PROTEOLYTIC EVENTS OF WOUND-HEALINGCOORDINATED INTERACTIONS AMONG MATRIX

METALLOPROTEINASES (MMPs), INTEGRINS,

AND EXTRACELLULAR MATRIX MOLECULES
Bjorn Steffensen*
Department of Periodontics-MSC7894, University of Texas Health Science (enter at San Antonio, 7703 Floyd (url Drive, San Antonio, TX 78229-3900; *corresponding author,

steffensenb@uthscsa.edu
Lari Hakkinen
Hannu Lariava
Department of Oral Biological and Medical Sciences, University of British Columbia, Vancouver, BC, Canada

ABSTRACT: During wound-healing, cells are required to migrate rapidly into the wound site via a proteolytically generated
pathway in the provisional matrix, to produce new extracellular matrix, and, subsequently, to remodel the newly formed tissue
matrix during the maturation phase. Two classes of molecules cooperate closely to achieve this goal, namely, the matrix adhesion and signaling receptors, the integrins, and matrix-degrading and -processing enzymes, the matrix metalloproteinases
(MMPs). There is now substantial experimental evidence that blocking key molecules of either group will prevent or seriously
delay wound-healing. It has been known for some time now that cell adhesion by means of the integrins regulates the expression of MMPs. In addition, certain MMPs can bind to integrins or other receptors on the cell surface involved in enzyme activation, thereby providing a mechanism for localized matrix degradation. By proteolytically modifying the existing matrix molecules, the MMPs can then induce changes in cell behavior and function from a state of rest to migration. During wound repair,
the expression of integrins and MMPs is simultaneously up-regulated. This review will focus on those aspects of the extensive
knowledge of fibroblast and keratinocyte MMPs and integrins in biological processes that relate to wound-healing.
Key words. Matrix metalloproteinases, MMP, integrins, extracellular matrix, wound-healing.

(I) Introduction
1ecent advances in the understanding of wound-healing
have demonstrated clearly that the temporal and spatial
events in this process are associated with characteristic
changes in the expression of the integrins, a central family of
cell-surface receptors for many extracellular matrix molecules.
The interactions between the integrins and their ligands have
been found to elicit "outside-in" and "inside-out" signaling
events. Such signaling events are fundamental for controlling
cell migration in the wound-healing process, and the signaling
and resulting cell behavior are modified following partial
degradation of the ligands. Thus, initial degradation of extracellular molecules may induce cell migration over the wound
surface, whereas migration may not occur over the intact molecules. The family of matrix metalloproteinases (MMPs) is
central to tissue homeostasis and remodeling. In addition,
these enzymes may also be required for cell motility in the
process of wound-healing. Indeed, the secretion and activation of the MMPs appear to be closely linked to interactions
between integrins and extracellular matrix molecules and the
associated signaling events.
We have reviewed the available research on the contributions of MMPs and integrins to wound-healing. This discussion
commences with overviews of MMPs and integrins and is followed by sections on integrin-MMP interactions in re-epithelialization and connective tissue wound-healing. Throughout,
we have aimed to provide a picture which describes the coordi12(5)373
398 ((2001)
200 1 )
12( 5 )373-398

nation of the integrins and their ligands as these relate to the

control of MMPs and cell behavior in wound-healing.

(II) Matrix Metalloproteinases

(A) GENERAL ASPECTS
In 1962, Gross and Lapiere observed that the rapidly remodeling tadpole tails possessed collagen-degradative activity at
neutral pH. Subsequent research showed that the enzyme
involved had the ability to cleave type I collagen molecules in
characteristic 3/4 and 1/4 fragments (Gross and Nagai, 1965).
Initial reports on collagenases in man came from studies of
human gingiva and bone (Fulmer and Gibson, 1966). Together,
these initial observations provided the basis for intense
research that has led to the isolation and cloning of a continuously increasing number of matrix metalloproteinases (MMPs),
now reaching approximately 28 (Table 1). Evolutionary analyses based on sequence and structural analyses have established
firmly that the MMPs form a distinct subfamily of the metallo-

endopeptidase family (Woessner, 1994).

The main characteristics of MMPs are that: (1) they are
secreted or released in latent forms as zymogens and can be
activated by proteinases or by organomercurials with a reduction in molecular mass due to loss of a prodomain; (2) their catalytic mechanism depends on zinc in the active site; (3) their
activity is inhibited by tissue inhibitors of metalloproteinases;
(4) the MMPs cleave one or more of the components of the

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TABLE 1
Substrate Specificities of the Members of the MMP Family
Common Name

Collagenases
Interstitial

Neutrophil
Collagenase 3

Collagenase 48,22

MMP Number

Substrates

MMP-1

Col 12, I, ,III Vill, X, entactin, TN, aggrecan, proGel A,

proGel B
MMP-8
Col 1,1 II, III, aggrecan
MMP-13
Col 1, II, 1113, IV, IX, X, XIV, TN, FBR18, FN15, gelatin3,
proGel B21
MMP- 1 88/-1 922 TN, aggrecan, (gelatin)22

Gelatinases
Gelatinase A

MMP-2

Gelatinase B

MMP-9

Col I, IV, V, VII, X, gelatins, FBR18, FN, LM, aggrecan,

elastin, proGel B, proColl-320, LM-528
Col 19, 1119, IV, V, gelatins, elastin, FBR18, entactin,
aggrecan

Stromelysins, matrilysin, metalloelastase

Stromelysin 1
MMP-3
Col II, IV, IX, X, XI, gelatin, LM, FBR18, FN, elastin, TN,
aggrecan, proCol, proGel B, neutrophil proCol
Stromelysin 2
Stromelysin 3
Matrilysin

MMP-10
MMP-1 1
MMP-7

Matrilysin 231
Macrophage
metalloelastase

MMP-26

Col IV, LM, FN, elastin, aggrecan

Col IV, FN, LM, aggrecan
Col IV, gelatin, LM, FN, entactin, elastin, aggrecan,
TN, proGel A, proGel B, proCol
Gelatins, casein31; FN, Fb, VN32; cxl-proteinase inhibitor33

MMP-12

Elastin, FBR18, Col IV, gelatin, FN, LM, VN, PG28

Membrane-type metalloproteinases
MT1 -MMP

MMP- 1 4

MT2-MMP'
MMP-15
MT3-MMP5
MMP-1 6
MT4-MMP6
MMP-1 7
MT5-MMp25
MMP-24
MT6-MMP26 leukolysin MMP-25

Others
Envelysin
Enamelysin I I 1
Epilysin34

MMP-20
MMP-21 /2230
MMP-2324
MMP-28

Progel A; yelatin, casein, elastin, FN, LM, VN, DSPG4;

Col I, II, 1II 7; TN, nidogen, aggrecan, perlecan,
proTNF(xc9; proColl-320
ProGel A16; FN, TN, LM, aggrecan, perlecan, proTNF&19
ProGel A
Gelatin, proGel A29
ProGel A
Gelatin

Gelatin; amelogenin23

Activities now identified as MMPs

Telopeptidasel 2
MMP-4
(likely MMP1 3) Col I, FN
3/4-collagen
MMP-5
endopeptidase13
(rat MMP-2)
Col 1, gelatin; 3/4-fragments col 1, 11, III
Acid MMP' 4
MMP-6 (MMP-3) Cartilage proteoglycan, insulin B chain

Table information expanded from reviews by Sang and Douglas (1996), Birkedal-Hansen et al. (1993), and
Matrisian (1992). Updated sources are referenced as footnotes.
Abbreviations: Col, native collagen; FBR, fibrillin; FN, fibronectin; LM, laminin; proCol, procollagenase;
proGel, progelatinase; TN, tenascin; VN, vitronectin; PG, proteoglycan; DSPG, dermatan sulfate proteoglycan; and TNF, tumor necrosis factor.
Sources: 3Knauper et al. (1 996a), 4Pei and Weiss (1996), 5Takino et al. (1 995), 6Puente et al. (1 996), 7Will and
Hinzmann (1995), 8Cossins et a/. (1996), 90kada et a1. (1992), 1 Bartlett et a!. (1996), 1 'Overall and Limeback
(1988), 12Nakano and Scoft (1987), 13Overall and Sodek (1987), 14Azzo and Woessner (1986), 15Knduper et al.
(1 997a), I6Butler et al. (1997), 170huchi et al. (1997), 18Ashworth et al. (1999), 19d'Ortho et al. (1997), 20Knauper
etal. (1 996b), 21Kn6uper etal. (1 997b), 22Pendas etal. (1997), 23Llano eta!. (1997), 24Velasco etal. (1999),
25Llano et al. (1999), 26Pei (1999), 27Chandler et al. (1996), 28Koshikawa et al. (2000), 29Wang et al., 30Gururajan
et al. (1998), 31de Coignac et al. (2000), 32Marchenko et al. (2001), 33Park et al. (2000); 34Lohi et al. (2001).

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extracellular matrix; and (5) they

have structural and sequence
similarities that include the
prodomain cysteine switch sequence PRCGVPD and the zinc-

binding HEXGHXXGXXHS/T
sequence (Woessner, 1991, 1994).

The MMPs are detectable

with high frequency in cells, tissues, and interstitial fluids and
have been implicated in a wide
variety of biological functions in
both health and pathological
conditions (Birkedal-Hansen et
al., 1993). In health, growth and
development require substantial
remodeling of the extracellular
matrix and cellular movement.
The transcripts and expressed
MMPs and MMP inhibitors
have been demonstrated during

ovulation, embryonic growth

and differentiation, trophoblast
invasion, skeletal growth and
remodeling, organ and tooth
development, and post partum
involution (Sodek and Overall,
1992; Werb et al., 1992; BirkedalHansen et al., 1993; Kleiner and
Stetler-Stevenson, 1999; Nelson
et al., 2000). In the present
review, we present a significant
volume of literature on the
MMPs in the wound-healing
processes. Several MMPs have
also been implicated in inflammatory tissue conditions such as
arthritis and periodontal disease
(Matrisian, 1992; Sodek and

Overall, 1992; Birkedal-Hansen,

1993). Because MMPs interact
with growth factors, examples
of MMPs in the release of
growth factors are presented
later. Importantly, recent data
have now demonstrated that
one of the MMPs, MMP-2,
cleaves the monocyte chemoattractant protein-3, resulting in
a

dampened inflammatory

re-

sponse (McQuibban et al., 2000).

Whereas the ability of MMPs to
degrade a variety of extracellular matrix components is of benefit in the developmental and
remodeling processes in health,
the presence of high levels of
tumor-cell-associated MMPs
has been associated with the
expansion and tissue penetration of tumor cells, ultimately
leading to formation of metas12(5):373

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1 2(5):373-398 (2001

tases (Zucker et al., 1987; Liotta and Stetler-Stevenson, 1991;

Birkedal-Hansen et al., 1993; Stetler-Stevenson et al., 1993).
The MMPs may be mobilized from cells by at least three
mechanisms. Thus, transcription may be regulated in response to
growth factors and cytokines, MMPs may be constitutively
expressed (i.e., gelatinase A), or MMPs (i.e., gelatinase B) may be
released on demand from cellular granule storage sites in polymorphonuclear leukocytes (Birkedal-Hansen et al., 1993). As we
will discuss later, it appears increasingly likely that MMPs, rather
than exerting their activities freely in tissue fluid compartments,
function in the proximity of cells and may be positioned, activated, and released as needed from the cell surfaces by several mechanisms (Basbaum and Werb, 1996; Steffensen et al., 1998).
The MMPs are secreted in latent forms and require activation for catalytic activity. The activation involves disruption of
the bond between the active site Zn++ and the Cys residue in the
conserved prodomain sequence PRCGVPD and the introduction
of a molecule of water as the fourth ligand (Van Wart and
Birkedal-Hansen, 1990). This mechanism has been referred to as
the "cysteine switch" (Springman et al., 1990; Windsor et al., 1991),
and the importance of the coordination has been further substantiated by spontaneous enzyme activation upon site-directed
mutations that introduce changes in the conserved propeptide
sequence (Sanchez-Lopez et al., 1988). Disruption of the bond
may also be achieved chemically by various agents, including
organomercurials, metal ions, thiol reagents, and oxidants
(Birkedal-Hansen et al., 1993). Because the detergent sodium
dodecyl sulfate (SDS) has a similar effect, this can be utilized in
zymography analysis of MMPs (Birkedal-Hansen and Taylor,
1982). Several proteases (trypsin, chymotrypsin, plasmin, plasminogen activators [u-PA and t-PA], neutrophil elastase, and
plasma kallikrein) may activate MMPs. Such proteolytic processing generally removes a segment of the prodomain from a position that is NH2-terminal to the final cleavage site and is followed by an autocatalytic cleavage which generates the final
active form of the enzyme (Nagase et al., 1990; Birkedal-Hansen
et al., 1993). Although the position of the final cleavage site varies
between the activated MMPs, it is generally at the tyrosine or
phenylalanine residues approximately eight residues downstream from the conserved prodomain cysteine residue (BirkedalHansen et al., 1993). MMPs may also proteolytically activate other
MMPs. Examples are the activation of procollagenase by
stromelysins (Murphy et al., 1987; Nicholson et al., 1989; Quantin
et al., 1989) and activation of progelatinase B by stromelysin-1
(Goldberg et al., 1992; Ogata et al., 1992) and gelatinase A
(Fridman et a!., 1995). In addition, cell-surface-bound membranetype (MT)-MMPs (Sato et al., 1994; Strongin et al., 1995; Butler et
al., 1997) initiate the activation of pro-gelatinase A, followed and
completed by autoactivation (Bergmann et al., 1995).
The control of the MMPs' catalytic activities is closely associated with the tissue inhibitors of metalloproteinases (TIMPs).
These are secreted proteins that are widely distributed in tissues and fluids and serve as specific inhibitors for the MMPs.
In addition to several TIMPs from various species, four human
TIMPs have been cloned (Carmichael et al., 1986; De Clerck et
al., 1989; Stetler-Stevenson et al., 1989; Wick et al., 1994; Kishnani
et al., 1995; Greene et al., 1996). The active forms of the MMPs
are bound tightly by TIMPs (Kds of 10-9 - 10-10 M) with a stoichiometry of 1:1. In addition, TIMP-1 and TIMP-2 complex
with the latent forms of gelatinase B and A, respectively
(Goldberg et al., 1989; Stetler-Stevenson et al., 1989; Ward et al.,
1991b). Whereas it was earlier proposed that the binding to the
12(5
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1 2( 5):-373-398 (1200 )

latent enzymes might serve to delay autocatalytic conversion to

the active form of gelatinase A (Howard et al., 1991), it is now
clear that this binding also serves in a cell-surface-associated
MT-MMP/TIMP-2/progelatinase A activation complex (Cao et
al., 1995; Strongin et al., 1995). Disruption of TIMP activity may
cause tissue damage and an expanded rate of tumor-invasive
growth (Liotta and Stetler-Stevenson, 1991; Edwards et al.,
1996). Also, transfection of normal cells with plasmids inducing
antisense TIMP mRNA to reduce TIMP expression conferred an
oncogenic phenotype on the cells (Khokha et al., 1989), and
TIMP-knock-out transgenic mice show an accelerated growth
and dispersion of tumors (Khokha et al., 1995).
An additional, naturally occurring inhibitor of MMPs is the
a02-macroglobulin. By a bait-entrapment mechanism (S0ttrupJensen and Birkedal-Hansen, 1989), upon cleavage of one or
more bonds in the bait region of c2-macroglobulin by the MMP,
a conformational change is initiated, leading to the entrapment
of the MMP through a covalent cross-linkage to the inhibitor
(Birkedal-Hansen et al., 1993). Interestingly, this binding does
not block access to the active site of the MMP by low-molecularweight substrates, suggesting that inhibition of cleavage of
high-molecular-weight substrates is the result of steric hindrance. The inhibitor-proteinase complexes are subsequently
cleared from the circulation by the liver or locally by tissue fibroblasts that possess functional receptors for oL2-macroglobulin
that may be involved in intemalization and destruction of the
complexes (Van Leuven et al., 1979; Dickson et al., 1981).

(B) MOLECULAR BASIS

FOR MMP-LIGAND INTERACTIONS
To understand the molecular basis for MMP interactions with
matrix molecules, we will review the MMP domain structure,
the contributions of distinct domains to ligand binding, and
finally the special situation of MMP-2 cell-surface localization
and activation.
(1) MMP domains
The MMPs are organized in distinct structural domains. There is
significant homology between the domains in the different
MMPs, but there is variation in the number of domains present
in each enzyme (Fig. 1) (Birkedal-Hansen et al., 1993). Following
the signal peptide, which is removed in the endoplasmic reticulum, all MMPs have a prodomain (77-87 residues), which is the
NH2-terminal domain of the secreted latent enzyme, and a catalytic domain. A COOH-terminal domain (-200 residues) that is
a constant finding in all MMPs except matrilysin (MMP-7) connects via a flexible, variable-length (5-50 residues), proline-rich
hinge region to the catalytic domain. The COOH-terminal
domain consists of four repeats with homology to vitronectin
and hemopexin, a heme-binding protein (enne and Stanley,
1987). Each repeat forms a blade of the four-bladed 3-propeller
three-dimensional structure (Libson et al., 1995).
The catalytic domain contains a Zn++ binding site with
three histidine residues in the highly conserved active site
sequence HEXGHXXGXXH (Woessner, 1994). In the latent form
of the enzyme, a fourth ligand site is occupied by a cysteine
contained in the conserved PRCGXPD motif of the prodomain.
Disruption of the bond to this cysteine (cysteine switch) and
introduction of a water molecule to occupy the fourth binding
site are key elements of enzyme activation (Springman et al.,
1990; Van Wart and Birkedal-Hansen, 1990).
Unlike other members of the matrix metalloproteinase

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375

Call binding sites have been proposed in

glutamine-asparagine-rich regions of the
catalytic domain (Lepage and Gache, 1990).
There is also recent evidence that Call
bound centrally in the COOH-terminal
domain of gelatinase A (Libson et al., 1995)
is required for interaction with extracellular matrix (ECM) proteins (Wallon and
Overall, 1997). In addition, the COOH-terminal domain may also bind a Zn++.

Collagenases

f...
l.IJ
Gelatinase A
}1 Pro
Gelatinase B

Hemopexin

Ca '

( Hemopexin

c
!1J Pro le~t
iHemopexini
)

(2) Ligand-binding domains

and functional properties of MMPs
Analyses of recombinant MMPs with
Stromelysins
domain deletions and studies of isolated
recombinant domains or modules have
=
Pr y
( HemoDexin
made it possible to assign functional contributions to distinct domains and modules
within the individual MMPs.
Membrane-type metalloproteinases
The COOH-terminal domains of several
MMPs provide binding to type I collagen, as
Catal ic
}ii1FPro
Hemopexin _)I. I
illustrated by the interaction between the
purified isolated autocatalytic COOH-terminal fragment of collagenase and native type
I
collagen (Bigg ct al., 1994). This interaction
Others
concurs with the observations that catalytic
domains alone from human fibroblast and
Figure 1. Domain structure of the major matrix metalloproteinase subgroups. Abbreviations neutrophil collagenases do not cleave native
are: Pre, signal peptide; Pro, prodomain; Fu, putative furin cleavage site; CBD, collagen- type I collagen (Clark and Cawston, 1989;
binding domain; Zn, Znr+-binding active site; TM, transmembrane domain; and V, type V Windsor et al., 1991; Murphy ct al., 1992a;
collagen-like region. The catalytic (Catalytic) and COOH-terminal hemopexin-like Schnierer ct al., 1993). In comparison, recom(Hemopexin) domains are also shown.
binant truncated human collagenase and
stromelysin-1 with deletion of the COOHterminal domaiins maintained their ability to degrade casein,
family, the gelatinases A and B contain three 58-amino-acidgelatin, and a p(
substrate (Murphy et al., 1992a). Because
residue long modules that are highly homologous to the
stromelysin alsco can bind to collagen by its COOH-terminal
fibronectin type l1-like modules and inserted into the catalytdomain, a hybriid enzyme was engineered which consisted of
ic domain (Collier et al., 1988; Wilhelm et al., 1989). In addition
the catalytic dornain of collagenase combined with the COOHto fibronectin and gelatinases of the MMP family, fibronectin
terminal domair of stromelysin-1. The catalytic properties of this
type l-like modules have been identified in the blood coaguhybrid collagem corresponded to those of the collagenase catlation factor XII (MacMullen and Fujikawa, 1985), the manalytic domain al one, but, in spite of binding to native type I colnose receptor (Taylor ct al., 1990), the mannose-6-phosphate
lagen, the hybri i did not have the capability to cleave this subreceptor (Lobel ct al., 1987), the insulin-like growth factor II
strate. These line's of evidence show that the collagenase COOHreceptor (Morgan et al., 1987), and the bovine seminal fluid
terminal domair is critical for positioning the cleavage site of the
proteins PDC-109 and BSP-A3 (Esch et al., 1983; Seidah et al.,
triple-helical typpe I collagen molecule with a precise orientation
1987). Gelatinase B contains an additional 54-amino-acidrelative to the ca talytic site of collagenase (Murphy ct al., 1992a).
residue proline-rich sequence located adjacent to the zincDeletions in the COOH-terminal domain of neutrophil collagebinding domain on the carboxyl-terminal side (Wilhelm et al.,
nase have ideni
a 16-amino-acid sequence as the critical
1989; Huhtala et al., 1991). The length of this sequence and a
region for colla gen binding, whereas a larger 62-amino-acid
30-55%, identity with a portion of the helical region of the colregion affected the efficiency of the collagenolytic activity
lagen u2(V) chain suggest that the appearance of this domain
(Hirose et al., 19')3).
may result from a recombinatorial event between the enzyme
Contrary to that observed for the collagenases, the COOHprecursor gene and a collagen gene (exon length in collagen
terminal domaii of gelatinase A has little or no contribution to
genes is commonly 54 bp) (Wilhelm et al., 1989).
the collagen-binnding properties of the enzyme, as determined
The six membrane-type metalloproteinases (MT-MMPs)
in binding assa:ys with the isolated rC domain (Overall et al.,
contain both a characteristic hydrophobic region that plays a
2000). This likely explains why gelatinase A with COOH-tercritical role in positioning the MT-MMPs in the cell membrane
minal domain dleletions retains full enzymatic activity on this
(Sato et al., 1994; Takino et al., 1995; Will and Hinzmann, 1995;
substrate (Fridn ian ct al., 1992). In addition to collagen, isolated
Puente et al., 1996), and an insertion of nine residues between
recombinant C( DOH-terminal domain of gelatinase A binds
the propeptide and the catalytic domain ending with the
plasma fibronec
RXR/KR consensus sequence that, in stromelysin-3, is essential
which is a substrate of the enzyme, and
heparin (Wallonn and Overall, 1997; Overall ct al., 2000).
for activation by furin (Pei and Weiss, 1995).

Ii3 [i,

?ptide
iise
i

fified

-i

The MMPs require Ca++ ions for activity, and putative

376

-tin,
Tissue inhil jitors of metalloproteinases form 1:1 stoichio-

Crit Rev Oral Biot Med

12 (50 73-398

(2001 )

metric complexes with activated MMPs by

interactions with both the catalytic and the
COOH-terminal domains (Birkedal-Hansen
et al., 1993; Bigg et al., 1994). The contribution
of the MMP COOH-terminal domains to the
binding of TIMPs is illustrated in COOH-terminal domain deletion enzyme mutants
which show a reduced inhibition of gelatinase
A by TIMP-2 and a lower affinity of
stromelysin-1 for TIMP-1 (Fridman et al.,
1992; Baragi et al., 1994). Isolated gelatinase A
rC-domain binds TIMP-2 (Fridman et al.,
1992; Bigg ct al., 1997; Wallon and Overall,
1997). In addition, pro-gelatinases A and B
can form complexes with TIMP-1 (Wilhelm ct
al., 1989) and TIMP-2 (Goldberg et al., 1989;
Ward et al., 1991b; Fridman et al., 1992;
Willenbrock ct al., 1993), respectively. Binding

Cell

tce Activation of Pro-Gelatinase A

Surfa

w
w

-Inegrin
.Y active
7 latent

--\T MT-MMI`
Cell memnbrane
V TIMP-2
Figure 2. Propo: sed model for cell membrane activation of pro-MMP-2. Extracellular sigextracellular environment
nals induce the c to produce pro-MMP-2 that is secreted to the
is in a complex of pericellular collagen bound to the cell membrane
(1) One site of s ?)torage
from which the enzyme may be released when needed. The extracelluby
integrin
laten(, t MMP-2 molecules (3) may combine with TIMP-2 (4) and enter the MTpooi
ar an
ao
MMP-mediated activation pathway (5). While TIMP-2 bridges the pro-MMP-2 COOH-terminal domain a nd the prodomain of one molecule of MT-MMP, a second MT-MMP can
cleave the MMP-'2 prodomain followed by the release of the activated MMP-2 (6).

:ell

of TIMP-2 to pro-gelatinase A is important for

enzyme(Lao
cell membrane activation of the enzyme
(Cao
et al., 1995), an event which does not occur in
COOH-terminal domain deletion mutants
(Murphy et al., 1992b; Ward et al., 1994).
Little is known about the function of the
54-amino-acid proline-rich domain that has some homology
to the collagen chain o2(V) and is found only in gelatinase B
(Wilhelm et al., 1989). However, gelatinase B with deletion of
the type V collagen-like domain cleaved denatured type I collagen as well as native types V and XI collagen
(Pourmotabbed, 1994).
In gelatinase A and B, the predominant collagen-binding
properties reside in the collagen-binding domains (CBDs)
(Collier et al., 1988; Wilhelm et al., 1989; Banyai and Patthy, 1991;
Collier ct al., 1992; Banyai et al., 1994; Murphy et al., 1994;
Steffensen ct al., 1995) that are composed of three fibronectin
type II-like modules. Characterization of isolated rCBD from
human gelatinase A has demonstrated that this domain
accounts for most, if not all, of the native and denatured type I
collagen-binding properties of human gelatinase A and also
enables the enzyme to bind to denatured type II, III, IV, V, and X
collagens, native type III, V, and X collagens, elastin, and
heparin (Steffensen ct al., 1995; Abbey ct al., 1997; Overall ct al.,
2000; Steffensen and Martin, 2001). Recent studies to delineate
the contributions of the three individual CBD modules to ligand-binding support the concept of module cooperation in the
interactions with enzyme substrates and other extracellular
matrix proteins by the observation that truncated collagen-binding domains consisting of modules 1+2 or 2+3 have reduced collagen-binding properties and do not bind elastin (Abbey ct al.,
1997; Overall ct al., 2000). CBD deletion mutants of gelatinase A
lost the binding to type I collagen but not to TIMP-1. Although
the catalytic activity in the mutant enzyme corresponded to that
of the wild-type gelatinase A for a peptide substrate, the activity against casein was reduced by 50%, and that against type I collagen by 90%, (Murphy et al., 1994). A CBD deletion mutant of
gelatinase B also abolished the type I gelatinolytic activity
(Pourmotabbed, 1994). These results stress the importance of
CBD for substrate positioning and successful cleavage. Furthermore, after deletion of the CBD that provides gelatinase A with
elastin-binding properties (Steffensen ct al., 1995), gelatinases A
or B mutants neither bound gelatin nor retained elastinolytic
activity (Shipley et al., 1996).
12(5)373-398 (2001 )

^7

(3) Cell membrane localization and activation

of progelatinase A
Progelatinase A (proMMP-2) is unique among the MMPs in
that it can be activated by a cell-membrane-associated mechanism. Initially, it was observed that progelatinase A was
processed to 62-, 59-, and 43-kDa active forms following stimulation of human fibroblasts with Con A (Overall and Sodek,
1990, 1992; Brown et al., 1993) or of tumor cells with TPA
(Brown et al., 1990; Azzam and Thompson, 1992; Emonard ct al.,
1992). For localization of the site of activation, it was subsequently found that whole-cell lysates and plasma membranes
of Con A-stimulated fibroblasts, but not the conditioned medium from the same cells, had the capacity to process progelatinase A (Ward et al., 1991a). This suggested that a component of
the cell membrane was involved in the endogenous activation
of progelatinase A. This component was later identified as
MT1-MMP (Sato et al., 1994), and an additional five MT-MMPs
have been cloned (Takino et al., 1995; Will and Hinzmann, 1995;
Puente ct al., 1996; Llano et al., 1999; Pei, 1999). Recently, it has
also been demonstrated that the recombinant catalytic domain
of MT2-MMP can initiate activation of progelatinase A (Butler
et al., 1997). The MT-MMPs possess a hydrophobic domain that
anchors the enzymes in the cell membrane (Cao ct al., 1995; Pei
and Weiss, 1996). One model for cell-surface positioning of
progelatinase A during activation entails the TIMP-2 bridging
of the COOH-terminal domain of progelatinase A and MTlMMP (Strongin ct al., 1995) and explains the requirement for
the progelatinase A COOH-terminal domain for membrane
activation (Murphy et al., 1992b; Ward ct al., 1994) (Fig. 2). The
observation that both TIMP-2 binding and gelatinase A activation require an MT1-MMP with an intact prodomain (Cao et al.,
1998) supports the involvement of a second molecule of MT1MMP in the catalytic cleavage of the gelatinase A prodomain.
This model is supported by the finding of reduced cell-membrane binding as well as activation of progelatinase A in the
presence of excess TIMP-2 or isolated gelatinase A rC domain,
but not TIMP-1 (Ward ct al., 1991a, 1994; Overall ct al., 2000). In

Crit Rev Oral Biol Med

377

lar penetration of basement

membrane matrices and the
expression of gelatinase A
(Seftor et al., 1992, 1993). It is
not yet known whether occupation of the av133 receptor is
Integrin Function
Wound Normal
associated with altered gelatiin Regulation of MMPs
Intearin KCa Epithelial KCb Licand
nase A expression. However,
incubation of cells with other
+c
+
Oc1[31
n idg
CollI,IV,V,VI
integrin ligands-such as the
+
+
0L2131
Coll i, III, IV, V, VI, denatured Coll Iriduction of MMP-1 expression
fibronectin CS1 and heparin+
+
R egulao of M - e
LM-5, denatured Coll I?
AP3131
+
+
Regulation of MMP-9 expression
FN
o513 1
peptides-or
binding
to thewith
blocking antibodies
131,
+
+
TN
c91 1
nd
d
and
integrin
x5131
o3,
o4A3l,
+
+
n
LM-5
oL6p34
profoundly affects cellular
+
Oxv
nd
+/-d FN, VN
d
expression of several MMPs
+e
+e
ovl3I
n
FN, VN, [AP-TGF13
+
nd
(Werb etHuhtala
1989; Larjava et
+/- VN
0Lvp35
1993a;The al., et al., 1995). al.,of
-f
+
0Lv16
R
FN, LAP-TGFI, TN, VN
understanding
cell-membrane
localization of
a
Integrin expression in wound keratinocytes.
MMPs is developing, and it is
b Integrin expression in resting skin and oral mucosal epithelium.
becoming evident that several
Expressed in vivo.
direct and indirect modes of
d Variable expression in vivo.
e
interaction
between the enExpression shown only in vitro.
f
zyme and the cell-membrane
No expression in vivo.
g
nd, not determined.
components are utilized for
Other abbreviations used: Coll 1, type collagen; LM, laminin; FN, fibronectin; VN, vitronectin; TN, tenascin-C;
enzyme-binding. Biologically,
LAP-TGF13, latency-associated-peptide-TGF13 complex.
cell-surface-associated proteolytic activities may explain
addition, our investigations have demonstrated the presence of the tight regulatory mechanisms of MMP activities and also the
an additional mechanism for cell-surface localization of gelativery localized degradative processes (Basbaum and Werb, 1996).
nase A/progelatinase A that is mediated by the CBD interacting with cell-surface-bound collagen (Steffensen et al., 1998).
(III) Integrins
The relative importance of the CBD and the COOH-terminal
domain for progelatinase A complex formation with MTGENERAL ASPECTS
MMPs and activation has been demonstrated by the unaltered
activation in gelatinase A CBD deletion mutant enzyme but no
Integrins are known to play an important role in regulating a
activation in a COOH-terminal domain deletion mutant of the
wide range of cellular functions during growth, development,
enzyme (Murphy et al., 1992b, 1994; Ward et al., 1994).
differentiation, and the immune response (Giancotti, 1997; Aplin
Although CBD is not directly required for membrane-mediatet al., 1998; Miyamoto et al., 1998; Ruoslahti, 1999). In addition,
ed activation, excess rCBD enhanced cellular activation of
integrins serve a critical function in cell adhesion and signaling
gelatinase A, possibly by releasing cell-surface-bound progeduring wound-healing, where they are fundamental to relatinase A for entry into the MT-MMP-mediated activation
epithelialization and granulation tissue formation (Hakkinen et
pathway (Steffensen et al., 1998) (Fig. 2).
al., 2000a). Integrins are cell-surface transmembrane ux hetAdditional mechanisms for cell-surface localization and
erodimers that contain a large extracellular ligand-binding
activation of MMPs have been demonstrated or proposed. It has
domain, a transmembrane domain, and a relatively short intrabeen reported that TIMP-2 as well as a complex of progelatinase
cellular cytoplasmic tail that functions as a signal-transducing
A/TIMP-2 can bind directly to cell surfaces with a Kd of 2.5 nM
element (Aplin et al., 1998; Howe et al., 1998; Giancotti and
and 30,000 sites/cell (Emmert-Buck et al., 1995). TIMP-2 binding
Ruoslahti, 1999). At the moment, 24 cx4 heterodimers composed
to cells can further elicit signal transduction events, as illustratof 18 different oa- and 8 different 1-subunits are known. Both the
ed by the rTIMP-2-induced production of cAMP and cell-prolif(x- and 1-subunits are transmembrane glycoproteins that cooperative response that can be inhibited by an adenylate cyclase
erate in integrin binding to ligands. Functionally, many integrins
inhibitor (Corcoran and Stetler-Stevenson, 1995). Based on these
have overlapping ligand-binding functions. For example, both
results, it has been proposed that a specific TIMP-2 receptor can
cU131 and (X2131 can bind collagen, although their signaling funcmediate enzyme-binding (Emmert-Buck et al., 1995). In additions appear to be different (Ivaska et al., 1999). Indeed, certain
tion, the gelatinase A COOH-terminal domain can bind
cell types can express more than one of the integrins with binddirectly to the cellular oxvP33 integrin receptor (Brooks et al.,
ing specificity for the same ligand. This is exemplified in kera1996). The COOH-terminal domain of gelatinase A is homolotinocytes, which can express at least three fibronectin recepgous to vitronectin, which is a major extracellular matrix ligand
tors-namely, otv1l, oxvP6, and u5131-that may collaborate in
for the cxvP33 integrin receptor (Pytela et al., 1985; Suzuki et al.,
cell adhesion and migration (Koivisto et al., 1999) (Table 2). Some
1986). Interestingly, occupation in vitro of this cell-surface recepof the integrins, such as oxll and ix2131, contain a 200-aminotor by blocking antibodies or by vitronectin enhanced the celluacid I-domain that functions in ligand recognition. An addition-

TABLE 2
Putative Integrin Ligands and Integrin-mediated Regulation
of MMP Expression in Keratinocytes during Wound-healing

378

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12(.):373-3Q8

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12(5):373-398 (2001)

al level of complexity inherent in the integrin receptors is caused

by the fact that several integrins have splice variants with specific functions (de Melker and Sonnenberg, 1999). Among these are
multiple variants of at least a3, cx6, and P13 integrin cytoplasmic
tails that are expressed in a tissue-specific manner. A very interesting variant of the common 1P-subunit is the 131c that appears

to participate in controlling the cell cycle (Fornaro et al., 1998). We

have observed that this variant, which inhibits cell proliferation,
is up-regulated in migrating keratinocytes and fibroblasts of
wounds, thereby providing a potential mechanism to block the
cell cycle in these cells while they are migrating during wound

repair (Hakkinen et al., unpublished observation).

In addition to the functional diversity that may result from
altemative splicing, the function of integrins may be regulated at
multiple other levels (Aplin et al., 1998; Howe et al., 1998;
Giancotti and Ruoslahti, 1999). Integrins on the cell surface can
be in either an inactive or an active form. Two major control
mechanisms are central to regulating the state of activity of integrins, the "outside-in" and "inside-out" signaling. In the outside-in
activation, integrins become activated by ligand-binding, a

process that is dependent on the extracellular concentration and

balance of divalent cations, Call and Mg". Fine-tuning of the
signaling is also a stepwise process, which is demonstrated by
the fact that integrin affinity (the binding of integrins to ligands)
and avidity (integrin clustering by the ligands) can induce different signaling events (Miyamoto et al., 1998). In wound-healing, integrins are expressed not only on basal cells, but also on
suprabasal cell layers of the epithelium. Using an antibody specific to the active 13 integrin, we have demonstrated that 13
integrins in the uppermost keratinocyte layers are not active in
ligand-binding (Hakkinen et al., manuscript in preparation). The
potential for the presence of integrins at different stages of activation makes the interpretation of immunolocalization results
complex, since most of the available antibodies do not distinguish between ligand-binding and inactive integrins.
Interactions of integrins with their ligands lead to clustering
(high concentration of integrins in one area), which, in turn,
causes multiple intracellular proteins to accumulate in the area
(Miyamoto et al., 1998). Integrin clustering results in tyrosine
phosphorylation of focal adhesion kinase (FAK) and formation
of intracellular complexes containing pl3OCas, paxillin, and Src,
and several cytoskeletal proteins, including talin, (x-actinin, and
vinculin (Aplin et al., 1999). These intracellular complexes are in
direct contract with extracellular molecules via the integrins. Via
this organization of the cytoskeletal elements, integrin binding to
the ligand appears to activate small GTPases, RhoA, Cdc42, and
Rac, that regulate the formation of lamellipodia and filopodia,
directly (Defilippi et al., 1999). Therefore, these processes may
have direct impact on wound-healing, since migrating keratinocytes extend long filopodia/lamellipodia-like cytoplasmic
projections into the wound (Larjava et al., 1996). Embryonic epidermal cells do not move by extending lamellopodia into the
wound. Rather, they use actin filament cables to pull wound
edges together (Martin and Lewis, 1992).
As a result of "inside-out" signaling, which is distinct from
the "outside-in" signaling described above, the integrin ligandbinding site can be altered by changes in the cytoplasmic tail
(Leisner et al., 1999). These signaling pathways are best known
with leukocyte integrins but are now also being characterized in
other cell types. Specifically, blocking or activating various intracellular kinases is one way to alter the cytoplasmic interactions
of integrin tails, resulting in altered ligand-binding. Certain
12(5):373-398
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Crit Rev

growth factors can also participate in this process. Keratinocyte

growth factor, which is induced in wound-healing and stimulates keratinocyte growth, enhances keratinocyte adhesion and
migration by a process involving alterations in integrin avidity
(Putnins et al., 1999). It is likely that both signaling pathways are
involved during wound-healing for two reasons. First, wound
fluids contain several known and unknown biomolecules potentially capable of inside-out signaling. Second, the wound bed
extracellular matrix differs from the basement membrane and is
also being constantly remodeled by the MMPs and other
enzymes. In addition, there is a deposition of de novo matrix by
migrating cells by which information is conveyed into the cell
through the integrin outside-in signaling mechanism.
Integrins associate with several transmembrane proteins
that may critically regulate integrin function (Hemler, 1998).
Among these, the integrin-associated protein (IAP), members
of the transmembrane-4 superfamily (TM4SF), growth factor
receptors, and urokinase-type plasminogen activator receptor
(uPAR) are thus far the best-known of such proteins. TM4SF
proteins, such as CD151 and integrin a3131, are capable of forming a stable complex that may be required for OP31 integrin
function and cell migration. Another TM4SF protein, CD9,
associates with the c3I1 integrin in keratinocytes to form a
complex involved in regulating cell motility (Jones et al., 1996).
It is not known whether OP3131 integrin function is regulated
through this mechanism during wound repair. Growth factor
receptors also accumulate in the same complexes on cell surfaces as integrins and regulate integrin functions (Miyamoto et
al., 1998; Aplin et al., 1999).
For regulation of its growth, a cell integrates information
from the integrin-mediated cell adhesion, signals conveyed by
growth factor receptors, signals from integrin co-receptors
such as syndecans, and signals from cell-cell adhesion molecules (Aplin et al., 1999). In the context of wound healing, the
synergy between integrin and growth factor receptors is probably a key process in the regulation of cell proliferation.
Integrins can also bind growth factors directly. Recently, vP131
and ov16 integrins were shown to interact with transforming
growth factor 131 through its latency-associated peptide that
contains an arginine-aspartate-glycine (RGD) peptide
sequence (Munger et al., 1998, 1999). It was demonstrated further that this binding also activates TGF-13. The resulting focal
activation of TGF-131 may play an important role in the focal
deposition of connective tissue and expression of MMPs,
which are both critical for wound repair.

(IV) Re-epithelialization in Wound-healing

(1) INTRODUCTION
An essential feature of wound-healing is controlled proteolytic
degradation and remodeling of the extracellular matrix (for
related reviews, see Kahari and Saarialho-Kere, 1997; Shapiro,
1998; Murphy and Gavrilovic, 1999; Streuli, 1999). A growing
body of literature has enhanced our understanding of the
important role of MMPs and their inhibitors in wound-healing.
The emerging picture entails both spatial and sequential differences in the timing and level of expression of the MMPs and
TIMPs during normal wound-healing. In addition, regulation of
MMP expression and activation has recently been closely linked
to cell-adhesion events that are controlled by integrins (Tables 2
and 3, Figs. 3 and 4).
To re-establish tissue integrity after injury, re-epithelializaOral

Biol

Crit Rev Oral Biol Med

Med

379

379

atinocytes bordering the

wounds, but it is not
expressed by proliferating
keratinocytes (Stricklin et al.,
1993; Saarialho-Kere, 1998)
n
MMP
of
(Fig. 3). In both acute and
Expressior
FBLa
Ligands
Integrin
Integrin-medialted Regulation
chronic ulcers, the keratinocytes that express
+b
Coil I,I I,IVC, Vc, Vlc, LM 1c
ol 13
Up-regulation (of MMP- 1 3 expression
in three-dimensiional collagen matrix; MMP-2, -7, -'9f MMP-1 have been detected
in the migrating epithelial
+
Upregulation c)fMMP-1 and MMP-13
Coll I, Il1C IIIcl IV,vc, VI, VII,
a2131
front, where there is no basedenatured Coll Ic, LM-iC
expression in t*hree-dimensional collagen matrix
ment membrane (Saarialho+
FN, denatured Coll lc
(AP1
+d
1
FN (CS-1)
Suppression ofFMMP-1 and MMP-3 expression
Kere, 1998). These observations from dermal wounds
by FN fragmernt (CS- 1 )
+
Induction of M MP-1 and MMP-3 expression by
FN
are supported and extended
ot5p31
FN fragment ( 120 kDa)
by the finding that there is a
+
nde
FN, VN, FB, Fbg
otv
characteristic timing of colav13l
nd
+d
FN, LAP-TGF1
lagenase expression in
+d
nd
FN, VN, FB, Fbg, TN
acvP3
experimental human der+d
nd
VN
Pv135
mal wounds (Inoue et al.,
1995). Thus, analyses of
in fibroblasts during wound repair.
biopsies from in vivo-healing
FBL,
integrin
expression
b
wounds by in situ hyExpressed in vivo.
c
Shown in cells other than connective tissue fibroblasts.
and immunobridization
d
Expression shown in vitro only.
histochemistry
at various
e
nd, not determined.
time
between
days 1
points
f Up-regulated in dermis of col -integrin knock-out mice.
and 14 after wounding
Other abbreviations used: Coll 1, type collagen; LM, laminin; FN, fibronectin; VN, vitronectin; FB, fibrin; Fbg, fiboccurred showed peak collarinogen; TN, tenascin-C; and LAP-TGF1, latency-associated-peptide-TGF13 complex.
genase expression on day 1,
followed by a gradual
tion must occur as rapidly as possible. Keratinocytes dissolve
decrease toward no detectable enzyme at the time of complete retheir hemidesmosomal complexes, releasing them from their
epithelialization. A very similai r pattern of collagenase expression
contact with basement membrane and allowing quick migration
occurred in an in vitro experimeental model, where punch wounds
across the wound defect. There is clear evidence that modulation
were created in organ-cultured skin explants. Indeed, collagenase
of the wound matrix by several MMPs is required for migration
in migrating keratinocytes of thaese ulcers was already detected 4of keratinocytes, and that this process is regulated by integrin6 hrs after wounding occurred, demonstrated a peak at 12-24 hrs,
type cell-surface receptors. Keratinocytes in wounds migrate and gradually decreased until the time point when the wound
either on or through the provisional wound matrix, which is rich
was completely covered by epithelium (Inoue et al., 1995).
in fibronectin and fibrin, or they encounter a connective tissue
Expression of MMP-1 is also se(en in keratinocytes on partly intact
matrix consisting of various collagen types, especially type I colbasement membrane during rEepair of experimental suction blislagen (Woodley, 1996). In small gingival wounds, keratinocytes ter wounds as well as in bullae produced by several skin diseases
migrate through the fibrin-fibronectin matrix and may not come (Saarialho-Kere et al., 1995). ThIus, MMP-1-expressing cells locate
into contact with a type I collagen-containing matrix (Larjava et
close to the connective tissue matrix and lack contact with the
al., 1993b; Hakkinen et al., 2000a). In dermal wounds, however,
basement membrane.
In keratinocyte cultures, MMP-1 is expressed only if the
keratinocytes are believed to migrate under the clot in contact
with the dermal matrix (Woodley, 1996). The interactions of
cells are in contact with natiive type I collagen. Keratinocyte
keratinocytes with the matrix in these two scenarios require interaction with other matri:x proteins-such as fibronectin,
expression of different integrins, and the enzymatic modification laminin-1, laminin-5, basemen-t membrane matrix, and type III
of the matrix is also different. Migrating keratinocytes express
collagen-does not induce M[MP-1 expression (Sudbeck et al.,
the oL5131 major fibronectin-binding integrin, the o3131 and the
1997b). Therefore, contact betvveen keratinocytes and type I colc6P4 integrins, both of which bind to laminin-5, the u21l colla- lagen is apparently crucial for MMP-1 expression. Interaction of
gen receptor, the xvfl fibronectin receptor, and the o9131 keratinocytes with type I colIlagen is mediated by the ca2P1
tenascin receptor (Larjava et al., 1996; Hakkinen et al., 2000a).
i
integrin that also seems to be involved
directly in the induction
Interestingly, among these receptors, the cav135 vitronectin-bind- of MMP-1 expression, since a2! integrin-blocking antibodies preing integrin is expressed in dermal wounds but is absent from oral vent MMP-1 induction. Migrattion of keratinocytes on type I colwounds (Juhasz et al., 1993; Clark et al., 1996; Haapasalmi et al.,
lagen requires both a2131 integ ,rin and MMP-1 activity (Pilcher et
1996) (see also Table 2).
al., 1998). Keratinocytes do not migrate on collagen that has been
rendered resistant to cleavage by MMP-1 through introduction
(2) CELL ADHESION VIA INTEGRINS REGULATES
of specific mutations in the collagenase cleavage site, and their
MVP-1 EXPRESSION IN MIGRATING KERATINOCYTES migration also can be blocked by TIMP-1, MMP-1 antiserum, or
In normally healing ulcers and acute burn wounds, interstitial
antibodies to ox2p1 integrin (Piilcher et al., 1997). MMP-1 appears
t a2131 integrin on keratinocyte
collagenase (MMP-1) is consistently expressed by migrating ker- to be associated directly with the

TABLE 3
Putative Integrin Ligands and Integrin-mediated Regiulation
of MMP Expression in Fibroblasts during Wound-heailing

cx4P

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M -*(TIMPFC
C.

;.

)MMP -9* CD
(MtATMP-3)

(()>i
MMP- 2.
MMP-2
MMP-1,3*

MM\P-1
MMP- 1

(MMP-IO)
1)
(TIMP-3)

MMP-9

CT

MMP-2\
~/MMp-3
MMp-8
X

TIMP-1\

TIMP-3

C)~MMP-9

TIMP-2
MMP 1X
sepraseuA uPAA

MMP-l

Figure 3. Cellular expression of matrix-degrading proteases during mucosal and dermal wound-healing. The histological characteristics
of healing in human mucosal wounds after 3 days (Panel A) and 7 days (Panel B). On day 3, epithelial and connective tissue cells are
migrating into the wound space (Panel A), whereas, on day 7 (Panel B), the re-epithelialization and granulation tissue formation is ongoing. The corresponding panels C and D summarize schematically the cellular expression of matrix-degrading proteases at the stages of
wound-healing presented in panels A and B, respectively. Please see text of review for details and appropriate citations. (Panels A and C)
In early wounds, epithelial cells migrate into and through the fibrin clot to cover the wound space. During this process, keratinocytes at
the migratory front express MMP-1, -9, and -10, uPA, and TIMP1, whereas non-migratory keratinocytes located at the wound margin
express MMP-3 and -9, as well as TIMP-1 and TIMP-3. The MMPs expressed by keratinocytes at the migratory epithelial front likely serve
to generate a path for cell migration through the provisional wound matrix. In comparison, the MMPs generated by the cells at the wound
edge may be detaching cells from the basement membrane, permitting subsequent motility to occur. During the early phases of wound
repair, connective tissue fibroblasts from the wound margin start proliferating and migrating toward the provisional wound matrix. At this
time point, the fibroblasts express MMP- 1, -2, -13, and seprase that may serve to detach the cells from the matrix to promote cell migration into the wound provisional matrix and to modulate the structure and organization of the new extracellular matrix. (Panels B and D)
After re-epithelialization is complete, the keratinocytes cease to migrate and start to stratify and differentiate. Although the basal wound
keratinocytes continue to produce MMP-9, the expression of MMP-1, MMP-10, TIMP-1, and TIMP-3 is down-regulated in the epithelium.
At this time point, stromal cells, including fibroblasts, endothelial cells, macrophages, and inflammatory cells, have migrated into the rovisional wound matrix to form granulation tissue. As was observed for fibroblasts at the wound margin during early wound repair, fi roblasts in the granulation tissue express MMP-1, -2, -13, and seprase. Furthermore, granulation tissue cells start to express MMP-3, -8, 9, -11, -12, and -14, uPA, TIMP-1, and TIMP-3. At this time point, TIMP-2, presumably produced by the wound keratinocytes, localizes
in the granulation tissue immediately beneath the wound epithelium. MMP-8 in the granulation tissue is most likely produced exclusively
by neutrophils during the initial inflammatory phase of wound-healing, and MMP-1 2 in the deepest parts of the regenerating connective
tissue is probably primarily a product of macrophages located in this area. MMP-1 1 is detected specifically along the periphery of the
aranulation tissue, where new collagen is accumulating. The spatial and temporal co-localization of MMP-14 (MT1-MMP) and MMP-2 in
the granulation tissue fibroblasts presumably relates to the role of MMP-14 in the activation of MMP-2. It is not known how MMP-3, -10,
-1 1, -12, and -14 are expressed in oral mucosal wounds. The abundant expression of proteolytic enzymes most likely is required for successful remodeling of newly deposited immature granulation tissue matrix but also serves to modulate cell-matrix interactions that regulate
the cellular functions critical to wound repair, as is discussed in detail in the text of the review. Furthermore, these proteases may release
extracellular matrix-associated growth factors bound to extracellular matrix molecules and participate in their activation. Abbreviations
used are: E, epithelium; CT, connective tissue; FC, fibrin clot; and GT, granulation tissue. *MMP-9 or MMP-1 3 are expressed by human
mucosal but not by human dermal keratinocytes or fibroblasts, respectively. For additional details and references, see the text.
(2001)
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381

proteins

ACTIN

B
ACTI

[- focal adhesion focal adhesion

s
proroteins

focal adhesion

ACTIN

focal

adhfesion

oeins

focal adhesion

MEMBRANE

proteins

CG

Fxp

Cx3PI

ac31
a2,3?

a6p1
av,3

cxvP5

avp5?

Figure 4. Potential mechanisms of focalized proteolysis involving cell membrane-extracellular matrix interactions. From current knowledge on
interactions among integrins, MMPs, and extracellular matrix molecules, this Fig. presents potential mechanisms involved in the control oF cellmembrane-associated proteolytic events. See text for further details and citations. (A) Urokinase-type plasmin activator (uPA), complexing with
its cellular receptor (uPAR), co-localizes with integrins at the focal adhesions and may there modify interactions of integrins with the ligands that
are needed for cell migration through the provisional wound matrix. The uPA/UPAR complex also potentially participates with integrins in signaling inside the cell. For example, uPA/uPAR can associate with the cu5pl integrin at the focal adhesions on fibronectin, with the c3A13 and
ca631 integrins at focal adhesions on laminin, or with the cxvP33 or cavP35 integrins at focal adhesions on vitronectin. (B) Integrin ca211 potentially bids to MMP-1, thereby localizing the enzyme to cell adhesion sites on collagen. (C) MMP-2 and MMP-9 bind to an as-yet-unidentified
cell-surTace molecule to localize and store these MMPs at the cell membrane. (D) The integrin cXvP33 can bind MMP-2, which may in turn degrade
collagen that has been initially denatured by MMP-1. The integrin cavP3 can also serve as receptor for the cryptic RGD site exposed in collagen after denaturation (see also E). and thereby localize MMP-2 at the denatured collagen. (E) Denaturation of collagen by heat exposes the
cryptic binding sequence RGD that can be recognized by the cuvl33 and ca311 integrins and possibly also by the cr2131 and cav35 integrins.
(F) Heat denaturation of collagen also exposes a DGEA site in the collagen molecule that can serve as an alternative binding site for the cx2:31
integrin. (G) The integrin cr211 and MT1 -MMP can co-localize at focal adhesion sites where MT1 -MMP, directly or through activation of MMP2, may degrade matrix molecules and also transfer signals to the cytoplasm.
cell surfaces (Dumin et al., 1999, 2001). This interaction provides
a mechanism for bringing together the matrix (collagen), the
receptor (ca211 integrin), and the enzyme (MMP-1), resulting in
controlled and focalized proteolysis at the cell adhesion sites to
facilitate cell migration. Recently, it was demonstrated that only
the initial induction of MMP-1 expression in keratinocytes in
contact with type I collagen is mediated by the cu2131 integrin
(Pilcher et al., 1999). Sustained expression of MMP-1 and cell
migration also requires autocrine activation of the epidermal
growth factor receptor (Pilcher et al., 1999). This is another interesting example of how integrins and growth factor receptors act
together to regulate MMP-1 expression and cell migration.
Laminin-1, but not laminin-5, appears to be able to down-regulate MMP-1 expression effectively in keratinocytes that are in
contact with collagen (Sudbeck et al., 1997b). Laminin-5 is
expressed by migrating keratinocytes (Kainulainen et al., 1998),
whereas the deposition of laminin-1 does not occur until the reepithelialization is complete (Larjava et al., 1993b). In addition,
laminin-1 inhibits keratinocyte migration and is a poor adhesion
substratum for these cells (Woodley et al., 1988). It is an interesting possibility that deposition of laminin-1 during basement
membrane re-organization could be the signal to keratinocytes
to suppress MMP-1 expression. There is some evidence that the
cleavage of type I collagen by migrating keratinocytes causes
ca211 integrin-dependent re-organization of the collagen fibrils,

382

resulting in tracks for directional movements of migrating cells

(Pilcher et al., 1998). Recently, it was also shown that type I collagen digested by collagenase can promote disassembly of
smooth-muscle focal adhesions and promote cleavage of FAK,
paxillin, and talin (Carragher ct al., 1999). Whether this phenomenon is also true for human keratinocytes remains to be shown,
but it could provide a mechanism by which tight cell adhesions
to the collagen matrix are locally released and thereby allow for
cell migration. The collagen matrix processed by migrating keratinocytes could also facilitate the migration of epithelial cells
tracking behind the leading edge by exposing cryptic sites on
collagen for other potential receptors, such as the avP5 integrin
(Messent et al., 1998; Murphy and Gavrilovic, 1999). Migrating
keratinocytes express the a5Xf1 fibronectin-binding integrin.
Signaling through this receptor involved with regulation of
MMP-1 expression in Vitro is evident when cells are exposed to
fibronectin fragments containing the cell-binding site, i.c., integrin-binding site (Werb ct al., 1989). This is pertinent because
fibronectin fragments have been demonstrated in wound fluids
(Wysocki and Grinnell, 1990; Grinnell ct nl., 1992). It is quite possible that cells migrating through the fibrin-fibronectin provisional matrix could face fibronectin fragments that induce
MMP-1 expression. Keratinocytes would then be ready to come
into contact with and migrate on collagen after they have finished cutting their path through the provisional matrix. Whether

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this regulatory mechanism, first demonstrated with synovial

fibroblasts, can be applied to human keratinocytes remains to be
shown. Thus, expression of MMP-1 may be regulated by interactions between the extracellular matrix molecules and several different integrins. MMP-1 expression and activation in keratinocytes require tight regulation during normal healing, since
overexpression of this enzyme may cause a delay in wound reepithelialization (Di Colandrea et al., 1998). This model is supported by evidence for elevated MMP-1 expression in chronic,
poorly healing wounds, as is discussed below.

(3) PROTEOLYTIC PROCESSING OF LAMININ-5

BY PLASMIN AND MMP-2
Laminin-5 (LM-5) is an interesting basement membrane protein
that can have multiple and reverse functions, depending on the

processing of the molecule. Migrating keratinocytes express

and deposit LM-5 regardless of the wound model (Kainulainen
et al., 1998). The unprocessed intact (x3 subunit of LM-5 is found
under the migrating tips of keratinocytes and appears to promote keratinocyte migration in vitro (Goldfinger et al., 1999).
Both o31l and o6r4 integrins interact with LM-5, and both are
present on migrating keratinocytes in wounds (Larjava et al.,
1993b; Kainulainen et al., 1998). It is possible that these receptors cooperate in LM-5 binding during re-epithelialization. As
in wounds, cultured keratinocytes deposit LM-5 to support
their migration, regardless of which matrix proteins were used
as the original substratum. Integrin a31l-specific antibodies
block keratinocyte migration under these conditions, suggesting that the cx3 P1 integrin plays an important role in controlling
keratinocyte migration (Zhang and Kramer, 1996). In comparison, the oL6f4 integrin plays a major role in hemidesmosome
formation (Sonnenberg et al., 1991). Therefore, interaction of the
o6r4 integrin with LM-5 could be expected to provide a stopsignal to keratinocytes rather than support migration. This
would be true if LM-5 were not modified during wound-healing. However, LM-5, that promotes hemidesmosome formation, may be processed by plasmin (Goldfinger et al., 1998).
Moreover, the o-6f34 integrin function can be regulated by EGF,
which can promote keratinocyte migration toward laminins
(Mainiero et al., 1996). Recent evidence points to a role of cu2p1
integrin in transient binding of endogenously produced
unprocessed laminin-5 which promotes keratinocyte migration
(Decline and Rousselle, 2001). In contrast, binding of newly
sythesized laminin-5 by 03PI1 integrin could retard cell migration before hemidesmosomes are formed by action of ox6P34
(Decline and Rousselle, 2001).
MMP-2 can also cleave LM-5, thereby creating a fragment
that promotes cell migration. This has been shown for tumor
cells and normal breast epithelial cells (Giannelli et al., 1997,
1999) but not yet for normal keratinocytes. MMP-2 is found in
wound fluids, although its mRNA, that is present in the stoma in
vivo, is absent from both normal and wound keratinocytes
(Saarialho-Kere et al., 1993; Salo et al., 1994). Only occasionally
has MMP-2 protein been found in epidermal structures (Ashcroft
et al., 1997). Epithelial cells in normal and wounded rat cornea
express MMP-2 (Ye and Azar, 1998). It is possible that migrating
keratinocytes could bind MMP-2 to their cell surfaces, since cultured keratinocytes both express and interact with MMP-2
(Makela et al., 1998). In wounded Hacat keratinocyte cultures,
that do not express MMP-1, MMP-2 localizes to focal contact-like
structures in migrating keratinocytes that also deposit LM-5
(Mikela et al., 1999). MMP inhibitors can block keratinocyte
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migration in these cultures, supporting the view that MMP-2

might be used for focalized proteolysis of LM-5 in migrating keratinocytes, at least under culture conditions. MMP-2 is present in
"invadopodia" of tumor cells (Monsky et al., 1993) and interacts
specifically with the uvf33 integrin in endothelial cells, as discussed above. However, because keratinocytes do not express
this integrin, an interaction between MMP-2 and the cell surface
would depend on other integrins-an option that remains to be
demonstrated at this time. Whether other integrins are involved
in MMP-2 binding to the keratinocyte cell surface also remains to
be shown. Therefore, the evidence for an active role of MMP-2 in
in vivo re-epithelialization of dermal and mucosal wounds is
inconclusive. MT1-MMP that is involved in the activation of
MMP-2 (see above) has been recently shown also to cleave LM-5
with a pattern similar to that of MMP-2, suggesting that MT1MMP-either directly or indirectly through MMP-2 activationcleaves LM-5 to trigger cell migration (Koshikawa et al., 2000).
MT1-MMP may promote cell migration also by cleavage of the
CD44 hyaluronan receptor (Kajita et al., 2001).
(4) MMP-9 AND RE-EPITHELIALIZATION
Expression of MMP-9 has been studied in human skin by biochemical and in situ hybridization techniques (Pyke et al., 1992;
Oikarinen et al., 1993; Saarialho-Kere et al., 1993; Agren, 1994). In
contrast to epidermal healing, MMP-9 is expressed in nonwounded and in migrating keratinocytes of mucosal palatal
wounds (Salo et al., 1994). A strong signal for MMP-9 mRNA is
also detected in suction blister wounds (Oikarinen et al., 1993).
In rat corneal wounds, migrating epithelial cells express MMP9 (Ye and Azar, 1998). In rat skin, MMP-9 is also expressed in the
basal keratinocytes of the wound during early days (days 1-5) of
healing (Okada et al., 1997; Madlener et al., 1998). MMP-9
enzyme activity is also found in wound fluids (Salo et al., 1994).
In cultured keratinocytes that have been wounded, the distribution of MMP-9 appears diffuse, although in other cell types,
such as endothelial cells, MMP-9 has been localized into focal
contacts with integrins (Partridge et al., 1997; Makela et al., 1999).
Keratinocytes express relatively high amounts of MMP-9, and
this synthesis can be up-regulated by TGFI, TNFoT, or IL-1l3
(Salo et al., 1994). MMP-9 can also activate latent TGF3 on the
cell surface (Yu and Stamenkovic, 2000). MMP-9 binds to the
keratinocyte cell surface, but the binding is possibly through
CD44 and not via an integrin-mediated interaction (Makela et al.,
1998; Yu and Stamenkovic, 2000). Integrins may instead participate in the regulation of MMP-9 expression in keratinocytes.
Antibodies to cx3 and P1 integrin, but not those to cx2 or (x5, stimulate MMP-9 expression in keratinocytes regardless of the
matrix on which they were originally seeded (Makela et al.,
1994). Interestingly, it has been reported recently that the avP36
integrin cytoplasmic tail also could be involved in the regulation
of MMP-9 expression in cultured colon cancer cells (Niu et al.,
1998; Agrez et al., 1999). Wound keratinocytes express ovr36
integrin after re-epithelialization is complete (Haapasalmi et al.,
1996). However, it is not known whether this regulatory mechanism is associated with wound-healing, especially since the specific role of MMP-9 during re-epithelialization remains unclear.
In summary, MMP-9 could potentially play a role in cleaving
basement-membrane type IV collagen, the fibrin-fibronectin
clot, denatured collagen, and in processing proteins of the
regenerating basement membrane during wound-healing. Of
note, in wounded Hacat keratinocytes, MMP-9 expression did
not correlate with migration (Makela et al., 1999).

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(5) STROMELYSINS (MMP-3 AND -10) AND PLASMIN

IN RE-EPITHELIALIZATION

Stromelysin-1 and -2 (MMP-3 and -10) are also expressed by keratinocytes in the acute wound but by distinct cell populations
(Vaalamo et al., 1996). Whereas stromelysin-I is expressed at a
distance from the wound edge in proliferating keratinocytes,
stromelysin-2 mRNA is detected in migrating keratinocytes at the
migratory front of the epithelium, where it co-localizes with
MMP-1 (Saarialho-Kere et al., 1994; Vaalamo et al., 1996). At present, there is no evidence for binding of MMP-3 or MMP-10 to
extracellular matrix proteins or keratinocyte cell surfaces. Based
on the timing of the expression, stromelysin-2 (MMP-10) may
assist keratinocyte migration at the epithelial front and also contribute to remodeling of the newly formed provisional matrix or
matrix produced by keratinocytes. In comparison, stromelysin-1
(MMP-3) likely participates in restructuring of the newly formed
basement membrane. MMP-10 may also function in activating
other MMPs such as MMP-9. Like MMP-1 and MMP-10, the
urokinase plasminogen activator (uPA) is also present in keratinocytes at the migratory edge of the epithelium (Vaalamo et al.,
1996). Plasmin has been implicated in the in vivo activation of collagenase and stromelysin (He et al., 1989; Carmeliet et al., 1997).
The major role for uPA and plasmin during wound-healing, however, is more likely the degradation of the fibrin-fibronectin clot.
It has been shown elegantly that re-epithelialization and fibrin
degradation are delayed in uPA-deficient mice (Romer et al.,
1996). Interestingly, mice deficient in both uPA and fibrinogen
heal normally, demonstrating that uPA is primarily used for fibrinolysis during wound-healing (Bugge et al., 1996). Keratinocyte
migration is completely prevented when the activity of both
MMPs and uPA is blocked, but not when either one alone is
blocked (Lund et al., 1999). Also, broad-spectrum MMP inhibitors
decrease, but do not fully block, re-epithelialization in the domestic pig wound-healing model (Agren, 1999). Interestingly, uPA
and its receptor (uPAR) co-localize with the ctvP35 integrin in cultured Hacat keratinocytes (Reinartz et al., 1995). The axv35 integrin mediates cell locomotion on vitronectin (Kim et al., 1994), but
plasmin abrogates the cell adhesion function of the cxv5 integrin
(Reinartz et al., 1995). The importance of keratinocyte axvI35 integrins for wound healing has been questioned, because wounds in
atv35-deficient mice heal normally (Huang et al., 2000). If this
receptor is indeed required for wound-healing, the described regulation would be another example of how keratinocytes can
locally reduce their adhesion to matrix molecules, in this case vitronectin, to promote cell locomotion. Thus, keratinocytes possess
multiple integrins and proteolytic enzymes that interact in a coordinated fashion to balance cell adhesion and detachment to
acquire the proper state of migration. The repertoire of keratinocyte MMPs involved in re-epithelialization was recently
expanded by the novel MMP-28 (Epilysin) that localizes to the

migrating keratinocytes similarly to MMP-1 and MMP-10 (Lohi

al., 2001). However, the substrate specificity and functions for

this MMP during wound repair remain unknown.

et

(6) MMP ACTIVITY IS CONTROLLED BY TIMPs

DURING RE-EPITHELIALIZATION
There is mounting evidence that tissue degradation results from
localized cell-surface-associated events (Basbaum and Werb,
1996; Steffensen et al., 1998). Further, the balance between levels
of matrix metalloproteinases and their specific inhibitors, in
particular the TIMPs, may be crucial to successful wound-heal384

ing, as will be discussed further under "Chronic wounds".

During the initial phases of normal dermal wound-healing,
TIMP-1 and TIMP-3 mRNA localizes to basal keratinocytes at a
distance from the migrating edge (Vaalamo et al., 1999). At later
stages of wound-healing, TIMP-1 is present first in basal keratinocytes away from the wound edge (Vaalamo et al., 1996).
Subsequently, the inhibitor occurs in the stroma corresponding
to the base of the wound and around minor capillaries, where it
is possibly secreted from endothelial cells or macrophages
(Stricklin et al., 1993). As discussed above, TIMP-1 and -3 readily inhibit stromelysin-1 and collagenase. Therefore, coordinated
MMP and TIMP expression may contribute to the control of keratinocyte proliferation and also basement membrane organization (Vaalamo et al., 1999). TIMP-1 expression appears to be regulated through integrins, since its expression is stimulated in
keratinocytes cultured on type I collagen but is inhibited on
laminin-1 (Petersen et al., 1990). TIMP-2 protein has been detected around migrating keratinocytes in human wounds and is
believed, at least partially, to be produced by these cells
(Vaalamo et al., 1999). TIMP-2 could therefore participate in the
inactivation of both MMP-1 and MMP-9 and, possibly, MMP-2.

(V) Wound-healing Events

in the Connective Tissue Matrix
As described in the preceding section on re-epithelialization,
wound-healing of the connective tissue matrix also includes cell
migration, controlled degradation of the ECM, and the release
and activation of growth factors. Connective tissue repair
involves several different cell types, including inflammatory
cells, fibroblasts, and endothelial cells. This part of the review
will concentrate on the role of fibroblasts in wound repair. There
is increasing evidence that signals elicited by interactions
between ECM molecules and integrins regulate the expression
of proteinases in fibroblasts. In addition, modulation of the
ECM by localized proteolysis can modify the integrin-mediated
interactions between fibroblasts and ECM molecules, and thereby control cell function during wound-healing (Table 3, Figs. 3
and 4). These processes require spatially and temporally tightly
regulated expression and activation of proteinases.
(A) INTEGRINS MEDIATE FIBROBLAST MIGRATION
DURING EARLY PHASES OF WOUND-HEALING

The major ECM components of resting dermal and oral mucosal connective tissue are collagen and cellular fibronectin.
Quiescent fibroblasts in this connective tissue matrix express
the collagen receptors c131 and o2f1 and the major fibronectin
receptor cu531 integrin, which they use for adhesion to the
matrix (Welch et al., 1990; Gailit et al., 1996; Xu and Clark, 1996).
These cells also express (X3131 (Xu and Clark, 1996), u4Al (Gailit
et al., 1993), and integrin heterodimers of the (xv subfamily
(Hakkinen et al., manuscript in preparation) that also bind
fibronectin (Aplin et al., 1998). Although fibroblasts in culture
may express ov31, av33, and ov15 integrins (Gailit et al., 1994;
Hakkinen et al., 1994; Gailit and Clark, 1996), it is not completely clear which 3-subunits combine with the cxv to form the functional integrin heterodimers in vivo.
When wounding occurs, quiescent fibroblasts are activated
to migrate into the blood clot. Although fibroblasts reside in a
collagen-rich connective tissue matrix prior to migrating into the
wound, it is not known whether the cells migrate along collagen
fibers or interact with other molecules that cover or associate
with the collagen fibers. Such ECM molecules, which associate

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with collagen, include fibronectin and proteoglycans that are

known to support or modulate cell adhesion and migration. In
fact, there is evidence that, in early wound-healing, fibroblast
migration may be primarily mediated by fibronectin. Thus, cell
migration from an experimental three-dimensional collagen
matrix into a fibrin clot required the presence of fibronectin in the
matrix and could be blocked with antibodies against a5131 and
ocv33 integrins (Greiling and Clark, 1997). Accordingly, about
three to five days after tissue injury, fibroblasts migrating into the
blood clot express the primary fibronectin receptor u5f31 integrin
and a3r31 integrin, while cells at the wound margin down-regulate the expression of the collagen-binding oxl and -2 integrins
(Gailit et al., 1996; Xu and Clark, 1996). Three days after wounding occurs, fibroblasts at the wound margin also express xv
integrins that can interact with multiple ligands, including
fibronectin (Gailit et al., 1996; Xu and Clark, 1996). In addition to
binding fibronectin, fibroblasts can use the xvf33 and atv15 integrins for adhesion to vitronectin (Gailit and Clark, 1996) and cxvI3
for cell adhesion and migration on fibrin and fibrinogen (Gailit et
al., 1997; Greiling and Clark, 1997). At this time, it is not clear
how these integrins are used for cell migration in vivo, where
cells are exposed to a complex matrix composed of multiple celladhesion proteins. However, there is some evidence that the
composition of the ECM is one of the mechanisms that regulate
integrin expression during wound-healing. For example, fibroblasts cultured in fibrin and fibronectin-rich matrices, that resemble the early wound matrix, show selective up-regulation of x3
and o-5 integrin mRNAs, whereas cells in a collagen-rich matrix
show up-regulation of cx2 integrin (Xu and Clark, 1996).

(B) FIBROBLASTS EXPRESS MULTIPLE PROTEINASES

DURING WOUND-HEALING
Although fibroblasts have the capacity to express a remarkably
wide variety of MMPs in culture, the in vivo expression of these
proteinases and their inhibitors is tightly regulated spatially and
temporally. By the use of semi-quantitative reverse-transcriptase polymerase chain-reaction (RT-PCR), cultured human skin
fibroblasts were shown to possess high levels of mRNAs for
MMP-1, -2, -3, -10, and -18 as well as MT1-MMP, MT3-MMP,
and MT4-MMP, but lower levels of mRNAs encoding for MMP7, -11, and MT2-MMP (Giambernardi et al., 1998; Grant et al.,
1999). When in contact with collagen, human dermal and gingival fibroblasts also express MMP-13 (Ravanti et al., 1999a,b).
Analyses of healing sites, 1 to 3 days after wounding
occurred, by in situ hybridization showed transcripts for MMP-1
(Ravanti et al., 1999b), MMP-2 (Salo et al., 1994), and seprase
(Ghersi et al., submitted for publication) in fibroblasts near the
wound edge. During early granulation tissue formation and later
during remodeling of the wound matrix, stromal cells in the granulation tissue also express MMP-1, -2, -3, -9, -11, and 12 (Wolf et
al., 1992; Saarialho-Kere et al., 1993; Stricklin et al., 1993, 1994; Salo
et al., 1994; Vaalamo et al., 1996, 1997; Madlener, 1998; Ravanti et
al., 1999b), MT1-MMP (Okada et al., 1997), seprase (Ghersi et al.,
submitted for publication), urokinase plasminogen activator
(uPA) (Grondahl-Hansen et al., 1988; Schafer et al., 1994; Vaalamo
et al., 1999), and TIMP-1 and TIMP-3 (Vaalamo et al., 1999).
Histologically, MMP-1-positive cells in the granulation tissue are
sporadic and consist primarily of activated fibroblasts, but
macrophages and endothelial cells also express MMP-1 (Stricklin
et al., 1993, 1994; Vaalamo et al., 1996, 1997; Pilcher et al., 1999;
Ravanti et al., 1999b). Interestingly, subsets of fibroblasts at the
wound edge and in the granulation tissue of oral mucosal
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wounds expressed MMP-13 (Ravanti et al., 1999b), whereas

MMP-13 was not present in skin wounds healing normally
(Vaalamo et al., 1997). In porcine bum wounds, the expression of
MMP-2 is up-regulated in the regenerating dermis adjacent to
wound epithelium (Stricklin et al., 1994). However, in human resting oral mucosa, MMVP-2 mRNA appears to be constitutively
expressed in an inactive form in connective tissue cells (Salo et al.,
1994). Of note, mRNAs encoding MMP-2 and MT1-MMP are spatially and temporally co-localized primarily in the wound stromal
fibroblasts at the time of peak activation of MMP-2 in the granulation tissue (Salo et al., 1994; Okada et al., 1997). In light of the cellmembrane-associated activation mechanism for proMMP-2
described earlier, it is quite likely that the induction of MT1-MMP
expression results in activation of MMP-2 in the granulation tissue cells. The stromelysins MMP-3 and MMP-11 are expressed by
subsets of fibroblasts in cutaneous wounds, with MMP-11 localizing specifically to areas of scar formation (Wolf et al., 1992;
Vaalamo et al., 1996). In murine wounds, the macrophage metalloelastase (MMP-12) is not expressed during early wound repair
but localizes abundantly, presumably in macrophages, at the
deeper layers of regenerated dermis after re-epithelialization is
completed (Madlener, 1998). In addition to MMPs, several other
proteases are induced during wound-healing and likely assist
MMPs in focalized proteolysis. For example, seprase, which
belongs to the family of integral membrane serine proteinases,
localizes at lamellipodia and filopodia in migrating fibroblasts
and participates in collagen degradation (Chen, 1996; Ghersi et al.,
submitted for publication). That seprase expression by fibroblasts
is up-regulated in the connective tissue in proximity to the
wound suggests that this enzyme may be involved in the regulation of fibroblast migration into the wound area where it could
participate in collagen matrix degradation (Ghersi et al., submitted for publication). Also, uPA, which participates in activation of
MMPs and degrades fibrin and fibronectin (Birkedal-Hansen et
al., 1993; Schafer et al., 1994), is central for wound-healing.
In the granulation tissue, two MMP inhibitors, TIMP-1 and
TIMP-3, localize mostly near newly forming blood vessels
(Vaalamo et al., 1999), where they may limit the proteolysis to
the perivascular matrix and regulate angiogenesis during neovascularization (Johnson et al., 1994; Anand-Apte et a., 1997). In
comparison, TIMP-2 is expressed by stromal cells under the
regenerating wound epithelium (Vaalamo et al., 1999). In this
area, TIMP-2 may serve to control those MMPs which are produced by epithelial cells, and to limit the proteolytic activity to
the pericellular matrix of the migratory epithelial wound cells.

(C) INTEGRINS INTERACT WITH SEVERAL

PROTEINASES DURING CELL MIGRATION
(1) Introduction
In culture, cell migration involves attachment of the cytoskeleton
to the ECM by integrins, extension of lamellipodia, and generation of the force that drives the cell forward (Regen and Horwitz,
1992; Sheetz, 1994; Palecek et al., 1996, 1998, 1999). The cell adhesion to the ECM mediated by integrins plays a major role in cell
migration. The strength of adhesion to matrix molecules may be
modulated by changes in the number of ligands available, the
number and/or affinity of integrins, or by modulation of the
integrin-binding to ECM proteins resulting from interaction with
other matrix or cell membrane molecules or by proteolysis
(Palecek et al., 1997; Lukashev and Werb, 1998). Whereas cell
migration occurs under conditions of intermediate strength

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adhesion to the matrix, too weak or too strong adhesion inhibits

migration in vitro (DiMilla et al., 1993). Therefore, fibroblast
migration into a wound requires quite a delicate balance among
the factors that control the strength of cell adhesion.
Localized proteolysis at the cell membranes is a mechanism
that, in all likelihood, is central to initiating and sustaining
migration, and to processing the ECM molecules during wound
repair. It is also most likely that limited proteolysis exposes
cryptic cell-binding sites on the ECM molecules needed for cell
migration. Yet, excessive proteolysis can be detrimental to cell
migration, because degradation of signaling sequences in ECM
proteins and of cellular receptors may disrupt cell-matrix interactions (Parks and Mecham, 1998). Therefore, the proteinases
that are localized to and activated at the contact areas of the
ECM molecules and the plasma membrane appear to be the
best candidate enzymes for regulating cell migration.
(2) Collaboration of integrins and plasmin
Regulation of plasmin activation is a good example of how
molecules involved in proteolysis of ECM molecules and localized to the cell surface may be used to regulate cell migration
during wound repair. It also demonstrates mechanisms for
integrin interaction with the molecules that are involved in the
focalized proteolysis. For example, integrins regulate the cellsurface localization of urokinase, an activator of plasminogen
(P1llanen et al., 1987; Seftor et al., 1992). This is evidenced by the
finding that, in cell culture, uPA, cell membrane uPA receptor
(uPAR), soluble plasminogen activator inhibitor-I (PAI-1),
otv13 integrin, and, possibly, also vitronectin form a multi-molecular complex. This complex regulates the interactions of the
cell with the ECM molecules (Planus et al., 1997). Recently, it
was shown that soluble PAI-I inhibits vascular smooth-muscle
cell migration by regulating the accessibility of specific integrin-binding sites in vitronectin (Stefansson and Lawrence,
1996). Interestingly, localization of uPA at cell adhesion sites
may initiate proteolysis of the ECM but rather, by removing
PAI-1 from the matrix, may expose cryptic cell-binding sites
needed for cell migration (Stefansson and Lawrence, 1996).
Because a variety of proteinases can interact with PAI-I and
remove it from the matrix (Levin and Santell, 1987; Pintucci et
al., 1993), it is quite possible that mechanisms like those
described above may be common to additional proteinases.
At focal adhesion sites, uPA-uPAR complexes can also
co-localize with other integrins, including the cx3, ox6, x5otv,
and the 131, 133, and 135 subunits. This association depends
on the composition of the ECM (Xue et al., 1997; Yebra et al.,
1999). That uPAR co-precipitates with the integrin 131 subunit from cell-membrane extracts indicates that uPAR physically associates with the 131 integrin subunit on the cell
membrane (Xue et al., 1997; Yebra et al., 1999). Ligation of
uPAR with uPA elicits activation of intracellular signaling
pathways that regulate gene expression (Yebra et al., 1999),
suggesting that uPAR may also modulate the signaling
events from integrins at the cell-matrix adhesion sites.
Results from studies using gene knock-out animals suggest that the molecules involved in plasmin activation are also
important for cell migration during wound repair in vivo. For
example, urokinase-deficient mice developed non-healing
wounds (Carmeliet and Collen, 1995) as a result of delayed
wound closure (Romer et al., 1996). This suggests that cell
migration into the wound is aberrant in the urokinase-deficient mice. The observation that serine proteinase inhibitors

prevent fibroblast migration into fibrin clots further supports

the role of plasminogen and its activators in controlling cell
migration. One should note that these proteinases are not
required for cell migration along collagen-rich matrices prior
to reaching the fibrin clot (Greiling and Clark, 1997). Thus, it
appears that there are distinct proteolytic mechanisms regulating cell migration in the intact connective tissue, which may
not involve serine proteases, and additional mechanisms operating in the fibrin clot involving serine proteases of the plasminogen activation system (see below).

(3) Interactions between integrins and MMPs

There is evidence that MMPs also regulate cell migration. In contrast to serine proteases, which operate mainly in cell migration
in the fibrin clot, MMPs may regulate migration in the connective tissue and in the fibrin clot. Like the molecules involved in
the regulation of plasmin activation, MMPs also operate in collaboration with integrins at the cell surface. For example, as discussed under "Activation of gelatinase A", proMMP-2 may form
an activation complex with TIMP-2 and MT1-MMP on cell surfaces. Since MT1-MMP and TIMP are spatially and temporally
co-expressed in the granulation tissue (Okada et al., 1997), the
component requirement for proMMP-2 activation is satisfied in
this stage of wound-healing (Strongin et al., 1995; Butler et al.,
1998). Interestingly, there is also an aggregation of 131 integrins at
the cell membrane during MMP-2 activation, although the significance of this observation is not yet clear (Ellerbroek et al.,
1999). Both MMP-2 and MT1-MMP have collagenolytic activities
and can degrade blood clot components such as vitronectin and
fibronectin (Imai et al., 1995; Pei and Weiss, 1996; Ohuchi et al.,
1997). Furthermore, MT1-MMP can degrade cross-linked fibrin
in the clot (Hiraoka et al., 1998). One may speculate that a coordination of 131 integrins and MT1-MMP and MMP-2 activity at
cell membranes mediates migration of cells from the collagenand fibronectin-rich connective tissue into the fibrin clot during
early granulation tissue formation. In fact, in various cell types,
MT1-MMP and MMP-2 localize at lamellipodia and invadopodia that are important for cell migration in a three-dimensional
matrix (Nakahara et al., 1997; Sato et al., 1997; Belien et al., 1999).
Interestingly, fibroblasts from MTI-MMP-deficient mice are
unable to degrade the collagen matrix even when they are stimulated by TNFo or IL-113 in vitro (Holmbeck et al., 1999). The
MT1-MMP knock-out mice also developed progressive fibrosis
of their claws. Therefore, MT1-MMP seems to be important in
development and connective tissue homeostasis. It will be interesting to learn the precise role of MT1-MMP from wound-healing studies in the MTI-MMP-deficient mouse.
An additional integrin-MMP interaction that may regulate enzyme activation and fibroblast migration into the granulation tissue involves direct binding between MMP-1 and the
ax21I integrin I-domain-an interaction that appears to be
independent of u2131 integrin binding to collagen (Dumin et
al., 1999). Both the ox2131 integrin and MMP-1 are expressed in
fibroblasts in connective tissue at the wound edge (Welch et al.,
1990; Stricklin et al., 1993, 1994; Gailit et al., 1996; Vaalamo et al.,
1996, 1997; Xu and Clark, 1996; Ravanti et al., 1999b). In early
phases of wound repair, MMP-1 could localize at cell adhesion
sites for collagen and there promote cell detachment from the
connective tissue matrix to initiate cell migration (Murphy
and Gavrilovic, 1999).
Focalized proteolysis of ECM molecules may regulate cell
migration. For example, by its proteolytic processing of collagen

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fibers, collagenase exposes cryptic RGD-dependent integrinbinding sites (Davis, 1992; Messent et al., 1998), which promote
cellular responses different from those of the native molecule.
Binding of the c 13 and ox231 integrins to native collagen is
mediated via a non-RGD site, whereas denaturation of collagen
exposes a cryptic RGD-site that can be recognized by x2f31 and
ox3r31 integrins in vascular smooth-muscle cell cultures
(Yamamoto and Yamamoto, 1994; Yamamoto et al., 1995; Xu and
Clark, 1996). Denaturation of collagen also exposes a cryptic
DGEA site that is recognized by the ox231 integrin expressed by
arterial smooth-muscle cells (Yamamoto et al., 1995). It is not yet
completely clear whether similar mechanisms regulate fibroblast-collagen interactions. It is possible that co-localization of
integrins and proteolytic enzymes, capable of exposing cryptic
binding sites in the ECM molecules, constitutes part of a mechanism to regulate cell migration during wound repair.

(D) CONTRIBUTIONS OF TENSIONAL FORCES

TO THE RE-ORGANIZATION OF GRANULATION TISSUE
(1) Introduction
Once fibroblasts reach the wound, they re-organize the provisional matrix by degrading the clot components and producing
a new collagen-rich matrix. Initially, this new matrix contains an
abundance of molecules. Among these are the large tenascin-C
isoform (Chuong and Chen, 1991; Hakkinen et al., 2000b) and
those fibronectin isoforms of the alternatively spliced EDA and
EDB modules, which are generally present at very low levels in
mature tissues (ffrench-Constant et al., 1989). At early stages of
healing, collagen is deposited in a random orientation.
Therefore, selective degradation, synthesis, and re-organization
of molecules present in the provisional matrix and granulation
tissue are required to normalize the tissue structure and composition. Such modifications of the structure and organization of
the ECM molecules by, e.g., proteolytic degradation can potently regulate cell behavior. Conversely, the structural change of the
ECM can regulate cellular production of matrix-degrading
enzymes through integrin-mediated signals. In this complex
process, integrin-mediated signaling is also controlled by growth
factors and by tensional forces in the tissues (Juliano, 1996; Shyy
and Chien, 1997; Rosenfeldt et al., 1998).
(2) Tensional forces and structural organization
of ECM regulate MMP expression
through aol,l and a2,B1 integrins
An important process component of connective tissue repair is
matrix re-organization, which involves cell-mediated contraction
of the wound matrix. During this process, fibroblasts that have
populated the granulation tissue, especially myofibroblasts, attach
to the newly deposited collagen by means of integrins. During
subsequent contraction, the collagen network is re-organized, and
the wound edges are approximated (Schiro et al., 1991). As the
wound tissue contracts, cells experience a change in the tensional
forces that can potently modulate protein synthesis. Experimentally induced tension in three-dimensional collagen-rich
matrices require specific integrin-collagen interactions to generate
the signaling which results in specific gene activation (Riikonen et
al., 1995a). For example, cells grown in three-dimensional collagen
gels attached to culture vessels experience an isometric tension
similar in magnitude to that in skin wounds (Kolodney and
Wysolmerski, 1992; Grinnell et al., 1999). In such mechanically
loaded collagen matrices, fibroblasts show increased rates of pro12(5):373-398
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tein synthesis. Upon gel detachment, which resembles the relaxation experienced by granulation tissue after contraction, the protein synthesis is reduced to normal levels (Mochitate et al., 1991).
Specifically, upon stress release, fibroblasts in collagen gels downregulate their expression of several ECM molecules, including
tenascin-C and types I and XII collagen, up-regulate MMP-1 and
MMP-13, and maintain fibronectin levels relatively unchanged
(Mauch et al., 1989; Chiquet-Ehrismann et al., 1994, 1996; Langholz
et al., 1995; Riikonen et al., 1995b; Ravanti et al., l999a,b; Trachslin
et al., 1999).

Apparently, changes in collagen and MMP gene expression

in response to matrix relaxation are regulated by specific collagen receptors. Thus, the reduced collagen synthesis after stress
relaxation is mediated by the cx13 integrin. In comparison, the
up-regulation of MMP-1 and -13 involves the ox2131 alone or the
o1131 together with (x2X1 integrin, respectively (Langholz et al.,
1995; Riikonen et al., 1995b; Ravanti et al., 1999a,b). Interestingly,
fibroblasts from oU-integrin knock-out animals had increased
expression of MMP-13 (Gardner et al., 1999) as well as MMP-2, 7, and -9 (Pozzi et al., 2000). This suggests that the or1I31 integrin
may play an important role in the regulation of several MMPs
during wound-healing.
Cell shape and cytoskeletal actin organization also affect
the MMPs, because disruption of actin with cytochalasin D
induces expression of MT1-MMP and activation of MMP-2 in
fibroblasts (Tomasek et al., 1997). It is noteworthy that fibroblasts are induced to generate activated MMP-2 only when cultured in three-dimensional collagen gels and not in monolayer
cultures (Seltzer et al., 1994). In this process, signals are mediated through u2p1 integrins but independent of collagen gel contraction (Seltzer et al., 1994). This regulatory pathway may be
cell-type-specific, since a human osteosarcoma cell line (Saos)
produces and activates MMP-2 in three-dimensional fibrillar
collagen matrices, in spite of not possessing the cu2p1 integrin
(Hakkinen et al., manuscript in preparation).

(3) Role of MMPs in wound contraction

and ECM re-organization
MMP inhibitors can prevent collagen gel contraction exerted by
dermal fibroblasts (Scott et al., 1998) and Saos cells (Hakkinen et
al., manuscript in preparation) in vitro, indicating that MMPs are
fundamental to the process. However, the specific MMPs
involved in collagen gel contraction have not yet been fully identified. For example, the broad-spectrum MMP inhibitor Batimastat inhibited contraction in Saos cells (Hakkinen et al., manuscript in preparation), whereas specific inhibitors for MIP-1
(Broberg and Heino, 1996) or MMP-2 (Halkkinen et al., manuscript
in preparation) did not prevent contraction. Additional evidence
suggesting that MMP-1 is not central to collagen gel contraction
in these cells is derived from the observation that tyrosine kinase
inhibitors, that also block contraction, do not affect MMP-1
expression (Broberg and Heino, 1996). Finally, the observation
that scar contraction was impaired and wound repair delayed in
MMP-3-deficient mice (Bullard et al., 1999) strongly implicates a
role for MMP-3 in wound contraction.

(E) REGULATION OF MMP EXPRESSION

IN FIBROBLASTS BY THE COMPOSITION OF THE ECM
In addition to the three-dimensional organization of the
matrix, the molecular composition of the ECM affects MMP
expression by wound fibroblasts. This regulation proceeds
probably in two ways: (1) through direct induction of MMP
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387

gene expression by modulation of cell-ECM interactions, or (2)

by an indirect mechanism where cell-ECM interactions induce
expression of secreted intermediate molecules that regulate
MMP expression.
Wounding induces deposition of the large tenascin-C isoform in the granulation tissue (Chuong and Chen, 1991;
Hakkinen et al., 2000b). This large isoform is otherwise expressed
during embryonic development and mediates cellular functions
different from those mediated by the small isoform, which is primarily expressed in adult tissues (Matsuoka et al., 1990; Prieto et
al., 1990; Borsi et al., 1992). The large tenascin-C isoform is also
more susceptible to proteolytic cleavage than the small isoform.
It can be degraded by the MMPs -2, -3, and -7 (Siri et al., 1995).
When present in the matrix concurrently with fibronectin,
tenascin-C can induce expression of several MMPs, including
MMP-1, stromelysin, and MMP-9 by synovial fibroblasts, possibly as a result of signaling by integrins (Tremble et al., 1994). On
the contrary, MMPs may indirectly up-regulate tenascin-C
expression. If vascular smooth-muscle cells are cultured on collagen matrices that are under tension, cellular MMPs degrade
the matrix and thereby generate ligands for cxvI3 integrins.
Subsequently, avr3 interaction with tenascin-C alters cell shape,
increases epidermal growth factor (EGF) receptor clustering, and
enhances EGF-dependent growth. In comparison, if these cells
are cultured on relaxed collagen matrices, their MMP activity
and tenascin-C synthesis are suppressed, and they undergo
apoptosis in the presence of EGF (Jones et al., 1997).
Also, SPARC (osteonectin/BM40), a secreted protein that
modifies interactions between cells and ECM, is expressed in the
granulation tissue (Reed et al., 1993; Motamed, 1999). SPARC
promotes production of MMP-1, MMP-3, and MMP-9 when
present in the matrix along with collagens or vitronectin
(Tremble et al., 1993). This MMP regulation is, in part, a result of
direct induction of MMP gene expression by interactions of cells
with ECM containing SPARC, and in part caused by induction
of cellular production of a secreted intermediate, presumably a
cytokine (Tremble et al., 1993).

(F) PROTEOLYTIC DEGRADATION OF ECM

MOLECULES REGULATES MMP EXPRESSION
THROUGH INTEGRIN-MEDIATED SIGNALING
As discussed above under the epithelial aspects of wound-healing, laminin cleavage alters behavior of cells in contact with the
proteolytically processed laminin as compared with intact
laminin. Likewise, proteolytic fragmentation of fibronectin may
result from proteolysis by such MMPs as MMP1, -2, and -3, or
by elastase (Wysocki and Grinnell, 1990; Grinnell and Zhu, 1994;
Fukai et al., 1995). The resulting peptides regulate several cellular functions, including cell proliferation, chemotaxis (Kapila et
al., 1998), migration (Schor et al., 1999), apoptosis (Kapila et al.,
1999), and MMP expression in a manner that differs from that of
the full-length native molecule. For example, in synovial fibroblasts, fibronectin fragments, but not intact fibronectin, can
induce MMP-1 and MMP-3 expression by signaling through the
oa5il1 integrin (Werb et al., 1989; Damsky et al., 1992; Tremble et
al., 1992, 1995). Likewise, the central 120-kDa segment of
fibronectin, which also includes the RGD-containing cell-binding site, has significant MMP-inductive capacity. MMP expression is also stimulated by cell interactions with the V peptide
from the heparin-binding region of fibronectin (Kapila et al.,
1996), the NH2-terminal, or the collagen-binding domains
(Homandberg et al., 1992; Xie et al., 1994). In contrast to these

388

stimulatory effects, the peptide corresponding to the CS-1 region

suppresses MMP expression through interaction with the (A131
integrin (Huhtala et al., 1995).
Human periodontal ligament fibroblasts constitutively
express MMP-2, uPA, and TIMPs and are induced also to produce MMP-9 when exposed to intact fibronectin. Furthermore,
contact interaction of these periodontal cells with the 120-kDa
segment of fibronectin leads to the expression of MMP-1 and
stromelysin and to increased levels of uPA (Kapila et al., 1996).
Importantly, when spliced out from the intact fibronectin molecule, the alternatively spliced EDA fragment induces MMP-1,
MMP-3, and MMP-9 expression in synovial fibroblasts (Saito et
al., 1999). Contrary to the direct integrin-mediated mechanisms
of MMP expression induced by the 120-kDa and CS-1 fragments, the inductive effects of the EDA fragment are mediated
by an autocrine mechanism involving increased cellular production of IL-1, which in turn up-regulates MMP expression
(Saito et al., 1999). Thus, different segments of fibronectin have
the capacity to regulate MMP expression by fibroblasts and
potentially other cells of the connective tissue matrix differentially and to modulate cell behavior involved in cell migration
and matrix remodeling.

(G) DOES MMP-13 HAVE A SPECIFIC ROLE

IN ORAL MUCOSAL WOUND-HEALING?
Human gingival fibroblasts express MMP-13 if cultured on
two-dimensional collagen matrices (Ravanti et al., 1999b). This
is in contrast to observations for human skin fibroblasts that
express MMP-13 only if they are placed in three-dimensional
collagen gels (Ravanti et al., 1999a). The response to growth
factors also differs between these fibroblasts of dermal and
gingival origin. TGF-P3 significantly up-regulates MMP-13
expression in gingival fibroblasts but decreases this enzyme in
dermal fibroblasts (Ravanti et al., 1999a). In human gingival
wounds, MMP-13 is expressed by fibroblasts at the wound
edge during the early repair phase and by granulation tissue
fibroblasts during tissue remodeling (Ravanti et al., 1999b). In
contrast to gingival wounds, MMP-13 is not expressed in normally healing skin wounds (Vaalamo et al., 1997). These examples present strong evidence that MMP-13 expression is differentially regulated in skin and gingival fibroblasts both in
vivo and in vitro. Because MMP-13 has a wide substrate specificity (Freije et al., 1994; Fosang et al., 1996; Knauper et al.,
1996a, 1997a; Mitchell et al., 1996), this MMP may play an
important role in the matrix re-organization during gingival
wound repair. Ravanti et al. (1999b) have suggested that MMP13 is a factor in preventing scar formation because scarring is
minimal in gingival as compared with dermal wounds
(Sciubba et al., 1978; Yang et al., 1996).

(H) ROLE OF MMPS IN THE RELEASE

OF ECM-BOUND AND CELL-ASSOCIATED
GROWTH FACTORS
The ECM and basement membranes contain inactive growth
factors bound to the matrix molecules. When wounding occurs,
these ECM-bound growth factors may be released from the
matrix by proteinases and, subsequently, activated proteolytically or by other mechanisms (Taipale and Keski-Oja, 1997).
Proteases can also cleave growth factor receptors on cell surfaces
(Levi et al., 1996). These processes provide important mechanisms for spatial and temporal control of the release and activation of growth factors required for successful wound repair. For
example, during wound healing, TGF-[ is secreted in an inactive

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form complexed with the latency-associated peptide (LAP) and

latent TGF-13 binding protein (LTBP). LTBP mediates the binding
of latent TGF-13-complex to matrix molecules. This phase of production and storage of LTBP-containing latent complex is an initial stage in regulating the bioavailability of TGF-3 in tissues
(Taipale and Keski-Oja, 1997; Sinha et al., 1998). For example, in
cell culture, latent TGF-f co-localizes on the pericellular
fibronectin-rich fibrillar structures (Taipale et al., 1996), while in
connective tissue and regenerating skin, TGF-,B seems to localize
at epithelial basement membranes and elastin-associated
microfibrils (Roberts and Sporn, 1996; Raghunath et al., 1998).
One of the mechanisms for TGF-3 activation involves,
first, the release of the matrix-bound latent complex and, subsequently, the activation of the released growth factor by serine proteinases, including plasmin (Flaumenhaft and Rifkin,
1991; Munger et al., 1997). The activation of TGF-3 most likely
occurs at the cell membrane close to the cell-surface receptors
that bind the active TGF-1 (Munger et al., 1997). In addition to
binding to its cell-surface signaling receptor, the released,
active TGF-3 may also be sequestered and thereby inactivated
by binding to some ECM molecules, including type IV collagen, fibronectin, thrombospondin, heparan sulphate proteoglycan, decorin, or biglycan (McCaffrey et al., 1992; Ruoslahti
et al., 1992; Shultz-Cherry and Murphy-Ullrich, 1993).
Another growth factor, fibroblast growth factor-2 (FGF-2),
that associates with cell-surface or basement-membrane heparan
sulphate proteoglycans can be released in a biologically active
form by proteinases like plasmin and thrombin and by heparin
(Flaumenhaft and Rifkin, 1992; Benezra et al., 1993).
Alternatively, cleavage of the ectodomain of fibroblast growth
factor receptor 1 (FGFR-1) by MMP-2 releases the entire extracellular domain of this receptor, which can then bind and sequester
fibroblast growth factor (FGF) in the tissue. Therefore, solubilization of FGFR-1 by MMP-2 may contribute to modulating the
angiogenic and mitogenic activities of FGF (Levi et al., 1996).
Platelet-derived growth factor (PDGF), which is important
for proliferation and migration of fibroblasts, is also
sequestered on basement-membrane proteins or SPARC in the
connective tissue (Raines et al., 1992; Eming et al., 1999) but utilizes a different mechanism for release-namely, the association
of PDGF with SPARC is sensitive to pH changes, and PDGF
may be released as a result of lowered pH that occurs during
wound-healing (Raines et al., 1992; Eming et al., 1999).
In addition to FGF-2, MMPs can be involved in the release
and activation of other growth factors. For example, MMP-1, -2,
and -3 have the capacity to cleave insulin-like growth-factorbinding protein-3 and by this mechanism to control the bioavailability of insulin-like growth factor (Fowlkes et al., 1994).
Additionally, MMP-2, -3, or -7 can release decorin-bound TGF13 in vitro and thereby provide a putative mechanism to increase
the bioavailability of this growth factor during wound-healing
(Imai et al., 1997). These brief examples show that controlled and
localized release and activation of growth factors and release of
functional growth factor receptor ectodomains by proteinases
contribute to the mechanisms by which cells can rapidly target
growth factor activity to their cellular microenvironment.

(VI) Conclusions
To migrate in healing wounds, cells require mechanisms that
tightly control their adhesion to matrix components. Whereas
integrins provide important tools for cell attachment, MMPs
facilitate detachment of cells from the matrix. In this process,
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MMPs are transported to the matrix adhesion sites, where they

are then activated and locally degrade the matrix molecules. As
a result of this focalized proteolysis, cells are released for locomotion. Upon proteolytic fragmentation, matrix molecules are
recognized by the same or other integrins and provide signals
that may vary from those of the intact molecules and further
stimulate specific MMP expression. The proteolytic process
may also expose cryptic binding sites on the matrix molecules
for interaction with additional integrins, thereby inducing signaling events that are different from those occurring via the
integrins that originally positioned the cell in the non-cleaved
matrix. Therefore, cleavage of matrix molecules by MMPs has
the potential to induce migration or, alternatively, to reverse the
function of the matrix molecules from one of stimulating
migration to one of supporting stable anchorage. During
wound-healing, proteolytic activity may release growth factors
that are stored in the extracellular matrix. Therefore, the cell
coordinates signals from integrins involved in cell adhesion,
from growth factor receptors, and from integrin co-receptors. In
normal wounds, the expression of MMPs and TIMPs is wellbalanced, but there is evidence that an imbalance in favor of
proteolysis contributes to the pathogenesis of poorly healing
wounds. Therefore, an understanding of the molecular mechanisms of focalized MMPs activity is critical for deciphering
integrin-mediated cell behavior during wound-healing. Future
studies are likely to uncover novel mechanisms that will further elucidate the signaling pathways which control the assembly of integrins and MMP activities at the interfaces between
cells and extracellular matrix molecules.

Acknowledgments
The research and manuscript development were supported
by grants from the Medical Research Council of Canada to
Drs. Hannu Larjava and Lari Hakkinen, and by NIH grant
DE12818 and a South Texas Health Research Council Grant to
Dr. Bjorn Steffensen.

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