From the Inflammatory Diseases Research and § Chemical and Physical Sciences, The DuPont Pharmaceutical Company, Wilmington, Delaware 19880-0400
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ABSTRACT |
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A method was developed for generating soluble,
active "aggrecanase" in conditioned media from
interleukin-1-stimulated bovine nasal cartilage cultures. Using bovine
nasal cartilage conditioned media as a source of the aggrecanase
enzyme, an enzymatic assay was established employing purified aggrecan
monomers as a substrate and monitoring specific aggrecanase-mediated
cleavage products by Western analysis using the monoclonal antibody,
BC-3 (which recognizes the new N terminus, ARGS, on fragments produced
by cleavage between amino acid residues Glu373 and
Ala374). Using this assay we have characterized cartilage
aggrecanase with respect to assay kinetics, pH and salt optima, heat
sensitivity, and stability upon storage. Aggrecanase activity was
inhibited by the metalloprotease inhibitor, EDTA, while a panel of
inhibitors of serine, cysteine, and aspartic proteinases had no effect,
suggesting that aggrecanase is a metalloproteinase. Sensitivity to
known matrix metalloproteinase inhibitors as well as to the endogenous tissue inhibitor of metalloproteinases, TIMP-1, further support the
notion that aggrecanase is a metalloproteinase potentially related to
the ADAM family or MMP family of proteases previously implicated in the
catabolism of the extracellular matrix.
The aggregating cartilage proteoglycan, aggrecan, along with type
II collagen, is responsible for the mechanical properties of articular
cartilage. Aggrecan molecules are composed of two N-terminal globular
domains, G1 and G2, which are separated by an
interglobular domain (IGD),1
followed by a long central glycosaminoglycan (GAG)
attachment region and a C-terminal globular domain, G3
(1-2). These aggrecan monomers interact through the G1
domain with hyaluronic acid and link protein to form large molecular
weight aggregates which are trapped within the cartilage matrix (3-5).
Aggrecan provides normal cartilage with it properties of
compressibility and resilience, and is one of the first matrix
components to undergo measurable loss in arthritis. This loss appears
to be due to an increased rate of aggrecan degradation that can be
attributed to proteolytic cleavage within the IGD of the core protein.
Cleavage within this region generates large C-terminal, GAG-containing
aggrecan fragments lacking the G1 domain which are unable
to bind to hyaluronic acid and thus diffuse out of the cartilage matrix.
Two major sites of proteolytic cleavage have been identified within the
IGD: one between amino acid residues Asn341 and
Phe342 and the other between amino acid residues
Glu373 and Ala374. Matrix metalloproteinases
(MMP-1, -2, -3, -7, -8, -9, and 13) have been shown in vitro
to cleave within the IGD predominately at the
Asn341-Phe342 site (6-10). Identification of
G1 fragments formed by cleavage at the
Asn341-Phe342 site within human articular
cartilage (7, 11) and in synovial fluids (12) suggest a role for MMPs
in proteoglycan degradation in vivo.
The second cleavage site was first described a number of years ago
based upon identification of aggrecan fragments with an ARGS N terminus
(13-15), however, the enzyme responsible for cleavage at the
Glu373-Ala374 bond has not yet been identified.
This uncharacterized activity has been given the name "aggrecanase"
based on its ability to cleave the aggrecan core protein. Four other
potential aggrecanase sites have been identified within the C-terminal
region of aggrecan between G2 and G3 (14, 16),
although the Glu373-Ala374 cleavage site within
the IGD has been most widely studied. C-terminal fragments with the N
terminus, ARGSV ... , formed by cleavage between amino acid
residues Glu373 and Ala374 have been identified
in media from chondrocyte monolayer and cartilage explant cultures
undergoing matrix degradation (13-19). This sequence was found to be
present on a number of different size fragments, indicating that
aggrecanase was cleaving at the Glu373-Ala374
bond within the IGD to generate products with a single N terminus which
possessed variable C termini. N-terminal sequence analyses have
identified aggrecan fragments in synovial fluids of patients with
osteoarthritis, inflammatory joint disease, and joint injury (20, 21)
which have the ARGSV N terminus, and G1 fragments have also
been identified within the cartilage matrix with the NITEGE C terminus
(11) indicating that aggrecanase plays an important role in human
aggrecan catabolism.
Although evidence for induction of cleavage at the aggrecanase site
within the IGD has been demonstrated in a number of in vitro
tissue and cell culture studies (13-19), attempts to identify aggrecanase proteolytic activity in culture media or cell/tissue extracts from these models have been largely unsuccessful. Recent work
using an artificial recombinant protein comprising the IGD sequence of
aggrecan, rAgg1, as a substrate, has identified activity in media from
retinoic acid-stimulated rat chondrosarcoma cell cultures which cleaves
at the Glu373-Ala374 bond (22). However, the
protease responsible for this cleavage remains unidentified and uncharacterized.
In this paper we describe the generation of soluble, active aggrecanase
activity in conditioned media from interleukin-1 (IL-1)-stimulated bovine nasal cartilage. Using this source of aggrecanase, we have developed an enzymatic assay employing purified native aggrecan monomers as the substrate and detection of specific products of aggrecanase-mediated cleavage by Western analysis with the monoclonal antibody, BC-3 (23), which recognizes the new N terminus ARGSV on
fragments produced by cleavage at the aggrecanase site. We have
performed initial enzymatic characterization of the soluble cartilage
aggrecanase activity generated in response to IL-1. The work described
herein provides a method for generating soluble aggrecanase activity
and an assay for monitoring this activity which should serve as
important tools to enable the isolation, purification, and molecular
characterization of the enzyme.
Materials--
Dulbecco's modified Eagle's medium and fetal
bovine serum were from Life Technologies, Inc. (Grand Island, NY). The
IL-1 used was a soluble, fully active recombinant human IL-1 Cartilage Preparation--
Bovine nasal cartilage septa were
removed from bovine noses obtained fresh at the time of slaughter.
Uniform cartilage disks (1 mm thick, 8 mm in diameter) were prepared.
Prior to organ culture studies, disks were equilibrated in tissue
culture for at least 3 days in Dulbecco's modified Eagle's medium
supplemented with 5% heat-inactivated fetal calf serum, penicillin,
streptomycin, amphotericin B, and neomycin (100 IU/ml, 100 µg/ml,
0.25 µg/ml, and 50 µg/ml, respectively).
Cartilage Cultures--
Cartilage slices were incubated in
serum-free Dulbecco's modified Eagle's medium supplemented with
antibiotics as above at 37 °C in an atmosphere of 95% air, 5%
CO2 in the presence of 500 ng/ml IL-1 Glycosaminoglycan Assay--
Sulfated GAG in culture media or
cartilage digests were monitored by the amount of polyanionic material
reacting with 1,9-dimethylmethylene blue (27), using shark chondroitin
sulfate as a standard. GAG release is reported as micrograms of GAG per
mg wet weight cartilage or as percent of total GAG.
Gelatin Zymography--
Culture media were diluted 1:20 with
water and 15 µl mixed with an equal volume of sample buffer (0.5 M Tris-HCl, pH 6.8, 4% SDS, 0.005% bromphenol blue, 20%
glycerol) (Novex, San Diego, CA), incubated at room temperature for 10 min, and run on a 10% SDS-PAGE gels containing 0.1% gelatin in
running buffer (0.24 M Tris, 2 M glycine, 35 mM SDS, pH 8.3) for 90 min at 125 volts. The gels were
renatured in 2.5% Triton X-100 in water for 30 min at room temperature
and then incubated at 37 °C overnight in developing buffer (50 mM Tris, 0.2 M NaCl, 5 mM
CaCl2, 0.02% Brij 35, pH 7.6). After incubation, gels were
stained in 0.25% Coomassie Brilliant Blue R-250 for 4 h at room
temperature and destained in distilled water containing 30% methanol
and 10% glacial acetic acid to reveal zones of lysis within the
gelatin matrix. EDTA (5 mM), E64 (10 µg/ml), pepstatin (1 µg/ml), or benzamidine HCl (10 mM) or XS309 (1 µM) were added to the developing buffer to identify which
classes of proteinases were responsible for lysis of the gelatin.
Caseinase Enzymatic Assay--
Casein labeled with resorufin
(43.5 µM) was incubated with protease-containing media in
a final volume of 200 µl of 50 mM Tris-HCl, 5 mM CaCl2 at pH 7.8 at 37 °C as described
previously (29). The reactions were stopped and unclipped substrate
precipitated by adding trichloroacetic acid to a final concentration of
5%. Samples were filtered through a 96-well filtration plate
(Millipore Co., Bedford, MA) and filtrates collected and neutralized by
addition of 2 M Tris to each well. The absorbance was then
read at 575 nm. The concentration of the resorufin-labeled peptides in
the filtrate, as determined from a standard curve, was used as a
measure of proteolytic activity. Some conditioned medium samples were assayed in the presence of 2 mM 4-aminophenylmercuric
acetate (APMA) to activate latent matrix metalloproteinases. With some samples EDTA was included to inhibit metalloproteinase activity or
XS309 was included to inhibit matrix metalloproteinase activity.
Analysis of Aggrecan Catabolites--
For analysis of aggrecan
fragments generated by specific cleavage at the
Glu373-Ala374 site, proteoglycans and
proteoglycan metabolites were enzymatically deglycosylated with
chondroitinase ABC (0.1 units/10 µg of GAG) for 2 h at 37 °C
and then with keratanase (0.1 units/10 µg of GAG) and keratanase II
(0.002 units/10 µg of GAG) for 2 h at 37 °C in buffer
containing 50 mM sodium acetate, 0.1 M
Tris/HCl, pH 6.5 (23). The digests were monitored by measuring the
decrease in dimethylmethylene blue reactivity. After digestion, the
samples were precipitated with 5 volumes of acetone and reconstituted in an appropriate volume of SDS-PAGE sample buffer.
BC-3 Western Blot Analysis--
Equivalent amounts of GAG from
each sample were loaded on 4-12% gradient gels and then separated by
SDS-PAGE under nonreducing conditions, transferred overnight to
nitrocellulose, and immunolocated with 1:1000 dilution of the
monoclonal antibody BC-3 (22). Overnight transfer resulted in complete
transfer of low and high molecular weight standards and samples as
determined by evaluating the gel by colloidal Coomassie analysis
(Novex, San Diego, CA) following protein transfer; no detectable levels
of protein remained in the gel. Subsequently, membranes were incubated
with goat anti-mouse IgG alkaline phosphatase conjugate and aggrecan
catabolites visualized by incubation with the appropriate substrate
(Promega Western blot alkaline phosphatase system) for 10-30 min to
achieve optimal color development. BC-3-reactive aggrecan fragments
were then quantified by scanning densitometry. For quantitation, the
stained blots were captured on a UVP Imagestore 7500 Camera System
(Cambridge, UK) with GelBase Pro system software. The integrated pixel
density of the bands was quantified using IPLAB GEL software and data obtained only in the 10-fold linear detection range and reported as sum
total pixels.
Preparation of Aggrecan Substrate--
Bovine nasal cartilage
septa were removed from bovine noses obtained fresh at slaughter. The
aggrecan was isolated from the cartilage by extraction at 4 °C for
48 h with 4 M guanidine-HCl, 0.05 M sodium
acetate, pH 5.8, containing the protease inhibitors disodium EDTA,
6-aminohexanoic acid, phenymethanesulfonyl fluoride, and benzamidine
HCl. The aggrecan monomers were isolated by equilibrium density
gradient centrifugation in cesium chloride (30) and the bottom of this
gradient (d > 1.54 g/ml) containing the monomers was
dialyzed at 4 °C against water using Mr
10,000 cutoff membranes, lyophilized, and stored at Aggrecanase Enzymatic Assay--
Conditioned medium from bovine
nasal cartilage stimulated with IL-1 were filtered to remove any
particulate matter using a Corning 0.45-micron filter prior to use in
the enzymatic assay. Medium was then incubated with purified native
bovine aggrecan monomers with intact GAG chains in a final volume of
200 µl in 20 mM Tris, 100 mM NaCl, 10 mM CaCl2 buffer at pH 7.5. At the end of the
incubation the reaction was quenched with 20 mM EDTA, the
aggrecan was deglycosylated with chondroitinase ABC, keratanase, and
keratanase II, and the aggrecanase-generated products were detected by
BC-3 Western blot analysis as described above. In initial enzymatic
assays, product generation was quantitated by scanning all
BC-3-reactive bands. However, under the conditions used for the
enzymatic assay, quantitation of the predominant 250-kDa band, which
represents the initial product detected, gave equivalent results to
quantitation of both this band and the minor lower molecular mass
bands. Therefore, product generation was routinely assessed by
quantitation of the 250-kDa band. Based on kinetic analysis, the
following conditions were established for the assay: 50 µl of
aggrecanase-containing medium was incubated with 500 nM
aggrecan substrate in a final volume of 200 µl at 37 °C for 4 h as indicated above. pH activity profiles were performed in 50 mM MES, 100 mM NaCl, 10 mM
CaCl2 buffer and pH was adjusted using 5 N HCl.
Inhibitors were dissolved in dimethyl sulfoxide as a 10 mM
stock and added to the reaction mixture immediately prior to enzyme
addition. Dimethyl sulfoxide concentrations in the enzyme assay never
exceeded 1%; this concentration of dimethyl sulfoxide had no effect on
aggrecanase activity.
Aggrecanase Generation in Bovine Nasal Cartilage
Culture--
Incubation of bovine nasal cartilage with 500 ng/ml IL-1
for 6 days, with media replaced every 2 days, induced the degradation and release of aggrecan from the cartilage (Fig.
1). By monitoring the amount of GAG
remaining in the cartilage at the end of the incubation, percent
release of GAG from the tissue was determined at each time period;
greater than 95% release of GAG was achieved by day 6. Cartilage was
then incubated with IL-1 for an additional 10-12 days without media
change to allow aggrecanase to accumulate in the media. To evaluate the
time course of aggrecanase activity generation, samples of conditioned
media were taken from cultures at various times during incubation with
IL-1 and evaluated for aggrecanase activity. Incubation of this
conditioned media with aggrecan substrate for 4 h resulted in the
generation of fragments cleaved at the
Glu373-Ala374 site as detected by BC-3 Western
analysis (Fig. 2). Media quenched with
EDTA prior to incubation with aggrecan served as assay blanks to
control for fragments present in the media prior to enzymatic assay.
Incubation of the aggrecan substrate alone did not result in the
generation of BC-3-reactive fragments (data not shown).
Although BC-3 Western blot analysis was routinely used to monitor
aggrecan fragments produced by cleavage at the
Glu373-Ala374 bond, initially digests generated
by aggrecanase cleavage of exogenous aggrecan monomers were also
analyzed by NITEGE Western blot and by biotinylated hyaluronic acid
binding to confirm results with the BC-3 antibody. Time course analyses
showed that the G1-NITEGE fragment was generated
simultaneously with the BC-3 epitope supporting cleavage at the
Glu373-Ala374 bond by
aggrecanase.2 The band of
aggrecan fragments expressing the NITEGE epitope appeared as a doublet
between 64 and 70 kDa, consistent with the G1-NITEGE
products previously obtained following IL-1
High background levels of BC-3-reactive fragments produced in response
to IL-1-induced degradation of the cartilage matrix were observed in
media on day 2 and day 4 (Fig. 2). However, subtraction of background
levels of BC-3 from the levels present following the enzymatic assay
indicated that aggrecanase activity was present even at these early
time periods. By day 6 of culture, BC-3-reactive fragments were no
longer detected in conditioned media. Incubation of day 6 media with
500 nM aggrecan substrate for 4 h resulted in the
formation of a BC-3-reactive, aggrecanase-generated product of ~250
kDa. Aggrecanase activity accumulated in the media as the enzyme
activity was monitored from days 6 to 16. In media from cultures
stimulated for 10 days or greater, additional minor BC-3- reactive
bands that migrated between 100 and 250 kDa were detected.
Aggrecanase Enzymatic Assay Development--
In order to establish
appropriate assay conditions and evaluate aggrecanase kinetic
properties, the effect of incubation time, enzyme concentration, and
substrate concentration on product formation were evaluated. Linearity
of product formation was observed up to 4 h (Fig.
3A), and over enzyme
concentrations between 25 and 80 µl of conditioned media (Fig.
3B). Aggrecanase activity appeared to approach saturation
with respect to aggrecan substrate at 500-1000 nM (Fig.
3C). However, increasing viscosity precluded use of higher substrate concentrations.
Stability--
Aggrecanase activity was found to be stable during
storage at Characterization of Aggrecanase Activity--
Using the assay
conditions defined above, we studied the salt and pH optimum of crude
aggrecanase present in conditioned medium. Optimal activity was
achieved with 100 mM NaCl. At 250 mM NaCl, activity was decreased by ~50% and was completely lost at 500 mM NaCl or higher (Fig. 4).
The pH optimum for aggrecanase was found to be at 7.5 (Fig.
5). However, a rather broad pH range between 6.5 and 9.5 supported greater than 75% of the activity seen at
the pH optimum.
Effect of Protease Inhibitors--
Aggrecanase activity was
inhibited by the metalloprotease inhibitor, EDTA, while a panel of
inhibitors of serine, cysteine, and aspartic proteinases had no effect
(Table I). The ability of the endogenous
inhibitor of matrix metalloproteinase, TIMP-1, to block aggrecanase
activity was investigated using full-length recombinant bovine TIMP-1.
TIMP-1 caused a concentration-dependent inhibition of
aggrecanase activity (Fig. 6). The
IC50 was estimated from the concentration-response curve to
be 210 nM. However, TIMP-2 at concentrations up to 1 µM was inactive against aggrecanase (data not shown).
The peptidic hydroxamates, BB-16 (25) and XS309 (26), which are potent,
synthetic inhibitors with broad specificity against MMPs were tested
for their ability to block aggrecanase activity (Table
II). BB-16 resulted in a
concentration-dependent inhibition of aggrecanase activity,
while XS309 was inactive at concentrations up to 10,000 nM.
Previous studies from our laboratory suggested that although MMP-8
(neutrophil collagenase) has the ability to cleave native aggrecan at
the Glu373-Ala374 bond, it does not represent
the cartilage aggrecanase (31). To confirm this hypothesis we also
evaluated SA751 (26), a potent, selective synthetic MMP-8 inhibitor,
for its ability to inhibit aggrecanase activity. SA751 was inactive
against aggrecanase at concentrations up to 10,000 nM
(Table II), while it has a Ki of 2 nM
for inhibition of MMP-8.
To ascertain whether aggrecanase activity was associated with known
metalloproteinase activities, gelatinolytic and caseinolytic activities
were assayed in medium from IL-1-stimulated cartilage. Multiple bands
of gelatinolytic activity were observed in conditioned medium by
gelatin zymography. All of these gelatinases were shown to be
metalloproteases by the complete loss of gelatinolytic activity when
the zymogram was incubated with EDTA (Fig.
7), whereas E-64, pepstatin, or
benzamidine hydrochloride (which inhibit the cysteine, aspartate, and
serine protease classes, respectively) had no effect on gelatinase
activity under similar conditions (data not shown). In contrast to its
lack of effect on aggrecanase activity, XS309 resulted in a complete
inhibition of the gelatinolytic activity (Fig. 7). Similarly,
caseinolytic activity was also detected in the conditioned media. Both
active and APMA-activated caseinolytic activity were blocked by EDTA
(Table III), indicating that this activity was due to a metalloproteinase(s). Furthermore, this caseinolytic activity was completely blocked by XS309, the MMP inhibitor that was ineffective against aggrecanase.
Although several studies suggest that cleavage of the aggrecan
core protein at the Glu373-Ala374 bond plays a
key role in cartilage matrix degradation, the protease responsible for
this cleavage remains unidentified. Therefore, our current study had
three main goals: 1) to establish a method for generating active
aggrecanase; 2) to develop an assay to assess and follow this activity;
and 3) to characterize aggrecanase enzymatic activity.
Stimulation of chondrocyte cultures with retinoic acid or bovine nasal
cartilage with IL-1 for short time periods has been shown to result in
the production of aggrecan fragments formed by cleavage at the
Glu373-Ala374 bond (18, 32, 33). However,
aggrecanase activity as measured by cleavage of exogenous aggrecan was
not detected in media from these cultures (34). We had previously found
that induction of MMP-3 in cartilage cultures by IL-1 resulted in the
elution of only the inactive zymogen into the culture media at early
time points, although both the zymogen and active forms of the enzyme were present within the matrix (35). Only after depletion of aggrecan
from the matrix or in the presence of an active-site inhibitor was the
active form of MMP-3 protein detected in the culture media, suggesting
that the active enzyme was bound to a matrix component, possibly
aggrecan. We reasoned that this might also be the case for aggrecanase.
Therefore, in an attempt to generate and release active aggrecanase
from bovine nasal cartilage, we first stimulated the cartilage to
deplete the aggrecan from the matrix and then restimulated and
monitored the media for the presence of aggrecanase activity. This
procedure resulted in the generation of readily detectable aggrecanase
activity in conditioned media. In contrast to MMP-3, aggrecanase was
present in the conditioned media as the active form at days 2 and 4 of
incubation with IL-1. However, products generated during the enzymatic
assay were much easier to detect following depletion of the matrix when
background BC-3-reactive fragments were no longer present in the
conditioned media.
The conditioned media exhibited aggrecanase activity (i.e.
cleavage of aggrecan at the Glu-Ala bond) without activation. In fact,
incubation with APMA or chymotrypsin, which are frequently used to
activate pro-MMPs, did not increase this activity. In addition, XG076,
an inhibitor of pro-MMP activation, which is effective in blocking
aggrecanase-mediated cleavage in IL-1-stimulated bovine explant
cultures (33), did not inhibit aggrecanase in the enzymatic
assay,3 indicating that
aggrecanase is not being activated in some manner during the assay.
Although we cannot rule out that the active aggrecanase could be bound
to matrix molecules, specific binding and orientation within the matrix
is not required, since we observe activity using isolated aggrecan
substrate in solution. Thus, aggrecanase generated in conditioned media
from IL-1-stimulated bovine nasal cartilage is a soluble, activated
enzyme, free of association with the chondrocyte cell surface. These
characteristics are consistent with those reported in rat
chondrosarcoma cell cultures stimulated with retinoic acid, where
activity was detected in media using an artificial recombinant aggrecan
interglobular domain protein substrate (22). In contrast to earlier
studies (34), a recent publication demonstrated the ability of
membranes isolated from stimulated chondrocytes to generate aggrecan
fragments formed by cleavage at the
Glu373-Ala374 bond (36). This recent
identification of aggrecanase activity associated with chondrocyte
membranes, in addition to our demonstration of soluble activity in
culture media, open the possibility that aggrecanase may be an integral
membrane protein that is subsequently cleaved to a soluble form or that
it may be a soluble enzyme that is associated with a cell
surface-binding protein on the chondrocyte membrane.
Although other matrix metalloproteases are induced in the
aggrecanase-containing conditioned media from IL-1-stimulated
cartilage, none of these appear to be responsible for the BC-3-reactive
fragments produced by cleavage at the the
Glu373-Ala374 bond. Studies using purified
enzymes have shown that MMP-1, -2, -3, -7, -8, -9, and -13 cleave at
the Asn341-Phe342 bond and do not readily
cleave at the Glu373-Ala374 site (6-10).
Furthermore, we have shown that 1 µM XS309, a potent MMP
inhibitor with Ki values in the nanomolar range
against MMP-1, -2, -3, -8, and -9 that was inactive at concentrations up to 10 µM in inhibiting aggrecanase, completely blocked
all gelatinolytic activities in aggrecanase-containing media. These data indicate that aggrecanase does not cleave gelatin and that aggrecanase activity is not represented by these gelatinolytic MMPs
induced in response to IL-1 stimulation. Caseinolytic activity, detected in the conditioned media, was inhibited almost completely by 1 µM XS309. Since at 1 µM XS309 does not
inhibit aggrecanase, these data suggest that aggrecanase does not
cleave casein and that the caseinolytic activity generated in response
to IL-1 does not represent aggrecanase. Interestingly, XS309, which
completely blocked both the gelatinolytic and caseinolytic activity
generated in response to IL-1, was found to be inactive in blocking
IL-1-induced aggrecan degradation in cartilage explant
cultures,4 while BB-16, which
is active against aggrecanase enzymatic activity, was effective
(33).
The substrate in the enzymatic assays used to determine
Ki values of the synthetic MMP inhibitors against
the various MMPs was a fluorogenic peptide substrate. We have not
determined the Ki values for these compounds using
the natural substrates for the different MMPs or using aggrecan, to
compare with the Ki values determined using the
peptide substrate. Therefore, the possibility that this difference in
substrate may confound the comparison of the potency of these compounds
against MMPs with their potency in the aggrecanase assay, where native
aggrecan is used as the substrate, must be considered. However, when
XS309 and BB-16 were evaluated for inhibition of MMP-3 using the native aggrecan substrate, these compounds exhibited potency in the nanomolar range as was the case using the peptide substrate.
Using BC-3 Western blot analysis to monitor aggrecan fragments produced
by cleavage at the Glu373-Ala374 bond, we have
developed an enzymatic assay specific for aggrecanase-generated products. Although Western blot analysis does not provide a
particularly rapid means of product evaluation, quantitation is
possible, thus enabling characterization of aggrecanase activity. This
assay was used to assess product formation with respect to incubation time and enzyme and substrate concentration dependence. Aggrecanase displayed a sensitivity to salt, with activity being lost at NaCl concentrations above 500 mM. A broad pH optimum extending
into the alkaline range was observed, although maximum activity was seen at pH 7.5. Aggrecanase was also sensitive to elevated temperature with the activity being lost after treatment at 56 °C.
Inhibitor studies indicate that aggrecanase is a metalloendopeptidase
as it is inhibited by the metalloprotease inhibitor EDTA, but not by
inhibitors of the cysteine, serine, or aspartyl protease classes.
Inhibition by TIMP-1 opens the possibility that this protease may be a
member of the matrix metalloproteinase family of enzymes. However, the
IC50 for inhibition of aggrecanase by TIMP-1 is more than
100-fold higher than that reported for MMPs (37, 38). In addition, we
were not able to obtain inhibition with TIMP-2 at concentrations up to
1 µM. During the time this manuscript was in
review, similar results were reported for inhibition of aggrecanase
activity from chondrocytes using a recombinant aggregan interglobular
domain substrate (39). Aggrecanase could represent a member of the MMP
family that differs significantly from other MMPs in its interaction
with TIMP-1 or which may lack the C-terminal region that has been shown
to be important in binding to TIMP for some MMPs (40). Alternatively,
aggrecanase may be a member of a closely related non-MMP class of metalloproteases.
Although the MMP inhibitor, BB-16, is capable of inhibiting the
proteolytic activity of aggrecanase, not all MMP inhibitors are
effective in blocking this activity. In addition, some hydroxamates have been shown to inhibit other related metalloproteases, such as TNF
converting enzyme (41, 42), which is a member of the family of
mammalian adamalysins or ADAM (a
disintegrin and metalloproteinase domain) proteins (43). Our laboratory has recently demonstrated that
atrolysin C, a snake venom metalloproteinase with homology to the ADAM
proteins is capable of cleaving aggrecan at the
Glu373-Ala374 aggrecanase site (44). This
observation, taken together with the lack of inhibition by other potent
MMP inhibitors and the high concentrations of TIMP-1 required for
inhibition, support the hypothesis that aggrecanase may be a member of
a related class of metalloproteinases such as the ADAM family. Although
our data are consistent with aggrecanase representing a member of the
metzincin family of metalloproteases, further characterization awaits
purification and sequencing of the aggrecanase protein.
Time course studies indicated that aggrecanase activity accumulated in
conditioned media from IL-1-stimulated bovine nasal cartilage with
increasing time of incubation from day 6 through day 16. Although
activity in conditioned media was difficult to observe due to high
background levels of BC-3 fragments prior to day 6, quantitation of
epitope by scanning densitometry and subtraction of background levels
from levels present following the enzymatic assay indicated that active
aggrecanase was present in media at days 2 and 4. The formation of
BC-3-reactive products from endogenous aggrecan during the course of
matrix depletion confirm that active aggrecanase is present within the
tissue during the first 2 days of culture.
Upon incubation of aggrecanase with the aggrecan substrate an intense
BC-3-reactive band migrating at approximately 250 kDa was detected.
Subsequently, minor lower molecular mass BC-3-reactive bands were
detected, indicating further cleavage at the C terminus of the
molecule. In addition to the Glu373-Ala374
cleavage site within the IGD of the aggrecan core protein, four other
potential sites for cleavage by aggrecanase have been identified within
the C-terminal region of bovine aggrecan between G2 and G3 (14, 16). The mass of the secondary fragments produced by aggrecanase cleavage of the purified bovine aggrecan substrate, along with their BC-3 reactivity, are consistent with these fragments having undergone cleavage both within the IGD at the
Glu373-Ala374 bond and at site(s) corresponding
to those proposed for cleavage within the C-terminal region of the core
protein. Lack of effect by the potent MMP inhibitor, XS309, on the
appearance of the lower molecular mass BC-3-reactive bands at ~150
and 110 kDa3 indicate that they are not the result of
further cleavage of the 250-kDa fragment by MMPs in the media.
Although the pattern of BC-3-reactive fragments produced upon cleavage
of exogenous aggrecan substrate by the soluble aggrecanase in
conditioned media is similar to that generated during IL-1-induced aggrecan degradation in cartilage cultures, in Fig. 2 the lower molecular weight bands are under-represented in the enzymatic assays
due to the short incubation time (4 h) as compared with the 48-h
periods during which cleavage of the endogenous substrate took place.
In experiments where incubation of exogenous aggrecan with media is
allowed to continue for longer times, the lower molecular mass bands at
~150 and 110 kDa increased in intensity so that the pattern seen
looked extremely similar to that generated by cleavage of the
endogenous substrate in response to IL-1. Interestingly, the two lowest
molecular mass species (~75 and 64 kDa) are not generated upon
digestion of the exogenous substrate by aggrecanase even with
incubations of 48 h. We speculate that these bands are formed by
further cleavage of aggrecanase-generated fragments toward the C
terminus of the aggrecan molecule by MMPs. This hypothesis is supported
by studies showing that although there was little effect on
IL-1-induced aggrecan degradation in bovine nasal cartilage with the
MMP inhibitor, XS309, which does not inhibit aggrecanase, the
generation of these two BC-3-reactive bands was blocked.2
Isolation and purification of the aggrecanase enzyme requires a tissue
source of the protease and an enzymatic assay to follow activity during
purification. In the studies reported herein, we have developed a
method for generating soluble aggrecanase activity from bovine nasal
cartilage, performed initial characterization of this protease, and
developed an assay for monitoring this activity which should lead to
the isolation of the aggrecanase enzyme.
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
produced as described previously (24). The specific activity was 1 × 107 units/mg of protein, with 1 unit being defined as
the amount of IL-1 that generated half-maximal activity in the
thymocyte proliferation assay. Antibody BC-3 (23) which recognizes the new N terminus ARGSV ... on aggrecan degradation produced by
aggrecanase was provided by Dr. Bruce Caterson (University of Wales,
Cardiff, United Kingdom). Chondroitinase ABC lyase (Proteus
vulgaris) (EC 4.2.2.4), keratanase (Pseudomonas sp.)
(EC 3.2.1.103), and keratanase II (Bacillus sp.) were from
Seikuguku (Kogyo, Japan). Full-length bovine tissue inhibitor of
metalloproteases-1 (TIMP-1) was from Calbiochem (Cambridge, MA). The
protease inhibitors, antipain dihydrochloride, aprotinin, bestatin,
chymotrypsin, E-64, EDTA, leupeptin, Pefabloc, pepstatin, and
phosphoramidon were from Boehringer-Mannheim (Indianapolis,
IN). The hydroxamic acid MMP inhibitors, BB-16
((2S,3R)-2-methyl-3-(2-methylpropyl)-1-(N-hydroxy)-4-(o-methyl)-L-tyrosine-N-methyl amide), XS309 ([3S-[3R*,
2-[2R*,2-(R*,S*)]-hexahydro-2-[2-[2-(hydroxyamino)-1-methyl-2-oxoethyl]-4-methyl-1-oxopentyl]-N-methyl-3-pyridazinecarboxamide), and SA751
(N-[1(R)-Carboxyethyl]-a-(S)-(4-phenyl-3-butynyl)glycyl-L-O-methyltyrosine, N-methylamide) were synthesized at DuPont Pharmaceuticals as
described previously (25, 26). BB-16 and XS309 are potent nanomolar inhibitors of a number of MMPs, including MMP-1, -2, -3, -8, and -9, while SA751 is a selective MMP-8 inhibitor with a Ki of 2 nM against MMP-8 and a Ki of
>10,000 nM against MMP-3 and MMP-1.
. Media were
replaced every 2 days for the first 6 days of culture and saved at
70 °C for analysis. Cartilage was then incubated with IL-1 (500 ng/ml) from days 6 to 18 without media change to allow accumulation of
aggrecanase activity in the media. Media were sampled every 2 days and
saved at
70 °C for analysis. In some cases, at the end of
incubation cartilage was digested with papain and the GAG content of
the digest determined.
20 °C.
Aggrecan substrate was prepared by dissolving lyophilized aggrecan
monomers in buffer at a concentration based on the assumption of a
molecular weight of 1 × 10
6.
RESULTS
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Fig. 1.
Time course of IL-1-induced proteoglycan
release from bovine nasal cartilage. Bovine nasal cartilage slices
were incubated in media containing 500 ng/ml IL-1 and media were
replaced every 2 days and assayed for aggrecan release using the
dimethylmethylene blue assay for GAG. Data are plotted as cummulative
micrograms of GAG released/mg of cartilage wet weight.
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Fig. 2.
Time course of aggrecanase generation in
media from IL-1-stimulated bovine nasal cartilage. Bovine nasal
cartilage slices were incubated with 500 ng/ml IL-1 for 18 days with
media being replaced every 2 days for the first 6 days of culture.
Samples of media taken at various times during culture were assayed for
aggrecanase activity by incubating with aggrecan substrate (500 nM) for 4 h at 37 °C and evaluating products by
BC-3 Western blot analysis. Media quenched with EDTA prior to
incubation with substrate served as assay blanks (lanes labeled
B at each time point).
treatment of bovine
chondrocytes (18).
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Fig. 3.
Aggrecanase assay kinetics.
Aggrecanase-containing media was incubated with purified aggrecan
substrate at 37 °C. Aggrecanase-mediated cleavage at the
Glu373-Ala374 bond were monitored by BC-3
Western blot analysis and BC-3-epitope quantitated by scanning
densitometry. Fifty microliters of media were incubated with 1 µM aggrecan substrate for various times (A),
or various amounts of conditioned media were incubated with 1 µM aggrecan substrate for 4 h (B), or 50 µl of media were incubated for 4 h with various concentrations
of aggrecan substrate (C). BC-3-reactive product was plotted
versus time, media volume, or substrate concentration.
70,
20, or 4 °C in culture media; greater than 90%
of the activity was recovered after 14 days or longer storage at
70,
20, and 4 °C. Activity was also stable to repeated freeze-thaw cycles of
70 to 4 °C. Heating at 42 °C for 15 min did not
affect activity, but activity was completely lost following 15 min
heating at 56 °C or above.
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Fig. 4.
Affect of salt concentration on aggrecanase
activity. Aggrecanase-containing media were incubated with 500 nM aggrecan substrate at 37 °C for 4 h in the
presence of various concentrations of NaCl and product generation was
monitored by BC-3 Western blot analysis.
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Fig. 5.
Affect of pH on aggrecanase activity.
Aggrecanase-containing media were incubated with 500 nM
aggrecan substrate for 4 h in 50 mM MES, 100 mM NaCl, 10 mM CaCl2 buffer with pH
adjusted using 5 N HCl. Product was monitored by BC-3
Western blot analysis and activity plotted as a percent of maximal
activity versus pH.
Effect of protease inhibitors on aggrecanase activity
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Fig. 6.
Inhibition of aggrecanase activity by
TIMP-1. Aggrecanase-containing media were incubated with 500 nM aggrecan substrate for 4 h at pH 7.5 in the absence
or presence of various concentrations of bovine TIMP-1 and product
formation monitored by BC-3 Western blot analysis (A).
Percent inhibition of product formation was plotted versus
TIMP-1 concentration (B).
Effect of synthetic matrix metalloproteinase inhibitors on aggrecanase
activity
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Fig. 7.
Gelatinolytic activity in media from
IL-1-stimulated bovine nasal cartilage. Conditioned medium from
day 16 of culture was analyzed for gelatinolytic activity by gelatin
zymography without or with the inclusion of 5 mM EDTA or 1 µM XS309 in the developing buffer.
Effect of inhibitors on caseinolytic activity of conditioned media
DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Robert Copeland for MMP Ki values and Liana Bauerle for expert technical assistance.
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FOOTNOTES |
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* 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: Principal Research
Scientist, Inflammatory Diseases Research, The DuPont Pharmaceutical Co., Experimental Station E400/4239, P. O. Box 400, Wilmington, DE
19880-0400. Tel.: 302-695-7078; Fax: 302-695-7873.
2 M. D. Tortorella, M. A. Pratta, and E. C. Arner, unpublished data.
3 E. C. Arner and M. D. Tortorella, unpublished data.
4 E. C. Arner and M. A. Pratta, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: IGD, interglobular domain; GAG, glycosaminoglycan; MMP, matrix metalloproteinase; PAGE, polyacrylamide gel electrophoresis; IL, interleukin; TIMP-1, tissue inhibitor of metalloproteinases; APMA, 4-aminophenylmercuric acetate; MES, 4-morpholineethanesulfonic acid.
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REFERENCES |
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