From the Departments of Biochemistry and § Anatomy/Histology, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 17033
Received for publication, October 24, 2000, and in revised form, December 13, 2000
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ABSTRACT |
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Matrix vesicles (MV) play a key role in the
initiation of cartilage mineralization. Although many components in
these microstructures have been identified, the specific function of
each component is still poorly understood. In this study, we show that
metalloproteases (MMP), MMP-2, -9, and -13 are associated with MV
isolated from growth plate cartilage. In addition, we provide evidence
that MV contain transforming growth factor- Matrix vesicles (MV)1
are membrane-enclosed microstructures released from the plasma membrane
of hypertrophic growth plate chondrocytes (1-3). These extracellular
microstructures have the critical role of initiating the mineralization
process in growth plate cartilage (1-3). They contain various
proteins, including annexins II, V, and VI, and alkaline phosphatase
(4-6) The annexins are Ca2+ channel-forming proteins,
which enable Ca2+ influx into MV and the formation of the
first crystal inside the vesicles (7). Alkaline phosphatase is an
enzyme that generates inorganic phosphate from organic phosphate
compounds (3). In addition, extracellular matrix proteins, such as
aggrecan, link protein, and types II and X collagen, are associated
with the outer surface of MV (5, 8, 9).
During mineralization of the growth plate, the first crystal phase
forms and grows inside the vesicle lumen. Once these intralumenal crystals have reached a certain size, they rupture the membrane and
grow out into the extracellular matrix (3). The process by which this
occurs has yet to be defined, but it is plausible to assume that MMPs
are likely involved in the degradation and remodeling of the
extracellular matrix for subsequent mineralization. This hypothesis is
supported by a previous report suggesting the presence of MMPs in MV
isolated from chondrocyte cultures (10). Furthermore, we have reported
previously an increased production of active TGF- Chondrocyte Isolation and Serum-free Cell
Culture--
Prehypertrophic chondrocytes were isolated from day 17 avian upper sterna utilizing microsurgical techniques as described previously (11). Tissue fragments were incubated in 0.25% trypsin and
0.1% crude collagenase mixture in Hanks' buffered saline solution for
3-4 h at 37 °C. Resulting cell suspensions were filtered through a
Nitex filter, counted, and resuspended at a density of 5 × 106 cells/ml. Agarose cultures were prepared as described
earlier in 2 ml of serum-free Dulbecco's modified Eagle's medium,
high glucose (Life Technologies, Inc.), containing 50 µg/ml
penicillin/streptomycin, 2 mM L-glutamine, 1 mM cysteine, and 1 mM sodium pyruvate (complete medium) (11). For alginate bead cultures, cells were resuspended in
0.15 mM NaCl, pH 7.2, and alginate (Keltone
LVCRTM, Kelco, NJ) (13-15). Bead cultures were prepared by
extruding the alginate/chondrocyte suspension dropwise through an
18-gauge needle into 102 mM CaCl2 resulting in
~1 × 105 chondrocytes per bead. Beads were rinsed
with 0.15 M NaCl, placed into 35-mm Petri dishes, and
covered with 2 ml of complete medium. Conditioned medium was collected
and stored at Isolation and Treatment of MV--
MV were isolated from
juvenile avian tibiotarsal growth plate cartilage by mild trypsin and
collagenase digestion and purified by ultracentrifugation as described
previously (5). To remove matrix proteins and other components attached
to the outer surface, MV were sequentially treated with 0.25 units of
hyaluronidase (Type IV-S, Sigma), 1 M NaCl and 5 units of
pure bacterial collagenase from Clostridium histolyticum
(Form III, Advanced Biofacture, Lynbrook, NJ) per 150 µg of total MV
proteins. To disrupt the vesicle membrane, vesicles were treated with
phosphate-buffered saline containing 0.1% CHAPS (Sigma). After each
treatment, MV were pelleted by ultracentrifugation, and aliquots were
removed for analysis.
Determination of Alkaline Phosphatase Activity--
Alkaline
phosphatase activity was measured in each fraction utilizing
p-nitrophenyl phosphate as substrate (Sigma 104, Sigma) as
described previously (5).
Determination of TGF- Electron Microscopy--
For transmission electron microscopy
(TEM), 150 µg of each MV fraction was pelleted, fixed in Karnovsky
fixative, embedded in plastic, sectioned, and viewed and photographed
with a JEM100CXII transmission electron microscope as described
previously (6).
Substrate Gel Zymography and Immunoblot Analysis--
Total
protein concentration for each MV fraction was determined by the BCA
assay (Pierce). 100 µg of each MV fraction were run on 8%
SDS-polyacrylamide gels containing 0.1% gelatin as substrate. After
washing with 1% Triton X-100 to remove SDS, the gel was incubated at
37 °C for 1 h in 100 mM Tris, 1 mM
CaCl2, pH 8.0. Coomassie staining resulted in areas of
clear bands corresponding to the molecular weight sizes of proteases
present in the sample. For immunoblot analysis, 100 µg of each MV
fraction were separated on 8-16 or 4-20% Tris glycine,
SDS-polyacrylamide gradient gels (NOVEX, San Diego, CA). After
electrotransfer to nitrocellulose membranes, the membranes were
incubated with primary antibodies followed by secondary
peroxidase-conjugated antibodies. Specific reactive bands were viewed
with the Renaissance Chemiluminescence kit (PerkinElmer Life Sciences)
and Kodak X-Omat Blue film. Polyclonal antibodies specific for avian
MMP-2 were a kind gift of Dr. James Quigley; polyclonal antibodies
specific for avian MMP-13, annexins II, V, and VI were produced as
described previously (18, 19); polyclonal antibodies specific for
TGF- Fluorescence-labeled Gelatin Substrate Assay--
MV fractions
after hyaluronidase treatment (200 µg of total protein) were mixed
with a fluorescence-labeled gelatin substrate (Molecular Probes,
Eugene, OR). Samples with or without inhibitors were incubated for
2 h at 37 °C, and the fluorescence was measured at an
excitation wavelength of 495 nm and an emission wavelength of 515 nm in
a Photon Technology International fluorimeter (South Brunswick, NJ).
Inhibitors used include CMT-8, at inhibitor concentrations specific for
MMP-13 (this inhibitor was kindly provided by Dr. Brad Zerler,
CollaGenex Pharmaceuticals, Newtown, PA)); doxycycline (Sigma), a
general inhibitor of collagenases and gelatinases; and 1,10 phenanthroline (Molecular Probes, Eugene, OR), a general inhibitor of
MMP activities.
Hypertrophic Chondrocytes Increased the Production of Active
TGF- Authentic MV Contain MMP-2, -9, and -13--
A previous study has
provided evidence that MV isolated from chondrocyte cultures contain
MMPs (10). An additional study suggested that MV might be involved in
TGF-
Matrix vesicles were isolated from juvenile chicken growth plate
cartilage by mild trypsin and collagenase digestion and purified by
ultracentrifugation as described previously (5). TEM analysis revealed
that MV were round to oval in size with diameters between 100 and 300 nm (Fig. 2A, untreated
MV). Immunoblot analysis of the MV fractions confirmed the
presence of annexins II, V, and VI (Fig. 2B, untreated
MV) and high levels of alkaline phosphatase activity (Fig.
2C, untreated MV). In addition, previous studies have provided evidence that MV are associated with extracellular matrix
proteins, including proteoglycans, types II and X collagen (5, 8, 9).
These extracellular matrix components can be selectively removed from
the vesicle surface without destroying vesicle integrity. Hyaluronidase
treatment was shown to remove surface-attached proteoglycans, whereas
treatment with 1 M NaCl and pure bacterial collagenase
removes surface-attached types II and X collagen (5, 8, 9).
As shown in Fig. 2A, sequential treatment with
hyaluronidase, NaCl, and collagenase left the MV structure intact.
Immunoblot analysis revealed the presence of annexins II, V, and VI in
the MV fractions after the various treatments (Fig. 2B).
Furthermore, alkaline phosphatase activity was similar in the untreated
MV fraction and MV fractions after the various treatments (Fig.
2C). However, amino acid analysis revealed a significant
reduction in hydroxyproline content (0.074 nM) after
sequential treatments with hyaluronidase, NaCl, and collagenase
compared with untreated MV (0.1324 nM). Finally, we
incubated hyaluronidase/NaCl/collagenase-treated vesicles with CHAPS to
rupture the MV membrane and expose the nucleational core complex and
other intralumenal components. The nucleational core complex has been
shown to contain Ca2+, Pi, acidic
phospholipids, and associated proteins, such as annexins (24; see also
Fig. 2B), and some alkaline phosphatase activity (see Fig.
2C). TEM analysis confirmed that no intact MV were present after detergent treatment (Fig. 2A,
Hyl/NaCl/Coll/CHAPS MV).
When MV fractions were subjected to gelatin zymography, bands with
molecular weights consistent with MMP-2, -9, and -13 were present (Fig.
3, untreated MV). The band for
MMP-13 is rather weak in the zymogram shown in Fig. 3A.
However, a longer incubation of the gels in Tris/NaCl buffer was not
possible because of high levels of activities of MMP-2 and -9 in the MV
fractions. Thus, Fig. 3B represents the same area of the gel
shown in Fig. 3A enlarged and contrast adjusted to help
visualize the MMP-13 band. Whereas MMP-13 activity was present in MV
fractions after hyaluronidase treatment (Fig. 3B,
Hyl), MMP-13 activity was lost after combined treatment with
hyaluronidase and NaCl (Fig. 3B, Hyl/NaCl). In contrast, MMP -2 and -9 activities were still retained in the pellet
fraction even after sequential treatments with hyaluronidase, NaCl,
collagenase, and detergent (Fig. 3A, Hyl, Hyl/NaCl,
Hyl/NaCl/Coll, and Hyl/NaCl/Coll/CHAPS). The zymograms
also revealed one band of activity above and one band below MMP-9
activity. By molecular weight, these bands are the same size as
bacterial collagenase which was used to isolate MV from growth plate
cartilage. In fact, antibodies against bacterial collagenase recognized
bands of these exact molecular weights in MV isolated by our
protocol.2
To assess the proportion of active MMP-2, -9, and -13 present in
association with MV, MMP activities present in MV fractions were
measured in the absence and presence of various MMP inhibitors using a
fluorescent gelatin substrate. In these experiments
hyaluronidase-treated MV were used which were proteoglycan-depleted,
but still contained MMP-2, -9, and -13 activities (see Fig. 3). CMT-8,
which at a concentration of 15 µM is a specific inhibitor
for MMP-13 (25), resulted in 9% inhibition of the total activity
present in MV (Table I). A higher dose of
CMT-8, which inhibits other collagenases and gelatinases in addition to
MMP-13 (25), reduced the MMP activity by 23%. Doxycycline, an
inhibitor of MMP-2, -9 and -13, inhibited 56% of the total activity,
whereas 1,10-phenanthroline, a metal ion chelator and general inhibitor
of MMP activity, depleted 61% of the activity (Table I). These data
demonstrate that the majority of MMPs associated with MV are
active.
Immunoblot analysis with antibodies against avian MMP-13 and -2 confirmed the existence of these MMPs in MV (Fig.
4, A and B,
untreated MV). We did not confirm the identity of MMP-9 by immunoblot analysis because antibodies against avian MMP-9 are not
currently available. However, size, substrate specificity, and EDTA
sensitivity in the zymograms clearly indicate the presence of MMP-9 in
the MV preparations.
Interestingly, immunoblot analysis revealed the presence of both the
58-kDa MMP-13 and smaller degradation products in MV fractions after
sequential treatments with hyaluronidase, NaCl, and collagenase where
no detectable activity was visible on the zymograms (compare Figs.
3A and 4A, Hyl/NaCl and
Hyl/NaCl/Coll). Immunoreactivity for MMP-13 was lost in MV
fractions treated with hyaluronidase, NaCl, collagenase and detergent
(Fig. 4A, Hyl/NaCl/Coll/CHAPS) suggesting that
MMP-13 is directly associated with MV, but its activity was lost and
the protein was partially degraded after the sequential treatments with
hyaluronidase, NaCl, and collagenase. This is consistent with our
previous findings showing that the bulk of MMP-13 produced by
hypertrophic chondrocytes is rapidly degraded to inactive smaller forms
(18). In contrast, immunoblot analysis and zymography revealed the
presence of MMP-2 in MV pellets after all treatments (compare Figs.
3A and 4B). In addition, as shown in Fig.
3A, MMP-9 activity is retained in the MV pellet fraction
after all treatments, indicating that MMP-2 and -9 appear to be
integral components of MV and that the various treatments do not affect
their activities.
MV-associated MMPs Activate TGF-
The preferred storage form of TGF- In this study, we demonstrate that authentic MV isolated from
chicken growth plate cartilage contain active collagenase, MMP-13, and
active gelatinases, MMP-2 and -9. Furthermore, authentic MV not only
contain latent and active TGF- Although Dean et al. (26) have shown that MV isolated from
chondrocyte cultures contain MMP-2, -9, and -3, the location of these
MMPs within the vesicles was not discerned. Vesicles in their study
were isolated from avian chondrocytes passaged several times and
cultured in monolayer (26), and thus it is not clear whether these MMPs
are integral components of MV or whether these MMPs are associated with
extracellular matrix proteins that are present in the vesicle
preparations. In this study, we isolated authentic MV from in
vivo growth plate cartilage and characterized the major MMP
components and their location within these MV fractions. Location of
these MMPs was determined using sequential treatments with
hyaluronidase, NaCl, and pure bacterial collagenase. These treatments
were shown to selectively remove extracellular matrix proteins from the
vesicles (5). MMP-2 and -9 activity was retained in the pellet fraction
even after detergent treatment revealing that these MMPs are integral
components of MV. MMP-13 activity was lost after hyaluronidase and NaCl
treatment. However, immunoblot analysis revealed the presence of
degraded products of MMP-13 in the pellet fraction after sequential
treatment with hyaluronidase, NaCl, and collagenase, suggesting that
MMP-13 is also an integral component of MV. The protein probably got degraded during the sequential treatments, which is consistent with
previous findings from our laboratory and other laboratories showing
that MMP-13 is very sensitive to in vitro manipulation and
is rapidly degraded to inactive smaller forms (18, 27). Thus, our data
clearly indicate that MMP-2, -9, and -13 are the major MMPs present in
MV and that they are integral components of the vesicles.
MV have been shown to have the critical role of initiating the
mineralization process in growth plate cartilage (3). The first crystal
phase forms and grows inside the vesicle lumen in a protected
environment before the crystals rupture the MV membrane and grow out
into the extracellular matrix (3). Extensive matrix remodeling is
required for the outgrowth of the crystals from the vesicles into the
extracellular matrix. MMP-13 is an interstitial collagenase with high
affinity for type II collagen and was shown to cleave type II and X
collagen (27). MV-associated MMP-2 and -9 can then further degrade the
resulting collagen fragments. The combined action of these
MV-associated collagenase and gelatinases might allow rapid matrix
remodeling and the release of matrix-bound Ca2+, thereby
enabling rapid mineralization of the extracellular matrix.
We have shown previously that MMP-13 mRNA is already produced by
early hypertrophic chondrocytes, but active MMP-13 protein is only
found in late stage hypertrophic chondrocytes (18). MMP-2 is involved
in a cascade resulting in the activation of pro-MMP-13 zymogen (28,
29). Thus, it is possible that MV after being released from the plasma
membrane of hypertrophic chondrocytes contain MMP-2 and pro-MMP-13
zymogen. MV-associated MMP-2 might then be involved in activating the
MV-associated pro-form of MMP-13. Thus, the sensitive and easily
degradable active form of MMP-13 would be only activated at the site
where its activity is required.
Boyan et al. (22) have reported that MV isolated from
chondrocyte cultures were capable of activating latent TGF- In conclusion, this study reveals the presence of active MMP-2, -9, -13, and TGF- (TGF-
) and that
MV-associated MMP-13 is capable of activating latent TGF-
. To
determine whether MMPs are associated directly with MV, vesicles
isolated from growth plate cartilage were sequentially treated with
hyaluronidase, NaCl, and bacterial collagenase to remove matrix
proteins and other components attached to their outer surface. Finally,
the vesicles were incubated with detergent to rupture the MV membrane and expose components that are inside the vesicles. Each treated MV
fraction was subjected to substrate zymography, immunoblotting, and
substrate activity assay. Whereas active MMP-13 was lost after combined
treatment with hyaluronidase and NaCl, MMP-2 and -9 activities were
still retained in the pellet fraction even after detergent treatment,
suggesting that the gelatinases, MMP-2 and -9, are integral components
of MV. In addition, MV contain TGF-
in the small latent complex, and
MMP-13 associated with the MV surface was responsible for activation of
TGF-
. Since the amount of TGF-
activated by hypertrophic
chondrocytes increased with mineral appearance in serum-free
chondrocyte cultures, a role for active MV-associated MMPs is suggested
in activation of TGF-
seen during late chondrocyte hypertrophy and
mineralization of growth plate cartilage.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
by hypertrophic
chondrocytes and the involvement of MMP-13 in this process (11, 12). In
this report we isolated authentic MV from chick growth plate cartilage
and analyzed them for the presence of MMPs and their location within
the vesicles. Furthermore, we tested the role of MV, especially
MV-associated MMPs, in activation of TGF-
in hypertrophic growth
plate cartilage.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C until assayed for TGF-
.
Present in Conditioned Medium from
Chondrocytes Cultures--
Active TGF-
present in conditioned
medium from chondrocytes cultures was determined as stimulation of a
TGF-
-responsive plasminogen activator inhibitor-1 promoter construct
linked to a luciferase reporter, and values represent luciferase
reporter gene activity. This promoter construct was stably transfected in mink lung epithelial cells (PAI/L cells) (16). Amounts of luciferase
activity were determined using Promega Luciferase Assay System
(Promega, Madison, WI) with the Optocomp I luminometer (GEM
Instruments, Pineville, NC). To determine the concentration of
total TGF-
(latent and active) present in conditioned medium, luciferase reporter gene activity was measured before and after heat
inactivation of TGF-
present in conditioned medium at 85 °C for
12 min (17). To calculate the concentration of latent and active
TGF-
present in conditioned medium, standard curves were generated
by measuring luciferase reporter gene activity after treating cells
with defined concentrations of commercially available active TGF-
2
(R & D Systems, Minneapolis, MN).
2 were purchased from Santa Cruz Biotechnology, Inc., Santa
Cruz, CA.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Prior to Mineralization--
Previously, we demonstrated that
hypertrophic chondrocytes maintained in serum-free agarose cultures
produced ~20% active TGF-
after 7 days in culture (11). Increased
production of active TGF-
was unexpected since most cell types
studied to date produce TGF-
in a latent form (17, 20, 21). This
result suggests that an increase in active and total TGF-
produced
by hypertrophic chondrocytes may be indicative of a role for TGF-
in
chondrocyte maturation. To test this hypothesis, pre-hypertrophic chondrocytes were grown in long term serum-free agarose culture, and
conditioned medium was isolated at various time points and assayed for
TGF-
production using the PAI/L assay. Since only the active form of
TGF-
stimulates PAI-1 promoter activity, we first incubated
conditioned medium with PAI/L cells to determine the amount of active
TGF-
present in conditioned medium. To determine the total amount of
TGF-
(latent and active), conditioned medium was first incubated at
85 °C for 12 min to heat-activate latent TGF-
and then incubated
with PAI/L cells. A standard curve was generated by incubating PAI/L
cells with various defined concentrations of active TGF-
(16, 17).
After 42 days, mineralization in these cultures was evident; bright
field microscopy revealed dark areas in these cultures indicative for
mineral deposits (Fig. 1A).
When these cultures were maintained for an extended period (up to 82 days), the amount of total TGF-
produced steadily increased and
reached a maximum between days 42 and 56 in culture (Fig. 1B). Notably, the proportion of TGF-
produced in an
active form steadily increased to reach 100% of the total produced
TGF-
by chondrocytes after 49 days in culture. This increased
production of active TGF-
also coincides with the induction of
mineralization seen in these cultures at day 42 (Fig. 1A).
Mineralization was accompanied by greater than 2-fold increase in total
TGF-
produced by the chondrocytes, which peaked at day 56 and slowly
returned to basal levels (~ 1.5 ng TGF-
/5 × 106
cells/48 h) at 82 days in culture (Fig. 1B). Interestingly,
at the time of onset of mineralization, ~100% of the total TGF-
was produced in an active form (Fig. 1B). The percentage of
active TGF-
in these cultures remained high even when the total
TGF-
levels returned to basal levels (Fig. 1B, day
82). These data indicate that the maturation process of
prehypertrophic chondrocytes to hypertrophic mineralizing chondrocytes
is accompanied by increased production of active TGF-
(11).
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Fig. 1.
Increase of active
TGF- during mineralization of hypertrophic
growth plate chondrocytes. Prehypertrophic chondrocytes were
cultured at high density under serum-free agarose conditions as
described under "Experimental Procedures." Conditioned medium from
these cultures was collected every 48 h through an 82-day culture
period. A, phase contrast micrograph showing the presence of
dark areas indicative of mineral deposits (arrows) in
chondrocyte cultures grown under serum-free agarose conditions for 56 days (magnification × 200). B, the concentration of
active and latent TGF-
present in conditioned medium of these
cultures after various days was determined by measuring luciferase
reporter gene activity after treating PAI/L cells with
non-heat-activated and heat-activated conditioned medium and measuring
luciferase reporter gene activity as described under "Experimental
Procedures." Standard curves were generated by measuring luciferase
reporter gene activity after treating PAI/L cells with defined
concentrations of commercially available active TGF-
. Data are the
average of four different experiments.
activation (22). Thus, it is possible that the persistent
increase in active TGF-
, which accompanies the initiation of
mineralization, may be due to the involvement of MV-associated
proteases. To test this hypothesis, we determined which MMPs are
associated with authentic MV isolated from hypertrophic growth plate cartilage.
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Fig. 2.
Characterization of authentic MV after
various treatments. MV isolated from juvenile avian growth plate
cartilage were sequentially treated with hyaluronidase, 1 M
NaCl, and bacterial collagenase to remove attached extracellular matrix
components. Finally, MV were treated with CHAPS to disrupt the MV
membrane and to expose intravesicular components. A, each MV
fraction was pelleted and fixed in Karnovsky fixative for transmission
electron microscopy. Electron micrographs show untreated MV, MV
following sequential treatments with hyaluronidase, NaCl, and
collagenase (Hyl/NaCl/Coll MV), and MV preparations
following sequential treatment with hyaluronidase, NaCl, collagenase,
and CHAPS (Hyl/NaCl/Coll/CHAPS MV). Magnification × 87,000. B, 100 µg of total protein of each MV fraction
were run on 8-16% Tris glycine gradient gels for immunoblot analysis
of annexins II, V, and VI. Untreated, untreated MV
preparations; Hyl, MV after hyaluronidase treatment;
Hyl/NaCl, MV after sequential treatment with hyaluronidase
and NaCl; Hyl/NaCl/Coll, MV after sequential treatment with
hyaluronidase, NaCl, and collagenase; Hyl/NaCl/Coll/CHAPS,
MV after sequential treatment with hyaluronidase, NaCl, collagenase,
and CHAPS. C, specific alkaline phosphatase activity of each
MV fraction.
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Fig. 3.
Metalloproteases associated with
authentic MV. 100 µg of total protein of each MV fraction were
run on an 8% SDS-polyacrylamide gel containing 0.1% gelatin substrate
and analyzed by zymography. Coomassie staining revealed clear areas
corresponding to active protease in the sample. A,
untreated, untreated MV preparations; Hyl, MV after
hyaluronidase treatment; Hyl/NaCl, MV after sequential
treatment with hyaluronidase and NaCl; Hyl/NaCl/Coll, MV
after sequential treatment with hyaluronidase, NaCl, and collagenase;
Hyl/NaCl/Coll/CHAPS, MV after sequential treatment with
hyaluronidase, NaCl, collagenase, and CHAPS. B, the area of
the gel represented with the bracket in A was rescanned to
improve the contrast and visualization of the band representing MMP-13
activity.
Active MMPs associated with authentic MV isolated from growth plate
cartilage
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Fig. 4.
Immunoblot analysis for MMPs associated with
authentic MV. 100 µg of total protein of each MV fraction were
run on a 4-20 or 8-16% Tris glycine SDS-polyacrylamide gradient gels
and electrotransferred onto nitrocellulose membranes, and the membranes
were immunostained with antibodies specific for MMP-13 (A)
and MMP-2 (B). Untreated, untreated MV
preparations; Hyl, MV after hyaluronidase treatment;
Hyl/NaCl, MV after sequential treatment with hyaluronidase
and NaCl; Hyl/NaCl/Coll, MV after sequential treatment with
hyaluronidase, NaCl, and collagenase; Hyl/NaCl/Coll/CHAPS,
MV after sequential treatment with hyaluronidase, NaCl, collagenase,
and CHAPS.
--
To determine whether MV
are the site of TGF-
activation, we first investigated whether
TGF-
is associated with MV. The total amount of TGF-
present in
the MV fractions was assayed with the PAI/L assay after incubation of
MV fractions at 85 °C to heat-activate TGF-
as described above
(16, 17). Equivalent amounts of TGF-
were present in the MV fraction
before and after sequential treatments with hyaluronidase, NaCl, and
collagenase (0.164 pg/µg MV protein versus 0.183 pg/µg
MV protein), whereas following detergent treatment the levels decreased
to 0.05 pg/µg MV protein (Fig.
5A). Immunoblot analysis
of MV fractions after sequential treatments with hyaluronidase, NaCl,
and collagenase revealed the presence of active TGF-
25-kDa homodimer and the small latent N-terminal peptide of TGF-
(Fig. 5B, lane 1). After detergent treatment the immunoreactive
bands for TGF-
were lost (Fig. 5B, lane 2). These
findings suggest that the small latent TGF-
complex is an integral
component of MV.
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Fig. 5.
Presence of TGF- in
authentic MV. A, the amount of total TGF-
present in
MV was determined by a modified mink lung epithelial cell line stably
transfected with a plasminogen activator inhibitor promoter construct
driving a luciferase reporter (PAI/L). MV fractions were incubated at
85 °C for 12 min to heat-activate any TGF-
present, and the
concentration of total TGF-
was calculated from a standard curve
derived from various defined concentrations of active human recombinant
TGF-
2 as described under "Experimental Procedures." The
concentration of total TGF-
is reported in pg per µg of total MV
protein. B, 100 µg of total protein of each MV fraction
were run on 4-20% SDS-polyacrylamide gradient gels. After
electrotransfer to nitrocellulose membranes, the membranes
were immunostained with primary antibodies against TGF-
2. Lane
1, MV after sequential treatments with hyaluronidase, NaCl, and
collagenase; lane 2, MV after sequential treatments with
hyaluronidase, NaCl, collagenase, and CHAPS. LAP,=
latency-associated peptide of the small latent TGF-
complex.
in the growth plate is as a
latent complex (20). In addition, in preliminary studies we showed that
MMP-13, but not MMP-2 or -9, is capable of activating TGF-
from this
complex (12). The ability of MV-associated MMPs to activate latent
TGF-
was analyzed by assaying the formation of active TGF-
from
the latent complex present in conditioned medium from serum-free
chondrocyte alginate bead cultures in the presence of MV fractions.
Conditioned medium was mixed with MV fractions (150 µg of total
protein), incubated overnight at 37 °C, and the amount of active
TGF-
determined by the PAI/L method. As shown in Fig.
6, addition of MV to conditioned medium
caused a 1.8-fold increase in the amount of active TGF-
compared
with conditioned medium incubated without MV. However, addition of hyaluronidase/NaCl-treated MV to conditioned medium did not lead to a
significant increase in the levels of active TGF-
. We have demonstrated above that hyaluronidase/NaCl-treated MV showed MMP-2 and
-9 activity but no MMP-13 activity. Thus, it is plausible that because
of the loss of MMP-13 activity, hyaluronidase/NaCl-treated MV are not
able to activate latent TGF-
. To explore this possibility further,
we determined whether CMT-8 inhibits MV-mediated activation of latent
TGF-
. As shown in Fig. 7, the addition
of CMT-8 in concentrations of 5 and 15 µM (specific for
inhibition of MMP-13) resulted in a significant inhibition of
MV-mediated activation of TGF-
.
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Fig. 6.
TGF- activation by
authentic MV. 150 µg of total protein of untreated MV,
hyaluronidase, 1 M NaCl-treated MV (Hyl/NaCl
MV), and hyaluronidase, 1 M NaCl/bacterial
collagenase/CHAPS-treated MV (Hyl/NaCl/Coll/CHAPS MV) were
incubated at 37 °C with conditioned medium (Cond.Med.,
CM) from chondrocyte cultures containing latent TGF-
. The
amount of active TGF-
after 12 h of incubation was measured by
the PAI/L assay. Data are the average of three experiments with three
different MV preparations; values are means ± S.D.
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Fig. 7.
MV-associated MMP-13 activates
chondrocyte-produced latent TGF- . MV (150 µg of total protein) were incubated at 37 °C overnight with
conditioned medium from chondrocyte cultures containing latent TGF-
in the presence of various concentrations of CMT-8, an inhibitor of
MMP-13. The amount of active TGF-
present after 12 h of
incubation was determined by the PAI/L assay. Data are the average of
four experiments with two different MV preparations; values are
means ± S.D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, but these particles are able to
activate latent TGF-
present in conditioned medium from chondrocyte
cultures. TGF-
activation by authentic MV was inhibited by CMT-8, at
concentrations specific for MMP-13 inhibition, suggesting that
MV-associated MMP-13 is involved in activating TGF-
in hypertrophic
growth plate cartilage.
. In this study, we confirm and expand their findings and demonstrate that authentic MV isolated from growth plate cartilage are capable of
activating latent TGF-
produced by hypertrophic chondrocytes. Furthermore, our data reveal that active TGF-
2 and
latency-associated peptide of the small latent TGF-
2 complex are
present in authentic MV fractions even after sequential treatment with
hyaluronidase, NaCl, and collagenase, suggesting that small latent
TGF-
may be directly associated with the outer surface of authentic
MV and that the complex may be in close association with MV-associated MMP-2 and -13. In addition, we provide evidence that CMT-8 at concentrations, which specifically inhibit MMP-13 activity,
significantly suppressed MV-mediated activation of TGF-
, suggesting
that MMP-13 plays a major role in activating TGF-
from its small
latent complex. Thus, MV might not only be the site for initiation of
mineralization, but they might also be the site for activation of
TGF-
in hypertrophic mineralizing growth plate cartilage. Active
TGF-
has been shown to be an important factor that promotes early
stages of osteoblast differentiation (30-32). Thus, MV-associated
MMP-2 is involved in activating MV-associated MMP-13. The active form
of MMP-13 releases active TGF-
from the MV-associated small latent
form of TGF-
. The active TGF-
may then facilitate the
differentiation of cartilage-invading preosteoblastic cells to
osteoblastic cells, leading to the replacement of cartilage by bone.
in authentic MV isolated from hypertrophic growth
plate cartilage and suggests potential roles for these MMPs in matrix
remodeling and TGF-
activation during mineralization and bone
formation. Thus, MV released from terminal differentiated chondrocytes
contain essential components required for initiation of mineralization,
matrix remodeling, and subsequent bone formation. For example, they
contain the Ca2+ channel-forming annexins II, V, and VI,
essential components for formation of the first intralumenal crystal
phase; MMPs, required for matrix remodeling; and latent TGF-
which
after being activated by MV-associated MMPs may be required for
osteoblastic differentiation and the replacement of cartilage by bone.
The exact roles of these and other MV components and their potential
interactions, however, still have to be elucidated.
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ACKNOWLEDGEMENTS |
---|
We thank Gerald Harrison for help with isolation of authentic MV, Sylvia Decker for TEM expertise, and Dr. Brad Zerler (Collagenex) for providing us with CMT-8.
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FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants AR 43732, AR 46245 (to T. K.), DE 11876, DE 05676 (to M. D'A.), and AR 40075 (to P. S. L.).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.
Current address: Anatomy and Cell Biology, Temple University
Medical School, 3400 North Broad St., Philadelphia, PA 19140.
¶ To whom correspondence should be addressed: Dept. of Orthopaedics and Rehabilitation, H089, Penn State College of Medicine, Hershey Medical Center, 500 University Drive, Hershey, PA 17033. Tel.: 717-531-7788; Fax.: 717-531-1607; E-mail: tkirsch@psu.edu.
Published, JBC Papers in Press, January 5, 2001, DOI 10.1074/jbc.M009725200
2 G. Harrison, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are:
MV, matrix vesicles;
MMP, metalloproteases;
TGF- , transforming growth factor-
;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
TEM, transmission electron microscopy.
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