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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2002;22:914.)
© 2002 American Heart Association, Inc.

Vascular Biology

Monocyte Chemoattractant Protein-1 Induces Proliferation and Interleukin-6 Production in Human Smooth Muscle Cells by Differential Activation of Nuclear Factor-B and Activator Protein-1

Christiane Viedt; Judith Vogel; Thomas Athanasiou; Weili Shen; Stephan R. Orth; Wolfgang Kübler; Jörg Kreuzer

From Innere Medizin III (C.V., J.V., T.A., W.S., W.K., J.K.) Universität Heidelberg, Germany, and Innere Medizin, Abteilung für Nephrologie/ Hypertonie, (S.R.O.), Inselspital, Bern, Switzerland.

Correspondence to Dr J. Kreuzer, Innere Medizin III, Universität Heidelberg, Bergheimer Str. 58, 69115 Heidelberg, Germany. E-mail joerg_kreuzer{at}med.uni-heidelberg.de

	Abstract

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Abstract— Inflammatory response and chemotaxis of vascular wall cells play an important pathogenic role in the development of atherosclerosis. Monocyte chemoattractant protein-1 (MCP-1) is a potent chemoattractant for monocytes. Besides the induction of monocyte recruitment, it has been suggested that MCP-1 may directly activate smooth muscle cells. We investigated whether MCP-1 affects the proliferation and cytokine production of human vascular smooth muscle cells (VSMCs) and determined the underlying signal transduction pathways. Stimulation of VSMCs with MCP-1 induced proliferation and resulted in a concentration- and time-dependent release of the proinflammatory cytokine interleukin-6 (IL-6). Pretreatment with pertussis toxin, GF109203X, and pyrrolidine dithiocarbamate inhibited MCP-1–dependent IL-6 release, suggesting the involvement of G_i proteins, protein kinase C, and nuclear factor-B (NF-B). MCP-1 also induced extracellular signal–regulated kinase, which, along with IL-6 release, was inhibited by pertussis toxin. PD98059 prevented MCP-1–induced extracellular signal–regulated kinase activation and cell proliferation. MCP-1 stimulated the binding activity of NF-B and of activator protein-1 (AP-1). As demonstrated by cis element double-stranded (decoy) oligodeoxynucleotides, NF-B was involved in IL-6 release by MCP-1, whereas proliferation was dependent on AP-1. The results clearly demonstrate that MCP-1 induces differential activation of NF-B and AP-1 in VSMCs. Thus, our data propose a new mechanism for the proatherogenic effect of MCP-1.

Key Words: atherosclerosis • monocyte chemoattractant protein-1 • interleukin-6 • nuclear factor-B • activator protein-1

	Introduction

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An important role in atherosclerotic lesion formation has been attributed to monocyte chemoattractant protein-1 (MCP-1).¹ Increased MCP-1 expression has been detected in atherosclerotic lesions but not in normal arteries.² Elevated levels of MCP-1 have been found in patients with myocardial infarction³ and systemic inflammation.⁴ MCP-1 is the prototype of the C-C chemokine ß subfamily and exhibits its most potent chemotactic activity toward monocytes and T lymphocytes.⁴ In addition to promoting the transmigration of circulating monocytes, MCP-1 exerts various effects on monocytes, including cytokine production and adhesion molecule expression.¹ MCP-1 expression is induced by inflammatory cytokines, growth factors, or complement factors in monocytes, endothelial cells, and vascular smooth muscle cells (VSMCs).^5–7

Besides the induction of monocyte recruitment, it has been suggested that MCP-1 can lead to gene induction in VSMCs and endothelial cells. MCP-1 can induce ß₂-integrin upregulation and tissue factor expression in VSMCs^8,9 and trigger cell migration in endothelial cells.¹⁰ Furthermore, CCR2, the major receptor for MCP-1, has been recently identified on VSMCs.^11,12

Thus, the weight of available evidence indicates that MCP-1 is a key factor that initiates the inflammatory process of atherogenesis and sustains the proliferative response in the vessel wall. Despite the evolving body of evidence revealing MCP-1 expression as 1 progression factor for atherogenesis, no information is available concerning its gene-regulatory mechanisms in VSMCs. We now provide evidence that MCP-1 can increase the proinflammatory response of VSMCs and induce cell proliferation via a mechanism involving the transcription factors nuclear factor-B (NF-B) and activator protein-1 (AP-1).

	Methods

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Human recombinant MCP-1, tumor necrosis factor- (TNF-), platelet-derived growth factor (PDGF) BB, interleukin-6 (IL-6) ELISA, anti–MCP-1, and anti CCR-2 antibody were purchased from R&D Systems. Pertussis toxin (PTX), pyrrolidine dithiocarbamate (PDTC), and PD98059 were from Calbiochem. GF109203X was from Biomol. Phenylmethylsulfonyl fluoride and NP-40 were from Sigma Chemical Co. Anti–-actin antibody, Pefabloc, and E-64 were purchased from Roche. Anti–phospho-ERK1/2 (where ERK is extracellular signal–regulated kinase) and anti-ERK1/2 antibodies were obtained from New England Biolabs. NF-B and AP-1 oligonucleotides were from MWG. Cell culture media and supplements were from GIBCO-BRL.

Cell Culture
Human VSMCs were grown by explant technique from unused portions of saphenous veins harvested for coronary artery bypass surgery, as described previously.¹³ More than 95% of the cells reacted with the smooth muscle -actin antibody that selectively recognizes VSMCs. Before the experiments, cells from passage 3 to 6 were washed with PBS and grown in serum-free medium (0.1% BSA in DMEM) for 24 hours to render VSMCs quiescent. Proliferation was measured by counting the cells and by [³H]thymidine incorporation. Dedifferentiation and differentiation of SMCs was induced as described, and the differentiation state was verified by detection of smooth muscle myosin and calponin.¹⁴

IL-6 Release
Cells were grown to confluence in 96-well plates and kept in serum-free medium for 24 hours before stimulation. IL-6 was determined in cell culture supernatants by ELISA, according to the manufacturer’s instructions.

Immunoblot
VSMC lysates (10 µg per lane) were subjected to 12% SDS-PAGE and subsequent immunoblotting, as described previously.¹⁵ Proteins were detected by using enhanced chemiluminescence (ECL, Amersham). Exposures were recorded on Hyperfilm (Amersham) for different time points and quantified by the use of a densitometer (Bio-Rad).

Reverse Transcription–PCR
Total RNA from VSMCs and monocytes was extracted, and specific cDNA was reverse-transcribed from 1 µg RNA, as described.¹¹ Primers were synthesized according to published sequences.¹⁶ Polymerase chain reaction (PCR) products were analyzed by gel electrophoresis and sequenced by MWG.

AP-1 EMSA
An electrophoretic mobility shift assay (EMSA) was carried out as described previously.¹⁵ Nuclear extracts (2 µg each) were incubated with [-³²P]ATP–labeled oligonucleotide probes and resolved on a 4% native polyacrylamide gel, which after dehydration was exposed to x-ray film for 12 to 24 hours. For the supershift assay, rabbit polyclonal antibodies against c-Jun and c-Fos were incubated with samples after the initial binding reaction between nuclear protein extracts and consensus oligodeoxynucleotide (ODNs).

NF-B EMSA
EMSA was carried out as described previously.¹³ Protein extracts (10 µg each) and [-³²P]ATP–labeled ODNs were incubated for binding of active NF-B for 20 minutes at room temperature and separated from unbound ODNs by electrophoresis on a native 5% polyacrylamide gel, and autoradiography was performed. Specificity of NF-B/DNA binding was tested with antibodies against p65 or p50 subunits of NF-B.

Decoy ODNs Technique
NF-B decoy ODNs, AP-1 decoy ODNs, and mutated controls used in the present studies have been described previously.¹⁷ VSMCs were preincubated with 10 µmol/L double-stranded ODNs for 6 hours. The ODNs-containing medium was then removed, and cells were washed twice with medium and incubated in fresh medium containing the stimuli for the indicated time.

Statistical Analysis
Multiple comparisons were evaluated with ANOVA, followed by the Fisher protected least significant difference method. Data are presented as mean±SD. Values of P<0.05 were considered statistically significant.

	Results

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Expression of CCR2 mRNA and Protein in VSMCs
The presence of a specific receptor binding site would be a key indication for the direct activation of VSMCs by MCP-1. Reverse transcription–PCR was conducted to amplify RNA isolated from VSMCs and mononuclear cells by the use of specific primers for the MCP-1 receptor, CCR2. The PCR products were analyzed by agarose gel electrophoresis (Figure 1A). The resultant CCR2 sequence was verified by sequencing the PCR product (data not shown). The presence of CCR2 protein was confirmed by immunoblots of VSMC lysates demonstrating a band migrating at 38 kDa, corresponding to the molecular weight of CCR2 protein (Figure 1B). CCR2 protein was present in VSMCs grown in FCS and under serum-free conditions as well as in dedifferentiated and differentiated VSMCs. Differentiation of VSMCs was verified through the detection of an increased expression of smooth muscle myosin and calponin (data not shown).

View larger version (29K): [in this window] [in a new window]   Figure 1. A, VSMCs express CCR2 mRNA. PCR for CCR2 was performed by using cDNA from VSMCs (lane 1) and monocytes (lane 2). Lane 3 is the negative control. PCR products were analyzed by agarose gel electrophoresis. Data are from 3 representative experiments. B, CCR2 protein is expressed in VSMCs. Lysates from VSMCs that were grown with 10% FCS (lane 1) or in serum-free medium for 24 hours (lane 2), from monocytes (lane 3), or from dedifferentiated (lane 4) or differentiated VSMCs (lane 5) were subjected to 10% SDS-PAGE and subsequent immunoblotting by using an anti-CCR2 antibody. Results are representative of three independent experiments.

MCP-Induced Proliferation of VSMCs
Stimulation with MCP-1 (1 ng and 10 ng/mL) significantly increased cell proliferation (Figure 2A). [³H]Thymidine incorporation was used to verify the cell numbers obtained by counting and demonstrated similar results (data not shown). Pretreatment of VSMCs with PTX, which inhibits G_i protein binding, with GF109203X, a protein kinase C (PKC) inhibitor, and with PD98059, a selective inhibitor of mitogen-activated protein kinase kinase (MEK), inhibited the proliferative effect of MCP-1 (Figure 2B). To exclude a paracrine effect of IL-6 on VSMC proliferation, VSMCs were incubated with antibodies against IL-6 and MCP-1 when stimulated with MCP-1. After 48 hours, the MCP-1 antibody but not the IL-6 antibody significantly inhibited cell proliferation (Figure 2B).

View larger version (39K): [in this window] [in a new window]   Figure 2. MCP-1 induced proliferation of VSMCs. A, VSMCs were stimulated with MCP-1, FCS (10%), or PDGF BB, as indicated, and cell number was counted. B, MCP-1–induced proliferation is Gi protein, PKC, and ERK1/2 dependent. VSMCs were preincubated with PTX (100 ng/mL, 16 hours), GF109203X (2x10-6 mol/L, 60 minutes), or PD98059 (30 µmol/L, 60 minutes), followed by stimulation with MCP-1 (10 ng/mL) for 48 hours in serum-free medium containing 0.1% BSA. For antibody experiments, VSMCs were incubated with antibodies against IL-6 and MCP-1 (each 10 µg/mL) when stimulated with MCP-1 (10 ng/mL). Data are shown as (mean±SD) fold change compared with baseline control from 6 independent experiments *P<0.05.

Induction of IL-6 Release by MCP-1 in VSMCs
To assess the inflammatory capacity of MCP-1 in atherogenesis, IL-6 release was determined. Stimulation of VSMCs with recombinant MCP-1 resulted in a time-dependent IL-6 secretion that began at 8 hours and reached a plateau with a significant (6-fold) increase at 24 hours (Figure 3A). The specificity of MCP-1–induced IL-6 release from VSMCs was established by conducting inhibition experiments with the use of neutralizing, anti–MCP-1, and anti–CCR-2 antibodies, which significantly reduced MCP-1– but not TNF-–dependent IL-6 release (Figure 3A). The dose-dependent release of IL-6 was confirmed by exposing VSMCs to increasing concentrations of MCP-1 for 24 hours, and then IL-6 was determined in the supernatants (Figure 3B).

View larger version (33K): [in this window] [in a new window]   Figure 3. MCP-1 induces IL-6 secretion from VSMCs. A, Cells were stimulated with MCP-1 (10 ng/mL) or TNF- (50 U/mL), and supernatants were assayed for IL-6. For antibody experiments, VSMCs were incubated with antibodies against MCP-1, CCR-2, or control IgG (each 10 µg/mL) when stimulated with MCP-1 (10 ng/mL). IL-6 release is shown as (mean±SD) fold change compared with unstimulated controls from 8 independent experiments, *P<0.05. B, Dose effect of MCP-1 on IL-6 synthesis is shown. VSMCs were exposed to increasing doses of MCP-1 (0.001 to 100 ng/mL) for 24 hours. Values are mean±SD (n=6). *P<0.05 compared with control. MCP-1–induced IL-6 release involves Gi proteins, PKC, and NF-B. VSMCs were preincubated with PTX (100 ng/mL, 16 hours), GF109203X (2x10-6 mol/L, 60 minutes), PDTC (10 µmol/L, 2 hours), or PD98059 (30 µmol/L, 60 minutes), followed by stimulation with MCP-1 (10 ng/mL). The supernatants were collected after 24 hours and assayed for IL-6 concentration by ELISA. Values are mean±SD (n=7). *P<0.05 compared with 10 ng/mL MCP-1.

Mechanisms of MCP-1–Induced IL-6 Secretion
To investigate the signal transduction pathways leading to MCP-1–mediated IL-6 release, VSMCs were preincubated with PTX, which inhibited the IL-6 release without affecting basal levels of the cytokine. MCP-1–induced IL-6 secretion was also inhibited by GF109203X or PDTC, an inhibitor of the IL-6 transcription regulator NF-B. In contrast to MCP-1–induced cell proliferation, pretreatment with PD98059 had no effect on IL-6 release (Figure 3B).

MCP-1 Induces Activation of ERK1/2
Measurements of ERK activity were used to gauge the ability of MCP-1 to activate VSMCs. MCP-1 caused a time-dependent transient activation of ERK1/2. ERK1/2 activity peaked 20 minutes after stimulation, with a 5.5-fold increase above baseline (Figure 4A). Reprobing of the Western blot with an antibody against total ERK1/2 was used to control equal protein loading (Figure 4A). The stimulatory effect of MCP-1 was concentration dependent, as shown in Figure 4B.

View larger version (26K): [in this window] [in a new window]   Figure 4. Activation of ERK1/2 by MCP-1. A, VSMCs were stimulated with MCP-1 (10 ng/mL) for the indicated periods of time. Cell lysates (15 µg) were subjected to 12% SDS-PAGE and subsequent immunoblotting. The activity of ERK1/2 was assayed with a phospho-specific anti-ERK1/2 antibody. Equal protein loading was ascertained by immunoblotting with an antibody against nonphosphorylated ERK1/2. Phospho-ERK1/2 (p-ERK1/2) band intensities were quantified by densitometry (bottom panel). The activities of ERK1/2 are shown as (mean±SD) fold change from 4 independent experiments compared with unstimulated controls. *P<0.05. B, Activation of ERK1/2 by different concentrations of MCP-1 is shown. VSMCs were stimulated for 20 minutes with MCP-1. *P<0.05 (n=4).

Mechanisms of MCP-1–Induced Activation of ERK1/2
To assess the pathways by which MCP-1 activates ERK1/2, VSMCs were incubated with specific inhibitors. Pretreatment with PTX, PD98059, and GF109203X inhibited maximal ERK activation by 78%, 77%, and 63%, respectively (P<0.05).

MCP-1 Activates the Transcription Factors NF-B and AP-1
The effects of MCP-1 on NF-B and AP-1 as downstream effectors were examined. Active NF-B was already present after 30 minutes of stimulation, and maximal NF-B activation was found after 2 hours (Figure 5A). Excess unlabeled ODNs reduced the signal intensity of the band associated with active NF-B, confirming specificity of the DNA-protein interaction. The addition of NF-B subunit p65–specific antibody to the binding reaction resulted in a shift of the binding complex, and p65 was identified as the prevailing protein in the activated NF-B complexes (data not shown). PDTC significantly inhibited MCP-1–induced NF-B activation, but PD98059 had no effect (Figure 5A). Accordingly, the MCP-1–induced DNA-binding activity of AP-1 was also investigated and found to be increased after stimulation for 30 minutes, with a peak activation lasting 1 to 2 hours (Figure 5B). Excess unlabeled AP-1 consensus sequence reduced the signal. The addition of c-Jun–, c-Fos–, and Fra-1–specific antibodies to the AP-1 binding reaction resulted in a shift of the binding complex and identified c-Jun as the prevailing protein in the AP-1 complexes (data not shown). PD98059 attenuated the MCP-1–induced AP-1 activation, whereas PDTC did not (Figure 5B).

View larger version (76K): [in this window] [in a new window]   Figure 5. Effect of MCP-1 on the DNA binding activity of NF-B. A, VSMCs were stimulated with MCP-1 (10 ng/mL) or TNF- (50 U/mL, 1 hour) for the indicated times, and EMSA for NF-B was performed as described in Methods. Competition of MCP-1–induced NF-B binding activity with a 100-fold excess unlabeled consensus NF-B ODNs (cold excess) demonstrated specificity of MCP-1–induced DNA binding complexes. B, Effect of MCP-1 on the DNA binding activity of AP-1 is shown. VSMCs were stimulated with MCP-1 (10 ng/mL) or PDGF (10 ng/mL, 2 hours) for the indicated times, and EMSA for AP-1 was performed as described in Methods. Competition of MCP-1–induced AP-1 binding activity with a 100-fold excess unlabeled consensus AP-1 ODNs (cold excess) served as a control. For inhibition experiments, cells were preincubated with PDTC (10 µmol/L, 2 hours) or PD98059 (30 µmol/L, 60 minutes), followed by stimulation with MCP-1 (10 ng/mL) for 2 hours (NF-B) or 1 hour (AP-1). Data are from 3 representative autoradiograms.

Significance of NF-B and AP-1 Activation for the MCP-1–Induced IL-6 Synthesis and Proliferation
The role of NF-B and AP-1 in MCP-1–mediated IL-6 gene regulation was elucidated further by the use of cis element double-stranded (decoy) ODNs, which scavenge active transcription factors, thereby blocking their binding to the promoter regions in target genes. Pretreatment with NF-B decoy ODNs, but not control ODNs or AP-1 decoy ODNs, specifically inhibited IL-6 secretion in response to MCP-1 (please refer to online Figure IA, which can be accessed at http://atvb.ahajournals.org). In contrast, cell proliferation was specifically inhibited by AP-1 decoy ODNs but not control ODNs or NF-B decoys ODNs (see online Figure IB).

	Discussion

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We demonstrate that apart from its effect on monocytes, MCP-1 activates 2 different signaling pathways in VSMCs: 1 leading to release of the proinflammatory cytokine IL-6 and 1 leading to cell proliferation. MCP-1 potently activated the transcription factors NF-B, a transcription factor system commonly involved in inflammatory and immune responses, and AP-1, a transcription factor involved in inflammatory and growth responses. This is the first report demonstrating that MCP-1 could induce cellular responses via transcription factor activation in VSMCs.

MCP-1 is produced by various cell types within the arterial wall, including macrophages, VSMCs, endothelial cells, and fibroblasts.¹ Studies using transgenic mice provide compelling evidence for a direct role of MCP-1 and its CCR2 receptor in monocyte recruitment and lesion progression during atherogenesis.^18,19

Our results show that VSMCs express CCR2 mRNA and protein. CCR2 protein was detected independently of the differentiation state or proliferative activity of VSMCs. Functional specific activity of CCR-2 receptor in SMCs could be clearly demonstrated by inhibition experiments with a CCR-2–neutralizing antibody. The expression of CCR2 has been previously described in human VSMCs^11,12 and human endothelial cells.¹⁰ In contrast to these findings, Schecter et al⁹ could not detect CCR2 in VSMCs on the basis of PCR studies. Nevertheless, their study clearly demonstrated the binding of MCP-1 and the induction of tissue factor.⁹ The reasons for differing results with respect to CCR2 RNA detection in VSMCs are still unclear. However, it is possible that the CCR2 mRNA is differentially regulated according to cell type and/or experimental conditions.

Previous studies have mostly focused on the induction of MCP-1 in VSMCs by inflammatory agents. Data in the present study now suggest that through the production of IL-6 in response to NF-B and AP-1 activation, MCP-1 itself functions as an inflammatory mediator for VSMCs. Inflammatory responses mediated by cytokines are presumably important at all stages of atherosclerosis. Monocyte adherence to endothelium and infiltration of the vessel wall are probably the first steps leading to the development of the fatty streaks, which depend on endothelial expression of adhesion molecules, an event that is regulated by cytokines.²⁰ In the advanced stages of atherosclerosis, cytokines may promote destabilization and rupture of plaques by the induction of matrix-degrading enzymes, ultimately leading to thrombosis and complete obstruction of the vessel.²¹ Induction of IL-6 by MCP-1 could contribute to these events. IL-6 production in the vessel wall may be an important factor in local and generalized inflammatory reactions in the evolution of acute coronary syndromes, because IL-6 regulates the expression of adhesion molecules and other cytokines,^8,22 which potentially enhance the inflammatory reaction. Stimulation of lymphocytes by IL-6 may also be important, because activated T lymphocytes are present in human atheromas and probably contribute to ongoing inflammation within the plaque.^23–25

In the present study, stimulation with MCP-1 induced not only cytokine release but also VSMC proliferation, which may also contribute to lesion progression. Previously ambiguous results have been reported for MCP-1–dependent VSMC proliferation. MCP-1 has been shown to be both a positive and negative regulator of rat VSMC proliferation.^26,27 The reasons for the different findings have yet to be identified but may include species and cellular phenotype dependence.

Seven transmembrane domain receptors couple via heterotrimeric G proteins to effect a wide spectrum of cellular responses, and as such, it was of interest to determine the signaling mechanisms. PTX blocked the MCP-1–induced proliferation, suggesting the involvement of G_i proteins. This finding is in accordance with data from Myers et al²⁸ and Schecter et al,⁹ who have shown that signal transduction of the human MCP-1 receptor can be blocked by PTX. The inhibition of PKC reduces MCP-1–dependent proliferation and IL-6 release. PKC has also been shown to be important for MCP-1 signaling in monocytes and T cells,^29,30 indicating a central role for this pathway in MCP-1 signal transduction.

Cells can respond to extracellular stimuli by activating signaling cascades that are mediated by members of the mitogen-activated protein kinase family, such as ERK, which has been shown to be involved in the activation of AP-1³¹ and NF-B.³² The present study demonstrates that MCP-1 rapidly activates ERK1/2 in VSMCs. MCP-1–induced ERK activation was G_i protein, PKC, and MEK dependent. To investigate the role of ERK in the MCP-1–dependent cellular response, ERK phosphorylation was inhibited, which completely inhibited cell proliferation, whereas IL-6 release was not affected.

IL-6 production, as well as the synthesis of other cytokines, and cell proliferation are regulated at the transcriptional levels. Previous reports indicate that the NF-B binding site located between positions -72 and -63 on the IL-6 gene is important for the induction of IL-6.³³ NF-B dimers do not promote gene transcription by themselves but as a part of a complex of several coactivators.³⁴ Moreover, NF-B interacts with a variety of other transcription factors in a positive or negative manner. One of the factors most commonly involved in the activation of NF-B target genes is AP-1, which is also involved in the regulation of the IL-6 transcription, with a consensus binding sequence found in position -283 to -277 in the IL-6 promoter.³⁵

In the present study, we could demonstrate that MCP-1 increased IL-6 expression and stimulated NF-B and AP-1 activation. Therefore, we reason that MCP-1 stimulates IL-6 gene expression through the NF-B and AP-1 complexes. Although recent studies have shown the involvement of the ERK pathway in NF-B activation,^32,36 we observed no inhibitory effects of PD98059 on MCP-1–induced NF-B binding activity in VSMCs. The finding that the selective ERK inhibitor PD98059 did not inhibit IL-6 release and activation of NF-B suggests that MCP-1 induces IL-6 induction via a pathway distinct from activation of ERK and AP-1. However, ERK and AP-1 were found to be crucial for proliferation.

The present findings indicate that NF-B and AP-1 induce distinct cellular responses. To prove this hypothesis, we used the decoy approach against NF-B and AP-1 binding sites. As shown in a previous report by our group,¹⁷ gel mobility shift assays demonstrated that decoy ODNs against NF-B or AP-1 binding sites specifically competed, whereas control ODNs did not. On examination of the functional coupling between NF-B and AP-1 activation and MCP-1–induced IL-6 synthesis, our results showed that only NF-B, but not AP-1, decoy ODNs effectively inhibited IL-6 production in response to MCP-1. Control decoy ODNs had no effect. This clearly shows that NF-B plays an important role in MCP-1–induced IL-6 secretion. In contrast, MCP-1–induced cell proliferation was largely dependent on AP-1 activation, whereas NF-B activation did not play a role in MCP-1–induced cell proliferation.

In conclusion, our results demonstrate that apart from its chemotactic property for monocytes, MCP-1 promotes proinflammatory responses in VSMCs by activation of transcription factors. Thus, MCP-1 should be regarded as more than "just" a chemokine; it should also be regarded as a proinflammatory mediator capable of inducing the production of inflammatory cytokines and cell proliferation. This may be one of the underlying mechanisms by which local or systemic MCP-1 expression can foster atherogenesis.

	Acknowledgments

 
This study was supported by a grant from the "Deutsche Forschungsgemeinschaft" Bonn/Bad Godesberg and by a grant from the "Thyssen Stiftung," Köln, Germany. We thank the Department of Cardiac Surgery, University of Heidelberg, for supplying human saphenous veins and Katharina Hanna for excellent technical assistance.

Received February 15, 2002; accepted March 6, 2002.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Terkeltaub R, Boisvert WA, Curtiss LK. Chemokines and atherosclerosis. Curr Opin Lipidol. 1998; 9: 397–405.[CrossRef][Medline] [Order article via Infotrieve]

2. Ylä-Herttuala S, Lipton BA, Rosenfeld ME, Sarkioja T, Yoshimura T, Leonard EJ, Witztum JL, Steinberg D. Expression of monocyte chemoattractant protein-1 in macrophage-rich areas of human and rabbit atherosclerotic lesions. Proc Natl Acad Sci U S A. 1991; 88: 5252–5256.[Abstract/Free Full Text]

3. Matsumori A, Furukawa Y, Hashimoto T, Yoshida A, Ono K, Shioi T, Okada M, Iwasaki A, Nishio R, Matsushima K, Sasayama S. Plasma levels of the monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 are elevated in patients with acute myocardial infarction. J Mol Cell Cardiol. 1997; 29: 419–423.[CrossRef][Medline] [Order article via Infotrieve]

4. Rollins BJ. Monocyte chemoattractant protein 1: a potential regulator of monocyte recruitment in inflammatory disease. Mol Med Today. 1996; 2: 198–204.[CrossRef][Medline] [Order article via Infotrieve]

5. Taubman MB, Rollins BJ, Poon M, Marmur J, Green RS, Berk BC, Nadal-Ginard B. JE mRNA accumulates rapidly in aortic injury and in platelet-derived growth factor-stimulated vascular smooth muscle cells. Circ Res. 1992; 70: 314–325.[Abstract/Free Full Text]

6. Torzewski J, Oldroyd R, Lachmann PJ, Fitzsimmons CJ, Proudfoot D, Bowyer DE. Complement-induced release of monocyte chemotactic protein 1 from human SMC: a possible initiating event in atherosclerotic lesion formation. Arterioscler Thromb Vasc Biol. 1996; 16: 673–677.[Abstract/Free Full Text]

7. Ueda A, Okuda K, Ohno S, Shirai A, Igarashi T, Matsunaga K, Fukushima J, Kawamoto S, Ishigatsubo Y, Okubo T. NF-kappa B and Sp1 regulate transcription of the human monocyte chemoattractant protein-1 gene. Immunology. 1994; 153: 2052–2063.

8. Ikeda U, Ikeda M, Seino Y, Takahashi M, Kasahara T, Kano S, Shimada K. Expression of intercellular adhesion molecule-1 on rat vascular smooth muscle cells by pro-inflammatory cytokines. Atherosclerosis. 1993; 104: 61–68.[CrossRef][Medline] [Order article via Infotrieve]

9. Schecter AD, Rollins BJ, Zhang YJ, Charo IF, Fallon JT, Rossikhina M, Giesen PL, Nemerson Y, Taubman MB. Tissue factor is induced by monocyte chemoattractant protein-1 in human aortic smooth muscle and THP-1 cells. J Biol Chem. 1997; 272: 28568–28573.[Abstract/Free Full Text]

10. Weber KS, Nelson PJ, Grone HJ, Weber C. Expression of CCR2 by endothelial cells: implications for MCP-1 mediated wound injury repair and In vivo inflammatory activation of endothelium. Arterioscler Thromb Vasc Biol. 1999; 19: 2085–2093.[Abstract/Free Full Text]

11. Denger S, Jahn L, Wende P, Watson L, Gerber SH, Kubler W, Kreuzer J. Expression of monocyte chemoattractant protein-1 cDNA in vascular smooth muscle cells: induction of the synthetic phenotype: a possible clue to SMC differentiation in the process of atherogenesis. Atherosclerosis. 1999; 144: 15–23.[CrossRef][Medline] [Order article via Infotrieve]

12. Hayes IM, Jordan NJ, Towers S, Smith G, Paterson JR, Earnshaw JJ, Roach AG, Westwick J, Williams RJ. Human vascular smooth muscle cells express receptors for CC chemokines. Arterioscler Thromb Vasc Biol. 1998; 18: 397–403.[Abstract/Free Full Text]

13. Kranzhofer R, Schmidt J, Pfeiffer CA, Hagl S, Libby P, Kubler W. Angiotensin induces inflammatory activation of human vascular smooth muscle cells. Arterioscler Thromb Vasc Biol. 1999; 19: 1623–1629.[Abstract/Free Full Text]

14. Su B, Mitra S, Gregg H, Flavahan S, Chotani MA, Clark KR, Goldschmidt-Clermont PJ, Flavahan NA. Redox regulation of vascular smooth muscle cell differentiation. Circ Res. 2001; 89: 39–46.[Abstract/Free Full Text]

15. Viedt C, Soto U, Krieger-Brauer HI, Fei J, Elsing C, Kübler W, Kreuzer J. Differential activation of mitogen-activated protein kinases in smooth muscle cells by angiotensin II: involvement of p22phox and reactive oxygen species. Arterioscler Thromb Vasc Biol. 2000; 20: 940–948.[Abstract/Free Full Text]

16. Charo IF, Myers SJ, Herman A, Franci C, Connolly AJ, Coughlin SR. Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails. Proc Natl Acad Sci U S A. 1994; 91: 2752–2756.[Abstract/Free Full Text]

17. Viedt C, Hänsch GM, Brandes RP, Kübler W, Kreuzer J. The terminal complement complex C5b-9 stimulates interleukin-6 production in human smooth-muscle cells through activation of transcription factors NF-B and AP-1. FASEB J. 2000; 14: 2370–2372.[Abstract/Free Full Text]

18. Boring L, Gosling J, Cleary M, Charo IF. Decreased lesion formation in CCR2-/- mice reveals a role for chemokines in the initiation of atherosclerosis. Nature. 1998; 394: 894–897.[CrossRef][Medline] [Order article via Infotrieve]

19. Aiello RJ, Bourassa PA, Lindsey S, Weng W, Natoli E, Rollins BJ, Milos PM. Monocyte chemoattractant protein-1 accelerates atherosclerosis in apolipoprotein E–deficient mice. Arterioscler Thromb Vasc Biol. 1999; 19: 1518–1525.[Abstract/Free Full Text]

20. Bevilacqua MP, Pober JS, Mendrick DL, Cotran RS, Gimbrone MA. Identification of an inducible endothelial-leukocyte adhesion molecule. Proc Natl Acad Sci U S A. 1987; 84: 9238–9242.[Abstract/Free Full Text]

21. Libby P, Sukhova G, Lee RT, Galis ZS. Cytokines regulate vascular functions related to stability of the atherosclerotic plaque. J Cardiovasc Pharmacol. 1995; 25 (suppl 2): S9–S12.

22. Le J, Vilcek J. Interleukin 6: a multifunctional cytokine regulating immune reactions and the acute phase protein response. Lab Invest. 1989; 61: 588–602.[Medline] [Order article via Infotrieve]

23. Hansson GK, Holm J, Jonasson L. Detection of activated T lymphocytes in the human atherosclerotic plaque. Am J Pathol. 1989; 135: 169–175.[Abstract]

24. Libby P. Molecular bases of the acute coronary syndromes. Circulation. 1995; 91: 2844–2850.[Free Full Text]

25. van der Wal AC, Becker AE, van der Loos CM, Das PK. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaque is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation. 1994; 89: 36–44.[Abstract/Free Full Text]

26. Porreca E, Di Febbo C, Reale M, Castellani ML, Baccante G, Barbacane R, Conti P, Cuccurullo F, Poggi A. Monocyte chemotactic protein 1 (MCP-1) is a mitogen for cultured rat vascular smooth muscle cells. J Vasc Res. 1997; 43: 58–65.

27. Ikeda U, Okada K, Ishikawa S, Saito T, Kasahara T, Shimada K. Monocyte chemoattractant protein 1 inhibits growth of rat vascular smooth muscle cells. Am J Physiol. 1995; 268: H1021–H1026.[Abstract/Free Full Text]

28. Myers SJ, Wong LM, Charo IF. Signal transduction and ligand specificity of the human monocyte chemoattractant protein-1 receptor in transfected embryonic kidney cells. J Biol Chem. 1995; 270: 5786–5792.[Abstract/Free Full Text]

29. Sozzani S, Luini W, Molino M, Jilek P, Bottazzi B, Cerletti C, Matsushima K, Mantovani A. The signal transduction pathway involved in the migration induced by a monocyte chemotactic cytokine. J Immunol. 1991; 147: 2215–2221.[Abstract]

30. Cai JP, Hudson S, Ye MW, Chin YH. The intracellular signaling pathways involved in MCP-1-stimulated T cell migration across microvascular endothelium. Cell Immunol. 1996; 167: 269–275.[CrossRef][Medline] [Order article via Infotrieve]

31. Karin M, Liu ZG, Zandi E. AP-1 function and regulation. Curr Opin Cell Biol. 1997; 9: 240–246.[CrossRef][Medline] [Order article via Infotrieve]

32. Vanden Berghe W, Plaisance S, Boone E, De Bosscher K, Schmitz ML, Fiers W, Haegeman G. p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways are required for nuclear factor-kappaB p65 transactivation mediated by tumor necrosis factor. J Biol Chem. 1998; 273: 3285–3290.[Abstract/Free Full Text]

33. Shimizu H, Mitomo K, Watanabe T, Okamoto S, Yamamoto K. Involvement of a NF-B like transcription factor in the activation of the interleukin-6 gene by inflammatory lymphokines. Mol Cell Biol. 1990; 10: 561–599.[Abstract/Free Full Text]

34. Sheppard KA, Rose DW, Haque ZK, Kurokawa R, McInerney E, Westin S, Thanos D, Rosenfeld MG, Glass CK, Collins T. Transcriptional activation by NF-kappaB requires multiple coactivators. Mol Cell Biol. 1999; 19: 6367–6378.[Abstract/Free Full Text]

35. Vanden Berghe W, De Bosscher K, Boone E, Plaisance S, Haegeman G. The nuclear factor-kappaB engages CBP/p300 and histone acetyltransferase activity for transcriptional activation of the interleukin-6 gene promoter. J Biol Chem. 1999; 274: 32091–32098.[Abstract/Free Full Text]

36. Briant L, Robert-Hebmann V, Sivan V, Brunet A, Pouyssegur J, Devaux C. Involvement of extracellular signal-regulated kinase module in HIV-mediated CD4 signals controlling activation of nuclear factor-kappa B and AP-1 transcription factors. J Immunol. 1998; 160: 875–885.

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