Activation of Mitogen-Activated Protein Kinase Is Required for Migration and Invasion of Placental...

Vascular Biology, Atherosclerosis and Endothelium Biology

Activation of Mitogen-Activated Protein Kinases byLysophosphatidylcholine-Induced MitochondrialReactive Oxygen Species Generation inEndothelial Cells

Nobuo Watanabe,* Jaroslaw W. Zmijewski,†

Wakako Takabe,*‡ Makiko Umezu-Goto,§

Claire Le Goffe,† Azusa Sekine,* Aimee Landar,†

Akira Watanabe,* Junken Aoki,§ Hiroyuki Arai,§

Tatsuhiko Kodama,* Michael P. Murphy,¶

Raman Kalyanaraman,� Victor M. Darley-Usmar,†

and Noriko Noguchi* **From the Research Center for Advanced Science and

Technology,* and Graduate School of Pharmaceutical Sciences,§

University of Tokyo, Tokyo, Japan; Chugai Pharmaceutical Co.

Ltd.,‡ Tokyo, Japan; the Center for Free Radical Biology and

Department of Pathology,† University of Alabama, Birmingham,

Alabama; the MRC-Dunn Human Nutrition Unit,¶ Cambridge,

United Kingdom; the Medical College of Wisconsin,� Milwaukee,

Wisconsin; and the Faculty of Engineering,** Doshisha

University, Kyoto, Japan

Lysophosphatidylcholine (lysoPC) evokes diverse bio-logical responses in vascular cells including Ca2� mobi-lization, production of reactive oxygen species, and ac-tivation of the mitogen-activated protein kinases, butthe mechanisms linking these events remain unclear.Here, we provide evidence that the response of mito-chondria to the lysoPC-dependent increase in cytosolicCa2� leads to activation of the extracellular signal-regu-lated kinase (ERK) mitogen-activated protein kinasethrough a redox signaling mechanism in human umbil-ical vein endothelial cells. ERK activation was attenu-ated by inhibitors of the electron transport chain pro-ton pumps (rotenone and antimycin A) and anuncoupler (carbonyl cyanide p-trifluoromethoxyphe-nylhydrazone), suggesting that mitochondrial innermembrane potential plays a key role in the signalingpathway. ERK activation was also selectively attenuatedby chain-breaking antioxidants and by vitamin E tar-geted to mitochondria, suggesting that transduction ofthe mitochondrial hydrogen peroxide signal is medi-ated by a lipid peroxidation product. Inhibition of ERKactivation with MEK inhibitors (PD98059 or U0126)

diminished induction of the antioxidant enzyme hemeoxygenase-1. Taken together, these data suggest arole for mitochondrially generated reactive oxygenspecies and Ca2� in the redox cell signaling path-ways, leading to ERK activation and adaptation of thepathological stress mediated by oxidized lipids suchas lysoPC. (Am J Pathol 2006, 168:1737–1748; DOI:

10.2353/ajpath.2006.050648)

Lysophosphatidylcholine (lysoPC) is a naturally occurringbioactive lipid molecule thought to be important in thepathophysiology of atherosclerosis. It can be formed as aby-product of lecithin cholesterol acyltransferase-medi-ated cholesterol esterification with phosphatidylcholine inhigh-density lipoprotein or produced through phospho-lipase A2-mediated hydrolysis of phosphatidylcholine inplatelet membranes during inflammation.1 LysoPC canalso be generated from oxidation of low-density lipopro-tein (LDL), accounting for nearly 50% of the phosphati-dylcholine equivalents in oxidized LDL.2 This is particu-larly important because accumulation of oxidized LDL inthe arterial intima has been suggested to play a role in thepathogenesis of atherosclerosis.3 In addition to the pro-atherogenic responses to lipid oxidation products, theendothelium adapts to this stress by induction of antiox-idant protective pathways including glutathione-depen-

Supported by “the special coordination funds for promoting science andtechnology” and the Academic Frontier Research Project on “New Fron-tier of Biomedical Engineering Research” from the Ministry of Education,Science, Culture, Sports, and Technology in Japan; the National Institutesof Health grants ES10167 and HL58031(to V.M.D.-U.); the Program ofFundamental Studies in Health Sciences of the National Institute of Bio-medical Innovation; and Focus 21 Project of New Energy and IndustrialTechnology Development Organization.

N.W. and J.W.Z. contributed equally to this work.

Accepted for publication January 30, 2006.

Address reprint requests to Noriko Noguchi, #34, Research Center forAdvanced Science and Technology, University of Tokyo, 4-6-1 Komaba,Meguro, Tokyo 153-8904. E-mail: [email protected].

American Journal of Pathology, Vol. 168, No. 5, May 2006

Copyright © American Society for Investigative Pathology

DOI: 10.2353/ajpath.2006.050648

1737

dent pathways and enzymes such as heme oxygenase-1(HO-1).4–6 HO-1 catalyzes the breakdown of pro-oxidantheme with the release of carbon monoxide and antioxi-dant products.7 The impact of lysoPC on these pathwaysor the mechanisms of signal transduction have not beenexamined in depth. Previously, lyso-lipids have beenshown to causes cell lysis above their critical micelleconcentration (40 to 50 �mol/L),8 but in blood, it is un-likely that these concentrations of the free lipid areachieved because of binding of albumin. In cell culturemodels, lysoPC exposure leads to increased expressionof adhesion molecules, pro-inflammatory cytokines, andother potentially pro-atherogenic genes.9 Some biologi-cal actions of lysoPC have been ascribed to the activa-tion of G protein-coupled receptors, namely, GPR 410,11

and G2A,12 as well as the platelet-activating factor re-ceptor.13,14 However, recent reports have cast doubt ontheir role as lysoPC receptors.15–17 In addition, the abilityof lysoPC to incorporate into cell membranes18 results inmembrane perturbations that blunt a number of cellularresponses to various vasoactive ligands.19–21 Previousstudies have revealed that lysoPC can cause an increasein cytosolic Ca2�,21,22 induce reactive oxygen species(ROS),23 and activate a number of protein kinase path-ways including the mitogen-activated protein kinase(MAPK) pathways,24 multiple isoforms of protein kinaseC,25 and tyrosine kinases.26 Nevertheless, the mecha-nisms underlying those cellular signaling events and themechanistic links between them remain unclear.

ROS can be generated in various cells in response to avariety of stimuli including growth factors, cytokines, andphysicochemical stress. A growing body of evidence sug-gests that ROS can act as second messengers in a numberof signal transduction pathways.27 The mechanisms areonly now emerging and include modulation of the thiolproteome, thereby controlling the redox tone that regulatesthe sensitivity of redox signaling pathways to ROS-depen-dent activation, inactivation of phosphatases, and activationof tyrosine kinases.27,28 Several sources of vascular ROSproduction that could lead to cell signaling have been iden-tified, including the NAD(P)H oxidases (NOXs), uncoupledendothelial nitric-oxide synthase, and most recently mito-chondria.29 In this respect, the NOX enzymes are potentialcandidates because endothelial cells express subunits ofthe phagocyte NAD(P)H oxidase, including gp91 (NOX2)and its homologs, NOX1, NOX4, and NOX5.30 In contrast,ROS production by mitochondria, although long regardedas a pathological by-product of respiration, is increasinglybeing implicated in redox cell signaling pathways.31–38 Sev-eral mechanisms/molecules have been suggested as directregulators of ROS production in mitochondria, includingmodulation of the membrane potential (��),39 nitric oxide,and Ca2�.40,41 In isolated mitochondria, it has been shownthat the mitochondrial complexes I, II, and III are potentialsources of ROS formation. Precise molecular mechanismsremain unclear but could involve the posttranslational mod-ification of mitochondrial proteins such as the recently de-scribed S-thiolation of complex I.42

In this study, we investigated the mechanism of ROSproduction by lysoPC, signaling pathway activation, andtheir pathophysiological significance in human umbilical

vein endothelial cells (HUVECs). The data revealed thatROS production by lysoPC occurred predominantly in themitochondrion and that this was associated with activationof extracellular signal-regulated kinase (ERK) but not c-JunN-terminal kinase (JNK). The mechanisms of transductionof the ROS signal remain unclear but could involve theformation of a lipid peroxidation product within the or-ganelle. Finally, we found that ERK activation by lysoPCleads to increased levels of the anti-atherogenic enzymeHO-1, consistent with a cytoprotective role of this mitochon-drial ROS signaling pathway.

Materials and Methods

Cells and Reagents

HUVECs were purchased from Clonetics (San Diego,CA). Cells were maintained in endothelial basal medium2 (Clonetics) supplemented with 2% fetal bovine serum,growth factors, and antibiotics, all of which were suppliedby the manufacturer (Clonetics), under a humidified at-mosphere containing 5% CO2 at 37°C. L-�-Palmytoyl-lysophosphatidylcholine (16:0) was purchased fromSigma (St Louis, MO). Unless otherwise noted, all otherchemicals were used as obtained from the manufacturer.

1-[2-(5-Carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]-2-(2�-amino-5�-methyl phenoxy)-ethane-N,N,N�N�-tetraacetic acid pentaacetoxymethyl ester (Fura-2-AM),Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnT-BAP), and PD98059 were from Calbiochem (San Diego,CA). U0126 was from Promega (Madison, WI). MitoSOXand 2�,7�-dichlorodihydrofluorescein diacetate (DCFH-DA) were from Invitrogen (Carlsbad, CA). Phospho-specific antibodies against ERK, JNK, and pan-ERK wereobtained from BD Biosciences (San Jose, CA). Anti-HO-1antibody and anti-CuZnSOD antibody were from Stress-Gen (Victoria, BC, Canada) and Santa Cruz Biotechnol-ogy (Santa Cruz, CA), respectively. The monoclonal anti-phosphotyrosine antibody 4G10 was provided by Dr.Tanaka at the University of Tokyo. All other chemicalswere of analytical grade.

Western Blotting Analysis of the Activation Stateof MAPK and the Levels of HO-1

HUVECs were cultured in serum-containing medium until50 to 70% confluency and then made quiescent by incu-bating with low-serum medium (0.5% fetal bovine serumin endothelial cell basal medium) for 14 to 16 hours. Next,the quiescent cells were incubated with lysoPC (0 to 40�mol/L) for 0 to 90 minutes or 6 hours for the induction ofHO-1. The inhibitors of mitochondrial respiratory chain,iron and calcium chelators, or lipophilic antioxidants,mito-E or diphenyleneiodonium (DPI), were applied for 15to 60 minutes before exposure of the cells to lysoPC(unless otherwise indicated). Cells were then washedtwice with phosphate-buffered saline and lysed (50mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 0.5% [v/v] TritonX-100, 1 mmol/L ethylenediamine tetraacetic acid, 1

1738 Watanabe et alAJP May 2006, Vol. 168, No. 5

mmol/L Na3VO4, 50 mmol/L NaF, 0.5 mmol/L phenyl-methylsulfonyl fluoride, 2 �g/ml leupeptin, 2 �g/ml pep-statin A, and 2 �g/ml aprotinin). The cell lysates werecollected in centrifuge tubes, vigorously vortexed, andcentrifuged at 10,000 � g for 15 minutes at 4°C. Proteinconcentration was determined by Bradford reagent (Bio-Rad, Hercules, CA) with bovine serum albumin (BSA) asa standard. Samples were mixed with Laemmli samplebuffer and boiled for 5 minutes. Equal amount of proteinswere resolved by 10% sodium dodecyl sulfate-polyacryl-amide gel electrophoresis and transferred onto polyvi-nylidene difluoride membranes (Immobilon P; Millipore,Billerica, MA). The membranes were probed with respec-tive anti-phospho-MAPK antibody, anti-phosphotyrosineantibody, or an anti-HO-1 antibody, followed by detectionwith horseradish peroxidase-conjugated anti-mouse orgoat anti-rabbit IgG followed by visualization by en-hanced chemiluminescence (ECL plus; Amersham Bio-sciences, Little Chalfont, Buckinghamshire, UK). Proteinbands were quantified by densitometry, and each exper-iment was performed three or more times.

Measurement of Cellular ROS

Quiescent cells in four-well chambered coverslip (Nalge,Naperville, IL) were incubated with 2�,7�-dichlorodihy-drofluorescein diacetate (DCFH-DA) (20 �mol/L) for 60 min-utes followed by treatment with various concentrations oflysoPC for 30 minutes at 37°C. Cells were also incubatedwith MitoTracker Deep Red 633 (0.5 �mol/L) for 15 minutes.To examine the effects of iron or calcium chelators or mito-chondrial complex inhibitors, cells were pre-incubated withN,N�-bis-(2-hydroxybenzyl)ethylenediamine-N,N�-diaceticacid (HBED) for 60 minutes or 1,2-bis(2-aminophenoxy)-ethane-N,N,N�,N�-tetraacetic acid (BAPTA), ethylene gly-col-bis(2-aminoethylether)-N,N,N�,N�-tetraacetic acid(EGTA), ruthenium red (RuRed) or rotenone, thenoyltri-fluoroacetone (TTFA), or DPI for 30 minutes before ap-plying lysoPC. Subsequently, cells were washed twicewith culture medium, and images were acquired fromthree or more randomly chosen fields using an invertedepifluorescence microscope (IX70; Olympus, Tokyo, Ja-pan). The levels of 2�,7�-dichlorofluorescein (DCF) fluo-rescence were quantitated using SIMPLEPCI software(Compix, Cranberry Township, PA). The subcellularsource of ROS generation was assessed by single bidi-rectional scans of live cells using a Leica DMIRBE in-verted epifluorescence/Nomarski microscope with LeicaTCS NT Laser Confocal optics. The levels of mitochon-drial superoxide in HUVECs induced by lysoPC were alsomeasured using the mitoSOX probe. Briefly, cells weregrown in a 4-well chambered coverslip until 70 to 80%confluent. Cells were then loaded with the mitoSOX probe(5 �mol/L) for 15 minutes followed by treatment with orwithout lysoPC (5 �mol/L) or rotenone (5 �mol/L) for 60minutes at 37°C. Images were merged and processedusing IPLab Spectrum and Adobe Photoshop (AdobeSystem).

Ca2� Assay

Subconfluent HUVECs were loaded with 5 �mol/L Fura-2-AM for 30 minutes at 37°C in Ringer buffer (150 mmol/LNaCl, 4 mmol/L KCl, 2 mmol/L CaCl2, 1 mmol/L MgCl2, 5.6mmol/L glucose, and 5 mmol/L HEPES-NaOH, pH 7.4) con-taining 0.1% BSA, and cells were harvested by trypsiniza-tion. The cell density of the suspension was adjusted to 1 �106/ml in Ringer buffer without BSA. Changes in the fluo-rescence emission were monitored on a spectrophotometer(CAF-100; Jasco, Tokyo, Japan) at 37°C with excitationwavelengths at 340 and 380 nm and an emission wave-length at 500 nm. Intracellular Ca2� concentration was cal-culated according to the following equation

�Ca2�]i � Kd � ([F � Fmin]/[Fmax � F]) � (Sf/Sb)

where Kd is 224 nmol/L, F is the ratio of the fluorescenceemission at 340 nm/fluorescence emission at 380 nm,and Fmax and Fmin are obtained in the presence of 0.5%Triton X-100 and subsequently 37.5 mmol/L EGTA, re-spectively, at the end of each assay. Sb and Sf areemission values at 380 nm corresponding to Fmax andFmin, respectively. In assays with EGTA, coefficients ob-tained without EGTA were used for calculation.

Small Interfering RNA (siRNA) Transfection

The siRNA double-strand targeting p22phox DNA se-quence 5�-AAGTACATGACCGCCGTGGTG-3� (211–231,GenBank accession number BT006861; sense, 5�-GUA-CAUGACCGCCGUGGUGdTdT-3�; antisense, 5�-CACCA-CGGCGGUCAUGUACdTdT-3�) and a control nonsilencingdouble-strand targeting DNA sequence 5�-AATTCTC-CGAACGTGTCACGT-3� were purchased from QIAGEN(Dusseldorf, Germany) Cells grown in 60-mm plates around50% confluency were transfected with either nonsilencingRNA or siRNA against p22phox using Lipofectamine 2000(Invitrogen). Briefly, siRNA (240 pmol) and Lipofectamine2000 (12 �l) were mixed in OPTI-MEM (750 �l) to allowcomplex formation for 20 minutes at room temperature.Next, the complex was added dropwise into a HUVECculture (3 ml of antibiotic-free serum containing medium).The medium was changed 8 hours after transfection, and allexperiments were performed between 48 and 72 hoursafter transfection. p22phox could be completely sup-pressed at the protein level in the siRNA-transfected cellsduring this treatment.

Results

Induction of Phosphorylation of ERK, JNK, andTyrosine Residues in High Molecular Proteins

Previous studies have shown that MAPK pathways areactivated by lysoPC in endothelial cells,24 and in the caseof smooth muscle cells, it is dependent on ROS produc-tion.43 In the first series of experiments lysoPC-depen-dent phosphorylation of ERK and JNK in endothelial cellswas confirmed. Cells were incubated for a period of 14 to

Mitochondrial ROS Activate MAPKs 1739AJP May 2006, Vol. 168, No. 5

16 hours under conditions of 0.5% serum to achieve aquiescent state before the addition of lysoPC for therange of concentrations and times shown (Figure 1). Asignificant increase in pERK1/2 phosphorylation was ev-ident at a concentration of lysoPC of 10 �mol/L andreached a maximum at 40 �mol/L lysoPC over a 15- to30-minute time period (Figure 1A). It is evident from Fig-ure 1B that this transient ERK1/2 and JNK phosphoryla-tion occurs over a similar time course and concentration.In contrast to ERK and JNK phosphorylation, lysoPCcaused robust and prolonged tyrosine phosphorylation ofhigh-molecular weight proteins (Figure 1B).

LysoPC Induces Ca2� Influx

Possible mechanisms for lysoPC-induced ERK and JNKactivation may involve the reported elevation of intracellularcalcium that occurs on exposure of cells to this lipid.21,22

Cells were loaded with Fura 2-AM (5 �mol/L) and harvestedby trypsinization, and the suspensions were exposed tolysoPC. Exposure of HUVECs to lysoPC (20 to 30 �mol/L)provoked robust elevations of intracellular Ca2�, which per-sisted for at least 5 minutes (Figure 2). To examine whetherthe lysoPC-dependent increase in Ca2� was derived fromintracellular stores or an extracellular pool, cells were stim-ulated by lysoPC in the presence of EGTA. Under theseconditions, the lysoPC-induced increase in intracellularCa2� level was significantly decreased, suggesting that theCa2� is derived from an extracellular source (Figure 2).

Ca2� Influx by LysoPC Plays a Key Role in theActivation of the ERK1/2 Pathway

To determine whether lysoPC-induced ERK1/2 activationwas associated with Ca2� mobilization, the effects onERK1/2 phosphorylation of the Ca2� chelators and theinhibitor of the mitochondrial Ca2� uniporter (RuRed)were assessed (Figure 3). The cytosolic Ca2� chelator1,2-bis(2-aminophenoxy)-ethane-N,N,N�,N�-tetraaceticacid acetoxymethyl ester (BAPTA-AM; 20 �mol/L), extra-cellular Ca2� chelator EGTA (2 mmol/L), and RuRed(10–50 �mol/L) significantly inhibited ERK1/2 phosphor-

ylation (60 to 70%) (Figure 3, A–D). Simultaneous addi-tion of equimolar Ca2� abolished the inhibitory effect ofEGTA (Figure 3C). Notably, these treatments had littleeffect on lysoPC-dependent JNK phosphorylation, sug-gesting that the activation of this signaling cascade pro-ceeds through a different mechanism. Taken together,these results suggest that extracellular Ca2� influx in-duced by lysoPC exposure leads to ERK1/2, but not JNK,activation in endothelial cells through a mechanism in-volving mitochondrial Ca2� uptake.

Figure 1. LysoPC increases phosphorylation of ERK1/2, JNK, and tyrosineresidues in high-molecular weight proteins. Representative immunoblots oftotal ERK, phospho-ERK1/2, phospho-JNK, and tyrosine-phosphorylatedproteins in HUVECs induced by increasing concentrations of lysoPC (0 to 40�mol/L) for 15 minutes (A) or the kinetics of their appearance (0 to 90minutes) measured at 30 �mol/L (B).

Figure 2. Ca2� mobilization by lysoPC in HUVECs. Representative trace ofcytosolic levels of Ca2� evoked by lysoPC treatment. HUVECs were loadedwith Fura-2 (5 �mol/L) for 30 minutes, harvested by trypsinization, and thenstimulated with indicated concentrations of lysoPC (10 or 30 �mol/L) at 37°C.A significant decrease in the level of lysoPC-induced Ca2� mobilization wasevident in the presence of EGTA (2.5 mmol/L; added 1 minute beforetreatment with 20 �mol/L lysoPC). Similar results were obtained from threeindependent experiments.

Figure 3. Effects of Ca2� chelators on lysoPC-induced phosphorylation ofERK and JNK. HUVECs were pre-incubated with RuRed (50 �mol/L),BAPTA-AM (BAPTA, 20 �mol/L) for 30 minutes (A) or with EGTA (2mmol/L) in the presence or absence of CaCl2 (2 mmol/L) for 15 minutes (C).Next, cells were stimulated with lysoPC (30 �mol/L) for 15 minutes. Supple-mentation of medium with CaCl2 reversed the inhibitory effect of EGTA (C).The histograms in B and D show the intensity of each band for p-ERK in Aand C, respectively, obtained by densitometric analysis wherein lysoPC-dependent increase of p-ERK in the control group was set at 100%. *P � 0.05compared with lysoPC treatment alone or lysoPC plus dimethylsulfoxide(DMSO); #P � 0.05 compared with EGTA alone (mean � SEM, n � 3).


Ca2� Mobilization Regulates Mitochondrial ROSGeneration

Previously, induction of DCF fluorescence in cells ex-posed to lysoPC has been reported,44 although themechanism remains unclear. We hypothesized that mito-chondrial Ca2� uptake could lead to an increase in ROSformation in the organelle. To test this hypothesis, weused DCFH-DA (20 �mol/L) to measure ROS formation inthe cell. This compound is converted to DCFH inside thecell, and on exposure to either hydrogen peroxide orperoxynitrite, it is oxidized to the fluorescent product DCFthrough mechanisms involving transition metal ions orcellular peroxidases as catalysts.45 In the first series ofexperiments, cells were exposed to lysoPC (20 �mol/L)for 30 minutes, then the medium was changed, and im-ages were acquired. In agreement with previous studies,lysoPC treatment resulted in increased DCF fluores-cence, and this was significantly (40%) inhibited byRuRed (50 �mol/L) (Figure 4). To further examine therelationship between Ca2� influx and ROS/reactive nitro-gen species generation by lysoPC, the effects of theCa2� chelators BAPTA-AM (20 �mol/L) and EGTA (2mmol/L) on DCF fluorescence were measured. Treatmentof cells with either of these compounds resulted in nearlya 40 to 60% decrease in the lysoPC-dependent DCFfluorescence (Figure 4). These data suggest that the

mitochondrial lysoPC-dependent ROS generation is as-sociated with an increase in mitochondrial Ca2� uptake.

As an alternative approach to measuring ROS gener-ation induced by lysoPC, the mitoSOX Red probe wasused. This compound is a mitochondrial targeted form ofdihydroethidium. Exposure of cells to lysoPC or rotenoneincreased mitoSOX fluorescence six- and fivefold, re-spectively (Figure 4, C and D). These data support thefindings with DCF that lysoPC can initially induce mito-chondrial superoxide generation.

As a further test for the mitochondrial origin of lysoPC-dependent ROS formation, HUVECs were loaded withDCFH-DA (5 �mol/L) followed by treatment with lysoPC(5 �mol/L) for 30 to 60 minutes with the mitochondrialspecific marker MitoTracker (500 nmol/L) applied for 15minutes before imaging. After washing with serum-freemedium, images were acquired with single bidirectionalscanning. As shown in Figure 5, lysoPC-dependent DCFfluorescence colocalized with MitoTracker, consistentwith a significant contribution of the organelle to lysoPC-dependent ROS formation. To gain some mechanisticinsight into the site of mitochondrial ROS production bylysoPC, DCFH oxidation was also examined in the pres-ence of inhibitors of the mitochondrial electron transportchain. In isolated mitochondria, inhibition of complex I byrotenone is known to increase ROS production at this sitebut would inhibit ROS formation arising from reversed

Figure 4. Effects of Ca2� chelators on lysoPC-induced ROS generation in HUVECs. A: HUVECs were loaded with DCFH-DA (20 �mol/L) in the presence orabsence of RuRed (50 �mol/L), EGTA (2 mmol/L), or BAPTA-AM (20 �mol/L) for 30 minutes. Then, cells were further incubated with or without lysoPC (20�mol/L) for 30 minutes. Cells were washed with culture medium, and live cells were imaged on an inverted fluorescence microscope. C: HUVECs were loadedwith mitoSOX (5 �mol/L) followed by the treatment with lysoPC (5 �mol/L) or rotenone (5 �mol/L) for 60 minutes, and images of live cells were acquired. Band D: Average of DCF or mitoSOX fluorescence of cells treated as indicated in A or C was obtained from three randomly chosen fields and is expressed as pixelintensity/cell (mean � SEM, n 3; *P � 0.01 compared with lysoPC treatment alone.


electron transport through complex I.46 Rotenone treat-ment alone resulted in increased DCF fluorescence (Fig-ure 6) but did not affect the ability of lysoPC to furtherinduce DCF fluorescence. In addition to rotenone, weused the flavoprotein inhibitor DPI, which has beenshown to inhibit mitochondrial complexes at low concen-trations in endothelial cells.47 DPI substantially inhibitedDCF fluorescence induced by lysoPC (Figure 6). In arecent study we have shown that the complex II inhibitorTTFA does not inhibit ROS formation by the extracellularsource of hydrogen peroxide, glucose oxidase.48 In ad-dition, the complex II inhibitor TTFA substantially inhibitedlysoPC-induced ROS generation.

Activation of the ERK1/2 Pathway by LysoPC IsChanneled through Mitochondria

To test whether NOXs participate in the regulation ofthe lysoPC-dependent ERK activation in HUVECs, ansiRNA directed against p22phox was used. Previousstudies have demonstrated that the abrogation of thefunction of NOX family of enzymes by transfection witha dominant-negative p47phox decreased lysoPC-in-duced ERK1/2 activation in vascular smooth muscle-like cells.43 The Western blotting analysis revealedessentially complete elimination of the level of p22phoxsubunit (22 kd) in HUVECs transiently transfected withp22phox siRNA (Figure 7A), whereas the nonsilencing

siRNA had no effect. Next, the level of lysoPC-inducedERK1/2 phosphorylation was determined. As shown inFigure 7B, phosphorylation of ERK1/2 induced by ly-soPC was not decreased in the p22phox-depletedcells. Thus, involvement of the NOX enzymes in ly-soPC-induced ERK1/2 activation in HUVECs seemsunlikely. In a further series of experiments, a role ofendothelial nitric oxide synthase was examined by ap-plication of the inhibitor L-NAME (100 �mol/L) to themedium before activation by lysoPC. The NOS inhibitorshowed no effect on either ROS formation or activationof the MAPK (data not shown). Moreover, we couldobserve no inhibitory effect on lysoPC-induced ERKactivation in cells in which endothelial nitric-oxide syn-thase protein had been diminished by an siRNA di-rected against the enzyme (data not shown).

To investigate whether lysoPC-induced ERK1/2 activa-tion depends on mitochondrial function, the inhibitors ofmitochondrial oxidative phosphorylation were used. Inhi-bition of complex I or complex III by rotenone (2 �mol/L)or antimycin A (10 �mol/L), respectively, or dissipation ofthe proton gradient by the uncoupler carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP; 10 �mol/L)significantly attenuated ERK1/2 phosphorylation inducedby lysoPC (30 �mol/L) (Figure 8). In contrast, the com-plex II inhibitor TTFA (50 �mol/L) or the complex V (ATPsynthase) inhibitor oligomycin (10 �g/ml) has no signifi-cant effect on ERK1/2 phosphorylation. Western blot

Figure 5. LysoPC induces ROS generation in mitochondria. Cells were loaded with DCFH-DA (5 �mol/L) for 60 minutes followed by incubation with or withoutLysoPC (5 �mol/L) for 30 to 60 minutes. Cells were also incubated with MitoTracker (0.5 �mol/L) for 15 minutes before imaging. Images were acquired on aconfocal scanning microscope (see Materials and Methods). The exposure of HUVECs to lysoPC increased the intensity of DCF fluorescence that colocalized withthe mitochondrial marker.


analysis also demonstrated that none of the inhibitorssignificantly attenuated the lysoPC-dependent increasein JNK phosphorylation. These data suggest that theactivity of complex I and complex III in the electron trans-port chain and the mitochondrial inner membrane poten-tial (��) contribute to lysoPC-dependent ERK1/2phosphorylation.

It is possible that mitochondrial inhibitors prevent ERKactivation by an indirect mechanism, for example by ATPdepletion.49 To test for this, the effects of the FCCP (10�mol/L), RuRed (50 �mol/L), or rotenone on phorbol 12-myristate 13-acetate (PMA)-induced ERK activation weredetermined. No inhibitory effect could be observed withFCCP or RuRed in PMA-induced phosphorylation ofERK1/2 (Figure 9). The treatment with rotenone slightlyattenuated ERK1/2 phosphorylation by PMA, but the ex-tent was far less than that observed under lysoPC stim-ulation. Thus, it is unlikely that changes in the levels ofATP induced by the electron transport inhibitors or un-

Figure 6. Effects of inhibitors of mitochondrial oxidative phosphorylation onlysoPC-induced ROS generation. Cells were pre-incubated with rotenone (10�mol/L), TTFA (10 �mol/L), or DPI (20 �mol/L) for 30 minutes followed bytreatment with lysoPC (20 �mol/L) for additional 30 minutes. The imageswere acquired (A), and DCF fluorescence was averaged (B) as describedabove (Figure 4). The rotenone treatment alone increased DCF fluorescence,which was additive to DCF fluorescence obtained after treatment with ly-soPC. *P � 0.05 compared with lysoPC treatment alone; #P � 0.05 withrotenone compared with control cells (mean � SEM, n � 3).

Figure 7. Effect of siRNA-mediated knockdown of p22phox on the phos-phorylation of ERK1/2 and JNK by lysoPC. HUVECs were transfected witheither nonsilencing control siRNA (n.s.RNA) or p22phox siRNA (detailed inMaterials and Methods). At 48 hours after transfection, cells were serum-starved for 16 hours and stimulated with lysoPC (30 �mol/L) for 15 minutes.A: Western blot analysis shows almost complete disappearance of p22phoxin cells treated with p22phox siRNA compared with control or n.s.RNA-treated cells. The cell lysates of naı̈ve and retinoic acid-treated HL60 cellswere used as an indicator of the p22phox relative electrophoretic mobility. B:Effects of silencing of p22phox on lysoPC-induced phosphorylation ofERK1/2 or JNK. The Western blots shown are representative of the threeindependent experiments.

Figure 8. Effects of inhibitors of oxidative phosphorylation on lysoPC-in-duced MAPK activation. Representative immunoblots of phospho-ERK1/2,phospho-JNK and total ERK (A) and quantitative data obtained by densito-metric analysis (B). A: Cells were pre-incubated with DMSO (0.1%), rotenone(2 �mol/L), TTFA (50 �mol/L), antimycin A (10 �mol/L), FCCP (10 �mol/L),or oligomycin (10 ng/ml) for 30 minutes and then exposed to lysoPC (30�mol/L) for 15 minutes. *P � 0.05 compared with lysoPC treatment alone(mean � SEM, n � 3).


coupler were sufficient to suppress the activation of ERKinduced by lysoPC.

Activation of the ERK Pathway by LysoPC IsROS-Dependent

Involvement of ROS in ERK activation was examined. Thelevel of ERK1/2 phosphorylation was almost completelyinhibited by DPI (20 �mol/L) and the membrane-perme-able SOD mimetic MnTBAP (250 �mol/L) (Figure 10, Aand B). However, the effects of those compounds on JNKactivation and tyrosine phosphorylation of high-molecularweight proteins were not significant (Figure 10). Ebselen,a glutathione peroxidase mimetic, also preferentially at-tenuated the lysoPC-induced phosphorylation of ERK1/2.Furthermore, as shown in Figure 10C, the lipid peroxylradical scavengers �-tocopherol, �-tocotrienol, and asynthetic radical scavenging antioxidant BO65350 couldalso mitigate ERK1/2 without affecting JNK phosphoryla-tion. Thus, the activation of the ERK pathway is potentiallyassociated with ROS production. Taking into account thespecificity of the lipid radical scavengers and their mini-mal ability to scavenge both superoxide and hydrogenperoxide, a role for the formation of lipid peroxides in ERKactivation is suggested.

Targeting Mitochondria with Vitamin E Alters theLevel of LysoPC-Dependent ROS Formationand ERK1/2 Phosphorylation

The results thus far suggest that activation of the ERKpathway by lysoPC involves the induction of mitochon-drial ROS production, leading to intraorganelle lipid per-oxidation. To further evaluate this possibility, cells werepretreated with mitochondrially targeted tocopherol de-rivative 2-(3,4-dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-1-benzopyran-2-yl)ethyl]triphenylphosphonium bromide(MitoVitE). This compound can accumulate in mitochon-dria according to the mitochondrial inner membranepotential (��).51 Figure 11A demonstrates that level oflysoPC-induced DCF fluorescence was significantly in-hibited in cells pretreated with MitoVitE (500 nmol/L) for60 minutes. Under the same conditions, MitoVitE alsoinhibited lysoPC-dependent ERK activation.

Recently, it has been shown that DCF fluorescenceinduced by exogenous hydrogen peroxide can be inhib-ited by addition of an iron chelator such as HBED.52 Inthis study, lysoPC-induced DCF fluorescence was par-tially (30%) decreased in cells pre-incubated withHBED at 100 �mol/L (Figure 11A), suggesting a role foriron in lysoPC-induced DCF fluorescence. Additionally,pretreatment with HBED also decreased the level ofERK1/2 phosphorylation (Figure 11C). Taken together,these data suggest that generation of lipid peroxides,which results from mitochondrial ROS production andparticipation of iron, could preferentially regulate ERKactivation by lysoPC in HUVECs.

ERK Activation by LysoPC Is Coupled withInduction of Heme Oxygenase 1

The regulation of pro-atherogenic genes in response tolysoPC is well documented, but these proteins are notknown to have mitochondrial component in their regula-tion.9,24 In contrast, the antioxidant enzyme HO-1 hasrecently been shown to be regulated, in part, by ROS,and in a separate series of studies, ERK has also beenimplicated.5,35,53 These data suggest that an adaptation

Figure 10. Effects of antioxidant agents on lysoPC-induced ERK or JNKphosphorylation. In A, cells were pre-incubated with or without DMSO(0.1%), DPI (20 �mol/L), MnTBAP (250 �mol/L), or ebselen (20 �mol/L) for30 minutes and then treated with lysoPC (30 �mol/L) for 15 minutes. B:Quantitative data were obtained from three or more of independent exper-iments. *P � 0.05 compared with lysoPC treatment alone. C: Cells werepre-incubated with the lipophilic antioxidants (each 50 �mol/L) for 16 hoursfollowed by treatment with lysoPC (30 �mol/L) for 0 to 20 minutes.

Figure 9. Effects of selected inhibitors of oxidative phosphorylation onPMA-induced phosphorylation of ERK1/2. Representative immunoblots (A)and densitometric analysis (B) of phospho-ERK1/2 and total ERK. Cells wereincubated with rotenone (2 �mol/L), RuRed (50 �mol/L), or FCCP (10�mol/L) and then treated with PMA (1 �mol/L) in parallel with exposure tolysoPC (30 �mol/L) for 15 minutes. *P � 0.05 compared with lysoPC treat-ment alone (mean � SEM, n � 3).


to the stress of lysoPC exposure that leads to the activa-tion of ERK could result in increased levels of HO-1. Insupport of this hypothesis, we found that lysoPC was apotent inducer of HO-1 protein expression in HUVECs,and this effect could be attenuated by the inhibitors ofMEK (PD98059 or U0126) (Figure 12).

Discussion

Recent evidence suggests that mitochondrially producedROS may contribute to the regulation of various physiolog-ical processes including metabolic rate,31,32 tumor necrosisfactor-�,54 leptin,33 endothelin cell signaling,55 and me-chanical stress.34,56 A number of exogenous stimuli impor-tant in the pathophysiology of cardiovascular disease, suchas lipid peroxidation products, have been shown to induceROS in cells and to induce cell signaling, but a role for themitochondrion has not been investigated.57,58 For example,involvement of ROS in a range of lysoPC-induced cellularresponses, from activation of the MAPKs and tyrosine ki-nases to the expression of several genes, has been sug-gested.43,44,59 In this study, we confirm that lysoPC inducesthe transient activation of MAPKs in endothelial cells underconditions that do not induce cytotoxicity (Figure 1). Onesource of the ROS formed in response to lysoPC in HUVECsand vascular smooth muscle cells is thought to be theNADPH oxidases, which are members of the NOX familyof proteins.43,44 Using an siRNA approach, it was shownthat the p22phox subunit of the NOX enzymes doesnot significantly contribute to ERK activation in HUVECs(Figure 7).

Figure 11. Effects of MitoVitE or HBED on lysoPC-induced DCFH oxidation and phosphorylation of ERK1/2. The level of DCF fluorescence was demonstratedin cells pre-incubated with MitoVitE (500 nmol/L) and HBED (500 nmol/L) for 60 minutes followed by treatment with lysoPC (20 �mol/L) for an additional 30minutes. The representative images were acquired with epifluorescence microscopy (A), and quantitated analysis of DCF fluorescence (B). C: In parallelexperiments, the level in ERK1/2 phosphorylation was determined, and results of densitometric analysis are shown. *P � 0.05 compared with lysoPC treatmentalone (mean � SEM, n � 3).

Figure 12. Effects of the inhibitors of the ERK pathway on HO-1 induction bylysoPC. Serum-starved cells were pre-incubated with PD98059 (30 �mol/L)or U0126 (20 �mol/L) for 30 minutes followed by treatment with lysoPC (25�mol/L) for additional 6 hours. A: A representative blot; B: quantitative dataobtained from five independent experiments. *P � 0.05 compared withlysoPC treatment alone.


Although it is not clear at present whether lysoPC initiallyacts at the cell surface through binding to its proposedreceptors,10–14 or other mechanisms such as membraneperturbation,60 lysoPC brings about a robust influx of extra-cellular Ca2�. In this study, a role for Ca2� influx in lysoPC-dependent ERK activation is supported by the finding thatEGTA, BAPTA, and RuRed all result in a substantial inhibi-tion of ERK activation (Figure 3). This response is not due toa general dysfunction in cell signaling because JNK is notaffected in a similar manner. Taken together, these datasuggest that Ca2� enters the cell via the plasma membraneand is taken up by the mitochondrion, which then leads toERK activation. The fact that ERK can be activated by ROSand mitochondria are capable of generating ROS suggeststhat mitochondria may be a link between Ca2� influx andERK activation. When mitochondria accumulate cytosolicCa2�, the Ca2�-dependent matrix dehydrogenases are ac-tivated, increasing NADH levels and the potential to formROS.41,61,62 For the detection of intracellular ROS, weelected to use DCF-dependent fluorescence, which wehave recently used to demonstrate that oxidized low-den-sity lipoprotein is capable of eliciting mitochondrial ROSformation in endothelial cells.48 We next determined that theCa2� influx inhibitors that suppressed ERK activation alsosuppressed lysoPC-dependent DCF fluorescence (Figure4). In addition we determined by confocal microscopy thatthis DCF formation was localized to the mitochondrion (Fig-ure 5). Measuring ROS formation using DCF has a numberof drawbacks that have been well recognized in the litera-ture (reviewed in Ref. 45). Therefore, we confirmed mito-chondrial ROS formation using MitoSOX, which detects su-peroxide through different mechanisms than DCF.

To test for the respiratory chain as a potential source ofthe mitochondrial ROS, inhibitors were used. First, we usedthe complex I inhibitor rotenone, which increased DCF flu-orescence alone but did not prevent a further increase inresponse to lysoPC (Figure 6). In contrast, TTFA, which is acomplex II inhibitor, decreased DCF fluorescence by ap-proximately the same extent as the Ca2� chelators, sug-gesting a role for complex II in lysoPC-dependent ROSformation. DPI, a flavoprotein inhibitor, also inhibited ly-soPC-dependent ROS formation, but these results cannotbe used to confirm the role of a specific protein becauseDPI acts via pleiotropic mechanisms.

ERK activation by lysoPC was blocked by rotenone, thecomplex III inhibitor antimycin A, and the protonophoreFCCP. Unexpectedly, TTFA had no significant effect onERK activation (Figure 8). Thus, lysoPC-dependent ERKactivation requires a contributory activity from both com-plexes I and III and an inner-membrane impermeable toprotons. To explain the apparently anomalous effect ofTTFA, we considered the relationship between mitochon-drial ROS formation under normal conditions and in thepresence of respiratory chain inhibitors. In the normal func-tioning of the mitochondrion, the production of ROS in-creases as a function of ��,39 but this relationship is de-coupled in the presence of inhibitors such as rotenone,TTFA, and antimycin A.63,64 For this reason, we cannot drawconclusions regarding the link between lysoPC-induced mi-tochondrial ROS formation and ERK activation on the basisof these data. However, it is possible to address the role of

the mitochondrion in both ROS formation and ERK activa-tion with the data using calcium influx inhibitors. BlockingCa2� uptake into the mitochondrion inhibits both lysoPC-dependent ERK activation and ROS formation, suggestingthat these two events are mechanistically connected.

In the next series of experiments, we used selectedantioxidants to determine the potential mechanismthrough which mitochondrial ROS formation contributesto ERK activation. Because it is difficult to rationalize howhydrogen peroxide, which is freely diffusible, can exertdifferential effects when formed at different sites in therespiratory chain, we propose the formation of a second-ary mediator. A logical candidate would be lipid peroxi-dation, which can generate a family of molecules, suchas 4-hydroxy-2-nonenal, that can initiate signal transduc-tion events including MAPK activation.65

In support of this concept, the lipid peroxyl radical scav-engers tocopherol, tocotrienol, and BO653 and the mito-chondrially targeted �-tocopherol analog MitoVitE inhibitedERK activation to the same extent as the Ca2� chelators.Because the iron chelator HBED inhibited ERK activation,then it is possible that the hydrogen peroxide and ironderived from the electron transport chain proteins initiatelipid peroxidation (Figure 11). Consistent with this interpre-tation, both ebselen and MnTBAP have also been shown toinhibit lipid peroxidation66,67 and lysoPC-dependent ERKactivation (Figure 10). Overall, these data suggest that mi-tochondria transduce the hydrogen peroxide signal to alipid-derived signaling molecule.

It is also now becoming clear that in addition to thecytotoxic pathways induced by lipid oxidation products,protective antioxidant pathways are also activated.4–6,53

These include the transcriptional regulation of antioxidantdefenses such as glutathione and the antioxidant enzymeHO-1.4–6,53 This is important, because it has been demon-strated that HO-1 overexpression by an adenovirus-medi-ated gene transfer can attenuate atherosclerotic lesion for-

Figure 13. Potential signaling mechanism for ERK activation by lysoPC. Ca2�

influx from the extracellular stores induced by lysoPC exposure leads to uptakeof the ion in mitochondria where it causes ROS production, possibly at multiplesites (respiratory chain complexes and TCA cycle dehydrogenases). Lipid per-oxides generated as a result of membrane oxidation activate the ERK MAPKpathway. Impairment of electron transport with rotenone or antimycin A de-creases mitochondrial Ca2� uptake and thereby attenuates ERK activation.


mation in ApoE knockout mice,68 whereas genetic ablationof HO-1 exacerbates lesion formation in the same animalmodel.69 Interestingly, mitochondrial ROS has been shownto play a regulatory role in controlling levels of HO-1.35 Inthese experiments, mitochondrially generated hydrogenperoxide, but not ERK activation, was shown to be essentialfor HO-1 up-regulation. However, other studies have shownthat ERK activation can play an important regulatory role inHO-1 expression in response to other oxidized lipids.5,53

These findings led us to test the potential of lysoPC toinduce mitochondrial ROS formation in endothelial cells andexamine the possible link to ERK activation and HO-1 ex-pression. We found that lysoPC induced the expression ofHO-1 in an ERK-dependent manner, suggesting thatmitochondrial-dependent activation of ERK may be an im-portant cytoprotective pathway in vascular endothelial cells(Figure 12).

In conclusion, the present study revealed that the ly-soPC-dependent activation of ERK has a significant contri-bution derived from Ca2�-dependent mitochondrial ROSformation, which can be readily detected on exposure ofendothelial cells to the lipid (Figure 13). The mechanisms oftransduction of the hydrogen peroxide signal appear toinvolve the formation of a lipid peroxidation product withinthe mitochondrion. The mitochondrial ROS-dependent ac-tivation of ERK in turn plays a key role in the induction of theanti-atherogenic enzyme HO-1.

Acknowledgments

We thank Dr. Kumiko Saeki of International Medical Cen-ter of Japan for providing HL60 cells and Dr. Keiko Ho-riuchi for technical supports for siRNA experiments.

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