Endothelin is a potent inhibitor of matrix metalloproteinase-2
secretion and activation in rat mesangial cells
Jian
Yao,
Tetsuo
Morioka,
Bing
Li, and
Takashi
Oite
Department of Cellular Physiology, Institute of Nephrology, Niigata
University School of Medicine, Niigata 951 - 8510, Japan
 |
ABSTRACT |
We examined the
effects of endothelin (ET) on the activity of matrix
metalloproteinase-2 (MMP-2) in cultured MCs. Addition of the
ETA receptor antagonists or neutralizing anti-endothelin antibody into MC cultures markedly augmented the secretion and activation of MMP-2. On the contrary, addition of the exogenous ET-1
into MC culture significantly inhibited the synthesis of MMP-2 in both
basal and cytokines (tumor necrosis factor-
and interferon-
) plus
lipopolysaccharide-stimulated conditions. Furthermore, pretreatment of
cells with exogenous ET-1 obviously prevented cytochalasin
D-elicited activation of MMP-2, an effect that was completely abolished by ETA receptor antagonist, FR139317.
In addition, ET-1 was found to be able to suppress the expression of
membrane type-1 MMP (MT1-MMP) and promote the conversion of tissue
inhibitor of matrix metalloproteinase-2 (TIMP-2) from cell associated
form to secreted form. The addition of recombinant TIMP-2 into the
culture abrogated dose-dependently the cytochalasin D-elicited activation of MMP-2. These results suggest that
ET is a potent inhibitor of MMP-2 secretion and activation in MCs. These novel findings may help us understand the subtle regulation of
the synthesis and activation of MMP-2 in MCs. It also provides us with
further insight into the pathophysiological mechanisms involving ET in
the regulation of matrix turnover in glomerulus.
endothelin receptor antagonist; tissue inhibitor of matrix
metalloproteinase-2; zymography; cytochalasin D; cytokines
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INTRODUCTION |
THE EXTRACELLULAR MATRIX
(ECM), which includes the glomerular basement membrane (GBM) and
the mesangial matrix, fulfills an important structural and regulatory
function in the renal glomerulus. In the numerous pathological
processes involved in the glomerulus, the integrity of GBM and
mesangial matrix is frequently disrupted. Proteinuria, partially due to
proteolytic destruction of the basement membrane, and mesangial
expansion, resulting from the abnormal accumulation of matrix
molecules, are two major hallmarks of glomerular diseases. Thus
knowledge of the mechanisms and processes regulating the synthesis and
breakdown of GBM or mesangial ECM is critical for our understanding of
the pathogenesis of glomerular diseases.
Located in the center of the glomerulus, the intrinsic mesangial cell
(MC) plays a key role in both synthesis and degradation of the
glomerular ECM (35). There have been extensive reports about the ability of cultured MCs to synthesize a broad variety of
glomerular ECM proteins (18, 35). Recently, the
expressions of proteolytic activity by MCs and its role in glomerular
pathophysiology have been the focus of numerous studies (4, 12,
14, 20, 31, 35, 37, 39). Matrix metalloprotease-2 (MMP-2, also called gelatinase A or collagenase IV) is the predominant MMP synthesized by MCs (12, 13) and plays an essential role in the remolding of ECM under various physiological and pathological conditions (10). Altered expression and activation of
MMP-2 in a variety of glomerular diseases, in both human beings and experimental animal models, have been reported (14, 31,
37). In addition to its collagen-degrading activity, it has been
recently suggested that active MMP-2 may play a role in the
proliferation and in the expression of an inflammatory phenotype of rat
mesangial cells (39). A wide variety of compounds, such as
inflammatory cytokines and mediators [interleukin-1 (IL-1), tumor
necrosis factor-
(TNF-
), interferon-
(IFN-
),
PGE2, nitric oxide], growth factors (TGF-
), and tumor
promoting agent phorbol 12-myristate 13-acetate (PMA), as well as
nonphysiological agents such as cytochalasin D and concanavalin A, have
been demonstrated to be able to upregulate MMP-2 expression and/or
activation in MCs (1, 2, 13, 26, 27, 29, 38, 42). However,
factors involved in the downregulation of MMP-2 activity in MCs have
not been studied in any depth.
Endothelin (ET) is one of the major pathogenic factors implicated in
the abnormal accumulation of ECM in the kidney. Increased expressions
of ET as well as ET receptors are found in a variety of glomerular
diseases with matrix deposition (7, 8). Treatment with a
specific ETA receptor antagonist attenuates ECM
accumulation and glomerulosclerosis in several models of kidney injury
(5, 6). Furthermore, induction of the synthesis of
multiple extracellular matrix components via ETA receptor
by ET in cultured MCs has been reported (18). Because
matrix turnover represents both matrix synthesis and degradation, an
alternative way of ET action on matrix accumulation might be the
inhibition of matrix degradation, via regulation of the synthesis
and/or activation of proteolytic enzymes, released by MCs or other cell
types. In this respect, the suppressive effects of ET on the activity
of fibrinolysis via upregulation of plasminogen activator inhibitor in
cultured MCs have been documented (21). In addition,
exogenous ET-1 was found to be able to inhibit the activity of
collagenase in cultured cardiac fibroblast (19).
Conceivably, ET might also be able to modulate the activity of MMP-2 in
cultured MCs. This study was designed to test this hypothesis. Our
results demonstrate that ET is a potent inhibitor of both MMP-2
secretion and activation in cultured rat MCs.
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MATERIALS AND METHODS |
Materials.
Gelatin, lipopolysaccharide (LPS), polyclonal rabbit anti-MMP-2
antibody, anti-tissue inhibitor of metalloproteinase-2 (TIMP-2) antibody, prestained molecular weight markers for SDS-PAGE, DMEM, and
synthetic human endothelin-1 were obtained from Sigma (St. Louis, MO).
Monoclonal antibody to endothelin-1, -2, and -3 was purchased from
Biogenesis. BQ-123 and IRL-1038 were from Alexis (San Diego, CA).
FR139317 was a gift of Fujisawa Pharmaceutical (Osaka, Japan).
Recombinant hTIMP-2 was purchased from Fuji Chemical (Toyama, Japan).
An MMP-2 activity assay kit was obtained from Amersham Pharmacia
(Piscataway, NJ). Microcon and Immobilon polyvinylidene difluoride
membrane were supplied by Millipore (Bedford, MA). Enhanced
chemiluminescence (ECL) reagents were obtained from Amersham (Arlington
Heights, IL).
Rat MC culture.
MC isolation and culture were performed as described previously
(40, 41). In brief, the renal cortices of male Wistar rats
(150 g) were homogenized under sterile conditions and passed over three
sieves with pore sizes of 200, 100, and 75 µM. Glomeruli, which were
retained on the 75-µm sieve, were seeded in DMEM containing 20% FCS,
insulin (5 µg/ml), penicillin (100 U/ml), and streptomycin (100 U/ml). After three to four passages in DMEM containing 20% FCS, pure
MC populations were obtained. MCs were characterized by the following
criteria: positive immunocytochemical staining with antibodies against
Thy-1.1 (32) and smooth muscle
-actin, absence of
staining for antigen factor VIII, and cytokeratins. MCs were used for
experiments between passages 5 and 20.
Preparation of conditioned medium.
MCs were seeded into a 24-well culture plate and allowed to grow in
20% FCS-DMEM until 90% confluence; then MCs were thoroughly washed
with serum-free DMEM and starved in the same medium for 1 day.
Afterward, the medium was replaced with fresh serum-free medium with or
without the addition of particular test agents, and the culture was
continued for the indicated time intervals. The conditioned medium was
collected, centrifuged, aliquoted, and stored at
70°C until required.
Zymography.
To analyze gelatinolytic activity, aliquots of MC conditioned medium
(20 µl) were mixed with 5 µl 5× nonreducing sample buffer (0.5 M
Tris · HCl, pH 6.8, 10% SDS, 50% glycerol, 0.5% bromphenol blue). Electrophoresis was performed in a 0.75-mm-thick 7.5%
polyacrylamide/SDS gel containing 1 mg/ml gelatin as substrate. After
electrophoresis the gels were incubated in 2.5% Triton X-100 and 50 mM
Tris · HCl, pH 7.5, for 1 h at room temperature, followed
by 16-20 h at 37°C in a collagenase buffer containing 50 mM
Tris · HCl, pH 7.5, 100 mM NaCl, and 10 mM CaCl2.
Thereafter, the gels were stained with Coomassie blue, and zones of
lysis were visualized. Standard protein markers were utilized for
assignment of molecular mass.
Detection of MMP-2 activity by antibody capture assay.
To quantitate the MMP-2 activity secreted by MCs, a specific MMP-2
activity assay was conducted by using an antibody capture method
(11). For this assay, purified MMP-2 or MC culture
supernatants were added to wells of a 96-well microliter plate
previously coated with a monoclonal MMP-2 antibody (RPN2631; Amersham
Pharmacia). After incubation overnight at 4°C, plates were washed
vigorously with phosphate buffer solution containing 0.05% Tween 20. Next, p-aminophenylmercuric acetate (1 mM), an
organomercurial, was added to activate any captured MMP-2, after which
an enzyme substrate solution containing 50 mM Tris · HCL, 1.5 mM NaCl, 0.5 mM CaCl2, 1 µM ZnCl2, 0.01%
BRIJ 35, and chromogenic peptide substrate S-2444 (Amersham Pharmacia
Biotech) was introduced. The reaction was allowed to proceed at 37°C
for 2 h, and then the absorbance at 405 nm was recorded. Cleavage
of chromogenic substrate produced a linear increase in absorbance with
increasing concentrations of MMP-2 standards. MMP-2 activity and
concentration in MC culture supernatants were expressed as nanograms
per milliliter based on the results obtained with purified MMP-2 standards.
Western blots.
MCs were seeded onto 60-mm culture plates and allowed to grow in 20%
FCS-DMEM until 90% confluence. MC were then starved in serum-free DMEM
for 2 days, before being stimulated with different agents for various
periods of time. The reaction was terminated by washing cells rapidly
with cold PBS at 4°C. The cells were lysed with RIPA lysis buffer (50 mM Tris · HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1%
deoxycholate, 0.1% SDS) containing 25 µg/ml aprotinin, 2 mM sodium
orthovanadate, 25 µg/ml leupeptin, 2 mM phenylmethylsulfonyl
fluoride, and 50 mM sodium fluoride for 30 min on ice. Lysates were
clarified by centrifugation at 13,000 rpm for 15 min at 4°C, and
protein concentrations were determined by using a Bio-Rad protein assay
kit. Equal amounts of cellular lysates or concentrated culture
supernatants were separated in 7.5% SDS-polyacrylamide gels and
electrotransferred to 0.4 µM polyvinylidene difluoride membranes. The
membranes were blocked with 3% BSA in PBS-0.1% Tween 20, pH 7.4, overnight at 4°C. After washing with PBS-0.1% Tween 20, membranes
were incubated with either anti-MMP-2 antibody (1:1,000) or anti-TIMP-2
(1:1,000) antibody at room temperature for 1 h. After extensive
washing with three changes of PBS-0.1% Tween 20, membranes were
incubated for 1 h with horseradish peroxidase-conjugated sheep
anti-rabbit IgG or rabbit anti-mouse IgG at 1:10,000 dilution in
blocking buffer. After washing, immunoreactivity was detected by using the ECL system.
RT-PCR.
Total RNA was extracted from mesangial cells by using a kit of RNA STAT
from Tel-Test B (Friendswood, TX). First-strand cDNA was synthesized by
a T-Primed First-Stand Kit from Amershan Pharmacia. PCRs were performed
and optimized according to standard protocols by using a kit from
TaKaRa Shuzo (Shiga, Japan). Primers used for PCR were custom
synthesized (GIBCO-BRL), and the sequences of each primer were as
follows: 1) MMP-2, forward, 5'-ATCTGGTGTCTCCCTTACGG and
reverse, 5'-GTGCAGTGATGTCCGACAAC; 2) TIMP-2, forward,
5'-CAAAGGACCTGACAAGGAC and reverse, 5'-TTGATGCAGGCAAAGAAC;
3) MT1-MMP, forward, 5'-ATTGATGCTGCTCTCTTCTGG and reverse,
5'-GTGAAGACTTCATCGCTGCC; and 4) GAPDH, forward,
5'-TCCCTCAAGATTGTCAGCAA and reverse, 5'-AGATCCACAACGGATACATT. The
predicted sizes of the amplification products are 150, 182, 348, and
308 bp for MMP-2, MT1-MMP, TIMP-2, and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), respectively.
Statistical analysis.
Statistical analyses were performed by the Student's
t-test. Data are presented as means ± SD. P
values of <0.05 were considered statistically significant.
 |
RESULTS |
ET is an endogenous inhibitor of MMP-2 secretion and activation.
We first examined whether endogenous ET had any effect on the basal
secretion of MMP-2 by cultured MCs. For this purpose, a neutralizing
monoclonal antibody against ET-1, -2, and -3 was added to MC cultures,
and this produced a dose-dependent enhancement of MMP-2 activity, as
revealed by zymography (Fig.
1A). The effect was not
observed after addition of an irrelevant, isotype-matched control IgG.

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Fig. 1.
Effects of neutralizing anti-endothelin-1 (ET-1), -2, and
-3 monoclonal antibody and ETA receptor antagonists on
matrix metalloproteinase-2 (MMP-2) activity as revealed by
zymography. Mesangial cells (MCs) were incubated with the indicated
concentrations of neutralizing anti-ET antibody (A,
C) or ETA receptor antagonists (B,
C) for periods of up to 72 h. Culture supernatants were
harvested and assayed for gelatinase activity as described in
MATERIALS AND METHODS. Similar results were obtained in 2 additional experiments.
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ETA receptor antagonist has been reported to be effective
in attenuating ECM accumulation and glomerulosclerosis in several models of kidney injury, as well as in blocking ET-elicited synthesis of ECM in cultured MCs (5, 6, 18). We therefore asked whether ETA receptor antagonist could also enhance the
activity of MMP-2 via blocking of the functional receptor of ET. As
depicted in Fig. 1B, incubation of MCs with ETA
receptor antagonist FR139317 resulted in an obvious increase in MMP-2
activity. This action of FR139317 was concentration dependent and most
obvious at 24 h (Fig. 1B). A similar effect was
observed with another ETA receptor antagonist, BQ123 (Fig.
1C).
In longer term cultures, there was a spontaneous activation of MMP-2,
as revealed by the appearance of an additional band of molecular mass
about 62 kDa in the zymogram (Fig. 1). In the presence of
ETA receptor antagonists or anti-ET antibody, this band
appeared earlier and clearly increased in intensity (Fig. 1). These
results suggest that ET is an endogenous inhibitor of MMP-2 secretion
and activation in MCs.
The zymographic data were further confirmed by Western blot by using a
specific anti-MMP-2 antibody. As shown in Fig.
2, the neutralizing anti-ET antibody
(Fig. 2A) and ETA receptor antagonist (Fig.
2B) increased the protein secretion of MMP-2 in a
dose-dependent fashion.

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Fig. 2.
Effects of the neutralizing anti-ET-1, -2, and -3 monoclonal antibody and ETA receptor antagonist on MMP-2
protein secretion by Western blot analysis. MCs were incubated with the
indicated concentrations of neutralizing anti-ET antibody
(A) or ETA receptor antagonists (B)
for 24 h. Equal amounts of supernatants were concentrated and
analyzed for MMP-2 protein by using immunoblotting as described in
MATERIALS AND METHODS. Blots shown are 1 representative
experiment from a series of 3, with similar results.
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Exogenous ET-1 inhibits the secretion of MMP-2.
If ET is an endogenous inhibitor of MMP-2, addition of exogenous ET-1
into cultures should result in a reduction of MMP-2 activity. As shown
in Fig. 3, the addition of exogenous ET-1
into MC culture slightly lowered the basal level of MMP-2 secretion as
revealed by zymography (Fig. 3). To clearly demonstrate the inhibitory
action of exogenous ET-1 on MMP-2 production, we examined the effects
of ET in a system where the activity of MMP-2 was amplified by the
addition of different combinations of TNF-
, IFN-
, or LPS into MC
culture. As indicated in Fig. 4, in the presence of stimulants, the activity of MMP-2 was obviously enhanced, and this enhancement could be partially prevented by pretreatment of MC
with exogenous ET-1 (10
7 M) (Fig. 4). Because zymography
is only a semi-quantitative assay for MMP-2, to further confirm the
above results and to accurately quantitate the change of MMP-2 in the
presence of exogenous ET-1, additional quantitative assays for MMP-2
were employed. By using an antibody capture assay for MMP-2 activity,
we selectively examined the effects of ET-1 on cytokines (INF-
and
TNF-
) plus LPS-stimulated releasing of MMP-2 by MCs. It was found
that under the stimulated condition, MCs increased the secretion of
MMP-2 more than twofold that of control, whereas treatment of MCs with
ET-1 significantly inhibited both the basal and stimulated secretion of
MMP-2 (Fig. 5). Very similar results were
obtained in Western blot studies by using a specific antibody against
MMP-2, as revealed in Fig. 6. Consistent
with the reduction of MMP-2 activity and protein secretion, we also
found a decreased expression of MMP-2 at mRNA level in the presence of
ET under both basal and stimulated conditions. This is reflected by the
decreased amount of the amplication product of MMP-2 gene, but not of
the product of the housekeeping gene GAPDH in RT-PCR (Fig.
7).

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Fig. 3.
Effects of ET-1 on MMP-2 activity. A representative
zymographic analysis of MMP-2 activity in culture supernatants of MCs
after treatment for 24 h with increasing concentrations of ET is
shown. Similar results were obtained from an additional experiment.
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Fig. 4.
Suppressive action of ET-1 on stimulant-induced
enhancement of MMP-2 activity. MCs either untreated or treated with
10 7 M of ET-1 for 45 min were exposed to different
combinations of lipopolysaccharide (LPS) (10 µg/ml), tumor necrosis
factor- (TNF- , 50 ng/ml), and interferon- (IFN- , 100 U) for
48 h. Culture supernatants were harvested and assayed for
gelatinase activity by zymography. Similar results were obtained in 3 additional experiments.
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Fig. 5.
Effects of ET-1 on basal and stimulant-induced activity
of MMP-2 as revealed by antibody capture assay. MCs treated with or
without 10 7 M of ET-1, were exposed to the mixed
stimulant (10 µg/ml LPS, 50 ng/ml TNF- , and 100 U IFN- ) for
48 h. Supernatants were collected and assayed for MMP-2 activity
by using a commercial kit as described in MATERIALS AND
METHODS. Data are expressed as means ± SD
(n = 4). Similar results were obtained from an
additional experiment. *P < 0.01.
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Fig. 6.
Inhibitory effects of ET-1 on the basal and
stimulant-induced protein secretion of MMP-2 as indicated by Western
blot. A: MCs, with or without treatment with
10 7 M of ET-1, were exposed to the mixed stimulant (10 µg/ml LPS, 50 ng/ml TNF- , and 100 U IFN- ) for 48 h. Equal
amounts of culture supernatants were concentrated and analyzed for the
presence of MMP-2 protein by using immunoblotting. B:
densitometric analysis of data from A. Values represent the
means ± SD of 3 separate experiments and are expressed as percent
untreated controls. * P < 0.01.
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Fig. 7.
RT-PCR analysis of MMP-2. MCs, with or without treatment
with 10 7 M of ET-1, were exposed to the mixed stimulant
(10 µg/ml LPS, 50 ng/ml TNF- , and 100 U IFN- ) for 24 h.
RNA extraction and RT-PCR analysis were performed as described in
MATERIALS AND METHODS. Result shown is a representative of
the 3 separate experiments.
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It is worth mentioning that, in addition to the obvious increase of
MMP-2 secretion, cytokines could elicit the activation of MMP-2 by
inducing an additional band of 62-kDa molecular mass in the zymogram
and Western blot (Figs. 4 and 6). ET treatment, however, greatly
lessened the intensity of this band (Figs. 4 and 6).
Exogenous ET-1 inhibits the activation of MMP-2.
MMP-2 is known to be secreted in latent form and must be activated to
exert catalytic action. Therefore, it is important to understand the
effects and the mechanisms of endothelin on the activation of MMP-2.
For these purposes, a well-established model of MMP-2 activation
induction by cytochalasin D was employed (1, 2). Mesangial cells were exposed to cytochalasin D
in the presence or absence of ET-1 for 18 h, and the culture
supernatant was then subjected to gel zymography. As indicated in Fig.
8, cytochalasin D elicited
the activation of MMP-2, as demonstrated by the appearance of one major
band of ~62 kDa. This action of cytochalasin D was dose
dependent (Fig. 8B). In the presence of ET-1, the conversion of MMP-2 from the latent to the active form by cytochalasin
D was significantly inhibited as revealed by zymography
(Fig. 8) and Western blot (Fig. 9).
Desitometric analysis of data from four separate experiments by Western
blot indicated that the percent active MMP-2 in total MMP-2 was
significantly decreased, from 49.5 ± 8.8 in cytochalasin
D-treated cells to 34.2 ± 8 in cells pretreated with
ET-1 (Fig. 9B). This action of ET-1 could be
observed in a wide range of concentrations tested (Fig. 8A)
and was most probably mediated by ETA receptors, because
ETA receptor antagonist FR139317 almost completely blocked
this effect of ET-1 (Fig. 10).

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Fig. 8.
Suppressive effects of ET-1 on cytochalasin D (Cyto
D)-elicited activation of MMP-2. A: MCs, with or
without treatment with the indicated concentrations of ET-1, were
exposed to 1 µg/ml of Cyto D for 18 h. Culture
supernatants were harvested and assayed for MMP-2 activity by using
zymography. B: MCs were pretreated with 10 7 M
of ET-1 before stimulation with the indicated concentrations of Cyto
D.
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Fig. 9.
Western blot showing the suppressive effects of ET-1 on
Cyto D-elicited activation of MMP-2. A: MCs were
pretreated with 10 7 M of ET-1 before exposure to Cyto
D (1 µg/ml) for 18 h. Equal amounts of supernatants
were concentrated and analyzed for MMP-2 protein by using
immunoblotting as described in MATERIALS AND METHODS.
B: desitometric analysis of data from A. Values
represent the means ± SD of 4 separate experiments and are
expressed as percent active MMP-2 in total MMP-2. #P < 0.05.
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Fig. 10.
Blocking of the suppressive effects of ET-1 on Cyto
D-induced activation of MMP-2 by ETA receptor
antagonist. MCs were either left untreated or pretreated with 5 × 10 5 M ETA receptor antagonists for 1 h
before exposure to ET-1 (10 7 M), as well as Cyto
D (1 µg/ml), for an additional 18 h. Culture
supernatants were harvested and assayed for MMP-2 activity by using
zymography. Similar results were obtained in 2 additional
experiments.
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The activation as well as activity of MMPs are strictly regulated by
endogenous inhibitors, delineated as TIMPs. MMP-2 activity in
particular is controlled by TIMP-2 through a direct protein-to-protein interaction (10). We therefore studied the effects of ET
on the secretion and expression of TIMP-2 by MCs. As demonstrated in
Fig. 11, treatment of MC with
cytochalasin D inhibited dose-dependently the secretion of
TIMP-2 into the medium, whereas the treatment increased the level of
cell-associated TIMP-2 (Fig. 11). Conversely, ET exerted an
exactly opposite effect and partially counteracted the action of
cytochalasin D on TIMP-2 redistribution. The increment of
the soluble form of TIMP-2 by ET could contribute to the inhibition of
MMP-2 activation, because the direct addition of recombinant TIMP-2
into the culture suppressed concentration-dependently the cytochalasin
D-elicited activation of MMP-2 (Fig.
12).

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Fig. 11.
Effects of ET-1 on tissue inhibitor of
metalloproteinase-2 (TIMP-2) protein distribution as analyzed by
Western blot. MCs were either left untreated or pretreated with
10 7 M of ET-1 before the addition of the indicated
concentrations of Cyto D (up to 1 µg/ml) for 18 h.
Equal amounts of supernatants (top) and cellular lysates
(bottom) were harvested and analyzed for TIMP-2 protein
expression by using immunoblotting. Similar results were obtained in 2 additional experiments.
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Fig. 12.
Prevention of Cyto D-elicited activation of
MMP-2 by the recombinant TIMP-2. MCs were exposed to Cyto D
(1 µg/ml) with or without the simultaneous addition of the indicated
concentrations of recombinant TIMP-2 (µg/ml) for 18 h. Culture
supernatants were harvested and assayed for MMP-2 activity by using
zymography. Result shown is a representative of the 3 separate
experiments.
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Because MMP-2 is considered to be activated on the cell surface by the
membrane type 1 matrix metalloproteinase (MT1-MMP) (1,
20), we also examined the effects of ET on the mRNA expression of MT1-MMP by using semi-quantitative RT-PCR. As indicated in Fig.
13, cytochalasin D
obviously increased the levels of the amplication product of MMP-2 and
MT1-MMP in RT-PCR, compared with the respective nontreated controls. On
the contrary, ET exerted an opposite effect under both basal and
cytochalasin D-stimulated conditions. Interestingly, neither cytochalasin D nor ET had any influence on the
expression of TIMP-2 (Fig. 13). As an internal control, the expression
of the housekeeping gene GAPDH was not altered.

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Fig. 13.
RT-PCR analysis of MMP-2, membrane type 1 matrix
metalloproteinase (MT1-MMP), and TIMP-2. MCs were either left
untreated or pretreated with 10 7 M of ET-1 before the
addition of Cyto D (1 µg/ml) for 18 h. RNA
extraction and RT-PCR analysis were performed as described in
MATERIALS AND METHODS. Result shown is a representative of
the 3 separate experiments.
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DISCUSSION |
The suppressive action of exogenous ET-1 on collagenase activity
has been previously reported in cardiac fibroblasts (19). However, the role of endogenous ET on collagenase activity as well on
the activation of collagenase and the underlying mechanisms implicated
has not been fully examined so far. Here, we reported several novel
findings related to the regulation of MMP-2 by ET in cultured MCs.
First, ET was demonstrated to be an endogenous inhibitor of MMP-2
secretion and activation in cultured MCs. Second, ET was found to be
able to inhibit the cytokines (IFN-
and TNF-
) plus LPS-elicited
production of MMP-2. Third, ET had the ability to block the conversion
of pro-MMP-2 to active MMP-2, possibly via suppressing the MT1-MMP
expression and increasing the secreted form of TIMP-2.
Addition of ETA receptor antagonists into MC cultures
caused an obvious increase of MMP-2 activity into the medium. Similar effects were produced by neutralizing anti-ET monoclonal antibody. The
results suggest that the action of ETA receptor antagonists was through blocking of the function of endogenous ET, rather than a
direct induction of MMP-2 expression. Besides the obviously augmented
activity of pro-MMP-2 in zymography, the amount of activated MMP-2 was
also increased by ETA receptor antagonists or by the neutralizing anti-ET antibody. This was reflected by the earlier appearance as well as the enhanced intensity of the 62-kDa band, representing activated MMP-2. These results clearly indicate that ET is
a potent endogenous inhibitor of both MMP-2 secretion and activation in
rat MCs. This idea is further strengthened by the fact that exogenous
ET was able to inhibit the synthesis and activation of MMP-2 in both
basal and stimulated conditions. It should be noted that the
accumulation of the main substrate of MMP-2, collagen IV, in cultured
rat mesangial cells in the presence of ET, has been previously reported
(18). Furthermore, production of ET by MC
(36) and the autocrine actions of ET on MC behavior have also been well documented (23, 24).
The inhibitory action of exogenous ET on basal MMP-2 secretion as
detected by zymography was marginal, whereas by using Western blot or
quantitative MMP-2 activity assay, a 30% reduction of MMP-2 was found;
the discrepancy of these results may reflect the different
sensitivities of the assay systems employed. Zymography is only
a simple, semi-quantitative assay for MMP-2. It is worth mentioning
that the detection and comparison of MMP-2 activity in this study were
based on the equal volume of culture supernatants. The possible
influence of increased MC numbers under ET stimulation was not taken
into account. Because ET is a potent mitogen for MCs and there was a
constant increase of MC numbers in the presence of ET (data not shown),
the actual suppressive effect of ET on MMP-2 activity is, on a
cell-for-cell basis, greater than that expressed by the data.
The mechanisms by which ET exerts its effect on the activity of MMP-2
appear to be both complicated and multiple. ET could act by inhibiting
the mRNA and protein expression of MMP-2, as indicated in this study by
RT-PCR, Western blot, and the specific antibody capture assay. It could
also control the activation of MMP-2 by regulating the expression
and/or activation of molecules involved in MMP-2 activation. In this
respect, we demonstrated that ET was able to suppress the mRNA
expression of MT1-MMP and to enhance the proportion of the secreted
form of TIMP-2. The increased level of TIMP-2 in the culture medium may
contribute to the suppression of MMP-2 activation by ET. This is
supported by the observation that the addition of recombinant TIMP-2
into medium concentration dependently inhibited the cytochalasin
D-elicited activation of MMP-2. A previous study has
reported that ET has the ability to upregulate plasminogen
activator inhibitors and suppress the activity of fibrolysis in MCs
(21). Several studies have suggested a close interaction
between MMPs and the plasminogen/plasmin system. For example, a role of
plasmin in the activation of latent MT1-MMP, which in turn could
activate latent MMP-2, has been proposed (33). Addition of
plasmin into MC cultures can lead to the conversion of latent MMP-2
into active MMP-2 (3, 4). Thus it is likely that part of
the action of ET on MMP-2 activation could be a secondary phenomenon,
resulting from its effects on the plasminogen/plasmin system.
Mesangial cells have both ETA and ETB
receptors. The available data support the concept that ETs act on MC
via two different receptors (23). In this study, we
demonstrated that ETA receptor antagonists potentiated the
secretion and activation of MMP-2 in MCs, indicating that the
ETA receptor is responsible for the regulation of MMP-2
activity. The complete blockade of the suppressive effects of ET on
cytochalasin D-elicited activation of MMP-2 by the
ETA receptor antagonist FR139317, provided additional
evidence supporting this conclusion. Several investigators have
reported that ET induces mesangial matrix synthesis via ETA
receptor (18). Furthermore, ETA receptor
antagonist treatment of rats with various kidney diseases reduces the
glomerular deposition of ECM proteins compared with untreated controls
(5, 6, 17). In addition, the suppressive action of
exogenous ET-1 on collagenase activity in cultured cardiac fibroblasts
has also been demonstrated to be mediated by ETA receptors
(19). Thus it seems likely that ET acts on matrix turnover
via ETA receptors.
It is worth noting that some of the actions of ET on matrix synthesis
in MCs are reported to be mediated by TGF-
(18). TGF-
itself has proved to be a potent inhibitor in ECM degradation in cultured MCs (3). Therefore, it can be assumed that the effects of ET on MMP-2 activity might also be via TGF-
. However, most of the previous studies have implicated TGF-
as a promoter rather than an inhibitor of MMP-2, releasing in MCs as well as in other
cell types (26, 30, 34). Thus the exact role of TGF-
in
ET-induced suppression of MMP-2 activity remains to be addressed.
What are the potential in vivo pathophysiological implications of this
study? As a predominant MMP involved in the degradation of major
components of the GBM and mesangial matrix, MMP-2 plays an important
role in the turnover of ECM in the renal glomerulus. The production of
MMP-2 under steady-state conditions has been reported. Studies on renal
biopsies demonstrated the presence of small amounts of MMP-2 in the
normal glomerulus (14). The expression of MMP-2 in
quiescent mouse, rat, and human mesangial cell lines has also been
shown (25, 27, 28). Physiological levels of ET, secreted
by MCs as well as other cell types within the glomerulus, may
negatively regulate the activity of MMP-2, keeping its proteolytic
potential within acceptable limits. Interestingly, one of the
substrates of MMP-2 is reported to be big ET-1. MMP-2 cleaves big ET-1
to yield a novel and potent vasoconstrictor, ET-1 (15). It
is highly possible that the autoregulatory loop between MMP-2 and ET
might play an important role in the regulation of vascular responses.
Under inflammatory conditions, ET expression is upregulated by a
variety of cytokines, including IL-1, TNF-
, and IFN-
(22,
24). The enhanced level of ET might, in turn, counteract the
action of these cytokines on MMP-2 activity, thus lessening the
abnormal degradation of ECM and damage to glomerular structures. On the
other hand, in diseases characterized by the accumulation of glomerular
extracellular matrix, such as diabetic glomerulosclerosis, the
increased expression of ET and decreased activity of MMP-2 have been
reported (8, 9, 16, 37). Suppression of the activity of
the protein-degrading enzymes by ET could contribute to
glomerulosclerosis. In this context, augmentation of MMP-2 activity by
ET receptor antagonists may be one of the important mechanisms by which
such agents act therapeutically to slow progressive glomerulosclerosis.
In conclusion, our results demonstrate that ET is a potent inhibitor of
MMP-2 secretion and activation in MCs. This finding may help us
understand the subtle regulation of the synthesis and activation of
MMP-2 in MCs. It also provides us with further insight into the
pathophysiological mechanisms involving ET in the regulation of matrix
turnover in glomerulus.
 |
ACKNOWLEDGEMENTS |
The authors thank Dr. S. Batsford, Institute of Medical
Microbiology and Hygiene, Freiburg University, Germany, for critical review of and advice on the manuscript and K. Kamata for excellent technical assistance.
 |
FOOTNOTES |
This study was supported by grants from the Ichiro Kanehara Foundation
and Naito Foundation (97) and a Grant-in-Aid for
scientific research (C: No. 10670988) as well as for encouragement of
young scientists (A: No. 11770594) from the Ministry of Education,
Science, Sports, and Culture, Japan.
Address for reprint requests and other correspondence: Dr.
Takashi Oite, Dept. of Cellular Physiology, Institute of Nephrology, 1-757 Asahimachi-dori, Niigata 951-8510, Japan
(E-mail: oite{at}med.niigata-u.ac.jp).
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.
Received 28 June 2000; accepted in final form 19 December 2000.
 |
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