Monocyte Chemoattractant Protein-1 Induces Proliferation and Interleukin-6 Production in Human Smooth Muscle Cells by Differential Activation of Nuclear Factor-κB and Activator Protein-1
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 Gi proteins, protein kinase C, and nuclear factor-κB (NF-κB). 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-κB and of activator protein-1 (AP-1). As demonstrated by cis element double-stranded (decoy) oligodeoxynucleotides, NF-κB 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-κB and AP-1 in VSMCs. Thus, our data propose a new mechanism for the proatherogenic effect of MCP-1.
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 β 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 β2-integrin upregulation and tissue factor expression in VSMCs8,9⇓ 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-κB (NF-κB) and activator protein-1 (AP-1).
Human recombinant MCP-1, tumor necrosis factor-α (TNF-α), 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–α-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-κB and AP-1 oligonucleotides were from MWG. Cell culture media and supplements were from GIBCO-BRL.
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 α-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
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.
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).
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.
An electrophoretic mobility shift assay (EMSA) was carried out as described previously.15 Nuclear extracts (2 μg each) were incubated with [γ-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).
EMSA was carried out as described previously.13 Protein extracts (10 μg each) and [γ-32P]ATP–labeled ODNs were incubated for binding of active NF-κB 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-κB/DNA binding was tested with antibodies against p65 or p50 subunits of NF-κB.
Decoy ODNs Technique
NF-κB 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.
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.
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).
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 Gi 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).
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-α–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).
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-κB. 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.
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-κB and AP-1
The effects of MCP-1 on NF-κB and AP-1 as downstream effectors were examined. Active NF-κB was already present after 30 minutes of stimulation, and maximal NF-κB activation was found after 2 hours (Figure 5A). Excess unlabeled ODNs reduced the signal intensity of the band associated with active NF-κB, confirming specificity of the DNA-protein interaction. The addition of NF-κB 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-κB complexes (data not shown). PDTC significantly inhibited MCP-1–induced NF-κB 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).
Significance of NF-κB and AP-1 Activation for the MCP-1–Induced IL-6 Synthesis and Proliferation
The role of NF-κB 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-κB 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-κB decoys ODNs (see online Figure IB).
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-κB, 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 VSMCs11,12⇓ 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-κB 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 Gi 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-κB.32 The present study demonstrates that MCP-1 rapidly activates ERK1/2 in VSMCs. MCP-1–induced ERK activation was Gi 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-κB binding site located between positions −72 and −63 on the IL-6 gene is important for the induction of IL-6.33 NF-κB dimers do not promote gene transcription by themselves but as a part of a complex of several coactivators.34 Moreover, NF-κB 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-κB 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-κB and AP-1 activation. Therefore, we reason that MCP-1 stimulates IL-6 gene expression through the NF-κB and AP-1 complexes. Although recent studies have shown the involvement of the ERK pathway in NF-κB activation,32,36⇓ we observed no inhibitory effects of PD98059 on MCP-1–induced NF-κB binding activity in VSMCs. The finding that the selective ERK inhibitor PD98059 did not inhibit IL-6 release and activation of NF-κB 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-κB and AP-1 induce distinct cellular responses. To prove this hypothesis, we used the decoy approach against NF-κB 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-κB or AP-1 binding sites specifically competed, whereas control ODNs did not. On examination of the functional coupling between NF-κB and AP-1 activation and MCP-1–induced IL-6 synthesis, our results showed that only NF-κB, 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-κB 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-κB 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.
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; revision accepted March 6, 2002.
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