Oxidized LDL Induces Transcription Factor Activator Protein–1 but Inhibits Activation of Nuclear Factor–κB in Human Vascular Smooth Muscle Cells
Abstract Oxidized LDL (Ox-LDL) has been implicated in the development of atherosclerotic lesions, mainly due to its enhanced uptake by macrophages and its ability to alter gene expression in arterial cells. In the present study we demonstrated that Ox-LDL activates activator protein–1 (AP-1), a transcription factor generally induced by mitogenic substances. Lysophosphatidylcholine, which is generated during oxidation of LDL, stimulated AP-1 in a dose-dependent manner. In contrast, the radical-dependent transcription factor nuclear factor–κB (NF-κB) was not activated by Ox-LDL, and at a concentration of 50 μg/mL, Ox-LDL inhibited lipopolysaccharide-induced activation of NF-κB. Oxysterols but not lysophosphatidylcholine inhibited lipopolysaccharide-induced NF-κB activation, suggesting that they may be responsible for the inhibitory effect of Ox-LDL. In conclusion, Ox-LDL has opposing effects on the activities of NF-κB and AP-1, suggesting involvement of mechanisms for transcriptional regulation that are strongly affected by lipid oxidation products.
- oxidized LDL
- transcription factor activator protein–1
- nuclear factor–κB
- Received February 10, 1995.
- Accepted June 22, 1995.
Oxidative modification of LDL particles in the arterial intima is believed to be an early key event in the development of atherosclerotic lesions.1 This modification leads to the formation of intimal macrophage foam cells by rendering LDL susceptible to uptake by the scavenger receptor pathway. Ox-LDL also influences the secretory activities and functional properties of endothelial cells, SMCs, and macrophages, suggesting a complex role for lipoprotein oxidation in atherosclerosis. The biological actions of Ox-LDL include both stimulatory and inhibitory effects on gene transcription as well as general cytotoxicity and have been attributed to the formation of reactive molecules such as lipid peroxides, lysoPC, aldehydes, and oxysterols.2 3 However, the interactions between products generated by lipid peroxidation and intracellular signaling remain poorly understood.
Transcription factor NF-κB is present in most cell types as a cytosolic heterodimer composed of NFKB1 (p50) and RelA (p65) subunits bound to an inhibitor protein, I-κB. NF-κB regulates the inducible expression of a variety of genes involved in inflammatory and immune responses.4 Following activation, NF-κB dissociates from I-κB and is translocated to the nucleus. A wide variety of agents, including cytokines, UV light, antibodies to cell-surface proteins, LPS, and H2O2, activate NF-κB. The finding that substances that generate radicals activate NF-κB and the observation that several antioxidants inhibit the stimulatory effect of almost all known NF-κB activators have led to the conclusion that intracellular radicals have an integrative role in NF-κB activation.5 Oxidation of LDL is associated with the generation of substances with radical properties such as lipid peroxides,6 suggesting radical-mediated activation of NF-κB as a possible mediator of the biological effects of Ox-LDL. Some indirect support for this hypothesis comes from studies demonstrating that NF-κB activates transcription of VCAM-17 and E-selectin8 9 genes in endothelial cells and that an increased endothelial expression of VCAM-1 is the first identifiable response of the vessel wall to diet-induced hypercholesterolemia in experimental animals.10
The transcription factor AP-1 consists of homo- or heterodimers of the proteins encoded by the fos and jun gene families and is believed to regulate genes involved in the control of cell growth and differentiation.11 Exposure to H2O2 results in a marked accumulation of c-jun and c-fos mRNA but only a weak stimulation of the DNA binding capacity of AP-1.5
Proliferation of SMCs in the arterial intima is one of the major factors responsible for the development of atherosclerotic lesions.12 This proliferation is believed to be initiated by growth factors secreted by activated SMCs, macrophages, and endothelial cells within the developing lesion and may be further enhanced by the presence of Ox-LDL.13 The observation that SMC DNA synthesis is stimulated by radical generators such as H2O2 and xanthine/xanthine oxidase14 suggests that lipid oxidation products may act as mediators of the mitogenic activity of Ox-LDL. In the present study we have analyzed the effects of native and Ox-LDL and several lipid oxidation products on the DNA binding activity of transcription factors NF-κB and AP-1.
l-α-LysoPC (egg yolk) was purchased from Sigma. [γ-32P]dATP (185 TBq/mmol) was from Amersham. Antibodies against NFKB1 (p50), RelA (p65), and the oligonucleotides for mobility shift assays were obtained from Santa Cruz Biotechnology, Inc. 25-Hydroxycholesterol, 7α-hydroxycholesterol, and cholestan-5α,6α-epoxy-3β-ol purified by high-performance liquid chromatography15 were a generous gift of Dr Ingemar Björkhem, Karolinska Institute, Stockholm, Sweden.
Human arterial SMCs originally isolated by explant technique were kindly provided by Dr Ulf Hedin (Karolinska Institute). The cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS, penicillin (50 U/mL), and streptomycin (50 μg/mL). The cells showed positive α-actin immunoreactivity (HHF35 antibody was kindly provided by Dr Allan Gown, University of Washington, Seattle). Cells in the 12th through the 18th passages were used in the experiments. Prior to experiments, confluent cells in 100-mm dishes were serum-starved two times in Ham’s F-12 medium for 24 hours. RPMI-1640 medium was used for treatments with H2O2 because this medium does not contain transition metal ions, which catalyze the decomposition of H2O2 into hydroxyl radicals.
Preparation of Nuclear Extracts
Cells in 100-mm plastic dishes were rinsed with ice-cold PBS and harvested in 5 mL PBS by scraping. Nuclear extracts were prepared essentially as described by Alksnis et al.16 Briefly, the cells were washed with 1 mL PBS and resuspended in 100 μL hypotonic buffer (in mmol/L: HEPES 10, pH 7.3, KCl 10, MgCl2 1.5, DTT 1, and PMSF 1). After centrifugation, cells were lysed by resuspension in 300 μL lysis buffer (10 mmol/L HEPES, pH 7.3, 10 mmol/L KCl, 1.5 mmol/L MgCl2, 0.4% Nonidet P-40, 1 mmol/L DTT, 1 mmol/L PMSF, 1 μg/mL leupeptin, and 15 μg/mL aprotinin). After a 10-minute incubation at 4°C nuclei were collected by centrifugation for 1 minute at 8000g, and the pellets were washed once in 1 mL of 20 mmol/L KCl buffer (20 mmol/L HEPES, pH 7.3, 22% glycerol, 20 mmol/L KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L PMSF, 1 μg/mL leupeptin, and 15 μg/mL aprotinin). The isolated nuclei were resuspended in 15 μL of 20 mmol/L KCl buffer, and 60 μL of 0.6 mol/L KCl buffer (20 mmol/L HEPES, pH 7.3, 22% glycerol, 0.6 mol/L KCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 1 mmol/L DTT, 1 mmol/L PMSF, 1 μg/mL leupeptin, and 15 μg/mL aprotinin) was added. Nuclear proteins were extracted by incubation on ice for 30 minutes. After centrifugation for 15 minutes at 8000g, the supernatant containing nuclear proteins was transferred to a precooled microcentrifuge tube, and an aliquot of the extract was diluted 40 times with 484 mmol/L KCl buffer (mixture of those above) for protein assay. Protein concentration (in micrograms per milliliter) was determined spectrophotometrically17 according to the following equation: Protein Concentration=184×A(230 nm)−81.7×A(260 nm), where A is absorbance.
Equal amounts of protein from nuclear extracts (1 to 3 μg) were incubated on ice with 2 μg poly(dI-dC) and 1 μg acetylated bovine serum albumin in binding buffer (giving the final concentrations stated below) for 10 minutes. The oligonucleotide probe (50 000 cpm in 5 μL) was added, and the reaction mixture (25 μL) was incubated for 30 minutes at room temperature. Final concentrations in binding reactions were as follows: 10% glycerol, 10 mmol/L HEPES, pH 7.9, 60 mmol/L KCl, 5 mmol/L MgCl2, 0.5 mmol/L EDTA, 1 mmol/L DTT, and 1 mmol/l PMSF. DNA-protein complexes were separated from unbound DNA probe on native 7% polyacrylamide gels in low-ionic-strength buffer (22.3 mmol/L each Tris and borate and 0.5 mmol/L EDTA, pH 8). The sequences of the double-stranded oligonucleotide probes labeled with T4 kinase and [γ-32P]dATP were as follows: κB consensus, 5′-AGT TGA GGG GAC TTT CCC AGG C-3′; κB mutant, 5′-AGT TGA GGC GAC TTT CCC AGG C-3′; and AP-1 consensus, 5′-CGC TTG ATG AGT CAG CCG GAA-3′.
Preparation of LDL
Blood was drawn from fasting, healthy volunteers, and plasma was recovered by centrifugation at 1400g for 20 minutes at 1°C. The isolated plasma was adjusted to d=1.10 kg/L by the addition of NaCl. A density gradient consisting of 3 mL of 1.10 kg/L–density plasma and 3 mL of 1.065, 1.020, and 1.006 kg/L NaCl solutions, respectively, was then formed in cellulose nitrate tubes (Ultraclear tubes, Beckman). The gradient was centrifuged in a Beckman L8-55 ultracentrifuge at 40 000 rpm in a Beckman SW-40 swinging-bucket rotor at 20°C overnight.18 The VLDL and IDL fractions were aspirated from the top 3 mL, and LDL was harvested from the next 4 mL of the tube. EDTA and excess salt were subsequently removed by dialysis against EDTA-free PBS. The protein content of the LDL preparation was determined as described by Lowry et al.19 Endotoxin levels in both LDL and Ox-LDL were below 5 ng/mg LDL protein as determined by Limulus assay (Pharmacia).
Oxidation of LDL
Copper oxidation was performed by incubating the LDL at 37°C in 5% CO2 in the presence of 5 μmol/L CuSO4 for varying times, typically 15 hours.20 Total amounts of aldehydes in Ox-LDL were determined by colorimetric assay (Bioxytech S. A.). The aldehyde content of LDL oxidized for 0, 2, 6, or 24 hours was 8.6, 8.6, 13.1, and 15.2 nmol/mg LDL protein, respectively.
Activation of NF-κB and AP-1 in SMCs by H2O2 and LPS
Serum-starved SMCs contained small or undetectable amounts of active NF-κB. Exposure of SMCs to H2O2 in serum-free RPMI-1640 medium resulted in a dose-dependent activation of NF-κB (Fig 1⇓). The DNA binding activity of AP-1 was also increased by H2O2, but not as strongly as that of NF-κB (data not shown). Maximal activation of both transcription factors was obtained at an H2O2 concentration of 400 μmol/L. EMSAs revealed two distinct bands that were specific for NF-κB (Fig 2⇓). The addition of excess unlabeled mutant κB oligonucleotide (with one point mutation in the binding site) to the binding reactions resulted in the disappearance of unspecific binding activity, whereas specific bands were unaffected. When unlabeled probe was used as a competitor, the specific complexes were more susceptible to competition than the unspecific ones (Fig 2⇓). NF-κB–specific complexes with higher mobility (in the lower band) were retarded by antibodies against NFKB1 (p50). Antibodies directed against RelA (p65) retarded the slower migrating complex as well, leaving a minor portion of the complexes with higher mobility unaffected. Taken together, these data suggest that the major component of NF-κB in SMCs consists of heterodimers between p50 and p65. There seemed to be small amounts of p50 homodimers as well as smaller quantities of a complex that did not react with antibodies to p50 or p65. The supershift assays also suggested the existence of at least one additional NF-κB–related complex in the extracts. Incubation of SMCs with LPS resulted in an activation of both NF-κB and AP-1 (Fig 3⇓). Maximal activation of NF-κB was obtained at LPS concentrations between 500 and 750 ng/mL, whereas AP-1 required higher concentrations for maximal binding activity.
Effect of Ox-LDL on NF-κB Activity in SMCs
The effect of Ox-LDL on NF-κB activity was analyzed by using either native LDL or LDL oxidized by exposure to copper for 2 to 16 hours. None of the preparations of native or Ox-LDL stimulated the activity of NF-κB in quiescent SMCs (Fig 4⇓). In contrast, Ox-LDL inhibited LPS-induced activation of NF-κB. The inhibitory effect increased with the extent of oxidative modification; LDL oxidized by incubation with copper for 16 hours completely abolished the activation of NF-κB caused by 1 μg/mL LPS (Fig 4⇓) as well as that induced by H2O2 (data not shown). The inhibitory effect of Ox-LDL was concentration dependent. A lower concentration of copper-oxidized LDL, 10 μg/mL, neither inhibited nor activated NF-κB as determined by EMSA (Fig 5⇓ and data not shown).
Effect of Ox-LDL on AP-1 Activity in SMCs
At a concentration of 50 μg/mL Ox-LDL activated AP-1 (Fig 6⇓). AP-1 activation by Ox-LDL was not significantly affected by the low LPS concentration (5 ng/mL) that in itself did not activate AP-1 in these cells. When CuSO4 and PBS were added to the culture medium at concentrations similar to that resulting from the addition of copper-oxidized LDL, neither NF-κB nor AP-1 were affected (Figs 5⇑ and 6⇓ and data not shown). Analysis of the influence of degree of oxidation revealed that LDL only marginally modified by exposure to copper for 2 hours was as effective in activating AP-1 as LDL extensively modified by incubation with copper for 16 hours (Fig 7⇓). The nuclear extracts used to analyze AP-1 activity were the same as those used for NF-κB analysis (Fig 4⇑), in which the biological activities of the different preparations of copper-oxidized LDL were clearly different.
Effect of LysoPC on NF-κB and AP-1
Phosphatidylcholine is the major phospholipid in LDL. During oxidation of LDL up to approximately 50% of this phospholipid can be converted into lysoPC.20 Studies in our laboratory have demonstrated that lysoPC is responsible for most of the mitogenic activity of Ox-LDL (A. Stiko, et al, unpublished data, 1994). Incubation of SMCs with lysoPC resulted in a dose-dependent activation of AP-1 with a maximal effect obtained at a concentration of 15 μg/mL (Fig 8⇓). Only minor amounts of active NF-κB were detected in cells exposed to lysoPC (Fig 9⇓). At higher concentrations of lysoPC, serum-induced NF-κB binding activity was slightly inhibited (Fig 9⇓).
Effect of Oxysterols on NF-κB
Ox-LDL at a protein concentration of 50 μg/mL can be expected to contain 1 to 12 μg oxysterols/mL.20 At a concentration of 5 μg/mL all of the three oxysterols (cholestan-5α,6α-epoxy-3β-ol, 7α-hydroxycholesterol, and 25-hydroxycholesterol) inhibited LPS-induced NF-κB activity (Fig 10⇓), but this inhibition was weaker than that obtained by 50 μg/mL LDL oxidized by exposure to copper for 16 hours (Fig 4⇑). As measured by laser densitometry, treatment of cells with oxysterols resulted in a 50% inhibition of the LPS-induced activation of NF-κB.
The radical-sensitive transcription factor NF-κB may function as a mediator of the biological effects of oxidized lipoproteins. Experimental studies provide some support for this hypothesis. LDL mildly oxidized by exposure to soybean lipoxygenase activates NF-κB in cultured endothelial cells.21 In rabbits fed a cholesterol-enriched diet, activation of VCAM-1 expression appears to be the initiating factor responsible for the recruitment of circulating monocytes and the formation of early inflammatory lesions.10 Diet-induced hypercholesterolemia is associated with an increased endothelial production of superoxide radicals.22 Taken together with the findings that activation of endothelial VCAM-1 expression is effectively blocked by antioxidants7 and that the promoter of the VCAM-1 gene contains two NF-κB binding sites, these observations suggest that hypercholesterolemia is associated with a radical-dependent, NF-κB–mediated activation of endothelial VCAM-1 expression. However, Maier et al23 report that neither NF-κB nor any of the adhesion molecules (VCAM-1, E-selectin, intracellular adhesion molecule–1, or P-selectin) were activated by Ox-LDL despite the fact that Ox-LDL caused an increase in monocyte binding to endothelial cells.
We have shown in this study that LDL oxidized by exposure to copper sulfate did not activate NF-κB in SMCs. Instead, at a concentration of 50 μg/mL Ox-LDL, a marked inhibition of NF-κB activation could be observed. Simultaneously, Ox-LDL stimulated the DNA binding activity of AP-1. LPS-mediated activation of NF-κB was inhibited by three different oxysterols, suggesting that these substances may be partially responsible for the inhibitory effect of Ox-LDL.
In contrast to its effect on NF-κB, Ox-LDL was found to stimulate the DNA binding activity of AP-1. This effect was dependent on concentration and to a lesser extent on the degree of oxidation, suggesting that the activation was due to a substance that was formed during oxidation of LDL. Several lines of evidence suggest that this substance may be lysoPC. LysoPC stimulated AP-1 activity in a dose-dependent manner, and oxidation of LDL results in the formation of lysoPC at concentrations equivalent to those found to activate AP-1. For example, if Ox-LDL contains 400 nmol lysoPC/mg protein,20 the lysoPC concentration in medium containing Ox-LDL at a concentration of 50 μg protein/mL will be 10 μg/mL. LysoPC also activates protein kinase C, a potent mediator of AP-1 activation.24 25 26 Preliminary studies in our laboratory have demonstrated that lysoPC is a potent mitogen for SMCs (A. Stiko, et al, unpublished data, 1994). Therefore, it is likely that the mitogenic activity of Ox-LDL is at least partially explained by a lysoPC-dependent activation of AP-1. Several other observations also indicate that lysoPC may play a role in atherogenesis. LysoPC is abundantly present in atherosclerotic plaques,27 functions as a selective chemoattractant for mononuclear leukocytes,28 induces expression of VCAM-1 in endothelial cells,29 and stimulates the synthesis of heparin-binding epidermal growth factor–like growth factor in endothelial cells30 and monocytes.31 Whether these effects are mediated by activation of AP-1 remains to be determined.
The possibility that the inhibitory effect of Ox-LDL on NF-κB activation is due to general cytotoxicity should be taken into account. However, the parallel activation of AP-1 and the identification of single-lipid molecular species (lysoPC and oxysterols) that are capable of activating AP-1 or inhibiting activation of NF-κB argue against this. Furthermore, no cell detachment could be observed after treatment with Ox-LDL for up to 6 hours.
In a study on cultured endothelial cells, Parhami et al21 have shown that LDL minimally oxidized by incubation with soybean lipoxygenase enhanced NF-κB activity. To analyze how the extent of oxidation influences the effects of Ox-LDL, we exposed SMCs to LDL incubated with copper for 2, 4, 6, and 16 hours. No stimulatory effect on NF-κB activity could be observed with the partially oxidized LDL preparations obtained by a shorter exposure to copper, but these preparations were less potent inhibitors of NF-κB than fully oxidized LDL. Other researchers20 have determined the peroxide content of Ox-LDL. If these peroxides are not considerably more potent activators of NF-κB than exogenous H2O2, their concentration is probably too low to induce NF-κB activation. LDL oxidized with copper for 4 hours should contain a maximum amount of lipid peroxides,20 but we could not detect NF-κB activation with any of the copper-oxidized LDL preparations oxidized for 2, 4, or 6 hours.
Among the genes that could be affected by altered levels of AP-1 and NF-κB are the inflammatory cytokines tumor necrosis factor–α and IL-1β. Even though NF-κB regulates the expression of tumor necrosis factor–α and IL-1β, a number of reports32 33 34 35 suggest that AP-1 sites are more important for the activation of these genes. AP-1 may be involved in the transcriptional regulation of several other cytokines as well,32 including IL-2, IL-3, IL-6, colony stimulating factor–1, and transforming growth factor–β1. It is also interesting to note that the mitogenic response of fibroblasts to tumor necrosis factor–α requires active AP-1.36 In bacteria the NF-κB–related, radical-activated transcription factor oxyR controls the expression of antioxidant enzymes.37 38 If NF-κB plays a similar role in human SMCs, the inhibitory effect of Ox-LDL could impair the survival of cells in lesions under oxidative stress. However, further studies to clarify which SMC genes are controlled by NF-κB are required before the putative role of Ox-LDL–mediated inhibition of NF-κB activation can be evaluated.
In summary, the present study shows that oxidative modification of LDL is associated with the formation of substances that inhibit the activation of NF-κB but stimulate the activity of AP-1 in human SMCs. These effects may be of importance in atherogenesis, but their possible pathophysiological role remains to be determined.
Selected Abbreviations and Acronyms
|EMSA||=||electrophoretic mobility shift assay|
|FCS||=||fetal calf serum|
|Ox-LDL||=||oxidatively modified LDL|
|SMC||=||smooth muscle cell|
|VCAM-1||=||vascular cell adhesion molecule-1|
This study was supported by grants from the Swedish Medical Research Council (8311), the King Gustaf V 80th Birthday Fund, the Nordic Academy for Advanced Study (94.30.024/00), the Nanna Svartz Fund, the Swedish Heart and Lung Foundation, and the Wallenberg Foundation.
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