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
Trang 1Regulation of matrix metalloproteinase activity in health and disease
Elin 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 domahemopex-in that is lhemopex-inked 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.
Trang 2other 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 specificactiv-ity, 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
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 N Zn
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.
Trang 3MMP 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 Proteolytic cleavage of the pro- and HPX-domains B Allosteric activation
Zn N SH
Zn SH
1
Zn
2
Zn
3
Zn
4
Zn SH N
1a
SH Zn
2a Auto-cleavage
Zn
3a Auto-cleavage
Proteases
Mercurials SH-reactive agents Chaotropic agents ROS
Detergents
Zn N SH
Zn
N SH
1
Zn SH N
Zn SH
1a
2a
Zn SH
3a
HgCl 2 / 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 HgCl 2 , 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 HgCl2or 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).
Trang 4formation 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 actiacti-vation-
activation-temperature as well as trypsin concentration and the two additives, Brij-35 and Ca2+ [22] At 37C, 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 implicaactiva-tions 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⁄ Trp90 versus 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
Trang 5prodomain 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 facregula-tor 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 HgCl2 and 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,
Trang 6peroxynitrite 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 removal
of 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 inteinte-grin 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
Trang 7the 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
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 the formation of heterodimers and complexes
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 subsub-strates ProMMP-9 forms a complex with one or several CSPG core
Trang 8proteins 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-chains
covalently 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
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 collagendegrading MMPs (MMP2, 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
Trang 9determinants 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 Exo-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 through
compartmentalization
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
Trang 10gelatinolytic 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-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]