Lysophosphatidylcholine Stimulates Monocyte Chemoattractant Protein-1 Gene Expression in Rat Aortic Smooth Muscle Cells
Objective— Monocyte chemoattractant protein (MCP)-1 is a proatherogenic factor that is responsible for ≈60% of plaque macrophages in mouse models of atherosclerosis. We investigated whether lysophosphatidylcholine (LPC), enriched in oxidized low density lipoprotein, can modulate the expression of MCP-1 in arterial wall cells.
Methods and Results— LPC induced a 3-fold increase in MCP-1 mRNA in rat vascular smooth muscle cells (VSMCs) in a time- and dose-dependent manner. Nuclear runon analysis showed that this increase was attributable to increased MCP-1 gene transcription. There was a 2-fold increase in MCP-1 protein in the conditioned media of cells treated with LPC. LPC-associated increases of MCP-1 mRNA and protein were similar to those produced by platelet-derived growth factor-BB, a known inducer of MCP-1. Analyses of the MCP-1 promoter in transiently transfected VSMCs indicated an LPC-responsive element(s) between base pairs −146 and −261 (relative to transcription initiation). Further studies suggested that LPC-induced MCP-1 expression partially involves mitogen-activated protein kinase/extracellular signal–regulated kinase, a tyrosine kinase(s), and (to a lesser extent) protein kinase C but not the activation of the platelet-derived growth factor receptor.
Conclusions— LPC stimulates MCP-1 expression at the transcriptional level in VSMCs, suggesting a molecular mechanism by which LPC contributes to the atherogenicity of oxidized low density lipoprotein.
- monocyte chemoattractant protein-1
- smooth muscle cells
- tyrosine kinase
- mitogen-activated protein kinase/extracellular signal–regulated kinase
Oxidized LDL (oxLDL) is involved in the pathogenesis of atherosclerosis. Lysophosphatidylcholine (LPC) is a prominent component of oxLDL. During oxidation, 40% of LDL phosphatidylcholine can be converted to LPC by LDL-associated phospholipase A2,1 an enzyme that is an independent predictor of coronary heart disease.2 LPC is also enriched in another atherogenic lipoprotein, β-VLDL.3
A number of proatherothrombotic effects of these lipoproteins have been attributed to the inflammatory effects of LPC, including (1) disturbance of vascular tone,4 (2) induction in endothelial cells (ECs) of adhesion molecules5,6⇓ and chemoattractants,7 (3) stimulation of vascular smooth muscle cell (VSMC) migration8 and proliferation,9 and (4) inhibition of endothelial migration after injury.10 Recently, LPC has been shown to bind to G-protein–coupled receptors (GPRs) in lymphocytes and various tissues, including the aorta, to induce receptor internalization, mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) activation, and chemotaxis.11,12⇓ Thus, LPC can trigger signal transduction cascades involved in the initiation and development of atherosclerosis.
Another important proatherogenic molecule is monocyte chemoattractant protein (MCP)-1. MCP-1 is an immediate early gene13 and is induced by growth factors14 and inflammatory cytokines.15 MCP-1 recruits monocytes, precursors of foam cells, into the arterial wall16 and has been shown to mediate oxLDL-induced monocyte chemotaxis in cocultures of VSMCs and ECs.17 In mouse models of atherosclerosis, deficiency of MCP-118,19⇓ or its receptor, CCR2,20 led to an ≈50% to 80% reduction in lesion size, and overexpression of MCP-1 accelerated atherosclerosis progression,21 thereby providing direct evidence of the pathophysiological importance of MCP-1.
In addition to ECs and macrophages, VSMCs are another major source of vessel wall MCP-1. For example, MCP-1 secreted from cultured VSMCs was responsible for all of the platelet-derived growth factor (PDGF)-induced monocyte chemotactic activity.22 MCP-1 mRNA was found in VSMCs of human atherosclerotic plaques in vivo.23 In addition, MCP-1 secreted by VSMCs overlaid by ECs contributed to the monocyte chemotactic activity in response to oxLDL.17 Although LPC-induced monocyte chemotaxis could result from a direct chemotactic effect of LPC in vitro,24 an indirect mechanism involving MCP-1 induction is still quite important because of the central role that MCP-1 plays in atherogenesis,18,19,21⇓⇓ which may represent a final common pathway for many proatherogenic factors.
Given the foregoing and the findings that secretory phospholipase A2,25 LPC,26 and functional LPC receptors11 are all present in the arterial wall, we studied the regulation of MCP-1 by LPC in VSMCs, the most abundant arterial cell type. We found that LPC stimulates the production of MCP-1 by VSMCs at the level of transcription through a mechanism that involves MEK/ERK, tyrosine kinase, and (to a lesser extent) protein kinase C (PKC) activities. Therefore, a similar mechanism may contribute to the proatherogenic effects of LPC in the arterial wall.
The Methods section can be found online at http://www.atvb.ahajournals.org.
High concentrations of LPC are known to be cytotoxic.27 To determine the maximal subtoxic concentration, rat VSMCs were treated with increasing concentrations of LPC. As assessed by a measure of mitochondrial function (Methods), rat VSMC metabolic activity was 100.0±1.9%, 102.8±1.3%, 100.8±1.3%, 78.3±2.9%, and 23.4±2.3% (relative to untreated cells, mean±SD) after treatment with 0, 20, 50, 100, and 200 μmol/L LPC, respectively. Thus, up to 50 μmol/L LPC did not affect rat VSMC metabolic activity. In addition, treatment with up to 50 μmol/L LPC did not change cell counts, nor did we observe any changes in the cell morphology as described by others27 during the treatment period (data not shown). Therefore, concentrations up to 50 μmol/L were considered subtoxic and were used in all subsequent experiments. A concentration of 50 μmol/L is comparable to that reported in the circulation.28
Induction of MCP-1 mRNA and Protein by LPC
Figure 1A shows a representative Northern blot analysis of MCP-1 mRNA abundance in quiescent subconfluent rat VSMCs treated with 0 to 50 μmol/L LPC. The signal intensities for MCP-1 mRNA are summarized in Figure 1B. MCP-1 mRNA was detectable in untreated VSMCs, as was MCP-1 protein in the conditioned media of untreated cells (≈7.8 ng/mL per 106 cells). There was a dose-dependent increase in MCP-1 mRNA after 4 hours of LPC treatment. At the highest LPC concentration, MCP-1 mRNA appeared to plateau at 3-fold the value in untreated cells. The induction of MCP-1 mRNA by LPC (50 μmol/L, 4 hours) was associated with a ≈2-fold increase in MCP-1 protein secretion (Figure 1C). The extent of LPC induction of MCP-1 mRNA (below) or protein (Figure 1C) was comparable to that by PDGF-BB (20 ng/mL for 4 hours).
Time-Dependent Induction of MCP-1 mRNA in VSMCs by LPC
Figure 2A shows a representative slot-blot analysis of MCP-1 mRNA abundance in quiescent subconfluent rat VSMCs treated with 50 μmol/L LPC for 0 to 24 hours. PDGF-BB treatment (20 ng/mL) served as a positive control. The signal intensities are summarized in Figure 2B. Note that in LPC-treated cells, MCP-1 mRNA abundance peaked at 4 hours and declined to baseline after 8 hours. This pattern was different from that induced by PDGF-BB treatment, which resulted in a more rapid induction and return to baseline, consistent with previous observations.14
Time-Dependent Induction of MCP-1 mRNA Synthesis by LPC
To determine whether LPC induction of MCP-1 mRNA was the result of increased transcription, we performed nuclear runon assays (Figure 3). In untreated cells, there was detectable transcription of the MCP-1 gene (Figure 3A), consistent with the basal levels of MCP-1 mRNA. MCP-1 transcript levels were not changed at 15 minutes, 30 minutes (data not shown), or 1 hour after treatment but then increased to 200% of baseline 3 hours after treatment, with a return to baseline by 4 hours.
LPC Responsive cis-Acting Elements of the Rat MCP-1 Promoter
Transfection experiments were conducted to determine the LPC-responsive elements in the MCP-1 promoter. As summarized in Figure 4, serial deletions from the 5′ end of the MCP-1 promoter (−59, −146, −261, −1053, and −2565 bp from transcription start site) were ligated upstream from the firefly luciferase gene.29 Known cis elements for other agonists in the promoter region are also indicated in Figure 4 and include the following: AP-1/Sp1 binding sites (−54 to −39),30,31⇓ nuclear factor (NF)-κB binding sites (−2287 to −2278 and −2261 to −2252),31 and PDGF-responsive elements (−146 to −128 and −84 to −59).14
Deletion of the region containing the NF-κB sites in the MCP-1 promoter did not decrease LPC-induced luciferase activity. After the MCP-1 promoter was truncated to <146 bp, LPC induction was lost, indicating the existence of an LPC-responsive element(s) in the region −146/−261. Further removal of a −146 to −59 region containing a PDGF-responsive element14 did not have an additional effect on LPC induction. The promoter was not further truncated, because we14 and others30 have shown that basal promoter activity would be lost after deletion of the AP-1/Sp1 sites.
Involvement of MEK/ERK, PKC, Tyrosine Kinase, and PDGF Receptors in LPC-Induced MCP-1 Expression
A functional LPC receptor, GPR4, has recently been demonstrated in the aorta and has been shown to activate MEK/ERK on LPC binding.11 To determine whether the GPR4-MEK/ERK cascade is involved in LPC-induced MCP-1 expression, we treated cells with PD098059, a specific MEK/ERK inhibitor, before and during the treatment with LPC. PD098059 (at 30 μmol/L) partially inhibited LPC-induced MCP-1 mRNA accumulation (≈38%, Figure 5A) and MCP-1 secretion (≈50%, average of 2 determinations). At this inhibitor concentration, PDGF-BB–induced (data not shown) and angiotensin II–induced MCP-1 accumulation is completely blocked.32
The lack of complete inhibition in LPC-induced MCP-1 accumulation may reflect the involvement of other signaling pathways in addition to GPR4-MEK/ERK. PKC has been shown to be involved in LPC-induced MCP-1 expression in ECs.7 To investigate the role of PKC in VSMCs, the cells were pretreated with phorbol 12,13-dibutyrate for 24 hours to downregulate PKC before LPC-treatment.33 As shown in Figure 5B, downregulation of PKC completely aborted the induction of MCP-1 by another phorbol ester, phorbol 12-myristate 13-acetate (PMA), but had only a minor effect (≈25%) on LPC-induced MCP-1 expression.
Because tyrosine kinases are important mediators of chemokine-GPR–induced signal transduction,34 we examined the effect of the general tyrosine kinase inhibitor genistein on LPC induction of MCP-1 mRNA. As shown in Figure 5A, genistein (at 30 μmol/L) completely inhibited the effect of LPC. The PDGF receptor is also a known tyrosine kinase. The characteristics of LPC induction (ie, the comparable extent to which MCP-1 expression was induced by LPC and PDGF-BB and the lag in the peak of MCP-1 transcript synthesis induced by LPC relative to the peak with PDGF-BB14) were compatible with an initial stimulation of PDGF production by LPC, which then activates the PDGF pathway. This would also be consistent with the findings of Chai et al,9 who have reported that LPC activates a variety of growth factor receptors, such as fibroblast growth factor-2. Therefore, we tested the effects of the blockade of the PDGF receptor on LPC induction. As shown in Figure 6, pretreatment with 2E1A2, the monoclonal antibody that specifically recognizes the β-PDGF receptor35,36⇓ (the only isoform present in rat VSMCs37), abolished PDGF-BB induction of MCP-1 mRNA, but there was no effect on LPC induction. Taken with the genistein results, this indicates that a tyrosine kinase(s) other than the PDGF receptor is involved in LPC-induced MCP-1 expression.
We have examined whether a mechanism contributing to the chemotactic activity for monocytes17 of oxLDL is the LPC-induced transcription of the MCP-1 gene in VSMCs. This would be expected to have significant atherogenic consequences because VSMCs express MCP-1 and because they are the most abundant cells in the arterial wall. We found that LPC at concentrations comparable to those found in mammalian plasma28 induces MCP-1 mRNA abundance and protein secretion in a dose- and time-dependent manner. The accumulation was mainly due to increased transcription, involving an LPC-responsive element(s) in the ≈−261 to −146 region of the promoter. On the basis of inhibitor- and receptor-blocking experiments, the induction process appeared to partially involve MEK/ERK, PKC, and a tyrosine kinase(s) other than the PDGF receptor.
LPC has many effects that are expected to play important roles in atherogenesis.38 For example, LPC stimulates cytokine,39,40⇓ chemokine,7 adhesion molecule,5,41⇓ and growth factor production8 at the transcriptional level in vitro. The present study now demonstrates that 1 mechanism by which LPC may contribute to the recruitment of monocytes into the arterial wall by atherogenic lipoproteins is the transcriptional upregulation of MCP-1 in VSMCs, which is different from the direct chemotactic effect of LPC.24 Because MCP-1 is responsible for ≈60% of the monocyte/macrophage area in mouse models of atherosclerosis,19,20⇓ the indirect chemotactic effect of LPC (through MCP-1) may be quite significant in vivo.
ECs are another source of LPC-induced MCP-1 production.7 The greater cell mass of VSMCs compared with ECs and the abundance of monocytes/macrophages deep within the atherosclerotic plaque (ie, adjacent to the medial smooth muscle layer) would argue that VSMCs may be a major contributor to the concentration gradient for monocyte chemotaxis. In fact, MCP-1 from VSMCs of the tunica media was implicated in early lesion formation in diet-induced hypercholesterolemic primates,42 whose lipoproteins consist mainly of β-VLDL enriched with LPC.
We have previously identified cis-acting elements responsible for PDGF-induced MCP-1 expression in VSMCs at positions −146 to −128 and −84 to −59 of the rat MCP-1 promoter.14 However, these elements are not likely to be involved in the response to LPC. Instead, an LPC-responsive element(s) appears to be in the region −261 to −146 (Figure 4). Database analysis (Institute for Transcriptional Informatics, Pittsburgh, Pa, which can be accessed at http://www. ifti.org) identified a number of consensus sequences for enhancers, including a PEA3 site (also responsible for LPC-induced endothelial NO synthase expression43), a W element (in the interferon-γ–responsive region of the human class II major histocompatibility complex gene DPA44), and an element in the intron region of murine fibroblast growth factor-8 gene.45 Demonstration of the roles of these candidates in the transcription regulated by LPC will require additional studies.
It is notable that the removal of an upstream 1512-bp MCP-1 promoter segment containing the NF-κB sites, previously shown to be involved in MCP-1 expression induced by other stimuli,31,46–49⇓⇓⇓⇓ did not decrease the induction by LPC. In fact, there was a moderate, but not significant, increase (Figure 4). Because there could be cis elements within the deleted segment that are negative transcription factors, our results do not completely rule out the possibility of NF-κB involvement. Also of interest were cis-acting sequences for peroxisome proliferator–activated receptor (PPAR)-α, because a previous report50 indicated that MCP-1 expression from human ECs induced by oxLDL was mediated by PPARα. However, no PPARα consensus sequences were found in the rat MCP-1 promoter up to position ≈3657 bp.49 Thus, if there are PPARα-responsive elements, they must be further upstream.
We were interested in the signaling process by which LPC activates MCP-1 expression. LPC could stimulate PDGF expression, which then would activate its own receptor in an autocrine or paracrine fashion. Besides the kinetic evidence compatible with this scenario (summarized in Results), LPC has been shown to stimulate growth factor production in other contexts.8 However, the data from the antibody blockade experiment did not support this model (Figure 6).
Takahara et al7 showed that in ECs, LPC-induced MCP-1 expression was significantly (53%) inhibited by the PKC inhibitor staurosporine. Our results with a highly specific PKC downregulator were less dramatic in LPC-treated VSMCs (Figure 5B). Whether this quantitative difference represents cell background effects or the known lack of specificity of staurosporine is not clear, but in any case, PKC mediation of LPC induction of MCP-1 appears to be a minor pathway in VSMCs.
Another signaling possibility is suggested by the recent demonstration of LPC-activated GPR family receptors (resulting in cell chemotaxis) in T lymphocytes12 and in a number of tissues, including the aorta.11 On ligand binding, mitogen-activated protein kinase,11,12⇓ tyrosine kinase,51 and/or other mediators are activated. Our results (Figures 5A) indicate that MEK/ERK and tyrosine kinase activities are involved in the LPC induction of MCP-1. The tyrosine kinase activity appeared to be upstream from MEK/ERK because genistein was completely effective, whereas the MEK/ERK inhibitor only partially blocked the induction. The involvement of a tyrosine kinase activity has also been implicated in LPC-induced expression of intercellular adhesion molecule-1 in human umbilical ECs.6
This putative tyrosine kinase activity is not likely to be the PDGF receptor, given the evidence noted above against its participation in effects of LPC. One candidate is Janus kinase, which has been shown as downstream from tyrosine kinase for a number of chemokine receptors, including CCR2 and CCR5,51 and has been shown to be activated by LPC in ECs.52 Whether this kinase or other receptor or Src-related kinases are responsible for LPC-induced MCP-1 expression requires further studies.
In summary, our results suggest that the atherogenic effects of LPC in vivo include the induction of MCP-1 expression in VSMCs at the level of transcription. It is also possible that in other cell types that express MCP-1 in the arterial wall (macrophages and ECs), a similar induction occurs, further augmenting the influence of LPC. Because LPC is a major component of oxLDL, further elucidation of the pathways by which LPC induces MCP-1 production will increase our knowledge of the molecular mechanisms by which this modified lipoprotein exerts its potent atherogenic effects.
This work was supported by National Institutes of Health grants DK-44498 (Dr Fisher), HL-61814 (Dr Fisher), HL-61818 (Dr Taubman), and American Heart Association (AHA) Grant-in-Aid (Dr Berman). Dr Rong was awarded an AHA Heritage Affiliate Postdoctoral Fellowship (grant 0020480T). We thank Drs Nathalie A Lokker, Millennium Pharmaceuticals, Inc, for kindly providing the β-PDGF receptor monoclonal antibody 2E1A2; Dr Kiyoshi Nose, Showa University School of Pharmaceutical Sciences, Japan, for kindly providing the MCP-1 promoter-CAT plasmid; Dr Vladimir Y. Bogdanov, Mount Sinai, for kindly providing the −1053, −261, −146, and −59 luciferase reporter constructs and helpful advice; and Lillie Lopez, Albert Einstein College of Medicine, for expert technical assistance.
Received March 1, 2002; revision accepted August 9, 2002.
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