(Received for publication, May 30, 1995)
From the
Matrix metalloproteinases (matrixins) constitute a group of
extracellular proteinases belonging to the metzincin superfamily. They
are involved in both physiological and pathological tissue remodeling
processes, including those associated with cancer progression.
Stromelysin-3, which is expressed in most invasive human carcinomas, is
a matrix metalloproteinase with unusual functional properties. In
particular, its mature form does not cleave any of the major
extracellular matrix components. To define critical structural
determinants involved in controlling stromelysin-3 proteolytic
activity, we have used site-directed mutagenesis. We show that the
deletion of at least 175 C-terminal amino-acids is sufficient to endow
mouse stromelysin-3 with activities against casein, laminin, and type
IV collagen. In the case of the human enzyme, however, a further and
single Ala-235 Pro substitution is necessary to observe similar
activities. Ala-235, which characterizes human stromelysin-3 among
matrixins, is located immediately after the C terminus of the
``Met-turn,'' which forms a hydrophobic basis for the
catalytic zinc atom in the metzincin family. We conclude that human
stromelysin-3 has gained specific functional properties during
evolution by amino acid substitution in the catalytic zinc environment,
and that it represents an attractive target for specific inhibitors
that may be used to prevent cancer progression.
Matrix metalloproteinases (matrixins) constitute a group of
extracellular proteinases of related primary structure, including
collagenases, gelatinases, stromelysins, and their
activators(1, 2, 3) . Matrix
metalloproteinases are believed to be mediators of physiological and
pathological remodeling processes occurring during development,
involution, repair, and cancer
progression(4, 5, 6, 7) . In
particular, these enzymes have been shown through both correlative and
direct inhibitor studies to be essential for cancer cell
invasion(8, 9, 10) . However, the
demonstration that stromelysin-1 plays a role in morphogenesis during
development (11) and the observation that a matrix
metalloproteinase-like enzyme is implicated in tumor necrosis
factor- processing (12) suggest that the contribution of
matrix metalloproteinases to tumor progression may be not limited to a
role in cancer invasion and also involve other aspects of the malignant
phenotype. In agreement with this possibility, matrix metalloproteinase
expression is not specific to invasive carcinomas and is also observed
in a number of precursor lesions ( (13) and references
therein).
Stromelysin-3 (ST3) ()was identified through
its overexpression in fibroblastic cells of invasive breast carcinomas (14) , and similar observations were thereafter made in most
other human carcinomas(13) . ST3-expressing fibroblastic cells
are specifically detected in the immediate vicinity of cancer cells,
suggesting that ST3 may play a role in stromal-epithelial interactions
during carcinoma progression, thereby contributing to tumor growth.
Indeed, Engel et al.(15) have found that recurrent
breast carcinoma was more frequent in patients showing tumors with high
ST3 RNA levels than in those with low ST3 RNA levels. Although ST3 has
a protein domain organization similar to that of other
stromelysins(14, 16) , it was found to display both
unusual structural and functional properties(17, 18) .
In particular, the putative mature form of human ST3 is unable to
hydrolyze any of the major extracellular matrix components of basement
membranes or interstitium(19) .
The present study was initiated to identify structural determinants controlling ST3 enzymatic activity. By site-directed mutagenesis, we demonstrated that deletion of the 175 C-terminal amino acids is necessary to endow mouse ST3 with proteolytic activities toward casein and some extracellular matrix molecules, while a similar deletion is ineffective with the human enzyme. However, when Ala-235 flanking the ``Met-turn'' (20) is replaced by a proline, the C-terminally truncated human protein exhibits proteolytic activities similar to those of the mouse enzyme. These findings add further support to the concept that human ST3 is a matrix metalloproteinase with unique functional properties and that it represents a potential target for specific inhibitors that may lead to the development of new anticancer agents(21, 22) .
C-terminally truncated forms for human (h) and mouse (m) ST3 were
obtained by introducing a second mutagenesis in the cDNA described
above, using the following primers in order to creat 3`-stop codons
(italic) followed by an unique XhoI restriction site for
cloning (underlined); the numbers following EH indicate the number
of amino acids that were C-terminally deleted: hE
H216,
5`-ACCCCAGCCCTGGGCTGACTCGAGGGGATAGACACCAAT-3`; hE
H158,
5`-TACCCAGCATAATGATCTCGAGACTGGCAGG-3`; mE
H216,
5`-GCCCCAACTTTGAGCTGATAGCTCGAGACAGATACC-3`; mE
H205,
5`-GAGATTGCATAGTAGCTCGAGGAAACCCCGCCA-3`; mE
H195,
5`-CCAGATGTCTGATAGCTCGAGTTCGACGCG-3`; mE
H183,
5`-TCCACCATCCGATGATAGCTCGAGTTCTTCAAGGCA-3`; mE
H175,
5`-TTCAAGGCATGATAGCTCGAGAGGCTGCGC-3`; mE
H167,
5`-CTGCGCAGTTGATGACTCGAGCCCGGGTAT-3`; mE
H158,
5`-TATCCTGCTTAGTAACTCGAGGCACTGGCA-3`.
Plasmids were then digested with NdeI and XhoI restriction enzymes, and the resulting ST3 DNA fragments were ligated into the corresponding sites of the pET-3b vector modified by introduction of a polylinker containing BstEII, KpnI, NdeI, and XhoI restriction sites.
The EH216 ST3 constructs were
further modified by site-directed mutagenesis for substitution of
Ala-235 by Pro-235 in the human enzyme (primer
5`-GCCCTGATGTCCCCGTTCTACACCTTT-3` was designed to remove an AciI cleavage site) and for substitution of Pro-239 by Ala-239
in the mouse enzyme (primer 5`-GCCCTCATGTCCGCGTTCTACACCTTC-3`
was synthesized in order to create a BstUI restriction site,
underlined). After mutagenesis, plasmids were digested with NdeI and XhoI restriction enzymes, and the resulting
ST3 cDNA fragments were ligated into the corresponding sites of the
pET-3b vector. All recombinant plasmids were sequenced with an
automated sequencer (Epicentre Technologies, Madison, WI) in order to
check that no mutation was introduced during plasmid construction.
In some experiments, EH216 ST3 preparations were extracted
from bacterial inclusion bodies as described above, adjusted to 6 M urea, and loaded onto a Q-Sepharose anion-exchange column (3 ml)
previously equilibrated with 50 mM Tris-HCl, pH 8.5,
containing 100 mM DTT and 6 M urea. Elution from the
column was performed with a NaCl gradient (0-500 mM) in
50 mM Tris-HCl, pH 8.5, containing 6 M urea. The
100-250 mM NaCl fractions were pooled, and purified
E
H216 ST3 was renatured by dialysis as described above.
For casein zymography, samples were
electrophorezed under nonreducing conditions in SDS-polyacrylamide (12
or 15%) gels containing 2 mg/ml /
-casein (Sigma, C-7891).
After electrophoresis at 4 °C, SDS was exchanged by three washes
with Triton X-100 (2.5%) in order to regenerate caseinolytic activity.
Gels were then incubated at 37 °C in 50 mM Tris-HCl, pH
7.5, containing 5 mM CaCl
and 1 µM ZnCl
. After 48 h of incubation, gels were stained with
Coomassie Brillant Blue R250 and destained in order to visualize clear
bands representing caseinolytic enzymes on a dark background.
-casein degradation was quantified using
C-labeled
-casein (Sigma, C-6034), as described previously by Murphy et
al.(29) with incubation at 37 °C for 18 h. In some
experiments,
-casein (Sigma, C-6905) degradation was
semiquantitately evaluated after incubation with ST3 in Tris assay
buffer at 37 °C for 18 h, followed by SDS-PAGE (15%) and silver or
Coomassie Blue staining. Similar tests were performed to evaluate ST3
activities toward human type IV collagen (provided by K. Kuhn,
Martinsried, Germany), laminin (extracted from EHS tumor according to
Timpl et al.,(30) ), and human plasma
1-proteinase inhibitor (provided by J.-P. Martin, Rouen, France).
Figure 1:
Electrophoretic analysis of human and
mouse ST3 forms expressed in E. coli. A, schematic
representation of human (h) and mouse (m) putative
mature ST3 (E) (lacking the prodomain) and of C-terminally ST3
truncated forms (EH), lacking part (E
H158)
or most (E
H216) of the ST3 hemopexin-like domain.
H158 and
H216 indicate that the 158 and 216
C-terminal amino acids have been deleted, respectively. Numbers above
or below frames refer to the first and last amino acids for each form,
according to Basset et al.(14) and Lefebvre et
al.(16) . C-terminal amino acids for each form are also
indicated inside frames by using the single-letter code. B, Coomassie Blue staining of crude ST3 preparations
after SDS-PAGE. Whole bacterial cells transformed with ST3 cDNAs
defined in panelA and cloned into the pET-3B vector
were collected after 3 h of
isopropyl-1-thio-
-D-galactopyranoside induction, lyzed,
and analyzed by SDS-PAGE (12%) under reducing conditions. pET-3b
corresponds to bacteria transformed with the pET-3b vector alone. The
mobilities of standard molecular size (kDa) proteins are
indicated.
Recombinant ST3 being
predominantly found in the insoluble protein fraction of E.
coli, bacterial inclusion bodies were treated with 8 M urea in the presence of 100 mM DTT. After inclusion body
solubilization, the different ST3 forms were refolded by slowly
dialyzing out the urea in the presence of CaCl and
ZnCl
ions, as described under ``Materials and
Methods.'' SDS-PAGE showed that while most bacterial contaminants
were eliminated during refolding, and the ST3 preparations obtained
after urea removal comprised both protein species at the expected
molecular weight and other species at lower molecular weights, the
latter being shown to correspond to ST3 degradation products using
monoclonal antibody 5ST-4C10 (Fig. 2, A and B and data not shown). Although the proportions of these low
molecular weight ST3 species were found to vary from one preparation to
another, they were usually more abundant with mouse than with human
ST3.
Figure 2:
Electrophoretic analyses of human and
mouse recombinant ST3 forms after extraction from bacterial inclusion
bodies and refolding. Human (h) (A and C) and mouse (m) (B and D) ST3
forms (2 µg) solubilized from bacterial inclusion bodies were
analyzed after protein refolding by SDS-PAGE (12%) under reducing (A and B) or nonreducing conditions (C and D). A and B, Coomassie Blue staining. C and D, /
-casein zymography. E, E
H158 and E
H216 ST3 forms are defined in Fig. 1and its legend. The mobilities of standard molecular size
(kDa) proteins are indicated.
Figure 3:
Electrophoretic analyses of recombinant
ST3 expressed in MCF7 cells stably transfected with a mouse ST3 cDNA.
Media conditioned by parental MCF7 cells (parental) or MCF7
cells transfected with a mouse ST3 cDNA (mST3) were collected
and concentrated about 50-fold by ammonium sulfate precipitation, and
analyzed by SDS-PAGE (12%) under reducing (A) or nonreducing (B) conditions. A, Western blot revealed with
monoclonal antibody 5ST-4C10 raised against the ST3 catalytic domain. B, /
-casein zymography. The high molecular weight
species detected by Western blot did not cleave casein, in contrast to
a lower molecular weight form. The mobilities of standard molecular
size (kDa) proteins are indicated.
In order to define the
structural determinants present in the hemopexin-like domain and
preventing mouse ST3 to digest casein, we performed sequential
deletions of the mouse ST3 C-terminal portion (Fig. 4A). After extraction from bacterial inclusion
bodies and protein refolding, these mouse ST3 preparations were
analyzed by SDS-PAGE (Fig. 4B) and /
- casein
zymography (Fig. 4C). While the mouse E
H167 form
itself was unable to digest casein, all other C-terminally truncated
mouse ST3 forms examined were found to display caseinolytic activity.
These findings indicate that the structural determinant preventing
mouse ST3 to digest casein corresponds to part or totality of the
hemopexin-like domain between Gly-318 and Arg-492 (Fig. 1A and 4A, and their legends).
Figure 4:
Electrophoretic analyses of C-terminally
truncated forms of mouse recombinant ST3 expressed in E. coli. A, schematic representation of putative mature ST3 forms
truncated at the C terminus. Numbers following EH indicate the number of amino acids that were deleted, while those
below frames indicate the position of the last C-terminal amino acid in
each form, according to Lefebvre et al.(16) .
C-terminal amino acids in each C-terminally truncated ST3 forms are
indicated inside frames by using the single-letter code. B,
Coomassie Blue staining of refolded recombinant ST3 forms (2 µg)
analyzed by SDS-PAGE (12%) under reducing conditions. C,
/
-casein zymography of the same ST3 forms (2 µg). The
mobilities of standard molecular size (kDa) proteins are
indicated.
While the deletion of the
C-terminal part of the hemopexin-like domain endowed mouse ST3 with
caseinolytic activity, a similar deletion was ineffective in the case
of human ST3 (Fig. 2C). This observation was unexpected
since the human and mouse ST3 catalytic domains exibit 94% amino acid
identity(16) . However, human ST3 is the sole matrixin so far
identified having an alanine (residue 235) instead of a proline
C-terminal to the ``Met-turn'' (Table 1), which is
believed to provide a hydrophobic environment necessary for the
catalytic zinc atom(20) . Although the presence of Ala-235 in
human ST3 was initially deduced from a breast cancer cDNA(14) ,
its presence has been confirmed both by sequencing the ST3 gene ()and a cDNA obtained from a human placenta cDNA library
(data not shown). Thus, we hypothesized that Ala-235 could be
responsible for the inability of human ST3 to digest casein. Indeed,
the substitution of Ala-235 by Pro was sufficient to endow human
E
H216 ST3 with caseinolytic activity by zymographic analysis (Fig. 5). Conversely, when mouse E
H216 ST3 was mutated to
replace Pro-239 by Ala, its caseinolytic activity was strongly reduced.
Figure 5:
Electrophoretic analyses of human and
mouse EH216 ST3 forms. Wild-type (h Ala 235; m Pro
239) and mutated (h Pro 235; m Ala 239)
recombinant forms (2 µg) of human (h) and mouse (m) E
H216 ST3 (see Fig. 1for definition)
extracted from bacterial inclusion bodies were analyzed after protein
refolding by SDS-PAGE (15%) under reducing (A) or nonreducing (B) conditions. A, Coomassie Blue staining; B,
/
-casein zymography. The mobilities of standard
molecular size (kDa) proteins are
indicated.
Figure 6:
Electrophoretic analysis of purified human
and mouse EH216 ST3 forms. Human (lanes1-3) and mouse (lanes4-6)
E
H216 ST3 forms (see Fig. 1for definition) were extracted
from bacterial inclusion bodies, and purified by Q-Sepharose
chromatography as described under ``Materials and Methods.''
Samples (2 µg of protein) were analyzed by SDS-PAGE (15%) under
reducing conditions and Coomassie Blue staining. Lanes1 and 4, crude material solubilized in 8 M urea; lanes2 and 5, unbound materials; lanes3 and 6, refolded proteins after elution from
Q-Sepharose with NaCl (100-250 mM fractions). The
mobilities of standard molecular sizes (kDa) proteins are
indicated.
In
agreement with the observation made using casein zymography, human
EH216 ST3 could not degrade
C-labeled
-casein (Fig. 7A). When its activity against
-casein was
evaluated using a more sensitive assay in which proteolytic products
were analyzed by SDS-PAGE (see ``Materials and Methods''),
only a weak activity could be evidenced (data not shown). However,
mouse E
H216 ST3 could degrade
C-labeled
-casein
almost proportionally to the amount of added enzyme between 2 and 8
µg/ml protein (Fig. 7A). The specific activity of
mouse ST3 toward
C-labeled
-casein was found to be 20
± 5 units/mg versus 350 ± 30 units/mg for
trypsin-activated stromelysin-1 (data not shown). Caseinolytic activity
was inhibited by 2 mM 1,10-phenanthroline or 1 mM EDTA (Fig. 7A), as well as by the specific
matrixin inhibitors TIMP-1, TIMP-2, and TIMP-3 (Fig. 7B). Inhibition (95%) was achieved with a
ST3/TIMP-1 molar ratio of 1:1. However, the caseinolytic activity of
mouse E
H216 ST3 was not abolished by 100 µM Pro-Leu-Gly hydroxamate, an inhibitor of human interstitial
collagenase ( (31) and data not shown).
Figure 7:
Quantitation of caseinolytic activities of
recombinant human and mouse EH216 ST3 forms. Quantitation was
performed using
C-labeled
-casein (400 µg/ml) as
substrate and incubation at 37 °C for 18 h. A, activities
of human (h) and mouse (m) E
H216 ST3 (see Fig. 1for definition) (2-8 µg/ml) in the absence or in
the presence of 5 mM EDTA or 2 mM 1,10-phenanthroline (1,10 phen). Results are expressed as the amount of
C (counts/min) remaining in the supernatant after
precipitation with 3% trichloroacetic acid at 0 °C for 30 min.
Counts/min observed in the absence of ST3 (average 800) have been
subtracted. B, inhibition of mouse E
H216 ST3 caseinolytic
activity by tissue inhibitors of metalloproteinases, TIMP-1, TIMP-2,
and TIMP-3. Mouse E
H216 ST3 (8 µg/ml) was incubated with
increasing amounts of TIMPs (0-8 µg/ml), at 37 °C for 18
h. Results are expressed as percentage of ST3 activity observed in the
absence of TIMP.
Purified mouse and
human EH216 ST3 forms were then tested against laminin, type IV
collagen, and plasma
1-proteinase inhibitor. Mouse E
H216 ST3
was able to cleave laminin and type IV collagen at 37 °C (Fig. 8A), thus confirming previous observations
obtained with C-terminally truncated mouse ST3 forms produced by using
an eucaryotic expression system(18) . In the same experimental
conditions, human E
H216 ST3 was unable to degrade laminin or type
IV collagen (Fig. 8A). However, both mouse and human
E
H216 ST3 showed activity toward plasma
1-proteinase
inhibitor, although the mouse enzyme was more potent than the human
enzyme ( Fig. 9and data no shown). Full cleavage of
1-proteinase inhibitor (2 µg) was achieved with 250 ng of
mouse and 2 µg of human E
H216 ST3, respectively. These
activities were completely inhibited by the addition of EDTA, TIMP-1,
or TIMP-2 ( Fig. 8and Fig. 9, and data not shown).
Figure 8:
Degradation of laminin and type IV
collagen by human and mouse EH216 ST3 forms. Human and mouse
E
H216 ST3 forms (see Fig. 1for definition), purified by
Q-Sepharose chromatography before protein refolding, were incubated
with laminin or type IV collagen at 37 °C for 18 h. The products
were analyzed by SDS-PAGE (8%) under reducing conditions followed by
silver staining. A, laminin (2 µg) (lanes1-5) or type IV collagen (3 µg) (collagen
IV) (lanes6-10) were incubated alone (control, lanes1 and 6) or with
human (2 µg) (lanes2, 3, 7,
and 8) or mouse (2 µg) (lanes4, 5, 9, and 10) E
H216 ST3 forms. Where
indicated (lanes3, 5, 8, and 10), incubation was performed in the presence (+) of
TIMP-1 (2 µg). B, type IV collagen (3 µg) was
incubated alone (lane1, control) or with
human (lane2, h Ala 235) and mouse (lane4, m Pro 239) wild-type ST3 forms (2
µg) or with the corresponding mutated human (lane3, h Pro 235) and mouse (lane5, m Ala 239) E
H216 ST3 forms (2
µg).
Figure 9:
Degradation of 1-proteinase inhibitor
by human and mouse E
H216 ST3 forms. Plasma
1-proteinase
inhibitor (3 µg) was incubated at 37 °C for 18 h alone (control, lane1) or with human (lanes2 and 3) or mouse (lanes4 and 5) E
H216 ST3 (see Fig. 1for definition) (2
µg). Incubation was performed in the absence (-) (lanes1, 2, and 4) or presence (+) (lanes3 and 5) of TIMP-2 (2 µg). After
incubation, products were analyzed by SDS-PAGE (8%) followed by
Coomassie Blue staining. I and C indicate intact and
cleaved
1-proteinase inhibitor, respectively. The mobilities of
standard molecular size (kDa) proteins are
indicated.
Importantly, the substitution of Ala-235 by Pro in human EH216
ST3 was sufficient to endow the human enzyme with activity against
laminin and type IV collagen (Fig. 8B and data not
shown). Conversely, the replacement of Pro-239 by Ala in mouse
E
H216 ST3 led to an enzyme that has lost part of its ability to
degrade laminin and type IV collagen. Altogether, these observations
emphasize the importance of the proline/alanine residues that
C-terminally flank the Met-turn in controlling ST3 functional
properties.
In previous work using an eucaryotic expression system, we had demonstrated that C-terminal truncation of the putative mature form of mouse ST3 was necessary to observe stromelysin-like activity(18) . In this respect, mouse ST3 differs from the other stromelysins whose enzymatic activity is not substantially affected by C-terminal truncation (32, 33) and exhibits partial analogy with interstitial (34, 35) and neutrophil collagenases(36, 37) , which acquire stromelysin-like activity after C-terminal truncation. The present study was undertaken to determine 1) whether the previous observations made using an eucaryotic expression system can be reproduced using a procaryotic expression system, 2) whether human ST3 behaves similarly to the mouse enzyme, and 3) the nature of the critical structural determinants controlling the unusual functional properties of ST3.
Putative mature forms of ST3 in which the prodomain had been deleted were expressed in E. coli using the pET-3b vector and tested for proteolytic activity in casein zymography. Neither the human nor the mouse ST3 forms thus produced exhibited activity by casein zymography, as had been observed for the ST3 forms generated by eucaryotic expression systems (18, 19 and the present study). However, when at least 175 C-terminal amino acids were deleted, we found that mouse ST3 could degrade casein. On the other hand, the deletion of the C-terminal hemopexin-like domain was not sufficient to endow human ST3 with caseinolytic activity. In the case of the human protein, detection of caseinolytic activity required a further mutation in which Ala-235 was replaced by a proline. Ala-235 was thought to represent a critical residue in human ST3 because all other matrix metalloproteinases, including mouse and frog ST3, contain a proline residue at this position(1, 2, 3, 38, 39) . Ala-235 C-terminally flanks the Met-turn, which is believed to provide a hydrophobic base beneath the catalytic zinc atom(20) . Furthermore, by analogy with the structure determined for human neutrophil collagenase(40) , Ala-235 should belong to the outer wall of the putative S1` pocket of the active-site cleft. Our finding that the substitution of Ala-235 by Pro is sufficient to endow the catalytic domain of human ST3 with caseinolytic activity, while the corresponding replacement of Pro-239 by Ala in the mouse enzyme decreases proteolytic activity, is the first direct evidence demonstrating the functional importance of this amino acid residue.
The critical role of Ala-235 is further demonstrated by the
observation that the human ST3 catalytic domain, in contrast to the
mouse one, cannot digest laminin and type IV collagen, while after
substitution of Ala-235 by a proline, the human enzyme also degrades
laminin and type IV collagen. The inability of human recombinant ST3
produced in E. coli to digest laminin and type IV collagen is
consistent with the observation recently made by Pei et al.(19) using recombinant enzyme produced in COS-7 cells.
These data raise the possibility that human ST3 is not a functional
proteinase, but a molecule that has evolved to acquire a new biological
function. This has been observed in the case of hepatocyte growth
factor(41, 42) , haptoglobin(43) , and protein
Z(44) , which are all similar in structure to serine
proteinases, although two residues of the catalytic site have been
replaced with amino acids that could not support
catalysis(45) . Alternatively, human ST3 may be an enzyme with
a highly restricted substrate specificity. Pei et al.(19) have proposed that some serine proteinase inhibitors,
including 1-proteinase inhibitor, are physiologically relevant ST3
substrates. Our finding that
1-proteinase inhibitor, in contrast
to other substrates, is cleaved by both human ST3 forms having a
proline or an alanine at position 235, also supports this hypothesis.
However, all matrix metalloproteinases can cleave
1-proteinase
inhibitor, and in some cases with an apparently much higher efficiency
than ST3(46, 47) . Thus, the most likely hypothesis is
that ST3 is a matrix metalloproteinase with functional properties
distinct from all other matrix metalloproteinases and whose
physiological substrate is presently unknown. In this respect, we note
that mouse ST3 with an intact C-terminal domain, in contrast to the
corresponding catalytic domain, cannot cleave casein, thereby
suggesting that the ST3 hemopexin-like domain is also involved in
endowing ST3 with a restricted substrate specificity.
The specific
expression of the ST3 gene in fibroblastic cells immediately
surrounding cancer cells in invasive human carcinomas ( (13) and (14) and references therein) has raised the
possibility that ST3 contributes to tumor progression. This possibility
is supported by clinical observations showing that recurrent breast
carcinoma is more frequent in patients having tumors with high ST3 RNA
levels (15) and by experimental data showing that ST3
expression favors tumor take in nude mice. ()In this
context, it appears reasonable to include ST3 among those matrix
metalloproteinases that might represent targets for therapeutic
intervention using synthetic matrix metalloproteinase
inhibitors(21, 22) . Such inhibitors have been found
in preclinical studies to be efficient and well
tolerated(9, 48, 49) , and the clinical
evaluation of some of them has been recently initiated(21) .
The demonstration that human ST3 possesses unusual functional
properties adds further support to the possibility of obtaining
inhibitors with appropriate selectivity to target ST3 for the treatment
of human carcinomas.