Signaling Mechanisms of Nuclear Factor-κB-Mediated Activation of Inflammatory Genes by 13-Hydroperoxyoctadecadienoic Acid in Cultured Vascular Smooth Muscle Cells
Oxidatively modified low density lipoprotein (LDL) has been implicated in the pathogenesis of atherosclerosis. LDL oxidation may be mediated by several factors, including cellular lipoxygenases. The lipoxygenase product of linoleic acid, 13-hydroperoxyoctadecadienoic acid (13-HPODE), is a significant component of oxidized LDL and has been shown to be present in atherosclerotic lesions. However, the mechanism of action of these oxidized lipids in vascular smooth muscle cells (VSMCs) is not clear. In the present study, we show that 13-HPODE leads to the activation of Ras as well as the mitogen-activated protein kinases, extracellular signal-regulated kinase 1/2, p38, and c-Jun amino-terminal kinase, in porcine VSMCs. 13-HPODE also specifically activated the oxidant stress-responsive transcription factor, nuclear factor-κB, but not activator protein-1 or activator protein-2. 13-HPODE-induced nuclear factor-κB DNA binding activity was blocked by an antioxidant, N-acetylcysteine, as well as an inhibitor of protein kinase C. 13-HPODE, but not the hydroxy product, 13-(S)-hydroxyoctadecadienoic acid, also dose-dependently increased vascular cell adhesion molecule-1 promoter activation. This was inhibited by an antioxidant as well as by inhibitors of Ras p38 mitogen-activated protein kinase and protein kinase C. Our results suggest that oxidized lipid components of oxidized LDL, such as 13-HPODE, may play a key role in the atherogenic process by inducing the transcriptional regulation of inflammatory genes in VSMCs via the activation of key signaling kinases.
Oxidatively modified LDL (oxLDL) has been implicated in the pathogenesis of atherosclerosis.1,2 Evidence shows that oxLDL as well as its lipid components can exert potent effects on cellular functions. LDL oxidation may be mediated by several factors, including cellular lipoxygenases (LOs).3 Furthermore, 15-LO was shown to be colocalized with epitopes of oxLDL in macrophage-rich areas of atherosclerotic lesions.4 The LO enzyme catalyzes the hydroxylation of arachidonic or linoleic acids.5 The LO product of linoleic acid, 13-hydroperoxyoctadecadienoic acid (13-HPODE), is a significant component of oxLDL and has been shown to be present in atherosclerotic lesions.6 Evidence suggests that 13-HPODE has pathological effects in endothelial cells, vascular smooth muscle cells (VSMCs), and monocytes.7–9 LO products can induce the binding of monocytes to endothelial cells.10 Earlier studies by us and others have shown that LO products have potent growth and chemotactic effects in VSMCs.11,12 LO products could also mediate the growth and chemotactic effects of high glucose as well as that of growth factors, such as angiotensin II and platelet-derived growth factor.12–14 However, the signal transduction pathways and gene regulation mechanisms initiated by the lipid peroxidation products of the LO pathway, such as 13-HPODE in VSMCs, are not clear. We have tested the hypothesis that 13-HPODE can lead to transcriptional regulation of inflammatory genes, such as vascular cell adhesion molecule-1 (VCAM-1), in VSMCs via increased activation of Ras, the mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun amino-terminal kinase (JNK), and the oxidant stress-responsive transcription factor nuclear factor-κB (NF-κB). Our results suggest that oxidized lipid components of oxLDL, such as 13-HPODE, may play a key role in the atherogenic process by inducing the transcription of inflammatory genes in VSMCs via key signaling kinases.
Cell Culture and Treatment With 13-HPODE
Primary cultures of porcine VSMCs (PVSMCs) were obtained as described earlier15 and cultured in DMEM containing 10% FCS. All experiments were performed between passages 2 and 5. Quiescent PVSMCs in 100-mm dishes were preincubated for 60 minutes in DMEM containing 0.2% BSA and then treated with 13-HPODE for the indicated time periods. Cells were then processed for either nuclear or cytosolic protein extraction, Western blots, or chloramphenicol acetyltransferase (CAT) assays as described below. 13-HPODE was prepared according to the method described.16 In some experiments, we also used 13-HPODE and LDL, obtained as a generous gift from Dr S. Parthasarathy (Emory University, Atlanta, Ga).
Cells were grown to ≈70% confluence on chamber slides and arrested for 16 to 18 hours. Washed cells were treated with 13-HPODE (10 μmol/L) or vehicle (ethanol) for 10 minutes at 37°C. In some experiments, cells were pretreated for 16 hours with the farnesyl transferase inhibitor (FPT III, 10 μmol/L, BioMol Corp). Cells were then washed, fixed in 4% paraformaldehyde, and visualized with anti-Ras antibody by use of confocal microscopy as described.17
Activation of ERK1/2 and p38 MAPKs was determined by immunoblotting with antibodies specific to the phosphorylated forms of these kinases as described.18 Probing with antibody to α-actin or the nonphosphorylated kinase was used to correct for protein loading. JNK activity was measured by using immune-complex kinase assays with substrate glutathione S-transferase-c-Jun as described.18
Preparation of Cytosolic and Nuclear Extracts
After the incubations, cells were washed, scraped into 1 mL PBS, and spun down at 4°C. Cell pellets were lysed as described.19 Cell lysates were centrifuged for 10 minutes at 1600g on a 1-mL cushion of 1.0 mol/L sucrose. Nuclear pellets were washed once, and nuclear and cytosolic protein fractions were prepared as described by us.19
Immunoblotting to Detect the p65, p50, and IκB-α Subunits of NF-κB
To determine nuclear or cytosolic levels of NF-κB subunits, protein extracts (5 to 10 μg) were resolved on 8% SDS-polyacrylamide gels and electrotransferred to polyvinylidine difluoride membranes. After the blocking of nonspecific binding, blots were incubated with polyclonal antibodies to the p65, p50, or IκB-α subunits of NF-κB (1:1000, Santa Cruz Biotech), followed by horseradish peroxidase-conjugated secondary antibody (1:30 000), and visualized by the enhanced chemiluminescence detection system (Tropix).19 To verify equal protein loading, blots were stripped and probed with an antibody to α-actin (for cytosolic proteins) and histone-H1 (for nuclear proteins). Bands were quantified on a laser densitometer.
Electrophoretic Mobility Shift Assay
Synthetic oligonucleotides corresponding to 2 NF-κB consensus sequences in the human VCAM-1 promoter region (5′-CTGCCCTGGGTTTCCCCTTGAAGGGATTTCCCTCCGCCT-3′) were used as probes for electrophoretic mobility shift assay (EMSA).20 We also used oligonucleotides containing consensus binding sequences for activator protein (AP)-1 and AP-2 transcription factors (Santa Cruz) as controls for DNA binding. [γ-32P]ATP labeling of the oligonucleotides and EMSAs to evaluate DNA binding of nuclear proteins were performed as described earlier.19 For supershift analyses, nuclear extracts were preincubated for 1 hour with antibodies (2 μg each) against the NF-κB subunit proteins before probe addition. Gels were dried, protein-DNA complexes were visualized on a PhosphorImager (Molecular Dynamics), and quantification of radioactivity in each complex was carried out by use of ImageQuant software.
Transient DNA Transfections and CAT Assays
These methods were carried out as described previously.19,21 Briefly, PVSMCs were split 24 hours before transfections to give 60% confluence in 100-mm dishes and transfected with 10 μg of the reporter plasmid p85VCAM-CAT or pSV2CAT (positive control).20 Cells were then treated with 13-HPODE or vehicle. Sixteen to 18 hours later, cell extracts were assayed for CAT activity as described.19,21 In some experiments, cells were also pretreated with inhibitors, such as the antioxidant N-acetylcysteine (NAC, 100 μmol/L, Sigma Chemical Co), protein kinase C inhibitor (calphostin C, 100 nmol/L, Calbiochem), ERK1/2 inhibitor (PD98059, 25 μmol/L, New England BioLabs), Ras inhibitor (FPT-III, 10 μmol/L, BioMol Corp), and p38 MAPK inhibitor (SB202190, 1 μmol/L, Upstate Biotechnology) for 15 minutes before 13-HPODE addition.
Data are expressed as mean±SEM of multiple experiments. Paired Student t tests were used to compare 2 groups, or ANOVA with the Dunnett post hoc test was used for multiple groups with the use of PRISM software (Graph Pad). Bands from Western blots and kinase assays were quantified on a laser densitometer, and EMSAs were performed by a PhosphorImager with NIH ImageQuant software. Statistical significance was detected at the 0.05 level.
Activation of Ras by 13-HPODE
Because Ras, the small GTP binding protein, is a key common upstream activator of the MAPKs, we examined whether 13-HPODE can activate Ras. Quiescent VSMCs were pretreated with the Ras inhibitor (FPT III, 10 μmol/L) or vehicle alone and then stimulated for 10 minutes with 13-HPODE (10 μmol/L). Cells were then stained with a Ras antibody, and Ras translocation was examined by confocal microscopy. Figure 1 shows that in cells stimulated with 13-HPODE, there is a marked increase in the translocation of Ras to the cell membrane and that this was completely blocked by FPT III. Vehicle alone had no effect. Furthermore, 12(R)-hydroxyeicosatetraenoic acid, a cytochrome P-450 metabolite of arachidonic acid, had no effect on Ras. These results demonstrate that 13-HPODE activates Ras in VSMCs.
Activation of MAPKs by 13-HPODE
To evaluate the signal transduction mechanisms of action of 13-HPODE, we examined whether it could activate the MAPK family members. Serum-depleted VSMCs were treated with 13-HPODE (10 μmol/L) for 5, 10, and 30 minutes, and the cell lysates were assayed for MAPK activation. Figure 2 indicates that 13-HPODE could activate ERK1/2 (top blot), p38 MAPK (second blot), and JNK (third blot) with similar kinetics; ie, activation was observed by 5 minutes and remained elevated until 30 minutes. Blots were stripped and probed with antibody to α-actin as the loading control (bottom blot). These results demonstrate that 13-HPODE significantly activates ERK1/2, JNK, and p38 MAPKs (Figure 2, bar graph).
Activation of NF-κB DNA Binding Activity by13-HPODE in VSMCs
Oxidant stress and peroxides have been shown to induce the activation of NF-κB in various cell types, including VSMCs. Because 13-HPODE is a lipid hydroperoxide, we examined whether it could activate NF-κB in VSMCs. Serum-depleted PVSMCs were treated for 30 minutes with vehicle (0.1% ethanol) alone or with various concentrations of 13-HPODE (0.1 to 25 μmol/L). Nuclear extracts were subjected to EMSA with use of an oligonucleotide probe containing NF-κB consensus sequence from the VCAM-1 promoter. As shown in Figure 3 (top), 13-HPODE increased NF-κB DNA binding activity in a dose-dependent manner with maximum activity at 10 μmol/L, whereas vehicle alone had no effect (lane 1 [leftmost]). The same nuclear extracts were also tested for binding to a labeled oligonucleotide-containing consensus sequence for the transcription factor AP-1. 13-HPODE had no effect on AP-1 DNA binding activity (Figure 3, bottom) or AP-2 (not shown), indicating the specificity of 13-HPODE for NF-κB activation.
We also determined the time course of NF-κB activation. 13-HPODE (10 μmol/L) could induce NF-κB activation as early as 5 minutes, peaking at ≈30 minutes and remaining elevated until 2 hours after stimulation (results not shown). Thus, 13-HPODE increased NF-κB activation but had no effect on AP-1 or AP-2 DNA binding activity in PVSMCs.
To determine the physiological implications (namely, whether 13-HPODE in oxLDLs can be a VSMC activator), we compared NF-κB activation in cells treated for 30 minutes with 13-HPODE (10 μmol/L), 13-HPODE complexed to unmodified LDL (100 μg/mL), or unmodified LDL alone. Representative EMSA data from 2 separate experiments are as follows: 13-HPODE 3.2-fold over control, 13-HPODE+LDL 2.8-fold, and LDL 1.1-fold. Thus, 13-HPODE complexed to LDL could effectively activate NF-κB similar to 13-HPODE alone, whereas unmodified LDL alone was ineffective.
Subunit Composition of 13-HPODE-Induced NF-κB DNA Binding Complex
NF-κB is a heterodimeric protein consisting of p50, p65, Rel-B, or other subunits. To identify the composition of the 13-HPODE-induced NF-κB complex, nuclear extracts were subjected to supershifting analyses with p65, p50, or Rel-B antibodies as described.19 We noted that a p50 antibody clearly supershifted the complex, and a p65 antibody caused a decrease in the complex, whereas the combination also supershifted the DNA-protein complexes in 13-HPODE-stimulated cells (results not shown). However, Rel-B antibodies were without any effect. Thus, 13-HPODE-induced NF-κB is composed of p50 and p65 subunits.
Nuclear Translocation of NF-κB and Depletion of Cytosolic IκB-α in 13-HPODE-Treated Cells
On cellular stimulation, cytosolic NF-κB subunits dissociate from phosphorylated IκB and translocate to the nucleus.22,23 Free IκB-α in the cytosol is targeted to proteasomes for degradation.23 To test nuclear translocation of p50 and p65 subunits, nuclear extracts from 13-HPODE-stimulated PVSMCs were analyzed by immunoblotting with p65 or p50 antibodies. Figure 4 shows that 13-HPODE increased nuclear levels of p65 by 15 minutes, and these levels remained higher than basal levels until 2 hours after stimulation by 13-HPODE (Figure 4, top left panel). Nuclear levels of p50 were also increased up to 2 hours after stimulation (not shown). The blots were then stripped and probed with antibodies to histone-H1 as loading control for nuclear proteins. As shown (Figure 4, bottom left panel), histone levels did not change in 13-HPODE-stimulated cells.
Cytosolic extracts from these cells were immunoblotted with an antibody to IκB-α, and the results showed that IκB-α levels were reduced in 13-HPODE-stimulated cells up to 30 minutes and then returned to basal levels after 2 hours (Figure 4, top right panel). The blots were stripped and probed with actin as a loading control for cytosolic proteins (bottom right panel). Thus, nuclear translocation of NF-κB with concomitant depletion of cytosolic IκB-α levels further demonstrated that 13-HPODE stimulated NF-κB in VSMCs.
Involvement of Oxidative Stress and Signaling Kinases in 13-HPODE-Induced NF-κB Activation
To understand the signaling mechanisms involved in NF-κB activation by 13-HPODE, VSMCs were pretreated with inhibitors of protein kinase C (PKC), p38 MAPK, or the antioxidant NAC for 20 minutes. The cells were then stimulated with 13-HPODE, and NF-κB DNA binding was determined by EMSA. Radioactivity in each DNA-protein complex was quantified by PhosphorImager, and results are shown in Figure 5. The PKC inhibitor calphostin C (100 nmol/L) completely abolished 13-HPODE-induced NF-κB DNA binding activity, whereas the p38 MAPK inhibitor SB202190 (1 μmol/L) had no effect on DNA binding. NAC (100 mmol/L) also had inhibitory effects. These results suggest that PKC activation and oxidant stress are involved in 13-HPODE-induced NF-κB DNA binding activity.
Transcriptional Activation of Human VCAM-1 Promoter by 13-HPODE
Transcription of human VCAM-1 in response to inflammatory cytokines is regulated by NF-κB binding sites in the promoter.20 To examine the effect of 13-HPODE on NF-κB-induced transcription, PVSMCs were transiently transfected with a plasmid containing the CAT reporter gene driven by the VCAM-1 promoter (p85VCAM-CAT). Cells were then treated with 13-HPODE, the hydroxy product 13-(S)-hydroxyoctadecadienoic acid (13-HODE, 5 to 60 μmol/L), or tumor necrosis factor (TNF)-α (10 μg/mL) as a positive control. As seen in Figure 6, 13-HPODE (5 to 60 μmol/L) stimulated VCAM-1 promoter activation, whereas the nonperoxy compound, 13-HODE, was ineffective under the same conditions. As expected, TNF-α robustly stimulated VCAM-1 transcription. The effects of 13-HPODE at 7.5 and 30 μmol/L were significant (3.8±0.3-fold and 5.4±0.2-fold over control, respectively; n=3, P<0.001). Thus, 13-HPODE can also induce NF-κB-dependent functional gene expression in PVSMCs. In the same experiment, PVSMCs were also transfected with pSV2CAT, in which CAT expression is driven by simian virus 40 (SV40) promoter without NF-κB sites. 13-HPODE had no effect on CAT expression driven by the SV40 promoter (data not shown).
Signal Transduction Events in 13-HPODE Induced VCAM-1 Transcriptional Activation
PVSMCs were transiently transfected with p85VCAM-CAT and pretreated with various pharmacological inhibitors for 30 minutes. Then the cells were stimulated with 13-HPODE (30 μmol/L), and CAT activity was determined. Results showed that NAC (100 μmol/L) and inhibitors of PKC (calphostin C, 20 nmol/L), p38 MAPK (SB202190, 1 μmol/L), and Ras (FPT III, 10 μmol/L) clearly inhibited transcriptional activation of p85VCAM-CAT (80% to 90% inhibition in each case). However, a phosphatidylinositol-3-kinase inhibitor (LY294002, 25 μmol/L) or ERK inhibitor (PD98059, 25 μmol/L) had no effect under these conditions (data not shown). Thus, 13-HPODE activation of VCAM-1 transcription involves the activation of multiple closely related and interacting pathways, including oxidant stress, Ras, PKC, and p38 MAPK.
It is now well known that biologically active oxidized lipids play an important role in the development of atherosclerosis. OxLDL as well as its component lipids can have potent biological effects in various cell types. However, the specific mechanisms by which they act are still unclear. In particular, there is increased interest in determining the signal transduction mechanisms of action of these oxidized lipids.24,25
The present study demonstrates that oxidized lipids, such as 13-HPODE, can lead to potent cellular effects that could play key roles in atherosclerosis. We showed that 13-HPODE can significantly activate the MAPKs (ERK, p38, and JNK) in porcine VSMCs. We also noted for the first time that 13-HPODE treatment of VSMCs could lead to Ras activation, as determined by the translocation of Ras to the membrane. Although several studies have shown that oxidized lipids can activate members of the MAPK pathway, the mechanism has been unclear. Ras activation by 13-HPODE can be a novel mechanism for MAPK activation. However, additional studies are needed to determine whether 13-HPODE induces Ras translocation via activation of a putative cell surface receptor or novel membrane perturbation mechanisms. Recent studies have suggested that oxidant species can lead to downstream signaling and kinase activation by inactivating tyrosine phosphatases.26 Studies in rat VSMCs showed that linoleic acid and 13-HPODE activate ERK1/2 and also increase DNA synthesis.8 Although we noted that 13-HPODE had weak hypertrophic effects, it did not increase DNA synthesis (authors’ unpublished data, 2001). This discrepancy may be due to differences in the cell types.
We also noted that 13-HPODE could lead to the activation of the oxidant responsive transcription factor, NF-κβ. This was specific for NF-κB inasmuch as 2 other transcription factors, AP-1 and AP-2, were not activated. In addition, 13-HPODE-induced NF-κB activation was functionally associated with the transcriptional regulation of the inflammatory VCAM-1 gene as assessed by reporter gene expression studies. In unpublished observations, we noted that this was also associated with VCAM-1 protein expression in human VSMCs treated with 13-HPODE. That study was performed by ELISA in human VSMCs because antibodies to porcine VCAM-1 were unavailable. The NF-κB sites on the VCAM-1 promoter play an important role in gene regulation.20,27 We also observed that 13-HPODE-induced VCAM-1 promoter activation was similar in the p85VCAM-CAT construct (with 2 NF-κB sites) as in the longer p248VCAM-CAT construct (unpublished), suggesting that NF-κB may play a key role in 13-HPODE-induced VCAM-1 transcription similar to that seen with TNF-α.20 However, detailed mutation studies are needed to fully resolve these issues.
13-HPODE-induced NF-κB DNA binding was blocked by the antioxidant NAC as well as the PKC inhibitor calphostin C but not by the p38 MAPK inhibitor SB202190. On the other hand, 13-HPODE-induced VCAM-1 transcription (by CAT assays) was blocked by NAC, by calphostin C, and also by SB202190. This indicates a key role for PKC and oxidant stress in 13-HPODE-induced gene regulation. It also illustrates a novel new role for p38 MAPK in VCAM-1 regulation, wherein it does not interfere with NF-κB DNA binding but appears to have a nuclear role in VCAM-1 transcription. Further studies are needed to determine this nuclear role of p38 MAPK. One possibility is that p38 MAPK can augment the transactivating capabilities of p65 by phosphorylating it. In our studies, the ERK/MAPK kinase inhibitor PD98059 was ineffective (not shown). Overall, our observations suggest that p38 MAPK can mediate the pathological effects of oxidized lipids and further underscore the role of p38 MAPK in inflammatory gene expression.
It is possible that p38 MAPK activation and oxidant stress are downstream from PKC activation.28,29 Our results suggest that a key consequence of Ras activation by 13-HPODE is the activation of PKC, downstream MAPKs, and oxidant stress.
We also showed that 13-HPODE complexed to native LDL (but not native LDL alone) could activate NF-κB to a similar extent as 13-HPODE. This supports our reasoning that HPODE in oxLDL could be a stimulant for VSMCs. An important aspect to consider when examining the cellular effects of oxidized lipids is the extent of their uptake and metabolism by the cells as well as whether there are specific receptors for these lipids. A recent study by Auge et al30 demonstrates that 13-HPODE is poorly taken up by VSMCs, endothelial cells, and macrophages and that this small uptake is independent of the oxLDL receptor CD36. They concluded that the biological effects of oxidized lipids, such as 13-HPODE, may involve a direct interaction with cell surface components or that the small amounts that do enter the cells may have potent cellular effects. Our data are compatible with both these possibilities. Furthermore, some effects of 13-HPODE may be due to its conversion to 13-HODE, which can also be incorporated into cells and have potent effects.31 In addition, certain effects of 13-HPODE may be secondary to the oxidant stress induced by it. This is supported by our observation that many effects of 13-HPODE were blocked by antioxidants and also by recent studies showing that 13-HPODE can lead to hydrogen peroxide generation.32 Overall, our data suggest that multiple closely related pathways mediate 13-HPODE-induced inflammatory gene regulation. However, whereas the p38 MAPK pathway seems to play a key role in 13-HPODE-induced VCAM-1 regulation, the ERK1/2 pathway seems less important. This does not rule out the possibility that the ERK1/2 and JNK pathways mediate other cellular effects of 13-HPODE.
The induction of genes such as VCAM-1 by 13-HPODE may play a role in atherosclerosis because this can result in augmented recruitment as well as the retention of monocytes in the subendothelial space. The importance of 13-HPODE in atherogenesis was also recently demonstrated by Navab and colleagues.33,34 They noted that LDL oxidation requires seeding molecules, such as 13-HPODE,33 which also increases the formation of biologically active phospholipids.34
The present results indicate that oxidized lipids of the LO pathway, which are also key constituents of oxLDL, have potent cellular effects. Hence, maneuvers to block their formation may be therapeutically beneficial. We recently showed that a specific molecular inhibitor of LO, namely, an LO ribozyme delivered in cationic liposomes, could block platelet-derived growth factor-induced VSMC migration and also fibronectin expression.35 Furthermore, this 12-LO ribozyme could also significantly attenuate neointimal thickening in a rat carotid artery model of balloon injury.35
This work was supported by National Institutes of Health (NIH) grants PO1-HL-55798 and the Juvenile Diabetes Research Foundation (to R.N.), Clinical Investigator Award NIH KO8-HL-03137 (to B.V.K.), and NIH grant HL-19134-26 (to K.U.M.).
Received May 15, 2001; revision accepted June 13, 2001.
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