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Critical Reviews in Biochemistry and Molecular Biology

ISSN: 1040-9238 (Print) 1549-7798 (Online) Journal homepage: http://www.tandfonline.com/loi/ibmg20

Biochemistry and molecular biology of gelatinase


B or matrix metalloproteinase-9 (MMP-9): The next
decade
Jennifer Vandooren, Philippe E. Van den Steen & Ghislain Opdenakker
To cite this article: Jennifer Vandooren, Philippe E. Van den Steen & Ghislain Opdenakker
(2013) Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9
(MMP-9): The next decade, Critical Reviews in Biochemistry and Molecular Biology, 48:3,
222-272
To link to this article: http://dx.doi.org/10.3109/10409238.2013.770819

Published online: 02 Apr 2013.

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ISSN: 1040-9238 (print), 1549-7798 (electronic)
Editor: Michael M. Cox
Crit Rev Biochem Mol Biol, 2013; 48(3): 222272
! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10409238.2013.770819

REVIEW ARTICLE

Biochemistry and molecular biology of gelatinase B or matrix


metalloproteinase-9 (MMP-9): The next decade
Jennifer Vandooren, Philippe E. Van den Steen, and Ghislain Opdenakker

Abstract

Keywords

Research on matrix metalloproteinases (MMPs) and in particular on gelatinase B, alias MMP-9,


has grown exponentially in the decade 20032012. Structural details about flexibility of MMP-9
monomers, together with glycosylation, oligomerization, heterogeneity and instability of the
wildtype enzyme explain why crystallography experiments have not yet been successful for the
intact enzyme. MMP-9 may be viewed as a multidomain enzyme in which the hemopexin,
the O-glycosylated and the catalytic domains yield support for attachment, articulation and
catalysis, respectively. The stepwise proteolytic activation of the inactive zymogen into a
catalytically active form becomes gradually better understood. Priming of activation by MMP-3
may be executed by meprins that destabilize the interaction of the aminoterminus with the
third fibronectin repeat. Alternatively, autocatalytic activation may occur in the presence of
molecules that tightly bind to the catalytic site and that push the cystein residue in the
prodomain away from the catalytic zinc ion. Thanks to the development of degradomics
technologies, substrate repertoires of MMP-9 have been defined, but it remains a challenge to
determine and prove which substrates are biologically relevant. The substrate repertoire has
been enlarged from extracellular to membrane-bound and efficient intracellular substrates,
such as crystallins, tubulins and actins. Biological studies of MMP-9 have tuned the field from
being primarily cancer-oriented towards vascular and inflammatory research. In tumor biology,
it has been increasingly appreciated that MMP-9 from inflammatory cells, particularly
neutrophils, co-determines prognosis and outcome. Aside from the catalytic functions
executed by aminoterminal domains of MMP-9, the carboxyterminal hemopexin (PEX)
domain of gelatinase B exerts non-catalytic anti-apoptotic signaling effects. The recognition
that gelatinase B is induced by many pro-inflammatory cytokines, whereas its inhibitors are
increased by anti-inflammatory cytokines, has generated interest to target MMP-9 in acute
lethal conditions, such as bacterial meningitis, sepsis and endotoxin shock, and in acute
exacerbations of chronic diseases. Previously described transcriptional regulation of MMP-9 is
complemented by epigenetic checkpoints, including histone modifications and microRNAs.
Because activation of proMMP-9 may be executed by other MMPs, the therapeutic dogma that
MMP inhibitors need to be highly selective may be keyed down for the treatment of lifethreatening conditions. When inflammation and MMP-9 fulfill beneficial functions to clear
damaging protein complexes, such as in systemic autoimmune diseases, therapeutic MMP
inhibition has to be avoided. In Mmp9 gene knockout mice, specific spontaneous phenotypes
emerged with effects on the skeletal, reproductive and nervous systems. These findings not
only have clinical correlates in bone growth and fertility, but also stimulate research on the
roles of MMPs and MMP-9 in endocrinology, immunology and the neurosciences. Mmp9deficient mice are valuable tools to define MMP-9 substrates in vivo and to study the role of this
enzyme in animal models of inflammatory, vascular, neoplastic and degenerative diseases.
Future challenges include solving the crystal structure, definition of the functions of covalent
oligomers and heteromers in biology and pathology, life-imaging of MMP-9 activity, substrate
determination in situ and the study of inhibitor effects on fertility, cancer and inflammation and
in neurobiology and regenerative medicine. Such studies will better define conditions in which
inhibition of MMP-9 is beneficial or has to be avoided.

Gelatinase B, MMP-9, pathology, physiology,


regulation, structure
History
Received 7 December 2012
Revised 24 January 2013
Accepted 24 January 2013
Published online 2 April 2013

20
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Laboratory of Immunobiology, Rega Institute for Medical Research, University of Leuven, KU Leuven, Belgium

Introduction

Address for correspondence: Ghislain Opdenakker, Laboratory of


Immunobiology, Rega Institute for Medical Research, KU Leuven,
Minderbroedersstraat 10, B-3000 Leuven, Belgium. E-mail: Ghislain.
Opdenakker@rega.kuleuven.be

Scientific progress is made by critically questioning obtained


data, by building new models, by reflection on existing
paradigms and, eventually, by overturning previous theories
that prove to be wrong. Scientific progress is also based
on investments of time, energy and resources into the

Gelatinase B/MMP-9

DOI: 10.3109/10409238.2013.770819

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Figure 1. Evolution of the PubMed literature on all MMPs, MMP-2, MMP-9, ATPsynthase, t-PA and caspases. The past decades, MMPs have
been increasingly studied. Following a contemplation period after the turn of the century, MMPs again attract attention and curiosity. MMP-2
and MMP-9, the two gelatinases, are the most studied MMPs. In comparison, the caspases are another enzyme family with caspase-3 being most
publicized.

development of new technologies and applications. In comparison with academia, industry has somewhat lost interest
in matrix metalloproteinases (MMPs) as druggable targets a
decade ago (Coussens et al., 2002). Possibly this is based
on expectations of obtaining a lucky strike, rather than on
critically analyzing obtained data to make the right choices
for industrial success. Excellent MMP inhibitors have been
developed meanwhile and all the present-day omics tools
provide us with clearer views on expression, regulation and
eventually altered activities of MMPs in pathological versus
normal conditions. These elements are stepping stones for
renewed industrial interests.
It is clear that applications of MMP inhibitors and MMP
diagnostics in vascular and inflammatory diseases are
approaching (Hu et al., 2007). Cancer therapy with MMP
inhibitors may also revive, when with the aid of new
technologies a broader, wider and deeper picture of what
happens in the tumor micro-environment is casted (Gerg
et al., 2008; Kruger et al., 2010; Nakasone et al., 2012;
Overall & Kleifeld, 2006).
The progress in fact revival and the shortcomings in
MMP research are best illustrated by comparisons of all MMP
literature in PubMed and that of some well-studied enzymes:
ATPsynthase (Lau & Rubinstein, 2012; von Ballmoos
et al., 2008), tissue-type plasminogen activator and the
caspases. ATPsynthase is an excellent example of a multidomain enzyme attracting multidisciplinary interests, ranging
from chemistry to biochemistry, from molecular biology
to physiology and from genetics to endocrinology. Tissue
plasminogen activator (t-PA) is an important example of
a proteinase that gained status from its clinical use in

thrombolysis as life-saver after myocardial infarction and


stroke (Elijovich & Chong, 2010; Tsui et al., 2005), whereas
caspases confront us with the fact that (programmed) death
is inherent to life (Feinstein-Rotkopf & Arama, 2009).
From Figure 1 it can be deduced that (i) MMP research
attracts increasing interest from biomedical scientists and thus
has grown exponentially in the last decade (ii) within the MMP
literature, studies on MMP-2 and MMP-9 are overrepresented
(iii) MMP-9, alias gelatinase B, tops at more than 50% of all
MMP literature. Although this picture is skewed and is a result,
probably an artifact, of the commonly used and picogramsensitive technology of gelatin zymography (Vandooren et al.,
2013), it also implies that filtering the literature to generate
holistic insights or generally applicable paradigms in the MMP
field becomes increasingly difficult. For this reason, the present
update of literature on gelatinase B/MMP-9 is built starting
from a previous critical review (Van den Steen et al., 2002a),
that still forms a primary resource for scientists entering the
field. Here, we try to complement the literature on gelatinase B/
MMP-9 since 2002. During this last decade, approximately
12 600 manuscripts about MMP-9 were entered into the NIH
PubMed database. Consequently, it is impossible to summarize
or mention all entries. This also implies that we apologize to
those scientists, who have contributed to stimulate the field, but
whose work could not be included here.
The MMPs are a family of more than 20 metallopeptidases
in Clan MA of proteolytic enzymes (Barrett et al., 2012;
Rawlings et al., 2010). The MMP family includes gelatinases,
collagenases, stromelysins, matrilysins and membrane-type
MMPs (Nagase et al., 2006). In Figure 2, we illustrate
the modular domain structure of prototypic family members,

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MMP

Locus

Gelatinase B

20q11.2-q13.1

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16p13.3

MT-MMPs

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Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

Stromelysins

Matrilysins

3, 10
11
7
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signal peptide
propeptide
active site
Zn2+-binding domain
hemopexin domain
fibronectin repeats
O-glycosylated domain
membrane anchor

11q22.3
22q11.23
11q21-q22
11p15

Figure 2. The modular domain structure of prototypic MMP family members. The MMPs are ordered by decreasing domain complexities and
their chromosomal locations in the human genome. Genome locations were obtained from the Entrez Gene Database (Maglott et al., 2011). The color
codes for individual domains will be used throughout the whole manuscript and is the same in all figures.

as ordered by decreasing domain complexities, and show the


chromosomal locations in the human genome. Further details
about nomenclature, gene synteny in the human species,
latency, activation and catalytic mechanisms have been
illustrated in previous reviews (Cauwe & Opdenakker,
2010; Cauwe et al., 2007; Parks et al., 2004; Van den Steen
et al., 2002a) and are not reiterated here. The MMP family
exerts its functions in biology and pathology in a network
alongside and together with other enzyme families, e.g. serine
proteinases and thiol proteases (Barrett et al., 2012), other
subfamilies, including, A disintegrin and metalloproteinases
(ADAMs) (Klein & Bischoff, 2011; Murphy, 2008) and
a disintegrin and MMP with thrombospondin motifs
(ADAMTSs) (Apte, 2009) and unique members such as
t-PA. This has been recognized as the protease network, the
protease web or the protease internet (Overall & Dean, 2006).
The origin of the name gelatinase B has previously been
explained and was later complemented with the name
MMP-9 to give insight into the catalytic principle (Cauwe
& Opdenakker, 2010). As for many other MMPs, extracellular
matrix substrate cleavage by gelatinase B/MMP-9 is only one
of the many functions of MMP-9. Throughout this manuscript, we will use gelatinase B and MMP-9 as true synonyms,
since both names complement each other and emphasize
different aspects of the same molecule.
While first studied as an enzyme involved in ECM
remodeling by degradation of mainly denatured collagens
(gelatins) (Collier et al., 1988) and other matrix-associated
substrates such as elastin (Senior et al., 1991) and aggrecan
(Fosang et al., 1992), it became clear that this is only a notion
of the catalytic functions of MMP-9 and not necessarily a real
in vivo executed function. Cytokines and chemokines may be
converted by MMP-9 into (more) active (pro-IL-1b, IL-8) or

inactive (CTAP-III, PF-4, GROa) immune signals


(Opdenakker et al., 2001b; Schonbeck et al., 1998; Van den
Steen et al., 2000). Similarly, membrane-bound proteins or
molecules at the interface between membranes and the
extracellular milieu may be processed (Cauwe et al., 2007)
by MMP-9. For example, degradation of myelin basic protein
(MBP) potentiates neuroinflammation and generates encephalitogenic peptides (Proost et al., 1993). Truncation of
ICAM-1 leads to inactivation of its adhesion function and
protects tumor cells from clearance by cytotoxic T cells and
NK cells (Fiore et al., 2002). MMP-9 regulates the permeability of epithelial barriers by degrading occludins in the
tight junctions connecting adjacent cells (Caron et al., 2005;
Giebel et al., 2005; Pflugfelder et al., 2005). Intriguingly, also
a broad range of intracellular proteins located in vesicles,
mitochondria, the nucleus and cytoplasm can be processed by
MMP-9 (Cauwe & Opdenakker, 2010). Important substrates
include cytoskeletal proteins such as actins and tubulins
(Cauwe et al., 2009), that are critical in providing shape to
cells and in molecular trafficking. In addition, MMP-9 is able
to induce aggregation of tau protein and this forms one of the
suggested mechanisms of Alzheimers disease (Nubling et al.,
2012). Finally, also non-catalytical signaling properties of
MMP-9 are being discovered, for example in cell apoptosis
(Redondo-Munoz et al., 2010). As a conclusion, MMP-9 or
gelatinase B is the prototype of a multi-domain enzyme
family with many functions in biology and pathology.

Recent insights in MMP-9 structure and activity


MMP-9 monomer as a multi-domain enzyme
MMPs are multidomain enzymes. The active site and the
metal binding site form the catalytic domain which is kept

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225

Figure 3. Amino acid sequence of MMP-9 with the indication of demonstrated posttranslational modifications by N-linked glycosylation, cysteine
bridging, oligomerization and proteolytic truncation, including activation of the zymogen form. All these processes are executed by enzymatic
reactions. The attachment of multiple O-linked oligosaccharides on the O-glycosylated domain is not indicated because site-specific annotation is not
yet known. The two free cysteine residues (in the O-glycosylated domain and in the hemopexin domain, indicated in red) are the candidates for the
formation of oligomers and covalent complexes with neutrophil gelatinase B-associated lipocalin (NGAL or lipocalin 2). Color code of domains is the
same as in Figure 2. a; meprin a, b; meprin b, NE; neutrophil elastase, b-hem; b-hematin, KK; tissue kallikrein, KLK7; kallikrein-related peptidases 7.

inactive by an aminoterminal propeptide domain (Nagase &


Woessner, 1999). Most MMPs, except the smaller matrilysins,
have a COOH-terminal hemopexin domain (PEX). This
domain is involved in substrate specificity and interacts
with inhibitors and cell surface receptors (Piccard et al.,
2007). The two gelatinases (gelatinase A/MMP-2 and
gelatinase B/MMP-9) contain three fibronectin (FN) repeat
which facilitate the degradation of (large) gelatinous
substrates (Pourmotabbed, 1994; Vandooren et al., 2011).
Uniquely, MMP-9 comprises a central O-glycosylated (OG)
domain, previously called the collagen V-like domain
(Wilhelm et al., 1989), which is a flexible 64 AA linker
between the catalytic domain and the PEX domain (Van den
Steen et al., 2006). A cartoon model for the multidomain
structure of intact MMP-9, expressed in insect cells and
thus including insect oligosaccharides, is shown in Figures 3
and 4 (panel D). In addition, panel A of Figure 4 illustrates in
the first model (based on the crystal structure of MMP-2) how
the attached oligosaccharides may contribute to the size and
shape of gelatinase B. In particular, the O-glycosylated

domain was first represented as a bar, as its shape was


hypothesized to be highly elongated due to steric effects of the
O-linked sugars (Opdenakker et al., 2001b). Panel B demonstrates a refinement of the original model structure with
experimentally obtained data of occupancy of N-glycosylation
sequons, structural glycan analysis and sedimentation coefficients from ultracentrifugation analysis (Van den Steen et al.,
2006). Low-resolution molecular analysis was obtained with
the use of small angle X-ray scattering (SAXS) experiments
(Rosenblum et al., 2007b) and the contour of one conformation with a detailed view of known crystallized fragments
is depicted in panel C. Finally, atomic force microscopy
analysis demonstrated the flexibility of the O-linked
domain (Rosenblum et al., 2007b). Because gelatinase B is
a glycosylated enzyme, possessing both N- and O-linked
oligosaccharides, molecular presentations represent only
single glycoforms. It needs to be stressed that glycosylated
MMP-9 preparations are always mixtures of glycoforms and
that the type and the culture conditions of the producer cells
determine the attached oligosaccharide compositions and

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site-specific glycosylation (or the glycotypes) (Dwek, 1996).


The fact that purified samples of MMP-9 are mixtures of
several different glycoforms implies that on SDS-PAGE
analysis the enzymes are visualized as broad and often
unsharply delineated protein bands. Figure 4 also represents
how scientific progress in structural analysis is made
when crystallography experiments are not yet successful: by
generating a model that integrates all information about
compositional analysis of the protein and its posttranslational
modifications, by reiterative refinements of the model on the
basis of further details into glycosylation site occupancy and
comparisons with other glycoproteins, by complementation
with the use of biophysical methods that yield information
about molecular shapes, including ultracentrifugation analysis, atomic force microscopy and SAXS analysis and by
bringing together multiple expertises.
Currently, only partial MMP-9 crystal structures are
available (Figure 4, panel C). A truncated MMP-9 form
containing the FN repeats, zinc ion binding domain, the active
domain and propeptide has been crystallized and imaged
(PDB ID: 1L6J) (Elkins et al., 2002). Also, a deletion mutant
containing only the active domain and Zinc-binding site was
crystallized (Rowsell et al., 2002). The recombinant
expressed MMP-9 hemopexin domain has also been crystallized and characterized (PDB ID: 1ITV) (Cha et al., 2002).
Finally, with the use of SAXS, Rosenblum et al. have
established the first low-resolution picture of an inactive
mutant of full size pro-MMP-9 (Rosenblum et al., 2007b).
The contour and the specific domains of one conformation of
this structure are shown in panel C of Figure 4. Presently, this
is the highest resolution three-dimensional structure of the
MMP-9 monomer, which lends itself to generate a useful
cartoon model.
The catalytic domain of a true MMP
The catalytic domain is formed by combining the metalbinding site and the active site. The catalytic cleft of MMP-9
is closely similar to that of other MMPs and contains a
catalytically essential Glu402 and a Zn2 ion. Nevertheless,
unique amino acids in the active site are important for
enhancing the catalytic efficiency towards gelatin and peptide
substrates. Leu397 and Ala406 are important for general
catalytic activity, whereas Asp410 and Pro415 specifically
contribute to enhancing the ability to cleave type V collagen
and gelatin, respectively (OFarrell & Pourmotabbed, 2000).
With the use of phage display technology, three major
substrate families have been identified. The largest group has
a Pro-X-X-Hy-(Ser/Thr) consensus sequence (X represents
any residue and Hy is a hydrophobic residue). The second
group has a Gly-Leu-(Lys/Arg) motif and the third group of
substrates has two subsequent Arg residues (Arg-Arg) (Kridel
et al., 2001). More than 20 cleavage sites were also identified
in denatured collagen type II (Van den Steen et al., 2002b).
In recent years, degradomics studies have revealed a broad
range of new MMP-9 substrates (Cauwe et al., 2008, 2009;
Greenlee et al., 2006; Prudova et al., 2010; Vaisar et al., 2009;
Xu et al., 2008) and detailed overviews have been published
about the full spectrum of MMP-9 substrates (Cauwe &
Opdenakker, 2010; Cauwe et al., 2007; Prudova et al., 2010;
Van den Steen et al., 2002a).

Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

The propeptide domain is essential for latency


Propeptide domains in MMPs are aminoterminal sequences of
approximately 80 amino acids that are important for enzyme
control. These contain the cysteine switch PRCXXPD
consensus sequence, which interacts with the catalytic zinc
ion, thereby keeping the enzyme inactive (Springman et al.,
1990; Van den Steen et al., 2002a). Activation of the enzyme
requires either proteolytic processing of the propeptide
domain or physical or steric disruption of the propeptide
conformation. Proteolytic activators of MMP-9 include MMP3 (Ogata et al., 1992), kallikrein (Desrivieres et al., 1993),
plasmin (Lijnen et al., 1998) and neutrophil elastase (NE)
(Ferry et al., 1997). Interference with the propeptide involves
modification of the cysteine residue and its interaction with
the catalytic site zinc ion. Examples include oxidation
(Meli et al., 2003; Paquette et al., 2003), S-nitrosylation
(Gu et al., 2002) or S-glutathiolation (Okamoto et al., 2001).
Figure 5 shows detailed information on the structure of the
propeptide of MMP-9. The propeptide contains an occupied
N-glycosylation site and two MMP-3 cleavage sites.
N-glycosylation at Asn38 has been described (Kotra et al.,
2002) and might protect proMMP-9 from proteolytic degradation or autocatalysis. Removal of this glycan did not affect
the activation by MMP-3 (Van den Steen et al., 2001).
Although several enzymes are able to process the propeptide
domain, MMP-3 is most often used for in vitro activation
studies (Geurts et al., 2008, 2012a; Olson et al., 2000).
It introduces an initial truncation between Glu59/Met60
which reveals a second truncation site between Arg106/
Phe107, resulting in an active 82 kDa MMP-9 (Ogata et al.,
1992). Activation with the catalytic fragment of stromelysin1/MMP-3 is preferred for biological experiments above
organomercurial activation with APMA, the latter of which
is extremely toxic and difficult to remove after sample
activation.
The fibronectin repeats yielding protein affinity
Between the active site and the metal binding site, a sequence
is inserted that consists of a cluster of three fibronectin (FN)
repeats. The three repeats are almost identical and each
includes two intramolecular disulfide bonds (Figure 3)
(Elkins et al., 2002). The FN repeats are important for the
binding and catalysis of large substrates such as elastins
(Shipley et al., 1996) and denatured collagens or gelatins.
This was demonstrated in several independent studies (LauerFields et al., 2008; Shoji et al., 2011; Steffensen et al., 1995;
Vandooren et al., 2011; Xu et al., 2005). Tyr320, Arg307,
Asp309, Asn319 and Asp323 were shown to be important for
the binding to gelatins (Collier et al., 1992).
The O-glycosylated domain and enzyme flexibility
The OG domain in human MMP-9 is a unique linker sequence
which connects the active site to the hemopexin domain
(Figures 3 and 4). The linker is only 64 amino acids long and
is rich in proline, serine and threonine residues. For a certain
time this linker domain has been named the collagen type Vlike domain because of its homology with the collagen type V
sequence (Wilhelm et al., 1989). Later, it was found that this

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Figure 4. The multidomain structure of MMP-9. The following MMP-9 domains are shown; the propeptide (green), the active site (yellow), the three
fibronectin repeats (blue), the metal binding site (orange) with the catalytic zinc ion (grey), the OG domain (brown) and the PEX domain (red). Three
different representations are shown. Panel A; model of MMP-9 from 2001 (Opdenakker et al., 2001b). Panel B; A refined model for MMP-9, based on
compositional and site-specific glycan analysis and sedimentation data, generated by Dr. Mark Wormald, Oxford University Glycobiology Institute
(Van den Steen et al., 2006). Panel C; a contour model of one MMP-9 conformation based on the observations made with the use of atomic force
microscopy and SAXS analysis (Rosenblum et al., 2007b). The colored protein segments are based on the analysis of the two presently available crystal
structures of MMP-9 (PDB ID: 1L6J and 1ITV). It needs to be emphasized that the O-glycosylated domain and its attached oligosaccharides yield
molecular flexibility and heterogeneity. As a consequence, the picture represents one conformation of many possible shapes. Panel D; cartoon model,
illustrating the fingertip interaction between the aminoterminus of the prodomain with the third fibronectin repeat, the flexibility of the O-glycosylated
domain, the structural separation of the catalytic part and the hemopexin domain and the open spaces occupied by highly mobile oligosaccharides
(not indicated). The color code for the indication of domains is the same as in Figures 2 and 3.

domain is rich in O-glycans, with similarity to mucins and


therefore was renamed as O-glycosylated domain or OG
domain (Van den Steen et al., 2006). Several studies indicated
that this domain is indispensable for correct MMP-9 function.
For example, deletion of the OG domain reduces MMP9-mediated cell migration (Dufour et al., 2008), significantly
lowers the efficiency of degradation of large gelatinous
substrates (Vandooren et al., 2011) and disrupts inhibition
by TIMP-1 and internalization by cargo receptors LRP-1 and
LRP-2 (Van den Steen et al., 2006). In addition, structural
studies indicated that this O-glycosylated domain yields
flexibility to intact MMP-9 (Rosenblum et al., 2007b).
Two types of flexibility may be distinguished in MMPs:
intradomain and interdomain flexibility. For example, intradomain flexibility relates to different conformations of the
substrate binding pockets in the catalytic domain. Interdomain
flexibility involves the orientation of the protein domains
relative to each other, for example, the orientation of
the catalytic site relative to the hemopexin domain. The
OG domain lends the MMP-9 molecule a high degree of
interdomain flexibility, by allowing independent movements
of the terminal hemopexin domain and catalytic domain.
In general, two conformations are found, the extended and
the contracted conformation, respectively, separating and
combining the hemopexin and catalytic domains (Rosenblum
et al., 2007b). This feature is implicated in the degradation of
denatured collagens (Overall & Butler, 2007; Rosenblum
et al., 2010). Although collagens are highly stable structures
(Ramachandran & Kartha, 1954), resistant to most proteases,
they can be degraded by MMP-9 after an initial cleavage by
one of the three collagenases (MMP-1, MMP-8 or MMP-13).
Characteristically, collagenases generate g and fragments

(Van den Steen et al., 2004) and, upon binding, MMP-9


will crawl along the collagen fibrils towards the free
collagen tails (Rosenblum et al., 2010; Saffarian et al., 2004).
It was suggested that the intradomain flexibility of the
O-glycosylated domain may be involved mechanistically in
this process (Overall & Butler, 2007). Upon arrival at the site
where collagenase has cleaved the segment, MMP-9 further
induces unwinding of the collagen fibril, a feature that does
not require catalysis, because it also happens with a catalytically dead mutant of MMP-9 (Rosenblum et al., 2010).
The hemopexin domain as a lifebuoy
Sequence alignment of the hemopexin domains of all human
MMPs shows that the hemopexin domain of MMP-9 forms a
separate cluster (Massova et al., 1998). In fact, it shares only
2533% amino acid identity with hemopexin domains from
other MMPs (Dufour et al., 2011). This finding adds to the
uniqueness of MMP-9 within the MMP family. During the
last decade, further insights into both structure and function
of the hemopexin domain have been established.
Structurally, this carboxyterminal domain of MMP-9
consists of a four-bladed b-propeller with a disulfide bridge
(between Cys516 and Cys704) connecting blades I and IV.
This covalent linkage is critical for the structural integrity
of the domain (Cha et al., 2002). Recombinant MMP-9
hemopexin domains have been produced as monomers and
reduction-sensitive multimers. Further investigations indicated that the multimers were not formed by intermolecular
disulphide linkages but predominantly by hydrophobic interactions. For this reason some authors have suggested a role for
the hemopexin domain in the formation of MMP-9 multimers

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J. Vandooren et al.

(Cha et al., 2002; Dufour et al., 2008). It remains a point of


discussion whether the disulfide bridges in the hemopexin
domain are the same for monomers and multimers.
Four functions have already been attributed to the
hemopexin domain: interaction with substrates, binding to
inhibitors, binding to cell surface receptors and induction
of auto-activation. It interacts with substrates such as gelatin,
collagen type I, collagen type IV, elastin and fibrinogen
(Burg-Roderfeld et al., 2007; Roeb et al., 2002). Remarkably,
the affinity of this interaction is higher for full-length
proMMP-9 than for activated MMP-9 without the propeptide
(Burg-Roderfeld et al., 2007). Besides substrate binding,
the hemopexin domain also interacts with inhibitors, for
example with the carboxyterminus of TIMP-1 (Goldberg
et al., 1992) and with TIMP-3 (Butler et al., 1999). However,
the exact interaction interfaces are not yet known. The
hemopexin domain also mediates interaction with receptors
at cell surfaces, such as the cargo receptors low-density
lipoprotein receptor related-protein-1 (LRP-1) and megalin/
LRP-2 (Hahn-Dantona et al., 2001; Van den Steen et al.,
2006). Through this interaction, endocytosis and catabolism
of the MMP-9 enzyme is potentiated (Piccard et al., 2007).
In other cases, the hemopexin interaction is a way to
localize MMP-9 to the cell membrane, for example by the
interaction with the Ku70/Ku80 heterodimer (Monferran
et al., 2004).
Recently, a new feature of the PEX domain was discovered. Several substrates induce proMMP-9 autocatalysis
and thereby potentiate activation of the enzyme. In analogy
to the binding of heme with hemopexin, b-hematin interacts
with the MMP-9 PEX domain and induces autocatalytic
processing of proMMP-9 (Geurts et al., 2008). Likewise,
a macrophage cell line (THP-1) secretes a complex of MMP-9
with a chondroitin sulfate proteoglycan (CSPG) (Winberg
et al., 2000). This MMP-9/CSPG complex is covalently linked
through the hemopexin domain with autocatalytic properties
(Winberg et al., 2003).
Posttranslational modifications introducing
structural variation
Posttranslational modifications alter specific parts of a
protein/enzyme after synthesis, thereby inducing extra levels
of structural and functional diversity. Posttranslational modifications may be executed upon many different amino acid
residues and thus exist in great variety. A well-known
posttranslational modification in MMP-9 is the formation of
disulfide bonds between two cysteine residues within one
chain. This modification fixes structural conformations within
protein domains. The proMMP-9 sequence (without the signal
peptide) contains 17 cysteine residues (Figures 3 and 5).
However, only seven intramolecular cysteine bridges are
formed. Each of the three fibronectin repeats contains two
cysteine bridges and these connections are necessary for the
secretion of proMMP-9 by cells (Khan et al., 2012). The PEX
domain has a single cysteine bridge interconnecting hemopexin blade I with blade IV (Cha et al., 2002). Furthermore,
disulfide bridges may also be formed between several MMP-9
monomers: this leads to the formation of MMP-9 multimers
(Van den Steen et al., 2006). These may involve cysteine

Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

468 in the O-glycosylated domain and cys674 in the


hemopexin domain.
In addition, cysteine 99 in the propetide, which interacts
with the catalytic zinc ion and thereby keeps the enzyme
inactive, is prone to several types of posttranslational
modifications that most often result in activation of the
enzyme. For example, interaction of this cysteine thiol with
nitric oxide results in S-nitrosylation, irreversibly modifying
the residue into a sulfinic (SO2H) or sulfonic (SO3H) acid
and activation of the enzyme (Gu et al., 2002).
MMP-9 contains both O-glycans and N-glycans (Mattu
et al., 2000; Rudd et al., 1999). The most densily packed
glycans on MMP-9 are O-glycosylations, situated in the
O-glycosylated domain which contains 14 potential glycosylation sites (Mattu et al., 2000; Van den Steen et al., 2006).
Three possible N-glycosylation sites, with the sequons
Asn-Xaa-Ser or Asn-Xaa-Thr (in which Xaa is any amino
acid except proline), are available in the MMP-9 sequence,
of which only two are occupied (Figures 3 and 5). Nglycosylation is mediated by the oligosaccharyl transferase at
Asn38 and Asn120, respectively, in the propeptide and in the
catalytic domain (Kotra et al., 2002; Van den Steen et al.,
2006). In the endoplasmic reticulum (ER) of the cell,
underglycosylated 85 kDa MMP-9 is altered into an 89 kDa
intermediately glycosylated form and finalized into the
92 kDa MMP-9 when passing through the Golgi apparatus
(Hanania et al., 2012; Olson et al., 2000). N-glycosylation
happens cotranslationally on the nascent protein (Dwek,
1996), whereas O-glycosylation occurs during later stages in
the Golgi and trans-Golgi when the protein domains are
already folded (Van den Steen et al., 1998).
Oligomers, complexes and truncated forms
In human tissues and biological fluids, MMP-9 is found as
monomers and oligomers, in complexes with other molecules
and as low-molecular weight truncated forms. In most
manuscripts on MMP-9, the authors have placed emphasis
on or only mention the monomeric MMP-9 form. It remains a
point of discussion whether such simplification is justified.
Are the oligomers functionally identical to monomers,
i.e. mechanistically superfluous, or do these exert specific
functions? Are these and other forms identically or differently
regulated in physiology and pathology? Do truncation forms,
complexes and oligomers possess similar or different substrates, receptors, inhibitors than monomers? These and other
questions are examples to illustrate that still much has to
be discovered about the various forms of MMP-9. In Figure 6,
various forms of MMP-9, present in biological samples, are
illustrated by zymography analysis.
Oligomers
MMP-9 oligomers are simultaneously produced with MMP-9
monomers, but usually in minor quantities. Since the monomers are more abundant, these are also most easily observed
with the use of gelatin zymography analysis. In all biological
samples, we have always observed multimers together with
the monomers, but sometimes samples needed to be
concentrated to visualize the oligomers. Several publications
specifically mention homodimers as the reduction-sensitive

Gelatinase B/MMP-9

229

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DOI: 10.3109/10409238.2013.770819

Figure 5. Domain structure of MMP-9 and details of the propeptide. The propeptide contains the cysteine switch consensus sequence PRCXXPD
(underlined) which contains Cys99 with which it blocks the enzyme active site. The propeptide sequence is subject to several types of posttranslational
modifications including N-glycosylation. The secondary structure of the propeptide consists of three perpendicular a-helices. At the aminoterminus,
the propeptide has hydrophobic interactions and forms a hydrogen bridge with residues from the third fibronectin repeat. Centrally, the domain tightly
interacts with the active site and the metal binding site (Elkins et al., 2002).

230

J. Vandooren et al.

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Complexes

Figure 6. Gel zymographic analysis of bronchial alveolar lavage fluid


samples from non-cystic fibrosis bronchiectasis patients and illustration
of relative abundances of monomers versus oligomers and of truncation
forms. Besides MMP-2, that is present in all biological fluids or extracts,
several forms of MMP-9 are detected. In the high molecular weight
region, MMP-9 oligomers are typically seen at the top of a 7.5%
acrylamide gel. The covalent MMP-9/NGAL complexes are found in
between the MMP-9 monomers and oligomers, and are indicative for the
presence of neutrophils in the biological sample. Often, truncated MMP9 forms are found at the bottom of the gel. The uses of zymographic
methods has recently been reviewed (Vandooren et al., 2013). (Samples
are by the courtesy of Dr. P. Goeminne, Division of Pneumology,
University Hospitals Gasthuisberg, KU Leuven).

oligomeric MMP-9 forms (Collier et al., 2011; Olson et al.,


2000). However, in our hands as well as in other studies, the
migration of the oligomers on SDS-PAGE analysis exceeded
the theoretical value of dimers (Opdenakker et al., 2001a;
Piccard et al., 2007; Van den Steen et al., 2006) and
corresponded more with trimers than with dimers. Till
clear structural evidence for dimers becomes available, for
instance, from atomic force microscopy, SAXS, cryo-electron
microcopy or X-ray diffraction analysis, we advocate to
use the terminology of multimers or oligomers instead of
dimers.
Oligomerization is thought to require the formation of
intermolecular cysteine bridges. Thus, for dimers one disulfide bridge is sufficient, whereas for trimers, at least two such
covalent linkages are necessary. It was proposed that the
free cystein residue within the hemopexin domain (Cys674,
Figure 3) mediates MMP-9 oligomerization. However, this
residue was claimed to be buried within the protein. Indeed,
recombinant MMP-9 hemopexin domains were produced as
non-covalent oligomers in vitro (Cha et al., 2002). In addition,
a study with MMP-9 deletion mutants showed that also the
O-glycosylated domain is essential for the formation of
oligomers. Based on these observations, it is tempting to
assume that Cys468, which is located in the OG-domain,
is involved in the formation of MMP-9 multimers. However,
directed mutagenesis studies showed that without cysteine468
the formation of oligomers was still possible (Van den Steen
et al., 2006).
In functional studies, Dufour et al. showed that MMP-9
multimers interact with CD44, leading to the activation
of EGFR and consequently the MAPK (ERK1/2) pathway,
thereby mediating cancer cell migration (Dufour et al.,
2008, 2010, 2011). By targeting a compound to the
hemopexin domain, the formation of multimers could
be hindered and cancer cell migration was abolished
(Dufour et al., 2011).

Neutrophil gelatinase-associated lipocalin (NGAL) in association with MMP-9 is the best known heteromeric complex
of MMP-9. In the human species, it has a molecular weight
of 125 kD and is formed by covalent linkage of MMP-9
and NGAL (Triebel et al., 1992). NGAL belongs to the
superfamily of lipocalins (Kjeldsen et al., 1993), which
are biochemical markers for a variety of diseases (Xu &
Venge, 2000). The NGAL/MMP-9 complex is mainly
secreted by neutrophils. Possibly for these reasons, the
NGAL/MMP-9 complex is useful as a maker for several
diseases (Hatipoglu et al., 2011; Tsai et al., 2011).
Functionally, complex formation with NGAL is thought to
protect MMP-9 from proteolytic degradation (Yan et al.,
2001b). Another high-molecular weight (300 kDa) MMP-9
complex is produced by human macrophages. As mentioned
above, the leukemic macrophage cell-line THP-1 secretes
a MMP-9/CSPG complex (Winberg et al., 2000) with altered
biochemical properties (Malla et al., 2008).
Truncated forms
Besides the active 82 kDa MMP-9 (Ogata et al., 1992),
MMP-3 also generates a 65 kDa form of MMP-9, lacking both
the aminoterminal propeptide and the carboxyterminal
hemopexin domain (Okada et al., 1992). Since the carboxyterminal domains of MMP-9 are required for a high affinity
binding with the natural inhibitor TIMP-1 (OConnell et al.,
1994), 65 kDa MMP-9 is able to escape inhibitor control.
65 kDa MMP-9 has been purified from body fluids and was
less susceptible to inhibition by TIMP-1. In addition, KLK7
and meprin-a are also able to remove the carboxyterminal
domains from MMP-9 (Bellini et al., 2012; Geurts et al.,
2012a; Ramani et al., 2011).

Regulation of MMP-9 levels and activity


The general principles and the various levels at which MMP-9
activity is regulated were outlined already a decade ago
(Opdenakker et al., 2001a). These are reiterated briefly here
and novel findings from recent literature are highlighted.
A first level is the regulation of MMP-9 under the control
mechanisms of a variety of signaling pathways that stimulate
or reduce the transcription of the MMP9 gene. Second, MMP9 is regulated at the level of mRNA and translation into the
preproenzyme. Several signaling pathways are able to stimulate the degradation of MMP-9 mRNAs thereby reducing the
final amount of preproMMP-9. One novel principle at this
level is regulation by microRNAs and this will be highlighted
here. Next, proMMP-9 may be stored in secretory vesicles of
neutrophils, making secretion of proMMP-9 a third level of
regulation. When MMP-9 is secreted as a proenzyme by fast
(within minutes to 1 h) degranulation of preformed and stored
MMP-9 from neutrophils or by classical and slower secretion
mechanisms of all other cell types (a process that takes at least
612 h), proMMP-9 has to be activated by various mechanisms (vide supra) in the extracellular milieu. Finally, once
activated this activity is regulated by various MMP-9
inhibitors. The different levels of MMP-9 regulation are
illustrated in Figure 7. We will now discuss these various

DOI: 10.3109/10409238.2013.770819

levels of regulation in more detail and with an emphasis of


novel findings from the last decade. For insights into the
literature of MMP-9 regulation from before 2002, the reader
is referred to a previous resource (Van den Steen et al.,
2002a). An overview of recently discovered molecules known
to stimulate or block MMP-9 is shown in Table 1. The method
and levels of regulation are included.
Transcriptional regulation of MMP-9

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General considerations: receptors and signaling


Both gelatinases (MMP-2 and MMP-9) are encoded by genes
located on different human chromosomes. Whereas most
MMPs are encoded by human chromosome 11 (Van den
Steen et al., 2002a) and MMP-2 is generated from human
chromosome 16 (Murphy & Crabbe, 1995), MMP9 is syntenic
with MMP24 on human chromosome 20q11.2 (Figure 2). It is
a well-documented fact that many members of the MMP
family are transcriptionally controlled by cytokines, growth
factors, hormones and cell interactions (Nagase et al., 2006)
and MMP-9 follows this rule (Van den Steen et al., 2002a).
Gelatinase B is produced by a range of immune cells,
e.g. monocytes, macrophages, lymphocytes and dendritic
cells, and also by other cells such as osteoblasts, fibroblasts
and endothelial cells (Opdenakker et al., 2001b). An
updated overview of MMP-9 secreting cells can be found
in Table 2.
In general, MMPs are regulated by the mitogen-activated
protein kinases (MAPKs). This family of kinases has three
major members: the extracellular signal-related kinases
(ERKs) which are activated by mitogens and phorbol esters,
the c-Jun N-terminal kinase (JNK)/stress-activated protein
kinases and p38 which are triggered by cellular stress and
inflammatory cytokines. MMP-9 is mainly regulated by
ERK1/2. However, a vast range of additional signaling
pathways have been discovered. Among more recently
discovered pathways are those mediated by farnesoid receptors, peroxisome proliferator-activated receptors, integrin
interactions and metabolic sensors, including the sirtuins,
and galectins. An overview of the regulation of MMPs and
their inhibitors was by Clark et al., including an extensive
discussion on the promoter organization of all MMPs (Clark
et al., 2008).
Often suppression of MMP-9 transcription is a result of
interference with the binding of essential transcription factors
such as NF-kB and AP-1 to the promoter region. Several
other factors were shown to interfere with the basic MMP-9
promoter activation machinery. Examples include Kiss1
(Lee & Welch, 1997), RECK (Takagi et al., 2009), EGR-1
(Bouchard et al., 2010), LZAP (Wang et al., 2007) and the
ATXN1 protein family (Lee et al., 2011). Repressor
complexes have also been documented. For example, a
complex of SP2 and KLF6 binds to the Sp1 site in the
MMP-9 gene promotor and thereby suppresses MMP-9
transcription. Upon activation of the farnesoid X receptor
(FXR) signaling pathway the SP2/KLF6 repressor complex is
disrupted by interaction with SHP (Das et al., 2006).
Bile acids were found to induce MMP-9 production
by binding to nuclear FXR which subsequently binds to the
MMP-9 promoter region and induces transcription.

Gelatinase B/MMP-9

231

In addition, this FXR-MMP-9 pathway potentiates focal


adhesion kinase (FAK) activation and attenuates AP-1
MMP-9 upregulation (Das et al., 2009). Rosiglitazone, a
peroxisome proliferator-activated receptor-g (PPAR-g) agonist, inhibits MMP-9 expression by activating glycogen
synthase kinase (GSK)-3b. In vascular smooth muscle cells
(VSMCs) this leads to reduced proliferation and survival
(Lee et al., 2009). MMP-9 expression can be upregulated by
MEK5 through AP-1 (Mehta et al., 2003)
A role has been suggested for the MMP-2. MT1-MMP
complex in MMP-9 regulation. Binding of MMP-2 to MT1MMP is thought to decrease the expression levels of MMP-9
(Esparza et al., 1999). This might be in line with reported
compensation mechanisms in MMP-2-knock out (KO) mice
(Esparza et al., 2004). In a mouse keratinocyte cell line
(MK cells), it was demonstrated that MMP-9 levels are
elevated via signaling through a a3b1 integrin and MEK/
ERK-dependent pathway (Iyer et al., 2005). Upon induction
of Ras, MMP-9 levels were upregulated. Galectin-7 is
able to induce de novo MMP-9 mRNA synthesis (Demers
et al., 2005).
Increased epigenetic expression of AP-1 and NF-kB by
histone-4 acetylation may occur as a result of SIRT1
inhibition (Nakamaru et al., 2009) and forms a way to link
cellular metabolism to gene expression. AP2a is able to
downregulate MMP-9 expression by binding to the MMP-9
promotor region and thereby interfering with transcription
factors AP1 and Sp-1 (Schwartz et al., 2007).
Downregulation of SCC-S2, an NF-kB-inducible transcription factor associated with enhanced breast cancer cell
invasion and metastasis, coincided with the decreased
expression of MMP-9 and MMP-1 (Zhang et al., 2006).
Few proteins are known to down-regulate MMP-9 expression. One of them is Kiss-1, known as a suppressor of cancer
cell proliferation and metastasis (Li et al., 2012). It was found
to be a suppressor of MMP-9 expression by reducing NF-kB
binding to the promoter region (Yan et al., 2001a).
Differentiating-repression factor-1 (DRF-1) also negatively
regulates MMP-9 expression by binding to KRE in the MMP9 promoter region (Kobayashi et al., 2004). A newly
discovered negative regulator of MMP-9 is transgelin, a
2225 kDa actin-binding protein which is situated in the cell
membrane and cytoplasm (Nair et al., 2006).
When cells migrate out of blood vessels through the layer
of endothelial cells and basement membranes, they encounter
a pleiotropy of ECM molecules and these may influence
the production of MMP-9. For T-cells it was shown that
fibronectins can upregulate the production of both MMP-2
and MMP-9 (Esparza et al., 1999). In addition, a4b1integrinmediated adhesion to VCAM-1 also induces MMP-2 and
MMP-9 production (Yakubenko et al., 2000).
Exogenous stimuli of MMP-9 expression have been added to
the long list of agonists and these may be physical, biochemical
or biological stimuli, whereas also novel elements of cell
transformation have been linked to MMP-9 regulation.
Infrared (IR) radiation from natural sunlight leads to
heat shock response and induces the production of MMP-1
and MMP-9 in human keratinocytes by inducing ERK, JNK
and p38 kinase signaling (Shin et al., 2008). Lipoteichoic acid
(LTA), a component of Gram-positive bacterial cell walls, is

PPAR-g agonist, inhibition of ERK and activation of GSK-3b, results in reduced expression of MMP-9
Inhibition of the RhoA/ROCK pathway but increased MMP-9 mRNA levels
Inhibition of expression
Induces MMP-9 expression
Induces expression of MMP-9
T-cell binding to VCAM-1, mediated through a4b2 integrin
Induces MMP-9

Rosiglitazone
Simvastatin
Tetracyclins
TGF-b
TNF-a
VCAM-1
VEGF

Inhibition of secreted levels of MMP-9, was suggested to involve destabilization of the actin cytoskeleton

Induces autocatalytic activation of proMMP-9 by interacting with the hemopexin domain

Simvastatin

Regulation of activation
B-hematin

(Geurts et al., 2008)


(continued )

(Khan et al., 2012)


(Oh et al., 2001; Rhee & Coussens, 2002; Takahashi
et al., 1998)
(Turner et al., 2005; Turner et al., 2007)

(Ingraham et al., 2011)


(Bonacci et al., 2011)
(Chen et al., 2011)
(Fayard et al., 2009)
(Cheng et al., 2006)
(Oh et al., 2001; Rhee & Coussens, 2002; Takagi
et al., 2009; Takahashi et al., 1998)
(Lee et al., 2009)
(Turner et al., 2005; Turner et al., 2007)
(Salo et al., 2006)
(Lamar et al., 2008a)
(Nee et al., 2004; Porter et al., 2004)
(Yakubenko et al., 2000)
(Hiratsuka et al., 2002)

(Steenport et al., 2009)

(Wang et al., 2010)


(Wang et al., 2007)
(Tai et al., 2010)
(Steenport et al., 2009)

(Zhang et al., 2011)


(Huwiler et al., 2003)
(Lee et al., 2011)
(Huang et al., 2011)
(Das et al., 2006)
(Rajapakse et al., 2006)
(Ganor et al., 2009; Huang et al., 2011; Major et al.,
2002)
(Yagi et al., 2009)
(Esparza et al., 1999; Yakubenko et al., 2000)
(Demers et al., 2005)
(Cheung et al., 2006; Chou et al., 2003)
(Straat et al., 2009)
(Gschwandtner et al., 2008)
(Quiney et al., 2006b)
(Purwar et al., 2008)
(Nee et al., 2004; Wang et al., 2005; Wu et al., 2004;
Wu et al., 2009)

References

J. Vandooren et al.

Regulators of compartmentalization and secretion


PDI
Mediates the formation of intermolecular disulfide bonds, required for secretion of MMP-9
RECK
Prevents secretion of MMP-9

Low O2 level
OA-NO2
oxLDL
PN-1
Radiation
RECK

MMP-3

LTA
LZAP
Melatonin
MMP-1

Decreases MMP-9 mRNA levels


Induces expression of MMP-9
Induction of de novo mRNA synthesis
Induces expression of MMP-2 and MMP-9
Reduces MMP-9 mRNA levels
Induces MMP-9 production , H1R receptor signaling
Reduces MMP-9 expression
Higher mRNA levels
Abolishes the stimulating effect of TNF-a.
Enhances MMP-9 secretion (MCF-7 cells) through SHP-2-dependent pathway.
Induces MMP-9 expression in astrocytes through caMKII/JNK/c-JUN cascade.
Induces expression via calcium/caM/caMKII-dependent transactivation of PDGFR pathway
Selective inhibition of NF-B
Reduces transcription by inhibition of p-ERK1/2, activated protein factor-1, and protein kinase C
Induces MMP-9 expression in macrophages by stimulating the COX-2/PGE2/EP4 signaling pathway and inducing
TNF-a production
Induces MMP-9 expression in macrophages by stimulating the COX-2/PGE2/EP4 signaling pathway and inducing
TNF-a production
Reduced O2 levels result in induction of MMP-9 expression via HIF-1a and the Wnt pathway
Inhibition of expression by binding to PPARg
miRNA mediated upregulation
Increased expression of MMP-9
Increased expression of MMP-9 by inducing the PI3K/Akt pathway
Reduces expression of MMP-9 by reducing promoter activity

Induces MMP-9 production through activation of ERK, p38, mitogen-activated protein and NF-kB
Stabilizes MMP-9 mRNA upon induction with IL-1b
Represses the transcription factor ETV4
Inhibits p38 phosphorylation and thereby blocks the p38 signaling pathway
Bind to FXR and MMP-9 promoter region
Decreases expression of MMP-9 through inhibition of AP-1
Induces MMP-9

Mechanism (inhibition in italic font)

Edaravone
Fibronectin
Galectin-7
GnRH
HCMV infection
Histamine
Hyperforin
IL-13
IL-1b

Regulators of transcription
AGE
ATPgS
ATXN1
Berberine
Bile acids
CCOS
EMMPRIN/CD147

Regulator

Table 1. Update of regulators of MMP-9.

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232
Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

Prevents APMA-induced activation, possibly due to S-thiolation at the propeptide cysteine


Activation
Reduces the levels of uPA
Potentiate activation of MMP-9 by MMP-3 by processing of the propeptide
Activates MMP-9, induces MMP-9
Activates MMP-9
Prevents APMA-induced activation, possibly due to S-thiolation at the propeptide cysteine
Partially removes the propeptide and activates proMMP-3
Directly irreversibly activates proMMP-9 through S-nitrosylation of the cysteine switch
Promotes activation by binding to the thiol group of the cysteine switch mechanism and thereby induces
autocatalysis of the prodomain
Removal of the propeptide

Mechanism (inhibition in italic font)

Direct inhibition of MMP-9 activity


Reduces the levels of TIMP-1, shift in TIMP-1/MMP-9 balance

(Berton et al., 2001)


(Straat et al., 2009)
(Nee et al., 2004)
(Tai et al., 2010)
(Itoh & Nagase, 1995; Jackson et al., 2010)
(Oh et al., 2001; Rhee & Coussens, 2002; Takahashi
et al., 1998)
(Paemen et al., 1996; Salo et al., 2006)
(Nee et al., 2004)

(Gong et al., 2008; Lijnen, 2001; Liu et al., 2005)

(Pei et al., 2006)


(Peppin & Weiss, 1986)
(Tai et al., 2010)
(Geurts et al., 2012a)
(Steenport et al., 2009)
(Yamamoto et al., 2004; Zhao et al., 2003)
(Pei et al., 2006)
(Itoh & Nagase, 1995; Jackson et al., 2010)
(Gu et al., 2002)
(Bonacci et al., 2011)

References

AGE: advanced glycation end product, AP-1: activator protein 1, APMA: 4-aminophenylmercuric acetate, ATPgS: Adenosine 50 -O-(3-thio)triphosphate, CaM: Calmodulin, CaMKII: Ca2/calmodulin-dependent
protein kinases II, CCOS: carboxylated chito-oligosaccharides, COX: cyclo-oxygenase, EP4: Prostaglandin E receptor 4, ERK: extracellular-signal-regulated kinases, FXR: Farnesoid X receptor, GnRH:
gonadotropin-releasing hormone, GSH: Glutathione, GSK-3b: Glycogen synthase kinase-3 beta, H1R: Histamine H1 Receptor, HCMV: Human cytomegalovirus, HClO: hypochlorous acid, HIF: hypoxia
inducible factor-, IL-: Interleukin-, JNK: c-Jun N-terminal kinases, LTA: lipoteichoic acid, MCF-7: Michigan Cancer Foundation7, NAC: N-acetylcysteine, NE: neutrophil elastase, NF-kB: nuclear factor
kappa-light-chain-enhancer of activated B cells, NO: Nitric oxide, OA-NO2: Nitro-oleic acid, oxLDL: oxidized low-density lipoprotein, PDGFR: platelet-derived growth factor receptor, PDI: protein disulfide
isomerase, p-ERK: phosphorylated ERK, PGE2: prostaglandin E2, PI3K: Phosphoinositide 3-kinase, PN-1: protease nexin-1, PPARg: peroxisome proliferator-activated receptor-g, RECK: reversion-inducing
cysteine-rich protein with kazal motif, RhoA: Ras homolog gene family, member A, ROCK: Rho-associated protein kinase, SHP-2: Src Homology protein-2, TGF-b: Transforming growth factor beta, TIMP-1:
tissue inhibitor of metalloproteinases-1, TNF-a: tumor necrosis factor alpha, uPA: urokinase-type plasminogen activator, VCAM-1: vascular cell adhesion molecule 1.
This table is an extension of information provided in an other review (Van den Steen et al., 2002a).

Tetracyclins
TNF-a

Regulation by inhibition of activity


Fatty acids
Direct inhibition by binding to the fibronectin repeats
HCMV infection
Induces TIMP-1 mRNA levels
IL-1b
Reduces the levels of TIMP-1, shift in TIMP-1/MMP-9 balance
Melatonin
Increases the levels of TIMP-1, shift in TIMP-1/MMP-9 balance
NE
Degrades TIMP-1
RECK
Direct inhibition of MMP-9

Plasmin/plasminogen

GSH
HClO
Melatonin
Meprins
MMP-3
MMP-26
NAC
NE
NO
OA-NO2

Regulator

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Gelatinase B/MMP-9
233

234

J. Vandooren et al.

Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

Table 2. Update on cellular origins of MMP-9.

Cell type
Astrocytes (RBA-1)

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B-CLL cells
Bladder cancer cells
(HTB9, HTB5)
Breast cancer cells (MCF-7,
MDA-MB-231, 168FARN)
Cardiac myofibroblasts
Endothelial cells (HUVEC)
Hepatocellular carcinoma cells
(HCC)
Keratinocytes
(MK cells, HaCaT Cells)

Macrophages (MPM, RAW264.7)

Location of MMP-9
Cytosol, cell membrane, nucleus
and associated with the cytoskeleton (actin and microtubules), LAMP-2, molecular
motor proteins (myosin V,
kinesin). Secreted in 400500 nm vesicles with or without
TIMP-1
On cell membrane and in podosomes, found in cell lysates and
cell culture medium
Secreted into cell culture medium,
in an SHP-2-dependent way
Secreted into cell culture medium

Stimulus for production/


inhibition (in italic)

References

LPS, TNF-a, IL-1b, LTA

(Sbai et al., 2010; Wang et al.,


2010; Wu et al., 2004;
Wu et al., 2009)

CXCL12, a4b1 integrin


stimulation

(Bauvois et al., 2002; Kamiguti


et al., 2004; Redondo-Munoz
et al., 2006)
(Kumar et al., 2010)

P38 MAPK/MAPKAPK2
signaling
IL-1b, alternatively spliced CD99,
PN-1
TNFa
Simvastatin
Bile acids
Radiation, LPA

Secreted into cell culture medium

a3b1 integrin and MEK/ERKdependent pathway, IL-13, Heat


Shock (IR), TGF-b, histamine

Unique Golgi-derived cytoplasmic


vesicles, associated with stable
microtubules through kinesin
5B and 3B. Found together with
calreticulin, PDI.
Increased secretion of MMP-2 and
MMP-9 by activation of the
GnRH receptor

LPS, IFN-g, MMP-1, MMP-3, C5a


Infection with HCMV, berberine

(Byun et al., 2006; Fayard et al.,


2009; Wang et al., 2005)
(Porter et al., 2004)
(Das et al., 2009)
(Cheng et al., 2006; Park et al.,
2011)
(Gschwandtner et al., 2008; Iyer
et al., 2005; Lamar et al.,
2008a; Purwar et al., 2008; Shin
et al., 2008; Xue & Jackson,
2008)
(Hanania et al., 2012; Huang et al.,
2011; Speidl et al., 2011;
Straat et al., 2009; Steenport
et al., 2009)

GnRH

(Cheung et al., 2006)


(Huwiler et al., 2003)

Schwann cells

Induction of MMP-9 expression


upon stimulation with inflammatory cytokines (IL-1b, TNFa)
LPS, TNF-a

Stem cells
Smooth muscle cells (VSMC,
HASMC)

Decreased O2 levels
OxLDL
Rosiglitazone

Ovarian cancer cells


Renal mesangial cells

(Chattopadhyay & Shubayev,


2009)
(Ingraham et al., 2011)
(Chen et al., 2011; Lee
et al., 2009)

B-CLL; B-cell chronic lymphocytic leukemia, CD99; cluster of differentiation 99, CXCL; CXC chemokine ligand, CXCR; CXC chemokine receptor,
ERK; extracellular-signal-regulated kinases, GnRH; gonadotropin-releasing hormone, HaCaT; cultured human keratinocyte, HASMC; human aortic
smooth muscle cells, HCC; hepatocellular carcinoma, HCMV; human cytomegalovirus, HUVEC; human umbilical vein endothelial cell, HTB;
heterotopically transplanted rat urinary bladder, IFN-g; interferon-g, IL-; interleukin-, IR; infra-red light, LAMP-1; lysosomal-associated membrane
protein 1, LPA; Lysophosphatidic acid, LPS; lipopolysaccharide, MAPK; mitogen-activated protein kinase, MAPKAPK; MAPK-activated protein
kinase, MCF-7; Michigan Cancer Foundation7, MEK; MAPK or Erk kinases, MK; mouse keratinocyte, MPM; mouse peritoneal macrophages,
oxLDL; oxidized low-density lipoprotein, PDI; protein disulfide isomerase, PN-1; protease nexin-1, RAB27a; Ras-related protein, SHP-2;
Src Homology protein-2, TGF-b; Transforming growth factor beta, TLA; lipoteichoic acid, RBA; rat brain astrocytes, TIMP-1; tissue inhibitor
of metalloproteinases-1, TNF-a; tumor necrosis factor alpha, VCAM-1; vascular cell adhesion molecule 1, VSMC; vascular smooth muscle cell.
This update complements a previous review (Van den Steen et al., 2002a) and is not exhaustive.

able to induce MMP-9 expression by activating a calmodulin


kinase II (CaMKII)-dependent phosphatidyl inositol triphosphate kinase (PI3K)/Akt-JNK pathway and transactivation
of platelet-derived growth factor receptor (PDGFR). This
also leads to increased cell migration (Wang et al., 2010).
Berberine, a natural extract from Rhizoma coptidis, reduces
MMP-9 and extracellular matrix MMP inducer (EMMPRIN)
expression in PMA-stimulated macrophages by suppressing
the activation of p38 pathway (Huang et al., 2011). In
hepatocellular carcinoma (HCC) cells, lysophosphatidic
acid (LPA) activates LPA receptor 1 and subsequently the
production of MMP-9. Both PI3K/Akt and PKCd/p38 MAPK
pathways are required for LPA-induced upregulation of

MMP-9 (Park et al., 2011). In cervical carcinoma-associated


myeloid cells, a STAT3-dependent molecular cascade was
identified which leads to MMP-9 induction (Schroer et al.,
2011). Stem cells brought in a low oxygen level environment,
produce high levels of MMP-9 and this was shown to happen
through hypoxia-inducible transcription factor (HIF-1a) signaling and subsequent activation of the canonical Wnt
pathway. This results in increased cell proliferation and
migration. The migratory and proliferating capacities could be
blocked by MMP-9 inhibition (Ingraham et al., 2011).
Cannabinoid receptor 2 (CB2R) signaling in dendritic cells
reduces AMP levels, leads to subsequent decrease in ERK
activation and reduced binding of c-Fos and c-Jun to the

DOI: 10.3109/10409238.2013.770819

promoter AP-1 site resulting in reduced production of MMP-9


(Adhikary et al., 2012).
MMP-9 can be upregulated with a carboxyterminal
11 residue fragment (EKQKVDLSTDC) of avb6 integrin.
The upregulation of either MMP-9 or MMP-2 is dependent
on the tissue context in which this peptide is presented
(Morgan et al., 2004).
Regulation of MMP-2 and MMP-9 in bladder cancer
cells was mediated by p38MAPK-driven MAPKAPK2 and
resulted in stabilization of MMP-2 and MMP-9 transcripts
(Kumar et al., 2010).

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Regulation by cytokines
A number of studies have reinforced earlier findings from
1991 that cytokines induce MMP-9 production (Masure et al.,
1991; Opdenakker et al., 2001b). For instance, the invasive
potential of human breast cancer cells was enhanced by
adding IL-1b which acts through an SHP-2-dependent
signaling pathway. Activation of this pathway results in
higher levels of secreted MMP-9 (Wang et al., 2005). As
outlined above, also in bronchial epithelial cells, MMP-9
expression can be activated by the activation of NF-kB,
whereas PPAR activators were shown to inhibit the
expression of MMP-9 by counteracting NF-kB (Shishodia
et al., 2003).
Synergy and antagonism within the cytokine network
determine the immunological and physiological outcomes.

Gelatinase B/MMP-9

235

The MMP9 gene, responsive to the action of cytokines, and


the upstream signaling cascades follows this paradigm. For
example, while interferons tend to inhibit MMP-9 production
(Bartholome et al., 2001), IL-1b and IFN-g together synergistically induce MMP-9 production in tuberculosis(TB)infected macrophages (Harris et al., 2007). In addition,
TNF-a is able to induce MMP-9 expression in proximal
tubular cells, whilst IL-1b counteracts this effect (Nee
et al., 2004).
Astrocytes stimulated with proinflammatory cytokines
such as TNF-a and IL-1b produce proMMP-9 (Wu et al.,
2004, 2009). The involved mechanisms included IL-1b
binding to its receptor (IL1R), induction of an influx of
Ca2 into the cytoplasm, resulting in activation of c-Jun
through the caMPII/JNK pathway (Wu et al., 2009).
IFN-g was found to inhibit TNF-a-induced MMP-9
(Balasubramanian et al., 2011). Activation of the IFNg/Stat1 signal pathway suppresses PMA-induced Mmp-9 and
Vegf gene expression in mouse peritoneal macrophages
(Nosaka et al., 2011). These findings echo previously
reviewed studies (Van den Steen et al., 2002a).
Regulation by growth factors and hormones
TGF-b can induce the expression of MMP9 genes (Salo et al.,
1991) and acts by activating a heteromeric serine/threonine
kinase receptor complex which subsequently activates the
so-called Smads (Conidi et al., 2011; Zhu & Burgess, 2001).

Figure 7. Illustration of the different levels of MMP-9 regulation. At the transcriptional level MMP-9 is regulated by several pathways including
the Smad pathway, the MAPK pathway, the NIK/NEMO/IKK pathways, the STAT pathways and nuclear receptor pathways. The MMP-9 promoter
region has several regulatory elements, including AP-1 and NF-kB. Upon transcription, dynamic mRNP complexes control mRNA degradation
and stabilization, e.g. with the help of nucleolin. Once secreted, proMMP-9 is activated into MMP-9 by proteases such as MMP-3, plasmin
and trypsin. Futhermore, MMP-9 binds to several cell surface molecules, e.g. Ku, LRP1/2, integrins and CD44, forming an MMP-9 cell surface
complex.

236

J. Vandooren et al.

Recently it was shown that TGF-b and a3b1 integrin


cooperatively induce MMP-9 expression in immortalized
keratinocytes and that this feature was part of the immortalized phenotype (Lamar et al., 2008a). It is clear that in
cell types, different from keratinocytes and having other
receptors and signaling molecule levels, this regulation may
be completely different (Figure 7).
MMP-1 and MMP-3 can
indirectly induce the expression of MMP-9 in macrophages by
triggering the release of TNF-a. The release of TNF-a
induces the expression of COX-2 and PGE2 secretion. PGE2
subsequently binds to EP4 in the cell membrane and
stimulates MMP-9 production through MAPK/ERK1/2 signaling (Steenport et al., 2009). Because many members of the
TNF family need to be cleaved from a membrane-anchored
trimer form into a free cytokine, it is expected that more of
these indirect proteolytic regulatory mechanisms will be
discovered.

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Regulation by other proteases.

Signaling by neurotransmitters and hormones. Several neuro-

transmitters and hormones induce MMP-9 expression.


Histamine induces MMP-9 production in human keratinocytes by signaling through the histamine H1 receptor (H1R).
Histamine-induced MMP-9 leads to the destruction of type IV
collagen present in the basement membrane of healthy skin.
This finding gives an interesting insight into skin pathologies
(Gschwandtner et al., 2008; Harvima, 2008). Serotonin
receptor-4 (5-HT4R) signaling upregulates MMP-9 and has
been implicated in the formation of soluble amyloid-b protein
precursor alpha and a reduction of amyloid-b peptide (Ab)
deposition (Hashimoto et al., 2012). Adrenalin is also able to
induce the production of MMP-9. In a human colon
carcinoma cell line, supplementation with adrenalin resulted
in stimulation of cell proliferation via both b(1)- and b(2)adrenoceptors by a COX-2-dependent pathway and increased
levels of MMP-9 were registered (Wong et al., 2011). In
addition, noradrenaline induces MMP-2 and MMP-9 expression in the mouse neuroendocrine hypothalamus and in
nasopharyngeal carcinoma tumor cells (Maolood et al., 2008;
Yang et al., 2006). Stimulation of muscarinic acetylcholine
receptors in a human breast tumor cell line and in mouse
neuroblastoma cells results in increased MMP-2/9 expression
(Anelli et al., 2007; Pelegrina et al., 2012).
Epigenetic regulation. The field of epigenetic research is in
full expansion and will yield better insights and maybe
additional explanations of the previously found paradox why,
against expectations, MMP inhibitors yield severe side-effects
in individual cancer patients. Recently, an overview has been
published on the epigenetic regulation of MMP9 gene
expression (Labrie & St-Pierre, 2012). Epigenetic mechanisms are critical in the control of MMP9 in both normal and
disease conditions. Epigenetic regulation includes mechanisms such as histone modification, DNA methylation and noncoding RNAs (ncRNAs).
Inhibition of histone deacetylases (HDACs) with HDAC
inhibitors (iHDACs) results in either higher (Mayo et al.,
2003) or lower (Estella et al., 2012; Kaneko et al., 2004;

Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

Kuljaca et al., 2007; Lee et al., 2010; Mitmaker et al., 2011)


expression levels of mmp-9, depending on the cell type. The
MMP9 gene is also regulated by DNA methylation. Inhibitors
of DNA methylation induce mRNA and protein levels of
MMP-9 (Chicoine et al., 2002; Sato et al., 2003). For
example, in primary human aortic smooth muscle cell
(HASMC) oxLDL regulates MMP-2 and MMP-9 expression
by inducing miRNA (miR-29b) that subsequently downregulates DNA methyltransferase 3b (DNMT3b). This study
indicates that epigenetic regulation of MMP-9 might be a
novel mechanism in atherosclerosis (Chen et al., 2011)
(Table 3). Since ncRNAs cover a broad range of regulatory
functions and levels, these will be discussed in the next
section.
Regulation of mRNA
Although messenger RNA levels are often used as surrogate
parameter of protein amounts, steady-state amounts are
the dynamic result of positive (stabilization) and negative
(destabilization) regulation. RNA-binding proteins and
ncRNAs bind to the mRNA cis-acting elements and thereby
determine the degradation or stabilization of the a particular
mRNA (Wu & Brewer, 2012).
Regulation by RNA-binding proteins
Upon transcription, mRNAs reside as so called messenger
ribonucleoprotein complexes (mRNPs). This is a dynamic
complex of mRNA with proteins. The proteins are mediators
of posttranscriptional events such as capping, splicing, quality
control and trafficking (Hieronymus & Silver, 2004). Upon
treatment of HT1080 fibroblasts with 2,2-dipyridyl, MMP-9
protein levels increased very rapidly without any significant
changes in mRNA concentration. It was shown that this
enhanced MMP-9 synthesis was due to enhanced recruitment
of MMP-9 mRNA from the cytoplasm to the RER and
subsequent translation. Furthermore, MMP-9 mRNA was
shown to interact with two forms of the RNA-binding factor,
nucleolin, in contrast with MMP-2 whose mRNA did not
associate with nucleolin (Fahling et al., 2005).
The 30 untranslated (30 UTR) region of mRNAs may contain
the so-called adenylate-uridylate-rich elements (AREs) which
are binding zones for mRNA stabilizing factors and
de-stabilizing factors (Chen & Shyu, 1995). MMP-9 mRNA
has four AREs in its 30 UTR region. A specific mediator of
MMP-9 mRNA stability is the ELAV-like RNA-binding
protein HuR. Specificially, adenosine 50 -O-thiotriphosphate
(ATPgS), a stable ATP analogue, stimulates the binding
of HuR to MMP-9 AREs, thereby increasing the final
concentration of MMP-9 expressed by renal mesangial cells
(Huwiler et al., 2003).
Regulation by ncRNAs
In recent years, an explosion in research on ncRNAs occurred.
ncRNAs are RNAs that are not translated into proteins, but
rather have an alternative biological function. These RNAs
are further divided into other types of RNA according to their
function and features, e.g. microRNA (miRNA), small
interfering RNA (siRNA), piwi interacting RNA (piRNA),
small modulatory RNA (smRNA), small nuclear RNA

Gelatinase B/MMP-9

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(snRNA), small nucleolar RNA (snoRNA), etc. Although the


list of ncRNAs is continuously growing, our understanding of
their cellular mechanism is incomplete. In general, snRNAs
have regulatory functions on several levels of regulation,
including gene silencing, DNA demethylation and RNA
interference (Costa, 2007). Since RNA interference is mostly
studied, we will discuss ncRNAs at the level of translational
regulation.
Natural silencing machineries were selected during natural
evolution and exist as control mechanisms of mRNA. Small
RNA molecules from short-interfering RNAs (siRNAs) to
microRNAs (miRs) are even capable of moving between
cells and through the vasculature under the forms of silencing
RNAs (siRNAs) (Chitwood & Timmermans, 2010; Moazed,

237

2009). MMPs are susceptible to direct regulation by miRNAs


and the importance of this type of regulation has been
demonstrated in recent years. MiR-9 regulates MMP-14
expression thereby inhibiting the invasion, metastasis, and
angiogenesis of neuroblastoma (Zhang et al., 2012). MiR-155
and miR-146a directly downregulate MMP-16 and reduce
migration of human cardiomyocyte progenitor cells (hCMPs)
(Liu et al., 2012) and lower motility of differentiated Caco-2
cells (Astarci et al., 2012), respectively. MiR-143 reduces
lung metastasis of human osteosarcoma cells and targets
MMP-13 (Osaki et al., 2011). MMP-3 is downregulated
by miR-152, which reduces glioma cell invasion and angiogenesis (Zheng et al., 2012). HCC angiogenesis, invasion and
metastasis can be regulated by miR-29b which targets MMP-2

Table 3. miR sequences with potential regulatory effect on MMP-9 mRNA.


miR
type

Site
type

hsa-miR-3713

PC

hsa-miR-1224-3pA
hsa-miR-4690-3pA
hsa-miR-3123A
hsa-miR-1286A
hsa-miR-330-3pA
hsa-miR-183A
hsa-miR-4802-3pA
hsa-miR-4666-5pA
hsa-miR-4450A
hsa-miR-491-5pA,B

PC
PC
PC
PC
PC
V
PC
PC
PC
M

hsa-miR-4281A
hsa-miR-133bA
hsa-miR-133aA
hsa-miR-296-3pA

miRNA
A

Size

Predicted position
(of 30 UTR)

Total context
score

8mer

7380

0.39

C
C
C
PC
PC
PC
PC
PC
PC
PC

7mer-m8
7mer-m8
8mer
7mer-m8
7mer-m8
7mer-m8
7mer-A1
7mer-A1
8mer
7mer-m8

117123
120126
179186
814
1218
2228
2531
2632
3239
3440

0.19
0.14
0.25
N/A
N/A
0.20
0.10
0.04
0.37
0.24

PC
V
V
M

PC
PC
PC
PC

7mer-A1
7mer-m8
7mer-m8
7mer-m8

4248
4349
4349
4753

0.21
0.11
0.11
0.12

hsa-miR-1915A
hsa-miR-2355-5pA
hsa-miR-3667-3pA

PC
PC
PC

PC
PC
PC

7mer-m8
8mer
7mer-A1

5056
5259
5662

0.23
0.33
0.10

hsa-miR-4448A
hsa-miR-149A

PC
M

PC
PC

7mer-m8
7mer-m8

5864
6167

0.15
0.20

hsa-miR-892bA
hsa-miR-4470A
hsa-miR-3149A
hsa-miR-4773A
hsa-miR-4691-5pA
hsa-miR-1238A
hsa-miR-3124-3pA
hsa-miR-204A
hsa-miR-211A
hsa-miR-4287A
hsa-miR-4685-3pA
hsa-miR-483-3pA
hsa-miR-1253A
hsa-miR-3613-3pA
hsa-miR-3065-5pA
hsa-miR-3145-5pA
hsa-miR-3691-3pA
hsa-miR-3925-5pA
hsa-miR-1303A

PC
PC
PC
PC
PC
PC
PC
V
V
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC

PC
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC
PC

7mer-m8
7mer-m8
7mer-A1
7mer-m8
8mer
7mer-A1
7mer-m8
7mer-m8
7mer-m8
7mer-m8
7mer-m8
7mer-m8
8mer
7mer-m8
7mer-m8
7mer-m8
7mer-A1
7mer-A1
7mer-m8

6268
6773
7682
9197
97104
99105
100106
103109
2228
104110
104110
109115
134141
148154
151157
155161
173179
180186
181187

0.18
0.13
0.01
0.14
0.33
0.08
0.02
0.07
0.07
0.15
0.15
0.14
0.25
0.02
0.12
0.24
0.16
0.10
0.18

hsa-miR-548mA

PC

PC

7mer-A1

187193

0.10

Other target MMPs


MMP-2,-7,-8,-10,-11,-13,-14,-15,-16,-19,-20,-24,
-25,-26,-28
MMP-2,-11,-14,-15,-17,-19,-24,-27,-28
N/A
MMP-26
MMP-2,-3,-7,-8,-13,-14,-15,-16,-24,-26
MMP-1,-2,-3,-7,-14,-15,-16,-24,-28
MMP-3,-7,-10,-11,-13,-16,-19,-24,-27,-28
N/A
N/A
N/A
MMP-1,-2,-3,-7,-8,-10,-11,-13,-14,-15,-16,-17,-19,
-20,-21,-24,-25,-26,-27,-28
MMP-1,-8,-10,-11,-14,-15,-16,-17,-19,-20,-24,-28
MMP-2,-11,-14,-15,-16,-19,-21,-24,-25,-27,-28
MMP-2,-11,-14,-15,-16,-19,-21,-24,-25,-27,-28
MMP-1,-2,-3,-7,-8,-10,-11,-12,-14,-15,-16,-19,-20,
-21,-24,-25,-27,-28
MMP-2,-13,-14,-15,-16,-17,-19,-20,-24,-25
MMP-11,-25
MMP-2,-10,-11,-13,-14,-15,-16,-17,-19,-21,-24,
-25,-28
N/A
MMP-1,-2,-3,-8,-10,-11,-14,-15,-16,-17,-19,-21,24,-25,-28
MMP-14,-15,-16,-19,-20,-24,-25,-28
N/A
MMP-1,-2,-13,-16,-17,-28
N/A
N/A
MMP-8,-10,-11,14,-15,-16,-19,-24,-25,-28
MMP-2,-14,-15,-20,-21
MMP-8,-14,-15,-16,-17,-19,-24,-25,-28
MMP-2,-11,-14,-15,-16,-17,-19,-24,-28
MMP-2,-8,-10,-15,-19,-25,-28
N/A
MMP-2,-3,-8,-11,-13,-14,-15,-19,-27
MMP-2,-11,-13,-14,-16,-17,-19,-20
none
MMP-13,-14,-16,-17
N/A
MMP-1,-2,-7,-8,-11,-15,-16,-19,-20,-24
MMP-3,-13,-15,-19,-20,-25,-28
MMP-1,-2,-3,-10,-11,-14,-16,-17,-19,-20,-21,-24,
-25,-27,-28
MMP-11,-21

V; miRNA family broadly conserved among vertebrates, M; miRNA family conserved only among mammals, PC; poorly conserved, C; conserved,
7mer-m8; exact match to positions 28 of the mature miRNA, 7mer-A1; exact match to positions 27 of the mature miRNA followed by an A,
A
; predicted miR, B; experimentally identified miR.

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238

J. Vandooren et al.

(Fang et al., 2011). The above examples illustrate silencing


of MMP mRNAs, different from that of MMP-9. As MMPs
work together in a network of interactions (Van den Steen
et al., 2002a), this form of silencing may result in indirect
effects on MMP-9 biology.
Unfortunately, direct MMP-9 silencing has been described
in only few cellular systems. However, from theoretical point
of view many more studies are to be expected in the future,
because the target sequences for many miRs are present
in the MMP-9 sequence (NCBI Reference Sequence:
NM_004994). Tabel 3 displays potential human MMP-9
miRNAs as predicted with TargetScan (Grimson et al., 2007;
Lewis et al., 2005) or as identified experimentally. Other
potential target MMP for these miRNAs were predicted with
RNA22-HAS (Miranda et al., 2006). Till now, only miR-4915q has been experimentally validated as an MMP-9 targeting
miRNA. In fact, this miRNA was found to be correlated with
the MMP-9 expression pattern in glioblastoma multiforme
(GBM) patients and might be a potential target for antiinvasion therapy in GBM (Yan et al., 2011). These findings
illustrate the importance of miRNA regulation, but also
indicate that this part of MMP-9 biology requires urgently
further investigation.
In addition, artificial silencing may be obtained with the
use of antisense RNAs and ribozymes. For instance, in a
pioneering study the metastatic behavior of tumor cells
in specific experimental animal models was modulated by
inhibition of MMP-9 mRNA with a hammerhead ribozyme
(Hua & Muschel, 1996).
Regulation through compartmentalization
and secretion
Intracellular MMP-9
In analogy with other glycoproteins it is suggested that
the attachment of N-linked oligosaccharides occurs
co-translationally in the endoplasmic reticulum together
with the folding of gelatinase B (Dwek, 1996). The presence
of an N-linked sugar in the prodomain of MMP-9 has
therefore been associated with correct protein folding
(Kotra et al., 2002).
The cysteine bridges are then formed by protein disulfide
isomerase (PDI) yielding folded monomers. Whether the
oligomers are also associated at this point is so far unclear
(vide infra). When the N-glycosylated and folded proteins
move to the Golgi apparatus and the trans-Golgi, O-linked
oligosaccharides are sequentially added by specific sugar
transferases (Van den Steen et al., 1998). How the latter
process is controlled, whether only exposed serines and
threonines in the O-glycosylated domain may act as landing
sites for the sugar transferases or whether the protein
flexibility (Rosenblum et al., 2007b) also enables multiple
conformations remain open questions. Site-specific O-linked
glycosylation analysis may resolve such questions and
other enigmas. Are always the same amino acids used
for O-glycosylation? Is O-glycosylation dependent on
the cellular source? Do physiological or pathological
conditions alter the O-glycosylation and the flexibility of
the enzyme? Is the N- and O-glycosylation similar or
different in monomers and oligomers? All these questions

Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

will drive the development of new technology at the cutting


edge and will yield novel insights in the glycobiology of
gelatinase B.
Currently many unresolved questions thus exist about
the intracellular trafficking of MMP-9. Interestingly, trafficking of MMP-9 after translation is cell type dependent.
MMP-9 produced by neutrophils is stored in zymogen
granules, ready to be secreted upon an inflammatory
stimulus (Van den Steen et al., 2002a). Rab27a, a GTPase
involved in specific vesicle trafficking is known to
co-localize with neutrophil MMP-9, regulating the secretion of MMP-9 (Brzezinska et al., 2008). However, in
macrophages, MMP-9 secretion has a completely different
timing from that of neutrophils and is sorted differently.
While mature neutrophils have MMP-9 prestored in granules, which are released upon stimulation within minutes,
macrophages rely on de novo synthesis prior to the secretion
of MMP-9, a process that takes several hours at least
(Opdenakker et al., 2001a,b). As shown only recently, upon
activation, macrophages produce MMP-9 which can be
found intracellularly in small Golgi-derived cytoplasmic
vesicles, together with calreticulin and PDI. These vesicles
are associated with stable microtubules and the kinesin
transport proteins (5B and 3B isoforms). The association
is dependent on Rab3D, a GTPase involved in membrane
transport and exocytosis, and requires the formation of stable
(acetyl-a-tubulin) microtubules (Hanania et al., 2012).
Depending on the differentiation level of cells, further
specialization in MMP-9 trafficking has been found. In
astrocytes, proteinases and their inhibitors use different
pathways for trafficking and secretion. MMP-2 and MMP-9
are distributed in different vesicles in both LPS-treated and
control-treated astrocytes, whereas TIMP-1 co-localizes with
both MMP-2 and MMP-9 containing vesicles. Interestingly,
both gelatinases were found not only in the cytosol but
also in cytoskeletal, membrane and nuclear fractions of
astrocytes. The presence of MMP-9 within vesicles correlated
with LAMP-2 expression, which is a marker for late
endosomes and lysosomes. MMP-9 containing vesicles were
associated with actin and microtubules in a linear distribution
together with molecular motor proteins such as myosin V
(39%) and kinesin (91%). Outside of the astrocytes,
400500 nm vesicles, representing natural nanoparticles,
were detected, containing MMP-2 or MMP-9 with TIMP-1
(Sbai et al., 2010).
In addition, correct folding and PTMs of the MMP-9
enzyme are also a requirement for secretion. As stated before,
the intramolecular disulfide bonds in the fibronectin domain
and a correct folding of the prodomain are required for
secretion of proMMP-9. Therefore, an important role for
PDI in the secretion of proMMP-9 has been suggested
(Khan et al., 2012).
Neutrophils versus the rest
It is important to recognize the differences between neutrophils and other cell types (Borregaard, 2010). Mature
neutrophils make a special case of MMP-9: they produce
MMP-9 in association with neutrophil gelatinase B-associated
lipocalin (NGAL, vide supra). Neutrophils store, aside from

Gelatinase B/MMP-9

DOI: 10.3109/10409238.2013.770819

NGAL-MMP-9 complexes, monomers and oligomers as


proenzymes in granules, ready to be quickly released
(within less than 1 h) (Masure et al., 1991) after appropriate
stimulation. In addition, and in sharp contrast with all other
leukocyte types and other cells, neutrophils do not constitutively produce MMP-2 and do not make TIMP-1 (Opdenakker
et al., 2001a,b). This neutrophil TIMP-1-free MMP-9 recently
gained more attention, because it was demonstrated that this
MMP-9 form is pro-angiogenic (Ardi et al., 2007, 2009;
Bekes et al., 2011). These findings imply that TIMP-1 or
TIMP-1-mimicking selective inhibitors of MMP-9 may block
neutrophil-mediated angiogenesis.

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Gelatinase B anchored to the cell membrane


Once MMP-9 is secreted it may diffuse into tissues and ECM
or stick to receptors at the surface of cells. Some MMPs have
an additional protein domain to be anchored into membranes
or may be modified with a glycosylphophatidylinositol anchor
at the carboxyterminus (Figure 2). In fact, these membranetype MMPs (MT-MMPs) may be regarded as cell receptors
for their substrates or inhibitors. Therefore, it is relevant to
define systematically the anchoring sites of MMP-9 to cells:
CD44, LRP-1, LRP-2, Ku and integrins.
When MMPs are anchored to the cell membrane they can
target their catalytic activity to specific substrates within the
pericellular space. MMP-9 can bind to CD44 (Chakraborti
et al., 2003; Yu & Stamenkovic, 2000). This complex is found
on the cell membrane of many tumor cells from various
species, e.g. murine mammary carcinoma cells and human
B-CLL cells. The expression of CD44 correlated with
invasive capacity of these cells in vitro and in vivo and
with increased angiogenesis in tumor tissue. Details of the
interaction of MMP-9 with LRP-1 and LRP-2 have been
discussed above (Van den Steen et al., 2006) and were
reviewed in the context of the functions of the hemopexin
domain (Piccard et al., 2007). MMP-9 binds also integrins,
that are integral membrane receptors on many cell types,
mediating immune functions and playing roles in tumor
biology. Two types of evidence have been provided that
secreted MMP-9 can bind to integrins. Redondo-Munoz and
colleagues demonstrated signaling mediated by binding of the
MMP-9 hemopexin domain to a4b1-integrin (in conjunction
with CD44v) in B-CLL (Redondo-Munoz et al., 2008, 2010).
Dufour et al. demonstrated that the interaction of MMP-9 with
CD44 had an effect on cell migration and that peptide
inhibitors interfering with this binding had anti-cancer
activity (Dufour et al., 2010). Finally, peptide-mediated
interference with the binding of the hemopexin domain of
MMP-9 on a4b1-integrin also prevented signaling in B-CLL
(Ugarte-Berzal et al., 2012).
Ku is a heterodimer (Ku70/Ku80) known for its role in
repair of dsDNA. However, Ku is also found on the cell
surface, where its role remains elusive. Intriguingly, Ku80 can
interact with the hemopexin domain of MMP-9 on the
cell surface of highly invasive hematopoietic cells. It was
postulated that Ku acts as an MMP-9 docking molecule
(Monferran et al., 2004; Paupert et al., 2008).
Astrocyte cell fractioning experiments and subsequent
zymography analysis of the fractions indicated that proMMP-

239

9 and activated MMP-9 was associated with the cell


membrane (Sbai et al., 2010). In addition, MMP-9 has been
found on the cell surface of polymorphonuclear neutrophils,
an interaction which renders the protease considerably
resistant to TIMP-1 inhibition (Owen et al., 2003).

Activation of progelatinase B
The cysteine switch
An aminoterminal propeptide is present in all members of
the MMP family. It consists of approximately 80 amino acids
and caps the zinc-containing catalytic domain (Figure 5).
This propeptide domain contains the cysteine switch
PRCXXPD consensus sequence (Rosenblum et al., 2007a).
The cysteine switch explains the latency of pro-MMP-9 and
indicates that any means that can pull the cysteine99 away
from the Zn2 ion will result in activation and catalytic
activity. A decade ago it was already clear that activation
of MMP-9 occurs in a network of enzyme interactions.
At that time the following enzyme activators were described:
MMP-1, MMP-2, MMP-3, MMP-7, MMP-10, MMP-13,
MMP-26, trypsin, NE, cathepsin G and tissue kallikrein
(Van den Steen et al., 2002a). Meanwhile a number of new
details about gelatinase B activation have emerged and these
are briefly discussed here.
Occlusion of the active site by the propeptide domain and
coordination of the Zn2 with the sulfhydryl of Cys99 result
in a suppression of MMP activity. In vivo the MMP
zymogens are activated by proteases including kallikrein,
trypsin and other MMPs such as MMP-3 (Ogata et al.,
1992). In vitro this can also be achieved by chemical agents
such as aminophenyl mercuric acetate and other S-reactive
agents, reactive oxygen, detergents and heat. Since ROS are
also found in vivo, this is also a potential mechanism of
proMMP-9 activation, already described long ago (Peppin &
Weiss, 1986). Detergent-mediated denaturation, e.g. by SDS,
explains why pro-MMP-9 may be visualized by gelatin
zymography. If the SDS can be removed completely during
the renaturation process and pro-MMP-9 refolds completely,
then the zymogen form is not visible anymore on
zymography (Vandooren et al., 2013).
Zymogen activation releases the propeptide domain by
sequential proteolysis. When activated with MMP-3, the
first cleavage occurs in the loop connecting two helices of the
pro-peptide and the second cleavage occurs eight amino
acid residues downstream from the zinc-coordinated cysteine,
resulting in an active MMP (Ogata et al., 1992; Rosenblum
et al., 2007a). In the active MMP the Cys99 residue is
replaced by a H2O molecule. The group of Irit Sagi
(Rosenblum et al., 2007a) showed that stabilization of the
catalytic Zn2 ion (in three steps) is much faster than the
completion of the proteolytic event.
A graphical representation of the propeptide structure
and possible post-translational modifications is shown in
Figure 5. It is generally accepted that proMMP-9 is activated
by proteolytic cleavage of the propeptide. However, in line
with the effects of S-reactive reagents (see above), it has also
been demonstrated that proMMP-9 can be activated without
undergoing proteolysis (Bannikov et al., 2002).

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Artificial activation by 4-aminophenylmercuric acetate


The organomercurial reagent 4-aminophenylmercuric acetate
(APMA) causes a conformational alteration that allows a
stepwise autolytic cleavage of the propeptide of both
gelatinase A and gelatinase B. Human progelatinase B
(92 kDa) is fully activated by incubation with 2 mM APMA
for 2 hr at 37  C. Activation may be followed by the
enzymatic activity assay or sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis. The procedure of activation by APMA, however, results in an additional autolytic
cleavage that removes the carboxyterminal domain and
generates a 65 kDa enzyme (Okada et al., 1992) which will
affect the interaction with TIMP-1. Therefore it is recommended to use activated stromelysin-1/MMP-3 instead of
APMA. Other experimental evidence (Okada et al., 1992) that
the 67 kDa form is formed by cleavage of both aminoterminal
and carboxyterminal parts of gelatinase B was provided.
Finally, it needs to be stressed that activation of mouse
pro-MMP-9 by APMA is less efficient than human MMP-9,
but the explanation of this difference is unknown.
Activation by trypsin
Since the early days of MMP research, trypsin has been used
to activate latent pro-enzyme forms to yield catalytic activity
(Masure et al., 1990). However, this in vitro activation was
not associated with biological effects. Nevertheless, activation
by trypsin may be so far one of the few relevant in vivo
mechanisms. Indeed, trypsin is produced as trypsinogen by
exocrine pancreas acinar cells. This zymogen is normally
activated in the gut by enterokinase. In an animal model of
acute pancreatitis, it was shown that trypsinogen is activated
into trypsin and only under these conditions and in such tissue
samples pro-MMP-9 was activated into the 82 kDa form. It is
clinically well known that in situations of acute pancreatitis,
surgery has to be avoided because of the problem of tissue
autodigestion and difficult to control fistulization (Descamps
et al., 2004).
Activation by plasmin
ProMMP-9 can be activated into MMP-9 with plasmin.
Plasminogen, the plasmin zymogen form, is an ubiquitous
proenzyme and may be associated with the ECM and upon
activation to plasmin, it degrades several ECM proteins and
can activate MMPs. Plasminogen can be activated into
plasmin by uPA and tPA and this activation can be inhibited
by plasminogen activator inhibitor1 (PAI-1) and PAI-2 and
by a2-antiplasmin (Lijnen, 2001; Liu et al., 2005; Werb et al.,
1977). In vivo, plg KO mice have less activated MMP-9 and
migration of macrophages though ECM is decreased. These
observations suggest that plasminogen regulates macrophage
migration through activation of MMP-9 (Gong et al., 2008).
Activation by gelatinase A (MMP-2)
The activation of proMMP-9 by active MMP-2 was demonstrated in vitro in 1995 (Fridman et al., 1995). Since then, a
key question was how proMMP-2 is activated. This mechanism was elaborated as the MT1-MMP/MMP-2/TIMP-2

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paradigm (Itoh & Seiki, 2004; Itoh et al., 2001).


Subsequently, this network of interactions was demonstrated
as the MMP-9/MMP-2/TIMP-2 axis in which the role played
by TIMP-2 was demonstrated (Toth et al., 2003).
Activation by stromelysin-1 (MMP-3)
Incubation at 37  C for 2 h at a 40:1 molar ratio of progelatinase
B versus stromelysin-1 will remove the propeptide to yield
the 82 kDa active gelatinase B (Bannikov et al., 2002; Ogata
et al., 1992). In the presence of purified plasma membrane
fractions, this activation occurs more efficiently, suggesting
that, in vivo, activation of proMMP-9 is favoured in the
pericellular space (Toth et al., 2003).
Activation by urokinase-type plasminogen activator (uPA)
uPA bound to a cell surface receptor (urokinase-type
plasminogen activator receptor (uPAR)) provides a mechanism for the cell to activate an array of proteases which are in
close proximity of the cell surface. This restricts their activity
to only a portion of the cell surface (Chakraborti et al., 2003).
The uPA/plasmin/MMP cascade is relevant in many biological systems and has been discussed in relation to
neurological and vascular diseases and cancer (Cuzner &
Opdenakker, 1999; Lijnen, 2001).
Inhibition of activation
Pei et al. describe the inhibition of APMA-induced activation of MMP-9 by reduced glutathione (GSH) and
N-acetylcysteine (NAC). They state that GSH, but not
oxidized GSSG, renders pro-MMP-9 refractory to APMAinduced activation. The release of the thiol propeptide from
the zinc ion is prevented due to s-thiolation at the propeptide
cysteine (Pei et al., 2006).
Activation by substrate binding or allosteric interactions
Bannikov et al. (2002) suggested that proMMP-9 acquires
activity upon binding to gelatin or type IV collagen. Later,
it was shown that the activation of proMMP-9 by MMP-3
could be enhanced by the addition of b-hematin (4 h without
b-hematin and 30 min with b-hematin), a core constituent of
hemozoin or malaria pigment. It was postulated that
proMMP-9 binds b-hematin through its hemopexin domain
and that the sulfhydryl group of the propeptide interacts with
hematin and is followed by MMP-9 autocatalysis of the prodomain, and resulting in a truncation between Glu40/Met41
and Leu52/Leu53. Interestingly, the autocatalytic cleavage
(Glu40/Met41) is in accordance with the first cleavage site
induced by MMP-3, explaining why activation time by MMP3 is reduced in the presence of hemozoin (Geurts et al., 2008).
Activation by kallikrein-related peptidase 7 (KLK7)
KLK7 introduces a truncation in the carboxyterminal part of
MMP-9 (between Tyr443 and Gly444) and this results in an
active 51 kDa form, lacking the O-glycosylated and hemopexin domains. This cleavage by KLK7 was specific for
MMP-9 since MMP-2 was not activated by KLK7 (Ramani
et al., 2011).

Gelatinase B/MMP-9

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Activation by matrilysin-2 or MMP-26


The 51 kDa MMP-9 form obtained after processing by KLK7
resembles in domain structure matrilysin (with the addition of
the gelatin-specific fibronection repeats) (Figure 2). In two
separate studies, it was demonstrated that proMMP-9 can be
activated by MMP-26, also known as matrilysin-2 (Yamamoto
et al., 2004; Zhao et al., 2003).
Activation by human neutrophil elastase

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Human neutrophil elastase (HNE) activates or primes activation of proMMP-9 by introducing a cleavage between
Val58 and Ala59, Ala59 and Glu60 (Figures 3 and 5) in the
MMP-9 prodomain. In addition, HNE may degrade TIMP-1
and thereby inactivate the MMP-9 inhibition (Jackson
et al., 2010).
Priming of activation by meprins
Recently, activation of MMP-9 by meprin a and meprin b has
been described (Geurts et al., 2012a). Meprins are increasingly studied as convertases of proproteins, both in the
cytokine and in the protease fields (Jefferson et al., 2012;
Villa et al., 2003).
Meprin a was found to clip the tip of the gelatinase B
aminoterminus, thus destabilizing the proform (Figures 4
and 5). This resulted in priming for activation by MMP-3.
Remarkably, this meprin-mediated primed pro-MMP-9 form
corresponds with the natural protein produced by neutrophils
and for which the processing enzyme remained elusive for
20 years (Masure et al., 1991; Opdenakker et al., 1991a,b).
Furthermore, meprin also truncated MMP-9 at the carboxyterminus into a 68 kDa form. Maybe this form corresponds
to the 65 kDa hemopexin-less form described in earlier studies
(Bellini et al., 2012).
Inhibition of gelatinase B
Enzyme activity of MMPs leads to proteolysis of structural
(bone, cartilage) and functional (cytokines, hormones, receptors) molecules and these effects need to be tightly controlled
to keep the physiological balances in the host, whether this is
within the blood circulation or in tissues. The proteins
that execute these checks are inhibitors of MMPs such as
a2-macroglobulin in the circulation and the four specific
tissue inhibitors of MMPs (TIMP14). Several other proteins,
acting as inhibitors of MMPs, have gained attention. These
include the membrane-bound protein RECK (Rhee &
Coussens, 2002).
Inhibition by TIMPs
Both monomers and multimers of MMP-9 are inhibited by
TIMP-1. The major TIMP-1 binding site of pro-MMP-9 is
located in the hemopexin domain (OConnell et al., 1994).
Another study showed that proMMP-9 multimers have two
high affinity binding sites for TIMP-1 (Olson et al., 2000),
most probably these are also in the hemopexin domain
(OConnell et al., 1994). The hemopexin domain of MMP-9
interacts with the carboxyterminus of TIMP-1 (Goldberg
et al., 1992) and the aminoterminus of both TIMP-1 and

241

TIMP-2 can interact with the MMP-9 catalytic domain. As an


illustration of protease load, i.e. the balances between MMPs
and TIMPs, COS-1 cells co-expressing MMP-9 and TIMP-1
have a reduced capacity of cell migration, compared to COS-1
cells solely expressing MMP-9 (Dufour et al., 2008). It needs
to be stressed that the regulated expression of MMP-9 by most
cell types, coincides with expression of TIMP-1. Neutrophils
form a notorious exception to this rule, because these
terminally differentiated cells do not produce TIMP-1 (vide
supra). In addition, HNE proteolytically inactivates TIMP-1
when already in complex with MMP-9. This allows proMMP9 to be activated more easily by MMP-3. Trypsin is also
known to degrade TIMP-1 but not when complexed to
proMMP-9 (Itoh & Nagase, 1995).
In general, the focus of TIMP research is on the inhibitory
function against MMP activity. However, free TIMPs play
other functional roles: cell growth control, blocking angiogenesis and induction of oligodendrocyte differentiation.
For these functions to be executed, binding of TIMP-1 to
cell surface receptors is necessary. Therefore, alternative
perspectives on the TIMP/MMP balance were formulated. For
example, while under normal conditions, a balance exists
between TIMPs and MMPs, disease states are associated with
an imbalance. This may result in either excessive proteolysis
due to the presence of free active MMPs or in reduced
proteolysis due to excessive inhibition of MMP activity.
Alternatively, excessive expression of MMPs might deplete
the population of free TIMP molecules, resulting in reduced
TIMP functionality. As stated in this way, MMPs are viewed
to act as inhibitors of TIMPs (Moore & Crocker, 2012).
Similarly, under conditions of TIMP-1 degradation, for
example by NSE (Jackson et al., 2010), the unique TIMP-1
functionalities are also lost. It is also important to mention
that TIMP-1 has a paradoxical effect by promoting cancer
cell metastasis, an effect that may be the result of interactions
of MMPs and TIMPs within the protease web (Kruger
et al., 2010).
Finally, an often made misunderstanding is that TIMP-1
is a specific inhibitor for MMP-9. It should be clear
all TIMPs can inhibit the active forms of MMP-9.
Nevertheless, TIMP-1 (and TIMP-3) complexes preferentially
interact with the C-terminal hemopexin domain of proMMP-9
(Baker et al., 2002).
Inhibition by fatty acids
Fatty acids inhibit the activity of gelatinase A and gelatinase B.
The inhibition is dependent on the alkyl chain length and the
presence of unsaturated linkages. These tend to increase
inhibition and polyunsaturated fatty acids are more efficient
inhibitors of gelatinase B than of gelatinase A. Although it
was first thought that the inhibition by fatty acids was due to
the terminal carboxylate group and by chelation of the active
site zinc ion, Berton et al., showed that Zn2 chelation was
not the main determinant. The high inhibitory potential of
fatty acids against gelatinases is linked to the fact that these
MMPs have a deep hydrophobic S10 active site pocket. This
hydrophobic pocket may also accommodate biphenylalanine
in a peptide inhibitor, selected from a library of 10 000
synthetic heptapeptides. This heptapeptide inhibitor

242

J. Vandooren et al.

mimicked the natural sequence of the propeptide of MMP-9


(Hu et al., 2005a).
Furthermore, it was shown that an MMP-2 form, lacking
the fibronectin domain, possessed a different inhibition
profile by fatty acids compared to full-length MMP-2.
Therefore, the inhibitory potential of fatty acids may
involve binding to the fibronectin-like repeats, which are
only present in MMP-2 and MMP-9 (Berton et al., 2001)
(Figures 2 and 3).

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Inhibition by chemicals
Many MMP inhibitory drugs have been developed with the
original idea to use these as peroral treatment for invasive and
metastatic cancers. After the failure of clinical trials of MMP
inhibitors for cancer therapy, we suggested that many of these
drugs may be better candidates for inflammatory and vascular
diseases (Hu et al., 2007; Muroski et al., 2008).
Whereas the search for peptide and peptidomimetic MMP
inhibitors continues, gradually more preclinical studies are
emerging about the usefulness of these inhibitors in inflammation (Hu et al., 2005b, 2006; Dejonckheere et al., 2011;
Qiu et al., 2012a,b).
Although these applications are discussed in forthcoming
sections about the role of MMP-9 in diseases, it is critical to
notice that tetracyclines and chemically modified tetracyclines (CMTs) have been most studied and applied (Paemen
et al., 1996; Sorsa & Golub, 2005). Within this group of
molecules, doxycycline and minocycline, MMP-9 inhibitory
tetracyclines (Paemen et al., 1996) have been most evaluated
(Koistinaho et al., 2005; Xue et al., 2010; Yong et al., 2004;
Zabad et al., 2007). Tetracyclines lower the levels of MMP-9
secretion and also function as inhibitors of MMP-9 activity
(Salo et al., 2006).
Inhibitory chemicals have also been covalently bound to
resins to enable to purify MMPs in their active form. After
activation, MMP-9 is a rather unstable enzyme. With the
use of inhibitor tethered resin, however, active MMP-9 in
biological samples can be purified and detected with such
reagents (Hesek et al., 2006).
Other control mechanisms of gelatinase B activity
As is the case for all enzymes, temperature and pH control
catalytic activity. For MMP-2 and MMP-9 the environmental
pH may differ intracellularly and outside cells. In the
extracellular milieu, which is in equilibrium with body
fluids, the pH is mostly neutral under physiological conditions and this pH corresponds to the optimal one of MMP-9.
However, under conditions of inflammation, the extracellular
pH may decrease considerably and this has consequences for
the activity of MMP-9 and other enzymes. Finally, catalysis
by MMP-9 may also change in patients with fever or
undercooling, when the temperature gradually shifts from
the optimal one at 37 C (Fasciglione et al., 2000).
Complex formation
Homomultimerisation of MMP-9 is an intracellular enzymatic
process: even after prolonged incubation in vitro and in
the presence of glutathione as oxidation-reduction couple,

Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

monomers will not form multimers. However, multimers


produced by cells are reduction-sensitive and will be
converted in vitro to monomers in the presence of reducing
chemicals (Van den Steen et al., 2000). The multimer form
shows altered biochemical properties.
Although little is known about MMP-9 homomultimerisation, Bannikov et al. showed that the specific activity of
monomeric MMP-9 is lower than that of multimeric MMP9 (Bannikov et al., 2002). In contrast, Olson et al. showed
that MMP-9 monomers and MMP-9 multimers had similar
catalytic efficiency in hydrolyzing both a fluorogenic
peptide substrate (MOCAcPLGLA2pr(Dnp)-AR-NH2) and
gelatin (fluorescein-labeled DQ gelatin) (Olson et al.,
2000). A common and difficult problem with such studies
is the reliability of enzyme titrations. Most often colorimetric assays are used to determine the quantities of
purified enzyme preparations and these are subject to many
artifacts.
Another control mechanism of MMP-9 activity is localization to specific compartments. Integrins interact with the
carboxyterminal part (Bjorklund et al., 2004) of MMP-9 and
thus may bind the enzyme to the cell surface (vide supra).
The described integrins are leukocyte functional antigen-1
(LFA-1, also called a1b2-integrin) and a4b2-integrin
(Redondo-Munoz et al., 2006, 2008, 2010; Stefanidakis
et al., 2003).
MMP-9 also binds to intercellular cell adhesion molecule1 (ICAM-1, the LFA-1 ligand), or a complex containing
ICAM-1 on the cell surface and subsequently cuts the
extracellular part of this transmembrane protein (Fiore
et al., 2002; Sultan et al., 2004).
Finally, MMP-9 interacts with ECM collagens and,
dependently on the MMP-9 concentration, contraction of
collagen gels was shown to differ. Low MMP-9 concentrations promoted gel contraction and high MMP-9 concentrations resulted in lowered gel contraction (Defawe
et al., 2005).
From single substrates to substrate repertoires
A decade ago, it was possible to generate a shortlist of MMP9 substrates (Van den Steen et al., 2002a). As stated in the
introductory section, technology drives scientific progress.
If we know today much more about MMP-9 substrates, this
is mainly due to technology developments. Degradomics,
the definition of all substrates of a specific enzyme, has
complemented the technology of proteomics. Technical
innovations in protein labeling and mass spectrometry
analysis, often coming from the laboratory of Chris
Overall (Dean & Overall, 2007; Overall & Dean, 2006;
Overall et al., 2004; Prudova et al., 2010; Schilling & Overall,
2008) have advanced the field considerably. Differential gel
electrophoresis (DIGE) (Vierstraete et al., 2004) and identification of MMP substrates by multidimensional (Cauwe
et al., 2009) and ultrahigh sensitive chromatography techniques (Xu et al., 2008) have also broadened the spectra of
MMP-9 substrates.
With the use of various techniques, substrate repertoires
were thus identified. We reviewed membrane-bound substrates of MMPs (Cauwe et al., 2007). After the discovery

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of efficient intracellular substrates of MMP-9, including


heat shock and lens crystallins (Descamps et al., 2005;
Starckx et al., 2003), we systematically analyzed intracellular
MMP substrates (Cauwe et al., 2009; Cauwe & Opdenakker,
2010). In doing so, we found that MMP-9 may also
be an MMP of the intracellular matrix (ICM) (Cauwe &
Opdenakker, 2010).
Importantly, only in a limited number of studies, the newly
discovered substrates were validated in vivo, e.g. with the use
of MMP-9-deficient mice (Cauwe et al., 2011; Descamps
et al., 2005; Greenlee et al., 2006; Heissig et al., 2002;
Xu et al., 2010). This type of studies are instrumental to
yield biological insights into the vast sets of data generated by
degradomics research and to direct future pharmaceutical
research on MMP inhibitors.

243

Gonadotropin releasing hormone (GrH) was found to


increase the levels of MMP-2 and MMP-9 mRNA in human
decidual cells of the endometrium (Chou et al., 2003).
Two hormones, corticotropin-releasing hormone (CRH) and
urocortin are believed to induce the local secretion of MMP-9
in placenta and fetal membranes, which contributes to
membrane rupture and triggers onset and progression of
human labor (Li & Challis, 2005; Xu et al., 2002). However,
many fundamental questions remain about the role of MMP-9
in reproduction. For example, it is not yet known whether
the subfertility phenotype is due to male, female or double
subfertility problems. The subfertility phenotype (Dubois
et al., 2000) is subtle, because we did not observe it in a leaky
Mmp9 KO mouse line. With the present technologies of RNA
sequencing and proteomics analysis it will be possible to
study this aspect in-depth.

Physiological and pathological processes


Whereas MMP-9 was first considered as a modifier of ECM
proteins, in the past decade different roles of MMP-9 have been
found for substrates attached to the cell surface (Cauwe et al.,
2007) or even within cells (Cauwe & Opdenakker, 2010). New
MMP-9 substrates are being discovered at a high pace (Prudova
et al., 2010; Vaisar et al., 2009; Xu et al., 2008), adding more
pathways to the MMP-9 degradome. In addition, as mentioned
already, new functions of MMP-9 were discovered that
provided insights into how its domain structure contributes to
functional activities. As a comparison, immunoglobulin G
(IgG) is composed of 2 heavy and 2 light chains, each built from
individual immunoglobulin folds. This structure yields at the
aminoterminal region two antigen binding sites (Fragment with
the antigen binding sites, Fabs) and at the opposite carboxyterminal site the anchor for binding to receptors (Fragment
crystallizable, Fc). Furthermore, the Fabs and Fc of IgG are
connected by a flexible hinge region. The MMP-9 monomer
structure may be viewed in a similar way (Figure 4) and its
major functions, catalysis mediated by the aminoterminus and
binding to receptors through the carboxyterminal hemopexin
domain, are kept nicely separated by the O-glycosylated
domain. This central theme about structures and functions
needs to be kept in mind in the next sections about catalytic and
non-catalytic functions in physiology and pathology.
Physiological functions
Reproduction
Subfertility was noticed as a spontaneous phenotype in
MMP9 deficient mice (Dubois et al., 2000). Meanwhile
more than 100 publications have linked MMP-9 to reproduction. Because MMPs and their inhibitors are involved in the
preparation of the human endometrium for pregnancy, in
implantation into the uterus and for embryo development, it is
surprising that MMP-9 knockout mice are able to reproduce.
Many of the biological aspects of MMP-9, including catalytic
and non-catalytic activities, specific substrates involved in
reproduction and the roles of inflammatory cells (neutrophils)
have been studied in reproduction biology (Alexander et al.,
1996; Daimon & Wada, 2005; Martinez-Hernandez et al.,
2011; Whiteside et al., 2001).

Growth and development


Many studies on blood
vessel functions in wildtype and MMP-9 null mice have been
published (reviewed by Hu et al., 2007) and functional links
between MMP-9 and the formation, structure and remodeling
of new blood vessels have emerged. Vascular phenotypes are
thought to involve catalytic activity of MMP-9 resulting either
in the cleavage of ECM components (such as native and
denatured collagens) and processing of various angiogenic
chemokines such as CXCL5/ENA78, CXCL6 (granulocyte
chemotactic protein-2), and CXCL8 (interleukin-8) or release
of angiogenic cytokines such as VEGF and FGF. Catalytically
active, TIMP-free, hemopexin domain-containing, full-length
enzymes are required for angiogenesis induction (Ardi et al.,
2007). MMP-9 induces a differential influx of additional
protein components into angiogenic tissue and proteolytically
triggers release of bioactive basic FGF (FGF-2). The released
bioavailable FGF-2, acting through its cognate receptor FGFR2, is the actual inducer of angiogenesis downstream of activated
MMP-9. The VEGF/VEGFR pathway may function downstream of the generated FGF-2 (Ardi et al., 2009).
Human umbilical vein endothelial cells shed vesicles of
300 to 600 nm, containing MMP-2 and MMP-9 in both
proforms and active forms. External addition of these vesicles
to umbilical vein endothelial cells may stimulate the cells to
move through a matrigel, a layer of reconstituted basement
membrane. Levels of vesicular MMP-2 and MMP-9 were
increased by the addition of FGF and VEGF (Taraboletti
et al., 2002). Tumstatin, a fragment of the a3 chain of
collagen IV, is a suppressor of pathologic angiogenesis and
can be generated upon cleaving collagen IV with active
MMP-9 (Hamano et al., 2003).
Angiogenesis and vascular remodeling.

The first MMP-9 KO


mice were generated in 1998 and showed a phenotype of
growth retardation with bones that were 10% shorter than
those of WT mice (Vu et al., 1998). MMP-9 has an essential
role in the maintenance of bone structure, more specifically,
of the trabecular bone architecture. Lack of MMP-9 in mice
resulted in improved density of the trabeculae but more brittle
femurs (Nyman et al., 2011). Normal bone development is

Bone development and remodeling.

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maintained by osteoblasts (bone formation) and osteoclasts


(bone resorption). A good balance between both cell types is
essential for normal bone development. This issue as well as
matrix remodeling in endochondral bone formation were
nicely reviewed (Ortega et al., 2004).
During the development of the mouse, MMP-9 is highly
expressed in osteoclasts (Reponen et al., 1994). Bone
resorption requires several steps: migration of precursor
osteoclasts, invasion of osteoclasts out of the circulation into
the tissue, homing to bone and the differentiation of precursor
osteoclasts into active multinucleated osteoclasts (Yu et al.,
2003). SDF-1, a CXC chemokine, is responsible for the
homing of precursor osteoclasts to bone and also stimulates
the production of MMP-9, necessary for the transmigration
of the pre-osteoclasts. The differentiation of precursor osteoclasts into active osteoclasts can be induced by receptor
activator of nuclear factor kappa-B ligand (RANKL), a cytokine of the TNF cytokine family. Once the differentiation
process is initiated by RANKL, the differentiating osteoclasts
continuously produce MMP-9 (Yu et al., 2003). Once
stimulated with RANKL or SDF-1, the cells show an
increased capacity to migrate through a collagen matrix and
this activity could completely be abolished by administrating
a general MMP inhibitor (Yu et al., 2003).
MMP-9 is also indispensable for the migration of osteoclasts (OCs) through collagen in long bones (Blavier &
Delaisse, 1995) and is an essential component for the
migration of newly formed pre-osteoclasts into primitive
long bones. Once a binding between CD44 and hyaluronan
(HA) is established, the production of MMP-9 by OCs is
halted and OC migration, which may result in more bone
resorption, is prevented. It is suggested that HA-CD44
interaction may act as a stop signal for bone-resorbing cells
(Spessotto et al., 2002).
Wound healing
Epithelial regeneration
Normal regeneration of the epithelial barrier requires cell
migration, proliferation, formation of a multilayered structure
and restoration of interactions with the underlying tissue.
Upon injury, general MMP expression is up-regulated.
Although MMP-9 is not commonly expressed in epithelial
cells, the expression of MMP-9 is induced during the
process of wound healing. Additional MMP-9 is expressed
and provided by infiltrating inflammatory cells. Indeed,
the inhibition of MMPs has been linked to aberrant
re-epithelialization and incomplete restoration of cell adhesions (Mohan et al., 2002).
Paradoxically, MMP-9 KO mice have an increased rate of
corneal and skin re-epithelialization after surgical removal
of a portion of the epithelial layer. This effect could be
completely undone by rescue experiments (exogenous addition of proMMP-9). Therefore it is thought that MMP-9 has
a rather differential role on corneal epithelial regeneration.
Evidence was provided that the faster epithelial resurfacing
is due to a higher rate of cell proliferation of peripheral
cells. In addition, increases of IL-1a were registered in the
healing corneal wounds of MMP-9 deficient mice which is
consistent with the earlier onset of infiltration with

Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

inflammatory cells. In addition, a strikingly larger deposit


of fibrinogen was present in the wound bed of MMP-9 KO
mice, which suggests an impaired resorption of provisional
matrix which is deposited during wound healing. In the
cornea, this might interfere with the transparency which is
needed for appropriate light transmission (Mohan et al.,
2002). This contrasts clearly with findings in a cutaneous
wound healing mouse model, in which wound healing
was delayed in MMP-9 KO mice due to delayed reepithelization and reduced clearance of fibrin clots (Kyriakides
et al., 2009).
In human keratinocytes (Xue & Jackson, 2008) and airway
smooth muscle cells (Johnson & Knox, 1999), MMP-2 is
shown to promote cell survival and inhibit differentiation
through autocrine signaling, while autocrine MMP-9 counteracts MMP-2 and promotes cell differentiation (Xue &
Jackson, 2008).
Liver repair requires angiogenesis and mobile endothelial
cells for vascular reconstruction. In this context, bile acids
were found to induce MMP-9 production by binding to the
nuclear receptor Farnesoid X receptor (FXR) and subsequent
binding to the MMP-9 promoter region and induction of
mRNA production. Human umbilical vein endothelial cells
(HUVECs) incubated with the bile acid chenodeoxycholic
acid (CDCA) had increased motility and angiogenic capacity,
dependent on the FXR-MMP-9 signaling pathway. In addition, both FXR and MMP-9 are required for the formation of
focal adhesions (FA), established by phosphorylation and
activation of focal adhesion kinase (FAK) (Das et al., 2009).
Cell migration and tissue maintenance
The role of MMP-9 in cell migration, in particular of
leukocytes, has been a constant point of discussion. After
many studies it became clear that the read-outs, the animal
model and the type of cells being investigated differed too
much to obtain a clear view (Betsuyaku et al., 1999; DHaese
et al., 2000). Meanwhile, it is clear that the proteolytic
activity of MMP-9 is essential for progenitor cell recruitment
from the bone marrow, an important issue applied in stem cell
mobilization for transplantation and for regenerative medicine
(Heissig et al., 2002; Jin et al., 2006). Again, neutrophils and
neutrophil-derived MMP-9 may be a critical factor in this
process (Pruijt et al., 1999, 2002; Velders et al., 2004).
Similarly, during infections and in inflammation, MMPs and
ADAMs play pivotal roles (Murphy et al., 2008), and
leukocyte migration under the influence of chemotactic
factors contributes to host defense or may cause collateral
damage by expression of MMPs. As a consequence, deletion
of the MMP9 gene or inhibition of the protein may aggravate
infections (Calander et al., 2006; Letellier et al., 2010). The
fact that MMP-9 levels are increased during the course of
infections does not necessarily imply a beneficial role. This
may be the consequence of activation of leukocytes by
microbial products and even contribute to the immunopathology, as is the case in many autoimmune diseases (vide infra)
(Bergin et al., 2008; De Palma et al., 2008). In many such
instances, the inflammation, rather than the infection per se,
leads to tissue damage and pathology (Cuenca et al., 2006;
De Palma et al., 2009). Specifically, dependent on the animal

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model of infection (viral, bacterial, fungal or parasitic) or


inflammation (acute versus chronic) used, MMP-9 may have
beneficial or detrimental effects. One should have a clear
picture that currently used animal models of infection and
inflammation represent a broad spectrum form hyperacute
lethal infections to chronic sterile conditions. Consequently,
the corresponding immunopathology may vary from exclusive
neutrophil involvement (within minutes to hours) to slow
giant cell formation by fusion of macrophages (weeks to
months). Therefore, it is difficult to generate a general rule
about whether MMP-9 inhibition may be useful as adjunct
treatment of infection and inflammation (McMillan et al.,
2004; Renckens et al., 2006). This implies that for clinical
trials and other uses of MMP inhibitors the inclusion criteria
will have to be well defined.
We advocate that more studies are needed to obtain a better
and more general overview. When from preclinical studies
a congruent view is obtained, however, MMP-9 may be
therapeutically targeted with inhibitors. As a first step, clear
distinctions between infections and sterile inflammatory
reactions are critical (MacLauchlan et al., 2009; Moore
et al., 2011; Vermaelen et al., 2003). Second, in acute
infections with viruses and bacteria entering the circulation, a
general triggering of the most abundant circulating neutrophils leads to massive release of neutrophil collagenase/
MMP-8 and MMP-9 and to life-threatening shock conditions.
It is clear that MMP inhibition may help to save lives in such
conditions (Qiu et al., 2012a). At the other extreme of the
spectrum, in the case of chronic stages of (sterile) autoimmune diseases, in which the regenerative action of MMP-9
may be beneficial (vide infra), it is clear that MMP-9
inhibition has to be avoided. In addition, under sterile
conditions, even recruited neutrophils and neutrophil
MMP-9 may be beneficial and contribute to regenerative
angiogenesis (Heissig et al., 2010).
It was recently shown that MMP-9 activation by plasminogen is a requirement for macrophage trans-ECM migration (Gong et al., 2008). MMP-9 is required for dendritic cell
migration (Adhikary et al., 2012). These studies are in line
with previous ones on Langerhans cells in the skin and in
models of organ transplantation (Campbell et al., 2005;
Fernandez et al., 2005; Kobayashi, 1997).
Coincident with the boost
of regenerative medicine, MMP-9 has been studied in various
aspects of stem cell research. As previously mentioned for
IL-8/CXCL8mediated induction of hematopoietic progenitor
cells (Pruijt et al., 1999), MMP-9 is critical for accelerating
hematopoietic reconstitution after depleting hematopoietic
cells with 5-fluorouracil (5-FU). The mechanism is by
cleaving membrane associated Kit-ligand (mKitL) into
soluble Kit-ligand (sKitL) and thereby inducing a rapid
release of sKitL facilitating progenitor cell recruitment and
hematopoietic reconstitution (Heissig et al., 2002). Mobilized
peripheral blood stem cells (PBSCs) are valuable tools in
transplant surgery since they are a source of hematopoietic
stem cells (HSC). The release of these PBSCs from the bone
marrow can be induced with several factors such as granulocyte colony-stimulating factor (G-CSF) or certain chemokines
(e.g. CXCL2). However, also neutrophil-derived proteases

Stem and progenitor cell migration.

245

such as NE, cathepsin G and MMP-9 are indispensable in this


process. Synergistic mobilization (induction with G-CSF and
CXCL2) can be completely blocked by anti-MMP-9 (Pelus
et al., 2004). The OG domain and in particular the hemopexin
domain are important for MMP-9-induced cell migration.
MAPK and PI3K inhibitors can inhibit MMP-9-induced
cell migration and the JNK pathway can be linked to
MMP-9-induced cell migration (Dufour et al., 2008).
Upon brain injury (e.g. ischemia) adult neural stem/
progenitor cells (aNPCs) differentiate into neuroblasts and
migrate to the site of injury. Factors such as stromal cellderived factor 1 (SDF-1/CXCL12) and vascular endothelial
growth factor (VEGF) act as aNPC chemoattractants, a
process which requires aNPCs to produce MMP-3 and
MMP-9 (Barkho et al., 2008).
Stem cells brought into a low oxygen level environment
produce high levels of MMP-9 and this occurs through
HIF-1a signaling and subsequent activation of the canonical
Wnt pathway. This results in increased cell proliferation and
migration. The migratory and proliferating capacities were
blocked by MMP-9 inhibition (Ingraham et al., 2011).
By controlled proliferation and ECM
remodeling cardiac fibroblasts are responsible for maintaining
the structural integrity of the heart. During this process,
cardiac fibroblasts are activated into myofibroblasts which
start proliferating and invading heart tissue. In vitro cardiac
myofibroblast proliferation and invasion can be induced by
supplementing the cells with TNF-a. This increase in invasive
potential is thought to involve MMP-9 as its expression
levels are also upregulated upon TNF-a stimulation (Porter
et al., 2004).
Tissue maintenance.

Learning, memory and maintenance of the neuronal network


Probably the first study about the role played by MMP-9 in
brain activity was published as an abstract (Lim et al., 1998).
It was mentioned that MMP-9 knockout mice had impaired
learning and behaviour. One Hineininterpretierung is that
MMP-9 is an important enzyme to control behaviour and to
keep good memory. With the present aging of populations and
the intrinsic epidemic of neurological diseases such as stroke
and Alzheimers disease in elderly people, the studies on
memory and memory loss gain attention (vide infra). The role
of MMPs in diseases of the central nervous system have been
extensively reviewed (Opdenakker et al., 2003; Yong, 2005;
Yong et al., 1998). In recent years, normal functions of MMPs
have come into spotlights and several review articles on the
normal roles of MMPs in the CNS have been published
(Dziembowska & Wlodarczyk, 2012; Dzwonek et al., 2004;
Tonti et al., 2009).
Synaptic plasticity, learning and memory. Functional circuits

in the brain can be established by long-term potentiation (LTP),


a process by which long-term stimulated synapses result in
spine growth. In contrast, spine shrinkage can also occur in
long-term unstimulated synapses or long-term depression
(LTD). These processes of changing morphology of dendritic
spines are thought to lie at the basis of synaptic plasticity,
learning and memory. The exact mechanisms for LTP and LTD

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are not yet fully understood, however, it has been speculated


that proteases acting on the extracellular matrix are involved
(Agrawal et al., 2008; Michaluk et al., 2011).
Indeed, MMP-9 is necessary for LTP mediated enlargement of spines (Wang et al., 2008) and only catalytically
active MMP-9 causes dendritic spines to become longer and
thinner via integrin b1 (Michaluk et al., 2011). In addition,
ECM molecules such as fibronectin can determine morphological oligodendrocyte differentiation by influencing the net
activity and distribution of MMP-9 (Siskova et al., 2009).
For these reasons MMP-9 is an important molecule for
synaptic plasticity, learning and memory. In response to
neuronal activity, MMP-9 activity increases and results
in cleavage of b-dystroglycans (Michaluk et al., 2007;
Sbardella et al., 2012). The cleavage of b-dystroglycans by
MMP-9 and MMP-2 has also been studied in Schwann cells
(Zhong et al., 2006) and in brain pathologies such as
experimental autoimmune encephalomyelitis (Agrawal
et al., 2006), an animal model of multiple sclerosis, the
latter of which also leads to cognitive deficits. TIMP-1 can
also be produced by activated neurons and acts as a
controller of ECM degradation during late LTP. This
mechanism might be important to prevent further LTP of
other circuits in the same area of the activated neuron
(Okulski et al., 2007).
Regulation and maintenance of compartments in Schwann
cells. The link between the cortical cytoskeleton and the

basement membrane in Schwann cells is formed by a


dystroglycan-dystrophin complex. This complex basically
exists of the intracellular dystrophin which binds to the
actin filaments, the transmembrane b-dystroglycan that forms
the link between dystrophin and the extracellular space, and
a-dystroglycan which connects b-dystroglycan to extracellular matrix components such as laminins and proteoglycans.
The extracellular part of b-dystroglycan can be cleaved by
MMP-9 which results in the shredding of a-dystroglycan
(Agrawal et al., 2006; Michaluk et al., 2011; Sbardella et al.,
2012; Zhong et al., 2006).
MMP-9 suppresses Schwann cell proliferation and induces
differentiation functions (e.g. migration and myelin protein
maintenance) in vivo by stimulating MAPKp44/42 signaling
via activation of IGF-1, ErbB4 and PDGF tyrosine kinase
receptor and the Ras/Raf/MEK pathway (Chattopadhyay &
Shubayev, 2009). For this reason, MMP-9 functions as a
modulator of Schwann cell signaling and induces remodeling
after nerve injury.
Pathological roles of gelatinase B
Increased levels of MMP-9 have been associated with many
inflammatory, autoimmune, degenerative and neoplastic diseases, but this association does not necessarily imply a
functional role in these pathologies. Often MMP-9 levels are
measured by ELISA. It needs to be mentioned that with
ELISA one measures immunoreactivity in biological samples
and this may include proMMP-9, activation forms, degradation products, monomers, oligomers, heterocomplexes and
even non-covalent complexes with TIMP-1.
Secondly, when gelatin zymography is used as a quantitative method, complex samples may need to be prepurified

Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

first (Descamps et al., 2002). Against intuition and as


misinterpreted in many studies on biological samples, gelatin
substrate zymography analysis does not yield information on
enzyme activity, because it also detects (inactive) proforms
and because MMP-9/TIMP-1 complexes dissociate during
electrophoresis (Vandooren et al., 2013).
When increased levels of MMP-9 are associated with
pathology, a number of possibilities exist to obtain insights
into causes or effects and detrimental or beneficial roles.
These distinctions are important, because in the case of a
causal detrimental role of MMP-9, enzyme inhibition may
constitute an important way to combat the disease. If animal
models of the pathology exist, the use of (inducible) gene
knockout animals may give further insights (vide supra)
(Descamps et al., 2005; Greenlee et al., 2006; Xu et al.,
2010). A valuable overview of Mmp KO phenotypes, with
for each study information about the genetic background is
available (Hu et al., 2007).
Alternatively, the use of MMP inhibitors may be indicative
of a role of the enzyme and may cure diseases. Unfortunately,
often the lack of specificity of the inhibitors results in
inhibition of off-target MMPs and leads to unacceptable sideeffects. For this reason, new technologies, such as expression
microarrays and RNA sequencing experiments, will yield
broader views into pathology, the MMPs and natural
inhibitors involved and will define new treatment options.
Lung conditions
In recent years, MMP-9 has been extensively studied as a
key player in airway inflammation and remodeling. Lung
diseases are common and range from hyperacute hypersensitivity reactions, such as asthma and acute respiratory
distress syndrome (ARDS), to chronic diseases, such as
chronic obstructive pulmonary disease (COPD) and from
common genetic disorders, e.g. cystic fibrosis, to lung
cancer and interstitial lung fibrosis. In addition, lungs are
being transplanted with increasing success and these conditions are also associated with intrinsic problems in which
MMP-9 may be involved. A delicate balance exits between
deposition of ECM components and their degradation. An
imbalance may result in, for example, pulmonary fibrosis.
Since MMP-9 is involved in ECM degradation, its role has
been studied in several lung conditions (Profita et al., 2004).
Bronchial epithelial cells express MMP-9 upon stimulation
with TNF-a through activation of nuclear transcription
factor NF-kB (Hozumi et al., 2001). In addition, in
inflammatory lung diseases, leukocytes contribute to the
MMP-9 load. In lung cancer, the tumor cells and the
associated leukocytes may be producers of MMP-9
(Hanahan & Weinberg, 2011; Piccard et al., 2012).
Asthma and ARDS are well-known acute lung diseases in
which MMP-9 expression has been observed (Fligiel et al.,
2006; Kong et al., 2011; Profita et al., 2004). MMP-9/TIMP-1
ratios are important factors in the pathogenesis of ARDS. As
with many conditions, the imbalance between MMP-9 and
TIMP-1 is associated with differential airway remodeling that
leads to either short- or long-course ARDS. A predictive
value for MMP-9/TIMP-1 in ARDS prognosis is suggested
(Lanchou et al., 2003).

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Many forms of ARDS occur as consequences of infections,


ranging from viral and bacterial to parasitic infections. The
inflammatory triggers, often pathogen-associated molecular
patterns may be quite different, but their receptor mechanisms
converge into abovementioned intracellular signaling cascades leading to NF-kB-dependent activation of the MMP9
gene. In addition, other MMP genes may be activated.
For instance, detailed information about general MMP and
specific MMP-9 regulation has been recently reviewed for
Gram-negative bacteria (Vanlaere & Libert, 2009) and for
protozoan parasites (Geurts et al., 2012b). One example
relates to elevated MMP-9 levels in induced sputum samples
from patients with allergic bronchopulmonary aspergillosis.
MMP-9 ELISA levels correlated with the severity of airflow
obstruction and IL-8 levels (Gibson et al., 2003).
Elevated MMP-9 expression and activity levels in peripheral lung tissue of COPD patients were related to disease
severity. MMP-9 promoter activation could also be correlated
with disease severity. SIRT1 plays a critical regulatory role in
suppressing MMP-9 expression, as deduced from MMP-9
promoter analysis of peripheral lung tissue and studies using
macrophage-like U937 cells. mRNA, protein, and activity of
SIRT1 were significantly down-regulated with increasing
severity of COPD in lung tissue and PBMCs. High levels of
oxidative and nitrative stress are found in patients with
COPD, and increased expression of nitric oxide synthases and
of 4-hydroxy-2-nonenal, a signature of lipid peroxidation, are
observed in peripheral lung tissue of patients with COPD.
In U937 cells, oxidative stress decreased SIRT1 activity
without any change in the protein level in vitro. As stated
above, SIRT1 is a NAD-dependent protein deacetylase
and metabolic sensor, and oxidative stress reduces cellular
NAD levels via PARP-1 activation (Nakamaru et al., 2009).
Elevated levels of MMP-9 were also detected in exhaled
breath condensates from children with bronchiectasis.
TIMP-1 levels, however, showed no significant differences
(Karakoc et al., 2009).
Cigarette smoke contains several carcinogenic components
such as free radicals (superoxide radicals, hydroxyl radicals,
hydrogen peroxide) and benzo[a]pyrene which can activate
signal transduction pathways, resulting in lung inflammation
and malignancies. These toxins are thought to activate NF-kB
signaling and MMP-9 induction (Nakamaru et al., 2009;
Shishodia et al., 2003). Indeed, cigarette smoke can induce
the formation of emphysema by attracting immune cells
which release proteases. These proteases trigger a local
imbalance between proteases and their inhibitors, which leads
to ECM destruction and emphysema (Churg et al., 2007).
However, MMP-9 KO mice develop the same degree of
smoke-induced airway inflammation and airspace enlargement as control mice. Although MMP-9 is found in
emphysematous lungs, no correlation can be made with the
development of emphysema making MMP-9 inhibition
strategies less feasible (Atkinson et al., 2011).
Cystic fibrosis implies inflammation and remodeling of the
airways. Therefore, protease activity seems of significant
importance in the pathogenesis of CF. Increased levels of
active MMP-9 and decreased levels of TIMP-1 have been
found in the lungs of CF patients. In addition, high levels of
HNE are also found in sputum samples and these levels

247

correlate with the levels of activated MMP-9 (Gaggar et al.,


2007). Recently, it was shown that HNE is capable of
degrading TIMP-1 and cleaving the MMP-9 prodomain
(Jackson et al., 2010).
Lung transplantation is becoming routine treatment for
patients with end-stage lung diseases of various pathogenesis.
Upon transplantation, inflammation and rejection may occur
and specific drugs may be helpful to reduce these risks.
In a recent study it was shown that azithromycin reduces
rejection rates, an effect that was correlated with lower
levels and activities of MMP-9 in transplant lungs (Verleden
et al., 2011).
Inflammatory diseases
The discrimination between infections and inflammatory
diseases is fading out, in particular if one takes into account
that host microbiomes are gaining importance as contributing
factors that enhance the susceptibility to develop autoimmune
diseases. In principle, autoimmune diseases, such as rheumatoid arthritis and multiple sclerosis are characterized by
progressive life-long (chronic) inflammation. In many
patients these diseases flare up with acute inflammation. In
a simplified view, myeloid cells are major contributors in the
acute phases, whereas the chronic and progressive phases are
more orchestrated by lymphocytes. The reader is referred for
background information about these aspects to previous
reviews (Opdenakker & Van Damme, 2011; Opdenakker
et al., 2001a, 2003; Van den Steen et al., 2002a). Here we
discuss recent insights into diabetes and systemic lupus
erythematosus as systemic autoimmune diseases and glomerulonephritis, carditis and pemphigus as organ-specific autoimmune disorders.
Diabetes. One of the most common autoimmune diseases is

type I diabetes. It results from loss of insulin-producing


pancreatic b-cells and a subsequent b-cell-destructive autoimmune response. It has been suggested that MMP-9 is a
diabetogenic factor through proteolytic cleavage of insulin
and generation of immunodominant insulin peptides, thereby
triggering an autoimmune response (Descamps et al., 2003;
Opdenakker & Van Damme, 1994).
In both hyperacute and subacute mouse models of type I
diabetes, proMMP-9 is upregulated and associated with
disease activity. However, MMP-9 is not the causative
factor in this animal model but, when activated by trypsin,
it is a permissive factor for insulin degradation and diabetes
(Descamps et al., 2004).
In an induced diabetes mouse model (induction with
alloxan), increased plasma levels of MMP-9 are registered. In
Mmp9 KO mice induction of diabetes resulted in an increase
of MMP-2 as a compensatory mechanism. Increased levels of
MMP-9 upon induction of diabetes could be associated with
endothelial dysfunction and apoptosis (Camp et al., 2003).
In the mentioned animal models of diabetes, so far only
short-term effects were measured. In addition, in a xenotransplant model of islets of Langerhans, inflammation was
associated with rejection and MMP-9, from locally recruited
leukocytes, had a detrimental effect (Lingwal et al., 2012).
However, in a syngeneic islet transplantation model, MMP-9

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derived from a specific subset of myeloid cells contributed to


islet revascularization with beneficial effects (Christoffersson
et al., 2012). These examples clearly show that the contexts of
inflammation and the molecules and cells that are locally
recruited determine the success of transplantation. MMP-9
levels are increased in type 2 diabetic patients with CAD. The
antidiabetic drug PPARg-activator rosiglitazone significantly
reduces MMP-9, TNF-a and SAA serum levels (Marx
et al., 2003).
Aside short-term (weeks) effects of diabetes, hyperglycemia leads to advanced glycation end products (AGE).
Through interaction with receptors of AGE (RAGE), present
on many cell types, including myeloid cells, long-term effects
of diabetes become evident. This receptor interaction leads to
cytokine, chemokine and MMP induction. Alternatively, high
glucose levels enhance TGF-b and Smad signaling, increase
cell surface expression levels of TGF-b receptors and induce
MMP-mediated activation of latent TGF-b. Consequently,
glucose leads to cell hypertrophy and inhibition of MMP-9/
MMP-2 may reverse this process (Wu & Derynck, 2009). Lack
of glycemia control leads to long-term clinical effects of diabetes with neuropathy, nephropathy and retinopathy as notorious examples (Stitt, 2010; Sugimoto et al., 2008; Tang et al.,
2011). Increased levels of MMP-9 have been associated with
these three vascular complications of diabetes and may become
a biomarker of disease state (Bhatt & Veeranjaneyulu, 2010;
Kowluru et al., 2012; Lauhio et al., 2008).
Diabetic retinopathy and the role of MMP-9 are further
discussed in the section about eye diseases (vide infra). The
balance between MMP and TIMP concentrations and AGEinduced angiopathy play crucial roles in the process of
diabetic wound healing. For example, MMP-9 and TIMP-1
show pathologic effects on skin damage and ulcer healing.
The dynamic changes in MMP-9 and TIMP-1 in the diabetic
foot were studied in a rat model (Yang et al., 2009).
Systemic lupus erythematosus. The role of MMP-9 as a disease
marker of disease activity in systemic lupus erythematosus
(SLE) has been compared with other autoimmune diseases and
well reviewed (Ram et al., 2006). In SLE MMP-9 has a dual
role (disease-promoting by generating remnant epitopes for T
cell reactivity and generation of autoantibodies and diseaselimiting by clearance of abundant intracellular proteins after
cell death, vide infra). By clearing autoepitopes in immunogenic substrates, systemic antibody-mediated autoimmunity is
suppressed (Cauwe et al., 2011). This interpretation is based on
two types of evidence. First, MMP-9 cleaves efficiently many
intracellular substrates into remnant epitopes, against which
autoantibodies are formed in SLE (Cauwe et al., 2008, 2009). In
addition, in an animal model of lupus, showing increased
apoptosis, knocking out the Mmp9 gene led to increased
immunopathology, lymphoproliferation and autoantibody formation against DNA, small nuclear ribonucleoproteins and
many intracellular proteins. Autoantibodies in these mice were
remarkably similar in reactivities to those observed in patients
with SLE (Cauwe et al., 2011). MMP-9 thus has a protective
effect in this animal model of SLE.

Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

glomerulonephritis, autoimmune carditis, bullous pemphigoid


and multiple sclerosis. During an episode of glomerulonephritis, the immune system is triggered, resulting in the release of
soluble factors by both resident cells and infiltrating immune
cells. This process results in interstitial fibrosis and thickening
of the ECM. For this reason, the balances between MMP-9 and
TIMP-1 may be important in this process. TNF-a results in an
increase of MMP-9 levels in proximal tubular cell cultures and
both TNF-a and IL-1b lowered the TIMP-1 levels. When
combining TNF-a and IL-1b, however, MMP-9 levels were
not increased (Nee et al., 2004). Further studies about the
expression of all MMPs and TIMPs are needed to understand
better the pathology and to evaluate whether exogenous
inhibitors may become useful drugs.
In experimentally-induced autoimmune carditis (EAC),
both MMP-2 and MMP-9 become upregulated in the heart.
Initially the gelatinases are derived from infiltrating macrophages and in a later stage from cardiomyocytes. EAC could be
suppressed by treatment with minocycline, which inhibits predominantly MMP-9. Inhibition of MMP-2 did not suppress the
pathology which is in line with a significant role for MMP-9 in
the pathogenesis of EAC (Matsumoto et al., 2009).
Bullous pemphigoid (BP) develops when proteases such as
NE and MMP-9, released from e.g. polymorphonuclear
leukocytes (PMN), degrade hemidesmosomal and ECM
components in the basement membrane. This process results
in separation of dermis from epidermis and visual occurrence
of skin blisters. Mice deficient in MMP-9 are resistant to
experimentally-induced BP (Liu et al., 1998) and MMP-9 is
able to inactivate the a1-proteinase inhibitor, which is an
NE inhibitor, resulting in more active NE (Liu et al., 2000).
In addition, proMMP-9 can be activated by plasmin during
the initial phases of BP development (Liu et al., 2005).
These data are instrumental to define specific inhibitors
(Paemen et al., 1996), including monoclonal antibodies, for
treatment of pemphigus and related diseases (Shimanovich
et al., 2004).
In experimental autoimmune encephalomyelitis (EAE)
mouse models of multiple sclerosis, knocking out of MMP2 resulted in a more severe disease. The underlying mechanism was thought to involve MMP-9 since a significant
increase in MMP-9 levels was detected (Esparza et al., 2004).
These studies complemented those of EAE in MMP9-deficient mice (Dubois et al., 1999). A second complementation came with the identification of b-dystroglycan
cleavage as a major contributing element in EAE development. This cleavage is executed by both MMP-2 and MMP-9
and double MMP-2/MMP-9 knockout mice were completely
resistant against EAE. These data suggest that inhibitors with
dual specificity (against gelatinase A and B) may become new
drugs for the treatment of multiple sclerosis (Agrawal et al.,
2006). Since minocyclin and doxycyclin are gelatinase
inhibitors, recent clinical studies in MS demonstrate the
applicability of MMP inhibition as adjunct therapy (Metz
et al., 2009).

In allergic immunopathology, histamine is secreted


by mast cells and basophils upon allergen binding of their IgE
receptors. The released histamine binds to four

Allergy.
autoimmune inflammation. Organ-specific
autoimmune diseases with a presumed role of MMP-9 include

Organ-specific

Gelatinase B/MMP-9

DOI: 10.3109/10409238.2013.770819

transmembrane G-protein coupled receptors (H1R, H2R, H3R,


H4R) which are found in several cell types involved in
inflammation (Bongers et al., 2010). Histamine can trigger an
inflammatory state in keratinocytes through H1R signaling. In
keratinocyte cell cultures, H1R signaling induces the secretion
of MMP-9. In addition, healthy skin samples, challenged with
histamine lead to degradation of the basement membrane
(Gschwandtner et al., 2008). In experimentally-induced
asthma, TLR2 activation of neutrophils leads to the release
of MMP-9 and this protects against allergen-induced disease
(Page et al., 2009).

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Dentistry
The role of MMPs in dental pathology is known since long.
Importantly, the use of chemically modified tetracyclines as
treatment of periodontitis is an example of successful drug
development of MMP inhibitors (Sorsa & Golub, 2005). Even
at the interface between normal dentin tissue and artificial
materials, MMPs may play a remodeling role and inhibitors of
MMPs may be useful to prevent disease (De Munck et al.,
2009; Gu et al., 2011).
Muscle disease
Four groups of proteases have been shown to be involved
in skeletal muscle degeneration: the ubiquitin-proteasome
system, lysosomal proteases, calcium-dependent proteases,
and MMPs. However, only the MMPs are directly related to
degradation of the ECM (Liu et al., 2010).
Gelatinase A and B are highly upregulated in a model of
disuse-induced muscle atrophy. But only gelatinase A null
mutant knockout mice show significantly reduced muscle
atrophy as compared to wildtype littermates. With these
findings, the authors (Liu et al., 2010) suggest that
gelatinase A, and not gelatinase B, plays a critical role in
disuse-induced skeletal muscle atrophy. This may be due to
the difference in substrate specificity of both gelatinases.
MMP-2, but not MMP-9, is capable of digesting type I
collagen, which is the dominant type of collagen in the
muscle ECM (Liu et al., 2010).
Tumor necrosis factor-related weak inducer of apoptosis
(TWEAK) (Chicheportiche et al., 1997) was shown to be
involved in muscle atrophy (Dogra et al., 2007) and to induce
MMP-9 by upregulating its promotor through NF-kB and
AP1. In addition, after injecting mice with TWEAK, inflammation, necrosis, basement membrane degradation and
muscle loss were significantly attenuated in MMP-9 KO
mice, proving an important role for TWEAK-induced MMP-9
in myopathy (Li et al., 2009).
Skin conditions
MMP-9 is involved the shedding of desmoglein-3 (dsg-3)
from keratinocytes which can result in alterations of tissue
architecture and the formation of epithelial blisters
(Cirillo et al., 2007). Expression of MMP-9 is induced
in primary skin keratinocytes by IL-13 and MMP-9 and
IL-13 are coexpressed in acute lesions of eczema (Purwar
et al., 2008).
Infrared radiation, present in natural sunlight, causes an
increase in temperature in the human skin. This heat shock

249

induces the production of ROS, MMP-1 and MMP-9 in


human keratinocytes (Shin et al., 2008). Bullous pemphigoid
has been mentioned under the heading of autoimmune
diseases (vide supra).
Cardiovascular diseases
Cardiovascular diseases have an enormous impact on global
health and remain a primary target for the pharmaceutical
industry. Understanding the mechanisms of disease and the
molecules involved may lead to life-saving therapies and
novel drugs that reduce cardiovascular morbidity. We here
review a limited number of recent studies.
Atherosclerosis and restenosis
have been associated with localized remodeling of the ECM
and migration and proliferation of smooth muscles cells. For
this reason, matrix remodeling enzymes such as MMPs, and
in particular gelatinases which have the ability to degrade
gelatins, have been implicated (Whatling et al., 2004).
Vulnerable atherosclerotic plaques typically have a thin
fibrous cap, a reduced number of smooth muscle cells and a
large lipid core. The rupture of an atherosclerotic plaque
occurs more frequently at regions containing high amounts of
monocyte derived macrophages (MDM) and foam cells
(Speidl et al., 2004). Therefore, it was thought that macrophages are able to induce plaque rupture by secreting matrix
degrading proteases. Indeed, mouse macrophages overexpressing active MMP-9 significantly enhanced plaque rupture
(Gough et al., 2006).
MMP-9 and MMP-12 are able to cleave N-cadherin,
resulting in the proliferation of VSMCs through b-catenin
signaling (Dwivedi et al., 2009). VSMC proliferation contributes to intimal thickening. MMP-9 has a key role in the
migration of vascular smooth muscle cells (Mason et al.,
1999; Whatling et al., 2004).
In aortic valve stenosis tissue, gelatinases (MMP-2 and
MMP-9) are detected (Edep et al., 2000; Salo et al., 2006).
Also, MMP-9/TIMP-1 imbalances have been implicated
(Satta et al., 2003) and CMTs inhibited pathological remodeling of human aortic valves (Salo et al., 2006).
In a patient genotyping study, long microsatellites in the
promoter region of MMP-9 correlated with carotid atherosclerosis with thin fibrous cap plaques. However, microsatellite length did not correlate with plasma levels of MMP-9
(Fiotti et al., 2006).
MMP-9 and several other MMPs (MMP-1, MMP-2 and
MMP-3) are expressed in atherosclerotic tissue. Upon
activation they may contribute to vascular remodeling and
plaque rupture. In this context, oxidized low-density lipoproteins upregulate MMP-9 expression and downregulate TIMP1 expression in monocyte-derived macrophages. This may
contribute to matrix degradation in atherosclerotic plaques
(Lijnen, 2001). In addition, it was found that MMP-9
production in monocytes is upregulated by factors such as
epinephrine, norepinephrine (post-operative stress) and LPS
(Speidl et al., 2004).
It is thought that inappropriate vascularization is the
main cause of restenosis following angioplasty. In this
pathological vascular remodeling, VSMCs migrate into

Atherosclerosis and restenosis.

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J. Vandooren et al.

the lumina, promoted by degradation of the extracellular


matrix by matrix metalloprotenases. In a rat model, the
proinflammatory IL-17 induces migration of VSMC
cells and induces MMP-9 expression via p38 MAPK- and
ERK1/2-dependent NF-kB and AP-1 activation. TIMP-1
and 2 were not significantly affected by IL-17 suggesting
that IL-17 alters MMPs/TIMPs balances in favor of MMP
expression and induces ECM degradation (Cheng et al.,
2009).
Several gene polymorphisms were found in the promoter
(Van den Steen et al., 2002a), coding and 3-end untranslated
regions of the human MMP-9 gene. One polymorphism was
shown to predispose people to development of coronary
atherosclerosis. In contrast, a haplotype was found which
resulted in a protective effect against atherosclerosis (Morgan
et al., 2003).
During percutaneous coronary intervention, MMP-9 and
IL-6 are released from plaques. In addition, MMP-9 activity is
increased. MMP-9 functions as a biomarker to determine
plaque instability (Robertson et al., 2007).
In atherosclerotic plaques, complement components are
upregulated (Yasojima et al., 2001) and the complement
component C5a induces mRNA levels of MMP-1 and MMP-9
in human macrophages (Speidl et al., 2011). Berberine,
a natural extract from Rhizoma coptidis, reduces MMP-9
and EMMPRIN expression in PMA-stimulated macrophages
by suppressing the activation of the p38 pathway in
PMA-induced macrophages (Huang et al., 2011). Since
MMP-9 and EMMPRIN are expressed in atherosclerotic
samples (Major et al., 2002), this compound shows potential
as a treatment of atherosclerosis.
In primary HASMCs, oxLDL can regulate MMP-2 and
MMP-9 expression, necessary for cell migration, by miRNAmediated epigenetic regulation which might be a novel
mechanism in atherosclerosis (Chen et al., 2011). The
formation of neointima after carotid ligation was recently
shown to involve upregulation of MMP-9 activation by
MMP-3 (Johnson et al., 2011).
In general, MMPs have been implicated in the
development of aneurysms by an increase in proteolysis of
extracellular matrix proteins. However, MMP-2 polymorphisms were not associated with coronary aneurysms (Lamblin
et al., 2002).
Abdominal aortic aneurysm (AAA) presents as a
permanent dilatation of the abdominal aorta and involves
upregulation of proteolytic pathways, loss of the arterial
wall ECM, inflammation, oxidative stress and apoptosis
(Nordon et al., 2011), especially MMP-9 and MMP-12
have been implicated in AAA. This was based on both
in vitro activity tests and in vivo expression data. In
addition, it was shown that AAA degeneration and rupture
can be prevented with MMP inhibitors (synthetic tetracycline derivatives) or by overexpression of TIMP-1 in a
rat model (Lijnen, 2001). Also, Plg KO mice, which have
less MMP-9 activation and less macrophage migration
through the ECM, were protected against AAA and this
effect was abolished by adding active MMP-9. These
findings suggest an important role for the plasminogen/

Aneurysms.

Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

MMP-9 cascade in the inflammatory response and AAA


development (Gong et al., 2008).
In diabetes, AGEs induce MMP-9 through activation of
ERK, p38 mitogen-activated protein and NF-kB, a pathway
that is antagonized by TGF-b. This finding and previously
mentioned AGE functions in inflammation suggest that
AGE-neutralizing therapies may be effective in the prevention
of human AAA development and progression (Zhang
et al., 2011).
Left ventricular hypertrophy. Left ventricular hypertrophy is

an adaptation of the heart to high blood pressure, which may


progress into heart failure. The process involves remodeling
of the myocardial ECM and thus MMPs such as MMP-9.
MMP inhibition may be beneficial in the treatment of this
condition (Heymans et al., 2005). Hypertension and aortic
stiffness are associated with increased MMP-9 and serum
elastase activity (Yasmin et al., 2005).
Stroke. Increased blood levels of MMP-9 were correlated

with brain injury in stroke patients (Rosell et al., 2005). In


infarcted and hemorrhagic areas of the brain, strong infiltrations can be seen of MMP-9 containing neutrophils,
correlating with basal lamina collagen IV degradation and
degradation of the BBB. These findings relate MMP-9 levels
with hemorrhagic complications after stroke (Rosell et al.,
2008). Edaravone, a free radical scavenger used for neurological recovery after brain infarct suppresses the mRNA
expression and protein levels of MMP-9 and inhibited NF-kB
activation (Yagi et al., 2009).
It was shown that irradiation of an
ischemic site, promotes vascular regeneration. Low doses of
radiation up-regulate MMP-9 and thereby result in higher
levels of sKitL which stimulates progenitor cell migration
and incorporation of new mast cells in the ischemic tissue.
In addition, the radiation induces VEGF release from mast
cells resulting in more mast cell recruitment and further
up-regulation of MMP-9 (Heissig et al., 2005). Upon ischemia
induction in mice, higher levels of active MMP-9 were
measured. When treated with melatonin these levels
decreased significantly and coincided with reduced brain
damage and hemorrhagic transformation. The decreased
levels of MMP-9 could be correlated with decreased levels
of uPA and increased levels of TIMP-1 and PAI-1 (Tai
et al., 2010).

Ischemia and reperfusion.

Thrombosis results from an imbalance between


blood clotting and fibrinolysis, in which MMP-9 probably
contributes to the latter. IFN-g plays a detrimental role in the
resolution of deep vein thrombosis by suppressing MMP-9
and VEGF expression (Nosaka et al., 2011).

Thrombosis.

Transplantation biology
Upon experimental autologous and allogeneic bone marrow
transplantation, recombinant growth factors, such as G-CSF
and chemotactic factors including IL-8, are administered for
repopulation purposes and to mobilize HSCs (vide supra).

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DOI: 10.3109/10409238.2013.770819

In this context, it was observed that both cytokine types lead


to increased serum levels of MMP-9 (Carstanjen et al., 2002)
and that MMP-9 antibodies block mobilization (Pruijt et al.,
1999), suggesting an important role for MMP-9 in HSC
mobilization. Nevertheless, Mmp9 KO mice do not have
impairment in HSC mobilization upon a G-CSF or Flt-3 L
challenge, suggesting a role for compensatory enzymes
(Robinson et al., 2003).
Coronary artery bypass autografting (CABG) of the
saphenous vein is often performed in order to revascularize
myocardium. Unfortunately, occlusions in these grafts often
occur. Simvastatin, a commonly used statin, is able to reduce
the levels of MMP-9 in tissue and to inhibit the migration
of saphenous vein SMC (Porter et al., 2002) and human
cardiac myofibroblast cells (Turner et al., 2007). Simvastatin
disrupts the actin-based cytoskeleton by inhibition of
geranylgeranylation of RhoA and thus disruption of the
RhoA/ROCK pathway (Turner et al., 2005, 2007). Altered
levels of MMP-9 after lung transplantation were previously
mentioned as a surrogate marker of rejection-associated
inflammation (Verleden et al., 2011). Furthermore, alterations
in the levels of MMP-9, the cellular origin of the enzyme and
its functions in allograft rejection of tracheal cartilage
and transplanted hearts was demonstrated by comparisons
between wildtype and MMP-9-deficient mice (Campbell
et al., 2005; Fernandez et al., 2005).
Bone pathologies
Mutations in the MMP-9 gene have been associated with
genetic bone diseases called metaphysical anadysplasia
(Lausch et al., 2009). In addition, a single nucleotide
polymorphism (SNP) in the human MMP-9 gene is associated
with lumbar disc herniation (Hirose et al., 2008). Decreased
expression of the MMP9 gene in tibial dyschondroplasia
lesions has been observed in chickens. In this disease,
a decrease in vascularization is observed, related to the
expression of MMP9 (Velada et al., 2011). These data are in
line with the phenotypic characteristics of Mmp9-deficient
mice (Vu et al., 1998) that also have chondrodysplasia. Recent
functional data about normal bone turnover and the cellular
origin of MMP-9 after bone fracture yield multiple roles
of MMP-9 in differentiation and regulation of periosteal
and enchondral bone formation. The effects of inhibitors of
MMP-9 activity on normal and pathological bone remodeling
need to be further investigated (Ortega et al., 2010; Wang
et al., 2012).
Proliferative diseases
In recent years our view on tumor development has significantly changed. While originally mainly the tumor cells were
studied, more recently the emphasis has shifted to the study of
surrounding stroma cells, the ECM and components of the
immune system (Radisky & Bissell, 2004). With this in mind,
also our insights into the importance of MMPs in tumor
development have changed. For example, it was found that
tumor-surrounding stromal cells often are major producers of
tumor-associated proteases (Overall & Lopez-Otin, 2002;
Stuelten et al., 2005). In general, MMPs are involved in the
early stages of tumor development. They degrade the ECM

Gelatinase B/MMP-9

251

and basement-membrane and in doing so, contribute to the


formation of an ideal environment for further tumor development. MMPs further promote tumor development by
releasing/activating other tumor promoting agents. In later
stages, MMPs may promote metastasis (Overall & LopezOtin, 2002) for example by modifying tumor cell integrins
(Ranuncolo et al., 2002). By proteolysis of the ECM, cell
migration and thus tumor invasion may be promoted.
These complexities in the tumor micro-environment have
become better understood and provided explanations for the
failure of MMP inhibitors against invasion and metastasis of
tumor cells (Coussens et al., 2002; Kessenbrock et al.,
2010). The basic idea of MMP inhibition was good, but the
MMP inhibitors were moved to the clinic too quickly. More
basic research is needed to understand the role of MMPs in
tumor biology. We have already addressed the issue that
MMP inhibitors may constitute excellent drugs for specific
inflammatory and vascular diseases in which the genetics of
the host are stable (Hu et al., 2007). Even with a high
genetic instability of tumors (Hanahan & Weinberg, 2011),
the day will come when it may be possible to evaluate the
genetic (in)stability of specific tumors by analyzing multiple
markers. In a similar way as the resistance to chemotherapeutic drugs may be analysed and used to fine-tune cancer
therapy, it may become possible to evaluate which cancer
patients may benefit form MMP inhibition. We here review
recent literature about basic mechanisms related to this
paradigm. Then we address recent data about specific
tumors.
Basic mechanisms. Originally, the direct production of

proteinases by tumor cells was studied. Tumor promoting


and carcinogenic agents, as well as oncogenic proteins,
growth factors and hormones may contribute to the regulation
of MMP-9 production by various cancer cells (Van den Steen
et al., 2002a). In the last decade, the production of MMP-9 by
surrounding cells under the influence of tumor cells has
become an important study topic. For instance, expression of
MMP-9 in tumor surrounding fibroblasts can be induced by
the tumor cells (Stuelten et al., 2005). The study of signaling
within cells, after surface receptor triggering by soluble or
membrane-bound agonists is another key research interest,
because it may constitute a point for interference with new
drugs. Indeed, cells expressing specific transcription factors
have altered invasive properties (Jorda et al., 2005). MMP9-mediated proteolysis may also have beneficial effects.
For instance, tumstatin and endostatin are collagen fragments with antitumorigenic activity (OReilly et al., 1997;
Maeshima et al., 2002; Radisky & Bissell, 2004).
Inflammation has become one of the hallmarks of cancer,
but its effect may be antitumoral or protumoral (Hanahan &
Weinberg, 2011). Tumor-associated macrophages and neutrophils are therefore now discriminated on the basis of
markers into antitumoral macrophages (M1) and neutrophils
(N1) and antitumoral myeloid cells, respectively M2 and N2
(Allavena et al., 2008; Piccard et al., 2012).
In the tumor microenvironment, MMP-9 is often found as a
secretory product of recruited inflammatory cells, mainly
neutrophils and macrophages (Bergers et al., 2000; Coussens
et al., 2000; Nielsen et al., 1996). It is thought that tumor cells

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J. Vandooren et al.

are able to use this MMP-9 for invasion and metastasis


(Masson et al., 2005).
MMP-9, secreted in the tumor microenvironment by
stromal cells originating from the bone marrow, potentiates
the release of VEGF, resulting in increased serum VEGF
levels and further attraction of bone marrow-derived cells.
It is postulated that removing MMP-9 from the tumor
microenvironment, breaks the VEGF/bone marrow/MMP-9
loop, results in reduced serum levels of VEGF and therefore
reduces angiogenesis and myelopoiesis. This hypothesis was
successfully tested with an amino-biphosphonate inhibitor
(Melani et al., 2007). Neutrophils and macrophages express
MMP-9 differentially and have a differential influence on
vasculogenesis and angiogenesis (vide supra). As demonstrated with Mmp9 KO mice, MMP-9 is required for tumor
vasculogenesis. This effect could be undone with transplantation of wild-type bone marrow and could be attributed to
CD11b-positive myelomonocytic cells. Therefore, it is suggested that MMP-9 may become a target enzyme for adjunct
therapy in radiotherapy for cancer (Ahn & Brown, 2008). Fast
growing tumors often have areas of low oxygen content. This
environment stimulates the production of HIF1 that in turn
induces SDF-1/CXCL12 in tumor cells and recruits MMP-9
producing monocytic cells from the bone marrow. These cells
are sufficient to generate an angiogenic switch in glioblastoma (Du et al., 2008).
In order for a small tumor to grow, it needs the formation
of tumor-associated vascular structures. This event, tumor
cells starting producing proangiogenic factors in order to form
these vascular structures, is often referred to as the angiogenic
switch (Bergers et al., 2000; Folkman, 1992). In this
switching process, MMP-9 was shown to act as a proangiogenic factor in several cancer models and Mmp-9 KO mice
(Giraudo et al., 2004).
Besides adhesion receptors, mediating physical interactions between tumor cells and host tissue cells, and besides
ECM and cytokines/growth factors, promoting tumor cell
survival and growth, MMPs are a third class of molecules
involved in tumor-associated tissue remodeling (Yu &
Stamenkovic, 2000). The basement membrane and transmembrane proteins provide signals for cell survival and
growth. Loss of these signals may result in cell death or
suppression of proliferation in both normal and cancerous
cells. Whereas most MMP isoforms have been shown to
contribute to cancer progression and metastasis, these might
also negatively regulate cancer cell survival. For example,
MMP-9 suppresses the proliferation of T lymphocytes
through disruption of interleukin-2Ra signaling (Biswas
et al., 2010).
MMP-9-degranulating neutrophils have been linked with
the onset of the angiogenic switch in developing tumors.
Inflammatory neutrophils may serve as an immediate and
major source of MMP-9 in pre-angiogenic tissue. This
angiogenic proMMP-9 is released from human neutrophils
in a unique, TIMP-free form, which is in contrast to the
TIMP-1-complexed proMMP-9 produced by most other cell
types, including macrophages and tumor cells (Opdenakker
et al., 2001a). It appears to be the TIMP-free nature of
neutrophil proMMP-9 that determines its potent proangiogenic potential, as this distinct proMMP-9 form constitutes

Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

the major proangiogenic component of the entire human


neutrophil-released contents (Ardi et al., 2007, 2009).
Yu and Stamenkovic demonstrated that proteolytically
active MMP-9 is bound at the cell surface to CD44
(hyaluronan receptor) and locally cleaves and activates
TGF-b. These observations yielded an explanation for the
question why expression of the CD44-MMP-9 complex
correlates with TA3 cell invasiveness in vitro and in vivo
(Yu & Stamenkovic, 2000).
In recent years, the study of MMP-9 inhibitors has shifted
from synthetic molecules to investigations of natural inhibitors, such as RECK and TIMPs and to MMP-9-inducing
EMMPRIN. When screening a human fibroblast cDNA
expression library the RECK (reversion-inducing cysteinerich protein with Kazal motifs) gene was discovered for
inducing a flat reversion when expressed in a v-Ki-rastransformed NIH 3T3 cell line. It encoded a 110 kDa
membrane-anchored glycoprotein which was able to inhibit
MMP-9 secretion from cells and MMP-9 catalytic activity
(Takahashi et al., 1998). Meanwhile, it has been established
that both membrane-bound and solubilized RECK directly
inhibit catalytic activity of MMP-2, -9 and -14. By inhibiting
MMP-14, RECK also prevents the formation of the proMMP2/TIMP-2/MMP-14 complex and the activation of proMMP-2
into MMP-2 (Oh et al., 2001; Rhee & Coussens, 2002).
Furthermore, in patient studies, RECK mRNA levels were
correlated with the amount of activation of MMP-2, but not
MMP-9 (van der Jagt et al., 2006). In contrast, only
membrane-bound RECK protein inhibited secretion of
proMMP-9 (Oh et al., 2001; Rhee & Coussens, 2002). In
several cancer studies, higher RECK levels were correlated
with less invasiveness, less metastasis and lower recurrence
rates (Furumoto et al., 2001; Takeuchi et al., 2004; van der
Jagt et al., 2006). These data indicate that RECK is a
membrane-bound MMP inhibitor, distinguishable from other
MMP inhibitors. Its properties may create a local zone of high
inhibition of proteolysis around the cell (Welm et al., 2002).
Paradoxical data exist on TIMP expression and tumorigenesis, both in mouse models and human cancers. TIMPs
might have MMP-independent functions in tumor biology
(Welm et al., 2002).
Originally TIMP-1 was shown to possess antimetastatic
effects (Kruger et al., 1998). Later, TIMP-1 was discovered as
a factor that contributes to liver metastasis (Kopitz et al.,
2007). The mechanism is by cell signaling, in which
hepatocyte growth factor, hypoxia-inducible factor-1a and
microRNA are involved (Cui et al., 2012).
Similar paradoxical effects as those for TIMP-1 have also
been observed for MMP-9. For example, in a number of
studies with downregulation or gene knock-out of MMP-9,
increased tumor development, progression and metastasis
in vivo was observed (Deryugina et al., 2005; Roy et al.,
2007). Alternatively, in various tumor cell types (Chung et al.,
2003; Ito et al., 1999; Shieh et al., 2005; Sun et al., 2004;
van Ginkel et al., 2004; Wu et al., 2002), AXL protein
tyrosine kinase is upregulated. In normal circumstances, the
GAS6/AXL signaling cascade is involved in regulating
vascular cells (Melaragno et al., 1999). Recently this GAS6/
AXL signaling was shown to activate MMP-9 expression
through ERK/MEK, NF-kB and Brg-1. MMP-9 was shown to

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be a prerequisite for AXL-induced enhancement of invasion


(Tai et al., 2008).
Extracellular matrix MMP inducer (EMMPRIN, basigin,
CD147) is a heavily glycosylated protein that belongs to
the immunoglobulin superfamily (Biswas et al., 1995). The
protein is enriched on the surface of tumor cells where it
promotes tumor growth, invasion, metastasis and angiogenesis (Nabeshima et al., 2006). Signaling through EMMPRIN
induces the expression of several MMPs, including MMP-9
(Tang et al., 2004; Yang et al., 2003; Yang et al., 2012).
In addition, increased expression of MMP-9 and EMMPRIN
is associated with poor prognosis for patients with several
types of cancer (Piao et al., 2012; Zhong et al., 2008) and
increased invasion and metastasis of cancer cell types
(Yu et al., 2009).
Further basic research questions that were recently
addressed in tumor biology include specific ways of delivery
of MMP-9 in the tumor environment, local effects on
cell surface molecules and escape of tumors from immune
mechanisms and from therapeutic interventions such as
radiotherapy.
The shedding of vesicles containing MMP-9, MMP-2 and
uPA was shown for human breast carcinoma cells (Dolo et al.,
1994), human fibrosarcoma cells (Ginestra et al., 1997) and
human ovarian cancer cells (Dolo et al., 1999). Whether these
vesicles are similar to or different from those shed from
normal cells, such as astrocytes (Sbai et al., 2010), remains an
open question.
When MMP-9 is secreted into the extracellular milieu or
becomes membrane-bound, it may exert cleavage of adhesion
molecules involved in the immunological synapse and thus
limit antigen presentation in the activation of helper and
cytotoxic T lymphocytes. The shedding of the extracellular
domain of ICAM-1 by active MMP-9 is believed to be
involved in tumor cell evasion of immune surveillance. Upon
MMP-9-dependent ICAM-1 processing, tumor cells were also
more resistant to natural killer (NK) cell-mediated toxicity
(Fiore et al., 2002).
Radiotherapy induces cell necrosis and tissue hypoxia.
Both mechanisms induce IL-8 and neutrophil recruitment
and activation and leads to local inflammatory reactions.
Sublethal doses of radiation enhanced invasiveness of
hepatocellular carcinoma cells but not in normal hepatocytes.
Irradiation induced MMP-9 mRNA levels, protein levels
and activity by activating the PI3K/Akt/NFkB pathway
(Cheng et al., 2006).
Specific types of cancer
NGAL/MMP-9 complexes were found in
about 90% of the urine samples of patients with breast
tumors but were practically absent in samples from healthy
women (Bolignano et al., 2010). Furthermore high expression of MMP-9 was shown to be significantly associated
with shorter survival rates in patients with breast cancer
(Dufour et al., 2011). Increased levels of MMP-9, NGAL
and the MMP-9/NGAL complex were measured in the
serum of patients with invasive ductal carcinoma. These
parameters were correlated with disease severity
(Provatopoulou et al., 2009).

Breast cancer.

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253

In a breast cancer cell line, the cancer cells require


protease nexin-1 (PN-1) to disseminate to distant organs.
PN-1 is a serine protease inhibitor (serpin) (Gloor et al., 1986)
which binds and inactivates several proteases (e.g.trypsin,
prostasin, factor XIa, uPA, tPA, thrombin (Knauer et al.,
2000; Stone et al., 1987)). The PN-1/protease complex binds
LRP-1 and activates ERK signaling which leads to increased
expression of MMP-9 and contributes to tumor metastasis
(Fayard et al., 2009). Expression of MMP-2 and MMP-9 in
breast cancer is partially related to the expression of the
transcription factor AP-2 and HER2 oncogene. MMP-9
expressing stromal cells are related with poor prognosis in
hormone-responsive small tumors. In contrast, MMP-9
expression in carcinoma cells often relates to survival
(Pellikainen et al., 2004).
The invasive potential of human breast cancer cells can be
enhanced by adding IL-1b which acts through a SHP2-dependent signaling pathway. Activation of this pathway
also results in higher levels of secreted MMP-9 (Wang
et al., 2005).
An alternatively spliced variant of CD99 was shown to
result in elevated motility, fibronectin binding, invasiveness
and MMP-9 expression in two human breast cancer cell lines
(Byun et al., 2006). Moreover, Mmp9 gene transfer experiments indicate that MMP-9 has a positive role in the treatment
of established breast cancers. Overexpression of MMP-9
resulted in the release of antiangiogenic endostatin (Bendrik
et al., 2008). However, MMP-9 inhibition experiments in a
mouse breast cancer model revealed different outcomes
depending on the genetic background of different mouse
strains (Martin et al., 2008), again emphasing that information
about the genetic background of mouse strains is crucial.
Adhesion receptor integrin avb3 promotes metastasis of
human breast cancer cells in cooperation with MMP-9. Only
cells with activated integrin avb3 had activated 82 kDa MMP9 in the cell culture supernatant and these cells also showed
increased migration towards fibrinogen substrates. Since the
increase in metastatic potential is substrate-specific, the
authors suggest that fibrinogen degradation by active MMP9 might trigger additional pathways which promote migration,
e.g. attract neutrophils, and that the combination with
activated integrin avb3 results in a switch from firm adhesion
to dynamic migration (Ranuncolo et al., 2002).
Bladder cancer. MMP-9 has been implicated in bladder
cancer (Sier et al., 2000) and urinary MMP-9, MMP-2 and
TIMP-2 were found to be useful parameters for non-invasive
diagnosis of bladder cancer (Eissa et al., 2007). Migration and
invasion of bladder cancer cells is linked to p38 MAPK activity
and to production of increased levels of MMP-2 and MMP-9.
Moreover, the regulation of MMP-2 and MMP-9 was mediated
by p38MAPK-driven MAPKAPK2 and resulted in stabilization of MMP-2 and MMP-9 transcripts (Kumar et al., 2010).
Cervical cancer. In a preclinical mouse model for human

papillomavirus (HPV)-induced cervical cancer, MMP expression analysis with the use of RT-PCR, immunohistochemistry
and gelatin zymography techniques revealed that only MMP-9
expression was significantly upregulated during tumor progression (Giraudo et al., 2004). In this cervical cancer model,

254

J. Vandooren et al.

MMP-9 expression was pinpointed to stroma and tumorinfiltrating macrophages (Giraudo et al., 2004). In a clinical
analysis, increased MMP-9 expression levels were correlated
with poor prognosis for the patients (Sheu et al., 2003). In
cervical carcinoma-associated myeloid cells, a STAT3dependent molecular cascade was identified which leads to
MMP-9 induction (Schroer et al., 2011).

Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

The balance between RECK and MMP-9 expression


levels can be used as a prognostic indicator for colorectal
cancer (Takeuchi et al., 2004). In the late stages of colorectal
cancer, transcription factor AP2a expression is absent,
resulting in high expression levels of MMP-9 (Schwartz
et al., 2007).
Laryngeal cancer
(LC) tissue contains high levels or MMP-9 and Foxp3
Tregs compared to normal tissue. LC-derived MMP-9 is
capable of generating tolerogenic dendritic cells that are able
to induce the development of immunosuppressive Tregs
capable of suppressing LC-specific CD8 T cells. This could
also mean a suppression of CD8 cytotoxic T cells which are
necessary for immune surveillance. It is thought that MMP-9
is able to activate TGF-b inside dendritic cells and thereby
allowing it to generate Tregs (Wang et al., 2011).
Nasopharyngeal carcinoma tumor cells were more invasive
after stimulation with the stress hormone norepinephrine,
concomitant with an upregulation of MMP-2 and MMP-9
release (Yang et al., 2006).
Laryngeal and nasopharyngeal cancer.

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Skin and oral epithelial cancers. In recent years researchers

have been searching for reliable markers to predict metastasis


of squamous cell carcinoma (SCC). Non-invasive oral
carcinomas lacked MMP-7, MMP-9 and MMP-12, compared
to invasive oral carcinomas, and these MMPs were used as
prognostic markers (Impola et al., 2004). An important
feature in the progression of skin cancer is the epithelial-tomesenchymal transition (EMT) of keratinocytes. This transition into an invasive phenotype is believed to require TGF-b
signaling. Interestingly, whereas TGF-b has a rather negative
influence in later stages of cancer cell development, it plays a
protective role in early stages (Wang, 2001). In immortalized
keratinocytes, TGF-b induces MMP-9 expression (Salo et al.,
1991). Moreover, integrin a3b1 is able to potentiate MMP-9
activation by TGF-b (Lamar et al., 2008a). Keratinocyte
immortalization by p53-null mutation leads to altered a3b1function and induces the expression of MMP-9 potentiating
tumor cell invasion (Lamar et al., 2008b).
The migration of keratinocytes during tumor progression
may also be regulated by TNF-a via an MMP-9- and avb6
integrin-dependent pathway. This process is also involved in
wound healing (Scott et al., 2004).
In a mouse study with malignant keratinocytes, tumor
vascularization and invasion was inhibited in knock-out mice
lacking both MMP-2 and MMP-9 at the same time (Masson
et al., 2005). In a mouse keratinocyte cell line (MK cells),
secreted MMP-9 levels were elevated by increased Ras
oncogene expression. The signaling event was by MEK/
ERK and was a1b3 integrin-dependent. In addition, it
was shown that a1b3 integrin has the capacity to stabilize
MMP-9 mRNA and to enhance MMP-9 protein levels
(Iyer et al., 2005).
In esophageal squamous cell
carcinoma (ESCC), cytoplasmic expression of MMP-9 is
predominantly observed in carcinoma cells at the invasion
front. Comparable to prostate cancer, MMP-9 expression
was correlated with the expression of matrilysin-2/MMP-26,
which is an activator of proMMP-9 (vide supra). Concomitant
expression of matrilysin-2 and MMP-9 at the cancer invasive
front was correlated with poor prognosis (Yamamoto et al.,
2004). In pancreatic cancer, neutrophil-derived MMP-9 acts
as a potent and direct VEGF-independent angiogenic factor
(Bausch et al., 2011). Radiation-enhanced cell invasiveness of
hepatocellular carcinoma (HCC) cells was studied in relation
to MMP-9 expression. Radiation was found to increase MMP9 mRNA levels, protein amounts and activity. Interestingly,
this effect was not observed in normal hepatocytes (Cheng
et al., 2006). Lysophosphatidic acid (LPA) induced coordinated increases in MMP-9 expression and HCC cell invasion
(Park et al., 2011).

Cancers of the digestive system.

Inhibition of MMP-9 production with siRNA


successfully inhibited medulloblastoma tumor growth in an
intracranial injection model. Transfection of MMP-9 siRNA
resulted in cell cycle arrest (Rao et al., 2007). In a patient
study of meningiomas, higher expression of Ets-1, MMP-2
and MMP-9 was correlated with recurrence of meningioma.
In addition, Ets-1 expression was correlated with expression
of both MMP-2 and MMP-9. These findings show that Ets-1
might be involved in meningioma recurrence by up-regulating
MMP-2 and MMP-9 (Okuducu et al., 2006). In mice, glioma
tumor growth was successfully inhibited by adenoviral
mediated transfer of an antisense-MMP9 gene sequence
(Lakka et al., 2002b).
Promising results were obtained with simultaneous inhibition of cathepsin B and MMP-9 gene expression with the use
of an expression vector encoding two hairpin siRNAs. The
technique showed potential for use as glioma therapy by
inhibition of cancer cell invasiveness and of development of
cancer-associated vascular structures (Lakka et al., 2004).
An ERK-dependent pathway regulates MMP-9-mediated
glioma invasion. Cells with blocked ERK contained less
MMP-9 mRNA, protein and activity. In addition, the levels
of transcription factors AP-1 and NF-kB were reduced.
(Lakka et al., 2002a).

Brain tumors.

MMP-2 and MMP-9 are expressed and


secreted by prostate cancer cells, resulting in decreased
prostate cancer cell proliferation and increased sE-cad
shedding. The b3-integrin-ERK pathway was involved in
this PDK1-mediated expression and secretion of MMP-2 and
MMP-9 (Biswas et al., 2010). High expression levels of
MEK5 have been associated with proliferation and metastasis
of prostate cancer. MEK5 was also associated with higher
expression levels of MMP-9 mRNA by stimulating the
MMP9 gene promoter through AP-1 (Mehta et al., 2003).
In prostate carcinoma tissue, co-expression of matrilysin-2
and proMMP-9 were found to play an important role in tumor

Prostate cancer.

Gelatinase B/MMP-9

DOI: 10.3109/10409238.2013.770819

progression since matrilysin-2 is an activator of proMMP-9


(Zhao et al., 2003).
In the course of
B-CLL development, malignant cells infiltrate into lymphoid
tissue. MMP-9 is thought to be involved in this process. Cell
surface MMP-9 is bound to a4b1 and CD44v solely on
malignant B-cells, but not on normal B lymphocytes. Indeed,
a4b1 and CD44v are MMP-9 docking molecules on malignant B-CLL cells, necessary for cell migration (RedondoMunoz et al., 2008). B-CLL leukemia cells spontaneously
produce proMMP-9 (Bauvois et al., 2002) and high MMP-9
serum levels have a predictive value in leukemia patients
(Molica et al., 2003). MMP-9 is the major MMP in B-CLL
cells (Redondo-Munoz et al., 2006) and is a key player in
B-CLL cell invasion and trans-endothelial migration. Its
expression by B-CLL cells is regulated by a4b1-integrin and
CXCL12 (Redondo-Munoz et al., 2006). Elevated intracellular levels of MMP-9 correlate with advanced stage and poor
survival of patients with B-CLL. ProMMP-9 and mature
MMP-9 also play a role in B-CLL survival. a4b1 integrin and
a 190 kDa CD44 variant (CD44v) constitute a docking
complex for (pro)-MMP-9 at the B-CLL cell surface and
the MMP-9 hemopexin domain is required for this interaction,
hereby promoting B-CLL cell survival and migration
(Redondo-Munoz et al., 2010).
The expression of MMP-9 is dependent on the activity of a
p38 MAP kinase and blocking of this kinase and MMP-9
resulted in impaired survival of the B-CLL cells (Ringshausen
et al., 2004). The p38 kinase becomes activated by stress
signals such as LPS and inflammatory cytokines.
Hyperforin, the biochemically active component of the
herb Hypericum perforatum (St Johns wort) stimulates
apoptosis of cultured B-CLL cells from patients (Quiney
et al., 2006a). Hyperforin also inhibited the production of
MMP-9 and VEGF by B-CLL cells, resulting in an
antiangiogenic effect. (Quiney et al., 2006b).
MMP-9 expression has also been observed in T cell
lymphomas and their stromal cells (Aoudjit et al., 1997;
Ganor et al., 2009). Patients with MMP-9-expressing lymphoma have poor survival rates (Sakata et al., 2004). However,
in MMP-9 KO mice, the development of primary T-cell
leukemia cannot be prevented (Roy et al., 2007).
The previously mentioned MMP-9/Ku complex was
found on the cell surface of a subset of human leukemia
cells. It was suggested that this cell surface complex
mediates invasiveness. (Monferran et al., 2004; Paupert
et al., 2008).
Galectin-7 expressing lymphoma cells have accelerated
tumor development and show increased metastatic behavior.
Galectin-7 induces de novo MMP-9 mRNA synthesis
(Demers et al., 2005). Upon close contact with lymphoma
cells, stromal cells induce the expression of EGR-1 through
epidermal growth factor (EGF) signaling, a process which
also results in decreased expression of MMP-9 and subsequently leads to decreased growth of thymic lymphoma
(Bouchard et al., 2010).
In patients with primary central nervous system lymphoma, serum levels of YKL-40 and MMP-9 are associated
with radiographic disease status. Increase in serum levels of

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Lymphoma, leukemia and multiple myeloma.

255

YKL-40, but not MMP-9, predicts survival (Hottinger et al.,


2011).
Progression of multiple myeloma (MM) involves the
migration of MM cells from the bone marrow to the blood.
During this process, the cells have to pass through layers of
endothelial cells and basement membranes. For this reason,
MMP-9 might be of particular importance in MM cell
migration. In vitro, primary MM cells isolated from patients
have the ability to migrate through a matrigel, only if they
secrete MMP-9 (Vande Broek et al., 2004). In addition,
bone marrow endothelial and stromal cells can stimulate
MMP-9 secretion by MM cells, through the production of
hepatocyte growth factor and the chemokine CXCL12/SDF-1
(Parmo-Cabanas et al., 2006; Vande Broek et al., 2004).
Finally, MMP-9 activity was evaluated as an activator for
prodrug targeting in MM (Van Valckenborgh et al., 2005).
The MMP-9 production by fibrosarcoma cells
was down-regulated with Carboxylated chitooligosaccharides
(CCOS) (Rajapakse et al., 2006). However, in vivo studies
with siRNA show paradoxically increased intravasation and
metastasis in MMP-9 downregulated conditions (Deryugina
et al., 2005).

Fibrosarcoma.

Lung cancer. In one approach, an adenovirus expressing

antisense urokinase-type plasminogen activator receptor


(uPAR) and antisense MMP-9 was used. The adenovirus
significantly decreased the in vitro invasion and in vivo tumor
growth and metastasis by lung cancer cells (Rao et al., 2005).
Ovarian cancer. Ovarian cancer cells could be stimulated
with gonadotropin-releasing hormone (GnRH) to produce
MMP-2 and MMP-9 by signaling through c-jun. This also
resulted in increased cell motility and cell invasiveness
(Cheung et al., 2006).
In conclusion, many recent studies about MMP-9 in
various tumors indicate tumor-promoting effects, either by
increasing direct or indirect (by bystander cells) tumor cell
invasion and metastasis or by altering cell survival. In some
model systems paradoxical effects of MMP-9 or TIMP-1 have
been observed. For example, tumstatin, a fragment of the a3
chain of collagen IV can be generated upon cleaving collagen
IV with active MMP-9. The fragment can be detected in the
circulation of normal mice and was shown to be an
endogenous inhibitor of pathologic angiogenesis depending
on aVb3 integrin (Hamano et al., 2003).

Eye diseases
MMPs are involved in essentially every physiological process
in the various eye structures (Sivak & Fini, 2002). Increased
levels of both MMP-2 and MMP-9 have been detected in
human choroidal neovascularization (CNV) during agerelated macular degeneration (AMD). In a laser-induced
mouse model for CNV, Mmp2 KO and Mmp9 KO mice had a
lower incidence of CNV and lower disease severity. These
results were also corroborated by overexpression of TIMP-1
and TIMP-2 or injection of MMP inhibitors. The inhibition of
MMP-2, MMP-9 and MT1-MMP may be a promising strategy
for preventing AMD (Lambert et al., 2003).

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J. Vandooren et al.

When N-methyl-D-aspartate (NMDA) is intravitrealy


injected in rats (excitotoxic stimulation), gelatinolytic
activity is increased in retinal ganglion cells, but not in the
glial cells. This activity was linked with NO production by
nNOS and concomitant S-nitrosylation and activation of
MMP. Inhibition of MMP activity was shown to protect
retinal ganglion cells from excitotoxic damage (Manabe et al.,
2005). MMP-9-deficient mice are better protected against
degradation of the retina upon optic nerve ligation than
control animals (Chintala et al., 2002). One of the potential
substrates of MMP-9 in photoreceptor cells are the cyclic
nucleotide-gated channels. Cleavage may lead to subunit
modification with biophysical consequences (Meighan
et al., 2012).
MMP-9 was locally increased in various inflammatory eye
diseases, ranging from proliferative vitreoretinal to various
forms of conjunctivitis (Abu El-Asrar et al., 1998, 2000,
2001). Cytokines regulate MMP-9 in corneal epithelial cells
(Gordon et al., 2009). Furthermore, the activated form of
MMP-9 was associated with vitreous hemorrhage in diabetes
(Descamps et al., 2006) and MMP-9 levels correlated
with other inflammatory markers in autoimmune diseases
with eye involvement, such as Behcet disease and VogtKoyanagi-Harada disease (Descamps et al., 2008).

Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

contradictory results (Dewil et al., 2005; Kiaei et al., 2007),


hopeful results were obtained in studies with an MMP
inhibitor in transgenic ALS mice, in which MMP-2 and
MMP-9 levels were increased (Fang et al., 2010; Lorenzl
et al., 2006).
Chronic neuropathic pain is caused by lesions in the
central nervous system (spinal cord and thalamus) or in
peripheral nerves. The early and late development of neuropathic pain in dorsal root ganglia and in the spinal cord is
mediated by MMP-2 and MMP-9. After sciatic nerve damage,
which constitutes an animal model for neuropathic pain,
cytokines, chemokines and MMPs are altered. Moreover, in
Mmp9 KO mice IL-1b activation after nerve injury is altered.
These studies suggest that inhibition of MMP-2/MMP-9,
eventually with TIMP-1 or/and TIMP-2, may be beneficial for
the treatment of neuropathic pain (Ji et al., 2009).
Recently, MMPs have been studied in the proteolytic
processing of specific substrates in the central nervous system
(Cauwe et al., 2007). One example of such studies relates to
Huntingtons disease, in which it was found that MMP-9
possibly may assist in the cleavage of the huntingtin protein
(Miller et al., 2010).
Practical applications of MMP-9

Neurological disorders

MMP-9, MMP-9/NGAL complex and TIMPs as prognostic


indicators

The beneficial role of MMP-9 in neurodevelopment and


neuronal plasticity and its detrimental effects by dystroglycan
cleavage in neuropathology of multiple sclerosis and vascular
diseases of the central nervous system have already been
mentioned. However, in a variety of other neurological
disorders MMP-9 seems involved. Elevated levels of neurotransmitter or excessive stimulation of neuroreceptors, such as
glutamate receptors, lead to excitotoxicity by excessive Ca2
influx and the production of nitric oxide (NO) by neuronal
NO synthase (nNOS). In this context, Gu et al. showed
that NO can directly and irreversibly activate MMP-9 by
S-nitrosylation of the cyteine present in the propeptide
domain. Through this mechanism, S-nitrosylated MMP-9
contributes to cell detachment and leads to anoikis (Gu
et al., 2002).
In experimentally-induced epilepsy, MMP-9 induces apoptosis of hypocampal cells by cleaving b1-integrin and thereby
disrupting the integrin-mediated survival signals (Kim et al.,
2009). In patients with Guillain-Barre syndrome (GBS),
elevated plasma levels of MMP-9 are correlated with
electrophysiological abnormalities and altered cerebrospinal
fluid protein levels. Patients with demyelinating GBS had
higher levels of MMP-9 than patients with non-demyelinating
GBS, suggesting a role for MMP-9 in this demyelination
process (Sharshar et al., 2002). The cerebrospinal fluids of
patients with vascular dementia have also increased MMP-9
levels. (Adair et al., 2004).
Intense depolarization of neurons and glia cells initiates a
cascade (cortical spreading depression) which disrupts the
BBB in an MMP-9-dependent manner (Gursoy-Ozdemir
et al., 2004). MMP-9 was also studied in patients with
amyotrophic lateral sclerosis (ALS) and in preclinical animal
models. Although studies with MMP-9 knockout mice yielded

The development of biomarkers for cancer and inflammation


and also of prognostic and therapy follow-up and resistance
markers has become an industrial market under the umbrella
of personalized medicine. Classical markers, including
C-reactive protein for inflammation and oncofetal antigens
for cancer, are being complemented with other molecules.
Assays, ranging from single ELISA via multi-analyte ELISA
to protein and mRNA micro-arrays, are being commercialized
with increasing pace. As may be clear from the previous
sections, the analysis of the levels of MMP-9, MMP-9/NGAL
complexes and TIMPs may complement the marker arena
for several diseases. A critical analysis will address how to
measure these molecules, which molecular forms are detected
with the different techniques and how specific and selective
the used tests are. For MMP-9 we advocate to use at least two
assays: zymography and ELISAs, because both tests yield
complementary information. Indeed zymography analysis,
eventually after sample prepurification (Descamps et al.,
2002), gives information about all molecular forms, including
monomers, multimers, activation and degradation products,
but is in most cases a semi-quantitative method (Vandooren
et al., 2013). For ELISA, we suggest to use sandwich ELISA
with two different antibodies. For monoclonal antibodies
against MMP-9, we recommend to use antibodies against
different MMP-9 domains, as this will enhance the possibility
that only intact molecules are detected (vida supra). For the
detection of MMP-9/NGAL complexes, we recommend to use
an antibody against NGAL as capturing agent and an antibody
against MMP-9 for detection, or vice versa. It is clear that
ELISA will yield quantitative information that can be
enhanced with the inclusion of good standard preparations.
For reasons of compliance, in most of the clinical studies only
ELISAs were used. Unfortunately, this is not justified.

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DOI: 10.3109/10409238.2013.770819

Although zymography analysis may take more time, it yields


much better insights into the levels of the various molecular
forms of MMP-9. A negative point, about publications in
which zymography is used, is the classical misinterpretation
that enzyme activities were measured (Vandooren et al.,
2013).
NGAL/MMP-9 complexes were found in about 90% of the
urine samples of patients with breast tumors but were
practically absent in samples from healthy women. In cerebral
tumors, tumor excision was immediately followed by the
normalization of NGAL/MMP-9 levels, suggesting that the
measurement of this parameter might not only be an excellent
non-invasive diagnostic tool, but also a useful prognostic
indicator of response to treatment. In gastric carcinomas
tissue, the expression of the NGAL/MMP-9 complex in the
neoplastic tissue was also significantly higher than that in
non-neoplastic tissues. An important hyper-expression of
NGAL/MMP-9 was recently described by other authors in
biopsy samples of ESCC, where areas of simple hyperplastic
or dysplastic mucosa presented only a slight positive signal
(Bolignano et al., 2010; Moses et al., 1998).
Analysis of the potential prognostic value of MMP-9
levels, as well as those of TIMPs, in breast carcinoma
yielded some conflicting results. MMP-2 and MMP-9 levels
were analysed in gynecological neoplasias, prostatic neoplasia, bladder and renal carcinomas, lung carcinoma, neck
carcinoma, gastrointestinal cancer, melanoma, brain neoplasias (Turpeenniemi-Hujanen, 2005). Studies of concomitant
expression of MMP-9 and its inhibitors or activators
yields valuable information on the prognosis of diseases
involving MMP-9. For example, in ESCC, expression of both
matrilysin-2/MMP-26 and MMP-9 at the cancer invasive front
was correlated to poor prognosis (Yamamoto et al., 2004).
During percutaneous coronary intervention MMP-9 and
IL-6 are released from plaques. In addition, MMP-9 activity is
increased. MMP-9 may function as a biomarker to determine
plaque instability (Robertson et al., 2007).
High levels of MMP-9 in pleural fluids were associated
with development of pleural tuberculosis. The MMP-9
secretion was significantly higher in patients with tuberculous
effusions than in patients with malignant pleural disease
(Sheen et al., 2009). This citation represents one example of
many studies about increased levels of MMP-9 in serum, body
fluids or tissue extracts from patients with infections or
animal models in which microbes were used. Tuberculosis is
an interesting example because tuberculous granuloma formation is enhanced by a bacterial virulence factor that
induces MMP-9 and disruption of MMP-9 function attenuated
granuloma formation and growth of Mycobacteria (Volkman
et al., 2010).
Since the establishment of a role of MMP-9/TIMP in
neurological processes, such as learning and memory, the
study of the levels of MMP-9 in psychiatric disorders might
become rewarding (Okulski et al., 2007).
Gelatinase B inhibitors, from failures toward success
At the time when initial trials with broad-spectrum MMP
inhibitors showed negative outcomes in the therapy of invasive
cancers, our knowledge of MMPs was poor: all MMPs were not

Gelatinase B/MMP-9

257

yet discovered and the biology was not known. The use of
inhibitors resulted in side-effects, owing to inhibition of closely
related enzymes, such as the ADAMs and ADAMTSs (Apte,
2009; Overall & Lopez-Otin, 2002), lack of specificity, poor
pharmacokinetic studies, toxicity, and the inability to assess
inhibitory efficacy. The roles of MMPs are complex. As we
write, investigations towards tight control of proteolytic
activities are being done (Sela-Passwell et al., 2010).
Selective control of MMP-9 activity may be achieved by
exosite interactions (allosteric inhibitors), by transcriptional
regulation, by reinvestigations of known drugs, with natural
compounds and by highly selective monoclonal antibodies.
MMP-9 can be regulated either by controlling its
transcription and secretion or by direct inhibition of
enzyme activity. Xanthine-derivatives, nonsteroidal antiinflammatory drugs (NSAIDs) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors are
well known drugs that block MMP-9 transcription and
secretion (Opdenakker et al., 2001b). MMP-9 enzyme activity
can be blocked by compounds such as D-penicillamine,
hydroxamates and tetracyclines which all have a broad range
of inhibition of MMPs (Hu et al., 2007).
REGA-3G12 is an MMP-9 inhibitory antibody which
selectively targets the MMP-9 active site (Martens et al.,
2007). The most recent breakthrough in MMP-specific
monoclonal antibody technology was the development of
the so-called metallobodies. These are engineered antibodies
generated against the Zinc ion coordinated with a tripod
having three histidines and thus mimicking the zinc-binding
site of MMPs. Further immunizations of mice with recombinant MMP will select for highly selective monoclonal
antibodies. This technology was used to generate dualspecific antibodies that were effective in animal models of
inflammatory bowel diseases (Sela-Passwell et al., 2012).
This study is in line with data from other examples of
inflammatory diseases that were successfully treated with
MMP-9 inhibitors, including tetracyclines and many other
compounds (Hu et al., 2007; Qiu et al., 2012a).
The future of MMP-9 inhibition for the treatment of
cancers might lie in finding inhibitors of MMP-9 production
by cancer cells. Such inhibitors have already been found, for
example in prostate cancer (Kong et al., 2007). This goal for
cancer treatment can also be achieved by silencing strategies
for RNAs. MMP-9 siRNA was successfully used in arresting
medulloblastoma tumor growth in an intracranial model.
Silencing of MMP-9 resulted in a cell cycle arrest by ERKmediated p16 expression (Rao et al., 2007).
The use of MMP-9 inhibitors now seems feasible for
neurological disorders and for the treatment of cardiovascular
diseases (Matsumoto et al., 2009). We here address some
additional issues about MMP-9 inhibitors.
Thiol-containing compounds. Thiol-containing compounds

are known for their ability to chelate the active site zinc
(Freskos et al., 1999). The same biochemical principle has
been used with cysteine-containing peptides, that mimick the
peptide sequence of the cysteine switch in the propeptide
(Figure 5) (Hu et al., 2005a,b; Qiu et al., 2012b). Such
cysteine-containing peptides may form the basis to develop
future orally active peptidomimetics.

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Angiotensin-converting enzyme (ACE) inhibitors such as


captopril are used worldwide for the management of hypertension and heart failure (Jin et al., 2007). Interestingly,
captopril also effectively inhibits angiogenesis (Volpert et al.,
1996) and is a gelatinase inhibitor (Sorbi et al., 1993; Volpert
et al., 1996). Several other ACE inhibitors have the ability to
inhibit MMP-9. For example, imidapril and lisinopril have
been studied for their MMP-9 inhibitory potential (Yamamoto
et al., 2007).
Amifostine is a phosphorothioate that has been approved by
the US Food and Drug Administration for clinical use as a
cryoprotector in cancer therapy. It inhibits metastasis formation in a murine sarcoma Sa-NH Mouse model. Amifostine
inhibits the enzyme activities of MMP-9 and MMP-24 as a
function of increasing dose and time (Grdina et al., 2002).

Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

decade will be as proliferative for MMP research as the


previous one depends on new breakthroughs, development of
new techniques and scientific interests from pharmaceutical
and other industries. Applications of MMP-9 are not limited
to medical research. For instance, solving the crystal structure
and being able to grasp and understand the flexibility of this
model enzyme may yield fundamental insights into biology
and may be instrumental for other molecules, including
carbohydrate and nucleic acid modifying enzymes.

The use of MMP inhibitors


in cancer and arthritis treatment have been plagued by the
occurrence of musculoskeletal side-effects. It has been
proposed that some symptoms are related to non-specific
binding and inhibition of other MMPs (Rush & Powers,
2004). The question of using TIMPs as natural inhibitors has
not yet been properly addressed, maybe because proteins need
to be injected. However, the market of recombinant cytokines
and monoclonal antibodies is enormous, both for inflammatory and neoplastic diseases and all these (glyco)protein
drugs need to be injected. Neutralizing monoclonal antibodies
against TNF have become a classical treatment option for
rheumatoid arthritis and inflammatory bowel diseases.
Because of wide use of such reagents, more therapy-resistant
patients, who will need alternative treatments, are discovered.
Similarly, in the field of cancer treatments, therapy-resistance
is a common problem and new options for therapy should
be developed. We therefore stimulate the revival of MMP
inhibitor research, in the first instance for therapy of
genetically stable inflammatory disorders. In a second
instance, we continue and promote inhibitor research for
use in cancer therapy. At this level, TIMP-1 and MMP9-selective antibodies (and other drugs) will only be useful
in those neoplasms that show overexpression of the enzyme.
As a comparison, Trastuzumab (Herceptin) is beneficial in
Her-positive (positive for the receptor of human epidermal
growth factor) breast cancer treatment and it is not a major
obstacle to test for Her-positivity on tumor biopsy material.
In those types of cancer, in which the MMP-9 is bound to
the tumor cells, e.g. by CD44 or integrins, an additional
advantage of treatment with monoclonal antibodies is evident.
If complement-activating immunoglobulins are used, synergistic immune activation by the complement system
will assist in tumor cell eradication. Of course, the same
reasoning may be made for the use of monoclonals that are
directed against membrane-type MMPs on the surface of
tumor cells.

Overemphasis has
been placed on the structure and function of MMP-9
monomers. Although further investigations on the monomer
need to continue, the time is ripe to invest also efforts in the
study of the other molecular forms. Are the oligomers dimers
or do these assemble into other multimers? What controls the
subcellular formation, relative abundancy of monomers,
oligomers and heteromers with NGAL? Is the production of
MMP-9 forms cell- or tissue-specific and are these associated
with diseases? Do the oligomers possess similar or differential
functions towards substrates, inhibitors and receptors? Are
these similarly or differentially activated in tissues or body
fluids? Do monomers and multimers have different in vitro
enzyme kinetics, TIMP-1 affinities and inhibition profiles,
receptor binding properties and in vivo pharmacokinetics?
The common idea is to view TIMP-1 as the natural
inhibitor of MMP-9. However, much like MMP-9 itself with
multiple functions associated with individual protein
domains, TIMP-1, with two protein domains, possesses cell
signaling functions in addition to its role as inhibitor. This
implies that high-levels of MMP-9 might also result in a
decrease of free TIMP molecules and therefore a decrease in
TIMP-mediated signaling (Moore & Crocker, 2012). Another
possibility to consider is to target the TIMP-1/MMP-9 binding
interface. Unlike for other MMPs, TIMP-1 is able to bind both
the active site of active MMP-9 and the hemopexin domain.
Therefore, excess levels of proMMP-9 can also result in
TIMP-1 scavenging and have pathological effects.
A popular tool for the study of MMP-9 function is the use of
inhibitors. However, since no specific MMP-9 inhibitors exist,
care should be taken when interpreting results with presently
available inhibitors (Hu et al., 2007). An alternative powerful
tool, however, is the use of Mmp9 knockout mice and WT mice
with an identical genetic background (Whatling et al., 2004).
However, appropriate validation of genetic backgrounds is
indispensable, as has been shown in previous studies (Geurts
et al., 2011). Differences in the genetic backgrounds may be
one of the possible explanations for discrepancies observed
with mouse models of ALS (Dewil et al., 2005; Kiaei et al.,
2007). A further issue about the use of Mmp9 gene knockout
mice is the observation of compensation mechanisms. This
may be avoided with the use of organ- or tissue-specific and
conditional MMP-9 knockout mice.

The future for MMP-9 investigations

Inhibitor development and alternative strategies

The definition of future directions of MMP-9 research for the


next decade is straightforward at three levels: linking structure
with function, development of control mechanisms of MMP-9
activities and alternative uses of MMP-9. Whether the next

Exploitation of differences between individual MMPs. Figure 2

Natural inhibitors and antibodies.

Linking structure with functions of MMP-9.

illustrates the domain comparisons between the MMPs. Some


of these enzymes have been characterized at the atomic level
and for others, including MMP-9, specific domains have been

Gelatinase B/MMP-9

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DOI: 10.3109/10409238.2013.770819

expressed and crystallized. This information may be used by


specialists in molecular modeling to compare and complement data from X-ray crystallography, NMR and modeling
analysis (Rush & Powers, 2004). In fact, the progress made in
the definition of the molecular structure of the MMP-9
monomers is illustrated in Figure 4, which shows how, over
the last decade, a reiterative process of feeding constantly new
information from biophysical data leads to refinements of the
original model and yields already a reasonable idea about
conformations and provides a useful cartoon. Global molecular variation between MMP-9 conformations is experimentally demonstrated (Rosenblum et al., 2007b). By
comparisons of structures from different MMPs in relation
to substrate specificities, it was observed that the sizes and
shapes of the S1 pocket and the residues in the loop region
containing the structural zinc binding site were major
determinants in understanding the differences between individual enzymes (Rush & Powers, 2004).
MMP-9 and MMP-2 have a unique fibronectin domain
which promotes their interaction with substrates such as
gelatins. Since this domain has no importance in the cleavage
of small peptides (Nagase et al., 2006), it might be a tempting
exosite target for selective (partial) inhibition of catalysis
of a range of gelatinase substrates. An in depth study of
MMPs and their interactions with substrates led to the
development of an exosite binding triple-helical peptide
that selectively inhibits MMP-9 type V collagen-based
activities, compared with interstitial collagen-based activities
(Lauer-Fields et al., 2008).
Molecular biological research on MMP-9 has progressed a
lot since the cloning of the human cDNA (Wilhelm et al.,
1989) and the mouse gene (Masure et al., 1993), the latter
of which made it possible to generate different gene KO
constructs (Dubois et al., 1999; Vu et al., 1998). The
comparison of human and mouse phenotypes needs to be
mirrored by better comparisons of the human and mouse
glycoprotein structures. The latter will help to define
structural and functional similarities and, more importantly,
to key down and yield a scientific understanding of essential
differences.
Overcome structure mobility. Although MMP-9 has been

extensively studied over the past 10 years, so far vital


information for drug development remains elusive. While an
abundance of data on upregulation or down regulation of the
MMP-9 enzyme or its expression in diseased states exists,
clear and complete pathways for MMP-9 action, interaction
and expression remain unknown. These pathways are necessary for predicting the outcome of MMP-9 inhibition and
possible side-effects.
One unexpected problem that was evidenced by structural
analysis is the extensive mobility in the MMP active site.
However, the elasticity of the MMP active-site combined with
inhibitor mobility enables compounds predicted to be poor
binders based on static models to inhibit MMPs with highaffinity (Rush & Powers, 2004).
An alternative to overcome problems with the low
selectivity of inhibitors is the use of genetic techniques.
One mentioned example illustrates how to overcome structural heterogeneity. In mice tumor growth in gliomas was

259

successfully inhibited by adenoviral mediated transfer of an


antisense-Mmp9 gene sequence (Lakka et al., 2002b). In one
approach, an adenovirus expressing an antisense construct
encoding urokinase-type plasminogen activator receptor
(uPAR) and antisense MMP-9 was used. The adenovirus
could significantly decrease the in vitro invasion and in vivo
tumor growth and metastasis in lung cancer cells (Rao
et al., 2005).
MMP-9 inhibitors and cancer a complicated story. The

applicability of MMP-9 inhibitors for the treatment of cancers


is questionable. For example, it was shown that tumstatin,
a collagen fragment generated by MMP-9 proteolysis is
involved in the control of pathological angiogenesis such as
in tumor growth (Hamano et al., 2003). Complete inhibition
of MMP-9 might in this case have a tumor promoting
effect, by allowing the growth of pathological blood vessels,
especially in the later stages of tumor development.
The FDA-approved biphosphonate zoledronic acid (ZA)
was used in a model for papillomavirus-induced cervical
cancer and inhibits MMP-9 activity and production by
infiltrating macrophages, thereby reducing the association
of VEGF with angiogenic endothelial cell receptors. ZA
holds promise as an unconventional MMP-9 inhibitor for
antiangiogenic therapy of cervical cancer or diseases involving MMP-9 expression by infiltrating macrophages (Giraudo
et al., 2004). Another example relates to treatment of multiple
myeloma. In the case of MM, MMP-9 activity was evaluated
as an activator of prodrugs (Van Valckenborgh et al., 2005).
Indirect targeting of MMP-9. Some successes have been

booked by indirectly targeting proMMP-9 activation through


blocking of one of the upstream pathways. For example,
the plasminogen/MMP-9 cascade is an attractive target for
regulating inflammatory responses and the development of
AAA (Gong et al., 2008). Targeting of the PN-1/protease/
LRP-1 pathway reduces the secretion of MMP-9 (Fayard
et al., 2009).
Alternative uses of MMP-9
Use of MMP-9 producing cells for the treatment of
fibrosis. One of the experimental therapies for muscular

dystrophy patients includes transplantation of mesoangioblasts (Minasi et al., 2002; Sampaolesi et al., 2003,
2006). For optimal therapy, the transplanted cells need to be
delivered to the skeletal muscles, which is often difficult in
late stage muscular dystrophy due to sclerosis and fat
infiltration into the skeletal muscles. In a recent study, these
problems were overcome by engineering tendon fibroblasts to
produce MMP-9 in combination with the angiogenic factor
PLGF. The engineered fibroblasts were injected into late
stage dystrophic muscles and resulted in restored microcirculation and a reduction in connective tissue deposition. In
addition, subsequent cell therapy was equally efficient as in
early stage dystrophic mice (Gargioli et al., 2008).
Recently, it was
demonstrated that TIMP-1-free MMP-9 from neutrophils is a
potent pro-angiogenic factor (Ardi et al., 2007, 2009). This

Use of MMP-9 for inducing angiogenesis.

260

J. Vandooren et al.

may have potential applications in regenerative medicine, in


particular in conditions in which local angiogenesis is a
critical factor: burns, atonic wounds in elderly and diabetes
patients. If such clinical trials would be successful, one may
even try preclinical applications in tissue engineering and
stem cell work. Thanks to genetic engineering technology, the
production of medically applicable preparations in sufficient
quantities is possible.

Crit Rev Biochem Mol Biol, 2013; 48(3): 222272

declare no conflict of interest. The present study was


supported by funding from the European Union Seventh
Framework Programme (FP7/2007-2013) under grant agreement no 263307, by Geconcerteerde OnderzoeksActies
GOA 2012 017 and GOA 2013 014, the Fund for Scientific
Research of Flanders (FWO-Vlaanderen) and the University
of Leuven Research Fund. JV holds a doctoral fellowship of
the FP7 SaveMe project and PEVdS is a research professor of
the KU Leuven.

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Conclusions and future perspectives


During the decade 20032012, research on MMP-9 has
increased exponentially, making this enzyme the prototype
of the MMP family and one of the most studied enzymes.
MMP-9 is involved in fundamental biological processes
including development, angiogenesis, apoptosis, inflammation and cancer. Therefore, regulation of MMP-9 activity
has consequences for normal biological processes and in
pathological conditions. Whereas the activation mechanism of
proMMP-9 is biochemically understood, the relevant in vivo
activation processes need further studies. Regulation by
transcription is well undertsood, but future studies about
epigenetic control and natural or artificial silencing of MMP9 mRNA translation will enter into the spotlights.
The secretion pathways and molecular forms of MMP-9
differ between neutrophils and all other cell types.
Neutrophils do not produce MMP-2 or TIMP-1 and secrete
within 1 h after stimulation a covalent complex between
MMP-9 and NGAL. All other tested leukocyte types and body
cells produce constitutively MMP-2, often co-produce TIMP1 and take a considerable time to manufacture and secrete
MMP-9. A last level of control of MMP-9 activity is by
inhibition, executed by natural inhibitors or by man-made
(glyco)proteins, such as monoclonal antibodies, nanobodies,
metallobodies, synthetic peptides and small molecule drugs.
Structural research on MMP-9 is heading towards crystallography of the full enzyme, comparisons of monomers,
oligomers and covalent complexes, site-specific analysis of
posttranslational modifications, such as O-linked glycosylation, and model building of the intact enzyme forms with ECM,
membrane-bound and ICM substrates, inhibitors and receptors.
Functional research of gelatinase B will be directed
towards its role in male and female fertility, stem cell
research and regenerative medicine and neurobiology. In the
latter area, regulation of MMP-9 activities by neurotransmitters, ion pumps and channels, role in neuroplasticity, memory
and the aging brain, as well as in diseases such as ALS, MS
and neurodegeneration will become dominant. An uncultivated and open field for MMP research is at the level of
mental ilnesses.
The development and use of MMP-9 inhibitors will
continue and undergo a revival, once the pharmaceutical
industry will see the upcoming success of small enterprises
that take advantage of the many possible applications in
inflammatory diseases and the science-based selected uses
for cancer therapy.

Declaration of interest
This manuscript is submitted to Critical Reviews in
Biochemistry and Molecular Biology exclusively. The authors

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