Lysophosphatidylcholine Activates Extracellular Signal-Regulated Kinases 1/2 Through Reactive Oxygen Species in Rat Vascular Smooth Muscle Cells
Lysophosphatidylcholine (lysoPC) acts on vascular smooth muscle cells (VSMCs) to produce a mitogenic response through the activation of extracellular signal-regulated kinases 1/2 (ERK1/2). In the present study, we examined the importance of reactive oxygen species (ROS) in lysoPC-stimulated ERK1/2 activation in cultured rat VSMCs. Treatment with lysoPC for 3 minutes caused a 2-fold increase in intracellular ROS that was blocked by the NADH/NADPH oxidase inhibitor, diphenylene iodonium (DPI). Antioxidants, N-acetyl-l-cysteine, glutathione monoester, or α -tocopherol, inhibited ERK1/2 activation by lysoPC. Almost identical results were obtained in the VSMC line A10. Pretreatment of VSMCs with DPI but not allopurinol or potassium cyanide (KCN) abrogated the activation of ERK1/2. The Flag-tagged p47phox expressed in A10 cells was translocated from the cytosol to the membrane after 2 minutes of stimulation with lysoPC. The overexpression of dominant-negative p47phox in A10 cells suppressed lysoPC-induced ERK activation. The ROS-dependent ERK activation by lysoPC seems to involve protein kinase C- and Ras-dependent raf-1 activation. Induction of c-fos expression and enhanced AP-1 binding activity by lysoPC were also inhibited by DPI and NAC. Taken together, these data suggest that ROS generated by NADH/NADPH oxidase contribute to lysoPC-induced activation of ERK1/2 and subsequent growth promotion in VSMCs.
- vascular smooth muscle cells
- extracellular signal-regulated kinases 1/2
- reactive oxygen species
- signal transduction
Several lines of evidence suggest that oxidatively modified LDL plays a key role in atherogenesis.1,2⇓ Lysophosphatidylcholine (lysoPC) is one of the major phospholipid components that is increased during the oxidation of LDL.3 The concentration of lysoPC is elevated in atherosclerotic lesions in animals fed an atherogenic diet.4 LysoPC exerts various types of biological effects relevant to arteriosclerosis. It stimulates the growth and migration of vascular smooth muscle cells (VSMCs),5 induces adhesion molecules in endothelial cells,6 and impairs endothelium-dependent vasorelaxation.7 Previously, we reported that lysoPC stimulated the activation of p42/44 mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase 1/2 (ERK1/2) and the expression of the transcription factor c-Fos and c-Jun in cultured VSMCs. ERK1/2 is a member of the serine/threonine kinases, which are activated by a variety of stimuli involved in cell growth and differentiation.8,9⇓ Thus, lysoPC-induced ERK1/2 activation could be involved in the pathogenesis of arteriosclerosis. However, the mechanism by which lysoPC activates ERK1/2 remains unknown.
Oxidative stress also has long been implicated in atherogenesis. Reactive oxygen species (ROS) show numerous effects on cell functions, including induction of growth, regulation of kinase activity, and inactivation of endothelial-derived relaxation factor, nitric oxide.10–12⇓⇓ Moreover, ROS play important roles as second messengers in signal transduction pathways.13– 15⇓⇓ In VSMCs, ROS activate several tyrosine kinases and MAPKs, including ERK1/2, and induce proto-oncogenes, such as c-fos and c-myc.16–19⇓⇓⇓ ROS includes free radicals, hydroxyl radicals (OH·), a super oxide anion (· O2-), and a nonradical derivative, such as hydrogen peroxide (H2O2). In VSMCs and endothelial cells, NADH/NADPH oxidases represent the most important source of ·O2-.20,21⇓ NADPH oxidase catalyzes the NADPH-dependent reduction of oxygen to ·O2-, which in turn leads to the production of secondary derivatives such as OH· and H2O2. NADPH oxidase is a multicomponent enzyme. The plasma membrane-associated flavocytochrome b558 consists of two subunits, gp91phox and p22phox.21 Flavocytochrome b558 is the key catalytic component responsible for the direct transfer of electrons from NADPH to molecular oxygen.22,23⇓ The essential cytosolic components are p47phox, p67phox, and a second low molecular weight GTP-binding protein, Rac.21,24,25⇓⇓ These cytosolic proteins translocate from the cytosol to the membrane during NADPH oxidase assembly.26 In VSMCs, the translocation of p47phox is critical for NADPH oxidase activation.27 LysoPC has been reported to produce ROS in VSMCs,28 suggesting the possible involvement of ROS in signal transductions of lysoPC. In the present study, we addressed the question in VSMCs concerning whether ROS mediate lysoPC-induced ERK1/2 activation and whether NADH/NADPH oxidase is involved in the generation of ROS by lysoPC. Here, we show that inhibition of NADPH oxidase abrogates lysoPC-induced ROS generation and that several antioxidants and a NADPH inhibitor suppress ERK1/2 activation. These results indicate that lysoPC-induced ERK1/2 activation is mediated by ROS that are generated through the NADH/NADPH oxidase.
VSMCs were prepared from the thoracic aorta of 12-week-old Sprague-Dawley rats by the explant method as previously described.28 Subcultured VSMCs from passages 3 to 15 were used in the experiments. A10 cells (American Tissue Culture CRL-1476) were grown in DMEM containing 10% fetal bovine serum. For subsequent experiments, cells at ≈80% confluence in culture wells were used 1 day after serum depletion.
Measurements of ROS
The generation of ROS was measured by the flow cytometry by using dihydrorhodamine 123 (DHR) as the indicator.19 VSMCs were stimulated after incubation with 2 mmol/L DHR for 1 hour. The intracellular DHR was irreversibly changed into the green fluorescent compound rhodamine 123 (wave length 500 to 540 nm) by the ROS generated inside the cells. Cells were fixed for 20 minutes in 1% formaldehyde, and the cellular rhodamine 123 fluorescence intensity of 10 000 VSMCs was measured for each sample by flow cytometry with the excitation source at 488 nm.
Preparation of Cell Extracts and Western Blotting
Cellular proteins were isolated, and Western blotting was performed with indicated antibodies as described previously.29
ERK1/2 Activity Assay
After stimulation, cells were lysed with ice-cold lysis buffer as described previously.30 After a brief sonication, the samples were centrifuged for 5 minutes at 14 000g, and the supernatant was assayed for ERK1/2 activity with the BIOTRAK enzyme assay kit (Amersham).
Plasmid Constructions and Transfection
pGEX2-p47phox (wild type) and -p47phox W193R were generous gifts from Dr. H. Sumimoto (Kyushu University).31 Each of them were subcloned into pCDNA3 with an N-terminal Flag tag (pCDNA3F). A10 cells were transfected with the constructs by using Superfect reagent (Qiagen). pCDSRT7 ERK2 were kindly provided by S. Ohno (Yokohama City University).32
Immune Complex Kinase Assay
ERK2 was immunoprecipitated by the incubation of cell extracts (100 μg) from pCDSRT7 ERK2- and/or pCDNA3F p47phox-transfected A10 cells with a monoclonal antibody specific for T7. The immune complexes were resuspended in MAP kinase reaction buffer (25 mmol/L HEPES, pH 7.5, 10 mmol/L magnesium acetate, and 50 μmol/L unlabeled ATP) containing myelin basic protein (2 μg) and [γ-32P]ATP (1 μCi, 6000 Ci/mmol) and incubated at 30°C for 20 minutes. The reaction mixtures were boiled in SDS sample buffer, resolved by 15% SDS-PAGE, and visualized by autoradiography.
Twenty-four hours after the transfection, cells were stimulated by lysoPC, and soluble and particulate fractions were separated as described previously.33
Northern Blot Analysis
Total RNA was isolated, and Northern blot analysis was performed as described previously30 with c-fos as a probe.
Preparation of Nuclear Extracts and Electrophoretic Mobility Shift Assay (EMSA)
Nuclear extracts were prepared from VSMCs, and EMSA was performed with a commercial kit (Promega) as described previously.34 AP-1 consensus oligonucleotides (5′ -CGC TTG ATG AGT CAG CCG GAA-3′) were used in EMSA.
Data are expressed as mean±SD. Differences between data sets were evaluated by an unpaired Student t test. A level of P<0.01 was accepted as statistically significant.
NADH/NADPH Oxidase Mediates ROS Production by lysoPC
To demonstrate ROS generation in response to lysoPC in VSMCs, a fluorescent cell-sorting method with DHR123 was used. As shown in Figure 1A, the mean fluorescence in 12.5 μmol/L lysoPC-treated cells relative to untreated cells increased as early as 1 minute and peaked at 3 minutes. The increase in intracellular ROS was dependent on the concentration of lysoPC, with a maximal generation of ROS occurring at 6.125 to 12.5 μmol/L lysoPC (data not shown). The ROS produced by lysoPC was completely eliminated by dephenylene iodonium (DPI), an inhibitor of flavin-containing oxidative enzymes (Figure 1B) indicating that lysoPC-induced ROS formations are mediated through the NADH/NADPH oxidase.
Inhibition of lysoPC-Induced ERK1/2 Phosphorylation by Antioxidants
In response to lysoPC, there was an apparent phosphorylation of both ERK1 and ERK2 (p44 and p42 MAPK), with peak activity at 7 minutes before returning to the baseline as previously described.29 To gain insight into the mechanism of ERK activation by lysoPC, we examined the involvement of ROS by using several potent antioxidants. N-acetylcysteine (NAC) has been used extensively to study the role of ROS in several signaling pathways.13,35⇓ NAC directly scavenges ROS and increases the intracellular levels of reduced glutathione (GSH). GSH is a hydroxyl radical scavenger and a substrate of glutathione peroxidase, which degrades H2O2. The lysoPC-stimulated ERK phosphorylation was inhibited by NAC (20 mmol/L; Figure 2A). The inhibition by NAC was concentration-dependent, starting at 5 mmol/L and maximally at 20 mmol/L (Figure 2B). To verify that the inhibitory effect of NAC is attributable to its ability to scavenge ROS, we examined the effects of other antioxidants, GSH monoester (GSE) and α-tocopherol. α-tocopherol is a lipid soluble antioxidant that is also highly reactive toward lipid-soluble radicals. Both GSE and α-tocopherol markedly inhibited lysoPC-induced ERK1/2 phosphorylation (Figure 2C).
Effect of Antioxidants on lysoPC-Induced ERK1/2 Activation
We next examined the effects of the antioxidants on lysoPC-induced ERK1/2 activity as assessed by a kinase assay using their specific substrate. LysoPC stimulated the activity of ERK1/2 approximately 5-fold as compared with the control (see online Figure IA; which can be accessed at http://atvb.ahajounals.org). The ERK activation induced by lysoPC was significantly suppressed by NAC, GSE, and α-tocopherol. In A10 cells, a cell line of VSMCs, lysoPC also activated ERK1/2, and the activation was similarly suppressed by pretreatment with NAC, GSE, and α-tocopherol (see online Figure IB). These results indicate that the signal transduction of lysoPC-induced ERK1/2 activation involves ROS in VSMCs.
Involvement of NADH/NADPH Oxidase in lysoPC-Induced ERK1/2 Phosphorylation
To ascertain whether lysoPC-induced ERK1/2 activation is due to the activation of NADPH/NADH oxidase, we examined the effect of DPI on ERK1/2 phosphorylation. As shown in Figure 3A, DPI dose-dependently inhibited lysoPC-induced ERK1/2 phosphorylation. Although xanthine oxidase and mitochondria have been reported to generate ROS,36 neither allopurinol (xanthine oxidase inhibitor; 200 mmol/L) pretreated for 24 hours nor potassium cyanide (KCN) (mitochondria toxin; 0.2 mmol/L) pretreated for 30 minutes inhibited lysoPC-induced ERK1/2 phosphorylation (data not shown). Thus, ROS mainly generated by NADH/NADPH oxidase are required for lysoPC-induced ERK1/2 activation.
Translocation of p47phox Is an Essential Component for ERK Activation by lysoPC
To investigate whether lysoPC activates p47phox in VSMCs, we examined the translocation of p47phox from cytosol to the membrane. A10 cells expressing Flag-tagged p47phox were stimulated with lysoPC. As shown in Figure 3B, p47phox detected in the membrane fraction was increased within 1 minute of exposure to lysoPC, suggesting that the NADPH oxidase complex involving p47phox is activated by lysoPC. To determine the role of p47phox on lysoPC-stimulated ERK1/2 activation, we examined the effect of the empty vector or a dominant-negative p47phox, p47phox W193R, on cotransfected pCDSRT7 ERK2 activation by lysoPC. As shown in Figure 3C, lysoPC increased the ERK2 activity in the control A10 cells transfected with empty vector, whereas activation of ERK2 by lysoPC was significantly suppressed by the transfection of the pCDNA3F p47phox W193R. These data strongly indicate that p47phox is essential for ERK activation by lysoPC.
Involvement of PKC and Ras in lysoPC-Induced Raf-1 and ERK1/2 Phosphorylation
Growth factors, such as angiotensin II (Ang II) and platelet-derived growth factor-BB (PDGF-BB) have been reported to increase ROS production in VSMCs and activate ERK.14,21⇓ Protein kinase C (PKC) is reported to induce the phosphorylation of p47phox,37,38⇓ and we have shown that lysoPC-induced ERK activation partially involves PKC in VSMCs. Thus, we studied the role of PKC in ROS-mediated ERK1/2 activation by these growth factors or lysoPC. We have shown that pretreatment of GF109203X (GFX), a specific PKC inhibitor, did not affect Ang II-induced ERK1/2 activation.29 In contrast, pretreatment with GFX markedly suppressed lysoPC and phorbol 12-myristate 13-acetate (PMA) but not PDGF-BB-induced ERK1/2 phosphorylation (Figure. 4A). PKC could directly activate raf-1 and induce ERK1/2 activation. Thus, we examined the effect of GFX on lysoPC-induced raf-1 activation. As shown in Figure 4B, lysoPC, PMA, and AngII-induced phosphorylation of raf-1. Pretreatment of GFX completely inhibited raf-1 phosphorylation by PMA and partially by lysoPC. However, GFX had no effect on Ang II-induced phosphorylation of raf-1. Therefore, similar to the ERK activation, lysoPC-induced raf-1 activation seems to partially require PKC. Next, we examined the relationship between ROS and PKC to mediate lysoPC-induced raf-1 activation. Pretreatment with DPI markedly suppressed phosphorylation of raf-1 by PMA as well as lysoPC (Figure 4C). H2O2 markedly stimulated phosphorylation of raf-1. However, GFX had only a minor inhibitory effect on H2O2-induced raf-1 phosphorylation (Figure 4C). These data indicate that PKC is upstream of ROS-dependent raf-1 activation by lysoPC. Farnesyltransferase inhibitors, FPTIII, decrease Ras farnesylation, resulting in the loss of its function.39 LysoPC-induced raf-1 phosphorylation was partially inhibited with FPTIII, whereas it was completely inhibited by the combination of FPTIII and GFX (Figure 4D). PMA-induced raf-1 phosphorylation was also markedly but not completely inhibited by FPTIII (Figure 4D). Taken together, these data suggest the unique feature of lysoPC to activate ERK in that ROS-dependent raf-1 and ERK activation involve both PKC (as upstream of ROS) and Ras (as downstream of ROS).
Role of H2O2 in lysoPC-Induced ERK1/2 Activation
Catalase has been used widely to block the biological activities of H2O2.14 As shown in Figure IIA (which can be accessed at http://atvb.ahajounals.org), pretreatment of VSMCs with catalase (3000 U/mL) abolished ERK1/2 activation by H2O2. As shown in Figure IIB, pretreatment with catalase markedly inhibited lysoPC-stimulated ERK1/2 activation, implicating the involvement of H2O2 in ROS-dependent ERK1/2 activation by lysoPC.
Effect of NAC and DPI on c-fos mRNA Expression and AP-1 Binding Activity
As we have reported previously,30 Northern blot analysis revealed that lysoPC increased c-fos mRNA levels compared with control (see online Figure IIIA, which can be accessed at http://atvb.ahajounals.org). Pretreatment with DPI or NAC markedly inhibited the c-fos mRNA induction. EMSA showed that AP-1 binding activity was enhanced by lysoPC. DPI eliminated the lysoPC-induced increase of AP-1 binding activity (see online Figure IIIB). These results suggest that lysoPC-induced protooncogene c-fos expression and subsequent AP-1 activation are also mediated through the generation of ROS in VSMCs.
The major findings of the present study are as follows: (1) lysoPC generates intracellular ROS in VSMCs; (2) these ROS formations were abrogated with DPI; (3) lysoPC-induced ERK1/2 activation was inhibited by antioxidants; (4) pretreatment of VSMCs with DPI or overexpression of dominant-negative p47phox abrogated the activation of ERK1/2; and (5) induction of c-fos expression and enhanced AP-1 binding activity by lysoPC were also inhibited by DPI and NAC.
Although many enzymatic sources for generation of ROS depend on cell types, NADH/NADPH oxidases represent the most important source of · O2- in VSMCs.20,22⇓ To determine which enzyme systems are involved in lysoPC-induced ROS generation, we took advantage of inhibitors known to selectively inhibit NADPH/NADH oxidase, xanthine oxidase, and a toxin for mitochondria. We found that DPI abolished the increase in ROS by lysoPC. Moreover, lysoPC induced the translocation of p47phox from cytosol to the membrane. However, allopurinol and KCN had no effect on ROS production. Thus, we conclude that lysoPC increases intracellular ROS through the activation of NADH/NADPH oxidase in VSMCs. Our objective was to determine whether ROS are involved in ERK1/2 activation by lysoPC. In the present study, preincubation of GSE and NAC block the ERK1/2 phosphorylation/activation by lysoPC. In contrast, in vivo depletion of GSH by BSO enhanced phosphorylation of ERK1/2 by lysoPC. We further showed that dominant-negative p47phox as well as DPI inhibited ERK activation by lysoPC. These data strongly support our theory that ROS produced through NADH/NADPH oxidase are essential components for ERK activation by lysoPC. Quite recently, α-tocopherol has been reported to inhibit the phosphorylation and translocation of p47phox.40 Thus, the suppressive effect of α -tocopherol on lysoPC induced ERK1/2 activation may be due to the impairment of assembly of NADH/NADPH oxidase.
Activation of NADH/NADPH oxidase generates · O2-. In general, ·O2- is rapidly dismutated to H2O2. Both ·O2- and H2O2 have been reported to activate ERK1/2.16,41⇓ Which oxidant species play a dominant role in ERK1/2 activation by lysoPC? The present data showed that catalase-inhibited lysoPC stimulated ERK1/2 phosphorylation, implicating a major contribution of H2O2. The concentration of catalase used in the present study is sufficient to suppress H2O2-induced ERK activation. However, the inhibitory effect of catalase on lysoPC-induced ERK activation is weaker than that seen in the H2O2 experiment, whereas DPI almost completely inhibited ERK1/2 activation by lysoPC. These results suggest that not only H2O2 but also · O2- is involved in ERK1/2 activation by lysoPC in VSMCs.
Growth factors, such as Ang II or PDGF-BB, activate ERK via ROS in VSMCs.14,21⇓ Is there a mechanistic difference between growth factors or lysoPC-induced ERK1/2 activation via ROS? As shown in the present study, lysoPC-induced raf-1 and ERK1/2 activation seems to involve PKC, which is compatible with our previous findings.30 However, Ang II- and PDGF-BB-induced ERK1/2 phosphorylation were not dependent on PKC (Figure 4 and Eguchi et al29), suggesting the unique feature of lysoPC to activate ERK1/2. However, lysoPC-induced raf-1 and ERK1/2 phosphorylation were not completely inhibited with GFX. We have shown that long-term pretreatment of PMA only partially inhibited lysoPC-induced ERK activation in VSMCs.30 Thus, there seems to be a PKC-dependent and -independent pathway involved in lysoPC-induced ERK1/2 activation. Although PKC can directly activate raf-1 as reported previously,42 the present findings showed that PMA as well as lysoPC-induced phosphorylation of raf-1 were completely inhibited by DPI, whereas GFX had only a minor inhibitory effect on H2O2-induced raf-1 activation (Figure 4C). These data suggest that both PKC-dependent and -independent ERK1/2 activation pathways converge at the point of ROS induction. The discrepancy concerning the direct (PKC-raf) and indirect (PKC-ROS-raf) activation of raf-1 by PKC may be due to the cell type studied and/or the involvement of Ras as described below.
ROS play a role upstream of Ras,19 both lysoPC- and PMA-induced raf-1 activation are markedly blocked by a Ras inhibitor, FPTIII, in the present study. Therefore, lysoPC-induced raf-1 and ERK activation via PKC and ROS could be mediated, at least in part, through a Ras- dependent mechanism. Because pretreatment of both FPTIII and GFX completely blocked raf-1 activation by lysoPC, we further suggest that there may be several possible pathways in which ROS mediate raf-1 activation by lysoPC. The PKC-dependent pathway may partially involve Ras, whereas the PKC-independent pathway may be strictly through Ras, as illustrated in Figure 5. Future studies should be considered when addressing the mechanisms involved in the PKC-independent pathway of ERK activation by lysoPC and why this pathway is under control of Ras, even though both pathways similarly require ROS.
On activation, ERK1/2 translocate to the nucleus, where they phosphorylate transcription factors, such as TCF/ElK-1, which are bound to the c-fos promoter. Increased c-Fos synthesis results in elevated AP-1 activity. AP-1 is a sequence-specific transcriptional activator composed of Jun and Fos subunits that is involved in mitogenesis, differentiation, transformation, and inflammation. The present study showed that lysoPC enhanced c-fos mRNA expression and AP-1 activity. Both enhancements were abolished by the pretreatment of NAC or DPI. Elevated expression of the c-fos gene via alteration of the redox state has been shown to accompany cell proliferation. Thus, ROS might be involved in lysoPC-induced cell proliferation through ERK1/2, c-fos, and AP-1 activation.
In conclusion, ROS generated through NADH/NADPH oxidase are essential for the growth-promoting signals activated by lysoPC in VSMCs, suggesting that ROS might be good targets for preventing the atherosclerosis associated with hyperlipidemia.
This work was supported in part by the research grants HL58205, HL03320, DK20593 from the National Institutes of Health. In addition, Dr. Yamakawa was supported by Yokohama City University research grants and Dr. Eguchi was supported by an AHA Scientist Development Grant and a Vanderbilt University Diabetes Center Pilot and Feasibility Proposal.
Received May 10, 2001; revision accepted March 4, 2002.
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