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The Journal of Immunology, 2000, 165: 5788-5797.
Copyright © 2000 by The American Association of Immunologists

Amplification of IL-1{beta}-Induced Matrix Metalloproteinase-9 Expression by Superoxide in Rat Glomerular Mesangial Cells Is Mediated by Increased Activities of NF-{kappa}B and Activating Protein-1 and Involves Activation of the Mitogen-Activated Protein Kinase Pathways1

Wolfgang Eberhardt, Andrea Huwiler, Karl-Friedrich Beck, Sebastian Walpen and Josef Pfeilschifter2

Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe Universität, Frankfurt am Main, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The modulation of cell signaling by free radicals is important for the pathogenesis of inflammatory diseases. Recently, we have shown that NO reduces IL-1{beta}-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{beta}-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{beta}-triggered nuclear translocation of p65 and c-Jun and, in parallel, increased DNA binding activities of NF-{kappa}B and AP-1. Mutation of either response element completely prevented MMP-9 promoter activation by IL-1{beta}. 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{beta}-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-{kappa}B.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mesangial cells are known as specialized cells sharing a variety of smooth muscle cell properties, and under inflammatory conditions they also display features of macrophages (1). Once primed and activated by proinflammatory cytokines such as IL-1{beta} and TNF{alpha}, 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{beta} 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-{kappa}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-{kappa}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
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

Human rIL-1{beta} 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-{kappa}B-site (GGAATTCCCCC to GGAATTGGCCC) to generate pGL-MMP-9-{Delta}NF-{kappa}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 {Delta}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 I{kappa}B 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 [{gamma}-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. I{kappa}B 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, I{kappa}B, 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).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exogenously applied superoxide enhances the lytic activity of MMP-9 in rat MC

To evaluate possible effects of O2- on the activity and/or expression of MMP-9, MC were treated with IL-1{beta} 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{beta}-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. 1Go 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{beta}. 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. 1GoB). As reported recently, the NO donor SNAP significantly reduces the secretion of MMP-9 in the cellular supernatants (Fig. 1GoA). By contrast, simultaneous treatment of MC with IL-1{beta} and 8 mU/ml HXXO results in a marked potentiation of IL-1{beta}-stimulated gelatinase content (Fig. 1GoA).



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  		FIGURE 1. Modulation of gelatinolytic activities of MMP-9 and MMP-2 in MC by NO and ROS. Migration properties were determined using standard m.w. markers. A, Thirty-six hours after stimulation, 10 µl of supernatant was subjected to SDS-PAGE zymography. The concentrations of substances were: IL-1{beta}, 2 nM; SNAP, 500 µM; hypoxanthine, 50 µM; and xanthine oxidase, 8 mU/ml. The data shown are representative for three similar experiments. B, In vitro activation of latent MMP-9 by APMA. Supernatant (100 µl) from cells treated for 36 h with IL-1{beta} (2 nM) was incubated for an additional 3 h with the indicated concentrations of APMA before being subjected to SDS-PAGE zymography.

 
To evaluate whether HXXO directly alters the gelatinolytic activity of MMP-9, we performed in vitro zymography. Conditioned media from cytokine-stimulated MC were treated for 24 h with different concentrations of HXXO. None of the tested concentrations of HXXO altered MMP-9 gelatinolytic activity, thus indicating that O2--mediated alterations of zymogen activity are due to changes in MMP-9 expression and not to direct changes in MMP-9 activity (data not shown).

Superoxide potentiates the IL-1{beta}-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{beta} (2 nM) in the presence of vehicle or different concentrations of HXXO. As shown in Fig. 2GoA, HXXO dose-dependently augments steady state levels of MMP-9 mRNA in IL-1{beta}-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. 2GoA). We also tested DMNQ for modulatory effects on IL-1{beta}-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{beta} and 5 µM DMNQ (Fig. 2GoB).



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  		FIGURE 2. Effects of different ROS on IL-1{beta}-induced MMP-9 and TIMP-1 mRNA steady state levels. A, Quiescent MC were treated for 24 h with vehicle (control), IL-1{beta} (2 nM), or IL-1{beta} plus the indicated concentrations of HXXO (mU/ml) before being harvested for total mRNA preparation. Total cellular RNA (20 µg) was hybridized to a 32P-labeled cDNA insert from KS-MMP-9. Equivalent loading of the RNA was ascertained by rehybridization to a GAPDH probe. Similar results were obtained in another independent experiment. B, Northern blot analysis demonstrating that HXXO and DMNQ both amplify MMP-9 steady state mRNA induced by IL-1{beta}. The concentrations used are indicated. The data shown are representative of three independent experiments that gave similar results. C, Northern blot analysis demonstrating that the effects seen by HXXO are reversed by coincubation with SOD or the cell-permeable SOD mimic MnTBAP. Coincubation with the H2O2 generator glucose oxidase did not enhance the IL-1{beta}-mediated MMP-9 mRNA accumulation. The agents were used at the indicated concentrations. Equivalent loading of total RNA was ascertained by hybridization with an 18S ribosomal RNA probe.

 
In the next step we evaluated potential effects of peroxynitrite on cytokine-induced MMP-9 mRNA levels to check the possibility that the modulatory effects on MMP-9 mRNA observed with O2- were actually mediated by peroxynitrite. Generation of peroxynitrite is likely to result from the simultaneous production of NO and O2-, as it occurs in cytokine-treated MC (2, 3, 4, 5). To this end we used SIN-1, which is a metabolite of the vasodilator molsidomine and is considered to be a peroxynitrite donor (23). However, in none of the concentrations tested did SIN-1 have any discernible effect on IL-1{beta}-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-{beta}-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{beta} plus HXXO and SOD (10 U/ml) largely prevented the amplification of cytokine-induced MMP-9 expression by HXXO (Fig. 2GoC). 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{beta} (Fig. 2GoC).

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. 2GoC, glucose oxidase had no stimulatory effect on the IL-1{beta}-mediated MMP-9 mRNA accumulation. Similar results were obtained by costimulation of MC with IL-1{beta} 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{beta} is potentiated by superoxide and critically depends on NF-{kappa}B and AP-1 binding sites

To further evaluate whether the observed effects of HXXO on IL-1{beta}-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. 3Go). 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-{kappa}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{beta} exposure (Fig. 4GoA). Treatment of cells with HXXO further increased IL-1{beta}-mediated luciferase activity dose-dependently up to 5.5-fold (Fig. 4GoA), 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-{kappa}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{beta}-mediated MMP-9 promoter induction, point-mutated MMP-9 promoter constructs were tested for inducibility by IL-1{beta}. To this end we inserted two base pair mutations in either the NF-{kappa}B or AP-1 binding sites (Materials and Methods). As shown in Fig. 4Go, IL-1{beta}-driven luciferase activity was totally lost in both promoter mutants (Fig. 4Go, B and C), thus indicating that both AP-1 and NF-{kappa}B binding sites are indispensable for MMP-9 promoter activation by IL-1{beta} 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. 4Go). These results indicate that the O2- amplification of IL-1{beta}-mediated MMP-9 expression critically depends on intact NF-{kappa}B and AP-1 binding sites within the MMP-9 promoter region.



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  		FIGURE 3. Sequence of the 5'-flanking region of the rat MMP-9 gene. Putative binding sites for NF-{kappa}B and AP-1 transcription factors were further investigated in this study; the TATA box and translation initiation site are underlined. The major transcription start site (+1) was predicted from the high grade of homology to mouse and human genes, respectively.

 


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  		FIGURE 4. Dose-dependent modulation of IL-1{beta}-induced wild-type and point-mutated MMP-9 promoter constructs by HXXO. The upper panel schematically shows the MMP-9 promoter constructs used for testing the remaining luciferase activities. Mutations introduced into the NF-{kappa}B or AP-1 binding sites of pGL-MMP-9wt (wt, wild type) by two base pairchanges are described in Materials and Methods. MC grown on six-well plates were cotransfected with 0.4 µg of pGL-MMP-9wt (A) or pGL-MMP-9 {Delta}NF-{kappa}B (B) or alternatively with pGL-MMP-9 {Delta}AP-1 (C) and 0.1 µg of pRL-CMV. After overnight transfection, MC were treated for 24 h with vehicle (control) or alternatively with IL-1{beta} or IL-1{beta} plus the indicated concentrations of HXXO before harvested for measuring dual luciferase activities. Values for beetle luciferase were related to the values for Renilla luciferase and are depicted as relative luciferase activities. Data are the mean ({square}) ± SD ({blacksquare}). n = 3.

 
Superoxide facilitates nuclear translocation of p65 and c-Jun initiated by IL-1{beta}

To further confirm the contributions of NF-{kappa}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-{kappa}B and AP-1 transcription factors, respectively. As shown in Fig. 5Go, treatment of MC with IL-1{beta} 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{beta}-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. 5Go).



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  		FIGURE 5. Time course of IL-1{beta}-induced nuclear accumulation of p65 and c-Jun is modulated by superoxide. Nuclear extracts (30 µg) from MC stimulated for the indicated time points were immunoblotted with the anti-p65- and anti-c-Jun-specific Ab, respectively. The cytokine-mediated appearance of both proteins in the nucleus is dose-dependently accelerated by cotreatment with HXXO. Similar results were obtained in two independent experiments.

 
Superoxide increases the IL-1{beta}-mediated DNA binding activity of AP-1 and NF-{kappa}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-{kappa}B. We recently reported a time-dependent induction of NF-{kappa}B in IL-1{beta}-stimulated rat MC (19). Nuclear extracts were prepared 30 min after stimulation, the point of maximal effects of HXXO (Fig. 5Go). As shown in Fig. 6GoA, increasing concentrations of HXXO caused a gradual augmentation of IL-1{beta}-mediated DNA binding of NF-{kappa}B-bound complexes. HXXO given alone had only a weak effect on NF-{kappa}B binding, which in none of the experiments was comparable to that evoked by IL-1{beta}. Supershift analysis indicated that the upper complex that is induced by IL-1{beta} contained p50/p65 proteins, as this complex was partially supershifted by anti-p50 Abs and was completely supershifted by p65-specific Abs (Fig. 6GoA, 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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  		FIGURE 6. IL-1{beta}-induced DNA binding activity of NF-{kappa}B (A) and AP-1 (B) in rat MC is potentiated by HXXO. A, Activation of NF-{kappa}B binding was analyzed by EMSA using a NF-{kappa}B consensus oligonucleotide as described in Materials and Methods. MC were stimulated with vehicle, IL-1{beta} (1 nM), or IL-1{beta} (1 nM) plus different concentrations of HXXO (mU/ml) as indicated for 30 min. The specificity of DNA-bound complexes was determined by supershift analysis. Data are representative of three independent experiments. For supershift analysis p50- and p65-specific Abs were included to the binding reactions. B, Activation of AP-1 DNA binding in rat MC by IL-1{beta} and HXXO. Activation of AP-1 binding was assessed by EMSA using a consensus oligonucleotide for AP-1. To determine the specificity of the DNA binding complexes the EMSAs were performed with either radioactively labeled sense (wt-oligo) or mutated (mt-oligo) oligonucleotides. NS, Nonspecific complex.

 
EMSA analysis with the same extracts, probed with an AP-1-specific oligonucleotide revealed DNA binding of two different complexes (Fig. 6GoB). 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{beta} and dose-dependently augmented by HXXO. The IL-1{beta} effects on DNA binding in all the EMSA experiments were weak, as we used low, suboptimal concentrations of IL-1{beta} to optimize the modulatory effects of superoxide. HXXO on its own did not induce AP-1 binding (Fig. 6GoB). The specificity of the IL-1{beta}-inducible complexes was tested using an oligonucleotide with two base changes within the AP-1 binding motif (CA to TG). As shown in Fig. 6GoB 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. 6GoB).

IL-1{beta}- induced degradation of I{kappa}B{alpha} is accelerated by superoxide

Activation of NF-{kappa}B requires dissociation and rapid degradation of the inhibitory protein I{kappa}B{alpha} (25). Western blot analysis displayed a marked decrease in I{kappa}B{alpha} protein by IL-1{beta}, which was further enhanced by increasing concentrations of HXXO (Fig. 7Go). HXXO alone reduced basal I{kappa}B{alpha} 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{beta} alone (Fig. 7Go, right panel). In contrast the level of JNK-p54 protein remained constant, thus indicating that changes in I{kappa}B{alpha} level were not due to unequal loading (see also Fig. 9GoA). In summary, the increase in IL-1{beta}-mediated NF-{kappa}B activation by O2- is paralleled by enhanced proteolytic degradation of I{kappa}B{alpha}.



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  		FIGURE 7. I{kappa}B{alpha} degradation in cytokine-treated MC is enhanced by cotreatment with superoxide. Quiescent MC were treated with vehicle, IL-1{beta} (2 nM), or IL-1{beta} plus the indicated concentrations of xanthine oxidase (mU/ml) and 50 µM hypoxanthine for 15 min. Protein lysates (100 µg) from cytoplasmic fractions were prepared as described in Materials and Methods and were subjected to SDS-PAGE and immunoblotted using an anti-I{kappa}B{alpha} (MAD-3)-specific polyclonal Ab. The right panel shows an overexposure of a blot to clarify the different effects of IL-1{beta} and those of HXXO on I{kappa}B{alpha} degradation. Equal protein loading was ascertained by Ponceau-S staining. The lower panel shows the same blot stripped and reincubated with anti-JNK-2 Ab. Blots are representative of three similar experiments.

 


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  		FIGURE 9. Effects of superoxide on IL-1{beta}-induced phosphorylation of p38 MAPK, JNK/SAPK (A), and p42/p44 MAPK (B). Quiescent MC were treated with vehicle, IL-1{beta} (2 nM), or IL-1{beta} plus xanthine oxidase (8 mU/ml) and 50 µM hypoxanthine for 15 or 60 min as indicated. Protein lysates from whole cell extracts were prepared as described in Materials and Methods. Protein lysates (100 µg) were subjected to SDS-PAGE and immunoblotted using phosphorylation state-specific Abs. To ascertain that the total level of each MAPK remained unchanged, blots were stripped and reprobed with the Abs raised against the corresponding phosphorylation-independent MAPK. Equal protein loading was ascertained by Ponceau-S staining. Blots are representative of two similar experiments. A, The results were derived from the same blot, successively incubated with the indicated Abs. B, The immunoblot was derived from the same extracts used for A, but blotted on a separate filter.

 
Inhibition of IL-1{beta}-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{beta}-primed MMP-9 mRNA accumulation as well as the amplification of MMP-9 mRNA accumulation caused by O2- (Fig. 8Go). 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{beta}-primed accumulation of MMP-9 mRNA as well as the rise in MMP-9 mRNA level caused by O2- (Fig. 8Go). Evidently both the p38-MAPK and the ERK pathways are specifically involved in IL-1{beta} 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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  		FIGURE 8. PD98059 and SB203580 partially inhibit cytokine- and superoxide-mediated induction of MMP-9 mRNA steady state levels. Quiescent MC were treated for 24 h with vehicle (control), IL-1{beta} (2 nM), or IL-1{beta} plus HXXO (8 mU/ml) in the presence or the absence of PD98059 (20 µM), SB203580 (10 µM), or a combination of both inhibitors. The inhibitors were given 30 min prior to the addition of cytokines and HXXO. Total cellular RNA (20 µg) was hybridized with a 32P-labeled cDNA insert from KS-MMP-9. Equivalence of RNA loading in different lanes was ascertained by rehybridization to a GAPDH probe. The blot is representative of two similar experiments.

 
Superoxide modifies the phosphorylation state of the different MAPKs activated by IL-1{beta}

In the next step we examined the activation of the different MAPK cascades by IL-1{beta} 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. 9GoA). IL-1{beta}-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. 9GoA). Western blot analysis with a phosphorylation-independent Ab revealed that p38 protein remained unchanged under all conditions tested (Fig. 9GoA, 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{beta} at both time point tested. Interestingly, HXXO on its own induced phosphorylation of JNK at intensities comparable to those produced by IL-1{beta}. Treatment of cells with IL-1{beta} 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. 9GoA).

Finally, we monitored the phosphorylation status of p42/p44 ERKs that are activated by IL-1{beta} in rat MC (31). Treatment of MC with 8 mU/ml of HXXO induced phosphorylation of mainly p42 at both time points examined (Fig. 9GoB). 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. 9GoB, lower panel). In conclusion, IL-1{beta} and HXXO both act at least additively on the activation of the three MAPK cascades in MC.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we have addressed the question of whether ROS modulate the signal transduction cascades involved in the up-regulation of MMP-9 by proinflammatory cytokines in MC, which is a hallmark of inflammatory processes in the glomerulus. Previously, we have demonstrated inhibitory effects of NO on IL-1{beta}-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-{kappa}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{beta}-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{beta}, 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. 1GoB). 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-{kappa}B and AP-1 (41), which may contribute to the modulation of IL-1{beta}-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{beta}-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{beta}-stimulated MMP-9 expression in MC is critically dependent on NF-{kappa}B and AP-1 transcription factors, as was shown by transfections of a dominant negative mutant of NF-{kappa}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{beta}-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{beta}-initiated signals. Activation of NF-{kappa}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{beta}-triggered signals by O2-, as we observed IL-1{beta}-stimulated nuclear translocation of p65 and c-Jun proteins with subsequent DNA binding affinities being substantially increased by O2-. The augmentation of NF-{kappa}B-DNA binding was paralleled by an increase in IL-1{beta}-induced degradation of I{kappa}B{alpha}. The question of whether O2- is able to directly interact with redox-sensitive proteins that regulate kinases upstream from I{kappa}B, 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{beta}-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{beta} was affected by O2-. Recently, we and others have demonstrated activation of this MAPK pathway by IL-1{beta} and NO in rat MC (17, 46, 47). The finding that IL-1{beta}-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-{kappa}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.


   Acknowledgments
 
We thank Martina Apel, Ute Schmidt, and Simone Dorsch for technical assistance.


   Footnotes
 
1 This work was supported by the Deutsche Forschungsgemeinschaft (SFB553 and HU842/2-1) and a grant from the Paul and Ursula Klein-Stiftung (Frankfurt, Germany). Back

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. Back

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. Back

Received for publication April 19, 2000. Accepted for publication August 17, 2000.


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