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Arteriosclerosis, Thrombosis, and Vascular Biology. 1998;18:568-576

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1998;18:568-576.)
© 1998 American Heart Association, Inc.

Original Contributions

Biphasic Regulation of Transcription Factor Nuclear Factor-{kappa}B Activity in Human Endothelial Cells by Lysophosphatidylcholine Through Protein Kinase C–Mediated Pathway

Seigo Sugiyama; Kiyotaka Kugiyama; Nobuhiko Ogata; Hideki Doi; Yasutaka Ota; Masamichi Ohgushi; Toshiyuki Matsumura; Hideki Oka; ; Hirofumi Yasue

From the Division of Cardiology, Department of Medicine, Kumamoto University School of Medicine, Japan.

Correspondence to Kiyotaka Kugiyama, MD, Division of Cardiology, Department of Medicine, Kumamoto University School of Medicine, Honjo 1–1-1, Kumamoto City, Kumamoto, Japan 860. E-mail kiyo{at}gpo.kumamoto-u.ac.jp


*    Abstract
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 *Abstract
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Abstract—Lysophosphatidylcholine (lysoPC), which is generated in oxidized LDL (Ox-LDL) and abundantly exists in atherosclerotic arterial walls, has been shown to alter various endothelial functions and induces several endothelial genes expressed in atherosclerotic arterial walls. Nuclear factor-kappa B (NF-{kappa}B), a pleiotropic transcription factor, plays an important role in regulation of expression of various genes implicated in atherosclerosis. We have previously reported that lysoPC transferred from Ox-LDL to endothelial surface membrane activates endothelial protein kinase C (PKC), leading to modulated endothelial functions. This study was aimed at determining whether lysoPC could modulate activity of transcription factors in cultured human umbilical vein endothelial cells (HUVECs) by using electrophoretic mobility shift assay. LysoPC was found to increase DNA-binding activity of NF-{kappa}B in HUVECs within 15 minutes, which peaked at 1 to 2 hours and subsequently declined to the baseline level at 6 hours. Lower concentrations (5 to 15 µmol/L) of lysoPC markedly increased NF-{kappa}B activity, but higher concentration (50 µmol/L) of lysoPC inhibited the activity. Phorbol 12-myristate 13-acetate, a potent activator of PKC, also augmented NF-{kappa}B activity in HUVECs, mimicking the effects of lysoPC; furthermore, calphostin C and chelerythrine chloride, specific PKC inhibitors, and {alpha}-tocopherol, a clinically potent PKC inhibitor, suppressed the lysoPC-induced NF-{kappa}B activation. These results indicate that lysoPC regulates NF-{kappa}B activity in a biphasic manner dependent on its concentrations and incubation time in human endothelial cells and the endothelial PKC activation may in part be involved in the lysoPC-induced NF-{kappa}B activation. Thus, the time course and the positive and negative biphasic regulatory actions of lysoPC on NF-{kappa}B activity in endothelial cells might exhibit a unique effect of lysoPC in arterial walls on the different stages of atherosclerosis.


Key Words: endothelium • atherosclerosis • signal transduction • oxidized LDL • {alpha}-tocopherol


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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Atherosclerosis is associated with alteration of various endothelial functions.1 2 3 We have shown that Ox-LDL, which abundantly exists in atherosclerotic arterial walls,4 plays an important role in the endothelial functional alterations,5 6 7 and lysoPC is one of the active molecules generated during oxidative modification of LDL.5 8 Furthermore, we and others have shown that lysoPC induces expression of ICAM-1, vascular cell adhesion molecule-1, P-selectin, plasminogen activator inhibitor-1, macrophage chemoattractant protein-1, cyclooxygenase-2, and growth factors in endothelial cells.9 10 11 12 13 14 15 Although lysoPC could induce gene transcription of a great variety of molecules expressed in endothelium of atherosclerotic arteries, there are few reports explaining the intracellular mechanism(s) for diverse effects of lysoPC. Previously, we have shown that lysoPC activates PKC,16 17 leading to expression of ICAM-1 in isolated porcine coronary arterial endothelium.9

NF-{kappa}B, a pleiotropic transcription factor, plays an important role in regulation of expression of many inducible genes,18 and Brand et al19 have recently demonstrated that the activated form of NF-{kappa}B is present in human atherosclerotic arterial walls. The promoter regions of the lysoPC-inducible genes in endothelial cells have several binding sites for transcription factors such as NF-{kappa}B and AP-1, and the activities of NF-{kappa}B and AP-1 are known to be in part modulated by the PKC signal-transduction system.20 21 Thus, lysoPC could regulate DNA-binding activities of these transcription factors through the mechanism(s) of a PKC-mediated pathway, leading to the induction of a number of endothelial genes implicated in atherosclerosis. Therefore, in the present study, we have examined whether lysoPC could induce NF-{kappa}B activation in human vascular endothelial cells; if so, we have determined the possible involvement of a PKC-mediated pathway in the lysoPC-induced NF-{kappa}B activation.


*    Methods
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 up arrowIntroduction
 *Methods
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Cell Culture
Primary cultures of HUVECs were obtained by collagenase digestion and were incubated in medium 199 with 15% FCS, endothelium growth supplement, heparin, penicillin, and streptomycin at 37°C in a humidified atmosphere of 95% air/5% CO2 as previously described.12 16 The medium was replaced every 3 days, and HUVECs at passages 1 to 3 were used in the present study. Confluent cultures of HUVECs exhibited the typical cobblestone morphology, and most of those cells contained factor VIII–related antigen, as determined using indirect immunofluorescence as previously described.12 16

Experimental Protocols
After reaching confluence in 100-mm plastic dishes, the incubation medium of HUVECs was replaced with medium 199 containing 5% FCS and antibiotics without growth factors, and then the HUVECs were incubated for 4 hours. After that, the HUVECs were rinsed with serum-free medium 199 and further incubated in serum-free medium 199 for 6 hours before the experiments. The preincubated HUVECs were then rinsed with medium 199 and incubated in serum-free medium 199 with various concentrations of lysoPC or PMA in the presence or absence of a PKC inhibitor or other additives for the indicated time. In some experiments, the confluent HUVECs were incubated with or without {alpha}-tocopherol (50 to 200 µmol/L) for 24 hours before the serum reduction, and then stimulated by lysoPC in the presence or absence of various concentrations of {alpha}-tocopherol. LysoPC was dissolved in PBS by sonication just before use for the experiments. After the incubation, the HUVECs were washed twice with warm PBS and immediately frozen by liquid nitrogen and stored at -80°C until nuclear protein extraction.

Preparation of Nuclear Extracts
Nuclear extracts of HUVECs were prepared by the modified miniscale detergent treatment procedure as described by Schreiber et al22 and Chowdhury et al.23 Briefly, the HUVECs cultured in a 100-mm plastic dish were harvested into a 1.5-mL tube by scraping in 800 µL of Buffer-A (mmol/L: HEPES 10, pH 7.9; KCl 10; EDTA 0.1; EGTA 0.1; DTT 1.0; PMSF 1.0; pepstatin A 2 µg/mL) at 4°C and incubated on ice for 15 minutes, after which 50 µL of 10% Nonidet NP-40 was added into the tube, and the endothelial cells were homogenized by vigorous vortexing for 10 seconds. The homogenate was centrifuged at 3000 rpm in 4°C for 10 minutes to prepare endothelial nuclei, and the isolated nuclei in the pellet were resuspended in 10 µL of ice-cold Buffer-B (mmol/L: HEPES 20, pH 7.9; NaCl 400; EDTA 1.0; EGTA 1.0; DTT 1.0; PMSF 1.0; pepstatin 2 µg/mL) and further homogenized with a hand homogenizer at 4°C. Endothelial nuclear proteins were extracted by incubation of the nuclei homogenate on ice for 30 minutes, and then the supernatant containing nuclear protein was collected after centrifugation at 8000 rpm in 4°C for 15 minutes. The nuclear protein preparation was transferred into a new precooled microtube and stored at -80°C until use. Protein concentration was determined by the method of Bradford24 using bovine serum albumin as a standard.

EMSA
Double-stranded synthetic oligonucleotides were 5' end labeled with [r-32P]ATP and T4 polynucleotide kinase and used as hot probes. The sequences of the probes are as follows: NF-{kappa}B, 5'-CCAGAGGGGACTTTCCGAGAGG-3'; AP-1, 5'-CGCCGCAAGTGACTCAGCGCGGGG-3'; Sp-1, 5'-GATCGGGGCGGGGCGATCGGGGCGGGGCGATC-3'; CREB, 5'-CTCCTTGGCTGACGTCAGAGAGAGAG-3'; and C/EBP, 5'-CAAATGTAGTCTTATGCAATACACTTGTAGTCTTGCAACAG-3', (a kind gift from Dr Takiguchi, Department of Molecular Genetics, Kumamoto University).

Standard binding reaction was carried out in 20 µL of mixture containing 25 mmol/L HEPES-KOH (pH 7.6), 50 mmol/L KCl, 1 mmol/L EDTA, 1 mol/L DTT, 0.5 mmol/L spermidine, 0.5 mmol/L PMSF, 10% glycerol, 0.1 mg/mmol/L poly (dI-dC), 1 fmol probe (about 2x103 cpm), and 5 µg of the extracted nuclear protein as previously described.23 For competition experiments, the unlabeled cold oligonucleotides were derived into each reaction mixture before addition of nuclear extracts, and for supershift assay, the reaction mixture minus the labeled probe was incubated with 3 µL of a specific antibody raised against p50, p65, RelB, c-Rel, or control nonimmune serum at 4°C overnight. The nuclear protein/DNA binding reaction was performed by incubation of the final reaction mixture on ice for 30 minutes. After the incubation, 5 µL of Ficoll dye was added into the reaction tubes, and the samples were loaded onto 4.5% to 7.5% polyacrylamide gels made in a buffer containing 45 mmol/L Tris, 45 mmol/L boric acid, and 1.0 mmol/L EDTA. Electrophoresis was performed at constant voltage of 100 V for 90 minutes at 4°C. After the electrophoresis, the gels were dried and autoradiographed at -80°C for several days, and 32P radioactivity in the bands of NF-{kappa}B was quantified by using a Bio-Image Analyzer BA100 and the relative activity derived by dividing by the counts in the bands of control sample as reported previously.23

Reagents
Synthetic {alpha}-palmitoyl-lysoPC, {alpha}-stearoyl-lysoPC, {alpha}-tocopherol, and other chemicals were obtained from Sigma Chemical Co. Calphostin C, chelerythrine chloride, and PMA were from Calbiochem Novabiochem Co. Poly (dI-dC) was from Pharmacia Biotech. T4 polynucleotide kinase was from TAKARA Biomedicals. Antibodies raised against p50, p65, RelB, and c-Rel were from Santa Cruz Biotechnology Inc. PAF receptor blocker (WEB 2086) was a gift from Boehringer Mannheim (Mannheim, Germany). PMA, calphostin C, chelerythrine chloride, and {alpha}-tocopherol were prepared and diluted in DMSO. The final concentration of DMSO in the culture medium was 0.1%, and control incubation contained an identical amount of DMSO.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Effects of LysoPC on NF-{kappa}B/DNA Binding Activity in HUVECs
Treatment of HUVECs with lysoPC leads to induction of two distinct complexes that form on the NF-{kappa}B oligonucleotide probe in the low-concentration (4.5%) polyacrylamide gel as shown in Fig 1ADown (indicated by arrow). In the present study, HUVECs basally contained very little active NF-{kappa}B in the nucleus, and both {alpha}-palmitoly-lysoPC (C16:0) and {alpha}-stearoly-lysoPC (C18:0) equally increased NF-{kappa}B-binding activity. PMA, which is known to be one of the NF-{kappa}B activators, also induced double bands of NF-{kappa}B in HUVECs, mimicking the effect of lysoPC. Fig 1BDown shows the amount of activated NF-{kappa}B quantified by the Bio-Image Analyzer. Both {alpha}-palmitoyl-lysoPC and {alpha}-stearoyl-lysoPC induced approximately twofold increase in NF-{kappa}B activity, and PMA induced approximately fourfold increased activity. As shown in Fig 2ADown, nuclear extracts from lysoPC-treated HUVECs form a single band in the high-concentration (7.5%) polyacrylamide gel. In the competition experiments, the lysoPC-induced bands of protein/DNA complex were gradually competed by unlabeled cold NF-{kappa}B oligonucleotide probe in a dose-dependent manner (x5 to x100; 5 to 100 fmol) and were not affected by C/EBP oligonucleotide (100 fmol) (data not shown). Thus, the bands of protein/DNA complex (indicated by arrow) seem to indicate the specific NF-{kappa}B/DNA complex (Fig 2ADown). To examine the subunits of lysoPC-induced NF-{kappa}B complexes, we performed supershift assay using specific antibodies raised against p50, p65, c-Rel, and RelB. Fig 2BDown shows that the lower band of NF-{kappa}B complex was completely reacted with p50 antibody and mostly reacted with p65 antibody and slightly supershifted by RelB antibody; at the same time, the upper band was supershifted by p65 and p50 antibodies and partially reacted with c-Rel and RelB antibodies. Both upper and lower bands were not reacted with nonimmune serum. These results indicate that the lysoPC-induced NF-{kappa}B complexes include mainly p50 and p65, and a little of c-Rel and RelB.



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  		Figure 1. A, Electrophoretic mobility shift assay showing the effects of lysoPC and PMA on the NF-{kappa}B/DNA binding activity. Nuclear extracts from HUVECs after treatment for 60 minutes with or without {alpha}-palmitoyl-lysoPC, {alpha}-stearoyl-lysoPC, or PMA were analyzed for protein binding to the NF-{kappa}B consensus sequence by EMSA (4.5% gel). Lane 1, treated with vehicle; lane 2, treated with {alpha}-palmitoyl-lysoPC (15 µmol/L); lane 3, treated with {alpha}-stearoyl-lysoPC (15 µmol/L); lane 4, treated with PMA (50 nmol/L); lane 5, treated with PMA (50 nmol/L)+ x100 competitor (cold NF-{kappa}B oligonucleotide probe). Arrow indicates the bands of NF-{kappa}B/DNA complex. The result shown is representative of five independent experiments. B, Quantitative analysis of NF-{kappa}B/DNA binding activity in the nuclear extracts from lysoPC or PMA-treated HUVECs. Shown are control, vehicle-treated, {alpha}-palmitoyl-lysoPC (15 µmol/L), {alpha}-stearoyl-lysoPC (15 µmol/L), and PMA (50 nmol/L)-treated HUVECs. Radioactivity of the bands corresponding to NF-kB/DNA complex was counted by the Bio-Image Analyzer. NF-{kappa}B/DNA binding activity in control was set to 1.0, and the relative activity was derived by dividing by the counts in the bands of control sample and the mean and SDs were determined from five independent experiments and indicated.



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  		Figure 2. A, Competitive analysis of lysoPC-induced NF-{kappa}B/DNA binding complex using unlabeled cold NF-{kappa}B oligonucleotide probe. Nuclear extracts from HUVECs treated for 60 minutes with or without {alpha}-palmitoyl-lysoPC (15 µmol/L) were analyzed for protein binding to the NF-{kappa}B consensus sequence by EMSA (7.5% gel) in the presence of various concentrations of cold NF-{kappa}B probe (competitor). Lane 1, vehicle-treated HUVECs+competitor (-); 2, treated with lysoPC+competitor (-); 3, treated with lysoPC+competitor x5 (5 fmol); 4, treated with lysoPC+competitor x10 (10 fmol); 5, treated with lysoPC+competitor x50 (50 fmol); 6, treated with lysoPC+competitor x100 (100 fmol). Arrow indicates the band of NF-{kappa}B/DNA complex. The result shown is representative of three independent experiments. B, Supershift assay of lysoPC-induced NF-{kappa}B complexes using specific antibodies against p50, p65, c-Rel, and RelB. Nuclear protein was extracted from HUVECs after treatment for 60 minutes with lysoPC (15 µmol/L) and the supershift assay was performed as described in "Methods." Samples were run on 4.5% gel. Lane 1, lysoPC-treated HUVECs; lane 2, lysoPC-treated HUVECs+x100 cold NF-{kappa}B probe. Nuclear proteins were incubated with anti-p50 antibody (lane 3), anti-p65 antibody (lane 4), anti-c-Rel antibody (lane 5), anti-RelB antibody (lane 6), or nonimmune serum (NIS, lane 7). Black arrow indicates bands of NF-{kappa}B/DNA complexes and white arrow indicates supershifted bands. The result shown is representative of three independent experiments.

Time Course of Endothelial NF-{kappa}B Activation by LysoPC
To examine the kinetics of lysoPC-induced endothelial NF-{kappa}B activation, HUVECs were incubated with 15 µmol/L {alpha}-palmitoyl-lysoPC for the indicated time, and the nuclear protein was then prepared at each time point. As shown in Fig 3Down, lysoPC transiently induced NF-{kappa}B activation in HUVECs. The lysoPC-induced NF-{kappa}B activity was first detected within 15 minutes after the stimulation, peaked at 60 to 120 minutes, and subsequently declined to nearly the baseline level at 6 hours.



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  		Figure 3. Time course of NF-{kappa}B/DNA binding activity after stimulation with lysoPC. HUVECs were treated for the indicated time with {alpha}-palmitoyl-lysoPC (15 µmol/L), and nuclear extracts from the treated HUVECs were examined by EMSA (6% gel). A, Gel image of NF-{kappa}B/DNA binding activity. Time indicates minutes after stimulation. Lane 1, 0 minutes; lane 2, 15 minutes; lane 3, 30 minutes; lane 4, 60 minutes; lane 5, 120 minutes; lane 6, 240 minutes; lane 7, 360 minutes. Arrow indicates the bands of NF-{kappa}B/DNA complex. The result shown is representative of three independent experiments. B, Quantitative analysis of NF-{kappa}B/DNA binding activity.32P Radioactivity in the bands of NF-{kappa}B/DNA complex was quantified by the Bio-Image Analyzer. Data are calculated as relative ratio by time 0 minutes control setting at 1.0, and the relative activity was derived by dividing by the counts in the bands of time 0 minutes sample.

Concentration-Response Effect of LysoPC on NF-{kappa}B Activity
To examine the concentration-response effects of lysoPC on NF-{kappa}B activity, HUVECs were incubated with various concentrations of {alpha}-palmitoyl-lysoPC (5 to 50 µmol/L) for 60 minutes, and then we analyzed the NF-{kappa}B activity by EMSA. As shown in Fig 4Down, low concentrations of lysoPC (5 to 15 µmol/L) increased NF-{kappa}B activity in HUVECs, and the maximum activation was observed at the concentration of 15 µmol/L lysoPC. The stimulatory effect of lysoPC on NF-{kappa}B activation was overwhelmed with higher concentrations of lysoPC (30 to 50 µmol/L); furthermore 50 µmol/L lysoPC inhibited the NF-{kappa}B activity to a comparable level with the basal condition. These results indicate that lysoPC regulates endothelial NF-{kappa}B activity in a biphasic manner, dependent on its concentrations. Trypan blue staining revealed that no cell death was found after the incubation for 60 minutes with lysoPC at concentrations up to 50 µmol/L, suggesting that nonspecific cytotoxicity by lysoPC may not be involved in the inhibitory effect of higher concentrations of lysoPC.



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  		Figure 4. Effects of different lysoPC (LPC) concentrations on NF-{kappa}B/DNA binding activity in HUVECs. HUVECs were incubated for 60 minutes with various concentrations of {alpha}-palmitoyl-lysoPC (5 to 50 µmol/L) and nuclear proteins from the treated HUVECs analyzed by EMSA (5% gel). A, Gel image of NF-{kappa}B/DNA binding activity. Lane 1, treated with vehicle; lane 2, treated with 5 µmol/L lysoPC; lane 3, treated with 10 µmol/L lysoPC; lane 4, treated with 15 µmol/L lysoPC; lane 5, treated with 30 µmol/L lysoPC; lane 6, treated with 50 µmol/L lysoPC. Arrow indicates the bands of NF-{kappa}B/DNA complex. The result shown is representative of three independent experiments. B, Quantitative analysis of NF-{kappa}B/DNA binding activity.32P radioactivity in the bands of NF-{kappa}B/DNA complex was quantified by the Bio-Image Analyzer. Data are calculated as relative ratio by vehicle-treated control setting at 1.0, and the relative activity was derived by dividing by the counts in the bands of control sample.

Role of PKC-Mediated Pathway in LysoPC-Induced NF-{kappa}B Activation
We have previously reported that lysoPC activates endothelial PKC, leading to modulation of several endothelial functions.9 16 17 Therefore, we examined whether the PKC-meditated pathway is involved in the lysoPC-induced NF-{kappa}B activation in human endothelial cells. Fig 1Up shows that PMA (50 nmol/L), a potent PKC activator,25 significantly induced NF-{kappa}B activation in HUVECs. Next, we pretreated HUVECs with calphostin C26 (50 to 400 nmol/L) or chelerythrine chloride27 (5 to 10 µmol/L) for 15 minutes, and then lysoPC (15 µmol/L) was added to the incubation medium in the presence of the PKC inhibitor. After 60 minutes' incubation with lysoPC and the PKC inhibitor, the nuclear extracts from the treated HUVECs were examined by EMSA. As shown in Fig 5ADown, calphostin C, which specifically inhibits the regulatory domain of PKC, significantly inhibited the lysoPC-induced NF-{kappa}B activation in a dose-dependent manner (50 to 400 nmol/L), and calphostin C (200 nmol/L) also effectively inhibited the PMA-induced NF-{kappa}B activation (Fig 5CDown). Chelerythrine chloride, which specifically inhibits the catalytic domain of PKC, also inhibited the lysoPC-induced activation of NF-{kappa}B (Fig 5BDown).



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  		Figure 5. Effects of PKC inhibitors on lysoPC- or PMA-induced NF-{kappa}B/DNA binding activity in HUVECs. HUVECs were incubated for 60 minutes with lysoPC (15 µmol/L) or PMA (50 nmol/L) in the presence or absence of PKC inhibitors. After the incubation, the nuclear proteins were extracted and analyzed by EMSA (A, 5.5%; B, 7%; C, 5% gel). A, Effects of calphostin C on lysoPC-induced NF-{kappa}B activation. Lane 1 (control), treated with vehicle; lane 2, treated with lysoPC alone; lane 3, treated with calphostin C (50 nmol/L)+lysoPC; lane 4, treated with calphostin C (100 nmol/L)+lysoPC; lane 5, treated with calphostin C (200 nmol/L)+lysoPC; lane 6, treated with calphostin C (400 nmol/L)+lysoPC. B, Effects of chelerythrine chloride (CC) on lysoPC-induced NF-kB activation. Lane 1 (control), treated with vehicle; lane 2, treated with lysoPC alone; lane 3, treated with chelerythrine chloride (5 µmol/L)+lysoPC; lane 4, treated with chelerythrine chloride (10 µmol/L)+lysoPC. C, Effects of calphostin C on PMA-induced NF-{kappa}B activation. Lane 1 (control), treated with vehicle; lane 2, treated with PMA alone; lane 3, treated with calphostin C (200 nmol/L)+PMA. Arrow indicates the bands of NF-{kappa}B/DNA complex. The results shown are representative of three independent experiments.

Effects of {alpha}-Tocopherol on LysoPC-Induced NF-{kappa}B Activation
It has been demonstrated that vitamin E ({alpha}-tocopherol) has beneficial effects on vascular functions28 and the development of coronary artery diseases,29 and {alpha}-tocopherol is also known to be a clinically useful potent PKC inhibitor.28 30 We investigated the effects of {alpha}-tocopherol on lysoPC-induced NF-{kappa}B activation. As shown in Fig 6Down, treatment with {alpha}-tocopherol attenuated the lysoPC-induced NF-{kappa}B activation in a dose-dependent manner (50 to 200 µmol/L).



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  		Figure 6. Effect of {alpha}-tocopherol on lysoPC-induced endothelial NF-{kappa}B activation. HUVECs were incubated for 60 minutes with lysoPC (15 µmol/L) in various concentrations of {alpha}-tocopherol. After the incubation, the nuclear proteins were extracted and analyzed by EMSA (6% gel). Lane 1 (control), treated with vehicle; lane 2, treated with lysoPC alone; lane 3, treated with {alpha}-tocopherol (50 µmol/L)+lysoPC; lane 4, treated with {alpha}-tocopherol (100 µmol/L)+lysoPC; lane 5, treated with {alpha}-tocopherol (200 µmol/L)+lysoPC. Arrow indicates the bands of NF-{kappa}B/DNA complex. The result shown is representative of three independent experiments.

Effects of LysoPC on Transcription Factors in Human Endothelial Cells
We further examined the effects of lysoPC on several transcription factors such as AP-1, CREB, and Sp-1 in HUVECs. Treatment of HUVECs with lysoPC (15 µmol/L) for 1 hour increased AP-1 and CREB activity but did not affect on Sp-1 activity (Fig 7ADown, 7BDown, and 7CDown). Fig 8ADown shows the time course of AP-1, CREB, and Sp-1 activities when cells were incubated with 15 µmol/L lysoPC. LysoPC increased AP-1 and CREB activities from 1 hour and peaked at 4 hours after the stimulation but had no significant effect on Sp-1 activity, and the AP-1 and CREB activities were still higher than the baseline level after incubation with lysoPC for 6 hours. As shown in Fig 8BDown, lysoPC increased AP-1 activity from a concentration of 10 µmol/L, and the lysoPC-induced AP-1 activation was in a dose-dependent manner up to 50 µmol/L lysoPC. LysoPC also increased CREB activity from concentration of 15 µmol/L, and the lysoPC-induced CREB activity peaked at 30 µmol/L. As shown in Fig 7ADown and 7BDown, calphostin C slightly inhibited the lysoPC-induced AP-1 activity, but it had no effect on the lysoPC-induced CREB activity.



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  		Figure 7. Effects of lysoPC on endothelial AP-1, CREB, and Sp-1/DNA binding activities. HUVECs were incubated for 60 minutes with lysoPC (15 µmol/L), and then nuclear proteins were extracted and analyzed by EMSA (7% gels) using each oligonucleotide probe. A, AP-1/DNA binding activity. Lane 1 (control), treated with vehicle; lane 2, treated with lysoPC alone; lane 3, treated with calphostin C (200 nmol/L)+lysoPC. B, CREB/DNA binding activity. Lane 1 (control), treated with vehicle; lane 2, treated with lysoPC alone; lane 3, treated with calphostin C (200 nmol/L)+lysoPC. C, Sp-1/DNA binding activity. Lane 1 (control), treated with vehicle; lane 2, treated with lysoPC alone. Arrow indicates the bands of AP-1, CREB or Sp-1/DNA complex. The results shown are representative of three independent experiments.



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  		Figure 8. A, Time course of AP-1, CREB, and Sp-1/DNA binding activities after stimulation with lysoPC. HUVECs were treated for the indicated time with lysoPC (15 µmol/L), the nuclear proteins were extracted, and AP-1, CREB, and Sp-1/DNA binding activities analyzed by EMSA.32P radioactivity in the bands of NF-{kappa}B/DNA complex was quantified by the Bio-Image Analyzer, and the data are calculated as relative ratio by time 0 minutes control setting at 1.0, and the relative activity was derived by dividing by the counts in the bands of time 0 minutes sample. B, Effects of different lysoPC concentrations on AP-1, CREB, and Sp-1/DNA binding activities in HUVECs. HUVECs were incubated for 60 minutes with various concentrations of lysoPC (5 to 50 µmol/L), nuclear proteins were extracted, and AP-1, CREB, and Sp-1/DNA binding activities were analyzed by EMSA.32P radioactivity in the bands of NF-{kappa}B/DNA complex was quantified by the Bio-Image Analyzer. The data are calculated as relative ratio by vehicle-treated control setting at 1.0, and the relative activity was derived by dividing by the counts in the bands of vehicle-treated sample.

LysoPC-Induced NF-{kappa}B Activation is Not Mediated Through PAF Receptor
PAF has structural similarities to lysoPC, and it has been recently shown that PAF can activate NF-{kappa}B through PAF receptor.31 Therefore, to determine the possible involvement of a PAF receptor-mediated pathway in the lysoPC-induced NF-{kappa}B activation in the present study, we stimulated HUVECs by lysoPC (15 µmol/L) in the presence of WEB 2086, a specific PAF receptor antagonist,32 for 1 hour. Fig 9Down shows that WEB 2086 (5 and 10 µmol/L) did not affect the lysoPC-induced NF-{kappa}B activation in the present study.



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  		Figure 9. Effect of PAF-receptor blocker on lysoPC-induced endothelial NF-{kappa}B activation. HUVECs were incubated for 60 minutes with lysoPC (15 µmol/L) in the presence of WEB 2086 (5.0 or 10.0 µmol/L), which is a specific PAF-receptor blocker. After the incubation, the nuclear proteins were extracted and analyzed by EMSA (7.5% gel). Arrow indicates the bands of NF-{kappa}B/DNA complex. Lane 1 (control), treated with vehicle; lane 2, treated with lysoPC alone; lane 3, treated with WEB 2086 (5.0 µmol/L)+lysoPC; lane 4, treated WEB 2086 (10.0 µmol/L)+lysoPC. The result shown is representative of four independent experiments.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The major finding of this study is that lysoPC, an atherogenic lysophospholipid, can induce biphasic regulation of transcription factor NF-{kappa}B activity in human vascular endothelial cells, and its effect is partly mediated through a PKC-dependent pathway. We here showed that lower concentrations (5 to 15 µmol/L) of lysoPC increased endothelial NF-{kappa}B activity, but higher concentration (50 µmol/L) of lysoPC conversely inhibited it. Furthermore, lysoPC (15 µmol/L) significantly stimulated endothelial NF-{kappa}B activity within 15 minutes, which peaked at 1 to 2 hours and subsequently declined to the baseline level at 4 to 6 hours after the stimulation.

Atherosclerosis is associated with increase in endothelial expression of ICAM-1,33 VCAM-1,34 and MCP-1,35 most of which have been shown to be induced in endothelial cells by lysoPC.9 10 13 Furthermore, these genes are known to have possible NF-{kappa}B-binding sites in their promoter region. Recently, Brand et al19 have demonstrated that the activated form of NF-{kappa}B is present in human atherosclerotic arterial walls. These previous data indicate that transcription factor NF-{kappa}B could play an important role in pathogenesis and development of atherosclerosis. In most cell types, NF-{kappa}B is present in the cytosol as an inactive complex form binding to an inhibitor protein, I-{kappa}B, and following activation by many stimuli, NF-{kappa}B is activated by dissociation from I-{kappa}B and translocated into the nucleus.36 37 A wide variety of agents, including cytokines, free radicals, and lipopolysaccharides, can possibly activate NF-{kappa}B in atherosclerotic arterial walls.21 Previously, Parhami et al38 and Peng et al39 showed that minimally modified LDL and Ox-LDL enhanced NF-{kappa}B activity in cultured endothelial cells. We have previously demonstrated that lysoPC, which abundantly exists in atherosclerotic arterial walls8 40 and is generated in Ox-LDL by the hydrolysis of phosphatidylcholine via LDL-associated phospholipase A2 activity,41 participates in alteration of endothelial functions by Ox-LDL.5 9 12 17 Herein, we demonstrated that lysoPC increases NF-{kappa}B activity in HUVECs, suggesting the possible involvement of lysoPC in endothelial NF-{kappa}B activation in atherosclerotic lesions.

Previously, we and others have shown that lysoPC stimulates activity of purified PKC in-vitro42 and it increases PKC activity in the particulate fraction of endothelial cells16 17 and plateles.11 The precise mechanisms of PKC activation by lysoPC are not fully determined. It can be speculated that the amphiphilic lysoPC in atherosclerotic arteries is transferable to accessible membranous and macromolecular acceptors through the aqueous phase43 and the transferred lysoPC is slowly translocated to the inner plasma membrane and then metabolized. During this transmembrane movement, lysoPC might be capable of accessing and directly activating PKC in the plasma membrane. We have previously demonstrated that lysoPC increased expression of ICAM-1, which has an NF-{kappa}B binding site in its promoter region, in the coronary endothelium through the PKC-mediated pathway.9 Furthermore, the present study showed that calphostin C26 and chelerythrine chloride,27 specific PKC inhibitors, significantly attenuated lysoPC-induced NF-{kappa}B activation in HUVECs, and further, PMA, a potent activator of PKC,25 increased NF-{kappa}B activity, mimicking the effects of lysoPC. Therefore, these results indicate that PKC activation by lysoPC could partly be involved in the mechanism of lysoPC-induced NF-{kappa}B activation, leading to induction of transcription of some genes, including ICAM-1, in human endothelial cells. It has been well known that PKC is an important signal transducer in cellular responses to various biologically active extracellular stimuli.44 The NF-{kappa}B activation process is controlled by several intracellular signaling pathways, including phosphorylation and proteolytic degradation.36 37 45 Originally, Sen and Baltimore46 showed that NF-{kappa}B can be translocated into nucleus by PKC activation, and others also demonstrated the possible involvement of a PKC-mediated pathway in NF-{kappa}B activation.47 48 PKC plays a pivotal role in regulation of proliferation, differentiation, and gene expression, and PKC can interact upstream of other intracellular signaling networks, including activation of p21ras and Raf,49 50 and it has been reported that activation of Raf and p21ras is involved in NF-{kappa}B activation,51 52 suggesting the possibility that PKC or PKC-dependent intracellular signaling cascades might be implicated in the lysoPC-induced NF-{kappa}B activation.

In the present study, lower concentrations (5 to 15 µmol/L) of lysoPC activate but higher concentration (50 µmol/L) of lysoPC conversely inhibits NF-{kappa}B activity. These concentration-dependent opposite effects of lysoPC have been demonstrated in previous reports on endothelial adhesion molecule expression53 and monocyte and T-cell chemotactic response.54 We also have shown that lysoPC modulated purified endothelial PKC activity in a biphasic manner, ie, lower concentration of lysoPC activated, whereas higher concentration of lysoPC suppressed PKC activity.17 Furthermore, we and others recently demonstrated that lysoPC can biphasically regulate PKC activity in intact cells,11 16 suggesting that the concentration-dependent effects of lysoPC on PKC activity could participate in the biphasic regulation of NF-{kappa}B activation by lysoPC, as shown in the present study. Here, we reported that lysoPC rapidly stimulated endothelial NF-{kappa}B activity, which peaked at 1 to 2 hours and subsequently declined to the baseline level at 4 to 6 hours. In the previous reports, a similar time course was observed in lysoPC-induced endothelial gene expression of MCP-113 and cyclooxgenase-2,14 both of which have NF-{kappa}B binding sites in their 5' region. The time course of the lysoPC-mediated rapid NF-{kappa}B activation seems to be correlated with the early response to lysoPC-induced PKC activation in endothelial cells in our previous study,16 and the early lysoPC-induced NF-{kappa}B activation could be a possible initiator of the inducible gene expression in atherosclerotic arterial walls. The precise mechanisms of the late-phase decline of NF-{kappa}B activity and suppressive effect of higher concentration of lysoPC remain unclear at the present time; however, it is unlikely that nonspecific cytotoxicity of lysoPC may cause the suppression of NF-{kappa}B activity after the longer incubation and after the incubation with higher concentration of lysoPC, because no cell death was found by trypan blue exclusion test after 2 hours' incubation with 50 µmol/L lysoPC (maximum concentration tested) and after 6 hours incubation (maximum incubation time tested) with 15 µmol/L lysoPC. Furthermore, the lysoPC-induced AP-1 activation was in a dose-dependent manner of lysoPC (up to 50 µmol/L), and our previous study showed that protein synthesis rate was increased in a time and concentration-dependent manner.12 It is possible that intracellular cAMP level could be involved in the biphasic nature of the lysoPC-induced NF-{kappa}B response, since it has been demonstrated that elevated cAMP can inhibit NF-{kappa}B activity.55 However, cAMP may not play a major role in the biphasic regulation, because lysoPC could not significantly increase endothelial intracellular cAMP levels in the present study (data not shown). It has been reported that the activated NF-{kappa}B can induce expression of I-{kappa}B, an inhibitory factor of NF-{kappa}B, in endothelial cells,56 suggesting a possible feedback regulation by the lysoPC-activated NF-{kappa}B in HUVECs. Recently, Johnson et al57 have demonstrated that TNF activates NF-{kappa}B persistently, but IL-1 and PMA activate NF-{kappa}B transiently, with less persistence in HUVECs. They showed that this different kinetics may result from the sustained reduction in I{kappa}B-ß levels by TNF but not by IL-1 and PMA. We need to examine the effect of lysoPC on the time course of I{kappa}B degradation in regard to the biphasic regulation of the NF-{kappa}B response further. It has been shown that expressions of lysoPC-inducible genes such as ICAM-1 and MCP-1 on the luminal surface of arterial endothelium at early stages of atherosclerosis are increased, but their expressions are rare in the advanced atherosclerotic endothelium.33 35 Thus, the time course and the positive and negative biphasic regulatory actions of lysoPC on NF-{kappa}B activity in endothelial cells might express a sequence of expression of lysoPC-induced genes in arterial walls on the different stages of atherosclerosis.

We demonstrated that lysoPC simultaneously activated several transcription factors, NF-{kappa}B, CREB, and AP-1 in HUVECs in the present study, implying that multiple gene transcription cascades could be turned on. It has been shown that ICAM-1 promoter region has both NF-{kappa}B and AP-1 binding sites, and these transcription factors play an important role in ICAM-1 gene expression,58 59 suggesting that lysoPC could induce endothelial ICAM-1 expression by the simultaneous activation of NF-{kappa}B and AP-1. Gene expression is controlled by many transcription factors, and NF-{kappa}B has been shown to synergize with other different transcriptional proteins.60 Thus, lysoPC could induce endothelial gene expression via synergistic effects with NF-{kappa}B. And other transcription factors and activation of multiple transcription factors by lysoPC in a different manner from that of NF-{kappa}B may explain the diverse and complex effects of lysoPC. The lysoPC-induced activation of AP-1 was dose dependent (up to 50 µmol/L), and the peak of the activation was observed at 4 hours after the stimulation, indicating that the kinetics of AP-1 activation by lysoPC seem to be different from those of NF-{kappa}B. There is a possibility that the late phase of the lysoPC-induced AP-1 activation could be mediated through new AP-1 protein synthesis stimulated by lysoPC.

It is known that lysoPC modulates activities of various membrane-associated enzymes, such as adenylate and guanylate cyclase61 and (Ca2+, Mg2+)-ATPase.62 Ochi et al63 have previously shown that high concentrations of lysoPC (50 to 100 µmol/L) induce endothelial adhesion molecule expression through a PKC-independent pathway and Inoue et al reported that lysoPC can induce Ca2+ influx in endothelial cells.64 Therefore, there is a possibility that another intracellular signaling pathway(s) activated by lysoPC besides a PKC-mediated pathway may be involved in the mechanisms of lysoPC-induced NF-{kappa}B activation in endothelial cells. Recently, Zhu et al65 have demonstrated that lysoPC can activate NF-kB through tyrosine kinases but not a PKC-mediated pathway in HUVECs. The reasons for the discrepant results between their study and ours remain unknown. However, the incubation condition of HUVECs with lysoPC and the concentrations of lysoPC seem to be different between the studies. They stimulated HUVECs in the presence of 5% FCS with a higher concentration of lysoPC (100 µmol/L), which is more than micellar concentrations and may exhibit detergent-like properties. On the other hand, we stimulated endothelial cells with 5 to 50 µmol/L lysoPC without serum after the gradual serum-reducing periods in the present study, since lysoPC has various actions on vascular cells in the subendothelial space, where LDL is oxidized under serum-free condition. Serum contains a number of bioactive components, including growth factors that can activate tyrosine kinases and some transcription factors. It is thus possible that lysoPC might exert synergistic action with serum factors, leading to the PKC-independent activation of NF-{kappa}B in their study. It has been shown that PAF has structural similarities to lysoPC and PAF induces NF-{kappa}B activation through the PAF receptor,31 raising a possible involvement of a PAF receptor–mediated pathway in lysoPC-induced NF-{kappa}B activation. In the present study, WEB 2086, the specific PAF receptor antagonist,32 did not affect the lysoPC-induced NF-{kappa}B activation, indicating that a PAF receptor–mediated signaling pathway may not play a major role in the effect of lysoPC in HUVECs. Ares et al66 demonstrated that lysoPC stimulates AP-1 activity but not NF-{kappa}B in cultured human smooth muscle cells, but we reported here that lysoPC induces both NF-{kappa}B and AP-1 activation in human endothelial cells, raising the possibility that the effect of lysoPC on NF-{kappa}B activity can be different between smooth muscle cells and endothelial cells.37

There is increasing evidence that vitamin E, {alpha}-tocopherol, has beneficial effects on development and pathogenesis in cardiovascular diseases.29 {alpha}-Tocopherol has been known as a lipid-soluble antioxidant abundant in human plasma67 and has many biological effects on vascular cells.28 30 In the present study, we demonstrated that {alpha}-tocopherol attenuated the lysoPC-induced NF-{kappa}B activation in clinically possible doses. It has been reported that {alpha}-tocopherol can inhibit PKC activity in smooth muscle cells30 and endothelial cells in vitro.28 It can be speculated that {alpha}-tocopherol may attenuate the effect of lysoPC via regulation of PKC activity, and it could modulate endothelial inducible gene expression through regulating NF-{kappa}B activity in atherosclerotic arteries. We cannot deny a possibility that {alpha}-tocopherol could inhibit the lysoPC-induced NF-{kappa}B activation by its antioxidant effect, because {alpha}-tocopherol has been known as a lipophilic powerful antioxidant, lysoPC can induce superoxide production in HUVECs (K.K., unpublished data, 1997) and from rabbit aorta,68 and H2O2 can induce NF-{kappa}B activation.69 Further investigations are required to examine whether lysoPC could induce NF-{kappa}B activation in human endothelial cells through an oxidative stress–mediated pathway.

In conclusion, lysoPC, an atherogenic lysophospholipid, can induce biphasic regulation of transcription factor NF-{kappa}B activity in human vascular endothelial cells, and its effect is partly mediated through a PKC-dependent pathway. LysoPC could play an important role in the pathogenesis of atherosclerosis by modulating expression of some endothelial genes through regulating the activation of transcription factor NF-{kappa}B in atherosclerotic arterial walls.


*    Selected Abbreviations and Acronyms
 
AP-1 = activator protein-1
EMSA = electrophoretic mobility shift assay
HUVEC = human umbilical vein endothelial cell
ICAM-1 = intercellular adhesion molecule-1
lysoPC = lysophosphatidylcholine
NF-{kappa}B = nuclear factor-{kappa}B
Ox-LDL = oxidatively modified LDL
PAF = platelet-activating factor
PKC = protein kinase C
PMA = phorbol 12-myristate 13-acetate


*    Acknowledgments
 
This work was supported in part by a Grant-in-Aid for Scientific Research on C07670793 from the Ministry of Education, Science, and Culture of Japan, and by a Smoking Research Foundation Grant for Biomedical Research, Tokyo, Japan. We thank Masaki Takiguchi (Department of Molecular Genetics, Kumamoto University) for helpful discussion and for each oligonucleotide probe. We also thank Todd Bourcier (Vascular Medicine and Atherosclerosis Unit, Brigham and Women's Hospital, Harvard Medical School) for his helpful suggestions and discussion. Platelet activating factor receptor blocker (WEB 2086) was a gift from Boehringer Mannheim (Mannheim, Germany).


*    Footnotes
 
Presented in part at the 69th Scientific Sessions of the American Heart Association, New Orleans, La, November 10–13, 1996.

Received April 3, 1997; accepted November 14, 1997.


*    References
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