37

Critical Reviews in Oral Biology and Medicine, 4(2): 197-250 (1993)
Matrix Metalloproteinases: A Review*

H. Birkedal-Hansen,1'2 W. G. I. Moore,1 M. K. Bodden,36 L J. Windsor/
B. Birkedal-Hansen,4 A. DeCarlo,57 J. A. Engler27
Department of Oral Biology,1 Research Center In Oral Biology,2 Department of Restorative Dentistry,3
Department of Diagnostic Sciences,4 Department of Periodontics, University of Alabama School
of Dentistry,5 Department of Pathology,6 and Department of Biochemistry,7 University of Alabama
at Birmingham, Birmingham, Alabama
* Address all correspondence to: Dr. Henning Birkedal-Hansen, Department of Oral Biology, SDB Box 54,
University of Alabama School of Dentistry, University of Alabama at Birmingham, Birmingham,
Alabama 35294, (205) 934-6154.
ABSTRACT: Matrix metalloproteinases (MMPs) are a family of nine or more highly homologous Zn++endopeptidases that collectively cleave most if not all of the constituents of the extracellular matrix. The present
review discusses in detail the primary structures and the overlapping yet distinct substrate specificities of MMPs
as well as the mode of activation of the unique MMP precursors. The regulation of MMP activity at the
transcriptional level and at the extracellular level (precursor activation, inhibition of activated, mature enzymes)
is also discussed. A final segment of the review details the current knowledge of the involvement of MMP in
specific developmental or pathological conditions, including human periodontal diseases.
KEY WORDS: matrix metalloproteinases, extracellular matrix, collagenase, regulation of tissue destruction,
human periodontal diseases.
I. METABOLIC DEGRADATION OF THE

EXTRACELLULAR MATRIX*
At present it is possible to identify elements
of four to five distinct pathways for degradation
of extracellular matrices as summarized in Table
1. Structural macromolecules of interstitial connective tissues and basement membranes may be
degraded by matrix metalloproteinase (MMP)dependent, by plasmin (Pln)-dependent, and by
polymorphonuclear (PMN) leukocyte serine proteinase-dependent reactions, and in some cases
by an apparently distinct phagocytic pathway
based on intracellular digestion of internalized
material by lysosomal cathepsins. Mineralized
matrices are degraded by an entirely different
mechanism based on release of acidic thiol proteinases to a sealed microenvironment on the

matrix surface (osteoclastic pathway). This review focuses specifically on matrix metalloproteinases and their role in the metabolic degradation of the extracellular matrix in health and
disease and will only briefly summarize other
pathways.
At the outset it is important to recognize that
molecular âssembly of matrix macromolecules
follows entirely different pathways than molecular assembly. Consequently, the assembly process in itself is of little help in understanding how
the dissolution of tissue structures is brought about.
On the other hand, a certain level of insight into
the structure of natural fibril systems is necessary
Abbreviations used throughout the text are listed after the Acknowledgments section and before the reference list (page
231).
1045-4411/93/150
1993 by CRC Press, Inc.
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197
TABLE 1
Pathways for Metabolic Degradation of Extracellular Matrix
Pathway
Tissue degraded
Effector enzymes
Cellular location
Pig-dependent
pathway
Interstitial connective tissues,

basement membranes
Pin
Peri/extracellular
MMP pathway
Interstitial connective tissues,

basement membranes
MMPs
Peri/extracellular
PMN serlne proteinase

pathway
Insterstitial connective tissue,

basement membranes
PMN elastase, cathepsin G
Peri/extracellular
Phagocytic pathway
Insterstitial connective tissue
Cathepsins
Intraceliular
Osteoclastic pathway
Bone, cementum, dentin
Cathepsins
Peri/extracellular
to begin to understand those factors that dictate

whether a particular macromolecule is degraded
by one pathway or another. Proteolytic attack on
solid-phase matrix structures requires an initial
extracellular step to initiate the fragmentation
process. Because extracellular proteolysis proceeds
in an environment with considerable molar excess of either plasma or cellular proteinase inhibitors, it is necessary to compartmentalize the process, that is, to isolate the area to be destroyed and
to selectively transport and retain reactants at the
site of action. The importance of compartmentalization is perhaps most clearly seen in the case of
the osteoclast that seals off the bone surface to be
degraded and creates a transiently closed microenvironment that can be fully controlled (Vaes,
1988). However, vectorial transport is not limited
to the osteoclast, and it is reasonable to assume
that most cells can selectively direct the movement of reactants (Unemori et aL, 1990). This
effect is reinforced by mechanisms to retain the
enzymes at the site of action based on specific
binding affinities either for the substrate surface
(binding of tissue-type plasminogen activator
[t-Pa] and plasminogen to fibrin; binding of
gelatinases to collagen chain fragments) or for the
cell surface through specific receptor-ligand interactions (binding of urokinase-type plasminogen activator (u-Pa) and plasmin to cell surface
receptors) (Plow etai, 1986; Vasalli etaU 1985).
II. ALTERNATE PATHWAYS

A. Pig-Dependent Pathway
A body of evidence suggests that the Pindependent pathway plays an important role in the
remodeling of the extracellular matrix in cell

migration, trophoblast and tumor cell invasion,
metastasis, embryonic development, and growth.
A number of recent reviews have summarized the
chemistry and biology of this pathway (Dan0 et
al, 1985; Kruithof, 1988; Lijnen and Collen, 1988;
Moscatelli and Rifkin, 1988; Saksela and Rifkin,
1988; Takada and Takada 1988; Fears, 1989;
Gerard and Meidell, 1989; Laiho and Keski-Oja,
1989; Andreasen et al., 1990; Testa and Quigley,
1990).
Dissolution of susceptible extracellular matrix proteins is mediated by cleavage by plasmin, a broad-spectrum serine proteinase that is
converted from its inactive circulating precursor form, plasminogen (Pig), to catalytically
active form by specific activating enzymes, Pig
activators. The concentration of Pig in plasma,
interstitial fluids, and lymph is in the range of
100 to 200 |ig/ml (1 to 2 \iM)9 which represents
a phenomenal destructive potential. By comparison, our best estimates suggest that tissue
concentrations of MMPs are at least 10- to 100fold lower. The circulating Mr 92,000 singlechain Glu-Plg is activated by two cleavages
that result in truncation of the molecule to yield
a Mr 86,000 species composed of a Mr 63,000
heavy-chain disulfide bonded to a Mr 25,000
light chain. The activated enzyme (Pin) cleaves
Lys and Arg peptide bonds exposed on the surface of a wide range of native protein substrates, although at highly varying rates. Fibrin
and fibronectin are cleaved rapidly at multiple
sites, whereas type I and II collagens are not
cleaved at all. In general terms, the Pin-dependent pathway appears to mediate the degradation of provisional and rapidly remodeling
matrices but to leave intact the more slowly
198
remodeling scaffold structures, such as the type

I and II collagen fibrils.
Like other serine proteinases of the fibrinolytic, coagulation, and complement cascades,
Pig has evolved from a primordial prototype
trypsin-like proteinase by addition of regulatory
domains. The five so-called "kringle domains"
that encode the affinity for Lys residues are
thought to help retain the proteinase at the substrate surface by binding to COOH-terminal Lys
residues generated by cleavage of Lys-X bonds.
The Pig activating enzymes, u-Pa and t-Pa, are
also serine proteinases that have evolved by the
addition of regulatory domains to a prototype
trypsin-like proteinase domain. U-Pa (Mr 55,000)
contains an epidermal growth factor (EGF)-like
domain and a kringle domain, whereas t-Pa (Mr
70,000) lacks the EGF-like domain but contains
two kringles.
Pin-dependent proteolysis is initiated by secretion of one or both activating enzymes at
local tissue sites. Most cell types, including those
that dominate the human periodontal tissues (fibroblasts, keratinocytes, endothelial cells, PMN
leukocytes), may be induced to express one or
both activating enzymes. The activity of Pin and
of the activating enzymes is maximized by binding to the substrate (Pig and t-Pa) or the cell
surface (u-Pa and Pig) (recently reviewed by
Vaheri et ai, 1990). This arrangement serves to
concentrate the reactants at the site of cleavage
and under certain circumstances to protect against
capture by plasma [a-2-macroglobulin (a2M),
ocl-antiproteinase, oc2-antiplasmin] and cellular
[plasminogen activator inhibitor-1 (PAI-1), and
-2 (PAI-2), protease nexin-1 (PN-1)] inhibitors.
U-Pa is secreted as a latent precursor that is
activated outside the cell, presumably by limited
proteolysis, by conversion of the single-chain
precursor to a two-chain form. Pin can mediate
this conversion in vitro, but it is uncertain how
pro-u-PA and Pig interact to initiate the activation reaction when both are in the precursor
form.
B. Neutrophil Serine Proteinases

A body of evidence suggests that PMN leukocytes may mediate the degradation of extracel-
lular matrix macromolecules by release of two

granule serine proteinases, neutrophil elastase and
cathepsin G. These proteinases are capable of
cleaving a variety of extracellular matrix proteins
including type IV collagen (but not type V collagen), laminin, fibronectin, and heparan sulfate
and cartilage proteoglycans (Weiss, 1989; Jasin
and Taurog, 1991; Heck et al, 1990; Janusz and
Dogerty, 1991).
C. Phagocytic Pathway
The preponderance of evidence for degradation of extracellular matrix by a phagocytic pathway (reviewed by Melcher and Chan, 1981)
comes from ultrastructural studies that have unequivocally shown fragments of cross-striated
collagen fibrils completely enclosed inside cells
and in some cases associated with lysosomal
enzymes (Deporter and Ten Cate 1973, Garant
1976, Schellens et al, 1982). Although it has
been possible to replicate elements of this process in vitro (Yajima and Rose, 1977; Svoboda
et al\ 1979), it has not yet been possible to
clearly define the component molecular reactions. Selective inhibition studies suggest that
lysosomal cathepsins but not MMPs, are involved
in the process (Everts et a/., 1985, 1989, 1990).
At present it is difficult to reconcile the extracellular and intracellular pathways, but it is possible that fragments of fibrils may be excised
perhaps by a collagenase-dependent reaction and
that these fragments are subsequently internalized by the cell for digestion in phagolysosomes
mainly by thiol-proteinases of the cathepsin family. Phagocytic collagen degradation is particularly prevalent in areas of rapid collagen turnover such as the involuting uterus (Parakkal,
1969) and the periodontal ligament (Melcher
and Chan, 1981; Schellens et al, 1982). The
phenomenon has been identified in human and
animal periodontal ligaments in vivo (Listgarten,
1973; Melcher and Chan, 1981; Beertsen et al,
1978; Schellens et al, 1979), in healing wounds
(McGaw and Ten Cate, 1983), and in several
cultured cells including epithelial cells (Birek et
al, 1980), fibroblasts (Svoboda et al, 1979),
and macrophages (Parakkal, 1969; Deporter,
1979).
199
D. Osteoclastic Bone Resorption

The enzymatic mechanisms by which the
organic matrix of bones and teeth is removed
are still incompletely understood, but a body of
evidence recently reviewed by Vaes and collaborators (Vaes, 1988; Vaes et aL, 1992) suggests that both MMPs and lysosomal thiol-proteinases are involved. Osteoblasts respond to
parathyroid hormone and other resorption-inducing stimuli by expression of a typical interstitial collagenase (Civitelli et aL, 1989; Heath
et aL, 1985; Delaisse et aL, 1988), yet collagenase has no proteolytic activity under the acidic
conditions generated by the osteoclast. This and
other findings have led to the hypothesis that
osteoblasts initiate the resorptive process by
dissolution of the layer of osteoid using a collagenase-dependent (neutral) proteolytic process and thereby expose the underlying mineralized bone surface. Osteoclast precursors attracted perhaps by signals emitted by the vacating osteoblasts or released from the dissolving
bone surface populate the denuded mineral surface and differentiate. The microenvironment
below the osteoclast is sealed off, and the undulating membrane promotes the mixing of ingredients released on the bone surfaces. Because of the enormous capacity for acidification that permits a single adherent osteoclast to
lower pH below its plasma membrane from 7.0
to 3.0 in 6 min (Silver et aL, 1988), it is commonly believed that the mineral is disposed of
by a simple acidic dissolution process. It is
envisioned that acidic thiol-proteinase(s) released by the osteoclast are capable of dissolving the collagenous matrix at the low pH. A
promising candidate, a cathepsin B-like enzyme
from chicken osteoclasts, has been isolated and
sequenced (Blair et aL, 1986; Blair, personal
communication). The detailed enzymatic action of this and other thiol proteinases against
the irreversibly cross-linked collagenous matrix of bone and dentin is not yet well understood. Studies by Etherington (1977) and
Etherington and Birkedal-Hansen (1987) have
shown that the collagenous network of bone is
somewhat susceptible to cleavage by cathepsins at acidic pH, particularly in high Ca2+concentrations.
200
III. MATRIX METALLOPROTEINASE

PATHWAY
For recent reviews, the reader is referred to
Birkedal-Hansen (1987), Tryggvason et aL (1987),
Sakamoto and Sakamoto (1988), Vaes (1988),
Weiss (1989), Emonard and Grimaud (1990),
Goldberg et aL (1990), Matrisian (1990), Jeffrey
(1991), and Woessner (1991). A comprehensive
review of the field including a complete bibliography of the MMP literature through 1990 compiled by Woessner (1992) was published recently
(Birkedal-Hansen et aL, 1992a).
The MMP gene family encodes nine or more
metal-dependent endopeptidases with activity
against most if not all extracellular matrix macromolecules (Table 2). Eight human MMPs have
been cloned and sequenced, and the proteolytic
activity of the natural or recombinant forms verified for all but one, stromelysin-3 (SL-3) (MMP11). Recently, a novel MMP, a metalloelastase
from murine macrophages, has been added to the
list (Shapiro et aL, 1992). The enzymes share a
number of common structural and functional features but differ somewhat in terms of substrate
specificity. For instance, the ability to cleave fibrils
of types I and II collagens, which is characteristic
for the PMN-type and fibroblast-type collagenases (PMN-CL, FIB-CL), is not shared by other
members of the family. The physiologic basis for
this difference, however, is not well understood,
nor is the apparent redundancy of evolution of
nine enzymes with overlapping substrate specificities.
A. Modular Structure of MMPs

The nine members of the MMP family include (1) interstitial collagenases (FIB-CL and
PMN-CL), (2) stromelysins (SLs) (SL-1 and
SL-2), (3) gelatinases (Mr 72,000 gelatinases/type
III collagenase [Mr 72K GL] and Mr 92,000
gelatinase/type IV collagenase [Mr 92K GL]),
and (4) other MMPs (putative metalloproteinase
[PUMP-1], SL-3, macrophage metalloelastase
[MME]). The amino acid sequences of 16 human
and animal (rat, mouse, rabbit, pig) MMPs deduced from cDNA data are shown in Appendix 1.
The enzymes may be regarded as derivatives of
TABLE 2
Matrix Metalloproteinase Family
Enzyme
Abbreviation
Fibroblast-type
collagenase
FIB-CL
PMN-type
coliagenase
PMN-CL
Stromelysin-1
SL-1
MMP#
Mr
Extracellular matrix substrates
MMP-1
57,000/
52,000
Collagen I, II, III, ( l l l l ) , VII, VIII, X;

gelatin; PG core protein
MMP-8
75,000
Same as FIB-CL ( l l l l )
MMP-3
60,000
55,000
PG core protein; fibronectin;

laminin; collagen IV, V, IX, X;
elastin; proCL
Stromelysin-2
SL-2
MMP-10
60,000/
55,000
Same as SL-1
Stromelysin-3
SL-3
MMP-11
n.d.
n.d.
Macrophage metalloelastase
MME
53,000
Elastin
Mr 72K gelatinase
type IV collagenase
Mr 72K GL
MMP-2
72,000
Gelatin; collagen IV, V, VII, X, XI; elastin;

fibronectin; PG core protein
Mr 92K gelatinase/
type IV collagenase
Mr 92K GL
MMP-9
92,000
Gelatin; collagen IV, V; elastin; PG core

protein
Putative metalloproteinase-1
PUMP-1
MMP-7
28,000
Fibronectin, laminin, collagen IV, gelatin,

proCL, PG core protein
Note: n.d.: not determined.

MMP numbering according to Nagase, H. A. J. Barrett and J. F. Woessner, Jr. et aL: Matrix. Spec. Supp. No.
1:421-424 (1992).
the five-domain modular structure characteristic

of collagenases and SLs formed either by addition
or deletion of domains (Figure 1). The 17-29
residue hydrophobic signal sequence is followed
by a 77-87 residue propeptide that constitutes the
NH7-terminal domain of the secreted MMP precursor, a catalytic domain that contains the catalytic machinery including the Zn2+-binding site,
and a 5-50 residue proline-rich hinge region that
marks the transition to the -200 residue
hemopexin- or vitronectin-like COOH-terminal
domain that appears to play a role in encoding
substrate specificity. PUMP-1 does not possess
this last domain and therefore is considerably
smaller than the other members of the family. The
two gelatinases contain a single insert in the catalytic domain consisting of three tandem repeats of
fibronectin type II modules that endow the active
and latent enzymes with gelatin-binding properties (Goldberg et a/., 1989; Collier et aL, 1992).
Each of the enzymes contains a putative tridentate
Zn2+-binding site believed to constitute the active
site and to play a role in maintenance of catalytic
latency of the precursor form (Springman et aL,
1990, Van Wart and Birkedal-Hansen, 1990).

Although no MMP crystal structures are available, a body of evidence based on homology with
other Zn2+-binding enzymes suggests that the His
residues in the HEXGH sequence constitute two
of the ligands of the Zn2+-binding site (Vallee and
Auld, 1992). It is likely that the third ligand that
completes the HEXGHXXGXXH sequence is the
highly conserved His located six residues downstream (Appendix 1). A fourth ligand site is presumably occupied either by H2O (in the mature
active enzyme) or by a single unpaired Cys residue in the propeptide (in the latent precursor). All
MMPs contain a highly conserved Glu- and Asprich region between the Zn2+-binding site and the
hinge region that is likely to constitute a Ca2+binding site (Lepage and Gache, 1990). Another
Glu/Asp-rich region, again a potential Ca2+-binding site, is found in front of the fibronectin type II
domain inserts (Table 3). The COOH-terminal
domain consists of four repeats (Appendix 1) that
share some limited sequence homology with
modules also found in hemopexin and in
vitronectin (Matrisian et al. 1986b; Jenne and
201
PROTOTYPE
Fibronectin type II
Domain Inserts
MMP-9
Mr92KGL
MMP-2
Mr 72K GL
MMP-1
FIB-CL
MMP-8
PMN-CL
MMP-3
SL-1
MMP-10
a :: I II
I 11
MMP-11
MMP-?
MME
Domain structure of MMPs.
Stanley, 1987). The four tandem modules are held

together by a single disulfide bond composed of
two Cys residues that flank the hemopexin-like
domain (Figure 1; Appendix 1).
B. MMP Gene Structure

MMP genes show a highly conserved modular structure (Figure 2). The CL, SL-1, and SL-2
genes each contain ten exons and nine introns in
8 to 12 kbp of DNA (Matrisian et al., 1985;
Breathnach et al, 1987; Fini et al, 1987; Collier
et al., 1988). Based on cDNA sequences, it may
202
SL-3
PUMP-1
MMP-7
FIGURE 1.
SL-2
be surmised that PUMP-1 lacks exons 7-10, which

encode the hemopexin-like domain, as well as all
or most of exon 6, which encodes the hinge region. Mr 72K GL and Mr 92K GL genes are
considerably larger (26-27 kbp) and contain three
additional exons, which encode the three
fibronectin type II domains (Figure 2). The extended hinge region of M r 92K GL is encoded
entirely in exon 6 (Huhtala et al, 1990a; 1991).
The CL and SL-1 genes are located on the long
arm of chromosome 11 (Spurr et al., 1988); the
Mr 72K GL gene is located on chromosome 16
(Huhtala et al, 1990b).
TABLE 3
Asp- and Glu-Rich, Putative Ca2+-Binding Sequences
Sequence
Protein
DE H E R W T N N F T E
DD D E Q W T K D T T G
H-FIB-CL; site 1
H-SL-1
QD D 1 D G 1 Q A 1 Y G
QD D 1 N G 1 Q S L Y G
DS D D D G D D 1 T T 1
H-FIB-CL; site 2
H-SL-1
Sea urchin hatching
enzyme
EF
DV
DR
DL
DL
EK
DA
DK
N1
DD
DQ
DD~
DA
DD
S
N
T
D
D
D
D
D
Q
D
N
D
D
D
D
G
G
P
Q
G
G
G
G
G
N
R
D
_
D
D
D
D
N
N
N
N
D
D
D
D
D
1
D
G
G
G
G
G
G
G
C
G
G
G
G
T
L
R
Y
Y
T
T
Y
E
1
1
-
E
D
P
N
P
1
1
1
V
P
1
1
1
D
D
Q
D
D
T
D
S
N
D
D
D
-
F
L
E
V
L
T
F
A
Y
D
K
-
V
L
V
A
1
K
P
A
E
A
V
G
1
V
E
E
E
E
- D
D D
D E
T D D
FN-R 1
FN-R 2
FN-R 3
FN-R 4
FN-R 5
Calmodulin 1
Calmodulin 2
Calmodulin 3
Calmodulin 4
Thrombospondin
Myosin light chain
Troponin C consensus
Parvalbumin consensus
Uvomorulin Repeat B1
Lactalbumin
Human CL and SL-1 sequences are from Appendix 1; sequences for

fibronectin receptor (FN-R) sites 1-5, myosin light chain, troponin C,
calmodulin, and parvalbumin compiled by Rouslahti, E.: Am. Rev.
Biochem. 57:375-413 (1988); sequences for sea urchin hatching
enzyme, thrombospondin, human lactalbumin, and uvomorulin repeat
B1 compiled by Lepage T. and C. Gache EMBO J. 9:3993-3012
(1990); calmodulin sequences are from Cheung, W. Y.: Fed. Proc.
41:2253-2257 (1982).
Catalytic Domain
Propeptide
FN-type II Inserts
Hinge Region
Pexin-like domain
HUMAN Mr 92K GL
L_13_
HUMAN Mr 72K GL
10
HUMAN FIB-CL
10
RAT SL-1
FIGURE 2.
Exon structure of human FIB-CL, Mr 72K GL, and Mr 92K GL and rat SL-1 (Redrawn from Huhtala,
P., A. Tuuttila, L. T. Chou, J. Lohi, J. Keski-Oja and K. Tryggvason: J. Biol. Chem. 266:16485-16490 (1991). With
permission.)
203
C. MMP Substrate Specificity

Substrate specificity studies have provided
information about the catalytic properties of the
enzymes and in a few instances yielded clues to
biologic function that may help us answer two
connected questions: what are the natural substrates for individual MMP and how are specific
MMPs degraded in the tissues? On both protein
and peptide substrates, the MMPs have somewhat
overlapping substrate specificities (Table 2; Tables
7 to 11). For instance, virtually all of the enzymes
cleave gelatin and fibronectin at some rate, and
most cleave type IV and V collagens at sufficiently high temperatures. On the other hand, the
unique ability of FTB-CLs and PMN-CLs to cleave
interstitial collagens is not shared by other members of the family and suggests that this property
mirrors biologic function. MMPs cleave a wide
range of largely hydrophobic bonds in native proteins as detailed under the description of the individual enzymes (Tables 7 to 11). Synthetic peptides carrying these sequences are also cleaved by
the enzymes and often at considerable rates (Fields
et al, 1987; Fields et al, 1990; Mallya et al,
1990). Detailed analysis of the peptide cleavage
rates have shown that although the MMPs have
overlapping substrate specificities, sufficient differences exist to permit construction of optimized
peptide substrates that can discriminate between
the enzymes (Netzel-Arnett etal, 1991b). Single
amino acid substitutions in the P 4 -P' 4 sites may
yield greater than 20-fold differences in the
kcat/KM values for these substrates, and multiple
substitutions appear to be additive (Fields et al,
1987; Netzel-Arnett et al, 1991a).
The molecular disassembly of intact solidphase substrates such as natural collagen fibrils,
proteoglycan aggregates, and basement membranes is considerably more complex than the
cleavage of isolated soluble or reaggregated molecules exposed to a single enzyme, and these
processes are still incompletely understood. Two
examples illustrate this problem. The 3U -lU fragments of interstitial collagens that are produced
by collagenases at temperatures considerably below the midpoint melting temperature (Tm) have
not been unequivocally identified in any biologic
system. It is possible that they do exist as shortlived intermediates, but it is also possible that
further degradation takes place already at the fibril

level. A recent study on natural fragments generated during the degradation of the major cartilage
proteoglycan, aggrecan, has underscored this complexity. When isolated proteoglycan molecules
are exposed to SL-1, fragments are generated that
identify the cleavage site as a Asn-Phe bond in the
IPEN*FFGV sequence in the G1/G2 interglobular
domain, and evidence based on analysis of human
cartilage extracts suggests that this cleavage also
occurs in vivo (Fosang et al, 1991; Flannery
et al, 1992) (Table 10). Bovine cartilage
slices induced to degrade in culture, however,
do not carry the Phe-Phe terminal sequence expected from SL-1-mediated cleavage but carry
instead an Ala-Arg-Gly sequence located
approximately 30 residues downstream
(PLPRNXTEXE*ARGXVILTXK) (Sandy et al,
1991). The enzyme responsible for this cleavage
has not been identified.
D. Transcriptional Regulation of MMP
Genes
In the intact organism, degradative tasks are
accomplished both by growth factor/cytokinedependent and independent mechanisms. Among
the nine members of the MMP gene family, it is
possible to identify two pairs of enzymes (PMNand FIB-CL, Mr 72K GL and Mr 92K GL) with
almost identical substrate specificity but with different transcriptional regulation. One member of
each pair responds to growth factors and cytokines
whereas the other one does not. Growth factorresponsive MMPs (FIB-CL, SL-1, SL-3, and Mr
92K GL) are regulated by closely related mechanisms. In contrast, the Mr 72 GL seems to be
widely expressed by most cell types, at least in
vitro, and appears to be only moderately induced
or repressed (usually two to four-fold) (Salo et
al, 1991; Overall etal, 1991a). The regulation of
expression of MMP in the PMN is uniquely different from that of other cell types. Synthesis of
PMN-CL and Mr 92K GL is already completed
by the time the PMN enters the vasculature, and
any further regulation is mediated by granule release rather than by transcriptional events. In spite
of major differences in the transcriptional regulation, storage, and utilization of the enzyme, PMNs
appear to utilize the same gene for Mr 92K GL as
204
mesenchymal cells and keratinocytes. Transcription of MMP genes gives rise to mRNAs that are
relatively stable in the intracellular milieu, with
half lives ranging from 12 to 150 h (Brinckerhoff
etal, 1986; Overall etal, 1991a). Although most
of the regulatory mechanisms described in the
following are transcripional in nature, modulation
of mRNA half-life may also play a role.
Brinckerhoff et al. (1986) noted that FIB-CL
mRNA half-life in rabbit synovial fibroblasts
(t1/9 = 12 to 36 h) was significantly increased in
response to TPA, and Overall et al (1991a) found
that Mr 72K GL mRNA t1/2 in human fibroblasts
increased from 46 to 150 h after TGF-P stimulation, whereas those of CL (53 h) and tissue inhibitor of metalloproteinase-1 (TIMP-1) (60 h) remained unchanged.
1. Growth Factors and Cytokines

Stimulation or repression of growth factorand cytokine-responsive MMP genes in many
cases results in 20- to 50-fold changes in mRNA
and protein levels. It is possible to broadly distinguish between largely catabolic, interleukin-1
(IL-1), tumor necrosis factor-a (TNF-a); largely
anabolic transforming growth factor-(3 (TGF-p);
and hybrid growth factors and cytokines with
variable anabolic/catabolic effects, epidermal
growth factor (EGF), TGF-a, platelet-derived
growth factor (PDGF), basic fibroblast growth
factor (bFGF). For example, transcription of the
FIB-CL and SL-1 genes (and in some cells the
SL-3 and Mr 92K GL genes as well) is induced by
IL-lp, TNF-a, PDGF, TGF-a, EGF, bFGF, and
nerve growth factor (NGF) and with few exceptions (Salo et al., 1991) abrogated by TGF-P as
summarized in Table 4, In spite of the redundancy, some level of specificity appears to be
encoded in the process in that (1) different growth
factors and cytokines induce overlapping yet distinct repertoires of MMP and inhibitors and (2)
different cell types respond to the same growth
factors and cytokines by expression of unique and
distinct combinations of MMP and inhibitor genes.
For example, IL-lp induces expression of FIBCL and SL-1 in human fibroblasts but not in
foreskin keratinocytes (MacNaul et al, 1990;
Petersen et al, 1987); Mr 92K GL is repressed by
TGF-P in fibroblasts (Kerr et al, 1988b) but stimulated in keratinocytes (Salo et al, 1991); human
rheumatoid synovial fibroblasts respond to IL-lp
by induction of SL-1, whereas TNF-a induces
primarily FIB-CL (MacNaul et al, 1990); TNFa, TGF-a, and EGF as well as 12-0-tetradecanoylphorbol-13-acetate (TPA) induce high-level expression of SL-1 in fibroblasts but SL-2 in
keratinocytes (Birkedal-Hansen et al., unpublished).
Several growth factor and cytokine regulatory pathways converge at the AP-1 binding site,
which also constitutes the phorbolester-responsive element (TRE) (Angel et al, 1987a,b).
AP-1 complexes are heterodimers of proteins of
the two proto-oncogene families (Jun and Fos)
that bind to a ATGAGTCA concensus sequence
in the 5' upstream flanking region (-70 bp of the
translation start site of the human FIB-CL gene).
Oncogene and phorbol ester induction of FIB-CL
proceeds along a c-fos dependent pathway
(Schonthal et al, 1988) as does the induction of
SL-1 by PDGF but not by EGF (Kerr et al,
1988a). AP-1 binding sequences have been identified in the 5' flanking region of the FIB-CL,
SL-1, and Mr 92K GL genes but are missing in the
Mr 72K GL gene (Angel et al, 1987b; Schonthal
et al, 1988; Huhtala et al, 1991). Based on the
observation that expression of SL-3 (Basset et al,
1990) and SL-2 genes (in human keratinocytes,
Birkedal-Hansen et al, unpublished) can be induced by TPA, we predict that these genes also
contain AP-1 binding sequences. Sirum and
Brinckerhoff (1989) identified a putative AP-1
binding site in the human fibroblast SL-2 promoter (ATGAATCA) but speculated that the single
base change at position 5 (A for G) and perhaps
a substitution of T for A in position 9 immediately
following the consensus sequence might account
for the apparent lack of response of this promoter
to TPA in fibroblasts. The promoter region of
the human keratinocyte SL-2 gene, which is
highly responsive to TPA, has not yet been analyzed. The AP-1 site is a necessary but not sufficient element for transcriptional activation of
FIB-CL/SL-1 genes (Auble and Brinckerhoff,
1991; Gutman and Wasylyk, 1990; Buttice et al,
1991). The FIB-CL promoter contains a TPAand oncogene-responsive unit (TORU) composed
of at least two elements, the AP-1 site and the
205
TABLE 4
Stimulation and Repression of MMP Expression
Induction:
growth factors
and cytokines
IL-1 oc,p
TNF-oc
TGF-cc
EGF
PDGF
bFGF
NGF
TGF-p
Ref.
1, 2
2,3,4
5
6
6, 7
8, 9
10
11, 12
Induction:
other
Ref.
Repression
Ref.
TPA
Okadaic acid
Bacterial LPS
PGE2
Con A
cAMP
PTH
8, 13, 14, 15
16,
17, 18
19
20
21, 22
23
Glucocorticoids
Progesterone
1,24
25
9,26
27,28
29, 30
31,32
TGF-p
Retinoids
cAMP
IFN-y
Data from: (1) Frisch and Ruley, 1987; (2) MacNaul et al., 1990; (3) Dayer et al., 1985;
(4) Brenner et al., 1989b; (5) Lin and Birkedal-Hansen, unpublished; (6) Kerr et al., 1988a;
(7) Bauer et al., 1985; (8) Basset et al., 1990; (9) Edwards et al., 1987; (10) Machida et al.,
1991; (11) Salo et al., 1991; (12) Overall et al., 1991 a; (13) Aggeler et al., 1984; (14) Angel
etai, 1987a; (15) Wilhelm et al., 1989; (16) Kim et al., 1990; (17) Wahl et al., 1974; (18)
Cury etai, 1988; (19) Wahl et al., 1977; (20) Overall and Sodek, 1990; (21) McCarthy et
al., 1980; (22) Matrisian et al., 1986b; (23) Civitelli et al., 1989; (24) Jonat et al., 1990;
(25) Newsome and Gross, 1977; (26) Kerr et al., 1990; (27) Brinckerhoff, 1990; (28)
Nicholson etai., 1990; (29) Takahashi etai., 1991; (30) Kerr etai., 1988b; (31) Andrews
etai., 1990; (32) Wahl etai., 1990.
PEA3 binding site, which act synergistically to

induce maximal levels of transcriptional activation, by TPA and nonnuclear oncogenes (v-src,
Py middle T, Ha-ras, v-ra/, w-mos, and c-fos +
c-jun) (Gutman and Wasylyk, 1990). PEA3, a
transcription factor that also binds to the polyoma
virus enhancer, binds to the GAGGATGT sequence only a few bases from the AP-1 site:
5'93TCGAGAGGATGTTATAAAGCATGAGTCAG 3'
Although /<95-transcription is an early event

in the TPA-, IL-1 -, and TNF-oc-induction of MMP,
induction of fas does not necessarily result in
expression of growth-factor-responsive MMP
genes. This is supported by two lines of evidence:
(1) repression of MMP expression by TGF-P is
also /os-dependent but is independent of the
AP-1 site (Kerr et al, 1990) and (2) induction of
fas and of MMP by IL-1 can be uncoupled. An
IL-ip point mutant, IL-1(5127R_G, which binds to
the IL-1 receptor, stimulates transcription of earlyresponse genes fas and jun but fails to stimulate
transcription of the late genes such as FIB-CL and
SL (Conca et al, 1991). These findings show that

fas and jun expression is not necessarily accompanied by increased transcription of genes containing the AP-1 site and that at least two distinct
events are required for IL-1-mediated late gene
induction in fibroblasts. Auble and Brinckerhoff
(1991) recently showed that the smallest element
that confers TPA inducibility is 127 bp in length
and that this element contains an AP-1 site, a
PEA3-like element, and a sequence that includes
5' TTCA-3'. These studies also showed that although there is a great deal of homology between
the FIB-CL and SL-1 promoters, there are also
important structural and functional differences
(Sirum-Connolly and Brinckerhoff, 1991). A
46 bp fragment of the SL-1 promoter located
between nucleotides -54 and -100 upstream of
the transcription start site is required for both
phorbol and IL-1 induced transcription. This sequence contains two regulatory elements, an
AP-1-like and a nuclear inhibitor protein (NIP)
binding site. Both the AP-1 site and the NIP
binding sequence are involved in binding of
phorbol- and IL-1-induced nuclear proteins. On
206
the other hand, the PEA 3 element, which plays

an important role in transcriptional activation of
the FIB-CL gene by phorbolesters and oncogenes
(Gutman and Wasylyk, 1990; Auble and
Brinckerhoff, 1991), does not appear to play a
major role in SL-1 induction.
The AP-1 site is also a target for repression
of MMP expression by glucocorticoids. These
compounds ablate transcription of responsive
MMP genes through formation of complexes
between the glucocorticoid receptor (GCR) and
the JunlFos complex in a manner that prevents
the DNA-binding activities of both (Jonat et al,
1990; Yang-Yen et al, 1990). It is of interest in
this context that the AP- 1-like binding site in the
a-fetoprotein promoter (_160TGAACATAA)
is located within the half-site of the GCR
consensus binding site centered at position-160
(_ 166 TGTCCTTGAACATAAG_ 151 ) (Zhang
et al, 1991). In spite of the close functional and
spatial relationship between the elements that
mediate glucocorticoid repression and phorbol
ester induction, it is possible to uncouple these
transcriptional effects. Mutation of the TTAA
sequence at position -102 to -99 of the rabbit
CL promoter results in loss of dexamethasone
repressibility but does not affect phorbol inducibility (Auble and Brinckerhoff, 1991).
TGF-(3 and interferon-y (IFN-y) predominantly down-regulate MMP gene expression.
TGF-(3 represses both the constitutive and
cytokine-induced expression of CL and SL-1 in
fibroblasts through a c-/<9s-dependent mechanism
that involves a specific 10 bp TGF-p-responsive
element unrelated to the AP-1 site and located in
the -700 bp region (Edwards et al, 1987; Kerr
et al., 1990). The TGF-(3-responsive sequence
identified in the rat SL-1 promoter is
709GAGTTGGTGA
(consensus sequence: GNNTTGGtGa)
The same element is present in the promoter

regions of FIB-CL ( 246GAATTGGAGA) and of
other TGF-|3l-responsive proteins such as u-Pa,
elastase, proliferin, and c-Myc. The binding of a
Fos protein complex at this site counteracts the
positive regulation that occurs downstream at the
AP-1 site. It is not yet clear whether this is simply
a competition for c-Fos or whether c-Fos is modified to serve both as a negative and positive regula-
tor (Kerr et al, 1990). Recent studies suggest

that IFN-y suppresses the IL-1-induced secretion of FIB-CL by human articular chondrocytes,
human monocytes, and rat fetal osteoblasts, but
the mechanism has not been clarified (Shen et
al, 1988; Andrews et al, 1990; Wahl et al,
1990).
The AP-1 site also appears to be the target
for repression of MMP transcription in fibroblasts by retinoids (Brinckerhoff et al, 1980;
Brinckerhoff, 1990; Lafyatis et al, 1990). The
complex formed between retinoic acid and the
retinoic acid receptor represses the effect of
positive regulatory factors on transcription of
the SL gene, but the complex does not appear to
bind to the AP-1 site or to c-Fos or c-Jun
(Nicholson et al, 1990).
2. Hormonal Regulation
Degradation of the extracellular matrix is
modulated by hormones in several developmentally regulated processes, and it is likely that
this effect is mediated via transcriptional regulation of MMP expression. Injection of rats
with estradiol immediately postpartum significantly slows and retards the loss of collagen
(Woessner, 1969; Ryan and Woessner, 1971,
1974). Somewhat at variance with these findings, Jeffrey and collaborators showed that
progesterone and the synthetic analogue
medroxyprogesterone, but not other reproductive hormones, inhibit production of collagenase in explants of uterine tissues (Jeffrey et
al, 1971a,b; Koob and Jeffrey, 1974).
Medroxyprogesterone is also a potent inhibitor
of matrix remodeling outside the uterus and
completely blocks neovascularization and
collagenolysis in rabbit cornea (Newsome and
Gross, 1977; Gross et al, 1981). FIB-CL synthesis by osteoblasts, UMR 106 osteosarcoma
cells, and whole calvaria is induced or stimulated by parathyroid hormone (PTH) and 1,25
dihydroxy vitamin D 3 (1,25 di(OH)D3) (Delaisse
et al, 1988; Civitelli et al, 1989, Quinn et al,
1990), but it is not quite clear to what extent
this response is linked to the bone-resorbing
activity of the hormones in vivo.
207
3. Cell Shape, Cell-Substrate Adhesion

Induction or stimulation of MMP expression
may also occur in response to signals or events
that are physical rather than chemical in nature,
including phagocytosis of particulate matter (latex beads, urate and mycostatin crystals [Werb
and Reynolds, 1974; Birkedal-Hansen et al,
1976; Brinckerhoff et al, 1982]), heat-shock
(Vance et al, 1989), or treatment with cytochalasin B, an actin cytoskeleton-disrupting agent
(Harris et al, 1975; Werb et al, 1986). The
observation that induction of MMP expression
by phorbol esters and by proteinases (Werb and
Aggeler, 1978) is also associated with profound
changes in cell shape (rounding) prompted Werb
and co-workers to examine the relationship between cytoskeletal rearrangement and regulation of MMP expression (Aggeler et al, 1984;
Werb et al, 1986; Unemori and Werb, 1986).
Their studies indicated that cell-shape changes
often, but not invariably, induce MMP expression (Aggeler et al, 1984; Werb et al, 1986)
and that it is the reorganization of polymerized
actin rather than cell rounding per se that is
linked to induction of MMP expression
(Unemori and Werb, 1986). Somewhat at variance with these findings, Kuter et al (1989)
concluded that, at least in homogenous primary
rabbit corneal stromal cells, expression of FIBCL correlates with changes in cell shape only
in the presence of TPA or cytokines and that
alteration of cell shape induced by a variety of
factors is neither sufficient nor necessary for
induction of FIB-CL expression.
Because cell-shape changes are often dictated by cell-substrate adhesion, several studies
have suggested that the substrate alone can
modulate MMP expression, and the answer
appears to be affirmative. Engagement or crosslinking of integrin receptors by monoclonal
antibodies or fibronectin fragments (but not
intact fibronectin) results in transcription of
MMP genes (Werb et al, 1989). Other studies
have shown that seeding on collagenous matrices of either type I collagen, type IV collagen,
or matrigel stimulates expression of FIB-CL in
keratinocytes and fibroblasts (Emonard et al,
1990; Petersen et al, 1992). Contact with
laminin stimulates expression of gelatinases by
sarcoma cells, and this effect has been attributed to a 19 amino acid sequence of the laminin
A chain (Turpeenniemi-Hujanen et al, 1986;
Kanemoto et al, 1990).
4. Second Messenger Signaling

The signaling pathways that lead to induction of MMP expression are still incompletely
understood, but certain patterns are beginning to
emerge. Recent studies have implicated protein
kinase C (PKC), the major cellular receptor for
phorbol esters such as TPA, as an important
messenger in the transcriptional regulation of
growth factor-responsive MMP genes
(McDonnell et al, 1990). However, PKC does
not appear to be involved in all cases. SL-1
induction by NGF in rat PC 12 pheochromocytoma cells requires multiple protein kinases
acting on a number of postreceptor steps but
probably not PKC (Machida et al, 1991). Moreover, okadaic acid, a non-TPA-type tumor promoter that does not activate PKC but induces
"apparent" activation of protein kinases by inhibition of protein phosphatases, also induces FIBCL expression through an AP-1-dependent pathway (Kim et al, 1990). The role of 3'-5' cyclic
adenosine monophosphate (cAMP) remains enigmatic in that a rise in cytoplasmic cAMP in
some systems leads to stimulation of MMP expression and in others to repression. cAMP stimulates expression of FIB-CL and SL-1 by guinea
pig peritoneal macrophages, rat UMR-106
osteosarcoma cells, and rat fibroblasts (McCarthy
et al, 1980; Civitelli et al, 1989; Matrisian
et al, 1986b) but represses the constitutive expression of FIB-CL by human GM637 fibroblasts (Angel et al, 1992) and the IL-1-, EGF-,
and oncogene-induced transcription of FIB-CL
and SL-1 genes by rat and human fibroblasts
(Kerr et al, 1988b; Takahashi et al, 1991).
Recent studies suggest that cAMP activates a
different transcriptional machinery than either
TPA or growth factors/cytokines. A rise in intracellular cAMP induces transient expression of
Jun-B, whereas both c-Jun and FIB-CL are repressed. Jun B, like c-Jun, is capable of binding
to the AP-1 site but fails to activate the FIB-CL
promoter (Angel et al, 1992).
208
E. Activation of Metalloproteinase
Precursors
The biologic activation of MMP is still incompletely understood. This gap in knowledge
represents perhaps the most important obstacle to
our understanding of how cells utilize
metalloproteinases to degrade the extracellular
matrix. A body of evidence suggests that the la-
tency of the virgin enzyme is maintained, at least

in part, by virtue of a putative Cys-Zn2+ bond that
links the unpaired propeptide Cys residue (Cys73
in human FIB-CL) to the active site Zn i +
(Springman et al., 1990; Van Wart and BirkedalHansen, 1990) (Figure 3) and displaces the H2O
molecule, which is necesssary for catalysis. The
Cys residue remains cryptic in the latent form of
the enzyme but is exposed during activation as a
CL(APMA)
LATENT (Mr 52K)
ACTIVE (Mr 52K, 46K, 43K)
ACTIVE (Mr 41K)
FIGURE 3.
Latency/activation mechanism of human FIB-CL precursor. TRY:
trypsin cleavage site; PKK: plasma kallikrein cleavage site; CL (PKK, PL): autolytic
cleavage site following initial cleavage by plasma kallikrein or plasmin (PL); CL
(APMA): autolytic cleavage site following reaction with 4-aminophenylmercuric
acetate; SL: stromelysin cleavage site. Location of cleavage sites is from Suzuki,
K., J. J. Enghild, T. Morodomi, G. Salvesen, and H. Nagase: Biochemistry.
29:10261-10270 (1990).
209
result of conformational change (Lyons et al.,

1991a). Disruption of this putative Cys-Zn2+ bond
that stabilizes the propeptide in its "docked" position may be achieved by seemingly disparate
chemical and physical means (Figure 3).
Organomercurials, metal ions, thiol reagents, and
oxidants presumably interact directly with the
propeptide Cys-residue to shift the equilibrium
between the "closed" and the "open" form toward
the open form; chaotropic agents (3M KI, 3M
PROTEOLYTIC
ENZYMES
NaSCN) and detergents (1 to 2% SDS) induce

polypeptide chain conformational changes that
also shift the equilibrium entirely toward the open
form; proteolytic enzymes excise a portion of the
propepeptide so that the switch opens, most likely
because of an entropy effect (Figure 4). Once
stabilized in the open form, the enzyme catalyzes
several autolytic cleavages to generate the fully
processed form (Grant et al., 1987; Nagase et al.,
1990; Suzuki et al., 1990). Detailed analyses of
CONFORMATIONAL
PERTURBANTS
THIOL-REACTIVE AGENTS
LMW - ACTIVE
FIGURE 4.
Activation of MMP. The reversible opening of the cysteine switch plays a central
role in different activation pathways. The equilibrium may be shifted towards the open form by
proteolytic cleavage of the propeptide (left column) or by reaction of the propeptide Cys residue
with metal ions, organomercurials, oxidizing agents, and thiol reagents (right column).
210
the successive activation cleavages of human

proCL and proSL-1 (Figures 3 and 5) have shown
that exogenous proteinases (trypsin, Pin, chymotrypsin, neutrophil elastase, and plasma kallikrein)
attack a short basic sequence exposed on the surface of the molecule (Nagase et al, 1990; Suzuki
et al, 1990). This initial cleavage is sufficient to
permit a second, autolytic cleavage 5 to 8 residues
upstream of the unpaired Cys residue (SL-1C s75,
FIB-CLC 73 ). Exposure to organomercurials also
leads to intramolecular cleavage of the region, 5
to 8 residues upstream of the Cys residue (Nagase
et al, 1990; Stricklin et al, 1983; Grant et al,
1987). The final mature form of the enzyme is
produced autolytically by mtermolecular trimming
of 14 to 18 residues from this intermediate, including the unpaired Cys residue.
autolytic cleavage of its own hinge region already

inside E. coli but more slowly, if at all, the autolytic activation cleavages of the propeptide. Even
after complete selfcleavage at the hinge region
site, the molecule still contains an intact propeptide
and escapes capture by oc2M for a period of several hours and, by that criterion, is essentially
latent (Windsor et al, 1991). These findings raise
the interesting question of whether it is possible
for the enzyme to be "latent" on some substrates
(oc2M bait region, FIB-CL propeptide) and "active" on others (FIB-CL hinge region).
A puzzling observation is the so-called
"superactivation" of proFIB-CL by several other
MMPs including SL-1, SL-2, and PUMP-1
(Murphy et al, 1987; He et al, 1989; Nicholson
etal, 1989; Quantinâ/., 1989; Birkedal-Hansen
NH 2 -terminus
Catalytic Domain
PKK TRY, PKK
CL(PKK,PL) CL(A?MA)
CL(APMA)
FPATLETQEQDVDLVQKYLEKYYNLKNDGRQVEKRRNSGPVTO^
FIB-CL
SL-1
HNE
CT I PKK
i\\r
SL-l(APMA)
S L-1
YPLDGAARGEDTSM^VQKYLENYYDLKKDVKQFVRRKDSGPWKK
Exogenous
Proteinase
Clevage Site
SL-1
Autolytic
Cleavage
Sitel
Autolytic
Cleavage
Site 2
FIGURE 5.
Propeptide cleavages associated with activation of human FIB-CL and SL-1. Arrows identify
cleavage sites by exogenous proteinases (plasma kallikrein [PKK], trypsin [TRY], chymotrypsin [CT], human
neutrophil elastase [HNE], and human stromelysin-1 [SL-1]) and autolytic cleavage sites after activation is
initiated either by plasma kallikrein (CL [PKK]) or APMA (CL [APMA]; SL-1 [APMA]). Data from Suzuki, K., J. J.
Enghild, T. Morodomi, G. Salvensen and H. Nagase: Biochemistry. 29:10261-10270 (1990) and Nagase, H.,
J. J. Enghild, K. Suzuki and G. Salvesen: Biochemistry. 29:5783-5789 (1990).
The general sequence of events outlined above

is supported by mutational analyses. Replacement
of the Cys residue and of several other residues
(Arg, Gly, Val, and Pro) in the highly conserved
PRCGVPDV propeptide sequence (Appendix 1)
results in mutant enzymes that appear to be partially or fully active (Windsor et al, 1991; Park et
al, 1991; Sanchez-Lopez et al, 1988). The state
of activity of these mutants, however, remains
somewhat enigmatic. For example, the Cys-Ser
mutant of human FIB-CL rapidly catalyzes an
et al, unpublished). ProFIB-CL is capable of

completing all of the necessary autolytic activation cleavages, either after excision of the first 35
residues of the propeptide or after exposure to
organomercurials. The final cleavage of the sequence -Asp-Val-Ala-Gln-Phe-Val-Leu-, which
marks the transition from the propeptide to the
mature activated enzyme, yields either a Val or
Leu amino terminus (Val-FIB-CL; Leu-FIB-CL)
(Figure 3; Table 5). In the presence of proSL/SL,
the predominant mature form, however, is Phe211
CL, which has a 5 to 8-fold higher catalytic activity than either of the other two forms (Susuki
et al9 1990). These findings have prompted speculation about whether SL plays a role in the biologic activation of proFIB-CL, but the question
has not yet been resolved fully.
In the continuing search for a general, biologically relevant MMP activation mechanism,
the short basic propeptide sequences that are the
sites for the initial cleavage in trypsin/Pln-induced
activation of FIB-CL and SL-1 have attracted
considerable attention (Grant et al, 1987; Stricklin
TABLE 5
Human MMP Sequences Surrounding the Autolytic Activation Sites
K V M K Q P R C G V P D V A Q - - D
E
E
S
E
E
K
M M K K P R C G V
V M R K P R C G V P
V M R K P R C G V
S L R P P R C G V P
I M Q K P R C G V
T M R K P R C G N
*A*M R T P R C G V
P D
D V
P D
D P
P D
P D
P D
S
G
V
S
V
V
L
G
H
G
D
A
A
G
G - - - - - - - _ _ _ _ _ _ _
H - - - - - - G L S A R N R
E - - - - - - N - - - - - - R - - - - - - -
F *V *L T E G N P R W FIB-CL
- - F M * L T P G N P K W
PMN-CL
SL-1
_ _ *F R T F P G I P K W
- - - F S S F P
G M P K W SL-2
Q K R F V L S G G - R W SL-3
- - - Y S L F P N S P
K W PUMP-1
- -*Y N F F P R K P
K W M f 7 2 KG L
- - - F Q T F E G D L
K W M r 92K GL
Note: Asterisks indicate autolytic cleavage sites verified by sequence analysis. The NH2-terminal sequences of the
autolytic activation products of SL-2, SL-3, and PUMP-1 are not known.
Data from: FIB-CL: Grant, G. A., A. Z. Eisen, B. L Marmer, W. T. Roswit and G. I. Goldberg: J. Biol. Chem.
262:5886-5889 (1987); Suzuki, K., J. J. Enghild, T. Morodomi, G. Salveson, and H. Nagase: Biochemistry.
29:10261-10270 (1990); PMN-CL: Mallya, S. K., K. A. Mookhtiar, Y. Gao, K. Brew, M. Dioszegi, H. Birkedal-Hansen
and H. E. Van Wart: Biochemistry. 29:10628-10634 (1990); SL-1: Nagase, H., J. J. Enghild, K. Suzuki, and G.
Salveson: Biochemistry. 29:5783-5789 (1990); Mr72K GL: Stetler-Stevenson, W. G., H. C. Krutzsch, M. P. Wacher,
I. M. K. Margulies, and L. A. Liotta: J. Biol. Chem. 264:1353-1356 (1989a); Mr 92K GL: Wilhelm, S. M., I. E. Collier,
B. L. Marmer, A. Z. Eisen, G. A. Grant and G. I. Goldberg: J. Biol. Chem. 264:17213-17221 (1989); Tschesche,
H., V. Knauper, S. Kramer, J. Michaelis, R. Oberhoff and H. Reinke: Matrix. Spec. Suppl. No. 1: 245-255 (1992).
Organomercurial activation of the Mr 72K

GL results in autolytic cleavage, which removes
a Mr 8K peptide by hydrolysis at the Tyrg5 Asn86 bond, at a site homologous to that cleaved
in other metalloproteinases, eight residues downstream from the conserved propeptide Cys residue (Table 5) (Stetler-Stevenson et ai, 1989a).
The activation of the Mr 92K GL appears to proceed somewhat differently and with a different
endpoint. The enzyme is activated rather slowly
by either organomercurials or trypsin (Lyons et
al., 1991a). Trypsin-activation of human PMN
Mr 92 GL results in cleavage of a Lys-Ala bond
and yields an NH 2 -terminal sequence of
AMRTPRCGVD (Tschesche et al, 1992). Organomercurial-induced autoactivation of this enzyme
results in cleavage only one residue downstream,
namely at the Ala-Met bond, which yields a
MRTPRCGVD NH2-terminal sequence (Wilhelm
et al, 1989). In either case, the thiol-bearing sequence appears to be retained in the mature enzyme.
et al, 1983; Suzuki et al, 1990). Sequences surrounding the trypsin-sensitive site are only partially conserved, and it is not immediately apparent why some but not all MMPs are amenable to
activation by trypsin and Pin (Appendix 1). For
example, PUMP-1 (KNAN) is activated quite well
by trypsin, PMN-CL (RKNG) and M 92K GL
(KSLG) are activated only slowly, and Mr 72K
GL (KDTL) not at all under the same conditions.
The observation that activation of SL-1 also may
be initiated by cleavage by chymotrypsin (PheVal) and human neutrophil elastase (Val-Arg)
suggests that the conformation around this site
in addition to its sequence plays an important
role in encoding the susceptibility to proteolysis.
The question of whether cultured cells that secrete the proenzymes possess all of the necessary
components to complete their activation has not
been fully resolved. In fact, it has even proven
quite difficult to achieve MMP activation in cell
culture systems without addition of exogenous
proteinases such as Pig or trypsin. MMP precur-
212
sors secreted in cell culture "spontaneously" activate very slowly, if at all, even at elevated temperatures, perhaps because of coexpression of
MMP inhibitors (TIMP-1 and TIMP-2). In fibroblast cultures, partial activation of precursors of
FIB-CL and of Mr 72K GL may be achieved by
addition of the ionophore A23187 (Unemori and
Werb, 1988) and by Concanavalin A (Con A)
(Overall and Sodek, 1990; Ward et ai, 1991).
The latter investigators also provided evidence
that activation of the Mr 72K GL is mediated by
a cell surface bound "activator". Our own studies
have shown that activation of proFIB-CL in serum-free cultures of fibroblasts (which express
SL-1) and of keratinocytes (which express SL-2)
is very slow and incomplete for the first 3 to 5 d
of incubation and that collagen breakdown is
blocked during that period (Birkedal-Hansen et
al., 1992b; Birkedal-Hansen et a/., 1989; Lin et
aL, 1987; Lyons et a/., 1991a).
The degree of overlap between the Pig- and
metalloproteinase-dependent pathways, in general,
and the role of Plg/Pln in the metabolic activation
of MMP precursors, in particular, remain uncertain. Several studies using in vitro model systems
suggest that MMP-dependent proteolysis is greatly
accelerated in the presence of Pig (Mignatti et aL,
1986, He et al, 1989; Birkedal-Hansen et al.y
1992b). The stimulating effect of Pig, while not
analyzed fully in detail, is believed to reside in the
ability of Pin to initiate the autolytic activation of
several MMP zymogens, including FIB-CL and
SL-1. The reluctance to accept a generalized model
for pericellular proteolysis of extracellular matrix
substrates in which Plg/Pln play a role is focused
on two major points: (1) the lack of specificity of
Pin cleavage reactions is not easily reconciled
with the fact that most regulatory proteinase cascades are composed of highly specific enzymes
and (2) several MMPs are not activated by Pin.
Moreover, examples do exist of cells that degrade
model matrices by seemingly Pig-independent
processes. Such is the case for the degradation of
gelatin and collagen by PMN leukocytes (Peppin
and Weiss, 1986; Weiss et a/., 1985). These investigators provided strong evidence that, at least
in the PMN leukocyte, activation of PMN-CL and
Mr 92K GL may be achieved by oxidative pathways, presumably by oxidation of the unpaired
propeptide Cys residue by HOC1.
IV. INDIVIDUAL MATRIX

METALLOPROTEINASES
A. Interstitial Collagenases
The PMN-CL gene is only expressed by PMN
leukocytes, whereas that of FIB-CL is expressed
(after appropriate stimulation) by fibroblasts from
many different sources (skin, synovium, mucosa,
cornea, uterus), keratinocytes (Lin et ai, 1987;
Petersen et ai, 1987), endothelial cells (Moscatelli
et ai, 1980; Herron et aL, 1986), monocytes and
macrophages (Welgus et ai, 1985a; Campbell et
ai, 1987), chondrocytes (Lefebvre et ai, 1990),
and osteoblasts (Otsuka et ai, 1984; Quinn et ai,
1990). The protein cores of PMN-CL and FIB-CL
are of virtually identical size (Appendix 1), but
the PMN enzyme is more highly glycosylated and
has a considerably larger molecular mass than
FIB-CL (Mr 75,000 vs. 57,000/52,000). It is speculated that the carbohydrate moiety of PMN-CL
encodes targeting signals that direct the enzyme
to granule storage sites. FIB-CL is partially
glycosylated (Mr 57,000 form) and two potential
Asn-X-Thr N-glycosylation sequences have been
identified, but the preponderance of the molecules
(70 to 80%) remain unglycosylated (Nagase et
a/., 1981,1986; Wilhelm etal, 1986; Goldberg et
al, 1986). The function of the FIB-CL carbohydrate moiety is unknown, but it does not appear to
be important for catalytic activity. The two enzymes also differ in terms of activation mechanisms in that FIB-CL is readily activated by trypsin
and Pin whereas the PMN-CL is not. Examination of the primary structures (Appendix 1) reveals that the tribasic KRR sequence in fibroblast-type collagenase is altered to an RKN sequence, which apparently fails to confer the same
level of trypsin/Pln activatability.
The two collagenases differ markedly in terms
of transcriptional regulation. PMN-type collagenase is released instantly from granule storage
sites of triggered PMNs, and it is uncertain whether
PMN-CL expression is subject to transcriptional
regulation in the bone marrow. In contrast, FIBCL is not stored in cells but produced on demand
by initiating transcription of the gene (for instance by the action of growth factors and
cytokines). Because this process is dependent on
activation of a complex transcriptional apparatus,
213
the accumulation of the enzyme at the local site is

delayed by 6 to 12 h. The significance of this
difference is evident by comparison of the effect
of TPA on unleashing the destructive potential of
these two cell types. In the PMN leukocyte, TPA
gives rise to immediate release of most or all of
the enzyme (ever) produced by the cell whereas
in fibroblasts, 6 to 12 h elapse before any enzyme
is released, but once that happens, production and
secretion can be sustained for several days. The
PMN leukocyte is capable of responding in full
force instantly but not of sustaining any destructive activity beyond minutes.
1. Cleavage of Interstitial Coiiagens

The cleavage of interstitial coiiagens by collagenases has been studied in greater detail than
any other MMP-catalyzed reaction, but a number
of questions that pertain to the substrate specificity of these enzymes have been only partially
resolved. These include (1) the structural basis for
the unique ability of collagenases, but not other
MMP, to cleave coiiagens type I, II, and III and
(2) the structural basis for the susceptibility of the
collagenase-sensitive Gly-Ile bond but not other
potentially cleavable bonds along the triple helix.
scission of the Gly775-Ile776 or Gly775-Leu776 bonds

of the component a-chains (Table 6). The primary structure of the collagenase-sensitive site is
an important factor in determining the susceptibility to collagenases, and Wu etal. (1990) showed
that most substitutions in and around the scissile
bond by site-directed mutagenesis are unfavorable and yield either uncleavable or poorly susceptible collagen molecules. However, studies
using synthetic peptides modeled after the collagenase-sensitive region have shown that the unique
specificity cannot be accounted for entirely by the
amino acid sequence and that there are several
Gly-Leu-Ala and Gly-Ile-Ala sequences throughout the triple helix of interstitial coiiagens that are
not cleaved (Fields et al, 1987; Netzell-Arnett et
al, 1991a). In search of an explanation for the
unique susceptibility of the scissile Gly-Ile/GlyLeu bonds, several investigators have suggested
that the collagenase-sensitive region is a locus of
minor resistance that more readily unfolds and
relaxes its triple helical structure than other parts
of the molecule (Wang et al, 1978; Fields et al,
1987; Birkedal-Hansen, 1987). In support of this
evidence is the finding that several bonds in the
collagenase-sensitive region of type III collagen
are cleaved by trypsin, thermolysin, pronase, and
PMN elastase (Wang et al, 1978; Miller et al,
TABLE 6
Collagenase Cleavage Sites in Interstitial Coiiagens
Substrate
Calf and chick cc1(l)
Calf o2(l)
Chick o2(l)
Human oc1 (II)
Human a1(lll) (skin)
Human oc1 (III) (liver)
Calf a1 (III)
Sequence
Gly-Pro-Gln-Gly-Ile-Ala-Gly-Gln
Gly-Pro-Gln-Gly-Leu-Leu-Gly-Ala
Gly-Pro-Gln-Gly-Ile-Leu-Gly-Ala
-Ile-Ala-Gly-Gln
-Leu-Ala-Gly-Leu
Gly-Pro-Leu-Gly-Ile-Ala-Gly-Ile
Gly-Pro-Leu-Gly-Ile-Ala-Gly-Leu
Ref.
1,2,3
4
5
6
7
8
6
Data from: (1) Gross etal., 1974; (2) Highberger etal., 1982; (3) Glanville et
al., 1983; (4) Bomstein and Traub, 1979; (5) Dixit etal., 1979; (6) Miller et
al., 1976; (7) Seyer and Kang, 1981; (8) Lang etal., 1979. (Modified from
Netzel-Arnett, S., G. Fields, H. Birkedal-Hansen and H. E. Van Wart: J. Biol.
Chem. 266:6747-6755 (1991a).
At temperatures well below the denaturation

temperature, type I, II, and III coiiagens in solution are cleaved exclusively at a single locus by
214
1976; Mainardi etal, 1980a; Birkedal-Hansen et

al, 1985). It is more difficult to demonstrate a
similar proteinase-sensitive region in type I col-
lagens, but it can be done under narrowly defined

conditions (Ryhanen et al., 1983). The higher
rates of cleavage of type III collagen by nonspecific proteinases appears to mirror its faster cleavage also by collagenases, at least in some species
(Tables 7 and 8). These observations when held
together suggest that the collagenase-sensitive
region in most species unfolds more readily in
type III collagen than in type I. That the collagenase-sensitive site represents a locus of minor
resistance also in type I collagen is indirectly
supported by the observation that this site is the
preferred cleavage site by trypsin/chymotrypsin
(Ryhanen et al., 1983) and by unrelated
collagenolytic enzymes from the microorganism
Achromobacter iophagus (Lecroisey and Keil,
1979), the fiddler crab (Uca pugilator) hepatopancreas (Welgus and Grant, 1983), and the dermal larva Hypoderma lineatum (Lecroisey and
KeiL 1979). The hypothesis that the collagenasesensitive site has a greater degree of molecular
mobility than other sites is indirectly supported
by microcalorimetric studies, which have shown
that a segment between 3 and 30 triplets in length
starts to unfold as much as 10C below the denaturation temperature (Privalov et al., 1979). Although these data do not permit us to identify the
location of the unfolding segment, it is highly
likely, when viewed in the context of the extraordinary proteinase susceptibility of type III collagen, that unfolding is initiated around the collagenase-sensitive site.
The kinetic parameters of FIB-CL from several different laboratories are summarized in Table
7. Although the data were obtained by use of
different enzymatic assays, the consistency of
certain key determinations such as (1) the rates of
cleavage of type I collagens in solution at 25 to
30C, (2) the rates of cleavage of types I and III
collagen in solution at 37C, and (3) the KM values on collagen substrates, suggest that meaningful comparisons may be made from this unique
data set. The cleavage of type I collagen at 25 to
30C is a rather slow reaction by any criteria in
that less than one molecule per minute is cleaved
per molecule of enzyme (16 to 53 h"1). By comparison, casein is cleaved 10-fold, and oc2M 50fold, faster than type I collagen. The three interstitial collagens are cleaved at very different rates
by FIB-CL in the same temperature range (25 to
30C). For most species, there is in essence a tenfold difference between collagen types III and I
(=300 hr1 and =30 h"1) and again between collagen types I and II (-30 h"1 and - 3 h"1). The
cleavage of interstitial collagens is highly temperature-dependent particularly in the
subdenaturation temperature range (Tm = 40 to
42C). A rise in temperature between 25 to 30C
and 35 to 37C increases the rate of cleavage of
type I collagen by 50 to 80-fold (16 to 53 h"1 vs.
1600 to 1700 tr 1 ) and all but eliminates the rate
difference between types I and type III collagens.
It is also apparent that native triple helical collagen (1600 to 1700 h"1) is a much better substrate
than denatured collagen (230 to 750 h"1) at the
same temperature (37C). Not only is the rate of
cleavage 2 to 7-fold higher, but kM is 4 to 8-fold
lower so that, based on kcat/KM comparisons, triple
helical type I collagen is a 10 to 60-fold better
substrate for FIB-CL than either of its component
random coil a-chains. The rates of cleavage of
reconstituted fibrillar substrates are much lower
than those of soluble collagen molecules. At 35 to
37C, both type I and III collagen fibrils are cleaved
at rates in the range of 11 to 60 h"1 when compared with 1600 to 1700 hr1 for the same collagens in solution. It is likely that this difference
is due in part to the increased thermal stability of
fibrillar structures (Tm flbnls = 47 to 49C; Tm sol =
40 to 41C) (Birkedal-Hansen et al, 1985). It is
interesting to note that the difference in catalytic
rates between types I and III collagen, which
exists at 25 to 30C, all but disappears at 35 to
37C whether in soluble or fibrillar form. The
data shown in Table 7 also reveal several interesting species differences, particularly with respect
to cleavage of type III collagen. Whereas human,
dog, and cat type III collagens are cleaved very
rapidly (350 to 627 h"1), those of guinea pig and
chick are not; in fact, chick type III collagen is
cleaved about as slowly as collagen type II. PMNCL is a considerably more efficient enzyme (10 to
30-fold) than FIB-CL on virtually all substrates
except for type III collagen (Hasty et al, 1987;
Netzell-Arnett et al., 1991a). A comparison of the
catalytic properties of FIB-CL and PMN-CL is
shown in Table 8. The proteolytic activity of collagenases is not limited to collagen and collagenlike synthetic peptides. Recent studies have shown
the collagenases cleave a variety of susceptible
215
TABLE 7
Catalytic Properties of Human Fibroblast-Type Coliagenase
kcat(h"1)
Type 1 collagen, rat, soln

Type 1 collagen, rat, soln
Type 1 collagen, human, soln
Type 1 collagen, human, soln
Type 1 collagen, calf, soln
Type 1 collagen, guinea pig, soln
Type 1 collagen, dog, soln
Type 1 collagen, cat, soln
Type 1 collagen, chick, soln
Type II collagen, calf, soln
Type II collagen, calf, soln
Type II collagen, human, soln
Type II collagen, rat, soln
Type III collagen, human, soln
Type III collagen, human, soln
Type III collagen, dog, soln
Type III collagen, cat, soln
Type III collagen, guinea pig, soln
Type III collagen, chick, soln
Type 1 collagen, calf, soln
Type III collagen, calf, soln
Type III collagen, calf, fibrils
Type III collagen, human, fibrils
Type III collagen, dog, fibrils
Type III collagen, cat, fibrils
Type III collagen, guinea pig, fibrils
Type III collagen, chick, fibrils
Type 1 collagen, calf, fibrils
Type 1 collagen, dog, fibrils
Type 1 collagen, guinea pig, fibrils
Type 1 collagen, guinea pig, soln
Type 1 gelatin, oc1(l), guinea pig
Type I gelatin, a2(l), guinea pig
Type I gelatin, rat
P-casein
P-casein
Gly-Pro-Gln-Gly-lle-Ala-Gly-GIn
Gly-Pro-Gln-Gly-Leu-Ala-Gly-GIn
G!y-Pro-Leu-Gly-lle-Ala-Gly-G!n
Gly-Pro-Gln-Ala-lle-Ala-Gly-GIn
oc2-M, human
Ovostatin, chick
16
19.5
44
53.4
34.2
22.5
32.2
22.8
35.1
3.2
2.7
1.0
4.5
350
565
472
627
18.0
5.4
1653
1477
13.1
45.7
53.9
60.7
4.1
0.9
11.4
21.0
25.0
1700
230
750
24
420
160
730
970
1200
4500
1739
2.1
K M (HM)
0.8
0.9
0.8
0.8
0.8
0.9
1.5
1.1
1.0
2.4
1.6
2.1
1.1
1.7
1.4
1.1
1.8
0.7
1.2
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.9
7.0
3.7
9.8
n.d.
710
3300
2800
3600
2800
0.17
0.32
1(
c A P
'
1 h
"
1 )
Temp. (C)
Ref
30
25
30
25
25
25
25
25
25
30
25
25
25
30
25
25
25
25
25
35
35
35
37
37
37
37
37
35
37
37
37
37
37
30
30
30
30
30
30
30
25
25
1
2
1
2
2
2
3
3
3
1
2
2
2
1
2
3
3
2
3
4
4
4
3
3
3
3
3
4
3
5
6
6
6
7
8
7
9
9
9
10
11
11
19.0
21.7
54.0
66.8
42.8
25.0
21.5
20.7
35.1
1.3
1.7
0.48
4.1
205.9
403.6
429
348
25.7
4.5
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1888.9
32.9
202.7
2.5
n.d.
0.23
0.22
0.35
0.33
1.6
10229
6.6
Note: n.d.: not determined.

Data from: (1) Mallya etal., 1990; (2) Welgus etal., 1981; (3) Welgus etal., 1985c; (4) Birkedal-Hansen
etai, 1985; (5) Welgus etal., 1980; (6) Welgus etal., 1982; (7) Fields etal., 1990; (8) Windsor etal.,
1991; (9) Fields eta/., 1987; (10) Netzel-Arnett etal., 1991a; (11) Enghild etal., 1989.
216
TABLE 8
Kinetic Parameters for the Hydrolysis of Soluble Types I, II, and III Collagens and
Synthetic Peptides by Human FIB-CL and PMN-CL at 30 C
FIB-CL
Substrate
Rat type 1
Human type 1
Calf type II
Human type III
GPQGIAGQ
GPQAIAGQ
cat h " 1
KM,M
16
44
3.2
350
730
4,500
PMN-CL
Kcat/KM,M-h-i
0.8
0.8
2.4
1.7
3,300
2,800
19
54
1.3
210
0.22
1.6
690
460
130
1.0
1.0
2.3
200
39,000
87,000
2.5
6,900
4,800
690
460
57
80
5.7
18
From Netzel-Arnett, S., G. Fields, H. Birkefal-Hansen and H. E. Van Wart: J. Biol. Chem.
266:6747-6755 (1991a).
peptide bonds in several different proteins, as

summarized in Table 9. Collagenases also cleave
(3-casein quite readily (Table 7), but the susceptible bond(s) have not been identified.
B. Stromelysins
The stromelysin group of MMPs includes at
least two members, SL-1 and -2. It is more uncer-
TABLE 9
Collagenase Cleavage Sites in Noncoilagenous Proteins
Substrate
Sequence
Ref.
Human oc2-M
Human PZP
Human PZP
Human PZP
Rat a1 M
Rat ot1 M
Rat a2M
Rat oc2M
Rat a1-l3, variant 2
Gly-Pro-Glu-Gly-Leu-Arg-Val-Gly
Tyr-Gly-Ala-Gly-Leu-Gly-Val-Val
Ala-Gly-Leu-Gly-Val-Val-Glu-Arg
Ala-Gly-Leu-Gly-Ile-Ser-Ser-Thr
Glu-Pro-Gln-Ala-Leu-Ala-Met-Ser
Gln-Ala-Leu-Ala-Met-Ser-Ala-Ile
Ala-Ala-Tyr-His-Leu-Val-Ser-Gln
Met-Asp-Ala-Phe-Leu-Glu-Ser-Ser
Glu-Ser-Leu-Pro-Val-Val-Ala-Val
1
1
1
1
1
1
1
1
1
Rat a1-l3, variant 1

Human-FIB-CL, hinge
Human-FIB-CL, propeptide
Human-PMN-CL, propeptide
Human-oc1PI
Human-oc1PI
Human alACT
Chick-ovostatin
Ser-Ala-Pro-Ala-Val-Glu-Ser-Glu
Pro-Val-Gln-Pro-Ile-Gly-Pro-Gln
Asp-Val-Ala-Gln-Phe-Val-Leu-Thr
Val-Ala-Gln-Phe-Val-Leu-Thr-Glu
Ala-Gln-Phe-Val-Leu-Thr-Glu-Gly
Gly-X -Phe-Met-Leu-Thr-Pro-Gly
Gly-Ala-Met-Phe-Leu-Glu-Ala-Ile
Glu-Ala-Ile-Pro-Met-Ser-Ile-Pro
Leu-Leu-Ser-Ala-Leu-Val-Glu-Thr
Leu-Asn-Ala-Gly
1
2
3
3
3
4
5, 6
6, 7
5, 6
8
Abbreviations: PZP: pregnancy zone protein; a1M: alpha-1-macroglobulin; OC1I3:
alpha-1 -inhibitor-3; <x1-PI: alpha-1-proteinase Inhibitor, alpha-1-antitrypsin; a l ACT: alpha-1-antichymotrypsin.

Data from: (1) Sottrup-Jensen and Birkedal-Hansen, 1989; (2) Birkedal-Hansen
etai, 1988; (3) Grant et ai, 1987; (4) Mallya et ai, 1990; (5) Mast et ai, 1991;
(6) Desrocher et ai, 1991; (7) Knauper et ai, 1990; (8) Enghild et ai, 1989.
Modified from Netzel-Arnett, S. Fields, H. Birkedal-Hansen and H. E. Van Wart:
J. Bioi Chem. 266:6747-6755 (1991a).
217
tain whether the novel SL-3 (Bassett et al, 1990)

merits inclusion in this group or in a separate,
new group. SLs share with collagenase the typical
five-domain structure. The only major structural
difference is a slightly longer hinge region in SLs
(26 residues) than in collagenases (16 residues)
(Appendix 1). Comparison of the enzymatic properties of n-SL-1 and r-SL-2 (Nicholson et al,
1989) suggests that the two enzymes have virtually identical substrate specificities and cleave a
wide range of extracellular matrix protein substrates, including proteoglycan core protein, type
IV and V collagen, the nonhelical NH2 and COOHterminal peptides of type II collagen, fibronectin,
and laminin (see Table 2). Peptide bonds in natural proteins cleaved by SL-1 are shown in Table
10.
keratinocytes in the human, but an SL-1 (or

SL-2) homologue is induced in murine skin epidermis by phorbolester treatment (Matrisian et
al, 1986a; Wilhelm et al, 1987). SL-1 has been
extracted and purified from human patellar cartilage (Gunja-Smith et al, 1989).
SL-2 transcripts appear to be expressed less
abundantly than those of SL-1. Sirum and
Brinckerhoff (1989) found little or no SL-2 mRNA
in cultured human foreskin and synovial fibroblasts and were unable to significantly stimulate
mRNA levels either by growth factors (EGF and
IL-1) or by phorbolesters. On the other hand,
Muller et al (1988) observed that SL-2 message
was 4 to 5 times more abundant than that of
SL-1 in RNA extracts of human head and neck
tumors, and Breathnach et al (1987) identified rat
TABLE 10
Stromelysin Cleavage Sites in Natural Proteins
Substrate
SL-1
SL-1
PG core protein
PG link protein,
human
PG link protein,
rat
ori-PI
a1-ACT
AT-III
Sb P
cc2M
cc2M
Ovostatin
Sequence
Asp-Thr-Leu-Glu-Val-Met-Arg-Lys
Asp-Val-Gly-His-Phe-Arg-The-Phe
Ile-Pro-Glu-Asn-Phe-Phe-Gly-Val
Arg-Ala-Ile-His-Ile-Gln-Ala-Glu
Ref.
1
1
2
3
His-Ile-Gln-Ala-Glu-Asn-Gly-Pro
Glu-Ala- Ile-Pro-Met- Ser-Ile-Pro
Leu-Leu- -Ser-Ala-Leu- Val-Glu-Thr
Ile-Ala- Gly-Arg-Ser- Leu-Asn-Pro
Lys-Pro- Gln-Gln-Phe- Phe-Gly-Leu
Gly-Pro- -Glu-Gly-Leu- Arg-Val-Gly
Arg-Val- Gly-Phe-Tyr- Glu-Ser-Asp
Leu-Asn- Ala-Gly-Phe- Thr-Ala-Ser
4
4
4
5
6
6
6
Abbreviations: PG: proteoglycan; a1-PI: alpha-1-proteinase inhibitor,

alpha-1-antitrypsin; oc1-ACT: alpha-1-antichymotrypsin; AT-III:
antithrombin-lll; Sb P: substance P.
Data from: (1) Nagase et al, 1990; (2) Fosang et al., 1991;
(3) Nguyen et al, 1989; (4) Mast et al, 1991; (5) Harrison et al,
1989; (6) Enghild et al, 1989.
SL-1 is expressed by stromal cells either constitutively or after induction by growth factors/
cytokines (IL-1, EGF, TNF-oc, PDGF) or phorbol
esters (Chin et al, 1985; Herron et al, 1986;
Wilhelm et al, 1987). The enzyme does not appear to be expressed by PMN leukocytes and
SL-2 transcripts in Rous sarcoma virus transformed rat embryo fibroblasts. Moreover, SL-2
transcripts were inducible by TPA in the rat in the
presence of some, but not all, fetal bovine sera.
We have recently observed that TPA, TGF-a,
TNF-a, and EGF, which induce expression of the
218
SL-1 gene in human fibroblasts, induce expression exclusively of the SL-2 gene in human
keratinocytes (Birkedal-Hansen et ai, unpublished).
C. Gelatinases/Type IV Collagenases
The Mr 72K GL is perhaps the most widely
distributed of all MMP and has been identified in
skin fibroblasts (Seltzer etai, 1981), keratinocytes
(Salo et ai, 1991), chondrocytes (Lefebvre et ai,
1991), endothelial cells (Kalebic et ai, 1983),
monocytes (Garbisa et ai, 1986), osteoblasts
(Overall and Sodek, 1987), and in a number of
other normal and transformed cells (Sang et ai,
1990; Arthur et ai, 1989; Liotta et ai, 1979; Salo
et ai, 1983). Mr 72K GL does not appear, however, to be expressed by PMN leukocytes but is
present in a circulating form in plasma (Johansson
and Smedsrod, 1986). The Mr 92K GL is produced by keratinocytes (Wilhelm et ai, 1989;
Salo et ai, 1991), monocytes and alveolar macrophages (Mainardi et ai, 1984), PMN leukocytes (Hibbs et ai, 1985; Murphy et ai, 1989a)
and in a number of malignant or transformed cells
(Lyons et ai, 1991a; Wilhelm et ai, 1989; Moll
et ai, 1990, Davis and Martin, 1990). It is interesting to note that PMN, which express a unique
and distinct collagenase gene, utilize the same
gene for the Mr 92K GL, although in a manner
that yields a storable rather than a secreted gene
product.
The two gelatinases are similar in many characteristics, including high affinity for gelatin both
in the latent and activated form. The main structural difference is the extended 54 amino acid
hinge region sequence in the Mr 92K GL that
shares some homology with the oc2 chain of type
V collagen (Wilhelm et ai, 1989) (Appendix 1).
Another difference is that the Mr 72K GL precursor is tightly associated with TIMP-2, whereas
the Mr 92K GL proenzyme is associated with
TIMP-1 (Goldberg et ai, 1989; Wilhelm et ai,
1989). Howard et ai (1991a) showed that TIMP2 remained bound to the gelatinase even after
mercurial activation, but was able to retard
autoactivation.
Mr 72K GL is expressed constitutively by
most cells in culture but is only moderately responsive (two- to four-fold) to TPA and growth
factors that induce the Mr 92K GL (Salo et ai,

1985;Templetonâ/., 1990; Huhtala <tf a/., 1991).
It is interesting to note that TGF-p, which abrogates transcription of collagenase and SL-1
(Edwards et ai, 1987; Kerr et ai, 1990),
upregulates Mr 72K GL by two- to four-fold and
Mr 92K GL by up to eight-fold (Salo et ai, 1991;
Overall et ai, 1991a). These findings are of considerable interest because TGF-p in general appears to downregulate rather than stimulate MMP
expression. Examination of the promoter regions
of the two genes has only partially explained
these differences (Huhtala et ai, 1990a; 1991).
The Mr 72K GL gene contains no TPA-responsive elements (AP-1 binding sites), whereas the
Mr 92K GL promoter has two such elements. In
addition, the Mr 92K GL promoter contains a
sequence 5' GGTTTGGGGA 3', which matches
the consensus sequence of the TGF-P inhibitory
element 5' GNNTTGGNGN 3', but this element
fails to confer inhibition by TGF-p for reasons
that are not apparent (Huhtala et ai, 1991).
The Mr 72K GL and Mr 92K GL cleave a
number of peptide bonds such as Gly-Val, GlyLeu, Gly-Glu, Gly-Asn, and Gly-Ser in denatured
collagen to yield small peptides (Seltzer et ai,
1981; Seltzer et ai, 1990) (Table 11). Studies on
synthetic peptide substrates showed that Gly-Ile
was a preferred site of cleavage, and Trp or Leu
were good substitutions for Ala at the P' t site
(Netzel-Arnett et ai, 1991a). Besides gelatin and
types IV and V collagen, gelatinases also cleave
type VII collagen, found in anchoring fibrils (Seltzer et ai, 1989), cartilage type X collagen (Gadher
et ai, 1989; Welgus et ai, 1990), and elastin
(Senior et ai, 1991). Pepsin-solubilized type IV
collagen is degraded by the gelatinases (Mainardi
et ai, 1980b), whereas Mackay et ai (1990)
found that full-length type IV collagen apparently is resistant to proteolysis by both
gelatinases. These findings have raised questions as to whether gelatinases actually function
as true "type IV collagenases" in vivo. The initial cleavage site of soluble type IV collagen by
Mr 72K GL and Mr 92K GL is located approximately XU of the distance from the NH2-terminus
(Fessler et aiy 1984; Murphy et ai, 1989a), and
Hostikka and Tryggvason (1988) suggested that
the scissile bonds are Gly446-Ile447 [al(IV)] and
Gly464-Leu465 [a2(IV)], which are located in a
presumably triple helical domain.
219
TABLE 11
Mr 72K GL Cleavage Sites
Sequence
Ref.
Gly-Pro-Gln- Gly-Val- Arg-Gly-Glu

Gly-Pro-Ala- Gly-Val- Gln-Gly-Pro
Gly-Pro-Ser- Gly-Leu- -Hyp-Gly-Pro
Gly-Pro-Ala- Gly-Glu -Arg-Gly-Ser
Gly-Pro-Ala- Gly-Glx Asp-Gly-Pro
Gly-Ala-Lys- Gly-Leu- -Thr-Gly-Ser
Gly-Pro-Ala- Gly-Phe -Ala-Gly-Pro
Gly-Pro-Ile- Gly-Asn -Val-Gly-Ala
Gly-Pro-Hyl- Gly-Ser -Arg-Gly-Ala
Asp-Val-Ala- Asn-Tyr -Asn-Phe-Phe
1
1
1
1
1
1
1
1
1
2,3
Substrate
Gelatin,
Gelatin,
Gelatin,
Gelatin,
Gelatin,
Gelatin,
Gelatin,
Gelatin,
Gelatin,
Mr 72K
oc1(l)-CB8
oc1(l)-CB8
a1(l)-CB8
oc1(l)-CB8
oc1(l)-CB8
a1(l)-CB8
cc1(l)-CB7
cc1(l)-CB7
oc1(l)-CB7
GL
Data from: (1) Seltzer et al., 1990; (2) Stetler-Stevenson et al.,

1989a; (3) Howard et al., 1991a.
D. Other MMPs
SL-3 has been studied only at the message
level. SL-3 mRNA is expressed in human mammary tumors by mesenchymal cells adjacent to
invading epithelial tumor cells. The observation
that mRNA transcripts could be induced by TPA
and by growth factors in embryonic fibroblasts
suggests that the transcriptional regulation of this
enzyme is similar to that of FIB-CL, SL-1, and Mr
92K GL. The enzyme protein has not yet been
identified or isolated, but the deduced amino acid
sequence is consistent with the notion that it encodes a functional metalloproteinase. SL-3 has
the same principal domain structure as FIB-CL,
SL-1, and SL-2 but differs by an insert of 10
residues at the autolytic activation site (Basset et
al., 1990) (Table 5).
PUMP-1, which lacks the entire pexin-like
domain as well as the hinge region, cleaves a
wide range of substrates including fibronectin,
laminin, casein, gelatin, and proCL. The only
existing sequence information comes from cleavage of the insulin P chain (Woessner and Taplin,
1988):
FVNQHLCGSHLVEALYLVCGERGFFYTPKA
PUMP-1 cleaves Ala14-Leu15 and Tyr16-Leu17 in
the middle of the p chain. The enzyme is expressed in gingival fibroblasts (Overall and Sodek,
1991a) and has been isolated from the involuting
rat uterus and from rectal carcinoma cells
(Woessner and Taplin, 1988; Miyazaki et al.,

1990). PUMP-1 is induced by Con A and TPA in
human fibroblasts (Overall and Sodek, 1990).
E. Expression of Recombinant MMP
Expression of recombinant wild-type or mutant forms of MMP has proven to be a powerful
tool in analyzing structure-function relationships
and catalytic properties of the enzymes. Several
MMP may be expressed in E. coli with minimal
refolding efforts. These include FIB-CL (Windsor
et al., 1991), a truncated version of this enzyme
lacking the pexin domain (mini-CL), PUMP-1
(Windsor et al., unpublished), and a truncated
form of SL-1 (mini-SL-1) (Marcy et al, 1991).
The full-length forms of SL-1 and of the two GLs
can be obtained by expression in E. coli, but the
enzymes are not catalytically active, suggesting
that refolding is more involved. Expression is
driven from the bacteriophage T7 promoter by
introduction of the plasmid carrying the cDNA
construct into E. coli DE3 cells, which contain a
chromosomal copy of the T7 polymerase gene
under lac control. Induction with isopropyl P-Dthiogalactoside (IPTG) results in T7 RNA polymerase production, which allows for transcription
of mRNA and thus translation of the protein. It is
interesting to note that the recombinant enzymes
form intact latent constructs. This suggests that
the latent structure forms spontaneously during
and after synthesis.
220
Recombinant full-length proSL-1 and proCL

have also been expressed in eukaryotic systems
such as Cos cells and mouse mammary tumor
C127 cells using either the SV40 late promoter or
the mouse metallothionein I promoter with the
SV40 early or late polyadenylation site (Murphy
et al., 1987; Park et al, 1991). The enzymes
behave essentially as their natural counterparts,
except that r-FIB-CL displays lower activity on
collagen than the natural enzyme (Murphy et al,
1987; Docherty and Murphy, 1990), an observation also made when FIB-CL is expressed in E.
coli (Windsor et al, 1991). Quantin et al (1989)
expressed PUMP-1 as a fusion protein with the
staphylococcal protein A IgG-binding domain in
Cos cells using the plasmid pPROTA containing
the SV40 early gene promoter. This construct
permits easy purification of enzymes by passage
of the culture medium over IgG-Sepharose followed by elution with buffer containing
organomercurials. The recombinant PUMP-1 protein was shown to degrade the same substrates as
the native enzyme and to enhance or activate
proFIB-CL/CL. Full-length proSL-1 was recently
expressed in Hela cells by coinfection with a
vaccina virus vector containing the T7 promotor
and a T7 RNA polymerase construct (Galazka et
al, unpublished data). The recombinant enzyme
is fully latent yet catalytically competent. Rabbit
proCL has been expressed in baby hamster kidney (BHK) cells using the mouse metallothionein
I promoter (Brinckerhoff et al., 1990). The recombinant protein produced in this system, as its
natural counterpart, required SL-1 for maximal
activation. Devarajan et al. (1991) expressed the
52K unglycosylated form of human PMN-CL in
vitro in a reticulolysate system.
Several studies have been aimed at identifying the role of the various domains of MMP by
site-directed mutagenesis (Windsor et al., 1991;
Sanchez-Lopez et al, 1988; Park et al, 1991).
These studies have shown that the conserved
propeptide sequence PRCGVPDV is instrumental in maintaining the latency of the enzyme and
that the hemopexin-like domain plays a major
role in determining substrate specificity. "r-MiniCL", formed by deletion of the pexin-like domain
by mutagenesis (rather than autolysis), is inactive
against collagen yet has retained essentially full
catalytic activity against unfolded substrates such
as casein. If disulfide bond formation of the pexin-
like domain is abolished by site-directed mutagenesis, the activity against casein is only moderately
affected but collagen-cleaving activity is lost
(Windsor et al, 1991). Collier et al (1992) recently made alanine scanning mutations in the
fibronectin type II domains of the human 92K
GL. Several of these mutations destroyed gelatinbinding properties but did not adversely affect the
rate of cleavage of gelatin. This observation suggests that the gelatin-binding domains are not
required for catalytic activity against unfolded
collagen chains.
V. INHIBITION OF
METALLOPROTEINASE ACTIVITY
A. Synthetic Inhibitors
Chelating agents that interact with (or remove)
Zn2+ at the active site such as 1,10-phenanthroline
and EDTA are potent inhibitors of MMP but show
little if any selectivity and are therefore of limited
analytical or therapeutic potential. Several approaches have been employed to utilize substrate
specificity information to generate more selective
inhibitors based, in general, on synthesis of short
substrate analogue peptide sequences linked to
suspected chelating moieties such as hydroxamate,
thiol, phosphonamidate, phosphinate, and
phosphoramidate groups. The most potent of these
have reached ICs in the low nanomolar range. A
Leu-Phe-Ala thiol derivative synthesized by Gray
etal. (1986, 1987):
HS-CH 2 -(R,S)-CH-[CH 2 -CH(CH 3 ) 2 ]-CO-L-Phe-L-Ala-NH 2
reached IC50 values <1 nM for Mr 72K GL and 20

nM for collagenase. The terminal thiol group is
believed to form a coordination bond with the
active site Zn2+. Schwartz et al (1991) used detailed analysis of MMP peptide substrate specificities to optimize sulfur-based inhibitors against
human PMN- and FIB-CLs. The most potent inhibitors were thiols with IC50 in the 40 to 70 nM
range:
HS-(CH2-R-Leu)-Phe-Ala-HN2 and HS-(CH2-R-Leu)-Trp-Ala-NH0
Replacement of the chelating moiety with disulfide, sulfonate, sulfinate, sulfide, sulfoxide, and
221
sulfone analogs gave less-potent inhibitors.

Hydroxamate inhibitors have been produced by
Johnson et al. (1987) the best with a K{ of 5 nM:
HONH-CO-CH2-CH(Bu)-CO-NH-CH(Bu)-CO-NH-CH(Me)-COOEt
It is speculated that the terminal hydroxyl group

and the vicinal carbonyl group together form a
bidentate Zn2+ ligand. Phosphonamidate and
phosphoramidate peptide-analogue inhibitors
against FIB-CL and PMN-CL, which span the
scissile Gly-Leu or Gly-Ile bond and in which the
Gly carbonyl group is replaced by a P(=O) (OH),
have reached K{ values down to 20 to 30 nM
(Kortylewicz and Galardy, 1990; Mookhtiar et
al, 1987; Galardy etal, 1992). These include the
commercially available:
phthaloyl-Glyp-Ile-Trp-NHBzl
with a Kj of 25 nM with FIB-CL (Kortylewicz
and Galardy, 1989). Substitution of the scissile
peptide bond with a phosphinate linkage (PO2CH2) also gives potent inhibitors such as:
hydroxamate inhibitor of Mr 72K GL and Mr 92K

GL, HONHCO-CH 2 CH(i-Bu)CO-Tyr(OMe)NHMe (Searle SC39026; K. 2 nAf [Reich et al,
1988]) blocks trophoblast invasion in vitro,
whereas the noninhibitory analog (f-butoxy-LeuTyr (OMe)-NHMe) does not.
The studies summarized above have shown
that potent inhibitors of MMP can be produced
based on existing knowledge of substrate specificity coupled with trial-and-error experimentation. The best of these, with low nanomolar Ki
values, are clearly potent enough to hold therapeutic potential. It has proven more difficult to
target specific enzymes for inhibition while leaving others unaffected, although recent studies have
shown promise for this line of investigation. For
example, Netzel-Arnett et al (1991b) showed that
it is possible to optimize synthetic peptide substrates to yield as much as a 200-fold difference
in Kcat/KM between two highly homologous enzymes such as FIB-CL and PMN-CL. This finding inspires confidence that it will be possible to
construct synthetic inhibitors that for all practical
purposes target only a single MMP.
napthoyl-Glyp-cLeu-Trp-NHBzl
with K{ values near 10 nM (Galardy et al, 1992).
Tetracyclines and certain synthetic analogues
without antibiotic activity inhibit PMN-CL with
Kj in the micromolar range (Golub et al, 1983,
1985, 1987). The mechanism of inhibition is not
known, but it is suspected that it depends on the
chelating properties of the compounds. Tetracyclines are considerably less effective against FIBCL, but the reason for this selectivity is not known.
MMP inhibitor efficacy has mostly been determined with isolated enzyme preparations but a
number of studies suggest that these inhibitors
may have utility also in cell- or organ-based systems and may ultimately have therapeutic potential in intact organisms. A synthetic collagenase
inhibitor, ^-[-^-(benzyloxycarbonyO-amino-1 (R)-carboxypropyl]L-Leu-Tyr(OMe)-NHMe(CI1; SC 40827), produced by Searle, inhibits ovulation in perfused rat ovaries, whereas its inactive
(S) stereoisomer (CI-2; Searle SC40844) does not
(Brannstrom et al, 1988). This inhibitor also
blocks bone resorption in tissue culture (Delaisse
et al, 1985). Librach et al (1991) showed that a
222
B. Inhibiting Antibodies
Production of blocking antibodies represents
another attractive approach to the design of effective and specific inhibiting reagents. Affinitypurified polyclonal antibodies as well as monoclonal antibodies are both highly effective and
attain IC50 in the 1 to 10 |ug/ml range (20 to 200
nM). The best of these are almost as potent as
good synthetic inhibitors and are frequently highly
specific and readily discriminate between closely
related enzymes such as FIB-CL and PMN-CL
(Birkedal-Hansen etal, 1988). The production of
inhibiting monoclonal antibodies has been successfully accomplished for FIB-CL, Mr 72K GL,
and Mr 92K GL (Hoyhtya et al, 1988; BirkedalHansen et al, 1988; Hoyhtya et al, 1990; Lyons
etal, 1991a). The inhibition obtained with monoclonal antibodies to collagenase on collagenous
substrates is virtually complete (>90%) but similar antibodies against GLs on gelatin substrates
appear to be less effective (40 to 60% inhibition)
(Hoyhtya et al, 1988; Birkedal-Hansen et al,
1992c). Inhibiting antibodies have been shown to
block matrix degradation by live cells in several

model systems (Birkedal-Hansen et al, 1989;
Hoyhtya et al, 1990; Librach et al, 1991;
Gavrilovics et al, 1985; Meikle et al, 1989).
C. a-Macroglobulins
a-Macroglobulins inactivate susceptible proteinases by entrapment following cleavage of the
bait region (Sottrup-Jensen, 1989). The proteinase cleaves one or more bonds in the 40 residue
bait region and thereby initiates a conformational
change that leads to entrapment of the proteinase
(Sottrup-Jensen et al, 1989). In all a-macroglobulins except chicken ovostatin, this conformational change leads to hydrolysis of one internal
thiolester bond [-C(=O)-S-] per subunit and to
generation of a highly reactive glutamyl residue
(Sottrup-Jensen et al, 1989). The nascent glutamyl
residue reacts with a lysyl side chain exposed on
the surface of the attacking proteinase to covalently
cross-link the proteinase to the inhibitor by an
-lysyl-y-glutamyl bond.
Pro-Tyr-Gly-Cys-Gly-Glu-Glu-Asn-Met-Val
i
I
oo
Pro-Tyr-Gly-Cys-Gly-Glu-Glu-Asn-Met-Val
I
I
SH
OO
I
NH
I
Proteinase
Sottrup-Jensen et al. (1983) identified five lysine
residues located on patches on opposite sides on
the surface of the molecule as targets for e-lysyly-glutamyl crosslinks in the ct2M-trypsin complex. The trapping mechanism does not block the
active site per se and still permits access of lowmolecular-weight substrates. On high-molecularweight substrates, however, inhibition is complete because of steric hindrance. Although covalent capture following the initial entrapment appears to be an integral part of the inhibition mechanism by a2M, evidence suggests that the covalent
cross-linking of the proteinase is not necessary
for inhibition. Chicken ovostatin lacks the

thiolesters and therefore fails to covalently crosslink the proteinase yet has retained inhibitory
activity (Nagase and Harris, 1983). Moreover, it
is possible under certain conditions to retain the
inhibitory activity after disruption of the thiolesters
by treatment with nucleophilic agents such as
methylamine. The entire complement of cleavage
sites identified in the oc-macroglobulin bait region
is summarized in Figure 6. No information is yet
available on the conformation of the inhibitor or
its bait region, but the extraordinary susceptibility
to proteolysis suggests that the bait region consists of a readily accessible and highly flexible
stretch of peptide that is able to adapt to most
proteinases and present one or more scissile bonds.
Bait region cleavages catalyzed by FIB-CL and
SL-1 have been analyzed recently (Sottrup-Jensen
and Birkedal-Hansen, 1989; Enghild eftf/., 1989).
Some of these bonds are quite predictable (such
as the Gly-Leu bond of human oc2M), whereas
others are more surprising (Table 9,10; Figure 6).
The proteinase-inhibitor complexes formed with
oc2M are rapidly cleared from circulation in the
liver by uptake by receptors that recognize the
complex but not the unreacted inhibitor. Receptor-mediated clearing may also take place locally
in the tissue because fibroblasts, and perhaps other
cell types as well, possess functional oc2M receptors and are capable of internalizing and destroying the enzyme-inhibitor complexes (Van Leuven
et al, 1979; Dickson et al, 1981).
The cleavage of the human oc2M bait-region
by FIB-CL is extremely fast (rate constant >106
M ~ls~l) and results in complete and rapid capture
of the proteinase [tl/2 in the order of seconds
[Enghild et al, 1989; Sottrup-Jensen and BirkedalHansen, 1989]). The action of SL-1 is approximately 100-fold slower (kcat 5.8 x 10~3 s"1 vs.
480 x 10-3 s"1) (Enghild et al, 1989). Based on
k2/Ki or kcat/KM comparisons, these investigators
concluded that ot2M is a 150-fold better substrate
for FIB-CL than triple helical collagen type I and
perhaps the best existing substrate for this enzyme. ot2M is therefore a particularly effective
inhibitor of FIB-CL in the extracellular milieu.
The related inhibitors pregnancy zone protein
(PZP), ovostatin, and a-l-inhibitor-3 (a 113) are
also targets for cleavage by FIB-CL and presumably by other MMP as well (Nagase et al, 1983;
223
tr,pl,th,
su,st,tl
su,sb
he pa
Human cx2-M PKMC|PQLQQYEMHGPEGLRVGFYESDVMGRGHARLVHVEEPHT,tl,pe
ETVR
Human PZP PKSCSVIPSVSAGAVGQGYYGAGLGWERPYVPQLGTYNVIPLNNEQSSGPVP

tr,th,pa
tr,th,pa,
ct,pe,su,tl
pe,pa
Rat al-M
tl,pa,th
Ct,8U |
J pa su
pa I
8U,tl
I FIB-CL |
ii
sp
I I
PKVCtERLRDNKGIPAAYHLVSQSHMDAFLESSESPT
ct, su
RataiD
sp
PRYC|PMYQAYPPLPYVGEPQALAMSAI PGAGYRSSNIRTSSMMMMGASEVAQEVEVRJETVR
tr,th
Rat cc2-M
su
ETRR
pe
ii
PTYC|SFTDYDMVPLAVPAVALDSSTDRGMYESLPVVAVKSPLPQEPPRKDPPPKDPVI|ETIR
variant 2
ct,su
Rat alI3
variant 1
I FIB-CL [
tr,tb
sp
pe,ct
pe tr
I II I
PTYC -YEMNMWLSAPAVESELSPRGGEFEMMPLGVNKSPLPKEPPRKDPPPKDPVItETIR
FIGURE 6. Proteinase cleavage sites in a-macroglobulins. Boxes delineate bait regions. Location of cleavage
sites is from Sottrup-Jensen, L , 0 . Sand, L. Kristensen and G. H. Fey: J. Biol. Chem. 264:15781-15789 (1989),
Sottrup-Jensen, L. and H. Birkedal-Hansen J. Biol. Chem. 264:393-401 (1989), and Enghild, J. J., G. Salvesen, K.
Brew and H. Nagase: J. Biol. Chem. 264:8779-8785 (1989). cc: Clostridium histolyticum collagenase; eg: human
cathepsin G; cs: bovine chymosin; ct: bovine chymotrypsin; he: human PMN elastase; pa: papain; pe: porcine
pancreatic elastase; pi: human plasmin; sb: Streptomyces griseus proteinase B; sp: Staphylococcus aureus
proteinase; st: Streptomyces griseus trypsin; su: subtilisin Novo; th: bovine thrombin; tl: thermolysin; tr: bovine
trypsin.
Enghild etal, 1989; Sottrup-Jensen and BirkedalHansen, 1989).

A body of evidence suggests that oc-macroglobulins play an important role in the regulation of MMP activity. The rapid capture rates,
particularly with collagenase, render the reaction all but instantaneous. Moreover, Cawston
and Mercer (1986) showed that in a mixture of
TIMP and a2M, collagenase binds preferentially
to oc2M. It is also of note that the concentration
of oc2M in the interstitial fluid is quite considerable and often approaches plasma values
(2.2 mg/ml or 3 |iM, Tollefsen and Saltvedt,
1980). oc2M concentrations in synovial fluids
from rheumatoid arthritis patients range from
0.7 to 1.1 mg/ml whereas those of osteoarthritis
patients are slightly lower, 0.5 to 0.6 mg/ml,
(Virca et al, 1982, Abe et ai, 1973). Similar
224
determinations on gingival crevicular fluids

(GCFs) from variously inflamed sites have
yielded values from 0.2 to 2.4 mg/ml (Schenkein
and Genco, 1977; Tollefsen and Saltvedt, 1980;
Condacci et al, 1982; Sengupta et al, 1988).
These values suggest that a2M concentrations
in interstitial fluids are as much as 100- to 1000fold greater than those of TIMPs (Cawston et al,
1987; Hayakawa et al, 1991).
D. TIMPs
TIMPs form classic noncovalent bimolecular
complexes with the active forms of MMPs and
under certain circumstances with their latent forms
as well (as follows). TIMPs inhibit the activity of
the fully competent MMP and also appear to block
or retard MMP precursor activation (DeClerck et

aL, 1991a; Howard et aL, 1991a). The role played
by TIMPs in regulating matrix degradation may
therefore be exerted not only by classic proteinase
elimination but also by a perhaps even more potent effect, namely, the interceptive blockage of
MMP activation. Two members of the TIMP family, TIMP-1 and TIMP-2 (Carmichael^ a/., 1986;
Docherty et aL, 1985; Stetler-Stevenson et aL,
1989b; Boone et aL, 1990), have been cloned and
sequenced, but it is possible that still others exist
(Herron et aL, 1986). TIMPs appear to be distributed widely in tissues and fluids (Murphy et aL,
1991a; Welgus et aL, 1986) and to be expressed
by many different normal and transformed cell
types from skin (Hembry and Ehrlich, 1986), lung
(Welgus et aL, 1985a; Burnett et aL, 1986;
Horowitz et aL, 1989), mucosal membranes
(Geiger and Harper, 1981, Larivee et aL, 1986,
Drouin et aL, 1988), synovial tissues (Cawston
et aL, 1990; MacNaul et aL, 1990), cartilage
(Lefebvre et aL, 1990), dental pulp (Kishi and
Hayakawa, 1984), and ovaries (Curry etaL, 1990,
Freudenstein et aL, 1990).
TIMP-1 is a Mr 28K mannose-rich
sialoglycoprotein with a Mr 20K protein core
(Stricklin and Welgus, 1983; Carmichael et aL,
1986). The cDNA encodes a 23-residue signal
sequence, followed by a 184-residue inhibitory
domain (Docherty et aL, 1985). Human TIMP-1
contains 2 N-glycosylation sites, one at the triplet
N-Q-T (residues 30 to 32) and the second at
N-R-S (residues 86 to 88), but glycosylation does
not appear to be important for the inhibitory function of the molecule (Stricklin, 1986; Caterina et
aL, unpublished). TIMP-1, like most other proteinase inhibitors, is highly stable due in part to
six disulfide bonds (Williamson et aL, 1990), but
it is inactivated by limited proteolysis by broadspectrum serine proteinases such as trypsin, chymotrypsin, and elastase but appears to be resistant
to Pin (Okada et aL, 1988). The inhibitor forms
complexes with active FIB-CL, but not proFIBCL, in a 1:1 stoichiometry with Kds ranging from
10"9 M (Welgus et aL. 1985b) to 1.4 x 10~10 M
(Cawston et aL, 1983). Complex formation does
not appear to involve cleavage of the inhibitor,
and fully functional inhibitor can be recovered
from the complex (Murphy etaL, 1989b). Several
studies have suggested that TIMP-1 has erythroid
potentiating activity (EPA), but the physiologic

relevance of this observation has not been established (Docherty etaL, 1985; GassonetaL, 1985;
Huebner et aL, 1986; Golde et aL, 1980; Alitalo
etaL, 1990; Lotz and Guerne, 1991). Recent studies have shown that TIMP-1 also forms a complex with the zymogen form of Mr 92K GL
(Goldberg et aL, 1989; Howard et aL, 1991a,b).
TIMP-2, a Mr 22,000 unglycosylated protein,
was first described in rabbit brain capillary endothelial cells (Herron et aL, 1986) and bovine aortic endothelial cells (De Clerck et aL, 1989). The
inhibitor forms a complex with the zymogen form
Mr 72K GL (Goldberg et aL, 1989; StetlerStevenson et aL, 1989b). TIMP-2 can be isolated
in fully active form from its complex with Mr 72K
GL (Moore et aL, 1992; Ward et aL, 1991). TIMP-2
is 2 to 10 fold more effective than TIMP-1 against
the two GLs, whereas TIMP-1 appears to more
effectively inhibit FIB-CL (Howard et aL,
1991b). The sequence reveals a 26-residue leader
sequence followed by a 194 residue inhibitory
domain (Boone et aL, 1990; Stetler-Stevenson et
aL, 1990).
The TIMP-1 sequences from four species (rabbit, mouse, bovine, human) and TIMP-2 sequences
from two species (human, bovine), as well as a
partial sequence from chick TIMP (not yet classified as TIMP-1 or -2), are shown in Appendix 2.
Human TIMP-1 and -2 show an overall homology of 66% with 41% identity of residues (StetlerStevenson et aL, 1989b, 1990). The first 24 Nterminal residues are also highly conserved between TIMPs and across species, including the
VIRAK sequence recently noted by Woessner
(1991). Another highly conserved sequence exists around the CLW site (residues 155 to 157) in
the carboxy-terminal region. The identity of the
"active site" of the inhibitor remains speculative.
Because no bonds are cleaved as a result of complex formation, the identification of structures
that determine the binding properties of the
inhibitor is not straightforward. Deletion of the
COOH domain (past residue 134) does not appear
to abolish inhibitory activity or the ability to form
complexes with activated MMP (Murphy et aL,
1991b). Coulombe and Skup (1988) expressed
deletion mutants of murine TIMP-1 in an in vitro
reticulocyte lysate translation system and found
that residues 1 to 133 failed to form active TIMP-1
225
as did the COOH-terminal fragment (residues 125

to 192) (Appendix 2). These studies together with
those of Murphy et al (1991b) suggest that deletions after Cys134 (Coulombe and Skup, 1988,
residue 122) result in active inhibitor, whereas
deletions before this residue destroy inhibitory
activity. It is therefore most likely that the intact
inhibitory machinery of TIMP-1 is encoded by the
first 134 amino acid residues (Figure 7).
1989b; Murphy et al, 1989b; Stricklin and

Welgus, 1983). Complex formation is blocked by
small peptide inhibitors and therefore appears to
involve the MMP active sites (Lelievre et al,
1990). By contrast, complexes formed with the
latent zymogens of the Mr 72K GL and Mr 92K
GL apparently do not involve the enzyme active
site because the zymogens can be fully activated
by organomercurials while bound to the inhibitor
FIGURE 7.
Primary structure and disulfide bond assignment of human TIMP-1 and TIMP-2. Sequence data are from
Appendix 2. Disulfide bond assigment of TIMP-1 is from Williamson, R. A., F. A. 0. Marston, S. Angal, P. Koklitis, M.
Panico, H. R. Morris, A. F. Came, B. J. Smith, T. J. R. Harns and R. B. Freedman: Biochem. J. 268:267-274 (1990).
Proposed disulfide assignment of TIMP-2 is based on homology with TIMP-1.
7. Complex Formation
The noncovalent bimolecular complexes
formed between TIMP-l/TIMP-2 and catalytically active MMPs are entirely different from the
covalent complexes formed with oc-macroglobulins by the "venus-fly trap" mechanism described
in an earlier section (Stetler-Stevenson et al,
(Goldberg et al, 1989; Howard et al, 1991a).

Based on the observation that TIMP complexes
with most activated MMP are stable in 0.1 % SDS,
Murphy et al (1989b) and DeClerck et al (1991 a)
recently developed a capture method that permits
more detailed studies on the MMP intermediates.
Their findings showed that TIMP-2 captures the
nascent "switch open" form of 52K proCL and
226
prevents or retards its further autolytic conversion

to the 42K form. When Pin is used as the activator, 52K proCL was converted to the 46K intermediate, which is complexed by the inhibitor so
that further conversion to Mr 42K CL, which
usually follows formation of the Mr 46K intermediate, is blocked.
2. Regulation of TIMP Expression
The two TIMP genes are differently regulated. TIMP-1 expression is stimulated by a
variety of agents including growth factors (EGF,
TNF-oc, IL-1, TGF-(3), phorbol esters, retinoids,
and glucocorticoids (Clark et al., 1987; Edwards
et al, 1987; Coulombe and Skup, 1988; Nomura
et al, 1989; Mawatari et al, 1989; Overall and
Sodek, 1990). The human and murine TIMP-1
genes are located on the X chromosome whereas
the TIMP-2 gene is located on chromosome 17 in
man and chromosome 11 in the mouse (Huebner
et al, 1986; Jackson et al, 1987; Willard et al,
1989; Stetler-Stevenson, personal communication). Campbell et al (1991) identified a 38 bp
conserved sequence located in the first intron of
the murine TIMP-1 gene following the first
(untranslated) exon, which confers TPA-, serum-,
and TGF-P inducibility. This sequence contains a
near perfect AP-1 binding site (5' _843TGAGTAA
3'). Expression of TIMP-1 is stimulated somewhat by phorbolesters, although not nearly as
dramatically as the TPA-responsive MMPs
(Campbell et al, 1991). Expression of the TIMP-2
gene is downregulated by TGF-|3 and fails to
respond to phorbol esters (Stetler-Stevenson et
al, 1990). Both TIMP genes contain two or more
transcription start-sites and give rise to mRNAs
of varying size (Edwards et al, 1986; Campbell
et al, 1991; Stetler-Stevenson et al, 1990). Serum stimulation selectively induces shorter TIMP1 transcripts that lack the AUG start codon but
appear to be translated three-fold more efficiently
than the longer transcripts (Waterhouse et al,
1990).
3. Recombinant TIMPs
Recombinant TIMPs have been expressed
both in eukaryotic and prokaryotic systems. Ex-
pression of TIMP-1 in E. coli was successfully

accomplished by Carmichael and co-workers, who
also devised mechanisms to refold the protein
(Kohno et al, 1990). The refolding of TIMP from
E. coli extracts, however, is far from trivial, and
it is not certain that the final product, although
apparently fully active, is identical to the natural
inhibitor. More recently, TIMP-1 and TIMP-2
have been expressed by eukaryotic systems by
several laboratories either in murine myeloma
cells, monkey kidney cells (COS), Chinese hamster ovary cells, and vaccinia virus-infected HeLa
cells (DeClerckefa/., 1991a; Murphy etal, 1991b;
Williamson et al., 1990; Caterina, personal communication).
VI. BIOLOGIC FUNCTION OF MMPs

A. General Considerations
The high frequency with which MMP or inhibitor transcripts or protein can be detected in
cells, tissues, and interstitial fluids suggests that
these enzymes play a major role in the remodeling of the extracellular matrix. Yet, it is important
to recognize that there is still only little direct
evidence of the specific role of the various enzymes and their inhibitors in well-defined biologic processes. Likewise, the relationship of
MMP-dependent pathways to other pathways
mentioned at the beginning of this review remain
largely unresolved. Evidence in support of involvement of any or all of the MMPs have come
largely from association studies that link the expression of a particular enzyme to a particular
process, either in time or in locale, and from
studies on in vitro and in vivo perturbation of cell
behaviors (ability to degrade, invade, and metastasize) associated with up- or downregulation of
MMP or inhibitors. Because of the limited opportunity that exists for effectively modulating expression of single MMP genes in vivo, the evidence is largely indirect, and when the complete
body of evidence is viewed, somewhat incomplete. Major gaps exist in terms of identifying the
biologic activation mechanisms of MMP and the
manner in which they are utilized and regulated
once outside the cell. It has also proven difficult
to identify the natural substrates for most of the
227
MMPs in part because of their relatively broad

and overlapping substrate specificities (Table 2).
Only in the case of interstitial collagenases is
there strong evidence for involvement in a specific catabolic reaction, the cleavage of interstitial
collagen fibrils. For each of the other enzymes,
the biologic substrates are less clearly defined.
The existing body of evidence suggests that MMP
are mobilized by cells in at least three different
ways: (1) by initiating transcription of growth
factor-responsive MMP genes (collagenase, SL1, Mr 92K GL, and PUMP-1 by fibroblasts, endothelial cells, macrophages, and keratinocytes),
(2) by constitutive expression of MMPs that are
largely unresponsive to growth factors and
cytokines (Mr 72K GL in most cell types), and (3)
by the triggered release of prepackaged MMP
from granule storage sites (PMN-CL, Mr 92K GL
in PMN leukocytes). The enzymes are initially
released into the peri- and extracellular environment and are perhaps under certain conditions
capable of (re)associating with the plasma membrane (Chen et al, 1984; Chen and Chen, 1987;
Zuckeref a/., 1987, 1990).
B. Growth, Development, and Involution

Growth and development are associated with
rapid cell movement and with restructuring and
reshaping of the extracellular matrix. A number
of studies have provided evidence for involvement of MMPs and their inhibitors in developmentally regulated processes (recently reviewed
by Werb et al, 1992). These include ovulation
(Brannstrom et al, 1988), embryonic growth and
differentiation (Brenner et al, 1989a; Nomura
et al., 1989), trophoblast invasion (Librach et al.,
1991), parturition (Rajabi et al, 1988, 1990),
skeletal growth and remodeling (Sellers et al,
1978; Dean et al, 1985; Delaisse et al, 1988;
Nomura et al, 1989; Flenniken and Williams,
1990), development of organs including salivary
glands (Nakanishi et al, 1986; Hayakawa et al,
1992), tooth germs (Nomura et al, 1989) and
ovaries (Nomura et al, 1989), and tadpole tail
and uterine post-partum involution (Gross and
Bruschi, 1971; Koob and Jeffrey 1974; Ryan and
Woessner, 1971; Woessner and Taplin; 1988;
Jeffrey, 1991). The evidence for involvement of
MMPs in these processes is based on identification of MMPs and inhibitors, or their transcripts,
during particular stages of development or on
perturbation or covariation studies that have shown
that modulation of expression of either the process or the gene product affects the other. For
example, Delaisse et al (1988) showed that PTH
(and several other resorption-inducing agents) at
the same time increased the rate of resorption of
cultured calvaria and the level of collagenase activity that can be extracted from the specimens. In
a few cases, functional blocking experiments using synthetic MMP inhibitors have strengthened
the evidence, that is, by inhibition of ovulation in
perfused rat ovaries (Brannstrom et al, 1988), of
resorption of bone explants (Delaisse et al, 1985),
and of trophoblast invasion in vitro (Librach et
al, 1991).
C. Tumor Growth and Metastasis

Invasive growth of primary tumors and
metastases are dependent on destruction and remodeling of stromal architecture, and a body of
evidence suggests that expression of one or more
MMPs is common among malignant tumors. The
complement of MMPs varies from tumor to tumor, and not all tumors of the same type express
even the most abundant MMP. Two recent studies illustrate this heterogeneity. Pajouh et al (1991)
found PUMP-1 transcripts in tumor cells of 14 of
18 prostate adenocarcinomas but only in 3 of 11
normal prostate glands; Mr 72K CL was expressed
in 6 of 10 adenocarcinomas but in none of 4
normal samples; FIB-CL transcripts were not identified in any of 9 adenocarcinomas and 4 normal
samples; and no evidence of SL-1 was found in
any of 13 adenocarcinomas (Pajouh et al, 1991).
In another recent study, Basset et al (1990) found
SL-1/2 transcripts in four of ten human breast
carcinomas, whereas nine contained SL-3 transcripts. SL-3 transcripts were also found abundantly in larynx carcinomas and less so in carcinomas from colon, ovary, kidney and lung. In the
same material, FIB-CL, Mr92K GL, and PUMP1 transcripts were found in 50 to 70% of the
tumors, and all of the tumors expressed low levels
oftheM r 72KGL.
228
1. Stromelysins
Transin (rat SL-1) mRNA was first discovered in transformed cells by a subtraction hybridization approach (Matrisian et al, 1985, 1986a).
A number of studies have suggested that SLs are
expressed more abundantly in malignant than in
benign tumors or normal tissues from the same
location. Breathnach and collaborators found SL
message in eight of nine murine squamous cell
carcinomas induced by carcinogen treatment
(Matrisian et al, 1986a) and showed that SL transcript levels are elevated in invasive, nonmetastatic
mouse skin carcinomas when compared with normal skin (Ostrowski et al, 1988). The authors
also reported that highly metastasizing tumors
generated by repeated carcinogen treatment
showed uniformly high levels of SL-1 message.
The incidence of SL expression in human
tumors varies greatly with tumor type. Muller et
al (1988) found that 6 of 50 primary carcinomas
contained SL transcripts. No SL message was
found in any of 32 adenocarcinomas or in two
sarcomas, nor were SL transcripts detected in any
"normal" tissues. The authors also noted that tumors that contained SL transcripts appeared to be
rapidly evolutive and poorly differentiated. Although no attempt was made to systematically
discriminate between SL-1 and SL-2 transcripts,
screening of a pool of mRNAs from all positive
tumors revealed that SL-2 mRNA was four to five
times more abundant than that of either SL-1 or of
two other members of the MMP gene family,
collagenase and PUMP-1.
2. Gelatinases/Type IV Collagenases
Expression of Mr 72K GL and Mr 92K GLs is
often associated with invasive and metastatic tumors (Liotta et al, 1979, 1980; Garbisa et al,
1987; Nakajima et al, 1987; Bernhard et al,
1990). The observation that GLs cleave soluble
type IV collagen (Salo et al, 1983, 1985) has
raised the question of whether these enzymes play
a role in the biologic degradation of basement
membranes and thereby endow cells with invasive
properties. Recent studies seem to indicate that
intact type IV collagen is much less susceptible to
GLs(Mackay?ftf/., 1991; Chen et al, 1991), and
it is possible that cleavage of type IV collagen is

not the only, or perhaps the most, important role
for these enzymes in tumor development. In support of the notion that GLs (or other MMPs) play
a role in invasiveness are the findings that several
MMP inhibitors modulate the invasive or metastatic potential of tumor cells. Inhibitors of CL,
u-Pa, and Pin inhibit invasion of amnion membranes by B16 melanoma cells (Mignatti et al,
1986). Highly metastatic cells show lower constitutive expression of TIMP than their nonmetastatic
counterparts (Ponton et al, 1991), and
downregulation of TIMP-1 expression by antisense
constructs increases the tumorogenicity of 3T3
cells (Khokha et al, 1989). Exposure to recombinant and native TIMP-1 or TIMP-2 blocks the
invasive properties of malignant cells and reduces
metastasis (Schultz et al, 1988; Alvarez et al,
1990; KMmetal, 1991; DeClerckera/., 1991b).
A similar effect is obtained by inhibiting antibodies to Mr 72K GL (Hoyhtya et al, 1990).
In the aggregate, a considerable body of evidence suggests that tumor cell behaviors such as
invasion through reconstituted or authentic membranes, and metastasis, are more commonly associated with high levels either of Mr 72K or Mr
92K expression. On the other hand, these enzymes are not tumor specific. Mr 72K GL is
expressed constitutively by most cell types in
vitro and is also present in normal plasma at a
concentration of 300 to 500 ng/ml (Collier et al,
1988; Johansson and Smedsrod, 1986; Zucker et
al, 1992). Mr 92K GL is expressed constitutively
in PMN leukocytes (Bergmann et al, 1989), is
inducible by TPA and growth factors/cytokines in
normal keratinocytes, macrophages and endothelial cells (Salo et al, 1991, Goldberg et al,
1989), and is present in high concentrations (340
ng/ml) in human saliva (Davis, 1991). The evidence summarized above suggests that MMP
expression is more often associated with aggressive tumors but that it is not a sufficient and
perhaps not even a necessary requirement for
malignancy. It is likely that MMPs constitute but
one of several tools available to tumor cells to
remodel the extracellular matrix as a necessary
step in expansive, invasive, and metastatic growth.
Existing data suggest that there are at least two
different ways to activate MMP expression in the
coordinated tumor/stromal growth process: either
229
by expression of MMPs by the tumor cells themselves (SL-1 in carcinogen-induced mouse squamous cell carcinomas; PUMP-1 in prostate
adenocarcinomas) or by induction of MMP in
adjacent stromal cells (SL-3 in human breast carcinomas). These findings are of considerable interest because they suggest that tumor cells may
orchestrate remodeling of the extracellular matrix
not only by expression of MMP but also by inducing MMP expression in adjacent stromal cells.
D. Inflammatory Diseases
1. Rheumatoid Arthritis
The continuous or intermittent destruction of
rheumatoid joints has been the subject of several
investigations aimed at establishing a link between disease progression and MMP expression.
Several studies have shown increased collagenase activity in rheumatoid synovial fluids (Harris et al, 1969; Cawston et al, 1984; Hayakawa
et al, 1991) and in culture media from rheumatoid synovial tissues and cells (Evanson et al,
1968; Woolley etal, 1978; McNaul etal, 1990).
Collagenase and SL-1 have been identified near
the areas of destruction at the cartilage-pannus
junction and in the synovial lining cells (Woolley
etal, 1977; Okadaef a/., 1989,1990; McCachren
et al., 1990). In a comparative study, Case et al.
(1989a) found higher levels of SL-1 mRNA and
protein in human synovium from patients with
rheumatoid arthritis than in patients with
osteoarthritis. SL-1 transcripts and SL-1 protein
were detected in lining cells, and in the underlying stroma, and in chondrocytes and osteoclasts
of the joints of rats with streptococcal cell wall
arthritis but not in the same locations of athymic
rats (Case et al, 1989b). The development of
polyarthritis in these animals was antagonized by
TGF-P, presumably by downregulation of MMP
expression or upregulation of TIMP-1 (Brandes
et al, 1991). TIMP-1 levels are sigificantly elevated in synovial fluid from patients with rheumatoid arthritis when compared with controls (1.5
|ng/ml vs. 357 ng/ml) (Hayakawa etal, 1991) and
TIMP levels in synovial fluids of rheumatoid arthritis patients are as much as four-fold higher
than in serum (4.6 (ig/ml vs. 1.3 |Ltg/ml) (Cawston
etal, 1987).
2. Human Peridontal Diseases

Collagenase that appears to be predominantly
of the PMN-type is frequently detected in GCF
in natural human periodontitis and in experimental periodontitis in dogs and monkeys (Overall et al, 1991b; Sorsa et al, 1988). Enzyme
activity generally increases with disease severity
(Ohlssonefa/., 1973; Golub and Kleinberg, 1976;
Kowashi et al, 1979; Villela et al, 1987), as
does the ratio of active to latent enzyme
(Kryshtalskyj and Sodek, 1987; Kryshtalskyj et
al, 1986). Treatment aimed at reducing the level
of inflammation again lowers GCF collagenase
activity (Larivee et al, 1986). A Mr 92K GL that
is probably also of PMN origin has been detected in GCF as well (Overall et al, 1991b;
Gangbar et al, 1990). It is interesting to note
that neither Mr 72K GL or the SL-1 that might
serve as markers of stromal cell involvement
have been identified in GCF. The data summarized above are supportive of involvement at
some level of MMP in inflammatory diseases of
the human periodontium, yet the observation that
the enzymes are predominantly, and perhaps exclusively, of PMN origin and presumably carried to the GCF in migrating PMNs raises the
question of to what extent metabolically important enzymes in the periodontal tissue can be
measured in a meaningful way in GCF.
Immunolocalization studies by Woolley and
Davies (1981) have suggested that collagenase
is in fact expressed in the stromal cells of inflamed human gingiva, but the enzyme does not
appear to reach the GCF in quantities that are
amenable to analysis.
A number of potential mechanisms and scenarios may explain the important pathogenic elements of human periodontal diseases, but it has
not yet been possible to unequivocally identify
those microbial-host cell interactions that lead to
attachment loss. A body of evidence has established that expression or activity of MMP may
be stimulated in a variety of cultured cells derived from the human periodontal tissues. In
addition to PMNs, these include fibroblasts
(Overall et al., 1991), macrophages (Wahl et al.,
1974, \91T,Cxuyetal, 1988), and keratinocytes
(Linetal, 1987; Salo et al, 1991). MMP activity may be stimulated either directly by micro-
230
bial products from the bacterial plaques that

colonize the teeth and their surroundings (Uitto
and Raeste, 1978; Robertson et al., 1974) or
indirectly by inflammatory mediators generated
in response to oral microorganisms (Lyons et
al., 1991b). Candidate microbial products that
are likely to play a role in the transcriptional
activation of endogenous degradative pathways
include bacterial lipopolysaccharide (LPS) (Wahl
et al, 1974), proteinases (Birkedal-Hansen et
al, 1984; Uitto et al, 1989), and perhaps lectins
(Overall and Sodek, 1990). Analysis of GCF
have provided evidence that mediators such as
IL-la,(3 and TNF-a which are potentially capable of inducing MMP expression, are present
in GCF in physiologically meaningful concentrations (Masada et al, 1990; Rossamando et al,
1990).
ACKNOWLEDGMENTS
The expert assistance of Mr. Milton Pierson,
Mr. Greg Harber, and Ms. Gypsy Goebel is gratefully acknowledged. This project was supported
by USPHS grants DE 08228, DE 06028, DE
09122, DE 08580 (WGIM), DE 00283 (MKB),
and DE 00279 (ADC).
ABBREVIATIONS: 1,25 di(OH)D3:1, 25

dihydroxy vitamin D3; aII3: a-1 -inhibitor-3;
a2M: oc-2-macroglobulin; bFGF: basic fibroblast
growth factor; cAMP: 3'-5' cyclic adenosine
monophosphate; Con A: concanavalin A; EGF:
epidermal growth factor; FIB-CL: fibroblast-type
collagenase; GCF: gingival crevicular fluid;
IFNy. interferon-y; IL-I: interleukin-1; IPTG: isopropyl (3-D-thiogalactoside; LPS: lipopolysaccharide; MME: macrophage metalloelastase; MMP:
matrix metalloproteinase; Mr 72K GL: Mr 72,000
gelatinase/type IV collagenase; Mr 92K GL: Mr
92,000 gelatinase/type IV collagenase; NGF: nerve
growth factor; NIP: nuclear inhibitory protein;
PAI-1/2: plasminogen activator inhibitor-1/2,
PDGF: platelet-derived growth factor; PKC: protein kinase C; Pig: plasminogen; Pin: plasmin;
PMN: polymorphonuclear; PMN-CL: PMN-type
collagenase; PN-1: protease nexin-1; PTH: parathyroid hormone; PUMP-I: putative metallopoteinase-1; SL-1/2/3: stromelysin-1/2/3; t-Pa:
tissue-type plasminogen activator; TGF: transforming growth factor; TIMP-1/2: tissue inhibitor
of metalloproteinases-1/2; Tm: midpoint melting
temperature; TNF: tumor necrosis factor; TPA:
12-O-tetradecanoyl-phorbol-13-acetate; u-Pa:
urokinase-type plasminogen activator.
231
APPENDIX 1
MMP Sequences
The sequences are aligned to maximize homology. Asterisk indicates signal peptide cleavage site.
MMP sequence data from: human FIB-CL: Goldberg et al, 1986; Whitham et al, 1986; Templeton et
al, 1990; rabbit FIB-CL: Fini et al, 1987a; pig FIB-CL: Clarke et al, 1990; rat FIB-CL: Quinn et al,
1990; human PMN-CL: Hasty et al, 1990; Devarajan et al, 1991; human SL-1: Wilhelm et al, 1987;
Saus etal, 1986; rabbit SL-1: Fini etal, 1987b; mouse SL-1: Ostrowski etal, 1988; rat SL-1: Matrisian
et al, 1985; human SL-2: Muller et al, 1988; rat SL-2: Breathnach et al, 1987; human SL-3: Basset et
al, 1990; mouse MME: Shapiro et al, 1992; human PUMP-1: Muller et al, 1988; human Mr 72K GL:
Collier et al, 1988; human Mr 92K GL: Goldberg et al, 1989.
SIGNAL PEPTIDE
PROPEPTIDE
BASIC SEQUENCE
*
Z+.
"
CYSSEQUENCE
VDLVQKYLEK-Y-YNLKNDGRQVE
KRRNSGPV-YEKLKQMQEFFGLKVTGKPDAETLKVMKQPRCGVPDVAQ
PPLLLLLF---WGWSHS''FPAT--LETQEQD
VEMVQKYLEN-Y-YNLKDDWRKIP
KQRGNGLA-VEKLKQMQEFFGLKVTGKPDAETLKMMKQPRCGVPDVAQ
LPLLLLLL---WGVGSHG''FPAA--SETQEQD
ETQEQD
VEIVQKYLKN-Y-YNLNSDGVPVE
KKRNSGLV-VEKLKQMQQFFGLKVTGKPDAETLNVMKQPRCGVPDVAE
AEHYLKS-Y-YHPVT-LAGIL
KKSTVTST-VDRLREMQSFFGLDVTGKLDDPTLDIMRKPRCGVPDVGV
ATFFLLSWTHCWSLPL-P''YGDDDDDDLSEEDLEF
VSSKEKN
TKTVQDYLEKFY-QLPSNQYQST
RKNGTN-VIVEKLKEMQRFFGLNVTGKPNEETLDMMKKPRCGVPDSGG
MFSLKT
LPFLLLL
HVQISKA't FP
RRGPQ-PW---HAALPSSPAPAPATQE-APRPASSLRPPRCGVPDPSD
MAPSAAA-RALLPPMLLLLLQPPPLLARA LPPD--VHHLHAE
t
RRKDSN-LIVKKIQGMQKFLGLEVTGKLDTDTLEVMRKPRCGVPDVGH
MMHLA
FLVLLCLPVC
SA^ YPLSGAAK--EED--SNKDLAQQYLEK-Y-YNLEKDVKQF
MKS
LPILLLLCVAVC
SA't YPLDGAAR--GEDTS--MNLVQKYLEN-Y-YDLKKDVKQFV
RRKDSGPV-VKKIREMQKFLGLEVTGKLDSDTLEVMRKPRCGVPDVGH
MKT
LPTLLLLCVALC
SA^'YPLDGASR--DADTTN-MDLLQQYLEN-Y-YNLEKDVKQFV
KRKDSSPV-VKKIQEMQKFLGLEVTGKLDSNTLEVIRKPRCGVPDVGH
EN-Y-YGLAKDVKQFI
KKKDSS-LIVKKIQEMQKFLGLEMTGKLDSMTMELMHKPRCGVPDVGG
MKG
LPVLLWLCTAVC
SS'k YPLHG-S
EEDAG--MEVLQKYLEN-Y-YGLEKDVKQFT
KKKDSSPV-VKKIQEMQKFLGLKMTGKLDSNTMELMHKPRCGVPDVGG
ME
PLAILVLLCFPIC
SA'^YPLHGAVR---QDHST-MDLAQQYLEI-Y-YNFRKNEKQFF
KRKDSSPV-VKKIEEMQKFLGLEMTGKLDSNTVEMMHKPRCGVPDVGG
FLMMIVFLQVSAC
GA APMND-S---EF--AEW
YLSRFYDYGKDRIPMTKTKTNRNFLK
EKLQEMQQFFGLEATGQLDNSTLAIMHIPRCGVPDVQH
MK
MRLT
VLC-AVCLLPGSLA :^LPLP-QEAGGMS-ELQWEQA-QDYLKRFYLYDSET
KNANSLEAKLK---EMQKFFGLPITGMLNSRVIEIMQKPRCGVPDVAE
MEALMARGALTGPLRALCLLGCLLSHAAA k APSPIIK
FPGD-VAPKTDKELAVQYL-NTFYGCPKESCNLFVLKDT
LKK---MQKFFGLPQTGDLDQNTIETMRKPRCGNPDVAN
MSLWQ-PLV-LVLLVLGC---C
FA t APRQRPSTLVLFPGDLRTNLTDRQLAEEYLYRYGYTRVAEMRGES--K---SLGPALLLL---QKQLSLPETGELDSATLKAMRTPRCGVPDLGR
MHSF
MPG
HUMAN FIB-CL
RABBIT FIB-CL
PIG FIB-CL
RAT FIB-CL
HUMAN PMN-CL
HUMAN SL-3
HUMAN SL-2
HUMAN SL-1
RABBIT SL-1
MOUSE SL-1
RAT SL-1
RAT SL-2
MOUSE MME
HUMAN-PUMP-1
HUMAN Mr 72K GL
HUMAN Mr 92K GL
CATALYTIC DOMAIN
ACTIVATION SITES
CA-BINDING SITE I
N-NFTEYN
K-DFRNYN
FVLTPGNPRWENTHLTYRIENYTPDLSREDVDRAIEKAFQLWSNVSPLTFTKVSEGQADIMISFVRGDHRDNSPFDGPGGNLAHAFQPGPGIGGDAHFDEDERWTK-NFRDYN
FMLTPGNPKWERTNLTYRIRNYTPQLSFAEVERAIKDAFELWSVASPLIFTRISQGEADINIAFYQRDHGDNSPFDGPNGILAHAFQPGQGIGGDAHFDAEETWTN-TSANYN
GLSARNRQKRFVLSGG--RWEKTDLTYRILRFPWQLVQEQVRQTMAEALKWSD^
E-DASGTN
K-DTTGTN
[K-DTTGTN
-FSTFPGSPKWRKSHITYRIVNYTPDLPRQSVDSAIEKALKVWEEWPLTFSRISEGEADIMISFAVGEHGDFVPFDGPGWLAHAYAPGPGTNGDAHFDDDERWTE-DVTGTN
D-DVTGTN
G-PSGTN
TK-SFQGTN
UN
GGDSHFDDDELWTLGEGQVRV-KYGN
T
-FQTFEGDLKWHHHNITYWIQNYSEDLPRAVIDDAFARAFALWSA^/TPLTFTR /YSRDADIVIQFGVAEHGDGYPFDGKDGLLAHAFPPGPGIQGDAHFDDDELWSLGKGVWPTRFGN
232
HUMAN FIB-CL
RABBIT FIB-CL
PIG FIB-CL
RAT FIB-CL
HUMAN PMN-CL
HUMAN SL-3
HUMAN SL-2
HUMAN SL-1
RABBIT SL-1
MOUSE SL-1
RAT SL-1
RAT SL-2
MOUSE MME
HUMAN-PUMP-1
HUMAN Mr 72K GL
HUMAN Mr 92K GL
CATALYTIC DOMAIN
FIBRONECTIN TYPE II REPEATS
HUMAN F I E - C L
RABBIT F I E - C L
FIE-CL
PIG
PAT FIB-CL
HUMAN PMN-CL
HUMAN S L - 3
HUMAN SL-2
HUMAN S L - 1
RABBIT S L - 1
MOUSE S L - 1
RAT S L - 1
PAT s L - 2
MOUSE MME
HUMAN-PUMF-1
-.
-.
FrFLFNGreYNSZTDTGRSDGFLWCSTTYNFEIOXSKYGFCPHEALFTMGGNA^
F'r? IFElFSYSACTTrCRSDCLPWCSTTANYDTDDFPGFCPSERLYTRIXÂDGKFCQFPFIFCGQSYSACTTDGRSDGYRWCATA-
HINGE REGION
CATALYTIC DOMAIN
FIBRONECTIN TYPE II REPEATS
HUMAN Mr ~2K GI
HUMAN Mr 52K 31
CA-BINDING SITE 2
ZN-BINDING SITE
LHRVAA-HELGHSLGLSHSTDIGALMYPSY-TFS--GDVQLAQDDIDGIQAIYG
LYRVAA-HELGHSLGLSHSTDIGALMYPNYM-FS--GDVQLAQDDIDGIQAIYG
LYRVAA-HELGHSLGLSHSTDIGALMYPNYI-YT--GDVQLSQDDIDGIQAIYG
LFIVAA-HELGHSLGLDHSKDPGALMFPIYT-YTGKSHFMLPDDDVQGIQSLYG
LFLVAA-HEFGHSLGLAHSSDPGALMYPNYA-FRETSNYSLPQDDIDGIQAIYG
LLQVAA-HEFGHVLGLQHTTAAKALMSAFYT-FRYPL--SLSPDDCRGVQHLYG
LFLVAA-HELGHSLGLFHSANTEALMYPLYNSFTELAQFRLSQDDVNGIQSLYG
LFLVAA-HEIGHSLGLFHSANTEALMYPLYHSLTDLTRFRLSQDDINGIQSLYG
LFLVAA-HELGHSLGLFHSANPEALMYPVYNAFTDLARFRLSQDDVDGIQSLYG
LFLVAA-HELGHSLGLYHSAKAEALMYPVYKSSTDLSRFHLSQDDVDGIQSLYG
LFLVAA-HELGHSLGLEHSANAEALMYPVYKSSTDLARFHLSQDDVDGIQSLYG
LFLVAA-HELGHSLGLFHSNNKESLMYPVYRFSTSQANIRLSQDDIEGIQSLYG
LFLVAV-HELGHSLGLQHSNNPKSIMYPTYR-YLNPSTFRLSADDIRNIOSLYG
FLYAATHELGHSLGMGHSSDPNAVMYPTYGN-GDPQNFKLSQDDIKGIQKLYG
HUMAN FIB-CL
RABBIT FIB-17
PIG FIB-CL
PAT FIB-CL
HUMAN PMN-CL
HUMAN S L - 3
HUMAN SL-2
HUMAN SL-1
RABBIT SL-1
MOUSE S L - 1
PAT SL-1
PAT SL-2
MOUSE MME
HUMAIi-PUMP- i
G"rAÂTTAI\LCCFi-W3FCPDQGYSLFLVAA-HEFGHAMGLEHSQDPGAUlAPIY-TYT--KNFRLSQDDIKGIQELYG
^ _ . z.T^SN-ZSEl'i^GFCPDQGYSLFLVAA-HEFGHALGLDHSSVPFJU^YPMY-
HUMAN Mr ~LY.
HUMAN Mr "rCr"
___
PEXIN DOMAIN I
HINGE REGION
- ---
RSSNFVQPIGPQTPKACDSKLTFDAITTIRGEVMFFKDRFYMRTNPFY--PEVEI^
PSQNPSQPVGPOTPKVCDSKLTFDAITTIRGEIMFFKDF^YMR^
PSENPVQPSGPOTPQVCDSKLTFDAITTLRGEIJ4FFKDRFYMRTNSFY--^
PGDEDPNPKHPKTPEKCDPALSLDAITSLRGETMIFKDRFFWRLHPQQVEPELFL--TKS--FWPELPDHVDAAYEHPSRDLMFIFRGRKFWAL
LSSNPIQPTCPSTPKPCDPSLTFDAITTLRGEILFFKDRYFVmRHPQL--QRVEMN-FISL-FVJPSLPTGIQAAYEDFDRDLIFLFKGNQYWAL
-CPWPTVTSRTPALGPQAGIDTOEIAPLEPDAPPDACE-A-SFIAVSTIRGELFFFKAGFVWRLRGGQ--LQPGYPALASR-m
PFPASTEEP-LVFTKSVPSGSEMPMCDPALSFDAISTLRGEYLFFKDRYFWRRSHWN--PEPEFH-LISA-FV/PSLPSYLDAAYEVNSRDTWIFKGNEFWAI
PPPDSPETP-LVPTEPVPPEPGTPANCDPALSFI^VSTLRGEILIFKDRHFWRKSLRK--LEPELH-LISS-FWPSLPSGVDAAYEVTSKDLWIFKGNQFWAI
PAPASPDN---VPMEPVPPGSCTPVMCDPDLSFDAISTLRGEILFFKDRYFWR^^
TPTASPDVLVLVPTKSNSLEPETSPMCSSTLFFDAVSTLRGEVLFFro
PFTESPDVLV-VPTKSNSLDPETLPMCSSALSFnAVSTLRGEVLFFKDRHFWRKSLR-T-PEPGFY-LISS-FWPSLPSNME^YEVTO
__
ARPSSDATV-VPVPSVSPKPCTFVKCDPALSFDAVTOLRGEFLFFKDraF
AFvT.PPSL
TKPSSP-PST--FCHQSLSFDAVTIVGEKILFFKDI(^FWWKLPG---SPATNITSISSI-WPSIPSAIQAAYEIESRNQLFLFia)EKYWLI
--
KRSNSRKK
ASPDIDL-GTGFTPTLGPVTPEICKQDIVFDGIAQIRGEIFFFKDRFIWRTWPR-DKPMG-PLL
r.PTGPPSAGPTGPPTAGPSTA-TTVPLSPVDD
ACNVNI-FnAIAEIGNQLYLFKTCKYWRFSEGRGSRP^-PFLIADKWPA-LPRKLDSVFEEPLSKKLFFFSGRQVWVHi'
PEXIN DOMAIN 2
FKEI'iSFKIIHSFhTIHR-
PEXIN DOMAIN 3
HUMAN FIB-CL
RABBIT FIB-CL
PIG FIB-CL
PAT FIB-CL
HUMAN PMN-CL
HUMAN S L - 3
HUMAN SL-2
HUMAN S L - 1
RABBIT S L - 1
MOUSE S L - 1
PAT S L - 1
RAT SL-2
MOUSE MME
HUMAN - PUMP-1
HUMAN Mr 7 2K :
HUMAN Mr 92K :
PEXIN DOMAIN 4
LSEENTGKTYFF/ANKYWRYDEYKRSMDPGYPKMIAHDFPGIGHKVDAVFMK-DGFF- -YFFHGTRQYKFDPKT-KRILTL---QKANS--WFNCRKN
GFPRSVNHIE^VSEEDlX3KTYFPyANKYWRYDEYKRSMDAGYPKMIEYDFPGIGNKVDAVFKK-DGFF- - YFFHGTRQYKFDPKT-KRILTL -QKANS- -WRJCRKN
3FPSr/KNIDAAVFEEDTGKTYFP/AHECWRYDEYKQSMDTGYPKMIAEEFPGIGNKVnAVFQK-DGFL- -YFFHGTRQYQFDFKT-KRILTLQKANS--WFHCRKN
IFPK^vVJlLSAA'/HFEDTGKTLFFSGNHVWSYDDANQTMDKDYPRLIEEEFPGIGDKVDAVYEK-NGYI- -YFFM5PIQ--FEYSIWSNRIVRVM--PTNS--LLWC
lFPSS\'QAIDAAVFYRS--KTYFPyNDQFWRYDNQRQFMEPGYPKSISGAFPGIESKVDAVFQQ-EHFF- -HVFSGPRYYAFDLIA-QRVTRV---ARGNK--WLMCRYG
JL'/Pi^Pv^-AAL'/WGPEKIJKIYFFRGFUDYWRFHPSTRRVDSPVPRR-ATDWRGVPSEIDAAFQDADGYA- -YFLRGRLYWKFDPVK-VKALEGFPRLVGPD--FFGCAEPANTFL
JFPPTIRKIDAAVSDKEKKKTYFFAADKYWRFDENSQSMEQGFPRLIADDFPGVEPKVDAVLQA-FGFF- -YFFSGSSQFEFDPNA-RMVTHI
LKSNS--WLHC
SFPFT/RKIDM ISDKEKNKTYFF/EDKYWRFDEKRNSMEPGFPKQIAEDFPGIDSKIDAVFEE-FGFF- -YFFTGSSQLEFDPNA-KKVTHT---LKSNS--WLNC
r
SF?STIPÎD/ÎSDKERJCK'nTFv EDKYWRFDEKRQSLEPGFPRHIAEDFPGINPKIDAVFE-AFGFF- -YFFSGSSQSEFDPNA-KKVTHV---LKSNS--WFQC
JLPATvrKKIDA^JSN?^KJTYFF\/EDKYWRFDEKKQSMEPGFPRKIAEDFPGVDSRVDAVFE-AFGFL- -YFFSGSSQLEFDPNA-KKVTHILKSNSWR1C
^LPET.'iKIDA^JSLKDQKKTYFF/EDKFWRFDEKKQSMDPEFPRKIAENFPGIGTKVDAVFE-AFGFL --YFFSGSSQLEFDPNA-GKVTHI---LKSNS--WFHC
JFPPT^TJ<IDA^.WEKEKKKTYFFvrGDKYWRFDETRQLMDKGFPRLITDDFPGIEPQVDAVLH-AFGFF YFFCGSSQFEFDPNA-RTVTHT---LKSNS--WLLC
.FsA^Tao.ÂAVFDPLRQKWFFVDKHYWRYDVRQELMDPAYPKLFSTHFPGIKPKIDAVLYFKRH-- -YYLFQGAYQLEYDPLF-RRVTKT---LKSTSWFGC
- * -: ^3-L^-LPFCVQRV-DA^NON'SraiKKTYIFAGDKFWRYNFATOaMD^
VrFFl,EFLlL^VAwV-TG;iLRSGR-GKMLLFSGRRLWRFDVKAQMVDPRSASEVDRMFPGVPLI7^
233
APPENDIX 2
Primary Structures of TIMPs 1 and 2

Sequences are aligned to maximize homology. TIMP sequence data are
from: human TIMP-1: Docherty et al, 1985; Carmichael et al, 1986; rabbit
TIMP-1: Freudenstein et al, 1990; mouse TIMP-1: Gewert et al, 1987; calf
TIMP-1: Freudenstein et al, 1990; human TIMP-2: Stetler-Stevenson et al,
1989b; Boone et al, 1990; calf TIMP-2: Boone et a/., 1990; chicken TIMP:
Staskus et al, 1991.
20
10
rabbit-TlMP-l
mouse-TlMP-1
calf-TIMP-1
human-TlMP-1
human-TIMP-2
calf-TIMP-2
chick-TIMP
C
C
C
C
C
C
7
T
S
T
T
S
S
T
C
C
C
C
C
C
C
30
V P P H P Q T A F C N S D L V I R A K F V G A P E V
A P P H P Q T A F C N S D L V I R A K F M G S P R I
V P P H P Q T A F C N S D V V I R A K F V G T A E V
V P P H P Q T A F C N S D L V I R A K F V G T P E V
S P V H P Q Q A F C N A D V V I R A K A V S E K E V
S P V H P p Q A F C N A D I V I R A K A V N K K E V
V P I H P Q D A F C N S D I V I R A K V V G K K I M
N
N
N
N
D
D
40
50
60
- H T T L Y Q - - - - - R Y E I K T - T K M F K G F D A L
- E T T L Y Q - - - - - - R Y K I K M M T K M L K G F K A V
- E T A L Y Q - - - - . - - R Y E I K M - T K M F K G F S A L
rabbit-TIMP-1
mouse-TlMP-1
calf-TIMP-1
human-TIMP
human-TIMP
calf-TIMP-2
- Q T T L Y Q - - - - - - R Y E I K M - T K M Y K G F Q A L
S G N D I Y G N P I K R I Q Y E I K Q I K M F K G P E K S G N D I Y G N P I K R I Q Y E I K Q - I K M F K G P D Q -
rabbit-TIMP-1
mouse-TIMP-1
calf-TIMP-1
human-TlMP-1
human-TIMP-2
calf-TIMP-2
G
G
R
G
-
H A T
D A A
D A P
D A A
- - -
D
D
D
D
-
rabbit-TIMP-1
mouse-TIMP-1
calf-TIMP-1
human-TIMP-1
human-TIMP-2
calf-TIMP-2
F
F
F
F
Y
Y
L
L
L
L
L
L
I
I
I
I
I
I
A
T
A
A
A
A
rabbit-TIMP-1
mouse-TIMP-1
calf-TIMP-1
human-TIMP-1
human-TIMP-2
calf-TIMP-2
Q
Q
Q
Q
Q
Q
R
Q
R
R
K
K
S
R
R
R
K
K
rabbit-TIMP-1
mouse-TIMP-1
calf-TIMP-1
human-TIMP-1
human-TIMP-2
calf-TIMP-2
S
S
S
S
S
S
D
D
D
G
P
P
rabbit-TIMP-1
mouse-TIMP-1
calf-TIMP-1
human-TIMP-1
human-TIMP-2
calf-TIMP-2
P
L
P
P
D
D
G
G
G
G
G
G
70
Y T
Y T
Y T
Y T
I Y
I Y
P
P
P
P
T
T
A
V
A
A
A
A
M
M
M
M
P
P
E
E
E
E
S
A
S
S
S
S
S
A
V
L
V
V
A
A
C
C
C
C
V
V
G
G
G
G
C
C
80
Y S
Y A
Y F
Y F
G G -
H
H
H
H
V
V
K
K
R
R
S
S
S
S
S
S
L
L
Q
Q
Q
H
D
D
G Q L R
G R L R
G Q L S
G K L Q
G K A E
G K A E
100
- N G
- N G
- N
- D G
G D G
G N G
L
K
G
L
K
N
L
F
H
L
M
M
H
H
L
H
H
H
I
I
H
I
I
I
T
N
I
T
T
T
T
A
T
T
L
L
C
C
T
C
C
C
S
S
C
S
D
D
110
F V V
F L V
S F V
F V A
F I V
F I V
P
P
A
P
P
P
W
W
P
W
W
T
120
N S L S F S
R T L S P A
W N S M S S A
N S L S L A
D T L S T T
D T L S A T
G
V
G
G
S
S
F
F
F
F
L
L
T
S
T
T
N
N
K
K
K
K
H
H
T
K
T
T
R
R
N
-
Y
Y
Y
Y
Y
Y
A
S
A
T
Q
Q
A
A
A
V
M
M
G
G
G
G
G
G
C
C
C
C
C
C
D
G
E
E
-
M
V
E
E
E
E
C
C
C
C
C
C
T
T
T
T
K
K
V
V
V
V
I
I
F
F
F
F
T
T
C
C
C
C
C
C
A
L
S
L
P
P
S
S
S
S
M
M
I
I
I
I
I
I
P
P
P
P
P
P
C
C
C
C
C
C
H
K
K
K
Y
Y
T
T
T
T
D
D
H
H
H
H
E
E
C
C
C
C
C
C
L
L
L
L
L
L
W
W
W
W
W
W
T
T
T
T
M
M
D
D
D
D
D
D
160
S S
Q L
Q L
Q L
W V
W V
L
L
L
L
T
T
V
T
Q
E
E
G
G
G
G
K
K
S
S
S
S
N
N
D
E
D
E
I
I
K
D
K
K
N
N
G
G
G
G
G
F
Y
F
F
H
H
170
Q S R
Q S R
Q S H
Q S R
Q A K
Q A K
H
H
R
H
F
F
L
F
L
L
F
F
A
A
A
A
A
A
C
C
C
C
C
C
L
L
L
L
I
P Q
P R
P R
P R
K R
IK
L
L
L
L
S
S
C
C
C
C
C
C
A
T
T
T
A
A
W
W
W
W
W
W
E
R
Q
Q
Y
Y
S
S
S
S
R
R
L
L
L
L
G
G
190
R P R
G A R
R A Q
R S Q
A A P
A A P
I
I
I
I
D
D
R
R
R
R
I
I
F
Y
F
F
E
E
V
A
I
V
F
F
130
N
N
N
N
V
I
R
R
R
R
G
G
S
S
S
S
G
G
E
E
E
E
K
K
140
A
P
P
P
R
R
150
K D
M
I
P
P
90
E
E
E
E
K E
K E
A
A
K Q E F L D I E D P
K Q E F L D I E D P
234
L
L
L
L
I
I
E
E
Q
Q
S
S
180
E
N
E
E
S
RS
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Critical Reviews in Oral Biology and Medicine, 4(2): 197-250 (1993)

Matrix Metalloproteinases: A Review*

I. METABOLIC DEGRADATION OF THE

mechanism based on release of acidic thiol proteinases to a sealed microenvironment on the

Interstitial connective tissues,

Interstitial connective tissues,

PMN serlne proteinase

Insterstitial connective tissue,

PMN elastase, cathepsin G

Insterstitial connective tissue

Bone, cementum, dentin

to begin to understand those factors that dictate

II. ALTERNATE PATHWAYS

remodeling of the extracellular matrix in cell

remodeling scaffold structures, such as the type

B. Neutrophil Serine Proteinases

lular matrix macromolecules by release of two

D. Osteoclastic Bone Resorption

III. MATRIX METALLOPROTEINASE

A. Modular Structure of MMPs

Extracellular matrix substrates

Collagen I, II, III, ( l l l l ) , VII, VIII, X;

PG core protein; fibronectin;

Gelatin; collagen IV, V, VII, X, XI; elastin;

Gelatin; collagen IV, V; elastin; PG core

Fibronectin, laminin, collagen IV, gelatin,

Note: n.d.: not determined.

the five-domain modular structure characteristic

1990, Van Wart and Birkedal-Hansen, 1990).

Domain structure of MMPs.

Stanley, 1987). The four tandem modules are held

B. MMP Gene Structure

be surmised that PUMP-1 lacks exons 7-10, which

Human CL and SL-1 sequences are from Appendix 1; sequences for

C. MMP Substrate Specificity

further degradation takes place already at the fibril

1. Growth Factors and Cytokines

PEA3 binding site, which act synergistically to

Although /<95-transcription is an early event

SL (Conca et al, 1991). These findings show that

the other hand, the PEA 3 element, which plays

(consensus sequence: GNNTTGGtGa)

The same element is present in the promoter

tor (Kerr et al, 1990). Recent studies suggest

3. Cell Shape, Cell-Substrate Adhesion

4. Second Messenger Signaling

tency of the virgin enzyme is maintained, at least

LATENT (Mr 52K)

ACTIVE (Mr 52K, 46K, 43K)

ACTIVE (Mr 41K)

result of conformational change (Lyons et al.,

NaSCN) and detergents (1 to 2% SDS) induce

the successive activation cleavages of human

autolytic cleavage of its own hinge region already

The general sequence of events outlined above

et al, unpublished). ProFIB-CL is capable of

Organomercurial activation of the Mr 72K

IV. INDIVIDUAL MATRIX

the accumulation of the enzyme at the local site is

1. Cleavage of Interstitial Coiiagens

scission of the Gly775-Ile776 or Gly775-Leu776 bonds

At temperatures well below the denaturation

1976; Mainardi etal, 1980a; Birkedal-Hansen et

lagens, but it can be done under narrowly defined