Transcriptional Down-Regulation of Thromboxane A2 Receptor Expression via Activation of MAPK ERK1/2, p38/NF-<symbol unicode="03BA" ascii="kappa"></symbol>B Pathways

Background: We have developed an in vitro model by organ culture of rat mesenteric arteries to imitate vascular smooth muscle cell (VSMC) receptor changes in cardiovascular disease. By using this model, alteration of VSMC thromboxane A 2 (TP) receptors was studied. Methods and Results: After organ culture of the arteries, VSMC TP receptors were studied by using myography, real-time PCR and immunohistochemistry. We observed that organ culture for 24 and 48 h resulted in depressed TP receptor-mediated contraction in the VSMC, in parallel with decreased TP receptor mRNA and protein expressions. Phosphorylation of extracellular signalregulated kinase 1 and 2 (ERK1/2), p38 and nuclear factorB (NFB) was seen by Western blot within 1–3 h after organ culture. Inhibition of ERK1/2, p38 or NFB reversed depressed contraction as well as decreased receptor mRNA expression. Actinomycin D abolished decreased TP receptor-mediated contraction, while inhibition of translation, cyclooxygenase or nitric oxide synthase had no effect. TP receptor mRNA stability was unchanged during organ culture. Conclusions: The present study has demonstrated for the first time that organ culture of rat mesenteric arteries down-regulates VSMC TP receptor expression through activation of ERK1/2 and p38/NFB signal pathways. Copyright © 2008 S. Karger AG, Basel Received: October 28, 2007 Accepted after revision: March 18, 2008 Published online: September 4, 2008 Prof. Cang-Bao Xu, MD, PhD Division of Experimental Vascular Research, BMC A13, WNC Institute of Clinical Science in Lund, Lund University SE–221 84 Lund (Sweden) Tel. +46 46 222 0825, Fax +46 46 222 0616, E-Mail [email protected] © 2008 S. Karger AG, Basel 1018–1172/09/0462–0162$26.00/0 Accessible online at: www.karger.com/jvr D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /2 9/ 20 17 6 :1 6: 30 P M Down-Regulation of Thromboxane A 2 Receptor Expression J Vasc Res 2009;46:162–174 163 transcription is reported to be suppressed by PPAR and retinoids in VSMCs [10, 11] . Although ovariectomized rats exhibit enhanced vasoconstrictor responses to U46619, oestrogen replacement reduced TP receptor-mediated vasoconstriction [15] . Thus, it is important to obtain further knowledge about the molecular mechanisms responsible for alterations in TP receptor expression in VSMCs. G protein-coupled receptor (GPCR)-mediated VSMC contraction and proliferation are key elements in the development of cardiovascular diseases [16] . Organ culture of arterial segments has been developed as a model for studying dynamic GPCR changes in VSMCs [17] which mimic receptor changes seen in vascular diseases [18] . We have revealed that organ culture of arteries results in up-regulation of 5-hydroxytryptamine (5-HT) 1B/1D [19– 21] and endothelin B (ET B ) [22–24] receptors, and similar changes were observed in experimental stroke [25] as well as in patients [26] . De novo transcription and synthesis of new receptors through activation of intracellular mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1 and 2 (ERK1/2) are responsible for the up-regulation of these receptors [27, 28] . TP receptors are key elements in the development of cardiovascular diseases, which mediate VSMC proliferation and contraction, and platelet aggregation [29] . Therefore, the present study was designed to investigate the molecular mechanisms that are responsible for the regulation of TP receptors in VSMCs. We hypothesize that there is a transcriptional down-regulation of TP receptors in VSMCs which occurs through activation of MAPK/nuclear factor B (NF B) signal pathways. Our results demonstrate that the down-regulation of TP receptor during organ culture, which is seen in the vasculature of diabetic rats, could be reversed by inhibition of ERK1/2, p38/NFB pathways. Thus, these intracellular signal events may provide new options for modulation of the vascular complications in diabetes. Materials and Methods Tissue Preparation and Organ Culture Procedure Male Sprague-Dawley rats (weighing 300–350 g) were anaesthetized with CO 2 and exsanguinated. The superior mesenteric artery (about 1 mm in outer diameter) was removed gently, immersed into cold buffer solution (for composition, see below) and dissected free of adhering tissue under a microscope. The endothelium was denuded by perfusion of the vessel for 10 s with 0.1% Triton X-100 followed by another 10 s with a physiological buffer solution [17] . The removal of the endothelium was verified by no dilatory response to acetylcholine (10 –6 M ) on 5-HT (10 –5 M ) precontracted segments, and confirmed by immunohistology. The vessels were then cut into 1-mm-long cylindrical segments, used directly (fresh group) or incubated at 37 ° C in humified 5% CO 2 in air for different time points (organ culture group). The segments for organ culture were placed in a 24-well plate, 1 segment in each well, containing 1 ml Dulbecco’s modified Eagle’s medium containing L -glutamine (584 mg/l), supplemented with penicillin (100 U/ml) and streptomycin (100 g/ml). For the inhibition experiments, different inhibitors were added to the culture medium. The same volume of vehicle served as control. The experimental protocol was approved by the Lund University Animal Ethics Committee (M217-03). The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). Buffer Solutions Standard buffer solution (m M ): NaCl 119; NaHCO 3 15; KCl 4.6; MgCl 2 1.2; NaH 2 PO 4 1.2; CaCl 2 1.5; glucose 5.5. Analyticalgrade chemicals and double-distilled water were used for preparing all solutions. Dulbecco’s modified Eagle’s medium, penicillin and streptomycin were purchased from Gibco BRL (Paisley, Scotland). In vitro Pharmacology Fresh or incubated segments were immersed in a temperaturecontrolled (37 ° C) myograph system (Organ Bath Model 700MO; J.P. Trading, Aarhus, Denmark) containing 5 ml bicarbonate buffer solution. The solution was continuously aerated with 5% CO 2 in O 2 , resulting in a pH of 7.4. The arterial segments were mounted for continuous recording of isometric tension by the Chart software (AD Instruments, Hastings, UK). A resting tone of 2.5 mN was applied to each segment and the segments were allowed to stabilize at this tension for at least 1.5 h before being exposed to a potassium-rich (60 m M K + ) buffer solution with the same composition as the standard solution except that NaCl was replaced by an equimolar concentration of KCl. The potassium-induced contraction was used as a reference for the contractile capacity, and the segments were used only if potassium elicited reproducible responses over 1.0 mN. Concentration-response curves for vasoconstrictors were obtained by cumulative administration of the reagents. Real-Time RT-PCR Smooth muscle cells were isolated from fresh or cultured artery segments and homogenized in 1 ml of RNApro solution (Qbiogene, Carlsberg, Calif., USA) by using a FastPrep instrument (Qbiogene). The total RNA was extracted following a protocol from the FastRNA Pro kit supplier and purity was verified by a GeneQuant Pro (Amersham Biosciences, Cambridge, UK) spectrophotometer. Reverse transcription of total RNA to cDNA was carried out using TaqMan reverse transcription reagents (PE Applied Biosystems, Foster City, Calif., USA) in a Perkin-Elmer 2400 PCR machine (Perkin-Elmer, Waltham, Mass., USA) at 42 ° C for 30 min. Real-time quantitative PCR was performed with the GeneAmp SYBR Green PCR kit in a GeneAmp 5700 sequence detection system (Perkin-Elmer). The system automatically monitors the binding of a fluorescent dye to double-strand DNA by realtime detection of the fluorescence during each cycle of PCR amplification. D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /2 9/ 20 17 6 :1 6: 30 P M Zhang /Zhang /Edvinsson /Xu J Vasc Res 2009;46:162–174 164 PCR was performed in a 50l volume and started at a temperature of 50 ° C for 2 min, 95 ° C for 10 min and the following 40 PCR cycles were done with 95 ° C for 15 s and 60 ° C for 1 min. Dissociation curves were run after the real-time PCR to identify the specific PCR products. All primers were designed using the Primer Express 2.0 software (PE Applied Biosystems) and synthesized by TAG Copenhagen A/S (Copenhagen, Denmark). Specific primers for the rat TP receptor (gene ID: NM_017054) were designed as follows: TP receptor: forward: 5 -ATCTCCCATCTTGCCATAGTCC3 ; reverse: 5 -CCGATGATCCTTGAGCCTAAAG-3 . Elongation factor-1 (EF-1, gene ID: BC072542) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, gene ID: NM_ 023964) mRNA were used as references, since they are the product of a housekeeping gene, continuously expressed to a constant amount in cells. The EF-1 and GAPDH primers were designed as follows: EF-1: forward: 5 GCAAGCCCATGTGTGTTGAA-3 ; reverse: 5 -TGATGACACCCACAGCAACTG-3 . GAPDH: forward: 5 -GGCCTTCCGTGTTCCTACC-3 ; reverse: 5 -CGGCATGTCAGATCCACAAC-3 . Data were analysed with the comparative cycle threshold (C T ) method. To evaluate the amount of mRNA in a sample, EF-1 and GAPDH mRNA were assessed in the same sample simultaneously. The C T values of EF-1 and GAPDH mRNA were used as references to quantify the relative amount of TP receptor mRNA. The relative amount of mRNA was calculated with the C T values of TP receptor mRNA in relation to the C T values of EF-1 or GAPDH mRNA in the sample. TP Receptor mRNA Stability To study if a post-transcriptional mechanism of mRNA stability was involved in the down-regulation of TP receptors after organ culture, the segments were incubated in the presence of actinomycin D (AcD) for 0.5, 6 and 24 h, so that de novo synthesis of receptor mRNA was inhibited. Real-time RT-PCR was used to determine the amount of TP receptor mRNA expression. Immunohistochemistry Fresh or cultured arterial segments were immersed in a fixative solution consisting of 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 3 h at 4 ° C. After fixation, the specimens were dehydrated in 20% sucrose of phosphate buffer (0.1 M , pH 7.4) for 24 h at 4 ° C, and then frozen in Tissue-Tek (Sakura Finetek Europe B.V., Zoeterwoude, The Netherlands) and stored at –80 ° C. Sections were cut at 10m thickness in a cryostat and mounted on SuperFrost Plus slides. Immunohistology staining with primary antibody against rat TP receptors from rabbits (Alexis Biochemicals, Lausen, Switzerland) and secondary antibody goatanti-rabbit IgG conjugated to fluorescein isothiocynate (Alexis Biochemicals) was used to demonstrate TP receptor proteins. Briefly, the sections were incubated with the primary antibody (1: 150 dilution) overnight at 4 ° C, thereafter, the secondary antibody (1: 200 dilution) was applied for 1 h at room temperature in the dark. In the control experiments, either the primary or the secondary antibody was omitted. The stained arterial segments were observed under a confocal microscope (C1plus; Nikon Instruments Inc., Melville, N.Y., USA) and analysed by the Image J software (http://rsb.info.nih.gov/ij) [26] . The measurement was based on positive staining in the smooth muscle cells. For each vessel, 6 sections were studied and the values obtained were mean fluorescence in the areas selected (there were 6 preset areas per section), and analysed blindly as to the treatment protocol. Western Blot After organ culture, vessels were collected and frozen in liquid nitrogen, and homogenized in denaturing cell extract buffer (BioSource, Carlsbad, Calif., USA) with phosphatase inhibitor cocktail and protease inhibitor cocktail (Sigma, St. Louis, Mo., USA). Protein concentration was measured by a Bio-Rad protein analysis kit and a Tecan Infinite M200 microplate reader. Proteins (40 g) were loaded and separated on 4–15% Ready Gel Precast Gels (BioRad, Hercules, Calif., USA) and followed by One-Step Complete Western Kit (GenScript Corporation, Piscataway, N.J., USA) according to the manual. Briefly, membranes were incubated with Pretreat Solution on shaker for 5 min at room temperature, and then with primary antibody overnight at 4 ° C. After washing 3 times for 10 min, the membranes were developed with Working Solution (LumiSensor A + B) for 3 min, visualized using a Fuji film LAS-1000 Luminiscent Image Analyzer (Fujifilm, Stamford, Conn., USA), and then analysed with Image Gauge 4.0 (Fuji Photo Film Co. Ltd., Tokyo, Japan). The antibodies for phospho-p44/42 MAPK (Thr202/Tyr204), phospho-p38 MAPK (Thr180/Tyr182), phospho-NFB p65 (Ser536) and -actin were from Cell Signaling Technology (Beverly, Mass., USA). All antibodies were used at 1: 1,000 dilution and the experiments were repeated 3 times. Intracellular Signal Inhibitors and Drugs To link intracellular events to functional changes, a set of inhibitors was administered during organ culture. The MAPK pathway consists of 3 major members: ERK1/2, JNK and p38 [30] . NFB, one of its downstream transcriptional factors, is a key inflammatory signal molecule [31, 32] . In the present investigation, inhibitors including PD98059 and U0126 (inhibitors of ERK1/2) [33, 34] , SB203580 and SB239063 (inhibitors of p38) [35, 36] , SP600125 (inhibitor of JNK) [37] and wedelolactone (inhibitor of NFB) [38] were employed to examine the involvement of different MAPK subtypes and the NFB pathway. AcD and cycloheximide (CHX) were used as general transcriptional and translational inhibitors, respectively. Cyclooxygenase (COX) inhibitor, indomethacin, and nitric oxide synthase (NOS) inhibitor, N  -nitroL -arginine methyl ester ( L -NAME), were used separately or in combination to investigate whether COX and NOS were involved. All drugs were purchased from Sigma. U46619 was dissolved in ethanol to a stock concentration of 10 m M and further diluted in distilled water; GR32191b (selective antagonist for TP receptors) and L -NAME were dissolved in distilled water; indomethacin was dissolved in 95% ethanol; the others were dissolved in dimethyl sulfoxide. Data Analysis All data are expressed as mean values 8 SEM. The responses to U46619 are presented as percentage of contraction relative to 60 m M K + or the absolute values (mN) in the inhibition experiments, since the inhibitors might affect the reference-induced contraction. The amount of receptor mRNA is expressed as percentage of control group. One-way or two-way ANOVA with Bonferroni post-test was used for comparisons of more than 2 data sets. p ! 0.05 was considered to be significant. D ow nl oa de d by : 54 .7 0. 40 .1 1 10 /2 9/ 20 17 6 :1 6: 30 P M Down-Regulation of Thromboxane A 2 Receptor Expression J Vasc Res 2009;46:162–174 165 Results In vitro Pharmacology Organ culture of rat mesenteric arterial segments for 24 h ( fig. 1 a) or 48 h ( fig. 1 b) resulted in significantly depressed contractile responses to the selective agonist for TP receptor U46619 [39] , compared with fresh segments. For the endothelium-denuded group, the pEC 50 value was significantly decreased from 7.74 8 0.03 (fresh) to 7.00 8 0.05 (organ culture for 24 h; n = 8, p ! 0.001). The segments with intact endothelium had the same contractility and similar response to those without endothelium in both fresh and organ culture groups ( fig. 1 a). Since the present study was designed to investigate alteration of VSMC TP receptor expression and in particular the molecular mechanisms involved, only endothelium-denudated segments were used for further studies. In the endothelium-denuded segments, the reference (60 m M K + )-induced contractions (absolute values, mN) and the maximal contractile response (E max , % of K + ) to U46619 were not significantly changed within groups (n = 8, p 1 0.05; table 1 ). Pharmacological Characterization In order to characterize the TP receptors in the VSMCs, the contractile response to U46619 in fresh segments was antagonized by GR32191b, a selective TP receptor antagonist [40] . GR32191b caused concentration-dependent, parallel rightward shift of the concentration-response curve to U46619 without significantly altering E max ( fig. 2 a). Schild analysis gave a slope of 0.82 8 0.21, and a pK B value of 8.45 8 0.06 was obtained when the slope was constrained to unity in fresh segments ( fig. 2 b). SimOrgan culture 24 h, Endo+ Organ culture 24 h, Endo–