Nitrolinoleic acid: An endogenous peroxisome proliferator-activated receptor ␥ ligand

Francisco J. Schopfer*†‡, Yiming Lin‡§, Paul R. S. Baker*†, Taixing Cui§, Minerva Garcia-Barrio§, Jifeng Zhang§, Kai Chen§, Yuqing E. Chen‡§¶, and Bruce A. Freeman*†‡¶

*Department of Anesthesiology and †Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, AL 35294; and §Cardiovascular Research Institute, Morehouse School of Medicine, Atlanta, GA 30310

Edited by Louis J. Ignarro, University of California School of Medicine, Los Angeles, CA, and approved January 4, 2005 (received for review November 10, 2004)

Nitroalkene derivatives of linoleic acid (nitrolinoleic acid, LNO2) are from methanol (vehicle), linoleic acid (LA)-, and LNO2-treated formed via nitric oxide-dependent oxidative inflammatory reac- human aortic smooth muscle cells indicated that LNO2 specif- tions and are found at concentrations of Ϸ500 nM in the blood of ically and potently regulated the expression of key inflammatory, healthy individuals. We report that LNO2 is a potent endogenous cell proliferation and cell differentiation-related proteins (data ligand for peroxisome proliferator-activated receptor ␥ (PPAR␥; Ki not shown). Multiple peroxisome proliferator-activated receptor Ϸ133 nM) that acts within physiological concentration ranges. This ␥ (PPAR␥) target genes were significantly regulated, suggesting ␥ nuclear hormone receptor (PPAR ) regulates glucose homeostasis, that LNO2 serves as an endogenous PPAR␥ ligand. lipid metabolism, and inflammation. PPAR␥ ligand activity is spe- PPAR␥ is a nuclear hormone receptor that binds lipophilic cific for LNO2 and not mediated by LNO2 decay products, NO ligands. Downstream effects of PPAR␥ activation include mod- donors, linoleic acid (LA), or oxidized LA. LNO2 is a significantly ulation of metabolic and cellular differentiation genes and more robust PPAR␥ ligand than other reported endogenous PPAR␥ regulation of inflammatory responses (e.g., integrin expression ligands, including lysophosphatidic acid (16:0 and 18:1), 15-deoxy- and lipid transport by monocytes), adipogenesis, and glucose 12,14 ⌬ -PGJ2, conjugated LA and azelaoyl-phosphocholine. LNO2 homeostasis (10, 11). In the vasculature, PPAR␥ is expressed in activation of PPAR␥ via CV-1 cell luciferase reporter gene expres- monocytes, macrophages, smooth muscle cells, and endothelium sion analysis revealed a ligand activity that rivals or exceeds (12) and plays a central role in regulating the expression of genes synthetic PPAR␥ agonists such as and , is related to lipid trafficking, cell proliferation, and inflammatory coactivated by 9 cis-retinoic acid and is inhibited by the PPAR␥ signaling (13). Although synthetic such as ␥ antagonist GW9662. LNO2 induces PPAR -dependent macrophage rosiglitazone and ciglitazone are appreciated to be the most CD-36 expression, adipocyte differentiation, and glucose uptake potent PPAR␥ ligands yet described, considerable interest and also at a potency rivaling thiazolidinediones. These observations debate remains focused on the identity of endogenous PPAR␥ reveal that NO-mediated cell signaling reactions can be trans- ligands because of therapeutic potential and their intrinsic value duced by fatty acid nitration products and PPAR-dependent gene in understanding cell signaling. At present, tissue and plasma expression. levels of putative PPAR ligands are frequently not precisely defined and when so, are found in concentrations sometimes fatty acid ͉ nitric oxide ͉ free radical ͉ adipocyte differentiation ͉ redox orders of magnitude lower than those required to activate specific ␣, ␥,or␦ PPAR subtypes (14–16). Herein we report that he reaction of nitric oxide with tissue-free radical and nitroalkene derivatives of fatty acids are robust endogenous Toxidative intermediates yields secondary oxides of nitrogen PPAR␥ ligands that act within physiological concentration that mediate oxidation, nitration, and nitrosation reactions (1, ranges to modulate key PPAR␥-regulated signaling events in- 2). Of present relevance, the reaction of NO and NO-derived cluding adipogenesis, adipocyte glucose homeostasis, and CD36 species with oxidizing unsaturated fatty acids is kinetically rapid expression in macrophages. and exerts a multifaceted impact on cell redox and signaling reactions. NO readily outcompetes lipophilic antioxidants for Materials and Methods 13 the scavenging of lipid radicals, resulting in the inhibition of Materials. LNO2 and [ C]LNO2 were synthesized and purified by peroxyl radical-mediated chain propagation reactions (3). Both using LA (NuCheckPrep, Elysian, MN) and [13C]LA (Spectra the catalytic activity and gene expression of eicosanoid biosyn- Stables Isotopes, Columbia, MD) subjected to nitroselenylation thetic enzymes are also regulated by NO, affirming a strong as described (9). LNO2 concentrations were quantified spectro- linkage between NO and fatty acid oxygenation product synthe- scopically and by chemiluminescent nitrogen analysis (Antek sis and signaling (4, 5). Consistent with this latter precept, fatty Instruments, Houston) by using caffeine as a standard (9). acid nitration products generated by NO-derived species inhibit Anti-CD36 Ab was kindly provided by F. Debeer (University of multiple aspects of inflammatory cell function, indicating that Kentucky Medical Center, Lexington, KY); anti-PPAR␥ and nitrated fatty acids are both by-products and mediators of anti-␤-actin Abs were from Santa Cruz Biotechnology; and redox-signaling reactions (6–8). anti-aP2 Ab was from Chemicon. Horseradish peroxidase-linked Recently, the structural characterization and quantitation of nitrolinoleic acid (LNO2) in human red cells and plasma re- vealed this unsaturated fatty acid derivative to be the most This paper was submitted directly (Track II) to the PNAS office. abundant bioactive oxide of nitrogen in the vasculature. Net Abbreviations: LNO2, nitrolinoleic acid; PPAR, peroxisome proliferator-activated receptor; Ϸ PPRE, PPAR response elements; LA, linoleic acid; RXR, retinoic X receptor; azPC, 1-O- blood levels of 80 and 550 nM free and esterified LNO2, hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine; 9-oxoODE, 9-oxo-10(E),12(Z)-octadec- respectively, were measured in healthy humans (9). The obser- adienoic acid; 13-oxoODE, 13-oxo-9(E),11(Z)-octadecadienoic acid; LPA, lysophosphatidic vation that NO-dependent oxidative inflammatory reactions acid. yield nitroalkene derivatives of unsaturated fatty acids displaying ‡F.J.S, Y.L., Y.E.C., and B.A.F. contributed equally to this work. cGMP-independent cell signaling properties (5) motivated iden- ¶To whom correspondence may be addressed: E-mail: [email protected] or echen@ tifying a receptor that might transduce LNO2 signaling. Af- msm.edu. fymetrix oligonucleotide microarray analysis of cRNA prepared © 2005 by The National Academy of Sciences of the USA

2340–2345 ͉ PNAS ͉ February 15, 2005 ͉ vol. 102 ͉ no. 7 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408384102 Downloaded by guest on September 24, 2021 goat anti-rabbit IgG and Coomassie blue were from Pierce. temperature three times with freshly prepared KRPH buffer (5 3 [ H]rosiglitazone was from American Radiolabeled Chemicals mM phosphate buffer͞20 mM Hepes͞1 mM MgSO4͞1mM 3 (St. Louis). 2-deoxy-D-[ H]glucose was from Sigma. ScintiSafe CaCl2͞136 mM NaCl͞4.7 mM KCl, pH 7.4). The buffer was Plus 50% was from Fisher Scientific. Rosiglitazone, ciglitazone, replaced with 1 ␮Ci͞ml 2-deoxy-D-[3H]glucose in KRPH buffer 12,14 15-deoxy-⌬ -PGJ2, conjugated LA (CLA1 and CLA2), and for 10 min at room temperature. The treated cells were rinsed GW9662 were from Cayman Chemical (Ann Arbor, MI). The carefully three times with cold PBS, lysed overnight in 0.8 M 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate [16:0 lysophos- NaOH (0.4 ml per well), and neutralized with 13.3 ␮lof12M phatidic acid (LPA)], 1-O-9-(Z)-octadecenyl-2-hydroxy-sn- HCl. Lysate (360 ␮l) was added to 4 ml of ScintiSafe Plus 50% glycero-3-phosphate (18:1 LPA), 1-O-hexadecyl-2-azelaoyl-sn- in a scintillation vial, and the vials were mixed and counted. glycero-3-phosphocholine (azPC), and 1-palmitoy-2-azelaoyl- sn-glycero-3-phosphocholine (azPC ester) were from Avanti RNA and Protein Preparation and Analysis. RNA and protein ex- Polar Lipids. pression levels were analyzed by quantitative real-time PCR and Western blot analysis as described in ref. 20. Cell Transient Transfection Assay. CV-1 cells from American Type Culture Collection were grown to Ϸ85% confluence in DMEM͞ LNO2 Decay. LNO2 decay was induced by incubating 3 ␮M LNO2 F12 supplemented with 10% FBS and 1% penicillin– in medium plus serum at 37°C. At different times, samples were streptomycin. Then, cells were transiently cotransfected with a removed and analyzed for bioactivity or for LNO2 content by 13 plasmid containing the luciferase gene under regulation by four Bligh and Dyer extraction in the presence of 1 ␮M[ C]LNO2 as Gal4 DNA-binding elements (UASG ϫ 4 TK-luciferase, a gift an internal standard. Nondecayed LNO2 was quantified by from Ronald M. Evans, The Salk Institute, La Jolla, CA), in means of triple quadruple mass spectrometric (MS) analysis concert with plasmids containing the ligand-binding domain for (Applied Biosystems͞MDS Sciex, Thornhill, Ontario, Canada) the different nuclear receptors fused to the Gal4 DNA-binding as described in ref. 9. domain. For assessing full-length PPAR receptors, CV-1 cells were transiently cotransfected with a plasmid containing the Results luciferase gene under the control of three tandem PPAR To characterize LNO2 as a potential ligand for a lipid-binding response elements (PPRE) (PPRE ϫ 3 TK-luciferase) and nuclear receptor [e.g., PPAR␣, PPAR␥, PPAR␦, androgen hPPAR␥, hPPAR␣, or hPPAR␦ expression plasmids, respec- receptor, glucocorticoid receptor, mineralocorticoid receptor, tively. In all cases, GFP expression plasmid was cotransfected as progesterone receptor, and retinoic X receptor ␣ (RXR␣)], the control for the transfection efficiency. After the transfection CV-1 reporter cells were cotransfected with plasmids containing (24 h), cells were cultured for another 24 h in OPTIMEM the ligand-binding domain for these nuclear receptors fused to (Invitrogen). Then, cells were treated with different compounds the Gal4 DNA-binding domain and the luciferase gene under as indicated in the figure legends for 24 h in OPTIMEM. regulation of four Gal4 DNA-binding elements. LNO2 (1 ␮M) Reporter luciferase assay kits from Promega were used to induced significant activation of PPAR␥ (620%), PPAR␣ measure the luciferase activity, according to the manufacturer’s (325%), and PPAR␦ (221%), with no impact on androgen, instructions, with a luminometer (Victor II, Perkin–Elmer). glucocorticoid, mineralocorticoid, progesterone, or RXR␣ re- Luciferase activity was normalized by GFP units. Each condition ceptor activation (Fig. 1A). To further explore PPAR activation was performed with n Ն 3 for each experiment. All experiments by LNO2, CV-1 cells were transiently cotransfected with a were repeated at least three times. plasmid containing the luciferase gene under three PPRE in concert with PPAR␥, PPAR␣,orPPAR␦ expression plasmids. PPAR␥ Competition-Binding Assay. Human PPAR␥1 cDNA was Dose-dependent activation by LNO2 was observed for all PPARs inserted into pGEX (Amersham Pharmacia Biosciences) con- (Fig. 1B), with PPAR␥ showing the greatest response at clinically taining the gene encoding GST. GST-PPAR␥ protein induction relevant LNO2 concentrations. and receptor binding was assessed as described in ref. 17. PPAR␥ activation by LNO2 rivaled that induced by ciglitazone 12,14 and rosiglitazone and exceeded that of 15-deoxy-⌬ -PGJ2 (21, The 3T3-L1 Differentiation and Oil Red O Staining. The 3T3-L1 22), which only occurred at concentrations three orders of preadipocytes were propagated and maintained in DMEM magnitude greater than found clinically (Fig. 1C and Inset). containing 10% FBS. To induce differentiation, 2-d postconflu- Several other reported endogenous PPAR␥ activators added in ent preadipocytes (designated day 0) were cultured in DMEM equimolar concentration with LNO2 [LA, conjugated LA containing 10% FBS plus 3 ␮M LNO2 for 14 d. The medium was (CLA1, CLA2), LPA (16:0, 18:1), azPC, and azPC ester; 1 and changed every 2 d. Rosiglitazone (3 ␮M) and LA (10 ␮M) were 3 ␮M], displayed no significant activation of PPAR␥ reporter used as the positive and negative controls, respectively. The gene expression when compared with vehicle control (Fig. 1C) differentiated adipocytes were stained by Oil red O as described (23–25). LNO2-mediated PPAR␥ activation was inhibited by the in ref. 18. PPAR␥-specific antagonist GW9662 and enhanced Ϸ180% by coaddition of the RXR␣ agonist 9-cis-retinoic acid, which facil- The 2-Deoxy-D-[3H]glucose Uptake Assay in Differentiated 3T3-L1 itates PPRE promoter activation via heterodimerization of Adipocytes. The 2-Deoxy-D-[3H]glucose uptake assay was per- activated RXR␣ with PPAR␥ (Fig. 2A). formed as described in ref. 19. 3T3-L1 preadipocytes were grown LNO2 slowly undergoes decay reactions in aqueous solution, in 24-well tissue culture plates; 2-d postconfluent preadipocytes displaying a 30- to 60-min half-life (Fig. 2B), yielding NO and an ␮ ͞ ␮ were treated by 10 g ml , 1 M dexamethasone, and 0.5 array of oxidation products (data not shown). The activation of BIOCHEMISTRY mM 3-isobutyl-1-methylxanthine (all from Sigma) in DMEM PPAR␥ paralleled the presence and concentration of the LNO2 containing 10% FBS for 2 d, and then cells were kept in 10 ␮g͞ml parent molecule, as determined by concomitant receptor acti- insulin also in DMEM containing 10% FBS for 6 d (changed vation analysis and MS quantitation of residual LNO2 in reaction medium every 3 d). After induction of adipogenesis (8 d), test mixtures undergoing different degrees of aqueous decomposi- compounds in DMEM containing 10% FBS were added for an tion before addition to CV-1 cells (Fig. 2B). Affirmation that additional 2 d (changed medium every day). The PPAR␥- activation of PPAR␥ is LNO2-specific, rather than a conse- specific antagonist GW9662 was included 1 h before other quence of LNO2 decay products, was established by treatment of additions. After two rinses with serum-free DMEM, cells were PPAR␥-transfected CV-1 cells with NO donors and oxidized LA incubated for3hinserum-free DMEM and rinsed at room derivatives. Additionally, the fatty acid oxidation products 9-oxo-

Schopfer et al. PNAS ͉ February 15, 2005 ͉ vol. 102 ͉ no. 7 ͉ 2341 Downloaded by guest on September 24, 2021 Fig. 1. LNO2 is a potent PPAR ligand. (A) CV-1 cells, transiently cotransfected with different nuclear receptor ligand-binding domains fused to the Gal4 DNA-binding domain and the luciferase reporter gene under the control of four Gal4 DNA-binding elements, were incubated with vehicle (methanol) or LNO2 (3 ␮M,2h,n ϭ 4). (Inset) Dose response of LNO2-dependent PPAR␥ ligand-binding domain activation (n ϭ 4). (B) Dose response of LNO2-dependent PPAR␥, ␣, and ␦ activation (n ϭ 4). The luciferase reporter gene was under the control of three PPRE. (C) Response of CV-1 cells transfected with PPAR␥ and a luciferase reporter under the control of PPRE after exposure to LNO2 and other reported PPAR␥ ligands [1 and 3 ␮M each of ciglitazone, 1-palmitoyl-2-hydroxy-sn-glycero- 3-phosphocholine (LPA 16:0), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine (LPA 18:1), 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine (AzPC), 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (AzPC ester), ⌬9,11-conjugated LA (CLA1), and ⌬10,12-conjugated LA (CLA2), with n ϭ 3–5]. For 15-deoxy- 12,14 ⌬ -PGJ2, the concentration was 3 and 5 ␮M. ‘‘Vector’’ indicates empty vector. (Inset) By using the same reporter construct, the dose response of PPAR␥ ⌬12,14 ϭ Ϯ Ͻ activation by LNO2, rosiglitazone, 15-deoxy- -PGJ2, and LA was measured (n 3). All values are expressed as mean SD. *, significantly different (P 0.05) from vehicle control, using Student’s t test. All experiments were repeated at least three times.

10(E),12(Z)-octadecadienoic acid (9-oxoODE) and 13-oxo- with reported values (Fig. 2D, 40–50 nM; ref. 28). LNO2 9(E),11(Z)-octadecadienoic acid (13-oxoODE) are reported en- displayed an estimated Ki of 133 nM and LA an estimated Ki of dogenous PPAR␥ stimuli (26, 27). The 13-oxoODE had no effect Ͼ1,000 nM (previously reported as 1.7–17 ␮M; ref. 28). This 3 on PPAR␥-dependent reporter gene expression (Fig. 2C). Only reveals that the Ki for LNO2 displacement of [ H]rosiglitazone high and nonphysiological concentrations of 9-oxoODE, and the from PPAR␥ is comparable to this highly avid ligand. concerted addition of high concentrations of 13-oxoODE and The PPAR␥ agonist actions of LNO2 also were examined in S-nitrosoglutathione or spermine-NONOate [(Z)-1-{N-[3- a biological context, by using cell models noted for well estab- Aminopropyl]-N-[4-(3-aminopropylammonio)butyl]-amino}- lished PPAR␥-dependent functions. The scavenger receptor diazen-1-ium-1,2-diolate], resulted in modest PPAR␥ activation CD36 is expressed in diverse cell types, including platelets, (Fig. 2C). The NO donors added individually did not activate adipocytes, and macrophages. In macrophages, CD36 is a re- PPAR␥, even at high concentrations, eliminating the possibility ceptor for oxidized low-density lipoprotein, with expression that PPAR␥-mediated signaling by LNO2 is due to NO deriva- positively regulated by PPAR␥ (29). Treatment of mouse tives released during LNO2 decay (Fig. 2C). RAW264.7 macrophages with LNO2 induced greater CD36 Competititive PPAR␥ binding analysis quantified the dis- receptor protein expression than an equivalent rosiglitazone placement of [3H]rosiglitazone by unlabeled rosiglitazone, concentration, a response partially inhibitable by the PPAR␥- LNO2, and LA. The Ki for rosiglitazone was 53 nM, consistent specific antagonist GW9662 (Fig. 3A). Moreover, quantitative

2342 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408384102 Schopfer et al. Downloaded by guest on September 24, 2021 Fig. 2. Characterization of the PPAR␥ ligand activity of LNO2.(A) By using CV-1 cells cotransfected with PPAR␥ and PPRE-controlled luciferase expression plasmids, the activation of PPAR␥ by LNO2 was evaluated in the presence of PPAR␥-specific antagonist GW9662 added 1 h before LNO2 addition or upon coaddition of the RXR coactivating ligand 9-cis-retinoic acid (n ϭ 3). PPAR␥ activation by LNO2 was inhibited in a dose-dependent manner by GW9662 and was enhanced in the presence of the coactivator 9-cis-retinoic acid. (B) The action of LNO2 as a PPAR␥ ligand was compared with LNO2 decay products. Effective LNO2 13 concentrations after selected decay periods were measured by liquid chromatography-MS with electrospray ionization by using [ C]LNO2 as internal standard (9). PPAR␥ activation was assessed by means of PPRE reporter analysis in CV-1 cells (n ϭ 3). (C) Potential PPAR␥ ligand activity of LNO2 decay products was measured 3 by means of PPRE reporter analysis (n ϭ 4). (D) Competition of LNO2, linoleate, and unlabeled rosiglitazone for PPAR␥-bound [ H]rosiglitazone. For A–C, all values Ϯ Ͻ are expressed as mean SD. *, significantly different (P 0.05) from vehicle control; #, significantly different from LNO2 alone, using Student’s t test. All experiments were repeated at least three times.

real-time PCR revealed that LNO2-dependent PPAR␥ transac- thiazolidinediones widely used as insulin-sensitizing drugs. Ad- tivation induced a dose-dependent increase in CD36 mRNA dition of LNO2 (1–10 ␮M) to differentiated adipocytes induced expression in these macrophages (data not shown). a dose-dependent increase in glucose uptake (Fig. 3D Left). The PPAR␥ plays an essential role in the differentiation of adi- impact of LNO2 on glucose uptake was greater than that pocytes (30, 31). In support of this precept, selective disruption observed for equimolar 15-deoxy-PGJ2, equivalent to rosiglita- of PPAR␥ results in impaired development of adipose tissue (18, zone and similarly inhibited by the PPAR␥-specific antagonist 32). To define whether LNO2 induces PPAR␥-activated adipo- GW9662 (Fig. 3D Right). In aggregate, these observations es- genesis, 3T3-L1 preadipocytes were treated with LNO2, rosigli- tablish that LNO2 induces well characterized PPAR␥-dependent tazone, or LA for 2 wk. Adipocyte differentiation was assessed signaling actions toward macrophage CD36 expression and both morphologically and by means of Oil red O staining, which adipogenesis. BIOCHEMISTRY reveals the accumulation of intracellular lipids. Vehicle and LA did not affect differentiation, whereas LNO2 induced Ͼ30% of Discussion 3T3-L1 preadipocyte differentiation (Fig. 3B). Rosiglitazone The identification of bona fide high affinity endogenous PPAR␥ treatment affirmed a positive PPAR␥-dependent response. ligands has been a provocative issue that, when resolved, will LNO2- and rosiglitazone-induced preadipocyte differentiation advance our understanding of PPAR␥ modulation and reveal also resulted in expression of specific adipocyte markers new means for intervention in diverse metabolic disorders and (PPAR␥2 and aP2), an event not detected for LA (Fig. 3C). disease processes. This will also shed light on the broader PPAR␥ ligands play a central role in glucose metabolism, with contributions of this nuclear hormone receptor family to both

Schopfer et al. PNAS ͉ February 15, 2005 ͉ vol. 102 ͉ no. 7 ͉ 2343 Downloaded by guest on September 24, 2021 Fig. 3. LNO2 induces CD36 expression in macrophages and adipogenesis of 3T3-L1 preadipocytes. (A) Mouse RAW264.7 macrophages at Ϸ90% confluence were cultured in DMEM with 1% FBS for 16 h and then treated with various stimuli for 16 h as indicated. The PPAR␥-specific antagonist GW9662 was added 1 h before the treatment. The cell lysate was immunoblotted with anti-CD36 and anti-␤-actin Abs. (B and C) After reaching confluence (2 d), 3T3-L1 preadipocytes were cultured for 14 d and stained by using Oil red O (18) (B) or treated with various stimuli as indicated, and the cell lysate was immunoblotted with anti-PPAR␥, 3 anti-aP2, and anti-␤-actin Abs (C). (D) LNO2 increases 2-deoxy-D-[ H]glucose uptake in 3T3-L1 adipocytes. (Left) The dose-dependent effects of LNO2 on 2-deoxy-D-[3H]glucose uptake in 3T3-L1 adipocytes. (Right) PPAR␥-specific antagonist GW9662 was added 1 h before the treatment. 2-deoxy-D-[3H]glucose uptake assay was performed as described in Materials and Methods. All experiments were repeated at least three times. Values are expressed as mean Ϯ SD (n ϭ 6). Statistical analysis was done by using Student’s t test (*, P Ͻ 0.05 vs. vehicle control; #, P Ͻ 0.05 vs. GW9662 untreated groups). Veh, vehicle; Rosi, rosiglitazone; 15-d-PGJ2, 15-deoxy-PGJ2.

the maintenance of tissue homeostasis and the regulation of cell orders of magnitude below their binding affinities (1–15 ␮M), and organ dysfunction. Of relevance, the generation of PPAR- and are not expected to result in significant receptor occupancy activating intermediates from complex lipids often requires and activation in vivo. This latter category includes free fatty hydrolysis by phospholipase A2, with the identity of phospholipid acids, eicosanoids, 9- and 13-oxoODE, platelet-activating factor, 12,14 sn-2 position fatty acid derivatives that serve as PPAR agonists and 15-deoxy-⌬ -PGJ2 (14). For example, although LPA is a still forthcoming (33, 34). Thus, the fact that Ϸ80% of LNO2 notable PPAR␥ ligand of relevance to vascular, inflammatory, present in a variety of tissue compartments is esterified encour- and cell-proliferative diseases, the plasma concentration of LPA ages the notion that this pool of high affinity PPAR ligand is well below 100 nM (35, 36) and its binding affinity for PPAR␥ activity will be mobilized upon the phospholipase A2 activation has not been established. Also, the in vivo downstream vascular that occurs during to regulate the secondary signaling actions of LPA are inconsistent with its proposed formation of cell signaling molecules. PPAR␥ ligand activity (24, 37–39). Future studies using mice Comparison of PPAR␥-dependent gene expression induced genetically deficient for PPAR␥ and NO synthase isoforms by LNO2 with that of thiazolidinediones and putative fatty acid- should assist in defining the mechanisms of formation and and phospholipid-derived PPAR␥ ligands affirmed the robust endogenous PPAR ligand activity of nitrated fatty acids. ␥ activity of LNO2 as a PPAR ligand. Although a number of In summary, we show that LNO2 is a high-affinity ligand for endogenous lipophilic species are proposed as PPAR␥ ligands, PPARs, especially PPAR␥, that activates both reporter con- their intrinsically low binding affinities and in vivo concentra- structs and cells at physiological concentrations. Fatty acid tions do not support a capability to serve as physiologically nitration products, generated by NO-dependent reactions, thus relevant signaling mediators. Presently reported endogenous are expected to display broad cell signaling capabilities as PPAR␥ agonists include free fatty acids, components of oxidized endogenous nuclear receptor-dependent paracrine signaling plasma lipoproteins (9- and 13-oxoODE, azPC), conjugated LA molecules with a potency that rivals thiazolidinediones (Fig. 1C). derivatives (CLA1 and CLA2), products of phospholipase hy- Present data reveal that LNO2 mediates cell differentiation in drolysis of complex lipids (LPA), platelet-activating factor, and adipocytes, CD36 expression in macrophages, and inflammato- eicosanoid derivatives such as the dehydration product of PGD2, ry-related signaling events in endothelium (e.g., inhibition of ⌬12,14 15-deoxy- -PGJ2 (14, 22, 23). Herein, we observed minimal vascular cell adhesion molecule-1 expression and function; data or no activation of PPAR␥ reporter gene expression by 1–3 ␮M not shown) with an important contribution from PPAR␥- concentrations of these putative ligands, in contrast to the dependent mechanisms (Fig. 3). Nitroalkene derivatives of fatty ␥ dose-dependent PPAR activation by LNO2 that, for PPAR , was acids thus represent a unique class of receptor-dependent cell significant at clinically relevant concentrations as low as 100 nM. differentiation, metabolic, and anti-inflammatory signaling mol- A dilemma exists in that some putative endogenous PPAR␥ ecules that serve to converge NO and oxygenated lipid redox agonists have only been generated by aggressive in vitro oxidizing signaling pathways. conditions (e.g., Cu-mediated low-density lipoprotein oxidation) and have not been clinically quantified or detected (for instance, We thank Dr. Carlos Batthyany for helpful input. This work was azPC and conjugated LA). Other lipid derivatives proposed as supported by National Institutes of Health Grants HL58115 and PPAR ligands are present in Ͻ100 nM tissue concentrations, HL64937 (to B.A.F.) and HL068878, HL075397, HL03676, and

2344 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408384102 Schopfer et al. Downloaded by guest on September 24, 2021 S06GM08248 (to Y.E.C.). F.J.S. and Y.L. were supported by postdoc- filiate. P.R.S.B. was supported by National Institutes of Health Cardio- toral fellowships from the American Heart Association Southeast Af- vascular Hypertension Training Grant T32HL07457.

1. Schopfer, F. J., Baker, P. R. & Freeman, B. A. (2003) Trends Biochem. Sci. 28, 22. Kliewer, S. A., Lenhard, J. M., Willson, T. M., Patel, I., Morris, D. C. & 646–654. Lehmann, J. M. (1995) Cell 83, 813–819. 2. Hogg, N. (2002) Annu. Rev. Pharmacol. Toxicol. 42, 585–600. 23. Davies, S. S., Pontsler, A. V., Marathe, G. K., Harrison, K. A., Murphy, R. C., 3. Rubbo, H., Radi, R., Anselmi, D., Kirk, M., Barnes, S., Butler, J., Eiserich, J. P. Hinshaw, J. C., Prestwich, G. D., Hilaire, A. S., Prescott, S. M., Zimmerman, & Freeman, B. A. (2000) J. Biol. Chem. 275, 10812–10818. G. A., et al. (2001) J. Biol. Chem. 276, 16015–16023. 4. Marnett, L. J., Wright, T. L., Crews, B. C., Tannenbaum, S. R. & Morrow, J. D. 24. McIntyre, T. M., Pontsler, A. V., Silva, A. R., St Hilaire, A., Xu, Y., Hinshaw, (2000) J. Biol. Chem. 275, 13427–13430. J. C., Zimmerman, G. A., Hama, K., Aoki, J., Arai, H., et al. (2003) Proc. Natl. 5. O’Donnell, V. B. & Freeman, B. A. (2001) Circ. Res. 88, 12–21. Acad. Sci. USA 100, 131–136. 6. Coles, B., Bloodsworth, A., Eiserich, J. P., Coffey, M. J., McLoughlin, R. M., 25. Zhang, C., Baker, D. L., Yasuda, S., Makarova, N., Balazs, L., Johnson, L. R., Giddings, J. C., Lewis, M. J., Haslam, R. J., Freeman, B. A. & O’Donnell, V. B. Marathe, G. K., McIntyre, T. M., Xu, Y., Prestwich, G. D., et al. (2004) J. Exp. (2002) J. Biol. Chem. 277, 5832–5840. Med. 199, 763–774. 7. Coles, B., Bloodsworth, A., Clark, S. R., Lewis, M. J., Cross, A. R., Freeman, 26. Nagy, L., Tontonoz, P., Alvarez, J. G., Chen, H. & Evans, R. M. (1998) Cell 93, B. A. & O’Donnell, V. B. (2002) Circ. Res. 91, 375–381. 229–240. 8. Rubbo, H., Radi, R., Trujillo, M., Telleri, R., Kalyanaraman, B., Barnes, S., 27. Von Knethen, A. & Brune, B. (2002) J. Immunol. 169, 2619–2626. Kirk, M. & Freeman, B. A. (1994) J. Biol. Chem. 269, 26066–26075. 28. Ferry, G., Bruneau, V., Beauverger, P., Goussard, M., Rodriguez, M., Lamamy, 9. Baker, P. R., Schopfer, F. J., Sweeney, S. & Freeman, B. A. (2004) Proc. Natl. V., Dromaint, S., Canet, E., Galizzi, J. P. & Boutin, J. A. (2001) Eur. Acad. Sci. USA 101, 11577–11582. J. Pharmacol. 417, 77–89. 10. Lee, C. H. & Evans, R. M. (2002) Trends Endocrinol. Metab. 13, 331–335. 29. Chawla, A., Barak, Y., Nagy, L., Liao, D., Tontonoz, P. & Evans, R. M. (2001) 11. Marx, N., Duez, H., Fruchart, J. C. & Staels, B. (2004) Circ. Res. 94, Nat. Med. 7, 48–52. 1168–1178. 30. Tontonoz, P., Hu, E., Graves, R. A., Budavari, A. I. & Spiegelman, B. M. (1994) 12. Wang, N., Verna, L., Chen, N. G., Chen, J., Li, H., Forman, B. M. & Genes Dev. 8, 1224–1234. Stemerman, M. B. (2002) J. Biol. Chem. 277, 34176–34181. 31. Tontonoz, P., Hu, E. & Spiegelman, B. M. (1994) Cell 79, 1147–1156. 13. Chen, Y. E., Fu, M., Zhang, J., Zhu, X., Lin, Y., Akinbami, M. A. & Song, Q. 32. Evans, R. M., Barish, G. D. & Wang, Y. X. (2004) Nat. Med. 10, 355–361. (2003) Vitam. Horm. 66, 157–188. 33. Delerive, P., Furman, C., Teissier, E., Fruchart, J., Duriez, P. & Staels, B. 14. Bell-Parikh, L. C., Ide, T., Lawson, J. A., McNamara, P., Reilly, M. & (2000) FEBS Lett. 471, 34–38. FitzGerald, G. A. (2003) J. Clin. Invest. 112, 945–955. 34. Pawliczak, R., Han, C., Huang, X. L., Demetris, A. J., Shelhamer, J. H. & Wu, 15. Rosen, E. D. & Spiegelman, B. M. (2001) J. Biol. Chem. 276, 37731–37734. T. (2002) J. Biol. Chem. 277, 33153–33163. 16. Tzameli, I., Fang, H., Ollero, M., Shi, H., Hamm, J. K., Kievit, P., Hollenberg, 35. Saulnier-Blache, J. S., Girard, A., Simon, M. F., Lafontan, M. & Valet, P. A. N. & Flier, J. S. (2004) J. Biol. Chem. (2000) J. Lipid Res. 41, 1947–1951. 17. Fu, J., Gaetani, S., Oveisi, F., Lo, V. J., Serrano, A., Rodriguez, D. F., 36. Xu, Y., Shen, Z., Wiper, D. W., Wu, M., Morton, R. E., Elson, P., Kennedy, Rosengarth, A., Luecke, H., Di Giacomo, B., Tarzia, G., et al. (2003) Nature A. W., Belinson, J., Markman, M. & Casey, G. (1998) J. Am. Med. Assoc. 280, 425, 90–93. 719–723. 18. Zhang, J., Fu, M., Cui, T., Xiong, C., Xu, K., Zhong, W., Xiao, Y., Floyd, D., 37. Chen, Z., Ishibashi, S., Perrey, S., Osuga, J., Gotoda, T., Kitamine, T., Tamura, Liang, J., Li, E., et al. (2004) Proc. Natl. Acad. Sci. USA 101, 10703–10708. Y., Okazaki, H., Yahagi, N., Iizuka, Y., et al. (2001) Arterioscler. Thromb. Vasc. 19. Mukherjee, R., Hoener, P. A., Jow, L., Bilakovics, J., Klausing, K., Mais, D. E., Biol. 21, 372–377. Faulkner, A., Croston, G. E. & Paterniti, J. R., Jr. (2000) Mol. Endocrinol. 14, 38. Claudel, T., Leibowitz, M. D., Fievet, C., Tailleux, A., Wagner, B., Repa, J. J., 1425–1433. Torpier, G., Lobaccaro, J. M., Paterniti, J. R., Mangelsdorf, D. J., et al. (2001) 20. Zhang, J., Fu, M., Zhu, X., Xiao, Y., Mou, Y., Zheng, H., Akinbami, M. A., Proc. Natl. Acad. Sci. USA 98, 2610–2615. Wang, Q. & Chen, Y. E. (2002) J. Biol. Chem. 277, 11505–11512. 39. Collins, A. R., Meehan, W. P., Kintscher, U., Jackson, S., Wakino, S., Noh, G., 21. Forman, B. M., Tontonoz, P., Chen, J., Brun, R. P., Spiegelman, B. M. & Evans, Palinski, W., Hsueh, W. A. & Law, R. E. (2001) Arterioscler. Thromb. Vasc. Biol. R. M. (1995) Cell 83, 803–812. 21, 365–371. BIOCHEMISTRY

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