Biochemical and Biophysical Research Communications 398 (2010) 653–658
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a-Lipoic acid activates dimethylarginine dimethylaminohydrolase in cultured endothelial cells Woo Je Lee a, Seung-Hwan Kim b, Geun Hyang Kim b, Sung Min Han a, Jong Chul Won c, Chang Hee Jung a, Hye-Sun Park d, Do Sook Choi d, Ki-Up Lee a, Joong-Yeol Park a,* a
Department of Internal Medicine, University of Ulsan College of Medicine, Seoul, Republic of Korea Department of Pharmacology, University of Ulsan College of Medicine, Seoul, Republic of Korea Department of Internal Medicine, Inje University College of Medicine, Seoul, Republic of Korea d Asan Institute for Life Sciences, University of Ulsan College of Medicine, Seoul, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 24 June 2010 Available online 11 July 2010 Keywords: ADMA a-Lipoic acid DDAH Endothelial cell STAT3
a b s t r a c t Asymmetric dimethylarginine (ADMA) is a risk factor of cardiovascular diseases. a-Lipoic acid (ALA) was shown to improve vascular dysfunction, and to decrease plasma ADMA level. In this study, we investigated whether ALA activates dimethylarginine dimethylaminohydrolase (DDAH), the metabolizing enzyme of ADMA, in cultured endothelial cells. ALA significantly decreased ADMA level in culture media of endothelial cells. ALA increased the gene expression and activity of DDAH, and signal transducer and activator of transcription (STAT)3 phosphorylation. Transfection of STAT3 increased DDAH II promoter activity, and ALA amplified it. ALA-induced increase in DDAH II promoter activity was attenuated in the promoter that had mutation in putative STAT3-binding site. These results suggest that ALA reduces ADMA level by enhancing DDAH activity and DDAH II gene expression, thus providing a novel mechanism by which ALA regulates endothelial function. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Decreased nitric oxide (NO) bioavailability is one of the major features of metabolic syndrome and cardiovascular diseases [1,2]. Decreased endothelial nitric oxide synthase (NOS) activity and increased oxidative degradation of NO are two major causes of decreased NO bioavailability. ADMA is an endogenous competitive inhibitor of NOS and increased plasma concentration of ADMA has been described in a variety of conditions associated with endothelial dysfunction, including hypercholesterolemia, hypertension, diabetes mellitus, obesity, and atherosclerosis [3]. ADMA is synthesized by protein arginine methyltransferases (PRMTs) and metabolized by dimethylarginine dimethylaminohydrolase (DDAH) [4]. Reduced degradation of ADMA due to decreased DDAH activity has been proposed as a main mechanism for endothelial vasodilator dysfunction observed in hypercholesterolemia [5] and diabetes [6]. ALA is a naturally occurring short chain fatty acid with sulfhydryl groups that has a potent anti-oxidant activity. We previously reported that ALA reduces body weight [7] and improves insulin sensitivity in skeletal muscle [8] by regulating AMP-activated pro-
tein kinase (AMPK). In vascular cells, ALA improved endothelial function in obese rats by increasing NO bioavailability and activating AMPK [9]. In addition, ALA was shown to decrease plasma ADMA levels in patients with end-stage renal disease (ESRD) [4,10]. Although ALA’s anti-obesity action is independent from leptin or leptin receptor signaling [7], ALA has much similarity to leptin in that both of them increase AMPK in skeletal muscle and decrease it in the hypothalamus [7,8,11,12]. Signal transducer and activator of transcription (STAT)3 is a well-known downstream signal of leptin [13]. In this regard, STAT3 might play a role as a downstream signal of ALA. Recently, it was reported that the promoter region of DDAH II has putative binding sites for STAT [14]. In the present study, we examined whether ALA regulates the activity and mRNA expression of DDAH, and whether STAT3 is involved in the regulation of DDAH by ALA in cultured endothelial cells.
2. Materials and methods 2.1. Cell culture and treatment
* Corresponding author. Address: Department of Internal Medicine, University of Ulsan College of Medicine, Asan Medical Center, Song-Pa P.O. Box 145, Seoul 138600, Republic of Korea. Fax: +82 2 3010 6962. E-mail address:
[email protected] (J.-Y. Park). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.06.127
Murine endothelioma cells (bEnd.3) were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
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W.J. Lee et al. / Biochemical and Biophysical Research Communications 398 (2010) 653–658
10% fetal bovine serum (FBS) and antibiotics. Human aortic endothelial cells (HAECs) were obtained from BioWhittaker Inc. (Walkersville, MD) and cultured in endothelial basal medium (EBM, BioWhittaker) supplemented with 2% FBS and various growth factors. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2. ALA was provided by Bukwang Pharmaceutical Company (Seoul, Korea) and DMSO was used as vehicle. Cells were transferred to medium containing 0.5% FBS and incubated in media containing a various concentrations of ALA for the indicated time. All-trans-retinoic acid (atRA), a DDAH activator [14], was obtained from Sigma (St. Louis, MO). Compound C (Sigma) was used as an AMPK inhibitor. 2.2. Determination of ADMA concentration Concentration of ADMA in the conditioned media was measured by high-performance liquid chromatography (HPLC) and precolumn derivatization with o-phthaldialdehyde (OPA) was adopted as previously described [15]. 2.3. Determination of DDAH activity The activity of DDAH in endothelial cells was estimated by two different methods. First, we measured DDAH activity by comparing the concentration of ADMA before and after the enzyme inactivation by adding 30% sulfurosalicyclic acid. The difference of the ADMA concentrations reflected the DDAH activity [6]. Second, [3H]-L-NG-monomethyl-L-arginine ([3H]-L-NMMA), which is obtained from American Radiolabeled Chemicals (St. Louis MO), was used as a substrate and [3H]-L-citrulline was detected as product. [3H]-L-citrulline concentration in endothelial cells was determined as described [16]. 2.4. Northern blot analysis Total RNA was isolated from cells using TRIzol Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Aliquots of total RNA (20 lg) from each sample were loaded onto 1.0% formaldehyde-agarose gels. After electrophoresis, RNA was transferred to Hybond-N+ nylon membranes (Amersham Biosciences, Little Chalfont, UK) followed by UV cross-linking (UV cross-linker 1800, Stratagene, La Jolla, CA). The probes for DDAH II were labeled with [a-32P]dCTP using a random-primer DNA-labeling system (Amersham Biosciences). Membranes were hybridized with 32P-labeled probes at 65 °C overnight. The membranes were washed in wash solution I (2 SSC, 0.05% SDS solution) and subsequently in wash solution II (0.1 SSC, 0.1% SDS solution), air dried, and exposed to autoradiography film. Densitometric measurements of bands were made using the digitizing scientific software program, UN-SCAN-IT (Silk Scientific Corporation, Orem, UT). 2.5. Western blot analysis
pGL3-basic vector. Putative STAT3-binding site (from 1187 bp to 1179 bp) in DDAH II promoter was mutated by PCR overlap extension from 50 -TTTCGGCAA-30 to 50 -CCCAGGCAA-30 . DDAH II promoter and its mutant were confirmed by sequencing. Plasmid encoding cMyc-tagged forms of dominant-negative a1 and a2 AMPK (DNAMPK) were kind gifts from Dr. J. Ha (Department of Molecular Biology, Kyung Hee University College of Medicine, Seoul, Korea). Recombinant adenovirus was prepared as described previously [17]. 2.7. In vitro transient transfection and reporter assays Cells were transfected with 100 ng of reporter gene, and 100 ng of STAT3-expression vector using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA). b-Galactosidase plasmids were co-transfected as an internal control. Twenty-four hours after the transfections, the cells were left unstimulated or stimulated with 0.5 mM ALA and were harvested 24 h later. Luciferase activity was detected using a SIRUS Luminometer (Berthold, Pforzheim, Germany) and normalized to b-galactosidase activity. 2.8. Electrophoretic mobility shift assay (EMSA) bEnd.3 cells were homogenized in 400 ll of ice-cold buffer A (10 mmol/l NaCl, 4.5 mmol/l KCl, 7 mmol/l Na2HPO4 and 3 mmol/ l KH2PO4) and incubated on ice for 30 min. After treating with 25 ll of NP-40, the homogenate was layered onto buffer C (5 mmol/l HEPES, 26% glycerol, 1.5 mmol/l MgCl2, 0.2 mmol/l EDTA, 0.5 mmol/l DTT and 0.5 mmol/l PMSF) and centrifuged at 24,000g for 20 min. After centrifugation, the supernatant (nuclear extract) was collected and protein concentration was measured using a protein assay kit (Bio-Rad, Richmond, CA). Nuclear extracts (6 lg) were incubated with 60,000 cpm of the 32P-labeled DDAH II oligonucleotides for 20 min at 20 °C. For competition experiment, nuclear extracts were incubated on ice for 15 min with unlabeled oligonucleotides before addition of labeled probes. For supershift analysis, nuclear extracts were preincubated with STAT3 antibody for 60 min at room temperature. 2.9. Data analysis All data are shown as mean ± SEM. Comparisons between two groups were analyzed using unpaired Student’s t tests, and among multiple groups by ANOVA followed by a post hoc analysis using the Tukey’s multiple comparison test. A P value
