Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining a novel function for this important lipid regulator.Synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication. The membrane fusion process is mediated by a conserved set of SNARE proteins: vesicular synaptobrevin and plasma membrane syntaxin and SNAP-25. Recent data suggest that the fusion process may be subject to regulation by local lipid metabolism. Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin. We show that sphingosine, a releasable backbone of sphingolipids, activates synaptobrevin in synaptic vesicles to form the SNARE complex implicated in membrane fusion. Consistent with the role of synaptobrevin in vesicle fusion, sphingosine upregulated exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and hippocampal neurons, but not in neurons obtained from  Neurotransmission occurs when synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane causing release of their content. This neuronal exocytosis requires three SNARE  proteins: synaptobrevin-2  on the synaptic vesicle, and syntaxin-1 with SNAP-25 on the plasma membrane . FormatiIt is critical for neuronal physiology that synaptic vesicle exocytosis takes place with high spatial and temporal precision. Preventing uncontrolled formation of SNARE complex can be an important point for regulation of vesicle fusion and thereby of neurotransmission. Interestingly, synaptobrevin in synaptic vesicles does not readily react with syntaxin and SNAP-25 . Two altFor screening of lipids with a potential role in exocytosis, we purified synaptic vesicles from rat brain by a flotation procedure  and incu50 ∼10 μM (top fraction) following the sphingosine treatment.Titration experiments demonstrated that sphingosine allows synaptobrevin to form ternary SNARE complexes in a dose-dependent manner, with an EC0 ∼10 μM C. Althoum), a parameter linearly related to the plasma membrane area, which increases upon exocytosis , which express synaptobrevin-2 . We thenocytosis . The cyt% ± 8.6% A and 2B. 8.6%  with radioactive sphingosine for 10 min and after a wash step disrupted synaptosomes by hypo-osmotic shock. Following separation of synaptic fractions by centrifugation, distribution of sphingosine in synaptic membranes, synaptic cytosol and synaptic vesicles was analyzed by liquid scintillation A. Sphingsynaptobrevin knockout model (−/−Syb2). Mice deficient in synaptobrevin-2 die immediately after birth; however, embryonic neurons from these animals survive in culture . Nevertheless, in some cells one can detect occasional responses to stimulation and sphingosine effects were tested in these responding cells. The responses recorded from −/−Syb2 synapses did not show an increase after sphingosine treatment  to directly interact with negatively charged phospholipid membranes . We anal of 6 μM C. To pro of 6 μM . GST-syn of 6 μM D. Sphing of 6 μM D demonst of 6 μM . This in of 6 μM . Figure To confirm the ability of sphingosine to affect synaptobrevin properties on the membranes, we performed a limited proteolysis experiment. We noticed that the V8 protease at 0.1 μM concentration cannot readily digest synaptobrevin in synaptic vesicles. Addition of sphingosine allowed almost complete proteolysis of synaptobrevin but not of a control protein, synaptophysin F. In conA number of recent studies highlighted the physiological significance of sphingolipid metabolism in vesicle exocytosis. Sphingomyelinase activity has been linked with neurotransmitter release from cerebellar and mesencephalic neurons and also PC12 cells . A recenOur results suggest that sphingosine can relief membrane-mediated inhibition of the cytoplasmic part of synaptobrevin, thereby allowing formation of the ternary SNARE complex. It is conceivable that the positively charged sphingosine can locally disrupt electrostatic and hydrophobic interactions of the cytoplasmic part of synaptobrevin with vesicular membranes. Of note, the cytoplasmic part of synaptobrevin carries basic amino acids centered around hydrophobic tryptophans, all of which can contribute to its interaction with negatively charged phospholipid membranes . RecentlPreparation of vesicles for fusion with the plasma membrane requires a series of steps: tethering, docking and priming. It is well established that phospholipase C-mediated diacylglycerol production plays an important role in priming of syntaxin for SNARE-mediated vesicle fusion . Our datIn summary, our study defines a new mechanism for synaptic regulation: increased sphingosine production results in activation of synaptobrevin for SNARE complex assembly thereby facilitating synaptic transmission. We demonstrated that the unique properties of sphingosine are required for relieving the amphipathic cytoplasmic part of synaptobrevin from its association with the phospholipid membrane. This step is followed by synaptobrevin engagement of syntaxin/SNAP-25 resulting in SNARE assembly and an augmented pool of readily-releasable vesicles. Such a mechanism could explain, for example, stimulation of neurotransmitter release by growth factors that activate sphingomyelin hydrolysis . ImportaTo gain a better understanding of sphingosine signaling in subcellular settings, it will be useful to develop fluorescent reporters that can sense local release of this lipid metabolite similarly to a recent utilization of a protein domain in defining sites of diacylglycerol production . FurtherS-transferase (GST) fusion proteins of rat syntaxin 1A (aa 1–261), synaptobrevin-2 (aa 1–96 and full-length), wild-type SNAP-25B and SNAP-25B devoid of cysteines were described (n-octylglucoside (Sigma). Brain syntaxin and SNAP-25 were purified as described  were incubated with syntaxin1/SNAP-25 (2 μg) in the presence of 50 μM lipids (Biomol) for 30 min at 22°C. Reactions were analyzed by SDS-PAGE followed by immunoblotting using a synaptobrevin antibody .Liposomes were prepared as described . The bea3H-labeled sphingosine (Perkin-Elmer) for 10 min at 37°C and purified again on the Ficoll gradient. Synaptosomes were disrupted by the hypo-osmotic shock . Synaptic membranes were isolated by centrifugation . Synaptic vesicles were purified as described (Synaptosomes were prepared from rat brains using Ficoll (Sigma-Aldrich) gradient as described . To probescribed . Radioacescribed . For lim2PO4, 1.3 MgCl2, 0.15 Na2EGTA, 1 MgSO4, 5 NaHCO3, 10 D-glucose. Synaptosomes (1 mg protein/ml) were incubated for 10 min at 37°C with an equal volume of synaptosomal buffer B containing glutamate dehydrogenase  and 3 mM NADP (Sigma) in the presence of either sphingosine solution or vehicle, DMSO. Glutamate release was induced by addition of KCl (35 mM) in the presence of 2 mM CaCl2 and monitored by following fluorescence  was added to synaptosomes 15 min before addition of Ca2+/KCl. N-oleoylethanolamine  was added to synaptosomes 15 min prior to addition of sphingomyelinase. Six nanomolar botulinum neurotoxin D was added to synaptosomes 30 min prior to further additions.Freshly-isolated synaptosomes were resuspended in synaptosomal buffer B containing (in mM) 132 NaCl, 5 KCl, 20 HEPES, 1.2 NaHrescence . S. aurem) was measured at 22°C using the uncompensated whole-cell technique with a dual-phase lock-in patch-clamp amplifier . Melanotrophs were voltage clamped at −70 mV. Whole cell currents were stimulated with lock-in frequency of 1591 Hz and were collected by Cell software (Celica). The recording bath solution consisted of (mM) 130 NaCl, 10 HEPES/NaOH (pH 7.2), 10 glucose, 8 CaCl2, 1 MgCl2, and 5 KCl. Patch-clamp pipettes had a resistance of 1.5–4 MΩ and were filled with a solution containing (mM): 150 KCl, 2 MgCl2, 10 HEPES/KOH (pH 7.2), 2 Na2ATP, 2 EGTA, 1.74 CaCl2, yielding free [Ca2+] of 1 μM. Secretory responses were measured as a change in Cm (%) relative to the resting Cm determined immediately after the establishment of the whole-cell recording. The average steady-state currents were −12 ± 8 pA (control), −6 ± 16 pA (10 μM sphingosine), and −25 ± 16 pA (50 μM sphingosine); these values were not statistically different .Animals were sacrificed in accordance with the European Communities Council Directive (86/609/EEC). Melanotroph cell cultures were prepared from rats as described . Cells w2+ current peak (ICa) was recorded in voltage-clamped cells under whole cell configuration at 25°C. Cells were dialyzed with a solution containing (mM): 10 NaCl, 100 CsCl, 20 TEA-Cl, 0.1 EGTA, 20 HEPES, 5 MgATP, 0.3 NaGTP (pH 7.2). During recordings, cells were perfused with the Tyrode solution containing (mM) 137 NaCl, 1 MgCl2, 10 CaCl2, 10 glucose, 10 HEPES (pH 7.4). For patching the cells, pipettes of 3–5 MΩ resistance were used. Electrophysiological data were acquired with an EPC-9 amplifier using the Pulse software (HEKA Elektronik). Cm changes were estimated by the Lindau-Neher technique. A 400 ms sinusoidal wave  was given before the depolarizing protocol followed by a 2 s sinusoidal wave of the same characteristics, allowing calculations of Cm changes. Membrane current was sampled at 20 kHz. Cells were held at −80 mV and depolarizing pulses at +10 mV were applied at 1 min intervals. ICa was analyzed after the initial 5 ms of each depolarizing pulse. To eliminate interference from Na+ channel gating (m) was calculated by subtracting the basal mean Cm obtained 400 ms prior to depolarization from that obtained 50 ms after the end of the depolarizing pulse. Initial leak currents were not significantly different: −11 ± 5 pA (control), −10 ± 8 pA (10 μM sphingosine), and −9 ± 4 pA (50 μM sphingosine).In bovine chomaffin cell experiments, Cal gating , exocytisynaptobrevin-2-deficient dissociated cultures (courtesy of Dr. Thomas C. Südhof) were prepared as described (2 in a humidified incubator. We used a modified Tyrode's solution that contained (mM): 145 NaCl, 4 KCl, 2 MgCl2, 10 glucose, 10 HEPES, 2 CaCl2 (pH 7.4). Pyramidal neurons were whole-cell voltage clamped at −70 mV with borosilicate glass electrodes (3–5 MΩ). Electrode solutions contained (in mM) 105 Cs-methanesulphonate, 10 CsCl, 5 NaCl, 10 HEPES, 20 TEA-Cl, 4 Mg-ATP, 0.3 GTP, 0.6 EGTA, 10 QX-314 . Hippocampal cultures (10–21 days in vitro) were treated with lipids (50 μM) for 10 min at 22°C, washed thoroughly and then recordings were performed. Field stimulation was achieved by applying 20 mA pulses with a 1 ms duration using a bipolar platinum electrode; excitatory postsynaptic currents (EPSCs) were measured in the presence of 50 μM picrotoxin. Hypertonic sucrose responses were recorded by infusing modified Tyrode's solution containing 500 mM sucrose, 1 μM tetrodotoxin, 50 μM picrotoxin for 30 s. For enzymatic experiments, rat hippocampal cultured neurons were pretreated for 15 min with NOE (150 μM) followed by 10 min with sphingomyelinase (1 unit/ml) before the application of hypertonic sucrose.Control and escribed . The hip2, 1 MgCl2, 11 glucose, 12 NaHPO4, 2.4 NaHCO3 . Recordings of spontaneous miniature end-plate potentials (MEPPs) were performed using glass microelectrodes as described (Experiments on neuromuscular junctions were carried out using adult mouse diaphragm at 22°C. Muscles were continuously perfused with a solution containing (mM): 130 NaCl, 5 KCl, 2 CaClescribed . The rec2 and 100 units/ml benzonase (Novagen). Proteins were assessed by SDS-PAGE of boiled protein samples (10 μg protein) followed by immunoblotting using indicated antibodies.Following 30 min incubation with botulinum neurotoxins neurons were washed with PBS, and lysed in SDS-containing sample buffer supplemented with 10 mM MgCl


Vascular endothelial cells (ECs) constantly experience fluid shear stresses generated by blood flow. Laminar flow is known to produce atheroprotective effects on ECs. Nrf2 is a transcription factor that is essential for the antioxidant response element (ARE)-mediated induction of genes such as heme-oxygenase 1 (HO-1). We previously showed that fluid shear stress increases intracellular reactive oxygen species (ROS) in ECs. Moreover, oxidants are known to stimulate Nrf2. We thus examined the regulation of Nrf2 in cultured human ECs by shear stress.2) induced Nrf2 nuclear translocation, which was inhibited by a phosphatidylinositol 3-kinase (PI3K) inhibitor, a protein kinase C (PKC) inhibitor, and an antioxidant agent N-acetyl cysteine (NAC), but not by other protein kinase inhibitors. Therefore, PI3K, PKC, and ROS are involved in the signaling pathway that leads to the shear-induced nuclear translocation of Nrf2. We also found that shear stress increased the ARE-binding activity of Nrf2 and the downstream expression of HO-1.Exposure of human umbilical vein endothelial cells (HUVECs) to laminar shear stress (12 dyne/cmOur data suggest that the atheroprotective effect of laminar flow is partially attributed to Nrf2 activation which results in ARE-mediated gene transcriptions, such as HO-1 expression, that are beneficial to the cardiovascular system. Vascular endothelial cells (ECs) are in direct contact with blood flow and are constantly exposed to blood flow-generated shear stresses. Accumulated data in the literature reveal that laminar shear stress is beneficial for the endothelium . Numerou2O2), a major reactive oxygen species (ROS), is another Nrf2 stimulator. A recent study demonstrated that NO also induces Nrf2 nuclear translocation [Nuclear factor erythroid 2-related factor 2 (Nrf2) is a cap'n'collar (CNC) basic leucine zipper transcription factor . Evidenclocation . So far,location ,37. It wlocation ,39. Mitolocation ,41. Althlocation ,43, studlocation . In this2O2 to stimulate ECs were carried out, and similar results were obtained. Our data suggest that in some way, the atheroprotective role of laminar shear stress may be attributed to Nrf2 activation which results in initiation of ARE-mediated gene transcription, including HO-1 expression, that is beneficial to the cardiovascular system.In a previous study, we showed that shear stress increases intracellular ROS and antioxidant activity in ECs . Generat2 balanced with air. The following day, HUVECs were rinsed with buffer and grown to confluence within 2 to 3 days. The cells were subcultured on fibronectin-coated glass slides. One day prior to the shear stress experiments, the concentration of FBS in the culture medium was reduced to 2%, and this was used throughout the shear stress experiments.Primary cultures of HUVECs were harvested from umbilical cord veins by collagenase isolation. The harvested cells were resuspended in culture medium ), plated on 10-cm culture dishes, and then incubated at 37°C in 5% CO2 balanced with air.Exposure of HUVECs to shear stress was conducted in a parallel-plate flow chamber as previously described . ContinuTo prepare nuclear protein extracts, HUVECs were washed with cold phosphate-buffered saline (PBS) and then removed by scraping in detachment buffer . After centrifugation of the cell suspension at 2000 rpm, the cell pellets were resuspended in cold buffer A containing KCl (10 mM), EDTA (0.1 mM), dithiothreitol (1 mM), and phenylmethylsulfonyl fluoride (1 mM) for 15 min. The cells were lysed by adding 10% Nonidet P-40 and then centrifuged at 6000 rpm to obtain a pellet of nuclei. The pelleted nuclei were resuspended in cold buffer B containing HEPES (20 mM), EDTA (1 mM), dithiothreitol (1 mM), and phenylmethylsulfonyl fluoride (1 mM), as well as NaCl (0.4 mM), and then vigorously agitated from time to time, followed by centrifugation. The supernatant containing the nuclear proteins was used for Western blot analysis or an electrophoretic mobility shift assay (EMSA).Proteins were extracted in sodium dodecylsulfate (SDS) buffer and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). After being transferred onto a nitrocellulose membrane, antigens were analyzed by specific antibodies. Antigen-antibody complexes were detected using an ECL detection system (Pierce).BC011558) was designed using the GCG system, and the GADPH primer was provided by Dr. H. H. Chen . Nrf2 primers were synthesized by MB Mission Biotech  and consisted of 5'-ACA CGG TCC ACA GCT CAT CAT-3' (forward) and 5'-TTG GCT TCT GGA CTT GGA AC-3' (reverse); GAPDH primers were synthesized by the same company and consisted of 5'-TGG TAT CGT GGA AGG ACT CAT GAC-3' (forward) and 5'-ATG CCA GTG AGC TTC CCG TTC AGC-3' (reverse). When total RNA was isolated from HUVECs, a Superscript III one-step RT-PCR kit was used for the reverse transcription. After 25 cycles, the end products were subjected to 1% agarose electrophoresis to analyze the target RT-PCR products.The primer of Nrf2 (GenBank accession no.: The supernatant containing the nuclear proteins was used for the EMSA. The probe for EMSA was a synthetic 24-mer oligonucleotide (5'-GGG ACT GGT GAC TCA GCA AAA TCT-3') containing the ARE binding site within the promoter region and about 4 kb upstream of the human HO-1 gene . The pro2 which resembles physiological blood flow conditions in atherosclerotic plaque-free regions. Shear stress substantially increased the total cellular levels of Nrf2 protein over time  is mediated through PKCdelta/Nrf2/ARE signaling that leads to the upregulation of HO-1 [It was found that protein kinase C (PKC) phosphorylates Nrf2 and regulates concomitant ARE-mediated transcription in response to oxidative stress ,39. A re of HO-1 . These fChen et al. determined that dietary chemopreventive compounds induce Nrf2 translocation through MAPK pathways . Owuor eIn a previous study we showed that shear stress increases intracellular ROS which participate in cellular responses and signal transduction in endothelial cells . Since NOn the other hand, a recent study demonstrated that NO can also induce Nrf2 nuclear translocation . It is k2O2 also increased the ARE-binding activity of Nrf2, and this event was significantly suppressed by NAC  containing the ARE-binding site within the promoter region lying about 4 kb upstream of human HO-1 gene was used as a probe in the EMSA ,59. In t2O2 also increased HO-1 protein levels in a time-dependent manner  are a major source of ROS ,64, and 2O2, a major ROS, as the stimulus, and similar results were obtained. These results provide evidence that shear-induced Nrf2 regulates HO-1 expression via binding to the ARE in the promoter region, and this regulation involves PI3K and ROS.Results obtained from the gel shift assay further revealed that shear stress enhanced the ARE-binding activity of Nrf2 through the involvement of ROS Figure . Shear-i2 induced Nrf2 nuclear translocation, and we found that PI3K, PKC, and ROS, but not MAPKs (p38 and JNK), were involved in the signaling pathway. We also found that shear stress increased the ARE-binding activity of Nrf2 and HO-1 expression. HO-1 has been shown to have remarkable antioxidant abilities and is responsible for diverse protective functions against diseases such as atherosclerosis. Our data suggest that the atheroprotective role of laminar shear stress is partly attributed to Nrf2 activation which results in ARE-mediated gene transcription, including HO-1 gene expression, which is beneficial to the cardiovascular system.In the present study, we examined the regulation of shear stress on Nrf2 in HUVECs. We demonstrated that a laminar shear stress of 12 dyne/cmARE: antioxidant response element; ECs: endothelial cells; eNOS: endothelial nitric oxide synthase; HO-1: heme-oxygenase 1; HUVECs: human umbilical vein endothelial cells; MAPKs: mitogen-activated protein kinases; NAC: N-acetyl cysteine; NO: nitric oxide; Nrf2: nuclear factor erythroid 2-related factor 2; PI3K: phosphatidylinositol 3-kinase; PKC: protein kinase C; ROS: reactive oxygen species.The authors declare that they have no competing interests.CYH and HYH contributed equally to this study. CYH and HYH designed and performed the experiments, participated in the discussion of the results, and drafted part of the manuscript. WYW performed the inhibitor experiments and participated in the discussion of the results. CAL performed the EMSA experiments, participated in the discussion of the results, and helped to draft part of the manuscript. YCT participated in the experimental design and discussion of the results, and helped to draft part of the manuscript. YJC coordinated the experiments and participated in the discussion of the results. DLW co-conceived and co-designed the study, participated in the experimental design and interpretation of the results, reviewed and helped to revise the manuscript. HJH conceived and designed the study and the experiments, coordinated the execution of the study, interpreted the results, wrote the manuscript and also revised it. All authors read and approved the final manuscript.


We have recently established that the TPβ isoform of the human TXA2 receptor (TP) undergoes rapid, agonist-induced homologous desensitization of signalling largely through a G protein-coupled receptor kinase (GRK) 2/3-dependent mechanism with a lesser role for protein kinase (PK) C. Herein, we investigated the mechanism of desensitization of signalling by the TPα isoform. TPα undergoes profound agonist-induced desensitization of signalling  in response to the TXA2 mimetic U46619 but, unlike that of TPβ, this is independent of GRKs. Similar to TPβ, TPα undergoes partial agonist-induced desensitization that occurs through a GF 109203X-sensitive, PKC mechanism where Ser145 within intracellular domain (IC)2 represents the key phospho-target. TPα also undergoes more profound sustained PKC- and PKG-dependent desensitization where Thr337 and Ser331, respectively, within its unique C-tail domain were identified as the phospho-targets. Desensitization was impaired by the nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC) and PKG inhibitors l-NAME, LY 83583 and KT5823, respectively, indicating that homologous desensitization of TPα involves nitric oxide generation and signalling. Consistent with this, U46619 led to rapid phosphorylation/activation of endogenous eNOS. Collectively, data herein suggest a mechanism whereby agonist-induced PKC phosphorylation of Ser145 partially and transiently impairs TPα signalling while PKG- and PKC-phosphorylation at both Ser331 and Thr337, respectively, within its C-tail domain profoundly desensitizes TPα, effectively terminating its signalling. Hence, in addition to the agonist-mediated PKC feedback mechanism, U46619-activation of the NOS/sGC/PKG pathway plays a significant role in inducing homologous desensitization of TPα.Thromboxane (TX) A In addition, (ii) TPβ also undergoes more profound and sustained agonist-induced desensitization involving GRK 2/3-phosphorylation followed by internalization and we proposed a model whereby phosphorylation of Ser357 within the C-tail domain led to the recruitment and orientation of GRK2/3 which, in turn, facilitated its phosphorylation of Ser239 within IC3 thereby disrupting Gq coupling by TPβ. Hence, GRK2/3 binding is dependent on sequences within the unique C-tail domain of TPβ whilst Gq uncoupling is due to GRK phosphorylation of Ser239 within IC3 and, to a lesser extent, PKC phosphorylation of Ser145 within IC2.As stated, agonist-induced homologous desensitization of GPCRs is a central mechanism whereby the cellular responses to the receptor's own ligand is dynamically modulated and is most often initiated through phosphorylation of the ligand-engaged, conformationally active GPCR such as by members of the G protein-coupled receptor kinases (GRKs) and/or by the second-messenger kinases, such as PKA or PKC 2 mimetic U46619. Our data established that TPα is subject to homologous desensitization of signalling through two key mechanisms involving receptor phosphorylation: one involving a partial PKC-dependent mechanism and another involving a profound and sustained PKG-dependent mechanism engaging NO signalling and endothelial (e) nitric oxide synthase (NOS) activation. Unlike that of TPβ, we found no role for the GRK/β-arrestins in homologous desensitization of TPα. Hence, taken together, these studies demonstrate critical differences in the mechanisms of agonist-induced desensitization of signalling between the TP isoforms and add to the increasing lines of evidence for distinct physiologic roles for TPα and TPβ in the mediation and the regulation of signalling by the potent autocoid TXA2 in humans.In view of these findings, the aim of the current studies was to investigate the mechanism of homologous desensitization of signalling by TPα in response to the TXA22.132P] orthophosphate (8000–9000 Ci/mmol) and [3H]SQ29,548 (50.4 Ci/mmol) was from NEN Life Sciences Products. [3H] IP3 (20–40 Ci/mmol) was obtained from American Radiolabelled Chemicals Inc. Anti-HA 101R monoclonal antibody and anti-HA-3F10-peroxidase conjugated antibody were obtained from BABCO and from Roche Molecular Biochemicals, respectively. Monoclonal anti-eNOS antibody (Clone 3) was obtained from Transduction Laboratories. Anti-phospho-eNOSSer1177(Human)/1179(Bovine) antibody was purchased from Cell Signalling Technology. Thrombin was obtained from Sigma Chemical Co. FURA2/AM and d-myo-inositol 1,4,5-trisphosphate, 3-deoxyhexasodium salt, U46619, N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquino-linesulfonamide, 2HCL (H-89) and 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimide (GF 109203X), NG-Nitro-l-arginine Methyl Ester, Hydrochloride (l-NAME), KT 5823, LY 83583  were obtained from Calbiochem.SQ29,548 and I-BOP were from the Cayman Chemical Company. Anti-Gαq  antibody was obtained from Santa Cruz Laboratories.  SQ29,548 for Scatchard analysis or in the presence of 20 nM [3H] SQ29,548 for saturation radioligand binding experiments as previously described Cells were harvested by centrifugation at 500×2.52+]i) in FURA2/AM preloaded HEK 293 cell lines (2 × 106 cells/dish), each transiently co-transfected with pADVA (10 μg), and pCMV:Gαq (25 μg), was carried out as previously described 2+]i mobilization, HEK.TPα or HEK.TPβ cells (2 × 106 cells/dish) were co-transfected with pADVA (10 μg), pCMV:Gαq (25 μg) along with pRK5:βARK1495–689 (25 μg), encoding a dominant negative form of GRK2/βARK12+/Mg2+-free Hank's buffered salt solution, containing 10 μM HEPES, pH 7.67, 0.1% bovine serum albumin (BSA)) buffer at 107 cells/ml and incubated in the dark with 5 μM FURA2/AM for 45 min at 37 °C. Cells were collected by centrifugation , washed once in an equal volume of HBSSHB, and were finally resuspended in HBSSHB buffer at 107 cells/ml and kept at room temperature in the dark for 40 min. For each measurement of [Ca2+]i mobilization, aliquots of cells were diluted to 0.825 × 106 cells/ml in HBSSHB, 1 mM CaCl2 and FURA2 fluorescence was recorded  at 37 °C with gentle stirring with a Perkin Elmer-Cetus LS50-B spectrofluorometer at excitation wavelengths of 340 nm and 380 nm and emission wavelength of 510 nm, respectively.Measurement of intracellular calcium mobilization  were stimulated with 1 μM U46619 (Primary stimulation) or, as a control, with the vehicle for 4 min; thereafter, cells were collected by centrifugation ; were washed twice in 2 ml HBSSHB, 1 mM CaCl2; and finally resuspended in 2 ml HBSSHB,1 mM CaCl2 and were left to recover over various time intervals (ranging from 0 to 60 min); thereafter, cells re-stimulated with 1 μM U46619 (Secondary stimulation) and changes in [Ca2+]i mobilization were monitored as a function of time. To assess the role of protein kinase (PK) A, PKC or PKG on U46619-mediated desensitization of TPα signalling, cells were pre-incubated with the PKA inhibitor H-89 (10 μM), the PKC inhibitor GF 109203X (50 nM) or the PKG inhibitor KT 5823 (50 nM), for 15 min and respective kinase inhibitors were maintained throughout the primary/secondary U46619-stimulations and during the washes. To assess the role of the nitric oxide (NO) pathway on U46619-mediated desensitization of TPα signalling, cells were pre-incubated with the NO synthase inhibitor l-NAME (1 μM) or the soluble guanylate cyclase (sGC) inhibitor LY 83583 (1 μM) for 15 min and respective inhibitors were maintained throughout the primary/secondary U46619-stimulations and during the washes.Cells were stimulated with the TP agonist U46619 (1 μM) and changes in [Ca2+]i mobilization. Calibration of the signal was performed in each sample by adding 0.2% Triton X-100 to obtain the maximal fluorescence ratio (Rmax) and then 1 mM EGTA to obtain the minimal fluorescence ratio (Rmin). The ratio of the fluorescence at 340 nm to that at 380 nm is a measure of [Ca2+]iKd of 225 nM Ca2+ for FURA2/AM. The results presented in the figures are representative data from at least three or four independent experiments and are plotted as changes in Ca2+i mobilized (Δ[Ca2+]i (nM)) as a function of time (s) upon ligand stimulation. Changes in [Ca2+]i mobilization were determined by measuring peak rises in [Ca2+]i mobilized (Δ[Ca2+]i) and were calculated as mean changes in Δ[Ca2+]i ± S.E.M. (nM).In all cases, the drugs (agonist or inhibitors in 0.01% ethanol in HBS) were diluted in the vehicle HBSSHB such that addition of 20 μl of the diluted drug/inhibitor to 2 ml of cells resulted in the correct working concentration. The vehicle had no effect on U46619-mediated  orthophosphate (8000–9000 Ci/mmol) at 37 °C, 5% CO2. Where appropriate, the PKG kinase inhibitor KT 5823 (50 nM) or its vehicle was added during the labelling period. Thereafter, U46619 (1 μM) or an equivalent volume of vehicle  were added and cells were incubated for 10 min at 37 °C, 5% CO2. Reactions were terminated by transferring the dishes to ice and aspirating the labelled medium and HA-epitope tagged TP receptors were processed and immunoprecipitated using the anti-HA 101R antibody as previously described 32P-labelled proteins. Thereafter, blots were subject to phosphorimage analysis, and the intensities of agonist-induced phosphorylation were expressed in arbitrary units relative to basal (vehicle-stimulated) levels. In parallel experiments, cells were incubated under identical conditions in the absence of [32P] orthophosphate; HA-tagged TP receptors were immunoprecipitated from the same cell lines using the anti-HA 101R antibody and immunoblots were screened using the anti-HA 3F10 horseradish peroxidase-conjugated antibody Agonist-mediated TP phosphorylation in whole cells (1.8–2 × 102.8e) nitric oxide synthase, NOS, NOS, F-[5′-GAG GAA GTG GGC AGG AGA ATG-3′], R-[5′-GTA GTA GAA AGG GGA CAG GAC-3′], amplicon 294 bp; RNA extracted from HEK 293 cells (1.4 μg in 25 μl reactions) were converted to first strand (1°) cDNA using MMLV RT essentially as previously described 6 cells/10-cm dish) were plated some 48 h prior to experimentation. Cells were incubated with vehicle, 1 μM U46619 or 10 U/ml Thrombin for 5 min at 37 °C and aliquots of whole cell protein (100 μg/lane) were analysed by SDS-PAGE/western blot. Blots were initially screened using the anti-phospho eNOS1177 as per supplier's instructions  and, following stripping, were subsequently re-screened using the anti-eNOS (Transduction Laboratories).In order to assess eNOS phosphorylation, HEK.TPα, HEK.TPβ or HEK 293 cells . Statistical analyses were carried out using the unpaired Student's 33.12 receptor 2+]i mobilization and IP3 generation) in response to the TXA2 mimetic U46619 by comparing it to that of the previously characterised TPΔ328We have recently investigated the mechanism of agonist-induced homologous desensitization of signalling by the TPβ isoform of the human thromboxane (TX) A2 mimetic U46619 resulted in efficient mobilization of intracellular calcium . Moreover, U46619-desensitization of TPα signalling was sustained and did not significantly recover even at 60 min following the primary stimulation (p = 0.0001). On the other hand, pre-treatment of HEK.TPα cells with the vehicle, as opposed to U46619 itself, during the primary stimulation followed by washout confirmed that the cells retained the ability to fully signal in response to subsequent stimulation with U46619 . Moreover, desensitization of U46619-mediated [Ca2+]i mobilization by TPΔ328 was transient such that its signalling had fully recovered showing no desensitization at 60 min (Compare p = 0.22). Consistent with these data, primary stimulation of HEK.TPα and HEK.TPΔ328 cells with U46619 also yielded efficient increases in IP3 generation (3 generation by TPα following its secondary restimulation (p = 0.0049), it had no significant effect on IP3 generation by TPΔ328 at 60 min following the primary stimulation (p = 1). Moreover, similar agonist-induced desensitization of [Ca2+]i mobilization and IP3 generation occurred when I-BOP was used as the TP-stimulatory ligand (data not shown). Taken together these data confirm that like TPβ, TPα is subject to near complete and sustained agonist-induced homologous desensitization while, consistent with previous reports Δ328 undergoes partial and transient desensitization suggesting that the major target site(s) involved in homologous desensitization of TPα may be located within its unique C-tail domain.While primary simulation of HEK.TPlization D, consismulation H, p = 1.2+]i mobilization were investigated. Pre-incubation of HEK.TPα or HEK.TPΔ328 cells with H-89 had no significant effect on [Ca2+]i mobilization in response to their primary or secondary stimulation with U46619 (2+]i mobilization by TPα or TPΔ328 in response to primary agonist stimulation (Δ328 cells with GF 109203X fully impaired agonist-induced desensitization in response to its secondary U46619 stimulation (Compare To investigate whether the second messenger-regulated protein kinases (PKs) may play a role in mediating homologous desensitization of TPα, the effects of the PKA and PKC inhibitors H-89 and GF 109203X, respectively, on U46619-induced i mobilization in response to its secondary stimulation with U46619 at 15 min following the initial stimulation (p= 0.55). Taken together, these data confirm that whilst Ser145 within IC2 represents the phospho-target residue involved in partial U46619-induced, GF 109203X-sensitive homologous desensitization of TPΔ328145 also plays a minor though significant role in the homologous desensitization of TPα. However, the major determinant(s) of U46619-induced desensitization of TPα signalling appear to be located within its unique C-tail domain and that PKC may also have a partial role in that desensitization.Hence, it is evident that TPlization E and IP3type TPα E, pre-stresponse E. Simila3.2495–689, a dominant-negative form of GRK2/βARK1495–689 had no significant effect on primary U46619-induced [Ca2+]i mobilization by TPα or TPβ  , it had no effect whatsoever on agonist-induced desensitization of signalling by TPα  family are widely reported to play a central role in homologous desensitization of various GPCRs  Compare B. Thus, 239 has been identified as a critical site in the GRK2/3-mediated homologous desensitization of TPβ 3 domain of TPα but is clearly not targeted through GRK2/3  confirming that the mutation per se did not affect the basic ligand binding or signalling properties of TPαS239A. Similar to that of the wild type TPα  and maximal radioligand binding (Bmax) for each of the HEK 293 cell lines stably over-expressing the respective variant receptors compared well to those previously reported for wild type TPα . Moreover, while both TPαT337A . These data clearly indicate that mutation of Ser239, located within IC3, in combination with either Ser331 or Thr337 does not affect the overall level of TPα desensitization and hence, consistent with our previous data , in each case the level of agonist-induced desensitization was not significantly different to that of their respective single mutants TPαS331A , consistent with our previous data involving TPαS329  in response to U46619. However, pre-stimulation with agonist only partially desensitized signalling by TPαS331,T337A . Thus, taken together it appears that TPα is subject to GF 109203X-sensitive desensitization, where Ser145 and Thr3372 and C-tail domains, respectively, have been identified as the putative PKC-phospho-targets. In addition, Ser331, a site previously identified as a cGMP-dependent PKG site Consistent with the other mutant cell lines, U46619-induced stimulation of HEK.TPα3.4S331A phosphorylation in whole cells/in vivo. Initially, the specificity of the anti-HA antibodies to immunoprecipitate TPs from the respective cell lines, but not from the parent HEK 293 cells, was confirmed  did not interfere with their ability to mobilize iper se  promoting the conversion of  Compare C, it par Compare D. In the Compare E but sig Compare E and F. e)NOS and neuronal (n)NOS but not inducible (i)NOS transcripts  are regulated and established that GRK2/3 plays a major role in that homologous desensitization, with an additional minor role for PKC 357 within the unique C-tail domain of TPβ provides a docking site to recruit and orientate GRK2/3, facilitating its phosphorylation of Ser239 within IC3, impairing Gq coupling, desensitizing TPβ signalling and promoting its agonist-induced internalization 357 determined to be critical for desensitization of TPβ are not found in TPα clearly implying that the mechanism of agonist-induced desensitization of the TP isoforms may indeed differ. Moreover, consistent with that hypothesis, TPβ but not TPα undergoes agonist-induced internalization largely through a GRK/β-arrestin-dependent mechanism 2 mediated by the TPα isoform are specifically regulated thereby aiming to elucidate the mechanism of homologous desensitization of TPα.The platelet-derived prostanoid TXA2+]i mobilization and IP3 generation in response to the TXA2 mimetic U46619. Desensitization of TPα is sustained showing no significant recovery following agonist wash-out and re-stimulation some 60 min following the primary response. On the other hand and consistent with our previous data Δ328, the truncated derivative devoid of the divergent residues of TPα and TPβ, underwent partial and transient desensitization with its signalling fully recovered to that of its primary response at 60 min. Moreover, desensitization of TPΔ328 was blocked by pre-incubation with the PKC inhibitor GF 109203X or by mutation of Ser145 within IC2 to generate TPS145A,Δ328145 within IC2 to generate TPαS145A, a residue common to both TP isoforms and previously identified as the critical PKC phospho-target of both TPΔ328 and TPβ S145A was partially impaired, signalling to 24% of its primary response following secondary agonist stimulation. Hence, collectively these data suggested that whilst Ser145 within IC2 represents the PKC phospho-target residue involved in transient and partial desensitization of TPΔ328145 also plays a minor though significant role in the homologous desensitization of TPα. However, these data also suggest that the major determinant(s) of desensitization of TPα signalling appear to be located within its unique C-tail domain.It was initially confirmed that, similar to TPβ, TPα was subject to complete agonist-induced homologous desensitization as assessed by measurement of i mobilization by TPαS331A and TPαT337A following their secondary stimulation corresponded to 39% and 25% of their primary responses, respectively. Moreover, secondary agonist stimulation of TPαS331,T337A or TPαS145,S331,T337A yielded responses corresponding to 33% and 55% of their primary responses, respectively. The involvement of Thr337, a site previously identified as a target for PKC phosphorylation 145 and Thr337 within the IC2 and C-tail domains are functional PKC targets. In terms of Ser331, it was previously established that TPα, but not TPβ, is subject to desensitization in response to the potent vasodilator nitric oxide (NO) that occurs through direct type 1 PKG-mediated phosphorylation where Ser331 was identified as the PKG phospho-target S331A or TPαS331,T337A. Moreover, pre-incubation with KT 5823 reduced agonist-induced desensitization of signalling by TPα such that in its presence the level of [Ca2+]i mobilized following secondary agonist stimulation corresponded to ~ 38% of the primary response. Similarly, the sGC and NOS inhibitors LY 83583 (30%) and l-NAME (40%), respectively, yielded significant impairments in agonist-induced desensitization of TPα. On the other hand, consistent with previous findings that TPβ is not a target for NO-mediated heterologous desensitization or PKG phosphorylation l-NAME had any measurable effect on TPβ signalling following its primary or secondary agonist stimulation. Phosphorylation of eNOS at Ser1177 (Human)/1179 (Bovine) by phosphatidylinositol 3-kinase (PI3-K)-dependent AKT or, as more recently demonstrated, by Gq/PLCβ/PKCδ pathway, such as in response to vascular endothelial growth factor 1177 to levels comparable, if not greater, than those in response to thrombin stimulation.On the other hand, mutation of Ser3 generation and mobilization of [Ca2+]i. This, in turn, triggers the rapid and dynamic activation of Ca2+/calmodulin-sensitive NO synthases (NOS), such as through Ser1177 phosphorylation and activation of eNOS, promoting the synthesis of NO from l-Arg. NO in turn activates sGC leading to a rise in intracellular cGMP and, in turn, activation of cGMP-dependent PKG, or cGK 331 represents the phospho-target. As TPβ is not a target of PKG phosphorylation 145 and Thr337 contributing, in part, to the desensitization of TPα signalling. Thus, agonist-induced homologous desensitization of TPα is mediated, at least in part, through both PKG and PKC-catalysed mechanisms where Ser331 and Ser145/Thr337 located within IC2 and C-tail domains have been identified as the respective phospho-targets. It is noteworthy that collective mutation of those three sites to generate TPαS145,S331,T337A did not eliminate desensitization of TPα, clearly indicating that other factors may also contribute to agonist-induced desensitization of TPα.Hence, based on data presented herein, we propose a model for the mechanism of homologous desensitization of TPα signalling, as outlined in 2 and C-tail domains. The involvement of PKC in the agonist-induced desensitization of TPα is in keeping with studies by Spurney et al., involving the single murine TP where it was determined that mutation of three putative PKC phospho-Ser residues within its C-tail domain significantly attenuated that desensitization 2+ mobilization, that contributes to vasoconstriction and platelet activation status for example, are regulated by the NO/cGMP signalling cascade, mainly through the direct- or indirect-action of PKG. Amongst the key molecular targets of NO-regulated PKG thus far identified is the IP3 receptor 3-evoked Ca2+ release from intracellular stores 3 receptor-associated cGMP kinase substrate or IRAG 1177/1179 in bovine aortic endothelial cells in a mechanism involving Gq/PLCβ/PKCδ phosphorylation and Ca2+-dependent activation of eNOS 1177 in HEK 293 cells, we have not explored whether that eNOS activation occurs through PKCδ or through the classic PI-3K or other mechanism The most striking feature of the proposed mechanism of homologous desensitization of TPα presented herein is that it mainly involves feedback loops whereby the second messenger kinases PKC and PKG activated in response to its primary signalling bring about desensitization through phosphorylation of critical residues within its IC2 in other cell/tissue types, such as within the pulmonary microvasculature where the local release of NO acts as a compensatory mechanism in response to the proinflammatory and vasoactive effects of TXA22+ dependent eNOS or nNOS isozymes can occur in response to a diverse array of agonists that signal through GPCRs, including the widely documented bradykinin and muscarinic acetylcholine receptors signalling pathways, in addition to TP 2+]i, thereby providing another counter-regulatory mechanism between GPCR/Ca2+- and NOS/cGMP-regulated signalling systems in vitro and in vivo by PKC at Thr58, generating a partially active kinase that is more sensitive to cGMP levels 2-mediated PKC activation contributes to PKG1α activation and TPα phosphorylation/desensitization through the latter mechanism remains to be investigated.Whilst our finding herein that agonist-induced desensitization of TPα entails a closed feedback loop involving eNOS phosphorylation/activation, NO generation and subsequent PKG phosphorylation is a mechanism not typically associated with homologous desensitization of GPCRs in general, it is also in agreement with our previous studies demonstrating that TPα is indeed a direct target of NO/PKG phosphorylation and heterologous desensitization 2 by the counter-regulatory NO, while TPβ remains unchecked by that mechanism. These findings coupled to the fact that TPα, but not TPβ, is also a direct target of prostacyclin-induced heterologous desensitization Hence, in summary, it is indeed evident that there are fundamental differences in the mechanisms of homologous desensitization of TPα and TPβ adding further to the increasing lines of evidence that the TP isoforms may indeed have distinct physiologic roles. A critical difference between those two mechanisms is that agonist-mediated desensitization of TPα does not involve its internalization while desensitization of TPβ involves its recruitment to an intracellular compartment(s), making it susceptible to down-regulation or, alternatively, potentially available for participation in other signalling systems


Our data indicated that treatment of Chinese propolis 6.25 and 12.5 μg/ml for 12 hours increased VEC viability obviously. Exposure to Chinese propolis 6.25, 12.5, and 25 μg/ml for 6 and 12 hours significantly decreased PC-PLC activity and p53 level, and ROS levels were depressed by Chinese propolis 12.5 μg/ml and 25 μg/ml dramatically. The Δψm of VECs was not affected by Chinese propolis at low concentration but disrupted by the propolis at 25 μg/ml significantly, which indicated that Chinese propolis depressed PC-PLC activity and the levels of p53 and ROS in VECs but disrupted Δψm at a high concentration.To understand the mechanisms underlying the anti-inflammatory action of Chinese propolis, we investigated its effect on the activity of phosphatidylcholine-specific phospholipase C (PC-PLC) that plays critical roles in control of vascular endothelial cell (VEC) function and inflammatory responses. Furthermore, p53 and reactive oxygen species (ROS) levels and mitochondrial membrane potential (Δ Propolis is a resinous substance collected by honeybees from the bud and bark of certain trees and plants. It has been used in folk medicine from ancient times in many countries. Recently, it has been reported to possess various biological activities, such as antibacterial , antivirIn recent years, the anti-inflammatory action of propolis has been widely documented –16, and −/− mice; as well, inhibition of PC-PLC activity in vascular endothelial cells (VECs) reduced the expression of vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and monocyte chemotactic protein-1 induced by oxidized low-density lipoprotein contributing to the progression of atherosclerosis [Phosphatidylcholine-specific phospholipase C (PC-PLC), an important member of phospholipase C family, hydrolyzes phosphatidylcholine to produce phosphocholine and diacylglycerol in a number of receptor-stimulated cells. Phosphocholine and diacylglycerol are the most important second messengers that have been implicated in a wide range of cellular response such as cell growth, differentiation, senescence, and apoptosis of mammalian cells , 18. Preclerosis .μg/mL) affected PC-PLC activity in VECs deprived of FGF-2 and serum. Furthermore, we investigated the effects of Chinese propolis on p53 and reactive oxygen species (ROS) levels and mitochondrial membrane potential which were regulated by PC-PLC in VECs.However, the effect of Chinese propolis on PC-PLC activity in VECs deprived of basic fibroblast growth factor (FGF-2) and serum, which leads to inflammation in the vessel wall, is not known. Considering the important roles of PC-PLC in endothelial apoptosis and its proinflammatory properties, we investigated whether Chinese propolis . Medium 199 and fetal bovine serum (FBS) were from Hyclone Lab Inc. (USA). FGF-2 was from EssexBio Group, China. L-α-phosphatidylcholine, sulforhodamine B (SRB), and 2′,7′-dichlorodihydrofluorescin (DCHF) were from Sigma Co. (USA). JC-1 was from Invitrogen (USA). Primary antibodies against p53, β-actin, and secondary antibody (horseradish peroxidase) were from Santa Cruz Biotechnology (USA). All other reagents were ultrapure grade.Chinese propolis was obtained from colonies of honeybees,  Propolis samples were extracted with ethanol at room temperature for 24 hours The ethanol suspension was filtered under reduced pressure, and the filter liquid was then concentrated in a rotary evaporator under reduced pressure at 40°C until reaching a constant weight and then redissolved in ethanol. The prepared propolis was stored under a dry condition at 4°C.2 at 37°C. The cells were seeded on plates coated with 0.1% gelatin and allowed to grow to confluence before experiments. Human umbilical vein endothelial cells (HUVECs) were obtained by using the method of Nachman and Jaffe . HUVECs μg/mL). Chinese propolis was dissolved in ethanol, with final concentration of ethanol in the culture medium <0.1% (v/v). Ethanol at 0.1% (v/v) did not affect cell viability. The morphological changes of cells were observed under a phase contrast microscope  at 6, 12, and 24 hours, respectively.When the VEC cultures reached confluence, the medium was replaced with basal M199 medium (without FBS and FGF-2). Then the cells were divided for treatment: (a) culture in M199 medium with FBS and FGF-2 , (b) deprived of serum and FGF-2 but cultured in M199 medium with ethanol at <0.1% (v/v) (control), and (c) culture in basal M199 medium with Chinese propolis . At 6, 12, and 24 hours, cells were precipitated for 1 hour at 4°C with 100 μL 10% trichloroacetic acid and stained with SRB. The optical density was measured at 540 nm after reconstitution of the dye in 100 μL 10 mM Tris base [VECs were seeded in 96-well cell culture plates and grown to confluence then washed once with basal M199 medium. Then the cells were deprived of serum and FGF-2 and treated with Chinese propolis  by intracellular ROS . After tTo measure mitochondrial membrane potential, the fluorescent dye JC-1 was used. JC-1 exists as a monomer at low mitochondrial membrane potential and emits green fluorescence but forms aggregates and emits red fluorescence at high mitochondrial membrane potential . After tt test by SPSS v11.5 . A P < .05 was considered statistically significant.All experiments were performed in duplicate and repeated at least 3 times. Data are expressed as means ± SD. Statistical analysis involved use of the paired μg/mL for 6, 12, and 24 hours, the cell viability was examined by SRB assay. At 12 hours, Chinese propolis 6.25, and 12.5 μg/mL significantly increased VEC viability , and the propolis 12.5 and 25 μg/mL also depressed p53 level at 6, 12, and 24 hours . It significantly decreased the level of ROS at 25 μg/mL from 6 to 24 hours (**P < .01). However, Chinese propolis 6.25 μg/mL had no effect on ROS level .We used the lipophilic cation JC-1 to evaluate the effect of Chinese propolis on mitochondrial membrane potential in VECs. Chinese propolis at 6.25 and 12.5 otential ; howeverPropolis usually contains a variety of chemical compounds, such as polyphenols , terpenoids, steroids, and amino acid. The composition of propolis depends on the vegetation at the site of collection. Its chemical constituents and antioxidant activities of Chinese propolis have been studied in detail . In addiPC-PLC is a key upstream signal molecule. Miao et al. previous studies showed that PC-PLC played a significant role during VEC senescence and apoptosis , 31. VECp53 is another key protein in VEC associated with the activity of PC-PLC and plays an important role in VEC apoptosis signal transduction pathways. Apoptosis induced by p53 is firmly established as a central mechanism of tumour suppression . Cheng eμg/mL was excessively decreased from 6 to 24 hours; at the same time, mitochondrial membrane potential was decreased, which indicated that excessive decrease of ROS disrupted mitochondrial membrane potential. Thus, the balance of intracellular ROS might be important in cell survival. Very low level of ROS might have a harmful effect as very high level of ROS. And Chinese propolis preparation used may not contain any significant amounts of glucose oxidase that generates H2O2, which can role on to generate hydroxyl radical, and several of the polyphenols present in our preparation may, like quercetin, be taken up by cells which could affect the fluorescence data.ROS are normally generated in the mitochondria and have been identified as important mediators that regulate signal transduction , 35. As μg/mL was more effective than that of 6.25 μg/mL. Chinese propolis at high concentrations might be harmful, so it should be used at safe and effective doses according to different situations. We would like to further investigate the anti-inflammatory effects of mixtures of polyphenols known to be present on propolis.To our knowledge, this is the first report to reveal the effect of Chinese propolis on PC-PLC activity in VECs. Our present results showed that one major mechanism underlying the effect of Chinese propolis on PC-PLC activity might be through regulating p53 and ROS . PC-PLC


Individual tyrosines were then mutated to phenylalanine and their relevance for cellular adhesion and migration was tested experimentally. Functionally important tyrosine residues include two sites within the folded hSH3 domains of ADAP and two at the C-terminus. Furthermore, using a peptide pulldown approach in combination with stable isotope labeling in cell culture (SILAC) we identified SLP-76, PLCγ, PIK3R1, Nck, CRK, Gads, and RasGAP as phospho-dependent binding partners of a central YDDV motif of ADAP. The phosphorylation-dependent interaction between ADAP and Nck was confirmed by yeast two-hybrid analysis, immunoprecipitation and binary pulldown experiments, indicating that ADAP directly links integrins to modulators of the cytoskeleton independent of SLP-76.TCR stimulation leads to an increase in cellular adhesion among other outcomes. The adhesion and degranulation promoting adapter protein (ADAP) is known to be rapidly phosphorylated after T cell stimulation and relays the TCR signal to adhesion molecules of the integrin family. While three tyrosine phosphorylation sites have been characterized biochemically, the binding capabilities and associated functions of several other potential phosphotyrosine motifs remain unclear. Here, we utilize  Formation and maintenance of the “immunological synapse” rely on integrins, adhesion molecules that are indirectly regulated by TCR or chemokine receptor stimulation Simultaneous mutation of both YDDV motifs results in reduced cell conjugate formation and loss of LFA-1 polarization in stimulated Jurkat T cells in vitro phosphorylation to map tyrosine phosphorylation sites in ADAP (486–783) comprehensively. Mass spectrometric analysis reveals several sites of modification that comprise previously identified as well as novel sites. Two of these motifs are located in the folded hSH3 domains of ADAP at helix-sheet interfaces. Peptide pulldown experiments with a linear pYDDV-containing peptide show that several SH2 domain-containing proteins can bind to this motif in addition to SLP-76. Amongst these, actin cytoskeleton modulators Nck1 and Nck2 interact with the ADAP motif in a phosphorylation-dependent manner, thereby providing a direct link between integrin regulation and cytoskeletal rearrangements. Functionally, tyrosine to phenylalanine mutations of SLP-76/Nck interaction sites, of tyrosines in the hSH3 domains and in the C-terminus lead to an attenuation of Jurkat T cell adhesion and migration. This demonstrates that tyrosine phosphorylation of ADAP is more intricate than previously anticipated.Here, we use Antibodies were used for detection of phosphotyrosine , Fyn , Nck , ADAP , and SLP-76 . For immunoprecipitation of ADAP sheep antiserum was used N (aa 486–579), and hSH3C (aa 683–768) were expressed in E. coli and purified utilizing a His6-tag or a GST-tag that was eventually removed. Tyrosine to phenylalanine mutants were created by PCR amplification of the complete pEFBOS vector containing ADAP cDNA using primers with a central missense mutation. GST-Nck1 SH2 and GST-Nck2 SH2 expression clones for in vitro binding assays were kindly provided by Gianni Cesareni and Bruce Mayer 6 expression constructs.ADAP constructs were derived from isoform2 (GenBank NM_199335.2). ADAP-C (aa 486–783), hSH32, 0.5 mM EGTA, 0.1 mM sodium orthovanadate, 150 mM NaCl, 2 mM DTT, and 1 mM ATP at 20°C for 1 h. Tyrosine phosphorylation was detected after SDS-PAGE and western blotting with an anti phosphotyrosine antibody on the Odyssey® infrared imager .Recombinant proteins (45 µM) were phosphorylated by incubation with Fyn kinase (2.3 µM) in 50 mM Tris·HCl, (pH = 7.5), 10 mM MgClOdysseySoftware 2.1 (LI-COR) was used for quantification of phosphorylation signals. Statistical significance was tested with unpaired Student's t-test in Origin software. PyMOL was used for visualization of protein structures 3, 2 mM ac-Ala-Gln  supplemented with 10% FBS (Biochrom) without antibiotics in a humidified atmosphere with 5% CO2. For transfections 2×107 cells were pelleted, washed and resuspended in 350 µl PBS containing Ca2+ and Mg2+ (Biochrom). After addition of 30 µg of DNA a 230 V, 950 µF pulse  was applied in 4 mm cuvettes . Cells were cultured in a mixture of equal amounts of medium from passaged cells and fresh medium for 24 h before analysis. For stable isotope labeling by amino acids in cell culture (SILAC)12/13C6]L-lysine·2HCl, 0.2 g/l [12/13C6]L-arginine·HCl , and 2 mM L-glutamine (Invitrogen). Cells were cultured in SILAC media for nine population doublings.Jurkat cells  were incubated with OKT3 (TCR) for 30 min at 37°C prior to the adhesion assay. Cells were then allowed to adhere for 30 min at 37°C, unbound cells were carefully washed off with 3×1 ml Hanks buffered saline  and bound cells were fixed with 3.5% paraformaldehyde in PBS. The bound cell fraction was determined with the aid of an ocular reticle. Migration assays 5 cells in 200 µl migration assay medium were added to the upper chamber. After 2.5 hours of incubation at 37°C, cells that had migrated through the filter were collected and counted.Adhesion assays were performed as previously described in vitro phosphorylation for MS-analysis according to manufacturer's protocol except for elution which was achieved with 5×500 µl high phosphate buffer .Phosphoprotein Purification Kit  was used for affinity enrichment of phosphorylated proteins from 7 Jurkat T cells were left unstimulated, stimulated with OKT3 (2 µg/ml in 2.5 ml serum-free medium) or SDF-1α (100 ng/ml) at 37°C for 5 min. Ice cold PBS was added to stop stimulation. Pelleted cells were lysed in lysis buffer  with 1% (v/v) NP-40, 1 mM Na3VO4, protease inhibitor cocktail  on ice for 30 min. Cleared lysates were adjusted to [protein] = 5 g/l and incubated with ADAP or Nck antiserum (1∶150) at 4°C overnight. Immune complexes were precipitated with Protein-G-agarose (Sigma) for 2 h at 4°C and washed three times with TBS in centrifugal filter devices . Bound proteins were eluted by boiling in SDS sample buffer.For immunoprecipitation 5×10Cosedimentation of purified Nck and ADAP was investigated by phosphorylation of purified ADAP constructs (4.5 nmol) with Fyn (0.23 nmol) for 1 h. 2.3 nmol of the phosphorylated ADAP protein were then incubated with GSH-sepharose coupled GST-Nck1/2 SH2 (1.6 nmol) for 2 h at 4°C. The matrix was washed three times with TBS and bound proteins were eluted by boiling in SDS sample buffer.8-GAL1TATA-lac, LYS2::(lexAop)4-HIS3TATA-HIS3MATa, his3Δ200,trp1-901, leu2-3,112, ade2, lys2-801am, GAL4, gal80, cyh2, can1, ura3::(lexAop)] was co-transformed with a plasmid (pBTM117c) encoding the LexA-ADAP-C fusion and a plasmid (pASZ11-DM) encoding Fyn kinase domain (aa 251–520). Prey proteins were cloned as Gal4-AD fusions (pACT4-DM) and used to transform MATalpha strain L40ccα. Yeast strains were mated on YPD for 36 h. Diploid yeast were grown on SD media supplemented with histidine and uracil (SD2) for 3d. Interacting proteins were identified by growth on selective plates  and lacZ reporter gene activation assays after 5–7 days Y2H experiments were performed as described previously 586PIEDDQEV(p)YDDVAE600 were synthesized by standard solid-phase peptide synthesis (Fmoc chemistry). The N-terminal cysteine was attached to the ADAP sequence 586–600 to allow for covalent immobilization of the peptides on agarose beads using SulfoLink® Coupling Gel (Pierce) in accordance with manufacturer's instructions. Peptide binding assays were performed as duplicates in a crossover manner. Hence, each form of matrix-bound peptide (phosphorylated and non-phosphorylated) was incubated with either labeled or unlabeled cell lysates resulting in two independent pull down experiments. Incubation of 20 µl agarose beads (15 nmol peptide) was performed with 1.5 mg of total protein for 1 h at RT followed by four washing steps to remove unspecific binders. Bound proteins were eluted from the matrix with SDS sample buffer at 95°C for 5 min. Labeled and unlabeled samples were combined and proteins separated by PAGE on Tris-glycine gradient gels (4–20%).Isotope labeled Jurkat T cells were washed three times with ice cold PBS before lysis (see above) for 30 minutes on ice. Cell debris was removed by centrifugation at 16,000×g at 4°C for 10 minutes. Lysates were used immediately for peptide binding assays. Peptides of the type CRThe Coomassie-stained gel lane was cut into 40 slices. These were washed with 50% (v/v) acetonitrile in 50 mM ammonium bicarbonate, shrunk by dehydration in acetonitrile and dried in a vacuum centrifuge. The gel pieces were re-swollen in 10 µl of 50 mM ammonium bicarbonate containing 50 ng trypsin . After 17 h of incubation at 37°C, 10 µl of 0.5% (v/v) trifluoroacetic acid in acetonitrile was added, samples were sonicated for 5 min, and the separated liquid was taken to dryness under vacuum. Samples were reconstituted in 6 µl of 0.1% (v/v) TFA, 5% (v/v) acetonitrile in water.Peptides were analyzed with a reversed-phase LC system coupled to an LTQ-Orbitrap instrument. Details of LC-MS/MS procedures are provided as supporting text, Quantification was carried out using the Mascot Distiller Quantitation Toolbox  and was based on calculations of isotope intensity ratios of at least two arginine- or lysine-containing tryptic peptides with individual MASCOT scores indicating at least homology. Relative protein ratios were calculated from the intensity-weighted average of all peptide ratios. Proteins displaying enrichment factors >5 in two independent replicates were considered to be specific binding partners. Standard deviations of the quantification for individual proteins were obtained from Mascot Distiller. Analytical reproducibility was determined by multiple LC-MS measurements of selected samples showing that the experiment-to-experiment deviation of protein ratios is less than 25%.Enzymatic in-gel digestion with either trypsin and AspN or multiple proteases followed by nanoLC-MS/MS analysis were performed with Q-TOF or MALDI-TOF mass spectrometers as previously described in vitro phosphorylation of ADAP-C (aa 486–783) with Fyn kinase and mass spectrometric analysis. ADAP-C contains 13 of 16 tyrosines including the known phosphorylation sites, the two folded hSH3 domains, and all tyrosines with proposed functions . In addition, the tyrosine phosphorylation site at Y571 of hSH3N that is located at the very end of the domain . Proteomic approaches in Jurkat cells, which relied on pervanadate treatment are in agreement with several of the sites defined in our study (2 = 54∶44∶2) resemble the charge distribution of the inner leaflet of the plasma membrane. Of course, a more dramatic influence of phosphorylation in the context of cellular lipid composition and membrane curvature cannot be ruled out but we believe that phosphorylation is not triggering membrane dissociation. More likely, phosphorylation of the YGY motif might attract novel binding partners. For SH2 domain binding to this motif, the domains would have to unfold (C maintains its fold (data not shown), however lipid binding might shift this equilibrium towards an unfolded conformation. Future experiments have to show the extent of structural changes imposed by phosphorylation and whether phosphorylation-dependent protein complex formation exists for hSH3C.In this report, we comprehensively describe tyrosine phosphorylation sites within the T cell protein ADAP and demonstrate their role for T cell adhesion and migration. Individual phosphorylation sites and their functional role had been described in Jurkat T cells ur study . Our funo unfold , since tStrikingly, we find that the pYDDV motif selectively attracts proteins that take part in TCR proximal signaling which hints towards a function of this ADAP motif as a central hub for phosphorylation-dependent interactions. Six out of the seven highly enriched proteins found in our experiments are known constituents of the TCR proximal signaling complex and interestingly they were identified from a pulldown of unstimulated Jurkat cells. This indicates that the SH2 domains of these proteins are in a receptive conformation and are not blocked by intramolecular inhibition.3 and diacylglycerol. Neither of these two second messengers binds with significant affinity to the lipid-binding hSH3C domain of ADAP (data not shown). In contrast, the educt of the reaction, phosphatidylinositol--bisphosphate, interacts with hSH3C with a ∼20 µM KD when assuming a simplified two-state binding model 3 by PI3-kinase may counteract the depletion of PIP2 thereby keeping ADAP at the membrane at sites of high PIP3 concentration. However, whether such rearrangements and geometrical confinements are of relevance for cellular behavior remains to be investigated. The identification of RasGAP as a potential binding partner of ADAP raises the question whether there is a connection to the Ras signaling pathway or whether this molecule may act on Rap1 in the context of T cell signaling. The exchange factor RasGrp1 has been reported to bind to SKAP55, albeit the outcome of SKAP55 depletion in T cells remains controversial For the proteins listed in 771DDI774 of ADAP resulted in the identification of the same interaction partners (data not shown), indicating that such a dynamic exchange of interactors might be of importance for complex formation.Since the amount of immobilized peptide is high, relatively low affine SH2:peptide interactions might be captured by our approach . Howeverin vitro binding of Nck SH2, and the yeast two-hybrid experiments clearly argue for a direct interaction between ADAP and Nck. Recently, Lettau et al. identified the interaction of ADAP and Nck-SH2 in a screen for Nck binding partners C domain. This domain is 38 amino acids downstream of the second YDDV motif and would allow for a close proximity between the adapter and the membrane. This, in turn could promote the anchorage of the actin cytoskeleton at membrane attachment sites. Notably, there is an Ena/VASP/EVL interaction site in between the two YDDV motifs and VASP was previously described as colocalizing with ADAP at the T cell:APC junction and the membranes of the nascent phagosomes The experiments using SLP-76 deficient Jurkat cells, −/− T cells may be explained by a functional redundancy between Fyn and Lck Several of the other phosphorylation sites identified in our study might also harbor interesting functions. The motif around Y571 resembles an ITIM with predicted binding to CRKL, SHIP-2 or Tec SH2 domains but none of these interactions has been experimentally verified so far. Likewise, Y780 could represent a cognate motif for SH2 domains of T cell signaling proteins SH2D2A, HSH2D, and GRAP. The complexity of phosphorylation dependent signaling is further increased by parallel targeting of one protein by several kinases. For instance, a GPS2.0 based kinase prediction File S1Supporting text.(0.04 MB DOC)Click here for additional data file.Figure S1Kinetics of ADAP phosphorylation by Fyn do not depend on substrate concentration. A ADAP-C (486-783) phosphorylation during incubation with Fyn. [Fyn] = 0.29 µM, molar ratios of ADAP as indicated. Time course of endpoint-normalized fluorescence intensities after Western blotting and immunodetection of phosphotyrosine. Average of three experiments ± SD. B Curve shape parameter a from non linear curve fitting plotted against substrate concentration. Results from individual results (open symbols) and average ± SEM.(0.39 MB TIF)Click here for additional data file.


Chronic exposure to inorganic arsenic (iAs) found in the environment is one of the most significant and widespread environmental health risks in the U.S. and throughout the world. It is associated with a broad range of health effects from cancer to diabetes as well as reproductive and developmental anomalies. This diversity of diseases can also result from disruption of metabolic and other cellular processes regulated by steroid hormone receptors via aberrant transcriptional regulation. Significantly, exposure to iAs inhibits steroid hormone-mediated gene activation. iAs exposure is associated with disease, but is also used therapeutically to treat specific cancers complicating an understanding of iAs action. Transcriptional activation by steroid hormone receptors is accompanied by changes in histone and non-histone protein post-translational modification (PTM) that result from the enzymatic activity of coactivator and corepressor proteins such as GRIP1 and CARM1. This study addresses how iAs represses steroid receptor-regulated gene transcription. PTMs on histones H3 and H4 at the glucocorticoid receptor (GR)-activated mouse mammary tumor virus (MMTV) promoter were identified by chromatin immunoprecipitation analysis following exposure to steroid hormone±iAs. Histone H3K18 and H3R17 amino acid residues had significantly different patterns of PTMs after treatment with iAs. Promoter interaction of the coactivator CARM1 was disrupted, but the interaction of GRIP1, a p160 coactivator through which CARM1 interacts with a promoter, was intact. Over-expression of CARM1 was able to fully restore and GRIP1 partially restored iAs-repressed transcription indicating that these coactivators are functionally associated with iAs-mediated transcriptional repression. Both are essential for robust transcription at steroid hormone regulated genes and both are associated with disease when inappropriately expressed. We postulate that iAs effects on CARM1 and GRIP1 may underlie some of its therapeutic effects and as well be associated with its toxic effects. We found that iAs represses GR-mediated chromatin remodeling and transcription initiation and that methylation and acetylation at histone H3R17 and H3K18 respectively, decreased within minutes of iAs addition. Both of these histone PTMs are associated with transcriptional activation at steroid hormone-regulated promoters. Additionally, it was determined that CARM1 was absent from the promoter after treatment with iAs. Unexpectedly, while CARM1 may be a target for iAs, GRIP1 is also a probable target even though unlike CARM1 it was still associated with the promoter when cells were treated with iAs. Finally, the data suggest iAs-inhibited transcription is mediated through an indirect effect on one or both of these coactivator proteins that may be via deregulation of a cell signaling pathway.To determine whether iAs inhibits GR-mediated transcription from a stably integrated MMTV-chloramphenicol acetyltransferase (MMTV-CAT) reporter gene, 1470.2 cells were treated with 100 nM Dex±iAs at a range of concentrations that can be found in drinking water. In contrast to a transiently expressed reporter gene, stably integrated MMTV-CAT is associated with histone proteins in a regularly spaced chromatin conformation To determine whether iAs inhibited transcription at initiation or elongation cells were treated with 5 nM Dex±8 µM iAs and the amount of initiated CAT transcript from the MMTV promoter was determined by nuclear run-on analysis. Treatment with 5 nM Dex was used to slow the transcription process to enable evaluation of early events in activation. Dex alone increased transcript initiation with the peak at about 120 min and decreased initiation by 180 min, indicative of transcriptional repression . In contAccumulated CAT mRNA was measured by qRT-PCR and by 2 hours there was significantly more CAT mRNA with Dex alone than with Dex plus 8 µM iAs , in agreGRs are predominantly cytoplasmic prior to ligand binding and translocate to the nucleus and to targeted GREs when ligand is bound to the receptor To determine whether iAs affects the DNA-binding kinetics of ligand-bound GR, electromobility shift assay (EMSA) competitions were done. Nuclear extracts (NEs) made from cells treated for 30 min with 50 nM Dex alone or 50 nM Dex plus 8 µM iAs were incubated with a radiolabeled consensus GRE Activation and repression of transcription is accompanied by changes in histone PTMs and such modifications occur at the MMTV promoter on both histones H3 and H4 Following treatment with 5 nM Dex±8 µM iAs there were no significant differences between treatments in acetylation at histone H4, at amino acids H4K5, H4K8, or H4K12 or on histone H3 at H3K14 and H3K4 (data not shown). These amino acids were targeted because they have been shown by others to undergo PTM changes upon activation of transcription. Histone H3K18 acetylation (H3K18ac) occurs with H3R17 methylation (H3R17me) on the estrogen-regulated pS2 promoter To determine whether H3R17me correlates with H3K18ac in response to activation by GR, cells were treated with 5 nM Dex±8 µM iAs. H3R17me increased by 15 min of treatment with Dex alone, but not in the presence of iAs . TogetheBoth CBP and p300 are protein acetyltransferases that interact with the MMTV promoter To determine whether over-expression of CBP could restore transcription in cells treated with Dex+iAs, cells were transfected with an expression plasmid for CBP and after recovery were treated for 24 hours with 5 nM Dex±8 µMiAs. Over-expressed CBP was unable to restore transcription in iAs-treated cells when compared to transcription seen with Dex alone . Thus, aCARM1 is a protein methyltransferase (PRMT 4) that specifically targets H3R17 for methylation The p160 coactivator GRIP1 interacts with GR and CARM1 interacts with the promoter by binding to the C-terminal domain of GRIP1 In vitro pull-down assays further tested CARM1 interaction with the MMTV promoter in response to Dex + iAs  analysis of NEs (data not shown). There was no decrease in CARM1 bound at any concentration of iAs added  cells treated with Dex+iAs compared to Dex alone . In cellTranscription from the SGK promoter was inhibited by iAs treatment similarly to that at the MMTV promoter . To deteNucleosomal Methylation Activator Complex (NUMAC) on an ER-target gene where they physically interact with each other Although we see little difference in initiated transcripts after 60 minutes of treatment with 5 nM Dex + 8 µM iAs compared to Dex alone  we see aGRIP1 was found at NucB on the MMTV promoter following Dex + iAs treatment but CARM1 was not which suggested that iAs may inhibit transcription by disrupting the CARM1-GRIP1 interaction . FurtherTrim24 forms a complex with CARM1 and GRIP1 and its role may be in stabilizing the CARM1-GRIP1 interaction The GRIP1-CARM1 interaction domain on either protein may be a target for iAs resulting in CARM1 dissociation from GRIP1, or alternatively, another protein that stabilizes the CARM1-GRIP1 interaction could be an iAs target. The first possibility would be consistent with iAs mediated changes in interaction domain PTMs that affect CARM1-GRIP1 protein-protein interaction. CARM1 associates with promoters by binding to the C-terminal AD2 domain of GRIP1/SRC2 via CARM1s central domain The stability of CARM1 at promoters may also be affected by acetylation of H3K18 and H3K23 The apparent level of H3K18ac and H3R17me in response to iAs in these experiments was below basal levels . It is pin vitro system 7 µM iAs was effective in inhibiting Dex binding to GR. However, this concentration of iAs is not an achievable intracellular concentration because it would be toxic and result in cell death. In our analyses by ICP-MS of intracellular As+3 we found that treatment of cells with 8 µM iAs for 15 minutes to 1 hour, resulted in only 0.1 to 1.5 µM intranuclear As+3 with a correlation between the length of time of treatment and the amount of As+3 detected (data not shown). Note that these are the concentrations used in the in vitro system to determine whether iAs is acting directly or indirectly on proteins at the promoter  and an androgen 2. It is possible that ATO has a different affect on coactivator interaction than NaAsO2 but this is unlikely since both forms are sources of trivalent inorganic arsenic (As3+). It is also certain that after 24 hours of treatment the promoter-associated proteins would be very different than at 30 min after treatment. Because of the significant differences in the experimental approaches used it is difficult to determine from these data whether the dynamics of GRIP1/TIF2 interaction are the same or different in response to iAs and steroid hormone at GR and AR-regulated promoters.Because both CARM1 and one of the p160 coactivators  are essential to transcriptional regulation at all steroid hormone receptor-regulated promoters, identification of CARM1 and GRIP1 as players in iAs-mediated transcriptional inhibition raises the possibility that iAs may repress transcription by other steroid hormone receptors similarly. In support of this, we have found that iAs-mediated inhibition of an estrogen-responsive promoter in MCF7 breast cancer cells is functionally related to iAs effects on CARM1 and on SRC3/AIB1 a GRIP1 homologue that interacts with ERs (manuscript in preparation). While this manuscript was in preparation it was reported that TIF2, the human homologue of GRIP1/SRC2, does not bind to an AR-regulated promoter in LNCaP cells 24 hours after treatment with arsenic trioxide  contributing to possible mechanisms in iAs-mediated remission in acute promyelocytic leukemia (APL) 2) (ScienceLab.com).The 1470.2 cell line derived from the mouse adenocarcinoma parent line, C127i, constitutively expresses GR and has multiple copies of stably integrated MMTV-chloramphenicol acetyl transferase (MMTV-CAT) Cells were cross-linked with 1.5 mM Ethylene Glycol-bis (Succinimidylsuccinate) (EGS)  at 25°C for 25 min followed by 1.0% formaldehyde at 25°C for 10 min  or with formaldehyde only (Histone PTMs). Nuclei were isolated, and DNA digested to predominantly monosomes with micrococcal nuclease (0.0375 U/µg nucleic acid)  37°C for 6 min. ChIP was as described by the Upstate Biotechnology ChIP protocol (Upstate Millipore.com). Incubation with specific antibody or non-immune IgG was overnight at 4°C, protein A sepharose or magnetically coupled Protein G beads  were used to isolate immune complexes. In Sequential ChIPs antibody-antigen complexes from the first Ip were eluted by incubation in 10 mM DTT for 30 min at 37°C. Samples were diluted in 20 volumes 1% triton X-100, 2 mMEDTA, 150 mM NaCl, 20 mM Tris 8.0 and antibody added. Wash buffers were High salt , Low Salt , LiCl wash . Elution from beads was in 50 mM NaCl, 10 mM Tris 7.5, 5 mMEDTA with 0.5% or 1.2% SDS for 15minutes at room temp. DNA was purified with MinElute columns (Qiagen) and quantified by fluorimetry with PicoGreen  on a Synergy HT plate reader  or by NanoDrop spectrophotometer . Non-immune rabbit IgG was used to determine non-specific antibody interactions and was subtracted from specific interactions. Primers to an unrelated genomic region, ribosomal protein L30 (RPL30)  or to 5S ribosomal DNA were used to determine that the changes seen were specific to the promoter.ChIP DNA was analyzed by quantitative real-time (qPCR) with a TaqMan MGB Probe with a 6-FAM reporter and primers to MMTV Nuc B  , nuclei were isolated/extracted in Buffer C  and dialysis was for 2 hr against Buffer D . Protein was measured by Bradford assay .Mini-Dignam NEs were made 32P]-end-labeled wtGRE, 5′-GATCCGGTacaATCtgtTCTA-3′EMSAs included 20 µg of NE in Buffer D with 30–60 fmol of -UTP (3000 Ci/mmol) . and incubated for 45 min 30°C. RNA was extracted with TRI Reagent LS  and equal counts were added to nitrocellulose filters  previously blotted with PCR-amplified CAT reporter DNA (1 µg of 1 kB coding region), an equal amount of pUC18 DNA for background subtraction, and 5 s DNA for normalization. Hybridization was at 42°C in 50% formamide, 5x SSPE, 3x Denhardt's, 100 µg/ml yeast tRNA and 0.5% SDS for 12–16 h. Visualization/Quantification was on a Storm Typhoon PhosphorImager and ImageQuant analysis .Isolated nuclei (0.5 mg) were resuspended in 100 µl , 10 mM EDTA, 0.5% SDS, 100 µg/ml proteinase K, and digested overnight at 37°C to extract genomic DNA. DNA was prepared for qPCR by Min-Elute columns (Qiagen) and quantified by NanoDrop spectrophotometer . qPCR was with 15–30 ng total DNA and primers that flank the SacI site in NucB (see Table SI). A PCR product indicated chromatin inaccessibility because the SacI site was not accessible. Quantification of PCR product was by the Comparative C(t) method with normalization to β-Actin. Percent Accessibility = (1− (Treated/Untreated)) x100.Nuclei were harvested from treated cells and a portion quantified after proteinase K digestion (InVitrogen). 50 µg nuclei were digested with 10 U/µg SacI, New England Biolabs, for 15 minutes at 37°C in 50 µl of 50 mM NaCl, 50 mM Tris pH 8.0, 1 mM MgCl2, 50 µM each dTTP aS, dGTP aS, dCTP aS , 18 µM Biotin-14-dATP , 10 U Klenow  at 25°C for 15 min and attached to streptavidin-coated magnetic beads using the Dynabead Kilobase Binder Kit  in 200 µl Kit binding buffer as described by Fletcher et al A 1.8 kb Sph1/Nco1 fragment of the MMTV LTR from the pGEM3ZFM-LTRCAT plasmid that includes Nucs A-F of the MMTV promoter, was biotinylated , 0.5% Sarkosyl). Wash 2: SB with 200 mM KCl. Wash 3: SB with 2 mg/ml BSA. Washes 1 and 3 were at 25°C and wash 2 at 4°C. Assembled DNA was stored in SB with protease inhibitor cocktail  no more than 2 days at 4°C before use.Bead DNA (Nucs A–C) was assembled with HeLa core histones using Active Motif's (Carlsbad CA) Chromatin ATP-dependent Assembly Kit that utilizes purified recombinant human chromatin assembly complex ACF, and histone chaperone NAP-1 with modifications. Post assembly, 3×200 µl washes were done in Wash 1: Storage Buffer (SB)(10 mM HEPES (pH 7.8), 10 mMKCl, 1.5 mMMgCl2, 0.1% NP-40, 1 mM DTT, 0.5 mM PMSF, 1∶100 protease inhibitor cocktail  and resuspended at 0.01 ug/ul. Reaction components were 31 ul Pull-down buffer, 2 ul 50 mg/ml acetylated BSA , 0.5 ul poly (dI:dC). poly (dI:dC) (Pharmacia) 1.0 ug/ul), 60 ug Nuclear Extract, 0.05 ug bead DNA, Buffer D to 50 ul on ice in a 96 well round-bottom polypropylene plate (Costar). After incubation at 30°C for 15 min immobilized bead DNA was magnetically immobilized  to remove reaction mix and 2X 50 ul washes in cold Pull-down buffer. Bead DNA-bound proteins were separated on denaturing 4–20% Tris-Glycine gels , followed by Western Blot analysis.Chromatin assembled bead DNA was washed twice in Pull-Down buffer  and 4 mM acetyl CoA , extracted with ethyl acetate and CAT activity was measured by thin layer chromatography. Acetylated products were visualized on a Molecular Dynamics PhosphorImager and analyzed with ImageQuant . CAT activity was expressed as percent conversion .Five micrograms protein was incubated for 30 min at 37°C with [SEM. Statistical significance was determined by two-tailed t-test assuming equal variances.Data are expressed as means and Table S1PCR Primer sequences(0.04 MB DOC)Click here for additional data file.Table S2Antibodies(0.00 MB DOC)Click here for additional data file.