From the Department of Biochemistry, the University of Washington,
Seattle, Washington 98195 and the Department of
Vascular Biology, American Red Cross, Rockville, Maryland 20855
Received for publication, September 29, 2000, and in revised form, December 6, 2000
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ABSTRACT |
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We have recently shown that the adhesive defect
observed in dermal fibroblasts derived from thrombospondin 2 (TSP2)-null mice results from an increase in matrix metalloproteinase 2 (MMP2) levels (Yang, Z., Kyriakides, T. R., and Bornstein, P. (2000) Mol. Biol. Cell 11, 3353-3364). Adhesion was
restored by replacement of TSP2 and by inhibitors of MMP2 activity. In
pursuing the observation that TSP2 and MMP2 interact, we now
demonstrate that this interaction is required for optimal clearance of
extracellular MMP2 by fibroblasts. Since TSP2 is known to be
endocytosed by the scavenger receptor, low density lipoprotein
receptor-related protein (LRP), we determined whether interference with
LRP function affected fibroblast adhesion and/or extracellular MMP2
levels. Addition of heparin, which competes for the binding of TSP2 to
LRP coreceptor proteoglycans, inhibited adhesion of control but not
TSP2-null cells, and a blocking antibody to LRP as well as the LRP
inhibitor, receptor-associated protein, also inhibited adhesion and
increased MMP2 levels only in control fibroblasts. TSP2 did not inhibit
active MMP2 directly and did not inhibit the activation of
pro-MMP2. Finally, the internalization of 125I-MMP2
was reduced in TSP2-null compared with control fibroblasts. We propose
that clearance of MMP2-TSP2 complexes by LRP is an important
mechanism for the regulation of extracellular MMP2 levels in
fibroblasts, and perhaps in other cells. Thus, some features of the
phenotype of TSP2-null mice, such as abnormal collagen fibrillogenesis,
accelerated wound healing, and increased angiogenesis, could result in
part from increased MMP2 activity.
Thrombospondins (TSP)1 1 and 2 are large extracellular macromolecules whose diverse functions
reflect their ability to bind to multiple cell-surface receptors,
cytokines, growth factors, and proteases, and to structural components
of the matrix (1, 2). TSP2-null mice display a complex phenotype that
is characterized by changes in connective tissues, particularly in
response to injury, an increase in vascular density and endosteal bone
growth, and a bleeding defect (3-5). Dermal fibroblasts, isolated from adult animals, also show an adhesive defect in vitro that is
most evident when cells are plated on a variety of pure protein
substrates in the absence of serum. Adhesion was restored by prolonged
(48 h) incubation of TSP2-null cells with recombinant mouse TSP2 or by
transfection with a TSP2 cDNA gene (6). The basis for this adhesive
defect was recently investigated and was shown by zymography to result
from an increase in matrix metalloproteinase 2 (MMP2) levels in both
the conditioned media and cell layers of cultured cells (6). Although
virtually all of the enzyme that was analyzed was in the zymogen or
pro-MMP2 form, an increase in active MMP2 in TSP2-null cells was
inferred from the observation that inhibitors of MMP2, including TIMP2
and a neutralizing anti-MMP2 antibody, corrected the adhesive defect
(6).
Both TSP2 and its close relative, TSP1, are known to interact with the
scavenger receptor, low density lipoprotein-related receptor protein
(LRP), an interaction that results in the endocytosis and lysosomal
degradation of the TSP (7-11). This interaction is mediated by the
NH2-terminal heparin-binding domain of the TSP and is
competed by heparin (7, 9-11). TSP1 and TSP2 also interact with a
number of serine proteases, including plasmin, cathepsin G, and
neutrophil elastase, and function as competitive inhibitors of these
enzymes (12). In view of the fact that LRP is capable of binding and
endocytosing both In this study we find that although TSP2 binds both pro-MMP2 and MMP2
directly, the protein does not function as a direct binding inhibitor
of the active protease nor does it prevent the activation of pro-MMP2.
However, TSP2-null cells were defective in the uptake of extracellular
MMP2. Furthermore, inhibitors of LRP function reduced adhesion of
control skin fibroblasts and, correspondingly, increased MMP2 levels in
these cells. We therefore propose that the interaction of MMP2 with
TSP2, and possibly also with TSP1, and the subsequent uptake of the
protein-enzyme complex by LRP serve as a means of regulating
extracellular MMP2 levels. These results have implications not only for
the phenotype of the TSP2-null mouse but also for the control of
processes such as collagen fibrillogenesis, wound healing, and angiogenesis.
Enzyme-linked Immunosorbent Assay--
Recombinant mouse
full-length TSP2 was produced in insect cells and purified as described
previously (14). Human TSP1 was purchased from Hematologic Technologies
(Essex Junction, VT). Human MMP2 was a kind gift of Dr. Christopher
Overall (University of British Columbia, Canada) or was purchased from
Chemicon (Temecula, CA). Rabbit anti-MMP2 polyclonal antibody was
purchased from Chemicon. For the direct-binding enzyme-linked
immunosorbent plate assay, 96-well microtiter plates (Linbro®, Flow
Laboratories, McLean, VA) were coated with TSP1, TSP2, gelatin
(Fisher), and asialofetuin (Sigma) at 10 µg/ml, 50 µl/well in 0.1 M Tris-HCl, pH 7.2, at 4 °C overnight. The plates were
blocked with 1% bovine serum albumin in the same buffer containing 5 mM CaCl2 for 1 h at room temperature. MMP2
(4 µg/ml) in blocking solution was then added to wells for 2 h.
Subsequently, rabbit anti-MMP2 antibody (1 µg/ml) was added for
another 2 h, followed by alkaline phosphatase-conjugated goat anti-rabbit IgG (Sigma). Color was developed with
p-nitrophenyl phosphate substrate (1 mg/ml) in 10 mM diethanolamine, pH 9.5, containing 0.5 mM
MgCl2. Between each incubation, the wells were washed with
Tris-HCl buffer to remove unbound protein.
A405 was measured in a microplate reader
with SOFTmax®PRO software (Molecular Devices, Sunnyvale, CA). Each
determination represents the average of four wells.
Gelatinolytic Assay--
MMP2 activity was determined by a
gelatinolytic assay with soluble gelatin as a substrate (15). The
gelatin was radiolabeled with [3H]acetic acid according
to Cawston and Barrett (16). Two µg of human MMP2 was incubated in
100 µl of reaction buffer containing 50 mM Tris-HCl, pH
7.5, 50 mM NaCl, 5 mM CaCl2, and
0.01% Brij-35 at 37 °C for 2 h, with or without activation by
1 mM 4-aminophenylmercuric acetate (APMA). In experiments
that tested the ability of TSP2 to inhibit MMP2, TSP2 was added in a
chain molar ratio of 1 to 1 with respect to MMP2. Fifty µg of
radiolabeled gelatin was heat-denatured at 60 °C for 15 min, cooled
to 37 °C, and added to the incubation mixture in a final volume of
200 µl. The reaction was allowed to proceed for 24, 48, 72, and
96 h at 37 °C in the presence of 0.03% toluene to prevent
bacterial contamination. Undegraded gelatin was precipitated at 4 °C
with a mixture of 4% trichloroacetic acid and 0.8% tannic acid. The
reaction mixture was centrifuged at 10,000 × g for 15 min at 4 °C, and aliquots of the resulting supernatants were counted
for radioactivity in a liquid scintillation counter.
Cell Culture--
Dermal fibroblasts were isolated by
collagenase treatment of skin taken from the backs of adult
(2-3-month-old) mice. Each cell preparation was derived from an
individual mouse. Briefly, after removal of hair and subcutaneous
tissues, skin was treated with 0.25% trypsin and antibiotics in a
calcium- and magnesium-free solution. After overnight incubation at
4 °C, the epidermis was stripped off the dermis by holding the
dermis with forceps and gently scraping off the epidermis with a
scalpel. The isolated dermis was then digested in 0.25% bacterial
collagenase (Sigma) in DMEM containing 0.02% CaCl2 and
0.01% MgSO4 at 37 °C for 1-2 h until the cells were
completely dissociated from the digested tissue. Cells derived from a
2-cm2 segment of skin were collected by centrifugation and
washed twice with DMEM, supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin G, and
100 µg/ml streptomycin before plating on a 100-mm tissue culture dish
in the same medium. Unattached cells and debris were removed, and the
medium was replaced after 2 days of plating. Cells were passaged at
confluence. After 2-3 passages, the cell population appeared, by light
microscopy, to be composed entirely of fibroblasts.
Iodination and Uptake of MMP2 by Fibroblasts--
MMP2 was
labeled with 125I using IODO-BEADS (Pierce) according the
manufacturer's protocol. Briefly, 1 mCi of Na125I
(Amersham Pharmacia Biotech) was incubated with IODO-BEADS for 5 min in
50 mM sodium phosphate buffer, pH 6.5, and then 20 µg of
MMP2 was added to a final volume of 200 µl. After 10 min of incubation, the reaction supernatant was removed and added to another
tube containing 0.5 ml of 0.1% bovine serum albumin as a carrier
protein. The IODO-BEADS were washed twice with 150 µl of reaction
buffer, and the pooled iodinated protein was gel-filtered on a PD-10
column (Amersham Pharmacia Biotech) to remove free 125I.
Fibroblast monolayers in 12-well tissue culture plates were cultured in
serum-free medium for 1 h, and then 30 nM of
125I-MMP2 was added (0.5 ml/well).
125I-MMP2-containing media were removed hourly over a 5-h
period, and the cells were washed three times with DMEM, dissolved in 0.5 ml of 0.1 N NaOH, and neutralized with 30 µl of 10%
glacial acetic acid. In a separate experiment, fibroblasts in 6-well
tissue culture plates were incubated in serum-free medium containing 30 nM of 125I-MMP2 as described above. After a 5-h
incubation, cells were washed three times with DMEM and then either
dissolved in NaOH and neutralized or treated with 0.25% trypsin at
37 °C for 10 min. The cells were then washed twice with DMEM,
dissolved in NaOH, and neutralized with acetic acid. Aliquots of the
resulting lysates were counted for radioactivity in a liquid
scintillation counter to measure the internalization of
125I-MMP2 by fibroblasts.
Cell Attachment Assays and Zymography--
Analyses for
attachment of fibroblasts on fibronectin-coated 96-well plates were
performed as described previously (6). Fibronectin was coated at a
concentration of 5 µg/ml in phosphate-buffered saline. Dermal
fibroblasts were incubated in cell culture medium containing heparin (5 µM), anti-LRP IgG (50 µg/ml), or RAP (1 µM) for 48 h prior to analysis. The polyclonal
rabbit anti-LRP functional blocking antibody (17) was affinity-purified
(10), and recombinant human RAP, expressed in bacteria as a fusion
protein with glutathione S-transferase, was prepared and
purified as described previously (18). Normal rabbit IgG was purchased
from Vector Laboratories (Burlingame, CA) and was used as a control.
Zymography of conditioned media was performed as described by Yang
et al. (6).
Western Blotting--
Serum-free conditioned media or lysates of
mouse skin fibroblasts were subjected to SDS-PAGE in 7.5 or 4-15%
gradient gels. A buffer composed of 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholate, and
0.1% SDS was used to extract cellular proteins from fibroblasts.
Separated proteins were transferred electrophoretically (19) from
polyacrylamide gels to nitrocellulose membranes in a mini trans-blot
cell (Bio-Rad) for 1 h at 100 V, followed by Western blot analysis
with anti-LRP IgG or anti-TSP2 polyclonal antibody (14). The resulting
antigen-antibody complexes were detected by incubation with alkaline
phosphatase-conjugated antibody (Sigma) and alkaline phosphatase
substrate (Bio-Rad) for determination of LRP expression. Incubation
with horseradish peroxidase-linked antibody (New England Biolabs,
Beverly, MA) and enhanced luminol reagent (PerkinElmer Life Sciences)
and exposure to x-ray film were used to determine TSP2 levels.
TSP2 Does Not Inhibit MMP2 Activity or the Activation of Pro-MMP2
by APMA--
We recently reported that TSP2-null fibroblasts have a
marked adhesive defect when plated on a number of protein substrates, and we showed that this defect resulted from a 2-fold increase in MMP2
protein in the conditioned media of the cells (6). The adhesive defect
was corrected by inhibitors of MMP2, by transfection of a full-length
TSP2 cDNA gene into the cells, or by long term preincubation with
recombinant TSP2 but not by adsorption of TSP2 to tissue culture
plastic. Furthermore, we and others (6, 20) have found that TSP2
interacts directly with pro-MMP2. TSP2 could modulate MMP2 activity in
a number of ways. TSP2 could function as a direct binding inhibitor of
MMP2, as has been shown for the binding of both TSP2 and TSP1 to a
number of serine proteases (12). TSP2 could also serve to sequester
MMP2 in the extracellular matrix and reduce its bioavailability since
TSP1, and probably TSP2 in view of its very similar sequence, bind to
fibronectin and to a number of collagens and proteoglycans (2, 21).
This explanation seems unlikely in our cell culture system since not only MMP2 activity, but also protein, was increased in the absence of
TSP2 (6). Finally, TSP2 could facilitate the clearance of TSP2 from the
pericellular environment.
As shown in Fig. 1, both the zymogen,
pro-MMP2, and APMA-activated MMP2 bound equally well to TSP1 and TSP2
in a direct binding solid phase assay. The extent of binding was only
about 60% that to gelatin, but this difference is in keeping with a
postulated role of TSP2 as a modulator of MMP2 activity rather than as
a substrate for the enzyme. In preliminary experiments, the
Kd value for binding of pro-MMP2 to TSP2 was found
to be in the micromolar range.2 However, as revealed
by a gelatinolytic assay with soluble [3H]gelatin as a
substrate, the binding of TSP2 to pro-MMP2 did not inhibit its
activation by APMA (Fig. 2). Furthermore,
TSP2 did not inhibit the activity of APMA-activated MMP2. Fig. 2 also shows that the small amount of activity associated with preparations of
pro-MMP2 was not inhibited by TSP2.
Heparin Inhibits the Attachment of Control but Not TSP2-null
Fibroblasts--
The accumulation of MMP2 protein in the conditioned
media of TSP2-null cells argues strongly for a role for TSP2 in
clearance of MMP2 from the pericellular environment of fibroblasts.
Both TSP1 and TSP2 are known to be endocytosed and catabolized by the LRP scavenger receptor (7-11). This process probably involves heparan
sulfate proteoglycans as coreceptors since cellular uptake of the
heparin-binding domain of TSP1 was inhibited by heparin, and
degradation of this domain was reduced in Chinese hamster ovary cells
that lacked heparan sulfate glycosaminoglycans (8). We hypothesized
that TSP2-MMP2 complexes might also be bound and endocytosed by the LRP
receptor. If so, the addition of heparin to wild-type cells in culture
should lead to reduced adhesion, whereas a much smaller effect should
be seen with TSP2-null cells. We therefore added heparin at 5 µM to the culture media of dermal fibroblasts 48 h
prior to determination of their attachment properties. As shown in Fig.
3, treatment of wild-type fibroblasts
decreased their attachment to fibronectin by 57% and led to a level of
attachment that equaled that of TSP2-null cells, whereas heparin
reduced the attachment of TSP2-null cells by only 15%. These findings, together with the known ability of TSP2 to interact with LRP and MMP2,
suggest that TSP2 could play a major role in regulating MMP2 levels and
adhesion in mouse dermal fibroblasts.
Anti-LRP Antibodies and RAP Reduce Attachment and Increase MMP2 and
TSP2 Levels in Control Mouse Dermal Fibroblasts--
LRP was first
identified as a homologue of the low density lipoprotein receptor (22)
and is now recognized as a member of a family of at least six
homologous mammalian receptors that include the low density lipoprotein
receptor, LRP, LRP-DIT, gp330 (megalin), very low density lipoprotein
receptor, and apoER-2. It was subsequently shown that LRP is identical
to a receptor that had been described as responsible for the
internalization of
As a more stringent test of our hypothesis that the uptake of TSP2-MMP2
complexes by LRP serves as a means of regulating extracellular MMP2
levels, we treated dermal fibroblasts with a polyclonal rabbit anti-LRP
functional blocking antibody or with the LRP inhibitor, RAP. RAP is a
39-kDa protein that is located in the endoplasmic reticulum but binds
and antagonizes the function of LRP and other members of the low
density lipoprotein receptor family (13). Since RAP and LRP are found
in different cellular compartments, its mode of action is unclear.
Willnow et al. (28) have proposed that RAP functions as a
chaperone to prevent ligand-induced aggregation and degradation of LRP
in the endoplasmic reticulum. In any event, extracellularly
administered RAP has been found to be effective in inhibiting the
internalization and degradation of both intact TSP1 and its
heparin-binding domain (7, 8, 11). Treatment of fibroblasts with
anti-LRP IgG at 50 µg/ml or RAP at 1 µM significantly decreased the attachment of wild-type cells to fibronectin to levels
that are close to those of TSP2-null cells, but had essentially no
effect on the attachment of TSP2-null cells (Fig.
5). These results are consistent with
those of heparin treatment (Fig. 3). As predicted by our postulate that
anti-LRP or RAP inhibited the binding and internalization of a
TSP2-MMP2 complex, zymography of serum-free conditioned media from
dermal fibroblasts demonstrated that treatment with these agents
significantly increased MMP2 levels in wild-type but not in TSP2-null
cells (Fig. 6). Analysis of the zymogram
in Fig. 6 indicated that 79 and 76% of the difference in MMP2 levels
between wild-type and TSP2-null cells were restored by anti-LRP and
RAP, respectively. Furthermore, SDS-PAGE and Western blot analysis of
conditioned media revealed that the extracellular level of TSP2 in
wild-type cells increased significantly after treatment with anti-LRP
IgG or RAP (Fig. 7). Since tissue
inhibitor of metalloproteinases 2 (TIMP2) levels, as determined by
Western blot analysis, did not differ appreciably between wild-type and TSP2-null cells,3 these
experiments establish the TSP2/LRP system as a major regulator of
extracellular MMP2 levels and activity.
There is precedence for the involvement of LRP in the cellular uptake
of matrix metalloproteinases. Recently, Barmina et al. (29)
reported that collagenase-3 (MMP13) bound to a receptor, tentatively
identified as being identical to a member of the mannose-receptor type
C lectin family and that the internalization of the enzyme-receptor complex was in turn mediated by LRP. To determine whether MMP2 could
bind directly to LRP, solid phase assays were employed using purified
reagents. These experiments revealed that MMP2 was not able to bind
with high affinity to LRP-coated microtiter
wells4 and confirmed that
MMP2 does not interact directly with LRP.
Internalization of MMP2 by Dermal Fibroblasts Is Reduced in
TSP2-null Cells--
As a final test of our hypothesis, we compared
the uptake of exogenous MMP2 by wild-type and TSP2-null dermal
fibroblasts. Iodinated MMP2 was incubated with mouse dermal fibroblasts
in serum-free medium for 5 h. The cellular uptake of
125I-MMP2 was then measured by liquid scintillation
counting of cell lysates after removal of media containing unbound
125I-MMP2. As shown in Fig.
8A, the association of
125I-MMP2 with TSP2-null fibroblasts was significantly
lower than that for control cells at each time point of the experiment
(p < 0.01). At the end of the 5-h incubation period,
the association of 125I-MMP2 with cells lacking TSP2 was
only 67% that for wild-type cells.
As a control to determine what fraction of cell-associated MMP2 was
internalized, cells were incubated for 5 h with
125I-MMP2 and then treated with trypsin prior to counting.
As shown in Fig. 8B, 55% of the radioactivity associated
with wild-type cells in monolayer culture, and 39% of that associated
with TSP2-null cells, was retained after trypsinization. The
125I-MMP2 released by trypsin presumably includes both
surface-bound MMP2 and enzyme that was trapped nonspecifically in the
monolayer. Nevertheless, the reduction in internalization of MMP2 by
TSP2-null cells, compared with controls, is clearly preserved after
trypsinization. In this experiment the uptake of MMP2 by TSP2-null
cells was only 48% of controls, a greater difference than that seen at
5 h in Fig. 8A. In unpublished experiments3
we have also shown that prior incubation of wild-type cells with 1 µM RAP reduces uptake of MMP2 to the same extent as
incubation with heparin, as would be predicted if LRP serves as the
major mechanism for internalization of TSP2-MMP2 complexes. The basis for the non-TSP2-mediated uptake of MMP2 is not known. It is not likely
to result from binding to TSP1 and LRP-mediated endocytosis of
TSP1-MMP2 complexes since the attachment of TSP2-null cells is not
reduced much further by prior addition of heparin to the culture medium
(Fig. 3).
Bein and Simons (20) have reported recently that human TSP1 does not
directly inhibit the degradation of type IV collagen by MMP2 in an
in vitro assay. These authors also showed that TSP1 inhibited the activation of pro-MMP9 by MMP3 in vitro.
However, their conclusion, based on cell culture experiments with
bovine aortic endothelial cells, that TSP1 inhibits the activation of pro-MMP2 is open to the alternative explanation that TSP1 increased the
clearance of both pro-MMP2 and active MMP2 by the very low density
lipoprotein receptor in these endothelial cells. Furthermore, a role
for TSPs in inhibition of pro-MMP2 activation is not easily reconcilable with an increase in not only MMP2 activity but also MMP2
protein, as shown by Yang et al. (6) in TSP2-null cells.
Implications of Increased MMP2 Levels in Fibroblasts for the
Phenotype of the TSP2-null Mouse--
A number of features of the
TSP2-null mouse, including abnormal collagen fibrillogenesis and
increased angiogenesis in skin and in subcutaneous tissues in response
to injury, remain unexplained. In recent experiments we have found
that, despite the ready demonstration that TSP2-null dermal fibroblasts
in culture accumulate increased levels of MMP2 (see Ref. 6 and this
study), analyses of extracts of uninjured dermis by gelatin zymography
failed to show a difference in MMP2 levels between normal and TSP2-null
tissue.3 This apparent discrepancy can be explained by the
fact that the TSP2 content of uninjured adult mouse skin is very low,
at least by the criterion of immunohistochemistry (14). These findings are also in accord with increasing evidence that matricellular proteins
such as TSP2 are expressed predominantly during development and growth
and in response to injury (2). Indeed, cells in culture in
serum-containing medium, which synthesize considerable amounts of TSP2,
display many aspects of a reaction to injury, a phenomenon that has
been termed "culture shock" (30). Recently our laboratory has shown
that, in contrast to normal dermis, the MMP2 content of the provisional
matrix or granulation tissue formed in a healing excisional skin wound
is markedly increased.5
Much of the newly formed TSP2 in a healing wound is associated with
collagen fibers. It is therefore possible that TSP2 does play a role,
either directly or indirectly, in normal collagen fibrillogenesis and
that the abnormally sized and shaped dermal and tendon collagen fibrils
seen by electron microscopy in the TSP2-null mouse (3) reflect the
absence of TSP2 and consequent increase in MMP2 during this critical
period of morphogenesis. More recent, albeit circumstantial, evidence
for this view comes from electron microscopic examination of 4- and
8-day postnatal mouse hindlimb flexor tendons. It was found that the
tendon fibroblast processes that delimit developing collagen fibril
bundles in the growing tendon were less regular and not as closely
apposed to the collagen fibrils in TSP2-null as compared with normal
tissues (31). Although by no means diagnostic, such changes are
compatible with alterations in MMP activity.
There is a substantial literature that documents a role for MMP2 in
angiogenesis (32) and in tumor growth and metastasis (33). A direct
demonstration is seen in MMP2 knockout mice that manifest reduced
angiogenesis and tumor progression (34). It is therefore possible that
the increased dermal vascular density observed in TSP2-null mice
results, in part, from increased MMP2 levels. Since LRP has not been
found in umbilical vein endothelial cells (7) and may not exist in
other endothelial cells, TSP2-MMP2 complexes may be bound and
internalized by another member of the LRP receptor family, notably the
very low density lipoprotein receptor that has been documented to bind
and internalize TSP1 (11). In tissues such as skin, endothelial cells
may also be influenced by the paracrine effects of fibroblasts and
pericytes.6
Conclusions and Directions for Future Research--
The LRP
scavenger receptor has been implicated in the binding and
internalization of a number of protein inhibitor-enzyme complexes of
the fibrinolytic pathway (13, 35), in the cellular uptake of
chylomicron remnants (36), and Pseudomonas toxin A (37), in
the regulation of cell surface levels of the urokinase receptor and
tissue factor (38, 39), and in the processing of the
A major issue that requires additional study is the means by which
increased MMP2 activity reduces the adhesion of fibroblasts, and
possibly other cells, in vitro and perhaps in
vivo. A common explanation that is given is that MMP2 is capable
of degrading many of the extracellular proteins to which cells attach,
including fibronectin and several collagens (41). However, this
explanation may not provide the whole answer since reduced attachment
of TSP2-null cells can be documented in assays that are performed for
1 h in the absence of serum, conditions that are not likely to
support the deposition of a substantial matrix. Another possibility is that increased MMP2 activity may alter the cellular surface, perhaps by
proteolysis of adhesion receptors, as suggested by Ray and Stetler-Stevenson (42). Finally, it is not excluded that MMP2 could
interact with cell-surface receptors and generate changes in
intracellular signaling that in turn compromise focal adhesions. These
possibilities are currently under study in our laboratories.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
2-macroglobulin proteinase and
plasminogen activator inhibitor 1-tissue plasminogen activator and -urokinase-type plasminogen activator complexes (13), we postulated
that TSP2-MMP2 complexes might also be cleared by the scavenger receptor.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Both pro-MMP2 and activated MMP2 bind to
TSP2. The interaction of MMP2 with substrate-bound proteins was
determined in a direct binding solid phase assay. Pro-MMP2 and
APMA-activated MMP2 bound equally well to TSP2 and TSP1, but the extent
of binding was only about 60% that to gelatin. Binding to asialofetuin
served as a negative control. This experiment is representative of
three independent experiments.
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Fig. 2.
TSP2 neither inhibits the activation of
pro-MMP2 by APMA nor the activity of active MMP2. Pro-MMP2
activity, with or without activation by 1 mM APMA, was
determined in a gelatinolytic assay using soluble
[3H]gelatin as a substrate. Preincubation of pro-MMP2
with TSP2 did not inhibit its activation by APMA: compare pro-MMP2/TSP2 + APMA with MMP2/APMA. Also, the gelatinolytic activity of activated
MMP2 was not affected by TSP2: compare MMP2/APMA + TSP2 with
MMP2/APMA.
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Fig. 3.
Effect of heparin on cell attachment of mouse
dermal fibroblasts. Wild-type and TSP2-null mouse dermal
fibroblasts were incubated in the presence of 5 µM
heparin for 48 h prior to determination of attachment on
fibronectin. Treatment of fibroblasts with heparin decreased the
attachment of wild-type cells to the level of that of TSP2-null cells
but had a much smaller effect on TSP-null cells. This experiment is
representative of three experiments with cells derived from different
individual mice.
2-macroglobulin-proteinase complexes
(23, 24). LRP is synthesized as a single chain that is cleaved to form
515- and 85-kDa subunits (25). The distribution of LRP in human tissues
and its subcellular location in human keratinocytes and fibroblasts has
been described (26, 27). Although LRP is known to be expressed in
embryonic mouse fibroblasts (17) it has not, to our knowledge, been
reported in adult mouse dermal fibroblasts. Cell lysates of dermal
fibroblasts were therefore subjected to SDS-PAGE and Western blot
analysis with an anti-LRP polyclonal antibody. As shown in Fig.
4, two specific components with molecular
masses of 515 and 85 kDa, corresponding to
- and
-subunits of
LRP, respectively, were detected in dermal fibroblasts. It can be seen
that the levels of LRP protein, as judged by Western blot analysis, are
at least as high in TSP2-null fibroblasts as in wild-type cells. Thus a
defect in internalization of TSP2-MMP2 complexes would not appear to
result from a deficiency of LRP.
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Fig. 4.
LRP expression in TSP2 mouse dermal
fibroblasts. Equal amounts of dermal fibroblast lysate proteins,
obtained directly from cell monolayers or from trypsinized cells, were
subjected to SDS-PAGE in a 4-15% gradient gel and Western blot
analysis with polyclonal anti-LRP antibody. Two specific components
with molecular masses of 515 and 85 kDa corresponding to the - and
-subunits of LRP, respectively, were detected in dermal fibroblasts.
The expression of LRP in TSP2-null fibroblasts was at least equal to
that in wild-type cells. Molecular masses were estimated from
concurrently run molecular mass standards.
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Fig. 5.
Effect of anti-LRP and RAP on attachment of
mouse dermal fibroblasts. Wild-type and TSP2-null mouse dermal
fibroblasts were incubated with polyclonal anti-LRP rabbit IgG (50 µg/ml) or RAP (1 µM) in cell culture media for 48 h prior to the attachment assay. Treatment of cells with anti-LRP or
RAP reduced the attachment of wild-type cells to fibronectin to the
level of TSP2-null cells but had no significant effect on attachment of
TSP /
cells. This experiment is representative of three independent
experiments.
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Fig. 6.
Zymography of conditioned media from mouse
skin fibroblasts treated with anti-LRP or RAP. Mouse skin
fibroblasts were cultured in the 10% serum-containing medium for
48 h in the presence of anti-LRP rabbit IgG (50 µg/ml) or RAP (1 µM). The medium was then changed to serum-free medium
containing the same reagents and culture resumed for another 20-24 h.
Equal amounts of conditioned medium protein were then applied to
SDS-PAGE, 0.1% gelatin under nonreducing conditions. The gelatinolytic
activity of MMP2 was significantly increased in media from wild-type
fibroblasts treated with anti-LRP IgG or RAP and was almost equal to
that of TSP2-null fibroblasts. Treatment of TSP2-null fibroblasts did
not have a significant effect on the activity of MMP2. This experiment
was performed twice with very similar results.
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Fig. 7.
TSP2 levels in conditioned media of dermal
fibroblasts treated with anti-LRP IgG or RAP. Equal amounts of
protein from conditioned media of dermal fibroblasts treated with
anti-LRP IgG or RAP were subjected to SDS-PAGE and Western blot
analysis with anti-TSP2 antibody. The levels of TSP2 in wild-type
conditioned media were significantly increased after cells were treated
with these agents. As expected, no TSP2 was detected in TSP2-null
cells.
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Fig. 8.
Uptake of 125I-MMP2 by mouse skin
fibroblasts. A, mouse skin fibroblasts were incubated with
30 nM 125I-MMP2 in serum-free media for periods
up to 5 h. At each time point, media containing unbound
125I-MMP2 were removed; aliquots of cell lysates were
measured by scintillation counting, and the results were normalized to
cell protein. The association of 125I-MMP2 with TSP2-null
fibroblasts was significantly lower than that for wild-type cells. Each
data point represents the average of four determinations. For most time
points the error bars do not extend beyond the symbols on
the graph. This experiment was performed twice with very similar
results. B, after a 5-h incubation, cell monolayers were
either lysed directly after washing or treated with 0.25% trypsin to
remove noninternalized 125I-MMP2, and the cells were lysed
for scintillation counting. The internalization of
125I-MMP2 was substantially reduced in TSP2-null cells, as
compared with that in controls.
-amyloid
precursor protein (40). Our study and that of Barmina et al.
(29) now implicate LRP, directly or indirectly, in the clearance of
MMP2 and MMP13 from the pericellular environment. It seems likely that
extracellular levels of other members of the MMP family will also be
regulated in a similar fashion, either by LRP or by other members of
the LRP family. Internalization and lysosomal degradation of
protein-MMP complexes represent a definitive mechanism for disposal of
excessive extracellular proteolytic activity, a function that cannot be
provided by the noncovalent, albeit high affinity, interaction between
MMPs and TIMPs.
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ACKNOWLEDGEMENT |
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We thank members of our laboratories for helpful discussions and a careful reading of the manuscript.
![]() |
FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AR 45418 and HL 50784.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 Biochemistry, Box 357350, University of Washington, Seattle, WA 98195. Tel.: 206-543-1789; Fax: 206-685-4426; E-mail: bornsten@u.washington.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M008925200
2 C. Overall, Z. Yang, and P. Bornstein, unpublished data.
3 Z. Yang, D. K. Strickland, and P. Bornstein, unpublished data.
4 E. A. Hahn-Dantona and D. K. Strickland, unpublished data.
5 T. R. Kyriakides and P. Bornstein, unpublished data.
6 L. C. Armstrong and P. Bornstein, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: TSP, thrombospondin; LRP, low density lipoprotein-related receptor protein; MMP2, matrix metalloproteinase 2; RAP, receptor-associated protein; APMA, 4-aminophenylmercuric acetate; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium.
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