Matrix metalloproteinases and their inhibitors in connective

tissue remodeling
J. FREDERICK WOESSNER, JR.1
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami Florida 33101, USA

ABSTRACT Matrix metalloproteinases are an impor- important disease processes such as joint destruction in rheu-
tant group of zinc enzymes responsible for degradation of matoid and osteoarthritis, tumor invasion, and periodontitis.
the extracellular matrix components such as collagen and The history of this field has been reviewed by Birkedal-
proteoglycans in normal embryogenesis and remodeling Hansen (3) and brief general reviews have appeared that em-
and in many disease processes such as arthritis, cancer, phasize the enzymology (4) and molecular biology of the
periodontitis, and osteoporosis. A matrixin family is metalloproteinases (5, 6).
defined, comprising at least seven members that range in In the 28 years since collagenase was discovered there has
size from Mr 28000 to 92000 and are related in gene se- been a burgeoning of this field, which culminated in the First
quence to collagenase. All family members are secreted as International Symposium on Matrix Metalloproteinases
zymogens that lose peptides of about 10,000 daltons upon held at Destin, Florida, in September of 1989. The proceed-
activition. Latency is due to a conserved cysteine that ings will appear as a supplement to the journal Matrix
binds to zinc at the active center. Latency is overcome by (7). In the bibliography I prepared for this issue there
physical (chaotropic agents), chemical (HOC1, mercurials), were 2700 references to metalloproteinases and their inhibi-
and enzymatic (trypsin, plasmin) treatments that separate tors. Obviously this vast material cannot be covered in de-
the cysteine residue from the zinc. Expression of the tail. Rather, I will review the properties of the proteinases
metalloproteinases is switched on by a variety of agents and their inhibitors with particular attention to the mecha-
acting through regulatory elements of the gene, particu- nisms that have arisen for the control of these enzymes. It is
larly the AP-1 binding site. A family of protein inhibitors easy to appreciate the potential danger to the organism that
of Mr 28,500 or less binds strongly and stoichiometri- might ensue upon secretion of proteinases that can digest its
cally in noncovalent fashion to inhibit members of the supporting framework. Therefore, there is strict regulation
family. The serum protein a2-macroglobulin and rela- of secretion, which occurs only at moments of need; there are
tives are also strongly inhibitory.-Woessner, J. F., Jr. multistep activation processes for the zymogen forms, and
Matrix metalloproteinases and their inhibitors in con- there are multiple tissue and blood inhibitors to check pro-
nective tissue remodeling. FASEBJ. 5: 2145-2154; 1991. teinase action before it goes too far.

Key Words: ext racellular malt-jr proteinose (metallo) collagenose

TIJt’fP - connective tissue THE MATRIX METALLOPROTEINASE FAMILY
(MATRIXINS)

Foa A TUMOR CELL TO METASTASIZE, IT must break away from The chief characteristics of the matrix metalloproteinases
its neighbors, force its way through the surrounding stroma, are:
and penetrate the basement membrane in order to enter the
circulation. When it arrives at its destination, these steps #{149}
The catalytic mechanism depends on zinc at the active
must be repeated in reverse order. These steps require exten- center.
sive degradation of the extracellular matrix components in- #{149}
The proteinases are secreted in zymogen form.
cluding interstitial collagens, basement membrane collagen #{149}
The zymogens can be activated by proteinases or by or-
(type IV), fibronectin, laminin, and various proteoglycans ganomercurials.
(1). The cell is able to accomplish this task because it is #{149}
Activation is accompanied or followed by a loss of Mr of
provided with a battery of metalloendopeptidases that are about 10,000.
secreted from the cell and are able to digest a wide range of #{149}
The cDNA sequences all show homology to that of col-
proteins of the extracellular matrix. However, such capabil- lagenase.
ity is not a peculiar property of tumor cells, but is almost #{149}
The enzymes cleave one or more components of the extra-
universally distributed among mesenchymal cells of all types cellular matrix.
and is also found in certain epithelial and endothelial cell #{149}
Activity is inhibited by tissue inhibitor of metallopro-
types. teinases (TIMP).2
The first member of the metalloproteinase family, col-
lagenase, was discovered by Gross and Lapi#{232}re (2) in 1962 in
the tail of the metamorphosing tadpole. Here the enzyme
was critical for the normal resorption of the tail connective ‘To whom correspondence should be sent, at: Department of Bio-
chemistry and Molecular Biology, R-127, University of Miami
tissue that accompanies maturation of the frog. Since that in-
School of Medicine, P.O. Box 016960, Miami, FL 33101, USA.
itial discovery a number of matrix metalloendopeptidases
2Abbreviations: AP-1, activator protein-l; IL 1, interleukin 1,
have been described. These are involved in many normal
MMP, matrix metalloproteinase; pump 1, putative metallopro-
remodeling processes such as embryonic development, post- teinase 1; T3F, transforming growth factor; TIMP, tissue inhibitor
partum involution of the uterus, bone and growth plate of metalloproteinase; TNF, tumor necrosis factor; TPA, 12-0-tetra-
remodeling, ovulation, and wound healing as well as in some decanoylphorbol-13-acetate; TRE, TPA responsive element.

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 I have proposed the name matrixin for this family (8), one or more of these domains; the simplest member (MMP-7)
which is often referred to as the collagenase gene family. has Mr 28000 and lacks the fibronectin, collagen, and
Currently there are seven well-characterized matrixins. hemopexin domains, which are not required for proteolytic
Their properties and interrelationships are illustrated in activity (13, 14). The proenzyme domain contains a cysteine
Table 1 and Fig. 1. Table 1 presents the use of the names residue that is involved in activation (see below). The fibro-
Matrix MetalloProteinase # abbreviated as MMP-1, MMP-2, nectin-like domain is believed to facilitate binding of the two
etc. This system was originally proposed by Okada et at. (9) gelatinases to their substrate gelatins. The function of the
and has been updated by an unofficial nomenclature com- collagen-like domain is unknown. The hemopexin-like do-
mittee (8). MMP-4 (10) and MMP-6 (11) have thus far been main is believed to help determine substrate specificity.
described in only one laboratory, and sequence data are not Thus, for collagenase this hemopexin-like domain can be re-
available. MMP-5 is identical to MMP-2. The value of such moved by autolysis, leaving a catalytically active domain that
systematic names is emphasized by the multiplicity of terms digests various proteins but can no longer attack collagen
that have been used for individual members such as strome- (15). On the other hand, MMP-3 can lose this domain, with
lysin (MMP-3) and the difficulties of naming proteinases of a reduction in Mr of the active enzyme from 45000 to
broad specificity after single substrates. A glossary of animal 28000, without any change in specificity or specific activity
metalloproteinases that do not fit the criteria of the matrixin (16).
family, e.g., snake venom proteinases, enkephalinase, and
procollagen propeptidases, is also found in ref 8.
The structural interrelationships among the family mem- ACTIVATION OF MATRIX METALLOPROTEINASES
bers are illustrated in Fig. 1. It is seen that the largest mem-
ber (MMP-9) has seven domains: a signal peptide, a propep- The amino acid sequence of two regions of importance is
tide domain that is lost on activation, a domain of the active shown in Table 2 for the 11 enzymes for which the cDNA se-
enzyme that is separated from the putative zinc-binding quence is available. Unfortunately, no X-ray structure is
domain by an intervening fibronectin-like domain, a curious known for any member, so the zinc-binding site identifica-
type V collagen-like domain, and a COOH-terminal hemo- tion is a putative one based on the similarity of the HExGH
pexin-like domain (12). Other members of the family lack sequence to the HExTH zinc-binding sequence in thermoly-

TABLE 1. Nomenclature and natural substrates of the matrix metalloproteinases”

Groups Enzyme names Molecular mass, kDa Matrix substrates

I Interstitial collagenase 52 Deduced Collagen types I, II, III, VII, X

MMP-1 (EC 3.4.24.7) (57)/52 Latent
Vertebrate collagenase (48)/42 Active Gelatins
Fibroblast collagenase 24 Modified

Neutrophil collagenase 53 Deduced Collagens I, II, III

MMP-8 85 Latent
65 Active

II 72-kDa gelatinase 72 Deduced Gelatin type I

MMP-2 72 Latent Collagens, IV, V, VII, X
Type IV collagenase 66 Active Fibronectin, elastin

92-kDa gelatinase 80 Deduced Gelatins I, V

MMP-9 92 Secreted Collagens IV, V
Type V collagenase 84 Active

III Stromelysin 52 Deduced Proteoglycan, link protein

MMP-3 (EC 3.4.24.17) (60)/57 Secreted Fibronectin, laminin
Transin (50)/48 Active Gelatins I, III, IV, V
Proteoglycanase 28 Active Collagens III, IV, V, IX
Procollagenase activator Procollagen peptides
Coll’ase activator protein Activates procollagenase
Weak elastin digestion
Stromelysin-2 53 Deduced Gelatins I, III, IV, V
MMP-10 53 Secreted Weak on collagens III, IV, and V
Transin-2 47 Active
28 Active Activates procollagenase
Fibronectin

Uterine metalloproteinase 28 Deduced Gelatins I, III, IV, V

MMP-7 28 Secreted Proteoglycan
Pump-i (21)/19 Active Fibronectin
Activates procollagenase

This table presents recommended names for the matrixins, together with synonyms. The molecular weights are those deduced from cDNA (minus
the signal peptide) and those of the secreted form (variably glycosylated) and active forms (or forms). Minor forms are in parentheses.

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 SIGNAL P1(1 ACTIVE FIB80ICCTIN ZINC 2(V)
IEMOPEXIN
PEPTIDE PEPTIDE(S) ENZYP( 0LLAGEN

92 kDa LATINASE 87 C 176 #{149} 210

72 kDa LATINASE 80 C 112 I-[ 202

INTERSTITIAL 80 c’II 112 209 I

LLAGENASE

ST4ELYSIN
ElI 82 c’II 112
213

El 1 i c’I I 113
1601
Figure 1. Domain structure of the matrixins of human origin. The number of amino acid residues is shown within each domain. Where
domains are deleted, the connection of remaining domains is indicated by a single line. C indicates the approximate position of the cysteine
switch. Sequence data may be found in ref 5.

sin and related microbial proteinases (20). It is seen that agents can cause the peptide containing the cysteine to fold
there are two His residues in the highly conserved consensus back, breaking the cysteine-zinc contact. In either event, the
sequence: Ala-Ala-His-Glu-hydrophobe-Gly-His (Table 2). enzyme can then attack this peptide autolytically and cleave
The recent review of zinc proteins by Vallee and Auld (20) it (by both intra- and intermolecular mechanisms). It is
emphasizes the importance of such a pair of His residues in likely that in vivo activation proceeds most frequently by
zinc binding. However, the third ligand in other zinc pro- proteolysis. Thus, enzymes such as plasmin may cleave to
teins is typically 13-160 residues distant. For example, in the left of the cysteine but still remove sufficient length of
thermolysin, this third ligand is Glu, separated by 19 resi- peptide so that the cysteine is no longer held in tight apposi-
dues from His in the COOH-terminal direction. Nothing tion to the zinc atom. Autolysis again ensues to produce the
comparable has been found in the matrixins; some suspicion cleaved, permanently active form of the enzyme.
may attach to the third conserved His residue six residues to As an illustration, collagenase is considered in more detail
the right (Table 2), but this residue is preceded by a highly (22). A partial sequence is shown for the region of interest:
variable residue.
R RN S !T LXV M Q PR C’3C VP DV A Q’#{176}P
V L TX C
It has long been a puzzle as to how collagenase (and other
matrixins) can be activated by so many different agents:
aminophenylmercuric acetate, HOd, sodium dodecyl sul-
fate, chaotropic agents, and various proteinases such as tryp- If one mixes latent collagenase from human rheumatoid
sin and plasmin. It appeared that a relatively inaccessible fibroblasts with human plasma kallikrein, the earliest
sulfhydryl group was involved. The sequence data (Table 2) cleavage is seen to the right of Arg35 or Arg36; trypsin (23)
reveal that there is a highly conserved cysteine residue in the and plasmin cleave at position 2. After this step, the active
proenzyme domain of each enzyme. Van Wart’s group (21) center of collagenase becomes exposed and produces an auto-
has proposed a cysteine switch model that is illustrated in lytic cleavage at position 3. The resultant collagenase is now
Fig. 2. It is believed that the conserved cysteine is coordi- active, but not fully so. Full activity is achieved only after
nated to the zinc atom at the active center, thereby excluding further cleavage beyond the Cys73 residue at position 5, ex-
water, which must be the fourth substituent in the active en- posing an NH2-terminal Phe group. This cleavage is
zyme (20). Various reagents can react with this cysteine, con- produced only by addition of stromelysin (MMP-3), which
verting it to a nonbinding form. Alternatively, chaotropic is therefore postulated to be important in the in vivo activa-

TABLE 2. Amino acid sequence of putative zinc binding region and cysteine switch region of matrix m#{128}talloproteinases

MMP-# Source and enzyme Cysteine switch region Putative zinc binding region Reference

MMP-1 Human fibroblast collagenase 68-MKQPRCGVPDVA 193-LHRVAA . HELGHS LGL SHST 5

MMP-1 Rabbit fibroblast collagenase 68-MKQPRCGVPDVA 193-LYRVAA . HELGHS LGL SHST 16
MMP-2 Human 72-kDa gelatinase 69-MRKPRCGNPDVA 364-LFLVAA . HEFGHAMGL EHSQ 5
MMP-3 Human stromelysin 70-MRKPRCGVPDVG i96-LFLVAA . HEIGHS LG L FHSA 5
MMP-3 Rabbit stromelysin 72-I RKPRCGVPDVG i97-LFLVAA. HELGHS LGL FHSA 17
MMP-3 Rat transin 68-MHKPRCGVPDVG 194-LFLVAA.HELGHSLGLFHSA 18
MMP-7 Human pump-i 65-MQKPRCGVPDVA 190- . FLYAATHELGHS LGMGHSS 5
MMP-8 Human neutrophil collagenase 66-MKKPRCGVPDSG 191-LFLVAA . HEFGHS LGLAHSS 19
MMP-9 Human 92-kDa gelatinase 75-MRTPRCGVPDLG 376-LFLVAA . HEFGHALGLDHSH 5
MMP-10 Human stromelysin-2 69-MRKPRCGVPDVG 195-LFLVAA . HELGHS LGL FHSA 5
MMP-10 Rat transin-2 70-MHKPRCGVPDVG 196-LFLVAA . HELGHS LGL SHSN 18

Residues numbers from N-end of proenzyme, omitting signal peptide. Identities shown in boldface.

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 lular, and minor collagens. However, the best substrate
found to date for interstitial collagenases is a2-macroglobulin.
The value of kcatlKm (M . h X 10k) 5 280 for this sub-
strate compared with values in the range of 0.1-li for various
types and species of collagen, and 0.01 for synthetic octapep-
tides (25). Collagenase also digests casein and many species
of this enzyme will digest gelatins, some at rates similar to
those for collagen (26). A systematic study of collagenase
specificity has been mounted by Van Wart’s group (26, 27);
a series of 16 octapeptides was synthesized as variants on the
sequence Gly-Pro-Gln-Gly5IIe-Ala-Gly-Gln found at the site
cleaved in the al(I) chain of collagen. Collagenase showed
kcat5 for these compounds that were higher than those for
collagen by factors of as much as 40; however, the Km values
were 4000-fold higher. The large size of the collagen
molecule and its triple helical structure are important in en-
zyme binding. The best octapeptide substrates have two
hydrophobic residues on either side of the scissile bond.
However, a2-macroglobulin is cleaved in the sequence
which partly matches
the collagenase susceptible sequence Gly-Pro-Xaa-Gly5Leu-
of the a2(I) collagen chain. A series of 10 members of the
macroglobulin family is cleaved by collagenase; the se-
quences cleaved are listed by Sottrup-Jensen (28) together
with sequences cut by autolysis within the collagenase
molecule. The only clear pattern that emerges is that the P1’
residue is invariably hydrophobic (typically Leu, Ile, Vat)
and that P1 is usually Gly or a hydrophobic residue.
Figure 2. Cysteine switch mechanism for activation of metallopro- Further enzymes were named after other types of collagen,
teinases as proposed by Springman et al. (20). Cysteine in the e.g., type IV collagenase and type V collagenase. These
proenzyme domain (Fig. 1) contacts zinc to maintain latency of the
names are much less suitable as these high-numbered colla-
enzymes. Physical agents such as sodium dodecyl sulfate (SDS) or
gens contain triple helical domains with interruptions such
chaotropic agents can unfold the structure to expose zinc. Reagents
as the omission of Gly in a triplet. As a result, these collagen
that react with sulibydryl groups will inactivate the cysteine:
types can be cleaved by nonspecific enzymes such as throm-
N-ethylmaleimide (NEM), oxidized glutathione (GSSG), hypo-
bin (28) as well as by other matrixins such as MMP-3 and
chlorous acid (HOCI), and organomercurials such as aminophenyl-
mercuric acetate (APMA). Alternatively, proteolytic enzymes can
MMP-7. Furthermore, there is no case in which these other
collagenases have been shown by sequence determination to
cleave the propeptide, even ahead of the cysteine. In a second step,
these active forms can be autolytically cleaved by the activated cleave collagen in a helical sequence. Another source of con-
metalloproteinase to remove the propeptide and confer permanent fusion is the finding that type IV collagenase actually acts
activity. more readily on type V collagen (30). Therefore, the name
72-kDa gelatinase is perhaps preferable. However, MMP-7,
and to a lesser extent MMP-3, digest gelatins. The use of the
tion of collagenase. An alternative activation of the enzyme name MMP-2 helps to avoid these ambiguities.
with APMA causes the cysteine to become dissociated from Almost every enzyme in Table 1 has been found to cleave
the zinc (Fig. 2) and the collagenase then cleaves itself at an octapeptide containing the collagen sequence GPQGIAGQ
position 4. Prolonged incubation leads to further inter- (or minor variant). Additional studies of the B-chain of insu-
molecular autolysis with cleavage at positions 6 and 7. For lin show that several of these enzymes cleave the Ala-Leu and
reasons not yet understood, cleavage that removes Phe81 Val-Leu bonds (11, 14). Moreover, certain synthetic peptide
results in an enzyme with only 40% of the full activity. inhibitors (see below) inhibit some of these proteinases with
Therefore, stromelysin produces a form of collagenase that similar efficacy. We find that collagenase, MMP-3, and
has the highest specific activity yet noted. The human neu- MMP-7 are inhibited by the peptide hydroxamate SC 44463
trophil collagenase can be readily activated by hypochlorous (31). Finally, all of the enzymes are inhibited by TIMP.
acid that is released as part of the neutrophil activation These results lead to the conclusion that the specificities of
process (24), so it is not dependent on the presence of the matrixins are closely related. There is no case in which
MMP-3. all these enzymes are produced by a single cell, so the earlier
concept of three members (collagenase, proteoglycanase, and
gelatinase) taking care of most situations of matrix degrada-
SPECIFICITY OF THE MATRIXINS tion is perhaps reasonable. Various cell types might then use
different members from each group to accomplish similar
Members of this family generally possess quite broad spe- ends in different tissues.
cificity, a point that was not fully appreciated when many of However, these three classes of matrixins (Table 1) do not
the members were named. Thus, collagenase was named for account for one of the major matrix components-elastin.
its ability to cleave collagen in the triple helix domain. More No member of the family digests elastin with reasonable
recently the name has been modified to interstitial col- efficiency, compared for example with neutrophil elastase,
lagenase, not to specify the localization of the enzyme but to although MMP-3 has a weak action (32). However, there is
indicate that it can cleave the three interstitial collagens a macrophage elastase that has moderate activity against
(types I, II, III) which are distinguished from other, pericel- elastin (33). This enzyme is small in size similar to MMP-7

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 and is inhibited by TIMP (33). In the absence of data on TIMPs appear to be highly selective in their inhibition of the
latency and sequence, it is not yet possible to assign this en- various matrixins. Therefore, the necessity of having multi-
zyme to the matrixin family. ple sizes or forms of TIMP remains unclear.

TISSUE INHIBITORS OF MATRIX TIMP MECHANISM OF INHIBITION IS UNKNOWN

METALLOPROTEINASES (TIMPs)
There is no information on the mechanism of inhibition by
It is becoming increasingly clear in recent years that the con- TIMP and its relatives nor on which region of the molecule
trol of extracellular proteolysis is critically important to the is involved in the noncovalent binding to metalloproteinases.
organism. Thus, a large family of serpins has been un- I have considered the five known sequences of TIMPs (Fig. 3)
covered, concerned with holding serine proteinases in check and note the highly conserved region at positions 16-22
after they leave neutrophils or become activated in blood (Leu’7 is replaced by Ile or Vat in two cases). In view of the
clotting (34). Similarly, tissues contain members of the specificity of the matrixins for hydrophobic groups, they
TIMP family (35). TIMP (sometimes called TIMP-i) is a might bind to this region. For example, a good substrate for
protein of Mr 28,500 that is found in many tissues (or cul- MMP-3 has Phe-Phe-Gly-Leu to the right of the scissile
tures thereof). The sequence of its protein backbone is bond (45). If the S1’-S2’-S3’ region of the enzyme interacted
known; there are 184 amino acids for an Mr of about 21,000, with the Leu-Val-Ile in positions 17-19 of TIMP, the COOH
but glycosylation brings the mass to the higher value. There group of Asp’6 residue might then be positioned to interact
are 12 highly conserved cysteine residues (Fig. 3) which form with the zinc to render the enzyme inactive. At present this
6 disulfide bonds, organizing the molecule into two distinct must be considered purely speculative. A similar but less
domains (36). TIMP forms a 1:1 stoichiometric complex highly conserved region is found at position 82-87 (Fig. 3.
with MMP-1 and MMP-3. The enzymes bind reversibly Glu is positioned ahead of four hydrophobic groups (Phe
with high-affinity K0 = 10b0 M (35). The complex can be is replaced by Tyr in two cases). Coulombe and Skup (46)
dissociated by treating with EDTA at acid pH (40) or by truncated the cDNA for mouse TIMP and expressed pro-
SDS-PAGE. TIMP is quite stable to acid and boiling, but is teins consisting of Me(23-Cys99 or TIMP lacking Cys1-Arg9#{176}
sensitive to reducing agents and can be inactivated by reduc- (corresponding to Gln9#{176} in Fig. 3); neither product had any
tion and alkylation (40). inhibitory effect. This was interpreted as indicating that the
Additional members of the TIMP family are smaller pro- full molecule was necessary for activity. However, the S-S
teins referred to as IMP or TIMP-2 (41, 42). When MMP-2 bond locations were not recognized at this time; perhaps the
is produced by cultured tumor cells it is recovered in the disulfide bond Cys99-Cys3 or other pairs could not form in
medium as a proenzyme form with TIMP-2 firmly bound to these truncated versions, distorting the domain structures.
it, although presumably not at the active center (42, 43).
MMP-9 is also secreted as a complex of zymogen with
TIMP (12). The original TIMP is often referred to as OTHER INHIBITORS OF MATRIXINS
TIMP-1 to distinguish it from TIMP-2. Sequence analysis
of TIMP-2 of human cDNA shows a protein with 38% se- A second line of defense against rampaging metalloprotein-
quence identity to human TIMP-i; in this case the authors ases is found in the serum in the form of a2-macroglobulin.
refer to the inhibitor as metalloproteinase inhibitor (MI) As mentioned previously, this protein serves as an excellent
(37). This protein remains at a small size of about 20,000, substrate for collagenase (and the other matrixins, as far as
rather than becoming highly glycosylated as does TIMP. A tested). Cleavage occurs in the bait region, inducing a con-
larger TIMP-like inhibitor (LIMP) of 76 kDa has recently formational change in the inhibitor, which then entraps and
been purified from fibroblast culture medium (44). It covalently binds the enzyme through rearrangement of a
forms complexes with MMPs 1, 2, and 3 and is not recog- thiolester bond so that it cannot cleave collagen or other
nized by TIMP antibodies. It is similar, or identical, to matrix macromolecules. In the rat there are further forms of
large inhibitors reported earlier (44). Thus far, none of the macroglobulin: a1-macroglobulin and a,-inhibitor-3 that
also act as potent inhibitors of collagenase (29) and MMP-7
(J. F. Woessner, Jr., unpublished results). In the chicken egg
80
RNH 5RHF YGCV
70
SEMAPTYVFR
60 55
I DAAOGIAQFGKYNKTNK
40
IE Y
a related protein, ovostatin or ovomacroglobulin, serves a
,\Es
F Ii 10 20 30
a
3 similar function and acts by a similar trapping mechanism
H2N-C I CV P P HP Q I A F C N sFrV7R F S G I P 6 V N a i L (26). Indeed, this inhibitor prefers metalloproteinases over
IA 90
0KLCIDGLLHITTC5F
Ilso I
GCE
iso
WOSL850IA-O01 proteinases of the other classes. As the chicken protein lacks
V
A I
V t
C-C
I the thiolester, inhibition is not dependent on formation of a
P 1203 1 1 covalent bond to the metalloproteinases. In spite of the rapid
V I V 0
N K 13SF cleavage of a2-macroglobulin by collagenase, we were able to
5 1 P
I F IC EllO detect active collagenase in human serum that was not
5 6 SI N
110 LR p trapped by the inhibitor (47); this may be caused by the low
K L 160 level of enzyme that may result in a low frequency of fruitful
I CALHRSQFG6
1400
S
I
0
collisions with the inhibitor.
GTH CL VT DOL
150
10 Synthetic inhibitors for the matrixins have been prepared
in many labs, with a view to inhibiting disease processes such
Figure 3. Primary structure of TIMP. The sequence of human as metastasis and osteoarthritis. In general, the most success-
TIMP is shown, together with its six disulfide bridges (36). ful inhibitors have been ones that lack a scissile bond and
Residues in bold face are those which are identical in five forms of have in its place a carboxyl, sulfhydryl, or hydroxamate
TIMP: human (37), mouse (38), rabbit (39), and human and bo- group that can coordinate to the zinc atom to block catalysis
vine metalloproteinase inhibitor (MI, ref 37). Boxes enclose possi- (48). Thiol compounds (49) and phosphoramidates (50) have
ble sites of enzyme binding. proved effective in vitro. The inhibitor SC-40827, a modified

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 Leu-Tyr peptide with a propyl carboxy function on the amino phorbol-responsive element or a TATA box in the promoter
side of Leu, is an effective inhibitor of metalloproteinases in region; however, there is a potential binding site for AP-2 in
resorption of cultured bone and in ovum release from per- the first exon. Possible TPA-responsive elements (AP-1) have
fused ovaries (51). been found in the 5 ‘-flanking region of rabbit stromelysin
and rat transin 1; cAMP-responsive elements (AP-2 sites) in
the same cDNA and in the eDNA for human collagenase.
MOLECULAR BIOLOGY OF THE Glucocorticoid-responsive elements are seen in rabbit stro-
METALLOPROTEINASES melysin cDNA.

Eleven matrixins have been studied as cDNA clones to pro-

vide the sequences tabulated in Table 1. Such studies have REGULATION OF METALLOPROTEINASE
led to identification of putative proteinases from the cDNA PRODUCTION
without accompanying evidence of catalytic activity. Such
was the case for human MMP-7 (putative metalloprotein- The matrixins are not stored in most cell types (neutrophils
ase) (pump 1) and MMP-iO (stromelysin 2) and for rat tran- and macrophages are exceptions) and are not synthesized
sin 2 (in transfected cells) (18, 52). The cDNAs for MMP-7 and secreted into the matrix until a clear signal arrives that
and 10 were subsequently expressed in COS cells, and the they are needed. When the signal ceases or negative signals
resultant proteins possessed the predicted proteolytic activity arrive, synthesis is stopped or falls to a low level. These
(13, 53). regulatory signals or agents work almost exclusively at the
The gene structure has been ascertained for five of the level of gene transcription, as just discussed. Table 3 presents
matrixins: human MMP-1, human MMP-2 (54), rat MMP-3 a long, but not exhaustive, list of factors that can affect the
(18), rat MMP-10 (18), and rabbit MMP-1 (17). All these production of collagenase and/or stromelysin. Although the
genes consist of 10 exons and 9 introns except MMP-2, synthesis of these two enzymes is often coordinated, this is
which has 3 additional exons (#5, 6, 7) corresponding to the not always the rule. Most of these observations have been
three 58-amino-acid sequences constituting the fibronectin- made for cultured fibroblasts and chondrocytes; other cell
like domain. The various genes range in length from 8,200 types may respond differently. Fibroblasts respond to the ex-
to 30,000 base pairs. The human MMP-2 gene is on chro- tracellular matrix, as indicated by effects of various recon-
mosome 16, whereas collagenase and stromelysin are close stituted collagen types and of antibody to integrin receptors.
together on the long arm of chromosome 11 (55). Factors that affect the cell shape and influence the actin
Studies of matrixin gene regulation have been fruitful in cytoskeleton are particularly apt to induce metalloproteinase
two respects: they have helped to uncover the mechanisms of gene expressions (62). Many of the factors in Table 3 are im-
gene regulation in general as well as specific mechanisms for plicated in pathological processes that are accompanied by
the regulation of metalloproteinases. The group of Rahms- matrix breakdown. Thus crystals are involved in diseases of
dorf (56) has examined the 5’ upstream sequences of human the articular joint such as chondrocalcinosis; lipopolysaccha-
collagenase genomic eDNA and identified the phorbol re- rides induce inflammation; several of the factors are involved
sponsive element (TRE) as the AP-1 binding site. As in tumor induction or cell transformation. Growth factors
reviewed by Krane et al. (57) two proto-oncogene products, generally stimulate enzyme production, presumably in
c-Jun and c-Fos, are among the protein components of the ac-
tivator protein (AP-l) that binds to this site. The two proteins
TABLE 3. Factors inducing/stimulating collagenase and
form a heterodimer held together by the leucine zipper
stromelysin production”
mechanism. Synthesis of c-Fos is essential for phorbol (TPA)
induction of collagenase and also for the cell’s response to
IL 1, TNF-a, and serum factor. Cyclohexamide blocks the Factors acting at cell surface: Physical agents acting on cell:
response to IL 113 and TNF-a, which suggests that the Calcium ionophore A23l87 Heat shock (4)
Cell fusion UV irradiation
response is dependent on the synthesis of c-Fos and c-Jun.
Collagen types in substratum
Both c-Fos and c-Jun mRNA levels are increased by TNF-a Concanavalin A (4) Cytokines/growth factors:
(58). The requirement for c-Fos in collagenase induction by Crystals: urate (4) Epidermal growth factor
oncogenes was established by Sch#{246}nthalet at. (59) by use of hydroxyapatite Fibroblast growth factor b
a plasmid to transfect cells with antisense Fos sequences; this calcium pyrophosphate Interferons a, , y (4)

completely abolished the collagenase response. Recently, a lntegrin receptor antibody Interleukin Ia,
Iron (4) Platelet-derived growth factor
binding site for the transcription factor PEA3 has been
Phagocytosis Tumor necrosis factor a (4)
shown nine bases upstream from the AP-l site in the col- Polyhydroxyethylmethacrylate
lagenase gene; this protein acts synergistically with AP-i Other:
after phorbol induction. This regulatory unit has been Chemical agents: Viral transformation, oncogenes
dubbed TORU (TPA and oncogene responsive unit (60). For Cyclic AMP Autocrine agents (64)
the rabbit collagenase gene, Brinckerhoff (61) has shown the Colchicine (63) Aging of fibroblasts (65)
Cytochalasin B, D
presence of regulatory elements that respond positively to Lipopolysaccharide (63) Repressive factors:
phorbol esters and heat shock and negatively to dexametha- Mitomycin C Retinoic acid
sone and retinol. Response to TPA is enhanced only 14-fold Pentoxifylline (4) Glucocorticoids (63)
when 250 bp are present in the 5-flanking region, but Phorbol diesters Adenovirus-5 EIA gene (66)
39-fold when 1200 bp are present. This indicates the exis- Prostaglandin E (63) Estrogen (63)
tence of further modulation well upstream from the TRE Trifluoperazine Progesterone (63)
site.
Transforming growth factor fi (63)
5’-Flanking sequences are also known for stromelysin,
72-kDa gelatinase, and rat transin I and 2 (reviewed in ref “Unless otherwise indicated, factors are referenced in Frisch and Werb
62). The 5-flanking region of MMP-2 does not show a (62).

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 preparation for tissue remodeling that may accompany ratios of 1:10 stromelysin:collagenase as well as fairly high
growth. Recently Brinckerhoff (64) has shown that fibro- levels of enzyme (22). No one has yet recovered active col-
blasts produce two protein autocrine factors related to serum lagenase from any tissue to determine the amino group ex-
amyloid and /32-microglobulin that regulate MMP-1 pro- posed.
duction.
Repressive factors that inhibit production of matrixins in-
clude retinoids, glucocorticoids, and TGF-13; the latter is par- BIOLOGICAL ROLE OF THE MATRIXINS
ticularly repressive when given in combination with a
stimulatory agent such as IL 1 (67). It has recently been A number of biological systems have been studied in great
shown (68-70) that glucocorticoids inhibit collagenase syn- detail. There is space here only to point to reviews of these
thesis by interfering with AP I activity through a mechanism topics. The central role of matrixins in basement membrane
that does not involve protein synthesis or binding of the hor- and matrix breakdown in tumor invasion and angiogenesis
mone receptor to DNA. Rather, mutual binding of the re- has been reviewed by Moscatelli and Rifkin (1). The rela-
ceptor protein and the c-jun protein prevents either one from tionships of collagenase to bone resorption during normal
binding to DNA. growth and turnover and in various disease states has been
detailed by Sakamoto and Sakamoto (74), who also cover
uterine involution, tadpole metamorphosis, ovulation,
IN VITRO VS. IN VIVO STUDIES OF THE wound healing, and periodontal disease. Destruction of ar-
METALLOPROTEINASES ticular cartilage by metalloproteinases is an important fea-
ture of both rheumatoid and osteoarthritis. Dean et at. (75)
Few studies have concerned activities of the matrixins in review the concept of an imbalance of metalloproteinases
vivo, and for an important reason. Tissue metalloproteinases and TIMP as contributing to the pathogenesis of osteoar-
are normally found only in tissues undergoing remodeling or thritis; this is an important concept in many disease pro-
breakdown, and then only in low levels on the order of 1-10 cesses. Closely allied to this topic is the involvement of metal-
ig per gram wet tissue. Moreover, the enzymes are difficult loproteinases in cartilage breakdown preparatory to calcifi-
to extract. Because the enzymes are not dislodged by deter- cation of the epiphyseal growth plate (76). Matrisian (6) has
gents, it was thought that they might be bound to matrix discussed the role of metalloproteinases in blastula implanta-
components such as collagen. I developed a method of ex- tion in the uterus and in embryogenesis of organs and tis-
traction based on the concept of melting the collagen at 60#{176}C sues. Tissue destruction by neutrophils is reviewed by
and dislodging collagenase with 0.1 M CaCl2 (71). Recently, Weiss (24).
other enzymes such as MMP-3 (72) and MMP-7 (14) were However, there are other ways to degrade extracellular
found to be extracted by the same regimen, although there matrix in addition to digestion by matrixins (74). Thus, the
is no reason to believe they would bind to collagen. Possibly neutrophil has a battery of enzymes such as elastase and
there are matrix receptors for the matrixins that have yet to cathepsin G (serine proteinases) that can degrade much of
be identified. If so, their interaction with the active form of the matrix. In tissues such as the involuting uterus there is
MMP-7 would suggest that the binding site on the various extensive phagocytosis of collagen and other matrix compo-
enzymes would lie within the minimal 19,000-dalton nents by macrophage-like cells that continue digestion intra-
domain. cellularly within the lysosomal system. Intracellular diges-
Although a great deal of information on MMP stimulation tion of collagen is important in osteoclastic remodeling of
and repression has been gathered, as illustrated in Table 3, bone as well.
this pertains in almost every case to cells and tissues in cul-
ture. Whether the same effects occur in living animals re-
mains to be explored in most cases. A cautionary example is FUTURE PROSPECTS
provided by the involuting postpartum uterus of the rat (73).
Collagenase production has been studied by direct assay in A vast amount of work remains to be done in this field. One
vivo and in cells cultured from this organ. In both cases, col- of the first requirements is for X-ray diffraction crystal struc-
lagenase was found to be produced in significant amounts tures. This will permit us to see the active center, the location
during the involution process. However, hormonal respon- of the zinc atom, the origin of latency, and the mode of bind-
sivity was quite different: estradiol suppressed the in vivo ing to TIMP. More important, it will facilitate the design
levels of collagenase and retarded collagen loss from the of inhibitors. At present only interstitial collagenase has been
uterus whereas it had no effect on cultured uterine cells. crystallize#{231}l. However, resolution has been disappointing
Progesterone, on the other hand, strikingly inhibited col- (about 4 A, ref 77) presumably because of partial autolysis
lagenase production in cultured uterine smooth muscle cells of the protein. A great deal remains to be learned about en-
but had little effect in vivo. Such differences might be due to zyme specificities: are the higher-numbered collagens IV
differences in hormone receptors on cultured cells vs. cells in and above cleaved in helical regions, are there peptide sub-
vivo or to further metabolism of hormones in either situation. strates that will distinguish among the various matrixins,
How do the matrixins become activated in vivo? Although what are the specificities for any of the enzymes besides cot-
organomercurials and trypsin will not be present in the tis- lagenase? This in turn could lead to the rational design of in-
sues, plasminogen may be expected to enter from the blood hibitors that would be useful in treating diseases such as
vessels and the mesenchymal cells can produce plasminogen cancers and the arthritides.
activators. Activation by plasmin is possible, but whether this Almost nothing is known about the evolution of the
is the pathway actually followed or there are other activating matrixin family. Collagen first makes its appearance in the
proteinases remains to be proven. A further question is coelenterates. Collagenase has not yet been observed in
whether the full activity of collagenase is reached in vivo by forms lower than the frog, although the sea urchin hatching
action of stromelysin. Is stromelysin present in sufficiently protease shows considerable homology with collagenase and
high concentration in the tissue to react with collagenase in stromelysin (78). Is collagenase to be expected in the lower
a reasonable time frame? The in vitro experiments used forms, or if not, what mechanisms exist for the breakdown

MATRIX METALLOPROTEINASES 2151

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 of collagen in these forms? Did MMP-7 evolve first, with 15. Clark, I. M., and Cawston, T. E. (1989) Fragments of human
subsequent addition of domains, or has it lost domains origi- fibroblast collagenase. Purification and characterization.
nally found in its progenitor? Is the zinc binding site con- Biochern. j 263, 201-206
served from the microbial proteinases or is this a case of con- 16. Okada, Y., Harris, E.
D., Jr., and Nagase, H. (1988) The
vergent evolution? precursor of a metalloendopeptidase from human rheumatoid
synovial fibroblasts. Purification and mechanisms of activation
The techniques of molecular biology hold great promise
by endopeptidases and 4-aminophenylmercuric acetate. Bio-
for the future of this field. The understanding of promoter c/tern. J. 254, 731-741
and enhancer regions of the MMP genes is still in its infancy. 17. Fini, M. E., Plucinska, I. M., Mayer, A. S., Gross, R. H., and
A great number of factors can stimulate or repress enzyme Brinckerhoff, C. E. (1987) A gene for rabbit synovial cell col-
expression; how are their effects produced on the gene? Why lagenase: a member of a family of metalloproteinases that de-
and how is the cell cytoskeleton so intimately linked with this grades the connective tissue matrix. Biochemistry 26, 6156-6165
expression? Site-directed mutagenesis can be used to learn 18. Breathnach, R., Matrisian, L. M., Gesnel, M. -C., Staub, A.,
about the active center, zinc binding sites, substrate specifi- and Leroy, P. (1987) Sequences coding for part of oncogene-
city, role of cysteine in activation, etc. We would particularly induced transin are highly conserved in a related rat gene.
Nucleic Acid Res. 15, 1139-1151
like to understand the role of these enzymes in living organ-
19. Hasty, K. A., Pourmotabbed, T F., Goldberg, G. I., Thomp-
isms and to know which regulatory factors are important in son’ J. P., Spinella, D. G., Stevens, R. M., and Mainardi, C. L.
vivo. These problems may begin to yield to the methods of (1990) Human neutrophil collagenase: a distinct gene product
in situ hybridization, introduction of antisense RNA into with homology to other matrix metalloproteinases. J. Biol.
cells, and use of transgenic mice. C/tern. 265, 11421-11424
20. Vallee, B. L., and Auld, D. S. (1990) Zinc coordination, func-
This work was supported by the National Institutes of Health tion, and structure of zinc enzymes and other proteins. Bio-
grants AR-16940 and HD-06773, and by Miles, Inc. chemistry 29, 5647-5659
21. Springman, E. B., Angleton, E. L., Birkedal-Hansen, H., and
Van Wart, H. E. (1990) Multiple modes of activation of latent
REFERENCES human fibroblast collagenase: evidence for the role of a CysT3
active-sitezinc complex in latency and a “cysteine switch”
1. Moscatelli, D., and Rifkin, D. B. (1988) Membrane and matrix mechanism for activation. Proc. Nail. Acad. Sci. USA 87, 364-368
localization of proteinases: a common theme in tumor cell inva- 22. Suzuki, K., Enghild, J. J., Morodomi, T., Salvesen, G., and
sion and angiogenesis. Biochim. Biophys. Acta 948, 67-85 Nagase, H. (1990) The mechanism of activation of tissue procol-
2. Gross, J., and Lapi#{232}re,C. M. (1962) Collagenolytic activity in lagenase by matrix metalloproteinase 3 (stromelysin). Biochemis-
amphibian tissues: a tissue culture assay. Proc. NatI. Acad. Sci. try 29, 10261-10270
USA 54, 1197-1204 23. Grant, G. A., Eisen, A. Z., Marmer, B. L., Roswit, W. T., and
3. Birkedal-Hansen, H. (1988) From tadpole collagenase to a Goldberg, G. I. (1987) The activation of human skin fibroblast
family of matrix metalloproteinases. j Oral Patiiol. 17, 445-451 procollagenase. Sequence identification of the major conversion
4. Emonard, H., and Grimaud, J. -A. (1990) Matrix metallo- products. J. Biol. C/tern. 262, 5886-5889
proteinases. A review. CelL Mol. BioL 36, 131-153 24. Weiss, S. J. (1989) Tissue destruction by neutrophils. New EngI.
5. Docherty, A. J. P., and Murphy, G. (1990) The tissue metallo- j Med. 320, 365-376
proteinase family and the inhibitor TIMP: a study using 25. Enghild, J. J., Salvesen, G., Brew, K., and Nagase, H. (1989) In-
cDNAs and recombinant proteins. Ann. Rheurn. Dis. 49, teraction of human rheumatoid synovial collagenase (matrix
469-479 metalloproteinase 1) and stromelysin (matrix metalloproteinase
6. Matrisian, L. M. (1990) Metalloproteinases and their inhibitors 3) with human a2-macroglobulin and chicken ovostatin.j Biol.
in matrix remodeling. Trends Genet. 6, 121-125 Chem. 264, 8779-8785
7. Birkedal-Hansen, H., et al., eds) Proc. First. mt. Syrap. Matrix 26. Fields, G. B., Netzel-Arnett, S. J., Windsor, L. J., Engler, J. A.,
Metalloproteinases, Destin, Florida, September, 1989. Matrix Birkedal-Hansen, H., and Van Wart, H. E. (1990) Proteolytic
(Suppl) 11 In press activities of human fibroblast collagenase: hydrolysis of a broad
8. Nagase, H., Barrett, A. J., and Woessner, J. F., Jr. (1991) range of substrates at a single active site. Biochemistry 29,
Nomenclature and glossary of the matrix metalloproteinases. 6670-6677
Matrix (Suppl.) In press 27. Fields, G. B., Van Wart, H. E., and Birkedal-Hansen, H. (1987)
9. Okada, Y., Nagase, H., and Harris, E. D., Jr. (1986) A metallo- Sequence specificity of human skin fibroblast collagenase. Evi-
proteinase from human rheumatoid synovial fibroblasts that dence for the role of collagen structure in determining the cot-
digests connective tissue matrix components. Purification and lagenase cleavage site. j Biol. C/tern. 262, 6221-6226
characterization. j Biol. C/tern. 261, 14245-14255 28. Sottrup-Jensen, H., and Birkedal-Hansen, H. (1989) Human
10. Azzo, W., and Woessner, J. E, Jr. (1986) Purification and char- fibroblast collagenase-a-macroglobulin interactions. Localiza-
acterization of an acid metalloproteinase from human articular tion of cleavage sites in the bait regions of five mammalian a-
cartilage. J. Biol. C/tern. 261, 5434-5441 macroglobulins. J. BioL C/tern. 264, 393-401
11. Nakano, T, and Scott, P. G. (1987) Partial purification and 29. Sage, H., Pritzl, P., and Bornstein. P. (1981) Susceptibility of
characterization of a neutral proteinase with collagen telopep- type V collagen to neutral proteases: evidence that the major
tidase activity produced by human gingival fibroblasts. Biochern. molecular species is a thrombin-sensitive heteropolymer,
Cell BioL 65, 286-292 [al(V)]2a2(V). Biochemistry 20, 3778-3784
12. Wilhelm, S. M., Collier, I. E., Marmer, B. L., Eisen, A. Z., 30. Okada, Y., Morodomi, T., Enghild, J. J., Suzuki, K., Yasui, A.,
Grant, G. A., and Goldberg, G. I. (1989) SV4O-transformed hu- Nakanishi, I., Salvesen, G., and Nagase, H. (1990) Matrix
man lung fibroblasts secrete a 92-kDa type IV collagenase metalloproteinase 2 from human rheumatoid synovial fibro-
which is identical to that secreted by normal human macro- blasts. Eur. J. Biochern. 194, 721-730
phages. j BioL C/tern. 264, 17213-17221 31. Woessner, J. F., Jr., Morioka, N., Zhu, C., Mukaida, T., Butler,
13. Quantin, G., Murphy, G., and Breathnach, R. (1989) Pump-i T, and LeMaire, W. J. (1989) Connective tissue breakdown in
eDNA codes for a protein with characteristics similar to those ovulation. Steroids 54, 491-499
of classical collagenase family members. Biochemistry 28, 32. Galloway, W. A., Murphy, G., Sandy, J. D., Gavrilovic, J.,
5327-5334 Cawston, T. E., and Reynolds, J. J. (1983) Purification and
14. Woessner, J. E, Jr., and Taplin, C. J. (1988) Purification and characterization of a rabbit bone metalloproteinase that de-
properties of a small latent matrix metalloproteinase of the rat grades proteoglycan and other connective tissue components.
uterus. j Biol. C/tern. 263, 16918-16925 Biochem. j 209, 741-752

2152 Vol. 5 May 1991 The FASEB Journal WOESSNER

w.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on September 15, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
 33. Banda, M. J., and Werb, Z. (1981) Mouse macrophage elastase. 51. Br#{228}nnstr#{246}m,
M., Woessner, J. F, Jr., Koos, R. D., Sear,
Purification and characterization as a metalloproteinase. Bio- C. H. J., and LeMaire, W. J. (1988) Inhibitors of mammalian
c/tern. j 193, 589-605 tissue collagenase and metalloproteinases suppress ovulation in
34. Carrell, R. W., and Boswell, D. R. (1986) Serpins: the super- the perfused rat ovary. Endocrinology 122, 1715-1721
family of plasma serine proteinase inhibitors. In Proteinase Inhibi- 52. Muller, D., Quantin, B., Gesnel, M.-C., Milton-Collard, R.,
tors (Barrett, A. J., and Salvesen, G., eds) pp. 403-420, Elsevier, Abecassis, J., and Breathnach, R. (1988) The collagenase gene
Amsterdam family in humans consists of at least four members. Biochern. j
35. Cawston, T. E. (1986) Protein inhibitors of metallo-proteinases. 253, 187-192
In Proteinase Inhibitors (Barrett, A. J., and Salvesen, G., eds) pp. 53. Nicholson, R., Murphy, G., and Breathnach, R. (1989) Human
589-610, Elsevier, Amsterdam and rat malignant-tumor-associated mRNAs encode stromelysin-
36. Williamson, R. A., Marston, F. A. 0., Angal, S., Koklitis, P., like metalloproteinases. Biochemistry 28, 5195-5203
Panico, M., Morris, H. R., Came, A. F., Smith, B. J., Harris, 54. Huhtala, P., Chow, L. T., and Tryggvason, K. (1990) Structure
T. J. R., and Freedman, R. B. (1990) Disulphide bond assign- of the human type IV collagenase gene. j Biol. Chem. 265,
ment in human tissue inhibitor of metalloproteinases (TIMP). 11077-11082
Biochem. J. 268, 267-274 55. Huhtala, P., Eddy, R. L., Fan, Y. S., Byers, G., Shows, T. B.,
37. Boone, T. C., Johnson, M. J., De Clerck, Y. A., and Langley, and Tryggvason, K. (1990) Completion of the primary structure
K. E. (1990) eDNA cloning and expression of a metallopro- of the human type IV collagenase preproenzyme and assign-
teinase inhibitor related to tissue inhibitor of metalloprotein- ment of the gene (CLG4) to the q21 region of chromosome 16.
ases. Proc. NatL Acad. Sci. USA 87, 2800-2804 Genomics 6, 554-559
38. Edwards, D. R., Waterhouse, P., Holman, M. L., and Den- 56. Angel, P., Baumann, I., Stein, B., Delius, H., Rahmsdorf,
hardt, D. T (1986) A growth-responsive gene (16C8) in normal H. J., and Herrlich, P. (1987) 12-O-Tetradecanoyl-phorbol-13-
mouse fibroblasts homologous to a human collagenase inhibitor acetate induction of the human collagenase gene is mediated by
with erythroid-potentiating activity: evidence for inducible and an inducible enhancer element located in the 5-flanking region.
constitutive transcripts. Nucleic Acids Res. 14, 8863-8878 Mol. CelL Biol. 7, 2256-2266
39. Horowitz, S., Dafni, N., Shapiro, D. L., HoIm, B. A., Notter, 57. Krane, S. M., Conca, W., Stephenson, M. L., Amento, E. P.,
R. H., and Quible, D. J. (1989) Hyperoxic exposure alters gene and Goldring, M. B. (1990) Mechanisms of matrix degradation
expression in the lung. Induction of the tissue inhibitor of in rheumatoid arthritis. Ann. NY Acad. Sci. 580, 340-354
metalloproteinases mRNA and other mRNAs. j BioL C/tern. 58. Brenner, D. A., O’Hara, M., Angel, P., Chojkier, M., and
264,7092-7095 Karin, M. (1989) Prolonged activation of Jun and collagenase
40. Murphy, G., Koklitis, P., and Came, A. F. (1989) Dissociation genes by tumor necrosis factor-a. Nature (London) 337, 661-663
of tissue inhibitor of metalloproteinases (TIMP) from enzyme 59. Sch#{246}nthal,A., Herrlich, P., Rahmsdorf, H. J., and Ponta, H.
complexes yields fully active inhibitor. Biochem. j 261, (1988) Requirement for fos gene expression in the transcrip-
1031-1034 tional activation of collagenase by other oncogenes and phorbol
41. Herron, G. S., Werb, Z., Dwyer, K., and Banda, M. J. (1986) esters. Cell 54, 325-334
Secretion of metalloproteinases by stimulated capillary endo- 60. Gutman, A., and Wasylyk, B. (1990) The collagenase gene
thelial cells. II. Expression of collagenase and stromelysin ac- promoter contains a TPA and oncogene-responsive unit encom-
tivities is regulated by endogenous inhibitors.j BioL Chem. 261, passing the PEA3 and AP-1 binding sites. EMBO j 9,
2814-2818 2241-2246
42. Stetler-Stevenson, W. G., Krutzsch, H. C., and Liotta, L. A. 61. Brinckerhoff, C. E., and Auble, D. T. (1990) Regulation of col-
(1989) Tissue inhibitor of metalloproteinase (TIMP-2). A new lagenase gene expression in synovial fibroblasts. Ann. NY Acad.
member of the metalloproteinase inhibitor family. j Biol. Chem. Sci. 580, 355-374
264, 17374-17378 62. Frisch, S. M., and Werb, Z. (1989) Molecular biology of colla-
43. Goldberg, G. I., Marmer, B. L., Grant, G. A., Eisen, A. Z., gen degradation. In Collagen: Molecular Biology (Olsen, B. R.,
Wilhelm, S., and He, C. (1989) Human 72-kilodalton type IV and Nimni, M. E., eds) Vol. IV, pp. 85-108, CRC Press, Boca
collagenase forms a complex with tissue inhibitor of metallo- Raton, Florida
proteases designated TIMP-2. Proc. NatL Acad. Sci. USA 86, 63. Werb, Z. (1989) Proteinases and matrix degradation. In Textbook
8207-8211 of Rheumatology, 3rd ed (Kelly, W. N., Harris, E. D., Jr., Ruddy,
44. Cawston, T. E., Curry, V. A., Clark, I. M., and Hazelman, S., and Sledge, C. B., eds) pp. 300-321, W. B. Saunders,
B. L. (1990) Identification of a new metalloproteinase inhibitor Philadelphia
that forms tight-binding complexes with collagenase. Biochern. j 64. Brinckerhoff, C. E., Mitchell, T. I., Karmilowicz, M. J., Kluve-
269, 183-187 Beckerman, B., and Benson, M. D. (1989) Autocrine induction
45. Teahan,J., Harrison, R., Izquierdo, M., and Stein, R. L. (1989) of collagenase by serum amyloid A-like and 132-microglobulin-
Substrate specificity of human fibroblast stromelysin. Hydroly- like proteins. Science 243, 655-657
sis of substance P and its analogues. Biochemistry 28, 8497-8500 65. Sottile, J., Hoyle, M., and Millis, A. J. T. (1988) Differential
46. Coulombe, B., and Skup, D. (1988) In vitro synthesis of the ac- response of early and late passage fibroblasts to collagenase
tive tissue inhibitor of metalloproteinases encoded by a com- stimulatory factor in conditioned media. Collagen ReL Res. 8,
plementary DNA from virus-infected murine fibroblasts. 361-374
J. BioL C/tern. 263, 1439-1443 66. Frisch, S. M., Reich, R., Collier, I. E., Genrich, L. T., Martin,
47. Rajabi, M., Dean, D. D., and Woessner, J. F., Jr. (1987) High G., and Goldberg, G. I. (1990) Adenovirus EIA represses pro-
levels of serum collagenase in premature labor - a potential bio- tease gene expression and inhibits metastasis of human tumor
chemical marker. Obstet. GynecoL 69, 179-186 cells. Oncogene 5, 75-83
48. Caputo, C. B., Wolanin, D. J., Roberts, R. A., Sygowski, L. A., 67. Chandrasekhar, S., and Harvey, A. K. (1988) Transforming
Patton, S. P., Caccese, R. G., Shaw, A., and DiPasquale, G. growth factor-13 is a potent inhibitor of IL-I induced protease
(1987) Proteoglycan degradation by a chondrocyte metallopro- activity and cartilage proteoglycan degradation. Bioc/zern. Bio-
tease. Effects of synthetic protease inhibitors. Bioc/tem. Phar- phys. R#{128}s. Comrnun. 157, 1352-1359
macol. 36, 995-1002 68. Jonat, C., Rahmsdorf, H. J., Park, K. -K., Cato, A. C. B.,
49. Darlak, K., Miller, R. B., Stack, M. S., Spatola, A. F, and Gebel, S., Ponta, H., and Herrlich, P. (1990) Antitumor promo-
Gray, R. D. (1990) Thiol-based inhibitors of mammalian col- tion and antiinflammation: down-modulation of AP-l (Fos/Jun)
lagenase. Substituted amide and peptide derivatives of the leu- activity by glucocorticoid hormone. Cell 62, 1189-1204
cine analogue 2-[(R,S)-mercaptomethyl]-4-methylpentanoic acid. 69. Yang-Yen, H. -F, Chambard, J. -C., Sun, Y. -L., Smeal, T., and
j BioL C/tern. 265, 5199-5205 Schmidt, T. J., Drouin, J., and Karin, M. (1990) Transcrip-
50. Kortylewicz, Z. P., and Galardy, R. E. (1990) Phosphoramidate tional interference between c-Jun and the glucocorticoid recep-
peptide inhibitors of human skin fibroblast collagenase. j Med. tor: mutual inhibition of DNA binding due to direct protein-
Chem. 33, 263-273 protein interaction. Cell 62, 1205-1215

MATRIX METALLOPROTEINASES 2153

w.fasebj.org by Univ of So Dakota Lommen Hlth Sci Library (192.236.36.29) on September 15, 2018. The FASEB Journal Vol. ${article.issue.getVolume()}, No. ${article.issue.getIssueNum
 70. Sch#{252}le, R., Rangarajan, P., Kliewer, S., Ransone, L. J., Bolado, lagenolysis and bone resorption. Mol. Aspects Med. 10, 301-428
J., Yang, N., Verma, I. M., and Evans, R. M. (1990) Functional 75. Dean, D. D., Martel-Pelletier, J., Pelletier, J. -P., Howell, D. S.,
antagonism between oncoprotein c-Jun and the glucocorticoid and Woessner, J. F, Jr. (1989) Evidence for metalloproteinase
receptor. Cell 62, 1217-1226 and metalloproteinase inhibitor imbalance in human osteo-
71. Weeks, J. G., Halme, J., and Woessner, J. F, Jr. (1976) Extrac- arthritic cartilage. j Clin. Invest. 84, 678-685
tion of collagenase from the involuting rat uterus. Biochirn. Bzo-
76. Howell, D. S., and Dean, D. D. (1991) The biology, chemistry
phys. Ada 445, 205-214
and biochemistry of the mammalian growth plate. In Disorders
72. Gunja-Smith, Z., Nagase, H., and Woessner, J. F., Jr. (1989) of Bone and Mineral Metabolism (Coe, F L., and Favus, M. J., eds)
Purification of the neutral proteoglycan-degrading metallopro-
Raven, New York
teinase from human articular cartilage tissue and its identifica-
tion as stromelysin matrix metalloproteinase-3. Biochem. j 258, 77. Lloyd, L. F., Skarzynski, T., Wonacott, A. J., Cawston, T. E.,
115-119 Clark, I. M., Mannix, C. J., and Harper, G. P. (1989) Crystalli-
73. JelTrey, J. J. (1986) Biological regulation of collagenase activity. zation and preliminary X-ray analysis of porcine synovial col-
In Regulation of Matrix Accumulation (Mecham, R. P., ed) pp. lagenase. j Mol. Biol. 210, 237-238
53-98, Academic, Orlando, Florida 78. Lepage, T., and Gache, C. (1990) Early expression of a
74. Sakamoto, S., and Sakamoto, M. (1988) Degradative processes collagenase-like hatching enzyme in the sea urchin embryo.
of connective tissue proteins with special emphasis on col- EMBOJ. 9, 3003-3012

2154 Vol. 5 May 1991 The FASEB Journal WOESSNER

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