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INTRODUCTION |
Dysregulation of extracellular matrix turnover is an important
feature of glomerular inflammatory processes and may result in the loss
of the mechanical and functional integrity of the glomerulus (1-3).
Physiologically, the balance between synthesis and degradation of
matrix proteins is guaranteed by the action of a family of
zinc-dependent, neutral proteinases designated matrix
metalloproteases (MMPs).1 A
tight regulation of these proteases is accomplished by different mechanisms, including the regulation of gene expression, the processing of the inactive zymogenes by other proteases, and finally, the inhibition of the active enzymes by the action of endogenous inhibitors of MMPs, the TIMPs (for review, see Ref. 4). Cultured mesangial cells
(MC) respond to proinflammatory cytokines such as tumor necrosis factor
or interleukin-1
(IL-1) with the production of several MMPs,
including MMP-9 (gelatinase-B), mainly due to an increase in gene
transcription (5, 6). The transcriptional regulation of the rat MMP-9
gene by proinflammatory cytokines is localized to a 0.7-kb region
upstream from the transcriptional start site and critically depends on
the binding sites for activator protein-1 (AP-1) and nuclear factor
B (NF-B) transcription factors, respectively (5, 7). Besides
MMP-9, MC under inflammatory conditions can express a variety of genes
coding for potent inflammatory mediators, such as inducible
nitric-oxide synthase (iNOS), the cytosolic and secreted types of
phospholipases (PLA-2), and cyclooxygenase 2, respectively (8-11).
Metabolites of these enzymes themselves can have modulatory effects on
MMP-9 expression, as we have previously reported a NO-mediated
suppression of cytokine-induced MMP-9 expression (6).
Pharmacologically, the transcriptional activation of MMP-9 is modulated
by agonists of the glucocorticoid receptor mainly through interference
with NF-B and members of the Ets/PEA transcription factor
family.2 We now searched for
a possible modulation of MMP-9 expression by agonists of another member
of the nuclear receptor superfamily, the peroxisome
proliferator-activated receptors (PPARs) (for review, see Ref. 12).
Three distinct PPAR subtypes have been identified, PPAR
, -/
,
and -
, with each subtype showing a specific distribution, physiologic function, and a specific set of endogenous as well as
synthetic ligands. Physiologically, PPARs are key players in lipid and
glucose metabolism, and therefore, disturbances of PPAR function have
been implicated in a variety of metabolic disorders such as
dyslipidemia and diabetes (13). In addition, PPAR, similar to
PPAR, has been reported to exert potent anti-inflammatory activities
in various cell types by inhibiting the expression of inflammatory
genes such as COX-2 (14), IL-6 (15), endothelin-1 (16), and various
acute-phase proteins (17) just to name a few of them. In accordance
with these in vitro data, PPAR-deficient mice display an
exacerbated response to inflammatory stimuli (14, 15). Moreover,
PPAR null mice exhibit a higher degree of kidney damage in response
to ischemia/reperfusion injury when compared with wild type controls,
thus indicating that PPAR, in addition to its antiinflammatory
actions, exhibits also cytoprotective properties in the kidney (18,
19). Interestingly, recent observations demonstrate a role of
glucocorticoids in PPAR gene expression in the development of rat
kidney, suggesting a cross-talk between both nuclear hormone receptor
pathways (20).
Although many studies have addressed the question of how PPARs directly
activate gene expression via PPREs, less information exists about
negative mechanisms that mostly involve interference with
transcriptional activators. In this study we provide an additional mechanism by which PPAR agonists, independent of direct
transcriptional effects, indirectly, via amplification of nitric oxide
production, can efficiently regulate the level of cytokine-induced
MMP-9 expression.
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EXPERIMENTAL PROCEDURES |
Reagents--
Human recombinant IL-1 was from Cell Concept
(Umkirch, Germany). The PPAR activators were WY-14,643 and
LY-171883, and the NO donors were DETA-NONOate and
S-nitroso-D-penicillamine. The NOS inhibitor was
NG-monomethyl L-arginine
(L-NMMA), Actinomycin-D (from Streptomyces species) and cycloheximide were purchased from Alexis Biochemicals (Grünberg, Germany). Ribonucleotides and restriction enzymes as
well as modifying enzymes were purchased from Roche Diagnostics.
Cell Culture--
Rat glomerular MC were grown in RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 5 ng/ml insulin, 100 units/ml penicillin, and
100 µg/ml streptomycin. Serum-free preincubations were performed in
Dulbecco's modified Eagle's medium supplemented with 0.1 mg/ml fatty
acid-free bovine serum albumin for 24 h before cytokine treatment.
For experiments, 3.0-5.0 × 106 of MC/10-cm culture
dish were used between passages 8 and 19. All supplements were
purchased from Invitrogen. The amount of dead cells was determined by
trypan blue exclusion, and the amount of cells was determined by use of
a Neubauer chamber. Cell cytotoxicity was measured by the use of a
cytotoxicity detection kit (Roche Diagnostics).
cDNA Clones and Plasmids--
cDNA inserts for rat MMP-9
and TIMP-1 were generated as described recently (6). A cDNA for rat
TIMP-2 was generated using internal primers of coding sequence of
rat TIMP-2 mRNA (GenBankTM accession no AJ409332). The
following primers were used: 5'-CGG GAA TGA CAT CTA TGG CAA CC-3'
(sense) and 5'-AAA GCT GGA CCA GCC TCG ATG TC-3' (antisense).
A glyceralaldehyd-3-phosphate dehydrogenase cDNA clone was
generated using the internal primers of the coding sequence of rat
glyceralaldehyd-3-phosphate dehydrogenase mRNA (accession number
NM017008). A cDNA insert from mouse 18 S rRNA was from Ambion
(Austin, TX).
Cloning of Rat MMP-9 Promoter and Transient
Transfections--
The 5'-flanking region of the rat MMP-9 gene was
cloned utilizing the Genome Walker kit (CLONTECH
Laboratories, Heidelberg, Germany) using internal (upstream) and
external (downstream) primers from the rat MMP-9 cDNA (accession
number U36476) as follows: MMP-9 internal primer,
5'-AGGGGCAGCAAAGCTGTAGCCTAG-3' and MMP-9 external primer,
5'-TTTCAGGTCTCGGGGGAAGACCACATA-3'.
A 1.8-kb fragment from a EcoRV-cut library was isolated by
PCR under stringent conditions. The fragment was subsequently subcloned into pBluescript-II KS+ and sequenced using the automated
sequence analyzer ABI 310 (PE Applied Biosystems, Weiterstadt,
Germany). The sequence has been deposited in the
GenBankTM/EMBL databases (accession number A7438266).
Subsequently, the 1.8-kb fragment of MMP-9 promoter was subcloned into
pGL-III Basic vector coding for beetle luciferase (Promega, Mannheim,
Germany) using XbaI and XhoI restriction sites.
Introduction of a double point mutation into a putative distal
PPRE-like site (GT to CA) to generate pGL-MMP-9-
PPRE-1 was performed
using the forward primer 5'-ATG GAG ACT CAA GCA CAC CTA TGT GT-3'
(corresponding to a region from 
1763 to 1738). Generation of a
double transition into a second proximal-lying PPRE-like binding site
to generate pGL-MMP-9 PPRE-2 was done using the forward primer
5'-TCC CAT CCA GCA CAC CCC GAG GCT TA-3' (corresponding to a region
from 896 to 871). All mutant constructs were generated using the
QuikChange site-directed mutagenesis kit (Stratagene). Transient
transfections of MC were performed using Effectene reagent (Qiagen,
Hilden, Germany). Transfections were performed following the
manufacturer's instructions. The transfections were performed as
triplicates and repeated at least three times to ensure reproducibility
of the results. Transfection with pRL-CMV coding for Renilla
luciferase was used to control for transfection efficiencies.
Luciferase activities were measured with the dual reporter gene system
(Promega) using an automated chemiluminescence detector (Berthold, Bad
Wildbad, Germany)
Northern Blot Analysis--
Total cellular RNA was extracted
from MC using the Tri reagent (Sigma). Procedures for RNA hybridization
were as described previously (6).
SDS-PAGE Zymography--
Assessment of gelatinolytic activity of
proteins from cellular supernatants was performed as described
previously (6). To exclude the possibility that alterations in
gelatinolytic contents were due to differences in cell numbers, we
routinely determined total cell numbers under each of the experimental
conditions. Proteins with gelatinolytic activity were visualized as
areas of lytic activity on an otherwise blue gel. Migration properties of proteins were determined by comparison with that of prestained full
range rainbow protein markers (Amersham Biosciences).
Cell-free Incubation Experiments--
In these experiments we
tested the effects of the PPAR agonist WY-14,643 on MMP activities
in the conditioned culture medium harvested from MC. Incubations were
carried out in a total volume of 100 µl. After incubation (16 h) at
room temperature, samples were mixed with 2× sample buffer (4% SDS,
0.005% bromphenol blue, and 20% glycerol) and loaded directly on gels
for SDS-PAGE zymography.
Electrophoretic Mobility Shift Assay (EMSA)--
Preparation of
crude nuclear extracts from cultured mesangial cells and subsequent
EMSA was performed as described previously (21). The primers used for
EMSA are as depicted in Table I. Competition experiments were done by
coincubation with different dilutions of a primer stock solution
corresponding to 10-, 100-, and 1000-fold excess (10, 100, 1000 pmol)
of unlabeled double-stranded oligonucleotide in the DNA-protein binding
reaction. Wild type and mutant consensus oligonucleotides for
competition experiments were from Santa Cruz Biotechnology. The
sequences for wild type and mutant (forward) PPRE oligonucleotides are
depicted in Table I.
PPAR- and --specific antibodies used for supershift experiments
were purchased from Affinity Bio Reagents (Golden, CO). Polyclonal
antibodies specific for p50 and p65 were purchased from Santa Cruz
Biotechnology. For supershift analysis, 2 µl of the antibody were
preincubated overnight in a cold room before the binding reaction.
Transcription Assay--
For each assay nuclei from ~3 × 107 cells were isolated by lysing cells in ice-cold 10 mM Tris (pH 7.4), 10 mM NaCl, 3 mM
MgCl2, 0.5% Nonidet P-40. Nuclei were isolated by spinning
at 800 × g and, finally, resuspended in 50 mM Tris (pH 7.4), 5 mM MgCl2, 0.1 mM EDTA, and 40% glycerol. For further procedures we
combined the classical run-on protocol with that of the more sensitive RNase protection assay and proceeded as followed. Nuclei suspensions were mixed with an equal volume of 2× reaction buffer containing 100 mM Hepes (pH 8.0), 10 mM MgCl2, 300 mM KCl, 200 units of RNasin and 1 mM of each
ATP, GTP, CTP, and UTP and incubated for 30 min at 30 °C.
Transcription was stopped by adding 20 µg of DNase I followed by 80 µg of proteinase K. Subsequently the nuclear RNA was isolated by
using the Tri reagent and by subsequent chloroform precipitation after
simple ethanol precipitation. A cDNA clone coding for rat MMP-9
(pKs-MMP-9 rat) was linearized with PvuII and used as a
template for MMP-9 antisense RNA. The MMP-9 antisense transcript was
synthesized in vitro using T7 RNA polymerase and [-32P]UTP (3000 Ci/mmol). Nuclear RNA and 100,000 cpm
of the labeled T7-derived MMP-9 antisense transcript were
co-precipitated by ethanol precipitation and hybridized at 42 °C
overnight in 30 µl of FAB hybridization buffer containing 80%, 1 mM EDTA, 40 mM PIPES (pH 6.4), and 400 mM NaCl. After hybridization, samples were digested with
RNase A and T1 for 1 h at 30 °C. RNA samples were again
extracted with phenol/chloroform, and protected double-stranded RNA
hybrids were ethanol-precipitated. The protected fragments were
separated on a 5% acrylamide, 8 M urea gel and analyzed
using a phosphorimaging. The amount of MMP-9 RNA signals was normalized by hybridizing the same set of RNA samples with a radioactively labeled
antisense RNA coding for rat glyceralaldehyd-3-phosphate dehydrogenase.
Western Blot Analysis--
The total cellular levels of iNOS
protein were analyzed by Western blot analysis using total cellular
extracts (50 µg) and probed with a polyclonal antibody specific for
rat N-terminal iNOS (22). Total cellular levels of MMP-9 were assessed
by use of a polyclonal antibody specific for rat MMP-9 (Chemicon,
Hofheim, Germany).
Nitrite Measurements in MC Supernatants--
Nitrite contents of
cellular supernatants were measured as a readout for NOS activity. 100 µl of supernatants were mixed with 100 µl of Griess reagent
(Merck). The absorbance at 540 nm with a reference wavelength at 595 nm
was measured, and nitrite concentration was determined using a
calibration curve with sodium nitrite standards.
Statistical Analysis--
Results are expressed as the
means ± S.D. The data are presented as x-fold induction compared
with control conditions or compared with IL-1-stimulated values (#).
Statistical analysis was performed using Student's t test
and analysis of variance for significance. p values < 0.01 (** or ##) were considered significant.
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RESULTS |
Effect of the PPAR Activator WY-14,643 on Cytokine-induced MMP-9
Activity and mRNA Steady-state Levels of MMP-9, TIMP-1, and
TIMP-2--
The family of gelatinases includes the two genetically
distinct 72- and 92-kDa type IV collagenases, MMP-2 and MMP-9,
respectively, both of which are expressed in MC. Whereas MMP-2 shows a
constitutive expression, MMP-9 expression is highly induced by
proinflammatory cytokines such as IL-1 (6, 7). Because the
expression of MMP-9 in MC after treatment with IL-1 reaches a
maximal level after 24 h we chose incubation times of 24 h.
To evaluate possible effects of PPAR activators on the proteolytic
activity secreted into the conditioned media from cytokine-treated
cells, MC were treated with IL-1 (2 nM) in the presence
or absence of different concentrations of WY-14,643, a potent activator
of PPAR (12).
The gelatinolytic content of conditioned medium of MC withdrawn after
24 h of stimulation was tested by zymography using gelatin as a
substrate. WY-14,643 reduced in a dose-dependent manner the IL-1-stimulated MMP-9 activity with a maximal inhibition seen at 100 µM. WY-14,643 alone had no effects on enzyme secretion (Fig. 1A). Higher
concentrations used in 24-h incubations caused cytotoxic effects as
monitored by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test (data not shown). The levels of the lytic activity of
latent and active MMP-2, represented by the two lytic bands at 68 and
72 kDa, remained unchanged under all experimental conditions, consistent with the constitutive expression of MMP-2 in rat MC (7).
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Fig. 1.
Inhibition of cytokine-induced MMP-9 and TIMP
levels in MC by PPAR agonists.
A, dose-dependent inhibition of the
IL-1-induced gelatinase activity by WY-14,643. MC were
simultaneously treated with vehicle (control) () or with IL-1 (2 nM) (+) in the presence of the indicated concentrations of
WY-14,643. 24 h after stimulation, 10 µl of cell supernatant was
subjected to SDS-PAGE zymography (zym.) using gelatin as a
substrate. The migrating properties of lytic bands, corresponding to
inactive pro-MMP-9 (92 kDa), and the inactive and active forms of MMP-2
(72 and 68 kDa) are indicated by migration properties determined with
molecular mass markers. The data are representative for three
independent experiments giving similar results. B, Northern
blot analysis (n.b.), demonstrating a
dose-dependent modulation of IL-1-induced MMP-9, TIMP-1,
and TIMP-2 mRNA steady-state levels by WY-14,643. MC were treated
for 24 h as indicated and subsequently extracted for total
cellular RNA. 20 µg of total RNA were hybridized to
32P-labeled cDNA inserts from rat MMP-9, TIMP-1, and
TIMP-2 genes. Equivalent loading of RNA was ascertained by
rehybridization to an 18 S ribosomal probe. The data are representative
for three independent experiments giving similar results. C,
dose-dependent inhibition of the IL-1-induced MMP-9
mRNA steady-state levels by LY-171,883. Quiescent MC were
coincubated for 24 h with IL-1 (2 nM) and the
indicated concentrations of the PPAR activator LY-171,883. Total RNA
(20 µg) were hybridized to 32P-labeled cDNA inserts
from rat MMP-9 and TIMP-1 and TIMP-2 cDNAs, and equivalence of
loading was ascertained by rehybridization to a
glyceralaldehyd-3-phosphate dehydrogenase (GADPH) probe. The
data are representative for three independent experiments giving
similar results.
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To evaluate whether the reduction of the lytic content of MMP-9 is due
to a decrease in the amount of the corresponding MMP-9 mRNA, we
performed Northern blot analysis using a cDNA probe from the rat
MMP-9 gene (6). As shown in Fig. 1B, WY-14,643
dose-dependently attenuated the cytokine-induced MMP-9
mRNA level with a maximal inhibition seen at 100 µM
WY. WY-14,643 given alone had no effects on the basal MMP-9 mRNA
levels. These data indicate that the alterations of cytokine-induced
zymogen activity by WY-14,643 predominantly result from changes in the
MMP-9 expression levels. We next checked mRNA levels of TIMP-1 and
TIMP-2, the endogenous inhibitors of MMP-9. Most interestingly, TIMP-1
followed a similar expression pattern as MMP-9, showing a strong
increase of basal mRNA levels by IL-1 and a
dose-dependent attenuation of IL-1-induced steady-state mRNA levels by WY-14,643 (Fig. 1B). Similarly to MMP-9,
basal TIMP-1 mRNA levels were not affected by WY-14,643. In
contrast, TIMP-2 displayed high basal steady-state mRNA levels that
were attenuated by IL-1 and furthermore attenuated by the highest concentration (100 µM) of WY-14,643 (Fig.
1B).
Furthermore, we tested other PPAR activators on MMP-9, TIMP-1, and
TIMP-2 steady-state mRNA levels. The concentrations used were in a
range known to maximally stimulate PPAR activation in cell cultures. As
shown in Fig. 1C, the prototypic PPAR ligand LY-171883, in a
way similar to WY-14,643, dose-dependently inhibited the
amounts of IL-1-induced MMP-9 and TIMP-1 steady-state mRNAs, respectively. The reduction of MMP-9 and TIMP-1 mRNA levels again was paralleled by a decrease of MMP-9 activity in the conditioned media
(data not shown). In contrast, the high basal levels of TIMP-2 mRNA
were reduced by IL-1 and further attenuated by increasing concentrations of LY-171,883 (Fig. 1C). These data
demonstrate that the inhibition of MMP-9 levels is not paralleled by an
increased expression of TIMPs.
WY-14,643 Does Not Alter Gelatinolytic Activity of
Pro-MMP-9--
To evaluate whether PPAR agonists are able to alter
the activity of secreted MMP-9, we performed in vitro
zymography. Conditioned media from cytokine-treated MC were incubated
for 16 h with or without WY-14,643 (100 µM). The
lytic band at 92 kDa, which is not detectable under control conditions
but is inducible by IL-1, corresponds to the inactive proform of
MMP-9 as is demonstrated by cleavage to the active 86-kDa form by
treatment with different concentrations of
p-aminophenylmercuric acetate (APMA) (Fig.
2B). As shown in Fig.
2A, WY-14,643 does not alter the gelatinolytic activity of
pro-MMP-9, thus indicating that the observed WY-mediated reduction of
zymogen activity is due to a reduction of IL-1-mediated expression
and secretion of pro-MMP-9.
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Fig. 2.
A, WY-14,643 does not affect
extracellular IL-1-induced gelatinolytic activity of latent MMP-9.
The conditioned media from MC stimulated for 24 h with IL-1
were incubated for 16 h with WY-14,643 (100 µM) at
room temperature and subsequently assayed by SDS-PAGE zymography. The
results are shown as triplicates. B, in
vitro activation of latent MMP-9 by
p-aminophenylmercuric acetate (APMA). The
IL-1-inducible band corresponds to the inactive proform of MMP-9,
which is cleaved to the active 86-kDa form by treatment with
p-aminophenylmercuric acetate. Supernatants form MC treated
for 24 h with IL-1 (2 nM) were incubated for an
additional 3 h with the indicated concentrations of
p-aminophenylmercuric acetate before being subjected to
SDS-PAGE zymography.
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Modulation of Cytokine-induced MMP-9 Promoter Activity by PPAR
Agonists--
To evaluate whether the PPAR-mediated inhibition of
IL-1-induced MMP-9 steady-state mRNA levels resulted from an
inhibition of MMP-9 gene transcription, we cloned a 1.8-kb promoter
fragment of the rat MMP-9 gene by genome walking using MMP-9
gene-specific antisense primers as described under "Experimental
Procedures." In addition to a multitude of putative elements involved
in the cytokine-mediated regulation of MMP-9 expression, we found by computational analysis two regions that displayed a high homology to
PPRE-like motifs and which were denoted as PPRE-1 and PPRE-2, respectively (Fig. 3A).
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Fig. 3.
A, sequence of the upstream 1.8 kb of
the rat MMP-9 gene promoter. Potential binding sites involved in the
signaling of IL-1 are underlined. The numbers
in parentheses indicate the degree of homology to consensus
sequences of transcription factor binding sites. Putative binding sites
for PPARs (PPREs) are framed. The transcriptional start site
(+1) was predicted as described previously (7). Schematic
representation of a 1.8-kb wild type pGL-MMP-9 promoter construct
(B) and PPRE-mutant analogs (C and D)
and their corresponding promoter activation by IL-1 and WY-14-643
is shown. Potential binding sites for transcription factors involved in
cytokine signaling of MMP-9 are indicated. Point mutations within
putative PPREs are indicated. Luciferase activities of the different
MMP-9 promoter constructs are shown in the lower panels
(B-D). Subconfluent MC were transiently cotransfected with
0.4 µg of pGL-MMP-9 (1.8) (B) or with pGL-MMP-9 PPRE-1
(D) or pGL-MMP-9 PPRE-2 (C) and with 0.1 µg
of pRL-CMV coding for Renilla luciferase. After an overnight
transfection, MC were treated for 24 h with vehicle (control),
IL-1 (2 nM), WY-14,643 (100 µM), or a
combination of IL-1 and WY-14,643 as indicated. The values for
beetle luciferase were related to values for Renilla
luciferase and are depicted as relative luciferase activities. Data are
the means ± S.D. (n = 6). *, p  0.05; **, p 0.01, compared with vehicle or
IL-1-stimulated conditions (##, p 0.01).
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Transient transfection of MC with pGL-MMP-9 (1.8 kb), comprising the
1.8-kb promoter fragment fused to a luciferase reporter gene, was
followed by a 24-h treatment with either vehicle, IL-1 (2 nM), WY-14,643 (100 µm), or both compounds in combination
and assayed for luciferase activity. IL-1 significantly stimulated luciferase activity (2.67-fold, p < 0.01), and
surprisingly, this was further amplified by the addition of WY-14,643
(4.1-fold, p < 0.01), which on its own had a moderate
stimulatory effect on MMP-9 promoter activity (1.8-fold,
p < 0.01, Fig. 3B). We recently have
identified AP-1 and NF-B response elements as being crucially involved in the IL-1-mediated transcriptional activation of MMP-9 (7). To test for a functional role of the two putative PPREs in the
amplification of cytokine-induced MMP-9 promoter activity by PPAR
ligands, each PPRE was point-mutated by an exchange of two base pairs
and tested for remaining promoter inducibility (Table
I). Mutation of the proximal PPRE at
888/872 (PPRE-2) had no effect on promoter inducibility independent
of which stimulus was used (Fig. 3C). In contrast, mutation
of a distal-lying PPRE at 1752/1738 (PPRE-1) prevented the
potentiation of cytokine-induced MMP-9 promoter activity by WY-14,643
without affecting promoter activation by IL-1 (Fig. 3D).
These data suggest that PPRE-1, in contrast to PPRE-2, is functionally
involved in the amplification of cytokine-induced MMP-9 promoter
activity by PPAR without affecting cytokine-induced transcription of
MMP-9. Furthermore, these data demonstrate that the modulation of
cytokine-induced mRNA steady-state levels by PPAR agonists
cannot be explained by regulatory events occurring in the upstream
1.8-kb MMP-9 promoter context.
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Table I
Oligonucleotides used in EMSA and for point mutation generation
Consensus binding sites are underlined, and mutations are bold and
italicized.
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PPARa Activators Enhance the Cytokine-induced DNA Binding to
PPRE-1--
To further confirm the functionality of a cognate
PPRE-promoter binding site in the transcriptional amplification of
MMP-9, we performed EMSA using a 32P-labeled
oligonucleotide comprising the critical PPRE-1 binding site. Treatment
of cells with IL-1 (2 nM) or WY-14,643 (50 and 100 µM) caused binding of a single slow migrating complex
(Fig. 4A, left
panel). Importantly, the intensity of DNA binding was strongly
increased when both reagents were given in combination (Fig.
4A, left panel). Similar results were obtained
with a PPRE consensus oligonucleotide (data not shown). The identity of
the DNA-bound complex was confirmed by shifting the EMSA band through the addition of a PPAR-specific antibody (Fig. 4A),
whereas the addition of a PPAR-specific antibody had no effects on
DNA binding (Fig. 4A, right panel).
Interestingly, the disappearance of the PPRE-1-positive band after the
addition of the PPAR antibody was paralleled with the appearance of
a strong band with faster migration properties (arrow in
Fig. 4A). Probably, the binding of the antibody triggers a
conformational change that allows for the binding of a further
transcription factor to this promoter binding site that is normally
competed by PPAR. Furthermore, the specificity of the DNA-bound
complex was underlined by competition assays. The addition of different
concentrations of unlabeled wild type consensus PPRE oligonucleotide
(wt PPRE) dose-dependently impaired DNA binding,
whereas the addition of cold mutant PPRE oligonucleotide only in a very
high concentration (1:10) competed with the labeled PPRE-1 probe, which
is most probably due to an unspecific competition by the
oligonucleotide (Fig. 4A, right panel).
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Fig. 4.
Differential modulation of
IL-1-induced binding to MMP-9-specific
PPRE and NF-B binding sites by
WY-14,643. DNA binding was analyzed by EMSA using gene specific
oligonucleotides (Table I). The conditions for binding were as
described under "Experimental Procedures." A,
cytokine-induced PPAR binding is enhanced by WY-14,643. For EMSA,
serum-starved MC were stimulated with either vehicle (), IL-1 (+)
(2 nM), WY-14,643 (50 and 100 µM), or IL-1
plus WY (100 µM) as indicated for 5 h before being
harvested for nuclear extract preparations. The experiments were
performed three times, and representative experiments are shown.
Supershift analysis is shown identifying PPAR but not PPAR as
IL-1-/WY-inducible complex in MC (right panel). For
supershift analysis the antibodies were preincubated overnight at
4 °C before the addition of the labeled oligonucleotide. The altered
migration property of the supershifted complex is indicated by an
arrow. The same gel shows a representative competition study
by using different molar excess (depicted as different dilutions of an
oligonucleotide stock solution) of unlabeled wild-type (wt
PPRE) or mutated PPRE (mut PPRE) consensus
oligonucleotides (sequences are shown in Table I). B,
cytokine-induced NF-B binding is inhibited by WY-14,643. Supershift
analysis is shown identifying an IL-1-inducible p50 and
p65-containing complex (right panel).
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WY-14,643 Inhibits the IL-1-induced DNA Binding of
NF-B--
PPAR agonists can inhibit the transcription of
proinflammatory genes indirectly by interfering with the activation of
NF-B as it has been shown for the human IL-6 promoter (15).
Previously we have demonstrated that NF-B is critically involved in
the activation of a 0.6-kb promoter portion of MMP-9 by IL-1 (7). To
test whether activators of PPAR could modulate the activation of
NF-B binding in MC, we performed EMSA using a MMP-9 gene-specific oligonucleotide described in Table I. Treatment of MC with IL-1 (2 nM) induced DNA binding of a single complex and a weaker
binding by WY-14,643 (Fig. 4B, left panel).
Supershift analysis revealed that the cytokine-induced complex contains
both p50 and p65 subunits, and the p65 antibody totally inhibited DNA
binding. This indicates activation of a p50-p65 heterodimeric complex
that mediates the potent transactivating activity of NF-B (Fig.
4B, right panel). The formation of this
IL-1-induced complex was markedly attenuated in cells that were
simultaneously treated with WY-14,643, indicating that
PPAR-dependent signaling cascades interfere with the DNA binding
of NF-B (Fig. 4B, left panel). It should be
noted that WY-14,643 alone induced a pronounced increase in
NF-B binding which, however, was significantly less than the effects
observed with IL-1. Taken together these results demonstrate that
activators of PPAR show mixed effects on NF-B activity depending
on the presence or absence of a simultaneous cytokine stimulus.
However, their positive effects through PPRE dominate and result in the potentiation of MMP-9 gene expression.
Inhibition of Cytokine-induced MMP-9 Expression by PPAR Agonists
Is Switched to a Potentiation in the Presence of the NOS Inhibitor,
L-NMMA--
Because the data of the promoter studies do
not reflect the reduction of the cytokine-induced MMP-9 mRNA
steady-state levels by PPAR agonists, we hypothesized that PPAR
activators may alter posttranscriptional events regulating MMP-9
mRNA stability. Previously, we have described the inhibitory
effects of NO on the IL-1-induced MMP-9 steady-state mRNA levels
in rat MC. Correspondingly, we found that inhibition of iNOS leads to a
marked potentiation of cytokine-induced MMP-9 mRNA levels, thus
proving a potent inhibitory action of endogenously produced NO (6). To
test whether the inhibitory effects of the PPAR activators on the
IL-1-induced MMP-9 mRNA levels depends on NO production, MC were
treated with IL-1 and different PPAR agonists in the presence or
absence of the NOS inhibitor L-NMMA. We first measured by
zymography the gelatinolytic content of conditioned medium of MC
withdrawn 24 h after stimulation. Strikingly, the reduction of
cytokine-induced MMP-9 content, most prominently seen with the highest
concentration of the PPAR agonists WY-14,643 (100 µM)
or bezafibrate (500 µM), was not only reversed but even
potentiated in the presence of L-NMMA (Fig.
5A). The amplification of
cytokine-mediated zymogen contents by L-NMMA was ~3-fold,
demonstrating that PPAR activators in the absence of
cytokine-triggered NO generation potently augment the cytokine-induced
levels of MMP-9 (Fig. 5A). Similar to the amplification of
gelatinolytic contents, the addition of PPAR agonists plus
L-NMMA results in a strong amplification of the IL-1-mediated MMP-9 steady-state mRNA levels (Fig.
5B), whereas L-NMMA by its own had no effects on
the basal MMP-9 mRNA levels (data not shown). Similarly to MMP-9,
cytokine-induced TIMP-1 mRNA levels were inhibited by both PPAR
agonists but were strongly amplified in the presence of
L-NMMA (Fig. 5B). In contrast, the high level of
basal TIMP-2 mRNA was weakly reduced by IL-1, and the reduction
by IL-1 was weakly enhanced by the addition of WY-14,643, whereas
bezafibrate had no further modulatory effect on this attenuation of
TIMP-2 steady-state mRNA levels. Furthermore, the presence of
L-NMMA had no effects on the TIMP-2 mRNA levels, thus
demonstrating that TIMP-1, but not TIMP-2 expression, similarly to
MMP-9, is negatively affected by PPAR agonists in a
NO-dependent manner.
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Fig. 5.
Dose-dependent effects of
WY-14,643 and bezafibrate on IL-1-induced
MMP-9 secretion (A) and MMP-9, TIMP 1, and TIMP-2
mRNA steady-state levels in the presence or absence of the iNOS
inhibitor, L-NMMA. MC were incubated for 24 h
with vehicle (control), IL-1 (2 nM), IL-1 plus the
indicated concentrations of WY-14,643 or bezafibrate. In the presence
of IL-1 and the highest concentration of PPAR activator, cells
were additionally treated with (+) the NOS inhibitor L-NMMA
(3 mM). The data shown in A and B are
representative for three independent experiments giving similar
results. A, 10 µl of aliquots from cell supernatants were
assessed by SDS-PAGE zymography, and the migration property of the
lytic band was determined using standard molecular mass markers.
B, Northern blot analysis was performed using 20 µg of
total RNA from the very same cells assayed for lytic activity that were
successively hybridized with specific DNA probes for rat MMP-9, TIMP-1,
and TIMP-2. Equivalent loading of RNA was ascertained by a final
rehybridization to an 18 S ribosomal probe.
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To further confirm that the modulation of cytokine-induced MMP-9
mRNA levels by PPAR activators negatively correlates with endogenous NO production, we stimulated MC with increasing
concentrations of IL-1 to gradually increase iNOS expression (23,
24) in the presence of a fixed concentration of WY-14,643 (100 µM) (Fig. 6). Whereas the
low dose of IL-1 (0.1 nM) was not able to induce detectable levels of MMP-9 mRNA, concentrations between 0.5 and 2 nM IL-1 caused a strong induction of MMP-9 mRNA that
was paralleled by a dose-dependent increase of nitrite
accumulation in the cell culture supernatants (Fig. 6). Interestingly,
although the addition of WY-14,643 to all IL-1 concentrations caused
a further increase in nitrite levels, WY differentially affected the
IL-1-induced MMP-9 mRNA levels depending on the concentration of
IL-1 applied (Fig. 6). Whereas at low concentrations of IL-1 (0.1 and 0.5 nM) WY-14,643 clearly enhanced the cytokine-induced
MMP-9 mRNA levels in the presence of higher concentrations of
IL-1 (2 nM), WY-14,643 caused a significant reduction of
cytokine-induced MMP-9 mRNA levels.
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Fig. 6.
IL-1-induced MMP-9
steady-state mRNA levels are differentially modulated by WY-14,643,
depending on the concentration of cytokine used for stimulation.
Quiescent MC were coincubated for 24 h with the indicated
concentrations of IL-1 in the presence (+) or absence () of a
constant concentration of WY-14,643 (100 µM). Northern
blot analysis demonstrates a dose-dependent modulation of
cytokine-induced MMP-9 mRNA steady-state levels. 20 µg of total
RNA were hybridized to a 32P-labeled cDNA insert from
KS-MMP-9. Equivalent loading of RNA was ascertained by rehybridization
to an 18 S ribosomal probe. The stable end product of NO was measured
by the Griess assay, and the amount of total nitrite release by MC is
given at the bottom of the figure. The experiment was performed three
times, and one representative experiment is shown.
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In summary, these data indicate that WY-14,643 principally potentiates
the level of cytokine-induced MMP-9 mRNA, but this effect is
antagonized by the negative effect of NO produced endogenously in large
amounts upon stimulation with high concentrations of IL-1.
The Cytokine-induced Increase in Nitrite Production Is Amplified by
WY-14,643 and Is Due to Enhanced iNOS Expression--
The experiments
with L-NMMA clearly demonstrate a causal role of NO in the
PPAR-dependent repression of MMP-9 mRNA steady-state levels. To elucidate whether activators of PPAR, similarly to MMP-9,
also influence iNOS expression, we performed Western blot analysis
using an N-terminal iNOS-specific antibody (22). Treatment with IL-1
for 24 h results in the appearance of an iNOS band at 130 kDa,
which was not detectable in the extracts from untreated or
WY-14,643-treated MC (Fig. 7, upper
panel). When cells were simultaneously treated with IL-1 plus
WY-14,643, cytokine-triggered iNOS expression was strongly enhanced and
paralleled by an amplification of nitrite production (Fig. 7,
lower panel). These results indicate that PPAR activators
potentiate cytokine-mediated iNOS expression and subsequent nitrite
production.
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Fig. 7.
Cytokine-induced expression of inducible NO
synthase is potentiated by WY-14,643 and results in an amplification of
nitrite release. Quiescent MC were treated for 24 h with
vehicle (), IL-1 (2 nM), WY-14,643 (100 µM), or IL-1 plus WY-14,643. Total cellular protein
lysates (50 µg) were subjected to SDS-PAGE and immunoblotted using an
anti-iNOS N-terminal antibody (22) (upper panel). The
lower panel shows the nitrite levels in the corresponding
cell supernatants as measured by the Griess assay. Equal loading of
protein was ascertained by Ponceau S staining. Similar results were
obtained in two independent experiments.
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The Amplification of Cytokine-induced MMP-9 mRNA Levels by
WY-14,643 in the Presence of L-NMMA Is Independent of the
1.8-kb MMP-9 Promoter Context--
Next, we tested whether the
modulation of MMP-9 promoter activity by WY-14,643 was affected by NO.
To this end MC were transiently transfected with pGL-MMP-9 (1.8 kb) and stimulated with IL-1 to trigger endogenous NO
formation or with DETA-NONOate as an exogenous source of NO. As shown
before, treatment of MC with IL-1 in the presence of WY-14,643
caused a potentiation of cytokine-dependent pGL-MMP-9
luciferase activity (Fig. 8A).
Moreover, the addition of either L-NMMA or DETA-NONOate did
not affect cytokine or cytokine plus PPAR-induced MMP-9 promoter
activity (Fig. 8A) and also had no effects on the basal
promoter activity of pGL-MMP-9 (1.8 kb) (data not shown). These
data suggest that not a transcriptional but a posttranscriptional
mechanism mediates NO suppression of cytokine-stimulated MMP-9 mRNA
steady-state levels.
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Fig. 8.
Exogenous and endogenous NO do not affect the
potentiation of cytokine-induced MMP-9 promoter activity by WY-14,643.
A, subconfluent MC were transiently cotransfected with 0.4 µg of pGL-MMP-9 (1.8) and with 0.1 µg of pRL-CMV, coding for
Renilla luciferase. After an overnight transfection, MC were
treated for 24 h with vehicle or with IL-1 (2 nM)
in the presence or absence of WY-14,643 (100 µM),
L-NMMA (3 mM), or DETA-NONOate (500 µM) as indicated. The values for beetle luciferase were
related to values for Renilla luciferase and are depicted as
relative luciferase activities. Data represent the means ± S.D.
(n = 6). *, p 0.05; **,
p 0.01, compared with vehicle or IL-1-stimulated
conditions (#, p 0.05; ##, p 0.01). B, WY-14,643 increases the content of nuclear MMP-9
mRNA. RNase protection assay demonstrating that the
cytokine-induced content of nuclear MMP-9 mRNA is further amplified
by WY-14,643. MC were either treated with vehicle (control) or
stimulated with IL-1 (2 nM), WY-14,643 (100 µM), or both agents in combination for 4 h. The
amount of nuclear MMP-9 transcripts in isolated nuclei was determined
by hybridizing the RNA transcripts with a radiolabeled antisense RNA
from rat MMP-9. Equal counts of radiolabeled samples were loaded on
acrylamide/urea gels and analyzed by phosphorimaging analysis. A band
that possibly corresponds to hybridization of a smaller MMP-9-splicing
variant is indicated by an asterisk. Similar results were
obtained in two independent experiments.
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Nuclear MMP-9 mRNA Levels Are Increased by the PPAR Agonist
WY-14,643--
The promoter studies indicate that PPAR agonists
have a positive effect on the transcriptional activity of
IL-1-triggered MMP-9. However, the use of partial promoter fragments
in reporter gene assays does not necessarily reflect the real
transcriptional regulation. Therefore, in addition to promoter studies,
we used a modified transcription assay with intact nuclei. MC were
treated for 4 h with either vehicle (control), IL-1 (2 nM), or WY-14,643 (100 µM) or both in
combination before cells were lysed for nuclear fractionation. The
quality of nuclei was tested by microscopy, and only fractions
containing pure and intact nuclei were used for the assay. We used a
combination of classical nuclear run-on with that of RNase protection
assay since the sensitivity of RNase protection assay is severalfold
higher than that of conventional protocols involving hybridization of
labeled RNA to blots containing immobilized cDNA probes. As shown
in Fig. 8B, MMP-9 shows a relatively high basal expression
that corresponds well with the high basal promoter activities of the
MMP-9 promoter constructs and which suggests a strong
posttranscriptional regulation that might explain the observed low
basal MMP-9 steady-state mRNA levels. Concomitantly, IL-1
treatment caused a moderate increase of MMP-9 gene expression that was
further amplified by the addition of WY-14,643. In the protection assay
we observed a second band with faster migrating properties (Fig.
8B, asterisk), most probably reflecting a
splicing variant of nascent MMP-9 mRNA also observed in the
Northern blots (Fig. 6). In agreement with the data from reporter gene
assays, WY-14,643 by its own caused an increase in nuclear MMP-9
mRNA levels. Because posttranscriptional events regulating mRNA
stability mainly occur in the cytoplasm, these results clearly
demonstrate that the increase in nuclear MMP-9 mRNA levels results
from an increased transcription rate.
In summary, these data clearly demonstrate that the inhibitory effects
by PPAR agonists on the cytokine-induced steady-state MMP-9 mRNA
levels result from indirect, posttranscriptional events.
Effect of NO on MMP-9 mRNA Stability--
To test whether NO
could affect the stability of cytokine-induced MMP-9 mRNA, we used
the transcription inhibitor actinomycin D. MC were stimulated for
20 h with IL-1 (2 nM) before transcription was blocked by actinomycin D (5 µg/ml). Subsequently, cells were treated with vehicle or different NO donors. The reduction in MMP-9
mRNA observed under cytokine-stimulated conditions occurred with a
half-life of ~8 h (Fig. 9A,
upper panel). Interestingly, the addition of NO by either
DETA-NONOate (Fig. 9A) or
S-nitroso-D-penicillamine (Fig. 9B)
markedly decreased MMP-9 mRNA stability, with a half-life of ~4
h. This is most impressively documented by the complete loss of the
MMP-9 transcripts 8 h after the addition of the NO donors.
Importantly, WY-14,643, in a similar experimental setting, did not
affect the mRNA half-life of MMP-9 (data not shown).
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Fig. 9.
The stability of cytokine-induced MMP-9
mRNA is strongly diminished by the NO donors DETA-NONOate
(A) and
S-nitroso-D-penicillamine
(B). Quiescent MC were treated for 20 h with
IL-1 (2 nM). After incubation, cells were washed twice
and incubated with actinomycin D (5 µg/ml). After a short
preincubation of 30 min, cells were additionally treated for the
indicated time points without (NO) or with
(+NO) the different NO donors (500 µM). At the
time points indicated after the start of actinomycin D treatment, cells
were harvested and extracted for total cellular RNA. 20 µg of total
cellular RNA were hybridized to a 32P-labeled MMP-9 probe.
The equivalent loading of RNA was ascertained by subsequent
hybridization with an 18 S rRNA probe. The densitometric analysis of
three independent experiments with DETA-NONOate is shown in the
upper panel of Fig. 8A. The MMP-9 mRNA levels
from MC treated with IL-1 in the absence (filled bars) or
presence (open bars) of NO donor after 30 min of
actinomycin-D (0 h) were set as 100%. Data in B are
representative for two experiments giving similar results.
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Furthermore, we addressed the question of whether endogenously produced
NO was, similar to exogenously added NO donors, able to reduce the
stability of MMP-9 mRNA. To this aim, MC were treated with IL-1
(IL-1) for 20 h either in the presence or absence of the NOS
inhibitor L-NMMA (3 mM) before de
novo transcription was blocked by actinomycin D (Fig.
10). As expected from the effects observed for exogenous NO, inhibition of endogenous NO production by
L-NMMA caused a significant retardation of MMP-9 mRNA
degradation (Fig. 10A). These data clearly demonstrate that
endogenous NO levels similar to exogenous applied NO are sufficient to
induce inhibitory effects on MMP-9 mRNA degradation.
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Fig. 10.
A, the stability of cytokine-induced
MMP-9 mRNA is inhibited by endogenously produced NO. Quiescent MC
were treated for 20 h with IL-1 (2 nM) in the
presence or absence of the NOS inhibitor L-NMMA (3 mM) as indicated. After incubation cells were washed twice,
incubated with actinomycin D (5 µg/ml), and harvested for RNA
extraction after the indicated time points. 20 µg of total cellular
RNA were hybridized to a 32P-labeled MMP-9 probe. The
equivalent loading of RNA was ascertained by subsequent hybridization
with an 18 S rRNA probe. Similar results were obtained in two
independent experiments. n.b., Northern blot analysis.
B, the stability of MMP-9 protein and zymogen
(zym.) activity of secreted MMP-9 is not affected by
endogenously produced NO. Quiescent MC were treated for 20 h with
IL-1 (2 nM) in the presence or absence of NOS inhibitor
L-NMMA (3 mM) as indicated. After incubation,
cells were washed twice, incubated with cycloheximide (10 µg/ml), and
harvested for isolation of total protein lysates after the indicated
time points. Protein lysates (100 µg) were subjected to SDS-PAGE and
immunoblotted (w.b.) using a MMP-9-specific antibody
(upper panel). Equal protein loading was ascertained by
Ponceau S staining. The blot is representative of two independent
experiments giving similar results. In parallel, 10 µl of cell
culture supernatants were collected after the indicated time points and
subjected to SDS-PAGE zymography (lower panel). The
indicated band migrated at 92 kDa and corresponds to the latent form of
MMP-9.
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In a further approach we tested whether iNOS inhibition affects MMP-9
protein stability and/or activity. MC were treated for 20 h with
IL-1 (2 nM) in the presence or absence of
L-NMMA (3 mM) before protein synthesis was
blocked by the addition of cycloheximide (10 µg/ml). After the
indicated time points, conditioned media were collected for measurement
of gelatinase activity, and protein levels were assayed by Western blot
analysis using a specific MMP-9 antibody (Fig. 10B).
We observed that the stability of MMP-9 protein is quite high when
compared with its corresponding mRNA since even a blockade of
protein synthesis of 24 h did not change the total content of
MMP-9 protein. Similarly to the cellular content of MMP-9, cycloheximide had no effects on the lytic content in the cell supernatants (Fig. 10B, lower panels). Most
interestingly, there was no change in the MMP-9 protein contents when
cells were grown in the presence of the NOS inhibitor
L-NMMA, thus demonstrating that NO has no effects on the
protein stability and gelatinolytic content of MMP-9.
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DISCUSSION |
Increased expression of matrix-metabolizing enzymes is a hallmark
of many inflammatory processes and may lead to irreversible alteration
of tissue architecture. Particularly MMP-9 has been shown to be
centrally involved in the dysregulation of extracellular matrix
turnover associated with severe pathologic conditions such as
rheumatoid arthritis (25) or fibrosis of lung, skin, and kidney (26,
27, 1). The inflammatory cytokines IL-1 and tumor necrosis factor
are among the most potent inducers of MMP-9 gene expression, mainly
through the involvement of mitogen-activated protein kinase pathways,
leading to increased activity of NF-B and AP-1 transcription factors
(5, 7). Interference with these signaling cascades, therefore, has been
suggested as a promising strategy to prevent aberrant matrix turnover.
Here we have focused on possible modulatory effects of PPAR agonists
on cytokine-induced MMP-9 expression since the PPAR-signaling
pathway exerts critical control functions in acute inflammation as
shown by the exacerbated inflammatory response in PPAR null mice
(18). We demonstrate that various structurally different PPAR
agonists such as WY-14,643, LY-171883, and fibrates potently suppress
cytokine-induced MMP-9 expression in renal MC. A down-regulation of
lipopolysaccharide-induced MMP-9 secretion by the PPAR agonist
fenofibrate was recently documented in the human monocytic THP-1
cell line without addressing the underlying mechanisms (28). The
anti-inflammatory action by PPAR transcription factors is well
established and in many cases occurs by antagonizing the
pro-inflammatory action of NF-B (14, 15, 29) as well as AP-1 (15).
In line with these observations, we demonstrate that in MC the
cytokine-induced binding of a p50/p65-containing complex to a
MMP-9-specific NF-B binding site at 560/550 is substantially
reduced by PPAR agonists. However, the reduction of cytokine-induced
NF-B binding was not complete and somehow contrasts with the
stimulatory effects on NF-B binding by the PPAR agonist alone
(Fig. 3B). Obviously, this reduced level of NF-B activity
is still sufficient to allow for a potentiation of the
cytokine-triggered MMP-9 expression. Activation of NF-B is crucially
involved in IL-1-mediated MMP-9 promoter activation (7). Whether the
inhibition in DNA binding is caused by a physical interaction of
PPAR with Rel-A transcription factors or indirectly, by the
increased expression of the inhibitor of NF-B (IB) by PPAR
activators, remains to be evaluated. Recently, Delerive et
al. (15) have shown that both AP-1 and NF-B activation are
targeted by PPAR. In line with these considerations, the inhibition
of NF-B DNA binding by PPAR agonists in the context of a 1.8-kb
portion of rat MMP-9 promoter did not influence the PPAR-dependent amplification of IL-1-triggered MMP-9
promoter activity by the PPRE-like binding site at 1752/1738.
Interestingly, the PPRE sequence motif, which is defined as two direct
AGG(T/A)CA repeats and separated by 1-5 nucleotides is shared by other
nuclear receptors such as the thyroid hormone and the vitamin D
receptors (12). Like PPARs these receptors bind to direct repeats in a complex with the retinoic acid receptor (RXR). It is worth mentioning that although the PPRE-like motif within the MMP-9 promoter contains only one half-site, which completely matches a PPRE sequence motif, EMSA and supershift analysis indicate PPAR binding comparable with that
observed with a complete consensus motif. The observation that PPAR
agonists by their own increased the nuclear content of MMP-9
transcripts and have a weak stimulatory effect on MMP-9 promoter
activity paralleled by increased DNA binding to PPRE and NF-B sites
somewhat contrasts with the finding that PPAR activators do not
affect the basal MMP-9 mRNA steady-state and zymogen levels.
Obviously, the PPAR-triggered signals do not completely cover the
cytokine-induced signaling pathways in rat MC. Moreover, in the
complete promoter context additional regulatory events affecting
transcriptional activators or repressors may explain the lack of MMP-9
mRNA increase in the absence of cytokine-induced signals. In
line with these observations, the potentiating effects of PPAR
agonists on cytokine-induced sPLA2 promoter activity in rat MC, similar
to their effects on MMP-9 expression, cause activation of the sPLA2
promoter without having significant effects on the basal sPLA2 mRNA
steady-state level (30).
In summary, these experiments clearly demonstrate that PPAR agonists
synergistically with IL-1 amplify MMP-9 promoter activity through a
PPRE-dependent DNA binding despite a partially reduced NF-B signaling. As found by a modified nuclear run-on assay, the
PPAR-mediated decrease in MMP-9 mRNA steady-state and zymogen levels is not attributable to an inhibition of MMP-9 gene expression but points to possible posttranscriptional regulatory events.
In addressing this hypothesis we most interestingly found that the
reduction in cytokine-induced MMP-9 mRNA and zymogen levels by
PPAR agonists is switched to strong potentiation in the presence of
an inhibitor of NO synthesis. Furthermore, we demonstrate that treatment with PPAR agonists causes a strong increase of
cytokine-induced iNOS expression and subsequent NO formation. These
data suggest that PPAR-dependent effects on MMP-9
expression levels primarily result from alterations in NO production.
This is in a line with our previous finding that NO, either given
exogenously or endogenously by stimulation of iNOS expression, potently
inhibits the mRNA steady-state levels of cytokine-induced MMP-9 in
MC (6). The negative modulation of MMP-9 expression has been confirmed
in other cell types and suggests a general mechanism of NO-triggered tissue remodeling (31-33).
When testing a 1.8-kb fragment of the promoter region of the rat MMP-9
gene by reporter gene assay we found that NO had no direct effects on
cytokine-induced MMP-9 promoter activity, although the expression of
many genes has been shown to be transcriptionally modulated by NO in
rat MC (34, 35). However, a transcriptional control of MMP-9 gene
expression seems unlikely since in our hands none of the NO-sensitive
candidate transcription factors, including AP-1 and NF-B (36-38),
were significantly affected by NO (data not shown). Whether regulatory
regions upstream from 1.8 kb of the MMP-9 gene may be negatively
influenced by NO is the subject of ongoing investigations.
The 3'-untranslated region of the rat MMP-9 gene bears several AUUUA
motifs, allo
Agonists
Is Indirect and Due to a NO-mediated Reduction of mRNA
Stability*
§¶,
**,
TOP
ABSTRACT
INTRODUCTION
