Lipids (2014) 49:457–466 DOI 10.1007/s11745-014-3893-8
ORIGINAL ARTICLE
Nitrooleate Mediates Nitric Oxide Synthase Activation in Endothelial Cells Eunju Shin • Eunju Yeo • Jihye Lim • Yun Hee Chang • Haeryun Park • Eugene Shim • Haeyon Chung • Hye Jin Hwang • Jiyeon Chun • Jinah Hwang
Received: 14 August 2013 / Accepted: 18 February 2014 / Published online: 25 March 2014 Ó AOCS 2014
Abstract Nitrated lipids such as nitrooleate (OLA-NO2) can act as endogenous peroxisome proliferator-activated receptor gamma (PPARc) ligands to exert vascular protective effects. However, the molecular mechanisms regarding nitric oxide (NO) production and its regulation are not fully defined in the vasculature. Here, we show that OLA-NO2 increased endothelial NO release by modulating activation of endothelial nitric oxide synthase (eNOS) in endothelial cells. Treatment with OLA-NO2 (3 lM) increased NO release in a time-dependent manner. OLA-NO2 decreased protein expression of eNOS and caveolin-1 (Cav-1) but increased heat shock protein 90 (Hsp90) expression. Immunoprecipitation analysis confirmed that OLA-NO2 replaced eNOS/Cav-1 with eNOS/ Hsp90 interaction, resulting in increasing eNOS activity. OLA-NO2 also induced eNOS phosphorylation at Ser633 and Ser1177 and eNOS dephosphorylation at Ser113 and Thr495. In addition, OLA-NO2 induced phosphorylation of Akt and extracellular signal-regulated protein kinase
E. Shin E. Yeo J. Lim Y. H. Chang H. Park J. Hwang (&) Department of Food and Nutrition, College of Natural Sciences, Myongji University, YongIn 449-728, Korea e-mail: [email protected] E. Shim H. Chung Department of Food and Nutrition, Soongeui Women’s College, Seoul, Korea H. J. Hwang Department of Food and Nutrition, Dongeui University, Busan, Korea J. Chun Department of Food Science and Technology, Sunchon National University, Sunchon, Jeonnam, Korea
(ERK1/2), which might contribute to eNOS activation. Collectively, these results substantiate a new functional role for nitrated fatty acid, demonstrating that OLA-NO2 exerts vascular protective effects by increasing NO bioavailability through eNOS phosphorylation/dephosphorylation and interaction with associated proteins such as Hsp90 and Cav-1. Keywords HUVEC
Nitrooleate eNOS Hsp90 Caveolin-1
Abbreviations OLA-NO2 Nitrooleate PPARc Peroxisome proliferator-activated receptor gamma NO Nitric oxide eNOS Endothelial nitric oxide synthase Cav-1 Caveolin-1 Hsp90 Heat shock protein 90 Akt Protein kinase B ERK1/2 Extracellular signal-regulated protein kinase HO-1 Heme oxygenase-1 15d-PGJ2 15-Deoxy-D12,14-prostaglandin TZD Thiazolidinediones VEGF Vascular endothelial growth factor BAEC Bovine aortic endothelial cells HUVEC Human umbilical vein endothelial cells OLA Oleic acid NO2Nitrite MAPK Mitogen-activated protein kinase IP Immunoprecipitation AIS Autoinhibitory sequences CaM Calmodulin CHIP C-terminus of the Hsp70-interacting protein PI3K Phosphatidylinositide-3 kinase
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MKP
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Mitogen-activated protein kinase phosphatases
Introduction Nitroalkene derivatives such as nitrooleate (OLA-NO2) have emerged as unique classes of nitric oxide (NO) and fatty acid-derived signaling molecules. Recent studies have shown that OLA-NO2 and nitrolinoleic acid are the largest pools of bioactive nitrogen oxides in the vasculature at the concentrations of *1 lM in human circulation [1]. Emerging evidence suggests that nitrated lipids have been considered pluripotent cell signaling molecules because they induce vasorelaxation of rat aortic rings, prohibiting aggregation of human platelets and vascular smooth muscle cell proliferation, and attenuate lipopolysaccharidemediated macrophage cytokine releases, superoxide generation, and integrin expression of human neutrophil [1–3]. Additionally, nitrated fatty acids can act as Peroxisome proliferator-activated receptor gamma (PPARc) ligands within physiological concentrations in PPARc-dependent and PPARc-independent manners [1–3]. Nitrated lipids are expected to exert the vascular protective effects by acting as PPARc ligands [3, 4]. Recent studies indicate that PPARc regulates vascular functions such as anti-inflammatory response and cell recruitment in vascular endothelial and smooth muscle cells [5, 6] and mouse models [7]. Activation of PPARc by both endogenous and synthetic ligands in endothelial cells significantly increased activity of endothelial nitric oxide synthase (eNOS), Cu/Zn superoxide dismutase and heme oxygenase-1 (HO-1) and decreased NADPH oxidase, resulting in vascular protection [7, 8]. Moreover, nitrated fatty acids have the electrophilic property of the b-carbon adjacent to the nitro-bonded carbon [2, 3, 9]. It can facilitate Michael addition reactions with biological nucleophiles such as cysteine, histidine, and lysine residues of target proteins [9]. For example, 15-deoxy-D12,14-prostaglandin (15dPGJ2) is a potent natural ligand for PPARc and exerts a variety of biological functions through PPARc-dependent and PPARc-independent pathways [1–4] due to the electrophilic double bond of cyclopentenone ring [1, 2]. In addition, nitrated lipids were recently reported to play a role in inhibiting lipopolysaccharide-induced inflammatory responses, accompanied by nitroalkylation of NF-jB p65 protein [3]. PPARc ligands such as 15d-PGJ2 and thiazolidinediones (TZD) have been reported to increase NO release and bioavailability through modulation of eNOS activity [5, 10]. eNOS is a key molecule in normal vascular biology
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and pathophysiology. NO produced by eNOS mediates vasorelaxation and inhibits vascular tone, smooth muscle cell proliferation, platelet aggregation and leukocyte adherence to the vascular wall [5, 11–14]. Regulation of eNOS is controlled by eNOS-interacting protein interactions, phosphorylation, subcellular localization, fatty acid acylation, and cofactor availability [11, 12]. For example, specific stimuli such as vascular endothelial growth factor (VEGF), bradykinin, and shear stress have shown to induce eNOS activity by facilitating replacement of caveolin-1 (Cav-1), eNOS-inhibitory protein, with heat shock protein 90 (Hsp90), eNOS-stimulatory protein [13, 14]. In addition to eNOS–protein interactions, phosphorylation/dephosphorylation of eNOS also increases the enzyme activity through induction of electron flux from the reductase to the oxygenase domain of eNOS [11, 12]. Recently, OLA-NO2 was reported to increase eNOS activity by phosphorylation of eNOS-ser1179 in bovine aortic endothelial cells (BAEC) and mouse models [15]. However, the precise molecular mechanisms of OLANO2-stimulated endothelial NO release remain to be defined. The current study was designed to further characterize the molecular mechanisms underlying OLA-NO2stimulated eNOS activity to induce NO bioavailability and vascular protection in human endothelial cells. Therefore, we first defined the effects of OLA-NO2 on multi-site phosphorylation/dephosphorylation of eNOS and eNOSinteracting proteins in NO production and potential components, i.e. Akt and ERK1/2, of eNOS activity.
Materials and Methods Cell Culture and Reagents Human umbilical vein endothelial cells (HUVEC; Lonza) monolayers were grown and maintained at 37 °C in 5 % CO2 in endothelial cell basal medium-2 according to protocols provided by the manufacturer (Lonza). In all experiments, cells were plated on 0.5 % gelatin-coated plates and confluent HUVEC (passages 2–8) were treated with vehicle (0.1 % ethanol), 3 lM OA-NO2, or oleic acid (OLA). OLA was used as a negative control. NO Production Analysis NO release was determined by the accumulation of nitrite (NO2-), the stable oxidation product of NO, using the NOspecific fluorometric assay kit (Cayman). Briefly, nitrate reductase was added to 100 ll of media and incubated at room temperature for 30 min for the conversion of nitrate into nitrite. After incubation, 2,3-diaminonaphthotriazole was added for an acidic solution, followed by NaOH which
Lipids (2014) 49:457–466 Table 1 PCR primers and conditions
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Gene
Primers
Tm (°C)
Cycle
Cav-1
Forward 50 -GACTTTGAAGATGTGATTGC-30
56
30
57
34
61
28
58
35
60
30
60
30
Reverse 50 -AGATGGAATAGACACGGCTG-30 eNOS
Forward 50 -CAGCACCTTGGCAGAAGAG-30 Reverse 50 -TTAGCCACGTGGAGCAGAC-30
Hsp90
Forward 50 -CATGAAAGCCCAGGCACTTCG-30 0
Reverse 5 -CTTCCATGCGAGACGCATCC-3 PPAR-c
0
Forward 50 -GAAATGACCATGGTTGACAC-30 Reverse 50 -CAGGACTCTCTGCTAGTACA-30
Actin
Forward 50 -AGAAAATCTGGCACCACACC-30 Reverse 50 -CTCCTTAATGTCACGCACGA-30
GAPDH
Forward 50 -ACCACAGTCCATGCCATCAC-30 Reverse 50 -TCCACCACCCTGTTGCTGTA-30
enhances the detection of the fluorescent product, 1(H)naphthotriazole. Absorbance was measured at 360 nm for excitation and 430 nm for emission using a fluorescence spectrophotometer (Perkin Elmer). Each sample was normalized to cell protein content and a serial dilution of nitrite standard was prepared for each experiment.
peroxidase-conjugated secondary antibody (diluted 1:5,000 in Tris-buffered saline with TweenÒ 20). Immunodetection was performed using a chemiluminecence method (Pierce). After immunodetection, relative densities of bands were quantified using an NIH ImageJ program. All data were normalized to the actin content of the same sample.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis
Immunoprecipitation (IP)
Total RNA was isolated using the Trizol reagent (Invitrogen). Total RNA (2 lg) was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (Invitrogen). PCR was performed using Taq polymerase (Takara) and the primer pairs (Bioneer). Relative mRNA levels were quantified using the NIH ImageJ program. Primer sequences and PCR conditions were described in Table 1.
After experimental treatments, cells were washed with in PBS and lysed with IP buffer [50 mM Tri-HCl (pH 7.4), 150 mM NaCl, and 1 % NP-40] containing protease inhibitors and PMSF. Whole cell lysates (200 lg protein) were incubated with a monoclonal antibody to eNOS (#610297; BD Transduction Laboratories) and successively with protein G-agarose beads (Roche). Immunoprecipitates were immunoblotted as described in western blot analysis. Statistical Analysis
Western Blot Analysis Whole cell lysates (20 lg of protein/lane) derived from HUVEC treated with either 3 lM OLA-NO2, or OLA were resolved in 10 % Bis–Tris gels and transferred onto a polyvinylidene difluoride membrane. After the membrane was blocked in 5 % skim milk, the membrane was probed overnight at 4 °C with primary antibodies (diluted 1:1,000 in 5 % bovine serum albumin solution) specific to eNOS (#610297), Hsp90 (#610418), Cav-1 (#610057), eNOSThr495 (#612707), eNOS-Ser633 (#612665), eNOSSer1177 (#612393) (BD Transduction Laboratories), eNOS-Ser113 (#9575), Akt (#9272), Akt-Ser473 (#9271), P44/42 MAPK (ERK1/2; #9102), phsopho-p44/42 MARK (#9101) (Cell Signaling) and actin (#sc-1616) (Santa Cruz). After being washed, the membrane was incubated for 1 h at room temperature with the corresponding horseradish
All data are presented as means ± SD. The data were analyzed with nonparametric methods due to small sample sizes using SPSS computer-based statistics programs (Ver. 20). The Mann–Whitney test and the Wilcoxon rank sum test were performed for statistical differences between two groups and one-way analysis of variance, respectively. The level of statistical significance was taken as p \ 0.05.
Results OLA-NO2 Increases NO Production in Endothelial Cells To determine the effect of OLA-NO2 on endothelial function, we investigated the NO generation in HUVEC.
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OLA-NO2 Increases eNOS Activity Through Phosphorylation/dephosphorylation of eNOS
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NO production (%Control)
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*
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Fig. 1 Effects of OLA-NO2 on NO production. HUVEC were treated with vehicle (control), 3 lM OLA-NO2 or OLA. After each treatment, NO production was measured by the Griess method. Each bar represents the mean ± SEM (n = 4); *p \ 0.05 vs. the control group
Compared to the control group, treatment with 3 lM OLANO2 significantly increased NO production by 50–100 % in a time-dependent manner (Fig. 1). 5 lM 15d-PGJ2 as a positive control significantly increased NO production for 8 h by 50 %. NO production of OLA-NO2 was consistently greater than that of 15d-PGJ2, which has already shown NO release as a PPARc ligand in previous studies [5, 6]. The corresponding native OLA for 4 h had no stimulatory effect on NO production. This result indicated that OLANO2 increased NO production like the other PPARc ligand, 15d-PGJ2 in endothelial cells. OLA-NO2 Regulates mRNA and Protein Expression of eNOS-related Genes To determine whether OLA-NO2 regulates eNOS and eNOS-related genes to increase NO release, we examined mRNA and protein expression of eNOS, Hsp90, Cav-1, and PPARc. HUVEC were treated with either 3 lM OLA-NO2, or OLA. In terms of mRNA levels, OLA-NO2 had no effect on eNOS but to a somewhat lesser extent, increased Hsp90 and decreased Cav-1 mRNA levels. However, OLA-NO2 significantly increased PPARc mRNA level at early times (2–8 h) (Fig. 2a). With respect to protein expression, treatment with 3 lM OLA-NO2 significantly decreased eNOS and Cav-1 expression, while OLA-NO2 markedly increased Hsp90 and PPARc expression in time-dependent (Fig. 2b) and dose-dependent manners (data not shown) when normalized to actin expression. The molecular effects of native OLA were similar to those of the control group (Fig. 2a, b).
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Because OLA-NO2 increased NO release without increasing eNOS expression, post-translational mechanisms of eNOS activity were examined. Despite that OLA-NO2 decreased total expression, OLA-NO2 increased phosphorylation of eNOS-Ser1177 at early times (2–4 h) and then gradually increased phosphorylation of eNOS-Ser633 until 16 h. However, OLA-NO2 gradually increased dephosphorylation of eNOS at Ser113 and Thr495. The corresponding native OLA had no effects on phosphorylation and dephosphorylation of eNOS (Fig. 3a). These results suggested that OLA-NO2 stimulated eNOS activity for NO production through changes in eNOS phosphorylation/dephosphorylation. OLA-NO2 Alters Interactions of eNOS with Associated Proteins Since proportion of Hsp90 and Cav-1 to eNOS association determines eNOS activation, we examined physical interactions between eNOS, Hsp90 and Cav-1 using immunoprecipitation (IP). In spite of eNOS reduction, treatment with OLA-NO2 significantly decreased the overall cellular content of Cav-1 and instead, increased that of Hsp90 associated eNOS in a time-dependent manner. Effects of native OLA were similar to those of the control group (Fig. 3b). Therefore, this result suggested that OLA-NO2 stimulated eNOS activity for NO production through changes in physical interactions of eNOS with associated proteins. OLA-NO2 Induces Phosphorylation of Akt and ERK1/2 Protein Kinases Several kinases such as phosphatidylinositide-3 Kinase (PI3K)/Akt (Protein Kinase B), extracellular signal-regulated kinase (ERK), AMP-activated protein kinase, protein kinase G, CaM-dependent kinase, and protein kinase A have been shown to be involved in eNOS phosphorylation [11, 12]. Therefore, the possibility that OLA-NO2 activates eNOS through Akt-dependent and ERK1/2 (P44/P42 MAPK)-dependent changes in the phosphorylation levels of eNOS was examined. OLANO2 increased the phosphorylation levels of Akt-Ser473 (Fig. 4a) and ERK1/2 (P44-Thr202/P42-Tyr204) (Fig. 4b). Phosphorylation of Akt-Ser473 was induced by threefold at 2 h treatment, followed by phosphorylation of ERK1/2 with peak induction (sixfold) at 4 h. After peak induction, each kinase gradually recovered basal levels. Native OLA had no effects on phosphorylation of Akt and ERK1/2 (Fig. 4a, b). This result indicates that
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Fig. 2 Effects of OLA-NO2 on mRNA and protein expression. HUVEC were treated with vehicle (control), 3 lM OLANO2 or OLA. a RT-PCR was performed using each genespecific primer. b Cell lysates were prepared and subjected to SDS-PAGE, followed by immunoblotting for eNOS, Hsp90, Cav-1, PPARc and actin. Each bar represents the mean ± SEM (n = 4). *p \ 0.05 vs. the control group
3 µM OLA-NO2
A 0
2
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OLA 16
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(hr) eNOS Hsp90
Cav-1 PPARγ Actin
100 80
*
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40 20 0
0
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3 µM OLA-NO2
Cav-1/actin (% Control)
Hsp90/actin (% Control)
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(hr)
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* *
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*
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16 5
3 µM OLA-NO2
Akt and ERK1/2 are potential upstream components of eNOS signaling.
Discussion Previous studies [2–4, 15] reported that OLA-NO2 acted as an endogenous PPARc ligand and induced
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*
120 100 80 60 40 20 0 10
46 OLA
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34
48
16 5
3 µM OLA-NO2
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80
*
160
OLA
PPAR γ/actin (% Control)
B
eNOS/actin (% Control)
GAPDH
(hr)
*
250
64
(hr)
OLA
*
*
200 150
*
100 50 0 10
22
34
48
16 5
3 µM OLA-NO2
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(hr)
OLA
eNOS activity by phosphorylation of eNOS-ser1177 by MAP kinase-dependent signaling in BAEC. In the present study, we demonstrate that OLA-NO2 regulated reciprocal interactions of eNOS/Hsp90 and eNOS/ Cav-1 and multisite phosphorylation of eNOS through Akt and ERK1/2 signaling, leading to eNOS activity and subsequent NO production in human endothelial cells.
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A
3 µM OLA-NO2
0
2
4
8
B
OLA 16
4
IP : eNOS
(hr) eNOS pS113
3 µM OLA-NO2
eNOS pT495
0
4
8
OLA 16
4
(hr)
eNOS pS633
eNOS (140 kDa)
eNOS pS1177
Hsp90 (90 kDa)
Total eNOS (140 kDa)
Cav-1 (22 kDa)
Fig. 3 Effects of OLA-NO2 on site-specific eNOS phosphorylation and on interactions of eNOS/Hsp90 and eNOS/Cav-1. HUVEC were treated with vehicle (control), 3 lM OLA-NO2 or OLA. a Cell lysates were prepared and subjected to SDS-PAGE, followed by immunoblotting for phosphorylated sites (pS113, pT495, pS633 and
pS1177) of eNOS or total eNOS and b immunoprecipitated with monoclonal antibody to eNOS. Immunoprecipitates were then subjected to SDS-PAGE and immunoblotting for eNOS, Hsp90 and Cav-1 antibodies
B 350
*
300 250
*
200 150 100 50 0 0
2
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3 µM OLA-NO2
16
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(hr)
P-ERK/ERK (% Control)
A P-Akt/Akt (% Control)
2
OLA
1000
*
800
*
600 400 200 0 0
2
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3 µM OLA-NO2
16
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(hr)
OLA
Akt pS473
ERK1/2 pT202/Y204
Total Akt (60 kDa)
Total ERK1/2 (44/42 kDa)
Fig. 4 Effects of OLA-NO2 on site-specific Akt and ERK1/2 phosphorylation. HUVEC were treated with vehicle (control), 3 lM OLA-NO2 or OLA. Cell lysates were prepared and subjected to SDS-PAGE, followed by immunoblotting for phosphorylation of a Akt and b ERK1/2
eNOS is located in small invaginations of the plasma membrane called caveolae and produces NO in response to the stimuli. NO production in endothelial cells plays pivotal roles in vascular biology. Impaired NO leads to endothelial dysfunction, which is associated with risk factors against vascular diseases including hypercholesterolemia, diabetes mellitus, insulin resistance and obesity. Therefore, modulating NO production is a critical thing for prevention or treatment of endothelial dysfunction. Antidiabetic agents such as TZD drugs are used to boost NO production. The effects of these drugs are known to be mediated, in part, through their binding to PPARc. For example, both of endogenous (OLA-NO2 and 15d-PGJ2) and synthetic (rosiglitazone and ciglitazone) PPARc ligands, have been reported to increase endothelial NO production [5, 6]. However, numerous side effects such as hepatotoxicity, edema, heart failure, and weight gain demand the introduction of PPARc ligands and/or new therapy.
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Since OLA-NO2 acts as an endogenous PPARc ligand with an electrophilic center, OLA-NO2 has been reported to have similar characteristics to those of 15d-PGJ2 [2–4, 9]. 15d-PGJ2 was reported to decrease only eNOS protein expression, but not mRNA levels after 24 h treatment of 15d-PGJ2 in endothelial cells [2, 5]. In our previous study [16], 15d-PGJ2-induced eNOS reduction was strongly related to Hsp70 induction and eNOS translocation from soluble to insoluble fractions due to the biological activities of its electrophilic center but was not related to either transcriptional regulation, proteasomal degradation or apoptosis. In contrast to this result, it was reported that 15d-PGJ2 decreased both expression and activity of eNOS in HUVEC by inhibiting eNOS mRNA and protein synthesis, but this decrease was observed only after long-term treatment of 15d-PGJ2 for 48–72 h [17]. To our knowledge, there is only one report regarding the OLA-NO2induced alteration of eNOS expression in BAEC [15]. They reported that OLA-NO2 increased eNOS expression due to
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phosphorylation of eNOS-Ser1179 rather than transcriptional regulation with no difference in promoter activity. The differences in eNOS down-regulation with previous reports may be attributed to different culture conditions, such as cell sources and media systems. Interestingly, OLA-NO2-induced eNOS reduction is associated with Hsp90 induction and Cav-1 reduction in a time-dependent manner in HUVEC. In other words, Cav-1 reduction was replaced with Hsp90 induction. Hsp90, a molecular chaperone protein, is a crucial regulator of eNOS because it cooperatively enhances the affinity of eNOS for binding calmodulin (CaM) and balances output of NO and superoxide [11, 12]. Previous evidence has shown that the association of eNOS with Hsp90 enhances eNOS activity. For example, various stimuli, including VEGF, fluid shear stress, and estradiol, enhance the interaction between eNOS and Hsp90 to increase NO production [13]. Cav-1, the scaffolding protein of caveolae, is another regulator of eNOS and inhibits its activity by occupying its CaMbinding site in the basal state [14]. Various stimuli dissociate eNOS from Cav-1 and increase NO synthesis. For example, plasma NO levels were substantially increased in Cav-1-knockout mice [18]. Collectively, it is suggested that OLA-NO2 regulates the molecular expression and association of Hsp90 and Cav-1 with eNOS. Dissociation of eNOS from Cav-1, as a prerequisite for eNOS activation, may be compromised by the amount of Hsp90 associated with eNOS as well as by the molecular alteration. Since it has been controversial whether nitrated lipids can act as a PPARc ligand within physiological concentrations in PPARc-dependent and PPARc-independent manners [1–4], PPARc expression was examined. OLANO2 significantly increased mRNA and protein levels of PPARc at early times (2–8 h) and mRNA level of PPARc returned to that of the control group at later time points (8–16 h) (Fig. 2a, b). Based on the molecular alteration of PPARc, PPARc induction may be responsible for OLANO2 action. The activity of eNOS is regulated not only by the amount of expression but also by post-translational modifications including its phosphorylation, subcellular localization, and interactions of eNOS with associated proteins [11–14]. Precise regulation of eNOS activity involving multisite phosphorylation of specific serine and threonine residues has emerged. The five eNOS serine/threonine phosphorylation sites have been identified, including Ser613, Ser615, Ser633, Ser1177 and Thr 495 at human eNOS. Although the functional effects of each known site is controversial, it has been shown that eNOS activity was increased by phosphorylation of Ser615, Ser633, and Ser1177 but also dephosphorylation of Ser113 and Thr 495 [11, 12]. Besides confirming that nitrated fatty acids stimulated NO production through the phosphorylation of
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eNOS at Ser1179 in BAEC [15], we extended that OLANO2 stimulated NO production through four multisite regulations of eNOS phosphorylation. Since phosphorylation of eNOS at Ser633 and Ser1177 coincides with dephosphorylation of eNOS at Ser113 and Thr495 in this study, we confirmed that OLA-NO2 did stimulate eNOS activity and led to an increase in NO production, in part, through multisite coordination of eNOS phosphorylation. Interestingly, phosphorylation of Ser633 is a later event than phosphorylation of Ser1177. Phosphorylation of Ser1177 has been suggested to be critical for eNOS activation in response to several stimuli, such as shear stress, VEGF, and 8-bromo cAMP, which are known to increase NO bioavailability [19]. All three stimuli consistently induced slower phosphorylation of Ser635 than that of Ser1179, suggesting a priming effect of phosphorylation of Ser1177 for a subsequent phosphorylation of Ser633. Location of Ser633 and Ser117 in each of the two autoinhibitory sequences (i.e., AIS I and AIS II) might explain differential phosphorylation of eNOS. The Ser633 resides in the flavin mononucleotide binding domain in AIS I. Phosphorylation of Ser1177 in AIS II seems to dismiss the blockage of electron transfer within the C termini of the two eNOS monomers [20]. Structurally, phosphorylation of Ser633 is consistently preceded by phosphorylation of Ser1177 after removal of the hindrance imposed by AIS II [21]. Instead, later phosphorylation of Ser633 maintained persistent eNOS activity at the intracellular calcium level after its initial activation [22]. eNOS activity is regulated by direct interactions of the enzyme with cellular proteins, such as Hsp90, CaM, Cav-1 and G protein-coupled receptors [11, 12]. As described above, Hsp90 is an effective activator of eNOS activation because of the displacement of eNOS from inhibitory interaction with Cav-1. Conversely, Cav-1 is a potent inhibitor of eNOS activation because its scaffolding domain (amino acids 82–101) binds with oxygenase domain of eNOS [11–13]. IP analysis demonstrated that eNOS activity was strongly related to Hsp90 induction and partly to Cav-1 reduction, irrespective of eNOS reduction, suggesting that eNOS activity is most likely caused by direct Hsp90 induction. Although this study has demonstrated that OLA-NO2 increased eNOS activity through modulation of multisite phosphorylation of eNOS and eNOS-interacting proteins, there has been speculation about OLA-NO2-induced eNOS activity. In spite of eNOS reduction, enhanced eNOS activity is probably attributed to Hsp70 induction and eNOS translocation from soluble to insoluble fractions due to the biological activities of its electrophilic center [16]. The electrophilic property of the b-carbon adjacent to the nitro-bonded carbon can form covalent adducts with cysteine residues of cellular proteins [9]. Several cysteine
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residues (including 29 and 99 residues) of eNOS might be targets of covalent bonding [23], causing disruption of natural protein folding and subsequent eNOS protein aggregates in the insoluble fraction [16]. C-terminus of the Hsp70-interacting protein (CHIP) is another possible mechanism of eNOS trafficking. For example, 15d-PGJ2 re-distributed eNOS from the Golgi and plasma membrane to the cytoplasm instead of decreasing eNOS protein content, suggesting that 15d-PGJ2 might enhance eNOS-CHIP interactions and subsequent translocation [16]. The precise mechanism of OLA-NO2-induced eNOS phosphorylation remains to be defined but activity of kinase and phosphatase could be involved. Particularly, Akt pathway is predominantly involved in the regulation of Hsp90-mediated eNOS phosphorylation [13, 24] (Fig. 5).
Fig. 5 Potential mechanisms underlying OLA-NO2-mediated increases in endothelial NO release. In this model, OLA-NO2 stimulates endothelial NO release, in part by modulating posttranslational mechanisms of eNOS regulation, including multisite eNOS phosphorylation and eNOS–protein interactions. For example, OLANO2 increases phosphorylation of eNOS-Ser633 and eNOS-Ser1177 and dephosphorylation of eNOS-Ser113 and eNOS-Thr495. It also decreases the association of eNOS with Cav-1, and instead, increases the association of eNOS with Hsp90. Taken together, these observations demonstrate how OLA-NO2 directly enhances endothelial NO bioavailability, an effect that may contribute to the reported vascular protective effects of OLA-NO2
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Various stimuli such as estrogen, VEGF, and shear stress increased eNOS activity through PI3K-dependent Akt activation and subsequent eNOS-Ser1177 phosphorylation in endothelial cells [11, 12]. There is only one report regarding the effects of OLA-NO2 on the MAP kinase signaling pathway. Khoo et al. [15] reported that OLA-NO2 increased eNOS-Ser1179 phosphorylation and NO production via Akt-dependent, p38-dependent, and ERK1/2dependent signaling in BAEC. Enhanced eNOS activity can be imitated with recombinant Akt and overexpressed constitutively active Akt in intact cells [25, 26]. Treatment of 15d-PGJ2, not ciglitazone, also induced Akt phosphorylation, causing the cardioprotective signaling in male Wistar rats [27]. Besides, Cav-1 decreased the small GTPase Rac1, which in turn modulated the PI3K/Akt/ eNOS pathway [28]. Whereas numerous studies have demonstrated the functional role of Akt in the context of eNOS activation and NO release, the role of mitogen-activated protein kinase phosphatases (MKP) is quite controversial. MKP, dual-specificity phosphatases, play important roles in the regulation of ERK1/2, p38 kinase, and JNK signaling pathways. For example, bradykinin-induced ERK1/2 phosphorylation led to reduced eNOS activity in BAEC [29] and 15d-PGJ2induced ERK1/2 phosphorylation was involved in 15dPGJ2-induced apoptosis in cancer cells [30, 31]. In contrast to these findings, Cai et al. [32] reported that H2O2 treatment induced phosphorylation of ERK1/2 and PI3K/Akt, and subsequent eNOS-Ser1179 phosphorylation and NO production in BAEC. They suggested the possible mechanisms; (1) numerous potential ERK1/2 phosphorylation sites in eNOS (2) indirect relationship between ERK1/2 and eNOS through eNOS–Hsp90 interaction (3) involvement of ERK1/2 in the inhibitory interaction of eNOS/Cav-1. To our knowledge, it is the first to demonstrate that OLA-NO2 can stimulate NO production through four multisite regulations of eNOS phosphorylation and eNOS–proteins interactions via Akt and ERK1/2 signaling. In summary, our findings demonstrate that activation of OLA-NO2 in vascular endothelial cells provides a novel mechanism for stimulating endothelial NO release and hence NO bioavailability. OLA-NO2 increased phosphorylation of Akt and ERK1/2 and subsequent eNOS activation through reciprocal interactions of eNOS/Hsp90 and eNOS/Cav-1 and phosphorylation/dephosphorylation of eNOS, leading to NO production (Fig. 5). Collectively, these results demonstrate how OLA-NO2 has the potential to directly modify vascular endothelial function and to modulate the production of NO, a critical mediator in maintenance of normal vascular physiology. These findings further refine our understanding of nitroalkenes, novel targets for pharmacological intervention in the vascular system.
Lipids (2014) 49:457–466 Acknowledgments This study was supported by the Basic Science Research Program through the National Research Foundation of Korea Grant (NRF) funded by the Ministry of Education (No. 2010-0187 and 2011-0156).
References 1. Baker PR, Lin Y, Schopfer FJ, Woodcock SR, Groeger AL, Batthyany C, Sweeney S, Long MH, Iles KE, Baker LM, Branchaud BP, Chen YE, Freeman BA (2005) Fatty acid transduction of nitric oxide signaling; multiple nitrated unsaturated fatty acid derivatives exist in human blood and urine and serve as endogenous peroxisome proliferator-activated receptor ligands. J Biol Chem 280:42464–42475 2. Hwang J, Lee KE, Lim JY, Park SI (2009) Nitrated fatty acids prevent TNFalpha stimulated inflammatory and atherogenic responses in endothelial cells. Biochem Biophys Res Commun 387:633–640 3. Cui T, Schopfer FJ, Zhang J, Chen K, Ichikawa T, Baker PR, Batthyany C, Chacko BK, Feng X, Patel RP, Agarwal A, Freeman BA, Chen YE (2006) Nitrated fatty acids: endogenous antiinflammatory signaling mediators. J Biol Chem 281: 35686–35698 4. Li Y, Zhang J, Schopfer FJ, Martynowski D, Garcia-Barrio MT, Kovach A, Suino-Powell K, Baker PR, Freeman BA, Chen YE, Xu HE (2008) Molecular recognition of nitrated fatty acids by PPAR gamma. Nat Struct Mol Biol 15:865–867 5. Calnek DS, Mazzella L, Roser S, Roman J, Hart CM (2003) Peroxisome proliferator-activated receptor gamma ligands increase release of nitric oxide from endothelial cells. Arterioscler Thromb Vasc Biol 23:52–57 6. Hwang J, Kleinhenz DJ, Lasse`gue B, Griendling KK, Dikalov S, Hart CM (2005) Peroxisome proliferator-activated receptorgamma ligands regulate endothelial membrane superoxide production. Am J Physiol Cell Physiol 288:C899–C905 7. Hwang J, Kleinhenz DJ, Rupnow HL, Campbell AG, Thule´ PM, Sutliff RL, Hart CM (2007) The PPARgamma ligand, rosiglitazone, reduces vascular oxidative stress and NADPH oxidase expression in diabetic mice. Vascul Pharmacol 46:456–462 8. Wright MM, Schopfer FJ, Baker PR, Vidyasagar V, Powell P, Chumley P, Iles KE, Freeman BA, Agarwal A (2006) Fatty acid transduction of nitric oxide signaling: nitrolinoleic acid potently activates endothelial heme oxygenase 1 expression. Proc Natl Acad Sci USA 103:4299–4304 9. Batthyany C, Schopfer FJ, Baker PR, Dura´n R, Baker LM, Huang Y, Cerven˜ansky C, Branchaud BP, Freeman BA (2006) Reversible post-translational modification of proteins by nitrated fatty acids in vivo. J Biol Chem 281:20450–20463 10. Polikandriotis JA, Mazzella LJ, Rupnow HL, Hart CM (2005) Peroxisome proliferator-activated receptor gamma ligands stimulate endothelial nitric oxide production through distinct peroxisome proliferator-activated receptor gamma-dependent mechanisms. Arterioscler Thromb Vasc Biol 25:1810–1816 11. Dudzinski DM, Michel T (2007) Life history of eNOS: partners and pathways. Cardiovasc Res 75:247–260 12. Mineo C, Shaul PW (2012) Regulation of eNOS in caveolae. Adv Exp Med Biol 729:51–62 13. Balligand JL (2002) Heat shock protein 90 in endothelial nitric oxide synthase signaling: following the lead(er)? Circ Res 90:838–841 14. Garcı´a-Carden˜a G, Martasek P, Masters BS, Skidd PM, Couet J, Li S, Lisanti MP, Sessa WC (1997) Dissecting the interaction between nitric oxide synthase and caveolin. Functional
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significance of the nos caveolin binding domain in vivo. J Biol Chem 272:25437–25440 Khoo NK, Rudolph V, Cole MP, Golin-Bisello F, Schopfer FJ, Woodcock SR, Batthyany C, Freeman BA (2010) Activation of vascular endothelial nitric oxide synthase and heme oxygenase-1 expression by electrophilic nitro-fatty acids. Free Radic Biol Med 48:230–239 Hwang J, Lee HI, Chang YS, Lee SJ, Kim KP, Park SI (2007) 15-deoxy-Delta12, 14-prostaglandin J2-induced down-regulation of endothelial nitric oxide synthase in association with HSP70 induction. Biochem Biophys Res Commun 357:206–211 Jo´zkowicz A, Nigisch A, Winter B, Weigel G, Hukb I, Dulaka J (2004) 15-Deoxy-D12,14-prostaglandin-J2 inhibits expression of eNOS in human endothelial cells. Prostaglandins Other Lipid Mediat 74:11–28 Zhao YY, Liu Y, Stan RV, Fan L, Gu Y, Dalton N, Chu PH, Peterson K, Ross J Jr, Chien KR (2002) Defects in caveolin-1 cause dilated cardiomyopathy and pulmonary hypertension in knockout mice. Proc Natl Acad Sci USA 99:11375–11380 Boo YC, Hwang J, Sykes M, Michell BJ, Kemp BE, Lum H, Jo H (2002) Shear stress stimulates phosphorylation of eNOS at Ser(635) by a protein kinase A-dependent mechanism. Am J Physiol Heart Circ Physiol 283:1819–1828 Michell BJ, Harris MB, Chen ZP, Ju H, Venema VJ, Blackstone MA, Huang W, Venema RC, Kemp BE (2001) Identification of regulatory sites of phosphorylation of the bovine endothelial nitric-oxide synthase at serine 617 and serine 635. J Biol Chem 277:2344–42351 McCabe TJ, Fulton D, Roman LJ, Sessa WC (2000) Enhanced electron flux and reduced calmodulin dissociation may explain ‘‘calcium-independent’’ eNOS activation by phosphorylation. J Biol Chem 275:6123–6128 Boo YC, Sorescu GP, Bauer PM, Fulton D, Kemp BE, Harrison DG, Sessa WC, Jo H (2003) Endothelial NO synthase phosphorylated at SER635 produces NO without requiring intracellular calcium increase. Free Radic Biol Med 35:729–741 Ravi K, Brennan LA, Levic S, Ross PA, Black SM (2004) S-nitrosylation of endothelial nitric-oxide synthase is associated with monomerization and decreased enzyme activity. Proc Natl Acad Sci USA 101:2619–2624 Sato S, Fujita N, Tsuruo T (2004) Modulation of Akt kinase activity by binding to Hsp90. Proc Natl Acad Sci USA 97:10832–10837 Dimmeler S, Fleming I, Fisslthaler B, Hermann C, Busse R, Zeiher AM (1999) Activation of nitric oxide synthase in endothelial cells by Akt-dependent phosphorylation. Nature 399:601–605 Dimmeler S, Assmus B, Hermann C, Haendeler J, Zeiher AM (1998) Fluid shear stress stimulates phosphorylation of Akt in human endothelial cells: involvement in suppression of apoptosis. Circ Res 83:334–341 Zingarelli B, Hake PW, Mangeshkar P, O’Connor M, Burroughs TJ, Piraino G, Denenberg A, Wong HR (2007) Diverse cardioprotective signaling mechanisms of peroxisome proliferatoractivated receptor-gamma ligands, 15-deoxy-Delta12,14-prostaglandin J2 and ciglitazone, in reperfusion injury: role of nuclear factor-kappaB, heat shock factor 1, and Akt. Shock 28:554–563 Gonzalez E, Nagiel A, Lin AJ, Golan DE, Michel T (2004) Small interfering RNA-mediated down-regulation of caveolin-1 differentially modulates signaling pathways in endothelial cells. J Biol Chem 279:40659–40669 Bernier SG, Haldar S, Michel T (2000) Bradykinin-regulated interactions of the mitogen-activated protein kinase pathway with the endothelial nitric-oxide synthase. J Biol Chem 275: 30707–30715
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ORIGINAL ARTICLE
Nitrooleate Mediates Nitric Oxide Synthase Activation in Endothelial Cells Eunju Shin • Eunju Yeo • Jihye Lim • Yun Hee Chang • Haeryun Park • Eugene Shim • Haeyon Chung • Hye Jin Hwang • Jiyeon Chun • Jinah Hwang
Received: 14 August 2013 / Accepted: 18 February 2014 / Published online: 25 March 2014 Ó AOCS 2014
Abstract Nitrated lipids such as nitrooleate (OLA-NO2) can act as endogenous peroxisome proliferator-activated receptor gamma (PPARc) ligands to exert vascular protective effects. However, the molecular mechanisms regarding nitric oxide (NO) production and its regulation are not fully defined in the vasculature. Here, we show that OLA-NO2 increased endothelial NO release by modulating activation of endothelial nitric oxide synthase (eNOS) in endothelial cells. Treatment with OLA-NO2 (3 lM) increased NO release in a time-dependent manner. OLA-NO2 decreased protein expression of eNOS and caveolin-1 (Cav-1) but increased heat shock protein 90 (Hsp90) expression. Immunoprecipitation analysis confirmed that OLA-NO2 replaced eNOS/Cav-1 with eNOS/ Hsp90 interaction, resulting in increasing eNOS activity. OLA-NO2 also induced eNOS phosphorylation at Ser633 and Ser1177 and eNOS dephosphorylation at Ser113 and Thr495. In addition, OLA-NO2 induced phosphorylation of Akt and extracellular signal-regulated protein kinase
E. Shin E. Yeo J. Lim Y. H. Chang H. Park J. Hwang (&) Department of Food and Nutrition, College of Natural Sciences, Myongji University, YongIn 449-728, Korea e-mail: [email protected] E. Shim H. Chung Department of Food and Nutrition, Soongeui Women’s College, Seoul, Korea H. J. Hwang Department of Food and Nutrition, Dongeui University, Busan, Korea J. Chun Department of Food Science and Technology, Sunchon National University, Sunchon, Jeonnam, Korea
(ERK1/2), which might contribute to eNOS activation. Collectively, these results substantiate a new functional role for nitrated fatty acid, demonstrating that OLA-NO2 exerts vascular protective effects by increasing NO bioavailability through eNOS phosphorylation/dephosphorylation and interaction with associated proteins such as Hsp90 and Cav-1. Keywords HUVEC
Nitrooleate eNOS Hsp90 Caveolin-1
Abbreviations OLA-NO2 Nitrooleate PPARc Peroxisome proliferator-activated receptor gamma NO Nitric oxide eNOS Endothelial nitric oxide synthase Cav-1 Caveolin-1 Hsp90 Heat shock protein 90 Akt Protein kinase B ERK1/2 Extracellular signal-regulated protein kinase HO-1 Heme oxygenase-1 15d-PGJ2 15-Deoxy-D12,14-prostaglandin TZD Thiazolidinediones VEGF Vascular endothelial growth factor BAEC Bovine aortic endothelial cells HUVEC Human umbilical vein endothelial cells OLA Oleic acid NO2Nitrite MAPK Mitogen-activated protein kinase IP Immunoprecipitation AIS Autoinhibitory sequences CaM Calmodulin CHIP C-terminus of the Hsp70-interacting protein PI3K Phosphatidylinositide-3 kinase
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MKP
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Mitogen-activated protein kinase phosphatases
Introduction Nitroalkene derivatives such as nitrooleate (OLA-NO2) have emerged as unique classes of nitric oxide (NO) and fatty acid-derived signaling molecules. Recent studies have shown that OLA-NO2 and nitrolinoleic acid are the largest pools of bioactive nitrogen oxides in the vasculature at the concentrations of *1 lM in human circulation [1]. Emerging evidence suggests that nitrated lipids have been considered pluripotent cell signaling molecules because they induce vasorelaxation of rat aortic rings, prohibiting aggregation of human platelets and vascular smooth muscle cell proliferation, and attenuate lipopolysaccharidemediated macrophage cytokine releases, superoxide generation, and integrin expression of human neutrophil [1–3]. Additionally, nitrated fatty acids can act as Peroxisome proliferator-activated receptor gamma (PPARc) ligands within physiological concentrations in PPARc-dependent and PPARc-independent manners [1–3]. Nitrated lipids are expected to exert the vascular protective effects by acting as PPARc ligands [3, 4]. Recent studies indicate that PPARc regulates vascular functions such as anti-inflammatory response and cell recruitment in vascular endothelial and smooth muscle cells [5, 6] and mouse models [7]. Activation of PPARc by both endogenous and synthetic ligands in endothelial cells significantly increased activity of endothelial nitric oxide synthase (eNOS), Cu/Zn superoxide dismutase and heme oxygenase-1 (HO-1) and decreased NADPH oxidase, resulting in vascular protection [7, 8]. Moreover, nitrated fatty acids have the electrophilic property of the b-carbon adjacent to the nitro-bonded carbon [2, 3, 9]. It can facilitate Michael addition reactions with biological nucleophiles such as cysteine, histidine, and lysine residues of target proteins [9]. For example, 15-deoxy-D12,14-prostaglandin (15dPGJ2) is a potent natural ligand for PPARc and exerts a variety of biological functions through PPARc-dependent and PPARc-independent pathways [1–4] due to the electrophilic double bond of cyclopentenone ring [1, 2]. In addition, nitrated lipids were recently reported to play a role in inhibiting lipopolysaccharide-induced inflammatory responses, accompanied by nitroalkylation of NF-jB p65 protein [3]. PPARc ligands such as 15d-PGJ2 and thiazolidinediones (TZD) have been reported to increase NO release and bioavailability through modulation of eNOS activity [5, 10]. eNOS is a key molecule in normal vascular biology
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and pathophysiology. NO produced by eNOS mediates vasorelaxation and inhibits vascular tone, smooth muscle cell proliferation, platelet aggregation and leukocyte adherence to the vascular wall [5, 11–14]. Regulation of eNOS is controlled by eNOS-interacting protein interactions, phosphorylation, subcellular localization, fatty acid acylation, and cofactor availability [11, 12]. For example, specific stimuli such as vascular endothelial growth factor (VEGF), bradykinin, and shear stress have shown to induce eNOS activity by facilitating replacement of caveolin-1 (Cav-1), eNOS-inhibitory protein, with heat shock protein 90 (Hsp90), eNOS-stimulatory protein [13, 14]. In addition to eNOS–protein interactions, phosphorylation/dephosphorylation of eNOS also increases the enzyme activity through induction of electron flux from the reductase to the oxygenase domain of eNOS [11, 12]. Recently, OLA-NO2 was reported to increase eNOS activity by phosphorylation of eNOS-ser1179 in bovine aortic endothelial cells (BAEC) and mouse models [15]. However, the precise molecular mechanisms of OLANO2-stimulated endothelial NO release remain to be defined. The current study was designed to further characterize the molecular mechanisms underlying OLA-NO2stimulated eNOS activity to induce NO bioavailability and vascular protection in human endothelial cells. Therefore, we first defined the effects of OLA-NO2 on multi-site phosphorylation/dephosphorylation of eNOS and eNOSinteracting proteins in NO production and potential components, i.e. Akt and ERK1/2, of eNOS activity.
Materials and Methods Cell Culture and Reagents Human umbilical vein endothelial cells (HUVEC; Lonza) monolayers were grown and maintained at 37 °C in 5 % CO2 in endothelial cell basal medium-2 according to protocols provided by the manufacturer (Lonza). In all experiments, cells were plated on 0.5 % gelatin-coated plates and confluent HUVEC (passages 2–8) were treated with vehicle (0.1 % ethanol), 3 lM OA-NO2, or oleic acid (OLA). OLA was used as a negative control. NO Production Analysis NO release was determined by the accumulation of nitrite (NO2-), the stable oxidation product of NO, using the NOspecific fluorometric assay kit (Cayman). Briefly, nitrate reductase was added to 100 ll of media and incubated at room temperature for 30 min for the conversion of nitrate into nitrite. After incubation, 2,3-diaminonaphthotriazole was added for an acidic solution, followed by NaOH which
Lipids (2014) 49:457–466 Table 1 PCR primers and conditions
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Gene
Primers
Tm (°C)
Cycle
Cav-1
Forward 50 -GACTTTGAAGATGTGATTGC-30
56
30
57
34
61
28
58
35
60
30
60
30
Reverse 50 -AGATGGAATAGACACGGCTG-30 eNOS
Forward 50 -CAGCACCTTGGCAGAAGAG-30 Reverse 50 -TTAGCCACGTGGAGCAGAC-30
Hsp90
Forward 50 -CATGAAAGCCCAGGCACTTCG-30 0
Reverse 5 -CTTCCATGCGAGACGCATCC-3 PPAR-c
0
Forward 50 -GAAATGACCATGGTTGACAC-30 Reverse 50 -CAGGACTCTCTGCTAGTACA-30
Actin
Forward 50 -AGAAAATCTGGCACCACACC-30 Reverse 50 -CTCCTTAATGTCACGCACGA-30
GAPDH
Forward 50 -ACCACAGTCCATGCCATCAC-30 Reverse 50 -TCCACCACCCTGTTGCTGTA-30
enhances the detection of the fluorescent product, 1(H)naphthotriazole. Absorbance was measured at 360 nm for excitation and 430 nm for emission using a fluorescence spectrophotometer (Perkin Elmer). Each sample was normalized to cell protein content and a serial dilution of nitrite standard was prepared for each experiment.
peroxidase-conjugated secondary antibody (diluted 1:5,000 in Tris-buffered saline with TweenÒ 20). Immunodetection was performed using a chemiluminecence method (Pierce). After immunodetection, relative densities of bands were quantified using an NIH ImageJ program. All data were normalized to the actin content of the same sample.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) Analysis
Immunoprecipitation (IP)
Total RNA was isolated using the Trizol reagent (Invitrogen). Total RNA (2 lg) was reverse-transcribed using Moloney murine leukemia virus reverse transcriptase (Invitrogen). PCR was performed using Taq polymerase (Takara) and the primer pairs (Bioneer). Relative mRNA levels were quantified using the NIH ImageJ program. Primer sequences and PCR conditions were described in Table 1.
After experimental treatments, cells were washed with in PBS and lysed with IP buffer [50 mM Tri-HCl (pH 7.4), 150 mM NaCl, and 1 % NP-40] containing protease inhibitors and PMSF. Whole cell lysates (200 lg protein) were incubated with a monoclonal antibody to eNOS (#610297; BD Transduction Laboratories) and successively with protein G-agarose beads (Roche). Immunoprecipitates were immunoblotted as described in western blot analysis. Statistical Analysis
Western Blot Analysis Whole cell lysates (20 lg of protein/lane) derived from HUVEC treated with either 3 lM OLA-NO2, or OLA were resolved in 10 % Bis–Tris gels and transferred onto a polyvinylidene difluoride membrane. After the membrane was blocked in 5 % skim milk, the membrane was probed overnight at 4 °C with primary antibodies (diluted 1:1,000 in 5 % bovine serum albumin solution) specific to eNOS (#610297), Hsp90 (#610418), Cav-1 (#610057), eNOSThr495 (#612707), eNOS-Ser633 (#612665), eNOSSer1177 (#612393) (BD Transduction Laboratories), eNOS-Ser113 (#9575), Akt (#9272), Akt-Ser473 (#9271), P44/42 MAPK (ERK1/2; #9102), phsopho-p44/42 MARK (#9101) (Cell Signaling) and actin (#sc-1616) (Santa Cruz). After being washed, the membrane was incubated for 1 h at room temperature with the corresponding horseradish
All data are presented as means ± SD. The data were analyzed with nonparametric methods due to small sample sizes using SPSS computer-based statistics programs (Ver. 20). The Mann–Whitney test and the Wilcoxon rank sum test were performed for statistical differences between two groups and one-way analysis of variance, respectively. The level of statistical significance was taken as p \ 0.05.
Results OLA-NO2 Increases NO Production in Endothelial Cells To determine the effect of OLA-NO2 on endothelial function, we investigated the NO generation in HUVEC.
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OLA-NO2 Increases eNOS Activity Through Phosphorylation/dephosphorylation of eNOS
300
NO production (%Control)
250
* *
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*
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0
0
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PGJ2
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Fig. 1 Effects of OLA-NO2 on NO production. HUVEC were treated with vehicle (control), 3 lM OLA-NO2 or OLA. After each treatment, NO production was measured by the Griess method. Each bar represents the mean ± SEM (n = 4); *p \ 0.05 vs. the control group
Compared to the control group, treatment with 3 lM OLANO2 significantly increased NO production by 50–100 % in a time-dependent manner (Fig. 1). 5 lM 15d-PGJ2 as a positive control significantly increased NO production for 8 h by 50 %. NO production of OLA-NO2 was consistently greater than that of 15d-PGJ2, which has already shown NO release as a PPARc ligand in previous studies [5, 6]. The corresponding native OLA for 4 h had no stimulatory effect on NO production. This result indicated that OLANO2 increased NO production like the other PPARc ligand, 15d-PGJ2 in endothelial cells. OLA-NO2 Regulates mRNA and Protein Expression of eNOS-related Genes To determine whether OLA-NO2 regulates eNOS and eNOS-related genes to increase NO release, we examined mRNA and protein expression of eNOS, Hsp90, Cav-1, and PPARc. HUVEC were treated with either 3 lM OLA-NO2, or OLA. In terms of mRNA levels, OLA-NO2 had no effect on eNOS but to a somewhat lesser extent, increased Hsp90 and decreased Cav-1 mRNA levels. However, OLA-NO2 significantly increased PPARc mRNA level at early times (2–8 h) (Fig. 2a). With respect to protein expression, treatment with 3 lM OLA-NO2 significantly decreased eNOS and Cav-1 expression, while OLA-NO2 markedly increased Hsp90 and PPARc expression in time-dependent (Fig. 2b) and dose-dependent manners (data not shown) when normalized to actin expression. The molecular effects of native OLA were similar to those of the control group (Fig. 2a, b).
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Because OLA-NO2 increased NO release without increasing eNOS expression, post-translational mechanisms of eNOS activity were examined. Despite that OLA-NO2 decreased total expression, OLA-NO2 increased phosphorylation of eNOS-Ser1177 at early times (2–4 h) and then gradually increased phosphorylation of eNOS-Ser633 until 16 h. However, OLA-NO2 gradually increased dephosphorylation of eNOS at Ser113 and Thr495. The corresponding native OLA had no effects on phosphorylation and dephosphorylation of eNOS (Fig. 3a). These results suggested that OLA-NO2 stimulated eNOS activity for NO production through changes in eNOS phosphorylation/dephosphorylation. OLA-NO2 Alters Interactions of eNOS with Associated Proteins Since proportion of Hsp90 and Cav-1 to eNOS association determines eNOS activation, we examined physical interactions between eNOS, Hsp90 and Cav-1 using immunoprecipitation (IP). In spite of eNOS reduction, treatment with OLA-NO2 significantly decreased the overall cellular content of Cav-1 and instead, increased that of Hsp90 associated eNOS in a time-dependent manner. Effects of native OLA were similar to those of the control group (Fig. 3b). Therefore, this result suggested that OLA-NO2 stimulated eNOS activity for NO production through changes in physical interactions of eNOS with associated proteins. OLA-NO2 Induces Phosphorylation of Akt and ERK1/2 Protein Kinases Several kinases such as phosphatidylinositide-3 Kinase (PI3K)/Akt (Protein Kinase B), extracellular signal-regulated kinase (ERK), AMP-activated protein kinase, protein kinase G, CaM-dependent kinase, and protein kinase A have been shown to be involved in eNOS phosphorylation [11, 12]. Therefore, the possibility that OLA-NO2 activates eNOS through Akt-dependent and ERK1/2 (P44/P42 MAPK)-dependent changes in the phosphorylation levels of eNOS was examined. OLANO2 increased the phosphorylation levels of Akt-Ser473 (Fig. 4a) and ERK1/2 (P44-Thr202/P42-Tyr204) (Fig. 4b). Phosphorylation of Akt-Ser473 was induced by threefold at 2 h treatment, followed by phosphorylation of ERK1/2 with peak induction (sixfold) at 4 h. After peak induction, each kinase gradually recovered basal levels. Native OLA had no effects on phosphorylation of Akt and ERK1/2 (Fig. 4a, b). This result indicates that
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Fig. 2 Effects of OLA-NO2 on mRNA and protein expression. HUVEC were treated with vehicle (control), 3 lM OLANO2 or OLA. a RT-PCR was performed using each genespecific primer. b Cell lysates were prepared and subjected to SDS-PAGE, followed by immunoblotting for eNOS, Hsp90, Cav-1, PPARc and actin. Each bar represents the mean ± SEM (n = 4). *p \ 0.05 vs. the control group
3 µM OLA-NO2
A 0
2
4
8
OLA 16
4
(hr) eNOS Hsp90
Cav-1 PPARγ Actin
100 80
*
60
*
*
40 20 0
0
2
4
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16
3 µM OLA-NO2
Cav-1/actin (% Control)
Hsp90/actin (% Control)
120
4
(hr)
100
* *
60
*
*
40 20 0 10
22
34
48
16 5
3 µM OLA-NO2
Akt and ERK1/2 are potential upstream components of eNOS signaling.
Discussion Previous studies [2–4, 15] reported that OLA-NO2 acted as an endogenous PPARc ligand and induced
140
*
120 100 80 60 40 20 0 10
46 OLA
22
34
48
16 5
3 µM OLA-NO2
120
80
*
160
OLA
PPAR γ/actin (% Control)
B
eNOS/actin (% Control)
GAPDH
(hr)
*
250
64
(hr)
OLA
*
*
200 150
*
100 50 0 10
22
34
48
16 5
3 µM OLA-NO2
46
(hr)
OLA
eNOS activity by phosphorylation of eNOS-ser1177 by MAP kinase-dependent signaling in BAEC. In the present study, we demonstrate that OLA-NO2 regulated reciprocal interactions of eNOS/Hsp90 and eNOS/ Cav-1 and multisite phosphorylation of eNOS through Akt and ERK1/2 signaling, leading to eNOS activity and subsequent NO production in human endothelial cells.
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A
3 µM OLA-NO2
0
2
4
8
B
OLA 16
4
IP : eNOS
(hr) eNOS pS113
3 µM OLA-NO2
eNOS pT495
0
4
8
OLA 16
4
(hr)
eNOS pS633
eNOS (140 kDa)
eNOS pS1177
Hsp90 (90 kDa)
Total eNOS (140 kDa)
Cav-1 (22 kDa)
Fig. 3 Effects of OLA-NO2 on site-specific eNOS phosphorylation and on interactions of eNOS/Hsp90 and eNOS/Cav-1. HUVEC were treated with vehicle (control), 3 lM OLA-NO2 or OLA. a Cell lysates were prepared and subjected to SDS-PAGE, followed by immunoblotting for phosphorylated sites (pS113, pT495, pS633 and
pS1177) of eNOS or total eNOS and b immunoprecipitated with monoclonal antibody to eNOS. Immunoprecipitates were then subjected to SDS-PAGE and immunoblotting for eNOS, Hsp90 and Cav-1 antibodies
B 350
*
300 250
*
200 150 100 50 0 0
2
4
8
3 µM OLA-NO2
16
4
(hr)
P-ERK/ERK (% Control)
A P-Akt/Akt (% Control)
2
OLA
1000
*
800
*
600 400 200 0 0
2
4
8
3 µM OLA-NO2
16
4
(hr)
OLA
Akt pS473
ERK1/2 pT202/Y204
Total Akt (60 kDa)
Total ERK1/2 (44/42 kDa)
Fig. 4 Effects of OLA-NO2 on site-specific Akt and ERK1/2 phosphorylation. HUVEC were treated with vehicle (control), 3 lM OLA-NO2 or OLA. Cell lysates were prepared and subjected to SDS-PAGE, followed by immunoblotting for phosphorylation of a Akt and b ERK1/2
eNOS is located in small invaginations of the plasma membrane called caveolae and produces NO in response to the stimuli. NO production in endothelial cells plays pivotal roles in vascular biology. Impaired NO leads to endothelial dysfunction, which is associated with risk factors against vascular diseases including hypercholesterolemia, diabetes mellitus, insulin resistance and obesity. Therefore, modulating NO production is a critical thing for prevention or treatment of endothelial dysfunction. Antidiabetic agents such as TZD drugs are used to boost NO production. The effects of these drugs are known to be mediated, in part, through their binding to PPARc. For example, both of endogenous (OLA-NO2 and 15d-PGJ2) and synthetic (rosiglitazone and ciglitazone) PPARc ligands, have been reported to increase endothelial NO production [5, 6]. However, numerous side effects such as hepatotoxicity, edema, heart failure, and weight gain demand the introduction of PPARc ligands and/or new therapy.
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Since OLA-NO2 acts as an endogenous PPARc ligand with an electrophilic center, OLA-NO2 has been reported to have similar characteristics to those of 15d-PGJ2 [2–4, 9]. 15d-PGJ2 was reported to decrease only eNOS protein expression, but not mRNA levels after 24 h treatment of 15d-PGJ2 in endothelial cells [2, 5]. In our previous study [16], 15d-PGJ2-induced eNOS reduction was strongly related to Hsp70 induction and eNOS translocation from soluble to insoluble fractions due to the biological activities of its electrophilic center but was not related to either transcriptional regulation, proteasomal degradation or apoptosis. In contrast to this result, it was reported that 15d-PGJ2 decreased both expression and activity of eNOS in HUVEC by inhibiting eNOS mRNA and protein synthesis, but this decrease was observed only after long-term treatment of 15d-PGJ2 for 48–72 h [17]. To our knowledge, there is only one report regarding the OLA-NO2induced alteration of eNOS expression in BAEC [15]. They reported that OLA-NO2 increased eNOS expression due to
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phosphorylation of eNOS-Ser1179 rather than transcriptional regulation with no difference in promoter activity. The differences in eNOS down-regulation with previous reports may be attributed to different culture conditions, such as cell sources and media systems. Interestingly, OLA-NO2-induced eNOS reduction is associated with Hsp90 induction and Cav-1 reduction in a time-dependent manner in HUVEC. In other words, Cav-1 reduction was replaced with Hsp90 induction. Hsp90, a molecular chaperone protein, is a crucial regulator of eNOS because it cooperatively enhances the affinity of eNOS for binding calmodulin (CaM) and balances output of NO and superoxide [11, 12]. Previous evidence has shown that the association of eNOS with Hsp90 enhances eNOS activity. For example, various stimuli, including VEGF, fluid shear stress, and estradiol, enhance the interaction between eNOS and Hsp90 to increase NO production [13]. Cav-1, the scaffolding protein of caveolae, is another regulator of eNOS and inhibits its activity by occupying its CaMbinding site in the basal state [14]. Various stimuli dissociate eNOS from Cav-1 and increase NO synthesis. For example, plasma NO levels were substantially increased in Cav-1-knockout mice [18]. Collectively, it is suggested that OLA-NO2 regulates the molecular expression and association of Hsp90 and Cav-1 with eNOS. Dissociation of eNOS from Cav-1, as a prerequisite for eNOS activation, may be compromised by the amount of Hsp90 associated with eNOS as well as by the molecular alteration. Since it has been controversial whether nitrated lipids can act as a PPARc ligand within physiological concentrations in PPARc-dependent and PPARc-independent manners [1–4], PPARc expression was examined. OLANO2 significantly increased mRNA and protein levels of PPARc at early times (2–8 h) and mRNA level of PPARc returned to that of the control group at later time points (8–16 h) (Fig. 2a, b). Based on the molecular alteration of PPARc, PPARc induction may be responsible for OLANO2 action. The activity of eNOS is regulated not only by the amount of expression but also by post-translational modifications including its phosphorylation, subcellular localization, and interactions of eNOS with associated proteins [11–14]. Precise regulation of eNOS activity involving multisite phosphorylation of specific serine and threonine residues has emerged. The five eNOS serine/threonine phosphorylation sites have been identified, including Ser613, Ser615, Ser633, Ser1177 and Thr 495 at human eNOS. Although the functional effects of each known site is controversial, it has been shown that eNOS activity was increased by phosphorylation of Ser615, Ser633, and Ser1177 but also dephosphorylation of Ser113 and Thr 495 [11, 12]. Besides confirming that nitrated fatty acids stimulated NO production through the phosphorylation of
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eNOS at Ser1179 in BAEC [15], we extended that OLANO2 stimulated NO production through four multisite regulations of eNOS phosphorylation. Since phosphorylation of eNOS at Ser633 and Ser1177 coincides with dephosphorylation of eNOS at Ser113 and Thr495 in this study, we confirmed that OLA-NO2 did stimulate eNOS activity and led to an increase in NO production, in part, through multisite coordination of eNOS phosphorylation. Interestingly, phosphorylation of Ser633 is a later event than phosphorylation of Ser1177. Phosphorylation of Ser1177 has been suggested to be critical for eNOS activation in response to several stimuli, such as shear stress, VEGF, and 8-bromo cAMP, which are known to increase NO bioavailability [19]. All three stimuli consistently induced slower phosphorylation of Ser635 than that of Ser1179, suggesting a priming effect of phosphorylation of Ser1177 for a subsequent phosphorylation of Ser633. Location of Ser633 and Ser117 in each of the two autoinhibitory sequences (i.e., AIS I and AIS II) might explain differential phosphorylation of eNOS. The Ser633 resides in the flavin mononucleotide binding domain in AIS I. Phosphorylation of Ser1177 in AIS II seems to dismiss the blockage of electron transfer within the C termini of the two eNOS monomers [20]. Structurally, phosphorylation of Ser633 is consistently preceded by phosphorylation of Ser1177 after removal of the hindrance imposed by AIS II [21]. Instead, later phosphorylation of Ser633 maintained persistent eNOS activity at the intracellular calcium level after its initial activation [22]. eNOS activity is regulated by direct interactions of the enzyme with cellular proteins, such as Hsp90, CaM, Cav-1 and G protein-coupled receptors [11, 12]. As described above, Hsp90 is an effective activator of eNOS activation because of the displacement of eNOS from inhibitory interaction with Cav-1. Conversely, Cav-1 is a potent inhibitor of eNOS activation because its scaffolding domain (amino acids 82–101) binds with oxygenase domain of eNOS [11–13]. IP analysis demonstrated that eNOS activity was strongly related to Hsp90 induction and partly to Cav-1 reduction, irrespective of eNOS reduction, suggesting that eNOS activity is most likely caused by direct Hsp90 induction. Although this study has demonstrated that OLA-NO2 increased eNOS activity through modulation of multisite phosphorylation of eNOS and eNOS-interacting proteins, there has been speculation about OLA-NO2-induced eNOS activity. In spite of eNOS reduction, enhanced eNOS activity is probably attributed to Hsp70 induction and eNOS translocation from soluble to insoluble fractions due to the biological activities of its electrophilic center [16]. The electrophilic property of the b-carbon adjacent to the nitro-bonded carbon can form covalent adducts with cysteine residues of cellular proteins [9]. Several cysteine
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residues (including 29 and 99 residues) of eNOS might be targets of covalent bonding [23], causing disruption of natural protein folding and subsequent eNOS protein aggregates in the insoluble fraction [16]. C-terminus of the Hsp70-interacting protein (CHIP) is another possible mechanism of eNOS trafficking. For example, 15d-PGJ2 re-distributed eNOS from the Golgi and plasma membrane to the cytoplasm instead of decreasing eNOS protein content, suggesting that 15d-PGJ2 might enhance eNOS-CHIP interactions and subsequent translocation [16]. The precise mechanism of OLA-NO2-induced eNOS phosphorylation remains to be defined but activity of kinase and phosphatase could be involved. Particularly, Akt pathway is predominantly involved in the regulation of Hsp90-mediated eNOS phosphorylation [13, 24] (Fig. 5).
Fig. 5 Potential mechanisms underlying OLA-NO2-mediated increases in endothelial NO release. In this model, OLA-NO2 stimulates endothelial NO release, in part by modulating posttranslational mechanisms of eNOS regulation, including multisite eNOS phosphorylation and eNOS–protein interactions. For example, OLANO2 increases phosphorylation of eNOS-Ser633 and eNOS-Ser1177 and dephosphorylation of eNOS-Ser113 and eNOS-Thr495. It also decreases the association of eNOS with Cav-1, and instead, increases the association of eNOS with Hsp90. Taken together, these observations demonstrate how OLA-NO2 directly enhances endothelial NO bioavailability, an effect that may contribute to the reported vascular protective effects of OLA-NO2
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Various stimuli such as estrogen, VEGF, and shear stress increased eNOS activity through PI3K-dependent Akt activation and subsequent eNOS-Ser1177 phosphorylation in endothelial cells [11, 12]. There is only one report regarding the effects of OLA-NO2 on the MAP kinase signaling pathway. Khoo et al. [15] reported that OLA-NO2 increased eNOS-Ser1179 phosphorylation and NO production via Akt-dependent, p38-dependent, and ERK1/2dependent signaling in BAEC. Enhanced eNOS activity can be imitated with recombinant Akt and overexpressed constitutively active Akt in intact cells [25, 26]. Treatment of 15d-PGJ2, not ciglitazone, also induced Akt phosphorylation, causing the cardioprotective signaling in male Wistar rats [27]. Besides, Cav-1 decreased the small GTPase Rac1, which in turn modulated the PI3K/Akt/ eNOS pathway [28]. Whereas numerous studies have demonstrated the functional role of Akt in the context of eNOS activation and NO release, the role of mitogen-activated protein kinase phosphatases (MKP) is quite controversial. MKP, dual-specificity phosphatases, play important roles in the regulation of ERK1/2, p38 kinase, and JNK signaling pathways. For example, bradykinin-induced ERK1/2 phosphorylation led to reduced eNOS activity in BAEC [29] and 15d-PGJ2induced ERK1/2 phosphorylation was involved in 15dPGJ2-induced apoptosis in cancer cells [30, 31]. In contrast to these findings, Cai et al. [32] reported that H2O2 treatment induced phosphorylation of ERK1/2 and PI3K/Akt, and subsequent eNOS-Ser1179 phosphorylation and NO production in BAEC. They suggested the possible mechanisms; (1) numerous potential ERK1/2 phosphorylation sites in eNOS (2) indirect relationship between ERK1/2 and eNOS through eNOS–Hsp90 interaction (3) involvement of ERK1/2 in the inhibitory interaction of eNOS/Cav-1. To our knowledge, it is the first to demonstrate that OLA-NO2 can stimulate NO production through four multisite regulations of eNOS phosphorylation and eNOS–proteins interactions via Akt and ERK1/2 signaling. In summary, our findings demonstrate that activation of OLA-NO2 in vascular endothelial cells provides a novel mechanism for stimulating endothelial NO release and hence NO bioavailability. OLA-NO2 increased phosphorylation of Akt and ERK1/2 and subsequent eNOS activation through reciprocal interactions of eNOS/Hsp90 and eNOS/Cav-1 and phosphorylation/dephosphorylation of eNOS, leading to NO production (Fig. 5). Collectively, these results demonstrate how OLA-NO2 has the potential to directly modify vascular endothelial function and to modulate the production of NO, a critical mediator in maintenance of normal vascular physiology. These findings further refine our understanding of nitroalkenes, novel targets for pharmacological intervention in the vascular system.
Lipids (2014) 49:457–466 Acknowledgments This study was supported by the Basic Science Research Program through the National Research Foundation of Korea Grant (NRF) funded by the Ministry of Education (No. 2010-0187 and 2011-0156).
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