From the Department of Vascular Biology, Holland
Laboratory, American Red Cross, Rockville, Maryland 20855, the
¶ Department of Biochemistry and Molecular Biology and Institute
for Biomedical Sciences, George Washington University Medical Center,
Washington, D. C. 20037, and § Department of Biochemistry,
University of Washington, Seattle, Washington 98195
Received for publication, January 5, 2001, and in revised form, January 30, 2001
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ABSTRACT |
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Members of the matrix metalloproteinase (MMP)
family of enzymes participate in matrix remodeling and share a number
of structural and functional features. The activity of this family of
proteinases is carefully regulated at the level of zymogen activation
and by a family of specific inhibitors termed tissue inhibitors of metalloproteinases (TIMP). It is now becoming clear that levels of
certain MMPs are modulated by their association with cellular receptors
that mediate their rapid internalization and degradation. In the
current investigation we report that the amount of MMP-9 in conditioned
cell culture medium is significantly increased when mouse embryonic
fibroblasts are grown in the presence of the 39-kDa receptor-associated
protein (RAP), an antagonist of ligand binding to low density
lipoprotein receptor-related protein (LRP). In vitro assays
reveal that the MMP-9·TIMP-1 complex binds to LRP with high affinity
and that the binding determinant for LRP appears to reside on MMP-9.
Cell lines expressing LRP mediate the internalization of
125I-labeled MMP-9·TIMP-1 complexes, whereas cell lines
genetically deficient in LRP show a diminished capacity to mediate the
cellular catabolism of MMP-9·TIMP-1 complexes. The results
demonstrate that LRP is a functional receptor for MMP-9 and suggest a
major role for LRP in modulating remodeling of the extracellular matrix by regulating extracellular proteinase activity.
Members of the matrix metalloproteinase
(MMP)1 family of enzymes
participate in matrix remodeling and share a number of structural and
functional features. All require zinc and calcium ions for activity and
are synthesized as inactive zymogens that need to be cleaved to become
active enzymes (1-3). One member of this family, MMP-9, plays an
important role in controlling angiogenesis (4, 5) and in extracellular
matrix remodeling after myocardial infarction (6). Thus, mice in which
the MMP-9 gene has been deleted exhibit an abnormal pattern of skeletal
growth plate vascularization and ossification (5). Growth plates from
MMP-9-deficient mice in culture show a delayed release of an angiogenic
activator. Together, these data establish a role for this proteinase in
controlling angiogenesis. Using a transgenic mouse model of multistage
carcinogenesis Bergers et al. (4) conclude that MMP-9
triggers an angiogenic "switch" that renders normal pancreatic
islets angiogenic by releasing increased amounts of vascular
endothelial growth factor. In another transgenic model of epithelial
carcinogenesis, Coussens et al. (7) observe that mice
lacking MMP-9 show reduced keratinocyte hyperproliferation at all
neoplastic stages and a decreased incidence of invasive turmors.
Like most proteinases, the activity of MMP-9 is tightly regulated. MMP
activity is modulated by a family of specific inhibitors, termed tissue
inhibitors of metalloproteinases (TIMP) (8). TIMPs regulate MMP
activity during extracellular matrix turnover, and ablation of TIMP
gene expression results in enhanced extracellular matrix proteolysis
concomitant with up-regulation of cell invasive activity (9, 10).
Recent studies demonstrate a unique role for TIMPs independent of their
ability to inhibit MMPs by demonstrating that TIMP-2 can directly
regulate tyrosine kinase-type growth factor receptor activation
(11).
An additional pathway capable of regulating the levels of MMP-13 was
recently discovered when Barmina et al. (12) identified cellular receptors that are able to bind MMP-13. One of these receptors, the low density lipoprotein receptor-related protein (LRP),
mediates the internalization and degradation of this proteinase. LRP, a
member of the LDL receptor superfamily, also modulates levels of MMP-2
by binding and internalizing MMP-2-thrombospondin 2 (TSP2) complexes
(13). In both of these studies, the catabolism of MMPs was inhibited by
a 39-kDa receptor-associated protein (RAP). This molecule binds
reversibly to LRP (14) and other members of the LDL receptor family
such as gp330/megalin (15) and the VLDL receptor (16) and
inhibits ligand binding by these receptors (14, 17). RAP is found
primarily in the endoplasmic reticulum, where it functions as a
molecular chaperone by assisting in receptor folding and processing
(18) and preventing the association of newly synthesized receptors with
endogenous ligands (19). Due to its high affinity for LRP and ability
to antagonize ligand binding, exogenously added RAP constitutes a
powerful tool to study LRP-mediated mechanisms. In the current
investigation, we report that the levels of MMP-9 in conditioned cell
culture media are increased significantly when cells are grown in the
presence of RAP. Furthermore, we demonstrate that MMP-9 directly binds with high affinity to LRP and that this receptor mediates the internalization and subsequent degradation of this enzyme. Based on
these studies, we propose that clearance of MMP-9 by LRP is an
important mechanism for regulating extracellular levels of this proteinase.
Proteins--
Human RAP was expressed in bacteria as a fusion
protein with glutathione S-transferase as described
previously (14). Cleavage with thrombin and purification of recombinant
RAP was carried out as described (14) with modification. After elution
from glutathione S-transferase-Sepharose, RAP was placed on
a MonoS column equilibrated with 0.02 M sodium phosphate,
pH 5.5, and eluted with a gradient from 0 to 2 M NaCl over
30 min at a flow rate of 1 ml/min. The RAP was then recycled three
times over AffinityPakTM Detoxi-GelTM
endotoxin-removing gel (Pierce). LRP was purified by ligand affinity chromatography followed by ion exchange chromatography on MonoQ HR
(Amersham Pharmacia Biotech) as described (20). MMP-9·TIMP-1 and
MMP-2·TIMP-2 complexes were purchased from Chemicon (Temecula, CA). A
fragment containing the eight ligand binding repeats of the human VLDL
receptor (VLDLr1-8) was generated as a GST fusion protein as described
(21). After purification and digestion with thrombin to release the
VLDLr1-8, refolding and purification of the soluble receptor fragment
was accomplished as described (21). Anti-MMP-9 and anti-MMP-2
monoclonal antibodies were purchased from Calbiochem. Anti-LRP
monoclonal antibody 8G1 (22) was produced and purified as described
previously. Goat anti-mouse IgG conjugated to either horseradish
peroxidase or alkaline phosphatase was purchased from Bio-Rad.
Cell Culture Conditions and RAP Treatment--
Mouse embryonic
fibroblasts (PEA-10, LRP(+/ Gelatin Zymography--
Gelatin gels (10%) and Zymogram
developing buffer (10×) were purchased from Novex (San Diego, CA) and
used as described (24, 25). Briefly, samples were diluted in
nonreducing SDS sample buffer and electrophoresed on 10% gelatin gels.
After electrophoresis, the gels were washed 2× 30 min in 2.5% Triton
X-100 and incubated 16 h at 37 °C in 1× Zymogram developing
buffer. After incubation, gels were stained with Coomassie Brilliant
Blue (0.3% in 50% methanol, 10% glacial acetic acid) and destained
with 10% glacial acetic acid, 10% isopropanol, 5% glycerol. The gels
were scanned using an AFGA scanner and converted to TIFF files,
and optical density analysis was performed on a Macintosh computer
using the public domain NIH Image program.
Enzyme-linked Immunoabsorbent Assays--
Ligands (MMP-9, MMP-2,
and BSA) were coated on microtiter wells at 4 µg/ml in 50 mM Tris, 150 nM NaCl (TBS) with 1 mM CaCl2 for 16 h at 4 °C. Wells were
washed 3× with TBS, 1 mM CaCl2 and blocked
with 0.3% BSA in TBS, 1 mM CaCl2 for 1 h
at room temperature. Subsequently, wells were incubated for at 4 °C
in increasing concentrations of LRP in TBS, 1 mM
CaCl2, 0.05% Tween 0 with 0.3% BSA. The wells were washed
3× with 0.3% BSA in TBS, 1 mM CaCl2, 0.05%
Tween 20. Bound LRP was detected with monoclonal antibody 8G1 (1 µg/ml) and goat anti-mouse IgG conjugated to alkaline phosphatase.
The wells were then incubated in phosphatase substrate (Sigma 104) in
0.1 M glycine, 1 mM MgCl2, 1 mM ZnCl2, pH 10.4,until color development. The
absorbance for each sample was measured at 405 nm. Data were analyzed
by nonlinear regression analysis using the equation,
Ligand Blotting Experiments--
Ligand blotting experiments
were carried out as previously described (26), with slight
modifications. Briefly, LRP or VLDLr1-8 were subjected to SDS-PAGE on
4-12% gradient gels (Novex, San Diego, CA) under nonreducing
conditions and transferred to nitrocellulose filters. The filters were
blocked with 3% nonfat milk and incubated with 20 nM
MMP-9·TIMP-1 for 16 h at 4 °C in TBS containing 3% nonfat
milk, 5 mM CaCl2, and 0.05% Tween 20. In some
experiments, 500 nM RAP was included. After three washes,
the filters were incubated with an anti-MMP-9 monoclonal antibody (1.0 µg/ml) for 1 h at 25 °C and then incubated with a goat
anti-mouse IgG horseradish peroxidase conjugate for 1 h at
25 °C. MMP-9 binding was visualized using the Super Signal West Pico
chemiluminescence kit (Pierce).
Iodination of MMP-9--
MMP-9·TIMP-1 complex was labeled with
[125I]iodine to a specific activity ranging from 2-10
µCi/µg of protein using Iodogen (Pierce).
Cell Internalization and Degradation Assays--
Mouse embryonic
fibroblasts (LRP(+/+)) and LRP-deficient mouse embryonic fibroblasts
(PEA-13, LRP( MMP-9 Accumulates in the Conditioned Medium Collected from Cells
Treated with the LDL Receptor Family Inhibitor, RAP--
Previous
reports demonstrated that LRP can directly bind and mediate the
cellular catabolism of MMP-13 (12) and regulates levels of MMP-2 as
well (13). To further investigate the role of LRP in the catabolism of
the MMP gelatinases MMP-2 and MMP-9, LRP(+/
In the current experiments, we noted that control levels of MMP-2 were
high and that very little change in MMP-2 levels occurred when the
cells were cultured in the presence of RAP, whereas in another study
using mouse dermal fibroblasts, an increase in MMP-2 levels in the
presence of RAP was noted (13). As this accumulation did not occur in
dermal fibroblasts isolated from mice in which the TSP2 gene was
deleted, we previously concluded that MMP-2 catabolism by LRP is
mediated by TSP2. Indeed the embryonic fibroblasts used in the current
study produce barely detectable levels of TSP2, as determined by
immunoblot of conditioned
media.2
MMP-9 Binds Directly to LRP--
To investigate the mechanism by
which LRP modulates MMP-9 levels, we initiated experiments to determine
if MMP-9 can directly bind to LRP. The preparation of MMP-9 used in the
current experiments was composed of MMP-9 in complex with its cognate
inhibitor TIMP-1. Our initial experiments examined the interaction of
the MMP-9·TIMP-1 complex with LRP by ligand-blotting experiments.
Purified human LRP and a soluble VLDLr fragment containing the ligand
binding region of this receptor (VLDLr1-8) were first subjected to
SDS-PAGE under nonreducing conditions, transferred to nitrocellulose,
and then incubated with 20 nM recombinant human
MMP-9·TIMP-1 complex. The binding of MMP-9·TIMP-1 complex to LRP
was then visualized using an anti-MMP-9 IgG. The results demonstrate
that MMP-9·TIMP-1 binds to LRP (Fig.
2A) but not to VLDLr1-8 (Fig.
2B). The binding to LRP was blocked when excess RAP was
incubated along with MMP-9, confirming the specificity of the
interaction.
We next wished to ascertain if the presence of TIMP-1 is required for
binding of MMP-9 to LRP. To investigate this, a "receptor blot" was
performed. In this experiment, RAP (as a positive control), the
MMP-9·TIMP-1 complex, and the MMP-2·TIMP-2 complex were first subjected to SDS-PAGE under nonreducing conditions (Fig.
3, A-C) to separate MMP-9 and
MMP-2 from TIMP-1 and TIMP-2, respectively. After electrophoresis, the
proteins were transferred to nitrocellulose and incubated with purified
LRP, and receptor binding was visualized with an anti-LRP IgG. The
results demonstrate that LRP recognized RAP (Fig. 3B,
lane 1) and TIMP-free MMP-9 (Fig. 3B, lane
2) but not TIMP-1, MMP-2, or TIMP-2. Binding of LRP to MMP-9 was
abolished by the addition of excess RAP (Fig. 3C),
confirming the specificity of the interaction. MMP-9 is known to be
composed of domains stabilized by disulfide bonds, and consequently we
investigated the binding of LRP to MMP-9 subjected to SDS-PAGE under
reducing conditions. The results (Fig. 3D, lane
2) indicate that LRP failed to bind to MMP-9 when the enzyme was
subjected to reduction before SDS-PAGE. In contrast, when RAP was
subjected to SDS-PAGE under reducing conditions, no impact on the
ability of LRP to bind RAP was noted (Fig. 3D, lane
1), consistent with the fact that RAP contains no cysteine
residues. These results indicate that MMP-9 directly binds to LRP even
in the absence of TIMP-1 and that reduction of MMP-9 abolishes its
ability to bind LRP.
To measure the affinity of LRP for MMP-9·TIMP-1 complexes, a solid
phase assay was employed in which the MMP-9·TIMP-1 complex was first
immobilized on microtiter wells. Increasing concentrations of LRP were
then incubated with the immobilized MMP-9·TIMP-1 complex, and the
extent of LRP binding was measured using the anti-LRP monoclonal
antibody 8G1. An apparent KD of 0.6 nM
for the binding of LRP to MMP-9·TIMP-1 was estimated by nonlinear regression analysis of the data (Fig. 4,
closed triangles). In contrast, MMP-2·TIMP-2 complexes
demonstrated much lower affinity for LRP in the same assay (Fig. 4,
closed circles), with an apparent KD
value estimated to be at least 10-100-fold weaker than that of MMP-9.
No binding of LRP to microtiter wells coated with BSA was observed
(Fig. 4, open squares). In the same experiment, the apparent
affinity of LRP for microtiter wells coated with RAP was determined to
be 0.07 nM (data not shown).
We also investigated the binding of MMP-9·TIMP-1 to microtiter wells
coated with LRP. Increasing concentrations of MMP-9·TIMP-1 in the
absence or presence of excess RAP were added to the microtiter wells,
and after washing, binding was determined by using a specific antibody
recognizing MMP-9. The results (Fig. 5)
demonstrate that MMP-9·TIMP-1 binds to solid phase LRP with an
estimated KD value of 20 nM determined
by nonlinear regression analysis. The binding appeared specific for
LRP, because the binding was completely inhibited when excess RAP was
included in the assay. In addition, no binding of MMP-9·TIMP-1 to
microtiter wells coated with BSA was observed. In the same experiment,
the apparent KD for RAP binding to microtiter wells
coated with LRP was determined to be 4 nM (data not shown).
Together, the experiments in Figs. 4 and 5 confirm that LRP binds MMP-9
with high affinity. Curiously, we noted that the apparent affinity of
LRP for MMP-9·TIMP-1 and RAP was considerably higher when these
ligands were coated to microtiter wells. The reason for this
discrepancy is not apparent, but we suspect that the increased affinity
for RAP when this ligand is coated on microtiter wells might be due to
the presence of multiple binding sites on LRP for this molecule (27),
resulting in multivalent attachment. Therefore, LRP may also contain
multiple binding sites for the MMP-9·TIMP-1 complex as well.
LRP Mediates the Cellular Catabolism of MMP-9--
To determine
the physiological relevance of the interaction between MMP-9 and LRP,
LRP(+/+) and LRP(
We next examined the time course of internalization and degradation of
125I-MMP-9·TIMP-1 complex in LRP-expressing and
LRP-deficient cells (Fig. 7).
LRP-expressing cells demonstrated a time-dependent
accumulation of radioactivity (Fig. 7A), which was prevented
when the cells were incubated with excess RAP. In contrast,
LRP-deficient cells only internalized small amounts of
125I-labeled MMP-9·TIMP-1 complexes (Fig. 7B).
LRP-mediated internalization of 125I-labeled MMP-9·TIMP-1
complexes was associated with their cellular-mediated degradation (Fig.
7C), as confirmed by blocking with chloroquine, an inhibitor
of lysosomal proteases. Virtually all of the cellular-mediated degradation was blocked by RAP, and no degradation was detected in cell
lines deficient in LRP (Fig. 7D). These data confirm that LRP mediates the cellular uptake of MMP-9·TIMP-1 complexes, leading to their degradation.
In the current investigation, we have demonstrated that murine
fibroblasts cultured in the presence of RAP accumulate MMP-9 in their
media, indicating that a RAP-sensitive receptor contributes to the
catabolism of this molecule. In vitro binding studies and cellular uptake experiments confirm that this receptor is LRP. These
studies established that LRP binds MMP-9 specifically and with high
affinity. MMP-9 is secreted as a complex with its specific inhibitor,
TIMP-1, and both the MMP-9·TIMP-1 complex and free MMP-9 bind to LRP,
suggesting that a LRP binding determinant is primarily located within
the MMP-9 molecule. A role for LRP in mediating the cellular catabolism
of MMP-9 was confirmed by demonstrating that murine fibroblasts lacking
LRP are inefficient in mediating the cellular internalization and
degradation of MMP-9. Together, these data confirm that LRP is a
cellular receptor for MMP-9 and functions to regulate the levels of
MMP-9.
LRP is a member of the LDL receptor family of endocytic
receptors that also includes the LDL receptor, the VLDL
receptor, apoE receptor 2, gp330/megalin, and LRP-1b. Deletion
of the LRP gene in mice revealed an essential but undefined role for
this receptor during development (28). The biological activity of LRP
was initially characterized as a clearance receptor for chylomicron remnants (29) and complexes of In the case of MMP-9, tight regulation of its activity and levels are
likely to be important, since this MMP has important biological
properties. Thus, studies using mice deficient in MMP-9 have
demonstrated roles for this enzyme in embryonic bone development (5),
inflammation (36), and tumor progression and metastasis (37).
Interestingly, two recent studies (4, 7) that examine the role of MMP-9
in tumor development implicate secretion of the enzyme by macrophages,
which are also rich in LRP. These in vivo models indicate
that MMP-9 may function to proteolyze the extracellular matrix,
resulting in the release of growth factors such as vascular endothelial
growth factor, which are normally thought to be sequestered in this
matrix. Additionally, MMP-9 may also function to activate transforming
growth factor LRP is known to regulate the levels of another proteinase system
involving urokinase-type plasminogen activator (uPA) and its cellular
receptor, the uPA receptor. The ability of LRP to modulate uPA receptor
levels (41) and uPA activity (42) has been offered as a mechanistic
explanation to account for the role that LRP plays in cellular
migration and invasion. For example, in human umbilical vein smooth
muscle cells, both RAP (43, 44) and anti-LRP antibodies (43)
significantly inhibit their migration. In a different model employing
mouse embryonic fibroblasts, Weaver et al. (41)
observed that LRP-deficient cells migrated nearly twice as rapidly as
the LRP-expressing cells. The migration difference was only noted on
culture wells that were pre-coated with serum or vitronectin but not in
wells coated with type I collagen or Matrigel. In this study, the
contribution of LRP to cellular migration was attributed to its effect
on the activity of the uPA/uPA receptor system (41). In light of the
findings of the current study that LRP is a receptor for MMP-9 and
therefore capable of regulating its extracellular levels, it will be
important to reevaluate the mechanism by which LRP impacts cellular
migration and invasion.
In summary, the studies presented in this manuscript demonstrate that
LRP is a functional receptor for MMP-9 and confirm that LRP is capable
of mediating the cellular uptake of at least three MMPs (MMP-2, MMP-13,
and MMP-9). These findings implicate a major role for LRP in modulating
excessive extracellular proteinase activity and, by doing so, in
regulating remodeling of the extracellular matrix. An investigation of
the implications of these interactions in vivo is ongoing.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
); PEA-13, LRP(
/
); mouse embryonic
fibroblasts, LRP(+/+)) (23) were grown in Dulbecco's modified Eagle's
medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. In the experiments involving RAP
treatment, subconfluent cells were cultured in the absence (control) or
presence of 500 nM recombinant RAP (RAP-treated) for
24 h in serum-free medium supplemented with
Nutridoma® -NS media supplement (Roche Molecular
Biochemicals). This 24 h-conditioned medium was then analyzed by
gelatin zymography to detect both MMP-2 and MMP-9.
where A is the absorbance at 405 nm,
Amax is the absorbance value at saturation,
Amin is the background absorbance in the absence
of ligand, [ligand] is the molar concentration of free ligand, and
KD is the dissociation constant. Since the free
ligand concentration is unknown in these experiments, the use of this
equation assumes that the total amount of added ligand is far greater
than the amount of ligand bound to the microtiter wells.
(Eq. 1)
/
)) were plated onto 12-well plates at ~1 × 105 cells/well and grown overnight at 37 °C, 5%
CO2. Cells were then washed twice with assay medium
(Dulbecco's modified Eagle's medium containing Nutridoma media
supplement; 20 mM HEPES, pH 7.4, and 1.5% BSA) and
incubated in this medium for 1 h at 37 °C. The cells were then
incubated with assay medium containing 5 nM
125I-MMP-9·TIMP-1 for the indicated times at 37 °C.
After incubation, the cells were washed three times with assay medium
and detached with 0.5 ml of trypsin/proteinase K (to dissociate
LRP·125I-MMP-9·TIMP-1 complexes from the cell surface)
and then pelletized by centrifugation (3000 rpm for 5 min). The amount
of internalized 125I-MMP-9·TIMP-1 complex was determined
by measuring the radioactivity associated with the cell pellet in a
counter. Nonspecific uptake was assessed by measuring
125I-MMP-9·TIMP-1 internalization in the presence of an
excess amount of the LRP antagonist RAP (1 µM).
Degradation was determined by measuring the radioactivity in the
trichloroacetic acid-soluble fraction of the culture supernatant in a
counter. In all experiments a control was included in which the
amount of degradation products generated in the absence of cells was
also measured.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) mouse embryonic
fibroblasts were cultured in serum-free medium for increasing lengths
of time in the absence or presence of RAP. RAP was first identified
when it was found to co-purify with LRP (20). Subsequent work (14, 17)
revealed that this molecule binds tightly to LRP and prevents binding
of all known ligands to this receptor. In the current experiments, the
conditioned media were collected and analyzed for the presence of
gelatinases by gelatin zymography (Fig.
1). The results indicate that LRP(+/
) cells treated with RAP contain similar amounts of 72-kDa MMP-2 when
compared with parallel cultures treated with BSA. However, the
RAP-treated cultures contain significantly more of the 105-kDa MMP-9 in
their media than the control-treated cells. The increased levels of
MMP-9 in the conditioned medium of RAP-treated cells suggest that LRP
regulates the levels of MMP-9 present in cell culture media.
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Fig. 1.
MMP-9 accumulates in conditioned media of
cells treated with RAP. LRP(+/ ) murine embryonic fibroblasts
were cultured in serum-free Dulbecco's modified Eagle's medium in the
absence or presence of 500 nM RAP for the times indicated.
After incubation, the conditioned medium was collected from each
culture, and the cells were counted. Aliquots of conditioned media (30 µl/106 cells) were analyzed for the presence of
gelatinases (MMP-2 and MMP-9) by gelatin zymography (top
panel). C, control treatment BSA (20 µg/ml).
Zymographs from two experiments were scanned and converted into TIFF
files, and the pixel density of the zones of clearing that correspond
to MMP-9 and MMP-2 was analyzed using NIH Image. The average pixel
densities for both the 105-kDa mouse MMP-9 and the 72-kDa MMP-2 bands
in the control-treated cells at 24 h were set to 1.0, and all
other densities were normalized to these values.
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Fig. 2.
Ligand blot analysis reveals that MMP-9 binds
LRP but not VLDLr1-8. Purified LRP (A, 2 µg) or
VLDLr1-8 (B, 3.5 µg) were subjected to SDS-PAGE under
nonreducing conditions and transferred to nitrocellulose. The blots
were stained by Ponceau S (lanes 1 and 4) and
incubated with human MMP-9·TIMP-1 (20 nM) overnight at
4 °C in the absence (lanes 2 and 5) or
presence (lane 3) of 500 nM RAP, as indicated.
The blots were washed and incubated for 2 h with an anti-MMP-9 IgG
(2 µg/ml). After further washing, the blots were incubated with a
goat anti-mouse IgG conjugated to horseradish peroxidase, and ligand
binding was visualized using chemiluminescence. sVLDLr,
soluble VLDLr.
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Fig. 3.
Binding of LRP to MMP-9 does not require
TIMP-1. RAP, MMP-9·TIMP-1 complexes, and MMP-2·TIMP-2
complexes were subjected to SDS-PAGE on 4-20% gradient gels under
either nonreducing (A, B, and C) or
reducing conditions (D). Ponceau S staining of the
nitrocellulose membrane after transfer is shown in panel A.
The blots were incubated overnight at 4 °C with LRP (10 nM) in the absence (B) or presence of 500 nM RAP (C) as indicated. Receptor binding was
detected using monoclonal anti-LRP antibody 8G1 and goat anti-mouse IgG
conjugated to horseradish peroxidase and visualized using
chemiluminescence.
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Fig. 4.
LRP binds to immobilized MMP-9·TIMP-1
complexes. Purified human MMP-9·TIMP-1 ( ), MMP-2·TIMP-2
(
), and BSA (
) were coated on microtiter wells at 4 µg/ml,
blocked with BSA, and then incubated overnight at 4 °C with
increasing concentrations of LRP. The amount of protein bound was
detected using monoclonal anti-LRP antibody 8G1 and goat anti-mouse IgG
conjugated to horseradish peroxidase. Each data point represents the
average of duplicate determinations. The solid curves
represent the best fit to Equation 1 determined by nonlinear
regression.
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Fig. 5.
Binding of MMP-9·TIMP-1 complexes to
immobilized LRP is inhibited by RAP. Microtiter wells were coated
with LRP ( ,
) or BSA (
) at 4 µg/ml. The wells were blocked
with BSA and then incubated overnight at 4 °C with increasing
concentrations of MMP-9 in the absence (
) or presence (
) of 2 µM RAP. The bound MMP-9 was detected using a monoclonal
anti-MMP-9 antibody and goat anti-mouse IgG conjugated to alkaline
phosphatase. Each data point represents the average of duplicate
determinations. The solid curve represents the best fit to
Equation 1 determined by nonlinear regression.
/
) cells were incubated with
125I-MMP-9·TIMP-1 complex for 6 h. After incubation,
the extent of radioactivity associated with the cell surface (Fig.
6A) or internalized (Fig.
6B) was measured. The results indicate that the
125I-MMP-9·TIMP-1 complex associates with the cell
surface and is internalized in LRP-expressing cells, and the amount of
125I-MMP-9·TIMP-1 complex associated with the cell
surface or internalized was greatly reduced when the cells were
incubated with excess RAP. In contrast, very little
125I-MMP-9·TIMP-1 complexes were associated with the cell
surface or internalized in cells deficient in LRP. Interestingly, a
small amount of 125I-MMP-9·TIMP-1 internalization was
observed in LRP-deficient cells that were inhibited by RAP (Fig.
6B), suggesting that another as yet unidentified member of
the LDL receptor family may also recognize the MMP-9·TIMP-1
complex.
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Fig. 6.
Cellular-mediated uptake of
125I-labeled MMP-9·TIMP-1 complexes reguire LRP.
LRP(+/+) and LRP( /
) mouse embryonic fibroblasts were plated into
culture wells (1 × 105 cells/well).
125I-Labeled-MMP-9·TIMP-1 complexes (5 nM)
were added in the absence (solid bars) or presence
(open bars) of RAP (1 µM). After 6 h, the
radioactivity associated with the cell surface (A) and
internalized (B) was determined as described under
"Experimental Procedures." Each bar represents the
average of triplicate determinations.
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Fig. 7.
Time course of internalization and
degradation of 125I-labeled-MMP-9 by LRP(+/+)
and LRP( /
) cells. LRP(+/+) (A and
C) and LRP(
/
) mouse embryonic fibroblasts (B
and D) were plated into wells (1 × 105
cells/well), and 125I-labeled-MMP-9·TIMP-1 complex (5 nM) was added to each well in the absence
(circles) or presence of 1 µM RAP
(triangles). At the indicated times, the extent of
internalization (A and B) and degradation
(C and D) were determined as described under
"Experimental Procedures." In panels C and D,
100 mM chloroquine (squares) was employed to
determine cellular-mediated degradation. Each point represents the
average of triplicate determinations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-macroglobulin with
proteinases (22). Subsequent work has revealed that this receptor
recognizes several classes of ligands, including serine proteinases
(30, 31), proteinase-inhibitor complexes (32, 33), and the
matricellular proteins TSP1 (26) and TSP2 (34). The ability of LRP to
bind MMP-9 now reveals that this receptor plays a major role in
regulating levels of certain MMP family members as well. Barmina
et al. (12) demonstrate that the catabolism of MMP-13
(collagenase-3) was found to involve two receptors: a specific
collagenase-3 receptor that acts as a primary binding site on cells,
and LRP, which is required for internalization of this enzyme. Yang
et al. (35) found an adhesive defect in dermal fibroblasts
derived from TSP2-null mice that results from accumulation of MMP-2 in
the cell media. Anti-LRP IgG and RAP both inhibited adhesion and
increased MMP-2 levels in conditioned media from wild type, but not
TSP2-null cells, confirming that LRP is also involved in catabolism of
MMP-2 as well (13). Since the clearance of MMP-2 was shown to be
TSP2-dependent, it was suggested that clearance of
MMP-2·TSP2 complexes by LRP is an important mechanism for the
regulation of MMP-2 levels (13). The ability of LRP to modulate three
MMPs (MMP-2, MMP-13, and MMP-9) indicates a major role for this
receptor in removing excessive extracellular proteolytic activity.
(38). Both of these molecules are able to promote
angiogenesis and tumor progression. Curiously, LRP levels and activity
are known to be substantially decreased in tumors (39, 40), which would
decrease catabolism of MMP-9, leading to higher levels of this enzyme
at the tumor site. Of interest in this regard, Kancha et al.
(40) investigated the expression of LRP in invasive and noninvasive subclones derived from tumor cells. They observed a 2-3-fold decrease in LRP activity in invasive subclones compared with noninvasive subclones derived from human prostate PC-3 and DU 145 and melanoma A2058 cells. This study suggested a correlation between invasive phenotype and low LRP expression in different tumor cells.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HL50784, HL54710, and AR 45418.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Vascular
Biology, Holland Laboratory, American Red Cross, 15601 Crabbs Branch
Way, Rockville, MD 20855. Tel.: 301-738-0726; Fax: 301-738-0465; E-mail: strickla@usa.redcross.org.
Published, JBC Papers in Press, February 2, 2001, DOI 10.1074/jbc.M100121200
2 Z. Yang and P. Bornstein, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are: MMP, matrix metalloproteinase; BSA, bovine serum albumin; LDL, low density lipoprotein; LRP, LDL receptor-related protein; RAP, receptor-associated protein; TIMP, tissue inhibitor of metalloproteinase; TSP, thrombospondin; VLDL, very low density lipoprotein; VLDLr, VLDL receptor; uPA, urokinase-type plasminogen activator; TBS, Tris-buffered saline; PAGE, polyacrylamide gel electrophoresis.
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