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-Induced Matrix Metalloproteinase-9 Expression by Superoxide in Rat Glomerular Mesangial Cells Is Mediated by Increased Activities of NF-
B and Activating Protein-1 and Involves Activation of the Mitogen-Activated Protein Kinase Pathways1
Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe Universität, Frankfurt am Main, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced matrix metalloproteinase (MMP-9) expression
in glomerular mesangial cells (MC). Here we report that exogenously
administrated superoxide, generated by the hypoxanthine/xanthine
oxidase system (HXXO) or by the redox cycler
2,3-dimethoxy-1,4-naphtoquinone, caused a marked amplification of
IL-1-primed, steady state, MMP-9 mRNA level and an increase in
gelatinolytic activity in the conditioned medium. Superoxide generators
alone were ineffective. Cytokine-induced steady state mRNA levels of
TIMP-1, an endogenous inhibitor of MMP-9, were affected similarly by
HXXO. Transient transfection of rat mesangial cells with 0.6 kb of the
5'-flanking region of the rat MMP-9 gene proved a transcriptional
regulation of MMP-9 expression by superoxide. HXXO augmented the
IL-1-triggered nuclear translocation of p65 and c-Jun and, in
parallel, increased DNA binding activities of NF-
B and AP-1.
Mutation of either response element completely prevented MMP-9 promoter
activation by IL-1. Moreover, specific inhibitors of the classical
extracellular signal-regulated kinase (ERK) pathway and p38
mitogen-activated protein kinase (MAPK) cascade, partially reversed the
HXXO-mediated effects on MMP-9 mRNA levels, thus demonstrating
involvement of ERKs and p38 MAPKs in MMP-9 expression. Furthermore,
IL-1-triggered phosphorylation of all three MAPKs, including
p38-MAPK, c-Jun N-terminal kinase, and ERK, was substantially enhanced
by superoxide. Our data identify superoxide as a costimulatory factor
amplifying cytokine-induced MMP-9 expression by interfering with the
signaling cascades leading to the activation of AP-1 and
NF-B. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and TNF
, which are
released from professional inflammatory cells, mesangial cells
(MC)3 themselves
secrete a variety of inflammatory mediators, including NO (2, 3) and reactive oxygen species (ROS) (4, 5).
Superoxide, a prominent member of ROS, is synthesized by different
enzymes, most prominently the NADPH oxidases, xanthine oxidase,
cyclo-oxygenases, lipoxygenases, and the cytochrome P450 oxidases.
Comparable to NO, ROS are considered key players in inflammatory
processes including those of the kidney (6). A further
important feature of glomerular inflammation comprises dysregulation of
extracellular matrix (ECM) turnover, which considerably affects the
mechanical and functional integrity of the glomerulus (7).
Deposition of mesangial ECM represents a tightly regulated balance
between synthesis and degradation of matrix proteins, the latter being
predominantly regulated by the action of metal-dependent, neutral
proteinases, designated matrix metalloproteinases (MMPs).
Physiologically, a tight regulation of MMP activity is necessary and
accomplished at different levels, including gene expression and
processing of the inactive proenzyme and by the action of endogenous
tissue inhibitors of metalloproteinases (TIMPs). Cultured MC upon
stimulation with IL-1 produce high levels of MMP-9 (gelatinase B)
mainly due to an increase in gene transcription (8, 9, 10).
Based on the knowledge that MC under inflammatory conditions are
exposed to high levels of ROS, we investigated whether ROS, in
particular superoxide (O2-),
could influence the expression and/or activity of MMP-9 in a manner
comparable to the recently reported action of NO (10). The
mechanisms underlying the ROS-mediated response may involve direct
alterations of protein kinases or transcription factors by mechanisms
that are not completely understood (6, 11).
Cytokine-mediated up-regulation of MMP-9 expression critically depends
on the activation of NF-B and AP-1 both binding to the corresponding
regulatory elements within the promoter region of the MMP-9 gene
(12, 13). Both transcriptional activators are commonly
altered by changes in the cellular reduction-oxidation (redox) status
and are highly inducible by oxidative stress (14). A
prominent signaling pathway that processes cellular stress signals is
that of the mitogen-activated protein kinases (MAPKs), an evolutionary
highly conserved protein family (15, 16). Members of this
family include the stress-activated protein kinase (SAPKs), also
referred to as c-Jun N-terminal kinases (JNKs), p38-MAPK, and the
classical MAPK, also denoted extracellular signal-regulated kinases
(ERKs). All three subgroups of MAPK can be activated by proinflammatory
cytokines (15, 16, 17). However, the biological outcome of
MAPK activation is highly divergent and critically depends on the cell
type investigated. Our data suggest that the integration of different
signaling pathways activated by oxidative stressors may critically
control the expression of MMP-9 by modulating the activation status of
transcription factors such as AP-1 and NF-B. This study supports the
idea that ROS can function as important trigger of matrix degradation
in areas exposed to high oxidative stress. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human rIL-1 was obtained from Cell Concept (Umkirch,
Germany). Manganese-tetrakis(4-benzoic acid)porphyrin (MnTBAP) and
S-nitroso-N-acetyl-D,L-penicillamine
(SNAP) were obtained from Alexis (Grunberg, Germany). Xanthine oxidase
and hypoxanthine were purchased from Roche (Mannheim, Germany).
Dimethoxy-1,4-naphtoquinone (DMNQ), glucose oxidase (from
Asperigillus niger), superoxide dismutase (SOD; from bovine
erythrocytes), SB203580, and PD98059 were purchased from Calbiochem
Novabiochem (Bad Soden, Germany). All other chemicals were purchased
from Sigma (Deisenhofen, Germany)
Cell culture
Rat glomerular MC were cultured as described previously (18). MC were grown in RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mM glutamine, 5 ng/ml insulin, 100 U/ml penicillin, and 100 µg/ml streptomycin. Serum-free preincubations were performed in DMEM supplemented with 0.1 mg/ml of fatty acid-free BSA for 24 h before cytokine treatment. For experiments 3.0–5.0 x 106 of MC/10-cm culture dish were used between passages 8 and 19. All supplements were purchased from Life Technologies (Eggenstein, Germany). The amount of dead cells was determined by trypan blue exclusion.
cDNA clones and plasmids
cDNA inserts for rat MMP-9 and TIMP-1 were generated as described previously (10).
A GAPDH cDNA clone was generated using internal primers of coding sequence of rat GAPDH mRNA (accession no. NM 017008). A cDNA insert from mouse 18S ribosomal RNA was obtained 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 using the Genome Walker kit (Clontech, Heidelberg, Germany) with internal (upstream) and external (downstream) primers from the rat MMP-9 cDNA (accession no. U36476) as followed: MMP-9 internal primer, 5'-AGGGGCAGCAAAGCTGTAGCCTAG-3'; and MMP-9 external primer, 5'-TTTCAGGTCTCGGGGGAAGACCACATA-3'.
A 0.65-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) and has been deposited in the GenBank/EMBL
databases (accession no. AJ 293580). The forward and reverse primer
sequences used for subcloning into pGL-III Basic vector coding for
beetle luciferase (Promega, Mannheim, Germany) were as
followed: 5'-GGAGTCAGCCTGCTGGGGTTAG-3' (forward) and
5'-TGAGAACCGAAGCTTCTGGGT-3' (reverse). Introduction of a double-point
mutation into the NF-B-site (GGAATTCCCCC to GGAATTGGCCC)
to generate pGL-MMP-9-
NF-B was performed, using the following
(forward) primer: 5'-GGGTTGCCCCGTGGAATTGGCCCAAATCCTGC-3'
(corresponding to a region from -572 to -541). Creation of a double
transition within the AP-1 binding site (CTGAGTCA to
CTGAGTTG) to generate pGL-MMP-9 AP-1 was performed using
the following (forward) primer:
5'-CACACACCCTGAGTTGGCGTAAGCCTGGAGGG-3' (corresponding to a region
from -98 to -65). The mutants were generated using the Quik Change
Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). Transient
transfections of MC were performed using the Effectene reagent (Qiagen,
Hilden, Germany). Transfections were performed following the
manufacturer’s instructions. The transfections were performed in
triplicate and were repeated at least twice to ensure reproducibility
of the results. Transfection with pRL-CMV coding for Renilla
luciferase was used for control of 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 TRIzol reagent (Life Technologies). Procedures for RNA hybridization were described previously (10).
SDS-PAGE zymography
Assessment of gelatinolytic activity of proteins from cellular supernatants was performed as described previously (10). 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 a prestained full range rainbow protein marker (Amersham Pharmacia Biotech, Freiburg, Germany). Photographs of the gels were scanned by an imaging densitometer system from Bio-Rad (Munich, Germany).
EMSA
Preparation of crude nuclear extracts from cultured mesangial
cells was performed as described previously (19). The
cytoplasmic fractions were separated by centrifugation and used for
detection of IB protein levels. Consensus oligonucleotides used in
the binding reactions were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA) The cDNA strands were end labeled by T4 polynucleotide
kinase using [
-32P]ATP (3000 Ci/mmol;
Amersham Pharmacia Biotech). Binding reactions were performed for 30
min on ice with 5 µg of protein in 20 µl of binding buffer
containing 4% Ficoll, 20 mM HEPES (pH 7.9), 50 mM KCl, 1 mM EDTA, 1 mM
DTT, 1 mM PMSF, 0.25 mg/ml BSA, 2 µg of poly(dI-dC), and
20,000–25,000 dpm of 32P-labeled
oligonucleotide. DNA-protein complexes were separated from unbound
oligonucleotide by electrophoresis through native 4.5% polyacrylamide
gels using 0.5x Tris-borate-EDTA. Following electrophoresis, the gels
were fixed and analyzed using a phosphorimager (Fujifilm, Fuji, Tokyo,
Japan).
Competition experiments were performed by coincubation with a 50- to 100-fold excess (10–20 pmol) of unlabeled double-stranded oligonucleotide in the DNA-protein binding reaction.
Polyclonal anti-p50 and anti-p65 Abs used for the supershift experiments were purchased from Santa Cruz Biotechnology. For supershift analysis 2 µl of the Ab was preincubated for 30 min at room temperature before the binding reaction.
Western blot analysis
Nuclear cell extracts (20–50 µg) were used for assessing
nuclear import of p65 and c-Jun, respectively. IB protein level were
analyzed using 50–100 µg of total protein from the corresponding
cytoplasmic fractions. For detection of (phospho-) MAPKs whole cell
lysates were prepared. Cells were lysed in SDS sample buffer (62.5 mM
Tris-HCl (pH 6.8), 2% (w/v) SDS, 10% glycerol, 50 mM DTT, 1 mM PMSF,
and 0.1% (w/v) bromophenol blue), extracted by sonifying, and
subsequently heated to 95°C for 5 min. After a final centrifugation
step, 50 µl of protein extract was directly subjected to gel
electrophoresis. Proteins were separated by 10% SDS-PAGE and
transferred to Immobilon polyvinylidene difluoride membranes
(Millipore, Eschborn, Germany). Equal loading of protein was confirmed
by Ponceau-S staining. The membranes were preincubated for 1 h at
room temperature in Tris-buffered saline, pH 7.6, containing 0.05%
Tween-20 and 2% fatty acid-free BSA (Roth, Karlsruhe, Germany).
Filters were incubated overnight at 4°C with the primary Abs, which
were detected by an HRP-conjugated polyclonal Ab (1/10,000; Santa Cruz
Biotechnology). For detection of the immunoreactive protein, we used
the ECL system (Amersham-Pharmacia). Polyclonal Abs specific for p65,
c-Jun, IB, p38, and JNK-2 were obtained from Santa Cruz
Biotechnology. Polyclonal Abs raised against phospho-SAPK/JNK,
phospho-p38, and phospho p42/p44 ERK were purchased from New England
Biolabs (Beverly, MA). The p42/p44 (ERK)-specific antiserum
was generated as described previously (20, 21).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To evaluate possible effects of
O2- on the activity and/or
expression of MMP-9, MC were treated with IL-1 in the presence of
xanthine oxidase in combination with its substrate hypoxanthine
(hereafter referred to as HXXO). For comparison, cells were treated
simultaneously with the NO donor SNAP, as we recently have shown that
NO reduces MMP-9 levels in cell culture supernatants from
IL-1-stimulated MC (10). The gelatinolytic content of
conditioned medium of MC withdrawn after 36 h of stimulation was
tested by zymography using gelatin as a substrate. As shown in Fig. 1
supernatants of MC under stimulatory
conditions contained both gelatinases, MMP-2 and MMP-9, as detected by
lytic zones in the zymogen gels. The lower migrating bands at 72 and 68
kDa are representative for latent (pro-) MMP-2 and active MMP-2,
respectively. In contrast, MMP-9 always displayed only one lytic band,
which is not detectable under control conditions but is induced by
IL-1. This band corresponds to the inactive proform of MMP-9, which
is cleaved to the active 86-kDa form by treatment with 1 mM
p-amino phenylmercuric acetate (APMA), thereby confirming
the capacity of the inactive enzyme to convert to the active form (Fig. 1B). As reported recently, the NO donor SNAP significantly
reduces the secretion of MMP-9 in the cellular supernatants (Fig. 1A). By contrast, simultaneous treatment of MC with IL-1
and 8 mU/ml HXXO results in a marked potentiation of IL-1-stimulated
gelatinase content (Fig. 1A).
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Superoxide potentiates the IL-1-induced increase in MMP-9 and
TIMP-1 mRNA levels
We performed Northern blot analysis using a rat-specific cDNA
probe from the rat MMP-9 gene. MC were stimulated for 24 h with
IL-1 (2 nM) in the presence of vehicle or different concentrations
of HXXO. As shown in Fig. 2A,
HXXO dose-dependently augments steady state levels of MMP-9 mRNA in
IL-1-treated MC. We next examined the action of HXXO on mRNA levels
of TIMP-1, the endogenous inhibitor of MMP-9. TIMP-1 acts via binding
to the latent proenzyme, thereby preventing its activation
(22). Similarly to MMP-9, TIMP-1 mRNA levels were
amplified by O2- (Fig. 2A). We also tested DMNQ for modulatory effects on
IL-1-induced MMP-9 mRNA steady state levels. As with HXXO we found a
marked increase in MMP-9 mRNA levels when MC were simultaneously
treated with IL-1 and 5 µM DMNQ (Fig. 2B).
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-induced MMP-9 mRNA (data not shown), thus excluding
peroxynitrite action on MMP-9 expression. Because the
hypoxanthine/xanthine oxidase system produces not only
O2- but also
H2O2, which in aqueous
solutions is generated by dismutation of
O2-, we evaluated whether the
effects seen with ROS were related to
H2O2 formation. For this
purpose we first checked whether the amplification of IL--induced
MMP-9 expression by HXXO could be modulated by coincubation with SOD,
which removes O2- and converts
it to H2O2. Coincubation of
MC with IL-1 plus HXXO and SOD (10 U/ml) largely prevented the
amplification of cytokine-induced MMP-9 expression by HXXO (Fig. 2
C). At a concentration of 100 U/ml SOD, we observed a
further decrease in the mRNA level of MMP-9. These data suggest that
O2-, but not
H2O2, contributes to
amplification of MMP-9 mRNA accumulation. To further corroborate these
findings we also tested MnTBAP, a membrane-permeable SOD mimetic.
Again, we observed a strong inhibitory effect on MMP-9 mRNA
accumulation in cells exposed to IL-1 (Fig. 2C).
In a further approach we tested whether incubation with glucose
oxidase, a H2O2-generating
system, is able to mimic the costimulatory effects seen with HXXO. As
shown in Fig. 2C, glucose oxidase had no stimulatory effect
on the IL-1-mediated MMP-9 mRNA accumulation. Similar results were
obtained by costimulation of MC with IL-1 and
H2O2 at concentrations
between 20 and 200 µM, respectively (data not shown). In summary,
these data identify O2- as the
ROS responsible for the potentiation of MMP-9 expression in
cytokine-stimulated MC.
Activation of a 0.6-kb MMP-9 promoter region by IL-1 is
potentiated by superoxide and critically depends on NF-B and AP-1
binding sites
To further evaluate whether the observed effects of HXXO on
IL-1-mediated MMP-9 expression occur on a transcriptional levels, we
cloned a 0.6-kb promoter fragment of the rat MMP-9 gene by PCR using a
5' gene-walking kit with specific primers from the cDNA from the rat
MMP-9 gene as described in Materials and Methods (Fig. 3). Sequence analysis revealed a high
degree of sequence homology (87%) to a corresponding region of the
mouse MMP-9 gene. Especially binding sites for AP-1, Sp-1, and NF-B,
which are essentially involved in the induction of the MMP-9 gene by
12-O-tetraphorbol 12-myristate 13-acetate and cytokines
(12), were highly conserved in the promoters of human,
mouse, and rat genomes. A homologous promoter region bears the
cis-acting elements sufficient for AP-1-mediated MMP-9 gene
induction by v-Src in HT-1080 and HepG2 human tumor cell lines
(24). Transient transfection of MC with pGL-MMP-9 wt,
comprising 0.6 kb of the 5'-flanking region of the rat MMP-9 gene,
revealed a 2.0- to 2.3-fold increase in luciferase activity upon
IL-1 exposure (Fig. 4A).
Treatment of cells with HXXO further increased IL-1-mediated
luciferase activity dose-dependently up to 5.5-fold (Fig. 4A), thus indicating that this region of the MMP-9 promoter
bears superoxide-sensitive regulatory elements. Recent work has
demonstrated dual regulation of MMP-9 expression by NF-B and AP-1
transcription factors in rat MC (13). To confirm the
functional role of these redox-sensitive transcription factors in the
IL-1-mediated MMP-9 promoter induction, point-mutated MMP-9 promoter
constructs were tested for inducibility by IL-1. To this end we
inserted two base pair mutations in either the NF-B or AP-1 binding
sites (Materials and Methods). As shown in Fig. 4, IL-1-driven luciferase activity was totally lost in both promoter
mutants (Fig. 4, B and C), thus indicating that
both AP-1 and NF-B binding sites are indispensable for MMP-9
promoter activation by IL-1 in rat MC. In all the transfection
experiments we observed an overall reduced basal activity of both
mutated promoter constructs, which possibly implies that both binding
sites equally contribute to a basal promoter activity in the reporter
gene assays. Moreover, exogenously given
O2- no longer had a
potentiating effect on the mutated reporter genes (Fig. 4). These
results indicate that the O2-
amplification of IL-1-mediated MMP-9 expression critically depends
on intact NF-B and AP-1 binding sites within the MMP-9 promoter
region.
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To further confirm the contributions of NF-B and AP-1
transcription factors to
O2--mediated MMP-9 gene
expression, we tested whether
O2- affects nuclear
translocation of p65 and c-Jun, the active subunits of NF-B and AP-1
transcription factors, respectively. As shown in Fig. 5, treatment of MC with IL-1 for
5 h caused an increase in nuclear p65 protein levels, whereas no
significant changes could be observed at a very early time point (30
min). Remarkably, coincubation of IL-1-treated cells with increasing
concentrations of O2- elevated
nuclear translocation of p65-specific proteins at all time points
tested. Superoxide alone did not modulate the basal levels of p65
protein in the nucleus (data not shown). Similar to p65, nuclear import
of c-Jun protein was efficiently increased by HXXO at both time points
tested (Fig. 5).
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-mediated DNA binding activity of
AP-1 and NF-B
To test whether the increased translocation of p65 and c-Jun
evoked by HXXO is functionally linked to a rise in DNA binding capacity
we performed EMSA analysis using consensus oligonucleotides for AP-1
and NF-B. We recently reported a time-dependent induction of NF-B
in IL-1-stimulated rat MC (19). Nuclear extracts were
prepared 30 min after stimulation, the point of maximal effects of HXXO
(Fig. 5). As shown in Fig. 6A,
increasing concentrations of HXXO caused a gradual augmentation of
IL-1-mediated DNA binding of NF-B-bound complexes. HXXO given
alone had only a weak effect on NF-B binding, which in none of the
experiments was comparable to that evoked by IL-1. Supershift
analysis indicated that the upper complex that is induced by IL-1
contained p50/p65 proteins, as this complex was partially supershifted
by anti-p50 Abs and was completely supershifted by p65-specific Abs
(Fig. 6A, right panel). The binding activity of
the lower migrating complex was strongly reduced by
anti-p50, but was not affected by the anti-p65 Ab, thus
indicating that this is most likely a p50-p50 homodimeric complex
(19).
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B). The faster migrating complex was constitutively bound
under all conditions tested, whereas DNA binding of a second slower
migrating complex was slightly induced by IL-1 and dose-dependently
augmented by HXXO. The IL-1 effects on DNA binding in all the EMSA
experiments were weak, as we used low, suboptimal concentrations of
IL-1 to optimize the modulatory effects of superoxide. HXXO on its
own did not induce AP-1 binding (Fig. 6B). The specificity
of the IL-1-inducible complexes was tested using an oligonucleotide
with two base changes within the AP-1 binding motif (CA to TG). As
shown in Fig. 6B incubation of nuclear proteins with the
mutated AP-1 oligonucleotide resulted in a strongly reduced DNA binding
of the slower migrating complex, but did not affect the binding
capacity of the fast migrating complex (NS in Fig. 6B).
IL-1- induced degradation of IB is accelerated by
superoxide
Activation of NF-B requires dissociation and rapid degradation
of the inhibitory protein IB (25). Western blot
analysis displayed a marked decrease in IB protein by IL-1,
which was further enhanced by increasing concentrations of HXXO (Fig. 7). HXXO alone reduced basal IB
protein to levels that reached maximally 70% of control conditions.
However, in none of the experiments did the suppressive effects of HXXO
reach the extent achieved with IL-1 alone (Fig. 7, right
panel). In contrast the level of JNK-p54 protein remained
constant, thus indicating that changes in IB level were not due
to unequal loading (see also Fig. 9A). In summary, the
increase in IL-1-mediated NF-B activation by
O2- is paralleled by enhanced
proteolytic degradation of IB.
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-induced MMP-9 mRNA accumulation by SB203580
and PD98059
To examine a possible involvement of MAPKs in the
O2--mediated amplification of
cytokine-promoted MMP-9 expression, we tested SB203580 and PD98059,
specific inhibitors of the p38-MAPK and ERK cascades, respectively
(26, 27, 28). Both inhibitors when preincubated for 30 min
substantially reduced IL-1-primed MMP-9 mRNA accumulation as well as
the amplification of MMP-9 mRNA accumulation caused by
O2- (Fig. 8). However, neither SB203580 nor PD98059
was able to completely abrogate MMP-9 mRNA induction, although both
inhibitors efficiently blocked the specific MAPKs pathways in rat MC at
10 µM (data not shown). The combination of both inhibitors inhibited
in an additive manner the IL-1-primed accumulation of MMP-9 mRNA as
well as the rise in MMP-9 mRNA level caused by
O2- (Fig. 8). Evidently both
the p38-MAPK and the ERK pathways are specifically involved in IL-1
induction of MMP-9 as well as in the potentiating action of
O2-, and both signaling
pathways are indispensable for full activation of MMP-9 gene
expression. Another class of MAPK that is most prominently involved in
the activation of AP-1 transcription factor is the JNK pathway, which
could not be addressed in these series of experiments due to the lack
of specific inhibitors.
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In the next step we examined the activation of the different MAPK
cascades by IL-1 and the potential modulating action of
O2-. We found that
phosphorylation of p38 MAPK, which is assumed to reflect activation of
the enzyme, appears after 1 h of stimulation but not at the early
time point (15 min; Fig. 9A).
IL-1-induced p38 phosphorylation was further enhanced by HXXO,
whereas HXXO on its own showed only a moderate effect on the
phosphorylation status of p38 MAPK (Fig. 9A). Western blot
analysis with a phosphorylation-independent Ab revealed that p38
protein remained unchanged under all conditions tested (Fig. 9A, lower panel). Next, we checked for changes in
endogenous JNK phosphorylation by Western blotting using an Ab that
specifically recognizes phosphorylation on
Thr183/Tyr185. Dual
phosphorylation at these residues is essential and sufficient for
kinase activity (29, 30). Phosphorylation of SAPK/JNK was
induced by IL-1 at both time point tested. Interestingly, HXXO on
its own induced phosphorylation of JNK at intensities comparable to
those produced by IL-1. Treatment of cells with IL-1 plus HXXO
further enhanced JNK phosphorylation, which in all cases was at least
additive to those reached by either stimulus alone. Stripping the blot
and incubation with a phosphorylation-state independent Ab proved that
differences did not result from differences in the JNK protein level
(Fig. 9A).
Finally, we monitored the phosphorylation status of p42/p44 ERKs that
are activated by IL-1 in rat MC (31). Treatment of MC
with 8 mU/ml of HXXO induced phosphorylation of mainly p42 at both time
points examined (Fig. 9B). Compared with the phosphorylation
of p38 MAPK, augmentation of the ERK pathway appeared 15 min after
stimulation, indicating that the different kinase cascades follow
different kinetics of stimulation in MC. The steady state protein level
of total p42/p44 MAPKs remained unchanged under the conditions tested
(Fig. 9B, lower panel). In conclusion, IL-1
and HXXO both act at least additively on the activation of the three
MAPK cascades in MC.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced MMP-9
expression in rat MC (10). Because both radicals are
produced in high quantities under proinflammatory conditions in MC
(2, 3, 4, 5), we were interested in whether
O2- generators in a way similar
to NO would affect expression and activity of MMP-9. Most surprisingly,
we found that both radicals have opposing effects on MMP-9 mRNA levels,
with O2- amplifying and NO
attenuating the response. By contrast, we have identified superoxide
and NO as costimulatory factors amplifying cytokine-induced
inducible NO synthase gene expression in rat MC (32, 34).
These apparently inconsistent observations are unexpected, as a common
set of transcription factors, including AP-1 and NF-B is critically
involved in the regulation of both proinflammatory genes inducible NO
synthase (19, 34) and MMP-9 (13).
The interaction of reactive nitrogen species and reactive oxygen
species is thought to be of high relevance for the regulation of
proinflammatory genes in cells containing NO as well as
O2--generating enzymes. In this
context it is important to note that the ratio between NO and
O2- formation determines
whether MC live or die by either apoptosis or necrosis (35, 36). The free radicals NO and
O2- react with each other at a
rate close to that limited by diffusion and outcompete most other
targets of the radicals found in a cell (37). Simultaneous
production of NO and O2- in
vivo also leads to the generation of peroxynitrite (38),
which thus is a further candidate for modulation of MMP-9 expression.
However, testing SIN-1, a peroxynitrite-releasing compound
(23), we found no change in the IL-1-induced MMP-9 mRNA
levels, thus indicating that the amplification of MMP-9 expression in
MC is most likely not mediated by peroxynitrite. MC have a high
capacity to produce MMP-9 upon stimulation with proinflammatory
cytokines, especially IL-1, as previously described (10, 14). Here we show that MMP-9 is secreted as latent inactive
enzyme, as shown by the in vitro conversion to the shorter migrating
68-kDa form by APMA (Fig. 1B). In vivo the conversion from
the latent to the active enzymes is executed by the actions of soluble
proteases, most prominently tissue plasminogen activator and the
membrane-bound collagenase MT1-MMP (39, 40). However,
under cell culture conditions these proteases may either not be present
or may be adequately active to convert high levels of the latent
enzyme. The final proof of an involvement of proteolytic activators in
vivo has yet to be presented and does not exclude the possibility of
alternative pathways of MMP activation.
A further crucial step regulating MMP-9 activity is the inhibition by
TIMPs, a family of polypeptides that noncovalently bind to active or
latent MMPs, thereby inhibiting their enzymatic activity. Here we
report that, similar to that of MMP-9, the cytokine-induced expression
of TIMP-1 is modulated by ROS. The 5'-regulatory region of the rat
TIMP-1 gene contains putative binding sites for NF-B and AP-1
(41), which may contribute to the modulation of
IL-1-activated TIMP-1 expression by ROS in a way similar to that
described for the expression of MMP-9. A parallel regulation of
IL-1-induced expression of MMPs and TIMP was observed in human
fibroblasts (42) and rat MC (10). In more
general terms, a coordinate expression of proteases and their
inhibitors by inflammatory cytokines and ROS will allow a fine-tuned
regulation of tissue proteolysis and protect against overwhelming
tissue destruction.
Activation of vascular MMP-2 and MMP-9 by ROS in vitro was recently demonstrated in macrophage-derived foam cells (43). This activation probably depends on a direct reaction of ROS and the thiol groups within the catalytic site of MMPs. In contrast, in rat MC, HXXO was unable to modulate MMP-9 activity in vitro, thus indicating that the capacity of ROS to directly affect MMP enzymatic activity is not generally applicable.
IL-1-stimulated MMP-9 expression in MC is critically dependent on
NF-B and AP-1 transcription factors, as was shown by transfections
of a dominant negative mutant of NF-B and by a c-Jun-antisense
construct (13). Our data using reporter gene assays
confirm these results. Mutation of either binding site abrogated the
IL-1-driven induction of a rat MMP-9 promoter fragment as well as
the amplification by O2-. This
is consistent with the finding that HXXO on its own did not induce
MMP-9 mRNA and suggests that the effect of HXXO on MMP-9 essentially
requires additional IL-1-initiated signals. Activation of NF-B
and AP-1 is highly sensitive to changes in the cellular redox state. In
this study we provide evidence for the involvement of both
transcription factors in the amplification of IL-1-triggered signals
by O2-, as we observed
IL-1-stimulated nuclear translocation of p65 and c-Jun proteins with
subsequent DNA binding affinities being substantially increased by
O2-. The augmentation of
NF-B-DNA binding was paralleled by an increase in IL-1-induced
degradation of IB. The question of whether
O2- is able to directly
interact with redox-sensitive proteins that regulate kinases upstream
from IB, e.g., the IKK enzyme complex, is under current
investigation. An ROS-induced modification of redox-sensitive proteins
has recently been demonstrated for the apoptosis signal-regulating
kinase (ASK1) (44). Furthermore, our data reveal an
interference of O2- with the
p38-MAPK and ERK-dependent signaling pathways, because amplification of
IL-1-mediated MMP-9 expression by
O2- was substantially reduced
by the p38-MAPK inhibitor SB203580 and by the ERK kinase inhibitor
PD98059, respectively. Recently, the involvement of both MAPK cascades
has been demonstrated for the regulation of MMP-9 expression and the in
vitro invasion properties of the human squamous carcinoma cell line
UM-SCC-1 by phorbol esters (45).
Finally, we examined whether the activation of SAPK/JNK by IL-1 was
affected by O2-. Recently, we
and others have demonstrated activation of this MAPK pathway by IL-1
and NO in rat MC (17, 46, 47). The finding that
IL-1-mediated phosphorylation of JNK is strongly enhanced by
O2- indicates that JNK or
its upstream activators may function as further targets of
O2- action in MC. A concerted
activation of the ERK and JNK pathways was also found to be necessary
for the differential regulation of cell motility and MMP-9 production
in human epidermal keratinocytes (48).
It is important to note that the ERK as well as the JNK and p38 pathways are involved in coordinate AP-1 activation by distinct mechanisms, the up-regulation of c-Fos or c-Jun, respectively (49). Activation of the JNK pathways is commonly responsible for activation of c-Jun, whereas the ERK pathway in most cell types leads to the phosphorylation and activation of ELK-1, a transcription factor mediating the induction of the c-fos gene by ternary complex factors (50).
In this context our studies suggest that
O2- can principally deliver
signals into all major MAPK cascades activated by proinflammatory
cytokines in renal MC. Activation of these cascades finally regulates
the activity of key transcription factors, such as c-Jun or NF-B,
leading to further augmentation of cytokine-triggered MMP-9 gene
expression. These processes may result in elevated glomerular MMP-9
expression and thus contribute to the progression of disease and
correlate with the structural glomerular damage seen in a variety of
inflammatory glomerular diseases (51, 52). Amplification
of cytokine-mediated MMP-9 gene expression by
O2- may explain how increased
levels of ROS, mainly produced by inflammatory cells, are functionally
linked with the increased levels and duration of proteolytic activity
within the inflamed glomerulus. The simultaneous production of
O2- and NO by MC exposed to an
inflammatory environment and the opposite effects of both radicals on
MMP-9 expression may provide an additional level of modulation with a
subtle change in the ratio of
O2-/NO production resulting in
quite dramatic shifts in MMP-9 expression, thus constituting a
switch-like mechanism. Our studies provide insight into the molecular
mechanisms underlying dysregulated MMP expression and may provide new
therapeutic strategies for the treatment of inflammatory diseases
associated with pathological MMP-9 overproduction.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Josef Pfeilschifter, Zentrum der Pharmakologie, Klinikum der Johann Wolfgang Goethe Universität, Theodor Stern Kai 7, D-60590 Frankfurt am Main, Germany.
3 Abbreviations used in this paper: MC, mesangial cell; APMA, p-amino phenylmercuric acetate; ECM, extracellular matrix; DMNQ, dimethoxy-1,4-naphtoquinone; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; MMP-9, matrix metalloproteinase-9; MnTBAP, manganese-tetrakis(4-benzoic acid)porphyrin; ROS, reactive oxygen species; SAPK, stress-activated protein kinase; SNAP, S-nitroso-N-acetyl-D,L-penicillamine; SOD, superoxide dismutase; TIMP, tissue inhibitor of matrix metalloproteinases.
Received for publication April 19, 2000. Accepted for publication August 17, 2000.
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) ± SD (
).
n = 3.
