To isolate cell-synthesized MMP-9/CSPG complex, the cells were washed three times in serum-free medium and then cultured for 72 h in serum-free RPMI 1640 medium with 0.1 lMphorbol 12-myr
Trang 1Calcium-induced activation and truncation of promatrix
metalloproteinase-9 linked to the core protein
of chondroitin sulfate proteoglycans
Jan-Olof Winberg1, Eli Berg1, Svein O Kolset2and Lars Uhlin-Hansen1
1
Department of Biochemistry, Institute of Medical Biology, University of Tromsø, Norway;2Institute of Nutrition Research, University of Oslo, Norway
In the leukemic macrophage cell-line THP-1, a fraction of
the secreted matrix metalloproteinase 9 (MMP-9) is linked
to the core protein of chondroitin sulfate proteoglycans
(CSPG) Unlike the monomeric and homodimeric forms
of MMP-9, the addition of exogenous CaCl2 to the
proMMP-9/CSPG complex resulted in an active gelatinase
due to the induction of an autocatalytic removal of the
N-terminal prodomain In addition, the MMP-9 was
released from the CSPG through a process that appeared
to be a stepwise truncation of both the CSPG core protein
and a part of the C-terminal domain of the gelatinase The
calcium-induced activation and truncation of the MMP-9/
CSPG complex was independent of the concentration of
the complex, inhibited bythe MMP inhibitors EDTA,
1,10-phenanthroline and TIMP-1, but not bygeneral
inhibitors of serine, thiol and acid proteinases This
indi-cated that the activation and truncation process was not
due to a bimolecular reaction, but more likelyan intra-molecular reaction The negativelycharged chondroitin sulfate chains in the proteoglycan were not involved in this process Other metal-containing compounds like amino-phenylmercuric acetate (APMA), NaCl, ZnCl2and MgCl2 were not able to induce activation and truncation of the proMMP-9 in this heterodimer On the contrary, APMA inhibited the calcium-induced process, whereas high con-centrations of either MgCl2 or NaCl had no effect Our results indicate that the interaction between the MMP-9 and the core protein of the CSPG was the causal factor in the calcium-induced activation and truncation of the gel-atinase, and that this process was not due to a general electrostatic effect
Keywords: gelatinase B; MMP-9; proteoglycan; activation; calcium
The superfamilyof matrixins or matrix metalloproteinases
consists of at least 18 different mammalian zinc- and
calcium-dependent metalloproteinases (MMPs) [1–4], of which
monocytes/macrophages can express several types [5–9]
Together, the MMPs are able to degrade most extracellular
matrix proteins [3,4,10], as well as regulating the activityof
serine proteinases bydigesting various serpins (serine
proteinase inhibitors) [11], and the growth factor activityof
insulin-like growth factor (IGF) bythe abilityto degrade
IGF binding protein (IGHBP) [12] Thus MMPs have broad
substrate specificity, and have been shown to be involved in
various regulatoryprocesses in normal and pathological
conditions in different tissues and organs
The activityof MMPs is regulated at the transcriptional,
translational and post-translational levels Most of the
MMPs are synthesized in their latent pro-form, and must
be converted to their active forms in the extracellular space The cysteine in the conserved PRCG(V/N)PD sequence in the pro-domain binds to the active site zinc
as a fourth ligand, and hence is involved in the mainten-ance of the latencyof the enzymes [3,13] During the activation, either parts of or the entire N-terminal pro-domain are removed This process can be performed by various agents in vitro, including p-aminophenylmercuric acetate (APMA), SDS, urea, chaotropic agents, heat treatment and byproteinases [3,10,13–17] A model for the latencyand activation of MMPs has been proposed, called the cysteine-switch or velcro model, which suggests that there is an equilibrium between a switch-open and a
switch-closed form of the pro-enzyme [16] The reaction
of the free thiol group in the switch-open form with for example organomercurials has been suggested to drive the equilibrium toward the open form, which then undergoes
an autolytic conversion to an active form Once activated, the activityof MMPs can be regulated byendogenous inhibitors such as a2-macroglobulin and tissue inhibitors
of MMPs (TIMPS) [3,4,10,13,18]
MMP-9 has been found as a monomer as well as in various dimeric forms [19–23] In the homo- and hetero-dimeric forms, the proteins are either covalentlylinked to each other through disulfide bonds [20,22,23] or through
a strong noncovalent and predominantlyhydrophobic
Correspondence to J.-O Winberg, Department of Biochemistry,
Institute of Medical Biology, University of Tromsø, 9037 Tromsø,
Norway Fax: + 47 77 646222, Tel.: + 47 77 645488,
E-mail: janow@fagmed.uit.no
Abbreviations: APMA, amino-phenylmercuric acetate; cABC,
chon-droitin ABC lyase; CS, chonchon-droitin sulfate; GAG, glycosaminoglycan;
MMP, matrix metalloproteinase; PG, proteoglycan; TIMP, tissue
inhibitors of MMP; SG, serglycin; SBTI, soybean trypsin inhibitor.
(Received 1 July2003, revised 4 August 2003, accepted 8 August 2003)
Trang 2dimerization contact [24] Recently, we discovered that
the leukemic monocyte cell line THP-1 produced a new
type of reduction-sensitive heterodimer, in which MMP-9
is stronglylinked to the core protein of a chondroitin
sulfate proteoglycan (CSPG) [23] Proteoglycans (PGs)
constitute a distinct class of glycoconjugates,
character-ized bya core protein substituted with highlynegatively
charged glycosaminoglycan (GAG) chains of which
chondroitin sulfate (CS) is a major type [25–27] The
expression of various types of PG in THP-1 cells are not
extensivelystudied, but it has previouslybeen reported
that serglycin is the major CSPG released from these
cells [27] As most cells produce several types of PG, one
can expect that THP-1 cells also express various PG core
proteins such as versican and syndecans At present, it is
not known which of these PG core proteins are bound
covalentlyto MMP-9 The discoveryof complexes of
MMP-9 covalentlylinked to the core protein of CSPG
expanded our view of how MMP-9 can interact with
PGs, as it was known previouslythat both MMP-9 and
MMP-2 binds to the negativelycharged GAG chains in
PGs through positivelycharged clusters in the C-terminal
hemopexin-like domains of the MMPs [28,29] Because
calcium is known to maintain the structural integrityof
MMPs including MMP-9 [30,31] and to be of
import-ance for the activityin APMA activated MMP-9 [32,33],
we investigated if either APMA, APMA in combination
with calcium, or calcium alone could induce an in vitro
activation of the proMMP-9/CSPG heterodimer and
eventuallyinfluence on the enzymatic activityof the
complex The results presented show that proMMP-9
bound covalentlyto the CSPG has different
character-istics compared to other MMP-9 forms
Experimental procedures
Materials
Safranin O (number S-2255), cetylpyridinuim chloride,
phorbol 12-myristate 13-acetate, Triton X-100, chondroitin
sulfate C, trypsin, soybean trypsin inhibitor (SBTI),
EDTA, 1,10-phenanthroline, gelatin, calf skin collagen,
pepstatin, leupeptin, N-ethylmaleimide and alkaline
phos-phatase-conjugated antibodywere purchased from Sigma
Pefabloc was from Pentapharm Ltd Human recombinant
TIMP-1, Q-Sepharose, Sephadex 200, Sephadex G-50
(fine), Gelatin-Sepharose, Heparin-Sepharose, Amplify,
14C-labeled RainbowTMprotein molecular mass standards,
[35S]sulfate and mouse monoclonal antibodies against
human MMP-9 (#IM10L) were obtained from Amersham
Pharmacia Biotech According to the manufacturer, the
MMP-9 antibody(#IM10L) detects onlythe latent
92 kDa form under both reducing and nonreducing
conditions Polyclonal antibodies against TIMP-1,
MMP-7 and the C-terminal region and the hinge region
of MMP-9 were obtained from Chemicon International,
Inc Chondroitin ABC lyase (proteinase free) was
pur-chased from Seikagaku Kogyo Co CDP-StarTM
chemi-luminescent substrate was obtained from New England
Biolabs Unlabeled molecular mass standards were from
Bio-Rad Cronex 4 medical X-rayfilm was obtained from
Sterling Diagnostic Imaging
Cells The human leukemic macrophage cell-line THP-1 was a kind gift from K Nilsson, Department of Pathology, Uppsala University, Sweden The cells were cultured in RPMI 1640 medium with 10% fetal bovine serum,
100 lgÆmL)1of streptomycin, and 100 UÆmL)1of penicillin
To isolate cell-synthesized MMP-9/CSPG complex, the cells were washed three times in serum-free medium and then cultured for 72 h in serum-free RPMI 1640 medium with 0.1 lMphorbol 12-myristate 13-acetate, and the conditioned medium was thereafter harvested and treated as described earlier [23] This medium was then used directlyfor analyses
or purification of CSPG (see below)
Purification of proMMP-9/CSPG complexes The proMMP-9/CSPG complex was purified byQ-Seph-arose anion-exchange chromatographyas described previ-ously[23] Briefly, the column (bed volume 2 mL) was washed with 50 mL of 0.05 M sodium acetate, pH 6.0, containing 6M urea and 0.35M NaCl During these conditions, both the 92 and 225 kDa forms of the MMP-9 passed through the column Bound material was eluted with 1.5MNaCl in 0.05Msodium acetate, 6Murea,
pH 6.0 The CSPG-containing fractions, detected bythe Safranin O method (see below), were pooled and diluted with 0.05Msodium acetate/6Murea to give a final NaCl concentration of 0.35M The material was then re-subjected
to another column of Q-Sepharose After extensive wash with the buffer containing 0.05Msodium acetate, 6Murea and 0.35M NaCl, bound material was eluted with a gradient of 0.35–1.5M NaCl in 0.05M sodium acetate,
6Murea, pH 6.0 The column was run at a flow rate of 0.5 mLÆmin)1 and fractions of 1 mL were collected The fractions containing most CSPGs, as determined bySafr-anin O, were pooled and desalted on Sephadex G-50 (fine) columns run in H2O The volume was reduced in a Speed Vac (Savant)
In other experiments, the Q-Sepharose purified proMMP-9/CSPG complex was further purified byfirst incubating the complex in the presence of 10 mMof EDTA for 30 min at 4C The EDTA and EDTA extracted material was then separated from the intact proMMP-9/ CSPG complex on gel permeation chromatography Two hundred microlitres of this material was added to an
800· 0.4 cm Sephacryl 200 column, and fractions of 50 lL were collected The intact proMMP-9/CSPG complex was eluted in the void-volume
Purification of MMP-9 from the THP-1 cells The proMMP-9 in conditioned medium from the THP-1 cells was partlypurified bysubjecting the culture medium to
a Gelatin-Sepharose column Both the MMP-9 monomer and dimer forms bound to the column, while the MMP-9/ CSPG complex was detected in the pass-through fractions Prior to elution of the bound proMMP-9 from the column with 10 mM of dimethylsulfoxide, the column was thor-oughlywashed with 0.1MHepes buffer, pH 7.5 The eluted and pooled MMP-9 fractions were passed over a Sephadex G-50 (fine) column, run in 0.1 Hepes, pH 7.5
Trang 3Degradation of PG-bound CS-chains by chondroitin
ABC lyase (cABC) treatment
The PG-bound CS-chains were degraded bydigestion for
2 h at 37C with 0.2–1.0 units of cABCÆmL)1of 0.05M
Tris/HCl, pH 8.0, containing 0.05M sodium acetate In
some experiments, the degraded CS-chains were removed
from the proMMP-9/PG core protein complex bygel
chromatographyon a Sephadex G-50 column In other
experiments, the resulting proMMP-9/core protein complex
was separated from remaining intact complex or other
impurities on either a new Q-Sepharose column
pre-equilibrated with 0.35MNaCl, or on a Gelatin-Sepharose
column, or alternativelya combination of these two
columns During the conditions used, the
proMMP-9/PG-core protein complex did not bind to the Q-Sepharose
column The proMMP-9/core protein complex was bound
to the Gelatin-Sepharose column, and was eluted from the
column using 10% dimethylsulfoxide, while the intact
proMMP-9/CSPG complex passed through this column
Detection of PG-bound CS-chains
PG-bound CS-chains were quantitated
spectrophotometri-callybythe Safranin O method [34] as described previously
[23] Briefly, 30 lL was mixed with 300 lL of 50 mM
sodium acetate, pH 4.75, containing 0.02% Safranin O
The mixture was subjected to a microsample filtration
manifold using the slot-blotting process After filtration
through nitrocellulose filter (Millipore HA 0.45 lm), each
sample was washed twice with 100 lL H2O byfilling the
wells and reapplying the vacuum The nitrocellulose filter
was removed and the individual dots were cut out and
transferred to tubes containing 200 lL of 10%
cetylpyridi-nium chloride in H2O The precipitates were solubilized by
incubation at 37C for 30 min Vortexing was performed
every10 min during the incubation The absorbance of the
solubilized color was measured in a Pharmacia Ultrospec III
spectrophotometer at 536 nm The amount of GAGs in
each sample was estimated from a standard curve of
4–40 lgÆmL)1of chondroitin sulfate C
Gelatin zymography
SDS/substrate PAGE was carried out as described
previ-ously[23] with gels (7.5· 8.5 cm · 0.75 mm) containing
0.1% (w/v) gelatin in both the stacking and the separating
gel, 4 and 7.5% (w/v) of polyacrylamide, respectively The
gelatin zymograms were calibrated with both human
gelatinase standards from capillarywhole blood as
des-cribed previously[35], protein standards and the
condi-tioned serum-free THP-1 medium Ten microlitres of
conditioned medium or purified CSPG was mixed with
3 lL of loading buffer (333 mM Tris/HCl, pH 6.8, 11%
SDS, 0.03% bromophenol blue and 50% glycerol) Eight
microlitres of this nonheated mixture was applied to each
gel, which was then run at 20 mA at 4C Thereafter, the gel
was washed twice in 100 mL of washing buffer (50 mMTris/
HCl, pH 7.5, 5 mM CaCl2, 1 lM ZnCl2 and 2.5% (v/v)
Triton X-100) and then incubated in 100 mL of assaybuffer
(50 mM Tris/HCl, pH 7.5, 5 mM CaCl2, 1 lM ZnCl2 and
1 mMAPMA) for approximately20 h at 37C Gels were
stained with 0.2% Coomassie Brilliant Blue R-250 (30% methanol) and destained in a solution containing 30% methanol and 10% acetic acid Gelatinase activitywas evident as cleared (unstained) regions The area of the cleared zones was analyzed with the GelBase/GelBlotTMPro computer program from Ultra Violet Products
Western immunoblotting analysis Purified CSPG was electrophoresed on SDS/PAGE 4% (w/v) in stacking gel and 7.5% (w/v) in separating (gel) and electroblotted to a poly(vinylidene difluoride) membrane After blockage of nonspecific binding sites with non fat milk (5% in Tris-buffered saline), blots were incubated for 1 h at room temperature with the appropriate antibodyagainst human MMP-9 After washing, the blots were incubated for
1 h at room temperature with an alkaline phosphatase-conjugated antibody, diluted 1 : 20 000 in blockage solu-tion and developed with CDP-StarTM chemiluminescent substrate All procedures were performed according to the manufacturer The area and intensityof the stained bands was also analyzed with the GELBASE/GELBLOTTM PRO computer program from Ultra Violet Products
Activation of latent gelatinases The gelatin-degrading enzymes are secreted from THP-1 cells into the culture medium in a latent form and require proteolytic activation The trypsin-titration of the latent enzymes in the THP-1 conditioned medium was mainly achieved as described previously[36], except that the activation time in the present work was between 15 and
30 min at 37C
The gelatinases associated with CSPG were activated by incubating the proMMP-9/CSPG complex at 37C with 0.1–100 mM of CaCl2 for 2 h In some experiments, the complex was incubated with either 1 mMAPMA, 10 mM MgCl2, 0.001–10 mMZnCl2or 10–200 mMNaCl for 2 h at
37C To test for a possible prevention of the activation process, either 10 mM EDTA, 1 mM 1,10-phenanthroline, 1–20 nM human recombinant TIMP-1, 0.1–4.5M urea,
1 mM pefabloc, 2 lgÆmL)1leupeptin, 1 lgÆmL)1pepstatin
or 1 mMN-ethylmaleimide was added to the proMMP-9/ CSPG complex prior to incubation with 10 mMof CaCl2 for 2 h at 37C
3 H-labeling of calf skin collagen Acid-soluble calf skin collagen was labeled with tritium by reductive methylation of the amino groups as described previously[37] Collagen denatured for 5–10 min at 90C resulted in gelatin
Gelatinolytic proteinase activity Briefly, 50 lL of activated or non activated cell-conditioned medium or, activated or non activated purified MMP-9/ CSPG (5 lg), was mixed with 50 lL of 0.1MHepes buffer,
pH 7.5 and 50 lL of the 3H-labeled gelatin solution (2.3 mgÆmL)1 or 107c.p.m.Æmg)1) In inhibition experi-ments, either 10 mMEDTA, 1 mM1,10-phenanthroline or 3–20 n of human recombinant TIMP-1 was added to
Trang 4CaCl2 activated MMP-9/CSPG complex The gelatinase
assays were carried out at 37C for approximately20 h
Twentymicrolitres of the supernatants were subjected to
SDS/PAGE Thereafter, the gel was soaked in Amplifyand
dried Nondegraded and degraded [3H]gelatin were detected
with autoradiography
Results
Calcium-induced activation of the MMP-9/CSPG
complex
Experiments were performed to determine whether purified
MMP-9/CSPG complex was active and able to degrade
gelatin.3H-labeled gelatin was incubated with the MMP-9/
CSPG complex at 37C in the absence or presence of
exogenouslyadded CaCl2 After 24 h the sample was
applied to SDS/PAGE and the gel was analy zed by
autoradiography No fragmentation of gelatin could be
detected in the absence of CaCl2, while in the presence of
10 mM CaCl2, the gelatin was degraded to smaller
frag-ments (Fig 1A, lane 3 and Fig 1B, lane 4) When
APMA-treated MMP-9/CSPG complex was incubated in the
absence or presence of 10 mM CaCl2, no degradation of
gelatin was obtained (Fig 1B, lanes 3 and 5)
The CS chains were not involved in the activation of
the complex, as the same results were obtained when the
CS-chains were enzymaticallydegraded bycABC lyase and removed from the complex bySephadex G-50 gel chroma-tographyprior to incubation with gelatin
The gelatinase activity of the CaCl2activated MMP-9/ CSPG complex was totallyinhibited in the presence of either 10 mM of EDTA (Fig 2A) or 1 mM of 1,10-phenanthroline (data not shown) Also human recombinant TIMP-1 (3–20 nM) inhibited the gelatinase activityin a concentration-dependent manner (Fig 2B)
The addition of calcium released low molecular size forms of MMP-9 from the MMP-9/CSPG complex
As shown earlier [23], gelatin zymography of the MMP-9/ CSPG complex reveal bands in the stacking gel and a band around 300 kDa (Fig 3A, lane 1) When the MMP-9/ CSPG complex was incubated with 10 mMCaCl2for 2 h
at 37C prior to electrophoresis, both these bands either disappeared or were stronglyreduced, and new bands with lower Mrappeared (Fig 3A, lane 2) Two weak bands at 80 and 85 kDa were seen along with a strong doublet at 74/76 kDa The same pattern occurred when the MMP-9/ CSPG complex was incubated for 2 h at 37C with varying CaCl2concentrations from 0.1 to 100 mM(data not shown) However, the process was concentration dependent The
Fig 1 Activation of proMMP-9/CSPG with CaCl 2 Purified CSPG
was incubated for 2 h at 37 C with (+) or without (–) CaCl 2 (10 m M ),
cABC or APMA (1 m M ) as indicated under each lane Five
micrograms of the CSPG was then mixed with [3H]gelatin and
incu-bated for 24 h at 37 C as described in the method section These
samples (20 lL per lane) were then separated on a 7.5% SDS/PAGE
gels, and the radioactivityof the labeled gelatin and its degradation
products were detected byautoradiography Lane 1 in (A) and (B)
shows a [ 3 H]gelatin control, and both gels contained [ 3 H]gelatin
incubated with either trypsin-activated THP-1 conditioned serum-free
medium or trypsin as a positive control (not shown) At the left in each
figure is shown the position of the rainbow standard markers and their
M r in kDa The arrowheads indicate the bottom of the application
well.
Fig 2 Inhibition of theCaCl 2 -activated MMP-9/CSPG complex (A) Five micrograms of purified MMP-9/CSPG was incubated for 2 h
at 37 C in the presence of 10 m M of CaCl 2 and then mixed with [3H]gelatin, either with (+) or without (–) EDTA (10 m M ) or human recombinant TIMP-1 as indicated under each figure These mixtures were thereafter incubated for 24 h at 37 C as described in the Experimental procedures section (B) The amount of TIMP-1 used was 3.3 n M (lane 5), 6.7 n M (lane 6) and 20 n M (lane 7) In (A) and (B), negative controls are shown in lane 1 ([ 3 H]gelatin) and in lane 3 ([3H]gelatin incubated with 5 lg of unactivated MMP-9/CSPG) Lane
2 shows a positive control of [3H]gelatin degraded bytrypsin-activated THP-1 conditioned serum-free medium At the left in each figure is shown the position of the rainbow standard markers and their M r in kDa The arrowheads indicate the bottom of the application well.
Trang 5low Mr bands were much weaker at 0.1 mM calcium
compared to the higher concentrations, and at 0.01 mM
calcium, no low Mrbands appeared This calcium-induced
conversion of the MMP-9/CSPG complex to lower Mr
forms was not affected bythe presence of 0.05% Brij-35, a
compound known to inhibit autoactivation and autolytic
degradation of MMP-2 and MMP-9 [38] Thus, treatment
of the MMP-9/CSPG complex with calcium resulted in
proteolytic cleavage and the release of the gelatinase from
the complex When the complex was incubated for 2 h at
37C in the presence of either 0.001–10 mMZnCl2, 10 mM
MgCl2 or 10–200 mM of NaCl, no conversion to low
molecular size forms could be detected in gelatin
zymogra-phy(data not shown) The calcium-induced activation and
truncation of the MMP-9/CSPG complex was not affected
bythe presence of either 10 mM MgCl2 or 200 mMNaCl
(data not shown) Thus, the salt-induced processing of the
complex is not due to a general electrostatic or ionic strength
effect, but appears to be a unique effect of the chemical
properties of calcium
As expected, the degradation and removal of CS-chains
from the complex resulted in the disappearance or a large
reduction of the bands in the stacking gel and the band at
300 kDa, and new bands around 120–150 kDa appeared (Fig 3A, lane 4) Treatment of this MMP-9/PG-core protein complex with 10 mM of CaCl2 for 2 h at 37C prior to electrophoresis resulted in the appearance of new bands with the same Mr as the bands from the cABC untreated material (Fig 3A, lane 5) The same result was obtained if the degraded CS-chains were removed or not removed from the sample, demonstrating that the CS-chains were not involved in the calcium-induced processing and release of the gelatinase from the complex The CaCl2-induced conversion of the MMP-9/CSPG complex to lower molecular size forms was inhibited bythe presence of either 10 mMof EDTA (Fig 3A, lanes 3 and 6), 1–20 nMof human recombinant TIMP-1 (Fig 3B) or 1 mM
of 1,10-phenanthroline (data not shown) The addition of these inhibitors to the complex after calcium activation, but prior to electrophoresis gave the same pattern as without inhibitors (data not shown) The CaCl2induced conversion
of the MMP-9/CSPG complex to lower molecular size forms was not inhibited bythe presence of either pefabloc, leupeptin, N-ethylmaleimide or pepstatin (data not shown), i.e general inhibitors of serine, thiol and acid proteinases
As EDTA inhibited the calcium-induced conversion of the MMP-9/CSPG complex to lower Mrforms, we used this inhibitor to investigate the kinetics of the processing of the complex The CaCl2-induced conversion to lower molecular size forms was stopped by10 mMEDTA after 1, 5, 10, 15,
30, 60 and 120-min incubation, which showed that the bands at 80, 85 and 100 kDa were intermediates, with maximum intensitybetween 5 and 15 min (Fig 4A) The intensityof the 74/76 kDa doublet increased during the entire incubation period (Fig 4A) However, in a few other preparations of the MMP-9/CSPG complex, the induced conversion to lower Mrforms byCaCl2was slower, and the conversion seemed to involve at least one additional step, i.e the formation of a transient species at 180 kDa (Fig 4B) As with the other preparations, the intensityof the 74/76 kDa bands increased during the entire incubation period
The released forms of MMP-9 from the proMMP-9/CSPG complex were N- and C-terminally truncated
To determine whether the different bands obtained after CaCl2 treatment lacked either the N- or the C-terminal regions, Western blots were performed using various antibodies against MMP-9 Under reducing conditions, onlythe 92 kDa band was obtained with all antibodies used (Fig 5) When the complex had been incubated for 2 h in the presence of CaCl2, this 92 kDa band was weaker than in the untreated control (Fig 5) As no bands with an Mr lower than 92 kDa appeared in the blots treated with antibodies against the proform of MMP-9 (Fig 5A), these results show that the calcium-induced truncated forms of MMP-9 must lack the N-terminal pro-domain A strong band at approximately70 kDa appeared in the CaCl2 -treated material when the polyclonal antibody that recog-nizes the hinge region was used (Fig 5B, lane 2) Intact MMP-9 was onlyweaklystained bythis antibody(Fig 5B, lane 1) As proMMP-9 in the serum-free conditioned medium from THP-1 cells also was weaklystained bythis antibody(data not shown), it appears that the epitope in the hinge region is partlyhidden in the intact enzyme The fact
Fig 3 Activation of proMMP-9/CSPG with CaCl 2 results in the
release of low M r forms of thegelatinase Gelatin zymography of 4 lg
per lane of MMP-9/CSPG, which has been incubated for 2 h at 37 C
with (+) or without (–) cABC, CaCl 2 (10 m M ), EDTA (10 m M ) or
human recombinant TIMP-1 as indicated under the figure (A)
Arrowheads show the 74/76 kDa doublet and 80 and 85 kDa forms of
gelatinase in the CaCl 2 treated material (B) The amount of TIMP-1
used was 1 (lane 3), 10 (lane 4) and 20 n M (lane 5) In (A) and (B), at
the left side is shown the position of the 225 and 92 kDa forms of
proMMP-9 in serum-free culture medium of THP-1 cells and the
72 kDa form of proMMP-2 in serum-free culture medium of human
skin fibroblasts Arrow shows the border between the stacking and the
separating gel Due to the high glycosylation of the proMMP-9/CSPG
complex, the proteins migrate as if theyinitiallyare distributed to the
edges of the stacking gel well, and two spots in the separating gel
appears instead of a clear band This is typical for highly glycosylated
proteins as described byCarlsson [50].
Trang 6that no band was detected at 80/84 kDa in the CaCl2
-treated sample, but onlya strong band at approximately
70 kDa indicates that also a certain degree of C-terminal
processing is necessaryto fullyexpose the epitope The
70 kDa band was not detected bythe polyclonal antibody
against the C-terminal region of MMP-9 (Fig 5C, lane 2),
showing that the 74/76 kDa bands lack large parts of their
C-terminal region This antibodydetected a weak band
at around 80 kDa in the CaCl treated material (Fig 5C,
lane 2) Thus, calcium induced both an N- and a C-terminal truncation of CSPG bound proMMP-9
As a control, the proMMP-9/CSPG complex was treated with either 10 mM EDTA, 1 mM 1,10-phenanthroline or various amounts of TIMP-1 (50, 100 and 200 nM) prior to incubation of these mixtures with 10 mMof CaCl2in 2 h at
37C These samples were thereafter treated with 0.1M dithiothreitol and subjected to SDS electrophoresis and analyzed by Western blotting, using the two polyclonal antibodies against the hinge and C-terminal region, respect-ively As seen in Fig 5B,C (lanes 3 and 4), only the 92 kDa species is seen in the samples treated with either EDTA or 1,10-phenanthroline, and the intensitycorresponds to the untreated sample Increasing concentrations of TIMP-1 resulted in a successivelyreduced amount of the 70 kDa form of the enzyme (using the antibody against the hinge region, data not shown)
The released forms of MMP-9 from the heterodimer are active
The proMMP-9/CSPG complex treated with 10 mMCaCl2 for 2 h at 37C was applied to Q-Sepharose chromato-graphy The released truncated forms of MMP-9 were collected in the flow-through fraction, whereas the MMP-9 bound to CSPG were eluted from the column by1.5M NaCl The released forms of MMP-9 degraded [3H]-labeled gelatin, both in the presence and absence of 10 mMCaCl2 (Fig 6A, lanes 5 and 6), whereas the MMP-9 complexed to the CSPG needed CaCl2for activity(Fig 6A, lanes 7 and 8) Gelatin zymography revealed that this second addition
of CaCl2resulted in a further release of low Mrforms of the gelatinase from the complex (data not shown)
The flow-through fraction from the Q-Sepharose column
of the CaCl2-treated proMMP-9/CSPG complex contained the 74/76 kDa, 80/85 kDa and 100 kDa forms of MMP-9
To determine which of these forms were active in solution, proMMP-9/CSPG complex was incubated with CaCl2at various time intervals and then mixed with a2-macroglo-bulin, an inhibitor known to bind and trap active MMPs but not the proform of the enzymes [39] Gelatin zymography (Fig 6B) showed that all the released forms of MMP-9 (74/76, 80/85 and 100 kDa) reacted with a2-macroglobulin, which resulted in a partial or total disappearance of the gelatinolytic zones This indicated that all four forms were active in solution However, no change in intensityof the MMP-9 complexed to CSPG was observed in the presence
of a2-macroglobulin
CaCl2-induced activation of the MMP-9/CSPG complex was not abolished by removal of potential contaminating proteins bound to the CS-chains
Some MMP-9/CSPG preparations needed about 20–24 h incubation with 10 mMof CaCl2at 37C to release low Mr forms from the complex, while for most preparations release
of low Mrforms was obtained after 2 h incubation It is known that CS chains can bind various proteins [40] The variation in time needed for activation of the proMMP-9 in different preparations could therefore be due to different amounts of contaminating proteins that inhibits the CaCl-induced activation of the proMMP-9 in the CSPG
Fig 4 Time dependent release of low M r forms of thegelatinaseduring
theactivation of theproMMP-9/CSPG complex with CaCl 2 Gelatin
zymography of 4 lg per lane of CSPG (A and B represent two
dif-ferent CSPG preparations), which has been incubated with 10 m M of
CaCl 2 for various time points at 37 C as indicated under the figure.
The activation was stopped at the indicated time points bythe addition
of 10 m M EDTA to the reaction mixture At the right is shown the
position of the 225 and 92 kDa forms of proMMP-9 in serum-free
culture medium of THP-1 cells and the 72 kDa form of proMMP-2 in
serum-free culture medium of human skin fibroblasts Arrow shows
the border between the stacking and the separating gel.
Trang 7complex Alternatively, the activation could be due to the
presence of another metalloproteinase that cleaves and
activates the proMMP-9 in the complex in the presence of
CaCl2
As TIMP-1 inhibits the CaCl2-induced activation of the
MMP-9/CSPG complex, we investigated whether the
THP-1 cells produced TIMP-1 Western blots showed that
the serum-free conditioned THP-1 medium contained
TIMP-1 (Fig 7, lane 2), and from ELISA the amount of
TIMP-1 in the conditioned medium was estimated to be
approximately8.4 lgÆmL)1 We also investigated whether
some of the TIMP-1 was bound to the proMMP-9/CSPG
complex in spite of the dissociating conditions used to avoid
unspecific binding during the isolation procedure In some
of the purified MMP-9/CSPG preparations, a small amount
of TIMP-1 was detected (Fig 7, lane 1), while no TIMP-1
was detected in other preparations (data not shown) The
various amounts of TIMP-1 in the different proMMP-9/
CSPG preparations maytherefore explain the variations in
the time needed to activate the complex with CaCl2
During purification, 6M urea was present to prevent
aggregation and to remove proteins reversiblybound to the
proMMP-9/CSPG complex Urea and salts were finally
removed bygel filtration, followed bya concentration step
of the purified complex If urea and salts were not properly
removed in all preparations, this might affect the CaCl2
-induced conversion of the complex to lower Mr forms of
the gelatinase, as well as the cABC degradation of the
CS-chains of the PG The presence of 4.5M urea in the
proMMP-9/CSPG preparation had no effect on the
posi-tion of the two main gelatinase bands (Fig 8, lanes 1 and 2),
suggesting that neither the band seen in the stacking gel nor
the 300-kDa band at the top of the separating gel are due
to aggregation However, 4.5 urea inhibited both the
CaCl2-induced conversion of the complex to lower Mr forms (Fig 8, lanes 5 and 6) and also cABC-degradation of the CS-chains (Fig 8, lanes 3 and 4) Up to 0.5Murea had
no effect on the CaCl2-induced conversion to lower Mr forms, while a concentration-dependent effect was seen from 1Mand up to 4.5M(data not shown) In contrast to this, it was first at a concentration of 4.5Mthat urea had an effect on the cABC degradation of the CS-chains Thus, remnants of urea in some preparations mayexplain the delayin the kinetics of the CaCl2-induced conversion of the proMMP-9/CSPG complex to lower Mrforms
Previously, we have shown that conditioned THP-1 medium did not contain MMP-1, MMP-2 and MMP-8 [23] The two former enzymes are known activators of MMP-9 [13] Another MMP that is known to activate MMP-9 is matrilysin (MMP-7) [13] that is also known to bind strongly
to GAG-chains, especiallyto heparin and heparan sulphate [41] However, neither the THP-1 medium nor the purified proMMP-9/CSPG complex revealed bands between 18 and
30 kDa in zymograms of gels containing either gelatin, casein or carboxymethylated-transferin (data not shown); nor was MMP-7 detected with Western blots (data not shown)
The proMMP-9/CSPG complex was treated in ways that were expected to result in the release and separation of potential contaminating proteins bound to the CS-chains of the complex In those experiments both intact and cABC-treated proMMP-9/CSPG complexes were subjected to the same chromatographycolumns, but in different experi-ments Conditions were used so that onlythe intact or the cABC-treated complexes bound to the column Intact proMMP-9/CSPG bound to the Q-Sepharose column, while the proMMP-9/PG-core protein passed through this column The opposite was the case when these complexes
Fig 5 Western blots showing N- and C-terminal truncation of the low M r forms from theCaCl 2 -activated MMP-9/CSPG complex Purified CSPG (30 lg) was treated for 2 h at 37 C with (+) or without (–) 10 m M CaCl 2 , 10 m M EDTA, 1 m M 1,10-phenanthroline (OP) as indicated under the figure Western blots were run under reducing conditions, i.e samples were treated with 0.1 M dithiothreitol prior to electrophoresis In A, the monoclonal MMP-9 antibody(IM10L) from Amersham was used This antibodydetects onlythe pro-form of the enzyme In B, a polyclonal antibodyagainst the hinge region of MMP-9 was used In C, a polyclonal antibodyagainst the C-terminal region of MMP-9 was used M r standard markers are shown at the left, and the arrowhead indicates the position of the 92 kDa band in the serum-free culture medium from THP-1 cells.
Trang 8were applied to Gelatin-Sepharose and Heparin-Sepharose columns In other experiments, intact proMMP-9/CSPG was subjected to gel chromatography(Sephacryl 200) in the presence and absence of EDTA In all these experiments, the obtained proMMP-9/CSPG complex and proMMP-9/ PG-core protein complex could be activated bythe addition
of CaCl2 However, none of the complexes could be activated byAPMA or APMA plus CaCl2 as shown by zymography and degradation of 3H-labeled gelatin (data not shown) This stronglyindicates that the calcium-induced activation of the proMMP-9 complexed to CSPG was not due to anyCS-chain bound contaminants
Fig 8 Urea inhibits both the CaCl 2 -induced release of low M r forms of thegelatinaseand cABC degradation of theCS-chains in thecomplex Four micrograms of purified CSPG was incubated for 2 h at 37 C with (+) or without (–) 4.5 M urea, cABC and 10 m M CaCl 2 as indi-cated under the figure Arrow shows the border between the stacking and the separating gel At the left side is shown the position of the 225 and 92 kDa forms of proMMP-9 in serum-free culture medium of THP-1 cells and the 72 kDa form of proMMP-2 in serum-free culture medium of human skin fibroblasts.
Fig 7 Western blots showing TIMP-1 in purified MMP-9/CSPG and
in serum-free THP-1 conditioned medium Both the purified CSPG (30 lg) and the conditioned medium was treated with 0.1 M dithio-threitol prior to electrophoresis M r standard markers are shown at the left The position of commercial human recombinant TIMP-1 was identical with the bands seen in the figure.
Fig 6 Released forms of MMP-9 from the CaCl 2 -treated heterodimer
areactive (A) Purified CSPG was treated with CaCl 2 for 2 h at 37 C,
and thereafter applied to a Q-Sepharose column as described in
Experimental procedures MMP-9 forms that were released from the
CSPG were collected in the flow through (F) fractions, whereas the
MMP-9 that was still bound to CSPG (B) was attached to the column.
The bound material was eluted with 1.5 M NaCl The flow through
material (F), the bound material (B) and the starting material of intact
CSPG (U) was either treated for 2 h at 37 C with (+) or without (–)
10 m M CaCl 2 as indicated under the figure, and then incubated with
[ 3 H]gelatin for approximately24 h at 37 C as described in
Experi-mental procedures At the left is shown the M r of the rainbow M r
standard markers in lane 1, and the arrowhead indicates the bottom of
the application well Lane 2 shows the negative control of nondegraded
[3H]gelatin, and lane 3 shows a positive control of trypsin digested
[3H]gelatin In lane 5, the bands at 30 kDa and below appear as two
spots instead of a band due to a crack in the dried gel (B) Gelatin
zymography of CSPG treated with CaCl 2 at different time intervals as
indicated under the figure At the indicated time points, each sample was
either untreated (–) or treated (+) with 800 lgÆmL)1of
a2-macro-globulin (a2MG) for 10 min at 37 C, after which 10 m M EDTA was
added to the reaction mixture to stop the activation At the left is shown
the position of the 225 and 92 kDa forms of proMMP-9 in serum-free
culture medium of THP-1 cells and the 72 kDa form of proMMP-2 in
serum-free culture medium of human skin fibroblasts The arrow shows
the border between the stacking and the separating gel.
Trang 9The calcium-induced activation of proMMP-9 is restricted
to the proMMP-9 covalently bound to CSPG
If the calcium-induced activation of proMMP-9
com-plexed to CSPG was due to anycontaminations, it should
be expected that such contaminants also could activate the
monomeric/homodimeric proMMP-9 Monomeric and
homodimeric forms of proMMP-9 were therefore purified
from THP-1 conditioned medium As shown in Fig 9, the
same pattern occurred when proMMP-9 was incubated
with either CaCl2 treated or untreated proMMP-9/CSPG
complex in the presence or absence of
1,10-phenanthro-line This was also the case when these mixtures were
treated with or without Brij-35 (0.05%) or SBTI (data not
shown) These experiments show that the MMP-9/CSPG
complex in the presence of calcium is not able to activate
and process externallyadded monomeric/homodimeric
proMMP-9, which indicates that the calcium-induced
activation and processing of the proMMP-9 is restricted
to proMMP-9 complexed to CSPG Further, the results
stronglysuggest that the calcium-induced activation does
not involve anycontaminating calcium-dependent
pro-teinase, but is due to an autoproteolysis of the
proMMP-9/CSPG complex
To investigate if the calcium-induced activation of the
proMMP-9/CSPG complex is a bimolecular reaction or
not, various concentrations of the complex were incubated
with and without 10 mMof exogenous CaCl2for 0–4 h at
37C As shown in Fig 10, the calcium-induced activation
was concentration independent, which stronglysuggests
that the autoactivation and the truncation process is not a
bimolecular reaction, but rather an intramolecular reaction
Discussion
Previously, we have shown that a significant amount of the MMP-9 produced byTHP-1 cells is linked to the core protein of CSPG [23] Western blots indicated that it was the
92 kDa proform that was bound to the CSPG In the present work we have investigated the activityand condi-tions for inducing activation of the MMP-9 bound to the CSPG core protein The enzyme in the complex was inactive
in the soluble activityassayin the absence of exogenously added calcium, supporting that the synthesized complex contains onlythe 92 kDa pro-form of the gelatinase The addition of exogenous calcium resulted in the generation of N- and C-terminallytruncated forms of MMP-9 which were enzymatically active Inhibition studies showed that the truncation and activation of the pro-MMP-9 in the complex was due to a metalloproteinase, and most likelya matrix metalloproteinase as TIMP-1 inhibited the activation Our results exclude calcium-induced truncation and activation as
a bimolecular process that involves either another metallo-proteinase or an N-terminallytruncated form of MMP-9 The process is more likelyto be due either to an intramolecular autoactivation process (i.e within one het-erodimer) or to two MMP-9/PG molecules existing as dimers where the MMP-9 in each subunit might proteo-lytically cross-activate each other Although the autoacti-vation in the latter scenario involves two distinct MMP-9 molecules, the process will not follow the kinetics of a bimolecular reaction as the two MMP-9 molecules that act
on each other occurs within the same dimer This conclusion
Fig 10 Calcium-induced truncation and release of MMP-9 from the proMMP-9/CSPG complex was not dependent on the concentration of thecomplex Various concentrations of the proMMP-9/CSPG com-plex were incubated for 2 h at 37 C in the absence (–) or presence (+)
of CaCl 2 (10 m M ) as indicated in the figure, after which 2.1 lg of CSPG was withdrawn and loaded to the gel Shown are the released forms from 74 to 100 kDa in the calcium-treated material, while a very faint band is seen in the untreated material To the left is shown the position of the 92 kDa form of proMMP-9 in serum-free culture medium of THP-1 cells and the 72 kDa form of proMMP-2 in serum-free culture medium of human skin fibroblasts At the bottom of the figure is shown the relative amount (± SD) of the CaCl 2 -induced 74–100 kDa forms from four independent experiments The amount released from the CSPG with the lowest concentration (0.5 mgÆmL)1) was set to 100%.
Fig 9 Neither proMMP-9/CSPG nor calcium-activated proMMP-9/
CSPG activates endogenously added proMMP-9 monomer and
homodimer The monomer (92 kDa) and dimer (225 kDa) forms of
proMMP-9 was isolated from conditioned THP-1 medium as
des-cribed in Experimental procedures The partlypurified proMMP-9
was incubated for 24 h at 37 C with 3 lg of purified CSPG in the
presence (+) or absence (–) of 10 m M CaCl 2 and 1 m M
1,10-phen-anthroline (OP) as indicated under the figure Aliquots corresponding
to 0.27 lg of CSPG were then added to the gel, in order to prevent the
appearance of the released forms of MMP-9 from the complex The
arrowhead shows the border between the stacking and separating gels,
and the arrow shows the position of the 72 kDa form of proMMP-2 in
serum-free culture medium of human skin fibroblasts Similar results
appeared when the CSPG was treated with CaCl 2 for 2 h at 37 C
prior to mixing with the partlypurified proMMP-9.
Trang 10is based on the following observations: (a) neither
calcium-treated nor -uncalcium-treated proMMP-9/CSPG activated
exo-genouslyadded monomeric or homodimeric proMMP-9;
(b) the calcium-induced activation of the proMMP-9 in the
complex was not dependent on the concentration of the
complex; (c) calcium-induced activation took place even in
preparations of the proMMP-9/CSPG and proMMP-9/
PG-core protein complexes that were treated in such ways
that possible CS-chain bound activators were released and
removed from the complex Large amounts of
endogen-ouslyproduced TIMP-1 in the conditioned medium
prob-ablyexplains whythe proMMP-9 in the complex was not
activated and processed to low Mrforms alreadyduring the
72 h cell synthesis period, and hence before the isolation
procedure started
In the present work, it is shown that the interaction
between proMMP-9 and the CSPG core protein causes
changes in the proMMP-9 with respect to its abilityto
autoactivate The first example is the response to urea
During the purification of the complex, large amounts of
urea was added to the preparation to dissolve and remove
reversiblybound contaminants from the complex Although
urea is known to induce autoactivation and processing of
92 kDa proMMP-9 to lower Mrforms [42], the treatment
of the proMMP-9/CSPG complex with urea during the
purification procedure did not induce activation and
processing of the proenzyme in the complex Only the
92 kDa pro-form of MMP-9 was detected in the purified
material under reducing conditions
The second example is the response to APMA alone or in
combination with CaCl2, which did not result in truncation
and activation of the enzyme in the proMMP-9/CSPG
complex Previous studies have shown that treatment of
calcium-depleted proMMP-9 with APMA resulted in an
inactive enzyme that had lost approximately 8–9 kDa of its
N-terminal prodomain, with Met75 as the N-terminal
residue [32,33] This form of the enzyme had retained the
78PRCGVPD sequence that blocks the active site
[32,43,44] However, in the presence of Ca2+the
APMA-treated enzyme was active This was due to a further
processing of the enzyme, such that its C-terminal end was
autocatalytically removed [32,43,44] It has been suggested
that calcium induced a conformational change in the
N-terminallytruncated enzyme and unblocked the active
site Site-directed mutagenetic studies indicate that calcium
interacted with Asp432 and probablya residue in the
remaining prodomain or in the catalytic domain [33]
Several reports show that the APMA-induced
autoactiva-tion of various proMMPs is a complicated process To
achieve an understanding of the mechanism behind the
APMA induced activation, Cys75 was chemically modified,
the prodomain was successivelydeleted and the amino acids
in the 73PRCGVPD were changed through site-directed
mutagenesis of proMMP-3 [45–47] These studies indicated
that APMA was first bound to residues other than Cys75 in
the prodomain and induced a conformational change prior
to binding to the Cys75, followed by autoactivation It was
also shown that if 63 or more amino acids in the prodomain
of MMP-3 were deleted, addition of APMA no longer
accelerated, but rather inhibited the autoactivation process
The third example that shows the interaction between
proMMP-9 and the PG-core protein alters the abilityof the
gelatinase to autoactivate is the effect of calcium, which is a stabilizer of proMMP-9 and other MMPs, but induces truncation and activation of the proMMP-9/CSPG com-plex The various and complex effects metals exert on MMPs can be visualized bya recent study, where it was shown that calcium and zinc, but neither of the metals alone, could activate a truncated form of human
proMMP-3 that lacked the first proMMP-34 N-terminal amino acids and the entire C-terminal hemopexin domain [48] The difference in response between proMMP-9 and the proMMP-9/CSPG complex to the treatment of urea, APMA, APMA in combination with calcium, and calcium alone is most likely due to the interaction between the enzyme and the core protein of the CSPG, and not through a general electrostatic effect involving the CS-chains as 200 mM of NaCl alone could not induce activation of the complex, nor did high salt concentrations affect the calcium-induced activation Our observations that the ionic strength is not of importance for the activation was also reflected bythe fact that the activation was equallyas effective at 100 mMas at 10 mMof calcium The metal-induced activation of the MMP-9/ CSPG complex was specific for calcium as the activation process could not be mimicked byeither NaCl, MgCl2, ZnCl2or mercury(APMA) Thus, the interaction between proMMP-9 and the CSPG core protein probablygenerates
a binding site for exogenous calcium that causes destabili-zation in the proenzyme and allows for autoactivation of the enzyme This interaction must also hide the epitopes that are normallyinvolved in the APMA-induced activation of MMP-9, and expose epitopes that results in an APMA-induced inhibition of the calcium-APMA-induced activation Recentlyit has been shown that the various forms of proMMP-9 are not equallysusceptible to activation The monomeric and homodimeric forms of MMP-9 respond differentlyto MMP-3-induced activation of these enzymes [22], while the proMMP-9/NGAL heterodimer was more effectivelyactivated bymercurial compounds in the pre-sence of human neutrophil lipocalin (HNL) than both the monomeric and homodimeric forms of proMMP-9 [49] Thus, formation of various dimers of MMP-9 results in enzyme variants with altered biochemical properties, which expand the biological properties and function of the enzyme that might be optimal under various conditions
The calcium-induced activation of the proMMP-9/CSPG complex resulted in truncated variants of MMP-9 that had lost both the N-terminal domain and large parts of the C-terminal domain The truncated variants that were released from the CSPG had lost their N-terminal part and were active in solution, as theyreacted with a2-macro-globulin and degraded3H-labeled gelatin In addition, the smallest truncated variants contained the hinge region that connects the catalytic site and the C-terminal hemopexin-like domain, but at least a part of the C-terminal domain was lacking These results indicate that the interaction between MMP-9 and the CSPG core protein must involve the most C-terminal part of the MMP-9 hemopexin-like domain Some of the truncated variants that had lost their N-terminal pro-domain, were active and reacted with a2-macroglobulin, and had an Mrthat was larger than the
92 kDa proform of MMP-9 In those variants, a part of the core protein from the CSPG must still be linked to the N-terminal truncated MMP-9 molecule Thus, the