From the
The precursor of matrix metalloproteinase 9 (pro-MMP-9) forms a
complex with the tissue inhibitor of metalloproteinases (TIMP)-1
through the C-terminal domain of each molecule, and the N-terminal
domain of TIMP-1 in the complex interacts and inhibits active MMPs. We
have reported that a catalytic amount of MMP-3 (stromelysin 1)
activates pro-MMP-9 (Ogata, Y., Enghild, J. J., and Nagase, H.(1992) J. Biol. Chem. 267, 3581-3584). To
activate pro-MMP-9 in the complex, however, an excess molar amount of
MMP-3 is required to saturate the TIMP-1 in the complex. The aim of
this study was to test the hypothesis that the requirement for excess
MMP-3 can be circumvented by specific destruction of TIMP-1 by
non-target proteinases. We have tested trypsin, plasmin, cathepsin G,
neutrophil elastase, and chymotrypsin as possible inactivators of
TIMP-1 and found that neutrophil elastase inactivates TIMP-1 in the
complex without significant destruction of pro-MMP-9. Once TIMP-1 is
inactivated, pro-MMP-9 can be readily activated by a catalytic amount
of MMP-3. These results suggest that neutrophil elastase may
participate in connective tissue destruction at the inflammatory sites
not only by its direct action on matrix macromolecules but also by
rendering pro-MMP-9 in the pro-MMP-9
Matrix metalloproteinase 9 (MMP-9),
MMP-9, like other MMPs, is secreted from cells as an inactive
zymogen (pro-MMP-9). Thus, activation of pro-MMP-9 is one of the key
steps involved in control of its enzymic activity in the extracellular
space. Pro-MMP-9 consists of a propeptide domain, a catalytic domain
that contains the zinc-binding
HEXXHXXGXXH motif, three repeats of
fibronectin type II-like domain and type V collagen-like domain, and a
C-terminal hemopexin/vitronectin-like domain(1, 2) . The
treatment of pro-MMP-9 with trypsin and 4-aminophenylmercuric acetate
(APMA) activates the zymogen, but a most likely candidate of pro-MMP-9
activator in vivo is thought to be MMP-3 (stromelysin
1)(21, 22, 23) . However, the recent finding
that pro-MMP-9 forms a specific complex with an endogenous MMP
inhibitor, TIMP-1(16) , has introduced complexity in the
activation of pro-MMP-9 by MMP-3. TIMP-1 binds non-covalently to
pro-MMP-9 through the C-terminal domains of the two
molecules(22, 24) , and the N-terminal domain, which has
inhibitory activity, is exposed for interaction with other active MMPs.
Therefore, the activation of pro-MMP-9 in the complex by MMP-3 requires
more than a molar stoichiometric amount of MMP-3 or blockage of the
TIMP-1 by other MMPs.
In this communication,
we report another possible mechanism by which pro-MMP-9 in the complex
may become readily activable by MMP-3. This results from a specific
inactivation of TIMP-1 in the complex by human neutrophil elastase
(HNE). This observation, together with the ability of HNE to activate
MMP-3 from its precursor(25) , suggests that HNE plays a key
role in tissue destruction in inflammatory sites in vivo.
Pro-MMP-9 is activated in vitro by treatment with
mercurial compounds, SDS as demonstrated by SDS-containing gelatin
zymography, and trypsin. Morodomi et al.(27) reported
that pro-MMP-9 can be activated by a relatively high concentration (10
µg/ml) of cathepsin G and plasmin, but the levels of activation
were 22-30% and 10%, respectively, while Okada et al.(23) reported more effective activation by these enzymes.
Nonetheless, the most likely activator of pro-MMP-9 in vivo is
MMP-3 since a catalytic amount of MMP-3 is sufficient to activate this
zymogen (21-23). MMP-3 processed the propeptide of pro-MMP-9 in a
stepwise manner by initially cleaving the Glu
TIMP-1, on the other hand, can be proteolytically
inactivated by a number of non-target proteinases such as HNE and
trypsin(32) . In this report, we have demonstrated that HNE
preferentially cleaves TIMP-1 and inactivates the inhibitor without
affecting the integrity of pro-MMP-9 significantly. The action of HNE
on TIMP-1 in the complex is probably on the Val
Although
trypsin inactivates TIMP-1 by degrading it into several fragments (data
not shown), it did not destroy TIMP-1 in the pro-MMP-9
In
conclusion, we have demonstrated that HNE preferentially inactivates
TIMP-1 in the pro-MMP-9
The complex (20 µg/ml) was treated with HNE (5
µg/ml) for indicated periods. After inactivation of HNE by 2 mM Dip-F, the samples were incubated with 0.2 or 0.5 molar ratio of
MMP-3 at 37 °C for indicated periods of time. MMP-9 activity was
measured using
We thank Dr. Keith Brew for critical reading of the
manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
TIMP-1 complex activable by
MMP-3 as well as activating pro-MMP-3.
(
)also
designated gelatinase B, is a member of the matrixin family(1) .
The enzyme readily digests heat-denatured collagen (gelatins), but it
also degrades collagen types IV, V, XI, laminin, elastin (see Refs. 1
and 2 for review), entactin(3) , aggrecan core
protein(4) , and cartilage link protein(5) . The enzyme
was first found in neutrophils (6) and shown to be
immunologically identical to the gelatinase in macrophages (7-9).
Recent studies, however, have demonstrated that MMP-9 is also produced
in articular chondrocytes(10, 11, 12) , synovial
fibroblasts(13) , T-lymphocytes(14, 15) , HT-1080
fibrosarcoma cells, monocytic leukemia cell lines (16) when they
are stimulated with interleukin-1, tumor necrosis factor
, and/or
a phorbol ester. Elevated expression of MMP-9 in
cytotrophoblasts(17) , osteoclasts in developing
embryos(18) , osteoarthritic chondrocytes(12) ,
macrophages in rheumatoid synovium (19), and in invasive cancer cells
(see Ref. 20 for review) suggests that the enzyme may play an important
role in cellular migration, invasion, and tissue remodeling and
catabolism under certain physiological and pathological conditions.
(
)
Materials
APMA, Brij 35, diisopropyl
phosphorofluoridate (Dip-F),
12-O-tetradecanoylphorbol-13-acetate (TPA), trypsin (bovine),
chymotrypsin (bovine), plasminogen (human), urokinase (human), and
alkaline phosphatase-conjugated donkey anti-(sheep IgG) IgG were from
Sigma. Dulbecco's modified Eagle's medium (DMEM),
antibiotics, fetal calf serum (FCS), Hanks' balanced salt
solution (HBSS), and lactalbumin hydrolysate were from Life
Technologies, Inc. Human fibrosarcoma cell line HT-1080 was obtained
from American Type Culture Collection. HNE and human neutrophil
cathepsin G were from Athens Research Technology Inc. Human pro-MMP-3
was purified from the culture medium of rheumatoid synovial fibroblasts
and activated as described previously(25) . Human pro-MMP-1
(interstitial collagenase) was purified from the medium of the
TPA-treated U-937 cells according to Suzuki et
al.(26) . Pro-MMP-1 was activated with plasmin and MMP-3,
and MMP-3 and plasmin were removed by chromatography on anti-MMP-3 IgG
coupled to Affi-Gel 10 and Sephacryl S-200, respectively. TIMP-1 was
purified from the medium of U-937 cells(27) . Antiserum against
human pro-MMP-9 and reduced TIMP-1 were raised in sheep using purified
antigens as described previously(28) . Both antisera were shown
to be specific by Western blotting analysis using the concentrated
crude culture medium of HT-1080 cells.
Cell Cultures
HT-1080 cells were cultured in DMEM
containing 10% FCS. After confluency the cells were washed with HBSS
and treated with TPA (20 ng/ml) in serum-free DMEM supplemented with
0.2% lactalbumin hydrolysate for 2 days. This conditioned medium was
harvested and used for purification of the pro-MMP-9TIMP-1
complex.
Purification of Pro-MMP-9
Pro-MMP-9TIMP-1
Complex
TIMP-1 complex was purified from
conditioned medium of HT-1080 cells. First, culture medium was passed
through a column of gelatin-Sepharose 4B equilibrated with TNC buffer
(50 mM Tris-HCl (pH. 7.5), 0.15 M NaCl, 10
mM CaCl
, 0.02% NaN
). The column was
washed with the same buffer, and the enzyme was subsequently eluted
with the same buffer containing 2% dimethyl sulfoxide. The eluted
protein was pooled, dialyzed against TNC buffer, concentrated with an
Amicon YM-10 membrane, and subjected to gel permeation chromatography
on Sephacryl S-300. The protein peak containing pro-MMP-9 was mostly a
complex with TIMP-1. Fractions from the front edge of the peak were
pooled as the pro-MMP-9
TIMP-1 complex.
Enzyme Assays
All enzyme assays were carried out
in TNC buffer containing 0.05% Brij 35. The collagenolytic activity of
MMP-1 was measured using C-acetylated type I collagen
(guinea pig) according to the method of Cawston and Barret(29) .
The gelatinolytic activity of MMP-9 was measured using heat-denatured
C-acetylated type I collagen (guinea pig)(30) . One
unit of collagenolytic and gelatinolytic activity degraded 1 µg of
collagen or gelatin per min at 37 °C.
TIMP Assay
TIMP activity was measured against the
41-kDa MMP-1. Various concentrations of the samples were incubated with
a constant amount of MMP-1 at 37 °C for 30 min, and residual
collagenolytic activity was measured against C-acetylated
type I collagen (guinea pig).
Determination of Concentrations of Pro-MMP-9
The amount of the 41-kDa MMP-1 and the
45-kDa MMP-3 was determined by titration with TIMP-1. The amount of the
pro-MMP-9TIMP-1
Complex, MMP-1, and MMP-3
TIMP-1 complex was determined by titration of the TIMP-1
in the complex with MMP-1 or MMP-3, assuming the molar ratio of
pro-MMP-9 to TIMP-1 in the complex is 1:1.
Western Blotting and Zymography
Western blotting
analysis was carried out as described previously(31) . Sheep
anti-(human MMP-9) IgG was used as a primary antibody at a
concentration of 5 µg/ml and sheep anti-(human TIMP-1) serum at a
1:1000 dilution, and alkaline phosphatase-conjugated donkey anti-(sheep
IgG) IgG was used for a secondary antibody. Zymography was conducted
with SDS-polyacrylamide gel containing gelatin (0.8 mg/ml) as described
previously(27) . Enzymic activity was visualized as negative
staining with Coomassie Brilliant Blue R-250.
Inactivation of TIMP-1 in the Pro-MMP-9
Five different proteinases (bovine trypsin,
bovine chymotrypsin, HNE, human cathepsin G, and human plasmin) were
tested for their ability to inactivate the TIMP-1 component of the
pro-MMP-9TIMP-1
Complex by HNE
TIMP-1 complex. The treatment of the complex with
trypsin, HNE, and chymotrypsin diminished apparent TIMP-1 activity in a
time-dependent manner, but plasmin or cathepsin G had little effect (Fig. 1). SDS-PAGE and immunoblotting analysis of the
pro-MMP-9
TIMP-1 complex after treatment with these proteinases
showed that only HNE degraded TIMP-1 in a time-dependent manner (Fig. 2). HNE also cleaved some of the pro-MMP-9, but the amount
was low in comparison with TIMP-1 degradation. The degradation products
of TIMP-1 had molecular masses of 17 and 16 kDa, identical to those of
TIMP-1 treated with HNE. The degradation rates of TIMP-1 in the complex
and that of free TIMP-1 were almost identical. Although the treatment
with trypsin and chymotrypsin also diminished the TIMP-1 activity of
the complex, Western blotting analyses indicated that the TIMP-1
molecule was intact. Instead, pro-MMP-9 was processed into lower
molecular weight species. TIMP-1, when not bound to pro-MMP-9, is
susceptible to degradation by trypsin or chymotrypsin(32) , but
both enzymes activate pro-MMP-9(23, 27) . Thus, the loss
of MMP inhibitory activity of TIMP-1 in the complex after trypsin or
chymotrypsin treatment is likely to result from the formation of the
complex through the inhibitory site of TIMP-1 and active site of
activated MMP-9, so that the TIMP-1 molecule is no longer available to
inhibit other MMPs (MMP-1 in Fig. 1). To verify this, the trypsin
or chymotrypsin-treated complex was applied to an anti-TIMP-1 affinity
column. The complex, after treatment with trypsin (10 µg/ml) for 1
h at 37 °C, exhibited no TIMP activity, whereas the complex, after
treatment with chymotrypsin (10 µg/ml) for 4 h, retained about 34%
TIMP activity. However, no gelatinolytic activity was detected in
either case (data not shown). Application of these samples to the
anti-(TIMP-1) IgG-Affi-Gel 10 column indicated that all low molecular
weight species of MMP-9 generated by trypsin or chymotrypsin bound to
the column and eluted with 6 M urea together with TIMP-1 (Fig. 3). These results indicate that the activated MMP-9 formed
a complex with TIMP-1 through the catalytic site of the enzyme and the
N-terminal inhibitory domain of the inhibitor. Plasmin and cathepsin G
had very little effect on TIMP-1 and pro-MMP-9.
Figure 1:
Inactivation of TIMP-1 in the
pro-MMP-9TIMP-1 complex by proteinases. Purified
pro-MMP-9
TIMP-1 complex (20 µg/ml) was incubated with HNE (5
µg/ml) (
), bovine chymotrypsin (10 µg/ml) (
), bovine
trypsin (10 µg/ml) (
), human plasmin (10 µg/ml)
(
), or human cathepsin G (10 µg/ml) (
) at 37 °C for
indicated periods of time. After incubation, the reactions were
terminated with 2 mM Dip-F, and the samples were subjected to
the assay for TIMP activity to inhibit the purified 41-kDa MMP-1 as
described under ``Experimental
Procedures.''
Figure 2:
Western blotting analysis of the
pro-MMP-9TIMP-1 complex treated with proteinases. The samples
from Fig. 1 was analyzed by Western blotting using sheep anti-(human
MMP-9) and sheep anti-(human TIMP-1) antibodies as a first antibody. C, pro-MMP-9
TIMP-1 complex without treatment; CT, chymotrypsin.
Figure 3:
Complex formation between the trypsin- or
chymotrypsin-activated MMP-9 and TIMP-1. Pro-MMP-9-TIMP-1 complex (25
µg/ml) was treated with trypsin (10 µg/ml) or chymotrypsin (CT) (10 µg/ml) at 37 °C for 1 or 4 h, respectively.
After terminating the reaction with 2 mM Dip-F, the sample
(100 µl) was applied to an anti-(TIMP-1)IgG-Affi-Gel 10 column (1
ml) equilibrated with TNC buffer. The unbound materials were collected
into a 2-ml fraction, and the bound materials were eluted with TNC
buffer comtaining 6 M urea into the same volume. Then 1 ml of
each fraction was precipitated with 4.5% trichloroacetic acid with 5
µg of human fibronectin as a carrier. The precipitates were then
dissolved in 30 µl of loading buffer containing
-mercaptoethanol and applied to Western blotting analysis using
sheep anti-(human MMP-9) and anti-(human TIMP-1) antibodies. S, starting materials; U, unbound fraction; B,
bound fraction.
Activation of Pro-MMP-9 in the HNE-treated Complex by
MMP-3
Incubation of the pro-MMP-9TIMP-1 complex and MMP-3
at a molar ratio of 1:0.2 or 1:0.5 at 37 °C failed to activate
pro-MMP-9 in the complex even after 24 h (). This was due
to the inhibition of MMP-3 by TIMP-1 of the complex. A very small
amount of pro-MMP-9 was converted to an intermediate 86-kDa form after
incubation with a 0.5 molar ratio of MMP-3 for 24 h (Fig. 4).
This species is an initial product of MMP-9 generated by MMP-3, but it
does not have enzymic activity(21) . When the HNE-treated
complex was incubated with a catalytic amount of MMP-3, pro-MMP-9 was
activated in a time- and a dose-dependent manner. The level of
pro-MMP-9 activation was dependent on the degree of TIMP-1 degradation
by HNE (, Fig. 4). After a 4-h treatment with HNE,
about 30% of TIMP-1 activity was left (Fig. 1). Under these
conditions, the addition of a 0.2 molar ratio of MMP-3 to the complex
did not activate pro-MMP-9, but a 0.5 molar ratio proportion of MMP-3
did activate the zymogen generating the fully active 82-kDa form (Fig. 4). The lack of activation at a 0.2 molar ratio of MMP-3
can be attributed to the inhibition of MMP-3 by the intact TIMP-1 that
was present in the complex. After incubation of the complex with HNE
for 8 h, residual TIMP-1 activity was 15% of the original, and a 0.2
molar amount of MMP-3 was able to activate the complex. The maximal
activity of MMP-9 detected was about 75% of that generated by treatment
of the complex with HNE for 4 or 8 h. A decrease in MMP-9 activity
after incubation with a 0.5 molar ratio of MMP-3 at 37 °C for a
longer incubation time was due to degradation of MMP-9 either by
autolysis or by MMP-3.
Figure 4:
Activation of the HNE-treated
pro-MMP-9TIMP-1 complex by MMP-3. The same samples in Table I
were applied to zymography. Pro-MMP-9 and MMP-9 bands were visualized
by negative staining with Coomassie Brilliant Blue
R-250.
-Met
bond and then the Arg
-Phe
bond for
complete removal of the propeptide(21) . However, this action of
MMP-3 is readily inhibited when pro-MMP-9 binds to TIMP-1(22) .
This is evident from the relatively high affinity between TIMP-1 and
MMP-3 as shown by the K
value of 0.1
nM and k
value of 1.9
10
M
s
(33) .
Pro-MMP-9 was found as a complex in the conditioned medium of HT-1080
cells, U937 cells, H-Ras-transformed human fibroblasts, and human
alveolar macrophages treated with lipopolysaccharide(34) . When
these complexes are treated with APMA or trypsin, pro-MMP-9 in the
complex is processed to an active species, but it is readily inhibited
by TIMP-1 in the complex.
MMP-3 is unable to activate
pro-MMP-9 in the complex unless TIMP-1 is saturated with an active
MMP.
-Cys
bond
(
)in the N-terminal domain of the
inhibitor as SDS-PAGE analysis of the HNE-treated complex and free
TIMP-1 showed fragments with the same molecular masses. After treatment
of the complex with the HNE, a catalytic amount of MMP-3 activated
pro-MMP-9. After a longer incubation time (e.g. 24 h), less
activity of MMP-9 was detected. This resulted from the degradation of
the activated MMP-9, possibly by MMP-3. A similar observation was
reported recently by Shapiro et al.(35) .
TIMP-1
complex (Fig. 2). Under the experimental conditions that we
employed, trypsin preferentially activates the zymogen, which in turn
binds to the inhibitory site of TIMP-1. It is notable that TIMP-1, in
this complex, is resistant to proteolysis by trypsin, suggesting that
the trypsin cleavage sites in TIMP-1 are protected by MMP-9.
TIMP-1 complex and renders pro-MMP-9
activable by MMP-3. Shifts of the balance between proteinases and
proteinase inhibitors produced by the inactivation of proteinase
inhibitors by non-target enzymes are an important consideration in
tissue destruction. Examples of this are described for a number of
serpins, which are inactivated by bacterial
proteinases(36, 37) , cysteine proteinases(38) ,
and MMPs(39) . The present study suggests that HNE may play an
important role in connective tissue destruction under inflammatory
conditions not only by its direct action on connective tissue matrix
components but also by converting pro-MMP-3 to active MMP-3 (25) and rendering pro-MMP-9 activable by the selective
destruction of TIMP-1. The activated MMPs through these mechanisms may
further accelerate tissue damage by their direct action on the matrix
as well as inactivation of the endogenous elastase inhibitor,
-proteinase inhibitor(39) .
Table: Expression of gelatinolytic activity in the
pro-MMP-9 TIMP-1 complex treated with HNE by catalytic amount
of MMP-3
C-labeled guinea pig type I gelatin.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.