growth plate cartilage cultureshave been shown to produce matrix vesicles that contain both pro- and active MMP-2 andMMP-3, as well as TIMP-1 and TIMP-2. Chondrocytes from growth zones produce mem-brane vesicles with higher MMP content than chondrocytes from resting zones, indicatingthat theses enzymes are involved in ECM remodeling at the hypertrophic cell zone in thegrowth plates of long bones (Schmitz et al., 1996).
The intracellularly activated MMP-9 had a slightly higher molecular weight than APMAactivated MMP-9, which may represent intermediate forms that are more susceptible to fullactivation after secretion.
In melanoma cells, MMP-2 and MMP-9 have been detected in a high number of small,vesicular organelles organized along the microtubular network. The two enzymes werenot colocalized, but were often found in close proximity to each other. A high degree ofoverlapping distribution was seen between the MMP-2 positive vesicles, the motor proteinkinesin and a-tubulin within the cells. Treatment of the cells with a microtubule-interferingdrug impaired the secretion of MMP-2 and MMP-9 (Schnaeker et al., 2004). Taken together,these studies indicate that various cell types can store pro- and active MMP-2 and MMP-9,as well as their activators, intracellularly in small exocytic vesicles. These vesicles may beactively propelled along microtubules towards the plasma membrane by the motor proteinkinesin. Shedding of such vesicles may be a way of achieving rapid, directional proteolysisduring cell migration, invasion or during 3D morphological organization in the process ofangiogenesis.
3. The novel and unexpected nuclear localization of MMPs
An emerging concept in MMP regulation is their intra/extracellular location because bothsecreted and membrane bound MMPs have been found localized at various subcellular sites.Proteolytic enzymes typically function intracellularly, extracellularly or intranuclearly, but
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not in all locations. In particular, the MMPs have been viewed up-to-now as bulldozers,destroying the extracellular matrix to permit normal remodeling, to turnover of the most ofthe ECM components, and to contribute to pathological tissue destruction and tumor cellinvasion (Morrison et al., 2009; Kessenbrock et al., 2010; Ugalde et al., 2010). Recently, theidentification of specific matrix and non-matrix substrates for MMPs and the elucidation ofthe biological consequence of cleavage indicates that several MMPs are not only just formatrix anymore, demonstrating their novel and unexpected nuclear localization.
Although the discovery of nuclear localization/compartmentalization of MMPs might beat first dismissed as sloppy protein targeting, the identification of a nuclear localization signalof some MMPs suggests that this alternate localization represents an important, heretoforeunderappreciated, aspect of MMP functions, opening a new field in the cell biology andbiochemistry, beginning the dissection of such relevant down-stream targets.
In fact, there are increasing strong evidences that the novel subspecialized and compart-mentalized nuclear MMPs may participate in many physiological and pathological cellularprocesses, in which they can act as both constitutive, regulatory and inducible proteinases.The transcriptional and translational control of MMP expression, the nuclear localizationduring healthy and pathological conditions, the regulation of intracellular and intranucleussorting, the nuclear substrates, the transport and trafficking of MMPs (including local acti-vation and inhibition steps) will be the new routes to identify and understand the specializedroles of nuclear MMPs, a gamble with the next future.
3.1. MMPs in the nuclei of various cell types during physiological andpathological events: internalization mechanism and/or nuclear translocation?
Cell membrane proteins, growth factors, and an increasing amount of ECM and nonECMpeptides have also been identified as MMP substrates, expanding the potential importanceof this family to include direct effects on cell-cell signaling and intercellular interactions. Inparticular, the MMP substrates identified and validated in complex biological functions referto proteins belonging the cytokines, chemokines, growth factors, cell motility and adhesionmolecules, metabolic enzymes, and blood clotting cascade (Rodriguez et al., 2010): no datawas reviewed about nuclear proteins as MMP substrate or nuclear localization and functionof MMP.
Since 1995 several MMPs and TIMPs have been demonstrated as active proteases local-ized in nuclei of various human and animal cell types, including heart myocytes, brainneurons, breast and endothelial cells, fibroblasts and hepatocytes (Table 1).
3.1.1. Tissue inhibitor of metalloproteinases, TIMPsThe first evidence was referred to the preliminary nuclear localization of TIMP-1 in
cultured human gingival fibroblasts (Gin-1 cells) by immunohistochemistry (Li et al., 1995).The discovery was then confirmed characterizing the TIMP-1 maximum accumulation inthe nuclei of Gin-1 cells during the S-phase of the cell cycle (Zhao et al., 1998). Theunequivocally demonstration of nuclear TIMP-1 localization at concentrations significantlyhigher than that found in cytosolic compartment, suggested the possibility of an activetransport mechanism for the passage of cytosolic TIMP-1 to nucleus against a concentration
MMP-10Stromelysin-2 Breast cancer tissue (human)MMP-13Collagenase-3 Cortical neuronal cells (rat), neurons and oligodendrocytes
(human and rat), cancer breast cells (human)MMP-14 MT1-MMP Ischemic cells (mouse), normal and cancer liver cellsMMP-15 MT2-MMP Breast cancer cells (human)TIMP-1 Gin-1 cells (human), MCF-7 cells (human)
gradient (Zhao et al., 1998). Moreover, the absence of TIMP-2 and TIMP-3 suggested thatTIMP-1 may participate in regulating cell cycle by inhibiting unknown nuclear MMPs (likestromelysin/MMP-3, cited but never published).
Chinese hamster ovary cells (stably transfected with the TIMP-1/enhanced green fluores-cent protein expressing plasmid) were co-cultured with several human cell lines, showingthat the chimeric fusion TIMP-1/EGFP protein bound to the membrane surface of MCF-7breast cancer cell line but not to non-neoplastic HBL-100 breast epithelial cells (Ritter et al.,1999). The localization of TIMP-1/EGFP in the nucleus of MCF-7 cells after 72 hours ofco-culture suggested that TIMP-1 may be taken up only by malignant breast cells in whicha nuclear trafficking exists. The novel nuclear uptake of internalized proteins indicated forthe first time that TIMP-1 may affect cellular proliferation of cancer cells also influencingnuclear functions (such as replication and/or transcription).
Unfortunately, the very interesting field of research about nuclear localization and func-tions of TIMP family proteins is lacking of further studies, neglecting the potential rolethat these proteins (with unique biomolecular and cellular functions other than the MMPinhibitory properties)(Mannello and Gazzanelli, 2001; Troeberg and Nagase, 2007) mightalso have in nuclear processes.
3.1.2. Matrix metalloproteinases, MMPsSeveral classes of MMPs (including MMP-2, -3, -9, -11, -13 and MT1-MMP/MMP-
14) have been identified and characterized in nuclei of various cell types from humantissues and in animal models. Although the mechanisms of nuclear translocation of thedifferent MMPs are generally poorly characterized, to facilitate the search of nuclear MMPcompartmentalization, we present the findings according to cell and tissue localization.
3.1.2.1. Brain. Starting from the first preliminary evidence showing that MMP-2 and MMP-9 were predominantly activated in SOD1-/− mice after focal cerebral
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ischemia-reperfusion with clear gelatinolytic activity in the ischemic cortex neuronal nuclei(Gasche et al., 2001), several studies highlighted that the gelatinolytic activity of someMMPs appeared in the nuclear compartment of neurons in the brain at different stagesfollowing transient focal cerebral ischemia.
Through multiple approaches, it has been demonstrated the spatio-temporal activationof MMP-2 and -9 gelatinases in the brain of rats subjected to transient middle cerebralartery occlusion (MCAo)(Amantea et al., 2007; Amantea et al., 2008). In particular, theauthors demonstrated that gelatinolytic activity was significantly increased in the nuclei ofischemic core regions, as early as 15 min after reperfusion following MCAo, increasinggradually with the progression of reperfusion; gelatinolytic activity also increased in thecytosolic compartment, reaching significance after 22 h of reperfusion. Nuclear, cytosolicand ECM MMP-2 and -9 gelatinolytic activities detected in the controlateral hemisphereof rats subjected to MCAo were not significantly different from enzyme activity found insham-operated mice (Amantea et al., 2008), suggesting that the gelatinolysis occurring inneuronal nuclei may be linked to MMP-dependent cell death triggering neuroinflammatoryreactions.
MMP-13 (or collagenase-3), a member of collagenase subfamily of MMPs with crucialrole in the MMP activation cascade (Mannello et al., 2005b; Rosenblum et al., 2007a;Mannello, 2011), has been found in activated form within the cell nuclei of neural cells aftercerebral ischemia (Cuadrado et al., 2009). This effect was also reproduced in vitro in ratcortical neuron cultures exposed to oxygen and glucose deprivation, suggesting that MMP-13 activation and its nuclear translocation is an early consequence of an ischemic stimulus.In fact, the authors demonstrated that also MMP-13 was already found in cell nucleus at30 min of brain ischemia whereas at 2 h after MCAo the gelatinolytic activity appearedalso in the cell cytoplasm. Noteworthy, they found that active MMP-13 was up-regulatedboth in rats and humans after stroke, mostly in neurons and oligodendrocytes; althoughthe different cytoarchitecture of rats and humans may explain the different localization ofischemic lesions (in rats neurons and oligodendrocytes are found together especially instriatum, whereas in human these cells are more abundantly found in white-matter areas),the authors found that rat oligodendrocytes showed high nuclear reactivity for MMP-13,in humans they were not able to detect them. On the other hand, they found that nuclearMMP-13 was mainly produced in neurons, clearly seen in both rats and humans, suggestingthat MMP-13 may have novel functions within cell nucleus (probably liked to the apoptoticcascade as part of ischemic stimulus).
Bearing in mind the results obtained up-to-now, two major questions arise: 1) what wouldbe the function of nuclear MMPs during ischemia and/or after stroke? 2) how these MMPscan enter in the nucleus (as they are synthesized in the cytoplasm and normally secreted ormembrane anchored)?
To partially answer these questions, a recent study demonstrated that in brain shortly afterthe injury, some MMPs in a nuclear fraction of ischemic tissue are able to cleave nuclearproteins which facilitate accumulation of oxidized DNA at an early stage after an ischemicinsult (Yang et al., 2010). In particular, the authors found that intense MMP-2 and MMP-9gelatinase activity was seen in the neuronal nuclei of rat brain cells underwent to MCAoafter 3 h of reperfusion, detecting also the proteolytic activity of gelatinases in the cellnucleus within the infarction in brain parenchyma samples from deceased stroke patients.
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Starting from the evidence that both MMP-2 and -9 were involved in the nuclear gelatinaseactivity, the authors found in both cellular membrane and nucleus of the ischemic cellsthe co-localization of MT1-MMP or MMP-14 (the membrane type-1 metalloproteinase,known as the major activator of MMP-2). Interestingly, the authors also found that MT1-MMP co-localized with MMP-2 in the ischemic nuclei and in nuclear extracts, detectingthe expression and localization of furin, a well known MT1-MMP activator.
Noteworthy are the findings that ischemic nuclear extracts were able to significantlyincrease the release of MMP-dependent cleavage products of both poly-ADP-ribosepolymerase-1 (PARP-1) and X-ray cross-complementary factor 1 (XRCC1), nuclear pro-teins playing crucial roles in DNA fragmentation, DNA base excision repair and cellapoptosis. The involvement in degradation of these nuclear substrates via MMP-2 and -9activated by furin-enhanced MT1-MMP activity during ischemic insult was fully inhibitedthrough specific MMP-inhibitor, demonstrating that the early nuclear MMP-2/MMP-9/MT1-MMP gelatinase proteolysis is directly involved in the early reduction of bothPARP-1 and XRCC1 induced by transient focal cerebral ischemia, and then in the enhancednuclear accumulation of oxidative DNA damage in the ischemic rat brain with reperfusion.Interestingly, the effective inhibition of the early intranuclear MMP gelatinolytic activitycould reduce neuronal DNA fragmentation and cell death at a later stage after ischemicinsult, opening new possible therapeutic strategy (Fig. 3).
Some issues remain unsolved: 1) how excessive nuclear MMP gelatinase proteolysis inneurons could affect the balance between detrimental and beneficial role of PARP1; 2) donuclear TIMPs and/or local furin control MMP-2 activation and proteolysis in nuclei ofneurons during the early stage of post-ischemic reperfusion both in human stroke and inanimal model? 3) do the nuclear MMP-2, -3, -9 and -13 protease activation occur after anischemic insult independently of blood supply restoration?, taking into account that it may becritical in the human setting as stroke patients remain with artery occlusion for long periodsunless spontaneous or therapeutic reperfusion occurs, and that we do not know actually thereal artery perfusion status at the death for human brain samples; last but not least, 4) howthe cytosolic and/or membrane-bound MMPs can enter in the nucleus compartment?
3.1.2.2. Heart. Starting from the preliminary evidence highlighting a strong signal forMMP-2 in the left ventricular porcine myocyte, along the sarcolemma surface and in perin-uclear zones (Coker et al., 1999), it has been clearly demonstrated that in both rat and humanheart there were a significant presence of MMP-2 within the myocyte nuclei in a relativelyhomogeneous distribution with a degree of association with the condensed chromatin (Kwanet al., 2004). Interestingly, in human heart nuclear extract, both MMP-9 and MMP-2 wereidentified; in particular, zymogenic, activated and complexed forms of MMP-2 (molecularmass of 72, 64 and 130 kDa, respectively) were immunocharacterized. Moreover, MMP-2 gelatinase activity and protein isoforms have been identified in rat heart ventricles andhuman heart, co-localizing with PARP which show a peculiar MMP-dependent proteolyticdegradation pattern. This cleavage would inactivate PARP in a manner similar to caspase,playing possible protective/beneficial role when PARP is overactivated (excess of PARPmay be removed sparing the cell from ATP depletion) or a detrimental one in preventing itfrom repairing DNA (e.g., hindering DNA strand break repair). Noteworthy is that close tothe C-terminal of MMP-2, the authors found two small very basic patches of aminoacids
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Fig. 3. Hypothetical diagram of the events linking intranuclear MMPs and oxidative DNA damagein brain cells during ischemia, reperfusion and stroke. In ischemic brain tissue (shortly after theinjury), some nuclear MMPs are able to cleave nuclear proteins which facilitate accumulation ofoxidative damaged DNA at an early stage after an ischemic insult. In particular, MT1-MMP co-localized with MMP-2 in the ischemic nuclei and in nuclear extracts, jointly with the expression offurin, a well known MT1-MMP activator. In ischemic nuclear extracts were found a significantlyincrease of MMP-dependent cleavage products of both poly-ADP-ribose polymerase-1 (PARP-1)and X-ray cross-complementary factor 1 (XRCC1), nuclear proteins playing crucial roles in DNAfragmentation, DNA base excision repair and cell apoptosis. The degradation of these nuclear proteinsvia MMP-2 and -9 activated by furin-enhanced MT1-MMP activity during ischemic insult enhancednuclear accumulation of oxidative DNA damage proning the ischemic rat brain with reperfusion toapoptosis.
separated by a variable spacer; this pattern is characteristic for the typical nuclear localiza-tion sequence (NLS), suggesting a possible mechanism involved in the nuclear translocationand localization (Kwan et al., 2004).
3.1.2.3. Liver. While investigating MMP-3 expression in hepatocellular carcinoma, it hasbeen observed a prominent homogeneous nuclear immunostaining for MMP-3 in about 90%of hepatocellular carcinoma specimens (Si-Tayeb et al., 2006). Adjacent hepatocytes andmyofibroblasts also exhibited nuclear MMP-3 staining, although plasmocytes featured the
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expected cytoplasmic staining. This finding was confirmed with two MMP-3 antibodiesdirected at the hinge and proximal hemopexin domains but not with antibodies recognizingN- or C-terminal regions of MMP-3. The nuclear form(s) of MMP-3 appeared to haveundergone additional processing or to have concealed certain epitopes; western blot analysisof the HepG2 hepatocellular carcinoma cell line revealed 45- and 35-kd immunoreactiveMMP-3 proteins in nuclear fractions, consistent with mature (45-kd) and truncated versionsof the enzyme. Surprisingly, casein zymography and casein affinity purification studies inHepG2 nuclear extracts revealed only the 35-kd form, suggesting that the 45-kd MMP-3 maybe maintained in an inactive state by forming a complex with another protein. The absenceof full-length MMP-3 in nuclear extracts raises the possibility that processing is required toexpose the nuclear localization signal for nuclear transport. Accordingly, to discern whetherMMP-3 enter the nucleus through the nuclear pore via a mechanism involving recognition ofNLS by transporter proteins, the authors detected the putative NLS (PKWRKTH) at position107 to 113 in amino acid sequence, demonstrating also that the deletion of two amino acidsof the putative NLS led to a large decrease in the nuclear localization of chimeric MMP-3.All these data demonstrated that MMP-3 NLS is functional and that the nuclear translocationof MMP-3 in hepatocytes is operated by an NLS-dependent mechanism. Finally, the authorsunderline that the nuclear localization of MMP-3 is also associated with an increased rateof apoptosis, as revealed by the significant reduction of the proapoptotic effect obtainedwith the mutated form of MMP-3 (devoid of catalytic activity) and using a specific MMP-3inhibitor.
It is possible that nuclear MMP-3 could also damage the genomic environment, causingapoptosis of some cells, whereas damaged cells defective for apoptosis will be selected fortumorigenesis; this ideas may be consistent with the frequent finding of nuclear MMP-3in samples of cirrhosis, a well-known precancerous lesions, suggesting that a persistentexpression of nuclear MMP-3 in hepatic tumor cells may further damage the nuclear matrixand contribute to the accumulation of genetic and epigenetic abnormalities (Monvoisinet al., 2002; Si-Tayeb et al., 2006).
However, some questions remain to be solved: 1) on the basis of the NLS functionalityand interaction with the nuclear pore complex, why no pro-MMP-3 form was found inthe nucleus? 2) why a short MMP-3 form is found in the nucleus (alternative splicing ofthe primary pro-MMP-3 transcript or alternative promoter usage)? 3) do have other MMPsthe putative NLS suggesting a common ability to translocate to the nucleus? 4) could pro-MMP-3 be cleaved intracellularly and then translocate into nuclei without NLS interactionwith the nuclear pore complex (e.g., via importin or carrier proteins)? 5) does nuclear shortMMP-3 form cleave PARP in vivo during the apoptosis induction in hepatic cells?
On the basis of the previous studies demonstrating that rat liver nuclear extracts diddisplay zymogen and activated forms of MMP-2 and proMMP-9 gelatinolytic activities(Kwan et al., 2004), it has been detailed in hepatocellular carcinoma the subcellularlocalization and survival of MT1-MMP (or MMP-14)(Ip et al., 2007). In particular, theauthors found that MT1-MMP expression remained minimal in normal livers, but up-regulated in livers adjacent to tumors, linking the presence of MT1-MMP in the nucleiof tumor cells with aggressive large tumors and poor overall survival, but not with classicpathological characteristics (like tumor stages, infiltration, gender, age of virus serology).Noteworthy, MT1-MMP (containing caveolin scaffolding domains (Annabi et al., 2001))
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and caveolin-1 demonstrated concomitant presence in the perinuclear regions of hepatocel-lular carcinoma, suggesting that caveolae-mediated endocytosis may be involved in nucleartranslocation/trafficking of MT1-MMP in hepatocellular carcinoma. Moreover, as MT1-MMP is closely related to MMP-2 activation and coexist in the same hepatoma cells (Ogataet al., 1999), the co-localization in hepatocellular carcinoma nuclei of MT1-MMP andMMP-2 may explain the novel functions triggering the MMP proteolytic cascade inside thenucleus; in fact, MMP-2 may be activated by MT1-MMP and active MMP-2 may cleave theMT1-MMP into active fragments, suggesting a dual functional repertoire influencing thenuclear processes linked to apoptosis and carcinogenesis (e.g., the specific preferences insubcellular fractions of latent and/or active MT1-MMP and MMP-2 may infer the possibilityof different functionally distinct pools of MMPs translocated within the nucleus).
3.1.2.4. Lung. It is well-known that cigarette smoke-induced activation of proteases suchas MMPs contributes to lung alveolar destruction due to cell death. It has been found thatcigarette smoke-induced apoptosis, enhanced annexin V binding and cleaved PARP wellcorrelated with increased gelatinase activity, revealing that the levels of pro-MMP-2 andactive MMP-2 were increased in cytosolic and nuclear fractions isolated from cigarettesmoke-exposed pulmonary artery endothelial cells (Aldonyte et al., 2009). In particular,MMP-2 protein co-localized with gelatinolytic enzyme activity in the nucleus of cigarettesmoke-exposed pulmonary artery endothelial cells undergoing apoptosis. These observa-tions support the notion that MMP-2 contributes to cigarette smoke-induced gelatinaseactivity, which localizes in the nuclear region primarily and correlates with annexin V bind-ing and PARP cleavage, suggesting a novel function of MMP-2 in the degradation of thenuclear matrix in cigarette smoke-induced endothelial apoptosis.
3.1.2.5. Breast. In a comprehensive bio-molecular and immunocytochemical study aboutthe expression of all known human MMPs in healthy and cancer breast tissues, it has beendescribed that for MMP-1 the immunostaining showed a clear predominance for the nucleiof tumor cells with a slight additional staining in the tumor cell’s cytoplasm (Kohrmannet al., 2009), whereas no staining of MMP-1 was found in normal breast tissue (Mannello,2011). Nuclei of tumor cells were also stained with MMP2. MMP-2 was also found in thecytoplasm of normal breast endothelial cells, albeit not in their nuclei. For MMP-10, nucleiand cytoplasm of tumor cells were positive, whereas no staining was found for stromal andimmune cells. For MMP-13 a weak to moderate staining of the cytoplasm and nuclei oftumor cells and, in addition of the cytoplasm of normal breast cells was found. MMP-15showed a strong staining in the cytoplasm of tumor cells with additional slightly stainingof few stromal cells and in some nuclei of tumor cells. Finally, weak cytoplasmic positivitywas seen in all vital steroid receptor negative MDA-MB-486 breast cancer cells for MMP-3,which in addition was found in the majority of the nuclei, too. The nuclear localization ofthese MMPs was associated with aggressive tumor features including poor prognosis. Nomechanisms or functions were provided/suggested about the role of nuclear MMPs in thebreast cancer processes.
3.1.2.6. Connective tissue. Active MMP-3 in the nuclei of chondrocytic cells in cul-ture and in nuclei of normal and osteoarthritic chondrocytes in vivo has been shown to be
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involved in transcriptional gene regulation (Eguchi et al., 2008). Nuclear MMP-3 boundto a transcription enhancer sequence (TRENDIC) in the connective tissue growth factor(CCN2/CTGF) promoter and activated transcription of CCN2/CTGF. This growth factorpromotes physiological chondrocytic proliferation and ECM formation. Pro- and activeMMP-3 could activate the CCN2/CTGF promoter, where various domains of the MMP par-ticipated in the activation. Both the HPX and the Cat-Hinge regions activated the promoter,whereas the prodomain and the hinge-region alone had no effect on the activation. Comparedto the wild type MMP-3, lower promoter activation occurred in the presence of catalyti-cally dead MMP-3 mutants. This suggested that MMP-3 can regulate the CCN2/CTGFpromoter activity by two completely different mechanisms. One involves proteolytic pro-cessing of one or several nuclear proteins, whereas the other is independent of the processingcapacity of the proteinases and involves the HPX domain. A DNA-binding domain wasfound in the HPX domain, as an anti- MMP-3 HPX antibody blocked the protein-DNAinteractions. The hinge region contains proline-rich sequences found in some transcrip-tion factors. The properties of MMP-3 as a transcription factor was evaluated by analyzingnuclear MMP-3 associated proteins (NuMAPs). Several NuMAPs were detected, such asheterochromatin proteins, transcription coactivators/corepressors, RNA polymerase II andnucleosome/chromatin assembly protein. One of the NuMAPs, HP1c, was demonstrated tointeract with MMP-3 and to co-activate the CCN2/CTGF promoter with MMP-3. Anotheridentified NuMAP was the transcription repressor NCoR1, suggesting that MMP-3 mightdegrade NCoR1 to prevent transcription repression of the CCN2/CTGF promoter.
Noteworthy, this study demonstrated several new aspects of MMP-3, clarifying crucialaspect about the unexpected functions of nuclear MMPs (Fig. 4): MMP-3 acted as a tran-scription factor in cell nuclei; the MMP3 gene encodes a TRENDIC-binding factor; MMP3was identified and localized in the nuclei both in vitro and also in vivo; both the nuclearuptake of extracellular MMP3
and the nuclear targeting ability of putative NLSs in MMP3 were shown. All of the datatogether demonstrated that MMP3 is translocated to the cell nucleus and, once there, transactivates the CCN2 gene. The nuclear MMP3 may function when chondrocytes are in aphysiological or pathological status.
MMP3 is known as a secretory proteinase that degrades ECM proteins such as pro-teoglycans and collagens, while MMP3 also cleaves and activates other MMPs, such asgelatinases MMP-2 and MMP-9, and growth factors, such as TGF-�. The authors suggestthat a partition trafficking may occur, hypothesizing that some MMP3 would be locatedin the cytosol for further transport to the nucleus, while other MMP3 would be secretedthrough the ER-Golgi complex. This may result in both direct nuclear transportation fromthe cytoplasm and the internalization-nuclear transportation of MMP3.
The authors also found six putative NLSs in MMP3 for nuclear entry through the nucleo-pores; interestingly, externally added recombinant human MMP3 translocated to the nucleusin 30 min, whereas the amount was decreased at 60 min after the addition, suggesting thatthis loss of nuclear MMP3 may have been caused by degradation or nuclear export. There-fore, MMP3 can shuttle back to the cytoplasm after being translocated to the nucleus. Ofnote, it is possible that multiple NLSs are recognized synergistically for efficient nucleartranslocation, exposing on the surface of the molecule to be transported for recognition byimportins for the nuclear translocation from the cytoplasm. In fact, the posttranslational
F. Mannello, V. Medda / Progress in Histochemistry and Cytochemistry 47 (2012) 27–58 49
Fig. 4. Schematic representation of the multiple roles of extracellular, intracellular and intranuclearMMP-3 in chondrocytes. Based on numerous previous reports, the stromelysisn-1 (classified as MMP-3) has been recognized as a matrixin functioning as regulatory proteinases degrading substrates presentin extra- and intracellular milieu, but also modulating gene expression and cleaving peculiar proteinsin the nuclei. In fact, the canonical presence of MMP-3 in extracellular space (by the well-knownsecretion pathway mainly related to the N-terminal signal peptide of MMPs) is associated to the enzy-matic degradation of Types IV and IX collagens, laminin, fibronectin, elastin and proteoglycans. Afterthe translocation from ECM to cytosol (through a putative receptor), the degradation of nonmatrixcytosolic proteins inside the cells have been also characterized, mainly linked to the activation ofseveral biological precursors like proTNF-�, proIL-1�, IGFBPs, myelin basic protein, pro-MMP1,HB-EGF, pro-TGF-� etc. After translation or via the protein-mediated nuclear transportation, thenovel roles of MMP-3 have been identified as gene regulator (after the binding of MMP-3 with theTRENDIC motif for trans regulation of the CCN2/CTGF gene) and as nuclear proteinases cleavingpeculiar proteins (such as PARP and XRCC1). These evidences support the involvement of MMP-3 inapoptosis and gene regulation of tissue remodeling during physiological and pathological conditions.
modification of MMP3 can remove or hide the primary NLS and expose another NLS onthe molecular surface, suggesting the differential use of multiple NLSs.
Starting from the well-known evidence that MMP3 interacts with TIMPs in the cellsas well as in the extracellular environment (Mannello and Gazzanelli, 2001), it is pos-sible to speculate that an appropriate amount of nuclear MMP3 is under the control ofnuclear TIMPs, so that it can work properly as a transcription factor, since the authors also
50 F. Mannello, V. Medda / Progress in Histochemistry and Cytochemistry 47 (2012) 27–58
detected TIMP-1 in the nuclear fraction of cells (Eguchi et al., 2008). About the mecha-nism(s) of the apoptosis elicited by MMP3, other possibilities are that the vast amount ofcellular/nuclear MMP3 induces apoptosis by digesting both cytoplasmic and intranuclearprotein components or that the nuclear MMP3 induces the overexpression of CCN2/CTGF,which subsequently induces cellular apoptosis as previously demonstrated (Hishikawa et al.,2000).
Of note, MMP2 was found in the nuclei and capable of cleaving in vitro several nuclearproteins (like PARP1 and XRCC1, crucial for chromatin DNA maintenance) which canbe inactivated or activated by MMP3 itself, or MMP2 can be activated by MMP3. Thecleavage and inactivation of PARP result in the inhibition of poly(ADP-ribosyl)ation andexpose the DNAase hypersensitive region. The exposure of such naked DNA can supply anMMP3-accessible region on DNA, proning to genetic and epigenetic modifications, likelyto the MT1-MMP cleavage functions of centrosomal pericentrin causing in human cellschromosome instability (Golubkov et al., 2005).
Starting from the unequivocal demonstration that MMP3 from outside of the cells and inthe cytoplasm was translocated into the nucleus, where MMP3 bound to DNA/chromatin andtrans activated a crucial apoptosis-related CCN2 gene (Eguchi et al., 2008), and accordingto numerous past reports highlighting the novel nuclear localization and function of someMMPs, it is also possible speculate a multiple role for MMP3, i.e., its functioning at extra-,intracellular and intranuclear levels. In fact, MMP3 acts to degrade ECM components out-side (like the structural proteins fibrillar collagens and elastin, substrate adhesion moleculesas fibronectin, laminin and collagen IV and proteoglycans), degrades and activates severalintracellular peptides (such as MMPs 1, 7, 8, 9 and 13, tenascin, fibrin/fibrinogen, insulingrowth factor binding proteins, connective tissue growth factor, pro-IL-1�) whereas inducesCCN2 and alters indirectly (and/or directly?) other matrix-related proteins in the nucleus(like PARP and XRCC). This multiple-functioning MMP3 may play an important role inthe development and matrix remodeling of cartilage and bone, and the abnormalities in theMMP3 dynamics may be involved in the MMP-related matrix diseases, e.g., osteoarthritisand rheumatism, and in fibrotic diseases such as systemic sclerosis and atherosclerosis.
4. Concluding remarks and outlook
Recent studies have uncovered multiple divergent roles for different MMPs in a varietyof physiological and pathological processes (Malemud, 2006). From the recent findings ofnuclear localization in the different cells and tissues discussed above, it has become clearthat MMPs serve as regulatory enzymes beyond acting as simple housekeeping proteasesand harbor important functions to degrade ECM components outside, to cleave and activateseveral intracellular peptides whereas may unexpectedly induce gene expression and alter(indirectly and/or directly) matrix-related proteins in the nucleus in healthy and diseaseconditions.
MMP-2, -3, -9, -13, MT1-MMP and TIMP-1 have been demonstrated in the nucleus ofvarious cell types, including heart myocytes, brain neurons, breast epithelial cells, endothe-lial cells, fibroblast and hepatocytes. Although the mechanisms of nuclear translocationof the different MMPs are generally not fully characterized, studies indicate that different
F. Mannello, V. Medda / Progress in Histochemistry and Cytochemistry 47 (2012) 27–58 51
MMPs may have distinct trafficking route (e.g., MMP-2 has a typical nuclear localizationsequence close to the C-terminus that might be involved in the nuclear localization (Kwanet al., 2004); nuclear signaling sequences are found in the catalytic domain of MMP-3 (Si-Tayeb et al., 2006; Eguchi et al., 2008); for MT1- MMP, has been suggested a endocytosismechanism via internalization and nuclear translocation as a result of the colocalization ofcaveolin-1 and MT1-MMP in perinuclear regions (Ip et al., 2007)).
For what concerns the novel functions of nuclear MMPs, they have been associatedwith apoptosis in several studies (Tonti et al., 2009). In fact, increased nuclear gelatinolyticactivity, colocalized with MMP-2, has been demonstrated in pulmonary endothelial cellsundergoing apoptosis. MMP-2 activation in these cells was suggested to be induced byreactive oxygen and nitrogen species produced by cigarette smoke (Aldonyte et al., 2009).Intranuclear gelatinolytic activity has also been observed in rat brain neurons after post-ischemic reperfusion, and this gelatinolytic activity was associated with DNA fragmentation(revealing colocalization among MMP-2, MMP-9, and MT1-MMP in conjunction to thewell known activator furin)(Yang et al., 2010). In both cardiac myocytes and pulmonaryendothelial cells, as well as in brain neuronal cells, nuclear gelatinolytic activity is correlatedwith the processing of two important factors in the DNA repair machinery (i.e. the DNArepair enzyme PARP and the factor XRCC1, which protect cells from apoptosis): they wereshown to be processed by MMP-2 and MMP-9 (Kwan et al., 2004; Aldonyte et al., 2009;Yang et al., 2010). Thus, nuclear MMP activity may contribute to the apoptotic process afterischemic injuries by processing PARP and XRCC1, hence interfering with the oxidativeDNA repair system (Yang et al., 2010). In addition to MMP-2 and MMP-9, expression ofactive MMP-13 was also increased in the nucleus of neural cells after cerebral ischemia inboth rats and humans, with a nuclear translocation mechanism promoted by oxygen andglucose deprivation in the cells following ischemia (Cuadrado et al., 2009).
At this stage, an obvious question arises: what do the MMPs not do? Starting fromthe important axiom about the substrate discovery and the search for biological roles forMMP proteases: “Just because it can, does not mean it does, biochemical evidence cannotnecessarily be translated to cell culture nor to animal models and certainly not necessarilyto man (Overall and Butler, 2007; Schilling and Overall, 2007), what is now clear is that themajority of MMP substrates are non-matrix molecules. Indeed, it is reasonable to proposenow that the major functions of MMPs (beyond acting as simple housekeeping proteases)harboring to the well known degradation of almost all ECM components outside the cell,cleaving and activating several intracellular peptides like cytokine and growth factor bindingproteins, whereas are also unexpectedly inducing gene expression and altering nuclearmatrix-related proteins.
Future studies are required to delineate the MMP polymorphisms and translational mech-anisms (Mannello, 2009), leading also to the generation of the truncated forms of nuclearMMPs and TIMPs, and structural insights should aid the drug development of exogenouscompounds that may act in an allosteric manner for crucial nuclear MMPs and TIMPs, andtherefore may be more specific for individual MMP forms than currently available inhibitors(Mannello and Tonti, 2009).
The understanding of the nuclear balance between MMPs and TIMPs and their recentlyidentified substrates continues to be an expanding area in cell and molecular biology (Tontiet al., 2009). More research is still needed to increase the understanding of the localization of
52 F. Mannello, V. Medda / Progress in Histochemistry and Cytochemistry 47 (2012) 27–58
MMPs and TIMPs, in vivo binding partners, substrate processing, involvement of exosites insubstrate processing and the regulation of enzyme activity by binding partners. Last but notleast, they should provide starting points for the development of novel selective therapeuticmodalities for various human diseases.
Acknowledgements
The Dr. Susan Love Research Foundation (Santa Monica, CA, USA) is kindly acknowl-edged for their support to Prof. Ferdinando Mannello (DSLRF Grant Award 2011).
We apologize to those authors whose work could not be cited as a result of oversightsand/or space limitations.
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