(Received for publication, March 22, 1995; and in revised form, May 26, 1995)
From the
The precursor of matrix metalloproteinase 9 (proMMP-9,
progelatinase B) noncovalently binds to tissue inhibitor of
metalloproteinases (TIMP)-1 through the C-terminal domain of each
molecule. We have isolated the proMMP-9
Matrix metalloproteinases (MMPs), ( To date, 11 members of
the matrixin family have been identified, and their enzymic activities
have been
characterized(1, 2, 4, 5, 6, 7, 8) .
These include three collagenases (interstitial collagenase (MMP-1),
neutrophil collagenase (MMP-8), and collagenase 3
(MMP-13)(4) ), two gelatinases (gelatinase A (MMP-2) and
gelatinase B (MMP-9)), two stromelysins (stromelysin 1 (MMP-3) and
stromelysin 2 (MMP-10)), matrilysin (MMP-7), stromelysin 3
(MMP-11)(5, 6) ), macrophage metalloelastase
(MMP-12)(7) , and membrane-type MMP (MMP-14)(8) . Among
them, MMP-2 and MMP-9 are unique in that their zymogen forms can form a
complex with TIMP-2 and TIMP-1,
respectively(9, 10, 11, 12, 13, 14, 15, 16) .
The progelatinase-TIMP complexes are formed through the interaction of
the C-terminal hemopexin/vitronectin-like domain of the zymogen and the
C-terminal domain of the
inhibitor(16, 17, 18, 19, 20, 21) .
Since the inhibitory activity of TIMPs resides in the N-terminal
domain(17, 22) , the proMMP-2 In this report, we have isolated the
human proMMP-9
Figure 1:
SDS-PAGE analysis of the
proMMP-9
Figure 2:
SDS-PAGE and zymographic analyses of the
proMMP-9
Figure 3:
SDS-PAGE and zymographic analysis of the
proMMP-9
The lack of proteolytic activity in
the APMA- and the trypsin-activated complexes was further examined for
their abilities to bind to
Figure 4:
Lack of binding of the APMA- or
trypsin-treated proMMP-9
Figure 5:
Loss of MMP-1 inhibition activity of the
proMMP-9
Figure 6:
Activation of the proMMP-9
Figure 7:
Transfer of TIMP-1 from the
proMMP-9
Figure 8:
Transfer of TIMP-1 from the
proMMP-9
Activation of matrixin zymogens is an important regulatory
step in connective tissue matrix turnover. Most proMMPs are activated in vitro by treatment with proteinases, sulfhydryl-reacting
agents, mercurial compounds, chaotropic agents, HOCl, low pH, and heat (46, 47, 48, 49) . The recent
finding of the progelatinase-TIMP complexes have introduced further
complexity in progelatinase activation since TIMP present in the
complex can inhibit MMP activities that are required for the final
activation step. In this report, we have investigated the mode of
activation of the human proMMP-9
Figure S1:
Scheme 1Steps involved in activation of
the proMMP-9
When the proMMP-9 The behavior of the
proMMP-2 A likely candidate for the in vivo activator of proMMP-9 is
MMP-3(21, 31, 32) , but MMP-3 fails to
activate the proMMP-9 Once the
N-terminal domain of TIMP-1 is coupled with an active MMP, a catalytic
amount of MMP-3 can process proMMP-9 to an active 80-kDa MMP-9. Under
these conditions, active MMP-9 may be partially bound to the
TIMP-1
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
TIMP-1 complex from the
medium of human fibrosarcoma HT-1080 cells and investigated the
activation processes of the complex by 4-aminophenylmercuric acetate,
trypsin, and matrix metalloproteinase 3 (MMP-3, stromelysin 1). The
treatment of the proMMP-9
TIMP-1 complex with
4-aminophenylmercuric acetate or trypsin converts proMMP-9 to lower
molecular weight species corresponding to active forms, but no
gelatinolytic activity is detected. The lack of enzymic activity
results from binding of TIMP-1 to the activated MMP-9. The treatment of
the proMMP-9
TIMP-1 complex with a possible physiological proMMP-9
activator, MMP-3, does not reveal any gelatinolytic activity unless the
molar ratio of MMP-3 to the complex exceeds 1. This is due to the
inhibition of MMP-3 by TIMP-1 forming a ternary
proMMP-9
TIMP-1
MMP-3 complex. The formation of the ternary
complex weakens the interaction between proMMP-9 and TIMP-1, resulting
in partial dissociation of the complex into proMMP-9 and the
TIMP-1
MMP-3 complex. When MMP-3 is in excess, the propeptide is
completely processed, and the full activity of MMP-9 is detected.
Similarly, the proMMP-9
TIMP-1 complex inhibits MMP-1
(interstitial collagenase) and in turn renders the proMMP-9 activable
by a catalytic amount of MMP-3. These results suggest that formation of
the proMMP-9
TIMP-1 complex regulates extracellular matrix
breakdown in tissue by switching the predominant MMP activity from one
type to another.
)also designated
as matrixins, are a group of structurally related zinc
metalloendopeptidases capable of degrading extracellular matrix
components (for review, see (1) and (2) ). These
enzymes are considered to play a key role in normal tissue remodeling
and in pathological destruction of the matrix in many connective tissue
diseases such as arthritis, periodontitis, and tissue ulceration and in
cancer cell invasion and
metastasis(1, 2, 3) . Activities of the MMPs
are precisely controlled, not only by their gene expression in various
cell types but also by activation of their precursors (proMMPs) and
inhibition by endogenous inhibitors, such as
-macroglobulin (
M) and tissue
inhibitors of metalloproteinases
(TIMPs)(1, 2, 3) .
TIMP-2 and the
proMMP-9
TIMP-1 complexes are capable of inhibiting active
MMPs(10, 15, 23, 24) . This
introduces complexity in activation of progelatinase-TIMP complexes.
proMMP-2 free of TIMP-2 can be readily activated by a mercurial
compound, 4-aminophenylmercuric acetate (APMA)(25) , and by a
specific cell surface activator expressed in fibroblasts and cancer
cells treated with concanavalin A or a phorbol
ester(26, 27) . Recently, membrane-type MMP has been
identified as an activator of proMMP-2(8) ; however, the
activation of proMMP-2 by a cell surface activator is prevented in the
presence of TIMP-2(28, 29) . The proMMP-9
TIMP-1
complex is also resistant to activation by MMP-3(21) , while
proMMP-9 free of TIMP-1 is readily activated by MMP-3 (21, 30, 31) . On the other hand, many
investigators reported that treatment of the proMMP-2
TIMP-2
complex or the proMMP-9
TIMP-1 complex with APMA yielded
gelatinolytic activity with concomitant conversion of the proenzyme to
active forms, although the specific activities of the activated
complexes were considerably lower than those of TIMP-free
gelatinases(11, 12, 21, 23, 24, 34) .
By contrast, our studies on the activation of the proMMP-2
TIMP-2
complex by APMA have indicated that the activated proMMP-2 is inhibited
by the TIMP-2 before it loses the propeptide by
autocatalysis(24) . To attain the enzymic activity, masking of
the N-terminal inhibitory domain of TIMP-2 with another active MMP is
prerequisite(24) .
TIMP-1 and investigated the molecular
rearrangements that occur in the complex upon activation with APMA and
trypsin. To address a possible biological implication of the
proMMP-9
TIMP-1 complex formation to extracellular matrix
catabolism, we have also investigated the conditions that are required
for the activation of the complex by MMP-3, a likely proMMP-9 activator in vivo.
Materials
APMA, Brij 35,
diisopropylphosphorofluoridate (DFP),
12-O-tetradecanoylphorbol-13-acetate (TPA), trypsin (bovine),
plasminogen (human), urokinase, and alkaline phosphatase-conjugated
donkey anti-(sheep IgG) IgG were from Sigma. Dulbecco's modified
Eagle's medium, RPMI 1640 medium, antibiotics, fetal calf serum,
and lactalbumin hydrolysate were from Life Technologies, Inc.
Gelatin-Sepharose 4B, Sephacryl S-200, and S-300 were from Pharmacia
Biotech Inc. DE-52 (DEAE-cellulose) was from Whatman. Green A Matrex
gel and YM-10 membrane were from Amicon Corp. Affi-Gel 10 was from
Bio-Rad. Human M was a gift from Dr. Jan J. Enghild,
Duke University Medical Center. Human fibrosarcoma cell line HT-1080
cell was obtained from American Type Culture Collection.
Cell Cultures
HT-1080 cells were cultured in
Dulbecco's modified Eagle's medium containing 10% fetal
calf serum and antibiotics. After confluency, cells were washed with
Hanks' balanced salt solution and treated with TPA (20 ng/ml) in
serum-free Dulbecco's modified Eagle's medium supplemented
with 0.2% lactalbumin hydrolysate for 2 days. The conditioned medium
was harvested and used for purification of the proMMP-9TIMP-1
complex.
Anti-(human TIMP-1) IgG-Sepharose
Polyclonal
anti-(human TIMP-1) antibodies were prepared in sheep by intramuscular
injection of the purified human TIMP-1 from U-937 cells (35) emulsified with complete Freund's adjuvant once and
twice with incomplete Freund's adjuvant as described
previously(36) . The antiserum obtained was shown to be
specific for human TIMP-1 by Western blotting analysis. Sheep
anti-(human TIMP-1) IgG isolated by ammonium sulfate precipitation (40%
saturation) and DE-52 fractionation was coupled to Affi-Gel 10 and used
as immunoadsorbent beads.Enzyme Assays
All enzymic assays were carried out
in TNC buffer (50 mM Tris-HCl (pH 7.5), 0.15 M NaCl,
10 mM Ca, 0.05% Brij 35, 0.02%
NaN
). The collagenolytic activity of MMP-1 was measured
using
C-acetylated type I collagen (guinea pig) according
to Cawston and Barrett(37) . The gelatinolytic activity of
MMP-9 was measured using heat-denatured
C-acetylated type
I collagen (guinea pig)(38) . One unit of collagenolytic and
gelatinolytic activity degraded 1 µg of collagen or gelatin/min at
37 °C.
Purification of proMMP-9
The
proMMP-9TIMP-1 Complex
TIMP-1 complex was purified from the culture medium of
TPA-treated HT-1080 cells. The medium was applied to a
gelatin-Sepharose 4B column equilibrated with 50 mM Tris-HCl
(pH 7.5), 0.4 M NaCl, 10 mM CaCl
, 0.02%
NaN
. The column was washed with the same buffer, and
proMMP-9 bound was eluted with same buffer containing 2% (v/v) dimethyl
sulfoxide. The fractions were pooled, concentrated with an Amicon YM-10
membrane, and applied to Sephacryl S-300. SDS-PAGE analysis indicated
that most of the proMMP-9 eluted from the gelatin-Sepharose was in a
complex with TIMP-1. Earlier fractions from the column of Sephacryl
S-300 were pooled and used for experiments as the proMMP-9
TIMP-1
complex. A possible contamination of free proMMP-9 was examined by
applying the purified complex (100 µg/ml, 100 µl) onto a column
of anti-(human TIMP-1) IgG/Affi-Gel 10 (0.5 ml) equilibrated with TNC
buffer. The unbound fraction (2 ml) and the bound material, eluted with
2 ml of 50 mM Tris-HCl (pH 7.5), 6 M urea, 10
mM CaCl
, 0.02% NaN
, were analyzed by
SDS-PAGE. The purified complex was essentially free of free proMMP-9.
Purification of MMP-3 and MMP-1
Human proMMP-3 was
purified from the culture medium of rheumatoid synoviocytes and
activated with chymotrypsin as described previously(39) . Human
proMMP-1 was purified from TPA-treated U-937 cell culture medium and
activated by plasmin and MMP-3 according to Suzuki et
al.(40) . After activation, plasmin was inactivated by 2
mM DFP, and the 41-kDa MMP-1 was isolated by chromatography on
anti-MMP-3 immunoadsorbent and on Sephacryl S-200. MMP-1 was free of
MMP-3 and plasmin.Estimation of the Amount of the proMMP-9-TIMP-1 Complex,
MMP-1, and MMP-3
The amounts of the 41-kDa MMP-1 and the 45-kDa
MMP-3 were estimated by titration with a known amount of TIMP-1. The
amount of the proMMP-9TIMP-1 complex was estimated by titration
of TIMP-1 in the complex with the known amount of MMP-1 or MMP-3,
assuming the molar ratio of proMMP-9 to TIMP-1 of the complex is 1.
Electrophoresis, Zymography, and Western
Blotting
SDS-PAGE was performed according to the method of
Bury(41) , and proteins were stained with silver
nitrate(42) . Zymography was conducted using SDS-polyacrylamide
gels containing gelatin (0.8 mg/ml) as described
previously(35) . Enzymic activity was visualized as negative
staining with Coomassie Brilliant Blue R-250. Western blotting was
carried out according to Ito and Nagase(43) . Sheep anti-(human
proMMP-9) IgG, sheep anti-(human TIMP-1) serum, sheep anti-(human
MMP-3) serum, and/or sheep anti-(human MMP-1) serum were used as
primary antibody, and alkaline phosphatase-conjugated donkey
anti-(sheep IgG) IgG was used as secondary antibody.
Isolation of the proMMP-9
The conditioned medium of TPA-stimulated HT-1080 cells
contained the proMMP-9TIMP-1
Complex
TIMP-1 complex as well as a small amount of
free proMMP-9 and free TIMP-1. The gelatin-Sepharose step isolated the
complex and free proMMP-9. The separation of the complex from free
proMMP-9 was attained by gel permeation chromatography on Sephacryl
S-300. Application of the final product to an anti-(human TIMP-1)
immunoadsorbent column showed that the purified complex was virtually
free of free proMMP-9 (Fig. 1). A very small amount of proMMP-9
(<3%) was found in the unbound fraction of the anti-TIMP-1 column (Fig. 1, lane2), but it is not known whether
this is due to partial dissociation of the complex or contamination of
free proMMP-9. Nevertheless, neither case affects the interpretation of
our studies, as the amount of free proMMP-9, if any, is very small.
TIMP-1 complex. The purified proMMP-9
TIMP-1 complex
was applied to anti-(TIMP-1) IgG-Sepharose affinity chromatography to
examine the presence of free proMMP-9. The samples before and after the
chromatography were analyzed by SDS-PAGE under reducing conditions. Lane 1, the purified proMMP-9
TIMP-1 complex before
anti-(TIMP-1) IgG-Sepharose; lane 2, the fraction unbound to
anti-(TIMP-1) IgG-Sepharose; lane 3, the fraction bound to
anti-(TIMP-1) IgG-Sepharose. See ``Experimental Procedures''
for details. The gel was stained with silver
nitrate.
Lack of Enzymic Activity of the proMMP-9
proMMP-9 is
activated by APMA and trypsin(21, 31, 32) .
In contrast, when the proMMP-9TIMP-1
Complex upon Treatment with APMA or Trypsin
TIMP-1 complex was treated with
these agents, the gelatinolytic activity detected was negligible (Table 1). This suggests that TIMP-1 in the complex may inhibit
the activated MMP-9 or prevent the activation of proMMP-9. SDS-PAGE
analysis of the APMA-treated complex under reducing conditions
indicated that proMMP-9 was mostly converted to an 84-kDa species and
to a lesser extent an 82-kDa species (Fig. 2A).
Zymographic analysis of the samples under nonreducing conditions
indicated that only a small portion of the 82- and 80-kDa species
showed gelatinolytic activity, suggesting that most of the converted
MMP-9 was inhibited (Fig. 2B). In contrast, when
proMMP-9 free from TIMP-1 was activated with APMA, the 80-kDa species
was further converted to another active form of 68-kDa by an autolytic
reaction as demonstrated previously(35) . Absence of the 68-kDa
species in the APMA-treated complex further supports the notion that
the enzymic activities of the 82- and the 80-kDa species are inhibited
by TIMP-1. Treatment of the proMMP-9
TIMP-1 complex with trypsin
converted proMMP-9 to 82 and 68 kDa and four other smaller fragments (Fig. 3A). The TIMP-1 molecule was intact under these
conditions (Fig. 3A). The 82- and 68-kDa species are
identical to those generated with free proMMP-9 treated with
trypsin(35) . Similar to APMA treatment, trypsin-treated
samples exhibited only a low gelatinolytic activity by zymography (Fig. 3B). Since no significant gelatinolytic activity
was detected with these samples using
C-acetylated gelatin
in solution, a small activity detected by zymography might have
resulted from partial dissociation of the active MMP-9
TIMP-1
complex due to SDS treatment. Indeed, when the APMA- or trypsin-treated
complexes were subjected to Western blotting analysis for TIMP-1 under
nonreducing conditions, a majority of TIMP-1 was located at around
90-100 kDa (data not shown), suggesting that the activated MMP-9
formed a complex with TIMP-1.
TIMP-1 complex treated with APMA. The complex (10
µg/ml) was treated with 1 mM APMA at 37 °C for the
indicated periods of time. The samples were then subjected to SDS-PAGE
under reducing conditions (A) and zymography under nonreducing
conditions (B).
TIMP-1 complex treated with trypsin. The complex (10
µg/ml) was treated with trypsin (10 µg/ml) at 37 °C for the
indicated periods of time. After inactivating trypsin with 2 mM DFP, the samples were subjected to SDS-PAGE under reducing
conditions (A) and zymography under nonreducing conditions (B).
M. Only proteolytically
active enzymes are able to bind to
M and form a large
enzyme-inhibitor complex(44) . We previously demonstrated that
an active MMP-9 bound to
M stoichiometrically, which
resulted in the shift of gelatin-lysis zones of activated MMP-9 in
zymography(35) . Inability of the enzyme to bind to
M is indicative of the lack of proteolytic activity.
When the APMA- or trypsin-activated proMMP-9
TIMP-1 complex was
reacted with a 4-fold molar excess of
M at 37 °C
for 1 h prior to zymographic analysis, all species exhibiting
gelatinolytic activity by zymography failed to bind to
M (Fig. 4), indicating that the APMA- or
trypsin-activated proMMP-9
TIMP-1 complex does not possess
proteolytic activity.
TIMP-1 complex to
M. The
proMMP-9
TIMP-1 complex (10 µg/ml) was treated with 1 mM APMA for 24 h or trypsin (10 µg/ml) for 30 min at 37 °C.
Trypsin was inactivated with 2 mM DFP. The samples were then
diluted 10-fold with TNC buffer, reacted with
M (500
µg/ml) at 37 °C for 1 h, and applied to zymographic analysis
under nonreducing conditions. C, the control sample without
activation; APMA, the complex treated with 1 mM APMA
for 24 h; Trypsin, the complex treated with 10 µg/ml of
trypsin for 30 min.
Loss of MMP Inhibition Activity of the
proMMP-9
The
proMMP-9TIMP-1 Complex after APMA or Trypsin Treatment
TIMP-1 complex was able to inhibit active MMPs. Fig. 5shows the stoichiometric inhibition of MMP-1 (interstitial
collagenase) by the complex. When the complex was treated with APMA or
trypsin, however, the inhibitory activity of the complex against MMP-1
was completely lost. This indicates that MMP-9 and TIMP-1 form a
complex through their reactive sites after activation of proMMP-9 and
the inhibitory domain of TIMP-1 is no longer available for other MMPs.
TIMP-1 complex upon treatment with APMA or trypsin. The
proMMP-9
TIMP-1 complex was treated with APMA or trypsin, and then
its ability to inhibit MMP-1 (1.1 pmol) was measured by determining the
residual collagenolytic activity of MMP-1.
, the complex without
treatment;
, the complex treated with 1 mM APMA for 24 h
at 37 °C; ▪, the complex treated with trypsin (10 µg/ml)
for 1 h at 37 °C followed by inactivation of trypsin with 2 mM DFP.
Activation of the proMMP-9
A catalytic amount of MMP-3 activates proMMP-9 in a
stepwise manner(31) ; however, activation of the
proMMP-9TIMP-1 Complex by
MMP-3
TIMP-1 complex by MMP-3 is not readily
achieved(21) . This is anticipated, as MMP-3 can be readily
inhibited by the complex. Therefore, we postulated that activation of
the complex might be possible if TIMP-1 present in the complex was
saturated with an active MMP. To investigate this, the complex was
incubated with an increasing amount of MMP-3 for 1 and 4 h at 37 °C
and the MMP-9 activity was measured against
C-acetylated
gelatin. The gelatinolytic activity of MMP-3 is 0.2% of that of MMP-9 (45) ; thus, the contribution of MMP-3 in this assay system is
negligible. As shown in Fig. 6, gelatinolytic activity was
observed only when the molar ratio of MMP-3 to the complex exceeded 1.
The maximal specific activity of MMP-9 detected by incubation of the
complex with MMP-3 at a 1:2 molar ratio for 4 h was 20,000 units/mg,
about the same activity of the fully activated
MMP-9(31, 35) . Zymographic analysis of each
incubation mixture showed that the active 80-kDa (82-kDa with
reduction) species was generated only after a molar ratio of MMP-3 to
the complex exceeded 1. proMMP-9 was partially processed to 84-kDa
(86-kDa with reduction) below a 1:1 molar ratio. This species is not
proteolytically active, and the conversion of the 84-kDa species to the
active 80-kDa form requires the specific hydrolysis of
Arg
-Phe
by MMP-3(31) . Lack of
proteolytic activity of the 84-kDa species as well as proMMP-9 was
demonstrated by their inability to bind to
M, whereas
most of the 80-kDa species bound to
M and shifted to
the top of the gel where
M-proteinase complexes run on
SDS-PAGE under nonreducing conditions (Fig. 6B). A
small amount of activity remaining at 80-kDa is probably due to partial
dissociation of the enzyme from the
M complex by SDS
treatment(35) .
TIMP-1
complex by 45-kDa MMP-3. The complex were incubated with the 45-kDa
MMP-3 at various molar ratios at 37 °C for 1 and 4 h. The samples
were subjected to measurement of gelatinolytic activity (A)
and zymographic analysis (B).
Interaction of the proMMP-9
The inability of MMP-3 to activate proMMP-9 bound to
TIMP-1 when the amount of MMP-3 was below a 1:1 molar stoichiometry
suggests that MMP-3 preferentially interacts with TIMP-1. We then
examined whether active MMP-3 forms a stable
proMMP-9TIMP-1 Complex with
MMP-3
TIMP-1
MMP-3 ternary complex. To investigate this,
the proMMP-9
TIMP-1 complex was reacted with MMP-3 at different
molar ratios, and the mixtures were applied to a gelatin-Sepharose
column. Since both proMMP-9 and MMP-9 bind to the column, any molecules
that are recovered in the bound fraction are associated with either
proMMP-9 or MMP-9. As shown in Fig. 7A, TIMP-1 of the
complex was recovered in the bound fraction, but MMP-3 was not. When
MMP-3 was reacted with the complex at molar ratios to the complex of
0.5:1 and 1:1 at 37 °C and the mixtures were applied to the column
immediately or 1 h after the reaction, MMP-3 was recovered in both the
unbound and the bound fractions in approximately equal amounts in both
cases (Fig. 7B). proMMP-9 recovered in the bound
fraction remained primarily as a zymogen. The recovery of TIMP-1 in the
gelatin-unbound fraction together with MMP-3 indicates that the ternary
complex was partially dissociated into the MMP-3
TIMP-1 complex
and free proMMP-9 by weakening of the interaction between proMMP-9 and
TIMP-1. This dissociation was more prominent in a higher salt
concentration (data not shown). When the proMMP-9
TIMP-1 complex
was reacted with a one molar excess of MMP-3 for 1 h at 37 °C, all
proMMP-9 was converted to an 82-kDa species (80-kDa without reduction) (Fig. 7B). While a larger portion of TIMP-1 was
recovered in the gelatin-unbound fraction, about 35% of TIMP-1 was in
the bound fraction together with MMP-3. The molecular composition
recovered in the latter fraction is therefore the active MMP-9 coupled
with an MMP-3
TIMP-1 complex. A similar ternary complex and a
partial dissociation of proMMP-9 and TIMP-1 were also observed with
MMP-1 (Fig. 8). Like in the case of MMP-3, TIMP-1 did not
dissociate from proMMP-9 completely even after reaction with one molar
excess of MMP-1. Although proMMP-9 remained as a zymogen because MMP-1
is incapable of activating proMMP-9, addition of a catalytic amount of
MMP-3 to these samples could fully activate proMMP-9 (data not shown).
TIMP-1 complex to active MMPs. The complex (0.1 nmol) was
incubated with 45-kDa MMP-3 at 1:0.5, 1:1, and 1:2 molar ratio at 37
°C. After incubation for an indicated period of time, the samples
were immediately applied to a gelatin-Sepharose 4B column (1 ml).
Unbound (U) or bound (B) fraction were pooled,
concentrated with 5% (w/v) trichloroacetic acid, and subjected to
Western blotting analyses under reducing conditions (panelB). The first antibodies used were a mixture of sheep
anti-(human MMP-9), anti-(human TIMP-1) and anti-(human MMP-3)
antibody. PanelA shows the results when the
proMMP-9
TIMP-1 complex and MMP-3 were applied to the column
separately.
TIMP-1 complex to MMP-1. The proMMP-9
TIMP-1 complex
was reacted with MMP-1 at the molar ratio indicated for 2 h. The
samples were then analyzed as in Fig. 7except that sheep
anti-(human MMP-1) serum was used instead of sheep anti-(human MMP-3)
serum.
TIMP-1 complex by APMA and
proteinases and the role of TIMP-1 during these processes. The steps
involved in the proMMP-9
TIMP-1 complex are summarized in Fig. S1.
TIMP-1 complex by APMA and proteinases. (I), the
proMMP-9
TIMP-1 binds to and inhibit MMP-3 and other MMPs. The
resulting proMMP-9
TIMP-1-MMP ternary complex dissociates
partially into free proMMP-9 and the TIMP-1-MMP complex. (II), proMMP-9
of the complex is activated by MMP-3 when TIMP-1 is saturated with
active MMP. (III), the treatment of the proMMP-9
TIMP-1 complex
with APMA or trypsin processes proMMP-9 to active forms, but their
enzymic activity is inhibited by TIMP-1.
TIMP-1 complex was treated
with APMA or trypsin, no gelatinolytic activity was detected, although
proMMP-9 was converted to lower molecular mass species, which
correspond to active forms generated from proMMP-9 free of
TIMP-1(35) . The lack of enzymic activity after these
treatments was due to the inhibition of the activated MMP-9 by TIMP-1
in the complex (Fig. S1). A low gelatinolytic activity (<6%)
detected after the treatment of the complex with APMA is most likely
due to the presence of free proMMP-9. Alternatively, it may be due to
the contamination of other proMMPs, which bind to TIMP-1 in the complex
after APMA treatment, allowing for an equivalent amount of proMMP-9 to
be activated. This also emphasizes that the detection of the seemingly
active forms of MMP-9 by SDS-PAGE or zymographic analysis alone does
not assure that the functionally active enzyme is generated. In this
regard, a test for their abilities to interact with
M
is a useful means to identify proteolytically active enzyme
species(31, 35) .
TIMP-2 complex upon APMA treatment is different from that
of proMMP-9
TIMP-1. In the former case, the disruption of the
Cys
-Zn interaction by APMA is sufficient for TIMP-2 to
bind to the catalytic domain of the activated proMMP-2, and the
propeptide of proMMP-2 is not removed(24) . On the other hand,
when the proMMP-9
TIMP-1 complex is activated by APMA or trypsin,
proMMP-9 is processed to low molecular weight active forms, which are
then inhibited by TIMP-1. Since the inhibitory N-terminal domain of
TIMP-1 in the proMMP-9
TIMP-1 complex is free, it is possible that
TIMP-1 bound to the C-terminal domain of activated MMP-9 may interact
with another activated MMP-9 harboring TIMP-1. Such an interaction may
form either a tetrameric complex ((MMP-9
TIMP-1)
) with
2-fold symmetry or an oligomeric complex
((MMP-9
TIMP-1)
). However, both the
proMMP-9
TIMP-1 complex and the activated complex were eluted at
the same elution position on the gel permeation chromatography (data
not shown), indicating that neither tetrameric nor oligomeric complexes
were formed. Nonetheless, our studies do not reveal whether the
formation of the active MMP-9
TIMP-1 complex is a result of an
intra- or intercomplex rearrangement. In the latter case, transient
oligomerization should occur, but it is evident that the C-terminal
interaction of the proMMP-9
TIMP-1 complex becomes weaker when the
N-terminal domain of TIMP-1 is occupied by other MMPs (see Fig. 7and 8). This is a contrast to the proMMP-2
TIMP-2
complex, which largely remains as a stable ternary complex with another
active MMP when it is treated with APMA (24, 50) .
TIMP-1 complex when the amount of MMP-3 is
less than a molar stoichiometry (Fig. S1). Under these
conditions, MMP-3 is readily inhibited by the complex. Goldberg et
al.(21) reported that some of the secreted proMMP-9 forms
a covalently bound homodimer or a complex with proMMP-1. These forms of
proMMP-9 do not bind to TIMP-1, and they can be readily activated by
MMP-3(21) . When the proMMP-9
TIMP-1 complex is reacted
with a substoichiometric amount of an active MMP, APMA or trypsin was
able to generate proteolytically active MMP-9 in proportion to the
amount of TIMP-1 bound to an active MMP. On the other hand, the ability
of MMP-3 to activate proMMP-9 depends on the balance between TIMP-1 and
MMP-3; the saturation of TIMP-1 is prerequisite for MMP-3 to activate
proMMP-9 of the complex. The saturation of TIMP-1 may be attained by
other active MMPs as shown with MMP-1 in this study.
MMP complex ( Fig. 7and Fig. 8and Fig. S1), but it does not give much influence on the enzymic
activity of MMP-9. This mode of activation suggests a shift of
predominant MMP activity from one type to another. The former MMP
activity may be regulated by the TIMP-1 in the proMMP-9
TIMP-1
complex, which in turn renders proMMP-9 to be activable. Thus, the
expression of specific MMP activities are closely related not only to
the temporal gene expression of different types of proMMPs and TIMPs
by specific cell types but also to the subsequent activation of proMMPs
and the involvement of TIMPs during this process. Such interactions are
probably important parameters for determination of precise regulations
of extracellular matrix turnover in vivo.
M,
-macroglobulin; APMA,
4-aminophenylmercuric acetate; DFP, diisopropylphosphorofluoridate;
TPA, 12-O-tetradecanoylphorbol-13-acetate; PAGE,
polyacrylamide gel electrophoresis; IgG, immunoglobulin G.
We thank Dr. John L. Fowlkes for critical reading of
the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.