MINIREVIEW

Regulation of matrix metalloproteinase activity in healthand diseaseElin Hadler-Olsen, Bodil Fadnes, Ingebrigt Sylte, Lars Uhlin-Hansen and Jan-Olof Winberg

Department of Medical Biology, Faculty of Health Sciences, University of Tromsø, Norway

Introduction

Matrix metalloproteinases (MMPs) are a subfamily of

zinc- and calcium-dependent enzymes belonging to the

metzincin superfamily. Characteristic for this super-

family is the HEXXHXXGXXH zinc-binding motif

and a conserved methionine located C-terminal to the

zinc-ligands, which forms a Met-turn [1]. In humans,

there are 24 MMP genes, but only 23 MMP proteins

because MMP-23 is coded by two identical genes at

chromosome 1. MMPs are built up by various

domains (Fig. 1). All MMPs contain an N-terminal

signal peptide that directs the enzymes to the secretory

pathway, a prodomain with a conserved PRCGXPD

sequence that confers the latency of the enzymes and a

catalytic domain with the catalytic zinc localized in the

large and relatively shallow active site cleft. In addi-

tion, all MMPs except the two matrilysins (MMP-7

and -26) and MMP-23 contain a C-terminal hemopex-

in (HPX)-like domain that is linked to the catalytic

domain through a hinge region. In most MMPs, this

hinge region consists of 10–30 amino acids, whereas, in

MMP-9, this linker contains approximately 64 amino

acids and is heavily O-glycosylated [2]. In six of the

membrane-anchored members of the MMP family, the

HPX region ends in either a type I transmembrane

domain with a short intracellular sequence or a glycosyl-

phosphatidylinositol moiety. MMP-23 differs from the

Keywords

activation; compartmentalization;

complexes; exosite; heteromers; inhibition;

matrix metalloproteinases

Correspondence

J.-O. Winberg, Department of Medical

Biology, Faculty of Health Sciences,

University of Tromsø, 9037 Tromsø, Norway

Fax: +47 77 64 53 50

Tel: +47 77 64 54 88

E-mail: jan.o.winberg@uit.no

(Received 30 April 2010, revised 4 October

2010, accepted 18 October 2010)

doi:10.1111/j.1742-4658.2010.07920.x

The activity of matrix metalloproteinases (MMPs) is regulated at several

levels, including enzyme activation, inhibition, complex formation and

compartmentalization. Regulation at the transcriptional level is also impor-

tant, although this is not a subject of the present minireview. Most MMPs

are secreted and have their function in the extracellular environment. This

is also the case for the membrane-type MMPs (MT-MMPs). MMPs are

also found inside cells, both in the nucleus, cytosol and organelles. The role

of intracellular located MMPs is still poorly understood, although recent

studies have unraveled some of their functions. The localization, activation

and activity of MMPs are regulated by their interactions with other pro-

teins, proteoglycan core proteins and ⁄or their glycosaminoglycan chains, as

well as other molecules. Complexes formed between MMPs and various

molecules may also include interactions with noncatalytic sites. Such exo-

sites are regions involved in substrate processing, localized outside the

active site, and are potential binding sites of specific MMP inhibitors.

Knowledge about regulation of MMP activity is essential for understanding

various physiological processes and pathogenesis of diseases, as well as for

the development of new MMP targeting drugs.

Abbreviations

APMA, p-aminophenylmercuric acetate; CS, chondroitin sulfate; FnII, fibronectin II; GAG, glycosaminoglycan; GSH, glutathione;

Hp, haptoglobulin; HNL, human neutrophil lipocalin; HPX, hemopexin; MMP, matrix metalloproteinases; MMPI, metalloproteinase inhibitor;

MT-MMP, membrane-type matrix metalloproteinase; NuMAP, nuclear MMP-3 associated protein; PG, proteoglycan; SIBLING, small integrin-

binding ligand N-linked glycoprotein; TIMP, tissue inhibitor of metalloproteinase; TnI, troponin I.

28 FEBS Journal 278 (2011) 28–45 ª 2010 The Authors Journal compilation ª 2010 FEBS

other MMPs by lacking the HPX domain, which is

replaced by a C-terminal cystein array region and an

immunoglobulin G-like domain, and, instead of the

N-terminal signal peptide, this enzyme contains an

N-terminal type II transmembrane domain. In two of

the secreted MMPs, MMP-2 and MMP-9, the catalytic

domain also contains a module of three fibronectin II

(FnII)-like inserts. A recent review described the molec-

ular interactions of the HPX domains of the various

human MMPs [3]. The various domains, modules and

motifs in the MMPs are involved in interactions with

other molecules, and hence affect or determine the activ-

ity, substrate specificity, and cell and tissue localization,

as well as activation, of MMPs.

Activation of proMMPs requires physical delocaliza-

tion of the prodomain from the catalytic site, the

so-called cystein-switch model [4]. Two main mecha-

nisms are involved in the activation of MMPs. One is

proteolytic cleavage and removal of the prodomain

[5,6] and the other is allosteric activation where the

prodomain is displaced from the catalytic site of

the enzymes without being cleaved (Fig. 2). Most of

the MMPs are secreted as proenzymes and their acti-

vation occurs in the pericellular and extracellular

space. By contrast, all membrane-type (MT)-MMPs

and three of the secreted MMPs contain a unique

sequence (RX[K ⁄R]R) at the C-terminal end of the

prodomain (Fig. 1). These MMPs can be activated

intracellularly by furin, a serine proteinase belonging

to the convertase family, which can cleave the prodo-

main at this unique sequence [5,6].

Together, the MMPs are able to degrade most extra-

cellular matrix (ECM) proteins. In addition, they can

process a large number of non-ECM proteins, such as

growth factors, cytokines, chemokines, cell receptors,

serine proteinase inhibitors and other MMPs, and

thereby regulate the activity of these compounds as

summarized recently [7]. An increasing number of

studies have shown that processing of some protein

and peptide substrates by MMPs requires that the sub-

strates not only interact with the active site, but also

regions outside the active site. Such regions are

referred to as noncatalytic sites or exosites, which can

be motifs localized in the catalytic domain or in one of

the other domains. An important role of the exosites

may be to orient the substrate properly for cleavage

and, for some substrates, exosite-binding is an absolute

requirement for degradation. A recent review [8]

focused on current knowledge with respect to struc-

tural and functional bases for allosteric control of

Secreted MMPs Membrane-anchored MMPs

Fibronectin II-like domain

MMP-2

MMP-9

Minimal domain

MMP-7, -26

MMP-11, -21, -28

Simple HPX domain

MMP-1, -3, -8,-10, -12, -13,-19, -20, -27

Furin-activated

MMP-23

MT1, -2, -3, -5-MMP

MT4, -6-MMP

Zn

N

C

Zn

N

Zn NZn

N

Zn

N

Zn

N

Zn N

C

C

N

Zn

Zn

N

Catalytic domain

Propeptide domain

Hinge-region

HPX like domain

FnII like module

RX[K/R]R motif

C Type I transmembrane domain

N

C

Type II transmembrane domain

GPI-membrane anchor

C-terminal Ca-Ig domain

Cell membrane

O-glycosylated hinge-region

Fig. 1. Domain structure of secreted and membrane-anchored MMPs. Most MMPs contain a propeptide domain, a catalytic domain, a linker

(hinge-region) and a HPX domain. The hinge region in MMP-9 is heavily O-glycosylated. The three furin-activated MMPs and all of the mem-

brane-anchored MMPs have a basic RX[K ⁄ R]R motif at the C-terminal end of their prodomains. This motif can be cleaved inside the cells by

furin-like proteinases. The two gelatinases (MMP-2 and -9) contain three FnII-like repeats in their catalytic domain, N-terminal to the catalytic

Zinc-binding site. Four of the six MT-MMPs are anchored to the cell membranes through a type I transmembrane domain and the other two

through a glycosylphosphatidylinositol moiety. The seventh membrane-anchored MMP, MMP-23, has an N-terminal type II transmembrane

domain. The two minimal domain MMPs and MMP-23 lack the HPX domain and, in the latter enzyme, this domain is replaced by a C-termi-

nal cystein array (Ca) and an immunoglobulin-like (Ig) domain.

E. Hadler-Olsen et al. Regulation of MMP activity

FEBS Journal 278 (2011) 28–45 ª 2010 The Authors Journal compilation ª 2010 FEBS 29

MMP activities. Previous reviews have described new

techniques that can be used in the search for exosites

and examples of exosites derived from the use of these

techniques [9,10]. Although our knowledge of specific

exosites in the various MMPs is still very limited, these

sites will become of increasing importance as targets

for future drug development. Hopefully, future drugs

will not affect all substrate degradation by a given

enzyme, but only the processing of selected substrates.

Some of the substrates that MMPs are known to

process are localized intracellularly [7]. Although all

MMPs contain a signal peptide that directs them to

the secretory pathway, an increasing number of reports

have found various MMPs localized also inside cells.

This may partly explain the ability of some MMPs to

process intracellular proteins and further demonstrates

the complex roles of MMPs under physiological and

pathological conditions. Among the earliest intracellu-

lar MMP substrates detected are troponin I (TnI) [11],

aB-crystallin [12] and lens bB1 crystallin [13]. In vivo,

MMP cleavage of these substrates was linked to health

and disease. MMP-2 degradation of TnI is associated

with diminished contractive function of the heart [11],

MMP-9 degradation of aB-crystallin with multiple

sclerosis [12] and MMP-9 cleavage of lens bB1 crystal-

lin with cataract [13].

MMPs interact with various cell surface and pericel-

lular molecules that alter the function of the enzyme,

as well as affect cellular behaviour [14]. MMP-induced

cleavage and degradation of ECM and non-ECM mol-

ecules may either prevent or provoke diseases such as

cancer [15], as well as cardiovascular, autoimmune,

neurodegenerative and various connective tissue dis-

eases. Knowledge about the regulation of MMP activ-

ity is therefore important for understanding various

physiological processes, as well as the pathogenesis of

a large number of diseases. In addition, such know-

ledge is also important for the development of novel

treatment strategies. A number of excellent reviews on

MMPs and their functions are available. The present

review focuses on the regulation of MMP activity with

an emphasis on post-translational modifications, the

A BProteolytic cleavage of the pro- and HPX-domains Allosteric activation

Zn

NSH

Zn

SH

1

Zn

2

Zn

3

Zn

4

Zn

SH

N

1a

SH

Zn

2a Auto-cleavage

Zn

3a Auto-cleavage

Proteases

MercurialsSH-reactive agentsChaotropic agentsROSDetergents

Zn

NSH

Zn

N

SH

1

Zn

SH

N

Zn

SH

1a

2a

Zn

SH

3a

HgCl2 / APMA

NGAL (HNL)

Gelatin / Collagen IV / Collagen VI (α2-chain) / SIBLING

Auto-cleavage

Fig. 2. Proteolytic and allosteric activation of MMPs. (A) Proteolytic cleavage of the pro- and HPX domain of MMPs by various proteinases,

including serine proteases such as trypsin or other MMPs. Steps 1–4 represents partly to fully processed propeptide, HPX and hinge

regions. Processing of the proMMP by another proteinase can be facilitated by the interaction of the target proMMP with other macromole-

cules that present the inactive proenzyme to its activator (not shown). Binding of mercurial compounds such as APMA or HgCl2, other SH

reactive agents, reactive oxygen species (ROS), chaotropic agents and detergents such as SDS results in conformational changes of the pro-

enzyme (step 1a) followed by activation through successive autocleavage of the propeptide (steps 2a and 3a). This may also be followed by

autoprocessing of the HPX region (not shown). (B) Allosteric activation in which the propeptide remains intact (step 1), as suggested for the

binding of proMMP-9 to gelatin or collagen IV, binding of proMMP-2 to collagen VI (a2 chain), as well as binding of individual SIBLINGS to

specific MMPs (see text). Binding of HgCl2 or APMA to proMMP-9 results in a conformational change (step 1a) followed by autocleavage

that did not remove the conserved PRCGV sequence from the enzyme (step 2a). This truncated enzyme had a low specific activity. Neutro-

phil gelatinase associated lipocalin (NGAL) ⁄ human neutrophil lipocalin (HNL) bound to the new N-terminus without further processing of the

enzyme (step 3a), resulting in a fully active enzyme (see text).

Regulation of MMP activity E. Hadler-Olsen et al.

30 FEBS Journal 278 (2011) 28–45 ª 2010 The Authors Journal compilation ª 2010 FEBS

formation of heterodimers and complexes, compart-

mentalization, and the role of exosites in substrate deg-

radation and enzyme inhibition.

Activation mechanisms

To induce activation of a proMMP, the prodomain

must be physically delocalized from the catalytic site

(Fig. 2). There are various ways to achieve such a

delocalization followed by activation. One is through

S-reactive agents, organomercurials and reactive oxy-

gen species, interacting with the conserved cysteine in

the prodomain. Another is the induction of conforma-

tional changes through binding of chaotropic agents

and detergents such as SDS. In all cases, the confor-

mational changes (Fig. 2A, step 1a) are followed by an

autocatalytic stepwise degradation of the prodomain

(Fig. 2A, steps 2a and 3a) [5,6]. Proteinases can cleave

the prodomain in one or several steps, producing an

active MMP with reduced molecular size. A large

number of proteinases such as serine and metallopro-

teinases are involved in the activation of proMMPs. In

some cases, one enzyme generate a partly active

enzyme that can be fully activated by a second enzyme

removing one or more amino acids from the prodo-

main, as described for MMP-1 [5,6]. Thus, it is not

sufficient to remove the zinc-binding motif in the

prodomain of the MMP to obtain a fully active

enzyme; the catalytic efficiency of the activated MMP

also depends on the cleavage site C-terminal to this

motif.

Both MMP-2 and MMP-9 have been shown to be

activated in vivo by serine proteinases such as chymase

and trypsin, suggesting a biological relevance. Using

knockout mice, it was also shown that mast cell chym-

ase had a key role in the activation of proMMP-9 and

proMMP-2 [16]. In mice with acute pancreatitis,

trypsin induced the activation of proMMP-2 and

proMMP-9. ProMMP-9, when activated by endoge-

nous trypsin, was reported to be a permissive factor

for insulin degradation and diabetes [17]. Similarly, a

significant association between high endogen concen-

trations of trypsin and activation of proMMP-9 was

found in ovarian tumor cyst fluids [18]. Trypsin has

been shown to be an efficient activator of most proM-

MPs in vitro [6]. Instead of activating proMMP-2, it

was reported that trypsin induced degradation of the

enzyme [19]. Other studies showed that trypsin could

activate proMMP-2, although less efficiently compared

to compounds such as p-aminophenylmercuric acetate

(APMA) [20–23]. There is no contradiction in these

results. We have shown that the balance between acti-

vation and degradation is dependent on the activation-

temperature as well as trypsin concentration and the

two additives, Brij-35 and Ca2+ [22]. At 37 �C, the

presence of 0.05% Brij-35 and 10 mm Ca2+ mainly

prevented both activation and degradation, whereas a

lack of these two compounds resulted in trypsin-

induced degradation. However, at intermediate concen-

trations of Brij-35 and Ca2+, trypsin induced the

activation of proMMP-2. Different modes of activa-

tion can have implications for the biochemical proper-

ties of the enzymes depending on the cleavage site. In

the trypsin-activated MMP-2, the N-terminal residue

was either Lys87 or Trp90 [22], with the former being

identical to the cleavage site generated by human tryp-

sin-2 [23]. The N-terminal residue was Tyr81 in mem-

brane-type1 MMP (MT1-MMP) or APMA-activated

enzyme [6]. The slightly shorter N-terminus in the

trypsin-activated enzyme resulted in reduced catalytic

efficiency and weaker tissue inhibitor of metallopro-

teinase (TIMP)-1-binding compared to the enzyme

activated by MT1-MMP or APMA [22]. Docking stud-

ies of TIMP-1 revealed that the slightly weaker binding

of the inhibitor to the trypsin-activated MMP-2 could

be attributed to its shorter N-terminus (Lys87 ⁄Trp90versus Tyr81) because Phe83 and Arg86 interacted

directly with the inhibitor.

Activation through domain specific interactions

A proMMP can be presented to its activator protein-

ase by interactions with other proteins or glycosamino-

glycans (GAGs). In addition, interactions between a

proMMP and other molecules can result in an active

MMP without proteolytic processing of the propep-

tide, allosteric activation (Fig. 2B). It is sufficient that

the propeptide is distorted away from the active site,

which leaves an open active site that can bind and pro-

cess substrates. Removal of the binding partner causes

a reversion into an inactive proenzyme. Below, we

review some of the recent literature that has focused

on the role of various proMMP-binding partners

involved in activation.

Allosteric activation

Gelatinase interactions with collagen and gelatin

Binding of macromolecules or specific thiol-binding

reagents to an MMP with an intact or a partially

cleaved prodomain can induce enzyme activation

despite the presence of the conserved PRCGXPD

sequence. ProMMP-9 bound to either a gelatin or type

IV collagen-coated surface could cleave a fluorogenic

peptide substrate, as well as gelatin, even if the

E. Hadler-Olsen et al. Regulation of MMP activity

FEBS Journal 278 (2011) 28–45 ª 2010 The Authors Journal compilation ª 2010 FEBS 31

prodomain of the enzyme remained intact [24]. The

specific activity of the proenzyme bound to the gelatin-

coated surface was approximately 10% of the active

MMP-9 bound to the same surface. Furthermore, the

enzymatic activity of both enzyme forms was inhibited

by TIMP-1 with comparable kinetics. Similar observa-

tions were made for proMMP-2. The proenzyme could

degrade DQ-gelatin in the presence of low concentra-

tions of the triple-helical domain of the a2 chain of the

microfilamentous collagen VI [25]. The above examples

are illustrated in Fig. 2B (step 1).

Interactions with small integrin-binding ligand

N-linked glycoprotein (SIBLING)

Individual members of the SIBLING family are known

to bind strongly to both pro- and active forms of

specific MMPs. Bone sialoprotein binds MMP-2,

osteopontin binds MMP-3 and dentin matrix protein-1

binds MMP-9, all with a 1 : 1 stoichiometric ratio and

binding constants in the nanomolar range [26]. These

SIBLINGs and MMPs are also co-expressed and colo-

calized in salivary glands of humans and mice [27].

The interaction between the SIBLING and its partner

proMMP resulted in an active MMP without autocata-

lytic removal of the propeptide [26]. Studies indicated

that binding of a SIBLING to a proMMP induced

large conformational changes in the enzyme, suggest-

ing that the propeptide is physically removed from the

catalytic site, thereby allow substrate binding (Fig. 2B,

step 1). Furthermore, the three SIBLINGs have a ten-

to 100-fold higher affinity for the complement regula-

tor factor H than for their partner MMPs. The proM-

MP ⁄SIBLING complex was dissociated in the presence

of factor H and a re-inactivation of the catalytic

activity by the still attached propeptide [26]. The same

research group also showed that the amino-terminal

region, especially exon 4, is essential for bone sialopro-

tein-mediated activation of proMMP-2 [28]. It appears

that bone sialoprotein also can regulate the activity of

active MMP-2 by modulating the inhibitory effect of

TIMP-2 and synthetic MMP-inhibitors [28,29]. The

findings of a recent study challenged the view that

certain SIBLINGs are able to bind and induce alloste-

ric activation of specific MMPs [30].

Interactions between proMMP-9 and neutrophil

gelatinase associated lipocalin

Human neutrophil lipocalin (HNL), also called neutro-

phil gelatinase associated lipocalin, is known to form a

strong reduction sensitive heterodimer with proMMP-9

[31,32]. Mercurial compounds such as APMA and

HgCl2 are known to partly activate the 92 kDa proM-

MP-9 in several constitutive steps that generate an

83 kDa form of the enzyme with the M75RTPRCGV

peptide as the N-terminal sequence [6]. Hence, the con-

served Cys80 that interacts with the catalytic zinc is

not removed (Fig. 2B, steps 1a and 2a). Treatment of

proMMP-9 with an excess of HNL also induced a

partial activation of the proenzyme with an identical

N-terminus as the HgCl2 exposed enzyme [33]. When

the enzyme was activated with a combination of HgCl2and HNL, this resulted in a fully active enzyme with

an activity comparable to trypsin activated MMP-9

[33]. Despite the full activity of the HgCl2 and HNL

activated enzyme, this had an N-terminus identical to

the HgCl2 activated enzyme (Fig. 2B, step 3a) [33],

whereas trypsin activation of MMP-9 caused removal

of the entire propeptide, with Phe88 as the N-terminal

residue (Fig. 2A, steps 1 and 2) [6]. Similar results were

obtained with isolated proMMP-9 homodimer and

proMMP-9 ⁄HNL heterodimer when activated with

HgCl2 and an excess of HNL. By contrast, HNL had

no effect on trypsin-activated MMP-9. Kallikrein is a

plasma proteinase that can partially activate proMMP-

9, and the presence of an excess of HNL resulted in a

synergistic effect with a 30–50% increase in activity

compared to kallikrein activation alone. Altogether,

this suggested that the N-terminus of the partially acti-

vated proenzyme is entrapped in the hydrophobic-

binding pocket of HNL and the propeptide–HNL

complex is thereby detached from the catalytic site,

generating a fully active enzyme without further trun-

cation (Fig. 2B, step 3a) [33].

Activation by peroxynitrite and glutathione

Enzymatic activity of intact proMMPs against physio-

logical substrates has also been detected in the pres-

ence of peroxynitrite and glutathione (GSH) [34].

Examples of this are proMMP-1 and -8 processing of

triple helical collagen I, and proMMP-9 processing of

gelatin. One of the products generated when GSH

reacts with peroxynitrite is GSNO2. It was shown that

this product most likely activates the proenzymes

through S-glutathiolation of the cystein in the con-

served PRCGXPD sequence of the propeptide by

forming a stable disulfide S-oxide [34]. Peroxynitrite

can also induce activation of proMMP-2 without loss

of the prodomain. This activation appeared to be con-

centration-dependent and was attenuated by GSH [35].

Other studies have reported activation of proMMP-2

by peroxynitrite, although the activation was followed

by a cleavage of the enzymes prodomain, resulting in

an enzyme with a reduced molecular size [36,37]. Thus,

Regulation of MMP activity E. Hadler-Olsen et al.

32 FEBS Journal 278 (2011) 28–45 ª 2010 The Authors Journal compilation ª 2010 FEBS

peroxynitrite as well as GSH along with peroxynitrite

may activate the MMPs by two completely different

mechanisms: one being allosteric and the other com-

prising an autocatalytic removal of the prodomain.

Peroxynitrite has also been shown to inactivate

TIMP-1 [38]. Thus, it appears that peroxynitrite poten-

tiates MMP activity not only by the direct activation

of proMMPs, but also by preservation of MMP

activity after it is generated. There appear to be a

controversy whether GSNO is able to directly induce

activation of MMPs by modulating the conserved Cys

in the enzyme prodomains [39].

Activation through proteolytic removalof the prodomain

TIMP regulation of MT1-MMP-induced activation

of proMMP-2

MT1-MMP-induced activation of proMMP-2 is a two-

step process involving the MMP inhibitor, TIMP-2,

which has been described in detail in several reviews.

Briefly, it has been shown that the TIMP-2 enhancement

of the MT1-MMP-induced activation of proMMP-2 is a

result of the formation of a ternary complex where the

inhibitor acts as a link between the two enzymes. In this

complex, the MT1-MMP is inactive as a result of its

interaction with the N-terminal part of TIMP-2,

whereas the C-terminal part of the inhibitor binds to the

HPX domain of proMMP-2. Another MT1-MMP mol-

ecule can now cleave the proMMP-2 in the complex and

generate a 64 kDa inactive intermediate. This intermedi-

ate is further autocatalytically processed into the fully

active 62 kDa form of MMP-2 [40,41]. The step at

which TIMP-2 is involved when it enhances the

MT1-MMP-induced activation of proMMP-2 has been

questioned because studies have shown that the inhibi-

tor enhances the autoactivation step, but is not neces-

sary for the first cleavage step [42–44]. The other

TIMPs, TIMP-1, -3 and -4, can also regulate the MT1-

MMP-induced activation of proMMP-2 [43], where

TIMP-1 only prevents the second step (autoactivation)

and locks the enzyme in an inactive intermediate form

[43,45]. These examples demonstrate the complexity of

the MT1-MMP-induced activation of proMMP-2 and

how the activation process can be differently regulated

by various TIMPs.

MT-MMP-induced activation of proMMP-2

Other MT-MMPs can also activate proMMP-2,

although this activation does not involve TIMP-2.

Both the MT2-MMP and the MT3-MMP-induced

activation of proMMP-2 required a proMMP-2 with

an intact HPX domain [46,47]. Both MT3-MMP and

proMMP-2 bind to chondroitin sulfate (CS) chains of

cell surface proteoglycans (PGs). This interaction

enhances the activation of proMMP-2, probably by

presenting the gelatinase to its membrane-bound acti-

vator [46]. Both the catalytic and the hinge region of

the MT3-MMP interacted with the CS-chains, whereas

proMMP-2 interacted through the HPX domain.

Furthermore, CS-chains with the sulfate attached to

the 4-position of the GAG-chains (C4S) but not to the

6-position (C6S) enhanced the activation in the pres-

ence of suboptimal concentrations of MT3-MMP.

Binding of proMMP-2 to the CS-chains without

MT3-MMP did not result in activation. The complex

interactions of various proteins involved in MT-MMP-

induced activation of proMMP-2 are further elucidated

by the involvement of claudins, which are tetraspan

membrane proteins. MT-MMP mediated proMMP-2

activation was enhanced in the presence of claudin-1,

-2, -3 and -5 [48]. Claudins not only replaced TIMP-2

in the MT1-MMP-induced activation of proMMP-2,

but also enhanced the activation of proMMP-2 by all

MT-MMPs. Claudin-1 binds to both MT1-MMP and

proMMP-2, and this binding appears to involve only

the catalytic domains of the two enzymes. Another

membrane protein shown to enhance MT1-MMP-

induced activation of proMMP-2 was avb3 integrin

[49–51]. MMP-2 binds through its HPX domain to an

MT1-MMP-cleaved and activated form of avb3 inte-

grin [49–52]. This activated integrin enhanced the sec-

ond autocatalytic step of the activation by binding to

the 64 kDa intermediate form of MMP-2 [52]. Binding

and activation of MMP-2 was abrogated in the pres-

ence of avb3 integrin-binding macromolecules such as

vitronectin and HKa (two-chain high molecular weight

kinogen) [50,52,53]. Binding of MMP-2 to avb3 inte-

grin appears to be controversial because the findings

of another study did not support an interaction

between MMP-2 ⁄PEX and avb3 integrin [54].

Activation through interactions with elastin,

heparin and CD151

ProMMP-2 and active MMP-2 binds to soluble and

insoluble elastin through the FnII module of the cata-

lytic domain [55]. When proMMP-2 binds to insoluble

elastin, this induces a fast autoactivation of the proen-

zyme followed by inactivation [56]. A similar phenom-

enon of enhanced autolysis has also been observed

when proMMP-2 binds to heparin, although this bind-

ing involves the enzymes C-terminal HPX domain [57].

These are just two examples of how an interaction of

E. Hadler-Olsen et al. Regulation of MMP activity

FEBS Journal 278 (2011) 28–45 ª 2010 The Authors Journal compilation ª 2010 FEBS 33

the proenzyme with various ECM components regu-

lates the activity of the enzyme.

One of the two minimal domain MMPs, proMMP-7,

can also be captured and activated at cell membranes.

The proMMP-7 propeptide can interact with the

C-terminal extracellular loop of the transmembrane

protein CD151 [58]. The interaction between the

propeptide and CD151 was suggested to induce confor-

mational changes followed by autocatalytic activation.

Formation of proMMP-9 dimers affect activation

of the enzyme

MMP-9 is known to form various types of dimers

including homo- and heterodimers that involve the

C-terminal HPX-like domain of the enzyme [31,59–63].

The proMMP-9 monomer is more rapidly activated by

MMP-3 than the homodimer [61]. The interaction

between the C-terminal domain of proMMP-9 and a

CSPG core protein has also been shown to affect the

activation of proMMP-9 [64]. By contrast to the

proMMP-9 monomer and homodimer, the proMMP-

9 ⁄CSPG complex was not activated by the organomer-

curial compound APMA [64]. On the other hand,

Ca2+ which is known to stabilize but not activate

MMPs, induced a concentration independent and

hence intramolecular autoactivation of the proMMP-9

bound to the CSPG. The Ca2+-induced activation

resulted in a proteolytic removal of the propeptide

from the complex bound proMMP-9. In the presence

of Ca2+, activated enzyme forms were also released

from the complex. This was the result of cleavage of a

part of the PG core protein and at least a part of the

C-terminal HPX domain of proMMP-9, leaving the

hinge region bound to the enzyme [64]. A large reduc-

tion of the HPX is likely to alter substrate specificity

because several specific substrate exosites in the HPX

domain may have been removed. Only the proMMP-9

in the CSPG complex was activated when Ca2+ was

added to a mixture of purified proMMP-9 and proM-

MP-9 ⁄CSPG complex. Furthermore, a mixture of

Ca2+ and APMA did not activate the proMMP-

9 ⁄CSPG complex [64], although Ca2+ is known to

participate and enhance APMA induced activation of

proMMP-9 [62,65–67].

During hemolysis and ⁄or hemorrhage, Hb is released

into the circulation and ⁄or into surrounding tissues.

Heme, the prostetic group of Hb, is released from the

protein and converted to hemin, the Fe3+ oxidation

product of heme. During malaria infection, Hb inside

the red blood cells is digested by parasites. This results

in the production of the chemically inert crystalline sub-

stance, hemozoin, which is released into the circulation

when the red blood cells burst. Hemozoin is indentical

to b-hematin, the synthetic form of hemozoin. The

HPX domain of proMMP-9 can bind to both hemin

and b-hematin, which results in an autocatalytic

truncation of parts of the enzyme’s prodomain [68]. The

truncation in the presence of hemin results in two

enzyme forms with Arg17 and Thr64 as N-terminal

residues. b-hematin induced truncation results in two

enzyme forms with Arg42 and Leu54 as new N-terminal

residues, with the former identical with the first cleavage

by MMP-1,-2,-3,-7 and -13 [6]. These partly truncated

forms of MMP-9 are inactive. The presence of the hinge

region of the enzyme accelerated the truncation process.

b-hematin, but not hemin, accelerated MMP-3 induced

activation to the fully active 82 kDa MMP-9 with

Gln89 as N-terminal.

Activity regulated through theformation of heterodimers andcomplexes

Some of the dimers formed with MMP-9 are detected

in SDS ⁄PAGE under nonreducing conditions, but not

under reducing conditions. Hence, these dimers are

reduction sensitive and assumed to be linked through

one or several disulphide bridges. The formation of

different MMP-9 complexes results in altered biochem-

ical properties of the enzyme. In cells that produce

both proMMP-9 and TIMP-1, these two molecules are

bound together through their C-terminal domains, and

the presence of TIMP-1 affects the activity of the

enzyme [69]. When proMMP-9 forms a dimer with col-

lagenase, binding to TIMP-1 is prevented [70]. There

are conflicting data concerning whether the proMMP-9

homodimer is able to form a complex with TIMP-1

[59,61,71].

In its heterodimer form with neutrophil gelatinase

associated lipocalin, proMMP-9 can bind TIMP-1 and

form a ternary complex [72] and the enzyme is pro-

tected from degradation [73]. Two members of the

cystatin family, fetuin-A and cystatin C, bind to

MMP-9 and protect the enzyme from autolytic degra-

dation [74]. The above examples show that there are

different ways by which the activity of an MMP can

be regulated and preserved.

Both MMP-9 and MMP-2 interact with gelatin as

well as collagen through the three FnII-like modules in

their catalytic domain [70,75–84]. This interaction is

important for the ability of these enzymes to degrade

these physiological substrates, although it has no effect

on their degradation of several other physiological sub-

strates or chromogenic peptide substrates. ProMMP-9

forms a complex with one or several CSPG core

Regulation of MMP activity E. Hadler-Olsen et al.

34 FEBS Journal 278 (2011) 28–45 ª 2010 The Authors Journal compilation ª 2010 FEBS

proteins through its HPX domain [64]. When proM-

MP-9 is bound to CSPG core proteins, the enzyme

cannot bind gelatin, suggesting that the gelatin-binding

sites in the FnII-like modules of the enzyme are

masked [85]. Complex formation involving more than

one domain in the enzyme is likely a result of the high

structural flexibility of the large hinge region. The

extreme flexibility of MMP-9 was demonstrated by

atomic force microscopy combined with small-angle

X-ray scattering and analytical ultracentrifugation [86].

The interaction between proMMP-9 and CSPG core

proteins has resulted in changes of several biochemical

properties of the enzyme. On this basis, it is tempting

to assume that active MMP-9 still attached to the

CSPG core protein will have altered biochemical prop-

erties compared to unbound active MMP-9. Such

properties may include substrate specificity, catalytic

efficiency and ability to interact with inhibitor mole-

cules, hence giving rise to altered regulation of enzyme

activity.

Haptoglobulin (Hp) is a plasma protein mainly

expressed in the liver, and belongs to the family of

acute-phase proteins that is induced during the inflam-

matory process. Hp consists of a dimer of ab-chainscovalently linked by disulphide bonds, as well as oligo-

mers [87]. Hp have a high affinity for Hb

(Kd = 10)12m), and is considered to be involved in

the clearance of Hb. The HPX region of MMP-9 has

been shown to form a strong reduction sensitive com-

plex with Hp [88]. Gelatin was reported to bind more

strongly to the proMMP-9 ⁄Hp complex than to either

proMMP-9 monomer or homodimer, although the spe-

cific activity against gelatin was similar for the active

MMP-9 ⁄Hp complex and the active MMP-9 monomer.

Furthermore, binding of proMMP-9 to Hp did not

influence the activation of the enzyme by MMP-3.

Binding of MMP-9 to Hp may comprise a method of

regulating MMP-9 activity because Hp is known to

bind cellular receptors followed by internalization and

degradation.

Role of exosites in regulation of activity

The complex substrate specificity of individual MMPs

is not only determined by their substrate-binding sub-

sites on each side of the catalytic zinc, but also by sub-

strate-binding to motifs outside this region (exosites).

The role of exosites has been recognized for a long

time for enzymes acting on polymer biomolecules such

as the restriction endonucleases [89], although was not

reported until 1989 for MMPs [90]. It was observed

that stored MMP-1 was autocatalytically truncated,

which resulted in a processed enzyme lacking the

C-terminal HPX domain. This truncated enzyme was

no longer able to cleave triple helical collagen I, but was

able to degrade gelatin (denatured collagen). The HPX

region was also found to be necessary for the cleavage

of the triple helical region in interstitial collagen by

other collagen-degrading MMPs (MMP-2, -8, -13 and -

14) [91–95]. The active site region in the MMPs is too

narrow (5 A) to allow a triple helical collagen (15 A) to

enter the active site. The HPX region in the collagenases

locally unwinds the triple helical collagen, and then a

single a-chain can enter the catalytic site and be cleaved

[96]. In addition, it was shown that a small segment in

the catalytic domain, R183WTNNFREY191, is necessary

for the enzymes ability to cleave triple helical collagen

[96]. Production of MMP-3 ⁄MMP-1 chimeras revealed

that additional unique structural elements in the cata-

lytic domain are involved.

In the two gelatinases, MMP-2 and MMP-9, the

FnII-like repeats in the catalytic site of the enzymes

can interact with elastin, type I, III, IV, V, X and XI

collagens, as well as gelatins. This may facilitate the

localization of these enzymes to connective tissue

matrices. This interaction appears to be of importance

for the degradation of macromolecules such as elastin,

gelatin and collagens IV, V and XI, but does not influ-

ence the degradation of chromogenic substrates or

other macromolecules [70,75–84]. Hence, the FnII-like

module in the gelatinases contains important exosites

for the degradation of some substrates.

Many potent small molecule MMP inhibitors

(MMPIs) have been entered into clinical trials for can-

cer treatment, although most of them have been dis-

continued as a result of a lack of specificity and

selectivity. Successful cancer therapy based on MMPIs

must not only be selective against MMPs validated as

targets, but also spare MMPs validated as antitargets

[97,98]. To develop new therapeutic MMPIs, it is of

pivotal importance to understand the structural basis

of recognition, binding and cleavage of substrates, as

well as the recognition and binding of natural inhibi-

tors (TIMPs). Recent data indicate that subtype spe-

cific inhibitors may also lead to new treatment of acute

and chronic inflammatory and vascular diseases [99].

Most known MMPIs are targeting the catalytic

region and the catalytic zinc, which are very similar

between the MMPs. Designing specific small molecular

MMPIs targeting the catalytic site is therefore prob-

lematic [99]. MMPIs targeting less conserved binding

sites outside the prime subsites of MMPs are consid-

ered to be more specific. Within the MMP family, dis-

tinct preferences for collagen types are seen, which

must reflect structural differences in MMP collagen-

binding [100]. Exosites are considered to be important

E. Hadler-Olsen et al. Regulation of MMP activity

FEBS Journal 278 (2011) 28–45 ª 2010 The Authors Journal compilation ª 2010 FEBS 35

determinants for these differences in specificity by

introducing contact regions between the substrate and

the MMP outside the primary specificity subsites. Exo-

sites are regarded as novel binding sites that represent

unique opportunities for designing subtype selective

inhibitors. Efforts have been put into both high

throughput screening [101] and the design of inhibitors

targeting exosites without interfering with the catalytic

zinc [8]. Such inhibitors are considered to act selec-

tively against the degradation of a specific substrate,

and represent a novel therapeutic approach with puta-

tive reduced side effects.

Binding of the collagen triple helix is necessary for

collagenolysis. Some studies have taken advantage of

potential substrate exosites in MMP-2 and MMP-9

collagenolytic behaviour by designing triple helical

substrate and triple helical transition state analogues.

One such study indentified inhibitors with high selec-

tivity for the gelatinases (MMP2- and MMP-9) com-

pared to other MMPs [102]. Furthermore, the FnII

insert of MMP-9 was suggested to contain exosites

involved in the binding of type V collagen model sub-

strates and inhibitors. A triple helical peptide that

incorporates an FnII insert-binding sequence was con-

structed and found to give selective inhibition of

MMP-9 type V collagen-based activity [103].

Exosites related to collagenolysis have also been iden-

tified in the active site cleft [104] and the catalytic

domain [105] of MMP-1, and were also suggested in

analogous regions of MMP-8 and MMP-13 [106].

Recently, a highly selective MMP-13 inhibitor was

reported that did not chelate the catalytic zinc, but

instead bound in the S1¢ pocket [107]. This structural

region shows diversity among MMPs. A recent study

has further elucidated the role of the specificity loop for

selective MMP-13 inhibition by indentifying the steric

requirements for binding to this region [108]. Other

studies have also described selective MMP-13 inhibitors

that do not interfere with the catalytic zinc [101,109].

Regulation of activity throughcompartmentalization

Through their motifs and modules, the secreted MMPs

are directed to various compartments in the extracellu-

lar environment as well as to cell membranes. Among

their binding partners in these compartments are colla-

gens, laminins, fibronectin, elastin, core proteins and

GAG-chains of PGs. This compartmentalization regu-

lates the MMP activity by locating and concentrating

them close to or on potential substrates. The interac-

tion with their binding partners varies in 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 [57,72,110–117], where the interaction with hep-

arin occurs through the HPX domain of MMP-1, -2

and 9 [110,112,116]. MMP-7 lacks the HPX domain

and interacts through the catalytic and the prodomain.

This MMP binds much stronger to the GAG-chains

than the other MMPs [117]. MMP-7 could be

extracted from tissues by heparinase digestion or by

using extraction buffer containing heparin, heparan

sulfate or protamin [117]. Similarily, it was necessary

to use various extraction conditions to quantify the

amount of gelatinases in mouse kidneys [118]. Binding

of secreted MMPs to cell membranes is another way

of regulating their activity. This may lead to the acti-

vation of the enzymes, as discussed above, and pro-

mote cell migration and cell invasion through

basement membranes and tissues. Binding of MMPs to

cell membranes may also activate intracellular signal-

ing cascades, an effect independent of their proteolytic

activities [119–123]. Cell surface associated enzymes

can also be internalized and either directed to the lyso-

zymes for destruction or be a source of intracellular

activity. An emerging concept in MMP regulation is

their intra ⁄ extracellular location because both secreted

and membrane bound MMPs have been found local-

ized to various intracellular sites. In the following part

of present minireview, we focus on the subcellular

location, processing of intracellular substrates and

putative physiological relevance of this activity.

Nuclear localization

MMP-2, -3, -9, -13 and MT1-MMP have been demon-

strated in the nucleus of various cell types, including

heart myocytes, brain neurons, endothelial cells, fibro-

blast and hepatocytes. The mechanisms of nuclear

translocation of the different MMPs are generally

poorly characterized. MMP-2 has a typical nuclear

localization sequence close to the C-terminus that

might be involved in the nuclear localization [124].

A nuclear signaling sequence is also found in the cata-

lytic domain of MMP-3, which appeared to be essen-

tial for the translocation to the nucleus. Full-length

MMP-3 was absent from the nucleus, suggesting that

processing is required to expose the nuclear localiza-

tion signal for nuclear transport [125]. For MT1-

MMP, a caveolae-mediated endocytosis has been sug-

gested as a mechanism of internalization and nuclear

translocation as a result of the colocalization of caveo-

lin-1 and MT1-MMP in perinuclear regions [126].

Nuclear localization of MMPs has been associated

with apoptosis in several studies. Increased nuclear

Regulation of MMP activity E. Hadler-Olsen et al.

36 FEBS Journal 278 (2011) 28–45 ª 2010 The Authors Journal compilation ª 2010 FEBS

gelatinolytic activity, colocalized with MMP-2, has

been demonstrated in pulmonary endothelial cells

undergoing apoptosis. MMP-2 activation in these cells

was suggested to be induced by reactive oxygen and

nitrogen species produced by cigarette smoke [127]. In-

tranuclear gelatinolytic activity has also been observed

in rat brain neurons after post-ischemic reperfusion,

and this activity was associated with DNA fragmenta-

tion. Furthermore, this gelatinolytic activity colocalized

with MMP-2 and MMP-9, and was reported to be

markedly reduced in the presence of a general MMP

inhibitor or by MMP-2 and MMP-9 antibodies. MT1-

MMP as well as furin, a MT1-MMP activator, was also

found in the nucleus of the ischemic rat brain neurons,

suggesting a possible mechanism for intracellular acti-

vation of MMP-2 by MT1-MMP [128].

In both cardiac myocytes and pulmonary endothelial

cells, as well as in brain neuronal cells, nuclear gelatin-

olytic activity is correlated with the processing of two

important factors in the DNA repair machinery (i.e.

the DNA repair enzyme poly-ADP-ribose polymerase

and X-ray cross-complementary factor 1, which protect

cells from apoptosis). These two factors were shown to

be processed by MMP-2 and MMP-9 [124,128]. Thus,

nuclear MMP activity may contribute to the apoptotic

process after ischemic injuries by processing poly-

ADP-ribose polymerase and X-ray cross-complemen-

tary factor 1 and hence interfere with the oxidative

DNA repair system [128]. In addition to MMP-2 and

MMP-9, expression of active MMP-13 was also

increased in the nucleus of neural cells after cerebral

ischemia in both rats and humans. The nuclear trans-

location of MMP-13 was promoted by oxygen and

glucose deprivation in the cells following ischemia,

although the biological relevance of this is not known

[129].

Active MMP-3 in the nuclei of chondrocytic cells in

culture and in nuclei of normal and osteoarthritic

chondrocytes in vivo has been shown to be involved in

transcriptional gene regulation [130]. Nuclear MMP-3

bound to a transcription enhancer sequence (TREN-

DIC) in the connective tissue growth factor

(CCN2 ⁄CTGF) promoter and activated transcription

of CCN2 ⁄CTGF. This growth factor promotes physio-

logical chondrocytic proliferation and ECM formation.

Pro- and active MMP-3 could activate the

CCN2 ⁄CTGF promoter, where various domains of the

MMP participated 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. Compared to the wild-

type MMP-3, lower promoter activation occurred in

the presence of catalytically dead MMP-3 mutants.

This suggested that MMP-3 can regulate the

CCN2 ⁄CTGF promoter activity by two completely dif-

ferent mechanisms. One involves proteolytic processing

of one or several nuclear proteins, whereas the other is

independent of the processing capacity of the protein-

ase and involves the HPX domain. A DNA-binding

domain was found in the HPX domain, as an anti-

MMP-3 HPX antibody blocked the protein-DNA

interactions. The hinge region contains proline-rich

sequences found in some transcription factors. The

properties of MMP-3 as a transcription factor was

evaluated by analyzing nuclear MMP-3 associated pro-

teins (NuMAPs). Several NuMAPs were detected, such

as heterochromatin proteins, transcription co-activators ⁄corepressors, RNA polymerase II and nucleosome ⁄chromatin assembly protein. One of the NuMAPs,

HP1c, was demonstrated to interact with MMP-3 and to

co-activate the CCN2 ⁄CTGF promoter with MMP-3.

Another identified NuMAP was the transcription

repressor NCoR1, suggesting that MMP-3 might

degrade NCoR1 to prevent transcription repression of

the CCN2 ⁄CTGF promoter [130].

Cytosolic and vesicle localization

A study on dopaminergic neurons suggested a pro-

apoptotic role of active intracellular MMP-3. During

apoptosis, the proform of MMP-3 was cleaved to a

catalytically active form (48 kDa) by a serine protein-

ase [131]. Lack of intracellular MMP-3 activity pro-

tected the dopaminergic cells from apoptosis.

Inhibition of the MMP-3 activity attenuated the

activation of caspase-3, the executioner enzyme in

apoptosis.

By contrast to the apoptosis-promoting effects of

cytosolic MMP-3 and the MMPs localized in the

nucleus, perinuclear MMP-1 appeared to prevent

apoptosis [132]. Intracellular MMP-1 has been demon-

strated in various cell types, including glia cells, epithe-

lial cells and fibroblasts. At an early state of apoptosis,

both the pro- (57 kDa) and the active (45 kDa) forms

of MMP-1 colocalized with mitochondria that clus-

tered around the nucleus. At later stages, it accumu-

lated around the nucleus and nuclear fragments,

suggesting a possible role in the breakdown of the

nuclear envelope. Furthermore, the intracellular levels

of MMP-1 varied with cell cycle progression and were

highest during the M phase. These observations sug-

gest that intracellular association of MMP-1 to mito-

chondria and nuclei have implications for the control

of cell growth, and may contribute to the well-known

association of this enzyme with tumor cell survival and

spreading [133,134].

E. Hadler-Olsen et al. Regulation of MMP activity

FEBS Journal 278 (2011) 28–45 ª 2010 The Authors Journal compilation ª 2010 FEBS 37

Intracellular MMP-2 activity has been shown to be

a mediator of acute myocardial (ischemia ⁄ reperfusion)stunning injuries, characterized by a reversible loss of

contractile function during the post-ischemic reperfu-

sion phase [11]. In vitro and in vivo studies suggested

that this was a result of MMP cleavage of the contrac-

tile protein regulatory element, TnI, and the cytoskele-

tal protein a-actinin [11,135,136]. Other possible

MMP-2 substrates in cardiac myocytes are desmin and

myosin light chain-1 [136,137]. The most probable

mode of MMP-2 activation inside cardiac myocytes

undergoing ischemia-reperfusion injuries is via peroxy-

nitrite.

Unlike the other members of the MMP family, and

despite the presence of the N-terminal signal peptide,

most of the MMP-26 (matrilysin-2 ⁄ endometase) pro-

duced is reported to be retained inside the cell

[138,139]. The conserved PRCGXPD motif in the

prodomain involved in the latency of other MMPs is

replaced by the unique PH81CGVPD motif in MMP-

26. This motif, along with other atypical structures, is

assumed to facilitate autocatalytical activation of the

enzyme inside the cell [140]. Furthermore, it has been

reported that MMP-26 has one high-affinity and one

low-affinity calcium-binding site [141]. Normal intra-

cellular calcium-levels probably maintain MMP-26 in

an inactive state and the active enzyme may only be

seen during transient intracellular calcium influx. An

increased level of MMP-26 in breast cancer has been

found to correlate with longer patient survival [142].

This positive effect of intracellular MMP-26 is

assumed to be a result of its capacity to process the

estrogen receptor b [142].

Storage in exocytic vesicles

Polymorphonuclear leukocytes and mast cells can store

MMPs, as well as other proteinases and PGs, in exocy-

tic vesicles and release them into the extracellular envi-

ronment upon activation of the cells. Recent studies

have shown also that endothelial cells, chondrocytes

and various cancer cells can store MMPs in intracellu-

lar vesicles.

Endothelial cells could release MMP-2, MMP-9,

MT1-MMP, TIMP-1 and TIMP-2 very rapidly, sug-

gesting that they originate from intracellular storage

compartments. The vesicle content of both pro- and

active MMPs was increased by stimulation with the

angiogenic factors fibroblast growth factor-2 or vascu-

lar endothelial growth factor. The addition of isolated

vesicles to endothelial cells increased their ability to

invade and form capillary-like structures in vitro [143].

Growth plate cartilage cultures have been shown to

produce matrix vesicles that contain both pro- and

active MMP-2 and MMP-3, as well as TIMP-1 and

TIMP-2. The MMP activity was strongly increased by

treatment with the vitamin D metabolite 1,25-

(OH)2D3. Chondrocytes from growth zones produce

membrane vesicles with higher MMP content than

chondrocytes from resting zones, indicating that theses

enzymes are involved in ECM remodeling at the

hypertrophic cell zone in the growth plates of long

bones [144,145].

Ovarian carcinoma ascites-derived membrane vesi-

cles have been shown to contain both pro- and active

forms of MMP-2 and MMP-9, active urokinase-like

plasminogen activator, MT1-MMP and urokinase-like

plasminogen activator receptor. Ascites from patients

with late stage cancers had higher vesicle content and

contained more active enzymes than ascites from

patients with non-malignant lesions or early stage can-

cer. Purified ascites vesicles were found to stimulate

the invasion of cultured ovarian cancer cells through

matrigel, and this invasion was markedly inhibited by

the addition of either MMPI or serine proteinase

inhibitors [146]. Furthermore, fibrosarcoma cells are

also shown to shed membrane vesicles containing both

pro- and active forms of MMP-2 and MMP-9, as well

as urokinase plasminogen activator [147].

In oral carcinoma cells, both pro- and processed

forms of MMP-9 have been found in cytoplasmic

vesicular structures often co-compartmentalized with

trypsin-2, an activator of proMMP-9. In addition, the

same carcinoma cells expressed enterokinase, which is

an activator of trypsinogen, the zymogen form of tryp-

sin-2. This suggests the existence of an intracellular

cascade where enterokinase can activate trypsin-2,

which may further activate proMMP-9. The intracellu-

larly activated MMP-9 had a slightly higher molecular

weight than APMA activated MMP-9, which may rep-

resent intermediate forms that are more susceptible to

full activation after secretion [148].

In melanoma cells, MMP-2 and MMP-9 have been

detected in a high number of small, vesicular organ-

elles organized along the microtubular network. The

two enzymes were not colocalized, but were often

found in close proximity to each other. A high degree

of overlapping distribution was seen between the

MMP-2 positive vesicles, the motor protein kinesin

and a-tubulin within the cells. Treatment of the cells

with a microtubule-interfering drug impaired the secre-

tion of MMP-2 and MMP-9 [149]. 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 be actively propelled along

Regulation of MMP activity E. Hadler-Olsen et al.

38 FEBS Journal 278 (2011) 28–45 ª 2010 The Authors Journal compilation ª 2010 FEBS

microtubules towards the plasma membrane by the

motor protein kinesin. Shedding of such vesicles may

be a way of achieving rapid, directional proteolysis

during cell migration, invasion or during 3D morpho-

logical organization in the process of angiogenesis.

Concluding remarks

Post-translational regulation of MMP activity is com-

plex and involves various macromolecular interactions.

These interactions may direct the enzymes to specific

compartments in the extracellular environment, to the

cell surface or to intracellular sites. Furthermore, such

interactions may concentrate the enzymes close to or

on target substrates, and can also affect the activation

of inactive proenzymes. The binding of MMPs to

other macromolecules may also regulate the activity

of the enzymes either through stabilization or through

induction of autodegradation. Enzyme activity may be

regulated through binding partner interactions that

includes noncatalytic or exosites, and thereby inhibit

or prevent the processing of a specific substrate.

MMPs are involved in a large number of physiologi-

cal and pathological processes. An MMP may prevent

disease by processing one substrate, whereas the

same enzyme may promote disease by process-

ing another substrate. More research is needed to

increase the understanding of the localization of

MMPs, in vivo binding partners, substrate processing,

involvement of exosites in substrate processing and

the regulation of enzyme activity by binding partners.

On the basis of such information, new specific MMPI

targets for novel drugs may be discovered that are

both MMP- and substrate-specific. Hopefully, such

specific MMPIs can be used in therapy against rele-

vant diseases and result in less side effects compared

to the present MMPIs.

Acknowledgements

The Northern Norwegian Regional Health Authorities

are acknowledged for their support of Bodil Fadnes.

We apologize to those authors whose work could not

be cited as a result of space limitations.

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