Potent vasodilation effect of amurensin G is mediated through the phosphorylation of endothelial nitric oxide synthase

Biochemical Pharmacology 84 (2012) 1437–1450

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Potent vasodilation effect of amurensin G is mediated through the phosphorylation of endothelial nitric oxide synthase Tran Thi Hien a,b, Won Keun Oh a, Bui Thu Quyen b, Trong Tuan Dao a, Jung-Hoon Yoon c, Sei Young Yun d, Keon Wook Kang b,* a

BK21 Project Team, College of Pharmacy, Chosun University, Gwangju 501-759, Republic of Korea College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 151-742, Republic of Korea Department of Oral & Maxillofacial Pathology, College of Dentistry, Daejeon Dental Hospital, Wonkwang University, Daejeon 302-120, Republic of Korea d Department of Pharmaceutical Cosmetics, Kwangju Womens’s University, Gwangju 506-713, Republic of Korea b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 29 June 2012 Accepted 5 September 2012 Available online 13 September 2012

Endothelial nitric oxide synthase (eNOS) has important regulatory functions in vascular tone, and impaired endothelium-dependent vasodilatation is a key event in diabetes and atherosclerosis. Vitis amurensis grapes containing resveratrol oligomers are consumed as wine and fruit and have antioxidative and neuroprotective effects. In this study, our goal was identify the most potent eNOSactivating compound among six stilbenes and oligostilbenes found in V. amurensis and to clarify its molecular mechanism. Among the six tested compounds, amurensin G most potently relaxed endothelium-intact aortic rings and increased eNOS phosphorylation and nitric oxide (NO) production. Amurensin G increased both estrogen receptor (ER) phosphorylation and ER-dependent gene transcription, and ERa or ERb inhibition suppressed amurensin G-mediated eNOS phosphorylation. Amurensin G enhanced the activities of phosphatidylinositol 3-kinase (PI3K) and Src and their chemical inhibitors suppressed amurensin G-stimulated eNOS phosphorylation. Moreover, amurensin G activated AMP-activated protein kinase (AMPK), and amurensin G-stimulated eNOS phosphorylation and PI3K activation were reversed by AMPK inhibition. ER inhibition reversed AMPK-dependent PI3K activation in response to amurensin G. Amurensin G-mediated endothelium-dependent relaxation was blocked by inhibition of AMPK, ER, Src, or PI3K. These results suggest that amurensin G enhances NO production via eNOS phosphorylation in endothelial cells, and ER-dependent AMPK/PI3K pathways are required. Amurensin G would be applicable to prevent atherosclerosis. ß 2012 Elsevier Inc. All rights reserved.

Keywords: Amurensin G AMPK eNOS Estrogen receptor PI3-kinase/Akt

1. Introduction Endothelial dysfunction and subsequent vasodilatation impairment are key events in atherosclerosis and type II diabetes [1,2]. Endothelial nitric-oxide synthase (eNOS) is important for the

Abbreviations: ACC, acetyl-CoA carboxylase; ACh, acetylcholine; AICAR, 5-aminoimidazole-4-carboxamide-1-b-D-ribofuranoside; AMPK, adenosine 50 -monophosphate-activated protein kinase; CA-AMPK, constitutive active AMPK; CaMK II, calmodulin-dependent protein kinase II; DAF-2, DA (4,5-diaminofluorescein diacetate); DN-AMPK, dominant negative mutant of AMPK; eNOS, endothelial nitric oxide synthase; ER, estrogen receptor; ERK, extracellular signal-regulated kinase; GR, glucocorticoid receptor; JNK, c-Jun activated protein kinase; LKB1, liver kinase B1; MAPK, mitogen-activated protein kinase; L-NAME, NG-nitro-L-arginine methyl ester; MPP, Methyl-piperidino-pyrazole; NO, nitric oxide; NOS, nitric oxide synthase; PBS, phosphate buffered saline; PI3K, phosphatidylinositol 3-kinase; PP2, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyramidine; THC, tetrahydrochrysene. * Corresponding author at: College of Pharmacy, Seoul National University, Daehakro 1, Gwanak-gu, Seoul 151-742, Republic of Korea. Tel.: +82 2 880 7851; fax: +82 2 872 1795. E-mail address: [email protected] (K.W. Kang). 0006-2952/$ – see front matter ß 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bcp.2012.09.004

regulation of vasculature basal tone. In response to diverse chemicals and physical stimuli (e.g., vascular endothelial growth factor and shear stress), nitric oxide (NO) is generated by constitutively expressed eNOS via L-arginine oxidization [3]. As eNOS-mediated NO production simultaneously decreases vascular tone and inhibits low-density lipoprotein oxidation [4], safe natural compounds that potently activate eNOS may be beneficial for patients with hyperlipidemia and atherosclerosis. Plant-derived compounds are becoming of increasing interest as potential anti-atherosclerosis therapeutics. Some natural compounds in fruits, vegetables, oil seeds, and herbs have lipidlowering effects and reduce atherosclerotic lesions [5–7]. Resveratrol is a natural polyphenolic stilbene frequently found in grapes [8]. Resveratrol is the main active component of red wine, and its intake is inversely correlated with the incidence of chronic cardiovascular disease [9,10]. However, people are consuming resveratrol without the adequate published evidence for its effects in humans [11]. According to the literature search in scientific databases, the effect of resveratrol on coronary heart diseases was investigated in 118 papers, whereas only one paper was identified

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investigating effect in humans [11]. Supplementation of resveratrol dose-dependently increased cerebral blood flow in healthy adults, but the compound did not enhance cognitive function [12]. Ghanim et al. have shown that glucose, insulin and insulin resistance scores remained unchanged after 40 mg resveratrol supplementation for 6 weeks in healthy individuals [13]. Hence, published evidence is not enough to justify the use of pharmacological doses of resveratrol to humans. Vitis amurensis (Vitaceae), a wild grape, is distributed in East Asia and is widely used for juice and wine. It has been reported that isolated oligostilbenes from the root possess anti-inflammatory activity, and are structurally classified as resveratrol oligomers [14]. Resveratrol increases eNOS activity via the estrogen receptor (ER)a–Src–caveolin-1 interaction [15], and chronic resveratrol administration restores vascular responsiveness of cerebral

arterioles in type I diabetes [16]. In this study, we assessed vasorelaxation- and eNOS phosphorylation-causing effects of six stilbenoids and oligostilbenoids from V. amurensis. We found that amurensin G most potently elicited endothelium-dependent relaxation and eNOS phosphorylation in human endothelial cells. eNOS activity is regulated by diverse pathways, including calcium/calmodulin-dependent activation, transcriptional and posttranslational mechanisms, and through eNOS phosphorylation. Many signaling cascades are involved in eNOS phosphorylation and activation. ERs localized in the cell membrane, which transduce nongenomic estrogen effects, crosstalk with G-proteincoupled receptors and receptor tyrosine kinases coupled with Src, phosphatidylinosiol-3-kinase (PI3K)/Akt, or mitogen-activated protein kinase (MAPK) family members [17]. Activated ERs elicit PI3K/Akt to activate eNOS, which results in enhanced NO release

Fig. 1. (A) Structures of stilbenes and oligostilbenes isolated from V. amurensis. (B) Comparison of effects of six compounds from Vitis amurensis on vasodilatation. Endothelium-intact aortic rings were anchored to the organ chamber and each compound (10 mg/ml)-mediated vascular relaxation was monitored in the precontracted aortic rings by 1 mM phenylephrine. Data are expressed as relative relaxation % to acetylcholine (ACh, 1 mM)-mediated relaxation and represent the mean  SD of 4 separate experiments. (C) Concentration-dependent vasorelaxation effect by amurensin G and effect of NOS inhibitor (L-NAME) on amurensin G-mediated vasorelaxation. Endotheliumintact aortic rings were anchored to the organ chamber and preincubated with or without 10 mM L-NAME for 30 min and 1–10 mg/ml amurensin G mediated vascular relaxation was monitored in the precontracted aortic rings by 1 mM phenylephrine. Data are expressed as relative relaxation % to acetylcholine (ACh, 1 mM)-mediated relaxation (right upper panel tracing) and represent the mean  SD of 4 separate experiments. (D) Effect of amurensin G on eNOS phosphorylation in aortic rings. Rat aortic rings were incubated with 10 mM amurensin G for the indicated time (5–90 min) and the homogenates of aortic rings were subjected to phosphorylated eNOS immunoblottings. Relative densitometric scanning results were shown in bottom of the representative blot. The result was confirmed by two independent experiments.

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that may lead to vasodilatation in the vasculature [18]. A series of recent studies revealed that AMP-activated protein kinase (AMPK) increases eNOS activity via Ser 1177 phosphorylation [19]. We attempted to identify cellular signaling pathways involved in eNOS phosphorylation in response to amurensin G. 2. Materials and methods 2.1. Compounds from Vitis amurensis Three stilbenoids [trans-resveratrol, piceatannol, (+)-ampelopsin-F] and three oligostilbenoids [(+)-ampelopsin-A, amurensin G, e-viniferin] were isolated from V. amurensis as previously described [20] (Fig. 1A) and donated from Dr. Bae K (Chungnam National University, Deajeon, Korea). Purity of amurensin G (>95%) was confirmed by high performance liquid chromatography (Supplementary Fig. 1A). 2.2. Materials Antibodies against eNOS, Ser 1177 phospho-eNOS, phosphoAkt, Akt, phospho-p38 kinase, p38 kinase, phospho-extracellular

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signal regulated kinase (ERK), ERK, phospho-c-Jun N-terminal kinase (JNK), JNK, phospho-ERa (Ser118 and Ser167), ERa, phospho-Src, phospho-liver kinase B1 (LKB1), LKB1, phosphoAMPK, AMPK, phospho-acetyl CoA carboxylase (ACC, downstream target of AMPK), ACC, horseradish peroxidase-conjugated antimouse and anti-rabbit IgG antibodies were purchased from Cell Signaling Technology (Beverly, MA). ERb antibody was obtained from Abcam (Cambridge, MA). Control and ERa/b siRNAs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). LKB1 siRNA was obtained from Dharmacon, Inc. (Lafayette, CO). LY294002 (PI3K inhibitor), ICI-182780 (ER antagonsit), 4-amino5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2, Src inhibitor), pertussis toxin (Ptox, Gai inhibitor), compound C (AMPK activator) and NG-nitro-L-arginine methyl ester (L-NAME, eNOS inhibitor) were purchased from Calbiochem (La Jolla, CA). Methylpiperidino-pyrazole (MPP, ERa antagonist) and tetrahydrochrysene (THC, ERb antagonist) were obtained from Tocris Bioscience (Ellisville, MI). Actin antibody and other reagents used for molecular studies were obtained from Sigma (St. Louis, MO). Dominant negative mutant (DN-AMPK) or constitutive active form (CA-AMPK) of AMPK overexpression plasmids was kindly donated by Dr. Ha JH in Kyunghee University (Seoul, Korea).

Fig. 1. (Continued ).

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2.3. Organ chamber study

2.6. Reverse transcription-polymerase chain reaction (RT-PCR)

Male Sprague-Dawley rats (270–330 g) were sacrificed and thoracic aortas were carefully removed and placed in a modified Krebs–Ringer-bicarbonate solution containing (in mM) NaCl, 118.3; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2; CaCl2, 2.5; NaHCO3, 25.0; Ca2+ EDTA, 0.016 and glucose, 11.1 (control solution). The aortas were cleaned of loose connective tissue and then cut into rings (2 mm wide), The aortic rings were suspended horizontally between two stainless steel stirrups in the organ chambers filled with 5 ml of control solution (37 8C, pH 7.4) and bubbled with 95% O2 and 5% CO2. The change in tension was measured isometrically with Grass FT03 force transducers (Grass Instrument Co., Quincy, MA), and data were acquired and analyzed with a PowerLab 8/30 Data Acquisition System and LabChart pro software (AD Instruments, Colorado Springs, CO). The rings were stretched progressively to the optimal tension (2 g) before the addition of 1 mM phenylephrine. Once the plateau of the contraction elicited by phenylephrine was obtained, the aortic rings were rinsed three times with warm control solution and 1 mM acetylcholinemediated relaxation was tested in the precontracted rings by 1 mM phenylephrine to check endothelium-dependent relaxation responsiveness. In some experiments, rings were incubated for 30 min with LY294002 (10 mM) [21], ICI-182780 (100 nM) [22], PP2 (10 mM), PTX (100 nM) [23], MPP (10 mM), THC (10 mM) or compound C (20 mM) [24]. Because endothelial cells in aortic rings are directly exposed to Krebs–Ringer-bicarbonate buffer, we assumed that the concentrations of chemical inhibitors in organ chamber study could be identical to those in endothelial cell culture study. After 30-min incubation, 10 mg/ml amurensin G was added and the relaxation response was monitored in the precontracted aortic rings after addition of 1 mM phenylephrine. eNOS phosphorylation in aortic rings was also detected using Western blot analyses. The aortic rings were placed in a serum free DMEM media for 6 h and then exposed to amurensin G for indicated time. The aortic rings were homogenized in PBS by micro-homogenizer, transferred to microtube and centrifuged at 10,000  g. The supernatants were subjected to protein assays and immunoblottings. Protein levels were determined by using Promeasure kit (Intron Biotech., Sungnam, Korea). Each lane was loaded with 20 mg of the sample lysates. Equal protein loading was verified using actin as an internal standard.

Total RNA was isolated from the cells using a total RNA isolation kit (RNAgents, Promega, Madison, WI). The total RNA (1.0 mg) was reverse transcribed using an oligo(dT) 18-mer and Moloney murine leukemia virus reverse transcriptase (Bioneer, Eumsung, Korea). PCR was performed using selective primers for human eNOS (forward, 50 -GCCAGAACACAGCCCAGCTC-30 ; reverse, 50 CCCAGTTCTTCACACGAGGGAAC-30 ) and S16 ribosomal protein (S16r) genes (forward, 50 -TCCAAGGGTCCGCTGCAGTC-30 ; reverse, 50 -CGTTCACCTTGATGAGCCCATT-30 ). The PCR products were electrophoresed through a 2% agarose gel and visualized by ethidium bromide staining. The band intensities of the amplified DNA were compared after visualization with an FLA-7000 (Fujifilm, Tokyo, Japan).

2.4. Cell culture ECV 304 cells were obtained from the American type culture collection (Bethesda, MD). The cells were maintained at 37 8C in an incubator with a humidified atmosphere of 5% CO2 and cultured in DMEM containing 10% fetal bovine serum. Primary cultured HUVEC were purchased from Innopharmascreen (Asan, Chungnam, Korea) and cultured in M199 medium containing 10 units/ml heparin, 20% fetal bovine serum and 20 ng/ml fibroblast growth factor. The used compounds were dissolved in dimethylsulfoxide (DMSO) and the stock solutions were added directly to the culture media. Control cells were treated with DMSO only. The final concentration of solvent was always <0.1%. 2.5. Cytotoxicity test Viable adherent cells were stained with MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] (2 mg/ ml) for 4 h. Media were then removed and the formazan crystals produced were dissolved by adding 200 ml of dimethylsulfoxide. Absorbance was assayed at 570 nm using a microplate reader (LB941, Berthold Technologies, Wild Bad, Germany) and cell viabilities were expressed as ratios versus untreated control cells.

2.7. Reporter gene analysis A dual-luciferase reporter assay system (Promega, Madison, WI) was used to determine gene promoter activity. Briefly, cells were plated in 12-well plates and transiently transfected with 1 mg pERE-Tk-Luc plasmid and 10 ng phRL-SV plasmids (hRenilla luciferase expression for normalization; Promega) using Hillymax reagent (Dojindo Molecular Technologies). The cells were then incubated in culture medium without serum for 6 h. Firefly and hRenilla luciferase activities in the cell lysates were measured using a luminometer (LB941, Berthold Technologies, Bad Wild, Germany). The relative luciferase activity was calculated by normalizing the promoter-driven firefly luciferase activity to the hRenilla luciferase activity. 2.8. Western blot analysis After treatment, cells were collected and washed with cold phosphate buffered saline (PBS). The harvested cells were then lysed on ice for 30 min in 100 ml lysis buffer [120 mM NaCl, 40 mM Tris (pH 8), 0.1% NP40 (Nonidet P-40)] and centrifuged at 12,000 rpm for 30 min. Supernatants were collected from the lysates and protein concentrations were determined using protein assay kit (Pierce, Rockford, IL). Aliquots of the lysates (30 mg protein) were boiled for 5 min and electrophoresed on 10% SDSpolyacrylamide gels. Proteins in the gels were transferred onto nitrocellulose membranes, which were then incubated with primary antibodies or mouse monoclonal actin antibodies. The membranes were further incubated with secondary antibodies. Finally, protein bands were detected using an enhanced chemiluminescence Western blotting detection kit (Pierce Biotechnology, Rockford, IL). 2.9. Measurement of NO production Production of NO was assessed using the NO-specific fluorescent dye 4,5-diaminofluorescein diacetate (DAF-2 DA; Cayman Chemical, Ann Arbor, MI). Briefly, ECV 304 cells were grown to 95% confluence in chamber slides (Lab-Tek, Rochester, NY) and serum-starved overnight. Cells were then loaded with DAF-2 DA (final concentration, 2 mM) for 30 min at 37 8C, rinsed 3 times with DMEM media and kept in the dark. Cells were then treated without or with amurensin G as indicated in the figure legends. The cells were fixed in 5% paraformaldehyde for 5 min at 4 8C. Fixed cells were visualized using a fluorescence microscope (Axiovert 200M; Carl Zeiss, Germany) and an inverted epifluorescence microscope with an attached chargecoupled device camera using appropriate filters with a peak excitation wavelength of 480 nm and a peak emission wavelength of 510 nm.

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2.10. Statistical analysis One-way analysis of variance (ANOVA) procedures were used to assess significant differences between treatment

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groups. When treatment was found to have a significant effect, the Newman–Keuls test was used to compare multiple group means. Statistical significance was accepted at either p < 0.05.

Fig. 2. eNOS phosphorylation and NO production by amurensin G in ECV 304 cells and HUVEC. (A) Concentration-dependent eNOS phosphorylation by amurensin G. ECV 304 cells were treated with amurensin G (1–10 mg/ml) for 1 h and the total cell lysates were subjected to immunoblottings with antibody against Ser-1177 phosphorylated eNOS or total eNOS. Insulin was used as a representative activator for eNOS phosphorylation. Relative changes in the eNOS phosphorylation were assessed by scanning densitometry. Data represent the means  SD of 3 separate experiments (significant as compared to control, *p < 0.05; control level = 1). (B) Time course of eNOS phosphorylation by amurensin G. ECV 304 cells were treated with amurensin G (10 mg/ml) for the indicated time (5–60 min). Relative changes in the eNOS phosphorylation were assessed by scanning densitometry. Data represent the mean  SD of 3 separate experiments (significant as compared to control, *p < 0.05; control level = 1). (C) NO production by amurensin G. ECV 304 cells were serum-starved overnight and loaded with DAF2-DA as described under Section 2. Cells were then stimulated with amurensin G (0.5–10 mg/ml) for 60 min. After amurensin G treatments, cells were fixed in 5% paraformaldehyde and visualized with an epifluorescent microscope. Emission of green fluorescence is indicative of NO production. Insulin was used as a representative activator for eNOS-mediated NO production. (D) eNOS mRNA changes by amurensin G. ECV 304 cells were treated with amurensin G (10 mg/ml) for 3–48 h. eNOS mRNA was detected by RT-PCR analysis. (E) Concentration-dependent eNOS phosphorylation by amurensin G in HUVEC. HUVEC were treated with amurensin G (1–10 mg/ml) for 1 h and Ser-1177 phospho eNOS was detected by immunoblottings. Relative changes in the eNOS phosphorylation were assessed by scanning densitometry. Data represent the mean  SD of 3 separate experiments (significant as compared to control, *p < 0.05; control level = 1).

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in the 1–10 mg/ml concentration range for the subsequent experiments.

3. Results 3.1. Effects of stilbenes and oligostilbenes from Vitis amurensis on vascular tone and eNOS phosphorylation eNOS-activated NO production plays a protective physiological role in the vasculature. In order to screen drug candidates against atherosclerosis, we determined the efficacy of each of six stilbenoid and oligostilbenoid compounds (piceatannol, (+)ampelopsin-A, (+)-ampelopsin-F, amurensin G, trans-resveratrol, e-viniferin, Fig. 1A) from V. amurensis on endothelium-dependent relaxation in rat aortic rings. Because Elı´es et al. had reported that 30 mM (9 mg/ml) resveratrol increased NO production in human endothelial cells [25], we selected 10 mg/ml concentration for the primary screening. All six compounds evoked endotheliumdependent relaxation in phenylephrine-precontracted aortic rings at a 10 mg/ml concentration (Fig. 1B). Among them, amurensin G most potently elicited vasodilatation with a calculated EC50 of 5.08 mg/ml (Fig. 1C, left panel). Because amurensin G-mediated relaxation was completely reversed by 10 mM L-NAME treatment (Fig. 1C, right lower panel), we assumed that NO production through eNOS activation was involved in the observed vasorelaxation. Because eNOS phosphorylation is a key regulatory mechanism for its enzymatic activity, we then determined the effects of the six compounds on eNOS phosphorylation in ECV 304 human endothelial cells. Immunoblot analyses revealed that 10 mg/ml amurensin G potently increased eNOS phosphorylation in ECV 304 cells (Supplementary Fig. 1B). Immunoblot analysis using aortic ring homogenates also confirmed that amurensin G increased the level of phosphorylated eNOS at 15–60 min after 10 mg/ml amurensin G exposure (Fig. 1D), which suggests that eNOS phosphorylation and subsequent NO production by amurensin G is directly coupled with vascular relaxation. We then assessed potential cytotoxicity of amurensin G in ECV 304 cells by MTT assay. Amurensin G (0.3–30 mg/ml) did not cause cytotoxicity in ECV 304 cells (Supplementary Fig. 1C). Thus, we used amurensin G

3.2. Amurensin G increases eNOS phosphorylation and NO production in ECV 304 cells and human umbilical vein endothelial cells (HUVECs) We then examined concentration- and time-dependent effects of amurensin G on eNOS phosphorylation. When ECV 304 cells were treated with 1–10 mg/ml amurensin G for 1 h or incubated with 10 mg/ml amurensin G for the indicated time points (5–60 min), the levels of phosphorylated eNOS increased in a concentration- or timedependent manner, respectively (Fig. 2A and B). To examine whether eNOS phosphorylation by amurensin G stimulated NO production, ECV 304 cells were loaded with DAF-2 DA, a dye that upon binding to an oxidized NO species fluoresces. As shown in Fig. 2C, amurensin G treatment increased green fluorescence (indicative of NO production) in a concentration-dependent manner. We further determined eNOS mRNA levels after exposure of ECV 304 cells with 10 mg/ml amurensin G. eNOS mRNA expression slightly increased at 3–12 h and peaked at 24 h (Fig. 2D). To confirm these results, HUVEC were exposed to amurensin G (1–10 mg/ml), and eNOS phosphorylation was determined. As expected, eNOS phosphorylation was increased by amurensin G treatment in HUVECs (Fig. 2E). 3.3. Role of the PI3-kinase/Akt pathway in eNOS phosphorylation by amurensin G PI3K/Akt and mitogen-activated protein kinase (MAPK) (i.e., p38 kinase, ERK, and JNK) pathways are directly or indirectly involved in cellular signaling of vascular relaxation and NO production [26]. To clarify the upstream signaling pathway(s) of amurensin G-mediated eNOS phosphorylation, we determined the activities of PI3K, ERK, p38 kinase, and JNK in amurensin G-treated ECV 304 cells. Amurensin G treatment caused sustained phosphorylation of Akt, but not p38 kinase, ERK, or JNK (Fig. 3A). To address the involvement

Fig. 2. (Continued ).

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of PI3K/Akt activation in eNOS phosphorylation by amurensin G, the effect of LY294002 (a PI3K inhibitor) was assessed. Fig. 3B shows that PI3K inhibition significantly reduced amurensin G-induced eNOS phosphorylation. 3.4. Involvement of the ER-dependent PI3K/Akt pathway in eNOS phosphorylation by amurensin G Recent evidence suggests that stilbenoids and oligostilbenoids exert estrogen-like activity, raising the possibility that they may

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directly or indirectly activate ERs [27,28]. Moreover, ERa directly interacts with PI3K and modulates its activity in human vascular endothelial cells [29]. To further determine whether amurensin Gmediated eNOS phosphorylation was linked to ER signaling, we determined whether amurensin G affected ER-dependent [estrogen response element (ERE)-reporter)] transcriptional activity. Amurensin G treatment significantly increased ERE reporter activity, but the intensity increase was weaker than that induced by 100 nM 17b-estradiol (Fig. 4A). In addition, ERa phosphorylation at Ser 118 or Ser 167 was enhanced by amurensin G, implying that amurensin

Fig. 3. Role of PI3K/Akt pathway in amurensin G-stimulated eNOS phosphorylation. (A) Effect of amurensin G on the activities of PI3-kinase, ERK, JNK and p38 kinase. ECV 304 cells were treated with 10 mg/ml amurensin G for the indicated times and then immunoblotted with phosphorylation-specific antibodies that recognize phospho-Akt (p-Akt), phospho-ERK (p-ERK), phospho-p38 kinase (p-p38) and phospho-JNK (p-JNK). Parallel immunoblots were analyzed for total kinase levels with anti-Akt, ERK, p38, and JNK antibodies. The result was confirmed by two independent experiments. (B) Effect of PI3K inhibitor on amurensin G-stimulated eNOS phosphorylation. ECV 304 cells were 30 min preincubated with 10 mM LY294002 and then cells were incubated with 10 mg/ml amurensin G for additional 60 min. Relative changes in the eNOS phosphorylation were assessed by scanning densitometry. Data represent the mean  SD of 3 separate experiments (significant as compared to control, *p < 0.05; significant as compared to amurensin G-treated sample, #p < 0.05; control level = 1).

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Fig. 4. Role of estrogen receptor signaling in amurensin G-mediated PI3K/Akt activation and eNOS phosphorylation. (A) Amurensin G-induced increase in ER-dependent transcription. ECV 304 cells were transiently transfected with pERE-Tk-Luc plasmid as described in Section 2. Following transfection, the cells were incubated with or without amurensin G (0.3–10 mg/ml) or 17-b-estradiol (E2, 100 nM) for 24 h. Data represent the mean  SD of 4 separate samples (significant as compared to control, *p < 0.05). (B) Effect of amurensin G on the phosphorylation of ERa. ECV 304 cells were treated with 10 mg/ml amurensin G for the indicated times and then immunoblotted with phosphorylationspecific antibodies that recognize Ser 118 phospho-ERa and Ser 167 phospho-ERa. Parallel immunoblots were analyzed for total ERa or ERb levels with anti-ERa and ERb antibodies. (C) Effect of ER antagonist on eNOS and Akt phosphorylation. ECV 304 cells were pretreated with 100 nM ICI-182780 (ICI) for 30 min and the incubated with 10 mg/ml amurensin G for additional 60 min. Phosphorylated eNOS and Akt were detected by Western blot analyses. Relative changes in the eNOS phosphorylation were assessed by scanning densitometry. Data represent the mean  SD of 3 separate experiments (significant as compared to control, *p < 0.05; significant as compared to amurensin G-treated sample, # p < 0.05; control level = 1). (D) Effects of inhibitors targeting PI3K, ER and eNOS on amurensin G-stimulated NO production. ECV 304 cells were pretreated with LY294002 (LY, 10 mM), ICI (100 nM) or L-NAME (10 mM) for 30 min and then stimulated with amurensin G for 60 min.

G may trigger ER signaling in a ligand-independent manner (Fig. 4B). We also found that both ERE luciferase reporter activity and ERa phosphorylation were increased by amurensin G in HUVEC (Supplementary Fig. 2A and B). To test the role of ER activation in eNOS phosphorylation and PI3K activation by amurensin G, ECV 304 cells were exposed to ICI-182780, a pure ER antagonist. ICI-182780 reduced the phosphorylation of eNOS and Akt in response to amurensin G (Fig. 4C). The data indicate that amurensin Gstimulated eNOS activation is coupled with the ER-dependent PI3K/Akt pathway. We then confirmed the effects of an ER antagonist and PI3K inhibitor on amurensin G-induced NO production. As shown in Fig. 4D, amurensin G-mediated NO production (shown by DAF-2 fluorescence) was suppressed by ICI-182780, LY294002, or L-NAME (NOS inhibitor) pre-treatment. The pleiotropic effects of estrogen are mediated through the differential expression of two ER subtypes, ERa and ERb [30]. We recently showed that ECV 304 cells express both ERa and ERb, and their selective ligands increase eNOS phosphorylation [31]. Both ERa and ERb are simultaneously involved in the regulation of eNOS activity in endothelial cell caveolae [32,33]. To further investigate which ER subtype was associated with amurensin

G-mediated eNOS activation, specific antagonists or siRNAs targeting ERa and ERb were used. Fig. 5A shows that amurensin G-stimulated eNOS phosphorylation was diminished in ECV 304 cells transfected with ERa or ERb siRNA. Moreover, MPP (ERa antagonist) or THC (ERb antagonist) treatment caused the same results (Supplementary Fig. 2C). These results support the notion that both ERa and ERb regulate eNOS phosphorylation, presumably by the PI3K/Akt pathway. Several biological actions of estrogen are associated with intracellular regulatory cascades, such as cytosolic tyrosine kinase (Src) and Gai protein coupling [34,35]. Crucial evidence of ER-Gaiprotein coupling to eNOS came from the observation that pertussis toxin (GaI inhibitor) blocks estradiol-mediated eNOS activation [36]. In addition, Src kinase is essential for PI3K/Akt-dependent eNOS activation in endothelial cells [37]. Here, we found that amurensin G increased the active form of Src (Tyr 416-phosphorylated Src; Fig. 5B); amurensin G-induced eNOS phosphorylation was significantly suppressed by inhibitors targeting Src (PP2) or Gai (Ptox; Fig. 5C and D). DAF-2 fluorescence analysis confirmed that amurensin G-stimulated NO production was reduced by inhibitors targeting ERa, ERb, Src, or Gai protein (Supplementary Fig. 2D).

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3.5. Role of AMPK in phosphorylation of eNOS by amurensin G Many natural compounds containing stilbene or oligostilbene moieties activate AMPK, and they have beneficial effects on metabolic diseases [38]. Because Ser 1177 phosphorylation of eNOS is also controlled by AMPK [39], we further determined whether amurensin G activated AMPK in endothelial cells. Representative AMPK activation markers (i.e., phosphorylation of AMPK and ACC) were both increased by amurensin G in a

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concentration- (left) or time-dependent manner (right) in ECV 304 cells (Fig. 6A). The same results were also observed in HUVEC (Fig. 6B). We then tested whether AMPK activation was required for amurensin G-induced eNOS phosphorylation. ECV 304 cells were pre-treated with compound C, an AMPK inhibitor, and we examined amurensin G-dependent eNOS phosphorylation. Amurensin G-stimulated eNOS and ACC phosphorylation was significantly impaired in compound C-pre-treated ECV 304 cells (Fig. 6C), suggesting that AMPK is also involved in eNOS activation by

Fig. 5. Involvement of ERa, ERb, Src and Gai pathways in amurensin G-mediated eNOS phosphorylation. (A) Effects of siRNAs for ERa and ERb on eNOS phosphorylation induced by amurensin G. ECV 304 cells were transfected with 60 pmol control siRNA, ERa siRNA or ERb siRNA and then the cells were incubated with 10 mg/ml amurensin G for 60 min. Relative changes in the eNOS phosphorylation were assessed by scanning densitometry. Data represent the mean  SD of 3 separate experiments (significant as compared to control, *p < 0.05; significant as compared to amurensin G-treated sample, #p < 0.05; control level = 1). (B) Effect of amurensin G on Src-phosphorylation in ECV 304 cells. ECV 304 cells were exposed to 10 mg/ml amurensin G for 0–60 min. (C) Effect of Gai inhibitor (pertussis toxin, Ptox, 10 nM) or (D) Src inhibitor (PP2, 10 mM) on eNOS phosphorylation induced by amurensin G. ECV 304 cells were preincubated with 10 nM Ptox or 10 mM PP2 and then the cells were incubated with 10 mg/ml amurensin G for 60 min. Relative changes in the eNOS phosphorylation were assessed by scanning densitometry. Data represent the mean  SD of 3 separate experiments (significant as compared to control, *p < 0.05; significant as compared to amurensin G-treated sample, #p < 0.05; control level = 1).

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Fig. 6. AMPK activation by amurensin G and its role in eNOS phosphorylation. (A) Concentration- (left) and time-dependent (right) AMPK activation by amurensin G in ECV 304 cells. (Left panel) ECV 304 cells were incubated with amurensin G (1–10 mg/ml) for 1 h and phosphorylated AMPK and ACC were determined by Western blot analyses. 2 mM 5-amino-1-b-D-ribofuranosyl-imidazole-4-carboxamide (AICAR) was used as a representative AMPK activator. (Right panel) ECV 304 cells were incubated with 10 mg/ml amurensin G for the indicated time points. The results were confirmed by two independent experiments. (B) AMPK activation by amurensin G in HUVEC. HUVEC

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amurensin G. To confirm these results, ECV 304 cells were transfected with a DN-AMPK or CA-AMPK overexpression vector. DN-AMPK transfection prior to amurensin G treatment significantly reduced amurensin G-stimulated ACC and eNOS phosphorylation (Fig. 6D). Conversely, CA-AMPK transfection alone markedly increased phosphorylation levels of ACC and eNOS, but did not further enhance eNOS phosphorylation in response to amurensin G (data not shown). 3.6. Cross-talk between the AMPK pathway and ER-dependent PI3K/ Akt pathway Structurally, amurensin G is classified as a resveratrol trimer, a known AMPK activator [40]. A recent study suggested that AMPK activation restores estrogen responsiveness in human endothelial cells [41]. Furthermore, 17-b-estradiol activates AMPK in endothelial cells and C2C12 cells [42]. Because both ER-dependent PI3K/ Akt and AMPK pathways were separately required for eNOS phosphorylation by amurensin G, we further tested possible crosstalk between the two pathways. Inhibition of ER signaling by ICI182780 suppressed amurensin G-induced ACC phosphorylation (Fig. 7A). However, DN-AMPK overexpression failed to inhibit the Ser 167 phosphorylation of ERa and ER-dependent transcription by amurensin G treatment (Fig. 7B). Based on these results, we concluded that ligand-independent ER activation by amurensin G occurs before AMPK activation. We also found that AMPK blocked by DN-AMPK overexpression decreased amurensin G-induced Akt phosphorylation (Fig. 7C). In contrast, PI3K/Akt inhibition by LY294002 did not affect ACC phosphorylation in response to amurensin G (Fig. 7D). The data imply that PI3K/Akt is activated under the control of ER/AMPK pathways and acts on eNOS phosphorylation (Fig. 8). 3.7. Amurensin G-mediated endothelium-dependent relaxation is reversed by inhibition of ER, PI3K, AMPK, Src, or Gai protein We performed organ chamber studies to assess whether ER/ AMPK/Akt signaling was involved in amurensin G-mediated endothelium-dependent relaxation. Incubation of aortic rings with LY294002 (PI3K inhibitor), ICI-182780 (ER antagonist), compound C (AMPK inhibitor), THC (ERb antagonist), PP2 (Src inhibitor), or Ptox (Gai protein inhibitor) significantly suppressed amurensin G-mediated vasorelaxation. Compound C (AMPK inhibitor) and MPP (ERa antagonist) also significantly reversed amurensin G’s relaxation effect; however, the inhibition intensities were weaker than that of LY294002, PP2 or ICI-182780 (Fig. 7E and Supplementary Fig. 3). 4. Discussion Resveratrol is classified as a natural stilbene derivative and is the principal pharmacologically active component of red wine, the intake of which is inversely related with the incidence of chronic cardiovascular diseases [9]. Stilbenes and oligostilbenes are plentiful in V. amurensis, and they have diverse bioactivities, such as antioxidation [43], cancer chemoprevention [44], anti-inflammatory [9], and anti-allergy effects [45]. Previous studies have shown that eNOS is activated by red wine polyphenols [25,46].

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Further, resveratrol activates eNOS via Src-dependent eNOS phosphorylation and improves cerebral vascular function [15,16]. Using six stilbenes and oligostilbenes from V. amurensis, we found for the first time that amurensin G potently activates eNOS phosphorylation in human endothelial cells. The PI3K/Akt pathway is a key kinase axis for Ser 1177 or Ser 1179 eNOS phosphorylation, and is essential in regulating eNOS activity and NO production in physiological circumstances [47,48]. The current experiments demonstrated that amurensin G strongly activated PI3K/Akt, and that eNOS phosphorylation and NO production in response to amurensin G were attenuated by PI3K inhibition. The PI3K/Akt pathway is under the control of ERdependent signaling [49] and ERa modulates PI3K activity via direct interaction in vascular endothelial cells [29]. We and other groups showed that resveratrol, a phytoestrogen, stimulates ERdependent transcription in breast cancer cells [50,51]. Because amurensin G is a resveratrol trimer, we then focused on the role of ERs in amurensin G-stimulated eNOS phosphorylation. Amurensin G increased ERE reporter activity, but did not change ERa and ERb expression levels. Interestingly, we found that phosphorylation of Ser 167 or Ser 118 residue of ERa of was enhanced by amurensin G treatment. Even in the absence of estrogen, ERa can be activated in a ligand-independent manner. Specifically, ERa Ser 118- or Ser 167-phosphorylation by ERK or Akt stimulates ER-dependent transcription of target genes [52]. Hence, amurensin G-mediated ER activation may result from ligand-independent activation. However, we cannot exclude the possibility that amurensin G directly binds to ERa or ERb, as resveratrol has weak agonistic activities on ERa (7.7 mM) and ERb (29 mM) [53]. ERa and ERb are localized in the plasma or caveolae membranes, and mediate nongenomic signaling for eNOS activation in endothelial cells [32,33]. We previously showed that ECV 304 cells express both ERa and ERb, and specific ligands acting on both receptor subtypes stimulate eNOS phosphorylation [31]. In this study, we demonstrated that amurensin G-induced eNOS and Akt phosphorylation was suppressed by a putative ER antagonist (ICI-182780) or by blocking ERa and ERb. These data suggest that activation of both ERa and ERb is involved in PI3K/Akt-mediated eNOS phosphorylation by amurensin G. ERs located in the plasma membrane can form a molecular complex with Src, Shc, multiple receptor tyrosine kinases, and G-protein isoforms [54]. eNOS stimulation by 17-b-estradiol requires plasma membrane ERa coupling to Gai and activated Gai mediates the requisite downstream signaling events [36]. In addition, Haynes et al. suggested that membrane ER-induced rapid eNOS activation is mediated by a sequential Src/PI3K/Akt cascade in endothelial cells [37]. Here, we found that PP2 (Src inhibitor) as well as Ptox (Gai inhibitor) potently abrogated eNOS phosphorylation and endothelium-dependent relaxation in response to amurensin G. Hence, both Src tyrosine kinase and Gai signaling may be actively involved in the ER-dependent cascades of PI3K/Akt/eNOS phosphorylation by amurensin G. Although PI3K/Akt plays a central role in regulating eNOS phosphorylation, eNOS phosphorylation can be affected by other kinases, including AMPK [39] and protein kinase A [55]. AMPK is activated either by an increase in the AMP/ATP ratio during metabolic stress or by activation of upstream kinases, such as LKB1 and Ca2+/calmodulin-dependent protein kinase (CaMK) II [56]. A

were incubated with amurensin G (1–10 mg/ml) for 1 h and phosphorylated AMPK and ACC were determined. (C) Effect of compound C on amurensin G-induced eNOS phosphorylation. ECV 304 cells were pretreated with 10 mM compound C (AMPK inhibitor) for 30 min and then incubated with 10 mg/ml amurensin G for additional 60 min. Phosphorylated eNOS and ACC were detected by Western blot analyses. The bottom panel shows a relative densitometric changes in the eNOS phosphorylation. Data represent the mean  SD of 3 separate experiments (significant as compared to control, *p < 0.05; significant as compared to amurensin G-treated sample, #p < 0.05; control level = 1). (D) Effect of DN-AMPK transfection on amurensin G-induced eNOS phosphorylation. ECV 304 cells were transfected with DN-AMPK or pcDNA control for 24 h, the cells were then treated with 10 mg/ml amurensin G for 60 min. Phosphorylated eNOS and ACC were detected by Western blot analyses. The result was confirmed by two independent experiments.

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Fig. 7. Crosstalk between ER, PI3K and AMPK pathways for the amurensin G-mediated eNOS phosphorylation. (A) Effect of ER antagonist on amurensin G-induced AMPK activation. ECV 304 cells were pretreated with 100 nM ICI-182780 (ICI, ER antagonist) for 30 min and the incubated with 10 mg/ml amurensin G for additional 60 min. (B) Effect of DN-AMPK transfection on amurensin G-induced Ser 167 ERa phosphorylation. ECV 304 cells were transfected with DN-AMPK and pcDNA control for 24 h, the cells were then treated with 10 mg/ml amurensin G for 60 min. (C) Effect of DN-AMPK transfection on amurensin G-induced Akt phosphorylation. ECV 304 cells were transfected with DN-AMPK and pcDNA control for 24 h, the cells were then treated with 10 mg/ml amurensin G for 60 min. (D) Effect of PI3K inhibitor on amurensin G-stimulated AMPK activation. ECV 304 cells were pretreated with 10 mM LY294002 (LT, PI3K inhibitor) for 30 min and then incubated with 10 mg/ml amurensin G for additional 60 min. All phosphorylated forms of ACC, ERa and Akt were detected by Western blot analyses. The results were confirmed by at least two independent experiments. (E) Effects of specific inhibitors targeting PI3K, ERa/b and AMPK on amurensin G-mediated vasorelaxation. Endothelium-intact aortic rings were anchored to the organ chamber and preincubated with or without 10 mM LY, 100 nM ICI, 10 mM MPP, 10 mM THC, 10 mM PP2, 10 nM Ptox or 20 mM compound C for 30 min and 10 mg/ml amurensin G mediated vascular relaxation was monitored in the precontracted aortic rings by 1 mM phenylephrine. Data are expressed as relative relaxation % to acetylcholine (ACh, 1 mM)mediated relaxation and represent the mean  SD of 4 separate experiments (significant as compared to amurensin G alone-treated sample, *p < 0.05). Representative tracings were shown in Supplementary Fig. 3.

recent AMPK knockout mouse study clearly demonstrated that the beneficial effects (e.g., anti-obesity and increased insulin sensitivity) of resveratrol depend on its AMPK activation [57]. Here, we found that amurensin G activated AMPK and AMPK inhibition suppressed eNOS phosphorylation by amurensin G. Previous studies demonstrated that LKB1 acts as an upstream kinase of the AMPK pathway and regulates eNOS phosphorylation [58]. In our study, LKB1 was activated by amurensin G. Furthermore, introduction of LKB1 siRNA reduced amurensin G-induced AMPK

activation and eNOS phosphorylation (Supplementary Fig. 4). These results suggest that the AMPK pathway is alternatively required for amurensin G-stimulated eNOS phosphorylation, which may be mediated through LKB1 activation. AMPK lies upstream of Akt in the pathway leading from receptor activation to eNOS stimulation [59]. Because inhibition of either PI3K or AMPK simultaneously blocks amurensin Gstimulated eNOS phosphorylation, we further tested whether cross-talk between PI3-kinase/Akt and AMPK was associated with

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Amurensin G

ERα/β AMPK

Src

Gαi

Endothelial cells PI3K/Akt Ser 1177

P

eNOS

NO Vascular smooth muscle relaxation Potential beneficial effects against chronic vascular diseases Fig. 8. Proposed mechanism for amurensin G’s eNOS activation.

eNOS phosphorylation by amurensin G. We found that Aktphosphorylation induced by amurensin G treatment was attenuated by an AMPK inhibitor, but that PI3K inhibition did not affect amurensin G-mediated AMPK phosphorylation. In addition, a putative ER antagonist decreased the amurensin G-stimulated AMPK phosphorylation. Thus, it is plausible that amurensin G first activates ERs, and that this is responsible for further serial activation of the AMPK/PI3K/Akt pathway and subsequent eNOS phosphorylation. In summary, the present study showed that amurensin G activated eNOS via eNOS phosphorylation, and that the AMPK/ PI3K/Akt pathway, which is under the control of the ER pathway, played a critical role in amurensin G-mediated eNOS phosphorylation. Considering the plentiful amount of resveratrol- or amurensin G-like (oligo)stilbenoids in V. amurensis grapes, amurensin G or the plant extracts could be applicable in preventing cardiovascular diseases. Collectively, our observations have important implications for the elucidation of the pharmacological mechanism of amurensin G. Acknowledgment This work was supported by National Research Foundation of Korea (NRF) grants funded by the Korea government [grant nos. 2010-0001707 and 2011-0029091]. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bcp.2012.09.004. References [1] Widlansky ME, Gokce N, Keaney JJ, Vita JA. The clinical implications of endothelial dysfunction. J Am Coll Cardiol 2003;42:1149–60. [2] Fatehi-Hassanabad Z, Chan CB, Furman BL. Reactive oxygen species and endothelial function in diabetes. Eur J Pharmacol 2010;636:8–17. [3] Moncada S, Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med 1993;329:2002–12.

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