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Methods in Molecular Biology TM

VOLUME 151

Matrix
Metalloproteinase
Protocols
Edited by
Ian M. Clark

HUMANA PRESS
MMPs and TIMPs – An Historical Perspective 1

1
MMPs and TIMPs

An Historical Perspective

J. Frederick Woessner, Jr.

1. Introduction
The matrix metalloproteinase field has a clearly defined starting point, the
seminal study of Jerome Gross and Charles Lapière in 1962 (1) in which frag-
ments of resorbing tadpole tail were cultured on reconstituted collagen gels. A
collagenolytic enzyme was recovered from the culture medium which could
attack native collagen fibrils. Within a short time these studies were extended
to show a similar activity in a wide variety of tissues, that the collagen mol-
ecule was cut into 3/4- and 1/4-length fragments, and that the enzyme activity
was dependent on metal ions. The tissue inhibitors of metalloproteinases were
discovered in the following decade in tissue culture (2) and serum (3). It was
soon recognized that the matrix metalloproteinases (MMPs) are involved in a
great many processes of normal development and growth and in pathological
processes such as arthritis and cancer metastasis as tabulated in Woessner, 1998
(4). This has resulted in a tremendous outpouring of studies now numbering
about 10,000 references with 1,000 appearing each year. There have been many
recent reviews of the MMPs, including an entire book—Matrix Metallopro-
teinases—edited by Parks and Mecham (5) (1998) and detailed chapters in The
Handbook of Proteolytic Enzymes (6) and Methods of Enzymology Vol. 248
(7). These will be cited at appropriate points below.
There are now at least 18 distinct vertebrate MMPs. This group was
originally defined on the basis of dependence on zinc for catalytic activity
and the presence of a large propeptide responsible for latency. The recognition
of other enzymes with such properties (e.g., reprolysins) has led to the defini-
tion of the group on the basis of DNA sequence. As outlined in the Handbook
From: Methods in Molecular Biology, vol. 151: Matrix Metalloproteinase Protocols
Edited by: I. Clark © Humana Press Inc., Totowa, NJ

1
2 Woessner

of Proteolytic Enzymes (6) pp. 1144 – 1147, the matrixin subfamily and the
serralysin subfamily are grouped together in family M10 of the metal-
loproteinase clan MB. The closely related family M12 contains the astacins
and the reprolysins. The close relationship is not in the sequence but in the
protein fold—the so-called metzincin fold (8). Of these 4 subgroups, only the
MMP subfamily is inhibited by tissue inhibitor of metalloproteinases (TIMPs),
whereas all members are susceptible to hydroxamate inhibitors originally
developed for the MMPs.
Table 1 lists the currently known MMPs and related forms from lower organ-
isms. The MMPs have a very wide range of domains and segments that can be
added to the basic core found in matrilysin: the propeptide and catalytic domains.
These may be selected from the furin cleavage site RXKR, the fibronectin-like
repeats, the hinge region, the hemopexin domain and the transmembrane domain.
Chapter 4 provides more detail about the size and functions of the various
domains and inserts. One consequence of these variations is that the MMPs may
range in Mr from 105,000 for the largest proenzyme to 19,000 for the smallest
active enzymes. There are three web sites that provide useful data about the
MMPs. Neil Rawlings and Alan Barrett maintain a site named MEROPS that
gives detailed information about each proteolytic enzyme; it is found at http://
www.bi.bbsrc.ac.uk/MEROPS/. Those enzymes that are listed in the Enzyme
Commission list (see Table 1) can be found at http://alpha.qmw.ac.uk/~ugca000/
iubmb/enzyme/. Finally, each MMP that has been sequenced can be found
by name and animal species in the SwissProt protease databank at http://
expasy.hcuge.ch/sprot/sprot-top.html.

2. A Brief Historical Overview of the Individual MMPs

2.1. Interstitial Collagenase (MMP-1; Collagenase 1)
As mentioned previously the first discovery of a vertebrate collagenase was
that of Gross and Lapière (1). Although the major emphasis of their paper was on the
frog enzyme, activity was also shown in tissues of rat, pig, chick, and mouse. The rat
and mouse enzymes are likely, in retrospect, to have been MMP-13 whereas the frog
enzyme could have been MMP-1 or MMP-18 or a mixture. The frog enzyme was
shown to cleave the collagen molecule into a 3/4-length piece from the N-end and a
1/4-length piece from the C-end by the unusual method of reconstituting these frag-
ments into Segment Long Spacing structures that could be viewed in the electron
microscope (9). A complete purification of skin MMP-1 was achieved in 1970 (10).
Oddly, it was not recognized until 1971 that the collagenases occurred as zymogens
(11) that could be activated by trypsin (12) or organomercurials (13). The first com-
plete sequence, based on cDNA was reported in 1986 (14). Reviews of collagenase 1
are found in (15–17).
MMPs and TIMPs – An Historical Perspective 3

Table 1
The Matrix Metalloproteinases
MMP-No. Name E.C. number Other names/notes

MMP-1 Collagenase 1 3.4.24.7 Interstititial collagenase

MMP-2 Gelatinase A 3.4.24.24 72 kDa gel’ase,
type IV collagenase
MMP-3 Stromelysin 1 3.4.24.17 Transin, proteoglycanase, CAP
(MMP-4 Procollagen peptidase — Discontinued = MMP-3)
(MMP-5 3/4-Collagenase — Discontinued = MMP-2)
(MMP-6 Acid metalloprotease — Discontinued = MMP-3)
MMP-7 Matrilysin 3.4.24.23 Pump-1
MMP-8 Collagenase 2 3.4.24.34 Neutrophil collagenase
MMP-9 Gelatinase B 3.4.24.35 92 kDa gel’ase,
type V collagenase
MMP-10 Stromelysin 2 3.4.24.22 Transin 2
MMP-11 Stromelysin 3 Furin motif
MMP-12 Macrophage elastase 3.4.24.65 Metalloelastase
MMP-13 Collagenase 3 Rodent intersititial
collagenase
MMP-14 Membrane type matrix MT1-MMP, furin motif
metalloproteinase 1
MMP-15 Membrane type matrix MT2-MMP, furin motif
metalloproteinase 2
MMP-16 Membrane type matrix MT3-MMP, furin motif
metalloproteinase 3
MMP-17 Membrane type matrix MT4-MMP, furin motif
metalloproteinase 4
MMP-18 Collagenase 4 Xenopus
MMP-19 No trivial name RASI-1
MMP-20 Enamelysin
XMMP No trivial name Xenopus, furin motif
MMP-C31 (also H19, Y19) Caenorhabditis elegans
— Envelysin 3.4.24.12 Sea urchin
— Soybean MMP Glycine max, no cDNA
— Fragilysin 3.4.24.74 Bacteroides fragilis

2.2. Gelatinase A (MMP-2, Type IV Collagenase)

Sellers et al. in 1978 (18) were the first to separate a gelatinase activity from
collagenase and stromelysin in culture medium from rabbit bone. A similar
enzyme, acting on basement membrane type IV collagen was reported by Liotta
et al. the following year (19). Gelatinase was purified from human skin (20),
4 Woessner

mouse tumor cells (21), rabbit bone (22), and human gingiva (23). The com-
plete sequence of the human enzyme except for the signal peptide was reported
by Collier et al. (24). Gelatinase A has a triple repeat of fibronectin type II
domains inserted in the catalytic domain; these participate in binding to the
gelatin substrates of the enzyme (25). Recent reviews of gelatinase A are found
in (26–28).
2.3. Stromelysin 1 (MMP-3)
A neutral proteinase distinct from collagenase was first noted in 1974 by
Sapolsky et al. in human cartilage (29) and Werb and Reynolds in rabbit bone
fibroblast culture (30). The enzyme was metal-dependent and lost about 20,000
Daltons upon activation of the latent form (31). Purification of the rabbit bone
enzyme (32) permitted its detailed characterization and it was referred to as
proteoglycanase. The name ‘stromelysin’ was introduced by Chin et al. (33)
but the enzyme has also been named transin (34) and collagenase activating
protein (35). The first cDNA sequence was obtained for rat transin in 1985
(34). Recent reviews are found in (36–38).
2.4. The Missing Enzymes (MMP-4, -5, and -6)
These three enzymes were given numbers soon after their discovery, but
it was subsequently determined that they were identical to other known MMPs.
MMP-4 or collagen telopeptidase was described by Scott in 1983 in pig and
human gingival extracts (39,40). Nakano et al. (41) proposed the name MMP-4
for the human enzyme that removes the C-telopeptides from type I collagen. The
subsequent demonstration that stromelysin cleaves the N-telopeptide, and prob-
ably the C-telopeptide (42), from type II collagen suggested that MMP-4 was
actually MMP-3. It is possible that MMP-13 may have been involved as well.
Overall and Sodek (43) described a 3/4-collagenase that acted on fragments
of type I collagen produced by MMP-1. However, this was subsequently iden-
tified as MMP-2 (44). Sapolsky et al. (45) extracted an aggrecan-degrading
activity from human cartilage with an acid pH optimum of 5.5. This activity
was purified (46) and shown to increase in osteoarthritis (47). However, it was
subsequently demonstrated (48,49) that this acid pH optimum was actually a
property of MMP-3.
2.5. Matrilysin (MMP-7)
Matrilysin is the smallest of the MMPs, having only the propeptide and cata-
lytic domains. It was first identified in the postpartum involuting uterus of the
rat by Sellers and Woessner in 1980 (50). However, it was not until 1988 that
the enzyme was purified and characterized from rat uterus (51) and cloned and
MMPs and TIMPs – An Historical Perspective 5

sequenced from a human library (52). Expression of the human cDNA estab-
lished that this enzyme, up until then known as putative matrix metal-
loproteinase or Pump-1, was a protease with properties similar to the rat
enzyme (53). The enzyme was also independently discovered as the kidney
activator of urokinase (54). Reviews of the enzyme and its properties may be
found in (55–57).
2.6. Neutrophil Collagenase (MMP-8, Collagenase 2)
A collagenase activity from human neutrophils was first reported by Lazarus
et al. in 1968 (58,59). This could be activated by a factor in synovial fluid (60).
The enzyme was located in the specific granules (61) and released upon phago-
cytosis (62). The enzyme was first purified in 1983 (63) and shown to be a
distinct gene product from MMP-1 by antibody reaction (64). Cloning and
sequencing was accomplished in 1990 (65). Review articles are found in
(16,66,67). An early and lengthy series of papers in the 1970s purporting to
describe neutrophil collagenase were finally shown by Ohlsson to refer to a
serine enzyme and not to MMP-8 (68).
2.7. Gelatinase B (MMP-9, Type V Collagenase)
Harris and Krane in 1972 (69) detected a gelatinase activity in rheumatoid
synovial fluid. In retrospect this is likely to have been MMP-9. Sopata et al.
(70) described a gelatinase from human polymorphonuclear leukocytes. This
was shown to be latent and activated by organomercurials (71). Rabbit mac-
rophages produce a very similar enzyme which is able to digest type V col-
lagen (72). The neutrophil collagenase and gelatinase were resolved in 1980
(73). Purification was achieved in 1983 (74) and sequencing of the cDNA in
1989 (75). An interesting phenomenon, still not fully understood, is the bind-
ing of TIMP-1 to proMMP-9 to form a complex that further regulates the
activation and subsequent activity of the enzyme (76). Human neutrophil
MMP-9 commonly occurs as a complex with lipocalin (77). A series of papers
concerning a 95 kDa protein in plasma that binds to gelatin culminated in the
identification of this protein as MMP-9 (78).
2.8. Stromelysin 2 (MMP-10, Transin 2)
This is the first of the MMPs to be discovered by cloning and sequencing,
in this case as rat transin 2 (79), which indicated an enzyme 71% identical to
transin 1 (MMP-3) This was soon followed by the cloning of the human enzyme
(52). Both the rat and human forms were shown to have proteolytic activity
when they were expressed (80); they were inhibited by TIMP and activated
latent proMMP-1. Review articles are found in (36,38,81).
6 Woessner

2.9. Stromelysin 3 (MMP-11)

The putative protease was first identified as a cDNA clone by Basset et al. in
1990 (82). Several years went by before Murphy et al. (83) demonstrated that
the expressed protein showed weak proteolytic activity. Pei and Weiss (84)
first showed respectable catalytic action of the enzyme in digesting α1-pro-
teinase inhibitor; α2-macroglobulin was also attacked. This MMP contains an
RKRR sequence at the end of the propeptide and it was shown by Pei (85) that
there is indeed intracellular activation of the proenzyme by the action of furin.
In this respect, stromelysin 3 is quite distinct from the other two stromelysins
and the evolutionary tree also emphasizes this distinction (4). The relative pau-
city of substrates has resulted in the lack of extensive reviews of this enzyme.
Basset et al. (86) present a good review of the role of MMP-11 in cancer
progression.
2.10. Macrophage Elastase (MMP-12)
Mouse macrophages were found to produce both serine and metallo proteases
acting on elastin (87). Banda and Werb (88) established the metalloprotease nature of
the elastase and showed it was not blocked by α1-proteinase inhibitor, which was in
fact a substrate. The mouse enzyme was purified in 1981 (89) and the studies of its
specificity (90) were the earliest of any MMP except MMP-1. The mouse enzyme
was cloned, sequenced, and assigned to chromosome 9 (91) followed in a few years
by the human enzyme sequence and chromosome assignment to 11q22.2-22.3 (92).
Review articles are found in refs. 93,94.
2.11. Collagenase 3 (MMP-13)
In the first paper of Gross and Lapière (1) collagenase was noted in the
involuting rat uterus and in mouse and rat bone. It was assumed at that time
that all the collagenases were the same and it was not recognized that the rat
and mouse lacked the gene for MMP-1. When the rat uterine collagenase was
sequenced (95) it was noted that the homology to human MMP-1 was unex-
pectedly remote. But it wasn’t until the human MMP-13 was sequenced (96)
that the explanation emerged; the rat enzyme was very much closer
to the human MMP-13 than to MMP-1. There is a long history of the
rat/mouse enzyme starting with purification from bone (97) and uterus
(98,99). The uterine enzyme is one of the earlier MMPs to have been
extracted directly from tissue (100). The human enzyme has not yet been char-
acterized in much detail. Reviews may be found in (16,101).
2.12. Membrane Type Matrix Metalloproteinase 1 (MT1-MMP)
Sato et al. in 1984 (102) cloned and sequenced a novel MMP that appeared
to have a transmembrane domain close to its C-terminus and coming after the
MMPs and TIMPs – An Historical Perspective 7

hemopexin domain; this enzyme could activate proMMP-2 also bound to the
cell surface. Further cloning and sequencing of the human enzyme followed
shortly in 3 labs (103–105). The early reports used the nomenclature MT-
MMP-1, however, this proved very confusing as many thought it meant
MMP-1 (i.e., collagenase) inserted in the membrane. Reviews of this enzyme
are found in (106,107).

2.13. Membrane Type Matrix Metalloproteinases 2, 3,

and 4 (MT2-MMP, MT3-MMP, MT4-MMP)
In view of the recent discovery of these enzymes, there is relatively little litera-
ture at the moment. There is a report of the cloning and sequencing of MT2-MMP
by Takino et al. in 1995 (108). However, they were unaware of the cloning of a
second membrane type enzyme by Will and Hinzmann (109). Therefore, the Will
enzyme should be designated MT2 and the Takino enzyme is MT3. Proteolytic
activity was demonstrated for expressed MT2-MMP (110). MT3-MMP is subject
to alternate splicing, yielding two sizes of enzyme (111). MT4-MMP was
sequenced by Puente et al. (112). Reviews of these additional MT-MMPs are found
as part of the articles cited for MT1-MMP.

2.14. MMP-18 and Higher

There is a single reference to MMP-18 or collagenase 4 (113). The cDNA
was isolated from a Xenopus library and represents the first amphibian MMP
actually sequenced. The expressed protein is a collagenase. A brief review is
presented in (114).
The sequence of MMP-19 was first presented in 1996 (115); the authors
were unaware of the Xenopus enzyme and so used the name MMP-18 in their
paper. However, it is more appropriately designated MMP-19; there is no trivial
name assigned to this enzyme yet. Two further sequences were reported the
following year (116,117), the latter group worked with a rheumatoid arthritis
synovial inflammation and so used the designation RASI-1 for their enzyme.
The protein has been identified in tissues (118) but there is only sparse infor-
mation on the enzyme activity (116). The enzyme is similar to the other 55 kDa
MMPs, but has a long hinge region between the catalytic and hemopexin
domains.
Enamelysin (MMP-20) was noted as a metal-dependent activity in the devel-
oping enamel of pig and rat (119,120) and cow (121). The pig enzyme was cloned
and sequenced in 1996 (122) and the human, the following year (123).
Finally, mention may be made of XMMP, a novel enzyme from Xenopus
which has a long insert between the propeptide and the catalytic domain,
including an RRKR motif indicative of furin processing (124).
8 Woessner

2.15. MMPs from Invertebrates, Plants, and Bacteria

The sea urchin contains a metalloproteinase active in the hatching process,
this is now known by the name envelysin. Although a protease had been impli-
cated in hatching for about 50 years, it was only in 1989 that a metalloprotease
of about 54 kDa was purified (125). The sequence followed in the next year
(126) and the enzyme was found to be homologous to MMP-1. The propeptide
was somewhat long at 148 amino acids and the catalytic plus hemopexin
domain contained a more typical 421 residues. A second species was sequenced
by Nomura et al. (127) and they introduced the current name. A review is avail-
able in (128).
The nematode Caenorhabditis elegans has a number of sequences that
appear to correspond to MMPs. However, there is only a single recent report
on the nature of these enzymes, designated MMP-C31, MMP-H19, and MMP-
Y19 (129). The last two have the RXKR motif suggesting furin processing.
The first two are inhibited by TIMP-1.
Soybean leaves were found to contain a metalloproteinase (130) that was
purified in 1991 (131). A sequence of the active form, obtained by classical
amino acid sequencing, revealed a 20 kDa active domain related to MMP-1
(132). It is inhibited by TIMP and hydroxamate compounds. A recent update is
found in (133).
The intestinal bacterium Bacteroides fragilis produces an enterotoxin that is
important in human and animal disease. It was purified by van Tassell (134),
sequenced by Moncrief et al. (135) and shown to be a metalloproteinase related
to MMP-1. The expressed protein was capable of causing disease in lamb, rat,
and rabbit (136). A recent review is found in (137).

3. Tissue Inhibitors of Metalloproteinases (TIMPs)

The release of proteolytic enzymes into the extracellular matrix outside the
cell poses certain risks of untrammeled breakdown. A number of mechanisms
have evolved to help avoid uncontrolled proteolysis. First, the cells produce
most MMPs only after receiving an appropriate signal requesting de novo syn-
thesis. Second, the enzymes are in a proform that requires activation, presum-
ably by controlled processes such as urokinase activation of plasminogen that
in turn activates MMPs or by cell-surface MT-MMPs. Finally, there are gen-
eral inhibitors in the matrix such as α2-macroglobulin family members and the
more specific TIMPs. There are currently 4 known TIMPs, although the reasons
for this multiplicity are not yet clear. In addition to maintaining control over the
activity of MMPs, TIMP-1 and -2 are able to bind directly to the hemopexin
domain of MMP-9 and MMP-2, respectively, exerting further control over the
activation process. There are also other roles of TIMPs that do not seem to be
MMPs and TIMPs – An Historical Perspective 9

directly attributable to protease inhibition such as growth factor activity, steroido-

genesis, and cell morphology modulation.
3.1. Tissue Inhibitor of Metalloproteinases 1 (TIMP-1)
It was first noted by Bauer et al. in 1975 (2) that cultured human fibroblasts
produced an inhibitory protein. In the same year Woolley (3) noted a serum
inhibitor dubbed β1-anticollagenase. Cartilage was shown to have an extract-
able inhibitor (138). A complete purification was achieved in 1979 (139) from
skin fibroblasts, followed later by protein from rabbit bone (140) and cow aorta
(141). An important feature of the inhibitor is its resistance to high tempera-
ture, but sensitivity to reduction and alkylation (141). A sequence was obtained
in 1985 (142). This provided the surprise that the protein was already known in
a completely different context.
Work by Gauwerky et al. in 1980 (143) showed that human T-lymphoblasts
produce a factor that potentiates erythroid factor. This erythroid potentiating
factor was cloned and sequenced by Gasson et al. (144) and it is that sequence
that TIMP was subsequently found to match (142). But TIMP has been redis-
covered on a number of other occasions: fibroblasts were shown to express a
gene upon stimulation by serum, the gene sequence matched EPF/TIMP (145).
Phorbol stimulation of mouse cells produced a tumor promoting factor TPA-
S1 which also had the sequence of TIMP (146); this appears in a number of
literature reports under the name phorbin. Newcastle disease in mice induces a
gene that matches TIMP (147) and bovine cumulus cells produce embryo-
genin-1 which is also TIMP (148). Sertoli cells produce a stimulator of ste-
roidogenesis that is a complex of TIMP-1 and cathepsin L (149). Some of these
additional growth factor effects are reviewed in (150,151). Finally, TIMP-1 is
able to bind to the hemopexin domain of proMMP-9 (152); here it is not react-
ing with the active center, which is concealed by the propeptide, and so it is
believed to have an important regulatory role.

3.2. Tissue Inhibitor of Metalloproteinases 2 (TIMP-2)

The second form of TIMP was first detected during the development of the
technique of reverse zymography (153). Sheep cervix was noted to contain both a
28 kDa and a 20 kDa form of TIMP (154). De Clerck et al. (155) purified both
forms from bovine endothelial cell culture and showed by sequence of the N-ter-
minus that there was 51% identity. A partial sequence was also obtained by
Goldberg et al. (152) who also observed the complex between TIMP-2 and
proMMP-2. This complex is believed to have a regulatory role, particularly in the
activation of proMMP-2 by cell-surface MMP-14 (104). A complete sequence
was obtained by Edman degradation (156) followed shortly by cDNA sequenc-
10 Woessner

ing (157). As with TIMP-1, there have been several re-discoveries based on
growth effects. Thus, a mouse factor that promoted survival of pituitary cells
in culture proved to have the sequence of TIMP-2 (158) as did an autocrine
factor that stimulated SV-40 transformed human fibroblasts (159). A factor
stimulating osteosarcoma cells also proved to be TIMP-2 (160). As in the case
of TIMP-1, it is unlikely that these effects can all be explained on the basis of
blocking proteolysis. A review of TIMP-1 and TIMP-2, particularly with
respect to their assay, is presented in (161).
3.3. Tissue Inhibitor of Metalloproteinase 3 (TIMP-3)
Blenis and Hawkes in 1983 (162) found expression of a protein in the extra-
cellular matrix produced by transformed chick embryo fibroblast cells. This
protein of Mr 21,000 binds tightly to the matrix (163) but was not identified as
a TIMP until 7 years later (164). The name assigned to the protein based on
cDNA sequence was ChiMP-3 (165). The human and mouse TIMP-3 clones
were sequenced by Apte and colleagues (166,167). Considerable interest has
been aroused by the discovery that Sorsby’s fundus dystrophy involves muta-
tions in this protein (168).
3.4. Tissue Inhibitor of Metalloproteinases 4 (TIMP-4)
This inhibitor was first identified upon cloning (169), mRNA was seen in heart
but was low in kidney, placenta, colon, and testes and absent in many other tissues.
TIMP-4 is similar to TIMP-2 in its ability to bind to proMMP-2 (170). The mouse
and rat proteins have also been sequenced (171,172). Expressed TIMP-4 has been
shown to inhibit at least MMP-1, -2, -3, -7, and -9. In general, most TIMPs appear
to be able to inhibit most active MMPs, but there are considerable differences in
binding affinities. An overview of homologies among 17 species of the 4 TIMPs is
presented in (173).

4. Conclusion
MMPs continue to be discovered. Highly specialized MMPs such as
enamelysin, found only in the embryonic tooth enamel may have corre-
sponding undiscovered counterparts in other tissues or organs. The human
genome project will no doubt uncover further examples. In the case of TIMPs,
there is an example from the C. elegans sequence of a TIMP that seems to
consist solely of the N-terminal inhibitory domain. Similar inhibitors might
exist in higher organisms. There have been several reports of small IMPs, below
the size of TIMP, seen on zymograms (174).
A great deal still remains to be learned about the MMPs and their inhibition.
We know almost nothing about the natural substrates of the MMPs in vivo,
about how the cell senses and regulates the level of enzymes and inhibitors out
MMPs and TIMPs – An Historical Perspective 11

in the matrix, and about the ability of the cell to substitute one enzyme for
another in knockouts. The clear involvement of the MMPs in the major disease
processes of cancer, cardiovascular disease, and arthritis suggests that there
will be significant funding for research on these enzymes. Attention will no
doubt focus on ways to regulate the activities through gene manipulation and
through specific inhibitors of enzyme expression or activity.

Acknowledgment
Supported by NIH Grant AR-16940.

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 Another Random Document on
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