(Received for publication, October 21, 1994; and in revised form, January 18, 1995)
From the
Matrix metalloproteinase 7 (MMP-7) has been purified as an
inactive zymogen of M 28,000 (proMMP-7) from the
culture medium of CaR-1 human rectal carcinoma cells. The
NH
-terminal sequence of proMMP-7 is
Lys-Pro-Lys-Pro-Gln-Glu, which is identical to that of matrilysin. The
zymogen is activated by 4-aminophenylmercuric acetate (APMA), yielding
an intermediate form of M
21,000 and an active
species of M
19,000 which shows the new
NH
-terminal sequence of
Tyr
-Ser-Leu-Phe-Pro-Asn-Ser. Although trypsin fully
activates the zymogen, the activation rate by plasmin or leukocyte
elastase is confined to
50%. ProMMP-7 can be activated by MMP-3
(stromelysin 1) to its full activity in a single-step mechanism and
generates the same NH
terminus obtained by APMA activation,
whereas MMP-1 (tissue collagenase), MMP-2 (gelatinase A), and MMP-9
(gelatinase B) do not have such an effect. On the other hand, proMMP-1
is activated by MMP-7 to an activity similar to that obtained by APMA
and the activation by MMP-7 is enhanced up to
6.5-fold in the
presence of APMA. This enhanced activity is donated by specific
cleavage at the Gln
-Phe
bond of proMMP-1.
MMP-7 can also activate proMMP-9 up to
50% of the full activity
with a new NH
terminus of
Leu
-Arg-Thr-(Asn)-Leu. Incubation of proMMP-2 or proMMP-3
with MMP-7 results in no activation of these proMMPs. MMP-7 degrades
type IV collagen, laminin-1, fibronectin, proteoglycan, type I gelatin,
and insoluble elastin. These results suggest that in vivo MMP-7 may play a role in degradation of extracellular matrix
macromolecules in concert with MMP-1, -3, and -9 under pathological
conditions.
The matrix metalloproteinases (MMPs) ()are a gene
family of Zn
endopeptidases that can digest various
extracellular matrix macromolecules. Ten members have been identified
and classified into four major types of structurally and functionally
related metalloproteinases: 1) interstitial collagenases (tissue
collagenase = MMP-1; neutrophil collagenase = MMP-8;
collagenase 3 = MMP-13), which degrade fibrillar collagens; 2)
gelatinases/type IV collagenases (72-kDa metalloproteinase =
gelatinase A = MMP-2; 92-kDa metalloproteinase =
gelatinase B = MMP-9), which hydrolyze gelatins and type IV and
V collagens; 3) stromelysins (stromelysin 1 = MMP-3; stromelysin
2 = MMP-10), which are active against a wide range of substrates
including proteoglycans, laminin, fibronectin, type IV collagen, and
telopeptides of other collagens; 4) other MMPs (matrilysin =
MMP-7; stromelysin 3 = MMP-11; macrophage metalloelastase
= MMP-12)(1, 2) . Recent studies on MMP-11 have
demonstrated a weak activity against fibronectin, laminin,
proteoglycan, and gelatin(3) . MMP-12 is known to digest
insoluble elastin although no information is available about the
activity against other extracellular matrix macromolecules(4) .
MMP-7 is unique in size for lack of the COOH-terminal domain. The
proteinase can cleave a wide range of substrates such as fibronectin,
laminin, proteoglycan, elastin, gelatin, type IV collagen, aggrecan,
and
entactin(5, 6, 7, 8, 9, 10) ,
and the substrate specificity is reported to be similar to that of
MMP-3 (8) . It has been shown that the zymogen of MMP-7
(proMMP-7) is activated by 4-aminophenylmercuric acetate (APMA) and
trypsin following the stepwise mechanisms proposed for other proMMPs
such as proMMP-3 (11) . However, little is known about
activation by other factors including serine proteinases and active
oxygen metabolites which are capable of activating many proMMPs and are
present in pathological conditions(1) . A growing body of
evidence has disclosed intermolecular activation of proMMPs. It is well
known that MMP-3 is an effective activator for proMMP-1, -8, and
-9(12, 13, 14, 15, 16) .
Suzuki et al.(13) have examined the precise
mechanisms of proMMP-1 activation and demonstrated that MMP-3 fully
activates proMMP-1 by cleavage of the Gln
-Phe
bond of the proenzyme. Although similar activation of proMMP-1 is
reported to occur with MMP-7(8) , no studies have been made on
the molecular mechanisms. In fact, very limited information about the
interactions of MMP-7 with other MMPs is available.
In this study, we have purified proMMP-7 from the culture medium of CaR-1 human rectal carcinoma cells and studied the activation of the zymogen, interactions with other MMPs, and the enzymic properties. The present studies demonstrate that proMMP-7 is activated by MMP-3 to its full activity, and conversely MMP-7 can activate proMMP-1 and proMMP-9. We also show that MMP-7 has a wide range of substrate specificity, which is different from that of MMP-3 in specific activity.
APMA-activated MMP-7 was stable without loss of
the activity even after storage at 23 °C for 4 weeks, although
proMMP-7 showed spontaneous activation (up to 30%) in the same
condition. However, there was no decrease in the activity or
spontaneous activation of proMMP-7 when stored at 4 °C for the same
period. On the other hand, proMMP-7 became the active species of M
19,000 when thawed after storage for a month at
-20 °C. Inhibitor studies indicated that MMP-7 is a typical
metalloproteinase. The activity of MMP-7 was completely inhibited by
chelating agents including EDTA, EGTA, and 1,10-phenanthroline, TIMP-1
and TIMP-2, and thiol compounds (data not shown). SDS and
2-mercaptoethanol were also inhibitory. However, inhibitors of serine,
cysteine, or aspartic proteinases did not show any significant effect.
Figure 1:
Time course of
proMMP-7 activation by APMA and conversion of proMMP-7 during the
activation. A, proMMP-7 (55 ng) was incubated with () or
without (
) 1 mM APMA for up to 24 h at 37 °C. At the
incubation times indicated, the samples were subjected to the assay
using [
H]Cm-Tf for 30 min at 37 °C. B, ProMMP-7 (700 ng) incubated with 1 mM APMA was
also analyzed by SDS-PAGE (12.5% total acrylamide) under reducing
conditions. After electrophoresis the gel was stained with silver
nitrate. C0 and C24, proMMP-7 incubated at 37 °C with
buffer alone for 0 and 24 h, respectively. Protein standards are
phosphorylase b (94 kDa), bovine serum albumin (68 kDa),
ovalbumin (43 kDa), carbonic anhydrase (29 kDa), soya-bean trypsin
inhibitor (21 kDa), and lysozyme (14 kDa).
During activation with 1 mM APMA, proMMP-7 of M 28,000 was processed initially to a molecule of M
21,000 and then converted to the M
19,000 species (Fig. 1B). When
these MMP-7 species were compared with the activation curve, generation
of the M
19,000 species was correlated with the
activity, indicating that the M
19,000 form is an
active species. The NH
terminus of the active species of
MMP-7 was Tyr
-Ser-Leu-Phe-Pro-Asn-Ser.
Figure 2:
Time course activation and conversion of
proMMP-7 by trypsin. ProMMP-7 (55 ng) was incubated with trypsin at 37
°C at 10 µg/ml (), 1 µg/ml (
), 0.1 µg/ml
(
), and 0 µg/ml (
). The MMP-7 activity was measured
against [
H]Cm-Tf for 30 min at 37 °C after
termination of the reaction with 2 mM DIFP. Activity achieved
by incubation of proMMP-7 with 1 mM APMA for 4 h at 37 °C
was taken as 100% activity. Inset, conversion of proMMP-7 by
trypsin activation. A mixture of
I-labeled and unlabeled
proMMP-7 (55 ng) incubated with trypsin (1 µg/ml) up to 8 h was
electrophoresed on SDS (12.5% total acrylamide) gel. After
electrophoresis the gel was dried and autoradiographed. Upper
arrow, proMMP-7 of M
28,000; middle
arrow, a species of MMP-7 with M
22,500; lower arrow, a doublet of MMP-7 forms with M
21,000 and 20,000. C0, proMMP-7 incubated with buffer
alone for 0 h at 37 °C.
Figure 3:
Activation of proMMP-7 by plasmin or
leukocyte elastase in the presence or absence of cathepsin G. ProMMP-7
(55 ng) was treated with plasmin (A) or leukocyte elastase (B) at 37 °C at 10 µg/ml (), 1 µg/ml
(
), or 0.1 µg/ml (
) in the absence of cathepsin G, and
at 10 µg/ml in the presence of 10 µg/ml cathepsin G (
).
The reaction was stopped with 2 mM DIFP, and MMP-7 activity
was assayed against [
H]Cm-Tf for 30 min at 37
°C. Activity obtained by incubation of proMMP-7 with 1 mM APMA for 4 h at 37 °C was taken as 100% activity. No
activation occurred with proMMP-7 incubated with buffer alone up to 8 h
(data not shown). Inset, M
changes in
proMMP-7 during activation with 10 µg/ml plasmin (A) or 10
µg/ml leukocyte elastase (B). The reaction products were
analyzed on SDS-PAGE as described in Fig. 2. A: upper arrow, proMMP-7 of M
28,000; lower arrow, a species of M
19,000. B: upper arrow, proMMP-7 of M
28,000; lower arrow, a species of M
20,500. C0, proMMP-7 incubated with buffer alone for 0
h.
To examine the effect
of active oxygen metabolites on proMMP-7 activation, the zymogen was
reacted with various concentrations of HOCl or
HO
. However, both reagents did not activate
proMMP-7 (data not shown). The possibility of proMMP-7 activation by
acid exposure, another activation mechanism shown with
proMMP-9(30, 35) , was also examined by incubation of
the zymogen in the range from 2.4 to 9.7. However, no activation
occurred by the treatment (data not shown).
Figure 4:
Activation of proMMP-7 by MMP-3. A, proMMP-7 (550 ng) was incubated with active MMP-3 in the
presence (,
) or absence (
,
,
,
,
) of 1 mM APMA. The incubation was performed at
different molar ratios of MMP-3 to proMMP-7;
and
, 0.1
molar;
and
, 0.5 molar;
and
, 1 molar;
and
, 2.5 molar;
and
, 5 molar. Since the
activation curve of proMMP-7 by MMP-3 in the molar ratios of
0.5-5 in the presence of APMA was very similar, it is indicated
by
. The total activity was measured using
[
H]Cm-Tf substrate for 30 min at 37 °C, and
the MMP-7 activity was calculated by subtraction of the activity
generated by active MMP-3 in each assay. The full activity of MMP-7 was
taken from that of proMMP-7 activated with 1 mM APMA (
)
at 37 °C for 4 h.
shows proMMP-7 incubated with buffer
alone. B, conversion of proMMP-7 during incubation with MMP-3.
Five molar excess amount of MMP-3 was incubated with proMMP-7 (500 ng)
in the absence of APMA at 37 °C for 2 h (lane 2), 4 h (lane 3), 8 h (lane 4), and 24 h (lane 5). Lanes 1 and 6 show proMMP-7 incubated with buffer
alone for 0 and 24 h, respectively. The reaction products were
subjected to SDS-PAGE (15% total acrylamide) under reducing conditions
after termination of the reaction with 20 mM EDTA and the gel
was stained with silver nitrate. Arrow and arrowhead indicate active MMP-3 of M
45,000 and 28,000,
respectively. The protein bands of MMP-3 are more faintly stained than
that of MMP-7 species in the gel.
Figure 5:
Activation of proMMP-1 by MMP-7. ProMMP-1
(137 ng) was incubated with MMP-7 in the presence (,
,
) or the absence (
,
,
) of 1 mM APMA.
MMP-1 activity was assayed using [
C]collagen for
1 h at 37 °C. Since maximal activation of proMMP-1 with 1 mM APMA (
) was obtained after 4 h of incubation at 37 °C
during a period of 0-24 h, the incubation was performed up to 4
h. The molar ratios of MMP-7 to proMMP-1 are 0.1 (
,
), 0.5
(
,
), and 1 (
,
). Incubation of proMMP-1
with buffer alone (
) shows no
activation.
ProMMP-9 was also activated by
MMP-7, although the activation rate was confined to 50% of the full
activity (Fig. 6A). The activation occurred in a
dose-dependent manner, and maximal activation was seen after 4 h of
incubation with MMP-7 in a molar ratio of 1:1, the activity of which
declined to 20% after 24 h (Fig. 6A). In the
presence of both MMP-7 and 1 mM APMA, proMMP-9 was activated
to its full activity, and the activation was faster than that by APMA
alone (Fig. 6A). Incubation of
I-labeled
proMMP-9 with MMP-7 in a 1:1 molar ratio converted the zymogen of M
92,000 to a fragment of M
78,000 with intermediate forms with M
83,000
and M
80,000 (Fig. 6B). As
previously reported by us(16) , proMMP-9 was processed to a M
67,000 active form with the
NH
-terminal amino acid sequence of
Met
-Arg-Thr-Pro-Arg through an intermediate species of M
83,000 during APMA activation. When proMMP-9
activation was performed in the presence of MMP-7 and 1 mM APMA, the active form of M
62,000 was
generated via an intermediate species of M
83,000
(data not shown). The M
62,000 species of MMP-9
was associated with
70% of the full activity, whereas only
25% activity was seen with the M
78,000
species. NH
-terminal amino acid sequence analyses
demonstrated that the M
78,000 species has
Leu
-Arg-Thr-(Asn)-Leu sequence and the M
62,000 species Met
-Arg-Thr-Pro-Arg and
Phe
-Gln-Thr-Phe-Glu sequence in a 1:1 molar ratio.
Immunoblot analyses using monoclonal antibodies specific to
NH
-terminal (residues 31-49) or the COOH-terminal
domain (residues 643-661) of proMMP-9 (16) showed that
the M
78,000 species lacks the COOH-terminal
domain and the M
62,000 species both
NH
- and COOH-terminal domains (data not shown).
Figure 6:
Time course activation and conversion of
proMMP-9 by MMP-7. A, proMMP-9 (215 ng) was incubated with
activated MMP-7 in the presence (,
,
) or the
absence (
,
,
) of 1 mM APMA. Activity of
proMMP-9 incubated with 1 mM APMA (
) for 24 h at 37
°C was taken as the full activity. Incubation of proMMP-9 with
buffer alone (
) shows negligible activation. The incubation was
performed in three different molar ratios of MMP-7 to proMMP-9:
and
, 0.1 molar;
and
, 0.5 molar;
and
, 1 molar. B, a mixture of
I-labeled and
unlabeled proMMP-9 (860 ng) was incubated with MMP-7 in 1:1 molar ratio
at 37 °C for various times as indicated. After termination of the
reaction with 20 mM EDTA, the samples were subjected to in
SDS-PAGE (9% total acrylamide), and the gel was autoradiographed.
Protein standards are as in Fig. 1B. C0,
proMMP-9 incubated with buffer alone for 0
h.
In contrast to the effect of MMP-7 on proMMP-1 and proMMP-9, MMP-7 did not activate proMMP-2 even in a 5:1 molar ratio and no definite processing of the proMMP-2 molecule was observed. Treatment of proMMP-3 with MMP-7 resulted in neither activation nor processing of the zymogen (data not shown).
We have purified the precursor of MMP-7 to homogeneity from
the culture medium of CaR-1 human rectal carcinoma cells. The
NH-terminal sequence analyses of the precursor and
APMA-activated MMP-7 and immunoblotting data indicate that the enzyme
purified in this study is identical to a zymogen of pump-1, matrilysin (38) .
Like other proMMPs, the enzymic activity was detected
only after activation of proMMP-7 with APMA. Our previous studies have
demonstrated that the autocatalytic activation of proMMP-2 and proMMP-3
by APMA is concentration dependent and the maximal activation occurs
with 1.0 mM APMA for proMMP-2 (17) and 1.5 mM APMA for proMMP-3(18) . Compared with these proMMPs,
proMMP-7 is different in that it can be fully activated by a wide range
of APMA concentrations, i.e. 0.1-1 mM APMA.
Since proMMP-7 has only one cysteine residue in the propeptide region
of the molecule and lacks the COOH-terminal hemopexin-like domain, it
may be possible that the conformational changes of the molecule by APMA
can readily be induced by low concentrations of APMA and become active.
During activation with APMA, proMMP-7 of M 28,000
was converted to an active species of M
19,000
with an intermediate form of M
21,000.
NH
-terminal sequence analysis indicated that cleavage of
the Glu
-Tyr
bond results in the fully active
MMP-7 and is identical to the site for activation of recombinant
MMP-7(11) . This bond is located at a position three amino
acids downstream from the highly conserved
Pro-Arg-Cys-Gly-Val/Asn-Pro-Asp sequence of the propeptide of all
proMMPs. The hydrolysis of corresponding bonds, the
Gln
-Phe
bond in proMMP-1(13) , the
Asn
-Tyr
bond in proMMP-2 (17, 39) , the His
-Phe
bond
in proMMP-3 (40) , and the Arg
-Phe
bond in proMMP-9 (15) , is also an essential event for
activation of these proMMPs, although proMMP-9 activation by APMA is
achieved by sequential processing of both NH
-terminal
propeptide at a position four amino acids upstream from the conserved
sequence and COOH-terminal domain(16) . The step-wise
activation, as shown in the present studies, is a common feature of the
APMA-mediated activation of proMMPs and has been reported with human
recombinant proMMP-7 (promatrilysin)(11) .
Trypsin is an
effective activator of proMMP-7 as shown here and
previously(11) . However, unlike proMMP-1, -3, and -9, proMMP-7
was not effectively activated with other serine proteinases. Only
plasmin and leukocyte elastase activated proMMP-7 up to 50% of the
activity. Although cathepsin G had the ability to accelerate the
activation processes by plasmin or leukocyte elastase, the activation
rate was unchanged. In addition, exposure of proMMP-7 to acid or HOCl,
which is known to cause activation of proMMP-8 (41) and
proMMP-9(16, 42) , did not show any effects on the
activation. These data suggest that serine proteinases and active
oxygen metabolites derived from plasma and inflammatory cells may play
a limited role in the activation of proMMP-7. Thus, other mechanisms
should be necessary for proMMP-7 activation in vivo.
It has
been reported that MMP-3 is a good activator of
proMMP-1(12, 13) , proMMP-8(14) , and
proMMP-9(15, 16) . In the present studies we have
demonstrated for the first time that MMP-3 can fully activate proMMP-7
and generate an active species of M 19,000, which
has the same NH
terminus as that obtained by APMA
activation. The activation of proMMP-7 was not associated with
formation of intermediate species and was dependent on the
concentrations of MMP-3. In addition, this activation process required
enzymic activity of MMP-3 because neither proMMP-3 nor MMP-3
inactivated by heat or 1,10-phenanthroline treatment activated the
zymogen. Thus, it seems probable that proMMP-7 is directly activated
via the cleavage of the Glu
-Tyr
peptide bond
by the action of MMP-3. A similar mechanism has been reported with
proMMP-8 activation by MMP-3(14) . Recent studies on tissue
localization of MMP-7 have disclosed that MMP-7 is expressed in various
carcinoma tissues including stomach, colon, head and neck, lung and
prostate carcinoma(43, 44, 45) , glomerular
mesangial cells(34) , endometrial gland
epithelium(46) , and peripheral blood monocytes(47) .
On the other hand, proMMP-3 is also produced by cancer
cells(44, 48, 49) , fibroblasts(50) ,
rheumatoid synovial cells(51) , and endometrial stromal cells (46) and can be stored in the extracellular milieu since both
latent and active forms of MMP-3 bind to collagen fibrils(52) .
CaR-1 cells used in the present study did not produce MMP-3. However,
simultaneous production of MMP-7 and MMP-3 has been demonstrated in
human glioma cell lines(49) . Although previous studies showed
no coordinated mRNA expression of MMP-7 and MMP-3 in stomach and colon (43) , prostate (45) and breast
carcinomas(53) , the co-expression of MMP-7 and stromelysins
(MMP-3 and/or MMP-10) has been reported in squamous cell carcinomas of
the lung, and head and neck(44) . Actually, our
immunolocalization studies on the human colon and lung carcinoma
tissues have demonstrated that some carcinoma cells occasionally
produce both MMP-7 and MMP-3. (
)In addition, concomitant
expression has been reported in mesangial cells (54) and
endometrial tissue(46) . It is well known that proMMP-3 is
readily activated by various serine proteinases such as plasmin,
leukocyte elastase, cathepsin G, and plasma kallikrein(18) .
Thus, these suggest the possibility that MMP-7 participates in the
degradation of the extracellular matrix macromolecules in vivo through activation by interaction with MMP-3.
The present
studies have demonstrated that MMP-7 can enhance MMP-1 activity
6.5-fold more than that achieved by APMA activation, confirming
the previous study using recombinant human MMP-7(8) . This
phenomenon was first reported for proMMP-1 activation by
MMP-3(12, 55) and then for proMMP-8 activation by
MMP-3(14) . In these experiments, the hydrolysis of the
specific bonds located three amino acids downstream from the conserved
Pro-Arg-Cys-Gly-Val/Asn-Pro-Asp sequence, Gln
-Phe
for MMP-1 and Gly
-Phe
for MMP-8, is
observed(13, 14) . Although the previous study (8) did not determine the cleavage site of proMMP-1 after
activation with MMP-7, our study revealed the cleavage at the same
position, i.e. Gln
-Phe
bond, as that
reported with MMP-3 (13) . Thus, it can be concluded that the
hydrolysis of the Gln
-Phe
bond of proMMP-1 is
essential for full activation. Recent crystallographic studies on MMP-8
demonstrated that the NH
-terminal ammonium group of
Phe
forms a salt bridge with the side chain carboxylate
group of Asp
and suggested that this linkage may be
related to the full activity of MMP-8 and MMP-1(56) .
We
have also demonstrated that MMP-7 can partially activate proMMP-9.
Removal of NH-terminal propeptides is believed to be
indispensable to formation of active MMP species. However, our previous
studies demonstrated that the sequential processing of both
NH
- and COOH-terminal peptides from the proMMP-9 molecule
is essential for full activation, leading to formation of the active
species of M
67,000 with the NH
terminus of
Met
-Arg-Thr-Pro-Arg-Cys-Gly-Val-Pro-Asp(16) . On
the other hand, O'Connell et al.(57) have
recently reported that removal of the COOH-terminal domain is
unnecessary for the activation from data using the
COOH-terminal-truncated proMMP-9. ProMMP-9 activation by MMP-7 in the
present study was associated with removal of both the first
NH
terminal 15 amino acid residues and COOH-terminal domain.
Since Ogata et al.(15) have reported that no enzymic
activity is seen with the intermediate species of MMP-9 generated by
cleavage at the Glu
-Met
bond, it seems
unlikely that removal of 15 amino acid residues from the
NH
-terminal propeptide of proMMP-9 is responsible for the
activity up to
50%. Thus, proMMP-9 activation by MMP-7 may be
achieved through the conformational changes due to the cleavage in both
NH
- and COOH-terminal peptides of the zymogen. A similar
mechanism is proposed for activation of proMMP-11 (prostromelysin
3)(3) . As the activation rate of proMMP-9 by MMP-7 is confined
to
50%, the biological function of the activation is unclear.
However, it might be possible that proMMP-9 activation is accelerated
by interaction with both MMP-3 and MMP-7.
MMP-7 can digest cartilage
proteoglycan, type IV collagen, type I gelatin, fibronectin, laminin-1,
and insoluble elastin. This substrate specificity of MMP-7 appears to
be analogous to that of MMP-3(29) . However, the digestion
patterns of type IV collagen, type I gelatin, fibronectin, and
laminin-1 by MMP-7 were different from those with MMP-3 of M 28,000 which is a low molecular weight form of
MMP-3 lacking both NH
- and COOH-terminal
domains(18, 29) . In addition, proteoglycan degrading
activity of MMP-7 was 1.3-fold higher than that of MMP-3(29) .
Among MMP-1, -2, -3, -7, and -9 MMP-7 had the highest activity against
insoluble elastin,
11-fold more active than MMP-3. These results
indicate that although MMP-7 and MMP-3 share substrates they have
different specific activities against each macromolecule. Elastin is
the highly cross-linked extracellular matrix component of elastic
connective tissues such as blood vessels, lung, and skin. The elastic
lamina is present beneath the serosal mesothelial cell lining of the
stomach and colon and can function as a barrier to carcinoma cell
invasion. Since the enhanced expression of MMP-7 has been reported in
stomach and colon cancers(43) , it may be possible that in
concert with other MMPs such as MMP-3, MMP-7 produced by the cancer
cells plays a part in the breakdown of the elastin as well as other
extracellular matrices, which facilitates tumor cell dissemination to
the abdominal cavity.