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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:2527.)
© 2000 American Heart Association, Inc.

Vascular Biology

Mitogen-Activated Protein Kinases Mediate Matrix Metalloproteinase-9 Expression in Vascular Smooth Muscle Cells

Aesim Cho; Jonathan Graves; Michael A. Reidy

From the Department of Pathology (A.C., M.A.R.) and the Department of Immunology (J.G.), University of Washington, Seattle.

Correspondence to Aesim Cho, PhD, Department of Pathology, Box 357335, University of Washington, Seattle, WA 98195. E-mail ascho{at}u.washington.edu

	Abstract

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Abstract—Expression of matrix metalloproteinase (MMP)-9 has been linked to the progression of plaque rupture and intimal formation in arterial lesions. In this study, we determined which factors and signaling pathways are involved in regulating the MMP-9 gene. Rat carotid arterial smooth muscle cells treated with tumor necrosis factor (TNF)- showed a marked increase in MMP-9 activity and mRNA level, whereas platelet-derived growth factor (PDGF) showed a slight induction of the MMP-9 mRNA level. TNF- treatment caused an increase in c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38 MAPK), and extracellular signal–regulated kinase (ERK) activities, whereas PDGF treatment caused an increase in ERKs and p38 MAPK activities without any effect on JNK activity. Treatment with either SB203580 (inhibitor of p38 MAPK) or U0126 (inhibitor of the ERK pathway) downregulated the TNF-–induced MMP-9 expression in a dose-dependent manner. Treatment of cells with TNF- and PDGF together stimulated the MMP-9 expression at a level higher than that observed with either factor alone, suggesting that TNF- and PDGF have a synergistic effect on MMP-9 expression in arterial smooth muscle cells. Furthermore, suboptimal inhibitory concentrations of SB203580 and U0126 together almost completely inhibited the MMP-9 expression. These results suggest that p38 MAPK and ERK pathways contribute to the transcriptional regulation of MMP-9 in arterial smooth muscle cells.

Key Words: mitogen-activated protein kinases • p38 mitogen-activated protein kinase • extracellular signal–regulated kinase • matrix metalloproteinase-9 • tumor necrosis factor

	Introduction

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Degradation of extracellular matrix by matrix metalloproteinases (MMPs) is thought to be important in the progression of atherosclerosis and plaque rupture. MMP-1, MMP-3, and MMP-9 have been identified in human atherosclerotic lesions, and the enhanced expression of MMP-9 at the shoulders of these lesions has been linked to plaque rupture.¹ In experimental animal models, MMPs are also shown to be important for smooth muscle cell migration into the intima. In a rat arterial injury model, after the initial medial smooth muscle cell replication, medial smooth muscle cells migrate and first appear in the intima 4 days after injury.² MMP-9 is expressed within 6 hours after injury in rat carotid arteries and continues to be expressed up to 6 days, whereas MMP-2 activity is markedly increased after 4 days of injury.³ The importance of these MMPs in the migration of smooth muscle cells is illustrated by the finding that the administration of MMP inhibitor almost completely inhibits the number of smooth muscle cells migrating into the intima.³ Although MMPs play an important role in lesion growth, little is known about the signaling mechanism involved in regulating MMP expression in vascular smooth muscle cells.

Several growth factors and cytokines, such as basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), tumor necrosis factor (TNF)-, and interleukin (IL)-1, induce MMPs, including interstitial collagenase (MMP-1), stromelysin (MMP-3), and type IV collagenase (MMP-2 and MMP-9) in a variety of cell types.^{4 5 6} Likely intracellular signaling transduction pathways activated by these factors are mitogen-activated protein kinase (MAPK) pathways, of which there are 3 distinct groups: extracellular signal–regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. The ERK pathway is mainly activated by growth factors and has been linked to cell proliferation, cell growth, and differentiation.⁷ JNK and p38 MAPK are known as stress-activated protein kinases, inasmuch as they are strongly activated in response to stressful stimuli, such as osmotic shock, UV light, and cytokines.⁷ JNK and p38 MAPK have been linked to apoptosis, cell survival, transformation, development, cell migration, and immune activation.^{8 9} These MAPKs are activated by sequential kinase cascade; eg, ERKs are activated by MAPK kinase kinase (MKK)1 and MKK2, JNK is activated by MKK4 and MKK7, and p38 MAPK is activated by MKK3 and MKK6. Once activated, ERKs act on p90rks, Elk-1, c-Fos, and cAMP response element binding protein, and JNK phosphorylates activating transcription factor (ATF)-2 and c-Jun.^{10 11} Activated p38 MAPK can phosphorylate ATF-2, C/EBP-homologous protein (CHOP), and MAPK-activated protein kinase (MAPKAPK)-2, which activates small heat shock proteins and cAMP response element binding protein.^{12 13}

In the present study, we investigated the signaling pathways involved in the regulation of MMP-9 expression in vascular smooth muscle cells, and our data show that the activation of ERKs and p38 MAPK contributes to the induction of MMP-9 expression in vascular smooth muscle cells.

	Methods

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Cell Culture
Arterial smooth muscle cells, isolated from carotid arteries of Sprague-Dawley rats, were grown in Waymouth complete media supplemented with 10% calf serum, 200 U penicillin/mL, and 200 mg streptomycin/mL (GIBCO-BRL Laboratories). At confluence, cells were serum-starved for 24 hours and then treated with TNF- (R & D Laboratories), transforming growth factor (TGF)-ß1 (R &D Laboratories), PDGF (gift from Hoffman La Roche, Basel, Switzerland), bFGF (gift from Scios Nova, Sunnyvale, Calif), SB203580 (p38 MAPK inhibitor, Calbiochem), U0126 (MEK1 inhibitor, Promega), and anisomycin (Sigma Chemical Co) as indicated in each experiments. At the end of treatment, conditioned media were collected for zymography and Western blot, and cells were rinsed once with ice-cold PBS and collected for RNA extraction, kinase assays, and Western blot analysis. Cells were used between passages 5 and 25.

Zymography
Conditioned media were subjected to zymography according to the procedure previously described.¹⁴ Equal volumes of conditioned media were loaded onto 8% polyacrylamide gels containing 0.1% of type I gelatin (Sigma Chemical Co) and electrophoresed at a constant voltage. After electrophoresis, gels were rinsed in 2.5% Triton X-100 for 30 minutes, incubated for 16 to 18 hours at 37°C in a buffer (50 mmol/L Tris [pH 8.0], 2.5 mmol/L CaCl₂, and 0.02% sodium azide), rinsed in 10% trichloroacetic acid, and stained in rapid Coomassie stain. The stained gels were visualized by Eagle-Eye Image (Stratagene), and the relative amounts of MMP-9 and MMP-2 were quantified by NIH Image Analysis software.

Northern Blot Analysis and Quantitative PCR
Cells from culture experiments were scraped and lysed in Trizol (GIBCO-BRL), and RNA was extracted by chloroform extraction and isopropanol precipitation. Fifteen micrograms of total RNA was separated on a 0.5% formaldehyde–1.2% agarose gel in 1x MOPS buffer and transferred to nylon membranes. The membranes were hybridized with cDNA probe for rat MMP-9 (800 bp, a gift from Dr Birkedal-Hansen, University of Alabama, Birmingham)¹⁵ labeled with [³²P]dCTP by random primer extension (Multi-Prime, Amersham) for several days, washed, and then exposed to Hyperfilm-MP (Amersham). Two micrograms of total RNA from each sample was treated with DNase I (Promega), reverse-transcribed by use of Superscript reverse transcriptase (Promega), and polymerase chain reaction (PCR)-amplified quantitatively for MMP-9 expression with a Taqman Sequence Detection Assay (PE Biosystems). In this assay, we used a fluorogenic probe (5'-TTGCAGGGCCCCTTT-CTTATTGCC-3') and primers (5'-AGTTCTCGAATCACGGA-GGAAA-3' and 5'-CGGATCCTCAAAGGCTGAGTT-3') to detect the specific MMP-9 PCR product as it accumulates during PCR at 60°C annealing temperature. Similarly, the same RNA samples were PCR-amplified for 18S ribosome, and the MMP-9 expression was normalized to the 18S expression.

Western Blot Analysis
Cells from culture experiments were scraped and collected into kinase lysis buffer as described above. Ten micrograms of total protein of each extract was separated on a 10% SDS-PAGE gel and transferred onto nitrocellulose membranes (PROTRAN, Schleicher & Schuell). The membranes were blocked with 5% nonfat dry milk in 10 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, and 0.1% Tween 20 (TBS-T), incubated with primary antibody for 1 hour, washed with TBS-T, incubated with secondary antibody conjugated to horseradish peroxidase (Amersham) for 1 hour, washed again, and detected by enhanced chemiluminescence (New England Biolabs Inc). For detection of MMP-9, ERKs, phospho-p38 MAPK, phospho-JNK, phospho-ERK, and phospho-p90rsk, rabbit polyclonal antibodies raised against MMP-9 (kindly provided by Dr Moon, Flanders Institute of Biotechnology, Leuven, Belgium), ERKs, and phosphorylated p38 MAPK, JNK, ERK, and p90rsk (New England Biolabs Inc) were used as primary antibodies. For detection of c-Jun, goat polyclonal antibody raised against c-Jun (Santa Cruz Biotechnology) was used as primary antibody.

	Results

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Effect of Growth Factors and Cytokines on MMP-9 Expression
To determine which MAPK pathways might contribute to the regulation of MMP-9 gene expression in vascular smooth muscle cells, we examined the effect of growth factors and cytokines known to be present in injured arteries when MMP-9 is expressed. Rat arterial smooth muscle cells were treated with various concentrations of PDGF-BB, bFGF, TNF-, or TGF-ß1, and with the exception of TNF-, the conditioned media from smooth muscle cells showed no detectable MMP-9 gelatinolytic activity (Figure 1A). This induction of MMP-9 activity by TNF- was completely abolished in the presence of a soluble receptor for TNF- (data not shown). The gelatinolytic activity of MMP-2 was not significantly affected by any of the factors tested. Northern blot analysis showed that mRNA transcripts corresponding to MMP-9 gene were significantly increased with TNF- treatment, whereas TGF-ß1 and bFGF had little effect (please see Figure I, published online at http://atvb.ahajournals.org). PDGF showed a slight induction of the MMP-9 mRNA level at a concentration of 50 ng/mL (please see Figure I, published online at http://atvb.ahajournals.org).

View larger version (59K): [in this window] [in a new window]   Figure 1. Gelatinolytic activity (A) and phosphorylation level (B) of arterial smooth muscle cells treated with TNF-, TGF-ß1, PDGF, and bFGF for 24 hours (A) and for 0, 5, 10, and 20 minutes (B). Conditioned media were run on a gelatin zymogram, and kinase activities of cells were measured by Western blot analysis for phospho-JNK, -p38 MAPK, and -ERKs. Minutes after treatment are given. Figure is representative of 5 independent experiments.

Activities of MAPKs in Vascular Smooth Muscle Cells
To determine which signaling pathway is involved in the activation of MMP-9 expression, we examined the activities of 3 members of the MAPKs (ERKs, JNK, and p38 MAPK) in vascular smooth muscle cells treated with TNF-, bFGF, and PDGF. The kinase activities were assessed by measuring their degree of regulatory phosphorylation by using phosphospecific antibodies. bFGF and PDGF treatment showed very little increase in the phosphorylation of JNK, whereas TNF- markedly increased JNK phosphorylation (Figure 1B). The phosphorylation of p38 MAPK was slightly, but not significantly, increased with bFGF treatment, whereas its phosphorylation was markedly upregulated with PDGF and TNF- (Figure 1B). ERK phosphorylation was markedly elevated when cells were treated with bFGF and PDGF (Figure 1B). Interestingly, TNF- treatment also moderately increased the phosphorylation of ERKs. We also measured the activities of p38 MAPK, JNK, and ERK directly and observed a result similar to that shown in Figure 1B (data not shown). These data suggest that the induction of the MMP-9 mRNA level by TNF- and PDGF may be regulated by elevated activities of ERK, JNK, and p38 MAPK.

Effect of MAPK Activities on MMP-9 Expression
To examine directly whether MAPKs regulate MMP-9 expression, cells were stimulated in the presence of specific MAPK inhibitors. In the first experiment, Western blot analysis of the conditioned media of cells pretreated with SB203580 showed that the TNF-–induced expression of MMP-9 was inhibited by SB203580 in a concentration-dependent manner (please see Figure II, published online at http://atvb.ahajournals.org). The p38 MAPK is thought to be important for transcriptional and translational events, and so its effect on MMP-9 transcription was examined. Quantitative PCR showed that SB203580 inhibited TNF-–induced MMP-9 mRNA in a similar dose-dependent manner (Figure 2A). In addition, anisomycin, a known activator of the JNK and p38 MAPK pathway,^{16 17} significantly upregulated the MMP-9 mRNA level, which was also inhibited by SB203580 in a dose-dependent manner (Figure 2B). These data suggest a link between p38 MAPK activation and MMP-9 expression.

View larger version (23K): [in this window] [in a new window]   Figure 2. Quantitative PCR amplification of MMP-9 mRNA level of arterial smooth muscle cells pretreated with SB203580 (0.1, 0.3, 0.75, 1.25, 2.5, 5, 10, and 20 µmol/L) or dimethyl sulfoxide (0.1%, 0 µmol/L SB203580) for 45 minutes and then incubated with 100 ng/mL TNF- (A) or 10 µg/mL anisomycin (B) for 24 hours. Data are representative of 3 independent experiments, and values are expressed as mean (±SE) number of MMP-9 cDNA copies normalized to number of 18S cDNA copies.

SB203580 reversibly binds to active p38 MAPK and prevents phosphorylation of its substrates. Therefore, we measured the activity of MAPKAPK-2, a specific substrate of p38 MAPK, to confirm the action of SB203580. As shown in Figure 3A, a 50% inhibition was observed with an 0.5 µmol/L concentration of SB203580, which is similar to the observations of others.^{18 19} Complete inhibition of the activity of MAPKAPK-2 induced by TNF- or anisomycin was observed at concentrations of 5 to 10 µmol/L. These data indicate that activation of p38 MAPK makes a critical contribution to TNF-–induced MMP-9 expression in vascular smooth muscle cells.

View larger version (16K): [in this window] [in a new window]   Figure 3. MAPKAPK-2 activity of arterial smooth muscle cells pretreated with SB203580 (0.1, 0.3, 0.75, 1.25, 2.5, 5, 10, and 20 µmol/L) or dimethyl sulfoxide (0.1%, 0 µmol/L SB203580) for 45 minutes and then incubated with TNF- (100 ng/mL) for 10 minutes or with anisomycin (10 µg/mL) for 45 minutes. The MAPKAPK-2 activity is expressed as counts per minute (CPM) of [32P]ATP incorporated to MAPKAPK-2 substrate peptide. Experiments were repeated 3 times.

To exclude the possibility that SB203580 affected JNK and ERK pathways, the phosphorylation level of JNK and ERK was measured after treatment with the SB203580 compound. SB203580 had no effect on TNF-–induced JNK and had no effect on the phosphorylation of ERKs except at the high dose (20 µmol/L) (please see Figure III, published online at http://atvb.ahajournals.org). We also examined the downstream substrates of the MAPKs and found that the activity of c-Jun, a transcription factor considered to be the JNK target, was not affected with increasing concentration of SB203580 in cells treated with TNF- (Figure III). The phosphorylation level of p90rsk, a specific target of activated ERKs, also showed no change with SB203580 (Figure III). SB203580 also had no effect on the activity of JNK, c-Jun, ERK, and p90rsk induced by anisomycin (please see Figure IV, published online at http://atvb.ahajournals.org). These results clearly indicate the inhibitory specificity of SB203580 on the p38 MAPK pathway.

Others have suggested that ERKs are important for MMP-9 regulation, and we show that TNF- causes a moderate increase in ERK activity. Therefore, we examined the role of activated ERKs in the MMP-9 regulation in arterial smooth muscle cells by using inhibitors of the ERK pathway. The U0126 compound, a selective inhibitor of MEK1/MKK1, almost completely inhibited ERK activity at a concentration of 2.5 µmol/L (Figure 4A) and decreased the TNF-–induced MMP-9 gelatinolytic activity in a dose-dependent manner (Figure 4D). This suggests that the ERK activity contributes to the upregulation of the MMP-9 gene in arterial smooth muscle cells. U0126 had no effect on activation of c-Jun, but the phosphorylation of p38 MAPK was slightly decreased to the same extent with all concentrations of the inhibitor (Figure 4B and 4C).

View larger version (43K): [in this window] [in a new window]   Figure 4. Phosphorylation level of ERKs (A), p38 MAPK (B), and c-Jun (C) and gelatinolytic activity in conditioned media (D) of arterial smooth muscle cells pretreated with U0126 (1, 2.5, 5, 10, and 20 µmol/L) or dimethyl sulfoxide (0.1%, 0 µmol/L U0126) for 45 minutes and then treated with TNF- (100 ng/mL) for 10 minutes (A, B, and C) and for 24 hours (D). UT indicates untreated smooth muscle cells. Data are representative of 3 independent experiments.

Additive Role of p38 MAPK and ERKs on MMP-9 Expression
Because inhibition of either ERK or p38 MAPK activity downregulated the TNF-–induced MMP-9 gene expression, we were interested in examining whether ERK and p38 MAPK cooperate in the regulation of MMP-9. Thus, cells were pretreated with suboptimal inhibitory concentrations of SB203580 and U0126, and the gelatinolytic activity of MMP-9 in response to TNF- was measured. SB203580 (5 µmol/L) and U0126 (2.5 µmol/L) individually partially decreased the MMP-9 gelatinolytic activity, but together these inhibitors almost completely blocked MMP-9 (Figure 5).

View larger version (28K): [in this window] [in a new window]   Figure 5. Gelatinolytic activity of MMP-9 (top) in conditioned media from arterial smooth muscle cells pretreated with SB203580 (5 and 10 µmol/L [SB5 and SB10, respectively]), 2.5 µmol/L U0126 (U), and combination of SB203580 and U0126 for 45 minutes and then treated with TNF- (100 ng/mL) for 24 hours. The relative expression of MMP-9 is quantified from zymograms of 3 independent experiments and expressed as mean (±SE) percent of dimethyl sulfoxide (DMSO)-treated sample (bottom). Bonferroni/Dunn post hoc ANOVA was used to test for significant differences between inhibitor-treated samples and DMSO-treated sample. Data were considered significant at P<0.05.

TNF- and PDGF individually activate p38 MAPK and ERKs and induce MMP-9 in vascular smooth muscle cells (Figure 1). Therefore, we asked whether MMP-9 expression in smooth muscle cells stimulated with TNF- and PDGF together could still be blocked by these inhibitors of the p38 MAPK and ERK pathways. The addition of TNF- and PDGF together induced MMP-9 gelatinolytic activity to a higher level than did treatment with TNF- or PDGF alone (Figure 6A). This induction of MMP-9 by TNF- and PDGF together was only slightly inhibited by SB203580 or U0126 alone but was almost completely blocked when both inhibitors were added together (Figure 6B). Together, these data suggest that p38 MAPK and ERKs are the most important signaling pathways and that both are required for the synergistic induction of MMP-9 by cytokines and growth factors.

View larger version (42K): [in this window] [in a new window]   Figure 6. A, Gelatinolytic activity of MMP-9 in conditioned media treated with TNF- (100 ng/mL), PDGF (50 ng/mL), and combination of TNF- and PDGF for 24 hours (top). The relative expression of MMP-9 is quantified from zymograms of 4 independent experiments and expressed as an arbitrary unit (bottom). B, Gelatinolytic activity of MMP-9 in conditioned media from smooth muscle cells pretreated with 5 µmol/L SB203580 (SB5), 2.5 µmol/L U0126 (U2.5), and combination of SB203580 and U0126 (SB+U) for 45 minutes and then treated with TNF- (100 ng/mL) and PDGF (50 ng/mL) for 24 hours (top). The relative expression of MMP-9 is quantified from zymograms of 3 independent experiments and expressed as mean (±SE) percent of DMSO-treated sample (bottom). Bonferroni/Dunn post hoc ANOVA was used to test for significant differences between the growth factor–treated samples and the untreated sample (A) and between the inhibitor-treated samples and the DMSO-treated sample (B). Data were considered significant at P<0.05.

	Discussion

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Previous studies have shown that MMPs, specifically MMP-2 and MMP-9, are important for smooth muscle cell migration in balloon catheter arterial injury lesions.^{3 20} However, the factors and signaling mechanisms involved in the expression of MMPs in vascular smooth muscle cells are poorly understood. In the present study, we demonstrated that TNF- induces the expression of MMP-9 in vascular smooth muscle cells and that this induction is regulated by activation of the MAPKs, p38 MAPK and ERK.

Individually the ERK and the p38 MAPK pathways have been shown to regulate MMP-9 expression. For example, Gum et al²¹ showed that overexpression of dominant-negative MEK1 inhibits the MMP-9 expression in PMA-treated carcinoma cells. In a separate study, Simon et al²² showed that p38 MAPK is involved in PMA-induced MMP-9 secretion with use of the SB203580 compound. However, none of these studies addressed the potential role of ERK and p38 MAPK acting together in the regulation of MMP-9 gene expression. Our data show that complete inhibition of either p38 MAPK or ERK alone or partial inhibition of both pathways is able to totally downregulate MMP-9 expression. This means that when there is a complete inhibition of one pathway, activation of the other pathway is not sufficient to induce MMP-9 gene. Presumably, therefore, each pathway might contribute to different transcription factors necessary for activation of the MMP-9 promoter. If correct, this would explain why the absence of signal from either pathway would result in the complete inhibition of MMP-9. Thus, we believe that both ERK and p38 MAPK are necessary for MMP-9 expression. One caveat with this conclusion is the finding that anisomycin, a potent activator of p38 MAPK and JNK, does not activate ERK and yet strongly induces MMP-9 expression. This might suggest that activation of p38 MAPK to superphysiological levels can by itself stimulate MMP-9 expression; however, ERK as well as p38 MAPK signals are required when a physiological activator such as TNF- is used.

It has been widely accepted that SB203580 is a highly selective inhibitor of p38 MAPK.^{18 19} A recent study by Clerk and Sugden²³ demonstrated that SB203580 is able to inhibit JNK2-related isoforms in cardiac myocytes with 50% inhibition at concentrations of 3 to 10 µmol/L and thus raises a question concerning the specificity of this compound. However, these authors observed that even at 30 µmol/L SB203580, JNK activity was not completely inhibited (70% inhibition), whereas the MAPKAPK-2 activity was completely blocked at a concentration of 10 µmol/L, similar to previous observations by others.^{18 19} This shows that SB203580 is a more effective inhibitor of p38 MAPK than of JNK2. In the present study, SB203580 had no effect on JNK activity induced by TNF- or anisomycin, as demonstrated by its phosphorylation level and the activation of c-Jun (specific target of JNK). Furthermore, a similar range of SB203580 concentrations (10 µmol/L) completely inhibited MMP-9 expression and p38 MAPK activity. Thus, p38 MAPK activity, but not JNK activity, correlates with the regulation of MMP-9 gene expression.

U0126, an inhibitor of the ERK pathway, has been reported to be a potent and specific inhibitor of MEK1 (an upstream kinase of ERK 1/2); however, Favata et al²⁴ reported a 50% inhibition of p38 MAPK at 20 µmol/L U0126 in PMA-treated COS cells. We found U0126 to be a very potent inhibitor of the ERK pathway in arterial smooth muscle cells, as demonstrated by the complete inhibition of ERK phosphorylation; however, we did note a small inhibitory effect (40%) on TNF-–induced p38 MAPK activity. We do not think this finding significant because U0126 completely inhibits ERK activity and MMP-9 expression when p38 MAPK activity is still significantly elevated. Thus, we conclude that ERK and p38 MAPK are both necessary for MMP-9 expression.

Our data clearly show that TNF- is a potent regulator of MMP-9 in arterial smooth muscle cells. This finding is in accord with previous studies in which TNF- increased the MMP-9 production in human saphenous vein smooth muscle cells and human microvascular endothelial cells.^{25 26} We also show that TNF- and PDGF in combination can synergistically upregulate MMP-9 in vascular smooth muscle cells, and a similar finding was observed in skin fibroblasts.²⁷ This observation has an important implication to injured arteries, where these factors are likely to be present after balloon catheter injury. For instance, platelets adhering to the denuded subendothelial layer of the vessel wall immediately after injury would be a major source of PDGF release in injured arteries, and TNF- is expressed in rat arteries within 6 hours of injury and activates smooth muscle cell migration in vitro.^{28 29} Furthermore, several signaling kinases, including the p38 MAPK and ERKs, are activated early after injury before the onset of MMP-9 expression in rat arteries.³⁰ Thus, these cytokines and growth factors, released by balloon catheter injury, may control MMP-9 gene expression and smooth muscle cell migration by regulating the activation of p38 MAPK and ERKs.

It is not clear how the activation of p38 MAPK and ERK pathways results in the induction of MMPs. Several studies have shown that activator protein-1, nuclear factor (NF)-B, stimulatory protein-1, Ets, and retinoblastoma binding elements are involved in the regulation of the human MMP-9 gene.^{21 31} We found that TNF- increases the transactivation activity of NF-B, which is inhibitable by SB203580, thus suggesting that NF-B activity may play a role in the p38 MAPK–mediated regulation of MMP-9 (data not shown). Possibly, NF-B and activator protein-1, regulated by p38 MAPK and ERK, respectively, may cooperate on the activation of MMP-9 gene. Further studies will be required to identify the full complement of the transcription factors that are involved in the p38 MAPK–mediated and ERK-mediated control of the MMP-9 gene in vascular smooth muscle cells.

In summary, we showed that PDGF and TNF- synergistically upregulate MMP-9 expression and that activation of p38 MAPK and ERK pathways is necessary in the TNF-–induced regulation of MMP-9 in arterial smooth muscle cells.

	Acknowledgments

 
This study was supported by National Institutes of Health grant RO1 HL-41103. Dr Cho was supported by a postdoctoral fellowship from the Heart and Stroke Foundation of Canada. Authors would like to thank Frank Dastvan and Jeff Kozlowski for technical assistance with Northern blot and Western blot analysis and N. Eric Olson for helpful discussions for the manuscript.

Received April 19, 2000; accepted September 18, 2000.

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