AJP - Heart Information on EB 2010
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 290: H1777-H1787, 2006. First published December 30, 2005; doi:10.1152/ajpheart.01087.2005
0363-6135/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/5/H1777    most recent
01087.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (35)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Landar, A.
Right arrow Articles by Darley-Usmar, V. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Landar, A.
Right arrow Articles by Darley-Usmar, V. M.

Interaction of electrophilic lipid oxidation products with mitochondria in endothelial cells and formation of reactive oxygen species

Aimee Landar,1,2 Jaroslaw W. Zmijewski,1,2 Dale A. Dickinson,2,3 Claire Le Goffe,1 Michelle S. Johnson,1 Ginger L. Milne,4 Giuseppe Zanoni,6 Giovanni Vidari,6 Jason D. Morrow,4,5 and Victor M. Darley-Usmar1,2

1Department of Pathology, 2Center For Free Radical Biology, 3Department of Environmental Health Sciences, University of Alabama at Birmingham, Birmingham, Alabama; Departments of 4Pharmacology and 5Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee; and 6Department of Organic Chemistry, University of Pavia, Pavia, Italy

Submitted 13 October 2005 ; accepted in final form 21 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Electrophilic lipids, such as 4-hydroxynonenal (HNE), and the cyclopentenones 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2) and 15-J2-isoprostane induce both reactive oxygen species (ROS) formation and cellular antioxidant defenses, such as heme oxygenase-1 (HO-1) and glutathione (GSH). When we compared the ability of these distinct electrophiles to stimulate GSH and HO-1 production, the cyclopentenone electrophiles were somewhat more potent than HNE. Over the concentration range required to observe equivalent induction of GSH, dichlorofluorescein fluorescence was used to determine both the location and amounts of electrophilic lipid-dependent ROS formation in endothelial cells. The origin of the ROS on exposure to these compounds was largely mitochondrial. To investigate the possibility that the increased ROS formation was due to mitochondrial localization of the lipids, we prepared a novel fluorescently labeled form of the electrophilic lipid 15d-PGJ2. The lipid demonstrated strong colocalization with the mitochondria, an effect which was not observed by using a fluorescently labeled nonelectrophilic lipid. The role of mitochondria was confirmed by using cells deficient in functional mitochondria. On the basis of these data, we propose that ROS formation in endothelial cells is due to the direct interaction of these lipids with the organelle.

15-deoxy-{Delta}12,14-prostaglandin J2; isoprostane; cyclopentenone; 4-hydroxynonenal; atherosclerosis; rho0

LIPID PEROXIDATION can generate a broad range of reactive compounds that modify cell function through the activation of signaling pathways. This has been demonstrated most clearly with specific lipid oxidation products, particularly those containing an electrophilic carbon center (13, 14, 17, 18, 23, 31). The mechanisms entail the direct modification of nucleophilic protein residues by electrophilic lipids and/or the secondary formation of reactive oxygen species (ROS) in the cell (11, 19, 23, 29, 31, 38). Of particular interest are compounds formed by lipid peroxidation, such as 4-hydroxynonenal (HNE), which possess both an aldehydic function and an electrophilic carbon. In contrast, the cyclopentenones, which can be generated through both specific enzymatic pathways and nonspecific lipid peroxidation, possess only an electrophilic carbon. Various lines of evidence support the concept that both these classes of compounds are formed during the inflammatory process (27, 30, 32, 37).

With the use of biotin tagging techniques, a number of protein targets for the electrophilic cyclopentenones, such as 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2), have been identified that participate in redox cell signaling (23, 33, 38, 39). This electrophile-responsive proteome includes important signaling molecules such as ras, thioredoxin, and the regulator of key antioxidant defenses in the cell, Kelch-like erythroid cell-derived protein 1 (Keap-1) (5, 12). For example, modification of thiols in Keap-1 by reactive electrophiles leads to release of the transcription factor NF-E2-related factor 2 (Nrf2) and subsequent increase in transcription of genes under the control of the electrophile response element (23). The fact that electrophilic lipids target a number of redox-sensitive proteins suggests that these lipids induce a coordinated cellular response through several mechanisms (17). Furthermore, the overall effect of lipid electrophiles is also determined by their concentration. At low concentrations, the cyclopentenones and similar oxidized lipids present in oxidized low-density lipoprotein (oxLDL) induce the synthesis of cytoprotective antioxidant enzymes, including those that control glutathione (GSH) synthesis and heme oxygenase-1 (HO-1) (1, 13, 28). At high concentrations, lipid electrophiles can induce apoptosis through a number of mechanisms, including both the extrinsic and intrinsic pathways (10, 19, 22, 36).

Many of the biological responses induced by oxLDL are also found with purified individual lipid electrophiles (16, 23). This is of particular interest in understanding the response of vascular endothelial cells to exposure to oxidized lipids during the atherosclerotic process. In a recent study, we demonstrated that the generation of ROS and reactive nitrogen species (RNS) from the endothelial cell on exposure to oxLDL has a substantial mitochondrial contribution (42). This finding leads to the hypothesis that electrophilic lipid oxidation products may have a direct interaction with the mitochondrion. A role for the mitochondrion as a target for reactive lipid electrophiles has not been examined in a cellular context. This is important because it is now becoming evident that the organelle can transduce a number of oxidative and nitrosative signals and impact on redox cell signaling (3, 4, 7, 8, 35). For example, our recent studies have identified some members of the electrophile responsive proteome in the mitochondrion that may contribute to the cytotoxic effects of these compounds (20).

In this study, we selected for investigation the cyclopentenone electrophilic lipids 15d-PGJ2 and 15-J2-isoprostane (15-J2-IsoP) and the structurally distinct electrophilic aldehyde HNE (Fig. 1). These compounds then represent products derived from the enzymatic reactions of cyclooxygenase (15d-PGJ2) and two examples of products from nonspecific lipid peroxidation, HNE and a member of the isoprostane family (15-J2-IsoP). Evidence for the formation of these lipid oxidation products in vivo has been presented in a number of studies, although the free concentrations are not known due to their rapid metabolism and propensity to form protein adducts (9, 30, 32, 37). Here we have determined both the source of electrophile-dependent ROS and compared their ability to induce HO-1 and GSH in bovine aortic endothelial cells (BAEC). To monitor the distribution of electrophilic lipids within the cell, 15d-PGJ2 was conjugated to the fluorescent probe BODIPY (BD) and its distribution within the cell determined. We show that all the electrophilic lipids induced ROS generation in mitochondria and that BD-tagged 15d-PGJ2 localizes to the organelle.


Figure 1
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 1. Structures of compounds used in the study. A: 15-deoxy-{Delta}12,14-prostaglandin J2 (15d-PGJ2). B: BODIPY (BD)-15d-PGJ2. C: representative structure of 15-J2-isoprostanes (15-J2-IsoPs), which consist of a mixture of 4 isomers (stereochemistry is not indicated). D: hydroxynonenal (HNE). E: PGE2. F: BD-PGE2. Electrophilic carbons are depicted by asterisks.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. MitoTracker Deep Red 633 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine, hydrochloride (BODIPY FL EDA), 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA), 5-(and 6) carboxy-2',7'-dichlorofluorescein diacetate (carboxy DCF-DA), and anti-BD antibody were purchased from Molecular Probes (Eugene, OR). Glucose oxidase was obtained from Sigma (St. Louis, MO). All other reagents used were of analytical grade.

Cell culture and preparation of pseudo rho0 cells in BAEC. BAEC were prepared as described previously and used between passages 4–12 (14). To create mitochondrial DNA-depleted pseudo rho0 cells, BAEC were incubated in medium containing ethidium bromide (250 ng/ml), uridine (50 µg/ml), and sodium pyruvate (110 µg/ml) for 7–14 days (42). Pseudo rho0 cells were characterized as having decreased mitochondrial membrane potential ({Delta}{Psi}) and a decreased expression level (10–20% of control) of the mitochondrially coded subunit I of cytochrome-c oxidase (42).

Measurement of HO-1 and GSH. BAEC or rho0 cells were grown on six-well plates until confluent, followed by incubation in media containing 0.5% serum for 24 h. Cells were then treated with electrophilic lipids for 16 h at the concentrations indicated, lysed, and then resolved by SDS/PAGE, followed by Western blotting using anti-HO-1 antibody (StressGen). Total glutathione (GSH + GSSG) was determined by using the same lysate (28). Protein content was assayed by the Bradford method (protein assay kit; Bio-Rad, Hercules, CA).

Synthesis of BD-15d-PGJ2 and BD-PGE2. A BD moiety was added to 15d-PGJ2 and PGE2 via a carbodiimide-mediated condensation reaction with BODIPY FL EDA. The protocol is a modification of a previously reported method (23). Briefly, the reaction mixture consisted of 1 mg 15d-PGJ2 (Cayman Chemical), 1 mg EDC (Pierce), and 1 mg 5-(biotinamido)pentylamine BODIPY FL EDA in 97% acetonitrile. The reaction was incubated 18 h at room temperature, and the product was purified by HPLC by using a C18 Luna column (Phenomenex) with a linear gradient from 10% acetonitrile, 0.24% acetic acid to 95% acetonitrile. After extraction with chloroform, solvent was evaporated under N2 and the product reconstituted in 100% ethanol. BD tagging of the lipids was confirmed by electrospray mass spectrometry, and the concentration was measured by absorbance at 504 nm by using the extinction coefficient 76,000 M–1·cm–1.

Cytochemistry and detection of BD-15d-PGJ2 protein adducts. BAEC or rho0 cells were grown in four-well chambers (Nalge, Naperville, IL) until confluent, followed by incubation in media (0.5% serum) for 16 h. Cells were incubated with electrophile for 30 min with MitoTracker Deep Red 633 (0.5 µM) added 15 min before being imaged. The subcellular localization of BD and BD-tagged lipids was assessed by four bidirectional scans of live cells with the use of a Leica DMIRBE inverted epifluorescence/Nomarski microscope outfitted with Leica TCS NT laser confocal optics (filters absorption and emission: BD FL 505/513 nm; MitoTracker 644/665 nm). Images were merged, and colocalization efficiency of BD, BD-lipids, and MitoTracker was estimated by using SimplePCI software (Compix, Cranberry Township, PA). Digital processing of the images was also undertaken by using Adobe Photoshop (Adobe System). For the competition experiment (unlabeled 15d-PGJ2 with BD-15d-PGJ2), treated cells were washed twice with PBS and fixed with paraformaldehyde (2%) for 15 min and images acquired as described above.

For detection of BD-15d-PGJ2 protein adducts, BAEC were incubated as described in Cell culture and preparation of pseudo rho0 cells in BAEC and then washed twice (PBS) and lysed [50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% (vol/vol) Triton X-100, 1 mM EDTA, 1 mM Na3VO4, 50 mM NaF, 0.5 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, and 2 µg/ml aprotinin]. After separation by SDS-PAGE, samples were transferred to nitrocellulose and protein adducts detected with an anti-BD antibody.

Detection of dichlorodihydrofluorescein fluorescence. Cells were grown in four-well chambered coverslips as described in Cytochemistry and detection of BD-15d-PGJ2 protein adducts and then treated with electrophiles for 4 h. Cells were loaded with DCFH-DA (5 µM) for 60 min and incubated with MitoTracker Deep Red 633 (0.5 µM) for 15 min before being imaged. Furthermore, cells were washed twice with culture medium and images acquired by using epifluorescence or confocal microscopy. In some studies, a structural analog of DCF, carboxy DCF-DA (5 µM), was added to cells after preincubation with MitoTracker for 10 min, followed by image acquisition at 0, 2.5, and 15 min.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Electrophilic lipids induce cytoprotective pathways in BAEC. It has been shown that electrophilic lipids can induce cytoprotective pathways by binding to Keap-1, leading to the translocation of Nrf2 to the nucleus and activation of the electrophile response element (23, 26). This results in the upregulation of a number of cytoprotective genes, including glutathione synthetic enzymes and HO-1 (21). In the first series of experiments, we examined three distinct electrophilic lipids, HNE, 15-J2-IsoP, and 15d-PGJ2, for their ability to increase GSH and HO-1 levels (Fig. 2). The cyclopentenone lipids 15-J2-IsoP and 15d-PGJ2 were most potent, with a significant induction in both GSH and HO-1 levels achieved with ~1–2.5 µM, whereas 10 µM were required for HNE to exert similar effects. We have previously shown that lipids lacking an electrophilic carbon center, such as PGE2, do not induce GSH (23). The fact that HNE exerts similar effects on GSH induction as the electrophilic cyclopentenone lipids, whereas the nonelectrophilic lipid (PGE2) does not, is consistent with a requirement for an electrophilic carbon for these biological responses.


Figure 2
View larger version (20K):
[in this window]
[in a new window]
  		Fig. 2. Effect of different lipid electrophiles on GSH and heme oxygenase-1 (HO-1) induction. Bovine aortic endothelial cells (BAEC) were treated for 16 h with either 15d-PGJ2, 15-J2-IsoP, or HNE at concentrations noted. A: GSH was measured and normalized to total protein. Values are expressed as fold change relative to control (means ± SE; n = 3). B: representative Western blot analysis demonstrating a dose response of HO-1 induction by 15-J2-IsoP. C: quantification of HO-1 protein induction by HNE, 15d-PGJ2, or 15-J2-IsoP. Results are expressed as fold induction vs. control and represent means ± SE (n = 3). All values for treatment groups in A and C are significantly different from control (P > 0.001).

 
Electrophilic lipids stimulate ROS production in BAEC. To test for the formation of ROS in BAEC, cells were treated with 15d-PGJ2, HNE, or 15-J2-IsoP at concentrations selected from the previous experiments which caused a similar degree of GSH induction. Treatment with 15d-PGJ2, HNE, and 15-J2-IsoP increased DCF fluorescence in BAEC, consistent with increased ROS formation (Fig. 3). HNE increased DCF fluorescence but to a lesser extent than the cyclopentenone lipids 15d-PGJ2 or 15-J2-IsoP. Treatment with the nonelectrophilic lipid PGE2 (10 µM) did not increase DCF fluorescence, consistent with the hypothesis that this effect is specific for lipids containing electrophilic carbon centers.


Figure 3
View larger version (50K):
[in this window]
[in a new window]
  		Fig. 3. Effects of electrophilic lipids on reactive oxygen species (ROS) formation in BAEC. Confluent BAEC were treated with 15d-PGJ2 (4 µM), HNE (5 µM), 15-J2-IsoP (2.5 µM), and PGE2 (10 µM) for 4 h. Live cells were loaded with 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA, 2 µM) 60 min before being imaged. A: cells were then washed and imaged on an inverted fluorescence microscope. CTL, control. B: average of dichlorofluorescein (DCF) fluorescence intensity of cells treated as indicated in A. Data are means ± SE; n = 3. *P < 0.01 compared with CTL.

 
Lipid electrophiles induce formation of ROS in mitochondria. To test for a mitochondrial contribution to ROS generation induced by oxidized lipids, we used DCF fluorescence in conjunction with a mitochondria-specific marker and confocal microscopy. BAEC were exposed to 15d-PGJ2 (4 µM), HNE (5 µM), or 15-J2-IsoP (2.5 µM) for 4 h. Cells were loaded with DCFH-DA 60 min before being imaged. To assess colocalization with mitochondria, cells were also treated with MitoTracker. As shown in the left column in Fig. 4, all electrophiles caused an increase in the level of DCF fluorescence when compared with control. Merging of the DCF signal with the MitoTracker image indicates that DCF fluorescence colocalizes with the mitochondria (yellow in Fig. 4, right column). However, less intense DCF fluorescence was also present in nonmitochondrial compartments.


Figure 4
View larger version (123K):
[in this window]
[in a new window]
  		Fig. 4. Electrophilic lipids induce generation of ROS in mitochondria. BAEC were grown in 4-well chambers until confluent, followed by treatment with 15d-PGJ2 (4 µM), 15-J2-IsoP (2.5 µM), or HNE (5 µM) for 4 h. Images were acquired by using a confocal scanning microscope. Left to right: DCF fluorescence (green), MitoTracker (red), and merged image (yellow).

 
The association of DCF fluorescence with mitochondria could arise from a number of indirect mechanisms. One possibility is that the electrophile-dependent formation of DCF occurs at some other location in the cell and is then accumulated by the mitochondria. To test for this possibility, cells were exposed to 15d-PGJ2 (4 µM) for 4 h and then loaded with carboxy DCF-DA (5 µM). As carboxy DCF-DA enters the cell, fluorescence increases with time over 2–15 min. At 15 min, a substantial amount of carboxy DCF has diffused from the cell, but at neither time point is a pattern resembling the MitoTracker staining observed (Fig. 5A). This is in marked contrast to the pattern of DCF fluorescence evident after exposure to electrophiles (Fig. 4). The yellow color in the merge of the red and green fluorescence images in Fig. 5 is solely due to the nonspecific overlap of each stain. As an additional control, an extracellular source of hydrogen peroxide derived from the enzyme glucose oxidase was used and DCF colocalization with mitochondria assessed. As shown in Fig. 5B, glucose oxidase induces DCF fluorescence, but this shows no mitochondrial association at 15 min. We have previously demonstrated that the DCF fluorescence induced by the mitochondria-specific generator of ROS, myxothiazol, is clearly mitochondrially associated (42).


Figure 5
View larger version (78K):
[in this window]
[in a new window]
  		Fig. 5. Subcellular distribution of DCF generated by extracellular hydrogen peroxide and carboxy DCF. A: confluent BAEC were treated with 15d-PGJ2 (4 µM) for 4 h, followed by incubation with MitoTracker (0.5 µM/10 min), and then loaded with carboxy DCF-DA (5 µM). Confocal images of live cells were acquired in 0, 2.5, and 15 min. A, left to right shows fluorescence images of representative cells: carboxy DCF (green), MitoTracker (red), and merged image. B: cells were loaded with DCFH-DA (5 µM) for 60 min, followed by exposure to glucose oxidase-generated hydrogen peroxide (20 mU/ml) for 0–15 min. Cells were washed and confocal images acquired. B, left to right shows representative fields of cells and fluorescence images: DCF (green), MitoTracker (red), and merged image.

 
Association of 15d-PGJ2 with mitochondrion. In the next series of experiments, we synthesized fluorescently labeled lipids to determine whether the mitochondrial ROS formation observed in Figs. 3 and 4 was associated with the localization of 15d-PGJ2 to the mitochondrion (structures in Fig. 1). No significant difference in the capacity of BD-15d-PGJ2 to induce GSH was found when compared under identical conditions with untagged 15d-PGJ2, suggesting that the BD tag did not modify the biological response of cells to the native lipid (data not shown). Next, to examine subcellular localization of lipids, BAEC were incubated with BODIPY dye (BD), the labeled electrophilic lipid BD-15d-PGJ2 (10 µM), or the nonelectrophilic lipid BD-PGE2 (10 µM) and MitoTracker (0.5 µM). The confocal microscopy revealed a relatively diffuse pattern for the BODIPY dye alone interspersed with a punctate pattern that was not significantly associated with MitoTracker. In contrast, BD-15d-PGJ2 showed a discreet filamentous pattern of staining that predominantly colocalized with the mitochondrial reticular structure characteristic of these cells (Fig. 6A). A similar mitochondrial localization was observed with concentrations of BD-15d-PGJ2 as low as 1 µM (data not shown). The structurally similar lipid BD-PGE2, which lacks electrophilic carbons, exhibited a diffuse fluorescence and did not show accumulation in the organelle.


Figure 6
View larger version (63K):
[in this window]
[in a new window]
  		Fig. 6. Subcellular localization of BD-tagged 15d-PGJ2 and PGE2. BAEC were treated with BD (10 µM), BD-15d-PGJ2 (10 µM), or BD-PGE2 (10 µM) for 30 min. Cells were also incubated with MitoTracker (0.5 µM) for 15 min before being imaged. A, top to bottom shows confocal images of live cells treated with BD, BD-15d-PGJ2, or BD-PGE2: BD (green), MitoTracker (red), and merged BD and MitoTracker images. B: cells treated as described in A were lysed and the proteins were analyzed by Western blotting with anti-BD antibody. C: quantitative analysis where results are expressed as percentage of BD-15d-PGJ2 labeling and represent means ± SE. *P < 0.01 indicates statistically significant differences compared with CTL BAEC.

 
Formation of stable electrophilic lipid-protein adducts in endothelial cells. If the reactive electrophilic carbon center in the lipid is participating in the localization to the mitochondrion, then formation of stable adducts with proteins is predicted (23). To test for this, cells were treated with BD and BD-tagged lipids (10 µM) for 30 min, and a cell lysate was subjected to SDS-PAGE followed by Western blotting analysis with an anti-BD antibody. As seen in Fig. 6, B and C, significant labeling of proteins was only found after treatment with BD-15d-PGJ2. The low level of labeling observed with BD-PGE2 may be due to some conversion of BD-PGE2 to small amounts of the electrophilic lipid BD-PGA2 by nonenzymatic dehydration within the cells. If specific protein targets for electrophilic lipids are present in the mitochondrion, it suggests that preincubation of the cells with untagged lipid would block these sites and prevent localization to the organelle. To test for this possibility, cells were preincubated with 15d-PGJ2 (20 µM, 2 h) followed by exposure to BD-15d-PGJ2 (2.5 µM, 30 min). As can be seen in Fig. 7, pretreatment with 15d-PGJ2 resulted in a more disperse intracellular pattern of BD-15d-PGJ2 and less intense yellow color when the green BD-15d-PGJ2 and the red MitoTracker images are overlaid, indicating less mitochondrial localization of the BD-15d-PGJ2.


Figure 7
View larger version (70K):
[in this window]
[in a new window]
  		Fig. 7. 15d-PGJ2 competes with BD-15d-PGJ2 for localization to mitochondrion. BAEC were treated with or without unlabeled 15d-PGJ2 (20 µM) for 2 h, followed by 30 min exposure to BD-15d-PGJ2 (2.5 µM). Cells were washed and fixed, and images were acquired by using confocal microscopy. Top: mitochondrial localized BD-15d-PGJ2 (green), MitoTracker (red), and merged images in CTL cells. Bottom: effects of preincubation with unlabeled 15d-PGJ2 on mitochondrial localization of BD-15d-PGJ2.

 
Effects of respiratory chain on mitochondrial localization of electrophilic lipids. In the next series of experiments, we prepared BAEC that have been depleted of a functional mitochondrial respiratory chain by treatment with ethidium bromide (pseudo rho0 cells). These cells are characterized by having decreased mitochondrial {Delta}{Psi} and low levels of mitochondrially encoded respiratory chain subunits as compared with normal BAEC (42). Normal BAEC and pseudo rho0 cells were exposed to BD-15d-PGJ2, and colocalization to the mitochondria was assessed. The tagged electrophilic lipid colocalized to the mitochondria in normal BAEC, as observed in previous experiments (Fig. 8A). However, in pseudo rho0 cells, colocalization was substantially decreased (Fig. 8B), suggesting a requirement for a functional respiratory chain for electrophilic lipids to target the organelle. In addition, measurement of total cell DCF fluorescence was substantially decreased in the pseudo rho0 cells on exposure to 15d-PGJ2 (Fig. 9).


Figure 8
View larger version (104K):
[in this window]
[in a new window]
  		Fig. 8. Lack of mitochondrial association of BD-15d-PGJ2 in rho0 cells. BAEC (A) or rho0 (B) cells were incubated with BD-15d-PGJ2 (10 µM) for 30 min. MitoTracker (0.5 µM) was added for 15 min before cells were imaged. Bottom panels in A and B show a magnified field of images indicated by the white borders in the top panels. Left to right: BD-15d-PGJ2 (green), mitochondria stained with MitoTracker (red), and merged images.

 

Figure 9
View larger version (87K):
[in this window]
[in a new window]
  		Fig. 9. Loss of 15d-PGJ2-induced ROS generation in rho0 cells. BAEC or rho0 cells were incubated with 15d-PGJ2 (4 µM) for 3 h followed by loading with DCFH-DA (5 µM) for 1 h, and cells were then washed and imaged. Representative images of DCF fluorescence (green) selected from three randomly chosen fields are shown.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Although it has been known for some time that lipid oxidation products can stimulate ROS formation in cells, the sources and mechanisms involved still remain unclear (15, 39, 41, 42). In this respect, the mitochondrion is of particular interest because it is known that the organelle can produce ROS in response to stress (2). Furthermore, it has recently become evident that mitochondria may be a regulated source of ROS and so contribute to redox cell signaling (4). It is therefore important to investigate the signals leading to mitochondrial ROS formation. In this respect, lipid oxidation products are of particular interest because they contain families of molecules that are hydrophobic and are highly reactive with the nucleophilic centers present on proteins, particularly thiols. The mitochondrion is a likely site of reaction of electrophiles because of its extensive membranous network and abundance of proteins containing reactive thiol residues (24). Whether electrophilic lipids can penetrate the cytosolic milieu and localize to mitochondria in intact cells has not been previously addressed. This is potentially important because the literature describing experiments with isolated mitochondria suggests that lipid electrophiles, such as HNE, can modify mitochondrial function (2, 41).

One of the compounds we have used as a model electrophile in this study is the cyclopentenone prostaglandin 15d-PGJ2. Several lines of evidence suggest a mitochondrial interaction with exogenously added 15d-PGJ2. For example, high proapoptotic concentrations of 15d-PGJ2 increase ROS formation in a transformed breast cancer cell line, and this effect can be inhibited by the mitochondrial complex I inhibitor rotenone (34). Similar increases in ROS formation have also been reported in isolated mitochondria treated with 15d-PGJ2, and an electrophile responsive proteome in the mitochondria is composed of proteins with reactive thiols (20, 25). We have also shown that exposure of cells to low, nontoxic concentrations of 15d-PGJ2 induces the activity of complex I in the mitochondrion (6). However, because of the lack of availability of specific tags to monitor the fate of electrophiles in the cell, mitochondrial localization has not been previously investigated.

To test for a mitochondrial contribution to the increased ROS production after exposure to electrophilic lipids, endothelial cells were treated in conjunction with the mitochondrial label, MitoTracker. The concept that the electrophilic carbon center is required for mitochondrial ROS formation is supported by the fact that the electrophilic lipids 15d-PGJ2, 15-J2-IsoP, and HNE showed a strong mitochondrially localized formation of DCF fluorescence (Fig. 4). In contrast, the nonelectrophilic lipid (PGE2) did not induce DCF fluorescence (Fig. 3). Carboxy DCF was not accumulated in the mitochondrion after formation, and an extracellular source of hydrogen peroxide did not show a mitochondrial localization at early time points of exposure (Fig. 5). Nevertheless, the interpretation of DCF fluorescence is complicated by the fact that it requires the action of intracellular esterases, peroxidases, as well as extracellular iron (40). It is then possible that an indirect effect of the electrophilic lipids in the cytosol could lead to iron release or GSH depletion in the organelle and the observed mitochondrial association of DCF. Despite these limitations, it can still be used to effectively detect ROS/RNS such as hydrogen peroxide and peroxynitrite in cells (40). One important advantage of this technique is that it offers the possibility of localizing the source of ROS production within the cell.

To address whether exogenous electrophilic lipids can impact on mitochondrial proteins through a direct association, we designed a novel series of fluorescently tagged electrophilic and nonelectrophilic lipids. These compounds were used in combination with MitoTracker, and the results support the hypothesis that an electrophilic carbon center leads to the association of these reactive lipids with the organelle (Fig. 6). As expected, protein adducts were formed only with the electrophilic lipid (Fig. 6). The fact that mitochondrial localization could be blocked by preincubation with untagged 15d-PGJ2 suggests that there is a saturable component involved in mitochondrial localization (Fig. 7). Interestingly, localization of the electrophilic lipid to the mitochondrion and induction of ROS were dependent on the presence of a functional respiratory chain (Figs. 8 and 9). The reasons for this association are not clear at present but may be related to the fact that the mitochondrion contains a large concentration of proteins with reactive nucleophilic centers in a range of metabolic and regulatory enzymes.

In summary, we have shown that specific electrophilic cyclopentenones and HNE are capable of inducing mitochondrial ROS formation. It appears that the electrophilic lipids can diffuse to the organelle from an exogenous source and accumulate within the mitochondrion in a manner that requires a functional respiratory chain. This may be of biological significance under conditions of chronic inflammation, such as atherosclerosis, which are associated with the production of exogenous electrophilic lipid oxidation products due to the oxidation of low-density lipoprotein.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health (NIH) Grants ES-10167 and HL-58013 (to V. Darley-Usmar), NIH Grants DK-48831, CA-77839, ES-00267, and GM-15431 (to J. D. Morrow), and Philip Morris External Research Grant (to D. A. Dickinson).


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. Darley-Usmar, Dept. of Pathology, Univ. of Alabama at Birmingham, Biomedical Research Bldg. II, 901 19th St. South, Birmingham, AL 35294 (e-mail: Darley{at}path.uab.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Agarwal A, Balla J, Balla G, Croatt AJ, Vercellotti GM, and Nath KA. Renal tubular epithelial cells mimic endothelial cells upon exposure to oxidized LDL. Am J Physiol Renal Fluid Electrolyte Physiol 271: F814–F823, 1996.[Abstract/Free Full Text]
  2. Brand MD, Affourtit C, Esteves TC, Green K, Lambert AJ, Miwa S, Pakay JL, and Parker N. Mitochondrial superoxide: production, biological effects, and activation of uncoupling proteins. Free Radic Biol Med 37: 755–767, 2004.[CrossRef][Web of Science][Medline]
  3. Brookes PS, Levonen AL, Shiva S, Sarti P, and Darley-Usmar VM. Mitochondria: regulators of signal transduction by reactive oxygen and nitrogen species. Free Radic Biol Med 33: 755–764, 2002.[CrossRef][Web of Science][Medline]
  4. Cadenas E. Mitochondrial free radical production and cell signaling. Mol Aspects Med 25: 17–26, 2004.[CrossRef][Medline]
  5. Ceaser EK, Moellering DR, Shiva S, Ramachandran A, Landar A, Venkartraman A, Crawford J, Patel R, Dickinson DA, Ulasova E, Ji S, and Darley-Usmar VM. Mechanisms of signal transduction mediated by oxidized lipids: the role of the electrophile-responsive proteome. Biochem Soc Trans 32: 151–155, 2004.[CrossRef][Web of Science][Medline]
  6. Ceaser EK, Ramachandran A, Levonen AL, and Darley-Usmar VM. Oxidized low-density lipoprotein and 15-deoxy-{Delta}12,14-PGJ2 increase mitochondrial complex I activity in endothelial cells. Am J Physiol Heart Circ Physiol 285: H2298–H2308, 2003.[Abstract/Free Full Text]
  7. Chang SH, Garcia J, Melendez JA, Kilberg MS, and Agarwal A. Haem oxygenase 1 gene induction by glucose deprivation is mediated by reactive oxygen species via the mitochondrial electron-transport chain. Biochem J 371: 877–885, 2003.[CrossRef][Web of Science][Medline]
  8. Chen K, Thomas SR, Albano A, Murphy MP, and Keaney JF Jr. Mitochondrial function is required for hydrogen peroxide-induced growth factor receptor transactivation and downstream signaling. J Biol Chem 279: 35079–35086, 2004.[Abstract/Free Full Text]
  9. Chen Y, Morrow JD, and Roberts LJ II. Formation of reactive cyclopentenone compounds in vivo as products of the isoprostane pathway. J Biol Chem 274: 10863–10868, 1999.[Abstract/Free Full Text]
  10. Clay CE, Monjazeb A, Thorburn J, Chilton FH, and High KP. 15-Deoxy-{Delta}12,14-prostaglandin J2-induced apoptosis does not require PPAR{gamma} in breast cancer cells. J Lipid Res 43: 1818–1828, 2002.[Abstract/Free Full Text]
  11. Dickinson DA, Iles KE, Watanabe N, Iwamoto T, Zhang H, Krzywanski DM, and Forman HJ. 4-Hydroxynonenal induces glutamate cysteine ligase through JNK in HBE1 cells. Free Radic Biol Med 33: 974, 2002.[CrossRef][Web of Science][Medline]
  12. Dickinson DA, Levonen AL, Moellering DR, Arnold EK, Zhang H, Darley-Usmar VM, and Forman HJ. Human glutamate cysteine ligase gene regulation through the electrophile response element. Free Radic Biol Med 37: 1152–1159, 2004.[CrossRef][Web of Science][Medline]
  13. Dickinson DA, Moellering DR, Iles KE, Patel RP, Levonen AL, Wigley A, Darley-Usmar VM, and Forman HJ. Cytoprotection against oxidative stress and the regulation of glutathione synthesis. Biol Chem 384: 527–537, 2003.[CrossRef][Web of Science][Medline]
  14. Go YM, Levonen AL, Moellering D, Ramachandran A, Patel RP, Jo H, and Darley-Usmar VM. Endothelial NOS-dependent activation of c-Jun NH2-terminal kinase by oxidized low-density lipoprotein. Am J Physiol Heart Circ Physiol 281: H2705–H2713, 2001.[Abstract/Free Full Text]
  15. Hsieh CC, Yen MH, Yen CH, and Lau YT. Oxidized low density lipoprotein induces apoptosis via generation of reactive oxygen species in vascular smooth muscle cells. Cardiovasc Res 49: 135–145, 2001.[Abstract/Free Full Text]
  16. Hubatsch I, Mannervik B, Gao L, Roberts LJ, Chen Y, and Morrow JD. The cyclopentenone product of lipid peroxidation, 15-A(2t)-isoprostane (8-isoprostaglandin A2), is efficiently conjugated with glutathione by human and rat glutathione transferase A4–4. Chem Res Toxicol 15: 1114–1118, 2002.[CrossRef][Web of Science][Medline]
  17. Ishii T, Itoh K, Ruiz E, Leake DS, Unoki H, Yamamoto M, and Mann GE. Role of Nrf2 in the regulation of CD36 and stress protein expression in murine macrophages: activation by oxidatively modified LDL and 4-hydroxynonenal. Circ Res 94: 609–616, 2004.[Abstract/Free Full Text]
  18. Itoh K, Mochizuki M, Ishii Y, Ishii T, Shibata T, Kawamoto Y, Kelly V, Sekizawa K, Uchida K, and Yamamoto M. Transcription factor Nrf2 regulates inflammation by mediating the effect of 15-deoxy-{Delta}12,14-prostaglandin J2. Mol Cell Biol 24: 36–45, 2004.[Abstract/Free Full Text]
  19. Kondo M, Shibata T, Kumagai T, Osawa T, Shibata N, Kobayashi M, Sasaki S, Iwata M, Noguchi N, and Uchida K. 15-Deoxy-{Delta}12,14-prostaglandin J2: the endogenous electrophile that induces neuronal apoptosis. Proc Natl Acad Sci USA 99: 7367–7372, 2002.[Abstract/Free Full Text]
  20. Landar A, Shiva S, Levonen AL, Oh JY, Zaragoza C, Johnson MS, and Darley-Usmar VM. Induction of the permeability transition and cytochrome c release by 15-deoxy prostaglandin J2 in mitochondria. Biochem J 394: 185–195, 2006.[CrossRef][Web of Science][Medline]
  21. Lee TS, Tsai HL, and Chau LY. Induction of heme oxygenase-1 expression in murine macrophages is essential for the anti-inflammatory effect of low dose 15-deoxy-{Delta}12,14-prostaglandin J2. J Biol Chem 278: 19325–19330, 2003.[Abstract/Free Full Text]
  22. Levonen AL, Dickinson DA, Moellering DR, Mulcahy RT, Forman HJ, and Darley-Usmar VM. Biphasic effects of 15-deoxy-{Delta}12,14-prostaglandin J2 on glutathione induction and apoptosis in human endothelial cells. Arterioscler Thromb Vasc Biol 21: 1846–1851, 2001.[Abstract/Free Full Text]
  23. Levonen AL, Landar A, Ramachandran A, Ceaser EK, Dickinson DA, Zanoni G, Morrow JD, and Darley-Usmar VM. Cellular mechanisms of redox cell signalling: role of cysteine modification in controlling antioxidant defences in response to electrophilic lipid oxidation products. Biochem J 378: 373–382, 2004.[CrossRef][Web of Science][Medline]
  24. Lin TK, Hughes G, Muratovska A, Blaikie FH, Brookes PS, Darley-Usmar V, Smith RA, and Murphy MP. Specific modification of mitochondrial protein thiols in response to oxidative stress: a proteomics approach. J Biol Chem 277: 17048–17056, 2002.[Abstract/Free Full Text]
  25. Martinez B, Perez-Castillo A, and Santos A. The mitochondrial respiratory complex I is a target for 15-deoxy-{Delta}12,14-prostaglandin J2 action. J Lipid Res 46: 736–743, 2005.[Abstract/Free Full Text]
  26. McMahon M, Thomas N, Itoh K, Yamamoto M, and Hayes JD. Redox-regulated turnover of Nrf2 is determined by at least two separate protein domains, the redox-sensitive Neh2 degron and the redox-insensitive Neh6 degron. J Biol Chem 279: 31556–31567, 2004.[Abstract/Free Full Text]
  27. Milne GL, Musiek ES, and Morrow JD. The cyclopentenone (A2/J2) isoprostanes–unique, highly reactive products of arachidonate peroxidation. Antioxid Redox Signal 7: 210–220, 2005.[CrossRef][Web of Science][Medline]
  28. Moellering DR, Levonen AL, Go YM, Patel RP, Dickinson DA, Forman HJ, and Darley-Usmar VM. Induction of glutathione synthesis by oxidized low-density lipoprotein and 1-palmitoyl-2-arachidonyl phosphatidylcholine: protection against quinone-mediated oxidative stress. Biochem J 362: 51–59, 2002.[CrossRef][Web of Science][Medline]
  29. Moos PJ, Edes K, Cassidy P, Massuda E, and Fitzpatrick FA. Electrophilic prostaglandins and lipid aldehydes repress redox sensitive transcription factors p53 and HIF by impairing the selenoprotein thioredoxin reductase. J Biol Chem 278: 745–750, 2003.[Abstract/Free Full Text]
  30. Morrow JD. Quantification of isoprostanes as indices of oxidant stress and the risk of atherosclerosis in humans. Arterioscler Thromb Vasc Biol 25: 279–286, 2005.[Abstract/Free Full Text]
  31. Mullally JE, Moos PJ, Edes K, and Fitzpatrick FA. Cyclopentenone prostaglandins of the J series inhibit the ubiquitin isopeptidase activity of the proteasome pathway. J Biol Chem 276: 30366–30373, 2001.[Abstract/Free Full Text]
  32. Palinski W, Rosenfeld ME, Yla-Herttuala S, Gurtner GC, Socher SS, Butler SW, Parthasarathy S, Carew TE, Steinberg D, and Witztum JL. Low density lipoprotein undergoes oxidative modification in vivo. Proc Natl Acad Sci USA 86: 1372–1376, 1989.[Abstract/Free Full Text]
  33. Perez-Sala D, Cernuda-Morollon E, and Canada FJ. Molecular basis for the direct inhibition of AP-1 DNA binding by 15-deoxy-{Delta}12,14-prostaglandin J2. J Biol Chem 278: 51251–51260, 2003.[Abstract/Free Full Text]
  34. Pignatelli M, Sanchez-Rodriguez J, Santos A, and Perez-Castillo A. 15-Deoxy-{Delta}-12,14-prostaglandin J2 induces programmed cell death of breast cancer cells by a pleiotropic mechanism. Carcinogenesis 26: 81–92, 2005.[Abstract/Free Full Text]
  35. Rodriguez AM, Carrico PM, Mazurkiewicz JE, and Melendez JA. Mitochondrial or cytosolic catalase reverses the MnSOD-dependent inhibition of proliferation by enhancing respiratory chain activity, net ATP production, and decreasing the steady state levels of H2O2. Free Radic Biol Med 29: 801–813, 2000.[CrossRef][Web of Science][Medline]
  36. Saito S, Takahashi S, Takagaki N, Hirose T, and Sakai T. 15-Deoxy-{Delta}12,14-prostaglandin J2 induces apoptosis through activation of the CHOP gene in HeLa cells. Biochem Biophys Res Commun 311: 17–23, 2003.[CrossRef][Web of Science][Medline]
  37. Shibata T, Kondo M, Osawa T, Shibata N, Kobayashi M, and Uchida K. 15-Deoxy-{Delta}12,14-prostaglandin J2. A prostaglandin D2 metabolite generated during inflammatory processes. J Biol Chem 277: 10459–10466, 2002.[Abstract/Free Full Text]
  38. Shibata T, Yamada T, Ishii T, Kumazawa S, Nakamura H, Masutani H, Yodoi J, and Uchida K. Thioredoxin as a molecular target of cyclopentenone prostaglandins. J Biol Chem 278: 26046–26054, 2003.[Abstract/Free Full Text]
  39. Straus DS and Glass CK. Cyclopentenone prostaglandins: new insights on biological activities and cellular targets. Med Res Rev 21: 185–210, 2001.[CrossRef][Web of Science][Medline]
  40. Tampo Y, Kotamraju S, Chitambar CR, Kalivendi SV, Keszler A, Joseph J, and Kalyanaraman B. Oxidative stress-induced iron signaling is responsible for peroxide-dependent oxidation of dichlorodihydrofluorescein in endothelial cells: role of transferrin receptor-dependent iron uptake in apoptosis. Circ Res 92: 56–63, 2003.[Abstract/Free Full Text]
  41. Uchida K. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog Lipid Res 42: 318–343, 2003.[CrossRef][Web of Science][Medline]
  42. Zmijewski JW, Moellering DR, Le Goffe C, Landar A, Ramachandran A, and Darley-Usmar VM. Oxidized LDL induces mitochondrially associated reactive oxygen/nitrogen species formation in endothelial cells. Am J Physiol Heart Circ Physiol 289: H852–H861, 2005.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Cardiovasc ResHome page
S. M. Nadtochiy, P. R.S. Baker, B. A. Freeman, and P. S. Brookes
Mitochondrial nitroalkene formation and mild uncoupling in ischaemic preconditioning: implications for cardioprotection
Cardiovasc Res, May 1, 2009; 82(2): 333 - 340.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
Z. Han, S. Varadharaj, R. J. Giedt, J. L. Zweier, H. H. Szeto, and B. R. Alevriadou
Mitochondria-Derived Reactive Oxygen Species Mediate Heme Oxygenase-1 Expression in Sheared Endothelial Cells
J. Pharmacol. Exp. Ther., April 1, 2009; 329(1): 94 - 101.
[Abstract] [Full Text] [PDF]

Home page
 CarcinogenesisHome page
E.-H. Kim, H.-K. Na, D.-H. Kim, S.-A. Park, H.-N. Kim, N.-Y. Song, and Y.-J. Surh
15-Deoxy-{Delta}12,14-prostaglandin J2 induces COX-2 expression through Akt-driven AP-1 activation in human breast cancer cells: a potential role of ROS
Carcinogenesis, April 1, 2008; 29(4): 688 - 695.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
D. A. Andersson, C. Gentry, S. Moss, and S. Bevan
Transient Receptor Potential A1 Is a Sensory Receptor for Multiple Products of Oxidative Stress
J. Neurosci., March 5, 2008; 28(10): 2485 - 2494.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Pathol.Home page
Y.-M. Go, P. J. Halvey, J. M. Hansen, M. Reed, J. Pohl, and D. P. Jones
Reactive Aldehyde Modification of Thioredoxin-1 Activates Early Steps of Inflammation and Cell Adhesion
Am. J. Pathol., November 1, 2007; 171(5): 1670 - 1681.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
D. X. Zhang and D. D. Gutterman
Mitochondrial reactive oxygen species-mediated signaling in endothelial cells
Am J Physiol Heart Circ Physiol, May 1, 2007; 292(5): H2023 - H2031.
[Abstract] [Full Text] [PDF]

Home page
 Cancer Res.Home page
P. S. Brookes, K. Morse, D. Ray, A. Tompkins, S. M. Young, S. Hilchey, S. Salim, M. Konopleva, M. Andreeff, R. Phipps, et al.
The Triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-oic Acid and Its Derivatives Elicit Human Lymphoid Cell Apoptosis through a Novel Pathway Involving the Unregulated Mitochondrial Permeability Transition Pore
Cancer Res., February 15, 2007; 67(4): 1793 - 1802.
[Abstract] [Full Text] [PDF]

Home page
 Circ. Res.Home page
J. Gutierrez, S. W. Ballinger, V. M. Darley-Usmar, and A. Landar
Free Radicals, Mitochondria, and Oxidized Lipids: The Emerging Role in Signal Transduction in Vascular Cells
Circ. Res., October 27, 2006; 99(9): 924 - 932.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
M. P. Murphy
Induction of mitochondrial ROS production by electrophilic lipids: a new pathway of redox signaling?
Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1754 - H1755.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/5/H1777    most recent
01087.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (35)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Landar, A.
Right arrow Articles by Darley-Usmar, V. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Landar, A.
Right arrow Articles by Darley-Usmar, V. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.